The parapoxvirus orf virus encodes a novel soluble protein inhibitor of ovine granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2). The GM-CSF- and IL-2-inhibitory factor (GIF) gene was expressed as an intermediate-late viral gene in orf virus-infected cells. GIF formed homodimers and tetramers in solution, and it bound ovine GM-CSF with a Kd of 369 pM and ovine IL-2 with a Kd of 1.04 nM. GIF did not bind human GM-CSF or IL-2 in spite of the fact that orf virus is a human pathogen. GIF was detected in afferent lymph plasma draining the skin site of orf virus reinfection and was associated with reduced levels of lymph GM-CSF. GIF expression by orf virus indicates that GM-CSF and IL-2 are important in host antiviral immunity.

BackgroundThe apicomplexan parasite Cryptosporidium represents a threat to water quality and public health. An important zoonotic species involved in human cryptosporidiosis from contaminated water is Cryptosporidium parvum (C. parvum), the main reservoirs of which are known to be farm livestock particularly neonatal calves, although adult cattle, sheep, lambs and wildlife are also known to contribute to catchment loading of C. parvum. This study aimed to establish Cryptosporidium prevalence, species and genotype in livestock, deer and water in a catchment with a history of Cryptosporidium contamination in the public water supply.MethodsA novel method of processing adult ruminant faecal sample was used to concentrate oocysts, followed by a nested species specific multiplex (nssm) PCR, targeting the 18S rRNA gene, to speciate Cryptosporidium. A multilocus fragment typing (MLFT) tool was used, in addition to GP60 sequencing, to genotype C. parvum positive samples.ResultsA very high prevalence of Cryptosporidium was detected, with speciation identifying a predominance of C. parvum in livestock, deer and water samples. Four GP60 subtypes were detected within C. parvum with the majority IIaA15G2R1 which was detected in all host species and on all farms. Multilocus fragment typing further differentiated these into 6 highly related multilocus genotypes.ConclusionThe high prevalence of Cryptosporidium detected was possibly due to a combination of the newly developed sample processing technique used and a reflection of the high rates of the parasite present in this catchment. The predominance of C. parvum in livestock and deer sampled in this study suggested that they represented a significant risk to water quality and public health. Genotyping results suggested that the parasite is being transmitted locally within the study area, possibly via free-roaming sheep and deer. Further studies are needed to verify particular host associations with subtypes/MLGs. Land and livestock management solutions to reduce Cryptosporidium on farm and in the catchment are planned with the aim to improve animal health and production as well as water quality and public health.

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...

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The population of red squirrels (Sciurus vulgaris) in the British Isles is in decline and is being supplanted by the grey squirrel (Sciurus carolinensis). It has been suggested that parapoxvirus-associated disease has caused significant mortality in red squirrels and that grey squirrels are the source of the virus. A direct enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of antibody to squirrel parapoxvirus. We tested 140 sera from red squirrels and 223 from grey squirrels from different populations in the UK. A high percentage (61%) of apparently healthy grey squirrels, were found to have been exposed to the parapoxvirus. Only 2.86% (4/140) of red squirrels had antibody and three of these animals had parapoxvirus-associated disease. We postulate that the grey squirrel may act as a reservoir host for the virus.

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.

Twenty, eight-day-old specific pathogen-free (SPF) lambs were vaccinated by a single scarification approximately 4 cm in length on the inner right thigh with a double-pronged applicator. The titre of live virus in the vaccine was 10(7.2) TCID50/ml and the estimated dose per lamb was 0.04 ml. Three months and six months later 10 of the vaccinated lambs and five age-matched unvaccinated control specific pathogen free lambs were challenged by a single scarification with virulent virus on the inner left thigh in the same way. After the vaccination all 20 lambs developed lesions characteristic of orf virus infection that had largely resolved four weeks later, when they all had reciprocal ELISA antibody titres > or = 3200 that persisted in all but one of them until they were challenged. After the challenge, the development of lesions in the vaccinated and unvaccinated sheep was compared daily for four weeks by means of a clinical scoring system. Both groups of vaccinated lambs had significantly lower (P < 0.01) total clinical scores after challenge at three months and six months than the unvaccinated lambs.

The native red squirrel Sciurus vulgaris population in Britain has been on the decline for many years. A poxvirus associated with the introduced American grey squirrel S. carolinensis has been recognized as having a major role in the reduction of red squirrel numbers by causing a deleterious disease, known as squirrelpox, from which they seldom recover. In Ireland, red squirrel numbers have also been reducing while the grey squirrel population, first introduced in 1911, has been expanding. Until now, no poxvirus-associated disease had been found in Irish red squirrels and therefore, the role of squirrelpox in the displacement of red squirrels by grey squirrels in Ireland has been questioned. Here we report, for the first time, confirmed squirrelpox disease in two populations of red squirrels in Northern Ireland. In addition, we present serological evidence of the extent of poxvirus infection in the grey squirrels from both the Republic of Ireland and Northern Ireland, including an apparent increase in the seroprevalence of antibodies against the virus in grey squirrels over the period of the study, and discuss the implications of our findings for the conservation tactics employed to protect red squirrels.