Generation of killed but metabolically active (KBMA) Edwardsiella tarda and evaluation of its potential as a protective vaccine

A technology for inactivation of pathogens in human blood products by treatment with amotosalen hydrochloride (S-59) in combination with long wavelength ultraviolet light (UVA) to decrease transfusion-mediated sepsis has been applied to make safe vaccines against human pathogenic bacteria, and the resultants were called killed but metabolically active (KBMA) bacteria. In the present study, we first generated KBMA Edwardsiella tarda and evaluated its potential as a protective vaccine in olive flounder (Paralichthys olivaceus). To prevent the restoration of division ability by removal of psoralen adducts in the bacterial chromosome through the nucleotide excision repair (NER), the uvrA and uvrB genes knock-out E. tarda (ΔuvrAB E. tarda) was produced by the allelic exchange method. The optimal condition for generation of KBMA E. tarda was exposure of the ΔuvrAB E. tarda to 100 ng/ml of S-59 and 2.8 J/cm2 of UVA irradiation. The KBMA E. tarda could not replicate but showed a high metabolic activity (measured by lactate dehydrogenase activity) that was comparable to the wild-type E. tarda. In comparison of survival rates between groups vaccinated with the same dose of bacteria, fish immunized with KBMA E. tarda showed significantly higher survival rates than fish immunized with formalin-killed cell (FKC) E. tarda. Furthermore, fish immunized with 1 × 107 CFU/fish of KBMA E. tarda showed no mortality, while PBS-injected fish showed 100% mortality. The serum agglutination titer was sharply increased by 107 CFU/fish of KBMA E. tarda compared to those of fish immunized with 106 CFU/fish of KBMA E. tarda or 107 CFU/fish of FKC E. tarda. The consistently lower serum agglutination titers against KBMA E. tarda than against FKC E. tarda in both KBMA and FKC E. tarda immunized groups suggest that some factors secreted from KBMA E. tarda might inhibit the serum agglutination activity. In conclusion, the present results showed the higher potential of KBMA E. tarda than FKC E. tarda as a prophylactic vaccine.