AQ

61

Black tongue in dogs and its relation to sprue, pellagras and per-
t clear

tudies of tropical sprue in Porto Rico made i

nicious anemia. The &
that an experimental method would be of great value in studying the nature
and treatment of that condition. Moreover, it was thought that animal tests
mignt show a relationship between spruc, pellagra, and pernicious anemia.
Several dogs kept on diets low in protein and Vitamin B developed a condition
characterized by stomatitis, diarrhea, and central nervous system disturbances
the last, the condition

With the exception of

e to eight weeks.
preliminary observatt

in from fiv
ons indicate

resembled strikingly acute tropical sprue-
that disturbances of the gastric function are associated with the other syup-
toms. It 18 proposed to test the activity of the gastric juice of these dogs
for power to relieve pernicious anemia in human beings. As 4t has been found
possible to prevent the disturbances of the central nervous system, dogs may é
be kept in a state of chronic 4{llness similar to tropical sprue, and thus it 4
may be ascertained whether this chronic disease leads to experimental perni-

cious anemia.

Goebel, Dubos, francis, Babers, Goodner,

(prs. Avery: Stillman,

subjects dealt with in the report are:

ell). The special
accharide of Type ITI

of the capsular polys

Alloway» and TerT

The decompo sition

pneumococcus by a bacterial enzyme.

Methods of purification and concentration of the specific
enzyme,
ymal infection of rab-

 

protective action of the enzyme in de

pits with Type III pnevumococcus,

ype III) in monkeys-

production of pnevmococcus pneumonia (t
————E_ ti es

AR2

62

Isolation of microorganisms decomposing the capsular po lysaccha-
rides of other types of pneumococcus.
Chemo-immuno logical studies on conjugated sugar-proteins,
The specificity of antigens prepared by combining oO and 8
glucosides of glucose with protein,
The synthesis of glucosides of the disaccharides, cellobiose,
and maltose.
Studies on the interconvertibility of the specific types of pneu-
mococcus,
The degree and duration of active and passive immunity to pneumo-
coccus in rabbits.
There follows a general account on the {mmuno-chemistry of the
pneumococcus, which is presented in place of the more detailed and technical

descriptions contained in the report to the Scientific Directors.

Specific carbohydrates in pneumococcus infection (Oswald T. Avery).
San important advance in the study of infectious diseases came with the know.
ledge that pacteria, though simple in form and structure, exhibit differences
in biological epecificity as sharply defined as are those characteristic of
the more complex forms of life. The study of the {mmno logical specificity
of microorganisms is not only necessary in the elucidation of the biological
relationships existing between varieties of the same species of bacterium,
but is essential to the working out of epidemiological problems and to the de-
velooment of methods useful in the control of infectious diseases by specific
therapeutic and prophylactic measures.

‘Leaving out of consideration the promising but difficult field of

chemotherapy, the problems of specific cure and prevention of infection lie

 
423

63 ‘

 

in the attempt to interpret and imitate by artificial procedures certain pro-
tective processes of nature which constitute that which we call immnity. In
order to imitate successfully the natural processes involved in spontaneous
recovery from disease, it is necessary to know the nature of the svecific re-
actions between the infecting agent and the body tissues of the host. The
specif icity of these biological processes is advantageously studied by means
of the so-called immini ty reactions. These serve as a measure of the capaci-
ty of the animal organism to produce protective substances, and they afford
a means of studying the interaction between these specific antibodies and the
infectious microorganism when both are brought together in the animal body or
in the test tube.

"Investigations on the specificity of these reactions have added
mach to our knowledge of the many and diverse problems of infection. The
study, however, 4s one of varying complexity; the methods suitable in one in-
stance fail utterly when applied to another type of infection. While the mil
mechanism of the interaction between host and parasite has to 4 certain ex-
tent been exposed, the immediate problem dies in reconstructing for each mi-
crobe a more precise knowledge of the biological properties peculiar to it
and the specific reactions which the body develops against it. One approach
to this problem is the attempt to relate specific differences in biological
behavior to fundamental gifferences in the function and chemical composition
of the component parts of the bacterial cell and to determine the character

of the tissue responses to these separate constituents. The total immune

 

response of the host comprises not alone a reaction to the parasite a8 4
whole, but, in addition, the specific and individualized responses to the

chemically distinct and immunologically specific bacterial constituents. i

 
a)

 

64

"For the past several years we have been seeking to acquire a more
intimate knowledge of the relation between the immano-chemistry and the bio-
logical activities of pneumo cOccuss the most frequent and one of the most fa-
tal of the microbic incitants of pneumonia in man.

"JZ shall review one phase of the studies, namely, the role of spe-
cific carbohydrates in pneumococcus infection and immnity. Renewed impetus
was given to the study of pneumonia by the working out of the biological clas-
sification of pneumococci which made possible the recognition of sharply de-
fined and specific types within this previously confused species of microor-
ganisms. This has made possible the determination of the frequency of occur- ‘at
rence of specific types of pneumonia, and the recognition of differences in
the severity and mortality of the diseases they produce. A study of the pres~
ence of pneumococci in the mouth secretions of healthy individuals proved the
dissemination of the disease-producing types by healthy carriers and convales-
cents and suggested a new interpretation of the epidemiology of the disease,
while knowledge of type-specificity among pneumococci provided the only ra-
tional basis for the possible development of specific treatment by immune
serum, which in the case of type I infections, at least, has proved of dis-
tinct value.

tPpevmococcus ig a unicellular microorganism which is surrounded
by an envelope of material known as the cell capsule. This capsular layer ‘is
particularly well developed in the case of pneumococe! ® capable’ of growing and |
multiplying in the animal body. During growth these encapsulated cells ‘elabo-
rate in the medium of their environment 4 aiffusible substance which in solu-

Sle form retains the tyoe-svecificity of the bacterial cells from which it is

 

derived. This soluble specific substance is found not only in the filtrates

of young cultures, but also in the body fluids of animals exverimentally in-
A25
65

fected, and in the blood and urine of patients during the course of pneumo-
coccus pneumonia. The function of elaborating this specif ic material is most
highly develoned in the most virulent organisms. There are grounds for the
yelief that the capsule of these virulent cells is commosed largely of this
soluble specific substance. Thus, there is disposed peripherally about the
cell an outer layer of capsular substance woich reacts svecifically with the
serum of immune animals. ‘The reaction is remarkably specific, occurring only
when the antiserum and the reacting substance are both of the same specific
type. These immunological reactions form the basis of the original classifi-
cation and were worked out before there was any ynowledge of the chemical na-
ture of the substances upon which tyve-svecificity depends. The actual isola-
tion of these specific substances in purified form, the determination of their
chemical constitution and their relationsiip to the 4mmuno logical properties
of the cell as a wnole are problems to whicn I shall direct attention.

ithe type-specific capsular substances of pneumococcus, first
chemically isolated by Dr. Heidelberger, have been found in each instance to
velong to the class of sugar-like substances, namely the carbohydrates. No
matter from what type of oneumococcus these specific substances are recover-
ed they all possess in common the chemical properties of complex sugars -~
tue polysaccharides. ° Sut, interestingly encvehy the caosular polysaccharide

derived from each gvecific type of organism is chemically a@istinct, each: pos—

sessing unique chemical properties which serve to differentiate it sharply. 7 -

from the others. ‘Moreover, solutions of these capsular polysaccharides, in.
chemically purified form, exhibit immnologically the same specificity as do
the bacteria of which they originally formed a part. Some idea of how re-

markably reactive these sugars are may be judged from the fact that, by the

use of an appropriate serum, their presence may be detected in dilutions as

 

 

 

 
high as 1:5,000 ,000.

66

ta study of the chemistry of the capsular polysaccharides has

snown that these substances ar

e unusual compounds of simple sugars and uronic

acids. Although possessing many properties in common, they exhivit character-

{etic differences. For exampl

e, of the specific substances of tne first

three types of oneumococcus, the type I polysaccharide differs sharply from

the other two in containing ni

in possessing basic as well as

trogen as an integral part of the molecule, and

acidic properties; on the other hand the type

II nolysaccharide is a dextrorotatory weal acid and the type III a levorota-

tory strong acid, neither of which contains any nitrogen in the molecule. The

fact that the particular const
ly a carbohydrate is the more

been considered exclusively th

ituent determining type-svecificity is chemical-
striking since immunity reactions have hitherto

e function of proteins. Of equal importance is

he fact that this selective specificity 4s in each instance determined by

the chemical constitution of the particular polysaccharide in the capsule and

that the presence of this morphological structure conditions both the inva-

siveness of the parasite and the immune response of the host.

"The fact that poly

saccharides elaborated by bacteria growing in

the focus of disease may be found in the blood and urine, unchanged in spe-

cificity, indicates that the body possesses no enzymes capable of breaking

them down into simpler sugars.
terial sugars as_such are dire
ing the infection. So far as
not in the sense of true bacte
indicate that indirectly at le
natural processes of recovery.

tibodies, they tend to neutral

There is no evidence that these complex bac-
etly responsible for the intoxication accompany
is known, they are not primarily toxic, at least
rial toxins. There are facts, however, which
ast they may have a harmful effect upon the

Because of their specific capacity to bind an-

Sze the immune substances in the blood and thus

 

 
427 |

67

prevent the protective antibodies from reaching the infected areas. Moreover,
the capsular polysaccharides are known to exert an inhibiting action on phago-
cytosis, one of the most important cellular defenses of the body against pneu-
mococcus infection.

“Theoretically, at least, there is no apparent reason why these
complex sugars as isolated substances should not by themselves be capable of
stimulating the formation of antibodies in the animal body. Assuming that
they are of sufficient molecular size, they possess in complexity of structure
and colloidal behavior certain vroperties generally considered essential to
the antigenicity of proteins. Indeed, Francis and Tillett have found that in
the present state of purity the capsular polysaccharides are capable of incit-
ing antibody production when injected in minute amounts into the skin of con-
valescents and normal individuals. However, with few exceptions all attempts
to evoke any immune response in animals with the highly purified polysaccha-
rides alone have been uniformly unsuccessful. On the other hand, the more 1b
these carbohydrates are chemically purified, the more reactive they become in
the specific serum of immune animals. Under these conditions it appears that,
removed from the bacterial cells, the capsular polysaccharides still retain un-
impaired the property of binding with antibodies, although in this form they
become quantitatively less active in stimulating antibody production in ani-
mals. In this respect they may rightly be included in the group of immano-
logically important substances which Landsteiner has called haptens, —~ sub-

stances which have lost more or less completely their antibody-stimlating

 

function without impairment of the property of specifically combining with
antibodies.
tte elaboration of the capsular polysaccharide is an important

fonction of the cell. When this function is suppressed or inhibited, as it

 
428

68

may be under certain experimental conditions of growth, the capsule is no
longer formed. As a result the organisms lose their type-specificity and ex-
hibit only the common, undifferentiated characters of the species. On the ba-
sis of colony differentiation these degraded organisms are spoken of as the
"R" or rough forms, and the original encapsulated types are referred to as the
smooth or "S" organisms. The unencapsulated R forms of pneumococci, irrespec~
tive of their type derivation, are no longer capable of invading the animal
body; they have lost their virulence, and are readily taken up and destroyed
by the phagocytes of the host; immunologically they exhibit only the species-
snecificity common to all the degraded R forms of pneumococcus. This trans-
formation resulting in a loss of specific characters may occur in the animal
body as well as in the test tube. However, these degraded, avirulent variants
do not necessarily remain the harmless saprophytes they were originally
thougat to be, since it is now known that under suitable conditions they may
regain all the specific characteristics that distinguished the original para~
gitic type from which they came.

"of even greater piological interest 4s the phenomenon of the inter-
convertibility of the specific types of pneumococcus. Griffith of London first
showed experimentally by a special technic in mice that R forms derived from
one specific type of pneumococcus may be caused to acquire the characteristics
of another specific type. This important fact has been confirmed. by a number
of investigators. In addition, Dawson and Sia by special cultural methods,
nave found that the actual change from one specific type of pneumococcus to
another may be brought about in the test tube outside the animal body.

‘The experimental evidence now available seems to indicate that any
R strain of pneumococcus has potentially the function of elaborating any one

of the specific cansular polysaccharides; —~ the particular one being deter-

 

 
lll" ~
429

69

mined by a particular stimulus of a specific nature. Alloway has recently
found that this potential function latent in the living R cells may be specif-
ically activated by the addition to an appropriate medium of a bacterial ex-
tract prepared from a given specific type of pneumococcus. Under these con-
ditions, the R forms irrespective of their type derivation again elaborate @
capsular material identical in specificity with that of the type of pneumo-
coccus from which the ‘extract was prepared.

“There is at present no certain proof that transformations of this
kind ever occur spontaneously in nature. Nor is there as yet any epidemio-
logical or clinical evidence that this form of reversible adaption is a factor
in the origin of human infection. However, the experimental evidence leaves
no doubt that the non-invasive, non-encapsulated R cells under favorable cir-~
cumstances are potentially capable of again developing into highly virulent
organisms, and that tne acquisition of virwlence is invariably associated
with the restoration of the function of elaborating the specific capsular car-
bohydrates. Indeed, it is most significant that no matter whether one con-
siders pneumococcus from the viewpoint of virulence, antigenicity, or its ca-
pacity to undergo variation, the single determining factor associated with
all these characters ie the function of synthesizing the specific cansular
polysaccharides. Scarcely less important is the fact that the immuno logical
specificity of each of the specific types of pneumococcus depends upon the

chemical individuality of the particular carbohydrate in the cell capsule.

 

‘ag chemical substances, separate and apart from the bacterial
cells, these carbohydrates have been found to incite specific reactions in the
tissues of sensitized animals and in the sicin of patients convalescent from
pnevmococcus pneumonia. Guinea ples passively sensitized with the precipitat-

ing serum of an immune rabbit suffer violent anaphylactic shock and die within
430

70

three to four minutes following the intravenous injection of as little as .055
mz. of the homo logous polysaccharide. The anaphylactic reactions are strictly
type-specific. There is now ample evidence to support the view that protein-
free, even nitrogen-free, carbohydrates may induce acute anavhylaxis in specif-
ically sensitized animals.
uP llett and Francis have found that the injection of 0.01 mg. of

svecific polysaccharide into the skin of patients recovering from pneumococcus
pneumonia may evoke an inmediate local reaction in the form of a wheal sur-
rounded by a zone of erythema. The cutaneous reactions develop rapidly within
fifteen minutes and subside completely in from one to two hours; they are
elicited only by the specific polysaccharide derived from the same type of
pneumococcus as that causing the infection in the vatient. The capacity of
the skin to react to the specific bacterial sugar is intimately associated
with recovery and closely parallels the occurrence of type-specific antibodies
in the patient's serum. The results indicate that this specific skin test has
prognostic significance and may become of value in determining the therapeutic
dosage of antipneumococcus serum.

studies on ‘synthetic antigens' prepared by chemically combining
derivatives of glucose and galactose with proteins have snown that even these
simple sugars exert a determining influence on the immmological specificity
of compounds of which they form a part. The newly acquired specificity of

these artificially conjugated sugar-proteins is in each instance determined

 

by the chemical structure of the carbonydrate irrespective of the protein to
which it is attached. It is especially significant in the case of glucose

and galactose that the two sugar derivatives differ from each other only in
the spatial arrangement of the hydrogen and hydroxyl grouns on @ single carbon

atom. Itisa remarkable fact that the mere rotation of this carbon atom

ee sean | sneer nea inmate netted FT A

 
—
481

through an angle of 180° suffices to change completely the antigenic specifici-
ty of two substances otherwise chemically identical. In the case of these ar-
tificially prepared sugar-proteins, the two isomeric sugar derivatives can be
selectively differentiated one from the other by serological methods. These
observations on the immano-chemistry of carbohydrates confirm the original
studies of Landsteiner on the specificity of azo-proteins and furnish addition-
al evidence of the general dependence of immunological specificity upon the
chemical constitution of the reactive substances. It is evident, therefore,
that simple sugars, which by themselves are non-antigenic, may, when coupled

to a protein, specifically determine the immune response of treated animals,
and that the antibodies thus engendered reflect the orienting influence of the
sugar radical on the specif icity of the antigen as @ whole.

‘prom these results we were led to test the possibility of 'synthe-
sizing! an artificial bacterial antigen. For this purpose the capsular poly-
saccharide of type III pneumococcus was chosen, since it contains no nitrogen
and in its present state of purity may be regarded as & definite chemical en-
tity. Moreover, if results were obtained with this particular polysaccharide
they would be the more significant, since the free substance by itself has
never been found to elicit any immme response in rabbits, and even the ori-~
ginal bacterial celle from which it is derived fail ina majority of instances
to incite specific antibodies in these animals. From & chemical point of view,

the difficulty lay in synthesizing the appropriate derivative of this comp lex

 

 

sugar. It mst be one capable of being coupled to protein and one in which
the chemo-specific groups of the polysaccharide are not masked by the chemical
procedures. Dr. Goebel succeeded in synthesizing the amino-benzyl-ether of
the type III polysaccharide and in coupling the dilazonivum derivative with a

foreign protein, namely, the globulin from horse serum. This soluble antigen

 
72

therefore has in common with type III pneumococcus only the specific capsular
polysaccharide. Rabbits injected with this artificial antigen uniformly de-
veloped in their serum type-specific antibodies. The antiserum thus produced
not only precipitates the original polysaccharide, put agglutinates living cul-
tures of type III pneumococcus and protects animals against infection with vir-
ulent organisms of the homologous type.

"Ynowledge of the chemical nature and significance of the capsular
polysaccharides in pneumococcus infection and immunity led us to search for en-
zymes capable of decomposing these’ specific carbohydrates. A mumber of en-
zymes of animal and plant origin as well as cultures of various bacteria,
yeasts, molds and soil actinomycetes, many of which were known to decompose
cellulose and other complex carbohydrates, were tested without success. My
associate, Dr. Dubos, {solated from peat soil a bacillus which possesses an
enzyme that acts specifically on the capsular polysaccharide of type III pneu-
mococcus. From these bacilli the active enzyme has been extracted in soluble
form. By technical procedures, active preparations of the enzyme have been
purified and concentrated without appreciable loss in potency.

| "Tn view of the marked aifferences in the chemical composition of
the various capsular polysaccharides, it is not surprising to find that the
enzyme decomposes only the type TIT substance and has no effect upon any of
the other bacterial sugars thus far tested. In this respect, the selective ac~

tion of the enzyme is as specific as in the immne reaction between the type

 

III polysaccharide and ite homologous antibody. fhe polysaccharide acted upon

 

by the enzyme loses its serological specificity and is no longer precipitable
by type III antipneumococcus serum. This enzyme not only acts on the chemi-
cally isolated sugar, but it specifically decomposes this substance in the na~ Oy

tive form in which it exists in the capsules of the living cells, ‘When a
433

73

sterile solution of active enzyme is added to a growing culture of type III
pneumococci, the organisms lose their specific agglutinability and the soluble
capsular polysaccharide is serologically no longer demonstrable in the culture
fluid. Under these conditions, the enzyme decomposes the capsular substance
as rapidly as it is formed without impairing the viability of the decapsulated
organisms.

“The action of the enzyme does not result in a loss of the function
of elaborating the capsular substance, since pneumococci so treated promptly
regain their capsules when transferred to an enzyme-free mediun. The active
enzyme, therefore, represents a specific agent which by itself is neither bac~
tericidal nor bacteriolytic but which, by decomposing the capsular structure,
completely alters the biological behavior of the bacterial cell.

"In view of these findings, experiments were carried out to deter-
mine whether the enzyme would favorably influence the course of experimental
infection in mice with type III pneumococcus. It was found that a single in-
jection of an active preparation of enzyme protected mice against infection
with a million times the number of virulent organisms invariably fatal in the
untreated animals. The protective action of the enzyme is type-specific; just
as in the test tube 1t decomposes only the type III polysaccharide, so in the
animal body it is effective only against infection with type III pneumococcus.

"Experimental evidence indicates that in mice the enzyme also has —
a carative action when administered in the course of an infection already well
established at the time of treatment. The administration of the specific
agent as late as eighteen hours after the onset of infection has brought about
the recovery of mice infected with multiple lethal amounts of a virulent cul-
ture of type IIT pneumococcus. Experiments carried out in collaboration with

Dr. Goodner and Dr. Dubos have shown that the enzyme has also a marked curative

 

 

it
iu

 
Se

434

74

action in the disease brought about by infecting rabbits intradermally with a
highly virulent strain of type III pneumococcus. The experimental disease is
characterized by the rapid development at the site of inoculation of an in-
tense inflammatory lesion with spreading edema, marked cellular infiltration
3 and hemorrhagic necrosis, accompanied by fever and the early invasion of the
blood stream with increasing numbers of pneumococci. The infection ordinari-
ly terminates fatally within three to four days in 95 per cent of untreated
rabbits. Following the intravenous injection of an adequate amount of active
enzyme, the bacteremia promptly disappears; the local lesion, freed of bac-
teria, undergoes the natural processes of healing, and recovery occurs in 95
per cent of the animals so treated.

"The experimental results support the view that the primary action
of the enzyme lies in its capacity to decompose the capsular polysaccharide
of the invading pneumococci. The prpqess of decapsulation brought about by
the direct action of the enzyfme strips the bacteria of their capsular defense
and thereby exposes their naked and unprotected bodies to direct attack by
the phagocytes of the host. Thus phagocytosis, ineffective against the en-
capsulated forms, now becomes the important mechanism in the final destruction
of organisms from which the capsular substance has been removed by the action

of the enzyme.

"Tn this sense, the enzyme may be said to initiate a protective re-

 

action, the successful issue of which depends upon the effective phagocytic
response of the host. For these reasons, it at once becomes apparent that
the curative action of the enzyme is subject to the limitations imposed by the
variations that occur in the cellular defense of the infected animal.

“these studies suggest that the capsule -- long recognized as a

defense mechanism on the part of virulent bacteria -- is a decisive factor in
435
75

determining the fate of pneumococci in the animal body and that this structure

4s vulnerable to attack by agents other than specific antibodies."

Studies on Hemophilus influenzae (Drs. Cole and Pittman). The
study of bacteria of the genus Hemophilus described in the last report has been
continued. Strains of all species isolated from the respiratory tract are
still being studied; the greater emphasis, however, has been placed on an in-~
vestigation of the {mmunological relationship of S$ and R variants which were
derived from these S strains, the production in a horse of a specific anti-
serum egainst Type b bacilli, and a method of determining the protective po-
tency of this antiserum.

Type b H. influenzae antiserum has been produced in a horse. Sev-
eral hospitals have been supplied with the serum, and a few patients suffering
with influenzal meningitis have been treated with it. While the results have
thus far been unsatisfactory, it has not been used in a sufficient number of
cases to form definite conclusions concerning its efficacy. At present
strains from 22 cases of influenzal meningitis have been studied, and they
have all been of Type b with the exception of one, which was not type~specif~
ic and which produced rough (R) colonies.

That the Type b antiserum contains a protective substance has been
definitely shown by the prevention of the lesion (injuries) which Type b or-
ganisms induce in .the skin of rabbits. This protective substance can be quite
accurately measured. ‘The smallest amounts of the sera from the three large
successive bleedings that would protect against the "standard skin lesion
dose" were, respectively, 1/200, 1/600 and 1/800 cc. The "standard skin le-
sion dose" of culture is ten times the smallest amount of culture which will
induce a lesion that remains positive for three days. It is of interest to

note that the proteetive titre of the serum has steadily increased during a

 

 

 

 

 
—eeeeeeeeeeeee
436

76

course of immnigzation of more then a year. The serum is apparently type-
specific in its action, since it only prevents the skin lesion induced by
Type b organisms. fhe development of the lesions following the inoculation
with other type-specific strains and the R variants is apparently not in-

q fluenced by the immune serum.

(prs. Rivers, Berry, Benjamin, Sprunt, and Schwentker). Patients
with a variety of infectious diseases have been admitted to the ward for clin-
ical studies, particularly in regard to the changes 4n the blood picture as .
revealed by the supravital method of staining, and for the purpose of obtain~
ing material with which animals and tissue cultures were inoculated. The case
of agranulocytosis proved to be of interest because it occurred following ton-
sillectomy and responded favorably to intravenous injections of adenine sul-
phate.

Cultivation of vaccine virus for use in man, In a previous report
a simple medium for the artificial cultivation of vaccine virus was descerjbed.
The results of the work indicated that vaccine virus is capable of maltiplica-
tion in the presence of minced chick embryo tissue suspended in Tyrode's solu-
tion, The experiments, however, were conducted with a neurovaccine virus,
and, although the active agent obtained caused typical vaccinal lesions in
‘> rabbits, it was deemed best not to test it in human beings. To obtain a cul-
ture virus for Jennerian prophylaxis in man, it seemed advisable to adapt a
dermal strain of vaccine virus to our method of cultivation. The report deals

with the results of this work.

 

Vaccine virus, Lot 611, prepared by the New York City Board of
Health was used to initiate the cultures. Having rid the dermal vaccine vi-

rus of bacteria it remained for ws to determine whether it was possible to