Onderstepom·t J ou1·nal of Tl eterinary Sc'ience and _-l ni mal Industry, Volume 23, Numbe'rs 1 and 2, ilfw·ch, 1948.
'Printed in the Union of South Afrira by the
j : '
Government Printer, Pretoria.
The Effect of lnflamma t ion on of Immunity to Anthrax in
the Development Guinea Pigs.
By :MAX STEHKE, Sectwn of Bactenology, Unde1·stepoort.
The effect of infiammation on the virulence of anthrax in guinea pigs was discussed in the preceding paper. 'l'he effects on immunity, which are of more immediate practical importance, will be considered here. Most of this type of work on anthrax has been clone in ('Onnedion with the saponin vaccines of Mazzucchi (1929) and Hruska (1931). These workers usefl anthrax strains suspenlled in .saponin solution as vaecines, and 8howed that the intense inflammation which followed inoculation resulted in a decrease in virulence and in an increased production of immunity. Some years ago [Sterne (1939)
J,
I reviewed in detail the conflicting reports on .:\i(azzucchi's va.e<:ine, and confirmed, in the accompanying paper, that a saponin excipient increased the immunizing· power of anth1·ax vaccine.'l'he work reported on here makes clearer the circumstances in which this increased immunizing power occurs.
ExrERBIENTS.
'l'he " vaecine " used in these experiments was a sporulated culture of an immunizing, avirulent, uncapsulated Yariant of a virulent anthrax strain. [Sterne (1945)]. The dose used to test immunity \Hts approximately 100 lethal doses of a guinea-pig virulent strain.
1, Th8 genentl Effect of acute Inflammation on the Jlrudw·tion of lnwnmity to Anthra.v in Gu,inea Pigs.
It "Vvas shown in the preceding paper that an acute inflammation any- where in the body retanled the deYelopment of a killing strain of anthrax.
It should, therefore, also retard the developrneut of a vaecine strain and so lower the degree of immunity produced. Preliminary experiments indicated that this \YUS the case, and, accordingly, the following experim.ent - summarized in table 1- was arranged to illustrate this point. One lot of guinea pigs received 0·5 e.c. of a
±
per cent. saponin solution subcutan- eously on the abdomen, followed 24 hours later by 0·1 c.c. of a sub- immunizing dose of vaccine subcutaneously in a hind-limb. Another lot of guinea pigs received vaccine \Yithout any prelimin:ny treatment with saponin. Four weeks later. the guinea pig-s in both groups were inoculated w1th about a 100 lethal doses of the vuulent test strain. 'rhe result;;(which conformed to the results with similar tests on killing strains) sho"Vvecl that significantly fewer guinea pigs were immunized in the saponin- prepared group.
INFLAMMATIOX OK DEVELOl'MEKT OF IMMUKITY 'J'O AKTHR.\X.
Thus the development of a.nthrax i,; retarded by an acute inflammation situated r~t a distan!'e from the site of infection. 'The inYasiYenPss of virulent strains and the immllllllllllg' power of aYiruleut strains are rechu:!-'d.
'l'AliLJ:: ].
The effect. of injfo111111ai'ion caused by suponin on tl1e tll17n1tnt:nng fiOtCf"l'
of a.ntlrra:c injel"led at 11 different site.
Lived.
Xo. of
fnHammation Test.ed
Guinea
provoker! by Immuni,ed with ~ weeks latf'.r
Pigs. with
Ko. I Perecn-
I
tage.50 O·iicc. 1/,,th per cent. Small dose avirulent 100 m.l.d. spore; 13 26 saponin in fore-leg spores in hind-leg
:)0 No inflammation Nmall dose avirulent 100 m.l.d. spotes 33 66 spores in hi.nd-h•g
10 ~0 inA~tmmation Xot immunized 100 m.l.d. spores 0 0
-
2. The i!)ffecf.s of different E.cc!'[Jienfs on the lnwwnizing Powe1· of a·c·irulent Vacr;l~ne Sf tains.
From thP results with Yindent strnius in the pret·ediJlg- paper, it woultl Le expel'ted that the excipient would han little eftect on the imnnmiJ~ing
power of large tloses of Yaceine, but 11·ould iunease materially the immuuit.v elieited by small Closes. This was found to be the ras·e. Large doses imrnuuized so well, with or without any special excipient, that signifieant differences between groups '"ere not perceptible. \Vhen ,-nuall immunir.in;r doses, which diil not elicit a solid inunnnil.y to the challeng·ing rluse, 11·ere usetl all the irritant e:s:cipieni~ tested enhaneecl the immunizing powe1·.
The eff·ects of inoculating g-uinea pig-s IYith sub-immunizing amounts of vaecine suspended in tliffereut excipient~-; are ,;ummarized in table 2. The results paralleled those obtained with small doses o£ virulent spore;; in 1·arious exeipients. All tl1e initauts fnvometl the multiplication of the Yaucine (a;; eonlrl be tested by puncturing- and examining· the lesion), :md cons.equeutly iurreased the immtmizing- power. This improYement was propo1·tional to t h P amount of reaction protl ueecl by t h·e i rri tan t. ~a po nin, CaClz, 20 per cent. Na.,SO,, 20 per cent. :XaCl, had the gTeatest effect in inneasing- the immunizing· power, 50 per cent. glycerine had a noticeable effed, r~lum \HI!' le>'s eftediYe, allll ag·ar hatl no clenwustrahle eff.ect, iu the concPn tra tions used.
Discrssro)l'.
The >York reported in this and in the preceding paper sho11·c; ihat f'evern l factors must he eonsiclerecl wheu d isl'ussing the-effects of i nHanww- tion on innnuniiy in r~nthrax.
An arute inflammation pl'OYoker1 anywhere in the hotly hm; the samP genetal rPtanling eftect on the tleYelopment of a Yaecine Rtrain that it has on the development of a virulent 1<hain. ln the first case the result rs a tlimiwsheti immunity arul in ilw ~eeoud n dimini~hed vi1 nlence. 'l'o this
166
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'l'ABLE 2. ]<)!feet of vm·im1s etvcizJients on tfte Hl1111U1Hz£ng power of :;111all doses of arindPnl w~thra,v vacnne. No. :Jf Guinea Pigs. 20 20 20 54 28 20 62 52
[mmunized with Sma 11 close a ,·irulent spores Small dose avirulent spores Small dose avirulent spores Small close avirulent spores Sma.ll dose avirulent sp01·es Small dose avirulent spores Small dose avirulent spores Not immunized ......
Vaccine suspenrlecl in 2 per cent. saponin ........ l per cent. saponin ........... ~-per cent. saponin .......•... t per cent. saponin ........... ~ per cent. saponi11. ............ 1/;6 per cent. sa.ponin .............. 0·85 per cent. ~aCI. .................
Immunity teEted 4 \reeks later with 100 m.l.cL. ...... . 100 m.l.d ........ . JOO m.l.d .. . 100 m.l.d .. 100 m.l.d .. 100 m.l.d .. . 100 m.l.cl ..... . 100 m.l.cl ..... .
Lived. Ko.
I
Percentage. 19I
91) 20 100 20 100 .~2 I 96 23
I
82 13 65 15I
24 0 0 ---1 1--- 6 20 24 20 32 32Small close avirulent spores Small dose avirulent spores Small dose avirulent spore3 Small close avirulent spores Small close avirulent spores Not immunized ............ .
:!0 per cent. XaCJ................. 100 m.l.d......... {i 100 20 per cent. NaCI............... 100 m.l.d..... 19 95 10 per cent. NaCI.............. 100 m.l.d.... 13 !i4 !\ per cent. NaCI..... . . . . . 100 m.l.cl.... 1 !\ 0·81) per cent. NaC:l....... .. .. .. . 100 m.l.d......... 4 13 100 m.l.d......... 0 0 --------! I I 1---1--- 46 20 :JO 48 48 24 20 20 13 17 06 75
Small close avirulent spores Sma.ll dose avirulent. spores Small dose <Wimlent spores Small dosfl avimlent spores Not immuni7.ed ...... . Small dose avirulent snores Sma.ll dose avirulent spores Small dose avimlent spores Small dose avirulent spores Small dose avimlent sp:)res Small dose avirulent spore> Not immunized ..... .
!SO per cent. glyce•·ine .............. . 25 per cent. glycerine .......... . lO pet· cent. glycerine ............. . 0 · 85 per cent. NaCl. ............ . 2·5 per cent. CaCI2 ••••••••••. 20 pet· cent. Na2SO, ... . 130 per cent. glyce•·ine+ 10 per cent. Narl } per cent. alum ........ . O·J per cent. agar .... .. 0·85 per cent. XaCJ. .. . 100 m.l.rl. ....... . 100 m.l.d .. 100 m.l.d ... lOO m.l.cl .. 100 m.Ld .... . lOO m.l.d .. 100 m.l.cl.. 100 m.l.cl .. 100 m.l.cl ...... . 100 m.l.cl .... . 100 m.l.d ... . 100 m.l.d ..
:30 65 3 lfi 2 10 !) 19 0 0 ------- 18 75 19 !Vi I! I) f) 6 46 2 12 :30 31 0 0
::::: 1>- ~ Ul '""' t--: I=" '2: t-;
IIS"FL.UHIATIOl'\ O" DEVELOI'MEXT OF IMMUl'IITY TO .-I.IS"THILIX.
Pxtent, the use of an initant excipient whidt causes a ma1:ked oedematous reaction lowers the immunizing power oi ~a vaccine. Ho\Yever, the effect is counterbalanced, and in appropriate ~ircumstan<.;es more than counter- balanced, by the incTeased rate of multiplication of ~pores o1· bacilli in
damaged tissue. ·
This stimulation JS bbt demonstrated by injecting subliminal immunizing doses of spores m'acle "\IP in a:u irritant excipient. The iiicreat>e in immunizing power is considerable; lnit, it must bf' emphasized, this is only evident when small closes of vaccine are used. The process is analo- gous to that seen with···smaH doses of virulent spmes. The subliminal immunizing dose multiplies rapidly in the area a:fi:eded by the irritant and attains the threshhold n'ecessary for immunization before the defence mechanism i, full:v m·obilized. This incTement is rf'a(lily perceptible, far more so than a siinilar . increment to a: large immunizing dose, which immunizes solidly, ii'tespecti ve of the excipient used.
The antagonistic action between early stimulation due to tissue (leshuc- tion and later inhibition due to the development of acute inflammation is the key to the interpretation of the effects· o.f inflammation on immunity m anthrax. \Vith an irritant such as saponin, which eauses extensive necrosis ac<.;ompan ied by oed.ema, both .factors opei'ate. HmYever, when small doses of immunizing spores are used, the early stimulus, which con- verts a fraction of an immuizing dose into· a full immunizing dose, out- weighs the general inhibition developing- later. ·when large doses of spores are used, the inhibitory effect becomes . the more prominent. It is not unreasonable to (lraw analog-y between these experiments and those d(J'ne p1·eviously with virulent strains, and to rlecluce that the. secondary inhibi- tion is not induced· hy initants such ns concentrated NaC'l, which do not provoke oedema formation. ·
Saponin is widely used in practice as an excil)ient for autluax vaccines.
!fazzuchi claimed success for the use of. virulent strains suspended in 20 per cent. saponin. 'l'he very violent reaction this cam;ecl greatly limited virulence. Had M:azr.ucchi used attenuated ·straiiJS he wou~rl have found, as others found, that immunizing J>Ower \v~s a·lso decteasecl. He happened to strike a precarious balance between the high immunizing power of a virulent strain aud the· very marked. inhibitory effect of the reaction caut>e(l by the high concentration of saponin used. _Staub (1932), and Hamon anrl Staub (1936), who· suspended .attenuated strains in con'centrated Rapouin obtained poor immunity. They were working in the inlhibiting range of saponin activity without the compensation of using a. virulent strain.
Workers who used lower concentrations o£ i;laponin with Rubstantial numbers of ~pores we1·e unable to detect .any advantage, as they were using spore doses too large to be appreciably benefitted by the saponin. N everthelesR, the use of saponin spreri.d, because the concentration of saponin that gave tolerable reactions in practicE),. and the dose of spores uRually included in vaccines were in the range withi'n which stimulation occurs.
Ramon and Staub (1935,--Hl3&) strongly recommended excipients of lauolin and olive oil_, or of a mixture of alum and agar. Ramon considered the action analoo·oils to that of alun1 on _formol-toxoids-slow absorption reRnlting in a prolonged antigenic stimulus~ I have been unable to find any
?vidence for this contention. The increased immunity is clue to the rapid
~ncrease of t:he inoculum in damaged tissue, and is elicited by any Rufficiently unta.nt exc1pient.
168.
)L\,X STBlH\E.
It i:; worth noting that ,-JO per cent. glycerine (used in the Onderste- poort vaccine as a presen-ative) has a fitimula.ting action on immunity.
although this is less marked tlHm that of saponin or 20 per cent. NaCl.
'l'he simplicity and cheapness of the 20 per cent. Ka.Cl excipient have encouraged us to start field trials. It has aclvantageH over ::mponin in that it does not foam and also acts as a preserva.tive. Its lack of inhibitory action might be a disadvantage with Pasteur type vaccine, hut is probably not important '"ith the avirulent :;hains used at Onclerstepoort.
There was no evidence that the henencial action of saponin \\·as (]ue to any effect on the bacilli. [HomotoY (19=36)
J.
The action on the tis:mes is sufficient to explain the main fin(lings. 'l'he dose of spores usually inclucled in vac:ciJteH falls in the range appreciably benefitted hy an irritaut PXCipient.Sc;~DL\RY.
J. An aeute inflammation has tlw same general inhibitor~· efted on
imnnmi:r.ing power that it hac; on the virulence of anthrax strain:-;.
2. Irritant excipients have the same local stimulating effect on small immuni:r.ing closes of nntlm1x th<tt they lwn• on small virulent doses.
;}. 'l'he apparent <lifl'eren<"e in tlte eftect of irritants on small and ou Lng·p immunizing doses of ;;pores is he(·ause the rai.:-;iug of a sub-immunizing dose to a full innm.111 i :r.ing i,.; far more pen·eptible th8n the Rame order of i nnease of a large dose.
4. 'l'he probable achantage:- of nsing- 20 per ('ent. XaCl solution as an t>X!'ipient nre pointed out.
The bearing ·of these findings on earht>r reports IS diseu,.;sed.
l~EFERENOES.
HO~fOTOV, P. (1936). T.e ro1P de Ia Rapoqin rlans le vaccination anticharhonneuse. Ann.
lnst. Past .. Vol. 56. No. 5, pp. 535-!)83.
HRUSKA, C. (1931). Vaccination contre le charbon bactel'idien avec le vims non attenu£'.
Compt. Rend. Ara.J. Sr., Vol. 192, pp. 822-823.
~'fAZZUOOHf, M. (1929). Risultati rli un nuovo metodo di vaccinazione antiearhonchiosa eon alte rlosi di germi c rli spore l'irulPnti. La Olin. Veter., Vol. 02, pp. 662-671.
L~A?.lON, G. AND Wl'AUR. A. (1931\). Essais sur Ia 1•accination charhonneuRfl. Cornpt. Rend.
Soc. Rio/., Vol. 119, pp. 10'73-1076.
RA\IOCif, fJ. .\Kn STA UR, A. ( l936). Ess:1is sur I' immunisation contre le charho11 'll" unc nouvelle formule de 1·a~cination charbonncu,e. Bul. Acad. Veter., France, Vol. 9, :No. 7, pp. 375-3S7.
STAUB, A. (1932). Snr· ilt vaccination antirharbonncuse. Crnnpt. Rend. Soc. Biol., Vol. J 10, pp. 12l4-12l,i.
STERNE, M. (1939). The use of saponin spore vaccine for inoculation against anthrax in South Afric·a. Onderslepoort Journal Vet~r. Sc. anrl Anim. Jwlusl., \-ol. 12, pp. 279-362.
RTERNE, iVJ. (1945). Avirulent anthmx vaccine. Onde1·stepoort Journal Veter.
s,·
and Anim.lndu.•t:, Vol.· 21, pp. 41-43,.
