Prevention of disease due to hypervirulent Aeromonas hydrophila (vAh) and a global survey of vAh in farmed fish

Abstract

This dissertation documents research focusing on hypervirulent Aeromonas hydrophila (vAh) regarding its geographical distribution, potential transmission among freshwater fish species, vaccine control, and probiotic control. Chapter one reviews A. hydrophila, a Gram-negative bacterium widely recognized as an opportunistic pathogen causing Motile Aeromonas Septicemia (MAS) in diverse animal species. The review discusses the literature on the emergence of vAh as a primary pathogen in farmed fish and previous research on disease biocontrol, including vaccines and probiotics. The literature review on vAh research included geographical distribution, transmission, potential hosts, and interactions between vAh and fish. This incorporates a discussion of using vaccines and probiotics to protect fish against vAh, including their development, advantages and disadvantages, limits, and further studies. There is an emphasis on how to apply Bacillus spp. as a feed additive to protect fish against disease and/or to promote fish growth. In chapter two, a global survey of vAh among farmed freshwater fish was conducted to further understand its geographic distribution and fish hosts. Potential vAh isolates were collected from diseased fish and other environmental sources in SE Asia, South Asia, Europe, Central America, and West Africa. Carp (Cyprinus spp., Ctenopharyngodon spp., Hypophthalmichthys spp.) farmed in China, and channel catfish (Ictalurus punctatus) farmed in the US were the previously identified hosts for vAh, while striped catfish (Pangasianodon hypophthalmus) and pangas catfish (Pangasius pangasius) were identified in this global survey as susceptible hosts for this deadly pathogen in Vietnam and Cambodia. Multiple phylogenetic analyses based on vAh core genomes demonstrated the close relationship between newly identified vAh strains from Cambodia and Vietnam and carp isolates previously identified in China. These data indicate that vAh has the ability to spread in various freshwater fish species, further demonstrating its significant threat to modern aquaculture and the need for biosecurity measures to prevent its spread and effective ways to prevent disease. Chapter three evaluated an orally-delivered vaccine for vAh control in channel catfish. To protect channel catfish against vAh, an inactivated vaccine based on the A. hydrophila ML09-119 gfcD mutant was developed and tested in channel catfish fingerlings vial oral delivery, followed by immersion challenge with vAh ML09-119. The application of this orally-delivered vaccine on fish feed significantly reduced the mortality rate of channel catfish when compared to catfish fed with control feed. In addition, the vaccine protected against unique vAh strains isolated from catfish with MAS farmed in Alabama and Mississippi. Chapter four presented data on the use of B. velezensis AP193 for vAh disease control. B. velezensis AP193 was previously isolated as a probiotic due to its antibacterial effects in vitro and in vivo. It was previously published that feeding channel catfish with B. velezensis AP193 enabled increased survival after challenges with other bacterial pathogens (e.g., Edwardsiella ictaluri), resulting in pond water quality improvement and growth promotion. In this chapter, B. velezensis AP193 mutants were selected for streptomycin resistance, and some of the isolated mutants had increased production of difficidin. This secondary metabolite was previously shown to be responsible for the direct inhibition of vAh growth. The streptomycin-resistant mutant AP193strR produced 394% difficidin compared to B. velezensis AP193. Specific genes were identified from B. velezensis AP193strR that contained mutations compared to B. velezensis AP193. Further, B. velezensis AP193strR was adapted to enhanced growth on catfish feed. The isolated mutant B. velezensis AP193strR-CFA was hypothesized to result in an adapted probiotic strain that would grow more readily within the catfish gastrointestinal tract when fed with the same soy-based feed. Mutated genes were also identified for B. velezensis AP193strR-CFA that were predicted to confer enhanced growth on soy-based catfish feed. Chapter five evaluated the function of B. velezensis AP193 and its mutants as probiotics in channel catfish with in vivo pathogen challenge trials. To develop more solutions to protect channel catfish against vAh and to promote fish growth, B. velezensis AP193, B. velezensis AP193 commercial preparation, B. velezensis AP193strR, and B. velezensis AP193strR-CFA were orally delivered to channel catfish fingerlings for 30-72 days, followed by the immersion challenge with vAh ML09-119, E. ictaluri S97-773, and F. covae ALG-00-530. B. velezensis AP193 and its mutants slightly reduced fish mortalities when challenged with vAh ML09-119 and F. covae ALG-00-530 but didn’t result in significant differences in mortality reduction or growth promotion. B. velezensis AP193 strains recovered from catfish feed demonstrated 72.3% to 98.7% inhibitions against vAh ML09-119 in vitro, indicating significant potential in disease control. Still, their interactions with fish and aquatic pathogens remain for further studies. In summary, this research has deepened our understanding of vAh, including its global distribution and evaluation of vaccines and probiotics for vAh control. This hypervirulent pathogen has caused significant economic impacts on important farmed fish, like carp, channel catfish, and striped catfish. Research on its geographic distribution, potential transmission, and the development of new vaccines and probiotics based on the existing strains has provided some possible solutions for vAh control that can be used with many different fish species.