Analysis Of The Main Exotoxin Genotypes In The Different Strains Of Aeromonas Hydrophila And Immunoproteomics Of Extracellurlar Proteins

Aeromonas hydrophila is the causative agent of fatal hemorrhagic septicemia in fish and can cause a wide variety of human infections. A.hydrophila virulence involves multiple factors, especially several extracellular products, which play an important role in its pathogenesis and immunogenicity.In order to analyze the distribution of virulence factors in 58 strains of Aeromonas hydrophila, five main extracellular factors of A. hydrophila, including aerolysin,β-hemolysin, cytotoxic enterotoxin, thermo-sensitive exoproteinase and cytotonic enterotoxin, were investigated and the primers were designed. On the basis of the detection results of four exotoxin genes, all the strains were divided into five exotoxin genotypes: 79.3% of strains were Aer~+β-hly~+ Act~+ Alt~+; 8.6% were Aer~+β-hly~+ Act~+ Alt~-;3.4% were Aer~+β-hly~+ Act~- Alt~-;3.4% were Aer~+β-hly~- Act~- Alt~-;5.2% are Aer~-β-hly~- Act~- Alt~-.It suggested that the dominant strains were those virulent strains carry with all these four exotoxin genes.In order to study the correlation between the virulent characters and the distribution of the main exotoxin genotypes in different strains of A. hydrophila, seventeen representative strains of Ah in five virulence genotypes were chosen to detect the haemolysis, cytotoxicity and pathogenicity. The results showed that: in the main virulent genotype, Aer~+ *******-hlty~+ Act~+ Alt~+,the hemolytic titer was 1:128, cytotoxic titer was 1:256 and the lethal rates in mouse and Swordtail fish (Xiphophorus helleri) were 100%. With the decrease of the virulent genes, the hemolytic titers, cytotoxicity and lethal rates also decreased.A. hydrophila is an important bacterial pathogen in modern aquacultures. The ideal antigenic proteins are important for preparing the genetic engineering subunit vaccine of Ah. An immunoproteomic assay was used to identify antigenic proteins from the total extracellular proteins of the virulent strain J-1. Eighteen protein spots on PVDF membrane were recognized using the holo-bacterial antiserum of rabbit. Antigenic proteins on a duplicate gel were excised and identified by mass spectrographic analysis. PMF or MS/MS of the protein spots were performed using the MASCOT server. Ten antigenic proteins, including some known antigenic proteins, were identified, such as hemolysin, aerolysin, nuclease, elastase and so on. Two genes of the interesting proteins, outer membrane protein(OMP) and flagellin A(FlaA), were detected among 50 strains of Ah by PCR amplification. The results showed the two genes were widely distributed. This study may become the candicate antigen for vaccine.