大菱鲆源副乳房链球菌的分离鉴定及其毒力基因型  

Isolation,identification and virulence gene of Streptococcus parauberis from cultured turbot Scophthalmus maximus

作　　者：王鹤 李战军 黄华 徐晓莹 贺加贝 曹亚男 赵强 WANG He;LI Zhanjun;HUANG Hua;XU Xiaoying;HE Jiabei;CAO Ya nan;ZHAO Qiang(Yantai Marine Economic Research Institute,Yantai 264006,China;Shandong Fisheries Development and Resources Conservation Center,Yantai 264003,China)

机构地区：[1]烟台市海洋经济研究院,山东烟台264006 [2]山东省渔业发展和资源养护总站,山东烟台264003

出　　处：《大连海洋大学学报》2021年第4期563-572,共10页Journal of Dalian Ocean University

基　　金：烟台市重点研发计划项目(2019MSGY127);山东省现代农业产业技术体系鱼类创新团队(SDAIT-12-10)。

摘　　要：为研究2019年山东省烟台市某大菱鲆Scophthalmus maximus养殖场出现的以突眼、腹水、红肿为主要特征的疾病发病原因,用平板划线法从患病濒死大菱鲆(体质量为195~535 g)肝、肾、脾、心等组织中分离获得1株优势菌,采用形态学观察、16S rRNA序列分析、PCR特异性扩增和人工回归感染试验等方法对优势菌进行鉴定,检测其血清型、毒力基因携带情况,并用纸片扩散法(K-B)检测分离株对23种抗生素的敏感性。结果表明:从病鱼分离获得的菌株为γ溶血的革兰氏阳性球菌,生理生化特征为能水解七叶苷,不能水解精氨酸、赖氨酸等,能利用蔗糖、甘露醇、葡萄糖产酸,不能利用枸橼酸、鼠李糖、尿素等;经16S rRNA序列比对、链球菌特异性引物扩增等鉴定其为副乳房链球菌Streptococcus parauberis;该菌株的毒力基因型为scp B--bca--bac--lmb--fbs A--cfb--cps D--pav A--bib A+-sip+-cyl E--hyl B+;将分离菌株制备成6.3×10^(7)、6.3×10^(8)、6.3×10^(9)、6.3×10^(10) CFU/mL 4个浓度,腹腔注射健康大菱鲆,得到菌株对受试鱼的半致死浓度(LD 50)为6.9×10^(8) CFU/mL;药敏试验显示,该菌株对β-内酰胺类、喹诺酮类、四环素类、大环内酯类、多肽类、利福霉素类等6类14种抗生素类药物敏感,对氨基糖苷类、磺胺类及呋喃类等3类6种抗生素耐药,对诺氟沙星、强力霉素、多粘菌素B中度敏感。本研究中首次报道了中国鱼源副乳房链球菌自然感染发病,可为鱼源副乳房链球菌分子特性研究、致病机理、药物防治等提供科学参考。In order to explore the causes of diseases characterized by exophthalmos and sepsis in turbot Scophthalmus maximus with body mass of(195-535)g farmed in Yantai,Shandong Province in 2019,predominant bacterial strains were isolated from moribund individual tissues including kidney,spleen,and brain,and identified by morphological observation,16S rRNA gene sequence analysis,and specific PCR amplification.Genetic characteristics of the isolated strain were analyzed by virulence genes,and the susceptibility of the isolated strain to 10 categories including 23 antimicrobial dugs was determined using Kirby-Bauer diffusion method.The results showed that the strains isolated from the visceral tissues as Gram-positive cocci named as BH2-4 was identified as Streptococcus parauberis by the physiological and biochemical characteristics of non-hemolytic,acid from sucrose,mannitol,glucose,without being produced from citrate,rhamnose,hydrolysed aesculin,and by 16S rRNA sequence alignment and specific amplification of Streptococcus.The virulence genotype was found to be scp B--bca--bac--lmb--fbs A--cfb--cps D--pav A--bib A+-sip+-cyl E--hyl B+.The strain BH2-4 had median lethal concentration(LD 50)of 6.9×10^(8) CFU/mL determined in the healthy turbot injected with hormone interaperitoneally at a dose of 6.3×10^(7),6.3×10^(8),6.3×10^(9),and 6.3×10^(10) CFU/mL.The strain BH2-4 was shown to be high susceptible to 6 categories and 14 antibiotics includingβ-lactams,quinolones,tetracyclines,macrolides,peptides and rapamycin,resistant to 3 categories and 6 antibiotics,including aminoglycosides,sulfonamides and nitrofurans,and intermediately sensitive to norfloxacin,doxycycline and polymyxin B.The findings were reported for the first time the onset of natural infection of fish-sourced Streptococcus parauberis,and the identification of its characteristics can provide basic support for the research of fish-sourced Streptococcus parauberis disease surveillance,precision prevention,and drug control.

关 键 词：大菱鲆 副乳房链球菌 分离鉴定 药敏试验 毒力基因 

分 类 号：S941.42[农业科学—水产养殖] Q938.8[农业科学—水产科学]