A competitive ELISA for the detection of group-specific antibody to equine encephalosis virus.

A polyclonal antibody-based, group-specific, competitive ELISA (C-ELISA) for the detection of antibodies to equine encephalosis virus (EEV) was developed. The assay measures the competition between a specific guinea pig antiserum and a test serum, for a pre-titrated EEV antigen. The C-ELISA detected antibodies to the seven known EEV serotypes. Reference antisera raised against other arboviruses did not cross react with EEV antigen. Negative sera from horses in the United Kingdom were used to establish the baseline for a negative population. Negative and positive populations of South African horses, selected on the basis of virus neutralisation were assayed subsequently. Optimal test parameters, where sensitivity≅specificity≅100%, were calculated by two-graph receiver operator characteristic (TG-ROC) analysis to be at a cut-off value of 29.5% inhibition. Results show the EEV C-ELISA described to be sensitive, specific and reliable. Used in conjunction with ELISAs available for African horse sickness virus (AHSV), differential serological diagnosis between EEV and AHSV can be achieved.

No evidence of bluetongue virus in Switzerland.

We report the results of the first survey for antibody against bluetongue virus (BTV) that was conducted in Switzerland in the year 2003. In a nationwide cross-sectional study with partial verification, 2437 cattle sera collected from 507 herds were analysed using competitive enzyme-linked immunosorbent assays (c-ELISA). To adjust for misclassification, 158 sera, including 86 that were recorded equivocal in Switzerland, were sent to the Office Internationale des Epizooties designated regional reference laboratory in the UK for confirmation. No BTV antibody was detected in any of these samples, confirming the absence of BTV from Switzerland in 2003. The specificity of the c-ELISA used in Switzerland for individual Swiss cattle was calculated to be 96.5%. The mean herd sensitivity achieved in our survey ranged from 78.9% to 98.8% depending on the with-in herd prevalence and test sensitivity used for the calculations. The cumulated confidence level achieved with the survey based on a minimal expected prevalence of 2%, was 99.99% and therefore it was concluded that there was no evidence of BTV circulation in Switzerland in 2003.

Evidence for the presence of bluetongue virus in Kosovo between 2001 and 2004.

In 2001, clinical cases of bluetongue were observed in Kosovo, and in that year and in 2003 and 2004, serum samples were collected from cattle and small ruminants and tested for antibodies to bluetongue virus. The results provide evidence that bluetongue virus was not present in Kosovo before the summer of 2001, but that the virus circulated subclinically among the cattle and sheep populations of Kosovo in 2002, 2003 and 2004.

Live attenuated bluetongue vaccine viruses in Dorset Poll sheep, before and after passage in vector midges (Diptera: Ceratopogonidae).

The aim of this study was to address concerns associating with the use of BTV attenuated commercial vaccines in European sheep. These concerns include development of viraemia, possibility of transmission by vectors, reversion to virulence and re-assortment with wild-type viruses. The two vaccine viruses (BTV 2 and 9) replicated in two species of Culicoides subsequent to oral infection reaching titres suggesting transmission would occur. Viraemia in Dorset Poll sheep inoculated with either vaccine or insect passaged vaccine viruses persisted for up to 17 days, recording titres that ranged from 2.5 to 6.25 log(10)TCID(50)/ml, which is easily sufficient to infect vector Culicoides. Moderate to severe clinical signs of BT, albeit short lived, were observed in sheep following vaccination. However, to date there is no evidence of increasing virulence following two sequential passages through the vectors.

A comparison of the susceptibility of Culicoides imicola and C. bolitinos to oral infection with eight serotypes of epizootic haemorrhagic disease virus.

The mechanisms involved in introduction, maintenance and perpetuation of epizootic haemorrhagic disease virus (EHDV) in South Africa are not fully understood. This paper reports on the susceptibility of South African livestock associated Culicoides (Diptera: Ceratopogonidae) species to oral infection with eight EHDV serotypes. Virus was recovered from eight of 17 field-collected Culicoides species 10 days after oral feeding on blood/virus mixtures. Six EHDV serotypes were recovered from C. (Avaritia) imicola Kieffer, and seven serotypes were recovered from C. (A.) bolitinos Meiswinkel. Virus recovery rates in C. imicola ranged from 0.4% for EHDV 2 to 14.4% for EHDV 7, and in C. bolitinos from 0.6% for EHDV 6 to 12.3% for EHDV 2. There was a significant difference in virus recovery rates between serotypes in both species. Other Culicoides species that yielded EHDV after 10 days extrinsic incubation included C. (Meijerehelea) leucostictus Kieffer, C. (Culicoides) magnus Colaço, C. (Beltranmyia) nivosus de Meillon, C. (A.) gulbenkiani Caeiro, C. (Hoffmania) zuluensis de Meillon and C. onderstepoortensis Fiedler. Culicoides midges shown in this study to be susceptible to oral infection with EHDV are widely distributed in South Africa but differ considerably in their abundance, host preference and breeding sites.

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.

Incidence and isolation of bluetongue virus infection in cattle of the Santo Tomé Department, Corrientes Province, Argentina.

Sentinel herds were monitored for the detection of bluetongue (BT)-specific antibodies and virus over two periods, namely: June 1999 to August 2000 and September 2000 to April 2001. Herds were located in Santo Tomé (Herds 1 and 2) where BTV activity was known to occur. From June 1999 to August 2000, the cumulative incidence (CI) of bluetongue virus (BTV) infection was 0% and 35% in Herds 1 and 2, respectively. In the second period, the CI of BTV infection was 10% and 97% in Herds 1 and 2, respectively. The virus was isolated from red blood cells of animals that seroconverted and was identified as serotype 4. Averages of the monthly maximal temperatures were always above 19 degrees C. However, averages of the monthly median temperatures were below 19 degrees C and averages of the monthly minimal temperatures were below 15 degrees C from May 2000 to August 2000. There was no viral activity detected at that time. Culicoides insignis was identified as the predominant potential vector species (99%) trapped near sentinel herds. Although clinical disease has never been reported in Argentina, viral activity was detected and the virus has been isolated in sentinel herds.

Bluetongue virus antigen and antibody detection, and the application of laboratory diagnostic techniques.

Despite the significant advances that have been made in molecular techniques, the traditional approach using biology-based test procedures is still the mainstay for the laboratory confirmation of clinical diagnoses. The serological and virological techniques available for the detection and identification of bluetongue virus and antibody fall into two categories; those that are serotype-specific and those that are serogroup-specific. Although several assay methodologies have been described and used, thought should always be given to their use in different epidemiological situations and to the interpretation of results obtained therein.

A serological survey of ruminant livestock in Kazakhstan during post-Soviet transitions in farming and disease control.

The results of a serological survey of livestock in Kazakhstan, carried out in 1997--1998, are reported. Serum samples from 958 animals (cattle, sheep and goats) were tested for antibodies to foot and mouth disease (FMD), bluetongue (BT), epizootic haemorrhagic disease (EHD), rinderpest (RP) and peste des petits ruminants (PPR) viruses, and to Brucella spp. We also investigated the vaccination status of livestock and related this to changes in veterinary provision since independence in 1991. For the 2 diseases under official surveillance (FMD and brucellosis) our results were similar to official data, although we found significantly higher brucellosis levels in 2 districts and widespread ignorance about FMD vaccination status. The seroprevalence for BT virus was 23%, and seropositive animals were widespread suggesting endemicity, despite the disease not having being previously reported. We found a few seropositives for EHDV and PPRV, which may suggest that these diseases are also present in Kazakhstan. An hierarchical model showed that seroprevalence to FMD and BT viruses were clustered at the farm/village level, rather than at a larger spatial scale. This was unexpected for FMD, which is subject to vaccination policies which vary at the raion (county) level.

A group-specific, indirect sandwich ELISA for the detection of equine encephalosis virus antigen.

A polyclonal antibody-based, group-specific, indirect, sandwich ELISA (S-ELISA) for the detection of equine encephalosis virus (EEV) antigen was developed. Purified EEV particles were titrated in the S-ELISA and the limit of detection was determined to be approximately 9.0 ng of antigen/ml (0.45 ng/well). Positive S-ELISA reactions were recorded with seven serologically distinct EEV serotypes. No cross-reactions were recorded with other arboviruses including African horse sickness virus (AHSV) serotypes 1-9, bluetongue serotypes 1-24, epizootic haemorrhagic disease serotypes 1-8 and isolate 318, and selected isolates of Palyam, Eubenangee, Corriparta, Warrego, Akabane and bovine ephemeral fever viruses. The assay proved to be sensitive and specific for the rapid detection of EEV in cell cultures and in homogenated suckling mouse brain (MB). The data generated in this study suggest that the ELISA will be valuable for epidemiological studies of EE and will assist in making a reliable differential diagnosis between EEV and AHSV infections.

Determination of the oral susceptibility of South African livestock-associated biting midges, Culicoides species, to bovine ephemeral fever virus.

A total of 10 607 Culicoides midges (Diptera: Ceratopogonidae) were fed on either sheep or horse blood containing not less than 6.5 log10 TCID50/ml of bovine ephemeral fever virus (BEFV). Insects were collected during two consecutive summers from two distinct climatic areas. Two seed viruses, originating from either South Africa or Australia, were used separately in the feeding trials. Blood-engorged females were incubated at 23.5 degrees C for 10 days and then individually assayed in microplate BHK-21 cell cultures. Of the 4110 Culicoides that survived, 43% were C. (Avaritia) imicola Kieffer and 27% were C. (A.) bolitinos Meiswinkel. The remainder represented 18 other livestock-associated Culicoides species. Although BEFV was detected in 18.9% of midges assayed immediately after feeding, no virus could be detected after incubation. The absence of evidence of either virus maintenance or measurable replication suggests that most of the abundant livestock-associated Culicoides species found in South Africa are refractory to oral infection with BEFV. Future studies should be carried out using species of mosquitoes that are associated with cattle in the BEF endemic areas.

First evidence of bluetongue virus in Kazakhstan.

We report the results of the first serological survey for bluetongue virus in Kazakhstan. We analysed blood samples collected from 958 livestock and 513 wild saiga antelopes over a large area of the country, and found 23.2% seroprevalence in livestock and 0% in saigas. Seroprevalence in livestock did not vary by species, but increased significantly with age. There was no evidence for variation in seroprevalence at the regional level, but there was significant clustering at the farm level. Bluetongue has never before been reported in Kazakhstan, yet our results suggest that it may be endemic. We found seropositive animals at the furthest known northern limits of the bluetongue virus in this region of the world. Recorded vectors are not known to be present in Kazakhstan, so a novel vector is likely to be operating. The lack of evidence for bluetongue virus in saigas is unexpected and suggests a need for further investigation.

African horse sickness epidemiology: vector competence of south african Culicoides species for virus serotypes 3, 5 and 8.

The oral susceptibilities of 17 Culicoides species to infection with African horse sickness virus (AHSV) serotypes 3, 5 and 8 were determined by feeding field-collected midges on AHSV infected horse blood. The mean titres of virus in the bloodmeals for the three serotypes of AHSV were between 5.7 and 6.5 log10 TCID50/ml. Virus was detected, after 10 days incubation at 23.5 degrees C, in the Culicoides imicola Kieffer (Diptera: Ceratopogonidae) that had fed on blood containing AHSV 5 (8.5%) and 8 (26.8%), and in the Culicoides bolitinos Meiswinkel that had fed on AHSV 3 (3.8%), 5 (20.6%) and 8 (1.7%). Although 44.4% of the C. imicola were shown to have ingested AHSV 3 immediately after feeding, no virus was detected in 96 C. imicola after incubation. The relatively high titres of virus recorded in individual midges of both species after 10 days incubation suggested a fully disseminated infection. Previously, C. imicola was considered to be the only field vector of AHSV in Africa. Identifying C. bolitinos as a potential vector for AHSV is an important finding, which if proven will have a significant impact on our understanding of the epidemiology of AHS. No AHSVs could be detected in the other 15 species of Culicoides assayed, which suggests that some of the southern African Culicoides species are refractory to AHSV infection. However, further work with larger numbers of each species will be necessary to confirm this observation.

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.

Identification and differentiation of the nine African horse sickness virus serotypes by RT-PCR amplification of the serotype-specific genome segment 2.

This paper describes the first RT-PCR for discrimination of the nine African horse sickness virus (AHSV) serotypes. Nine pairs of primers were designed, each being specific for one AHSV serotype. The RT-PCR was sensitive and specific, providing serotyping within 24 h. Perfect agreement was recorded between the RT-PCR and virus neutralization for a coded panel of 56 AHSV reference strains and field isolates. Serotyping was achieved successfully with live and formalin-inactivated AHSVs, with isolates of virus after low and high passage through either tissue culture or suckling mouse brain, with viruses isolated from widely separated geographical areas and with viruses isolated up to 37 years apart. Overall, this RT-PCR provides a rapid and reliable method for the identification and differentiation of the nine AHSV serotypes, which is vital at the start of an outbreak to enable the early selection of a vaccine to control the spread of disease.

Serological and virological responses in mules and donkeys following inoculation with African horse sickness virus serotype 4.

Two groups, comprising 4 donkeys and 4 mules (group 1) and 4 donkeys and 3 mules (group 2), were used to determine the duration of viraemia and to monitor the development of antibodies following inoculation with African horse sickness virus (AHSV). One group of animals was given a single dose of attenuated AHSV serotype 4 (AHSV 4) vaccine. The second group was inoculated with a virulent field strain of AHSV 4. Both groups were subsequently challenged with the virulent field strain of AHSV 4, 51 and 58 days, respectively, after their primary inoculation. Blood and serum samples, collected on alternate days after the primary inoculations and also after subsequent challenge, were assayed for virus and antibodies. Seven of the 8 AHSV vaccinated (group 1) and 7 of the 7 AHSV inoculated (group 2) animals showed humoral antibody responses after primary inoculation. Although no infectious virus could be isolated from either group for the duration of the study, reverse transcription-PCR data obtained for the second group did show the presence of AHSV viral RNA from as early as day 5 in mules and day 9 in donkeys after the primary inoculation. Viral RNA was detected consistently up to day 47 in some animals and intermittently thereafter. There was no evidence of a second viraemia in any of the animals after challenge. The detection of specific antibodies, against AHSV 4 NS3 protein, in all animals confirmed that both donkeys and mules were infected and that the virus had replicated.

Donkeys as reservoirs of African horse sickness virus.

Investigations have been carried out to elucidate the possible role of the donkey in the epidemiology of African horse sickness (AHS). These studies have shown that despite the absence of pyrexia or other observable clinical signs, donkeys become infected with virulent AHS virus serotype 4 (AHSV 4) and that they develop a viraemia which can persist for at least 12 days, albeit at a comparatively lower titre than that recorded for similarly infected ponies. AHSV 4 showed a similar tissue tropism in the pony and donkey but the virus appeared to replicate less efficiently in donkey tissues. The only gross pathological changes observed in the donkeys post mortem were increased fluid accumulation in the serosal lined compartments, particularly the peritoneal cavity, and petechial and ecchymotic haemorrhages on the left hepatic ligament. The absence of infectious virus or viral antigens in any of the tissues collected at 14 and 19 days post inoculation (dpi) from 6 experimental donkeys suggest that, though susceptible to infection, the donkey is unlikely to be a long term reservoir for AHSV. Although AHSV 4 was detected in all 6 donkeys following the primary inoculation, no virus could be isolated from blood collected from two donkeys subsequently challenged with a second virulent virus, AHSV 5. Data generated from virus neutralisation tests showed a second primary antibody response, against AHSV 5, in these donkeys at 12 dpi. In contrast, the boost in antibody levels detected from 5 dpi, as measured by ELISA, was probably due to an anamnestic response against the AHSV group-specific viral proteins. Homogenised spleen tissue, collected post mortem from a donkey 7 dpi with AHSV 4, caused a lethal, cardiac form of AHS when inoculated into a susceptible pony.

Immunohistochemical demonstration of African horse sickness viral antigen in tissues of experimentally infected equines.

African horse sickness virus (AHSV) antigen was demonstrated immunohistochemically in formalin-fixed, paraffin-embedded sections of tissues collected from three ponies suffering from the peracute form of the disease and from one pony affected by the fever form. The pattern of the antigen distribution indicated a particular organ tropism characterised by an accumulation of AHSV antigen in cardio-pulmonary tissues of the animals with the peracute disease and in the spleen of the pony with the fever form. AHSV antigen was identified in endothelial cells of small blood vessels, particularly capillaries and in large mono-nuclear cells resembling macrophages or reticular cells of lymphatic tissues. Occasional circulating mononuclear cells with the morphology of monocytes were also positively stained within the larger vessels. The immunohistochemical results confirm earlier work suggesting that AHSV may have different tropisms to particular organs during various forms of the disease and that different target cell populations exist in vivo. Immunohistochemistry may be an additional useful method for diagnostic and research purposes in AHS.