Isolation And Identification Of Bacteria In Procambarus Clarkii And Analysis Of Pathogenic Characteristics Of Isolated Vibrio

Procambarus clarkii is an important cultured species.The aquaculture of crayfish suffered great losses due to frequent disease every year.There are few microorganisms in all tissues of crayfish,and conditionally pathogenic bacteria may cause disease when the natural environment changes or low immunity of the animal.Under certain conditions,the bacteria in vivo can cause the disease of crayfish,which is a potential threat to the outbreak of disease.Therefore,it is necessary to isolate and identify the bacteria from P.clarkii,especially the possible conditional pathogens.In this study,98 strains of bacteria were isolated from P.clarkii(53 strains were isolated from the intestine,45 strains were isolated from the haemolymph)according to the morphological differences of the colonies,and the 98 strains were classified into18 genera according to the bacterial 16 S r RNA.Eight Vibrio strains with significantly different colony morphology were found in the isolated bacteria.Based on 16 S r RNA multiple alignment,O1 and O139 serotype analysis,physiological and biochemical identification and specific fragment detection,5 Vibrio were identified as Vibrio mimicus,3 Vibrio were identified as Vibrio cholerae.Based on oral feeding of isolated Vibrio,intestinal pathological changes were analyzed.The experimental results showed that different Vibrio caused different damage in intestine,and the Vibrio I4(V.mimicus)and H7(V.cholerae)caused the most obvious increase in the number of bacteria in haemolymph of crayfish.The analysis of virulence genes showed that the 8 Vibrio strains all carried omp U and tox R,indicating that they may have the ability to tolerate harsh environment.Different gene of enterotoxin were detected isolated Vibrio,which indicated that they might have different enterotoxicity.Further studies showed that all the 8 Vibrio strains could degrade perineal membrane proteins,and the cell-free culture medium of V.mimicus could degrade perineal membrane proteins in a short time,while the cell-free culture medium of V.cholerae could not degrade perineal membrane proteins in the assay.In summary,98 strains of bacteria were isolated from P.clarkii in this study,and the intestinal damage caused by isolated Vibrio was analyzed,and the possible role of Vibrio in the formation of haemolyphatic flora of crayfish was evaluated.In this study,we added the data of the potential pathogen of P.clarkii,and warned that Vibrio may lead to the outbreak of disease of crayfish,which should be followed up with epidemiological investigation.