VETERINARY 
BACTERIOLOGY 
A TREATISE ON THE 
BACTERIA, YEASTS, MOLDS, AND PROTOZOA 
PATHOGENIC FOR DOMESTIC ANIMALS 
BY 
ROBERT EARLE BUCHANAN, Ph.D. 
PROFESSOR OF BACTERIOLOGY IN THE IOWA STATE COLLEGE 
OF AGRICULTURE AND MECHANIC ARTS; ■ BACTERIOLOGIST OF 
THE IOWA AGRICULTURAL EXPERIMENT STATION 
ANO 
CHARLES MURRAY, B. Sc., D. V. M. 
I 
PROFESSOR OF VETERINARY RESEARCH. IOWA STATE COLLEGE 
THIRD EDITION 
THOROUGHLY REVISED 
PHILADELPHIA AND LONDON 
W. B. SAUNDERS COM PA NY 
1922 Copyright, 1911, by W. B. Saunders Company. Revised, reprinted, and recopyrighted 
September, 1916. Reprinted July, 1917. Revised, reprinted, and 
recopyrighted January, 1922. 
Copyright, 1922, by W. B. Saunders Company 
MADE IN U. S. A. 
PRESS OF 
W. B. SAUNDERS COMPANY 
PHILADELPHIA PREFACE TO THE THIRD EDITION 
A second revision of the Veterinary Bacteriology has been 
necessitated by the advances in this field in the last several 
years. A more adequate discussion of the classification and re- 
lationships of microorganisms has been included. The newer 
conceptions of acidity on the basis of hydrogen ion concentra- 
tion has received increased emphasis. Several of the chapters 
on specific pathogenic bacteria have been completely rewritten, 
and others extensively revised. 
R. E. Buchanan. 
Charles Murray. 
Iowa State College, Ames, Iowa. 
January, 1922. PREFACE 
The present volume is a revision of the lectures on veterinary 
bacteriology given during the past six years to classes in the Division 
of Veterinary Medicine in the Iowa State College. It constitutes 
a serious attempt to put in usable form that fund of knowledge 
concerning bacteriology which the student of veterinary medicine 
should master. It is in no sense a text on pathology, and discus- 
sion of purely pathological subjects has been minimized as much as 
possible. The intention has been to confine attention as far as 
practicable to those topics that unquestionably lie in the province 
of bacteriology. This has been defined to include a discussion 
of immunity and of the pathogenic bacteria, yeasts, molds, and 
protozoa. 
The book is not intended to serve as a manual of laboratory 
practice, hence detailed discussion of methods and technic has 
been omitted. Methods of significance in diagnosis or treatment 
are given in greater detail in the discussion of specific organisms. 
Several organisms causing diseases of man not transmissible 
to lower animals have been included. In all cases they are closely 
related to organisms having significance to the veterinarian, they 
cause diseases which are commonly confused with somewhat similar 
diseases of lower animals, or they are valuable as illustrations 
of methods of immunization, treatment, or diagnosis. Such 
organisms are relatively few in number. 
A group system of discussion of the pathogenic bacteria has 
been adopted. The classification used has proved very helpful in 
my own classwork. The groupings used are not entirely satis- 
factory, in part due to the fact that some of the species have not 
been adequately described and differentiated in the literature. 
An effort has been made to point out the deficiencies in our present 
knowledge, both to give a better balanced presentation of the sub- 
ject and to stimulate interest in the solution of the problems. 
9 10 
PREFACE 
The pathogenic protozoa constitute a group which is particu- 
larly difficult to treat adequately, largely due to the rapid growth 
of the subject. Relatively few of the forms, moreover, are of 
immediate interest to the North American student. 
R. E. Buchanan. 
Iowa State College, Ames, Iowa. CONTENTS 
SECTION I 
MORPHOLOGY, PHYSIOLOGY, AND CLASSIFICATION 
OF BACTERIA 
PAGE 
Chapter I.-Introduction 17 
Definition of Bacteriology, 17.-Scope of Bacteriology,17.-Scope of Veterinary 
Bacteriology, 19.-The Microscope and Its Influence, 19.-Nature and Classi- 
fication of Microorganisms , 21.-Spontaneous Generation, 21.-Relationships 
of Microorganisms to Fermentation and Decay, 22.-Relationships of Microor- 
ganisms to Disease, 22.-Development of Laboratory Methods, 23.-Develop- 
ment of Theories of Immunity, 24.-Development of Sanitary Science and Pre 
ventive Medicine, 24. 
Chapter II.-Morphology and Relationships of Microorganisms 
Concerned in Disease Production 26 
Position of Pathogenic Microorganisms, 26.-Differentiation of Animals and 
Plants, 26.-Subdivisions of the Thallophytes, 27.-Morphology of Bacteria, 28. 
-Shape of Bacteria, 28.-Grouping of Bacterial Cells, 29.-Size of Bacteria, 
31.-Histology and Structure of Bacteria, 32.-Reproduction in Bacteria, 36. 
-Morphology of the Yeasts, Saccharomycetes, and Blastomycetes, 38.-Form, 
Size, and Grouping of Yeasts, 39.-Histology and Structure of the Yeasts, 39. 
--Yeast Protoplasm and Cell Inclusions, 39.-Reproduction in Yeasts, 40.- 
Morphology oj the Hyphomycetes or Molds, 41.-Form and Size of Hyphomy- 
cetes or Molds, 41.-Histology and Structure of Molds, 42.-Reproduction of 
Molds, 43.-Morphology of the Protozoa, 44.-Form and Size of Protozoa, 45.- 
Histology, 45.-Reproduction, 45. 
Chapter III.-Physiology of Microorganisms 46 
Food Relationships of Microorganisms, 46.-Composition of the Cell, 46.- 
Sources and Kinds of Foods, 46.-Moisture Relationships of Microorganisms, 
47.-Respiration of Microorganisms, 48.-Growth Rates and Death Rates, 49. 
-Rates of Growth, 49.-Rates of Death, 50.- Temperature Relations of Micro- 
organisms, 51.-Optimum Temperature, 51.-Minimum Temperature, 52.- 
Maximum Growth Temperature, 52.-Growth Temperature Range, 52.- 
Thermal Death-point, 52.-Light Relationships of Microorganisms, 53.-Effect 
of Electricity on Bacteria, 54.-Relationships of Microorganisms to Chemicals. 
54.-Chemotaxy, 55.-Tropisms, 56.-Influence of Reaction of Medium on 
Growth, 56.- Antiseptics and Disinfectants, 56.-Theories of Action of Anti- 
septics and Disinfectants, 57.-Characteristics of an Ideal Disinfectant, 57.- 
Disinfectants and Antiseptics in Common Use, 59.-Alkalies, 60.-Salts of the 
Heavy Metals, 60.-Adjustment of Organisms to Osmotic Pressure, 62.-Sym- 
biosis, Antibiosis, and Commensalism, 63.--Pigment Production by Microorgan- 
isms, 63.-jLight Production by Microorganisms, 64.-Fermentation and Enzyme 
Production, 64. 
Chapter IV.-Changes of Economic Significance Brought About 
BY NoN-PATHOGENIC ORGANISMS 68 
Production of Alcohol, 69.-Production of Acids, 69.-Decay and Putrefac- 
tion, 71.-Reduction Processes in Inorganic Compounds, 73.-Oxidation of 
Inorganic Compounds, 74.-Miscellaneous Changes, 81. 
Chapter V.-Classification of Microorganisms 82 
Discussion of Principles of Nomenclature, 82.-Classification of Bacteria, 83.- 
Order Eubacteriales. The True Bacteria, 84.-Key to the Families of the 
Eubacteriales, 85.-The Family Coccacese. The Spherical Bacteria, 85.- 
Key to the Genera of the Coccacese, 85.-The family Bacillaceae. The Spore 
Bearing Bacteria, 87.-The Family Bacteriaceae, 88.-Key to the Genera of 
Bacteriaceae, 88.-The Family Pseudomonadaceae, 89.-The Family Spiril- 
laceae, 89.-Order Actinomycetales. The Thread Bacteria, 90.--Key to the 
Genera, of Actinomycetales, 90.-Order Spirochae tales, 92.-Classification of 
Yeasts, 93.-Classification of the Molds, 93. 
11 12 
CONTENTS 
SECTION II 
PAGE 
Chapter VI.-Sterilization 94 
Sterilization by the Flame, 94.-Sterilization by Hot Air, 94.-Sterilization by 
Streaming Steam, 95.-Sterilization by Steam Under Pressure, 96.-Steriliza- 
tion at Temperatures Lower than Boiling-point, 98.-Sterilization by Addition 
of Chemicals, 98.-Sterilization by Filtration, 98. 
Chapter VII.-Culture-media and Their Preparation 100 
Adjustment of the Reaction of Media, 100.-Nature of Nutrients Required by 
Bacteria, 104.-Liquid Media, 104.-Bouillon or Beef Broth from Meat, 104.- 
Bouillon or Broth from Beef Extract, 105.--Sugar-free Broth, 105.-Sugar 
Broth, 105.--Glycerin Broth, 105.-Serum Broth, 105.-Dunham's Solution, 
105.-Beerwort, 105.-Milk, 105.-Synthetic Media, 106.-Liquefiable Solid 
Media, 106.-Nutrient Gelatin, 106.-Other Gelatin Media,106.-NutrientAgar 
107. - Blood-serum Agar, 107.-Chocolate Agar, 107.-Hormone Agar, 107.- 
Other Agar Media, 108.--Endo-agar-fuchsin Medium, 108.-Simplified Endo 
Agar, 108.-Eosin-methylene-blue Agar, 109.-Non-Liquefiable Media, 110.- 
Potato, 110. - Other Vegetable Media, 110. - Blood-serum, 110. - Egg 
Medium, 111. 
Chapter VIII.-Biochemical Tests 112 
Acid and Alkali Production. Determination of Changes in Hydrogen Ion 
Concentration, 112.-Determination of Acid Production, 113.-Gas Produc- 
tion, 114.-Reduction Processes, 115.-Alcohol Production, 116.-Aldehyde, 
116.-Acetyl Methyl Carbinol, 117.-Determination of Oxygen Relationships, 
117.-Indol Production, 118.-Thermal Death-point, 118.-Efficiency of Dis- 
infectants, 119.-Phenol Coefficient, 119. 
Chapter IX.-Microscopic Examination and Staining Methods. . 121 
Oil Immersion Objectives, 121.-Measuring Bacteria, 122.-Examination of 
Living Bacteria-Hanging Drops, 122.--Staining Methods, 123.-Mordants, 
123.-Formulas of Some of the Commonly Used Stains, 124.-Preparation of 
a Stained Mount, 124.-Spore Stain, 125.-Stain for Acid-fast (Acid-proof) 
Organisms, 126.-Wirth's Stain for Much Granules, 126.--Flagella Stain, 127. 
-Gram's Staining Method, 128.-Stabilized Gentian Violet, 128.-Capsule 
Stains, 128.- Muir's Stain, 128.-Johne's Stain, 129.-Raebiger's Stain, 129. 
-Blood and Protozoan Stains, 129.-Wright's Stain, 129.-Giemsa's Stain, 
130.-Negri Bodies, 130.-Lentz Method, 130.-India-ink Method, 131. 
Chapter X.-Methods of Securing Pure Cultures of Bacteria. . 132 
Dilution Method, 132.-Isolation by Smearing, 132.--Direct Isolation, 132.- 
Isolation by Plating, 133.-Isolation by the Use of Heat, 134.--Isolation by 
the Use of Differential Antiseptics or Disinfectants, 134.-Isolation by Animal 
Inoculation, 134. 
Chapter XI.-Study of Bacterial Cultures 135 
Cultural Characters, 135.-Agar Stroke, 135.-Potato, 135.-Blood-serum, 136. 
-Gelatin Stab, 136.-Nutrient Broth, 136.-Milk, 136.-Litmus Milk, 137.- 
Gelatin Plate Colonies, 137.-Colonies on Agar Plates, 138.-Physiologic 
Characters, 140. 
LABORATORY METHODS AND TECHNIC 
SECTION III 
BACTERIA AND THE RESISTANCE OF THE ANIMAL 
BODY TO DISEASE 
Chapter XII.-Microorganisms and Disease 141 
Infectious Diseases, 141.-Contagious Diseases, 142.-Avenues of Infection, 
142.-Virulence, 144.-Koch's Postulates, 144.-Animal Inoculation, 145.- 
Methods of Animal Inoculation, 146.-Types of Infectious Diseases, 147. 
Chapter XIII.-Immunity. General Discussion 149 
Immunity, 149.-External Resistance to Infection, 149.-Variations of Indi- 
viduals in Susceptibility to Disease. Predisposing Factors,150.-Types of Im- 
munity, 150.--Natural Immunity, 150.-Acquired Immunity, 151.-Active 
Acquired Immunity, 151.-Acquired Passive Immunity, 153.--Theories of Im- 
munity, 153.-Theory of Exhaustion, 153.-Noxious Retention Theory, 154.- 
Metchnikoff's Theory of Phagocytosis, 154.-Ehrlich's Humoral Theory, 154. 
-Duration of Immunity, 155.-Antigens and Antibodies, 155.-Antibodies as 
Factors in Acquired Immunity, 155. CONTENTS 
13 
PAGE 
Chapter XIV.-Antitoxins and Related Antibodies 157 
Antibodies of Ehrlich's First Order, 157.-Toxins, 157.-Antitoxins, 159.- 
Constitution of the Toxin, 162.-Constitution of Antitoxin, 162.-Diagram- 
matic Representation of Toxin and Antitoxins, 163.--Preferential Union of 
Toxins with Body-cells, 163.-Antitoxins of Commercial Importance, 164.- 
Manufacture of Diphtheria Toxin and Antitoxin, 164.-Preparation of Tetanus 
Toxin and Antitoxin, 171.-Preparation of Other Toxins and Antitoxins, 173. 
Chapter XV.-Agglutination and Precipitation 174 
Antibodies of Ehrlich's Second Order, 174.-Differentiation of Precipitation and 
Agglutination, 174.-Agglutination, 174.-Precipitins, 182. 
Chapter XVI.-Cytolysins, Including Bacteriolysins, and Hemol- 
ysins 185 
Antibodies of Ehrlich's Third Order, 185.-Cytolysins, 185.-Group Cytolysins, 
187.-Bacteriolysins, 188.-Hemolysins, 189.-Fixation of Complement and 
Its Utilization, 190.-Cytotoxins, 192.-Conglutination, 192. 
Chapter XVII.-Opsonins and Phagocytosis 194 
Opsonins, 195.-Opsonic Index, 197.-Autogenic Vaccines, 201.-Passive Op- 
sonic Immunization, 203.-Leukocytic Extracts, 203.-Aggressins, 203.-Sen- 
sitized Vaccines, 204. 
Chapter XVIII.-Anaphylaxis and Hypersusceptibility 206 
Phenomenon of Arthus, 206.--Serum Sickness in Man, 206.-Theobald Smith 
Phenomenon, 207.--Vaughn's Split Proteins, 207.-Mechanism of Anaphy- 
laxis, 207.-Relationship of Anaphylaxis to Certain Body Reactions, 210.- 
Bacterial Anaphylaxis, 210. 
SECTION IV 
PATHOGENIC MICROORGANISMS EXCLUSIVE OF THE 
PROTOZOA 
Chapter XIX.-Groups of Pathogenic Microorganisms 212 
Chapter XX.-The Group of Streptococci. The Genus Strepto- 
coccus 214 
Classification of Streptococci, 214.-Holman's Classification of Species of 
Streptococci, 21.5.-Streptococcus Pyogenes, 216.-Streptococcus Equi, 224.- 
Streptococcus Gallinarum, 227.-Streptococcus Vaginitidis, 229.:-Strepto- 
coccus Mastiditis Sporadicre, 231.- Streptococcus Sp., 231.-Streptococcus 
Lacticus, 231. 
Chapter XXI.-The Group of Specific Pyogenic Cocci. The 
Genera Diplococcus and Neisseria 235 
Diplococcus Pneumonia;, 23.5-Neisseria Meningitidis, 238.-Neisseria Intra- 
cellularis Equi, 241.-Neisseria Gonorrhoea, 241. 
Chapter XXII.-Staphylococcus Group 244 
Staphylococcus Aureus, 244.-Staphylococcus Albus, 248.-Staphylococcus 
Citreus, 249.-Staphylococcus (.Pyogenes) Bovis, 249.-Staphylococcus Asco- 
formans, 249. 
Chapter XXIII.-Anthrax Group. The Genus Bacillus 252 
The Group of Aerobic Spore-producing Bacilli, 252.-Bacillus Anthracis, 253. 
Chapter XXIV.-Blackleg-Tetanus Group. The Genus Clos- 
tridium 264 
Key to the More Important Pathogenic Members of the Tetanus-Blackleg 
Group of Bacteria, 265.-Clostridium Tetani, 265.-Clostridium Chauvsei, 271. 
Clostridium Gastromycosis Ovis. 277.-Clostridium Welchii, 278.-Clostridium 
CEdematis, 282.-Clostridium of Ghon-Sachs, 285.-Clostridium Sporogenes 
Metchnikoff, 286.-Clostridium Botulinum, 286. 
Chapter XXV.-Pasteurella, or Hemorrhagic Septicemia Group 290 
Common Characters of Group, 291.-Pasteurella Cholera Gallinarum, 293.- 
Suisepticus, 297.-Pasteurella Bovisepticus, 301.-Pasteurella Cuniculicida, 
302.- Pasteurella Pestis, 302. 
Chapter XXVI.-Glanders Group. The Genus Pfeifferella. . 306 
Pfeiff erella Mallei, 306.-Bacilli of Selter, Babes, and Kutscher, 317. 14 
CONTENTS 
PAGE 
Chapter XXVII.-Intestinal or Colon-typhoid Group. The 
Genus Bacterium 318 
Subgroup I.-Colon Subgroup, 319.-Bacterium Coli, 321.-Bacterium Acidi 
Lactici, 325.-Bacterium Communior, 325.-Bacterium Coscoroba,325.-Bac- 
terium Aerogenes, 325.-Bacterium Cloaca, 327.-Organisms Associated with 
Calf Scours or Calf Diarrhea, 327.-Capsulated Pathogenic Organisms, 328.- 
Bacterium Pneumonia, 329. 
Subgroup II.-Intermediate Hog-cholera, Enteritidis, or Gartner Subgroup, 330. 
-Bacterium Enteritidis, 330.-Bacterium Cholera Suis, 333.-Bacterium 
Typhi Suis, 337.-Bacterium Para-typhi, 338.-Bacterium Typhi Murium, 
339.-Bacterium Pullorum, 340. 
Subgroup III.-Typhoid-Dysentery Subgroup, 342.-Bacterium Typhosum, 
342.-Bacterium Dysenteria, 345. 
Chapter XXVIII.-Abortion-Malta Fever Group. The Genus 
Brucella 349 
Brucella (Bacterium) Abortus, 349.-Brucella (Bacterium) Melitensis, 353. 
Chapter XXIX.-Dog Distemper Group. The Genus Bacterium. 356 
Bacterium Bronchisepticum (.Bronchicanis), 356. 
Chapter XXX.-Fluorescent Group. The Genus Pseudomonas. 358 
Pseudomonas Pyocyanea, 358. 
Chapter XXXI.-Diphtheria-pseudotuberculosis Group. The 
Genus Cornebacterium , 361 
Cornebacterium Pseudotuberculosis, 362.-Cornebacterium Diphtheria,- 
367.-Cornebacterium HoSmanni, 372.-Cornebacterium Xerosis, 372. 
Chapter XXXII.-Swine Erysipelas Group. The Genus Erysi- 
pelothrix 373 
Erysipelothrix Rhusiopathia, 373.-Erysipelothrix Muriseptica, 377. 
Chapter XXXIII.-Hemophilic, or Influenza Group. The Genus 
Hemophilus -. 379 
Hemophilus Pyogenes, 379.-Hemophilus Influenza, 383.-Hemophilus Per- 
tussis, 384. 
Chapter XXXIV.-Acid-fast Group. The Genus Mycobacterium 385 
Mycobacterium Tuberculosis, 385.-Mycobacterium Paratuberculosis, 406.- 
Mycobacterium Lepra, 407.-Non-pathogenic Acid-fast Bacteria, 408. 
Chapter XXXV.-Vibrio Group 410 
Vibrio Metchnikovi, 410.-Vibrio Cholera. 412.-Vibrio Fetus, 414.-Non- 
pathogenic Spirilla, 416. 
Chapter XXXVI.-Spirochete Group 417 
Cultivation of Spirochetes, 420.-Classification of Spirochetes, 420.-Spiro- 
chata Obermeieri, 421.-Spirochata Duttoni, 423.-Spirochata Kochi, 425.- 
Spirochata Anserina (or gallinarum), 425.-Spirochata Theileri, 427.-Spiro- 
chata Pallida, 428.-Spirochata Pertenuis, 431.-Other Spirochetes, 431. 
Chapter XXXVII.-Actinomyces Group 432 
Classification of Actinomyces, 432.-Actinomyces Bovis, 434.-Actinomyces 
Nocardii, 437.-Actinomyces Capra, 439.-Actinomyces Necrophorus, 440.- 
Actinomyces Madura, 445.-Actinomyces of Other Infections, 445. 
Chapter XXXVIII.-Blastomycetes 446 
Blastomyces Farciminosus, 447.-Blastomyces Dermatitidis, 449.-Blasto- 
myces Coccidioides, 452. 
Chapter XXXIX.-Mold or Hyphomycete Group 454 
Aspergillus, 455.-Aspergillus Fumigatus, 457.-Aspergillus Flavus, 460.- 
Aspergillus Niger, 461.-Other Species of Aspergilli, 462.-Penicillium, 462.-- 
Fusarium, 463.-Fusarium Equinum, 464.-Sporotrichum, 466.-Sporotrich- 
um Beurmanni, 466.- Trichophyton, 470.-Trichophyton Granulosum, 472.- 
Trichophyton Felineum, 472.-Trichophyton Equinum, 472.-Trichophyton 
Caninum, 473.-Microsporum, 473.-Microsporum Lanosum, 474.-Micro- 
sporum Felineum, 474.-Microsporum Equinum, 475.-Achorion, 475.-Acho- 
rion Muris, 475.-Achorion Gallina, 475.-Oidium Albicans, 477. CONTENTS 
15 
SECTION V 
PAGE 
Chapter XL.-Structure, Relationships, and Classification of 
the Protozoa 478 
Structure of the Protozoa, 478.-Classification of the Protozoa, 480. 
Chapter XLI.-Pathogenic Protozoa of the Flagellata (Exclu- 
sive of the Spirochetes) 481 
The Genus Trypanosoma, 418.-Morphology, 482.-Cultivation of Trypano- 
somes, 484.-Method of Disease Production, 484.-Examination and Staining 
Methods, 484.-Trypanosoma Equiperdum, 485.-Trypanosoma Evansi, 487. 
-Trypanosoma Brucei, 488.-Trypanosoma Equinum, 490.-Trypanosoma 
Dimorphon, 492.-Trypanosoma Conglense, 492.-Trypanosoma Pecaudi, 493. 
-Trypanosoma Cazalboui, 494.-Trypanosoma Theileri, 495.-Trypanosoma 
Gambiense, 495.-Trypanosoma Lewisi, 496.-Trypanosoma Hippicum, 497. 
-The Genus Herpetomonas, 498.-Herpetomonas Donovani, 498.-Leishmania 
(Herpetomonas?) Infantum, 499.-Leishmania Tropica, 499. 
Chapter XLII.-Pathogenic Protozoa of the Class Rhizopoda. . 500 
The Genus Entamoeba, 501.-Staining Methods, 502.-Methods of Isolation and 
Cultivation, 502.-Entamoeba Coli, 503.-Entamoeba Histolytica, 504.-Enta- 
moeba Tetragena, 507. 
Chapter XLIII.-Sporozoa 508 
The Genus Piroplasma, or Babesia, 509.-Babesia Bigemina, 509.-Babesia 
Mutans, 511.-Babesia Equi, 511.-Babesia Ovis, 512.-Babesia Canis, 512. 
-Babesia (Piroplasma) Gibsoni, 514.-Babesia (Piroplasma) Commune, 514. 
-Theileria, 516.-Theileria Parva, 516.- The Genus Plasmodium, 516.-Plas- 
modium Vivax, 516.-Plasmodium Malari®, 518.-Plasmodium Immacula- 
tum and Falciparum, 518.- The Genera Proteosoma, Halteridium, and Hemopro- 
teus, 519.-The Genus Anavlasma. 519.-Anaplasma Marginale, 519.- The Gen- 
us Leukocytozoon, 521.-Genus Sarcocystis, 521.-The Genus Eimeria (Cocci- 
dium), 522.-Eimeria Avium, 523.-Eimeria Stiedae, 525.-Eimeria Bovis, 525. 
-Eimeria Faurei, 526.-Isospora Bigemina, 527. 
Chapter XLIV.-Parasitic Protozoa of the Ciliata 528 
Protozoan Commensals of the Rumen and Cecum, 528.-Balantidium Coli, 
531. 
PATHOGENIC PROTOZOA 
SECTION VI 
INFECTIOUS DISEASES IN WHICH THE SPECIFIC 
CAUSE IS NOT CERTAINLY KNOWN 
Chapter XLV.-Diseases Produced by Unknown Organisms 533 
Bacterial or Protozoan Relationships of Ultramicroscopic Organisms, 534.- 
Virus of Pleuropneumonia, 535.-Virus of Foot-and-mouth Disease, 536.- 
Virus of Rinderpest or Cattle Plague, 537.-Virus of Hog-cholera, 539.-Virus 
of Horse Sickness, 514.-Virus of Infectious Anemia of the Horse, 541.-Virus 
of Dog Distemper, 542.-Virus of Fowl Plague. 543.-Virus of Epithelioma 
Contagiosum. 544.-Virus of the Poxes, 545.-Virus of Yellow Fever, 546.- 
Virus of Rabies, 546.-Virus of Infectious Agalactia of Sheep and Goats, 550.- 
Virus of Guinea-pig Plague, 551.-Virus of Fowl Leukemia, 551.-Virus of Cer- 
tain Chicken Tumors, 551. 
SECTION VII 
BACTERIA OF WATER AND FOOD 
Chapter XLVI.-Bacteria of Water and Water Purification. .. 552 
Water Purification, 557. 
Chapter XLVII.-Milk. Its Constituents, Contamination, and 
Examination 561 
Influences Which Determine the Ultimate Bacterial Content, 567.-Standards 
for Production and Distribution of Certified Milk, 569.-Hygiene of the Dairy 
Under the Supervision and Control of the Veterinarian, 570.-Transportation, 
575.-Veterinary Supervision of the Herd, 575.-Bacteriologic Standards, 577. 
Bibliographic Index 579 
Index ..585 VETERINARY BACTERIOLOGY 
SECTION I 
MORPHOLOGY, PHYSIOLOGY, AND CLASSIFICATION 
OF BACTERIA 
CHAPTER I 
INTRODUCTION 
Bacteriology is commonly defined to include a considera- 
tion of three distinct groups of minute living organisms: the true 
bacteria, the molds, and the pathogenic protozoa. Some authorities 
have objected to the inclusion of a discussion of forms other than 
the true bacteria under the heading of bacteriology, and have 
proposed that the term microbiology be used instead. In accord- 
ance with this latter conception, the term microbe includes the 
bacteria, the molds, and the protozoa, and the science of microbiology 
includes the corresponding subdivisions, bacteriology, mycology, 
and protozoology. As most commonly used, however, the term 
"bacteriology" is regarded as a synonym of "microbiology," and in 
this text this usage will be followed. 
The reasons for including the bacteria, the molds (particularly 
those pathogenic to animals), and the pathogenic protozoa under 
bacteriology may be stated briefly as follows: All three groups 
must be studied by very similar methods. All three contain or- 
ganisms which are capable of bringing about pathologic conditions 
in man and animals. The microscopic unicellular animals, known 
as protozoa, are known to produce disease in some cases, and have 
been studied largely by means of the laboratory technic developed 
17 18 
VETERINARY BACTERIOLOGY 
by the bacteriologist. The boundaries between the groups of 
the bacteria on the one hand and protozoa on the other are like- 
wise far from distinct. In many cases it would prove impossible 
from a clinical examination alone of a new disease to determine 
whether it is caused by the invasion of true bacteria or of protozoa. 
It is evident that there are many practical difficulties in differen- 
tiating the bacteria and protozoa. 
Furthermore, the line of demarcation between the bacteria and 
fungi (including the molds, mildews, smuts, rusts, toadstools, 
puffballs, etc.) is very poorly defined. Particularly is it easy to 
find all intergradations between the bacteria and those groups of 
fungi commonly known as yeasts and molds. It appears, there- 
fore, that because of difficulties in differentiation and because of 
the relationships indicated that a consideration of bacteria, yeasts, 
molds, and protozoa may all be included under the title of bac- 
teriology. 
Parasitology is that branch of science commonly defined to in- 
clude a discussion of animal parasites, such as the mites, lice, 
nematodes, and similar types. Sometimes it is held to include con- 
sideration of the pathogenic protozoa and the parasitic molds. 
To this extent the fields of bacteriology and parasitology would 
therefore overlap. 
Although bacteriology is one of the youngest of the sciences, 
nevertheless it has grown so rapidly that many divisions and sub- 
divisions have come to be recognized. 
Medical bacteriology may be defined as that branch of bac- 
teriology which treats of those microorganisms (including the true 
bacteria, molds, and protozoa) that produce disease in the animal 
body or are related directly or indirectly to the maintenance of 
health. Agricultural bacteriology, with its subdivisions-soil bac- 
teriology, dairy bacteriology, and plant bacteriology-includes a con- 
sideration of all organisms of importance in soil fertility, in pro- 
duction and distribution of dairy products, in disease causation in 
plants, and to a certain degree in domestic animals. Sanitary 
bacteriology has for its field the methods of disease prevention based 
upon the knowledge of the organisms causing disease and the man- 
ner in which they spread. Systematic bacteriology discusses the 
classification and relationships of organisms. Immunology con- INTRODUCTION 
19 
siders the resistance and susceptibility of the body to disease and 
the means which may be used to increase such resistance. 
It is evident from the preceding definitions that the various 
divisions of bacteriology overlap to a considerable degree. 
Veterinary bacteriology may be defined to include that por- 
tion of medical bacteriology which concerns those microorganisms 
(bacteria, yeasts, molds, or protozoa) which affect the health of 
domestic animals. The history of veterinary bacteriology is 
closely linked with that of general medical bacteriology, for many 
of the diseases of man are transmissible to animals and vice versa. 
It should be remembered that both are merely subdivisions of a 
great science, concerning which it is important that the student 
should gain something of a perspective view, particularly with ref- 
erence to its history and development. This development has 
Fig. 1.-Leeuwenhoek's drawings of bacteria: A, B, Bacilli, probably; C-D, 
path of movement; E, cocci; F, Leptotrichia, probably; G, spirillum. 
been so rapid, and so many of the important discoveries have been 
made within recent years, that it is frequently difficult to deter- 
mine their relative importance. However, certain facts and 
personalities stand out so conspicuously that they are deserving of 
brief consideration. 
The Microscope and its Influence.-The existence of living 
plants or animals smaller than can be seen by the unaided eye 
was conjectured by several of the Greek philosophers and phy- 
sicians who used such theories in their speculations on the origin 
and cause of fermentation and disease. Until the discovery 
of the microscope such speculations were without any basis in 
fact. 
Leeuwenhoek (1632-1723), in the course of his examinations 
of a great variety of natural objects by means of the somewhat 20 
VETERINARY BACTERIOLOGY 
crude lenses of his own manufacture, chanced to observe the 
presence of motile and motionless microorganisms in the tartar 
from teeth and in various decaying organic materials. His 
correspondence with the Royal Society of London and the figures 
published leave no doubt but that he actually observed bacteria. 
These drawings are of such historic interest that they are here 
reproduced. 
Each advance in the efficiency of the microscope was followed 
by an advance in our knowledge of the microorganisms, although 
speculation frequently outran the ability to see clearly. The com- 
pound microscope has proved to be indispensable in the study of 
these forms. Since the introduction of this instrument the degree 
of magnification, the clearness of definition, and the mechanic ar- 
rangements for accurate focusing have been gradually improved 
until at the present time the homogeneous oil immersion objective, 
the compensating ocular, and the Abbe condenser are in constant 
use in the laboratory, and enable us to secure readily magnifica- 
tion to 1500 diameters or more. During the last several decades 
there has been little increase in magnification, due to two reasons. 
The greater the magnification the more convex and consequently 
the smaller must be the lenses used in the objectives, and the more 
difficult becomes their grinding and adjustment. Furthermore, 
the physicist tells us that a clear view, with determination of the 
size and shape of microscopic objects, cannot be obtained when the 
objects examined are smaller than one-half the wave length 
of the rays of light in which they are examined. There is thus 
a seemingly insurmountable barrier set to an indefinite increase 
in magnification. 
A recent advance has been made through the development of 
the ultramicroscope. This has made visible objects much smaller 
than those which had been observed previously. A bright gleam 
of light from an arc or similar source is passed across the darkened 
field of the microscope, and the light is reflected to the eye from 
any particles that may be in suspension. These objects are 
seen in the same manner that minute particles of dust are made 
visible in a bright ray of light that enters a darkened room. The 
use of the ultramicroscope has not as yet added many facts of 
value to our knowledge of the bacteria. INTRODUCTION 
21 
Nature and Classification of Microorganisms.-Leeuwenhoek, 
whom we have seen to be the first observer of bacteria, contributed 
very little to a knowledge of their essential nature. F. Muller 
(1786) worked out a simple classification, but did not differentiate 
between bacteria and protozoa. To him we owe several of the 
group names applied to bacteria, such as bacillus, vibrio, spirillum. 
Ehrenberg (1795-1875), with the improved microscope and 
lenses at his command, prepared the first logical classification of 
bacteria. Cohn (1828-1898) elaborated and modified Ehren- 
berg's classification. He differentiated the true bacteria from 
the protozoa, and his arrangement is the basis for the classi- 
fication used most extensively at present. With the continued im- 
provement in the microscope and laboratory technic, more careful 
studies of structure, form, and relationships have been rendered 
possible, and many classifications and groupings for bacteria 
have been suggested. The difficulty in finding morphologic 
characters that are accurate indices to true relationships 
has made the subject a troublesome one. 
In recent years several attempts have been made to 
secure a satisfactory classification of the genera of bacteria. 
The one used here is an adaptation of that suggested by 
a committee on nomenclature of the Society of American 
Bacteriologists. 
Spontaneous Generation.-In ancient times and even during 
the middle ages it was generally held by the philosophers and 
scientists that living things, animals, and plants, could arise de 
novo. Among the first observations that created doubt in man's 
mind as to the validity of this belief was that of Francisco Redi, 
who covered meat with gauze to protect it from flies, and found that 
maggots did not develop in it spontaneously, but arose from the 
eggs which the flies deposited on the screen. This pointed the 
path for other similar studies, and it was not long before the idea 
of spontaneous generation of the higher forms of life was aban- 
doned. When the microscope revealed the presence of myriads of 
microorganisms in all decaying or putrefying materials, it was 
concluded that these organisms arose without progenitors of their 
own kind, but directly from the organic materials of their sur- 
roundings. Boiling was believed to certainly destroy all life, 22 
VETERINARY BACTERIOLOGY 
yet it was found that boiled decoctions would not always remain 
free from microorganisms. The theory of spontaneous generation 
of these bacteria was opposed by some and supported vigorously by 
others of the best scientists of the time. Experiments were care- 
fully planned and a great variety of materials used, paving the way 
for the development later of the laboratory technic of the bacteriol- 
ogist. The sterilizing action of heat, the antiseptic action of 
certain chemicals, and the value of the cotton plug as a bacterial 
filter were demonstrated. The theory of spontaneous generation 
as a topic of contention practically disappeared about 1860. 
This was largely due to the efforts of Pasteur, who, by a long 
series of ingenious experiments, overthrew the last defense of the 
supporters of the theory. The dictum, omne vivum ex vivo (all 
life from life), is universally accepted at the present time, and the 
controversy has little but historic interest. 
Relationships of Microorganisms to Fermentation and Decay.- 
As has been previously noted, some of the early philosophers 
hazarded the opinion that decay might be caused by invisible 
living beings of some kind. The causal relationship of micro- 
organisms to decay and particularly to fermentation was first 
definitely established by the work of Louis Pasteur (1822-1895). 
He found the production of alcohol and carbon dioxid from sugar 
was due to a yeast, that milk soured because of the activity of 
bacteria, and that many of the familiar changes in organic sub- 
stances were accomplished by microorganisms. His conclusions 
were strenuously opposed and ridiculed by the great German 
chemist, Liebig. Doubtless the necessity for meeting the attacks 
of the latter and of establishing his points beyond possibility 
of refutation led him to devise and develop many of the laboratory 
methods in common use at the present time. As a result of 
Pasteur's work the fundamental importance of bacteria in the trans- 
formation of nitrogen and carbon compounds in nature, the 
disposal of waste, the purification of water, the enriching of the 
soil, and many of the changes in the manufacture of foods have 
been established. 
Relationship of Microorganisms to Disease.-The probable 
causal relationship of microorganisms of some kind to disease 
was argued a$ long ago as 1762 by Plenciz, of Vienna. His INTRODUCTION 
23 
theories were not generally accepted, and it was not until 1840 
that Henle proposed what we have come to call the germ theory 
of disease. He never succeeded in proving his point satisfactorily 
because of the lack of proper methods and technic. Many 
other writers within the next few years discussed the theory 
and numerous facts were adduced in favor of it. The majority 
of medical practitioners, however, put very little faith in it. The 
argument that certain organisms were always present was met 
with the statement that these organisms were the result and not 
the cause of the disease. Davaine (1863) practically demon- 
strated by inoculation experiments the causal relationship of a 
bacillus he found in the blood of diseased animals to anthrax. 
Pasteur (1865) proved the cause of a silkworm disease to be a 
protozoan parasite. Koch and Pasteur later cultivated the 
anthrax organism in the laboratory and showed beyond a doubt 
its relationship to the specific disease. Improved laboratory 
technic cleared up the cause of many diseases within the next de- 
cade or two. The discovery of the bacillus of tuberculosis by 
Koch (1882) marks the real beginning of bacteriologic science. 
The knowledge of protozoa as a cause of disease lagged somewhat 
behind that of bacterial infections. Evans (1880) described 
the trypanosome of surra and transmitted the disease by inocula- 
tion experiments. In 1882 Laveran observed the Plasmodium 
malarice, the cause of malaria. 
Development of Laboratory Methods.-Progress was delayed 
in the study of objects as minute as the bacteria because of the 
lack of proper methods for their isolation, observation, and identi- 
fication. Culture-media in which the pathogenic microorganisms 
could be grown were used by Pasteur and Koch. To the latter 
we are indebted (1882) for our knowledge of the solid media which 
can be used for the isolation of organisms from mixed cultures. 
The importance of this contribution can hardly be overestimated, 
for the use of pure cultures lies at the very foundation of all 
modern bacteriologic investigation. This one discovery accounts 
in large measure for the rapid advance made during the next 
two decades in the identification of the organisms producing 
disease. The use of aniline dyes in rendering cells and their struc- 
ture more plainly visible under the microscope we owe to Weigert, 24 
VETERINARY BACTERIOLOGY 
but the application to bacteriology was made by Koch. Since 
their introduction successive advances in staining technic have in 
every instance been followed by the discovery of new organisms 
related to disease. The microscope, liquefiable media, and 
anilin dyes constitute the trio of most important factors in the 
development of the science of bacteriology. 
Development of Theories of Immunity.-Knowledge that one 
attack of certain diseases generally prevented a recurrence and 
that diseases could not be indiscriminately transferred to all 
species of animals has existed ever since the foundation of medi- 
cine. Many theories have been advanced to account for this 
phenomenon. A few of the names of investigators who have put 
the facts into logical form for presentation and study should 
be considered. Metchnikoff conceived that the white blood-cor- 
puscles and<some other body cells acted as scavengers and des- 
troyed microorganisms in the blood. This theory in modified 
form furnishes to-day the logical basis for many of the operations 
of the practitioner. Von Behring (1890) published results of stud- 
ies on the diphtheria bacillus in which he recounted the discovery 
of the specific toxin of this organism and a specific antitoxin. 
He laid the foundation for the present-day " humoral " theory 
of immunity, which supplements so well the phagocytic theories 
of Metchnikoff. Ehrlich has correlated and coordinated the vari- 
ous facts of the humoral theory and has made substantial additions 
to it. He has created a terminology which has been quite generally 
accepted and has proved most useful in discussion of the sub- 
ject. So extensive have been researches in the field of immunity 
during the last two decades (since 1890) that it has assumed 
almost the rank of a coordinate science. 
Development of Sanitary Science and Preventive Medicine.- 
In 1858 Murchison formulated the so-called pythogenic theory of 
disease-i.e., that disease is caused by the emanations arising from 
decaying or putrefying organic matter, and by the consumption 
of such materials in water and food. This theory was quite com- 
monly accepted, and its practical applications form the basis for 
our modern sanitary science. The disposal of sewage and refuse, 
the purification of drinking-water, and adequate systems of plumb- 
ing were advocated and adopted before the germ theory of disease INTRODUCTION 
25 
had been well formulated and established. The rapid accumula- 
tion of evidence in favor of the germ theory gave rise to the art 
of preventive medicine. Lister (1875) advocated the use of 
antiseptics in the treatment of wounds and wound infection as a 
direct method of combating the activity of undesirable bacteria. 
The discovery of the means of transmission in most of the infectious 
diseases has enabled man to take measures for their eradication. 
Yellow fever, malaria, diphtheria, bubonic plague, and many other 
diseases are now kept in check by the use of preventive measures 
indicated by our knowledge of the manner in which they spread. 
It is probable that greater advance will be made within the next 
few years in the domain of preventive medicine, for mankind 
is fast coming to realize that prevention is better than cure. CHAPTER II 
MORPHOLOGY AND RELATIONSHIPS OF MICROORGANISMS 
CONCERNED IN DISEASE PRODUCTION 
Position of Pathogenic Microorganisms 
Differentiation of Animals and Plants.-The distinctions we 
commonly recognize as differentiating plants from animals in 
large measure disappear among the microscopic forms of life. 
It is worth while, therefore, to discuss the factors that are taken 
into consideration in the assignment of a particular microorgan- 
ism to the animal or the plant kingdom. The difficulty arises 
particularly in the differentiation of the bacteria and the protozoa. 
Bacteria, as will be shown later, probably have in all cases a cell- 
wall which in function is closely related to that of plants. A 
cell-wall is frequently absent in protozoa, the limiting mem- 
brane usually consisting of the ectoplast (see below) alone. The 
composition of the cell-wall is in some cases like that of many 
animals (chitin), in others like plants (cellulose). In shape 
and habit of growth and reproduction the bacteria resemble 
very closely certain undoubted plants among the blue-green 
algae and the mold fungi much more than they do animal forms. 
On the other hand, there are some types which intergrade with the 
protozoa, so that there is at present doubt as to their correct 
systematic position. In the matter of food supply and food 
utilization some bacteria resemble higher plants, others resemble 
animals. The possession of organs of motion by bacteria has 
no direct bearing on the subject, inasmuch as undoubted plants 
of the group of algae and many of the protozoa have them. The 
evidence, on the whole, is much in favor of classification of the 
true bacteria with plants. 
Before beginning a study of the morphology or structure of 
these microorganisms, their position and relationships to the 
other groups of plants and animals should be understood. Fol- 
26 MORPHOLOGY AND RELATIONSHIPS OF MICROORGANISMS 
27 
lowing is a discussion of these various groups, with particular 
emphasis upon the groups or subgroups of significance in medical 
bacteriology. 
The plant kingdom is generally divided into four great groups, 
the Spermatophytes, or seed-bearing plants, the Pteridophytes, or 
ferns and fern-like plants, the Bryophytes, or moss plants, and the 
Thallophytes, including all plants low in the scale of evolution, 
that have not become highly differentiated and that never have 
roots, stems, and leaves. All of the plant-like organisms to be con- 
sidered belong to this lowest group, Thallophytes. 
The animal kingdom may be divided into the Metazoa, or 
higher differentiated types, made up of many cells, and the Pro- 
tozoa, or unicellular forms. To the latter group belong all the 
animal-like organisms to be considered. 
Subdivisions of the Thallophytes.-Following is a key or out- 
line of the principal subgroups of the Thallophyta: 
A. Unicellular forms, multiplying only by splitting of the cells 
to form two equal daughter-cells. Never any sex cells. 
I. Cells containing blue-green coloring-matter. 
1. Schizophycece (Cyanophy cese or blue-green algae). 
II. Cells not containing blue-green coloring-matter. 
2. Schizomycetes (Bacteria). 
B. Unicellular or multicellular, multiplication by some method 
other than simple fission. Frequently sexual repro- 
duction occurs. 
I. Cells containing green coloring-matter (chlorophyll). 
3. Alga (sea-weeds, pond scums, etc.). 
II. Cells without green coloring-matter. 
4. Fungi (yeasts, molds, mildews, rusts, smuts, toadstools, 
puffballs, mushrooms, etc.). 
Of the last group (Fungi) only two subgroups are of especial 
pathogenic significance for the veterinarian, the yeasts and the 
molds. These two, with the bacteria, constitute the three types 
of microorganisms belonging to the plant kingdom that contain 
forms pathogenic for animals. 28 
VETERINARY BACTERIOLOGY 
Morphology of Bacteria 
Shape of Bacteria.-Bacteria may be classified according to 
shape into spheres, straight rods, bent or spiral rods, and filaments. 
A spherical form is called a coccus. Although the cocci are 
theoretically spherical, there are many that appear somewhat 
flattened or ovoid when in groups or chains. 
Fig. 2.-Types of bacteria': Cocci, bacilli, and spirilla (Jordan). 
A straight rod is called a bacillus. 
A curved or spiral rod is called a spirillum. 
The filamentous bacteria are those in which the organism is 
greatly elongated. The name trichobacteria has been given to 
such forms. Frequently the filamentous type exhibits branching 
and in other ways resembles the mycelium of the higher fungi or 
molds. 
Fig. 3.-Involution forms of bacteria: 1, Rhizobium leguminosarum. 
a, Normal rods; b, bacteroids. 2, Mycobacterium tuberculosis. 3, Bacillus 
subtilis. 4, Acetobacter aceti. 5, Pasteurella pestis. 6, Actinomyces sp. 
7, Spirillum sp. 8, Staphylococcus aureus: a, Normal forms; b, involution 
forms. 
When grown under unfavorable conditions, or as a result of the 
action of certain stimulation, many bacteria assume unusual and 
abnormal shapes. Cells of this type are called involution forms. 
Such cells are not necessarily incapable of continued growth and MORPHOLOGY AND RELATIONSHIPS OF MICROORGANISMS 
29 
reproduction of the more usual or normal type when brought 
under favorable conditions. Frequently, however, these cells 
soon die and can no longer reproduce. Various bacteria differ 
considerably with respect to the ease with which they produce 
involution forms. 
Grouping of Bacterial Cells.-The cells of bacteria are frequently 
surrounded by a gelatinous material (capsule, see below) which 
causes them to cling together in groups. This grouping in the 
various types is so constant that it is used to differentiate various 
genera from each other, and in some cases the species within 
Fig. 4.-Development of groups of cocci' 1, Development of strepto- 
coccus; 2, development of micrococcus; 3, development of sarcina; 4, 
development of staphylococcus. 
the genus. Bacterial cells divide normally at right angles to the 
longest axis of the cell. This allows of but little variation in the 
grouping of elongated types, but as the cocci have no "longest 
axis" they divide in various planes. 
Some cocci divide constantly in the same plane or in a plane 
parallel to the first. This results in the formation of a chain 
of cocci. An organism showing this grouping is called a strepto- 
coccus (pl. streptococci). 
Other cocci divide alternately in two planes at right angles 
to each other. Such an organism will be found in twos (diplo- 
coccus) or in fours {tetrad or tetracoccus). Diplococci may also 30 
VETERINARY BACTERIOLOGY 
be produced by the breaking up of chains of streptococci into 
pairs. 
Cocci sometimes divide in three planes at right angles to each 
other. This results in the formation of cubes or packets of cocci. 
A packet of this kind is called a sarcina (pl. sarcince). 
Fig. 5.-Shapes and groups of cocci: 1, Single coccus (micrococcus); 2, 
cocci in an irregular mass (staphylococcus); 3, spheric diplococci; 4, flat- 
tened diplococci; 5, coffee-bean-shaped diplococci; 6, lanceolate diplo- 
cocci; 7, streptococcus with short chains; 8, streptococcus with long 
chains; 9, a streptococcus made up of diplococcus elements; 10, cap- 
sulated micrococci; 11, sarcina. 
A staphylococcus (pl. staphylococci') is a coccus whose planes 
of division are not at right angles, or which divides at different 
intervals with a consequent irregular grouping of the cells much 
resembling grapes in a cluster. 
Fig. 6.-Types of bacilli. 
Bacilli occur either singly or in chains. The latter are some- 
times known as streptobacilli. 
Spirilla usually occur singly, although short chains of two or 
three individuals are sometimes observed. 
In a few bacteria the gelatinous envelop of the cell is greatly MORPHOLOGY AND RELATIONSHIPS OF MICROORGANISMS 
31 
thickened, and the bacteria, either cocci or bacilli, are embedded in 
the mass. Such a mass of cells is called a zodglea. 
Size of Bacteria.-The unit of microscopic measurement is 
the micron and is abbreviated by the Greek letter p. It is the 
Fig. 7.-Types of spirilla. 
TTHnr Part a millimeter or, approximately, the ^q00 part of an 
inch. 
Bacteria vary considerably in size, from forms 0.1 or less 
in diameter, barely visible under the microscope, to forms 
Fig. 8.-Types of filamentous bacteria: A, Leptotrichia; B, Cladothrix; C, 
Nocardia; D, Actinomyces or Streptothrix. 
100 U or more in length. Most bacteria are between 0.5 and 
5 in diameter and 0.5 U and 10 U in length. Some bacteria 
are undoubtedly too small to be seen with the highest powers of 
our microscopes, hence less than 0.1 u in diameter. We know of the 
existence of these organisms by their effects. The organism caus- 32 
VETERINARY BACTERIOLOGY 
ing hog cholera, for example, is so small that it will pass through 
the pores of a fine porcelain filter, and will cause disease when in- 
jected into a healthy pig. Such an organism is frequently spoken 
of as ultramicroscopic or, preferably, as a filterable virus. 
Histology and Structure of Bacteria.-This topic may be treated 
under four subheads, the cell wall with its related sheaths and cap- 
sules, the protoplasm, the cell inclusions, and the flagella. 
Cell W all. The bacterial cell is in all cases surrounded by a 
definite membrane that morphologically resembles the cell wall 
of higher plants. When tested chemically with various reagents 
and examined microscopically, it is sometimes found to give 
the reactions characteristic of chitin, the material which makes 
up the hard outer shell of insects, and is found as a cell mem- 
Fig. 9.-Capsulated bacteria. 
brane in many animals. Chemically chitin is an amino-sub- 
stitution product of a carbohydrate. The fact that the cell 
walls in bacteria so frequently resemble in composition those 
of certain animals has been used as an argument for the animal 
relationships of the bacteria. This is negatived, however, by 
the fact that in numerous molds and other fungi, undoubted 
plants, the cell walls are made up of a similar substance. 
The cell wall in bacteria is usually covered by a layer of mucil- 
aginous material, in most cases so thin that the most careful technic 
must be employed in its demonstration, in other cases a thick 
coating or capsule. The nature of this capsular substance has 
been a fertile subject for dispute. A few bacteriologists have 
claimed that it is composed of living protoplasm, the majority, MORPHOLOGY AND RELATIONSHIPS OF MICROORGANISMS 
33 
with seeming justification, believe that this is either an excreted 
material or merely an outer swollen and differentiated portion 
of the cell wall. Chemically this material differs in the various 
capsulated bacteria. In some cases it is composed of mucin, a 
slimy material made up of a protein-like substance united with 
a carbohydrate, and resembling the mucus secreted by certain 
body cells. In other cases the capsule is made up of pure carbo- 
hydrates and is closely allied to certain of the vegetable gums, such 
as gum arabic and gum tragacanth. The capsule of some bacteria 
is partially or wholly soluble in water. When such an organ- 
ism grows in suitable nutrient solution it renders the medium 
slimy or gelatinous. Slimy milk, bread and whey are caused by 
the luxuriant growth of such organisms. 
Fig. 10.-Bacteria showing sheaths. 
Some of the filamentous bacteria produce a firm sheath or tube 
outside of the cell, this sheath usually inclosing an entire filament 
or chain of cells. In composition it probably closely resembles 
the cell wall. In some cases it is impregnated with iron oxid. 
It is possible that the sheath is a modified type of capsule. 
Cell Protoplasm.-The living material within the cell wall 
is called the protoplasm. Structurally it may usually be dif- 
ferentiated into two layers, an outer thin layer lying closely 
appressed to the cell wall, and an inner portion. The outer layer 
or ectoplast performs one of the most important functions of the 
cell, as this is the membrane, and not the cell wall, that determines 
what materials in solution may enter and what may leave the 
cell; through it must pass by diffusion all the food that enters the 
cell. When certain bacteria, as the cholera spirillum, are placed 
in a strong solution of some salt which does not readily pass through 34 
VETERINARY BACTERIOLOGY 
this ectoplast, the water from the cell in part passes out, the 
protoplasm shrinks away from the cell wall, and the cell is said to 
be plasmolyzed (noun, plasmolysis). Such a plasmolyzed cell 
shows clearly the ectoplast separated from the cell wall. When 
a cell of certain species is placed in distilled water or a concentra- 
Fig. 11.-Plasmolysis and plasmoptysis of bacterial cells: A, Plasmo- 
lyzed bacterial cells; B, cells showing plasmoptysis, the protoplasm has burst 
the cell wall and is escaping. (Adapted from Fischer.) 
tion of salt considerably less than that to which it has been accus- 
tomed, the cell takes up water, the cell wall bursts, and part of the 
protoplasm escapes. This phenomenon is called plasmoptysis. 
The protoplasm of the cell is commonly homogeneous in appear- 
ance, and stains best with the basic aniline dyes. Either a definite 
Fig. 12.-Bacterial cell inclusions: A, Vacuoles in the cell, polar staining 
rods; B, vacuolate spirilla; C, fat globules; D, glycogen granules; E, meta- 
chromatic granules; F, sulphur granules. 
nucleus is not present, or the nuclear material is so scattered 
as to make the entire mass functionally a nucleus. Some bac- 
teria have been described as possessing a primitive type of 
differentiated nucleus, but such structures cannot be discerned 
in others. MORPHOLOGY AND RELATIONSHIPS OF MICROORGANISMS 
35 
Cell Inclusions.-Bacterial cells sometimes contain vacuoles, 
or spaces in the protoplasm filled with cell sap or some non-staining 
or non-refractive substance. A large vacuole near the center of 
the cell may crowd the protoplasm to the ends of the cell, and 
such organisms, when stained, are said to show polar staining. 
In other forms, as the diphtheria bacillus, granules are formed 
that stain much more intensely with the basic aniline dyes than 
does the remainder of the protoplasm. These are called meta- 
chromatic granules. The function of these granules is not clear. 
Certain species of bacteria living in water containing hydrogen 
sulphid are found to contain granules of free sulphur in their 
protoplasm. Still others have food materials in the form of oil 
globules or granules of glycogen. 
Fig. 13.-Distribution of the flagella of bacteria: A, Non-motile or atrich- 
ous bacilli, spirilla, and cocci; B, monotrichous flagella of bacilli, spirilla, and 
cocci; C, lophotrichous flagella of bacilli and spirilla; D, amphitrichous flagella 
of bacilli and spirilla; E, peritrichous flagella of bacilli and spirilla. 
Flagella.-Many bacteria are motile by means of whip-like 
threads of protoplasm which extend from their surfaces. These 
threads are known as whips or flagella (sing, flagellum). These 
flagella are observed with difficulty in the living organism ex- 
cept with dark field illumination and require peculiar stain- 
ing technic and careful treatment to make them visible in a 
stained mount. Comparatively few cocci, many of the bacilli, 
and most of the spirilla are flagellated. The distribution of 
flagella on the surface of the cell has been used as a basis for 
grouping. Atrichous bacteria have no flagella; monotrichous 
bacteria have a single flagellum at one end; lophotrichous, a group 
of flagella at one pole; amphitrichous, flagella at both ends; and 36 
VETERINARY BACTERIOLOGY 
peritrichous, flagella on all sides. Old cells and cells transferred 
from one medium to another are very apt to loose their flagella. 
A young culture is most suitable for determination of motility. 
True motility must not be confused with Brownian movement, 
which is a vibrating or oscillating motion of finely divided par- 
ticles of almost any kind suspended in water, and visible when 
examined under the microscope. This motion has not been 
satisfactorily explained, but it is probably due to rapid changes 
in surface tension of the liquid at the point of contact with the 
particles. 
Reproduction in Bacteria.-Multiplication in all bacteria is a 
simple process. The cell commonly elongates or enlarges, a cell 
wall develops across the middle, and the two cells separate. 
This operation may occur with considerable rapidity. Some organ- 
isms in favorable medium can grow to their full size and divide to 
form two individuals in the course of twenty minutes to an hour. 
If the organism could multiply in this geometric ratio for a short 
time the number of resultant organisms would be practically 
incalculable. For example, if a bacterium should divide every 
half-hour, at the end of two days the progeny would be represented 
by 296, a number having twenty-eight figures. Such rapid multi- 
plication is never long continued, for food supply is never long 
favorable, and waste products of the bacterial growth tend to 
accumulate and diminish the rate. Nevertheless, the rapidity 
of increase of bacteria accounts in a large measure for the consider- 
able changes they bring about in a short time, as in the souring 
of milk or invasion of the body in disease. 
Many bacteria also reproduce by means of spores. These 
are of two types: endospores, produced inside the bacterial cell, 
and arthrospores, consisting of entire differentiated cells. The 
former are produced by certain bacilli and spirilla, the latter by 
certain of the filamentous forms or trichobacteria. 
Endospores are formed by many bacilli, and possibly by 
some spirilla. They are produced in response to some definite 
stimulus, such as unfavorable conditions of the environment, 
accumulation of waste products, or change in reaction of the 
medium. The spore is essentially a portion of the protoplasm 
of the cell which has expelled most of its water and shrunken in MORPHOLOGY AND RELATIONSHIPS OF MICROORGANISMS 
37 
size until it occupies a portion only of the space within the cell 
wall, and has then surrounded itself with a heavy wall, probably 
chitinous in nature. In practically all cases there is but 'one 
spore in a cell. The spore may be equatorial or polar in position, 
and of less or greater diameter than the cell which produces it. 
The term clostridium is sometimes used to indicate a spore-bearing 
Fig. 14.-Development of endospores in a bacillus. (After Fischer.) 
rod in which the spore is equatorial and of greater diameter than 
that of the cell, resulting in a spindle. Endospores contain only 
about 20 per cent, of water as compared with 80 to 90 per cent, 
in the cells which produced them. An organism without a spore 
is usually differentiated by the term vegetative rod or vegetative 
cell. Spores are much more resistant to desiccation, heat, light, 
and chemicals than the vegetative cells. They are of use in 
Fig. 15.-Bacterial spore types: A, Equatorial spores of a diameter less 
than the cell; B, polar spores of a diameter less than the cell; C, equatorial 
spores of a diameter greater than the cell (clostridium type); D, drumstick or 
polar spores of a diameter greater than the cell. 
tiding the organism over unfavorable conditions. Spore-bear- 
ing bacteria are abundant in the soil, where they often are exposed 
to great ranges of moisture, temperature, and light. When a 
spore again comes under favorable conditions for growth, it 
germinates and produces a cell typical of its species. Germina- 
tion is accomplished either by stretching or bursting the spore 
wall. 38 
VETERINARY BACTERIOLOGY 
Arthrospores are bacterial cells set apart for purposes of repro- 
duction, and are usually differentiated appreciably from the normal 
cell. Several investigators have claimed that they are produced 
by some of the cocci, but this has never been satisfactorily estab- 
Fig. 16.-Germination of spores: A, Bacillus subtilis (Prazmowski); B, Bacil- 
lus anthracis (deBary); C, Clostridium sp. (deBary). 
lished. The filamentous bacteria or trichobacteria produce 
arthrospores or conidia, as they are sometimes called, by the dis- 
integration of some of the filaments into short rods or spheres 
which are capable of reproducing the parent type or by a process 
Fig. 17.-Arthrospores: A, Crenothrix polyspora (Cohn); B, Gallionella ferru- 
ginea, showing conidia formation (Ellis); C, Leptothrix ochracea (Ellis). 
of budding. In many cases the threads which break up into 
the spores are somewhat differentiated from the normal cells of 
the plant, and are aerial, resembling closely some of the molds. 
Morphology of the Yeasts, Saccharomycetes, and Blasto- 
MYCETES 
Yeasts, from the standpoint of the systematic botanist, are 
placed at some distance from the bacteria, for there are many 
differences between typical yeasts and typical bacteria. On the 
other hand, there are forms which intergrade between the two, MORPHOLOGY AND RELATIONSHIPS OF MICROORGANISMS 
39 
and are sometimes assigned to one group, sometimes to another. 
The yeasts and the molds also show intermediate types. 
Form, Size, and Grouping of Yeasts.-Yeast cells are usu- 
ally spherical, oval, ellipsoid, or cylindrical. For the most part 
they are larger than bacterial cells, although there are excep- 
tions. The true yeasts multiply not by fission, but by a process 
of budding. The cells commonly remain united for a time, giv- 
ing rise to masses consisting of many individuals. Sooner or 
later they break apart. The relative shape, size, and groupings 
of the yeast cells are used in the differentiation of species. In 
some species part of the cells become considerably elongated and 
form a kind of false mycelium resembling that of the molds. 
This character is not always constant in a given species, it may 
Fig. 18.-Types of yeast cells and groupings 
appear when the organism is growing in one kind of medium and 
not appear in another. 
Histology and Structure of the Yeast.-The very young cell 
has no cell wall, but by the time it is one-third grown the wall 
appears as a delicate membrane. In old cells it is sometimes of 
considerable thickness. Its composition has not been certainly 
determined, probably it is a carbohydrate or related compound, 
and not chitinous, as are the walls of bacterial and mold cells. 
To this substance the name yeast cellulose has been given. It 
has not been prepared free from nitrogen, so that it is possible 
that it may be nitrogenous in nature. It is sometimes surrounded 
by a gelatinous excretion or capsule, as is the case with bacteria. 
The yeast cells are never motile. 
Yeast Protoplasm and Cell Inclusions.-The contents of the 40 
VETERINARY BACTERIOLOGY 
yeast cell are more highly differentiated than are those of bacteria. 
The ectoplast, or limiting membrane of the protoplasm, is easily 
demonstrated by plasmolyzing the cell. This ectoplast (German 
Hautschicht) is the only membrane of the young cell, and the 
cell wall is probably secreted by it. The protoplasm is differ- 
entiated definitely into a nucleus and cytoplasm. The nucleus 
is not as easily demonstrated as in the higher plants and animals, 
Fig. 19.-Diagram of budding yeast cells and their contents: a, Glycogen 
granules; b, vacuoles; c, nucleus; d, dividing nucleus in bud formation. 
but may be shown by proper staining methods. The cytoplasm 
usually contains one or more vacuoles, spaces filled with cell sap 
and not taking the stain as does the cytoplasm. Older cells 
may also show oil globules or glycogen granules. 
Reproduction in Yeasts.-Yeasts commonly multiply vege- 
tatively by budding. A bit of the protoplasm protrudes on one 
side of the mother cell, the nucleus divides, and one part goes to 
Fig. 20.-Spores (ascospores) of the yeast (Hansen). 
the bud, the other remains within the cell, the bud enlarges, 
develops a cell wall, and is constricted off as a distinct individual. 
Many yeasts may also under favorable conditions produce spores. 
The development of the spores in the yeast cell differs considerably 
from that in the bacteria. The latter typically have but one spore 
developed within the cell, while a yeast cell usually produces two, 
four, six, or even eight spores. The nucleus divides several times 
to form a number of nuclei, each of which, together with the MORPHOLOGY AND RELATIONSHIPS OF MICROORGANISMS 
41 
protoplasm lying in contact with it, becomes surrounded by a 
membrane. A cell of the yeast (and certain other fungi) when 
filled with spores is called the ascus (pl. asci) or sac. The spores 
are called ascospores (Fig. 20). This method of spore production 
relates the yeasts quite definitely to some of the higher fungi. 
In some cases there is a primitive type of sexual reproduction 
or fertilization associated with the development of the spores. 
The spores are more resistant to an unfavorable environment than 
the vegetative cells. When brought under favorable conditions 
they germinate and develop into the typical yeast plant. In 
old yeast cultures some cells develop heavy cell walls, are filled 
with granular reserve food materials, and become potentially 
spores. Such cells are likewise probably resistant to unfavorable 
conditions, and serve to tide the yeast over periods of desiccation 
or poor food supply. They resemble the chlamydospores produced 
by many molds. 
Morphology of the Hyphomycetes or Molds 
The molds or hyphomycetes do not constitute a homogeneous 
group in the eyes of the systematic botanist, but belong to vari- 
ous subdivisions of the group of fungi. Some are related to 
the algae and are grouped under the Phycomycetes, others belong 
to the sac fungi or Ascomycetes, others are related to the smuts, 
rusts, and toadstools, or Basidiomycetes, and the largest number 
belong to the group of imperfect fungi or Fungi Imperfecti. From 
the viewpoint of the bacteriologist these botanic relationships 
are not significant; all the fungi, regardless of kinship, that agree 
in having the plant body made up of threads usually more or 
less branched, and forming more or less loose or cottony masses, 
in short, those that answer to the popular conception of molds, 
are grouped together as Hyphomycetes, Such a classification is 
scientifically justifiable only because of the great complexity of 
the various members of the family of fungi, and the fact that 
it is not the systematic position but the economic importance of 
the forms that is of significance. 
Form and Size of Hyphomycetes or Molds.-A mold may be 
differentiated from the yeasts and bacteria in that it is multi- 
cellular, with the cells united to form more or less branched 42 
VETERINARY BACTERIOLOGY 
threads called hypha (sing, hypha). The whole mass of threads or 
hyphae which go to make up the plant body of the mold is called 
the mycelium. In most molds certain threads are differentiated 
for the production of spores. The mycelium of the mold may 
extend over a considerable area, growing deep into the substratum 
for food or into the air to develop spores. 
Histology and Structure of Molds.-The mycelium in some 
molds is continuous throughout its length, possessing no cross 
walls which might separate the cells from each other. In the 
majority of forms, and in all those of economic importance to 
the veterinarian, the hyphae are divided by cross walls or septa 
Fig. 21.-Mold hyphae A, B, Non-septate hypha; of the Phycomycetes; C, 
tip of a non-septate hypha, showing numerous nuclei and vacuoles; D, septate 
branching hypha;; E, a single cell of a septate hypha, showing nucleus and vac- 
uoles. 
(sing, septum). The cell wall is composed of true cellulose in 
a few molds, in the majority it is chitinous as in the bacteria. 
The almost universal presence of chitin in the cell walls of the 
fungi is frequently lost sight of by those who regard its presence 
in the cell walls of bacteria as evidence of animal affinities. The 
protoplasm of molds, as in the yeasts, is made up of cytoplasm and 
nucleus. The outer layer of the cytoplasm or ectoplast is readily 
demonstrated in most molds by plasmolysis. In the forms that 
do not have septa dividing the hyphae into cells, the numerous 
nuclei are imbedded in the common cytoplasm. Functionally 
each nucleus with its bit of surrounding cytoplasm constitutes a MORPHOLOGY AND RELATIONSHIPS OF MICROORGANISMS 
43 
cell, although the statement is often made that the entire mold 
filament in the non-septate type is a single cell. 
Reproduction of Molds.-It is impracticable to go into detail 
concerning the reproduction of molds. Spores of many different 
types are produced (Fig. 22), sometimes three or four kinds by 
a single species. The spores exhibit every conceivable shape and 
coloring, are sometimes unicellular, at other times multiseptate. 
Hundreds of genera and thousands of species are known. The 
names applied to the different parts of the molds concerned in 
reproduction and the manner in which the spores are borne in 
some of the commoner molds may, however, be briefly discussed. 
Molds may be divided, for convenience, into those which 
bear the spores enclosed in a spore case or sporangium and those 
in which they are not so inclosed. This sporangium is commonly 
Fig. 22.-Types of mold spores. 
borne at the tip of a hyphal thread differentiated for the pur- 
pose, called a sporangiophore. Spores not produced inside of 
a sporangium and not the result of fertilization (i. e., asexual 
spores) are termed conidia (sing, conidium). Conidia are usually 
developed at the tip of specialized branches called conidiophores. 
Sometimes they are formed by the breaking up of the mycelial 
threads or hyphae, and are then called oidia (sing, oidium), 
in other cases they develop within the hyphse and are surrounded 
by it as by a sheath. When one of the cells in a hypha becomes 
enlarged and surrounded with a heavy wall it is called a chlamydo- 
spore. Some molds develop spores as a result of the union of 
sex cells (sexual spores). These are called ascospores when pro- 
duced in sacs (asci) and zygospores when formed by the union of 
two like cells as in certain Phycomycetes. 
Spores of the molds are commonly born on hyphae that extend 44 
VETERINARY BACTERIOLOGY 
into the air away from the moist surface of the medium in which 
they are growing. This facilitates their dispersal by the air 
Fig. 23.-Types of the spores and the spore-bearing organs of the molds-■ 
1, Sporangium of the Mucor: a, columella; 6, sporangiophore; c, spores; d, 
sporangium wall. 2, Sporangia of Sporodinia: a, sporangiophore; b, sporangia 
containing spores. 3. Ascus of an ascomycete, Peziza: a, ascus or spore sac; 
b, spore; c, sterile threads or paraphyses. 4, Oidium spore formation; the 
hyphae are segmenting to form spores or oidia. 5, a, Chlamydospores formed 
in the hypha of a Chlamydomucor. 6, Zygospore of a Mucor: a, hypha; b, 
suspensor; c, zygospore. 7, Conidiophore and conidia of Penicillium: a, con- 
idiophore; b, verticillate branches of the conidiophore; c, chains of spores or 
conidia. 8, Aspergillus: a, conidiophore; b, inflated tip of the conidiophore; c, 
sterigmata; d, chain of spores. 9, a, Hypha; b, poorly differentiated conidio- 
phore; c, chain of conidia. 
currents. When they fall upon a suitable medium they ger- 
minate and soon develop the typical mold plant. 
Morphology of the Protozoa 
The protozoa are unicellular and bear much the same relation 
to the higher animals or metazoa that the bacteria do to the higher 
plants. Notwithstanding that they are reckoned among the 
simplest forms of life, they are, nevertheless, greatly diversified 
in shape, size, and structure. Only the barest outline of their 
structure can be given here. For a more detailed account the 
student is referred to the section on Protozoa. MORPHOLOGY AND RELATIONSHIPS OF MICROORGANISMS 
45 
Form and Size of Protozoa.-The pathogenic protozoa vary 
in size from those visible to the naked eye to those barely visible 
with the highest powers of the microscope. Some are pos- 
sibly ultramicroscopic, the organism causing yellow fever, for 
example. In form and shape the greatest diversity is to be noted. 
Some are without definite shape, and are apparently only lumps of 
protoplasm, others are highly differentiated and have as great 
variety of organs (organella') as some of the higher animals. 
Histology.-A true cell wall, as found in the bacteria, yeasts, 
and fungi, is frequently not present in the protozoa. When 
found, it is chitinous in nature. The ectoplast forms the limit- 
ing membrane of the cell in the majority of cases. The protoplasm 
is differentiated into nucleus (sometimes two, a micronucleus 
and a macronucleus') and endoplasm or cytoplasm. Power of move- 
ment is possessed by many forms. This may be due to the 
development of pseudopodia, of flagella, or of cilia. 
Reproduction.-Asexual reproduction is accomplished in many 
cases by a simple process of fission, in others the procedure is 
much more complex. Sexual reproduction is quite general, but 
here again the complexity is so great as to render brief treatment 
impracticable. The relationship and structure of these forms 
will be considered in greater detail under the heading of Patho- 
genic Protozoa in Section V. CHAPTER III 
PHYSIOLOGY OF MICROORGANISMS 
Physiology has been defined by Barnes to include " a study 
of the behavior of plants (and animals) of all sorts, and of the 
ways in which this is affected by external agents of every sort." 
In our discussion of the physiology of microorganisms we shall 
have to deal principally with the interrelationships existing 
between these microorganisms and their environment. 
Food Relationships of Microorganisms 
A food is any substance which a living organism may make 
a part of its living material or use as a source of growth energy. 
The term is frequently used very loosely to include all the sub- 
stances of which an organism may make any use. For example, a 
distinction is sometimes made between green plants and animals 
on the basis of food used. The former are said to live on inorganic 
foods and the latter on organic. This distinction is erroneous. 
The difference is simply that green plants can manufacture their 
own foods out of inorganic material by the aid of the energy 
secured from the sun's rays through the green coloring-matter or 
chlorophyll, while animals make use of food already prepared. 
The materials of which some microorganisms make use are no more 
foods than the rays of the sun are a food for green plants. 
Composition of the Cell.-The food utilized by any micro- 
organism must contain the elements needed for the building up 
of the cell substance. The analysis of such cells shows them 
to be made up of the same elements as those of higher plants 
and animals, namely, carbon, oxygen, nitrogen, hydrogen, and 
smaller amounts of phosphorus, iron, magnesium, calcium, and 
some other elements. The foods utilized by organisms must, 
therefore, contain these elements likewise. 
Sources and Kinds of Foods.-Some bacteria, like the green 
plants, are capable of manufacturing their own food. For this 
purpose a source of energy is necessary. Some species utilize the 
46 PHYSIOLOGY OF MICROORGANISMS 
47 
energy of the rays of sunlight in much the same manner probably 
as do green plants. The coloring-matter in these forms, however, 
is purple or red (^acteriopurpurin). Other forms living in water 
which contains hydrogen sulphid, as in the sulphur springs, oxidize 
the hydrogen sulfid to free sulphur and even sulphuric acid and 
gain energy for the manufacture of their foods from carbon dioxid, 
water, and other compounds by this process. Still other forms 
are believed to make use of iron, ammonia, nitrites, and other 
inorganic substances, and by their oxidation secure the necessary 
energy. Organisms which can manufacture their own food out 
of inorganic substances are said to be prototrophic. The proto- 
trophic microorganisms so far as known are all bacteria or molds. 
Most microorganisms utilize organic matter in a dead or living 
condition for food. Those which utilize dead organic matter are 
called metatrophic, while those requiring living material or complex 
protein foods are called paratrophic. The latter are frequently dis- 
ease producing. It must not be supposed that these division lines 
are strictly drawn. For example, certain bacteria seem to make use 
of the oxidation of carbohydrates and other organic substances to 
enable them to take up the nitrogen from the air and to convert it 
into usable form. Such are both prototrophic and metatrophic. 
The peculiar food requirements of the different species must 
be kept in mind in the preparation of nutrient media for their 
growth. Some organisms will not grow in the presence of organic 
materials, while others require such specialized media as blood- 
serum or hemoglobin. 
A second grouping of microorganisms commonly used is based 
upon the relationship to other living organisms. Those which 
do not require a living host (animal or plant) are called sapro- 
phytes if bacteria, yeasts, or molds, and saprozoites if protozoa; 
those which require a living host are called parasites. Those 
parasites which do not produce disease are termed commensals. 
Moisture Relationships of Microorganisms 
Microorganisms require considerable amounts of water for 
their development. The optimum condition for growth in 
most cases is saturation. There is great variation in ability 
to resist desiccation. The spores of some bacteria and fungi 48 
VETERINARY BACTERIOLOGY 
and the encysted cells of some protozoa will live for years, while 
other forms are destroyed if allowed to become completely dried. 
Respiration is frequently defined as the taking up of oxygen 
and the elimination of carbon dioxid. This definition is entirely 
inadequate when we come to a discussion of microorganisms, 
if, indeed, it can be applied in any case even to higher animals 
and plants. Respiration seems fundamentally to be the process 
whereby energy is generated in the cell. Energy when evolved in 
the cell always originates from chemical changes in the compounds 
within the cell. Whether or not this energy may be gained by 
the oxidation of food materials when taken into the cell, or whether 
they must be first built up into protoplasm and this then broken 
down, is a matter of dispute at present among scientists. In 
any event the presence of free oxygen is certainly not neces- 
sary to this release of energy, for many bacteria as well as other 
plants and animals live in the absence of free oxygen. Organ- 
isms that grow only in the presence of oxygen are called aerobic; 
those which will grow only in the absence of free oxygen, anaerobic, 
and those which will grow either with or without free oxygen, facul- 
tative. It is probable that most of the so-called anaerobes grow 
better in the presence of minute quantities of oxygen. The 
end-products of respiration are found to differ with the type, 
aerobic bacteria usually produce carbon dioxid and water; anae- 
robic forms, less highly oxidized substances, such as alcohol and 
butyric acid. 
The oxygen requirements of anaerobic bacteria must be recog- 
nized in the laboratory if they are to be successfully cultivated. 
The air of the culture-tube or flask may be removed by a stream 
of hydrogen, nitrogen, or some other inert gas, the oxygen may be 
absorbed by the use of alkaline sodium pyrogallate, the air may 
be exhausted by an air-pump, the oxygen may be excluded by 
covering the medium with oil or some similar material, or the 
organism may be mixed with some aerobic form which will use 
up the oxygen and allow growth of the anaerobe. Probably 
the latter is the common method whereby anaerobes are able to 
grow in nature. An organism may be an obligate aerobe under 
certain conditions or in certain media and facultative under 
Respiration of Microorganisms PHYSIOLOGY OF MICROORGANISMS 
49 
different conditions. For example, many bacteria will grow in 
the absence of oxygen providing there is available some compound 
from which oxygen may be readily obtained. Some will use 
nitrates for this purpose, others may use sulphates, reducing 
them to nitrites and sulphids respectively. Many will grow 
under anaerobic conditions providing suitable carbohydrates 
which they can ferment are present. 
Growth Rates and Death Rates 
The various physical and chemical agencies to be discussed, 
manifest their action upon microorganisms by their effect upon 
the rate of growth on the one hand and the rate of death on the 
other. Before they can be discussed adequately, therefore, 
certain general considerations concerning rates of growth and 
death must be presented. 
Rates of Growth.-When bacteria or other organisms are 
planted in a suitable culture medium, and optimum conditions 
for growth maintained, the bacteria soon reach a maximum rate of 
growth. This can best be measured by determining the average 
length of time required for an organism to grow to its full size, 
divide, and form two individuals. This has been termed the 
generation time. It is the average length of time elapsing between 
cell divisions. It may be most conveniently determined in any 
given case, not by watc.hing the bacteria under the microscope, 
but by counting the number of living cells present in a given 
amount of culture after varying lengths of time. It is evident 
that the shorter the generation time is found to be, the more 
rapid is the rate of multiplication. Anything therefore which 
tends to shorten the generation time increases the rate of growth. 
The actual length of the generation time may be determined 
if it is certain that the bacteria are multiplying at a uniform rate, 
that is, that the cultures are not too young or too old, by count- 
ing the bacteria at the beginning and at the end of a given period. 
It is evident that if one starts with a single organism, at the 
end of the first generation period there will be two, at the end 
of the next four (22), at the end of the next eight (23) and so on. 
If n represents the number of generations, then there will be 
at the end of the nth generation period 2" bacteria. If B 50 
VETERINARY BACTERIOLOGY 
represents the number of bacteria at the beginning, and b the 
number at the end, then 
6 = B2". 
If b and B are known, their values may be substituted into 
the equation, and the value of n determined. 
If t is the total time, and g is the generation time, it is evident 
that the generation time must equal the total time divided by 
the number of generations 
t 
Q = 
n 
Careful studies of bacteria growing in culture media show 
that the length of the generation time does not remain a constant 
throughout the time of cultivation. When bacteria are first 
planted in a culture, particularly if they have been taken from an 
old culture, they frequently grow slowly at first. As they become 
accustomed to their environment they multiply more rapidly, 
that is, the value of g decreases. After a time the maximum rate 
of growth occurs, and the value of g is at a minimum. Still 
later the bacteria become crowded and the rate of growth slows 
down, g increases and finally the bacteria cease to grow at all and 
begin to die off. 
It is evident that a fair comparison of effect of any influence 
upon bacteria can be determined by comparing the values of g 
under different conditions. 
Rates of Death.-It is equally important to know something 
of the laws which govern the rates at which bacteria die off 
under unfavorable conditions. In most of the cases which have 
been carefully studied, the bacteria die off in accordance with 
a definite law, which may be stated as follows: with a given kind 
of organisms under uniform unfavorable conditions, the number of 
bacteria present in a culture will always be reduced by one half in 
equal periods of time, that is no matter how many bacteria there 
are at the beginning of a definite period of time, one half that 
number will always be alive at the end of the proper interval. 
For example, suppose that two cultures of the same organism, 
one containing a million bacteria and the other a thousand 
bacteria are subjected to the same unfavorable conditions. It is physiology of microorganisms 
51 
found that at the end of a definite period of time, say ten minutes, 
the bacteria in the less concentrated suspension number five 
hundred. It will be found that in the same period of time the 
number in the other culture has also been halved, that is, there 
are 500,000 bacteria left. Another way of stating is this, during 
each equal interval of time a definite percentage of those bacteria 
living at the beginning of the period will be killed. If we wish to 
compare unfavorable conditions in their effect upon the death of 
microorganisms we may compare the length of time required to 
reduce the numbers of bacteria by a definite percentage, say one 
half. If at one temperature, for example, half of the bacteria 
are killed in ten minutes, and at another temperature one half 
the bacteria are killed in five minutes, it is evident that the second 
temperature is far more destructive than the first. It will be 
noted that time required to kill half the bacteria is mathe- 
matically the converse of the generation time. 
In summary it may be emphasized that all effects of environ- 
ment upon microorganisms may be manifested in growing cul- 
tures by changes in the length of the generation time, changes 
in morphology, and in the physiological and cultural reactions. 
Likewise the effect of environment will be noted upon the rate 
of death by comparing the length of time necessary to kill a 
definite percentage of the microorganisms present. 
Temperature Relations of Microorganisms 
Optimum Temperature.-The optimum growth temperature 
is that which most favors the development of the microorgan- 
isms, that is, that temperature which gives the shortest generation 
time. The optimum varies with the species. A few organisms 
found in the ocean, in cold waters, alpine regions, etc., prefer a 
low temperature, from 0° to 15°. These are called psychrophilic. 
Those which prefer a somewhat higher temperature are called 
mesophilic. These latter may be again subdivided into those that 
prefer a "room" temperature of 18° to 25°, and those that 
prefer blood heat (man 37.5°) for the most parasitic forms. Tem- 
peratures such as are found in hot springs, interior of compost 
heaps (50° to 70°) favor the development of thermophilic bacteria. 52 
VETERINARY BACTERIOLOGY 
Minimum Temperature.-The lowest temperature at which 
an organism will continue growth is said to be its minimum. 
This temperature varies for different species. Some organisms 
will multiply in brine held at temperatures lower than the freez- 
ing-point of water. 
Maximum Growth Temperature.-The highest temperature 
at which an organism will multiply is called its maximum. This 
must not be confused with the thermal death point (see below). 
The majority of bacteria cannot grow above 45°. 
Growth Temperature Range.-The differences between the 
minimum and maximum growth temperatures vary within rather 
wide limits. Those organisms which exhibit considerable adap- 
tability and are able to grow through a wide range of temperature 
are called eurythermic. Most of the saprophytic organisms 
belong here. The parasitic types which have minima and 
maxima varying but little from the optima are stenothermic. 
Thermal Death Point.-The thermal death point of an 
organism is usually defined as that temperature which under 
given conditions will certainly destroy all the cells. It has been 
noted above that bacteria do not die off instantaneously. The 
following factors must be taken into consideration in the deter- 
mination of the rate of death of bacteria: 
1. The Absence or Presence of Spores.-Spores are much more 
resistant to high temperatures than are the vegetative cells. Forms 
having spores, therefore, have two rates of death, one for the 
vegetative cells and the other for the spores. 
2. Presence or Absence of Moisture.-Bacteria are more resis- 
tant to dry than to moist heat. The thermal death point is 
probably the temperature at which incipient coagulation of the 
albuminous protoplasm occurs, resulting in an inability to 
function. Water is necessary for this coagulation. The follow- 
ing table from Frost and McCampbcll illustrates this point: 
Egg albumen + 50 per cent, water coagulates at 56° C. 
Egg albumen + 25 " " " 74-80° C. 
Egg albumen + 18 " " " 80-90° C. 
Egg albumen + 6 " " " 145° C. 
Egg albumen + no water " 160-170° C. PHYSIOLOGY of microorganisms 
53 
This fact is emphasized by the laboratory methods of sterilization. 
The autoclave, with live steam at temperature of 120°, will destroy 
in ten minutes the most resistant spores, while in the hot-air 
oven a temperature of 150° to 170° for an hour is necessary. 
3. Reaction and Composition of Medium.--The reaction and 
composition of the medium has been found to exert a marked influ- 
ence on the thermal death point. Particularly should the hydro- 
gen-ion concentration of the medium, that is, the actual acidity, 
be noted. It is a well known fact, for example, that acid fruits 
are much more easily sterilized than are the more nearly neutral 
vegetables. 
4. Time of Exposure.-In general, the higher the temperature 
the shorter the period required to destroy life in the cells. Math- 
ematic formulas have been developed giving the time as a func- 
tion of temperature for some forms. Ten minutes' exposure is the 
usual standard. 
5. Specific Character of Organism.-Intrinsic variations in 
the character of protoplasm of different species make it necessary 
to determine the thermal death point for each species. 
Light Relationships of Microorganisms 
A few bacteria possessing bacteriopurpurin require light 
for their development. For most other microorganisms, particu- 
larly the bacteria and the pathogenic protozoa, darkness is the 
optimum light condition. Light, particularly the direct rays of the 
sun, will destroy all but the most resistant bacteria if exposed 
for a sufficient length of time. Sunlight when passed through 
a prism is readily broken up into its constituent colors, the least 
refracted rays, or the reds and yellows, at one end, and the more 
highly refracted rays, the blues and the violets, at the other. 
Exposure of bacteria to these various colored rays has shown 
the blues and violets to be most powerful, while the reds and 
yellows of the other end have little or no effect. It will be re- 
membered that the blue and violet rays are the ones which affect 
the photographic plate most intensely. The germicidal action 
of light on the pathogenic bacteria is of the greatest practical 
importance. It renders infection through the medium of the air 
in most cases a remote possibility. 54 
VETERINARY BACTERIOLOGY 
Even more powerful than the visible blue and violet light rays 
are the invisible ultra-violet rays. Lamps have been devised which 
give a maximum of ultra-violet light, and these have been found to 
be quite efficient in certain types of sterilization. The lamp com- 
monly used is a modification of the Cooper-Hewitt mercury vapor 
arc, in which the, glass has been replaced with quartz, for experi- 
ment has shown that most types of glass which permit of the pas- 
sage of visible light rays are at least partially opaque to the ultra- 
violet, while quartz will allow the free passage of such rays. When 
clear liquids are exposed in relatively thin layers to the rays from 
such a lamp, the bacteria present are quickly killed; the liquid is 
sterilized. The process is not efficient, however, when the liquid 
to be treated is .quite turbid, or opaque, or opalescent, as milk. 
It is probable that the ultra-violet light produces a coagulation 
or some other irreversible change in the protoplasm of the bacterial 
cells, for solutions of proteins exposed are precipitated. 
Sterilization of water by ultra-violet light has been used suc- 
cessfully in the purification of water supplies, and of water used in 
swimming-pools, etc. Apparently where properly used it is effi- 
cient. 
Effect of Electricity on Bacteria 
Strong direct currents of electricity passed through a solution 
containing bacteria will sterilize it. It is difficult, however, 
to dissociate the physical effect of the current directly upon the 
bacteria from the action of the chemicals produced by electrolysis. 
No practical use lias been made of the destructive action of 
electricity upon microorganisms, as the method is difficult to apply 
and is inefficient at best. The Rbntgen rays (x-rays) do not 
destroy bacteria even when the latter are exposed for consid- 
erable periods. 
Relationships of Microorganisms to Chemicals 
Microorganisms are profoundly affected both in growth and 
movement by the chemicals with which they come in contact. 
They may be attracted or repulsed, stimulated to increased growth, 
their development inhibited,or they may be destroyed when certain 
substances are present. PHYSIOLOGY OF MICROORGANISMS 
55 
Chemotaxy.-Motile microorganisms are attracted or repulsed 
by certain chemicals. The first is known as positive chemotaxy, 
the latter as negative. Certain protozoa and bacteria are attracted 
by oxygen and may be observed to swim about air bubbles under 
Fig. 24.-Chemotaxy: a, Spirilla attracted by a green algal cell which is 
giving off oxygen, aerotaxis; &, a leukocyte containing several bacteria which 
it has engulfed; c, capillary pipette containing a solution of beef extract, and 
at 2 an air bubble, placed in a drop of water containing motile bacteria. 
The latter are attracted in large numbers to the mouth of the tube; d, an air 
bubble surrounded by two concentric circles of organisms, the inner one 
bacteria, the outer protozoa. Each remains in the concentration of oxygen 
most favorable to its growth. 
the microscope. From their movements it is evident that differ- 
ent species prefer varying amounts of oxygen. This results in a 
grouping of the different kinds in concentric circles about the 
bubble. This type of chemotaxy is called aerotaxy (not aero- 
tropism) . The avidity of certain bacteria for oxygen has been used 
in the laboratory for their isolation from water, particularly the 
Asiatic cholera organism. Peptones and meht extractives at- 
tract many kinds of bacteria. This phenomenon may be readily 
demonstrated by introducing the tip of a minute capillary tube 
filled with such a solution into water containing numerous bacteria. 
These will be found to congregate in great numbers about the 
mouth of the tube and to enter it. Probably chemotaxis accounts 
for the flocking of the leukocytes or white blood-corpuscles to 
any part of the body attacked by certain bacteria. Micro- 
organisms are not always attracted by food stuffs and repelled 
by harmful substances. A mixture of peptone and mercuric 
chlorid will attract bacteria and then destroy them. 56 
VETERINARY BACTERIOLOGY 
Tropisms.-Organisms which are not free to move in response 
to a chemotactic stimulus may nevertheless be influenced in their 
direction of growth. Such a response in the direction of growth 
to an external stimulus is called a tropism. Mold hyphae will 
often grow toward a moist medium, while the conidiophores which 
they bear rise at right angles to its surface and seek to produce 
the spores as far as possible from a moist surface. These phenom- 
ena are known respectively as positive and negative hydrotropism. 
Fig. 25.-Chemotropism: a, b, Mold hyphae and conidiophores, showing the 
negative hydrotropism of the latter; c, an air bubble in a medium with four 
germinating mold spores. The hyphae are growing toward the air, showing 
positive aerotropism. 
The influencing of the direction of the growth by the action of 
chemicals is called chemotropism. The forms of mold and 
bacterial colonies, when growing upon artificial media, are 
largely determined by this factor. For example, many molds 
radiate in practically straight lines from their point of origin in 
a medium,and every branch quite exactly bisects the angle between 
the two filaments on either side. This mutual repulsion of the 
hyphae is doubtless due to certain of the products excreted by the 
cells. Heliotropism, or the influence of light on the direction of 
growth, is also observed in some forms. 
Influence of Reaction of Medium on Growth.-Many organisms 
are quite exacting in their requirements as to the reaction of the 
medium in which they are grown. Some forms grow best in a 
medium slightly acid, others refuse to develop except in one 
which is slightly alkaline. The majority of bacteria, however, 
grow well in a medium that is neutral to litmus. 
Antiseptics and Disinfectants 
An antiseptic is usually defined as anything that will inhibit 
the growth of microorganisms without necessarily destroying PHYSIOLOGY OF MICROORGANISMS 
57 
them. Inasmuch as anything which completely stops growth 
of bacteria must also kill them more or less rapidly an antiseptic, 
strictly speaking, is any substance which produces a relatively 
slow rate of death in bacteria. In the broadest sense, this term 
would include such physical agencies as the action of cold and 
heat, but in practice it is generally confined to chemical sub- 
stances. A disinfectant is a substance that will destroy patho- 
genic bacteria. Inasmuch as pathogenic and non-pathogenic 
bacteria are both destroyed by the same substances, there is 
little real difference in the meaning of disinfectant and germicide 
(something that will destroy all bacteria). A disinfectant, as 
distinguished from an antiseptic, kills bacteria rapidly. A 
deodorant is any substance that masks disagreeable odors or 
eliminates them entirely by removing their cause. A deodorant 
may or may not be a disinfectant or antiseptic. These latter 
terms are relative ones only, for any disinfectant if sufficiently 
diluted becomes an antiseptic. 
Theories of Action of Antiseptics and Disinfectants.-Germ- 
icides may destroy microorganisms by forming compounds with 
the protoplasm, by dissolving or coagulating the protoplasm, or 
by oxidation and complete destruction of the cells. Our most 
efficient disinfectants are those which destroy in the manner first 
named. The activity and efficiency of many disinfectants 
depend upon the ionization of the compound in solution. This is 
particularly true with the salts of heavy metals, such as mercuric 
chlorid (corrosive sublimate). It is the ionized mercury that is 
poisonous to bacteria. Mercuric chlorid does not ionize in pure 
alcohol, hence it is not poisonous to bacteria when in solution in 
that substance. The addition of various other chemicals may 
increase or decrease the ionization of the disinfectant and 
enhance or diminish its destructive action. 
Characteristics of an Ideal Disinfectant.-Certain character- 
istics may be listed as belonging to an ideal disinfectant. To the 
extent that a particular disinfectant measures up in its character- 
istics to those of the ideal, it is valuable for general use. The 
more important of these characteristics are as follows: 
1. Germicidal Power.-The ideal disinfectant should possess 
high germicidal power, that is, it should kill bacteria in dilute 58 
VETERINARY BACTERIOLOGY 
solution. The ability of a disinfectant to kill microorganisms is 
usually compared with that of phenol. In making such compari- 
sons it is customary to use the Bacterium typhosum, the cause 
of typhoid fever, as the test organism. Most commercial 
disinfectants, particularly the coal tar products, are sold upon 
the basis of their phenol coefficient. 
2. Stability - The disinfectant to be most valuable should be 
relatively stable in the presence of organic matter. Some of 
the most powerful of the disinfectants combine with organic 
matter forming insoluble compounds and pass out of solu- 
tion relatively completely. The strength of the disinfectant 
may be thereby rapidly decreased to a point where it no longer 
destroys microorganisms. 
3. Homogeneity.-Disinfectants should be homogeneous in 
composition. Substances which may be bought in pure condi- 
tion or in crystalline form such as mercuric chlorid are ideal from 
this point of view. Many of the commercial disinfectants, 
particularly those prepared from coal tar, may vary considerably 
in their composition from time to time, and consequently in 
their germicidal value. 
4. Solubility.-The ideal disinfectant is one which will dis- 
solve in all proportions in water, as alcohol, for example. 
5. Non-toxic to Higher Life.-An ideal disinfectant would be 
non-poisonous to man and to the higher animals. Obviously 
disinfectants which will kill one kind of cell and not injure 
another are not the most common. Most of the valuable disin- 
fectants are more or less injurious to tissues. Certain disinfec- 
tants, however, may be injected into the blood, exerting a more 
harmful influence upon microorganisms than upon tissues of 
the body, destroying the former without seriously injuring the 
latter. Such, for example, is the arsphenamine used in the 
treatment of syphilis. 
6. Non-corrosive.-Inasmuch as disinfectants must frequently 
be used in contact with metals, fabrics, etc. it is highly desirable 
that they should not attack metal, injure fabrics, leave stains 
or bleach color. 
7. Penetration.-Disinfectants differ decidedly in their power PHYSIOLOGY OF MICROORGANISMS 
59 
to penetrate materials to be sterilized. An ideal disinfectant is 
one which penetrates rapidly and efficiently. 
8. Economy.-The ideal disinfectant should be low in cost. 
Certain valuable disinfectants, because they are so expensive, 
can be used only in limited quantities and for special purposes. 
The high cost of salts of silver for example limits the use of 
silver practically to medicine. If the entire water supply of a 
city is to be sterilized obviously a disinfectant must be chosen 
which is relatively inexpensive. 
9. Power to Remove Dirt and Grease.-A film of oil or grease 
over the surface of certain materials may wholly prevent the 
action of many disinfectants. Those which have power to dis- 
solve or remove grease and all kinds of dirt are naturally more 
efficient. 
10. Deodorizing Power.-A disinfectant which can combinq 
with and destroy malodorous substances is preferable to one 
which does not. 
Disinfectants and Antiseptics in Common Use.-The addition 
of acid to any solution containing bacteria with the consequent 
increase in hydrogen ion concentration increases markedly the 
rate of death. 
Bacteria which grow well in a relatively high hydrogen ion 
concentration are termed acidophiles. The ability of these or- 
ganisms to grow in the presence of high concentrations of acid 
is sometimes utilized in their isolation. 
Acids are most commonly used as preservatives. The more 
important are the acetic (usually in the form of vinegar) and 
the lactic acids, the latter the preservative agent in sour milk, 
sauer kraut, and in silage. 
Strong acids are those which ionize most completely. Weak 
acids are those which ionize poorly. Lactic and acetic acids are 
relatively weak acids, and must be present in relatively large 
amounts in order to secure a high concentration of hydrogen 
ions. For example, tenth normal hydrochloric acid contains 
about ten times as many hydrogen ions as normal acetic acid. 
In a few acids either the anion or undissociated acid molecule 
may also exert a disinfection action. In some, this action is 
much more important than that of the hydrogen ion. For 60 
VETERINARY BACTERIOLOGY 
example, benzoic acid is a comparatively weak acid and yet it 
has relatively high antiseptic or disinfecting value. 
Alkalies.-High alkalinity, that is high concentration of 
hydroxyl ions, likewise exerts a destructive action upon micro- 
organisms. Lime owes its value as a disinfectant to this fact. 
Unslaked lime (calcium oxide) added to water is converted 
into calcium hydrate and when used in strong solutions, as 
in white wash, it has a marked disinfecting action. It is a 
valuable disinfectant to mix with stools or body excreta in 
order to destroy disease-producing bacteria. It should be 
noted, however, that upon exposure to air calcium hydrate 
is gradually converted into calcium carbonate which is no longer 
germicidal. 
Some bacteria will grow in relatively alkaline solutions. 
For example, the organism causing Asiatic cholera, the Vibrio 
cholera, will grow in much higher hydroxyl ion concentration, 
that is, in more alkaline solutions, than will most other species 
of bacteria. Use is made of this fact in isolating this organism 
from feces, the medium being made so alkaline as to inhibit 
the growth of most other microorganisms rendering the securing 
of this organism in pure culture comparatively easy. 
Salts of the Heavy Metals.-The salts of gold, silver, copper, 
and mercury are all active disinfectants. Copper sulphate is 
sometimes used in an effort to free water reservoirs and other 
city supplies from growths of objectional algse. Mercury is the 
most efficient of the metals and its salts are most commonly 
used. It acts by forming a mercuric albuminate of the 
protoplasm. When used in any solution containing consider- 
able quantities of protein or similar materials, as in feces, 
it must be added in excess and thoroughly mixed, for it is 
apt to form an insoluble coating over the surface of the 
solid particles and protect the bacteria in the interior from 
destruction. Mercuric chlorid is usually used in solutions of 
1:1000 or 1:2000. 
Phenol, or carbolic acid, C6H5OH, and the methyl phenols or 
cresols, C6HiCH3OH, either pure or in the trade mixtures, such 
as kreso, tricresol, creolin, etc., are among the most efficient 
and useful of disinfectants. They are most frequently used in PHYSIOLOGY OF MICROORGANISMS 
61 
1 to 5 per cent, solutions, and will destroy bacteria even in the 
presence of quantities of organic matter. 
Sulphur Dioxid and Sulphurous Add -When sulphur is 
burned it yields sulphur dioxid, a gas that has been much used 
in fumigation. It is powerless to destroy bacteria unless moisture 
is present, with which it may unite and form sulphurous acid. 
The latter is a very active bleaching and corrosive agent, hence 
it should not be used except where it can do no harm. One 
pound of water (about 1 pint) should be vaporized in a room 
for every 5 pounds of sulphur burned. This amount should 
efficiently disinfect 1000 cubic feet. Insects and other vermin 
are destroyed by the sulphur fumes. 
Alcohol is frequently used as an antiseptic, and sometimes as a 
disinfectant. The efficacy of this material is dependent upon the 
concentration. Beyer has shown that 70 per cent, alcohol is most 
powerful, proving to be thirty times as efficient as 60 per cent, and 
forty times as powerful as 80 per cent. Concentrations below 60 
per cent, and above 80 per cent, are apparently almost valueless. 
Formaldehyd, HCHO.-Formaldehyd is the gas used most 
widely in fumigation and disinfection. It is very soluble in 
water and is commonly sold as formalin, a 40 per cent, solution 
of formaldehyd. Like sulphur dioxid, formaldehyd is efficacious 
only in the presence of moisture, but, unlike it, does not bleach 
fabrics or injure materials. Formaldehyd may be evolved in 
gaseous form for disinfection in a variety of ways. Incomplete 
combustion of methyl alcohol according to the reaction 
2CH3OH + O2 = 2HCH0 + 2H2O 
is utilized in a number of lamps upon the market. When properly 
carried out the method may be efficient, but it has several 
disadvantages, i. e., expense and presence of a fire in a closed 
room. Heating the formalin over an open flame will liberate a 
part of the formaldehyd readily, but under these conditions 
it polymerizes and some of the polymers (paraformaldehyd) are 
insoluble. If the evaporation is continued to dryness, all of these 
will again be broken up and given off as formaldehyd. ' The 
same result can be reached more quickly by the addition of glyc- 
erin or some salt which will raise the boiling-point of the solu- 62 
VETERINARY BACTERIOLOGY 
tion above the dissociation temperature of the paraformaldehyd. 
An autoclave or closed vessel in which the solution is heated con- 
siderably above the boiling-point of water will serve the same pur- 
pose. Twelve ounces of formalin should be used for every 1000 
cubic feet to be fumigated. A convenient method for fumigating 
small rooms is to pour formalin over crystals of potassium per- 
manganate in an earthen vessel that is a poor conductor of heat. 
The permanganate is an active oxidizing agent and converts part 
of the formaldehyd into carbon dioxid and water, with the 
liberation of sufficient heat to vaporize a large portion of the 
remainder. The solid paraformaldehyd or paraform may be 
heated and is thereby converted into formaldehyd gas. On 
account of its cheapness and effectiveness formaldehyd is used 
much more commonly at present than any other of the gaseous 
disinfectants. 
Adjustment of Organisms to Osmotic Pressure.-Any crystal- 
loid in solution behaves within the limits of the solution like a 
gas, and the same laws of diffusion and diffusion pressures are 
applicable. Every organism when growing is surrounded by 
water containing substances in solution, and it also contains 
certain salts dissolved in the " cell sap " or water in the pro- 
toplasm. The ectoplast or limiting membrane of the protoplasm 
lying just within the cell wall is certainly in most, probably all, 
cases a semipermeable membrane, i. e., it will allow some sub- 
stances to pass through readily, as water; others pass through 
slowly, and still others, although in true solution, cannot pass at 
all. This ectoplast, in short, serves as an osmotic membrane 
and determines what substances may enter and leave the cell. 
An active cell always maintains within its sap a greater con- 
centration of solutes than the surrounding medium, the pressure 
on the inside of the membrane is greater than on the outside, 
and the cell is said to be in a state of turgor. When such a cell 
is placed in water containing a greater percentage of solutes than 
does the cell sap, water leaves the latter until the concentra- 
tion on the inside and outside again becomes the same. This 
means a shrinking of the protoplasm; it withdraws from the cell 
wall and the cell is said to be plasmolyzed. After a time the 
cell may readjust the amount of solutes in the cell sap and regain PHYSIOLOGY OF MICROORGANISMS 
63 
its turgor. For every cell, however, there is a limit beyond 
which the organism cannot go. Some yeast cells have been found 
to develop slowly in a solution containing 35 per cent, of cane- 
sugar. A solution of this concentration exerts a pressure of more 
than 350 pounds per square inch. It is apparent that such a cell 
must be profoundly modified. The fact that concentration 
of solutes inhibits the growth of microorganisms is utilized in 
the preservation of many food stuffs. Such foods as syrups, 
jellies, and candied fruits are preserved by the high osmotic 
pressure of the solutes. The action of sugars, salts, etc., is in 
the nature of a physical antiseptic. Physiological salt solution 
is one having the same concentration of salt as do the body cells 
of the particular organism to be studied. It usually contains 
.85 per cent, of sodium chlorid. 
Two organisms that live together and which are mutually 
beneficial are said to live in symbiosis. Each organism is called 
a symbion or symbiont. The symbionts are not necessarily closely 
related forms and may belong to the most widely separated groups 
of plants, as, for example, bacteria and members of the bean family 
of the flowering plants. 
Antibiosis is that condition which obtains when organisms 
prove inimical to each other's development. The growth of one 
species of organism in a cultured-medium may completely inhibit 
the development of some other type. For example, the organism 
(Streptococcus lacticus) which ordinarily sours milk prevents the 
development of most other species. 
An organism which uses the by-products of another as food, 
in other words, is parasitic without producing disease, is called a 
commensal. Many of the bacteria found on the skin, in the mouth, 
and in the intestinal tract of man and animals are of this character. 
All degrees of intergradation between symbiosis, true para- 
sitism, and commensalism have been described for different 
species. 
Symbiosis, Antibiosis, and Commensalism 
Pigment Production by Microorganisms 
Molds, yeasts, and bacteria are frequently found to be chromo- 
genic, that is, capable of producing pigments or coloring-matter. 64 
VETERINARY BACTERIOLOGY 
The colors produced range through all the colors and even shades 
of color of the spectrum. A few only of the pathogenic forms 
produce pigments. Many organisms, particularly molds and 
bacteria, excrete a pigment-forming substance which diffuses 
through the culture-medium and colors it. Such an organism 
is the Pseudomonas pyocyanea, which produces a diffuse pigment, 
one that changes the medium first to a green, then to a brown. 
Some organisms produce pigment granules outside the cell, such 
are said to be chromoparous, for example, Erythrobacillus prodi- 
giosus, which produces a red pigment. The cell walls of some 
bacteria (such as B. violaceus) and of many of the molds are 
colored. 
Pigments are generally produced only in the presence of free 
oxygen. Cultivation at high temperatures causes some organ - 
isms to lose the power of pigment production. Some pigments 
are soluble in water, others in alcohol, and others in ether and 
various fat solvents. They are of little economic importance, 
but are of value to the systematic bacteriologist in the separation 
and identification of species. 
Light Production by Microorganisms 
Several species of bacteria and fungi are known that give 
off light. These are said to be photogenic. Bacteria of this 
type are found commonly in the water of the ocean, and are 
easily isolated from salt fish. When grown in a test-tube, they 
are sometimes sufficiently luminous, so that they may be photo- 
graphed by their own light. 
Fermentation and Enzyme Production 
All microorganisms have their protoplasm bounded by the 
ectoplast, a semipermeable membrane, as previously shown. The 
cell wall, when present, seems to be a mechanical protection and 
support, and is readily permeable to most substances in solution, 
so may be disregarded in discussion. Whatever food or other mate- 
rials are taken into the protoplasm must pass by diffusion through 
the ectoplast. This membrane, on the other hand, must prevent 
any valuable constituent of the cell from leaving by diffusion. 
Its action is, therefore, selective.^ Microorganisms do not always 
find the potential food materials with which they are surrounded PHYSIOLOGY OF MICROORGANISMS 
65 
suitable for food, for they may be in a solid form or, if in solution, 
of such a character that they cannot pass through the ectoplast. 
Many organisms find it necessary to so change this food and 
digest it that it may be assimilated. Once inside the cell, it 
usually is not of such a character that it can be built up directly 
into the protoplasm and further changes are necessary; or if the 
Fig. 26.-A bacterial lamp. The inner wall of the flask is coated with a 
medium on which there is growing Bacterium phosphoreum. Photographed by 
its own light (Molisch). 
material is used simply as a source of energy and not incorporated 
into the cell substance, it is essential that the cell have some means 
of developing this energy. All cells accomplish these changes by 
means of enzymes (Gr. en, within, zyme, leaven). A dis- 
tinction was once made between the so-called organized and 
unorganized ferments. The former was held to be living cells 
which could bring about a change or fermentation, the latter any 
cell secretion which could bring about such changes. In other 66 
VETERINARY BACTERIOLOGY 
words, organized ferments were supposed to owe their activity 
directly to the protoplasm, the unorganized, to substances secreted 
by the protoplasm. This distinction is no longer maintained, 
as it seems altogether probable that fermentative changes of 
whatever kind are brought about by the secreted enzymes or 
unorganized ferments. Enzymes may be intra- or extracellular. 
The intracellular enzymes are extracted from the protoplasm 
with difficulty, and during the life of the cell do not leave it. 
Such an enzyme is that of bread or brewer's yeast (the zymase), 
which converts dextrose into alcohol and carbon dioxid. Extra- 
cellular enzymes are usually digestive in their action. Different 
kinds attack different substances. Microorganisms are known 
which produce enzymes that will break down cellulose, starch, 
sugars, fats, and proteins into simpler substances. The action 
of an enzyme is said to be specific; a given enzyme will in gen- 
eral change only one type of material. 
Enzymes are said to be organic catalysts (Gr. kata, down, 
lyo, to dissolve), that is, they bring about changes without 
themselves becoming part of the final product. Many inorganic 
catalysts, such as finely divided platinum, are known to the 
chemist. Although catalysts do not form a part of the final 
products, they certainly are a part of some of the intermediary 
products in many cases, but become free again when the action has 
been completed. Enzymes, then, are peculiar in that they are 
not used up in the using. Theoretically the amount of change that 
can be brought about by a given enzyme is limited only by the 
time and conditions under which it must act. 
Most enzymes produce changes that are hydrolytic in nature, 
that is, they bring about the incorporation of water into the organic 
molecule with resultant disintegration. The digestion of gelatin 
by the bacterial enzyme gelatinase, the conversion of starch into 
maltose by ptyalin and diastase, the digestion of proteins by 
pepsin, the conversion of saccharose or cane-sugar into invert 
sugar by invertase, and the clotting of milk by rennet are a few 
examples of such hydrolytic changes. The following reaction 
illustrates the hydrolytic cleavage of saccharose by the invertase 
produced by yeast: 
^12H220u 
Saccharose. 
+ H2O + Invertase Ilz; C0H12O0 
Dextrose. 
+ C0H12O8 
Levulose. 
+ Invertase. PHYSIOLOGY OF MICROORGANISMS 
67 
Other enzymes are active oxidizers. Changes of color in 
dead or injured plant and animal tissues are sometimes due to 
such oxidases. For example, potatoes turn black and apples 
become brown when the cells are bruised. Some other enzymes 
are said to be splitting. One of the best examples of these is the 
zymase of yeast, which converts dextrose into alcohol and carbon 
dioxid. 
C6H12O8 
Dextrose. 
+ Zymase = 2C2H5OH 
Alcohol. 
+ 2CO2 
Carbon 
dioxid. 
+ Zymase. 
Although alcohol and carbon dioxid represent the end-products, 
it is by no means certain that intermediate hydrolytic products 
are not formed, and this splitting action may be essentially hydro- 
lytic. Reducing enzymes have also been demonstrated in plant 
and animal tissues and undoubtedly occur in microorganisms. 
The autolytic (Gr. auto, self, lyo, dissolving) enzymes de- 
serve particular mention. Enzymes are known to occur in 
most animal and plant cells that will, at least partially, digest 
the cells in which they occur. The rigor mortis or stiffening of the 
tissues of an animal after death is due to such an autolytic enzyme 
which coagulates the muscle protoplasm. The softening of the 
tissues which occurs later, the so-called 11 ripening " of meat, is due 
in part to the action of another proteolytic enzyme which carries the 
digestion somewhat further. Microorganisms contain such en- 
zymes, and when the cells die, as in an old culture, they are then 
partially digested. This autolytic action we shall find to be of 
some practical significance in a discussion of disease production 
and immunity, as by it certain poisonous substances may be 
released from the cell. CHAPTER IV 
CHANGES OF ECONOMIC SIGNIFICANCE BROUGHT ABOUT BY 
NON-PATHOGENIC ORGANISMS 
Microorganisms bring about many changes, both analytic 
and synthetic, in the media in which they are cultivated. In 
any such medium growth products of many kinds will be found. 
These fermentative products may originate from the activity of 
extracellular enzymes, may consist of substances excreted from 
the cell as the product of intracellular enzymes, or as a result 
of the metabolic activity of the cell; they may be products of syn- 
thetic action (as the slimes and gums produced by a solution of 
the bacterial capsule), or they may be substances produced by the 
autolytic activity of intracellular enzymes after the death of the 
cell. Naturally, the products will vary greatly with the species 
of organism, the medium in which it is grown, and the character 
of the physical environment. 
From the standpoint of the veterinarian, microorganisms 
are of most importance because many of them produce disease. 
It would, however, give a false impression of the place and function 
of microorganisms in nature to neglect at least a brief consid- 
eration of some of the other changes which they can bring about. 
In this chapter some of the more important will be considered. 
The well-nigh universal distribution of bacteria and other 
microorganisms should be emphasized. They are to be found in 
the soil in great numbers, rich surface soil containing from 100,000 
to 5,000,000 bacteria to every dry gram. Their dried bodies and 
spores are constantly present free or attached to dust particles in 
the air. They are to be found in all surface waters in considerable 
numbers and are present even in the water from deep wells. 
They grow upon the surface of the skin of animals, and the mouth 
and digestive tract support a large and varied flora. It is apparent 
that whenever conditions are favorable for the growth of micro- 
organisms they will be present to begin growth. 
68 CHANGES BROUGHT ABOUT BY NON-PATHOGENIC ORGANISMS 
69 
The changes brought about by bacteria in nature are of such 
importance that but for their continuance plant and animal 
life on earth would quickly cease to exist. The fertility of the 
soil and the consequent production of all food stuffs is directly 
due to certain of the microorganisms present. 
Production of Alcohol.-The various alcohols, but more par- 
ticularly ethyl alcohol, are produced by certain bacteria, yeasts, 
and molds. It has been shown that in the yeast the ability to 
bring about this change is resident in the intracellular enzyme, 
zymase. Probably similar enzymes are present in the alcohol- 
producing bacteria and molds. The common bread or brewer's 
Fig. 27.-Brewer's yeast, Saccharomyces cerevisice. 
yeast is the form most commonly used in the manufacture of 
alcoholic beverages, but certain molds have been found very 
useful in the production of alcohol for industrial purposes. Alco- 
hol is commonly produced by the fermentation of one of the 
hexose monosaccharids, such as dextrose. The reaction may be 
given as follows: 
c0h12o6 
Dextrose. 
= 2C3H5OH 
Alcohol. 
+ 2CO2. 
Carbon 
dioxid. 
Yeast is utilized for its other product of fermentation, carbon 
dioxid, by the baker in bread making. Higher alcohols, such as 
butylic and amylic, are also formed. The yeasts which produce 
this change are quite widely distributed in nature, being par- 
ticularly abundant on the surface of fruits and in saccharine 
liquids. Fruit juices and other solutions containing sugar if 
allowed to stand, therefore undergo " spontaneous " fermenta- 
tion, with production of ciders, wines, and similar beverages. 
Production of Acids.-Several of the organic acids are com- 
monly produced by fermentative organisms. Three of these are 70 
VETERINARY BACTERIOLOGY 
of particular economic importance, namely lactic, acetic, and 
butyric. A great variety of others are occasionally produced, 
usually in small quantities only. 
Fig. 28.-Lactic acid bacteria: A, Streptococcus lacticus; B, Lactobacillus 
bulgaricus. 
Lactic Acid.-Dextrose and some other monosaccharids are 
converted into lactic acid by several common organisms. The 
reaction may be empirically represented as follows: 
c6h12o6 
Dextrose. 
= 2C2H;OH-COOH. 
Lactic acid. 
The reaction occurs most frequently in milk which is allowed to 
stand. In this case the lactose or milk-sugar is first broken 
down into the monosaccharids before being converted into lactic 
acid. 
C12H22O)! 
Lactose. 
+ h2o = c0h13o0 
Dextrose. 
+ C0H12O6. 
Galactose. 
The formation of lactic acid in milk is of the greatest economic 
importance, as the organisms which produce this acid are the ones 
Fig. 29.-Acetic acid organism, Acetobacter aceti: a, Normal individuals; b, 
involution forms. (Adapted from Hansen.) 
which are necessary to the development of proper flavors and 
quality in butter and cheese. This acid is also produced in 
the manufacture of sauer-kraut and to some extent in silage. 
The lactic acid formed in milk is instrumental in preventing the 
growth of putrefactive and other undesirable bacteria. CHANGES BROUGHT ABOUT BY NON-PATHOG ENIC ORGANISMS 
71 
Acetic Acid.-Acetic acid is the most important and the 
characteristic constituent of vinegar. It is produced by several 
species of bacteria by the oxidation of ethyl alcohol according 
to the following reaction: 
c2h5oh 
Alcohol. 
+ o2 = ch3cooh 
Acetic acid. 
+ h3o. 
Any solution containing alcohol, if left in contact with free oxygen, 
will commonly undergo this fermentation spontaneously, as 
Acetobacter aceti, the organism usually responsible, is ubiquitous. 
To insure rapid and efficient fermentation the cider or other 
alcoholic solution is sometimes inoculated with mother of vinegar, 
a mass of the organism which commonly forms a mat upon the 
surface of the fermenting liquid. 
Fig. 30.-Butyric acid bacteria, Clostridium butyricum. (Adapted from 
Fischer.) 
Butyric Acid.-Under anaerobic conditions saccharine solu- 
tions are apt to undergo butyric acid fermentation as a result of 
the development of the Clostridium butyricum or a related form. 
The reaction may be represented as follows: 
c6h12o6 
Dextrose. 
= C3H7COOH + 
Butyric acid. 
2CO2 + 2H2. 
Butyric acid has an exceedingly disagreeable odor and taste, 
hence the growth of this organism in any saccharine or starchy 
food substance renders it unfit for use. Inasmuch as these 
organisms are all spore producers, they resist heat well, and as 
they are anaerobic they will grow when sealed in a can and all 
air excluded. Rancidity in butter is sometimes due, in part 
at least, to the development of butyric acid. 
Decay and Putrefaction.-A distinction is sometimes made 
between the terms decay and putrefaction. The former is said to 72 
VETERINARY BACTERIOLOGY 
be decomposition of organic matter brought about by the aerobic 
bacteria, the latter by the anaerobic types. This distinction 
is not always acknowledged and adhered to, however. The 
substances produced by the decomposition by bacteria depend, of 
course, quite largely upon the nature of the material to be de- 
composed. The carbohydrates and fats break down into alcohols, 
acids, and carbon dioxid, but the proteins are split into a great 
variety of substances. Other agents, as acids and alkalis, will 
break up the proteins in a similar manner and into many of the 
same substances as do bacteria. Chemists have in recent years 
demonstrated that proteins are made up of large numbers of 
molecules of the a-amino acids linked together. An a-amino acid 
is an organic acid that has the NH2 group in the alpha position, 
that is, next the carboxyl. For example, the amino acid corre- 
sponding to C2H5CH2COOH, butyric acid, is C2H5CHNH2COOH. 
When these constituent links of the protein molecule are forced 
apart, they usually appear in the form of one of about twenty com- 
pounds which have been grouped as primary protein derivatives. 
Some of these normal derivatives are further altered by bacteria. 
Among them have been found certain compounds called ptomains, 
some of which are known to be very poisonous. The splitting 
usually continues until much of the organic matter is reduced 
to comparatively simple compounds, such as H2S, CO2, CH(, and 
NH3. The process of protein disintegration is called proteolysis. 
It usually occurs in several distinct stages. The proteins are 
first broken down into relatively complex substances called prote- 
oses, these then are broken down into peptones. This is called 
peptonization, as it is essentially the change that may be brought 
about by the enzyme pepsin from the stomach. The process con- 
tinues and the peptones become amino acids and at last ammonia 
is liberated. From an economic point of view this liberation of 
ammonia has the greatest significance, for from this transformation 
comes all the nitrogenous material used by plants and indirectly 
by animals as food. This is the essential transformation that all 
organic nitrogenous fertilizers, such as barnyard manure and 
dried blood, will undergo before they can be of any use to higher 
plants. By such changes water contaminated by sewage purifies 
itself. CHANGES BROUGHT ABOUT BY NON-PATHOGENIC ORGANISMS 
73 
Some of the nitrogenous products of bacterial decomposition 
are worthy of note, inasmuch as they are used in the laboratory in 
the differentiation of certain species. The most important of 
these are indol and skatol. They are organic compounds having 
the following formulas: 
CH. 
NH 
CH 
C . 
NH 
CH3 
c0h4; 
c6h4< 
CH. 
Indol. 
Skatol. 
Indol is produced by certain bacteria when growing in a solution 
of peptone. It is identified by the addition of nitrous acid, with 
which it combines to form nitroso indol, a bright red compound. 
In making the test in the laboratory it is customary to add a 
Fig. 31.-Some decay-producing and putrefactive bacteria. 
few drops of concentrated sulphuric acid, followed by a dilute solu- 
tion of nitrite. The sulphuric acid breaks up the nitrite, with the 
formation of free nitrous acid, which then unites with the indol. 
Indol and skatol are also formed in the intestines by the activity 
of certain of the bacteria found there and are of considerable 
physiological significance. 
Reduction Processes in Inorganic Compounds.--Changes similar 
to those just discussed are sometimes brought about by bacteria 
in inorganic compounds. When nitrates are in solution together 
with organic substances and under anaerobic conditions, the 
bacteria present in many cases will reduce the nitrates to nitrites 
and the nitrites to free nitrogen, apparently in order to utilize 
the oxygen. This process is usually called denitrification because 
the medium loses nitrogen, but is more correctly a reduction or 
deoxidation. Sulphates are reduced to sulphites and even to 
sulphids under similar conditions. For example, the sewage from 74 
VETERINARY BACTERIOLOGY 
a city whose water supply contains a large percentage of sulphates 
will develop hydrogen sulphid in considerable quantities if it is 
put under anaerobic conditions. Other reductions of a similar 
nature have been described for chlorates. 
Fig. 32.-Denitrifying bacteria: A, Bacterium coli, which changes nitrates 
into nitrites; B, Pseudomonas denitrificans, which produces free nitrogen from 
nitrates. 
Oxidation of Inorganic Compounds.-Bacteria and other 
microorganisms that live in the presence of oxygen are sometimes 
active oxidizers of inorganic compounds, securing in this manner 
the energy that is necessary for their various growth processes. 
Fig. 33.-Sulphate reducing spirillum, Spirillum desulfuricans. 
Oxidation of Hydrogen Sulphid.-Waters containing hydrogen 
sulphid, as do many of the so-called mineral springs, usually 
contain bacteria which gain their energy for food manufacture 
Fig. 34.-Microorganisms that oxidize hydrogen sulphid: A, B, Beggiatoa 
sp.; C, Thiophysa volutans (Hinze). 
and growth from the oxidation of this substance. The slimy- 
black and white deposit commonly found in such waters, when 
examined microscopically, will be seen to be made up of masses of CHANGES BROUGHT ABOUT BY NON-PATHOGENIC ORGANISMS 
75 
Beggiatoa and similar organisms whose cells will be found packed 
with sulphur granules. Probably the following reaction accounts 
for this formation of free sulphur: 
2H2S + O2 = 2H3O + S2. 
The process is carried still farther if there is any deficiency of 
the hydrogen sulphid, and the free sulphur is converted into 
sulphuric acid and sulphates. 
S2 + 3O2 = 2SO3 
H3O + SO3 = H2SO4. 
The sulphuric acid is, of course, at once neutralized by the bases 
present in the water. 
Oxidation of Iron.-Many natural waters contain ferrous car- 
bonate or some similar salt of iron. Certain bacteria oxidize 
Fig. 35.-Microorganisms that oxidize ferrous to ferric iron: a, Lep- 
tothrix ochracea; b, Gallionella ferruginea; c, Spirophyllum ferrugineum. 
(Adapted from Ellis.) 
this to ferric hydrate, and deposit this insoluble material in 
their sheaths. The reaction may be represented as follows: 
2Fe2CO3 + 3H2O + O = Fe2(OH)6 + 2CO3. 
Probably these organisms make use of the energy obtained by 
this reaction in the same manner that the sulphur bacteria do the 
oxidation of the sulphur, to secure energy for the formation of their 
foods and to gain the energy needed for growth and development. 
These organisms are particularly apt to occur in well water or 
spring water laden with iron, and have in some cases caused 
considerable trouble by clogging the water pipes with their growth. 
It is known that the bog iron ore of Sweden and probably the 76 
VETERINARY BACTERIOLOGY 
great iron beds of northern Minnesota have been deposited by 
the activity of such organisms. 
Oxidation of Ammonia.-In most soils there are numerous 
bacteria that oxidize free ammonia to nitrous acid, and by neutral- 
ization with the soil bases form nitrites. These organisms do not 
develop well in the laboratory in the presence of organic matter. 
It seems evident that they utilize the energy secured from the 
Fig. 36.-Bacteria that oxidize ammonia and nitrous acid to nitrous 
and nitric acid respectively: a, Nitrosomonas europea; b, N. javensis; c, 
Nitrobacter (Winogradsky). 
oxidation, of the ammonia to build up their protoplasm out of 
simple materials. They are among the best examples of the 
strictly prototrophic bacteria. Organisms capable of bringing 
about this change are called nitroso-bacteria. The reaction may be 
represented as follows: 
NH3 + 2O2 = HNO3 + H2O. 
This is the first of the two steps in the process called nitrification 
in the soil. 
Oxidation of Nitrous Acid.-The nitrous acid formed in the 
soil and in water, etc., by the preceding group is further changed 
by another group of organisms called the nitrate bacteria. Like 
the preceding, they are widely distributed in water and soil, 
and complete the process called nitrification or, better, oxidation 
of nitrogen. The nitrates produced by their activity are the 
source of nitrogen for green plants. A few of the latter are able 
to make use of nitrogen in the form of ammonia compounds, but 
in nature this rarely occurs. The reaction may be represented as 
follows: 
2HNO2 + O3 = 2HNO3. 
It is probable that the fertility of the average soil is more largely 
determined by the maintenance of conditions favorable to the CHANGES BROUGHT ABOUT BY NON-PATHOGENIC ORGANISMS 
77 
development of these nitrifying organisms than by any other 
single factor. Their importance in nature as essentials for the 
growth of the higher plants and, therefore, of animals can scarcely 
be overestimated. 
Nitrogen Fixation.-The free nitrogen of the air is so inert 
that very few living plants are capable of making use of it in 
the building up of their bodies. None of the green plants can 
bring this about of themselves. Certain molds and bacteria 
are able to make use of this source of nitrogen, however, and 
are, therefore, of the greatest economic importance. The fer- 
tility of the soil is largely dependent upon the fixed nitrogen that 
it contains, and the taking up of the free nitrogen by these 
organisms ultimately renders it available to other forms of plants. 
This does not mean that the bacteria take up the nitrogen from the 
Fig. 37.-Free living or non-symbiotic nitrogen-fixing bacteria: A, 
Clostridium pasteurianium; B, Azotobacter; a, A. chroococcum; b, A. agilis. 
(A after Winogradsky, B after Beyerinck.) 
air and immediately transform it into nitrates for the use of the 
higher plants, but that it is built up into their protoplasm and 
ultimately is set free by the death and decomposition of the organ- 
isms. Microorganisms which make use of free nitrogen commonly 
utilize carbonaceous materials as a source of energy. 
Organisms capable of fixing nitrogen are subdivided into two 
general groups, those which live free in soils and those which live 
in or on the roots of certain plants in a kind of symbiosis. The 
free living organisms which fix nitrogen belong to three general 
groups: first, certain anaerobic types belonging to the general 
group of butyric acid bacteria; second, certain aerobic species; 
third, a few molds. The anaerobic organism known to fix the 
nitrogen is Clostridium pasteurianium. Probably this organism 
is not of the greatest importance, as the conditions for 78 
VETERINARY BACTERIOLOGY 
its development do not often obtain. Bacteria of the nitrogen- 
fixing aerobic type belong to the group called Azotobacter. These 
organisms are abundant in many soils and fix considerable quan- 
tities of nitrogen, gaining energy therefor by oxidizing the car- 
bonaceous materials from dead plant tissues. The addition of 
straw, for example, to a soil will furnish sufficient food so that 
these bacteria will bring about an appreciable increase in the 
nitrogen content. The importance of the molds in this connection 
is not fully understood, but several species have been described 
which are capable of fixing nitrogen. 
The microorganisms which fix nitrogen in symbiosis with 
higher plants may be divided into two groups, those bacteria 
which grow upon the roots of legumes and the molds which 
grow on the roots of certain other plants. All plants belonging 
Fig. 38.-Rhizobium leguminosarum: a, Normal bacillar form; b, bacteroids 
or involution forms. 
to the legume or pulse family, such as clover, alfalfa, peas, beans, 
etc., usually bear upon their roots tubercles or nodules which, 
when opened, are found to be made up of cells tightly packed with 
bacteria. It has been shown experimentally that these organisms 
growing within the roots in some way take up free nitrogen from 
the air and eventually turn it over to the host plant, so that the 
legumes are not dependent for their development upon nitrogen 
which may be present in the soil, but can make use of the 
free nitrogen of the air as well. These plants are, therefore, 
very important in agriculture in the maintenance and increase of 
soil fertility. This organism, known as Rhizobium legumino- 
sarum (Fig. 38), enters the young growing root through a root 
hair and causes a kind of tumor formation in the tissues of 
the root, resulting in the development of the nodule. The organ- 
ism is at first a straight rod, but later, when growing inside the CHANGES BROUGHT ABOUT BY NON-PATHOGENIC ORGANISMS 
79 
cells of the host, it becomes much enlarged and shows many 
involution forms. The other organisms growing symbiotically 
within or upon the roots of plants are all molds. They develop 
either upon the surface of the root, forming a white, cottony, 
floccose covering, or they grow in the tissues just below the 
epidermis. They sometimes cause nodules to develop, as is the 
case with the Russian olive and the alder, or they produce no 
characteristic overgrowth of tissues at any one point, but are 
Fig. 39.-Nodules on the root of a legume, Soy bean. (Moore, U. S. Dept. 
Agr.) 
found quite uniformly present upon the young growing roots. 
Certain trees, such as the oak and the pine, particularly when 
growing in nitrogen poor soils, show the development of this 
mycorrhiza (Gr. fungus and root). It has been shown that these 
molds are quite active in taking up free nitrogen from the air and 
are of benefit to the plant upon which they occur. 80 
VETERINARY BACTERIOLOGY 
The Nitrogen Cycle.-The relationship of microorganisms to 
nitrogen and its compounds has been noted in the preceding pages. 
These changes may be summarized as follows: Certain bacteria 
break down organic compounds containing nitrogen with the ulti- 
mate liberation of ammonia. Other species change the ammonia 
to nitrous acid and nitrites. Still other species transform the 
nitrites to nitrates, and these the higher plants take up from the 
soil and transform again into complex organic substances. These 
eventually again decay or are eaten by animals and converted into 
animal tissues. The nitrogen of both plant and animal tissues 
Fig. 40.-Nitrogen cycle. Changes brought about by bacteria indicated by 
solid lines, other changes by dotted lines. 
ultimately undergoes the change first noted and the nitrogen again 
appears as ammonia. This series of changes is called the nitrogen 
cycle. It is to be noted in addition that some bacteria are found 
which decompose nitrates with the formation of nitrites and 
liberate free nitrogen in the so-called process of denitrification. 
Other species, either alone or in symbiosis with higher plants, 
take up and fix free nitrogen from the air and eventually convert it 
into a form available for higher plants. These changes may be 
better understood by reference to the accompanying diagram. CHANGES BROUGHT ABOUT BY NON-PATHOGENIC ORGANISMS 
81 
Miscellaneous Changes.-Many changes are brought about 
by bacteria other than those which are here discussed. Micro- 
organisms are of importance in tanning, curing of tobacco, the 
preservation of food-stuffs, such as silage and sauer-kraut, the 
retting of flax and hemp, the curing of the so-called burnt or 
heated hay, and in many other ways. It is to be remembered that 
probably in all cases these changes are brought about by the 
enzymes produced by the bacteria. CHAPTER V 
CLASSIFICATION OF MICROORGANISMS 
It is necessary in a consideration of organisms belonging to 
either the plant or animal kingdoms to divide or separate them 
into groups, with their apparent relationships as the basis for the 
grouping. Microorganisms of pathogenic significance we have 
previously divided into the four groups-bacteria, yeasts, molds, 
and protozoa. A discussion of the classification of the last will 
be reserved to the chapter on Diseases Produced by Protozoa. 
The classification of micro-organisms is by no means in a satis- 
factory state. Many bacteriologists and others who have inves- 
tigated diseases have failed to recognize the importance of simple 
classifications and have introduced many new names needlessly. 
It is a principle of nomenclature accepted since the time of Lin- 
naeus that every plant and animal belonging to a distinct type or 
species shall receive a Latin name, this name to be made up of two 
words only. The second of these words is the species name, and 
is peculiar to the particular kind under consideration; the first is 
called the genus or generic name. For example, among higher plants 
there is the genus Quercus, or oak, which is subdivided into many 
species, such as white oak, red oak, swamp oak, etc. {Quercus alba, 
rubra, etc.). The generic name is applied to all those species which 
resemble each other, as do all of the oaks. Species of plants and ani- 
mals are given names which are understood to serve as convenient 
terms for their designation. It is an established principle that the 
name first given to a plant or an animal is the one which should 
always be used whenever that name is in accordance with certain 
rules. Some bacteriologists have made the mistake of believing 
that a scientific name should be a description or even a descriptive 
term. It is no more necessary that the species name of a bacterium 
should describe that bacterium than that the given or Christian 
name of an individual should describe him. Disregard of this rule 
has resulted in some very unwieldy names being given to micro- 
82 CLASSIFICATION OF MICROORGANISMS 
83 
organisms, for example, names such as the following have been 
applied to bacteria: Bacillus membranaceous amethystinus mobilis, 
Bacillus argenteus phosphorescens liquefaciens, and even the fol- 
lowing, Bacillus saccharobutyricus fiuorescens liquefaciens im- 
mobilis. Such names are given under the mistaken idea that 
the specific name should be a description of the species. This is 
not customary in naming any of the higher plants and animals, 
and it is certainly not more desirable in bacteria. The yeasts, 
molds, and protozoa much more commonly than the bacteria have 
been studied by those who have had technical training in nomen- 
clature, consequently the classification of these forms is on a much 
more satisfactory basis. The only justification for a specific name 
made up of more than two words is that the two words taken to- 
gether express but a single idea. 
In the classification of plants and animals, as has been noted, 
it is customary to unite species into genera. Related genera are 
likewise united into tribes, the tribes into families, the families 
into orders, and the orders into classes. Sometimes other groups 
are interpolated, for example, families are sometimes divided into 
subfamilies and these into tribes. In both botany and zoology 
these group names are characterized by certain terminations. 
All names of plant orders end in -ales; of families, in -acece; and of 
tribes, in -ece. 
Classification of Bacteria 
Many different classifications have been proposed for bacteria, 
but not one of these has come into general use. A careful exam- 
ination of different texts in bacteriology, particularly those 
devoted to the pathogenic bacteria, will show that different sys- 
tems and schemes of classifications are used in dealing with 
closely related organisms. Not only have the groups been fre- 
quently changed, but many different names have been applied 
to almost every one of the pathogenic bacteria. The consequence 
is that in studying any pathogenic organism it is necessary to 
give not only the name preferred by the author but also a list 
of the synonyms which have been used by others. It seems 
probable that a satisfactory system of nomenclature is yet to be 
devised. The system of bacterial classification which is here 84 
VETERINARY BACTERIOLOGY 
outlined is that suggested by the Committee on Nomenclature of 
the Society of American Bacteriologists. 
The complete classification of bacteria from the standpoint 
of the botanist contains many forms of little or no economic 
importance, most having no sanitary or medical significance. 
In the classification here given most of these unimportant forms 
have been eliminated. 
The bacteria or Schizomycetes constitute a class which may 
be divided into five orders: the slime-mold bacteria (Myxo- 
bacteriales), the true bacteria (Eubacteriales), the sulphur 
bacteria (Thiobacteriales'), the thread bacteria or mold bacteria 
(Actinomycetales), and the spirochetes (Spirochaetales). The 
first and third of these orders contain no organisms of im- 
portance in veterinary medicine; they will be omitted in the 
classification. 
The order Eubacteriales or true bacteria includes those organ- 
isms which are least differentiated and least specialized. They 
are never filamentous and branched (except rarely in involution 
forms) and are never flexuous though some are spiral. The 
order Actinomycetales or mold bacteria comprises those forms 
which normally produce elongate cells, tending to become fila- 
mentous, sometimes branched, and mycelum like. The order 
Spirochwtales includes protozoan like spirals, usually relatively 
slender and flexuous. These orders will be discussed in order 
and the genera of veterinary significance noted. 
Order Eubacteriales. The True Bacteria 
Most of the common bacteria belong in this group, including 
the majority of the forms of medical significance. The cells may 
be spherical, cylindrical or spiral, but not commonly filamentous 
and never branched, except in the so-called involution forms. 
They may be motile by means of flagella or non-motile. When 
spiral they are never notably flexuous. In other words, they are 
neither mold-like nor protozoan-like. 
Altogether seven families have been described in this order, 
of which five are of medical importance. They may be differen- 
tiated by reference to the following key: CLASSIFICATION OF MICROORGANISMS 
85 
Key to the Families of the Eubacteriales1 
A. Cells spherical 1. Coccaceae 
AA. Cells not spherical 
B. Cells cylindrical. 
C. Producing endospores 2. Bacillaceae 
CC. Not producing endospores 
D. Flagella when pres- 3. Bacteriaceae 
ent peritrichous 
DD. Flagella polar 4. Pseudomonadaceae 
BB. Cells spiral 5. Spirillaceae 
I. The Family Coccaceae. The Spherical Bacteria 
The cells of the organisms belonging to this family are usually 
spherical when free, though during division they may be some- 
what elliptical. If the cells remain in contact after division 
they are frequently flattened at the plane of contact. They may 
form chains, packets or irregular masses. Very few cocci are 
motile; none of the motile species are of economic importance. 
Spores are not produced. Most of the genera of this family are 
parasitic, many species are disease producing. A few are of 
importance because of the fermentative changes which they 
bring about. The most important genera are Neisseria, Strepto- 
coccus, Diplococcus, and Staphylococcus.2 
These genera may be differentiated by the following key: 
Key to the Genera of the Coccaceae 
A. Cells occurring in chains 1. Streptococcus 
AA. Cells not in chains 
B. Cells in irregular masses, Gram 
positive 2. Staphylococcus 
BB. Cells in pairs 
C. Gram positive 3. Diplococcus 
CC. Gram negative 4. Neisseria 
1 The other family Nitrobacteriacece includes a number of genera of agricul- 
tural importance. Among them may be noted Nitrosomonas including soil 
forms oxidizing ammonia to nitrites, Nitrobacter including forms which 
oxidize nitrites to nitrates, Acetobacter including the acetic acid or vinegar 
bacteria, Rhizobium including the nitrogen-fixing bacteria of the roots of 
leguminous plants, and Azotobacter, free living nitrogen fixers. 
2 Other genera are: Leuconostoc forming large gelatinous masses in sugar 
solutions; Rhodococcus including the cocci which produce a red pigment, 
Micrococcus including Gram negative cocci showing irregular masses and usu- 
ally yellow pigment and Sarcina in which the cocci occur in regular packets. 86 
VETERINARY BACTERIOLOGY 
1. Streptococcus.-This genus includes those spherical bacteria 
whose cells occur normally in chains. For the most part the 
cells are Gram positive. Certain of the species are important 
in that they bring about lactic acid fermentation in milk. Others 
are capable of producing diseases. Certain species, for example, 
are commonly associated with tonsilitis, rheumatism, with 
the disease strangles in the horse, wound infection, and pus pro- 
duction. 
Fig. 41.-Types of Streptococcus: a, d, Streptococci consisting of uniform ele- 
ments; b, Streptococcus consisting of diplococcus elements; c, Diplococcus. 
2. Staphylococcus.-In this genus the spherical cells are united 
in more or less irregular masses. They are Gram positive, 
and usually parasitic. They are commonly associated with pus 
production, wound infection, the development of boils, abscesses, 
and similar conditions in the bodies of man and animals. 
3. Diplococcus.-The organisms belonging to this genus 
are strict parasites, Gram positive, occurring in pairs, the cells 
usually somewhat pointed and capsulated. The most important 
species is the organism usually associated with pneumonia. 
Fig. 42.-Types of cocci: a, isolated cells; b, showing tetrads, forming 
plates of cells or merismopedia; c, cells in an irregular mass-Staphylo- 
coccus. 
4. Neisseria.-The organisms of the genus are strict parasites, 
usually growing rather poorly in artificial culture media. 
The cells usually occur in pairs, flattened at the proximal sides, 
usually coffee bean shaped, without capsules and Gram negative. 
Two of the diseases produced by organisms of this group are of 
importance, namely, cerebral spinal meningitis and gonorrhea 
in man. CLASSIFICATION OF MICROORGANISMS 
87 
2. The Family Bacillaceas. The Spore Bearing Bacteria 
The bacteria of this family are all rod-shaped and all produce 
endospores. The cells may be motile or non-motile, when 
motile with peritrichous flagella. Two genera are of importance, 
Bacillus and Clostridium. The genus Bacillus includes those 
forms which are aerobic or facultative; the genus Clostridium 
those which are anaerobic. 
1. Bacillus.-The organisms belonging to this genus are 
all rod-shaped, sometimes occurring in chains. Under the right 
conditions these bacteria are among the most active in nature 
producing decomposition of organic materials. One species pro- 
duces the disease anthrax in animals and man, and one species 
the disease termed foul brood of bees. 
Fig. 43.-Types of bacilli: a, b, Non-motile bacilli {Bacterium); c, mono- 
trichous bacillus {Pseudomonas); d, lophotrichous bacillus {Pseudomonas); 
e, f, peritrichous Bacillus. 
2. Clostridium.-The organisms of this genus are all rod 
shaped, producing endospores but not growing in the presence 
of free atmospheric oxygen. Frequently the rods are swollen at 
time of spore production, producing spindle shaped or club 
shaped cells. Some of the bacteria of this group are among the 
most active of the putrefactive forms, particularly in the produc- 
tion of malodorous compounds such as butyric acid. Other 
species are capable of producing disease, especially when intro- 
duced into wounds. Among the diseases produced are gaseous 
gangrene in man, malignant edema, blackleg, and bradsot in animals 
and tetanus in man and animals. 88 
VETERINARY BACTERIOLOGY 
3. The Family Bacteriaceae 
This is the largest of the families of bacteria, including some 
nine genera. Here are found those rod shaped forms whose 
cells are usually regular, which do not produce endospores and 
which when motile have peritrichous flagella. In all of the 
genera containing disease producing forms the cells are Gram 
negative. Four of the genera are discussed here, three including 
pathogenic forms and one including species of importance in 
milk. These genera are Bacterium, Pasteurella, Hemophilus 
and Lactobacillus. 
Key to the Genera of Bacteriaceas1 
A. Cells Gram negative. 
B. Requiring hemoglobin or blood 
serum for growth in media 1. Hemophilus 
BB. Growing on ordinary culture 
media. 
C. Polar staining marked 2. Pasteurella 
CC. Polar staining not pro- 3. Bacterium 
nounced 
AA. Cells Gram positive 4. Lactobacillus 
1. Hemophilus.-The organisms belonging to this genus are 
minute, parasitic, Gram negative rods, which grow in the labora- 
tory only in special media, preferably containing hemoglobin. 
The most important species are Hemophilus influenzce, the or- 
ganism which has been supposed to cause influenza and which 
is quite certainly associated with many cases of the disease, 
Hemophilus pyogenes, a common cause of suppuration in animals, 
and Hemophilus pertussis, the cause of whooping cough. 
2. Pasteurella- The organisms of this genus are all rod shaped 
cells, Gram negative, and show bipolar staining. They are 
parasitic with very slight powers of fermentation. Members of 
1 Other genera belonging to this family are:- 
Erythrobacillus, producing red pigment. 
Chromobacterium, producing violet pigment. 
Erwinia, White, slimy forms pathogenic for plants. 
Proteus, closely related to Bacterium but liquefying gelatin rapidly. 
Zo'pfius, Gram positive putrefactive forms. CLASSIFICATION OF MICROORGANISMS 
89 
this genus produce many diseases in man and animals including 
the bubonic plague of man and hemorrhagic septicemias of animals, 
such as fowl cholera, and related diseases in swine, sheep, cattle 
and horses. 
3. Bacterium.-The organisms belonging to this genus are 
Gram negative rods, frequently motile, and easily cultivable. 
Most species ferment certain carbohydrates with the formation of 
acid and frequently of gas. They are typically intestinal para- 
sites in man and higher animals. Some species are not infre- 
quent in the soil. A few species are pathogenic, including the 
organisms causing typhoid fever, paratyphoid fever, dysentery and 
certain types of food poisoning. One species, the Bacterium coli, 
is frequently used as an index for the determination of the 
presence of sewage in water. 
4. Lactobacillus.-The cells are rod shaped, often long and 
relatively slender, Gram positive, and non-motile. Some species 
have very short cells and are almost coccus-like. No endospores 
are developed. Acid, particularly lactic acid, is usually pro- 
duced in considerable quantities from carbohydrates. Many of 
the species prefer to grow in the absence of oxygen, although 
they are not strictly anaerobic. Included in this genus are 
many bacteria important in the souring of milk, the formation of 
lactic acid in silage, sauerkraut, etc. 
4. The Family Pseudomonadaceaz 
This family Contains a single genus, Pseudomonas. The cells 
are rod shaped, and usually motile by means of polar flagella. 
Frequently a fluorescent green or brown pigment is produced in 
culture media. In other species an insoluble yellow pigment is 
formed. Among those producing a green fluorescent pigment is 
the Pseudomonas aeruginosa, an organism sometimes found in 
wounds. Many of the yellow species produce diseases in 
plants. 
5. The Family Spirillaceae 
The organisms belonging to this group are curved rods. Two 
genera are included, Vibrio and Spirillum. The former only 
is considered as it alone has pathogenic species. 90 
VETERINARY BACTERIOLOGY 
Vibrio.-This genus included short curved rods, motile by 
means of one, two or three polar flagella. Many are intestinal 
parasites and some are capable of causing disease. The most 
important organism is the Vibrio cholera producing Asiatic 
cholera in man, and certain species isolated from fowls. 
Fig. 44.-Types of spirilla: a, Non-motile spirillum; b, monotrichous 
spirillum (Vibrio); c, lophotrichous spirillum with 2 or 3 flagella (Vibrio); 
d, lophotrichous spirillum (Spirillum). 
Order Actinomycetales, The Thread Bacteria 
The organisms belonging to this order usually have the cells 
somewhat elongated, frequently filamentous, and with a decided 
tendency to the formation of branches. In some genera a 
definite branched mycelium is developed. The cells frequently 
show swelling, or are clubbed or irregular in shape. Endospores 
are not produced, although conidia are formed by some genera. 
Many of the genera are Gram positive and all are non-motile. 
Some species are aerobic. All of the eight genera contain species 
which are parasitic in man or animals, many of them pathogenic. 
Key to the Genera of Actinomycetales 
A. Typically producing long filaments 
B. Branched mycelium formed 1. Actinomyces 
BB. No true mycelium 
C. Cells more or less branched 
D. Gram negative 2. Actinobacillus 
DD. Gram positive 3. Erysipelothrix 
CC. Cells never branch. 
Gram positive threads 
later fragmenting in- 
to rods 4. Leptotrichia CLASSIFICATION OF MICROORGANISMS 
91 
AA. Not typically producing long fila- 
ments 
B. Acid fast 5. Mycobacterium 
BB. Not acid fast 
C. Cells elongate, fusiform 6. Fusiformis 
CC. Cells not fusiform 
D. Gram positive 7. Corynebacterium 
DD. Gram negative 8. Pfeifferella 
1. Actinomyces.-The organisms belonging to this group 
form fine threads (or mycelium) which breaks up into short 
segments which function as spores or conidia. Some of the 
species are parasitic, one, the Actinomyces bovis, producing lumpy 
jaw in cattle. Many other species are widely distributed in the 
soils, apparently growing upon decaying roots and similar organic 
matter. 
Fig. 45.-A, Leptothrix; B, Cladothrix; C, Nocardia; D, Actinomyces. 
2. Actinobacillus.-This genus differs from Actinomyces in 
that no true mycelium is formed. The cells are Gram negative, 
and fragment to form rods. The principal species is Actino- 
bacillus Lignieresi, the cause of the disease actinobacillosis in 
cattle. 
3. Erysipelothrix.-This genus includes certain Gram positive 
bacteria in which the filaments break up into short rods. For the 
most part the species are microserophilic. Several species pro- 
duce disease in man and animals, notably Erysipelothrix rhusio- 
pathioe, the cause of swine erysipelas. 92 
VETERINARY BACTERIOLOGY 
4. Leptotrichia.-The organisms of this genus have been 
largely described from the human mouth. The unbranched 
filaments are Gram positive, later fragmenting into rods. None 
are positively known to be pathogenic. 
5. Mycobacterium.-These organisms are slender rods which 
are stained with difficulty, but when once stained are resistant to 
decolorization by acids, that is, they are termed acid fast. The 
cells are sometimes swollen, showing club shapes or forked 
forms, occasionally the cells are branched. They are non-motile 
and Gram positive. Some of the species are present in the soil, 
a few are pathogenic to man and animals. The most important 
of the diseases caused are tuberculosis and leprosy in man, and 
para-tubercular dysentery in cattle. 
6. Fusiformis.-These organisms are anaerobic or micro- 
aerophilic, the cells frequently elongate and fusiform, staining 
somewhat unevenly. Filaments are sometimes formed. Several 
species have been found associated with disease in man as for 
example in Vincent's angina. 
7. Corynebacterium.-The cells are slender, often slightly 
curved rods with a tendency toward the formation of clubs, 
and containing granules which give the cells when stained a 
barred or irregular appearance. They are not acid fast but are 
Gram positive and aerobic. Most species are parasites, the 
most important being Corynebacterium diphtheria, the cause of 
diphtheria in man. 
8. Pfeifferella.-These organisms are non-motile rods, slender 
and Gram negative, staining poorly, sometimes forming threads,, 
and showing a tendency toward branching. When grown 
upon potato in the laboratory they develop a characteristic 
honey-like growth. The most important species is Pfeifferella 
mallei, the organism which causes glanders in the horse. 
Order Spirochaetales 
The organisms belonging to this group in many respects 
resemble the protozoa, and may be regarded as intermediate 
between the true bacteria and the protozoa. They are all more 
or less curved rods, frequently very slender. Many species are 
motile but there has been no definite demonstration of flagella. CLASSIFICATION OF MICROORGANISMS 
93 
They multiply either by longitudinal or transverse fission. It is 
probable that in some cases they have a relatively complex life 
history. Several genera have been described, the most important 
being Treponema. 
Treponema.-The organisms belonging to this genus are 
•exceedingly slender spiral rods, motile by means of flexous bend- 
ing of the body. The most important species is Treponema 
pallida, the cause of the disease syphilis in man. 
Figs. 46 and 47.-Types of Spirochaeta?. 
Classification of Yeasts 
Mycologists recognize a number of different genera in the group 
of yeasts. It is probable that the yeasts do not constitute a 
homogeneous group. The genus Saccharomyces includes such 
forms as the common bread and brewer's yeast {Saccharomyces 
cerevisice) which produce spores and are active in alcoholic fer- 
mentation. The name Torula is sometimes given to similar yeasts 
that are not spore producing. This latter name, however, is 
incorrectly so applied, as it was previously and is now used to 
indicate a genus of molds. The name Blastomyces has been 
commonly accepted to indicate the yeasts pathogenic for man 
and animals. It is probable that there is little reason for separation 
of Saccharomyces and Blastomyces on the basis of their morphology, 
but such a separation on the basis of pathogenesis seems to be 
advisable. 
Classification of the Molds 
Several hundred genera and many thousands of species have 
been described. Of these, a few genera only contain species that 
are pathogenic for man and animals. For a discussion of classi- 
fication the student is referred to Chapter XXXIX. SECTION II 
LABORATORY METHODS AND TECHNIC 
CHAPTER VI 
STERILIZATION 
Sterilization is the process whereby glassware, media, or 
any of the materials or apparatus used in the laboratory are 
entirely freed from living organisms. It is evident that in the 
study of bacteria it is necessary that we deal with pure cultures, 
that is, that one kind of organism only be present in the material 
which we are studying. It is quite impossible to determine from 
mixed cultures which of the organisms present bring about 
observed changes. Bacteria are present upon the surface of 
all laboratory apparatus, in the dust, in soil, upon the hands- 
they are ubiquitous, hence the necessity for sterilization. 
Sterilization may be accomplished by physical or chemical 
means. In practice the latter is generally called disinfection, and 
is rarely used in the laboratory. The term sterilization, there- 
fore, as commonly used, indicates the destruction of micro- 
organisms by physical processes. 
Sterilization by the Flame.-The platinum wire used in the 
transfer of bacteria in the laboratory is sterilized by heating to 
a red or white heat in the flame of the Bunsen burner. Similar 
methods are sometimes used in the sterilization of other small 
pieces of laboratory apparatus, such as cover-glasses and slides. 
Sterilization by Hot Air.-Glassware is commonly sterilized 
by subjecting it to a temperature of 150°C. to 170°C. in a hot-air 
oven for an hour. All bacteria will be destroyed at this tempera- 
ture providing the material to be sterilized is of a nature such that 
the heat can penetrate readily to all parts. This method cannot 
94 STERILIZATION 
95 
be used, however, in the sterilization of liquids or of any organic 
material which might be decomposed at such a temperature. 
Sterilization by Streaming Steam.-It is found in practice that 
live steam is the most efficient sterilizing agent for many of the 
media used in the laboratory. Steam under atmospheric pressure 
at sea-level has a temperature of about 100°. Some type of 
apparatus is used such that the live steam comes in direct con- 
tact with the material to be sterilized. One type of the apparatus 
is called the Arnold steam sterilizer (Fig. 49). It consists essen- 
tially of a pan with a double bottom opening into the sterilizing 
Fig. 48.-Oven for sterilization by hot air (Jordan). 
chamber above. The water between the bottoms is quickly 
heated to boiling temperature and is automatically replaced 
from the supply on the exterior through small holes as rapidly 
as it boils away. A single exposure to live steam for fifteen 
minutes is sufficient to kill all vegetative bacteria, but spores 
are not thus destroyed. It is customary, therefore, to heat for 
fifteen minutes on one day, keep the medium for twenty-four 
hours at a temperature suitable for the germination and devel- 
opment of any spores present, then heat again for fifteen minutes 
in the same manner. Those spores which have germinated will 96 
VETERINARY BACTERIOLOGY 
be destroyed by this second heating. A third heating, twenty-four 
hours later, will quite certainly destroy all the bacteria which may 
have been present. This process is called intermittent steriliza- 
tion. It finds its principal application in the sterilization of 
materials which would be changed or broken down by heating 
at a higher temperature. Among such materials are media con- 
taining sugars which undergo incipient caramelization when 
heated too hot. 
Sterilization by Steam under Pressure.-This is generally 
accomplished in the autoclave or digester, which consists essentially 
Fig. 49.-Arnold steam sterilizer (Fowler). 
of a chamber into which steam under pressure can be introduced 
(Fig. 50). Many different types of these autoclaves have been 
put upon the market. Live steam under a given pressure unmixed 
with air has a constant temperature; therefore, if the pressure 
of the steam is known, one can determine easily the temperature 
as well. It is necessary, however, that all air be first eliminated. 
This is accomplished by allowing the stop-cock, which is always 
present upon the steam chamber in the autoclave, to remain open 
until all the air has escaped and the steam issues in a constant 
stream. This cock is then closed and the pressure caused to STERILIZATION 
97 
rise as quickly as possible to 15 pounds to the square inch, or 
one additional atmosphere. This gives a temperature of about 
121°. Material to be sterilized should be allowed to remain 
fifteen minutes usually. If large bulks, such as flasks of media, 
are to be sterilized, a longer period must be allowed in order that 
Fig. 50.-Autoclave for sterilizing by steam under pressure. 
the media may be completely heated through. If very small 
quantities of material are being sterilized, a shorter period may 
be used. When properly carried out, sterilization by this method 
will certainly destroy all the bacteria present. Its principal 
disadvantage is that certain organic substances may be decomposed 
at this temperature. 98 
VETERINARY BACTERIOLOGY 
Sterilization at Temperatures Lower than Boiling-point.- 
It is sometimes necessary to sterilize media, particularly blood- 
serum, at temperatures lower than the boiling-point of water. 
This is accomplished by placing the material to be sterilized in an 
apparatus where it may be heated to the desired temperature, 
usually 70°-80° for one to two hours on each of five or more 
successive days. If large numbers of spores of certain organisms, 
such as Bacillus subtilis, are present, it is almost impossible to 
sterilize efficiently by this method. However, if care is used in 
securing the blood-serum to prevent the introduction of such organ- 
isms, sterilization may be easily accomplished at this tempera- 
ture. 
Sterilization by Addition of Chemicals.-It is only under excep- 
tional conditions that chemicals are used to sterilize media. 
Fig. 51.-Apparatus for sterilization by filtration (McFarland) 
It has been found that the addition of soluble materials, such 
as lactose, in considerable quantities to media containing pure 
cultures of certain bacteria will destroy the organisms so that 
they may be used as a vaccine. This method does away with 
the destruction of any of the characteristic metabolic products 
by heat. 
Sterilization by Filtration.-Bacteria may be removed from a 
liquid by passing it through a filter with pores so fine that the 
organism cannot penetrate. Such filters are made up in a great 
variety of shapes and densities. Among the many used are the 
Berkefeld, the Pasteur, and the Chamberland. These are made of 
unglazed porcelain. In filtration through these it is necessary, of 
course, that all the apparatus used, particularly the vessel into 
which the filtrate runs and the filter itself, be sterilized before use. STERILIZATION 
99 
This method of sterilization is commonly used for the removal of 
bacteria from culture-media when it is desired to study their 
soluble metabolic products, and in the removal of bacteria from 
sera which contain antitoxins and other antibodies. It is not 
commonly used in the sterilization of media intended for the 
cultivation of bacteria. Filters of this character have been used 
extensively in the filtration of water for drinking purposes. When 
first installed, they are quite efficient, but it is found that the organ- 
isms rapidly penetrate, and in the course of time are found in the 
filtrate. Such filters must, therefore, be sterilized at intervals if 
they are to remain efficient. CHAPTER VII 
Microscopic examination alone is quite insufficient to 
differentiate species of bacteria. By the aid of a microscope 
one cannot readily recognize the differences, for example, between 
the organisms which cause typhoid fever and certain of the normal 
inhabitants of the intestinal tract. It is necessary, therefore, 
in a study and differentiation of species, that we make use of 
different kinds of culture-media in which the bacteria may be 
grown. By the term medium is meant any nutrient substance or 
mixture upon which or in which bacteria will multiply. The 
bacteria in their development on the various media show certain 
growth reactions which are very useful in their differentiation. 
Some produce acids, others gas, alkalis, and proteolytic and 
coagulative enzymes. 
Adjustment of the Reaction of Media.-The reaction of 
a medium, that is its relative acidity or alkalinity, may be desig- 
nated in one of two ways; first, by the amount of normal1 acid or 
CULTURE-MEDIA AND THEIR PREPARATION 
1A normal solution of a chemical may be defined as one in which there is 
one gram of replaceable (acid) hydrogen or its equivalent per liter of solu- 
tion. For example, if we wish to prepare a normal solution of HC1, we must 
so dilute the acid that it contains one gram of hydrogen per liter of solution. 
This is best accomplished in any substance that can be readily weighed by 
dissolving the molecular weight expressed in grams in sufficient water to 
make a liter of solution. If there is more than one atom of replaceable 
hydrogen in the molecule, it is necessary to divide the amount used by the 
number of such atoms. For example, the molecular weight of H2SO4 is 
approximately 98, but there are two replaceable acid hydrogen atoms. 
Therefore, half this molecular weight in grams, or 49 grams, of the II2SO4 is 
made up to a liter of solution and contains one gram of acid hydrogen. 
The same principle is adopted in the preparation of a normal solution of 
an alkali; in-this case, however it is necessary to divide the molecular weight 
by the number of atoms of the base present, which will replace hydrogen. 
For example, the molecular weight of NaOH is 40. It contains one atom 
only of sodium, and a normal solution, therefore, contains 40 grams to the 
liter. Dry sodium carbonate (Na2CO2) has a molecular weight of 106. 
Twq atoms of sodium are present; therefore it is necessary to divide by two, 
so that a normal solution of sodium carbonate contains 53 grams to the liter 
of boJutiori. b It is evident that a given volume of a normal solution of an 
acid will neutralize exactly an equal volume of a normal alkali. 
100 CULTURE-MEDIA AND THEIR PREPARATION 
101 
normal alkali required to bring one hundred cubic centimeters 
of the medium to the neutral point of some particular indicator, 
or second, by the designation of the true hydrogen ion concen- 
tration or, conversely, of the true hydroxyl ion concentration of 
the medium. While the first method is still the more commonly 
used, the second method is in most instances preferable. 
Bacteria, yeasts, and molds are usually quite sensitive to 
the presence of an excess of acid or alkali. Some grow best in a 
medium which is strictly neutral, others prefer one which is 
somewhat on the acid side of neutrality, still others on the alka- 
line side of neutrality. Careful adjustment of reactions is there- 
fore necessary in many cases. Some organisms, for example, will 
not grow unless the medium has almost exactly the same reaction 
as has the blood or the tissues of the body in which they are 
accustomed to grow. 
The actual acidity of any solution is determined by the pres- 
ence of free hydrogen ions and alkalinity is determined by the 
presence of free hydroxyl ions. A solution is truly neutral 
when equal numbers of hydrogen and hydroxyl ions are present in 
a given volume. Pure distilled water is neutral, that is, when a 
molecule of water dissociates it breaks up into equal numbers of 
hydrogen and hydroxyl ions. The chemists has discovered, 
furthermore, that pure water and therefore any neutral solution 
of materials in water contain approximately one ten-millionth of a 
gram of hydrogen ions to the liter. This may also be written 
10~7 grams hydrogen ions per liter, or still more conveniently 
expressed in terms of normality of hydrogen ions. A normal solu- 
tion of hydrogen ions is one which contains one gram of hydrogen 
ions per liter. A neutral solution, therefore, is one which has a 
concentration of hydrogen ions of 10-7 normal. It was noted 
above that in a neutral solution there is the same number of 
hydroxyl ions as hydrogen ions. The hydroxyl ion concentra- 
tion at neutrality must therefore be also 10-7 normal. 
The physical chemist has demonstrated that the product of 
the normality of hydrogen ions by the normality of hydroxyl ions 
in a solution is a constant number. What this number is for 
aqueous solutions may be determined by multiplying 10-7 (nor- 
mality of hydrogen ions in a neutral solution) by 10~7 (normality 102 
VETERINARY BACTERIOLOGY 
of hydroxyl ions in a neutral solution) giving 10-14. In other 
words, the product of the normality of the hydrogen ion con- 
centration of a solution by the normality of the hydroxyl 
ion concentration must always be approximately 10-14. If 
either the hydroxyl or hydrogen ion concentration is known one 
can at once determine the concentration of the other. For 
example, if we are dealing with a solution having an hydrogen 
ion concentration of 10-5 normal its hydroxyl ion concentration 
must be 10~9 normal. It is thus possible to arrange a scale to 
designate the acidity of any solution in terms of its hydrogen ion 
concentration as follows: 
10° io-1 io-2 io~310-4 io-5 io-° io-7 io-8 io-9 
1O~10 10-n 10-i2 10-i3 10-u 
On this scale it will be noted that the larger the numerical value 
of the exponent, the smaller is the hydrogen ion concentration. 
It will be recalled that 10~7 represents neutrality. Numbers to 
the right of 10~7 represent increasing values of alkalinity or 
decreasing hydrogen ion concentration. Numbers to the left 
represent increasing acidity. Inasmuch as this method of 
statement is somewhat cumbersome, it has been suggested by 
Sorensen that the exponents be used to make up a scale, using 
positive signs instead of negative. This gives the scale: 
0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14. 
Each of these numbers is termed the P^ of a solution. A solu- 
tion having PH of 0, for example, would have a normality of 
hydrogen ion concentration of 10° normal or 1. One having PH 
value of 7 would have a hydrogen ion concentration of 10~7 
normal, that is, it would be neutral. It is evident, therefore, 
that the smaller the number in this scale the higher the hydrogen 
ion concentration, that is, the greater the acidity; and the larger 
the number the greater the alkalinity. 
Indicators are chemical substances which are of one color 
in a certain range of PH values or hydrogen ion concentrations, 
and of another color in other ranges. The indicator most com- 
monly used is litmus, which has a lilac color at true neutrality, 
that is, at a PH value of 7. At a PH value of 8, that is in a more 
alkaline solution, it is blue. At a PH value of 5, that is in a CULTURE-MEDIA AND THEIR PREPARATION 
103 
stronger concentration of hydrogen ions, it is red. Other indica- 
tors change color at other PH values. For example, phenolph- 
thalein is colorless in all hydrogen ion concentrations having a 
PH value less than 8.2, in more alkaline solutions, it is red. 
Many other indicators are known which change color at other 
points in the hydrogen ion scale. This change of color is not 
instantaneous. In adding alkali, for example, to an acid solu- 
tion containing litmus there is not an instantaneous transforma- 
tion of red into blue. By the use of standards whose hydrogen 
ion concentration is known it is possible to determine approxi- 
mately the hydrogen ion concentration of any material which it is 
desired to test, by the use of the intensity of color of appropriate 
indicators. 
Most bacteria grow in a hydrogen ion concentration of IO-7 
to 10~8, that is in solutions having a PH value between 7 and 8. 
The Pn value of blood is usually about 7.35. This indicates 
therefore the hydrogen ion concentration most useful in culti- 
vating many species of pathogenic bacteria. Inasmuch as most 
media are on the acid side of true neutrality it is necessary to add 
alkali, usually potassium or sodium hydrate, until test shows that 
the hydrogen ion concentration has become satisfactory. 
The hydrogen ion concentration of a medium or solution 
depends not only upon the actual concentration of the acid itself 
but upon the concentration of substances which are termed 
buffers. A buffer is any substance in a solution which tends 
to prevent rapid changes in hydrogen ion concentration upon 
the additions of alkalies or acids. Certain salts, particularly 
the phosphates, and many organic substances, particularly the 
amino acids and peptones, act in this manner. For example, the 
addition of a small quantity of an acid to distilled water will 
give a marked change in the hydrogen ion concentration, but 
the same quantity of acid added to solutions containing consider- 
able quantities of buffers may result in very slight differences in 
hydrogen ion concentration. It is evident since microorganisms 
are affected far more by differences in hydrogen ion concentra- 
tion than they are by the total amount of acid present, that 
heavily buffered media are preferred for the growth of micro- 
organisms. 104 
VETERINARY BACTERIOLOGY 
It is noted above that media are sometimes standardized by 
determining the quantity of acid or alkali required to bring one 
hundred cubic centimeters to the neutral point of some indicator. 
The one usually chosen in bacteriology is phenolphthalein. 
A solution is said to be-1, for example, when it will require one 
cubic centimeter of a normal solution of acid to bring it to the 
neutral point of phenolphthalein. 
Nature of Nutrients Required by Bacteria.-It is found that 
practically the same elements are necessary for the nutrition of 
bacteria as are essential for higher plants and animals, but they 
may be used in quite different proportions. 
It is particularly important that the disease-producing bac- 
teria be cultivated whenever possible. Cultivation outside the 
body is quite necessary to a satisfactory proof of pathogenicity, 
to differentiate species, and to secure the organism in quantities 
sufficient for preparation of vaccines, antitoxins, etc. A few 
standard media are commonly used in the laboratory for the 
growth of bacteria, and a great variety of special types have been 
devised for certain species that do not grow upon these. It is 
impracticable even to enumerate the many special media that 
have been employed. 
Bouillon or Beef Broth from Meat.-This is the commonest 
of laboratory media and serves as a basis for the preparation 
of many others. 
Place 500 gm. chopped lean beef in a liter of water and allow 
it to stand in a refrigerator over night. The juice is then pressed 
out with a meat press, boiled for half an hour, the coagulated 
albumins filtered out, the liquid made up to a liter with water, 10 
gm. of peptone added, and heated sufficiently to dissolve. The 
reaction is adjusted to the proper point, usually + 1, by titra- 
tion, or the medium is simply neutralized by addition of nor- 
mal NaOH, using phenolphthalein paper as an indicator if a high 
degree of accuracy is not required. The broth is then autoclaved 
at 15 pounds pressure or boiled for fifteen minutes, allowed to 
cool, and then filtered. The cooling throws down a precipitate 
of magnesium ammonium phosphate, which may then be removed. 
Liquid Media CULTURE-MEDIA AND THEIR PREPARATION 
105 
In many cases this is not objectionable and filtration may be 
carried out while the solution is still hot. The finished bouillon 
or broth is placed in test-tubes and flasks, and sterilized in the 
autoclave under a pressure of 15 pounds for 15 minutes. 
Bouillon or Broth from Beef Extract.-It is customary, in 
much of the routine work of the laboratory, to substitute for the 
preceding a broth in which three grams of a beef extract, such 
as Liebig's, is substituted for the meat. 
Sugar-free Broth.-There is generally present in the preceding 
media a small amount of carbohydrate, largely dextrose. In 
some cases a sugar-free medium is required. Theobald Smith 
has devised a modification of the meat broth for this purpose 
which is commonly used. Several broth tubes containing vig- 
orous twenty-four-hour cultures of Bacterium coli are added to the 
meat infusion and kept at 37° for eighteen hours. In this time 
the bacteria will have used up all the sugar present. The broth 
is then prepared as above. 
Sugar Broth.-Sugar-free broth is generally modified by the 
addition of carbohydrates, such as dextrose, saccharose, and 
lactose, making 1 per cent, solutions. Such media must be 
subjected to intermittent sterilization in flowing steam and not 
in the autoclave, as the carbohydrates readily decompose. 
Glycerin Broth.-Five or 6 per cent, of glycerin added to 
broth makes it a much more favorable medium for many organisms. 
Serum Broth.-Blood-serum secured under strict aseptic pre- 
cautions may be added to sterile broth in various proportions. 
Tubes prepared in this manner should be incubated for a few 
days to determine whether or not the medium is sterile. Steril- 
ization can be effected only by filtration, as heating would coagulate 
the serum. 
Dunham's Solution.-This is a solution containing 1 per cent, 
peptone and 0.5 per cent, sodium chlorid in water. It is used in 
growing organisms for the determination of indol. 
Beerwort.-Unhopped beerwort is frequently used for the 
growth of yeasts and molds. 
Milk.-Fresh separated milk is tubed and subjected to intermit- 
tent sterilization. Commonly, litmus is added in sufficient quan- 
tities to make the milk a distinct blue. 106 
VETERINARY BACTERIOLOGY 
Synthetic Media.-It is sometimes desirable to prepare a 
medium in which the exact chemical composition of every ingre- 
dient is known. The nature of all the changes brought about by 
bacteria can be studied chemically in such a medium, and the 
food requirements determined by changes in the composition. 
Most synthetic media contain as a basis an aqueous solution 
of certain salts, among them potassium phosphate and sodium 
chlorid. Special media of this kind are used extensively in the 
study of the soil bacteria; it is only occasionally that such a 
medium proves serviceable in the study of pathogenic forms. The 
most commonly used of the synthetic media is Uschinsky's solution. 
Water, distilled 1000 c.c. 
Asparagin 4 gm.; 
Ammonium lactate 6gm.; 
Na2HPO4 2 gm.; 
NaCl 5 gm.; 
This medium is known as albumin free. 
Liquefiable Solid Media 
Nutrient Gelatin.-This is prepared by the addition of 10-15 
per cent, of gelatin to bouillon as prepared above. The gelatin 
should be the best "gold label." Care must be used in heating 
the solution while dissolving the gelatin or the latter will stick 
to the bottom of the vessel and burn. It is best to use an asbestos 
pad, a double boiler, or a rice-cooker. The gelatin is itself acid, 
so that it is necessary to adjust the reaction after it has dissolved. 
The medium is then cooled to 60°, and the white of an egg thor- 
oughly mixed with it. It is again heated to the boiling-point 
without stirring. The coagulation of the egg removes suspended 
particles and makes filtration easier. The nutrient gelatin is 
tubed and sterilized in the autoclave at 120° for ten to fifteen min- 
utes. It should be cooled at once after removal from the sterilizer. 
Care must be exercised not to heat the medium too long or it may 
fail to solidify when cooled. 
Other Gelatin Media.-Any of the liquid media already dis- 
cussed, with the exception of the milk and serum broth, may be 
made solid by the addition of 10 to 15 per cent, gelatin. Among 
the more commonly used are dextrose, lactose, and glycerin 
gelatin. CULTURE-MEDIA AND THEIR PREPARATION 
107 
Nutrient Agar.-This is prepared by the addition of 1.5 per 
cent, of shredded or powdered agar-agar to bouillon. Agar-agar 
is a carbohydrate-like material, probably related to the vegetable 
gums, which is prepared from certain of the seaweeds of the 
Pacific and Indian Oceans. The mixture must be boiled vigorously 
for half an hour to insure thorough solution of the agar. This 
medium does not burn as readily as does gelatin, and long- 
continued heating does not interfere with its solidification when 
cooled. The nutrient agar may be sterilized in the autoclave 
for fifteen minutes at 120°. 
Blood-serum Agar.-Liquid sterile blood-serum is heated to 
40° to 50° C. and mixed with liquid agar cooled to 40° to 50° C., 
equal parts or in mixture of 1 : 2. It is then solidified by cooling. 
It may be poured into plates before solidifying or may be left in 
test-tubes. Ascites fluid and hydrocele fluid may be substituted 
for the serum. 
Chocolate Agar.-10 c.c. fresh defibrinated sheep blood are 
added to every 100 c.c. of neutral beef infusion agar. It is 
important that the blood be added while the agar is hot; that is, 
immediately after the latter is taken from the Arnold sterilizer 
(95 F.). The medium should have a dark chocolate color. This 
medium is valuable for culturing material from cases of pneu- 
monia, broncho-pneumonia, etc. 
Hormone Agar.-(Huntoon.) 
Chopped beef heart or steak (must be comparatively fresh) 500 
Water 
Peptone (Bacto peptone gives the best results) 10 
Agar (Bacto or thread agar that has been soaked) 16 
Salt 5 
Whole Egg 1 
All of these ingredients are placed in an ordinary enamel- 
ware vessel, preferably a large coffee pot, and heated over an 
open flame with constant stirring until the red color of the meat 
infusion changes to brown, at a temperature about 68° C. Care 
should be taken not to run the temperature much above this 
point as the medium then begins to clot, which is undesirable at 
this time. The medium is now titrated by the addition of normal 
sodium hydrate until it is slightly alkaline to litmus paper, and 108 
VETERINARY BACTERIOLOGY 
then 1 c.c. per liter is added in addition. The vessel is covered 
and placed in the Arnold sterilizer or in a water bath at a temper- 
ature of 100° C. for 1 hour, removed, and the firm clot which has 
formed separated from the sides with' a rod and the vessel re- 
turned to the sterilizer or water bath at 100° C. for 1^ hours. 
It is now removed and allowed to stand at room temperature for 
about 10 minutes in a slightly inclined position; during this 
time the fluid portion separates and may be removed by pipetting 
or, in the case of the coffee pot, by simply pouring it off carefully. 
If it is poured through a fine wire sieve many small particles 
of meat clot may be caught. The product is allowed to stand in 
tall cylinders for 15 to 20 minutes until the fat present has risen 
to the surface when it can be removed. The medium is now 
tubed and sterilized by the intermittent method. Autoclaving 
is to be avoided. If the medium, although usually clear enough 
for practical purposes, seems too turbid, further clearing may 
take place by filtration through glass wool, asbestos wool, sedi- 
mentation or centrifugation. 
Other Agar Media.-Agar may be used as the solidifying 
agent for any of the liquid media described. It has the advan- 
tage over gelatin that it may be kept at blood heat, while gelatine 
under such conditions would liquefy. 
Endo-agar-fuchsin Medium: 
Beef infusion agar (3 per cent., neutral), made alkaline 
by addition of 10 c.c. 10 per cent, soda solution. . . 1000 gm. 
Lactose 10 gm. 
Fuchsin (saturated alcoholic) 5c.c. 
Sodium sulphite (10 per cent, solution) 25 c.c. 
In plates this medium should have the natural agar color or 
light rose. 
Typhoid and paratyphoid colonies are colorless or light rose; 
coli colonies are an intense red. 
Simplified Endo Agar.-(Levine) 
Distilled water 1000 c.c. 
Pepton (Difco) 10 gm. 
Dipotassium phosphate 2 to 5 gm. 
Agar 15 to 30 gm. CULTURE-MEDIA AND THEIR PREPARATION 
109 
Boil ingredients until dissolved and make up loss due to 
evaporation. Place measured quantities in flasks or bottles and 
sterilize in autoclave at 15 pounds for 15 minutes. Just prior 
to use add to each 100 c.c. of the melted agar 
Lactose 1 gram or 5 c.c. of 20 per cent, lactose solution 
Basic fuchsin (16 per cent, alcoholic solution) 46 c.c. 
Sodium sulphite (10 per cent, solution freshly prepared and brought 
to a boil over flame) 2^ c.c. 
Pour plates and allow to harden in incubator. Inoculate in 
streaks. 
Bacterium coli forms colonies 4 mm. or more in diameter, 
deep red and buttonlike showing a greenish metallic sheen in 
reflected light. Bacterium aerogenes colonies do not show the 
metallic luster, are lighter colored, markedly convex and con- 
siderably larger; colonies of the intermediate and typhoid group 
are transparent, colorless or faintly yellow or amber colored. 
Eosin-methylene-blue Agar.-(Levine) 
Distilled water 1000 c.c. 
Peptone (Difco) 10 gm. 
Dipotassium phosphate 2 gm. 
Agar 15 gm. 
Boil ingredients until dissolved, and make up loss due to 
evaporation. Place measured quantities in flasks or bottles and 
sterilize in autoclave at 15 pounds pressure for 15 or 20 minutes. 
Just prior to use add to each 100 c.c. of the melted agar the follow- 
ing: 
Lactose 1 gram or 5 c.c. of 20 per cent, lactose solution. 
Eosin (2 per cent, yellow aqueous solution) 2 c.c. 
Methylene blue (0.5 per cent, aqueous solution) 2 c.c. 
No adjustment of reaction is necessary. Pour medium into 
sterile petri dishes and allow to harden. Cultures to be tested 
are streaked on the surface. 
Bacterium coli forms flat, dark centered, button like colonies 
2 to 4 mm. in diameter, which by reflected light show a charac- 
teristic greenish metallic sheen. Bacterium aerogenes colonies are 
in general much larger, convex with brownish centers and rarely 
exhibit a metallic luster. 110 
VETERINARY BACTERIOLOGY 
Potato.-Cylinders are cut from potatoes by means of an apple- 
corer or special potato borer. These are divided by a diagonal 
longitudinal cut such that each half has one long sloping surface. 
Non-liquefiable Media 
Fig. 52.-Preparation of potato tubes: a, Potato cylinder cut diagonally; 6, 
side view in tube; c, front view. 
It is well to soak in running water for a few hours to prevent 
their turning dark when sterilized. They are placed with the 
sloping surface up in test-tubes with a bit of saturated absorbent 
cotton in the bottom, or in special potato tubes. The latter 
are tubes constricted a short distance from the bottom. The 
bulb thus formed is filled with water and the potato rests on the 
constriction above. This device enables one to keep the potatoes 
moist for considerable periods. They are sterilized in the auto- 
clave for fifteen minutes at 120°. 
Other Vegetable Media.-Carrots and other vegetables may 
be prepared in the same manner as potato. 
Blood-serum.-Solidified blood-serum has been found to be 
essential to the growth in the laboratory of certain of the patho- 
genic bacteria. It is best to avoid all the initial contamination 
of the serum possible, as it is difficult, by the methods used in 
sterilization, to rid the medium of all the spore producers when 
they are present in considerable numbers. The blood, usually 
from cattle, is allowed to clot, and the clear, straw-colored serum is 
removed. A clear, solidified serum may be prepared by heating 
the slanted tubes to 76° for an hour or more on five or six consecu- CULTURE-MEDIA AND THEIR PREPARATION 
111 
tive days. An opaque medium is secured by heating to a tempera- 
ture of 95°. The serum may be sterilized and yet remain liquid 
by placing in an incubator at 58° C., or in a water-bath at the same 
temperature for three or four hours on several consecutive days. 
If care has been used in drawing the serum the slight contamination 
that may have occurred will be overcome by this method. 
Loeffler's blood-serum is a mixture of three parts of the serum 
with one part of neutral 1 per cent, dextrose broth. It is solidi- 
fied in the same manner as the simple serum. 
Egg Medium.-This medium was developed by Dorset, of the 
U. S. Bureau of Animal Industry. It has come into common 
use for the growth of the Bacillus tuberculosis and has been used in 
recent years as a satisfactory substitute for blood-serum. Dorset's 
description of the method of preparation follows: "The egg 
shell is broken carefully, and the entire contents dropped into a 
wide-mouthed sterile flask. The yolk may be broken with a 
sterile platinum wire. Gentle shaking of the flask will serve 
to mix the white and yolk of the egg quite thoroughly. Care 
should be taken, however, not to shake the flask so that a foam will 
be produced, otherwise an uneven and unsatisfactory surface will 
be obtained when the medium is hardened. When the mixing 
is complete, the egg is poured into tubes, care being taken to avoid 
foaming, and the tubes containing about 10 c.c. of the medium are 
then inclined in a blood-serum oven and hardened at a temperature 
of 70° C. This hardening will usually require two days, four or 
five hours each day. Sterilization will be accomplished at the 
same time. A higher temperature may be used and the medium 
will be hardened more quickly. The growth of tubercle bacillus 
seems to be more vigorous when the egg is hardened at 70° to 
74° C., and, in addition, the prolonged heating probably insures 
a more certain sterilization. The medium after hardening is 
opaque and yellowish in color, and usually dry, there being 
practically no water of condensation in the tube. The egg tubes 
should be kept in an ice-box to prevent further drying. Just before 
inoculation, three or four drops of sterile distilled water should 
be added to each tube to supply the moisture required for the 
satisfactory development of the tubercle bacillus." CHAPTER VIII 
BIOCHEMICAL TESTS 
The physiological characteristics of bacteria are of consider- 
able importance in the differentiation of species. A knowledge 
of such characteristics is of assistance in the isolation and recog- 
nition of certain species, as in the detection of sewage bacteria 
in water. 
Acid and Alkali Production. Determination of Changes in 
Hydrogen Ion Concentration.-Many bacteria when grown in 
nutrient solutions, particularly those containing carbohydrates, 
bring about marked changes in hydrogen ion concentration. 
The amount of such change accomplished by a given organism 
depends upon several factors, the most important being the 
following: first the amount of acid produced, second the kind of 
acid produced, third the amount of buffer present in the nutrient 
medium, and fourth the amount of alkali (that is the concentration 
of hydroxyl ions) produced at the same time. Alkali may be 
developed either by the production of ammonia or by the trans- 
formation of the salt of a strong acid to the salt of a relatively 
weak or little dissociated acid. For example, certain bacteria 
may transform sodium citrate into sodium carbonate, the latter 
being decidedly alkaline in its reaction. 
Two methods are in common use for detecting changes in 
hydrogen ion concentration. The first is by a determination of 
the electric conductivity. The second is a colorimetric method. 
For usual laboratory routine the colorimetric method is the 
simpler and is the only one which will be discussed. In this 
method it is customary to add a suitable indicator either to the 
solution to be tested or to a portion of this solution diluted 
somewhat with distilled water. The color secured is then com- 
pared with the color produced by similar addition of indicator 
to standard solutions whose hydrogen ion concentration is known. 
The indicators most used in the bacteriological laboratory for 
112 BIOCHEMICAL TESTS 
113 
this purpose are those developed by Clark and Lubs. In the 
following table the name of each of these indicators is given 
followed by the color in its acid range, next the color in its alka- 
line range, and finally the range of PH values through which it 
changes color. 
Color Changes of Clark and Lubs Indicators 
Indicators 
Full acid 
color 
Full alkaline 
color 
Sensitive range. The in- 
dicator changes from 
the acid color to the 
alkaline color between 
the following Ph values 
Thymol blue (acid range) 
Red 
Yellow 
1.2-3.8 
Brom phenol blue 
Yellow 
Blue 
3.0-4.6 
Methyl red 
Red 
Y ellow 
4.4-6.0 
Brom cresol purple 
Y ellow 
Purple 
5.2-6.8 
Brom thymol blue 
Yellow 
Blue 
6.0-7.6 
Phenol red 
Yellow 
Red 
6.8-8.4 
Cresol red 
Yellow 
Red 
7.2-8.8 
Thymol blue (alkaline range). 
Yellow 
Blue 
8.0-9.6 
Phenolphthalein 
Colorless 
Red 
8.0-9.8 
Cresolphthalein 
Colorless 
Red 
8.2-9.8 
It is apparent that an approximate idea can be secured of 
the change in hydrogen ion concentration by using different indi- 
cators and determining which gives an acid color and which an 
alkaline color. For example, if it is found that phenol red gives a 
yellow reaction the medium is acid and must be below the PH 
value of 6.8. If methyl red, on the other hand, gives an alkaline 
color it must be of a PH value above 6. The exact PH value can 
be determined then by the use of Brom thymol blue, comparing 
the intensity of the color change from yellow to blue with stand- 
ard solutions whose PH values are known. 
For discussion of the methods of preparing standard solutions 
and colors for accurate determination for hydrogen ion concen- 
tration a suitable laboratory manual should be consulted. 
Determination of Acid Production.-What has been stated 
above concerning determination of hydrogen ion concentration 
will indicate the method of determining whether or not acid has 
actually been produced by an organism. It should be noted, 
however, that the determination of hydrogen ion concentration 114 
VETERINARY BACTERIOLOGY 
is not a determination of the total amount of acid produced. 
This can be determined only by a comparative titration. It is 
customary to titrate to a definite tint a sample of the sterile 
medium retained as a check, using phenolphthalein as an indi- 
cator. For this purpose, it is usual to place 5 c.c. of the sterile 
material mixed with 45 c.c. of distilled water in a porcelain 
evaporating dish, add a drop or two of alcoholic solution of 
phenolphthalein and add twentieth normal (N/20) alkaline 
until a definite pink color has been established. This is kept as a 
standard, and the amount of alkali required noted. A similar 
titration is made using the medium in which the organism to be 
studied has been grown. This is treated in exactly the same 
fashion and the twentieth normal alkali added until the same 
tint has been secured as with the check. The differences between 
the amounts of alkali required for bringing to the same tint of 
red will vary directly with the amount of acid formed. 
In some cases it is desirable to determine the kind of acid 
which has been produced. Simple chemical tests for the various 
organic acids have not in most instances been developed. Some- 
thing of their nature, however, may be determined by separating 
them into volatile and non-volatile. If a solution containing 
an acid is acidified with sulfuric acid and the material dis- 
tilled, certain acids, particularly acetic, propionic and butyric 
will pass over, while other acids, particularly lactic, will not. 
This makes it possible by titration of the distillate to determine 
the relative proportion of volatile and non-volatile acid. 
Gas Production.-A few pathogenic bacteria produce gas from 
proteins, but most gas-producing species require the presence of 
a carbohydrate. The gases most commonly formed are carbon 
dioxid and hydrogen. One or more species of cellulose-fermenting 
organisms can also produce methane, and some of the denitrifiers 
free nitrogen. 
The ability to produce gas may be determined by inoculation 
of the organism into a dextrose agar or gelatin tube. Gas-bubbles 
will appear in the medium if the organisms can ferment dextrose. 
This sugar is generally used, as it is more easily fermented than 
most other carbohydrates. The fermentation tube is commonly 
used for the study of gas production. The closed arm is entirely BIOCHEMICAL TESTS 
115 
filled, and the open arm partly filled, with broth containing 
the sugar to be tested. The gas found after inoculation col- 
lects in the closed arm and may be conveniently measured 
by means of a Frost gasometer. The approximate composition 
of the gas may be determined by filling the open arm with normal 
sodium hydrate and securely closing the opening with the thumb, 
mixing the gas with the alkaline solution by passing it several 
times from one arm to the other, finally returning it to the closed 
arm and removing the thumb. The liquid will then rise in the 
Fig. 53.-Fermentation tube and Frost gasometer (Heinemann). 
closed arm to replace the carbon dioxid absorbed. The remaining 
gas may be transferred to the open arm and tested by the flame. 
Hydrogen is indicated by a slight explosion. The relative pro- 
portion of carbon dioxid and hydrogen is sometimes of importance 
in the differentiation of species. More important still is the 
ability of a species to ferment different kinds of carbohydrates. 
Some ferment dextrose, but not lactose or saccharose-some fer- 
ment two only, and some all three. 
Reduction Processes.-Somebacteria, when living in the absence 116 
VETERINARY BACTERIOLOGY 
of free oxygen, can reduce certain chemicals, evidently securing 
oxygen for growth processes by this means. Litmus, methylene- 
blue, and other pigments may be decolorized. Nitrates are 
frequently reduced to nitrites. For this determination a broth 
made from 0.1 per cent, peptone and 0.02 per cent, potassium 
nitrate is inoculated and incubated for four days. It is then 
tested by the following reagent for the presence of nitrites: 
a. 5 N acetic acid 1000 c.c. 
Sulphanilic acid 8 gm. 
b. 5 N acetic acid 1000 c.c. 
Alpha-amidonaphthylene 5 gm. 
Add 2 c.c. of each solution to the tube to be tested. A red or 
rose color will indicate the presence of nitrite. A control in check 
tubes of uninoculated broth should always be tested at the same 
time. 
In some cases denitrification goes still further and the nitrogen 
is liberated in the free state. 
Other reduction processes have been described. Among the 
more important are the reduction of sulphates to sulphids, and of 
chlorates to chlorites. 
Alcohol Production.-Yeasts produce considerable quantities 
of alcohol, and smaller amounts are formed as a result of the 
growth of a few bacteria and molds. Occasionally smaller 
amounts of other alcohols may be developed such as amyl, 
butyl and propyl. With yeasts, alcoholic fermentation is accom- 
panied by the production of carbon dioxide. The presence of 
ethyl alcohol may be detected by placing a few cubic centimeters 
of the material to be tested in a suitable container such as a test 
tube, and adding a small crystal of iodine and several cubic 
centimeters of strong solution of sodium hydrate. If alcohol 
is present heating this material over a Bunsen flame will give a 
distinct odor of iodoform. The amount of alcohol developed 
may be determined by distillation and the determination of the 
specific gravity of the distillate. 
Aldehyde.-Certain microorganisms, particularly when grow- 
ing in carbohydrate solutions, produce aldehyde in sufficient 
quantity to give a distinctive reaction. The detection of the 
presence of aldehyde is best accomplished by the use of a fuchsin BIOCHEMICAL TESTS 
117 
indicator. If a solution of basic fuchsin is decolorized by the 
addition of sodium sulphite (or better sulphurous acid) until the 
color has just disappeared or until the material is of a very light 
pink, the color of the fuchsin is restored upon the addition of 
aldehyde. This is the principle made use of in the so-called 
Endo medium. Certain bacteria when growing upon this 
medium form red colonies; the fuchsin turns red because of the 
development of aldehyde and acid. Other species of bacteria 
grown upon this medium do not change the color at all. 
Acetyl Methyl Carbinol.-This compound is produced by 
certain bacteria growing in the presence of carbohydrates. It is 
recognized by the addition of strong alkali such as sodium hydrate 
or potassium hydrate. When allowed to stand for a few hours 
an eosin pink or red color will develop, particularly near the 
surface, providing there is some peptone present. This is fre- 
quently called the Voges-Proskauer reaction, after the men who 
first noted it. 
Determination of Oxygen Relationships.-Bacteria are fre- 
quently divided into two groups, those which require free oxygen 
of the air for their development and those which will grow with- 
out. The first are termed aerobes, and the second anaerobes. 
Those bacteria which can grow either in the presence or the 
absence of free oxygen are termed facultative anaerobes, and those 
which will not grow in the presence of free oxygen are termed 
obligate anaerobes. 
It is frequently necessary in the laboratory to determine 
accurately the oxygen preferences of a microorganism. The 
bacteria which will grow only upon the surface of a medium, but 
never grow in the closed arm of a fermentation tube under any 
conditions are the obligate aerobes. For the most part these are 
organisms which are actual oxidizers, changing carbonaceous 
materials to carbon dioxide and water, for example. The faculta- 
tive anaerobes are those which grow in contact with air, but at 
least under certain conditions will grow in the absence of free 
atmospheric oxygen. Most of the facultative organisms are 
obligate aerobes on certain media and facultative on others. 
Many bacteria, for example, which usually require atmospheric 
oxygen can grow in its absence providing nitrates are present to 118 
VETERINARY BACTERIOLOGY 
furnish an available supply of oxygen, though not in the free 
form. Other bacteria will grow in the absence of oxygen pro- 
viding they have suitable carbohydrates. For example, the 
organism known as Bacterium coli will grow only in the open arm 
of a fermentation tube if sugar is absent, but in the presence of a 
suitable sugar such as dextrose it grows both in the open and in 
the closed arm. 
The obligate anaerobes are those which will grow only in the 
absence of free oxygen or are definitely injured by its presence. 
It is evident that special cultural conditions are necessary for 
their study. 
A few bacteria have been termed microcer ophites because they 
tend to grow in a definite concentration of oxygen. When mixed 
with melted agar in a test tube and the agar allowed to solidify, 
they will grow in a definite zone some distance below the surface 
of the medium. 
Indol Production.-Indol is one of the products of protein 
decomposition formed by bacterial action. It is of importance 
principally because it may be demonstrated readily and because 
of the economic importance of some of the bacteria which produce 
it. It is not formed in the presence of sugars. Dunham's solu- 
tion is inoculated with the organism to be tested and incubated 
for several days. To the tube are added a few drops of concentrated 
sulphuric acid and a cubic centimeter of a 0.1 per cent, solution 
of sodium nitrite. The sulphuric acid decomposes the nitrite, 
freeing nitrous acid, which unites with the indol to form a bright 
red compound known as nitrosoindol. The appearance of this 
characteristic red color is evidence, therefore, of indol production. 
Indol is an organic compound of the empirical formula, C8H7N, 
and the structural formula 
C0H4 
CH 
NH 
:CH. 
It is one of the products formed in intestinal putrefaction, and 
is the principal product which gives rise, under these conditions, 
to the characteristic " fecal " odor. 
Thermal Death-point.-The accurate determination of the exact 
temperature that is necessary to destroy various species of bacteria 
is frequently of great economic importance. Efficient steriliza- 
tion and pasteurization can be accomplished only when these facts BIOCHEMICAL TESTS 
119 
are known. Many methods have been suggested. In the labora- 
tory the determination is frequently made by subjecting freshly 
inoculated tubes of broth to different temperatures in a water-bath 
for ten minutes each. For reliable results more accurate methods 
are needed. One of the commonest and best is the use of the 
Sternberg bulb. This is blown of thin glass. A definite amount of 
culture is introduced and the neck sealed in the flame. The bulbs 
are completely immersed in a water-bath and suspended by wires 
or by some other method, so that they do not come in contact 
with the walls of the bath, and heated. A number of bulbs are 
prepared and one heated five minutes, another ten minutes, at 50°. 
The temperature is raised two degrees and two more bulbs are ex- 
posed. For sporeless bacteria the test should be made to 70°, 
and still higher for those that produce spores. The bulbs are cooled 
quickly after their exposure, and their contents mixed with agar in 
a Petri dish, or added to a tube of other suitable medium. This 
is then incubated for several days. The minimum temperature 
required to destroy the bacteria can readily be determined by a 
comparison of the tubes. 
Efficiency of Disinfectants.-The efficiency of disinfectants 
is determined by testing their action on pure cultures of bacteria. 
Koch's method, which has been commonly used, consists in drying 
the organism on silk threads, immersing them for varying lengths 
of time in the disinfectant to be tested, washing in sterile water, 
and placing the threads upon the surface of agar to determine 
growth. 
Hill's method is a modification of that of Koch, and is rather 
more accurate and relatively simple. Sterilized glass rods are 
coated at the tip with the bacteria to be tested by dipping them 
into a broth culture to a depth of an inch. These are placed in 
test-tubes and carefully dried in a thermostat. They may then be 
immersed to a somewhat greater depth in the disinfectant to be 
tested for definite periods of time, rinsed carefully in sterile water, 
and placed in tubes containing broth. 
Phenol Coefficient.-It has in recent years become standard 
practice to rate disinfectants by comparing their disinfecting 
power with that of phenol. The method was first proposed by 
Rideal and Walker. They prepared various dilutions of the 120 
VETERINARY BACTERIOLOGY 
disinfectant to be tested, and of phenol, and determined the 
ratio of the dilution of the former which killed Bacterium ty- 
phosum in a certain length of time to the corresponding dilution 
of the latter. This ratio they termed the phenol coefficient. 
This method was modified by Anderson and McClintock of the 
Hygienic Laboratory. The following procedure is used: 
1. Various dilutions of phenol and the disinfectant to be tested are 
prepared. 
2. Each tube is inoculated with 3% c.c. of a broth culture of Bacterium 
ty phosum, so-called "Hopkins" strain. 
3. Transfers of one standard loopful are made at intervals of two and 
one-half minutes to tubes of +1.5 meat extract broth, and these are in- 
cubated at 37° C. for forty-eight hours. 
4. The average of the ratios between the lowest concentration of the 
disinfectant being tested and of phenol required to kill in two and one- 
half minutes, and fifteen minutes is termed the hygienic laboratory phenol 
coefficient. CHAPTER IX 
MICROSCOPIC EXAMINATION AND STAINING METHODS 
Objectives having a higher power than those commonly used 
in other work are required for the -examination of bacteria. A 
-pj-inch or 1.8-2 mm. oil-immersion objective is most commonly 
used. This lens differs from the low-power dry lenses in that it 
requires a layer of cedar oil between it and the object to be exam- 
ined. This oil is used upon the lens for the following reasons. In 
general, the higher the power of the objective, the smaller the 
Fig. 54.-Diagram showing the function of an oil-immersion objective (adapted 
from Gage). 
opening through which light may come to the eye. It is necessary, 
therefore, that all the light possible shall enter the lens in order 
that a well-illuminated field may result. The accompanying ex- 
aggerated diagrammatic representation of the objective and the 
stage of the microscope may be helpful in understanding the use 
of the oil. 
Let C represent the microscopic slide, H the drop of oil having 
the same refractive index as glass, and L the tip of the objective 
121 122 
VETERINARY BACTERIOLOGY 
with the opening F'F. The rays of light are focused upon the ob- 
ject to be examined by the mirror or Abbe condenser. Those 
rays of light, such as BN, that strike the glass perpendicularly 
pass through and enter the lens without any deflection. A ray of 
light, such as AB, striking the glass at a considerable angle, is 
refracted upward and toward the normal or in the direction of BD. 
Upon entering the air it would again be refracted and leave the 
glass in a direction parallel to the original ray, or DE, and would 
not enter the lens. If, on the other hand, a drop of oil having the 
same refractive index as the glass intervenes, there will be 
no refraction at D, but the ray will pass through to the opening 
of the lens. This is represented by the ray A'BD'F'. The use 
of the oil, therefore, results in a more brilliantly illuminated field 
and a clearer definition of the objects to be examined. 
Measuring Bacteria.-Bacteria may be measured under the 
microscope in one of several ways. A micrometer scale ruled on 
glass may be inserted in the ocular, and the distance between the 
lines determined by examination of a micrometer scale ruled upon 
the slide or cover-glass examined under the microscope. When 
the calibration has been effected, the ocular micrometer may be 
used to measure the bacteria directly. To illustrate the method 
of measuring bacteria by means of a micrometer proceed as follows: 
(a) Insert eye-piece micrometer in the microscope. 
(&) Examine bacteria on a slide and record their lengths in divisions of the 
eye-piece micrometer. 
(c) Remove the slide of bacteria and substitute for it the stage micrometer. 
(d) Determine the relation of the divisions of the eye-piece micrometer 
and those of the stage micrometer. 
(e) The divisions on the latter are of fixed length, usually mm. 
The unit of measurement of bacteria is the micron (symbol p), the tAo 
part of a millimeter, therefore the length stated above (t^ mm.) = lOp. 
Suppose the adjustment is such as to show two divisions of the 
eye-piece micrometer equal to one division of the stage micrometer. 
This means that each eye-piece division represents one-half division 
on the stage micrometer or mm. = 5/jl. 
Examination of Living Bacteria.-Hanging Drops.-The deter- 
mination of the motility of bacteria can best be accomplished by 
the examination of the living cells under the microscope. A hang- 
ing-drop preparation is commonly used for the purpose. A loop- MICROSCOPIC EXAMINATION AND STAINING METHODS 
123 
ful of broth culture of the organism to be tested is placed upon the 
center of a carefully cleansed and flamed cover-glass. Growth 
from an agar or other culture may be used by substituting a 
drop of physiological salt solution or sterile bouillon and introduc- 
ing a minute quantity of the growth on a platinum needle. This 
drop is then carefully inverted over the cavity in a hollow ground 
slide, and sealed with a little vaselin. It may be examined with 
a high-power dry lens or with the oil-immersion objective. The 
drop may most easily be brought into focus at its margin. The 
light must be carefully regulated by means of the mirror and the 
iris diaphragm of the Abbe condenser to make the bacteria most 
clearly visible. 
Quite as effective an observation may be made in many cases 
by placing a drop of the culture upon a glass slide and dropping 
a cover-glass upon it, using care to include a few air-bubbles. 
A film of liquid sufficiently thick for the free movement of the bac- 
teria will remain between the two glasses. The edges of the air- 
bubbles furnish a convenient object upon which to focus. 
Staining Methods 
Bacteria as well as the pathogenic protozoa are generally so 
transparent when examined in a living condition that the details 
of their morphology can be made out only with difficulty. It is 
customary to stain these organisms with various anilin dyes 
which render them distinctly visible. 
The stains used in biological work are, for the most part, known 
as anilin dyes, because they are derivatives of anilin, C6H5NH2. 
They are grouped as acid or basic, depending on whether the acid 
radical or the base possesses the tinctorial powers. Fuchsin, 
for example, is a basic stain, while ammonium picrate is an acid 
stain. The basic stains are the more useful in the study of bacteria; 
the acid stains are sometimes used as counterstains, particularly 
for tissues in which the organisms may be embedded. The 
anilin dyes are of all the colors of the rainbow. The most com- 
monly used are gentian-violet, methylene-blue, thionin blue, 
fuchsin, and Bismarck-brown. 
Mordants.-Anything which will cause a stain to penetrate an 
organism better or which causes it to set is termed a mordant. 124 
VETERINARY BACTERIOLOGY 
For example, carbolic acid or anilin added to certain stains makes 
them more intense. A solution of iodin in potassium iodid, a 
mixture of tannic acid and iron sulphate, and many other solu- 
tions are used under various conditions as mordants. 
Formulas of Some of the Commonly Used Stains.-There are a 
few stains which find constant use in the laboratory. The formulas 
of these will be given. There are, in addition, a great many others 
which have special applications. 
Loffler's methylene-blue: 
Saturated alcoholic solution of methylene-blue 15 c.c. 
Solution of potassium hydrate (1 : 1000) 50 c.c. 
Aqueous solution of gentian-violet: 
Saturated alcoholic solution of gentian-violet 2.5 C.C. 
Distilled water 47,5 c.c. 
Anilin gentian-violet (Ehrlich's): 
Saturated alcoholic solution of gentian-violet 6c.C. 
Absolute alcohol 5 c.c. 
Anilin water 50 c.c. 
Anilin water is prepared by adding 2 c.c. of anilin to 98 c.c. of 
distilled water and shaking vigorously for several minutes. It 
should then be filtered until clear. 
Carbol or phenol fuchsin (Ziehl's): 
Saturated alcoholic solution of fuchsin 5 c.c. 
Solution of phenol, 0.5 per cent 45 c.c. 
Bismarck-brown: This is commonly used as a saturated aqueous 
solution. 
Gabbett's methylene-blue: 
Methylene-blue, dry 2 gm. 
Sulphuric acid 25 c.c. 
Distilled water 75 c.c. 
Preparation of a Stained Mount.-A drop of water, blood-serum, 
or broth about the size of a pinhead is placed upon a clean cover- 
glass. With a sterile platinum needle remove a small portion of 
the material to be examined and mix thoroughly in the drop. 
When the bacteria are in bouillon or other liquid media the drop 
of liquid is unnecessary. This is then spread in a thin film over the' MICROSCOPIC EXAMINATION AND STAINING METHODS 
125 
surface of the glass and dried. The film is next fixed by passing 
the cover-glass, film up, through the flame of the Bunsen burner 
three times. The stain is placed upon the glass and allowed to act 
for a few seconds to ten minutes, depending upon the organism 
and the stain used. This is then washed in water until no more 
stain comes off. It is dried between filter-paper and placed film 
down upon a drop of water on a slide and examined under the 
microscope. If satisfactory, it may be floated off with water, dried, 
and placed film down on a drop of Canada balsam on the slide. 
In many laboratories the use of the cover-glass is largely 
dispensed with, and certain routine examinations of many kinds 
can be more conveniently made by means of films prepared 
directly upon the glass microscopic slides. The procedure is 
practically identical with that detailed above for cover-glass 
preparations except that the immersion oil may be placed directly 
upon the stained film and no cover-glass used. 
Spore Stain.-Bacterial spores stain with difficulty, but once 
stained, do not yield up the stain readily. Any one of the follow- 
ing methods will be found to give good results: 
Hansen Method.-1. Prepare a film, fix, and stain with steam- 
ing hot carbol-fuchsin for five minutes. 
2. Decolorize with 5 per cent, acetic acid until the film is a light 
pink, and wash in water. 
3. Stain three minutes with Loffler's methylene-blue. 
4. Examine. 
Moller's Spore Stain.-1. Air dry smear. 
2. Fix in flame, or for two minutes in absolute alcohol. 
3. Place in chloroform (to remove fat). 
4. Wash in water. 
5. Stain in carbol-fuchsin, heating for one minute. 
6. Decolorize in 5 per cent, sulphuric acid. 
7. Wash. 
8. Contrast stain with methylene-blue. 
9. Wash, dry mount, and examine. 
By either method the spores appear red and the cell-body blue. 
Klein's Method of Staining for Spores.-1. Prepare a suspension 
of sporulating material in physiologic salt solution. Place in a 
small watch-glass or test-tube. 126 
VETERINARY BACTERIOLOGY 
2. Mix this with an equal quantity of filtered Ziehl carbol- 
fuchsin. 
3. Heat the mixture until it steams for six minutes. 
4. Place small loopful on slide and spread. 
5. Air dry and fix in flame. 
6. Decolorize with 1 per cent, sulphuric acid a few (one to two) 
seconds. 
7. Wash in water. 
8. Counterstain in dilute methylene-blue three to four minutes. 
Stain for Acid-fast (Acid-proof) Organisms.-Certain bacteria 
are stained with difficulty, but when once stained, they resist 
decolorization with ac;ds. The most important of these organisms 
is Mycobacterium tuberculosis. 
Acid Alcohol Method.-1. Prepare film, fix in flame, and stain 
with hot carbol-fuchsin for two minutes. 
2. Wash in 2 per cent, hydrochloric acid in 95 per cent, alcohol 
until there is no color visible in the thinner portions of the film. 
3. Wash in water and stain with methylene-blue for contrast. 
4. Wash and examine. 
Gabbett's Method.-1. Prepare film and stain as above with car- 
bol-fuchsin. 
2. Wash in water. 
3. Stain with Gabbett's methylene-blue for one-half to one 
minute. 
4. Wash and examine. 
The acid-fast organisms will be red in a blue field. 
Herman's Stain for Tubercle Bacilli in Tissues.-Solutions 
required: 
1. One per cent, ammonium carbonate in distilled water. 
2. Three per cent, crystal violet in 95 per cent, ethyl alcohol. 
Sections to be stained are placed on a slide or cover-glass and the 
water removed. About 7 drops of a mixture of 3 parts of solution 
1 with 1 part of solution 2 are added, and placed on water-bath for 
one minute. Ten per cent, nitric acid is allowed to act for a few 
seconds, then the section is placed in 95 per cent, alcohol. The 
tissue may be counterstained with dilute fuchsin or eosin. The 
bacteria are stained purple. 
Wirth's Stain for Staining Much's Granules of Mycobacterium MICROSCOPIC EXAMINATION AND STAINING METHODS 
127 
tuberculosis.-1. Stain in carbol methyl-violet twenty-four hours 
at room-temperature: 
Saturated alcoholic solution methyl-violet lOc.c. 
Two per cent, carbol water lOOc.c. 
2. Place in iodin solution (Lugol's) five to fifteen minutes. 
3. Wash in 5 per cent. HN03 one minute. 
4. Wash in 3 per cent. HC1 ten seconds. 
5. Differentiate in a mixture of acetone and absolute alcohol. 
For a counter-stain use highly diluted carbol-fuchsin 1 drop in 
50 c.c. water. 
The mount is not permanent. 
Flagella Stain.-The flagella of bacteria are not visible in 
ordinary stained mounts, and can be demonstrated only by a 
special technic. Young, twelve- to eighteen-hour cultures of 
bacteria should be used for their demonstration. A tube con- 
taining a few cubic centimeters (5) is inoculated with sufficient 
quantity of the growth carefully removed from the agar surface 
to produce a slight turbidity. Incubate for an hour in the ther- 
mostat. Drop two or three drops without mixing or spreading 
on a clean cover-slip. Dry and then fix in the flame. Many 
methods of staining flagella have been suggested; the two follow- 
ing are probably the best: 
Van Ermengem's Method.-1. Place the film for one hour in the 
following solution: 
Osmic acid, 2 per cent 1 part 
Tannin, 10-25 per cent, solution . 2 parts 
2. Wash in water, then absolute alcohol, then place in the 
following solution for a few seconds only: 
Silver nitrate, 0.05 per cent, in distilled water. 
3. Wash in the following solution for a few seconds: 
Gallic acid 5 gm. 
Tannin 3 gm. 
Fused potassium acetate 10 gm. 
Distilled water 350 c.c. 
4. Wash in silver nitrate solution until film turns black. 
5. Wash in water and examine. 128 
VETERINARY BACTERIOLOGY 
Ldfiler's Method.-1. Prepare film, fix, and apply the following 
mordant, heating for five minutes over a water-bath: 
Tannic acid (25 per cent, aqueous solution) 10 parts 
Saturated solution ferrous sulphate 5 parts 
Fuchsin (saturated alcoholic solution) 1 part 
2. Wash and blot with filter-paper. 
3. Stain with hot anilin-gentian-violet or carbol-fuchsin over 
a water-bath for five minutes. 
4. Wash and examine. 
Gram's Staining Method.-This method was first used to 
demonstrate bacteria in tissues, the bacteria retaining and the tis- 
sues losing the stain. It was later found that not all bacteria 
could be stained by this method, and it has in consequence come 
into general use for separating bacteria into two groups, termed 
respectively gram-positive and gram-negative, the former retain- 
ing the stain and the latter losing it. 
1. Prepare film, dry, and fix. 
2. Stain one and one-half minutes in anilin-gentian-violet. 
3. Treat with Gram's iodin solution one and one-half minutes. 
lodin 1 gm. 
Potassium iodid 2 gm. 
Water 300 c.c. 
4. Decolorize with 95 per cent, alcohol for five minutes. 
5. Wash, dry, and mount. 
Stabilized Gentian Violet.1 
Anilin 28 c.c. 
Gentian violet 8 gm. 
95 per cent, alcohol 100 c.c. 
N hydrochloric acid 5 c.c. 
Dist. water q.s 1000 
Dissolve gentian violet in alcohol. Add HC1 to the anilin 
and dissolve in water to make 900 c.c. Filter the aqueous solu- 
tion and add alcoholic stain. Filter. 
Capsule Stains.-Muir's Capsule Stain.-1. Prepare film and 
dry. 
2. Apply carbol-fuchsin (hot) for thirty seconds. 
3. Wash quickly in 95 per cent, alcohol, then in water. 
1 Stovall and Nichols, Jour. A.M.A., 1916, 66, 1620-1621. MICROSCOPIC EXAMINATION AND STAINING METHODS 
129 
4. Apply following mordant for five or ten seconds: 
(a) Saturated aqueous solution HgCl2 2 parts. 
(6) Twenty per cent, aqueous tannic acid 2 parts. 
(c) Saturated solution potash alum 5 parts. 
5. Wash in water and apply 95 per cent, alcohol one minute. 
6. Wash in water and apply methylene-blue for thirty seconds. 
7. Dry and examine. 
Johne's Capsule Stain.-This is particularly suited to the 
demonstration of the capsules of the anthrax bacillus in smears 
from blood or tissues. 
1. Dry in air. 
2. Fix by passing three times through the flame. 
3. Stain with 2 per cent, aqueous gentian-violet, heating slightly 
for one-quarter to one-half minute. 
4. Wash quickly in water. 
5. Wash in 1 to 2 per cent, acetic acid six to ten seconds. 
6. Wash in water; mount in water to examine. 
Raebiger's Capsule Stain.-This has been used for demonstra- 
tion of the capsules of the anthrax bacillus in tissue and blood- 
smears. 
The formalin gentian-violet is prepared by adding 15 to 20 gm. 
of gentian-violet to 100 to 150 gm. of 40 per cent, solution of for- 
maldehyd (commercial formalin). This should be thoroughly 
mixed and allowed to stand for some hours. The solution is 
filtered. 
1. Prepare thin smear of material. 
2. Air dry (do not fix). 
3. Stain twenty seconds. 
4. Wash in water. 
5. Mount in balsam. 
The capsules should appear reddish violet, the bacilli dark 
violet. 
Blood and Protozoan Stains.--Many special stains have been 
devised for demonstrating the blood elements and protozoa in 
the blood and in tissues. The chief of these are the Romanowsky 
and Giemsa, each with numerous modifications. These may most 
profitably be purchased ready for use from a reliable dealer. 
Wright's Stain.-One of the most satisfactory blood stains for 130 
VETERINARY BACTERIOLOGY 
routine work is that of Wright. This stain can be purchased in 
liquid form ready for use, or in powder form from which a satu- 
rated solution is made up as a stock solution. This is prepared 
for use by adding to 20 c.c. of the filtered stock solution 5 c.c. of 
methyl alcohol. A blood-smear is made and allowed to air dry. 
The slide is then flooded with the Wright stain and allowed to 
stand one minute. Distilled water is then added drop by drop 
until a metallic luster appears on the surface. The stain is per- 
mitted to act for five minutes, when it is washed off with distilled 
water, dried, and examined with or without oil immersion. 
Giemsa's Stain.-1. Apply the following fixing agent to moist 
films for twelve hours. 
95 per cent, alcohol 1 part. 
Saturated aqueous HgCl2 2 parts. 
2. Wash in water for a few seconds. 
3. Apply Lugol's solution for five minutes. 
4. Wash in water, then in .5 per cent, sodium thiosulphate. 
5. Stain with Giemsa stain one to ten hours. 
6. Wash and mount. 
A modification of this method consists of mixing 1 drop of 
concentrated Giemsa in 20 drops of distilled water. After fixing 
the smear for five minutes in methyl alcohol it is immersed in the 
dilute stain and placed in a 37|° incubator for ten to twelve hours. 
It is then washed off with distilled water until the film has a slight 
pink tinge. This method is recommended for staining of brain 
tissue for Negri bodies. 
Negri Bodies.-Lentz Method.-Smears upon clean glass 
slides are made from (a) Ammon's horn, (6) the cerebellum, or (c) 
the cerebral cortex. Without allowing to dry, the smears are 
fixed for about 10 seconds in neutralized methyl alcohol (alcohol 
500 c.c. to which about 0.25 gm. of sodium carbonate has been 
added) to which 0.1 per cent, picric acid has been added. The 
excess of fixative is removed by blotting with fine filter paper. 
The fixed smears are stained in the following solution: 
Saturated alcoholic solution of fuchsin 0.3 gm. 
Saturated alcoholic solution of methylene blue 2.0 gm. 
Distilled water 30.0 gm. MICROSCOPIC EXAMINATION AND STAINING METHODS 
131 
This solution, which is a modification of the one proposed by 
Van Gieson, changes rather quickly at room temperatures, but 
kept in the ice box gives good results for an indefinite time. The 
stain is poured on the smear and held over the flame until it 
steams. The smear is then washed in tap water and dried with 
fine filter paper. Negri bodies appear magenta, the nerve cells 
blue, and red blood cells yellow or salmon. 
3. Stain in methylene-blue solution one minute. 
4. Wash in water. 
5. Dry between layers of filter-paper. 
6. Differentiate in alkaline alcohol until only a slight pink can 
be recognized. 
7. Differentiate in acid alcohol until the thinner part of the 
preparation shows no blue. 
8. Wash short time in absolute alcohol. 
9. Dry and examine with oil immersion. 
The Negri bodies appear crimson as distinct from the vermilion 
blood-corpuscles, and show within them one or more blue inclu- 
sion bodies. 
India-ink Method for Bacteria and Protozoa.-Mix the fluid 
containing the organisms to be examined with an equal quantity 
of India ink. Make a thin smear, dry, and fix. The organisms 
do not stain with the ink, but appear as transparent bodies in the 
black field. CHAPTER X 
METHODS OF SECURING PURE CULTURES OF BACTERIA 
Bacteria must be studied in pure culture if one is to deter- 
mine with certainty their cultural, physiological, or pathogenic 
characters. One of the first efforts made in the study of a disease 
or any other process brought about by bacteria is to separate its 
causal organism from all others. Many methods have been devised 
for this purpose, not any one of them applicable to every case. 
Dilution Method.-This method of securing pure cultures is 
of historic interest only. In the beginnings of the cultivation of 
microorganisms the culture-media commonly used were liquids, 
such as infusions from meat and vegetables, and beerwort. This 
method was used most commonly in securing pure cultures of 
yeasts. A long series of flasks was prepared with sterile media. 
The impure culture or mixture of organisms was mixed thoroughly 
with the contents of the first flask, and a definite amount transferred 
from this to another flask, from this to each of several others, from 
each of these into another group, and so on. The last dilution 
would, in general, remain sterile, but among some of the dilutions 
would be a group in which some flasks would show growth and 
others of the same dilution would not. The inference was that 
such a flask had been planted with but a single organism, and the 
flask contents, therefore, constituted a pure culture. This method 
is cumbersome, uncertain, and is rarely used. 
Isolation by Smearing.-If a loopful of a mixed culture of 
microorganisms be drawn across the surface of a solid medium in 
parallel streaks, the first portion will generally show a solid line 
of mixed growth, but farther along the growth is discontinuous. 
Many of the isolated colonies here will be found upon examination 
to consist of pure cultures. This method is used for the isolation 
of bacteria from the mouth and throat in some cases. 
Direct Isolation.-Barber has devised a capillary pipette method 
whereby it is possible to pick up a single bacterial cell and transfer 
it to a nutrient medium without any other organisms being carried 
132 METHODS OF SECURING PURE CULTURES OF BACTERIA 
133 
over. This method has been found useful in the study of develop- 
mental and evolutionary problems, but is not practicable for 
routine laboratory isolations. 
Isolation by Plating.-The development by Koch of the lique- 
fiable media furnished a ready means for the isolation in pure 
Fig. 55.-Isolation by successive streak cultures on an agar or gelatin 
plate: A, First streak solidly grown; B, second streak, discontinuous; C, 
third streak, having many isolated colonies. 
culture of most species of bacteria. Nutrient agar or gelatin 
or one of their modifications may be used. The medium is lique- 
fied by heat, then cooled in a water-bath to about 43°. The 
mixed culture of organisms from which it is desired to isolate 
pure cultures is inoculated into one of the tubes. From this 
Fig. 56.-Petri dish (McFarland). 
transfers are made by means of a sterile platinum loop to a second 
tube; this is thoroughly mixed and transfers made to a third tube, 
and from this even to a fourth. Each of these tubes of media 
is then poured into a sterile, flat, glass, covered dish called a Petri 
dish. These Petri dishes or 11 plates " are allowed to stand until 
the medium has solidified; they are then incubated and examined 134 
VETERINARY BACTERIOLOGY 
from time to time. The organisms are separated from each 
other by this process of dilution, and are held fast by the solidifica- 
tion of the medium. In most cases the conditions are favorable 
for growth, and development begins. Within a few days sufficient 
multiplication takes place, so that the mass of organisms that has 
developed from the single isolated individuals has reached a size 
that can be easily seen with the unaided eye. Such a mass of organ- 
isms is termed a colony. Transfers from such colonies will show 
only a single kind of organism present, and by making isolations 
from each type of colony, pure cultures may be secured of each 
species present. 
Isolation by the Use of Heat.-When it is desired to isolate a 
spore-producing organism from non-sporulating forms, the culture 
may be heated to 80° for fifteen minutes. This will not destroy 
the spores, but will eliminate all other cells. If one species of 
spore-forming organism only is present, this results in a pure cul- 
ture at once; if more than one species, plating becomes necessary. 
Isolation by the Use of Differential Antiseptics or Disinfect- 
ants.-Not all species of bacteria are affected alike by a given 
antiseptic or disinfectant, and it is sometimes possible to add a 
substance that will prevent the growth or kill one form without 
interfering seriously with the growth of others. A small amount 
of phenol added to bouillon will inhibit the growth of most bacteria, 
with the exception of certain members of the intestinal group. 
A still better example of such substance is antiformin, which, 
when mixed with sputum or other materials containing tubercle 
bacilli, destroys all other organisms than these, and enables one to 
secure a pure culture at once. This will be discussed in greater 
detail under the heading of Tuberculosis. 
Isolation by Animal Inoculation.-Some species of pathogenic 
bacteria develop very slowly upon artificial media, or require 
a special medium for their growth. When these occur mixed 
with other organisms, it is sometimes difficult to secure them in 
pure culture. This difficulty may in some cases be overcome by 
animal inoculation. The injection of the organism into a suitable 
susceptible animal results in the destruction bythe body of the other 
bacteria injected at the same time, and the characteristic organism 
may later be isolated in pure cultures from the lesions of the disease. CHAPTER XI 
STUDY OF BACTERIAL CULTURES 
Species of bacteria are frequently separable from each other 
on the basis of differences in cultural characters alone. It is, 
therefore, important that careful descriptions should be kept of 
the cultural characteristics of each of the species. For assistance 
in such descriptions the Society of American Bacteriologists has 
adopted a standard descriptive chart from which the following are 
adapted: 
Cultural Characters 
Agar Stroke.-This is prepared by drawing an inoculated needle 
from the base to the top of the slanted surface of an agar tube 
that has solidified in the sloping position. In this culture are to 
Fig. 57.-' 
-Types of growth on agar slants. 
be noted the abundance, form, elevation, luster, surface, and optical 
characters of the growth, its pigment production, odor, consistency, 
and any changes that have occurred in the medium. 
Potato.-The potato is inoculated and the growth character- 
istics studied in the same manner as the agar stroke. 
135 136 
VETERINARY BACTERIOLOGY 
Blood-serum.-This is inoculated in the same manner as the 
agar slope, and the same characteristics are to be noted, with the 
addition of liquefaction or digestion of the medium. 
Gelatin Stab.-This is prepared by running an inoculated plat- 
inum needle in a straight line from the surface of an erect tube 
of nutrient gelatin nearly to the bottom, and withdrawing it with- 
out cutting the medium by any lateral motion of the needle. 
Fig. 58.-Potato slant culture (Page, Frothingham and Paige, in "Journal 
of Medical Research"). 
The characters to be noted are abundance and uniformity of 
growth along the line of the stab, the form of growth, liquefaction, 
and other changes in the medium. 
Nutrient Broth.-This is inoculated by shaking an infected 
platinum needle in the medium. The characters to be noted are 
abundance and character of surface growth and character of 
sediment. 
Milk.-Milk is inoculated in the same manner as the nutrient STUDY OF BACTERIAL CULTURES 
137 
broth. The characters to be noted are presence or absence of 
coagulation, type of curd produced, whether or not whey is extruded, 
peptonization or digestion of the casein, acid production, con- 
sistency, and changes in color of the medium. 
Litmus Milk.-In addition to the preceding, acid or alkali 
production and reduction of the litmus are to be noted. 
Fig. 59.-Types of growth in stab cultures: A, Non-liquefying: 1, Fili- 
form (Bad. coll); 2, beaded (Str. pyogenes); 3, echinate (Bad. acidi ladici); 
4, villous (Erysipel. murisepticum); 5, arborescent (B. mycoides). B, Lique- 
fying; 6, Crateriform (Pr. vulgaris, twenty-four hours); 7, napiform (B. 
subtilis, forty-eight hours); 8, infundibuliform (Erythrobacillus prodigiosus); 
9, saccate (Vibrio Finkleri); 10, stratiform (Ps. fluorescens) (Frost). 
Gelatin Plate Colonies.-Two or three tubes of gelatin are 
melted, cooled to 40 °, and one inoculated with a small amount of 
the organism to be studied. The tube is rolled until the bacteria 
are thoroughly distributed, and with a platinum loop a transfer 
is made to a second tube, and from this to a third. The con- 
tents of each tube are then poured into a Petri dish and allowed 138 
VETERINARY BACTERIOLOGY 
to solidify. The plate showing a small number of colonies devel- 
oping is the one chosen for examination. The characters to be 
Fig. 60.-Portion of an agar plate culture showing a mold colony and five 
bacterial colonies. 
noted are rapidity of growth, form, elevation, and edge of the colony 
and type of liquefaction if it occurs. 
Fig. 61.-Ameboid colony on an agar plate (Lewton-Brain and Deerr). 
Colonies on Agar Plates.-Plates containing nutrient agar are 
prepared in the same manner as the gelatin plates described. The STUDY OF BACTERIAL CULTURES 
139 
Fig. 62.-Spreading colony on an agar plate (Lewton-Brain and Deerr). 
Fig. 63.-Colonies in an agar plate culture (Lewton-Brain and Deerr). 
characters to be noted are rapidity of growth, form, surface ele- 
vation, edge, and internal structure of the colony. 140 
VETERINARY BACTERIOLOGY 
Physiological Characters 
It is customary to determine gas and acid production in car- 
bohydrate media, development of ammonia, reduction of nitrates 
to nitrites, indol production, temperature relations, including 
optimum growth temperature and thermal death-point, resistance 
to desiccation and disinfectants, and pathogenic characters. The 
methods of study of these characters have already been discussed. SECTION III 
BACTERIA AND THE RESISTANCE OF THE ANIMAL 
BODY TO DISEASE 
CHAPTER XII 
MICROORGANISMS AND DISEASE 
Infectious Diseases.-An infectious disease is one which is 
caused by some microorganism. The mere presence of micro- 
organisms in the body, however, does not constitute infection. In 
general, this is not regarded as occurring unless the organisms grow 
or multiply in the body and produce pathological changes in the 
tissues and symptoms of disease. An individual thus infected is 
termed the host. Both infective and infected have been used to 
describe inanimate objects which harbor pathogenic organisms 
and which may aid in their distribution; of these, the term infective 
is preferable. Infective objects, such as the manger of a glandered 
horse or the clothing of a person with a readily transmissible 
disease, are called fomites. The term virus is commonly used to 
designate the causal organism of an infectious disease when such 
organism is not known. Sometimes it is used in a broader sense 
to designate any disease-producing organism. 
Diseases, that is, pathological conditions of the body, may be 
either infectious or non-infectious. Among the infectious diseases 
of animals and man are the following: Fistula, boils, abscesses and 
similar pyogenic infections, strangles, pneumonia, meningitis, 
Malta fever, gonorrhea, diphtheria, tuberculosis, paratuberculosis, 
pseudotuberculosis, swine erysipelas, glanders, dysentery, typhoid, 
hemorrhagic septicemia, plague, chicken-cholera, anthrax, milk 
sickness or trembles, infectious abortion, foot-rot of sheep, tetanus, 
blackleg, malignant edema, botulism, Asiatic cholera, actinomy- 
cosis, blastomycosis, aspergilloses and related mycoses, ring- 
141 142 
VETERINARY BACTERIOLOGY 
worm, amebic dysentery, trypanosomiasis, Leishmaniosis, relap- 
sing and tick fevers, Texas fever and related piroplasmoses, ana- 
plasmoses, malaria, yellow fever, coccidiosis, pleuropneumonia, 
foot-and-mouth disease, rinderpest, hog-cholera, horse sickness, 
equine infectious anemia, fowl plague, the poxes, such as small-pox 
and chicken-pox, infantile paralysis, rabies, and others. 
Non-infectious diseases are those which are caused by some 
'injury to the body or by some physiologic disturbance not due to 
microorganisms. Among such diseases may be mentioned cer- 
tain types of tumors and cancers, diabetes, azoturia, certain 
types of neuritis, arteriosclerosis, cardiac hypertrophy, etc. In 
some cases the infectious or non-infectious origin of a disease has 
not been satisfactorily determined. Such a disease is cancer. 
Contagious Diseases.-Any disease in which the causal organ- 
ism may be readily transferred from one individual to another by 
direct or indirect contact is said to be contagious. The terms in- 
fectious and contagious have been used very loosely, sometimes 
even interchangeably. The best usage, however, is strictly to 
limit the terms as here defined. Infectious has to do with the 
cause of a disease, and contagious with its ease and method of 
transmission. All infectious diseases, therefore, which are 
readily transmitted by contact, direct or indirect, are said to be 
contagious. All contagious diseases are necessarily infectious, 
but the reverse is not true; for example, malaria in man and 
Texas fever in cattle cannot be regarded as contagious, as they 
are transmitted only through the bites of certain insects. Every 
gradation between highly contagious and non-contagious dis- 
eases is known. It is customary to indicate the degree of 
contagiousness by use of modifiers. We speak, therefore, of 
highly contagious, slightly contagious, etc. 
Avenues of Infection.-The avenue through which an organism 
gains entrance to the body is called its portal of entry or its infec- 
tion atrium. An infection arising from contact with infective 
external objects is termed exogenous; one caused by organisms con- 
stantly or normally present in the body or on it is termed endogen- 
ous. Traumatic infection frequently occurs through a break in 
the continuity of the skin or mucous surfaces. Wounds caused 
by weapons, instruments, and similar objects, the bites of animals MICROORGANISMS AND DISEASE 
143 
and sucking insects, such as the mosquito, flea, tick, or bedbug, 
may introduce a pathogenic organism. Ordinarily, the skin is an 
efficient barrier against infection. Microorganisms may occa- 
sionally enter through the glands or hair-follicles. Some bacteria 
apparently may injure the unbroken mucous surface, as, for 
example, the diphtheria bacillus. Certain disease organisms enter 
through the digestive tract. Bacteria have been shown to pass 
unharmed through the intestinal walls and to enter the lymph- 
vessels and the thoracic duct. To what extent this is the common 
source of infection in certain diseases, such as tuberculosis, is at 
present a matter of dispute. 
The lungs constitute the infection atrium in pneumonia, prob- 
ably in many cases of tuberculosis and aspergillosis, and possibly 
in certain diseases whose cause has not been determined, such as 
small-pox. 
The genital organs are the common infection atria in the so- 
called venereal diseases, such as syphilis, chancroid, and gonorrhea 
in man, dourine in the horse, and possibly in contagious abortion 
in cattle. Disease organisms rarely pass from the blood of the 
mother through the placenta to the blood of the fetus. In a 
strict sense, none of the infectious diseases present at birth are 
inherited. 
Cryptogenic infections are those which occur without the possi- 
bility of determining the infection atrium in a particular case. 
Tetanus, for example, sometimes occurs when careful search fails 
to show the channel through which the organism reached the 
tissues. 
The ability of an organism to multiply in the body or produce 
disease frequently depends upon the channel through which it 
enters; the organism which produces Asiatic cholera in man, for 
example, evokes this disease with its characteristic symptoms only 
when entering through the alimentary tract. The type of disease 
caused by a particular organism may also vary with the infection 
atrium; the disease caused by the organism of human plague shows 
differences depending on whether the infection occurs through the 
skin, the alimentary or respiratory tracts. An organism may pro- 
duce no lesions at the point of entry, but invade some other tissue; 
the bacillus of tuberculosis is known to pass through the mucous 144 
VETERINARY BACTERIOLOGY 
membranes of the intestines without visible injury to them and 
to produce tuberculosis of adjacent glands. 
Virulence.-The virulence of an organism is its relative ability 
to combat the defences of the body against infection, to invade the 
tissues, and to produce disease. The degree of virulence on the 
part of the organism and the relative resistance to infection on the 
part of the animal determine whether or not disease will be pro- 
duced, as well as its course and termination. The virulence of 
organisms shows great variations even within the same species. 
It may be modified in various ways. Methods of immunization 
are in many cases based upon the possibility of attenuation. 
Proof of the Causal Relationship of a Microorganism to a Dis- 
ease-Koch's Postulates.--The proof of the germ theory of disease 
may be dated from 1876, when Koch succeeded in demonstrating 
the causal relationship of Bacillus anthracis to the disease anthrax. 
He later formulated the rules (known as Koch's rules or postulates) 
for the determination of the specific relationships of an organism 
to a disease. They may be stated as follows: 
1. The suspected organism must be found in every case of the 
disease under consideration. 
2. The organism must be isolated and grown in pure culture. 
3. Inoculation of the organism into suitable animals should 
reproduce the disease. 
4. The organism must again be isolated from such animals. 
Unfortunately, the demonstration by means of these rules of 
the causal relationships of specific organisms to many diseases 
has proved impossible, and is unsatisfactory in others. There are 
several reasons for this: 
(a) The organisms in some cases have been shown to be very 
small, perhaps even ultramicroscopic, and capable of passing 
through a fine porcelain filter, as, for example, those which cause 
fowl plague and hog-cholera. 
(b) Some organisms, although evidently not ultramicroscopic, 
have never been satisfactorily demonstrated under the microscope, 
possibly from lack of proper staining methods. 
(c) Some organisms are specific for man and do not reproduce 
disease of the same type when inoculated into animals. With MICROORGANISMS AND DISEASE 
145 
some of these, accidental or intentional inoculation into man has 
supplied the needed evidence. 
(d) The organism may be demonstrated microscopically, but 
will not grow upon the culture-media of the laboratory. Such 
are some of the protozoa. With a few of these the proof has 
been perfected by the study of the growth of the organism in 
an intermediate host, for example, the malarial parasite in the 
mosquito. 
Evidence of the relationship of an organism to a disease may 
frequently be secured by using the agglutination, precipitation, 
or some of the other tests discussed in the following chapters. Im- 
provements in staining technic and culture methods continually 
reveal new organisms. 
Animal Inoculation.-Experimental inoculation and injections 
are made in the bacteriological laboratory for a number of reasons: 
1. To determine the causal relationships of a specific organism 
to a disease in accordance with Koch's rules. 
2. To diagnose certain diseases. For example, one of the 
methods of diagnosing glanders is to inject some of the nasal 
discharge from a suspected animal into a male guinea-pig and 
note the development of acute orchitis and subsequent general 
body reaction. 
3. To isolate certain pathogenic bacteria. For example, if it is 
desired to isolate the organism causing tuberculosis from sputum or 
from milk, it may be accomplished most readily by inoculating 
the infective material into a suitable animal. All the non-patho- 
genic organisms will be destroyed and the specific organism may be 
isolated in pure culture from diseased tissue or lesions of the ani- 
mal so infected. The animal body is used in a sense as a filter for 
the removal of the non-pathogenic bacteria. 
4. To determine the strength or concentration of certain bio- 
logical products. As will be seen later, the only way that has been 
devised for the determination of the strength or potency of certain 
poisons, such as toxins ahd of their antitoxins, is animal injection. 
The animal is used by the bacteriologist in much the same manner 
as an indicator is used by the chemist in determining the acidity or 
alkalinity of a solution. 
5. For the production of certain so-called antibodies, such as 146 
VETERINARY BACTERIOLOGY 
antitoxins, and for the demonstration of certain characteristics of 
the blood-serum in studies of immunity. 
The animal most frequently used in experimental work is the 
guinea-pig or cavy; next in importance is the rabbit. Mice and 
rats, particularly the white varieties, are often used. When birds 
are necessary, the pigeon and domestic fowl are generally utilized. 
Some of the larger animals, as the goat or horse, are used for the 
production of serum where it is required in considerable quanti- 
ties, as, in the manufacture of antitoxins. The monkey has been 
used to some extent in the study of diseases peculiar to man. 
Heifers are utilized in the preparation of the vaccine against small- 
pox. Swine are used in the preparation of hog-cholera antiserum. 
Methods of Inoculation.-Animals may be inoculated just 
beneath the skin, or subcutaneously. The hair is shaved from the 
Fig. 64.-Ear veins of a rabbit: a, Posterior vein; b, point at which injections 
may be most easily made; c, median vein (adapted from Frost). 
area selected, the skin is washed with an antiseptic, and a hypo- 
dermic needle inserted into the subcutaneous tissue. In the 
inoculation of a solid material a little incision may be made in 
the skin, the material inserted, and the opening closed. Usually 
stitches to hold the skin are unnecessary. Intravenous inocula- 
tion is accomplished by inserting the needle into a vein. Usu- 
ally a rabbit is selected for this purpose. Reference to Fig. 64 
will show the vein on the posterior edge of the ear, into which in- 
jections are usually made. The large median vein is not suitable, 
as it is situated in loose connective tissue and, therefore, difficult 
to enter. The posterior vein, on the other hand, is embedded in 
firm connective tissue and cartilage, so that it does not give before 
the needle-point. 
Intraperitoneal injection is accomplished by thrusting the hypo- MICROORGANISMS AND DISEASE 
147 
dermic needle through the abdominal wall. Some care must be 
used not to penetrate too rapidly, as there is danger of injuring the 
intestines. Intrathoracic inoculation is rarely practised. Injec- 
tion directly into the heart (intracardiac) may be successfully car- 
ried out if sufficient care is used. Inoculation by scarification is 
accomplished by scraping off the outer layers of skin without draw- 
ing blood and rubbing the organism on the surface moistened by 
the exuded serum. Intracranial injections may be made by use of 
a trephine to penetrate the skull, after which subdural inoculations 
are made with the hypodermic. Intra-ocular injections have some- 
times been performed for the specific purpose of observing from 
day to day the development of lesions upon the iris or in other 
tissues of the eye. Infection by inhalation is accomplished by 
forcing the animal to breathe the organism in dust or as a fine 
spray. Infection by way of the digestive tract is accomplished by 
feeding or ingestion. 
Types of Infectious Diseases.-Mixed and secondary infections 
are those in which two or more organisms are responsible for dis- 
ease production in a single individual. In the strict sense a mixed 
infection is one in which two or more organisms enter the body at 
approximately the same time and are jointly responsible for the pro- 
duction of disease. Such, for example, is a fistulous wither in which 
several species of pyogenic bacteria are growing. In a secondary 
infection one organism has grown for a considerable time and has 
evoked the characteristic symptoms of the disease before infection 
with a second organism occurs. It is evident that in many cases 
there can be no distinct differentiation between mixed and second- 
ary infections. The organisms causing the secondary infection 
in many cases are themselves capable of causing primary infec- 
tions; in others the organisms are quite incapable of invading the 
body alone. The occurrence of mixed and secondary infections 
renders difficult in many cases the task of identifying the organisms 
primarily responsible for a given condition and the diagnosis of a 
disease. 
When once the body has been invaded, the type of organism, 
its virulence, the resistance of the individual and the infection 
atrium will determine the localization or distribution of the organ- 
isms and the type bf disease produced. 148 
VETERINARY BACTERIOLOGY 
Toxemias are diseases in which the causal organism tends to 
remain localized, but which produces a potent toxin or poison which 
causes injury to tissues in other parts of the body. Such diseases 
are diphtheria and tetanus, for in these infections the organisms 
do not commonly spread far from the original site of infection and 
do not invade the blood-stream or lymph-channels. 
Many organisms tend to remain more or less localized, but do 
not produce a potent toxin. Such are most pyogenic infections, 
local inflammations, tuberculosis, and pneumonia. Such infec- 
tions are sometimes termed phlogistic. 
Septicemia is an infection in which bacteria are present and 
multiply in the blood-stream. Some authors restrict this term to 
include only such infections as are due to the pyogenic cocci. 
Bacteremia is used as a synonym of septicemia, or by some authors 
to include those blood infections not due to the pyogenic cocci, 
such as anthrax. 
Pyemia is a metastatic pyogenic infection, that is, one in which 
organisms travel to parts of the body other than the original infec- 
tion atrium, and localize. 
Sapremia is the result of the absorption of poisonous putre- 
factive products resulting from the destruction of necrotic tissues, 
usually as the result of the growth of saprophytic or semiparasitic 
bacteria. Such absorption may take place as the result of gangrene, 
a retained placenta, etc. 
The exanthemata include those diseases which are characterized 
by skin eruptions, such as cow-pox, chicken-pox, small-pox, sheep- 
pox, scarlet fever, etc. For the most part the causal organisms 
for these diseases have not been satisfactorily identified. CHAPTER XIII 
IMMUNITY. GENERAL DISCUSSION 
Immunity.-Immunity is a term used to express relative resist- 
ance to disease. It is defined by Ricketts as follows: " By immun- 
ity we understand that condition in which an individual or a species 
of animals exhibits unusual or complete resistance to an infection 
for which other individuals or other species show a greater or less 
degree of susceptibility." The converse of immunity is suscepti- 
bility, or lack of resistance. 
Resistance by the body to infection by microorganisms is 
due to a considerable number of factors. These may be grouped 
into two classes-the external resistance, due to body coverings 
and protective devices, and internal resistance, due to tissue and 
body humor reactions. 
External resistance to infection is of the greatest practical 
importance. The skin covering the surface of the body is an excel- 
lent and effective barrier against bacterial invasion. The skin 
is constantly sloughing off at the surface and being replaced from 
below. Entrance to the tissues is sometimes effected by micro- 
organisms through the hair-follicles and the sweat-glands; this is, 
however, exceptional. The subcutaneous tissues likewise obstruct 
the inward growth of organisms that have penetrated the skin. 
The mucous membranes constantly secrete mucus, which is as con- 
stantly removed, and the bacteria which have been caught go with 
it. The membranes which line the air-passages catch upon their 
moist surfaces practically all the bacteria that enter with the 
inspired air, and few ever reach the ultimate ramifications of the 
bronchioles, much less the alveoli. The gastric juice of the stomach 
is markedly germicidal. Many, though not all, bacteria which 
enter the body with the food are destroyed there. The intestinal 
juices, particularly the bile, are mildly antiseptic and inhibit the 
growth of many forms, although they are without effect on others, 
among them the so-called normal intestinal bacteria. 
149 150 
VETERINARY BACTERIOLOGY 
Variation of Individuals in Susceptibility to Disease. Pre- 
disposing Factors.-The same individual varies greatly at times 
in his ability to resist infection by bacteria. Age frequently deter- 
mines resistance to certain infections. For example, there are 
diseases, such as diphtheria and whooping-cough, which are 
much more common among children than among adults. Black- 
leg rarely attacks adult cattle. Advancing age seems to bring 
increased resistance to such infections. The mechanism of this 
kind of resistance to infection is not well understood. Hunger 
and thirst reduce the resistance of the body and predispose to 
infection. Exposure to excessive heat or the chilling of the body 
surfaces by cold will also reduce the body resistance so that organ- 
isms that ordinarily cannot produce disease gain a foothold. 
Fatigue has been demonstrated experimentally to render animals 
and man more susceptible to infection. The classic example of 
this diminution of resistance by fatigue is that of the white rat, 
which is normally immune to anthrax, but which, when exhausted 
by work in a treadmill, becomes susceptible to the disease and will 
succumb to infection. 
Types of Immunity.-Immunity may be divided into two types, 
natural and acquired. The former may be subdivided again into 
racial or specific immunity and individual immunity. Acquired 
immunity may be either active or passive. 
Natural immunity is congenital, that is, it is not acquired after 
birth. A racial immunity is one possessed by all the members of 
a group of individuals. Disease frequently cannot be transmitted 
from one species of animal to another, for example, man does not 
acquire many of the diseases of animals, such as hog-cholera and 
dog distemper, and, on the other hand, many diseases of man, such 
as measles, whooping-cough, and typhoid fever, cannot be trans- 
mitted to the lower animals. It is also said that the Algerian alone 
among the breeds of sheep is naturally immune to anthrax. In 
New York City it has been found that the Russian and Polish 
Jews are much more resistant to tuberculosis than are certain other 
races, particularly the Irish and the negroes-at least the death- 
rate among the former due to this disease is much lower. Individ- 
uals are also found who are naturally immune to disease. This re- 
sistance is perhaps more seeming than real in many cases, and has IMMUNITY. GENERAL DISCUSSION 
151 
been induced by mild infections that have resulted in recovery 
without the development of definite symptoms of the disease. 
It is a well-known fact that frequently a small percentage of the 
hogs in a herd which becomes infected with hog-cholera do not 
contract the disease, and inoculation experiments show them to be 
relatively immune. 
Acquired Immunity.-Immunity to a disease may be acquired 
in many different ways. An active acquired immunity is one brought 
about by the development in the tissues of the animal of certain 
antibodies which prevent the growth or destroy or neutralize the 
products of growth of the invading microorganisms. Passive 
acquired immunity is conferred upon the animal by the injection 
of antibodies which have been prepared in another animal. A 
passively immunized animal takes no part in the production of 
the antibodies to which it owes its immunity. 
Active Acquired Immunity.-A development of antibodies 
and the consequent immunization of an animal may be brought 
about by the various methods illustrated in the following out- 
line: 
A. Injection of living microorganisms. 
1. In quantities smaller than the amount needed to produce 
a fatal infection. 
2. Attenuated in various ways- 
(a) By growing upon artificial culture-media. 
(6) By growing at unusual temperatures. 
(c) By heating. 
(d) By growing in the presence of substances inimical to 
growth, such as weak antiseptics. 
(e) By animal passage. 
B. By injection of dead organisms. 
C. By injection of the products of autolytic digestion of or- 
ganisms. 
D. By injection of toxins. 
Immunization by the injection of sublethal doses of pathogenic 
organisms has not been generally practised. With most pathogenic 
organisms it may be demonstrated experimentally that if the 
number of living cells injected be decreased below a certain mini- 
mum, they are no longer capable of producing disease. The method 152 
VETERINARY BACTERIOLOGY 
is dangerous because the minimum may vary with different 
individuals and the animal utilized may in some instances prove 
susceptible and succumb. Usually, however, increasing numbers 
of the organism may be given, and the animal will eventually 
become entirely immune. This method is of more theoretical 
than practical importance, and is rarely used. 
As noted above, microorganisms may be attenuated, that is, 
their ability to produce disease lessened in several ways. Several 
species of bacteria are known gradually to lessen their virulence 
when cultivated for a time upon artificial media; for example, 
the Streptococcus pyogenes, which produces many suppurative in- 
fections, when cultivated upon artificial media, will finally lose 
so much of its virulence that it proves entirely non-pathogenic 
when inoculated into suitable animals. Inoculation of such non- 
virulent types will increase the resistance of the body to the viru- 
lent forms. The converse of this is also true, for the continued 
growth and transfer of many organisms from one animal to another 
may greatly exalt the virulence. It is possible in a given species 
to secure in some cases every gradation from the wholly non- 
pathogenic type to forms that are exceptionally virulent. 
Cultivation of some bacteria at a temperature higher than 
the growth optimum results in a diminution of virulence. The 
anthrax bacillus, whose optimum is 38°, may be cultivated at a 
temperature of 42° for a time, when it loses much of its virulence. 
It may then be used in the form of injections to increase resistance 
to the disease anthrax in animals. The organism which causes 
blackleg in cattle is heated to a temperature just below its thermal 
death-point, and is found to lose many of its pathogenic proper- 
ties, although it still causes the production of immune substances 
when injected into the body. A closely related method is to grow 
bacteria in the presence of mild antiseptics. The anthrax bacillus 
grown in the presence of carbolic acid (1: 600) has been found to 
lose its virulence. The growth of certain microorganisms in the 
body of animals other than the species in which they normally 
produce disease in some cases results in a decrease of virulence 
for the first species. The small-pox organism, for example, is 
grown for a time in the bodies of cattle, and is found to lose much 
of its virulence for man by this means. The injection of dead IMMUNITY. GENERAL DISCUSSION 
153 
microorganisms also results in conferring immunity in much the 
same manner, perhaps, as does the injection of non-virulent 
cultures. The injection of bacteria, whether dead or alive, in an 
effort to increase the immunity of the body, is known as vaccina- 
tion. This is also defined as the production of an infection that 
will run a benign course. Bacterial cultures are sometimes 
allowed to stand until a considerable amount of digestion or 
autolysis has occurred. When the dead or living bacteria are 
filtered out by means of porcelain filters, the material remaining 
in solution or the filtrate may be used for animal injection in an 
effort to confer immunity. In other cases the bacteria are grown 
in solutions where they produce certain specific poisons called 
toxins. These latter are removed by filtration and used to inject 
animals to induce the development of an active immunity. 
Acquired Passive Immunity.-Immunity may be conferred by 
the injection of serum that contains suitable antibodies. These 
may be, as will be seen later, in the nature of antitoxins, bac- 
teriolysins, or opsonins. This method of conferring immunity 
should not be confused with vaccination. The animal passively 
immunized takes absolutely no part in the development of its 
immunity. 
Theories of Immunity.-Four principal theories of immunity 
have been held since the acceptance of the germ theory of disease. 
The first two proposed are now of historic interest only, but the 
other two are well founded on fact and are generally accepted 
at the present time. When first proposed, these latter were sup- 
posed to be antagonistic, but as subsequently modified, they 
have been found to supplement each other, some facts being 
explained by the one and some by the other. These theories 
are worthy of brief consideration and comparison. 
Theory of Exhaustion.-It was noted by early laboratory 
workers that bacteria, yeasts, and molds would grow rapidly 
when first planted upon a favorable medium, then the rate of 
growth would become slower, and finally cease. It was concluded 
naturally that growth stopped because all of certain of the nutrients 
needed were exhausted. This theory was applied to the growth of 
microorganisms in the body, and it was believed that immunity 
was established when certain requisite food materials were ex- 154 
VETERINARY BACTERIOLOGY 
hausted and the organism in consequence could grow no longer. 
This theory was soon disproved. It was shown, for example, that 
the Corynebacterium diphtherice would grow luxuriantly on steril- 
ized blood taken from a person immune to diphtheria. Chemical 
analyses also showed that not all nutrients were exhausted from 
the medium in the culture tube when the organism ceased 
growing. 
Noxious Retention Theory.-Further study revealed the fact 
that organisms ceased growing in a culture solution because they 
produced substances deleterious to themselves. The organism 
which sours milk, for example, produces acid until the concen- 
tration is so great that it can no longer develop. The same is 
true of yeasts in the production of alcohol, and of bacteria in the 
transformation of alcohol to acetic acid. The nature of the 
noxious material is known in very few cases. A logical explana- 
tion of immunity seemed to be that something of the same 
kind occurred in acquired immunity; that the organisms devel- 
oped in the body until they produced so much material inimical 
to their own growth that multiplication would cease, and the 
individual would thereafter be immune. This theory was dis- 
credited by subsequent workers, who proved that the substances 
that prevented bacterial growth were produced by the body and 
not by the bacteria. 
Metchnikoff's Theory of Phagocytosis.-The theory was 
advanced by Metchnikoff and his students that certain of the 
body-cells, particularly the leukocytes, would take up, digest, and 
destroy bacteria. Immunization accordingly consisted in a kind 
of stimulation or training of the leukocytes to destroy the patho- 
genic organisms. Cells capable of destroying microorganisms in 
this manner he called phagocytes (Gr. phagein, to eat; kytos, cell). 
Certain of Metchnikoff's ideas have not borne the test of time, but 
in the main his theory still is used to account for immunity of 
certain types. 
Ehrlich's Humoral Theory.-Ehrlich has advanced the theory 
that immunity is due to substances present in the body humors 
which antagonize the growth and development of pathogenic 
organisms or are capable of neutralizing their products. Such 
substances capable of conferring immunity he calls antibodies. 
and to their production by the body-cells he attributes the devel- IMMUNITY. GENERAL DISCUSSION 
155 
opment of immunity. This theory has been tested in a very 
great variety of ways, and seems to explain better than any other 
most of the facts known relative to immunity. 
Duration of Immunity.-Recovery from certain diseases, 
such as small-pox, confers a lasting immunity; from others, such as 
pneumonia, a temporary immunity, and in still others the im- 
munity disappears immediately upon recovery, as in influenza. 
In some cases recovery from an attack seems actually to predis- 
pose to a recurrence, as in erysipelas. This last probably means 
simply the complete or relatively complete disappearance of im- 
munity from a peculiarly susceptible individual. The duration 
of immunity may depend in some cases upon the type of anti- 
bodies produced, although this is certainly not always the case. 
Probably the antibodies, particularly those introduced in passive 
immunization, are eliminated in some of the secretions and ex- 
cretions, or they may, in some cases, be easily destroyed. 
Antigens and Antibodies.-It has been found experimentally 
that injections of many substances besides bacteria and their 
products cause reactions on the part of the tissues, with conse- 
quent production of antibodies. These injected substances are 
called antigens. An antigen is, therefore, that substance which, 
when introduced into the body, stimulates the tissues to the pro- 
duction of antibodies. An antibody may be more accurately de- 
fined as any substance present in the serum which is capable of 
neutralizing, antagonizing, precipitating, agglutinating, or dissolv- 
ing the substance (antigen) which has induced the formation of such 
antibody. For example, the toxin of the diphtheria bacillus, when 
injected in non-lethal doses, induces the production of antitoxin 
by the tissues: the toxin is the antigen, the antitoxin is the anti- 
body. Similarly, egg-white injected into an animal would be 
termed an antigen and the precipitating substance produced as 
a consequence in the blood-serum is termed the antibody. 
Antibodies as Factors in Acquired Immunity.-The somatic 
reactions to the presence of antigens are now generally considered 
of primary importance in acquired immunity. Certain bacterial 
poisons called toxins cause the production by the animal tissues 
of the antibody called antitoxin, which will neutralize the toxin. 
The presence of certain bacteria in the body causes the production 156 
VETERINARY BACTERIOLOGY 
of bacteriolysins, substances which will dissolve or destroy bacteria. 
Opsonins (Gr. opsonein, to prepare for eating) are antibodies 
which will unite with the bacteria and enable the phagocytes 
to take them up and destroy them. Acquired immunity may, 
therefore, be said to be either antitoxic, bacteriolytic (antibacterial), 
or opsonic, or a combination of any or all of these types. Three 
other types of body reactions are also generally considered in a 
discussion of immunity. Although they probably in no instance 
actually account for immunity, they are found very useful in 
diagnosis, and their consideration is quite essential to a full dis- 
cussion of theories of immunity. The blood-serum of individuals 
suffering from certain infections, particularly bacterial, acquires 
the property of agglutinating or clumping these organisms. The 
blood-serum from a horse having glanders when dropped into a 
culture of the glanders bacillus will cause the bacteria to clump 
together and settle out, leaving the medium clear. The phenom- 
enon of agglutination is used in the diagnosis of this and other 
diseases. A somewhat similar body reaction is provoked by the 
injection of soluble proteins derived from some other species of 
animal (or plant) into the body of an animal. The serum of such 
an animal acquires the property of precipitating the corresponding 
protein when mixed with it in a test-tube. This phenomenon 
of precipitation is made use of in the differentiation of many kinds 
of proteins. The presence of certain substances in the blood, 
usually proteins derived from any foreign source, may result in 
the sensitization of the body to such, rather than immunity. This 
phenomenon is known as anaphylaxis (Gr. an, against; phylasco, 
to guard) and has led to an explanation of many otherwise obscure 
pathological and bacteriological facts. CHAPTER XIV 
ANTITOXINS AND RELATED ANTIBODIES 
(Antibodies of Ehrlich's First Order) 
Toxins.-The word toxin has been used with a considerable 
variety of meanings. A substance is said to be toxic when it 
brings about an abnormal condition when introduced into the 
body. The discovery that certain microorganisms, particularly 
bacteria, produced their harmful effects by means of the poisons 
which they excreted led to the use of the term toxin to include all 
poisons produced by bacteria. Further study demonstrated that 
these bacterial poisons differed considerably in their method 
of action and in other characters. Ehrlich first clearly differen- 
tiated the bacterial poisons and used the name toxin to indicate 
a definite type. The name endotoxin is used to designate certain 
poisonous substances that are contained within the protoplasm 
of the cell, and are not excreted as are the true toxins. Some 
authors have included all these poisonous substances produced 
by bacteria under the name toxine, and recognize toxin as used in 
Ehrlich's sense. 
Characteristics of a Toxin (Ehrlich).-According to Ehrlich, 
the toxins constitute a group of substances having the following 
characteristics: 
1. Most true toxins are labile, that is, they are usually easily 
destroyed by heat, by acids, by exposure to light and air. 
2. The chemical nature of the toxins, with one possible excep- 
tion, is not understood. Beyond the fact that they are organic 
in origin and usually give some of the protein reactions, little is 
known of their chemistry. 
3. Biological tests (i. e., animal injections) have been found 
to be the only tests by which the toxins may be recognized and 
studied. These animal injections, as has been before stated, 
are quite as essential in determining the character and strength 
157 158 
VETERINARY BACTERIOLOGY 
of a toxin as are indicators to the chemist in his study of end- 
reactions. 
4. Toxins act upon the body by combining chemically with 
definite cells and tissues. This union is not effected at once 
in all cases. The evidences of damage, with the clinical symp- 
toms of poisoning, do not appear until after the lapse of a period 
of incubation. The length of this period varies with different 
toxins-with some of the snake venoms it is very short, in other 
toxins it is a matter of hours, or even days. 
5. The injection of non-lethal doses of toxin into a .suitable 
animal causes the tissues to react and to produce antitoxin, 
which will neutralize the toxin, and result in immunization. 
Toxins may be differentiated from most other poisonous sub- 
stances with which they may be confused by reference to the 
preceding. Poisonous alkaloids, such as strychnin, for example, 
do not cause the production of antibodies. Immunization against 
a toxin is, therefore, not to be confused with drug habituation. 
Sources of Toxins.-Toxins have been found to be produced 
by a considerable number of plants and animals. Certain of the 
flowering plants form powerful toxins, such as ricin in the castor- 
oil bean (Ricinus communis and R. zanzibarensis), abrin from the 
jequirity bean (Abrus precatorius), and robin from the bark of the 
locust (Robinia sp.). Among the fungi, certain poisonous 
mushrooms (or "toadstools"), as the Amanita, have been shown 
to contain toxins. Certain molds are stated to produce toxins, 
particularly Aspergillus. Toxins have been demonstrated in the 
animal kingdom in the venom of snakes, scorpions, and spiders, 
in the skin of certain reptiles, in the blood of the eel, and of cer- 
tain fish. A few species of bacteria have been shown to produce 
toxins, but in several cases the amount and character of the 
toxin are relatively unimportant. The bacteria which have 
been found to form appreciable amounts of toxin, and 
which produce lesions of the body tissues through the action of 
these toxins, are relatively few. The more important are the 
following: 
Corynebacterium diphtherice, the cause of diphtheria. 
Clostridium tetani, the cause of tetanus or lockjaw. ANTITOXINS AND RELATED ANTIBODIES 
159 
Clostridium botulinum, the cause of certain cases of botulism 
or meat-poisoning. 
The organisms in which true toxins have been demonstrated, 
but in which the toxin production does not seem to account for 
the lesions of the disease, are: 
Pseudomonas pyocyanea, associated with suppurative pro- 
cesses. 
Clostridium chauvcei, the cause of blackleg. 
Staphylococcus aureus, associated with suppurative processes. 
Staphylococcus albus, associated with suppurative processes. 
It should again be emphasized that the above list of bacteria 
does not include all those which may produce poisoning, but 
does include the most important known to produce true toxins. 
Many other species are known to produce endotoxins. 
Specificity of Toxins.-Toxins must combine with the cells 
or tissues of the body in order to injure them. Toxins do not all 
attack the same tissue, but show a selective action. In some cases 
a number of tissues may be injured, in others the damage is 
limited quite strictly to one type. The toxin produced by the 
bacillus of tetanus attacks the cells of the central nervous system 
and is called a neurotoxin. One of the toxins commonly present 
in snake venom destroys red blood-cells (hemotoxin). Probably 
some animals owe their immunity to certain toxins to the fact 
that some of the less important or non-vital body-tissues will 
combine with the toxin and prevent its union with more vital 
portions. 
Antitoxins.-Antitoxins are antibodies produced by the tissues 
of the body as a result of injection or presence of toxins. The 
fact of antitoxin production may be readily demonstrated by mix- 
ing the serum of an immune animal with toxin and injecting the 
mixture into a suitable animal. The normal toxic action will 
be found to be inhibited by the antitoxin of the serum. The 
most generally accepted and valuable of the explanations of the 
production of antitoxins by the body is that offered by Ehrlich, 
based upon his theory of cell nutrition, the lateral chain or side- 
chain theory of immunity. 
Ehrlich's Theory of Cell Nutrition.-Several explanations have 160 
VETERINARY BACTERIOLOGY 
been offered by physiologists for the phenomenon of cell nutrition. 
Food substances carried to the cell by the blood must pass through 
the vessel and cell-walls and be anchored there if they are to be 
used in any of the processes of cell metabolism. According to 
Ehrlich, the protoplasm must be made up of molecules having an 
affinity for a great variety of food materials. These molecules 
he conceives to be made up of a central portion surrounded by 
atomic groups which unite with certain food molecules and bind 
them to the cell. These atomic groups have affinity for certain 
food substances, and, therefore, are differently constituted. These 
atomic groups he calls side-chains or, better, cell receptors. The 
character of these receptors may be illustrated by a chemical 
analogy. Benzene has the following formula: 
H H 
c c 
H C 
C H 
c c 
H H 
Any one of these hydrogens may be substituted by some element 
or group. Suppose one such to be replaced by the carboxyl 
group (COOH), another by the amino group (NH2), another by 
the aldehyd group (CHO), and still another by a hydroxyl 
group (OH). Such a hypothetical compound might be illustrated 
as follows: 
H COOH 
c c 
OH C 
c nh2 
c c 
H 
CHO 
There are several possible ways in which such a compound 
might react. Should an alkali be brought into contact with it, 
the base would be taken up by the carboxyl atom group. Acids 
would be bound by the amino group. Other substances would be 
bound by other atom groups. The cell receptors must be con- ANTITOXINS AND RELATED ANTIBODIES 
161 
sidered in this manner, as being of different types, each one capable 
of uniting with some food substance-probably only one. To take 
up the variety of substances necessary for cell life and activity, 
it is evidently necessary that there should be a multiplicity of 
these receptors, and their action must be considered as very specific. 
It is found convenient to represent these cell receptors in a 
diagrammatic form. Such is scarcely necessary in a consideration 
of antitoxin, but will be found very helpful in a discussion of the 
more complex antibodies. 
Ehrlich's Theory of Antitoxin Production.--As has been stated 
previously, toxins are believed to combine with the tissue-cells 
before the latter can be injured. This union of toxin with the 
cell takes place through the medium of the cell receptors, some of 
which are thus diverted from their normal functions. As a result, 
if the cell is not too seriously injured by the toxin, it or the neigh- 
boring cells produce an increased number of receptors of the type 
thus used. This is in accordance with the general hypothesis 
enunciated by Weigert, that injury or irritation always results in 
an overgrowth of tissue-a hypercompensation. For example, 
rubbing the skin will produce a callus, leaky heart valves cause 
hypertrophy of the heart, and the cicatricial tissue of a newly 
healed wound is generally greater in amount than the tissue it 
replaces. These receptors are, therefore, produced in great 
numbers, and many are eventually displaced and escape into the 
cell-plasma and finally into the blood-stream. These freed cell 
receptors still retain their affinity for the toxin and constitute the 
antitoxin molecules of the serum. For each of the statements just 
made there seems to be a good proof. That the toxin actually 
unites with the body-cells has been shown by experiment, for 
example, the 'brain tissue of an animal mixed with tetanus toxin 
will absorb the latter and remove it from the solution, so that 
it is without effect when injected into animals. That there is an 
actual increase in the number of cell receptors may be shown by 
injecting a small amount of toxin into an animal, and before anti- 
toxins have appeared in the blood, injecting more toxin. The 
response to the second injection will be quicker than to the first, 
and the animal will succumb to what is not ordinarily a fatal 
dose. This indicates an increased power of fixation for the toxin, 162 
VETERINARY BACTERIOLOGY 
i. e., an increase in the number of the receptors. The appearance 
and increase in the freed cell receptors or antitoxins in the blood 
shows that these receptors are thrown off in large numbers. The 
antitoxin in the serum unites with the toxin probably in much the 
same manner as an acid neutralizes an alkali. The principal 
difference is that in carrying out the test, animal inoculations 
must take the place of the indicator of the chemist. The union 
between the toxin and antitoxin has been shown to obey the gen- 
eral laws of chemical union. 
Constitution of the Toxin.-Toxins are easily destroyed by 
heat, chemicals, light, and air. It will be shown later that the 
loss of toxicity does not result from a total destruction of the 
toxin molecule, for it still is able to unite with the antitoxin. 
It is evident that the toxin molecule is made up of two parts- 
a thermostabile portion, which unites with cell receptors, either 
in the cell or free as antitoxins, called the haptophore, or binding 
group, and a thermolabile group, called the toxophore, which causes 
the cell injury after union by means of the haptophore has taken 
place. When the toxophore group of a toxin has been destroyed, 
that which remains is called a toxoid. That toxoids exist may be 
demonstrated in two ways. The injection of a toxin solution 
that has been heated to 56 0 for half an hour into a suitable animal 
does not result in the development of symptoms of poisoning, but 
does cause the production of antitoxin; in other words, the toxoids 
retain the ability to unite with the cell receptors and to bring 
about their increase and elimination from the cell. An antitoxic 
serum may be mixed in suitable amounts with a solution of 
heated toxin (toxoid), and after a time mixed with virulent toxin, 
and the latter will be found to remain uncombined. The anti- 
toxin is completely neutralized by the toxoid, and the toxin 
added subsequently finds no antitoxin free with which it can 
unite. 
Constitution of Antitoxin.--Some antitoxins are more stable 
than the corresponding toxin. However, they are easily destroyed 
by a temperature of 60° if sufficiently prolonged. There is no 
reason to suppose that the antitoxic molecule is made up of more 
than one group. Inasmuch as it has a binding group, this may be 
called a haptophore. It has not as yet proved possible to separate ANTITOXINS AND RELATED ANTIBODIES 
163 
antitoxins from serum globulins; it is inferred, therefore, that they 
are similarly constituted. 
Diagrammatic Representation of Toxin and Antitoxins.-The 
preceding facts may in large part be recorded by diagrams. These 
are, of course, arbitrary in shape and appearance, but are helpful 
in an understanding of the reaction. The diagrams commonly 
used for this purpose are given in Fig. 65. 
Preferential Union of Toxins with Body-cells.-The union of 
antitoxin with toxin occurs apparently as readily within the 
Fig. 65.-Diagrammatic representation of toxins and of antitoxin pro- 
duction-1, Toxin molecule: a, Haptophore; b, toxophore. 2. Toxoid, a 
toxin molecule that has lost its toxophore. 3, Molecule of cell protoplasm 
showing the union of the toxin molecule: a, Free toxin; b, toxin attached to 
a cell receptor c; d, other cell receptors. 4 illustrates the overproduction of 
receptors by the cell and their elimination (6) into the blood-stream as anti- 
toxin molecules (c). 5, Neutralization or union of antitoxin with toxoid 
and toxin. 
body as without. When toxins are injected and antitoxins are 
present in the circulation, the latter will commonly unite with 
the former and prevent union with the body-cells. In some 
exceptional conditions, however, this is shown not to occur; for 
some reason the affinity of cell receptors still united to the cell 
seems to be greater than those free (antitoxin). In other words, 
the tissues seem to become hypersensitive to the presence of the 
toxin, so that the antitoxin no longer protects. Tissue immunity 164 
VETERINARY BACTERIOLOGY 
is/ therefore, not always the same as antitoxin immunity. This 
hypersensitiveness is of unusual occurrence. An animal that is 
being immunized against tetanus toxin by injections of increasing 
amounts may suddenly and unexpectedly show a marked reaction, 
or may even succumb, when additional amounts are injected, 
even though antitoxin may be demonstrated in considerable 
quantities in the blood. It is evident that, although this phe- 
nomenon is of infrequent occurrence, it is of some little importance. 
Possibly this may be related to the reactions to be described later 
under the heading of Anaphylaxis. 
Antitoxins of Commercial Importance.-Antitoxins specific 
for the venom of certain snakes and for diphtheria and tetanus 
toxins are prepared commercially. The manufacture and 
standardization of these antitoxins are of importance to the 
veterinarian for several reasons. The theory of immunity has 
been largely developed through a study of diphtheria toxins and 
antitoxins. The larger animals, such as the horse and goat, are 
generally used in the commercial manufacture of antitoxin. 
The tetanus antitoxin is used extensively in veterinary practice. 
A brief consideration of the production of diphtheria and tetanus 
toxin and antitoxin is, therefore, advisable. 
Manufacture of Diphtheria Toxin and Antitoxin.-Diphtheria 
toxin is prepared by growing the diphtheria bacillus in suitable 
broth. A sugar-free broth is prepared as described in Chapter 
VII. This is titrated and the reaction adjusted accurately to 
+ 0.5. It is then placed in flat-bottomed toxin flasks in a layer a 
few centimeters in thickness, and autoclaved. To each flask is then 
added sufficient sterile dextrose solution to make a 0.2 per cent, 
dextrose broth. It is found in experience that different strains of 
the diphtheria bacilli may vary considerably in toxin production. 
The strain used most frequently in the United States is the one 
known as the Park-Williams. The organism is grown in broth 
tubes, where it forms a film over the surface. Portions of this film 
are transferred carefully to the surface of the broth in the flasks and 
incubated at 37° for eight days. Microscopic examination of the 
culture should show it to be uncontaminated. To destroy the 
bacteria and prevent possible infection of those handling the 
material 5 c.c. of phenol or similar antiseptic is then added to ANTITOXINS AND RELATED ANTIBODIES 
165 
each liter. In some cases it is filtered through a porcelain filter, 
which will remove the bodies of the bacteria, but not the soluble 
toxins. The amount of toxin present in the solution must be 
next determined for two reasons: first, to insure the presence of 
toxins in sufficient concentration for efficient immunization, and, 
second, to determine the amount that may be injected into the 
horse without serious injury. The amount of toxin that, when 
injected subcutaneously, will kill a guinea-pig weighing 250 gm. 
in three days is called the minimum lethal dose for the guinea-pig 
(abbreviated M. L. D.). The broth should contain at least 1000 
Fig. 66.-Apparatus for the injection of considerable quantities of toxin 
into the horse: a, Graduated cylinder closed by a rubber cork, b. Air is 
pumped into the constant pressure bulb e, and from this passes into the cylinder. 
The toxin is forced out through the needle at i (Levaditi). 
M. L. D. per cubic centimeter. The horse is most commonly 
employed for the production of the antitoxin. Care is used that 
the animal is fairly vigorous and entirely free from any infectious 
disease. Injections of 100 M. L. D. are made subcutaneously, 
or, better yet, larger amounts, neutralized by antitoxin already 
prepared, or attenuated by mixing with Lugol's solution, are 
first used. The animal responds by fever and swelling at point 
of injection and by other minor symptoms. After a lapse of 
several days, or when the horse has recovered, a second injec- 166 
VETERINARY BACTERIOLOGY 
tion of a larger amount is given. Increasing doses of the toxin 
are injected at intervals until as much as 500 c.c. of the toxin 
may be administered at one time. Not all horses produce 
enough antitoxin in their blood to be commercially valuable; 
hence the antitoxin content is usually determined some time be- 
fore the process of immunization is complete. When the animal's 
blood contains the maximum amount of antitoxin, it is drawn 
by a sterile trocar from the right jugular vein into wide-mouthed 
sterile jars (Fig. 68). Usually a little less than one liter of blood 
for every hundred pounds weight of the horse is removed, as this 
Fig. 67.-Injection of a horse with toxin (Levaditi). 
amount may be withdrawn without appreciable injury. After 
a rest the horse may again be injected and bled. 
The jars of blood are allowed to stand until the clot has shrunk 
and the clear, straw-colored serum has separated. This contains 
the antitoxin and is the serum antidiphtheriticum of the Pharma- 
copeia. It must now be standardized, that is, the amount of 
antitoxin per cubic centimeter determined. Several methods 
of determining the potency of the antitoxin have been pro- 
posed. Inasmuch as the unit formulated by Ehrlich has been 
generally adopted (except by the French) its development will 
be briefly traced. Behring first proposed as an antitoxic unit, ANTITOXINS AND RELATED ANTIBODIES 
167 
the least amount of antitoxin which, when mixed with 100 M. L. 
D. of the toxin, would prevent a 250-gm. guinea-pig injected with 
the mixture from dying within four days. This implied the keep- 
ing of the toxin of a certain strength for the standardization of 
antitoxin. The toxin was found to be unstable, and the same 
toxin would at different times yield different results. Furthermore, 
antitoxins are neutralized by toxoids as well as toxins. The toxin 
cannot be preserved for long periods without deterioration, as 
would be necessitated if used as a government standard. Ehrlich, 
therefore, made use of a toxin which he had studied in his labora- 
tory to standardize a large quantity of serum. This serum he 
dried in a current of warm air in a partial vacuum. As a result, 
Fig. 68.-A trocar inserted into the jugular vein of the horse. The com- 
pressor causes a noticeable engorgement of the vessel (Kretz in Kraus and 
Levaditi). 
he secured a considerable amount of serum in the form of dried 
scales. The number of immunity units per gram of this dried 
material was very accurately determined. Exactly equal amounts 
by weight of this standard serum were placed in each of a large 
number of special tubes. The serum was placed in one arm 
and phosphorus pentoxid (P2O5), an active dehydrating agent, 
in the other. The air was then exhausted as completely as 
possible by an air-pump and the tubes sealed. They then were 
placed in a dark refrigerator, where a constant temperature was 
maintained. The antitoxin under these conditions was found 
to retain its potency undiminished for a long period. Ehrlich 168 
VETERINARY BACTERIOLOGY 
placed 1700 units in each tube. Each month a tube is opened 
and its content of serum dissolved in 1700 c.c. of a mixture of 
water and glycerin. Each cubic centimeter, therefore, contains one 
immunity unit of antitoxin. A careful study of the toxin which 
Ehrlich had used in preparing this standard showed him that it 
contained, in addition to the 100 M. L. D. of toxin, an equal 
amount of toxoid. Theoretically, therefore, this immunity unit 
prepared by him contains sufficient antitoxin to neutralize 200 
M. L. D. of a pure toxin. In view of the fact that all antitoxin 
Fig. 69.-A tube used for the preservation of antitoxin by the Hygienic 
Laboratory of the Public Health and Marine Hospital Service: a, Serum scales, 
or antitoxin; b, phosphorus pentoxid, an active dehydrating agent (M. J. 
Rosenau, Bulletin No. 21, Hygienic Laboratory). 
in this country is now standardized with reference to this U. I. 
of Ehrlich, the immunity unit for diphtheria antitoxin has been 
redefined as an amount of antitoxin equivalent to that contained 
in 1 c.c. of solution when the contents of the tubes prepared by 
Ehrlich are dissolved in 1700 c.c. of water. In the United States 
a similar set of tubes (Fig. 69) has been prepared by the. Hygienic 
Laboratory of the Public Health and Marine Hospital Service, 
and the standard serum is sent from that laboratory to the manu- 
facturers. 
The old antitoxin cannot be used directly to determine the 
potency of new antitoxin, but a lot of toxin must first be standard- 
ized. It is necessary to express the strength of the toxin in terms ANTITOXINS AND RELATED ANTIBODIES 
169 
of the standard antitoxin. Toxin is used which has been pre- 
served until the first rapid transformation of toxin to toxoid has 
ceased, and it has, therefore, become relatively stable. A series 
of syringe tubes is prepared (Fig. 70), each one containing one 
standard immunity unit of antitoxin, and to these are added 
varying amounts of the toxin to be tested; each is then injected 
into a 250-gm. guinea-pig. The amount of antitoxin will be more 
than sufficient to neutralize the toxin in some cases, and the 
animals injected will show no ill effects; in other cases the toxin 
will be in excess, and the animals will die. The amount of toxin 
Fig. 70.-Battery of syringe tubes for testing the potency of toxin and anti- 
toxin (Madsen). 
which, when thus mixed with 1 unit of antitoxin, will kill a 250-gm. 
guinea-pig in just four days is called the L+ dose. The amount of 
the toxin solution just necessary to neutralize an immunity unit, as 
evidenced by the almost complete lack of tissue reaction at the 
site of injection, is called the LO (limit zero) dose. In a solution 
containing toxins only (no toxoids) the difference between the L + 
and the LO dose would be 1 M. L. D. of the toxin, but such toxoid- 
free solutions cannot be obtained; hence the difference between 
the two is greater. The only reason that the LO dose is determined 
in practice is that a toxin solution, in which the L + and LO doses 
differ widely, is not suitable for carrying out the test and should 
be discarded. When the L+ dose of the toxin has been satis- 
factorily determined, it may then be used to determine the potency 170 
VETERINARY BACTERIOLOGY 
of fresh antitoxin. A series of syringe tubes, each containing one 
L+ dose of the toxin, is arranged, and the varying amounts of 
the antitoxin serum to be tested are added. The amount of 
serum which will prevent a 250-gm. guinea-pig from dying in 
less than four days contains 1 immunity unit. 
According to Ehrlich, diphtheria toxin is in reality made up of 
two poisons-the true toxin and toxone. The latter he holds to 
account for the paralyses that are frequent sequelae in diphtheria. 
The same antitoxin neutralizes both the toxin and the toxone. 
This toxone is of some theoretic and practical importance in the 
standardization of the toxin and the antitoxin. 
The amount of antitoxin present varies greatly in the serum 
produced by different horses. One which contains less than 
250 U. I. per cubic centimeter is rarely used. The greater the 
concentration, the more valuable is the serum for prophylaxis and 
cure. Many efforts to concentrate diphtheria antitoxin have been 
made, all based upon the fact that the blood-serum is a mixture of 
various proteins, and that the antitoxin seems to be inseparably 
bound up with certain ones only of these. The removal of the 
proteins having no antitoxic value results in a considerable con- 
centration of the antitoxin-holding proteins. Several methods 
have been devised for this purpose. All are based upon differences 
in solubility or coagulability of the various serum constituents. 
The methods of Gibson and of Banzhaf deserve mention. The 
former precipitates the proteins of the serum by the addition of an 
equal amount of a saturated solution of ammonium sulphate. 
This throws down the antitoxic and some other fractions of the 
serum, but does not precipitate certain of the non-antitoxic albu- 
mins. These are filtered out, the precipitate is dissolved in water 
to its original volume, and is again precipitated by ammonium sul- 
phate. This precipitate, when filtered, is relatively free from al- 
bumins. It is then stirred into a saturated solution of sodium 
ehlorid, which dissolves the pseudoglobulins with the antitoxin. 
The insoluble portions are filtered out and the clear filtrate acidified 
by the addition of 0.25 per cent, of 80 per cent, acetic acid. The 
precipitate which is thrown down is collected over hard filters, par- 
tially dried by pressure with bibulous filter-paper, and placed in a 
parchment bag for dialysis in running water. The acid is neu- ANTITOXINS AND RELATED ANTIBODIES 
171 
tralized by the addition of sodium carbonate, and dialysis is con- 
tinued until the soluble salts have practically disappeared. If 
care is used, the antitoxin will be found to be dissolved in a much 
smaller quantity of water than originally present in the serum. 
The concentration may be from two to three and one-half times 
the original. 
Banzhaf makes use of the various coagulation temperatures 
of the serum constituents in effecting their separation. The 
albumins and part of the non-antitoxic globulins are precipitated 
by heating for twenty-four hours at 58°. Sodium chlorid crystals 
are added to saturation, and much of the remaining globulin, 
transformed by heat, is precipitated, leaving the pseudoglobulins 
Fig. 71.-One type of filtration apparatus used for serum: a, Filter; b, 
test-tube within a filter flask from which the air is partially exhausted by 
the vacuum pump at d (Weidanz). 
and the associated antitoxin in solution. The clear filtrate is 
acidified with acetic acid, and the precipitate prepared as in the 
preceding method. By this method it is claimed a concentration 
of several times, the original may be obtained. 
The antitoxic serum is in all cases filtered through sterile un- 
glazed porcelain, and, after the addition of a small amount of 
preservative, placed in sterile containers and sealed. 
Preparation of Tetanus Toxin and Antitoxin.-The tetanus 
toxin is prepared by growing Clostridium tetani in broth under 
anaerobic conditions. This may be accomplished by the use of a 172 
VETERINARY BACTERIOLOGY 
hydrogen atmosphere, but more easily by covering the medium 
with a layer of paraffin or other neutral oil. The methods of pre- 
paring the toxin for use and of manufacture of the antitoxin do 
not differ materially from those used in the production of diph- 
theria antitoxin. 
Unlike diphtheria toxin, the tetanus toxin may be dried and 
preserved indefinitely without deterioration. It is, therefore, the 
toxin and not the antitoxin which is sent out from the Hygienic 
Fig. 72.-A filtration apparatus after Uhlenhuth and Weidanz. 
Laboratory to the serum laboratories for the purpose of standard- 
ization in the United States. A standard toxin has been prepared 
at this laboratory, and its M. L. D. for a 350-gm. guinea-pig care- 
fully determined. This is sent out in dried form, and is diluted 
before use, so that each cubic centimeter contains 100 M. L. D. of 
the toxin for a 350-gm. guinea-pig. The immunity unit is defined 
as follows: "The immunity unit for measuring the strength of ANTITOXINS AND RELATED ANTIBODIES 
173 
tetanus antitoxin shall be ten times the least quantity of antitetanic 
serum necessary to save the life of a 350-gm. guinea-pig for ninety- 
six hours, against the official test-dose of a standard toxin furnished 
by the Hygienic Laboratory of the Public Health and Marine 
Hospital Service." 1 
Another statement is that one-tenth of a unit, mixed with 
100 M. L. D. of the standard toxin, contains " just enough free 
poison in the mixture to kill the guinea-pig in four days after 
subcutaneous injection." The amount of toxoid has not been 
accurately determined in this test-toxin used; therefore, the stand- 
ardization test must in all cases be in terms of the Hygienic Labora- 
tory toxin, no other sample of toxin being suitable. The test-dose 
is called the L + dose, as in the diphtheria toxin. 
Preparation of Other Toxins and Antitoxins.-As has before 
been stated, antitoxins have been prepared for a large number of 
toxins. The two already discussed are by far of the greatest im- 
portance commercially. In the development of theories of im- 
munity considerable use has been made of antiricin and antiabrin. 
An antitoxin for pollen (called pollantin) has been used to some 
extent in hay-fever. Antitoxins against snake venom may be pur- 
chased upon the market. They are of considerable importance 
in certain tropical countries, particularly India, where poisonous 
snakes abound. 
1 Treasury Dept., Circular No. 61,''Oct. 25, 1907. CHAPTER XV 
AGGLUTINATION AND PRECIPITATION 
(Antibodies of Ehrlich's Second Order) 
Gruber, in 1896, discovered that the blood of animals immun- 
ized against Bacillus typhosus, or the blood of patients having the 
disease, when added to a liquid culture of the organism, caused 
the bacteria to cease moving and to clump together. This 
phenomenon has been named agglutination. Later it was found 
that the use of protein substances as antigens caused the pro- 
duction in the body of substances which, when mixed with the 
protein in solution, would form a precipitate. This is known as 
the precipitation phenomenon. The antibody responsible for 
agglutination is called an agglutinin; for precipitation, a precipitin. 
Differentiation of Precipitation and Agglutination.-The dis- 
tinction between agglutination and precipitation may be stated as 
follows: Agglutination occurs when the antigen is in suspension 
in the form of individual cells or finely divided particles. Pre- 
cipitation occurs when the antigen is a colloid in solution. 
Agglutination.-Agglutinins may be grouped into two classes, 
normal and immune. A normal agglutinin is one present in the 
body without any infection or systematic immunization. An 
immune agglutinin is one that is developed as a result of the pres- 
ence of an organism or its products in the body. There is no 
reason to suppose that the normal and immune agglutinins differ 
from each other in any essential particular. It is possible that all 
normal agglutinins are in reality produced as a result of an unde- 
tected infection or to the presence of the so-called group agglu- 
tinins to be considered later. 
The agglutination reaction is said to be specific; that is, the 
agglutinin will agglutinate, in general, only the homologous 
organism. The term homologous is used to indicate the relation- 
174 AGGLUTINATION AND PRECIPITATION 
175 
ship between an antigen and its specific antibody. The serum 
of a glandered animal is homologous for the glanders bacillus, but 
is heterologous for the typhoid bacillus. 
Agglutinins are formed by the body for most foreign cells which 
may enter or be injected. Red blood-cells, other body-cells, pro- 
tozoa or bacteria, may be the antigens which provoke agglutinin 
production. Under the right conditions the clumping will occur 
whether the cells be living or dead, motile or non-motile. 
Agglutinogen.-The antigen which causes the body to react and 
produce agglutinins is called an agglutinogen. It is evident that 
the agglutinogen is not the cell used for injection, but some sub- 
stance produced by it. A culture of Bacillus typhosus in broth 
may be passed through a porcelain filter, and the sterile filtrate 
will still cause agglutinin production when injected into the animal 
body. The agglutinogen is either something thrown off by the 
antigenic cell in the process of growth, or formed as a result of 
autolytic disintegration and digestion. Evidently some con- 
stituent of the cell excites the production of the antibodies or 
agglutinins, and these, therefore, unite with the corresponding 
material in the bacterial or other cell. 
Ehrlich's Theory of Agglutinin Production.-.According to 
Ehrlich, the agglutinogen unites with the receptors of the body- 
cells, which are diverted in this way from their normal function. 
As a result, there is an overproduction of the receptors and they 
are freed as agglutinins. These freed receptors or agglutinins 
differ in several ways from the antitoxins, for they not only com- 
bine with the antigen, but they bring about certain changes in it. 
Such receptors, to differentiate them from antitoxins and similar 
antibodies (receptors of the first order), are termed receptors of 
the second order. 
Constitution of the Agglutinin.-The agglutinin may be shown 
to consist of two portions-a binding group and an agglutinating 
group. The presence of the binding group, or haptophore, may be 
shown by mixing bacteria with a serum containing the specific 
agglutinin, and centrifuging. The supernatant liquid will be 
found to have lost its agglutinating power--that is, the agglutinins 
will all have united with the bacteria first added, and will be removed 
thereby from solution. The agglutinating group of the agglutinin 176 
VETERINARY BACTERIOLOGY 
is called the agglutinophore, the zymophore, or the zymotoxic group. 
This group is unstable, and may be destroyed by heating to a tem- 
perature of 60° to 75° and by acids and alkalis. It changes with 
age slowly. An agglutinin which has lost its zymotoxic group is 
called an agglutinoid. The agglutinoid still retains the capacity 
to unite with the antigen, but has lost the ability to act upon it. 
The presence of agglutinoids may be demonstrated by mixing a 
serum containing them with the homologous organism, and allow- 
ing the mixture to stand for a time. No agglutination will take 
place, nor will it occur when fresh agglutinin is added. The 
Fig. 73.-Agglutination and formation of agglutinins: 1, Diagrammatic 
representation of bacterial or other antigenic cells with fixed (a) and freed (6) 
agglutinogen groups. 2, Union of agglutinogen with cell receptors of the 
second order: a, Molecule of the cell protoplasm, with a cell receptor of the first 
order (e) and of the second order (6), showing its haptophore (c) and its aggluti- 
nophore or zymophore (d); f, an agglutinogen group united to the cell receptor. 
3, Overproduction of cell receptors b and freed receptors or agglutinin molecules 
at c. 4, Union of agglutinin with the bacterial or other cell: a, Bacterial cell; 
b, agglutinogen still attached as cell receptor to bacterial cell; c, agglutinin; 
d, agglutinin united to agglutinogen. 
agglutinoid unites with the cell and blocks the union of the ag- 
glutinin. Certain investigators have claimed to have produced 
antiagglutinins by the use of agglutinins as an antigen, inoculating 
them into another species of animal. These antiagglutinins, when 
mixed with the agglutinins, unite with them and prevent them from 
uniting with the homologous antigen when it is added. 
Body or Somatic and Flagellar Agglutinins.-It is probable that 
agglutinogen in motile organisms may originate in two ways-from 
the flagella or from the cell-bodies. Agglutinins have been differ- 
entiated in such cases into those that bring about agglutination AGGLUTINATION AND PRECIPITATION 
177 
by combining with the flagella (flagellar agglutinins') and those 
which unite with the cell-body (somatic or body agglutinins). 
Method of Agglutinin Action.-Common salt must be present 
in any serum which agglutinates. Its function is not thoroughly 
understood, nor is the general phenomenon of agglutination itself 
susceptible of a simple explanation. The older theories of the 
change whereby the organisms are made glutinous by the union 
of the agglutinin are to be discarded, and the true explanation is 
doubtless to be found somewhere in the field of colloid chemistry. 
Concerning the exact nature of the change in the cell we know 
little or nothing. The cells are certainly not seriously injured; 
Fig. 74.-Diagrammatic representation of group agglutination: Let 1, 2, 
and 3 represent the agglutinogen given off by three related species of micro- 
organisms. The organisms 1 and 2 have agglutinogens a and F in common, 
but in quite different proportions. No. 3 likewise has F in common with both 
of the others, and D in common with 1. If the area covered in the diagrams 
represents the relative proportions of agglutinogen of each kind, the readiness 
with which one species may be agglutinated by a heterologous serum may 
be understood. 
in fact, an organism may grow luxuriantly in a serum which agglu- 
tinates it. One of the delicate tests for agglutinability is to grow 
the organism in such a serum, and note the production of long 
threads (Pfaundler's reaction). 
Probably' the most helpful conception of the agglutination 
reaction is to be found in a comparison with the phenomenon 
termed salting out" of proteins. A study of a bacterial sus- 
pension made by passing an electric current will generally show 
the bacteria moving toward the positive electrode. This shows 
that the bacterial cells carry a negative charge and tend to repel 
each other and thus keep in suspension. If high concentrations 178 
VETERINARY BACTERIOLOGY 
of certain salts are added to many bacterial suspensions, the 
bacterial cells will flocculate. Saturated ammonium sulphate 
solution, for example, will flocculate or agglutinate typhoid 
bacilli. Apparently the electrical charge on the bacteria has 
been neutralized, they no longer repel each other, and tend to 
stick together and settle out. A study of the specific agglutina- 
tion of bacteria by serum indicates the probable function of the 
agglutinin to be sensitization to salt, the latter producing the 
actual clumping. It can be shown, for example, that a salt 
free serum containing a specific agglutinin will not agglutinate 
the homologous organisms until salt is added. 
Spontaneous agglutination of suspensions of bacteria some- 
times occur. For example, difficulty is'sometimes experienced with 
typhoid bacteria used for agglutination tests when suspended in 
physiological salt solution as a result of their spontaneous floccu- 
lation, that is, their agglutination in the' absence of specific 
antisera. This may usually be corrected by using a lower con- 
centration of salt. Suitable dilution of salt solution will give 
a concentration which will no longer cause agglutination except 
upon addition of the specific serum. 
Some attempt has been made to use the fact that many bac- 
teria agglutinate at definite hydrogen ion concentrations for pur- 
pose of identification. It has not proved practical. 
Group Agglutinins.-The statement has been made that agglu- 
tinins are specific. This must be somewhat modified. It has been 
shown that the serum homologous for a certain organism may 
clump to a less degree some other species or closely related forms, 
and in rare instances forms quite unrelated. This seems to be 
due to the fact that not all the agglutinogen given off^ by a 
particular organism is of one type; the agglutinins produced, 
therefore, are likewise of different types. It is entirely probable 
that closely related organisms should throw off some identical 
agglutinogen, and, therefore, have some common agglutinins. 
Agglutination of an organism by a heterologous serum is termed 
group agglutination. The agglutinins which are specific for the 
organism are called its chief agglutinins, and those common to two 
or more organisms are termed coagglutinins. It may sometimes 
be shown that differences exist between the agglutinins produced AGGLUTINATION AND PRECIPITATION 
179 
by different strains of the same organism. The importance is 
apparent, therefore, of using care in testing the agglutinating power 
of any serum to dilute to such an extent that the action of the 
coagglutinins may be negligible and that of the specific or chief 
agglutinins, recognized. 
Agglutination Tests in Disease Diagnosis.-The fact that an 
organism developing in the body generally excites the production 
of a specific agglutinin has led to the wide use of the fact in the 
diagnosis of certain infectious diseases. Not all diseases cause 
an appreciable production of agglutinin. The test is carried out 
by mixing serum from the suspect with the organism. If agglu- 
tination occurs in proper dilution, it is evident that the specific 
organism is or has been present in the patient. The test is fre- 
quently reversed, and serum from an animal showing high agglu- 
tinating power is used to differentiate between different species 
and races of bacteria. The principal disease organisms which cause 
the production of appreciable amounts of agglutinin are as follows: 
Bacterium typhi (typhoid fever), Bacterium paratyphi (paraty- 
phoid), Bacterium enteritidis (meat-poisoning), Bacterium dysen- 
tence (bacillary dysentery), Bacterium coli, Pseudomonas pyo- 
cyanea, Pfeifferella mallei (glanders), Pasteur ella pestis (bubonic 
plague), Mycobacterium tuberculosis (tuberculosis), Bacterium 
melitensis (Malta fever), Streptococcus pyogenes, and some other 
pyogenic cocci meningitidis (epidemic cerebrospinal meningitis), 
Vibrio cholera (Asiatic cholera) and others. The test is not 
commonly used in practice for the recognition of all of them- 
some are of experimental interest only. 
The diagnosis of disease by agglutination, particularly of ty- 
phoid, is commonly called the "Gruber-Widal," or simply the 
"Widal" test. The test may be made either by observation of a 
hanging drop or microscopically, or it may be made by naked-eye 
observation of the reaction in the test-tube, or macroscopically. 
Microscopic Widal, or Agglutination Test.-Dilutions of the 
serum are generally made 1:10, 1:20, and 1:40, or even more. 
To prepare a 1:40 dilution for a hanging drop, place 19 loops of 
physiological salt solution, separately, upon a clean microscopic 
slide, then add one loopful of serum to be tested, and mix thoroughly 
with the diluent. Place one loopful of a suspension of the organ- 
ism (broth culture or suspension from the surface of an agar slant 180 
VETERINARY BACTERIOLOGY 
in physiological salt solution) upon each of two clean cover-glasses; 
to one add one loopful of the serum dilution, to the other a loopful 
of sterile physiological salt solution. Invert over the cavity of a 
hollow-ground slide and examine microscopically. The check 
should show the organisms uniformly distributed over the field, 
and moving about actively, if the organism is motile. The organ- 
isms in the other may show no change, but if the serum has 
come from a patient infected with the organism, the bacteria will 
soon begin to clump together, and in the course of a few minutes 
to an hour practically all of the organisms will be found so 
Fig. 75.-The Widal or agglutination test of the typhoid bacillus, using 
serum from a typhoid patient: A, Check showing the uniform distribution 
of the bacilli; B, clumps of bacteria in the positive test (Jordan). 
clumped, very few or none remaining isolated in the field. Motility 
is lost in motile forms. The test is a very delicate one, as is evi- 
denced by the fact that the agglutination may sometimes be secured 
in dilutions as great as 1: 100,000. The higher the dilution 
at which agglutination occurs, the greater is the specificity of 
the reaction. As has been seen, the dilution must be great enough 
in every case to escape the activity of the normal and the common 
group agglutinins. Serum from an animal that has been immun- 
ized against a specific organism may be used in the recognition 
of that organism. Typhoid serum in this way can be used in the 
detection of the typhoid bacillus. Such a test also enables one to 
differentiate between closely related forms, as the varieties of the 
dysentery and of the paratyphoid bacilli. AGGLUTINATION AND PRECIPITATION 
181 
Macroscopic Widal, or Agglutination Test.-A series of small 
test-tubes is prepared, each tube containing a definite amount of a 
suspension of the organism. To these are added varying quantities 
of serum, making dilutions of 1: 10, 1: 50, 1: 100, 1: 200, and higher. 
A positive reaction is indicated by the appearance of small flocculi 
of bacteria, which soon settle to the bottom, leaving the super- 
natant liquid clear. The reaction may not be complete for several 
hours, and the tubes should be allowed to stand for twenty-four 
hours before making final observations. Check tubes should 
always be kept as controls, in which the liquid should remain 
uniformly turbid. Advantage is taken by certain manufacturers 
of the fact that dead bacteria, as well as the living, may be 
agglutinated. They make " agglutinometers " containing all 
the apparatus and materials for making a complete test. The 
bacterial suspension supplied will keep for a long period. 
Pfaundler's Reaction.-Pfaundler has called attention to the 
fact that a mixture of serum containing typhoid agglutinin with 
typhoid bacteria will cause the latter organisms, when incubated, 
to grow into threads. The diagnosis of typhoid as worked out by 
Mandelbaum is as follows: Mix the patient's blood with 15 parts 
of a broth culture of Bacterium typhi containing 1 per cent, 
sodium citrate. The mixture is drawn up into a bulb pipette and 
incubated for five hours. A microscopic examination of the 
supernatant liquid at the end of this time will show either actively 
motile bacilli or thread formation. In the latter case the diagnosis 
is positive. 
Significance of Agglutinins in Immunity.-Agglutination takes 
place in the body as well as without. Clumps of typhoid bacilli 
may be found in various capillaries in cases of typhoid. It is 
uncertain what significance is to be attached to the phenomenon. 
It can scarcely be of advantage, except possibly that it prevents 
to some degree the distribution of the bacteria through the blood. 
Hemagglutinins.-Certain bacteria, and certain toxic materials 
from plants and animals, contain substances which agglutinate 
red blood-cells. Such agglutinins are termed hemagglutinins. 
When this agglutination occurs in the blood, it results in the 
formation of emboli of the red blood-cells. These emboli are of 
considerable significance in some diseases. Hemagglutinins may 182 
VETERINARY BACTERIOLOGY 
also be formed by the injection of the red blood-cells of one species 
into another. 
Precipitins.-The precipitins are quite analogous to the ag- 
glutinins. They are formed as a result of the injection of a great 
variety of proteins or protein derivatives. An antigen which in- 
duces the development of precipitins is known as a precipitogen. 
The precipitogen is doubtless the protein molecule itself. It con- 
sists essentially of a binding or haptophore group only. The 
precipitin seems to be formed in a manner similar to that already 
described for agglutinins. It may be shown to consist of a 
zymophore or precipitating group, and a haptophore or binding 
group. The zymophore is easily destroyed by heat, but the hap- 
tophore is thermostabile. A precipitin that has lost its zymophore 
is known as a precipitoid. The precipitins are quite specific, but 
group precipitation will take place when related proteins are 
treated with a serum homologous to one of them. The blood- 
sera of various ruminants, for example, exhibit group precipita- 
tion. An antiserum homologous to human serum will precipitate 
the serum of anthropoid apes. 
The work of Nuttall has shown quite definitely the limits 
of group precipitation. He tested 900 kinds of blood, using in 
all 30 antisera, and made a total of about 16,000 combinations. He 
showed that, on the whole, the closer the relationship, the greater 
the amount of common or group precipitation. For example, 
he determined that the blood of apes of the old world would yield 
a heavier precipitate with human antiserum than would that of 
apes of the new world. Another exception to specificity has 
been found in the protein of the crystalline lens: an antiserum 
for the lens protein of man or the ox will produce a precipitate in 
solution containing the lens protein from other animals not at all 
closely related. It has been suggested that the cataract of the 
eye may be due to the formation of an autoprecipitin for the 
protein of the lens and its consequent partial coagulation or pre- 
cipitation in situ. 
The mechanism of precipitation is not well understood, but 
it is probably to be explained on the basis of certain facts of 
colloid chemistry. The test is so delicate that a positive reaction AGGLUTINATION AND PRECIPITATION 
183 
has been secured with a dilution of 1:100,000 of egg-white, while 
the ordinary protein tests of the chemist fail to show 1: 1000. 
Uses Made of the Precipitation Phenomenon.-Several practical 
applications have been made of precipitation in the differentiation 
of proteins. These are the recognition of blood-stains, the differ- 
entiation of meats from different species of animals, particularly 
horse-flesh from beef, and the diagnosis of many other protein- 
containing substances, including bacteria and their products, as in 
the diagnosis of glanders and of anthrax. 
Recognition of Blood-stains.-It is sometimes necessary in 
murder trials to determine with certainty the origin of a blood-stain. 
The fact that the stain has been produced by blood may be easily 
demonstrated by the chemist, but he has no ready means of telling 
with certainty whether the blood is of animal or human origin. 
Uhlenhuth was the first to call attention to the value of this test 
in legal medicine. The precipitation test, when properly carried 
out, enables the determination to be made with a high degree of 
certainty. An antiserum specific for human blood must be se- 
cured first by injections of human serum into a rabbit, at intervals 
of a few days, for a period of several weeks. A bit of the material 
with the blood-stain is placed in a watch-glass and 5 c.c. of sterile 
physiological salt solution is added. This is allowed to stand until 
some of the blood proteins have been dissolved. This may be 
shown by blowing into the liquid through a capillary tube, when a 
fairly permanent foam will be produced. If any dirt or sediment 
appears in the solution, it is removed by filtration. An effort 
is made to secure a dilution of the serum of about 1:1000. The 
diluted serum is placed in a series of test-tubes, and the specific 
antiserum is added. If the blood-stain is from human blood, the 
precipitate will make itself apparent as a clouding in the course of 
a few minutes. The reliability of the test has been recognized 
by the German courts, and the results have been accepted as 
evidence. By varying the procedure, the same method may be 
used in differentiating animal bloods. 
Differentiation of Meats.-Meat inspection, particularly in 
certain European countries, includes the differentiation of meats. 
In certain localities large quantities of horse-flesh are used for food, 
but the law forbids that horse-flesh be sold as beef. There are cer- 184 
VETERINARY BACTERIOLOGY 
tain chemical differences between the two,-differences in the com- 
position of the fat and possibly in the abundance of glycogen,-but 
these differences require careful chemical analysis and examination 
for their recognition. The precipitation test furnishes an easier 
and more reliable method for reaching the same end. Further- 
more, the testing may be extended to an examination of mixed 
meats, as sausages, and the various kinds of meat present deter- 
mined. Specific antisera must be prepared for each of the meats 
which it is desired to recognize by mincing the meat and soaking 
it in physiological salt solution. This material is then used in the 
immunization of a rabbit by repeated injections during several 
weeks. The flesh to be tested is likewise extracted with physio- 
logical salt solution, the solution filtered and tested as in the blood 
diagnosis with the various specific antisera. It will give a most 
prominent precipitate with its homologous antiserum, and the 
differentiation may thus be made. 
Differentiation of Bacteria.-It has been found that the injec- 
tion of the bacteria-free filtrates of liquid cultures of bacteria will 
induce the production of an antiserum specific for the filtrate 
from that particular type of organism. This method is not as 
reliable as the agglutination method of differentiating bacteria, 
but it may be used, and is just as specific. 
Similar tests have been made to differentiate from each other 
the proteins derived from certain plants and plant-seeds. It may 
be stated that, in general, the injection of any protein in abso- 
lutely pure condition will cause the production of the homologous 
specific antiserum in the animal injected. CHAPTER XVI 
CYTOLYSINS, INCLUDING BACTERIOLYSINS, AND HEMOLYSINS 
(Antibodies of Ehrlich's Third Order) 
The use of animal, plant, or bacterial cells as antigens has been 
found usually to cause the development, by the tissues, of specific 
antibodies, which have the power of destroying these cells, and 
in many cases of actually dissolving or digesting them. These 
antibodies are termed cytotoxins. In most cases the action is 
lytic or dissolving; the antibodies are lysins or cytolysins. Cyto- 
lysins are frequently subdivided with reference to their antigens, as 
bacteriolysins, hemolysins, nephrolysins, etc. Any substance which 
destroys bacterial cells is said to be bactericidal, and this expression 
is used when the method of cell destruction is not specified. Cyto- 
lysins are produced by certain bacteria, and are also found in 
certain snake venoms. 
Bacteriolysins were first noted by Pfeiffer. He found that 
when cholera spirilla were injected into the peritoneal cavity of the 
immune guinea-pig, the serous exudate rapidly dissolved and 
destroyed them. This lytic action of the blood-serum is called 
Pfeiffer's phenomenon. Later, it was discovered that this reaction 
would take place just as well in a test-tube (in vitro) as in the 
animal body. 
Cytolysins.-Cytolysins have been shown to be made up of two 
elements. When a cytolytic serum is heated to 56° for half an 
hour, or is allowed to stand for a time, it loses its lytic property. 
This is regained, however, when a little fresh normal serum is added. 
The normal serum is said to reactivate the immune serum. The 
normal serum alone is not lytic, nor is the immune serum, but when 
mixed, they will destroy cells. It is evident, therefore, that the 
cytolysin is made up of two constituents, neither of which can 
act without the other. The thermostabile constituent of the im- 
185 186 
VETERINARY BACTERIOLOGY 
mune serum is termed amboceptor;1 the thermolabile constituent 
of normal serum is termed complement? 
Action of Amboceptor.-It may be shown that the amboceptor 
unites with the antigenic cell with which it comes in contact. 
This may be demonstrated by the following experiment: A 
heated immune serum (i. e., one containing amboceptor only) 
is added to the homologous cell, allowed to stand for a time, then, 
by means of repeated centrifugation and washings with physiologi- 
cal salt solution, the serum may be completely removed. To the 
washed cells normal fresh serum (i. e., containing complement) 
is added, when cytolysis may be observed. This seems to show 
that the amboceptor unites with the cell and cannot be removed 
by washing, but cannot, on the other hand, destroy the cell until 
the complement is added. 
It is apparent that the essential action is the sensitization of 
the antigen to the action of the complement. 
Action of Complement.-Normal serum containing complement 
only shows no cytolytic activity. An experiment reciprocal to 
the preceding, the addition of complement containing serum to a 
suspension of cells, followed by centrifugation and washing, and 
the addition of amboceptor, will not result in cytolysis. The 
complement is evidently not bound to the cell, and can only be 
attached through the amboceptor. The amboceptor may be con- 
sidered as a structure which links or binds the complement to 
the cell and enables it to destroy such cell, or, perhaps better, as a 
substance which sensitizes the cell to the action of the complement. 
Specificity of Amboceptors and Complements.-The amboceptor 
is formed usually as the result of immunization, and is specific 
for its antigen. The amboceptor for the red blood-cells of one 
species of animal will not unite with those of an unrelated species. 
Inasmuch as normal serum will activate many different ambocep- 
tors, it has been argued that all complement is of a single type. 
Ehrlich has attempted to demonstrate that there are many com- 
plements, and the general activating power of certain fresh sera 
1 Synonyms of amboceptor are immune body, Immunkbrper, Zwischen- 
kbrper, substance sensibilisatrice, copula, desmon, philocytase, fixateur, 
preparator, Hilfkorper. 
2 Synonyms of complement are addiment, alexin, cytase. CYTOLYSINS, BACTERIOLYSINS AND HEMOLYSINS 
187 
for many amboceptors is due to the multiplicity of the comple- 
ments which they contain, but the weight of evidence is against 
this conception. 
Ehrlich's Conception of Formation of Amboceptor and Comple- 
ment.-Ehrlich calls an amboceptor a freed cell receptor of the 
third order. Such a receptor he believes exists in the form of a 
double haptophore, and unites first with food or other materials, 
then, by means of the other haptophore, with complement which 
is present in the serum. The latter doubtless normally brings 
Fig. 76.-Formation and action of cytolysins: 1, Bacterial or other cell, 
a, with receptors, b, which are thrown off as antigens, c; 2, protoplasmic mole- 
cule of the body a, with a receptor of the third order, b. This receptor, by 
means of its cytophilous haptophore, d, can unite with the antigen, e, and 
by means of its complementophilous haptophore, c, it can unite with the 
complement f. At g is shown a receptor with both haptophores occupied. 
At h is a freed cell receptor or amboceptor; 3, a bacterial or other cell, a, to which 
an amboceptor has united by means of the receptor b, and with a complement 
united to c. This completes the lytic system, and the cell may be destroyed 
by the complement. 
about changes of a digestive nature which enable the cell proto- 
plasm to make use of the food. The antigens used in immuniza- 
tion unite with such receptors and divert them from their normal 
function. Possibly the cell is injured; it is, at any rate, stimulated 
to an overproduction of these receptors, and they are thrown free 
in the blood as amboceptors. 
Group Cytolysins.-It may be shown that related cells contain 
some similar antigens, and that the cytolysins for one species of 
cell may dissolve in low dilutions the cells of another species. 
This phenomenon is similar to that of group agglutination, and 
seems to be based upon the same general facts. 188 
VETERINARY BACTERIOLOGY 
Bacteriolysins.-Bacteriolysins are normally present for certain 
bacteria in the blood of some animals, as bacteriolysins for the 
anthrax organism in dog's blood. They may be developed for 
many organisms by systematic immunization, or they may appear 
during the course of an infection. Their presence may be demon- 
strated in two ways-either by direct microscopic examination or 
by plating methods. In the first method the organisms are mixed 
with the serum and examined microscopically. They can be 
found, by actual observation, gradually to disintegrate and dis- 
appear. The second method offers certain advantages. The 
serum is mixed with the bacteria, and from time to time portions 
of the mixture are plated out, and the rapidity of the destruction 
determined by the relative number of colonies that develop. 
Neisser and Wichsberg have developed a technic which enables 
them to differentiate dead and living cells by the fact that leuko- 
bases are formed from methylene-blue during life and not after 
death. 
Bacteriolytic Sera Used in Practice.-By no means all bacteria 
will induce the production of bacteriolysins in any quantity in 
the body, as, for example, the pyogenic organisms and the pneu- 
mococcus. The members of the intestinal group and the spirilla, 
on the other hand, are readily destroyed by this means. Antisera 
have been prepared and used for several of the latter. In the 
manufacture of the antisera either living or dead organisms may 
be used. Methods of titration, whereby the actual bacteriolytic 
content of the serum used may be known, have not thus far been 
developed. The antisera generally have other antibodies devel- 
oped in addition to the bacteriolysins. Plague serum and that 
specific for swine erysipelas contain some bacteriolysins, and 
probably the same is true of the sera used in immunizing against 
hog-cholera and against rinderpest. 
Bacteriolytic sera for passive immunization are prepared by the 
methodical introduction of the organism into the animal body. 
Either the first injections must be made with dead organisms or 
an animal which has recovered from an attack of the disease 
must be chosen. The animal is hyperimmunized by increas- 
ing doses of the virulent organism after the first establishment of 
immunity. This results in the accumulation of considerable CYTOLYSINS, BACTERIOLYSINS, AND HEMOLYSINS 
189 
quantities of immune substances (amboceptors) in the blood. 
This serum may then be used in passive immunization. Vaccina- 
tion, or the injection of dead or living bacteria into the body, with 
the resultant development of an active immunity, owes its effi- 
ciency, in some cases at least, to the production, by the body, of the 
bacteriolysins specific for the organism. 
Bacteriolytic sera for passive immunization are not employed 
against many diseases. Some organisms, as has been stated, cause 
the production of little or no bacteriolysin. The bacteriolysin 
developed in the blood of one species is not always suitable for the 
passive immunization of another. There have been various 
reasons advanced for this fact: the injection of the foreign serum 
may cause the production of anti-amboceptors, or of anticom- 
plements, or of some other antibody which would inhibit the lytic 
action of the injected serum. Complement soon disappears from 
an immune serum; therefore the amboceptors are dependent for 
their activation upon the normal complement of the blood. This 
complement may not in all cases be suitable for combination with 
the particular amboceptor used, and the lytic activity be thus 
inhibited. When the antiserum to be used for passive immuni- 
zation comes from the same species of animal, as is the case in 
immunization against hog-cholera and rinderpest, these objections 
do not seem to obtain. 
Hemolysins.-Hemolysins for the red blood-cells of one animal 
usually may be obtained by injection of these into another. 
They are divided into three types, the classification being made 
upon the basis of relationship. Heterolysins are developed by the 
injection of the red blood-cells of one species into another; isolysins 
by the red blood-cells of one animal into another animal of the same 
species, and autolysins by an individual for his own red blood-cells. 
The last two, particularly the autolysins, are not easily produced 
or demonstrated. 
The study of hemolysis has proved of great value in two ways: 
First, hemolysis is a phenomenon that may be easily observed, 
and it has been used, therefore, more than any other, in the study 
of the nature and activity of cytolysins. Hemolysis is readily 
detected, because the hemoglobin escapes into the solution which 
remains permanently red, while unhemolyzed red blood-corpuscles 190 
VETERINARY BACTERIOLOGY 
soon sink to the bottom, leaving the blood-serum clear and color- 
less. Second, it is of indirect use in the diagnosis of certain dis- 
eases, and in the recognition of certain organic substances. This 
second use is called variously absorption or fixation of comple- 
ment, or, after the name of its discoverers, the Bordet-Gengou 
phenomenon. 
Fixation of Complement and Its Utilization.-The method of 
complement fixation is one that enables us to determine the pres- 
ence or absence of cytotoxic, cytolytic, or similar antibodies in a 
serum. Inasmuch as such substances are generally present in the 
serum of a diseased individual, the determination of their presence 
may constitute in such cases a diagnosis of the disease. A specific 
example, the demonstration of fixation of complement by use of 
serum from a glandered horse, will first be discussed. Five dif- 
ferent solutions are always needed in carrying out a test of this 
kind: 
1. Suspension of Pfeifferella mallei (the glanders bacillus 
antigen). 
2. Heated serum of glandered animal (amboceptor). 
3. Normal serum, usually taken from guinea-pig (complement). 
4. Red blood-cells. Those of sheep generally used (antigen). 
5. Heated serum of rabbit immunized against 4 (amboceptor). 
Suppose that 1 and 2 are mixed, and a small amount of 3 added. 
Evidently the complete bacteriolytic system is present and bac- 
teriolysis should occur. This is difficult to demonstrate micro- 
scopically, however, and would not be demonstrated at all in that 
manner if an excess of the first antigen or suspension of bacilli is 
used. It is evident that the complement added will be used up or 
" bound " by the combination of bacillus and amboceptor. No. 4 
and 5 are next added. They unite with each other, but, as all 
the complement has been used up, there can be no hemolysis. 
The fact that hemolysis does not occur is proof, therefore, that the 
complement has been removed. Since hemolysis does not occur, 
amboceptors for Pf. mallei must be present in the horse's blood, 
and the diagnosis of the disease would be positive. If hemolysis 
does occur, evidently the patient's serum lacks amboceptor specific 
for Pf. mallei, and the diagnosis would be negative. The test 
may be reversed also, and some organism suspected of being Pf. 
mallei may be substituted for No. 1, and the same technic carried CYTOLYSINS, BACTERIOLYSINS AND HEMOLYSINS 
191 
through. A lack of hemolysis would indicate that the organism 
had bound the amboceptor and complement, and, therefore, was 
Pf. mallei in fact, while hemolysis would indicate the reverse. 
The accompanying figure will make this clearer. In carrying 
out a test of this kind it is necessary to arrange a very complete 
series of checks. When properly managed the method is very 
accurate and has yielded results of value in many cases. 
Fig. 77.-Fixation or absorption of complement: A, Diagrammatic rep- 
resentation of-1, Bacterial cell, a (Pfeifferella mallei in text), with antigen 
b; 2, amboceptor for the antigen of (1) (the glanders serum); 3, normal com- 
plement of the guinea-pig's blood with the haptophore c and the zymophore 
b', 4, red blood-cell (sheep) with the antigenic receptors b; 5, amboceptor 
from the blood of the rabbit immunized against 4. B, 1, Bacterial cell 4- 
amboceptor + complement makes a complete lytic series; bacteriolysis 
occurs, and the complement is removed from solution; 2, the red blood-cell 
+ its amboceptor; no hemolysis, as the complement has been used by the 
other series. C, 1, Bacterial cell not Pf. mallei, to which amboceptor (2) 
cannot unite, hence the complement is not removed from the serum, and 
when the red blood-cells and their specific amboceptor are added, a complete 
hemolytic system (3) is formed and hemolysis occurs. 
This test has been most used in the diagnosis of syphilis, the 
antigen being secured from a macerated syphilitic fetus or from 
normal beef heart and the amboceptor from the blood of the 
suspect. This is known as the Wassermann syphilis test. A 
similar test may be used for diagnosis in other diseases. The 
same method may be used also in the differentiation of proteins. 
In this case the antigen used is the protein, and the first ambocep- 
tor is in the serum of an animal immunized against this protein. 
The method is even more reliable than the precipitation tests 192 
VETERINARY BACTERIOLOGY 
already discussed, but is more difficult to carry out, hence is less 
frequently used. It may be used for the identifications of blood- 
stains. 
Cytotoxins.-Antisera have been prepared for a number of 
different body-cells, as epithelial cells, spermatozoa, and leuko- 
cytes. Within limits they seem to be specific. It is believed 
that some pathological conditions in the body are produced by 
autocytotoxins, substances produced by the body poisonous for its 
own tissues. 
Conglutination.-In the course of experimentation by Ehrlich 
and Sachs it was observed that upon the addition of fresh unheated 
horse serum to a suspension of red blood-cells of the guinea-pig 
slight hemolysis occurred. The addition of heated ox serum to a 
suspension of the red corpuscles resulted in no hemolysis. An 
addition of both inactive ox serum and fresh horse serum resulted 
in very active hemolysis. The phenomenon is explained on the 
basis that the guinea-pig corpuscles are sensitized by the ambo- 
ceptor of the ox serum and thus made susceptible to the action of 
the complement in the fresh horse serum. 
In checking the experiments of the above investigators Bordet 
and Gay observed that the inactivated ox serum has but slight 
agglutinating power for the corpuscles of the guinea-pig, and that 
fresh horse serum likewise agglutinates them only slowly and 
slightly, whereas a mixture of the two sera causes rapid and com- 
plete agglutination. The ox serum seemingly increases the hemo- 
lysin and agglutinin of the horse serum, and Bordet and Gay 
therefore concluded that this property was due to a thermostabile 
substance peculiar to the ox serum. This they call conglutinin, and 
class it with the colloids. Their explanation is that guinea-pig 
corpuscles are affected by sensitizers (amboceptors) in both the 
horse and ox serum. After this sensitization the corpuscles are 
in a condition to fix the complement of the horse serum. This 
complement possesses but slight hemolytic power. But when 
the corpuscles have become sensitized and laden with the comple- 
ment their properties of molecular affinity are modified to the 
extent that they become capable of attracting the colloidal 
substance (conglutinin) of the ox serum which combines with CYTOLYSINS, BACTERIOLYSINS, AND HEMOLYSINS 
193 
them. The result is twofold: Strong agglutination occurs and 
the corpuscles are more readily destroyed by complement. 
Applying these principles, Stranigg, Pfeiler, Strenge, and others 
have made use of the so-called conglutination test for the diagnosis 
of dourine, glanders, syphilis, and various other diseases. The 
technic for the glanders conglutination test is much the same as 
that for complement fixation. A definite quantity (established by 
previous titration) of normal unheated horse serum is added to a 
tube containing inactivated blood-serum of the horse and an extract 
of the glanders bacillus (previously titrated). This mixture is 
incubated for one hour at 37|°. There are then added the 
inactivated ox serum (previously titrated) and a suspension of 
washed red blood-cells of the sheep (or other animal). A second 
incubation for one or two hours is then made. 
If the serum being tested is from a glandered animal the red 
blood-cells settle to the bottom, leaving the fluid above clear; if 
from an animal not glandered the corpuscles gather at first in 
clumps and later hemolyze slowly, making the fluid red. 
The advantages claimed for the conglutination test are that it 
is applicable in asses and mules which possess anticomplementary 
substances in their blood-serum, and that the ingredients of the 
test are more readily obtainable than are those for complement 
fixation. The test is considered even more delicate than the com- 
plement fixation and requires more careful titration of the reagents. 
Pfeiler claims for it a greater degree of accuracy, and cites cases in 
which it was positive where both agglutination and complement 
fixation were negative, subsequent postmortem verifying the accu- 
racy of the test. CHAPTER XVII 
It was observed by Parum as early as 874 that decay-producing 
bacteria quickly disappeared when 'ntxuduced into the body, and 
could not be demonstrated in tn z olood or other tissues. To 
Metchnikoff, however, must be yh/en the credit of elaborating the 
theory of phagocytosis. By the term phagocytes (Gr. phagein, to 
eat) is meant any body-c 11 which is capable of taking up and 
destroying other cells, o mally those of foreign origin. These 
phagocytes are in some cases fixed body-cells, but, for the most part, 
are leukocytes or white blood-cells of different kinds. Upon the 
phagocytic activity of the body-cells Metchnikoff established his 
theory of immunity. It may be stated briefly as follows: If an 
animal is either naturally or artificially immune to a disease, it 
means that the invasion of the body by organisms is followed by a 
struggle between the organisms and the phagocytes. These 
phagocytes ingest the organisms and render them harmless. 
Metchnikoff subdivides the phagocytes in two ways-first, on the 
basis of their morphological and functional behavior, and, second, 
on the basis of their relationship to the surrounding tissue, i. e., 
some are mobile, others are fixed. The leukocytes in the blood are 
in part the free-moving phagocytes. The small lymphocytes are 
not known to have any phagocytic power, and it is probable they 
are not endowed with active motion. Most cells have not been 
shown to be phagocytic. The polymorphonuclear and the large 
mononuclear leukocytes are the phagocytes par excellence. Certain 
fixed cells of the lymph-nodes and the spleen are likewise active 
phagocytes. 
Metchnikoff terms the polymorphonuclear leukocytes the 
microphages (Gr. micros, small; phagein, to eat), and the large 
mononuclear, the macrophages (Gr. macros, large; phagein, to eat), 
OPSONINS AND PHAGOCYTOSIS 
194 OPSONINS AND PHAGOCYTOSIS 
195 
and believes that they perform somewhat different functions in the 
body. 
Wright and Douglas, in 1902, published observations that 
threw much needed light on the theory of phagocytosis. They 
showed that, in most cases, at least, the bacteria would not be 
destroyed by the white blood-cells or phagocytes in the absence 
of blood-serum. They showed that the serum contained something 
that rendered the bacteria positively chemotactic or attractive 
for the phagocytes, and they accordingly named this something 
opsonin (Gr. opsonein, to prepare a meal). 
Opsonins.-Opsonins may be shown to be present in certain 
sera by the following experiment: Blood of a suitable animal is 
drawn into citrate solution and centrifuged, thus throwing the 
Fig. 78.-Phagocytosis by human leukocytes: 1, Staphylococcus aureus; 2, 
Bacterium dysenteries; 3, Bact. typhosum; 4, Mycob. tuberculosis; 5, Neisseria 
meningitidis (1, 2, and 3 adapted from Levaditi and Inman, 4 from Freeman, 
and 5 from Councilman). 
cellular elements to the bottom. The top layer of corpuscles, 
or " cream," containing a large proportion of leukocytes, is pipetted 
off, and mixed with physiological salt solution and again centrifuged. 
A second washing and centrifugation removes all traces of adher- 
ent serum. The corpuscles are then mixed with a suspension of 
the bacteria and incubated for fifteen minutes. At the end of 
that period mounts are prepared, stained with a suitable blood- 
stain, such as Jenner's or Wright's, and examined. The bacteria 
will not, in general, be engulfed by the phagocytes. A similar 
series carried through, but with the addition of a little immune 
serum, will exhibit marked phagocytosis, and considerable 
numbers of the microorganisms will be found within the leuko- 196 
VETERINARY BACTERIOLOGY 
cytes. Evidently the presence of the. serum, or rather the op- 
sonin which it contains, induces phagocytosis. Opsonins are 
believed so to change or alter the bacteria as to make them posi- 
tively chemotactic for the phagocytes. This does not mean that 
the bacteria are destroyed, for there is no evidence that they are 
in any way injured. That it is an alteration of the organisms, 
and not a mere stimulation of the phagocytic cell, may be shown as 
follows: A suspension of washed corpuscles prepared as before 
is treated with immune serum, allowed to stand, and then washed. 
When mixed with the bacterial suspension, no phagocytosis occurs; 
evidently the opsonin is not bound to the phagocytes, nor does it 
stimulate them when they are brought in contact with the bacte- 
rial cells. The converse of this experiment may be tried, the bac- 
teria added to the immune serum, and then centrifuged out and 
washed free from all the serum with salt solution. When these 
organisms are added to a suspension of the washed leukocytes, 
active phagocytosis occurs. The opsonin is bound to the bacterial 
cell, and makes it in a sense attractive to the leukocytes. The 
negative chemotaxis is converted by this union into a positive 
chemotaxis. The action of the opsonin has been likened to that 
of an amboceptor, for it links up the bacteria and the white blood- 
cells. The opsonins are, however, not identical with bacteriolytic 
amboceptors. 
Opsonins for some organisms are quite constantly present in 
the blood. For example, human blood contains opsonins for the 
organisms producing bubonic plague, Malta fever, pneumonia, 
dysentery, Asiatic cholera, and for the pyogenic organisms. 
These are called normal opsonins. Immune opsonins are those 
produced as a result of infection or immunization. It is also 
probable that some species of bacteria may be taken up by phago- 
cytes in total absence of opsonins. On the other hand, it is be- 
lieved that certain bacteria, when they invade the body, may 
develop a resistance to phagocytosis. The appearance of the 
capsule about the anthrax bacillus in the blood has been thought 
to be a protective agency of this character. Whether the im- 
mune and the normal opsonins are identical is a moot question. 
Probably they may differ, it being contended that the normal 
opsonin is simply the normal complement of the serum, for it has OPSONINS AND PHAGOCYTOSIS 
197 
been found to be thermolabile (58° to 60° destroys). The immune 
or specific opsonin is, on the other hand, relatively thermostabile. 
Opsonic Index.-It is sometimes advisable to compare the 
opsonic content of the serum of a diseased animal with that of a 
healthy individual. The ratio between the two sera may be 
determined by the relative effect they have on the rapidity of 
phagocytic action. It is customary, though not in all cases 
necessary, to use the leukocytes of the species of animal under 
investigation. The technic of the operation is as follows: 
Fig. 79.-Standardization of bacterial emulsion. A photomicrograph showing 
the bacteria and the red blood-cells mixed (Miller). 
Preparation of Leukocytes.-These may be secured by bleeding 
into citrate solution (1 per cent, in physiological salt solution) and 
centrifuging. Pipette off the serum and citrate solution, add physio- 
logical salt solution, mix, and again centrifuge; repeat the washing 
and centrifuging to remove the last traces of serum. Carefully 
pipette off the upper layer of corpuscles, rich in leukocytes, and 
keep in a small test-tube. Sometimes the defibrinated whole blood 
is used. In other cases, particularly with small experimental 
animals, an intraperitoneal injection of bouillon is made, and the 
leukocyte-rich exudate removed from the peritoneal cavity in the 
course of a few hours. 198 
VETERINARY BACTERIOLOGY 
Preparation of Bacterial Emulsion.-The organisms must be 
present in a perfectly homogeneous suspension. With most 
organisms a twenty-four-hour slant agar 
culture may be used, the growth scraped 
off into sterile physiological salt solution, 
triturated, and diluted until it shows only 
a faint opalescence. Filtration is sometimes 
necessary. This technic must be varied 
somewhat for different organisms. 
Preparation of Serum.-Both normal 
serum and serum from the animal to be 
tested are secured by bleeding, allowing 
the blood to clot, centrifuging, and taking 
off the serum with a pipette and transferring 
to a small tube. Before use, the serum is 
sometimes diluted-usually ten times. 
Fig. 80.-Opsonizing 
pipette containing the 
leukocytes, bacteria, 
and serum (Miller). 
Fig. 81.-Opsonic incubator (Miller). 
Technic of Test.-A capillary pipette with a fine bore is prepared 
from glass tubing, and a mark is made a short distance, usually OPSONINS AND PHAGOCYTOSIS 
199 
about 2 cm., from the end. The suspension of leukocytes is then 
drawn up to the mark, then an air-bubble is admitted, then the 
serum to be tested to the same mark, a second air-bubble, and 
finally an equal amount of the bacterial suspension. The contents 
of the tube are blown out upon a glass plate or into a watch-glass 
and thoroughly mixed. This mixture is again drawn some dis- 
tance into the capillary pipette and the end sealed in the flame. 
It is then kept at 37 ° for fifteen minutes, preferably in an opsonic 
incubator, where it may be rotated frequently to secure thorough 
mixture. The end is then broken from the pipette, and smears 
are made and stained with some good blood-stain, such as Wright's 
or Jenner's modification of the Romanowsky, which will stain 
bacteria. Special staining methods must sometimes be used, as in 
the examination of the tubercle bacillus. 
Determination of the Opsonic Index.-The total number of 
bacteria ingested by 100 polymorphonuclear leukocytes is 
counted and the average number per leukocyte calculated. This is 
termed the phagocytic index. This number is determined both 
with normal serum and the serum to be tested. The ratio between 
the number of organisms ingested by the leukocytes when treated 
with the specific serum and when treated with normal serum-that 
is, the ratio between the phagocytic indices-is called the opsonic 
index. In general, the opsonic index is found to be low (less 
than unity) in chronic bacterial infections. 
McCampbelVs Modification of Opsonic Test.-McCampbell has 
considerably shortened the technic in veterinary practice as fol- 
lows: The bacterial suspension containing 0.8 per cent, sodium 
citrate is drawn to the 0.5 mark on the leukocyte pipette of a 
hemocytometer, the specific blood to be tested is drawn to the 
same mark, then all drawn into the bulb, mixed, replaced in the 
capillary tube, and the whole wrapped with a rubber band and 
incubated. The same procedure is carried through with normal 
blood. The disadvantages of this method are the presence of 
sodium citrate, which interferes to a slight degree with action of 
opsonins, and the use of two lots of leukocytes from two animals, 
one diseased and the other normal. Smears and examinations 
are made as in the preceding. It is possible there may sometimes 
be intrinsic differences in these leukocytes, which render direct 200 
VETERINARY BACTERIOLOGY 
comparison between their activity somewhat misleading. These 
difficulties are more than outweighed by the increased ease of 
manipulation, and good results are commonly secured. 
The variation in the opsonic content of a patient's blood gives 
valuable information relative to the development of resistance. 
As stated above, in many chronic infections the opsonic index 
is below unity. In the treatment of tuberculosis in the human, 
by means of minute injections of tuberculin, the determination 
of the opsonic index has given much information of practical 
nature. The injection of bacterial vaccines or bacterins which 
have been carefully studied is followed at first by an initial lower- 
ing of the index. This is called the negative phase. Later, the 
index should rise above unity, when the positive phase is said to be 
established. If the index can be kept above unity, the prognosis 
is generally regarded as favorable. 
Methods of Opsonic Immunization.-Immunization due to 
increased opsonin is generally brought about by the introduction of 
bacteria or their products into the body. Such an immunity is, 
therefore, active. There is good reason to believe that injection 
of an opsonic serum in some cases confers some passive immunity. 
The most commonly practised method of increasing the opsonin 
content of the blood is by vaccination. This may be defined as 
the injection or inoculation with organisms or their products in 
such a way that the disease produced will run a benign course. 
Such a procedure in many cases induces the production of bac- 
teriolysins, in others opsonins, and in still others both. Vaccines 
may consist of either living or dead organisms. If the former, 
they may be either virulent or attenuated. Vaccination with viru- 
lent organisms is not frequently practised. Some organisms do 
not produce typical and serious diseases unless they enter the 
body by a certain channel. The injection of the Asiatic cholera 
organism subcutaneously is not followed by any serious results, 
but, when the alimentary tract is the infection atrium, the char- 
acteristic symptoms of the disease are produced. This fact has 
been used as the basis for practical vaccination against this dis- 
ease. Usually vaccines consist of attenuated organisms. Atten- 
uation or weakening or decrease of virulence is accomplished in 
several ways: in some cases by growing at high temperatures, as OPSONINS AND PHAGOCYTOSIS 
201 
for anthrax, heating to temperatures just below the thermal 
death-point, as in blackleg, by animal passage, as in small-pox, or 
by growth on artificial media, as with members of the hemorrhagic 
septicemia group. 
Vaccines consisting of dead organisms are called bacterins. 
These are prepared and sold commercially under various trade 
names. They usually consist of pyogenic organisms which have 
been killed by heat. The injection of these bacterins has been 
shown to increase the opsonic index for specific organisms when 
properly administered. It has been found, however, that there are 
many strains of some of the pyogenic bacterial species, for example, 
of Streptococcus pyogenes, and that vaccination against one strain 
does not always protect against another. Vaccines are, therefore, 
prepared containing organisms isolated from as many sources 
as possible and containing many races or strains. Such a vaccine 
is called polyvalent, a vaccine containing but one strain, univa- 
lent. 
Autogenic Vaccines.-Inasmuch as it cannot be readily deter- 
mined to what strain a particular organism causing a chronic 
bacterial infection, such as suppuration in a fistula, belongs, it is 
sometimes desirable to prepare a vaccine of this particular organ- 
ism for this single case. A vaccine prepared in this manner, to be 
used in the animal from which the organism was isolated, is said 
to be autogenic. The use of autogenic vaccines, carefully prepared 
and standardized, has proved successful in many cases in the treat- 
ment of stubborn and chronic suppurative conditions. The tech- 
nic of preparation and use follows. 
Isolation of Organism.-In many instances a slant agar culture, 
made directly by securing material on a sterile platinum loop 
from the deeper parts of the wound or abscess, will prove to contain 
only the causal organism. In most cases, however, it will be found 
advisable to plate directly in agar and incubate at blood-heat. 
An examination of the colonies will reveal the causal organism 
(or organisms in a mixed infection). 
Preparation of Vaccine (McCampbell).-Into each of two flat 
flasks or bottles, 100 c.c. of nutrient agar is placed, sterilized, and 
slanted. Streaks of the organism are made the full length of the 202 
VETERINARY BACTERIOLOGY 
slant and incubated twenty-four hours at blood-heat. The water 
of condensation is removed without disturbing the growth, and 
100 c.c. of sterile physiological salt solution added. The growth 
is carefully scraped from the surface with a long sterile glass rod; 
the suspension is placed in a sterile flask and shaken thoroughly. 
Standardization of the Vaccine.-Before injections are made, 
the number of bacteria present per given volume of the suspension 
must be known in order that accurate dosage may be determined. 
The standardization may be effected by direct count or by use of 
the nephelometer. In the first method, equal parts of the bac- 
terial suspension and of defibrinated human blood are thoroughly 
mixed, a smear prepared, and stained with a blood-stain or carbol- 
thionin. The number of red blood-cells on several fields and the 
number of bacteria on these fields are counted (see Fig. 79). The 
number of red blood-cells may be taken as 5,000,000 per cubic 
millimeter. The number of bacteria per cubic millimeter will be 
found by the following proportion: 
5,000,000 : x :: number red blood-cells per field : number bacteria per field. 
Suppose 20 red blood-cells per field, and 50 bacteria, the ratio 
becomes- 
5,000,000 : x :: 20 : 50 or x (number of bacteria per cubic cent ,erer) = 
12,500,000. 
To convert the number of bacteria per cubic centimeter, multiply 
by 1000, which in this case would give 12,500,000,000° It is cus- 
tomary to dilute the bacterial suspension so that each cubic cen- 
timeter will contain a number of bacteria that is r eadily reckoned, 
usually 50,000,000 per cubic centimeter. 
The nephelometer is an instrument described by McFarland, 
in which tubes containing varying amounts of barium sulphate 
in suspension in water are observed side by side with tubes of equal 
size containing the bacteria. After the tubes containing barium 
sulphate have been compared as to opacity with tubes containing 
known numbers of bacteria per cubic centimeter, they may be 
used in determining the number present in other bacterial suspen- 
sions. OPSONINS AND PHAGOCYTOSIS 
203 
Passive Opsonic Immunization.-It is probable that some anti- 
bacterial sera, as the antimeningococcic serum, owe in part their 
immunizing and their curative powers to the presence of opsonins. 
The part that these opsonins may play in passive immunization 
is not thoroughly understood. 
Leukocytic Extracts.-It has been found possible by Hiss 
and others to secure an antibacterial substance or endolysin 
from leukocytes by a process of autolysis. These leukocytic 
extracts have not been extensively used in practice, but they 
are of considerable theoretic importance. Leukocytes may be 
obtained by injecting 10 c.c. of a sterile solution of 5 per cent, 
aleuronat and 3 per cent starch into the pleural cavity of a rabbit. 
In twenty-four hours the animal may be killed and the pleural 
fluid rich in leukocytes removed, using every precaution to retain 
sterility. It is quickly centrifuged, the supernatant liquid re- 
moved, about 2 c.c. of sterile distilled water added, and the emulsion 
beaten up with a platinum spatula. Sterile water is then added, 
and the suspension incubated at 37° for eight hours. This allows 
time for relatively complete cell autolysis. Injection of such autol- 
ysate has been found to influence favorably the course of experi- 
mental pyogenic infections. The endolysins are not identical 
with bacteriolysins, but are relatively thermostabile, they cannot 
be reactivated when inactivated and are not increased by the 
process of immunization. 
Aggressins.-The term "virulence," as applied to microorgan- 
isms, is not readily defined. The reaction of an organism in the 
body cannot be predicted by its behavior in culture-media. The 
most virulent diphtheria bacillus, for example, is not necessarily 
the one that produces the largest amounts of toxin in culture- 
media. Concerning this mechanism of virulence there has been 
much discussion and speculation. 
Bail has developed what he has termed an aggressin hypothesis 
to account for these variations in virulence. He has defined an 
aggressin as a substance which is secreted by pathogenic organisms, 
when growing in the animal body, which will neutralize the efforts 
of the tissues to destroy the organism. The work on this subject 
has been done principally by Bail and his students, although many 
substantiating facts have been developed by other investigators. 204 
VETERINARY BACTERIOLOGY 
Sensitized Vaccines.-A method of vaccine preparation con- 
sisting of the suspension of living bacteria in their specific immune 
serum (serobacterin) has been proposed by Besredkaand Metchni- 
koff. The purpose of this is to sensitize the bacteria. According 
to the observations of these authors and others such vaccines 
produce only slight local and general reactions, the negative 
phase being practically eliminated, while there is a general and 
prompt response with antibody formation The specific immune 
serum may assist in the disintegration of the bacteria by furnish- 
ing either opsonin or a bacteriolytic amboceptor which with the 
natural complement of the tissues tends to cause a lysis of the 
bacterial cell. The experiments of Theobald Smith with toxin- 
antitoxin mixture, in which he showed that dissemination of 
toxin was much more general through the body when the mixture 
was administered than when toxin alone was given inclines Smith 
to the view that the same principle holds true with reference to 
sensitized bacteria. The theory is of practical importance, inas- 
much as it is claimed that the injection of bacteria-free aggressins 
into an animal results in the ultimate development of anti-aggres- 
sin and of an active immunity. 
Bail divides bacteria into three groups-true parasites, half- 
parasites, and saprophytes. The true parasites include such forms 
as the anthrax bacillus, in which the injection of a very small num- 
ber results fatally through a rapid multiplication of the organisms. 
The half-parasites are those having a lower degree of virulence- 
such that relatively large injections must be made to produce in- 
fection. The third group includes those totally devoid of patho- 
genic properties. The difference between these forms is a difference 
in the ability to produce aggressins. 
Aggressin production is demonstrated by Bail as follows: A 
guinea-pig is injected intraperitoneally with many times a fatal 
dose of culture of Bacterium ty phosum or Vibrio choleroe. The 
peritoneal exudate is removed after the death of the animal and 
centrifuged to remove the cellular elements, as well as most of the 
bacteria. The organisms remaining in the supernatant liquid are 
destroyed by a chemical disinfectant. If a small quantity of this 
exudate is injected into a guinea-pig, together with a quantity of 
the specific organisms that would usually be non-lethal, the animal OPSONINS AND PHAGOCYTOSIS 
205 
will die with an acute infection. The injected exudate apparently 
makes the organisms more virulent. The aggressins are contained 
in this exudate, and convert a mild infection into one that is fatal. 
The repeated injection of sterile aggressin causes the development 
in the animal body of an active immunity. According to Bail, 
anti-aggressins can be demonstrated in the blood-serum of such 
animals. One of the strongest links of corroborative evidence 
is the fact that the bacteriolytic power of a serum in vitro is not an 
index of the ability of the animal from which it was taken to resist 
infection. The modus operandi of the aggressin is to be sought 
in the inhibition of phagocytosis. Bail found that when typhoid 
bacilli are injected intraperitoneally, there is a marked increase in 
the number of phagocytes in the exudate within a few hours, but 
when they are injected with aggressin there is no such increase- 
the phagocytes are evidently not attracted. 
This theory has not been accepted in its entirety by many 
bacteriologists. The facts given by Bail are largely accepted, 
but the theory is not well established. Some authors claim to have 
shown that the so-called aggressin is simply endotoxin released in 
the body by the lysis of the bacterial cells. It is also claimed that 
there is no demonstrable difference between the endotoxins pro- 
duced in culture-media and the aggressins formed in the body. 
Peritoneal exudate containing aggressin, according to Bail, has been 
filtered through porcelain, and found to be toxic for guinea-pigs 
after filtration. Bail has attempted to develop practicable methods 
of immunization in several diseases by an application of the theory. 
None of his methods have come into general use. CHAPTER XVIII 
ANAPHYLAXIS AND HYPERSUSCEPTIBILITY 
The body normally takes its food through the intestinal tract,, 
or by enteral introduction. Any foreign proteins introduced in 
this manner are generally changed by digestion, so that they may 
be used by the body-cells or assimilated. When introduced by 
parenteral injection (outside the alimentary canal, as subcu- 
taneously, intravenously, etc.) antibodies of different kinds are 
produced. Thus far we have considered three antibodies which 
antagonize bacteria or other antigens which are introduced, but 
there exists still another type of body reaction, sensitization toward, 
rather than immunization against, an antigen. This phenomenon 
is exhibited only under certain conditions, and the body is then 
said to show anaphylaxis or hypersensitiveness to the antigen. 
The significance can best be understood by a study of specific 
examples. A number of these have been noted independently and 
are deserving of mention. 
Phenomenon of Arthus.-Arthus injected rabbits subcu- 
taneously with horse serum at six-day intervals. The first three 
injections were readily absorbed by the tissues, the fourth was 
followed by some edema at the site of injection, and, after the 
sixth or seventh injection, the skin at the site became gangrenous, 
and a deep abscess scar was finally formed. If the sensitized 
animal be injected intravenously with the serum, it appears rest- 
less, lies on its belly, and respiration frequently increases; it 
defecates frequently, finally falls upon its side, and commonly dies, 
all within the space of two or three minutes. 
Serum Sickness in Man.-Many observers have noted that the 
injection of antitoxic sera into man sometimes is followed by a 
fever, the appearance of a rash or urticaria, pains in the joints, etc. 
In some individuals the reaction is shown after the first injection, 
in most cases only after a second injection, given some time after 
206 ANAPHYLAXIS AND HYPERSUSCEPTIBILITY 
207 
the first. Evidently there may be an inborn or a developed sensi- 
tiveness in man to serum injection. 
Theobald Smith Phenomenon.-Theobald Smith made the 
observation that when guinea-pigs are injected with horse serum, 
and a second injection is given after the space of ten days or more, 
the pig will show signs of hypersensitiveness, and if a sufficient 
quantity, 5 or 6 c.c., is injected intraperitoneally, death will result 
in a few minutes. The first injection serves to sensitize against the 
second. This phenomenon in the guinea-pig is so striking that it 
has been used by many, investigators in a study of the reaction. 
The general phenomenon was given the name of anaphylaxis. 
This will be discussed, and its utilization in diagnosis and in ex- 
planation of certain hitherto obscure body reactions reviewed. 
Vaughn's Split Proteins.-Vaughn and Wheeler and their co- 
workers have succeeded in showing that proteins in general may be 
split by chemical means into a portion which when injected into an 
animal gives rise to symptoms resembling those of anaphylaxis, 
and a portion which is inert. The protein to be split is boiled in 
absolute alcohol containing 2 per cent, sodium hydrate. The toxic 
fraction is soluble in alcohol, the non-toxic insoluble. Each of 
these fractions gives the protein reactions and is to be classified 
as a protein or at least as a complex polypeptid or peptone. When 
injected into guinea-pigs these fractions behave quite differently. 
The alcohol-insoluble portion cannot produce anaphylactic symp- 
toms,. but can be used in sensitizing animals against the protein 
from which it was derived. The alcohol-soluble portion produces 
anaphylactic symptoms when injected, but does not sensitize 
against the protein. 
Mechanism of Anaphylaxis.-Many theories of the mechanism 
of the phenomenon of anaphylaxis have been proposed, but even 
yet many factors are unknown, and an entirely adequate ex- 
planation is lacking. The following facts seem to be firmly 
established: 
1. It is possible to sensitize a suitable animal by means of a 
great variety of proteins, such as blood-serum of various animals, 
egg-white, milk, plant proteins, bacterial proteins, and yeast pro- 
teins. Sensitization may be effected in many cases by the injec- 
tion of minute quantities of the proteins, in some cases only a frac- 208 
VETERINARY BACTERIOLOGY 
tion of a milligram is required. The protein may be heated without 
destroying its sensitizing power. 
2. It is possible to secure an anaphylactic shock in an animal 
that has been previously injected with a sensitizing dose of protein 
only after the lapse of a definite period following the first injection, 
the length depending in part upon the size of the sensitizing dose. 
3. The sensitization of an animal in general can be effected best 
by the parenteral introduction of the protein. Some cases are on 
record, however, of sensitization as the result of ingestion or rectal 
injections. 
4. The type of anaphylactic reaction secured differs with the 
species of animal, and to a less degree with the site and method of 
making the second injection, and the amount of protein used. In 
the guinea-pig the first sign of reaction is usually evidenced within 
a few seconds to a few minutes after injection. The animal then 
appears restless, and scratches its nose with its front feet. Usually 
urine and feces are passed. The breathing becomes more rapid and 
the animal falls on its side. There is evidence of dyspnea. Gener- 
ally the animal dies in convulsions, or in some cases quietly. Usu- 
ally the heart beats for a time after breathing ceases. If smaller 
quantities of protein are injected the animal may show the first 
symptoms, but gradually recovers. In acute anaphylaxis death 
may occur in two to five minutes. There is also a decided drop in 
temperature in animals showing anaphylactic shock, even though 
the reaction be a slight one. 
Rabbits show the anaphylactic reaction readily upon a second 
injection of the antigen. There is generally defecation, urination, 
and decided weakness, the body resting on the ground and the 
head drooping. Breathing becomes irregular and labored. Con- 
vulsion and death may follow promptly, or death may result only 
after several hours. 
In dogs the anaphylactic symptoms are; a decided fall in blood- 
pressure and evidence of cerebral anemia. The animals usually 
vomit, urinate, and defecate; they may gradually grow weaker and 
die. 
Anaphylactic shock has also been observed in cattle, horses, 
and goats. This is of some practical importance, as a repeated 
injection of a heterologous serum into one of these animals may ANAPHYLAXIS AND HYPERSUSCEPTIBILITY 
209 
give rise to severe symptoms or even produce death. For example, 
Bang attempted to immunize cows against infectious abortion by 
the injection of horse-serum broth-cultures of the organism at 
intervals. He found that the second injection was followed by 
very grave anaphylactic symptoms, necessitating the substitution 
of ox serum for horse serum in the preparation of the medium. 
5. The reaction is highly specific. An animal sensitized against 
a particular protein will react only when that protein is injected. 
6. A condition of passive anaphylaxis may be induced by the 
injection of blood-serum from a sensitized animal into a normal 
animal. Such an animal will react to the injection of the protein 
in the same manner as an actively sensitized individual. 
7. There is a very material diminution in the complement of the 
blood of an animal immediately following an anaphylactic shock. 
Experiments show that the complement itself takes some part in 
the production of the shock, for animals whose blood has been 
depleted of complement by other means do not react upon injection 
of the protein. 
8. A mixture of blood from a sensitized animal and of antigen 
will produce anaphylactic shock when injected into a normal ani- 
mal under certain conditions. It is probable, therefore, that the 
reaction is due primarily to the development of some poisonous 
substance in the blood as a result of the interaction of the protein 
and some antibody formed as a result of the sensitizing injection. 
Certain experiments, however, tend to show that in some cases at 
least there is an active participation of the body-cells in producing 
the reaction. 
9. The injection to produce the anaphylactic reaction gives 
the quickest results when made intravenously or into the heart. 
Intraperitoneal injection is somewhat less rapid in its action, and 
subcutaneous still less. 
10. An animal which has just recovered from an anaphylactic 
shock does not show the same symptoms when a second injection, 
is made soon after. Or if a small dose of protein is injected into 
the animal a large dose injected later may cause no reaction. It is 
possible that an explanation of this phenomenon of anti-anaphy- 
laxis, as it is called, is to be found in the fact that the first injection 
uses up all of the specific antibody formed. 210 
VETERINARY BACTERIOLOGY 
Many theories of anaphylaxis have been proposed, but none 
as yet seem adequately to explain all of the phenomena connected 
with it. The theory of Vaughn and Wheeler in many respects 
seems to fit conditions best. These investigators claim that the 
sensitizing dose of protein when injected is gradually broken 
up by the body into the two fractions corresponding to those 
secured by laboratory method (see above). The portion corre- 
sponding to their alcohol-insoluble fraction stimulates the tissues 
to the production of a specific antibody, a lysin, which can break 
down the protein readily. Gradually this lysin accumulates in the 
body fluids, and upon the injection of the second dose the protein is 
split rapidly and the toxic fraction formed so quickly and in such 
amounts as to give rise to the symptoms of poisoning. 
Relationship of Anaphylaxis to Certain Body Reactions.-The 
anaphylactic explanation of serum sickness in man has already been 
discussed. Several cases are on record where injection of diph- 
theria antitoxin has resulted in death within a few minutes. 
Rosenau and Anderson have recorded cases in which the prophy- 
lactic injection of antitoxic serum into normal individuals resulted 
in explosive manifestations of anaphylaxis, such as a prickling 
sensation in chest and neck, labored breathing, paralysis, convul- 
sions, and death within five minutes after injection. It seems 
evident that the individual was naturally hypersensitive. For- 
tunately, such cases are extremely rare. The prophylactic and 
curative value of diphtheria antitoxin far outweigh its dangers. 
Rosenau and Anderson have also recorded some evidence that 
certain of the toxemias of pregnancy, particularly puerperal eclamp- 
sia, are to be accounted for by a sensitization of the body by 
the cells of the placenta. It was found that guinea-pigs might be 
sensitized with the placenta of the same species by a single injec- 
tion, and a second injection later gave a typical anaphylactic 
reaction. 
Bacterial Anaphylaxis.-It has been shown by several investi- 
gators that proteins from bacteria may be used in sensitizing ani- 
mals against a second injection; in other words, they resemble 
proteins from other sources in this respect. Extracts from 
Bacterium coli, B. anthracis, Mycob. tuberculosis, Bad. typhosum, 
and others have been shown to sensitize. This fact has been ANAPHYLAXIS AND HYPERSUSCEPTIBILITY 
211 
deemed of considerable practical importance in disease diagnosis. 
There is reason to suppose that infection with certain bacteria 
will sensitize the body to the injection of the bacterial proteins or 
extracts or to the dead bodies of the organism. It has been 
found, for example, if the dead organisms, or extracts from them, 
be rubbed into the skin of an infected individual, an inflamma- 
tion, marked by some edema, redness, and frequently the forma- 
tion of papules, will occur. This is true in tuberculosis and some 
other diseases. That this is one of the manifestations of anaphy- 
laxis seems probable. The injection of tuberculin, consisting of 
dead tubercle bacilli and their products of growth, into an animal 
having tuberculosis will cause a characteristic rise in temperature, 
and is, therefore, of great diagnostic value. This reaction re- 
sembles the anaphylactic reaction in many ways. For example, a 
second injection following soon after the first will give no reaction; 
probably the body is in a state of anti-anaphylaxis. Further- 
more, advanced cases of the disease do not respond-perhaps 
they are either constantly anti-anaphylactic or show anaphylactic 
immunity due to the constant presence of large numbers of 
organisms in the body. On the other hand, there are some unex- 
plained differences between this reaction and that typical of 
anaphylaxis produced by other proteins. Many points remain 
still to be explained. That it is, however, of a similar nature 
seems to be altogether probable. A similar reaction may be 
secured with the emulsion of dead glanders bacilli (mallein) in 
glanders. SECTION IV 
PATHOGENIC MICROORGANISMS EXCLUSIVE OF THE 
PROTOZOA 
CHAPTER XIX 
The microorganisms which produce disease can best be studied 
after the related forms are classified into groups. The character- 
istics common to all members of a group are more easily remem- 
bered than the characteristics of each member of all the groups. 
A classification of microorganisms may be based upon the 
character of the disease produced, that is, it may be pathologic, or 
it may be based upon the characters of the organisms themselves, 
their morphology, physiology, and culture. A pathologic classi- 
fication is open to the objection that very different types of infec- 
tion may be produced under different conditions by the same or 
closely related organisms. Certain pus cocci, for example, may 
produce erysipelas, wound suppuration, septicemia, pyemia or 
local infection, and inflammation of any organ of the body. On 
the other hand, infections having somewhat similar clinical char- 
acters may be produced by very different organisms. 
The groupings used in this text will be based upon relation- 
ships rather than pathological resemblances. 
GROUPS OF PATHOGENIC MICROORGANISMS 
Main Divisions of Pathogenic Microorganisms 
The microdorganisms which cause disease may be separated 
into four principal groups: the bacteria, the yeasts, the molds, 
and the protozoa. The bacteria only will be subdivided here, 
the other main groups being reserved for later chapters. 
The pathogenic bacteria of veterinary interest may be divided 
into twenty-two groups. They may be differentiated by the 
following key: 
Principal Groups of Pathogenic Bacteria 
A. Cells spherical, cocci 
B. Cells occurring typically in chains 1. Streptococcus group 
BB. Cells not typically in chains 
212 GROUPS OF PATHOGENIC MICROORGANISMS 
213 
C. Cells typically in pairs 2. Specific pyogenic coccus 
group (Diplococcus and 
Neisseria) 
CC. Cells typically in irregular groups 3. Staphylococcus group 
AA. Cells not spherical 
B. Cells not forming long threads, not mycelium like 
C. Cells not spiral 
D. Cells not fusiform 
E. Producing endospores 
F. Aerobic 4. Anthrax group (Bacillus) 
FF. Anaerobic 5. Blackleg-tetanus group 
(Clostridium) 
EE. Not producing endospores 
F. Not acid fast 
G. Not hemoglobinophilic 
H. Gram negative 
I. Not producing 
a fluorescent pigment 
J. Polar stain- 
ing 6. Hemorrhagic septicemia 
group (Pasteurella) 
JJ. No polar 
staining 
K. Characteristic honey-like growth on 
potato 
7. Glanders group (Pfeiffer- 
ella) 
KK. Not as preceding 
L. Aerobic 
8. Intestinal group (Bac- 
terium) 
LL. Microserop hilic 
9. Bovine abortion group 
(Bacterium or Brucella) 
II. Producing a fluorescent pigment 
10. Fluorescent group (Pseu- 
domonas) 
HH. Gram positive 
I. Producing metachromatic granules. Aerobic 
11. Diphtheria group (Coryne- 
bacterium) 
II. No metachromatic granules 
J. Microserophilic 
12. Swine erysipelas group 
(Erysipelothrix) 
JJ. Hemoglobinophilic, or at least requiring 
serum for growth 
13. Influenza group (Hemo- 
philus) 
FF. Acid fast 14. Tuberculosis group 
( Mycobacterium) 
DD. Cells fusiform, elongate 15. Fusiform group (Fusi- 
formis) 
CC. Cells spiral 
D. Cells relatively short and plump 16. Vibrio group 
DD. Cells relatively long, slender and flexuous 
17. Spirochete group (Trepo- 
nema, etc.) 
BB. Producing long threads 14. Ray fungus group (Acti- 
nomyces, Actinobacillus, 
etc.) CHAPTER XX 
THE GROUP OF STREPTOCOCCI. THE GENUS STREPTOCOCCUS 
The Streptococcus group of bacteria includes all those forms 
whose cells are spherical and occur usually in chains of greater or 
less length. All of the species described are non-motile, and most 
are Gram-positive. Spores are never produced. The various 
species show differences in their ability to produce acid from various 
carbohydrates. In general, they give scant growth on the surface 
of culture-media, preferring anaerobic or micro-aerophilic condi- 
tions. 
The bacteria of this group are widely distributed, but are most 
common upon the skin of man and animals, in intestines, in milk, 
etc. They are not common as saprophytes in nature apart from 
animals or their excretions. 
A satisfactory classification of the streptococci has not yet been 
formulated, although a considerable amount of work has been 
done. 
Several species of Streptococcus are pathogenic. Many pyo- 
genic infections in man and more rarely in animals, mastitis of cer- 
tain types, brustseuche, strangles or distemper of equines, septi- 
cemia in birds, follicular vaginitis in cattle, petechial fever, possibly 
abortion in mares are to be ascribed to organisms of this group. 
As previously stated, no systematic effort to classify all of these 
forms on the basis of morphology and physiology has been suc- 
cessful. Lingelsheim in 1899 suggested that two types to be known 
as Streptococcus longus and Str. brevis be recognized, the more viru- 
lent forms he believed to produce longer chains usually of more 
than six cells, and the less virulent forms he found to form short 
chains. A strict adoption of this classification proved imprac- 
ticable because of variations in different media and under different 
environments. 
Schottmuller's classification proposed in 1903 the use of blood- 
agar in differentiation. The more virulent types he termed Strep- 
214 THE GROUP OF STREPTOCOCCI 
215 
tococcus longus or Str. erysipelatos. These formed long chains and 
hemolyzed the blood-cells. His Str. mitis or Str. vindans he found 
to be less virulent, to produce shorter chains, and to be devoid of 
the power of hemolysis. His Str. mucosus proved to be an encap- 
sulated form, and more closely related to the pneumococci. 
The classification proposed by Andrewes and Horder in 1906 is 
given here likewise, because it has been the basis of a considerable 
amount of recent work and because these names are frequently 
used. The summary of the groups as given by Buerger1 is as 
follows: 
(A) Streptococcus equinus: A saprophytic group in which most 
of the organisms ferment only saccharose and glucosides, Non- 
pathogenic. Found usually in horse dung. 
(B) Streptococcus mitis: Saprophytic, in the human saliva and 
feces, fermenting sucrose and lactose and not glucosides. 
(C) Streptococcus pyogenes: Long chained, hemolytic, acidifying 
milk without clotting; fermenting sucrose, lactose, and salicin. 
Most of the virulent streptococci belong here. 
(D) Streptococcus salivarius: Short chains, fermenting sucrose, 
lactose, and raffinose. Usually non-pathogenic. 
(E) Streptococcus anginosus, much like Str. salivarius. Clots 
milk, reduces neutral red, and produces acid in sucrose and 
lactose and often raffinose. 
(F) Streptococcus fcecalis: Short chain form which ferments lac- 
tose, sucrose, and mannite. Occasionally pathogenic, typically 
of intestinal origin. 
Holman's Classification of Species of Streptococci 
A. Non-hemolytic 
I. No acid from lactose 
(a) No acid from mannitol 
1. No acid from salicin Streptococcus ignavus 
2. Acid from salicin Str. equinus 
(b) Acid from mannitol 
1. No acid from salicin Str. non-hemolyticus III 
2. Acid from salicin Str. non-hemolyticus III 
1 Buerger, Leo, Jour, of Exp. Med., 9, 429, 1907, 216 
VETERINARY BACTERIOLOGY 
II. Acid from lactose 
(a) No acid from mannitol 
1. No acid from salicin Str. salivarius 
2. Acid from salicin Str. mitis 
(b) Acid from mannitol 
1. No acid from salicin Str. non-hemolyticus I 
2. Acid from salicin Str. fecalis 
B. Hemolytic 
I. No acid from lactose 
(a) No acid from mannitol 
1. No acid from salicin Str. subacidus 
2. Acid from salicin Str. equi 
(b) Acid from mannitol 
1. No acid from salicin Str. hemolyticus III 
2. Acid from salicin Str. hemolyticus II 
II. Acid from lactose 
(a) No acid from mannitol 
1. No acid from salicin Str. anginosus 
2. Acid from salicin Str. pyogenes 
(b) Acid from mannitol 
1. No acid from salicin Str. hemolyticus I 
2. Acid from salicin Str. infrequens 
It will be noted that the primary division in the preceding key is upon the 
basis of hemolytic power. 
The species of Streptococcus that will be considered here are 
Streptococcus pyogenes, Str. equi, Str. gallinarum, Str. vaginitidis, 
and Str. lacticus. 
Streptococcus pyogenes 
Synonyms.-Streptococcus erysipelatos; Str. puerperalis; Str. 
articulorum; Str. pyogenes malignis; Str. septicus; Str. scarlatinosus; 
The large amount of study devoted to the streptococci in recent years has 
led to many relatively elaborate classifications. That of Holman is impor- 
tant enough to justify inclusion. THE GROUP OF STREPTOCOCCI 
217 
Str. phlogogenes; Str. mastitidis; Str. agalactice vaccarum; Str. 
pyogenes boms. 
Pasteur first recognized the Streptococcus in pus, but Ogston, 
between 1880 and 1884, first definitely isolated and described it. 
Fehleisen, in 1883, found the organism in erysipelas, and Rosen- 
bach described it in detail and gave it its present name. 
The student will find no more puzzling group of organisms than 
the Streptococci. They have been found in connection with all 
types of inflammatory processes. Some strains are exceedingly 
Fig. 82.-Streptococcus pyogenes in lactose broth (Heinemann in "Journal of 
Infectious Diseases"). 
virulent, others wholly lack the power of disease production. 
Differences have been recorded in the cultural characters of isola- 
tions from different sources, and the species split into several on 
the basis of these variations. None of these classifications has 
proved to be wholly satisfactory, and, for the present, it is probably 
best to treat all the forms as varieties merely of one rather poly- 
morphic species. 
Distribution.-Streptococcus pyogenes does not adapt itself 
as readily to a saprophytic mode of existence as do the staphylo- 
cocci. It is commonly present upon the skin of man and animals, 218 
VETERINARY BACTERIOLOGY 
and has been isolated from a great number of different inflamma- 
tory and suppurative processes in both. 
Morphology and Staining Characters.-This organism is a 
coccus about 1 g in diameter, occurring in chains of greater or less 
length. Sometimes there is a tendency toward diplococcus 
formation, and the threads may be made up of many such diplo- 
cocci. Where two organisms approximate, they are more or less 
flattened. The organism is non-motile, does not produce spores, 
is easily stained by the common anilin dyes, and is Gram-positive. 
The length and character of the chains is variable in different 
media and under varying growth conditions, and likewise in strains 
isolated from different sources. This latter fact has been made use 
of by some investigators in an effort to perfect a classification. 
The typical form does not produce capsules, although capsulated 
varieties have been described. 
Isolation and Culture.-The organism may frequently be 
isolated in pure culture directly from the wound, but it is gen- 
erally necessary to pour plates and isolate from the colonies. 
Care must be used in this latter method not to overlook the 
colonies, for many strains produce minute colonies only, and 
in mixed infections they may be missed. Inasmuch as most 
strains ferment lactose, with the production of acid, the use of 
litmus-lactose-agar plates is sometimes helpful, the colony appear- 
ing surrounded by a zone of red. 
Growth occurs in most of the laboratory media, particularly 
upon the addition of a sugar, such as dextrose. The colonies 
upon agar and gelatin are small,-rarely larger than a pinhead, 
-at first transparent, and almost dewdrop-like. Later they 
may become somewhat opaque. The gelatin is not usually lique- 
fied, although saprophytic strains are known which possess this 
property. Whether these latter are typical Streptococcus pyogenes 
is uncertain. Upon agar slants this organism tends to grow in 
the form of discrete colonies. Bouillon is sometimes clouded by a 
uniform distribution of short chains; in other cases it remains clear, 
the organism growing in masses of long, tangled threads which 
remain as a sediment at the bottom. Blood-serum is unusually 
favorable as a medium. A growth is frequently produced upon the 
potato, though many strains refuse to develop on this medium. THE GROUP OF STREPTOCOCCI 
219 
Milk may or may not be coagulated, with acid production and no 
digestion of the curd. 
Physiology.-Streptococcus pyogenes is aerobic and facultative 
anaerobic. Its optimum temperature is about 37°, but growth 
will usually take place at room-temperatures. The thermal death- 
point differs in various strains-usually about 60° for fifteen 
minutes is sufficient to destroy. Antiseptics and disinfectants 
are efficient in destruction. No pigment is 
produced. No coagulating or proteolytic 
enzymes are developed in the typical strains, 
although, as indicated above, gelatinase has 
been reported in some saprophytic types. 
No indol is produced. No gas is developed in 
any medium. Acid is produced by most 
strains from many sugars, particularly dex- 
trose and lactose. Efforts have been made to 
classify the various types of Streptococcus on 
the basis of their acid production in various 
sugars. This seems to be helpful in determin- 
ing the origin of intestinal types in some cases. 
Pathogenesis.-Unexplained differences 
and variations in pathogenesis may be 
noted in the various strains which have 
been studied. Virulence for a given species 
of animal may be increased by passage 
through that species. 
Mechanism of Disease Production.-Little 
more is known of the mechanism of disease 
production with this organism than with the 
staphylococci. Neither the hemolytic streptolysin nor the endo- 
toxins, which have been described, seem adequately to explain its 
pathogenesis. Changes are usually brought about in tissues 
which are in more or less intimate contact with the organism. 
No class of infections shows better the necessity of virulence of 
an organism and lack of resistance on the part of the tissues in 
order to bring about pathologic changes. 
Experimental Evidence of Pathogenesis.-Inoculation experi- 
ments of animals have duplicated practically every infection with 
Fig. 83.-Streptococ- 
cus pyogenes on a slant 
agar culture (Frankel 
and Pfeiffer). 220 
VETERINARY BACTERIOLOGY 
which this organism has been found associated in man and ani- 
mals. The causal relationship of the organism to many infec- 
tions has been abundantly demonstrated. All laboratory animals 
may be infected with strains exhibiting sufficient virulence. 
Disease and Lesions Produced.-Streptococcus pyogenes is asso- 
ciated as primary cause with a long list of infections in both man 
and animals. As a secondary invader it is of great importance 
in many other diseases. Mixed infections with Staphylococcus 
aureus and other organisms are common. Some of the more 
important are worthy of note. 
Wound Infection and Suppuration.-Streptococcus pyogenes is 
not as common in surgical wounds and other traumata as the 
Staphylococcus aureus and 3. albus. Karlinski, in 1890, examined 
suppurative processes in man, animals, and birds, and found 
that Streptococcus was present in about 22 per cent, of the cases 
in man, 27 per cent, in lower animals, and 15 per cent, in birds. 
Staphylococci were present in about 70 per cent., 54 per cent., 
and 55 per cent, respectively. Lucet found Streptococci alone 
in 9 cases, and associated with other organisms in 10 cases, out of 
a total of 52 examinations of abscesses in cattle. According to this 
author Streptococcus pyogenes is not commonly found in suppuration 
in horses, and this result has been repeatedly confirmed. Pus 
production as a result of infection with Str. pyogenes in sheep and 
in swine is probably rare. 
Septicemia and Pyemia.-When an unusually virulent Strepto- 
coccus gains entrance to the blood-stream it may cause septicemia 
(blood-poisoning). Direct growth through a blood-vessel wall 
may result in the formation of an infected blood-clot, and later, 
when broken up, this produces thrombi and may lead to em- 
bolism. Abscess formation proceeds at the new foci of infection. 
Multiple metastatic abscess formation of this character is known 
as pyemia. 
Erysipelas.-This infection in man is characterized by a severe 
inflammation of the skin, in which this organism is present in the 
lymph-spaces of the subcutaneous tissue. Erysipelas seems to be 
due to an invasion with a peculiarly pathogenic organism and to a 
lack of resistance on the part of these tissues. Somewhat similar 
lesions have been noted upon lower animals. The erysipelas of THE GROUP OF STREPTOCOCCI 
221 
swine must not be confused with this disease, as it is caused by a 
totally different organism. 
Infection of Mucous Surfaces.-Tonsillitis in man, enteritis in 
children, non-diphtheritic anginas, and similar inflammations of 
mucous surfaces are commonly caused by Streptococcus. Puer- 
peral fever, an infection of a mucous surface following childbirth 
and its consequent septicemia, has been demonstrated in many 
cases to be due to Streptococcus infection, not infrequently con- 
tracted from a case of erysipelas. Less is known of the relation- 
ship of Streptococcus to related diseases in animals, though doubt- 
less it plays an important part. 
Peritonitis following an enterotomy is frequently due to infec- 
tion of the peritoneum with Streptococcus pyogenes, although other 
organisms are equally capable of giving rise to this condition. 
Pneumonia, particularly the traumatic pneumonia of the horse, 
may be caused by this organism. The so-called contagious pleuro- 
pneumonia of the horse is believed by many investigators to be 
due to an organism which has not been shown to differ materially 
from Streptococcus pyogenes, except for its exceptional virulence and 
mode of attack. The disease is contagious, and in this respect 
differs from most types of streptococcic infection. 
Ulcerative Endocarditis.-This affection is produced most com- 
monly by Streptococcus, although a Staphylococcus is found in 
some cases. Cauliflower-like excrescences of the heart-valves, 
with consequent valvular insufficiency and embolism due to the 
breaking off of these particles, are the characteristic lesions. 
Arthritis.-Inflammation of the bone covering (periostitis), 
infections of the bone-marrow (osteomyelitis), and of the joints 
(arthritis) are generally the result of streptococcic invasion. 
In all newborn animals there is danger of infection through the 
navel, and consequent production of "navel ill" (omphalophlebi- 
tis). The umbilical vein, according to Moore, becomes heavily 
infected with bacteria, which invade the joints by metastasis. 
The selective action of the organisms in infecting certain tissues 
at one time and others at another is not well understood. Rheu- 
matic fever in man (acute articular), and probably in animals, 
has been shown to be sometimes due to Streptococcus pyogenes. 
The infection atrium in man appears to be the tonsils. 222 
VETERINARY BACTERIOLOGY 
Suppurative Cellulitis-Under this heading Moore has described 
the streptococcic infections of the subcutaneous tissues, par-r 
ticularly of the lower extremities. In sheep it is called locally 
"foot-rot." 
Mastitis is commonly caused by Streptococcus. Here again 
the particular strain selects a particular organ, and may be trans- 
ferred from one animal to another by the hands of the milker. 
Several different species of the Streptococcus have been described 
as associated with garget or infectious mastitis, but there does not 
seem to be any good reason for believing them to be anything but 
specialized strains of the Streptococcus pyogenes. As will be seen 
later, the presence of Streptococcus in milk does not necessarily 
predicate mastitis, for non-pathogenic forms are common. Ac- 
cording to Sven Wallan Ernst the mastitis streptococci can be 
differentiated from the non-pathogenic forms by the possession of a 
thin capsule-like membrane, which in some cases becomes consid- 
erably thickened. Furthermore, the pathogenic strains do not 
sour milk. 
Septic Sore Throat.-Several epidemics in man of septic sore 
throat have been traced to milk, and in some cases to milk from 
cows suffering from mastitis. 
Petechial fever or Morbus maculosus of the horse has been com- 
monly ascribed to infection with Streptococcus pyogenes. 
Immunity.-A hemolytic toxin, streptolysin, may be demon- 
strated in some strains of Streptococcus pyogenes. For this an 
antitoxin has been produced. It seems probable that there is 
also a toxin which injures or destroys leukocytes. These toxins 
vary in amount, however, in cultures of different strains, and seem 
to vary independently of the virulence. They certainly do not 
account for the pathogenic nature of the organism. Agglutinins 
may occasionally be demonstrated, but are not of diagnostic 
importance. Endotoxins are produced by both virulent and non- 
virulent strains. Bacteriolysins are probably not important. 
Opsonins, normal and immune, and the phagocytosis induced by 
them probably explain any immunity which is exhibited by the 
body. This immunity, like others produced by opsonins, is not last- 
ing; in fact, it is so transient that it may be said that an attack of 
erysipelas, for example, renders one even more subject to recurrence. THE GROUP OF STREPTOCOCCI 
223 
Treatment by use of the bacterins, and particularly autogenic 
vaccines, has been found successful in chronic suppurations in 
both man and animals. In acute attacks it is of little or no value. 
It seems to be the consensus of opinion among investigators that 
the use of a polyvalent vaccine is more efficacious than a univalent 
when it is not autogenic. 
Antistreptococcic sera are produced for use by both the veter- 
inarian and the physician. The serum of Marmorek is prepared 
by the use of a culture having such virulence for rabbits that 
1,000,000 c-c- proves fatal. Horses are immunized by repeated in- 
jections of such broth cultures and their serum used. This seems 
to be of distinct benefit 
in immunizing passively 
an animal against the 
same strain, but does not 
protect against others. 
Polyvalent sera are pre- 
pared by immunizing 
horses against a con- 
siderable number of 
strains of the Strepto- 
coccus. The results from 
the use of such sera have 
not proved as successful 
as hoped, though some 
have reported excellent 
results. Such a serum 
doubtless owes its protective influence to its opsonic content, but 
Hektoen and Ruediger have shown that in some antistreptococcic 
sera on the market the opsonin content was below normal. The 
advisability of the use of antistreptococcic serum in general strep- 
tococcic infections in veterinary practice cannot be determined 
at present from the data available. It is possible that in some 
diseases, particularly of equines, a serum, either curative or pro- 
phylactic, prepared from a homologous organism, may prove prac- 
ticable and helpful. 
Diagnosis.-Smears of pus stained with methylene-blue or 
by Gram's method will usually reveal the organism if present 
Fig. 84.-Streptococcus pyogenes in pus 
(Frankel and Pfeiffer). 224 
VETERINARY BACTERIOLOGY 
in any numbers. Sometimes the pus seems to be practically 
sterile upon microscopic examination, but cultures prepared from 
it will reveal the presence of the Streptococcus. 
Transmission and Prophylaxis.-The constant presence of 
various strains of this organism upon the hair and skin makes the 
prevention of its entrance into wounds difficult. Cleanliness 
and the use of mild antiseptics are the only practicable methods 
of inhibiting its growth. In those infections which are character- 
ized by a specialized organism, and which are more or less con- 
tagious, isolation of infected animals and quarantine of those 
exposed, with disinfection of barns, particularly stalls and mangers, 
are the methods which must be used in checking the spread. 
Streptococcus equi 
Synonym.-Streptococcus coryzoe contagiosce equorum. 
Disease Produced.-Strangles or distemper in equines. 
The disease strangles in horses has a long recorded history. 
It was described by Solleysel in 1664, and its contagious nature 
determined by Lafosse in 1790. Schutz (1888) first isolated and 
described the organism now generally considered to be the specific 
cause. 
Distribution.-The disease is quite widely distributed both in 
Europe and America. It generally attacks young animals. 
Morphology and Staining Characters.-The organism is a 
Gram-positive Streptococcus, resembling the Streptococcus pyogenes 
in both morphologic and staining characters. Some authors 
state that the organism is Gram-negative, for it is decolorized if 
the alcohol remains too long in contact. Even forty-five seconds' 
exposure to alcohol is sufficient for decolorization. The chains 
in pus are usually long and twisted. 
Isolation and Cultural Characters.-The organism may be iso- 
lated in pure culture, in most cases from the deeper portion of the 
characteristic abscesses, by the same methods used for Strepto- 
coccus pyogenes. Preliminary purification by mouse injection may 
be necessary. Bureschello claims that in many cases strangles 
is a mixed infection of Staphylococcus aureus and Streptococcus equi. 
In such cases plating would be necessary to differentiate the species 
of organisms present. The media for growth of Str. equi must be THE GROUP OF STREPTOCOCCI 
225 
slightly alkaline to phenolphthalein. The addition of glycerin or 
glucose favors growth. The best growth is secured upon solidified 
horse serum, serum agar, serum broth, and sugar broth. On agar 
the organism produces thin, bluish, transparent colonies. In the 
water of condensation there is a whitish flocculent precipitate. 
In agar stabs characteristic outgrowths from the line of puncture 
develop. Hemolysis is produced on blood agar. Gelatin is not 
suitable as a culture-medium because of the temperature require- 
ments of the organism. In milk there is good growth without 
change in the appearance of the medium. On potato there is no 
visible surface growth. 
Physiology.-A temperature of 60° will destroy the organism 
in an hour; of 80°, in thirty minutes. It is destroyed readily by 
direct sunlight and by disinfectants. The organism does not 
persist in nature for a long period outside the animal body. It 
grows well only at blood-heat. According to Holth it produces 
acid from glucose, mannose, galactose, fructose, maltose, cello- 
biose, sucrose, glycogen, dextrin, soluble starch, salicin, and in 
slight degree from arbutin. No acid is formed from sorbose, xylose, 
arabinose, rhamnose, glycoheptose, trehalose, formose, gentio- 
biose, lactose, raffinose, inulin, • sorbit, mannit, dulcit, adonit, 
glycerin, erythrit, perseit, and amygdalin. It is claimed that the 
sugar reactions are sufficient to make certain the differentiation 
from other organisms found in the horse. 
Experimental Evidence of Pathogenesis.-Pus from abscesses 
in strangles kills white mice, with evidence of acute septicemia in 
two to four days, or after a longer period as a result of pyemia. 
An abscess generally develops at the point of inoculation. Rab- 
bits and guinea-pigs succumb to the injection of large quantities 
of the organism, but are not readily infected. Cattle, sheep, 
swine, dogs, and birds are very resistant. Subcutaneous inocu- 
lations of the horse will usually provoke abscess formation at the 
point of inoculation. Typical strangles in horses was caused by 
Schutz and others by the use of pure cultures. The introduction 
of the pure culture into the nose of young susceptible horses gave 
rise to the characteristic purulent catarrh with secondary abscess 
in the lymph-nodes. It seems evident, however, that there must 
be some predisposing factor to the disease in most cases. 226 
VETERINARY BACTERIOLOGY 
Inoculations upon the nasal mucous membrane frequently fail 
to infect, particularly when old cultures are used, for the organism 
rapidly loses its virulence in culture-media. Muller and others 
have shown that ingestion is the common method of infection. 
Some authors have been inclined to believe that the true cause of 
strangles has not been discovered, and that Streptococcus equi is a 
secondary invader, but the work of Muller, Schutz, and others in- 
dicates that this organism stands in strict etiologic relationship to 
the disease. Age is a predisposing factor, young animals are 
most commonly infected. Other predisposing causes are fatigue 
and exposure to cold, hard work, or any other factor that lowers 
the vitality. Variations in virulence wholly unexplained probably 
account for the epizootic character of the disease. 
Diseases and Lesions Produced.-The infection atria are 
probably the upper air-passages. The disease may be produced 
in simple or malignant form. A catarrhal discharge, with inflam- 
mation of the nasal mucous membranes, is generally first noted, 
followed quickly by a swelling of the adjacent lymphatic glands and 
of the submaxillary and pharyngeal lymph-nodes. These gener- 
ally develop into abscesses. The infection spreads through the 
lymph-channels, but generally remains localized in the tissues 
adjacent to the original point of infection. Metastatic infections 
may occur in practically any of the organs of the body, and in the 
chronic types of the disease great variations in localization will be 
found. Fatal termination is rare (0 to 3 per cent.), but sometimes 
occurs, due to septicemia, pyemia, or pneumonia. 
Immunity.-Toxins and antitoxins, agglutinins, and bacterio- 
lysins have not been satisfactorily demonstrated for this organism. 
Immunity is conferred by an attack of the disease, but is transitory, 
and the same animal may suffer from the disease a second time. 
Animals over five years old are quite generally immune. Prob- 
ably immunity can be best accounted for by increased opsonin 
content of the blood and consequent stimulation of phagocytosis. 
Immunization by vaccination with living and with dead cul- 
tures has not given wholly satisfactory results. Possibly the 
injection of autogenic cultures may have a protective influence. 
Todd has prepared a vaccine by growing the organism on blood- 
serum for twenty-four hours, then large flasks, containing 10 per THE GROUP OF STREPTOCOCCI 
227 
cent, serum broth, are inoculated and incubated a month. Six 
per cent, of sterile glycerin is added, and the material concentrated 
at 60° for two days over unslaked lime. The organism is destroyed 
by this means and the material evaporated to a thick paste. This 
is diluted and used as a vaccine. He has reported favorable 
results. Kitt prepared a bacterin by heating serum bouillon cul- 
tures to 53-55°. Marxer used a bacterial extract prepared by 
shaking a culture with 25 per cent, urea or galactose for four and 
one-half days. The use of the serum of animals recovered from the 
disease and hyperimmunized by repeated injections has been fol- 
lowed by even better results. Certain veterinarians have secured 
unusually good results by the prophylactic injection of such a 
serum. The immunity conferred is not permanent. The cura- 
tive effect seems sufficient to warrant its use in many cases. 
Jensen concludes that in incipient cases, where only catarrhal in- 
flammation of the nasal mucosa is in evidence, the disease is often 
aborted; but that the action in more advanced cases is relatively 
uncertain, particularly if suppurative lesions have developed; 
and that in pyemic and complicated cases it is quite valueless. 
Diagnosis.-Presumptive bacteriologic diagnosis may be made 
by identification of a Gram-positive Streptococcus from the lesions 
characteristic of the disease. 
Transmission probably occurs frequently by the use of com- 
mon drinking troughs and by ingestion of infective food, per- 
haps rarely by inhalation. The fact that the organism does not 
persist long outside of the body indicates that infection is frequently 
the result of direct contact. The work of Muller indicates that the 
most common infection atrium is probably the upper part of the 
alimentary tract, the organisms gaining access to the lymph- 
glands. It is probable that the organism may remain in the glands 
and follicles of the nasal mucosa after recovery of some animals, 
and such animals might easily infect others. 
Streptococcus gallinarum 
Synonyms.-Streptococcus of Norgaard and Mohler. Strep- 
tococcus capsulatus gallinarum (?). 
Disease Produced.-Apoplectiform and other septicemias in 
chickens. 228 
VETERINARY BACTERIOLOGY 
This disease was first described, and its cause isolated, by 
Nbrgaard and Mohler in 1902. The same disease was studied by 
Moore and Mack in 1905. Damman and Manengold (1906) 
recorded an epidemic of "fowl sleeping sickness," due to a Strep- 
tococcus, and later, in 1908, noted a similar outbreak. 
Distribution.-The disease has been reported from the original 
outbreak in Virginia, from northern New York, from Sweden, 
and a similar, if not iden- 
tical, disease from Ger- 
many. 
Morphology.-The or- 
ganism is a typical Strep- 
tococcus with chains of 
variable length, cells 0.6- 
0.8 in diameter, stains 
readily by the ordinary 
anilin dyes, and is positive 
to Gram's stain. With the 
exception of the doubt- 
ful difference in diameter, 
there appear to be no mor- 
phologic characters which 
differentiate it from Strep- 
tococcus pyogenes. The German type is described as forming 
capsules in the blood, and may be entirely distinct. 
Isolation and Culture.-The organism may be isolated from the 
blood and internal organs of affected fowls. It does not produce 
sufficient acid to coagulate milk, but differs culturally otherwise 
in no marked degree from Str. pyogenes. 
Pathogenesis.-Experimental Evidence.-Inoculations of pure 
cultures into fowls, rabbits, mice, and swine are fatal, while those 
into the guinea-pig, sheep, and dog are not. 
Disease and Lesions Produced.-The disease is a typical sep- 
ticemia, marked by parenchymatous degenerations and hemor- 
rhage. Nbrgaard and Mohler state that fowls frequently die in 
twelve to twenty-four hours after the first symptoms. 
Immunity.-Little is known relative to the causal organism 
and its products. Probably they differ little, if at all, from those 
Fig. 85.-Streptococcus gallinarum (Mag- 
nusson) . THE GROUP OF STREPTOCOCCI 
229 
of Str. pyogenes. The discoverers state that active immunity 
may be conferred by the injection of bouillon culture filtrates and 
by vaccination with killed cultures, and passive immunity by the 
injection of the blood-serum of an immunized animal. 
Bacteriologic Diagnosis.-The organism may be identified as a 
Gram-positive organism in smears from the blood and internal 
organs and by culture. 
Transmission.-According to the German authorities the 
disease is not very contagious, but its appearance in considerable 
numbers of fowls at one time remains unexplained. 
Streptococcus vaginitidis 
Synonym.-Streptococcus of Ostertag. 
Disease Produced.-Contagious granular or verrucose vaginitis 
in cattle. 
Ostertag, in 1898, isolated and cultivated a Streptococcus 
from the purulent discharge and from the deeper layers of the 
mucous membrane in cases of infectious vaginal catarrh. His 
findings have been confirmed by the work of Hecker, Raebiger, 
and Hess. The latter edited a report of the Swiss Veterinary 
Surgeons Society in 1903, which gives a very complete summary of 
the knowledge of the disease and its cause. 
Distribution.-The disease has been reported from all parts 
of Europe, and is wide-spread in North America. 
Morphology.-The organism occurs in short chains of six to 
nine individuals, held together by a delicate capsule. It stains 
readily with common anilin dyes, and is decolorized by Gram's 
method. This latter differentiates it sharply from the Str. pyog- 
enes. 
Isolation and Culture.-Isolation may be made upon agar or 
gelatin. Plate cultures are usually necessary, as there are gener- 
ally many other bacteria constantly present in the infected vagina. 
Growth occurs on gelatin (without liquefaction), blood-serum, 
and agar, particularly glycerinizecl. It produces a diffuse cloud- 
ing of bouillon. Acid production is so weak that milk is not 
coagulated. 
Physiology.-The organism is aerobic and facultative anaerobic. 
Acids are produced in small quantities, if at all, in carbohydrate 230 
VETERINARY BACTERIOLOGY 
media. The optimum growth temperature is blood-heat, but 
good growth occurs at room-temperature. 
Pathogenesis.-Experimental Evidence.-The organism is not 
pathogenic for any of the laboratory animals, nor can it produce 
disease in horses, hogs, sheep, or dogs when inoculated into the 
vagina. Inoculation of pure cultures into the vagina of heifers 
has been found to reproduce the disease, so that there seems to be 
little doubt of its etiologic relation to the disease. It seems to be 
an example of extreme specialization, such as is found in the organ- 
ism causing gonorrhea in man. 
Disease and Lesions Produced .-The disease in an acute form 
produces a swelling of the labia of the vulva, with increased secre- 
tion from the mucous membranes of the vagina. Later the dis- 
charge becomes purulent, then granules from the size of a pin- 
head to a rape-seed develop. These are the enlarged lymph-fol- 
licles of the mucous membrane. The acute stage lasts usually 
for several weeks, the discharge becomes less prominent, and 
finally the chronic stage sets in, and may persist indefinitely or 
gradually disappear. The organism may be isolated from the 
pus in the acute stage, or from the deeper layers of the mucosa 
in the later stages. In the bull the glans penis may show gran- 
ules similar to those of the vagina, or there may be a purulent 
catarrh of the prepuce. In the cow the disease may involve the 
uterus and possibly produce abortion and even sterility. 
Immunity.-Recovery from the disease presumably results 
from the development of an active immunity, perhaps opsonic 
in nature. No means- of artificial immunization have been 
employed. 
Bacteriologic Diagnosis.-The presence of a Gram-negative 
Streptococcus in the depths of the mucous membranes should be 
diagnostic of the disease. Identification in the discharges would 
necessitate cultures. 
Transmission.-The disease is probably most commonly trans- 
mitted by coition or by immediate contact with soiled litter and 
fodder. THE GROUP OF STREPTOCOCCI 
231 
Other Streptococci of Uncertain Significance 
Streptococcus mastitidis sporadicae.-{Str. agalactice conta- 
giosce; Str. der infektidsen Induration des Enters). 
Organisms of mammitis or mastitis in cattle have been found 
in all parts of the world, frequently associated with the disease in 
epidemic form. This organism was originally reported as Gram- 
negative, but the Str. mastitidis, Gram-positive, is reported by the 
local government board in England as the commoner type. There 
are no good differential characters other than pathogenesis to 
separate this latter form from Str. pyogenes and Str. lacticus. 
On account of the general occurrence of this latter species in milk, 
direct microscopic examination of the milk is often insufficient 
for the purpose of determining the condition of the udder, i. e., 
whether or not it is infected with garget. 
Streptococcus Sp.-Epizootic pleuropneumonia in equines. 
Stable pneumonia. 
A number of investigators have demonstrated a Streptococcus 
present in the lesions of certain pneumonias in the horse. The 
organisms isolated by different investigators have not always the 
same cultural, morphologic, and physiologic characteristics. By 
some the organism is regarded as identical with Str. equi, by 
others as a specific type, and by still others it is believed that 
the specific organism is still unknown, and the Streptococci de- 
scribed are but secondary invaders. An organism resembling the 
human pneumococcus, if not identical with it, has been isolated 
from some cases of equine pneumonia, and will be considered in 
connection with that organism. 
Streptococcus factious 
Synonyms.-Bacillus lactis acidi; Streptococcus lactis; Str. acidi 
lactici; Bacterium lactis acidi. 
Strangely enough, the organisms first isolated from milk, and 
described as the cause of its souring, are not the ones now regarded 
as most commonly associated with this change. These were mem- 
bers of the intestinal group, or bacilli that produce both acid and 
gas in milk. Leishman, in 1899, gave the name Bacillus lactis 
acidi to an organism which he isolated from soured milk, and re- 
garded as the common cause of this fermentation. Kruse later 232 
VETERINARY BACTERIOLOGY 
showed that Leishman had been mistaken in regarding it as a 
bacillus, and renamed the organism Streptococcus lacticus. Since 
then experimental data have accumulated, which seem to demon- 
strate quite conclusively that normal souring of milk is brought 
about in the vast majority of cases by this organism. The work of 
Heinemann in this country served materially to clear up the rela- 
tionship between this and other forms. 
Distribution.-Streptococcus lacticus is found generally in milk, 
butter, and cheese, upon the skin and hair of cattle, and in feces. 
Isolation, Morphology, and Culture.-This organism may be 
most easily isolated from milk by plating in litmus-lactose gelatin. 
The characteristic non-liquefying colonies surrounded by red 
may be easily recognized. Its morphologic characters differ 
in no marked degree from Streptococcus pyogenes. Culturally, 
Fig. 86.-Streptococcus lacticus (Heinemann, in "Journal of Infectious Dis- 
eases"). 
many strains of this latter organism appear to be identical with 
Str. lacticus. In fact, the resemblance is so close that there is a 
good reason to believe that Str. lacticus is a strain of Str. pyogenes 
which has adapted itself to a saprophytic life. 
Physiology.-Its physiologic characters differ in no noteworthy THE GROUP OF STREPTOCOCCI 
233 
degree from the preceding. Acid is produced from dextrose and 
lactose. According to Heinemann the dextro acid is produced 
by this organism and the levo by the B. lactis aerogenes and other 
members of the intestinal group. 
The heat resistance of Str. lacticus is of particular importance 
in connection with pasteurization of milk. It has been generally 
regarded that pasteurization would certainly kill all organisms 
of this type, and it has been believed that pasteurized milk would 
contain only the spores of putrefactive bacteria, and if improperly 
handled would "rot." Ayers and Johnson have shoym recently 
the falsity of this assumption. There are practically always 
present in any given sample of raw milk one or more strains of 
the lactic-acid organism which will resist the temperature of the 
pasteurizer, and will bring about normal souring in the milk 
pasteurized. 
The production of lactic acid in milk is of considerable im- 
portance in preventing the growth of undesirable bacteria. Milk 
entirely freed from these forms, as by heating to the boiling-point, 
does not sour, but undergoes a putrefactive fermentation, brought 
about by organisms ordinarily held in abeyance by the develop- 
ment of the lactic acid. 
Pathogenesis.-Heinemann, by a series of animal inoculations, 
has shown it possible to exalt the virulence of typical Str. lacticus 
so that it would kill rabbits as quickly as virulent Str. pyogenes. 
The close relationship of the two forms can scarcely be ques- 
tioned. Before the work of Heinemann it had been concluded 
by many workers that the presence of streptococci in milk was 
necessarily an indication of udder infection, and examination for 
streptococci was made a part of the routine work of certain board 
of health laboratories. Inasmuch as it is now known that the 
Str. lacticus is normally present in practically all milk, it is evident 
that such examinations are of little use. A streptococcic milk 
standard is not practicable. 
Utilization.-The statement is common in the literature that 
different strains of Streptococcus lacticus are used in pure culture 
as starters for the purpose of ripening cream in the manufacture 
of butter. The work of Hammer and others has recently 
demonstrated that this statement scarcely gives the exact status. 234 
VETERINARY BACTERIOLOGY 
It has been shown that the desired aroma and flavor characteris- 
tic of good butter are due to the adsorption by the butter fat of 
certain volatile acids produced by bacteria in the ripening cream. 
These are not produced by the Streptococcus lacticus itself, but by 
certain associated organisms, particularly by Streptococcus 
citrovorus. The latter organism produces the desirable volatile 
acids probably in large part from the citric acid of the milk and 
from the lactic acid produced by Streptococcus lacticus. A desir- 
able starter is one containing a suitable mixture of Streptococcus 
lacticus and. Streptococcus citrovorus. The desirable flavor is the 
result therefore of an associative action. CHAPTER XXI 
THE GROUP OF SPECIFIC PYOGENIC COCCI. THE GENERA 
DIPLOCOCCUS AND NEISSERIA 
The group of diplococci includes those organisms which when 
single are usually spherical or oval in shape, and whose cells are 
usually in pairs. All the organisms are non-motile and do not 
produce spores. They are usually strict parasites. 
The organisms of this group may be divided into two genera, 
the separation being made upon the basis of the reaction to the 
Gram stain. The Gram-positive form is Diplococcus pneumonice, 
the Gram-negative forms Neisseria meningitidis, N. gonorrhoea, 
and N. intracellular is equi. Not all of these produce disease in 
animals, but all are of sufficient importance to warrant brief dis- 
cussion here. 
Diplococcus pneumoniae 
Synonyms.-Pneumococcus; Streptococcus pneumoniai; Dip- 
lococcus lanceolatus; Micrococcus pneumonice; Micrococcus lanceo- 
latus. 
Disease Produced.-Pneumonia in man, and probably some 
animals, particularly the horse. 
Sternberg, in 1880, described this organism from normal 
sputum, but Frankel, in 1885, first definitely associated the organ- 
ism with croupous pneumonia. Considerable difficulty early 
arose, due to the confusion of two distinct organisms; namely, 
the form under consideration with the pneumobacillus of Fried- 
lander. A somewhat similar organism, possibly a Gram-negative 
variety of this form, has been found by Mayer associated with 
pneumonia in the horse. 
Distribution.-Throughout the world, in diseased and healthy 
individuals. 
Morphology and Staining.-Usually occurs in twos, more rarely 
in chains of four or six, spherical or more generally flattened at 
the point of contact, and with opposite side somewhat elongated 
and pointed, whence the name, lanceolatus. Capsules may be 
235 236 
VETERINARY BACTERIOLOGY 
demonstrated in the body, but do not appear in culture-media ex- 
cept in serum broth. It stains readily with ordinary anilin dyes 
and is Gram-positive. 
Isolation and Cultural Characters.-The organism may in some 
cases be isolated from the blood directly in pure cultures. It 
is most readily obtained from the sputum by animal passage. 
Growth occurs on most laboratory media except potato. Growth 
is never luxuriant, the organism developing as discrete, transparent, 
dewdrop-like colonies upon the surface of the medium. Bouillon 
is slightly clouded. Milk is 
acidified and coagulated. The 
addition of glycerin, and par- 
ticularly blood-serum, stimu- 
lates growth in most media. 
Physiology.-The optimum 
temperature is 37°; little or 
no growth will occur at lower 
temperatures. Desiccation for 
several months, particularly in 
sputum, does not always kill 
the organism. Twelve hours' 
exposure to direct sunlight is 
fatal. Acids are produced from 
many carbohydrates. • 
Pathogenesis.-The pneumococcus produces acute septicemia 
when injected into the mouse, guinea-pig, or rabbit. The produc- 
tion of typical pneumonia is attended with difficulty if, indeed, 
it has been satisfactorily demonstrated. Its principal claim to 
recognition as the etiologic factor in the disease rests upon its 
presence in the lesions and its general pathogenic relationship to 
animals. The fact that it is quite commonly present in the sputum 
of normal individuals seems to indicate that there are great differ- 
ences in disease-producing power among different strains. Whether 
or not the organism isolated by Mayer from pneumonia in the horse 
is identical with this organism cannot at present be determined, 
nor can its relationship to "Brustseuche" be said to be satisfac- 
torily proved. 
The tissues of the lung invaded by the organism become con- 
Fig. 87.-Diplococcus pneumoniae in 
pure culture (Weichselbaum) (Kolle 
and Wassermann). THE GROUP OF SPECIFIC PYOGENIC COCCI 
237 
gested, and blood-plasma is poured into the alveoli. The fib- 
rinogen coagulates and the lung becomes "hepatized," that 
is, liver-like in consistency. Frequently there is more or less 
hemorrhage, largely by diapedesis, and the lung becomes red- 
dened. Later leukocytes, particularly the polymorphonuclear 
type, invade, and the tissues become gray. Autolytic digestion 
of the fibrin and other exudates supervenes, and the material 
passes off through the air-passages or is resorbed. 
Metastatic infections 
with the pneumococcus are 
common in man. Inflam- 
mations of the endocar- 
dium, the pericardium, the 
pleura, and the meninges 
have been found to be due 
to this organism in a small 
percentage of cases. Otitis 
media is sometimes the 
initial infection, and may 
be followed by meningitis. 
Immunity.-No toxin 
has been demonstrated in 
this organism. Endotoxins 
may be demonstrated, but 
whether they account for 
its pathogenicity is uncertain. Agglutination of the pneumo- 
coccus occurs with the blood-serum of an infected individual, but 
not usually in greater dilutions than 1 :50. Opsonins, both 
normal and immune, have been demonstrated, and likewise there 
has been isolated from virulent Pneumococci a substance that 
inhibits phagocytosis. 
Immunity to pneumonia is transient; relapses frequently 
occur, possibly due to decrease in immunity during convalescence. 
Recovery in some cases seems to be followed by increased sus- 
ceptibility. The immunity developed is evidently opsonic in 
nature. 
Bacteriologic Diagnosis.-The most conclusive method of 
bacteriologic diagnosis is by isolation and cultivation of the organ- 
Fig. 88.-Diplococcus pneumoniae colony 
on agar plate (X100) (Frankel and 
Pfeiffer). 238 
VETERINARY BACTERIOLOGY 
ism. A demonstration of the characteristic lanceolate, capsulated, 
Gram-positive diplococci is often diagnostic. The agglutination 
test is not conclusive. 
Fig. 89.-Diplococcus pneumoniae. Stained preparations showing capsules 
(Buerger, in "Journal of Infectious Diseases"). 
The relationship of this organism to pleuropneumonia and 
contagious pneumonia in equines is uncertain, and further work 
needs to be done before the connection can be made clear. 
Neisseria meningitidis 
Synonyms.-Diplococcus intracellular is meningitidis; Micro- 
coccus weichselbaumii; Streptococcus meningitidis; meningococcus. 
Disease Produced.-Epidemic cerebrospinal meningitis in 
man. 
Weichselbaum, in 1887, first adequately described this organism 
from meningeal exudate, and proved its pathogenic nature by 
animal experimentation. It has since been observed repeatedly 
in many epidemics both in Europe and America. 
Morphology and Staining.-Neisseria meningitidis in stained 
smears of meningeal exudate usually appears as a diplococcus, 
or in groups of four. In culture-media it is about 1 m in diameter, 
and is usually in pairs, rarely in short chains. The latter fact 
would seem to indicate that this organism should be classed as a THE GROUP OF SPECIFIC PYOGENIC COCCI 
239 
Streptococcus, but the tetrad formation sometimes seen seems, 
on the contrary, to show that cell division may be in two planes, 
hence the use of the generic name, Micrococcus. No capsules 
are produced. It stains readily with the ordinary anilin dyes, 
but is Gram-negative. 
Isolation and Culture.-The organism may be obtained by a 
lumbar puncture with a sterile hypodermic needle, and transferred 
directly to artificial media. It is best to use a medium containing 
serum for the first isolation, for this bacterium frequently does not 
grow well on artificial media at the first. Upon blood-serum 
at 37° white, viscid, coherent colonies develop. Serum may be 
added to agar or bouillon and will be found to favor the growth. 
Milk is not changed. Frequent transfers are necessary to the 
preservation of cultures in artificial media. 
Physiology.-The meningococcus is killed almost immediately 
by desiccation. In culture-media autolysis rapidly occurs, and 
the organism soon disappears. No acids are produced in carbo- 
hydrate media. Proteolytic enzymes are not produced. 
Pathogenesis.-The meningococcus does not readily infect 
common laboratory animals unless intraperitoneal injections of 
comparatively large amounts of the culture be used. Even in 
this case the result seems to arise from the absorption of the toxic 
products rather than from an invasion of the tissues. Injection 
of pure cultures into the spinal cavity of the goat has been found 
to produce meningitis, and inoculation into the monkey has 
resulted practically in a duplication of the disease as it occurs in 
man. The relationship of the organism to the disease is, therefore, 
well demonstrated. 
The disease is an acute inflammation of the meninges, accom- 
panied by a purulent exudate. The organism sometimes, though 
rarely, enters the blood. Metastatic involvement of the lungs 
and other organs occurs in a few cases. 
Immunity.-No true toxins have been demonstrated; endo- 
toxins are probably produced, and released through the autolytic 
disintegration of the organism. Agglutinins are developed in 
sufficient quantity, so that the blood of a patient frequently ag- 
glutinates in a dilution of 1 : 50. Although no distinct bacterio- 
lysins have been demonstrated, specific amboceptors for the organ- 240 
VETERINARY BACTERIOLOGY 
ism may be shown to be present in the blood by the hemolytic 
absorption of complement test. Opsonins probably play the 
largest part in the development of immunity. All these anti- 
bodies mentioned have been determined to be present in the 
serum of immunized horses. 
Vaccination against the disease is not practised. Flexner 
and Jobling have prepared an immune serum from the horse by 
the injection of dead bacteria, followed by the injection of living 
Fig. 90.-Neisseria meningitidis in a preparation of pus from a brain abscess. 
Note that the organism is generally intracellular (Flexner). 
bacteria and the products of their autolytic digestion. The 
serum is injected directly into the spinal canal after the withdrawal 
of an equal amount of the purulent exudate. It comes in direct 
contact, therefore, with the organisms, and probably stimulates 
phagocytosis by its opsonin content. The use of this serum has 
proved highly successful; by its means mortality has been mate- 
rially reduced. 
Bacteriologic Diagnosis.-Lumbar puncture, with demonstra- 
tion in smears of a Gram-negative diplococcus, occurring prin- THE GROUP OF SPECIFIC PYOGENIC COCCI 
241 
cipally within the leukocytes, constitutes a satisfactory diagnosis. 
The agglutination test may be applied, but is not used in practice. 
Transmission and Prophylaxis.-How the organism gains en- 
trance to the spinal and brain cavities is not certainly known. It 
is found in the early stages of the disease upon the nasal mucous 
membranes. It is spread probably by the use of infected hand- 
kerchiefs or by the inhalation of infectious droplets. 
Synonyms.-Micrococcus intracellular is equi; diplococcus in- 
tracellularis equi. 
An organism in no important particular differing from the 
meningococcus has been reported by Johne, Ostertag, and others 
in epizootic or cerebrospinal meningitis, or Borna disease in 
horses. Ostertag succeeded in producing the disease in horses by 
subdural injections of pure cultures. Other organisms have been 
found in similar outbreaks by other investigators. Careful 
study of the relationship of these organisms to the diseases must 
be made before conclusions as to their importance as an etiologic 
factor would be justified. This epizootic infection should not be 
confused with the far more common type of so-called meningitis 
produced by forage poisoning. 
Neisseria intracelluiaris equi 
Neisseria gonorrhoeas 
Synonyms.-Micrococcus gonorrhoeas; gonococcus; diplococcus 
of Neisser. Diplococcus gonorrhoeas. 
Diseases Produced.-Gonorrhea and its sequelae in man. 
Neisser, in 1879, noted the occurrence of a characteristic coccus 
in gonorrheal pus. Bumm, in 1885, succeeded in obtaining the 
organism in pure cultures, and determined by inoculations into 
human subjects the causal relationship of the organism to the 
disease. 
Distribution.-Gonorrhea is a common disease in all classes of 
men, particularly in civilized countries. 
Morphology and Staining.-The gonococcus closely resembles 
the meningococcus. In stained preparations of gonorrheal pus 
the organisms occur generally in pairs inside the polymorphonuclear 
leukocytes. The cells are usually coffee-bean shaped. The in- 
dividual cells measure about 1.6 by 0.8 p,. There is little or no 
tendency to chain formation, the cells being arranged in irregular 242 
VETERINARY BACTERIOLOGY 
masses in culture-media. The organism stains readily with the 
ordinary anilin dyes and is Gram-negative. This latter fact is 
important, as it renders differential diagnosis between the com- 
mon staphylococci and the gonococcus possible. 
Isolation and Cultural Characters.-The organism may be iso- 
lated directly from gonorrheal pus, care being exercised to secure 
pus not contaminated with organisms from the skin. Consid- 
erable difficulty is sometimes experienced in securing cultures. 
Usually no growth will occur on ordinary agar or gelatin, although, 
.when considerable quantities of pus are spread over the surface, 
some colonies will develop. Wertheim's medium, composed of 1 
part of human serum to 2 parts of nutrient agar, is commonly 
Fig. 91.-Neisseria gonorrhwtE in pus (Gunther). 
found to be the one on which growth is most readily secured. 
The organism gradually adapts itself to growth on artificial media, 
and after a few transfers develops more luxuriantly. The colonies 
resemble those of the Str. pyogenes, being small, discrete, and trans- 
parent. 
Physiology.-The organism is aerobic. It is easily destroyed 
by desiccation, although when dried in pus it may retain its 
vitality for weeks. It must be transferred every two days if 
kept at thermostat temperatures, less frequently in the refriger- 
ator, to keep it alive. 
Pathogenesis.-Evidence of Pathogenesis.-That the gono- 
coccus causes gonorrhea is evident from the fact of its universal THE GROUP OF SPECIFIC PYOGENIC COCCI 
243 
occurrence in gonorrheal pus, and from inoculation experiments 
upon man. The laboratory animals do not contract the disease 
upon inoculation. 
Character of Disease and Lesions.-The organism ordinarily 
causes an acute urethritis in both sexes. It may also produce 
gonorrheal ophthalmia, particularly in the newborn. The ure- 
thritis may become chronic and lead to stricture. Secondary 
involvement of the Fallopian tubes, ovaries, and peritoneum in 
the female, and of the epididymis and bladder in the male, fre- 
quently occurs. Metastatic infections of the joints, causing gon- 
orrheal rheumatism; of the heart valves, causing endocarditis; 
of the brain and cord, causing meningitis, are not uncommon. 
The organism may persist for a long period in a dormant state. 
Immunity.-No true toxins are produced by the gonococcus, 
but endotoxins have been demonstrated, as have also specific 
agglutinins and precipitins. Little or no immunity is developed 
as the result of an infection. The presence of specific ambocep- 
tors in the blood has been shown by the method of fixation of 
complement. Vaccination with the autolytic products of the 
organism, and with cultures killed by heat, and by mixture with 
strong solutions of lactose or other sugars have been used with 
a moderate degree of success. Porrey has prepared an anti- 
gonococcus serum from the rabbit by immunization with living 
cultures, and claims to have secured favorable results from its use. CHAPTER XXII 
STAPHYLOCOCCUS GROUP 
The Staphylococcus group includes those Gram-positive cocci 
associated in irregular masses (not in chains) which in general pro- 
duce non-specific pyogenic infections. All the species are aerobic, 
non-motile, and, in general, liquefy gelatin and digest casein. 
The principal species are Staphylococcus aureus, St. albus, St. 
citreus, and St. (Botryomyces) ascoformans. These organisms are 
the most frequent cause of pyogenic infections in animals as well 
as in man. They are widely distributed in nature, but in general 
are parasitic or semiparasitic. 
Staphylococcus aureus 
Synonyms.-Micrococcus pyogenes aureus; Staphylococcus pyog- 
enes aureus; Micrococcus aureus. 
Staphylococci were definitely described as present in pus by 
Ogston (1881). Three years later Rosenbach (1884) cultivated 
them upon artificial media and differentiated several species, among 
them the one under consideration. Other investigators have fre- 
quently isolated this organism from pus in man and practically all 
domestic animals. 
Distribution in Nature.-Staphylococcus aureus occurs quite 
constantly upon the skin and hair of man and animals, in the nose 
and mouth of man, occasionally in human feces, and frequently in 
milk. It is often carried about in atmospheric dust, and is not un- 
common in water, especially when contaminated with sewage. 
Lucet found St. aureus associated with pus production in the horse 
three times in pure culture and forty-four times in association with 
other organisms out of a total of 93 cases. 
Morphology and Staining Characters.-The organism is spher- 
ical, sometimes slightly flattened where two cells are appressed. 
In pus and in the blood it is usually in masses like grape-clusters 
(whence the name, Staphylococcus); in culture-media the cells are 
244 STAPHYLOCOCCUS GROUP 
245 
grouped irregularly. The diameter of the cells is about 0.7 to 
0.9 p, rarely larger. It stains well with ordinary anilin dyes and 
is Gram-positive. 
Isolation and Culture.-Staphylococcus aureus may be fre- 
quently secured in pure culture by making cultures directly from a 
fistula or other suppurating focus, first cleansing the outer portion 
and securing material on a sterile platinum needle. In general, it is 
best, however, to plate out the. drop of pus obtained in this way. 
This is not only important in preventing contamination, but 
in order to diagnose mixed infections, especially in isolations made 
for the purpose of preparing autogenic vaccines. The organism 
Fig. 92.-Stained mount of the 
Staphylococcus aureus from agar (Gun- 
ther) . 
Fig. 93.-Colony of Staphylococcus 
aureus on agar (Heim). 
grows well in all the common laboratory media. The colonies 
upon gelatin appear as disks with smooth, definite edges, and with 
granular, dark interior. Within a few days the colony sinks in a 
cup of liquid. The liquid is cloudy, with a golden-yellow sediment. 
The growth on agar is abundant, shining, and well circumscribed. 
Upon the potato the growth is luxuriant, and the orange pigment 
is produced here in greatest abundance. Bouillon is clouded. 
Milk is curdled with a slight acid reaction, and the curd is eventu- 
ally digested. 
Physiology.-A study of the physiologic characters of this 
organism makes it apparent that there are many races of which 246 
VETERINARY BACTERIOLOGY 
account must be taken. It has not been satisfactorily demon- 
strated, however, that there is any relationship between these 
variations and pathogenicity. 
Pigment Production.-An orange-yellow pigment is produced. 
Fermentation.-A slight power to produce acid in milk has been 
noted. No gas is formed. Nitrates are reduced to nitrites. 
A proteolytic enzyme which digests casein is produced in milk. 
Gelatinase, rennin, and maltase have been demonstrated. 
Relation to Oxygen.-The organism is aerobic and facultative 
anaerobic. 
Vitality.-There is a marked resistance to desiccation, although 
it is not probable that the organism can remain alive for very long 
periods in this condition. Cultures upon media will remain alive 
for months. The optimum growth temperature is blood-heat, 
although growth is good at room-temperatures and below. The 
various isolated strains have shown great variation in heat resist- 
ance. Usually a temperature of 60° for half an hour suffices to 
destroy all the cells, but some require 80° for the same length of 
time. The cells are easily destroyed by common disinfectants. 
Pathogenesis.-Mechanism of Disease Production.-Staphylo- 
cocci have been shown to produce a leukocytotoxin called leuko- 
cidin. A stained mount of pus will frequently show that many of 
the leukocytes have been destroyed, and that the cells are begin- 
ning to disintegrate.. Staphylolysin, a hemolytic toxin, is also 
produced, particularly by the virulent strains. It seems to have 
been demonstrated, however, that these two toxins do not explain 
the pathogenic nature of the organism satisfactorily. Possibly 
anaphylaxis will explain some of the reactions secured, but there 
seems to be some poisonous property, possibly an endotoxin, 
which is not at present understood. We have no satisfactory 
explanation for its mechanism of disease production. 
Experimental Evidence of Pathogenesis.-The causal relation- 
ship of this organism to pus production and wound infection has 
been amply demonstrated. One-tenth c.c. of a twenty-four-hour 
culture of a moderately virulent strain will kill a rabbit, when 
injected intravenously, in four to eight days, and, upon post- 
mortem examination, abscesses containing the same organism will 
be found in many of the internal organs. STAPHYLOCOCCUS GROUP 
247 
Types of Natural Infection.-As has been stated previously, 
Staphylococcus aureus is most frequently found associated with 
wound infections, and is also a common cause of abscesses, 
carbuncles, boils, acne, and furuncles in man and animals, of 
poll-evil and fistula in the horse, and similar lesions in other ani- 
mals. The organism may gain entrance to the circulation and pro- 
duce septicemia or pyemia in man, rarely in animals. It has also 
been found in man as the cause of certain metastatic infections, 
particularly of the bone-marrow (osteomyelitis), and ulcerative 
endocarditis. These have also been produced experimentally in 
the laboratory animals. Inflammation of the udder (mastitis) 
in cows is occasionally caused by this organism. 
Immunity.-The production of two specific toxins, the hemo- 
lytic staphylolysin and the leukocidin, has already been noted, 
as also the probable 
importance of an endo- 
toxin. Antitoxins for 
the two first mentioned 
have been produced, 
but do not seem to 
confer immunity. Ag- 
glutinins have been 
demonstrated in normal 
as well as infected ani- 
mals; they seem, how- 
ever, to be of no diag- 
nostic value. The pre- 
cipitation reaction has 
likewise been obtained 
with the bacterial fil- 
trate. Bail has claimed 
that the production of aggressins accounts for pathogenicity, but 
his results are inconclusive. Bacteriolysins for Staphylococcus 
aureus are not produced in appreciable quantities, and are probably 
not of any immunizing value. Opsonins may be demonstrated in 
both normal and immune blood, and seem to be the most important 
serum component in determining immunity. 
Immunization.-Bacterins and vaccines, both univalent and 
Fig. 94.-Staphylococcus aureus in pus (Frankel 
and Pfeiffer). 248 
VETERINARY BACTERIOLOGY 
polyvalent, have been prepared, and have been extensively tested 
in cases of chronic suppuration. The results of repeated injections 
have been encouraging in many cases. A check has been kept on 
the development of immunity in man by repeated determinations 
of the opsonic index. Care is used to eliminate the negative phase 
as far as possible in making the injections. Autogenic vaccines 
have proved even more successful in both man and animals. 
The methods of preparation of such a vaccine have already been 
discussed. 
Bacteriologic Diagnosis.-A mount of pus stained by Gram's 
method reveals the organism distinctly, if present. Its character- 
istic morphology will, in general, render its recognition easy. 
This method has proved to be of particular value in differentiating 
this organism from certain other pyogenic forms, such as the 
gonococcus. Frequently a stained mount will reveal but a few 
bacteria, and cultures are then necessary. These must be made in 
any event where it is desired to make a positive diagnosis. 
Transmission and Prophylaxis.-The fact that the organism is 
constantly present on the skin and hair makes it particularly diffi- 
cult to prevent its entrance into wounds. The common prac- 
tices of aseptic surgery are the best preventives of infection. 
Staphylococcus albus 
Synonyms.-Micrococcus pyogenes albus; Staphylococcus pyog- 
enes albus. 
This organism differs principally from Staphylococcus aureus by 
being devoid of color. In certain cases of suppuration it has been 
found alone, and in many cases associated with the preceding. In 
all other respects what has been said with reference St. aureus 
will apply to this form. It is believed by some investigators that 
those two organisms are simply varieties of one form which they 
term St. pyogenes. Lucet found this organism associated with 
three-fourths of the pyogenic infections of the horse. He isolated 
it in pure culture from 11 cases and in mixed culture from 63 out of 
93 cases studied. STAPHYLOCOCCUS GROUP 
249 
Staphylococcus citreus 
Synonyms.-Micrococcus pyogenes citreus; Staphylococcus pyog- 
enes citreus; Micrococcus citreus. 
This organism was originally described by Rosenbach as present 
on the skin. It is doubtfully pathogenic and relatively uncommon. 
It differs principally from aureus in the production of a lemon-yel- 
low pigment on agar and potatoes and its lack of power to liquefy 
gelatin. In other respects it resembles the two preceding forms, 
and is possibly but a variety of them. 
Staphylococcus (pyogenes) bovis 
Synonym.-Micrococcus pyogenes bovis. 
This organism is somewhat smaller than Staphylococcus aureus, 
and does not liquefy gelatin. It has been claimed to be more 
commonly the cause of suppuration in cattle than the typical 
Staphylococcus aureus. It is doubtful whether there is a valid 
specific difference between the two. 
Synonyms.-Micrococcus botryogenus; Botryococcus ascofor- 
mans; Botryomyces ascoformans ; Zoogloea pulmonis equi; Micro- 
coccus ascoformans; Ascococcus Johnei; Discomyces equi. 
Disease Produced.-Botryomycosis. 
Animals Infected.-Equines, more rarely cattle and swine. 
The organism associated with botryomycosis was described 
in 1870 by Bollinger, and later investigated independently by 
Rivolta and Micellone in 1879, by Johne in 1885, and by Rabe 
in 1886. 
Distribution.-The disease is not uncommon in horses both in 
America and Europe. According to Eberlena, 5 per cent, of all 
horses admitted to the surgical clinic of the Berlin Veterinary 
School were affected with botryomycosis. 
Morphology.-The cocci in the tissues are relatively large. 
1 to 1.5 y in diameter. They are embedded in the thick capsules 
of the organism, forming a gelatinous mass of considerable size, a 
zooglea. These masses of cocci are frequently large enough to be 
visible to the naked eye as bunches of granules the size of tiny 
Staphylococcus ascoformans 250 
VETERINARY BACTERIOLOGY 
sand grains. Under the microscope they look like mulberries. The 
capsular material forms a sac-like membrane around the entire 
colony, and other colonies or masses appear to arise as though by a 
process of budding. 
Upon culture-media the capsule formation is not very evident, 
if present, and the cells are usually in pairs or tetrads grouped as a 
Sarcina, rather than as a Micrococcus, while in many respects it 
resembles the Staphylococcus aureus. It stains readily with anilin 
dyes and is Gram-positive. 
Isolation and Culture.-Staphylococcus ascoformans may be 
isolated in pure culture from the characteristic lesions, or in mixed 
infection it may be secured by plating. In most of its cultural charac- 
ters it resembles a weakened strain of Staphylococcus aureus. Gela- 
tin colonies are small, hemispherical, and sharply defined. They 
vary from silvery gray to gold gray. Glage characterized a thickly 
seeded plate as having the appearance of being sprinkled with pol- 
len. Slight liquefaction and cupping of the medium when at the 
surface is claimed by some authors and denied by others. In 
gelatin stab the growth is filiform and white, followed by a slow, 
crateriform liquefaction. The colonies on agar are scarcely visible 
in most strains, although more vigorous types have been described. 
On potato the growth is yellowish and has an aromatic odor. 
Physiology.-This organism produces a small amount of gelat- 
inase, as evidenced by the liquefaction of the gelatin. The tem- 
perature optimum is apparently 20° to 30°. At 37° growth is slower 
and the golden yellow color'is not produced. The cells are rela- 
tively resistant to desiccation. 
Pathogenesis.-Experimental Evidence.-Guinea-pigs injected 
with St. ascoformans generally succumb to septicemia. Mice 
are not susceptible. Sheep and goats develop ulcers at the point 
of inoculation. Injection into the horse usually results in sup- 
puration, but occasionally the typical botryomycomata are de- 
veloped. Whether or not this is a variety of St. aureus is un- 
settled, but it seems improbable that it is a distinct species. 
Character of Disease and Lesions Produced.-The lesions 
resemble superficially certain fibromata and other neoplasms. 
The infection usually takes place where the surface of the skin 
has been abraded, as by harness, through wounds at castration, STAPHYLOCOCCUS GROUP 
251 
on the udder, and elsewhere on the body. The tissues involved 
become grayish red, then lardaceous, and eventually form a mass 
resembling a fibroma. These sometimes reach considerable size. 
Metastatic infection through the lymph-channels may result in 
the involvement of considerable areas and infection of the internal 
organs, particularly the lungs. 
Immunity.-Methods of developing immunity have not been 
devised. It is possible that autogenic vaccination might be of 
value. 
Transmission.-As noted above, infection generally occurs 
through wounds or abrasions of the skin. CHAPTER XXIII 
ANTHRAX GROUP. THE GENUS BACILLUS 
The Group of Aerobic Spore-producing Bacilli 
The organisms which belong to this group resemble each other 
in that all are aerobic rods (some are also facultative anaerobes) 
and produce endospores. Practically all the species also liquefy 
gelatin and are Gram-positive. Many also show a decided tendency 
for the cells to remain united in chains. 
Many species of aerobic spore-bearing bacilli are known. 
They are often termed collectively the "hay bacillus" or Bacillus 
subtilis group. Some species are among the most abundant of 
bacteria in dust and soil. They are the organisms which are most 
active in the soil in bringing about the decomposition of organic 
substances, particularly those changes grouped together under the 
heading of Ammonification. They are, therefore, of great im- 
portance in the maintenance of soil fertility. Because of the 
resistance of the spores to high temperatures and desiccation these 
organisms are among those which most frequently contaminate 
culture-media in the bacteriologic laboratory. 
The various species are differentiated from each other by the 
size, shape, and position of the spores, the tendency to chain 
formation, motility, by differences in cultural and physiologic 
characters, and in pathogenesis. 
One species only exhibits sufficient disease-producing power 
to merit consideration here. This is Bacillus anthracis, the 
cause of anthrax. It can be separated with certainty from some 
of the common saprophytic soil species only by animal inocu- 
lations, by physiologic and serologic studies, or by careful 
observation of colony characters. 
252 ANTHRAX GROUP. THE GENUS BACILLUS 
253 
Bacillus anthracis 
Synonym.-Bacterium anthracis; Bacteridium anthracis. 
Disease Produced.-Anthrax. The disease is also known in 
cattle as splenic fever. (German, Milzbrand; French, Charbon.) 
In man the disease may be termed "malignant carbuncle" or 
"woolsorters' disease." The animals most commonly attacked 
are cattle, sheep, and swine, more rarely horses and man. 
Historical.-The anthrax bacillus was first noted in literature 
by the French physician Rayer, in 1850, though it is probable that 
Fuchs had seen the organism 
as early as 1842. The first 
accurate description was that 
of Pollender in 1855. The 
last author stated that he had 
observed the rods in blood in 
1849. 
The complete demonstra- 
tion of the causal relationship 
of this organism to the disease 
was furnished by the work of 
Robert Koch, published in 
1876. This is of interest his- 
torically as the first instance 
of the cultivation of an organ- 
ism on solid media in the laboratory, and the successful repro- 
duction of disease after repeated transfers in pure culture. 
Distribution.-The organism is a true parasite, and probably 
in nature does not increase outside of infected animals. The dis- 
ease has been recorded from all parts of the world. There are accu- 
rate records of its existence in Asia and Europe in ancient times. 
It is known at present from every continent. It has been 
reported from about one-third of the States in the United States, 
and probably occurs sporadically in most of them, though the dis- 
ease is at present not common except in a few localities. 
Morphology and Staining.-Bacillus anthracis is a large rod, 
straight, usually with truncate ends, 1 to 1.25 by 4.5 to 10 y, in 
short chains when examined in tissues or blood and in long chains 
in culture-media. It is non-motile. Capsules may be demon- 
Fig. 95.-Bacillus anthracis, rods with- 
out spores (Gunther). 254 
VETERINARY BACTERIOLOGY 
strated in smears from blood and other tissues. Spores are pro- 
duced only when the organism is grown in the presence of free oxy- 
gen. They do not, therefore, occur in the bacillus as found in the 
blood and the tissues. The single spore produced by a cell is oval 
or spherical, occupies the middle of the cell, and is of almost the 
same diameter. The spores are much more refractive than the 
protoplasm of the non-sporulating cells. The spores germinate 
on being brought under favorable growth conditions by breaking 
through the spore wall at the pole. They may be demonstrated 
by a contrast spore stain. The vegetative rod stains readily with 
the aqueous anilin dyes and is Gram-positive. Metachromatic 
granules may rarely be demonstrated. 
Many methods have been used for the demonstration of the 
capsules in blood-smears. The contrast stain of Klett or the single 
stain of Johne or of Rabiger (7. w.) are satisfactory. The Roman- 
owsky blood stain also shows capsules and granules well, the cap- 
sules being colorless, the granules red, and the protoplasm blue. 
The capsules can be best found in smears of blood from infected 
animals. They have been found, however, to develop in culture- 
media containing egg-white or blood-serum. Suitable staining 
technic may sometimes demonstrate capsules from pure cultures 
in other media. 
The more suitable spore stains are those of Moller and of Klein. 
Occasionally asporogenous strains of Bacillus anthracis have 
been discovered or have developed in the laboratory. Some of 
these have been found to possess undiminished virulence, but in 
most cases the pathogenicity was largely lost. 
Isolation and Culture.-Bacillus anthracis may be readily iso- 
lated in pure culture upon any of the common laboratory media by 
direct inoculation from the blood of an infected animal or from the 
internal organs, particularly the spleen or liver. Plate cultures in 
agar or gelatin are sometimes necessary when the organism is mixed 
with other forms. The colonies microscopically are found to con- 
sist of long chains of bacilli, which, under the low power, resemble 
tufts of curled hair. This appearance is quite characteristic, but 
is closely duplicated by certain soil organisms of the Bacillus sub- 
tilis group. In gelatin stabs a "spiking" occurs, i. e., filaments 
radiate from the line of puncture and give the appearance of an ANTHRAX GROUP. THE GENUS BACILLUS 
255 
inverted fir tree. The gelatin is liquefied slowly. The growth on 
potato is creamy in color and rather dry in consistency. Blood- 
serum is slowly liquefied. Milk is rendered slightly acid, curdled 
by a lab ferment, and the casein digested. In bouillon the organ- 
ism frequently forms a pellicle which readily settles to the bottom. 
Clouding of the medium does not usually occur. 
As will be noted from the preceding descriptions, the Bacillus 
anthracis grows readily on practically all the culture-media, par- 
ticularly if they are neutral or have a weakly alkaline reaction. 
All of the character- 
istics of the Bacillus an- 
thracis on culture-media 
can be duplicated in the 
growths of other mem- 
bers of the group which 
are strictly saprophytic. 
Care should, therefore, 
be used in the diagnosis 
of this organism from 
cultural characters alone. 
Physiology.-B acil- 
lus anthracis grows best 
in the presence of oxy- 
gen, and produces spores 
only■ under such condi- 
tions. Under anaerobic 
or semi-anaerobic conditions, as in lackmus molke and litmus 
agar, it decolorizes or acts as a reducing agent. 
The optimum growth temperature is 30-37°. Good growth 
may be secured at room-temperature or even below. At low tem- 
peratures the spores are not produced, the optimum for spore de- 
velopment being 25-32°. The maximum growth temperature 
is about 45°. 
The vegetative rods are readily destroyed by heat, five and one- 
half minutes at 65° sufficing. The spores are much more resistant, 
requiring 100° for from three to twelve minutes. When dried they 
have been heated to 140° for three hours before being killed. They 
likewise exhibit great resistance to desiccation. When dried upon 
Fig. 96.-Bacillus anthracis, with spores 
(Frankel and Pfeiffer). 256 
VETERINARY BACTERIOLOGY 
threads they have been known to retain their vitality for many 
years. Because they are probably the most resistant of the 
spores of pathogenic bacteria, they have been commonly used as 
test objects in determining the efficiency of disinfectants. Five 
per cent, phenol kills the spores only after prolonged contact, ac- 
cording to some investigators even forty days. 
Several other species of bacteria, particularly the Pseudomonas 
pyocyanea, inhibit the growth of B. anthracis. Pyocyanase (q. v.}, 
a preparation made from cultures of Pseudomonas pyocyanea, 
will dissolve B. anthracis in vitro. 
Fig. 97.-Bacillus anthracis colony 
(Gunther). 
Fig. 98. -Bacillus anthracis, stab 
culture in gelatin (Gunther). 
Indol is not produced. Small quantities of hydrogen sulphid 
develop in peptone solutions. The enzyme gelatinase, which 
digests gelatin, has been demonstrated in the bacteria-free filtrates 
from cultures. A rennet-like enzyme and others which digest ca- 
sein and blood-serum are produced. Some acid is formed in certain 
media, but it is not marked. The organism causes hemolysis 
when grown in blood-bouillon or on blood-plates. 
Pathogenesis.-Mice and guinea-pigs are highly susceptible 
to infection with anthrax, rabbits only slightly less so. These 
animals usually succumb in from twenty-four to forty-eight hours 
after inoculation. Rats are less susceptible, but show considerable 
variations. They usually die in about three days, but the disease 
sometimes assumes a subacute or chronic form and the animal may ANTHRAX GROUP. THE GENUS BACILLUS 
257 
live for several days to as many weeks. Sheep die quickly after 
the injection of minute doses of the organism, cattle are slightly 
more resistant, but usually succumb. Horses, swine, and goats 
may also be infected, but do not contract the disease spontaneously 
as frequently as do the other species. Dogs and cats are also some- 
what susceptible. Certain birds may sometimes be infected, but, 
in general, are relatively immune. Cold-blooded animals are re- 
fractory. Man is relatively susceptible. 
Character of Disease and Lesions Produced.-The type of dis- 
ease and the lesions produced vary somewhat with the animal 
infected. 
In cattle the disease is usually an acute febrile infection with 
no external localization. The temperature goes to 41 to 42°, 
there are difficulty in respiration, hematuria, and bloody discharges 
from the body openings. It is very quickly fatal usually; the 
animal sometimes dies within a few minutes or hours after the first 
symptoms; in other cases the animal may live for several days. 
Occasionally the disease manifests itself as an anthrax carbuncle, 
usually on the shoulder, breast, or neck. This type is not quite so 
uniformly fatal. 
Sheep die very quickly; usually the symptoms are not manifest 
more than a few hours. Carbuncles are very rare. Horses usu- 
ally have the disease in acute form, dying in a day or two. Occa- 
sionally in localized anthrax (carbuncles) they live for somewhat 
longer periods. Swine, being decidedly more resistant, show car- 
buncles of the mucous membranes and localized glandular infec- 
tions not infrequently chronic or terminating in recovery. Anthrax 
in the dog is much as in swine. 
Three methods of infection in man determine the three types 
of anthrax which may develop: Pulmonary anthrax or woolsorters' 
disease is contracted by the inhalation of anthrax spores. The 
possible presence of anthrax spores in hair, wool, and on hides neces- 
sitates careful disinfection. The disease runs its course as an 
atypical pneumonia, practically always fatal. Intestinal anthrax 
results from the ingestion of living anthrax bacilli. The disease 
is very rare, but usually fatal. In its earlier stages it is probably 
localized. Cutaneous anthrax, or malignant carbuncle, is the 
result of infection through scratches or cuts in the skin. At first 258 
VETERINARY BACTERIOLOGY 
the lesion resembles that of a common furuncle. Soon there is 
necrosis of tissues near the primary pustule, and blood infection and 
death frequently follow. However, in many cases the infection 
remains localized and eventually heals. 
The disease is in most animals a rapidly fatal septicemia. The 
most characteristic change to be noted upon autopsy is the great 
enlargement of the spleen, with distention of the capsule and soft- 
ening of the pulp, congested and usually dark in color. In the 
vicinity of areas of localized infection there are generally hemor- 
rhages and hemorrhagic exudate. The liver, kidneys, and lungs 
are usually congested and ecchymotic. The organism is found in 
great numbers in the blood-stream. 
Figs. 99 and 100.-Bacillus anthracis, stained mount of blood, showing the 
capsules of the bacilli (Preisz). 
Immunity.-No true toxin has been demonstrated for the 
anthrax bacillus; in fact, it is difficult to account for the patho- 
genicity of the organism on the basis of specific poison formation. 
Immune serum is claimed by some investigators to have a con- 
siderable specific agglutinative power, but others have failed to 
demonstrate this. It seems that this reaction is inconstant and 
may be entirely absent, even though the serum have a high im- 
munizing value. Certain normal bloods, as from the dog, show 
bacteriolytic power, but specific bacteriolysins cannot be demon- 
strated in most immune sera. Opsonins, both normal and immune, 
have been demonstrated. 
No wholly satisfactory explanation of the factors which de- 
termine immunity in animals naturally insusceptible and those ANTHRAX GROUP. THE GENUS BACILLUS 
259 
which have acquired immunity has been developed. Among the 
facts which apparently must be taken into consideration are the 
following: 
1. Immunity cannot be wholly due to the natural bactericidal 
power of the serum, for the serum of the highly susceptible rabbit 
has as much anthracidal power as that of the relatively resistant 
wild rat. There is apparently no direct relationship between the 
natural anthracidal power of a serum and the resistance of the 
animal. 
2. There is apparently some other factor in determining im- 
munity than phagocytosis, for the white blood-cells of susceptible 
animals may rapidly engulf virulent anthrax bacilli in vitro. 
3. The Bacillus anthracis when growing in the animal tissues 
usually produces capsules. This same character may be developed 
by growing the organisms in sera in the laboratory. Such organ- 
isms have been termed "animalized." It has been claimed by 
some authors that these capsulated organisms are far more resist- 
ant to the phagocytic and bactericidal action of immune blood 
than are the uncapsulated. Both points have been disputed by 
other writers. It seems doubtful if the development of the capsule 
really can account for the ability of the organism to invade the 
body of an animal and produce a disease. 
4. The work of Bail and others seems to show that in infection 
of a susceptible animal two distinct stages may be noted. The 
intraperitoneal injection of considerable numbers of anthrax bacilli 
into a guinea-pig is followed by a rapid diminution of these num- 
bers, due to phagocytosis and bacteriolysis. Some organisms ap- 
parently persist, and after a time begin multiplying rapidly in the 
tissues and blood-stream. Possibly these organisms are more 
resistant, or the defences of the body have been broken down. 
Bail explains the phenomenon by the assumption that the persist- 
ing bacteria produce an aggressin which effectively neutralizes 
the immune bodies of the tissues. The bacteria are thereupon 
free to multiply. He claims that a true active immunity may be 
developed only by causing the body to form an anti-aggressin, 
an immune body which will effectually neutralize the aggressins 
of the bacillus, thus enabling the other antibodies to destroy the 
bacteria. 260 
VETERINARY BACTERIOLOGY 
Active immunization by vaccination has been extensively prac- 
tised. The organism may be attenuated in a variety of ways. The 
first method developed was that of Pasteur, and it is still the one 
most commonly used. The organism is grown at a temperature of 
42° to 43° for varying lengths of time. The pathogenicity gradu- 
ally decreases until injections no longer kill the rabbit; longer 
growth attenuates it until the guinea-pig is not susceptible, and 
finally, even the mouse will not succumb. The exact length of 
time required for attenuation in each instance can be determined 
only by experimentation, as there are many unknown or uncon- 
trolled factors involved. 
In Russia the Zenkowsky modification of the Pasteur method 
has been used. The organisms are attenuated until 0.2 gm. of 
Vaccine I will not kill a 350-gm. guinea-pig when injected sub- 
cutaneously, but 0.01 gm. is regularly fatal for the white mouse. 
Vaccine II kills a guinea-pig on the third day, while 0.5 gm. will 
not kill an 800-gm. rabbit. The organisms are grown on agar 
slants until spores are produced. These are then mixed with 30 
to 40 per cent, glycerin. The advantage of this method of pre- 
paring lies in the fact that it may be kept for a longer period with- 
out deterioration. 
The immunity lasts about a year. Attempts to use killed 
cultures of anthrax bacilli or their sterile products in producing 
immunity have not yielded satisfactory results in practice. Bail 
claims that an active immunity may be established by the use of 
aggressins. The material used is the serous fluid from animals 
dead from anthrax. This is sterilized by phenol and injected into 
an animal to be immunized. The aggressin should preferably be 
secured from the same species of animal as the one to be immunized. 
The immunity is not established until after the lapse of ten days 
or more. When established, it is claimed that the animal will 
resist infection with fully virulent cultures. This method of im- 
munization with aggressins has not been thoroughly tested out in 
practice. 
Passive immunization by means of antisera has been tried by 
numerous investigators. Cattle, the horse, ass, and sheep have 
been used in the production of such sera. The animals are first 
immunized by Pasteur's method of vaccination, and in ten days ANTHRAX GROUP. THE GENUS BACILLUS 
261 
or two weeks from q to yy-p of a loop of a fully virulent 
culture is injected. Two or three weeks later a somewhat larger 
dose is given, and the dosage is gradually increased until many 
loopfuls-then entire cultures-are injected. In three to four 
months an immunity is developed such that the subcutaneous 
injection of several cultures from agar slants may be made 
without noteworthy reactions. The blood is drawn about 
two weeks after the last injection. The animal may then, after 
the lapse of two weeks, be again injected and again bled. The 
serum is pipetted off and preserved with 0.5 per cent, phenol. 
Exact methods of standardization such as are used with antitoxins 
cannot be employed. An approximation of the potency can be 
reached by the intravenous injections of varying amounts of serum 
into rabbits, followed five to ten minutes later by inoculating 
each subcutaneously with yjp of a loop of a virulent culture. 
A serum is regarded as usable if 2 out of 5 animals injected with 
2, 3, 4, 5, and 6 c.c. of the serum survive, and the remainder live 
longer than control animals. The serum shows in vitro no higher 
bacteriolytic power than serum from normal animals of the same 
species. The potency of the serum cannot be estimated by com- 
plement fixation reaction; nor can the activity of the serum be 
ascribed to its opsonin constant. Bail claims the activity to be 
due to the anti-aggressins present. Agglutinins are present, but 
cannot be used in determining the titer of the serum. The serum 
may be used both in prophylaxis and cure. It is of therapeutic 
value in man also. Ten c.c. is a prophylactic dose for sheep. 
The serum is of greatest use where it is necessary to immunize 
large numbers of animals quickly, as in a herd of sheep in which 
anthrax has made its appearance. 
Bacteriologic Diagnosis.-The disease may be diagnosed by the 
following bacteriologic methods: 1. Microscopic examination. 2. 
Cultures. 3. Animal inoculations. 4. Precipitation. 
Microscopic Examination -Fresh blood from animals having 
septicemic anthrax will show the organisms in the hanging drop. 
Mounts from blood or infected tissues, particularly the spleen, 
may be stained with methylene-blue, by Gram's method, or by a 
suitable capsule stain. 262 
VETERINARY BACTERIOLOGY 
Cultures.-Agar plates may be poured or streaked. The 
colonies showing the characteristic curled-hair margins should be 
fished and used for animal inoculation. 
Animal Inoculation.-A suspension of the suspected material 
should be injected into mice or guinea-pigs. 
Precipitation Test.-This test, first worked out by Ascoli and 
usually termed the Ascoli thermoprecipitation test, is based upon 
the fact that it is possible to secure precipitating sera of high titer 
from immunized animals for the anthrax bacillus audits soluble con- 
stituents. The test is most commonly employed in the recognition 
of anthrax in tissues and organs submitted for diagnosis. The task 
of securing a suitable precipitating serum is not always an easy one, 
as animals immunized in the same manner will show great differ- 
ences in the precipitin content of the serum. A portion (1 to 2 
gm.) of the organ to be examined (usually the spleen) is heated for 
five minutes in a tube with 5 c.c. physiologic salt solution, cooled, 
and filtered. The clear filtrate is placed in a narrow tube, and 0.5 
c.c. of the precipitating serum placed in the bottom by means of a 
capiljary pipette. A positive test is evidenced by the formation 
within fifteen minutes of a whitish cloud near or at the point of 
contact. Reactions which develop after two hours are not to be 
regarded as specific. This test is a useful adjunct to the other 
methods discussed. A reaction may often be secured even when 
putrefaction of the tissues is far advanced. 
The isolation of anthrax bacilli from soil, wool, hides, etc., is 
relatively difficult. The material may be heated to 60-70° for 
one-quarter to one-half hour to kill the vegetative cells, and the 
material used for animal inoculation. 
Transmission.-Anthrax is usually transmitted from one ani- 
mal to another by ingestion, more rarely through skin lesions. 
Cutaneous infection and infection by inhalation are most common 
in man. The organism does not sporulate within the body. Dead 
animals should be burned or buried deeply. The excretions from 
an infected animal, the feces in particular, contain many bacteria 
which can form spores on leaving the body. Pastures once in- 
fected may remain so for many years, as the spores are not readily 
destroyed by desiccation and may persist in the soil for a long 
time. Blood-sucking flies sometimes spread the disease from one ANTHRAX GROUP. THE GENUS BACILLUS 
263 
animal to another by direct inoculation. Care should always be 
used in dealing with infected animals, as the disease is fatal to 
man as well. CHAPTER XXIV 
BLACKLEG-TETANUS GROUP. THE GENUS CLOSTRIDIUM 
The bacteria of this group include all the anaerobic spore- 
producing bacilli. Most of the organisms are obligate anaerobes, 
some are micro-aeropholic, but none are aerobic. With few ex- 
ceptions the organisms are motile. They are, in general, Gram- 
positive, though there is considerable variation in the rapidity 
with which decolorization takes place. 
The following are the more important forms which have been 
described as species of this group: Clostridium tetani, causing 
tetanus in man and animals; Cl. chauvcei, causing blackleg in 
cattle; Cl. welchii, causing an infectious edema in man and 
animals; Cl. gastromycosis ovis, causing bradsot in sheep; Cl. 
botulinum, causing botulism or meat-poisoning in man; Cl. 
oedematis, causing malignant edema in animals and man; Cl. sp. 
of Ghon-Sachs, causing an emphysematous edema in swine and 
man, and the Cl. sp. of Hibler, causing gaseous edema. In addi- 
tion, Cl. sp. of Novy and the Cl. enteritidis sporogenes are capable 
of producing disease in experimentally infected laboratory ani- 
mals. Several non-pathogenic putrefactive and soil species of 
this group have been isolated, among them Cl. putrificus, Cl. 
amylobacter, and others. 
The separation of the various pathogenic and closely related 
non-pathogenic species is a matter of considerable difficulty. 
The first attempt to find criteria for adequate differential diagno- 
sis was that of Hibler.1 More recently the British committee 
appointed to study the bacteriology of war wounds have greatly 
clarified the problem. They showed that many of the irregu- 
larities and discrepancies of the older literature were due to the 
failure to have pure cultures. The work of Meyer, Hellei; and 
their associates has also served to indicate the organisms of this 
group which are of most importance in animal pathology. 
1 Untersuchungen liber die pathogenen Anaeroben, Jena, 1908. 
264 BLACKLEG-TETANUS GROUP. THE GENUS CLOSTRIDIUM 
265 
Key to the More Important Members of the Blackleg-tetanus 
Group of Bacteria 
A. Spores terminal and spherical, giving drumstick 
appearance to the cell. Clostridium tetani. 
AA. Spores not both spherical and strictly apical. 
B. Non-motile. Spores only in sugar free 
media. Clostridium welchii. 
BB. Motile. 
C. Sucrose fermented. Clostridium chauvoei. 
CC. Sucrose not fermented. 
D. Galactose and salicin fermented, 
glycerol not fermented. Clostridium oedematis. 
DD. Galactose and salicin not fer- 
mented, glycerol fermented. Clostridium botulinum. 
Clostridium tetani 
Synonyms.-Bacillus of Nicolaier; Plectridium tetani; Bacillus 
tetani. 
Disease Produced.-Tetanus or lockjaw in man and animals. 
(German, Starrkrampf.) 
Nicolaier, in 1885, observed the Clostridium tetani in pus from 
laboratory animals that had died following subcutaneous inocula- 
tion with small amounts of garden-soil. He cultivated the organ- 
ism, but did not succeed in securing it in pure culture. Kitasato, 
in 1889, succeeded in growing the organism in pure culture, and in 
transmitting the disease experimentally. Kitasato and Veyl, in 
1890, described the production of the tetanus toxin. 
Distribution.-The organism is found in all parts of the world. 
It is particularly common in street-dust and fertilized garden-soil, 
and is found quite constantly in the alimentary tract of herbivo- 
rous animals. Lukas found it present in the excrement of 16 out of 
17 horses which he examined. It is possible that it may for a time 
maintain a saprophytic existence and multiply in the soil under 
certain conditions. 
Morphology and Staining.-Clostridium tetani is a rather long, 
slender rod, 0.5 by 2 to 5 m with rounded ends, usually single, 
rarely in short chains. It is motile by means of numerous 
peritrichic flagella. Capsules are not produced. Spores are 
formed abundantly. Their size and position are so character- 
istic as to be practically diagnostic. They are spherical, two 
or three times the diameter of the rod, and terminal, giving 266 
VETERINARY BACTERIOLOGY 
the organism the appearance of a drumstick. The organism 
stains readily with the ordinary anilin dyes and is Gram- 
positive. 
Isolation and Culture.-The isolation in pure culture of Clostri- 
dium tetani is attended with considerable difficulty, largely on 
account of its being an obligate anaerobe. Kitasato first succeeded 
in isolating it by producing tetanus in experimental animals, 
then inoculating broth, and, after growth had taken place, heat- 
ing to a temperature of 80° for half an hour. This temperature 
should destroy all but spores. The broth may then be inoculated 
into agar or gelatin and kept under anaerobic conditions. If 
spores of other anaerobes are 
present, it may be necessary 
to make several consecutive 
animal inoculations and iso- 
lations. 
The colonies of the te- 
tanus bacillus upon gelatin 
plates show minute radiating 
lines of growth from a central 
nucleus resembling somewhat 
those of Bacillus subtilis. 
Gelatin stabs show an arbor- 
escent growth. The gelatin 
is slowly liquefied. Growth 
is favored by the presence of reducing substances, such as dex- 
trose or lackmus solution. Radiating filaments are also pro- 
duced in glucose agar stabs. Bouillon is clouded and a sediment 
forms. Blood-serum is liquefied. Milk is more or less com- 
pletely peptonized; it may or may not show coagulation. 
Hibjer's brain medium is made alkaline and blackened as the 
result of the formation of iron sulfid. 
Physiology.-The optimum growth temperature is 37.5°, but 
the organism multiplies rapidly at room-temperatures, though 
not below 14°. It is an obligate anaerobe, and in pure culture 
requires practically complete exclusion of oxygen. It will develop, 
however, under aerobic conditions when in mixed cultures with 
Fig. 101.-Clostridium tetani, rods and 
spores (Gtinther). BLACKLEG TETANUS GROUP. THE GENUS CLOSTRIDIUM 
267 
aerobes. The spores resist desiccation indefinitely. They are also 
much more than usually resistant to the action of disinfectants. 
Likewise, resistance to heat is so marked that Theobald Smith 
found in one case exposure to live steam for seventy minutes failed 
to destroy the organism, although usually a shorter period suffices. 
Acid is produced from carbohydrates, and a small amount of 
gas, consisting of methane and carbon dioxid from dextrose. En- 
zymes which liquefy gelatin and blood-serum have been demon- 
strated. 
Fig. 102.-Clostridium tetani, colo- 
nies in dextrose gelatin (Frankel and 
Pfeiffer). 
Fig. 103.-Clostridium tetani, deep 
stab culture in dextrose gelatin 
(Frankel and Pfeiffer). 
Pathogenesis.-Experimental Evidence.-Injection of pure cul- 
tures of Clostridium tetani into experimental animals causes the 
development of a typical tetanus. The white mouse is among 
the most susceptible of animals to inoculation. Infection of the 
animal is within one to three days followed by tetanic convulsions 
and death. Birds are not ordinarily susceptible to infection. The 268 
VETERINARY BACTERIOLOGY 
disease is most common in men and in the horse, although no 
mammalia are immune. The disease is not transmitted by inges- 
tion. The injection of the characteristic toxin is followed by the 
symptoms of the disease. 
Character of Disease and Lesions Produced.-The disease is a 
typical toxemia. There is rarely, if ever, a general invasion of the 
tissues. The organism remains localized at the seat of inoculation, 
and produces the toxin which brings about the characteristic symp- 
toms. The entrance of the organism into a wound is not always 
followed by the development of tetanus, for anaerobic conditions 
must obtain, and it has been found that tetanus spores entirely 
freed from toxin cannot germinate when introduced into the tissues 
in moderate numbers. It is evident that the organism has little 
initial pathogenic power. Frequently considerable amounts of dirt 
are introduced into the wound simultaneously with the organism 
in natural infections, and produce proper conditions for rapid de- 
velopment. When conditions are not favorable to the germination 
of the spores at the site of inoculation, they may remain alive in 
situ for considerable periods of time. They may also be taken 
up by leukocytes and transported in the body fluids to other tissues 
where they may remain dormant indefinitely, being incited to 
growth only by some tissue injury at their point of lodgment. 
Such localization and subsequent activation doubtless account for 
the so-called cryptogenic infections. The tetanus toxin produced is 
in part absorbed by the end-organs of the motor nerves, and travels 
to the nerve-cells of the central nervous system by way of the axis- 
cylinders of the peripheral nerves, and in part is carried to the 
central ganglion-cells by the blood-stream. The incubation period 
noted is due to the time required for toxin to be produced and for it 
to reach the central nervous system. That the toxin has a special 
affinity for nervous tissue, and may be bound by it, has already 
been noted in the discussion of toxins and antitoxins. The period of 
incubation in man averages about nine or ten days. In the horse it 
varies from four to twenty days. Under exceptional conditions this 
period may be much longer. Mortality is over 90 per cent, when 
there is a short period of incubation, and over 50 per cent, where 
the period is prolonged. The characteristic symptom in all ani- 
mals is a tetanus, or stiffening of the muscles. The muscles at the BLACKLEG TETANUS GROUP. THE GENUS CLOSTRIDIUM 
269 
site of inoculation may be the first, and in mild cases they may be 
the only ones, affected. In the horse the appearance of the tetanus 
or lockjaw, the retraction of the eyes and protrusion of the nicti- 
tating membrane, spasmodic contraction of other muscles of the 
head, and those of other parts of the body are diagnostic. A 
postmortem examination usually shows absence of gross lesions. 
Certain degenerative changes in the motor cells of the cord may be 
observed in stained sections. Hemorrhages in different organs are 
an inconstant accompaniment of the disease. 
Immunity.-For the preparation of toxin the organism is grown 
in bouillon under anaerobic conditions, i. e., in an atmosphere of 
hydrogen, with surfaces of the medium covered with paraffin or 
paraffin oil or with oxygen excluded in some other manner. After 
incubation for a period of one or two weeks the broth is filtered 
through porcelain. The toxin may be prepared in dried form by 
precipitation with an excess of ammonium sulphate. After stand- 
ing overnight the brown scum is removed and dried, first between 
hardened filter-paper, then in a desiccator, pulverized, and pre- 
served in a darkened refrigerator. Various methods of purifica- 
tion have been devised, such that a dried toxin may be prepared of 
which 0.00000025 gm. will prove quickly fatal to a white mouse. 
As has been said, this toxin has a peculiar affinity for the cells of 
the central nervous system. Two poisonous constituents of the 
toxin have been differentiated-tetanolysin, which lakes the red 
blood-cells, and tetanospasmin, which gives rise to the characteristic 
tetanus symptoms. 
In the preparation of antitoxin the unprecipitated toxic broth 
or a solution of the dried toxin is used. The smallest amount of 
toxin that will certainly kill a 350-gm. guinea-pig in three to four 
days is taken as the unit of toxicity. Increasing amounts of the 
toxin are injected at intervals into a horse. The blood-serum of the 
immunized horse contains the specific antitoxin. Many methods 
of standardization of the antitoxin have been used. In the United 
States it is titrated by guinea-pig injections against a standard 
toxin sent out by the Hygienic Laboratory of the Public Health 
and Marine Hospital Service. It is used in both human and veter- 
inary medicine, principally as a prophylactic. The tetanus anti- 
toxin has not taken the place in the treatment of tetanus that is 270 
VETERINARY BACTERIOLOGY 
occupied by the antitoxin specific for diphtheria in the treatment of 
that disease. It seems that the symptoms of the disease are ex- 
hibited only after the union of the toxin with nerve-cells; that is, 
after much of the damage has already in large measure been ac- 
complished. The injection of antitoxin at this time will doubtless 
neutralize any toxin present in the blood, but cannot remove the 
toxin already bound to the nerve-cells. The antitoxin is generally 
injected subcutaneously, but in severe cases intravenous, intra- 
neural, and intraspinal injections are made to insure the contact of 
antitoxin with the toxin present. Its use is doubtless indicated in 
all cases. As a prophylactic it has been found quite certainly to 
prevent the development of tetanus when injected before the ap- 
pearance of symptoms. In human medicine it is customary to 
make injections following severe wounds into which dust and dirt 
have gained entrance, such as Fourth-of-July wounds. The same 
may be said with reference to severe wounds, nail-punctures, and 
similar traumata in the horse. 
Bacteriologic Diagnosis.-The organism may sometimes be 
recognized in stained mounts of the pus from the wounds. The 
drumstick shape of the sporulating cells is quite characteristic. 
Isolation in pure culture and animal inoculation may also be used. 
The symptoms of tetanus are so distinctive, however, that these 
methods are rarely called into use. 
Transmission.-Tetanus is one of the best examples of a non- 
contagious, infectious disease. Infection occurs almost invariably 
directly through the skin. The almost universal presence of the 
organism about stables renders infection easy. Nail-punctures are 
particularly apt to result in tetanus, as they introduce the organism 
deep into the tissues; superficial healing and exclusion of air quickly 
take place, and conditions are then right for rapid multiplication. 
In some localities tetanus is a common disease following castration 
of domestic animals. Infection through the umbilical cord in the 
newborn sometimes occurs. It should again be emphasized that 
it seems very difficult for the tetanus bacillus to gain a foothold and 
proliferate except in tissues that have been injured. The constant 
presence of these organisms in the intestines does not produce dis- 
ease. So-called cryptic infections are not of uncommon occurrence, 
particularly in the horse. In these the point at which the organ- BLACKLEG TETANUS GROUP. THE GENUS CLOSTRIDIUM 
271 
ism gains entrance to the body frequently cannot be determined. 
Usually this comes either from the wound having healed super- 
ficially, so as to be difficult of recognition, or from the wound having 
been originally so insignificant as to have escaped notice. Some 
investigators believe that the organism may occasionally gain en- 
trance to the blood-stream from the intestines, but is unable to pro- 
duce an infection except when it lodges in tissue traumatically 
or otherwise injured, such as a broken bone or a bruise. Spores 
may be carried from a wound to othef parts of the body, and 
develop only when the tissue has been injured. 
Clostridium chauvaei 
Synonyms.-Bacillus feseri; Bacillus chauvoci; B. chauvoci; 
B. chauveaui; B. anthracis symptomatici. 
Diseases Produced.-Blackleg, symptomatic anthrax, quarter 
evil, quarter ill, Rauschbrand, charbon symptomatique; in cattle 
and rarely, in sheep and goats. 
Arloing, Cornevin, and Thomas, in 1889, described the Clostri- 
dium chauvcei as the cause of blackleg, and proved its etiologic 
relation to the disease. Kitasato (1889) first grew the organism 
in pure culture. It has also been studied extensively by Grass- 
berger and Schattenfroh and by Hibler. 
Morphology and Staining.-Clostridium chauvcei is a large bacil- 
lus with rounded ends, usually single, but occasionally in pairs, 
0.5 to 0.6 by 3 to 5 m. When stained smears from fresh serous 
exudates or muscles are examined the organism is found never 
to occur in long chains or filaments. This fact is of value in the 
differentiation of this organism from the related Cl. cedematis of 
Koch and from the Ghon-Sachs bacillus. It is motile by means 
of peritrichic flagella. Involution forms, consisting of greatly 
enlarged rods, are frequently encountered, particularly in old 
cultures. Capsules when produced in the body fluids are 
relatively thin. Spores are produced, sometimes central, but 
more frequently, near a pole, rarely quite terminal. They are 
long ellipsoidal in shape and are not generally more than twice 
the diameter of the rod. The ability of the organism to produce 
spores in media containing carbohydrates is of use in differentiat- 
ing it from Cl. welchii. Spore production is not abundant 272 
VETERINARY BACTERIOLOGY 
when the medium becomes acid. The organism is easily stained 
by the common aqueous anilin dyes and is Gram-positive. There 
is frequent occurrence of Gram-negative cells in old cultures. 
Smears prepared from muscles and treated with iodin potassium 
iodid show the presence in many cells of red stained granules 
termed uery thro granulose." 
Isolation and Culture.-The organism may in many cases be 
isolated in pure culture directly from the tissues infected. Growth 
in culture-media is quite de- 
pendent upon the reaction of 
the medium,' and to a less de- 
gree upon the presence of carbo- 
hydrates. For the best growth 
| per cent, soda content beyond 
Fig. 104.-Clostridium chauvcei 
(Kolle and Wassermann). 
Fig. 105.-Clostridium chauvcei, 
colonies in a dextrose gelatin shake 
culture (Frankel and Pfeiffer). 
neutrality is advisable. Body fluids or tissues except as they 
may act as reducing agents or contain carbohydrates do not 
increase the suitability of media containing them. Inoculation 
of bits of infected tissue into broth containing a bit of paren- 
chymatous tissue to act as a reducing agent will generally give 
a pure culture. Plates may be poured and kept under anae- 
robic conditions. The colonies are spherical, or somewhat 
irregular, with microscopic radiations. Dextrose gelatin is 
an exceptionally favorable medium. In a shake culture the BLACKLEG-TETANUS GROUP. THE GENUS CLOSTRIDIUM 
273 
colonies appear in the lower portion of the tube, each usually 
with its gas bubble, and surrounded by a liquefied area. 
These colonies show numerous radiating threads, or papillae, 
giving the appearance of a chestnut burr. Bouillon is clouded, 
gas is produced, and a flaky white deposit forms. Growth in 
Kibler's brain medium results in the development of a permanent 
acidity and no blackening, but with the evolution of considerable 
quantities of gas. Milk is not particularly favorable as a medium 
without the addition of serum or tissues. Little gas is formed 
in milk. Usually a flocculent curd is formed, but this is not 
digested. 
Physiology.-Serum media are not peptonized. The optimum 
growth temperature is about blood-heat, but good growth occurs at 
room-temperatures. The organism is a strict anaerobe. Con- 
cerning its other physiologic characters there is considerable dis- 
agreement among investigators. This may be due to the fact that 
there are strains which react very differently, and may constitute 
distinct varieties, or still more probably to the fact that impure 
cultures have been studied. Grassberger and Schattenfroh 
claim that the organism shows considerable variability, and that 
certain characters are easily lost. The spores are quite resistant 
to desiccation, living in this condition for years. Heating for six 
minutes at 100°, according to Hibler, is necessary certainly to 
destroy them. The dry spores are quite heat resistant. Gas is 
produced from many carbohydrates including glucose, levulose, 
maltose, lactose, sucrose, but not from mannitol, glycerol, dulci- 
tol, salicin and inulin. It is also produced in meat medium. 
Alkaline carbohydrate media is first neutralized and then becomes 
acid. 
Pathogenesis.-Experimental Evidence.-Inoculation of pure 
cultures into laboratory animals results in death, with production 
of many of the characteristic symptoms of blackleg, particularly 
the edema about the point of inoculation. Intramuscular injection 
of the guinea-pig is followed by the first symptoms in about fourteen 
hours. A soft inflamed swelling develops at the site of inoculation. 
In twenty-four to thirty hours the inflammation has spread to other 
muscles, and these have become emphysematous. Inoculation is 
usually fatal; occasionally the disease runs an atypical course and 274 
VETERINARY BACTERIOLOGY 
the animal recovers. Upon section, in typical cases, the tissues are 
found to be edematous and hemorrhagic, and the muscles to contain 
many gas bubbles. The body cavities usually show an abundant 
serous exudate. The disease may also be produced experimentally 
in cattle, so that there is no doubt as to the etiologic relationship 
of this organism to the disease. It has not been found in man. 
Character of Disease and Lesions Produced.-Rabbits, white 
mice, and white rats may be infected. The gray rat appears to be 
relatively immune. Blackleg in cattle is characterized by a 
swelling, edema, and emphysema of the muscles and the sub- 
cutaneous tissues of the infected part. Infection appears most 
commonly in the shoulder or hindquarter. The swelling increases 
rapidly in size, and the emphysema soon manifests itself by the 
crackling sound produced when the thumb is drawn firmly across 
the part. After death the organisms continue to grow and the 
body becomes distended with gas. The subcutaneous tissues of the 
infected part are edematous, even gelatinous, with blood and gas 
bubbles. The underlying muscles are dark brown or even blackish, 
whence the name, blackleg. The disease usually results fatally in 
cattle in from one to three days after the first appearance of the 
symptoms. 
Immunity.-The production of true toxins by Clostridium 
chauvcei is not well understood. According to some authors no 
toxin can be demonstrated. Others believe that there is a 
relatively thermostabile toxin produced which will endure a 
temperature of even 115°. Grassberger and Schattenfroh, who 
have made the most careful study of this problem, claim to have 
succeeded in producing broth cultures containing a toxin that, in 
doses as small as those employed with diphtheria toxin, will kill 
laboratory animals. This toxin is produced by certain strains 
of the organism only, but these they believe are the more patho- 
genic. This toxin they have shown to be thermolabile. They 
have worked out methods of standardization closely resembling 
those of Ehrlich for diphtheria. Antitoxin may be produced 
by the injection of increasing doses into suitable animals, particu- 
larly cattle, and this antitoxin has a protective influence when 
injected into other animals. This method of immunization has 
never come into general use. BLACKLEG-TETANUS GROUP. THE GENUS CLOSTRIDIUM 
275 
Animals that have recovered from an attack of the disease 
acquire immunity to a recurrence. Very young cattle and aged 
cattle have a considerable degree of natural immunity. To what 
the immunity developed may be due is not well understood. 
Probably it is in part opsonic. 
Active immunization of animals by vaccination is extensively 
practiced. Many methods of attenuation of the organism for the 
vaccine have been developed. The one so long in common use in 
the United States is the one adopted by the Bureau of Animal 
Industry, and is essentially that developed by Kitt. Fresh ma- 
terial is secured by macerating in a mortar the muscle tissue from 
a blackleg tumor and squeezing the fluid through a linen cloth. 
This is spread in a thin layer and dried to a brown scale at a 
temperature at about blood-heat. This dried virus retains its 
virulence for several years at least. The vaccine is prepared by 
mixing 1 part of this material with 2 parts of water and placing 
in a hot-air oven at a temperature of 95° to 99° for six hours. 
This dries the material and attenuates the organism. It is then 
pulverized and put up in packages containing a definite number 
of doses. Before use a cubic centimeter of water for each dose is 
added, and the material mixed and then filtered. The injection 
then is made with 1 c.c. The dried material, pressed into the 
form of tablets, is sometimes inserted under the skin without sus- 
pending it in water. In some cases threads soaked in suspen- 
sions of the attenuated organisms are drawn into the subcu- 
taneous tissues by means of a needle. Vaccination has proved 
quite satisfactory. 
Improved methods of more recent time include the use of 
aggressins and filtrates. Schobl showed by experiments that 
the edematous fluid derived from blackleg lesions when made 
sterile by filtration through porcelain, possessed remarkable 
immunizing properties. Following his method of treatment the 
Kansas Experiment Station tested the germ free fluid on many 
thousands of cattle and found it highly efficient. The affected 
tissues from blackleg calves were placed in a press and the 
juices expressed. These were then filtered, preservative added 
and used in doses of 8 to 15 c.c. and found to produce sufficient 
immunity to protect calves against doses of virus which promptly 
killed non-vaccinated calves. 276 
VETERINARY BACTERIOLOGY 
At present there is in successful use a product known as black- 
leg filtrate, which consists of the medium on which blackleg 
bacteria have been grown, passed through porcelain filters to 
make it germ free. The results obtained have been very satis- 
factory. The chief advantage claimed for these new immunizing 
agents is that there is no possibility of the disease being produced, 
a thing which frequently occurs when the attenuated vaccine is 
used. 
Bacteriologic Diagnosis.-A presumptive determination of 
the organisms may be made by smear preparations from the in- 
fected tissues. The lack of chains and filaments in smears from 
serous effusions is particularly characteristic, according to Hibler. 
Anaerobic cultures will demonstrate the specific organism in pure 
culture if inoculated with a bit of the tissue before decomposition 
has begun and before putrefactive bacilli have gained entrance. 
Animal inoculation, particularly subcutaneous inoculation into 
the guinea-pig, may prove useful. Usually the symptoms of the 
disease are so characteristic that a bacteriologic test is wholly 
unnecessary. 
Transmission.-It is believed that Clostridium chauvcei is widely 
distributed in nature. The disease occurs only in certain localities. 
There are districts which are never affected. Attempts have been 
made to correlate the topography of the country, such as character 
of soil, presence of marsh land, etc., with the prevalence of the dis- 
ease without marked success. Organisms closely related to Cl. 
chauvcei may be found widely in the soil, but, for the most part, 
they do not possess the peculiar pathogenic characters of this 
form. Infection is believed to occur through wounds. The dis- 
ease is rarely, if ever, contracted directly by one animal from 
another. It is not always possible to locate the point at which the 
organisms gained entrance-in fact, these cryptic infections con- 
stitute a considerable proportion of the cases. It is possible that 
the explanation sometimes offered for similar infections in tetanus 
will hold good here also; that is, that the organisms may occasion- 
ally gain entrance to the blood from the intestinal tract or from old 
wounds and that they cannot produce disease except when they 
lodge in some tissue that has been injured, as from a bruise. BLACKLEG TETANUS GROUP. THE GENUS CLOSTRIDIUM 
277 
Clostriditim gastromycosls ovis 
Disease Produced.-Braxy or bradsot in sheep. 
Distribution.-The disease is recorded from the northern por- 
tion of Europe, particularly in Ireland, the Faroes, the Shetlands, 
Scotland, portions of England, Norway, and North Germany. 
Gilruth claims the disease is also present in Tasmania. 
Historical.-The organism was first described in 1888 by Niel- 
sen, who clearly differentiated the disease from anthrax, with which 
it had previously been confused. The Highland and Agricultural 
Society and the British Board of Agriculture and Fisheries have 
instituted investigations as to the disease and its causal organism. 
Among the most careful studies have been those of Jensen. 
Morphology and Staining.-The organism is a large bacillus, 
about 1 by 2 to 6 ft with rounded ends. The cells are usually 
single, but in smears made from serous effusions chains and fila- 
ments are common, in this respect differing from the organism of 
blackleg. Spores are produced both in tissues and in cultures. 
They are ellipsoidal, usually central, only occasionally polar, and 
cause but little enlargement of the cells. They are motile by means 
of peritrichous flagella. Capsules are not recorded. The cells 
stain readily and are Gram-positive. 
Isolation and Culture.-This organism grows readily in most 
media under anaerobic conditions, particularly if some sugar is 
present. Agar-plate colonies, at first smooth and lens shaped, in 
forty-eight hours become covered with outgrowths resembling those 
of Clostridium chauvosi, giving the colony a felted appearance. 
Gelatin colonies are gradually surrounded by an area of liquefied 
medium. The addition of serum to the medium favors the 
growth of the organism. It grows well in milk, causing an acid 
coagulation, but no peptonization of the casein. 
Physiology.-The optimum growth temperature is between 
35° and 40°, although good growth takes place at room-tempera- 
tures. The spores are resistant to desiccation, but are readily 
killed by boiling. It resembles the bacillus of blackleg in that the 
most favorable reaction of the medium is slightly alkaline, little or 
no growth occurring in an acid medium. While growth is favored 
by the presence of sugar, it is soon stopped by the acids developed. 278 
VETERINARY BACTERIOLOGY 
Gas and acid are formed (according to Bahr) from the fol- 
lowing sugars: Dextrose, mannose, galactose, fructose, lactose, 
maltose, and glycerin, but not from saccharose, raffinose, sorbose, 
arabinose, xylose, rhamnose, mannite, dulcite, adonite, and ery- 
thrite. 
The ability of the organism to produce gas from pure proteins 
is not well demonstrated. Gelatinase is produced; other proteo- 
lytic enzymes are seemingly not formed. 
Pathogenesis.-The disease is primarily one of infection through 
the walls of the omasum. It is characterized by the presence of 
reddish slimy fluid in the abomasum, edema and swelling, and 
frequently extensive necrosis of the mucous membranes and sub- 
mucosa. Generally there is a marked hemorrhagic infiltration 
in the region of the pylorus. Edema and necrosis and often em- 
physema of other tissues may be noted. 
Subcutaneous inoculation of sheep gives rise to an infection 
closely resembling symptomatic anthrax or blackleg. The organ- 
ism has been shown to be pathogenic for sheep, goats, swine, calves, 
guinea-pigs, pigeons, and fowls, while mice and rabbits are more 
resistant. 
Immunity.-Small quantities of a toxic substance are present 
in culture filtrates. Whether true toxins are present has apparently 
not been conclusively demonstrated. 
Jensen has used several methods of immunization. The vac- 
cine most used is prepared similarly to that for blackleg by drying 
spore-bearing broth culture, then heating thirty minutes at 100° C. 
Suspensions of this material are injected subcutaneously. This 
vaccine has been used quite extensively and successfully. His 
serovaccine consists of a mixture of the above vaccine with dried 
immune serum. 
Transmission.-The site of the principal lesions of the disease 
leads to the assumption that the infection follows ingestion of the 
organism. Feeding experiments in general, however, have failed 
to produce the disease. A completely adequate explanation of sea- 
sonal prevalence, distribution, and method of infection has not 
been given. 
Clostridium welchii 
Synonyms.-Bacillus aerogenes capsulatus; Bacillus welchii; 
Bacterium welchii; B. phlegmonis emphysematosce; B. enteritidis BLACKLEG TETANUS GROUP. THE GENUS CLOSTRIDIUM 
279 
sporogenes; B. perfringens; Granulobacillus saccharobutyricus 
immobilis; B. anaerobicus cryptobutyricus; B. cadaveris butyricus; 
B. emphysematis vaginae. 
Disease Produced,-Gaseous edema in man, a secondary in- 
vader in various animal diseases. Welch and Nuttall, in 1892, 
described Bacillus aerogenes capsulatus from the body of a man 
who died from an aortic aneurysm. The internal organs and sub- 
cutaneous tissues showed considerable emphysema. Since that 
time it has been repeatedly isolated in Europe and America. It 
was independently described by Frankel in 1893 as B. phlegmonis 
emphysematosa! from 4 cases of gas gangrene. 
Distribution.-The organism is common in garden-soil, par- 
ticularly that contaminated with excreta. 
Morphology and Staining.-Clostridium welchii is a rod, 1 to 
1.5 by 4 to 8 n, with usually rather square ends. Very short, 
almost coccal forms, and long filaments may be found under 
certain growth conditions. Some strains show curved rods. 
Involution forms (club shapes, filaments, tadpole forms, granu- 
lar types, etc.) are found particularly in old cultures upon coagu- 
lated serum. It frequently occurs in chains, but may be found 
in pairs and small groups. The organism does not appear in 
serous infusions in chains or filaments. It is non-motile. In 
this respect it differs from the other members of this group. 
Spores are sometimes found in infected wounds, but are most 
readily formed in such media as casein broth, alkaline egg 
broth and coagulated serum, free from fermentable carbohy- 
drates. They are rarely seen in fermentable media. Individual 
strains vary in their spore producing ability. The spores are 
large, oval in shape and with slightly flattened ends, subterminal 
or central in position. They are central, and the cells develop 
as clostridia. Capsules may be demonstrated in the body fluids 
and in media containing serum. The organism stains readily 
with the common anilin dyes and is Gram-positive in young 
cultures with frequent occurrence of Gram-negative individuals 
in old cultures. Involution forms are frequent in artificial media. 
Isolation and Culture.-McCampbell has described a modifica- 
tion of Welch's method of isolation as follows: "1 gm. of soil is 
shaken in sterile NaCl solution (0.85 per cent.), and inoculated 280 
VETERINARY BACTERIOLOGY 
into sterile neutral litmus-milk tubes, which are covered with 25 
mm. of neutral paraffin oil, for the purpose of securing anaero- 
biosis, and then incubated for twenty-four hours at 37°. At the 
end of this time the milk in the tubes is coagulated and shows 
acids and gas. A subculture is made in a second litmus-milk 
tube under oil, and incubated for twelve hours in order to pre- 
vent the possible overgrowth of other bacteria. At the end of this 
time the milk usually shows coagulation, acid and gas production, 
as in the first instance ('stormy 
fermentation'); 0.5 c.c. of the 
whey in the subculture is then 
injected into the posterior au- 
ricular vein of a rabbit. In 
three or four minutes the animal 
is killed by a blow on the head, 
and the body is incubated at 
37° for eight to ten hours, at the 
end of which time the abdomen 
is markedly distended with gas. 
When ignited, this explodes and 
burns with a hydrogen flame. 
The thorax of the animal is 
carefully opened, and cultures made from the heart blood in dex- 
trose broth, covered by neutral paraffin oil. In from eight to 
twenty-four hours the culture tubes show a marked cloudiness, 
abundant gas production, and, in most instances, an odor of 
butyric acid." 
The colonies upon agar and gelatin plates are round, grayish, 
semitranslucent; they are usually nucleated, and resemble those 
of Clostridium tetani. Upon agar slants a thin, coalescent, 
yellowish-white growth occurs. Gelatin may or may not be 
slowly liquefied. Bouillon is clouded with a heavy precipitate. 
Little or no growth occurs on potato. Upon blood-serum the 
growth resembles that upon agar with no change in the medium. 
Milk is quickly coagulated, with gas and acid production. 
Physiology.-Growth occurs best at 37°. The thermal death- 
point for non-sporulating culture is 50° for ten minutes, for spores 
100° for fifteen minutes. Gas is produced from dextrose, levulose, 
Fig. 106.-Clostridium welchii 
(Jordan). BLACKLEG TETANUS GROUP. THE GENUS CLOSTRIDIUM 
281 
galactose, maltose, lactose, and saccharose, but not from mannitol, 
dulcitol, or salicin. Probably some differences are to be found in 
various strains. Gas is likewise produced from pure proteins, 
such as recrystallized egg-white. Butyric and lactic acids have 
been detected. 
Pathogenesis.-Experimental Evidence.-Intravenous injec- 
tion of the rabbit frequently, though not always, causes death, 
but subcutaneous inoculations are without effect. Guinea-pigs 
are susceptible, as are also pigeons and mice. 
Character of Disease and Lesions Produced.-Infections with 
Clostridium welchii among the lower animals have been noted in a 
few instances only, and then only in the rabbit and in the dog, as a 
result of severe injuries. However, it may quickly invade tissues 
after death, and give opportunity for mistaken diagnosis. It has 
been isolated from cattle dead of anthrax, swine that have suc- 
cumbed to cholera, together with Cl. chauvcei from blackleg lesions 
in cattle, etc. It has not been shown satisfactorily that it ever 
invades the tissues generally before death. It is a secondary 
invader in practically every instance of natural infection. It has 
been found in emphysema of many organs in the human body. 
Herter believes that the presence of large numbers of this organ- 
ism or its varieties in the intestines is responsible for the produc- 
tion of primary pernicious anemia, particularly in children. 
From the veterinary standpoint the organism is of principal 
interest, not so much because of its slight pathogenic power, as 
the fact that it may be confused upon isolation with other spore- 
bearing anaerobes. 
Immunity.-Frankel failed to secure immunity in laboratory 
animals by injections of killed cultures, though an immune serum 
was secured from a dog after a process of immunization. 
Cl. welchii produces a specific toxin which when injected into 
animals stimulates the production of a specific antitoxin. Well 
washed emulsions of the organism when introduced into experi- 
mental animals produce no infection. Combined, however, 
with a sub-lethal dose of toxin a fatal gas gangrene rapidly 
develops. Bull and Pritchett have proved that the necrosing 
and hemolytic properties of filtrates of Cl. welchii are due to the 
presence of an exotoxin, which is destroyed by heating to between 282 
VETERINARY BACTERIOLOGY 
60° and 70° C., and which is capable of stimulating the produc- 
tion of antitoxin. This antitoxic serum when employed under 
proper conditions inhibits and arrests infection with Cl. welchii. 
Opsonins are present in normal and in increased quantities in 
immune sera, as are also specific bactericidal substances. 
Bacteriologic Diagnosis.-The organism can be recognized 
certainly from tissues only by isolation, and a study of its mor- 
phology, physiology, and effect upon animals. Its Gram-positive 
staining characters, lack of motility, and the difficulty with which 
spores may be demonstrated are significant. By the use of 
specific antitoxin the bacteriologic diagnosis may be confirmed. 
A liquid culture of the organism under study may be mixed with 
varying doses of the specific antitoxin before injection into a suit- 
able laboratory animal. If infection is inhibited the organism is 
Cl. welchii. Or if an injection of the antitoxin renders an animal 
passively immune to infection with a particular organism, then 
that organism is Cl. welchii. 
Transmission.-The organism probably gains entrance to the 
body through wounds or after death invades the tissues from the 
intestines. 
Clostridium oedematis 
Synonyms.-Vibrion septique; Bacillus oedematis maligni, 
Koch. 
Diseases Produced.-Malignant edema; Malignes Edem, 
cedeme malin, septicemie gangreneuse, in various animals and in 
man. 
Pasteur, in 1877, found that the injection of putrid flesh into 
a rabbit was followed by an edema at the point of inoculation, and 
ultimately by the death of the animal, with changes in many of 
the internal organs. That these changes were due to a specific 
organism and not to the poisons of the putrid flesh alone, was 
shown by transfers from one animal to another, and by the isola- 
tion of an anaerobic bacterium. Koch later (in 1881) studied 
the disease. 
Morphology and Staining.-The Clostridium oedematis closely 
resembles the Clostridium chauvcei morphologically, and was 
long considered closely related to it but the work of Meyer seems BLACKLEG TETANUS GROUP. THE GENUS CLOSTRIDIUM 
283 
to prove that the two are quite distinct. The organism is a 
rod, 0.8 to 1 by 2 to 10 m, with rounded ends, single or in chains. 
Many of the cells are long and filamentous. This is particularly 
evident in smears made from serous effusions. It is motile, 
with numerous peritrichic flagella. Capsules have not been 
demonstrated. Spores are produced, usually polar, but some- 
times equatorial. The spore is short ellipsoidal. The rod is not 
greatly distended by the spore, although the snowshoe or Clos- 
tridium shape is usually 
evident. The organism is 
Gram-positive, though 
more readily decolorized 
by alcohol than are most 
Gram-positive forms, and 
stains readily with the 
common anilin dyes. 
Isolation and Culture.- 
The organism may be se- 
cured in pure culture with- 
out difficulty, under ana- 
erobic conditions, from the 
edematous tissues of the 
infected animal. Its cul- 
tural characters in many 
ways resemble those described for Clostridium chauvwi. In 
Hibler's brain medium alkalinity and blackening develop. 
Gelatin is digested, milk is curdled, and the casein digested is 
made alkaline. Coagulated serum is not liquefied. 
Physiology.-The organism is an obligate anaerobe. Growth 
is luxuriant at room-temperature as well as at blood-heat. The 
spores are resistant to desiccation and to heat. Hibler states that 
they can resist several hours' heating to 98°. The optimum tem- 
perature is about 37°, though good growth occurs at 18°. Gas 
is produced from dextrose, levulose, galactose, maltose, lactose 
and salicin, but not from sucrose, mannitol, dulcitol, glycerol or 
inulin, probably also from proteins. Enzymes that liquefy 
gelatin are present. Milk is clotted in 3 to 7 days. There is 
no proteolysis of meat or solid serum. 
Fig. 107.-Clostridium cedematis, 
spores and rods from an agar culture 
(Frankel and Pfeiffer). 284 
VETERINARY BACTERIOLOGY 
Pathogenesis.-Experimental Evidence.-Inoculation of pure 
cultures of the organism into the laboratory animals, and also 
into the horse and other 
domestic animals, will pro- 
duce a typical infection. 
The most infective mate- 
rial is 24-48 hour glucose 
broth. 
Character of Disease 
and Lesions Produced.- 
The tissues at the point of 
invasion, infiltrated with 
yellow or red serum, are 
usually hemorrhagic. The 
muscle becomes an intense 
deep red and is softened, 
but there is no putrid 
odor. Hemorrhages are 
generally to be found in the subcutaneous tissues. Many cases 
of the disease have been noted in man, and it is not uncommon 
in the horse and the sheep. 
It is probable that the cases 
of so-called symptomatic an- 
thrax in the horse have been, 
in reality, infections with this 
organism. Infection has also 
been observed in swine, dogs, 
and rabbits. 
Immunity.-Animals which 
recover from an infection are 
found to be thereafter im- 
mune. The organism is also 
known to produce a leukocidin 
which destroys white blood- 
cells. Antisera have been prepared, and have been shown to 
contain antibacterial substances and antitoxins. The antitoxic 
serum, unlike that of Cl. welchii, does not protect if given after 
the infection is once established. Agglutinins are produced in 
Fig. 108.-Clostridium oedematis, tissue 
smear showing rods without spores 
(Frankel and Pfeiffer). 
Fig. 109.-Clostridium oedematis, dex- 
trose gelatin culture (Gunther). BLACKLEG TETANUS GROUP. THE GENUS CLOSTRIDIUM 
285 
the blood of rabbits inoculated intravenously with heated, 
washed cultures. 
Transmission.-The organism usually gains entrance through 
wounds, although the possibility of a cryptic infection, such as is 
claimed to occur in tetanus, should not be ignored. In man the 
disease has been known to occur following injections in which an 
unclean hypodermic syringe was used, and a case has been 
reported in which the organisms were believed to have gained 
entrance to the body through the intestinal ulcers of typhoid 
fever. Infection may follow delivery, castration, shearing of 
sheep, use of unclean syringes or instruments, or dirty wounds of 
any kind. 
Clostridium of Ghon-Sachs 
Synonyms.-Bacillus wdematis maligni of Ghon and Sachs. 
Disease Produced.-Gaseous edema in man and animals. 
Distribution.-Like the other members of this group, it is 
probable that this organism is widely distributed in the surface 
soils. 
Historical.-The organism was isolated from a case of gaseous 
gangrene in man by the above named investigators and they 
assuming that the Cl. oedematis maligni was proteolytic, and 
finding that this non-proteolytic described it as a new organism. 
Recent investigation of the bacteria of war wounds seems to leave 
little doubt but that this organism was the same as the vibrion 
septique of Pasteur. It has been isolated from a variety of 
animals, particularly cows with puerperal infection, and from 
swine. 
Morphology and Staining.-The organism is a motile rod very 
similar in morphology to Clostridium chauvai. In smears from 
serous effusions, however, it occurs in chains and long filaments. 
The sporulating cells are somewhat swollen. The spores are 
ellipsoidal in shape. No capsules have been demonstrated. The 
cells stain readily and are Gram-positive. 
Culture.-The colonies in solid media resemble those of the 
blackleg bacillus. Milk is usually coagulated with gas and acid 
production, but with no digestion of the curd. Hibler's brain 
medium is acidified and not blackened. 286 
VETERINARY BACTERIOLOGY 
Pathogenesis.-The organism is pathogenic for guinea-pigs, 
rabbits, white rats, gray rats, and mice. In experimental inocu- 
lation an emphysematous edema without much hemorrhage is 
produced. 
Clostridium sporogenes Metchnikoff 
This organism was found present in a large proportion of war 
wounds in acute cases of gas gangrene as well as in conditions in 
which the wound was progressing satisfactorily. It is generally 
in association with other organisms. In general it is not lethal 
for laboratory animals in doses up to 4 c.c. Some strains are, 
however, capable of producing a putrid, perforating gangrene. 
The organism is of importance principally because of the possi- 
bility of confusion with other members of this group and because 
of its extraordinary capacity for persisting in the presence of 
other organisms. 
Clostridium botulinum 
Synonym.-Bacillus botulinus. 
Disease Produced.-Meat, sausage, and food poisoning in 
man, botulism (botulus, sausage). 
Van Ermengem, in 1896, isolated an organism from sausage, 
which he believed to be the cause of poisoning. The organism 
has since that time been several times isolated, and is, therefore, 
of some hygienic importance, particularly in meat inspection and 
in meat hygiene. This disease or poisoning should not be con- 
fused with that produced by the Bacterium enteritidis. 
Distribution.-A few well-authenticated reports of the isola- 
tion of the organism are on record, from European countries, 
many, within the past few years from the United States, from 
man, the horse, mule, cow and domestic fowls. The disease has 
been reported as caused by the eating of sausage, fish, lobsters, 
oysters and vegetables such as preserved beans and peas and in 
animals from eating spoiled ensilage and grain. 
Morphology and Staining.-Clostridium botulinum is a large 
bacillus, with usually rounded ends, 0.9 to 1.2 by 4 to 6 v. It is 
commonly single or in pairs, sometimes in short chains. Involu- 
tion forms frequently occur. It is motile by means of four to BLACKLEG-TETANUS GROUP. THE GENUS CLOSTRIDIUM 
287 
eight peritrichic flagella. Capsules have not been demonstrated. 
Small oval spores, somewhat greater in diameter than the bacillus, 
are produced in a subterminal position. The organism stains 
readily with the anilin dyes and is Gram-positive. 
Isolation and Culture.-Growth is sparse in media which 
contain no sugar. The colonies on dextrose gelatin are at first 
circular, transparent, light yellow, and soon liquefy the gelatin. 
Under the low power of the microscope they appear to consist of 
granules in constant motion. Later the colonies become brown 
and opaque. According to Van Ermengem, milk is not curdled 
and the organism grows spar- 
ingly. Graham suggests the 
following method for the isola- 
tion and identification of the 
organism. "A sample of 
watery extract of the feed pre- 
ceding final filtration is diluted 
and seeded under conditions 
favorable for the development 
of B. botulinus. Pork broth 
faintly alkaline plus 2 per cent, 
dextrose is then inoculated with 
various dilutions and placed in 
an atmosphere of hydrogen; 
or anaerobiosis may be ob- 
tained by covering the surface of the tubes with sterile paraffin 
oil. The pyrogallic acid or vacuum methods can also be em- 
ployed. Preceding the period of incubation, the inoculated tubes 
should be subjected to a temperature of 80° C. for fifteen or 
twenty minutes. After incubating in the dark at room tem- 
perature for ten days, pathogenicity of cultures is determined 
following oral administration of 0.5 mil of the mixed broth culture 
to guinea-pigs. The production of characteristic symptoms and 
death in guinea-pigs by the broth culture of obligate spore- 
bearing anaerobes, and facultative anaerobic spore-bearing 
bacilli is considered diagnostic. Immunological tests may be 
projected on guinea-pigs if toxic broth cultures are encoun- 
tered, antitoxic sera (A and B) being used to establish the 
Fig. 110.-Clostridium botulinum 
(van Ermengem in Kolle and 
Wassermann). 288 
VETERINARY BACTERIOLOGY 
relation, if any, of the intoxication induced to B. botulinus. 
Pure cultures are obtained by seeding in plain agar and fishing 
colonies to pork agar shake cultures containing 2 per cent, 
dextrose." 
Physiology.-The organism, is an obligate anaerobe. Its 
optimum growth temperature is 25 to 30°. Some strains grow 
little, if at all, at blood-heat, and when developing at this tem- 
perature produce numerous involution forms. Gas is produced 
from dextrose and lactose but not from saccharose. Acid, in 
part butyric, is produced in dextrose media. 
Pathogenesis.-Injections of the organism into the body of 
laboratory animals have revealed the fact that some strains of 
the organism are pathogenic only by virtue of the toxins that are 
elaborated outside of the body. It does not apparently increase 
in numbers in the tissues. Probably this may in part be ac- 
counted for by its normal optimum growth temperature. The 
toxin produced, on the other hand, is very poisonous, whether 
injected or ingested. The use of raw or imperfectly cooked 
animal foods or of infective canned foods may give rise in man 
to the symptoms of botulism in the course of twenty-four to 
thirty-six hours, often with fatal termination. 
Immunity.-The toxin produced by Clostridium botulinum is 
among the most powerful known-0.00005 to 0.0001 gm. is fatal 
in three to four days when injected subcutaneously into a guinea- 
pig, and 0.0001 to 0.0005 gm. will destroy a rabbit. A most 
striking characteristic of this toxin, and one which distinguishes 
it from those of diphtheria and tetanus, is its ability to produce 
poisoning when taken into the body by way of the alimentary 
tract, withstanding the gastric and intestinal digestive processes. 
Guinea-pigs and even apes are killed by the ingestion of 0.01 mil. 
of a dextrose broth-culture solution in which the organism has 
been grown. The toxin is destroyed by exposure to light and air. 
Heating to a temperature of 80° renders it non-toxic. Antitoxin 
has been prepared from the goat and from the horse by gradually 
increasing doses of the toxin. This antitoxin exerts both a 
prophylactic and a curative effect when injected. Immunologic 
tests with the antitoxin of Clostridium botulinum indicate that 
there are two types of the organism designated by Burke, Type BLACKLEG-TETANUS GROUP. THE GENUS CLOSTRIDIUM 
289 
A and Type B. The latter type according to Graham is most 
frequently encountered in animals; the Type A from canned 
fruits and vegetables and from ensilage. Chickens are not sus- 
ceptible to Type B, but are to Type A. The antitoxic serum 
prepared against one strain is not effective in neutralizing the 
toxin from the other strain nor in checking the disease produced 
by the other strain. This would suggest the use of a polyvalent 
antitoxic serum in the treatment of spontaneous cases. 
Bacteriologic Diagnosis.-This can be accomplished only by 
isolation and cultivation of the specific organism by serological 
tests with known antitoxin against suspected toxin and by the 
agglutination test. 
Transmission.-The organism has been isolated from poi- 
sonous meat, canned vegetables and fruits, ensilage and grains 
and from normal swine feces. The disease can be produced only 
by the ingestion of substances in which the organism has been 
growing. CHAPTER XXV 
PASTEURELLA, OR HEMORRHAGIC SEPTICEMIA GROUP 
The organisms belonging to this group are all aerobic, non- 
motile, Gram-negative bacilli that do not produce spores, and that 
show a decided tendency to polar staining. They exhibit compara- 
tively slight powers of fermentation. 
The bacteria of this group were first recognized as closely re- 
lated by Hueppe in 1886. He included in his species Bacillus 
septicemice hcemorrhagicce, the causal organism of chicken cholera, 
rabbit septicemia, swine plague, hemorrhagic septicemia of cattle 
and wild animals, and several others. Trevisan included all of 
these organisms as separate species in a new genus which he named 
Pasteurella, after Pasteur, who had studied the causal organism 
of chicken cholera. The name pasteurellosis is, therefore, fre- 
quently used to designate a disease caused by an organism of this 
group. The classification proposed by Lignieres in 1901 has been 
extensively used, particularly by the French bacteriologists. 
The organisms which are to be grouped with certainty here are 
those which cause the following animal diseases: Hemorrhagic sep- 
ticemia of cattle, of sheep, septic pleuropneumonia of calves, fowl- 
cholera, rabbit septicemia, and the "Loffler-Schutz" swine plague. 
Here is also to be included the organism which causes human 
plague. 
The great similarity of these various organisms to each other 
has led many writers to include them as varieties of a single species 
which has been variously named Bacillus bipolaris septicus, B. 
plurisepticus, Bacterium bipolare pluricida, Bacillus septicemice 
hcemorrhagicce, Coccobacillus, and Bacterium multicidum. 
There is still great need of careful comparative studies of the 
organisms belonging to this group. It is by no means certain how 
many species are included. It is probable that in certain of the 
diseases the organism is a secondary invader. Non-virulent strains 
and perhaps species are known to occur frequently in nature. 
290 PASTEURELLA, OR HEMORRHAGIC SEPTICEMIA GROUP 
291 
Since it has thus far not proved possible to differentiate the 
organisms associated with the various diseases by means of their 
morphologic or biologic characters, it is necessary to adopt 
tentatively a pathologic classification, and group them with 
reference to the animals naturally infected and the diseases pro- 
duced. The following list includes only the more important 
types that have been described, and is not complete: 
A. Organisms which produce disease in lower animals only: 
1. In birds. Pasteurella (aviseptica) choleras gallinarum. 
2. In swine. Pasteurella suiseptica. 
3. In cattle. Pasteurella boviseptica. 
4. In equines. Pasteurella equiseptica. 
5. In rabbits. Pasteurella cuniculicida. 
6. In rats and other rodents. Pasteurella pseudotuber- 
culosis rodentium. 
B. Organism producing disease in man: Pasteurella pestis. 
Because of the close resemblance among the organisms causing 
hemorrhagic septicemias of the lower animals, the discussion of 
morphologic, physiologic, cultural, and biologic characters common 
to all species will be given first, limiting the discussion under the 
species to the characters of value in differentiation and to problems 
of immunity and pathogenesis. 
Hemorrhagic Septicemia Group 
Distribution.-Organisms having all the morphologic and bio- 
logic characters and sometimes even the pathogenesis of the dis- 
ease-producing members of this group have been isolated many 
times from the mouth, nose, and alimentary tract of many animals. 
They are undoubtedly common as secondary invaders, and in some 
diseases have served to obscure the real causal organism by the 
regularity of their presence. It is contended by some writers that 
the sporadic nature of these diseases is due to the sudden acquisi- 
tion of virulence by forms already present in the body. Not only 
the relative virulence of these ubiquitous organisms is in need of 
study, but also careful comparative studies of their cultural and 
physiologic characters. 
Morphology and Staining.-The organisms are non-motile rods. 
In body fluids they are usually about 0.5 by 1 ju, and with aqueous 292 
VETERINARY BACTERIOLOGY 
anilin dyes show intensive coloration at the poles, the central 
portion of the cell staining faintly or not at all. Giemsa's stain 
shows the polar granules well. In culture-media there occurs con- 
siderable variation in size. There do not seem to be well-marked 
differences in size among the different species. In pure cultures 
the organisms may be cocci, diplococci, or short rods. The polar 
staining character is much more difficult to demonstrate in cul- 
tures than in body fluids. By the use of carbol-fuchsin applied for 
one-half to one second many cells with polar granules may be 
demonstrated. The organisms are uniformly Gram-negative. 
Cultural Characters.-All species are readily cultivated on 
suitable artificial media. 
Gelatin colonies are small, white, and usually irregular, without 
marked distinctive characters. Gelatin slants show numerous 
small, round, flat, gray to translucent dewdrop-like colonies, 
which later become gray white and opaque. The gelatin is 
never liquefied. 
On agar plates the colonies resemble those on gelatin. There 
is no tendency for the colonies to spread over the medium. The 
addition of blood-serum increases the luxuriance of growth, dex- 
trose and glycerin have no perceptible effect. 
The organism does not grow in Endo medium or on malachite 
green agar. On Drigalski's medium there is good growth without 
a change of color. Blood agar shows no hemolysis. 
Little or no growth occurs upon potato. 
In broth uniform clouding occurs with occasional clearing 
by sedimentation. Old broth cultures are often slimy. 
No change occurs in milk. 
Luxuriant growth occurs on Huntoon's hormone medium and 
on meat medium. 
Physiology.-The optimum temperature is about 37°, the 
minimum, 12° to 13°, and the maximum, 42° to 43°. 
The organisms are aerobic, growing little or not at all under 
anaerobic conditions. 
Gas is never developed in any sugars; small amounts of acid 
may be. 
No enzymes liquefying gelatin or blood-serum are produced. 
Indol is formed by some organisms, apparently not by others. 
Slight development of hydrogen sulphid and reduction of nitrates 
have been recorded. PASTEURELLA, OR HEMORRHAGIC SEPTICEMIA GROUP 
293 
Pathogenesis.-The most susceptible of the laboratory animals 
is the rabbit. Intravenous injections usually prove fatal in a few 
hours to a day. Autopsy reveals the development of a septicemia 
in which the lesions are not particularly characteristic. Petechial 
hemorrhages are commonly noted on the mucous membranes of the 
respiratory and digestive tracts, and there may be swelling of 
lymph-nodes and spleen, congestion and edema of the lungs, and 
hyperemia of the kidneys. 
Practically all species are also pathogenic for mice and for spar- 
rows. Guinea-pigs are somewhat more resistant. 
Other species of animals are much more distinctive in their 
reactions. Fowls, for example, are quite susceptible to the bacillus 
of fowl cholera, but quite resistant to infection with mammalian 
types. 
No true toxin has been demonstrated and, in consequence, no 
antitoxin. The disease in its acute form in any animal is a bacter- 
emia in which enormous numbers of bacteria are present in the 
blood-stream. 
Immunity.-An endotoxin is produced which goes into solution 
slowly as a result of the autolysis of the cells. Broth filtrates of 
young cultures are innocuous, while autolyzed cultures are toxic. 
The toxicity of various strains has been shown to vary, but it is 
apparently a variable quite independent of virulence. MacFad- 
yean has also demonstrated the presence of intracellular poisons 
by grinding the cells at the temperature of liquid air and by ex- 
tracting with a dilute alkali. 
Weil and Bail have sought to explain the virulence of this 
organism and the peculiarities of the infection by the use of the 
aggressin hypothesis. It seems evident that certain strains are 
capable of paralyzing the antibacterial defences of the body and 
thereby prevent phagocytosis. Citron and others have shown that 
such inhibiting substances may be dissolved from virulent cells. 
Pasteurella cholerae gallinarum 
Synonyms.-Bacillus choleras gallinarum; B. cholerce; Bacter- 
ium avicidum; Bacillus avisepticus. 
Diseases Produced.-Fowl or chicken cholera, in domestic 
fowls and other birds. 294 
VETERINARY BACTERIOLOGY 
Distribution.-The disease has a wide distribution in Europe 
and America. 
Historical.-Perroncito, in 1878, first discovered this organism. 
Pasteur, in 1880, succeeded in cultivating it, and with it performed 
many experiments on attenuation and immunization. Consider- 
able historical importance is attached to it as marking the beginning 
of the study of experimental immunity. 
Morphology and Staining.-Typical of the group. Consider- 
able variations in size of the organisms from different strains may 
occur. 
Isolation and Culture.- 
Characteristics of the group. 
Physiology. - Characteris- 
tics of the group. According 
to Hadley, indol may or may 
not be produced and nitrates 
are usually reduced. Some 
acid is produced from dex- 
trose, but not from saccharose, 
lactose, or mannite. 
Pathogenesis.-Great vari- 
ations in virulence have been 
noted among various strains. 
It may prove pathogenic upon 
inoculation or in some cases upon ingestion to fowls, geese, pigeons, 
mice, and rabbits, producing very rapidly fatal septicemias. 
Guinea-pigs are relatively immune. 
The pigeon is among the most susceptible of experimental birds. 
The injection of minute quantities of a virulent culture into the 
breast muscle will prove fatal in from twelve to twenty-four hours. 
At the site of inoculation there develop an induration and thick- 
ening of the skin. The subcutaneous tissues show a straw-colored 
exudate. Usually there is found an acute pericarditis, enlarge- 
ment of the spleen, and severe hemorrhagic enteritis. 
In domestic fowls there appears congestion of the heart with 
an accumulation of serum in the pericardial sac. The liver is also 
congested, frequently showing petechise. The spleen is enlarged 
and softened. The lungs usually are congested and may show areas 
Fig. 111.-Pasteur ella cholerce galli- 
narum, from an agar slant (X 1000) 
(Gunther). PASTEURELLA, OR HEMORRHAGIC SEPTICEMIA GROUP 
295 
of hepatization. Enteritis is usually demonstrable, frequently 
hemorrhagic. 
Rabbits are likewise susceptible and succumb in ten to twenty 
hours with characteristic symptoms. 
Infection may also be secured in the larger animals, producing 
in many cases typical hemorrhagic septicemias. 
Immunity.-No true toxin has been demonstrated for this 
organism. Endotoxins are produced. One attack of the 
disease with recovery confers immunity. Agglutination in dilu- 
tions of 1 : 6000 has been shown with blood of animals artificially 
Fig. 112.-Pasteurella cholerce gallinarum, in pigeon's blood (Frankel and 
Pfeiffer). 
immunized. The nature of this immunity is not certainly known, 
although opsonins have been demonstrated. 
Pasteur, in 1880, worked out a method of prophylaxis by the 
use of vaccines prepared by attenuating the organisms by long-con- 
tinued cultivation upon artificial media. Broth cultures were 
allowed to stand from three to ten months. Under these condi- 
tions the virulence is gradually lost, and inoculation into the fowl 
is followed by a mild local reaction only. This immunizes against 
subsequent injections of the virulent form. Pasteur believed that 
the attenuating factor was the abundant supply of oxygen, for 296 
VETERINARY BACTERIOLOGY 
cultures which he sealed from the free entrance of air he found to 
retain their virulence even after ten months. He also found that 
various strains showed great differences in their rate of attenuation. 
The Pasteur method of vaccination has never come into general 
use. Tests have shown that the use of the vaccine sent out by the 
Pasteur Institute was apt to produce typical cholera in some fowls. 
It has been shown that some degree of immunity is conferred by the 
injection of killed cultures of the organism. 
It has also been found that immunization against one of the 
members of the hemorrhagic septicemia group immunizes like- 
wise against others. Injections of the Pasteurella boviseptica, for 
instance, will protect against subsequent injection with Past, 
cholera gallinarum. Lignieres has prepared a polyvalent vaccine 
by growing at 42° to 43° organisms isolated from sheep, cattle, 
horses, dogs, hogs, and fowls in bouillon. When allowed to 
grow for five days it constitutes the Vaccine I; for two days only, 
Vaccine II. One-eighth c.c. of I is injected, and twelve to 
fifteen days later the same amount of Vaccine II. By this 
means he claims to be able to immunize against all types of 
hemorrhagic septicemia in animals other than fowls. This 
method has not been utilized in practice, although a few recorded 
tests have been favorable. 
Hadley has succeeded in isolating one strain (his No. 52) of a 
non-virulent organism which has very high immunizing value 
when used as a vaccine. He has found that vaccination with this 
strain confers an active immunity against all pathogenic strains 
tested. Hadley also concludes that the virulence of various strains 
of fowl-cholera organisms is subject to far less variation than has 
been supposed. 
Kitt and Mayr, in 1897, showed that it is possible to secure a 
protective serum from the horse and other animals, as goats and 
swine, by injections of living fowl-cholera organisms, and this 
serum, when injected in suitable quantities into susceptible animals, 
will protect them from injections of virulent bacilli. Schreiber, in 
1899, elaborated upon an observation of the preceding investiga- 
tion that animals immunized against swine-plague bacilli {Pas- 
teurella suiseptica) were likewise immune to fowl cholera. In 
1902 he gave the name "septicidin" to a polyvalent serum PASTEURELLA, OR HEMORRHAGIC SEPTICEMIA GROUP 
297 
which he prepared by immunization with Past, suiseptica and 
Pasteurella choleroe gallinarum. The reports relative to the effi- 
ciency of this serum are conflicting. It has not come into general 
use. Hertel, in 1902, reported that by intravenous injections of 
dead bacteria followed by living bacteria into an ass he secured 
a serum which in injections of 0.5 c.c. protected pigeons against 
10,000 times the normal lethal dose of the organism. Lignieres 
and Spitz have also prepared a polyvalent serum, using the 
Ligniere vaccine noted above. There is no record of a practical 
utilization of this method. Kitt and others, in 1904, have 
described sera which protect fowls experimentally inoculated 
with Past, cholera gallinarum, but, like the others described, 
these have not come into general use. It may be concluded, 
therefore, that while immunization against fowl-cholera, either 
vaccination or the use of antisera, has been shown to be pos- 
sible, it has not been proved practicable. 
Bacteriologic Diagnosis.-Stained mounts of the blood which 
reveal the presence of Gram-negative bacilli showing prominent 
bipolar staining are diagnostic. The bacillus may be readily iso- 
lated in artificial media. Whether or not serum reactions, particu- 
larly agglutination, might be utilized in diagnosis is not known. 
Transmission.-The disease is supposed to be transmitted from 
bird to bird by ingestion of food or water fouled with excretions 
containing the specific organism. 
Pasteurella suiseptica 
Synonyms.-Bacterium suicidum; Loffler-Schutz bacillus; Ba- 
cillus suicida; Bacillus suiseptica. 
Disease Produced.-Swine-plague, Schweineseuche. 
Distribution.-The organism has been isolated from swine 
many times in Europe and America. 
Loffler and Schutz, in 1886, published results which established 
the identity of swine-plague as a specific disease by the discovery 
of the causal organism. In the same year Smith isolated what 
proved to be the same organism from hogs in the United States. 
Since that time it has been isolated from animals in many parts of 
Europe and the United States. From the beginning the close 
relationship between the swine-plague and fowl-cholera bacilli 
was recognized. In the early literature of swine diseases in 
America there is much confusion relative to the use of the terms 298 
VETERINARY BACTERIOLOGY 
4'swine-plague" and "hog-cholera." The discovery that hog- 
eholera is caused primarily by a filterable virus has made neces- 
sary a very careful retraversing of the knowledge relative to 
swine-plague, and it has been urged that probably the Pasteurella 
suiseptica is a secondary invader merely, as is the hog-cholera 
bacillus, and that the two diseases differ not at all in their primary 
cause. The evidence at present seems to point, however, to a 
specific disease caused by 
Past, suiseptica and en- 
tirely distinct from hog- 
cholera. The question 
cannot be said to be 
satisfactorily settled at 
the present time. In the 
United States it seems 
probable that swine- 
plague, if such really 
exists, is relatively un- 
important in compari- 
son with hog-cholera. 
The situation is still fur- 
ther complicated by the 
fact that many writers 
have found in the respiratory tracts of normal swine numerous 
bacteria having all the characteristics of typical Past, suiseptica, 
even to pathogenicity. 
Morphology and Staining.-Typical of the group. 
Isolation and Culture.-Typical of the group. 
Physiology.-Typical. Slight acid production in dextrose and 
saccharose. Indol production and nitrate reduction appear to be 
variable. 
Pathogenicity.-The different strains show great fluctuations 
in virulence. In general, the pathogenicity for laboratory animals 
appears to be that of the group. The mouse and rabbit are particu- 
larly susceptible. The guinea-pig is somewhat more resistant. 
In fowls and pigeons large injections are usually required to 
produce infection. Experimental infection may also be secured 
in the horse, in cattle, sheep, dogs, and cats. The results in swine 
have proved quite variable. The injection of a highly virulent 
Fig. 113.- Past, suiseptica (after deSchwein- 
itz and McFarland). PASTEURELLA, OR HEMORRHAGIC SEPTICEMIA GROUP 
299 
culture may lead to the death of the animal within one to three 
days from septicemia, with an extensive hemorrhagic edema about 
the site of inoculation. The injection of less virulent cultures or 
the use of animals showing some degree of resistance may lead only 
to local edema followed by abscess formation, or to a more chronic 
infection which terminates fatally in a few weeks. In the latter, 
section shows extensive lung lesions including partial hepatization 
with necrotic areas, fibrinous pleuritis, 
and enlargement of the spleen. 
Intravenous injection of swine is 
usually followed by a fatal septicemia. 
A disease closely simulating the 
typical swine-plague has been produced 
in swine by intratracheal injections and 
by inhalation. 
The spontaneous infection of swine 
may occur as a septicemia which proves 
very quickly fatal, as a pneumo-enteritis 
characterized by the involvement and hepatization of consider- 
able lung areas, or as a chronic type. 
The evidence seems to point to the Past, suiseptica as a nor- 
mal inhabitant of swine. It is possible that, like the pneumo- 
coccus, it can produce infection and increase its virulence under 
certain conditions, decrease in body resistance, etc. 
Immunity.-As with the fowl-cholera bacillus, no true toxins 
have been demonstrated. Both active and passive immunization 
against the Past, suiseptica have been accomplished. Active 
immunization has been attempted in many different ways. The 
killed and living cultures have not, in general, proved satisfactory in 
immunizing the hog, although they have been successfully used in 
the preparation of antisera from the horse and other animals. 
Weil has elaborated the following technic, making use of the so- 
called "natural aggressins" for the establishment of immunity: 
A rabbit is injected intraperitoneally with 5 c.c. of bouillon con- 
taining a drop of twenty-four-hour culture of a highly virulent 
strain of the organism. The animal should die within the next 
tw'enty-four hours. The exudate, varying in amount from 1 to 20 
c.c., is pipetted off and sterilized by the addition of 0.5 per cent, of 
Fig. 114.-Pasteurella sui- 
septica in blood (after de- 
Schweinitz, Report Bureau 
of Animal Industry). 300 
VETERINARY BACTERIOLOGY 
phenol, then heated to 44° for three hours, then its sterility deter- 
mined by transfers to broth. If the broth shows no growth, the 
material is sterile and is ready for use. This may be used to inject 
laboratory animals and thereby establish immunity. The animal 
immediately after injection becomes more susceptible to the disease, 
presumably due to the presence of aggressin in the blood, but later 
a relatively permanent active immunity is produced. In practice 
it is found that in the immunization of hogs it is necessary that the 
exudate containing the aggressin be obtained from other hogs 
rather than from rabbits. Wassermann and Citron have de- 
veloped a somewhat similar method of immunization by the use 
of so-called "artificial aggressins" or bacterial extracts. These 
methods of immunization are of much more theoretic than prac- 
tical importance. 
Passive immunization by means of antisera has been studied by 
several investigators. A rabbit may be actively immunized by 
one of the preceding methods, and its serum may protect a mouse 
in doses of less than 0.1 c.c. against a fatal injection of a highly 
virulent organism. Wassermann and Ostertag and their pupils 
have shown that an antiserum specific for one strain of Past, 
suiseptica is not always effective for others. They, therefore, 
prepare serum by the systematic immunization of a horse against 
several strains of the organism until a serum of high potency is 
produced. Its strength is determined by injections into mice. 
Experiments upon young pigs with this serum are claimed to have 
been highly successful, but the method has not come into general 
use. Simultaneous injections of immune sera and of Past, 
suiseptica have also been advocated. 
In summary it may be said that immunization against swine- 
plague is still in the experimental stage, and that no completely 
satisfactory method has been evolved. 
Bacteriologic Diagnosis.-The identification of the causal 
organism by actual isolation is the only practicable method of 
bacteriologic diagnosis. 
Transmission.-The means by which the disease spreads natu- 
rally are not fully understood. It is possible that it is by ingestion, 
probably sometimes by inhalation. PASTEURELLA, OR HEMORRHAGIC SEPTICEMIA GROUP 
301 
Pasteurella boviseptica 
Synonyms.-Bacterium bovisepticum; B. bipolar e multicidum; 
Bacillus bovicida, B. vitulisepticus: Bacillus bovisepticus. 
Diseases Produced.-Hemorrhagic septicemia in cattle, buf- 
falo, and related wild animals; septic pleuropneumonia of calves; 
Rinderseuche, Wildseuche. 
The early descriptions of the disease refer to it as attacking 
cattle, wild animals, and swine. Bollinger, in 1878, first described 
it as Wild- and Rinderseuche, attacking wild boar and deer. Kitt, 
in 1885, isolated an organism belonging to the hemorrhagic septi- 
cemia group. Since that time numerous investigators have re- 
ported epizootics of the disease in many countries. Fernmore, in 
1898, first noted its presence in the United States. It has been 
repeatedly found since that time, particularly in the Mississippi 
Valley. 
Morphology and Staining-Typical of the group. 
Culture and Physiology.-Typical of the group. 
Pathogenesis.-The organism is usually pathogenic for the 
mouse, pigeon, rabbit, and sparrow. Highly virulent cultures 
produce a quickly fatal septicemia in calves. 
The lesions in the adult animals are characteristically petechiee 
in many of the body organs, particularly in the serous surfaces. 
Hemorrhages are quite uniformly present also in the subcutaneous 
tissues. In some cases these are quite extensive and involve a con- 
siderable portion of the body surfaces. 
Septic pleuropneumonia in calves is one of the most frequent 
types of infection. In this disease the pleura is often partially 
covered with thick fibrin layers, the interstitial connective tissues 
of the lungs show a serohemorrhagic infiltration, the remainder of 
the lung tissue is hemorrhagic. According to Jensen, in those cases 
in which the infection progresses more slowly, the exudate laid 
down in the connective tissue is responsible for the development 
of firm white yellow layers which surround the thickened lung 
lobules, which are dark red or mottled in color. 
Immunity.-Repeated injections of animals at first with non- 
virulent and later with virulent organisms will produce a relatively 
high degree of active immunity. The serum from such animals has 
considerable power to produce passive immunity. Some inves- 302 
VETERINARY BACTERIOLOGY 
tigators claim that there are many strains of hemorrhagic septi- 
cemia and, therefore, a polyvalent serum must be secured either 
by injecting many strains of organisms into the serum animal, or 
by mixing the serum secured from several animals each immun- 
ized against several strains. 
Bacterins prepared by killing cultures of Past, boviseptica 
by heat or antiseptic have been used for prophylactic purposes 
with apparently good results. Such bacterins are frequently 
polyvalent. 
Organisms belonging to this group have been isolated from 
a considerable number of animal diseases in addition to the 
ones which have already been described. Rabbit septicemia or 
rabbit plague (Pasteurella cuniculicida'), pneumo-enteritis, or 
hemorrhagic septicemia of sheep and of the horse, infectious 
pneumonia of goats, Biiffelseuche or pasteurellosis of the buffalo, 
dog typhoid or dog pasteurellosis, hemorrhagic septicemia of 
elephants, of geese, wild birds, and many other animals have been 
ascribed to Past, septicemice haemorrhagicce. As has been before 
stated, the evidence in some of these cases seems to be inconclusive. 
Other Hemorrhagic Septicemias of Animals 
Pasteurella pestis 
Synonyms.-Bacterium pestis; B. pestis bubonicce; Bacillus 
pestis. 
Disease Produced.-Bubonic plague in man and rodents. 
Yersin and Kitasato, in 1894, independently described the 
organism which causes bubonic plague. Since that time it has 
been isolated and described by many observers in numerous 
outbreaks. 
Distribution.-The disease is endemic in parts of China and 
India. At various times it has spread as an epidemic over the; 
entire civilized world. Cases have been reported within recent 
years in most of the civilized countries. 
Morphology and Staining.-The Pasteurella pestis morphologi- 
cally resembles the other members of this group. Involution 
forms are produced so readily upon appropriate culture-media, 
such as partially desiccated agar and salt agar, that their develop- 
ment has been regarded as diagnostic. Capsules may sometimes, 
be demonstrated on culture-media, but not in tissues. PASTEURELLA, OR HEMORRHAGIC SEPTICEMIA GROUP 
303 
Isolation and Culture.-Growth in general is typical of the 
group. One character found useful in diagnosis is the "stalac- 
Fig. 115.-Pasteur ella pestis (Wherry). 
tite" formation in broth covered with oil and allowed to remain 
without being disturbed. Under these conditions long delicate 
threads are produced which 
hang from the oil and resemble 
the stalactites found in caves. 
Physiology.-Reactions in 
general are those typical of the 
group. Dextrose broth is 
acidified, but no gas is pro- 
duced. Indol is not formed. 
Pathogenesis. - Experi- 
mental Evidence.-The organ- 
ism readily infects mice, rats, 
guinea-pigs, rabbits, dogs, 
cats, and monkeys when 
they are experimentally inocu- 
lated. The symptoms and 
lesions produced are entirely typical of bubonic plague in man. 
Accidental infection of man resulting in 4 cases of plague occurred 
Fig. 116.-Pasteur ella pestis, bacilli 
from a bubo (Gunther). 304 
VETERINARY BACTERIOLOGY 
in a Vienna laboratory at a time when bubonic plague did not exist 
elsewhere in Europe. The causal relationship of the organism to 
the disease may be held to be fully established. 
Character of Disease and Lesions Produced.-The disease in 
experimental animals may be a rapidly fatal septicemia, or in those 
animals which are somewhat resistant, as the rat, typical buboes 
(enlarged and suppurating lymph-nodes) or abscesses in the spleen 
or liver are produced. The disease in man may be one of three 
types-septicemic, usually rapidly fatal with the organisms gen- 
erally distributed through the blood and various tissues of the 
body; the pneumonic, also rapidly fatal; and the bubonic, the most 
common, in which the lymph-nodes are infected, become enlarged, 
and ulcerate. The bubonic type is less fatal, recovery taking 
place in a small percentage of cases. The septicemic type of the 
disease is often accompanied by extensive subcutaneous hemor- 
rhages, which gave the name "black death" to the epidemics of 
medieval Europe. 
Immunity.-No true toxin has been demonstrated for Pas- 
teurella pestis, although endotoxins have been shown to be present. 
Agglutinins for this organism may be found in the blood of ad- 
vanced cases and of convalescents, but they appear too late to be 
of any diagnostic value. The reaction is rather doubtfully specific. 
The reaction occurs only in low dilutions-rarely above 1 : 20. 
Agglutination in much higher dilutions may be secured with im- 
mune serum-sometimes as high as 1 : 1000 has been observed. 
Precipitins have also been noted in laboratory experimentation. 
Opsonins for Past, pestis have been demonstrated in normal 
human serum and in immune serum. Bactericidal substances are 
present in the serum of artificially immunized animals. 
Active immunization of man against bubonic plague has been 
quite extensively practised. The procedure consists in every case 
of injection of killed or attenuated bacilli or their products as a 
prophylactic measure. The use of the various substances has 
proved quite successful. Haffkine's vaccine has been used in India. 
It consists of a killed six-weeks' culture of plague bacilli in broth. 
Modifications of this method have been utilized by many investiga- 
tors. The immunity established is probably both opsonic and 
bactericidal. PASTEURELLA, OR HEMORRHAGIC SEPTICEMIA GROUP 
305 
Passive immunization by the injection of the serum of horses 
hyperimmunized against Pasteurella pestis has been highly suc- 
cessful, according to some, and of no material advantage accord- 
ing to others. Several procedures have been advocated. The 
following is that of Kolbe and Kumbein: A culture of Past, pestis 
is passed through rats to exalt its virulence. This is planted upon 
agar and incubated forty-eight hours at 30°, the growth washed 
off, and suspended in physiologic salt solution. The suspension 
is killed by heating to 70° for an hour and its sterility determined. 
The horse is injected at intervals of a few days with gradually 
increasing doses of the dead bacteria, until after seven or eight 
injections the bacteria from six or more culture-tubes are injected 
at one time. Injections of minute quantities of the living organ- 
ism are then begun, and finally, after repeated injections, the 
living organisms from 16 cultures are used. The interval be- 
tween injections is governed by the reaction of the animal. 
Usually it is from five to eight days. The animal is then bled, 
and the serum preserved by the addition of 0.5 per cent, phenol. 
Bacteriologic Diagnosis.-The organism may be recognized in 
stained mounts from the pus from a bubo as a small Gram-negative 
bacillus, with characteristic bipolar staining. It may also be isola- 
ted upon culture-media and identified by its growth characteristics. 
Transmission.-The pneumonic form of the disease may be 
transmitted by the inhalation of infectious droplets. Plague is 
not known to occur in the human following ingestion of the organ- 
ism. The bubonic or most common type is probably transmitted 
to man by the bite of fleas (or from their excretions scratched into 
the skin), which have left rats dead of the disease. An epidemic 
of plague in the human is commonly preceded by an epizootic 
among the rats of the community. It has been shown experiment- 
ally that a flea may transmit the disease from an infected to a non- 
infected individual. It is also known that the cannibalistic tend- 
ency of rats to eat their dead is in part responsible for the spread of 
the disease among vermin. The annihilation of the rat is the best 
prophylaxis known. The disease has in certain places, as about 
San Francisco, been found to spread to such rodents as the ground- 
squirrels and wood-rats. When a region once becomes thoroughly 
infected it is, therefore, difficult to stamp out the disease. CHAPTER XXVI 
GLANDERS GROUP. THE GENUS PFEIFFERELLA 
One organism only, Pfeifferella mallei, the cause of glanders 
and farcy in equines, is known to belong to this group. It should 
be noted that the so-called pseudoglanders and the causal organ- 
isms are treated under other chapter headings. These latter 
organisms are not related to the organism in question except in 
that they produce lesions which are sometimes confused with 
glanders clinically. Some of the pseudoglanders organisms be- 
long to such disease groups as the bacteria, the blastomycetes, 
and the hyphomycetes. 
Pfeifferella mallei 
Synonyms.-Bacterium mallei; Mycobacterium mallei; Bacillus 
mallei. 
Diseases Produced.-Glanders and farcy in equines; Rotz; 
morve. 
Loftier and Schutz, in 1882, demonstrated the presence of a 
characteristic rod (P/. mallei') in the nasal discharge of 'a horse 
affected with glanders. Kitt, in 1883, and Weichselbaum, in 
1885, confirmed these results and added to our knowledge of the 
organism. 
Distribution.-Glanders is known in practically every civil- 
ized country. 
Morphology and Staining.-The glanders bacillus is a short rod, 
usually straight, but sometimes somewhat curved. The ends are 
rounded. It is usually single, more rarely in pairs or short chains 
in artificial media. In tissues the organism is generally in pairs. 
The cells are usually thicker and shorter in broth than on solid 
media. In stained mounts from the caseated pus from the 
lymph-gland of a guinea-pig, or from a glanders nodule from the 
liver of a field mouse, the cells are thicker and show a decided 
tendency to polar staining. Involution forms are frequently pro- 
duced; enlarged cells, clubbed forms, filaments, and even branch- 
ing have been observed. This last fact has led to the grouping of 
306 GLANDERS GROUP. THE GENUS PFEIFFERELLA 
307 
this form with the higher fungi by some authors. The normal 
rods vary from 0.5 to 1.0 by 1..' to 5 n. The organism is non- 
motile, and does not produce spores or capsules. It stains with 
the ordinary anilin dyes, and still better with stains containing a 
mordant. It sometimes shows some granular differentiation of 
the cytoplasm resembling the diphtheria bacillus. It is not acid- 
fast and is Gram-negative. 
Isolation and Culture.-The glanders bacillus is rarely in pure 
cultures in the nasal discharges, so that for its isolation from such 
sources a special technic is 
necessary. It is customary 
to inject intraperitoneally 
a male guinea-pig with a 
small quantity of the dis- 
charge from an ulcer, mixed 
with a little bouillon or 
physiologic salt solution. 
Within two to four days 
the testes swell and give 
evidence of acute inflam- 
mation. The animal is 
then killed, a testis re- 
moved and opened under 
aseptic conditions, and the 
contents of one of the small 
abscesses or foci of inflammation removed on a sterile platinum 
needle to suitable media. 
The glanders bacillus grows upon the ordinary culture-media, 
particularly upon those that contain glycerin, upon blood-serum, 
and potato. The colonies upon agar and glycerin-agar plates are 
whitish or yellowish, glistening, usually circular. Upon the 
slanted medium the colonies are coalescent and form a moist, 
shining layer of a slimy consistency. In bouillon and glycerin 
bouillon it produces an initial turbidity, followed by sedimenta- 
tion; a shining white pellicle is likewise formed when the medium 
is not shaken. On blood-serum the colonies are first discrete, 
clear, yellowish, viscous, hemispheric drops which coalesce to 
form a transparent layer over the surface; this later becomes 
Fig. 117.-Pfeifferella mallei from glycerin 
agar (X 1000) (Frankel and Pfeiffer). 308 
VETERINARY BACTERIOLOGY 
gray and opaque. Gelatin is not liquefied. The growth upon 
potato is perhaps the most characteristic. It may be described as 
forming within forty-eight hours a yellow, honey-like, semitrans- 
parent growth that gradually becomes brownish or amber in tint. 
The potato itself is tinted greenish or greenish brown. This 
reaction is not characteristic if potatoes having too acid a reaction 
are used. They may be neutralized previously to inoculation by 
soaking in dilute sodium carbonate. 
Physiology.-The glanders bacillus is aerobic and facultative 
anaerobic. Its optimum growth temperature is 30° to 40°, but 
its growth limits, at least in 
freshly isolated cultures, are 
about 25° and 42°. Its 
thermal death-point is 55°, 
with ten minutes' exposure. 
Pathogene sis. -Experi- 
mental Evidence.-There is 
an abundance of evidence 
to prove that Pf. mallei is 
the cause of glanders. All 
the lesions of the disease 
may be duplicated by the 
experimental inoculation of 
pure cultures into labora- 
tory animals and the horse. 
The guinea-pig is very sus- 
ceptible. A subcutaneous 
inoculation is followed within a few days by local swelling and in- 
duration, which soon ulcerates and discharges to the surface. The 
disease spreads largely through the lymph-channels, and the 
lymph-nodes enlarge and suppurate. Various metastatic infections 
of the joints, the lungs, liver, and other organs occur. Death seems 
to be due to exhaustion. Infection may similarly be transmitted 
to the rabbit. The horse may be readily infected, as may sheep, 
goats, the cat, and the dog. Cattle and the house-rat do not con- 
tract the disease. It occurs in man through infection from gland- 
ered animals and through working with pure cultures in the 
laboratory. 
Fig. 118.-Pfeiff er ella mallei, in section 
from the spleen of a field-mouse (Frankel 
and Pfeiffer). GLANDERS GROUP. THE GENUS PFEIFFERELLA 
309 
Character of Disease and Lesions Produced.-The disease as 
found in equines may be either of an acute or a chronic type. 
The former is commoner in the ass and mule, and the latter in 
the horse. The acute type of disease is commonly ushered in with 
a chill, there is a mucopurulent discharge, and death usually occurs 
in from one to four weeks. The chronic type shows no marked 
characteristics in its early stages; the lymph-nodes in various 
parts of the body become infected and enlarge. This may exist 
for a long period in an animal, and may terminate finally in an 
acute attack. The lesions in the chronic type are generally 
present on the nasal mucosa, in the lungs, and in the lymph-glands. 
The nodular glanders of the nasal mucosa is the most frequent 
type. The nodules, small at first, enlarge to the size of a pea, 
then break down, suppurate, and form chronic ulcers. When 
healing of the deeper ulcers occurs, the star-shaped scar resulting is 
quite characteristic. In the lungs lesions are almost invariably to 
be found; these may be nodular, or consist of infiltration of con- 
siderable areas of tissue. In farcy or cutaneous glanders the nod- 
ules form in the skin; the lymph-vessels become swollen and feel 
like a string of beads or a knotted cord. These nodules occasion- 
ally break through to the surface and ulcerate. In man the organ- 
ism commonly gains entrance through abrasions or wounds in the 
skin, or by inhalation, and the infection produced is practically 
always fatal. 
Immunity.-No toxins have been demonstrated for Pf. 
mallei, although endotoxins are produced. Agglutinins are present 
in the blood-serum of normal animals, but in much greater con- 
centration in the blood of infected animals. Precipitins may also 
be demonstrated. Of the bactericidal and opsonic nature of sera 
less is known. 
Active Immunization.-Immunization by the use of suspensions 
of dead bacteria or their products (mallein) has been attempted 
both in prophylaxis and cure. Although some favorable results 
have been reached, the subject needs further study. No method 
of vaccination or active immunization has as yet been shown to be 
practical and successful. 
Passive Immunization.-The blood-serum of animals, such as 
the ox, naturally immune to glanders has been claimed to possess 310 
VETERINARY BACTERIOLOGY 
immunizing power when injected into smaller laboratory animals, 
as the rabbit. Nocard and Prettner have each attempted im- 
munization by the administration of serum from cattle that had 
been repeatedly injected with virulent cultures of the glanders 
bacillus, but found the method ineffective. Galtier observed 
that treatment with such serum only prolonged the course of the 
disease which had been induced by experimental inoculation. 
Bacteriologic Diagnosis.-A presumptive bacteriologic diag- 
nosis may be made by an examination of properly stained pus or 
sections of tissue, and a more positive diagnosis by the methods of 
animal inoculation, agglutination, precipitation, absorption of 
complement, and by the use of mallein. 
Examination of Pus and Tissues.-The nasal secretions and 
the pus from ulcers always contain a mixed bacterial flora, and are 
consequently unsatisfactory for microscopic examination. The 
bacilli of glanders are not readily recognizable and are, therefore, 
very difficult to differentiate from other bacteria. The nodules of 
the disease and the subcutaneous tissues are exceptions to the 
above rule, as are the foci in the submaxillary glands. These may 
be freshly incised and satisfactorily stained. For demonstration 
of the organism in tissues the method of Kiihne is recommended 
as giving good results. Carbol-methylene-blue (methylene-blue, 
1.5 gm.; alcohol, 10 c.c., and 5 per cent, aqueous phenol or carbolic 
acid, 100 c.c.) is used to stain the sections one-half hour; they are 
then washed in water, then in very dilute hydrochloric acid (10 
drops to 500 c.c. of water), and quickly transferred to a solution of 
lithium carbonate (8 drops of a saturated solution to 10 c.c. of 
water), then to distilled water, dehydrated in absolute alcohol con- 
taining a little methylene-blue, then cleared in anilin oil. The 
bacteria should show plainly, but are few in number and not always 
readily discovered. 
Diagnosis by Animal Inoculation.-Strauss' Reaction.-.A male 
guinea-pig is inoculated intraperitoneally with a. small amount of 
the suspected material. This is preferably obtained from the 
interior of encapsulated nodules or abscesses, or from the base of 
fresh ulcers. The nasal discharge is less satisfactory because of the 
presence of large numbers of other varieties of bacteria, which may 
cause the death of the animal prematurely from peritonitis or sep- GLANDERS GROUP. THE GENUS PFEIFFERELLA 
311 
ticemia. In from two to four days, exceptionally not until the 
twelfth, the testes become enlarged and tender, the skin above 
them is reddened and shiny. The animal, in case of a positive reac- 
tion, should be killed and the contents of the testes examined mi- 
croscopically to determine the presence of a Gram-negative charac- 
teristic bacillus. If not killed, abscesses which discharge large 
numbers of bacteria develop, and the animal emaciates rapidly, 
dying within two weeks. Other organisms may give the orchitic 
reaction, but they are Gram-positive, with the exception of 
Pseudomonas pyocyanea. A culture should always be made from 
the pus in the scrotum to make diagnosis certain. 
Agglutination Test for Glanders.-The serum of a normal horse 
will frequently agglutinate the glanders bacillus when in dilutions 
of 1:100, 1:500, rarely more. The following general rule as men- 
tioned by Hutyra and Marek is quite applicable and reliable: 
Agglutination, if appearing in dilutions of 1 : 400 or less, with 
few exceptions denotes freedom from infection; in dilutions of 1 : 
1000 or more, also with few exceptions, agglutination indicates 
the presence of infection; in dilutions of 1 : 2000 agglutination 
signifies recent infection. The organisms used in the agglutination 
test may be either living or dead. The latter are commonly used, 
as it does away largely with danger of infection to man. The bac- 
terial suspension is prepared by removing the growth from a young 
culture on agar and suspending it in physiologic salt solution con- 
taining 0.5 per cent, phenol. This is heated at 70° for two to four 
hours; this kills the bacteria, but does not interfere with the ag- 
glutination reaction. Equal amounts of this suspension are placed 
in a series of small test-tubes, and to these are added equal amounts 
of different dilutions of the serum to be tested, and the final dilu- 
tions of the serum determined. Dilutions are usually prepared 
1 : 100, 1 : 200, 1 : 400, 1 : 500, 1 : 800, 1 : 1000, and up to 1 :4000 
or more. The tubes are kept at 37° for from twenty-four to thirty- 
six hours, or the reading of the result may be hastened by centrif- 
ugation. A positive reaction is indicated by a film covering the 
entire bottom of the tube, a negative by no precipitate or a little 
sediment in the bottom of the convexity, not forming a film. 
Whether or not a positive reaction is accompanied by a complete 
clearing of the test fluid depends upon the concentration of the 312 
VETERINARY BACTERIOLOGY 
suspension and the dilution and potency of the serum used. The 
fluid may remain somewhat cloudy in a positive reaction in the 
higher dilutions, not all the organisms being agglutinated. The 
suspensions of killed organisms may be secured ready for use 
from some pharmaceutical houses, together with tubes and mate- 
rials for preparing the proper dilutions. The suspension when 
properly prepared and preserved in the dark will keep for a con- 
siderable time. The microscopic test for agglutination has not 
proved practicable, as normal serum agglutinates microscopically 
in high dilutions. When properly carried out the macroscopic 
test is claimed by some to be an even better diagnostic than mallein. 
Konew's Precipitation Test, or the Ring Test.-A solution of 
glanders bacilli prepared by adding 10 c.c. of an 8 per cent, anti- 
formin1 solution to the bacilli washed from the surface of a forty- 
eight-hour slant agar culture. The bacteria will go into solution 
within two hours. It is well to add even more of the organism if 
it appears to dissolve rapidly, as it is desirable to get as concen- 
trated a solution as possible. The solution must then be care- 
fully neutralized, preferably by the use of 5 per cent, sulphuric 
acid. This is then filtered through paper, then through a Berke- 
feld filter, to remove all undissolved bacteria. The filtered solu- 
tion is termed "mallease." A test-tube is filled to a depth of 3 cm. 
with mallease, and blood-serum from a suspected case is introduced 
by means of a pipette. The end of the pipette should be passed 
through the layer of mallease and should rest against the bottom of 
the tube before the serum is allowed to flow. A quantity of serum 
about equal to the mallease is introduced. The pipette is with- 
drawn quickly and carefully to prevent any mixture of the two 
liquids. The serum has a higher specific gravity and remains at 
the bottom, with the mallease as a distinct superficial layer. If 
the serum is from an animal free from the disease, there will be no 
reaction. A positive diagnosis of glanders is indicated by a white 
cloudiness that appears along the line separating the two liquids. 
This is due apparently to precipitation by the specific precipitins 
formed in the serum. In acute or well-marked cases the reaction 
occurs almost immediately, and usually in all cases within fifteen 
1 The composition of antiformin is given on p. 380 It is a patented dis- 
infecting solution, and may be purchased upon the market. GLANDERS GROUP. THE GENUS PFEIFFERELLA 
313 
minutes. Observations recorded up to the present indicate that 
this method is giving satisfactory results in most instances. The 
possibility of demonstrating precipitins in chronic cases presents 
an additional advantage for this method. 
Fixation of Complement Test.-Schutz and Schubert1 have de- 
scribed a satisfactory method of adapting Wassermann's syphilis 
test by fixation of complement to the diagnosis of glanders. 
Mohler and Eichhorn2 have tested out the method and found it 
highly satisfactory. Hemolytic amboceptor is prepared, prefer- 
ably by a recent method described by A. F. Coca. Rabbits are 
given two intravenous injections of washed erythrocytes of the 
sheep of 1 c.c. or at most 2 c.c., at intervals of not less than four 
days. At the end of five days after the last injection the rabbits 
are bled and their blood-serum obtained. Such amboceptor is usu- 
ally highly potent and the potency remains uniform for a long time. 
It must be inactivated by heating to 56° for thirty minutes before 
it can be used. 
Fresh guinea-pig serum is used as complement. The antigen 
used is an extract of glanders bacilli prepared from the growth on 
slant glycerin-agar tubes. The growth is washed off with physio- 
logic salt solution and heated to 60° for four hours to kill the bac- 
teria. The suspension of organisms is then placed in flasks and 
shaken in a shaking apparatus for four days. It is then centrifuged, 
the clear liquid removed, and 10 per cent, of a 5 per cent, solution 
of phenol added. This antigen may be preserved without material 
deterioration for several months if kept in a cool, dark place. 
It is necessary to titrate the rabbit serum and likewise the 
antigen in order to determine the amounts most suitable for carry- 
ing out the test. For each set of determinations of diagnosis fresh 
guinea-pig serum must be used. Blood-serum from the animal 
that is suspected of having glanders must be inactivated by heat- 
ing to 58° for thirty minutes. The materials necessary for the 
test are- 
1. Washed sheep corpuscles, 5 per cent, suspension (antigen 1). 
2. Inactivated serum from rabbit immunized against 1 (am- 
boceptor 1). 
1 Arch. f. Wiss. u. prakt. Tierheilkunde, Band 35, pp. 44-83, 1909. 
2 Bull. 136, Bureau Animal Industry, U. S. Dept, of Agriculture. 314 
VETERINARY BACTERIOLOGY 
3. Fresh guinea-pig serum (complement). 
4. Extract of glanders bacilli (antigen 2). 
5. Inactivated serum from suspected animal (amboceptor 2). 
The test is carried out in test-tubes. In tubes 1 and 2 there is 
placed 0.1 c.c. of the serum (No. 5, above), and in tubes 3 and 4, 
0.2 c.c. of the same. One c.c. of the established dilution of glanders 
bacilli (No. 4, above) is then added to tubes 1 and 3. To each tube 
is then added 1 c.c. of the dilution of fresh guinea-pig serum that 
has been established by preliminary test. Each tube is now made 
up to 3 c.c. with physiologic salt solution. They are then placed 
in the thermostat at 37° for an hour. They are then removed and 
to each tube is added 1 c.c. of the previously standardized rabbit 
serum (No. 2, above) and 1 c.c. of the sheep corpuscles (No. 1, 
above). The tubes are shaken and incubated for ten hours. 
A positive diagnosis is indicated by lack of hemolysis in tubes 1 and 
3 and complete hemolysis in tubes 2 and 4. Checks must be made 
to determine the hemolytic activity of each of the above con- 
stituents independently. 
This method is essentially a laboratory one and quite imprac- 
ticable for field work. There seems to be no reason why blood 
samples or, better, serum samples from suspected cases should not 
be sent to properly equipped laboratories for diagnosis and report. 
The method apparently is capable of giving good results, and seems 
to be more accurate than the mallein test. 
Conglutination Test for Glanders.-The conglutination reaction 
has been advocated by Anderson and others as a satisfactory 
method of diagnosis in glanders. The following materials are 
necessary in the test: 
1. Suspension of killed glanders bacilli 
2. Inactivated serum of horse suspected, in various 
0.05 c.c. 
amounts, from 
0.001-0.1 c.c. 
3. Normal horse serum (containing complement) 
0.1 c.c. 
4. Inactivated cattle serum (containing conglutinin). . 
5. Suspension of goat corpuscles in physiologic salt 
0.04 c.c. 
solution 
0.5 c.c. 
Each volume is made up to 2.5 c.c. with physiologic salt salution. 
Numbers 1, 2, and 3 are first mixed and allowed to stand one-half 
hour at 37°, then numbers 4 and 5 are added. The reaction is GLANDERS GROUP. THE GENUS PFEIFFERELLA 
315 
determined after standing one hour at 37°. The test has been 
claimed to be quite specific, but it has not been extensively tried 
out. (See page 192.) 
Mallein Test for Glanders.-Mallein is a suspension of killed 
glanders bacilli, together with the products of their autolytic disin- 
tegration. What the active principles in bringing about the char- 
acteristic reaction in a glandered horse may be is not known. 
Probably they are the soluble bacterial proteins, possibly true 
endotoxins. The various laboratories use different methods of 
preparing mallein. The most important of these are worthy of 
note. 
The mallein of Roux is prepared by the Pasteur Institute as 
follows: The virulence of the glanders bacillus used is increased 
by passage through rabbits, and is such that mice and rabbits are 
killed in less than thirty hours by intravenous injections. Flasks 
containing 250 c.c. of glycerin bouillon are inoculated and incu- 
bated a month at 35°. The cultures are killed by exposure to a 
temperature of 100° for thirty minutes in an autoclave, then 
evaporated to one-tenth the volume, and filtered through filter- 
paper ("papier Chardin"). The final product is a dark-brown, 
syrupy liquid, containing 50 per cent, glycerin. For use this is 
mixed with nine times its volume of 0.5 per cent, carbolic acid. 
The diagnostic dose is 2.5 c.c. of this dilution. 
The mallein of Vladimiroff, used in the Russian Empire, is pre- 
pared by inoculating a considerable number of flasks, each con- 
taining 600 to 800 c.c. of beef-broth, with a vigorous culture of B. 
mallei, and incubating for eight months at 37°. The flasks are 
shaken from time to time to cause the shiny, gray-white pellicle 
which forms to sink to the bottom. The culture is then examined 
for purity, sterilized in the autoclave at 110°, and filtered. This 
is concentrated and again diluted until the diagnostic dose for the 
horse is 1 c.c. 
The mallein or morvin of Babes is prepared by inoculating 
potato paste with the glanders bacillus, and incubating six weeks. 
It then is heated at 68° for three and one-half hours, emulsified 
with water, filtered through a Witt filter, and precipitated with 
alcohol. This precipitate is washed in alcohol, then in ether, and 
dried. The diagnostic dose is 0.02 to 0.03 gm. It is prepared for 
injection by dissolving in a mixture of glycerin and water. 316 
VETERINARY BACTERIOLOGY 
The malleinum siccum, or dried mallein, of Foth is prepared 
by growing the glanders bacillus in 4.5 per cent, glycerin broth. 
The cultures used are rendered virulent by passage through cats, 
guinea-pigs, and field-mice. The material is incubated at 37.7° 
for three weeks. It is concentrated, and the organism killed by 
evaporation at a constant temperature of 76° to 80° to one-tenth 
of its former volume. This is filtered and poured into absolute 
alcohol, in which a precipitate immediately forms. This pre- 
cipitate is washed in alcohol and dried in a desiccator. The 
final product is a white powder which readily dissolves in water. 
The diagnostic dose for the horse is 0.045 to 0.05 gm. 
The mallein prepared in the laboratories of the Bureau of 
Animal Industry consists of glycerinated broth in which the Pf. 
mallei has grown four to five months, has been heated, concen- 
trated, and filtered. It is diluted by the addition of one-half 
its volume of glycerin and one and one-half times its volume of 
1 per cent, phenol. The diagnostic dose is 1 c.c. 
No practicable method of standardizing mallein has been 
worked out other than trial upon a considerable number of healthy 
and infected animals. The variations in the methods of produc- 
tion of mallein given above are due to a desire to secure a very 
uniform product. 
There are three methods of applying mallein in use at the 
present time. They are the subcutaneous, ophthalmic, and cu- 
taneous. 
Subcutaneous Mallein Test.-Following subcutaneous injection 
of a suitable dose of mallein, animals suffering from glanders show 
a rise of temperature beginning usually between the fourth and 
eighth hour. From this time on to the fourteenth hour, in excep- 
tional cases later, the rise continues. After reaching the maximum 
the decrease is gradual until normal. Organic symptoms, such as 
increased respiration and heart action, muscular tremor, depression, 
dulness, and loss of appetite, may frequently be observed and are 
of value in diagnosis. The appearance of an inflamed, edematous 
swelling at the point of inoculation is also usually noted. In gen- 
eral, an increase of 2° C. rising above 40° C. is considered a positive 
reaction. Students are referred to texts on practice for complete 
discussion of this phase of the test. GLANDERS GROUP. THE GENUS PFEIFFERELLA 
317 
Ophthalmic Mallein Test.-This consists of introducing into 
the conjunctival sac of one of the eyes several drops of a spe- 
cially prepared mallein. This may be introduced either with the 
aid of a camel's-hair brush or a medicine-dropper. The reaction 
begins from the fourth to the sixth hour after application and 
may continue for twenty-four to thirty-six hours. It consists 
of a conjunctivitis marked by swelling of the lids, redness of the 
membrane, and a purulent secretion which collects at the inner 
canthus and on the hair below the eye. The test is recognized 
by the Bureau of Animal Industry as a reliable one. 
Cutaneous Mallein Tests.-These may be carried out either by 
scarification of the skin, followed by application of concentrated 
mallein, or by intradermal injection of concentrated mallein. 
Either case is marked by edematous swellings whose extent depends 
upon the quantity of mallein injected. The limited number of 
tests of this character furnish a very unsatisfactory basis for con- 
clusions regarding the reliability and accuracy. 
Transmission.-The disease is transmitted from one animal to 
another through infected food, mangers, drinking troughs, etc.; 
rarely through wounds or skin abrasions. Veterinarians and horse- 
men sometimes become infected through the skin, rarely by inhala- 
tion. 
Bacilli of Selter, Babes, and Kutscher.-Organisms morpho- 
logically similar to the preceding have been isolated from pus by 
Selter and by Babes, and from the nostrils of a healthy horse by 
Kutscher. They may be differentiated readily by their lack of 
pathogenesis, and would rarely, if ever, lead to mistakes in diag- 
nosis. CHAPTER XXVII 
INTESTINAL OR COLON-TYPHOID GROUP. THE GENUS 
BACTERIUM 
The organisms belonging to this group may be characterized 
as plump, Gram-negative rods, frequently though not always 
motile; they produce no spores, do not in general liquefy gelatin,, 
and in most cases ferment certain sugars, with acid and sometimes 
gas production. There is no distinct tendency to the formation 
of polar granules. The group contains many undoubted species 
that may be easily differentiated, but there are many intergrading 
types and forms showing similar morphologic and cultural charac- 
ters, but differing considerably in kind and in degree of virulence. 
These latter make a systematic presentation of the group as a whole 
difficult. The group name is given because of the prominence of 
these organisms in the intestinal flora in disease and health in both 
man and animals. They are, therefore, abundant in sewage and 
water contaminated thereby, and in soil, particularly that which 
has received additions of barnyard manure. They are less common 
in virgin soil and uncontaminated water. 
The members of this group are divided, for convenience in 
study, into three subgroups. This arrangement seems to represent 
evident relationships. The fermentative powers of the organisms 
are used as a basis upon which to make the groupings. The first 
of these is known as the colon bacillus subgroup, the second as the 
intermediate, hog-cholera or enteritidis subgroup, and the third 
as the typhoid-dysentery subgroup. The principal points of differ- 
ence among these subgroups may be summarized in the following 
table, giving the fermentation reactions in dextrose and lactose 
broth: 
Subgroup I. 
Subgroup II. 
Subgroup III. 
Colon subgroup. 
Intermediate sub- 
Typhoid-dysentery sub- 
group. 
group. 
Acid. 
Gas. 
Acid. Gas. 
Acid. Gas. 
Dextrose + 
+ 
Dextrose + + 
Dextrose ± - 
Lactose + 
+ 
Lactose - - 
Lactose - - 
318 INTESTINAL OR COLON-TYPHOID GROUP 
319 
The differences may be summarized as follows: The organisms 
of Subgroup I ferment both dextrose and lactose, with formation 
of both acid and gas; those of Subgroup II form acid and gas 
from dextrose, but not from lactose; and those of Subgroup III 
may or may not form acid from dextrose, but never from lactose, 
and gas from neither of the sugars. 
The fermentations of other carbohydrates and related com- 
pounds,, are used to differentiate species and varieties from each 
other. A few can be satisfactorily differentiated only by the ag- 
glutination reaction. Those organisms which produce gas in the 
fermentation tube grow in both the open and closed arm, as do those 
which produce acid, and those which do not ferment the sugar 
are usually confined to the open arm. The composition of the 
gas, that is, the relative proportion of CO2 and H2, is also of diag- 
nostic value. 
Subgroup L Colon Subgroup 
The organisms of the colon subgroup are characterized by their 
ability to ferment both dextrose and lactose with formation of 
both acid and gas. A satisfactory classification of the species has 
not been worked out as yet. Even the approximate number of 
species to be recognized is uncertain. Many characters to be 
noted in separation of species have been proposed; those which 
have proved helpful are the following: 
1. Motility and capsule production. 
2. Indol production. 
3. Carbon dioxid-hydrogen gas ratio. 
4. Acid production, particularly hydrogen ion concentration in 
various sugars. 
5. Gas production from different sugars. 
6. Production of acetyl-methyl-carbinol (Voges-Proskauer reac- 
tion). 
7. Utilization of uric acid as a source of nitrogen. 
The organisms of this subgroup are of interest because 
several are normal inhabitants of the intestinal tract, and their 
presence in water is, therefore, an evidence of fecal contamination 
and because some of the species are pathogenic. It is as yet im- 
possible to prepare a logical classification which includes all forms. 
The subgroup, for convenience, will be considered under three 
sections: 320 
VETERINARY BACTERIOLOGY 
1. Organisms primarily of sanitary significance. 
2. Organisms associated with infectious diseases of animals, 
particularly calf diarrhea. 
3. Organisms showing capsule formation, and sometimes patho- 
genic. 
These sections overlap; the same species in some cases must 
be considered under all three sections. 
The name Bacterium coli or Bacillus coli communis is often 
used to include all of the species here discussed, very often, in- 
deed, to include all forms of sanitary significance. 
Organisms of the Colon Subgroup Primarily of Sanitary 
Significance 
The organisms of this section are frequently used as an index 
of water pollution. They are all inhabitants of the intestinal 
tracts of man and most animals; certain species are not uncom- 
mon in soil, on grains, etc. 
The following key to some of the more important species is of 
assistance in their separation. It should be emphasized that the 
names Bacterium coli and B. coli communis are often used to in- 
clude all of these species. 
Key to the More Important Species of the Colon Group 
I. Not producing acetyl methyl carbinol (Voges-Proskauer reaction nega- 
tive); acid to methyl red, and carbon dioxide and hydrogen produced 
in approximately equal volumes from glucose. Cannot utilize uric 
acid as a source of nitrogen. 
A. Sucrose not attacked. 
1. Salicin fermented with acid and 
gas. 
Coli Section. 
1. Bad. coli. 
2. Salicin not attacked. 2. Bad. acidi-ladici. 
B. Sucrose fermented with acid and gas. 
1. Motile. 3. Bad. communior. 
2. Non-motile. 
(a) Salicin fermented*with acid 
and gas. 4. Bad. neapolitanum. 
(b) Salicin not fermented. 5. Bad. coscoroba 
II. Producing acetyl methyl carbinol (Voges-Proskauer reaction positive); 
alkaline to methyl red, and forms two or more times as much carbon 
dioxide as hydrogen from glucose. Capable of utilizing uric acid as 
a source of nitrogen. INTESTINAL OR COLON-TYPHOID GROUP 
321 
Aerogenes Section. 
A. Glycerol and starch fermented with 
acid and gas formation; non-motile, 
gelatin not liquefied. 6. Bact. Aerogenes. 
B. Glycerol and starch not fermented; 
motile, gelatin liquefied. 7. Bact. cloacae. 
Bacterium coli 
Synonyms.-Bacillus coli communis; B. pyogenes foetidus; 
Bacterium coli commune; colon bacillus. 
Emmerich, in 1885, isolated an organism which was later 
named Bacillus neapolitanus, from the feces of patients suffering 
from Asiatic cholera. Escherich, in 1886, isolated a related 
organism which he termed Bacterium coli commune, from normal 
feces. Since that time organisms of this group have been found 
to be constantly present in the < - 
intestines of man, most animals, 
and even some birds. The 
question of its occurrence in 
nature independent of fecal 
contamination is a moot one, 
but there is increasing ten- 
dency to consider that reports 
of its common occurrence in 
water and soils are due to 
confusion with Bacterium aero- 
genes. That it may maintain a 
saprophytic existence outside 
the body for some time seems 
to be well established, but the 
evidence that it does not usually long so maintain itself is 
increasing. 
Distribution.-Not all investigators are in agreement as to the 
proportion of lactose fermenting bacteria from the intestines 
which belong to the species Bacterium coli. MacConkey con- 
cluded about 38 per cent, from human feces and 25 per cent, 
from bovine feces were of this type. Clemesha gives 17 and 
9 per cent, respectively. Levine found 5 per cent, of Bad. coli 
in horse feces, 22 per cent, from the pig, 25 per cent, from the 
Fig. 119.-Bacterium coli, stained 
preparation from a twenty-four-hour 
agar slant (X 650) (Heim). 322 
VETERINARY BACTERIOLOGY 
cow, 5 per cent, from sheep, and 15 per cent, from man. The 
work of Ferriera, Horta, and Paredes indicated that this type 
is widely distributed in the feces of animals. 
Morphology and Staining.-The Bacterium coli is a rod, vary- 
ing from 0.4 to 0.7 by 2 to 4 m, sometimes shorter and almost coc- 
cus-like with rounded ends, usually single, but occasionally in 
short chains. It does not produce spores or capsules. It is 
rather sluggishly motile, at least in young cultures, usually with 2 
to 8 flagella, rarely more. It stains readily with the ordinary 
anilin dyes, sometimes showing some vacuolization and polar 
granules. It is Gram-negative. 
Isolation and Culture.-Bacterium coli may be readily isolated 
from feces or sewage by plating the material in various dilutions 
in litmus-lactose agar and incubating at blood-heat. The 
colonies of Bad. coli appear surrounded by a zone of red, due to 
the formation of acids from the lactose. The colonies must be 
fished and tested in various carbohydrate solutions, so that those 
of true Bad. coli may be differentiated from other members of the 
subgroup. Upon gelatin plates the colonies are moist, grayish 
white, opaque, becoming darker and more coarsely granular. 
Gelatin is not liquefied. Stab cultures in gelatin show a filiform 
growth along the line of puncture and a spreading growth at the 
surface. The agar cultures resemble those on gelatin. Bouillon 
is quickly clouded, sometimes with formation of a pellicle. On 
potato a moist, spreading growth occurs, and the potato is dark- 
ened. Milk is coagulated by the formation of acids; the curd 
shrinks, but is not digested. The various media used in the 
recognition of Bad. coli in water analysis will be discussed under 
that heading. 
Physiology.-Bacterium coli is aerobic and facultative anaero- 
bic. Its optimum growth temperature is 37°, but growth is 
luxuriant at room-temperature and even below. The thermal 
death-point is 60° for fifteen minutes. The following carbohy- 
drates are fermented: levulose, galactose, dextrose, maltose, 
lactose, salicin, glycerin, dulcite, but not saccharose. The gas 
formula from dextrose is approximately 
co2 
1 
. When 
grown in dextrose (0.5 per cent.) phosphate medium the solution 
h2 
1 INTESTINAL OR COLON-TYPHOID GROUP 
323 
becomes permanently acid to methyl-red. Indol is produced in 
Dunham's solution. Peptonizing and proteolytic enzymes have 
not been demonstrated. The Voges-Proskauer test is negative. 
It cannot utilize nitrogen from uric acid. 
Pathogenesis.-The following quotation from Jordan epito- 
mized our present estimate of the pathogenicity of Bacterium 
coli: 11 The common occurrence of agonal or postmortem invasion 
of the body by the colon bacillus tends to diminish the value of 
the supposed evidence 
derived from finding the 
colon bacillus in the in- 
ternal organs after death, 
and there can be no 
doubt that the role in 
human pathology as- 
signed to the colon bac- 
illus by some investiga- 
tors, notably certain 
French bacteriologists, 
has been greatly exag- 
gerated. Failure to dis- 
tinguish between the 
true colon group and the 
group of meat-poisoning 
bacilli is doubtless re- 
sponsible for some of the statements attributing pronounced 
pathogenic properties to Bad. coli. The frequent ascription 
of various inflammatory processes, particularly those occurring 
in the appendix and peritoneum, to the unaided activities 
of Bad. coli appears to be without sufficient justification. 
Many of the cases reported rest on the evidence derived from 
simple aerobic cultivation, and the possible concurrence of 
anaerobic or other organisms not growing by ordinary methods 
has not been excluded." The preceding was written with 
pathogenesis for the human body in mind, but the conclusions 
are even more true with reference to its pathogenesis for ani- 
mals. Many diseases in domestic animals have been ascribed 
to infection with varieties of Bad. coli from insufficient evi- 
Fig. 120.-Bacterium coli showing the flagella 
(Migula). 324 
VETERINARY BACTERIOLOGY 
dence. It has been shown that even in the normal body colon 
bacilli sometimes escape from the intestines, and are to be found 
in the mesenteric lymph-nodes, and occasionally in some of the 
other internal organs. 
Experimental Evidence of Pathogenesis.-The intraperitoneal 
injection of broth cultures of Bact. coli into the guinea-pig results 
in the death of the animal, usually within three days. Animal 
experimentation has demonstrated quite conclusively that there 
are considerable differences in virulence of colon bacilli isolated 
from different animals or from the same animal at different times. 
Character of Lesions and Disease Produced.-Bacterium coli has 
been isolated from suppurations in pure culture. In man it is 
known occasionally to invade the gall-bladder, and is a common 
cause of cholecystitis. It may serve as a nucleus for gall-stones, 
and is probably instrumental in their formation by the precipita- 
tion of cholesterin. Inflammation of the ureters and of the urin- 
ary bladder is commonly caused in many by organisms that can- 
not be differentiated from typical Bact. coli. It has been 
reported as the cause of calf diarrhea or white scours, and from 
malignant catarrh in cattle. These will be discussed later. 
Usually the colon bacillus does not give rise to putrefactive prod- 
ucts, and must be regarded as a harmless or possibly even useful 
commensal. 
Immunity.-A considerable degree of immunity to Bacterium 
coli may be induced by injections of cultures, killed or living. 
Agglutinins are present in normal serum, but may be greatly 
increased by systematic immunization. Specific precipitins for 
the bacterial proteins are present in the immune serum. Opso- 
nins are present in normal serum. The body has naturally a 
high degree of immunity against the Bact. coli. This may be 
accounted for by the presence of Bact. coli in the intestines and 
the continued opportunity for infection. The toxic properties of 
the organism are probably due to the presence of endotoxins. 
Bacteriologic Diagnosis.-The isolation of the characteristic 
colonies upon litmus-lactose agar from Endo- or from Conradi- 
Drigalski plates are all simple and quick methods of determining 
the presence of Bact. coli. The recognition and isolation of this 
organism from water will be discussed at greater length under the 
heading of Water Analysis. INTESTINAL OR COLON-TYPHOID GROUP 
325 
Bacterium acidi lactici 
This organism was first isolated from milk by Hueppe. It has 
been frequently confused because of similarity of name, original 
source of isolation, and the production of lactic acid with 
Lactobacillus ladis acidi, a totally unrelated form. In all essen- 
tial characteristics this organism resembles the Bad. coli except 
that it does not ferment dulcite with the production of gas. 
Organisms of this type are not uncommon in feces. Mac- 
Conkey found 34 per cent, in human feces and 16 per cent, in bo- 
vine, Clemesha found 53 and 40 per cent, respectively. Levine 
found 15 per cent, in horse dung, 45 per cent, from pig, 25 per 
cent, from cow, and 5 per cent, from sheep. 
It is possible that some of Jensen's calf-scours organisms be- 
long to this type. 
Bacterium communior 
Synonyms.-B. coli communior; Bacillus communior. 
This organism was named Bacillus coli communior by Durham 
because he believed it to be even more common in the intestines 
than his B. coli communis. It differs from Bad. coli in its ability 
to ferment saccharose. MacConkey found about 15 per cent, of 
this type in human and 48 per cent, in bovine feces. Clemesha 
gives 7 and 10 per cent, respectively. Levine found about 60 
per cent, in the horse, 20 per cent, in the pig, 25 per cent, in the 
cow, and 45 per cent, in sheep. Its occurrence in water has the 
same significance as Bad. coli. It is probable that the most com- 
mon of the calf-scours bacterial types of Jensen belong to this 
species. 
Bacterium coscoroba 
This organism has been several times reported. It resembles 
Bacterium acidi lactici, but ferments saccharose. 
Bacterium aerogenes 
Synonyms.-Bacterium lactis aerogenes; Bacillus pyogenes; 
Bacillus lactis aerogenes. The capsulated B. pneumoniae of 
Friedlander, B. rhinoscleromatis, and B. ozoence quite probably 
belong here or are closely related. 326 
VETERINARY BACTERIOLOGY 
Escherich, in 1885, described this organism which he isolated 
from sour milk. The typical Bad. aerogenes seems to be quite 
widely distributed in nature; it has been found commonly present 
on grains by several investigators, and has been found to be the 
predominant organism of this group in soils by Johnson and 
Levine. It is not so typically fecal in habitat as many other 
members of the group. 
Morphology and Staining.-Bacterium aerogenes differs mor- 
phologically from the Bad. coli principally by the lack of 
flagella and in the ability to produce capsules when grown 
in milk. 
Isolation and Culture.-This organism may be isolated in the 
same manner as Baderium coli upon litmus-lactose agar. The 
colonies upon agar and gelatin are larger, thicker, and more 
slimy than those of the colon bacillus. Milk is curdled more 
rapidly. In gelatin stabs the growth along the streaks is filiform; 
that at the surface is thick, convex, and circumscribed. The 
whole stab culture is frequently described as "nail-like." 
Physiology.-In most respects this organism resembles Bac- 
terium coli. It ferments dextrose, lactose, and saccharose and 
glycerol with production of both acid and gas. Many strains also 
ferment starch. Indol is not commonly produced in Dun- 
ham's solution. Dextrose (0.5 per cent.) phosphate medium 
becomes alkaline to methyl-red. The Voges-Proskauer reaction 
is positive, showing the development of acetyl-methyl-carbinol. 
This reaction is one of the most distinctive in the differenti- 
ation of this species from other coli-like organisms. Gelatin is 
not liquefied. Grows in media containing uric acid as sole 
source of nitrogen. 
Pathogenesis.-This organism is not known to possess patho- 
genic powers. It is of interest principally because of its .close 
relationship to Bacterium coli and association with it. In making 
water examinations in the past no distinction has ordinarily been 
made between Bad. coli and Bad. aerogenes, inasmuch as they re- 
semble each other so closely and it has been assumed that they 
come from the same sources. It seems probable as the result of 
more recent work that the presence of this organism has much 
less sanitary significance than the preceding species. INTESTINAL OR COLON-TYPHOID GROUP 
327 
Bacterium cloacae 
Synonym.-Bacillus cloaca. 
This organism was isolated from polluted water by Jordan, 
and has been repeatedly found by subsequent workers. It differs 
from Bad. aerogenes in the power to liquefy gelatin, is motile and 
glycerol is not fermented. It is not common in feces. Its sani- 
tary significance is uncertain. 
Organisms Associated with Calf Scours or Calf Diarrhea 
Organisms belonging to the first subgroup of the intestinal 
group are generally regarded as the causal organisms of calf scours 
or calf diarrhea. The principal work has been that of Jensen in 
Denmark. 
Morphology and Staining.-These appear to be typical of the 
colon subgroup. 
Culture and Physiology.-Jensen has noted considerable varia- 
tion in the colony types of different races of the calf-scours organ- 
isms. The most marked differences, however, were found in sugar 
fermentations. He recognized two main groups (termed A and B) 
with three races in the first and four in the second. All the races 
agree in producing both acid and gas in the following sugars: 
Glucose, arabinose, xylose, rhamnose, maltose, and lactose. The 
races are differentiated on the basis of variations in sugar reactions 
in sucrose, sorbose, dulcitol, and adonite. The following key 
shows the groupings: 
A. Producing acid and gas from sucrose. Adonite negative. 
Group A. 
1. Producing acid and gas from sorbose. 
(a) Producing acid and gas from dulcite Race A. I 
(b) Not producing acid and gas from dulcite Race A. II 
2. Not producing acid and gas from sorbose Race A. Ill 
B. Not producing acid and gas from sucrose. Adonite negative or positive. 
Group B. 
1. Producing acid and gas from sorbose. 
(a) Producing acid and gas from dulcite Race B. I 
(b) Not producing acid and gas from dulcite Race B. II 
2. Not producing acid and gas from sorbose. 
(a) Producing acid and gas from adonite. Dulcite negative. Race B. Ill 
(b) Not producing acid and gas from adonite. 
(1) Dulcite positive Race B. IV 
(2) Dulcite negative Race B. V 328 
VETERINARY BACTERIOLOGY 
The one found most commonly (A. I) seems to be closely re- 
lated in fermentation reactions to Bacterium communior. Much 
work still remains to be done on the relationships of these organ- 
isms. It is possible that some of these types may constitute dis- 
tinct species. Jensen also records cases in which apparently 
typical Bad. aerogenes was the causal organism. Jensen has 
noted that it is usually the same race that causes trouble year 
after year in different animals on the same farm. 
Pathogenesis.-The characteristic symptoms do not seem to 
differ as a result of infection with different races of coli. 
The disease usually appears soon after birth, frequently within 
forty-eight hours. The calf shows fever, loss of appetite, and 
colic. The feces are liquid, usually yellowish, foul, and often 
mixed with gas. They may also show blood resulting from intes- 
tinal hemorrhage. Death may result promptly or there may be 
a more or less persistent diarrhea. Autopsy shows the mucous 
membranes of the abomasum to be congested or hemorrhagic, as 
also the intestinal mucosa. The mesenteric glands are often swol- 
len and red. The spleen is usually more or less swollen. 
Immunity.-Effective means of prophylaxis and cure by means 
of sera is complicated by the fact that so many races of colon bacilli 
may be responsible. Antisera may be prepared either against 
single strains or the sera may be polyvalent. 
The polyvalent serum is prepared by repeated intravenous 
injection of the horse with numerous races of colon bacilli isolated 
from cases of calf diarrhea. By combining the serum from differ- 
ent horses Jensen has prepared a polyvalent serum against as many 
as forty strains. 
No accurate method of determining the potency of the serum 
has been evolved. 
Bacterins, both univalent and polyvalent, have been prepared 
and used for prevention of the disease in districts or on farms where 
the annual losses are high. 
Capsulated Pathogenic Organisms 
Several species of capsulated bacteria belonging to this group 
have been described. The most important of these are Bacterium 
pneumoniae, Bad. mucosum capsulatum, Bad. rhinoscleromatis, and 
Bad. ozcence. One only, the Bad. pneumoniae, will be discussed. INTESTINAL OR COLON-TYPHOID GROUP 
329 
Bacterium pneumoniae 
Synonyms.-Bacillus pneumonia; Bacillus capsulatus muco- 
sus; pneumobacillus; pneumococcus of Friedlander. 
Friedlander, in 1883, discovered this organism in the sputum 
from a case of croupous pneumonia, and it was believed by him 
to be the cause of the disease. He failed to discover the real 
cause of pneumonia because the pneumococcus of Frankel does 
not grow readily upon plate cultures prepared by the method 
used. It has since been found repeatedly in normal saliva, and 
is still believed to be an occasional cause of pneumonia. It 
sometimes is found in the feces and in sewage. 
Morphology and Staining.-The organism as it occurs in the 
sputum is sometimes so short as to resemble a coccus. Usually it 
is single, rarely in chains. It is surrounded by a capsule in 
sputum and in milk. It is non-motile. It resembles the pre- 
ceding organisms closely in all other respects. 
Isolation and Culture.-Isolation is accomplished by plat- 
ing upon gelatin. Growth upon most media resembles that 
of the Bacterium aerogenes. 
Milk is not coagulated, 
although litmus milk is 
reddened. 
Physiology.-The organ- 
ism shows markedly less 
fermentative power than Bac- 
terium aerogenes, but other- 
wise closely resembles it. 
Dextrose, lactose, and saccha- 
rose are all fermented, but 
usually not vigorously. 
Growth occurs best at blood- 
heat, but the organism de- 
velops well at room-temperature. The thermal death-point is 
about 56°. Indol is produced. 
Pathogenesis.-Bacterium pneumonia has a very low virulence 
-only exceptionally will it infect any of the lower animals. It 
has been isolated in pure culture from the vegetations upon the 
heart valve in endocarditis, from otitis media, and occasionally it 
Fig. 121.-Bacterium pneumoniae, show- 
ing capsules (Gunther). 330 
VETERINARY BACTERIOLOGY 
is believed to cause catarrhal or lobular pneumonia. It is noted 
here simply because of its obvious relationship to the preceding 
organisms of the group. 
Subgroup IL Intermediate, Hog-cholera, Enteritidis, or 
Gartner Subgroup 
The classification and relationships of the organisms belonging 
to this subgroup are much confused at present. Whether or not 
the various forms described are all distinct species is doubtful. 
The most important will be discussed under the names by which 
they are commonly known, but this uncertainty as to correct 
grouping must constantly be borne in mind. 
The different races or species belonging to this subgroup have 
been differentiated in many ways, but no classification has been 
worked out that is entirely satisfactory. A grouping proposed by 
Harding and Ostenberg has been found useful by some workers. 
It is based upon the production of red in fuchsin sulphate agar 
when various carbohydrates are present. 
Jordan differentiates the more important types as follows; 
I. Xylose not fermented. Bact. paratyphosum (Paratyphoid A). 
II. Xylose fermented with acid and 
gas. 
A. Arabinose and dulcitol rapidly 
fermented. Bact. enteritidis and Bact. schotmulleri 
(Paratyphoid B). 
B. Arabinose and dulcitol not 
attacked or fermented very 
slowly. Bact. choleras suis. 
Bacterium enteritidis 
Synonym.-Bacillus of Gartner. Bacillus enteritidis. 
Diseases Produced.-Meat-poisoning and enteritis in man 
and in cattle. 
Gartner, in 1888, studied an outbreak of meat-poisoning in a 
village in Saxony, and isolated from a fatal case and from the un- 
cooked flesh of a cow the organism now known as Bacterium 
enteritidis. This organism since that time has been found in 
similar outbreaks of meat-poisoning and associated with certain 
infections in cattle. The organism has been found in meat, or so- 
called " ptomain''-poisoning in the United States. INTESTINAL OR COLON-TYPHOID GROUP 
331 
Schmitz found as a result of routine investigations carried on 
four years on flesh of slaughtered animals that this organism had 
never been found in mature beef or pork, but that it had been 
found many times in veal. He regards the organism as having an 
etiologic relationship to calf diarrhea. Several investigators 
have isolated organisms of this type from rats. 
Morphology and Staining.-Bacterium enteritidis resembles B. 
coll morphologically. The organism is short and thick, some- 
times with a thin capsule, mo- 
tile by means of numerous or 
few flagella. It does not pro- 
duce spores. It stains well or 
irregularly with the anilin dyes 
and is Gram-negative. 
Isolation and Culture.-The 
organism has been isolated di- 
rectly from the blood-stream 
and the spleen, and from the 
intestinal contents by plate 
cultures. Malachite green 
dulcite broth has been used suc- 
cessfully by several investiga- 
tors as an enrichment medium, as this seems to discourage the 
growth of colon bacilli. The cultural characters as reported 
vary with different authors, probably because different strains 
were studied. Colonies upon gelatin and agar resemble those 
of Bacterium coli. Bouillon is clouded, a delicate pellicle may 
form, and in a few days a whitish sediment collects. A yel- 
lowish, glistening layer forms on potato, frequently turning 
brownish with age. Growth in milk seems to vary with the 
organism studied. Some have been described as coagulating 
milk, but the typical form does not. 
Physiology.-Bacterium enteritidis is aerobic and facultative 
anaerobic. Its optimum growth temperature is between 30° and 
40°, but it grows well at room-temperature also. The thermal 
death-point, as determined by Mohler and Buckley, is 58° for 
twelve minutes. Dextrose and dulcite are fermented with produc- 
tion of acid and gas. Lactose, saccharose, salicin, and glycerin 
Fig. 122.-Bacterium enteritidis (Kolle 
and Wassermann). 332 
VETERINARY BACTERIOLOGY 
are not fermented by the typical strains, although some strains 
have been reported by a few investigators to ferment lactose. 
Indol is not produced. Gelatin is not liquefied. Milk may show 
initial acidity, but this is followed by a permanent alkalinity. 
Pathogenesis.-Experimental Evidence.-Bacterium enteritidis 
is pathogenic for the guinea-pig, mouse, and pigeon, but not for 
the cat. The guinea-pig may be fatally infected by intraperi- 
toneal of subcutaneous injections and by ingestion. The same is 
true of the rabbit. Mohler and Buckley produced a fatal infec- 
tion in young house-rats, while other authors report the rat as 
immune. The same is true of the dog, though this animal is 
relatively resistant. Chickens are immune. Sheep are readily 
infected. The hog succumbs to intravenous injection, as well as 
through feeding. 
Type of Disease and Lesions Produced.-In man the infection 
is marked by a severe enteritis and enlargement of the lymph- 
follicles. and Peyer's patches, and by small hemorrhages. The 
mortality in infections is low-probably less than 5 per cent. 
The infection in cattle, as observed by Mohler and Buckley, 
was characterized by degeneration of the heart muscle (frequently 
fatty) and hemorrhages therein; in the liver parenchymatous 
degeneration was accompanied by localized hemorrhagic extrav- 
asations; the spleen was enlarged and hemorrhagic and the 
lesions of acute enteritis, with necrosis of the epithelium, were 
evident. 
Immunity.-The so-called 11 toxin" of the Bacterium enteritidis 
is probably an unusually soluble and potent endotoxin. It differs 
from true toxins in being exceptionally heat resistant. Meat 
which has been quite thoroughly cooked is sometimes found capa- 
ble of giving Tise to toxic symptoms when ingested. The bac- 
teria-free filtrates from bouillon cultures and cultures in which 
the organisms have been killed by heat will kill guinea-pigs when 
injected in suitable quantities. It is probable that this endo- 
toxin is responsible for the quick development of symptoms in 
those who are poisoned by eating infected food. Specific agglu- 
tinins are developed in the blood of infected individuals, as are 
also coagglutinins for other members of the intestinal group. 
Some differences in agglutinability of the different strains isolated INTESTINAL OR COLON-TYPHOID GROUP 
333 
have been noted. It has been proposed that meat may be tested 
for the presence of Bacterium enteritidis by expressing the juice 
and determining its agglutinating power. This has not been 
proved practicable. It is probable that the bacilli already 
present in the meat would in some cases fix all the agglutinins 
present if stored for any length of time. Practicable methods 
of prophylactic or curative immunization have not been 
demonstrated. 
Bacteriologic Diagnosis.-The organism may be demonstrated 
by inoculation of infected flesh into suitable enrichment medium, 
such as malachite green dulcite broth, followed by plating. In 
man the disease may be diagnosed by the agglutination test, al- 
though with difficulty, for, as has been noted above, the various 
strains agglutinate differently, and blood from a typhoid or a 
paratyphoid patient may show a marked capacity to agglutinate 
Bacterium enteritidis. 
Transmission and Prophylaxis.-Probably a large proportion 
of the cases of so-called ptomain-poisoning is due to infection with 
Bacterium enteritidis and to the toxic products of metabolism of 
this organism. Such infection undoubtedly occurs frequently 
enough to justify rigorous measures for its prevention. Meat or 
milk from animals showing severe gastro-intestinal disturbances 
should never be used, as the infection in the human has in several 
well-authenticated instances been traced directly to such prac- 
tices. Probably most cases of meat-poisoning originate from 
use of flesh of diseased animals, but the possibility of infection 
with the organism after the animal has been slaughtered should 
not be ignored. Experiments have shown that when fresh meat 
is inoculated upon the surface with a culture of Bad. enteritidis 
the organism rapidly penetrates the tissues, even at low tempera- 
tures. Such infection might easily occur in unsanitary abattoirs 
through flies and careless handling. 
It should be noted that certain types of the paratyphoid bacil- 
lus are very similar to this form, if not identical with it, and 
doubtless are the cause of meat-poisoning as well. 
Bacterium choleras suis 
Synonyms.-Bacillus suipestifer; B. cholerce suis. B. sal- 
moni; Salmonella. 334 
VETERINARY BACTERIOLOGY 
Salmon and Smith, in 1885, described this organism as the 
cause of the disease called by them swine-plague. In the fol- 
lowing year Smith discovered another organism associated with 
a different disease of swine. This led to a revision of terminology 
which has since come into common use, and the organism first 
described is now known as the hog-cholera bacillus. Smith re- 
covered this organism from the spleens of about 500 hogs affected 
with hog-cholera. It was quite generally accepted as the cause of 
the disease until deSchweinitz and Dorset reported an outbreak of 
hog-cholera in which the Bad. cholera suis was not the primary 
infecting agent. This 
was shown by the trans- 
mission of the disease 
by blood filtered through 
fine-grained porcelain 
bougies, a procedure 
which removed the 
bacillus completely, as 
shown by the fact that 
the filtrate was quite 
incapable of infecting 
culture-media. By the 
subsequent work of Dor- 
set, Bolton, and Mc- 
Bryde it was shown 
quite conclusively that 
the Bad. cholera suis is 
not the cause of hog-cholera in the Mississippi Valley, and 
but a secondary invader at most. Other investigators in the 
United States and in Europe have confirmed these results. Hog- 
cholera and its virus will be considered, therefore, under the 
heading of Diseases Caused by Filterable Virus. The Bad. 
cholera suis, however, doubtless plays some part in the disease as 
a secondary invader, and is, therefore, worthy of consideration. 
Uhlenhuth and his collaborators in a study of 600 swine found 
organisms indistinguishable from this form in the intestines of 
8.4 per cent. 
Fig. 123.-Bacterium cholerce suis, organisms 
from a young culture (deSchweinitz, Bureau 
Animal Industry). INTESTINAL OR COLON-TYPHOID GROUP 
335 
Morphology and Staining.-This organism differs morpho- 
logically in no essential character from Bacterium enteritidis. 
Isolation and Culture.-Bacterium cholerce suis may frequently 
be isolated at once in pure culture from the organs of infected 
animals, particularly from the spleen. It has likewise been iso- 
lated by plating the intestinal contents of normal and infected 
animals. The organism grows upon agar and gelatin, forming 
a grayish, glistening, non-viscid growth, which is not particu- 
larly characteristic. Upon Conradi-Drigalski agar blue colonies 
are formed. On Loffler's malachite green clouded but translu- 
Fig. 124.-Bacterium choleroe suis, showing flagella (deSchweinitz, Bureau 
Animal Industry). 
cent colonies appear, in whose vicinity the agar is made yellowish. 
Endo plates give colorless colonies. No growth occurs upon 
potatoes having a decided acid reaction, but upon those which 
are neutral or alkaline a thin, glistening, usually yellowish layer 
is formed. Bouillon is uniformly clouded; a slight pellicle may 
appear in time. A grayish, friable sediment is formed. Milk 
shows a slight initial acidity, but soon becomes alkaline, and 
gradually becomes opalescent, and finally translucent. In Pe- 
truschky's lackmus molke the medium is reddened, then it turns 
blue and becomes intensely alkaline. It will be noted that there 336 
VETERINARY BACTERIOLOGY 
are no marked cultural differences between Bact. enteritidis and 
Bad. cholera suis. 
Physiology.-Bacterium cholera suis is aerobic and facultative 
anaerobic. The optimum growth temperature is about 37°; it 
grows also, but more slowly, at room-temperature, and it will de- 
velop also at 45° C. The thermal death-point is 58°, with ten 
minutes' exposure. The organism will remain viable for several 
days when dried. Gas and acid are produced in dextrose broth, 
also from arabinose, xylose, fructose, galactose, mannose, maltose, 
dulcite, mannite, and sorbite. Lactose and saccharose are not 
fermented, and no growth occurs in the closed arm of the fermenta- 
tion tube containing these sugars. Glycogen, inulin, adonite, 
starch, erythrite, and raffinose are not attacked. Indol is not or- 
dinarily produced. Gelatin is not liquefied. Hydrogen sulphid 
is formed from peptone. 
Pathogenesis.-It should again be emphasized that the Bac- 
terium cholera suis is not the primary cause of hog-cholera, but 
that it is a secondary invader of importance, and may be occasion- 
ally the primary cause of disease in hogs, but this disease probably 
would possess, according to Dorset, Bolton, and McBryde, a low 
degree of contagiousness. 
Experimental Evidence of Pathogenesis.-Some differences in 
virulence have been observed in cultures obtained from different 
sources. Rabbits succumb to septicemia in five to eight days when 
inoculated with c.c. of a virulent bouillon culture. Guinea-pigs 
are more refractory, and die after seven to twelve days. Subcu- 
taneous and intravenous injections and feeding experiments rarely 
produce death in the hog. The animal may sometimes show fever 
and depression, particularly after intravenous inoculation, but the 
infection is rarely fatal unless 1 or 2 c.c. or more of culture are used. 
However, some highly virulent cultures have been described. 
Feeding with large quantities of culture or long-continued feeding 
sometimes proves fatal. 
Undoubtedly this organism sometimes causes spontaneous in- 
fection of swine independently of the filterable virus. Schern and 
Stange have suggested for this type of disease the name parapest. 
Character of Disease and Lesions Produced.-An examination 
of a rabbit killed by injections of Bact. cholera suis shows lesions INTESTINAL OR COLON-TYPHOID GROUP 
337 
differing in no material respect from those discussed under Bad. 
enteritidis. To just what extent the characteristic lesions in hog- 
cholera, particularly in the chronic types, are due to infection by 
this organism is uncertain. It is probable that in many cases, at 
least, it is responsible for the development of intestinal ulcers. 
Inasmuch as it is sometimes found in the blood of animals infected 
by hog-cholera, it is probable that death may be due directly to 
their activity. 
Immunity.-The topic of immunity against hog-cholera will be 
considered under that heading. The Bacterium cholerce suis pro- 
duces no true toxin, but there is considerable formation of endo- 
toxin. Agglutinins are present normally in the blood of the hog, 
and immune agglutinins may be produced by the systematic im- 
munization of animals by killed cultures. Group agglutination 
with other members of this subgroup has been demonstrated. 
It has also been shown that immunization of the hog against true 
hog-cholera results in a considerable increase of agglutinins for 
Bad. cholerce suis in the blood. Opsonins for Bad. cholerce suis 
have been shown to be present in normal serum. Since the dis- 
covery of the filterable virus efforts at immunization by the use 
of vaccines and sera prepared by the use of Bad. cholerce suis have 
been practically abandoned. 
Bacterium typhi suis 
Synonyms.-Bacillus of Glasser, probably also Bacillus suipes- 
tifer of Voldagsen. 
These are perhaps best regarded as strains of the Bacterium 
cholerce suis isolated by Glasser and by Voldagsen, and showing 
some special characteristics. Generally these organisms give a 
somewhat more delicate growth on culture-media than the typical 
Bad. cholerce suis. 
In milk frequently there is no change, or at least the change 
to alkalinity is relatively slow. Uhlenhuth and Haendel have 
concluded that the differences from Bad. cholerce suis are 
insignificant. 
The men who first worked with these organisms concluded that 
they have unusually high pathogenicity for swine particularly for 
animals not more than four months old. They ascribe to them a 
causal relationship to pig typhoid. 338 
VETERINARY BACTERIOLOGY 
Synonyms.-Paratyphoid or paracolon bacillus. Bacillus 
paratyphosus. 
Disease. Produced.-Paratyphoid in man, possibly similar 
infections in animals. 
Gwyn, in 1898, isolated from a clinical typhoid case an organ- 
ism which belonged to the intermediate subgroup of intestinal 
organisms rather than to the typhoid-dysentery subgroup. In 
1900 Schottmiiller isolated two types of organisms from some- 
what similar cases. He termed these paratyphoid A. and paraty- 
phoid B. Similar organisms have been isolated repeatedly since 
that time-in some instances from an atypical typhoid case, in 
others from cases that had all the clinical symptoms of typhoid, 
but that did not give the agglutination reaction. 
Morphology and Staining.-This organism corresponds closely 
in morphologic and staining characters to the Bacterium enteritidis 
and the Bad. cholera suis. 
Isolation and Culture.-The organism has been isolated in 
pure culture directly from the blood, and by plate cultures from 
the internal organs in disease, and particularly from the intesti- 
nal contents of man and of the lower animals. In general cul- 
tural characters the organism resembles the Bacterium enteritidis. 
It has been found in practice that two varieties may be differ- 
entiated, termed A and B respectively. Type A does not produce 
a terminal alkalinity in milk and dissolve the casein, and in that 
respect differs from Bad. enteritidis; it produces an almost invisi- 
ble growth on potato, lactose whey is made permanently acid. 
Type B grows more luxuriantly, even on potato, milk is made 
strongly alkaline and cleared, lactose whey is also made alkaline. 
Physiology.-Not markedly different from Bacterium cholera 
suis, but according to Harding and Ostenberg producing red colora- 
tion on fuchsin sulphite agar containing either arabinose or xylose. 
Pathogenesis.-Paratyphoid fever in man has been attended 
by a low mortality; in consequence few autopsies have been re- 
ported. In both animals and man infection partakes more of the 
nature of an acute enteritis than does typhoid fever; the lympha- 
tics are not generally invaded as in typhoid, and the Peyer's 
patches are not swollen and ulcerated. 
Bacterium paratyphi INTESTINAL OR COLON-TYPHOID GROUP 
339 
Immunity.-Probably an endotoxin, less soluble, but in some 
respects similar to that of Bacterium enteritidis, is produced by 
these organisms. Agglutinins are produced in infected individ- 
uals. The agglutination reactions of types A and B differ 
markedly. It was this difference which first suggested the exist- 
Fig. 125.-Bacterium typhi murium (Migula). 
ence of the two types. No method of practical immunization 
against the disease is known. 
Bacteriologic Diagnosis.-The differentiation of paratyphoid 
may be made clinically by the specific agglutination tests. The 
absence of a test in a case of clinical typhoid calls for a repetition 
with the two types of paratyphoid bacilli. 
Transmission.-It is probable that certain gastro-intestinal 
infections in cattle may be caused by organisms of this type, and 
that meat and milk may become contaminated from these 
sources. Milk and meat are probably the most common sources 
of infection, although water has been clearly shown, in some 
instances, to be the source of epidemics. 
Synonym.-Bacillus typhi murium. 
Loftier, in 1889, described an organism as the cause of an 
epidemic among the mice kept for experimental purposes. Danysz, 
in 1900, described a similar, probably identical, organism, and 
Bacterium typhi murium 340 
VETERINARY BACTERIOLOGY 
recommended its use in the destruction of rats. These forms have 
all the morphologic and cultural characteristics of the enteritidis 
group, but show some differences in pathogenicity and in formation 
of specific agglutinins. Cultures of these organisms have been 
widely exploited as specific for mice and rats, producing a rapidly 
fatal disease, but as harmless to the higher animals. Reports as 
to their efficacy when fed to the vermin are conflicting; the results 
seem in some cases to have been favorable. It evidently is true 
that the virulence of the organism is subject to considerable varia- 
tions, for the careful work of Rosenau showed the culture which 
he possessed to be worthless in the extermination of rats. On the 
other hand, there is some evidence that the organism is not so free 
from harmful effect on the human as has been supposed. Fatal 
infections in man have been reported from Japan. 
Bacterium pullorum 
Synonyms.-Bacillus pullorum. 
Disease Produced.-White diarrhea of chicks. 
Rettger and Harvey have described the Bacterium pullorum as 
the specific cause of a white diarrhea in young chicks. The 
adult fowl is also affected but the mortality is lower. As a 
source of infection of the young chick through the egg or by con- 
tact the diseased adult bird is of great importance. The type of 
organism found in the young stock differs slightly from that 
found in adults. Type A, found in young stock, shows a greater 
tendency to form acid in several sugars than does type B (adult 
stock), and is aerogenic (Hadley). 
Morphology and Staining.-Bacterium pullorum is a rod, 0.3 to 
0.5 by 1 to 2.5 m, with rounded ends. It occurs singly or very 
rarely in chains. It is non-motile, does not produce capsules or 
spores. It stains readily and uniformly with ordinary aqueous 
anilin dyes and is Gram-negative. 
Isolation and Culture.-The organism may be isolated from 
the infected chicks by opening the body with aseptic precautions, 
and making streaks upon the surface of agar slants with blood or 
the pulp of the spleen or liver. Upon the agar slant the colonies 
are discrete, and at first resemble the pin-point, translucent 
colonies of the Streptococcus. They enlarge later. Upon gela- INTESTINAL OR COLON-TYPHOID GROUP 
341 
tin the colonies resemble those of the typhoid bacillus. Little 
growth occurs upon potato. Milk is a suitable medium; but 
there is little change, no coagulation, and no proteolysis. 
Physiology.-The organism is aerobic and facultative 
anaerobic. The optimum growth temperature is about 37°. 
Dextrose and mannite are fermented, with the production of 
both acid and gas (Rettger). Maltose, lactose, and saccharose 
are not fermented. Inclol is not produced. 
Pathogenesis.-Experimental Evidence.-Rettger has isolated 
the specific organism in several outbreaks of the disease from the 
internal organs, particularly the livers of chicks that had died of 
the disease or were showing symptoms. He also isolated it from 
abnormal egg-yolks in the ovaries of hens, from freshly laid eggs, 
and from the yolk-sacs of fully developed chicks within the shell. 
He also succeeded in infecting chicks by feeding, but the disease 
was not always contracted. Subcutaneous injections always 
proved fatal. The disease has appeared as a highly fatal one in 
several flocks of adult chickens, the organism recovered being 
agglutinable by immune serum of the type found in young stock 
but differing from the latter mainly in its reactions in sugar as 
above mentioned. 
Characteristics of Disease and Lesions- The most noticeable 
antemortem characteristics are emaciation and wasting of the 
chick, and the white diarrhea. The lesions are confined princi- 
pally to the intestines, and are particularly evident in the cecum. 
The liver is sometimes congested in areas. In the adult the ova 
present are irregular in appearance, instead of showing the 
normal spherical shape, while the color is greenish yellow to green. 
Immunity.-Practicable methods of immunization have not 
been evolved. 
Bacteriologic Diagnosis.-The organism may be isolated in 
pure culture from the internal organs, particularly the liver. 
To determine whether organisms resembling this organism are 
common Rettger, Hadley and others have found diagnosis by 
means of the agglutination reaction accurate and reliable and by 
the application of this test have succeeded in eliminating the 
infected birds of a flock, thereby eradicating the disease. 
Transmission and Prophylaxis.-Rettger claims that the 342 
VETERINARY BACTERIOLOGY 
disease is sometimes present before hatching, the organism being 
present in the ovaries and oviduct, and that contamination of the 
food likewise results in infection. 
Subgroup III. Typhoid-dysentery Subgroup 
The three important organisms belonging to this subgroup-• 
Bacterium typhi, Bad. dysenteries, and Bad. fcecalis alkaligenes- 
are not ordinarily pathogenic for the lower animals. They are, 
however, pathogenic for man, and since many of our diagnostic 
methods for other diseases have been discovered through their 
study, they are discussed briefly. 
Bacterium typhosum 
Synonyms.-Bacillus typhi; B. typhi abdominalis; Eberth or 
Eberth-Gaffky bacillus, Bacillus typhosus. 
Disease Produced.-Typhoid fever in man. 
Eberth, in 1880, discovered the Bacterium typhosum in the 
spleen and other internal organs of the body of persons who had 
died of typhoid fever. Gaffky, in 1884, cultivated the organism. 
It is now generally conceded to be the cause of typhoid fever, 
although the experimental animals cannot ordinarily be infected. 
Distribution/-Typhoid fever is widely distributed throughout 
temperate and tropical countries. It is constantly present, fre- 
quently in epidemic form, in the United States. 
Morphology.-Bacterium typhosum is a short, plump rod, usu- 
ally varying between 0.5 and 0.8 m in diameter, and 1 to 3 m in 
length. It is motile by means of numerous flagella. It does not 
produce capsules or spores. It stains readily with aqueous anilin 
dyes. Granular staining is sometimes observed, although the 
cells usually stain uniformly. It is Gram-negative. 
Isolation and Culture.-The desirability of isolating Bacterium 
typhosum from contaminated water has led to the development 
of many media in an effort to accomplish this. A quantitative 
estimation of the typhoid bacillus from such sources does not 
seem to be practicable, but the qualitative determination of 
presence may be carried out. The methods used are to inhibit 
the growth of the purely saprophytic organisms present by the 
use of antiseptic substances, such as malachite green, caffein, INTESTINAL OR COLON-TYPHOID GROUP 
343 
and crystal violet, and to incubate such media at blood-heat. 
These media do not inhibit, in general, the growth of either 
Bad. typhosum or Bad. coli, and dependence is placed upon 
differences in colony characters and media reactions to separate 
them upon subsequent plating. 
The colonies upon gelatin are somewhat smaller and more 
delicate than those of the Bacterium coli. This organism was 
originally described as producing a thin, "invisible growth" upon 
potato. This is true upon 
potato with an acid reac- 
tion, but upon alkaline or 
neutral potato the growth 
is relatively abundant. 
Physiology.-Baderium 
typhosum develops best at 
a temperature of 37°, but 
will grow at room-tempera- 
ture.- It is an aerobe and 
facultative anaerobe. No 
indol is produced. Acid, 
but no gas, is formed from 
dextrose. Neither acid nor 
gas is produced from lactose 
or saccharose. Some 
strains ferment xylose. 
There may be slight initial acidity in milk, but there is never 
coagulation of the casein. Proteolytic enzymes are not devel- 
oped in cultures. 
Pathogenesis.-Experimental Evidence.-The lesions typical of 
typhoid in man are not produced either by injection or feeding 
experiments upon most laboratory animals. The symptoms after 
intraperitoneal injection of a guinea-pig do not differ greatly from 
those produced by the Bacterium coli. Feeding experiments with 
anthrapoid apes within recent years have shown the possibility of 
producing the typical lesions of the disease in these animals. In- 
fection with typhoid bacilli in laboratory workers has several times 
occurred following the accidental ingestion of pure cultures of the 
organism. 
Fig. 126.-Bacterium typhosum, clump 
in a section of a spleen (Frankel and 
Pfeiffer). 344 
VETERINARY BACTERIOLOGY 
Character of Lesions and Disease Produced.-Clinical diagnosis 
of typhoid is frequently difficult, as the characteristics of the disease 
are often not well marked. The organism invades the intestinal 
lymph-system, and particularly the Peyer's patches. The latter 
become ulcerated, and perforation of the intestinal wall is not an 
uncommon result. The spleen is swollen. The bacteria are usu- 
ally found in the blood, though not commonly in large numbers, 
but are abundant in the spleen. Cystitis, cholecystitis, and bone 
metastases are not uncommon sequelae to the infection. 
Immunity.-No true toxin has been demonstrated for Bac- 
terium typhosum, but an endotoxin is present. Agglutinins and 
Fig. 127.-Bacterium typhosum, 
showing flagella (Gunther). 
Fig. 128.-Bacterium typhosum, col- 
ony on agar (Gunther). 
precipitins specific for the organism are likewise produced. Bac- 
teriolysins may be demonstrated in the blood of animals that have 
been artificially immunized by injections of the typhoid bacillus. 
There is developed in the body of an individual that has recovered 
from typhoid a certain degree of immunity, but this disappears, 
so that it is not unusual for a person to have several attacks of 
the disease. This immunity is probably both bacteriolytic and 
opsonic in nature. 
The use of antisera in passive immunization against typhoid 
and in curing the disease has not proved successful. The injection 
of such sera has not been shown to have either an immunizing or a 
curative effect in man. Active immunization by the injection of 
dead or living bacteria or their products has, on the contrary, been INTESTINAL OR COLON-TYPHOID GROUP 
345 
quite successful. Usually the organisms are scraped from the sur- 
face of agar cultures, suspended in physiologic salt solution, and 
killed by heat, or a broth culture may be used. 
Bacteriologic Diagnosis.-The Widal or agglutination test is 
commonly used in the diagnosis of typhoid. A dilution of 1 :40 
and higher is generally made to minimize the effect of the normal 
agglutinins which may be present in the blood. Both microscopic 
and macroscopic tests are used; the former is the more delicate, but 
the latter somewhat more reliable. The agglutinins often appear 
early in the course of the disease-usually by the fifth day or rarely 
later. Blood or serum for making the test may be either liquid or 
dried. It is received in the latter condition by many of the state 
and municipal bacteriological laboratories. The bacteria may be 
cultivated directly from the blood of a patieht. Frequently the 
organisms can be found in the blood somewhat before the serum 
exhibits a marked agglutinating power. Isolation of the organisms 
directly from the feces is sometimes resorted to in an effort to 
determine the occurrence of the so-called "bacillus carriers." 
Transmission.-Typhoid fever is contracted from contami- 
nated drinking-water, milk and other foods, and by contact, the 
frequency being in about the order named. Flies probably are 
commonly instrumental in carrying the organism from dejecta of 
typhoid-fever patients to food materials. The term bacillus 
carrier, or germ-carrier, is used to designate an individual who still 
harbors a pathogenic organism in the body after convalescence. 
Such germ-carriers are particularly dangerous, as they may give 
rise to an epidemic that is almost impossible to trace to its source. 
The danger of milk infection is probably the greatest from indi- 
viduals that are employed in dairies. Several epidemics have been 
traced to this origin. 
Bacterium dysenterise 
Synonyms.-Bacillus of Shiga,; Bacillus of Flexner. Bacillus 
dy senterice. 
Disease Produced.-Bacillary dysentery in man. 
Shiga, in 1898, discovered in the feces of patients suffering 
from dysentery a bacillus which he believed to be the specific 
cause of the disease. Previous to this it had been shown that 346 
VETERINARY BACTERIOLOGY 
amebae may cause dysentery, and it was when examining stools 
for these protozoa that Shiga discovered this organism. In 1900 
Flexner published the results of work in Manila and described 
another type of organism. Since that time many epidemics 
have been studied, and it is generally believed that the type 
described by Shiga is the more common, but that the bacillus of 
Flexner occurs in a certain proportion of the outbreaks, more 
particularly in the tropical countries. Other authors, Hiss in 
particular, have differentiated even more groups 
Morphology.-Bacterium 
dysenterice and Bad. typhosum 
are practically indistinguishable 
under the microscope in stained 
mounts. The Bad. dysenterice, 
however, is non-motile. Spores 
and capsules are not produced. 
It stains uniformly and is 
Gram-negative. 
Isolation and Culture.-The 
organism may be isolated di- 
rectly from the dejecta by plat- 
ing. The cultural characters in 
general closely resemble those 
of Bacterium typhi. Milk is rendered permanently alkaline, 
however. 
Physiology.-The physiologic characters of Bacterium dysen- 
terice closely resemble those of Bad. typhosum. The ability to 
produce acid in solutions of various carbohydrates and of the 
related alcohols is used as a means of differentiation of the 
varieties. Otho listed some fifteen different types by this means. 
A more conservative and valuable classification is that of Hiss, 
as modified by Shiga. 
Recent work seems to indicate that the dysentery group 
may be divided into a number of species which may be differ- 
entiated by the following key. 
Fig. 129.--Baderium dysenterice 
(Kolle and Wassermann). 
I. Mannitol not fermented. 
A. Indol not formed: rhamnose not fermented. 1. Bad. shigoe. 
B. Indol positive; rhamnose fermented with acid. 2. Bad. ambiguum. 
Key to the Dysentery and Allied Bacilli INTESTINAL OR COLON-TYPHOID GROUP 
347 
II. Mannitol fermented forming acid. 
A. Xylose not fermented. 
1. Lactose not fermented; milk slightly acid 
then alkaline; indol usually positive; 
rhamnose usually not fermented. 3. Bad. flexneri. 
2. Lactose fermented with acid; milk rendered 
strongly acid and usually coagulated; 
indol not formed, rhamnose fermented 
with acid. 4. Bad. sonnei. 
B. Xylose fermented with acid. 
1. Lactose and sucrose fermented forming 
acid; milk acid and clotted; dulcitol not 
attacked. 5. Bad. dispar 
2. Lactose and sucrose not fermented; milk 
alkaline; dulcitol attacked with acid 
production. 6. Bad. alkalescens. 
Pathogenesis.-Experimental Evidence.-The belief that the 
varieties of Bacterium dysenteries are the important etiologic 
factors in the disease is based upon the following facts: 
1. This organism in some one of its varieties has been shown 
to be present with great constancy in the patient's excreta. 
2. Injections of the organisms and their products kill labora- 
tory animals, particularly rabbits, although typical dysentery is 
not readily induced by feeding experiments. 
3. The blood-serum of a patient will, in general, agglutinate 
in high dilution the strain isolated from the feces. 
4. Antiserum has been successfully used in the prevention 
and cure of the disease. 
Character of Disease and Lesions Produced.-The intestine, 
particularly the colon, is inflamed and is sometimes ulcerated, and 
may even show diphtheritic necrosis. With the exception of this 
there is little that is characteristic of the disease. Unlike the 
typhoid bacillus, it does not commonly invade the blood or the 
internal organs, with the exception of the mesenteric glands. 
The disease is rather a toxemia than a bacteremia. 
Immunity.-A soluble toxin has been demonstrated for the 
Shiga type, but repeated efforts have failed to show that any such 
is produced by the Flexner type. This poison was at first believed 
to be an unusually potent endotoxin. Conradi first demonstrated 
the toxin by growing the organism upon agar, then suspending it in 
physiologic salt solution, and allowing the bacteria to undergo 348 
VETERINARY BACTERIOLOGY 
autolysis. Later, Rosenthal and others showed that toxin will be 
produced in considerable quantities in an alkaline bouillon, but not 
in one that is neutral or acid. This bouillon is either filtered 
through porcelain filters, or 0.5 per cent, phenol is added and al- 
lowed to stand, and then filtered through paper until clear. The 
toxin may be precipitated by ammonium sulphate, and after dial- 
ysis and drying of such, 1 to 2 gm. may be a lethal dose for a kilo 
of rabbit. It is weakened by prolonged heating at 70°, and de- 
stroyed at 80 to 100°. The rabbit is very susceptible to the in- 
jection of the toxin, while the guinea-pig is relatively resistant. 
The effect upon the rabbit may be characterized as a hemorrhagic 
necrotic enteritis. 
Shiga first used antisera in the treatment of dysentery. He 
regarded its curative properties as wholly bactericidal. Todd, 
Koram, Doerr, and others have, by systematic immunization of a 
horse, secured a serum that neutralizes the toxin actively. This 
has been used with very favorable results in the treatment of dys- 
entery caused by the Shiga bacillus. 
Bacteriologic Diagnosis.-The disease may be recognized by 
the Widal or agglutination test, and the several types of organisms 
differentiated in the same manner. The organism may likewise 
be isolated directly from the stools by plating. 
Transmission.-Dysentery is spread in much the same manner 
as typhoid, and the same preventive measures must be used. CHAPTER XXVIII 
ABORTION-MALTA FEVER GROUP. THE GENUS BRUCELLA 
Brucella (Bacterium) abortus of Bang and Bacterium melitensis 
are placed in this group. It should be noted that other organisms 
besides the Bad. abortus one here discussed are doubtless occa- 
sionally responsible for abortion in cattle and other animals. 
Brucella (Bacterium) abortus 
Synonyms.-Abortion bacillus of Bang; Bacillus abortus; 
Bacillus abonionis. % 
Disease Produced.-Infectious abortion disease in the cow. 
Bang, in 1897, described a bacillus as the probable cause of 
infectious abortion in the cow. The specific organism was iso- 
lated with difficulty. It has been isolated since that time in 
Europe several times, and more recently in the United States. 
Several investigators in the United States have described mem- 
bers of the colon group and of other groups as present in 
infectious abortion, but it is doubtful whether in many cases 
appropriate cultural methods have been utilized for the isolation 
of this organism. 
Distribution.-Contagious abortion has been reported from 
many localities on the continent of Europe and in Great Britain. 
It is known to occur in many sections of the United States. 
Morphology and Staining.-Bacterium abortus is very small, 
1 to 2 u long, 0.3 to 0.8 u broad, and, according to Nowak, resem- 
bles the bacillus of chicken-cholera. Nowak, on the basis of its 
morphology, groups it with the Pasteurellas or hemorrhagic sep- 
ticemia bacilli. It is polymorphic in culture-media. Involution 
forms occur as branched and clubbed types. It is non-motile, 
and neither capsules nor spores have been demonstrated. It 
stains readily by the aqueous anilin dyes, frequently showing 
polar granules. Carbol thionin, methylene-blue, or borax 
methylene-blue and Giemsa stain furnish beautiful pictures of the 
organism. It is Gram-negative. 
349 350 
VETERINARY BACTERIOLOGY 
Isolation and Culture.-The isolation and cultivation of Bac- 
terium abortus are attended with peculiar difficulties. The or- 
ganism may be frequently obtained at once in pure culture from the 
heart blood or the intestines of an aborted fetus. Bang used a 
medium consisting of nutrient agar (f per cent.) and liquid gelatin 
(5 per cent.) to which was added an equal quantity of sterile liquid 
blood-serum. These tubes were inoculated with suspected mate- 
rial, mixed well, and kept at blood-heat. In the course of three 
days numerous small, punctiform to pin-form colonies developed 
in a definite stratum a few millimeters below the surface. As will 
be noted under the discussion of physiology, the organism has an 
unusual relationship to oxygen, and the amount of oxygen needed 
Fig. 131.-Bacterium abor- 
tus. Culture in serum agar 
showing the definite stratum 
in which the colonies 
develop (Nowak). 
Fig. 130.-Bacterium abortus (Nowak). 
for its development is to be found at the depth at which the 
colonies form. These colonies are compact, rounded, or somewhat 
irregular, sometimes showing a dense nucleus surrounded by a 
lighter zone. When the organism is present in impure culture, as 
in the vagina of the cow, other methods are necessary for its isola- 
tion. Nowak has described a procedure which has proved satis- 
factory in the hands of several investigators. Probably simpler 
methods will be devised in time, but this appears to be the best thus 
far developed. The material is smeared over the surface of suc- 
cessive tubes of serum agar or over the surface of this medium in 
Petri dishes. These are allowed to stand for several days, and the 
colonies which develop are marked, as they are not Bact. abortus. ABORTION MALTA FEVER GROUP. THE GENUS BRUCELLA 
351 
The plates are then placed in a desiccator whose cubic contents of 
air has been determined, and plates of agar thickly seeded with 
B. subtilis are introduced, so that about 16 sq. cm. of surface is 
present per every 240 c.c. of space. It has been found experi- 
mentally that this will diminish the oxygen pressure to a point 
where the Bad. abortus will develop. After three days the plates 
are examined and search made for the Bad. abortus in the spaces 
between other colonies on the original plates. This same device, 
of reducing oxygen pressure by means of cultures of B. subtilis, 
may be used in the study of growth-characters on other media. 
Schroeder and Cotton, Evans and Steck have isolated the Bad. 
abortus directly from milk in ordinary petri dishes, even in 
gelatin medium. Traum has isolated it directly from the 
amniotic fluid and from the liver of aborted {tigs in open unsealed 
slants of the beef-liver-peptic-digest agar of Meyer. After 
primary culture the organism soon adapts itself to the ordinary 
oxygen tension and grows readily. The organism will grow in 
agar without addition of serum, particularly at the surface, 
but the addition of serum is decidedly beneficial. The colonies 
develop at the surface in about three days at 37° as small, 
usually discrete, transparent dots. In shake cultures in serum 
agar they appear in about four days in a well-defined stratum 
about 10 to 20 mm. below the surface. The individual colonies 
may reach a diameter of 1 mm. when well separated from each 
other. Growth in gelatin at room-temperature is slow. Bouil- 
lon cultures show development some millimeters below the 
surface, the medium above this remaining clear. Milk is not 
coagulated. Little or no growth takes place on potato. 
Physiology.-The optimum growth temperature is 37°, 
although the organism is found to multiply slowly at room-tem- 
perature. The relationship to oxygen, which has already been 
discussed, classifies the Baderium abortus as a micro-aerophile 
rather than a strict anaerobe. Strangely enough, Bang reports 
that the organism will also grow in an atmosphere of pure oxygen; 
two oxygen optima are, therefore, evident. More recent work 
has shown that the organism may be cultivated under strict 
aerobic conditions after a few transfers on laboratory media. It 
is relatively resistant to desiccation. It will remain alive for 352 
VETERINARY BACTERIOLOGY 
months in a retained mummified fetus, and for a year or more 
in a culture-medium. Acids and gas are not produced from 
carbohydrates. 
Pathogenesis.-Experimental Evidence.-Bang demonstrated 
the etiologic relationship of the organism to the disease by intra- 
venous injection, and by injection into the vagina and uterus of 
pregnant cows, also by feeding experiments. Sheep were also 
infected both by injection and feeding. Preisz did not succeed 
in transmitting the disease by vaginal injections into the cow, 
guinea-pig, or rabbit. More recent work has shown that inocu- 
Fig. 132.-Bacterium abortus, colonies on serum agar (Nowak). 
lation of these animals results in a chronic infection characterized 
by specific changes in the liver and spleen, and by arthritis. 
Pregnant animals will abort. Nowak succeeded in producing 
typical abortion of dead feti in guinea-pigs and rabbits by intra- 
peritoneal and intravenous injections. He did not succeed by 
intravaginal injections. 
Character of Disease and Lesions.-There are few symptoms 
either preceding or following the expulsion of the fetus. It is 
characterized as a rule by an interference with the develop- 
ment of the fetus frequently resulting in its premature expulsion 
either dead or alive. There is also a frequent manifest inflamma- 
tion of the fetal membranes and of the maternal cotyledons to- 
gether with retention of the placental membranes. ABORTION-MALTA FEVER GROUP. THE GENUS BRUCELLA 
353 
Immunity/-It is known that cows that have aborted one or 
more times may become immunized against the disease. Vac- 
cination and serum treatments have been attempted, but their 
worth has not been thoroughly proved. Bang showed that it is 
possible to immunize sheep and goats by subcutaneous injections 
of increasing quantities of living cultures to such an extent that 
they can withstand severe infection through food. Killed cul- 
tures did not prove to be effective. His trials with heifers did not 
prove to be as successful, although vaccination in badly infected 
herds has been claimed to be successful in some cases. 
Passive immunization by the use of the serum from cows which 
have aborted two or more times and then calved normally has 
been attempted, but results are not conclusive. 
Bacteriologic Diagnosis.-This may be made certain by the 
isolation of the specific organism in culture as outlined above. 
A tentative diagnosis may be made by preparing stained mounts, 
and demonstrating the presence of a short, Gram-negative bacil- 
lus in the uterine exudate and in the blood and tissues of the fetus. 
The agglutination and complement fixation reactions have been 
extensively used in diagnosing this disease. Both have proved 
reliable. The German Imperial Board of Health recognizes as 
positive those tests in which agglutination occurs in serum dilu- 
tions of 1:100 or over; as negative those under 1:100. Pohle has 
shown that a precipitation test may be used. 
The presence of the organism in milk can best be recognized 
by injection of guinea-pigs, which will show the characteristic 
lesions. 
Transmission.-It has been proved that the Bad. abortus can 
enter the body through the digestive tract. It cannot be dis- 
puted that it may also enter through other channels, neither has it 
been definitely proved. 
Brucella (Bacterium) melitensis 
Synonym.-Micrococcus melitensis. 
Disease Produced.-Malta fever in the goat and in man; 
Mediterranean fever. 
Bruce, in 1887, discovered a microorganism in the spleen of 
men dead from Malta or Mediterranean fever. Since that time 354 
VETERINARY BACTERIOLOGY 
it has been studied carefully by numerous investigators, and its 
relationship to the disease is well established. 
Distribution.-The disease is known to occur in all countries 
bordering on the Mediterranean, in southern Asia, South Africa, 
the Philippines, some of the islands of the West Indies, and in 
Mexico and Texas. 
Morphology and Staining/-Until the work of Meyer and 
Shaw in 1920, this organism was considered a coccus. These 
investigators found by a study of 21 cultures identified as Micro- 
coccus melitensis by various laboratories in the United States, 
England, Algiers and Italy that, confirming the repeated 
observations at previous times, the organism in smears made 
from young cultures and even from tissues is a typical short rod. 
Stained with gentian violet, the organism appears as short, oval 
rods frequently tapered at both ends. Identical smears stained 
with dilute carbol fuchsin show the organism more coccoid in 
morphology. Frequently in the water of condensation or liquid 
medium, short chains of 4-10 single, elongated coccoid elements 
can be recognized. The organism stains well with ordinary 
anilin dyes, but is Gram-negative. 
Isolation and Culture.-It may be isolated from the spleen 
during life or after death in pure culture, or by plating. On 
agar slants a fine granular film appears in from 24-36 hours. 
After 3 days a slight brownish tinge changes the moist well de- 
fined growth. Growth continues on peptic digest agar for weeks, 
even at room temperature, until a stringy, greasy, rather thick 
layer covers the surface. In gelatin the organism produces dark 
brownish granular colonies after from 10-30 days, but never 
liquefies the medium. In infusion broth, there is a slight initial 
turbidity, followed by clearing and the deposit of a stringy, 
tenacious sediment, occasionally a ring or pellicle formation. 
Milk is not changed. Meyer reports beef liver peptic digest agar 
admirably suited for isolation and growth of the organism. 
Physiology.-The Bacterium melitensis does not produce acid 
from any of the carbohydrates. Desiccation does not destroy it 
quickly, for the organism has been found to remain alive and 
virulent when dried for a considerable time. Pasteurization is 
fatal. ABORTION-MALTA FEVER GROUP. THE GENUS BRUCELLA 
355 
Pathogenesis.-The disease is a true bacteremia. Inoculation 
of pure cultures reproduces the disease in the goat, cow, and 
monkey. Accidental laboratory infections have proved its power 
of producing disease in man. Infection probably usually arises 
through ingestion. The disease is characterized by its low mor- 
tality, its long duration in man, and the accompanying articular 
rheumatism. It is a dis- 
ease primarily of the goat, 
though it is possible that 
cattle may sometimes har- 
bor and transmit it. Horses 
have also been experiment- 
ally infected. In guinea- 
pigs and rabbits the disease 
runs a chronic course. 
Immunity.-N o toxins 
have been demonstrated. 
Agglutinins are present in 
the blood in infected indi- 
viduals, so that agglutina- 
tion may sometimes be 
secured with high dilutions-occasionally as high as 1 : 6000. 
This test is one of the readiest methods of diagnosing the 
disease. 
Bacteriologic Diagnosis.-Diagnosis may be made by isolation 
of the organism by a puncture of the spleen, or by demonstrating 
the presence of specific agglutinins in the serum in dilutions of 
1 : 50 or greater. Mohler and Eichhorn found that complement 
fixation is a satisfactory method of diagnosis in the disease,' and 
more reliable than agglutination. 
Transmission and Prophylaxis.-The organism is excreted in 
the feces, urine, and milk from infected animals. Most cases in 
the human are acquired by drinking the milk of infected animals. 
The disease infects, in either the acute or chronic form, so large 
a proportion of the animals in some countries that the use of 
unheated milk is always attended with danger. 
Fig. 133.-Bacterium melitensis (X 1200) 
(Jordan). CHAPTER XXIX 
DOG DISTEMPER GROUP. THE GENUS BACTERIUM 
Bacterium bronchisepticum (bronchicanis) 
Diseases Produced.-Canine distemper and similar diseases 
in other animals. 
Ferry, in 1910, published a report of the bacterial findings in 
canine distemper in which he described an organism associated 
with the disease and which he believed to be the primary cause. 
He gave to this organism the name Bacillus bronchicanis., subse- 
quently changing this to B. bronchisepticus. His findings have 
been corroborated by M'Gowan, Torrey, and Rabe. 
Ferry1 (1916) calls attention to the close morphologic, cultural 
and serological characters of the Bad. bronchisepticum and Bad. 
pertussis. Smith2 finds the Bad. bronchisepticum and Pseudo- 
monas pyocyanea closely related except in pigment production 
and action in gelatin. 
Morphology and Staining.-The organism is a short slender 
rod, 2.3 to 5 u long, usually found singly, but often in pairs. 
In primary inoculations in broth the organism may be larger and 
oval in shape. Here, too, filaments may be observed. No spores 
are produced. It stains best with methylene-blue, with a char- 
acteristic bipolar staining and is Gram-negative. Active and 
progressive motility is noted. 
Isolation and Culture.-The organism is frequently in mixed 
infections with staphylococci, so that isolation is difficult. It is 
found in the nasal secretions, blood, and internal organs. Ferry 
reports positive findings in blood-cultures in 28.5 per cent, of 
cases. Others have found it less frequently. One to 5 c.c. of 
blood are planted in a flask containing 50 c.c. of broth. After 
twenty-four hours incubation plates or slant agar cultures in suc- 
cessive tubes are made. On agar the colonies appear after 
twenty-four hours as very fine, dewdrop-like, later enlarging and 
becoming opaque. 
1Journ. Bact., 1918, 3, p. 193. 
2Journ. Med. Res., 1913, 29, p. 299. 
356 DOG DISTEMPER GROUP. THE GENUS BACTERIUM 
357 
Bouillon is clouded with a heavy stringy growth in old cultures. 
Physiology.-No gas is produced in any carbohydrates. Lit- 
mus milk remains strongly alkaline. No indol is produced. 
Pathogenesis.-Dogs, guinea-pigs, rabbits, and monkeys are 
susceptible to infection. Inoculations into the air-passages of 
young dogs produce the disease in its characteristic form. Older 
animals are resistant. 
Character of Disease.-The disease is marked by catarrh of the 
air-passages, intestinal catarrh, discharge from the eyes, pustules 
on the skin in a small percentage of cases, and nervous symptoms. 
Immunity.-Recovery from the disease confers an immunity. 
Ferry reports satisfactory results from bacterin and serum treat- 
ment and recommends the former as a prophylactic and the 
latter as a curative measure. Best results seem to be derived 
from a mixed bacterin of Bacterium bronchisepticum and 
staphylococci. 
Bacteriologic Diagnosis.-The serum of animals suffering from 
natural infection agglutinates in dilutions of from 1 : 40 to 1 : 800. 
Little use of the agglutination test for the diagnosis of the disease 
has been made. The finding of the characteristic organism in 
smears from the nasal mucous membranes is of value in diagnosis. 
Transmission.-The disease is readily transmitted from animal 
to animal, or through exposure in kennels where diseased animals 
have been kept. CHAPTER XXX 
FLUORESCENT GROUP. THE GENUS PSEUDOMONAS 
The group of fluorescent bacilli includes those forms which 
produce a water-soluble, diffusible bluish green or greenish pig- 
ment. All of the species are Gram-negative, do not produce 
spores, are aerobic and facultative, and usually motile by means 
of one or more polar flagella. 
Several species belonging to this group have been described, 
the more important being Pseudomonas fluorescens, with several 
varieties, and Ps. pyocyanea. The latter is the only form which 
has distinct pathogenic properties. 
Pseudomonas pyocyanea 
Synonyms.-Bacillus pyocyaneus; Ps. aeruginosa; bacillus of 
green or blue-green pus in man and animals. 
Gessard, in 1882, described the Pseudomonas pyocyanea from 
blue-green pus. Since that time it has been isolated and studied 
by numerous investigators both in Europe and America. 
Distribution.-This organism has been isolated from the feces 
of man and animals, from sewage and surface waters, from the 
soil, and from air and dust. It is usually saprophytic or com- 
mensal in its growth, and is only rarely pathogenic. 
Morphology and Staining.-Pseudomonas pyocyanea is a 
slender rod with rounded ends, about 0.6 by 2.6 m or smaller, 
usually single, rarely in chains of 2 to 6 individuals. It is motile 
by means of a single terminal flagellum. No spores or capsules 
are produced. It stains readily by the ordinary anilin dyes and 
is Gram-negative. 
Isolation and Culture.-This organism is readily isolated by 
plating out pus which contains it, as the colonies are quite charac- 
teristic on account of the green pigment which they diffuse. It 
grows readily on all the common laboratory media. Upon 
agar and gelatin plates the thin, poorly defined colonies are charac- 
terized by the fluorescent pigment surrounding them. Upon 
358 FLUORESCENT GROUP. THE GENUS PSEUDOMONAS 
359 
slant agar the color diffuses until the whole of the medium is a 
light green, then a darker blue green, and finally a brown or brown 
red. Gelatin is rapidly liquefied. Bouillon is clouded, a pellicle 
forms, and the fluorescent pigment diffuses from the top downward. 
Potatoes support a slimy growth and turn green, then brown. 
Milk is coagulated by a rennet-like enzyme and the curd pep- 
tonized. 
Physiology.-This organism is preferably an aerobe, and 
grows most luxuriantly in the presence of oxygen, but growth will 
continue under anaerobic conditions. Proteolytic ferments which 
will digest gelatin, fibrin, and casein are produced. Two pigments 
are usually formed-one green and fluorescent (fluorescin), soluble 
Fig. 134.-Pseudomonas pyocyanea (Kolle and Wassermann). 
in chloroform, the other (pyocyanin) bluish and insoluble in chlo- 
roform. Pigment is not produced in the absence of oxygen. In 
old cultures the pigments become yellow or brown. Autolytic 
disintegration of the cells takes place in old cultures. Mucin, a 
compound made up of a protein and a carbohydrate, has been 
found present in cultures. To this may be ascribed its slimy con- 
sistency on agar or even in bouillon. The organism is resistant to 
desiccation. 
Pathogenesis.-Experimental Evidence.-Injection of cultures 
of Ps. pyocyanea subcutaneously into the guinea-pig or rabbit 
causes rapidly spreading edema, suppuration, septicemia, and 
death within a day or two. Not all cultures are equally 
pathogenic. 360 
VETERINARY BACTERIOLOGY 
Character of Infection Produced.-The Ps. pyocyanea is usually 
a secondary invader, although in man it has been found causing 
primary infections. It has not yet been proved ever to cause 
suppuration alone in any of the domestic animals, but is not un- 
common in pus, to which it gives a green or blue-green color. In 
man it has been found in purulent otitis media, meningitis, broncho- 
pneumonia, infantile diarrhea, and generalized infections. Koske 
and others have ascribed to this organism an etiologic relationship 
to chronic rhinitis ("bullnose") in swine. 
Immunity.-A true toxin is produced by virulent cultures. 
Wassermann found 0.2 to 0.5 c.c. of this fatal for the guinea-pig. 
Poels has described a "pyocyaneus bacillosis" in calves in a single 
herd. The disease was characterized as an acute diarrhea. An 
antitoxin has been prepared for this pyocyaneus toxin. An endo- 
toxin has also been demonstrated. A leukocytic poison, leukocidin, 
and a hemolytic toxin, hemotoxin, have been differentiated. Im- 
munity has been experimentally produced by the injection of 
killed cultures. Emmerich and Low have proposed the name 
pyocyanase for the broth filtrate of old cultures of pyocyanea 
concentrated in a vacuum. This material has been found to be 
very high in proteolytic ferments and has been suggested for many 
clinical uses, among them the destruction of bacteria in vitro, the 
solution of diphtheritic membranes, the spraying of infected mem- 
branes to destroy the causal organisms, etc. While it has been 
extensively experimented with, it has never come into common use. 
Bacteriologic Diagnosis.-The organisms may be most readily 
determined by plating. CHAPTER XXXI 
DIPHTHERIA-PSEUDOTUBERCULOSIS GROUP. THE 
GENUS CORYNEBACTERIUM 
The organisms which belong to this group are all rod-shaped 
bacteria of moderate size. They are non-motile, do not produce 
spores, are Gram-positive, and not acid fast. All are aerobic 
and facultative. They are all characterized by the possession of 
granules which cause irregular or banded staining of the cell, and 
all show a decided tendency to the production of branched and 
club-shaped cells. 
A number of species of these so-called "diphtheroid" bacilli 
have been described. Some of these are pathogenic, others are 
not. The latter are important principally because of the difficulty 
in differentiating them from the pathogenic types in disease diag- 
nosis. 
The important pathogenic organisms of this group are the 
C orynebacterium pseudotuberculosis, the cause of bovine caseous 
lymphadenitis, equine ulcerative lymphangitis and bovine lymph- 
adenitis, and pyelonephritis, and the Coryn, diphtherice, the cause 
of human diphtheria. Coryn, xerosis, from the eye, Coryn, 
hoffmanni, the pseudodiphtheria bacillus, and some other diph- 
theroids less well known, belong here. 
Methods of separation of the organisms of this group from each 
other are not entirely satisfactory, but in some cases are quite 
necessary because of importance in disease diagnosis, particularly in 
diphtheria. Acid production in carbohydrates and development 
of a true toxin are among the tests which have been used. Mor- 
phology is also of much assistance. 
The four most common members of the group may be differen- 
tiated by means of their reactions in Hiss's carbohydrate serum- 
water medium. 
Acid production in 1 per cent. 
Dextrose. 
Glycerol. Saccharose. Dextrin. 
Corynebacterium diphtheria? 
+ 
+ - + 
Corynebacterium pseudotubercu- 
losis 
+ 
_ _ - 
Corynebacterium xerosis 
+ 
± + 
Corynebacterium hoffmanni 
- 
- - - 
361 362 
VETERINARY BACTERIOLOGY 
All of the organisms named except Corynebacterium hoffmanni 
also acidify maltose. Morse1 has named two other non-patho- 
genic members of this group B. hoagii and B. flavidus. The 
former produces acid from dextrose, the latter from dextrose, mal- 
tose, and glycerin. The latter also produces a yellowish pigment. 
Morphologic and cultural differences among these species 
may be noted, and means of differentiation by their aid will be 
discussed under the several headings. 
Corynebacterium pseudotuberculosis 
Synonyms.-Bacillus pseudotuberculosis ovis, Preisz; Mycobac- 
terium pseudotuberculosis; Bacillus lymphangitidis ulcerosa; B. 
renalis bovis; B. pseudotubercidosis murium, Kutscher; Corynethrix 
pseudotuberculosis murium, Bongert; Bacillus furunculosis ulcerosce, 
Kitt; Bacillus of Preisz; Preisz-Nocard bacillus. 
To differentiate from the slcid-fast bacteria resembling the 
tubercle bacillus, it has been suggested that the latter be called 
pseudotubercle bacilli, and the former pseudotuberculosis bacilli. 
There is not entire agreement among investigators as to the actual 
identity of many of the organisms listed above as synonyms. 
Some have believed that all constitute a single somewhat pleo- 
morphic species, others divide into two or more species upon the 
basis of pathogenicity, serum reactions, and culture. 
Diseases Produced.-The infections produced by this organism 
have received a variety of names depending upon the animals 
attacked and the lesions produced. In sheep the disease has been 
known as caseous lymphadenitis, pseudotuberculosis, and pyo- 
bacillosis; in equines, ulcerative lymphangitis (sometimes termed 
pseudofarcy and pseudoglanders); in cattle, caseous lymphadenitis, 
pyelonephritis, and pyobacillosis; and in rabbits and other rodents, 
pseudotuberculosis and lymphadenitis. 
Distribution.-The disease in sheep has been reported from 
France, Germany, the United States, Argentina, Australia. 
Somewhat similar infections in horses have been described from 
France and the United States. In Argentina 10 per cent, of sheep 
slaughtered and in Australia even 15 per cent, are affected. 
1 Morse, M. E., Jour. Inf. Dis., 1912, 11, p. 281. DIPHTHERIA-PSEUDOTUBERCULOSIS GROUP 
363 
Historical.-This organism was first described from the lesions 
of sheep by Preisz and Guinard1 and later by many other writers 
from France. 
Turski2 recorded the presence of the disease in Germany, and 
Norgaard and Mohler3 in the United States. 
The organism as a cause of "pseudofarcy" in horses was first 
noted by Nocard4 in 1892. Later he concluded his organism to 
be identical with that of Preisz from sheep. 
Dunkel,5 as the result of comparative studies of Bacillus 
pseudotuberculosis ovis and B. pyogenes suis, Grips, and B. pyogenes 
bovis, Kunnemann, came to the conclusion that the latter could be 
transformed into the former, and that they should all be classed as 
the same species. The more recent work of Priewe and of Glage 
indicates, however, that the last two species are quite distinct 
from the first and are far more closely related to the influenza 
group (q. vi). 
Morphology and Staining.-The organism is a non-motile 
bacillus about 0.4 by 1 to 3 p. It resembles the diphtheria bacillus 
in the formation on artificial media of thickened and club-shaped 
forms. It is non-motile and does not produce spores or capsules. 
It stains well with the ordinary anilin dyes and is Gram-posi- 
tive. 
Isolation.-Pure cultures may be obtained by smears upon suit- 
able culture-media directly from a caseous nodule. Growth is 
scant at first, but becomes better after a time as the organism 
adapts itself to artificial media. 
Cultural Characters.-On agar at 37° dry colonies are formed 
within two days, the maximum size being reached in six to eight 
days. The colony usually shows a folded or granular surface, fre- 
quently with concentric rings and a papillate center. Glycerin 
agar is unfavorable. 
On gelatin the organism grows sparingly when kept at 22°. 
Potato shows no growth according to some authors, others re- 
1 Preisz and Guinard, Jour, de Med. Vet., 1891, 42, p. 503. 
2 Turski, Zeitschr. f. Fleisch. u. Milchhygiene, 1897, p. 173. 
" Norgaard and Mohler, Sixteenth Annual Report Bureau of Animal 
Industry, 1899, p. 638. 
4 Nocard, Annales del' Institut. Pasteur, 1892. 
6 Diss., Giessen, 1908. 364 
VETERINARY BACTERIOLOGY 
port a grayish white, slightly moist, irregular film, often scarcely 
visible. 
Solidified serum shows characteristic colonies of creamy gold 
yellow or orange color, reaching a diameter of 1.5 mm. 
No change occurs in milk. 
Solidified egg-white is as suitable as blood-serum, though the 
yellow color does not appear. 
Sterile cattle serum gives abundant yellow flocculent growth, 
with decided clouding and yellowing of the serum. Finally, a 
heavy yellow sediment collects. 
Bouillon is not permanently clouded, a granular deposit forms, 
together with a dry surface pellicle. 
Figs. 135 and 136.-Gorynebacterium pseudotuberculosis, colony and mount 
(Norgaard and Mohler in Report of Bureau of Animal Industry). 
Physiology.-A temperature of 65° for ten minutes will destroy 
the organism. Its optimum growth temperature is 37°, but some 
growth occurs even at 18°. It is relatively resistant to drying. In 
culture-media it remains viable for many months. Virulence ap- 
parently remains unimpaired for years. The organism is easily 
destroyed by disinfectants. Toxins (see below) may be developed 
in broth. 
Coryn, pseudotuberculosis is aerobic. Gas is not produced 
from sugar. Acid is formed from dextrose, but not saccharose, 
glycerin, or dextrin. DIPHTHERIA-PSEUDOTUBERCULOSIS GROUP 
365 
Pathogenesis.-The organism is pathogenic for mice, guinea- 
pigs, rabbits, goats, sheep, and probably for the horse. Fowls 
and pigeons are immune. Although toxins may be demonstrated 
in cultures, they probably are not sufficient in quantity to account 
for the development of the disease. 
Intravenous injection of the guinea-pig results in death in from 
four to ten days, with foci of suppuration and caseation in various 
internal organs, particularly the lungs and the liver. Subcutaneous 
injection is followed by enlargement and caseation or suppuration 
of the lymph-glands, with fatal termination in from fifteen to 
twenty-eight days. Particularly characteristic is the develop- 
ment of orchitis in male guinea-pigs following intraperitoneal 
injection. This resembles in many ways that following the injec- 
tion of glanders bacilli. 
Mice are even more susceptible than guinea-pigs. They suc- 
cumb to ingestion of the organism in from two to four weeks. 
Rabbits have about the same degree of susceptibility as guinea- 
pigs. 
This organism has most frequently been reported as the cause 
of ovine caseous lymphadenitis, but it is probably identical with 
organisms isolated from similar lesions in cattle and from equine 
ulcerative lymphangitis. 
The disease in sheep is found chiefly in breeding ewes. It pro- 
gresses slowly, and is frequently not recognized until the animal 
is slaughtered. The lymphatics are usually affected. The glands 
enlarge, caseate, and are often encapsulated. In more advanced 
cases the various internal organs are also infected, nodules some- 
whatresembling those of tuberculosis appearing in the lungs, spleen, 
liver, and kidneys. The disease is not commonly found in young 
animals, probably because the lesions have not had time to develop 
sufficiently to cause enlargement of the glands noticeable on inspec- 
tion. 
The disease as described in the horse is an ulcerative lymphan- 
gitis, the subcutaneous lymph-nodes being chiefly affected. These 
enlarge and break through to the surface, producing a condition 
which may readily be mistaken for farcy. Involvement of deeper 
glands and of the internal organs may occur later in the progress 
of the infection. 366 
VETERINARY BACTERIOLOGY 
Bacteriologic Diagnosis.-Because of the character of the lesions 
produced the disease may be confused with tuberculosis in the 
sheep or true farcy in the horse. Smears from the lesions in sheep 
show the Gram-positive, non-acid-fast organism which is very 
distinct from the tubercle bacillus. The glanders bacillus is Gram- 
negative; a mount of pus from equine lymphangitis stained by 
Gram's method should show the characteristic organism. 
Care in the differential diagnosis of farcy based upon the ability 
of the glanders bacillus to produce an acute orchitis in the male 
guinea-pig should be used, for this reaction is quite as characteristic 
of Corynebaderium pseudotuberculosis. Pure cultures secured are 
readily differentiated both morphologically and culturally. 
The Coryn, pseudotuberculosis resembles the diphtheria bacillus 
morphologically in many ways, but may be easily distinguished by 
annual inoculation and sugar fermentation reactions. 
Immunity.-This organism produces a true toxin when grown 
in broth culture. This reaches a sufficient concentration so that 
1 c.c. of the culture filtrate will kill a guinea-pig in twenty-four 
hours. Rabbits are likewise susceptible, but mice, cats, and dogs 
are more refractory. Intravenous injection of sheep with 10 c.c. 
is fatal within eighteen hours. The toxin resembles that of the 
diphtheria bacillus in many respects. It is readily destroyed by 
heat. An antitoxic horse serum has been produced which will pro- 
tect sheep or laboratory animals against injection of the toxin. 
However, these animals are not protected against infection with 
the organism when introduced. This antitoxic immunity is, 
therefore, of little practical significance. It is also claimed that 
diphtheria antitoxin will at least in part neutralize the toxin of the 
Coryn, pseudotuberculosis. Carre found that sheep that had re- 
covered from infection were not affected by injection of the toxin. 
This investigator also developed a method of vaccinating lambs 
against the disease by means of two vaccines of different degrees of 
virulence. While he claimed this to be successful, it has never come 
into use. 
Transmission.-In many cases the disease foci are closed and 
the bacteria are not eliminated from the body. It is probable it 
usually takes place as the result of ingestion, or possibly the organ- 
ism may gain entrance through skin abrasions. DIPHTHERIA-PSEUDOTUBERCULOSIS GROUP 
367 
Corynebacterium diphtherias 
Synonyms.-Klebs-Loffler bacillus; Bacillus diphtheria; Bac- 
terium diphtheria; Mycobacterium diphtheria. 
Disease Produced.-Diphtheria in man, rarely in some animals. 
Historical.-Klebs, in 1883, described an organism present in 
the false membrane of diphtheria, which Loffler, in 1884, secured in 
pure culture and showed to be pathogenic. A similar organism was 
isolated by him from a healthy child, so that he was reluctant to 
conclude that he had found the true cause of the disease. Roux 
and Yersin, in 1888-1890, showed that the various pathologic con- 
Fig. 137.--Bacillus diphtheria (Fpstein in Journal of Infectious Diseases). 
ditions most characteristic of diphtheria could be duplicated in 
animals by injection of the broth filtrate containing the toxin. 
Distribution.-Occurs in epidemics, particularly among the 
young, in Europe and America. 
Morphology and Staining.-C orynebacterium diphtheria is 
closely related to some of the higher bacteria or even the 
fungi. It stains readily with the common anilin dyes and 
is Gram-positive. When stained with methylene-blue a smear, 
prepared directly from an infected mucous membrane, will show 
rods varying from 0.4 to 1 m in diameter and 1.5 to 3.5 /z in length, 368 
VETERINARY BACTERIOLOGY 
frequently slightly curved, sometimes pointed or club shaped, some- 
times staining uniformly, but usually containing metachromatic 
granules, which stain more deeply than the remainder of the cell, 
and give a barred or granular appearance to the cell contents. 
These same variations may be observed in the organism taken 
from suitable culture-media, particularly Lbffler's blood-serum. 
Occasionally branched forms may be observed. Demmy has 
shown that the diphtheria bacillus varies almost from hour to hour 
in its morphology when grown upon blood-serum. In five hours 
after the culture is made the cells take the stain uniformly; in 
eight hours some cells show vacuolization; in twelve hours the or- 
Fig. 138.-Corynebacterium diphtherias, Wesbrook's types: a, c, d, Granular 
types; a1, c1, d1, barred types; a2, c2, d2, solid types (X 1500) (McFarland). 
ganism is larger and stains unevenly, and within forty-eight hours 
irregular and clubbed forms are abundant. Wesbrook has con- 
structed a chart which is in common use in laboratories in the 
designation of the different types of bacilli observed. Upon media, 
where growth occurs more slowly than upon serum, the organism 
remains smaller and stains more uniformly. Whether or not the 
morphologic types of Wesbrook represent true varieties or differ 
in their pathogenic properties is a matter of dispute. Wesbrook 
claims the types which develop rapidly upon blood-serum and 
show distinct granulation are virulent, while the slower-growing, 
solid types are relatively non-virulent. It is claimed by others DIPHTHERIA-PSEUDOTUBERCULOSIS GROUP 
369 
that the latter simply represent pseudodiphtheroids. No spores 
or capsules are produced. The organism is non-motile. 
Isolation and Culture.-Diphtheria bacillus may usually be iso- 
lated directly from the throat of a diphtheritic patient in pure 
culture upon agar or, better, upon blood-serum. In mixed infec- 
tions glycerin-agar plates may be poured from the growth on 
blood-serum. It grows well upon most of the laboratory media. 
Upon Loffler's blood-serum distinct colonies are sometimes visible 
within twelve hours as minute, pin-point, translucent dots; these 
enlarge, and within twenty- 
four hours show as small, 
opaque, gray colonies, usu- 
ally discrete. The organ- 
ism develops somewhat less 
luxuriantly upon agar and 
gelatin, although repeated 
transfers tend to increase 
the luxuriance. Growth 
occurs in milk, with but 
little or no observable 
change in the medium. 
Broth may be clouded. A 
delicate film or pellicle 
forms on the surface after 
a time, and if transfers of 
this are made to fresh 
broth, the growth may be largely confined to pellicle production. 
Advantage is taken of this fact in growing the organisms in pro- 
duction of diphtheria toxin. 
Physiology.-This organism is aerobic. Upon culture-media it 
will remain alive for long periods. Desiccation of a diphtheritic 
membrane does not necessarily destroy the bacillus, and it has 
been isolated from such after a period of months. The heat of 
pasteurization, 60° for thirty minutes, destroys it. In the dried 
condition it is more resistant. Dextrose is fermented by virulent 
forms in general, with the production of acid, but no gas. Glycerin 
and dextrin are also fermented, but not saccharose or lactose. 
The non-virulent types are believed to show less power of acid 
Fig. 139.-Corynebacterium diphtherias, 
mount from blood-serum showing the 
characteristic metachromatic granules 
(Frankel and Pfeiffer). 370 
VETERINARY BACTERIOLOGY 
production. Nitrates are reduced to nitrites. No indol is pro- 
duced. Proteolytic enzymes are not formed; gelatin is not lique- 
fied. 
Pathogenesis.-Mechanism of Disease Production.-Diphtheria 
is one of the best examples of a toxemia, as the organism remains 
upon the surface of the mucous membranes and in the necrotic 
tissues, rarely, if ever, entering the blood-stream, and brings about 
the characteristic lesions of the disease by means of the toxins which 
it produces. These are absorbed into the blood and bring about 
changes in many of the organs of the body. Death is sometimes 
due directly to asphyxiation by the false membranes occluding the 
air-nassa^es. 
Experimental Evidence 
of Pathogenesis.-The typ- 
ical diphtheritic inflamma- 
tion of the mucous mem- 
branes may be reproduced 
in young animals by direct 
inoculation in the trachea, 
and in older animals as well 
if the membranes are in- 
jured previously. The his- 
tologic lesions of the body 
organs can be produced by 
injections of the toxin of 
the organism. Paralysis 
due to toxin-poisoning may 
be developed under certain conditions. That this organism is 
the specific cause of diphtheria is, therefore, definitely estab- 
lished. 
Character of Disease and Lesions Produced.-The pharynx is 
most commonly affected; the larynx and the nasal mucous mem- 
branes are sometimes involved. There may also be diphtheritic 
conjunctivitis, diphtheritic infections of the middle ear and of the 
mucous membranes of the genital organs. The organisms remain 
localized, rarely entering the blood-stream, cause necrosis and 
degeneration of the epithelial cells and the deeper tissues as well. 
Blood-serum and fibrin are exuded from the vessels and, together 
Fig. 140.-Corynebacterium diphtherias, 
twenty-four-hour colony on agar (X 100) 
(Frankel and Pfeiffer). DIPHTHERIA-PSEUDOTUBERCULOSIS GROUP 
371 
with fragments of the necrosed tissue, form the diphtheritic mem- 
brane. Acute interstitial nephritis, fatty degeneration of the 
myocardium, and sometimes of the nerve tissues are due to the ab- 
sorption of the toxins. There is no constant ratio between the 
extent of the diphtheritic process in the throat or elsewhere and 
the severity of the symptoms and lesions produced by the toxin. 
Immunity.-The diphtheria bacillus, according to Ehrlich, 
produces two types of poison-the true toxin and the toxone. 
The first is responsible for the acute symptoms of poisoning, the 
latter for the paralysis that frequently occurs during convalescence. 
The haptophores of both toxin and toxone are believed to be 
identical, and to be naturalized by the same antitoxin. A con- 
sideration of the production of diphtheria antitoxin has been taken 
up in the chapter on Toxins and Antitoxins, under the heading of 
Immunity. Agglutinins may develop in the blood, but are not 
constant, and are of no practical value in diagnosis. They may be 
produced in experimental animals by the injection of killed cultures 
of the diphtheria bacillus. Precipitins have likewise been produced 
by artificial immunization, but usually cannot be demonstrated in 
the blood of infected individuals. Bactericidal sera may be pre- 
pared by repeated injections of killed, washed cultures of diphtheria 
bacilli, and favorable results have been reported in the use of such a 
serum in freeing the throat of convalescents from the bacteria. 
Its clinical value can scarcely be said to be proved. Opsonins have 
not been demonstrated. 
The value of diphtheria antitoxin for prophylactic and cura- 
tive injections is well established. It has resulted in materially 
lessening mortality whenever it has been used. As much as 100,000 
units have been injected in some cases, but the usual curative dose 
is 8000 to 15,000 units. 
Bacteriologic Diagnosis.-Sterile swabs are used to swab out 
the throat and nose of the suspect, and are smeared over the sur- 
face of blood-serum. Mounts made in five to eighteen hours 
thereafter, stained with Lbffler's methylene-blue, should show the 
characteristic organism with its metachromatic granules. Diag- 
nosis may sometimes be made directly from smears taken from the 
throat. 
Transmission.-The organism doubtless sometimes gains en- 372 
VETERINARY BACTERIOLOGY 
trance into the body by the inhalation of infective droplets, but 
more commonly by the use of common drinking vessels and 
through fomites. 
Corynebacterium hoffmanni 
Synonyms.-Bacillus pseudodiphthericus; Bacterium pseudo- 
diphthericum; Hoffmann's bacillus; Bacillus clavatus, and Coryne- 
bacterium pseudodiphthericum, Bacillus hoffmanni. 
This organism is non-pathogenic and of importance only 
because of the possibility of confusion with the preceding forms. 
It has been isolated many times from the throats of normal and 
even in some cases from diphtheria patients. The principal 
points of difference are: 
1. The cells are, on the average, shorter and thicker than those 
of the diphtheria bacillus; they are usually straight and somewhat 
enlarged at one or both ends. 
2. They stain more uniformly, sometimes showing transverse 
bands, rarely more than one or two in number. 
3. Neisser's stain does not reveal polar granules. 
4. The organism grows more luxuriantly on culture-media 
than Coryn, diphtherias. In broth there is more of a tendency to 
clouding and less to film formation. 
5. In Hiss serum-water Coryn, hoffmanni produces acid from 
none of the sugars. 
6. No toxins are produced, and the organism is non-patho- 
genic to animals. 
Corynebacterium xerosis 
This organism is the cause of a type of conjunctivitis in man 
known as xerosis. It is of interest here because of its close re- 
semblance to the diphtheria bacillus. The principal difference 
is the ability of Coryn, xerosis to produce acid from saccharose, 
but not from dextrin, while Coryn, diphtheria produces acid from 
dextrin and not from saccharose. This organism does not pro- 
duce toxin and is not pathogenic for the laboratory animals. CHAPTER XXXII 
SWINE ERYSIPELAS GROUP. THE GENUS ERYSIPELOTHRIX 
Two organisms have been described as belonging to this 
group-the Erysipelothrix rhusiopathice and E. muriseptica. As 
will be noted later, there is good reason to believe that these 
organisms are identical. The group may be characterized as 
made up of very minute, slender, non-motile, non-spore-produc- 
ing, Gram-positive rods. 
Erysipelothrix rhusiopathiae 
Synonyms.-Bacillus rhusiopathice suis; B. erysipelatis suis; 
Erysipelothrix porci; Bacillus rhusiopathice. 
Disease Produced.-Swine erysipelas, red fever of swine, 
rouget, Rotlauf. 
Loftier, in 1885, first described the organism present in swine 
erysipelas. The disease had previously been differentiated from 
anthrax by Pasteur and Thuiller. 
Distribution.-The disease has been reported from most of the 
European countries, and an organism resembling the Erysipelo- 
thrix rhusiopathice has several times been reported from the 
United States, although there has been no satisfactory demon- 
stration of the presence of the disease in this country. In Europe 
the disease is of considerable economic importance. 
Morphology and Staining.-Erysipelothrix rhusiopathice is a 
slender rod, variously given as 0.2 to 0.4 by 1 to 2 m, as it occurs in 
the blood is usually straight, but sometimes somewhat curved, 
single or in chains. In culture-media the cells are more variable 
and may be filamentous. It is non-motile, does not produce 
spores or capsules. It stains readily with the anilin dyes and is 
Gram-positive. 
Isolation and Culture.-The organism may be secured in pure 
culture by plating the blood or pulp from some of the internal 
373 374 
VETERINARY BACTERIOLOGY 
organs, the spleen in particular. The colonies in the gelatin 
plate appear on the second or third day, and are quite character- 
istic. Each colony is found to be surrounded by a zone of much- 
branched threads. They permeate the medium, and are not found 
upon the surface, as is the case with anthrax and other forms which 
produce colonies with filamentous edges. 
Gelatin stab cultures develop only below the surface of the 
medium, showing the micro-aerophilic or semi-aerophilic growth 
characters of the organism. The 
mature stab has the appearance of a 
test-tube brush, streaks and disks 
of growth radiating from the center. 
Streak cultures do not develop well 
upon agar or blood-serum except by 
growth under anaerobic conditions, 
preferably by absorption of the 
oxygen by the alkaline pyrogallate 
method. Bouillon is clouded and 
produces a grayish-white sediment 
with no pellicle. Ordinarily no 
growth occurs upon potatoes even 
under anaerobic conditions. 
Physiology.-The E. rhusiopathice 
grows better anaerobically than aero- 
robically. It is unusually resistant 
for a non-spore-producing form. Des- 
iccation frequently fails to destroy the organism in several weeks. 
The thermal death-point is recorded by some authors as low as 52° 
for fifteen minutes, by others as high as 70°. The optimum growth 
temperature is 37°, but growth occurs well at room-temperatures. 
Gelatin is not completely liquefied, but generally softened. 
Pathogenesis.-Experimental Evidence.-Mice die of septicemia 
when inoculated with pure cultures. Death occurs usually within 
four days, frequently within forty-eight hours. Field-mice are 
immune, as are guinea-pigs, cattle, horses, and other equines, 
dogs, cats, chickens, and geese. Rabbits inoculated subcuta- 
neously develop an edema and redness at the point of inocula- 
tion, and this erysipelas-like lesion spreads to other parts of 
the body and the animal dies. Intravenous injection is quickly 
F i g. 141. - Erysipelothrix 
rhusiopathice, stab culture in 
gelatin-four days (Frankel 
and Pfeiffer). SWINE ERYSIPELAS GROUP. THE GENUS ERYSIPELOTHRIX 
375 
fatal through the development of a septicemia. The white rat, 
the sparrow, and the pigeon are likewise susceptible. The typi- 
cal disease may be produced in swine by inoculation of pure cul- 
tures. There is no doubt but that the Erysipelothrix rhusiopathice 
is the cause of the disease. There is some evidence that the infec- 
tion in a mild urticarial form is occasionally transmitted to the 
human. 
Character of Disease and Lesions Produced.-Small numbers of 
bacteria can generally be demonstrated in the blood, and abun- 
dantly in various of the body organs, particularly the spleen 
and the lungs. In the acute type of the disease the mucous mem- 
brane of the alimentary tract is reddened and shows petechial 
hemorrhages. The spleen and the lymph-nodes are somewhat 
enlarged and the lungs are usually congested. The most char- 
acteristic lesions are those of the skin, where numerous congested 
areas and local hemorrhages give rise to the spotted appear- 
ance. In chronic cases there is generally a verrucose or ulcerous 
endocarditis which is quite characteristic. 
Immunity.-No true toxin has been demonstrated for the Ery- 
sipelothrix rhusiopathice. The presence of specific amboceptors in 
immune serum may be shown by the method of complement 
absorption, but just what part these play in immunity has not 
been satisfactorily demonstrated. Opsonins are present and prob- 
ably account in part for the immunity. 
Very young animals-under three months-and those over 
one year rarely contract the disease. Animals that recover from 
the disease are thereafter immune. 
Active Immunization -It has been shown by the researches of 
Pasteur, later by Kitt and others, that passage of strains through 
certain animals, particularly the rabbit or the pigeon, leads to an 
attenuation such that the material may be used for vaccination 
of the hog. It is also known that continued cultivation usually 
reduces the virulence of the organism for mice. The virulence 
is subject to great and inexplicable variations. The Pasteur 
vaccine consists of an attenuated bouillon culture of the E. rhusio- 
pathice. The material is sent out in two packages, labeled Vaccine 
land Vaccine II. The contents are injected fifteen daysapart. Im- 
munity is established in two to three weeks after the second injeC- 376 
VETERINARY BACTERIOLOGY 
tion. The injected animals sometimes show the characteristic 
urticaria of the disease. The use of this method has led to varied 
results in different countries. Voges and Schutz have modified 
the Pasteur vaccine method by doing away with the Vaccine II, 
as they found the blood still contained bacilli at the time of the 
second injection, and concluded the latter, therefore, useless. 
It seems that a satisfactory immunization of the hog against the 
disease cannot be accomplished by injections of the killed organ- 
isms. 
Passive Immunization.-Emmerlich and Mastbaum, in 1891, 
described a method of preparing an immune serum which would 
protect animals against the disease. Their method was impractical 
in some respects, and was superseded by the method of Lorenz, and 
this by those of Leclainche, Voges and Schutz, and Lange. The 
antiserum is prepared by the intravenous injection of virulent bouil- 
lon cultures into the horse. Usually 100 c.c. constitutes the initial 
injection, and is followed at intervals by larger amounts-up to 
500 c.c. The injection produces a rise in temperature, and other 
reactions that disappear in twenty-four to forty-eight hours. The 
immune blood is drawn, and the serum used for passive immuniza- 
tion of swine. It is prepared on a large scale in several institutes 
in Europe, and is used extensively. Cattle and even buffalo are 
sometimes used instead of the horse in the preparation of the serum. 
Prettner claims that the use of cattle immune serum confers a more 
lasting immunity than that of the horse. Schreuber and Schubert 
studied the question of multiplicity of amboceptors, and came to 
the conclusion that a mixture of immune sera from horses and 
cattle would give a greater variety of amboceptors specific for the 
organism. A mixture of this kind is termed "double serum" 
(German, Rotlauf Doppelserum). The double serum has not 
proved in practice to be of any greater value than the serum from 
the horse alone. The immune serum is usually standardized by 
the use of the mouse, commonly by the method of Lorenz. A 
serum suitable for use should immunize a mouse in doses of 0.01 
gm. per 10 gm. of weight, against injection with 0.01 gm. of a 
virulent culture. Lorenz used mice weighing uniformly 15 gm. 
as a standard, and such a serum is said to have a titer of 0.015 gm. 
(mouse). Marx has modified the technic of Lorenz somewhat, SWINE ERYSIPELAS GROUP. THE GENUS ERYSIPELOTHRIX 
377 
but the principle is the same. Leclainche has advised the use of 
the pigeon, rather than the mouse, as a test-animal. The serum 
may be used curatively or prophylactically. Amounts up to 30 c.c. 
are used in curing the disease. In cases not too far advanced it 
arrests the disease, and has been shown to reduce the mortality 
materially. The injection of the serum prophylactically results 
in temporary immunity only, hence it is customary to establish an 
active immunity by injection of the specific organism, the culture 
and the serum being mixed together or injected separately at the 
same time. This method of immunization has proved of such 
value that it is extensively used in Europe. The active immunity 
developed as a result of the combined method lasts for periods of 
six months to a year or even longer. 
Bacteriologic Diagnosis.-Smears from the spleen, sometimes 
from the blood, will show the characteristic slender, Gram-positive 
bacillus. Isolation in gelatin plates gives a characteristic type of 
colony. The stab culture in gelatin is also diagnostic. 
Transmission.-The specific organism may be demonstrated 
in the feces of an infected individual. It may gain entrance di- 
rectly through the skin, but probably, in most instances, the infec- 
tion atrium is the alimentary tract. Typical virulent bacilli and 
non-virulent forms have been repeatedly isolated from healthy 
animals. It is evident that the interrelationships of this organism 
and the body are quite complex. 
Erysipelothrix muriseptica 
Synonym.-Bacillus murisepticus. 
Disease Produced.-Mouse septicemia. 
Koch, in 1878, called attention to the fact that the injection 
of putrid meat infusion into a mouse resulted in a septicemia due 
to a non-motile, minute rod. It is of importance chiefly because 
of its resemblance to the Erysipelothrix rhusiopathicc. It has been 
isolated from a variety of sources in nature, and has been known to 
cause epidemics in mice kept for experimental purposes. There 
are no marked cultural characters which may be used to differen- 
tiate the two organisms, and morphologically they are likewise 
very similar. It is claimed by some that the E. muriseptica is 
somewhat more slender than the E. rhusiopathice. It has been 
found possible to immunize the rabbit against the latter by injec- 378 
VETERINARY BACTERIOLOGY 
tions of the former. However, it has not been found possible to 
produce typical swine erysipelas by the injection of E. murisep- 
Fig. 142.-Erysipelothrix muriseptica, stained mount (Gunther). 
Fig. 143.-Erysipelothrix muriseptica, stab culture in gelatin (Gunther). 
tica. It is undoubtedly true that the two organisms are closely 
related. That they are varieties of one species, differing in viru- 
lence, is not improbable. CHAPTER XXXIII 
HEMOPHILIC, OR INFLUENZA GROUP. THE GENUS 
HEMOPHILUS 
The organisms belonging to this group resemble each other in 
being very small bacilli, aerobic, Gram-negative (in some cases 
decolorizing with some difficulty), non-motile, without spores, 
and capable of being cultivated only on media containing hemo- 
globin or serum (obligate paratrophic forms). 
Organisms of this group have been isolated from several dis- 
eases of man and animals, and several types have been found in 
the normal mouth and throat and in body secretions. Methods 
of differentiation of the several species described are not entirely 
satisfactory. 
The disease-producing organisms which attack animals or 
man and which require hemoglobin, serum, egg albumin or blood 
for their best growth are Hemophilus influenzae, for many years 
assumed to be the cause of influenza in man; Hem. pyogenes, 
a common cause of suppuration in swine, cattle, and occasion- 
ally other animals; Hem. pertussis, of pertussis or whooping- 
cough in man, and the Koch-Weeks bacillus of human conjuncti- 
vitis. The nonpathogenic forms are generally included under the 
head of pseudo-influenza bacilli. One of these is the Hem. 
haemoglobinophilis canis of Friedberger,1 isolated from the pre- 
putial secretion of the dog. 
Hemophilus pyogenes (Glage) 
Synonyms.-Bacillus pyogenes suis, Grips; B. pyogenes bovis, 
Kiinnemann. Bacillus pyogenes. 
Disease Produced.-Polyarthritis, pyobacillosis in swine and 
cattle, rarely in sheep, goats, and other animals. 
Historical.-This organism was first described byPoelsin 1897. 
Grips,2 in 1898, named an organism which he isolated from 
1Cent. f. Bakt. 1. Abt. Orig., 33, 1903, p. 401. 
2Zeitschr. f. Fleisch. u. Milch hygiene, 1898. 8, p. 166. 
379 380 
VETERINARY BACTERIOLOGY 
swine Bacillus pyogenes suis. Kiinnemann,1 in 1903, described a 
similar organism from cattle under the name B. pyogenes bovis. 
Ward2 (1917) has found the organism frequently in swine and 
cattle. Glage,3 in 1903, combined the two under the single 
specific name B. pyogenes. Dunkel concluded that this organism 
is identical with that which causes pseudotuberculosis of sheep 
(op v.}, but Glage and Priewe have shown them to be quite dis- 
tinct, and to be related to the influenza bacillus and members of 
the group of hemophilic organisms. 
Brown and Orantt4 state that the Hem. pyogenes is hemolytic 
but not hemoglobinophilic, though its growth is greatly favored 
by some higher protein material and regard the organism as be- 
longing to the Corynebacteria rather than to the influenza group. 
Morphology and Staining.-Hemophilus pyogenes is a relatively 
minute and slender rod, 0.2 to 3 m in length and 0.2 to 0.3 p in 
thickness. Microscopically it resembles the organisms of swine 
erysipelas and influenza, but is somewhat shorter and thicker 
than the former. The cells are sometimes bent, club-shaped, and 
tend to be somewhat polymorphic. Capsules are not produced. 
Spores are absent. The organism is non-motile. 
The cells are not as readily stained as those of many bacteria, 
though not acid fast. Carbol-fuchsin is a satisfactory stain. 
The organism is generally recorded as Gram-negative, but the 
cells decolorize so slowly that several authors regard it as Gram- 
positive. 
Cultural Characters.-The Hemophilus pyogenes resembles the 
group in refusing to grow satisfactorily except on media contain- 
ing serum hemoglobin, or in milk. Sometimes initial inocu- 
lations upon agar have been successful because enough of the 
body proteins have been carried over. 
The organism can be grown on serum agar, blood agar, or solidi- 
fied serum. In the latter medium at 37° colonies develop in from 
twenty-four to forty-eight hours. They are small, each in a 
small area of liquefied serum. The water of condensation 
remains unclouded, but develops a light sediment. Liquefaction 
1Archiv. f. wiss. u. prakt. Tierheilk, 1903, 29, p. 128. 
2 J. Bact., 1917, b, 11, 619. 
3Zeitschr. f. Fleisch. u. Milch hygiene, 1903, p. 166. 
4 J. Exp. Med., Vol. xxxii, No. 2, p. 219. HEMOPHILIC; OR INFLUENZA GROUP 
381 
of the serum continues until it is relatively complete. No putre- 
factive odor develops. Growth on blood agar is good and the 
blood is hemolyzed with small zones of hemolysis about very 
minute deep colonies. 
On serum agar small colonies with smooth edges are produced. 
Serum liquid media are not clouded, but a gray sediment col- 
lects. In milk incipient coagulation appears in forty-eight hours, 
later extending gradually from the bottom toward the top. A 
clear whey gradually separates, and in this the curd is suspended, 
later digesting in an acid medium. Casein appears to be essential 
to growth in milk, as the organism does not develop in whey. 
Potato, agar, gelatin, broth, etc., are not suited unless they 
contain serum. 
Physiology.-Hemophilus pyogenes grows best at blood-heat, 
the minimum being about 24° and the maximum 40°. The organ- 
ism retains its vitality in culutre-media for many weeks, but is 
easily killed by desiccation. It is sensitive to the action of 
antiseptics, and is easily destroyed by disinfectants. 
The organism grows either in the presence or absence of free 
oxygen, but the optimum condition is evidently a partial pres- 
sure of oxygen somewhat less than atmospheric. 
In fermented bouillon plus 10 per cent, horse serum plus 1 
per cent, of sugar, acid is produced from dextrose, saccharose- 
lactose and xylose. Indol is not formed. Nitrates, methylene- 
blue, and litmus are not reduced. 
Proteolytic and probably lab-ferments are produced. 
Pathogenesis.-The organism whether inoculated in the 
laboratory or found in practice is a typical pyogene causing chronic 
abscesses in various body organs, and occasionally a purulent 
catarrh. The pus developed in abscesses is usually creamy in 
consistency and greenish in color. Very frequently the organism 
is accompanied by other bacteria in mixed infections. Old ab- 
scess contents become caseous and dry. The pus in recent ab- 
scesses contains the organisms in great numbers. 
Rabbits are the most susceptible of laboratory animals to inoc- 
ulation. Local abscesses result from subcutaneous injections. 
Intravenous injection is followed by pyemia, usually with locali- 
zation in the joints. Intraperitoneal injection causes a purulent 
peritonitis, resulting in death in one to two weeks. Guinea-pigs 382 
VETERINARY BACTERIOLOGY 
are somewhat more refractory, but may be infected in the same 
manner. 
The organism is apparently non-pathogenic for rats. It pro- 
duces disease in the animals with divided hoofs, but not in the 
solipeds. 
Infection in swine is not infrequent, occurring in almost 
any part of the body and often taking the form of arthritis. 
Abscesses are most frequent in the neck, the thigh, and the 
mammary glands. Peritonitis, pleuritis, and pulmonary ab- 
scesses may occur, occasionally a purulent pneumonia. In the 
resultant pyemia the abdominal organs, the lymph-glands, and the 
joints may develop abscesses. In infection by Hemophilus pyo- 
genes, unlike tuberculosis, the lesions in lymph-nodes are relatively 
infrequent. Glage has contended that this organism may be the 
primary cause of swine plague, and has called attention to the re- 
semblance between this disease and its causal organism to the dis- 
ease influenza and its causal organism in man. Ostertag and others 
do not accept this interpretation, but regard the organism as a 
secondary invader. 
In cattle Kiinnemann found 38 infections with Hemophilus pyo- 
genes out of 56 suppurative inflammations, but only 15 times in 
pure culture. It has been found in peritonitis, pyemia, muscular 
abscesses, in wounds, pyelonephritis, mastitis, and polyarthritis 
following navel infection. The organism is far more pathogenic for 
young animals than for old. 
Chronic catarrhal mastitis and bronchopneumonia in cattle 
may also be caused by this organism. Concerning the former 
Glage states: "There develops either a purulent catarrhal mastitis, 
or abscesses varying from a walnut to an apple in size develop in 
the udder tissue, and are firmly encapsulated. The milk is at first 
watery and slimy, then purulent or bloody, viscous, greenish or 
gray, and often malodorous. Microscopically enormous numbers 
of small bacilli may be demonstrated. Ostertag and Weichel 
found this organism in 90 per cent, in pure culture, and mixed with 
colon bacilli and streptococci in the remaining 10 per cent." 
Whether or not this organism is the primary cause of certain 
types of bronchopneumonia in calves is unsettled, but it is un- 
doubtedly of importance, at least as a secondary invader. HEMOPHILIC, OR INFLUENZA GROUP 
383 
According to Olt, Hemophilus pyogenes may infect sheep and 
goats, with production of abscesses, pleuritis, pneumonia, pyemia, 
and mastitis. The organism should not be confused here with 
the more common pyogenic bacillus Coryn, pseudotuberculosis. 
Immunity.-Toxins apparently have not been demonstrated. 
Antisera containing agglutinins and bactericidal substances 
specific for this organism can be secured by the immunization of 
rabbits, calves, or dogs. The latter are preferable as abscesses 
do not develop in this animal as a result of the injection of the 
organisms. An antiserum for cure has been experimentally pre- 
pared, but has not come into use. Bacterins have been used by 
Ostertag and Weichel on goats, with some measure of success. 
However, in general feeding and subcutaneous injection of killed 
or living bacilli do not give rise to any considerable amount of 
immunity. Practical methods of immunization have not been 
worked out. 
Transmission.-Infection in many cases is traumatic. It is 
probable that the disease may be transmitted to calves or swine 
by milk from infected udders. 
Bacteriologic Diagnosis.-No accurate means of diagnosing 
infection with Hemophilus pyogenes has been developed other than 
isolation and recognition of the organism. The isolation of a 
small organism, which grows only upon media containing serum 
or, preferably, blood, from a' characteristic lesion would usually 
be sufficient. Attention should also be called to its low degree of 
pathogenicity for laboratory animals, and the presence of the 
organism in great numbers in stained mounts of the characteristic 
pus. 
Hemophilus influenzas 
Synonym.-Bacillus of Pfeiffer; Bacillus influenzae. 
Disease Produced.-Influenza in man (probably as a second- 
ary invader). 
The organism usually associated with influenza in man is 
of interest as the first bacterium of the hemophile group culti- 
vated. The bacillus is very minute, 0.2 to 0.3 by 0.5 m. It does 
not stain readily, is Gram-negative, and occasionally shows some 
tendency to bipolar staining. It grows only upon media con- 384 
VETERINARY BACTERIOLOGY 
taining hemoglobin and only at blood-heat. Studies of influenza 
during the epidemic of 1917-1918 seem to indicate that this 
organism is not the primary cause of the disease. 
Hemophilus pertussis 
Synonyms.-Keuchhusten bacillus, microbe de coqueluche. 
Bacillus pertussis. 
Disease Produced.-Whooping-cough in man. 
The organism culturally and morphologically resembles the 
influenza bacillus, though somewhat larger and plumper, and 
frequently showing a tendency toward bipolar staining. It may 
be cultivated upon blood agar. The disease is primarily an infec- 
tion of man only. \ CHAPTER XXXIV 
ACID-FAST GROUP. THE GENUS MYCOBACTERIUM 
The Mycobacterium tuberculosis, M. leprce, M. paratuberculosis, 
and certain common non-pathogenic bacteria isolated from hay, 
dung, milk, butter, and other sources, have common morphologic 
and staining characters which associate them as the acid-fast 
group. The crucial test for these forms is their ability to retain 
stains when treated with strong acids. They do not stain readily 
with the common anilin dyes, but when once stained they are 
"acid fast." Hot carbol-fuchsin is commonly used in determin- 
ing this character. 
The members of the acid-fast group of bacteria are all slender, 
non-motile rods, without capsules or spores, Gram-positive, and 
acid fast. Slight variations in morphology and cultural characters 
and considerable differences in pathogenesis serve to differentiate 
the various species. All the species may occasionally produce 
branched cells and threads, and it has been concluded by some in- 
vestigators that they belong rather with the fungi, or at least with 
the higher bacteria than with the lower bacteria. 
Care should be used not to confuse the term pseudotuberculosis, 
and particularly the bacteria associated with that disease, with the 
true tubercle bacilli and related forms. As has before been stated, 
the term pseudotuberculosis is purely pathologic, and refers to 
the type of lesions produced in the body, and not to any resem- 
blance of the organisms causing the disease to those of tuberculosis. 
The pseudotuberculosis bacilli are more closely related to the diph- 
theria group, and have already been discussed. 
Mycobacterium tuberculosis 
Synonyms.-Bacterium tuberculosis; Bacillus tuberculosis. 
Disease Produced.-Tuberculosis in mammals and birds. 
The disease in its various forms in man and animals has been 
known since ancient times. Villeman, in 1865, showed that the 
385 386 
VETERINARY BACTERIOLOGY 
disease could be transferred from one animal to another by injec- 
tion of the crushed nodules. He did not succeed in discovering the 
specific organism, however. In 1884 Dr. Robert Koch presented 
the results of his studies of the disease and described the organism 
which he had proved to be its etiologic factor. This work will 
always stand as a model of scientific research in bacteriology 
carried out under the most difficult circumstances. Staining 
methods and culture-media were both devised before the organism 
could be seen or grown. Koch succeeded in demonstrating its 
presence in the characteristic lesions, in growing it upon artificial 
media, and in reproducing the disease in animals by the inocula- 
tion of pure cultures. It was assumed in the beginning that tuber- 
culosis in all animals was caused by the same organism, but in 1896 
Theobald Smith called attention to the fact that certain differ- 
ences in cultural, morphologic, and pathogenic characters could 
be distinguished in the organisms isolated from human and bovine 
tuberculosis. Koch, in 1901, declared the two organisms to be 
distinct, and that the probability of human infection with bovine 
tuberculosis was remote indeed. Since that time the subject has 
been studied with varied results by many investigators. Prob- 
ably more has been written on this one disease than any ten or 
more other diseases. Journals devoted exclusively to tuberculosis 
are issued. Many points even yet are not fully understood, but 
the subject is upon a moderately sound basis at present. It seems 
best to differentiate three varieties of the tubercle bacillus-the 
human, the bovine, and the avian. A fourth type including forms 
isolated from cold-blooded animals may also be added. These 
possess many points in common, -and it is by no means certain 
that they cannot be transformed the one into the other, but 
they may, in general, be readily differentiated by specific 
morphologic and physiologic characters. They will be discussed 
together. 
Distribution.-Tuberculosis in man is known throughout the 
civilized world. In the United States over 110,000 deaths occur 
annually from this disease. It usually leads all other diseases in 
total number of deaths produced. The disease in cattle is nearly 
as widely distributed. It is found throughout Europe and America. 
Isolated localities free from the disease, however, are known. ACID-FAST GROUP. THE GENUS MYCOBACTERIUM 
387 
In the United States it is rarely encountered on the western ranges, 
but is relatively common in dairy and beef herds in other parts 
of the country. Whenever swine are allowed to follow tubercu- 
lous cattle they commonly become infected. The same is also 
true when they are fed upon unpasteurized milk from tuberculous 
animals. Tuberculosis occurs rarely in the horse. Sheep have 
been reported as tuberculous, but the disease is certainly rare; it 
is probably frequently confused with pseudotuberculosis or caseous 
lymphadenitis in this animal. Tuberculosis in domestic fowls is 
known to occur in many European localities, and in the United 
States has been reported from many states. 
Fig. 144.-Mycobacterium tubercu- Fig. 145.-Mycobacterium tubercu- 
losis in human sputum. Note the losis, human, mount from glycerin 
slender beaded character of the rods agar (Frankel and Pfeiffer). 
(X 1000) (Gunther). 
Morphology and Staining.-Mycobacterium tuberculosis is a 
slender rod, commonly somewhat bent, with rounded ends. It 
varies from 0.2 to 0.5 by 1.5 to 3.5 m, sometimes longer. Fre- 
quently the protoplasm takes the stain irregularly and gives a 
beaded appearance to the cell. No spores or capsules are pro- 
duced. The organism is non-motile. Branched and elongated 
forms resembling somewhat the actinomyces are sometimes 
observed. It is possible that these are involution forms, although 
many authors claim them to be developmental forms instead. 
The organism stains with difficulty, but when once stained 
is acid fast. It is possible that under certain conditions, 388 
VETERINARY BACTERIOLOGY 
in the animal tissues in particular, this acid-fast property 
may be temporarily lost. In very young cultures the bac- 
teria are sometimes not acid fast. In tissues and cultures con- 
taining tubercle bacilli that do not show the acid-fast character 
Much has demonstrated Gram-positive granules, which are prob- 
ably a growth stage of the tubercle bacillus. The acid-fast charac- 
ter is apparently due to the presence of a wax-like substance in the 
bacterial cell. The cells from which this has been removed by 
ether and benzol are no longer 
acid fast. 
Certain observers have 
claimed that there are cer- 
tain morphologic differences 
commonly to be observed 
between bovine and human 
tubercle bacilli. They have 
stated that the former are 
shorter, straighter, and 
thicker than the latter, and 
are less apt to show the ir- 
regular or granular staining 
noted above. These charac- 
ters are, of course, not suffi- 
cient to differentiate isolated 
bacteria of the two types, but cultures can sometimes be identified 
by an experienced observer. Whether or not one type may be 
transformed into the other type by animal inoculation or by 
cultural methods is questionable. Some investigators claim to 
have isolated typical human bacilli from animals that have been 
inoculated with bovine bacilli; others hold that there is no evidence 
of the transformation of the one type to the other. However this 
may ultimately be decided, it is certain that each type retains its 
characters with a considerable degree of constancy. The existence 
of two well-marked varieties is unquestioned. The bacillus 
of avian tuberculosis resembles the bovine type closely in its 
morphology and staining reactions. 
Isolation.-The isolation of Mycobacterium tuberculosis from 
lesions is attended with considerable difficulty. This is even 
Fig. 146.-Mycobacterium tuberculosis, 
bovine, in a section of the peritoneum 
(Frankel and Pfeiffer). ACID-FAST GROUP. THE GENUS MYCOBACTERIUM 
389 
more pronounced when an attempt is made to secure the organ- 
ism from the sputum or the feces where it exists in mixed culture. 
It may be isolated from infected organs by securing bits of the 
tissue and rubbing over the surface of inspissated blood-serum or 
other suitable medium. The method worked out by Theobald 
Smith has given excellent results in the hands of numerous in- 
vestigators. A dog is bled, using all aseptic precautions, from the 
femoral artery into a sterile vessel and the blood allowed to clot. 
The serum is removed by sterile pipettes to sterile test-tubes. 
These are slanted and heated to a temperature of 75° to 76° for 
about three hours, or until the serum is coagulated. The heating 
must be done in a saturated atmosphere and the medium stored so 
that there is no loss by evaporation. Bits of infected tissue are 
placed upon the surface and kept in a thermostat for several weeks. 
If no growth appears, the tissue is moved about and incubated 
again. A constant temperature of 37° and a saturated atmosphere 
must be maintained. 
A procedure somewhat simpler than the preceding has been 
described by Dorset and is found to give good results. The shell 
of fresh eggs is carefully broken, and the white and yolk dropped 
into a sterile flask, the yolk broken with a sterile rod or wire, and 
the contents of the flask shaken until the two are thoroughly mixed. 
Foaming is to be avoided. The mixture is placed in tubes, slanted, 
and heated at a temperature of about 70° for from four to five 
hours on two days. This coagulates and sterilizes the medium. 
The tubes should be stored where they will not lose water by 
evaporation. Several drops of distilled sterile water should be 
added to a tube just before inoculation. The isolation upon 
this medium is carried out as outlined above. A growth may 
generally be observed within ten days after inoculation with 
fresh tissue. 
Isolations from sputum or feces, milk, or other substances in 
which the organisms occur mixed with other forms, is attended with 
some difficulty. It is usually accomplished by injecting the mate- 
rial or the sediment yielded by centrifugation directly into a guinea- 
pig. The bacilli may later be isolated in pure culture from the 
nodules produced. Within recent years the use of "antiformin" 
and similar substances has considerably simplified this procedure. 390 
VETERINARY BACTERIOLOGY 
Antiformin is the trade-name given a disinfectant mixture having 
the following composition: 
Solution I. Sodium carbonate 12 gm. 
Chlorinated lime 8 gm. 
Distilled water 80 gm. 
Solution II. Sodium hydroxid 15 gm. 
Distilled water -. 85 gm. 
Equal quantities of the two solutions are mixed for use. The 
sputum or other material containing the tubercle organisms is 
placed in a centrifuge tube, and antiformin to about 20 per cent, 
of its bulk added. The tube is then corked, thoroughly shaken, 
and allowed to remain in a dark place for twenty-four hours. It is 
then centrifuged, the clear, supernatant liquid pipetted off, the 
tube filled with sterile physiologic salt solution, centrifuged, washed 
a second time, and the sediment smeared over the surface of 
serum slants. The antiformin destroys all other non-acid-fast 
bacteria present and dissolves the mucus and most of the cell 
elements, but when properly used seems to have little effect upon 
the tubercle bacilli, as they retain their vitality unimpaired. 
This is probably because of their chemical composition and waxy 
covering. 
Cultural Characters.-Mycobacterium tuberculosis does not 
grow readily when first isolated upon culture-media, and will 
grow only upon certain substances. After a few transfers it 
seems to become habituated to growth under these conditions and 
will develop through a much greater range of temperature and on 
other media. Development occurs best on media containing 
blood-serum, egg, or similar proteins, or to which glycerin has 
been added. 
The colonies upon blood-serum or glycerin agar appear in the 
course of ten days or two weeks as tiny grains barely visible to the 
naked eye. They gradually enlarge, and in subcultures may be- 
come confluent and cover the surface of the medium with a dry, 
rather'mealy, wrinkled growth; the colonies direct from lesions do 
not coalesce usually. The growth is white and lusterless, or rarely 
in old cultures cream or brown. In glycerin bouillon the growth 
generally occurs as a more or less continuous, heavy, wrinkled, 
white pellicle that breaks into pieces and sinks to the bottom when 
the medium is shaken. Similar growths occur upon other media 
which contain glycerin. In no other case is the growth so rapid. ACID-FAST GROUP. THE GENUS MYCOBACTERIUM 
391 
Cultural characters which may be used in the certain differen- 
tiation of the human, bovine, and avian tubercle bacilli are usually 
quite marked. The organisms isolated 
from the human and from the bird adapt 
themselves much more readily to arti- 
ficial media and grow more luxuriantly 
than does the bovine type. The former 
produce upon glycerin agar a relatively 
heavy wrinkled growth, the latter a 
much more delicate growth, frequently 
showing discrete colonies only. 
Physiology.-The tubercle bacillus 
is aerobic. Its optimum growth tempera- 
ture is about 37.5° for the human and 
the bovine types and somewhat higher 
for the avian. The growth temperature 
range is relatively narrow, no growth usu- 
ally being secured below 30° or above 42°. 
The thermal death-point is 60° for 
twenty minutes. This is, therefore, 
the minimum time and temperature for 
the efficient pasteurization of milk. Dry 
heat even at 100° does not kill the 
organism certainly within an hour. Sun- 
light destroys the organism quickly, but 
it is moderately resistant to desiccation. 
When dried in sputum cells have been 
known to live for at least two months. 
Most of the physiologic characters, such 
as acid, gas, pigment, and indol produc- 
tion, are negative. 
Theobald Smith has called attention 
to what appears to- be a very constant 
differential character between human 
and bovine tubercle bacilli. He found 
that in glycerin bouillon (2 per cent.), 
acid to phenolphthalein the human bacillus causes a permanent 
acid reaction, while with the bacillus of bovine origin the acidity 
Fig. 147.-Mycobac- 
terium tuberculosis, gly- 
cerin agar slant (Curtis). 392 
VETERINARY BACTERIOLOGY 
diminishes and the reaction becomes alkaline if the growth en- 
vironment of the culture is suitable. The tuberculin prepared 
from the human bacillus is acid, and from the bovine bacillus alka- 
line. This difference has been noted by other investigators since 
its first description, and seems to be one of the best methods of 
differential diagnosis. 
Pathogenesis.-Tuberculosis is characteristically a chronic 
disease. Even in experimental animals months are often required 
for it to run its course. As stated by Moore, "It does not destroy 
life by acute toxemia, but by a chronic and long-continued sys- 
temic poisoning and by the morbid changes brought about through 
the localization of these lesions in the organs necessary to life." 
Experimental Evidence of Pathogenesis.-The laboratory ani- 
mals are generally susceptible to infection with Mycobacterium 
tuberculosis. The constant presence of the organism in the lesions 
of the disease and its ability to reproduce the disease are sufficient 
evidence that it is the true etiologic factor. 
Important differences in pathogenesis are to be noted among 
the three varieties. The bovine bacillus is most pathogenic for 
laboratory animals, the human next, and the avian least (except 
for birds). Guinea-pigs inoculated subcutaneously with bovine 
bacilli generally succumb in less than fifty days; those inoculated 
with human bacilli generally live more than fifty days. Intra- 
peritoneal injection of the bovine type is fatal in seven to eighteen 
days, of the human type in from ten to thirty-eight days. The 
difference upon intravenous injection of the rabbit is even more 
marked-with bovine bacilli death occurs within three weeks, 
with human bacilli the animals usually live for several months and 
may even recover. The avian type ordinarily does not produce 
fatal infection in guinea-pigs, although rabbits succumb and fowls 
and pigeons contract the disease readily. Calves inoculated with 
bovine tubercle bacilli usually succumb promptly to generalized 
tuberculosis, while inoculation with human strains lead usually to 
local lesions which heal eventually. 
Character of Disease and Lesions Produced.-Almost any part 
of the body may be affected with tuberculosis. The disease, 
wherever found, generally involves the lymphatics. It is charac- 
terized by the development of nodules having an essentially similar ACID-FAST GROUP. THE GENUS MYCOBACTERIUM 
393 
structure in all tissues. The presence of the tubercle bacilli in a 
tissue causes a proliferation of the fixed connective-tissue cells to 
form the beginning of a miliary tubercle. Lymphocytes are gener- 
ally attracted and are present in the surrounding tissues in con- 
siderable numbers. A more or less definite layer of ''epithelioid" 
cells forms the boundary of the tubercle. Typical giant-cells 
with peripheral nuclei are found near the center. Coagulation- 
Figs. 148 and 149.-Tubercular hypertrophy of the intestinal wall in the 
bovine (Chausse). 
necrosis proceeds and the interior caseates. Encapsulation with 
fibrous tissue may occur, and the whole may eventually become 
calcified. These calcareous grains persist in healed tuberculous 
areas. Tubercles frequently are formed in masses. In the cow 
tubercular lymph-glands sometimes may equal or exceed the size 
of an orange. 
The arrangement of the nodules frequently shows clearly the 394 
VETERINARY BACTERIOLOGY 
path of the spread of the bacilli through lymphatic metastases. 
Direct growth through tissues with invasion of new areas probably 
rarely occurs. The organisms are not commonly found in the 
blood-stream, although they may sometimes be carried to other 
parts of the body by this means. Infection of the bones, joints, 
and meninges probably occurs in this manner. 
The organs most commonly the seat of lesions vary with the 
species of animal and the mode of infection. In the human, pul- 
monary infection (consumption) is most common, although in- 
testinal tuberculosis, infection of the lymphatics of the neck 
(scrofula), of the bones and joints (tubercular osteitis and arthritis), 
of the meninges, and of the liver, spleen, kidneys, and other organs 
of the body, and the serous membranes lining the cavities are not 
uncommon. Lupus or tuberculosis of the skin is of frequent 
occurrence in certain European countries. Cattle generally show 
nodules in the mesentery and in the peritoneum (Perlsucht or 
pearl disease). The lungs and the accompanying lymph-glands 
and the intestines commonly show lesions. In a certain small 
percentage of tuberculous cows, variously estimated from a frac- 
tion of 1 to 5 per cent., tuberculous lesions may be found in the 
udder. Any and all of the organs of the body may be infected. 
Swine are most commonly infected in the lymph-glands of the 
neck (swine scrofula), and in the abdominal organs and the lungs. 
Avian tuberculosis most frequently attacks the abdominal organs, 
particularly the liver and spleen, more rarely the lungs. 
Immunity.-No true toxin has been demonstrated for the 
tubercle bacillus. Endotoxins are produced. These are liberated 
from the cell with difficulty because of its composition and slow 
dissolution. Specific agglutinins and precipitins have been de- 
monstrated in the blood of infected individuals and in immune 
serum. Opsonins, both normal and immune, have been shown to 
occur. The development of bacteriolysins has not been satis- 
factorily demonstrated. 
Methods of active immunization are all dependent upon the use 
of killed or attenuated bacteria or their products. The name 
tuberculin is given to any suspension of dead tubercle bacilli or a 
solution of their products. As will be noted later, tuberculin is 
not only of therapeutic significance but of great diagnostic value. ACID-FAST GROUP. THE GENUS MYCOBACTERIUM 
395 
Many types have been prepared by various workers. Some of 
the more important will be described before a discussion of their 
use in immunization and in diagnosis is undertaken. 
Koch's Old Tuberculin (Alt Tuberculin).-Flat-bottomed 
flasks containing 5 per cent, glycerin-broth to a depth of 2 to 3 
cm. are inoculated with Mycobacterium tuberculosis. For veteri- 
nary practice tuberculin may be prepared from either the bovine 
or human type. The latter grows far more rapidly on artificial 
media and for this reason is generally if not universally used. 
The inoculating material is carefully placed on the glass 
Fig. 150.-Section of a tubercular intestinal wall showing the organisms and 
giant-cells (Chausse). 
at the surface of the liquid in order that it may spread out at once 
as a film over the surface. Unless the organism is at the surface 
little or no growth will occur. In the course of four weeks at blood- 
heat the surface of the broth is covered with a heavy, wrinkled 
pellicle. By the end of eight weeks it is ready for the preparation 
of the tuberculin. The contents of several flasks are then united 
and placed in a porcelain evaporating dish on a water-bath. The 
material is concentrated to about one-tenth of its original bulk, 
when the glycerin content becomes about 50 per cent, and is filtered 396 
VETERINARY BACTERIOLOGY 
through sterile paper or through porcelain filters. It is a brown 
viscous liquid which contains, in addition to the glycerin, salt, 
albumose, and the various specific growth products and extractives 
of the tubercle bacillus. This constitutes the tuberculin commonly 
used. 
Tuberculin of Denys -This investigator believed that the 
efficiency of the tuberculin was impaired by the heat used in con- 
Fig. 151.-Tuberculin flask showing the growth of Mycobacterium tubercu- 
losis (McFarland). 
centration. He filtered unheated broth cultures through porcelain 
and utilized the filtrate. 
The tuberculol of Landmann, the tuberculocidin or antiphthisin 
of Hirschfelder are all tuberculins prepared by various modifica- ACID-FAST GROUP. THE GENUS MYCOBACTERIUM 
397 
tions of the original Koch method, such as repeated extraction of 
bacilli at different temperatures, treatment with H2O2, etc. 
Several tuberculins for veterinary use have been prepared by 
German firms. Bovotuberkulol and phymatin are among these. 
The former is used in the same manner as the old tuberculin and 
the latter for the ophthalmic test. Klimmer and Wolff-Eisner 
give a list of 17 such products that have been proposed or used in 
diagnosis. 
New Tuberculin, or Bacillus Emulsion of Koch.-The cultures 
are prepared, dried, and ground as for the manufacture of T. R. 
They are then suspended in glycerinated physiologic salt solution. 
Some preservative, as phenol, is added. 
This by no means completes the list of preparations of the 
tubercle bacillus. Most of them are of laboratory significance 
only, the ones of any considerable practical importance being the 
old tuberculin and the purified product. 
Tuberculins have been standardized in various ways. One 
method is to inoculate subcutaneously a series of guinea-pigs weigh- 
ing 350-400 gms. with 0.5 mg. of tubercle bacilli. Such infection is 
usually fatal, if allowed to run its course, in about six weeks. Three 
weeks after inoculation, if the animals are decreasing in weight, an 
amount of tuberculin varying from 0.05 to 0.3 c.c. is injected, and 
the strength of the tuberculin determined by the amount necessary 
to kill such a guinea-pig within twenty-four hours. 
The use of tuberculin as a prophylactic or cure has not proved 
successful with the lower animals, nor has it in man when used 
in large doses. Within recent years it has come into common use 
in the treatment of human tuberculosis, minute injections being 
given and care taken that no febrile reaction shall follow. Deter- 
minations of the opsonic index from time to time have been used 
with success in the determination of the proper spacing of the in- 
jections. This method is intended to stimulate opsonin produc- 
tion and thus aid the body in ridding itself of the bacilli. 
Many methods of immunization against tuberculosis by means 
of injections of virulent or attenuated cultures of tubercle bacilli 
have been proposed and tested. Some of these are worthy of 
mention. 
Von Behring developed a method of immunization by the use of 398 
VETERINARY BACTERIOLOGY 
bovovaccine. This consists of dried human tubercle bacilli, mixed 
with water at the time of vaccination. Calves from two to twelve 
weeks old are injected intravenously with 4 mg. of bovovaccine, 
followed three months later by 20 mg. There is evidence that this 
causes the development of a considerable degree of immunity 
against infection with bovine tuberculosis. Although it has been 
tested out on a relatively large scale, the immunity has been found 
to be so transitory that the method has not proved of much prac- 
tical use. A somewhat similar vaccine, termed Tauruman, has been 
used by Koch and Schutz. Heyman enclosed human tubercle 
bacilli in a parchment sack surrounded by a gelatin capsule. By 
means of a trochar the sack was placed in the subcutaneous con- 
nective tissue, the theory being that the products of growth of the 
organism enclosed would diffuse out. Klimmer has advocated 
the use of a non-virulent organism, secured from salamanders that 
had been injected with human tubercle bacilli. The preparation 
is termed antiphymatol. It is probable that this organism may be 
of the type infecting cold-blooded animals. Klimmer has claimed 
his antiphymatol to be quite successful as a vaccine when com- 
bined with suitable hygienic measures. The vaccine of Friedmann 
is somewhat similar, the organism being derived in this case from 
the turtle; it has proved to be of no value as a curative agent in man. 
Antisera have been prepared by repeated injections of various 
animals with tuberculin T. R. and other products of the tubercle 
bacillus. None of them has proved successful in conferring passive 
immunity on other individuals. 
In summary it may be stated that up to the present time no 
practicable method of immunization against tuberculosis in cattle 
has been developed, although in man the use of carefully regulated 
injections of tuberculin is promising. 
Bacteriologic Diagnosis.-Tuberculosis may be diagnosed bac- 
teriologically by staining methods, animal inoculation, agglutin- 
ation, and the tuberculin reaction. 
Diagnosis by Staining Methods.-Tubercle bacilli may be 
readily recognized by their acid-fast character when examined 
microscopically. The presence of the characteristic acid-fast 
bacteria in the tissues or sputum is generally sufficient to establish 
diagnosis. This is not the case, however, with feces, milk, or ACID-FAST GROUP. THE GENUS MYCOBACTERIUM 
399 
other substances which may become contaminated with non-patho- 
genic acid-fast forms common in dust, in soil, etc. Resort must 
then be had to animal inoculation followed by isolation of the 
characteristic bacillus, or to isolation by the use of antiformin. 
The discovery of the bacilli in milk, sputum, and other body secre- 
tions may frequently be greatly facilitated by centrifugation and 
by the preparation of mounts from the sediment. Antiformin 
mixed with the material to be examined greatly aids in its sedimen- 
tation without interfering in any way with its staining properties, 
providing care is used in the washing as described under "isolation," 
or, better, by the addition of an equal quantity of untreated 
sputum before making the smear. 
Diagnosis by Animal Inoculation.-This is the most delicate 
method of determining the presence of tubercle bacilli. Intra- 
peritoneal injections of the suspected material into a guinea-pig 
will result in the development of the disease within a few weeks. 
Non-pathogenic acid-fast bacteria may give some of the patho- 
logic appearances of true tuberculosis, so that it is well to make 
certain of the diagnosis by isolation and cultivation of the organ- 
ism from the inoculated animal. An injection of tuberculin may 
be used to shorten the period of time necessary for diagnosis in the 
inoculated guinea-pig. 
Diagnosis by the Agglutination Test.-Although specific agglu- 
tinins for the tubercle bacillus may be demonstrated in the blood 
of those having the disease, the diagnosis by this method has not 
proved practicable. Great care is necessary to secure a homo- 
geneous suspension of the bacteria, and the agglutinins are rarely 
present in quantity. 
Diagnosis by the Tuberculin Tests.-Many methods of using 
tuberculin in diagnosis have been studied. The following are 
those which appear to be of the greatest importance. 
Subcutaneous injection of tuberculin into an infected animal 
causes a characteristic reaction. 
The test of cattle is made by injecting a standard dose of 
tuberculin. This as distributed by the Bureau of Animal Indus- 
try consists of old concentrated tuberculin so diluted that 4 mils, 
contain 0.5 gm. of old tuberculin. The normal temperature of 
the animal should be ascertained before injection. This is most 
accurately determined by taking the temperature every two 400 
VETERINARY BACTERIOLOGY 
hours on the day preceding the test, but in practice frequently 
but one or two preliminary determinations are made. After 
six or eight hours the temperature is again to be taken every two 
hours for the remainder of the twenty-four hours after injection. 
Care must be exercised that the animals are kept under normal 
conditions during the test, and that they remain quiet. Animals 
in heat or advanced in pregnancy or suffering from diseases should 
not be tested. A positive test should show an increase of at 
least 1.5° above the previous maximum recorded temperature. 
The temperature usually begins to rise in about eight hours, and 
reaches its maximum in from ten to eighteen hours after injec- 
tion, then gradually subsides. The rise in temperature is usually 
accompanied by a quickened pulse, and in milk cows a diminu- 
tion in the flow of milk. At the Eighth International Veterinary 
Congress the following rules were adopted for the thermic tuber- 
culin test: 
1. No animal should be subjected to the tuberculin test whose 
body temperature at the time of the test is above 39.5° C. 
2. In such animals a rise to a temperature of 40° C. or above is 
to be regarded as a positive reaction. 
3. Temperatures of 39.5° to 40° C. are to be regarded as 
questionable. 
There are many theories of the mechanism of the tuber- 
culin reaction, but it is now believed to be explained best on 
the basis of anaphylaxis. The fact that the amount of tuberculin 
used in the test is not appreciably poisonous to a healthy animal 
indicates that the infected animal has become sensitized against 
the bacterial constituents, probably proteins. The presence of a 
specific allergin has not been satisfactorily demonstrated, but some- 
thing of that nature is probably present and renders the tissues 
sensitive, principally about the lesions. That this sensitiveness 
extends to other tissues also is shown by the ophthalmic and other 
reactions to be described presently. It is a well-known fact that 
one injection of tuberculin will prevent a reaction to a second 
injection made shortly after. This fact is made use of by dishonest 
cattlemen in vitiating the tuberculin test. The probable explana- 
tion of this fact is that the body is in a condition of anti-anaphy- 
laxis, that the allergin has been exhausted by the preceding dose, 
and there has been insufficient time for the accumulation of a new ACID-FAST GROUP. THE GENUS MYCOBACTERIUM 
401 
supply. There is no evidence that the use of tuberculin as or- 
dinarily practised ever results in the permanent sensitization of the 
animal. This is an important point, as the test can be used repeat- 
edly in a herd of cattle, and the results may be relied upon. Ani- 
mals in advanced stages of the disease frequently fail to react. In 
such cases it is probable that the body is in a state of immunity to 
the proteins of the tubercle bacillus. As was stated in the discus- 
sion of anaphylaxis, the mechanism of this immunity is not well 
understood. This immunity to injections of tuberculin must not 
be confused with immunity to the disease, for it seems that these 
rest upon quite different factors. The diagnosis of tuberculosis 
in the human is generally accomplished by other means than in- 
jection, on account of the severity of the reactions. In cattle it 
maybe relied upon quite implicitly if the test is carried out properly. 
Failures in cattle are sometimes due to the extensive lesions, to the 
fact that the lesions are comparatively insignificant, calcified or 
encapsulated, or to previous injections of tuberculin, or to the use 
of antipyretics. 
Von Pirquet has described a cutaneous tuberculin reaction that 
is of diagnostic value in man, particularly in young children. The 
inner side of the forearm is washed with ether. One drop of old 
tuberculin is applied, and another at a distance of about 10 cm. 
The skin is slightly scarified and the drops rubbed with a bit of 
cotton. A positive diagnosis is evidenced by the development of a 
papule resembling that of vaccinia. The reaction is quite local, 
showing that the tissues of the body distant from the tuberculous 
lesions have been sensitized. The method has been extensively 
tested and has been found quite accurate. Von Pirquet secured 
positive reactions in 87 per cent, of the clinically tubercular, and in 
20 per cent, of clinically tubercular free. The reaction was found to 
be far more accurate during the first year of life than later. Lig- 
nieres modified this method by shaving the skin and avoiding scari- 
fication. Six drops of undiluted old tuberculin are applied and 
rubbed with a bit of absorbent cotton. The reaction is similar to 
the preceding. It has been termed the antituberculin reaction. 
The intradermal test is extensively used in the United States 
in the diagnosis of the disease in cattle. The technic is as follows: 
Intradermal Tuberculin Test.-One-tenth to two-tenths mils of 402 
VETERINARY BACTERIOLOGY 
50 per cent, tuberculin is injected into the tissue of the cutis with a 
short hypodermic needle. In cattle the point of injection that has 
proved most satisfactory is the caudal fold, stretching from the base 
of the tail to the anus. Care is used that the needle does not pass 
through the skin into the subcutaneous tissue. In swine the test is 
satisfactorily applied by injecting the tuberculin into the skin at 
the base of the ear near the median border on the dorsal surface. 
The reaction in cattle consists of an edematous, circumscribed 
swelling, reaching the size of a hulled walnut in twenty-four hours 
or more. This swelling does not subside for forty-eight to seventy- 
two hours after injection. In swine a positive reaction is indicated 
by a flat swelling with a reddened center appearing in about twenty- 
four hours. 
The test is coming into favor in this country, particularly where 
large numbers of animals are to be tested. The advantages over 
the thermal reaction are the ease of application, the fact that tem- 
peratures need not be taken and that observations can be made 
once or twice after inoculation without requiring the operator's 
presence in the meantime. Thus there is a great saving of time. 
The ophthalmo-reaction of Calmette and the conjunctival reac- 
tion of Wolff-Eisner have likewise found extensive use in recent 
years. A tuberculin specially prepared free from glycerin and 
other irritants is dropped into the conjunctival sac. Klimmer 
states that to secure this test satisfactorily in cattle a concen- 
trated (50 to 100 per cent.) tuberculin must be used, as cattle 
are relatively less sensitive to tuberculin than man. Wolff- 
Eisner claims that repeated instillation of the tuberculin into the 
same eye does not produce habituation, also that this reaction may 
be given when the thermic test fails in advanced cases of the dis- 
ease. A positive reaction consists in the appearance of a pro- 
nounced conjunctivitis, which shows itself in six to ten hours and 
disappears in two or three days. This reaction has not found as 
extensive use as the subcutaneous injection. 
Transmission and Prophylaxis.-Channels Through Which the 
Organisms Leave the Body.-These are determined largely in both 
man and animals by the localization of the lesions. The organism 
is to be found in the sputum in man and animals affected by pul- 
monary tuberculosis. The infectious droplets thrown out in ACID-FAST GROUP. THE GENUS MYCOBACTERIUM 
403 
coughing and the contamination of drinking-vessels are common 
sources of infection. Material coughed up from the lungs by 
cattle is commonly swallowed, and both pulmonary and intestinal 
tuberculosis in these animals results in large numbers of organ- 
isms being thrown off with the feces. This is probably the most 
important channel of exit in the cow. Tuberculosis in swine fol- 
lowing cattle is undoubtedly due to the ingestion of bacteria voided 
in this manner. Contamination of milk with tubercle bacilli is 
almost inevitable when they are constantly present in the feces. 
Milk is also found to contain tubercle bacilli when lesions are 
present in the udder. Whether or not they may be present when 
the udder is not tuberculous is a mooted question, but as the 
bacilli can rarely if ever be demonstrated in the blood, it is not 
probable that they can enter the milk direct when the udder lesions 
are absent. The urine may occasionally contain the bacteria. 
It should be emphasized that tuberculosis may exist either as a 
closed or an open type. In the former the organisms infect organs 
having no direct communication with the exterior of the body, such 
organisms are not therefore in a position to be eliminated with body 
excretions. In open tuberculosis, however, the organisms con- 
stantly or intermittently escape from the body. 
The disease is probably never inherited, the offspring being in- 
fected only when the disease infects the uterus and the placenta. 
This fact is of importance, for upon it the Danish veterinarian, 
Bang, has outlined a practicable method of building up herds free 
from tuberculosis. The calves are separated from their tubercu- 
lous mothers soon after birth and are fed only upon pasteurized 
milk. Those animals known to be tuberculous are separated from 
the others and a quarantine strict enough to prevent the transfer of 
the disease from infected to non-infected animals is established. 
Infection Atria in Tuberculosis.-1The portals of entry of 
Bacillus tuberculosis have been investigated at length in recent 
years, but there are still discrepancies in the results of investigators 
that are unexplained. One group of tuberculous infections, par- 
ticularly the pulmonary type in man, is probably due to inhalation 
of the organisms. This was at one time universally conceded, but 
the work of Calmette and others has shown that primary pul- 
monary tuberculosis may result from ingestion of the organisms. 404 
VETERINARY BACTERIOLOGY 
Certain investigators believe this to be far more common than in- 
fection by inhalation. It has been shown that the tubercle bacilli 
may be demonstrated in the thoracic duct of a dog fed upon the 
organisms within a few hours after their ingestion, and without 
any apparent lesion of the intestinal wall. The alimentary tract 
is undoubtedly the infection atrium in most cases of intestinal 
tuberculosis and of infection of the other abdominal organs, the 
mesentery and the peritoneum. The tonsils are believed with 
good reason to permit the infection of the neighboring lymph- 
glands and the consequent production of scrofula. Cutaneous 
lesions following abrasions or cuts of the skin have been observed, 
particularly in butchers, and in a few instances in veterinarians. 
Inter transmissibility of Human, Bovine, and Avian Tuberculosis. 
-Human tubercle bacilli do not readily infect cattle. When 
injected, they may cause local, but rarely general, infection. The 
fact that the human and bovine types of bacilli may be differen- 
tiated has already been emphasized. Both types of bacilli produce 
fatal infection in the monkey. Instances of probable cutaneous 
infections of man from cattle are on record. 
Park and Krumweide have outlined the factors which must 
be taken into consideration for differentiation of the bovine and 
human type of tubercle bacilli. In their own isolations of the 
tubercle bacilli from the human they have used the following 
criteria: "All cultures showing maximum luxuriance on glycerin 
egg in primary cultures are certainly human. All cultures not 
showing maximum luxuriance should then be transferred to 
glycerin egg, potato, and glycerin egg with bouillon added. Cul- 
tures which have failed on glycerin egg should also be transferred 
on plain egg, as glycerin egg may again fail. All cultures showing 
maximum luxuriance are quite certainly human." The forms 
which show a very sparse growth are as quite certainly bovine. 
Intermediate forms are tested upon rabbits, and, if necessary, upon 
calves. 
Swine readily contract bovine tuberculosis. They have also 
been known to become infected with avian tuberculosis through 
the consumption of birds dead from the disease. 
Birds contract the disease from each other by ingestion of 
excreted bacteria. Whether or not the disease may start from ACID-FAST GROUP. THE GENUS MYCOBACTERIUM 
405 
ingestion of bovine or human tubercle bacilli is not certainly 
known. 
It is evident that while each of the types of tubercle bacilli is 
generally associated with a specific host, on the other hand, each 
may, under appropriate conditions of infection and susceptibility, 
produce infection in other hosts. 
The following table, adapted from a paper by Park and Krum- 
weide, summarizes 1511 cases of human tuberculosis reported in 
which the bovine or human character of the organism was deter- 
mined. The age groupings are particularly interesting: 
Diagnosis. 
Adults, sixteen years 
and over. 
Children, five to 
sixteen years. 
Children under five 
years. 
Human. 
Bovine. 
Human. 
Bovine. 
Human. 
Bovine. 
Pulmonary tuberculosis. . 
778 
3 
14 
35 
1 
Axillary or inguinal tuber- 
cular adenitis 
3 
4 
2 
Cervical tubercular aden- 
itis 
3B 
1 
36 
22 
15 
24 
Abdominal tuberculosis. . 
16 
4 
8 
9 
10 
14 
Generalized tuberculosis 
of alimentary origin... . 
6 
1 
3 
4 
17 
15 
Generalized tuberculosis.. 
29 
5 
1 
74 
7 
Generalized tuberculosis 
and meningitis of ali- 
mentary origin 
1 
5 
10 
Generalized tuberculosis 
and meningitis 
5 
10 
76 
1 
Meningitis 
1 
3 
28 
4 
Tuberculosis of bones and 
joints 
32 
1 
41 
3 
27 
Other types 
34 
5 
6 
7 
3 
Totals 
940 
15 
131 
46 
292 
76 
There seems to be no reasonable doubt but that the bovine 
tubercle bacillus may infect the human. The above table shows 
it to be common enough in children under sixteen years to justify 
all reasonable precautions against the ingestion of infected meat 
and milk. The danger to the adult would appear to be almost 
negligible. It is estimated that less than 10 per cent, of fatal cases 
of tuberculosis in children are due to the bovine tubercle bacillus, 406 
VETERINARY BACTERIOLOGY 
but the incidence of the disease among children is much higher. 
The large proportion of bovine infections in scrofula and tubercu- 
losis of alimentary origin should be especially emphasized. 
Mycobacterium paratuberculosis 
Synonyms.-Bacillus of Johnes' disease; Bacillus paratuber- 
culosis. 
Disease Produced.-Chronic enteritis or paratubercular 
dysentery of cattle. 
Johnes and Frothingham, in 1895, described an acid-fast 
organism as the probable cause of chronic dysentery in cattle. 
They believed the organism to be identical with the avian tuber- 
cle bacillus, but this has been rendered improbable by subsequent 
investigations. 
Distribution.-The disease has been noted in various parts of 
Europe and in the United States. 
Morphology and Staining.-The organism closely resembles 
the Mycobacterium tuberculosis morphologically. It is a slender 
rod, usually 1 to 2 m in length, rarely longer. It is non-motile. 
Neither spores nor capsules have been observed. It does not 
stain readily with the aqueous anilin dyes unless mordanted. 
When once stained it is both acid fast and alcohol fast. The 
same staining technic may be used as with the tubercle bacillus. 
Isolation and Culture.-Twort and Ingram, Meyer, and others 
have succeeded in growing Mycobacterium paratuberculosis on both 
solid and liquid media that contain extracts from other acid-fast 
bacteria, such as M. tuberculosis and M. phlei. Meyer found a me- 
dium of equal parts of broth and tuberculin and 1 per cent, serum 
with 2 per cent, agar the most suitable for purposes of isolation. 
Antiformin may be used if necessary to destroy other organisms 
not acid fast in making the original isolation. Growth is slow, best 
at 37°. The organism tends to grow as dry, discrete colonies. 
Pathogenesis.-Experimental Evidence.--The disease has not 
been transmitted to any of the laboratory experimental animals. 
Intravenous injection into calves produces the typical disease after 
an incubation period of four to eight months. 
Character of Disease and Lesions Produced.-The disease is 
characterized by progressive emaciation and persistent chronic 
diarrhea, with commonly fatal termination. The lesions are ACID-FAST GROUP. THE GENUS MYCOBACTERIUM 
407 
confined to the intestines, small intestines primarily, and the 
colon secondarily. The mucosa shows thickening and wrinkling, 
but there is little evidence of congestion. The lesions are not 
sharply limited; there is no necrosis, although some of the villi 
may be denuded of epithelium. Giant-cells may rarely be demon- 
strated, but tubercle formation does not occur. 
Immunity.-Nothing is known relative to immunity to this 
disease. 
Bacteriologic Diagnosis.-The presence of acid-fast organisms 
in large numbers in the thickened mucosa in the absence of nodule 
formation is diagnostic on postmortem. The animal does not 
react to the injection of the standard tuberculin unless it is infected 
with tuberculosis. Bang and others have come to the conclusion 
that tuberculin prepared from avian tubercle bacilli can be used in 
a thermic test for this disease. Horne has also reported successful 
ophthalmic and cutaneous diagnosis with avian tuberculin. Meyer 
concluded that avian tuberculin was of little diagnostic value, but 
that a paratuberculin prepared from Mycobacterium paratuberculo- 
sis yielded better results. 
Transmission.-The disease is doubtless transmitted by 
ingestion. 
Mycobacterium lepras 
Synonyms.-Bacterium leprce; Bacillus leprce. 
Disease Produced.-Leprosy in man. 
Hansen, in 1872, discovered the bacillus of leprosy in the lesions 
of the disease. It was studied more at length by Neisser and 
Hansen, who published their report in 1880. 
Distribution.-Leprosy is common in Asia, northern Europe, 
in certain Pacific Islands, and is found occasionally in various 
parts of the United States. A similar, though probably riot 
identical, disease has been noted by Wherry and others in rats. 
Morphology and Staining.-Mycobacterium leprce resembles 
the B. tuberculosis. It is a slender rod, frequently as much as 6 y 
in length. The rods are usually straight, non-motile, do not 
produce spores or capsules. They stain somewhat more readily 
than the M. tuberculosis, but are distinctly acid fast. 
Isolation and Culture.-Several investigators claim to have 408 
VETERINARY BACTERIOLOGY 
cultivated the leprosy bacillus, but the results m every case need 
confirmation. 
Pathogenesis.-Experimental Evidence.-Lower animals, with 
the possible exception of the monkey, cannot be successfully 
infected with the Bacillus leprce. Arning succeeded in infecting 
a criminal in the Hawaiian Islands by subcutaneous implantation 
of leprous tissue. The belief in the pathogenicity of this organism 
rests principally upon its pres- 
ence in great numbers in the 
leprous tissues. 
Character of Disease and 
Lesions Produced.-The organ- 
isms may proliferate in the 
nerves, causing anesthetic lep- 
rosy; or in the subcutaneous 
tissues, producing nodules re- 
sembling superficially those of 
tuberculosis. The disease 
progresses slowly, the infected 
individual surviving for years 
or even several decades. 
Bacteriologic Diagnosis.-The disease may be diagnosed bac- 
teriologically by scraping a nodule and preparing a smear from 
the serum so obtained. The characteristic acid-fast organism 
may be demonstrated. In the hands of some investigators the 
complement binding reaction (using extracts from leprous organs 
as the antigen) has proved of diagnostic value. 
Fig. 152.-Mycobacterium leprae (Kolle 
and Wassermann). 
Non-pathogenic Acid-fast Bacteria 
Many species of non-pathogenic acid-fast bacteria have been 
described, and from a variety of sources. Some are normal 
inhabitants of the skin, others occur in dung and in soil. The 
presence of these organisms upon the body, in milk, etc., frequently 
renders a differential diagnosis necessary between them and the 
Mycobacterium tuberculosis. A few of the more important 
types will be briefly described. 
Mycobacterium smegmatis.-This organism has been repeat- 
edly observed in the smegma from the genitals of man and ACID-FAST GROUP. THE GENUS MYCOBACTERIUM 
409 
animals and from the skin of the axillae. Morphologically it 
resembles the tubercle bacillus. Its isolation is accomplished 
with considerable difficulty and only upon media containing 
blood-serum. The M. smegmatis is non-pathogenic. It is of 
importance principally because of the possibility of confusion 
with M. tuberculosis when it occurs in the urine. Although the 
smegma bacillus is acid fast, it may be decolorized by alcohol. 
Dung Bacillus, Grass Bacillus of Moeller, Butter Bacillus, etc. 
-Acid-fast bacteria not unlike the tubercle bacillus in mor- 
phology have been isolated from a great variety of sources. 
They may in general be easily differentiated from the tubercle 
Fig. 153.-Mycobacterium smegmatis, in a stained smear of preputial smegma 
(Frankel and Pfeiffer). 
bacillus, as they do not produce a generalized tuberculosis when 
inoculated into experimental animals. However, nodules resem- 
bling those of tuberculosis are found as a result of the injection of 
some species. The organisms when isolated upon culture-media, 
however, are found to develop luxuriantly even at room-tempera- 
tures. It is interesting to note that isolation of such bacteria 
from soil has been accomplished by the use of antiformin. The 
differential diagnosis of these forms from M. tuberculosis must 
always be accomplished by both animal inoculation and isolation 
upon culture-media. In making a diagnosis of tuberculosis 
from stained mounts the possible presence of these forms must be 
constantly borne in mind. CHAPTER XXXV 
VIBRIO GROUP 
One organism, pathogenic for animals, described by Smith as 
associated with abortion in cows, one from fowls, the Vibrio 
metchnikovi, one pathogenic for man, Vibrio cholera, and numer- 
ous non-pathogenic forms isolated from various sources have 
been described as belonging to this group. 
The organisms of this group are more or less bent rods, usually 
only a segment of a spiral, rarely showing one or more complete 
turns. All are motile, aerobic, Gram-negative, and without spores. 
Vibrio metchnikovi 
Synonyms.-Spirillum metchmkovi; Microspira metchmkovi. 
Disease Produced.-Septicemia of fowls. 
Gamaleia, in 1888, described this organism from an epizootic of 
fowl septicemia which occurred in Russia. What appears to be the 
same organism was later (1894) 
isolated by Pfuhl from water. 
Distribution.-This organ- 
ism does not seem to have 
been isolated from such a dis- 
ease by any investigator since 
the original work of Gamaleia. 
This latter, however, was suf- 
ficient to establish its etiologic 
relation to the disease. It is 
possible that the disease is 
more widely spread than the 
literature would indicate, inas- 
much as poultry diseases are in 
need of careful investigation. 
Morphology and Staining.-It is a curved rod, with rounded 
ends, or sometimes a spiral filament. It is actively motile by a 
single terminal flagellum, and is about 0.5 by 2 p. Neither capsules 
nor spores are produced. It stains readily, but is Gram-negative. 
Fig. 154.-Vibrio metchnikovi 
(Gunther). 
410 VIBRIO GROUP 
411 
Isolation and Culture.-The organism may be isolated from the 
intestinal contents of adult fowls and from the blood of younger 
fowls by plate cultures in nutrient gelatin. Upon these plates 
small white, punctiform colonies are developed in the course of 
twelve to sixteen hours; these enlarge rapidly, cause liquefaction 
of the gelatin, and are soon to be found in saucer-shaped depressions 
in the medium. Growth in a stab culture in gelatin is likewise 
rapid, and the gelatin is quickly liquefied in the form of a funnel. 
Upon agar slants a yellowish layer is developed. Bouillon is 
clouded, and a delicate pellicle may form. Milk is coagulated 
with acid reaction and without solution of the casein. 
Fig. 155.-Vibrio metchnikovi in the blood of a pigeon (X 1000) (Frankel 
and Pfeiffer). 
Physiology.-The organism is aerobic. It develops almost as 
rapidly at room-temperature as at blood-heat. The thermal 
death-point is 50° for five minutes. It is sensitive to the pres- 
ence of acids in culture-media, and soon dies in consequence of 
their production in milk. It produces acid from dextrose and 
lactose, but no gas. Indol is formed. Gelatinase is developed, 
but no enzyme which will digest casein. 
Pathogenesis.-Experimental Evidence.-Subcutaneous inocu- 
lations into the chicken, pigeon, and guinea-pig are quickly fatal; 
rabbits and mice succumb only to large doses. The disease ap- 
pears as a septicemia. According to Gamaleia, it may be com- 
municated to chickens by feeding pure cultures. 412 
VETERINARY BACTERIOLOGY 
Character of Disease and Lesions.- Chickens affected show 
many of the same symptoms as those infected with chicken-cholera, 
except that the temperature is little, if at all, elevated. Diarrhea 
is constantly present. There is a marked hyperemia of the ali- 
mentary canal, and a blood-tinged, grayish-yellow liquid is found 
in the small intestine. 
Immunity.-No true toxin has been demonstrated. Agglutin- 
ins are present in immune serum. Immunity may be established 
ip animals by repeated injections of killed cultures. 
Fig. 156.-Vibrio metchnikovi, gelatin stab (Frankel and Pfeiffer). 
Bacteriologic Diagnosis--This may be made on the basis of 
microscopic findings and isolation in pure culture. 
Transmission.-The disease is probably transmitted by the 
ingestion of food soiled by droppings from infected fowls. 
Vibrio choleras 
Synonyms.-Spirillum cholerce asiaticce; Microspira comma: 
Vibrio cholerce asiaticce. 
Disease Produced.-Asiatic cholera in man. 
Koch, in 1883, discovered the specific cause of Asiatic cholera 
in the rice-water stools of cholera patients. 
Distribution.-The disease is endemic in India and possibly 
parts of China. It has swept in epidemics over Europe several 
times within the last century. VIBRIO GROUP 
413 
Morphology and Staining.-The cholera spirillum is a short, 
slightly curved rod, whence the common designation of "comma 
bacillus." Longer filaments and involution forms are frequently 
observed. It is motile by means of a single polar flagellum. 
Spores and capsules are not produced. It stains readily with 
the common anilin dyes and is Gram-negative. 
Isolation and Culture.-It may be readily isolated from the 
stools of cholera patients by plating upon nutrient gelatin. 
The cultural characters are very similar to those of Vibrio 
metchnikovi. 
Physiology.-It is an aerobic organism. Growth occurs readily 
at room-temperatures, although the optimum is blood-heat. The 
Fig. 157.-Vibrio choleras (Kiihne- 
mann). 
Fig. 158.-Vibrio choleroe, showing 
flagella (Gunther). 
thermal death-point is 60°. Desiccation, disinfectants, and sun- 
light quickly destroy it. Blood-serum and gelatin are liquefied. 
Milk is not coagulated. Nitrates are reduced to nitrites. Indol 
is produced. The organism requires a neutral or slightly alkaline 
medium for its development. 
Pathogenesis.-Experimental Evidence.-Asiatic cholera, in the 
form found in man, usually cannot be transmitted to laboratory 
animals. The etiologic relation of Vibrio cholera to the disease, 
however, has been established by accidental and intentional infec- 
tion of several laboratory workers. Intraperitoneal injection of the 
guinea-pig results fatally. Ingestion of the organisms by young 414 
VETERINARY BACTERIOLOGY 
rabbits may result in the development of the symptoms and lesions 
of typical cholera as they are observed in man. 
Character of Disease and Lesions in Man.-Asiatic cholera is 
characterized by a severe diarrhea. The intestinal epithelium 
is desquamated, and gives rise to the appearance known as "rice- 
water" stools. The intestinal tract shows congestion and some- 
times extensive, even diphtheritic, necrosis. The loss of water 
from the blood results in a considerable, frequently fatal, diminu- 
tion in blood-pressure. 
Immunity.-True toxins have been demonstrated for the 
cholera spirillum. They are produced under peculiar cultural 
conditions. Antitoxin has also been produced. Agglutinins and 
precipitins are found in immune blood, and frequently in the blood 
of patients. Bacteriolysins may be readily demonstrated; in 
fact, it is with this organism that the classic demonstration of 
Pfeiffer's phenomenon was carried out. 
Active immunization against Asiatic cholera has been exten- 
sively practised. The vaccine consists either of cultures killed 
at 58° or of cultures attenuated by growth at 39°. 
Bacteriologic Diagnosis.-This may be accomplished by 
an examination of a stained mount from the rice-water discharges, 
by isolation of the organism, or by the agglutination test. 
Transmission.-The disease is transmitted through impure 
water and food, particularly vegetables, contaminated with the 
excretions of cholera patients. 
Vibrio fetus 
Synonym.- Spirillum fetus. 
Disease Produced.-Abortion of cattle. | 
Smith in 1919 in a study of the fetus, the fetal membranes 
and swabs from the uterus in 109 cases of abortion in cows, found 
62 (57 per cent.) associated with Bacterium abortus, 26 (23.8 per 
cent.) with spirilla, 2 (1.8 per cent.) with Hemophilus pyogenes 
and 19 (17.4 per cent.) sterile or without Bacterium abortus. He 
found Bacterium abortus associated with first pregnancies in 42 
cases, with the second in 14, with the third in 5, and with the 
fourth in 1. Spirilla were found in first pregnancies in 6, second 
in 9, third in 5 and fourth in 3. VIBRIO GROUP 
415 
Morphology and Stains.-Vibrio fetus varies more or less in 
length. Short forms consisting of 1 or 1| or more turns of 
about 2 u are found in young cultures and long ones of 2 to 4 
windings in old. The transverse diameter is probably not over 
0.2 to 0.3 m. Alkaline methylene blue gives satisfactory results 
when the staining is prolonged over night. Giemsa is less 
satisfactory. In fixed material, eosin-methylene blue is used 
with good results. With Lbffler's stain, a flagellum is found 
attached to one or both poles. The organism is Gram-negative. 
Granules in organisms stained 24 to 48 hours are not uncommon. 
Isolation and Culture.-The organisms may be obtained from 
the placenta of aborting cows and from the stomach, meconium 
and lungs of the fetus. 
If bits of tissue or particles of meconium or drop of stomach 
contents are added to slanted agar and the tube is hermetically 
sealed there will be seen after 3 or 4 days along the lateral margin 
of the slanted surface, between agar and glass, a narrow grayish 
line extending up from the water of condensation several centi- 
meters. After some days a thin, barely visible film of growth 
will extend from this line inward between agar and glass. The 
most suitable medium consists of ordinary agar slants to whose 
water of condensation a few drops of defibrinated horse blood are 
added. After several passages satisfactory growth occurs on 
plain agar. Growth is less vigorous than on blood agar. 
Growth on the slope surface is not so common as between glass 
and medium or in the condensation water. The latter becomes 
covered with a thick translucent mucus which owing to its 
viscidity is difficult to transfer. After strains become adapted 
to plain slanted agar, multiplication will occur in liquid media. 
Plain bouillon to which a few drops of defibrinated blood are 
added becomes clouded and a slight viscid sediment is formed. 
No recognizable multiplication occurs in milk. 
Physiology.-Dextrose, lactose and saccharose broth in fer- 
mentation tubes yield growth with uniform clouding, but neither 
acid nor gas is produced. Solidified blood serum is not liquefied. 
Pathogenesis.-The effect of the organism upon small labora- 
tory animals is apparently not harmful. Rats, mice, rabbits and 
guinea-pigs are refractory. 416 
VETERINARY BACTERIOLOGY 
Immunity.-Agglutinins are produced by repeated injections 
of rabbits with small doses of the organism. An agglutinating 
titer of 1 : 2560 has been obtained. 
Non-pathogenic Spirilla 
Many species of spirilla closely resembling the preceding in 
morphology and cultural characters, but lacking in pathogenicity, 
have been isolated from a variety of sources. Such are the 
Vibrio proteus or Spirillum of Finkler and Prior, isolated from the 
stools in a case of cholera nostras, Spirillum tyrogenum, or 
Deneke's spirillum from cheese, Spirillum phosphorescens, isolated 
from water, and many others. CHAPTER XXXVI 
There is probably more confusion relative to the classification 
of the members of this group of organisms than in any other 
group of bacteria or protozoa. In the first place, there is by 
no means an agreement among investigators as to whether these 
organisms should be included under the heading of bacteria or of 
protozoa. There seems to be more evidence in recent literature 
of protozoan rather than of bacterial 
relationships. Second, it is evident that 
the group is not homogeneous, and efforts 
have been made to separate the group 
into several genera. In not a single case 
have we a full and satisfactory know- 
ledge of the life-history, particularly in 
those forms which are transmitted from 
one animal to another by parasites. 
Until this is worked out it will be im- 
possible to make a separation of the dif- 
ferent types into genera on the basis of 
true relationships. In the discussion of 
the group the genus name of Spirochaeta 
is retained, the first of the synonyms 
given being the one which has been suggested by Blanchard in his 
revision of the group on the basis of protozoan relationships. 
The argument advanced for protozoan relationships may be 
summarized as follows. According to several observers, multipli- 
cation, frequently, though not invariably, takes place by longitu- 
dinal rather than transverse division of the cell. Morphologic- 
ally, it is believed that these organisms differ from the true bacteria 
by being in all cases flexible, and swimming with a sinuous motion. 
In several species undulating membranes similar to those of the 
protozoa have been demonstrated. Chromidia-like bodies, resem- 
bling the scattered nuclei in some protozoa, have been identified. 
SPIROCHETE GROUP 
Fig. 159.-Spirochaeta 
pinnae; A, B, Cells show- 
ing the undulating mem- 
brane; C, a coiled organ- 
ism (adapted from Gon- 
der). 
417 418 
VETERINARY BACTERIOLOGY 
According to Prowazek, plasmolysis does not take place with salt 
solutions several times as concentrated as those required for plas- 
molysis of bacterial cells. Certain spirochetes have been observed 
to enter red blood-cells and there assume a coiled condition. 
A multiplication of the organisms has been observed in the eggs 
of a tick which transmitted a spirochete disease, as have also 
certain forms which have been interpreted as stages in a more 
complex life-history. Leishman has found that certain spiro- 
Sp. dentium. 
Sp. inaequalis 
Sp. tenuis' 
Sp. denticola 
Sp. undulata 
Sp. dentium 
Sp recta 
Fig. 160.-Spirochaetae of different species. From a smear from a tonsillar 
lacuna stained by Burril's India-ink method (Gerber). 
chetes, when ingested by the tick, lose their motility and change 
morphologically, with resultant liberation of small bodies which 
stain like chromatin. These are of various shapes-rods, cocci, 
or spirals. They enter the lining cells of the malpighian tubules; 
they are found in the oviduct, the ovary, and the immature eggs, 
and in all stages of development of the young tick. Inoculation of 
material containing these bodies, but not true spirochetes, resulted 
in the development of tick-fever. The details of this life-history 
are in present need of elucidation. It has been claimed by Mar- SPIROCHETE GROUP 
419 
choix that the virulence of the spirochete of fowl septicemia can 
be preserved only by passing through the body of the tick which 
transmits the disease. Continual transfer of the organism from one 
fowl to another, without the intermediation of the tick, causes 
a gradual decrease in virulence. This would seem to indicate that a 
part of the life cycle of this organism must be passed in the body 
of the intermediate host or tick. None of this group of organisms 
may be cultivated upon the common laboratory media, and 
they can be induced to multiply in vitro only under very special 
conditions. All these facts would seem to make out a strong case 
for those who believe in the protozoan relationships. 
There are, on the other hand, investigators who believe quite 
as firmly that these organisms are bacteria. Novy and Knapp 
studied with great care a spirochete of relapsing fever. They 
concluded that division was always transverse, and that the 
longitudinal divisions reported by others were accidental associa- 
tions or intertwining of two organisms. Their results with the 
use of plasmolyzing agents received an interpretation quite the 
reverse of other investigators. Experiments in immunization, 
the resistance to heat, stability of form, and staining qualities of 
the flagellum all associated the organism with the bacteria. Borrel, 
Frankel, and others claim to have demonstrated the presence of 
numerous peritrichic flagella on certain forms, a condition which is 
different from any known protozoan. As before stated, there is so 
much discordance in the published work of the various investigators 
that definite conclusions cannot be reached. It has been suggested 
that these organisms form a group intermediate between the 
bacteria and the protozoa. This is not as probable as has been 
sometimes urged, and such a disposition is simply a confession of 
our lack of definite knowledge. It is by no means improbable 
that, some species of this group will be found to belong to the 
bacteria and others to the protozoa, and that supposed homologies 
are only superficial resemblances. 
Another line of evidence tending to relate the spirochetes to 
the bacteria has been the successful isolation and cultivation of 
certain free living forms from water. Wolbach and Burger passed 
pond water through a Berkefeld V filter, and added the filtrate to 
a neutral 1 per cent, solution of glucose in hay infusion. In forty- 420 
VETERINARY BACTERIOLOGY 
eight hours there developed a clouding due to the presence of a 
spirochete which was successfully grown in colony form on glucose 
hay infusion agar, the colonies resembling those of bacteria. The 
organism was named Spirochaeta elusa. Another species, Sp. 
biflexa was also isolated in a similar manner. 
Cultivation of Spirochetes.-Many attempts at cultivation of 
pathogenic spirochetes in blood-serum and other media were made 
without much success. The organisms might multiply for a time, 
but it proved impossible to get growth from continued transfers. 
Noguchi at last devised a method for securing pure cultures and 
continued growth. The method was first worked out for Spirochaeta 
pallida,--but has since been used for many other forms. A bit of 
fresh kidney tissue from a rabbit is dropped into a test-tube, care 
being used in the removal from the animal to prevent all contami- 
nation. A mixture of sterile ascitic fluid, 1 part, and slightly al- 
kaline agar, 2 parts, is added. The material from which the isola- 
tion is to be secured is triturated, and a deep stab-culture into the 
agar is made and the culture covered with paraffin oil. In many 
cases it is impossible in this first culture to free the spirochetes from 
other organisms. 
These contaminating bacteria grow along the line of the stab, 
as does also the spirochete. The fresh tissue absorbs the free oxy- 
gen of the medium and insures the anaerobic conditions so necessary 
for growth of these organisms. Usually the spirochetes will grow 
out into the medium as a hazy zone. By careful inoculation of 
fresh tubes from this hazy growth, it is generally possible by a few 
transfers to secure a pure culture. The essentials for development 
seem to be (1) a suitable ascitic fluid or, with some species, some 
other protein; (2) a weekly alkaline agar; (3) fresh tissue; and (4) 
anaerobic conditions. 
For the cultivation of certain blood spirochetes, which can be 
secured directly from the blood, liquid ascitic fluid and animal 
tissue covered with paraffin but without agar is a suitable 
medium. 
Classification of Spirochetes.-Certain species have been re- 
moved from the genus Spirochceta and new genera created for 
them by certain authors. These names are so common in literature 
that their meaning should be known. It may here be again SPIROCHETE GROUP 
421 
emphasized that the name Spirochaeta, as used in the discussion of 
the various species below, includes all of these genera. 
Spirochaeta (in Narrow Sense).-Organism with an exceedingly 
slender, spiral, flattened body, with a narrow, undulating mem- 
brane that surrounds the entire body in a spiral. No flagella and 
no spores are produced. Reproduction probably occurs by longi- 
tudinal division. 
Spiroschaudinnia.-Blood parasites. Minute wavy or spiral 
threads, with undulating membrane and no flagella. Free motile 
stage alternates with a stage in which the organism is coiled up in 
one of the host-cells. Developmental stages have been demon- 
strated in the intermediate host. The life-history is imperfectly 
known. 
Treponema.-Spiral body, not flattened, tapering at the ends. 
Flagellum at each pole. No undulating membrane. Multiplica- 
tion by longitudinal division. Does not stain with common 
aqueous anilin dyes: special staining methods are required. 
The following organisms described as belonging to this group 
are of sufficient economic importance to warrant their considera- 
tion here: Spirochaeta obermeieri, Sp. duttoni, Sp. pallida, Sp. 
pertenuis, in man; Sp. anserina {Sp. gallinarum), in geese and other 
fowls; Sp. theileri, in cattle; Sp. ovina, in sheep, and Sp. hyos, in 
swine. 
All the members of this group may be characterized as slender, 
spiral threads, motile by sinuous movements, cultivable only by 
special means and in some cases difficult to stain, requiring special 
technic. 
Spirochaeta obermeieri 
Synonyms.-Spiroschaudinnia recurrentis; Spirillum ober- 
meieri; Sp. recurrentis. 
Diseases Produced.-Relapsing fever, recurrent fever, spirillosis 
in man. 
Obermeier, in 1873, published his discovery of a large spiral 
organism in the blood of patients suffering from relapsing fever. 
Since that time it has been repeatedly observed, and several 
similar species have been described infecting man, differing prin- 
cipally in their pathogenicity for small animals, and the fact that 
immunity to one does not immunize against the other. 422 
VETERINARY BACTERIOLOGY 
Distribution.-The disease is known from Europe, and occurs 
in isolated cases in many parts of the world. 
Morphology and Staining.-Spirochaeta obermeieri is a very 
slender, tapering spiral. It is about 0.4 /z in diameter, and varies 
greatly in length. It is always many times as long as broad. 
There are from two to ten spirals or turns in the organism, as 
commonly observed. It is motile, with a very rapid, screw- 
like motion and a waving motion of the entire organism. The 
organism may be observed in the living condition. It is best 
stained by the Romanowsky method or some modification of it. 
Fig. 161.-Spirochaeta obermezeri from the blood of a rat (Novy and Knapp, 
in "Journal of Infectious Diseases"). 
Isolation and Culture.--Noguchi succeeded in cultivating this 
organism in ascitic fluid in which sterile rabbit kidney was im- 
mersed, the whole covered with paraffin oil and incubated at 37°. 
Hata claims to have secured a similar result by mixing half-coagu- 
lated horse serum with double the volume of physiologic salt 
solution, using this as a substitute for ascitic fluid. Further- 
more, he found that the reddish yellow coagulum which forms in 
such serum may be used in place of the kidney tissue. SPIROCHETE GROUP 
423 
Pathogenesis.-Experimental Evidence.-The organism is 
pathogenic for man, monkeys, mice, and rats. The most prac- 
ticable method of maintaining a culture is by repeated transfers 
of the organism from one animal to another. 
Character of Disease and Lesions.-The disease in man is char- 
acterized by pains in the head and back and by high fever. This 
is followed by a complete apparent recovery. A second attack, or 
relapse, occurs usually in about a week, then a third, and even 
more. The relapses tend to decrease in intensity. The spiro- 
chetes are to be found in large numbers during the relapses, though 
usually in diminished numbers. 
Immunity.-No specific toxin has been demonstrated. An 
attack of the disease confers an active immunity. Repeated in- 
jections of blood containing spirochetes result, in the rat, in the 
development of a hyperimmunity. Immune and hyperimmune 
blood may be used in conferring a passive immunity. The agen- 
cies responsible for the development of immunity are not well 
understood. 
Bacteriologic Diagnosis.-The organism may be found in 
fresh preparation or in stained mounts of the blood during a 
paroxysm. 
Transmission.-The disease is supposed to be transmitted by 
the bite of an infected bed-bug (Acanthia lectularia), possibly by 
the clothing louse (Pedicrdus vestimenti). Toyoda found that the 
organisms could penetrate the intestinal wall of the louse and 
multiply in the body cavity. 
Spirochaeta duttoni 
Synonyms.--Spiro schaudinnia duttoni; Spirillum duttoni. 
Disease Produced.-West African tick fever in man. 
Ross and Milne, and Dutton and Todd, working independently 
in 1904, demonstrated the presence of this spirochete in the blood 
of individuals infected with this disease. Morphologically it 
resembles the preceding closely, but is usually longer and more 
loosely coiled. It has been claimed to possess diffuse flagella. 
The disease is transmitted by the bite of a tick {Ornithodoros 
moubata). 
Noguchi has succeeded in cultivating this organism by the 424 
VETERINARY BACTERIOLOGY 
same methods used for Spirillum recurrentis. There is no visible 
alteration in the culture-medium except a faint opalescence. 
Life Cycle.-The work of Leishman, Todd, and others indi- 
cates that the spirochetes, when taken into the body of the tick, 
undergo a kind of nuclear fragmentation into chromatin granules, 
which find their way through the wall of the digestive tract into 
the various organs, principally to the malpighian tubules and the 
ovaries. These same granules may be demonstrated in the eggs 
Fig. 162.-Spirochaeta duttoni from the blood of a rat (Novy and Knapp, in 
"Journal of Infectious Diseases"). 
of infected females, and the nymphs are able to transmit the disease. 
Leishman believes that holding the tick at a temperature of 34°, 
or even blood-heat, causes these granules to develop into small 
spirochetes. They are exuded from the body through the coxal 
glands and the fluid secretion of the malpighian tubules, and gain 
entrance to the wound caused by the tick bite after the tick has 
loosed its hold, and not through the salivary glands. It is pos- 
sible that a cycle of changes of this type may account for the 
relapses that occur in the disease. SPIROCHETE GROUP 
425 
Immunity.-Leishman succeeded in establishing an active 
immunity in a monkey that recovered from the disease by causing 
infected ticks to feed upon it at intervals. 
Spirochaeta kochi 
This organism, causing East African tick fever, has been found 
to be distinct from that causing the West Coast fever. To the 
former type the name Spirochceta kochi has been given. Other 
related types of organisms causing relapsing fever have been 
described; that described by Novy and Knapp has been called 
Spirochata novyi. It is probable that a disease found in India is 
caused by still another organism. 
Spirochaeta anserina (or gallinartim) 
Synonyms.-Spirillum anserina; Spirochaeta gallinarum; Sp. 
Marchouxi; Sp. nicollei; Sp. granulosa. 
Disease Produced.-Spirochetosis, spirillosis, or septicemia in 
geese and domestic fowls. 
Sacharoff, in 1890, described the Spirochaeta anserina as the 
cause of an acute septicemia in geese, and the same organism was 
studied by Gabritschewsky in 1898. Marchoux and Salimbeni, 
in 1903, reported a septicemia or spirochetosis of domestic fowls 
in Brazil. Since that time similar organisms have been reported 
from many places. There is considerable difference of opinion 
as to the identity of the spirochetes isolated from the goose and 
the domestic fowl, and from the latter in various parts of the world. 
The peculiarities in virulence are such that the problem can be 
solved only with considerable difficulty. The organisms are 
morphologically identical. They are at least closely related, and 
are, therefore, grouped together. The name Sp. anserina was the 
one first used, and, therefore, has priority. The name Sp. gal- 
linarum, however, is more commonly met with in literature. 
Distribution.-The disease has been reported from Russia and 
the Caucasus, Hungary, northern, central, and southern Africa, 
southern Asia, and South America. It probably is wide-spread. 
Morphology and Staining.-The organism of fowl spirillosis is a 
tenuous spiral 10 to 20 p in length, with an average of one spiral 
per micron in length. It is actively motile. No flagella have 426 
VETERINARY BACTERIOLOGY 
been demonstrated. According to Balfour, the organism under- 
goes changes in the body of the intermediary host, the tick (Argas 
Persians') corresponding closely to those described above for Sp. 
duttoni in the tick (Ornithodorus moubata'). The organism pro- 
duces the same characteristic chromatin granules. Chicks in- 
oculated with material showing these granules, but no spirochetes, 
are found to develop typical spirochetosis. 
The organism may be easily demonstrated when living and 
motile. Carbol-fuchsin, Leishman, and Giemsa's stains may be 
used in the preparation of mounts. 
Fig. 163.-Spirochaeta anserina (gallinarim). An agglutinated group 
from the blood of a fowl (Novy and Knapp, in "Journal of Infectious Dis- 
eases"). 
Isolation and Culture.-Noguchi has cultivated this organism 
by his. ascitic fluid-tissue method. 
Pathogenesis.-Experimental Evidence-Many birds may be 
infected by inoculation, among them the goose, duck, fowl, guinea- 
fowl, turtle-dove, sparrow, and other birds; usually not the pigeon, 
although there is disagreement on this point. The rabbit, white 
mouse, guinea-pig, monkey, horse, and man are not susceptible. 
Each of the various types described have usually showed the 
greatest virulence for the species of bird from which it was originally SPIROCHETE GROUP 
427 
described, and many variations in virulence have been observed. 
It is believed that transfer through the intermediary host is neces- 
sary for the retention of virulence. 
Character of Disease and Lesions.-The disease is characterized 
as a true septicemia; Levaditi has shown that the organisms also 
invade the intercellular spaces in various organs. The disease 
runs an acute course, marked by fever. Young fowls may show 
relapses, but the adults rarely. In this respect the disease differs 
from that caused by Spirochaeta obermeieri in man. The organisms 
are present in the blood in great numbers during the crisis. They 
rapidly disappear during convalescence. The mortality varies 
from 80 per cent, or more in some outbreaks to a small percentage 
in others. 
Immunity.-It is probable that agglutinins are formed, but the 
difficulty of getting suspensions of the spirochete is so great that 
the question has not been adequately tested. Immunity is 
developed by an attack of the disease followed by recovery, but 
to what agencies this is due is not certainly known. Marchoux 
and Salimbeni heated blood from infected fowls for five minutes at 
55°, and succeeded in establishing immunity by its injection. 
Transmission.-The disease is commonly transmitted from one 
fowl to another by the ticks Argas persicus, A. miniatus, and pos- 
sibly A. reflexus. The louse is believed also, by Balfour, to be an 
intermediary host. The latter author found that spirochetes 
could be demonstrated in the salivary glands of the tick in fourteen 
days after injection. The necessity for a definite incubation period 
has not been shown. 
Spirochaeta theileri 
Synonyms.-Spirillum theileri; Sp. ovina; Spirochaeta ovis; 
Sp. equi. 
Disease Produced.-Spirillosis in cattle, sheep, and horses. 
Theilcr, in 1902, discovered a spirochete in the blood of cattle 
in South Africa. It has been found since that time in Cameroon, 
East Africa, and Annam. The organism is 0.25 to 0.4 p by 10 to 
30 p. It resembles the preceding morphologically. The disease 
is a benign infection, and the organisms soon disappear. It is 
transmitted by the bite of a tick (Rhipicephalus decoloratus). 428 
VETERINARY BACTERIOLOGY 
Theiler, in 1904, reported the occurrence of a spirochete similar 
to Spirochaeta theileri in the blood of sheep in the Transvaal. 
It was later found in northern Africa. Novy and Knapp have pro- 
posed the name Spirillum (Spirochceta) ovis for this form. The 
same investigator described a spirochete associated with a disease 
of the horse in South Africa, and later it was reported from the 
west coast. Novy and Knapp term this Spirochceta equi. Todd 
and others, by cross inoculations, have demonstrated that these 
organisms from the horse, sheep, and ox all belong to the same 
species. 
Spirochaeta pallida 
Synonyms.-Spirillum pallidum; Treponema pallidum. 
Disease Produced.-Syphilis in man. 
Schaudinn and Hoffmann, in 1905, described the organism 
which is now generally accepted as the cause of syphilis. Prior to 
that date many organisms had been described as possible causes, 
but all are now known either to be secondary invaders or commen- 
sals. 
Distribution.-The disease is widely distributed among all 
civilized peoples. 
Morphology and Staining.-The Spirochuta pallida is a slender 
organism, less than 0.5 p in diameter and 4 to 20 p in length. 
The spirals are quite regular, and vary in number from three to 
forty. A flagellum has been detected at each pole. The organism 
is actively motile. Multiplication is probably by transverse 
division, although longitudinal division is said to occur. The 
organism is so slender, and stains with such difficulty, that there 
are many unsettled points relative to its morphology. Whether 
or not it may pass through a cycle of changes that would mark it 
as certainly protozoan is not definitely known. 
The common anilin dyes used in bacteriologic work fail to 
demonstrate this organism in tissues or in smears. The India- 
ink method of demonstrating spirochetes may be used as a simple 
procedure for showing the organisms in smears. This consists in 
allowing a thin film of India ink to dry upon the smear. The 
organisms do not take up the ink, and may be recognized as 
transparent spirals in the black field. They may also be stained 
by Giemsa's eosin and azur stains. The stain must be freshly SPIROCHETE GROUP 
429 
prepared. Very satisfactory results are achieved by impregna- 
tion with silver, particularly for the demonstration of the organ- 
ism in tissues. 
Isolation and Culture.-Noguchi succeeded in securing pure 
cultures by his ascitic fluid-agar-tissue method. 
Pathogenesis.-Experimental Evidence.-The organism may 
always be found in the primary and secondary lesions of syphilis, 
and has been repeatedly demonstrated in the tertiary as well, 
although it is much more difficult to find in the latter stage. It 
Fig. 164.-Spirochaeta pallida in the lumen of a bronchus in congenital syphilis 
(Hedren). 
may be found in the internal organs of a syphilitic fetus. An 
infection has been produced in the cornea and the iris of the rabbit, 
and the organism shown to be present. The primary and secon- 
dary lesions of the disease have been produced in the monkey, 
particularly in the anthropoid apes, and the spirochetes found in 
each of the stages. The evidence is very strong, therefore, that 
Spirochaeta pallida is the cause of syphilis. Until the organisms 
can be injected in pure cultures and produce the disease this evi- 
dence cannot, however, become indisputable. 430 
VETERINARY BACTERIOLOGY 
Character of Disease and Lesions.-In man the primary lesion 
in the form of a chancre appears in about three weeks after in- 
fection, usually on or near the external genitalia. It is followed by 
invasion of the neighboring lymphatics and by progressive en- 
largement of the lymph-nodes as the disease progresses. Usually 
about six weeks elapse between the appearance of the primary 
and secondary lesions. These latter are probably dependent 
upon an invasion of the blood, and consist of localized skin erup- 
tions, falling of the hair (alopecia), and the symptoms of generalized 
infection, such as fever. This may last for several years and an 
immunity be established, which, however, may not be complete 
enough to prevent gradual sclerosis of blood-vessel walls and 
degenerations in the parenchymatous organs, and even the appear- 
ance of tertiary lesions. 
Immunity.-No practicable method of either active or passive 
immunization against the disease has been developed by the use of 
the organism or its products. 
Bacteriologic Diagnosis.-This may be accomplished by direct 
examination, stained mounts, the Wassermann test, or by chemical 
recognition of certain changes in the character and composition of 
the blood-serum. The organisms may be observed in the fluid 
expressed from fresh tissues by use of dark-field illumination. 
Smears may be prepared and stained with Giemsa's stain, or tissue 
sections may be used. 
The Wassermann test for syphilis has already been described 
in the discussion of fixation of complement in the section on 
Immunity. As an antigen, extracts from the organs of a fetus are 
used, for in these organs the spirochetes are found in the greatest 
numbers. The blood-serum of the suspected patient is tested for 
its possible content of specific amboceptor with fresh guinea-pig 
serum for complement, sheep red blood-cells, and the serum from a 
rabbit possessing hemolytic amboceptor for these erythrocytes. 
The test requires considerable care and must be checked at 
every step. It has been found in practice to give quite re- 
liable data. The test has been modified in many ways since first 
proposed. 
Noguchi has prepared an antigen from pure cultures of Spiro- 
chaeta pallida. He has found that he could obtain positive tests SPIROCHETE GROUP 
431 
only in isolated cases of long-standing syphilis which had been 
treated, and that the positive reactions obtained in active cases 
were not due to antibodies that combine specifically with such 
antigen. Craig and Nichols have shown that serum from un- 
treated cases of syphilis give positive reaction against syphilitic 
liver extract, but negative against the pallida antigen. This 
material, which Noguchi terms luetin, has also been used in a 
dermal test for syphilis. 
Various substances, such as 1 per cent, solutions of lecithin, 
sodium oleate, sodium glycocholate, and taurin have been found 
to give more or less characteristic precipitates with the blood of 
syphilitics. 
Transmission.-The disease is transmitted usually through 
sexual congress, rarely through infective drinking-vessels, closets, 
and by direct inoculation, as sometimes happens in surgical work. 
The disease may be present at birth; the organism may possibly 
enter through the ovum or the sperm, or pass from the circulation 
of the mother to that of the fetus. 
Synonym.- Treponema pertenue. 
Disease Produced.-Yaws in man. 
Castellani, in 1905, reported the occurrence of spirochetes in a 
tropical disease known as yaws. The organism resembles that of 
syphilis, but is probably distinct, as shown by inoculation experi- 
ments and study of specific antigens and antibodies in comparison 
with those of syphilis. 
Spirochaeta pertenuis 
Other Spirochetes 
Dodd, in 1906, found a spirochete in a disease of the pig in 
South Africa. It was associated principally with dark, hemor- 
rhagic lesions of the skin. 
Spirochetes may be found in considerable numbers in the 
mouth, and, under certain conditions, in the intestinal tract, upon 
the skin, and about the genitalia. They are, for the most part, 
believed to be harmless commensals. CHAPTER XXXVII 
ACTINOMYCES GROUP 
The members of this group are often called Trichomycetes or 
thread fungi. In many of their morphologic characters they 
resemble bacteria. Frequently they occur as short rods that 
cannot be differentiated by examination from true bacilli. Usu- 
ally, however, they occur in threads, which in some genera may be 
branched. These threads may show more or less differentiation 
into parts, and certain portions may develop into conidia or spores. 
These organisms show a more complex life-history, therefore, than 
do the true bacteria. On the other hand, they can scarcely be 
grouped with the true' molds, as they are much simpler in struc- 
ture. They may be considered as a group, therefore, related closely 
to both bacteria and molds and partaking of the nature of each. 
These organisms show such diversity of morphology in the 
animal body and in culture-media that a satisfactory classification 
into species and genera is a difficult problem. Many generic 
names have been proposed. They will all here be regarded as 
belonging to the genus Actinomyces. 
Several organisms are included in this genus, as here dis- 
cussed, that may be shown to belong to the bacilli and not to the 
trichomycetes. 
Many species of organisms of this group are known from the 
descriptions of a single author only. It is difficult to determine 
from these descriptions how many are valid species and how many 
merely synonyms. The facts seem to be that Actinomyces are 
widely distributed in nature. They may be isolated in abundance 
from most soils, and may be found to develop upon almost any 
plate of medium exposed to the air. Only under exceptional 
conditions are they pathogenic, but it is probable that the species 
usually described as such are normally saprophytes that can 
upon occasion proliferate in the tissues of the body and produce 
disease. The fact that cattle become infected through the gums 
or tongue, where the awns of certain grasses penetrate, that the 
barley testers, who bite the barley grain to determine its brewing 
432 ACTINOMYCES GROUP 
433 
quality, are most frequently infected among men in temperate 
climates, that injuries to the feet of natives of certain tropical 
countries (where no adequate protection is worn on the feet) are 
frequently followed by local infections, and that Actinomyces have 
Fig. 165.-Actinomyces calicolor, a non-pathogenic trichomycete from 
the soil. A ring colony on a semisolid medium showing filaments and 
aerial hyphae (Muller). 
been found causing infections in practically all domestic animals 
by one investigator or another is evidence of the wide distribution 
of the members of the genus. The species to be described are 
Actinomyces bovis and A. nocardia in cattle, A. caprce in goats, A. 
madurce, and A. eppingeri in man. 
The group, as a whole, may be characterized as consisting of 
slender, branching organisms, which may develop into colonies 
made up not only of threads but rods, cocci, and other cell forms. 
Frequently, in animal tissues, and sometimes upon artificial media, 
the ends of the threads may be clubbed. When grown upon the 
surface of artificial media some forms develop aerial hyphae, 
which segment into chains of conidia. All species retain the Gram 434 
VETERINARY BACTERIOLOGY 
stain to a greater or less degree. Some are aerobic, others facul- 
tative, and still others obligate anaerobes. Pigments are produced 
by some species. 
Actinomyces bovis 
Synonyms.-Streptothrix bovis; Cladothrix actinomyces; Str ept o 
thrix actinomyces; Discomyces bovis. 
Fig. 166.-Actinomyces ccelicolor. Colony on agar. This colony structure 
is quite typical of many species (Muller). 
Disease Produced.-Lumpy jaw and wooden tongue (actino- 
mycosis) in cattle, and probably related infections in other animals 
and man. 
Harz, in 1878, gave the name Actinomyces to the ray-fungus, 
which Bollinger, in the preceding year, had found present in the 
characteristic tumor-like growths in cattle. 
Distribution.-The infection is known from Europe and North 
and South America. 
Morphology and Staining.-In the infected tissues the organism 
forms minute yellowish granules, sometimes large enough to be 
readily observed by the unaided eye. These granules are made up 
of compact masses of the organisms. Branched filaments, with a 
more or less radial arrangement, are to be observed occupying 
the central portion, commonly mixed with coccus-like degeneration 
products. The margin of the granule or rosette, when examined 
in cross-section, is found to consist of club-like enlargements of 
the threads, showing a marked refractivity to light. The filaments ACTINOMYCES GROUP 
435 
are slender, usually about 0.5 /j. in diameter. It is believed that 
the formation of the clubbed ends is correlated in some way with 
the resistance of tissue to invasion. They have been variously 
regarded as degeneration products, involution forms, and as indi- 
cating a thickening of the sheath to protect the organism against 
antibodies produced by the tissues. Young colonies on artificial 
media consist of interlacing, branched threads, which tend to form 
compact masses. These commonly break up into bacillus-like 
segments, in a manner not unlike the formation of certain spores 
among higher fungi, by segmentation of the hyphal threads. 
Whether or not these correspond to the oidial type of spore pro- 
duced in the higher fungi, or represent spores at all, is not known. 
The clubbed type rarely develops in artificial media. The organism 
stains readily with the common anilin dyes and is Gram-positive. 
It is not acid fast. 
Isolation and Culture.- 
The organism is not easily iso- 
lated in pure cultures, par- 
ticularly when it occurs in 
mixed cultures with pyogenic 
cocci in the lesions. Wright 
has described a technic which 
he found quite uniformly suc- 
cessful. Pus or tissues con- 
taining the organism in fila- 
mentous rosettes is preferable 
to that containing only the 
clubbed type, as in the latter 
degeneration has gone so far 
that frequently no growth will 
occur. The granules are washed in sterile water, crushed be- 
tween sterile slides, and inoculated in varying amounts into 
tubes of melted 1 per cent, dextrose agar, and incubated at 37°. 
In his experience the colonies developed characteristically from 
5 to 12 mm. below the surface, but others have found them to 
form quite as well upon the surface of the medium. Isolated 
colonies may then be transferred to other media. In bouillon 
the organism forms distinct, solid, spherical, or mulberry-like 
Fig. 167.-Actinomyces bovis, tissue 
section showing the radial arrangement 
and the clubbing of threads (Gunther). 436 
VETERINARY BACTERIOLOGY 
masses at the bottom of the tube. Growth is secured with diffi- 
culty upon the surface of the medium, according to Wright, but 
other investigators have not experienced the same difficulty. It 
forms on agar and glycerin agar colonies, which at first resemble 
tiny drops of amber; these enlarge, and either remain discrete 
or coalesce to form a distinctly wrinkled, 'lichen-like" mem- 
brane, which frequently has a dusty appearance. Gelatin is 
slowly liquefied. 
Physiology.-The organism may be regarded as a facultative 
aerobe, as growth appears to take place best under anaerobic 
conditions. The optimum 
growth temperature is 37°. 
The organism is resistant 
to desiccation and will live 
for a long period, probably 
months, in a dried condi- 
tion. Gelatin is liquefied. 
There is no gas- or acid- 
production. A brown to 
black pigment may be 
produced. 
Pathoge nesis.-Experi- 
mental Evidence.-In the 
great majority of cases ex- 
perimental inoculation i s 
without result. Many ani- 
mals have been used - 
cattle, sheep, swine, dogs, cats, rabbits, and guinea-pigs. In 
relatively a few cases significant lesions have been developed. 
Musgrave, Clegg, and Polk have produced extensive suppura- 
tive lesions by intraperitoneal inoculation of the monkey, the 
infection terminating fatally in about three weeks. The com- 
mon lack of pathogenesis may be due to differences in resistance, 
to a diminution of virulence due to cultivation, or to the manner 
of inoculation. In cattle it may gain entrance with a grass awn, 
and this may protect it from the destructive agencies of the 
tissues until its pathogenicity is well established. 
Character of Disease and Lesions Produced.-A swelling or 
Fig. 168.-Actinomyces bovis (Strepto- 
thrix actinomyces), stained mount from 
culture-medium (Musgrave, Clegg, and 
Polk, in "Philippine Journal of Science"). ACTINOMYCES GROUP 
437 
tumor-like mass develops in cattle at the site of infection. This 
softens and ultimately discharges thick, yellowish pus. The 
discharge after the lesion has opened may become intermittent 
in character. When the tongue is the primary seat of infection 
it becomes swollen, indurated, and protrudes from the mouth in 
some cases. The bones of the jaw are often attacked. The in- 
fection is chronic. Animals rarely die from immediate effects. 
In a few instances metastatic infection of other parts of the body 
than the head and neck have been reported. 
In man the disease usually attacks the softer tissues, progresses 
more rapidly than in cattle, and is apt to terminate fatally from 
metastatic infection. Whether or not the organism isolated from 
human actinomycosis is the same as that found in cattle is uncer- 
tain. The same may be said of the forms that have been isolated 
from similar infections in other animals, among them the horse, 
dog, and pig. 
Immunity.-No method of immunization against the disease 
has been developed. 
Bacteriologic Diagnosis.-A microscopic examination of the 
unstained pus will usually reveal the characteristic granules, 
with the radial arrangement of clubs or of tangled bits of branched 
threads. A film stained by Gram's method will bring the latter 
out clearly when present in small numbers only. 
Transmission.-It is believed that the organism commonly 
enters the body through a trauma, through carious teeth, or by 
being carried into the tongue or the gum with the sharp awns of 
certain grasses and grains. So far as known the disease is wholly 
non-contagious. 
Actinomyces nocardii 
Synonyms.-Streptothrix nocardii; A ctinomyces farcinica;.Strep- 
tothrix farcinica; Nocardia farcinica. 
Disease Produced.-Bovine farcy. Farcin du bceuf. 
Nocard, in 1888, first described an Actinomyces or Strepto- 
thrix from the lesions of cattle in Guadeloupe suffering from a 
disease termed bovine farcy. The disease itself has not been 
adequately studied, although the organism has been investigated 
by several workers. There is no record of its occurrence in the 
United States. 438 
VETERINARY BACTERIOLOGY 
Morphology and Staining.-The organism is slender, much 
branched, and interwoven. In culture-media short, plump fila- 
ments with branches may occur, and in old cultures many ovoid 
cells are found. The organism is Gram-positive, and many por- 
tions, particularly in old cultures, are acid fast and also alcohol 
fast. 
Isolation and Culture.-It may be isolated in pure culture from 
lesions directly upon artificial media. The colonies upon agar 
Fig. 169.-Actinomyces nocardia, stained mount from culture (Musgrave, 
Clegg, and Polk, in "Philippine Journal of Science"). 
are small, white, irregular, raised, and opaque. Upon glycerin 
agar they are at first delicate, but soon coalesce and present 
a moist, meal-like growth. Bouillon is never clouded, but a 
grayish, flocculent mass forms at the bottom. Milk is un- 
changed. 
Physiology.-The organism is a facultative aerobe. It de- 
velops best at 37°. It is resistant to desiccation and maintains 
its virulence when cultivated. 
Pathogenesis.-Experimental Evidence.-Guinea-pigs are easily 
infected by intraperitoneal injections. The organism produces 
numerous nodules resembling tubercles upon the peritoneum 
and the abdominal organs, particularly the liver, spleen, and ACTINOMYCES GROUP 
439 
kidneys. Intravenous injection gives rise to a condition resembling 
generalized miliary tuberculosis. Intraperitoneal injection of the 
monkey gives rise to similar lesions. Cattle and sheep develop, 
at the point of a subcutaneous inoculation, an abscess which dis- 
Fig. 170.-Actinomyces caproe, stained mount from culture (Musgrave, Clegg, 
and Polk, in "Philippine Journal of Science"). 
charges, ulcerates, and may disappear, to reappear after an 
interval. 
Character of Disease and Lesions.-The disease in cattle is 
characterized by an enlargement of the superficial lymph-nodes, 
which ulcerate and have much the appearance of farcy in the horse. 
The internal organs may be affected, with a resultant pseudo- 
tuberculosis. 
Immunity.-Methods of immunization have not been de- 
veloped. 
Bacteriologic Diagnosis.-The organism may be recognized 
in preparations from the lesions, but, for differentiation from other 
Actinomyces or Streptothrices, culture and animal inoculation 
are necessary. 
Transmission.-The disease is probably transmitted by wound 
infection, but this is not certainly known. 
Synonyms.-Streptothrix caprce and possibly >S. canis. 
Actinomyces caprse 440 
VETERINARY BACTERIOLOGY 
Disease Produced.-Actinomycosis (streptothricosis) in goats, 
possibly in the dog. 
Silberschmidt, in 1899, publishes a description of an organism 
belonging to this group, which he isolated from a goat affected 
with a disease which closely simulated tuberculosis. It has been 
studied by several other investigators, who are in agreement that 
it should be regarded as a distinct species. 
Morphology and Staining.-Morphologically, it resembles the 
true bacteria more than other members of this group. The fila- 
ments are comparatively short and show little tendency to form 
tangled masses, but separate easily. Both in culture and in the 
lesions rod forms and cocci are predominant. It stains with the 
anilin dyes rather irregularly, and is alcohol and acid fast. 
Isolation and Culture.-The organism grows rather readily 
upon most of the laboratory media, so that isolation is not a matter 
of difficulty. Upon agar the growth appears in two to three days 
as small, brownish colonies. It is somewhat more luxuriant upon 
glycerin and maltose agar, the colonies coalescing to give the 
growth a moist, mealy appearance. The colonies are light brown 
in color. Growth upon potato is similar. In bouillon the colonies 
develop upon the surface as fine dry disks, and form a. pellicle, 
which finally settles as a sediment, the broth remaining clear. 
Physiology.-The organism is a facultative aerobe. 
Pathogenesis.-The organism produces tubercle-like lesions 
in the rabbit, guinea-pig, and monkey upon inoculation. It is not 
of any considerable economic importance. 
Actinomyces necrophorus 
Synonyms.-Bacillus diphtherice vitulorum; B. filiformis; 
Streptothrix cuniculi; Actinomyces cuniculi; B. necroseos; Strep- 
tothrix necrophora; Bacillus necrophorus. 
Diseases Produced.-A large number of diphtheritic and ne- 
crotic pathologic conditions in animals, necrobacillosis. 
Loffler, in 1894, described this organism from calf diphtheria. 
Later Schutz found it associated with the intestinal ulcerations of 
hog-cholera. It is now known to produce spontaneous disease in 
birds and in both domestic and wild animals, including cattle, 
sheep, goats, antelope, reindeer, horse, deer, roe, swine, kangaroo, 
guinea-pig, dog, monkey, and fowl. ACTINOMYCES GROUP 
441 
Distribution.-Bang succeeded in demonstrating the presence 
of this organism in the feces of normal hogs, but not in the intes- 
tinal contents of the cow. It is probably rather widely distributed 
in some localities. The infection has been described from various 
sections of Europe and America. 
Morphology and Staining.-The organism is a long, slender rod, 
usually bent more or less, although short rods and filaments may 
be observed. It is about 0.7 to 1.5 /z in diameter, and is generally 
thicker in cultures than in tissues. The filaments vary between 
2 and 100 /z. In the tissues and colonies the filaments are matted 
together, but definite branching has not been satisfactorily demon- 
strated. The stained rods are usually beaded, giving rise to a 
very characteristic appearance. Involution forms, as long clubs, 
frequently occur. The organism is non-motile and does not pro- 
duce spores or capsules. It stains readily with the common anilin 
dyes, but is Gram-negative. 
Jensen has developed the following method of staining the 
organism in tissues: The piece of tissue is fixed in Muller's fluid, 
washed, and hardened in alcohol. The sections are stained some 
minutes in toluidin-safranin, dehydrated with a concentrated alco- 
holic solution of safranin, and decolorized in a concentrated solution 
of fiuorescin in clove oil, then in clove oil, alcohol, counterstained in 
aqueous methyl-green, dehydrated by alcohol, xylol, and mounted 
in balsam. The necrosis bacilli are stained red, the tissue, green. 
None of the other bacilli studied by Jensen give this reaction. 
The peculiarities of staining, and the possession of easily stained 
granules, or of a vacuolate protoplasm, have caused some authors 
to group this germ with the diphtheria bacillus, but these appear- 
ances are even more characteristic of certain of the Actinomyces, 
particularly those isolated from soil. 
Isolation and Culture.-Actinomyces necrophorus is most 
easily isolated in pure culture by inoculating diseased tissue into 
rabbits or white mice. The pure culture may then be secured 
from the infected organs. The cultural characters are all modi- 
fied by the fact that the organism is a strict anaerobe. 
Colonies may develop upon the surface of serum agar plates if 
the oxygen is removed by the alkaline pyrogallate method, but not, 
according to Mohler and Morse, in an atmosphere of hydrogen 
or in a vacuum. They appear in forty-eight hours as minute, 442 
VETERINARY BACTERIOLOGY 
dirty white, round, opaque colonies, with gas bubbles developing 
below the surface. In seventy-two hours the colony appears wooly, 
and the central portion, upon microscopic examination, is shown 
to be a felted mass of threads with a border of long, wavy filaments. 
The addition of serum to media increases materially the luxuriance 
of the growth. Bouillon becomes turbid, and then gradually clears, 
with subsidence of the organism. Gelatin is not liquefied. 
Physiology.- Actinomyces necrophorus is an obligate anaerobe. 
Its temperature growth limits are between 30° and 40°, with an 
optimum at about 35°. The organism is readily destroyed by 
Fig. 170a.-Actinomyces necrophorus (Mohler, Bureau of Animal Industry, 
Circular No. 160). 
disinfectants. No pigment is produced. A very characteristic 
odor, "between the odor of cheese and of glue," may be noted in 
both cultures and lesions. No enzymes capable of liquefying 
gelatin or blood-serum are produced. Gas is formed in bouillon. 
Milk is not coagulated nor are acids formed. Indol is produced. 
Pathogenesis.-Experimental Evidence.-Rabbits may be read- 
ily infected with the Actinomyces necrophorus. A subcutaneous 
injection of a small amount of necrosed tissue results in the 
death of the rabbit in about a week. The inoculated area is 
necrotic to some depth, and to a distance along the surface of | to 
1 inch from the point of injection. The necrosis is complete and ACTINOMYCES GROUP 
443 
the tissues wholly disintegrated. In some cases gas bubbles may 
be observed. The inoculation of pure cultures results in death 
more slowly; frequently two weeks are required. The animal 
dies suddenly after a series of convulsions. Mice are readily 
infected. Guinea-pigs are much more refractory, but occasion- 
ally die as a result of inoculation. There seems to be abundant 
experimental evidence to connect the Actinomyces necrophorus 
with many types of necrosis in animals. There is no evidence 
that the organism enters the normal healthy unbroken skin. It 
is usually a secondary invader. 
Character of Disease and Lesions Produced.-Mohler and Wash- 
burn have given an excellent resume of the conditions under which 
this organism has been found. In many of these conditions it 
has not been satisfactorily established that this organism is the 
sole cause, for pyogenic cocci and other organisms may produce 
the same changes. More work is needed upon these infections. 
The possible presence of this organism in necrotic infections of all 
kinds must be borne in mind. The organism has been reported 
from the following infections, and probably in most cases is re- 
sponsible for the accompanying necrosis: necrotic dermatitis, ne- 
crotic scratches in the horse, necrotic pox in horses, cattle, goats, 
and hogs, several types of necrosis in rabbits, necrosis of the hoof 
in the horse, necrosis of the mouth and esophagus, ulcerative and 
necrotic vulvitis, vaginitis, and metritis, foot-rot of cattle, lip and 
leg ulceration of sheep, necrotic omphalophlebitis, and joint ill in 
young animals, necrosis in the alimentary tract and other viscera 
in many animals, and possibly even avian diphtheria. It is some- 
times of considerable economic significance, particularly in the 
so-called lip and leg ulceration of sheep. Some of the affections, 
particularly this latter, are known to be contagious. Much work 
still remains to be done, however, on the different infections and 
possible variations in virulence. 
The lesions produced in all tissues have many common charac- 
ters. They are essentially coagulation necroses with caseation. 
Metastatic infection is very apt to occur. The local lesion is 
described by Mohler and Morse as a "sharply circumscribed patch 
of yellowish or dull brown, sometimes greenish-white, homogeneous, 
structureless, dry, crumbly tissue debris of soft, cheesy consistence, 444 
VETERINARY BACTERIOLOGY 
resembling compressed yeast, and manifesting a characteristic 
stench. The line of demarcation between the living tissue and the 
dead mass is a narrow hyperemic zone." A false membrane is 
formed over the surface as a "result of coagulation necrosis of the 
inflammatory exudate and entanglement in its meshes of the hya- 
line degenerated tissue-cells and leukocytes." 
Immunity.-It has been suggested that the organism produces 
a true toxin because of its intense local destruction of tissue, and 
because of the death of laboratory animals with many of the symp- 
toms of a toxemia. No toxin has been isolated, however, in spite 
Fig. 171.-Actinomyces maduroe, stained mount from culture (Musgrave, Clegg, 
and Polk, in "Philippine Journal of Science"). 
of many attempts. Probably an endotoxin is produced. It is 
stated that intravenous injections of the organism into the goat 
confer an immunity. No practicable method of immunization 
has been developed. 
Bacteriologic Diagnosis.-The organism may be observed in 
mounts prepared from the tissue just surrounding the necrosed area. 
Its appearance is characteristic enough to differentiate it from other 
forms that may be present. Animal inoculations, preferably into 
the rabbit, are generally necessary to secure pure cultures. 
Transmission.-It is improbable that the organism ever gains 
entrance through the unbroken skin or mucous membrane. ACTINOMYCES GROUP 
445 
Scratches, wounds, abrasions, or injuries of other types supply an 
infection atrium. The disease, however, must be regarded as 
mildly contagious. 
Actinomyces madurae 
Synonym.-Streptothrix madurce. 
Disease Produced.-Madura-foot, mycetoma, streptothricosis 
in man. 
Vincent, in 1894, cultivated an Actinomyces from cases of 
mycetoma or madura-foot in man. This disease occurs in certain 
tropical countries, as southern Asia and the Philippines. It is 
undoubtedly a different species from those already described. It 
is not known to affect animals in nature, but will infect the monkey 
upon intraperitoneal inoculation. 
Actinomyces of Other Infections 
Probably about thirty or more other species have been described 
belonging to this genus. In most cases they have been reported 
but once or have been incompletely described. As has before 
been emphasized, careful work is still needed in order to deter- 
mine the true number of valid species and their relationship to 
disease. CHAPTER XXXVIII 
BLASTOMYCETES 
The genus name Blastomyces is used to designate a group of 
pathogenic fungi having many points in common with the members 
of the genera Saccharomyces and possibly Torula. It is not 
certainly known that the forms thus classified are closely related 
among themselves, for it is a well-known fact that many of the 
Hyphomycetes, when grown in certain culture-media, will assume 
a form indistinguishable from the yeasts. It is possible, therefore, 
that some of the forms described as members of the genus Blasto- 
myces may be only growth 
stages of higher forms. Here, 
again, as has been emphasized 
in other groups, there is need 
still for careful morphologic 
and cultural studies of the 
various species that have been 
described, for some of them 
are very imperfectly known. 
An understanding of the 
morphology of the Blasto- 
myces can best be obtained by 
a preliminary discussion of the 
Saccharomyces or true yeasts. 
The one character which sepa- 
rates this genus from the Hyphomycetes is the difference in the 
vegetative method of reproduction. This is accomplished by 
budding. The mother-cell is usually oval or round, and, at 
various points on its surface, produces small buds, which enlarge 
and soon separate as independent cells. Occasionally these cells 
may remain together and become considerably elongated. By 
continued budding from the tip, a chain of cells is formed simu- 
lating a mycelial thread of one of the Hyphomycetes. The 
Fig. 172.-Brewer's yeast, Saccharo- 
myces cerevisia (Gunther). 
446 BLASTOMYCETES 
447 
cell differs from that of a bacterium by the presence of a 
definite nucleus, which may be demonstrated by careful staining 
technic. 
Spores are produced by some yeasts when the cells are brought 
under the right conditions of moisture, oxygen pressure, and 
temperature. Generally, two, four, or six are produced within 
a single cell. This type of spore formation relates such forms 
definitely to the higher fungi, known as Ascomycetes or sac 
fungi. In these fungi the spores are borne in a sac or ascus, 
and the cell of the yeast, with its contained spores, is supposed 
to represent a simple type of ascus. Resting cells, consist- 
ing of heavily walled or encapsulated cells filled with protein, 
glycogen, or oil-granules, are formed by many yeasts. These 
granules may resemble spores and have doubtless many times 
been mistaken for them. When brought under favorable con- 
ditions the cell, as a whole, begins again to produce buds, 
showing conclusively that the granules cannot be regarded as 
spores. 
Among the true yeasts those which are not known to produce 
spores are sometimes placed in the form genus Torula. It is not cus- 
tomary to make this distinction among the pathogenic yeasts or 
Blastomyces, although it has been attempted by some authors. 
As here used, the term Blastomyces includes all those pathogenic 
forms which reproduce regularly by budding, and may or may not 
produce ascospores. 
The organisms belonging to this group are Blastomyces farci- 
minosus, B. dermatitidis, and B. coccidioides. 
Blastomyces farciminosus 
Synonyms.-Cryptococcus farciminosus; Leishmania farcimi- 
nosa. 
Disease Produced.-Blastomycetic epizootic lymphangitis or 
pseudofarcy in the horse. 
Rivolta, in 1873, first described the organism associated with 
this disease. Tokoshige, in 1897, cultivated the organism and 
determined its classification. It has been studied since that time 
by several investigators. Galli-Valerio contends that this organ- 
ism is a protozoan and not a Blastomyces. There is a clinically 448 
VETERINARY BACTERIOLOGY 
similar disease, since described in Europe and the United States 
as due to a member of the mold genus Sporotrichum. The organ- 
ism described by Tokoshige should be reinvestigated. It is pos- 
sible that it may prove to be a Sporotrichum also. 
Distribution.-The disease is known from Italy, Egypt, Tunis, 
England, France, northern Europe, Japan, India, the Philippines, 
and possibly the United States (North Dakota, Iowa). 
Morphology and Staining.-The organism as it occurs in tissues 
does not show budding forms ordinarily, but reproduces by a 
series of sporulations. In mounts prepared from the tissues it 
has a double refractive contour, which makes it stand out dis- 
tinctly from the remainder, even when unstained. It is usually 
spherical or ovoid, 3 to 4 ju in diameter. The cell contents may be 
homogeneous or granular. In culture-media the organism consists 
of hyphal and spherical forms. Cells 
with buds may be found, identifying 
the organism definitely with the Blas- 
tomycetes. Cells containing granules, 
and resembling closely the resting cells 
of the yeasts, are common. It has 
not been conclusively shown that true 
sporulation takes place in culture- 
media. The organism stains readily 
with aqueous anilin dyes and is Gram- 
positive. The latter method of stain- 
ing is useful in demonstrating the 
organism in pus or tissues. The alcohol must not remain too long 
in contact with the organism or it will lose color. 
Isolation and Culture.-The Blastomyces fardminosus is isolated 
upon culture-media with considerable difficulty. Several investi- 
gators, particularly those holding to the protozoan nature of the 
organism, deny that it has been accomplished. In view of the 
success which has attended the cultivation of an essentially similar 
organism in man, there seems no good reason to deny that Toko- 
shige and others have succeeded in securing it in pure cultures. A 
slightly acid medium is said to be more favorable than one which is 
alkaline. Growth is very slow in any event. 
Bouillon finally shows a white, flocculent deposit. Upon 
Fig. 173.--Blastomyces far- 
ciminosus, cells from culture- 
media (adapted from Toko- 
shige). BLASTOMYCETES 
449 
the surface of agar, gray-white granular colonies make their ap- 
pearance in the course of a month, and finally attain to a diameter 
of 1 to 4 mm. The colony is wrinkled, and can be removed only 
with difficulty. Growth upon gelatin is essentially similar. 
Potato seems somewhat more favorable, and growth occurs more 
rapidly, but is of the same character as on agar. 
Physiology.-The organism is aerobic. Growth occurs at 
room-temperature as well as at 37°. It does not liquefy 
gelatin. Sugars are not fermented with production of either 
acid or gas. 
Pathogenesis.-Experimental Evidence.-Guinea-pigs and rab- 
bits are not easily infected with pure cultures. Typical lesions 
have been produced in the horse by Tokoshige. They are readily 
produced by the injection of pus from natural infections. 
Character of Disease and Lesions.-The disease in the horse 
shows a marked superficial resemblance to farcy. The infection 
progresses through the subcutaneous lymphatics and forms dis- 
tinct nodules. These may suppurate. Metastatic infection of 
the internal organs occasionally occurs. 
Immunity.--No practicable method of immunization has been 
developed. 
Bacteriologic Diagnosis.-The organism may be readily ob- 
served in a mount of the pus from a lesion stained by Gram's 
method. 
Transmission.-It is supposed that infection is traumatic, that 
the organism gains entrance through cutaneous lesions. The 
disease not highly contagious. 
Blastomyces dermatitidis 
Synonyms.-Saccharomyces dermatitidis; O'idium dermatitidis. 
Disease.-Blastomycetic dermatitis in man. 
Busse, in 1894, first described an organism of this group as the 
cause of a fatal infection in man. Gilchrist, in 1896, found a 
similar organism as the cause of a dermatitis in man. Since that 
time the organism has been repeatedly isolated and studied. 
Distribution.-Blastomycetic dermatitis has been reported 
from the United States, the Philippines, and Europe. 
Morphology and Staining.-It is probable that several distinct 450 
VETERINARY BACTERIOLOGY 
species have been grouped together; that is, not all cases have 
shown morphologically identical organisms to be present. They 
have not as yet been sufficiently studied to justify their separation 
as distinct species, but will be treated rather as one polymorphic 
form. Careful morphologic and cultural studies are still needed. 
In the tissues the organisms appear almost invariably as 
budding forms. The cells are spherical or ovoid, from 10 to 17 g 
in diameter. They are distinctly double contoured. Several 
investigators have observed what they believe to be sporulating 
forms. The cells are frequently granular or vacuolate, resembling 
Fig. 174.-Blastomyces dermatitidis. Budding forms and mycelial growth 
from glucose agar (Irons and Graham, in "Journal of Infectious Diseases"). 
typical yeast cells in this respect. Upon culture-media numerous 
hyphal threads and budding cells are produced. 
The organisms do not stain very readily with the aqueous 
anilin dyes. 
Isolation and Culture.-Isolation of the organism is usually 
attended with considerable difficulty. Blood-serum slants are 
usually employed and inoculated with material from the lesion. 
Repeated trials are sometimes necessary before a growth is secured. 
After once accustomed to growth on artificial media, no difficulty 
is found in getting the organism to develop upon most of the 
common culture-media. BLASTOMYCETES 
451 
Small white colonies showing a mold-like surface, due to the 
formation of numerous aerial hyphae, develop upon the surface 
of agar. The addition of dextrose to the medium somewhat in- 
creases the luxuriance of the growth. In bouillon a fluffy, mold- 
like colony or a granular sediment develops without any evidence 
of the diffuse clouding generally found in yeast cultures. Gelatin 
is not liquefied. Milk may or may not show coagulation and 
slight digestion of the casein. Potato is a favorable medium. 
Fig. 175.-Blastomyces dermatitidis (Hamburger, in "Journal of Infectious 
Diseases"). 
Physiology.-Growth occurs at room-temperatures, but some- 
what more luxuriantly at 37°. The organism is aerobic and facul- 
tative anaerobic. Gas and acids are not produced in carbo- 
hydrate media. 
Pathogenesis.-Experimental Evidence.-Guinea-pigs and rab- 
bits may be infected, with production of either a local abscess or 
generalized blastomycosis. The lesions resemble in their essential 
characters those found in the human body. 
Character of Disease and Lesions.-In man a papule generally 
appears upon one of the extremities, the face, or more rarely, 452 
VETERINARY BACTERIOLOGY 
elsewhere. A viscid pus is exuded, and there is commonly con- 
siderable enlargement. Healing with an abundant formation of 
cicatricial tissue gradually occurs. Usually the lymphatics are 
not involved, the disease differing in this respect from the lymph- 
angitis of the horse. The course of the disease is usually chronic, 
and it may persist for years, new ulcers appearing successively on 
various parts of the body. Generalization has been reported in a 
considerable number of cases. The skin lesions have sometimes 
been confused with those of syphilis and tuberculosis. Primary 
infection of the lungs has been shown in several cases. 
Immunity.-No method of establishing immunity has been 
developed. 
Bacteriologic Diagnosis.-This may be accomplished by direct 
microscopic examination of the pus. Phalen and Nichols state 
that the organism may be most easily demonstrated by treating 
unstained sections with potassium hydrate and mounting in 
glycerin. 
Transmission.-It is supposed that infection sometimes occurs 
through wounds, but several instances have come to light in which 
the infection was primarily pulmonary and the skin lesions sec- 
ondary. 
Blastomyces coccidioides 
Synonym.-O'idium coccidioides. 
Disease Produced.-Blastomycosis, so-called coccidioidal gran- 
uloma in man. 
Posades and Wernecke, in 1892, first reported a case of so- 
called coccidioidal granuloma from Argentina. In the United 
States the disease has been recorded principally from California, 
particularly in the San Joaquin Valley. 
Morphology.-This form is of particular interest, because the 
budding or true blastomyces form very rarely occurs in the tissues 
and multiplication is almost wholly through sporulation. The 
organism in the tissues is spherical and doubly contoured. It may 
reach a diameter of 30 /j. or even more. Budding forms have been 
recorded from pus. In artificial media the organism resembles 
a mold, but budding forms may be observed. The method of 
reproduction in tissues, by the formation of spores within the 
mother-cell and their liberation by a rupture of the cell membrane, BLASTOMYCETES 
453 
has led some investigators to believe that the organism is really 
a protozoan. However, sporulation of this general type occurs 
in the yeasts. The question seems to be definitely settled in 
favor of the plant hypothesis by the culture forms on artificial 
media. 
Pathogenesis.-Whether this organism should be separated 
from the preceding is uncertain. Frequently no cutaneous lesions 
are produced; the infection is systemic and probably always fatal. 
Meningeal involvement is common. CHAPTER XXXIX 
MOLD OR HYPHOMYCETE GROUP 
The term Hyphomycete, as usually interpreted, is one of con- 
venience only, for within this group are included members of the 
four great divisions of fungi generally recognized by botanists. 
The members of the group are many times not closely related. 
They all resemble each other in having a plant body or mycelium, 
which consists of threads or hyphae made up, in the majority of 
forms, of chains of cells. Reproduction is not generally by budding, 
although this may sometimes occur. The hyphae themselves break 
up into spores, or spores are borne at the tips of hyphae that have 
been differentiated for the purpose. The hyphae may unite to 
form a more or less solid mass, sometimes tissue-like in appearance. 
This mass may remain viable when dried for a considerable time, 
and may function in much the same manner as a resistant spore 
in tiding the organism over unfavorable conditions. Such a 
structure is called a sclerotium. The names given to these various 
types of structures have already been discussed under the heading 
of Morphology in Section I. 
The Hyphomycetes, for the most part, belong to the division 
of fungi termed Fungi imperfecti by the botanist. The name is 
derived from the fact that these fungi are not known to produce 
perfect or sexual spores. Hundreds of genera and thousands of 
species have been described as belonging to this group. Many of 
these are doubtless simply developmental stages of forms that 
are known under other names. The life-history of some fungi 
has been found to be so complex, and consists of so many stages, 
that five or six names have been applied and the different stages 
put in different groups of fungi, until it was found that all were 
the same polymorphic species. Unfortunately, careful mor- 
phological study has not been made of the pathogenic members 
of this group, and there is the greatest confusion in the nomen- 
clature. The pathologists and bacteriologists who have de- 
454 MOLD OR HYPHOMYCETE GROUP 
455 
scribed the organisms have rarely paid any attention to their 
botanical relationships, and the organisms themselves, for the 
most part, have been ignored by the botanist in his classification. 
As before stated, the possession of a more or less definite 
mycelium, a more or less "mold-like" growth, and the general 
production of spores are all that is needed to include an organism 
in the group. Many of the organisms of the group are very com- 
mon in nature and are pathogenic only under exceptional condi- 
tions, while others have so adapted themselves to a parasitic 
existence that they may be regarded as obligate parasites. Many, 
too, have been noted once or twice only in certain pathologic 
conditions, and it is by no means certain that they were more than 
accidental parasites. 
The genera of molds containing species of known pathogenicity 
are- 
Aspergillus. 
Penicillium. 
Fusarium. 
Sporotrichum. 
Microsporon and Trichophyton. 
Achorion. 
O'idium or Oospora. 
The Genus Aspergillus 
The Aspergill! are widely distributed in nature. They are 
abundant in the soil and on decaying materials of all kinds. 
Their spores are common in the air, and cultures may readily be 
secured in most localities by simple plate exposure. They are not, 
however, present in such numbers as the genus next to be de- 
scribed, Penicillium. Several hundred species have been de- 
scribed, and by some authors the genus is subdivided into two 
genera, Aspergillus and Sterigmatocystis. 
Aspergillus is placed by the botanists among the Ascomycetes 
or sac fungi, because at one stage in the life-history sexual repro- 
duction occurs, resulting in the formation of sacs filled with spores. 
This phase of the life-history has been worked out in but few species; 
however, it is probable that it occurs in all when grown under the 
right conditions. 456 
VETERINARY BACTERIOLOGY 
The mycelium of Aspergillus is colorless and hyaline, much- 
branched, penetrating for a short distance into the substratum or 
medium, and usually sending up aerial hyphae, which give the 
colony a floccose or downy appearance. The hyphae are septate, 
that is, cross walls are formed and the cells are divided from each 
other by them. Asexual reproduction takes place by the forma- 
tion of enlarged, erect, spore-bearing hyphae, called conidiophores. 
These conidiophores are inflated at the tip and become covered 
Fig. 176.-Morphology of the Aspergillus glaucus: a, Mycelia and perithecia 
on the surface of the medium, with a single conidiophore; d, a very young peri- 
thecium; e, cross-section through a perithecium, somewhat older; f, cross- 
section through a mature perithecium, showing the asci and the ascospores 
(as); bb1, isolated asci; cc1, ascospores ripe and germinating (c b1 d e f after 
deBary, ab A after Wehmer). 
with papillae, which develop into short stalks, called sterigmata 
(singular, sterigma). The sterigmata may branch once or many 
times, giving rise to bunches of secondary sterigmata, or they may 
remain unbranched. The species with branched sterigmata are 
frequently grouped together into a genus Sterigmatocystis. From 
the tips of these sterigmata spores or conidia are abjointed and 
hang together to form long chains. The spore mass at the tip of 
the conidiophore is termed a head. The spores are usually colored 
green, brown, yellow, or black, or in a few species they are colorless. MOLD OR HYPHOMYCETE GROUP 
457 
They are spherical or oval in shape. Their surfaces are not easily 
wetted; they are easily detached, and are readily carried about 
by currents of air. This explains the readiness with which birds 
and some animals become infected in the respiratory tract when 
fed on moldy grain or fodder. When the spores come under 
favorable growth conditions they germinate and reproduce the 
mold. 
If growth conditions are right, careful observation will enable 
one to discover the sexual stages in the reproduction. Two 
filaments, somewhat differentiated, begin to twist together until 
they form a typical cork-screw. The cell contents fuse and fer- 
tilization is effected. A tangled mass of threads arises about 
this cell, forming a compact layer or covering termed the peri- 
thecium. The enclosed cell grows rapidly, and produces a con- 
siderable number of enlarged cells, each of which eventually is 
found to contain spores, usually eight in number. These cells 
or sacs are called asci (singular ascus), and the spores are termed 
ascospores. These, like the conidia, when brought under favorable 
growth conditions, reproduce the mold. An Aspergillus may con- 
tinue to multiply indefinitely without the sexual stage developing; 
it is not improbable that some species have altogether lost the 
power of reproducing other than by means of the conidia. 
Several species of Aspergilli have been described as pathogenic. 
Doubtless these are normally saprophytes, and only produce 
disease under exceptional conditions. 
Diseases Produced.-Aspergillosis of birds; pneumomycosis in 
man and many animals. 
The occasional presence of Aspergillus in lung infections has 
been known since early in the nineteenth century. Probably 
Mayer and Emmet, in 1815, were the first to note its presence in 
the lungs of a bird, in this instance a jay. Since that time the 
organism has been reported many times. In most cases no careful 
species determination was made, but the probabilities are greatly 
in favor of Aspergillus fumigatus being the species responsible. 
Aspergillosis has been reported from the stork, raven, flamingo, 
eider-duck, parrot, pigeon, chicken, hawk, bullfinch, plover, 
Aspergillus fumigatus 458 
VETERINARY BACTERIOLOGY 
pheasant, bustard, duck, goose, ostrich, swan, and turkey among 
birds, from the horse, dog, and cow among animals, and from man. 
It has been reported from Europe and the United States. 
Morphology.--In culture-medium it forms greenish or bluish 
gray or later brownish masses. The conidiophores are abundant, 
but short. The enlarged tip of the conidiophores is hemispheric, 
and 8 to 20 m in diameter, bluish-green, and later brown. The 
Fig. 177.-Aspergillus fumigatus: 1, Optical section through a conidiophore; 
2, conidiophore and conidia; 3, conidia; 4, a, perithecium; b, an isolated ascus; 
c, d, ascospores, front and lateral view; 5, swollen hyphse, bl, and conidiophores 
(4, a-d, after Grijns, remaining after Wehmer). 
perithecia with ascophores have been observed in culture-media. 
In the lung tissues the branching mycelium may be observed on 
microscopic examination, and the sporophores may be seen pro- 
jecting into the air-sacs, where the conidia are produced. These 
spores are never formed except in the presence of oxygen. 
Isolation and Culture.-The Aspergillus fumigatus may be 
readily isolated from the lesions upon almost any of the commonly 
used artificial media, particularly when spores are produced. For MOLD OR HYPHOMYCETE GROUP 
459 
the best development the medium should be slightly acid. It 
develops readily upon potato and bread. Colonies become visible 
in a day, usually as tiny, white, cottony growths, which, within 
a few days, turn green, due to the formation of the spores of that 
color. 
Physiology.-The Aspergillus fumigatus is an aerobe. The 
optimum growth temperature is from 35° to 40°. According to 
Mohler and Buckley, growth occurs, but spores do not form 
below 20°. The spores are resistant to high temperatures. They 
Fig. 178.-Aspergillus fumigatus from a culture on agar (Frankel and Pfeiffer). 
have been found to survive an exposure of seven hours at 65°. 
Twelve hours' contact with a 5 per cent, solution of phenol is not 
sufficient certainly to destroy them. They can withstand desicca- 
tion indefinitely. 
Pathogenesis.-Experimental Evidence.-The organism pro- 
duces death within a few hours or days when injected intravenously 
or intrathoracically into the chicken. The pigeon is particularly 
susceptible to injections. Rabbits and guinea-pigs likewise suc- 
cumb, usually from a generalized infection. There is sufficient 
experimental evidence to justify the conclusion that Aspergillus 
fumigatus may produce a primary and fatal infection in many 
animals. 460 
VETERINARY BACTERIOLOGY 
Character of Disease and Lesions Produced.-By far the greatest 
number of cases of aspergillosis have been reported from birds. 
The lesions are generally located in the lungs, air-sacs, and hollow 
bones, where the spores may readily lodge. In man and animals, 
particularly the horse, the usual picture is an infection of the lungs 
and air-passages, but occasionally of the mucous membranes of 
other parts of the body. Metastatic infection of other organs is 
not infrequent. The organism causes the development of nodules 
not unlike those of tuberculosis. In the lungs the tubes are fre- 
quently occluded by the green fructifications of the fungus. There 
is more or less necrosis of tissue immediately surrounding the or- 
ganisms. 
Immunity.-Several investigators claim to have produced toxic 
substances, if not true toxins, by the growth of the organisms in 
artificial media. These claims have not been sufficiently sub- 
stantiated, although there is considerable a priori evidence, 
from the character of the lesions and symptoms, that powerful 
toxic substances of some kind are produced. No method of suc- 
cessful immunization has been developed. 
Bacteriologic Diagnosis.-A diagnosis may usually be made 
by the character of the lesions and the appearance of the green 
spores. A microscopic examination of the scrapings of the in- 
fected mucous membranes should reveal the spores and character- 
istic conidiophores without difficulty. 
Transmission.-The organism doubtless grows on decaying 
organic matter outside the body. The feeding of moldy grain or 
fodder may give ample opportunity for infection by inhalation. 
The preponderance of pulmonary primary infections shows that 
inhalation probably is the common method of infection. 
This organism has been described by various investigators, 
who believed it to be, in part at least, responsible for blind staggers 
or meningo-encephalitis in horses. It occurs in great quantities 
on moldy corn and other grains. Although the complete data 
have not been presented, the work of Haslam indicates that it is 
of some pathogenic significance. 
Morphology.-The sterile hyphse are cobwebby and white. 
Aspergillus flavus MOLD OR HYPHOMYCETE GROUP 
461 
The conidiophores are erect. Conidia are 5 to 7 p in diameter, 
Fig. 179.-Aspergillus flavus: 1, 2, 3, 4, Various stages in the development 
of conidiophores; 5, section and surface of a hypha, showing the numerous 
colorless granules with which it is covered; 6, natural size of the fungus; 7, 
conidia (Wehmer;. 
globose. Spore masses are yellow and yellowish green. Sclerotia 
are small and dark. 
This organism occurs under conditions similar to the preceding, 
and is believed also to be pathogenic to horses and other animals 
that consume grain infected with it. 
Morphology.--The mycelium is at first white, then darker, 
abundant, and penetrates the medium to a considerable distance. 
The conidiophores are long and the spores borne up at some dis- 
tance from the surface of the substratum. The sterigmata are 
branched. The conidia are 3.5 to 4.5 p. in diameter, roughened. 
The spore masses ultimately become black, and may be readily 
differentiated in this manner from the two preceding. This 
Aspergillus niger 462 
VETERINARY BACTERIOLOGY 
Fig. 180.-Aspergillus niger: 1, 2, 3, 4, Stages in the development of the 
conidiophores; 5, conidia; 6, detail, showing the branched sterigmata; 7, 8, 
sclerotia; 9, natural size of the fungus (Wehmer). 
organism has also been found in the ear and in lesions in the 
lungs. 
Other Species of Aspergilli 
Several other species of Aspergilli have been reported as 
pathogenic. Among them are Aspergillus nigrescens, A. subfuscus, 
and A. glaucus. These produce nodular mycotic foci in the in- 
ternal organs of laboratory animals into which they have been 
injected. They do not commonly produce infection under natural 
conditions. 
The Genus Penicillium 
Penicillium is closely related to Aspergillus, the principal 
difference being the manner in which the asexual spores or conidia 
are borne. The conidiophores are erect and much branched at the 
tip, the branches arising in whorls and are not enlarged at the apex. 
From the end of each ultimate branch a chain of spores is abjointed. MOLD OR HYPHOMYCETE GROUP 
463 
giving to the organism under the microscope the appearance of 
being covered with little brooms. The sexual stage is essentially 
similar to that of Aspergillus. Penicillia are even more common 
than the Aspergilli. They occur as blue or green molds upon fruit, 
and upon a great variety of decaying materials. Of the hundreds 
of species of Penicillium that have been described, the majority 
are green or bluish-green in color, but white, gray, yellow, orange, 
and brown forms are known. 
Fig. 181.-Penicillium glaucum: a, a1, Tips of conidiophores showing the 
characteristic method of branching and the chains of spores; b, perithecium; 
c, asci; d, ascospores (Brefeld). 
None of the species of Penicillium are known to be harmful, 
but their constant presence in moldy silage and grain which has 
poisoned animals makes it necessary to consider them in any 
discussion of forage poisoning. 
The members of this genus are nearly all saprophytes or plant 
parasites. Fusarium is included among the Fungi imperfecti, 
as a sexual or perfect stage is unknown in the life-history of most 
The Genus Fusarium 464 
VETERINARY BACTERIOLOGY 
of the species. Webber has found an ascus stage in one species, 
and concludes that the genus Necomospora of the Ascomycetes is 
the perfect form; in other species it is the genus Gibberetta. 
Fusarium is characterized by its loose, spreading, cottony 
mycelium with numerous cross walls, i. e., septate. The conidio- 
phores are not markedly different from the sterile hyphae, and are 
usually branched. The conidia are borne at the tips of these 
branches. They are long, slender, sickle or crescent shaped 
usually, and divided into several or many cells by cross walls or 
septa. 
Several species of Fusarium are found commonly on grains and 
moldy corn. This fungus has been believed by some investigators 
to be of significance in forage poisoning. It is one of the several 
forms which must be considered in a determination of the poisonous 
properties of forage. 
One species, the Fusarium equinum, is believed to produce 
dermatitis in the horse. 
Fusarium equinum 
Disease Produced.-Itch disease, associated with sarcoptic 
dermatitis. 
Norgaard, in 1901, noted the presence of a Fusarium in a derma- 
titis of horses in the State of Oregon, and proposed the name 
Fusarium equinum for the fungus. Melvin and Mohler later 
studied the disease in greater detail. The disease has been re- 
ported only from this one locality, but in this instance affected 
several thousand horses on the Umatilla Indian reservation. 
Morphology.-The mycelium upon culture-media is septate and 
branched. Three forms of spores are produced. The microconidia 
are small and oval, one or two celled. The macroconidia are 
large, sickle shaped, three to five septate, and pointed at the 
ends. They are 25 to 55 y long by 2.5 to 4.5 n wide. Chlamydo- 
spores are formed in the mycelial threads by a cell rounding up to 
a diameter of 8 to 15 ju and becoming densely granular. The 
spores may be recognized in the hair-follicles of the diseased ani- 
mals. 
Isolation and Culture.-No difficulty was experienced in secur- 
ing a growth of the organism on artificial media. The more MOLD OR HYPHOMYCETE GROUP 
465 
favorable media are potato and bread, but good growth will take 
place on glucose or plain agar. The growth is white and cottony, 
and the spores are produced in abundance. 
Pathogenesis.-Inoculation experiments were unsuccessful, so 
that the evidence of pathogenesis rests entirely upon the constant 
occurrence of the organism in the disease in question. Itch- 
mites (Sarcoptes equi) were found, but the investigators believe 
their numbers insufficient to account for the disease. It is entirely 
Fig. 182.-Fusarium equinum, mycelium and conidia (Melvin and Mohler, 
Bureau of Animal Industry). 
possible that the organism is a secondary invader, or produces the 
disease in a kind of symbiotic relationship with the Sarcoptes. 
The fungus seems to enter the hair-follicles, penetrates between 
the epidermal cells, and involves the surrounding skin, causing 
an intense itching. The body becomes covered with a crust or 
scurf, at first gray and afterward darker. The presence of the 
organism in the hair-follicles causes the hairs to fall out, resulting 
in an almost complete alopecia. 466 
VETERINARY BACTERIOLOGY 
The Genus Sporotrichum 
Authorities differ greatly in the delimination of this genus. 
According to botanists, the genera Microsporon and Trichophyton 
are synonyms of Sporotrichum. Pathologists and bacteriologists 
in general, however, make a distinction between them. The 
classification of the latter will be adopted here and the term Sporo- 
trichum used in the narrow sense. 
Sporotrichum is distinguished by the production of definite 
hyphse, which are usually creeping and irregularly branched. 
Definite conidiophores are not developed, or consist only of small 
side branches. The conidia are borne either on the sides or ends 
of the hyphse, singly or in clusters. They are usually very numer- 
ous, ovoid or spherical in shape, and hyaline or rarely lightly 
colored. The molds belonging to this group are in need of careful 
study and revision, as there is great uncertainty concerning many 
of the species. 
One, or possibly two, species of Sporotrichum have been shown 
recently to be of considerable pathogenic significance. 
Sporotrichum beurmanni 
Synonym.-Possibly Sporotrichum schenkii. 
Disease Produced.-Sporotrichosis in man and animals, one 
type of epizootic lymphangitis in horses. 
Schenk, in 1898, and Hektoen and Perkins, in 1900, described 
a species of Sporotrichum causing multiple abscesses in man. 
de Beurmann, in 1903, described similar forms from France. The 
disease has been reported in man and rats from Brazil, from man 
in California, Argentina, Germany, and in the horse in the United 
States and Madagascar. This disease is probably quite wide- 
spread, but has not been recognized, or has been confused with 
others, until recently. It is to be differentiated sharply from the 
true epizootic lymphangitis of the horse. An excellent discussion of 
the organism in its relation to disease in the horse was contributed 
by Page, Frothingham, and Paige in 1910. 
Morphology.-The examination of the material from culture is 
most easily made in a hanging drop. The hyphae are slender and 
septate. Spores or conidia are borne at the tips of side branches; 
usually a number are formed successively and are found then MOLD OR HYPHOMYCETE GROUP 
467 
in clusters. The conidia are small, oval or spherical. They fre- 
quently bud to some extent, and resemble somewhat the cells of 
Blastomyces. The hyphae stain easily with the common anilin 
dyes and are Gram-positive. In using the latter stain the 
alcohol must not remain too long in contact, otherwise the 
stain will be removed. Whether or not the organism ever de- 
velops a perfect or sexual stage is not known, but it does not 
seem probable. 
Isolation and Culture.-The organism may readily be isolated 
from pus from the lesions. Potato is the most favorable medium. 
Fig. 183.-Sporotrichum beurmanni, from culture showing the mycelium and 
spores (Page, Frothingham, and Paige, in "Journal of Medical Research"). 
Original isolations show at the end of a week, transplants at the 
end of two or three days, as white, filamentous colonies. These 
enlarge, become darker at the center, and finally turn dark brown 
or black, frequently surrounded by a rim of white. The colony 
becomes wrinkled. Upon gelatin the growth remains white. 
Liquefaction begins in from three to ten days or even later. The 
addition of dextrose causes the center of the colonies to darken. 
In agar, and particularly in neutralized glycerin agar, growth is 
good, and the colonies remain white. Blood-serum is not lique- 
fied. In litmus milk growth occurs with little change in the 468 
VETERINARY BACTERIOLOGY 
medium, or coagulation without acid production may take place 
after the lapse of several weeks. In liquid media growth occurs 
in the form of more or less separated colonies, usually accompanied 
by a surface pellicle. 
Physiology-The organism is an obligate aerobe. Growth 
occurs best at 25° to 28°, but is not prevented at 37°. The spores 
resist desiccation for considerable periods. Acid is produced from 
dextrose, but not from lactose, mal- 
tose, saccharose, mannite, dulcit, ado- 
nit, inulin, or raffinose. Gas is not 
produced from any sugar. No indol 
is formed. Gelatin is slowly liquefied. 
The organism is destroyed at a tem- 
perature of 60° for five minutes. 
Pathogenesis.-Experimental Evi- 
dence.-There is an abundance of evi- 
dence that Sporotrichuni heurmanni is 
pathogenic for man and animals. Ac- 
cidental laboratory infections have 
taken place in man. Inoculation of 
pure cultures into mice and white rats 
gives rise to abscesses at the point of 
inoculation, and the infection gradu- 
ally extends. Guinea-pigs and rabbits 
are infected with greater difficulty. 
An infection somewhat resembling 
farcy develops upon inoculation into 
the horse. 
Character of Disease and Lesions 
Produced.-The infection is usually 
benign in character in the horse. 
There is no fever reaction during 
the course of the disease. Nodules develop which are gen- 
erally spherical and sharply delineated. These nodules scarcely 
rupture, but pus accumulates at the center, the skin above is 
thinned and softened, serum exudes from the surface, the hair is 
loosened, and a crust holding the hairs together is formed. The 
ulcers are crateriform, and usually contain a little creamy pus. 
Fig. 184.-Sporotrichum 
beurmanni, culture and col- 
onies on potato (Page, Froth- 
ingham, and Paige, in "Jour- 
nal of Medical Research"). MOLD OR HYPHOMYCETE GROUP 
469 
Healing is accomplished by granulation. Sections of nodules 
from laboratory animals reveal the organism in the tissues. 
Immunity.-No toxins have been demonstrated for this organ- 
ism. Widal has shown the presence of agglutinins for the spores in 
the blood of infected individuals. No method of immunization, 
based upon the organism or its products, has been developed. 
Bacteriologic D iagno sis. - This 
may be accomplished by animal in- 
oculation, thus securing the organism 
in pure culture. Widal claims that 
the agglutination of spores will take 
place in dilutions as high as 1: 800; 
but the same reaction takes place in 
lower dilutions with the blood-serum 
of individuals having actinomycosis. 
Bloch found that a bouillon filtrate 
from an old culture would in man give 
the von Pirquet cutaneous reaction, as 
was described in the chapter of Bacillus tuberculosis. Gougerot 
recommends for the skin reaction the use of sporotrichosin, a 
sterile suspension of killed Sporotrichum beurmanni in salt 
solution. 
Transmission.-The disease is transmitted by intimate contact 
usually. It has been found to be transferred from animals to man. 
Probably the organism usually gains entrance through abrasions 
of the skin. 
Fig. 185.-Sporotrichum 
beurmanni, in a section of a 
mesenteric abscess of a rat 
(adapted from Fielitz). 
The Genera Trichophyton, Microsporum, Achorion, and 
Oidium 
The organisms belonging to the two genera, Trichophyton and 
Microsporon, are frequently included together under the single 
genus Sporotrichum. The two names, Trichophyton and Micro- 
sporon, are also used very loosely and interchangeably. A care- 
ful study of the relationships of the various forms is needed. 
No very clear differentiation of this group from the preceding 
can be given. 
Organisms belonging to these genera are the cause of many 
skin and hair infections in man and animals. 470 
VETERINARY BACTERIOLOGY 
The Genus Trichophyton 
The species belonging to this genus and the two following genera 
have been studied most carefully by Sabouraud, and his classifica- 
tion has been extensively used. 
The various species described are all the causes of diseases in 
man and animals, known as herpes tonsurans and ringworms. 
All invade the hair, compactly filling its interior with a mass 
of parallel hyphse extending longitudinally. The hair may be ex- 
amined directly under the microscope by first immersing it for 
a few seconds in a warm solution of 30 grams of caustic potash in 
70 c.c. of water. The filaments are composed entirely of quad- 
rangular cells 4 to 5 /J. in length and about the same width. These 
chains are quite regular in their arrangement, and are straight. 
When branching occurs it is 
by dichotomy, but is not 
abundant. 
These organisms may be 
cultivated readily on special 
maltose (or other sugar) agar. 
Isolation and Culture.- 
Pure cultures are secured with 
difficulty, as the skin and hair 
are generally filled with bac- 
teria which overgrow the 
mold. Kral has suggested 
pulverizing hairs with fine sand 
or silicon powder and pouring 
gelatin plates of various dilu- 
tions. Kitt has taken advantage of the resistance of the organism 
to destruction by alkalies by washing the hairs and scales from an 
infected area with a solution of KOH, which removes most of the 
bacteria without materially injuring the mold spores. Potato 
is a favorable medium for growth. A rather velvety, wrinkled, 
relatively heavy membrane forms which may be white or colored. 
Gelatin is slowly liquefied. 
Sabouraud states that there is great variation in the appear- 
ance of cultures in different media. A microscopic examination 
of a mount from culture-media shows the development of a dense 
Fig. 186.-Trichophyton tonsurans from 
agar plate culture (Gunther). MOLD OR HYPHOMYCETE GROUP 
471 
mass of branched hyphse definitely septate, slender, and hyaline. 
The spores are borne laterally on the branches of the hyphse, 
usually sessile, one celled, and ovoid or spherical in shape. Some 
species also develop chlamydospores in the mycelium. 
The species of Trichophyton are divided into two principal 
groups, Endothrix and Ectothrix. In the former the chains of cells 
in the diseased hair are entirely within the hair, in the latter they 
are both within and without, forming a coating over the hair 
surface. 
Types of Endothrix.--The Endothrix group of species according 
to Sabouraud contains about fifteen species, all parasites on man. 
The best known of these are Trichophyton tonsurans (the Tr. 
crateriforme of Sabouraud), Tr. sulfurerum, and Tr. cerebriforme. 
Among 500 cases of dermatomycoses observed by Sabouraud the 
first named produced 115, the second 52, the third 39, the fourth 
13 of the infections. The other species were rare. Ninety-five 
per cent, of all trichophytoses in man were found to be due to the 
first three species listed. 
Types of Ectothrix.-The species of Ectothrix are divided into 
two groups termed Microides and Meyaspores. The spore-like 
cells which make up the mycelial threads as they affect the hair are 
small, 3 to 4 y in diameter in the former, and large, 8 y, in the 
latter. 
Species of Ectothrix microides.-Eight species of Microides are 
recognized by Sabouraud. They are placed in two subgroups, 
the type gypseum with six species, and the type niveum "with two 
species. The former all produce chalky colonies on media, the 
latter downy colonies. The first group contains species which 
attack man primarily, though readily transferred to laboratory 
animals, as the guinea-pig. One species, Trichophyton granulosum, 
attacks the horse. The two species of the niveum type are Tr. 
felineum (or Tr. radians) and Tr. denticulatum. The former is 
primarily a parasite of domestic animals transferable to man and 
will be discussed later, the latter is a human parasite primarily. 
Species of Ectothrix megaspores.-Seven species have been de- 
scribed, of which the following are parasitic on animals: Tricho- 
phyton equinum, Tr. caninum, Tr. verrucosum, Tr. discoides. The 
remainder are known only from man. 472 
VETERINARY BACTERIOLOGY 
Trichophyton granulosum 
Synonym.- Trichophyton gypseum. 
This species was described by Sabouraud in 1908 as the cause 
of a dermatomycosis of the horse. 
Morphology.-Typical of the Microades group. Spores on hairs 
3 to 4 p in diameter. 
Culture.-On maltose agar a powdery yellowish colony is 
developed, with relatively large granules on its surface. The 
center is usually irregularly umbilicate. 
Pathogenesis.-The disease in the horse is characterized by a 
development of small areas on which the hair is roughened. The 
hair over these areas is stuck together in small pencils. The 
hair falls with a scaly crust. The areas from which the hair falls 
remain naked and dry for some time; finally hair grows again 
slowly. The plaques are numerous and small. The disease is 
quite persistent. 
Inoculation on horses and guinea-pigs is successful. The 
disease is apparently occasionally transmitted to man. 
Trichophyton felineum 
Synonyms.- Trichophyton niveum; Tr. radians. 
Disease Produced.-Ringworm of cat, perhaps also in horses, 
cattle, sheep, and swine. 
Morphology.-Typical of group. The base of a parasitized 
hair shows large spores, 7 to 9 p in diameter, forming a collar ad- 
herent to the hair and to edge of hair-follicle. 
Culture.-On maltose agar a powdery white colony is formed, 
umbilicate and irregularly wrinkled on the interior and with a large 
number of slender rays about the margin. 
Pathogenesis.-According to Brumpt this organism attacks 
animals, producing a characteristic type of ringworm. It has 
been found by Sabouraud to produce disease in man as well. By 
some authors the cat is regarded as the principal host. 
Trichophyton equinum 
This organism was first isolated by Matruchot and Dassonville, 
in 1898, from a dermatomycosis of the horse. It was later studied 
by Sabouraud and by Gedoelst. The former regards it as the com- 
monest type of Trichophyton attacking the horse. MOLD OR HYPHOMYCETE GROUP 
473 
Morphology.-In the crust observed on the lesions of the horse 
the filaments are not abundant, generally rather straight or un- 
dulating. In the hairs the morphology is typical of the group, 
the large "spores surround the hair base. 
Culture.-On maltose agar a white colony is formed, but it is 
not powdery, but velvety in texture. On potato the growth is 
moist and of the color of yellow ochre. 
Pathogenesis.-The disease in the horse manifests itself in 
small areas more or less numerous on the rump, shoulder, and back. 
The largest patches are not more than 1| cm. in diameter. Man 
may also be infected, as may also the guinea-pig. 
Trichophyton canintim 
Matruchot and Dassonville, in 1902, described a folliculitis 
in the dog caused by this organism. The hair dropped from the 
infected areas. The organism is termed a true ectothrix large- 
spored Trichophyton. 
The Genus Microsporum 
The species of this genus all produce dermatomycoses in man 
or animals. In all types the hair appears frosted, encased for 
several millimeters with a delicate white sheath. Under the micro- 
scope this is found to be made up of small spores, 2 to 4 /z in di- 
ameter, not disposed in chains, but arranged as a mosaic over the 
surface. In culture-media the hyphae frequently show racquet- 
shaped cells arranged in rows, which eventually develop into inter- 
calary chlamydospores. The spores are borne much as in Tri- 
chophyton, usually they are somewhat more elongate. Many 
colonies show a peculiar multicellular, fusiform, relatively large 
spore, the septae of which are all parallel and transverse. These 
are also sometimes found in species of Trichophyton. Peculiar 
hyphae with outgrowths resembling a comb, the so-called pectinate 
hyphae, are also formed. 
The various species may be cultivated in the same manner as 
Trichophyton. 
Four species are known which produce disease primarily in 
man. They are relatively slow growers upon laboratory media and 
are inoculable upon animals with difficulty or not at all. Seven 474 
VETERINARY BACTERIOLOGY 
species primarily pathogenic for animals have been described. 
These grow readily upon laboratory media and often infect man. 
These species are: 
Microsporum lanosum 
Synonyms.-Microsporum caninum; M. canis. 
Disease Produced.-A dermatomycosis of the dog, also occa- 
sionally attacking the horse and children. 
The disease was first studied and the organism recognized by 
Bodin and Almy, in 1897. It is one of the common skin diseases 
of the dog. 
Morphology.-Relatively few of the hairs of the diseased area 
from the dog are found to be infected with the fungus. It may 
readily be found in the scales from the infected skin. The poly- 
hedric superficial spores on the hairs are of the characteristic 
type. 
Culture.-Growth occurs readily on maltose and other sugar 
media. The colony on maltose agar shows a central area which is 
glabrous and powdery, surrounding this a ring or annulus of velvety 
appearance. The central portion may be umbilicate. 
Pathogenesis.--Sabouraud described four distinct phases of 
the infection in the dog. The areas infected show first a roughening 
of the coat, followed by a loss of hair, leaving a smooth skin. Usu- 
ally there then appears a pustular folliculitis, followed finally by 
healing and recovery. 
Infection of man occurs readily, particularly in children, pro- 
ducing a ringworm of the scalp. 
The disease in man shows some clinical differences from that 
caused by the far more common Mierospovum adouini. 
Inoculations upon animals is successful; particularly suscep- 
tible are dogs and guinea-pigs. 
This species was first recognized by Fox and Blaxall, in England, 
in 1896, as causing a disease of the cat. Transmission to man was 
noted by these investigators as well as by Mewborn, of New York, 
in 1902. Morphologically and culturally the organism is typical 
of the genus. It has been experimentally transferred to the cat, 
the dog, and the guinea-pig. 
Microsporum fetineum MOLD OR HYPHOMYCETE GROUP 
475 
Microsporum equinum 
This species was described by Bodin and Delacroix, in 1896. 
The organism produces disease in the horse and in man, and may be 
developed in the guinea-pig by inoculation. 
The Genus Achorion 
This genus differs in minor details from Microsporum and 
Trichophyton. When growing inside a hair it seems to destroy 
the interior so that air enters, and when such hair is mounted for 
examination the air bubbles are quite characteristic, this fre- 
quently alone being sufficient to differentiate this causal organism 
as belonging to this genus. In the hair the filaments are not so 
closely massed as those of Trichophyton frequently are, and do not 
show the straight rows of spore-like cells. 
Upon culture-media the organism produces numerous heavy 
walled chlamydospores, both intercalary and apical, on short side 
branches. The pectinate hyphae are often formed as in Micro- 
sporum. Spores both single celled and multilocular (likewise re- 
sembling those of Microsporum) are also produced. The two 
genera are evidently closely related, if, in fact, they should be sep- 
arated at all. 
Culture upon media can be accomplished as for the two pre- 
ceding genera. 
One species, Achorion schonleinii is the common species attack- 
ing man. Three species, A. quinckeanum (A. muris), A. gypseum, 
and A. gallince attack animals or birds. 
Synonym.-Achorion quinckeanum. 
It has been recognized for more than a half-century that 
favus is a disease which may be transmitted from the mouse to 
man, producing an infection not unlike that of Achorion schon- 
leinii. 
Achorion muris 
Achorion gallinae 
Synonym.-Lopophyton gallins. 
Disease Produced.-Tinea cristse gallae. Fowl favus. 
The disease was first studied by Gerlach, in 1858. Since that 
date it has been described by numerous writers. 476 
VETERINARY BACTERIOLOGY 
Morphology.-A microscopic examination of the fungus collar 
which surrounds the base of feathers from infected areas shows 
a mass of tangled threads of the mycelium, much resembling chains 
of spores. 
Culture.-The organism may readily be secured in pure culture 
from the infected feathers. The colonies on maltose-agar are white, 
usually showing concentric rings and radial foldings. If grown at 
30° the colonv takes on a rose color. 
Fig. 187.-A chorion schonleinii, section showing the hyphae (Frankel and 
Pfeiffer). 
Pathogenesis.-In the fowl the disease may attack the head, 
the comb, the wattles, or the ear lobes. It usually begins by the 
development of one or many small white points. It appears as a 
pellicle adherent to the adjacent epidermis. The spots increase 
in size and fuse. When the lesion appears on a surface with 
feathers, a sort of collar or cone forms at the base of each 
feather, and the plumes fall, each carrying with it its fungus 
collar. The bird may recover, but the disease is commonly quite 
persistent. 
The disease may be inoculated into man, the rabbit, and the 
guinea-pig. MOLD OR HYPHOMYCETE GROUP 
477 
Oidium albicans 
Synonym.-Monilia candida. 
Disease Produced.-Thrush in infants; sometimes in the 
young of animals. 
Berg, in 1840, described this organism as the cause of thrush. 
It has since that time been repeatedly isolated and cultivated. 
It is known from most civilized countries. 
Morphology.-The mycelium of this organism is poorly de- 
veloped; frequently the whole growth consists of budding yeast- 
like cells. These may be spherical, elliptical, oval or cylindrical, 
the shorter cells about 4 /jl in diameter by 5 to 6 ai in length. The 
hyphal threads are much longer. There can be little differentia- 
tion in many cases between the conidia and the cells of the hyphae, 
but on artificial media the conidia are frequently definitely ad- 
jointed from the tip of the conidiophore. Chlamydospores may 
form in the hypha;. 
Isolation and Culture.-The organism may be isolated without 
difficulty from the lesions of the disease. A distinctly acid me- 
dium should be used. On most nutrient media it develops super- 
ficial, spherical, white, waxy to granular colonies. Gelatin is not 
liquefied, nor is blood-serum. 
Pathogenesis.-The organism, when injected intravenously 
into rabbits, produces a fatal infection not unlike a generalized 
infection with a Blastomijces. Typical thrush has been produced 
in the mouth of young animals and birds by inoculation. The 
disease generally occurs in the mouth of sucklings, usually as a 
benign infection. It is characterized by the formation of white 
patches on the mucous membrane, varying in size from points to 
considerable areas. The infection may extend to the pharyngeal 
or laryngeal mucosae; rarely metastatic infection of internal organs 
may occur. SECTION V 
PATHOGENIC PROTOZOA 
CHAPTER XL 
STRUCTURE, RELATIONSHIPS, AND CLASSIFICATION OF THE 
PROTOZOA 
A protozoan may be defined as a unicellular organism be- 
longing to the animal kingdom. Protozoa exist throughout their 
life-history as single-celled individuals, or as colonies of single 
cells; that is, the cells are not united to form tissues or organs, 
and never constitute a portion only of a multicellular form. 
The protozoa show some forms which intergrade with the bac- 
teria. The group is frequently defined as containing spiral forms, 
such as the Spirochaetae, some investigators believing that these 
have more bacterial than protozoan characteristics, others taking 
quite the opposite view. It is difficult, on the other hand, to draw 
a sharp line of demarcation between the protozoa and the multi- 
cellular animals or metazoa. Just when a group of cells ceases to 
be a mere group of independent units, and becomes a tissue which 
forms the whole or part of a multicellular form, it is difficult to 
determine. 
Although the protozoa are regarded as the simplest and most 
primitive of living things, nevertheless many are complex in struc- 
ture and have the cell divided into specialized parts, sometimes 
termed organella, somewhat similar in function to the organs of the 
higher forms. They frequently undergo many changes in form 
during their life. Their life-history is, therefore, relatively com- 
plex as compared with that of the bacteria. 
Structure of the Protozoa.-The body substance of a protozoan 
may be divided into the ectoplasm, or outer layer, which conies into 
478 STRUCTURE, RELATIONSHIPS, CLASSIFICATION OF PROTOZOA 
479 
contact with the material environment, and the endoplasm, with- 
in. Within the endoplasm there is generally a nucleus (in some 
forms two nuclei, a large and a small), and frequently inclusions 
of other sorts. 
The ectoplasm in some cases cannot be differentiated sharply 
from the endoplasm. These are the exception, however, and not 
the rule. The ectoplasm may become variously modified, and, by 
secretion, form shells or a heavy membrane for protection. Many 
of the pathogenic protozoa become encysted by the formation of a 
heavy, chitinous wall. 
The endoplasm is made up of a very delicate, foam-like or 
alveolar structure, the density of which varies greatly. It may 
enclose amorphous granules or crystals of different kinds, green 
granules or chromatophores, vacuoles, etc. 
None of the protozoa are certainly known to be entirely des- 
titute of nuclear material. The nucleus may be devoid of a 
nuclear membrane or distributed; generally a membrane is pres- 
ent. A single homogeneous nucleus is present in most forms, 
with the exception of the infusoria, which have, in general, a large 
or macronucleus and a small or micronucleus. At some stages in 
the life-history many nuclei may be present in a single cell. This 
is particularly true just before or during spore formation. 
The protozoa with few exceptions are motile, at least during 
certain stages in the life-history. The exceptions are among certain 
of the parasitic Sporozoa. Special organs of locomotion are 
frequently found. Pseudopodia are changeable processes of 
the protoplasm which are thrown out by the cell, the cell contents 
frequently flowing forward into the pseudopodia. Many or few 
may be present at one time. They may be short and blunt or 
long and slender. Usually the endoplasm as well as ectoplasm 
takes part in their formation. The protozoan flagella are derived 
from the ectoplasm; are usually unchangeable in shape, long, 
thin, and pointed; in general, longer than the cell itself. One, 
two, or many may be present. They propel the cell by a series 
of undulations or spiral or rotary motions. Cilia, when present, 
are found in considerable numbers. They are shorter, generally 
blunt, and, by striking the water in unison, resemble oars in their 
motion. 480 
VETERINARY BACTERIOLOGY 
The life-histories of the various protozoa show such complexi- 
ties that they may better be treated under .the separate subdi- 
visions. 
Classification of the Protozoa.-Authorities are not in entire 
accord with reference to the principal subdivisions of the protozoa. 
The classes here used are those proposed by Doflein in his "Lehr- 
buch." 
Key to Classes of Protozoa 
Subdivision I., Plasmodroma.--Protozoa motile by means of 
pseudopodia or flagella, with one or more vesicular nuclei, repro- 
duction isogamous or anisogamous, usually with two developmental 
cycles, in which sexual generations alternate with sexual. 
1. Motile by means of flagella Class I. Mastigophora. 
2. Motile by means of pseudopodia Class II. Rhizopoda. 
3. Variously motile, usually reduced through parasit- 
ism. Metagamic multiplication by means of 
numerous spores Class III. Sporozoa. 
Subdivision II., Ciliophora.-Protozoa with numerous cilia, 
with one or more principal nuclei and one or more vesiculate ac- 
cessory nuclei (or occasionally the latter only). Reproduction by 
means of an isogamous fusion, or by means of interchange of nu- 
clear material without fusion of the cell-bodies. Multiplication 
only as a result of simple division or by budding. 
1. Cilia present throughout the life of the cell. Food 
taken up by osmosis or through a cytostome.... Class IV. Ciliata. 
2. Cilia present only in the young cell; food taken up 
through funnel-like organella Class V. Suctoria. CHAPTER XLI 
PATHOGENIC PROTOZOA OF THE FLAGELLATA 
The pathogenic forms of the class Flagellata differ from 
the Rhizopoda in that they do not possess pseudopodia. In most 
cases the organism has a relatively definite form. The cells are 
motile by means of one or many flagella. 
This class contains many hundreds of species distributed 
among many genera and families. Most of these are non-parasitic. 
Many species are commensals in the intestines of man and animals, 
and have been suspected of producing intestinal disorders. 
The genera, Spirochaeta, Treponema, and Spiroschaudinnia, 
perhaps belong with the Flagellata, and show some distinct re- 
semblances to the genus Trypanosoma. Their position among 
the protozoa, however, is challenged by many bacteriologists. 
They are, therefore, treated as a separate group in a preceding 
chapter. 
(Exclusive of the Spirochetes) 
An undulating membrane and terminal flagellum 
present Trypanosoma. 
Undulating membrane not present Herpetomonas. 
The first recorded observations of trypanosomes are those of 
Valentine, who saw them in the blood of a salmon in 1841. Nu- 
merous observers found similar organisms in the blood of other 
fish, reptiles, and batrachia. Lewis, in 1878, observed the first 
trypanosome of mammalian blood in the rat. Evans, in 1880, 
noted them in the blood of animals infected with surra and be- 
lieved them to be the cause of the disease. Bruce, in 1894, de- 
scribed another form from Africa as the cause of nagana or tsetse-fly 
disease. Since that time the number of known species has in- 
The Genus Trypanosoma 
481 482 
VETERINARY BACTERIOLOGY 
creased rapidly. A small proportion only of those described are 
known to be pathogenic. Among the pathogenic forms, however, 
are to be found those that are among the most serious hindrances 
to the development of a live-stock industry, and even to human 
habitation in certain countries. 
Morphology.-Trypanosomes usually are elongated cells, more 
or less spindle shaped, rarely almost as broad as long, but always 
tapering more or less to the ends. Most of the pathogenic forms 
that have been described are several times as long as broad when 
observed in the blood. The anterior end of the cell is tipped with 
a flagellum. Along one side, extending longitudinally on the cell, 
is a thin membrane. The flagellum extends along this membrane 
Fig. 188.-Trypanosoma equiperdum, morphology of the trypanosome: 1, 
A rather thick, short trypanosome-a, Blepharoplast; b, protoplasm (cyto- 
plasm); c, nucleus; d, undulating membrane; e, flagellum attached to the edge 
of the membrane; f, anterior extension of the flagellum. 2, A longer cell. 
3, 4, Trypanosomes in process of longitudinal division (adapted from Gonder 
and Sieber). 
and forms its outer edge. The flagellum finally terminates in the 
cell-protoplasm near a granule which stains deeply. This granule 
may be situated in various parts of the cell, but is usually in the 
posterior portion. Its relative position is one of the characters 
used in the differentiation of species. It has been called by various 
names, as micronucleus, kinetonucleus, motor nucleus, centrosome, 
and blepharoplast. This last name is to be preferred, as it is the 
one that is most frequently used in the descriptions, and is com- 
monly used for similar structures found in many other flagellated 
protozoa. The flagellum is, in part, therefore, embedded in the 
protoplasm, in part is attached to the edge of the undulating mem- 
brane, and in part is free at the anterior end. A nucleus, usually PATHOGENIC PROTOZOA OF THE FLAGELLATA 
483 
situated near the center or anterior end, may be demonstrated in 
stained preparations. It is relatively large and granular in struc- 
ture. The entire body of the trypanosome is mobile, and the or- 
ganism may vary its shape to some degree. It swims about with 
the flagellum in front. 
Multiplication is accomplished by a preliminary division of the 
blepharoplast followed by that of the nucleus, and this by a longi- 
tudinal splitting of the cell to form two individuals. In cultures 
the cells may frequently be observed in the form of rosettes or 
clusters, with the flagellar ends pointing out. These rosettes do 
not occur in the blood. Transverse division does not occur. No 
conjugation or fertilization process in trypanosomes has been cer- 
tainly detected. 
Some investigators have believed that trypanosomes in the 
animal body may have an ultramicroscopic stage in their develop- 
ment. Bruce and Bateman, in a series of careful experiments, 
have shown that this does not occur. 
The existence of a regular cycle of changes in the life of a try- 
panosome is at present a somewhat mooted question. Some inves- 
tigators believe that they have established the existence of a rela- 
tively complex life cycle. Kleine, Bruce, and others believe that 
certain species of insects which transfer the disease must be con- 
sidered true hosts, and that they do not become infective for some 
days-in the case of Trijpanosoma gambiense about twenty-- 
after biting an infected individual. Rodenwalt and others have 
found that certain developmental changes take place in the gut of 
the insect; others have failed to find them. At present it may be 
concluded that the occurrence of developmental changes has not 
been satisfactorily demonstrated, although there is good reason to 
believe that such may occur. Carimi, Schaudinn, and others have 
recognized what they believe to be an endoglobular stage in the 
development of the organism in the body. Battaglio claims to 
have demonstrated for Tr. brucei, Tr. lewisi, and Tr. vespertilionis 
a developmental cycle in the blood, which includes sporulation of 
micro- and macrogametocytes, with formation of micro- and macro- 
gametes. These conclusions have been combated by others. 
Not all trypanosome infections are transmitted through insects. 
Certain transformations have been noted with some forms in the 484 
VETERINARY BACTERIOLOGY 
body itself. There is no evidence that an insect can transfer the 
organism to its progeny; that is, hereditary transmission through 
insects play no part in the life-history. This is in marked con- 
trast to certain other diseases, such as the piroplasmoses. 
Cultivation of Trypanosomes.-Novy and MacNeal, in 1903, 
gave an account of a method which they had successfully used in 
the cultivation of trypanosomes in artificial media. Equal parts of 
defibrinated rabbit blood and melted nutrient agar are mixed and 
the tubes are slanted and allowed to solidify. The water of con- 
densation is inoculated with a small amount of blood containing 
trypanosomes. The first culture of trypanosomes frequently 
develops slowly, but subsequent transfers more quickly. Not all 
trypanosomes can be cultivated in this manner with equal facility. 
Method of Disease Production.-The organisms are found 
typically in the circulating blood, and to a less degree in the other 
body-fluids. Anemia and emaciation are frequently associated 
with the various trypanosome infections. The spleen is quite 
commonly enlarged. 
Examination and Staining Methods.-Usually the examination 
under a cover-glass of a drop of blood from an infected animal will 
reveal the organism, particularly if made directly with the low- 
power objective of the microscope. The active motion of the 
organisms reveals their presence by movements of the blood-cells, 
and they may be thus located and then studied under the higher 
powers. Centrifugation of blood or body-fluids must be resorted 
to in some instances to concentrate the cells when they are present 
in small numbers only. Blood-films stained by Wright's method 
yield very satisfactory results. 
The vast number of tropical and subtropical diseases due to 
trypanosomes makes it impossible to discuss any except the most 
important of such parasites. Some of these trypanosomes are 
primarily important as the course of human diseases, secondarily 
as affecting animals. The most important of this class is the Try- 
panosoma gambiense, the cause of sleeping-sickness of certain 
African countries. Monkeys, dogs, cats, guinea-pigs, and rabbits 
are readily susceptible to this trypanosome. PATHOGENIC PROTOZOA OF THE FLAGELLATA 
485 
Trypanosoma equiperdum 
Synonym-Trypanosoma rougeti. 
Disease Produced.-Dourine: maladie du coit in horses (horse 
syphilis). 
Rouget, in 1896, first described this trypanosome. Other 
investigators have conclusively established its etiologic relation- 
ship to the disease. It is of particular interest as the only 
trypanosome disease of importance in Europe and in North 
America. 
Distribution.-The disease is known from Germany, Austria, 
France, and southern European countries, northern Africa, western 
Asia and India, Chile, Java, and several local outbreaks have oc- 
curred in North America (Illinois, Nebraska, Wyoming, South 
Dakota, Iowa, and northwestern Canada). 
Morphology (See Fig. 188).-Moore and Bredini, in a study of 
this trypanosome as it occurs in artificially infected rats, con- 
cluded that it passed through certain developmental stages, finally 
being converted into rounded bodies, with two long .delicate flagella. 
The organism, as it occurs in the lesions and blood of the horse, is 
a slender cell, usually about 25 to 28 p in length. There are no 
particular differentiating characters between this organism and the 
ones associated with other trypanosomiases of the horse. The 
blepharoplast is distinct, the membrane considerably folded, the 
nucleus central, and the free flagellum about f to | the length 
of the organism. Protoplasmic granules are never present in the 
organism directly originating from the horse. 
Cultivation.-Thomas and Breinl have succeeded in cultivating 
the organism in a modified Novy and MacNeal medium in one trial 
out of nineteen. 
Pathogenesis.-Experimental Evidence.--The disease may be 
transmitted experimentally to the horse, the ass, to fowls, and 
even to ruminants and apes, according to some observers, while 
according to others the latter animals are quite refractory. 
The disease occurs naturally only among the equines. 
Character of Disease.-The infected animal becomes emaciated, 
edematous swellings appear on the genitals, while whitish or chalk- 
like areas appear in the skin and mucosa of the external genitalia. 
Purulent foci occasionally develop in the testicular tissue. The dis- 486 
VETERINARY BACTERIOLOGY 
ease usually runs a chronic course. The animal frequently becomes 
gradually paralyzed. Recovery is infrequent. 
Immunity.-Animals that recover from the disease are thereby 
rendered immune to a second infection. They are not, however, 
rendered immune to infection with another trypanosome, such as 
that of surra. It appears that an immunity acquired during preg- 
nancy may be transmitted to the offspring. No practical method 
of immunization based upon the organism or its products has been 
developed. 
Watson reports the use of serum from chronic cases of dourine 
in horses in large doses, up to 300 c.c. as protecting against virulent 
infection. Similar observations upon laboratory animals were 
made by Rouget, Nocard, Uhlenhuth, Zwick, and Fisher and others. 
The latter observed that such inoculations protected against the 
dourine trypanosome only, not against the nagana trypanosome. 
Bacteriologic Diagnosis.-This may occasionally be accom- 
plished by a microscopic examination of the fluids from the lesions 
and the identification of the characteristic trypanosome in stained 
mounts. The organisms can rarely be found in the peripheral 
blood. The blood-tinged fluid secured from the plaques when they 
first appear is the most favorable material for their identification. 
They may be found in the blood-stream of artificially infected 
laboratory animals. In doubtful cases inoculation of blood or the 
serous fluid from plaques may be made into dogs, and examination 
of the swellings on such animals be subsequently made with 
positive findings. 
Agglutination Test.-Lange, Zwick, and Winkler and others 
report the agglutination test as satisfactory in determining virus 
carriers among stallions. They found that serum of infected 
animals would agglutinate dourine trypanosomes in homogeneous 
suspension in dilutions of from 1 :400 to 1 : 25,000, while that of 
non-infected animals did not agglutinate in dilution higher than 
1 : 50. The chief objection to this as a practical test is that other 
trypanosomes may be agglutinated, hence the test is of no value as 
a differentiating one. 
Complement-fixation Test.-Watson, of Lethbridge, Canada, 
makes report of 15,000 cases tested by this method. Concerning 
the reliability of the test he states that 100 per cent, of dourine- PATHOGENIC PROTOZOA OF THE FLAGELLATA 
487 
infected animals, whether in the active or latent stages of the 
disease, give positive reactions, provided two to three months 
incubation period has elapsed. In the less resistant animals one 
month is sufficient. 'He prepares his antigen by inoculating a 
number of white rats with Trypanosoma equiperdum, collecting 
blood from them when it is teeming with the trypanosomes, and 
centrifuging the blood at 1500 revolutions per minute to separate 
the trypanosomes from the blood-cells. Subsequent washing and 
centrifuging frees the trypanosomes from serum. Such antigen 
may be kept indefinitely if frozen solid. 
Conglutination Test.-This test has been used by a number of 
investigators. Wehrbein states that it is satisfactory, but is 
more sensitive to faulty technic, and consequently more difficult 
to employ than the complement-fixation test. 
Transmission.-The disease is commonly transmitted from one 
animal to another through coition. Whether or not the organism 
can enter through the intact mucosa is not certainly known, but 
appears probable. Blood containing the organism will infect a 
rabbit if placed in the conjunctival sac. Sieber and Gonder claim 
to have succeeded in transmitting the disease through the medium 
of the fly Stomoxys calcitrans, but this certainly is not the com- 
mon method. No developmental stages could be observed in the 
fly- 
Trypanosoma evansi 
Synonym.-Spirochaeta evansi. 
Disease Produced.-Surra in horses, mules, cattle, carabou or 
water buffalo, camels, elephants, clogs, goats, and sheep. 
The disease has been known for a long time from southern Asia. 
The organism was first described from India by Evans in 1880. 
Distribution.-The disease is known from India, China, the 
Philippines, Africa, and Australia. 
Morphology.-The organism as it occurs in the blood is actively 
motile. It is usually between 22 and 30 p in length, and from 1 to 2 
p in diameter. It tapers to the anterior end, but the posterior is 
somewhat blunt. The undulating membrane and the free flagel- 
lum are well differentiated. This organism can scarcely be separ- 
ated from Trypanosoma hrucei on the basis of morphology. Laveran 
and Mesnil, however, differentiate on the basis of the former being 488 
VETERINARY BACTERIOLOGY 
more slender, possessing a longer flagellum, and greater motility 
in hanging drop. 
Cultivation.-This organism has been cultivated on Novy and 
MacNeal's medium, but only after repeated trials. 
Pathogenesis.-The disease may be readily transmitted to sus- 
ceptible animals by the injection of blood containing the try- 
panosome. The disease itself is characterized in the horse as a 
relapsing fever, with eruptions, either generalized or localized in 
the skin. Petechial hemorrhages of the mucosae are frequent. 
The subcutaneous tissues are infiltrated and edematous. It is 
almost invariably fatal in horses. Cattle are relatively resistant 
to the disease, and in these animals recovery usually occurs, but 
the blood may remain infective for a considerable period. Buffalo 
frequently succumb. Camels, elephants, and dogs are not infre- 
quently infected. 
This disease resembles nagana clinically, and the causal organ- 
isms can scarcely be differentiated. Animals immunized against 
the one disease are susceptible to the other, which would seem to 
establish specific differences sufficient to separate the organisms as 
distinct species. 
Bacteriologic Diagnosis.-The organisms gradually increase in 
numbers in the blood during the onset of the disease, and have 
been found as numerous as 350,000 per cubic millimeter. They 
are frequently not present in the blood between the periods of 
fever. 
Transmission.-Fraser and Symons state that in the Federated 
Malay States four species of the fly genus Tabanus, particularly 
Tabanzis fumifer, are responsible for the spread of the disease. 
Probably other flies may carry the organism as well. Experiments 
seem to show that the transference in this case is merely mechan- 
ical, and that there is no developmental cycle in the intermediate 
host. Carnivorous animals may be infected by ingestion if there 
are lesions in the mucous membranes. 
Disease Produced.-Nagana or tsetse-fly disease in horses, 
cattle, camels, buffalo, antelopes, and related wild animals, pos- 
sibly the elephant. 
Trypanosoma brucei PATHOGENIC PROTOZOA OF THE FLAGELLATA 
489 
The fact that this disease follows the bite of the tsetse fly has 
long been known by African natives, and the early explorers con- 
firmed their belief. Bruce, in 1896, described the trypanosome 
which causes the disease. 
Distribution.-Known only in Africa, particularly in Zulu- 
land. 
Morphology.-The organism is sluggishly motile. According 
to Laveran and Mesnil, the organism from rats, mice, and guinea- 
pigs is 26 to 27 p long, including the flagellum, while in horses and 
mules it is 28 to 33 p. In breadth it varies between 1.5 and 2.5 p. 
The free part of the flagellum is shorter than that of Trypanosoma 
evansi, while the breadth is usually greater. Granules may gen- 
erally be observed in the protoplasm. Irregular forms occur 
in the blood after death, and in the lymphatic glands, spleen, bone- 
Fig. 189.-Trypanosoma brucei (adapted from Gonder and Sieber). 
marrow, liver, and lungs during life. Kleine believes, from his 
experiments with fly transmission, that there must be a develop- 
mental stage occurring in the insect. 
Isolation and Culture.-Novy and MacNeal succeeded in grow- 
ing the organism of nagana in the medium already described. Only 
a few tubes out of a large number were found to show growth. 
Pathogenesis.-Experimental Evidence.-Inoculation of the 
mouse, rat, dog, cat, and monkey results in an acute infection; 
of the rabbit, guinea-pig, equines, and swine, in a subacute infec- 
tion; and of cattle, goats, and sheep, in a chronic infection. 
Character of Disease and Lesions Produced.-The disease is of 
greatest importance in the equines. The incubation period is 
from three to twelve days (Theiler). There is a continued or re- 
mittent fever and a watery discharge from the eyes and nose. 
The animal becomes much emaciated before death, which usually 490 
VETERINARY BACTERIOLOGY 
occurs in from two weeks to three months. Edema of the ventral 
region is common. If the animal dies during a febrile attack the 
spleen is -acutely swollen, otherwise there is generally present a 
chronic tumor of the organ. Sometimes the spleen is entirely 
normal. The lymph-glands are generally enlarged. 
Immunity.-Rodet and Vallet believe that the organism is 
rapidly destroyed in the spleen. No practicable method of im- 
munizing against the organism has been developed. It has been 
found that the injection of human serum into laboratory animals 
at intervals will greatly prolong their life, but will not cure. There 
is probably some relationship between immunity of man and the 
trypaniciclal character of his serum. Goats, sheep, and cattle show 
a considerable percentage of cures and are thereafter immune. 
Their serum, however, has little immunizing power. 
Bacteriologic Diagnosis.-Stained mounts of the blood from 
infected animals will generally reveal the characteristic parasites. 
Centrifugation of the blood will frequently result in the collec- 
tion of the organisms in a layer just at the surface of the cor- 
puscles. 
Transmission -The disease is commonly transmitted from one 
animal to another by the bite of the tsetse fly (Glossina morsitans), 
and, according to Koch, in those regions where this fly is unknown 
by the closely related fly G.fusca. The herbivorous animals native 
to sections of the country where the disease is prevalent are almost 
invariably infected, and render infection of other animals easy. 
Kleine experimentally showed that another species of Glossina 
(G. palp Olis') could transmit the disease. He found that the flies 
did not become infective until eighteen days had elapsed after 
biting an infected animal. Other experimenters have found that 
the fly loses its infectiveness within a day or two after feeding 
upon an infected animal, and have concluded that transmission is 
wholly a mechanical affair. 
Trypanosoma equinum 
Synonym.- Trypanosoma elmassiani. 
Disease Produced.-Mal de caderas of the horse (Spanish 
caderas = rump or hindquarter). 
Elmassian, in 1901, announced his discovery of the specific PATHOGENIC PROTOZOA OF THE FLAGELLATA 
491 
trypanosome of this disease in Argentina. Voges and Lignieres 
confirmed this discovery in the following year. The disease is so 
prevalent in some sections that cattle exclusively are used for 
riding and driving. 
Distribution.-Parts of South America, particularly Brazil, 
Paraguay, and Argentina. 
. Morphology.-This trypanosome resembles those of surra and 
nagana, but the blepharoplast is so inconspicuous and stains so 
faintly that it may be readily overlooked. The cell is usually 
between 22 and 24 /z in length and 2 to 4 ju in width. Cells about 
to divide are somewhat larger. The difference in the blepharoplast 
of this and most other forms renders identification easy even in 
mixed infections. 
Pathogenesis.- -Experimental Evidence.-Inoculation of the 
organism causes a fatal infection in the horse; the mule and donkey 
are somewhat more resistant, as are mice, rats, and other rodents, 
rabbits, and other laboratory animals. Birds cannot be infected. 
The pig, sheep, goat, and ox may show transitory symptoms, but 
are highly refractory. 
Character of Disease and Lesions.-The disease differs from surra 
and nagana in the almost complete absence of edema, and is 
characterized by a paralysis of the hindquarters. There is a 
progressive emaciation, fever, and the hindquarters become weak; 
the horse in walking scarcely raises the hoof above the ground. 
Finally, the animal supports itself by leaning or falls to the ground. 
There are no lesions upon the genital organs. 
Immunity.--No method of practicable immunization against 
the organism has been developed. 
Bacteriologic Diagnosis.--The organism may be found in the 
blood, particularly during the fever paroxysms. 
Transmission.-The disease is evidently enzootic in certain 
parts of South America in rodents or other animals. One of these, 
the capybara {Hydrochaxus capybara), has been found to be in- 
fected, and it is said that stockmen can sometimes foretell an 
outbreak of the mal de caderas by the death of many of these ani- 
mals in the vicinity. The method of transmission is not certainly 
known. Flies have been supposed to act as carriers, but definite 
proof is lacking. 492 
VETERINARY BACTERIOLOGY 
Trypanosoma dimorphon 
Disease Produced.-Gambian horse sickness, trypanosomiasis 
in horses and other equines, cattle, sheep, and goats. 
Dutton and Todd, in 1902, reported the discovery of a specific 
trypanosome in a disease of horses in Senegambia. The same, or 
very similar trypanosome, has since that time been reported from 
many African localities in other animals as well. 
Distribution.-Various localities in Africa (French Guinea, 
Zanzibar, Sierra Leone, Mozambique, Zululand). 
Morphology.-The organism is characterized by the absence 
of a free flagellum, the flagellum terminating with the undulating 
membrane. It is dimorphic, some of the cells being 20 to 25 y 
in length, others only about 12 g. Transitional forms between 
these extremes may be found. The undulating membrane is not 
well developed. Protoplasmic granules are very rare or are absent 
in the cell. 
Pathogenesis.-Experimental Evidence.-The disease has been 
experimentally produced by the inoculation of the organism into 
sheep, cattle, goats, rabbits, horses, and white rats. 
Character of Disease and Lesions.-The infection is acute in the 
rat, acute or chronic in the rabbit and dog, and chronic in the ox, 
sheep, and in equines. It produces a severe anemia, with changes 
in the red blood-cells. In the horse there is progressive emacia- 
tion. A marked edema is rarely produced. Death does not occur 
usually for months after infection. Recovery sometimes occurs. 
Immunity.-No practicable method of immunization by means 
of the organism or its products has been developed. 
Bacteriologic Diagnosis. The organism may be found in the 
blood at certain periods, but repeated examination is sometimes 
necessary. Inoculation of other animals with the blood will never- 
theless show it still to be infective. 
Transmission.-Transmission supposedly occurs through the 
Glossina palpalis. Other flies are considered to be occasional 
carriers. 
Broden described a disease of horses in the Congo Free State 
which closely simulated nagana, but the trypanosome resembled 
rather Trypanosoma dimorphon. It is also reported from Rhodesia. 
Trypanosoma congolense PATHOGENIC PROTOZOA OF THE FLAGELLATA 
493 
Asses and dromedaries also are infected. Laveran, as a result of 
inoculation experiments, has concluded that this is a distinct 
species. It has been reported from several localities in southern 
Africa. It is fatal for cattle and sheep. The organisms are 10 
to 20 p in length and 1.5 to 2.5 p wide. The flagellum shows no 
free portion, hence animal inoculations are necessary to differen- 
tiate between this and Tr. dimorphon. Transmission is through 
the Glossina morsitans. 
Trypanosoma pecaudi 
Disease Produced.-Baleri, or trypanosomiasis in horses, cattle, 
sheep, and goats. 
This organism was described by Laveran from the blood of in- 
oculated sheep brought to Paris by Cazalbou. 
Fig. 190.-Trypanosoma pecaudi (Laveran). 
Distribution.-The disease is known from the French Sudan. 
Morphology.-The organism closely resembles Trypanosoma 
dimorphon. Two forms are described: long slender cells, 25 to 35 
p in length by about 1.5 p in width, with narrow, undulating mem- 
brane and fairly long flagellum, and short broad forms, 14 to 20 p by 
3 to 4 p, with no free flagellum and a wide, undulating membrane. 
Pathogenesis.-Character of Disease and Lesions.-In the horse 
the disease is characterized by repeated attacks of a severe fever, 
swellings in various parts of the body, injection of the conjunctiva, 
and a considerable degree of emaciation. Rats, mice, guinea- 
pigs, and dogs are susceptible to infection. 494 
VETERINARY BACTERIOLOGY 
Transmission.-Buffard claims the Glossina palpalis is the agent 
of its transmission, others that the G. longipalpis is the commoner. 
Trypanosoma cazalboui 
Disease Produced.-Souma or soumaya in cattle, sheep, horses, 
and mules. 
Distribution.-Africa, from the French Sudan, French Congo, 
Upper Nile. 
Morphology.-The organism, including its free flagellum, is 
about 21 by 1.5 p. The oval nucleus is centrally located. The 
undulating membrane is poorly developed and is little folded. The 
terminal portion of the flagellum is free. There are no marked 
characters differentiating the organism from Trypanosoma evansi 
Fig. 191.-Trypanosoma cazalboui (Laveran). 
Pathogenesis.-Experimental Evidence.-Dogs and small test 
animals seem to be relatively immune. The infection may be 
readily transmitted to horses and cattle and the smaller ruminants. 
Cross-inoculation experiments have shown the disease to be dis- 
tinct from surra. 
Character of Disease.-It generally attacks cattle. The disease 
leads to a progressive emaciation, weakness of hindparts, the skin 
is harsh, and there is a staring coat. In many individuals the lower 
surfaces of the body show marked edema. The temperature is 
variable. The disease may be acute lasting not over fifty days, 
while in other cases the course may be prolonged for a year or 
more. PATHOGENIC PROTOZOA OF THE FLAGELLATA 
495 
Bacteriologic Diagnosis.-The organisms are present in the 
blood, usually in small numbers only. 
Transmission.-Pecaud concludes that Glossina palpalis is 
responsible for the distant transmission of the organism, and that 
members of the fly-genera, Stomoxys and Tabanus, may produce 
immediate transmission. 
Trypanosoma theileri 
Disease Produced-Theiler at first believed this organism to 
be the cause of galziekte or gall-sickness in bovines, but later 
investigation has led him to class it as a harmless commensal in the 
blood of cattle. 
This organism was first noted by Theiler and was described at 
greater length by Laveran. Schnitt in Germany, Peters in South 
America, and Crowley in America have described very similar 
organisms in the blood of cattle. 
Distribution.-A large part of South Africa, possibly also in 
India. 
Morphology.-The organism associated with this disease is of 
unusual size, 30 to 70 y in length and 2 to 5 y in width. This 
alone is sufficient to differentiate it from other forms. There is a 
long, free flagellum. The nucleus is central, but the blepharoplast 
is a considerable distance from the posterior end. Many proto- 
plasmic granules may usually be seen. 
Pathogenesis.-The organism seems to be inoculable only into 
cattle. It resembles the rat trypanosome in being thus limited 
to a single host. 
Bacteriologic Diagnosis.-The organisms are quite common in 
the blood, but soon disappear. 
Transmission.-It has been found to be transmitted by the 
bite of the fly, Hypobosca rufipes. 
Trypanosoma gambiense 
Synonyms.-Trypanosoma ugandense; Tr. Castellani. 
Disease Produced.-Human trypanosomiasis, or sleeping 
sickness. 
Distribution.-The disease is endemic upon the western coast 
of Africa and in certain of the central portions. 496 
VETERINARY BACTERIOLOGY . 
Morphology.-The organism is 10 to 28 by 1.4 to 2 p.. Forms 
undergoing division are somewhat larger. The free flagellum may 
be one-fourth to one-third the length of the body. Rarely no 
free portion of the flagellum can be demonstrated. The undulating 
membrane is narrow. The blepharoplast is near the posterior end. 
Protoplasmic granules that stain like chromatin are commonly 
observed. 
Pathogenesis.-Experimental Evidence-The disease, with its 
characteristic clinical symptoms, may be reproduced by the injec- 
tion of blood containing the organisms into the monkey. Dogs, 
jackals, cats, guinea-pigs, and rabbits are readily infected. Mice 
frequently recover and are thereafter immune. Goats and sheep 
are relatively refractory, but sometimes succumb. It may pro- 
duce a mild chronic infection in the horse and in cattle. 
Character of Disease.-The disease is insidious in its onset. 
Two distinct stages may be recognized. These were for a long time 
supposed to be different diseases. In the first stage the organisms 
appear in the blood; there may or may not be fever. In the sec- 
ond stage pains in the back, tremors, and drowsiness supervene. 
Finally the patient dies in a comatose condition. In this second 
stage the organisms are present in numbers in the cerebrospinal 
fluid. The disease appears to be always fatal, but may run a 
chronic course, lasting several years. 
Immunity.-No practicable method of immunization has been 
developed. 
Bacteriologic Diagnosis.-The organisms are usually scanty in 
the blood, and centrifugation is necessary to find them. They may 
usually be demonstrated in the fluid secured by a lumbar puncture. 
They may also commonly be demonstrated by puncturing an en- 
larged lymphatic gland and examining the fluid secured. 
Transmission.-One of the tsetse flies, Glossina palpalis, has 
been found to transfer the disease. 
Trypanosoma lewisi 
Chaussat, in 1850, and Lewis, in 1877, noted the presence of 
a flagellate in the blood of rats. It is so commonly present in the 
blood of rats in many parts of the world that it has been frequently 
used in the laboratory for study and demonstration, although its PATHOGENIC PROTOZOA OF THE FLAGELLATA 
497 
pathogenic properties are almost nil. This trypanosome cannot 
be transmitted to any other genus of mammals so far as known; 
even the closely related genera of the rodents are refractory. It 
evidently is a highly specialized commensal. The organism, with 
its flagellum, measures about 24 to 25 p in length by 1.5 p in width. 
Protoplasmic granules are frequently present. 
Trypanosoma hippicum 
Disease Produced.-A disease of equines, known as murrina 
and derrengadera. 
The organism was described by Darling, in 1910, as the cause 
of disease in horses and mules. 
Distribution.-Panama, Venezuela, and Central America. 
Morphology.-In size the organism resembles Trypanosoma 
evansi and Tr. brucei. According to Darling, the length varies 
from 18 to 28 g, breadth, 11 to 3 y. The flagellum is usually short 
and not entirely free, though sometimes free. Nucleus is located 
posteriorly. Coarse granules are numerous in the protoplasm. 
The posterior end is blunt, never attenuated as is that of Tr. lewisi. 
Pathogenesis.-Besides horses and mules, most of the labora- 
tory animals can be infected. In the dog it produces lameness in 
the posterior extremities. Swine may be infected, but calves are 
refractory. Cattle never acquire the disease. 
Character of Disease.-Progressive emaciation, edema, anemia, 
febrile paroxysms, and conjunctivitis. 
Postmortem Changes.-Slight enlargement of the spleen, pe- 
techiated kidneys, hemorrhages on both endocardium and epi- 
cardium. 
Immunity.-No method of immunization has been developed. 
Bacteriologic Diagnosis.-The clearness of the blepharoplast 
characterizes the organism. The parasite is found in the peripheral 
blood quite generally, but not constantly. As soon as the tem- 
perature of an infected animal reaches 101°, blood examination will 
usually reveal the parasite. During the latter part of the paroxysm 
it may not be demonstrable. Inoculation of the more suscep- 
tible laboratory animals is an aid to diagnosis. Cross inoculation 
with other trypanosomes has shown the Trypanosoma hippicum 
to be distinct. 498 
VETERINARY BACTERIOLOGY 
Transmission.-This probably does not occur through the 
tabanids, as the disease occurs in localities where these flies are not 
found. Darling concludes from observations that flies probably 
act as mechanical carriers by alighting on gall sores and wounds of 
animals infected, and from these carrying the infectious material 
to non-infected animals. 
The Genus Herpetomonas 
This genus includes certain flagellates that have an essentially 
trypanosome-like structure without an undulating membrane. 
The cell is elongated in the typical He'rpetomonas; the closely 
related genus Crithridia comprises those forms in which the body 
is much shortened. The flagellum is anterior, the blepharoplast 
distinct, as in the trypanosomes, and the nucleus centrally located. 
Organisms of this genus have been frequently reported from 
the gut of mosquitoes and flies. Certain of the trypanosomes 
sometimes assume shapes that resemble closely the Herpetomonas. 
The genus assumes pathogenic significance, principally because 
of the tentative classification of certain protozoa known as the 
Leishman-Donovan bodies as members of this genus. Three 
species have been described. 
Herpetomonas donovani 
Synonyms.-Leishman-Donovan bodies; Leishmania donovani; 
Trypanosoma donovani. 
Disease Produced.-Kala-azar, cachexial or Dumdum fever 
in man. 
Leishman, in 1900, observed this parasite in smears from 
the spleen of a patient that had died of Dumdum fever. His 
account was published in 1903. 
Distribution.-Throughout southern Asia and northern Africa. 
Morphology.-The organism as ft occurs in the body is com- 
monly intracellular. It is found principally in the spleen, liver, 
bone-marrow, and lymph-glands. It is oval, spherical, or pear 
shaped, usually between 2 and 3.5 y in length and 1.5 to 2 y in 
width. Two staining granules occur in the interior, the larger 
spherical, and the smaller, and more deeply staining granule, 
somewhat elongated. The organisms multiply by a preliminary 
division of both of the chromatic granules (nucleus and blepharo- PATHOGENIC PROTOZOA OF THE FLAGELLATA 
499 
plast), followed by a constriction of the cell. In cultures typical 
flagellates are produced. The organism elongates somewhat, 
and a vacuole appears at one side of the blepharoplast, and from 
this a single flagellum develops. The body finally assumes an 
elongated form not unlike a trypanosome without an undulating 
membrane. This is the Herpetomonas stage. This may now di- 
vide longitudinally, frequently unequally, splitting off very slender 
cells. The systematic position of this organism is still somewhat 
in doubt. It may be that it should be regarded as belonging to a 
distinct genus, and the name Leishmania used instead of Herpeto-. 
monas. 
Pathogenesis.-The disease is characterized by enlargement of 
the spleen and by fever. 
Transmission.-It is believed that the parasite is transferred 
from the dog to man through some intermediate host. 
Leishmania (Herpetomonas ?) infantum 
Nicolle has described an organism similar to the preceding from 
a disease which he calls infantile kala-azar. The organisms re- 
Fig. 192.-Herpetomonas infantum: A, Organisms from the spleen of a 
child; B, a mononuclear containing the organisms, and C, an endothelial cell 
from the spleen; D, various stages in the development of the Herpetomonas 
form from the Leishman-Donovan bodies (Nicolle). 
semble the preceding, but are believed to constitute a separate 
species. The disease is primarily one of dogs, which may be trans- 
mitted to children. 
Leishmania tropica 
Synonyms.-Ovoplasma orientate; Helcosoma tropicum. 
Wright has described a similar organism as the cause of oriental 
sore or Delhi boil in man. It is probably transmitted likewise 
by some biting insect. The dog may be infected and show clinical 
symptoms similar to man. CHAPTER XLII 
PATHOGENIC PROTOZOA OF THE CLASS RHIZOPODA 
The Rhizopoda are protozoa having during adult life movable 
or changeable processes of protoplasm called pseudopodia. Repro- 
duction is accomplished through simple fission and by spore 
formation. 
Among the hundreds of genera and thousands of species of 
organisms belonging to this group that have been described there 
Fig. 193.-Ameba in culture (Schardinger). 
is one (possibly two or three) which has been found to be patho- 
genic. They are certainly known to be pathogenic in man only, 
but the frequent occurrence of the organisms of this genus is the 
500 PATHOGENIC PROTOZOA OF THE CLASS RHIZOPODA 
501 
intestines of the lower animals, and the possibility of confusing the 
adult stage with certain developmental stages of the sporozoa 
renders a discussion of these forms advisable in a veterinary text. 
The possibility of any of the forms being pathogenic for lower 
animals has not been sufficiently investigated. 
The Genus Entamceba 
The normal inhabitant of the intestinal tract of man was first 
known as Amoeba coli. Later, Schaudinn renamed it Entamoeba 
coli, and gave the name Entamoeba histolytica to the pathogenic 
type associated with the amebic dysentery. The genus Entamoeba 
was differentiated from Amoeba by the absence of a contractile 
vacuole and by the formation of multinucleated, cysts. 
Authorities differ greatly in their estimates of the number of 
species of amebae present in the intestines of man. Walker and 
Sellards state that eighteen forms have been described as separate 
species. The best classification, and the one most commonly used 
now, is that of Schaudinn. He recognizes two species at least- 
one a normal non-pathogenic form, Entamoeba coli, and one patho- 
genic for man, Entamoeba histolytica. More recently Hartmann 
and others have described a third species, E. tetragena, and Koid- 
sumi a fourth, E. nipponica, both from cases of dysentery. The 
possibility of any of the dysenteries of the lower animals being 
caused by amebae has not been sufficiently studied. Smith believes 
that certain diseases of turkeys and other fowls, particularly entero- 
hepatitis, are caused by an ameba termed Amoeba meleagridis, but 
others have contended this to be but a developmental stage in 
the life-history of a Coccidium. 
Examination of Living Amebce.-The amebae may be examined 
in the stools in a living condition by placing a portion of the 
liquid or a bit of the solider material moistened with physiologic 
salt solution on a slide, and pressing down a cover-glass not too 
firmly. Craig advises the use of a very weak solution of neutral 
red to stain the living organisms when* they are not present in too 
great numbers. For the specific determination of the amebae 
present an examination of this kind is frequently all that is neces- 
sary. The slide must be maintained at about blood-heat in order 
to detect motility. 502 
VETERINARY BACTERIOLOGY 
Staining Methods.-The organisms stain rather readily with the 
usual laboratory stains, but these are of little value in the differ- 
entiation of the parts of the cell and in separating species. Wright's 
stain in favorable specimens, if carefully used, gives the best 
differentiation of the parts of the cell. The organism in tissues 
is best stained by Heidenhain's iron-hematoxylon or Borrel's 
stain. The smears should be fixed from fifteen to twenty minutes 
in alcohol and ether, equal parts, for all stains but Wright's. For 
the latter no fixation is required. 
Methods of Isolation and Cultivation.-Amebae commonly 
use bacteria and related organisms as food. A culture of amebae 
must provide for the growth of bacteria, but this growth must not 
be so luxuriant as to overgrow and eliminate the amebae. The 
agar medium recommended by Musgrave and Clegg may be used. 
This contains agar, 20 gm.; NaCl, 0.03 to 0.05 gm.; beef-extract, 
0.3 to 0.5 gm.; water, 1000 c.c., made 1 per cent, alkaline to 
phenolphthalein. Variations in the materials used are sometimes 
necessary for some specialized saprozoites. This medium is 
melted, poured into a sterile Petri dish, and allowed to solidify. 
The surface of the medium is streaked with the material containing 
the amebae. The bacteria and amebae will usually both multiply 
if the plate be kept at a suitable temperature. Two operations are 
necessary to secure a culture in which it is known that all of the 
amebae are of one species and all of the bacteria of one kind. The 
mixed culture is placed upon the medium in the center of the 
Petri dish. Concentric circles of the organism with which it is 
desired to grow the ameba are placed about this. The amebae, 
as they crawl through the successive circles, gradually lose the 
original organisms with which they started and come to feed on the 
one kind. After one or more transfers a growth of the amebae 
may be secured with the one species of organism desired. It is not 
always possible to secure growth with every species of organism, 
as the different amebae have been found to require various species 
of bacteria. 
In order to secure a culture containing a single kind of ameba 
it is necessary to isolate a single individual. Ordinarily this may 
be accomplished by examination of the surface of an agar culture 
until an isolated ameba is found, and this is then transferred to a PATHOGENIC PROTOZOA OF THE CLASS RHIZOPODA 
503 
fresh plate. Musgrave and Clegg recommend running the tip of 
the lens against such an organism and removing it, attached to the 
lens, and inoculating the new medium by running the lens down 
in contact with it. 
Synonym.-Amoeba coll. 
Disease Produced.-The organism is probably non-pathogenic. 
The Entamoeba coli was probably seen and recognized by Lambl 
in 1860. Since that time it has been repeatedly noted by many 
investigators. Schaudinn, in 1905, first clearly differentiated 
the organism and traced its life-history. His work has been con- 
firmed and extended by Craig in the United States. The organ- 
ism is present in a large percentage (50 per cent., according to 
Entamoeba coli 
Fig. 194.-Entamoeba coli: A, Non-motile form; B, cell showing psudopodia; 
D, E, stages in cell division; F, G, H, I, J, stages in encystment and sporula- 
tion (adapted from Craig). 
Schaudinn) of healthy individuals. It is most easily recognized 
in the feces after the administration of a saline cathartic. 
Morphology.-The Entamoeba coli is a mass of protoplasm not 
possessing a definite cell wall, but with a nucleus containing usually 
one or more nucleoli. One (rarely more) non-contractile vacuole is 
occasionally present. The organism varies from 8 to 50 /z in 
diameter, but in the majority of cases is between 10 and 20 /a. 
When encysted it is usually between 10 and 15 /z. The organism is 
approximately spherical when not in motion. It is sluggishly 
motile, and usually is not moving when observed in a hanging 
drop. In this it is of a dull gray color. The protoplasm cannot 
be differentiated into endoplasm and ectoplasm when the organ- 
ism is at rest. When in motion the ectoplasm may sometimes be 
seen. This fact is of considerable diagnostic value. The endo- 
plasm is finely granular, and rarely contains more than a single 504 
VETERINARY BACTERIOLOGY 
vacuole. The nucleus is definite, spherical, and contains chroma- 
tin granules and one or more nucleoli; it is 5 to 8 y in diameter. 
When stained by Wright's method, the ectoplasm and endoplasm 
may be easily differentiated. The ectoplasm stains blue, the 
endoplasm violet, and the nucleus red. 
Multiplication is commonly accomplished in the intestines 
by simple division. The nucleus divides and the protoplasm con- 
stricts to form two individuals. This process is common in the 
liquid stools, but when drier and firmer, cystic reproduction is 
more common. A refractive hyaline cyst forms about the spher- 
ical organism and thickens. The protoplasm becomes homo- 
geneous in appearance and clearer than before. The nucleus then 
undergoes complicated divisions and recombinations, which ul- 
timately result in the formation of eight nuclei. When the wall 
is ruptured, these nuclei, with bits of the protoplasm, escape and 
constitute eight young amebae. 
Pathogenesis.-Repeated efforts to produce disease by injec- 
tion of Entamoeba coli into the colon of cats and other animals have 
failed. It must be regarded as a normal inhabitant of the intes- 
tines of man. Similar organisms have been found in the intes- 
tines of animals. 
Bacteriologic Diagnosis.-Entamoeba coli may be recognized 
in the feces by a direct microscopic examination as an ameba 
showing very slight motility, rounded pseudopodia, little or no 
observable differentiation between ectoplasm and endoplasm, 
comparative lack of vacuoles in protoplasm, and by the production 
of eight daughter-cells from each cyst. 
Entamoeba histolytica 
Synonym.-Amoeba dysenteries. 
Disease Produced.-Amebic dysentery in man. 
Loesch, in 1875, described Amoeba coli as the cause of a dysen- 
tery in man, and claimed that the rectal injection of feces con- 
taining the organisms into dogs produced dysentery. Robert 
Koch, in 1883, showed the organism to be present in the ulcerations 
of the intestines. Kartulis firmly established a probable etio- 
logic relationship by his studies in Egypt, published in 1886. 
Councilman and Lafleur studied the pathology of the disease in PATHOGENIC PROTOZOA OF THE CLASS RHIZOPODA 
505 
great detail. Schaudinn gave to the organism its present name of 
E. histolytica, and described its morphology with accuracy. 
The disease has been reported from all sections of the world. 
It seems to be much more prevalent in tropical than in temperate 
climates, but has been identified repeatedly in the United States. 
Morphology.-According to Craig, the morphology of this 
organism varies so greatly in culture-media that only the forms 
found in the feces can be regarded as typical. The organism under 
these conditions varies in diameter to 50 y or more, showing it 
to be larger than Entamoeba coli. At rest, the organism is spherical 
Fig. 195.-Entamoeba histolytica: 1, Organism in motion-a, Vacuoles; b, 
red blood-cells; c, pseudopodium composed chiefly of ectoplasm. 2, Organ- 
ism showing nucleus a, and vacuoles, b. 3. Preliminary changes upon begin- 
ning sporulation-a, Vacuoles; b, chromatin masses escaping from the nucleus 
and scattering through the cell. 4, Chromatin masses scattered through the 
cell, b; c, vacuole. 5, Cell budding off spores, the chromatin masses or new 
nuclei passing through the ectoplasm and escaping as spores. 6, Free spores 
(adapted from Craig). 
or ovoid; when in motion, its shape is extremely variable. It is 
usually actively motile, throwing out pseudopodia much more 
rapidly than E. coli. 
Color is absent, and the organism appears clear, or there may 
be a greenish tinge, due to the presence of hemoglobin from the 
blood in the feces and the blood-corpuscles engulfed. In the 
larger cells, rarely in the smaller, the endoplasm and ectoplasm 
may be quite sharply differentiated. The latter is hyaline, 
glass-like, and refractive, much firmer than the comparatively 
delicate membrane of Entamoeba coli, and the two are relatively 506 
VETERINARY BACTERIOLOGY 
easily distinguishable by this means. The ectoplasm apparently 
comprises about one-third of the protoplasm. One or more vacu- 
oles are always present, as many as ten sometimes being found. 
The nucleus is faint and difficult to distinguish in the unstained 
mount. It is about 5 to 6 jU in diameter. The chromatin is 
relatively sparse. With Wright's stain the ectoplasm stains 
more deeply than the endoplasm, the opposite of what is true in 
E. coli. 
Reproduction is accomplished in two ways. The first consists 
of a division of the nucleus, followed by a constriction of the cell 
to form two individuals. This process is essentially the same as 
that in Entamoeba coli. The method of spore formation, gemma- 
tion, or budding is quite different. When conditions arise unfa- 
vorable to continued vegetative existence, spores are produced. 
The nucleus, by a process of fragmentation, throws out chromatin 
granules or chromidia, which gradually collect under the ecto- 
plasm, form new nuclei, and are finally thrown off from the ex- 
terior, together with some of the protoplasm, as a spore or bud. 
These spores are round or oval, have a yellowish membrane, and 
measure 3 to 6 /i, usually about 4 y in diameter. The membrane 
which forms about these spores is resistant to the penetration and 
action of stains. 
Pathogenesis.-Experimental Evidence.-Schaudinn dried feces 
of dysentery patients after demonstrating that they contained 
Entamoeba histolytica, in the form of spores in large numbers, and 
that they were free from E. coli. These were fed to a kitten, and 
death resulted in fourteen days, with characteristic ulceration of 
the intestinal wall. Similar experiments have since been repeated 
many times. There is little or no doubt of the pathogenicity 
of the organism, and the disease produced may be considered as a 
clinical entity. 
Character of Disease and Lesions.-The disease produced is a 
chronic dysentery, marked by intestinal ulceration, and frequently 
by abscesses in the liver. The presence of the relatively firm 
ectoplasm is believed to account for the ability of the organism 
to force its way into the tissues between the cells. 
Immunity.-No method of immunization against the Entamoeba 
histolytica has been developed. PATHOGENIC PROTOZOA OF THE CLASS RHIZOPODA 
507 
Bacteriologic Diagnosis.-The Entamoeba histolytica may be 
recognized in the feces, and separated from the E. coli by its larger 
size, its distinct differentiation into a hyaline refringent ectoplasm 
and a more granular endoplasm, the presence of more than one 
vacuole usually, the common presence of erythrocytes in the pro- 
toplasm in the course of digestion, by the less prominent nucleus, 
and by formation of spores by a process of budding with encyst- 
ment. 
Entamoeba tetragena 
Synonym.-Entamoeba africana. 
This species was first described by Viereck in dysentery in 
Africa. Since that time the same organism has been noted by 
Hartmann, Werner, and others. It has been found capable of 
producing dysentery in cats. It resembles morphologically the 
Entamoeba coli more closely than the E. histolytica, but differs by 
the formation of four spores instead of eight within the cysts. CHAPTER XLIII 
SPOROZOA 
The Sporozoa stand alone among the protozoa, in that all 
the species are parasites. Many are harmless commensals, but a 
considerable number are pathogenic. The sporozoan cell contains 
typically a single nucleus, except in the Myxosporidia, which are 
multinucleate. Food is taken in by diffusion through the plasma 
wall. Gastric and contractile vacuoles are present. The adult 
form is non-motile; the young forms are frequently actively 
motile, either ameboid or flagellate. In most of the Sporozoa 
the differentiation of the protoplasm into ectoplasm and endo- 
plasm can be clearly made out. The reproduction of the Sporozoa 
is the principal character which differentiates this group from 
others. Spores are always produced in those forms in which the 
complete life-history is known. The details of spore formation 
vary greatly in the different forms, but the essentials are the 
same. 
The cell as a whole, in most forms, divides to form archispores 
or sporoblasts; each of the archispores forms spores, and each spore 
then produces one or more sporozoites. Many forms show addi- 
tional methods of reproduction. Formation of sex cells or gametes, 
with fusion of like or unlike cells, takes place in many forms, and 
serves to further complicate the life-history. 
Blood-smears may be stained by one of the Romanowsky 
stains or by Wright's method to demonstrate the protozoa of this 
group. Considerable care must be exercised in the examination 
of such blood mounts not to confuse the normal blood con- 
tents, such as blood-platelets, with developmental stages of the 
protozoa. 
The class Sporozoa contains many families and genera; only 
a few of the latter contain species that are of economic importance. 
The following artificial key will assist in differentiating the various 
genera of veterinary interest: 
508 SPOROZOA 
509 
A. Sporozoa found in the blood-cells. 
1. In erythrocytes. 
(a) At some stage occupying a considerable proportion of the interior of 
the cell. 
(1) In mammalian blood. 
(a) Well-differentiated, often two in a cell. In animals. 
1. Initial multiplication in the spleen, 
organisms in peripheral blood usu- 
ally rod shaped Theileria. 
2. Constantly in blood-cells. Usually 
pear shaped Babesia (Piroplasma). 
(b) Ameboid of first, finally filling the 
cell Plasmodium. 
(2) In avian blood Proteosoma (Haemoproteusb 
(b) Forming minute dots, seemingly entirely 
of chromatin Anaplasma. 
2. In leukocytes. 
(a) In mammals Leukocytogregarina. 
(b) In birds Leukocytozoon. 
B. Sporozoa occurring in muscles Sarcocystis. 
C. Sporozoa occurring in membranes (mucous or 
serous) Eimeria (Coccidium) and 
Isospora. 
The Genus Piroplasma, or Babesia 
An organism belonging to this genus was first noted by Babes 
and called by him Hcematococcus. Theobald Smith, in 1889, made 
the first observation which related one of these organisms to Texas 
fever. He called the organism Pyrosoma. This was later changed 
to Piroplasma by Patton, and still later by Starcovici to Babesia. 
The organisms of this genus occur in the red blood-cells of 
various mammals and produce several distinct diseases. They do 
not show pigmentation. The life-history of all the species has not 
been satisfactorily worked out. 
Babesia bigemina 
Synonyms.-Pyrosoma bigeminum; Apiosoma bigeminus; Piro- 
plasma bigeminum; Hcematococcus bovis; Ixidoplasma bigeminum. 
Disease Produced.-Texas fever, or tick fever in cattle- 
bovine piroplasmosis. 
Theobald Smith, in 1889, discovered the cause of Texas fever 
in cattle. His work was fundamental and remarkably complete. 
Since that time investigators have found the same organisms in 
many countries. 510 
VETERINARY BACTERIOLOGY 
Distribution.-Southern United States, Australia, South Amer- 
ica, Europe, India, Philippines, and Africa. 
Morphology.-In the blood of infected animals the organisms 
are generally in pairs. They are commonly piriform, one end being 
rounded and the other somewhat pointed. The acute ends are 
usually pointed toward each other. The organisms vary from 0.5 
to 2 /z in diameter, and 2 to 4 /z in length. The reproductive 
stages have not been thoroughly worked out, nor has the develop- 
ment in the tick. The organism stains readily with such dyes as 
alkaline methylene-blue and by Wright's method. 
Pathogenesis.-The relationship of the organism to the disease 
has been satisfactorily demonstrated by inoculation experiments. 
Fig. 196.-Babesia bigemina, infected red blood-cells with 1-4 parasites, a 
blood-platelet in the center (Sieber). 
The disease in cattle is characterized by fever and a hemoglobin- 
uria, with considerable destruction of red blood-corpuscles. In 
acute cases death often occurs in five to eight days after the 
symptoms are first noted. Those cases in which recovery takes 
place generally harbor still in their bodies the specific organisms, 
but remain perfectly well. 
Immunity.-As already noted, recovery from disease does not 
necessarily predicate the disappearance of the organism from the 
blood, and it may persist for years. It has been found that 
immunity against a fatal attack of this disease may be conferred 
by inoculation with the blood of immune animals. This results 
generally in a mild infection, which immunizes against one of a SPOROZOA 
511 
severer type. Such methods of vaccination are quite widely 
practised. 
Bacteriologic Diagnosis.-The organism may usually be 
demonstrated in the blood when stained with Lbffler's methylene- 
blue or Wright's stain. 
Transmission.-Natural infection takes place only through the 
bite of infected cattle ticks (Rhipicephalus annulatus or Bobphilus 
bovis) in the United States and closely related forms in other 
countries. The female tick becomes engorged with blood and falls 
to the ground, where, after a time, eggs are laid. They hatch in 
from nineteen days to five or six months, depending upon the tem- 
perature conditions. The young ticks crawl up the stems of grass 
and shrubs. They must get upon the body of an animal or die of 
starvation. The ticks from an infected mother are themselves 
infective, and may transmit the disease to the animal whose blood 
they suck. 
Synonym.-Piroplasma mutans. 
Theiler has established the presence of a form of bovine piro- 
plasmosis in southern Africa, due to a prototozoan which he has 
named Piroplasma mutans. It is smaller than the P. bigeminum, 
and animals immunized' against one will contract disease caused 
by the other. 
Babesia mutans 
Synonym.-Piroplasma equi. Possibly Babesia asini. 
Disease Produced.-Equine biliary fever. Equine piroplas- 
mosis. 
Guglienni, in 1899, discovered this organism in Italy, and 
Theiler later elaborated an account of the disease and the organism 
as it occurs in South Africa. 
Distribution.-The disease has been noted from South Africa, 
Central Africa, Algeria, Italy, Sweden, Russia, India, and Vene- 
zuela. It is evident that further observation may show an exten- 
sive distribution. 
Morphology.-The organism is smaller than Piroplasma bigemi- 
num, but resembles it. It occurs singly or in pairs, or rarely in 
rosettes, in the red blood-cells. Occasionally it is free in the 
plasma. The disease was first supposed to be non-transmissible by 
Babesia equi 512 
VETERINARY BACTERIOLOGY 
blood injection, but Theiler succeeded by intravenous injections of 
virulent blood. It also attacks the ass and the mule. 
Pathogenesis.-The disease is characterized by jaundice and a 
high fever. It may run an acute or a chronic course; frequently 
it is fatal within a few days. The lymph-nodes and the spleen are 
considerably enlarged. Animals born in infected districts are 
commonly immune. 
Transmission.-The disease is transmitted by ticks in South 
Africa by Rhipicephalus evertsi. The nymphs are infected and 
transmit the disease to other horses when they become adults. 
Babesia ovis 
Synonyms.-Piroplasma ovis; H cematococcus ovis; Amoebospo- 
ridium polyphagum. 
Disease Produced.-Hemoglobinuria, malarial catarrhal fever, 
or icterohematuria in sheep. 
Babes, in 1892, first noted the parasites in the blood-cells of 
sheep in Rumania at the time that he made his observations on 
piroplasmosis of cattle. 
Distribution.-It has been noted from Italy, France, Turkey, 
Venezuela, and the West Indies, South Africa, Rumania, German 
East Africa, and probably in the United States (Montana). 
Morphology.-It is similar to Piroplasma bigeminum, but 
smaller (1 to 1.8 p in diameter). It is commonly single, sometimes 
double, in the cells, and frequently occurs in the plasma. 
Pathogenesis.-The disease may be transferred by the injec- 
tion of blood containing the organisms into healthy animals. The 
incubation period is about eight to ten days. Other animals, 
including cattle, cannot be infected. The disease is commonly 
fatal. It is characterized by anemia, icterus, and frequently 
hemoglobinuria. 
Transmission.-The disease is transmitted through the bite of 
a tick (Rhipicephalus bursa). The adult only transfers the disease. 
Synonyms-Pyrosoma bigeminum vax.canis; Piroplasma canis. 
Disease Produced.-Biliary fever or malignant jaundice of the 
dog. 
Babesia canis SPOROZOA 
513 
Prani and Galli-Valerio, in 1895, first described the blood 
parasite of canine piroplasmosis. 
Distribution.-It has been reported in China, India, Italy, 
France, Hungary, South Africa, East Africa, and possibly from the 
United States. 
Morphology.-The organisms are morphologically almost 
identical with Piroplasma bigeminum, but are larger. They are 
generally 2 to 4 ju in diameter. They are sometimes found abun- 
dantly in the plasma, and in a single blood-cell there may be as 
many as sixteen of the organisms. The free organisms are spher- 
ical; those within the corpuscles are pear shaped or many angled. 
Multiplication is apparently by direct division. 
The life cycle is better known for this species than for other 
members of the genus. The pear-shaped bodies within the red 
Fig. 197.-Babesia canis: 1-11, Organisms in various developmental stages in 
red blood-cells in culture; 12, 13, organisms free in the plasma (Deseler). 
cells are relatively large, one end being regularly pointed. Usually 
two are found in a single cell, sometimes four, eight, or more. 
They multiply by longitudinal fusion. An ameboid stage occurs 
while the organism is on the exterior of the red cell, or while the 
organism is young. 
Breinl and Hindle showed that if a dog in an advanced stage of 
the disease is bled, the heart blood mixed with 2| per cent, of sodium 
citrate, motile cells with long flagella may be developed. These 
may be differentiated into gametes. Union of the gametes occurs 
in the alimentary tract of the tick. The zygote is elongate, and 
makes its way into the body of the tick, eventually infecting the 
blood of an animal bitten by the tick. 
Culture.-Thomson and Fantham have succeeded in cultivating 
Babesia canis in vitro. Heart blood is secured, and yw c.c. of 50 
per cent, solution of glucose in water added to 10 c.c., defibrinated, 514 
VETERINARY BACTERIOLOGY 
and kept at 37°. In the culture, oval, piriform, ameboid, and round 
parasites were found. Hemolysis occurs in all cultures. 
Pathogenesis.-The disease may be readily transferred by the 
injection of virulent blood. It cannot be transmitted to other 
species of animals. Nuttall and Graham-Smith did not succeed 
in reproducing the disease in the fox and jackal. .The period of 
incubation is three days or more. There is fever, and sometimes 
icterus and hemoglobinuria. Anemia is marked. The spleen is 
greatly enlarged, the gall-bladder is distended, and the kidneys 
are often ecchymotic. Chronic cases frequently recover. The 
acute cases are almost invariably fatal. Animals which have ap- 
parently recovered show the parasite in the blood for long periods 
and retain their infectivity. 
Bacteriologic Diagnosis.-Stained blood-films will demonstrate 
the organism if present. 
Transmission.-At least three species of tick, and probably one 
species of flea, have been found to act as carriers of the organism. 
Babesia (Piroplasma) gibsoni 
Patton has described an organism causing piroplasmosis in 
hounds in the Madras Hunt in India. Later it was discovered 
also in the blood of a native jackal. Its relationship to the Piro- 
plasma canis has not been satisfactorily determined. 
Babesia (Piroplasma) commune 
Phillips and McCampbell have described a species of Piro- 
plasma as the cause of an epizootic of dogs at Columbus, Ohio. 
Fig. 198.-Babesia commune, organisms in the red blood-cells (adapted from 
Phillips and McCampbell). 
The organisms found were similar to the Babesia canis, but these 
investigators were able to demonstrate the organism in the blood 
of guinea-pigs injected with virulent blood. The cat was also 
infected, but not the horse, cow, rat, or rabbit. This fact of SPOROZOA 
515 
Fig. 199.-Theileria parva, life cycle: 1, Agametes of the first generation 
(metagametes); 2, a and b, agamonts with one nucleus; 3, a and b, agamonts 
with several nuclei; 4, a and b, medium-sized agamonts; 5, a and b, large aga- 
monts with numerous nuclei; 6, a an4 b, agamonts undergoing schizogony; 7, 
a and b, agametes; 8, a and b, reduction forms of agamonts; 9, a and b, seg- 
mentation of reduction forms of agamonts; 10, a and b, young agamonts; 11, 
a and b, medium-sized gamonts with several nuclei; 12 and 13, a and b, large 
gamonts with numerous nuclei; 14, a and b, gamonts undergoing schizogony; 
15, free gametocytes; 16, gametocytes in the red blood-corpuscles; 17, micro- 
and macrogametes in the stomach of the tick; 18, copulation; 19, karyomyxis; 
20 and 21, formation of the ookinetes; 21, retort forms of ookinete; 22, ookinete 
(Gonder, in "Journal of Comparative Pathology and Therapeutics"). 516 
VETERINARY BACTERIOLOGY 
transmissibility led to the tentative adoption of a new specific 
name, as the true Piroplasma cdnis is not known to be transmissible 
to any other animals. The organisms found were round or pear 
shaped. The round type were from 0.5 to 1.5 p in diameter, and 
the piriform 1.5 by 2.3 p. Considerable pleomorphism was 
evident. 
Theileria parva 
Synonyms.-Piroplasma parva; Babesia parva. 
Disease Produced.-East African coast fever, Rhodesian red- 
water, Rhodesian tick fever in cattle. 
The organism and the disease have been studied by Theiler, 
Koch, and others. This protozoan is the smallest of the Piro- 
plasmas known. In the red cells it forms a small rod that has a 
chromatin granule at one end. Frequently ring forms are observed, 
never the pear-shaped types of P. bigeminum. Gonder has worked 
out in detail the life-history of the organism. This disease is 
peculiar, in that transference of blood containing the organism from 
one animal to another does not result in the transference of the 
disease. Repeated inoculations are without effect. It is trans- 
mitted by means of the brown tick {Rhipicephalus appendiculatus) 
and the black pitted tick (R. simus). The affected animals show 
high fever and swelling of the lymph-nodes. Anemia, icterus, and 
hemoglobinuria are rarely observed. Immunity to this disease 
does not immunize against Texas fever. What is probably the 
same disease has also been described from southern Russia and in 
Java. 
The Genus Plasmodium 
Malaria in man has been found to be due to three species of 
protozoa, usually placed in the genus Plasmodium. The organ- 
isms pass certain parts of their life-cycle in the blood-corpuscles 
in man, and the remainder in the gut and tissues of the mosquito. 
The organisms of malaria were first noted by Laveran in 1880, 
and the various types have been differentiated since that time. 
Plasmodium vivax 
Disease Produced.-Tertian malaria in man. 
Distribution.-This is the commoner malaria of temperate 
climates. SPOROZOA 
517 
Morphology and Life-history.-The organism when first 
recognized in the blood is small, with ameboid movements. It 
penetrates the red blood-corpuscle, and develops until the interior 
Fig. 200.-Diagram illustrating the life-cycle of the malarial parasite: 
A, Sporozoites entering a red blood-cell; B, C, D, E, the organism in various 
stages of development; F, G, the formation of sporocytes and their division 
into spores which infect new red blood-cells. The series A to H represents 
the cycle through which the organism passes in the human body; 7, infected 
red cell ingested by a mosquito. The organism may now develop through the 
series J', K', L', M', to form microgametocytes and microgametes or through 
J, K, L, M, to form a macrogamete which unites with a microgamete to form 
a fertilized ovum; P, the organism penetrates the stomach-wall of the mos- 
quito and develops through the stages Q, R, S, T, U. From U large numbers 
of slender spores are liberated into the body cavity. These pass to the salivary 
glands of the mosquito and are injected when the insect again bites (Rees). 
of the corpuscle is filled. When full grown, it may double the 
diameter of a blood-cell. The organism then segments to form a 
rosette of bodies, which round off to form small spores or merozo- 
ites. These are freed by the disintegration of the red blood-cell, and 518 
VETERINARY BACTERIOLOGY 
attach themselves to other cells and begin development anew. 
This, may be repeated several times. This is called the asexual 
phase of the life-history. The organism may be taken in by the 
mosquito (Anopheles), and here completes its life-cycle by passing 
through the sexual phase. Two types of cells are found to de- 
velop from the spores in the body of the mosquito. The male 
cell (known as microgametocyte) produces five to eight micro- 
gametes. The female cells (macrogametes) are larger and granu- 
lar. A microgamete fuses with one of the macrogametes to form 
what may be termed a fertilized "egg," copula, or ookinete. 
This burrows into the wall of the stomach of the mosquito, en- 
cysts, and enlarges greatly. The contents finally break up 
into a considerable number of spherical bodies known as sporo- 
blasts. These in turn produce great numbers of delicate fila- 
mentous bodies called sporozoites. These are liberated by the 
rupture of the cyst, and pass through the body cavity, and finally 
enter the poison or salivary gland, whence they are inoculated 
into the next victim of the mosquito. This cycle in the insect 
is completed in from eight to ten days, and during this time the 
insect is not infective. 
Pathogenesis.-The disease is characterized by chills, followed 
by fever, which occur every forty-eight hours. The infection is 
usually benign; a fatal termination is very rare. The chills and 
fever develop at the time of formation of the merozoites and the 
infection of new cells. 
Transmission.-The disease is transmissible only through the 
bite of the mosquito. The elimination of the possibility of this 
transfer is the all-important factor in efficient prophylaxis. 
Plasmodium malariaz 
This organism produces the quartan malaria in which the 
interval between paroxysms of fever is seventy-two hours, and the 
asexual cycle is completed in this time. This disease, like the 
preceding, is benign, and yields readily to quinin treatment. 
Plasmodium immaculatum and falciparum 
This type of malaria is usually tropical. It is malignant, 
and does not yield readily to treatment. Two types are known, SPOROZOA 
519 
a quotidian, which completes its asexual cycle in twenty four 
hours, and a tertian, which requires forty-eight. Whether or not 
these are distinct species is uncertain, but is probable. 
The Genus Proteosoma, Halteridium, and Hemoproteus 
These are genera of sporozoa which produce a malaria-like 
infection in birds. In some forms a part of the life-cycle is also 
spent in the body of the mosquito-in this case a Culex. None of 
the species are of any considerable economic importance. 
The Genus Anaplasma 
This genus was created by Theiler in 1910 to accommodate the 
organism described as "marginal points" in the erythrocytes 
of cattle. The protozoan consists of a tiny dot of chromatin- 
like material in the corpuscle, usually near the margin, never 
more than one-thirtieth to one-twentieth the size of the cell. 
The name Anaplasma comes from the apparent lack of any cyto- 
plasmic material. 
Anaplasma marginale 
Synonym.-Marginal points. 
Disease Produced.-Anaplasmosis, Galziekte, or gall sickness 
in cattle. 
This organism, according to Theiler, has been observed by 
several investigators, among them Smith and Kilborne, in their 
study of Texas fever. These observers have believed it to be a 
developmental stage of the Piroplasma (Babesia) bigeminum. 
Theiler has succeeded in demonstrating the distinction between 
the organisms, and defines Texas fever as a mixed infection of 
Anaplasma marginale and Piroplasma bigeminum. 
Distribution.-Known with certainty from South Africa; prob- 
ably widely distributed. 
Morphology and Staining.-The organism may be single in the 
corpuscles, or there may be several within a single cell. The para- 
sites usually lie near the periphery of the corpuscle, rarely free 
in the blood. They are small, spherical, rarely more than one- 
tenth of the diameter of the cell, frequently less. By appropriate 
staining methods the presence of a central granule surrounded by 
a less apparent capsule may be demonstrated. Sieber has ob- 520 
VETERINARY BACTERIOLOGY 
served multiplication of the organisms within the blood-cells by 
a simple type of division. 
Fig. 201.-Anaplasma marginale in red blood-cells. Note the irregularity in 
size and in the staining of these cells (Sieber). 
The Anaplasma marginale does not stain readily with the 
usual anilin dyes, but can be demonstrated easily by a stain 
Fig. 202.-Anaplasma marginale, stained by Heidenhain's hematoxylon: 
a, Single parasite; b, beginning of division, the diplococcus types; c, dumb-bell 
forms; d, completed divisions; e, free parasites (Sieber). 
such as Giemsa's. Heidenhain's iron hematoxylin also gives 
good results. It may be seen even in the living cells as a refrac- 
tive marginal granule. SPOROZOA 
521 
Pathogenesis.-The disease produced has an incubation period 
of sixteen to sixty days. The specific organisms may be first 
demonstrated from the spleen; later they become abundant in the 
blood. As many as 30 per cent, of the cells may be infected. The 
first evident reaction is irregularity and poikilocytosis of the red 
blood-cells, followed by more or less polychromasia and fragmenta- 
tion. The serum does not seem to acquire any hemolytic proper- 
ties. The febrile periods in the disease coincide with the presence 
of the greatest number of marginal points in the corpuscles. Cattle 
are susceptible to artificial infection. It is believed by Theiler 
and his coworkers that the primary symptoms of disease in Texas 
fever are due to Piroplasma bigemina, but that the secondary 
symptoms are due to this Anaplasma marginale, which requires 
a longer incubation period. 
Transmission.-The organism is transmitted through the tick 
(Bobphilus decolor alas}. 
The Genus Leukocytozoon 
Protozoa somewhat resembling the malarial parasites have 
been found in the white blood-corpuscles or leukocytes in birds 
Fig. 203.- Hepatozoon pernidosum, a leucocytozoon from the blood of 
a rat: a, Free parasite; b, parasites in the mononuclear leukocytes (adapted 
from Miller). 
by' several observers, and in the domestic fowl and the dog in 
Tonkin by Marhis and Leger. They are not known to be of any 
economic importance. 
The Genus Sarcocystis 
These sporozoa are usually elongated, tubular, oval, or even 
spherical. Cysts with a double membrane are formed, and in these 522 
VETERINARY BACTERIOLOGY 
are produced reniform or sickle-shaped sporozoites, with a polar 
capsule and a projectile thread. 
Species of this genus have been described from the muscles 
of a large number of vertebrates. In most cases the organism 
does not do any appreciable harm. Recently (1908) Watson has 
called attention to the prevalence of sarcosporidiosis in western 
Canada, particularly in animals suspected of loco-poisoning or 
infected with dourine. Six cases were found in cattle and two in 
horses suspected of being locoed, three in dourine-affected equines, 
and one in a filly showing cachexia. He concludes that these or- 
ganisms may sometimes be an important factor in disease. In 
some cases the entire musculature may be affected with serious 
and even fatal consequences. A close relationship between loco- 
poisoning and sarcosporidiosis is shown by the autopsy records. 
Recovery from this affection and from dourine may be prevented 
or retarded by the presence of the organisms. The species found 
in the horse is Sarcocystis bertrami; in sheep and goats, £. tenella; 
in swine, >8. miescheriana; in man, >8. lendemanni; in the mouse, 
>8. muris. 
The Genus Eimeria (Coccidium) 
This genus belongs to the sporozoan order Coccidiida. This 
order is characterized by having in the adult an oval or spherical 
form, not motile. Sporulation takes place within an endocellular 
cyst. The genus Eimeria is differentiated by the formation 
of four sporocysts or sporoblasts, each of which contains two 
sporozoites. The life-history is relatively complex, and varies 
in some details in different species. 
The organism is taken into the body with food in the form 
of a cyst, which ruptures and allows the escape of the spindle- 
shaped sporozoites. These penetrate the epithelial cells of the 
intestinal walls or other membranes. The sporozoite on entering 
the cells rounds up into a sphere, and then grows rapidly in size 
at the expense of the host cell. These growing organisms are 
called at this stage schizonts. The nucleus of the mature schizont 
fragments, and the protoplasm then breaks up into a considerable 
number of spindle-shaped cells, called merozoites, somewhat re- 
sembling the sporozoites. These break out of the mother schizont SPOROZOA 
523 
and infect new cells. They may then develop as schizonts and 
repeat the same cycle, or may develop into sexual reproductive 
cells. Some of these are of considerable size and correspond to 
an egg; these are termed the macrogametes. Others, called micro- 
gametocytes, develop similarly at first, then form a considerable 
number of very slender, thread-like cells called microgametes. A 
microgamete fuses with a macrogamete to form the oocyte. This 
then continues to enlarge, and secretes a chitinous wall; i. e., 
becomes encysted. When mature, the contents of the cyst divide 
to form four spherical bodies called sporoblasts. These become 
somewhat elongated and spindle shaped. In each of the sporo- 
blasts two still more slender fusiform sporozoites develop. The 
cyst is freed, and upon ingestion by a suitable host the cycle be- 
gins again. 
Coccidiosis occurs in many of the invertebrates, which do not 
seem to be seriously affected, but in the vertebrates serious and 
even fatal disease may be caused by the organisms. 
Eimeria avium 
Synonyms.-Psorospermium avium; Coccidium revolta; C. per- 
foratum; C. tenellum. 
Disease Produced.-Coccidiosis in domestic fowls and in birds. 
These diseases have been studied by a great number of inves- 
tigators, and many theories of their causation have been de- 
veloped. Hadley and others have seemed recently to show quite 
conclusively that they are cases of avian coccidiosis, and the various 
organisms described by others as causal were secondary invaders 
or developmental stages of the organism in question. 
Distribution.-The disease probably has a very wide distribu- 
tion over the United States and Europe, but adequate data are 
not at hand for a determination. It is certainly known from 
many localities in the eastern States. 
Morphology and Life-history.-The life-history is typical for 
Coccidium as outlined above. The adult coccidial cyst is oval 
or ellipsoidal. It measures about 14 by 21 p. 
Pathogenesis.-A considerable number of infections in the 
domestic fowls have been ascribed to this organism. A mild in- 
fection may not result in marked symptoms. An intestinal and 524 
VETERINARY BACTERIOLOGY 
cecal infection in the young chick has been found to be a potent, 
if not the principal, cause of a diarrhea, which causes such 
Fig. 204.-Eimeria avium, life-cycle: 1, Mature encysted Coccidium: 
2, division into four sporoblasts; 3, sporoblasts elongated and spindle shaped: 
4, two sporozoites have developed in each sporoblast; 4 a the cyst ruptured 
and the sporoblasts and the sporozoites escaping; 5, epithelial cell of the in- 
testinal tract; 5 a, 5 b, 5 c, 6, 6 a, stages in development within the cell, the 
schizont stage; 7, schizont dividing to form merozoites; 7 a, cell and schizont 
ruptured and merozoites escaping. These again infest the epithelial cells and 
may repeat the cycle 5-7 a. Others produce the sexual stages 8 and 8 a to 11; 
8, 9, infection of a cell with a merozoite and development of the macrogamete; 
10, cell ruptured, exposing the macrogamete; 8 a, infection of a cell with a 
merozoite and development of the microgametocyte; 9 a, formation of the 
microgametes; 10 a, liberation of the microgametes; 11, fusion of the micro- 
gamete with the macrogamete; 12, 13, 14, 15, development of the mature 
encysted coccidium (Cole, Hadley, and Fitzpatrick). 
heavy losses in certain localities. A similar infection in adult 
chickens may also prove fatal. This is particularly true of the SPOROZOA 
525 
turkey, which is unusually susceptible. The principal symptoms 
are three in number-diarrhea, progressive languor or stupor, and 
loss of appetite with pronounced emaciation. The disease may 
be acute or chronic, but is quite generally fatal. Some fowls 
may harbor the organisms for long periods without any apparent 
symptoms. 
Bacteriologic Diagnosis.-An examination of smears from in- 
fected membranes will show developmental stages of the organism. 
The cysts may usually be demonstrated in the feces. 
Transmission.-The disease is undoubtedly acquired by the 
ingestion of cysts which have been given off in the feces of diseased 
fowls. It is believed probable that wild birds may also become in- 
fected, and aid materially in the dissemination of the organism. 
Eimeria stiedae 
Synonyms.-Monocystis stiedce; Psorospermium cuniculi; Coc- 
cidium oviforme; C. perforans; Pfeifferia princeps. 
Disease Produced.-Coccidiosis of the rabbit. 
Rivolta, in 1878, first described this organism from the rabbit. 
It occurs in the intestinal epithelium. It may be present without 
evidence of disease, but is undoubtedly the cause of serious epi- 
zootics among tame and wild rabbits. 
Eimeria bovis 
Disease Produced.-Bovine coccidiosis. It has been re- 
ported most frequently from Switzerland. 
The organism was first noted by Zschokke in 1892. The dis- 
ease of young cattle characterized by enteritis with bloody feces 
has been reported by several European investigators. The organ- 
ism is morphologically typical of the genus, and has the same life- 
history. The disease has an incubation period of about three 
weeks. In the feces appear blood flecks of greater or less size; 
in severe cases there develops a bloody diarrhea. Occasionally 
the animal dies, but usually recovery takes place within a few 
weeks. 
The oocytes are abundant in the feces of affected animals, re- 
sembling those of Eimeria stiedoz. The organisms in all stages of 526 
VETERINARY BACTERIOLOGY 
development may be found in the epithelium of the large intestine 
and the rectum. The mucous membranes may be inflamed, even 
purulent and covered with a diphtheritic membrane. Petechial 
and larger hemorrhages are to be observed. 
Fig. 205.-Eimeria stiedce: a, b, c, Schizonts, with production of mero- 
zoites which may repeat the cycle or develop the sexual stage; e, f, g, develop- 
ment of the macrogamete; h, i, j, s, development of the microgametocytes and 
microgametes; k, mature coccidium, which encysts and divides to form four 
sporoblasts; I, formation of the sporozoites and their liberation by a rupture 
of the cyst (Schaudinn). 
Eimeria faurei 
Disease Produced.-Ovine coccidiosis. 
Moussu and Marotel have described an ovine coccidiosis, par- 
ticularly in lambs. The developmental cycle was typical. The 
same or a similar coccidiosis has been reported in the United States. SPOROZOA 
527 
Isospora bigemina 
Disease Produced.-Coccidiosis of the dog and cat. 
Stiles, in 1892, described a Coccidium bigeminum, as a parasite 
occurring in the intestinal villi of the dog and cat. It has also 
been reported in man. The ripe oocysts of this form produce 
two spores, each with four sporozoites (in contrast to Eimerid). 
The oocysts are 22 to 40 by 19 to 38 [jl. The spores are ellip- 
soidal, filling nearly the interior of the oocyte. The spores are 
10 to 18 n in length. CHAPTER XLIV 
PARASITIC PROTOZOA OF THE CILIATA 
The Ciliata are differentiated from other protozoa by the pres- 
ence of cilia during the entire life of the cell. In most species there 
is also a cytostome or mouth, though a few species take up their 
food by osmosis. The cells are relatively constant in shape, that is, 
never ameboid. In many cases the cells are complicated in struc- 
ture, possessing many different types of organella. Most forms 
possess one or more contractile vacuoles which are useful in species 
differentiation. Multiplication is usually through a process of 
splitting into two individuals, usually without the development 
of a resting stage. Many species become encysted to withstand 
unfavorable conditions. 
Only one or two species are known to be disease producers, 
but many are commensals in the alimentary tracts of animals, 
particularly in the rumen of the ruminants and in the cecum of 
the horse. 
Certain protozoa are quite constantly met in those parts of the 
alimentary tract of herbivorous animals where cellulose digestion 
occurs, in the rumen of cattle, sheep and goatSj and in the cecum 
of the horse. They are present in relatively large numbers. Some 
of the earlier investigators believed that perhaps one-fifth of the 
contents of these organs were protozoan cells. This is probably 
an exaggeration, but indicates their abundance. They seem to be 
destroyed for the most part when the food masses pass to other 
parts of the alimentary tract. They probably feed on bacteria and 
plant fragments. These organisms are, so far as known, neither 
useful nor harmful, they are true commensals. 
The following key to the most common species may be useful in 
the differentiation of these commensals in the search for patho- 
genic organisms in disease: 
Protozoan Commensals of the Rumen and Cecum 
528 PARASITIC PROTOZOA OF THE CILIATA 
529 
A. No spiral zone of cilia about the mouth. 
1. Cells oval or nearly spherical, anterior end trun- 
cate. Ectoplasm homogeneous, cuticle-like. 
Surface closely covered with very fine cilia ar- 
ranged in relatively wide longitudinal rows. 
Near the anterior end the cilia are longer. One 
vacuole containing a refractile concretion is 
found near the anterior end. Principal nu- 
cleus is large, spherical. No secondary nucleus 
evident Genus 1. Butschlia. 
2. Cells more elongate, ciliation very uniform. 
Front of cell rounded, posterior end somewhat 
pointed, slightly flattened on one side, no an- 
terior ring of longer cilia. Both principal and 
secondary nucleus present Genus 2. Isotrichia. 
B. Possessing a definite spiral zone of large cilia or 
membranella about the mouth. 
1. Body with cilia uniformly distributed. Cell 
oval or ellipsoidal, the anterior end somewhat 
blunted, cells capable of changing form to 
some degree. Usually longitudinally striped. 
Principal nucleus is kidney shaped, secondary 
nucleus vesicular Genus 3. Balantidium. 
2. Cilia absent from major portion of body, mem- 
branella about the mouth or grouped at 
definite points on the cell. 
(a) Possessing two spiral rows of membranella on 
anterior end. Cells relatively large, long oval, 
somewhat flattened. At posterior end there 
are three rows of membranes passing around 
the cell, each membrane with three points Genus 4. Ophryoscolex. 
(6) Possessing a single spiral row of membranella 
at anterior end. 
(1) Posterior end of cell extended into three 
points, one of which is rudder-like and much 
longer than the other two. The peristome 
or ring or adoral membranella are retrac- 
tile Genus 5. Entodinium. 
(2) Posterior end of cell entire. Adoral ring 
consists of twenty-four membranella, or large 
cilia. The mouth is in a blunt cone within 
the peristome. Near posterior end there 
occur two short tubular bodies from which a 
cluster of membranella protrude. These 
are used in swimming Genus 6. Cycloposthium. 
Butschlia.-Two species, Butschlia parva and B. neglecta, are 
common in the rumen of cattle. B. postciliata occurs in the cecum 
of the horse. 530 
VETERINARY BACTERIOLOGY 
Isotrichia.-The species Isotrichia prostoma and I. intestinalis 
are very common in the rumen of cattle, as is also a closely related 
form, Dasytricha ruminantium. 
Ophryoscolex.-Two species from the rumen of sheep are 
Ophryoscolex scolex and 0. inermis. 
Entodinium.-The species Entodinium caudatum, E. bursa, E. 
dentatum, E. rostratum, and E. minimum are all found in the paunch 
of ruminants. 
Fig. 206.-Balantidium coli in a blood-vessel of the submucosa of the intes- 
tines (Bowman, in "Philippine Journal of Science"). 
Cycloposthium.-The species Cycloposthium bipalmatum occurs 
in the cecum of the horse. 
Balantidium.-One species of Balantidium produces disease in 
man. 
Other species of protozoa which have been described by Braune 
from the ruminants are Trichomastix ruminantium, Trichomonas 
ruminantium, Callimastix frontalis. Awerinzeff and Mutafoua have 
described Diplodinium fiorentinii, Ophryoscolex intermixtus, 0. 
fasciculus, and 0. labiatus. PARASITIC PROTOZOA OF THE CILIATA 
531 
Trichomonas.-Hadley has reported the occurrence of tricho- 
monads in healthy and sick fowls throughout the intestinal tract. 
They are almost invariably present in the intestinal and cecal 
contents but are found in the greatest numbers in the mucous 
layers of the caeca, sometimes deep in the crypts, whereas cocci- 
dia multiply mainly in the duodenum and amoebae in the epithe- 
lial layer of the lower intestine and caeca. He has presented 
evidence to prove that entero-hepatitis (black head) of turkeys is 
due to Trichomonas and that the Amoeba meleagridis of Smith is 
simply one stage of this organism. He states that confusion with 
the amoebae arises when the Trichomonas becomes non-motile, 
loses its flagella, and becomes rounded or oval in shape. It was 
this stage which stains pink with eosin that he believes was mis- 
taken by Smith for an amoeba. 
This flagellate is described by Hadley1 as "pear shape, elon- 
gate, half moon or globular shaped depending upon the stage of 
development. In the free swimming stage it has a length of 
8 to 12 m. It manifests the usual Trichomonas characteristics, 
viz., three anterior and one posterior flagella, a vibratory mem- 
brane, axostyle, chromatic line, chromatin blocks, nucleus, 
vacuoles, peristome, blepharoplast and rhizoplast." Hadley 
finds that the pathological condition is complicated in the 
liver only rarely by amoebic infection, and in the caeca both by 
amoebae and coccidia. 
Balantidium coli 
Disease Produced.-A rare fatal enteritis in man. 
This organism is oval in shape-50 to 70 by 60 to 100 n-and 
possesses a funnel-shaped peristome which terminates at the mouth. 
Cilia cover the surface. The two nuclei and two contractile vacu- 
oles may commonly be made out. An excretory apparatus is evi- 
denced by the extrusion of waste material at a definite point in the 
cell surface. The life-history is relatively complex. The cell 
commonly multiplies by direct division. Conjugation and encyst- 
ment may occur. 
The Balantidium coli appears to be a common inhabitant of the 
intestinal tract of swine. A large number of instances are recorded 
1BuIl. Rhode Island Agr. Exper. Sta., 166 and 168. 532 
VETERINARY BACTERIOLOGY 
in which it has been found associated in man with a severe and 
even fatal type of diarrhea. The connection of this organism with 
the disease as a causal agent rather than as a commensal seems to 
be well authenticated. It appears that infection may follow the 
ingestion of the cysts produced by the organism. The disease has 
been reported from Europe, the United States, and the Philippines. SECTION VI 
INFECTIOUS DISEASES IN WHICH THE SPECIFIC 
CAUSE IS NOT CERTAINLY KNOWN 
CHAPTER XLV 
DISEASES PRODUCED BY UNKNOWN ORGANISMS 
Within the last two decades there have been described a 
number of diseases in which the causal organism is said to be 
ultramicroscopic. By this is meant that with the best powers of 
the microscope no definite organism can be distinguished. Such 
an organism is better called a filterable virus, because filtrates 
passed through porcelain filters retain their pathogenicity for sus- 
ceptible animals. 
It is a principle of optics that no object can be clearly differen- 
tiated that is smaller than one-half the wave-length of the light 
in which it is examined. This means that there is an apparently 
insuperable physical obstacle to the observation of some of these 
forms, as our best lenses approach moderately near to this limit 
in their magnification. Our reasons for believing that there may 
be organisms this small will be discussed under the heading of 
various diseases. 
There is in more or less general use an instrument known as the 
ultramicroscope, which renders visible objects more minute than 
had heretofore been observed. This instrument enables one to 
observe objects by making use of the principle worked out by 
Tyndall in determining the absence of floating dust particles in the 
air. He noted that when a ray of a very bright light was admitted 
to a darkened room or box, this ray could be distinctly seen as 
long as there were any floating .particles, but became invisible 
when these dust particles had completely subsided. The motes or 
particles, even when smaller than can usually be seen by the un- 
aided eye, became visible when thus illuminated. This principle is 
533 534 
VETERINARY BACTERIOLOGY 
applied to microscopic examination by sending the rays from an 
arc light or similar source, so that they are concentrated in a 
powerful beam, which is passed through the hanging drop or similar 
preparation from side to side. Exceedingly minute particles may 
thus be made visible. The use of this instrument has been found 
to be less helpful in the fields ,of biologic research than had been 
hoped. Very few facts concerning the organisms that cause 
disease, and particularly the ultramicroscopic organisms, have 
been discovered by its aid. 
An attempt has sometimes been made to compare the relative 
size of ultramicroscopic organisms by the use of porcelain filters 
of different degrees of density. It has been found that the virus 
of some diseases will pass through coarse filters, but not through 
the finer ones. It does not seem to be entirely a matter of the 
relative size of pores and organisms that may pass through the 
filter. It is probably a phenomenon analogous to an adsorption 
quite as much as mechanical filtration that removes the organ- 
isms. 
Bacterial or Protozoan Relationships of Ultramicroscopic 
Organisms.-There is no practicable method telling certainly 
whether or not a filterable virus should be ' grouped with the 
protozoa or with the bacteria. There are methods which may 
sometimes be used that will give inferences, however. It has been 
found possible in some cases to secure growth in culture-media, 
such as is used for bacteria. Such organisms are probably bac- 
teria. In others, the type of disease produced may resemble so 
closely some other infection produced by a known organism that a 
probable classification into protozoan or bacterial might be made. 
The type of immunity developed may likewise be of importance. 
In most instances these differences are not pronounced enough to 
allow of certainty in the classification. 
The more important diseases which have been described as 
due to filterable virus are contagious pleuropneumonia of cattle, 
rinderpest or cattle plague, foot-and-mouth disease, hog-cholera, 
horse sickness, dog distemper, fowl plague, equine anemia, fowl- 
pox, infectious agalactia in sheep and goats, fowl leukemia, 
guinea-pig plague, certain chicken tumors. DISEASES PRODUCED BY UNKNOWN ORGANISMS 
535 
Virus of Pleuropneumonia 
Disease Produced.-Pleuropneumonia, peripneumonia, lung 
plague of cattle and other bovines. 
Nocard and Roux described the causal organism in 1898. The 
disease itself has been known in Europe for several centuries. 
Distribution.-The disease is known from Europe, Africa, Aus- 
tralia, Asia, and has been imported into the United States, where, 
in 1886, it killed 10,000 animals in Illinois alone. 
Nature of Virus.-The causal organism is doubtless a bacterium 
that is just at the limit of visibility. In suitable fluids it may 
be observed under very high powers as a tiny motile point. Bouil- 
lon inoculated with the serous exudate from the pleura or from one 
of the areas of consolidation in the lungs, sealed in a collodion sac, 
and placed in the peritoneal cavity of a rabbit for two weeks, will 
show a slight clouding or opalescence. Transfers to new media 
similarly treated will likewise result in growth. This materia] 
may be shown to be infective. The organism may also be culti- 
vated in a mixture of bouillon and blood-serum outside the animal 
body. In two to three days it show's a very faint clouding. Agar 
containing serum develops delicate, transparent, almost invisible 
colonies. The optimum temperature is 37°; no growth occurs 
under 30°. 
Pathogenesis.-Injection of pure cultures of the organism into 
cattle results in infection. Intrapleural injections or inhalation 
of the organism results in a typical clinical picture of the disease. 
The disease is characterized by more or less extensive areas of he- 
patization in the lungs and an inflammation of the pleura, accom- 
panied by a serofibrinous exudate. The amount of fluid which 
collects may be very considerable. 
Immunity.-Recovery from the disease results in a relatively 
permanent immunity. Immunization against the disease by vac- 
cination with serum from the pleural cavity of infected animals has 
been practised. The material is injected subcutaneously. Its use 
is attended with danger, as from 0.5 to 5 per cent, or even more of 
those vaccinated have been killed by the vaccine. The method in 
question has been of some use in immunization, but a stamping- 
out process would appear to be more efficacious. 
Nocard and Roux have advised vaccination wTith pure cultures, 536 
VETERINARY BACTERIOLOGY 
and claim to have secured more favorable results than by the 
older method. Nocard has also produced a curative and pro- 
phylactic serum by the hyperimmunization of animals by the 
injection of increasing doses of pure culture until 6 liters have 
been used. In doses of 40 c.c. it served as an efficient prophylactic, 
and in larger amounts as a curative agent in the early stages of the 
disease. 
Transmission.-The method of natural spread of the disease 
is not certainly known. It is probably through inhalation of the 
causal organism. 
Virus of Foot-and-mouth Disease 
Disease Produced.-Foot-and-mouth disease, aphthous fever, 
Maul- and Klauenseuche in cattle and other bovines, sheep, goats, 
swine, deer, and occasionally horses, dogs, cats, and man. 
The disease has been known for over a century in Europe. 
Loffler and Frosch, in 1897, Hecker, in 1898, and others since 
that time have shown that the virus of foot-and-mouth disease 
may pass through Chamberland and Berkefeld filters, but not 
through the thicker Kitasato filter. 
Distribution.-The disease is known from most of Europe, 
Asia, and Africa. It has been introduced several times into the 
United States, but has been stamped out. It has also been 
reported from Argentina. 
Nature of the Virus.-Recently Stauffacher has claimed to 
have demonstrated the causal organism to be a protozoan which 
he names Aphthomonas infestans. He places it close to the genus 
Leishmania. For demonstration of the organisms in tissue sec- 
tions and blood-smears he fixed in 70 per cent, alcohol, stained 
from two to six hours in 0.2 per cent, aqueous acid fuchsin, rinsed 
in distilled water, and stained in Ehrlich's fuchsin methylene-blue 
for six to ten hours, rinsed in distilled water and placed in abso- 
lute alcohol until color was no longer extracted, cleared in xylol, 
and mounted in balsam. 
Stauffacher succeeded in cultivating the organisms in the con- 
densation water of Nicolle's blood-agar both from the blood and 
from vesicles of infected animals. The protozoan here was found 
in two forms, one shorter and more plump, the body 20 to 25 p in DISEASES PRODUCED BY UNKNOWN ORGANISMS 
537 
length and 3 in width, with long flagellum. The other type were 
long slender cells, even 120 in length. A type of spore produc- 
tion was also observed leading to the production of cells probably 
small enough to pass a filter. The disease was transmitted by the 
use of pure cultures and the same organism isolated. 
Pathogenesis.-The disease may be produced by the inocula- 
tion of susceptible animals with the filtrate through a porcelain 
filter. It is characterized by an acute fever, the appearance of a 
vesicular eruption on the mucous membranes of the mouth, on 
the feet, and between the toes. It is commonly not fatal, but is 
so contagious, and leads to such losses in flesh and milk, that it is 
among the most feared of cattle diseases. 
Immunity.-Vaccination by intentional infection of animals has 
sometimes been practised in an effort to "get it over with" as 
quickly as possible when it breaks out in a herd. An animal 
recovered is relatively immune. Such methods are attended by 
considerable risk. An efficient and safe method of immunization 
or vaccination has not been developed. 
Transmission.-The organisms gain entrance through contact 
of healthy animals with the saliva or other secretions of an in- 
fected animal. They may be transmitted to young animals or to 
man in milk. 
Virus of Rinderpest or Cattle Plague 
Disease Produced.-Cattle plague, rinderpest, or contagious 
typhus in cattle, rarely in sheep, goats, and camels. 
Nocard and, later, Tartakowsky observed that the body fluids 
in animals having this disease contained no visible microorgan- 
isms, but were infective. Nicolle and Adelbey established that 
these fluids, if thinned with water, could be passed through the 
coarser Berkefeld filters, but not through a fine-pored Chamber- 
land, without losing their virulence. 
Distribution.-The disease has been reported from a large 
portion of the area of Europe. It is endemic in southern Asia, 
is known in the Philippines, and has caused great losses in Egypt 
and in Southern Africa. It has not gained entrance to the United 
States. 
Character of Virus.-It is filterable and has not been culti- 
vated. Blood sealed hermetically in tubes is found to retain its 538 
VETERINARY BACTERIOLOGY 
virulence for months. The virus is destroyed by desiccation or 
by heating to 58° to 60°. It may survive in putrefying flesh 
for considerable periods. It is easily destroyed by disinfectants. 
Pathogenesis.-The virus is present in all the body tissues 
and excretions. One one-thousandth of a gram of blood from 
an infected animal at the height of the disease has been found 
sufficient to reproduce the disease in a susceptible animal. The 
infection is an acute, highly fatal fever, in which there are croupous 
diphtheritic lesions of the intestinal tract. It is typically a cattle 
disease, but occasionally attacks other animals. 
Immunity.-Animals which recover spontaneously from the 
disease are highly immune, and the blood has some power of pas- 
sive immunization when injected into another animal. Vac- 
cination with the nasal secretion of sick animals into the tails of 
others has been practised, and has been found in some instances to 
result in a low mortality. The method has been practically 
abandoned. Injections of the bile from animals having the disease 
has been advocated by Koch and extensively practised in South 
Africa, with good results. 
The common method of immunization against rinderpest is 
that developed by Kolle and Turner. Animals which have recov- 
ered spontaneously from an infection, or that have been im- 
munized by injections of virulent blood and gall, are hyper- 
immunized by repeated injections of virulent blood. The first 
injection is of a liter of virulent blood. After the subsidence of the 
reaction an injection of 500 c.c. is given, and later a third injec- 
tion of a liter. A fourth injection may be made. The blood is 
drawn from the jugular vein at three intervals, a week apart. 
Another injection of a liter of virulent blood is given, and later the 
animal is again bled. The serum, in amounts of 20 c.c., should 
protect an animal against injection of 1 c.c. of virulent blood. 
An injection of 50 to 100 c.c. of the serum so secured will protect 
an animal against infection for a space of two to four months 
usually. A more permanent immunity may be established by the 
use of what is termed the "serum simultaneous" method. The 
animal is injected on one side with 8 to 25 c.c. of the immune serum, 
and on the other with 1 c.c. of virulent blood. Some animals react 
by a distinct fever, others show no effect. The latter are rendered DISEASES PRODUCED BY UNKNOWN ORGANISMS 
539 
immune for several months only, while the former for much longer 
periods. The blood of the animals reacting is infective during the 
period of fever. The vaccination mortality is about 1 per cent. 
Transmission.-The disease is readily transmitted by means of 
soiled food, water, and by direct contact. 
Virus of Hog-cholera 
Disease Produced.-Hog-choleraj Schweinepest, swine fever. 
From the time of the researches of Salmon and Smith on this 
disease, published in 1885, until 1904, the cause of hog-cholera was 
believed to be the Bacterium cholerce suis (Bact. suipestifer}. In 
the latter year de Schweinitz and Dorset showed the typical hog- 
cholera in the United States to be due to a filterable virus. This 
has been confirmed by Hutyra, Uhlenhuth, and others in Europe. 
Distribution.-The disease is wide-spread in Europe and North 
America. 
Nature of Virus.-The organism causing hog-cholera is a filter- 
able virus. It passes readily through porcelain filters. It has never 
been cultivated. Fluid material will retain its virulence for a pe- 
riod of ten to fourteen weeks, at least when kept at room-tem- 
peratures. It is killed by exposure to 60° to 70° for an hour. 
Desiccation does not destroy it at once, but only after a lapse of 
several days. It may be destroyed by disinfectants, but is rela- 
tively resistant. 
Pathogenesis.-The subcutaneous injection of 1 to 2 c.c. of 
filtered blood-serum or body fluids results in the production of the 
disease. The animals may die of a very acute type of the disease, 
or it may assume a chronic form. The acute cases generally reveal, 
on autopsy, hyperemia and acute swelling of the internal organs, 
and hemorrhages on the serous and mucous membranes, and fre- 
quently a serous transudate into the pericardium. In the more 
chronic type ulcerated and necrotic areas are commonly found in 
the intestines, together with pneumonia. 
The disease cannot be transferred to other animal species. 
The Bacterium cholerce suis is probably a secondary invader, but 
the lesions produced by this organism may in some cases be of the 
greatest importance. 540 
VETERINARY BACTERIOLOGY 
Immunity-Animals that have recovered from an infection 
with hog-cholera are thereafter immune, and it has been shown 
that their blood has some immunizing power when injected into 
other susceptible individuals. 
Practical methods of immunization were developed through 
the work of Dorset, McBryde, and Niles in this country. The 
method devised by them and commonly used is as follows: It is 
necessary to start with an animal that has recovered from the 
disease or that has already been immunized. This animal is then 
hyperimmunized by the intravenous injection of virulent blood. 
Such blood must be of a strain of known virulence established by 
previous tests. It is preferably obtained from pigs weighing 
60 to 90 pounds which have been inoculated seven to ten days pre- 
viously, and which show well-marked symptoms and subsequent 
postmortem lesions of acute cholera. About 5 c.c. of the defibrin- 
ated virulent blood is injected for each pound of body weight. Ten 
days later the animal thus hyperimmunized is bled from the 
carotid artery or the tip of the tail. The blood is defibrinated 
and the clot removed. This defibrinated blood is preserved by 
the addition of | of 1 per cent, carbolic acid. Before use it is 
tested on susceptible pigs to determine its potency. For testing 
it is customary to use young pigs weighing from 50 to 60 pounds. 
A serum of satisfactory potency should protect, in a dose of 
15 c.c. or less, one of these animals against an injection of 2 c.c. 
of virulent blood. Permanent immunity is conferred by the use 
of the serum simultaneous method. In this method virulent 
blood (1 to 2 c.c.) is injected at the same time as the immune 
serum. This results in the development of an active immunity, 
which is relatively permanent in comparison with the immunity 
of four to six weeks conferred by antiserum injection alone. 
The use of this serum has been found to be highly successful in 
practice. 
It is not known what property of the antiserum is thus effec- 
tive, whether it is antitoxic, opsonic, or bactericidal. There is 
some evidence that it is the last, but proof is difficult to secure. 
Transmission.-The virus may be demonstrated in the blood, 
the tissues, and the urine of infected animals. It is probable that 
infection commonly takes place through ingestion. DISEASES PRODUCED BY UNKNOWN ORGANISMS 
541 
Virus of Horse Sickness 
Disease Produced.-African horse sickness, or Pferdesterbe. 
This disease, known from southern Africa for more than a 
century, was first shown by MacFadyen, in 1900, and later, in 
1901, by Nocard to be due to a filterable virus. The disease is 
characterized as an acute or subacute disease of solipeds, that ap- 
pears in epizootics during the hot months of the year. The prin- 
cipal lesions are edematous swellings and hemorrhages of the inter- 
nal organs. The virus will pass through a Berkefeld or a Chamber- 
land porcelain filter if the serum is diluted with physiologic salt 
solution. 
Immunization against the disease may be brought about by the 
use of serum from hyperimmunized animals. Koch hyperimmu- 
nized horses that had recovered from the disease by three to four 
injections of virulent blood at intervals of two to four weeks, as 
much as 2 liters being used for the last injection. Serum simul- 
taneous injections of this hyperimmunized serum and virulent 
blood into susceptible animals in correctly proportioned doses will 
immunize. Theiler reports the safest method to be the injection of 
300 c.c. immune serum intravenously and | c.c. virulent blood 
subcutaneously. Animals thus treated will show a rise in tempera- 
ture, upon which a second injection of 50 to 100 c.c. of serum is 
made. 
The disease has been found to be contracted generally at night, 
and the first frost puts an end to the epizootic for the year. Con- 
siderable quantities of the virus must be fed before an infection is 
produced, showing that natural infection is probably in some other 
manner than by ingestion. It is probable that mosquitoes, possibly 
flies, act as carriers. The disease cannot be regarded, therefore, 
in the strictest sense as contagious. 
Virus of Infectious Anemia of the Horse 
Disease Produced.-Infectious anemia, pernicious anemia, 
mud fever, swamp fever of the horse. 
This disease has been known as a clinical entity in Europe for 
three-quarters of a century. Carre and Villee (1904-1906) and 
Ostertag and Marek (1907) have demonstrated the disease to be 
due to a filterable virus. The disease has been studied in North 542 
VETERINARY BACTERIOLOGY 
America by several investigators, and the ultramicroscopic nature 
of the virus has been independently demonstrated. It is not 
certain that all the infections described under this name are 
identical, but there is considerable evidence tending to establish 
such as a fact. 
Distribution.-The disease is probably wide-spread, but has 
not always been clearly differentiated. It is known from Germany, 
France, Hungary, Switzerland, and Sweden in Europe; and from 
Saskatchewan, Manitoba, Minnesota, the Dakotas, Nebraska, 
Kansas, Colorado, Wyoming, Montana, Texas, and Nevada in 
North America. 
Nature of the Virus.-The causal organism is a filterable virus 
that cannot be differentiated by staining methods and has not been 
cultivated. It is found in the blood, the urine, and the feces of 
infected animals. It is destroyed at a temperature of 58°. It will 
withstand drying for several months, and liquids maintain their 
infectivity for months, even when decaying. 
Pathogenesis.-The virulent blood or. blood-serum will infect 
another animal upon subcutaneous injections of small quantities. 
The incubation period after injection varies from five to nine days, 
or even more. The initial symptom is a fever. The disease is more 
apt to be acute when the organism is introduced by injections than 
by ingestion. The disease may be characterized as an acute or 
chronic anemia which has the appearance of a septicemia in which 
there is a great destruction of blood-elements. The anatomic 
findings are characteristic. Mack, in his work on cases in Nevada, 
notes profound cardiac and respiratory disturbances. There is a 
progressive destruction of the red blood-cells, parenchymatous 
degeneration of the kidneys and liver, and extensive changes in the 
vascular system. The spleen is engorged and frequently degener- 
ated, and the bone-marrow undergoes profound degeneration. 
Immunity.-No method of immunization has been developed. 
Transmission.-European writers are of the opinion that in- 
fection arises through the ingestion of food soiled by excretions 
of infected animals. The mode of dissemination has not been satis- 
factorily established. 
Virus of Dog Distemper 
Disease Produced.-Dog distemper, Hundstaupe. DISEASES PRODUCED BY UNKNOWN ORGANISMS 
543 
Bacteria belonging to several different groups, particularly to 
the colon-typhoid and to the hemorrhagic septicemia groups, 
have been described as the cause of dog distemper. Carre, in 
1905, attributed the cause to a filterable virus. Ferry and others 
concluded that Carre must have been in error in his conclusions, 
inasmuch as they claim to have found Bacterium bronchisepticum 
(q. v.) to be the primary cause. 
Distribution.-Europe and America; probably in other parts 
of the world. 
Nature of the Virus.-Little is known of the virus beyond the 
fact that it can be passed through a porcelain filter. It has not 
been cultivated. 
Pathogenesis.-The disease is a highly fatal, acute infection 
of young carnivorous animals, characterized by an acute catarrh 
of the mucous membrane, and frequently a catarrhal pneumonia. 
In a small percentage of the cases nervous symptoms develop. 
The discharge from the mucous membranes is highly infective. 
Secondary infection with bacteria is common, and is believed by 
some investigators to account for many of the deaths. 
Immunity.-Many attempts have been made to prepare an anti- 
or immune serum that would be satisfactory in preventing or curing 
the disease. Several have been placed upon the market, but none 
has been shown to be efficacious. It is possible that success 
might be attained by the use of hyperimmune blood. 
Transmission.-The disease is transmitted by direct or indirect 
contact with infected individuals. 
Virus of Fowl Plague 
Disease Produced.-Fowl plague, chickenpest of domestic 
fowls. 
This disease has generally been confused with chicken cholera, 
which it closely resembles clinically, although Perroncito un- 
doubtedly described it in 1878. Centanni and Savonuzzi, in 1901, 
showed that the virus could pass through a porcelain filter. This 
has been amply confirmed by other writers. 
Distribution.-The disease is known only from northern Italy, 
the Tyrol, Germany, and France. 
Character of the Virus.-The organism is a filterable virus, and 544 
VETERINARY BACTERIOLOGY 
is probably ultramicroscopic, although Rosenthal, Kleine, and 
Schiffman have described bodies in the nerve-centers that are 
possibly protozoan in nature. The virus is found in the blood, the 
nasal secretion, and generally throughout the tissues. The blood 
retains its virulence for three months when sealed in tubes and 
kept in a dark place. The thermal death-point is 55° for thirty 
minutes or 60° for five minutes. The dried virus has been found 
to retain its virulence for two hundred days, and in glycerin and 
serum mixture for two hundred and seventy days. It is easily 
destroyed by disinfectants. 
More recent experimental work has shown that the virus of fowl 
plague is capable of passing the pores of fine-grained filters, and 
even the so-called ultra-filters. It is claimed by some authors that 
the virus must approach in size the protein molecule. Andriewsky 
and others contend that this is an example of a Contagium vivum 
fluidum, that is, does not consist of organized cells at all, but a 
self-perpetuating fluid living material. It has been claimed that it 
shows many of the reactions of the serum globulin. 
Pathogenesis.-Injection of as small amount as 1>ooJ,ooo c.c. of 
virulent blood or secretions is sufficient to infect. The virus 
is pathogenic for many birds besides fowls, but not for mam- 
mals. The disease is characterized by the hemorrhages into 
the serous membranes in acute cases, and in the less acute edema 
of the subcutaneous tissues and of the serous membranes, and the 
formation of a fibrous exudate upon the latter. 
Immunity.-Practicable methods of immunization have not 
been developed. 
Transmission.-The disease is transmitted by the ingestion of 
food soiled with the infective feces, nasal secretion, or blood of 
infected birds. 
Disease Produced.-Fowl pox, epithelioma contagiosum in 
domestic fowls, sore head, probably fowl diphtheria. 
Marx and Sticker, in 1902, determined the cause of fowl pox to 
be a filterable virus. Several other investigators subsequently 
confirmed their results. 
Distribution.-The disease is known to occur in Europe and in 
the United States. It is doubtless widely distributed. 
Virus of Epithelioma Contagiosum DISEASES PRODUCED BY UNKNOWN ORGANISMS 
545 
Nature of the Virus.-Marx and Sticker showed that when 
an epithelial nodule was triturated in physiologic salt solution 
the fluid which passed through a Berkefeld filter was infective. 
Some investigators have observed tiny spherical granules, less than 
0.25 ijl in diameter, in the emulsion of the virus, but it is by no means 
certain that these are the disease-producing organisms. The virus 
is relatively resistant to unfavorable conditions. The nodules 
may be dried for weeks without death of the virus. It is destroyed 
by heating to 60° for eight minutes. Mixed with glycerin it re- 
tains its infectivity for many weeks. It is easily destroyed by 
disinfectants. 
Pathogenesis.-The disease is a chronic, contagious infection, 
characterized by an initial catarrh of the mucosa of the head, 
followed by wart-like growths (epithelial hyperplasia) of the skin, 
especially of the comb and naked skin of the head, sometimes 
associated with a croupous diphtheritic condition of the mucosa 
of the head. This latter condition is one of those grouped under 
the general name of fowl diphtheria. The disease commonly ter- 
minates favorably in three to five weeks. 
Hadley and Beach claim to have been successful in securing 
active immunity by triturating the crust which forms on the skin 
and the membranes from the mucous surfaces in physiologic salt 
solution, heating at 55° C., and using this as a vaccine. 
Transmission.-The disease is transmitted by direct contact 
with infected fowls. 
Virus of the Poxes 
Disease Produced.-Small-pox in man, cow-pox, sheep-pox, 
horse-pox, swine-pox, goat-pox. 
There is much doubt relative to the position of the virus of the 
various poxes. They are included in this group tentatively, as it 
is found that the contents of the vesicles of the eruptions may be 
filtered through a thin, coarse porcelain filter under pressure with- 
out losing their infectivity. The causal microorganisms are, at 
some stages at least, filterable and possibly ultramicroscopic. 
Certain cell inclusions have been described as being probably of 
protozoan nature, but the subject cannot be said at present to be 546 
VETERINARY BACTERIOLOGY 
completely elucidated. The protozoan parasite has been named 
Cytorrhyctes vaccines. in man. 
Immunity.-Recovery from an attack of variola is accompanied 
by a relatively permanent immunity. Vaccination is, therefore, 
commonly practised, particularly against small-pox in man. The 
attenuated virus in this instance is secured by passage through 
an animal, usually a heifer. The vaccinating material is the lymph 
from the vesicles produced on the animals. It is inoculated into 
the skin by scarification. The virulence is apparently very 
greatly decreased by this method of inoculation, so that a relatively 
mild type of disease is produced which terminates in immunity 
being established. To what this immunity may be due is not 
known. 
Virus of Yellow Fever 
Yellow fever in man has been shown to be due to a filterable 
virus, possibly an ultramicroscopic organism. All efforts at 
cultivation have failed. The disease is spread only through the 
bites of mosquitoes that have taken virulent blood. The organ- 
ism evidently undergoes a part of its life-cycle in the blood of the 
mosquito (Stegomyia), for the latter does not become infective 
itself for several days. It is evidently more than a mere mechan- 
ical transfer of the organism by the mosquito; the latter serves: 
as a true intermediate host. The recent work of Noguchi would 
seem to indicate that a spirochete {Leptospira} is the causal 
organism, this organism being cultivable and microscopic. 
Virus of Rabies 
Disease Produced.-Rabies in animals. Hydrophobia in man. 
Lyssa. 
The disease has been studied at great length by many in- 
vestigators, and there is still great disparity of opinion as to the 
nature of the cause. Remlinger and Riff at Bey in 1903 showed 
that the virus could be passed through a porous Berkefield filter. 
This has been substantiated since by several workers. As will 
be seen below, this does not satisfactorily settle the problem, as 
those who hold to the protozoan nature of certain bodies in the 
nerve-centers in the disease contend that extremely minute plas- 
tic stages in the life-cycle of the organism might easily pass 
through. DISEASES PRODUCED BY UNKNOWN ORGANISMS 
547 
Distribution.-The disease is worldwide in distribution. 
Nature of the Virus.-Students of the etiology of this disease 
may be divided into two groups-those who believe in the presence 
of a specific ultramicroscopic organism, and those who believe 
in the presence of a protozoan with certain stages of development 
in which the organism is small enough to pass the pores of the filter. 
The latter theory has been developed by Negri. The organism has 
been named Neurorrhyctes hydrophobia . In 1903 he demonstrated 
the presence of specific bodies, which have been termed Negri 
bodies, in the larger ganglia-cells of Ammon's horn, as well as in 
other parts of the central nervous system. There is little ques- 
tion but what these bodies are characteristic of the disease; the 
disputed point is whether they are specific organisms or degenera- 
tion products of the cell. Williams and Lowden summarize the 
evidence of the protozoan nature of these bodies as follows: 
"They have definite characteristic morphology; this mor- 
phology is constantly cyclic, i. e., certain forms always predominate 
in certain stages of the disease, and a definite series of forms in- 
dicating growth and multiplication can be demonstrated; the 
structure and staining qualities, as shown especially by the smear 
method of examination, resemble those of certain known protozoa, 
notably of those belonging to the suborder Microsporidia." 
The Negri bodies in suitably stained preparations are found 
to vary in size from less than 0.5 to 25 y. This variation in size is 
markedly noticeable in different species of animals. In cattle 
they are very large and numerous in the hippocampus; in the 
horse very small and in limited areas; in cats mostly in the 
Purkinje cells of the cerebellum. In shape they may be spherical, 
ovoid, or ellipsoidal. The bodies shows a characteristic 
structure, a smooth hyaline margin, with inclusions of various 
kinds that resemble chromatin granules. 
They may be readily stained by Giemsa's method, or with 
eosin and methylene-blue. 
Pathogenesis.-The organism enters the body through wounds, 
usually bites of animals. It then passes slowly along the per- 
ipheral nerves to the central nervous system. The portion of 
this to which these nerves directly lead is the most seriously 
affected. Characteristic gross anatomic lesions are quite lacking 
in this disease. 548 
VETERINARY BACTERIOLOGY 
The period of incubation is variable; it is usually several 
weeks. It probably represents the period necessary for the virus 
to reach the central nervous system and develop there. The dis- 
ease is commonly fatal. It affects most mammals, including man, 
but is primarily a disease of the carnivora, particularly the dog. 
Immunity.-The Pasteur method of treatment is essentially a 
method of vaccination at intervals with attenuated virus. The 
virus is found, on experimentation, to be rather variable in its 
power to produce disease. The virulence is exalted by repeated 
inoculations of rabbits until it becomes the "fixed" virus of 
Pasteur, and will kill rabbits in six to seven days. This is then 
Fig. 207.-A Negri body. Note the circle of chromatoid granules about the 
central body (X 2000) (Williams and Lowden). 
injected into a rabbit, and upon its death the spinal cord is care- 
fully removed with all aseptic precautions, and suspended in a 
desiccator over caustic potash. It is kept at a constant tempera- 
ture of 23° in the absence of light for two weeks. The vaccine 
consists of an emulsion of this cord in physiologic salt solution. 
Later, injections are made with a cord that has been dried for a 
shorter period. Repeated injections are made. The fact that the 
disease has normally a long incubation period gives an oppor- 
tunity in the human for the use of this method. The active 
immunity established by the injection of the attenuated virus is DISEASES PRODUCED BY UNKNOWN ORGANISMS 
549 
sufficient to destroy the infecting organism. This method of 
treatment has been highly successful when commenced in time. 
It is still strictly an active immunity. To what principle it is due 
is not known. While the Pasteur method is extensively used in 
institutions for the treatment of rabies, as also by physicians, 
there have been developed other methods. One of these is 
coming into more general use. This is the " dilution method " of 
Hogges. A definite quantity of the spinal cord of a rabbit 
killed by fixed virus is emulsified in 100 c.c. of normal salt solu- 
tion. Dilutions of this solution are prepared and the patient is 
given first a dilution of 1 to 1000, and subsequent injections of 
increasing concentration until a dilution of 1-10 is reached. 
Fig. 298.-Removal of the spinal cord from a rabbit (Stimson, Bull. No. 65, 
Hygienic Laboratory). 
Bacteriologic Diagnosis.-The disease may be diagnosed by 
animal inoculation and by microscopic examination. For the 
former it is customary to inject an emulsion from the brain into a 
rabbit. The inoculation is usually made subdurally. Sections 
or smears may be made from the brain and stained to show the 
characteristic Negri bodies. Small portions of the gray substance 550 
VETERINARY BACTERIOLOGY 
are removed from the cerebral cortex in the region of the crucial 
sulcus, the cerebellar cortex, and the hippocampus major. These 
are crushed on a slide and a smear made by means of a cover-glass. 
These dried smears may be stained by Giemsa or other stains, 
perhaps most readily by the method described by Williams and 
Lowden: "To 10 c.c. of distilled water 3 drops of a saturated 
alcoholic solution of basic fuchsin and 2 c.c. of Lbffler's solution of 
methylene-blue are added. The smears are fixed while moist in 
Fig. 209.-Method of drying the spinal cord of a rabbit for the purpose of 
attenuation (Stimson, Bull. No. 65, Hygienic Laboratory). 
methyl alcohol for one minute. The stain is then poured on, 
warmed until it steams, poured off, and the smear is rinsed in water 
and allowed to dry." 
Transmission.-The saliva of diseased animals is found to be 
infective, and the disease is transmitted commonly through the bite. 
Virus of Infectious Agalactia of Sheep and Goats 
Celli and deBlasi in 1906 determined the infectious agalactia 
of sheep and goats to be due to a filterable virus, and their con- DISEASES PRODUCED BY UNKNOWN ORGANISMS 
551 
elusions were verified by CarrA The channel of infection as de- 
termined by experiments appears to be the alimentary tract. The 
disease results in a diminution or cessation of milk flow. Food is 
probably infected through the milk or from the abundant secretion 
from the eyes resulting from a keratitis characteristic of the disease. 
By hyperimmunization by repeated injections of virus it is pos- 
sible to secure a serum that possesses a decided immunizing power. 
deGaspari has described a guinea-pig disease characterized by 
loss of appetite, trembling, convulsions, and finally death. In the 
brain substance this investigator first found a filterable virus by 
the use of the Berkefeld filter. This reproduced the disease upon 
inoculation into healthy animals, the infection always proving fatal. 
The virus is present in the blood-serum and in all the body organs. 
The virus is relatively resistant. It may be destroyed by ex- 
posure for an hour to a temperature of from 70° to 72°. Six days in 
decaying material, fourteen days in pure glycerin, and ten days' 
drying did not suffice to destroy it. Five per cent, phenol killed 
the virus in half an hour. Other animals than the guinea-pig are 
not susceptible to infection. 
Virus of Guinea-pig Plague 
Virus of Fowl Leukemia 
Ehemann has claimed that the leukemia of the domestic fowl 
is due to a filterable virus. 
Virus of Certain Chicken Tumors 
Rous, Peyton, and Murphy have described three distinct types 
of tumors in the domestic fowl, each due to a filterable virus. One 
of these was a spindle-celled sarcoma, another an osteochondro- 
sarcoma, and a third a spindle-celled sarcoma with numerous 
blood sinuses. 
Repeated inoculation gradually raised the virulence of each 
strain, but each continued to produce its particular type of tumor 
upon inoculation. A certain amount of tissue injury at the site 
of inoculation seems to favor the development of the tumor. In- 
jection of the infusorial earth, for example, increases the percentage 
of "takes." SECTION VII 
BACTERIA OF WATER AND FOOD 
CHAPTER XLVI 
BACTERIA OF WATER AND WATER PURIFICATION 
Diseases of man and animals, particularly those of the ali- 
mentary tract, are frequently .transmitted through contaminated 
or impure water. The impurity, so-called, arises from the presence 
of sewage or surf ace-wash. This does not mean that every water 
containing sewage is necessarily harmful, but that the presence of 
the sewage is an indication of the possible and probable occasional 
presence of pathogenic forms. 
Bacteriologic examination of water is important for several 
reasons. Much smaller quantities of contaminating organic matter 
may be determined by bacteriologic than by chemical means. 
Its methods may be used in the determination of the potability 
of water-supplies, in tracing a typhoid or similar epidemic to its 
source, and in determining the efficiency of filters for water-supplies 
and of different types of sewage-disposal systems. 
Water may be examined bacteriologically, either quantitatively 
or qualitatively. In the former a determination of the total num- 
ber of bacteria present in the water is made; in the latter the tests 
are designed to determine the abundance of certain specific disease- 
producing bacteria, as Bacterium typhosum. The former is the 
most useful examination made in determining the potability of a 
water; the latter is rarely used. 
Quantitative Examination of Water.-In general, the greater 
the quantity of organic and decomposing matter present in water, 
the greater will be the number of bacteria present. However, 
it must be noted that changes in the environment, such as tem- 
552 BACTERIA OF WATER AND WATER PURIFICATION 
553 
perature, may cause great variations in bacterial content, even 
though the original water be contaminated. For example, water 
from a source quite above suspicion may have less than 100 bac- 
teria to the cubic centimeter. This same water, carefully sampled 
in a sterile bottle and allowed to stand at room-temperature, may 
show in twenty-four to forty-eight hours hundreds of thousands of 
bacteria to the cubic centimeter. It is important, therefore, that 
the sample taken for examination shall be typical, and that it be 
examined immediately, to prevent multiplication of the bacteria 
present. 
Media Used.-Either nutrient gelatin or agar may be used. It 
should be prepared according to the methods outlined by the 
American Public Health Association. The gelatin will, in general, 
give a somewhat higher count than the agar, but, when many 
liquefying species are present, the count on the gelatin must be 
made before all the slower-growing species have had a chance to 
develop. 
Methods.-Various dilutions of the water to be tested are 
placed in a series of sterile Petri dishes, and the melted medium to 
be used is poured in and thoroughly mixed. After the medium has 
solidified, the plates may be kept at room-temperature or, better, 
placed in a thermostat which maintains a temperature of about 
20° to 22°, or with agar, 37°. The number of colonies developing 
upon a given plate, multiplied by the dilutions introduced, will 
give approximately the number of organisms present per cubic 
centimeter in the original sample. The final count should be made 
after the lapse of forty-eight hours. Agar at 37° is counted in 
twenty-four hours. Discrepancies will usually be detected be- 
tween the numbers, as determined from the plates containing the 
lesser and the greater dilutions. It is customary to use the plate 
having the nearest to 200 colonies in making the final estimation. 
Where more than this number of colonies are present, it is probable 
that many more have failed to develop at all, or to a size that can be 
readily detected, on account of the overcrowding. The numbers 
of bacteria can, of course, be determined only approximately by 
the higher dilution; it is, therefore, customary to follow the mode 
of expression suggested by the committee on standard methods of 
the American Public Health Association: 554 
VETERINARY BACTERIOLOGY 
Numbers of bacteria from- 
1-50 shall be recorded to the nearest unit. 
51-100 " " " " 5 
101-250 " " " " 10 
251-500 " " " " 25 
501-1,000 " " " " 50 
1,001-10,000 " " " " 100 
10,001-50,000 " " " " 500 
50,001-100,000 " " " " 1000 
100,001-500,000 " " " " 10,000 
500,001-1,000,000 " " " " 50,000 
1,000,001-5,000,000 " " " " 100,000 
Interpretation of Results.-No standard for the number of 
bacteria that may be present in potable water can be set because 
of the various factors which may determine a high count. Stern- 
berg, however, has suggested a standard that is in general applic- 
able; water containing less than 100 bacteria per cubic centimeter 
is probably pure; one containing 500 bacteria is suspicious, and one 
with 1000 bacteria is quite certainly bad. The number of bac- 
teria normally present in unpolluted supplies of various kinds 
differs considerably; for example, that in the deep wells of a region 
from those of its lakes, and standards must, therefore, be es- 
tablished for each. Determination of numbers is probably most 
useful in systematic examination of the efficiency of filtration of 
public water-supplies. In some countries these tests are made 
daily, and the maximum bacterial content of the filtered water that 
may be used has even been fixed by law. 
When gelatin is used, a separate count may be made of the 
colonies which develop that are capable of liquefying the medium. 
Such organisms are particularly characteristic of the surface soil, 
and usually belong to the Bacillus subtilis or to the Proteus groups. 
The presence of such in large numbers is an index to the extent of 
the surface wash, and not in general of the extent of sewage pollu- 
tion. This determination may be of value in the examination of 
shallow wells. 
Agar plates may be incubated at blood-heat. The typical 
water bacteria develop very slowly, if at all, at this temperature. 
A count made in twenty-four hours of such a plate is a fair index of 
the amount of sewage contamination usually, as the organisms from 
this source thrive best at this temperature. This determination, BACTERIA OF WATER AND WATER PURIFICATION 
555 
however, is largely displaced by the use of the litmus-lactose-agar 
plates, as discussed under Qualitative Analysis. 
Qualitative Examination of Water.-As has before been stated, 
water may be examined for the specific pathogens it may contain, 
or for the presence of sewage and intestinal bacteria, particularly 
Bacillus coli. 
Isolation of Specific Pathogens.-Bacterium typhosum is the 
organism for which examinations have been most frequently 
made. It has been actually isolated from water in a few instances 
only. Frequently only a very small percentage of the colonies 
which develop from direct plating of typhoid stools are typhoid 
colonies. The chances of direct isolation by plating sewage or 
water from a supply is, therefore, remote, even though this 
organism be present in numbers such as to cause an epidemic of 
the disease among the consumers. 
Usually the search for the specific organism in the water-supply 
is not begun until there is an outbreak of the disease, and the 
probabilities are that the organism by that time has disappeared 
from the supply. Many methods of isolation have been devised, 
but are not commonly used. For the most part, they are dependent 
upon enrichment by placing the suspected water in broth contain- 
ing antiseptics which will inhibit the growth of other bacteria, but 
not of the intestinal, forms. This material is then plated and the 
typhoid-like colonies are fished out and tested one at a time, the 
crucial test usually applied being the ability to agglutinate with 
high dilution of typhoid antiserum. 
The specific organism of Asiatic cholera may be more readily 
isolated than that of typhoid. Flasks of peptone salt solution 
are inoculated with the suspected water, and incubated at blood- 
heat for twenty-four hours or less, and transfers are made to fresh 
flasks from the surface layer. The cholera spirillum has a consider- 
able avidity for free oxygen, and swarms just below the surface in 
much greater numbers than elsewhere in the medium. Plates 
made from this surface film should show the characteristic colonies. 
Isolation of Bacterium coli.-Advantage may be taken of the 
physiologic and cultural characteristics of the Bacterium coli to 
isolate it from water. In examination of large numbers of sam- 
ples it is often found useful to make what is termed a preliminary or 556 
VETERINARY BACTERIOLOGY 
presumptive test for the presence of the colon bacillus. Fermenta- 
tion tubes containing 1 per cent, lactose broth are inoculated 
with varying amounts of the water to be tested. If gas is not 
produced in any of the tubes, it is evident that Bad. coli is not 
present, at least in any considerable numbers. A negative result 
is, therefore, good evidence of the purity of the water examined. 
A positive test makes it probable that the water contains the 
colon bacillus, although further tests are necessary to establish 
the fact; hence the name, presumptive test. The evidence that 
Bad. coli is present is much strengthened if gas is formed to the 
amount of 30 per cent, and not more than 80 per cent. An 
approximation of the number of colon bacilli present may some- 
times be made by observing the dilutions of the water in which 
gas is produced. For example, if gas is produced in dilutions of 
1 : 0, 1 : 10, and 1 : 100, but not in higher dilutions, it may be 
inferred that there are between 100 to 1000 B. coli present per 
cubic centimeter in the original sample. Many investigators 
use lactose bile rather than broth in the presumptive tests. 
A determination of the number of Bacterium coli present in a 
given sample may be secured by plating different dilutions in 
agar containing 1 per cent, lactose, colored blue by litmus solu- 
tion, and incubating twenty-four to forty-eight hours at 37°. 
Organisms which can ferment lactose with acid production are 
surrounded by a red discoloration of the litmus. Such organisms 
are the Bad. coli, Bad. aerogenes, and Streptococcus. The first 
two may be considered together, as they are usually held to indi- 
cate the same facts, although recent work seems to indicate that 
Bad. aerogenes is not a true intestinal form, but found in soils, 
on grains, etc. The colonies of these organisms may usually be 
readily differentiated from those of Streptococcus by their larger 
size, their shiny appearance, and the frequent gas bubble accom- 
panying the colony if it lies below the surface. The Streptococ- 
cus colonies, on the other hand, are small, rarely larger than a 
pin-head, and never have gas bubbles. It is usually necessary 
to make transfers from colonies and carry them through the 
various media to complete the identification. A highly con- 
taminated water will commonly reveal acid colonies directly 
upon plating, but in presence of large numbers of other bacteria 
preliminary enrichment will often show presence of Bad. coli BACTERIA OF WATER AND WATER PURIFICATION 
557 
when direct plating would give negative results. Plates may 
be poured from fermentation tubes that show gas production. 
Bacterium coli is not uncommon in nature; its constant presence 
in the feces of most animals makes it widely distributed. It is 
entirely probable that the presence of small numbers of Bact. coli 
in water may, therefore, be without significance from the stand- 
point of potability. It is generally regarded in America that 
if Bact. coli can be constantly demonstrated in 1 c.c. samples of 
the water, this is an indication of recent sewage contamination. 
When its presence may be demonstrated only by the use of larger 
samples than 1 c.c. the evidence must be regarded as inconclusive. 
Water Purification 
Self-purification of Natural Waters.-Natural waters, both 
running and impounded (as in lakes and reservoirs), gradually 
free themselves from organic and bacterial contamination. The 
rapidity and efficiency of this cleansing process depend upon many 
factors. Bacterial purification is most rapid in impounded waters, 
and chemical purification in running streams. 
Sedimentation is probably the most potent factor in freeing 
a contaminated water from bacteria. The bacteria themselves 
have a slightly greater specific gravity than water, and tend 
to go to the bottom under the influence of gravity. This occurs 
more rapidly when other and larger solid particles are in suspen- 
sion; flocculation and more rapid sedimentation then frequently 
occur. Advantage is taken of this fact in the artificial purification 
of water, and coagulants of different kinds are added, which carry 
down the bacteria, together with other suspended material. Dimi- 
nution of food supply with consequent disappearance of many bac- 
teria is likewise important. Probably light destroys some organ- 
isms, and others are ingested by protozoa. Some species do not 
develop in the presence of certain other forms, that is, they exhibit 
antibiosis. Water-plants, algw, and natural obstructions of all 
kinds to water-flow exert a filtering action. Changes in temperature 
may inhibit the growth and even destroy some bacteria. A con- 
taminated stream is constantly diluted by the influx of ground- 
water and of tributaries. 
Purification of Drinking-water.-For domestic purposes water 558 
VETERINARY BACTERIOLOGY 
may be effectually purified by heating to the boiling-point for a 
few minutes. All the pathogenic bacteria are eliminated by this 
method. Berkefeld porcelain filters, if properly constructed, are 
also efficient. They must be cleaned and sterilized at short inter- 
vals, otherwise the organisms will grow through the pores of the 
filter, and the water passing through will be as contaminated as the 
original supply. Even more efficient is purification by means, of the 
ultra-violet rays. Where a city supply must be purified, it is com- 
monly pumped into reservoirs and allowed to settle, with or without 
the addition of coagulants. It is then passed through filters of 
various types, usually sand. Passage through a properly con- 
structed filter of this type has been shown to be exceptionally effi- 
cient. Such a system requires careful supervision. An efficient 
system will remove over 99 per cent, of the bacteria originally 
present. Some city supplies are pumped under pressure through 
sand-filters. This does not seem to be as efficient a means of rid- 
ding the water of bacteria as the other; as a filter, in any case, tn 
retain its highest efficiency, must remain for some time undisturbed, 
and filters of the latter type require frequent cleaning and washing. 
The installation of filtration plants for purification of city supplies 
has in many cases resulted in a marked diminution of the death-rate 
from typhoid and other intestinal diseases. 
Sewage Disposal.-The question of proper disposal of sewage 
is closely related to the topic of pure water for domestic purposes. 
Usually sewage is allowed to flow into a suitable stream, and is 
purified as it passes down stream. There is no valid objection 
to this, providing there is a sufficient and constant flow of water 
in the stream to insure dilution, and the water of this stream is 
not used as a city supply further down. Unfortunately, too little 
attention has been paid to this subject, and the high typhoid death- 
rates in some cities are due directly to the use of such sewage-pol- 
luted water. Berlin and Paris purify their sewage by using it in the 
irrigation of large tracts of land, and re-collecting the water in the 
underdrains. Such a system is highly efficient, but, as it requires 
a particular type of soil, large areas, and suitable conditions, it is 
not often practicable. For sewage disposal in small cities and 
towns, and even private residences or farms, some of the numerous 
modifications of the septic tank and filter-bed have been shown to BACTERIA OF WATER AND WATER PURIFICATION 
559 
be most efficient. The sewage is first carried to a septic tank, so- 
called-a large tank, usually of brick or concrete, and commonly 
covered. This tank is planned so that the sewage flow of twelve to 
twenty-four hours will fill it, or in other words, that a given portion 
of sewage will require that time to pass through. Here much of 
the solid material settles out. The dissolved oxygen, if any be 
present in the raw sewage, is quickly used up, and anaerobic condi- 
tions are established. Under such treatment the decomposition 
of the organic matter occurs rapidly. Most of the sediment 
of the septic tank is soon dissolved by bacterial action. Gases, 
particularly H2S, CH4, H2, and NH3, are produced. These rise 
to the top, and are there intercepted by the heavy scum which 
forms, and oxidized to H2SO4, or free S2, CO2, H2O, and HNO3; 
hence there is but little disagreeable odor to be noted about such a 
plant. The organic material is, for the most part, broken down 
into soluble, easily oxidizable substances. The sewage must not 
be held under these conditions for too long a period, otherwise the 
decomposition will go too far. The sewage then usually passes to 
a dosing chamber. This is simply a chamber which automatically 
discharges through one or more siphons whenever it becomes filled. 
The sewage passes from here either into contact beds or into filter- 
beds. The former consist of beds of crushed rock usually with 
water-tight walls. The sewage may be. sprinkled over the surface 
constantly and allowed to trickle through (the so-called trickling 
filter), or it may be poured on to the bed in bulk, and held in con- 
tact with the crushed stone for a time and then discharged. In 
either case the sewage becomes thoroughly aerated, and the aerobic 
bacteria rapidly oxidize the organic matter present. A filter-bed, 
on the other hand, is constructed of sand underlaid with gravel and 
stone. The sewage is spread out over the surface and is allowed to 
seep through. Opportunity for thorough aeration of the sand and 
gravel is given by the time elapsing between the discharges from 
the dosing chamber. Frequently several beds are used, and the 
sewage is discharged first upon one, then upon another. The or- 
ganic material is retained, probably by adsorption, and, as the 
sewage passes through, the bacteria are largely filtered out, and the 
organic material is, for the most part, quite completely oxidized. 
The water leaving the drains under these filter-beds is relatively 560 
VETERINARY BACTERIOLOGY 
pure, in some cases quite as pure as water from the average shallow 
well. As has been stated, there are many modifications of the 
type of disposal plant. It has been adapted to use for the farm- 
house as well as the city. 
Recently a marked advance has been made in sewage disposal 
by the use of the so-called activated sludge method. The sewage is 
run into tanks, either with intermittent or constant flow, and air is 
bubbled through rapidly. Gradually oxidizing bacteria multiply 
in the sewage, particularly in the sludge, and oxidation of the 
organic matter is comparatively rapid. When the tank has reached 
its maximum efficiency, sewage is purified within a few hours, being 
separated into two portions, a clear liquid which will prove stable 
and is comparatively free from bacteria and a flocculent sediment 
or sludge. This latter is in part removed from time to time, and 
may be used as a fertilizer. CHAPTER XLVII 
MILK. ITS CONSTITUENTS, CONTAMINATION, AND 
EXAMINATION 
Milk is a complex mixture consisting of dissolved substances 
forming a solution which contains suspended matter, this entire 
mixture in turn containing in emulsion certain undissolved sub- 
stances. The suspended and dissolved substances constitute the 
milk plasma which separates into coagulum and milk serum upon 
coagulation. The fat is in emulsion. Several salts and certain 
cells undissolved or precipitated are also contained. About 87 per 
cent, of milk is water and 13 per cent, solids. Approximately 4 
per cent, of the solids is fat, the balance solids not fat. The con- 
tent of the latter is about 3.3 per cent, protein (nitrogenous com- 
pounds), 0.7 per cent, ash, and 5 per cent, lactose. Caseinogen and 
albumen are the chief nitrogenous compounds, of which the former 
makes up about four-fifths of all. The following table, summarized 
from Van Slyke's analyses, shows the composition of milk: 
Albumen=0.7 
Casein =2.6 
Milk=100 
Water= 87.1 
Solids = 12.9 
Fat = 4.0 
Solids 
not fat= 8.9 
Proteins =3.3 
Milk-sugar=4.9 
Ash (salts)=0.7 
3.3 
100 
12.9 
8.9 
Various hydrolytic enzymes, such as oxidases, galactase, etc., are 
contained. 
Bacteria-free milk is very difficult to obtain without some 
process of sterilization. Bacteria capable of bringing about various 
changes in milk must, therefore, be considered. 
Changes from Normal to Decomposed Milk.-Koning distin- 
guishes seven types of important changes which may occur in milk. 
The milk will show most, if not all, of the following stages: 
1. Germicidal stage. 
2. Development of lactic-acid-producing microorganisms. 
561 562 
VETERINARY BACTERIOLOGY 
3. Neutralization of acid by alkali producers. 
4. Putrefaction. 
5. Lactic acid bacteria again multiply due to neutralization of 
acid by organisms in third stage. 
6. Development of fungi (O'idium lactis and others). 
7. Butyric acid production by bacteria and change of the food 
product into a stinking putrid fluid. 
The Germicidal Action of Milk.-Wolfhiigel and Riedel as early 
as 1886 claimed to have demonstrated bactericidal substances in 
milk by showing that the cholera vibrio multiplied much more 
rapidly in boiled milk than in unboiled. Fokker, in 1890, asserted 
that normal raw milk possessed germicidal properties. He cul- 
tured lactic acid bacteria in raw and in boiled milk, and showed that 
the former resisted spoiling for a longer period. Other investi- 
gators claimed that the bactericidal properties existed only for 
certain kinds of bacteria and not for others. These latter investi- 
gators maintained that the composition of milk favors certain bac- 
teria by creating conditions favorable for their development, while 
others are held in abeyance because of the lack of constituents 
favoring their growth, and also become injured by the products of 
the favored organisms. 
Bauer, Rullman, Tommsdorf, and others have proved that 
specific germicidal substances, such as amboceptors, leucins, alex- 
ins, etc., do exist in special kinds of milk, such as mammitis and 
colostral milk, while they contend that their existence in normal 
milk has not been satisfactorily demonstrated. Some would at- 
tempt to account for the apparent diminution in numbers of bac- 
teria by the fact that milk has an agglutinating action upon 
bacteria, and that, therefore, bacterial clumps rather than single 
bacteria originate the colonies on a plate. 
The decrease of bacteria in fresh milk as shown by bacterial 
counts seems, nevertheless, to be established. It is only after 
a few hours, generally, that rapid multiplication of bacteria 
begins. 
The presence of leukocytes in milk, and the fact that for a few 
hours subsequent to drawing the milk they are capable of ingesting 
bacteria, suggests the possibility that germicidal properties of milk 
are due in part to phagocytic action. This action plus that of MILK. ITS CONSTITUENTS, CONTAMINATION, EXAMINATION 
563 
the true bactericidal substances may account for the germicidal 
action of milk. The bactericidal property of milk persists for 
but a relatively short time. Under favorable cool temperatures 
it may last for some days, but in warm temperature it disap- 
pears after a few hours. A temperature exposure of 80° C. com- 
pletely destroys it. Under even the most favorable conditions 
no reliance should be placed upon it as a means of destroying all 
bacteria. 
Acid Production in Milk.-The organisms producing lactic 
acid primarily, belong for the most part to the genera Streptococ- 
cus and Lactobacillus. Organisms producing smaller amounts of 
lactic acid and larger amounts of volatile acids, proteolytic 
ferments, etc., in general belong to the genera Bacterium and 
Staphylococcus. 
Putrefactive bacteria are practically always present in milk. 
They rarely develop sufficiently, however, to become prominent 
because of the much more rapid growth of the lactic acid bacteria 
and the creation of an acid reaction unfavorable to putrefaction. 
Opportunity is therefore denied for the production of poisonous 
putrefactive substances. The complete destruction of all the 
lactic acid bacteria, as may occasionally occur in pasteurization, 
or the inhibition of their growth by holding milk for a long period 
at low temperatures may offer opportunities for the develop- 
ment of these putrefactive forms. It is probable also that the 
acid production inhibits to some degree the multiplication of 
pathogenic bacteria. 
The flavor and aroma of butter will depend very largely upon 
the type of organisms which have been growing in the cream; the 
butter fat absorbs considerable quantities of dissolved substances 
present in the fermenting cream; it is, therefore highly desirable 
that these should be of the proper character. If batches of cream 
are allowed to ripen spontaneously without the addition of any 
starter there is apt to be a marked lack in uniformity in the pro- 
duct. A much better and more uniform result is secured by 
inoculating the cream which is to be ripened with a considerable 
amount of desirable lactic acid bacteria. Such organisms are sold 
under the name of commercial starters. It is generally assumed 
that they are pure cultures, or practically pure cultures, of Strep- 564 
VETERINARY BACTERIOLOGY 
tococcus lacticus or some very closely related organism. This is 
introduced into pasteurized milk and allowed to grow until the 
milk has reached a satisfactory state of acidity. With this a 
larger bulk of milk is inoculated and finally the whole mass used 
a starter in the cream which is to be churned. A heavy inocu- 
lation with the desirable microorganisms and their growth prod- 
ucts usually overwhelms undesirable types of bacteria and leads 
to the development of a satisfactory product. 
Recent studies have made it evident that the changes brought 
about in a satisfactory starter are not the result simply of the 
growth of Streptococcus lacticus. Another organism closely 
related to it must ordinarily grow with it. Cream which has 
been ripened by means of a pure culture of the ordinary Strepto- 
coccus lacticus does not produce butter with as satisfactory a 
flavor and aroma as that produced by cream which has been 
inoculated with a mixture of these organisms. The ripening pro- 
duced by a starter is therefore an excellent example of associa- 
tive action. The true Streptococcus lacticus apparently produces 
very little change in milk or cream other than the development 
of the pure lactic acid from lactose. The associative organism, 
also a coccus (Streptococcus citrovorus) can by its own growth 
bring about very little change in milk. When grown in the 
presence of Streptococcus lacticus, however, the activity of this 
organism is greatly stimulated and small amounts of volatile 
acids are developed. It is to the absorption of these and perhaps 
of other growthoproducts that the butter owes its characteristic 
aroma and flavor. 
Neutralization of Acid.-Very little decomposition of sour milk 
occurs for several days if the milk be kept under anaerobic condi- 
tions, but upon exposure to air there occurs the development of 
molds (chiefly Oidium lactis) upon the surface, and the lactic acid 
is oxidized to carbon dioxid and water. Thus the acidity is de- 
stroyed and conditions favorable for the development of putre- 
factive organisms are created. Combination of acid with the 
caseinogen of the milk also assists in the neutralization. 
Putrefaction.-Putrefactive bacteria multiply rapidly as soon 
as excess of acidity is overcome. Proteus, Bacillus subtilis, Ps. 
fluorescens, B. mesentericus, and others, together with molds, 
complete the changes whereby the milk becomes putrescent. MILK. ITS CONSTITUENTS, CONTAMINATION, EXAMINATION 
565 
Some of the unusual changes that occur in milk due to certain 
bacteria give rise to such terms as ropy, blue, red, soapy, bitter, 
etc. Ropy milk results from slime-producing bacteria which may 
either produce capsules which are dissolved, act upon proteins 
with the formation of mucin-like substances, or upon the carbo- 
hydrates forming gums. Blue and red milk are the result of 
pigment formation by Pseudomonas cyanogenes, Erythrobacillus 
erythrogenes, and others. B. lactis saponacei and B. sapolacticum 
cause a soapy condition of milk; a sharp, rancid, soapy taste 
develops, and upon shaking a tenacious foam forms. 
Such undesirable bacteria are usually present because of un- 
sterilized milking utensils, and the application of milk of lime and 
hot soda solutions, with cleaning and disinfecting of stables usually 
result in their elimination. 
Contamination of Milk with Bacteria.-The possibility of milk 
contamination is recognized as from the following sources: Mam- 
mary gland and ducts of the same, hair and skin of the cow, air of 
stables, hands and clothing of the milker, milking utensils, handling 
of milk subsequent to milking. 
Mammary Gland.-The healthy tissue of the udder, like other 
normal tissues, is free from bacteria. Milk, therefore, as it is being 
secreted is sterile. In the ducts of the udder and particularly in 
the milk cistern it may come in contact with bacteria, so that when 
it leaves the teat the foremilk particularly may show a low bac- 
terial count. This usually is not above 100 per cubic centimeter, 
rarely as much as 500. Variations in different cows are noticeable, 
and some which appear normal may show a much higher bacterial 
content. 
Hair and Skin.-Lack of careful grooming results in the ac- 
cumulation on the hair of masses of filth, largely fecal matter, which 
at time of milking may fall into the pail and serve as an important 
source of milk contamination. Daily grooming at a time as remote 
from milking time as possible is recommended. Experiments by 
the New York Experiment Station do not indicate that clipping 
the hair is of advantage in preventing contamination; in fact, milk 
from clipped cows had a higher germ content than that from un- 
clipped. This was supposedly due to the fact that whereas in 
unclipped cows the dirt at the base of the hairs not removed by a 566 
VETERINARY BACTERIOLOGY 
superficial grooming is held there by the hair, in clipped ones this 
dirt readily became detached and fell into the pails. 
Air of Stables.-Dust particles which are floating in the air 
of the stable may play an important part in milk contamination. 
It is only under the most adverse conditions, such as during the 
handling of hay and feed or bedding or by continued exposure of the 
milk in the open pail, that contamination from air is to be consid- 
ered of great importance. These are the conclusions reached by 
the New York State Experiment Station, where it was found that 
"while the numbers of bacteria in stable air markedly exceeded 
those of city street or office air, the number falling into the milk 
during the ordinary milking time under conditions permissible in 
any respectable dairy is so small as to be negligible." 
The bacteria from this source are usually those of the putre- 
factive or the Bacillus subtilis types. 
Hands and Clothing of Milkers.-Personal cleanliness of milkers 
and the use of clean outer garments have much to do with the 
purity of milk. The chance for infection from unclean milkers 
is great, and such contamination is very apt to lead to serious re- 
sults, since organisms from this source are much more apt to pro- 
duce disease in man than those coming from the animal. 
Milking Utensils.-These doubtless contribute a greater num- 
ber of bacteria to milk than any of the other sources. Poorly 
soldered seams, rusted areas, and sharp angles at edges furnish 
favorable lodgment for the accumulation of milk, which at once 
becomes a favorable medium for the growth of bacteria. Exposure 
to a heavy fall of dust particles and bacteria from the air is avoided 
by the use of pails with small tops, rather than the old-style pail with 
flaring top. Vessels should be cleansed with sal soda and hot water, 
scrubbed with a brush to remove all adhering masses, rinsed thor- 
ougly in clear water, and steamed for fifteen minutes in a chest. 
Before placing in the chest the tops should be covered with cloth, 
and this should not be removed until the pail is to be used. 
Careless Handling of Milk.-Although milk may show a very 
low bacterial count up to the time it is ready for storage, there is 
still ample opportunity for contamination. If allowed to stand 
open in the cans or to remain for long periods in warm temperature 
the entrance and multiplication of bacteria will be great. Dipping 
from the cans in dippers which have been neglected as to proper MILK. ITS CONSTITUENTS, CONTAMINATION, EXAMINATION 
567 
sanitary care may result in the introduction of enormous numbers 
of bacteria not only with the milk thus removed, but into that 
remaining in the can. 
There are at least five important factors to consider as influenc- 
ing the bacterial content of milk. They are: Initial contamination; 
the time elapsing between the drawing of the milk and its con- 
sumption; the temperature to which it was lowered immediately 
following withdrawal and at which it has been maintained since; 
the care with which it has been handled; and whether it has been 
pasteurized. 
Initial contamination is to be kept at the minimum by proper 
care of stables, cows, and milkers. Furnishing as it does such a 
favorable medium for the growth of bacteria, it is important that 
the time milk is held before consumption should be as short as 
possible, for given much time the increase of bacteria will be enor- 
mous. Closely associated with this factor is that of temperature. 
Held even for but a comparatively short timp at a favorable tem- 
perature the increase will be rapid. Milk should be quickly cooled 
immediately after it is drawn, preferably to a temperature of 50° or 
less. It will keep thus for several days without serious deteriora- 
tion. Careful handling in transportation and in retail shops is im- 
portant in keeping the bacterial content low. Pasteurization 
results in a great reduction of the bacterial content, and pasteurized 
milk kept properly cooled will retain its favorable condition for a 
long time. 
Diseases Transmitted Through Milk.-There are two classes 
of disease of the human which may be transmitted through the 
agency of milk. The first includes those which come from the 
consumption of milk from animals suffering from mastitis, the sec- 
ond those due to specific microorganisms. 
At least three types of disease in the human traceable to con- 
sumption of milk from udders affected with infectious mastitis are 
recognizable: 
(1) Gastro-intestinal catarrh due to streptococci contained in 
such milk. This condition is characterized by fever, dulness, 
nausea, vomiting, diarrhea, fainting, and cramps. Numerous well- 
authenticated cases of this kind are on record, and careful inves- 
Influences Which Determine the Ultimate Bacterial Content 568 
VETERINARY BACTERIOLOGY 
tigations in all reported cases trace the condition back to the in- 
fected udder. 
(2) Epidemics of sore throat accompanied by swelling of the 
lymph-glands of the neck, fever, colic, and diarrhea. Several 
such epidemics in this country have been investigated, and here 
again the cause has been found to be streptococci of milk from mas- 
titis cases. 
(3) Enteritidis and paratyphoid infections, in which the evidence 
points strongly to milk infection. Reports of such conditions are 
few, and none can be said to trace with certainty to enteritidis, or 
paratyphoid mastitis, but such origin is not improbable. 
The specific diseases whose transmission through milk is recog- 
nized include typhoid and paratyphoid fever, cholera, diphtheria, 
tuberculosis, and scarlet fever. The occurrence of these disease- 
producing organisms in milk may be traced to the handling of milk 
by affected persons or by healthy persons who are bacillus carriers, 
or to infective material gaining access to the utensils through the 
use of polluted water. Typhoid fever epidemics due to a con- 
taminated milk supply are usually easily differentiated from those 
of other organs due to the fact that the disease is confined to house- 
holds having a common milk supply. While Levy claims to have 
isolated Bacterium typhosum from an abscess of the udder, it is. 
generally recognized that this organism is unable to produce 
disease in cattle, and its presence in milk is due to careless hand- 
ling, such as the use of contaminated water for washing vessels, 
or through bacillus carriers or convalescing patients. The possi- 
bility of transmission through bottles that have been returned 
unwashed from households where typhoid is present should also 
be recognized. 
Paratyphoid is transmitted in ways quite similar to typhoid. 
In addition, transmission through consumption of milk from cows 
with mastitis due to organisms of this type is held as possible and 
probable. Diphtheria and scarlet fever, while not so commonly 
traceable to contaminated milk supplies, are recognized as being 
transmitted in this way. The contamination in this case is from 
some infected individual who has handled the milk. Infection of 
milk with the Mycobacterium tuberculosis occurs readily and 
directly when the animal is suffering from tuberculosis of the MILK. ITS CONSTITUENTS, CONTAMINATION, EXAMINATION 
569 
udder. It may occur also, in an indirect way, through con- 
tamination of the udder or skin of the cow with feces in pul- 
monary or intestinal tuberculosis, the organisms being thus 
introduced when the infective material falls into the pail. The 
recognition of the bovine type of tuberculosis in lesions of 
tuberculosis both of children and adults is mentioned in another 
chapter (see p. 405). This strongly indicates the possibility of 
the bovine tubercle bacillus being transmitted to man, and sug- 
gests the necessity of careful supervision of herds supplying milk 
to the public. Tuberculin-tested and tuberculosis-free herds 
should be the rule, and where this is impossible, pasteurization 
should be required. 
A few diseases, such as anthrax, Malta fever, and foot-and- 
mouth disease, which are transmissible from animal to man are 
occasionally spread through milk, but their occurrence is rare 
enough as to be considered of slight importance. 
Standards for Production and Distribution of Certified Milk 
Certified milk, in the strict sense of the term, is milk produced 
under legal contract between a medical milk commission and a 
dairyman, and which conforms to the requirements of such com- 
mission. These requirements differ in different cities. Some cities 
have excellent milk ordinances which conform quite closely to the 
standards as adopted by the American Association of Medical Milk 
Commissions. Milk entitled to be certified is clean and wholesome, 
is obtained from healthy cows kept in sanitary quarters, fed 
wholesome feed, and given pure water. It is drawn from clean cows 
by clean healthy attendants into clean receptacles and in a clean 
atmosphere. It is handled in a clean manner, cooled quickly, put 
into sterile vessels, placed in cold storage, and iced in transporta- 
tion. 
Inspected milk comes from clean cows that are tuberculin- 
tested and is drawn and cared for under sanitary conditions, but 
does not meet all the rigid standards demanded for a certified 
milk. 
Pasteurized milk is milk heated for a time at a temperature 
below the boiling-point in order to destroy harmful bacteria. The 570 
VETERINARY BACTERIOLOGY 
so-called holder process is the one in most general use. In this the 
milk is heated to 60° to 65° C. and this temperature is maintained 
for about twenty minutes. 
The following provisions contained in the methods and stand- 
ards for production and distribution of certified milk, as adopted 
by the American Association of Medical Milk Commissions, May 
1, 1912, relate particularly to bacteriologic standards, veterinary 
inspection, and animal hygiene, and are of particular interest to 
veterinary students. 
Hygiene of the Dairy Under the Supervision and Control of the 
Veterinarian 
1. Pastures or Paddocks.-■Pastures or paddocks to which the 
cows have access shall be free from marshes or stagnant pools, 
crossed by no stream whiqh might become dangerously contami- 
nated, at sufficient distances from offensive conditions to suffer no 
bad effects from them, and shall be free from plants which affect 
the milk deleteriously. 
2. Surroundings of Buildings.-The surroundings of all build- 
ings shall be kept clean and free from accumulations of dirt, rub- 
bish, decayed vegetable or animal matter or animal waste, and the 
stable yard shall be well drained. 
3. Location of Buildings.-Buildings in which certified milk is 
produced and handled shall be so located as to insure proper shelter 
and good drainage, and at sufficient distance from other buildings, 
dusty roads, cultivated and dusty fields, and all other possible 
sources of contamination; provided, in the case of unavoidable prox- 
imity to dusty roads or fields, the exposed side shall be screened 
with cheese-cloth. 
4. Construction of Stables.-The stables shall be constructed so 
as to facilitate the prompt and easy removal of waste-products. 
The floors and platforms shall be made of cement or other non- 
absorbent material and the gutters of cement only. The floors 
shall be properly graded and drained, and the manure gutters shall 
be properly graded and drained, and shall be from 6 to 8 inches 
deep and so placed in relation to the platform that all manure 
will drop into them. 
5. The inside surface of the walls and all interior construction 
shall be smooth, with tight joints, and shall be capable of shedding MILK. ITS CONSTITUENTS, CONTAMINATION, EXAMINATION 
571 
water. The ceiling shall be of smooth material and dust-tight. 
All horizontal and slanting surfaces which might harbor dust shall 
be avoided. 
6. Drinking and Feed Troughs.-Drinking troughs or basins shall 
be drained and cleaned each day, and feed troughs and mixing 
floors shall be kept in a clean and sanitary condition. 
7. Stanchions.-Stanchions, when used, shall be constructed 
of iron pipes or hard wood, and throat latches shall be provided 
to prevent the cows from lying down between the time of cleaning 
and the time of milking. 
8. Ventilation.-The cow stables shall be provided with ade- 
quate ventilation either by means of some approved artificial device, 
or by the substitution of cheese-cloth for glass in the windows. 
Each cow to be provided with a minimum of 600 cubic feet of air 
space. 
9. Windows.-A sufficient number of windows shall be installed 
and so distributed as to provide satisfactory light and a maximum 
of sunshine, 2 feet square of window area to each 600 cubic feet of 
air space to represent the minimum. The coverings of such win- 
dows shall be kept free from dust and dirt. 
10. Exclusion of Flies, Etc.-All necessary measures should be 
taken to prevent the entrance of flies and other insects, and rats and 
other vermin into all the buildings. 
11. Exclusion of Animals from the Herd.-No horses, hogs, dogs, 
or other animals or fowls shall be allowed to come in contact with 
the certified herd, either in the stables or elsewhere. 
12. Bedding.-No dusty or moldy hay or straw, bedding from 
horse-stalls, or other unclean materials. shall be used for bedding 
the cows. Only bedding which, is clean, dry, and absorbent may 
be used, preferably shavings or straw. 
13. Cleaning Stable and Disposal of Manure.-Soiled bedding 
and manure shall be removed'at least twice daily, and the floors 
shall be swept and kept-free from refuse. Such cleaning shall be 
done at least one hour before the milking time. Manure, when 
removed, shall be drawn to the field or temporarily stored in con- 
tainers so screened as to exclude flies. Manure shall not be even 
temporarily stored within 300 feet of the barn or dairy building. 
14. Cleaning of Cows.-Each cow in the herd shall be groomed 
daily, and no manure, mud, or filth shall be allowed to remain upon 572 
VETERINARY BACTERIOLOGY 
her during milking; for cleaning, a vacuum apparatus is recom- 
mended. 
15. Clipping.-Long hairs shall be clipped from the udder and 
flanks of the cow and from the tail above the brush. The hair on 
the tail shall be cut so that the brush may be well above the 
ground. 
16. Cleaning of Udders.-The udders and teats of the cow shall 
be cleaned before milking; they shall be washed with a cloth and 
water, and dry wiped with another clean sterilized cloth-a sep- 
arate cloth for drying each cow. 
17. Feeding.-All foodstuffs shall be kept in an apartment sep- 
arate from and not directly communicating with the cow barn. 
They shall be brought into the barn only immediately before the 
feeding hour, which shall follow the milking. 
18. Only those foods shall be used which consist of fresh, palat- 
able, or nutritious materials, such as will not injure the health of 
the cows or unfavorably affect the taste or character of the milk. 
Any dirty or moldy food or food in a state of decomposition or 
putrefaction shall not be given. 
19. A well-balanced ration shall be used, and all changes of food 
shall be made slowly. The first few feedings of grass, alfalfa, en- 
silage, green corn, or other green feeds shall be given in small 
rations and increased gradually to full ration. 
20. Exercise.-All dairy cows shall be turned out for exercise 
at least two hours in each twenty-four in suitable weather. Exer- 
cise yards shall be kept free from manure and other filth. 
21. Washing of Hands.-Conveniently located facilities shall be 
provided for the milkers to wash in before and during milking. 
22. The hands of the milkers shall be thoroughly washed with 
soap, water and brush, and carefully dried on a clean towel im- 
mediately before milking. The hands of the milkers shall be rinsed 
with clean water and carefully dried before milking each cow. 
The practice of moistening the hands with milk is forbidden. 
23. Milking Clothes.-Clean overalls, jumper, and cap shall be 
worn during milking. They shall be washed or sterilized each 
day, and used for no other purpose, and when not in use they shall 
be kept in a clean place, protected from dust and dirt. 
24. Things to be Avoided by Milkers.-While engaged about the MILK. ITS CONSTITUENTS, CONTAMINATION, EXAMINATION 
573 
dairy or in handling the milk employees shall not use tobacco or 
intoxicating liquors. They shall keep their fingers away from their 
nose and mouth, and no milker shall permit his hands, fingers, lips, 
or tongue to come in contact with milk intended for sale. 
25. During milking the milkers shall be careful not to touch 
anything but the clean top of the milking stool, the milk pail, and 
the cow's teats. 
26. Milkers are forbidden to spit upon the walls or floors of 
stables, or upon the walls or floors of milk houses, or into the water 
used for cooling the milk or washing the utensils. 
27. Foremilk.-The first streams from each teat shall be re- 
jected, as this foremilk contains large numbers of bacteria. Such 
milk shall be collected into a separate vessel and not milked on to 
the floors or into the gutters. The milking shall be done rapidly 
and quietly, and the cows shall be treated kindly. 
28. Milk and Calving Period.-Milk from all cows shall be 
excluded for a period of forty-five days before and seven days after 
parturition. 
29. Bloody and Stringy Milk.-If milk from any cow is bloody 
and stringy or of unnatural appearance, the milk from that cow 
shall be rejected and the cow isolated from the herd until the cause 
of such abnormal appearance has been determined and removed, 
special attention being given in the meantime to the feeding or to 
possible injuries. If dirt gets into the pail, the milk shall be dis- 
carded and the pail washed before it is used. 
30. Make-up of Herd.-No cows except those receiving the same 
supervision and care as the certified herd shall be kept in the same 
barn or brought in contact with them. 
31. Employees Other than Milkers.-The requirements for milk- 
ers, relative to garments and cleaning of hands, shall apply to all 
other persons handling the milk, and children unattended by 
adults shall not be allowed in the dairy nor in the stable during 
milking. 
32. Straining and Strainers.-Promptly after the milk is drawn 
it shall be removed from the stable to a clean room and then emp- 
tied from the milk pail to the can, being strained through strainers 
made of a double layer of finely meshed cheese-cloth or absorbent 
cotton thoroughly sterilized. Several strainers shall be provided 
for each milking, in order that they may be frequently changed. 574 
VETERINARY BACTERIOLOGY 
33. Dairy Building.-A dairy building shall be provided which 
shall be located at a distance from the stable and dwelling pre- 
scribed by the local commission, and there shall be no hog-pen, 
privy, or manure pile at a higher level or within 300 feet of it. 
34. The dairy building shall be kept clean and shall not be 
used for purposes other than the handling and storing of milk 
and milk utensils. It shall be provided with light and ventilation, 
and the floors shall be graded and water-tight. 
35. The dairy building shall be well lighted and screened and 
drained through well-trapped pipes. No animals shall be allowed 
therein. No part of the dairy building shall be used for dwelling 
or lodging purposes, and the bottling room shall be used for no 
other purpose than to provide a place for clean milk utensils and 
for handling the milk. During bottling this room shall be entered 
only by persons employed therein. The bottling room shall be 
kept scrupulously clean and free from odors. 
36. Temperature of Milk.-Proper cooling to reduce the tem- 
perature to 45° F. shall be used, and aerators shall be so situated 
that they can be protected from flies, dust, and odors. The milk 
shall be cooled immediately after being milked, and maintained at 
a temperature between 35° and 45° F. until delivered to the con- 
sumer. 
37. Sealing of Bottles.-Milk, after being cooled and bottled, 
shall be immediately sealed in a manner satisfactory to the com- 
mission, but such seal shall include a sterile hood which completely 
covers the lip of the bottle. 
38. Cleaning and Sterilizing of Bottles.-The dairy building shall 
be provided with approved apparatus for the cleansing and steril- 
izing of all bottles and utensils used in milk production. All 
bottles and utensils shall be thoroughly cleaned by hot water and 
sal soda, or equally pure agent, rinsed until the cleaning water is 
thoroughly removed, then exposed to live steam or boiling water 
at least twenty minutes, and then kept inverted until used, in a 
place free from dust and other contaminating materials. 
39. Utensils.--All utensils shall be so constructed as to be easily 
cleaned. The milk pail should preferably have an elliptical open- 
ing 5 by 7 inches in diameter. The cover of this pail should be 
convex, so as to make the entire interior of the pail visible and 
accessible for cleaning. The pail shall be made of heavy seamless MILK. ITS CONSTITUENTS, CONTAMINATION, EXAMINATION 
575 
tin, and with seams which are flushed and made smooth by solder. 
Wooden pails, galvanized-iron pails, or pails made of rough, porous 
materials are forbidden. All utensils used in milking shall be kept- 
in good repair. 
40. Water-supply.-The entire water-supply shall be abso- 
lutely free from contamination, and shall be sufficient for all dairy 
purposes. It shall be protected against flood or surface drainage, 
and shall be conveniently situated in relation to the milk house. 
41. Privies, etc., in Relation to Water-supply.-Privies, pig- 
pens, manure piles, and all other possible sources of contamination 
shall be so situated on the farm as to render impossible the con- 
tamination of the water-supply, and shall be so protected by use 
of screens and other measures as to prevent their becoming breed- 
ing-grounds for flies. 
T ranspor tation 
42. In transit the milk packages shall be kept free from dust 
and dirt. The wagon, trays, and crates shall be kept scrupulously 
clean. No bottles shall be collected from houses in which com- 
municable diseases prevail, unless a separate wagon is used and 
under conditions prescribed by the department of health and the 
medical milk commission. 
43. All certified milk shall reach the consumer within thirty 
hours after milking. 
Veterinary Supervision of the Herd 
44. Tuberculin Test.-The herd shall be free from tuberculosis, 
as shown by the tuberculin test. The test shall be applied in accord- 
ance with the rules and regulations of the United States Govern- 
ment, and all reactors shall be removed immediately from the 
farm. 
45. No animals shall be admitted to the herd without first 
having passed a satisfactory tuberculin test, made in accordance 
with the rules and regulations mentioned; the tuberculin to be 
obtained and applied only by the official veterinarian of the com- 
mission. 
46. Immediately following the application of the tuberculin test 
to a herd for the purpose of eliminating tuberculous cattle, the cow 
stable and exercising yards shall be disinfected by the veterinary 576 
VETERINARY BACTERIOLOGY 
inspector in accordance with the rules and regulations of the United 
States Government. 
47. A second tuberculin test shall follow each primary test after 
an interval of six months, and shall be applied in accordance with 
the rules and regulations mentioned. Thereafter, tuberculin tests 
shall be reapplied annually, but it is recommended that the retests 
be applied semi-annually. 
48. Identification of Cows.-Each dairy cow in each of the certi- 
fied herds shall be labeled or tagged with a number or mark which 
will permanently identify her. 
49. Herd-book Record.-Each cow in the herd shall be registered 
in a herd book, which register shall be accurately kept, so that her 
entrance and departure from the herd and her tuberculin testing 
can be identified. 
50. A copy of this herd-book record shall be kept in the hands 
of the veterinarian of the medical milk commission under which the 
dairy farm is operating, and the veterinarian shall be made re- 
sponsible for the accuracy of this record. 
51. Dates of Tuberculin Tests.-The dates of the annual tuber- 
culin tests shall be definitely arranged by the medical milk com- 
mission, and all of the results of such tests shall be recorded by the 
veterinarian and regularly reported to the secretary of the medical 
milk commission issuing the certificate. 
52. The results of all tuberculin tests shall be kept on file by 
each medical milk commission, and a copy of all such tests shall be 
made available to the American Association of Medical Milk Com- 
missions for statistical purposes. 
53. The proper designated officers of the American Association 
of Medical Milk Commissions should receive copies of reports of 
all of the annual, semi-annual, and other official tuberculin tests 
which are made, and keep copies of the same on file and compile 
them annually for the use of the association. 
54. Disposition of Cows Sick with Diseases Other than Tuberculo- 
sis.-Cows having rheumatism, leukorrhea, inflammation of the 
uterus, severe diarrhea or disease of the udder, or cows that from 
any other cause may be a menace to the herd shall be removed from 
the herd and placed in a building separate from that which may be 
used for the isolation of cow's with tuberculosis, unless such build- MILK. ITS CONSTITUENTS, CONTAMINATION, EXAMINATION 
577 
ing has been properly disinfected since it was last used for this pur- 
pose. The milk from such cows shall not be used nor shall the cows 
be restored to the herd until permission has been given by the 
veterinary inspector after a careful physical examination. 
55. Notification of Veterinary Inspector.-In the event of the 
occurrence of any of the diseases just described between the visits 
of the veterinary inspector, or if at any time a number of cows 
become sick at one time in such a way as to suggest the outbreak 
of a contagious disease or poisoning, it shall be the duty of the 
dairyman to withdraw such sickened cattle from the herd, to de- 
stroy their milk, and to notify the veterinary inspector by telegraph 
or telephone immediately. 
56. Emaciated Cows.-Cows that are emaciated from chronic 
diseases, or from any cause that in the opinion of the veterinary in- 
spector may endanger the quality of the milk, shall be removed 
from the herd. 
Bacteriologic Standards 
57. Bacterial Counts.-Certified milk shall contain less than 
10,000 bacteria per cubic centimeter when delivered. In case a 
count exceeding 10,000 bacteria per cubic centimeter is found, 
daily counts shall be made, and if normal counts are not restored 
within ten days the certificate shall be suspended. 
58. Bacterial counts shall be made at least once a week. 
59. Collection of Samples.-The samples to be examined shall 
be obtained from milk as offered for sale, and shall be taken by a 
representative of the milk commission. The samples shall be 
received in the original packages, in properly iced containers, and 
they shall be so kept until examined, so as to limit as far as possible 
changes in their bacterial content. 
60. For the purpose of ascertaining the temperature, a separate 
original package shall be used, and the temperature taken at the 
time of collecting the sample, using for the purpose a standardized 
thermometer graduated in the Centigrade scale. 
61. Interval Between Milking and Plating.-The examinations 
shall be made as soon after collection of the samples as possible, 
and in no case shall the interval between milking and plating the 
samples be longer than forty hours. 
62. Plating.-The packages shall be opened with aseptic pre- 578 
VETERINARY BACTERIOLOGY 
cautions after the milk has been thoroughly mixed by vigorously 
reversing and shaking the container twenty-five times. 
63. Two plates at least shall be made for each sample of milk, 
and there shall also be made a control of each lot of medium and 
apparatus used at each testing. The plates shall be grown at 37° C. 
for forty-eight hours. 
64. In making the plates there shall be used agar-agar media 
containing 1.5 per cent, agar and giving a reaction of 1.0 to phenol- 
phthalein. 
65. Samples of milk for plating shall be diluted in the proportion 
of 1 part of milk to 99 parts of sterile water; shake twenty-five 
times and plate 1 c.c. of the dilution. 
66. Determination of Taste and Odor of Milk.-After the plates 
have been prepared and placed in the incubator, the taste and odor 
of the milk shall be determined after warming the milk to 100° F. 
67. Counts.-The total number of colonies on each plate should 
be counted, and the results expressed in multiples of the dilution 
factor. Colonies too small to be seen with the naked eye or with 
slight magnification shall not be considered in the count. 
68. Records of Bacteriologic Tests.-The results of all bacterial 
tests shall be kept on file by the secretary of each commission, copies 
of which should be made available annually for the use of the 
American Association of Medical Milk Commissioners. BIBLIOGRAPHIC INDEX 
Breinl and Hindle, 513 
Breinl and Thomas, 485 
Broden, 492 
Brown and Orantt, 380 
Bruce, 353, 481, 483, 489 
Bruce and Bateman, 483 
Brumpt, 472 
Buckley and Mohler, 331, 332, 459 
Buerger, 215, 238 
Bull and Pritchett, 281 
Bumm, 241 
Bureschello, 224 
Burger and Wolbach, 419 
Burke, 288 
Busse, 449 
Calmette, 402, 403 
Cari mi, 483 
Carre, 366, 543, 551 
Carre and Villee, 541 
Castellani, 431 
Celli and de Blasi, 550 
Centanni and Savonuzzi, 543 
Chamberland, 98 
Charbon, 253 
Chaussat, 496 
Chausse, 393, 395 
Chillees, 582 
Citron, 293 
Citron and Wassermann, 300 
Clark and Lubs, 113 
Clegg and Musgrave, 502, 503 
Clegg, Musgrave and Polk, 436, 438, 
439, 444 
Clemesha, 321, 325 
Coca, 313 
Cohn, 21 
Cole, Hadley, and Fitzpatrick, 524 
Conradi, 347 
Cornevin, 271 
Cotton and Schroeder, 351 
Councilman, 195 
Councilman and Lafleur, 504 
Craig, 501, 503, 505 
Craig and Nichols, 431 
Crowley, 495 
Curtis, 391 
Damman and Manengold, 228 
Danysz, 339 
Darling, 497, 498 
Abbe, 122 
Adelbey and Nicolle, 537 
Almy and Bodin, 474 
Alt. 395 
Anderson, 314 
Anderson and McClintock, 120 
Anderson and Rosenau, 210 
Andrewes and Horder, 215 
Andriewsky, 544 
Arloing, 271 
Arning, 408 
Arnold, 95 
Ascoli, 262 
Babes, 315, 509, 512 
Babes and Selter, 317 
Bahr, 278 
Bail, 203, 205, 247, 259, 261 
Bail and Weil, 293 
Balfour, 426, 427 
Bang, 349, 350, 351, 352, 353, 403, 
407, 441 
Banzhaf, 171 
Banzhaf and Gibson, 170 
Barber, 132 
Barnes, 46 
Bateman and Bruce, 483 
Battaglio, 483 
Bauer, 562 
Beach and Hadley, 545 
Behring, 166 
Berg, 477 
Berkefeld, 98 
Besredka and Metchnikoff, 204 
Beyer, 61 
Beyerinck, 77 
Blanchard, 417 
Blaxall and Fox, 474 
Bloch, 469 
Bodin and Almy, 474 
Bodin and Delacroix, 475 
Bollinger, 249, 301, 434 
Bolton, Dorset, and McBryde, 334, 
336 
Bordet, 190 
Bordet and Gay, 192 
Borrel, 419, 502 
Bowman, 530 
Braune, 530 
Bredini and Moore, 485 
Brefeld, 463 
579 580 
BIBLIOGRAPHIC INDEX 
Dassonville and Matruchot, 472, 473 
Davaine, 23 
de Bary, 38, 456 
de Beurmann, 466 
de Blasi and Celli, 550 
Deerr and Lewton-Brain, 138 
de Gaspari, 551 
Delacroix and Bodin, 475 
Demmy, 368 
Deneke, 416 
de Schweinitz, 299, 334, 335 
de Schweinitz and Dorset, 334, 539 
de Schweinitz and McFarland, 298 
Deseler, 513 
Dodd, 431 
Doerr, 348 
Doflein, 480 
Dorset, 111, 389 
Dorset and de Schweinitz, 334, 539 
Dorset, Bolton, and McBrvde, 334, 
336 
Dorset, McBryde and Niles, 540 
Douglas and Wright, 195 
Drigalski, 292 
Dunham, 105 
Dunkel, 363, 380 
Durham, 325 
Dutton and Todd, 423, 492 
Eberlena, 249 
Eberth, 342 
Ehrenberg, 21 
Ehrlich, 24, 124, 154, 157, 159, 160, 
161, 166, 167, 168, 170, 174, 
175, 185, 186, 187, 274, 371 
Ehrlich and Sachs, 192 
Eichhorn and Mohler, 313, 355 
Ehemann, 551 
Ellis, 38, 75 
Elmassian, 490 
Emmerich. 321 
Emmerich and Low, 360 
Emmerlich and Mastbaum, 376 
Epstein, 367 
Ernst, 222 
Escherich, 321, 326 
Evans, 23, 481, 487 
Evans and Steck, 351 
Fantham and Thomson, 513 
Fehleisen, 217 
Fernmore, 301 
Ferriera, Horta and Paredes, 322 
Ferry, 356, 357. 543 
Fielitz, 469 
Finkler and Prior, 416 
Fischer, 34, 37, 71 
Fisher, 486 
Fitzpatrick, Hadley and Cole, 524 
Flexner, 240, 346 
Flexner and Jobling, 240 
Fokker, 562 
Foth, 316 
Fox and Blaxall, 474 
Frankel, 235, 279, 281, 329, 419 
Frankel and Pfeiffer, 219, 223, 237, 
247, 255, 267, 272, 283, 284, 
295, 307, 308, 343, 369, 370, 
374, 387, 388, 409, 411, 412, 
459, 476 
Fraser and Symons, 488 
Freeman, 195 
Friedberger. 379 
Friedlander, 235, 329 
Friedmann, 398 
Frosch and Loffler, 536 
Frost, 146 
Frost and McCampbell, 52 
Frothingham and Johne, 406 
Frothingham, Page and Paige, 136, 
466, 467, 468 
Fuchs, 253 
Gabbett, 124, 126 
Gabritschewsky, 425 
Gaffky, 342 
Gage, 121 
Galli-Yalerio, 447 
Galli-Valerio and Prani, 513 
Gal tier, 310 
Gamalcia, 410 
Gartner, 330 
Gay and Bordet, 192 
Gedvelst, 472 
Gengou, 190 
Gerber, 418 
Gerlach, 475 
Gessard, 358 
Ghon and Sachs, 264, 271, 285 
Gibson and Banzhaf, 170 
Giemsa, 129, 130, 426, 520 
Gilchrist, 449 
Glage, 250. 363, 379, 380, 382 
Glage and Priewe, 380 
Glasser, 337 
Gonder, 417, 515, 516 
Gonder and Sieber, 482, 487, 489 
Gougerot, 469 
Graham, 287, 289 
Graham, and Irons, 450 
Graham-Smith and Nuttall, 514 
Gram, 128 
Grassberger and Schattenfroh, 271. 
274 
Grijns, 458 
Grips, 379 • 
Gruber, 174, 179 
Guglienni, 511 
Guinard and Preisz, 363 
Gunther, 242, 245, 253, 256, 284. 
294, 303, 329, 344, 378, 410. 
413, 435, 446, 470 
Gwyn, 338 BIBLIOGRAPHIC INDEX 
581 
Hadley, 294, 296, 340, 341, 523, 531 
Hadley and Beach, 545 
Hadley, Cole and Fitzpatrick, 524 
Haendel and Uhlenhuth, 337 
Haffkine, 304 
Hamburger, 451 
Hammer, 233 
Hansen, 40, 70, 125, 407 
Hansen and Neisser, 407 
Harding and Ostenberg, 330, 338 
Hartmann, 501, 507 
Harvey and Rettger, 340 
Harz, 434 
Haslam, 460 
Hata, 422 
Hecker, 536 
Hecker, Raebiger, and Hess, 229 
Hedrin, 429 
Heidenhain, 502, 520 
Heim, 245, 321 
Heinemann, 115, 217, 232, 233 
Hektoen and Perkins, 466 
Hektoen and Ruediger, 223 
Heller, 264 
Henle, 23 
Herman, 126 
Hertel, 297 
Herter, 281 
Hess, Hecker and Raebiger, 229 
Heyman, 398 
Hibler, 264, 266, 271, 273, 276, 283, 
285 
Hill, 119 
Hindle and Breinl, 513 
Hinze, 74 
Hirschfelder, 396 
Hiss, 346, 361 
Hoffmann and Schaudinn, 428 
Hogges, 549 
Holman, 215, 216 
Holth, 225 
Hopkins, 120 
Horder and Andrewes, 215 
Horne, 407 
Horta, Ferriera and Paredes, 322 
Hueppe, 290, 325 
Huntoon, 107, 292 
Hutyra, 539 
Hutyra and Marek, 311 
Ingram and Twort, 406 
Inman and Levaditi, 195 
Irons and Graham, 450 
Jenner, 195, 199 
Jensen, 227, 277, 278, 301, 325, 
327, 328, 441 
Jobling and Flexner, 240 
Johne, 129, 241, 249, 254 
Johne and Frothingham, 406 
Johnson and Levine, 326 
Jordan, 28, 95, 180, 280, 323, 327, 
330, 355 
Karlinski, 220 
Kartulis, 504 
Kilborne and Smith, 519 
Kitasato, 265, 266, 271 
Kitasato and Veyl, 265 
Kitasato and Yersin, 302 
Kitt, 227, 275, 297, 301, 306, 375,470 
Kitt and Mayr, 296 
Klebs, 367 
Klein, 125 
Klein and Moller, 254 
Kleine, 483, 489, 490 
Kleine, Rosenthal, and Schiffman, 
. 544 
Klett, 254 
Klimmer, 398, 402 
Klimmer and Wolff-Eisner, 397 
Knapp and Novy, 419, 422, 424, 425, 
426, 428 
Koch, 23, 24, 119, 133, 144, 253, 
271, 282, 377, 386, 395, 397, 
412, 490, 504,' 516, 538, 541 
Koch and Pasteur, 23 
Koch and Schutz, 398 
Kolbe and Kumbein, 305 
Kolle and Turner, 538 
Kolle and Wassermann, 236, 272, 
287, 331, 346, 359, 408 
Konew, 312 
Konig, 561 
Koram, 348 
Koske, 360 
Kral, 470 
Kraus and Levaditi, 167 
Kretz, 167 
Krumweide and Park, 404, 405 
Kruse, 231 
Kiihne, 310 
Kuhnemann, 413 
Kumbein and Kolbe, 305 
Kiinnemann, 363, 379, 380, 382 
Kutscher, 317 
Lafleur and Councilman, 504 
Lafosse, 224 
Lambl, 503 
Lange, 376 
Lange, Zwick and Winkler, 486 
Laveran, 23, 493, 494, 495, 516 
Laveran and Mesnil, 487, 489 
Leclainche, 376, 377 
Leeuwenhoek, 19, 21 
Leger and Marhis, 521 
Leishman, 231, 232, 418, 424, 425, 
426, 498 
Lentz, 130 
Levaditi, 165, 166, 427 
Levaditi and Inman, 195 
Levaditi and Kraus, 167 
Levine, 108, 109, 321, 325 
Levine and Johnson, 326 
Lewis, 481, 496 582 
BIBLIOGRAPHIC INDEX 
Lewton-Brain and Deerr, 138 
Liebig, 22 
Lignieres, 290, 296, 297, 401 
Lignieres and Spitz, 297 
Lignieres and Voges, 491 
Lingelsheim, 214 
Linnaeus, 82 
Lister, 25 
Loesch, 504 
Loftier, 124, 128, 339, 367, 373, 440 
Loffler and Frosch, 536 
Loffler and Schutz, 290, 297, 306 
Lorenz, 376 
Low and Emmerich, 360 
Lowden and Williams, 547, 548, 550 
Lubs and Clark, 113 
Lucet, 220, 244, 248 
Lugol, 127, 130 
Lukas, 265 
MacConkey, 321, 325 
MacFadyean, 293, 541 
Mack, 542 
Mack and Moore, 228 
MacNeal and Now, 484, 485, 488, 
489 
Madsen, 169 
Magnusson, 228 
Mandelbaum, 181 
Manengold and Damman, 228 
Marchoux, 418 
Marchoux and Salimbeni, 425, 427 
Marek and Hutyra, 311 
Marek and Ostertag, 541 
Marhis and Leger, 521 
Marmorek, 223 
Marotel and Moussu, 526 
Mastbaum and Emmerlich, 376 
Matruchot and Dassonville, 472, 473 
Marx, 376 
Marx and Sticker, 544, 545 
Marxer, 227 
Mayer, 235, 236 
Mayer and Emmet, 457 
Mayr and Kitt, 296 
McBrvde, Bolton and Dorset, 334, 
336 
McBryde, Dorset and Niles, 540 
McCampbell, 199, 201, 279 
McCampbell and Frost, 52 
McCampbell and Phillips, 514 
McClintock, 120 
McFarland, 98, 133, 202, 368, 396 
McFarland and de Schweinitz, 298 
Melvin and Mohler, 464, 465 
Mesnil and Laveran, 487, 489 
Metchnikoff, 24, 154, 194, 204 
Mewborn, 474 
Meyer, 264, 282, 351, 354, 406, 407 
Meyer and Shaw, 354 
M'Gowan, 356 
Micellone and Rivolta, 249 
Migula, 323, 339 
Miller, 197, 198, 521 
Milne and Ross, 423 
Milzbrand, 253 
Mohler, 442 
Mohler and Buckley, 331, 332, 459 
Mohler and Eichhorn, 313, 355 
Mohler and Melvin, 464, 465 
Mohler and Morse, 441, 443 
Mohler and Norgaard, 227, 228, 363, 
364 
Mohler and Washburn, 443 
Molisch, 65 
Moller, 125 
Moller and Klein, 254 
Moore, 79, 221, 222, 392 
Moore and Bredini, 485 
Moore and Mack, 228 
Morse, 362 
Morse and Mohler, 441, 443 
Moussu and Marotel, 526 
Much, 126 
Muir, 128 
Muller, 21, 226, 227, 433, 434 
Murchison, 24 
Murphy, Peyton and Rous, 551 
Musgrave and Clegg, 502, 503 
Musgrave, Clegg and Polk, 436, 438, 
439, 444 
Negri, 547 
Neisser, 241 
Neisser and Hansen, 407 
Neisser and Wichsberg, 188 
Nichols and Craig, 431 
Nichols and Phalen, 452 
Nichols and Stovall, 128 
Nicolaier, 265 
Nicolle, 499 
Nicolle and Adelbey, 537 
Nielsen, 277 
Niles, Dorset and McBryde, 540 
Nocard, 363, 437, 486, 536, 537, 541 
Nocard and Prettner, 310 
Nocard and Roux, 535 
Noguchi, 420, 423, 429, 430, 431, 546 
Norgaard, 464 
Norgaard and Mohler, 227, 228, 363, 
364 
Novy, 246 
Novy and Knapp, 419, 422, 424, 425, 
426, 428 
Novy and MacNeal, 484, 485, 488, 
489 
Nowak, 349, 350, 352 
Nuttall and Graham-Smith, 514 
Nuttall and Welch, 279 
Obermeier, 421 
Ogston, 217, 244 
Olt 383 
Orantt and Brown, 380 BIBLIOGRAPHIC INDEX 
583 
Ostenberg and Harding, 330, 338 
Ostertag, 229, 241, 382 
Ostertag and Marek, 541 
Ostertag and Wassermann, 300 
Ostertag and Weichel, 382, 383 
Otho, 346 
Page, Frothingham and Paige, 136, 
466, 467, 468 
Paige, Frothingham and Page, 136, 
466, 467, 468 
Paredes, Ferriera and Horta, 322 
Park, 164 
Park and Krumweide, 404, 405 
Parum, 194 
Pasteur, 22, 23, 98, 217, 260, 282, 
285, 290, 294, 295, 296, 315, 
375, 548, 549 
Pasteur and Koch, 23 
Pasteur and Thuiller, 373 
Patton, 509, 514 
Perkins and Hektoen, 466 
Perrone ito, 294, 543 
Peters, 495 
Petruschky, 335 
Peyton, Rous, and Murphy, 551 
Pfaundler, 177, 181 
Pfeiffer, 185 
Pfeiffer and Frankel, 219, 223, 237, 
247, 255, 267, 272, 283, 284, 
295, 307, 308, 343, 369, 370, 
374, 387, 388, 409, 411, 412, 
459, 476 
Pffeiler, 193 
Pfuhl, 410 
Phalen and Nichols, 452 
Phillips and McCampbell, 514 
Pirquet, 401 
Plenciz, 22 
Poels, 360, 379 
Pohle, 353 
Polk, Musgrave and Clegg, 436, 438, 
439, 444 
Pollender, 253 
Porrey, 243 
Posades and Wernecke, 452 
Prani and Galli-Valerio, 513 
Preisz, 258, 352 
Preisz and Guinard, 363 
Prettner, 376 
Prettner and Nocard, 310 
Priewe, 363 
Priewe and Glage, 380 
Prior and Finkler, 416 
Pritchett and Bull, 281 
Proskauer and Voges, 117, 319, 323 
Prowazek, 418 
Rabe, 249, 356 
Raebiger, 129, 254 
Raebiger, Hecker and Hess, 229 
Rayer, 253 
Redi, 21 
Rees, 517 
Remlinger and Riff, 546 
Rettger, 341 
Rettger and Harvey, 340 
Ricketts, 149 
Rideal and Walker, 119 
Riedel and Wolfhugel, 562 
Riff and Remlinger, 546 
Rivolta, 447, 525 
Rivolta and Micellone, 249 
Rodenwalt, 483 
Ro det and Vallet, 490 
Romanowsky, 129, 199, 254, 422 
Rosenau, 168, 340 
Rosenau and Anderson, 210 
Rosenbach, 217, 244, 249 
Rosenthal, 348 
Rosenthal, Kleine and Schiffman, 
544 
Ross and Milne, 423 
Rouget, 485, 486 
Rous, Peyton and Murphy, 551 
Roux, 315 
Roux and Nocard, 535 
Roux and Yersin, 367 
Ruediger and Hektoen, 223 
Rullman, 562 
Sabouraud, 470, 471, 472, 474 
Sacharoff, 425 
Sachs and Ehrlich, 192 
Sachs and Ghon, 264, 271, 285 
Salimbeni and Marchoux, 425, 427 
Salmon and Smith, 334, 539 
Savonuzzi and Centanni, 543 
Schardinger, 500 
Schattenfroh and Grassberger, 271, 
274 
Schaudinn, 483, 501, 503, 505, 506, 
526 
Schaudinn and Hoffmann, 428 
Schenk, 466 
Schern and Stange, 336 
Schiffman, Rosenthal and Kleine, 544 
Schmitz, 331 
Schnitt, 495 
Schobl, 275 
Schottm tiller, 214, 338 
Schreiber, 296 
Schreuber and Schubert, 376 
Schroeder and Cotton, 351 
Schubert and Schreuber, 376 
Schubert and Schutz, 313 
Schutz, 224, 225, 226, 440 
Schutz and Koch, 398 
Schutz and Lbffler, 290, 297, 306 
Schutz and Schubert, 313 
Schutz and Voges, 376 
Seilards and Walker, 501 
Seller and Babes, 317 
Shaw and Meyer, 354 584 
BIBLIOGRAPHIC INDEX 
Shiga, 345, 346, 348 
Sieber, 510, 519, 520 
Sieber and Gonder, 482, 487, 489 
Silberschmidt, 440 
Smith, T., 105, 204, 207, 267, 297, 
334, 356, 386, 389, 391, 410, 
414, 501, 509, 531 
Smith and Kilborne, 519 
Smith and Salmon, 334, 339 
Solleys el, 224 
Sorensen, 102 
Spitz and Ligniere, 297 
Stange and Schern, 336 
Starcovici, 509 
Starrkrampf, 265 
Stauffacher, 536 
Steck and Evans, 351 
Sternberg, 235, 554 
Sticker and Marx, 544, 545 
Stiles, 527 
Stimson, 549, 550 
Stovall and Nichols, 128 
Stranigg, 193 
Strauss, 310 
Strenge, 193 
Symons and Fraser, 488 
Tartakowsky, 537 
Theiler, 427, 428, 489, 495, 511, 516, 
519, 541 
Thomas, 271 
Thomas and Breinl, 485 
Thomson and Fantham, 513 
Thuiller and Pasteur, 373 
Todd, 226, 348, 424, 428 
Todd and Dutton, 423, 492 
Tokoshige, 447, 448, 449 
Tommsdorf, 562 
Torrey, 356 
Toyoda, 423 
Traum, 351 
Trevisan, 290 
Turner and Kolle, 538 
Turski, 363 
Twort and Ingram, 406 
Tyndall, 533 
Uhlenhuth, 183, 334, 486, 539 
Uhlenhuth and Haendel, 337 
Uhlenhuth and Weidanz, 172 
Uschinsky, 106 
Valentine, 481 
Vallet and Rodet, 490 
Van Ermengen, 127, 286, 287 
Van Gieson, 131 
Van Slyke, 561 
Vaughn, 207 
Vaughn and Wheeler, 207, 210 
Veyl and Kitasato, 265 
Viereck, 507 
Villee and Carre, 541 
Villeman, 385 
Vincent, 445 
Vladimiroff, 315 
Voges and Lignicres, 491 
Voges and Proskauer, 117, 319, 323 
Voges and Schutz, 376 
Voldagsen, 337 
Von Behring, 24, 397 
Von Pirquet, 401 
Walker and Rideal, 119 
Walker and Sellar ds, 501 
Ward, 380 
Washburn and Mohler, 443 
Wassermann, 191, 313, 360 
Wassermann and Citron, 300 
Wassermann and Kolle, 236, 272, 
287, 331, 346, 359, 408 
Wassermann and Ostertag, 300 
Watson, 486, 522 
Wehmer, 456, 458, 461, 462 
Wehrbein, 487 
Weidanz, 171 
Weidanz and Uhlenhuth, 173 
Weichel and Ostertag, 382, 383 
Weichselbaum, 236, 238, 306 
Weigert, 23, 161 
Weil and Bail, 293 
Welch, 279 
Welch and Nuttall, 279 
Wernecke and Posades, 452 
Werner, 507 
Wertheim, 242 
Wesbrook, 368 
Wheeler and Vaughn, 207, 210 
Wherry, 303, 407 
Wichsberg and Neisser, 188 
Widal, 179, 469 
Williams, 164 
Williams and Lowden, 547, 548, 550 
Winkler, Zwick and Lange, 486 
Winogradsky, 76, 77 
Witt, 315 
Wolbach and Burger, 419 
Wolff-Eisner, 402 
Wolff-Eisner and Klimmer, 397 
Wolfhtigel and Riedel, 562 
Wright, 129, 130, 195, 199, 435, 436, 
499 
Wright and Douglas, 195 
Yersin and Kitasato, 302 
Yersin and Roux, 367 
Zenkowsky, 260 
Ziehl, 124 
Zschokke, 525 
Zwick, 486 
Zwick, Lange and Winkler, 486 INDEX 
Abortion, 349 
bacillus of Bang, 349 
infectious, 349 
of cattle, 414 
Abrin, 158 
Absorption, 190 
Acetic acid, 59, 71 
as preservative, 59 
Acetobacter, 85 
aceti, 28, 70, 71 
Acetyl methyl carbinol, production 
of, 117 
Achorion, 469, 475 
gallinse, 475 
gypseum, 475 
muris, 475 
quinckeanum, 475 
schonleinii, 475 
Acid, acetic, 59, 71 
as preservative, 59 
alcohol stain for acid-fast bacteria, 
126 
as preservative, 59 
butyric, 71 
carbolic, 60 
lactic, 59, 70 
as preservative, 59 
neutralization of, 564 
nitrous, oxidation of, 76 
production, 69, 112 
determination of, 113 
in milk, 563 
of bacteria, determination of, 
113 
sulphurous, 61 
Acid-fast bacteria, 92 
non-pathogenic, 408 
stain for, 126 
group, 385 
Acidophiles, 59 
Acquired immunity, 151 
active, 151 
antibodies as factors in, 155 
passive, 153 
passive immunity, 153 
Actinobacillosis, 91 
Actinobacillus, 90, 91 
lignieresi, 91 
Actinomyces, 90, 91 
bovis, 91, 433, 434 
caprae, 433, 439 
Antinomyces cuniculi, 440 
eppingeri, 433 
farcinica, 437 
group, 432 
madurae, 433, 445 
necrophorus, 440 
nocardii, 433, 437 
of other- infections, 445 
Actinomycetales, 84, 90 
Actinomycosis, 434 
in goats, 440 
Action of amboceptor, 186 
of complement, 186 
Active acquired immunity, 151 
Aerobes, 117 
Aerobic bacteria, 48. 
rods, 252 
spore-producing bacilli, 252 
Aerotaxy, 55 
Aerotropism, 55 
African horse sickness, 541 
Agalactia, infectious, 550 
Agar, blood-serum, 107 
chocolate, 107 
endo, simplified, 108 
eosin-methylene-blue, 109 
hormone, 107 
media, 108 
nutrient, 107 
plates, colonies on, 138, 139 
stroke, 135 
Agglutination, 156, 174 
group, 178 
precipitation and, differentiation, 
174 
test, 179, 181, 486 
for glanders, 311 
in disease diagnosis, 179 
Agglutinin, 174 
action, method of, 177 
body, 176, 177 
constitution of, 175 
flagellar, 176, 177 
group, 178 
immune, 174 
in immunity, significance of, 181 
normal, 174 
production of, Ehrlich's theory 
of, 175 
somatic, 176, 177 
Agglutinogen, 175 
585 586 
INDEX 
Agglutinoid, 176 
Agglutinometers, 181 
Agglutinophore, 176 
Aggressin hypothesis, 203 
Aggressins, 203 
artificial, 300 
natural, 299 
Agricultural bacteriology, definition, 
18 
Air, hot, sterilization by, 94 
Albumin-free culture-media, 106 
Alcohol, 61 
production of, 69, 116 
Aldehyde, production of, 116 
Algae, 27 
Alkali, production of, 112 
Alkalies, 60 
Alt tuberculin, 395 
Amboceptor, 186, 192 
action of, 186 
formation of, Ehrlich's conception 
of, 187 
specificity of, 186 
Amebic dysentery, 504 
Ameboid colony on agar plate, 138 
Ammonia, oxidation of, 76 
Amoeba coli, 501, 503 
dysenterias, 504 
meleagridis, 501, 531 
Amcebosporidium polyphragum, 512 
Amphitrichous bacteria, 35 
Anaerobes, 117 
facultative, 117 
obligate, 117 
Anaerobic bacteria, 48 
spore-producing bacilli, 264 
Anaphylaxis, 156, 206, 207 
bacterial, 210 
mechanism of, 207 
relationship of, to certain body 
reactions, 210 
Anaplasma, 509, 519 
marginale, 519 
Anaplasmosis, 519 
Anemia, infectious, 541 
of horse, virus of, 541 
pernicious, 541 
Anginas, 221 
Anilin dyes, 24, 123 
gentian-violet stain, 124 
water, 124 
Animal body, resistance of, to 
disease, 141 
inoculation, 145 
Animals and plants, differentiation, 
26 
Anthrax, 87, 253 
cutaneous, 257 
group, 252 
intestinal, 257 
pulmonary, 257 
Anthrax, symptomatic, 271 
Antiabrin, 173 
Antibiosis, 63 
Antibodies, 154, 155 
and related antitoxins, 157 
as factors in acquired immunity, 
155 
of Ehrlich's first order, 157 
second order, 174 
third order, 185 
Antiformin, 312, 389, 390, 409 
Antigens, 155 
Antigonococcus serum, 243 
Antiphthisin, 396 
Antiphymatol, 398 
Antiricin, 173 
Antiseptics, 56 
in common use, 59 
theories of action of, 57 
Antitoxin, 155, 159 
and related antibodies, 157 
constitution of, 162 
diagrammatic representation of, 
163 
diphtheria, 371 
standardization of, 166 
manufacture of, 164 
for pollen, 173 
of commercial importance, 164 
production of, Ehrlich's theory of, 
161 
tetanus, preparation of, 171 
Antituberculin reaction, 401 
Aphthomonas infestans, 536 
Aphthous fever, 536 
Apiosoma bigeminus, 509 
Apoplectiform septicemia, 227 
Archispores, 508 
Arthritis, 221 
Arthrospores, 36, 38 
Arthus, phenomenon of, 206 
Artificial aggressins, 300 
Asci, 43, 457 
Ascococcus Johnei, 249 
Ascoli thermoprecipitation test, 262 
Ascomycetes, 41 
Ascospores, 40, 41, 43, 457 
Ascus, 41 
Asiatic cholera, 412 
Aspergillosis, 457 
Aspergillus, 455 
flavus, 460 
fumigatus, 457 
glaucus, 462 
niger, 461 
nigrescens, 462 
subfuscus, 462 
Atrichous bacteria, 35 
Atrium, infection, 142 
Autocytotoxins, 192 
Autogenic vaccines, 201 INDEX 
587 
Autolysins, 189 
Autolytic enzymes, 67 
Avenues of infection, 142 
Avian coccidiosis, 523 
tuberculosis, 385 
Azotobacter, 85 
Babesia, 509 
asini, 511 
bigemina, 509 
canis, 512 
equi, 511 
mutans, 511 
ovis, 512 
parva, 516 
(piroplasma) commune, 514 
gibsoni, 514 
Bacillaceae, 85, 87 
Bacillary dysentery, 345 
Bacillus, 28, 87, 252 
abortionis, 349 
abortus, 349 
aerobic spore-producing bacilli, 
252 
aerogenes capsulatus, 278 
anaerobic spore-producing, 264 
anaerobicus cyptobutyricus, 279 
anthracis, 252, 253 
symptomatici, 271 
avisepticus, 293 
bipolaris septicus, 290 
botulinus, 286 
bovicida, 301 
bovisepticus, 301 
bronchicanis, 356 
bronchisepticus, 356 
butter, 409 
cadaveris butyricus, 279 
capsulatus mucosus, 329 
carrier, 345 
chauvaei, 271 
chauveaui, 271 
cholerae, 293 
gallinarum, 293 
suis, 333 
clavatus, 372 
cloacse, 32>7 
coli communior, 325 
communis, 320, 321 
colon, subgroup, 318, 319 
communior, 325 
diphtheriae, 367 
vitulorum, 440 
dung, 409 
dysenteriae, 345 
emphysematis vaginae, 279 
emulsion of Koch, 397 
enteritidis, 330 
sporogenes, 279 
erysipelatis suis, 373 
feseri, 271 
Bacillus filiformis, 440 
flavidus, 362 
furunculosis ulcerosae, 362 
grass, 409 
hay, 252 
hoagii, 362 
hoffmanni, 372 
influenzse, 383 
lactis acidi, 231 
aerogenes, 233, 325 
leprse, 407 
lymphangitidis, ulcerosa, 362 
mallei, 306 
murisepticus, 377 
mycoides, 137 
neapolitanus, 321 
necrophorus, 440 
necroseos, 440 
oedematis maligni, 282 
maligni of Ghon and Sachs, 
285 
of Babes, 317 
of Flexner, 345 
of Gartner, 330 
of Glasser, 337 
of green pus, 358 
of Johnes' disease, 406 
of Kutscher, 317 
of Nicolaier, 265 
of Pfeiffer, 383 
of Preisz, 362 
of Selter, 317 
of Shiga, 345 
ozoenae, 325 
paratuberculosis, 406 
paratyphoid, 338 
perfringens, 279 
pertussis, 384 
pestis, 302 
bubonicae, 302 
phlegmonis emphysematosae, 278 
plurisepticus, 290 
pneumoniae, 329 
of Friedlander, 325 
pseudodiphthericus, 372 
pseudotuberculosis ovis, 362 
pullorum, 340 
pyocyaneus, 358 
pyogenes, 325, 379, 380 
bovis, 379, 380 
foetidus, 321 
suis, 379, 380 
renalis bovis, 362 
rhinoscleromatis, 325 
rhusiopathiae, 373 
suis, 373 
salmoni, 333 
septicemiae, haemorrhagicae, 290 
subtilis, 137, 252 
suicida, 297 
suipestifer, 333 588 
INDEX 
Bacillus suipestifer of Voldagsen,337 
suiseptica, 297 
tetani, 265 
tuberculosis, 385 
types of, 87 
typhi, 342 
abdominalis, 342 
murium, 339 
typhosus, 342 
violaceus, 64 
vitulisepticus, 301 
welchii, 278 
Bacteremia, 148 
Bacteria, 17, 27, 212 
acid production of, determination 
of, 113 
acid-fast, 112 
non-pathogenic, 408 
aerobic, 48 
amphitrichous, 35 
anaerobic, 48 
and disease, 141 
and resistance of animal body to 
disease, 141 
atrichous, 35 
capsulated, 32 
pathogenic, 328 
causal relationship of, to disease, 
proof of, 144 
cell inclusions of, 35 
protoplasm of, 33 
cell wall of, 32 
cells of, grouping of, 29 
changes in hydrogen ion concen- 
tration, 112 
chromogenic, 63 
chromoparous, 64 
classification of, 82 
composition of cell, 46 
cultural characters, 135 
decay-producing, 73 
denitrifying, 74 
differentiation of, 184 
distribution of, 68 
effect of electricity on, 54 
eurythermic, 52 
facultative, 48 
filamentous, 28 
types of, 31 
flagella of, 35 
distribution of, 35 
food relationships of, 46 
foods of, 46 
generation time of, 49 
growth temperature range of, 52 
histology of, 32 
in contamination of milk, 565 
india ink method for, 131 
influence of reaction of medium on 
growth of, 56 
involution forms of, 28 
Bacteria, light production by, 64 
relationships of, 53 
living, examination of, 122 
lophotrichous, 35 
measuring of, 122 
mesophilic, 51 
metatrophic, 47 
moisture relationships of, 47 
monotrichous, 35 
morphology of, 28 
nature and classification of, 21 
of nutrients required by, 104 
nitrate, 76 
nitroso-, 76 
of food, 552 
of water, 552 
oxygen relationships, 117 
paratrophic, 47 
pathogenic, groups of, 212 
position of, 26 
peritrichous, 36 
photogenic, 64 
physiological characters, 140 
physiology of, 46 
pigment production by, 63 
prototrophic, 47 
psychrophilic, 51 
rates of death, 50 
of growth, 49 
reduction processes of, 115 
relationships of chemicals to, 54 
to disease, 22 
to fermentation and decay, 22 
reproduction in, 36 
respiration of, 48 
shape of, 28 
showing sheaths, 33 
size of, 31 
sources of foods, 46 
spherical, 85 
spore bearing, 87 
staining of, 123 
stenothermic, 52 
structure of, 32 
temperature relations of, 51 
thermal death point of, 52 
thermophilic, 51 
thread, 90 
true, 84 
types of, 28 
Bacteriacese, 85, 88 
Bacterial anaphylaxis, 210 
cells, grouping of, 29 
inclusions, 34 
plasmoptysis of, 34 
content of milk, 567 
cultures, study of, 135 
lamp, 65 
relationships of ultramicroscopic 
organisms, 534 
spore types, 37 INDEX 
589 
Bacteridium anthracis, 253 
Bacterins, 201 
Bacteriology, agricultural, definition, 
18 
definition, 17 
medical, definition, 18 
sanitary, 18 
veterinary, 19 
Bacteriolysins, 156, 185, 188 
Bacteriolytic sera used in practice, 
188 
Bacteriopurpurin, 47 
Bacterium, 87, 88, 89 
abortus, 349, 414 
acidi lactici, 137, 325 
aerogenes, 325 
anthracis, 253 
avicidum, 293 
bipolare pluricida, 290 
bovisepticum, 301 
bronchisepticum (bronchicanis), 
356 
cholerae suis, 333, 539 
cloacae, 327 
coli, 74, 89, 137, 320, 321 
commune, 321 
communior, 325 
coscoroba, 325 
diphtheria), 367 
dysenteriae, 342, 345 
enteritidis, 330 
foecalis alkaligenes, 342 
lactis acidi, 231 
aerogenes, 325 
leprae, 407 
mallei, 306 
melitensis, 349 
mucosum capsulatum, 328 
multicidum, 290 
ozoenae, 328 
paratyphi, 338 
pestis, 302 
pneumoniae, 328, 329 
pseudodiphthericum, 372 
pullorum, 340 
rhinoscleromatis, 328 
suicidum, 297 
suipestifer, 539 
tuberculosis, 385 
typhi, 342 
murium, 339 
suis, 337 
typhosum, 58, 342, 568 
welchii, 278 
Balantidium, 529, 530 
coli, 531 
Baleri, 493 
Basidiomycetes, 41 
Beef broth, 104 
extract bouillon, 105 
Beer wort, 105 
Bile, 149 
Biliary fever, 512 
equine, 511 
Biochemical tests, 112 
Bipolare multicidum, 301 
Bismarck-brown stain, 124 
Black death, 304 
head, 531 
Blackleg, 87, 271 
Blackleg-tetanus group, 264 
Blastomyces, 93, 446 
coccidioides, 447, 452 
dermatitidis, 447, 449 
farciminosus, 447 
Biastomycetes, 38, 446 
Blastomycetic dermatitis, 449 
epizootic lymphangitis, 447 
Blastomycosis, 452 
Blind staggers, 460 
Blood and protozoan stains, 129 
parasites, 421 
Blood-serum, 110, 136 
agar, 107 
Blood-stains, recognition of, 183 
Blue milk, 565 
Body agglutinins, 176, 177 
Body-cells, preferential union of 
toxins with, 163 
Bordet-Gengou phenomenon, 190 
Botrycoccus ascoformans, 249 
Botryomyces ascoformans, 249 
Botryomycosis, 249 
Botulism, 286, 581 
Bouillon, 104 
beef extract, 105 
Bovine coccidiosis, 525 
farcy, 437 
piroplasmosis, 509 
Bovotuberkulol, 397 
Bovovaccine, 398 
Bradsot, 87, 277 
Braxy, 277 
Brewer's yeast, 69 
Broth, beef, 104 
beef extract, 105 
glycerin, 105 
nutrient, 136 
serum, 105 
sugar, 105 
sugar-free, 105 
Brownian movement, 36. 
Brucella (bacterium) abortus, 349 
melitensis, 353 
Bryophytes, 27 
Bubonic plague, 89, 302 
Buffers, 103 
Bullnose, 360 
Butschlia, 529 
neglecta, 529 
parva, 529 
postciliata, 529 590 
INDEX 
Butter bacillus, 409 
Butyric acid, 71 
Cachexial fever, 498 
Calcium oxide, 60 
Calf diarrhea, 327 
scours, 325, 327 
Callimastix frontalis, 530 
Canine distemper, 356 
Capsulated bacteria, 32 
pathogenic bacteria, 328 
Capsule, 32 
stain, Johne's, 129 
Muir's, 128 
Raebiger's, 129 
Carbol or phenol fuchsin stain, 124 
Carbolic acid, 60 
Carbuncle, malignant, 253 
Carrier, 345 
Caseous lymphadenitis, 362 
Catalysts, 66 
Catarrhal fever, malarial, 512 
Cattle plague, 537 
Cecum, protozoan commensals of, 
528 
Cell inclusions, 35 
of yeast, 39 
nutrition, Ehrlich's theory of, 159 
plasmolyzed, 34 
protoplasm, 33 
receptors, 160 
vegetative, 37 
wall, 32, 45 
Cellulitis suppurative, 222 
Cellulose, 26 
yeast, 39 
Cerebrospinal meningitis, epidemic, 
238 
Certified milk, 569 
Charbon, 253 
symptomatique, 271 
Chemicals, relationships of, to 
bacteria, 54 
sterilization by, 98 
Chemotaxy. 55 
Chemotropism, 56 
Chicken tumors, 551 
Chickenpest, 543 
Chitin, 26, 32 
Chlamydospore, 41, 43 
Chocolate agar, 107 
Cholera, Asiatic, 412 
Chromobacterium, 88 
Chromogenic bacteria, 63 
Chromoparous bacteria, 64 
Chronic enteritis, 406 
Ciliata, 45, 480, 528 
Ciliophora, 480 
Cladothrix actinomyces, 434 
Classification of bacteria, 82 
Clostridium, 37, 87, 264 
Clostridium amylobacter, 264 
botulinum, 264, 286 
butyricum, 71 
chauvafi, 264, 271 
enteritidis sporogenes, 264 
gastromycosis ovis, 264, 277 
oedematis, 264, 282 
of Ghon-Sachs, 264, 285 
of Hibler, 264 
of Novy, 264 
pasteurianium, 77 
putrificus, 264 
sporogenes Metchnikoff, 286 
tetani, 264, 265 
welchii, 264, 278 
Co agglutinins, 178 
Coccaceae, 85 
Cocci, types of, 86 
Coccidioidal granuloma, 452 
Coccidiosis, 523, 525 
avian, 523 
bovine, 525 
cat, 527 
dog, 527 
ovine, 526 
rabbit, 525 
Coccidium, 509 
bigeminum, 527 
oviforme, 525 
perforans, 525 
perforatum, 523 
revolta, 523 
tenellum, 523 
Coccobacillus, 290 
Coccus, 28 
Coefficient, phenol, 119 
Colon bacillus, 321 
subgroup, 318, 319 
Colonies, gelatin plate, 138 
on agar plates, 138, 139 
Colon-typhoid group, 318 
Comma bacillus, 413 
Commensalism, 63 
Commensals, 47, 63 
Complement, 186 
action of, 186 
fixation of, 190 
test in glanders, 313 
formation of, Ehrlich's conception 
of, 187 
specificity of, 186 
Complement-fixation test, 486 
Conglutination, 192 
test for glanders, 314 
Conglutinin, 192 
Conidia, 38, 43 
Conidiophores, 43, 456 
Conjunctival tuberculin test, 402 
Contagious diseases, 142 
typhus in cattle, 537 
Corrosive sublimate, 57 INDEX 
591 
Corynebacterium, 91, 92 
diphtheriae, 92, 361, 367 
hoffmanni, 361, 362, 372 
pseudodiphthericum, 372 
pseudotuberculosis, 361, 362 
xerosis, 361, 372 
Corynethrix pseudotuberculosis mu- 
rium, 362 
Cow-pox, 545 
Cryptococcus farciminosus, 447 
Cryptogenic infections, 143 
Cultural characters of bacteria, 135 
Culture-media, 23 
acidity of, 101 
adjustment of reaction of, 100 
agar, 108 
albumin-free, 106 
beef broth from meat, 104 
beerwort, 105 
blood-serum, 110 
agar, 107 
broth from beef extract, 105 
chocolate agar, 107 
Dunham's solution, 105 
egg, Hl 
endo-agar, simplified, 108 
endo-agar-fuchsin, 108 
eosin-methylene-blue agar, 109 
gelatin, 106 
glycerin broth, 105 
hormone agar, 107 
hydrogen ion concentration of, 
' 103 
liquefiable solid, 106 
liquid, 104 
milk, 105 
non-liquefiable, 11C 
nutrient agar, 107 
gelatin, 106 
potato, 110 
preparation of, 100 
serum broth, 105 
sugar broth, 105 
sugar-free broth, 105 
synthetic, 106 
Uschinsky's solution, 106 
vegetable, 110 
Cultures of bacteria, methods of 
securing, 132 
by dilution, 132 
by direct isolation, 132 
isolation by animal inocu- 
lation, 134 
by plating, 133 
by smearing, 132 
by use of differential 
antiseptics or disin- 
fectants, 134 
by use of heat, 134 
study of, 135 
stab, types of growth in, 137 
Cutaneous anthrax, 257 
malleih test, 317 
tuberculin reaction, 401 
Cyanophyceae, 27 
Cycloposthium, 529, 530 
bipalm atum, 530 
Cytolysins, 185 
group, 187 
Cytoplasm, 40, 45 
Cytorrhyctes vaccinee, 546 
Cytotoxins, 185, 192 
Dairy bacteriology, 18 
hygiene, 570 
Dasytricha ruminantium, 530 
Death, black, 304 
rates of bacteria, 50 
Decay, 71 
relationships of microorganisms 
to, 22 
Decay-producing and putrefactive 
bacteria, 73 
Delhi boil, 499 
Deneke's spirillum, 416 
Denitrification, 73 
Denys tuberculin, 396 
Deodorant, 57 
Deodorizing power of disinfectant, 59 
Dermatitis, blastomycetic, 449 
s arcop tic, 464 
Dermatomycosis, 472, 474 
Derrengadera, 497 
Desiccation, 47 
Dextrose, 69 
Diarrhea, white, of chicks, 340 
Differentiation of bacteria, 184 
of meats, 183 
Dilution method in treatment of 
rabies, 549 
of securing pure cultures, 132 
Diphtheria, 367 
antitoxin, 371 
fowl, 544 
toxin and antitoxin, manufacture 
of, 164 
Diphtheria-pseudotuberculosis group 
361 
Diphtheroid bacilli, 361 
Diplococcus, 29, 85, 86, 235 
gonorrhcese, 241 
intracellularis equi, 241 
meningitidis, 238 
lanceolatus, 235 
of Neisser, 241 
pneumoniae, 235 
Diplodinium norentinii, 530 
Direct isolation method of securing 
pure cultures, 132 
Discomyces bovis, 434 
equi, 249 
Disease and bacteria, 141 592 
INDEX 
Disease, contagious, 142 
diagnosis, agglutination tests in 
179 
infectious, 141 
types of, 147 
non-infectious, 141, 142 
predisposing factors to, 150 
produced by unknown organisms, 
533 
proof of causal relationship of 
microorganism to, 144 
relationship of microorganisms 
to, 22 
resistance of animal body to, 141 
susceptibility to, variation of 
individuals in, 150 
transmitted through milk, 567 
woolsorters', 253, 257 
Disinfectant, 56 
deodorizing power, 59 
economy of, 59 
efficiency of, standardization of, 
119 
germicidal power, 57 
homogeneity of, 58 
ideal, characteristics of, 57 
in common use, 59 
non-corrosive, 58 
non-toxic to higher life, 58 
penetration of, 58 
power to remove dirt and grease, 
59 
solubility of, 58 
stability of, 58 
theories of action of, 57 
Distemper, 224 
Dog distemper group, 356 
virus of, 542 
Dourine, 485 
Dried mallein, 316 
Drinking-water, purification of, 557 
Drops, hanging, 122 
Drug habituation, 158 
Dumdum fever, 498 
Dung bacillus, 409 
Dunham's solution, 105 
Dysentery, 89 
amebic, 504 
I bacillary, 345 
paratubercular, 406 
East African coast fever, 516 
tick fever, 425 
Eberth-Gaffky bacillus, 342 
Economy in disinfectants, 59 
Ectoplast, 33, 40, 45 
Ectothrix, 471 
megaspores, 471 
microides, 471 
Edema, gaseous, 279 
malignant, 87, 282 
Efficiency of disinfectants, stand- 
ardization of, 119 
Egg medium, 111 
Ehrlich's conception of formation of 
amboceptor and complement, 
187 
humoral theory of immunity, 154 
theory of agglutinin production, 
175 
of antitoxin production, 161 
of cell nutrition, 159 
of immunity, 159 
Eimeria, (coccidium), 509, 522 
avium, 523 
bovis, 525 
faurei, 526 
stiedse, 525 
Electricity, effect of, on bacteria, 54 
Endo agar simplified, 108 
Endo-agar-fuchsin medium, 108 
Endocarditis, ulcerative, 221 
Endogenous infection, 142 
Endolysin, 203 
Endoplasm, 45 
Endospores, 36, 252 
development of, in a bacillus, 37 
Endothrix, 471 
Endotoxin, 157 
Entamoeba, 501 
africana, 507 
coli, 501, 503 
histolytica, 501, 504 
nipponica, 501 
tetragena, 501, 507 
Enteritidis subgroup, 318, 330 
Enteritis, 221, 330, 531 
chronic, 406 
Entodinium, 529, 530 
bursa, 530 
caudaturn, 530 
dentatum, 530 
minimum, 530 
rostratum, 530 
Enzymes, 65, 66 
autolytic, 67 
production, 64 
splitting, 67 
Eosin-methylene-blue agar, 109 
Epidemic cerebrospinal meningitis, 
238 
Epithelioma contagiosum, 544 
Epizootic lymphangitis, 466 
pleuropneumonia in equines, 231 
Equine biliary fever, 511 
piroplasmosis, 511 
Erwinia, 88 
Erysipelas, 220 
swine, 373 
Erysipelatis murisepticum, 137 
Erysipelothrix, 90, 91 
muriseptica, 373, 377 INDEX 
593 
Erysipelothrix porci, 373 
rhusiopathise, 91, 373 
Erythrobacillus, 88 
prodigiosus, 64, 137 
Erythrogranulose, 272 
Eubacteriales, 84, 85 
Eurythermic bacteria, 52 
Exanthemata, 148 
Exhaustion, theory of, 153 
Exogenous infection, 142 
External resistance, 149 
Extracellular enzymes, 66, 68 
Facultative anaerobes, 117 
bacteria, 48 
Farcin du boeuf, 437 
Farcy, 306 
Fermentation, 64 
relationships of microorganisms 
to, 22 
Ferments, organized, 65 
unorganized, 65 
Fever, paratyphoid, 89 
puerperal, 221 
splenic, 253 
typhoid, 89, 342 
Filamentous bacteria, 28 
types of, 31 
Filterable virus, 32, 533 
Filtration, sterilization by, 98 
Fixation, nitrogen, 77 
of complement, 190 
test in glanders, 313 
Flagella, 45 
of bacteria, distribution of, 35 
stain, 127 
Flagellar agglutinins, 176, 177 
Flagellata, pathogenic protozoa of, 
481 
Flame, sterilization by, 94 
Fluorescent group, 358 
Fluorescin, 359 
Fomites, 141 
Food, bacteria of, 552 
poisoning, 89 
in man, 286 
relationships to bacteria, 46 
Foot-and-mouth disease, 536 
virus of, 536 
Foot-rot, 222 
Formaldehyd, 61 
Formalin, 61 
Foul brood of bees, 87 
Fowl diphtheria, 544 
favus, 475 
leukemia, 551 
plague, virus of, 543 
pox, 544 
septicemia, 227 
sleeping sickness, 228 
tumors, 551 
Fungi, 27 
imperfecti, 41, 454 
thread, 432 
Fusarium equinum, 464 
Fusiformis, 91, 92 
Gabbett's method for acid-fast 
bacteria, 126 
methylene-blue stain, 124 
Gall-sickness, 495, 519 
Galziekte, 495, 519 
Gambian horse sickness, 492 
Gangrene, gaseous, 87 
Gartner subgroup, 330 
Gas, production of, 114 
Gaseous edema, 279 
gangrene, 87 
Gastric juice, 149 
Gelatin, nutrient, 106 
plate colonies, 137 
stab, 136 
Generation, spontaneous, 21 
time of bacteria, 49 
Gentian-violet, aqueous solution of, 
124 
stabilized stain, 128 
Genus achorion, 475 
aspergillus, 455 
bacillus, 252 
bacterium, 318, 356 
brucella, 349 
Clostridium, 264 
corynebacterium, 361 
entamoeba, 501 
erysipelothrix, 373 
fusarium, 463 
hemophilus, 379 
herpetomonas, 498 
microsporum, 473 
mycobacterium, 385 
penicillium, 462 
pfeifferella, 306 
piroplasma, 509 
plasmodium, 516 
pseudomonas, 358 
sporotrichum, 466 
trypanosoma, 481 
Germ theory of disease, proof of, 144 
Germ-carrier, 345 
Germicidal action of milk, 562 
power, of disinfectants, 57 
Germicide, 57 
Germination of spores, 38 
Giemsa's stain, 129, 130 
Glanders, 306 
group, 306 
Globules, oil, 35, 40 
Glycerin broth, 105 
Glycogen, 35 
granules, 40 
Goat-pox, 545 . 594 
INDEX 
Gonococcus, 241 
Gonorrhea, 241 
Gram's staining method, 128 
Granules, glycogen, 40 
metachromatic, 35 
Granulobacillus saccharobutyricus 
immobilis, 279 
Granuloma, coccidioidal, 452 
Grass bacillus, 409 
Group agglutination, 178 
agglutinins, 178 
cytolysins, 187 
of pathogenic bacteria, 212 
precipitation, 182 
Growth temperature range, 52 
Gruber-Widal test, 179 
Guinea-pig plague, 551 
Gypseum, 471 
Habituation, drug, 158 
Haematococcus, 509 
bovis, 509 
ovis, 512 
Haemoproteus, 509 
Hair infections, 469 
Halteridium, 519 
Hanging drops, 122 
Hansen's method for spore stain, 125 
Haptophore, 162, 175, 182 
Hautschicht, 40 
Hay bacillus, 252 
Hay-fever, antitoxin for, 173 
Head, 456 
Helcosoma tropicum, 499 
Heliotropism, 56 
Hemagglutinins, 181 
Hemoglobinuria, 512 
Hemolysins, 185, 189 
Hemophilic group, 379 
Hemophilus, 88 
haemoglobinophilis canis, 379 
influenzae, 88, 379, 383 
pertussis, 88, 379, 384 
pyogenes, 88, 379 
Hemoproteus, 519 
Hemorrhagic septicemia, 89, 301 
group, 290, 291 
Hemotoxin, 159, 360 
Herman's stain for tubercle bacilli 
in tissues, 126 
Herpetomonas, 498 
donovani, 498 
Heterologous serum, 175 
Heterolysins, 189 
Hoffmann's bacillus. 372 
Hog-cholera, 298 
group, 318, 330 
virus of, 539 
Homogeneity of disinfectants, 58 
Homologous serum, 175 
Hormone agar, 107 
Horse sickness, 492 
virus of, 541 
syphilis, 485 
Horse-pox, 545 
Host, 141 
Hot air, sterilization by, 94 
Humoral theory of immunity, 24 
Ehrlich's, 154 
Hundstaupe, 542 
Hydrogen ion concentration, deter- 
mination of changes in, 112 
sulphid, oxidation of, 74 
Hydrophobia in man, 546 
Hydrotropism, 56 
Hygiene of dairy, 570 
Hyperimmunization, 188 
Hypersensitiveness, 206 
Hypersusceptibility, 206 
Hyphae, 42, 454 
Hyphomycetes, 41, 454 
form of, 41 
group, 454 
histology of, 42 
morphology of, 41 
reproduction of, 43 
size of, 41 
structure of, 42 
Hypothesis, aggressm, 203 
Icterohematuria, 512 
Ill, quarter, 271 
Immune agglutinin, 174 
opsonins, 196 
Immunity, acquired, 151 
active, 151 
antibodies as factors in, 155 
passive, 153 
agglutinins in, significance of, 181 
duration of, 155 
general discussion, 149 
natural, 150 
racial, 150 
side-chain theory of, 159 
theories of, 153 
development of, 24 
types of, 150 
Immunization, passive opsonic, 203 
Immunology, definition, 19 
Incubation, period of, 158 
India ink method for bacteria and 
protozoa, 131 
Indol, 73 
production of, 118 
Infantile kala-azar, 499 
Infected, 141 
Infection, 141 
atrium, 142 
avenues of, 142 
by ingestion, 147 
by inhalation, 147 . 
cryptogenic, 143 INDEX 
595 
Infection, endogenous, 142 
exogenous, 142 
mixed, 147 
phlogistic, 148 
secondary, .147 
traumatic, 142 
Infectious abortion, 349 
agalactia, 550 
anemia, 541 
of horse, virus of, 541 
disease, 141 
in which specific cause is not 
known, 533 
types of, 147 
Infective, 141 
Influenza, 383 
group, 379 
Ingestion, infection by, 147 
Inhalation, infection by, 147 
Injection, intracardiac, 147 
intracranial, 147 
intra-ocular, 147 
intraperitoneal, 146 
Inoculation, animal, 145 
by scarification, 147 
intrathoracic, 147 
intravenous, 146 
methods of, 146 
subdural, 147 
Inorganic compounds, oxidation of, 
74 
reduction processes in, 73 
Inspected milk, 569 
Intermediate subgroup, 318; 330 
Internal resistance, 149 
Intertransmissibility of human 
bovine and avian tuberculosis, 404 
Intestinal anthrax, 257 
group, 318 
Intracardiac injection, 147 
Intracellular enzymes, 66, 68 
Intracranial injections, 147 
Intradermal tuberculin test, 401 
Intra-ocular injections, 147 
Intraperitoneal injection, 146 
Intrathoracic inoculation, 147 
Intravenous inoculation, 146 
Iron, oxidation of, 75 
Isolation by animal inoculation, 
for securing pure cultures, 134 
by plating, for securing pure 
cultures, 133 
by smearing for securing pure 
cultures, 132 
by use of differential antiseptics 
or disinfectants, for securing 
pure cultures, 134 
of heat for securing pure 
cultures, 134 
direct, for securing pure cultures, 
132 
Isolysins, 189 
Isospora, 509 
bigemina, 527 
Isotrichia, 529, 530 
intestinalis, 530 
prostoma, 530 
Itch disease, 464 
Ixidoplasma bigeminum, 509 
Jaundice, malignant, 512 
Jaw, lumpy, 434 
Johne's capsule stain, 129 
disease, 406 
Kala-azar, 498 
infantile, 499 
Keuchhusten bacillus, 384 
Klebs-Loffler bacillus, 367 
Klein's method of staining for 
spores, 125 
Koch's old tuberculin, 395 
postulates, 144 
Koch-Weeks bacillus, 379 
Konew's precipitation test, 312 
Laboratory methods, development 
of, 23 
Lactic acid, 59, 70 
as preservative, 59 
Lactobacillus, 88, 89 
bulgaricus, 70 
lactis acidi, 325 
Lateral chain theory of immunity, 
159 
Leishman-Donovan bodies, 498 
Leishmania donovani, 498 
farciminosa, 447 
(herpetomonas) infantum, 499 
tropica, 499 
Lentz's stain for Negri bodies, 130 
Leprosy, 407 
Leptotrichia, 90, 92 
Leuconostoc, 85 
Leukemia, fowl, 551 
Leukocidin, 246, 360 
Leukocytic extracts, 203 
Leukocytogregarina, 509 
Leukocytozoon, 509, 521 
Light production by bacteria, 64 
relationships to bacteria, 53 
Lime as disinfectant, 60 
Liquefiable solid media, 106 
Liquid media, 104 
Litmus milk, 137 
Living bacteria, examination of, 122 
Lockjaw, 265 
Lbffler's flagella stain, 128 
methylene-blue stain, 124 
Loffler-Schutz bacillus, 297 
Lophotrichous bacteria, 35 
Lopophyton gallinse, 475 596 
INDEX 
Luetin, 431 
Lumpy jaw, 434 
Lung plague of cattle, 535 
Lymphadenitis, 362 
caseous, 362 
Lymphangitis, blastomycotic epi- 
zootic, 447 
epizootic, 466 
ulcerative, 362 
Lysins, 185 
Macrogametes, 523 
Macronucleus, 45 
Macrophages, 194 
Macroscopic Widal test, 179, 181 
Madura-foot, 445 
Maladie du coit, 485 
Malaria, 516 
quartan, 518 
tertian, 516 
Malarial catarrhal fever, 512 
Mal de caderas, 490 
Malignant carbuncle, 253 
edema, 282 
jaundice, 512 
Malignes edem, 282 
Mallease, 312 
Mallein, dried, of Foth, 316 
of Babes, 315 
of Roux, 315 
of Vladimiroff, 315 
test, cutaneous, 317 
glanders, 315 
ophthalmic, 317 
subcutaneous, 316 
Malleinum siccum, 316 
Malta fever, 353 
group, 349 
Marginal points, 519 
Mastigophora, 488 
Mastitis, 222 
Maximum growth temperature, 52 
McCampbell's modification of 
opsonic index, 199 
Measuring bacteria, 122 
Meat, differentiation of, 183 
Meat poisoning, 286, 330, 333 
Media, 100. See also Culture-media. 
Medical bacteriology, definition, 18 
Medicine, preventive, development 
of, 24 
Megaspores, 471 
Meningitis, epidemic cerebrospinal, 
238 
Meningococcus, 238 
Meningo-encephalitis, 460 
Mercuric albuminate, 60 
chlorid, 60 
Mercury, 60 
Mesophilic bacteria, 51 
Metachromatic granules, 35 
Metals, heavy, salts of, 60 
Metatrophic bacteria, 47 
Metazoa, 27, 44 
Metchnikoff's theory of phagocy- 
tosis, 154 
Microaerophiles, 118 
Microbe, 17 
de Coqueluche, 384 
Microbiology, 17 
Micrococcus, 85 
ascoformans, 249 
aureus, 244 
botryogenus, 249 
citreus, 249 
gonorrhoeas, 241 
intracellularis equi, 241 
lanceolatus, 235 
melitensis, 353 
pneumoniae, 235 
pyogenes albus, 248 
aureus, 244 
bovis, 249 
citreus, 249 
weichselbaumii, 238 
Microgametocytes, 523 
Microides, 471 
Micronucleus, 45 
Microorganisms. See Bacteria. 
Microphages, 194 
Microscope and its influence, 19 
Microscopic examination, 123 
Widal test, 179 
Microspira comma, 412 
metchnikovi, 410 
Microsporum, 469, 473 
adouini, 474 
caninum, 474 
canis, 474 
equinum, 475 
felineum, 474 
lanosum, 474 
Milk, 105, 136 
acid production in, 563 
bacteria in contamination of, 565 
bacterial content of, 567 
certified, 569 
changes in from normal to decom- 
posed, 561 
composition of, 561 
constituents of, 561 
contamination of, 561, 565 
diseases transmitted through. 567 
examination of, 561 
germicidal action of, 562 
inspected, 569 
litmus, 137 
pasteurized, 569 
putrefaction in, 564 
Milzbrand, 253 
Minimum temperature, 52 
Mixed infections, 147 INDEX 
597 
Moisture relationships to bacteria, 47 
Mold group, 454 
hyphae, 42 
spores, types of, 43, 44 
Molds, 17, 212 
chromogenic, 63 
classification of, 93 
form of, 41 
histology of, 42 
morphology of, 41 
reproduction of, 43 
size of, 41 
structure of, 42 
Moller's spore stain, 125 
Monilia candida, 477 
Monocystis stiedae, 525 
Monotrichous bacteria, 35 
Morbus maculosus, 222 
Mordants, 123 
Morvin of Babes, 315 
Mouse septicemia, 377 
Much's granules of mycobacterium 
tuberculosis, Wirth's stain for, 126 
Mucin, 33 
Mucous membranes, 149 
Mud fever, 541 
Muir's capsule stain, 128 
Murrina, 497 
Mycelium, 42, 91, 454 
Mycetoma, 445 
Mycobacterium, 91, 92 
diphtherias, 367 
leprae, 385, 407 
mallei, 306 
paratuberculosis, 385, 406 
pseudo tuberculosis, 362 
smegmatis, 408 
tuberculosis, 28, 126, 385, 568 
Mycology, 17 
Mycorrhiza, 79 
Myxobacteriales, 84 
Nagana, 488 
Natural aggressins, 299 
immunity, 150 
Navel ill, 221 
Necrobacillosis, 440 
Negative chemotaxy, 55 
hydrotropism, 56 
Negri bodies, Lentz's stain, 130 
Neisseria, 85, 86, 235 
gonorrhoeae, 235, 241 
intracellularis equi, 235, 241 
meningitidis, 235, 238 
Nephrolysins, 185 
Neurorrhyctes hydrophobias, 547 
Neurotoxin, 159 
Neutralization of acid, 564 
New tuberculin, 397 
Nitrate bacteria, 76 
Nitrification in soil, 76 
Nitrobacter, 85 
Nitrobacteriaceae, 85 
Nitrogen cycle, 80 
fixation, 77 
Nitroso-bacteria, 76 
Nitrosomonas, 85 
Nitrous acid, oxidation of, 76 
Niveum, 471 
Nocardia farcinica, 437 
Non-corrosive quality of disinfect- 
ants, 58 
Non-inf ectious diseases, 141, 142 
Non-liquefiable media, 110 
Non-pathogenic acid-fast bacteria, 
408 
spirilla, 416 
Non-toxic to higher life, quality of 
disinfectants, 58 
Normal agglutinin, 174 
opsonins, 196 
solution, definition of, 100 
Noxious retention theory, 154 
Nucleus, 40, 45 
Nutrient agar, 107 
broth, 136 
gelatin, 106 
required by bacteria, nature of, 
104 
Nutrition, cell, Ehrlich's theory of, 
159 
Obligate anaerobes, 117 
(Edeme malin, 282 
Oidium, 43, 469 
albicans, 477 
coccidioides, 452 
dermatitidis, 449 
lactis, 564 
Oil globules, 35, 40 
Old tuberculin, 395 
Omphalophlebitis, 221 
Oocyte, 523 
Ophryoscolex, 529, 530 
fasciculus, 530 
inermis, 530 
intermixtus, 530 
labiatus, 530 
scolex, 530 
Ophthalmic mallein test, 317 
Ophthalmo-tuberculin test, 402 
Opsonic immunization, passive, 203 
index, 197 
determination of, 199 
McCampbell's modification of, 
199 
preparation of bacterial emul- 
sion for, 198 
of leukocytes for, 197 
of serum for, 198 
technic of test, 198 
Opsonins, 156, 194, 195 598 
INDEX 
Opsonins, immune, 196 
normal, 196 
Optimum temperature, 51 
Organella, 45, 478 
Organisms, ultramicroscopic, 32 
Organized ferments, 65 
Oriental sore, 499 
Osmotic pressure, adjustment of 
organisms to, 62 
Ovine coccidiosis, 526 
Ovoplasma orientale, 499 
Oxidation of ammonia, 76 
of hydrogen sulphid, 74 
of inorganic compounds, 74 
of iron, 75 
of nitrous acid, 76 
Oxidizers, 67 
Oxvgen relationships, determination 
of, 117 
Papier Chardin, 315 
Paracolon bacillus, 338 
Paraformaldehyd, 61 
Parasites, 47 
blood, 421 
Parasitic protozoa of ciliata, 528 
Parasitology, definition, 18 
Paratrophic bacteria, 47 
Paratubercular dvsentery, 406 
Paratyphoid bacillus, 338 
fever, 89 
Passive immunity, acquired, 153 
opsonic immunization, 203 
Pasteurella, 88, 290 
boviseptica, 291, 296, 297, 301 
cholera) gallinarum, 291, 293, 296, 
297 
cuniculicida, 291, 302 
equiseptica, 291 
pestis, 28, 291, 302 
pseudo tuberculosis rodentium, 
291 
suiseptica, 291, 296, 297 
Pasteurellosis, 290 
Pasteurized milk, 569 
Pathogenic bacteria, groups of, 212 
microorganisms, position of, 26 
protozoa, 478 
of class rhizopoda, 500 
of flagellata, 481 
Penetrating quality of disinfectants, 
58 
Penicillium, 462 
Peptonization, 72 
Period of incubation, 158 
Peripneumonia, 535 
Perithecium, 457 
Peritonitis, 221 
Peritrichous bacteria, 36 
Pernicious anemia, 541 
Pertussis, 384 
Petechial fever, 222 
Petri dish, 133 
Pfaundler's reaction, 177, 181 
Pfeifferella, 91, 92 
mallei, 92, 306 
Pfeifferia princeps, 525 
Pfeiffer's phenomenon, 185 
Pferdesterbe, 541 
Phagocytes, 154, 194 
Phagocytosis, 194 
Metehnikoff's theory of, 154 
Phenol, 60 
coefficient, 119 
Phenomenon of Arthus, 206 
Theobald Smith, 207 
Phlogistic infections, 148 
Photogenic bacteria, 64 
Phycomycetes. 41 
Phymatin, 397 
Physiological salt solution, 63 
Pigment production by bacteria, 63 
Piro plasm a, 509 
bigeminum, 509 
canis, 512 
equi, 511 
mutans, 511 
ovis, 512 
parva, 516 
Piroplasmosis, equine, 511 
Plague, bubonic, 89, 302 
cattle, 537 
fowl, 543 
guinea-pig, 551 
lung, of cattle, 535 
Plant bacteriology, 18 
Plants and animals, differentiation, 
' 26 
Plasmodium, 509, 516 
falciparum, 518 
immaculatum, 518 
malarias, 518 
vivax, 516 
Plasmodroma, 480 
Plasmolysis, 34, 42 
Plasmolyzed cell, 62 
Plasmoptysis, 34 
of bacterial cells, 34 
Plec tridium tetani, 265 
Pleuropneumonia, 535 
epizootic, in equines, 231 
septic, 301 
virus of, 535 
Pneumobacillus, 329 
Pneumococcus, 235 
of Friedlander, 329 
Pneumomycosis, 457 
Pneumonia, 221 
stable, 231 
Poisoning, food, 89 
meat, 286, 330 
ptomain, 330, 333 INDEX 
599 
Polar staining, 35 
Pollantin, 173 
Pollen, antitoxin for, 173 
Polyarthritis, 379 
Polyvalent vaccine, 201 
Positive chemotaxy, 55 
hydrotropism, 56 
Potato, 110, 135, 136 
Power of disinfectant to remove 
dirt and grease, 59 
Precipitation, 156, 174 
agglutination and, differentiation, 
174 
group, 182 
test, Konew's, 312 
uses made of, 183 
Precipitin, 174, 182 
Precipitogen, 182 
Precipitoid, 182 
Predisposing factors to disease, 150 
Preisz-Nocard bacillus,. 362 
Preventive medicine, development 
of, 24 
Proteins, salting out of, 177 
split, Vaughn's, 207 
Proteolysis, 72 
Proteosoma, 509, 519 
Proteus, 88 
vulgaris, 137 
Protoplasm, cell, 33 
yeast, 39 
Prototrophic bacteria, 47 
Protozoa, 17, 27, 212 
classification of, 480 
form of, 45 
histology of, 45 
india ink method for, 131 
morphology of, 44 
parasitic, of ciliata, 528 
pathogenic, 478 
of class rhizopoda, 500 
of flagellata, 481 
reproduction of, 45 
size of, 45 
structure of, 478 
Protozoan and blood stains, 129 
commensals of rumen and cecum, 
528 
Protozoology, 17 
Pseudofarcy, 362, 363 
in horse, 447 
Pseudoglanders, 362 
Pseudomonadacese, 85, 89 
Pseudomonas, 87, 89 
aeruginosa, 89, 358 
denitrificans, 74 
fluorescens, 137, 358 
pyocyanea, 64, 256, 311, 356, 
358 
Pseudopodia, 45, 479 
Pseudotubercle bacilli, 362 
Pseudotuberculosis, 362 
bacilli, 362 
murium, 362 
Psorospermium avium, 523 
cuniculi, 525 
Psychrophilic bacteria, 51 
Pteridophytes, 27 
Ptomain-poisoning, 330, 333 
Ptomains, 72 
Puerperal fever, 221 
Pulmonary anthrax, 257 
Purification of drinking-water, 557 
of water, 552, 557 
Putrefaction, 71, 564 
Pyelonephritis, 362 
Pyemia, 148, 220 
Pyobacillosis, 362, 379 
Pyocyanase, 256, 360 
Pyocyaneus bacillosis, 360 
Pyocyanin, 359 
Pyogenic cocci, 235 
Pyrosoma, 509 
bigem inum, 509 
var. canis, 512 
Pythogenic theory of disease, 24 
Qualitative examination of water, 
555 
Quantitative examination of water, 
552 
Quartan malaria, 518 
Quarter evil, 271 
ill, 271 
Rabies in animals, 546 
Racial immunity, 150 
Raebiger's capsule stain, 129 
Rauschbrand, 271 
Reaction of media, adjustment of, 
100 
Receptors, cell, 160 
Recognition of blood-stains, 183 
Recurrent fever, 421 
Red fever of swine, 373 
milk, 565 
Reduction processes in inorganic 
compounds, 73 
of bacteria, 115 
Relapsing fever, 421 
Reproduction in bacteria, 36 
Resistance, 149 
external, 149 
internal, 149 
Retention, noxious, theory of, 154 
Rhizobium, 85 
leguminosarum, 28, 78 
Rhizopoda, 480, 500 
Rhodesian redwater, 516 
tick fever, 516 
Rhodococcus, 85 
Ricin, 158 600 
INDEX 
Rigor mortis, 67 
Rinderpest, 537 
Rinderseuche, 301 
Ring test, 312 
Ringworm, 472 
Robin, 158 
Rods, aerobic, 252 
vegetative, 37 
Romanowsky stain, 129 
Ropy milk, 565 
Rumen, protozoan commensals of, 
528 
Saccharomyces, 93 
cerevisiae, 69, 93 
dermatitidis, 449 
morphology of, 38 
Salmonella, 333 
Salt solution, physiological, 63 
Salting out of proteins, 177 
Salts of heavy metals, 60 
Sanitary bacteriology, definition, 18 
science, development of, 24 
Sapremia, 148 
Saprophytes, 47 
Saprozoites, 47 
Sarcina, 30, 85 
Sarcocystis, 509, 521 
bertrami, 522 
lendemanni, 522 
miescheriana, 522 
muris, 522 
tenella, 522 
Sarcoptes equi, 465 
Sarcoptic dermatitis, 464 
Sausage poisoning, 286, 581 
Scarification, inoculation by, 147 
Schizomycetes, 27, 84 
Schizonts, 522 
Schizophycese, 27 
Schweinepest, 539 
Schweineseuche, 297 
Sclerotium, 454 
Scrofula, swine, 394 
Secondary infections, 147 
Self-purification of natural waters, 
557 
Sensitization, 156 
Sensitized vaccines, 204 
Septic pleuropneumonia, 301 
Septicemia, 148, 220 
hemorrhagic, 89, 301 
group, 290 
in fowls, 410, 425 
mouse, 377 
Septicemie gangreneuse, 282 
Septicidin, 296 
Septum, 42 
Sera, bacteriolvtic, used in practice, 
188 
Serobacterin, 204 
Serum antidiphtheriticum, 166 
antigonococcus, 243 
broth, 105 
heterologous, 175 
homologous, 175 
sickness in man, 206 
simultaneous method, 538 
Sewage disposal, 558 
Sheath, 33 
Sheep-pox, 545 
Sickness, serum, in man, 206 
Side-chain theory of immunity, 159 
Side-chains, 160 
Skatol, 73 
Skin, 149 
Sleeping sickness, 495 
Small-pox in man, 545 
Soapy milk, 565 
Sodium chlorid, 63 
Soil bacteriology, 18 
nitrification in, 76 
Solubility of disinfectants, 58 
Somatic agglutinins, 176, 177 
Sore head, 544 
throat, septic, 222 
Souma, 494 
Spermatophytes, 27 
Spherical bacteria, 85 
Spirilla, 30 
non-pathogenic, 416 
types of, 31, 90 
Spirillacese, 85, 89 
Spirillosis, 421, 425, 427 
Spirillum, 28, 89, 90 
anserina, 425 
cholerae, asiaticse, 412 
duttoni, 423 
fetus, 414 
metchnikovi, 410 
obermeieri, 421 
of Finkler and Prior, 416 
ovina, 427 
ovis, 428 
pallidum, 428 
phosphorescens, 416 
theileri, 427 
tyrogenum, 416 
Spirochaeta, 93, 420, 421 
anserina, 421, 425 
biflexa, 420 
duttoni, 421, 423 
elusa, 420 
equi, 427, 428 
evansi, 487 
gallinarum, 421, 425 
granulosa, 425 
hyos, 421 
kochi, 425 
Marchouxi, 425 
nicollei, 425 
novyi, 425 INDEX 
601 
Spirochaeta obermeieri, 421 
ovina, 421 
ovis, 427 
pallida, 420, 421, 428 
pertenuis, 421, 431 
recurrentis, 421 
theileri, 421, 427 
Spirochete group, 417 
Spirochetes, 431 
of other infections, 431 
Spirochetosis. 425 
Spirochaetales, 84, 92 
Spiroschaudinnia, 421 
duttoni, 423 
recurrentis, 421 
Splenic fever, 253 
Split proteins, Vaughn's, 207 
Splitting enzymes, 67 
Spontaneous generation, 21 
Sporangiophore, 43 
Sporangium, 43. 
Spore case, 43 
stain, 125 
Spore-bearing bacteria, 87 
Spore-producing bacilli, anaerobic, 
264 
Spores, 36, 508 
germination of, 38 
of yeast, 40 
Sporoblasts, 508 
Sporotrichosin, 469 
Sporotrichosis, 466 
Sporotrichum, 466 
beurmanni, 466 
schenkii, 466 
Sporozoa, 480, 508 
Sporozoites, 508 
Stab cultures, types of growth in, 137 
Stability of disinfectants, 58 
Stabilized gentian-violet, 128 
Stable pneumonia, 231 
Stain, 124 
anilin gen tian-violet, 124 
aqueous solution of gentian-violet, 
124 
Bismarck-brown, 124 
blood, 129 
Giemsa's, 130 
Wright's, 129 
capsule, 128 
Johne's, 129 
Muir's, 128 
Raebiger's, 129 
carbol or phenol fuchsin, 124 
Ehrlich's, 124 
flagella, 127 
Loffler's, 128 
Van Ermengem's, 127 
dor acid-fast bacteria, 126 
acid alcohol method, 126 
Gabbett's method, 126 
Stain for negri bodies, Lentz's, 130 
Gabbett's methylene-blue, 124 
Giemsa, 129 
Gram's method, 128 
Herman's for tubercle bacilli in 
tissues, 126 
Loffler's methylene-blue, 124 
Muir's capsule, 128 
protozoan, 129 
Romanowsky, 129 
spore, 125 
Hansen's method, 125 
Klein's method, 125 
Moller's, 125 
stabilized gentian-violet, 128 
Wirth's for Much's granules of, 
mycobacterium tuberculosis, 
126 
Wright's, 129 
Ziehl's, 124 
Stained mount, preparation of, 124 
Staining methods, 123 
polar, 35 
Stalactite, 303 
Staphylococcus, 30, 85, 86, 244 
albus, 244, 248 
ascoformans, 249 
aureus, 28, 244 
(Botryomvces) ascoformans, 244 
citreus, 244, 249 
pyogenes, 248 
albus, 248 
aureus, 244 
bovis, 249 
citreus, 249 
Staphylolysin, 246 
Starrkrampf, 265 
Starter, 233 
Steam, streaming, sterilization by, 
95 
under pressure, sterilization by, 96 
Stenothermic bacteria, 52 
Sterigmata, 456 
Sterigmatocystis, 455, 456 
Sterilization, 94 
at temperatures lower than 
boiling-point, 98 
by addition of chemicals, 98 
by filtration, 98 
by flame, 94 
by hot air, 94 
by steam under pressure, 96 
by streaming steam, 95 
Strangles, 224 
Strauss' reaction, 310 
Streaming steam, sterilization by, 95 
Streptobacilli, 30 
Streptococcus, 29, 85, 86, 214 
acidi lactici, 231 
agalactiae contagiosae, 231 
vaccarum, 217 602 
INDEX 
Streptococcus anginosus, 215 
articulorum, 216 
brevis, 214 
capsulatus gallinarum, 227 
citrovorus, 234, 564 
classification of, 214 
coryzae contagiosa; equorum, 224 
equi, 216, 224, 231 
equinus, 215 
erysipelatos, 215, 216 
foecalis, 215 
gallinarum, 216, 227 
Holman's classification of, 215 
lacticus, 63, 70, 216, 231, 232, 564 
lactis, 231 
longus, 214, 215 
mastitidis, 217, 231 
sporadicae, 231 
meningitidis, 238 
mitis, 215 
mucosus, 215 
of Norgaard and Mohler, 227 
of Ostertag, 229 
phlogogeries, 217 
pneumonia), 235 
puerperalis, 216 
pyogenes, 137, 215, 216, 229, 231, 
232 
bovis, 217 
malignis, 216 
salivarius, 215 
scarlatinosus, 216 
septicus, 216 
vaginitidis, 216, 229 
viridans, 215 
Streptothricosis, 440, 445 
Streptothrix actinomyces, 434 
bovis, 434 
canis, 439 
caprae, 439 
cuniculi, 440 
farcinica, 437 
madurae, 445 
necrophora, 440 
Subcutaneous mallein test, 316 
Subdural inoculations, 147 
Suctoria, 480 
Sugar broth, 105 
Sugar-free broth, 105 
Sulphur dioxid, 61 
Sulphurous acid, 61 
Suppurative allulitis, 222 
Surra, 487 
Susceptibility, 149 
to disease, variation of individuals 
in, 150 
Swamp fever, 541 
Swine erysipelas, 373 
group, 373 
fever, 539 
red fever of, 373 
Swine scrofula, 394 
Swine-plague, 297, 298, 334 
Swine-pox, 545 
Symbion, 63 
Symbiont, 63. 
Symbiosis, 63 
Symptomatic anthrax, 271 
Synthetic media, 106 
Syphilis, 428 
horse, 485 
Wassermann test for, 191 
Systematic bacteriology, 18 
Taoruman, 398 
Temperature lower than boiling- 
point, sterilization at, 98 
relations to bacteria, 51 
Tertian malaria, 516 
Test, agglutination, 179, 181 
for glanders, 311 
complement-fixation, in glanders, 
313 
conglutination for glanders, 314 
cutaneous mallein, 317 
Gruber-Widal, 179 
mallein, for glanders, 315 
ophthalmic mallein, 317 
Pfaundler's, 181 
precipitation, Konew's, 312 
ring, 312 
Strauss', 311 
subcutaneous mallein, 316 
thermoprecipitation of Ascoli, 262 
Wassermann for syphilis, 191 
Widal, 179 
Tetanolysin, 269 
Tetanospasmin, 269 
Tetanus, 87, 265 
toxin and antitoxin, preparation 
of, 171 " 
Tetracoccus, 29 
Tetrad, 29 
Texas fever, 509 
Thallophytes, 27 
subdivisions of, 27 
Theileria, 509 
parva, 516 
Theobald Smith phenomenon, 207 
Theories of immunity, 153 
development of, 24 
Ehrlich's humoral, of immunity, 
154 
of cell nutrition, Ehrlich's, 159 
of exhaustion, 153 
of noxious retention, 154 
of phagocvtosis, Metchnikoff's, 
154 
Thermal death point, 52, 118 
Thermophilic bacteria, 51 
Thermoprecipitation test of Ascoli, 
262 INDEX 
603 
Thiobacteriales, 84 
Thread bacteria, 90 
fungi, 432 
Thrush, 477 
Tick fever, 509 
Tinea cristse gallae, 475 
Tongue, wooden, 434 
Tonsillitis, 221 
Torula, 93 
Toxemias, 148 
Toxin, 155, 157 
characteristics of, 157 
constitution of, 162 
diagrammatic representation of, 
163 
diphtheria, manufacture of, 164 
standardization of, 166 
preferential union of, with body- 
cells, 163 
sources of, 158 
specificity of, 159 
tetanus, preparation of, 171 
Toxine, 157 
Toxoid, 162 
Toxone, 170, 371 
Toxophore, 162 
Traumatic infection, 142 
Treponema, 93, 421 
pallidum, 93, 428 
pertenue, 431 
Trichobacteria, 28, 36 
Trichomastix ruminantium, 530 
Trichomonas, 531 
ruminantium, 530 
Trichomycetes, 432 
Trichophyton, 469, 470 
caninum, 471, 473 
cerebriforme, 471 
crateriforme, 471 
denticulatum, 471 
discoides, 471 
equinum, 471, 472 
felineum, 471, 472 
granulosum, 471, 472 
gypseum, 472 
niveum, 472 
radians, 471, 472 
sulfurerum, 471 
tonsurans, 471 
verrucosum, 471 
Tropisms, 56 
True bacteria, 84 
Trypanosoma, 481 
brucei, 483, 488 
castellani, 495 
cazalboui, 494 
congolense, 492 
dimorphon, 492 
donovani, 498 
elmassiani, 490 
equinum, 490 
Trypanosoma equiperdum, 485 
evansi, 487 
gambiense, 483, 484, 495 
hippicum, 497 
lewisi, 483, 491 
pecaudi, 493 
rougeti, 485 
theileri, 495 
ugandense, 495 
vespertilionis, 483 
Trypanosomiasis, 492, 493 
human, 495 
Tsetse-fly disease, 488 
Tubercle bacilli in tissues, Herman's 
stain for, 126 
Tuberculin, 394 
Alt, 395 
Koch's, 395 
New, 397 
of Denys, 396 
test, conjunctival, 402 
cutaneous, 402 
intradermal, 401 
ophthalmo, 402 
T. R., 397, 398 
Tuberculocidin, 396 
Tuberculol, 396 
Tuberculosis, 385 
Tumors, chicken, 551 
Turgor, 62 
Typhoid fever, 89, 342 
Typhoid-dvsentery subgroup, 318, 
342 
Typhus, contagious, in cattle, 537 
Ulcerative endocarditis, 221 
lymphangitis, 362 
Ultramicroscope, 20 
Ultramicroscopic organisms, 32 
bacterial relationships of, 534 
Univalent vaccine, 201 
Unorganized ferments, 65 
Uschinsky's solution, 106 
Vaccination, 153, 189, 200 
Vaccines, autogenic, 201 
isolation of organism, 201 
preparation of, 201 
standardization of, 202 
polyvalent, 201 
sensitized, 204 
univalent, 201 
Vacuoles, 35, 40 
Van Ermengem's flagella stain, 127 
Variation of individuals in sus- 
ceptibility to disease, 150 
Vaughn's split proteins, 207 
Vegetable culture-media, 110 
Vegetative cell, 37 
rod, 37 604 
INDEX 
Veterinary bacteriology, definition, 
19 
supervision of herd, 575 
Vibrio, 89, 90 
cholerse, 60, 90, 410, 412 
asiaticae, 412 
fetus, 414 
Finkleri, 137 
group, 410 
metchnikovi, 410 
proteus, 416 
Vibrion septique, 282 
Virulence, 144, 203 
Virus, 141 
filterable, 32, 533 
of dog distemper, 542 
of epithelioma contagiosum, 544 
of foot-and-mouth disease, 536 
of fowl plague, 543 
of guinea-pig plague, 551 
of hog-cholera, 539 
of horse sickness, 541 
of infectious agalactia of sheep 
and goats, 550 
of infectious anemia of horse, 541 
of pleuropneumonia, 535 
of poxes, 545 
of rabies, 546 
of rinderpest, 537 
of yellow fever, 546 
Voges-Proskauer reaction, 117 
Wassermann syphilis test, 191 
Water analysis, 552 
anilin, 124 
bacteria of, 552 
natural, self-purification of, 557 
purification, 552, 557 
quantitative examination of, 552 
West African tick fever, 423 
Whips, 35 
White diarrhea of chicks, 340 
Whooping-cough, 384 
Widal test, 179 
Wildseuche, 301 
Wirth's stain for staining Much's 
granules of mycobacterium tuber- 
culosis, 126 
Wooden tongue, 434 
Woolsorters' disease, 253, 257 
Wright's blood stain, 130 
Yaws, 431 
Yeasts, 212 
brewer's, 69 
cell inclusion of, 39 
cells and groupings, types of, 39 
cellulose, 39 
chromogenic, 63 
classification of, 93 
form of, 39 
grouping of, 39 
histology of, 39 
morphology of, 38 
protoplasm, 39 
reproduction in, 40 
size of, 39 
spores of, 40 
structure of, 39 
Yellow fever, 546 
Ziehl's carbol or phenol fuchsin 
stain, 124 
Zooglea, 31 
Zooglcea pulmonis equi, 249 
Zopfius, 88 
Zygospores, 43 
Zymase, 66, 69 
Zymophore, 176, 182 
Zymotoxic group, 176