Self-destructing attenuated adjuvant Salmonella strains: design, construction, characterization, and evaluation of safety and efficacy following in ovo inoculation of chicken embryos

ABSTRACT Self-destructing attenuated adjuvant Salmonella (SDAAS) strains are next-generation attenuated S . Typhimurium strains designed to be used as live adjuvants. They possess diverse means to prevent persistence in vivo , survival if excreted, and to enhance the induction of innate immunity. In this study, we investigated whether SDAAS strains can be safely delivered to chicken embryos by in ovo inoculation without compromising hatchability or hatchling health. We then evaluated their growth in different media, sensitivity to complement-mediated killing, motility, serum agglutination, flagellar and LPS profiling, their ability to attach to and invade Int-407 cells, and the activation of different pattern recognition receptors displayed by HEK-Blue cells. We also determined whether these strains could induce protective immune responses in chicks following homologous challenge at day of hatch with a virulent S . Typhimurium strain by reducing intestinal colonization and organ invasion. Our findings demonstrate that SDAAS strains can be safely administered in ovo in a dose-dependent manner. Family A strains exhibit strict requirements for 2,6-diaminopimelic acid and D-alanine, while Family B strains demonstrate absolute dependence on L-arabinose for growth in liquid and solid media. Adjuvant strains demonstrate increased activation of murine receptors NOD1, NOD2, TLR4, TLR5, TLR8, and TLR9 in HEK-Blue cells compared to the parent strain χ3761, despite having lower rates of cell adhesion to and invasion of Int-407 cells. Lastly, we observed that in ovo administration of SDAAS strains reduced titers of S . Typhimurium recovered from birds as they became older, suggesting a time-dependent enhancement of protective immunity induced by SDAAS strains.