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In September 2015, a large outbreak caused by epizootic hemorrhagic disease virus (EHDV) was identified in Israeli dairy and beef farms. The main clinical signs were reduced milk production, weakness, drooling, lameness and recumbency, fever, slight erythema of nasal and oral mucosae, weight loss, and abortion. Dyspnea, cachexia, and death were observed less frequently. The clinical diagnosis was confirmed by ELISAs and EHDV-specific real-time reverse transcription PCR (RT-rtPCR), followed by conventional RT-PCR of the VP2 gene and sequence analysis. According to the sequence and phylogenetic analysis of theVP2 gene, the 2015 Israeli EHD outbreak was caused by EHDV-6, which was found not only in clinically ill cattle, but also in aborted fetuses.

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.

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A previously described polymerase chain reaction (PCR) assay (amplification of a 238-bp frag- ment of ovine herpesvirus 2 (OHV-2) genomic DNA) for diagnosis of sheep-associated malignant catarrhal fever (MCF) was adapted for use on formalin-fixed, paraffin-embedded tissues. Variables affecting its use were examined. Archived tissues from cattle, white-tailed deer (Odocoileus virginianus), and bison (Bison bison) diagnosed with MCF by clinical signs or histologic lesions were obtained from 2 veterinary diagnostic labo- ratories. Tissues from healthy animals and from animals diagnosed with other common bovine viral diseases were examined as controls. A total of 86 blocks from 37 suspect MCF cases were examined. Forty-one blocks from healthy animals and animals with unrelated viral diseases were examined as controls. The assay was specific for sheep-associated MCF and did not yield false-positive signals from healthy animals or from cases of infectious bovine rhinotracheitis, bovine virus diarrhea, mucosal disease, or parainfluenza-3 virus infection. A wide variety of tissues were suitable substrates, including spleen, lymph node, intestine, brain, lung, and kidney. Extracted DNA provided a more suitable target than did unextracted tissue lysate. The highest levels of viral DNA were present in lymphoid organs and intestine, but the data indicate that in acute clinical cases, most organs contain sufficient viral DNA to serve as a suitable diagnostic specimen. Fixation of 0.5-cm 3 blocks of tissue in 10% neutral buffered formalin was deleterious to the target DNA, and PCR signals progressively diminished after fixation for .45 days. Detection of genomic DNA of OHV-2 by PCR was successful for archived tissues that were 15 years old. Sheep-associated malignant catarrhal fever (MCF) can pose a formidable diagnostic challenge to clini- cians and pathologists alike, in that its protean presen- tation can make it difficult to differentiate it from sev- eral other common viral diseases. The classical signs, most prominently corneal edema, fever, and general- ized lymphadenopathy, are not always evident, partic- ularly in acute cases in highly susceptible species such as deer, 21 moose, and bison (unpublished data). If pre- sent, the predominant microscopic lesions of MCF (lymphoproliferation, mucosal inflammation, and vas- culitis) are highly suggestive. However, they may be either absent or so mild that the diagnostician has dif- ficulty in differentiating them from similar lesions that can accompany bovine virus diarrhea (BVD)/mucosal disease, infectious bovine rhinotracheitis (IBR), and epizootic hemorrhagic disease. 5 In situ immunohisto- chemistry or immunofluorescence is of little diagnostic value because, in spite of many attempts, no successful demonstration of viral antigen in tissues has been re- ported. In addition, until very recently, the few labo- ratory tests available for MCF serologic diagnosis

Epizootic hemorrhagic disease (EHD) of deer is an arthropod-borne Orbivirus that causes infection in wild and domestic ruminants. Infection with EHD virus (EHDV) causes fatal disease, especially in white-tailed deer (Odocoileus virginianus), bighorn sheep (Ovis canadensis), mule deer (Odocoileus hemionus), pronghorn (Antilocapra americana), and possibly black-tailed deer (Odocoileus hemionus columbianus). In domestic ruminants, EHDV may induce clinical and pathologic signs similar to those of bluetongue (BT). However, in North America, natural infections caused by EHDV serotype 1 and serotype 2 have rarely been associated with overt clinical disease in cattle. Laboratory diagnosis is most often based on the detection of anti-EHDV antibodies in serum. Among several serologic assays, agar gel immunodiffusion (AGID) is most widely used but is highly subjective, consumes large quantity of reference reagents, and may be complicated by cross-reactions between EHDV serotypes and other orbiviruses, such as BT viruses (BTVs). As an alternative, enzyme-linked immunosorbent assays (ELISAs), both blocking (bELISA) and competitive (cELISA), have been developed in an attempt to overcome the above problems and to serve as large-scale screening tests with objective interpretation. Recently, a cELISA was developed that utilizes an EHDV group-specific monoclonal antibody (MAb) designated C.31, which is group specific and unreactive with antibodies against BTV. After some modifications, the performance of the this cELISA was compared with that of the standard AGID test for detection of anti-EHDV antibodies using bovine field sera collected in Canada (EHD free) and parts of the USA, where EHD is endemic, and deer sera collected in Georgia (USA). Antigen was prepared from baby hamster kidney cells in serum-free medium and infected with plaque-purified EHDV serotype 1, according to a previously described procedure. Sera from serial or paired blood samples from calves experimentally infected and/or challenged with EHDV serotypes 1 and 2, from a calf experimentally infected with BTV serotype 10, and from several calves exposed to South African BTV serotypes 1-16 and 18-20 and the US BTV serotypes 10, 11, 13, and 17 were used to study seroconversion values. 1-5 To establish negative value, 2 panels totalling 1,194

a2 that react with the major core protein VP7 5,7 to the reference Mab 3-17-A3 b in the cELISA. 3 Three hybridomas, 7D3A.2, 8A3B.6, and 8B1B.1, a2 were prepared against BTV17, and their Mab’s were found to be reactive with BT serotypes 1-20. The Mab’s bound to VP7 (designated VP9) 2 and did not react with epizootic hemorrhagic disease (EHD) virus. The BTV group antigen 5 was prepared using baby hamster kidney (BHK-21) cell cultures infected with BTV1 and used to coat an Immulon l c plate. Dilutions of the Mab’s were reacted on the coated plate, the plates were washed, and antimouse IgG conjugated to horseradish peroxidase d was added. After incubation, the plates were washed and the substrate and chromogen, hydrogen peroxide and 2-2’azino-di(3-ethyl benzthiazaline-6 sulfonic acid) e in a citrate buffer, were added. After 15 minutes, the optical densities (OD’s) at 405 nm were recorded. The dilution of Mab fluid to achieve an OD of 0.8-1.0 was determined. The test sera, diluted 1:2, 1:4, and 15, were used in the cELISA Mab system, adding them immediately before the Mab. Positive control antiserum from the bluetongue agar gel immunodiffusion (BT-AGID) test was further diluted 2-fold from 1:10 to 1:160. Results were reported as negative when the sample at a final dilution of 1:4 did not decrease the OD of the Mab by 60% or more. Titers were expressed as the reciprocal of the highest serum dilution giving a positive result. The BT-AGID test was conducted in accordance with the package insert in the licensed test kit. Results were reported as positive (+), weak positive (W+), negative (N), or question (?) for samples of undetermined status. The microtiter virus neutralization (VN) test

Bluetongue viruses (BTV) and epizootic hemorrhagic disease viruses (EHDV) are closely related orbivirus serogroups that infect both wild and domestic ruminants; both virus groups are vector-borne by Culicoides spp. 5 Although it is generally thought that these viruses cause disease in artiodactyls only, abortion and death due to BTV serotype 11 infection was recently reported in domestic dogs following vaccination with a contaminated modified live combination canine vaccine. 1,2 Dogs also are susceptible to African horsesickness viruses (AHSV), 3,9 another serogroup of orbiviruses. Although a high prevalence of antibodies to EHDV and BTV occurs in white-tailed deer (WTD, Odocoileus virginianus) in the southeastern United States, especially in the coastal plain, 6 little information is available regarding natural exposure of nonartiodacty ls such as dogs. In such an area, dogs might be naturally exposed to these viruses, either by vector transmission or, as in the case of AHSV, 9 through consumption of infected meat. The purpose of this study was to determine prevalence of antibodies to BTV and EHDV in dogs from the coastal plain of Georgia. Dog serum samples used in this study were from a larger randomized sample that was collected at veterinary clinics throughout Georgia from April 1992 to March 1993. Randomization was accomplished by requesting the participating veterinarians to obtain a blood sample from the first 4 dogs that entered the clinic on alternate Mondays. All samples selected for this study were from dogs from the coastal plain of Georgia; efforts were made to select dogs that were primarily outside dwellers. The following information was recorded for most of the dogs sampled: county of residence, age, sex, breed, and number of hours inside and outside. All samples were stored at -80 C until testing. Serum samples were tested for precipitating antibodies to BTV and/or EHDV serogroups using EHDV and BTV agar gel immunodiffusion (AGID) test kits a