中华绒螯蟹致病性弗氏柠檬酸杆菌的分离鉴定及其药敏特性  

Isolation,identification and antibiotic susceptibility of pathogenic Citrobacter freundii from Eriocheir sinensis

作　　者：黄晓东 周慧华 安健 杨先乐[1,2,3] 曹海鹏[1,2,3] HUANG Xiao-dong;ZHOU Hui-hua;AN Jian;YANG Xian-le;CAO Hai-peng(National Pathogen Collection Center for Aquatic Animals(Shanghai Ocean University),Shanghai 201306,China;Shanghai Engineering Research Center for Aquaculture(Shanghai Ocean University),Shanghai 201306,China;Shanghai Collaborative Innovation for Aquatic Animal Genetics and Breeding(Shanghai Ocean University),Shanghai 201306,China;Lianyungang City Oceanic and Fishery Development Center,Lianyungang,Jiangsu 222000,China)

机构地区：[1]国家水生动物病原库(上海海洋大学),上海201306 [2]上海水产养殖工程技术研究中心(上海海洋大学),上海201306 [3]水产动物遗传育种中心上海市协同创新中心(上海海洋大学),上海201306 [4]连云港市海洋与渔业发展促进中心,江苏连云港222000

出　　处：《南方农业学报》2019年第7期1613-1619,共7页Journal of Southern Agriculture

基　　金：江苏省海洋与渔业科技创新与推广项目（Y2018-8)

摘　　要：【目的】明确中华绒螯蟹黑鳃病的病原菌及其药敏特性,为该病的临床诊断和防控提供科学依据。【方法】采用传统方法从患黑鳃病的中华绒螯蟹肝胰腺组织中分离病原菌,通过人工回归感染试验确定病原菌的致病性,利用API 20E细菌鉴定系统和16S rRNA序列分析对病原菌进行鉴定,并以K-B药敏纸片扩散法测定其药敏特性。【结果】从患黑鳃病的中华绒螯蟹肝胰腺中共分离获得4株疑似病原菌株(C1、C2、C3和C4),人工回归感染试验结果表明,仅C1菌株感染中华绒螯蟹后出现死亡,死亡率达90%,且试验中华绒螯蟹出现黑鳃、肝胰腺呈浅黄色的病症,与自然发病的症状基本一致。依据C1菌株的生理生化特性及16S rRNA序列分析结果,可确定C1菌株为弗氏柠檬酸杆菌(Citrobacter freundii),其对健康中华绒螯蟹的半数致死剂量(LD50)为2.85×105 CFU/mL。药敏试验结果表明,C1菌株对新霉素、阿米卡星、庆大霉素、链霉素、多西环素、四环素、复方新诺明、氧氟沙星和恩诺沙星等9种抗生素高度敏感,对多粘菌素B中度敏感,对氨苄西林、羧苄青霉素和万古霉素3种抗生素已产生耐药性(不敏感)。【结论】弗氏柠檬酸杆菌感染可引起中华绒螯蟹黑鳃病,且对中华绒螯蟹具有较强毒力,实际养殖生产中可选用新霉素、多西环素等渔用抗生素进行防治。【Objective】The aim of the research was to clarify the pathogenic bacteria and drug sensitivity of Eriocheir sinensis black gill disease,and provide scientific basis for clinical diagnosis,prevention and control of the disease.【Method】Bacterial isolates were isolated from naturally infected E.sinensis using traditional methods and pathogenicity of the pathogen was determined by artificial infection test.The pathogenic isolate was identified by API 20E bacterial identification system and 16S rRNA sequence analysis.Its antibiotic susceptibility was tested by Kirby-Bauer disc diffusion method.【Result】Four suspected pathogenic strains(C1,C2,C3 and C4)were isolated from the hepatopancreas of E.sinensis with black gill disease.The results of artificial regression infection showed that only strain C1 died after infected with E.sinensis.The mortality rate was 90%.In addition,E.sinensis also showed black gill and yellowish hepatopancreas.The symptoms of the disease were basically the same with natural infection.The pathogenic strain C1 was identified as Citrobacter freundii based on physiology and biochemistry characteristics and 16S rRNA sequences analysis.It had a half lethal dose(LD50)of 2.85×105 CFU/mL for E.sinensis and showed high susceptibility to compound neomycin,amikacin,gentamycin,streptomycin,doxycycline,tetracycline,sulfamethoxazole,ofloxacin and enrofloxacin.It was moderately sensitive to polymyxin B,and resistant(insensitive)to antibiotics such as ampicillin,carbenicillin and vancomycin.【Conclusion】C.freundii infection can cause black gill disease of E.sinensis,and it is highly toxic to E.sinensis.Neomycin,doxycycline and other fishery antibiotics can be used to prevent and treat the disease.

关 键 词：中华绒螯蟹 黑鳃病 弗氏柠檬酸杆菌 分离鉴定 药敏特性 

分 类 号：S945.6[农业科学—水产养殖]