011rlerstepoort J ov.nutl of VeteJ·inar.IJ :icwnce a.wl dnt:mal lndv.str.IJ, T olunw 12, .\"umber 1, .7un1tlli)J, 19:39.
Printed in the Union of Soutl1 .-\ frica L~· the Government Printer, Pretoria.
The Preparatio n of Anthrax Spore Vaccines (for Cattle a nd Shee p) in South A frica .
By :J!A:X S'l'EHNE antl K M. lUIDl~S().\", St><'lion of Hul'lt>riolog·.\·, ( lndt·r~lt>pood.
l.'iTIIOilllCTIO::\".
AsTHIUX spore ,·a~·~·ines haYt" 1)(-'t'll u.setl .i11 South }, h i1·;1 for i he pa~t
~I"Yt>llll'eJJ .n~ar~ 11·itll, on tht> "·holt·, Yf'ry sali,.;Lu·tory rt>snlh. In tlw com·se of timt• tht· original mt>lhocL of JU't>Jl<lring tlH" ,.a1·cine ltaYt' het>n sOJ!lt'\Yhat modifie1l, antl IH' think tlw puhlishill.!-(' of ihe lllodifi<'alions re<'enilY introrlltl't>d may i11ten·st olht>r 11·orkt>rs in this
field. ·
\-.il.ioen, Cur.-son <tlld l'om·it· (JV:20), 11·ho.sP p<llJPl' \\'<Is i·onct'I'J!Pd with tht· ]'t•riod IH:!:2-IH20, rlist·usst•d the prP]J:trahon and ust' of
<tnihr<L\. nw<'ine in gn·nt ddail. A l11·id snllllllar\· of ilwi1· 111Piho1l of
)li'PJitll·in.~· \"HC('illP .is ~,!'1\-etl lH•lo\\·.
An attenu.ated strain (tht> 11·ell-knm1·11 Ho.·dwfl' slr<tin) was gT0\1'11 on nntrit·ni <lgar i11 \Yoo1lhead tlasks until sporub tio11 was
11·t'll ;Hhan<'ed. This look <lhOI;I <I 11·eeL Thf' fhsb 11·t·re left. <It
l'OOJII fenljWl'<iiUn-• for <Jil<JI'!lPl' \H'Pk, <Ill(] f]w ,!-(TOid h tht'll \\'<Jshed oil 11·ith s<1line. 'l\,·il·t· the anlOunt. lJy IH•ig-ht, of gly('(·rine "·",;then
;uldt•d to thP poolt·d siLSJIPllsiolls from a Jlllmlwr of \\'oodhPad flm:ks.
This 1·onc-eni r;Jietl sUSJlPnsiOII IY:ts teste1l <IS follo11· . ..;:- 1ahhii receiYPLl 0 1 l'.C. s ubcu b llt'0\1 sl.1·.
" 0·01 "
,!-('lllll8<1-plg 0·1
"
,, 0·01
"
0·001
The 1·abbits were e:xpeeterl to liYe and the g·mnea-Jngs to die.
PlU:l'.IRA'I'fON OF ANTHRAX SPORE VACCINES.
Thereafter sheep aJl(l goab Wt're i llOC ulrd eel a~ follmY~ :-
2 sheep each receiYecl :!0·0 l'.C. of thE' co 11 re11 tra terl suspensiOn,
~ulH;uh\ neously.
2 " " 0.]
2 " " " 0·02
2 " 0·0] ) J
2
o-oor:,
1 goat
" 10·0
,
".l "
.,
0"
1 " " 0·0:2
1 001
"
l 0 ·QQ.)
"
All these animal,; ,,.<'IE' E'SJH:'<·le<l (n ~lll'YII·e. including tht• ~lwep and g-on1s which got 20·(1 ;uHI 10·0 <·.('. 'l'hn-'l' ,,.<'<'l's lall'r nll these
aniJIIal~ 11·en~ in-octtbtPil 11·it h 1.000 :JI. L.J). of <I ,·irulerll ~port>
suspension, (o 1cst lhf•ir iJJllltuniJ_,-. 'L'he hatch 11·as Gonsidered goorl if nll the aJ1imals sun·iY<·cl. aYe!';Jg'E' if one m· h1·o clierl. <illd poor if mon• 1liPd. Voor batclws ll'l'l'e u~uall,'\· lliSP;JrrlPd.
Uoo1l Y;tccines 1·ause1l <l tempera ( urE' rea1·lion in DO per cl-)llt.. of the animal;; iesle1l. 'L'lw sub~cr1uent 1t>st 11·ith Yirul1:nt :iJlOI't'S, however, rarely caused any rPaction. Goorl hatche:;, that is thosP of whi1·h 0·00'J c.c. of tht> un1lilutecl ntuint> 1Jl'Ott>c1ed slteep, \Yere uiluted 1 :2QQ for issue; aYet·ag·p hatches J: jQQ, and poor hntclws, where 0 · 01 c.c. hacl faile1l to protect, IYere discal'llPil.
~~Te exantine<l the ptotol'oL of the hatdu•s pa.-;sell and i:;:;uell at this time and e:xtrnded 1he following additional informabo11.
'rhe O·J to 0·001 c.c. amounb of the uncliluterl var:cine killerl guinea-pig-s in 24 to 72 hom·~.
The Ya<:cin<" ou·a~iotwlly killr•cl 1·ablJiis :1ncl g-oaL.;, lniJ· 11·.a,; wwrl if it did llot kill ralll1its \l'hPu rdPslNl.
Sutnmari~ing· 1he;;e :;ta(emenh 11·e find (·hat the vaccinP 11·a,;
€Xpecteu t., be avirulent for shl'P]l, almost <ll·irulent for goal.-; <111(1 rabbits, aud very Yindent for g-uinea-pig~. At (hi;:; time i.IHJ 1nillion dosPs were iKscwd 1wr ,\'E'<IJ' ;1nd v1·ry fl':'· <·omplaints J'el'e:ivecl. As the demanrl for v.a1'1'iJJP inr-rpa,;<"rl <·omplain(,; \\·pre made ahout severe react ionK and rlea1 hs follo11·ing- inontl;dion. A possible renso11 for thi,; was that tilt' llOJ11tlarit~, of the 1ac('ine ;1ncl the propaganrla con1luc1ed by the Veteritnny Department resulterl in mauy fanners in or_; ula ting against an thra :x even 11· hPre the 1list>ase was not an im1ninent dang-Pr. \Vhere n nthrax 11·as a sl'riom; menac(), or·ec'tsional
;:;evere read ion,; and 1leal hs were clisrega nled: hut seq'l!ebe to inoculal.ion were not regarrled with equanilllity in nre:1s where the danger fron1 the natural di,;ease wa;:; slight. It is Jlossihle therefore that. the increasing- volume of COll1]Jlaints had more to 1lo with the changing nature of lhP inoculation than with :tny r:onsider:1ble change in the Yacr:ine.
10
.lUX STEH:'iE AND E. M. ROlliNSON.
To obviate such reactions, batc:hes v>c>re lah'r (1926) issued at higher dilutionR, such .as 1 ::~>00, antl this it> turn atlversel.v affected the immunity produced. A fmther tronblP t>xperiell('etl at this time wns irregular sporulat·ion a11d iht> ('OnsequPtll nlpid tlt'teriorat.on ~f son1e btliches. Qnin (personal com1nunication) Rtatnd that this in egularity ,,·as nttrihuted to the pt·pseJJ<·P of mucoid grmYth and that steps WPrc> iotkPn to ensm·e <.;nltures as fn'e from mucoid gTo\\·i:b an<l f·.ap,;ulatt·'rl orgnni,;ms n,; possible. (Gro11·th ai :1:2° C, sf'lectlon of non-mucoitl strnins). At this time abo pepton<' was omitted from t·he nntri<'nt agar, in the hope thai the lH>YPrly of thP l1H'<lium would iJilln!·!' lwl'ter sporulation. This peptont>-fn'e metliuJn w;ls HSe<l u11til U):Ji). In J!)~lO t.o J9T! ~'Wil grenh'r slress 11·as lnitl on tbe DPf'<l for li.Oil-mucoid euli nrt>s hy Coles (pPrsonal cotmnuni<:aLion), who mn in- tainecl that seYerP reactions in inoculatPd animals "·ert> usualh associated with batches c:ontaining mnn,v capsulated hacilli. BPhYPe~l 1929 to 1935 the tern pe1·a tu re of incn ba lion IY;l,; ra ise<l io :Ji)° C.
01Ying to mc11l:Y complaints of la('k of immunit,,- in Hl27 to 19:29, the virulence of thP sh.ailiS used 1vas innease<l again, in 1!)00; but ihe dilution .at whil'l1 i he vaccinP v.·as ic;sued \\·as hig-her. Neverthele~s,
some ,;eYPre rendions still oPcurred, and during this period there were 1·eports both of 1m due Yirnlence nncl of lac]< of immunizing- p011·er.
vVe haYC, in this intToduciLOJL, rathet· stT!'ssed the untoward results of immunization. :\.dually, con:;ide1·ing- tlw Ya:;t issue, Lhe vacl'ine \\·as Y<'ry satisfactory. 'l'herP 11·erP PI101Jg'h unfortunate incidents, however, to ('<!use pedm·hation :1mnng-d t hosP n'Sponsible for preparing thP Y;tC('i 11<'.
As a result. of research clone bt>h,·epn 19:14 and 19:38, much information was gaim'<l about the behaYiour of nilPnuated strains, and we h:ne bePn abll' to adopt a mme st·nnllardize<l technique and to obtaill far mort' COJlSioilent rPsult,; than formerly. \Ye thet·efore giYe belo11·, lnif'fty, the methods no11· used to prt>pare thP mdinary anthrax single spor<'-v.accine.
PltEPAILITIOX OF Y ACCIXE.
SPlechon of Faccine Strains.
The attemwtion of anthrnx strains hv tlw l'nsLPuri;ln rnethoJ is a tedious and often unsatisfadory procedure; for allbough it is uot difficult to cause a strain to lose Yirulen('e, it is clifhc11lt io find straius which mainb in this n('ll. leYel ·Of Yirulence. There are, however, enough stable, attl·nuatecl slrnins in <'xistcnl'e to mnh' the mHlm·- taking of new attenuation;; unnece..;sary. The Boshoff t;(ra.in, attenuated by Kind (J922), has given good J't'sults in t·his and other countries. \iV e have also found strains isolated from Ca rbozoo ltfailiinder and Carbozoo J,edeTle Yery suitable for vaceinP production.
'l'he virulencf' of these liTo strains, \Yhich are Yery much the same in apvearance an(l behaviour, can be easily exalte<l by guiJJea-pig passage, and rrclucecl by ageing the cultures on nutrient agar.
PREPARATION OF AKTill{AX SPOlm VACCINES.
Xevertheless, spore snspensions prepared from any of these strains remain remarkably stable at the level of virulence of the culture from which the spores arose. 'l'hus, within limits, Yaccines of different potencies can be prepared.
ChaTactPristics of raccine Sh'nins.
These must grow luxuriantly on nutrient agar. 'I' he edges of the grmYth should be slightly roug·h, and the surface glistening and shining, thus inllicating the presence of many capsulated bacilli.
Here our requirements differ radically hom those of the earlier workers at Onderstepoort, who wanted growths containing as few capsulated bacilli as possible. Coles contended that the capsulated bacilli were responsible for severe reactions and in this he was probably correct; for it has been shown, Bail (1915), Schaefer (l93G), Stamatin (1934), Sterne (1937), that the virulent phase of anthrax is en psul.atecl. Nevertheless since virulence for guinea-pigs was, and is, a criterion usen in assessing the value of a vaccine, there must be organisms in the capsulated phase in all the Yaccines.
'l'he earlier workers, by reducing the number of mucoid bacilli, but retaining the virulence standard for guinea-pigs, \\·ere obviously using a culture containing relatively few, but rather Yirulent bacilli in the capsulated phase. It appears that such Yaccines, if used on beasts nwre sensitive than ordinary, sometimes produced Yery marked reactions and occasional rleaths. Fm· this reason we, while retaining approximately the same Yirulence standard fm·
guinea-pigs, attain this by using cultures with large numbers of capsula ten bacilli which are, necessarily, more attenuated than formerly. This is, technically, an easier stanrlard to maintain, seeing that attenuated strains tend naturally to a mucoid habit of growth.
Jlled1>um.- Vve have reverted to using nutrient agar containing peptone. 'l'he (;om position is as follows :- Meat 500 gnn., peptone (Merck's) 10 gnn., sodium chloride 5 grn1., eli-sodium-hydrogen phos- phate (Na2HPO.,) 2 grn1., agar-agar 25 gnn., water 1,000 c.c. 'l'he pH is adjusted to 7 ·4. Exhaustive tests showen that the omission of peptone did not favour sporulation, but merely slowed down the rate of growth and lessened. the amount of culture ultima tel~: han esten. Good sporulation appeared to he associaterl rather with a vigorous than with a retarded growth. Each hateh -of medium is tested for its ability to support a vigorous growth of the vaccine strain, before being used.
lnoculahon.-A twenty-four-hour Mason's tube (Mason 1933) culture of the vaccine strain is scraped off into 500 c.c. nutrient broth.
'l'his fairly dense suspension is used, immediately, to inoculate a bout 50 Woodhead fiasks, of one litre capacity, each containing 250 c.c.
nutrient agar. Formerly, a twenty-four-hour broth culture was used as the inoculum, hut this technique was discarded owing to the tendency of a liquid medium to select unwanterl dissociants.
12
)lAX STBllKE AXD E. ) I . RODIKSON.
Growth.- 'rhe flasks are incubated at 37 ·6° to 38 · 6° C. 'l'his temperatm·e must be 1·igiclly arl hered to, as 11·e have found th.a t lower-
ing the temperature affect,; sporulation arh er,.;ely. Strains "·hich sporulate readil.1· at :J7·5° C. may show onl.1· nwsse,; of meta('hro- matic granules at Jf)O 0. or 36° C. vVe now realize that much of the ea1·lier difficulty with sporulation \Yas the result of lO\Yering the tem- perature to inhibit capsulation.
After h1·o days the surfare of the growth should be raised and slimy, and smears should show large numbers of capsulated bacilli as 11·ell a" a good proportion of spores. Later the mucoid appearance is grarlually lost, while the proportion of spores increases rapidl~·.
Sporulation ought to be complete by the fifth day, and the growth ready to 'wash off. If, as occasionally happens, spm·ulation at this time is poor, the batch is discarded, because such lots neYer make good Yaccine.
An aventge-sized batch consists of 200 \Yoodhead flasks.
Testing of Vaccine Batches.
Testing of virulence ·in [!1tinea-7n.r;s and rabbits.- Each \Vood- head flask yields 80-90 c.c. of gl~·cerinized spore suspension.
2 rabbits each recetve 0·1 c.c. su bcu tan eou sly, and must SUI'VI\'e.
3 gmnea-p1gs each recmve 0·1 c.c. subcutaneously, and should die.
3 guinea-pigs each rece1ve 0·01 c.c. subcutaneously, and should rlie.
3 gmnea-ptg::; each rece1Ye 0·001 c.c. subcutaneously, and shoul<l die.
The guinea-pigs are carefully seleeted and should weigh 600 grm.
each. In interpreting· these results, the survi,-al time after inocula- tion must be taken into consideration. If all the guinea-pigs die beh\·een 48 and 72 hours, or if all, irrespectiYe of the close, die at about the same time, the vaerine "·ill be too virulent. The differences in dosage, 0 ·1 to 0 · 001 <:.e. of the mother suspeusion, must show a well-marked effect. Batches where 0 ·1 to 0 · 001 c.c. kill guinea-pigs in 24 to 72 hours are discarrled, eYen if the rabbits surYive. This interpretation differs radieally from thnt of Viljoen au(l his ('()- workers. The deaths of the guinea-pigs should range from 3-i days after inoculation. Occasionally a guinea-pig will suniYe; this is not considered a bad sign. If any rabbit rlies of anthrax the batch is discarded. In practice rabbits do not die if the vaccine shows average virulence for guinea-pigs, so the latter test is regarded as the more important.
Safety and I mnumity test 1:n sheep and .t]oats .- 'l'he following table shows a typical and satisfactory test.
13
PllEPARATION OF ANTHRAX SPORE VACCINES.
TABLE I.-Safety and hwm;unity Test on Sheep and Goats.
Batch No. 99 (for Cattle and Sheep).
SAFE'rY TEST. IMMUNI'l'Y TEST.
No. of Certain
Amount of Killing Doses
Sheep. Undiluted Result. of Virulent Result.
Vaccine Spores Injected
Injected. 3 Weeks Later.
- -
1 20·0 c.c. ' L. 100 L.
2 20·0 c.c. L. , L.
3 0·1 c.c. L. , L.
4 0·1 c.c. L. , L.
5 0·1 c.c. L. , L.
6 0·1 c.c. L. , L.
7 0·01 c.c. L. , L.
8 0·01 c. c. L. , L.
9 0·01 c.c.
I L. , t (3)
10 0·01 c. c.
I
L. , L.Goats.
1 10·0 c.c. L. , L.
2 10·0 c.c. L. , L.
Control Sheep.
1 Nil. - 1 t (4)
2 Nil. - 10 t (2)
L = Lived. t (3) = Died on 3rd Day.
The immunity is tested "·itb a dilution of a stock glycerine- saline suspension of the virulent Boshoff strain. This suspension was prepared two-and-a-half years ago and has not yet started to lose virulence. As it is impossible to establish the M:.L.D. for sheep at a reasonable cost, we have adopted as our standard ten times an approximately determined :JI.L.D. This we call a Certain Killing Dose (C.K.D.).
A batch is never passed unless all the sheep and goats survive the safety test. It is passed, however, if one or two fail to withstand the virulent spores. IV e also deviate from the criteria formerly used in the following respects:-
(1) Not more than one or two of the eight sheep immunized with 0 ·1 and 0 · 01 c.c. should show temperature reactions.
(2) Most of these sheep sho-uld show temperature reactions to the virulent test dose.
These subsidiary criteria are important because judging on deaths and survivals only becomes a haphazard affair, when clone on so few animals. Sometimes, for example, a very solid immunity produced in sheep is the only indication that a batch may be too virulent.
14
~IAX STEitXE .-\N]) E. l\L lWBIKSOK.
If the iesb h a 1·e he en ,;at i sf.actmy, the cour·en ira ie<l su,;pe11 sion is diluted 1:20, 1:
,-)o
or ] :100 11·ith glycerine-snliuP rlepending on whether two, one m· no sheep died in the immunity test. 8ome latitu<le is allo"·e(l here. T£ the g-uinea-1Jig-,; bud died fuirly quickly, a batch woul<l lJP i::;sued at 1: 100 eYen it one or two of the sheepdi<~d in the immunih- te,;t. 'l'hP dosP for catlle i,; 1·0 c.c. and for sheep 0-5 c.<:. of the.diluteJ suspension. ~o balch is ever issued at dilutions hig-her til<J!l 1:100. H :1 lot of YflcciJu-· is rather virulent, as j11clged by t.he lahorator.v (pst, it is <lis<·.rn<l!:'ll, n-ot clilute<L 'l'hus most ·of these test·s are desig-tJP<l to :;elPd 1·accines of slightly lower Yirulence than tho~e formerly chosen.
Oulv a h-adion of t.he total amo11nt of Y.::t<·<·uJe issued io used on sheep. As \1·e haYe found these animals more susceptible than cattle, \Ye sele<·i sheep Y<Jccine from the mil<lPr cattle hatches.
Compar£son of test:; on sheep done befll'een 192()-1935 and betweeu 1936-1938.- Tahle II sumtuari~es the safety an(l immunity tests done ou sheep pr.ior to H:J:1!) an<l after 1 Wl5 11·hen the mmlifir:a lions men- tioned aboYe wPrP i.ntrorlu ced. T 11 ih e perio<l J 92G-19;l5 Llosa ges below 0 · 01 c.c. are not included, as tests with these smaller atHounts have no"· been d iscon tinue<l.
T.H1LE II.- Swnmmy of Tests on Tlaccine Butr·hes (1926-1938).
~======~~-~=-=-=-=---~===-~---~
:'llo. of Sheep Immunized.
139 139 149
Period over
"'hich Tests were done.
1926-1935
Co(TROLS NOT REG
36 1936-1938
71 78
No. of c.e.
No. which Per cent.
Concentrated Suspension received by each Sheep.
died after I dying after Immunity Te.,t. Immunity Test.
. I
20·0 l 0·7
0·1 I 22 i 15·8
0·01
I
61I
40·1UT~ARI.Y INCLUDE[) DURING THIS PERIOD.
20·0 0 I 0·0
0·1 8 11· 2
0·01 12 15·4
- - - - - - - ' - - - CONTROLS.
68 1
- ---- - -- - - -- - -- - -
1936-1938 I_ -
Nil. 68
I
____ l _ _
~-- 100-0From 192G-l935 batches were issued at dilutions of 1:100 to 1 : 500 of the concentrated suspension depending on whether the vaccine \1·as weak or strong. In spite of several batches in this series haviug- immuni~ed down to 0-001 c.c. there was, on the average, a big drop in immuni~ing power bebveen the 0-1 and 0 · 01 c. c. leveL Froin J93G"1938, altlwugh the batches were on the average less virulent than the earlier ones, there was n.11 insignificant drop in
15
PREPARATION OF ANTHUAX SPORE VACCINES.
lmmumz1ng power between the 0 ·1 and 0 · 01 c.c. level; the latter beino· the level at which the vaccine is issued. vVe believe the main reas;n for this more even protection to be the increased effecti-ve dosage, which has been brought about as follows:-
(1) The presence of large numbers of capsulated bacilli.
(2) The increase in harvests brought about by using peptone in the medium.
The concentration of the vaccine is, relatively, still higher, because we now dilute 1 : 25 to 1: 100 as compared with 1: 100 to 1:500 in former years. A point not br·ought out in Table II is that there was little variability between batches after 1936.
Field tests.
(1) oOO closes, at double strength, are injected into cattle in the field.
(2) If there are no, or very slight, reactions to the preliminary test, a further 10,000 doses are issued in small lots to different areas.
(3) If no complaints ale received, the routine issue of the batch may be commenced.
The yearly issue of vaccine is now six million doses. Eaeh batch contains about a million, and twelve to fifteen such lots are preparer1 during the year. More batches are actually started, but get discarded at different stage;; of manufaeture. Fr·om the t'n lve to fifteen successful hatches a final selection is made, mainly on field tests and reports.
If at any time adverse reports about a batch a1·e received j1·om different localities, the issue of that batch is suspenrled. Experience has shmn1 it wiser to assume that most untoward Tesults are caused by the Yaccine, than to blame the farmer, the needles, the condition of the eattle, the hot weather, the cold weather, the drought, the rain or the depression. We have stressed the statement that the reports shoulrl eome from different areas. This avoids confusion with post- inoculation accidents consequent on infections, such as those caused by Cl. chau?;oei or CZ. sezJticwn.
Discuss ToN.
Cmnparison of field and laboratOTy Testtlt,,.- I t must be emphasized that laboratory results give only a general indication of the way a vaccine will behave in the field, and that there is no sub- stitute for the large seale testing of new hatches, or of new types of vaccine. Patently dangerous methods of immunization have some- times been advoeated through workers not appreeiating the wide
~·tllf betwee~1 the stabled and the free-ranging animal. For example, m the routme tests of the last two years 50 goats have eaeh been inoeulated with 10·0 c.c. of undiluted eattle vaceine. Not one has died. If, however, such vaccines were to be used for goats under field conditions, a heavy mortality (20-70 per cent.) would occur with the ino?ulat.ion. of ev~n small amounts of vacci~1e. Every now and then th1s pomt IS forc1bly brought home to us, when people accidently use cattle or sheep vaccine for goats; the results are usually catastrophic.
16
~fAX STER~E AND E. M. l10Hl~SO~.
X eYert heless laboratory te~ts, ''"hen carefull~· interpreted, giYe
itHlispen~ahle informtdion.
Co11tpari:son of fi.eld results 11:itlt old and tc/tJ, 11eu· typl:' wccine.- The results of the larg-e ,;cnle field inoculation~ rlone after 1935 com- pare favourably \Yith those clone earlier. There haYe been no com- plaints about lack of immunity, although there has been plenty of positiYe eYidence of the vaccine's effi<·aey in ;;topping :uul preventing anthrax on heaYih· infected farms. Generallv, the immunity has been effeetiYe a1uf uniform, in spite of our u~<>ing- mort> atte1~uated strai11S.
A more detailed shuh of the results of immuni>mtion in the field ''"ill be giYen in another paper now in course of preparation.
Although there han" been a fe"· complaints of undue Yirulence, the number has been insignificant. The bat<·he~ responsible \YeJe
"·ithrlrawll and replaeerl h~- milder Yaceine. In no ease ,,-ere sucb hatches issued at higher rlilutions. In Hl35, 19:)6, and 1937, about 00 deaths from Yaccine anthrax were reported out of approximate]~
] G million <:a ttle i noc11la ted. F mh- of these deaths occurred with hi·o batc:hes u~ed on about 300,000 ~mimals and then withrlnnn1.
Thus the increase iu the <'On<·eutration of ;;pore,; iu ihP Yaceine, tog·ether "·ith the reduction in Yirult>nee of the strair1s 11se<l, enabled us to produce a Yaeciue g-iYinp; a uniform and good imm1mit.1·, 11·ithout causing· many seYere I'Padions or deaths.
_-I_ ~YotP o-n Types of Post-inoc1tlation Ar·r·idents.
(1) Death in 24-72 hours; bloo<l smear sho,Yiug- no anthrax bacilli. )fo~t s11<·h pro,-e to he Cl. cl,anroei infectiou.
(2) Death in 72-9G hours; anthrax bacilli in blood ::;mem·. Satund Yindent anthrax is usually blamed for sueh <leath::;; but they ma.;y be the result of inoculation. Such quiek death::; a,; the result of inoculation are more common in sheep. For example, of one lot of 2.50 sheep inoc11lated with a bateh of vaceine "·hich was subsequently withrlrawu, 8 died in 72 hour:; of typieal apopledie anthrax. Culture,;
from the blood sho\Ye<l organisms of the Ya<·cine t~·pe.
(:~) A swelling, <liffuse and oedematou,;, "hich may reach the
<le1dap, abdomen and udder. Sueh swellings mny take a "·eek or ten ,Jay:; to deYelop. )!ilk yield and eondi tion go clown fast aml death may -oceur in from 1 to 0 weeks. )fost animal:; affected in this way J·ec·oYer. This is the usual t~·pe of seYere reaction. The oedema i::;
almost inYaria bly -of a serous-g-elatinous nature, aud it is rare to find bacilli in blood smear~ frolll nuimals which haYe sneeumbed to this type of reaction.
HRI<'ERENCES.
B.-\lL. 0. (1915). Yeranderung der Bakterien im Tinkorper. ·c-ber die Korrelation Z11·isch,,n Kapselbildung, Sporenbildung 11ncl Tnfektiositat des ?l