中间球海胆黑嘴病病原菌的分离与鉴定  

Isolation and identification of pathogenic bacteria associated with black-mouth disease in sea urchin Strongylocentrotus intermedius

作　　者：刘岩松 张伟杰[1] 王中[1] 殷维骏 欧凡江 田文卓 刘雷 常亚青[1] LIU Yansong;ZHANG Weijie;WANG Zhong;YIN Weijun;OU Fanjiang;TIAN Wenzhuo;LIU Lei;CHANG Yaqing(Key Laboratory of Mariculture&Stock Enhancement in North China's Sea,Ministry of Agriculture and Rural Affairs,Dalian Ocean University,Dalian 116023,China)

机构地区：[1]大连海洋大学农业农村部北方海水增养殖重点实验室,辽宁大连116023

出　　处：《大连海洋大学学报》2022年第5期793-801,共9页Journal of Dalian Ocean University

基　　金：国家重点研发计划“蓝色粮仓科技创新”项目(2018YFD0901601);农业农村部农业科研杰出人才及创新团队项目;大连市高层次人才创新支持计划(2019RQ122);辽宁省教育厅科研项目(LJKZ0702)。

摘　　要：为确定中间球海胆Strongylocentrotus intermedius黑嘴病的病原,从患黑嘴病中间球海胆体腔液中分离出一株优势菌(记为D1),通过人工回感试验确定致病性后,对该致病菌进行电镜观察、革兰氏染色、生理生化鉴定和16S rRNA序列比对以鉴定其分类地位,并测定了D1菌株对30种抗生素的敏感性。结果表明:中间球海胆经D1菌株回感后,发病症状与自然状态下患黑嘴病海胆症状相同,并在回感海胆体腔液中分离出相同菌株;D1菌株长×宽为(2.10±0.31)μm×(0.68±0.08)μm,为革兰氏阴性菌;16S rRNA序列比对结果显示,D1菌株与棘皮动物弧菌Vibrio echinoideorum的一致性高达99.04%;生化鉴定结果显示,D1菌株与蔗糖、6%NaCl胨水和氧化酶反应均呈阳性;根据生理生化试验和16S rRNA分子生物学鉴定结果,确定该病原菌为棘皮动物弧菌;药敏试验显示,该菌株对氧氟沙星、多黏霉素B、复方新诺明、氨苄西林和羧苄西林等16种抗菌药物敏感,对麦迪霉素、庆大霉素等11种抗菌药物耐药。研究表明,中间球海胆黑嘴病的病原菌为棘皮动物弧菌,且该菌株对部分抗菌药物不敏感,具有一定的耐药性。In order to probe into the pathogen of black mouth disease in sea urchin Strongylocentrotus intermedius,a dominant strain(D1)was isolated from the coelomic fluid of the sea urchin with the black mouth disease and then was artificially challenged to the healthy sea urchin to confirm the pathogenicity.The pathogenic bacteria were subjected to electron microscopic observation,gram staining,physiological and biochemical identification and 16S rRNA sequence comparison to identify its classification.The sensitivity of the strain to 30 antibiotics was also detected.The results showed that after being alleloped by D1 strain,the symptoms of the sea urchin with bulbus intermedius are the same as those of the sea urchin with black mouth disease in the natural state and the same strain was isolated in the humor of the sea urchin cavity.It was found that the bacterium was(2.10±0.31)μm×(0.68±0.08)μm(length×width)in size,with gram negative.16S rRNA comparison showed that the bacterium was similar to Vibrio echinoideorum with as high as 99.04%of similarity.The bacterium reacted positively with sucrose,6%sodium chloride solution,and oxidase.The physiological and biochemical test and the molecular biology identification results of 16S rRNA revealed that the pathogen was V.echinoideorum.The susceptibility test showed that the strain was sensitive to 16 antibacterial drugs such as ofloxacin,polymyxin B,compound trimoxazole,ampicillin,carboxicillin,and cefazolin and resistant to 11 antibacterial drugs including medithromycin and gentamicin.The finding indicated that V.echinoideorum was the pathogen of the black-mouth disease in sea urchin,which is not sensitive to some antibacterial drugs and has certain drug resistance.

关 键 词：中间球海胆 黑嘴病 病原鉴定 

分 类 号：S947.9[农业科学—水产养殖]