山羊源伪结核棒状杆菌的分离鉴定及其生物学特性分析  

Isolation,Identification and biological characteristics of Corynebacterium pseudotuberculosis from goats

作　　者：林龙华 许秋 马彩萍 柴霁芸 侯杰 孙宏飞 杜素素 范翠龙 祝瑶 张万江[1] LIN Long-hua;XU Qiu;MA Cai-ping;CHAI Ji-yun;HOU Jie;SUN Hong-fei;DU Su-su;FAN Cui-long;ZHU Yao;ZHANG Wan-jiang(State Key Laboratory for Animal Disease Control and Prevention,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China;College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830052,China;College ofWildlife and Protected Area,Northeast Forestry University,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所动物疫病防控全国重点实验室/动物细菌病创新团队,黑龙江哈尔滨150069 [2]新疆农业大学动物医学学院,新疆乌鲁木齐830052 [3]东北林业大学野生动物与自然保护地学院,黑龙江哈尔滨150006

出　　处：《中国预防兽医学报》2025年第1期23-30,共8页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划(2021YFD1800401);中央级公益性科研院所基本科研业务费专项(1610302022001、1610302024001);黑龙江省自然科学基金(LH2024C061)。

摘　　要：为鉴定引起黑龙江省齐齐哈尔市某山羊养殖场山羊体表脓肿的病原,并分析其生物学特性,本实验无菌采集40份体表患有脓肿的山羊脓液进行细菌的分离鉴定、革兰氏染色;采用PCR扩增分离菌16S rRNA基因并测序,基于细菌16S rRNA基因序列构建分离菌的系统进化树;通过微量肉汤稀释法分析细菌对7类代表性抗菌药的敏感性;通过Illumina PE 150对细菌全基因组测序,并利用ResFinder数据库预测细菌的相关耐药基因,利用VFDB数据库预测细菌的毒力基因;通过脉冲场凝胶电泳(PFGE)对细菌分型鉴定;通过小鼠致病性试验分别对分离菌和ATCC 19410株的致病性进行评价。结果显示:分离菌在血琼脂平板上可形成狭窄的β溶血环,革兰氏染色可见蓝紫色、球杆状的细菌;16S rRNA基因的测序鉴定结果显示引起山羊体表脓肿的病原为伪结核棒状杆菌,经统计从40份病料样品中分离出10株伪结核棒状杆菌。16SrRNA系统进化树结果显示,分离菌和伪结核棒状杆菌聚为同一分支,与谷氨酸棒状杆菌、肾棒状杆菌等处于临近分支,表明分离菌为伪结核棒状杆菌。药敏试验结果显示,10株分离菌对所测试的6种抗菌药均敏感,1-1、24两株分离菌对庆大霉素敏感,其余8株分离菌对庆大霉素均耐药。耐药基因预测结果显示10株分离菌均未检测到耐药基因。毒力基因预测结果显示10株分离菌均检测到与黏附性、铁摄取与调节、毒素相关的毒力相关基因21个。PFGE将10株分离菌分为两种谱型,表明至少有两种谱型的伪结核棒状杆菌正流行于该羊场。小鼠致病性试验结果显示,将毒力最强的3-1菌株对小鼠进行腹腔感染后,最高剂量组死亡小鼠剖检可见肠道充血肿胀、肝脾肿大、边缘出血梗死;低剂量组死亡小鼠剖检可见腹腔脏器出现黄白色脓肿,其产生的病变与ATCC 19410标准株感染小鼠产生的病变一致,前两个高剂量3-1菌株�To investigate the etiology of caprine cutaneous abscesses on a goat farm in Qiqihar,Heilongjiang Province,40 abscess pus samples were collected aseptically for bacterial isolation,identification and Gram staining.The phylogenetic tree was constructed based on bacterial 16S rRNA gene sequence.The sensitivity of bacteria to 7 representative antibiotics was assessed via microbroth dilution method.Whole genome sequencing(Illumina PE 150)was performed to analyze the related antimicrobial resistance genes(ARGs)(ResFinder database)and virulence genes(VFDB database).Pulsed field gel electrophoresis(PFGE)was used for bacterial typing,and pathogenicity was evaluated through murine challenge experiments comparing the isolate with ATCC 19410 strain.The results showed that the isolate formed narrowβ-hemolytic rings on blood agar plate.Gram staining revealed blue-purple,bulb-shaped bacteria.16S rRNA sequencing identified Corynebacterium pseudotuberculosis(C.pseudotuberculosis)as the causative agent of caprine cutaneous abscesses,with 10 strains obtained from 40 samples.Phylogenetic tree clustered the isolate within the C.pseudotuberculosis clade,adjacent to C.glutamicum and C.renale,indicating that the isolate was C.pseudotu-berculosis.The results of antimicrobial sensitivity test(AST)showed that 10 isolates were sensitive to the 6 tested antibiotics,1-1,24 isolates were sensitive to gentamicin,and the other 8 isolates were resistant to gentamicin.The results of ARGs analysis showed that no ARGs was detected in 10 isolates.Virulence gene analysis showed that 21 candidate virulence genes related to adhesion,iron uptake,regulation and toxin were detected in all isolates.PFGE divided isolates into two spectral types,suggesting at least two circulating strains of C.pseudotuberculosis within the farm.The pathogenicity test in mice showed that after the intraperitoneal injection with strain 3-1,the autopsy of the dead mice in the highest dose group showed intestinal congestion and swelling,liver and spleen enlargement,and marginal

关 键 词：伪结核棒状杆菌 耐药基因 毒力基因 脉冲场凝胶电泳 致病性 

分 类 号：S852.61[农业科学—基础兽医学]