Testing of bulk tank milk from Northern Ireland dairy herds for viral RNA and antibody to bovine viral diarrhoea virus.

Bulk tank milk samples collected from 929 Northern Ireland dairy herds were classified according to their geographical location, somatic cell count and milk yield. Each sample was tested by ELISA for antibody levels to bovine viral diarrhoea virus (BVDV) and the herds were assigned to four groups with increasing antibody levels. In nine herds (1 per cent) no antibodies were detectable in the milk samples (group 1) and they were detectable at low levels in the milk from 90 herds (9.7 per cent, group 2), at moderate levels in 369 herds (39.7 per cent, group 3), and at high levels in the remaining 461 herds (49.6 per cent, group 4). The testing of samples from 90 herds in groups 1 and 2 after an interval of 12 months showed that the annual incidence risk for new infections with BVDV was in the range 0.133 to 0.477. There were significant relationships (P < 0.001) between the mean corrected optical density in the ELISA and herd location and somatic cell count, but not yield; the relationship with somatic cell count was linear (P < 0.01). Forty-three group 3 herds and 49 group 4 herds were selected at random and bulk milk samples were tested for BVDV by reverse transcriptase polymerase chain reaction. None of the group 3 herds was positive, but five group 4 herds were positive.

Genetic typing of ruminant pestivirus strains from Northern Ireland and the Republic of Ireland.

A study was performed to investigate the genotypes and sub-groups of pestiviruses present in ruminants in Ireland. These comprised one ovine and eighteen bovine pestiviruses from Northern Ireland and six bovine pestiviruses from the Republic of Ireland. A 288 base pair (bp) portion of the 5'-non coding region (5'-NCR) from each of 25 pestiviruses collected over a period of 31 years was amplified by reverse-transcription-polymerase chain reaction (RT-PCR) and the product directly sequenced. From each pestivirus, nucleotide sequences corresponding to bases 130 to 374 of the 5'-NCR of NADL were aligned and compared with each other and with the corresponding sequences of a number of reference, field or vaccinal strains of BVDV types I and II, border disease virus and classical swine fever virus. All of the 25 sequenced pestiviruses were found to be BVDV type Ia. These were closely related to the constituent viruses of the 2 inactivated vaccines currently licensed for use in Northern Ireland and to recent bovine isolates from England.

RFLP analysis of recent Northern Ireland isolates of infectious laryngotracheitis virus: comparison with vaccine virus and field isolates from England, Scotland and the Republic of Ireland.

Restriction fragment length polymorphism (RFLP) analysis was used to assist epidemiological investigations following the recent introduction of infectious laryngotracheitis virus (ILTV) to commercial poultry flocks in Northern Ireland (NI). A 4.9 kbp PCR product of the ILTV ICP4 gene was generated from each of 16 field isolates of ILTV originating from England, Scotland, NI and the Republic of Ireland (RoI) and of the single vaccine strain currently licenced for use within the United Kingdom. With the exception of isolate PV6/94 from RoI, all field isolates generated RFLP patterns, following digestion with HaeIII, similar to that obtained using the vaccinal strain. Following MspI digestion, NI isolates were indistinguishable from the vaccinal strain and recent English isolates. However, one English and one Scottish isolate, both made prior to the introduction of vaccination, and two isolates from RoI generated a second pattern following digestion with MspI.

Evaluation of an immunofluorescent antibody test to detect bovine herpesvirus 1-specific IgM.

An indirect immunofluorescent antibody test (IIFAT) was developed to detect bovine herpesvirus 1 (BHV-1)-specific IgM. All sera were treated with protein-G agarose prior to testing to eliminate the possibility of false-positive results due to IgM-isotype rheumatoid factor (IgM-RF). Specific IgM was first detected 8 days after experimental infection of 3 calves free of maternally derived antibody, with peak responses occurring 2-7 days later. Seroconversion was detected in all 3 calves using a single-dilution enzyme-linked immunosorbent assay. Following reinfection at 30 days postinfection, a low-level IgM response was detected in only 1 calf. Seroconversion was detected in 2 calves. There was no evidence of activation of IgM-RF by infection or reinfection with BHV-1. When 87 acute and convalescent serum pairs collected from 21 outbreaks of respiratory disease were tested, specific IgM was detected in 58 animals (66.6%) from 19 (90.5%) outbreaks. Seroconversion was detected in 44 of these animals (50.6%) from 17 outbreaks (81.0%). The correlations between these 2 assays on a calf and outbreak basis were 79.3% and 90.5%, respectively. Specific IgM was detected in 17/20 sera (85.0%) collected from an additional outbreak. No virus was detected by virus isolation or immunofluorescent staining in nasal mucus samples collected at the same time. Detection of specific IgM by IIFAT is a useful technique for the serodiagnosis of BHV-1 infection.

Evaluation of the suitability of a commercial bovine viral diarrhoea virus antigen capture ELISA for diagnostic testing.

The suitability of a commercial bovine viral diarrhoea virus (BVDV) antigen capture enzyme-linked immunosorbent assay (ELISA) for routine diagnostic testing of bovine serum samples was evaluated by comparing the ELISA results of 214 sera with those obtained after two passages in roller tube cultures of fetal bovine lung cells and immunofluorescent staining using fluorescein isothiocyanate-conjugated hyperimmune BVDV anti-serum. In addition, 208 of these samples were tested by virus isolation in a microtitre system followed by indirect immunoperoxidase staining using a pool of two non-competing pestivirus specific monoclonal antibodies. The sensitivity of the ELISA compared with virus isolation followed by immunofluorescent and immunoperoxidase staining was 47.8 and 45.8%, respectively. The corresponding figures of specificity and overall correlation were 95.3 and 95.1%, and 90.2 and 89.4%. Twenty-two of 24 pestivirus isolates from the positive blood samples were typed as BVDV-like by monoclonal antibodies, indicating that the poor sensitivity of the ELISA was not due to the presence of atypical pestiviruses in the test sample. These results suggest that this ELISA is not suitable for testing blood samples for BVDV in a diagnostic laboratory.

Evaluation of a single dilution ELISA system for detection of seroconversion to bovine viral diarrhea virus, bovine respiratory syncytial virus, parainfluenza-3 virus, and infectious bovine rhinotracheitis virus: comparison with testing by virus neutralization and hemagglutination inhibition.

A single-dilution quantitative enzyme-linked immunosorbent assay (ELISA) system, based on commercial ELISA kits, for the simultaneous detection of seroconversion to bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), parainfluenza-3 virus (PI3V), and infectious bovine rhinotracheitis virus (IBRV) was evaluated by testing acute and convalescent serum pairs from 564 cattle in 145 outbreaks of respiratory disease. Seroconversion to BVDV, BRSV, PI3V and IBRV was detected in 8.0%, 19.0%, 13.7%, and 7.4%, respectively, of serum pairs tested. Seroconversion was detected in 60.7% of herds and 34.6% of animals tested. Infection with 2 or more viruses was found in 46.6% of these herds and in 27.2% of these animals. The majority of BVDV infections (62%) were associated with other virus infections, suggesting that BVDV may potentiate infection with other agents rather than being a primary pathogen of the respiratory tract. The results were compared with those obtained by virus neutralization and hemagglutination inhibition testing, and the sensitivity, specificity, and overall correlation were calculated. Sensitivities of 92%, 95%, 100%, and 100% were obtained for BVDV, BRSV, PI3V, and IBRV, respectively. The corresponding specificity values were 89%, 92%, 86%, and 91%. The overall correlation for each virus was 90%, 93%, 90%, and 93%, respectively. These results demonstrate that this ELISA system may be used successfully to detect seroconversion in serum pairs, highlight the frequency of multiple viral infections in outbreaks of respiratory disease, and provide further evidence of an immunosuppressive role for BVDV infections.