Disease Prevention in Channel Catfish (Ictalurus punctatus) Through the Use of an Attenuated Aeromonas hydrophila Vaccine or the Probiotic Effects of Bacillus velezensis AP193.

Abstract

Since 2009, Motile Aeromonas Septicemia (MAS) has accounted for production losses of over 20 million pounds of channel catfish across the southeastern United States. The causative agent is a clonal population of highly virulent Aeromonas hydrophila (vAh.) The possible route of infection has previously been unknown, but this clonal population has been shown to have a novel O-antigen biosynthesis gene cluster. Further analysis of vAh strains has also indicated that this novel O-antigen cluster was conserved in vAh strains but was unique among described bacterial O-antigens. To determine the role of this novel O-antigen gene cluster in virulence, various single-gene deletions have been induced via the use of λ red recombinase mediated gene deletions. The genes encoding the O-antigen ligase (waaL), O-antigen polymerase (wzy), and a gene located adjacent to the O-antigen ligase, gfcD, were targeted for deletion and evaluated for their effect on A. hydrophila virulence. Each mutant was used to challenge fingerling catfish by intraperitoneal injection (IP) at a dose of 1x106 CFU/fish. Both the waaL and wzy mutants retained virulence, whereas the gfcD mutant was completely attenuated in its virulence. The gfcD mutant induced an adaptive immune response against wild-type vAh and resulted in significant protection against wild-type vAh. To validate the role of gfcD in vAh pathogenicity, the wild-type gfcD gene was used to complement the gfcD mutant, which resulted in a partial restoration of virulence. GfcD and the other gene products encoded by the gfcABCD operon are predicted to be involved in the export and assembly of an O-antigen capsule and the vAh gfcD mutant was found to lack an O-antigen capsule, significant reduction in biofilm formation, and reduced buoyancy compared to wild-type vAh. Furthermore, this gfcD mutant was shown to induce an adaptive immune response that protected catfish from subsequent vAh challenges when delivered intraperitoneally into fish. A replicated in-pond raceway trial also revealed that the protection elicited by IP injecting gfcD was sufficient at protecting channel catfish from a naturalistic induction of vAh. This protection could also be quantified in blood sera antibody titers six months after vaccination, without the administration of secondary boosters. Additionally, Bacillus velezensis AP193 has been shown to have the ability to reduce mortality associated with the bacterial pathogens A. hydrophila, Edwardsiella ictaluri, and Streptococcus iniae when fed to fish at a dose of 106-107 CFU/g of feed in aquaria models. A replicated pond study at the North Fisheries ponds demonstrated that probiotic AP193-fed channel catfish had a moderate increase in growth relative to control channel catfish, and a significant reduction in the pond water phosphorus levels. This led to the hypothesis that the probiotic is degrading phytate present in feed, resulting in improved water quality. Herein is data presenting two alternative forms of infectious disease prevention in channel catfish, an attenuated mutant vaccine delivered through IP injections and a Bacillus probiotic that can enhance water quality, reduce disease, and improve growth performance when delivered as a feed amendment.