Biological Characteristics Main Analysis And Immunoprotection Evaluation Of Extracellular Products Of Photobacterium Damselae

Photobacterium damselae is the main pathogen causing the pathogenesis of many aquatic animals and is now recognized as a new pathogen in aquaculture.In the early stage of this research group,a Photobacterium damselae strain was isolated from the dead turbot,and the biological characteristics and immune protection evaluation of the bacteria and extracellular products?ECP?were carried out to reveal the Photobacterium damselae.The disease mechanism and provide reference for the prevention and control of the disease.A dominant strain was isolated from the heart of the disease,and was identified as Photobacterium damselae by traditional identification method,biochemical identification and molecular biology method;The LD₅₀ for turbot was 6.3×10⁵cfu/mL,the strain was sensitive to kanamycin and florfenicol.The ECP of Photobacterium damselae was prepared by saturated ammonium sulfate ultrafiltration concentration method.The enzymatic activity of ECP was studied by agar plate diffusion method.The hemolytic spectrum and the effect of environmental physicochemical factors on extracellular enzyme activity were detected.The protein components were initially analyzed by SDS-PAGE electrophoresis and the protein components were identified by LC-MS/MS.The results showed that the ECP of Photobacterium damselae had hemolytic enzyme,protease,lipase and lecithin activity;the hemolytic titers of rabbit,sheep,goat and turbot erythrocytes were 2³,2⁴,2⁴,2⁶,respectively;Temperature,salinity and pH all affect ECP enzyme activity.When the culture time was 48 h,the temperature was37°C,the salinity was 4%,and the pH value was 7,the effect of ECP activity was the strongest;145 protein components were detected,which were mostly located in the cytoplasm and outer membrane,and there were a wide range of organisms.The pathogenicity test of Photobacterium damselae and ECP on turbot and Cynoglossus semilaevis Gunther showed that ECP had obvious pathogenicity to both,and the lesion characteristics caused by ECP were consistent with the lesion caused by strain infection;when the ECP concentration was 0.56 mg/mL,the mortality rates of turbot and Cynoglossus semilaevis Gunther were the higher and were 70%,80%,respectively;ECP had strongest immune protection against Cynoglossus semilaevis Gunther;Cynoglossus semilaevis Gunther was swollen and accompanied by bleeding.The structure of the sputum was disordered;the hepatocyte gap was widened and necrotic;the spleen was hemorrhage,the red blood cells were filled;the intestinal mucus was increased,and the intestinal villi were necrotic.Conclusion:The ECP of Photobacterium damselae had detected 26 protein components including 26 hypothetical proteins and 26 functionally unknown proteins,including hemolytic enzyme,protease,lipase and lecithin.The enzyme activity was affected by culture time,temperature,pH and the salt concentration;Photobacterium damselae and ECP were more pathogenic to Cynoglossus semilaevis Gunther and turbot,and had better immune protection against Cynoglossus semilaevis Gunther.