Development and application of a novel indirect ELISA based on recombinant ORFV114 protein for serodiagnosis of Orf virus in goats.

Orf, resulting from infection with the Orf virus (ORFV), is a zoonotic disease that threatens the health of both animals and people. Reliable serological detection methods are critical for managing and preventing Orf outbreaks. The ORFV114 protein, highly conserved across ORFV strains, serves as a promising antigen for diagnostic applications. In this study, we developed a novel indirect ELISA for ORFV serodiagnosis using a recombinant ORFV114 protein. The antigen was produced in Escherichia coli via the pET-32a-ORFV114 plasmid, then purified and refolded for use in the assay. Receiver operating characteristic (ROC) analysis of sera from 40 ORFV-negative and 30 ORFV-positive goats established an optimal cut-off value of 0.2938. At this threshold, the assay achieved a diagnostic sensitivity of 93.33% and a specificity of 95%, with an excellent area under the curve (AUC) of 0.9871 (P < 0.0001). The ELISA demonstrated high specificity, with no cross-reactivity against FMDV, GTPV, or PPRV, and showed good reproducibility (CV < 10%). Testing 168 serum samples from asymptomatic goats with the assay showed a 16.67% seropositivity rate for ORFV and an approximate 93.45% concordance with the virus neutralization test, demonstrating its practical applicability. This newly established indirect ELISA assay is a sensitive and specific tool for ORFV surveillance.