Recombinant Attenuated Vaccine Displaying Regulated Lysis to Confer Biological Containment and Protect Catfish against Edwardsiellosis.

We implemented a unique strategy to construct a recombinant attenuated vaccine (RAEV) with a biological containment phenotype that causes regulated bacterial cell wall lysis. This process ensures that the vaccine strain is not able to persist in the environment. The gene is responsible for the catalysis of one of the first steps in the biosynthesis of muramic acid, which is a crucial component of the bacterial cell wall. The regulated lysis phenotype was achieved by inserting the tightly regulated P cassette in place of the chromosomal promoter. Strains with this mutation require growth media supplemented with arabinose in order to survive. Without arabinose, they are unable to synthesize the peptidoglycan cell wall. Following the colonization of fish lymphoid tissues, the protein is no longer synthesized due to the lack of arabinose. Lysis is subsequently achieved in vivo, thus preventing the generation of disease symptoms and the spread of the strain into the environment. Vaccine strain χ16016 with the genotype ΔP::TT P is attenuated and shows a higher LD value than that of the wild-type strain. Studies have demonstrated that χ16016 induced TLR4, TLR5, TLR8, TLR9, NOD1 and NOD2-mediated NF-κB pathways and upregulated the gene expression of various cytokines, such as , , , and in catfish. We observed significant upregulation of the expression profiles of , and genes in different organs of vaccinated catfish. Vaccine strain χ16016 induced systemic and mucosal IgM titers and conferred significant protection to catfish against wild-type challenge. Our lysis RAEV is the first live attenuated vaccine candidate designed to be used in the aquaculture industry that displays this biological containment property.