ma Doc Ho 55386 P mmih HISAPQDAH'mftS • fab ttsr cornu® Uffutm mm&mm aiCTiou, obhdull staff/ ALLISP mUttLATOF $M SSCTIOB Translation Bequested by Theatre Intelligence, Target# Pate Be*14 AXIS 11 Feb 50 ascription of Contents* Foil translation of “Serological Studio# on Supersonic Have-Treated Polyvalent Cholera Vaccine,* by Army Medical College Spideaio- loflcal Laboratory, 16 Jun 42, Doc l?o 55366 0 Arey Statical & oil ago £pida« i *logical F.oaaarah Report Section 2. Jluafear 377 darolaglcal Stadias on f;av«-?raatad Polvvalsnt Cholara Tacclna ATVf Radical Collaga Xpidaa* olagy takorat'iry (r*J Om I3TII1, Caaw«&iliir) mm$ f9k*&i KoE-offlcial staff Section St Original Copy Glassification 43S-4 342-38 F «c«iv©d 16 Jus 42 Hajor (ifeUeal) SAITO, RySichi, 1b sharps. Doc Ho 5538$ £ Table of Contents fismtml Chapter 1, Inoculation materials and tost procedure. A. Inoculation materials. B. Test procedure. 1* Agglutination reaction. 2. Complement fixation reaction test. 3. Bacteriolysis. A* Immunisation test, C. Inoculation reaction. Chapter II* Twit rssult*. A. Besult* af experimentation os various antibodies In healthy IWi Xm Results on healthy ag«lutinln. 2, Feeults on test-tube bacteriolysis. 3* Complement fixation reaction results, km Ismmlz&tion. twit. B. Conntitutlenal and local symptoa® following inoculation. Cm Serological observations following inoculation. X. Agglutinin productivity tost results. 2* Basalts of tast-tube bacteriolysis. 3m aeults on productivity of complement fixation product*. km Iirsunlaation tost results. Stummrj and conclusions. Bibliography. Doe Mo 55360 D GflMnl Studies on ths antigenic properties of cholsra vaccines have progressed remarkably in recent years. Particularly outstanding have been those achievements spurred on by tbs rseent war. When compared to vaeeinss of the past considerable improvement is seen la antibody production especially in the reduction of seeondary effects! the antigenic properties of various antigen types are stronger. The supersonic wave*treated cholera antigen Is an antigen prepared by subjecting the cholera bacteria to the action of supersonic waves and thereby destroying their cells. Publications on supersonic wave*treated antigens are too numerous to mention. As the results on their agglutinin, bacteriolysis* production, complement fixation substances and immunisation strength proved superior to those of other antigen types, they will be elaborated below la tide report. Chapter I. Inoculation materials and test procedure. A. Inoculation material*i The teat vaccines consisted of the supersonic wave-tree ted vaccine (hereafter referred to ae f.S.V.) Mid the control cholera vaccine (hereafter referred to at K.V.) manufactured by this school. Laboratory personnel mere divided into two groups and inoculated with each of the vaccines. Comparative studies were performed on antibody productivity el thin the blood following the inoculation. 1. I’.S.V. Manufacture* The bacterial strains from which the polyvalent cholera vaccine was derived consisted of the Ishll, Ceguehi, Aka Tease strains for the original typeI the Hikojima and Chosho strains for tbs intermediate typej and the Dairen A7 and Ogawa 19 strain* for the variant types. A 10 BgTpsrxce suspension of each strain was prepared with a physio* logical saline solution aftsr culturing with agar (FH 7.6) at for 20 hours. Each suspension was subjected to a supersonic wave treatment <560 ke) for a 20-nlnute period. These were preserved in a refrigerator following a PH correction and were employed for the tests five days later. The suspensions were comprised of 120 ee of the Xshli strain, 40 cc each of the Geguehi, (Shanghai) Takiguohl, and Akatsuka strains, SO cc of the Inass strain and 20 cc each of the Hikojima, Cftosho, Dalrca A? and Ogawa strains. The bacterial content per cubic centimeter was 10 eg. The supersonic wave treatment time and the bacterial suspension concentration found to be west satisfactory in the previous experiment were adopted. Tlrulencs against else (± 12 g) was 0.3 eg. for the Ishii strain, 0.2 eg for the Hsguchi, Akatsuka and lease strains, over 0.5 m for strain, 0.3 m for the Hikojima strain, 0.2 mg for the Cb~aho strain and 0.2 eg far the Dairen iff and Ogawm strain*. 2. t. 7. nanufacturs* The He. 2 Laboratory was entrusted with the preparation of a polyvalent suspension consisting of a mixture of the nine strains described above. This was stored in a rsfrl germ tor after PH correction, 3. Test procedure# Laboratory personnel were divided Into two groups of five persons each (t.S.V. group and K. V. group) and given Inoculations of each vaccine type. The inoculation of toe Ho 55386 D the U.S.V. group was completed successfully but unfortuaetely oae person ia the K.V. group becas» ill and another retired froa the service during the course of the inoculation, * rlai presence of healthy antibodies van determined by takinv blood before the Inoculation. Preliminary testa on healthy sera canals tod of tha determination of agglutination, complement fixation test, test-tube bacteriolysis and ImamiEatioa teats* The first Inoculation aerie# consisted of A.C-a*? doses injected subcutaneously oa the upper are un 10 Apr 40; the second series (6.0-ng being performed on i£ April, derum van separated and readied for teats on tha ninth osy (25 April) folio** lag the second inoculation series* Antibody productivity following the injections ran examined by means of ag lutlniitlon reactions, coaplerant fixation reactions, test-tube bacteriolysis and irosunisation testa* 1* Arglutlnatloii reactiont The teat followed orthodox practices. Serum dilution started with a five-time dilution for the first test tube and ended with a I,2S0-tia» dilution. The anti vms were 1.0-ce and 0.3-cs bacterial suspension* of the Tehil, HikeJim and Ogar& strains each of which was cultured in agar for hour# at 37°C and diluted with a sterile physiolo- gical saline solution* On® cc of each antigen was used for the above serum. After being kept in an incubator for two hours at 37°T* and left standing overnight at room temperature the results of each were observed end evaluated. 2* Complement fixation reaction I rise teat was based m the Kobayashi method, Aa a preliaimjry test, tbs hemolytic titer and the aatigen*eo«plem«nt titer were measured. Hemolysins were of the goat series ooesessing a hemolytic titer of 6,4PO times. Four units were used for this test. The coiBpXement was derived by separating the sera taken ftm 10 marmots and used after standing for three hours at room temperature. The voluaa of the CCaploaeat to be used was measured each tims. Dilutions ranged from 10 to 16 tines. A test for antl- complswmtaTy effects was conducted before use. the use of the G,3,V, antigen followed the procedure outlined in previous reports. The nine bacterial strains were cultured in agar (PH 7.6) for 20 hours at 37°C and suspended in a physiological saline solution at a ratio of 10 mg per ee, These were treated with 560-fcc superscale waves far 20 stoat*** Bile was followed by the addition of 0,5 per cent carbolic acid and by refrigeration at The antigens displayed a self-retarding action1* at a dilution of eight tines when the fixation titer age.last inruns ©era was measured. It was proved that fixation ia Immune sera and ftsmn mrm was adequate. Teat sera were rendered inactive (56°C, 30 minutes) prior to th# test. In the wain test, 0,4 cc of the test serum and 0.6 cc of a physiological saline solution were placed ia the first test tube and 0.5 cc of a physiological saline solution was used ia diluting the contents of the second md subsequent teat tubes. 2 Dm Ho $$368 D One half cc cash of the antigen was poured into the second and subsequent test tubes and 0.5 ce each of the complement was added, storting with the first test tube, Those wore thoroughly shaken and placed 1» an incubator fop one hmr at 37**5. This was foilseed by the addition ef 1.0 ec of a 5.0 per cent sensitized bleed oell solution and a two-hoar incubation at The results were evaluated the following wonting after the oontents were stored in a rsfrifratss. 3. Test-tube bacteriolysisj The test sore were inactivated before use? the complement was derived from aarssot©. The bacterial solution employed was prepared by a 37°C, 20-hour culture and indi- cated a colony count ef 1,000 per cc when diluted to times with boi'illor in a Petri dish. Tide test was based on the Ssisser- Feebtberp method. The dilution for the first test tube was five tines, ending with a 2,560-tise dilution for the last test tube, ifce to a ehortage of initial blood sera all bacterial types could not be tested (test covered only the original type). Teels covering every typo were possible with the final blood sera. 4. Inramlsatlco teat* Tbs original, Intermediate and variant types were cultured la **sr for 20 hours, the developed bacteria were suspended In a rbjsiolsrlesl saline solution at a ratio of 0.7 my/0,4 co for the original type and the intermediate type and 0*5 mg/0.4 ee fer the variant type. Employing a tuberculin hypodermic syringe 0.1 co each of the test serum and 0.4 eo eaoh of the bacterial solution were drawn and intraperltoneally injected into a German nous* (12 #r). ?iee were observed for a three-day period. C. Inoculation reaction* Post-inoculation sywpton* of a constitutional nature such as chills, feverishness, vertigo, heaviness of head, general fatigue, arthralgia of the extremities, oppressive pains and pelvic pains and those of a local nature such as Inflammation, swelling, *sportfiinsoua p&lnse and oppressive pains were recorded. Tven syrptona occurring only once during the one-week observe t.ion period following the injections were recorded as being positive* Chapter H. Test results* A, fesclts of experlmstatioa oa various antibodies in healthy ssrumi reactions, bacteriolysis, complement fiction reactions nod iaammleation tests were performed op blood dr**wn from seven persons* the retained amount of anti- bodice is reported below* 1* Fssults tm hmithy ifglutinUt Tim afglu tins ties reaction results m the tntl% agglutinin from five T!*fi.V* cases •bowed for the original type,three casco positive at 5-10 tiass, two oases positive at 20 times, and one case positive at AD ties** Five eases of the variant type were positive at 5, 10 and 20 times9 fear eases at 40 times and two eases each at SO %»d 160 tines* The original and intermediate types were positive at 40 times and tike variant type at 160 times* healthy agglutinin was retained at.40 tires and bales but rarely at 160 times. (Sec 11 furs 2.) Agglutinin was absent In the f.V. or 1 final type. One ease of the Intermediate type was positive »t 40 times. One case Me No ?53*8 0 of the intermediate type m positive at AO timss. One case such of tha variant type was positive at 10-20 times ( ± )* Sxtrsmely •Mill amount# of healthy agglutinin were present, (See Figure 5.) 2. Results on teat*tube bacteriolysis* Bacteriolytic reaction* an tha T3.3.V* eaaoa war# limited to tha Initial aara of tha original typs* Tha results disclossd four cases indicating positive bacteriolysin at 10 tinea and t»o cases at 20 times before B.S.Y* Inoculation. The remaining two cases rare negative* Consequently, baetariolyain retention in healthy blood waa indicated at 20 tinea* Tha original and intermediate types of initial K.V. sera vara negative at 5 tinea* The variant type was positive at 5 tinea. Bacterial growth was unlimited in the other*. 3* Complement fixation reaction results* Complete hemolysis compared to the control was produced at 5 tinea* thro# eases vers positive at 10 tinea* i* Immunisation teat* The initial $*$•?• sera vara positive for two eases of the original type and mm ease of the intermediate type. The initial K.V. sera were positive for one ease of the intermediate type and tee ease* ef the variant type* In short, the defensive strength of mice against healthy ears was weak In tha ease of the Intermediate type and the variant type* B* Constitutional and loeal symptoms following inoculation* Secondary effects following the inoculations appeared to have been greatly influenced by the type and the individual characteristics of the antigen. The constitutional symptom following the first injection aerie# of B.S.V. were four eases of feverishness (approxi- mately 0.5WU00C) and general fatigue (all cases}* heaviness of tha bead (nervous symptom) was Indicated by three cases. One ease sash In the K.T. group showed general fatigue and loss of appetite. Heaviness of head and headaches (nervous symptoms) were Indicated by one cose each. The incidence of general fatigue and feverishness was higher than that contained in the initial re- port (July 1959)* local symptoms such as inflammation, swelling, "spontaneous pains** and oppressive pains were displayed by both vaccines* Secondary affects such as inflammation, swelling, "spontaneous pains" and oppressive pains were stronger compared to those shown by results in tha initial report. A sharp reduction In secondary effects compered to the first series was observed following the completion of the second Injection series. One case in the t.S.T* group developed inflammation, thirst, diarrhea and headaches when Inoculated following an attack by a cold. The others, however, indicated only general fatigue and heaviness of head (two eases}* Post-injection local symptoms such as Inflammation, swelling, "spontaneous pains" end oppressive pains were positive in every case. Consequently, both groups displayed, as a secondary effect, severe local reactions es well as constitutional symptoms such os general fatigue, inflammation, feverishness, headaches and heaviness of head, the Intensity of which decreased considerably in the second injection series. (See Table X.) The foregoing results Indicate the necessity for performing general studies on the detoxication of the B.S.T. vaccine* Injection mr In# First injection series BoM90iidt series Skiver- ing r«w iteeee Vertigo General Mi|u* loss of appetite Ihiret &Urrhee Arttwal- (d* (aU ;i ■■■ ties) los - K«0! He«&* Heart* #g «**!***» aou. MUNHN> JU'^g Vertigo fatigue Yiftilff of appetite IHtort l&eawfcea Irtoral- H<*.4 ate# He«v&» mm of he&d "Local's —r-^gr—r — — l^Shm^ am*&m lag ieo*WUI |3*i$&0* 11 «ive pair?# gi&(fo«r eiistrsssssfc— ties) SfcselX*- Xr£U&»» nation if4£ • ■ Hi i wppree- sive p&illQ P&ine** A — — ~h — 4 — — — — — — — 4 i 1 4- — : — — — — — - - i . j 4- IftoeuXfctlflQ B — — — — 4- — — -— — — — 4- 4— 4 14- 4 — r — — •— — — — -— — — 4~ 4- 4- 4- 8 C — — 4-- — -4- 1 — — — — — — — 4- 4- 44- -4- — — — — — -— — — —. 4- 4- 4- * 0 — — 4~ — 4“ — — — — — — 4- 4 4~ ■ ~j~. 4— — — — — 4- — — — — — — 4- > • — — 4- — 4~ — — — — — — 4- 4 4 4 4— — — 4— — 4- 4 4- — — — 4- •4_ —j— 4 4- to • J*n4 loaitlWB 0 0 4 0 5 0 0 0 0 0 0 3 5 9 $ 5 0 0 I 0 a 1 X 0 0 0 I 3 5 5 5 5 XOtal negative 5 $ X 5 0 $ 5 $ 5 5 9 a 0 0 0 0 % 5 h 5 3 4 4 5 5 5 4 a 0 0 0 0 Positive 0 0 m *o 0 100*0 0 0 0 0 0 0 60.0 MKMJ 100.0 icc.o 100.0 0 0 30.0 0 ¥2.0 ao.o m.0 0 0 0 m* o 60.0 loo*d 100,0 100.61 h 256.6 ret"ceri tAge negative 100.0 100*0 30*0 100.0 0 100.0 200.0 100.0 10D.0 100.0 30Cu0 40.0 0 0 0 0 3O&.0 100.0 ao.o 100.0 60.0 ao.o 90.0 300.0 100.0 100.0 SO.0 40.0 0 0 0 0 jl — — — — —— 4~ — — — — — — 4- -4 I-4- 4- — — — — — — — — ., f-.- ri n-^-- zr IT 4- tystoolm 0 — ■■■■ 4- ~' — ~v~- — — — _j— ■4- 4- — — — —. 4- — — — —- — — — -+- 4- 1 Total Fealtive 0 0 0 0 X 1 0 0 0 0 0 a t i I t a 0 0 # 0 X 0 0 0 0 0 0 0 2 2 —-J..-.— a a # WeMPWp Negative a a 2 a 1 1 a a a a t a 0 0 0 o a a a 2 I 2 a 2 2 a a 2 0 0 0 0 1 Milvi 0 0 0 0 50-0 50.0 0 o 0 0 0 0 100.0 100.0 UCJ0 300.0 0 0 o 0 50.0 0 0 Q 0 o“ 0 0 r ioo.o ioo.o K».o 106.0 JL Begetle* 100*0 100*0 100*0 100.0 90.0 50.0 100.0 100.0 100,0 1D0.0 100.0 — I0D.0 0 0 0 _ 0 100.0 100.0 100.0 10D.0 50.0 100.0 300.0 100.0 100.0 I00.0 300.0 100.0 0 0 0 0 Doc 3*o 553SS & Figure X* * jMg&owi mi4 mMMmi pwimi fcy cfcoXor* TO4&1&* and nM v*««ia»* Doc »o 55388 D Cm Serological observations following inoculation* The sec mad inoculation series followed the first serlea after a one-eeek interval. Quantitative tests wore conducted on the antibody productivity of blood takas a nook following the completion of the second series. 1* Agglutinin productivity tost results I According to OKITSD agglutinin for the 0.S.?. antigen indies tod mximm positivity st 900 tines on tbs seventh day and at 1,280 tivea on the tsnth day. The f.V,, however, was positive at 320 UaM for ths original, interstate and variant typos, differing narkodly fro® ths £.S,f • Ths following observations on agglutiulns aro classified according to ths antigen typo* a* tUS.V, Original typo* Five cases each positive at 5-320 tines; four eases at 640 timos end one case at 1,280 tines. Negative at 2,5dn timsm Intermediate typo* Five cases each positive at 5-160 tines; four cases at 320-640 tlf.es and two cases at 1,2B0 tines* He§stive at 2,$60 times* Variant type* Five cases positive at 5-640 times| four cases at 1,280 tima and two cases at 2,$60 times. (See ■Figure 2.) fe. K.Vm Original typo* Two cases positive at 5-320 times, negative at 640 tines* Intermediate type and variant type* Two cease positive at 5-80 tin*s and one ease at 160-320 time, Negative at 640 tioes» (Sea Figure 3,) The results of agential* productivity teats performed m the above are amnsarissA below* (1) Agglutinin for the U,S.F. was greatly aupsrior to that far the K.F, Agglutinin was positive at 160 tines In ease of the univalent antigen mentioned in earlier reports (April 1938, section dealing with inoculations). Agglutinin-production in the blood against polyvalent antigen was positive at 1,280-2,560 ties* which is the true titer for supersonic vave-trsated antigens, (2) The eera indicated mximm positivity at 160 times before inoculation and at 2,560 Unas after inoculation, an Increase of 16 times* (3) The initial I,?, ecra indicated positivity at 20 times. Positivity ms displayed at 320 times after inocula- tion, an increase of 16 tlms* (4) Annins t 320 times for £*?• antibodies the b,S.V. value me 2,560 times (eight times highest)• 0, Test results on ag lutinciicm titers showed, when combining the titers for three antibody types In the initial tJ.S,V. 6 Doc Mo 55388 0 sera, 12 eases each at 5*10 ties#, nine oases at 20 times, six oases at 40 times and two ease# cash at 80 and 160 times. Is the ease of the final sera, there were 15 esses each at 5*160 times, 14 ease# at 320 times, 13 cases at 640 times, seven case# at 1,280 time# and two cases at 2,560 times. The initial 8.V. sera shewed two eases at 5*10 time and one ease at 20 tisss, '"fe® final #er« Indicated six cases each at 5-80 times and four case# each at 160 and 320 times, d. figure 4 1# a graphic Interpretation of ag Intimation titers. The for the initial U,S,V. sera is between 5 to 10 times. ?hs ewrwe gradually descends fro? the point. The final mr& exatims® -m the sane level fro® 5 to l6o time# and descend mdrally, starting from 320 times and ending at 2, *60 times, (fee Tirana 4.) rigors 4, 4$clutination mfttfli r smalt# 5js 2J" ORAM Isz (1) - Initial E.S.V. ©era (2) • • Final tUS«?« cars (3) 0_._0 initial K.V, aera tl) o o Final **.?, sera The peak for the initial #era continue# frets 5 to 10 tUtt* before the «irwe deseed#. 'The final sera hold a pMl at 5-80 times, the enrr- dropping sharply at 16T-320 tlawt The ssntia&sas straight Hat indicated by 0,8,V, 1# a slrm of its superior prspsrtl#*, #. The Initial U.S.V. seram score for logarithmic exponent# of agglutination titer# is 12 points &t 10 ties#, 18 point# at. 20-40 tires, eight points at 80 tinea and 1? point# at IdO times or a total of 68 point#. The final serua score 1# 15 point# at 10 tine#| 50 polrta at 20 tlmss, 45 point# at 40 times, 60 point# at SO tin##, 75 points at 160 tines, 84 point# at 320 times, 84 point# at 640 tinea, 56 point# at 1,280 time# end 18 points at 2,560 times or a total score of 467 points. The Initial K.7. serum score la two point# at 10 times and three points at 20 tines or a total of fire points. The final serum score Is six points at 10 tlaaf, 12 point# at 20 times. 7 Do* Ho 55388 P I* point* at 40 Uim, 24 point* at 80 tia**, 20 point# at llO tint* and 24 point* at 32£ tin** or a total of 104 point*, Wban eoaparlng tfe# aoora* of n,5.V, and H.f.» tfe* fonsor 1* roughly four tie** hl*?h*r than th* lattor. (8** Figaro 5.) i 1 a 3 *1 li p j 1 6 is s |£ 1 in pi * a o <► | M . „ o \3> r 3> ft 3 3 18 £jS*)i t? J v > ? jr *j‘ s* & i! ? 1? |g| m |gf HI K 1 1 + « l+rlj Li*. #i£Pll 1 1 • e 3 g-1 1 1 r 1 1 I r sr f ol b ott 8 8uj IS ++4 iii fsf rf-1 f-4“4" i 4~+1 +4-4“ 44+ r “■ * o § b ot» 88k* tc . ■! ± ).. 4" t £ i + +HI ff i 4—(-- f g 08 • O ot« 1d8*o*>c. -j-^^ -f 11 -h i ‘ _ _ ; - ti i 4*4-4~ 4-41+ 8 Op oCJ b V* JS«* vN *111 f ff |+'4# £ 1 1 l*M 1 f 1 1 f f 1++11 s o§ • o H oG» gMffifmfi If++1 1 J _ 1 • . i 11 H-l i 111 f 11 8 O § ovn • o v* h* 0*WW | | • • v*> Kt “ J—r 3 p oi* o j o ■f 1 ! hr j 1 I 1 1H-H- + 1 1 i MINI 1 1 1 ; 1 ! 1 i 8G kit Cm 1 o»6J o |+ j | o lit 1 1 i | I ■ 1 II 1 11 III 1 1 1 i 11 r ## Km * o o oo o oo o III M 1 1 II 1 1 1 1 i m ii II 1 111 1 1 o o oo !g o*~ o oo c> b o oo oo o OO O oo o fT [ I O O O 0 m o o oo If p ooooooooocoooooo b o o O 0 g o o oo SownOOO *0*0 o o c oo c OOO OOO o H* O M 8 o o oo o So*- oo o o o o o o o • o OO o oo o j H it S o o j—' oo H o oo*- oo o * J o oo oo o OOO ! i H » 8 • ■ JM|-; - ? - .. ■ ' ’ g fS^| W(> W V* Ui On ' —r« ° b oocoooo0f'0°o0^0 H ■*— “~*“r ift m #*• 1 88 b b °bol?" °**H o o o o o o OOO OHO H | hi M J L ft 1 ss 0*& O OMH 0^»0HM u, f I M!0 O OM o w; m 1 NO OnU3 • • h «L.... • ts* *** s . _ & g °b0#~ £»> 8 i Q§ *° oK o ehSEw »»|Kp*V —i" 8 « 8 ,S «i o o OO o oo o oo o o o o o o o OOO OOO o o O O ? M 0 o o oo o oooooooooooo OOO oo o o o -4 O O b| 8 o • o vtO 1? o t* b tr* r* r’ rTI r m f* t* r* ir* v« & M ' ? o 8 • o Ovi c b,ow s** O X X . ._ m se sr P 1 8 o§ # o Okm . 8 pL* o n h ’ : is m m pi H | r4 - 8 €8 o o wv*» 8 owo r* * o . i « t** r* S _ 8€ ♦ • O O :■ ;■ O OM o tr « o m w in jr4 t*\ r» t* j tr* . V i h 8 20.0 ao.o 8 OM C tr • o r ■ | r I r*; tr* l’ O O OO o ow o r* ♦ o r* Ip* t4 Sr* V *• 8 In o o OO ■ 8 OXaS O ft* • o tr* tr* * t* r4 r* r* if o r\ B r*< r%Vaa- »r< • o ! l O o o OO , 8 ov* o r* • o r* r* t* r* r r* 1 o oi oo ooo© r» r« t*4' r*i' f* p* . g o o oo O OO O H r* t*1' tr* tr* tp* « -t r* fil og m c _ H Ov* 5 S» i i | 88 M II inn 1 1 + 1 II ii 1 5 1 o§ • o oK <3® M Vfw 1 1 T3M 1 1 nr i M l 1 1 1 + > 1; 1 1 c 1? # o 0^5* Cft w Ml ■.:•- i 1 K»» o a k\ 1 i-4 *4 *• t *A o c o »oo«oJi HI , k'i 1 *»l *4 *» i I i*-3 O g o « o g | >m3 *4 *4 *4 <« * o o » g O f* o g ||l *-]- ■ n j | M *4 *4 itt *4 O j o g j| 8[ *4 1-4 * 4 $4 w o o O go C4 O g li 1 i i4 N* *4 •** | ** O O' « $ > *4 Ml $ r- k$ *4 4*1 ogp n *r*< *s 3. c I | #4 *4 K o og- t4 4*4 O O ■ f | I «4 ** |l ttf <1 L o O Qf a O' : a |o| 1— < r — h #4 *4 hi |m ~J csr: o o gN o 5} ; |Ol « j- n *3 *4 ►3 : cv o a g« L o O j r| 1 ■1 | O O O O 0; o ooooo o ooooooo 1 ’ g | o a o o a OOO'O o o o o oooooo 18881*8 tl S?«8^P^'45i|>00 o _.o = 3 5 18a8ist= 1 o a * o 1 1 g«8i*s »RaiiiSf«rH . i O « 3 ii 3°; I 1 gw ill i?\ 5»w, ©31 o _ * o 9 J r ♦ # * * ! , . 1 f ' r , . 1 1 'T-; rn . *to*I b sm&i as fj r* f «* m'i 3 nRR: 4°WOHH«\r* J I sno • • 1S?S ! * * Kt j C1^ r n " I I I&9 ffi 4 **3 j o g *\«Jg i #}®;° ° ° ° ° ° i‘*vWi 0*CH0 ©s § r SR °. »f\ 44fj «oooeoao-*« • .o« o p _■ I | 1 1 1 1 1 1 MINI o< o o # * ! ► o go^og | 1 1 1 1 1 1 mini o< o > o o | M 1 1 1 1 i min o«* o r o o i O'OO t j 9 1 III III ill ill o< _ o > o j Q 1 mill + 11 1+4 OhC 0 o o > o •* \«n+• • 1 »a 1111II1 411444 o < o > o • 4* n • • r ft 1 1 1 III 1 ++4I+++ OnC i*a q SOO O * a Mini 444 444 O V« o q ij o gN© o g a ; 8 1 | +1 1 1 1 4-+44;++ ■Hu t*3\ nO V • »3C a© o i'Jo § j. a i++ 111 +++ +£4 ift JR5 8$ r c o § ° •* i ++ ii i ;+4 4 <-%vi .1 ; i L _ ■■ °. 4> O g O j i#f - ij ij ailllll llllll 1 + 1 1 /^v 4 1 Vy vy k\^ j+-1 ky v- 41 | iv* O f*. o 1 1 f* 1ft 1ft J |l Is It 3« s {•m S53SB I) Figure 3. Test resulfcfton K*V. inoculation. Doe Ho 55368 D Figure 5. Agglutination reaction exponential table Serna dllntio B {*) (-) 5 uo 20 40 80 160 T* 320 440 1280 2560 Total Wmtorn* positive Initial 0 12 12 9 6 2 2 0 0 6 oH • — a.s.*. Final 0 15 15 15 15 15 15 u 12 7 2 Nusber positIt® Initial 0 2 2 1 0 0 0 0 0 0 0 K.V. Final 0 $ 6 6 6 6 4 3 0 0 0 TUS.Va SCOT® Initial 0 0 12 IS 10 0 12 0 0 0 0 & Final 0 0 15 30 45 60 75 u H 56 10 447 K*Y. score Initial 0 0 a 0 0 0 0 0 0 5 Final 0 0 I 12 10 24 —i ■■■ j 20 24 0 0 — 0 104 2. Basalts of teat-tube bacteriolysis* A high bacterlolysin titer was the desired result in bacteriolysis but the growth of polyvalent anti gene proved to be inadequate for all bacterial types* The results are enumerated below* a* Bacteriolysis production in the blood following inoculations of C.S.Y. antigen was noted. Bacterial growth was indicated by one case of the original type at 320 tinea and by three eases at 640 tines* Bacteriolysis was not retained by five eases at 1,280 tines and above, the intermediate type showed one positive case at 160 tines, one ease at 320 tinea and three eaees at 640 tines1 all oases wore positive at 1,280 tines and above* Bacteriolysis production was detected in the blood following the R.Y. Inoculations. Bacterial growth for the original type was present in one ease each at 40 tines and 320 tinea. The intermediate type was positive at 20 tines (one ease) and 160 tinea (one case). The variant type was positive at 2 tines (TBs Sic)* Consequently, this vaccine possesses bacteriolysis at 40 tints. A comparison of the TJ.S.V. and the K.Y. is presented below. (1) The initial tJ.S.V. serum (original type) Is positive at 20 times and the final serum la positive at 160 times. Tha variant type 1* positive at 1,280 times. In short, the bacteriolysis production of the final stems la 8-64 tinea that of the initial serum. (2) The production Indicated by the original, intermediate and variant types of K.V, are four tines those of B.S.V. (3) The bacteriolytic titer of the intermediate types for both U.S.V. and K.Y. is one step lower than that of the original and variant types. 11 Doc So 55388 D (A) Tha values indicatod by the final E.S.F. sorun aro four tines higher than thooo of tho Initial soma. b. A graphic representation of tho bacteriolytic titere reveal* sharp fluctuations in tho Initial H.S.V. and tho Initial and final K.f. curves up to the 40-tins point. In othor words, bacterial growth occurs and bootariolysln production eoasoa beyond this point. On tho other band, tho bacteriolytic titor curve tar tho final U.S.V, aomn describes a stoop slope between tho 160- and 640-tins points. This shoos that the bacteriolytic titor of tho tf.S.V. Is ovorohsleliigly superior. (So# Figure 6.) Figure 6, Bacteriolysis results a*» QUPB Key (1) Initial Tf.S.V. torus (2) • Final fl.fi.V. sonm (3) °*~'° initial K.T. soru® (i) o---o Final K*?« soruffl o. Tho scores for tho logarithmic exponents of bacteriolytic titers are 172 points far tho Initial O.d.?* serum and 317 points for the final IUS.F, $ and 225 points for the initial K.V. oortm and 235 points for tho final mwwu Figure 7. Bacteriolysis exponential table dilution (*) 5 10 ao 40 80 1601 320 640 1280 2960 Fetal taafcar Initial 0 0 2 A A A A A A A poeltiv# final 0 0 0 0 0 1 3 9 » 15 inbnr Initial A 5 5 5 5 5 9 5 5 5 «•▼# Final 0 0 A 12 1A 20 2A m 32 3A C.S.T. Initial 0 0 A 12 16 20 2A m 32 36 172 Scot* Final 0 0 0 0 0 5 12 A3 66 136 317 K.7. Initial 0 5 10 15 20 25 30 35 40 A5 223^ ton Final 0 0 2 12 16 25 % A2 m 54 235 Soc »o 35m 8 3# lesults on productivity of complement fixation products* The euporlorlty of the supersonic wave-treated antigen in producing coaplanant fixing antibodies following ita inoculation baa already been established in reports published by this and other laboratories, however, shod lea concerned with fixation testa on polyvalent, wired antigens have not bssn reported. The comparative studies made on 0.8.V. and F.V. are described below. s. Three eases of the initial t.S.T. serum (five times central) ware positive at ten tinea. the final sent* (five tines control) was positive for five eases aseh st 10 and 20 tines, three cases at 40 times, two can©® st 80 tines and an* ease at 160 tines. Hemolysis was present in every ease with the Initial I.T. serum. The final serum (five ties* consol) was positive for two esses each at 10 and 20 times and for one case saeb at 40 and 80 times. The difference between 0.S.V. and K.7. was extremely alight, their ratios being 3*2»3.0« Post-lnoeuXation antibody production occurred at 170 tines compared to 30 times before inocu- lation. b. both 0.8.7. and 1.7. describe s sharp peak between 10 and 20 times when represented graphically. The 0.8*7. curve drops gradually fro® this point down to the 160-tiae point. The 1.7. curve drops abruptly, starting frost the 20-tins point and ending at the 80-tiae point. (See Figure 8.) Figure 8. Complacent fixation reaction reeulte 2|» Gum MX (1) •— # Final 0*8*7. esrusi (2) o—o Final K.7. aero® e. The scores obtained from the logarlthalc exponents of complement fixation reactions «r«f la the case of the final 0.S.7. ««nmt five potato at 10 tinea, 10 points at 20 tines, nlas points at 40 tines, #i$it points at 80 times and five point* at 160 times, or a total of 37 points. Th» scares for K.7. ara two points at 10 Uses, four painta at 20 times, thrao point* at 40 . Una* and four points at 80 tines, er a total of 13 points. the tf.8.7. in this respect la eoperlar. (8m 9.) Doe m 55m t> Figure 7. fwponentlal table of cowpleeent fixation titere S® yum dilution (*) 5 10 20 40 m 1«0 320 6i0 X2£0 2560 “~1 ratal MU *- ITOBwfr positive Initial 0 3 0 0 0 9 0 0 0 0 Fiml 0 5 5 5 3 2 I 0 0 0 Vtufear vtmiXXr* Initial 0 0 0 0 0 0 0 0 0 f) K.V. Final o 2 2 1 1 0 0 0 0 0 S.S*F. ue&m Initial a 3 0 0 0 0 <5 0 0 0 3 final 0 5 10 9 s 5 0 0 D 0 37 K#7* •CHflBP# Initial 0 0 0 0 0 0 0 0 0 0 0 Final o 2 4 3 4 0 9 0 0 0 13 —J 4* Immedmtiim test remits* «• Fo deaths occurred during the three-day observation period following the injection of sdeo with the fine I U.S.V. serum. In the test, one-unit (0.7 eg) end two-aait (1*4 »g) doses of the original type foiled to produce death* The amm recruits were ob- tained with equal doses of the Intermediate and variant types. Two isiee (variant type) and (TSJi Figure fleeing) nice (original end intermediate type) died on the flret day follewlnj..: the injection of final K.V. nn» b. One death each me produced on the first end second day with Initial tJ.S.V. sera of the original type* one death occurred on the third day with the intemodiate typo. One death per day resulted with each F.F. sstujs of the original, Intermediate and variant type. ■~Kree deaths occurred out of the eight cases treated with the fore- going initial tf.S.F. sera* ’’Time oat of sir cases died frora doses of Initial F.T. eera. The final F-S.F. seme meed ossa death out of a total of 15 casas* three deaths ont of sir eases resulted with the final K.V. sense. (See Figure 10.) Flgere V)* Itmmimlitm test results Observation period list day 2nd « fc*a.v. floai i 0 0 0 0 15 KtiEfcor positive Initial 3 0 0 3 6 K.V, Final 3 a 0 3 6 to ta Initial A X 1 6 u I Final 3 0 0 3 21 j Doe Ho 55388 D c* Through e graphic Illustration of the test results it will be observed that the Initial F.S.V. serum Indicates a value of 2.5 per cent (TKi Sle) over the entire period and that K.V. indi- cates 50 per cent on the first day. the value for the final $•£•?« serum ie negative, the final K.f* serum indicates 50-per cent fatality (three cases)« As a result the superior features of the 0.S.T, and the K.T. were determined on the third day of observation. (See Figure 11*) Figure 11* Xnmmiration test results 3* a* GRAFH III (1) 1st day (2) 2nd day (3) 3rd day (4) 1st day (5) 2nd day (8) 3rd day (7) Three-day period (8) Three-day parlod (9) Initial sera (10) Final sera (11) Initial earn (12) Final sera Summery and Conclusions Comparative experiments war© performed on the active antibody productivity of polyvalent O.S.T. and K.V* cholera antigens. The fact that antibod lac produced by the univalent tf.S.V. antigen are superior to 1,7. antibodies baa already been reported* Tbia has been established by the results of experiments on antibody productivity before and after inoculation* In the experiment the Inter-relationship between Idle antibodies (in the blood) of the Doc Ho 55388 D original, intermediate and itrilftt typos studied by means of agg lutination reactions, bacteriolysis, ©oroplemcnt fixation re- actions and immunisation tests* A* The secondary effects following an Inoculation of TT.S.V. vaccine are feverishness and amoral fatigue coupled with local reactions such as inflammation, swelling, "spontaneous pains0 and resolve pains* Approrisratsly similar result# are produced after second Injection aeries (on* case displayed loss of appetite, thirst and headaches as well). The symptoms displayed by K.T. cases are general fatigue, Xoes of appetite, headaches and heaviness of the head* Local re- actions Include Inflammation, swelling, 0spontaneous pains0 and oppressive pains* Reactions in the second injection series are practically confined to thosa of tho localised type. In view of the above results, tbs lessening of the secondary effects of tJ.S.V. presents an urgent problem. B. Healthy sera Indicate an agglutination titer of 40 times for the original and intermediate types and 160 tines for the variant types bacteriolysis at 20 timess complement fixation reaction at 10 time*} and positivity to immunization on tha second day. The mximm agglu tinla-antibody production for each final IT.S.V, serum type occurs at 1,280 tines (one ease) for the original type, 1,280 times (two casss) for the Intermediate type and 2,560 times (two eases) for the variant type* production in the ease of the K*V. serum occurs at 320 times (two cases) for ths original ’ype and at 320 times (one case each) for the remaining types. C. Bacteriolysin retention is observed In every type of initial r.S.T. sera at 20 times (two eases). Bacteriolysis is present in the final sera at 160 times (ona ease) for the original and variant types and at 80 times (one ease) for the intermediate type. The initial K.V. aerum Is positive at five times (one ease) and the final serum at ID times (six eases)* 0. Ths initial II.8.?* serum is positive si ID times (three eases) when tested for complement fixationi the final serum is positive up to 160 times (one case). The final K#T. serum is positive at 80 times. X. Immunisation testa reveal three deaths with initial tJ.S.V. sera but none with the final serai and three deaths each with the initial and final K*T. sera* This proves that the immunisation strength of H.S.?. Is high. Bibliography x. wmm and others, mvM mstrmrm mohimmn umm (Japan Journal of microbiology and Pathology) , Vol* 32, Ho. 10, October 1938* 2. mrml mmnf hibch Bixiimrmj m&iooui ussu, Vol* 32, Ho* 11, iovemfeer 1938* 3* rnBSHJ, I0H BM SUKXK6AXD (SixpUfUA Clinical Bacteriology), Part 0m* 4. mxmiCHi, *t«ujirof twm Ultmmm oimi mmmimm Do« No 553SB D (Modfina Bacteriology and Iraisunolofy) ♦ 5, KINDFA, Klyoahi, SATKINOATO MMtlQItt (Bacteriology and 6, Aray Group, amra SOSKIOAIt1 ROSZ (Array FioXd Spldosdolocy fcanuid). 7, ICHIJO, Taaokaoa and TOtAte, Army ?Sodlcal Collar tSpldoploloflcal Poaoarch Foport, Section 2f Subaaetion n7* 17