Development and validation of a recombinant Omp19+31-based indirect hemagglutination assay for serological diagnosis of bovine and ovine brucellosis.

BACKGROUND AND AIM: Brucellosis remains a major zoonotic disease affecting livestock production and public health worldwide, particularly in endemic regions such as Kazakhstan. Conventional serological assays used for animal brucellosis diagnosis are often limited by cross-reactivity associated with lipopolysaccharide-based antigens, leading to false-positive results and unnecessary culling of animals. The indirect hemagglutination assay (IHA) is a simple and economical serological method; however, its diagnostic specificity requires improvement. This study aimed to develop and validate a recombinant protein-based IHA using sheep erythrocytes sensitized with recombinant Brucella outer membrane and periplasmic proteins for the serological diagnosis of bovine and ovine brucellosis.

MATERIALS AND

METHODS

Recombinant Brucella proteins, including outer membrane protein (Omp)19, Omp25, Omp31, their chimeric combinations (Omp19+25, Omp19+31, and Omp25+31), and the periplasmic proteins Bp26 and Cu/ZnSOD, were produced in Escherichia coli BL21(DE3). Sheep erythrocytes were sensitized with recombinant proteins to prepare erythrocyte diagnostic reagents for IHA. A total of 1,440 serum samples from cattle and sheep, including samples from brucellosis-affected and brucellosis-free herds, were evaluated using IHA, indirect enzyme-linked immunosorbent assay (ELISA), and Rose Bengal plate test. In addition, biological samples from 32 seropositive cattle subjected to sanitary slaughter were examined by culture isolation and polymerase chain reaction. Diagnostic sensitivity, specificity, and accuracy were calculated with 95% confidence intervals (CI).

RESULTS

Among all tested antigens, the Omp19+31 chimeric protein demonstrated the highest diagnostic performance in IHA. The Omp19+31-based IHA achieved a sensitivity of 92.6% (95% CI: 85.5-96.4), specificity of 100% (95% CI: 99.2-100.0), and overall diagnostic accuracy of 98.8% (95% CI: 97.5-99.5). In the subset of 32 seropositive cattle, the assay detected Brucella-specific antibodies in all sera, including in culture-positive cases that were missed by commercial ELISA. The assay predominantly produced strong agglutination reactions and showed superior specificity compared with single-antigen formats. Cross-reactivity analysis confirmed that Omp19 and Omp31 exhibited high specificity toward Brucella spp. with minimal reactivity against related Gram-negative bacteria.

CONCLUSION

The recombinant Omp19+31-based IHA demonstrated excellent specificity, high diagnostic accuracy, and operational simplicity for the serological diagnosis of bovine and ovine brucellosis. The assay represents a promising complementary diagnostic tool for endemic and resource-limited settings where conventional lipopolysaccharide-based assays are limited by cross-reactivity. Further multi-center validation and assessment of differentiating infected from vaccinated animals potential are warranted before broader implementation.