Development and validation of a pentaplex assay for identification of antibodies against underdiagnosed diseases in cattle

Animal welfare and economic implications of infectious disease occurrence in cattle demand an efficient epidemiological monitoring as the foundation for the establishment of control and eradication programs. Bovine respiratory syncytial virus (BRSV), Parainfluenza virus type 3 (PI3V), Bovine Herpes virus-1 (BoHV-1), Bovine viral diarrhoea virus (BVDV), and Enzootic Bovine Leukosis virus (EBLV) cause common and underdiagnosed diseases in cattle that are endemic in most countries (WAHIS 2020). A hallmark of individual exposure to a viral pathogen is the presence of antibodies directed towards that virus. The aim of this study was to develop and validate a pentaplex assay to simultaneously detect and quantify serum antibodies against BRSV, PI3V, BoHV-1, BVDV and EBLV, as an efficient tool to yield epidemiological data. Monoplex assays were initially developed using either complete BRSV or BoHV-1 viral particles, or recombinant proteins for BVDV, EBLV or PI3V as capture antigens. In addition, 125 sera samples from unvaccinated cattle, which were classified as positive or negative towards each of the viruses by commercial ELISA kits, were used for validation. Conditions established for the monoplex assays were adopted for the pentaplex assay. The accuracy, determined by the area under the ROC curve, was greater than 0.97, and assay sensitivities and specificities were over 95% and 90% respectively for all antigens. Intra (r) and interassay (R) coefficients of variation were under 10% and 20%, respectively. Selectivity towards target viruses was shown by binding inhibition assays where unbound viruses reduced fluorescence intensities. Diagnoses agreement for samples analysed simultaneously in the monoplex and multiplex assays was almost perfect. In conclusion, a highly sensitive pentaplex assay was validated for the simultaneous identification of serum antibodies directed against BVDV, BOHV-1, PI3V, BRSV, and EBLV. The developed pentaplex assay complies with performance characteristics established by international guidelines for diagnostic tests (AOAC, Eurochem).