鲫鱼致病性维氏气单胞菌的分离鉴定及生物学特性分析  

Isolation,identification and biological characteristics analysis of pathogenic Aeromonas veronii from crucian carp

作　　者：齐燕玲 方琼亚 丁妍 杨炳坤[2] 黄淇星 薛智洋 陈思[1] 胡曼洁 王丽霞 张蕾[1,4] QI Yanling;FANG Qiongya;DING Yan;YANG Bingkun;HUANG Qixing;XUE Zhiyang;CHEN Si;HU Manjie;WANG Lixia;ZHANG Lei(College of Veterinary Medicine/College of Animal Science and Technology,Jilin Agricultural University,Changchun 130118,China;Jilin Academy of Fishery Sciences,Changchun 132109,China;Nongan County Center for Animal Disease Prevention and Control,Changchun 130299,China;Changchun Borui Agriculture and Animal Husbandry Group Co.,Ltd.,Changchun 130114,China)

机构地区：[1]吉林农业大学动物医学学院/动物科学技术学院,长春130118 [2]吉林省水产科学研究院,长春132109 [3]农安县动物疫病预防控制中心,长春130299 [4]长春博瑞农牧集团股份有限公司,长春130114

出　　处：《黑龙江畜牧兽医》2023年第4期64-71,130,131,共10页Heilongjiang Animal Science And veterinary Medicine

基　　金：财政部和农业农村部国家现代农业产业技术体系项目(CARS-45);吉林省科技发展计划项目(20210508006RQ)。

摘　　要：为了分离并鉴定吉林省松原市查干湖地区引起鲫鱼多种脏器出血导致死亡的病原菌,试验从患病鲫鱼的鳃及脏器中进行病原菌的分离纯化,对分离的病原菌进行形态观察、生化特性鉴定、16S rDNA和管家基因gyrB PCR扩增及序列鉴定、动物回归试验(锦鲫鱼和斑马鱼)、半数致死量(LD50)的测定、毒力基因的检测、生物学特性的检测及药敏试验。结果表明:从患病鲫鱼脾脏中分离纯化得到一株优势菌株,命名为PC-1,根据生化特性初步判断该菌株为维氏气单胞菌。分离菌株PC-1的16S rDNA基因与管家基因gyrB经PCR扩增后,分别得到大小约为1470 bp和1022 bp的目的条带,目的条带测序后分别与GenBank中已公布的序列进行BLAST比对分析,发现与其他维氏气单胞菌的核苷酸相似性在99%以上。构建的进化树显示分离菌株PC-1与其他维氏气单胞菌菌株处于同一分支。健康锦鲫鱼感染PC-1后的临床症状与自然发病鲫鱼相似,经计算,锦鲫鱼的LD50为2.0×10^(5)cfu/mL,斑马鱼的LD50为3.2×10^(4)cfu/mL。该菌携带Ⅳ型菌毛(tapA)基因、气溶素(aer)基因、细胞毒性肠毒素(act)基因、细胞兴奋性肠毒素(alt)基因、极性鞭毛(fla)基因、核酶(exu)基因、丝氨酸蛋白酶(ser)基因、弹性蛋白酶(ahyB)基因、酯酶(lip)基因9种毒力基因,不携带热稳定肠毒素(ast)基因。分离菌株PC-1培养24 h后形成较为稳定的生物被膜且生物被膜形成能力最强,同时OD600值在1.8以上。分离菌株PC-1对红霉素、羧苄西林和氨苄西林3种抗生素耐药,对米诺环素、庆大霉素和头孢氨苄3种抗生素中度敏感,对多西环素、四环素、新霉素等13种抗生素敏感。说明吉林省松原市查干湖地区鲫鱼感染的病原菌为维氏气单胞菌,该菌对鲫鱼具有较强的致病性。In order to isolate and identify the pathogenic bacteria causing multiple organ hemorrhages leading to death of crucian carp in Chagan Lake in Songyuan City,Jilin Province,the experiment was conducted to isolate and purify the pathogenic bacteria from the gills and visceras of diseased crucian carp.The isolated pathogenic bacteria were subjected to morphological observation,biochemical characterization identification,PCR amplification and sequence identification of 16S rDNA and housekeeping gene gyrB,animal regression test(Carassius auratus,zebrafish),determination of median lethal dose(LD50),virulence gene detection,biological characterization detection and drug sensitivity test.The results showed that a dominant strain,named PC-1,was isolated and purified from the spleen of diseased crucian carp,and the strain was preliminarily judged to be Aeromonas veronii based on its biochemical characteristics.The 16S rDNA gene of isolated strain PC-1 and the housekeeping gene gyrB were amplified by PCR,and the target bands of about 1470 bp and 1022 bp in size were obtained,respectively.The target bands were sequenced and analyzed by BLAST comparison with published sequences in GenBank,respectively,and nucleotide similarity with other Aeromonas veronii was found to be above 99%.The evolutionary tree results showed that the isolate PC-1 was in the same branch as other Aeromonas veronii strains.The clinical symptoms of healthy Carassius auratus infected with PC-1 were similar to those of naturally affected crucian carp,and the LD50was calculated to be 2.0×10^(5)cfu/mL for Carassius auratus and 3.2×10^(4) cfu/mL for zebrafish.The bacteria carried nine virulence genes,namely typeⅣpili(tapA)gene,aerosol(aer)gene,cytotoxic enterotoxin(act)gene,cellular excitatory enterotoxin(alt)gene,polar flagella(fla)gene,nuclease(exu)gene,serine protease(ser)gene,elastase(ahyB)gene,esterase(lip)gene,and didn’t carry heat-stable enterotoxin(ast)gene.The isolate PC-1 could form a more stable biofilm at 24 h of incubation and had the stron

关 键 词：鲫鱼 维氏气单胞菌 分离 鉴定 生物学特性 致病性 进化树 药敏试验 

分 类 号：S941.4[农业科学—水产养殖]