Validation of a Commercial Indirect ELISA Kit for the Detection of Bovine alphaherpesvirus 1 (BoHV-1)-Specific Glycoprotein E Antibodies in Bulk Milk Samples of Dairy Cows

SIMPLE SUMMARY: Recent studies have assessed the feasibility of using commercial indirect enzyme-linked immunosorbent assays (ELISA) on bulk milk (BM) samples. Today is a need to validate a diagnostic method for granting and maintaining a correct surveillance strategy for infectious bovine rhinotracheitis (IBR). However, the poor availability of reference materials for diagnostic tests on milk samples and, the low sensitivity of blocking glycoprotein E (gE) ELISAs using milk as a matrix, represent limitations for developing new assays to control infectious diseases in livestock. This study aimed to validate a commercial indirect ELISA kit to detect antibodies to gE of Bovine alphaherpesvirus 1 (BoHV-1) from BM samples, according to the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. The samples were collected from an IBR outbreak. The findings demonstrated high diagnostic performances. Furthermore, the validated kit is an easy-to-use and economical method to discriminate between BoHV-1 infected or immunised animals with gE-deleted markers vaccines using BM samples. Moreover, milk sampling represents a fast and non-invasive procedure for animals because it avoids negative effects, such as stress, and reduces the total cost of surveillance. ABSTRACT: In this study, we validated a commercial indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies to glycoprotein E (gE) of Bovine alphaherpesvirus 1 (BoHV-1) in bulk milk (BM) samples using the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. The assay performance characteristics were evaluated using a panel of positive (n = 36) and negative (n = 80) samples with known infectious bovine rhinotracheitis (IBR) status. The assay showed adequate repeatability (within-run and between-run), with a coefficient of variability (CV%) of replicates below 30%; only two 1:40 diluted samples had a CV% above 20%. Additionally, an agreement analysis of the qualitative results of replicates led to a Gwet’s agreement coefficient of 0.99 (95% confidence interval (CI): 0.96–1.00, p < 0.001). The estimated diagnostic sensitivity (DSe) and diagnostic specificity (DSp) were 100% (95% CI: 90.3–100%) and 97.5% (95% CI: 91.3–99.7%), respectively. Overall, a good level of agreement was observed between the assay results and the true IBR status of samples (weighted Cohen’s κ: 0.96, 95% CI: 0.78–1.00). The findings demonstrate that the indirect ELISA kit validated here is an easy-to-use and economical method to differentiate infected and gE-deleted marker vaccine-immunised animals using BM samples.