Development and evaluation of an indirect ELISA for detecting ovine rotavirus antibodies based on VP6 protein.

Abstract

BACKGROUND: Ovine rotavirus infection is a major viral cause of acute diarrhea in lambs, primarily affecting those aged 1 to 7 days, and can lead to mortality in both goats and sheep. Effective serological detection is crucial for disease surveillance and control. METHODS: The VP6 gene of ovine rotavirus was cloned and expressed as a recombinant protein (rVP6) in Escherichia coli (E. coli) using the pET-30a (+) vector. An indirect ELISA (iELISA) was subsequently developed using this rVP6 protein as the coating antigen. The assay conditions were systematically optimized. The performance of the iELISA was validated by evaluating its sensitivity and specificity using a panel of reference serum samples (n = 122), and its results were compared with those of an indirect immunofluorescence assay (IFA). Assay precision was assessed by calculating intra-assay and inter-assay coefficients of variation (CV). RESULTS: The rVP6 protein was successfully expressed. The optimized iELISA, using 50 ng of antigen per well and a 1:50 serum dilution, demonstrated a sensitivity of 86.96% and a specificity of 97.98%. Comparison with IFA yielded a Kappa value of 0.864, indicating almost perfect agreement. The assay showed good repeatability (intra-assay CV = 3.4%) and reproducibility (inter-assay CV = 4.53%). CONCLUSIONS: A sensitive, specific, and reproducible iELISA based on the rVP6 protein was successfully developed for detecting antibodies against ovine rotavirus. This assay provides a reliable and cost-effective tool suitable for large scale seroepidemiological surveillance of ovine rotavirus infection.