Protective Efficacy and DIVA Potential of a rfbG and galE Double-Deleted Salmonella Enteritidis Attenuated Live Vaccine in Chickens

Salmonella Enteritidis poses a significant threat to poultry health and production, leading to substantial economic losses worldwide. Transmission through contaminated poultry products poses a continuous risk to public health. Live-attenuated vaccines are a promising approach for controlling avian salmonellosis. However, achieving an optimal balance between safety, protective efficacy, and differentiating infected from vaccinated animals (DIVA) remains a major challenge. In the present study, a targeted double-gene deletion mutant designated Z11 ΔrfbGΔgalE was generated, and we systematically assessed its safety profile, protective efficacy, and DIVA potential were systematically assessed. The mutant exhibited a 1,243-fold reduction in virulence compared to the wild-type S. Enteritidis Z11 strain. Dose optimization experiments identified 5 × 10⁷ colony-forming units (CFU) as the minimum protective dose, which was subsequently used for vaccine evaluation. Following immunization, the chickens showed normal growth performance, no observable clinical abnormalities, and complete clearance of the vaccine strain from the liver and spleen within 21 d, thus presenting a favorable safety profile. Moreover, vaccination elicited strong humoral immunity, as evidenced by a significant increase in Salmonella-specific IgG levels following booster immunization. Notably, sera from chickens vaccinated with Z11ΔrfbGΔgalE were negative in a commercial slide agglutination assay, whereas sera from wild-type-infected chickens were positive, confirming its DIVA compatibility. Upon challenge with the wild-type strain, immunized chickens exhibited a 100% survival rate, compared with a 50% survival rate in the non-immunized group. In addition, vaccination significantly reduced bacterial colonization in the liver and spleen and prevented the development of clinical signs and pathological lesions. These findings indicate that the Z11 ΔrfbGΔgalE strain is a promising attenuated vaccine candidate that combines safety, immunogenicity, protective efficacy, and DIVA compatibility for the control of S. Enteritidis infection in poultry.