河南农业科学,2021,50(1):164-171Journal of Henan Agricultural Sciencesdoi :10.15933/ki.1004-3268.2021.01.021收稿日期:2020-07-06基金项目:四川省农业产业技术体系建设项目(SCCXTD -15)作者简介:邓龙君(1982-),男,四川梓潼人,高级工程师,硕士,主要从事水产动物病害研究㊂E -mail:denglongjun@大口黑鲈维氏气单胞菌的分离鉴定及其感染的病理损伤邓龙君(雅砻江流域水电开发有限公司,四川成都610051)摘要:为查明引起四川省某养殖场大口黑鲈(Micropterus salmoides )死亡的病因,采用传统方法从患病大口黑鲈内脏组织中分离病原菌,通过人工回归感染试验确定其致病性,利用细菌生化微量鉴定管和16S rRNA 序列分析鉴定病原菌,采用K -B 纸片琼脂扩散法测定病原菌的药敏特性,并采取石蜡切片技术观察病鱼病灶组织的病理特征㊂结果显示,从病鱼体内分离得到1株优势菌(MYLY01),经生理生化和16S rRNA 序列分析,确定其为维氏气单胞菌(Aeromonas veronii )㊂健康大口黑鲈感染病原菌后,出现与病鱼相似的临床症状和剖解变化,半数致死剂量(LD 50)为4.79ˑ108cfu /mL ㊂药敏试验结果显示,病原菌对头孢拉啶㊁头孢他啶㊁庆大霉素和新霉素等敏感,对多西环素㊁四环素和阿莫西林等耐药㊂病鱼临床症状表现为体表溃疡,鳃丝充血㊁出血,肝脏㊁脾脏和肾脏肿大,组织病理学观察肝脏㊁脾脏㊁肾脏和肌肉组织均表现为明显的变性㊁坏死和炎性细胞浸润㊂以上结果表明,维氏气单胞菌是引起此次大口黑鲈疾病的致病菌,通过引起器官组织病理变化造成机体损伤而死亡,养殖过程中可选用新霉素等渔用药物进行防治㊂关键词:大口黑鲈;维氏气单胞菌;分离鉴定;16S rRNA ;病理损伤中图分类号:S855.1㊀㊀文献标志码:A㊀㊀文章编号:1004-3268(2021)01-0164-08Isolation and Identification of Aeromonas veronii from Micropterus salmoides and Pathological Lesions of Its InfectionDENG Longjun(Yalong River Hydropower Development Co.,Ltd.,Chengdu 610051,China)Abstract :This study was conducted to investigate the bacterial pathogen that caused the death of Micropterus salmoides farmed in Sichuan Province.Pathogenetic bacterial was isolated from naturally infected Micropterus salmoides by using traditional methods,and the pathogenicity was determined by artificial infection test.The pathogenic isolate was identified by biochemical identification micro reagent tube and 16S rRNA sequence analysis.Its antibiotic susceptibility was tested by Kirby-Bauer discagar diffusion method,and the histopathological characterization of organs of the diseased Micropterus salmoides was further observed by conventional paraffin section technique.The pathogenic strain MYLY01was isolated from the diseased Micropterus salmoides and was identified as Aeromonas veronii based on phenotypic characteristics and 16S rRNA sequence analysis.It had an median lethal dose(LD 50)of 4.79ˑ108cfu /mL for healthy Micropterus salmoides ,and showed the same clinical symptom as the diseased Micropterus salmoides .Drug sensitivity tested results showed that the MYLY01was sensitive to cefradine,ceftazidime pentahydrate,gentamicin,neomycin and so on,but resistant to doxycycline,tetracycline,amoxicillin and so on.Clinical symptom of the diseased Micropterus salmoides showed ulcer in dorsum and side of the body,congestion and hemorrhage in gill,swelling in liver,spleen and kidney.Histopathological㊀第1期邓龙君:大口黑鲈维氏气单胞菌的分离鉴定及其感染的病理损伤observation showed obvious degeneration,necrosis and inflammation in liver,spleen,kidney and muscles. The findings confirm that A.veronii is the cause of this epidemic which induces the body damages and even death because of the pathological changes in organs,and the neomycin will be used for the control of this disease in the future.Key words:Micropterus salmoides;Aeromonas veronii;Isolation and identification;16S rRNA; Pathological lesions㊀㊀维氏气单胞菌(Aeromonas veronii)隶属气单胞菌科(Aeromonadaceae)气单胞菌属(Aeromonas),广泛分布于自然界中,有温和型(A.veronii biovar sobria)和维罗纳型(A.veronii biovar veronii)2个生物型[1],是一种可引起人㊁兽和水生生物共患的革兰氏阴性病原菌㊂现已证实,维氏气单胞菌可感染草鱼(Ctenopharyngodon idellus)[2]㊁中华鲟(Acipenser sinensis)[3]㊁西伯利亚鲟(Acipenser baeri)[4]㊁泥鳅(Misgurnus anguillicaudatus)[5]㊁花斑副沙鳅(Parabotia fasciata Dabry)[6]㊁鲱形白鲑(Coregonus clupeaformis)[7]㊁黄颡鱼(Pelteobagrus fulvidraco)[8]㊁湘华鲮(Sinilabeo decorus Tungting)[9]㊁大黄鱼(Pseudosciaena crocea)[10]㊁中华绒螯蟹(Eriocheir sinensis)[11]㊁凡纳滨对虾(Litopenaeus vannamei)[12]和大鲵(Andrias davidianus)[13]等数十种海淡水鱼虾蟹类和两栖动物类水产养殖动物,常引起出血㊁腹水和溃疡等症状,造成巨大的经济损失㊂人食用了被维氏气单胞菌污染的水产品后,可引起胃肠炎和败血症等,甚至可导致免疫力低下者死亡[14]㊂大口黑鲈(Micropterus salmoides)隶属鲈形目(Perciformes)太阳鱼科(Centrachidae)黑鲈属(Micropterus),原产于北美洲密西西比河水系,具有抗逆性强㊁适温性广㊁生长迅速㊁养殖周期短等优点,加之肉质鲜美㊁无肌间刺等特点[15],备受消费者青睐㊂自1983年引入我国以来,经多年人工养殖技术攻关与配合饲料的突破应用,大口黑鲈在我国南起广东㊁西至新疆㊁北达黑龙江等多个省份都有规模化养殖,并逐渐形成了繁育供苗㊁商品鱼养殖㊁饲料生产和食品加工等较为完善的产业链,已成为我国淡水养殖中最具前景的名优品种之一㊂随着养殖密度的不断增加㊁水体环境的恶化以及病原微生物的滋生传播,有关大口黑鲈疾病的报道日益增长㊂2019年9月,四川省某养殖场养殖的大口黑鲈发生以体表溃疡为特征的疾病,造成大量死亡㊂为明确其病因,对患病大口黑鲈进行病原菌分离鉴定,观察组织病理变化探究致病机制,药物敏感性试验筛选敏感药物,以期为大口黑鲈维氏气单胞菌病的科学诊断和有效防治提供参考㊂1㊀材料和方法1.1㊀试验动物患病大口黑鲈,体质量150g左右,采自四川省某养殖场;健康大口黑鲈,体质量180g左右,购自四川省新津县某养殖场㊂1.2㊀主要试剂脑心浸液琼脂(Brain heart infusion,BHI)购自北京欣经科生物技术有限公司;MH培养基㊁LB培养基㊁LB营养肉汤㊁细菌生化微量鉴定管购自杭州微生物试剂有限公司;细菌基因组DNA提取试剂盒购自天根生化科技(北京)有限公司;Taq DNA聚合酶和DNA Marker(DL2000)购自TaKaRa公司;药敏纸片购自杭州天和微生物试剂有限公司㊂1.3㊀剖解特征与组织病理学观察剖解病鱼,观察各组织器官的病理损伤情况,并取鳃丝㊁鳍条及体表黏液进行寄生虫检查㊂同时取患病鱼的肝脏㊁脾脏㊁肾脏㊁肠㊁鳃和肌肉等组织,经福尔马林固定,脱水㊁透明㊁石蜡包埋㊁切片㊁HE染色,中性树胶封片,在光学显微镜下观察组织损伤并拍照㊂1.4㊀病原菌的分离与形态特征观察在无菌条件下,从患病大口黑鲈的肝脏㊁脾脏㊁肾脏等组织取样,划线接种至BHI培养基,于28ħ恒温培养24~48h后,观察菌落生长形态㊁分布及一致性,然后挑取形状㊁大小㊁颜色均匀一致的优势单个菌落再次接种于LB培养基进行纯化培养,并观察菌落与细菌形态特征㊂纯化后的菌株于4ħ下保存备用㊂1.5㊀分离菌株生理生化特性及药敏试验分离菌株的生理生化特性采用微量生化法测定,参照‘常见细菌系统鉴定手册“[16]进行㊂药物敏感性试验采用K-B纸片琼脂扩散法测定㊂将分离菌株的单个菌落接种于LB营养肉汤,28ħ振荡培养24h,通过麦氏比浊法将菌液浓度调至1.5ˑ108cfu/mL㊂将新鲜菌液用无菌棉签均匀涂布于MH平板,并贴上药敏纸片28ħ培养24h,测定抑菌圈的直径并判定敏感度㊂敏感度根据杭州微生物试剂有限公司提供的标准进行判定㊂561河南农业科学第50卷1.6㊀分离菌株16S rRNA 基因序列测定及系统进化分析取新鲜菌液于12000r /min 离心2min,按照细菌基因组DNA 提取试剂盒操作说明提取DNA㊂采用16S rRNA 的通用引物[17]进行PCR 扩增㊂PCR反应程序:94ħ预变性3min;94ħ30s,56ħ30s,72ħ1.5min,共34次循环;最后,72ħ延伸5min㊂取5μL 扩增产物经1%的琼脂糖凝胶电泳检测,将PCR 产物送至成都擎科梓熙生物技术公司测序㊂将分离菌株的16S rRNA 序列与GenBank 数据库中已有气单胞菌属的序列进行BLAST 比对,采用DNA Star 软件进行多序列比对,并使用MEGA 7.1软件中的Neighbor -Joining 方法构建系统发育树㊂1.7㊀人工感染试验将分离菌株接种于LB 肉汤,28ħ振荡培养24h,参照麦氏比浊法调整菌液浓度至1.5ˑ109㊁1.5ˑ108㊁1.5ˑ107㊁1.5ˑ106C㊁1.5ˑ105cfu /mL㊂健康大口黑鲈在水温(28ʃ2)ħ中暂养7d 后,腹腔注射菌液0.2mL /尾,每组10尾;对照组注射等量的无菌生理盐水㊂接种后每隔24h 观察大口黑鲈的临床表现并统计死亡情况,连续观察14d㊂对感染后的病死鱼及时进行解剖,并进行细菌分离鉴定㊂2㊀结果与分析2.1㊀病鱼临床症状与剖解特征发病的大口黑鲈临床症状主要表现为游动缓慢,食欲降低,甚至不摄食㊂病鱼背部或体侧出现溃疡,并伴有水霉着生(图1A㊁B)㊂剖检发现,部分病A.背部溃疡;B.体侧溃疡;C.鳃丝充血㊁出血;D.肝脏肿大;E.脾脏肿大;F.肾脏肿大A.Ulcer in dorsum;B.Ulcer in side of the body;C.Congestion and hemorrhage in gill;D.Swelling in liver;E.Swelling in spleen;F.Swelling in kidney图1㊀患病大口黑鲈的剖解特征Fig.1㊀Clinical symptom of the diseasedMicropterus salmoides鱼鳃丝充血㊁出血,呈深红色(图1C);肝脏肿大,呈淡黄色或土黄色(图1D㊁E );脾脏㊁肾脏肿大(图1E㊁F),呈暗红色㊂鳃丝和体表黏液压片镜检未发现寄生虫㊂2.2㊀病原菌分离及形态观察从发病大口黑鲈的肝脏㊁脾脏㊁肾脏内分离获得1株优势菌株(编号为MYLY01),28ħ培养24h后,在LB 培养基上形成表面光滑,中央突起,边缘完整,直径1~2mm 的白色圆形菌落㊂经革兰氏染色,分离菌株为两端钝圆,多数单个排列,无荚膜,无芽孢的革兰氏阴性短杆菌(图2)㊂2.3㊀分离菌生理生化特性及药敏试验结果分离菌株的主要生理生化特性见表1㊂由表1可知,分离菌株与维氏气单胞菌的生理生化特性基本一致,初步判定其为A.veronii ㊂药敏试验结果显示,分离菌株对头孢呋辛㊁头孢他啶㊁多黏菌素B㊁新霉素㊁头孢噻呋钠㊁大观霉素㊁头孢拉啶㊁庆大霉素8种药物敏感;对四环素㊁复方新诺明㊁卡那霉素㊁阿莫西林㊁妥布霉素㊁多西环素㊁万古霉素㊁罗红霉素㊁克林霉素9种药物耐药(表2)㊂图2㊀分离菌株的革兰氏染色形态Fig.2㊀Micrograph of the isolated strainMYLY01by Gram staining2.4㊀分离菌人工感染试验结果菌液浓度为1.5ˑ109㊁1.5ˑ108㊁1.5ˑ107㊁1.5ˑ106cfu /mL 试验组的大口黑鲈在攻毒后第1天就表现出游动缓慢无力,鳍条出血等症状,第3天开始出现死亡;1.5ˑ105cfu /mL 试验组在攻毒后也出现不适反应,但未见死亡;整个试验期间对照组大口黑鲈未见异常㊂随着病情的发展,发病大口黑鲈的体表开始出现局部溃烂的典型症状㊂到试验期结束,各试验组死亡率分别为100%㊁60%㊁30%㊁10%㊁0%㊂根据寇氏法计算可得分离菌株对健康大口黑鲈的LD 50为4.79ˑ108cfu /mL㊂对人工感染发病的大口黑鲈体内再次分离细菌,获得与分离菌株形态㊁理化661㊀第1期邓龙君:大口黑鲈维氏气单胞菌的分离鉴定及其感染的病理损伤特性及16S rRNA 基因序列一致的菌株㊂2.5㊀分离菌16S rRNA 基因序列及系统发育分析通过16S rRNA 基因的通用引物扩增出1500bp的片段,纯化测序后提交NCBI 获得登录号为SUB6567018㊂将16S rRNA 基因序列与GenBank 中已有气单胞菌属的序列进行BLAST 比对,结果显示分离菌株MYHY01序列与A.veronii 的同源性最高㊂在基于分离菌株的16S rRNA 序列与数据库中已有气单胞菌属的序列建立系统进化树(图3),可判定该分离菌株为A.veronii ㊂表1㊀分离菌株MYHY01的生理生化特性Tab.1㊀Biochemical and physiological characteristics of the isolated strain MYHY01㊀Note:+means positive,-means negative.表2㊀分离菌株MYHY01的药敏试验结果Tab.2㊀Antibiotics sensitivitiesof the isolated strain MYHY01㊀Note:S means highly sensitive,I means moderately sensitive,R means insensitivity.图3㊀基于分离菌株MYHY01与相关菌株16S rRNA 基因序列构建的系统发育树Fig.3㊀Phylogenetic tree based on 16S rRNA gene sequence of the isolated strain MYHY01and other related species in Aeromonas761河南农业科学第50卷2.6㊀患病大口黑鲈组织病理特征观察病鱼肝脏肝细胞肿胀,颗粒变性㊁水泡变性,肝细胞索排列紊乱,肝血窦体积缩小(图4A)㊂脾脏淋巴细胞数量减少㊁坏死,黑素巨噬细胞中心明显,红髓淤血㊁出血(图4B)㊂肌纤维肿胀,排列紊乱,横纹消失,部分肌纤维溶解㊁断裂,炎症细胞浸润(图4C)㊂肾脏肾小管上皮细胞肿胀㊁变性㊁坏死,部分管腔内有均质红染的蛋白,形成管型;肾小球肿大,毛细血管扩张充血;肾间质疏松,造血组织细胞减少,巨噬细胞与中性白细胞浸润(图4D)㊂鳃小片上皮细胞肿胀,与毛细血管分离,毛细血管扩张㊁淤血㊁出血,鳃小片基部上皮下表增生,多量炎性细胞浸润(图4E)㊂皮肤真皮血管扩张淤血㊁出血,大量炎性细胞浸润(图4F)㊂A.肝脏肝细胞颗粒变性㊁水泡变性(箭头);B.脾脏淋巴细胞坏死(星号),红髓淤血㊁出血(箭头);C.肌纤维变性㊁坏死(箭头);D.肾小管上皮细胞变性㊁坏死(箭头),肾间质大量炎性细胞浸润(星号);E.鳃小片淤血㊁出血(箭头),上皮细胞增生(三角形)㊁炎性细胞浸润(星号);F.皮肤真皮淤血㊁出血(箭头),炎性细胞浸润(星号)A.Granular and vacuolar degeneration in hepatocytes(Arrow);B.Necrosis of lymphocyte (Asterisk ),hemorrhage of red pulp(Arrow )in spleen;C.Degeneration and necrosis in muscle (Arrow );D.Degeneration and necrosis in the renal tubular epithelial cells (Arrow )and infiltration of inflammatory cells inthe renal interstitium (Asterisk );E.Hemorrhage of branch leaf (Arrow),hyperplasia of epithelial cell(Triangle)and infiltrationof inflammatory cell(Asterisk)in gill;F.Hemorrhage of skin dermis ㊀(Arrow)and infiltration of inflammatory cells(Asterisk)in skin图4㊀感染维氏气单胞菌的大口黑鲈组织病理学损伤(HE 400ˑ)Fig.4㊀Histopathological lesions of the diseased Micropterussalmoides infected by A.veronii (HE 400ˑ)3㊀结论与讨论气单胞菌(Aeromonas sp .)感染在我国呈现广泛流行趋势,对水产养殖业造成严重危害㊂致病性气单胞菌分为3个复合群,即嗜水复合群㊁豚鼠复合群与温和复合群㊂嗜水复合群主要包括嗜水气单胞菌(A.hydrophila )和杀鲑气单胞菌(A.salmonicida )等,豚鼠复合群主要包括豚鼠气单胞菌(A.caviae )和中间气单胞菌(A.media )等,温和复合群主要包括温和气单胞菌(A.sobria )㊁维氏气单胞菌和简氏气单胞菌(A.jandaei )等[18]㊂维氏气单胞菌同嗜水气单胞菌㊁豚鼠气单胞菌被认为是气单胞菌中致病性最强的3种细菌,强致病性与其能产生气溶素㊁溶血素㊁肠毒素㊁胞外蛋白酶㊁鞭毛㊁菌毛㊁S 层蛋白㊁脂多糖和外膜蛋白等多种毒力因子有关[19]㊂研究证实,维氏气单胞菌感染草鱼[2]和花斑副沙鳅[6]分别具有6种和4种毒力因子基因㊂但本试验并未对维氏气单胞菌毒力因子进行检测,有待进一步研究㊂维氏气单胞菌是一种人㊁兽和水生动物共患的条件致病菌,现已被国家列为食品和水体安全的检测对象[20],应引起人们广泛关注和高度重视㊂维氏气单胞菌感染大口黑鲈剖解病变主要表现为体表溃疡,肝脏㊁脾脏与肾脏肿大;组织病理损伤主要表现为肌纤维呈波浪状㊁排列紊乱㊁肌纤维断裂㊁肌浆溶解,肝脏肝细胞肿胀㊁变性㊁坏死,脾脏淋巴细胞坏死㊁红髓出血,肾脏肾小管上皮细胞变性㊁坏死及肾间质大量炎性细胞浸润等,这与龙波等[21]报道的维氏气单胞菌感染加州鲈的病理损伤基本相同㊂但与维氏气单胞菌感染的其他宿主不尽相同,感染西伯利亚鲟引起心脏肿大,外表呈现凹凸不平的瘤状[4];感染中华绒螯蟹引起肝胰腺坏死[11];感染凡纳滨对虾引起软壳病,肝胰腺呈糊状[12];感染大鲵引起体表白斑㊁胃肠道出血[13]㊂维氏气单胞菌感染黄颡鱼[8]㊁中华绒螯蟹[11]㊁凡纳滨对虾[12]㊁金鱼[22]不同分离株的LD 50分别为3.98ˑ104㊁6.92ˑ104㊁8.7ˑ105㊁1.4ˑ107cfu /mL,毒力均高于本研究中的分离菌株㊂由此可见,维氏气单胞菌感染在不同宿主间会引起不同的病理损伤,这可能与宿主的种属特异性和不同菌株的毒力差异有关,毒力差异又与毒力因子种类及其数量有关,毒力因子中的气溶素则被认为是感染鱼类出血现象的主要因子[23]㊂本研究结果显示,维氏气单胞菌对头孢呋辛㊁大观霉素㊁新霉素㊁庆大霉素和多黏菌素B 等药物敏感,但对卡那霉素㊁多西环素㊁复方新诺明等药物耐药㊂结合‘水产养殖用药明白纸2019年2号“,推荐使用新霉素控制维氏气单胞菌感染疫情㊂本研究分离菌株的药敏试验结果与高彩霞等[2]㊁田甜等[3]㊁高通等[6]㊁龙波等[21]报道的维氏气单胞菌药861㊀第1期邓龙君:大口黑鲈维氏气单胞菌的分离鉴定及其感染的病理损伤敏试验结果存在差异,分析原因可能与不同菌株来源和不同地区的用药习惯有关㊂邓玉婷等[24]研究表明,广东主要水产养殖地区689株维氏气单胞菌对14种抗菌药物相对敏感,多重耐药率为1.74%;而耿昕颖等[25]研究表明,东北地区52株分离菌株中,多株对11种抗菌药物表现出5种多重耐药性, 6 10重耐药分别达到了9.62%㊁21.15%㊁40.38%㊁15.38%㊁13.46%㊂因此,在药物选择上应根据国家渔业用药准则,筛选敏感药物科学用药,避免耐药菌株的产生㊂鉴于维氏气单胞菌对水产养殖业的严重危害,针对性的免疫预防也已受到科研工作者们的重视,先后开展了DNA疫苗[26]㊁菌影疫苗[27]㊁菌蜕疫苗[28]㊁灭活疫苗[29]等的研究;另有研究表明,使用肠道益生菌对控制维氏气单胞菌感染具有一定作用[30];同时也有研究表明,五倍子㊁黄连的水煎剂对维氏气单胞菌有抑制作用[31]㊂这些研究为水产养殖上维氏气单胞菌感染的有效防控提供了重要的技术支撑,但还需开展其耐药机制研究,以期控制维氏气单胞菌耐药性的进一步产生㊂致谢:本研究得到了四川农业大学耿毅教授的大力帮助,在此表示衷心的感谢㊂参考文献:[1]㊀SREEDHARAN K,PHILIP R,SINGH I S B.Isolation andcharacterization of virulent Aeromonas veronii from asciticfluid of oscar Astronotus ocellatus showing signs ofinfectious dropsy[J].Diseases of Aquatic Organisms,2011,94(1):29-39.[2]㊀高彩霞,任燕,王庆,等.草鱼源致病性维氏气单胞菌的分离鉴定及药物敏感性分析[J].安徽农业大学学报,2018,45(3):409-415.GAO C X,REN Y,WANG Q,et al.Isolation,identification and antimicrobial susceptibility ofpathogenic Aeromonas veronii isolated from grass carp[J].Journal of Anhui Agricultural University,2018,45(3):409-415.[3]㊀田甜,张建明,杜合军.中华鲟源维氏气单胞菌的分离鉴定及其药敏特性[J].安徽农业大学学报,2017,44(6):1010-1016.TIAN T,ZHANG J M,DU H J.Isolation and identificationof Aeromonas 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