1株猪源A型产气荚膜梭菌的分离鉴定及致病性研究  

Isolation,Identification and Pathogenicity Analysis of a Strain of Swine-derived Clostridium perfringens Type A

作　　者：吴钰兴 邹潍力 程子馨 莫玉鹏 张凌源 纪宛彤 陈思宇[1,3] 郑浩东 麻润雯 李珣 李茂宁[1,4] 王晓晔[1] WU Yuxing;ZOU Weili;CHENG Zixin;MO Yupeng;ZHANG Lingyuan;JI Wantong;CHEN Siyu;ZHENG Haodong;MA Runwen;LI Xun;LI Maoning;WANG Xiaoye(Guangxi Colleges and Universities Key Laboratory of Prevention and Control for Animal Disease,Guangxi Key Laboratory of Animal Reproduction,Breeding and Disease Control,Guangxi Zhuang Autonomous Region Engineering Research Center of Veterinary Biologics,College of Animal Science and Technology,Guangxi University,Nanning 530004,China;Guilin Liyuan Group,Guilin 541012,China;Chongqing Yudong Health School,Chongqing 408300,China;Guilin Animal Disease Prevention and Control Centre,Guilin 541000,China;Dongguan Animal Disease Prevention and Control Centre,Dongguan 523000,China)

机构地区：[1]广西大学动物科学技术学院,广西壮族自治区兽用生物制品工程研究中心,广西畜禽繁育与疾病防控重点实验室,广西高校动物疫病预防与控制重点实验室,南宁530004 [2]广西桂林力源集团,桂林541012 [3]重庆市渝东卫生学校,重庆408300 [4]桂林市动物疫病预防控制中心,桂林541000 [5]东莞市动物疫病预防控制中心,东莞523000

出　　处：《中国畜牧兽医》2024年第6期2697-2706,共10页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金项目(32360902、32360864);广西自然科学基金项目(2021GXNSFAA196058);广西重点研发计划(AB21238003);南宁市重点研发计划(20212023)。

摘　　要：【目的】探究A型产气荚膜梭菌在仔猪腹泻中的致病性,为A型产气荚膜梭菌感染所致腹泻的诊断和治疗提供理论依据和参考。【方法】通过培养特性和革兰染色镜检对分离菌进行初步鉴定,通过生化试验、毒素基因PCR扩增、16S rRNA序列比对并构建进化树对可疑菌株进一步鉴定,利用药敏试验和动物回归试验研究分离菌株的耐药性和致病性。【结果】分离菌株在TSN平板上呈黑色菌落,在血平板上呈α、β双溶血环,革兰染色呈紫色粗大杆菌。生化试验结果显示,分离菌株葡萄糖、麦芽糖、蔗糖、乳糖、还原性硝酸盐试验、H_2S试验和明胶液化均呈阳性,甘露醇、吲哚试验和水杨酸结果呈阴性。16S rRNA序列比对结果显示,分离菌与NCBI数据库中已公布的产气荚膜梭菌16S rRNA基因序列相似性均>99%,鉴定该菌株为产气荚膜梭菌。PCR扩增出plc及cpb2基因,表明该菌株为携带β2毒素的A型产气荚膜梭菌。药敏试验结果显示,分离菌株对青霉素G、庆大霉素、林可霉素、磺胺异噁唑、环丙沙星、诺氟沙星耐药。动物回归试验结果显示,攻菌后仔猪粪便中可检出大量产气荚膜梭菌,该菌株可引起仔猪腹泻,死亡率达50%;死亡仔猪剖检可见小肠充血、出血、鼓气,从脾脏、小肠中可分离出产气荚膜梭菌,且在损伤严重的空肠肠段中分离出高载量A型产气荚膜梭菌。【结论】本试验成功分离出1株A型产气荚膜梭菌,可引起新生仔猪发病,致病性较强,该研究结果为A型产气荚膜梭菌感染仔猪的诊断、流行病学调查、治疗提供了参考依据。【Objective】The experiment was aimed to investigate the pathogenicity of Clostridium perfringens type A in piglet diarrhoea and provide experimental data and reference for the diagnosis and treatment of diarrhea caused by Clostridium perfringens type A infection.【Method】Preliminary identification was carried out by culture characteristics and staining microscopy,suspicious strains were further identified by biochemical tests,PCR amplification of toxin genes,16S rRNA sequence alignment and phylogenetic tree construction.Drug sensitivity and animal regression tests were conducted to assess drug resistance and pathogenicity of the isolated strain.【Result】The isolated strain displayed black colonies on TSN agar plates,αandβhemolytic zones on blood agar plates,and appeared as large purple rods in Gram staining.The results of biochemical tests showed that glucose,maltose,sucrose,lactose,reductive nitrate test,H 2S test and gelatin liquefaction were positive,while mannitol,indole test and salicylic acid test were negative.The 16S rRNA sequence alignment showed more than 99%identity with the 16S rRNA gene sequences of Clostridium perfringens in the NCBI database,the strain was identified as Clostridium perfringens.The plc and cpb 2 genes were amplified by PCR,indicating that the strain was Clostridium perfringens type A carrying theβ2 toxin.Drug sensitivity test revealed the isolate was resistant to penicillin G,gentamicin,lincomycin,sulfafurazole,ciprofloxacin,and norfloxacin.Animal regression test results showed that the strain could cause diarrhea in piglets,with a mortality rate of 50%.A significant amount of Clostridium perfringens was detectable in the feces post-infection.Necropsy of deceased piglets revealed congestion,hemorrhage,and distension in the small intestine,and Clostridium perfringens type A with high load was isolated from the severely injured jejunal segment.【Conclusion】This study successfully isolated a strain of Clostridium perfringens type A that could induce disease in neonatal p

关 键 词：A型产气荚膜梭菌 新生仔猪 分离鉴定 致病性 

分 类 号：S852.616.3[农业科学—基础兽医学]