Small ruminant lentiviruses (SRLVs) cause lifelong persistent infections in sheep and goats, impairing productivity and restricting livestock trade. Serological enzyme-linked immunosorbent assay (ELISA) testing is the primary method for SRLV epidemiological monitoring, and most commercial ELISAs use capsid (CA), envelope, or a combination of both proteins as antigens. We previously developed a CA-based indirect ELISA (iELISA) to detect SRLV antibodies. Here, we expressed full-length recombinant SRLV Gag protein and further developed an iELISA using Gag as the coating antigen. The cut-off value of the established Gag-based iELISA was determined to be 0.093 based on sample-to-positive (S/P) ratio analysis of 100 SRLV-negative sera. Parallel testing of 228 sera using the Gag-iELISA, CA-iELISA, and a commercial ELISA showed that the positive detection rate of the Gag-iELISA was 35.96 percentage points higher than that of the CA-iELISA and 4.39 percentage points higher than that of the commercial ELISA. All samples that tested positive by either the CA-iELISA or the commercial ELISA were also positive by the Gag-iELISA. Samples testing positive by the Gag-iELISA but negative by the CA-iELISA or the commercial ELISA were confirmed as SRLV antibody-positive by Western blot (WB). Concordance analysis of the Gag-iELISA and commercial ELISA showed 95.61% overall agreement. Endpoint dilution assays demonstrated that the Gag-iELISA showed twice the analytical sensitivity of the CA-iELISA and sensitivity comparable to that of the commercial ELISA. Furthermore, testing of 761 ovine sera from Hulunbuir during 2024-2025 revealed an overall SRLV seropositivity rate of 54.80%, with regional positive rates ranging from 0.00% to 84.44%. Collectively, this Gag-based iELISA is a sensitive and practical tool for large-scale SRLV serological surveillance.

Keywords: CAEV; ELISA; MVV; SRLV; serological surveillance; small ruminant.