【摘要】 为了查明导致河南省洛阳市某规模化奶牛场5头腹泻犊牛死亡的病原，并了解病原的耐药性，试验对病死犊牛进行剖检后采集肠系膜、肺脏、脾脏组织进行细菌的分离培养，对分离菌株进行生化鉴定、16S rDNA基因的PCR扩增与序列分析、毒素基因型判定、耐药性分析、耐药基因检测，并随机选取1株分离菌株进行小鼠致病性试验。结果表明：病死犊牛肝脏表面弥漫性出血，肾脏、脾脏肿大，脾脏被膜有出血点，肺脏肿大、出血，皱胃充血、坏死，整个肠道内充满大量酸臭气体，十二指肠坏死，结肠溃疡，肠系膜淋巴结肿大、出血，疑似产气荚膜梭菌感染。经分离、培养共获得5株细菌，分别来自5头病死犊牛，命名为LY-2、LY-7、LY-9、LY-10、LY-12。5株分离菌株的菌落形态、革兰氏染色结果和生化特性均与产气荚膜梭菌相符，且16S rDNA基因与GenBank中的产气荚膜梭菌参考菌株核苷酸序列相似性均在97.2%～100%之间，在遗传进化树中均与产气荚膜梭菌参考菌株聚为一支，与梭菌属其他菌株（尸毒梭菌、艰难梭菌、气肿疽梭菌等）处于不同分支；均检出α毒素和β毒素，未检出ε毒素和ι毒素，与C型产气荚膜梭菌相符。5株分离菌株对阿莫西林、卡那霉素、庆大霉素、复方新诺明耐药率均为100%,对氧氟沙星、恩诺沙星、氟苯尼考的耐药率均为0,对头孢曲松、氨苄西林、青霉素、链霉素、磺胺异口恶唑、环丙沙星的耐药率分别为25%、60%、40%、60%、80%、20%。5株分离菌株共检出4类抗生素中的6种耐药基因。其中β-内酰胺类耐药基因bla_CMY的检出率为100%,bla_TEM和bla_SHV均未检出；氨基糖苷类耐药基因aph（2″）-Ⅰb基因的检出率为80%,ant（6′）-Ⅰa、aph（2″）-Ⅰc和aph（2″）-Ⅰd均未检出；磺胺类耐药基因sul1、sul2、sul3的检出率分别为20%、40%、60%;氯霉素类耐药基因floR的检出率为80%;氟喹诺酮类耐药基因tet、LyrA和parC均未检出。分离菌株LY-2可致小鼠死亡，剖检死亡小鼠可见肾脏、肺脏、肝脏、脾脏和肠道均发生不同程度的病理改变。说明引起该奶牛场5头犊牛腹泻并死亡的病原均为C型产气荚膜梭菌，5株分离菌株对不同抗生素的耐药性存在差异，但均对氧氟沙星、氟苯尼考、恩诺沙星敏感，分离菌株LY-2对小鼠有明显的致病性。更多还原

【Abstract】 In order to identify the pathogen that caused the death of five calves with diarrhea in a large-scale dairy farm in Luoyang City, Henan Province, and to understand the drug resistance of the pathogen, the mesenteric, lung and spleen tissues of the diseased and dead calves were collected after autopsy in the experiment for bacterial isolation and culture. Biochemical identification of the isolated strains, PCR amplification and sequence analysis of the 16S rDNA gene, determination of toxin genotypes, drug resistance analysis, and detection of drug resistance genes were carried out. One isolated strain was randomly selected for the pathogenicity test in mice. The results showed that the liver surface of the dead calf was diffusely bleeding; the kidneys and spleen were enlarged; the spleen capsule had bleeding points; the lungs were swollen and bleeding; the abomasum was filled with bleeding and necrosis; the entire intestine was filled with a large amount of sour and foul-smelling gas; the duodenum was necrotic; the colon was ulcerated, and the mesenteric lymph nodes were swollen and bleeding. It was suspected to be Clostridium perfringens infection. After isolation and culture, a total of five bacterial strains were obtained respectively from five dead calves and named LY-2, LY-7, LY-9, LY-10, and LY-12 respectively. The colony morphology, Gram staining results and biochemical characteristics of the five isolated strains were all consistent with those of Clostridium perfringens. Moreover, the nucleotide sequence similarity of the 16S rDNA gene with the reference strain of Clostridium perfringens in GenBank was between 97.2% and 100%. In the genetic evolutionary tree, they all clustered together with the reference strain of Clostridium perfringens. It belonged to a different branch from other strains of the Clostridium genus（such as Clostridium cadaveris, Clostridium difficile, Clostridium chauvoei, etc.）. Both α toxin and β toxin were detected, while ε toxin and ι toxin were not detected, which was consistent with Clostridium perfringens type C. The resistance rates of the five isolated strains to amoxicillin, kanamycin, gentamicin and compound sulfamethoxazole were all 100%, while the resistance rates to ofloxacin, enrofloxacin and florfenicol were all 0. The resistance rates to ceftriaxone, ampicillin, penicillin, streptomycin, sulfadioxazole and ciprofloxacin were 25%, 60%, 40%, 60%, 80% and 20% respectively.A total of six drug resistance genes among four types of antibiotics were detected in five isolated strains.Among them, the detection rate of the β-lactam resistance gene bla_CMY was 100%, while bla_TEM and bla_SHV were not detected. The detection rate of the aminoglycoside resistance gene aph（2″）-Ⅰb gene was 80%, while ant（6′）-Ⅰa, aph（2″）-Ⅰc and aph（2″）-Ⅰd were not detected. The detection rates of sulfonamide resistance genes sul1, sul2 and sul3 were 20%, 40% and 60% respectively. The detection rate of the chloramphenicol resistance gene floR was 80%. The fluoroquinolone resistance genes tet, LyrA and parC were not detected. The isolated strain LY-2 could cause the death of mice. Upon autopsy of the deceased mice, it was observed that the kidney, lung, liver, spleen, and intestine all exhibited pathological changes to varying degrees. It is indicated that the pathogen causing diarrhea and death of five calves in this dairy farm is Clostridium perfringens type C. The resistance of the five isolated strains to different antibiotics is different, but they are all sensitive to ofloxacin, florfenicol and enrofloxacin. The isolated strain LY-2 have obvious pathogenicity to mice.更多还原