中华绒螯蟹致病性维氏气单胞菌的分离鉴定、药敏特性及其组织病理学观察

Isolation,identification and antibiotic susceptibility of pathogenic Aeromonas veronii in Eriocheir sinensis and its histopathological observations

【目的】查明引起江苏省连云港市某养殖场中华绒螯蟹死亡的病原菌及其药敏特性与组织病理特征,为该病的临床诊断和药物防控提供科学依据。【方法】采用传统方法从患病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其致病性,利用API ID32E细菌生化鉴定试剂条和16S rRNA序列分析对病原菌进行鉴定,以纸片扩散法测定病原菌的药敏特性,并通过常规石蜡切片观察患病中华绒螯蟹肝胰腺和肠道等病灶组织的病理特征。【结果】从患病中华绒螯蟹肝胰腺中分离获得1株具有致病性的病原菌株(HXH1),经生理生化特性鉴定和16S rRNA序列分析确定其为维氏气单胞菌(Aeromonas veronii)。HXH1菌株对健康中华绒螯蟹的半数致死剂量(LD50)为6.92×10^4CFU/mL,对复方新诺明、杆菌肽、奈替米星、氟苯尼考、多粘菌素B、新霉素、庆大霉素、氧氟沙星、诺氟沙星、恩诺沙星、卡那霉素、磺胺异噁唑、链霉素和甲氧嘧啶等14种抗生素高度敏感,对罗红霉素中度敏感,对多西环素、萘啶酸、阿莫西林、四环素和吡哌酸等5种抗生素已产生耐药性(不敏感)。与健康中华绒螯蟹相比,患病中华绒螯蟹的肝胰腺和肠道存在明显病理变化,具体表现为:肝胰腺细胞排列极其紊乱,局部可见坏死细胞,细胞核固缩深染;局部肠黏膜上皮组织溶解脱落至肠腔内。【结论】维氏气单胞菌对中华绒螯蟹具有较强的致病性,通过引起肝胰腺和肠道等器官组织的病理变化而造成机体损伤甚至死亡,养殖生产上可选用喹诺酮类、氨基糖苷类、磺胺类和酰胺醇类渔用抗生素进行防治。

【Objective】The present study was conducted to investigate the bacterial pathogen that caused the death of Eriocheir sinensis farmed in Lianyungang,Jiangsu and determine its antibiotic susceptibility and histopathological characterization in order to provide reference for clinical diagnosis and effective control of this disease in E. sinensis aquaculture.【Method】Bacterial isolates were isolated from naturally infected E. sinensis using traditional methods and the pathogenicity was determined by artificial infection test. The pathogenic isolate was identified by API ID32E biochemical identification reagent strip and 16S rRNA sequence analysis. Its antibiotic susceptibility was tested by disc diffusion method,and the histopathological characterization of pancreas and intestinal tract the diseased E. sinensis was further observed by conventional paraffin section technique.【Result】The pathogenic strain HXH1 was isolated from hepatopancreas of diseased E. sinensis and was identified as Aeromonas veronii based on phenotypic characteristics and 16S rRNA sequences analysis. It had an median lethal dose(LD50)of 6.92×10^4 CFU/mL for healthy E. sinensis and showed high susceptibility to compound sulfamethoxazole,bacitracin,netilmicin,florfenicol,polymixin B,neomycin,gentamycin,ofloxacin,norfloxacin,enrofloxacin, kanamycin,sulfisoxazole,streptomycin,trimethoprim,moderate susceptibility to roxithromycin and resistant( insensitivity)to doxycycline,nalidixic acid,amoxicillin,tetracycline and pipemidic acid. In addition,compared with healthy E. sinensis,the hepatopancreas and intestinal tract of diseased E. sinensis had obvious pathological change. The hepatopancreas cells of diseased E. sinensis were irregular-arranged,appeared local necrosis with pyknotic nucleus, local epithelial tissues of intestinal mucosa were dissolved and fell off into the intestinal cavity.【Conclusion】A. veronii has high pathogenicity for E. sinensis which induces the body damages and even death by causing pathological changes in hepatopancreas and intestine,fishery quinolones,aminoglycosides,sulfonamides and amphenicols antibiotics can be used as a reference for the control of this disease.