Development of a recombinant ELISA for ovine herpesvirus 2, suitable for use in sheep.

The minor capsid protein of ovine herpesvirus 2, identified as a potential antigen for serological testing, was over-expressed and purified to allow its assessment in ELISA. The corresponding gene sequence (OvHV-2 orf65, Ov65) was modified to incorporate epitope tags and internal restriction enzyme sites in an E. coli codon-optimised version of the gene. This codon-optimised gene was then subject to internal deletions to identify regions of the protein that could be removed while maintaining protein solubility and antigenicity. It was found that a derivative with deletion of the conserved 5'-end of the gene (Ov65delB) expressed a polypeptide that was soluble when over-expressed in bacteria and was detected by OvHV-2 specific sera. Proteomic analysis of the affinity purified Ov65delB showed that it contained multiple predicted Ov65 tryptic peptides but also showed contamination by co-purifying E. coli proteins. An indirect ELISA, based on this affinity-purified OV65delB, was optimised for use with sheep and cattle samples and cut-off values were established based on known negative serum samples. Analysis of groups of samples that were either presumed infected (UK sheep) or tested OvHV-2 positive or negative by PCR (cattle MCF diagnostic samples) showed that the assay had 95 % sensitivity and 96 % specificity for sheep serum; and 80 % sensitivity and 95 % specificity for cattle serum. The lower sensitivity with cattle samples appeared to be due to a lack of serological response in some MCF-affected cattle. This recombinant antigen therefore shows promise as the basis of an inexpensive, simple and reliable test that can be used to detect OvHV-2-specific antibody responses in both MCF-affected animals and in OvHV-2 reservoir hosts.

LOUPING-ILL VIRUS SEROSURVEY OF WILLOW PTARMIGAN (LAGOPUS LAGOPUS LAGOPUS) IN NORWAY.

In Norway, the Willow Ptarmigan (Lagopus lagopus lagopus) is experiencing population declines and is nationally Red Listed as Near Threatened. Although disease has not generally been regarded as an important factor behind population fluctuations for Willow Ptarmigan in Norway, disease occurrence has been poorly investigated. Both louping-ill virus (LIV) and the closely related tick-borne encephalitis virus are found along the southern part of the Norwegian coast. We assessed whether and where Norwegian Willow Ptarmigan populations have been infected with LIV. We expected to find infected individuals in populations in the southernmost part of the country. We did not expect to find infected individuals in populations further north and at higher altitudes because of the absence of the main vector, the sheep tick (Ixodes ricinus). We collected serum samples on Nobuto filter paper and used a hemagglutination inhibition assay for antibodies against LIV. We collected data at both local and country-wide levels. For local sampling, we collected and analyzed 87 hunter-collected samples from one of the southernmost Willow Ptarmigan populations in Norway. Of these birds, only three positives (3.4%) were found. For the country-wide sampling, we collected serum samples from 163 Willow Ptarmigan carcasses submitted from selected locations all over the country. Of these birds, 32% (53) were seropositive for LIV or a cross-reacting virus. Surprisingly, we found seropositive individuals from locations across the whole country, including outside the known distribution of the sheep tick. These results suggest that either LIV or a cross-reacting virus infects ptarmigan in large parts of Norway, including at high altitudes and latitudes.

Novel adenoviruses detected in British mustelids, including a unique Aviadenovirus in the tissues of pine martens (Martes martes).

Several adenoviruses are known to cause severe disease in veterinary species. Recent evidence suggests that canine adenovirus type 1 (CAV-1) persists in the tissues of healthy red foxes (Vulpes vulpes), which may be a source of infection for susceptible species. It was hypothesized that mustelids native to the UK, including pine martens (Martes martes) and Eurasian otters (Lutra lutra), may also be persistently infected with adenoviruses. Based on high-throughput sequencing and additional Sanger sequencing, a novel Aviadenovirus, tentatively named marten adenovirus type 1 (MAdV-1), was detected in pine marten tissues. The detection of an Aviadenovirus in mammalian tissue has not been reported previously. Two mastadenoviruses, tentatively designated marten adenovirus type 2 (MAdV-2) and lutrine adenovirus type 1 (LAdV-1), were also detected in tissues of pine martens and Eurasian otters, respectively. Apparently healthy free-ranging animals may be infected with uncharacterized adenoviruses with possible implications for translocation of wildlife.

One-step multiplex real time RT-PCR for the detection of bovine respiratory syncytial virus, bovine herpesvirus 1 and bovine parainfluenza virus 3.

BACKGROUND: Detection 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. RESULTS: A 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). CONCLUSIONS: The 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.