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.

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.

A new enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against foot-and-mouth disease virus. I. Development and method of ELISA.

A liquid-phase blocking sandwich ELISA has been developed for the quantification of antibodies against foot-and-mouth disease virus which may replace the virus neutralisation (VN) test. This test employs the incubation of a constant amount of antigen with a range of test serum dilutions in the liquid-phase before being assayed using a trapping ELISA. Thus it does not rely on the availability or growth of tissue culture cells. The assay is rapid and relatively simple to perform, reagents are used economically and results may be recorded within 24 h. The ELISA is sensitive and results are more specific and more reproducible than those obtained by VN. Results are expressed as reciprocal antibody titres which are analogous and of a similar order to those recorded by VN. Individual titres, therefore, may be easily assessed by workers in the field who are already familiar with VN.

A new enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against foot-and-mouth disease virus. II. Application.

The liquid-phase blocking sandwich ELISA has been evaluated for the serological study of antibodies against foot-and-mouth disease virus (FMDV). The titres recorded for sera from a population of more than 300 British uninfected, unvaccinated cattle which were examined against each of the seven immunologically distinct FMDV types were less than 1 in 40. A positive correlation between ELISA and VN titres was recorded for sera either vaccinated or involved in outbreaks of FMDV. The overall regression between the ELISA/VN data showed that 1 in 16 by VN was equivalent to 1 in 40 by ELISA. Thus ELISA is sensitive, specific and reproducible and results may be directly correlated to those recorded by VN. Serum titres may be interpreted as positive or negative and the number of sera which require retesting would be considerably less than by VN. It is suggested that this ELISA may be used as an alternative to the existing VN test for the quantification of antibodies against FMD virus.

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.

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.

Use of reverse transcriptase-polymerase chain reaction (RT-PCR) and dot-blot hybridisation for the detection and identification of African horse sickness virus nucleic acids.

A coupled reverse transcriptase-polymerase chain reaction assay (RT-PCR) for the detection of African horse sickness virus (AHSV) dsRNA, has been developed using genome segment 7 as the target template for primers. RNA from isolates of all nine AHSV serotypes were readily detected. The potential inhibitory effects of either ethylene diamine tetra acetic acid (EDTA) or heparin on the RT-PCR were eliminated by washing blood samples before lysis of the red blood cells and storage. There was a close agreement in the sensitivity and the specificity of the RT-PCR and an indirect sandwich ELISA. Confirmation of the presence of AHSV using RT-PCR and dot-blot hybridization on blood samples collected from horses experimentally infected with AHSV serotype 4 (AHSV 4) and AHSV serotype 9 (AHSV 9), was achieved within 24 hours, compared to the period of several days required for virus isolation. The RT-PCR and virus isolation methods showed similar levels of sensitivity when used for the detection of AHSV in 3 horses infected with AHSV 4, and in 2 out of 3 horses infected with a less virulent isolate of AHSV 9. Although viraemia was detected in the third horse by virus isolation, from 6 to 14 days after infection, this animal remained consistently negative by RT-PCR. Conversely, AHSV viral RNA was detected by RT-PCR in the blood of 4 donkeys and 4 mules up to 55 days after their experimental infection despite the absence of any detectable infectious virus. RT-PCR is a sensitive and rapid method for detecting AHSV nucleic acids during either the incubation period at the start of an African horse sickness (AHS) epizootic, or for epidemiological investigations in species where clinical signs may be inapparent.

VP7 from African horse sickness virus serotype 9 protects mice against a lethal, heterologous serotype challenge.

An established mouse model system was used to evaluate the effectiveness of the major outer core protein VP7 of African horse sickness virus (AHSV) serotype 9 as a subunit vaccine. Balb C mice were immunised with VP7 crystals purified from AHSV infected BHK cells. In groups of mice, each of which was immunised with > or = 1.5 micrograms of the protein in Freund's adjuvant, > or = 80% of mice survived challenge with a virulent strain of a heterologous AHSV serotype (AHSV 7), that killed > or = 80% of the mice in the uninoculated control groups. This level of protection was significantly greater than that observed in mice inoculated with equivalent amounts of either denatured VP7 (50% survival), or GST/VP7 fusion protein (50-70% survival), or which were vaccinated with AHSV 9 (40-50% survival). The VP7 protein folding, or its assembly into crystals, are thought to play some role in the effectiveness of the protective response observed. Titres of circulating antibodies against AHSV VP7 were determined by competitive ELISA but did not appear to correlate with the levels of protection observed. Passive transfer of these antibodies to syngeneic recipients also failed to protect Balb C mice from the AHSV 7 challenge. The observed protection is therefore unlikely to be due to an antibody mediated immune response.

Validation of ELISA for the detection of African horse sickness virus antigens and antibodies.

The mortality rate in susceptible populations of horses during an epizootic of African horse sickness (AHS) may be in excess of 90%. Rapid and reliable assays are therefore essential for the confirmation of clinical diagnoses and to enable control strategies to be implemented without undue delay. One of the major objectives of a recent European Union funded project was the validation of newly developed diagnostic assays which are rapid, sensitive, highly reproducible and inexpensive, for the detection of African horse sickness virus (AHSV) antigens and antibodies. The Laboratorio de Sanidad y Produccion Animal (LSPA) in Algete, Spain was charged with the responsibility of co-ordinating and supplying samples of viruses and antisera to the participating laboratories in Spain, France and the United Kingdom. The panels comprised 76 antigen samples for assay by indirect sandwich ELISAs and 53 serum samples for antibody detection by either indirect or competitive ELISAs. Results generated by ELISA for each laboratory were analysed in LSPA in terms of their relative sensitivities and specificities. There was a good agreement between the ELISAs used for either antigen or antibody detection. The participating groups agreed that any field sample giving a doubtful result would always be retested by ELISA and an alternative assay.

The use of ELISA for the detection of African horse sickness viruses in Culicoides midges.

The use of ELISA for the detection of African horse sickness viruses (AHSV) in midges preserved in 5.0% formalin was evaluated. No differences were detected by ELISA when testing AHSV infected batches of Culicoides midges collected in diluent with or without the addition of formalin. The ELISA was considered highly sensitive and easily distinguished between non-infected midges and batches containing varying numbers of infected and non-infected midges. Positive ELISA reactions were detected with formalin-preserved midges collected from the south of Spain during the 1988 AHSV epizootic. The assay, therefore, may be used in surveillance studies of either fresh or formalin-preserved midges to identify undisclosed and persistent AHSV foci. This information would be useful in helping to eradicate the virus from Europe and North Africa.

The use of African horse sickness virus VP7 antigen, synthesised in bacteria, and anti-VP7 monoclonal antibodies in a competitive ELISA.

A full-length cDNA clone of genome segment 7 of African Horse Sickness Virus, serotype 9 (AHSV9) was obtained using the PCR technique. The clone was sequenced and found to be 98.27% homologous to the previously published sequence of the equivalent cDNA clone from AHSV4 at the nucleotide level and to exhibit 99.7% identity at the amino acid level. The cDNA clone was transferred to pGEX-2T (Pharmacia), a bacterial expression vector, such that the reading frame of AHSV9 VP7 was continuous with that of the bacterial glutathione-S-transferase (GST) protein, under the control of the bacterial tac promoter. On induction with IPTG a fusion protein consisting of GST and VP7 was synthesised, which was readily purified on a GST-sepharose column (Pharmacia). The fusion protein reacted equally well in an indirect ELISA using monoclonal antibodies specific for AHSV9 VP7 or polyclonal guinea pig antisera raised against AHSV9 infectious sub-viral particles. This protein was also shown to be a suitable substitute for virus antigen, prepared from infected BHK cell extracts, in a competitive ELISA. Antibodies titres recorded for AHSV9 positive and negative horse sera were similar in the competitive ELISA using either bacterial AHSV VP7 or BHK extracted virus as the source of antigen, in combination with monoclonal or polyclonal antibodies, respectively, as the detectors.

An indirect sandwich ELISA for the identification of bovine enteroviruses.

An indirect sandwich ELISA is described for the detection of bovine enteroviruses. The assay was developed as an alternative to the complement fixation test and proved to be more sensitive and convenient. Ten bovine enterovirus prototype strains were easily discriminated. No cross-reactions were observed with other picornaviruses including foot-and-mouth disease viruses, swine vesicular disease virus, porcine enteroviruses and bovine rhinovirus.

A serogroup specific enzyme-linked immunosorbent assay for the detection and identification of African horse sickness viruses.

A serogroup specific, indirect, sandwich ELISA was developed for the rapid detection of African horse sickness virus and viral antigens in field samples or in infected tissue cultures. The assay was shown to be highly sensitive and capable of providing confirmation of clinical diagnosis within one day. The results demonstrated that this ELISA will be useful for epidemiological surveillance of insect and mammalian host populations.

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.

A rapid enzyme-linked immunosorbent assay for the detection of foot-and-mouth disease virus in epithelial tissues.

A rapid double sandwich enzyme-linked immunosorbent assay (ELISA) has been used for the identification and type differentiation of foot-and-mouth disease (FMD) viruses in epithelial tissue samples submitted for diagnosis from the field. No difficulty was experienced in the direct typing of freshly harvested epithelium from recently ruptured vesicles by the complement fixation (CF) test or ELISA. The ELISA was more sensitive and specific, but proved no more efficient than the traditional CF test in the direct typing of samples of poorer quality from many countries overseas where communications are often difficult. However, when both tests were used concurrently, FMD virus typings were confirmed in 27 more samples. Some possible reasons for the failure of ELISA to detect virus in certain cases are discussed.

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.

Neutralising antibodies to lumpy skin disease virus in African wildlife.

A total of 3445 sera from 44 different wild species collected between 1963 and 1982 in 11 African countries south of the Sahara, were examined for neutralising antibodies to Lumpy Skin Diseases (LSD) Virus (prototype Neethling). Antibodies were demonstrated in six species but were of low prevalence. It was concluded from the generally negative results, that wildlife in Africa probably does not play a very important part in he perpetuation and spread of LSD Virus.

Neutralising antibodies to wildebeest-derived malignant catarrhal fever virus in African wildlife.

A total of 2,722 sera collected between 1963 and 1983, from 43 different species of wildlife in 11 African countries was examined for neutralising antibodies against the wildebeest-derived strain of malignant catarrhal fever (MCF) virus. Antibodies were demonstrated in 10 species of Bovidae which included eight species from the sub-family Hippotraginae and one species each from Bovinae and Antilopinae. Neutralising antibodies were also recorded in hippopotamus. It is suggested that the high prevalence of antibodies recorded in sera from waterbuck and reedbuck indicate infection with MCF. However, titres in other species may be due to antigenically related viruses.