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.     

Bluetongue and related viruses in Nigeria: Experimental infection of West African dwarf sheep with Nigeria strains of the viruses of epizootic haemorrhagic disease of deer and bluetongue

West African dwarf sheep were inoculated by the subcutaneous route with epizootic haemorrhagic disease of deer (EHD) virus or bluetongue (BT) virus. No clinical disease was observed following primary EHD or BT infection, or subsequent challenge with either homologous or heterologous virus. However, viraemia was detected in non-immune sheep exposed to BT virus, but not in BT- or EHD-immune sheep challenged with either virus, or in non-immune sheep exposed to primary EHD virus infection. Complement fixing antibodies developed against both EHD and BT viruses following the primary infection with either virus, or subsequent challenge with homologous or heterologous virus. Following a primary infection, virus-neutralising (VN) antibodies developed only against the inoculated virus, while the detection of VN antibodied to both viruses followed the challenge of an EHD- or BT-immuned sheep with either the homologous or heterologous virus. These findings further support previous reports of a relationship between EHD and BT viruses. between EHD and BT viruses.     

Comparison of the epidemiology of epizootic haemorrhagic disease and bluetongue viruses in dairy cattle in Israel

An outbreak of epizootic haemorrhagic disease virus (EHDV) in cattle in Israel in 2006 enabled a comparison of the spatial distribution of epidemic exposure to EHDV with that of exposure to bluetongue virus (BTV), which is endemic in the country. The seroprevalence of both viruses was examined in 1650 serum samples collected from 139 farms representative of the spatial distribution of dairy cattle in Israel. A significant association between exposure to EHDV and BTV was demonstrated in both univariate and multivariate analyses. Recent exposure to BTV and EHDV (demonstrated by seroprevalence in calves) was clustered in different geographical locations, indicating that the two viruses had different patterns of spread, that of EHDV being influenced by winds and terrain barriers and that of BTV by herd immunity.     

Growth of bluetongue and epizootic hemorrhagic disease of deer viruses in poikilothermic cell systems

Continuous cell lines from the ticks Dermacentor variabilis, D. parumapertus, D. nitens, Rhipicephalus sanguineus and R. appendiculatus, the mosquitoes Aedes albopictus and Culex quinquefasciatus and the African toad Xenopus laevis were tested for their ability to replicate bluetongue (BT) and epizootic hemorrhagic disease of deer (EHD) viruses, and for their sensitivity as potential isolation systems. BT serotype 17 grew to peak titers of 10(4.5)-10(7.5) TCID50 ml-1 in all except one of the tick cell lines, EHD 2 virus attained titers similar to that of BT 17 in the mosquito and toads cells, but failed to replicate in tick cells. Only Aedes albopictus and Xenopus laevis cells were as sensitive to infection with low-passage BT 11 and EHD 2 viruses as control cultures of Vero and BHK cells. At 27 degrees C, persistent infection of Xenopus laevis cells occurred, producing low yields of BT 17 and EHD 2. When shifted to 32 degrees C, these cultures expressed virus in exponential increments. No cytopathic effect (CPE) was seen in any of the tick-virus systems, but infected mosquito and toad cells detached from the monolayer within 3-6 days after inoculation with either virus. In the toad cells, this CPE was presaged by the development of plaques within 48 h after infection. Potential applications of poikilotherm systems in orbivirus research are discussed.     

Bluetongue and epizootic hemorrhagic disease in white-tailed deer from Oklahoma: serologic evaluation and virus isolation

Blood samples were collected from 194 white-tailed deer from 27 locations in Oklahoma from 1977 through 1984. Sixty-eight (35%) of the deer had antibody against bluetongue virus (BTV) and 78 (40%) had antibody against epizootic hemorrhagic disease virus. Seropositive deer were detected in each of the 4 geographic quadrants of the state. Virus isolation was attempted in 40 deer from the northeast quadrant of Oklahoma (1983 through 1984); BTV was isolated from 11 deer, but epizootic hemorrhagic disease virus was not isolated. The isolation of BTV serotype 11 from these deer from 1983 through 1984 coincided with reported isolations of this serotype in other ruminants in Oklahoma during this time.     

流行性出血病病毒抗原捕获ELISA方法的建立

为建立流行性出血病病毒(EHDV)病原学检测方法,本试验用纯化处理的4种血清型病毒(EHDV-2、5、6和8)混合免疫兔和豚鼠,制备高免血清。以兔抗EHDV为捕获抗体,豚鼠抗EHDV为检测抗体,羊抗豚鼠IgG-HRP为酶标抗体,建立了EHDV抗原捕获ELISA(antigen capture ELISA,AC-ELISA)方法。通过反应条件优化,获得最佳工作条件:5%马血清为稀释液,包被抗体1∶15 000稀释,检测抗体1∶20 000稀释,酶标抗体1∶15 000稀释。通过检测60份阴性样品确定临界值,P/N值≥2为阳性、P/N值1.5为阴性;特异性试验表明该方法仅能检测出各血清型EHDV、蓝舌病病毒(BTV)、赤羽病病毒(AKAV)及中山病毒(CHUV)无交叉反应;敏感性试验表明该方法可以检出102.47±0.07 TCID50的病毒;批内和批间变异系数均10%;用RT-PCR与该方法同时对70份参考样品进行检测,符合率为92.86%。表明本试验建立的AC-ELISA方法为EHDV抗原检测提供了一种可靠实用的技术手段。     

Serological survey of ruminants in some Caribbean and South American countries for type-specific antibody to bluetongue and epizootic haemorrhagic disease viruses

The results of a serological survey of ruminant livestock in some countries of the Caribbean and South America for type-specific antibody to bluetongue virus are reported. Using the microneutralisation test with the international serotypes 1 to 22 of bluetongue virus, antibodies to several types were detected. Analysis of the data indicated that in 1981-82 bluetongue virus types 6, 14 and 17, or viruses closely related to them, were infecting ruminants in this region of the world. Antibody to the related virus of epizootic haemorrhagic disease (serotype 1) was also detected in cattle. The difficulty in interpreting the epidemiological significance of data generated by a serological survey of this kind is discussed.     

Development and evaluation of an enzyme-linked immunosorbent assay for detection of bovine antibodies to epizootic hemorrhagic disease of deer viruses.

An indirect enzyme-linked immunosorbent assay (I.ELISA) is described for detection of bovine serum antibody to epizootic hemorrhagic diseases of deer virus (EHDV). Serum samples, at a dilution of 1:200, were incubated with group-specific EHDV antigens, pre-adsorbed to microtiter plates. Bound antibodies were detected by a murine monoclonal antibody to bovine immunoglobulin (Ig)G1 (heavy-chain specific) conjugated with horseradish peroxidase. The performance of the I.ELISA in detecting antibodies to EHDV in sequential serum samples from calves experimentally infected with serotypes 1,2,3 and 4 was evaluated. The I.ELISA detected EHDV antibodies from 14 days postinfection when seroconversion by the standard agar gel immunodiffusion (AGID) test was also evident. The group-specific antibodies to EHDV increased exponentially during the first two to four weeks postinfection and remained relatively stable for about 12 months in some calves. Unlike observations with the AGID test, no reaction was seen in the I.ELISA between blue-tongue virus (BTV) antigen and sera from calves given a single dose of EHDV. The performance of the I.ELISA and AGID were compared using 3,135 AGID negative bovine field sera from herds in Ontario, Alberta and British Columbia and 130 AGID positive samples collected from cattle in 1987 and 1988 during and after outbreaks of EHD in the Okanagan Valley, British Columbia. The specificity and sensitivity of the assay relative to the AGID test were 99.3% and 91.5% respectively, with an overall agreement of 99.0% between the tests.(ABSTRACT TRUNCATED AT 250 WORDS)     

Simultaneous screening of bovine sera for antibodies to bluetongue and epizootic hemorrhagic disease of deer viruses by enzyme-linked immunosorbent assay.

An indirect enzyme-linked immunosorbent assay (I-ELISA) is described for simultaneous screening of bovine sera for detection of antibodies to bluetongue (BT) and epizootic hemorrhagic disease of deer (EHD) viruses (V). Optimal dilutions of BTV and EHDV antigens were combined and allowed to absorb on to the wells of microtiter plates. Appropriately diluted (1:100) bovine sera were allowed to incubate and the bound antibodies were detected by a murine monoclonal antibody (MAb) to bovine immunoglobulin (H-Chain) conjugated with horseradish peroxidase. The performance of the combined (C) I-ELISA in detecting antibodies to BTV and EHDV in sequential serum samples from calves experimentally inoculated with BTV, serotype 10, EHDV, serotype 1 (New Jersey) or EHDV serotype 2 (Alberta) was evaluated. Comparable antibody profiles were demonstrable by the CI-ELISA and separate I-ELISAs using either BTV or EHDV antigens. The results suggest that the CI-ELISA offers many advantages over the standard agar gel immunodiffusion (AGID) test and has potential application as a rapid, sensitive, inter-group-specific and inexpensive test for simultaneous screening of bovine sera for antibodies to BTV and/or EHDV.     

Survey for antibodies to viruses of bovine virus diarrhea, bluetongue, and epizootic hemorrhagic disease in hunter-killed mule deer in New Mexico

Sera from male mule deer (Odocoileus hemionus) collected in November 1977 in Otero County, New Mexico were tested fro antibodies to bovine virus diarrhea virus (BVDV), bluetongue virus (BTV), and epizootic hemorrhagic disease virus (EHDV). Neutralizing antibodies were detected in 26 of 76 (34%) sera tested for BVDV (titer greater than or equal to 1:16). Of 46 sera tested for antibodies to BTV and EHDV, 10 (22%) and 3 (7%), respectively, were positive. Three (7%) of 46 sera were suspect (titer < 1:20) for BTV, and 18 (38%) sera were suspect (titer < 1:20) for EHDV.     

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.     

A monoclonal antibody blocking ELISA detects antibodies specific for epizootic haemorrhagic disease virus.

The isolation of a monoclonal antibody (1G9/C9) with specificity for the epizootic haemorrhagic disease (EHD) serogroup has enabled the development of a highly sensitive and specific blocking ELISA (B-ELISA) for the detection of serum antibodies to EHD viruses. The assay was sensitive to blocking antibodies present in hyperimmune reference antisera to all six EHD serotypes tested but was unaffected by reference antisera to 19 South African and eight Australian serotypes of the related orbivirus bluetongue virus (BTV). The sensitivity of the EHD B-ELISA exceeded that of an indirect ELISA (I-ELISA) for EHD-specific antibody detection. Serum antibody titres to BTV and EHD in experimental and field sera, including a sentinel herd from which virus isolations were made, were examined in both the BTV and EHD B-ELISA tests. These results showed the B-ELISA was only sensitive to antibodies specific for the homologous serogroup in each case, even where sequential and mixed infections with each virus type occurred.     

Antibodies against certain bluetongue and epizootic haemorrhagic disease viral serotypes in Indonesian ruminants.

The orbiviruses contain several important viruses of livestock including bluetongue (BT) and epizootic haemorrhagic disease of deer (EHD) which share some group antigens. Preliminary screening of sera for antibodies to orbiviruses by the agar gel immunodiffusion (AGID) test has previously revealed widespread infections with the BT group in Indonesia. However serum neutralization (SN) tests give a more accurate estimate of exposure to each serotype in the BT and EHD groups, and in this study were applied to sera that had reacted previously in the AGID test. Five different serotypes of BT and one serotype of EHD virus were studied. Reactors to BT serotype 20 were the most prevalent, followed by EHD type 5 and BT types 21, 12, 1 and 17. Antibodies against BT serotype 20 were present in cattle, buffaloes, goats and sheep, but were most common in buffaloes. Buffaloes showed the highest exposure to the BT serotypes tested. Antibody to EHD type 5 occurred most frequently in cattle. Antibodies against all BT and EHD serotypes tested were found in buffaloes and cattle while goats had antibodies against BT types 20, 21 and EHD type 5 and sheep had antibodies only against BT type 20.     

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.     

An epizootic of adenovirus-induced hemorrhagic disease in captive black-tailed deer (Odocoileus hemionus).

Ten fawns and four adult black-tailed deer (Odocoileus hemionus) in a captive herd died as a result of adenovirus-induced hemorrhagic disease. Acute, systemic infections were characterized by hemorrhagic enteropathy, pulmonary edema, and occasional ulceration of the upper alimentary tract. Localized infections were limited to the upper alimentary tract and included stomatitis, pharyngitis, mandibular osteomyelitis, and rumenitis. In deer with acute, systemic infections, a diagnosis was made by histopathology and immunohistochemistry. The serum neutralization test was useful for confirming a diagnosis in animals with prolonged, localized infections. Deer originating from herds with a history of adenovirus infection should not be transferred to other captive herds or released into free-ranging populations because they may serve as carriers of adenovirus.     

Molecular and genetic comparisons of two serotypes of epizootic hemorrhagic disease of deer virus

The virus-specific double-stranded genome RNA of 2 serotypes of epizootic hemorrhagic disease of deer virus (EHDV) was evaluated by use of coelectrophoresis in polyacrylamide and agarose gel systems. The molecular weights of virion RNA segments were 0.32 to 2.57 X 10(6) for EHDV-1 and 0.33 to 2.54 X 10(6) for EHDV-2. Seven of 10 double-stranded RNA segments of the 2 serotypes had different electrophoretic mobilities in the polyacrylamide-gel electrophoresis system. Although the individual RNA segments of each serotype contained unique RNA sequences determined on the basis of 2-dimensional polyacrylamide-gel electrophoresis analysis of oligonucleotides, the corresponding segments of the 2 serotypes were found to be comparable and at least 1 pair of RNA segment was almost identical. Virus-specific polypeptides for the 2 serotypes were compared by use of gel electrophoresis. Eleven polypeptides were detected for EHDV-1 and 10 for EHDV-2. Six corresponding polypeptides of these 2 serotypes had different electrophoretic mobilities, indicating that these corresponding polypeptides differ in their molecular weights. A genetic relationship was not determined between the 2 EHDV serogroups and the blue-tongue serogroup viruses, using oligonucleotides mapping.     

Development of PCR-based tests for the identification of North American isolates of epizootic haemorrhagic disease virus.

A serogroup-specific polymerase chain reaction (PCR) assay and isolate identification strategies (restriction endonuclease analysis (REA) and nucleotide sequencing) were developed for the detection of North American isolates of epizootic haemorrhagic disease virus (EHDV). PCR primers (EHDV-pr4, EHDV-pr5) were designed to hybridize to the L3 gene of a North American isolate of EHDV serotype 1. Total nucleic acid was extracted from preparations of infected tissue culture and PCR was performed using a cDNA-PCR kit, according to the manufacturer's specifications. The PCR assay generated a 459 base pair product from North American isolates of EHDV serotypes 1 and 2, while bluetongue virus (BTV) serotypes 10, 11, 13, and 17, and cell controls, failed to demonstrate PCR products. Slight modifications allowed for the PCR detection of EHDV-1 and -2 in white-tailed deer blood (Odocoileus virginiatus); PCR fragments were not amplified from uninfected deer blood. A number of restriction endonucleases and sequencing primers were evaluated for their utility in isolate identification experiments. Specifically, REA employing HincII and cycle sequencing with an internal primer (EHDV-1-pr3) proved most successful for identifying isolate-specific genome markers. The techniques presented are expected to prove valuable for rapid and specific detection of possible future EHDV incursions in wild and domestic animal species.