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...

BackgroundDetection of respiratory viruses in veterinary species has traditionally relied on virus detection by isolation or immunofluorescence and/or detection of circulating antibody using ELISA or serum neutralising antibody tests. Multiplex real time PCR is increasingly used to diagnose respiratory viruses in humans and has proved to be superior to traditional methods. Bovine respiratory disease (BRD) is one of the most common causes of morbidity and mortality in housed cattle and virus infections can play a major role. We describe here a one step multiplex reverse transcriptase quantitative polymerase chain reaction (mRT-qPCR) to detect the viruses commonly implicated in BRD.ResultsA mRT-qPCR assay was developed and optimised for the simultaneous detection of bovine respiratory syncytial virus (BRSV), bovine herpes virus type 1 (BoHV-1) and bovine parainfluenza virus type 3 (BPI3 i & ii) nucleic acids in clinical samples from cattle. The assay targets the highly conserved glycoprotein B gene of BoHV-1, nucleocapsid gene of BRSV and nucleoprotein gene of BPI3. This mRT-qPCR assay was assessed for sensitivity, specificity and repeatability using in vitro transcribed RNA and recent field isolates. For clinical validation, 541 samples from clinically affected animals were tested and mRT-qPCR result compared to those obtained by conventional testing using virus isolation (VI) and/or indirect fluorescent antibody test (IFAT).ConclusionsThe mRT-qPCR assay was rapid, highly repeatable, specific and had a sensitivity of 97% in detecting 102 copies of BRSV, BoHV-1 and BPI3 i & ii. This is the first mRT-qPCR developed to detect the three primary viral agents of BRD and the first multiplex designed using locked nucleic acid (LNA), minor groove binding (MGB) and TaqMan probes in one reaction mix. This test was more sensitive than both VI and IFAT and can replace the aforesaid methods for virus detection during outbreaks of BRD.

The DRB gene of the domestic cat MHC appears to be highly polymorphic, with 71 alleles provisionally reported, based on exon 2 sequence. However, these alleles were reported prior to the adoption of strict criteria for allele identification. In this study, we investigated FLA-DRB exon 2 polymorphisms in a cohort of 33 British domestic cats by polymerase chain reaction (PCR) and clonal sequence analysis. Applying the strict criteria for assigning new alleles as used by the established mammalian MHC nomenclature committees, we defined 13 FLA-DRB alleles, including four previously unreported alleles. We identified many sequences that were one or two base pairs different from these 13 defined alleles, and have shown that these are most likely artefacts of PCR amplification. When the same criteria for allele acceptance were applied to the remaining previously reported sequences, 11 further alleles were confirmed. This suggests that to date there is good evidence for 24 FLA-DRB alleles fulfilling nomenclature criteria. Analysis of these 24 alleles reveals a similar pattern of MHC polymorphism to that seen in other mammals, with three regions of hypervariability.

A multiplex polymerase chain reaction to detect and differentiate Campylobacter fetus subspecies fetus and Campylobacter fetus -species venerealis: use on UK isolates of C. fetus and other Campylobacter spp. Aims: Subspeciation of Campylobacter fetus subsp. fetus (CFF) and Campylobacter fetus subsp. venerealis (CFV) is important for international animal import regulations. Phenotyping can be unreliable, and genotyping by techniques like pulsed field gel electrophoresis is difficult in routine diagnostic laboratories. A PCR subspeciation technique has been reported [Aust Vet J (1997) 75, 827]; we aimed to develop this PCR and investigate its use on UK C. fetus isolates. Methods and Results:We augmented the PCR with further primers, and tested 76 isolates of C. fetus and 16 isolates of other Campylobacter spp. PCR failed to correlate well with phenotyping, especially for CFV. We characterized the amplicon of the CFV-specific primers (reported as plasmid derived, but unavailable on the public databases); and predicted a parA gene sequence, anticipated to be plasmid-associated. However, although plasmid isolations from selected CFV isolates demonstrated the presence of several plasmids, there was no correlation between plasmid profile and PCR result. Further, the parA sequence was not detected by PCR in any of the plasmid bands. Conclusions: This PCR is not suitable for subspeciation of C. fetus in the UK. The results suggest that this is a reflection of the presence of an unusual clone of CFV currently present in cattle in this country. Significance and Impact of the Study: PCR cannot substitute for phenotyping of C. fetus isolates in the UK. The reasons for failure of PCR genotyping may reflect local strains and/or plasmid profiles. Further study is required to better elucidate molecular sub-speciation of C. fetus.

A trivalent (feline panleucopenia, feline herpesvirus, feline calicivirus), modified live, commercially available cat vaccine was used at either 6, 9 and 12 weeks of age (early schedule) or 9 and 12 weeks of age (conventional schedule), and the serological response to vaccination was assessed. The level of maternally derived antibody present at 6 weeks of age was also established. The use of early vaccination at 6 weeks of age induced an antibody response to each virus by 9 weeks of age in a significant proportion of kittens compared with unvaccinated littermates. There was no difference between the conventionally and early-vaccinated groups in terms of antibody response to any antigen by 12 and 15 weeks of age.