Vac &9 m 8^m , -U* K * y • *** V ASf , lutLtfm mmtmm* mmm, mmm* **mtj AUim Himm*mm km wtmm n* nwmm 2f«acl*Uon oooootoA % ti*«atrO Zatoll, forgot* 1>»U H#e*d '4fl8 U Itl ID Dooerlptton of Con to* tot Mi %rsnnXn%im of *l£ir]A«rljftMit«I t»»o«ro& on Start € JV«*ti**o of 8*$.w# 4feoi«p« V*oclaot • %ido*iolo«lo«l Moratory of Jonty ioAiool C*oUft8«a Iffco, Poe »o 533Smc Pedicel College Bpl deal ologl cel leanerch Soport Section 2. teeter If (Original Copy) Sxperloantel tee ear eh on foxle Fr«cttone of C.8, »r« Cholera Vaccine *edie«l Colleg# Ipldenialegloal Laborntory Colonel (eedieal) ISHII, ?hlro# Co«rs«ndlng Officer Laboratory We, X? TfCWIMO, BmnSi, on-official itaff Laboratory personnel Cept <*edlcal) WAIT?, *yoicM let tt (eedicel) SAWAlt fmaneo W0Of Talreehl, wt-offlclal ttaff Section J? Original Copy Classification Jh2 • JM S:f deceived 7 **0 y *oeber of Cop lee see Bo Table of Cantest* General Chapter I, statarials Cimpter II* TspariaentaX A. Yelidnsry taut* B. Hula te«t« I. *c«tlc add praaipltatloa treatment Aoetio acid praeloilata (A) 3. *c«t!c acid tolable fractiona Aeat-3*sa (1) 3. %dratad tMtCM *olobla fmnlm &thar extract (C) i£th«r l«i«llli« aahetaae* (D) Chapter III. Tlmleaea t#«tt an admit Chapter IT. A«h and ftlt*o*:«n content of fraction precipitate* Chapter T. Cfeterratioa* Bibliography u Boa so general i Scholars at hoae and abroad data that the true nature of bacterial toxin# can be found la proteins, particularly aueleoprotsinof and in carbohydrates. difference* and variations In the chemical • reportles of bacteria em be understood whoa it Is r«*ll?ed that their chealeal composition a* well a* their toxic properties vary with tho type of bacteria. the f»p|—CO advance* made recently on the study of bacterial carbohydrate* are coon* ad In# attention from other fields, > Ihc study of cholera toxin* cite ha* advanced, a* is evidenced by numerous publication* on the subject. Successive research on the virulence of carbohydrate* Is cholera bacteria are being : srformed, Recently KtJtOIA published hi* report on toxic proto.l* fraCtlsa*. A solution of the chemical properties of cholera toxin* will furnish sot only vitally Important bade data but will provide a guiding policy essential to vaccine aaocfaclure and preventive medicine* the specific mission of this laboratory I* to study supersede wave*treated cholera vaccines* Studio* on cholera toxin* haw been ex treacly rare. If supersonic wave-treated vaccine* arc to be improved in duality over tho old easier a vaccines it would ho Inadequate to ecUuteize merely the research concerned with the chealeal propertiee ef their toxins, full consideration nest be given toward providing Ideal condition* under which such studies cun be conducted. It has been reported already by this laboratory that the fc.B.W. cholera vaccine hoc proved tc be effective and hat produced excellent result* when examined for difficult insanity reaction*. Based on this, the first step in studying the toxins of the surersonic wave-treated cholera vecclue was the extraction of frectione by applying the relatively sicple precipitation reaction according to a fixed procedure. An examination of their toxic distribution revealed extressXy interesting signs. A part of the results will be presented la this report. Chapter I. lest materials A. Bacterial strain* fa# typo uaod In the experiment was tin XItool strain possessing a virulence of O.J ag. 1. faceIn* aamifactursl one vers prepared V adding the bacterlel culture (developed after ioeubetifig for 18 hour* at 37®C In an agar nedlu*) to a hycloloffital saline solution at the rate of 10 eg per cc. After subjecting the bacterial suspension tc the action of supersonic wave* (&00.000 cycle* r?#r second) for 15 oimttee. peptone and slant ««Xtares were cade to determine whether or not the bacterial cell* had been dsstroyed. those indicating cellular destruction were used In the experiment. Chapter II. Experimental procedure A. Prcliaiuary tests* la order to avoid the powerful precipi- tant reaction the *a*l«a» precipitation range at m fixed hydrogen ion concentration was determined by adding acetic acid or t rl chi 970*e* t Ic acid. Bo for* start lac the amis lasts the prel inlaary son bo 55 tests outlined b«lov w«rc completed* L Ibroolpl tetioa mage with acetic acid* argots of Pit a.o, 3.0. 3.5, Ko, Kb, S.o, 7.0 nad 8,0 were prepared with a Japan Pharmacopeia acetic acid solution. Tvo cc of each r««|ent was injected lute a sterile test tube stoppered with cotton* Aa enual toIumc (?,0 cc) of to*? bacterial *us. «B»iofi sa? rtdded tL* t«at tube* were ihakea thoroughly, After being allowed to stand for orer 18 honrt In a refrigerator or at rooa temperature the precipitation conditions war# eraelned. f Doc So it ?.o 3.0 3.5 Ko **•5 5.0 6.0 T.o s.o Room temp »ra tare (29.5®c) f \ _4_H_ Heavy precipita- tion at bottaa; top olear ill i -i i i ( \ ( ( > ( ) \' ) no chmxge rr+T Ixtremoly heavy precipi- tation at bottoia; top elonr ! 1 I Heavy oreclpita- tlon at bottom; top clear ~ T “ i-lght preelp l- tatlon at bottom; too turbid \ ' / Uniform turbidity / Uniform turbidity \ ) Uniform turbidity * / Uniform turbidity (—) j +~H I / ' > t. ) ( ) C ) Sefriterator ■—1— "i—r i V~ / \ ) \ J (h*c) Vo change bi*h% precipita- tion at bottoy; top turbid Heavy precipi- tation at bo t ton; top not eoMplote- ly clear Precipita- tion at bottom; top not completely clear Small amount of preci- pitation at bottom; top ooiae- what turbid Uniform turbidity Uniform turbidity Unifora turbidity Uniform turbidity Table 1. Preclpttatlo* results with acetic aclA of varying F8 values. (Decree of precipitation Indicated tgr-j- +f. 5 negative reclultatlon hy (-).) 3 3*« so Dm mtlH In fable I ehow * etrest idd agglutination** at ?l 3.0 - H,0 and e «U«r supernatant fluid* Vnlfvrm turbidity wee displayed at values «l«v« PH fc.O. the preliminary test v«» repented in order la detect p«MtU« errors la ia«t« results* She proper precipitation point wa* established at PS 3*S* 2. Precipitation rmgn with trichloroacetic »«IAi Dm trichloroacetic »c!4 test followed the «*m rmtdttr* ns that In the ae«li« »«iA ImI, litt appeared U indicate complete agglutination - prteiolUUmi In Si« PH 3*® * 3,4 range tt ran* tseparator© tat n |>oeitlve precipitation paint •nob as that resulting from the metis a©id test could not So attain**. B, Main tests* the onin tacts were oecmcneed after learntnit through preliminary tost* that collets agglutination - precipitation eonld to produced with * P% 3*5 eeetic sold solution* 1. Acetic add precipitation treatment* A 3&&»ec lapsilty separating funnel was fill** with 150 cc of Bacterial solution and 130 as of acetic acid solution* the mixture use allowed to stand after Bel no thoroughly shaken far several minutes. A cloud-like turbidity said partial precipitation resulted several Scare later* the greater portion precipitated after 2** hours | suspended particle# cessing turbidity gradually precipitated* this wet processed la a ‘ •kune type centrifuge on the third day* Approximately 10 ®<* *f the content was mmeured into each vessel and centrifuged at '4000 r.p.m* for 20 minutes. A slightly gray precipitate (l) and a transparent supernatant fluid wsre obtained. Acetic acid precipitate (A)i fen tine# by velum of alcohol wee added to the centrifuged precipitate (l) which was obtained through acetic acid reel citation described above* Bits css Allowed to stand In a refrigerator for three days, after which the elconol wee replaced. A snail velum of ether was added to the scdittwutt by centrifuging* an* auction filtration cat performd. A black powdery substance vac obtained us?on drying the eedlacntc* field was OAPM «• ?. Acetic mid soluble fraction* Acetone precipitate (B)i the supernatant fluid (IS), Measuring ep rovlaately 53b so, which renalned after the nestle acid precipitate was removed by centrifugal separation, wee placed In an evaporating dish end evaporated with the aid of an sloetrie fan. Precipitation was effected with acetous after the fluid had beer condensed to a volum of ubout $€ oo« Ibis wee allowed to stand la a refrigerator for e pU-hour period, Die supernatant acetone was removed by decantation. A suitebls to lam of acetone vmc added lamdlately to the sediments, this was stirred and the sunernetant fluid end sediment* (grey powder) were separated by euctloa filtration* the sedlneats were washed first with acetone and then with ether end dried la e vacuum deeelcator ever phosphoric anhydride, fhe tube tame was a grey powderi yield wee 0*fSf6 g* 3, Hydrated acetone soluble fractions* the sunermtaal fluid obtained by decnatation end the filtrate remaining after * no?, m 55i«ii suction filtration were eoabined aad evaporated, Btfeer vac added to the alnnte yield of sedlaeute, This w%« etirred and extraction was allowed to continue, Bwo fractions, the ether extract and the ether Ineolufele eedleant, were obtained after filtration. Ither extract (G)» A suall quantity of a %!*•&, reboot sediaent wfea produced w«er other waa nddad to the ether eolufele portion end evaporated with an electric fan. Held vac eerely 0.00t6 f, Ktner ineolufele sediaeat (B)l The ether insoluble eedleant wa» dried lute a powder, k yellowleh-erey ureeipitafce wee obtained; yield was Q.ftM (• Charter III. Tiruieacc teete on enlaale. A, Ueraan alee weighing 10 - IT f each were in looted for the virulence teete. tire elee couarlsed m grow*. B, Seat arterial* coneifted of 0,0? g each of the different powders dissolved In £♦> cc of physiological saline eolation, C, Injection# of the teet eolations were made Intraperltoaeally In 0,1 cc, 0,? co, 0,”* cc, 0,h cc, 0.*£ co, 0,6 ec, 0,? cc, O.t oe, 0,® ce and 1,0 cc doses, left reunite were evaluated after cheer*lag whether the ale# fend #urrlv*d or died during the throe-day ported following *he Injection*, B. Tlrnlence toil results, iVeelpltato (A) produced death with a I.* «g dose (¥B,)• teetone precipitate (8) which was obtained fey precipitat- ing the condensed supernatant fluid with acetone indicated a virulence of l,f ug (M,L,D, was equal to that of the noetic acid precipitate). Unfortunately tfele substance (B) wmo not analysed. In all probability thie higher eoapcaad w«e pert protein and contained a eaell mount of »oly saccharides, fhe ether eolufele substance (C), which Before eToporatioo reunified ae o supeiuetaat fluid in*teed of precipitating whom acetone woo added, failed to produce death with a 7.0 ng do**, fhe ether Insoluble portion (0) froa (C) did not cau«# doatll with a 7.0 m doee. ChapHr IT, Aeh and nitrogen content of fraction precipitate*. For the parpone at grasping a general picture concerning the toxicity of fraction preclpiteteo free the U.S.#, antigen, Acta on chemical properties wore obtained fey a*nearing the a*h and «itrosen contents of fraction* giving the highest yield*. Secau** of the »aalI yield analysis was not possible on fraction* other than the acetic add precipitate** 5 Sec to teenlto of mpmrimmt Acetic add precipltete (A) ntrot*» *9la*le stance $ &Ql$Lf consnap t lea it trogtm 6-73 m cc 10. 53* S*25 mg 3*28 oc lo.2?i tiio eth c onto at of the precipitate* wat 0.39 norcoat? the nltro 'tn contest of the organic »aHtr vac 10,Jg percent, *ltregen rdtto woe low compared to that ordinarily lad looted by proteine. Studiot on whetiftor they or* nncXeoprotolas or gl/eoordtolno and whether they coat an non-nitrogenous setter or e substance each ae « pol/eeeehftride «r« expected et » later dote. UROXA reported e at tregen mine of epuroxSaetely 13 percent for a toxic eubotance contained in a protein precipitated free an antolyelo prodaot of cholera bacteria, \ Kther extract (B)t the nitrogen teot aubetenee weighed 13*5 *&X «£j soh % esmceamtlon wa« not evident, Although practically the jior portion of the memataat fluid was believed to here oonalf ted of fatty cubetancea, detail e could not be learned dee to the eaall yield* Ihe nain points of the experiment are Hated in fable a tnmmnry of the procedure la gives la fable % 6 KHe *0 957«“ Cod* fraction Field Virulence Sltro*«m Atk 1 i U) ’>M%Uifei fro;a acetic acid ffiilpltcHife followed hy centrlfa*oll ratio*. o.l?7S« 1.* a* 10.JBt ’ 0.39* 1 (B) ice tone precipitate fron c«nd*ft«ad **l[ifnitout field la acetic eeld preciplta^ lion. 0.787% 1.8 m - (C) Ither soluble p*od«ctc a hydrated ace ton* extract of condensed superoctant fluid obtained Izi i cetio acid precipitation. 0.0036* Failed to cans* death at 7.0 m 0 - (»> fethor laeelobl# product of nboYe, | - — - 0.00U6g 11 Failed to cans* death at 7.0 *g « 1 I Table ?, Tlrmlonco i*»tt on froetlono T *>oc 553«» Chester T. Observation* The above fteii revsal that tbs acetic acid precipitate and the acetone precipitate nr* toxic and rostra l.S *f doses to kill mica. Since the bacterial strain frea which this antigen was derived pastas ms an of 0.3 s< It is astrsMM&r Interesting to not# that its virulence is approximately six ttat that of tha precipitates. An examination of the antigenic strength of thee# substances throat imamxilxatlofk tests is desired. If they crow to ha highly effective it will wean that a nethod for producing a superior antigenic substance has bass discovered. Ijumitatis* taste will be performed with nevly prepared antigenic material* whan the opportunity for continuing such a study is presented. the ether solable sabetfence and the ether insoluble substance failed to produce death with a f.O ag dote. ®ba shortage of antigenic materials prevented the tasting of higher dosages* this point mast be studied at a later date. s Doc »© 553S8K Virulence (l.S mg) Filtrates Spying—*■ Fther extraction—*- Filtration —- ® Drying-* Virulence (l.S ag) Vacuus*-dried powder— (A) 0.1?/8 % for analyst« (Black scale? powder) Mile J. beetle acid fraction test on tJ*8,W, vupertonic ware-treated vaccine 2 alcohol change* %ther • ■ | Supernatant fluid-* Wra >©ration ecFj ather-acetone-w £«fr Iteration—* < -notion filtration j 3-d«y refrigeration Virulence (bo death with 7*0 ag) rulence (a© death with 7,0 ag) U,S,¥, vaccine FH 3 Centrifaged sediment* Isuauni nation I maiuni ration (C) O.OO36 g for experiments (Hade snunlsr oswter)/ Sroeriaent (yellowl sh-grey «e dissent) m I I 8 a fi c « - Soluble substance' i (B) g 3 Sac Bo 553S8A Hhllogratite 1. OCR, 1885 (I88hj. 2. WWfPIOl, t»., ». kl. W. 28. 28. (IS10)| Nou«.( fh? nu. 3. BAIL, 0., Ktaehr. t.1 -a., ?h, 3f6, (191(0* IMd, ?6. 97 (1917)5 ma. ?7. l (m«). h. mnrm, ttickr. t. %*., 16, (xa$i»)t ma, is. i {189*0 i mt, ii, 393 U*9fi). 9. castas!, A., I?, 7*9 (issg). 6. 11‘AWf a, so,;,*, Jttaobr, f. %*.. 18, 17 (189*0. 7. CASSISM ct tOMfifl:., ». In Sw»4k. S. pat*. Mlcr->«■«.. (KCU>S, mm «. BMIWI) IT, 1. hb (1927). 8. ‘tecFAYDES, *• Is H«n»9, C?3 (1909). ?2, TSABWOTf, ». iarcb Salaottl (39). ?3. HOUBOiX, 8., La anarlaaatala, 5, 701 (IJIO), ?h. ClCCC.SA.TOI, t. Sarah Oalaottl (?9). 10 Doe No 553tvs. 2% 8UAASM* I., Aim, 3Tt> (15?0). go* JUiSfABl*!, #♦ imMU S#l $• 1* w#i»* **o€ (190?): IM4* 65# ?6 Clf^K g|* Laj09&I*31Iv I. «ad Jtsfliu, F.« J. «xa. *MU# **£t HI5 Uf*?). a** MAlsWtfO, I«ara, JHKKV lOARtf {**9**tmm%*k h«mIIgIim»)# 1$, 51? CWU. 23, »aiTJ>:, A* «l K»S 3*IiTO# 1., Sew d* 1m., 1*553 U935)s ifcl£* ?• U3 UfiOi !***♦ 3*319 (1917)I 1Wd* 4* (193*h *. 19? U93«). 33. KUK01 a* 4*»*fciko -fid SXSftHORA, jiro, IWAMAl M0*p riO-ETUJO XOKQKtf (Sn*fmh*l XocmmIi Unfcorafcwy %*&#*%) ?* f* (153*). 31, imo»?, *.V.* SXAA* ?.G. a«ld H1UA* XaA* J. ««*• 575 32. T08A* Aklhlfco, ■»« mUtXKMl tkmm {Sm*I *Wl*l of VmtmritmTf KmUcImi)* lb* 35$ (lf3?)» 13, XUX&fA* >«| USai#4-M* ******1 AAMfll rrmtlJO fPtarB (Summfcit s«?i*ae* *««Mrili l«Wtl«r ***>«**) *• 6 U9H>. u