Onderstepool"l Journal of retP7'lii01'Y Science and d.niowl lllrllrstry, rolume 6, ~rnmber l, l11ly, 1936.
The Lethal Dose of the Toxins of S ome An a erobes for Sheep.
By J. H. :JIAS05, F.R.C.Y.S., F. RS.E., J•;mpire ~Ia1·keting Board Re;;earch FellO\Y, Onclerstepoort.
A C'Oi\DlOX methocl of cletenni.ning- t11e immunity prollnc-ecl in sheep by the injection of antigens prrpa.recl hom the sporulating anarrobes is the i.ntnnnuscular or ~uhc-utn 11eons injection of liYin~r culture.
Pr·oYidell th:1t one ha~ had ronsiclerahle experience with the nutigen and the organism in qneRtion, liitle objertion can be raiRell- a nrugh answer, 1tS1tally sufficient for practical purposes, is obtai11e(l. How- ever, not infrequPntly the test culture, preparecl in the ;;ame manner as on Jll'eYimls occasions, fails to kill the eon.trob or test auiuwls that should llie, and thus the ex.per.iment is rellclerecl Yalneles,;.
\Yith the exception of ('l. dw?IUJPi (causing black quarter), the anae1·obes responsible for lliseases ill sheep are toxin procluct•r,;. 'l'hese are 07. septique (brax~·), the lamb clysentery bacillus or('/. 1celchii, Type B, \Yilsclou (lamb dysentery, bloeclpens), Cl. oedematifns (black
<li,;ease), <IIHl r'l. orito,til'us or Cl. lcelrlu·l, TypP ]), \Vilsllon (entero- toxaemia a 11 c1 ]1\tl py k icln ey cl i ~ea ,;e).
Since the toxins of the anaerobes are reallily stahilizell by pre- cipitating toxic filtrates with ammonium sulphate and llrying-, and, huiher, siure the injection of toxin is a more aceurate metholl of testing antitoxic immunity tlum is the injection of culture, tltP writer decided to COlll.[Jare the toxicity nf the Yariou;; toxins lll i:iheep and in mi('e alld g-uinea-pig,;. The results are prei:iented here, in the hope that they may serYe as a. guidP to others who have 110t had a suffic-ient number of ~heep at thei1· tli;;posal to e;;bblish the 1·atio behYeen toxicities in mice anll sheep.
All the organisms were· " :oingle-celled '· prior to the commence- ment of (he test. ~Iorpholog·ic.a 11)·, cnlturally, biocl1 emic-all.,-. nnd as regards their pathogenic effect ou gninea-pigs, they behaYe<l in the manne1· tle;;!'ribe<l fm t~'pic~tl -traius. The ('l. sepft!JIIf' and Cl.
oerlematiens cultnres were originally obtained from the :National Collection of 'rype Cultures, J..~ister Iustitute, the Cl. ovito,vicus strain (R2 ) hom Dr. Bennetts, and the lamb dysentery organi:;m was a suh-culttue of that originally isolated h.'' ~fajor Dalling·. It should he not eel that i his 'l'ype B straiu was of the " 19:·w ntriety "
()IontgomeriP a]l(l Rowlands, 1984; Dalling, 1934; :Jia:-:ou, 1934), an<l thus rlirl not lHorluce thP Ppsilon toxic fraction of GlPllll.'. et aha (1933).
All cultm·es wf're gTO\I'll at 37° C. in llohedsou's meat ln'oth (JwrsP flesh) fnr tlw £ollo,1·ing periorls: Typr B, 2U hours; Cl.
SC]Jtl:qlle, :JG houn;; Cl. oedelllatiens, 48 hours; aucl
rz.
ocito.ricus, 5 llays. After fi.ltrati(Jll, first through pulp and tlJen through aG l
LB'J'J-IAL HOSE CH' THE 'I.'OXIKS 01' SOC.lE .IJ>AEJWnl<:S "1'01! Sll"EI'.
Berkdt>ld camlle, the filtndp,; \YerP sut uratPtl with aimuon1um cml phatP, the precipitate blotted an<l finn ll:v clri t>cl 1·n T(l.('UO ove1·
H2SO,. For use 100 mg-JJJ. 1\'Pre <lissohP<l in ;)·0 r.c. of saline solution.
ThP miuimmn lethal <lose for mi('e and g·uinPa-11igs was estab- lishe<f in hun<he<l.-; of :111imals. 1'hP Cl. sepliqve and Tnw H toxins ha<l heen weparP<l seYPn .1·ear;; previousl~·, tlwir toxicit-ies remaining constant OYPr this pPrio<l. The oetlematiens toxin (toxieity 11·orke<l out hy thP intn.1musr·ulm injection of mirP) awl that of ovitoxieus
ll'f'l'E' of lf><'ent preparat-ion, iheir YalnPs being p:-;tahlishetl 01·er a periocl of months on approxin~<dPly 70 mire.
'J'lu'- ,\'/ieefi.- .All IH'H' :Yierinos, mostl~- hammeb and of tlifiel'eut ,,·eight:; <llHl :1ge:;. ::\early dl had heen i.hrougl1 :1 louping-ill experi- ment; SOlllf> tlnough blue-tongue, bla<'k t[LWl'teJ·, Ol' \'al'iOUS ]Jlani.- feetli.llg experiments. After the i njertion (ab ·ays intravenous) of toxin, at )past a week "·as allmYPd to Plapse hef{)l·e the result w.as l'et·onle<l. In somP ca,;ps, sheep rrrPiYetl tlll'ee tli.fferent toxins at tli:ffpJ·ent 1 imP~. However, Rheep 1Yhich Rurvive<l the injection of
r t.
l('('lrlu'i, 11~·ppn
toxin Jlf>\'f>l' l'e('eiYPtl that of ('7. lt'f'lclu'i, Type D antl rice 1'f'I'Sa.\Yhen experimeutaiiotl intlica!Ptl the approxima!P fatal tlose, furthel' tests werP put up in 11·hi('h "o much toxin prl' kilo-bocl~-
\Yeig-bt 1\'<lli g-iYell.
Hr:sn:rs.
C1. seJdique.
~umher of ,;heep u;;Ptl: :)8.
''T
Pi g-h ts Ya riecl hPhl-f'Pil 20 · 4 a IHl :)2 · 0 ki log-r.a m ~.TOXICITY.
-====;===== = = = = =
--,= ===-~---I rrespectivc of Weight.
!vlg1n. 1-tesult.
120 '!
110 t
100
tl
90
tti'l
80
ttttl
Per Kilogram Body weight.
:vrp:m. 1-tcsult.
4·~ -r
3·8 t
:3·5
tl
:~ ·3
tt
:3 ·0
ttttll
I
I
;<;fi'ect of injecting 2·6-3·3 mp:m.
per kilogram.
\\'eight (Kg.).
- -- - - --
I
Less than 22
tttl/11
22-25
tttt llll
25·3-27·5
tHttHi'/ I
28 Ol' more
ttHHII
72, 74, 7H
Htll
2·8til
+4.
70
tttl
2·6()6
ttl
2. :~liO, 62
ttl
2·254 'i'l I ·7
+6. 48, 50
't/1111
40
tl
:VLL.D. mice (1.5-18 gms.) 0·1-0·2 mgm.
}LL.D. guinea pigs (250-300 gms.) 3·0 mgm.
G2
tttttttt I I
·ntll I Ill
j· death.
I = live.
.1. If. ::-.1.\SO:\.
'l'u l1e !·erlai11 of killing· ;t mon~e, 0·:2 lllg'lll. "·a.~ llPI·e~~ar\·.
nlthongh 0·1 mglll. frPlptPntl,- di1l ~o. ThP .'ilnlP Yari;tliOJJ occunPd in shreep. 'Olll!' !wing bllPd ,,·ith '2·2 nJ~.flll. pPr kilogntm a11d some Ji.l·ing after haYillg' l'('CeiYell 0 (J_;j·:) lltg'lll. per kilog-ralll. lt \l·oulcl appe:n· ihal more !han :l·O mg-m. per l;ilog-r;tlll ,,-,\~ 11ecessnry to he rP;tsonahl_~- n·rlai11 of a ]d-lJal eftPd. T;1klug ihP h'tlJ<tl 1lo;:1·.~ ;ti 0·:.!
mg-m. anrl :1-.) mg-111. (Jer ki.log·rmn for the JilOU~e aud ~hePp n·~pP!·
tinly. the n1tio of mouse to kilogram ;:]Jeep-hod_,--\\·eight i~ l : 11· ,-1.
'J'hP time Plapsiug- het"·een injer-lion anrl 1le;tl h ,-,niPrl between four hour~ (:~-,-~ lllg'nl. p<'r kilogTalll) an1l :l() honrs (:l·(i lllp:lll. pe1·
kilog-ralll). Ho\\·e,·er. a~ a rnll-', ,;heep i11_jPr-led 1luring- the da_,- 11·ilh
<1 IPI h;tl do~!-' "Pl'P dt';Hl ne:-;t lllflrlli ng·. ('1. ,reft-lu·i, '!\·!)(' H.
::'luLillH•r of .~hPr'Jlli~Pd : IX.
\\'pight,; \·;niPd IJei\1-PPII 12 and :):2 kiJogT;\111~.
TO:\[(' I TY.
Per l.:i1ograrn hod_,. \n·ight.
~''·!!Ill. He,11lt. .\l.um.
~0-:!-1- [i· 1· 1-l . ;);)
Ill-I/ +-f·j··;--r II· /.)-0. !l
Iii () ;)-()·Iii)
10-1~ ·j··;·t II· ;];)-0 -ki
I
.:'II.L.IJ. mic·e (Ui-18 gms.) U-11~ m.c:m.
• \I.L.Il. g11irwa pit!' (~;)1)-:100 grm<.) 0 1.-l-ll ~ rngm.
·j·i[ +t-;-
i• dE<l.th .
li\-1'.
'1\tking- tlw 'hPep ::\J .L.n. to lw ·:2 Jll_glll. per kilogr<~m. llte
mou~e to ,;]wPp 1·alio i~ 1: fiO. The ininYal lH•I,,<'elt injPr·lion a)l(l death Yn r1ed lJeh~<'Pll OJ I<' hour (I ·l mgm. per kilog-ram) <\ltd four da.n (O·(i(j IJJg·JJJ. pPr blogr;\lll). Four ~h<-'ep died ,,·it!tiu four hmus, tlnee in hom 18 lo :JG hours, <liJ<t 1lneP i11 hnut :i to
+
d<tY~.('/. 1/'Clcll!l, Tqlf' ]).
10,
~
()
Xuml>(•1· of ~he1·p used: :l:i.
"'(>ig-ht~ varied lwi\\-I'Pll i..J- <llid :J!l TOXH'ITY
~\l~m.
1110
Iii. :W.
I. ~
-1-. :)
11-2 ;) I·H l. fl :J, () 7
i{(',;rrlt.
·i·
-t-o ·riii
·i·t
·r
·ft-f·f·f I
:.\lgm.
li·!j :2. t) 11·/. I) ·15. l II
0·:25
I)· l-1. (I· II O·O!l-0· I II· O;i. 0 · Oli5
O·()J 11·0:2ii .\I.L.Il. mic-e (15-18 grm.) 1!·11118 mgm.
()0
·i· death.
-;- tti" t +t
+-;- ttt·t·
II - lin·.
LETHAL DOSE OF THE TOXINS OF SOME Al'IAEROBES FOR SI-IEBI'.
Cl. wBlch?:'~, 'rype ]) toxin gave a sharper end poi.nt m sheep than either Cl. septtqtte or Cl. welchii, Ty1Je B, because, with the exception of one sheep, all \Yhich receiYed 0·1 mg-m. (or more) per kilogram died, whereas those injected with 0 · OG5 mgm. (or less) per kilogram lived. Taking 0 ·1 mgm. per kilogram as the lethal dosl', the mouse: sheep ratio is 1: 12 · 5.
The toxin killed quickly, 0 · 26 mgm. (or more) per kilogram killing in 1-4 hours. Doses around the ~f.L.D. killed in less than 24 hours, i.e. overnight the animals were dead, only one sheep (0·14 mgm. per kilogram) surviving this period, to che, howeYel·, within 48 hours. However, even after 24 hours this animal was moribund.
Cl. oPrlematiens.
Number of sheep used: G.
-weights vnriecl between 19 ~mel 24·6 kilogram~.
TOXICITY.
Irrespective of weight. Per kilogram body weight.
)fgm. Result. )Jgm. Result.
200 j· 8·5 t
150 t 8·0 t
150 t 7·5 -;-
150 I +·0 I
50 I 2·0 I
LO I 1·0 I
)I.L.I). mice (15-18 gms.) intmmuscular injection- O·OI:i-0·08 mi(m.
t = death. I = Jive.
The small number of sbeep used does uot allow the <hawing of condusions, hut assuming 7 · 0 mgm. per kilogram to he a lethal close for :-;beep, the mouse to sheep kilogram n1tio is in the region of 1 : 100.
The three animals which succumbed, died within 24 hours.
SuMMARY.
The lethal do;;e of the toxins of four anaerobes has heen :tscer- tained in mice, sheep, and in two instance;; in guinea-pig:-; abo. The
mou~e : kilogram-bmly-,,·eight--,:heep 1·atio io :ts follm1·s:-
Cl. septi(j'tt.e 1:17·5, Cl. welchii, 'J1ype H 1:60, Cl. 'tcelchi,l, Type D 1:12·5 ancl Cl. oNlematiens 1:100. The last ratio was anived at by the use of ouly six sheep a11rl is thus approximate.
B I~FgRENCES.
DALLlNG, T. (1934). A Xote following )fcntgomerie's ned Il01dumls' Article.
Yet . .T11l., Vol. 90, Ko. 8, p. 337.
GLJ•:i\'l\"Y, A. T., BARil, ~L, JONES, :vi. LL., DALLIXG, 'l'., A-:\'D ROSS, H. E. (HJ33). " ~[ultiple toxins produc-ed by some members of the Cl. ll·Plr/,ii group." .Tl. Path. Bact., Yol. 37, p. 53
MASON, J. H. (193-.i). Tlte Liquefaction of lnspissutecl Serum b~, the ]~nmb
Dysentery Bacillus. On.d'e1'Stepoo1·t Jnl., Vol. 2, :1\o. l, p. 33.
MONTGOMl~HlJ•:, H. F. Axn TIOWLANDS, W. T. (1934-). Lamb Dysentery in North "Vales. l'et. Jnl., Vol. 90, l\"o. 8, p. 323.
64
