Virulent Aeromonas hydrophila (vAh) is an important emerging bacterial pathogen causing motile Aeromonas septicemia (MAS) in farmed catfish. Understanding the pathogenicity of the disease is essential for the development of preventive measures. In this study, we aimed to develop a bioluminescent virulent A. hydrophila (BvAh) strain to understand the pathogen-host interactions during infection. To achieve this, a new bioluminescence expression plasmid, pAKgfplux3, was constructed and mobilized to vAh. Catfish were challenged with BvAh using immersion, injection, and adipose fin clip procedures, and bioluminescence signal was tracked in live catfish during infection. We developed a novel BvAh strain for the first time, conducted imaging of BvAh in live fish, detected infection routes and attachment sites of the pathogen, and determined target organs, which provided new insights on the pathogenesis of vAh. MAS progressed better in fish when protection of skin was bypassed. Abraded skin seems to provide a potential portal of entry during vAh infection.

Aeromonas hydrophila is a Gram-negative, rod shaped, facultative anaerobic bacterium that is ubiquitous to freshwater and slightly brackish aquatic environments and can cause infections in fish, humans, reptiles, and avian species. Recent severe outbreaks of disease in catfish aquaculture have been associated with a highly virulent Aeromonas hydrophila strain (VAH) that is genetically distinct from less virulent strains. Given that A. hydrophila is known to infect birds, we hypothesized that fish eating birds may serve as a reservoir for VAH and spread the pathogen by flying to uninfected ponds. Great Egrets were used in this transmission model because these wading birds frequently predate catfish farms. We found that Great Egrets that were fed VAH infected catfish shed VAH demonstrating their potential to spread VAH. Histologically there were changes found in selected tissue samples.

Virulent Aeromonas hydrophila (vAh) is a causative agent of motile Aeromonas septicemia (MAS) in catfish. There are limitations in the current therapeutic and preventative strategies against vAh. The pathogenesis of MAS as well as the immune response of catfish to vAh infection are poorly understood. The aim of this study is to: 1) develop a dual live attenuated vaccine against MAS and enteric septicemia of catfish caused by Edwardsiella ictaluri; and 2) evaluate the vAh bacterial load and gene expression patterns in catfish tissues following vAh infection. Previously, six recombinant vAh proteins (outer membrane protein, TonB-dependent receptor, three fimbrial proteins, and an ATPase) were identified to have vaccine efficacy against MAS, and live attenuated E. ictaluri vaccine strain ESC-NDKL1 was identified as an effective vector for expressing vAh antigens. A total of 29 recombinant ESC-NDKL1 strains have been constructed with the integration of one, two, or three genes encoding vAh antigens into the ESC-NDKL1 chromosome. Vaccine efficacy of the constructed strains was evaluated in channel catfish fingerlings. Four recombinant ESC-NDKL1 strains expressing two vAh antigens (ESC-NDKL1::atpase::fimMrfG, ESC-NDKL1::fim::fimMrfG, ESC-NDKL1::tdr::fimMrfG, and ESC-NDKL1::fim::ompA) showed significant protection against MAS with relative percent of survival (RPS) values of 55.72%, 60.18%, 61.74%, and 54.81%. Four triple recombinant ESC-NDKL1 strains (ESC-NDKL1::fimMrfG::ompA::fimA, ESC-NDKL1::atpase::fimMrfG::ompA, ESC-NDKL1::fim::fimMrfG::ompA and ESC-NDKL1::atpase::tdr::fim) showed the best protection with RPS values of 77.93%, 63.18%, 67.74%, and 82.35%. To gain a better understanding of vAh pathogenesis, catfish fingerlings were intraperitoneally injected with vAh strain ML09-119. The anterior kidney, liver, and spleen were collected for determination of vAh distribution and expression of thirteen pro-inflammatory, innate, and adaptive immune-related genes using real-time PCR. Results revealed that vAh spread into catfish tissues within 2 hours and peaked at 12 hours post-infection. vAh infection initiated a strong inflammatory response in catfish tissues. Additionally, our research revealed that surviving catfish were able to develop a primary immune response and possibly generation of memory B cells against MAS. Such information will facilitate the development of vaccines and therapeutic drugs for preventing and treating MAS outbreaks in catfish aquaculture.