Studies on the neurotropic virus of horsesickness III: The intracerebral protection test and its application to the study of immunity

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Onderstepom·t J ow·nal of V eten>na ry Science and A-nimal Ind-ustry, T'olltllle 4, lV111nbe1' 2, Ap-ril, 19:3;).

Studies on the Neurotropic Virus of Ho rse- sickness III.

The Intracerebral Protec tion Test and its Application to the Study of Immunity.

By R. A. ALEXAND:EH., B.Sc.Agric., D.V.Se., Empire :Marketing Board Research Fellow, Onderste}Joort.

Ix 1931 Sawyer and. Lloyd reported an intraperitoneal rn·otedion test.

iu mice in connection with their work on the virus of Yellow Fever.

This test has for its basis the fiwling that if mice . .are given an intra- veritoneal injection .of a ma;;sive tlose of Yellow Fever neurotropic virus and at the rmme time, an inert substance like ::;tarc.h in 2 per cent. solution is injec.t.ed intrac.erebrally to cause a traumatic. injury of the brain, a fatal specific. enc.epha.litis will ensue. If, how eYer, the Yirus it> mixe<l i11 'vitro with mouse sernm prior to injec.tiou, the neutralized mixture is incapable of setting up the di~:;ease in the majority of inst.anees. 'l'his test has been applied extensively to the study of immunity with considerable Rueeess (:Mahafty, Lloyd and l'emi.a, 1933; Hussel, 183~; Soper and de Andrade, 19:3:3); l.mt in reading the report::; of \\·orkers who have used it one eannot be other than impressed by certain limitations. Every mouse "·hich reeeives the same infeeting dose of virus intraperitoneally <loes not <lie awl w·hen the mortality is 100 per cent. the deaths are spread over a numher of days (5-10). The varying susceptibility of individual mice to infection by the intraperitoneal route necessitates the uo;e of comparatively large numbers for each serum or virus dilution iu titration experiments, and it appears to he difficult to compare (lUan- titatively the poteney of different samples of anti-sera with au.'- degTee of accuracy. Furt.hermore, it appears that the interjJretation of results, depending as it cloeo upon the stati~tic.al survey of the llerc.eutage of mice whieh surviYe injec.t.ion, and the percentage whieh llie within a period of from 4 to 10 days, is sometimes open to question.

In t.he ease of horsel'itlmess the adaptation of the method is c1uit.t>

nnsuitalJle heeause the injecti-on 0£ even thousan<ls of infectivt> doses of virus intraperitoneally with or \Yithout simultaTH'ons injury to the brain cannot be relied upon to set up infection in anything approach- ing a constant percentage in micE'. Consequently it he<~ame essential to develop a technique based npon intracerebral injection. This has been done and the te;;t has proYed inv.aluahle in the study of many problems assoeiatef1 with immunity ..

349

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\\'J,ile lilt• \\()]-~\ >Ya.' i11 ]'1'11!2TI''' ~L1x ThPil<'l' ( I!JTI1 n·poriPd hi' Yt>llow FPI'Pl' proiedi<lli le-;1 ll\ inlr<H'PI'Pilral ill,j<'t'iioJJ ··. It 11a.;

lillllll'dta1Pl,1· CljJJl<li'l'll\ tiLl[ 1 ill' IJ<t\\111~ of 1 lit• IIPll''ll\J'()jJit· Yll'll' , j

lwi':'Psirl;ne'~ JIPI'!llilied Jllodifit·a1 ion.; ,.,ipahlP

,,f

J'<'lldl'riJJg· tht> tv-1 lliOIP <t(:(:.lll'l11P i\llil .Je]ii'HtP.

J I ha,.; JWi'll :;iJII\r!l pn•Yioli.·d\· (jJ.t'\.iiiHlE'I', UJ:i.-1) 1 hat tlJ<• SU:ii'Cjll i- l>ilit,l of difrr·renl miu• <>f tilt• :;train in n.;p at tJndt•r . .;I<']J<'"J'i i,, n·lll<trk- al>l,· •·oJt,.;tani, lital lUll [H'I' ,.,,nl. of mil'e 11hil'h retTiYP a <'el·i:Jilt infectin~ tlu.;t~ ul ,·int,.; Jlltl'<I!'Pn·IIJ·alll· 11·ill di,·, <lltd that, l11 tlilutioJJ. tiiP iiirf' of a siock ,-j,·tts 'll::.]'f'll,iiiJ; Jlla.l· h · <l<'iPl'lllined to tllo-ft,],]

lillltt:i. ln <J:!clitinn th,• titn• ot H :ill'J'PIIsion of infPdil·,•lil'<lin Jll<tdt· l!JI iu Ill )11'1' l'l'lli. ,;tiilll' l't'ill<lill.; t'Oil:il:\111 f.,l' a Jll'l'itll! of :tllll<hl .;jx l!!llltllis II'IH•IJ slon·d i11 the l'l'i'l'ig·<'l':tlllr ;Ji " \i'IIIJl<'l':liun• .i11,.;l aL""'' frt•t•:t,illg·. T!J,•n·fun· ill!' fir.;t ''''1'111i:d,.; of :111 ilt'l'lll'illl' lt•.;t ill't' 1111- til!t-d. ll<lllll'l,l:

I . . \ 11·.;1 i !tl'p('j (·d. :Ill"

t!l'ill h.

allllll<il uf <·oJt"l :1111 i11 \\ liich a d1'<tl' !'111

,;tt"<·•·Ji!iLi!it\ 11hi<·h i.' ,.:t,li\

! . (.; l ( . t i ( ) ^{I :} ^{^'jl ·""' ll ( ';..; . i . ^{I ' .} :-..:. II ,. \. i \. ~ \ I ^{I ) ,.}

· I . \ ~i:tldt• ~llliig·t'll jll'l'il!illillg· ('Ol!]jl<tl'lc'tl!l IJI.'t\l't'l'll l'l'.'llil, oh!ili'IPd in ditf<•n'lll P'.;];t•;·t!IIPllh t'll!idUt·I•"J <11 diJh•rt•IJI I illl<'~.

::_ ....-\..t1 :tllti.~-~'li ^pPl'tJJitiin.!..l· titl·;.tti(JJt ttl L1irl_\- JJ:IJT~l\\. li1ait' ,,-ilb

CU!l:il:tllt :lt'l'lll'ili'.L

l<'or l'llll!J'let io11 11[ t ite IP'I it o11l.1 l't>!Hilin(•() lo i11• .;\tm1·11 1 h:1t I hP ,;,·1·a of .~11-'CI'Jllil,[e lwt•.;p.; tlo Hill c·lllltaill 1·il'll~ ne1Ill'<tl.i,.;JII;..!' ,;ull:il:tlll't'-. that the tleYt•lopmeni of illl1\l11lli1\ i,.; fcdlo\\·p,J 1.\ lhP "i'l'l'ill'illiCt' ,,f

<l<'tt•('Lthlt: Yirucidal <HIIi-hotlit•,.; i11 th,• SPI'Illll, rtud t\,:tt tit .. al'tion ot the.;p illli i-hodil'~ is ']IP!'itil'. Fl'lilll the daw gin'll Lelm>· it 11·ill Lt'

~1'1'11 lhai tlJPse l'l'it<·riil ha\'1' lll't'll tullill .. cl :111d that it 11·a:; ]'ussih!P to eL:I,or:lle;; suitahlt' letlluirl"" i11r <tltalllitatil·l· iu ,·ilm IIPlltl'aliza- t i ^(J11.

I. THE INTRACEREBRAL NEUTRALIZATION TEST.

'J.' ^{1·: C'l}l .'\ I (ll. I·

.l. l'n·;;untluut uf ..lnllljl'!l .

.Jlit:P an• Siii'J:ilwPd i}l t'.l'i;·r'IIIIS iiflt'l' illtl·:tr'l'illli;t! illjt·l'iiun 11t lli'II!'Viropi<· li:-;t•d Yiru.;. tl11• Lr:till.; !'ill't'll!ll\ lt'lllill'l'd 11ilh "''']'lir·

Jll'l'l'illllioll.', <tllll pi<ll'l'tl in .'lt'l'ile ,-Jil t'.t'. c·t•lltril:lg<· tuh<•,; filtt•d ll!lh ''"ik" . . \ itPJ' ihl' hr<~ilt '"IJ.;talll't' '"" lll'PII trilul':t1ed 11ith " g·b-;~ r"d or ,,,.,tlpel t.hf' inlH•.; III'P plat·p,J i11 tht• fn•t•:t,i!lg· c·ha11ilwr of" n•t'rig·pral"l' O\'t'l'IIig·ht. Se.xt nwrllillg· :Ill l'll!Ubiun iu ]1) Jll'l' 1'1'111. "l'J'IIlll salitiE: i~ pn•JW!'f.'<l in tht• u.;nal !!WII!LPI'. Tht• pro1·e,.;s of preliminary fn·Pzilt2

<llltl ,.;uddl'll iha\l·iug ha~ he<'ll i!dopiPrl hP!'illlSP 1111douhtt·rll,1· it airb ill n:lluLn haglltenLdi<>u '""] Lll·ilitille.; tborou!2·h Plllttlsific·;tlillll "f illl' bl'ain,.;. Tlte 1·in1~ ·'11~\)PII:>ioll i.' th(•Jl t·t•uil'ifuget! nt :2,;')()(1 J'l'\·ollttioll' }Jt'l' .lllilltill' fr1!' 1.'1 Illilllllt'"· JlJp :'llJlPl'll;ttillll )i<JIIj,j I'<II'Pfiill,\ tl(•c·Ulllt•d intiJ :t ~PI'Ollll iuliP and agai11 .;pun l1efore l,ei11g· dt>!'aniecl int11 a

~tPI'i)l' bottle fit1P<l \l'itJt il l'llhiJI'l' !'Ill']; <IIJtl ~lii!Pd Ill .jO ('. \\'ith t\it'

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11 . . \ . . \I.EX.\:\JlE;:.

pa,;,;;tg·e of timr• a fine J>rer·ipiia!e ,;e1tlE·:; nni to tbe lmttmn of iltP l>t~1tle bur thi, litH.' lw left quite mtdi-.;turhc>cl ,,.h<'ll J'PllloYillg :<<llllp]c>,;

<h re:1uirerl aut! cau~e~ no tle1PC't;thl.- <lP('rea,.;c> in tltP Yiru,.; titre.

It j,.; n•ali,;L·d thai· if "·"nld lw pt·deJ·al>iP to l't'IH"'·p all ti:''ll\"

tlebri" a11tl possible ugg'J'pg·:·tfion . ..; nf 1·iru." p<tl'\i('lt•s i>y til11'atiott hut

;tt tilt• tinH: the! Y:tlnt• ol' thr• :-:Pi1:r. tiltel' 11·:1." 1111t :tJ>Jli'P<·i;ltt'd <lttd lrl'ac!o1·1>l m••tnlll'att<'" 11·en· not <1YaiLthl1' ,;11 Y<'n· t llPI'tJug·h <'lll11L,itic·:t- tion <tnt! t'PIIIri.fng·atiotl 11a,; rPliPd IIJH>II for tiH• pl'ulllll·tillll of a lwnto- .!..!.<'IIPIJII" ,;u,;Jll'll."illll. 111 Jlil",;ill,!_!' it 111<1\. ],p lllt'll1i<>llt'd tJt;,( \'1'1'\

thc>I'Illtg·lt lrilul'atil>ll of th<' hrai11 Jll<lt;·ri<tl 11ilh ,;;tlld and gl.t:<:<

Jlitl'til·],,,.; a11d prolottgt•tl. .... ltal;illg in a tlll'..!t<ltlil·<~l ,;lt<ikt·t· 11 if], l>P:td' :tppP:tr,; lo ]t;I\'P tto IH'IIt>ti1·ial Pff1•d 11]>011 tlt\" 1dii11t<tf<' prodltl'i.

,\llttl<llliJ>IiLdiott,; an• canit•ll out 11·itl1 :t'Pj>tic· [>l'l'l':lldilllh. Allli-

!..!'c'tl,; haY<' ],1'\"11 ,;torerl for ~ix JJitlltlhs at 4:⁰ 11·it.hont tlH! :Hltli!ion of

~111_1· Jtl'E'"l't·Yati,-e <tlld at· n" tim1· h<t,; nn1· tlifti,·ult~- !JePn PxperiPJti·Prl 11·ith l>:lt'iPI'ial l'tlllt<llJtinalion. ThP _-;tnc·k <lll1ig·pn ha~ ltPen Jll't'Jl<ti'L'tl U:itJall1· 1<~ I'OlH)Iri~~· a11 approxintatP .:2 11er t:Pllt. -;nspeiJ,,;ion ·of brain

~n hsta II('!'. hut the actual colll'<'ll tndiun a JIJH'<1l''i to he of Ill> i m porta 11 CP J:n·o,-itletl it is 110! so hig-h I bat nn extf'lt'i"l' cli•po,;it ,;uh,.;equeni'l~·

fnrm'. ThP Yint' 1itrP i~ tht>ll cle!l•l'nt ine1l in t,,-~, -;tag·p,;: ·

J. ,\ [li'Plintin<~r:· J·uug-h titr<ttitJu lt_,-;;nial lPll-fol1l 1lil11tion.

:!. . . A. fi11:tl <tc·cHratP titration 1"-111-o-folcl dilution ltllllPr the> l'tJIHli-

tinn,; IISt'll for iu riiJ·O llPIIirnlizati'(,JI.

'l'hi-; lll<l\ Itt' illn,:lr:t1Pd Le.-;t lll n•h•n•ttt't' to till' n•,;u]h nhtaill('tl in an ;tl'lu<tt' <'Xfit't·itlt<'lli. Vonr I;H,IJ,;I' l11·:1ill., J'I'J>I't':it'ttlillg- l"l""ag·p .~·l'IJPr<ltion ,-111 of <lit<' ,;ir:till of Yin~,; 11-t'l'l' 1'1111Ji.-;ifil·ll ],_~- iltt• tPr·ltniqtJt' 1lel<lil,·ll ahoYP. A I'OII~ . .dt litr;tlitJn "!' thP ,-inh l't>ltlPn1 "f !Itt• :tJtli!.l·t•n

"·as l'llll in mice l1.v !et;-fold dil11tiun 11·ith 1lt1· l'oll""-ing· n•,;ult :- .

Fole

".f

.lfi('l' fnicl'ied 1rlllt !hr Ten-fulrl !Jil"ti""" u.f i'lnt.'.

'

lO. l : 1<1'. ]O". IU'. I : I o". : ]II''.

-;-

tlt·al h of mic·t'. -1 : ^;) + : ^.) ⁽: ^,) ^,⁾: ⁽⁾ ⁽⁾: ⁽⁾ II : II

\'q·n:. ln thi~ nnd all fttltil'<' li\.llli·:-:. till' llllllH.'ral ind.ivaii':O: tlH· d<l,\' nt'IC'I' inj('l'fion on\\ hit·h til<' lllOII~t· clit•d.. thu:-:. -~: .) l\\'cl lllil't' injtTlt•ll of \\lliC'h tlllt' dictl 011 tlu· lth

a.rHl on1· on t IH· .)1 h d;t \. <tftt·r i11 jt•t·t ion.

< l Jll(•an;-.; ~·.;Jin· i\·n.l. · .

x nH·;uJ.-: (l('nth dw· ltJ :-:olllt' ('iltl~l' otht'r than hor:-:.t·~it·knt·~~. u...:urdly injury to ln·ain af 11w titnl' of illjt·v1inll.

l>o"'t" 0· (I;) t'.t·. in! nu·t·n·ln·ally.

Front tltP. tig'IIJ'(•,.; it j_, ~fWil tlwt tltP iJtfPI'til'l-' iitre cd' tlw S11td;

PlllLLLion is po.-;,;ihly :tlllt\·e ;1 dilution 11f I: ]II' l>tl\ ltelll\1' l: 10.-.. Tht'l'P.- forP a ,;u..;pell~lOII t'ltll."i-;1i11g· of 1 part ol' th1• ,;til(']; ;tniig-Pn to 1()1) p:trb

:;.j}

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STGlJIES 0.'< THE C\KUWTHOPlC Vlltrs OF JlOHSESTCKKESS III.

of llihwnt conlll he ex11eded to !'OBtain in 0 ·00 c.1·. at least lOU minimal infective doses of Yirn~. For a neutralization test antigPn and antisPrnm would Le mixecl in ec1ual proportious ~o that for thP :fine titratiou, the clilutions illllicated below were mixed in er1ual quantit.ies with salint> before injection.

TABLE II.

Fate. of M·icc Injected u~ith Dil11tions of ,-lntz:,r;cn consisting of Oue Part of Stock Suspension to 100 pa.rts of Diluent then Mixed aa with Saline.

l>ilut ionH.

Antigen. l 10. I,:w. l -lO. 1/80. ^I WO. l/3:?0.

Death of mi,·P -l ; ii ; ;) ;) : ;) ; () -l; ii ; () ii; (i ; 0 (J ; II ; 0 1:1; II ; 0 0; 0; 0

It. is sPen tLPI'e:fore that the stol·k Pnndsion !lilntecl] t.o lOU results in antigen which after mixture with an equal quautity of anti-serum

WOUlJ contain in (J' 0i'J ^C.C.approximately .:f(} minimal infectiVP dor;e,;

since two out of three mice iiJjeded with the 1:40 dilution dit>d, awl injecton of the I :.SO dilution re~ultPd in no mortality.

The death on the 13th daY of one mouse \Yhil'h receiYed the 1:160 dilution was due to horse.~ickness as shown hY subinoculation.

Sueh deaths which appear to be quite out of pla!'·e clo oecur when material just above or just helo\Y the limit of infectivity is injected.

Inaecuracies in teehn ique ma~· he respon,;ihle for the a ppareut errm,.;

but a con,.;iclerable experience has shmvn that when consideration is paid only to those mice which llie within a period of three clays of the average resulting from the injection of 100 :M.I.]). of virus a dear cut picture i;; obtainPd. It is ,.;nlnnitte<l that the indusiou of thi~

time fador in thP iut.erpreiation of re~ult,.; has a definite preee1lt>nt i11 other neutralization work and is permissible.

In reeor1ling cleath::: eYery mon,.;p which succumbs during thP c;ritical period iR asRume!l to haYe !liell of horse.~irkness. This may be a ~ource of some slight PJT(ll' hut it ic; re!luceclio a minimum by using- healthy mice which haw heen a('eustonwrl to being· house!l mHler the conll it ions of the experiment.

fl. Collection of Anti-sera.

Bloocl is dra wu either into large test. tubes or Erlenmeyer flask,;

:fitterl with a ::;idP arm, and the serum is deeanted the follo~Yino· (laY nfte1· eontrartion of the dot. ~era havp been storP!l in the refrig;rato'r

usuall~' wii hout presenatiYe, but 0 · 7 per rent. tricresol-ether ha,.;

heen USPU without haYing any tlP-trim~ntal pffed upon its virucidal property. Sera WPre not filterPtl hut were t horoughlv centrifuged

heforP use. · ,.

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C. Tl1e Ne'lltm.Zi::ation Test Fro per.

After a considerable amount of ]_Jreliminary work on the mo,;t suitable method of carrying out the test it was decided to use falling dilutions of the anti-serum ,mder investigation against a constant antigen. Quite arbitrarily a dilution of antigen was selected to contain in 0·05 c.c. after mixture \Yith an equal volume of normal serum or saline between 40 aiH1100 )f.I.D. the exact titre being deter- mined at the time of the test.

:Monax agglutination tubes (length G · ;) ems., bore 0 · 7 5 em.) have been used, the tubes being selected to haw a uniform bore.

Aseptic precautions have been adopted throughout, i.e. all tubes an;

sterilized and are fitted with cotton wool plugs allll freshly boiled an(l cooled syringes are usecl for all measurements aJHl (lilutionR.

To each of a series of tubes placed in a row in a rack 1 c.c. of saline i:> adclerl. Two-fold dilutions of the serum are then prepared hv serial dilution in the usual manner. For this it has been found n~ost convenient to use syringe;< to each of which a long fine needle (B.vV. & Co. No. 205) is attached. The 1 c.c. -of fluid then may be forcibl;y squirted into the diluent without producing any foam since the point of the needle may he placed just helo"· the surface. Onlv the point of the needle is allowed to touch the fluid before a fresh syringe is used for thorough mixing and transfer.

The 1 e.e. of antig-en is then forcibly squirted into each tube containing serum dilution to ensure rapi<l and thorough mixing. The mixtures are incubated for two hours at !37° C. and stored o-.ernight in the 1·efrigerator. There is some evi<lence to show that the union between virus and anti-body is somewhat. loose at low temperatures, so, before injection next morning, all tubes are incubated at !37° C.

for a further }Jerioll of one hour.

It is preferable to use at least thrPe mil·e for the injection of each dilution. Unfortunately a serious shortage of mire has mane it necessary to use only two mice in a number of experiments.

It will he noticed that the serial <lilutionR of the anti-sera are made in saline and not in serum-saline. This proeerlure was adopted for the sake of sim})licity after it has been ascertaine<l that 5 per cent.

serum saline had no detrimental effect upon the virus--5 l)er cent.

being ihe minimum eorH:eutrntion posRible in a mixture of equal parts of saline anu antigen.

\.Vith each set of neutralization tests a control of the infeetive titre of the antigen is run by making serial <lilutions in 1 c.c. amounts and adding to each 1 c.e. of saline to take t1ie place of the anti-serum dilution.

ExrEn DIE NT _-\L

Since the antigen is the most important factor in the neutralization tests about. to be described the following results hom a series of different experiments have been l:olleded together to show that by the requisite <lilution of a stock suspension of virus a constant antigen can be prepared as requirell. Further it was of importanee to show

~)5!3

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~'lTDIF.S 0!\ THE KECHOTHOPH' YlHr~ OF IIOHSESICKXE~~ IJT.

that incubation for two hours at :no('. ancl storage overnight in tlw refrigerator followed l>y a fnrthPr one hour nt :wo hacl no cletrimental effect upon the virus.

An appHrs:imately 4 I>er rent. emubion of fonr hrnin,.; representing mm1se passag-e generation ()2 of ;;train 204-t<J was roughly titratecl in mice as follow,.;:-

lkath of micP

TABLE IlL

Hour;h 1'/tmtiun of Stock A11fi.r;en 111 Jl/1ee.

Yirus Dilutions.

1 : 4. 1:4x lo.¹!:4 l0².\1:4x lO'. I:4 10".11:-! 10^{5 •} l:4XIO".

4: 4 4; ;) ^;); ;) ll; () 0:0 _I_

0; I) 0:0

From thi,.; titration it '"ould be t>xpected that a 1 :40 clilution of the stoc.k antigen would c:ontain in 0·05 c.e. approximately J()O )I.L.D. of virus. This clilution was maclf' up hom time to time ancl testeJ with the results shown in Table I\-. l'rior to injection all clilution,.; wf'rf' innthatecl \UHler thP exact c·m1dition~ nf an i11 1·itro nf'ntrahzat.inn te~t.

Date.

J;j. 9. :J:l 26.\1.3:1 1a.1o.:J:J ::!7 .10. :Ja 4.1l.:Ja

:m. 11 . :J:J J.!. 12. :J:l ::!O.l2.:l:l ::!!!.12.:!:!

J7 .1.:!4 ::!:3. l.

:u

2.2.:1-!

9.2.:J+

::!7.2.34 J+.:J.:l4

TABLE ^1\'.

A.ccttratr 1'itratinn of A.11 t i.IJI'n.

Yirus Dilution,, Interim

days.

I

^I

A. l : 10. 1 : 20. l : 40. 1 : RO.

R 4; 4 ^;); ;) !5; :) 1); t') 4 ^;)

Ul -1-; .:!) i5; li ;) : 6 5 0

:Jn 4;4 4; f) 6; li :) ; 5 .~ I)

ij() !>;0 5; 0 ^f):8 0 ⁽⁾

;)8 4; 5 4; ^f) ^;); ;; ₅ ()

84 4;4 !} ; 5 5;!) 4; 4 6 0 98 4· 5 f>; 5 5:f> fi; 0 5 ⁽⁾

104 ^{;) ; !)} ^[);6 5; ^f) ^;)^;6 5 ⁽⁾

113 4;4 4; 4 4;4 ^;); 0 4 4 132 4; ^.') 4;!) 5;x ;) :X !) li 140 3; 4 -1-; ;) 4; ;) H;x ^~) ^f)

148 4; 4 4· 5 4;x 4;5 4 r;

15.5 4; 4 5; 6 4:4 ^;); :) 5 ^f) 17:l 4: 4 ;) : 6 5; ^ti ^{;} ; 6} 6 ⁽⁾ 188 4; ^;) :) ; [); 5 6;fl fj; n () 0

NoTE.- A-A.ntigen, i.e. 1:40 dilution of stock suspension.

1 : 160. l: 3~1.1.

D; 0 0;0

6:0 0;0

(); 0 0;0

0; 0 0;0

7; ⁽⁾ 0;0

0:0 0;0

0;0 0; 0

0;0 0;0

i): () 0;0

fi; () n;o 0; 0 II: 0

i j ; 0 ()· 0 0· 0 0; 0 0; 0 0; 0 0· 0 0; ⁽⁾

A cou;;i1lerat.ion uf the figures shmn thnt from a ~toek sn;;pt>lJSJOn str•red without preservative at

±

4° ( '. an antigen of reHtarlm lJiy

eon~tant infeetivity may he macle up. lt is true that Rome slight 354

(7)

H. A . . -\LEXAKDElL

variation occ1us up to the 5th mouth hut. this variation is well within the limits of experimental error. One particular titration, that carried out on the 50th clay, showed a marked deviation but ohvionsly this must have been clue to Rome gross technical error. After the .jth month there is a definite and decided tendeney for the titre to clE'crease. If i.ncnhation awl owrnight storage as indicated has any detrimental effect it is sufficientl v constant to make it neg·li!rihle for

comparative purposes. · ~ ,_

Coneurrently with the experiment detailed an ath:'mllt was made to use clessicatecl pow<lerecl hrain as the source of Yirus for the prepar~L

tion of antigen. It would he superfluous to give the figures but it may be statecl that the variations in titre were so g-reat, probably due to inaccuracies in weighing, and imperfect mixing, 1)owclering and sampling the dried brains that. it~ use in preference to virus stored in fluid form could not be contemplated.

l<'rom this experiment "·hich has heen confirmed repeatedly it is concluded that by the method described au antigen of constant titre may be made up as required. Thi~ antigen will keep unaltered for a periocl of approximately fiye months nfter which there is ~~

Lleci<lerl tendeney to deteriorate.

Having demonstrated the lHJssihility of preparing a suitable antigen it only remainecl to he shmYn that immune serum is capable of neutralizing the virtlH and that any neutralization in t•itro is specific. That this is the r-ase is clearly shown by referE'nr;e to the figures giYen in Table V.

TABLE V.

In Vitro N eutml1:::ation of 171:Tus.

~:ertun Dilution~.

SerunL

l .. ) l : 4. l :

s.

l : 16. l : 3:?. l : 64. l: 1:28. l: :?.-)13.

Immune horse ........ 0; 0 0· 0 4: 0 ^f);0 5; f5 4; 5

Immune horse ........ 0; 0 0; 0 0· 0 6; 0 0; 0 6 : ^;) ^;): 6 +: ^;) Sheep ............... 4; 5 5 ; 5 4 ; 5 4; 4

Susceptible foal ... 4; 4 4 ; 5 5; 5 4: .5 Susceptible horse (a.gecl) i) ; t) 4:

"

^4·^.5 ^4;^(j

i\fo•rE. -The antigen used was that. which was htratecl for infC'ct.ivity on 4.11. 33 in 'lahle IV above.

It is seen that the serum of the inmnute horse pv::>Set>::;es the property of neutralizing nem·otropic horsesickness Yirus in -rdro, the intracerebral iujection of mice being used a;.; thE' index of lo:>s of infectivity. Serum of a sheep, a susceptible foal and au aged suseE'l'- tible horse clid not possesg this property. It may he mentionecl that after a c·onsiderable experience there i~; no reaso11 to belieYe that the 11eutralizing- action is not entirely specific, and that the serum of

~usceptihle hor~e,.; contains no anti-boclieii.

35[)

(8)

STUDIES OK THE NEl"fWTHOl'IC VTHTS OF HORSESICKl'IESS Ill.

A consideration of the figures in Table V indicates further that a sharply defined encl-point e:s:actly indicating the neutralizing tit.re of a particular serum e<lnnot be anticipated. More accurate results are obtained from the use of larg·er number;; of mice but unfortunately the available supply waf: strictly limited.

At one time it wa~ thoug·ht that better results would he obtained from the addition of comp-lement in the form of fresh guinea- pig serum to each dilution. This has been tried on several occasions but it was apparent that the results rlid not justify the additional complication as a routine measure. At all events the results reported below will indicate that the method tlescribed dearly clemoust.rateil differences in neutrah1-ing power between samph~:-; c\f sera, "·hieh enables a quantitative cOlllLJarison to he clrawu.

K n:PlNG Qr.\LITIES oF ANTl-SEIU .

Frequently it is ueit.he1· ·convenient nor possible to earry out neutralization tests with immune ~era immediately after eollectio11.

Consequently it became necessar~· to aseerta in "·hether storage in the refrigerator at 4° C. with or "·ithout the addition of 0·7 per cen1.

lJheuol ether a,; pre;;enative ha.~ ally detrimental effect upon t.he viru- ci.clal ant.i-boClie:>. A. sample of serum obtained ·on 14.12.:33 from a

hor~e (20545) that had been innnnnizerl by the injeetiou of neurotropic virus 177 days previously was divided into two 1Jortions, to one of which pre,.;eJTative (0·7 per cent. phenol ether) "~as added.

N eutralizatiou te:;t:; were carried out with both these samvles on 20.12.3:) allCl 22.8.34, i.P. f'i:s: clays and. 251 rlay;; after hlee<ling respectively. 'l'he results are t>hO\vn in Table

YI.

It is seen that horsesidmeH<' anti-sera possesses remarkable keep- ing' qualities since no .apparent cleterimation was cletectable after 261 days storage at a temperature just. .above freezing. This is indeed fortunate becanse in the ensuing' work it wa:'l necessary frequently to iltore samples of ant.i-sera for more than 200 clays to ]1ermit. of relia hie quantitative e:s:1Jeriments by neutralization test~ against a single antig-en preparation. In ~ulclition 0·7 per cent. phenol ether as a preo3enative has no detrimental effed upon the viruc-idal anti- bodies.

II. THE APPLICATION OF THE TEST TO THE STUDY OF IMMUNITY.

The elaboration of a suitable techllique for the quantitative deter- mination {)f the antibody <:ontent of serum paver! the way for an i nten,;i ve study of the cl evelop men t of immunit~c in equines. \¥hen

th~ work was commenced the interesting nature of the results to be oht:1inerl wa8 not anticipated so that samples of ~er.a were not collected

a~ frequently or as regularly a.s could have been wished. Further, the slow pro,luction of antiborlies and the enormous accumulation of i-'amplef> of sera. has effectively prevented corroboration of every result hy duplicate series of experiments, bnt confirmator~~ tests have beeJ.J indurle11 at sufficiently frequent. int.enals to inclicate that the results rletailerl are not open to criticism on the gTouwlt< of .act:uraey.

35G

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'l'AHLE VI. 1\.er'Jilil.(j Q"llaldies of Anti-sera. Inter-Pre- Date. serva- val. tive. 1 : 2. 1 : 4..

Perum Dilutions. 1 : 8. 1 : 32. 1 : :;12. l : 11):!.!. 1 : Hi. 1 : 64. 1 : 1:!8. 1 : :!:)6. 1!133. Days. 20/12 0 0;0 0; 0 0;0 (); 0 fi; 6 fi; 6 .5; 6 r;; () 20/12 f) 0;0 0; 0 (); () fi:ll 0; () :3; 6 .~; .5 4; fi ]!134. 22/8 251 3; 0; 0; 0 6; (); (); () ();();();X (); (); (); X 4;:;; (); () :l; 4::;; () ;j; 4; :; ; () +; 4; 4; t) <XI 22/8 2iil 01 I); I) (); () ll; () (); () 4; fi ;j ; ;) 4;0 4;:; -l Vi1·us 'l'itration of Antigen.

I

Virus DilutionH. Antigen. ])at.c-. A. l : HI. l : 20. I 1 : 40. 1 : 80. I 160. l : :~:.!0. I : 1140. ;;:: ~ i); I) i); 6 ;) ; t) ,; ; 0 II; ll (); () 0:0 Generation 6:.!, strain 2044\l... . . . . . . . . . . . . 20 .1:.!. 33 ;) ; ;) ;.... ~ 4;fi 4; 5 (); fi 6;0 ll; II fi;O (); () Generation UO, strain 20449.. . . . . . . . . . . . . 22.8. :34 4: ;) ;.-.

...

2j ~ ...

(10)

ST1'DIES ON THE :1\El"HOTJ!OPIC VIHL'S OF IIOUSESICK:11ESS III.

A. ^THE^Al'iTlllOD^Y CoNTENT OI·' THE SElluM :FoLLO\YIKll lJ\Dl1TNIZATION.

On 23.6.33 two horses (2054,) and 20570) were giYen a subcu- taneous injection of 10 c.e. of a 0 ·1 }Jer cent. suspension of infective mouse brains representing passage generation 61 of t;train 20449. The first hor~e 20546 developed a mild febrile reaction from the 19th to the 23rd day after injection; the second horse (20570) showed no febrile disturbance. In neither animal did any elinical symptoms of horsesiclmess develop. Prior to inj ec.tion a sample of ::;e1·um was colleeted from both horses but unfortuuately at the time the value of the neutralization test was not appreciated so that bleedings at regular intervals were not commenced until about two and a half months later. This was an unfuduunate omission hut it "·ill be seen that the defect has been remedied in suhsequent \York. \Vith the se~a neutralizat.io11 tests were carried out the results being given in tabular form in Tables VII and VIII. The t.itrations of infectivity of each antigen employed are given in Table IX, from which it will be seen that. unit volume of anti-serum was required to neutralize approximately 100 111.L.D. of virus (bet.ween 80 and 160).

A critical survt>y of the result::; indicates that a sharply defined end ]JOint was seldom or never oLtaineLl in the titrations but the figure" clearlv imlicate t.he rise iu antihodv content and its tewlencv to fall <:<UlJseiiuently. An insnfficieJl t nmnb.er o£ mice was 11.--f'd fo'r the injection of eac·h tlilution to permit of a strictly accurate conception heing- obtained of the vinwidal potency of each ;;ample of anti- serum but it is believe!l that the figures in heavy black type represent the hig-hest concentration of semm just incapable of inactivating- tl1e test dose of antigen. It is ;;een that particular!~· "·ith horse 2()!)4:3 the rise in antibody eontent was rather slmY siJwe by the 70th day a

r~oncentration of 1:8 was required completely to neutralize the antigell used. At no time flid the antibody rise to a particularly high level but the peak was reaclu~d approximately six months after immunization. After the eighth month there was a !lecicled tendenl'y for the antibody content to fall.

As soon as the somewhat slow !levelopment of immunity was appreciated it was deeirled to confirm the fin(ling· by '\.Yet>ldy tests on the sera ·Of animals immunized by the serum-virus metholl. 'l'o over- come errors due to idiosyncrasie~ of indi>iclual horses two groups of three were selected from the sun·ivors of a batch of animals being· immunized, for the routine mass procl ucti on of hyperi mm une st>ruu{:

Group A were hyperimmuuized 69 clays after immunization and consequently ha!l to be discharged from the experiment. Group B was retained and it is proposed to continue tests of the vooletl sera at intervals for at least the next two years. All the horses received the first immunizing injection (5 e.e. X. virus plus 400 e.e. serum) on

~11.10.33, anrl the seronrl (5 r.e. 0 viru;; plu~ 400 c.c. serum) on 3.11.:33. The reactions varied in severity from " Yery mild " to

"severe''. The results are tabulated in TaiJles X awl

XI,

^th^{e anti-}

~en titrat.ions being given in the corresponding Tahle;; X A a.nd XI A.

In each group the sera from the three horses were mixerl in equal quantities for the nt>nhalization tests.

35R

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c.? 01 <:0

Date of test. 15.9.33 15.9.33 26.9.33 30.11.33 20.12.33 20.12.33 20.12. 33 25.1.34 25.1.34 9.2.34 27.2.34 14.3_.34 27.4.34 8.5.34 8.6.34 Date serum taken. 23.6.33 119133 22.9.33 15111133 29111133 71)2133 14.12. 33 21.12.33 12.1.34 1.2. 34 15.2.34 28.2.34 22.3.34 17.4.34 8.5.34 Interval after immti.nization. 0 days ... 70 " ... 92 "

. ... .

148 "

. . ...

162 " ... 170 "

· · ·· ·

177 " ··· 184 "

...

206 "

·· · ··

0 0 0 0 0; 0 5; 0 0 0 4;4 5;5 4;5 -

-

0 0 0 0 0; 0 0;0 0 0 5; 6 5;5 5;6 -- 0 X 0 0 0;0 5 ;6 6 0 4;5 4;5 4;4 -- -

rn H d 1:1 H i:'J ff. c

z

H ~ i:'J ~ d :::::

8

~ 0 "d H a <1

""

PJ cj rn 0 >:j ~ ~ rn i:'J Ul H a 1>1

z

i:'J rn if) H H H

(13)

C-:l -~ ...

TABLE IX. Titration of Virus Content of Antigen Used in Neutmlization Expe1·iments, Tables VII and VIII. Virus Dilutions. No. Date.

I l I I I I

A.

I

¹

: 10. 1 : 20. 1 : 40. l : 80. 1 : 160. l : 320. l : 640. l' 15.9. 33 .... 4;4 5 5 5 5 5;5 4;5 5 ;' 0 0;0 0;0 2 26.9.33 ..............

-

4 5 5 6 5;6 5;0 6;0 0; 0 0;0 3 30.11.33 .............. 4;4 5 5 5 5 4;4 5;0 0;0 0;0 0;0 4 20.12.33 .............. 5; 5 5 6 5 6 5;6 5;0 0;0 0;0 0;0 5 25.1.34 .. ' ......... 3; 4 4 5 4 5 6;x 5; 6 0;0 0; 0 0;0 6 2.2.34 .. ' ........... 4;4 4 5 4 X 4;5 4;5 6; 0 0;0 0 ;0 7 9.2.34 .. ; ........... 4;4 5 6 4 4 5;5 5;6 0;0 0;0 0;0 8 27 .2.34 ............ 4;4 5 6 5 6 5; 6 6; 0 0;0 0;0 0;0 9 20.4.34 .............. 4;4 4 5 4 5 4;5 5;0 0;0 0;0 0 0 lO 27.4.34 ........ 5; 5 5 5 4 4 4; 4; 5 4; 5; 0 0;0 0;0 0 0 ll 8.5.34 ...........

-

5 6 6 X 5 ;0 5; 6 0;0 0;0 0 0 12 8.6.34 .............. -4 4 4 5 4;4 6;0 0;0 0;0 0 0 ·------- NoTE.-Titration Nos. l-8 were carried out with mome passage 62, strain 20449. Nos. 9-12 with mouse passage 120 of the same strain.

t:::: ;.. ;.. H f:rj ~ ~ ~ ^t:;l

(14)

C<:> c:> 1.-:J

I

1 : 640. 8;0 0 0 0 0 7;0 0 0 0 0 6;0;0 0 0 0 0 6; 7 0 0 0 0

ffJ >-3 q t; >-< l'd ffJ 0

z

>-3 ~ l'd

z

l'd rj ~ 0

"'

::0 ~ >-< 0 <: ,_, ::0 d r:tl 0 "':: ~ 0 ::0 r:tl l'1 v; >-< 0 .... ,.., 2: t<J r:tl r:tl >-< >-< >-<

(15)

TABIJE XI.

T·itmt·ion of Ant·ibody Content Pooled Se·ra of G1·o·up B (20235, 20768, 20777). Interval after Serum d{lutions. Date of test.

Date serum taken. immuni-~

z::~~~

1 , 2. 1 1 , 4. 1 1 , 8. 1 1 , 16. 1 l , 32. 1 1 , 64. 11 , 128. 11 , 256. 11 , 512. 11 1024.11 , 2048 16.5.34 " 2:3.5.34 30.5.34 " 6.6.34 10.11.33 20.11.33 27 .11. 33 4.12.33 11.12.33 18.12.33 27 .12. 33 2.1. 34 8. l. 34 15.1. 34 22.1.34 29.1. 34

14 20 27 34 41 48 57 63 69 76 83 90 97

6;6 7;7 6;6 6;7 0 0 6 0 0 0 6;0 0;0 0 0 0 0 C<:l c;:, " C<:l 13.~:341.

5.2.34 12.2.34 19.2.34 26.2.34 14.3.34 28.3.34 10.4.34

104 111 ll8 134 148 170 " Date. A. 16.5.34 ... .. 5;5 23.5. 34 ... . 5;6 30.5.34 ... . 5; 5 6.6.34 ... . 5;5 13.6.34 ... . 5; 5

5; 5; 5; 6 6;0 7; 0; 0; 0 0; 0 0;0;0 0;0 x;O;O;O 0;0 0;0 0;0;0;0 0;0;0;0 0;0 0;0;0;0 0;0 0;0 0;0 x;O;O;O 0;0

5;5;6;6 6; 6 6;0;0;0 0;0 6;0;0;0 0;0 0;0;0;0 0;0 0;0;0;0 0;0;0;0 0;0;0;0 0;0 0;0;0;0 x;O 0;0 0;0 0;0;0;0 0;0

5;5;5;5 5;5;6;6 5; 6 5; 6 7;7;7;0 5;6;6;0 6 ;6 6; 7 5;6;0;0 5;5;6;0 6; 0 5; 0 5;0;0;0 5;0;0;0 0;0 5;7 5;0;0;0 6;0;0;0 0;0;0;0 6;6;0;0 0;0;0;0 6;0;0;0 0; 0 0; 0 5'0·0·0 0·0·0·0 o~o~o~o 6~7;o;o 7;0;0;0 5;6;6;0 0;0;0;0 7;0;0;0 7;0;0;0 6;7;0;0 seri.un infected. 0;0 0;0;0

TABLE XIA .

Virus Titrat·ion.

5; 5; 6; 7 5; 6 5;5;5;6 5;5 5 ·6 ·6

·o

'5 ;' 6' 5 6;7 0 5 5;5 0 0 0;0 0 x;O 5 0;0 0 6 7;0 0 6 6;6 6 7 0;0 0 5 6;7 0 6;0;0 Virus dilutions. 1 : 10. 1 : 20. _\ 5; 5 5; 5 . 6; 6 5; 5 5; 5 6; 6 5; 6 6; 7 5; 5 5;6

: 40. 5; 6 5;6 6;x 6; 7 5;0

1 : 80. 5;7 6; 7 5;0 0;0 6;6

-;-·---.-·---· 5; 6 5; 6 4;5;5;5 5;5 5;5;6;0 5; 6 5 6;6 0 5 5;6 0 6 0;0 0 7; 0 5 6;0 0 5 6;7 0 5 5;6 7 5 6;6 0 6 6;6 6 5; 6; 0

5;5;6;6 5; 5 5;6;6;6 5; 8 5;6;7;8 5;6;6;7 5 •6 ·6 •6 '5

i

1' 6 ;7 ;7 ;0 5;6;6;8 5;6;6;0 5;6;6;7 6;6;6;6 6; 6; 0 5;5;5;6 5; 5 5;5;5;6 5; 5 5;5;6;6 5;5;5;6 5; 6; 6; 7 6; 7 5;5;6;6 5;6;6;6 5; 5 5;5;6;6 5;5;6;6 5; 6; 6

5; 6 5;5 5; 6 5;6;6 1 : 160. 1 : 320. I : 640 .. 6; 0 0 ;0 0;0 6;0 8; 9

0;0 0;0 0;0 0; 0 0;0

0;0 0;0 0;0 0;0 0;0

id !"' :.. t< l'l ~ ~ tl i?j

""

(16)

STUDIES OK THE XEl"lWTHOI'IC VliU:S 01' l-IOl~SESICl~KES~ .Lll.

Results.

(h·oup .:-1.- It ^lS:;eeli that J± llay:; after injection uenn-.nlizing- antihoclir:; IYPre pre~eut in dPmon~trablP amouut. From thP 14th day thrre \1"1\.~ a slml' but steady innea,;e in litre nnti.l the WJth l1a,· ,,·hen

the horses \\Pt·e r1ischargr'll. .

(.;ro·u;J B.- In tlli;-; group antilJOdie:; 11ere not (lpnwn~trable on the 14th <lay hut \I'Pl'P present in nppreciahle .amount ou ihe 20ih clay.

F1·om thi~ point on ihPre "·as a gT<Hlual hut ,;leacl~- ri~P until a peak

\H1s ullaillPl1 about the 8:)1'(1 to ihr Uith daY. From the lOOth daY llwre \\'a:; a llistinct irwlenC'~- f01· the tih~ to deneche hui it w<:{s maintai11ecl at a fai1-l,1· high leYe] up to the J 70th <1uy, ihe last re!'orded titration.

Di.<CI/SSLU/1.

Agaiu it is Plllplw;-;izPll lhnt the results reronlel1 Rbonld not 1>f:- snutini.zec1 IYith au e1·p to tnailtematic·nl areurar·Y. JLttlter due C'Oll- silleration ::;hould lw ,;aid to 1 hP difiieulty of tihating a minute liYing enhiy (if <·I·ellPn('P is giYen to the gPneml conspnsus of opinion itt reg-ard i.o Yiruse;; to-l1ay) and to the u~~e a:; an indicator of a some11·!tnt gross biolog-ical test 11·bich (·:tnnot be otltPl' than approximatP unle,;~

mw.lyserl on a sbtisli!'al bnsi,;. ITo11·eYP.l'. the ri~e an<l lhe lendeJJc.\

of the antiboch coutPnt to hll is 11·el1 clefinPr1 so that Yie"·e<l in this light i.he results are of singular iulere~t. 'rlwy inrlic·ate:-

1. 'rhe rise ill ani ilHHlY content of i·he ;;entm of horses ahr1·

i m m uniza tio n is slm1·. .

2. After immuni<~ahou b.' tlw injection of an attenuatPd necno- lropi!' Yirus it take~ ahont (wo mouths fm the titre to reach n :;ig-ni:fi('anl fig-ure awl thP peak i;-; not ~dtainecl nntil ihe '200th <la.1·.

:]. .\fter immuniz:di011 ll\· ihe -.;erum ,-i ,·us meiho<l whiclt is hased upmt. the !'Onlrol i11 ci?:o h~- hyperimmunl' serum of a htll_,.

virulent Yiscerotropie Yints the iu it ial ri:;e in antiboc1,,- cotttPnt is considerably mm·e rapi(l anll lhe penk is reueherl after about 100 lhys. 4. At no time doPs the iihe of tlte auti-sermu obtail!Prl after immunization h)· ei! her methorl ai iai11 :1 high le,·el--nn it Yolume ot a 1:012 dilution of serum hc>iug-reqni1ecl to neulnthzP

±

100 }LL.D ..

and this figure is apJn·oximntPl_y thr same fm bolh lllethor1s of innnuni- zation.

;j . .r'dtPr the peak of anti-bod:,-formation h.a;; heett re:tched then' is a <lecicled tPJll1encY for it to fall but a consi<len<hle titre is maiJl- lainerl for :100 claY~ anrl J 70 <lays respectiwl_,. (the limits of tltr l'PSpedii'P tP::>tSJ.

G. As yrt there i.~ uo rlab to inrlic:ttP for how long thP unti-hod.~

(·ontent will be maintained. This .a,;pect is being stuc1ie(1 hnl 1·esnlts wi.ll no( lw antilahle for at lrast two ~·ears.

'l'hi s conception of slo''" immtut i iy llt'l'elopment has a" fa1· as the authOl' is mYare a sing-le prP('eclent in the literature, Yiz. the <lelayed immunological l'<'~poJtse in lwYiJte:> to the injection of C'ltltnres of

:)64