Studies on the neurotropic virus of horsesickness. V. The antigenic response of horses to simultaneous trivalent immunization

Onderstcz;oort Jovrnol of Vete1'1.11aJ'Y Science and Animal lnrlltstry, T"olume 1, 1Vwnbe1· 1, htly, 1936.

Studies on the N eu rotropic Virus of Horse- sickness V.

The Antigenic Response of Horses to Simul- taneous Trivalent Immunization.

By R. A. ALEXAXDER, Section of P1·otmwology and Yi rus Diseases, On rl ers tepoort.

I-r has been :>hown p1·eviously that there exists a plurality of antigen ically eli ffe1·eu t ;-;train,; of horsesic kness Yiru~. Con;-;idera tion of this important fact, in the light of results obtained \Yith the neuro- tropic Yinls Yaccine both in the laboratory ancl in the fielrl rluriug ihe season 1934-=-~5, has shown that the problem of inunuu i:la tion bas become one of the development of complete pol_vntlent i nnnun it_, ..

On pmel~· theoretical grounds it is reasonable to belieYe that this might he accomplished most :;atisfactorily by a serie~ of in.iedions euch of "·hich comprised a certain infecting rlose of a different Yirus strain, but the practical difficulties attached to suf'h a pror-t>dure are so Yast that it is essential to limit tbe number of in.iections to a minimum. If possible immunization should be coidiuecl to a single subcutaneous injection of a mixture of the clifte1ent strain~. This is the procedure \l·hich has been adopted but it is necessary to record the antigenic response in horses as <leterminecl by in Yiho :=:ernm neutralization tests and by in YiYo immunity tests.

From time to time during the course of routine Yaccine produc- tion by the method clescribe<l (this journal) horses were injected with material taken at random from col1l stm-age an<l kept at room temperature (±80° F.) for periods up to 14 clays so as to approximate the conditions under \Yhich immunization "-as carrie<l out in the fiel<l. Details of 5 such injections are the follo"-ing-:-

2 I 8 I 3-±. Horr-e :20987. 10 cc. subcutaneouslY Yarcine i>atch 8 p1 eparod 2417134. ..

'J/8/3,J. Horse 'JOU!:Jl. 10 c-c. subcutaneously ,·accine hatch 10 prepared 118!=!4.

0110134. Hon;p 20085. 10 ec. subcutaneously \·accine IJntch 2'~ prepared

2719134. .

3110134. Horsr> 20068. 10 c:c. subcutaneously vaccine hatch 27 prepared 3/10134.

22111/3-1. Horse 20941. 10 e;c. snbcutaneou;ly vaccine batch 40 prepared 20/10/34.

11

STUDIES ON THE KEL:lWTROPIC VIRUS OF HORSESICKNESS.

Each of the animals shmYed the anticipated mild febrile reaction to the Yacc.:ine and until required for bleeding "·ere stabled in company w1th a number of susceptible horses, exercise being restric.:tecl to nmnmg in a bare dry paddock from 10 a.m. to 3 p.m. each day. During this pe1;iod no cases of natural horsesickness occurred in the stables so that it is believed that between the time of injection and the time blood \Yas tapped for the collection of serum the horses were not exposed to natural infection.

Sinc.:e it has been shO\nl that the antibody content of serum reaches a maximum approximately G months, after the imnnu1izing injection, neutralization tests were c.:onducted \Yith serum colleded at thi:s :;tage using the intra-cerebral protection te~t in m1ce as described preYiously (Alexanrler, 1935). The results are g1ven m tabular form in Tables I, II an!l III on pages 11-13.

It ,,-ill be noticed that unit Yolume of the serum dilutions were requirerl to neutralize

±

100 minimal infective rloses of virus strain 449 but onlv +50 M.I.D. of strains 0 aml 4G4 B. In the tables no adjustment ·has been made as compensation for this slight Yariatiou in titre of the antigens since the results are so clear that this "·as considerecl hot h unnecessary and undesirable.

Consideralion of the tnhles s,ho11·s that each of the 5 horses developer] antibodies against each of the three vin1s strains. There

\\·as some slight Yariation in the respectiw ti!res notabl~· in the case of horse 20987 "·hose serum neuhalizecl 448 virus onlv to a titre

of ^{1/ " '} but in the case of horsesic kness even thiR lo"· ti tr:e in rl i en tes

that a solid immunity has been induced. 'l'he significance of these resnlts \Yill hecome clear if the tnbles are reall in conjunction 11·ith those indicating the antigenic diffrences of the virm; fitrains published preYiousl.\· (.-\.lexan der, 1935, pp. 3G9-17J).

~\fter the r:ompletiou of the experiment the 5 horses used "·ere exposed to natural horsesiclmess on the fann Kaalplaas "·hich is notorious as a bad horsesiekness fann. Throughout the season the.v rPmainerl in perfect health.

'l'o eon firm the result,.; of this experiment by rlirect immunity test 8 other l10rses \\·ere immunized in a similar manner. Four weeks after immuni;-:ntion the.v \Yere divided into 1 groups; group 1 (3 horses) recei1·erl 10 cc. of virulent 0 virus intravenously; group 2

(;~ horse~) receiwrl 10 cc. of virulent 449 virus; group 3 (2 horRes) rer-eivecl ]() cc. intravenouslv of a mixture of Rtrains 0 a1Hl 449. There \YE're no reactions. 'l'he virulence of the strains userl for the immunit.1· test IYas demonstrated subsequent!.\· hy routine paRsage through ~nsceplible horses all of which clierl. Unfortunately it was not possihle to c.:aiT.\' out an innnunit.\' teRt agninst strain 4G4 as this s,trnin in ib Yirulent form has been lost.

12

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ri'Am,Jo: I.-.Ye11tntli.zation of Vints Slrm:n 449. Antigell 'l'itration. Antigen Dilutions. D:tte. Virns Antigen. Cone. I \".

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1/20.

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l/40.

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20.3.351 Ho1·se 20968, bled 27.3.3:) ..... . 2!).3.3fi Horse 20()85, bled 27 .3.35 ..... . 22.:>.351 Horse--, bled 8.6.3/i ...... .

Neutrali<mtion Test . Serum Dilutions. 1/2. l/4. 1/8.

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]lak 26 .4.3:) 17.5.35 7.o.35 ... Ot Date. 26.4.35 26.4 .3:) l7.f>.35 17.5.35 7.6.35 'L'Aili.Jo: llL-.Vcutwli.:111ion uf \'irus Strr1111 4()4

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Antig-en Titration.

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.A ntigcn Dilutions.

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Cone.

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Virus Antigen. 1/80. 1 l/160. 1/320. I I /H40. Generation HiS, stmin :3450 4o4ll ..

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1/8. Horse 20087, bled 21 . J . 3:i ..... . Horse 20991, bled 21.1.:35 ..... .

llays.

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112 1 oo 172 1 oo Horse 209G8, bled 27.3. 3•> ..... . 173 1 oo H or·se 20985, bled 27.3. :3:> ...•.. ]7:) 00 Horse 20941, bled 8.5.3:) ....... I 180

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44 40 . 44 I 44 OOOx 4450 :)() 3456 45 46 4555 3450 4446 .:__ _ ____.: --- NoTK~Cone. of Anti!!en = Serum sc.linc dilution of stod< umrlsion. ]n the tables 'the numbers indicate the day after injection on which the mice died. 0 = Sur·vival for mol'C than 9 days. x = Death due to some cause other than horsrsickncss encephalitis usually injury at tim<· of injection. Thus, 4:i0x mNtns of 4 miPP injcetpd 1 <lied cby 4, 1 diN1 <lay i\, 1 died as iL dirPct n·snlt of injPct.ion ,,nd 1 Slii'Vil'!•d.

;:;; > > t; ~ > ~ tj M ;;:J

S1T DIES ON THE NEUROTJWl'IC VIRUS OF HOltSESICKNESS.

Drscu ssroK.

lt is not possible to generalize from the results obtained from so

~mall a number of animals but it "·ould appear that the simultaneous iujedion of 3 different strains of neurotropic attenuated Yirus results in the production of a wlid immunity against each. In other \Yonls, there is no antagonistic action of one strain against another. This finding is the justification for issuing a nccine which consists merely of a mixture of the different aYailable strains diluted in such a wa \' that the final product will contain not less than 100 infecting rlo~~s of eaeh strain per dose of Yaccine, and obviates the necessit)· of repeated injections in the field. Whether there ,\·ill be a similar antigenic response when a greater number than 0 strains are illclude<l i;; a point whi('h is being iuYestigated at the moment and will be reported in due course.

Suc-.nrAHY.

By in vitro awl in Yivo methods it has been sho"·n that the ,;im ulta neons injection .of 3 strains of neurotropic hOl'sesic lm ess n rus.

results in the produ('tion of a solid immunity against each.

REFERENCR.

ALEXANDER. H. A. (1936). Studies on the Neurotropic Yirus of Horse- sickness Hl. The 1 ntracerebral Protection Test and its Application to- the Study of Immunity. Understepoorf, J. of Vet. Sci. and A.n lnd.,.

VoL 4, So. 2, pp. 349-377.