The role of rpoS,hmp, and ssrAB in Salmonella enterica Gallinarum and evaluation of a triple-deletion mutant as a live vaccine candidate in Lohmann layer chickens

Salmonella enterica Gallinarum (SG) causes fowl typhoid (FT), a septicemic disease in avian species. We constructed deletion mutants lacking the stress sigma factor RpoS, the nitric oxide (NO)-detoxifying flavohemoglobin Hmp, and the SsrA/SsrB regulator to confirm the functions of these factors in SG. All gene products were fully functional in wild-type (WT) SG whereas mutants harboring single mutations or a combination of rpoS, hmp, and ssrAB mutations showed hypersusceptibility to H₂O₂, loss of NO metabolism, and absence of Salmonella pathogenicity island (SPI)-2 expression, respectively. A triple-deletion mutant, SGΔ3 (SGΔrpoSΔhmpΔssrAB), was evaluated for attenuated virulence and protection efficacy in two-week-old Lohmann layer chickens. The SGΔ3 mutant did not cause any mortality after inoculation with either 1 × 10⁶ or 1 × 10⁸ colony-forming units (CFUs) of bacteria. Significantly lower numbers of salmonellae were recovered from the liver and spleen of chickens inoculated with the SGΔ3 mutant compared to chickens inoculated with WT SG. Vaccination with the SGΔ3 mutant conferred complete protection against challenge with virulent SG on the chickens comparable to the group vaccinated with a conventional vaccine strain, SG9R. Overall, these results indicate that SGΔ3 could be a promising candidate for a live Salmonella vaccine against FT.