Production of RNase III-knockout, auxotrophic Edwardsiella tarda mutant for delivery of long double-stranded RNA and evaluation of its immunostimulatory potential.

The artificially synthesized polyinosinic-polycytidylic acid (poly IC) has been widely used to induce type I IFN responses in various vertebrates including fish. However, as poly IC is too expensive to use in aquaculture, the development of another economical long dsRNA producing method is needed to practically use long dsRNAs in aquaculture farms for the control of infectious diseases. In the present study, to produce long dsRNAs economically, we developed a novel long dsRNA production system based on the RNase III gene deleted auxotrophic mutant E. tarda (ΔalrΔrncΔasd E. tarda) and a long dsRNA-producing vector that was equipped with two modified λ phage PR promoters arranged in a head-to-head fashion. As the present genetically engineered E. tarda cannot live without supplementation of d-alanine and DAP, environmental and medicinal risks are minimized. Olive flounder (Paralichthys olivaceus) fingerlings administered the long dsRNA-producing auxotrophic E. tarda mutant (Δalr ΔrncΔasd E. tarda) showed significantly higher expressions of TLR22, Mx1, and ISG15 genes, indicating a potential to increase type I interferon responses.