The antigenic components of the toxins of Cl. botulinum types C and D

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Ond erstepom·t I ovrnaf of V etetinaty Science anrl Animal Industry, l'olume 0, .Ywnuet 1, J.11l'!}, 1U:35.

The Antigenic Components of th e Toxins of

Cl. botulinum Types C and D.

D.' J. H. :JL\80~, F .. Il.C.Y .S., F.Jl.S.E., J<:mpirP ~lnrkciing J1o::ml

l~esearch Fellow;

K :JI. lWHINSO~, F.ll.C'.Y.S., D.Y.Sc., Dr.Yet.:Jled., Suh- JhJ·ector of Yt>tPJinary Sen icPS, Ont!Pt>iepoorl.

INTRODUCTION.

l.:-iTOXLCATIO.\S caused IJy the toxin~ of tlte C and D type:;; of Cl. botulinwn have been re pori Pd from cliilere ut par( s of (he \\·orld (America. Ausiralia, Taslllania, antl South j..frica), the cli:;easPs produced usually lJPi n g aH,;oc i a tetl \\ i ( h the co llS1llll piion of carrion or rlPcomposecl material. Althouglt the fact that Lhe C an<l D t,vpe,;

tlo ca u ,;e intoxication,; in 1 lte tlo 111 e~i i e; animal,; is 11·e ll e:; (a hli~ heel, the rebtionship of 1 he (ox ins of (he· C type to another aml their n-•lation- sh it' to tl1c D type iR not in i he same posii ion. Benghon (I D2:2, J 92:3) 1·epoded i hai toxic fill n1h-•s of the (' type org-anism i!'ola.tc·<l hom fly lan·ae WPl'e uot ne11iTfllized b.1· ..A Or J3 type antitoxin Ullt[ i!Ja.t C anti- (oxin rlirl uot neutrali:r.e .\. or H toxiJJ;:;. SPddon ( 182:2), tli;-;cussing

hi~ parabotnlinus bacill11', notetl tlwt there \\.<ts no eros~ proteciio11 between it anrl the,\ orB lypPs. Pfenniger (1!324) slated that 0·2 c.c.

to ll·5 c.c. of C :mtitoxin neutralize<l h1·o fatal dose,; (for g-Hiuca-pig) of C ioxin, hut tllat this amount of toxin was no(· neu(ralizerl by A, 13, or Setlrlon a11tdoxinR. Pul'ther, Serlrlon toxin \Y.as lHmirali:~,ed

bY hiR C :mlitoxin, bui 110t h1· A 01 ]1.

Doyle (Hl23) noted that ihe (oxin as,;ociaied \\·iUt JimlJerneck in t:llickens, investigated h5· hilll, 11·aR no( neutrali:~,etl hy A or H tqw auhto:s:ins. Graham a1Hl H011ghion (1924) 11·ere able, ~Yiih antitoxins jllodu('ecl with type C ioxin, io llf'nhali:r.e Seddon (o:xin, a1ul, ful'ther, antito:xiu matle with ihe toxin p•·ochwetl by limhcntf'ck (type C) g-f'l'llb neui ralizP<l homologous, hf'if'rologou~, antl fly lan·.ae toxins.

;\~ alrearly rf'ported (ltohin,:on, J9:l0), one of u;-; (KM:.lL) r;ho,1·ed i hat the South African C t~·pe toxin (equine origin), a C type toxin (Graham, U.S.A.), awl i.he Seclclon toxin cross-11eutralizecl; how- PYer, D antitoxin tlicl noi UP1ltralize any of i.hese (oxi 11s. Further, in a lJPrsonal communication, Gra l1am statetl that his C antitoxin neu~ralizetl ('7. parabotulinmn toxin (i.e. (' type toxiu of South Afn can equine origin). M:eye1· an cl Gunni ,;011 (] 929), examining rnliures, receivetl from this institute, stated that the

n

^t^yp^e ^toxin

11·as not neutralized by A, B, ·or C antitoxins. Gunnison and ~Ie~·er

(1932), working with a clisease in dueks associated with Cl. bot1dimm1 type C, sho"·pd thai' ant.itoxins pre11arf'c1 from the <luck strain and

3 65

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.1:\TI(:E.'\lC C:O.:.IPO.'\E.'\TS OJ:' TJIJ.; TOXI.'\S 01' "CL. 1JO'l'L¹LI:"'L')l ''.

h·om ~train i);l(j Ca UPtLtralizc<l the duck, 5:.2G Ca, Sr<ldoH, and:):)() Ch ioxi ns, ,,·Jwrea~ Rrcl<lon (('b) antitoxin 11eul ralixed 011ly lite. honlOlo- g-ou,.; an<l lite '>-->0 ( 'b lox in~. BPnHrlts (HJ:l:J) ~howecl th.tt the toxm pr-epared hom an illlpllH' culltue of a t_qH' (':;train ,,·as 11eulral111ed by tbe AtLstrali:tl1 par:<holuli.11um antitoxin awl hy () an!Ito:-;_ul.

Theiler and Hobi11s011 (\IJ:.2SJ .•lnted that the ]) l.'JW anl1toxm (:J·O

<~.c.) di<l not 11rnlrahze l11·o leth.d dose,; of ibe Soullt Ahi~;an C, Lhc SecldoH, ~\met·i<·an C, m I he .\. t.~·pe loxins.

Ftom the fmegoing IIi, de:1t that u11:111imdy of OJlllllon <loe~ not P:s::ist as H'g:ud~ lhe JPlaiiOJhhi]r of the C loxinc,to Oil<' anothPI. '\'he possibihty exi,.;Ls lltal; •t ~;ans<' of lhi~ m:1_,. h<· lh:tt I he :1nlitoxi1b 11.~ed

\\'1'1"<' of Y<'r_\" low li.lrP. '\'hi~ opinion is :'tiJl]lOdcd l>.v ih<' f:u·t lhai l.argr a11t-n11.11l,.; of :111! iloxi11 (up lo 0·U c.c .. ) 11·erc reqtLin·<l lo IH'ulr:diz<' only a fe.~,- fatal <lo;-;e;-; of lo:-;in. Uowc>n•r, lh<' lilrralun• would indirale ilia! I hr• ]) (_,.Jl<' (Soulli Africa 11 bo,·in<', a:;~(H"ial<'<l "i I h lamsiekle) toxin is JIIOJIO~Jwr·inc.

I11 Yie11· of !he h c-k of U11iformi.l1· of n·,;ull.~ :tll<l us 11·ork 011 another group .of allaelnhe,.;, the Cl. ~velchii-r:l. oviLo.ric1ts group, sho11·ed th:1.t ihc loxins protluc-Prl hy indiYirlual nlr'nlher,; ma_,. ;;har(' lh<• same loxi<" h·aelions

l

\\"iLdon (HJ;)J), Olenn~· 1'/ rd?.rt (1!):\:{).

}fontgomPrie :lltrl no,dalllls (HJ:l..J.), Dulling (l£):1-J.), Ui\1 (1933)l, \\"Ork 11·ns conUllf'llCP<l on si:-; (' typr, one]) type. anrl ol!P A and one B ^(:qw loxins. 111 tile autl10rs' opinio1t, ihe res·ulh obbine<l ,;ho11· thnt in the C aurl D g'l'mlps there is, a~ in the Cl. 1ulrlu·; group, a shari11g of toxic antige11ic fractions.

METHODS.

Ori_qin of ('u7tw·cs.- The eultures 2GC, Serl<lon, and D \\·ere all impure, the ronlaminanb bring proteolytic non-paihogenic anae1·obes.

'rhat !he onl:,· toxin-prodnring germs coniainecl i.herein were of i.he boLulinus type was ;;hown h.v the fact i hat one cerl"a.i 11 lethal close of toxin (f01· mousP) \\;Is 11ot llf'lliralizr<l h~· 0·1 c.r-. of thP antitoxin~

o·E Cl. sr;Jt/rfllf', Cl. 'lrrlcl11·;, C7. Of'rlrmatieus, C/. l1istolyticu?n, ('/. !!'/ani, ('/. url!o.l·lr·us, and I he ·' l.amh rly;-;Pnlcry harillus ". or b1· a 1ni:d 1u·<· of 0·02=-l c.<". of en<"h of 1\wm. A <lrlerminPd atlct;lpL to ]JI!ri.l".'· ihP l> :-drnin failed. OnP of 11~ (1~."\f.TI.), :'0111<'

~-ear.-; ag·o, ^W<b ahlP t-o recli!<·r t-l1P numhPr of <·OJ!r·omitant lli!CJ·o- org-auic;mc; to l11·o, .a1ul :~ftpr furthN 11·ork PYI'nlually ,.;palerl ofr I hP

<"ulltH<' as pure. JTo\1-eYPI'. 011 -;uhniliUl·i llg' this ,;paled-off cull111·<' hvo )'('nn; laiPI', lhe presr'll('(' of :1t l<'asl liYO non-p:ilhog-elli(: :tnaerolH's

"·as noterl.

The ol her C an<l D strains, all receiYPrl from DL K. F. )Jpn:•r.

California, appearerl. on mirrosf'opical :lll<l cultural e::mminati011·, to be purr cnltnres.

The A anrl H strain::; gave no indication of impurity.

:2G C'.- .\ South African equine strain isolated at ihese lubOI·a- tories from a clecomposerl ral.

Seddon .- A ~train rece i ,-eel from Dr. Secl don, Australia.

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.J. JI. ~I.ISO.\" .IND E. )1. HOln.\"SO.\".

D.-A " hovi~ " st1 a in, isolated from a boYine clearl from bm- sie kte, Armoeclsv la Ide.

cl a.ncl B.- lteceiYed hom the Lister IJI~titule, London.

i)tr:tin 166, 1')·pe lJ: from K . . F. ,jieyer . LalJelle<l ·' South African BoYine ".

Strain 178, T,nH' (': hom K. F. )!eyer. Lahellecl "South :'dric:tn 550 ".

Strain 101, Type C: from .K. F. :Jiey<>r. La]JellPcl '· Be11gholl 5'2G ".

Strain J!J'2, Typ<> C: from K. 1:'. J[\'n··J·. L:dwllPd " Seddon

~traiJt h om J\u~(ndia ''.

Strain .200, T)]W C: from K. F. )Jeycr Lal1ellerl " B<>11ghon lJ 87 ".

Strain l(j;) ]) pro1·ed 11011-toxic, :b <li<l a hntl1er r.;ultme of th<•

~ame org·anism receiYPcl from Dr. _\IE'.~·er.

L're1wrc~tion of 1'on.ns.-JLober1.'0il'~ me:tt hrot h (lim·~<· 'llllL'icl<·

e:s:lrad, 1 per ct>nt. JIPptone, 0·':1 11er CE'IIt. :XnCl; the f!:tsk or tub(' containi11g boile<l meat to about Olle-thircl thr YolumP), plus .) per l'ent. sterile hon;p serum .'\':lS nsed in all ca~e:; to pro<luc<~ the (ox in.

The medium (pH 7·6) \\·as hoile<l for hH> hours, cooled quid:l.v, the :;erum added, iuoc:tLlated >Yith a 48-hours' <.;ultme of the germ, and incnlmted for se1·en d:tYs at ~17° C. The culture IY:ts ihen r·errdere<l

~terile by passing it thvrough, first, a paper pulp and then a Berke- feld filter ('anclle.

To render tht> toxin stable tbe toxic filtrate "'th saturaLe<l '"itl1 :tmmonium sulphate (about 55-60 g-rams per 100 c.e.) an<l the resul- i::lnt precipitate 1n·esse<l, dried 1:11 vacuo and pO\nlere<l. Sn<·h pO\Hler, rlissohecl in saline, maintained its otiginal (rlry) Yalue throug·hout the experiment.

Froductioll of clntito.cin.- Uo:tb proYerl suitalJle for this purpose, Leiug <.;heaJJ iu price anrl large enough to give a :;ufflcicn(·.\· of ,.;enrm for the uumber of te,.;(s carriPrl out. Formol-toxoi<l was employe(]

in ih.e initi:tl imllHIIIi~~,ation, lwing 1n·eparecl b)' a<lding O<l per ('(··ni'.

··ommerc:ial formalin (-1:0 JlE'I' r·eni'. formalrlehyrle) to tlr<> pulpe(l (not

•·an<llerl) toxin at pH 7·4. an<l incubating this :1t :no C. until 0·1 c.c.

clicl not kill a mouse 011 ~uh<.;utaneous inoculation. 'rhe iimP necr•,sa1·v for this to oc<·trr Yari.e<l l>ehH"en ] 311<1 i'1 \\eeks. ] t wa.-;. found ihat [t, 11':1.-< es-ential to re3(·h tb i:; rlegTef' of non-ioxicit.1· 1Jefore commencing i mmun i~~,ntion: 011 i \YO rH·c:t;;ions, tl1e lhf' of a C (South Afric~lll)

P<Juine toxin, t·hP ioxicih of 11·hil'h. ,,·as s1rch Hat 0·1 c.c. killed n mouse in three days (0 · 00 c.c. heing non-lethal), kille<l two goats 11hen a <lose of 2·0 c.c. \1":'\S reaclwrl. \Yhen a <losP of to:s:oirl behYeen :.?0 <·.c. and :l(J c:.c. had l)pen teachecl, one r·mrlcl 11·ith impunity rlouhle t lte dose weeld,~- or clLlnge o1·er to unmo<lifiPrl toxin. Tl1e :fiual G-8 i noc:ulations were of toxin, each close bring liei.\\'ePn 100 e.c. anrl :200 c.c. in amount. Unless the condition of the goat di<l not warr:<nt ii', the injection.-; wt>re made h1·ice 11·eekl,~·, tht> preYious close being- increased 50 per cent. and lnt·er 75 per eent. or 100 pE'r cent.

G7

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A:KTIGEKIC C01II'ONE!\TS O"F THE TOXINS 0.1:' " CL. llOTUU NUM " .

'l'he course of immunization lasted beh1·een i.'YO and three months.

Trial "·eekly titrati.om of bleedings "·ere made, commencing at about tbe second month; when no rise in antitoxin over the previous one was detected, the animal was exsanguinated, the serum collected from the clot and preserTecl ,,·ith 0·:) per cent. vhenol. One antitoxin, D(r), was prepared, wme years ago, hv one of us (E.~f.R.) in a horse h:v the use of the D ~train toxin.

T·itrat'io·ns of To.vin anrl Antito.ritt.-~Iice of about 15-18 gnuns were employed as test animals, the ro11te of injection being the sub- cutanemis. \Yhilst it is 1·ealizerl that the guinea-pig has been the test animal of ehoice for the titrahon of botulinns toxin and antitoxin, the mouse proverl to be quite as accuratt> an inclicai.or, a11d further was cheaper, mo1·e ea~il_,- houserl in large numlwrs, an<l obtainable in uniform hods· "·pigh t.

Dry toxin was <iissolwd in saline in the plO]Jortion of 100 mgm. to 5.0 c.<o. anl1 hom this ,;olnt ion dilution.~ ag-ain ma<le in saline. 'rhe app1·oximate minimal fat-al rlose was then worked out, anrl whilst no aHem pt ,,·a.-; macle to asce1 tain this ve1·;;r acc1Jr.atel_v, suffir.ieut mir,e were injectecl :no11nrl the probable L.D._,0 (TreY:lll) point to be reason- aNy certain that a Gertain fatal close could b<~ pr·e(lictPrl. The eml point was sufficiently sharp, "·ith all toxins .at ab01t!· the ~LL.D. level to allow a 30 per cent. drop in toxin amount to result in a " liYe"

or " paralysed " instead of a " death ".

For antitoxin htrahons, a (est dose of toxin was cho;:;en after a fe,,- preliminary trials. Beyond ensuring that this dose contained at least 10 sure fatal doses no attempt "·as made to have the same number of lethal closes in the test close of eaeh toxin. This dose, mixed "·i.th Yar;ying amonnh of anti.to:s:i.n, was left for one hour at room temperatnre (20°-26° C' .) and i njectecl into mice. \Vhere preliminary titrations showecl that a serum "·as poor or deficient in antitoxin, one ce1·tain fatal close of toxin ,,·as mixerl "·ith from 0 ·l c.c. to 0·5 e.G. of antitoxin and injederl.

In all cases, ohsen·ation was carrie<l on for seven days, re:mlh.;

being reconled a;; " liYed ", " par:dy;:;ed ·', or " clearl ".

EXPERIMENTAL.

Iu lhe first series of ti!r.atiOJJS, the nr>hn·alizing power of the antitoxins of 2G C, Secl<lon, and]) [indu(ling-D(r)J was tested against toxins marle from these strains. An t-xmnple of :mch a test is given in Table 1.

TAI!LE ] .

'l'oxin: 2oC. J[Lf): 0·000:3 c.c. '1.'. n. : 0·01 c. c.

Antitoxin: Seduon.

Dose (c.c.) D. ^1'. L.

0. 0007. . . ......... . + ^{0 0} 0 ·0008 ..................... . 3 1 0 0·001 .............. . 0 1 4 0·0012.. . ....... . 0 0 :l

(~I.L.D.=minimum lethal (lose. T.D.=test <lose. D= cliecl.

L =lived. P =paralysed, i.e. showed paralysis rhning the seven clays' observation but sunived ;;even <lays. In this titration 0 · 001 c.c. of antitoxin is taken as a nentrali zing rl ose.)

68

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.J. H. MASOK AND E. :M. ROlliNSON.

Similar titratiollS were can ied out with all the toxins aurl antitoxins, at least three anfl usltally six to eight mice being injected at those levels just around the neutral point. Table 2 summarises the titrations of the toxins of 26 C, Sefldon, and D against the antitoxins of these strains·. The central unenclosefl figure is the neutralizing dose of anti toxin.

TABLE 2.

===== =======--=-===-======

^~-

Toxin. • \LLil. T.D .

:2ti c ^0·0011~ 0·01

-~---

:-; 0·0001 0·01

0·00000:2 0 01

1 Antit.oxins.

1---,---,-- - - -

1 26 C. \ N.

I

^D.

I

^D^(r).

(:J-:!1) O·OO:l- 0·00:35

(I) 0 001

(50) 0·115

I (2oo-24oJ 0·1- 0·12

I

[1J [1J l 1J 1 [1]

-~---1---1----~---

(+) (L) (6(i) (!'i})

0·005- 0·00fi

[ l}-:2]

(lf) 0·02- 0·025

[ 600]

0 ·001:) [lU

(l) 0·03- 0·035

[ 1200]

ll· L

I [ 2]

I

( 1 / nOOJ-~

I

I 0

· l! ~ I

[ 2/5]

0·08 [+]

(1/HO) 0 ·02:>

[ l/8]

T.D. of D toxin v 2ti C and S antitoxins = 0 · 00001 aml 0 · 0001125 c.c·. rospecti voly. T. D. of 26 C toxin v D(r) antitoxin = 0 ·005 c.c·.

T. D. of 8 toxin ,. D(r) antitoxin = 0 · 001 c.c.

A II u nendosecl ligures = c. cs.

Figures in [ ] = ratio of one toxin to another.

Figures in ( ) = ratio of one >Lntitoxin to another.

Central unenf·losf'd figure = !l('lltralizing dose of antitoxin.

'l'he Gon~.:lusions to be (lr::nl'lt from 'rable 2 are (1) ihe toxins 26 C and Seddon are autigeniGally the Halllfl and (2) that D toxin contains some C antigen and the () a ncl Seddon toxins some D antigen, hut that the amouHt of heterologous antigen containerl in each toxin varies gTeatly, a,; does the amount of beterolog-011H antitoxin contain eel in each serum.

lH table 3 the 1·esults of titrating fresh bre\NS (precipated with ammonium sulphate and dried as before) of 26 C and Seddon toxins against antitoxins of 192 C, 178 C, 191 C, and 205 C are sum- mari,.;ed. 'rhe data given the1·ein confinns the resnHs shown in Table 2, Yi>':., that the toxins, 2G C', and Secl!lon, are antigr-nically the same.

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ANTIGENIC CO~IPO)IENTS 01:' THE TOXINS OF " CL. BOTULINUM " .

TABLE 3.

Antitoxins.

Toxins. iVILD. T.D.

192

I

¹⁷⁸

^I

¹⁹¹

26C

I

⁰^·001 ⁰^·01 0·006 ⁽¹²⁾ 0·0035 ⁽⁷⁾ ^I 0·006 ⁽¹²⁾

I

^[¹^] ^[¹^] I [1]

(8-10) (~-H) (8-10)

s

^0·00025 ⁰^·01 ^0·02- ^0·01- ^0·02-

I

0·025 0·015 0·025

I

^[⁴^] ^[⁴^] ^[⁴^]

Central unenclosed figure= neutralizing dose of antitoxin (c.c.).

All unenclosed figures = c.c. ·

:Figures in ( ) = rn,tio of one antitoxin to another.

I

²⁰⁵

(1) 0·0005

[1]

I (1)

I

⁰^·0025

I

^[⁵^] _-

In 'l'able 4 is summarized the titration of the toxins of 192 C, 178 C, 191 C, and 205 C against the six C antitoxins.

'l'.HlLE 4.

I

^I Antitoxins.

Toxins. :\ILD. T.D.

I I 26 c.

I

^Seddon.

_I

^1!.)2^c.

_I

¹⁷⁸^c.

_I

¹⁹¹^c.

T.D. =I 0·1 (3-4) 0·1 (1)

I

^0·025(27) 0·025(13-Hi)l 0·1 (10-12)

0·0025- 0·01- 0·02-

Hl2 C. I o ^-ooo5 ^O·O^l ^{0 ·00:3} ^0·017 ^0·012 ^0·03 I

[ 1/3] I[ 1(4-1/3] [ 1/3] [ 1/3] [ 1/4]

I

T.D. = 0 ·1 (:3!) 0 ·1 (l) 0·01 (25) 0·01 (10-12) 0·01 (17)

1 0·0001

0·01-

178 c. 0·035 0·01 0·025 0·012 0·017

I [1] [1] [ 1] [1]

I

^T.D.⁼

I [1"f\·]

-

₁₉₁

-

_c.

- ,

^0·01^0·035-⁽⁴⁾ ^{0 ·1} ⁽¹⁾ ⁰^{002 (5)} ⁰^·01 ⁽¹⁰⁾^I^o-01^0·00-!-^(t)

205 c.

I 0 ·0004 0·045 0·01 0·01 0·1 0·005

[ 1-1!] [1] [2] [ 5/6]

[

^v~o¹

I _T.D.₌ _0·₁ ₍₃₎ _0·₁ ₍₁₎ 0·01 (20-25) 0·01 (10-12) 0·01 (10-12) 0·012 1 0·012

I

^0·00015

_I

⁰0-035 ^·03- ^0·01-0·012 0·025

[1] [1] [1] [ 1]

:

All unenclosed figures = c.c.

Top unenclosed figures opposite T.D. = test doses used.

Figures in ( ) = ratio of one antitoxin to another.

Central unenclosed figures = neutralizing dose of antitoxin.

70

1[1]

I I ²⁰⁵c.

0·1 (lt-li) 0·004 [ 1/3]

0·1 (1t) 0·015 [1t]

0·01 (l) 0·0025 [ 1/5-1/4] 0. 01 (l-1',)

0·012 [1]

- - - -

(7)

.1. H. MASON AND E. M. ROlliKSON.

From Tables 3 and 4 the followino· information may be gathered:

the toxins 26 C, Seddon, 192 C, and 178 C are antigenically indis- tinguishable. As, however, 192 C and 178 C are merely purified cultures ·of 26 0 and Seddon respectively, one can say that, by the methods employed, no difference in the antigenic coustitutio~1 ~f their toxins can be elucidated. Further, reference to Table 2 mdl- cates that the amount of the D toxin fraction of their make-up does not differ much, if at all. These two toxins and the two Bengtson strains, 191 C, and 205 C, share two antigens, one of wh.ich may be termed the " main " and the other the " subsidiary " fraction. In the elimination of the 191 C toxin titration from Table 4, the sub- s·idiary fraction would remain unrevealed, and the other three toxins would be considered identical. It is obvious from the table that the expected ratios are not obtained with the use of 191 C toxin; three and possibly four figures are " out ", two of them beyond the limits of experimental error and of the n1.ethocl of testing. I£ the toxin molecule be considered as containing three fractions C" 02 , and D, then the apparent discrepancy obtained with the use of 191 C toxin may be explained. The particular brew employed contained an excess of the 0₂ fraction and, as follows, the antitoxin produced by it contained an excess of 02 antitoxin.

As there was the possibility that 191 C toxin readily dissociated,

·in vivo, from a toxin-antitoxin mixture, a number of tests were put up to check the point. On three different occasions, mixtures were made, and their effect on mice ascertained after they had stood for one hour at room temperahu·e and at 5° C. for 24 hours. The results did not differ materially from those recorded in 'l'able 4.

In table 5 are recorde<l the results of the titration of four C type toxins against the· D and D(r) antitoxins.

TAnLE 5.

Toxins.

Antitoxins.

T.D. 192 c. 178

c.

¹⁹¹c.

T.D. = 0·01 0·001 0·0025

[lJ [2t] _B _J

D 0·05 0·02 0·12

(1) (1) (1)

T.D. = 0·005 0·001 0·0005

[1] Ul ^[⁼ ^J

D(r) 0·1 0·15 >0·3

(!) (1/7) (= )

T.D. = Test dose of toxin used. (c.c.)

Central unenclosed figure= neutralising dose of antitoxin (c.c.).

Figures in [ J = ratio of one toxin to another.

Figures in ( ) = ratio of one antitoxin to another.

205 c.

0·005 B J 0·1

I ⁽¹⁾

0·001 [lJ 0·1

(1/5)

From the data given in 'l'able 5 it is apparent that the amounl of C antitoxin contained in the two D sera is not the same, in each ins•tance D(r) being much the poorer; it will be seen that 0 · 3 c.c.

71

(8)

ANTIGENIC COMPO:\'ENTS OF THE TOXINS OF ' ' CL. llOTULINUM ''.

of D(r) antitoxin was unable to neutralize o11e ~I.L.D. of 191 C toxin.

The possibility exists that this discrepant result may be explained by the amounts of the

cl

and

c2

fractions contained in the toxins and sntitoxins. As Table 4 shows, J 91 C toxin contains an excess of the C₂fraction; the lack of the anti-C₂ fraction in D(r) antitoxin woulcl explain its inability to neutl·alize.

Table 2 showed that cross-neutralizatio11 existed between the toxins and antitoxins of 26 C, Sedrlon, and D. Using a fresh brew of D toxin, a series of tests were put up to ascertain the neutralizing power of all the C .antitoxins against it. 'l'able G summarizes the result;;.

Toxin D (c. c.)

'l'ATILE G.

(~LL.D. of neTI· D toxi11 = 0·000002 c.c.) Antitoxins (c.c.).

2()

c.

s. 205 c. 192 c. 178 c.

0·1 0 ·l 0·1 0·5

I ^0·5

neutralized neutralized neutralized I

0·000007- 0·00001- 0·00001- Did not neutralize 0·00001 0 ·0000li5 0·000015 one ::\LL.D.

191 c.

0·5

-

'l'he antitoxins 26 C, Seddon, and 206 C in closes of 0 ·1 c.c. were able to neutralize a few ~I.L.D. of ]) toxin, but the three other C sera, even in closes of 0·0 c.c. \H're incapable of neutralizing onE' fatal close. 'l'he antitoxic haction of 191 C serum \Yas salted out bY half saturation with ammonium sulphate; the precipitate wa"8 clialysed against distillerl water for tlnee days and ihe inner fluid fanned down to one-([Uader of the orio·i11al volume. Although the anti-U (J 91) value of this ooneenhate was three times that of thE' m·iginal, 0 · .j c.c. was incapable of neutralizing one i~I.L.D. of D toxin. 'l'he possibility existed that the goats usell to produce 192, 178, and ] 91 antitoxins did 11ot 1·eceive enough of the D fraction (presumecl to be present in small quantity in the C toxic filtrate,;) to give rise to the prorluction of D antitoxin in clemonstrahle amount. Further, the C fraction of the toxin so preponclPraiecl over the D that unless the animal hocl:v ball exhausted its power of producing C antitoxin, there would he little ch.ance of ]) antitoxin being produced in appreciable quantity. Immunization \Yas therefore undertaken in three new g0ats. and carriecl on for three mDnths, finishing up the pr·ocess by injecting 100-200 c.c. toxin ten times within three weeks. 'l'he 192 goat m:u=: then bled out, but the other two \vere rested for a fortnight and then " shocked " by injections of 100-200 c.c. of toxin given every second clay for ten clays. By this method antisera were obtained containing demonstrable amounts of D antit0xin in the case of 191 and 178 antitoxins. Table 7 gives the results.

72

(9)

Toxin D

.J. H. MASON AND E. M. ROJlll\"SON.

'I' ^ABLE7.

(1'I.L.D. of D toxin=0·000002 c.c.) A ntito:c·ins.

191

c.

0·5 c.c.

neutralized 0 . 000004 c. c.- 0 . 000005 c. c.

178 c.

0 ·l c. c.

neutralized 0. 000015 c. c.

192 c.

0·5 c.c.

did not neutralize one )LL.D.

As a control to ihe large d{l8e of serum (0·5 c.c.) used, as· sh0wn m Table 7, experiments were carried out using

O·[j

c.c. of nor~nal goat serum and 0 · 5 c.c. of antitoxin prepared m goats agamst Cl. welch1:i, Type B, Cl. clvauvoei, Cl. ovito:cimts, and CZ.

sevptique. In no instance did this amount neutralize one sure let~al close of D toxin. 'I'he results given in Table 7 show· that the toxms of 191 0 and 178 0 contain a small amount of D toxin; the failure of 192 antitoxin may mean either that its toxin contains none of the D type toxin or that immunization vvas not carried far enough to allow of the production of demonstrable amounts of antitoxin.

The A and B type toxins appeared to be mouospecific. A test close (about 20 ~LL.D.) of A toxin was neutrali>~ed by 0 ·015 c.c. of its own antitoxin, whereas 0·2 c.c. of B, 0 (26 0, S, 191 0, 192 0, 178 0, and 205 0), and D antitoxins failed to neutralize one fatal close. Fmther, from 0 ·1 c.c. to 0 · 2 c.c. of A antitoxin did not neutralize one lethal close o£ the toxins of the B, D, and the variou::<

0 toxins. In the same "·ay, B toxin was neutralized by its own antitoxin, one tes·t close, about 100 lethal closes, being rendered non- toxic by 0·01 c.c. of antitoxin; however, its toxin (one nf.L.D.) was unaffected by the other antitoxins and its antitoxin had no neutralizing power on the other toxins.

DISCUSSION.

The results just recorded shmY that the toxins of the 0 and D botulinus group contain more than one toxic component. That there are three antigens concemed is faidy obvious-the 2 0 fractions, 0, and 02 , and the D. However, uo proof has been presented that these are the only antigens present, this investigation merely showing that, by ordinary methods and reasonably careful technique, some of the fractions making up the toxic molecule may be demonstrated. It is realized that a great deal more work would have to be done in order to arrive at anything approaching finality on the question. To explain an apparent discrepancy, the hypothesis h.as been formulated that the 0 toxin is composed of a 0, and a 02 fraction. AssuminO' this to be correct, there is th~ cle~nite possibility, arguing from the "'analogy of the CZ. u:elch.:u-ovttoxt~~~s group, that the proportion of the 0, and 02 fractions produced m culture would vary, depending upon the age of the culture. As an example it may be stated that CZ. welchii,

73

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ANTIGENIC COMPONENTS OF THE TOXINS OF " CL. BOTULINUM ".

'rype B, produces, in the early stages (12-24 hours) of growth only the CZ. paludis type of toxin, whereas if growth is allowed to continue, this diminishes greatly, to be replaced in large measure by the Cl. ovitoxictts type of toxin.

Further, as has been shown in the CZ. welchi'i group, the animal to be immunized also enters into the question; depending upon its

" natural " immunity and no doubt also on factors unknown, the antitoxin produced will differ qualitatively as well as· quantitatively.

Thus, it is obvious, arg1i.ing from such a standpoint, that this investigation serves. merely as a pointer to further work.

A second aud obvious explanation for the sharing of antigens is that the cultures were mixtures of more than one C and the D types.

This is a possible but extremely improbable explanation. Against it are the following facts: (1) the cultures originated in Aus•tralia, South Africa, and America; it is unreasonable to suggest that in each case the C (two varieties) and the D types were picked up; (2) there is some regularity in the anti-C coi_ttent -of D sera and the anti-D content of C sera ('rables 2, 5, 6, and 7). It will be seen that 0 · 02 c.c. to 0 ·1 c.c. of D antitoxin neutralizes 6 to 20 fatal doses of C toxin, whereas from 0·1 c.c. to 0·5 c.c. of the C antitoxins wa,;

necessary. to neutralize two to seven lethal doses of D toxin. In toxic filtrates made from a mixture, one would expect greater variation of the C and D fractions, as judg'ed by the anti-C and anti-D content of the sera. As, in the production of tl1e antitoxins, several (2 to 5) different brews of toxin were used to produce each antitoxin and as the anti-C content of the D sera and the anti-D content of the C sera was always very low, it would appear unlikely that a mixed culture is the explanation of the sharing of antigBns. Again with regard to the C toxins- if the irregularity obtained with 191 C toxin and antitoxin is to be explained by assuming contamination, then all the C cultures contained this contaminant. As before stated, such an occur- rence is extremely unlikely.

CoNcLusiONs.

Toxic broth filtrates of one A, one B, six C, and one D types Cl. botulint~m., and the antitoxins pr<lduced by injecting these filtrates into goats have been tested, from the standpoint of working out the antigenic " make up " of the toxins. The results obtained indi- cate:-

(1) (2) (3)

The A and the B types are Ul{)nospecific.

The C types contain three components (a) C_{1 ,} (b) C₂^, and D. The D fraction is contained in only very slight amount.

The D type contains chiefly the D, but also a small quantity of the C fraction.

REFERENCES.

BENGTSON, I. A. (1922). Prelimina1-y Note on a Toxin-producing Anaerobe isolated from the larvae of Lucilia CaesaT. Pu,bl. Health RepoTt Vol. 37

Part I, p. 164. · ' '

BENGTSON, I. A. (1923). A Toxin-producing Anaerobe isolated from Fly Larvae. Publ. Health RepoTt, Vol. 38, Part 1, p. 340.

74

(11)

J. H. MASOK A~D E. M. ROBIKSON.

BENNETTS, H. W. (1933). Botulism in Sheep in Western Australia and its Association with Sarcophagia. Jnl. of C01mc. Sci. and Jnrh~st. Res., Vol. 6, No. 3, p. '137.

DALLING, T. (1934). A Note following :VIontgomerie's and Rowlands' Article.

Vet. Jnl., Vol. 90, No. 8, p. 337.

DOYLE, L. P. (1923). Limberneck in Chickens. J. ilme1·. Vet. Med. tlss., Vol. 16, p. 754.

GILL, D. A. (1933). l'u]py Kidney Disease of Lambs (an Acute Entero- toxaemia of Bacterial Origin). Besw1·ch .Uepm·t, New Zealc~nd Dept. of Agric., Wellington.

GLEKNY, A. T.; BAl:Ul, 3.1.; JOXI~S, M. LL.; DALLING, 1'.; AND ROSS, H. E. (1933). Multiple 'l'oxins Produced by Some Organisms of the Cl.

welchii group. J. Path. and Bact., Vol. .37, p. 35.

G HAHA~f, H., ·IND 110UGHTON, I. D. (1924). Cl. bot1dinunt type C asso- cratecl "ith a L11nbe111eck-hke D1sease Ill Chickens ani! Ducks. J .. 4meT.

Vet. ivied. Ass. N.S., Vol. 17, p. 723.

GU~NISOK, J. B., AKD COLKHAK, G. E. (1932). Cl. botulintt11i type C associated with \Vestern Duck Disease. J. In/. Dis., Vol. 57, No. 3, p.

542.

IVmYER, K. F., A!\D GUNNISON, J. B. (1929). South African Strains of Cl.

bot1tlnwm and pcuabotuhm~111:; XXXVI~ \Vjth a cl,escription of Ul.

botulmum type D n. sp J. lnf. lJts., \ ol. 4o, p. 106.

l\IONTG03.1E1UB, R. F., ^AKD ROWLANDS, w. T. (1934). r~amb Dysentery in Korth \-Vales. Ve•t. Jnl., Vol. 90, No. 8, p. 323.

PFEKNIGER, \V. (1924). Toxico-immunologic ancl Serologic Helationship of B. bofulin?ts, type C and Pambot•itlimts " Seddon", XXII. J. In/. JJis., Vol. 35, p. 347.

ROBINSON, E. M. (19:10). The bacteria of the C'l. bot11linwn C ancl D types.

16th Bpt. DiT. Fet. Se?'V. Union of S.A..

SEDDON, H. H.. (1922). Tl1e Specific Identity of Bacillt~s pambotulimL.'!. J.

C'ornp. Path. and 'l.'heTap., Vol. 3.5, p. 275.

WILSDON, A. J. (1931). Observations on the Classification of Bacillus welchii.

Sec. Bept. DiTeet. Instit. tlnim. Path. Univ. C'arnbTidge, p. 53.

7)