Isolation And Identification Of Vibrio Neocaledonicus And Its Pathogenicity To Yesso Scallop (Patinopecten Yessoensis)

Abscess disease is a common bacterial disease in Yesso scallop（Patinopecten yessoensis）culture.Its typical symptom is orange or pink abscess produced by adductor muscle.The frequent occurrence of abscess disease caused huge economic losses in Yesso scallop aquaculture.Studies on Yesso scallop abscess disease were limited,with Vibrio splendidus and V.chagasii being reported as pathogens of the disease.In this study,pathogenic bacteria of Yesso scallop abscess disease were isolated and characterized.The pathogenicity of V.neocaledonicus strains and extracellular products on Yesso scallop,as well as the effects of V.neocaledonicus strains on the immune system of Yesso scallop were explored by microbiology,molecular biology,cell biology and other techniques.The main research results are as follows:1.A pathogen was isolated and characterized from the lesions of moribund Yesso scallops The dominant strain was isolated from the lesion of diseased Yesso scallop,and identified as V.neocaledonicus by sequencing.The experimental strain was named M-08.The bacteria culture and transmission electron microscopy showed that V.neocaledonicus M-08 could grow on TCBS AGAR.The bacteria were round,smooth and had a terminal flagella,which was not easy to be selected on TCBS AGAR.Physiological and biochemical characteristics analysis and drug sensitivity test showed that V.neocaledonicus M-08 had the characteristics of fermentative glucose,arabinose and mannose,but was not sensitive to p-nitro-β-d galactose,flphenicol and chloramphenicol.Haemolysis test showed that the strain had significantβhemolytic activity at8-36℃,and theβhemolytic activity was the highest at 32℃,suggesting its potential pathogenic ability.In the infection experiment,the half lethal dose（LD50）of M-08 was 3.54×10⁴ CFU Ml^-1.The infection experiments showed that the adductor muscle of the infected Yesso scallop had abscess and mucus.Histological results showed that there were pathological changes in adductor muscle,mantle,gill and hepatopancreas of infected individuals compared with healthy P.yessoensis,suggesting that V.neocaledonicus M-08 was pathogenic.2.The extracellular products of V.neocaledonicus are pathogenicThe M-08 strain was cultured at different temperatures and times to obtain the extracellular products,and the highest extracellular products were found at 28℃for 60 h.SDS-PAGE analysis showed that the protein bands of extracellular products collected under different culture conditions were basically the same,but the content of extracellular products varied greatly.The activities of protease,amylase and esterase of extracellular products were positive,but haemolysis activity was negative.Different concentrations of extracellular products can induce different degrees of mortality,including low concentration group（303.6μg/m L）10%,medium concentration group（786.0μg/m L）55%,high concentration group（1545.0μg/m L）100%.The median lethal dose（LD50）of extracellular products was 22.3μg protein/g body weight.The results showed that the extracellular products of M-08 strain were pathogenic to Yesso scallop.3.V.neocaledonicus M-08 can activate the immune system of Yesso scallopUnder the stimulation of M-08 strain,the activities of superoxide dismutase（SOD）and catalase（CAT）in serum,the contents of lysozyme（LZM）and malondialdehyde（MDA）and the m RNA expression levels of Py NF-κB and Py TNFR-1 in serum and apoptosis rate of haemocytes were significantly different under the stimulation of M-08 strain,indicating that M-08stimulation can activate the antioxidant system response of Yesso scallop and induce the apoptosis of haemocytes apoptosis at the same time.After M-08 strain stimulation,SOD and CAT activities in treatment group were significantly increased at 3 h and 6 h after stimulation,which were 2.05-fold（p<0.01）and 1.62-fold（p<0.01）of control group（sterile seawater）,respectively.LZM content was significantly increased at 12 h after stimulation,which was 2.66-fold of the control group（p<0.05）,MDA content was significantly increased at 3 h,6 h,12 h,24 h,48 h and 72 h,which were 2.15-fold（p<0.01）,4.3-fold（p<0.01）,2.49-fold（p<0.01）,2.57-fold（p<0.01）,2.14-fold（p<0.01）and 1.39-fold（p<0.05）of the control group;The m RNA expression of Py NF-κB was significantly decreased at 3 h and increased at 12 h after stimulation,which were 0.47-fold（p<0.05）and 3.29-fold（p<0.05）of the control group,respectively.The m RNA expression of Py TNFR-1 in treatment group was significantly increased at 24 h and 72 h,which were 2.03-fold（p<0.05）and 2.42-fold（p<0.05）of the control group,respectively.The apoptosis rate of haemocytes in the treatment group was significantly increased at 12 h and 24 h,which were 3.17-fold（p<0.05）and 3.08-fold（p<0.01）of the control group,respectively.In conclusion,V.neocaledonicus M-08 and its extracellular product are both pathogenic for Yesso scallop.M-08 can activate the immune system and antioxidant system,and induce and apoptosis of haemocytes.The results showed that V.neocaledonicus was a potential pathogen of Yesso scallop,which provided useful information for further understanding the pathogenicity of abscess disease and developing the prevention and control strategy of Yesso scallop abscess disease in aquaculture.