Incorporation of membrane-anchored flagellin into Salmonella Gallinarum bacterial ghosts induces early immune responses and protection against fowl typhoid in young layer chickens

The present study aimed to investigate whether the incorporation of flagellin, a TLR5 agonist, in the bacterial ghosts (BGs) of Salmonella Gallinarum can enhance protective immune responses against fowl typhoid, a septicemic disease of poultry, in chickens. BGs are empty cell envelopes derived from Gram-negative bacteria through the bacteriophage phiX174 gene E mediated lysis. In this study, the S. Gallinarum ghosts carrying flagellin were genetically constructed utilizing a lysis plasmid pJHL184-flagellin, designed for the coexpression of the flagellin and the lysis protein E. The adjuvant effect of flagellin was evaluated by immunizing seven day old brown nick layer chicks once orally with either S. Gallinarum-flagellin (SG-fliC) ghosts or S. Gallinarum (SG) ghosts alone. Our results showed that immunization with the SG-fliC ghosts elicited early and higher systemic (IgG) and mucosal (IgA) antibody responses compared to the SG ghosts alone, although not always statistically significant. Flow cytometric analysis of the CD3 + CD4+ and the CD3 + CD8+ T cell populations in peripheral blood mononuclear cells were higher in chickens immunized with the SG-fliC ghosts compared to the chickens vaccinated with the SG ghosts alone. Furthermore, the chickens immunized with SG-fliC ghosts exhibited significantly (p < 0.05) higher IL-6 and IFN-γ responses compared to the chickens vaccinated with the SG ghosts alone. On challenge with the virulent S. Gallinarum wild type strain at 28th day post immunization, 5 of 10 birds died (50%) in case of SG-fliC ghost group while 60% (6 of 10 birds died) mortality was observed in the SG ghost group. Collectively, these results suggest that the expression of flagellin in SG ghosts improves antigen-specific humoral and cell mediated immune responses, and can enhance protective efficacy of the BG-based vaccines against the virulent challenges.