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Serological Studies on Supersonic Wave-Treated Polyvalent Cholera

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Serological Studies on Supersonic Wave-Treated Polyvalent Cholera Doc Ho 55386 P ma mmih HISAPQDAH'mftS • fab ttsr cornu® Uffutm mm&mm aiCTiou, obhdull staff/ ALLISP mUttLATOF $M SSCTIOB Translation Bequested by Theatre Intelligence, Target# Pate Be* 14 AXIS 11 Feb 50 ascription of Contents* Foil translation of “Serological Studio# on Supersonic Have-Treated Polyvalent Cholera Vaccine,* by Army Medical College Spideaio- loflcal Laboratory, 16 Jun 42, Doc l?o 55366 0 Arey Statical & oilago £pida«i*logical F.oaaarah Report Section 2. Jluafear 377 darolaglcal Stadias on f;av«-?raatad Polvvalsnt Cholara Tacclna ATVf Radical Collaga Xpidaa* olagy takorat'iry (r*J Om I3TII1, Caaw«&iliir) mm$ 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 germtor 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* A rglutlnatloii 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 action 1* 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&lns e and oppressive pains were recorded.
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