狼源产气荚膜梭菌的分离鉴定及分子分型  

Isolation,identification and genotyping of Clostridium perfringens from wolf

作　　者：董秀梅[1] 李秀云[2] 刘念 杨巍[1] 邓爱怀[2] 吕文鹏[2] 关春宇[2] 张萍[1] 邹希明[2] 师东方[1] DONG Xiu-mei;LI Xiu-yun;LIU Nian;YANG Wei;DENG Ai-huai;LYU Wen-peng;GUAN Chun-yu;ZHANG Ping;ZOU Xi-ming;SHI Dong-fang(College of Veterinary Medicine,Northeast Agricultural University,Northeastern Science Inspection Station,China Ministry of Agriculture Key Laboratory of Animal Pathogen Biology,Harbin 150030,China;Harbin Northern Forest Zoo,Harbin 150322,China)

机构地区：[1]东北农业大学动物医学学院/农业部动物疫病病原生物学重点实验室东北科学观测实验站,黑龙江哈尔滨150030 [2]哈尔滨北方森林动物园,黑龙江哈尔滨150322

出　　处：《中国预防兽医学报》2022年第8期831-836,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(31672529);国家科技支撑计划项目(2012BAD12B03、2012BAD12B05);财政部和农业农村部:国家现代农业产业技术体系资助(CARS36)。

摘　　要：为探究黑龙江省某动物园狼急性死亡病例的病原菌及其生物学特性,本研究采集病死狼心血、肝、脾及颌下淋巴结样品,经细菌分离纯化,结果显示,上述样品接种鲜血琼脂培养基厌氧培养24 h后可见灰白色、双层溶血环的菌落,在强化梭菌培养基上形成黑色圆形菌落;革兰染色镜检可见单个或呈短链状排列的革兰阳性粗大杆菌。通过生化试验和16S rRNA基因的PCR扩增及序列分析对分离菌进行鉴定,并进一步经PCR扩增确定分离菌的毒素型。生化试验结果显示,分离菌能发酵葡萄糖、麦芽糖、乳糖、蔗糖,且还原性硝酸盐试验、明胶液化和牛奶爆裂发酵均呈阳性,且无运动性;16S rRNA基因、毒素基因的PCR扩增和遗传进化分析结果表明分离菌为A型产气荚膜梭菌,命名为HLJ15-2。经多序列位点分型分析(MLST)确定分离菌的ST型,结果显示分离菌为ST 221型,属于鸡源菌。采用K-B纸片扩散法对分离菌进行药敏试验,结果显示该菌对氟苯尼考、头孢呋辛钠、头孢噻肟等磺胺类和喹诺酮类抗菌药敏感。小鼠分别经腹腔接种10倍倍比稀释(5.21×10^(5) cfu/mL~1.52×10^(9) cfu/mL)的HLJ15-2菌液,计算该菌对小鼠的半数致死量(LD_(50)),采集病死小鼠肝、脾、肠进行组织病理学观察分析分离菌对小鼠的致病性,结果显示分离菌LD_(50)为2.26×10^(7) cfu/mL,剖检小鼠可见肝脏、脾脏均有出血,肠道膨隆,肠黏膜充血,组织病理切片观察可见肝内各级血管淤血,肝细胞条锁状排列消失,胞浆内出现大小不等的空泡;脾脏红髓比例增加,白髓部分细胞坏死;肠绒毛断裂,黏膜固有层上皮细胞增生、黏膜下层红细胞增多、充血、炎性细胞浸润。本研究首次报道了致病性A型产气荚膜梭菌可感染狼,并导致其急性死亡,为野生动物梭菌性传染病的诊断与防控提供参考。To explore the suspected pathogenic bacteria and its biological characteristics resulting in acute death of wolves in a zoo of Heilongjiang province,the heart blood,liver,spleen and submaxillary lymph nodes of dead wolves were collected for isolation and purification of bacteria.The results showed that the gray-white and double-layer hemolytic ring colonies were observed after anaerobic culture for 24 hours in the blood agar medium of heart blood,liver,spleen and submandibular lymph nodes,and black circular colonies were formed on the enhanced Clostridium medium.Gram-staining microscopy showed single or short-chain Gram-positive coarse bacilli.The isolates were identified by biochemical tests and PCR amplification and sequence analysis of 16S rRNA gene,and the toxin type of the isolates was further determined by PCR.The results of biochemical test showed that the isolate could ferment glucose,maltose,lactose and sucrose,and the reducing nitrate test,gelatin liquefaction and milk burst fermentation were all positive,and had no motility.The results of PCR amplification and genetic evolution analysis of 16S rRNA and toxin gene showed that the isolate was Clostridium perfringens type A(named HLJ15-2).The ST type of the isolate was determined by multilocus sequence typing(MLST),and it showed that the ST type of the isolate was 221,belonging to chickenderived bacteria.The antibiotics sensitivity of the isolate was detected by K-B paper diffusion method,and it showed that the isolate was highly sensitive to 7 drugs including florfenicol,cefuroxime sodium,cefotaxime,etc.The mice were inoculated intraperitoneally with 10-fold diluted(5.21×10^(5)cfu/mL-1.52×10^(9)cfu/mL)HLJ15-2 culture,and the pathogenicity was analyzed by challenge test in mice,and the liver,spleen and intestine tissues of the dead mice were collected for histopathological observation.The results showed that the median lethal dose(LD_(50))of the isolate was 2.26×10^(7)cfu/mL.Autopsy showed bleeding in both liver and spleen,intestinal distention and int

关 键 词：狼 产气荚膜梭菌 分离鉴定 16SrRNA 分子分型 

分 类 号：S852.61[农业科学—基础兽医学]