Challenges of BTV-Group Specific Serology Testing: No One Test Fits All.

A newly formatted enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to bluetongue virus (BTV) was developed and validated for bovine and ovine sera and plasma. Validation of the new sandwich ELISA (sELISA) was achieved with 949 negative bovine and ovine sera from BTV endemic and non-endemic areas of Australia and 752 BTV positive (field and experimental) sera verified by VNT and/or PCR. The test diagnostic sensitivity (DSe) and diagnostic specificity (DSp) were 99.70% and 99.20%, respectively, for bovine sera, and 97.80% and 99.50%, respectively, for ovine sera. Comparable diagnostic performances were noted for the sELISA compared to four competition ELISAs. While the sensitivity of the sELISA remained unaffected by BTV-15 positive sera, the cELISAs were not as sensitive. BTV-15 is endemic to Australia, and early warning depends on sensitive diagnoses of all serotypes: endemic or incurring. The sELISA failed to discriminate against epizootic hemorrhagic disease virus (EHDV) antibodies, the most serologically related orbivirus to BTV. The ACDP cELISA and the IDEXX kit showed cross-reactivity with some EHDV serotypes, with the least cross-reactive being the VMRD and the IDVet kits. Cross-reactivities, however, were also detected in sera raised experimentally from 10 isolates of the 21 known non-BTV orbiviruses. In this case, the sELISA was the least affected, followed equally by the VMRD and IDVet kits, and the IDEXX kit and the ACDP cELISA were the least discriminatory. In addition to exclusivity assessment of the ELISAs, an inclusivity assessment was made for all ELISAs using well characterized reference sera positive for antibodies to all serotypes BTV-1 to BTV-24.