Genome-wide comparison analysis of Brucella attenuated vaccine strain BA0711 and Brucella wild-type strain M28 and establishment of a duplex qPCR differentiation method.

Brucellosis, a zoonotic infectious disease caused by Brucella species, poses a serious threat to livestock production and public health. Vaccine immunization remains a key strategy to prevent and control the disease. This study aims to elucidate the genetic basis of virulence attenuation in the Brucella attenuated vaccine strain BA0711. We completed the whole-genome sequencing and functional annotation of the Brucella attenuated vaccine strain BA0711, and conducted a systematic genomic comparison with the Brucella wild-type strain M28. The results indicate that compared to the Brucella wild-type strain M28, the Brucella attenuated vaccine strain BA0711 exhibits 199 and 144 mutations, deletions, and unique gene fragments on chromosomes I and II, respectively. Although the Brucella attenuated vaccine strain BA0711 maintains strong host adaptability and viability and immunogenicity, its genome lacks genes encoding cold shock proteins and glutamine amidotransferases. Functional annotations reveal impairments in energy metabolism, stress defense, drug resistance, virulence expression, genomic plasticity, and evolutionary potential relative to the Brucella wild-type strain M28. A differential duplex TaqMan qPCR assay was developed based on genome-wide sequencing data. By targeting specific gene fragments of the Brucella attenuated vaccine strain BA0711, the assay displayed excellent specificity, high sensitivity (1 ×10¹ copies/μL) and great repeatability (intra and inter-assay coefficients of variation below 0.5%). Testing of 100 samples confirmed the method can accurately distinguish the Brucella attenuated vaccine strain BA0711, other Brucella strains and mixed infections. This study provides a reliable reference for epidemic surveillance, prevention strategy formulation, and clinical diagnosis and treatment, which will aid in mitigating the potential threats of brucellosis to public health and animal husbandry.