Abstract
Aquaculture is emerging as the fastest growing food-producing industry in the world because of the increasing demand for food fish consumption. However, diseases have caused the most destructive impact to aquaculture production and result in economic losses in the global aquaculture industry.1 Edwardsiella piscicida causes frequent disease outbreaks of edwardsiellosis in a variety of freshwater and marine fish species.2 Ichthyophthirius multifiliis (Ich), the causative agent of white spot disease, is a protozoan parasite that causes significant problems to the U.S. catfish industry.3 Vaccination would be the most effective method to prevent infectious diseases and their associated economic losses. Live recombinant immersion vaccines, which protect against several diseases by expressing protective antigens, have not yet been developed for the aquaculture industry. In this study, we have successfully designed and constructed a recombinant attenuated Edwardsiella vaccine (RAEV) vector system with regulated delayed attenuation in vivo attributes that synthesizes Ichthyophthirius multifiliis (Ich) protective antigens to enable vaccination of fish susceptible to white spot disease and edwardsiellosis. To eliminate use of plasmid vectors with drug resistance genes and to stabilize plasmid vectors in RAEVs in vivo, we characterized and deleted the E. piscicida asdA gene. The asdA mutant (χ16000) exhibits an obligate requirement for diaminopimelic acid (DAP) or a plasmid vector with the wild-type asdA gene to grow. This balanced-lethal plasmid vector-host system in E. piscicida enables synthesis of recombinant antigens to induce protective immunity against fish pathogens. The constructed RAEVs are, therefore, completely sensitive to all antibiotics. Recombinant protective antigen IAG52B from the fish pathogen I. multifiliis were synthesized by RAEV harboring Asd+ plasmids. The synthesis of i-antigen IAG52B by RAEV (RAEV-Ich) was confirmed by western blotting. Recombinant bacterial vaccines must be fully attenuated for fish hosts to avoid inducing disease symptoms while exhibiting a high degree of immunogenicity. We report here a means to achieve regulated delayed attenuation in vivo that is based on the substitution of a tightly regulated araC ParaBAD cassette for the promoters of the fur and crp genes of E. piscicida such that expression of these genes is dependent on arabinose provided during growth. Thus, following colonization of lymphoid tissues, the Fur and Crp proteins cease to be synthesized due to the absence of arabinose such that attenuation is gradually manifest in vivo to preclude induction of diseases symptoms. Our RAEV-Ich vaccine should protect teleost fish against both bacterial and parasitic infectious diseases.
Acknowledgements
University of Florida
* Presenting author
+ Student presenter
Literature Cited
1. FAO. The state of world fisheries and aquaculture, United Nations Food and Agriculture Organization, Rome. 2002.
2. Loch PT, Hawke JP, Reichley SR, Faisal M, Piero FD, Griffin MJ. 2017. Outbreaks of edwardsiellosis caused by Edwardsiella piscicida and Edwardsiella tarda in farmed barramundi (Lates calcarifer). Aquaculture. 481:202–210.
3. Dickerson H, Findly R. 2014. Immunity to Ichthyophthirius infections in fish: a synopsis. Developmental & Comparative Immunology. 43(2):290–299.