乌鳢诺卡氏菌病致病菌的分离、鉴定及组织病理学观察  

Isolation,identification,and histopathological observation of pathogen causing nocardiosis in Channa argus

作　　者：滕建 陈红菊[1] 薛良义[2] 李岩 路广金 周敏[1] 张冲 季相山[1] TENG Jian;CHEN Hongju;XUE Liangyi;LI Yan;LU Guangjin;ZHOU Min;ZHANG Chong;JI Xiangshan(College of Animal Science and Technology,Shandong Agricultural University,Taian 271018,China;College of Marine Sciences,Ningbo University,Ningbo 315832,China)

机构地区：[1]山东农业大学动物科技学院,山东泰安271018 [2]宁波大学海洋学院,浙江宁波315832

出　　处：《水产学报》2022年第5期836-847,共12页Journal of Fisheries of China

基　　金：山东省农业良种工程项目(2019LZGC013,2019LZGC020);山东省“双一流”奖补资金。

摘　　要：为研究乌鳢诺卡氏菌的致病机理,本实验通过病原分离鉴定、组织病理学和基因表达水平分析对病原菌的致病性、药物敏感性及乌鳢的免疫抗性进行了研究。结果显示,患病乌鳢主要感染了命名为SDAT 0011病原菌,通过菌落形态观察、革兰氏染色鉴定、生理生化鉴定、16S rRNA鉴定及诺卡氏菌特异序列扩增鉴定,结果均显示该病原菌为诺卡氏菌。将分离的SDAT 0011感染健康乌鳢后,1×10^(5)~1×10^(8) CFU/mL注射组的死亡率均为100%,感染乌鳢出现明显的诺卡氏菌病症状,如内脏出血,肝脏、脾脏和肾脏中有大量大小不等的结节,组织病理切片进一步检测发现,结节分界清晰,结节中有大量的淋巴细胞、受损或死亡的组织细胞。药物敏感性实验发现,诺卡氏菌对利福平等抗生素较为敏感,对青霉素等具有较强的抗性。基因表达分析显示,在感染初期(48 h)及中后期,Toll样受体2基因(TLR2)和Toll样受体13基因(TLR13)在脾脏和头肾的表达水平显著上调,而趋化因子受体9基因(CCR9)在脾脏和头肾中显著下调,这表明乌鳢Toll样受体和趋化因子受体信号通路可能在其抵抗诺卡氏菌感染中起重要作用。本实验为乌鳢诺卡氏菌病的治疗及其致病机理研究提供了重要的基础。To explore the pathogenic mechanism of nocardiosis in Channa argus,we studied the pathogenicity and drug sensitivity of the pathogen and the immune resistance of C.argus by isolation and identification of the pathogen,histopathology and gene expression analysis.The results showed that the diseased C.argus was mainly infected with a pathogen strain SDAT 0011.The strain SDAT 0011 was identified as Nocardia seriolae using the morphologic structure and staining observation,PCR amplification of species-specific primers,sequence analysis of16S rRNA gene,and physiological and biochemical tests.The challenge experiments showed that the isolated strains were pathogenic on C.argus and the mortality rate of the 1×10^(5)-1×10^(8) CFU/mL injection groups was100%.In addition,the diseased C.argus exhibited numerous marked white nodules(round/ovoid),ranging from0.1 to 0.2 cm in diameter on internal organs,especially the spleen,kidney,and liver.Histopathological observation and analysis showed that the structure of chronic granuloma was visible,with many lymphocytes,damaged or dead cells in the center.The antibiotic susceptibility assays of the strain SDAT 0011 showed that the strain was sensitive to rifampin,but resistant to penicillin,cefradine,and ampicillin,etc.Furthermore,the expression levels of toll-like receptors 2 gene(TLR2),toll-like receptors 13 gene(TLR13),and C-C chemokine receptor type 9 gene(CCR9)after infection were analyzed by real-time fluorescence quantitative PCR(qRT-PCR).In our study,following N.seriolae challenge,TLR2 and TLR13 were significantly up-regulated,while CCR9 was significantly downregulated at all examined time points in the spleen and head kidney.These results implied that some genes of the toll-like receptor signaling pathway and the chemokine signaling pathway have been activated in the early stages of infection to resist Nocardia infection.The results of this study will lay a foundation for the treatment of N,seriolae and studying it pathogenesis..

关 键 词：乌鳢 鰤诺卡氏菌 诺卡氏菌病 组织病理 免疫基因 

分 类 号：S941.42[农业科学—水产养殖]