Torstein Sandvik

Laboratory diagnostic investigations for bovine viral diarrhoea virus infections in cattle.

There are no pathognomonic clinical signs of infection with bovine viral diarrhoea virus (BVDV) in cattle. Diagnostic investigations therefore rely on laboratory-based detection of the virus, or of virus-induced antigens or antibodies in submitted samples. In unvaccinated dairy herds, serological testing of bulk milk is a convenient method for BVDV prevalence screening. Alternatively, serological testing of young stock may indicate if BVDV is present in a herd. In BVDV positive herds, animals persistently infected (PI) with BVDV can be identified by combined use of serological and virological tests for examination of blood samples. ELISAs have been used for rapid detection of both BVDV antibodies and antigens in blood, but should preferably be backed up by other methods such as virus neutralization, virus isolation in cell cultures or amplification of viral nucleic acid. Detailed knowledge of the performance of the diagnostic tests in use, as well as of the epidemiology of bovine virus diarrhoea is essential for identification of viremic animals in affected herds.

Detection and identification of ruminant and porcine pestiviruses by nested amplification of 5′ untranslated cDNA regions

Based on published gene sequences of bovine viral diarrhoea virus (BVDV) type I and classical swine fever virus (CSFV), genus- and species-specific primers were designed to detect and identify pestivirus cDNA sequences in a nested polymerase chain reaction (PCR). The PCR primers were validated using cDNA synthesized from 146 pestivirus isolates, comprising representatives of all four so far described genotypes (BVDV type I, BVDV type II, CSFV and border disease virus), as well as others of uncertain classification. PCR products of the predicted size were amplified from all viruses with the genus-specific primers. All 53 cattle isolates, including 5 typed antigenically as BVDV type II were amplified by the internal BVDV-specific primers, but not the CSFV-specific primers. The same result was found for other BVDV type I and II viruses isolated from sheep and pigs. Seventy-seven CSF viruses were amplified by their respective internal primers. Available information strongly indicate that 4 CSF viruses also amplified by the BVDV-specific primers had been contaminated with BVDV in cell cultures. Border disease viruses were mostly not detected by the BVDV-specific primers, but were detected weakly by the CSFV-specific primer pair. Using carrier RNA for extraction of viral RNA, the sensitivity of detection of the single and nested PCR was, respectively, 5 and 50 times higher than obtained with a cell culture assay. The RT-PCR also detected BVDV in all of 15 commercial batches of fetal calf serum examined, and verified three earlier diagnoses of CSFV by detecting specific gene sequences in 30 year old frozen archival organ samples.

Antigenic and genetic characterisation of border disease viruses isolated from UK cattle

Available empirical data on the natural occurrence of ruminant pestiviruses has shown that in cattle, bovine viral diarrhoea virus (BVDV) is nearly exclusively found, whereas both border disease virus (BDV) and BVDV can be isolated from sheep. During routine genetic typing of pestivirus RNA from UK cattle diagnosed as BVDV positive between 2006 and 2008, five samples that were classified as BDV positive yielded positive virus isolates in cell cultures. The samples originated from animals that had shown signs typical for BVD. Phylogenetic analysis of the bovine BDVs showed that two belonged to the BDV-1a group and three to the BDV-1b group, thereby matching the genetic diversity seen for previously described UK ovine BDVs. Antigenic typing with a set of monoclonal antibodies (MABs) showed that all bovine BDVs lacked one or more epitopes conserved among ovine BDV-1 isolates, and that they had gained reactivity with at least one BVDV-1 specific MAB. Serial passaging of two of the virus isolates in ovine cell cultures did not change the epitope expression pattern. These findings suggest that the presumed natural resistance of cattle against infection with BDV no longer holds. A consequence of this is that BVD diagnostic assays should be checked for their ability to also detect BDV, and also highlights the need for monitoring of the BDV status in sheep that may be in contact with cattle in areas with organised BVD control programmes.

Viral Dose and Immunosuppression Modulate the Progression of Acute BVDV-1 Infection in Calves: Evidence of Long Term Persistence after Intra-Nasal Infection

Bovine viral diarrhoea virus (BVDV) infection of cattle causes a diverse range of clinical outcomes from being asymptomatic, or a transient mild disease, to producing severe cases of acute disease leading to death. Four groups of calves were challenged with a type 1 BVDV strain, originating from a severe outbreak of BVDV in England, to study the effect of viral dose and immunosuppression on the viral replication and transmission of BVDV. Three groups received increasing amounts of virus: Group A received 102.55TCID50/ml, group B 105.25TCID50/ml and group C 106.7TCID 50/ml. A fourth group (D) was inoculated with a medium dose (105.25TCID50/ml) and concomitantly treated with dexamethasone (DMS) to assess the effects of chemically induced immunosuppression. Naïve calves were added as sentinel animals to assess virus transmission. The outcome of infection was dose dependent with animals given a higher dose developing severe disease and more pronounced viral replication. Despite virus being shed by the low-dose infection group, BVD was not transmitted to sentinel calves. Administration of dexamethasone (DMS) resulted in more severe clinical signs, prolonged viraemia and virus shedding. Using PCR techniques, viral RNA was detected in blood, several weeks after the limit of infectious virus recovery. Finally, a recently developed strand-specific RT-PCR detected negative strand viral RNA, indicative of actively replicating virus, in blood samples from convalescent animals, as late as 85 days post inoculation. This detection of long term replicating virus may indicate the way in which the virus persists and/or is reintroduced within herds.