A Duplex Fluorescent Microsphere Immunoassay for Detection of Bluetongue and Epizootic Hemorrhagic Disease Virus Antibodies in Cattle Sera.

Bluetongue virus (BTV) causes internationally reportable hemorrhagic disease in cattle, sheep, and white-tailed deer. The closely related, and often co-circulating, epizootic hemorrhagic disease virus causes a clinically similar devastating disease in white-tailed deer, with increasing levels of disease in cattle in the past 10 years. Transmitted by Culicoides biting midges, together, they constitute constant disease threats to the livelihood of livestock owners. In cattle, serious economic impacts result from decreased animal production, but most significantly from trade regulations. For effective disease surveillance and accurate trade regulation implementation, rapid, sensitive assays that can detect exposure of cattle to BTV and/or EHDV are needed. We describe the development and validation of a duplex fluorescent microsphere immunoassay (FMIA) to simultaneously detect and differentiate antibodies to BTV and EHDV in a single bovine serum sample. Performance of the duplex FMIA for detection and differentiation of BTV and EHDV serogroup antibodies was comparable, with higher sensitivity than commercially available single-plex competitive enzyme-linked immunosorbent assays (cELISA) for detection of each virus antibody separately. The FMIA adds to the currently available diagnostic tools for hemorrhagic orbiviral diseases in cattle as a sensitive, specific assay, with the benefits of serogroup differentiation in a single serum sample, and multiplexing flexibility in a high-throughput platform.

Sero-surveillance of emerging viral diseases in camels and cattle in Nouakchott, Mauritania: an abattoir study.

This study reports the monitoring of several emerging viral pathogens in Mauritania, which was carried out by the analysis of bovine and camel samples taken at the slaughterhouse of Nouakchott. Blood and serum were collected by random sampling from 159 camels and 118 cattle in March 2013 at the large animals abattoir in Nouakchott. Serological tests for Rift Valley Fever (RVF), Peste des Petits Ruminants (PPR), West Nile disease (WND), epizootic haemorrhagic disease (EHD) and African horse sickness (AHS) were carried out using commercial ELISA kits. The samples, which resulted positives for PPR, WND and AHS, were tested with the confirmatory virus neutralization test (VNT). According to ELISA results, serological prevalence of RVF was 45% (95% CI 52.3-37.7) in camels and 16% (95% CI 22.6-9.4) in cattle. The difference between the observed prevalences in camels and in cattle was significant (p value ≤ 0.01). PPR was absent in camels and had 12% prevalence (95% CI, 17.86-6.14) in cattle. Furthermore, camels showed 92% (95% CI, 96.1-87.9) prevalence of WNV, 73% (95% CI, 82.3-63.64) of EHD and 3% (95% CI, 5.6-0.4) of AHS. This data are of relevance since provided useful feedbacks on the circulation of the pathogens in field. Moreover, this survey provided new information on the susceptibility of camels to several emerging pathogens and on the possible use of this species as sentinel animal.

Evaluation of a commercial ELISA for detection of epizootic haemorrhagic disease antibodies in domestic and wild ruminant sera.

Bluetongue (BT) and epizootic haemorrhagic disease (EHD) are vector-borne viral diseases affecting domestic and wild ruminants. Both are notifiable under OIE rules. BT and EHD viruses (BTV and EHDV) are closely related Orbiviruses with structural, antigenic and molecular similarities. Both viruses can produce analogous clinical signs in susceptible animals. Serological tests are commonly used for BT and EHD diagnosis and surveillance. Competitive ELISA (c-ELISA) is the most widely used serological test for the specific detection of BTV or EHDV viral protein 7 (VP7) antibodies (Abs). The specificity and sensitivity of the BTV c-ELISA kits available on the market are recognized for the detection of BTV Abs. Concerning EHD, a single commercial EHDV c-ELISA kit (ELISA A kit) commonly used for diagnosis in Europe and Africa was available between 2011 and 2018 but is now no longer on the market. In this study, we evaluated a new commercial c-ELISA to detect ruminant EHDV VP7 Abs in 2,199 serum samples from cattle, sheep, goats, wild deer and zoo animals. The results showed that this ELISA kit is specific and can detect the presence of IgG anti-EHDV VP7 with a very good diagnostic specificity and a satisfactory sensitivity in domestic ruminants, zoo animals and wild deer. Therefore, the evaluated c-ELISA can detect the introduction of EHDV into an area where BTV-seropositive domestic animals are present. The performance of this kit is similar to that of the c-ELISA A kit and can thus be used for diagnosis.

Prospective Study of Epizootic Hemorrhagic Disease Virus and Bluetongue Virus Transmission in Captive Ruminants.

Bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) cause hemorrhagic disease (HD) in wild ruminants and bluetongue disease (BT) and epizootic hemorrhagic disease (EHD) in livestock. These viruses are transmitted by biting midges in the genus Culicoides (family Ceratopogonidae). Mortality from this disease can reach 90% in certain breeds of sheep and in white-tailed deer (Odocoileus virginianus). From January until December of 2012, we conducted a prospective study to determine the origin and routes of transmission of BTV and EHDV in captive deer and cattle. The objective was to determine the abundance of Culicoides spp. and BTV/EHDV infection prevalence in midges, cattle, and deer in an area experiencing an outbreak of BT and EHD. Agar gel immunodiffusion (AGID) tests to detect for EHDV and BTV antibodies were conducted on serum collected from cattle and deer, quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) was utilized for BTV/EHDV RNA detection in tissues from dead deer, and CDC miniature black light traps baited with dry ice were deployed to capture insects. The AGID results showed 19 out of 29 cattle and 18 out of 58 white-tailed deer seroconverted for these viruses during the vector season. Tradition gel-based reverse transcriptase polymerase chain reaction was utilized to determine serotype. Sixteen cows were positive for EHDV-2, EHDV-6, or BTV-12 and 15 deer positive for EHDV-1, EHDV-6, or BTV-12. Specimens from 14 species of Culicoides (Dptera: Ceratopogonidae) (Culicoides arboricola Root and Hoffman, Culicoides biguttatus Coquillett, Culicoides crepuscularis Malloch, Culicoides debilipalpis Lutz, Culicoides furens Poey, Culicoides haematopotus Malloch, Culicoides hinmani Khalaf, Culicoides nanus Root and Hoffman, Culicoides neopulicaris Wirth, Culicoides paraensis Goeldi, Culicoides stellifer Coquillet, Culicoides variipennis Coquillet, Culicoides villosipennis Root and Hoffman, and Culicoides venustus Hoffman) were captured and tested for BTV and EHDV using RT-qPCR assays. BTV viral nucleic acid was detected in three pools from three different species of midges: C. crepuscularis, C. debilipalpis, and C. stellifer.

Production of monoclonal antibodies binding to the VP7 protein of African horse sickness virus.

Monoclonal antibodies (MAbs) against AHSV were produced by immunising BALB/c mice with AHSV serotype 9 and six clones able to recognize specifically the VP7-AHSV with a strong reactivity were selected. The specificity of the MAbs was assessed in i-ELISA against a commercial VP7-AHSV and in immunoblot against a home-made VP7-AHSV, expressed by a Baculovirus expression system; potential cross-reactions with related orbiviruses (Bluetongue virus and Epizootic Haemorrhagic Disease virus) were investigated as well. One of the six MAbs selected, MAb 7F11E14, was tested in direct immunofluorescence and reacted with all nine AHSV serotypes, but didn't cross-react with BTV and EHDV. MAb 7F11E14 was also used to develop a competitive ELISA and was able to detect AHSV antibodies in the sera of AHS infected animals.

Inter-serotype reassortment among epizootic haemorrhagic disease viruses in the United States.

First described in 1955 in New Jersey, epizootic haemorrhagic disease (EHD) causes a severe clinical disease in wild and domestic ruminants worldwide. Epizootic haemorrhagic disease outbreaks occur in deer populations each year from summer to late autumn. The etiological agent is EHD virus (EHDV) which is a double-stranded segmented icosahedral RNA virus. EHD virus utilizes point mutations and reassortment strategies to maintain viral fitness during infection. In 2018, EHDV serotype 2 was predominantly detected in deer in Illinois. Whole genome sequencing was conducted for two 2018 EHDV2 isolates (IL41747 and IL42218) and the sequence analyses indicated that IL42218 was a reassortant between different serotypes whereas IL41747 was a genetically stable strain. Our data suggest that multiple strains contribute to outbreaks each year.

Novel serotype of bluetongue virus in South America and first report of epizootic haemorrhagic disease virus in Ecuador.

Bluetongue virus (BTV) and Epizootic haemorrhagic disease virus (EHDV) are closely related Orbiviruses that affect domestic and wild ruminants. In Ecuador previous serological studies reported the presence of BTV; however, no data are available about the presence of EHDV. In this study, 295 cattle without symptoms of infection were sampled from two farms located in Andean and Amazonian regions and from a slaughterhouse in the coastal region. ELISA analyses showed high prevalence of BTV (98.9%) and EHDV (81.3%) antibodies, and RT-qPCRs revealed the presence of EHDV (24.1%) and BTV (10.2%) genomes in cattle blood samples. Viral isolation allowed to identify EHDV serotype 1 (EHDV1) and BTV serotypes 9 (BTV9), 13 and 18. These findings suggest that BTV and EHDV are enzootic diseases in Ecuador.

A serosurvey of bluetongue and epizootic haemorrhagic disease in a convenience sample of sheep and cattle herds in Zimbabwe.

A convenience sample of sheep and cattle herds around the cities of Harare, Kwekwe and Bulawayo, located in the Highveld region of Zimbabwe, was used to estimate the seroprevalence and sero-incidence of bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) antibodies. A competitive enzyme-linked immunosorbent assay was used to identify serum antibodies against BTV and EHDV across three rainy seasons. The median sero-prevalence of BTV and EHDV antibodies in cattle was 62% (interquartile range [IQR]: 30-89) and 56% (IQR: 5-77), respectively. In sheep, the median sero-prevalence of BTV and EHDV was 41% (IQR: 19-63) and 0% (IQR: 0-21), respectively. Median sero-incidences of BTV and EHDV antibodies in cattle of 43% (IQR: 22-67) and 27% (IQR: 9-57) respectively were recorded. The median sero-incidence of BTV in sheep was 14% (IQR: 6-23). Based on these preliminary findings, animal health workers in Zimbabwe should continue to monitor the exposure rates of cattle and sheep to BTV and consider the possibility of strains emerging with increased pathogenicity. There are no previous published reports of antibodies against EHDV in Zimbabwe so the possibility of epizootic haemorrhagic disease existing in domestic livestock should now be considered by Zimbabwean animal health officials. Seroconversions to BTV and EHDV occurred predominantly at the end of each rainy season (March and April), which generally corresponds to high numbers of the Culicoides vectors. BTV isolations were made from three individual cows in two of the sentinel herds and all three were identified as serotype 3. This is the first time BTV serotype 3 has been recorded in Zimbabwe, although its presence in neighbouring South Africa is well documented.

Competitive enzyme-linked immunosorbent assay using baculovirus-expressed VP7 for detection of epizootic haemorrhagic disease virus (EHDV) antibodies.

Epizootic haemorrhagic disease (EHD) is a vector-borne infectious viral disease of domestic and wild ruminants. EHD could spread from infected northern African countries in free territories like the EU; therefore, the availability of diagnostic assays would represent key components for adequate surveillance and control programs. In this study, the gene encoding the VP7 protein of EHD virus (EHDV) was expressed into a baculovirus-infected insect cell system. With this unpurified protein we developed a home-made competitive ELISA (cELISA) and a total number of 275 serum samples, originating from domestic and wild ruminants, were tested. 74/275 were previously shown to be positive for EHDV antibodies by a commercially available ELISA kit. A "very good" agreement was demonstrated when compared to a commercial ELISA kit (Cohen's kappa value=0.832). Samples which caused disagreement between the two assays originated from wildlife which highlights the need for further validation by using serum samples from wild animals.

Characterization of genome segments 2, 3 and 6 of epizootic hemorrhagic disease virus strains isolated in Japan in 1985-2013: Identification of their serotypes and geographical genetic types.

We characterized genome segments 2, 3 and 6 (Seg-2, Seg-3 and Seg-6) of 11 Japanese strains of epizootic hemorrhagic disease (EHD) virus (EHDV) isolated in 1985-2013. The Japanese strains were divisible into two groups based on phylogenetic analyses of the nucleotide sequences of Seg-2 and Seg-6. In both of the phylogenetic trees based on Seg-2 and Seg-6, seven of the 11 Japanese strains were grouped together with EHDV-2 and EHDV-7 strains, and the other four Japanese strains were grouped with EHDV-1 strains. The phylogenetic analysis of Seg-2 among EHDV strains identified 10 of the 11 Japanese strains as EHDV-1, EHDV-2 or EHDV-7. The other Japanese strain, ON-4/B/98, isolated from an asymptomatic cow in 1998 was in the same group as the EHDV-2 and EHDV-7 strains in the phylogenetic trees based on Seg-2 and Seg-6, but the results suggested that the strain belongs to another serotype. We thus conducted a serum neutralization test to identify that serotype by using anti-EHDV-2 and anti-EHDV-7 rabbit sera. We observed that the ON-4/B/98 strain was not sufficiently neutralized by any of the antisera, which suggests that the strain could be assigned into a new serotype, tentatively named 'EHDV-10.' Sequences of Seg-3 were also determined, and all of the Japanese strains were grouped together with Australian strains, suggesting that the Japanese strains are a part of EHDV distributed in the Asia-Pacific region. The data obtained herein would be beneficial for the diagnosis and prevention of EHD in Japan and neighboring countries.

Generation of virus like particles for epizootic hemorrhagic disease virus.

Epizootic hemorrhagic disease virus (EHDV) is a distinct species within the genus Orbivirus, within the family Reoviridae. The epizootic hemorrhagic disease virus genome comprises ten segments of linear, double stranded (ds) RNA, which are packaged within each virus particle. The EHDV virion has a three layered capsid-structure, generated by four major viral proteins: VP2 and VP5 (outer capsid layer); VP7 (intermediate, core-surface layer) and VP3 (innermost, sub-core layer). Although EHDV infects cattle sporadically, several outbreaks have recently occurred in this species in five Mediterranean countries, indicating a potential threat to the European cattle industry. EHDV is transmitted by biting midges of the genus Culicoides, which can travel long distances through wind-born movements (particularly over water), increasing the potential for viral spread in new areas/countries. Expression systems to generate self-assembled virus like particles (VLPs) by simultaneous expression of the major capsid-proteins, have been established for several viruses (including bluetongue virus). This study has developed expression systems for production of EHDV VLPs, for use as non-infectious antigens in both vaccinology and serology studies, avoiding the risk of genetic reassortment between vaccine and field strains and facilitating large scale antigen production. Genes encoding the four major-capsid proteins of a field strain of EHDV-6, were isolated and cloned into transfer vectors, to generate two recombinant baculoviruses. The expression of these viral genes was assessed in insect cells by monitoring the presence of specific viral mRNAs and by western blotting. Electron microscopy studies confirmed the formation and purification of assembled VLPs.

Generation of virus-like particles for emerging epizootic haemorrhagic disease virus: Towards the development of safe vaccine candidates.

Epizootic haemorrhagic disease virus (EHDV) is an insect-transmitted pathogen which causes high mortality in deer populations and may also cause high morbidity in cattle. EHDV belongs to the Orbivirus genus and is closely related to the prototype Bluetongue virus (BTV). To date seven distinct serotypes have been recognized. However, a live-attenuated vaccine is commercially available against only one serotype namely EHDV-2, which has been responsible for multiple outbreaks in North America, Canada, Asia and Australia. Here we expressed four major capsid proteins (VP2, VP3, VP5 and VP7) of EHDV-1 using baculovirus multiple gene expression systems and demonstrated that three-layered VLPs were assembled mimicking the authentic EHDV particles but lacking the viral genomic RNA segments and the transcriptase complex (TC). Antibodies generated with VLPs not only neutralized EHDV-1 infection in cell culture but also showed cross neutralizing reactivity against two other serotypes, EHDV-2 and EHDV-6. For proof of concept, we demonstrated that EHDV-2 VLPs could be generated rapidly by expressing the EHDV-2 variable outer capsid proteins (VP2, VP5) together with EHDV-1 VP3 and VP7, the two inner capsid proteins, which are highly conserved among the 7 serotypes. Data presented in this study validate the VLPs as a potential vaccine and demonstrate that a vaccine could be developed rapidly in the event of an outbreak of a new serotype.

Host and Potential Vector Susceptibility to an Emerging Orbivirus in the United States: Epizootic Hemorrhagic Disease Virus Serotype 6.

Epizootic hemorrhagic disease viruses (EHDVs) are orbiviruses transmitted by Culicoides biting midges to domestic and wild ruminants. EHDV-1 and EHDV-2 are endemic in the United States, where epizootic hemorrhagic disease is the most significant viral disease of white-tailed deer (WTD;Odocoileus virginianus) and reports of epizootic hemorrhagic disease in cattle are increasing. In 2006, a reassortant EHDV-6 was isolated from dead WTD in Indiana and has been detected each subsequent year over a wide geographic region. Since EHDV-6 is not a historically endemic serotype in the United States, it is important to understand infection outcome in potential hosts. Specifically, we aimed to evaluate the pathogenicity of the virus in 2 primary US ruminant hosts (WTD and cattle) and the susceptibility of a confirmed US vector (Culicoides sonorensis). Five WTD and 4 cattle were inoculated with >10(6)TCID50EHDV-6 by intradermal and subcutaneous injection. All 5 WTD exhibited moderate to severe disease, and 3 died. Viremia was first detected 3 to 5 days postinfection (dpi) with surviving animals seroconverting by 10 dpi. Two of 4 inoculated cattle had detectable viremia, 5 to 10 dpi and 7 to 24 dpi, respectively. No clinical, hematologic, or pathologic abnormalities were observed. Antibodies were detected by 10 dpi in 3 of 4 cows.C. sonorensis were fed on WTD blood spiked with EHDV-6 and held for 4 to 14 days postfeeding at 25°C. From 4 to 14 days postfeeding, 19 of 171 midges were virus isolation positive and 6 of 171 had ≥10(2.7)TCID50EHDV-6. Although outcomes varied, these studies demonstrate the susceptibility of ruminant and vector hosts in the United States for this recently emerged EHDV serotype.

Inter-laboratory evaluation of the performance parameters of a Lateral Flow Test device for the detection of Bluetongue virus-specific antibodies.

Bluetongue (BT) is a viral vector-borne disease affecting domestic and wild ruminants worldwide. In this study, a commercial rapid immuno-chromatographic method or Lateral Flow Test (LFT) device, for the detection of BT virus-specific antibodies in animal serum, was evaluated in an international inter-laboratory proficiency test. The evaluation was done with sera samples of variable background (ruminant species, serotype, field samples, experimental infections, vaccinated animals). The diagnostic sensitivity was 100% (95% C.I. [90.5-100]) and the diagnostic specificity was 95.2% (95% C.I. [76.2-99.9]). The repeatability (accordance) and reproducibility (concordance) were 100% for seropositive samples but were lower for two of the seronegative samples (45% and 89% respectively). The analytical sensitivity, evaluated by testing positive sera at increasing dilutions was better for the BT LFT compared to some commercial ELISAs. Seroconversion of an infected sheep was detected at 4 days post infection. Analytical specificity was impaired by cross-reactions observed with some of the samples seropositive for Epizootic Haemorrhagic Disease Virus (EHDV). The agreement (Cohen's kappa) between the LFT and a commercial BT competitive ELISA was 0.79 (95% CI [0.62-0.95]). Based on these results, it can be concluded that the BT LFT device is a rapid and sensitive first-line serological test that can be used in the field, especially in areas endemic for the disease where there is a lack of diagnostic facilities.

Orbiviruses: A North American Perspective.

Orbiviruses are members of the Reoviridae family and include bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV). These viruses are the cause of significant regional disease outbreaks among livestock and wildlife in the United States, some of which have been characterized by significant morbidity and mortality. Competent vectors are clearly present in most regions of the globe; therefore, all segments of production livestock are at risk for serious disease outbreaks. Animals with subclinical infections also serve as reservoirs of infection and often result in significant trade restrictions. The economic and explicit impacts of BTV and EHDV infections are difficult to measure, but infections are a cause of economic loss for producers and loss of natural resources (wildlife). In response to United States Animal Health Association (USAHA) Resolution 16, the US Department of Agriculture (USDA), in collaboration with the Department of the Interior (DOI), organized a gap analysis workshop composed of international experts on Orbiviruses. The workshop participants met at the Arthropod-Borne Animal Diseases Research Unit in Manhattan, KS, May 14-16, 2013, to assess the available scientific information and status of currently available countermeasures to effectively control and mitigate the impact of an outbreak of an emerging Orbivirus with epizootic potential, with special emphasis given to BTV and EHDV. In assessing the threats, workshop participants determined that available countermeasures are somewhat effective, but several weaknesses were identified that affect their ability to prevent and control disease outbreaks effectively.

A Review of Knowledge Gaps and Tools for Orbivirus Research.

Although recognized as causing emerging and re-emerging disease outbreaks worldwide since the late 1800 s, there has been growing interest in the United States and Europe in recent years in orbiviruses, their insect vectors, and the diseases they cause in domestic livestock and wildlife. This is due, in part, to the emergence of bluetongue (BT) in northern Europe in 2006-2007 resulting in a devastating outbreak, as well as severe BT outbreaks in sheep and epizootic hemorrhagic disease (EHD) outbreaks in deer and cattle in the United States. Of notable concern is the isolation of as many as 10 new BT virus (BTV) serotypes in the United States since 1999 and their associated unknowns, such as route of introduction, virulence to mammals, and indigenous competent vectors. This review, based on a gap analysis workshop composed of international experts on orbiviruses conducted in 2013, gives a global perspective of current basic virological understanding of orbiviruses, with particular attention to BTV and the closely related epizootic hemorrhagic disease virus (EHDV), and identifies a multitude of basic virology research gaps, critical for predicting and preventing outbreaks.

Diagnostic Tools for Bluetongue and Epizootic Hemorrhagic Disease Viruses Applicable to North American Veterinary Diagnosticians.

This review provides an overview of current and potential new diagnostic tests for bluetongue (BT) and epizootic hemorrhagic disease (EHD) viruses compiled from international participants of the Orbivirus Gap Analysis Workshop, Diagnostic Group. The emphasis of this review is on diagnostic tools available to North American veterinary diagnosticians. Standard diagnostic tests are readily available for BT/EHD viruses, and there are described tests that are published in the World Organization for Animal Health (OIE) Terrestrial Manual. There is however considerable variation in the diagnostic approach to these viruses. Serological assays are well established, and many laboratories are experienced in running these assays. Numerous nucleic acid amplification assays are also available for BT virus (BTV) and EHD virus (EHDV). Although there is considerable experience with BTV reverse-transcriptase PCR (RT-PCR), there are no standards or comparisons of the protocols used by various state and federal veterinary diagnostic laboratories. Methods for genotyping BTV and EHDV isolates are available and are valuable tools for monitoring and analyzing circulating viruses. These methods include RT-PCR panels or arrays, RT-PCR and sequencing of specific genome segments, or the use of next-generation sequencing. In addition to enabling virus characterization, use of advanced molecular detection methods, including DNA microarrays and next-generation sequencing, significantly enhance the ability to detect unique virus strains that may arise through genetic drift, recombination, or viral genome segment reassortment, as well as incursions of new virus strains from other geographical areas.

Vaccines for Prevention of Bluetongue and Epizootic Hemorrhagic Disease in Livestock: A North American Perspective.

Bluetongue (BT) and epizootic hemorrhagic disease (EHD) are noncontagious, insect-transmitted diseases of domestic and wild ruminants caused by related but distinct viruses. There are significant gaps in our scientific knowledge and available countermeasures to control an outbreak of orbivirus-induced disease, whether BT or EHD. Both BT virus (BTV) and EHD virus (EHDV) cause hemorrhagic fevers in susceptible ruminants; however, BT is principally a disease of domestic livestock whereas EHD is principally a disease of certain species of wild, non-African ungulates, notably white-tailed deer. The live-attenuated (modified live virus [MLV]) vaccines available in the United States for use in small ruminant livestock do provide good protection against clinical disease following infection with the homologous virus serotype. Although there is increasing justification that the use of MLV vaccines should be avoided if possible, these are the only vaccines currently available in the United States. Specifically, MLVs are used in California to protect sheep against infection with BTV serotypes 10, 11, and 17, and a MLV to BTV serotype 10 is licensed for use in sheep throughout the United States. These MLV vaccines may need to continue to be used in the immediate future for protective immunization of sheep and goats against BT. There are currently no licensed vaccines available for EHD in the United States other than autogenous vaccines. If there is a need to rapidly develop a vaccine to meet an emerging crisis associated with either BTV or EHDV infections, development of an inactivated virus vaccine in a conventional adjuvanted formulation will likely be required. With two doses of vaccine (and in some instances just one dose), inactivated vaccines can provide substantial immunity to the epizootic serotype of either BTV or EHDV. This strategy is similar to that used in the 2006-2008 BTV serotype 8 outbreaks in northern Europe that provided vaccine to the field within 2 years of the initial incursion (by 2008). Further research and development are warranted to provide more efficacious and effective vaccines for control of BTV and EHDV infections.

Epizootic haemorrhagic disease virus in Reunion Island: evidence for the circulation of a new serotype and associated risk factors.

Bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) are members of the Orbivirus genus of the Reoviridae family transmitted between ruminants by the bites of Culicoides midges. BTV went undetected in Reunion Island between its first documented emergence in 1979 and two other serious outbreaks with both BTV-3 and EHDV-6 in 2003, and both EHDV-6 and BTV-2 in 2009. In these outbreaks, infected animals developed symptoms including hyperthermia, anorexia, congestion, prostration and nasal discharge. Samples were collected in 2011 to assess the prevalence of BT and EHD in ruminants native to Reunion Island by serological analysis. A cross-sectional study was undertaken on 67 farms, including a total of 276 cattle, 142 sheep and 71 goats. The prevalence rates of BT and EHD were 58% (95% CI [54.03-62.94]) and 38% (95% CI [33.85-42.63], respectively. Two further suspected outbreaks were confirmed to involve EHDV and BTV/EHDV. A new circulating EHDV serotype 1 of unknown origin was isolated. Our results confirm that the prevalence of both BT and EHD is high and that both are likely currently circulating. A high risk of BTV and EHDV infections was associated with the introduction of ruminants from neighbouring farms without quarantine, the presence of organic and other waste on the farm, and treatment against ectoparasites and insects.