The humoral and cellular immune response of sheep against Borna disease virus in endemic and non-endemic areas

Borna Disease (BD) is a mostly fatal disease of horses and sheep endemic in central Europe. Antibodies to Borna disease virus (BDV) have been described in sheep and other species living in BD non-endemic areas. Meaningful clinical BDV serology is hampered by difficulties in defining serological cut-offs, which require the investigation of populations from endemic areas. Here we studied BD serology in sheep from endemic and non-endemic areas of similar geography in Switzerland. Antibodies to BDV antigens were detected by ELISA and indirect immunofluorescence analysis (IFA) only in sera from 3 of 6 sheep with autopsy confirmed BD. One serum was positive by IFA but not by ELISA, while 2 sera were negative in both assays, indicating that not all diseased animals develop BDV specific antibodies. Six % of clinically healthy animals (6/106) from an endemic area and 2% from a non-endemic area (4/192) had serum antibody to either BDV p40 or p24 as detected by ELISA. None of the animals showed a cellular immune response to BDV p40. In some healthy sheep from the endemic area, serum antibody titers to BDV p24 antigen remained elevated over several months without onset of disease symptoms. Infections with either BDV or related viruses may thus occur at low frequency in sheep from non-endemic areas leading to the production of antibodies to BDV antigens. We further propose viral strain differences or environmental factor(s) may determine the clinical outcome.     

Seroprevalence of Borna disease virus in domestic animals in Xinjiang, China

To investigate the animals infected with Borna disease virus (BDV) in Xinjiang, China, we examined for BDV antibodies in the sera from groups of 20 horses, sheep and cattle, and from 165 wild rodents (18 species) by ELISA and immunoblot. The serological study disclosed the presence of antibodies to both BDV-p24 and -p40 in the horses (20%) and sheep (25%), whereas no apparent positive reaction was detected either in cattle or rodents. The results suggested that BDV is prevalent in horses and sheep in the district investigated.     

A monoclonal antibody capture ELISA to detect antibody to border disease virus in sheep serum.

An enzyme-linked immunosorbent assay (ELISA) has been developed to detect antibody to border disease virus (BDV) in sheep serum. A monoclonal antibody bound to 96-well microplates was used to capture antigen from detergent-solubilised BDV-infected cells. Single dilutions of test sera were then added to wells containing bound BDV antigen and control wells containing uninfected cell lysates. Specific antibody to BDV was detected by an anti-ovine IgG antiserum conjugated with horseradish peroxidase and the results expressed as ELISA units with reference to a standard curve. Sequential sera from 16 experimentally infected sheep and single sera from 103 sheep involved in a field outbreak were tested in the ELISA and for neutralising antibody. There was good qualitative correlation between the two tests.     

Prevalence of Antibodies to Coxiella burnetii Among Veterinarians and Slaughterhouse Workers in Nova Scotia

The complement fixation and the microimmunofluorescence tests were used to determine the prevalence of antibodies to Coxiella burnetii, the etiological agent of Q fever, among veterinarians and slaughterhouse workers in Nova Scotia. Seventeen percent of the 65 veterinarians and 12.5% of the 96 slaughterhouse workers tested had complement fixing antibodies to phase II C. burnetii antigen. Forty-nine percent of the veterinarians and 35% of the slaughterhouse workers had an antibody titer of ≥ 1:8 to phase II C. burnetii antigen using the microimmunofluorescence test while 30% of the veterinarians and 14.5% of the slaughterhouse workers had antibodies detected to phase I antigen. Male veterinarians had a significantly higher rate of antibodies to C. burnetii phase II antigen compared with female veterinarians (p < 0.0087). An univariate analysis revealed that positive antibody titers (microimmunofluorescence test) to phase II antigen among veterinarians were significantly associated with exposure to cow, sheep and goat placentas; to stillborn calves, newborn foals, lambs and kids. By multivariate analysis the risk was highest for male veterinarians exposed to sheep placentas.     

Toxoplasmosis and border disease in 54 Swedish sheep flocks. Seroprevalence and incidence during one gestation period.

Serum samples from 704 animals from 54 Swedish sheep flocks were analysed by ELISA twice during 1 breeding season for antibodies to Toxoplasma gondii and border disease virus (BDV). An ELISA, originally developed for the detection of antibodies to bovine viral diarrhoea virus (BVDV) in cattle, was assessed on sheep sera and the results were compared with those obtained in a virus neutralization test. The correlation between the 2 assays proved good. Before breeding, 132 (19%) sheep in 42 flocks had antibodies to T. gondii and 7 (1%) sheep in 5 flocks were seropositive to BDV. During the observation period 4 sheep seroconverted to T. gondii and 13 to BDV, giving an incidence rate of 0.7% and 1.9% respectively. No clinical signs due to the infections were observed. In 5 flocks the frequency of barrenness, abortion or stillbirths exceeded 5%, 5% and 8%, respectively, but there was no evidence that this was attributable to the agents studied. The proportion of BDV-positive flocks was significantly higher among flocks that had been in contact with cattle than among those that had not.     

The production and survival of lambs persistently infected with border disease virus.

From 1985 to 1989 lambs persistently infected with border disease virus (BDV) were produced for comparative immunological studies by infecting 57 susceptible pregnant ewes between 50 and 60 days' gestation with Moredun or Oban strains of BDV. Ewes were infected either by injection with virus grown in cell culture or by housing with lambs excreting BDV. There was no significant difference in the outcomes of these different methods of infection. There was a significant difference in the number of viable lambs born to ewes receiving the two viruses. Of 41 ewes infected with Moredun virus 21 produced 32 live lambs of which 17 were reared to 1 month old (53% viability). Of 16 ewes receiving Oban virus 10 gave birth to 17 live lambs of which 15 were reared to 1 month old (88% viability). All the lambs born to ewes infected with Moredun BDV had varying signs of tremor and increased hairiness ("hairy-shakers") while those born to ewes infected with the Oban virus had no obvious clinical signs. Survival of the lambs was poor. Up until February 1991, 14 Moredun and 10 Oban sheep between the ages of 4 months and 5.5 yr had died from a variety of causes. The two commonest causes were a chronic wasting syndrome and a mucosal disease-like syndrome which was associated with the recovery of cytopathic BDV. Mating of unrelated persistently infected sheep was largely unproductive although 2 lambs were reared.     

江苏部分地区羊群边界病流行情况调查

本实验室从安徽某羊场发生腹泻的山羊病料中检测分离到边界病病毒（borderdiseasevirus,BDV）,证明BDV在我国羊群中存在。为了进一步了解BDV在江苏羊群的流行情况,本研究收集江苏部分地区发生腹泻和健康羊群的血清和组织样品,采用RT—PCR方法进行检测,并对阳性样品进行病毒的分离鉴定,测定分离毒株5’-UTR基因片段,与其他已报道的毒株进行同源性比较并绘制进化树。结果表明有27．4％（29／106）样品呈阳性,不N羊场BDV阳性率为0～67％,共分离到4株不同的BDV毒株,它们之间的同源性为73．9％～95．6％,而与其他BDV毒株的同源性为66．2％～91．6％。进化分析表明AH12-02与其他各毒株均较远,形成单独的分支,另3个毒株与BDV3型毒株关系最近。采用ELISA试剂盒对血清样品BDV抗体进行检测发现不同羊场抗体阳性率存在较大差异（0～100%）,还有抗体阴性持续感染个体的存在。以上结果丰富了我国BDV分子流行病学数据,为进一步探索BDV在我国羊群中的流行情况奠定了基础。     

Comparison of a maedi-visna virus CA-TM fusion protein ELISA with other assays for detecting sheep infected with North American ovine lentivirus strains.

A maedi-visna virus CA-TM fusion protein ELISA (MVV ELISA) was evaluated for the detection of antibody in sheep infected with North American ovine lentivirus (OvLV). The results of the MVV ELISA were compared with other assays for OvLV antibody and with viral infection in an intensively studied group of 38 sheep with a high prevalence of OvLV infection and disease. The sensitivity, specificity, and concordance of assays for OvLV antibody (MVV ELISA, indirect ELISA, Western blot, and AGID), virus (virus isolation, PCR, antigen ELISA), and OvLV-induced disease in each animal were compared with OvLV infection status as defined by a positive result in two or more of the assays. Five sheep met the criteria for absence of OvLV infection. The sensitivity of the MVV ELISA in detecting OvLV infected sheep was 64%, whereas the sensitivity of the other three tests for antibody ranged from 85 to 94%. All the antibody assays were 100% specific in this group of animals. Of the assays for virus, the PCR test had the highest sensitivity and the best concordance with OvLV infection, but it also had the lowest specificity of any of the virus or antibody assays. Among the antibody tests, the concordance of the MVV ELISA compared most favorably with the AGID test for detecting OvLV-infected sheep. Analysis of serum samples from 28 lambs experimentally-infected with one of three North American strains of OvLV suggested that there were no significant strain differences detectable by antibody assay. Twenty virus-inoculated lambs were positive by both the MVV ELISA and the AGID test, five lambs were MVV ELISA negative and AGID test positive, and three lambs were MVV ELISA positive and AGID test negative. No pre-inoculation samples were positive by either assay. In a longitudinal study involving seven lambs, antibodies to OvLV were detected by AGID 3-5 weeks post-inoculation, but were not detected by MVV ELISA until 5-10 weeks post-inoculation. Among 128 naturally and experimentally-infected sheep that were seropositive in the AGID test, the overall sensitivity of the MVV ELISA was higher in the naturally infected sheep (84%) than in the experimentally infected sheep (69%). The data indicated that the MVV ELISA represents a less sensitive, but specific alternative for the detection of OvLV antibodies.     

Detection of antibodies to Borna disease virus in Turkish cats by using recombinant p40.

Recombinant p40 produced by baculovirus was used in an ELISA to screen samples of serum taken from 80 cats in Istanbul. The sera were also analysed for feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV). Antibodies to Borna disease virus- (BDV) p40 were detected in 34 (42-5 per cent) of the 80 cats. Seventy-three per cent of the sera which were positive for FIV and 26 per cent of the sera which were negative for FIV had antibodies to BDV. There was no difference in the percentage of sera which were positive for BDV between the cats that were positive or negative for FeLV. Three of the cats had neurological disease and two of these had antibodies to BDV. Six sera with low, medium or high optical densities (ODS) by ELISA were analysed by Western blotting. Only the sera with medium and high ODS reacted specifically with p40 at a dilution of 1 in 1,000.     

Epidemiological survey of Border disease virus among sheep from northern districts of Japan

The first epidemiological survey of Border disease virus (BDV) was undertaken in small ruminants in Japan. Ovine sera, collected from the northern prefectures of Hokkaido, Aomori and Iwate, were examined for the presence of antibodies against BDV using the neutralization peroxidase-linked antibody test. Twenty-nine (17.6%) of one hundred and sixty-five samples were seropositive for BDV. Results were specific, excluding cross-reactions with bovine viral diarrhea virus (BVDV). Only one sample (0.6%) was positive for BVDV, and was negative for BDV. Despite serological evidence of virus circulation, there have been no clinical cases of border disease in sheep in Japan. Although no diagnostic measures were performed, the infection did not appear to be associated with a reduction in ewe fertility nor with lamb mortality.     

边界病的研究进展

边界病（border disease,BD）是由边界病病毒（border disease virus,BDV）引起绵羊和山羊感染的一种传染性疾病,在世界范围内给畜牧业生产造成巨大的经济损失。该病临床表现为母羊生殖障碍,羔羊畸形、震颤、多毛等。BDV在绵羊、牛和猪的种间传播经常发生,给该病的诊断带来困难。目前,尚无有效的疫苗用于边界病的防控,因此,了解不同区域的边界病流行情况有助于防止其传播并优化控制措施。结合国内外近期研究报道,从病原学、流行病学、诊断措施等方面对边界病进行综述,以期为该病的防控工作提供参考。     

杜泊羊与蒙古羊及小尾寒羊杂交育肥试验研究

对内蒙古四子王旗地区在相同饲养条件下的杜寒及杜蒙90日龄、150日龄杂交育肥羔羊进行了随机取样称重,统计对比育肥羔羊平均体重和日增重,从而对杜寒、杜蒙杂交育肥羔羊在相同饲养条件下的育肥效果进行讨论。结果发现,杜蒙杂交育肥羔羊150日龄的平均体重、日增重以及60 d增重显著高于杜寒杂交育肥羔羊(P<0.05)。     

青海省海北州藏羊群中牛病毒性腹泻病毒和羊边界病毒的流行病学调查

为掌握青海省海北州藏羊群中牛病毒性腹泻病毒和羊边界病毒的感染情况,本研究采用RT-PCR方法分别对青海省海北州的161份健康藏羊血清样品和34份腹泻藏羊组织样品进行了BVDV和BDV的抗原核酸检测。结果显示：195份样品中BVDV和BDV总阳性率分别为29.74 %和14.36 %;161份健康藏羊血清样品中BVDV和BDV平均阳性率分别为26.71 %和11.80 %,BVDV/BDV混合感染率为4.35 %;34份腹泻藏羊组织样品中BVDV和BDV平均阳性率分别为44.12 %和26.47 %,BVDV/BDV混合感染率为17.65 %。本研究表明青海省海北州健康藏羊群和腹泻藏羊群中均存在BVDV、BDV的单独感染以及混合感染,且感染情况在个别养殖场(户)较为严重,本研究为青海藏羊的综合防控措施提供了指导依据,丰富了我国羊群中BVDV和BDV的流行病学资料。     

Border Disease Virus Transmitted to Sheep and Cattle by a Persistently Infected Ewe: Epidemiology and Control

In a Swedish sheep flock comprising 202 ewes and 13 rams, a pair of twin lambs born in the spring of 1990 demonstrated signs of border disease (BD) and were persistently infected (PI) with border disease virus (BDV). Investigation showed that BDV had been introduced in the preceding tupping period 5-6 months earlier by a bought-in ewe which, on the basis of immunoperoxidase- and polymerase chain reaction techniques, was shown to be PI with BDV. Only 7 of the ewes, all of which had been in close contact with the PI ewe, seroconverted during the subsequent gestation. Apart from the PI twin lambs the losses caused by BDV were restricted to 2 barren ewes. The twin lambs, the PI ewe and lambs from the other 4 ewes that seroconverted were removed from the flock. The flock was thereafter free from an ongoing infection with BDV as shown by the absence of seroconversion.In addition, 5 heifers in late pregnancy most probably seroconverted to bovine virus diarrhoea virus (B VDV) when kept in close contact with the same PI ewe during the winter of 1989-90. When these heifers were reintroduced to the BVDV-free dairy herd from which they originated, their serum antibody titres ranged between 1:250 and 1:1250. Neither these heifers - nor their calves–caused any spread of the infection in the herd, as indicated by the absence of seroconversion in 70 cows. The present investigation shows that in the control of both BDV in sheep and BVDV in cattle, it is important to ensure that the risk of transmission of pestivirus between the 2 species is minimized.     

Serological survey for antibodies against pestiviruses in sheep in Austria

The prevalence of antibodies to pestiviruses was investigated in 4931 sheep, in 377 flocks, in four federal states of Austria, by means of an indirect elisa that detected antibodies to Border disease virus (BDV) and bovine viral diarrhoea virus (BVDV). The mean flock prevalence was 62.9 per cent and the mean individual prevalence was 29.4 per cent. Comparative neutralisation studies on the elisa-positive samples with BVDV type 1 (BVDV-1), BVDV type 2 (BVDV-2) and BDV recorded 336 samples with higher titres (more than four times average) to BVDV-1, three samples with higher titres to BVDV-2 and 55 samples with higher titres to BDV. The other samples did not show clear differences in antibody titres against the strains of pestivirus tested because of cross-reactions. The seroprevalence of pestiviruses in sheep was significantly higher on farms with cattle. There were significant regional differences between the prevalences in flocks and individual sheep, the highest prevalences being in the region of Austria where communal alpine pasturing of sheep, goats and cattle is an important part of farming.     

Serum antibodies directed against classical swine fever virus and other pestiviruses in wild boar (Sus scrofa) in the Republic of Croatia

The presence of serum antibodies directed against classical swine fever (CSF) virus and other pestiviruses among the wild boar (Sus scrofa) population in Croatia was investigated. During 2003, serum samples from 214 wild boars were collected in 10 hunting areas in the continental part of the country.The sera were examined by enzyme immunoassay (ELISA) and in the virus neutralization test (VNT). Out of 214 sera tested 111 (51.87 %) were positive by ELISA and regarding neutralising antibodies, against CSFV 75 (35.05 %) samples were positive. In the VNT with the C-strain (conventional live vaccine strain China) and the strain Uelzen were used. Samples were also tested for neutralizing antibodies against border disease virus (BDV) using the strain 137/4 and against bovine viral diarrhoea virus (BVDV) using the NADL strain. Neutralizing antibodies against the C-strain were detected in 36 sera (16.82 %), against strain Uelzen in 17 sera (7.94 %) and in 22 sera (10.28 %) against both strains. In five sera (2.33 %) neutralizing antibodies against BVDV and BDV were found.     

Experimentally induced ovine border disease: extensive hypomyelination with minimal viral antigen in neonatal spinal cord

Border disease (BD) was experimentally induced in 9 lambs by inoculation of their dams with the BD-31 strain of border disease virus (BDV) at 50 days of gestation. These ewes developed serum-neutralizing antibody titers to BDV. The diagnosis of BD in their lambs was confirmed by hairy birthcoats, intrauterine growth retardation, tremors, and hypomyelination. Three clinically healthy age-matched control lambs, whose dams had been given an inoculum containing only BDV-free cell culture supernatant, were studied in parallel. There were significant differences in birth weights and in the lengths of the right tibiae and radii between the affected and the control lambs. There was a gradient in severity of clinical neurologic signs among the affected lambs, which directly correlated with the severity of hypomyelination in their spinal cords. However, the difference in severity of the neurologic deficits did not correlate with differences either in the precolostral serum-neutralizing antibody titers to BDV in these lambs or in the number of BDV antigen-containing cells in their spinal cords. Only approximately 0.01% to 0.3% of spinal cord cells, both in gray matter and white matter, were BDV antigen positive in the affected lambs. These results indicate that extensive infection of CNS cells with their subsequent destruction or functional alteration may not be a critical part of the pathogenesis of the hypomyelination in BD.     

A survey of antibodies to pestivirus in sheep in the Republic of Ireland

: Sera from 1,448 adult ewes in 91 flocks, representing all 26 counties in the Republic of Ireland, were examined for pestivirus antibodies using a commercially available ELISA which detected IgG1 antibody to border disease virus. Eighty-one sheep (5.6%) in 42 flocks (46.0%) were antibody-positive. Within infected flocks, the mean seroprevalence level was 11.4% with a range of 6.3% to 30.0%. The highest antibody prevalence was detected in sheep from central lowland counties of Ireland. Comparative neutralisation testing of 42 ELISA-positive sera detected geometric mean antibody titres of 136 to the NADL strain of bovine viral diarrhoea virus (BVDV), 92 to the Moredun strain of border disease virus and 21 to the 137/4 strain of border disease virus. These results suggest that BVDV may be the major ruminant pestivirus infecting sheep in Ireland. Although there are high numbers of infected flocks, many sheep within such flocks remain antibody-negative and are at risk of giving birth to lambs with congenital border disease.     

Seroprevalence of antibodies to ruminant pestiviruses in sheep and goats in Tyrol (Austria)

In this study 2058 blood samples from sheep of 150 flocks from the province of Tyrol were tested by ELISA and serum neutralisation tests for antibodies to ruminant pestiviruses. In the ELISA, positive results were obtained with 34.9% of individual sheep sera and in 89.3% of the sheep flocks. The prevalence in sheep and sheep flocks varied according to areas. Seroprevalence of pestiviruses was significantly (p < 0.05) higher in small ruminants pastured during summertime on the Alps. Comparative neutralisation studies were carried out on all positive blood samples with BVDV-1, BVDV-2 and BDV. 443 seropositive sheep samples exhibited clearly the highest titre against one of the pestivirus strains tested. 413 revealed the highest titres (2 or more fold) to BVDV-1, 6 to BVDV-2 and 24 to BDV. In some areas a very high rate of pestivirus seroprevalence could be found. This fact could be harmful to the BVDV-Elimination and Controlling Program in cattle in Austria.     

Differential diagnosis of classical swine fever and border disease: seroepidemiological investigation of a pestivirus infection on a mixed sheep and swine farm.]

During recent years neutralizing antibodies against Border Disease Virus (BDV) were found repeatedly in German pig herds. Consequently there was a demand for a differential diagnostic system. A permanent sheep cell line and BDV reference strain Moredun were chosen and were applied in a could be used case study. A pestivirus could be isolated from piglets on a mixed farm and was characterised as 'non-Classical Swine Fever' (CSF) by using monoclonal antibodies. Due to a CSF suspicion the pig herd was destroyed immediately. Serum samples of sheep from the same farm were used for further characterisation of the new virus isolate. A neutralization test of the sheep sera was performed against different pestiviruses and the new isolate. Neutralizing antibody titres against the new virus pig isolate were significantly higher than against all other pestiviruses. BDV strain Moredun recognised the antibodies clearly, whereas CSF viral strain Alfort 187 and several isolates of bovine viral diarrhoea virus (BVDV) strains scored the lowest cross reaction.