Application of Native Hapten Agar Gel Immunodiffusion Assay for Differentiating Brucella-Infected Cattle from Brucella abortus A19- Vaccinated Cattle

In the present study, a native hapten agar gel immunodiffusion assay (NH-AGID) was developed to differentiate Brucella infection-elicited antibodies from vaccine-elicited antibodies. The objective was to investigate the efficiency of NH-AGID in facilitating differential diagnosis between Brucella infection and Brucella abortus A19 vaccination and to utilise this method for monitoring Brucella excretion. The sensitivity and specificity of NH-AGID for Brucella detection were evaluated. To investigate whether NH-AGID can differentiate between natural Brucella infection-elicited antibodies and B. abortus A19 vaccination-elicited antibodies, 30 calves were subcutaneously immunised with a B. abortus A19 vaccine at the dose of 5 × 10 ¹⁰ CFU, and serum samples were collected at 7, 14, 21, 30, 60, 90, 120, 150, 180, and 250 days (d). Serum samples with a positive result in Rose Bengal test (RBT) were analysed by NH-AGID. A total of 1452 clinical B. abortus A19-immunised sera samples were tested by RBT and serum tube agglutination test(SAT), and serologically positive samples for Brucellosis were detected by NH-AGID. Milk samples or vaginal swabs corresponding to sera samples with positive NH precipitation bands were detected by Real-time PCR and AMOS-PCR. Although NH-AGID showed high specificity, its sensitivity was lower than that of RBT. The results of NH-AGID assay showed that the lipopolysaccharide antigen could be detected in B. abortus A19-immunised sera collected between 60d and 150d after immunization, with a positivity rate of 56.7–16.7%. Six milk and vaginal swabs tested positive for Brucella DNA in Real-time PCR analysis.The mean of cycle threshold(Ct) was 29.6–34.1. Among these samples, 498-bp target bands were amplified in four samples, and 750-bp target bands were amplified in two samples by AMOS-PCR. The results of product sequencing and identification by NCBI BLAST showed that 4 and 2 sequences belonged to B. abortus and B. melitensis , respectively. The present study revealed that RBT followed by NH-AGID is a simple and effective approach for serum diagnosis after 60 days of immunisation with B. abortus A19 vaccine. NH-AGID can also help identify animals with active Brucellosis or those excreting Brucella cells.