梅花鹿源A型产气荚膜梭菌的分离鉴定及耐药性分析  

Isolation and identification of Sika deer-derived Clostridium perfringens type A and drug resistance analysis

作　　者：温海燕 姜莉莉 刘若渊 万双秀 侯少阳 葛仕豪 史珍 陈穆贇 樊兆斌 WEN Haiyan;JIANG Lili;LIU Ruoyuan;WAN Shuangxiu;HOU Shaoyang;GE Shihao;SHI Zhen;CHEN Muyun;FAN Zhaobin(College of Pharmacy,Heze University,Heze 274015,China;College of Veterinary Medicine,Hebei Agricultural University,Baoding 071001,China)

机构地区：[1]菏泽学院药学院,山东菏泽274015 [2]河北农业大学动物医学院,河北保定071001

出　　处：《黑龙江畜牧兽医》2024年第22期118-123,132,138,139,共9页Heilongjiang Animal Science And veterinary Medicine

基　　金：山东省自然科学基金项目(ZR2019MC036,ZR2023QC302);兽药创新菏泽市工程研究中心项目;菏泽市禽免疫抑制病防控重点实验室项目;菏泽学院大学生创新训练项目;菏泽学院新兽药工程重点实验室项目。

摘　　要：2022年11月份,辽宁省某野生动物园梅花鹿陆续出现腹泻症状,甚至死亡,剖检发现皱胃黏膜出血明显,小肠黏膜出血,肠系膜淋巴结肿大,表现为肠毒血症特征,疑似产气荚膜梭菌感染。为进一步确定病原并了解其耐药性,试验首先无菌采集病死梅花鹿心脏、肝脏、肺脏、肾脏、脾脏和肠道内容物,进行细菌的分离培养;随后对分离菌进行生化鉴定、基因组DNA的提取与16S rDNA基因的PCR扩增,基于16S rDNA基因序列进行遗传进化分析;最后对分离菌进行了毒素基因的检测、小鼠致病性试验和耐药性分析。结果表明:从病料中分离纯化得到1株细菌,命名为LN202212。LN202212株在TSN琼脂平板上呈圆形、突起、光滑、湿润、中心环为黑色的菌落;在血琼脂平板上呈圆形、光滑、湿润灰白色菌落,菌落周围呈β溶血;革兰氏染色、镜检可见革兰氏阳性大杆菌,两端钝圆,呈单个或成对排列。LN202212株的酪蛋白、吲哚、脂酶、硝酸盐和甘露醇试验结果为阴性,卵磷脂酶、明胶、葡萄糖、麦芽糖和乳糖试验结果为阳性。LN202212株的16S rDNA基因序列与产气荚膜梭菌标准菌株ATCC 13124(登录号为MN326666.1)的核苷酸相似性为99.85%,与上海人源产气荚膜梭菌218002-301株(登录号为JQ940553.1)、印度豹源产气荚膜梭菌ATCC 13124株(登录号为MN326666.1)的亲缘关系较近。对LN202212株进行毒素基因检测,仅检测到α毒素cpa基因,其他6种毒素基因均未检测到,符合A型产气荚膜梭菌特点。小鼠接种分离菌24 h后陆续出现精神沉郁、行动迟缓、食欲减退、呼吸困难等症状,72 h后1只小鼠死亡(1/5),死亡小鼠肺脏明显出血,其他存活小鼠表现为肺脏出血,肝脏、脾脏淤血、肿大。病理切片可见部分肺泡破裂,肺泡腔内有血细胞,且有脱落的上皮细胞;肝细胞肿胀、变性,有出血和充血现象。LN202212株对恩诺沙星、先锋霉素V、头孢曲松敏感,�In November,2022,Sika deer successively showed diarrhea symptoms,and even died in a wild zoo in Liaoning Province autopsy found obvious wrinkled gastric mucosa hemorrhage,small intestinal mucosa hemorrhage and enlarged mesenteric lymph nodes,which manifested the characteristics of enterotoxemia,and was suspected to be infected with Clostridium perfringens.In order to further identify the pathogen and understand its drug resistance,in the experiment,the heart,liver,lung,kidney,spleen and intestinal tract contents of the diseased and dead Sika deer were first aseptically collected for isolation and culture of bacteria.Subsequently,the isolates were subjected to biochemical identification,genomic DNA extraction and PCR amplification of 16S rDNA gene;genetic evolution analysis was carried out based on the 16S rDNA gene sequence;and finally,the isolates were subjected to detection of toxin genes,mouse pathogenicity test and drug resistance analysis.The results showed that one bacterial strain,named LN202212,was isolated and purified from the diseased material.The strain LN202212 showed round,raised,smooth,moist colonies with a black center ring on TSN agar plates;which were round,smooth,moist grayish-white colonies withβ-hemolysis around the colonies presented on blood agar plates;the Gram-staining microscopic examination showed Gram-positive rods of coarse size,which were bluntly rounded on the ends and arranged singly or in pairs.The strain LN202212 was negative for casein,indole,lipase,nitrate and mannitol,and was positive for lecithinase,gelatin,glucose,maltose and lactose.The nucleotide similarity of 16S rDNA gene sequence of LN202212 and standard strain ATCC 13124(entry number was MN326666.1)was 99.85%.It was closely related to Shanghai human Clostridium perfringens 218002-301(entry number was JQ940553.1)and Indian leopard Clostridium perfringens ATCC 13124(entry number was MN326666.1).Toxin gene detection was performed on strain LN202212,and only theα-toxin cpa gene was detected,while the other six toxin gene

关 键 词：梅花鹿 A型产气荚膜梭菌 细菌分离 耐药性 致病性 

分 类 号：S852.61[农业科学—基础兽医学]