No evidence for involvement of sheep in the epidemiology of cattle virulent epizootic hemorrhagic disease virus

Epizootic hemorrhagic disease virus (EHDV) is an Orbivirus. While not previously considered as an important disease in cattle, several EHDV serotypes (EHDV-6 and 7) have recently been implicated in disease outbreaks. The involvement of sheep in the epidemiology of EHDV is still not understood. In this study we compared the prevalence of antibodies to EHDV and bluetongue virus (BTV) in sheep to their prevalence in cattle after an outbreak of EHDV that occurred in Israel during 2006. Sixty-six sheep and lambs scattered in seven herds were compared to 114 cows and calves scattered in 13 dairy cattle herds, matched to the sheep herds by location. While antibody prevalence to EHDV was high in cattle (35.2% within the outbreak zone) no evidence of exposure to EHDV was found in sheep (p<0.0001). Antibodies to BTV were apparent in both cattle and sheep though in the former it was significantly higher (63.2%, 16.7% respectively, p<0.0001), suggesting higher exposure of cattle to biting Culicoides midges. Taken together, these results imply that sheep have a negligible role in the epidemiology of EHDV.     

A survey of cattle for antibodies against bluetongue and epizootic hemorrhagic disease of deer viruses in British Columbia and southwestern Alberta in 1987.

In 1987 a serological survey of cattle for antibodies (Ab) to bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) was undertaken in British Columbia and southwestern Alberta after infection with the viruses was diagnosed in wild and domestic ruminants in the Okanagan Valley. Of 4610 cattle tested, five had Ab only to BTV, 125 had antibodies only to EHDV and 16 had Ab to both viruses. The Ab were identified as specific for BTV type 11 (BT-11) or EHDV type 2 (EHDV-2). All but one of the seropositive cattle originated in the Okanagan Valley of British Columbia. The remaining one seropositive animal which had Ab to EHDV-2 was pastured with a bull purchased from the Okanagan Valley.     

Exploratory spatial data analysis of regional seroprevalence of antibodies against epizootic hemorrhagic disease virus in cattle from Illinois and Indiana

OBJECTIVE: To estimate seroprevalence of antibodies against the serogroup of epizootic hemorrhagic disease viruses (EHDVs) and describe spatial distribution of antibodies against EHDV among cattle herds in Illinois and western Indiana. SAMPLE POPULATION: 9,414 serum samples collected from cattle in 60 herds over 3 transmission seasons. PROCEDURES: Serum samples were tested for antibodies against EHDV by use of an ELISA. Seroprevalence for 4 zones covering the length of Illinois and parts of Indiana were estimated. A multivariable mixed-effects logistic regression model with a random effect for herd was used to estimate seropositive risk for zone (1 through 4), age (yearling, adult), herd type (beef, dairy), transmission season (2000 to 2002), and zone by year interaction. Isopleth maps of seroprevalence at the herd level were produced. RESULTS: Antibodies against EHDV were detected in 1,110 (11.8%) samples. Estimated seroprevalence in 2000, 2001, and 2002 was 15.3%, 13.4%, and 5.2%, respectively. Seroprevalence was highest in the southernmost zone and lowest in the northernmost zone, but risk of seropositivity for EHDV among and within zones varied by year. Clusters of high seroprevalence in the south, low seroprevalence in the north, and outliers of high and low seroprevalence were detected. Risk mapping revealed areas of higher seroprevalence extending northward along the western and eastern ends of the study region. CONCLUSIONS: Seroprevalence of antibodies against EHDV in cattle was higher in the south than north; however, local complexities existed that were not observed in a serosurvey of antibodies against bluetongue virus from the same cattle population.     

Development of an enzyme-linked immunosorbent assay for the detection of antibody to epizootic hemorrhagic disease of deer virus.

An enzyme-linked immunosorbent assay has been developed to detect antibodies to epizootic hemorrhagic disease of deer virus (EHDV). The assay incorporates a monoclonal antibody to EHDV serotype 2 (EHDV-2) that demonstrates specificity for the viral structural protein, VP7. The assay was evaluated with sequential sera collected from cattle experimentally infected with EHDV serotype 1 (EHDV-1) and EHDV-2, as well as the four serotypes of bluetongue virus (BTV), BTV-10, BTV-11, BTV-13, and BTV-17, that currently circulate in the US. A competitive and a blocking format as well as the use of antigen produced from both EHDV-1- and EHDV-2-infected cells were evaluated. The assay was able to detect specific antibody as early as 7 days after infection and could differentiate animals experimentally infected with EHDV from those experimentally infected with BTV. The diagnostic potential of this assay was demonstrated with field-collected serum samples from cattle, deer, and buffalo.     

Expression and antigenic characterization of the major core protein VP7 of Chuzan virus, a member of the Palyam serogroup orbiviruses

The Palyam serogroup-specific antigen, VP7, of Chuzan virus strain K-47 was expressed in insect cells by a recombinant baculovirus. The expressed protein appeared as a single band of 38kDa corresponding to the predicted molecular mass of Chuzan virus VP7 by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). In immunoprecipitation analysis, the recombinant VP7 was not only recognized by all polyclonal antibodies against the Palyam serogroup viruses (PALV) tested in this study, but also by antisera to bluetongue virus (BTV) serotype 1, epizootic haemorrhagic disease virus (EHDV) serotypes 1 and 2. However, in Western immunoblot assay, no positive signals were observed between this protein and these antisera, even in the homologous reaction using antiserum to Chuzan virus. These findings demonstrate that the common antigenic determinants on the VP7 proteins of Chuzan virus and the other PALV serotypes are mainly conformational and that the proteins share some epitopes with those of BTV and EHDV beyond the serogroup. No cross-reactivities were detected between Chuzan virus VP7 and antisera to BTV and EHDV in agar gel immunodiffusion (AGID) and indirect ELISA tests, indicating that the recombinant VP7 is useful as a diagnostic reagent for serological tests of congenital abnormalities of cattle caused by PALV.     

Neutralizing antibody responses to bluetongue and epizootic hemorrhagic disease virus serotypes in beef cattle

Blood samples were obtained from sentinel beef cattle at monthly intervals, and the sera were tested for antibodies, using a bluetongue virus (BTV) immunodiffusion test (IDT) and virus-neutralization test (VNT), for 5 BTV serotypes (2, 10, 11, 13, and 17) and 2 epizootic hemorrhagic disease virus (EHDV) serotypes (1 and 2). The cattle tested were transported from Tennessee to Texas in 1984 and 1985. All cattle were seronegative by the BTV IDT at the initial bleeding in Texas in 1984 and 1985. In 1984, 16 of 40 (40%) cattle seroconverted as assessed by results of the BTV IDT. In the 16 seropositive cattle in 1984, neutralizing antibodies were detected to BTV serotypes 10 (n = 7), 11 (n = 3), and 17 (n = 11), and EHDV serotypes 1 (n = 1) and 2 (n = 7). In 1984, no cattle seroconverted to BTV-2 or BTV-13. In 1985, 10 of 36 (27.8%) cattle seroconverted as assessed by results of the IDT. Of the 10 seropositive cattle in 1985, neutralizing antibodies were detected to BTV serotypes 10 (n = 10), 11 (n = 10), 13 (n = 7), and 17 (n = 5), and EHDV serotypes 1 (n = 1) and 2 (n = 7). In 1985, no cattle seroconverted to BTV-2. Clinical diseases attributable to BTV or EHDV was not detected in these cattle in 1984 or 1985.     

Epizootic hemorrhagic disease virus serotype 7 in European cattle and sheep: Diagnostic considerations and effect of previous BTV exposure

Epizootic hemorrhagic disease virus (EHDV), an arthropod-borne orbivirus (family Reoviridae), is an emerging pathogen of wild and domestic ruminants that is closely related to bluetongue virus (BTV). The present study examines the outcome of an experimental EHDV-7 infection of Holstein cattle and East Frisian sheep. Apart from na?ve animals that had not been exposed to BTV, it included animals that had been experimentally infected with either BTV-6 or BTV-8 two months earlier. In addition, EHDV-infected cattle were subsequently challenged with BTV-8. Samples were tested with commercially available ELISA and real-time RT-PCR kits and a custom NS3-specific real-time RT-PCR assay. Virus isolation was attempted in Vero, C6/36 and KC cells (from Culicoides variipennis), embryonated chicken eggs and type I interferon receptor-deficient IFNAR(-/-) mice. EHDV-7 productively infected Holstein cattle, but caused no clinical signs. The inoculation of East Frisian sheep, on the other hand, apparently did not lead to a productive infection. The commercial diagnostic kits performed adequately. KC cells proved to be the most sensitive means of virus isolation, but viremia was shorter than 2 weeks in most animals. No interference between EHDV and BTV infection was observed; therefore the pre-existing immunity to some BTV serotypes in Europe is not expected to protect against a possible introduction of EHDV, in spite of the close relation between the viruses.     

Bluetongue and epizootic hemorrhagic disease in ruminants in Georgia: survey by serotest and virologic isolation

The frequencies of precipitating antibodies to bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) in domestic ruminants and white-tailed deer (WTD) in Georgia were 36% and 32%, respectively (n = 2,200). The frequencies of seropositivity to BTV and EHDV were high among cattle (47% and 42%, respectively [n = 1,068]) and less so in WTD (36% and 34% [n = 414]). The frequencies among sheep were 34% for BTV and 29% for EHDV (n = 286), whereas among goats, seropositivity was 8% for BTV and 7% for EHDV (n = 433). Serum samples from northeastern Georgia (1 of the 4 regions in the survey) had the highest frequency of precipitating antibodies for BTV (45%) and EHDV (38%). The lowest frequency was in southeastern Georgia, with 29% seropositivity for BTV and 24% seropositivity for EHDV. Of the 175 farms or herds in the serosurvey, 70% included animals that had BTV-precipitating antibodies, and 67% included animals which had EHDV-precipitating antibodies. Seventeen viral isolates were obtained from individual animals on 9 different farms. Fifteen of the isolates were BTV--8 from cattle, 4 from sheep, and 3 from WTD; 8 of them were serotype 11, and 7 were serotype 17. Viral isolates from each of 2 WTD were identified as EHDV serotype 1 and serotype 2. Of the total 17 isolates, 11 were from clinically healthy ruminants, and 6 were from animals with clinical signs of BT or EHD. Five of the viral isolates originated from northeastern Georgia, 7 from the northwestern region, and 5 from the southwestern region; none was obtained from specimens from the southeastern region.     

Antigenic relationship of Ibaraki,bluetongue, and epizootic Hemorrhagic disease viruses

Ibaraki virus, which causes a bluetongue-like disease of cattle in Japan, was compared antigenically with the four serotypes of bluetongue virus (BTV) found in the U.S. and with the two serotypes of epizootic hemorrhagic disease virus (EHDV). No antigenic relationship was found between Ibaraki virus and BTV serotypes 10, 11, 13, and 17 in tests for group or serotype-specific antigens. However, Ibaraki virus and EHDV were related antigenically. The agar gel precipitin and indirect fluorescent antibody tests for group antigens showed two-way cross relationships between Ibaraki virus and EHDV serotypes 1 and 2. The more restrictive serotype-specific neutralization test revealed that antigenic relatedness was stronger between Ibaraki virus and the serotype 2 (Alberta strain) of EHDV than between Ibaraki virus and the serotype 1 (New Jersey strain) of EHDV.     

Regional seroprevalence of bluetongue virus in cattle in Illinois and western Indiana

OBJECTIVE: To estimate seroprevalence of bluetongue virus (BTV) and the geographic distribution of seropositive cattle herds in Illinois and western Indiana. SAMPLE POPULATION: 10,585 serum samples obtained from cattle in 60 herds during 3 transmission seasons (2000 through 2002). PROCEDURES: In a longitudinal study, serum samples were tested for BTV antibodies by use of a competitive ELISA. Four geographic zones were created by use of mean minimum January temperature. A multivariable mixed-effects logistic regression model with a random effect for herd was used to estimate seropositive risk for zone, age of cattle, herd type, and transmission season. RESULTS: Overall, BTV antibodies were detected in 156 (1.5%) samples. Estimated seroprevalence in 2000, 2001, and 2002 was 1.49%, 0.97%, and 2.18%, respectively. Risk of being seropositive for BTV was associated with geographic zone and age. Seroprevalence increased progressively from northern to southern zones, with no evidence of BTV infection in the northernmost zone. In the southernmost zone, annual seroprevalence ranged from 8.65% to 11.00%. Adult cattle were 2.35 times as likely as juvenile cattle to be seropositive. CONCLUSIONS AND CLINICAL RELEVANCE: Overall seroprevalence was lower than has been reported for Illinois cattle. Bluetongue virus antibodies were distributed heterogeneously in this region. Only in the southernmost zone was seroprevalence consistently > 2%. Regionalization of BTV risk based on state borders does not account for such variability. Serologic data could be combined with landscape, climate, and vector data to develop predictive models of BTV risk within transitional regions of the United States.     

4种动物虫媒病病毒多重RT-PCR检测方法的建立

分别设计和合成4对特异性引物,通过对反应条件的优化,初步建立了鉴别蓝舌病病毒(BTV)、鹿流行性出血病病毒(EHDV)、水疱性口炎病毒(VSV)和赤羽病病毒(AKV)的多重RT-PCR检测方法,并对其特异性和敏感性进行了检测。结果表明,所设计的4对引物可对同一样品中的VSV、BTV、EHDV和AKV进行特异性扩增,所扩增的目的片段的长度分别为301、351、537、250bp。建立的四重RT-PCR检测方法能够检测出10-7稀释的细胞培养病毒液,4种病毒之间没有发生交叉反应,说明该检测方法特异性强,敏感性较高。所建立的多重RT-PCR方法可用于上述4种动物虫媒病病毒的快速鉴别诊断,在动物检疫、临床诊断和流行病学调查方面具有较好的应用前景。     

Evaluation of bluetongue virus diagnostic tests in free-ranging bighorn sheep

Five bluetongue virus (BTV) diagnostic tests were evaluated for use in free-ranging bighorn sheep. We sampled one bighorn sheep population four times between 1989 and 1995. The tests evaluated included virus isolation (VI), polymerase-chain reaction (PCR), serum neutralization (SN), agar-gel immunodiffusion (AGID), and competitive enzyme-linked immunosorbent assay (c-ELISA). The c-ELISA, AGID and SN tests had high levels of agreement in determining serogroup exposure in bighorn sheep. We used maximum-likelihood algorithms to estimate the parameters of each diagnostic test used. Although the c-ELISA and AGID had high sensitivity and specificity, the SN had perfect specificity but lower apparent sensitivity. Due to the potential of cross-reactions among multiple serotypes, results of the SN must be interpreted with caution when assessing serotype exposure in an area where multiple serotypes are endemic. The PCR assay delineated convalescent antibody titers from more-recent infections, and consequently, was pivotal in distinguishing a different exposure pattern between the bighorn sheep and cattle in an adjacent herd. Based on an increasing seroprevalence (50% to 100%), BTV circulated through this bighorn sheep population between 1989 and 1993. This increase in seroprevalence coincided with a bighorn die-off due to BTV infection in June, 1991. An adjacent cattle herd was sampled in 1995 for comparison. The bighorn sheep and adjacent cattle had different patterns of exposure to BTV between 1994 and 1995. There was no evidence that BTV circulated through the bighorn sheep population from 1994 to 1995. In 1995, seroprevalence to BTV decreased to 72%, none of yearling bighorn was seropositive, and all of the 39 bighorn sheep were PCR-negative. In contrast, all adult cattle were seropositive to BTV by c-ELISA and SN, and 4 of the calves were seropositive; 11 of the 24 cattle were PCR-positive, including all five calves. Overall, the pattern of temporal herd immunity in the bighorn sheep appeared to follow a classic epidemic curve, with the appearance and subsequent disappearance of herd immunity coinciding with the 1991 die-off in this population. As low levels of herd immunity and high proportions of susceptible animals are key factors in the development of epidemics, this population of bighorn sheep may be at increased risk for a BTV epidemic in the future.     

Re-emergence of bluetongue, African horse sickness, and other Orbivirus diseases

Arthropod-transmitted viruses (Arboviruses) are important causes of disease in humans and animals, and it is proposed that climate change will increase the distribution and severity of arboviral diseases. Orbiviruses are the cause of important and apparently emerging arboviral diseases of livestock, including bluetongue virus (BTV), African horse sickness virus (AHSV), equine encephalosis virus (EEV), and epizootic hemorrhagic disease virus (EHDV) that are all transmitted by haematophagous Culicoides insects. Recent changes in the global distribution and nature of BTV infection have been especially dramatic, with spread of multiple serotypes of the virus throughout extensive portions of Europe and invasion of the south-eastern USA with previously exotic virus serotypes. Although climate change has been incriminated in the emergence of BTV infection of ungulates, the precise role of anthropogenic factors and the like is less certain. Similarly, although there have been somewhat less dramatic recent alterations in the distribution of EHDV, AHSV, and EEV, it is not yet clear what the future holds in terms of these diseases, nor of other potentially important but poorly characterized Orbiviruses such as Peruvian horse sickness virus.     

Studies with enzyme-linked immunosorbent assays for the serodiagnosis of bluetongue and epizootic haemorrhagic disease of deer

An ELISA for the detection of serum antibody in sheep, cattle and goats to the viruses of bluetongue (BTV) and epizootic haemorrhagic disease of deer (EHDV) has been developed. Two methods of antigen preparation were analysed for efficacy in the ELISA and inter-group seroreactivity. A freeze-thaw (F/T) antigen appeared to have a narrower specificity than a cytoskeletal preparation from infected cells (P200) which contained all viral proteins. A higher background reactivity was seen when using the P200 antigen, suggesting that a F/T antigen, perhaps as a composite of serotypes, would be of greater value in an ELISA to replace current methods for antibody screening. The effect of multiple infections with unrelated orbiviruses was found to have no effect on the detection of antibody to BTV and EHDV by ELISA. The ELISA was able to demonstrate development and persistence of antibody to BTV in cattle over the course of 120 days.     

4种重要虫媒病的核酸液相芯片高通量检测方法的建立

为建立可检测鹿流行性出血热病毒(EHDV)、阿卡斑病毒(AKV)、蓝舌病病毒(BTV)和水泡性口炎病毒(VSV)的液相芯片快速检测技术,用DNAStar软件对GenBank中BTV的VP7基因、EHDV的VP7基因、AKV的N基因和VSV的NP基因序列进行序列分析,设计针对这些基因的特异性探针并标记生物素,分别与不同编号的荧光编码微球偶联后再与这些病毒相应基因的PCR产物杂交反应,用液相芯片检测仪(Liquichip 200)检测荧光信号建立了以上4种虫媒病的快速液相芯片检测方法。检测结果显示,该方法具有较好的特异性,偶联特异性探针的微球只与相应的病毒基因的PCR产物反应,而不与其他虫媒病病毒反应;检测灵敏度达到50~100个TCID50。本研究建立了可以同时检测鹿流行性出血热病毒、阿卡斑病毒、蓝舌病病毒和水泡性口炎病毒的快速高通量液相芯片技术,为其他类似病毒的快速高通量检测提供了借鉴和经验。     

Replication of bluetongue virus and epizootic hemorrhagic disease virus in pulmonary artery endothelial cells obtained from cattle,sheep, and deer

OBJECTIVE: To compare replication of bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) in pulmonary artery endothelial cells (ECs) obtained from juvenile cattle, sheep, white-tailed deer (WTD; Odocoileus virginianus), and black-tailed deer (BTD; O hemionus columbianus). SAMPLE POPULATION: Cultures of pulmonary artery ECs obtained from 3 cattle, 3 sheep, 3 WTD, and 1 BTD. PROCEDURE: Purified cultures of pulmonary artery ECs were established. Replication, incidence of infection, and cytopathic effects of prototype strains of BTV serotype 17 (BTV-17) and 2 serotypes of EHDV (EHDV-1), and (EHDV-2) were compared in replicate cultures of ECs from each of the 4 ruminant species by use of virus titration and flow cytometric analysis. RESULTS: All 3 viruses replicated in ECs from the 4 ruminant species; however, BTV-17 replicated more rapidly than did either serotype of EHDV. Each virus replicated to a high titer in all ECs, although titers of EHDV-1 were significantly lower in sheep ECs than in ECs of other species. Furthermore, all viruses caused extensive cytopathic effects and a high incidence of cellular infection; however, incidence of cellular infection and cytopathic effects were significantly lower in EHDV-1-infected sheep ECs and EHDV-2-infected BTD ECs. CONCLUSIONS AND CLINICAL RELEVANCE: There were only minor differences in replication, incidence of infection, and cytopathic effects for BTV-17, EHDV-1, or EHDV-2 in ECs of cattle, sheep, BTD, and WTD. It is not likely that differences in expression of disease in BTV- and EHDV-infected ruminants are attributable only to species-specific differences in the susceptibility of ECs to infection with the 2 orbiviruses.     

Characterisation of monoclonal antibodies to epizootic hemorrhagic disease virus of deer (EHDV) and bluetongue virus by immunisation of mice with EHDV recombinant VP7 antigen.

Immunisation of mice with recombinant VP7 antigen of epizootic hemorrhagic disease virus of deer (EHDV) induced serum antibody responses to EHDV. However, from the 19 monoclonal antibodies (Mab) produced from these mice, 15 were specific for EHDV and four for bluetongue virus (BTV). No Mabs were identified with the specificity for an epitope of VP7 shared by both EHDV and BTV in spite of the fact that they share a large portion of homology in VP7 amino acids composition. These Mabs were divided into five groups based on their specificity and interaction with each other. Group II Mabs, consisting of 13 Mabs, recognises a potential serogroup specific, linear epitope of EHDV VP7 antigen. One of the Mabs to BTV (Group V) was identified as BTV VP7 specific with the possibility of being the serogroup specific and recognizes a potential conformational epitope. Two Mabs from these VP7 specific groups were further analysed and found to be useful in a competitive enzyme-linked immunosorbent assay (C - ELISA) for detection of specific antibodies against EHDV and BTV in bovine sera.     

单一引物标记LUX荧光RT-PCR鉴别BTV和EHDV

采用在线LUXTM专业软件,根据BTV-NS3基因序列和EHDV-NS3基因序列,通过特异性单一引物序列3′末端的荧光标记,分别设计出两对BTV和EHDV的LUX荧光PCR引物,并采用BLAST软件对各引物进行匹配性和特异性分析,根据分析结果选择合适的引物合成。经过各反应条件的优化和特异性、敏感性试验,并对BTV、EHDV、VSV、PPRV、BVDV、AKV的BHK-21细胞培养物和临床样品检测,与常规RT-PCR进行对比检测,建立了能同时鉴别检测BTV和EHDV的二重LUXTM荧光PCR方法。该二重LUXTM荧光PCR的BTV和EHDV各自引物只对相应的病毒呈阳性反应,两者没有交叉反应现象,对健康牛、羊和猪基因组DNA、BKH-21细胞对照,以及其他几种相似疾病如VSV、PPRV、BVDV、AKV均呈阴性反应。该法对病毒的细胞培养液鉴别检测敏感性可达1TCID50C以上,比常规RT-PCR敏感性提高10倍以上,从样品核酸纯化到完成二重LUXTM荧光PCR反应和熔解曲线分析,仅需3h,在进出口动物检验检疫中快速鉴别BTV和EHDV具有实际应用价值。     

Bluetongue virus in the French Island of Reunion

This paper records the results of a bluetongue virus (BTV) serological survey and reports the first isolation of BTV on the French Island of Reunion. In January 2003, the French Island of Reunion, located off the coast of Madagascar, reported an outbreak of disease in cattle that resembled clinical bluetongue (BT) in sheep. The suspected causal agent was isolated and identified as epizootic haemorrhagic disease of deer virus (EHDV). However, because of the similarity in the clinical signs to those of BT, a retrospective survey against BTV was carried out using sera collected in 2002. Results revealed the presence of antibody in all sera tested indicating that BTV has been resident on the Island since 2002, and probably earlier. Although up to July 2003 no clinical BT had ever been reported in sheep, BTV viral RNA was amplified by RT-PCR from a single sheep blood collected in February that year, which strongly suggested that BTV was currently circulating on the Island. Following a second outbreak of disease in August 2003, this time involving a flock of Merino sheep, infectious BTV was finally isolated, and identified by both traditional and molecular techniques as serotype 3. The nucleotide and amino-acid sequences of the RT-PCR products amplified for BTV segments 7 and 10 from the sheep blood collected in February and August from different areas of the Island, were sufficiently diverse as to suggest that they were of different origins and/or different BTV serotypes.