Two auxotrophic genes that play essential roles in bacterial cell wall biosynthesis--
alanine racemase (alr) gene and
aspartate semialdehyde dehydrogenase (asd) gene--knock-out Edwardsiella tarda (Δalr Δasd E. tarda) was generated by the allelic exchange method to develop a
combined vaccine system.
Green fluorescent protein (GFP) was used as a model foreign
protein, and was expressed by transformation of the mutant E. tarda with
antibiotic resistant gene-free plasmids harboring cassettes for GFP and asd expression (pG02-ASD-EtPR-GFP). In vitro growth of the mutant E. tarda was similar to wild-type E. tarda when D-
alanine and
diaminopimelic acid (DAP) were supplemented to growth medium. However, without d-
alanine and/or DAP supplementation, the mutant showed very limited growth. The Δalr Δasd E. tarda transformed with pG02-ASD-EtPR-GFP showed a similar growth pattern of wild-type E. tarda when D-
alanine was supplemented in the medium, and the expression of GFP could be observed even with naked eyes. The virulence of the auxotrophic mutant E. tarda was decreased, which was demonstrated by approximately 10⁶ fold increase of LD₅₀ dose compared to wild-type E. tarda. To assess
vaccine potential of the present
combined vaccine system, olive flounder (Paralichthys olivaceus) were immunized with the GFP expressing mutant E. tarda, and analyzed protection efficacy against E. tarda challenge and antibody titers against E. tarda and GFP. Groups of fish immunized with 10⁷ CFU of the Δalr Δasd E. tarda harboring pG02-ASD-EtPR-GFP showed no mortality, which was irrespective to boost immunization. The cumulative mortality rates of fish immunized with 10⁶ or 10⁵ CFU of the mutant bacteria were lowered by a boost immunization. Fish immunized with the mutant E. tarda at doses of 10⁶-10⁷ CFU/fish showed significantly higher serum agglutination activities against
formalin-killed E. tarda than PBS-injected control fish. Furthermore, fish immunized with 10⁶-10⁷ CFU/fish of the mutant E. tarda showed significantly higher ELISA titer against GFP
antigen than fish in other groups. These results indicate that the present double auxotrophic genes knock-out E. tarda coupled with a
heterologous antigen expression has a great strategic potential to be used as
combined vaccines against various
fish diseases.