Detection Of Pathogenic Edwardsiella Tarda And Evaluation Of Immune Effect Of Its Extracellular Products

Edwardsiellar tarda(Et) is an important pathogenic bacteria for many animals,especially cultured freshwater and marine fish,which cause significant losses to aquaculture industry.ln this paper,a detection for pathogenic Et and its protective antigen were studied.In the article,a Dot-ELISA for detecting pathogenic Et w~ estabished,further the diagnostic kit was developed. With rabbit antiserum against ECP of reference strain JEL4,ECPs of 25 Et strains were detected by Dot-ELISA; additionally,alI strains had been detected both on their virulent factor Excellular Products, (ECPs) including the hemolysin and extracellular protease(ECPase), and on their pathogenicity to mice and Xiphophorus helleri. The results showed that the animal pathogenicity of Et had good correlation with its hemolysin other than with ECPase. The agreement between Dot-ELISA of JEL4 ECP and pathogenicity to animal was up to 100%.On the base of this,the ECP antiserum was lyophilized with media as the core detegent,and the condition of Dot-ELISA was optimalized to make the kit,which can detect the pathogenic Et quickly,simply and respectedly.In order to value the immune effect of Et, 150 mice and 300 Xiphophorus be/len were immunized with the formalin killed cells(FKC) and the extracellular products(ECP) of pathogenic Et JEL4 strain. .After 7 weeks,all are chanlleged with JEL4 and other five heterologous pathogenic Et strains.The results showed that the mice immunized with FKC and ECP were all protected (5/5)against all Ets(only one strain in ECP group was except which was 4/S).For immuned Xzphophorus belleni,the protect ratios of FKC and ECP to homogeneous strain were 60% and 100% respectly.,while can抰 protect all against heterologous strains,but the death procedure was put off significantly in ECP group.According to the gene sequence of hemolysin of Et newly found,600bp contained the hemolysin was amplified by PCR with specific primers from the plasmid pEDIO2.The PCR product was cloned with pcDNA3.1 vector to construct the recombinant eukaryotic expression vector pHL and transfected sp2/0 cell by lipofectamine.The SDS-PAGE showed the I6kD protein hemolysin was expressed.