Epizootic Haemorrhagic Disease Of Deer Research Articles

Dwarf bat's (Pipistrellus pipistrellus) lung diploid cell strains and their permissivity to orbiviruses (Reoviridae: Orbivirus) - pathogens of vector-borne animal diseases

Bat cell cultures are a popular model both for the isolation of vector-borne disease viruses and for assessing the possible role of these mammalian species in forming the natural reservoirs of arbovirus infection vectors. The goal of the research was to obtain and characterize strains of diploid lung cells of the bat (Pipistrellus pipistrellus) and evaluate their permissivity to bluetongue, African horse sickness (AHS), and epizootic hemorrhagic disease of deer (EHD) viruses. Cell cultures of the dwarf bat's lung were obtained by standard enzymatic disaggregation of donor tissue and selection of cells for adhesive properties. The permissivity of cell cultures was determined to bluetongue, AHL, and EHD orbiviruses. Diploid cell strains (epithelium-like and fibroblast-like types) retaining cytomorphological characteristics and karyotype stability were obtained from tissue of the bat's lung. Their permissivity to viruses of the genus Orbivirus of the Reoviridae family, pathogens of transmissible animal diseases, has been established. The permissivity of the obtained strains of bat's lung cells to bluetongue, AHL, and EHD viruses is consistent with the isolation of orbiviruses in bats of the species Pteropus poliocephalus, Pteropus hypomelanus, Rousettus aegyptiacus leachii, Syconycteris crassa, Myotis macrodactylus, and Eidolon helvum. Strains of diploid lung cells of the dwarf bat are permissive to orbiviruses of bluetongue, AHS, and EHD, which allows us to recommend them for the isolation of these viruses, and the species Pipistrellus pipistrellus to be considered as a potential natural reservoir and carrier of pathogens of these vector-borne diseases.

Diagnosing bluetongue virus in domestic ruminants: current perspectives.

This review provides an overview of current and potential new diagnostic techniques against bluetongue virus (BTV), an Orbivirus transmitted by arthropods that affects ruminants. Bluetongue is a disease currently notifiable to the World Organization for Animal Health (OIE), causing great economic losses due to decreased trade associated with bluetongue outbreaks and high mortality and morbidity. BTV cross-reacts with many antigenically related viruses including viruses that causes African Horse sickness and epizootic haemorrhagic disease of deer. Therefore, reliable diagnostic approaches to detect BTV among these other antigenically related viruses are used or being developed. The antigenic determinant for differentiation of virus species/serogroups among orbiviruses is the VP7 protein, meanwhile VP2 is serotype specific. Serologically, assays are established in many laboratories, based mainly on competitive ELISA or serum neutralization assay (virus neutralization assay [VNT]) although new techniques are being developed. Virus isolation from blood or semen is, additionally, another means of BTV diagnosis. Nevertheless, most of these techniques for viral isolation are time-consuming and expensive. Currently, reverse-transcription polymerase chain reaction (RT-PCR) panels or real-time RT-PCR are widely used methods although next-generation sequencing remains of interest for future virus diagnosis.

Establishment of Real-Time Fluorescent PCR Method for Detection of D Epizootic Hemorrhagic Disease of Deer

Epizootie hemorrhagic disease of deer virus (EHDV), is a member of the family Reoviridae virus (Orbi virus), and a double-stranded RNA virus from arthropods (mainly Culicoides), which is very similar in morphology and structure to Blue tongue virus (BTV). Serologically, there is a clear cross between the blue tongue virus and EHDV. EHD (caused by EHDV) is a seasonal, non-contact viral infectious disease. Under natural conditions, EHDV can cause infection of cattle, sheep and other domestic animals as well as many domesticated and wild ruminants such as white tail deer, Mi, large horned antelope and so on. The virus has been isolated from white-tailed deer, black-tailed deer, sheep, goats, cattle, bison and Culicoides. White tail deer infection is the most serious, and clinical characteristics are transient elevated body temperature, symptoms of shock, mucosal, serous bleeding and death in a coma [1]. At present, there is no effective vaccine and treatment for this disease. To control and eliminate EHD, it is necessary to spend a lot of manpower and material resources. Export trade is restricted, which brings great economic losses to the countries with EHD epidemic. Moreover, because of the complexity of antigen structure and the diversity of clinical symptoms of EHDV, it is difficult to diagnose in laboratory. Therefore, developing a quick and precise laboratory diagnostic method becomes more and more significant among researchers.

Bluetongue in white-tailed deer (Odocoileus virginianus) in Northeastern Mexico

Abstract Bluetongue (BT) and epizootic hemorrhagic disease of deer (EHD) are two distinct viral hemorrhagic diseases of domestic and wild ruminants caused by members of the family Reoviridae and transmitted by Culicoides midges. These conditions have been recognized in Canada and the United States for many years, but not in Mexico. Although in Mexico there is serologic evidence of EHD virus (EHDV) and bluetongue virus (BTV) in domestic and wild ruminants, to our knowledge, there have never been reports of clinical illness or fatalities attributed to either of these viruses. Two free ranging white-tailed deer (Odocoileus virginianus) in two licensed hunting ranches in Northern Mexico near the Texas border died unexpectedly. Postmortem and microscopic examinations revealed hemorrhagic lesions compatible with viral hemorrhagic disease (Reoviridae: Orbivirus). Tissues from one animal tested positive by RT-PCR for BTV but negative for EHDV. To our knowledge, this is the first time in Mexico where deer dying with hemorrhagic lesions consistent with Bluetongue tested positive for BTV by PCR. Key words: hemorrhagic disease, white-tailed deer, Orbivirus, Mexico.

Autres orbivirus : Mise à jour des informations sur la peste équine africaine et la maladie hémorragique épizootique en Europe et dans le bassin méditerranéen

Orbiviruses are vector-borne pathogens that can cause notifi­able diseases in animals, such as bluetongue (BT) and epizootic haemorrhagic disease of deer (EHD) in ruminants, or African horse sickness (AHS) in equines. The relatively recent expansion of BT in Europe to higher latitudes than expected has evidenced the need to explore the ways of introduction and exposure of other orbiviruses in Europe and in the Mediterranean Basin. AHS was successfully eradicated from Europe since the 1990s but continues to be endemic in many African countries. Of the nine AHS serotypes, two have been present in Mediterranean coun­tries: AHS-9 (1966) and AHS-4 (1987-1990). The last outbreaks (up to 2008) of AHS in Africa classified by serotype occurred in Senegal (AHS-9), Kenya (AHS-4), and Nigeria, Senegal and Ethiopia (AHS-2). EHD is caused by 10 serotypes and is notifi­able to the World Organisation for Animal Health (OIE) since 2008. It is present in America, Australia, Asia and Africa and is known to affect wild ruminants as well as cattle. EHD has been present in cattle in North Africa (EHD-9) and the Middle East (EHD-7) since 2006. Transport of infected Culicoides from Northern Africa to Southern Europe by wind is a proved way of orbivirus introduction. Import of infected asymptomatic animals from an endemic country also happened the first time AHS was introduced in Spain. Then, certain environmental conditions such as warm temperatures can favour perpetuation of the dis­ease in animals exposed to infected vectors. The frequent con­sideration of horses as expensive leisure animals can worsen the economic and social consequences of a possible outbreak. However, nowadays there are good diagnostic techniques for AHS. Eradication can be achieved with the available polyvalent live vaccines and control measures. This is not the case for EHD, because an effective vaccine is urgently needed and there have been cross-reactions in the diagnoses between BT and EHD. European countries can prepare against other orbivirus outbreaks by prevention through educational campaigns and inactivated vaccine banks for AHS, and by further research on the possible vectors, the overwintering capacity of certain orbiviruses, the infectivity in all affected species, the identification of other pos­sible reservoirs, and the development of risk assessments and modelling.

Diagnostic des orbivirus dans un pays d'endémicité

Diagnostic des orbivirus dans un pays d'endémicité

Serosurvey for selected infectious disease agents in free-ranging black and white rhinoceros in Africa.

Two hundred and eighty one serum samples collected from free-ranging black (Diceros bicornis) and white (Ceratotherium simum) rhinoceros, in the Republic of South Africa (RSA), Namibia, and Kenya from 1987-97, were examined for antibody to 16 different infectious agents. Positive antibody titers were detected against Akabane (59.8%), bluetongue (55%), African horse sickness (27.9%), epizootic haemorrhagic disease of deer (19.4%), parainfluenza type 3 (25.3%), bovine herpes virus 1 (3.1%), equine herpes virus 1 (8.8%) and bovine viral diarrhea (1.2%) viruses, and four serovars of Leptospira interrogans, (ranging 1.2 to 8.8%). No antibody was detected against Rift Valley fever virus, encephalomyocarditis virus, Brucella abortus, and Trypanosoma equiperdum. Interspecies differences were detected for African horse sickness, epizootic haemorrhagic disease of deer and parainfluenza type 3 viruses. There appeared to be some geographic variation in the prevalence of antibody for African horse sickness, bluetongue, epizootic haemorrhagic disease of deer, parainfluenza type 3, equine herpes virus 1 and Leptospira interrogans serovar bratislava.

Detection and differentiation of epizootic haemorrhagic disease of deer and bluetongue viruses by serogroup-specific sandwich ELISA.

A serogroup specific sandwich ELISA was developed for the detection of epizootic haemorrhagic disease of deer viruses (EHDV) in infected insects and tissue culture preparations. Polyclonal rabbit antiserum against purified EHDV core particles was used to capture viral antigen and specific binding detected using guinea pig antisera against EHDV core particles followed by anti-guinea pig immunoglobulin enzyme-labelled conjugate. The assay is EHDV specific and detects all 8 serotypes. No cross-reactions were found with related viruses such as bluetongue (BTV), Palyam, Tilligery or African horse sickness virus (AHSV). A similar serogroup specific sandwich ELISA was also developed for BTV. The assays showed a similar sensitivity in detecting the respective EHDV or BTV antigens in a pool of 500 midges where only 2 were infected. These assays allow a simple and rapid means of detecting and differentiating members of these closely related serogroups. The sensitivity of the tests will allow more extensive studies on vector competence and virus/vector distribution.

A rapid indirect ELISA for the serogrouping of Australian orbiviruses

This communication describes the development and evaluation of a simple and rapid method for the classification of Australian orbiviruses into one of seven established serogroups (i.e. bluetongue, epizootic haemorrhagic disease of deer, Palyam, Eubenangee, Corriparta, Wallal, Warrego) or an ‘ungrouped’ category. The Australian orbivirus serogrouping ELISA (SG-ELISA) utilised a sodium deoxycholate-treated cell lysate preparation from infected BHK cells which was subsequently probed in an indirect ELISA format with polyclonal antibodies representative of each serogroup. Bound immunoglobulin was detected by the use of a recombinant streptococcal protein G-HRPO conjugate and subsequent reaction with the chromogenic substrate. All reference orbiviruses tested in the SG-ELISA were identified and were in agreement with the serogroups originally designated. Minimal inter-serogroup cross-reactions were observed. One-way cross-reactions were observed between Warrego and Mitchell River viruses.

The smallest gene of the orbivirus, epizootic hemorrhagic disease, is expressed m virus-infected cells as two proteins and the expression differs from that of the cognate gene of bluetongue virus

The smallest gene (S10) of the virus of epizootic hemorrhagic disease of deer (EHD, serotype 2) is expressed as two proteins in virus-infected cells. By contrast, the non-structural proteins (NS3 and NS3A) encoded in the smallest gene of bluetongue (BT) viruses are difficult to detect in virus-infected cells. The nucleotide sequence of S10 of EHDV-2 contains two in-frame initiation codons which allow for translation of proteins of mol. wt. 25503 and 23921 analogous to NS3 and NS3A of BT viruses. The S10 genes of BT viruses are highly conserved (82%–99%); the nucleotide sequence similarity of S10 of EHDV-2 and BT viruses is about 64%. Some structural features of NS3 and NS3A are conserved in the two viruses, despite the divergence in the amino acid sequences of the proteins. The hydrophobic domains of the proteins and the putative transmembrane sequences are conserved, as are potential glycosylation sites in the proteins. A cluster of proline residues, which is conserved at residues 36–50 in all of the published sequences of NS3 of BT viruses, is conserved exactly in the alignment of the sequence of NS3 of EHDV-2 with that of the BT viruses. An explanation for the differences in expression of NS3/NS3A in EHD and BT viruses was not evident in comparing the nucleotide sequences of S10 of the viruses.

Temperature and the persistence of viruses in Culicoides spp. during adverse conditions.

The average daily maximum temperature for the coldest month during the winters of 1977-1978 and 1978-1979, when bluetongue (BT) virus overwintered in a cycle between Culicoides spp. and cattle in western Turkey (Aydin and Izmir provinces), was found to be 12.5 degrees C. This temperature agreed with the lowest temperatures found for Culicoides activity and for flight over long distances. Overwintering of BT virus in western Turkey and Lesbos (1979-1980), African horse sickness (AHS) virus in Spain (1987-1990) and BT and epizootic haemorrhagic disease of deer (EHD) viruses in British Columbia (1987-1988) was assessed using the following conditions for survival: maximum temperatures > or = 13 degrees C on 45% of days or more, a weighted "degree day" value of > or = 1.35 of 6 and an average daily maximum temperature of > or = 12.5 degrees C (all for the coldest month) together with a total of 40 days or fewer with maximum temperatures < 13 degrees C and < or = 10 consecutive days with maximum temperatures < 13 degrees C. Using these criteria, overwintering is unlikely to have occurred in Culicoides in Lesbos, Madrid, Toledo, Ciudad Real or British Columbia, but could have taken place in western Turkey (Aydin and Izmir) and southern Spain (Guadalquivir, Guadiana and Tagus Valleys). Isotherms of average daily maximum temperatures of 12.5 degrees C and 18 degrees C for the coldest month were drawn on maps covering various regions of the world, to indicate areas where virus and Culicoides activity could continue during the winter and to compare these areas with the known distribution of BT, EHD, AHS and Akabane infection.

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.

Molecular comparison of VP3 from bluetongue and epizootic hemorrhagic disease viruses.

The complete nucleic acid sequence of gene 3 from epizootic hemorrhagic disease of deer (EHD) virus serotype 1 was determined. The 2768 bp sequence encodes a single protein that contains 899 amino acids and has a molecular weight of 103 kDa. The predicted protein sequence has 94.7% identity with EHD virus serotype 2 and greater than 77% identity with the related bluetongue viruses serotypes 1, 10 and 17 VP3 proteins. The relevance of these data to studies of recombinant DNA diagnostics and genetic relatedness is discussed.

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. Antibodies 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.

Identifying bluetongue virus ribonucleic acid sequences by the polymerase chain reaction

A primer-directed nucleic acid amplification reaction, commonly known as the polymerase chain reaction (PCR), has been adapted to the identification of bluetongue virus (BTV) RNA. The protocol described in this article is designed for the detection of a purported globally-conserved, serogroup specific nucleic acid sequence within the BTV genome. Due to the double-stranded RNA composition of the BTV genome, the original polymerase chain reaction protocol has been modified to include chemical denaturation and reverse transcription steps that allow selective amplification from this unique template molecule. The amplification procedure yields a 210 base pair product when tested on samples of RNA from the prototypic strains of U.S. BTV serotypes 2, 10, 11, 13, 17. The amplification product is tentatively identified by agarose gel electrophoresis of the PCR samples. Final positive identification of the amplified BTV-specific product is determined by Southern blot hybridization of the PCR samples. RNA samples from uninfected cell culture controls and from cell cultures infected with two serotypes of epizootic hemorrhagic disease of deer (EHDV) yielded negative results. Preliminary experiments to quantify the threshold of sensitivity of this protocol, as described here, indicate positive detection of the BTV target RNA sequence at a level of less than 2 fg, or the amount of target sequence derived from less than 7500 viral particles. While this falls short of the theoretical limit of sensitivity of the PCR, the enhanced threshold of sensitivity of the PCR reaction compared to standard nucleic acid hybridization methodology suggests that rapid detection of BTV RNA directly within clinical specimens may be feasible with further refinements. Potential methods of enhancing this reaction are discussed.

Isolation of Arboviruses From Cattle and Insects at 2 Sentinel Sites in Queensland, Australia, 1979-85

Blood samples were collected regularly from two sentinel herds of cattle in northern and southern Queensland between 1979 and 1985. From 2660 samples, virus isolation attempts using baby hamster kidney (BHK21) and Aedes albopictus (AA) tissue cultures and suckling mice produced 308 viruses of which 243 (79%) were in the Palyam subgroup of orbiviruses. Mosquitoes and biting midges were collected at the southern sentinel herd site in January-February 1984 and processed for virus isolation in BHK2l and AA tissue cultures and by intrathoracic inoculation of Ae. aegypti mosquitoes. Totals of 14 338 midges of four species in 156 pools, and 9030 mosquitoes of 27 species in 232 pools, were processed and yielded 59 isolations. Of the 35 viruses isolated from Culicoides brevitarsis, 17 were members of the Palyam subgroup. Bovine ephemeral fever (BEF) virus was isolated once from Anopheles bancroftii, once from C. brevitarsis and 17 times from cattle. Akabane virus was isolated for the first time from C. wadai, as well as a further 10 times from C. brevitarsis and 20 times from cattle. Other viruses isolated from cattle included bluetongue serotype 1, and serotypes 5, 6 and 7 of epizootic hemorrhagic disease of deer (EHD). A new BEF group virus, tentatively called Oak Vale, was isolated nine times from Culex edwardsi mosquitoes. Of the orbiviruses, those in the Palyam subgroup were isolated almost exclusively in BHK2l tissue cultures but those in the bluetongue and EHD subgroups were isolated almost exclusively in AA cell cultures or after passage through Ae. aegypti. Of 22 rhabdovirus isolations from blood and insects (BEF, Kimberley and Tibrogargan), 16 were made only in AA cell cultures or after passage through Ae. aegypti.

Serosurvey of sahelian cattle for evidence of epizootic disease

Sera from Nigerian cattle (cattle from Niger) vaccinated against rinderpest and contagious bovine pleuropneumonia were examined for antibodies specific for six infectious diseases. The percentage positive was 1.7 for foot-and-mouth disease, 84.1 for bluetongue 78.1 for epizootic hemorrhagic disease of deer, 5.6 for Rift Valley fever and 85.7 for rinderpest. The data indicate that either or both bluetongue and epizootic hemorrhagic disease of deer virus infections are highly prevalent in Niger. The discovery of antibodies that reacted with Rift Valley fever antigens may mean that this disease is present in Niger, but cross-reactions with other phleboviruses cannot be ruled out on the basis of the hemagglutination inhibition test alone. Our inability to detect complement-fixing antibody to Mycoplasma mycoides mycoides agrees with the absence of acute clinical disease reports from Niger, but does not reflect on the immunogenicity of the vaccine. The high prevalence of antibody to rinderpest suggests that the vaccine used was effective.

Evaluation of a monoclonal antibody blocking ELISA for the detection of group-specific antibodies to bluetongue virus in experimental and field sera.

In order to overcome serological cross-reactions among orbivirus serogroups, which can hinder the accurate diagnosis of bluetongue virus (BTV) infection of livestock, a blocking ELISA (B-ELISA) incorporating a monoclonal antibody (20E9B7G2) with specificity for the BTV serogroup was developed. Experimental antisera raised to South African BTV serotypes 1 to 19 were tested in the B-ELISA and all blocked the binding of 20E9B7G2 to BTV antigen. The sensitivity and specificity of the assay was evaluated with a range of experimental and field sera and compared to a sensitive indirect ELISA (I-ELISA) for the detection of BTV-specific antibodies. The specificity of the B-ELISA was absolute for antibodies to BTV, showing no cross-reaction with experimental antisera to serotypes of the closely related orbivirus causing epizootic haemorrhagic disease of deer. The sensitivity of the B-ELISA exceeded that of the I-ELISA. In particular, the B-ELISA detected a BTV-specific antibody response much earlier after infection that the I-ELISA, while still exhibiting full sensitivity to BTV antibody titres several months after infection.

Rapid identification of bluetongue virus by nucleic acid hybridization in solution

A liquid phase hybridization format has been adapted for the identification of bluetongue virus (BTV) with respect to serogroup and serotype. In this paper we present experiments which demonstrate the identification of bluetongue virus with respect to both serogroup and serotype. BTV serogroup-specific and BTV serotype 17-specific single-stranded RNA probes labeled with 35S-CTP were used to identify BTV viral nucleic acids in infected cell culture lysates. Cell lysates were prepared for testing by rapid solubilization in a guanidine isothiocyanate/EDTA/dithiothreitol medium. Hybridizations were done in 12.5 μl reaction volumes for 2 h or overnight using nanogram quantities of probe. An RNAse solution was added subsequent to hybridization and incubated for 30 min. TCA-precipitable counts were collected and quantitated by scintillation counting. Specific hybridization signals were obtained in as little as 3 h in assays consisting of uninfected controls, cells infected with different BTV serotypes, and cells infected with a strain of epizootic hemorrhagic disease of deer (EHDV-1). By using asymmetric single-stranded RNA probes we were able to distinguish viral mRNA and viral double-stranded RNA components. We propose this technique as an ancillary or replacement procedure for the confirmation and classification of viral isolates using an appropriate battery of nucleic acid probes. Potential applications and methods of enhancing the technique are discussed.

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.