Aeromonas hydrophila is a reemerging 
pathogen of channel 
catfish (
Ictalurus punctatus); recent outbreaks from 2009 to 2014 have caused the loss of more than 12 million pounds of market size 
catfish in Alabama and Mississippi. 
Genome sequencing revealed a clonal group of A. hydrophila isolates with unique genetic and phenotypic features that is 
highly pathogenic in channel 
catfish. Comparison of the 
genome sequence of a representative catfish isolate (ML09-119) from this virulent clonal group with lower 
virulence A. hydrophila isolates revealed four fimbrial proteins unique to strain ML09-119. In this work, we expressed and purified four A. hydrophila fimbrial proteins (FimA, Fim, MrfG, and FimOM) and assessed their ability to protect and stimulate 
protective immunity in channel catfish fingerlings against A. hydrophila ML09-119 infection for vaccine development. Our results showed catfish immunized with FimA, Fim, FimMrfG, and FimOM exhibited 59.83%, 95.41%, 85.72%, and 75.01% relative percent survival, respectively, after challenge with A. hydrophila strain ML09-119. Bacterial concentrations in liver, 
spleen, and anterior 
kidney were significantly (p<0.05) lower in vaccinated fish compared to the non-vaccinated sham groups at 48 h post-infection. However, only the Fim immunized group showed a significantly higher 
antibody titer in comparison to the non-vaccinated treatment group (p<0.05) at 21 days post-
vaccination. Altogether, Fim and FimMrfG recombinant proteins have potential for vaccine development against virulent A. hydrophila infection. Genomic subtraction revealed three outer membrane proteins present in strain ML09-119 but not in the low 
virulence reference A. hydrophila strain; the major outer 
membrane protein OmpAI (OmpA1), TonB-dependent 
receptor (TonB-DR), and 
transferrin-
binding protein A (TbpA). Here, the genes encoding OmpAI, tonB-DR, and tbpA were cloned from A. hydrophila ML09-119 and were expressed into 
Escherichia coli. The purified recombinant OmpA, TonB-DR, and TbpA proteins had estimated molecular weights of 37.26, 78.55, and 41.67 kDa, respectively. 
Catfish fingerlings vaccinated with OmpA1, TonB-DR, and TbpA emulsified with non-
mineral oil adjuvant were protected against the subsequent A. hydrophila ML09-119 infection with 98.59%, 95.59%, and 47.89% relative percent survival (RPS), respectively. Furthermore, the mean liver, 
spleen, and anterior 
kidney bacterial loads were significantly lower in catfish vaccinated with the OmpA1 and TonB-DR than the non-vaccinated control group. ELISA demonstrated that catfish immunized with OmpA1, TonB-DR, and TbpA produce significant 
antibody response by 21 days post-immunization. Therefore, data generated during the study suggest that OmpAI and TonB-DR proteins could be used as potential candidates for vaccine development against A. hydrophila 
epidemic strain infection. However, TbpA 
protein failed to provide such strong protection. Recombinant 
ATPase from A. hydrophila also showed promise as a 
vaccine antigen. A live 
attenuated vaccine was prepared that combined the advantages of a live 
attenuated vaccine (ESC-NDKL1 (ΔgcvPΔsdhCΔfrdA) 
mutant of 
Edwardsiella ictaluri) against enteric septicemia of catfish (ESC) and three immunogenic recombinant proteins (Fim, FimMrfg, and 
ATPase) against A. hydrophila infection. Our results showed channel catfish fingerlings immersion-vaccinated with ESC-NDKL1::pETfim, ESC-NDKL1::pETmrfG, and ESC-NDKL1::pETATPase exhibited 100%, 91.67%, and 100% percent survival after challenge with the A. hydrophila ML09-119, which was significantly less than non-vaccinated group (88.89% mortality). In a second study, 
Catfish immunized with NDKL1::pETfim, ESC-NDKL1::pETmrfG, ESC-NDKL1::pETATPase had significantly (p<0.05) lower mortalities than sham-vaccinated group.