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Between 2001 and 2007, several outbreaks of disease associated with Border disease virus (BDV) infection were reported in the central Pyrenees (northeast Spain) and were associated with a major reduction in chamois (Rupicapra pyrenaica) populations. At the same time, wild boars (Sus scrofa) from the same area were found to be seropositive to this pestivirus, without showing clinical signs. The present study examines the susceptibility of domestic swine and the course of the infection with a BDV strain isolated from naturally infected chamois. Twenty pigs were inoculated with 1 x 10(7) TCID(50) (50% tissue culture infective dose) by oronasal route, and 16 control pigs received Eagles sterile Minimal Essential Medium. Serologic (enzyme-linked immunosorbent assay and virus neutralization test) and reverse transcription polymerase chain reaction assays were performed on serum samples obtained at 0, 3, 7, 10, 14, 21, and 31 days postinoculation (dpi). All infected pigs were viremic from 3 to 14 dpi. After 14 dpi, all infected animals developed an antibody response against the homologous virus. Clinical signs or histologic lesions were not observed in inoculated pigs. The present work demonstrates the susceptibility of domestic swine to a BDV strain of chamois origin.

Approximately 3,000 Pyrenean chamois (Rupicapra pyrenaica pyrenaica) died in northeastern Spain during 2005-2007. Border disease virus infection was identified by reverse transcription-PCR and sequencing analysis. These results implicate this virus as the primary cause of death, similar to findings in the previous epizootic in 2001.

In 2001 and 2002, an outbreak of a previously unreported disease, associated with a border disease virus (BDV), caused high mortality in the Southern chamois (Rupicapra pyrenaica) population in the Alt Pallars-Aran National Hunting Reserve in the Catalan Pyrenees (NE Spain). Between 2002 and 2006, sera and/or tissue samples taken from 116 healthy chamois shot during the hunting season, plus 42 from chamois affected by different diseases, were studied. A blocking enzyme-immunosorbent assay (ELISA) was used to study pestivirus seroprevalence in 114 healthy hunted and 31 diseased chamois, yielding positive results in 73.7 and 22.6% of the chamois, respectively. Comparative virus neutralization tests (VNT) performed on 42 seropositive samples with 6 pestivirus strains yielded statistically higher titres to BDV Spain 97, followed by BDV chamois, BDV 137/4, BDV Moredun, Bovine Diarrhoea virus-1 (BVDV-1) NADL and BVDV-2 atypical. Virological investigations for pestivirus detection were performed using an antigen ELISA test in 82 healthy and 18 diseased chamois, RT-PCR in 16 healthy and in all diseased chamois, and virus isolation in 14 diseased chamois. No viral antigen was detected in any of the healthy animals. A pestivirus, characterized as BDV by monoclonal antibodies, was detected in the 10 chamois showing clinical signs consistent with BDV infection. Sequence analysis in the 5' untranslated region (5'-UTR) revealed that they were grouped into the BDV-4 genotype. In the remaining chamois, infectious keratoconjunctivitis, pneumonia, trauma and contagious ecthyma were diagnosed. The cause of death was unknown in five chamois. The results suggest that the infection has become endemic in the population and that it could have a significant impact on chamois population dynamics.

An outbreak of a previously unreported disease affecting southern chamois (Rupicapra pyrenaica) in the central Pyrenees (NE Spain) was recorded in 2001 and 2002. There was a marked temporal distribution, most animals being found between February and June. After the outbreak, the population was found to have decreased by about 42%, most probably due to the disease. We examined 20 affected chamois. Clinical manifestations included depression, weakness and movement difficulties in all cases. Three chamois presented abnormal behaviour, with absence of flight reaction, and 16 showed different degrees of alopecia with skin hyperpigmentation. At necropsy cachexia was observed in all animals, four chamois had abscesses in different parts of the body, four had pneumonia, one had an extensive subcutaneous infection on the head and neck and one had severe orchitis. Microscopic lesions were found in the brain, mainly edema, gliosis, espongiosis, cariorrexis and neuronal multifocal necrosis. A perivascular mononuclear inflammatory infiltrate was present in three of them. Skin lesions included marked follicular atrophy, mild to moderate epidermal hyperplasia with orthokeratotic hyperkeratosis and follicular hyperkeratosis, and hypermelanosis. In 13 chamois there were haemosiderin deposits in the spleen, and in three individuals kidney "cloissone" was observed. Intraeritrocitic parasites were detected either by direct observation or PCR in 8 of 17 chamois. A pestivirus was isolated and detected by RT-PCR from 12 of 13 affected chamois and antigenic characterized as border disease virus by monoclonal antibodies. This is the first time a border disease virus has been associated with an outbreak of a high-mortality disease in a wild species.

Bulk-tank milk (BTM) samples from 154 sheep flocks were used to estimate BDV prevalence in the Basque Country in Spain using an ELISA and a RT-PCR test. The proportion of antibody-positive flocks was 68% but varied significantly between provinces and was 93% in Araba and 54-55% in Bizkaia and Gipuzkoa. Most ELISA-positive flocks had very low antibody inhibition percentage (AIP) indicating high seroprevalence and recent BDV exposure. However, only 9% flocks were PCR-positive suggesting few infected ewes were being milked at the time of sampling. Phylogenetic analysis of the 5' NCR sequences of BDV from seven infected flocks showed that all except one clustered within the group formed by BDV type C strains from a previous study in the region, whereas the remaining isolate was closest to BDV type A. These results suggest that BDV strains in most Basque flocks have a common origin and differences in prevalence between provinces are associated to recent events affecting BDV spread such as use of communal pastures and sheep trading. The widespread distribution of BDV in the region, advocates for the implementation of BDV control strategies and highlights the potential risk of sheep as a pestivirus reservoir for other species.

Between 2001 and 2002, samples from 1,413 animals in 21 Spanish small ruminant flocks, most of them with animals showing clinical signs compatible with Border disease (BD), were screened for the presence of Pestivirus antigen and antibodies by an indirect peroxidase monolayer assay (IPMA) and the virus neutralization test (VNT), respectively. Although all flocks harboured seropositive animals, virus could only be isolated from animals in five of the flocks. Between 4 and 11 months later all animals older than 6 months in three of the flocks were resampled. At this time, 51-83% of them had neutralizing antibodies. The prevalence of persistently infected (PI) animals within two of the flocks was 0.3 and 0.6%, respectively. The third flock presumably had eliminated all the PI animals. Fourteen virus isolates were obtained. The 5' untranslated region (5'UTR) was amplified by RT-PCR and directly sequenced. Phylogenetic analyses classified them as a group of Border disease viruses (BDV), separated from BDV-1, but showing a relatively low bootstrap value. Three of the 14 isolates were in the same subgroup as a set of formerly characterised Spanish isolates from the Basque Country, which were allocated to subgroup BDV-C. In addition, they were in the group with an isolate from chamois, which is currently allocated in group BDV-4. Because of its close relation to the chamois isolate, these isolates were tentatively reallocated in a subgroup BDV-4a. The remaining isolates generated a new subgroup, related but not in the same cluster as the chamois isolate, and was therefore tentatively assigned to a new subgroup BDV-4b. Our results show that classification and nomenclature of BDV needs to be harmonised.

Pestivirus infection was identified in 16 of 17 chamois during an outbreak of a previously unreported disease in Pyrenean chamois (Rupicapra pyrenaica pyrenaica) in northeastern Spain in 2001-02. By analysis of the 5' noncoding regions of the virus, we assigned it to the border disease virus cluster with pairwise similarity values ranging from 82.1% to 88.1%. It will be important to investigate the association of this pestivirus with disease in Pyrenean chamois.