The Brucella abortus A19ΔfeuPΔfeuQ double-mutant is highly attenuated and confers protection in BALB/c mice.

Brucellosis is a globally significant zoonotic disease that affects both animals and humans. Current vaccines against Brucella abortus (B. abortus), such as A19, suffer from several limitations, including residual virulence in animals and humans and the inability to serologically differentiate infected from vaccinated animals. Here, we describe attenuated strains that match the protective efficacy of the current vaccine but offer a substantially improved safety profile and enable differentiation from infection, addressing key limitations that have hampered current control tools. We constructed a double-gene deletion mutant (A19ΔfeuPΔfeuQ) from A19 by removing genes encoding a two-component regulatory system (TCS) located on chromosome II. The A19ΔfeuPΔfeuQ mutant exhibited a >1.5-log reduction in intracellular survival and BALB/c mice, indicating marked attenuation. Vaccination with this mutant induced significantly higher titers of IgG, and provided a 2.34-log greater reduction in bacterial burden at 4 weeks post-challenge. Additionally, the FEUP and FEUQ proteins served as specific antigens enabling serological differentiation between infected and vaccinated animals. These findings demonstrate that the highly attenuated A19ΔfeuPΔfeuQ mutant is a promising live vaccine candidate against bovine brucellosis, combining efficacy, improved safety, and diagnostic compatibility.