During investigations into recent population decreases in Pyrenean chamois (Rupicapra pyrenaica pyrenaica) 21 animals found dead or dying were necropsied. Immunohistochemistry revealed the presence of a pestivirus in organs from two of the 21 chamois. From one of these animals a pestivirus was isolated from the spleen, skin and serum. The virus had better growth in ovine than in bovine cells and was neutralized most effectively by an anti-border disease virus (BDV) reference antiserum. Using panpestivirus and genotype-specific primers selected from 59-untranslated region (UTR) of the pestivirus genome, BDV RNA was demonstrated by RT-PCR. Comparison of the chamois sequences from 59-UTR, entire N pro and E2 gene coding regions with those of other pestivirus genotypes revealed that this virus did not fall into any of the pestivirus genotypes identified so far. Results of phylogenetic analysis suggested that the chamois pestivirus was closely related to BDV and it was typed as BDV-4 genotype.Pestiviruses (family Flaviviridae) affect ruminants and suids. There are four accepted pestivirus species: Border disease virus (BDV), Bovine viral diarrhoea virus-1 (BVDV-1), BVDV-2 and Classical swine fever virus (CSFV); and an isolate tentatively classified as a pestivirus from a giraffe (Heinz et al., 2000). Genetic and antigenic characterization of new pestiviruses isolated from sheep has led to the proposal that BDV strains can be allocated into one of three genotypes, BDV-1 to -3 (Becher et al., 2003).The knowledge of pestivirus infections in wild animals is limited. Pestiviruses have been isolated from giraffe (Plowright, 1969), deer, buffalo, bison, bongo, alpaca and reindeer. The deer, buffalo, alpaca and bongo isolates had BVDV-1 genotypes. The bison and reindeer isolates were closer to BD virus (Becher et al., 1997(Becher et al., , 1999) and the reindeer isolate was classified into the BDV-2 genotype (Becher et al., 2003). Serological surveys have shown that many species of free-living ruminants have varying prevalence of antibody to pestiviruses (Nettleton, 1990).The Pyrenean chamois (Rupicapra pyrenaica pyrenaica) known locally as sarrio and isard, is a free-living ruminant grazing with domesticated cattle and sheep in the Pyrenean mountains, with a population of about 25 000 animals (Pérez et al., 2002). Recently, a population decrease has been observed in both the French and Spanish Central Pyrenees, and the possible involvement of pestiviruses has been reported (Guffond et Icre, 2003;Marco et al., 2003;Schelcher & Alzieu, 2003). The study reported here was undertaken in the Principality of Andorra and four hunting reserves in Aragon (Spain): Benasque, Los Circos, Viñamala and Los Valles. The area in which chamois deaths were excessive lies between Andorra to the east and Benasque reserve to the west.A serological survey was conducted to investigate the prevalence of pestivirus antibody in Pyrenean chamois. An ELISA was used to detect anti-pestivirus antibodies in 200 sera using a standard method employing the O...

The Principality of Andorra is surrounded by areas in which Pyrenean chamois (Rupicapra pyrenaica pyrenaica) populations were severely affected by infection with border disease virus (BDV) which caused disease outbreaks between 2001 and 2009. Nevertheless, the Andorran chamois populations were not affected during this period. In light of the severe impact of BDV on several of the neighboring Pyrenean chamois populations, we monitored local Andorran populations in an effort to detect pestivirus antibodies and BDV in wild ungulates. In addition, an episode of mortality between 2009 and 2010 in chamois was investigated. We analyzed samples (spleen or serum) from 175 Pyrenean chamois, 284 European mouflon (Ovis orientalis musimon), 13 roe deer (Capreolus capreolus capreolus), and five wild boars (Sus scrofa castilianus). With the exception of three dead chamois found between 2009 and 2010, all samples came from healthy animals hunted during the hunting season. A commercial blocking enzyme-linked immunosorbent assay (ELISA) was used to test sera for antibodies against pestivirus. Positive sera were tested with a comparative virus neutralization test (VNT) using three BDV strains and a bovine viral diarrhea virus strain. Reverse-transcription-polymerase chain reaction (RT-PCR) was performed on all sera and spleen homogenates. Antibodies against pestivirus were detected by ELISA in four of the 69 chamois (5%; 95% CI= 1.29-13.11). The VNT confirmed three of these chamois were infected with a BDV. Viral RNA was detected by RT-PCR in three chamois-one apparently healthy animal hunted in 2009 and two dead animals. Viral sequences showed that the three chamois were infected with a BDV-4, the same genotype that was involved in previous episodes of mortality in the Pyrenees. Although Pyrenean chamois from Andorra had had little contact with the pestiviruses until 2009, in this year BDV was associated with a severe disease outbreak.