Quantitative proteomics identifies GntR as a novel potential DIVA antigen for bovine brucellosis

Brucella is a zoonotic pathogen causing brucellosis. Current serological tests cannot differentiate infected from vaccinated animals (DIVA) due to antibody interference from live vaccines. This study used parallel accumulation-serial fragmentation (PASEF)-based quantitative proteomics to compare virulent S2308 and vaccine A19 strains of Brucella abortus. Among 2,131 identified proteins, 302 were differentially expressed, including 43 and 32 proteins exclusive to S2308 and A19, respectively. Bioinformatic analysis showed enrichment in metabolic and transport functions. The transcriptional regulator GntR family (GntR), with significantly high expression in S2308, was selected as a DIVA candidate antigen. Reverse transcription quantitative real-time PCR (RT-qPCR) and western blot confirmed significantly higher GntR transcription and expression in S2308 during logarithmic growth. Serological validation indicated stronger reactivity of GntR with sera from wild strain-infected cattle than from A19 vaccinated or negative cattle. An indirect enzyme-linked immunosorbent assay (iELISA) based on GntR was established and showed high sensitivity and specificity, consistent with standard bovine brucellosis diagnostics. Dynamic monitoring in a mouse model revealed that iELISA enabled DIVA differentiation from 0 to 21 days post-infection. Thus, GntR is a novel potential DIVA antigen, providing a robust serological tool to distinguish B. abortus wild strain infection from A19 vaccination in cattle. KEY POINTS: • PASEF-based quantitative proteomics to compare proteomic profiles of B. abortus S2308 and A19. • GntR transcription and expression are higher in Brucella S2308 than in A19 during logarithmic growth. • A novel potential DIVA antigen GntR was identified for bovine brucellosis.