WESSELSBRON DISEASE: VIROLOGICAL AND SEROLOGICAL STUDIES IN EXPERIMENTALLY INFECTED SHEEP AND GOATS

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1 Onderstepoort J. vet. Res (198) WSSLSBRON DSAS: VROLOGCAL AND SROLOGCAL STUDS N XPRMNTALLY NFCTD SHP AND GOATS A. THODORDS and J. A. W. COTZR Veterinary Researh nstitte Onderstepoort 1 1 ABSTRACT THODORDS A. & COTZR J. A. W Wesselsbron disease: virologial and serologial stdies in experimentally infeted sheep and goats. Onderstepoort Jornal of Veterinary Researh (198). Adlt sheep and goats and new-born lambs and kids were experimentally infeted with a Wesselsbron disease virs. The viraemia in lambs ommened approximately 7 h after infetion and lasted on the average for 5 h. A febrile reation whih was mostly biphasi ommened several hors after the viraemia and otlasted it by 5 h. The viraemia in adlt animals began abot 5 h after infetion and lasted for 3 h. The fever sally ommened several hors after the viraemia and as in 3 ases ot of 4 in lambs it otlasted the viraemia by at least 3 h. The virs old be reisolated in mie from every tisse examined in lambs althogh it has previosly been shown that pathologial lesions are restrited to the liver and lymphati tisses. Resme MALAD D WSSLSBRON: TUDS VROLOGQUS T SROLOGQUS SUR DS MOUTONS T DS CHYRS NFCTS ARTFCLLMNT Des hevres et des motons adltes ainsi qe des agneax et hevreax novea-nes ont ete infetes artifiiellement ave le virs de a maladie de Wesselsbron. La viremie hez les agneax ommenra approximativement 7 heres apres /infetion et dra en moyenne pendant ne periode de 5 heres. Une fievre prinipalement biphasiqe ommenra plsiers heres apres a viremie et persista pls longtemps qe elle-d por ne periode de 5 heres. La viremie hez!es animax adltes ommenra environ 5 heres apres /infetion et elle dra pendant 3 heres. La fievre ommenra habitellement plsiers heres apres a viremie et omme dans trois as sr qatre hez les agneax elle persista pls longtemps qe a viremie por ne periode da mains 3 heres. Le virs pet etre re-isole dans des soris a partir de haqe tiss examine dans les agneax malgre a observation faite anterierement qe les lesions pathologiqes sont limitees afoie et ax tisss lymphatiqes. NTRODUCTON The virs of Wesselsbron (WSL) disease belongs to the sbgrop Flavivirs of the family Togaviridae (Fenner 1976) and was isolated for the first time from a new-born lamb in the Wesselsbron distrit in the Orange Free State (Weiss Haig & Alexander 1956). Sbseqent work showed that WSL virs mainly ases mortality amongst new-born lambs and kids is an inapparent disease in adlt sheep goats and attle and may give rise to abortions in pregnant ewes (Weiss et a! 1956; Weiss 1957; Coetzer Theodoridis & Van Heerden 1978; Coetzer Theodoridis Herr & Kritzinger 1979; Coetzer & Theodoridis npblished observations). n addition to these effets the virs may also be responsible for teratology in the developing foets in sheep and pregnant attle (Coetzer & Barnard 1977; Coetzer eta!. 1979). The emphasis in the present stdy is on the repliation and distribtion of the virs in experimentally infeted lambs and kids and in adlt sheep and goats by determining the virs onentration in the blood and tisses over a period. Serologial stdies were also done on the animals that reovered from the infetion. MATRALS AND MTHODS Virs The WSL virs isolated in mie dring the otbreak (Coetzer et a!. 1978) and kept lyophilized at - o was sed to infet the experimental animals. The virs had been passaged 3 times in mose brain and had a titre of mose LD 5 /mf. nfetion of experimental animals Dorper sheep and goats were sed. All the adlt animals were tested for haemaggltinating antibodies to WSL virs and only the sseptible ones were Reeived 1 Jly 198-ditor infeted. The new-born ambs were kept with their mothers in a reasonably inset-free stable. Lambs and kids not older than 3 days were injeted intradermally with 5 mf of virs sspension and adlt sheep and goats sbtaneosly with 5 mf of the sspension. Virs assay in the blood and tisses Blood in an antioaglant (heparin) was periodially olleted from the infeted lambs and kids aording to the time shedle shown in Fig. 1. For infeted ambs were sarified at 4 h intervals after infetion and samples of varios tisses were removed aseptially. Three ewes and goats were killed after infetion at the time intervals shown in Fig.. n most instanes these samples had to be stored for a ople of days at - o before being tested. The blood samples were dilted : in bffered latose peptone (BLP) ontaining peniillin (5 U/mf) and streptomyin (5fLgfmt) and the tisses were maerated and grond to a homogenate. A 1% sspension in BLP was prepared ontaining the same onentration of antibiotis as was sed for the blood sspension. The spernatant was sed for the titration. Tenfold serial diltions of the above sspensions were prepared in BLP and one litter of day-old mie was injeted intraerabrally with 5 mt of the diltions. Deaths were reorded for 1 days the mose LD 5 being allated by the method of Reed & Menh (1938). Serologial tests To identify the reisolated virs with WSL positive sheep serm the serm-virs netralization test was arried ot in day-old mie sing the onstant serm-tenfold virs diltion method of Cnningham (196). At the same time sera of some of the experimental animals that reovered were tested for netralizing antibodies against WSL virs. * BLP: Final onentration of 1% peptone and 5% latose in 1 M phosphate bffer 1

2 WSSLSBRON DSAS: VROLOGCAL AND SROLOGCAL STUDS N NFCTD SHP AND GOATS 1 Lamb () " t f v.. " / / 1 " lamb Q 6 on N 1-5 o" _J 5l :J. 1Q-4 1-J. r ; t._" r a. 398 " CD " FG Viraemia and body temperatre of lambs infeted with Wesselsbron disease virs (---Temperatre) (-- Viraemia)

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4 WSSLSBRON DSAS: VROLOGCAL AND SROLOGCAL STUDS N NFCTD SHP AND GOATS 1 8 Kid " N 1Q- 1Q-5 _j " ::l 1Q r- " t " 1 1 " t " 1 v 46 4 ::l ;;; " m Kid A " 1Q-5 5 o" _j " ::l 1.C: " 5 " 1 3 " ".C: " m FG. 5-8 Viraemia and body temperatre of kids infeted with Wesselsbron disease virs (----Temperatre) (-- Viraemia) 4

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6 WSSLSBRON DSAS: VROLOGCAL AND SROLOGCAL STUDS N NFCTD SHP AND GOATS 1- we Q 6 46 " o_ Ql " :J.s;1"" " Ql 1 4 -" :J <ii 398 Q; 39.4 "-! CD 1-1- " " 1"" J 1Q- -.S 1-4 we " / / / " Ql :J " i " " 1Q- 1/ 1- v :J <ii 398 Q; " CD FG. 9-1 Viraemia and body temperatre of ewes infeted with Wesselsbron disease virs (--- - Temperatre) (-- -Viraemia)

7 A. THODORDS & J. A. W. COTZR 1" 1Q- 1()-< () J " :J.!: 1-. 1" we " f 1 l l v l " we ()-< J J 1()-<.!:. 1Q 3 1Q-...- / v" A.!: :J " 398 $ ll 39 1"

8 WSSLSBRON DSAS: VROLOGCAL AND SROLOGCAL STUDS N NFCTD SHP AND GOATS The haemaggltination inhibition test (HAl) was arried ot with all available sera from reovered animals by the tehniqe of Clarke & Casals (1958). A srose-aetone extrat of WSL virs-infeted mose brain was sed as antigen and the test performed at ph 6 4. RSULTS Viraemia and body temperatre in lambs and kids The viraemia in all 4 Jambs ommened at abot 7 h after infetion and was detetable for 5 h (Fig. 1-4) main peaks being observed at 3 h and 5 h. The body temperatre started rising between h in 3 Jambs (Fig. 1-3) dropped to normal at 55 h and rose again within hors. The fever ontined for at least 9 h in of the lambs (Fig. & ) and for abot 11 h in the other two (Fig. 3 & 4). WSL virs old be demonstrated within 18 h after inolation in the blood of all 4 kids (Fig & 8). Viraemia reahed a peak at abot 35 h after infetion in 3 of the kids (Fig. 5 7 & 8) while in the 4th kid it was higher at 5 h (Fig. 6). At abot 7 h the viraemia dropped in all for. The body temperatre rose between 35 and 45 h after infetion in all 4 kids registered main peaks (Fig. 6 7 & 8) and beame normal at abot 1 h in 3 of them (Fig. 5 6 & 7) whereas in the 4th kid (Fig. 8) it rose one more at 15 h. Virs distribtion in tisses of lambs At 4 h after infetion no virs old be deteted in the tisses of the lamb that was killed (Table 1). At 48 h and 7 h respetively virs was present in most tisses the highest onentration being in the blood and then in the adrenal and liver (Table 1). The virs was present in almost every tisse of the lamb that died at 96 h after infetion althogh the lesions were restrited mainly to the liver and lymphati tisse (Coetzer Theodoridis & Van Heerden 1978). forth (Fig. 1). The body temperatre started to rise at abot 7 h (Fig. 11 & 1) in 3 ewes and 55 h in the forth (Fig. 9). The highest temperatre was reorded at abot 1 h in 3 animals (Fig. 9 1 & 11) and at 7 h in the forth (Fig. 1). The fever otlasted the viraemia by at least 3 h in 3 ases ot of 4 (Fig. 9 1 & 11). Virs distribtion (assay) in tisses A stdy was made of virs onentration in the tisses of 3 sheep and goats (Table ). These animals were killed at varios intervals after infetion for the stdy of the pathology and the same material was sed for stdying the virs onentration. The highest virs titres were demonstrated in the liver and blood and then in the spleen lng and brain. WSL virs was present in fewer tisses in Sheep 4668 whih was killed earlier than in Sheep 371 whih was killed 3 h later (Table ). TABL Wesselbron disease virs onentration in logs 1 mose LD.o/ 5 mt in the tisses of experimentally infeted sheep and goats Sheep Goats Killed hors post-ino- ation Blood Liver Spleen < 5 16 Heart < Adrenal < 5 < 5 <5 Brain < 5 < Lng < Kidney < < 5 <5 Mesenteri lymph node TABL 1 Wesselsbron disease virs onentration in logs 1 mose LD. /5 mf in the tisses of experimentally infeted lambs Lamb Lamb Lamb Lamb* Xll Killed hors post-inolation * Blood < Liver < Spleen <5 <5 < 5 16 Heart <5 <5 < 5 Adrenal < Brain <5 14 < 5 5 Lng < Kidney <5 < 5 37 Presaplar lymph node <5 < 5 5 Mesenteri lymph node <5 < * Died shortly before post-mortem examination Viraemia and temperatre reation in ewes WSL virs old be demonstrated in the blood at abot 5 h in ewes (Fig. 1 & 11) at 58 h in the 3rd ewe (Fig. 9) and at 35 h in the 4th ewe (Fig. 1). Viraemia eased at abot 8 h after infetion giving an average dration of 3 h in 3 ewes and 5 h in the Development of antibodies to WSL virs All the virses reisolated from the blood and tisse samples of the inolated lambs were netralized by known WSL positive sheep serm. All the sera from the inolated animals tested at random netralized the known WSL virs (rslts not shown). With very few exeptions the majority of the inolated ewes goats lambs and kids showed high H Al titres in their sera when bled in the 3rd week (Table 3). The mean vale of the titre was the highest in the lambs and kids and then in the ewes and the goats. The latter had the lowest titre (Table 3). DSCUSSON The WSL virs isolated from sheep dring the most reent otbreak of the disease in 1974 ased fever in the majority of the infeted lambs kids ewes and goats. The mortality rate among lambs was 7% (Coetzer et a!. 1978) and nil among adlt animals. Viraemia was present for some time before the onset of fever bt eased several hors before the fever sbsided. However the overlapping period was long enogh to allow blood samples to qalify as a diagnosti medim. The graphs of this stdy an. be sed as a gide as to the optimm time for olletmg blood for diagnosti prposes. 8

9 A. THODORDS & J. A. W. COTZR TABL 3 Convalesent haemaggltination inhibition titres of experimentally infeted animals. Sera were taken 3-4 weeks after infetion wes Lambs Goats Kids Nos. Titre Nos. Titre Nos Titre Nos. Titre Mean vale wes Lambs Goats Kids The maximm virs titre in the blood of adlt sheep was 4 x mose LD 5 /mt with a mean vale of 4 x 1 4 Althogh this vale is low sheep old still be the speies responsible for the maintenane of the virs in the years of interval between epizootis. One shold also keep in mind the fat that nder natral onditions the environmental stress fators or serial passages of the virs in sheep may be responsible for higher virs onentration in the blood. Hrwever stdies on hostinset relationship are neessary to establish sh a possibility. One lambs beome infeted dring the orse of an epizooti inset transmission wold be easier sine they develop a higher level of viraemia than adlt animals. Serology data show that attle aqire WSL virs infetion between the epizootis withot any linial symptoms being observed. This was the ase with the sera of a sbstantial nmber of attle olleted from all provines dring the years while the WSL otbreak only started beoming notieable dring 1974 (Theodoridis npblished reslts). Similarly 3 years after WSL had sbsided among sheep yong attle were positive to this virs. Weiss (1957) also fond serologial evidene of infetion among attle after an epizooti in sheep. These serologial data indiate that attle an be sspeted as a natral reservoir of WSL virs. However this assmption is hallenged by the fat that while experimentally infeted attle failed to develop viraemia (one of 15) and linial symptoms they invariably seroonverted (Coetzer eta/. 1979). ACKNOWLDGMNTS The athors wish to express their appreiation to Mr S. T. Boshoff and Miss A. Hattingh for spplying the information on serology and to Miss A.. Bodenstein and Mr W. C. Holsher for their valable assistane in the laboratory. RFRNCS CLARK D. H. & CASALS J Tehniqes for hemaggltination and hemaggltination inhibition with arthropodborne virses. Amerian Jornal of Tropial Mediine and Hygiene COTZR J. A. W. & BARNARD B. J. H Hydrops amnii in sheep assoiated with hydranenephaly and arthrogryposis with Wesselsbron disease and Rift Valley fever virses as aetiologial agents. Onderstepoort Jornal of Veterinary Researh COTZR J. A. W. THODORDS A. & VAN B R DN ANNLN Wesselsbron disease. Pathologial haematologial and linial stdies in natral ases and experimentally infeted new-born ambs. Onderstepoort Jornal of Veterinary Researh COTZR J. A. W. THODORDS A. HRR S. & KRTZNGR L Wesselsbron disease: A ase of ongenital porenephaly and erebellar hypoplasia in alves. Onderstepoort Jornal of Veterinary Researh CUNNNGHAM C. H A laboratory gide in virology. 4th ed. Minnesota: Brgess Pblishing Co. FNNR F Classifiation and nomenlatre of virses. ntervirology RD L. J. & MUNCH H A simple method of estimating 5 per ent endpoints. Amerian Jornal of Hygiene WSS K.. HAG D. A. & ALXANDR R. A Wesselsbron virs-a virs not previosly desdribed assoiated with abortion in domestiated animals. Onderstepoort Jornal of Veterinary Researh WSS K Wesselsbron virs disease. Blletin of epizooti diseases of Afria