The Induction and Characterization of a Potential Rough‐Type <i>Brucella</i> Vaccine Candidate Strain RA343, and Its Safety, Protective Efficacy in Mouse and Guinea Pig Models

ABSTRACT Brucellosis is a zoonotic disease caused by Brucella spp. which seriously jeopardizes the health and safety of animals and human beings. Therefore, developing a live attenuated vaccine is a priority. In this study, a genetically stable Brucella rough RA343 strain was obtained by cross‐induction. The virulence and protective efficacy of RA343 were subsequently assessed, and RA343 showed reduction of survival ability in RAW264.7 cells and low pathogenicity in the murine model in vivo. Immunization with RA343 elevated expression levels of IFN‐γ and TNF‐α and a robust T‐cell immune response in mice. Guinea pigs were inoculated with RA343 at 1 × 10 9 CFU for single and booster immunization. After the single immunization of RA343, about 60% of guinea pigs could resist the attack of M28 or 2308 strain. The secondary immunization in guinea pigs confer 80% and 70% protection against M28 or 2308 challenges, respectively. Then, the gene expression profile of RAW264.7 cells infected with Brucella abortus A19 or RA343 was analyzed by RNA‐seq to investigate the cellular responses immediately after Brucella entry. The RNA‐seq analysis revealed that a total of 14,549 genes were significantly regulated by Brucella 1 h postinfection. The differential gene expression was predominantly associated with innate immune responses and many inflammatory pathways, such as MAPK, JAK‐STAT, and NF‐κB signaling ones. Our findings suggest that the RA343 strain is a promising novel vaccine candidate to protect animals from B. abortus and Brucella melitensis infection. Meanwhile, this study serves as a new reference for investigating the immune regulatory mechanisms of rough Brucella .