Iron is a fundamental element required by most organisms, including Brucella. Several researchers have suggested that the iron response regulator (irr) and rhizobial iron regulator (rirA) genes regulate iron acquisition by Brucella abortus, influencing heme synthesis by and virulence of this pathogen. However, little is known about another Brucella species, Brucella melitensis. In this research, we successfully constructed two mutants: M5-90Δirr and M5-90ΔrirA. The adhesion, invasion, and intracellular survivability of these two mutants were evaluated in RAW264.7 cells infected with 1 × 106 CFU of M5-90Δirr, M5-90ΔrirA, or M5-90. We also tested the sensitivity of cells to hydrogen peroxide and their ability to grow. In addition, the virulence of these two mutants was evaluated in BALB/c mice. The results showed that the ability of these two mutants to invade and adhere inside the murine macrophages RAW264.7 was attenuated but their ability to replicate intracellularly was strengthened, enhancing the resistance to hydrogen peroxide. The M5-90Δirr mutant showed stronger growth ability than the parental strain under iron-limiting conditions. No differences were observed in the number of bacteria in spleen between M5-90 and M5-90Δirr at 7 or 15 days postinfection. However, the number of M5-90ΔrirA in spleen reduced significantly at 15 days postinfection. The splenic index of the M5-90Δirr group is evidently lower than that of M5-90. This is the first report that irr and rirA genes of B. melitensis are associated not only with virulence but also with growth ability. Together, our data suggest that M5-90Δirr is a promising Brucella vaccine candidate.

This study aimed to investigate the causes of abortion in cows that were mixed fed with sheep and/or goats, and the presence of B. melitensis infection in cows. PCR of 34 (28%) specimens out of 120 samples were identified as B. melitensis. The traditional bacteriological tests identified all of the isolates from sheep/cow aborted fetuses and milk as B. melitensis biovar 3. This is the first study to demonstrate B. melitensis as the main etiological agent for cows mixed fed with sheep and/or goats in XUAR, northwest of China. This may cause severe infection in the local population, and pose a potential public health risk, especially when eating or drinking the products of contaminated milk.

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