凡纳滨对虾急性肝胰腺坏死病致病菌的分离鉴定、药敏特性及其组织病理学观察  

Isolation,identification and antibiotic susceptibility of bacterial pathogen associated with acute hepatopancreatic necrosis disease in Litopenaeus vannamei and its histopathological observations

作　　者：高晓华[1] 张海强[1] 安伟[1] GAO Xiao-hua;ZHENG Hai-qiang;AN Wei(Shanghai Fisheries Research Institute,Shanghai 200433,China)

机构地区：[1]上海市水产研究所,上海200433

出　　处：《淡水渔业》2022年第3期82-90,共9页Freshwater Fisheries

基　　金：上海市市级农口系统青年人才成长计划项目[沪农青字(2017)第3-9号]。

摘　　要：为查明上海市奉贤区某对虾养殖场疑似患急性肝胰腺坏死病的凡纳滨对虾(Litopenaeus vannamei)的病原菌,本研究从患病凡纳滨对虾肝胰腺组织分离纯化到1株优势菌FHBX-1,并通过人工回归感染试验确定其致病性,利用VITEK-2全自动微生物鉴定仪和16S rRNA、热休克蛋白HSP60基因序列分析进行综合鉴定,并检测其毒力基因。同时采用K-B纸片扩散法检测病原菌的药物敏感性并通过石蜡切片观察患病凡纳滨对虾肝胰腺组织的病理特征。结果显示:菌株FHBX-1经生理生化特征测定、16S rRNA和热休克蛋白HSP60基因序列分析,综合鉴定为副溶血弧菌(Vibrio parahaemolyticus),且携带了急性肝胰腺坏死病相关的毒力基因pirA^(VP)、pirB^(VP),未携带副溶血弧菌临床主要毒力基因耐热直接溶血毒素tdh和相对耐热直接溶血毒素trh基因。人工回归感染试验结果显示,凡纳滨对虾在菌液浓度为1.0×10^(6) CFU/mL浸泡感染试验组,7 d内累计死亡率为80%,患病凡纳滨对虾具有肝胰腺颜色变白、萎缩变小,胃肠变空等症状,从病虾再次分离的优势细菌与原攻毒菌FHBX-1特性相同。组织病理学观察显示,患病凡纳滨对虾肝胰腺上皮细胞严重脱落,肝小管崩塌,呈典型的AHPND病理特征。药敏试验结果表明,菌株FHBX-1对氟苯尼考、多西环素、恩诺沙星等11种抗菌药物敏感,对新霉素、红霉素中度敏感,对链霉素、庆大霉素等5种抗菌药物耐药。本研究可为对虾急性肝胰腺坏死病的流行病学研究及药物防控提供基础依据。The present study was conducted to investigate the bacterial pathogen that Litopenaeus vannameis with acute hepatopancreatic necrosis disease(AHPND)were farmed in Fengxian,Shanghai,A dominant strain FHBX-1 was isolated from hepatopancreas(HP)of diseased L.vannamei and pathogenicity was determined by artificial infection test.The strain FHBX-1was identified by the VITEK-2 Compact Identification System and the analysis of both 16S rRNA and HSP60 gene sequence,its virulence genes were detected by PCR methods.Meanwhile,its antibiotic susceptibility was tested by K-B disc diffusion method and the histopathological characterization of HP the diseased L.vannamei was observed by conventional paraffin section technique.The results showed that the pathogenic strain FHBX-1 was identified as Vibrio parahaemolyticus based on physiological and biochemical characteristics and the analysis of both of 16S rRNA and HSP60 gene sequence.It revealed positive amplification of the virulence genes pirA^(VP) and pirB^(VP) which is related to cause AHPND.However,it showed negative amplification results for the virulent gens V.parahaemolyticus strain markers-hermostable direct hemolyticus gene tdh and related hemolysin gene trh.The artificial infection test showed that the cumulative mortality rate of L.vannamei was 80%at a concentration of 1.0×10^(6) CFU/mL in 0.1 mL bacterial solution during seven days.The diseased L.vannamei appeared empty gut and HP became pale and atrophic,and the once more dominan bacteria isolated from diseased L.vannamei had the same characteristics of the strain FHBX-1.The histological analyses showed that HP tubules collapsed with massive sloughing of HP epithelial cells,which was the typical pathological characteristics of AHPND.The drug sensitivity test results showed that the strain was highly sensitive to florfenicol,doxycycline,enrofloxacin,and other 8 antimicrobial drugs,moderately sensitive to neomycin,erythromycin,and resistant to streptomycin,gentamicin,and other 3 antimicrobial drugs.The study in order

关 键 词：凡纳滨对虾(Litopenaeus vannamei) 急性肝胰腺坏死病 副溶血弧菌(Vibrio parahaemolyticus) 药敏特征 

分 类 号：S945[农业科学—水产养殖]