Classification, PCR Detection Of Pathogenic Edwardsiella Tarda And Functional Characterization Of EsaC Gene

Edwardsiella tarda is a Gram-negative enteric pathogen that has a wide host range. Edwardsiellosis caused by E. tarda is one of the most common fish diseases in aquaculture, causing huge economic losses. In this study, we identiified two E. tarda strains isolated from turbot (Scophthamus maximus), and established the PCR method for detection of pathogenic E. tarda, In addition, function of esaC gene was characterized, and a-triple-gene (evpH, esrB and aroA) deletion mutant was constructed for future screening of live attenuated vaccines. The results are presented as follows:1. Identification of E.tarda. Two bacteria strains, JN and MHK2 were isolated from diseased turbot from two fish farms. Artificial infection experiments in turbot showed that 50% lethal dose (LD₅₀) of JN and MHK2 were 2.51×10⁴ cfu/fish and 6.30×10³ cfu/fish, respectively. The 16S rRNA sequences of JN and MHK₂ showed 99% similarity with those of E. tarda, and these two stains showed high degree of identity with E. tarda in biochemical phenotypes. These resultes indicated that JN and MHK2 are identified as pathogenic E. tarda.2. PCR detection of pathogenic E. tarda. Type III secretion system (T3SS) is a major virulence factors of E. tarda. The T3SS apparatus gene esaV and the catalase gene katB were used as the target genes for PCR discrimination of pathogenic strains from non-pathogenic strains. Results showed that esaV was detected in all 11 pathogenic E. tarda strains but not in all 13 non-pathogenic strains; while katB was detected in all pathogenic E. tarda strains, and 4 of the non-pathogenic strains. Based on the above results, esaV can be used as a molecular marker to distinguish the pathogenic and the non-pathogenic E. tarda strains.3. Functional characterization of esaC gene: The E. tarda T3SS apparatus gene esaC was deleted by the in-frame deletion method. Westernblotting analysis showed that the esaC mutant lost its ability for secreting translocator proteins (EseB,EseC and EseD) into the culture medium. Further investigation showed that the mutant lost the autoaggregation phenotype in the culture medium. The LD₅₀ of the mutant was 10 times higher than the wild-type in the blue gourami (Trichogaster trichopterus) infection experiments. These results suggest that EsaC affects T3SS translocator proteins secretion and involved in the pathogenesis of E. tarda.4. Construction of a triple gene deletion mutant. The esrB gene encoding T3SS regulator, the evpH gene encoding one of the apparatus of T6SS (type VI secretion system) and the aroA gene encoding aromatic metabolites were deleted to obtain a tri-ple-gene deletion mutant in the genomic DNA of E. tarda. Artificial infection showed that virulence of this mutant significantly decreased with the LD₅₀>10⁸ cfu/ml. The mutant bacteria in the infected flounder (Paralichthys 0liuaceus) could not survive more than 18 days, and antibodies produced in the serum of infected fish could be detected after 21 days. These results show that the triple gene deletion mutant can be used as a candidate for further screening the live attenuated vaccine of E. tarda in the future.