The Isolation,Identification And Pathogenicity Research Of Shewanella Colwelliana From Patinopecten Yessoensis

Yesso scallop Patinopecten yessoensis is an important mariculture shellfish in northern China.In recent years,the abscess disease of Yesso scallop occured frequently,which caused huge economic losses.In this study,diseased Yesso scallop was taken as the research object,and the morphological characteristics,physiological and biochemical characteristics and hemolysis function of the potential pathogenic bacteria—Shewanella colwelliana,were studied through the isolation and identification of the bacteria in the focus.The protein components of its extracellular products were determined by the combination of LC-MSMS,and the potential pathogenic protein information of the strain was analyzed.The main results were summarized as follows:1.Isolation,identification and pathogenic characteristics of the pathogen of Yesso scallop.In this study,individual Yesso scallop with abscess was collected in the cultivation area of the North Yellow Sea,and 12 dominant potential pathogenic bacteria were obtained by bacterial isolation and culture from the muscle of the diseased Yesso scallops.The 16S rDNA gene sequence alignment analysis showed that 12 strains had the highest similarity with Shewanella colwelliana.The YD-1 strain that had the highest sequence similarity with S.colwelliana was cultured and observed by transmission electron microscopy.It was found that the YD-1 strain was a creamy yellow colony with a diameter of 1-1.5 mm and was rod-shaped with one terminal flagella.The YD-1 strain showed negative response by Gram staining.The results of antibiotic susceptibility test showed that the YD-1 strain was highly susceptible to cefotaxime,cefoperazone,chloramphenicol and azithromycin.The YD-1 strain exhibitedαhaemolytic activity at 28℃and37℃.The results of injection experiment showed that the YD-1 strain was highly pathogenic at16℃,with the median lethal dosage(LD₅₀)of 2.08×10⁶ CFU/mL.The infective symptoms included capsid atrophy and detachment,weak obturator muscle contraction,slow response to external stimulation and brown deposition on the shell edge,which were similar to the symptoms of abscess.2.Extraction and analysis of extracellular products of pathogens from Yesso scallop.In this study,the extracellular products of YD-1 strain were extracted using plate glass method,the results showed that the highest concentration of extracellular products（ECP）was obtained after 60 h of culture at 28℃.The enzyme activity of ECP was detected by the plate diffusion method,the results showed that YD-1 strain ECP had protease activity,amylase activity and lipase activity.The protein components of ECP were preliminarily identified by SDS-PAGE gel electrophoresis and SDS-PAGE showed that the protein bands mainly ranged from 14.4 kDa to116 kDa.The injection experiment of extracellular products was conducted at 16℃,the results showed that the the median lethal dosage of ECP was 75.1μg/mL.The infective symptoms included capsid atrophy and detachment,weak obturator muscle contraction,slow response to external stimulation and brown deposition on the shell edge,which were similar to the symptoms of artificial infection of YD-1 strain in the early stage.3.Main components of extracellular products of pathogens from Yesso scallop.The extracellular products of YD-1 strain were purified by column using glucan G100 as the media and identified by mass spectrometry.The identification results showed that the extracellular products contained 233 proteins,2611 peptides and the corresponding number of secondary mass spectrometry of proteins was 5025.According to Gene Ontology annotation analysis,the extracellular proteins of YD-1 strain were participate in three biological processes:protein folding,transcription,DNA templated,10 molecular functions including metal ion binding and dehydratase activity,and one cytoplasm.The extracellular proteins groEL,ClpB,HtpG,gmhA and ropB were potential pathogenic proteins.In conclusion,this study isolated and identified the pathogen of abscess Yesso scallop—S.colwelliana,which is pathogenic to Yesso scallop with the highly sensitive to cefotaxime,cefoperazone,chloramphenicol and azithromycin.The extracellular products of the YD-1 strain have protease,amylase and lipase activities.The pathogenic proteins groEL,ClpB,HtpG,gmhA and ropB in ECP may be involved in the infection of Yesso scallop by S.colwelliana.The results of this study can provide reliable basic information for the research of the etiology and infection mechanism of S.colwelliana,as well as the prevention and control the abscess of Yesso scallop.