摘要
为探究患病罗氏沼虾的病原及其感染的防治措施,本研究从浙江省某养殖场采集10只患病罗氏沼虾的肝胰腺、鳃及肌肉样品分离获得一株优势菌。通过对该优势菌经革兰氏染色镜检及生化鉴定,结果表明分离菌为弗氏柠檬酸杆菌(Citrobacter freundii)。采用PCR分别扩增分离菌的16S r RNA和gyr B基因序列,测序后经BLAST检索与这两种基因同源性均最高的菌株;通过MEGA 7.0软件中的Clustal W方法将分离菌与从Gen Bank中获得的同源性较高的菌株序列进行多序列比对,采用MEGA 7.0软件中的邻接法构建分离菌与上述菌株16S r RNA和gyr B基因的系统发育树。BLAST检索结果显示,分离菌与弗氏柠檬酸杆菌(KC210829.1)16S r RNA基因序列的同源性高达99.93%;与弗氏柠檬酸杆菌(KF156750.1)gyr B基因序列的同源性达98.76%。16S r RNA和gyr B基因的进化树结果显示,分离菌均与弗氏柠檬酸杆菌聚为一支,且支持率高达99%。进一步表明引起罗氏沼虾发病的病原为弗氏柠檬酸杆菌。将分离菌分别以1.02×10^(9)cfu/m L~1.02×10^(4)cfu/m L 6种浓度感染健康罗氏沼虾,每日观察并记录罗氏沼虾的发病症状及死亡率,根据Probit回归法计算分离菌对罗氏沼虾的半数致死量(LD50)。结果显示,1.02×10^(6)cfu/m L以上浓度的弗氏柠檬酸杆菌均能够导致罗氏沼虾出现体型消瘦、行动迟缓无活力的症状,1.02×10^(5)cfu/m L以下浓度的弗氏柠檬酸杆菌并不致罗氏沼虾出现症状。经统计该菌株对罗氏沼虾的LD50为1.01×10^(7)cfu/m L,表明该菌株对罗氏沼虾的致病性较强。采用PCR分别检测该分离菌株的定居因子基因(cfa)、尿素酶基因簇(ure G、ure F、ure E、ure D)、Vi抗原基因(via B)、外膜蛋白基因(omp X)等7种毒力相关基因,结果显示该菌株分别扩增到375 bp(cfa)、119 bp(ure G)、300 bp(ure F)、247 bp(ure E)、243 bp(ure D)、875 bp(via B)及250 bp(oomp X)的7种毒力基因。采用K-B纸片扩散法测定分离菌对10类共35种药物的敏感性,结果显示该分离菌对β-内酰胺类的头孢他啶和头孢曲松、多肽素类的多黏菌素B、氨基糖苷类的庆大霉素以及拉氧头孢这5种药物敏感,对β-内酰胺类的青霉素、氯霉素类的氟苯尼考、多肽类的万古霉素及苯唑醂、克林霉素和新生霉素等27种药物呈不同程度的耐药性,且为多重耐药菌。综上所述,本研究从患病罗氏沼虾中分离到了一株弗氏柠檬酸杆菌,该菌携带多种毒力基因,对罗氏沼虾的致病性较强,为进一步研究弗氏柠檬酸杆菌对水产动物的致病性及对罗氏沼虾弗氏柠檬酸杆菌病的防治提供参考依据。
To explore the pathogen of diseased Macrobrachium rosenbergii(M.rosenbergii)in a farm in Zhejiang Province and the prevention and control measures of its infection,the hepatopancreas,gill and muscle of 10 diseased M.rosenbergii were collected to isolate a dominant strain.The results of Gram staining and biochemical characterization showed that the isolate was a strain of Citrobacter freudii.The 16S rRNA and gyrB gene of the isolate were amplified by PCR,and the sequences of the strain with the highest similarity to the two genes were searched by BLAST after sequencing.The Clustal W method in MEGA 7.0 software was used to perform multiple sequence alignments between the isolate and the strains with high similarity obtained from GenBank.The phylogenetic tree of 16S rRNA and gyrB genes of the isolate and the above strains was constructed by the Neighbor-Joining method with MEGA 7.0 software.The results of BLAST search showed that the homology of 16S rRNA gene sequence between the isolate and C.fredii(KC210829.1)was as high as 99.93%.The homology of gyrB gene sequence with C.fredii(KF156750.1)was 98.76%.The phylogenetic trees indicated that the isolate was clustered with C.freundii,and the support rate was as high as 99%.It was further indicated that the pathogen causing the disease of M.rosenbergii was C.fredii.Moreover,the healthy M.rosenbergii were infected with the isolate at six concentrations from 1.02×109cfu/mL to 1.02×104cfu/mL.During the experiment,the symptoms of M.rosenbergii was observed and recorded daily,and the mortality of M.rosenbergii at different bacterial concentrations was counted,and the median lethal dose(LD50)of the strain was calculated according to the Probit regression method.The results showed that C.fredii at concentrations above 1.02×10^(6)cfu/mL could cause the symptoms of emaciation,slow action and inactivity of M.rosenbergii,while C.fredii at concentrations below 1.02×10^(5)cfu/mL could not cause the symptoms.According to the statistical results,the median lethal dose(LD50)of the strain to M.rosenbergii was 1.01×10^(7)cfu/mL,indicating that the strain was highly pathogenic to M.rosenbergii.Seven virulence-related genes of the isolate were detected by PCR,including colonization factor gene(cfa),urease gene clusters(ureG,ureF,ureE,ureD),virulence antigen gene(viaB)and outer membrane protein gene(ompX).The results showed that 7 virulence genes of 375bp(cfa),119 bp(ureG),300bp(ureF),247bp(ureE),243bp(ureD),875bp(viaB)and 250bp(ompX)were amplified from the isolate.In addition,the sensitivity of the isolated to 35 drugs in 10 categories was determined by the K-B disk diffusion method.The results showed that the isolate was sensitive to ceftazidime and ceftriaxone ofβ-lactams,polymyxin B of polypeptides,gentamicin of aminoglycosides and latamoxef.It also showed varying degrees of resistance to 27 drugs,such as penicillin ofβ-lactams,florfenicol of chloromycetins,vancomycin of polypeptides and benzimidazole mirin,clindamycin and novobiocin,and exhibited multiple drug resistance.In summary,a strain of C.freundii was isolated from the diseased M.rosenbergii in this study,which carried a variety of virulence genes.The pathogenicity test confirmed that the isolated strain had strong pathogenicity to M.rosenbergii,which provided a reference for further study on the pathogenicity of C.freundii to aquatic animals and the prevention and control of C.freundii disease in M.rosenbergii.
作者
赵梦瑶
陈静
姚嘉赟
袁雪梅
黄雷
彭先启
张海琪
ZHAO Meng-yao;CHEN Jing;YAO Jia-yun;YUAN Xue-mei;HUANG Lei;PENG Xian-qi;ZHANG Hai-qi(College of Life Science,Huzhou University,Huzhou 313000,China;Agriculture Ministry Key Laboratory of Healthy Freshwater Aquaculture,Key Laboratory of Fish Health and Nutrition of Zhejiang Province,Key Laboratory of Fishery Environment and Aquatic Product Quality and Safety of Huzhou City,Zhejiang Institute of Freshwater Fisheries,Huzhou 313001,China)
出处
《中国预防兽医学报》
北大核心
2025年第2期137-143,共7页
Chinese Journal of Preventive Veterinary Medicine
基金
浙江省“尖兵领雁+X”研发攻关计划项目(2024C02005)
湖州市重点研发计划项目农业“双强”专项(2023ZD2032)
浙江省科研院所扶持专项(2024YSZX01、2024YSZX04)
浙江省农业重大技术协调与推广计划项目(2024ZDXT16)
湖州市公益性应用研究项目(2023GZ31)。
关键词
罗氏沼虾
弗氏柠檬酸杆菌
致病性
毒力基因
药敏分析
Macrobrachium rosenbergii
Citrobacter freundii
pathogenicity
virulence genes
drug sensitivity analysis
