Development and validation of a VirB12-based indirect ELISA for differentiating Brucella abortus infection from A19-ΔVirB12 vaccination.

Brucellosis, a widespread zoonosis, requires reliable diagnostic tools capable of differentiating infected from vaccinated animals (DIVA), particularly with the emergence of DIVA-compatible vaccines such as Brucella abortus A19-ΔVirB12. VirB12, a key immunogenic component of the type IV secretion system, represents a promising serological target for this purpose. In this study, a VirB12-based indirect ELISA (iELISA) was developed and systematically evaluated for the detection of specific antibodies Recombinant VirB12 protein was successfully expressed in Escherichia coli, purified, and used to immunize mice, generating high-titer polyclonal antiserum (1:204,800). The established iELISA demonstrated excellent repeatability (intra- and inter-assay CVs <10%), high analytical sensitivity (detection up to a 1:3200 serum dilution), and high specificity with no cross-reactivity against other common bacterial pathogens. Clinical validation was performed using 244 bovine serum samples, including 37 Brucella-positive, 20 A19-ΔVirB12-vaccinated, and 187 negative samples.The iELISA exhibited superior overall concordance (98.36%) compared with the Rose Bengal Test (RBT, 90.98%) and showed a higher overall accuracy than a commercial cELISA (91.80%). Notably, although the commercial cELISA demonstrated high sensitivity for detecting positive sera, it failed to distinguish vaccinated from infected animals (0% identification rate), whereas the iELISA correctly identified 90.00% of vaccinated sera, highlighting its clear advantage in DIVA-based diagnostics. Collectively, this assay represents a practical, reliable, and DIVA-compatible diagnostic tool with strong potential for large-scale brucellosis surveillance and control programs.