The effect of inflammation on the survival of guinea pigs infected with anthrax

OnderstejJOOrt :lournal uf Veterinary Science a11d Animal Ind1tSt1)J, l'ul?tllte 20, Awn/;en 1 and 2, .llarr·lt, 1!.)48.

PrintL•d in the Union of South Africa by the GoH•n11nent Printer, Pretoria.

The Effect of Inflammation on the Survival of Guinea Pigs infected with Anthrax.

By MAX STERNE, Secti{)]t o£ Bacteriology, Onderstepoort.

Tu" exten;;ive lilen1hn·e on non-;;pecifi1· 1·e::;istance to bacterial infedions will not he revie1Yed in tletail, us goocl ;;mTey;; "·ere given by Philipson (1937) and Boquet and Stmnatin (18~~9). Most ·workers in this fiel<l Jwve in:jedeil bacteria, bacterial produd:s or extracts, or simplPr orgnnic or inorganic substances into animals and then ::;Lo,,·n that thPse anima]:; were mme resisbwt than controls to the injection of living het<:>rolog-ous lntderia into the prepared a1·e<L One might be more g-ent>ral an<l ~ay that the deYelop- meut o£ bacteria injected into an inflamed area is retanled to a lP;;ser or greater degree. Boquet and Stamatin emphasized that the inhibition was strictly lo<·al. HoweYer, Klein (189;~), Issaeff (1894), SobemhPim (1885), Bechhold (1822), Kepinov (1924), <d1<] other 11·orkers, have sho\\·n thnt non- specific proee<lure;; su(·h as those in(licnted aboYe raisPd the genera 1 re;;i;;tnu('e to in£ection.

The uommon bctor underlying the ui:fferent procedures appears to be that they all pliOvoke inflammatory uhanges, either b:· the d ire<·t injel'i ion of irritant chemicals or killed micro-organisms into the Rkiu or the peri- toneal cavity, or by the injection. of untigen into preYiOtmly senHiti:>:ed animals fPhil ipson (1937)

J

or by the production of an unrelatecl <lisP asP [Pullinger (1938)

J.

The work of Hruska (1831), :mel :Yla:>::mcchi (1828) 011

the effed o£ aucling saponin to anthr<lX Y<.lC<·ines is Ull important appli('aj ion in practice of the effects of inflammation on >irulence :mel innmlllity.

Mary "·orkers haYe uemonshated direct antagonism in viro behH•ell different mic1·o-org:1nisrns. HoweYer, such phenomena as the ilPstrudinn of lJ. anthmcis by Ps. pyocyaneou:;, or by sporulating af'rohes, or h.\· moulcls, fall outside the scope of this paper.

ExrERLi\11-:NTAL.

'l'his work W:1S UoQUe on anthrax in guinea pig·s. 'l'he i:e8t HpOl'f' SUSpeu- Rions were prepared hom Pasteur II stn1ins passaged £or several generations in guinea pigs to exalt their Yirulence. These suspensions kPpt for sen•ra l months with little deterioration.

l. The Gene1'al Effect of .lcute Inflammation on the Resistance of Guinea pigs to A ntl,·ra·.r.

The usual pr·ocedure was to inject an irritant into a fore-limb or intra peritoneally and a test dose o£ anthrax spores (50 to 100 letha 1 doiies) into a hind-limb. Table 1 shov;·" the effect of inje<·ting the irritant 24

157

1!\FL,\:MMATlO~ 0:\ ~l-H\"IY.\L 01-' c;rJJ\E-1 l'LG~ 1:\FEl"lEJJ \\Tl'II ANTJIJUX.

houn; before the anthrax ,;pores. 'l'h~ Jic;euse 11as appreciably retunleLl

"ith initant::: c;uch us tlll'Jlentine, saponin, and calcium chloride, \\"bile ini taut::: sue h as la dil: a c:i(l, L:OlJ ceu tra ted so eli um chloride, ancl c-oucen tra ted wdium ~ulphate, lwcl little etl'eci. .\11 these irritants ~-an:;ed extensi,-e ttenosi,;, Lut unl1· tile first group proYol\eJ the fonnation of all\" considerable tJedema. UeJJeralh- speabng-, the iultibitory eftect occ-mTeLl with initant::;

"llich Jn·o,··oked oeJema ±ormation in addition to necro,;i,;, an(l the magnitude of the i11 bihition 11·a,; 1e1-_y roughly propo1 tiona] to thf' e:--;tent of the oeclem<t.

The lo(·al antlu·;tx le::>iou IY<t::> uppreciabls smaller i11 the• animals in "·ltich au inflammation lwd lJee11 cau:;ed t h<nl in the c-ont-rols.

,\_ la1·•re uumLer of expe1·imeuts were also carrie1l iutenal b~t11·eeu tht> injediou of the irritant antl the The inhihitorv Pi'ft>d \YUS found 11·lle11 the irritant· 11·a,;

days before,

to

^{eig-ht honn; after the} <tllthras.

out in ''"hiclt 1 he ::;pon-•;.; \Ya::; Ya ried.

injedf'rl from four

Other tuetlw1ls of pr-oduci11g Lnge oedemas ~~-e1·e hied, a1Hl all 1·etardt>tl the deYelopment of anthrax. The geJtentl inhibitorY eft'ed wa,; not rel;ttetl to allY incre<tse or llecreclse of cin·ulatiug leucoc:des.

2. Tl1e Hlect ^of l11jectlny J nth·rn.c ^SjJIJI·es Into a11 ln.flomed Area. The lll<ljority of the experintettb 1lone h:v Boquet an1l Stamatin (19~·\U) ln-t·e of this type aJHl they sho11·e1l a marked iuhibitim1 of the c>eYerit.\· ot anthrax injected into the inflamed area>:. In table 2 [ll"P shmYn the efteds of iujecting <lll ini!ant :;uhcutaneouiily ,tnd follo11·ing thi" up ''"ith a larg-P test do;.;e of spores (50 to 100 m.l.1l.) iuto the same an'<l aftt~r Y;n·ious i11tenals. 'rl1ere ''""s inhibition of a fai1 h· high ouler, consi<lt·rahly higher titan ,;]to\Yll in the first se1·ie:s of expe1·imeuts. Tbi;.; 11·as sho11·u \n· infbm- uwtions hom 8 to 120 hours old, hut 1n1s at its nwximum 11·ith illflam- tuations 24- to -!8 houn; old. Tlw inhibitory pftect of inflalllmatiouc; younger tlwn 8 hour;; wa;; of the sante orde1· as th~ general pffed notecl pre-viously.

Again the irritants that cau;.;ecl ne<-rofiiH IYithout nnuke1l oerlt•ma lwtl little pft'ed on Yirulen ce (table 2 A).

Fp to this point, the result,; agreed 1•·ith those of Boquet aml Stamatin.

·\Yitt•n, however, the test <lo6e \Yac; reclll(·ecl to ] /600th of that previously given, the re,;ulb; ohLtinf'll ,,·erP Yer~· different. In tht• experiment set nut in table 2B. thi,; smnll close 11·as in.it>ded into a ;.;aponin inAamecl area ai 0 to 480 minutes after thP saponin ir.jedion. The zero time represent:;

saponin ancl ~pores g-iYen simultaneoush. The c:onhol g-roup 1·eceive(l the spores in a normal sit.-. It is dear, from the htble, tlwt the cleaths still ot'l·lute(l l:Ite1· iu tl1e saponin gToups: lmt tlte proportion of draths in the~P

gwup,; was con;;i(lerahly hip:hf'r than in the c·ontrols. This complete reYersal of tlw finding,; with larger closes of spnret< is fa1· more clearly shown in the

~erif's of ex]Jeriments sumntarisecl in table !1.

:J. The 7-:.fject of :1/i.rinr; .4uthro.r SJ701'es 11•itl, "0'.l'l'if1ients ('rl1lgin!}

Nen·os1·s nnrl lnflammotion.

ThP effect of Y<uious e:-;:c·ipients ^'lll the Yinllen1·P of lllt!·ro-organisms is of great practical importance. Hru:ska (1931) and :M:azzm·l'hi (192!)) and others claimed that anthr<lX spore~; in saponin were consiclerahly recluc·e1l in Yirulence. There has l1ePn a lack of unanimit~- on this point. In table :{

are set out the results of using cliffe1·eut exci]Jients ''"ith closes of anthrax spmes varying from npproximately 100 lethal cloiies to a fh-e-hundredth of thi:; amount.

15B

... ::,;1 <:.Z>

TAJH,J') 1. 1.!-'ffect of Inflammation P1·ovoked by Different Chemirals on 50 to 100 Lethal !JosPs Anthm.x Spores injected 2 i Hours Latn at A noth wr Site. No. of Guinea Pigs. 20 ... 20 ........ .. 20 ... .. 70 ......... . 20 ........ .. 20 ......... . 20 ..... . 3/i ......... . 4i'i ......... . 20 ......... . 20 ......... . 10 ..... . 240 ......... .

'allowing 0·5c.c. of chemic~tls s fore-lim ilcnt. in b. 2 pel' cent. sap on in pet· cent. sap on in } per cent. s~tp onin

±

per cent. sap on in } pet· cent. sap on in 1/ to per cent. s cp.:>nin 1/32 per· cent. s cponin 10 per cent. C Cl2 Turpentine 20 per cent Xa ,so, 20 per· cent. X .CI 10 per cent. Ia tie acid Nil 24 hours later 0 ·1c.c. spores in hind-limb. 2nd

I I

day. I W-100 m.l.d. :'i0-100 m.l.d. !i0-100 m.l.d. 50-100 m.l.d. l t'i0-100 m.l.d. 50-100 m.l.d. l !i0-100 m.l.d. (i !i0-100 m.l.rl.. :) I W-100 m.l.d. I 4 50-100 m.l.d. 20 50-100 m.l.rl. 19 150-100 m.l.d. 10 ;)0-100 m.l.d. 188

RESliLTS. D b 3rd

I

4th

I

5th

I

6th 7th day. day. day. day. day.

I

Lived. 5 ll 4 0 10 7 3 0 4 12 2 2 0 33 32 4 0 12 5 2 1 0 11 6 I 1 0 9 5 I 17 10 2 l

0 0 I 19 18 2 l 1 0 l 0 0 46 (i 0

l!:; ;... ~

"'

..., t=l i=tl ~ ~

,_. c., 0 '1'.\BLE :~. /Cjfcct uj Injecting 50 to 100 Lethal Dose" A.nth·ra:r: Sz1ores iulo lu.flatnmotions uj /Jijjl'rcut Llyes. -- R~;SlJL'l'S. Xo. of Inflammation 50-100 m.l.rl. Dead by I J,ivcd Guinea pmvoked by spores into Pigs. 0 · 5cc. subcut. inflamed tt~·ea I I I

I

I

I I I

I

of after 2nd 3r·cl 4th 5th 6th 7th 8~h *nth :1\o. I Per- day. day. day. day. clay. clay. day. I

day. centagc. 2!) ! per cent. saponin 120 hours 9 lO 3 1 I

2 8 36 :l: per cent. saponin 72 hours 5 10 3 1 1 l 15 42 25 ;} per cent. saponin 48 ,hours 3 l G 1 1 3 10 40 21 l per cent. saponin· 32 hours 2 2 2 lfi 71 JO;j :l per cent. saponin 24-hours 1 :2 7 13 9 5 ·1 64 61 41 ! per CPnt. saponin 8 hours l 4 II I

3 I

I 4 ;) 1:2 2(1 20 .:-per cent. saponin 2 hours 9 7 I

:) 1 0 0 83 Nil (controls) spores only 60 I 22 1

• I

0 0 I * = 9th and following clays. TABLE 2A. Com.parison of Effects of Injecting 50-100 m.l.d. :dnthraa· Spores into 24 !lom·s' Old .Yecroses flccomwmiecl /;y Oedema (fl), and Cnaccompawiecl b.t; OedPm.u. (B). I I RESULTS. 1\ o. of Iniiammation 50-100 rn.l.cl. Dead by I Lived. Guinea pr·ovokecl by in necrosecl Pigs. 0 ·lice. su bcut. area after I I I I I I

I

I

of 2nd 3rcl .Jth lith 6th 7th 8th *nth No.

I

Per- day. clay. clay. clay. day. clay. da.v. day. ccntagc I ** 105 A i per cent. saponin 24 hours I 2 7 13 9 ~3 4 64 61 40 A 10 per cent. CaCI2 24 hours 3 ~) 7 :1 li r; ;) 7 18 40 A Turpentine 24 hours I 3 6 7 6 :l 7 3 4 10 40 B 30 per· cent. NaCl 2-1 hours 13 18 5 2 2 0 0 10 ]3 20 per cent. Na2SO, 24 hours 7 3 I

0 I

0 78 ;Iii] (controls) Spores only ;)7 20 1 0 0 ; I -- *nth = 9th and following clf1ys. **A = oedema. **B = no oedema.

>-< ~ >,j r ;.... '-" ,..., <' ,.., :...

"' 8

~ ~ [fl Ci ;;:; <j >-< < > :-' ~ ~ c-: >-< ~ t=J )> ,. cr. .... ~ >,j ~·

s s:

, , """' '"" ,_ H > ~ '"" ~ ;:::1 ;... ;..:

TAl1LE 2B. The Effect of Injecting a Small Dose oj Anthrax SzJores (C'irca lf 10 of a Lethal Dose) into SitPs ptCjJrtred with Saponin. -----==-=-=-=== No. of Guinea Pigs.

Inflammation pro,·oked by O·lcc. of

1/ 10th m.l.d. spores injected into inflamed a•·ea after

RESULT~. Dead by 3•·d 4th 6th 7th 8th 9th nth day. day. 5th I day .. day. day. day. day. I

day. ... C':> 40 ... 40 40 40 40 40 40 40 40 40 JO 40 80

~ per cent. saponin 1-per cent. saponin k per cent. saponin 1 per cent. saponin §-per cent. saponin ~per cent. saponin 1 per cent. saponin } per cent .. saponin {-per cent. ~aponin 1 per cent. saponin -}per cent. s>tponin -~ per cent. saponin Nil (srores only).

480 min. 320 min. 240 min. 160 min. 120 min. 80 min. 60 min. 40 min. 20 min. 10 min. 5 n1in. 0 min.

2 l 6 2 3 l 7 3 ll 9 J 3 14 7 17 8 2 2 18 10 3 13 l() 2 17 10 4 22 13 2 ll 7 1 5 nth = lOth >tnd following da.ys.

2 3. 5 ll 8 ll 4 8 2 5 7 2 1 8 3 3 3 2 l l l l l I I 2 I 3 1 1 3 l

J,iYed Xo.

I

^{Per- ren}

tage . 19 48 7 lS 25 63 18 45 20 50 17 43 14 35 10 25 5 I :1 3 i-l 7 18 3 8 n~ 65 - .... '""

t<

[fJ ..., t:j P:> '2! t'l

I

I:\"FJ •. HHL\TlO:\ OK Sl"RVI\"AL OF <;t:IKE.\ l'l{iS IXl'J·:CTEJJ \\"l'lTI ,1.:\TJIU.IX.

The JeYelopme11t of the larger Joses was markedlY retarded bv excipieni.s th,, t eli<·ited oedema, but not bv excipient~ can Ring netTOBio; with little oe<lenw. The retarcling effed of i:hf' sDponin iB dearly evident. Whe11. the

<lose of spore" waB re<luce<l to a half and to a tPnth of a lethal cloRe nn enhrely different result appeared. All the excipients now used markecll:v increa8e<1 the 1·iruleuce of the spOl'es. Althoug·h saponin and eal<·ium chloricle, which provoke an oedemn n'sponse, <lelaved th·~ onset of cle;Jth, the end result

wa~ far more <leaths in the groups whPl'e .'lll irritant ex<·ipient \\·as em]Jloyed than in thP !'ontrols. A striking- result was obtained vdten small doses of Yegeta ti Ye ha!'illi "·ere used i11 stead of SlJores. An excipient con taiui ng a small amount of hiRtamine also ine1·enE.ed the Yirulen!'e of small <loRes of ,;pores.

The points emphasized are thnt ex<'ipients su<'ll <lfl saponin all<l calcium

<·hlori<le, whi<:h eausf\ 1wcrosis a11d oeclPma, slow dow11 the development of large anthr:1x ino!'ula, "·hile initants su<'h as concentrated Ralts, bdie a!'id, etc., whi!'h do not <>licit ¹¹¹¹ oe<lema baYe litth~ effect. HmYeveJ·, all these irritants, whether they retard thf' cleYelopment of large inot'ulu or not, ma1kedly inerease the killing power of small inocula. Even here -the initnnt.s. that provo];(:' ;111 ot>d<:'ma delay +he ·onsPt of <leaths.

Dv.;cussiOK.

'l'lw g·ellf'L'<ll actiou of inflammation accon1pani<:'d by !'Onsiderahle oedema on thP Yirulen!'e of :111tlnax is interesting; hut the <lis!'tu;sion of this and relnt<:'<l ph<:'nmll<:'Lla "·ill he left for a sep:n·Jt<:' papt>r.

The pffed~; of iHjeding anthrax spores into an inflamed area, or of iujPeting- spores or h:l<'illi with initant ex<·ipients, <lepend on tl1e nature of the initant an<l 011 the dose of spores ·or bacilli used. The apparently con1raclidory ohserYntions ma<le here that ex!'ipients sud1 as saponin innease thP ,-irnlence of small inocub and denPase that of brg·e inocula is one of the 1·easons fo1· tl1e conflic·ting- reports on the action of saponi11 fmmcl in the lite1·ature. The effect:-; of lo('al :.uul general bdon; eYol<ed by iuflamma- tion Y:uv with thP ^,;[;r,p of thP :mthrax ino<·ulmn. 'l'he initial t-iRtme <l<:'Rhuc- tiou <·at;sed by ;my i.nitant, fayours the nndtipli<;ation of small inocula of about 'f,,th to

t

a lethal doRe, ~.;o thai" the effed iw value of the ino('ulum is inl'rem;ed from a hadion to :1 full lethal close. Su!'h an increase iR verv :-;triking, fnr more so thm1 a similur iucrease imparted to sav <l 100 letlu;l doses, which alread~r ex('er!'ise a maximum pffect. Although thP initant acts in tlw same '"a~- ⁰¹¹ botl1 suwll and larg·e ino('ula, only tbe effed. on the former i:-; eYirlent. The w·nend retarding effect of inflammation also acts Oil both ;;mall and Lng-e inoeula. In the former, the general retarding efft>d, although shcmn b~· tbe delayed onset of dt>atbs, is more than counter- halnncecl by the in('reafie<l rate at which the hn!'illi multiply in the injured tis:-;ue. 'rhis stimulation is not Ro <1h1·ious \Yhen largP ino<·ub are usP<l; when the general rPtarcling eifect of inflammation on virulen('e bec·ome~

more prominent. All the initauts te~:;t<·<l inneasecl the killing power of snwll inocula. Tho;;e irritant,; that also provoked oedema formation caused a dt>lay in the onset of deathfi, eYen where the final tall:v showed that. tlw killing power of the inoculum was enhaJU·ed. Only those irritants evoking oe<lt>nw fonnatim1 PxCelTised an inl1ibitmy elfect on large inocula.

lf)2

No. of Guinea Pigs.

TABLE 3 Effect of Difje1·ent Errcipients un l'orious .clntluax lnoc·ula. Excipient. 0·2cc. 1} per cent. saponin 0 · 2cc. -} per cent. saponin 0 · 2cc.

±

per cent. saponin 0 · 2cc. ?. per cent. saponin 0·4cc. 20 per cent. Na.''l04 0·4cc. 30 per cent. NaCl 0 · 4cc. 0 · 85 per cent .. NaCl.

Dose spores. .~0 m.l.d. 50 m.l.d. 1\0 m.l.d. 50 m.l.d. iiO m.l.d. 50 m.l.d. 50 m.l.d.

I

RESHI.TS. 2nd day. IS 14 46

Dead by I

3rd 1 Jth 1 5th 1 6th 1 7th 1 nth 1 day. day. day. day. day. day. 1 8 I

5 I

2 :) 16 8 6 4 2 7 4 3 1 1 9 9 J 1 2 J 13 3 I 3

Livecl -- Xo.

' I

Percen ta(!e. -1-20 1 2~ 2 10 0 0 0 0 0 0 0 0 ---1---'---•---·---·---·---1---1---·---- 0 ·Icc. 2-! per cent. saponin 0 ·lee. 1 per cent. saponin 0 ·lee. } per cent. saponin O·lcc. {-per cent. ~aponin 0 ·lee. 30 per cent. NaCl 0 ·lee. 15 per cent. NaCL 0 ·lee. 10 per cent. NaOl 0 ·lee. 5 per cent. NaCL 0 ·lee. 50 per cent. glycerine 0 ·lee. 25 per· cent. glycet·irre 0 ·Icc. 5 per cent. glycerine 0 ·lee. 2 per cent. lactic acid 0 ·lee. 1/10 per cent. histam;ne O·lcc. 0·85 per cent. N>tCl O·lcc. ~pet· cent. saponin 0·1ce. 0·85 per cent. NaCI 0 · Lee. t per cent. saponin 0 ·Icc. ~" per cent. saponin O·lcr. 8 per cent. Ca.CI2 0 ·l cc. 50 per cent. l!iycerine O·lcc. 20 per cent. NaCI 0·1cc. 1/100 per cent. hi>t.amine

5 m.l.d. 5 m.l.d. 5 m.l.d. 5 m.l.d. 5 m.l.d. fi m.l.d. 5 m.l.d. fi m.l.rl. 5 m.l.d. ii m.l.d. 5 m.l.u. ii m.l.d. 5 m.l.d. 5 m.l.d. ! m.l.d. 1 m.l.d. 1/roth m.l.d. 1/10th m.l.cl. 1/10th m.l.d. 1/10th m.l.d. '/10th m.i.d. 1/,0th m.l.d. O·lcc. 0·85 per· cent. XaCI 1 1/10th m.l.d. --1---·---: O·lcc. 20 per cent. NaCl

l ' /,

0th m.l.d. (bacilli) O·lcc. 0·85 per cent. NaCl 1/",,th m.l.d. (bacilli)

H 9 16 5 9 6 3 6 7 28 2 11 nth = 8th and following d<ty<.

l 6 16 5 4 11 7 10 lO 13 8 36 4 10 1 8 21 40 5

I 8 10 fl 5 4 3 4 4 l 3 ll 33 9 22 38 37 28 10 8

2 5 3 .') 1 5 :) 4 J3 5 2 1 2

9 J 1 2 1 2 4 2 2 1

5 2 2 2

3 1 l I 2 1 2

3 3 1 0 4 0 0 0 0 0 1 0 l 4 7 20 3 -* 1 0 0 4

15 15 5 0 lO 0 0 0 0 0 5 0 5 4~ 14 40 8 s ') 0 0 20

-:; 1 ~ 1 + 1 -

⁵

1 - --1 -' 1

¹ :;

- i :; - "" ::

~

"'

~ ~ ~

IN:F'J •. UBL\TlOX OX SLitVIV.IJ. ()}' (;L'IXLI I'J(;S I:Sl'H'TI·:D \\Tl'll .INTlllUX.

tlDDLI.HY .\xn Co::snxsroxs.

(1) Tl1e deYelopment. of anthrax in g-uinea pig-Fi io; slowe<1 down if LUI acute inflannnahon accompanied by an oe<lema is proYoked elsewhere in the body. ThuF; tlw injection of saponin into a fore-limb m· into the peritoneal caYit_y 1·etanls an anthrax ino(·ulum in the bind-limb. 'l'issue destruction with incom;i<lerahle oedema, as caused hY (·oncenhated salt solution, does

not haYe this effPd. ·

(2) Large closes of :mthTax injectP<l into inflamed m·ea,; are retar<le<l and ·often <"ompletely inhibited. HoweY<'r, the killing p<nH'T of fractions of a leihal <lose are ellhance!l if injede<l into ear]~, inflammatory, oedematous le,;ions, although tlw on~et of <leath:; ma~· be clelaye<l. The effects of injecting Lng-<-' ino<'ulu into ne<To;;e<l 110JH>eclematous areas a1·e 1wither increar>ecl 110r diminished, \Yhile the killing po11·er of r>m:dl inocula ifi marke<llv increased.

(;~) Ex<"ipient;; F;uch ;~s saponi11 \Yhi<·h cause tisRue de;;hu<"tion <~<·c·oin­

punied h~- <"on;.;iclera bl e oedema ;;lo ,,. d mn1 the deYe lop men t of lm·ge dose;;

of anthrax. Snwll <lo;;e:-;, ±radium; of :1 lethal <lose, are, however, stimulatt>d and their killing- powe1· gTeatl~- innt>a~erl. althoug-h the onset of deaths ir>

some1dw.t <lebyed. Excipient,; such as concentrated salt, which cause neerosiH with little oe<lenw, hLne no appart>nt efft>l't on larg·e anthrax inocula, lmt increase the killiJl)!." p011·er of fradions of a letlwl close, without delayin~·

the 011set of deaths.

(4) 'L'lw appare-ntly anolllalou,; action of iuft.ammation ou small ancl on brge ino<"ula is expbinecl bY the fad that if stimulation 1·aiseR say a fifth of a lethal doRe to a full lethnl <lose, the effect is Yery obvious, whereaR the rai,.;ing of a lnmdrt>d lt>thal <lo;:;es an equiYalent amount "·ill he virtuall~­

undt>tedable.

l~EFFRE:\'CES.

BECHHOLO, If. (1922). Ticrexperimentelle Studien i.iber Kol!oidtherapie. Munch. Med.

Wochen, Vol. 69, No. 41, pp. 1+47--1449.

'ROQUET. A. A'<D STAMA'l'I~, X. (1939).

sur !'evolution du charbon bactcr·iclien.

Action empechantc des foyers inflammatoires Ann . .Tnst. Past., Vol 63, pp. 9-40.

HRUSKA, C. (1931). Vaccination contre le charbon bact6rirlien avec Jc virus non attenuc.

Compt. Rend. Acad. &., Vol. 192, pp. 822-823.

INSAEFl". (1894). Unter·suchungen i.ibcr die ki.instlichc Tmmunitiit gegen Chn1era. Zeit.

f. Hyq., Vol 16, pp. 287-:l:l8.

KEPINOV, T,. (19~4). !~tude sur l'immunitc nnn spcrifiqne. Action immunisantc des filtrats bact6rien non spi'cifique sur !'infection chol6riquc. Oomt. Rend. Soc. Bioi., Vol. 91, pp. 244-246.

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