Aeromonas hydrophila is a Gram-negative opportunistic pathogen that affects farm-raised catfish, which is the largest aquaculture industry in the U.S. Although it is historically considered a secondary pathogen, outbreaks caused by A. hydrophila as a primary pathogen began in 2009 and caused heavy losses of market-size catfish in Alabama and Mississippi. In particular, comparative genomics revealed that the vAh isolates have approximately 54 unique and distinguishable genomic features. Options for treatment of vAh outbreaks are limited. Only one antibiotic feed additive, oxytetracycline (Terramycin®), is currently labeled by the U.S. Food and Drug Administration for MAS. Improper or continuous antibiotic use has potentialto increase antimicrobial resistance in A. hydrophila.An effective vaccination method to control MAS caused by vAh in catfish aquaculture could reduce industry use of antimicrobials to treat the disease. Under two USDA AFRI grants funded through the Animal Health program, our group showed that several recombinant surface and extracellular proteins unique to vAh have vaccine efficacy. Examples include major outermembrane protein (OmpA), fimbrial biogenesis outer membrane usher protein (Fim), fimbrialprotein (MrfG), TonB dependent receptor (Tdr), and an ATPase. Furthermore, our group discovered that a patented attenuated Edwardsiella ictaluri vaccine strain (ESC-NDKL1; ΔsdhCΔgcvPΔfrdA mutant of E. ictaluri strain (93-146) is an effective carrier for these antigens using a recombinant expression system.The long-range goal of my work is to develop a stable recombinant live attenuated ESC-NDKL1 that acts as a dual vaccine to provide protection against MAS caused by vAh and ESC caused by E. ictaluri. My hypothesis is that expression of vAh surface proteins in a live attenuated E. ictaluri vaccine will provide significant protection against MAS, and that vAh stimulates significant inflammation in the catfish immune response.My first objectives for this project is to construct and evaluate a recombinant live attenuated vaccine strain as a dual strategy to prevent MAS and ESC. I will construct stable recombinant attenuated E. ictaluri with chromosomal integration of one, two, or three genes encoding vAh protective antigens under control of the T7lac promoter. I will evaluate vaccine efficacy of recombinant attenuated ESC-NDKL1 strain(s) as dual protection against MAS and ESC by immersion and oral vaccination methods.My second objective is to determine effects of vAh strain ML09-119 on the catfish immune response. I will determine the role of innate and adaptive immune components in the channel catfish response to vAh, and I will correlate this with vAh tissue distribution during infection.Important activities that will be completed in this project include:Insertion of one, two, and three vAh genes in live attenuated E. ictaluri chromosome.Real time PCR analysis to confirm the expression of the inserted vAh genes in live attenuated E. ictaluri chromosome.Fish challenge experiment to determine the efficacy of the recombinant vaccine strains to prevent MAS and ESC in catfish.Real time PCR analysis to determine the effects of vAh on expression of immune-related genes in catfish immune-competent tissues following infection with a sublethal dose of vAh.