Toward the Development of a Live Attenuated Vaccine: Construction and Evaluation of a Salmonella Enteritidis Mutant Strain

Abstract

This study developed a genetically attenuated Salmonella enteritidis (S. enteritidis) strain, designated as SD01ΔMg (deficient in asd, crp, rfaL, rffG, and rfbB), to enhance vaccine safety while maintaining immunogenicity. The virulence of the mutant strain was evaluated by determining the median lethal dose (LD₅₀). The deletions significantly diminished the virulence, as evidenced by a higher LD₅₀ in chicks, reduced proliferation in HeLa and RAW264.7 cells (p < 0.01), and reduced colonization in chick organs, with no detectable bacteria by day 11 post-inoculation. The strain also exhibited nutritional dependency, with an inability to survive without DAP nutrients. Immunologically, SD01ΔMg induced robust humoral (IgG), mucosal (IgA), and cellular (IL-6, IL-10, IFN-γ, TNF-α) immune responses post-vaccination comparable to wild-type and commercial vaccine strains. The animal infection test showed that gene deletion could lead to a significant decrease in the virulence of S. enteritidis in chicks, with an approximately 10 times higher LD₅₀ for chicks, and it demonstrated significantly lower colonization in chick tissues and organs. A toxicity protection experiment on one-day-old chicks demonstrated 80 % protection against high-dose wild Salmonella infection and organ damage mitigation. The findings confirm that targeted gene deletions effectively attenuate virulence without compromising immunogenicity. Collectively, our results underscore the strong potential of the SD01ΔMg strain for further development into a safe and efficacious live attenuated vaccine against S. Enteritidis infection.