of and Peter F. Nettleton, Janine A. Gilray, Pierre Russo, Elyess Dlissi

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Peter F. Nettleton, Janine A. Gilray, Pierre Russo, Elyess Dlissi. Border disease of sheep and goats. Veterinary Research, BioMed Central, 1998, 29 (3-4), pp.327-340. ￿hal-00902531￿

HAL Id: hal-00902531 https://hal.archives-ouvertes.fr/hal-00902531 Submitted on 1 Jan 1998

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Review article

Border disease of sheep and goats

Peter F. Nettletona Janine A. Gilraya Pierre Russo’ Elyess Dlissib

a Moredun Research Institute, International Research Centre, Pentiands Science Park, Bush Loan, Penicuik, Midlothian EH26 OPZ, Scotland, UK h Cneva Sophia-Antipolis, BP 1 I 1-06902 Sophia-Antipolis cedex, France C Institut De La Recherche Vétérinaire, La Rabta - 1006 Tunis, Tunisia

(Received 30 January 1998; accepted 13 March 1998)

Abstract - Border disease (BD) is a congenital disease of sheep and goats first reported inn 1959 from the border region of England and Wales. BD virus (BDV) is a in the genus and is closely related to virus and bovine virus diar- rhoea virus (BVDV). Nearly all isolates of BDV are non-cytopathogenic (ncp) in cell culture. There are no defined serotypes but isolated from sheep exhibit considerable antigenic diversity and three distinct antigenic groups have been identified. Distribution of the virus is world- wide. Prevalence rates vary in sheep from 5 to 50 % between countries and from region-to- region within countries. The disease in goats is rare and characterized by abortion. Clinical signs in sheep include barren ewes, abortions, stillbirths and the birth of small weak lambs. Affected lambs can show tremor, abnormal body conformation and hairy fleeces (so-called ’hairy-shaker’ or ‘fuzzy’ lambs). Vertical transmission plays an important role in the epidemiology of the dis- ease. Infection of fetuses can result in the birth of persistently infected (PI) lambs. These PI lambs are viraemic, negative and constantly excrete virus. The virus spreads from sheep to sheep with Pl animals being the most potent source of infection. Apparently healthy PI sheep resulting from congenital infection can be identified by direct detection of viral antigen or viral RNA in leukocytes or by isolation of ncp virus from blood or serum in laboratory cell cultures. Isolation of virus is unreliable in lambs younger than 2 months old that have received colostral antibody. The isolation of virus from tissues of aborted or stillbom lambs is difficult but tissues from PI sheep contain easily detectable levels of virus. To detect the growth of virus in cell cultures it is essen- tial to use an immune-labelling method. Acute infection is usually subclinical and viraemia is tran- sient and difficult to detect. Sheep may also be infected following close contact with cattle excreting the closely related BVDV. © Inra/E!sevier, Paris , border disease / border disease virus / pestivirus / sheep /

* Correspondence and reprints Tel.: (44) 131 445 5 111; fax: (44) 131 445 611 I; e-mail: nettpC!mri.sari.ac.uk Résumé - Border disease ou hypomyélinogenèse congénitale chez le mouton et la chèvre. La « border disease » (BD) est une maladie virale congénitale qui sévit chez le mouton et la chèvre. Elle a été décrite pour la première fois en 1959 à la frontière (en anglais, border, d’où le nom de la maladie, NDLR) entre l’Angleterre et le Pays de Galles. Le virus de la BD (V BD) est un pes- tivirus du genre Flaviviridae qui est fortement apparenté au virus de la peste porcine classique et au virus de la diarrhée bovine virale (VDBV). Presque tous les isolats de VBD sont non-cyto- pathogènes (ncp) en culture cellulaire. Il n’y a pas de sérotype défini, mais il existe une diversité antigénique considérable entre les différents pestivirus isolés du mouton, et trois groupes anti- géniques distincts ont été identifiés. Le virus se retrouve dans le monde entier. Les taux de pré- valence chez le mouton varient de 5 à 50 % selon le pays et même d’une région à l’autre dans un même pays. La maladie chez la chèvre est rare et se caractérise par un avortement. Les signes cli- niques chez le mouton incluent stérilité, avortements, mortinatalité, et naissance d’agneaux petits et faibles. Les agneaux affectés peuvent avoir des tremblements, une constitution physique anor- male, et un pelage très fourni. La transmission verticale joue un rôle important dans l’épidé- miologie de la maladie. L’infection de foetus peut entraîner la naissance d’agneaux infectés de manière persistante (IP). Ces agneaux IP sont virémiques, séronégatifs, et excrètent constamment le virus. Le virus se répand d’un mouton à l’autre, les animaux IP étant la source d’infection la plus importante. Les moutons IP, apparemment sains, mais infectés in utero, peuvent être iden- tifiés par la détection directe d’antigène viral, ou d’ARN viral dans les leucocytes, ou par l’iso- lement de virus ncp à partir du sang ou du sérum dans les cultures cellulaires de laboratoire. L’isolement du virus n’est pas fiable chez les agneaux de moins de 2 mois qui ont reçu les anti- corps du colostrum. L’isolement du virus à partir de tissus d’agneaux avortés ou mort-nés est dif- ficile, mais les tissus de moutons IP contiennent des niveaux de virus facilement détectables. Afin de détecter la croissance du virus dans les cultures cellulaires, il est essentiel d’utiliser une technique d’immuno-marquage. L’infection aiguë est généralement subclinique, et la virémie est transitoire et difficile à détecter. Les moutons peuvent également être infectés après un contact avec des bovins excrétant le virus, apparenté, de la diarrhée bovine virale. © Inra/Elsevier, Paris border disease / hypomyélinogenèse congénitale / pestivirus / mouton / chèvre 1. INTRODUCTION tivity with monoclonal and to their nucleotide sequences at selected Border disease in sheep and goats is genomic regions. have also been caused by the pestivirus, border disease tested by cross-neutralization experiments virus (BDV). BDV spreads naturally by with a view to identifying the best viruses the oro-nasal route and by vertical trans- for candidate vaccines. mission. It is a cause of congenital dis- ease in sheep and goats but can also cause Pestiviruses are enveloped, spherical acute and persistent infections. This paper particles approximately 50 nm in diameter. will principally describe BDV infection The pestivirus is a positive sin- in sheep. Infection in goats is rare and will gle-stranded RNA molecule, approxi- only be described where it is known to mately 12.5 kb long. There is a single open differ from infection in sheep. Compre- reading frame (ORF) flanked by a 5’-non- hensive reviews of the elucidation of the coding region (5’-NC) of 356-385 bases cause and pathology of the disease have and a 3’-non-coding region (3’-NC) of been published (Barlow and Patterson, 223-228 bases (figure /). The ORF 1982; Terpstra 1985). Major advances encodes composed of 4 000 since then have been in the molecular amino acids processed by viral and cellu- structure of the virus and the relationships lar enzymes. Of the proteins within the between sheep isolates and pestiviruses ORF the first is a non-structural from other species (Nettleton and Entri- autoprotease NPr° followed by the struc- can, 1995; Paton, 1995; Thiel et al., 1996). tural C nucleocapsid protein and glyco- proteins, Erns, E I and E2. Of the glyco- proteins, E2 is the immunodominant major 2. VIRUS PROPERTIES envelope protein. The remaining proteins are non-structural of which NS2-3 has The genus pestivirus within the family attracted most interest owing to its role in Flaviviridae has been divided into classi- cytopathogenity of pestivirus isolates. Vir- cal swine fever virus (CSFV), bovine virus tually all pestivirus isolates from sheep diarrhoea virus (BVDV) and border dis- and goats are non-cytopathic in cell cul- ease virus (BDV). The viruses were named ture. Two cytopathic sheep isolates have after the diseases from which they were been described, however, and in both these first isolated and traditionally pestiviruses it has been shown that they contain inser- isolated from pigs have been termed tions of cellular sequence within the NS2- CSFV, those from cattle BVDV and those 3 encoding region which results in its from sheep BDV. It is now known that cleavage to NS2 and NS3 (Becher et al., cross-infection between species occurs 1996). This is analogous to the similar readily and viruses have consequently process in BVDV viruses which is asso- been grouped more according to their reac- ciated with the development of mucosal disease in cattle. Such cattle are persis- type and one (R2727) was more similar tently infected with non-cytopathic (NCP) to viruses from the BVDV-1 genotype. BVDV following in-utero infection. Muta- There are few of tion of the virus RNA in the relatively reports persisting cross-neutralization involv- for NS2-3 can result in experiments region coding from and of NS3 which is correlated ing pestiviruses sheep goats. overproduction Nevertheless four with the development of mucosal disease. principal serological groups have been identified (Wensvoort From studies with monoclonal anti- et al., 1989), which correlate with the bodies and phylogenetic analysis of monoclonal antibody and genotype group- genomic sequences from the 5’-NC, Npro ings (Paton et al., 1995). As with the geno- C and E2 coding regions a consensus is typing results the giraffe and deer isolates that there are four emerging principal are serologically distinct from the other genotypes of pestiviruses: BVDV-l, four groups (Dekker et al., 1995). BVDV-2, BDV and CSFV (Paton et al., 1994; Becher et al., 1995; Tijssen et al., Due to the paucity of cross-neutraliza- tion results with we 1996). Two pestiviruses, one from a deer sheep pestiviruses selected ten isolates from and one from a giraffe, do not fit into any non-cytopathic different of the UK and of these groups and constitute separate regions compared virus types (Van Rijn et al., 1997). them with a cattle pestivirus. The source of the viruses is shown in table L All viruses While CSF viruses are predominantly were plaque-purified three times and pairs restricted to of the other pigs, examples of pestivirus-naïve lambs were infected three genotypes have all been recovered intranasally with 5 x 10! of each from et Exami- TCIDso sheep (Vilcek al., 1997). virus. Serum was collected from each lamb nation of three isolates has shown goat LO weeks later and tested in micro neu- them to be BVDV-1 et al., types (Becher tralization tests against approximately 100 1997). TCIDso of all the viruses. All the lambs The complete genomic sequence of the seroconverted, except one infected with American BDV reference strain, BD-31, R2727, and produced neutralizing anti- has recently been published (Ridpath and body titres between 256 and 2880 against Bolin, 1997). When compared with the their homologous virus. The geometric available complete genomic sequences of mean titres of neutralizing antibody pro- other pestiviruses the predicted amino- duced by pairs of sheep are shown in acid sequence identity varied from 711 table II. It can be seen that the BD viruses (BVDV-1) to 78 % (CSFV). Phylogenetic are serologically related but there is a spec- analysis segregated the pestiviruses into trum of antigenic cross-reactivity, with two branches, one containing BD31 and two clusters of more strongly related the CSFV strains and one containing strains. Thus G1480, JH2816, A1870, BVDV-1 and BVDV-2 strains. The L991 and G2048 (The Moredun BDV regions of highest sequence identity were group) appear to be closely related to each in the 5’-NC and the non-structural virus other but distinct from the second group polypeptide NS3. Comparison of the containing D861, B 1056 and Weybridge BD31 sequence with incomplete (The Weybridge BDV group). The two sequences from nine other BDVs showed strains G1305 and R2727 do not i mmedi- close homology with five including the ately fall into either group. The coefficient Australian reference strain X818 and the of antigenic similarity (R value) for the UK Moredun reference strain. Of the 11 viruses was determined as described remaining four viruses, three were more previously (Howard et al., 1987; Nettle- similar to viruses from the B VDV-2 geno- ton, 1987). The closer the value of R is to 100 the more closely related are the strains. 10 (26 %) belong to the BVDV-I group. A Values of R ;::: 25 depict a less than four- further five isolates (13 %) belong to the fold difference between strains which is BVDV-2 group (Vilcek et al., 1997).). not different within the con- significantly The relevance of all these results ditions of the neutralization tests. A value typing to vaccine development requires further of R < 5 signifies a > 20-fold reciprocal work. In there is a dearth of difference between the homologous and particular, cross-protection studies. The only cross- heterologous titres, and has been used to protection test used field brain define virus serotypes. published material to infect and challenge pregnant The R values shown in table III empha- ewes. The viruses recovered from these size further the two groups of serologi- field outbreaks were G 1480 (Moredun ref- cally distinguishable BDV isolates, the erence strain) a true BD virus and B 1056 one group related to Moredun BDV and a BVDV- isolate. In that thorough exper- the other to the Weybridge and BVD iment 12 pregnant ewes previously viruses. These antigenic differences cor- exposed to BVDV- were challenged relate well with genotyping results since all intramuscularly with heterologous BD the Moredun BDV related isolates type as virus on the 54th day of gestation; I ewes true BDVs whereas representatives of the (92 %) had diseased lambs. A further 11I other group all fit into what is now known pregnant ewes previously exposed to BDV as the BVDV-1 et were genogroup (Becher al.,., similarly challenged with the het- 1994; Vilcek et al., 1994). These results erologous BVDV-1 strain; one ewe together with recent genotyping studies aborted and five had diseased progeny, on a total of 38 UK sheep isolates show i.e. 55 % of the ewes had diseased lambs. that 23 (60 %) are true BD viruses and In contrast, similar sized groups of

immune ewes all had normal lambs when in fetuses infected early in gestation. Small challenged with the homologous virus to dead fetuses may be resorbed or their abor- which they had been previously exposed tion pass unnoticed since the ewes con- (Vantsis et al., 1980). tinue to feed well and show no sign of dis- This result could be expected given the comfort. As lambing time approaches, the abortion R values of the two viruses in table III and of larger fetuses, stillbirths and the births of weak lambs would imply that any BD vaccine should premature small, will be seen. The infection of contain at least one representative from pregnant severe and the BDV and BVDV-1 groups. The pro- goats produces placentitis high tection between BVDV-2 and these other death rates among kid fetuses (Barlow and two sheep-infecting groups will need to Patterson, 1982). be studied. During lambing, an excessive number of barren ewes will become apparent but it is the diseased live lambs that present the 3. CLINICAL DISEASE main clinical features characteristic of BD. The clinical signs exhibited by BD lambs are very variable and depend on the breed 3.1 Acute infections of sheep, the virulence of the virus and the time at which infection was introduced Healthy newborn and adult sheep into the flock (Barlow and Patterson, 1982; exposed to BDV experience only mild or Bonniwell et al., 1987; Roeder et al., inapparent disease. Slight fever and a mild 1987). Affected lambs are usually small leucopaenia are associated with a short- and weak, many being unable to stand. lived viraemia detectable between days 4 Nervous signs and fleece changes are often and 11 post-infection after which serum apparent. The nervous symptoms of BD neutralizing antibody appears. are its most characteristic feature. The Occasional BDV isolates have been tremor can vary from violent rhythmic contractions of the muscles of the hind shown to produce high fever, profound and back to detectable fine and prolonged leucopaenia, anorexia, con- legs barely of the ears junctivitis, nasal discharge, dyspnoea and trembling head, and tail. Fleece abnormalities are most obvious in smooth- diarrhoea, and 50 % mortality in young coated breeds which have fleeces lambs. One such isolate was recovered hairy on the neck and back. Abnormal from a severe epidemic of Aveyron dis- especially brown or black of the fleece ease among milk sheep in France in 1984 pigmentation also be seen in BD-affected lambs. (Chappuis et a]., 1986). A second such may isolate was a BDV contaminant of a live With careful nursing a proportion of CSFV vaccine (Wensvoort and Terpstra, BD lambs can be reared although deaths 1988). may occur at any age. The nervous symp- toms gradually decline and can have dis- appeared by 3 to 6 months of age. Weak- 3.2. Fetal infection ness, swaying of the hind-quarters together with fine trembling of the head may reap- The main clinical signs of BD are seen pear at times of stress. Affected lambs following the infection of pregnant ewes. often grow slowly and under normal field While the initial maternal infection is sub- conditions many will die before or around clinical or mild the consequences for the weaning time. Occasionally this is the first fetus are serious. Fetal death may occur presenting sign of disease when losses at at any stage of pregnancy but is commoner lambing time have been low and no lambs with obvious symptoms of BD have been Persistently viraemic sheep can be diag- born. nosed by virus isolation/detection in a blood sample. Viraemia is readily Some fetal infections occurring around detectable at any time except in the first 2 can result in lambs with mid-gestation months of life when virus is masked by severe nervous locomotor distur- signs, colostral and in animals older bances and abnormal skeletons. Such antibody than 4 old some of which lambs have lesions of cerebellar years develop hypopla- low levels of anti-BDV sia and and antibody (Nettle- dysplasia, hydranencephaly ton et al., 1992). Although virus detection porencephaly resulting from necrotizing in blood during an acute infection is dif- inflammation. The severe destructive ficult, persistent viraemia should be con- lesions to be immune mediated, appear firmed animals after an inter- and lambs with such lesions by retesting frequently val of at least 3 weeks. have high concentrations of serum anti- body to BDV. Most lambs infected in late Some viraemic sheep survive to sex- gestation are normal and healthy and are ual maturity and are used for breeding. born free of virus but with BDV antibody. Lambs born to these infected dams are Some such lambs, however, can be still- always persistently viraemic. Persistently born or weak and many die in early life. viraemic sheep are a continual source of infectious virus to other animals and their identification is a major factor in any con- 3.3. Persistent viraemia trol programme. Sheep being traded should be screened for the absence of BDV viraemia. When fetuses are infected before they Rams that are infected have any immune system and survive they persistently (PI) have infec- are born with a persistent viraemia. The usually poor quality, highly tive semen and reduced All rams ovine fetus can first respond to an anti- fertility. genic stimulus between approximately 60 used for breeding should be screened for and 85 d of its 150-d gestation period. In persistent BDV infection on a blood sam- fetuses infected before the onset of ple. Semen samples can also be screened immune competence viral replication is for virus but they are much less satisfac- than blood due to uncontrolled and 50 °!o fetal death is com- tory their toxicity for cell cultures. mon. In lambs surviving infection in early gestation, virus is widespread in all organs. Such lambs appear to be tolerant to the virus and have a persistent infection usu- 3.4. Late-onset disease in persistently ally for life. A precolostral blood sample viraemic sheep will be virus positive and antibody nega- tive. Typically, there is no inflammatory Some persistently infected sheep reaction and the most characteristic patho- housed apart from other animals sponta- logical changes are in the central nervous neously develop intractable scour, wast- system (CNS) and skin. At all levels in ing, excessive ocular and nasal discharges the CNS there is a deficiency of myelin. sometimes with respiratory distress. At This may be slight in lambs with mild or necropsy such sheep have gross thickening no symptoms but is severe in lambs with of the distal ileum, caecum and colon pronounced nervous symptoms. In the skin resulting from focal hyperplastic enteropa- there is an increased size of primary wool thy. Cytopathic BDV can be recovered follicles and fewer secondary wool folli- from the gut of these lambs. With no obvi- cles causing the hairy or coarse fleece. ous outside source of cytopathic virus it is most likely that such virus originates sibility of sheep and goats developing BD from the lamb’s own virus pool. Other from contact with other species of rumi- persistently infected sheep in the group nants since at least 52 species of captive or do not develop the disease. This syndrome, free-living ruminants are known to be which has also been suspected in occa- infected by pestiviruses (Nettleton, 1990). sional field outbreaks of BD, has several Pestiviruses are important contaminants similarities with bovine mucosal disease of modified live virus (MLV) vaccines. (Gardiner et al., 1983; Nettleton et al., All MLV vaccines produced in ovine, 1992; Monies and 1997). Simpson, bovine or porcine cell cultures or in media supplemented with serum from these species risk being contaminated with pes- 4. EPIDEMIOLOGY tivirus. Outbreaks of BD have been asso- ciated with the use of such vaccines; Border disease is widespread in Europe, sheep-pox and orf virus vaccines in sheep Australasia and North America and has and an orf virus vaccine in goats (Nettle- also been reported from Israel and North ton and Entrican, 1995). Africa. Antibody prevalence rates among adult sheep vary from 5 to 50 °!o between countries and from region-to-region within 5. DIAGNOSIS countries. The virus is not stable outside the host and its successful transmission is due to spread by PI sheep. Spread within 5.1. Virus isolation a flock can take years in sheep reared extensively at grass but trough-feeding or Pestiviruses are notorious contaminants other husbandry that allows close nose- of laboratory cell cultures with fetal bovine to-nose contact will hasten spread. In serum being the commonest way it is sheep reared intensively indoors spread introduced. It is essential that laboratories from PI animals occurs more readily and undertaking virus isolation have a guar- serious outbreaks of BD at lambing time anteed supply of pestivirus-free suscepti- can occur in sheep housed together in early ble cells and fetal bovine or equivalent pregnancy. serum which contains no antipestivirus and no virus. The Pestivirus exchange between sheep and activity contaminating virus can be isolated in a number of pri- cattle occurs readily, and outbreaks of BD or ovine cell cultures have been caused by transmission of virus mary secondary (e.g. testes, Ovine cell lines for from cattle to sheep (Carlsson, 1991). No kidney, lung). BDV are rare and report of natural disease in cattle due to growth commercially unobtainable. Semi-continuous cell lines spread of virus from sheep has been doc- derived from fetal lamb muscle or umented, but serial experimental exchange (FLM) choroid can be useful but of virus between PI cattle and sheep has sheep plexus different lines vary considerably in their been investigated. There was a high degree to virus. of genetic stability but the expression of susceptibility one or more epitopes on the E2 glycopro- From live animals the most sensitive tein appeared to depend on the host species way to confirm pestivirus viraemia is to (Paton et al., 1997). There are no reports of wash leukocytes three times in culture BD originating from pigs but an outbreak medium before co-cultivating them with of CSF-like disease in pigs was shown to susceptible cells for 7 d. Cells are frozen have been caused by BDV (Roehe et al., and thawed once and an aliquot passaged 1992). There remains the theoretical pos- on to further susceptible cells grown on flying coverslips. Three days later the cells testing leukocytes the antigen ELISA can are stained for the presence of pestivirus also be used on tissue suspensions, espe- using an immunofluorescence (IF) or cially spleen, from suspected PI sheep and, immunoperoxidase (IP) test. as an alternative to IF and IP methods, on cell cultures. From dead animals, tissues should be collected as 10 % weight by volume in Several pestivirus ELISA methods have virus medium. In the transport laboratory been published and a number of commer- they are ground up, centrifuged to remove cial kits are now available for detecting debris and the supernatant passed through BVDV in cattle. While some of these may filters. 0.45-!m Spleen, thyroid, thymus, be suitable for use in sheep further evalu- kidney, brain, lymph nodes and gut lesions ation is required. are the best organs for virus isolation. Semen can be examined for the pres- ence of BDV but raw semen is strongly 5.3 Nucleic acid detection cytotoxic and must be diluted usually at least 1:10 in culture medium. Since the Ovine can be detected threat of BDV-infected semen is pestiviruses by major the reverse chain from PI blood is a more reliable clin- transcriptase-polymerase rams, reaction (RT-PCR) which ical than semen for using primers sample identifying also detect from other such animals. pestiviruses species (Vilcek et al., 1994). While RT-PCR has not yet been evaluated for diagnostic pur- poses in it is to be of future detection sheep likely 5.2. ELISA for antigen value. The detection of viral RNA in fetal tissues may yet be an important application The first ELISA for pestivirus antigen since other methods are insensitive. The detection was described for detecting exquisite sensitivity of RT-PCR makes it viraemic This has now been mod- sheep. a valuable tool for detecting low level ified into a double monoclonal antibody virus contaminations as in cell culture con- ELISA for use in and (mab) capture sheep stituents or vaccines (Sandvik et al., 1997). cattle. Two capture mabs are bound to wells in microtitre plates and two other mabs to serve as conjugated peroxidase 5.4. tests detector mabs (Entrican et al., 1995). The Serological test is most commonly employed to iden- tify persistently infected viraemic sheep Antibody to BDV can be detected in using washed, detergent-lysed blood sheep sera using virus neutralization (VN) leukocytes. The sensitivity is close to that or an ELISA. Control positive and nega- of virus isolation and it is a practical tive reference sera must be included in method for screening high numbers of every test. These should give results within bloods. As with virus isolation, high lev- predetermined limits for the test to be con- els of colostral antibody can mask persis- sidered valid. Single sera can be tested to tent viraemia. The ELISA test is more determine the prevalence of BDV in a effective than virus isolation in the pres- flock, region or country. For diagnosis, ence of antibody, but may give false neg- however, acute and convalescent sera are ative results in viraemic lambs younger the best samples for confirming acute than 2 months old. The ELISA is usually BDV infection. Bleeds from one animal not sensitive enough to detect acute BDV should always be tested alongside each infections on blood samples. As well as other on the same plate. The choice of virus for use in the VN flocks the identification and disposal of test is difficult due to the antigenic diver- PI sheep may not be practicable. In which sity among pestiviruses. Reference strains case the level of flock immunity can be of cytopathic BD virus, e.g. Moredun or raised by deliberately exposing breeding cytopathic BVD viruses, e.g. Oregon stock to known PI lambs outwith the C24V or NADL can be used. No single breeding season. The rate of virus spread strain is ideal. Account should be taken will be increased by close herding indoors of the locally predominant genotype iso- for at least 3 weeks. lated from Local strains should be sheep. There is currently only one available tested to see which gives the highest anti- commercial vaccine for the control of titre with a of body range positive sheep BDV. This is a killed adjuvanted vaccine sera (Brockman et a]., 1988). The VN can which contains representative strains of also be with an NCP strain with performed BD and BVD-1 viruses (Brun et al., 1993). an IP used for the staining system being It should be administered to young ani- readout. mals before they reach breeding age in A monoclonal antibody capture ELISA order to maximize their immunity during for measuring BDV antibodies has been early pregnancy. Annual booster doses described. Two pan pestivirus mabs may be required. different on the detecting epitopes To prevent introduction of BD into a immunodominant non-structural protein flock, only home-bred replacement are to NS 2-3 used capture detergent-lysed females should be used. New rams should cell culture-grown antigen. The results be blood tested for virus before purchase correlate with the VN test qualitatively or in quarantine after arrival on the farm. (Fenton et al., 1991). If new ewes have to be bought they should also be blood tested to detect any PI virus carriers. In the absence of blood test- 6. CONTROL ing, and as an aid to control all infections of breeding, newly purchased ewes should The control of BDV in a sheep flock always be mated and kept separate from has two essential the iden- requirements: the rest of the flock until lambing time. tification of PI sheep and the prevention of Because of the risk of infection of sheep infection of ewes susceptible pregnant from PI cattle it is essential that pregnant the first half of especially during gesta- ewes are never mixed with cattle. tion. The control of BDV in infected flocks is difficult and will depend on the require- 7. CONCLUSION ments of farmers in relation to their farm- ing methods. In a flock which has recently The control of BD remains problem- had a sporadic outbreak of BD, the entire atical owing to the efficient spread of the lamb crop and the sheep that introduced virus by PI carrier-sheep and limited infor- infection must be removed to slaughter mation on cross-protection afforded by before the start of the next breeding sea- different virus genotypes. Further vaccine son. In endemically infected flocks of high development is required with candidate commercial value antibody testing of dif- vaccines being tested for efficacy in preg- ferent aged sheep will identify immune nant sheep. This approach has now been and susceptible groups and further blood adopted for efficacy testing of BVD vac- testing can be used to identify antibody cines in cattle (Brownlie et al., 1995). As negative, virus positive PI sheep. 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