Reprint from the American Journal of Veterinary Reearch. vol. 18, No. 66, January, 195?, pp. 162-166.

Vesicular Stomatit is in DeerLARS KARSTAD, D.V.M., and R. P. HANSON, Ph.D.

Madison, Wisconsin

Pnron ro 1943, horses and cattle were theonly known natural hosts of the virus ofvesicular stomatitis. In that year, an out-break of the disease occurred amons swinein an anti-hog cholera serum plant.i Sinee1952, vesicular stomatitis in swine andother animals has been recognized everyyear in certain areas of the southeasternstates. This poses a problem to individu-als concerned with the diasnosis and eradi-eat ion of vesieular exanlhe-a and foot-and-mouth disease.

fn June, 7955, a cooperative project wasset up between the U. S. Department ofAgriculture and the Wisconsin Agricul-tural Experiment Station to study theepizootiology of vesicular stomatitis inGeorgia and adjoining states. During thatsummer, a serological survey was conductedto obtain information relative to the num-bers and species of animals involved. Itwas found that over 50 per cent of thehorses, cattle, and swine tested in the re-

drained by the Altamaha River andIts tltibutaries earried specific virus-neu-

ng antibodies, indicating prior in-tion. Serum samples from raccoon and

in the area showed a high proportionpositive serum-neutralization titers., At

same time, numerous reports were re-ived of deer being observed with mouthLd. foot lesions. Some of these animalsre so debilitated that they were over-

n and killed by farmers' dogs. Sev-similarly affected deer had been shot

hunters and had been found unfit forAs a result of these observations. a

Paper NS 213 from the Department of Veterinary Sci, University of Wisconsin, Madison, publisheil rvithapproval of the director of the Wisconsin Agricul-

Experiment Station

Ihis stutly was supported in part by funils suppliedthe U,S. Departmetrt of Agriculture.

fl 'hie etuily is part of an epizootiological investigatione&r r ied ou t by the Wiscons in Agr icu l tu ra l Er

Station rvith the support of the AgricutturalService of the U.S.D.A. Coooeration of the

Disease and Parasite Research Branch and thenal Disease Eradication Branch of the U.S.D.A.,Fish anil Game Commission of Georgia, personnel of

Stowart, anil Dr. F. C. R,andall of Glennvil le. Ga..l ly acknowledged by the authors. The deer used

the experiment were supplied by the Wisconsin Con-

study was undertaken of experimentallyinduced vesicular stomatitis of deer.

nI . r r rnr , r ls , ' rxn ] l erHoosFive male deer * rvere exposetl to the virus of

vesicular stomatitis. Since the natural means ofexposure and the rclative susceptibility of thespecies nere unkrrown, several clifferent routes ofinoculation .rrere used initially to ensure infection.

The strains of virus were isolated from swinein Georgia and Louisiana cluring the summer of1955. These isolates tvere examinetl by means ofthe complement-fixation test at the U.S.D.A. Ani-mal Disease and Parasite Research Branch labo-ratory at Beltsville, Md., and founcl to be N,r-typevesicular stomatitis virus. VesiculAr tissue wasthen submitterl to the Wisconsin station where thevirus strains were propagated in embryonatingeggs, mice, ancl swine. Allantoic fluicls from thefirst ancl second passages in eggs rvere used forthe ileer inoculations. These fluids hacl a titerof 100 l.cl.un in chicken embryos.

A portable cattle-holiling chute rvas used forrestraint. Iloculatecl deer were kept in this forthe duration of the infection. In this way, ani-mals were confinecl sufficiently to permit the nec-essary handling ancl close observation, yet recum-bency anil limitecl forward ancl backward walkingrvas allowecl. A cliet of a good quality of alfalfa.hay, ground mixecl grain clairy concentrate, andwater was supplied at the anterior encl of thechute.

Inoculations were made with the animals underbarbiturate narcosis, For this purpose, pentothalsodium, pentobarbital soclium, or secobarbital so-dium were usecl. These clrugs rvere given intra-venously to efect.

Preinoculation bloocl samples were taken for se-rum-neutralization tests. Body temperatures wererecorcled where porsible. Three of the cleer werefouncl to be too excitable to permit readings to betaken without applying unrlue restraint. Virustitrations were carriecl out on 3 of the animalsto obtain information on the relative susceptibil-ity of cleer to vesicular stomatitis. Virus was in-oculatecl by intraclermal and intramucosal routesl0.1 m]. of inoculum rvas introducecl at each site.Six serial tenfokl dilutions of allantoic fluid wereinoculated at different sites on the tongue as amethod of virus titration. Similar ililutions wereplaeecl intradermally in the coronary bands or in-terdig i ta l sk in of the feet .

T*'o of the animals (D53, D54) were tlestroyed

* Wtri*t"it d,eer ( Od.ocoileus airginianus ) raised in\\'isconsin.Department.

[ 1 6 2 ]

1

Fig. f-Vesicle from tongue of deer D54 taken 50 hours postinoculation. Hematoxyli:r anileosin stain; x 240.

Fig. 2-Vesicle from muzzle of tleer D54 taken 50 hours postinoculation. Hematorylin antleosin stain; x 240.

| 1 6 3 I

164 I-.ans KnnsrAD AND R. P. HlNsoN AM. J. YET. Rrs.JANUARy 1957

at the peak of the rlisease (72 and b0 hours post-inoculation, respectively) to study the histopatho-logical changes procluced. Three cleer were heklsuffieiently long to observe specific antibocly re-sponse by measurement of convalescent serum-neutralization titers. Preinoculation and postin-oculation serum samples were examinecl for thepresence of specific virus-neutralizing antibocliesby inoculation of quantities of heat-inactivatedserum mixerl with equal amounts of selectecl virustlilutions into 9- or 10-day embryonating eggs.Serum-neutralization titers were expressecl inehieken embryo l.d.so units,

Recovery of virus was attemptecl from thebloocl, lungs, spleens, livers, lymph nocles, kicl-neys, and. tongues of the 2 destroyed animals.The method of recovery used was inoculation ofsuspensions of ground tissues into 8- to 10-clayembryonating eggs.

Euthanasia of animals at the end of the experi-ment was aecomplishecl by intraperitoneal injec-tion of one of the barbiturates or a solution ofchloral hyilrate.

A list of animals used ancl methocls of exoosureare given (table 1).

Rpsur,rsAll, preinoculation serum samples were

negative when examined for the presenceof reutralizing antibodies against NJ-typevesicular stomatitis virus. Febrile responsewas detected in the 2 animals on whichadequate body temperature records wereobtained. The body temperature of animalD51 rose from an apparent normal of101.3 to 101.8 F., to a peak of 104.3 F. at24 hours postinoculation. The decline fol-Iowing this was at first rapid to 102.6 F.

lowed exactly. The vesicles arose from con-fluence of lacunae which formed. betweenthe cells and from final cellular d.isinte-gration. The prickle cell layer apparentlywas first affected. Evidently, thi eells inthis area shrank and became rounded. Theintercellular bridges were disrupted anddestroyed. Eventually, many of the epi-thelial cells completely disintegrated andthe lacunae became confluent to form dis-tinct vesicles of signifi.cant size. Epithelialcell destruction usually extended. down tothe basal layer and sometimes even thiswas destroyed over small areas. Moderatenumbers of polymorphonuclear leukocytesand macrophages invaded the vesicles butvery few of these cells were present in thesubepithelial tissues. Congestion and evenhemorrhage of the subepithelal tissues wasa constant feature immediately under thevesicles but not elsewhere.

Surface epithelium remained intact fora relatively long time (fig. 2) as comparedwith observed vesicle development in cat-tle and swine, where fluid pressure insidethe vesicle eaused. bulging of the surfaeefollowed by early rupture of the vesicle.3Surfaee epithelium sometimes seemed toremain intact until cellular deseneratiorrin deeper layers became complete. Thensloughing sometimes occurred without thevesicle ever having ruptured.

Gross foot lesions were not observed. Asingle microscopic lesion, vesicular in na-ture, was observed in routine histologicalexamination of inoculated interdisital ikirrfrom the animal which was killed 50 hoursafter inoculation. It was thoueht thatpossibly vesiculation in the more- heavilyskinned coronary or interdigital regions wasa slower process which was being abortedby the rapid antibody response to tongueinfection. Aceordingly, an attempt wasmade to induce foot lesions in one animal(D55) by inoculation of coronary bands asthe only route of exposure. This animalmanifested a transient pyrexia followinginoculation but without development oigross foot lesions. Histolosical examina-tion was not made as the animal wasallowed to recover for demonstration ofantibody response.

Salivation was moderately inereased inthose animals whieh developed tongue ves-icles. Affected animals lost weiehf due totheir disinelination 1o eat when-mouth le-sions were present. At this time, alfalfa

at 40 hours, then more gradual to normalagain at 80 hours aftei inoculation. The

r deer, D55, showed an elevation oftemperature to 103.0 F. at 24 hours afterinoculation followed by a rapid return to

. Vesiculation was not observed inis animal.Three of the 4 animals exposed by intra-ucosal inoculation of the tongue devel-

tongue vesicles. These took the formsmall circumscribed areas at the sites of

lation, appearing first at 24 to 48rs postinoculation (fig. 1). Litt le vesic-

fluid developed in the affected. areas.surface of such areas was not raised

ove the level of the surroundins normalithelium but, on histological examina-

true vesicles were evident. Becauseearly stages of vesiculation were not

Av. J . Vr r . REs.JANUARY 1957

\rBsrcur,ln Srolreurrs rw Dnnn

hay was takeu in preference to finelyground dai ry eoncentrate. I l is to logicalsections made from lesions on the tongueof the deer killed. 72 hours postinocu.lationshowed small foreign bodies embedded. inthe degenerating epithelium (fig. 3). Ap-plication of the Hotchkiss-1\{cMannus dif-ferential stain n indicated that these parti-cles were of plant origin. Some of theseforeign bodies 'rvere found penetrating thesubmucosa.

One of the deer (D52) appeared "re-fractory" in that visible lesions did notdevelop follon'ing inoculations into thetongue, muzzle, and feet. As the deer wasan extremely nervous animal, his tempera-ture was not taken. A high serurn-nell-tralizing titerrvas found two rveeks afterexposure.

Virus rvas recovered only from vesiculartissues or f luids taken from inoculated ani-mals during the early stages of the dis-ease. Viremia may have existecl but virusrvas not detectable in blood taken at 50and 72 hours postinoculation. Lung, l iver,spleen, lymph node, and kidney tissuesgave negative results rvhen examined forvirus at 50 and 72 hours.

Virus titration by tongue inoculation rr.asattempted in anirnals D52, I)53, and D54.Titrations were read at 48 hours after in-oculation. Titers of 10't rD per mill i l i teru'ere obtained in animals D53 and D54.evidenced by thc development of lesions atthe sites of inoculation of 0.1-nl. quanti-t ies of allantoic fluid diluted to 10 5. Theseallantoie fluids titrated 10' l.d..n units ofvirus per mill i l i ter in S-day chicken ern-bryos. I la ter ia l f rom the same st ra in(Adamson), a different harvest, but onervhich also had an embryo titer of 10' l.d..olr,as titrated on the tongue of a mature co'wand in the coronary band of a 40-lb. pig.

A n i r r a lNo.

I n t r a m u c o s a l I n t r a d c r n r a lS e r i n o c u l a t i o n i n o ( ' u l a t i o n

Fig. 3-Vesicle fron tongue of cleer taken 72 hourspostino:ulation. Plant particles are the strawlikedarl<-staining botlies. Stainetl by Hotchkiss-Mc-

I '4 annus meihod: x 160.

The titer in the co\v was 10' and inpig 10a. Deer D52 gave no clinicalclence of infection.

Serums of the 3 deer which rvere helilsufficierrtly long to allow observation ofvirus-rieutralizing ti l t ibody response neu-tralizecl 10,000 I.d..n of NJ-type vesicularstornatit is virus rvhen examined 14 daysattcr c,xposure.

The rcsults of these experimental infec-t ions arc surnrnar izecl ( table 1) .

Drscussro-x

The observatious made iu this study en-able us to better undcrstatrd the role of

theevi-'

TABLE 1-Summary of Experimental Vesicular Stomatitis Virus Infections of Deer

I lou tc of e\posure

Grossles ions

C o n v a l e s c c n tF e b r i l e S N a n t i b o r l l '

responsc t l ler Disposition

D 5 1 6 n l o .

2 I r .

8 m o .

2 l 'r.

T o n g u e

T o n g u e

Tongue

Tongue

None

Ifuzzle, cau-dal folds, cor-onary bands

XIuu-zle, inter-digital skin

Interdigitalskin

Coronarybands

Coronarybands

Vcs ic les ontongue andtnu zzle

Noue

Vesicles on

Vesicles ontongue

None

++ 101 l .d. ;o

N o n e 1 0 { l . d . 5 o

Not deter- Not deter-m i n e d m i n e d

Not deter- Not deter-n i n e d r n i n e d

+ 1or l .d.so

I lekl for lurtherc\Deriments

Destroyed 2 wk,I)ostinoculatiol

Dcstroyed 72 hr.postinoculBtiol

Destroyed 50 hr.postinoculation

IIeld for furtherexperiments

D52

D 5 3

D 5 4

D 5 5

NI

}T

M

M

LEns I(.s.nsrAD AND R. P. HnNsoN AM. J . YEr . REs.JANUAR,Y 195?

deer in the epizootiology of vesicular sto-Tatitis.- Finding the serums of 60 per centof the deer in the enzootic area poiitive tothe serum-neutralizatiou test would leado.ne to suspect that deer are highly suscep_tible to vesicular stomatitis and

-tnat tt .

clisease produced is not fatal. This lvasfound to be the situation. Our experimen-tal findjngs help to explain the reports re-ceived from residents of the area that deerare seen with sore mouths and feet. Ourfailure to produce gross foot lesions doesnot prove that such lesions do not occur innatural infections. It is possible that en_virorrmental slress is arr impo"tant faetcrrin eausing these changes. Foot involve-ment in cattle has been difficult to pro_cluce experimentally, yet it is seen in heldeases of vesicular stomatitis.5

On the basis of l imi led observat ion. i t mavbe said that e l i r r iea l ly inapparent in fect ionsoccur. These may be even more commolt un-der conditions of rratural exposure. In suchcases, animals would subsequently earry vi-rus-neutralizing antibodies without havinsexperienced clinical disease. At the oppolsite extreme.- it is possible that the super-imposition of errvironmental stresses couldprecipitate the development of severe le-sions.. It is readily seen how a grazing a.ndbrowsing animal like the deer iiEht 5e fa-tally ineapacitated by severe mouth or foot

tal disease was short-lived and quicklv fol_lowed by high levels of virus-neutraiizinoantibodies. Howeve", deer may

"id il;;i;:

taining the disease in its present enzooticproportions by supplying a generation ofnew susceptible hosts each outbreak season.

Sulrlrany1) Vesicular stomatitis in swine was rec_

ognized as an enzootic disease in southeast-ern l]nited States in 1952.. 2) Detection of neutralizing antibodiesin deer from southern Georgia-and reportsof disease by hunters,leAto the considera_tion of deer as a ri6*F reservoir of ve_sieular stomatit is. tfT

3) Five deer were inoeulated bv sev-eral routes r,vith vesicular stomatitis virusltt-d developed mild, acute, or abortiveinfections.

4) Deer ar:e probably not important res_ervoirs of thrl virus, since reeovery is rapid.They may have a minor role in miintainingthe summer epizootic. The disease in deer-although mild under experimental condi_tions, may be more serious under certairrstress conditions existinq in the field.

References

l Schoen ing , H. W. , and Crarv fo rd , A , B . : Outbreak o tYesicular Stomatit is in Srvine and Its Differential Diag-nosis from Vesicular Exanthema and Foot-and-Mouth Dii.e a s e . U . S . D . A . C i r c . 7 8 4 , ( 1 9 4 b ) : 1 _ 1 4 .

. . : Kars tad ._L . . I { . ,

Adams, E . V . , Hanson. R. p . , audl 'e r r i s , D . H. : Ev ideuce fo r the Ro le o f Wi l r l l i fe in theEpizootics of Yesicular Stom&titis. J.A.V.M.A., t g9,( 1 9 5 6 ) : 9 5 - 9 6 .

_ 3Ch61y, T. L., Hanson, R. p., and trIcNutt, S. If.:

Pathology of Vesicular Stomatit is in Catile. proc. Book,A V M A ( 1 9 5 1 ) : 1 1 9 - 1 2 4 .

a Kligman, A. trI., and Mescon, If . : The periodic-Acid_Schiff Stain lor the Demonstration of Fungi in AnimalT i s s u e . J . B a c t . , 6 0 , ( 1 9 5 0 ) : 4 1 5 - 4 2 1 .

__ tRraud ly , C . 4 . , I {anson, R. p . , an i l Chorv , T . L . :

Vesicular Stomatit is u'ith particular l ieference to the1949 Wiscons in Ep izoot ic . Proc . Book , AVMA ( 1951 ) :61-69 .

involvement. The observed invasion of in-jured tissues by sharp plant particles maybe a compl i t .a t ing factor in r ra lura l in fec_

o^ns contributing to secondary bacterialffection. Therefore, one may suppose that,hile an uncomplicated caie oi

-vesicular

titis in a deer may be mild or evenpparent, secondary stresses could greatlyrease the seyerity of the attack.

It is unlikely that deer are reservoir hostsvesicular stomatitis, since the experimen-