Differentiating infected from vaccinated animals: Validation and application of NH-AGID assay in heifers immunized with Brucella abortus strain A19-∆VirB12

Differentiating infected from vaccinated animals is crucial for clinical diagnosis in brucellosis control. The objective of this study was to assess the performance of native hapten agar gel immuno-diffusion(NH-AGID) in differential identification of infected heifers from vaccined with strain A19-∆VirB12 and identifying shedding animals at the serologic level. 30 heifers were vaccined with A19-∆VirB12, and corresponding <b>s</b>erum and vaginal swab samples were collected at predetermined intervals. Serological assessments were conducted using the Rose-bengal test, serum agglutination test, competitive enzyme-linked immunosorbent assay and NH-AGID. <i>Brucella</i>-positive sera were further distinguished using differential identification ELISA and NH-AGID. Vaginal swab DNA was tested using AMOS-PCR and dual real-time fluorescence PCR. Results showed that NH-AGID demonstrated a high specificity and positive predictive values (PPV) for diagnosis of brucellosis. Additionally, the diagnostic accuracy of NH-AGID for infected and shedding animals was more than 90 %. Heifers vaccinated with A19-∆VirB12 were serologically confirmed as infected by NH-AGID and also tested positive by PCR. The application of NH-AGID effectively identified infected and shedding animals from vaccination with A19-∆VirB12, thereby supporting the brucellosis decontamination efforts.