• 中文核心期刊
  • CSCD来源期刊
  • 中国科技核心期刊
  • CA、CABI、ZR收录期刊

山羊伪结核棒状杆菌的分离鉴定及耐药性分析

Isolation, Identification and Antibiotics Resistance of Corynebacterium pseudotuberculosis from Goats

    摘要
  • 摘要:
      目的  分离、鉴定福建省某养殖场病死山羊的病原，明确山羊病死原因，为山羊伪结核棒状杆菌病的防治提供科学依据。
      方法  以无菌采集的病羊肺脏为材料，进行细菌分离纯化，鉴定分离菌株的形态特征及培养特性；提取菌株DNA，利用特异性引物和16S rRNA通用引物进行PCR，16S rRNA扩增片段送公司进行克隆测序并对序列进行分析；并进行生化、药敏和动物攻毒试验。
      结果  该分离菌株在含10%脱纤维绵羊血的琼脂培养基上，生长成直径1 mm左右、中央凸起、乳白色、边缘不整齐、伴有β溶血环的菌落，在营养琼脂培养基上生长不良，在麦康凯琼脂培养基上不能生长。16S rRNA测序结果经NCBI在线软件Blast后显示与GenBank上的伪结核棒状杆菌的同源性高达100%；山羊伪结核棒状杆菌特异性PCR有目的扩增条带；生化试验结果与伯杰氏细菌鉴定手册中伪结核棒状杆菌相符，表明分离菌为山羊伪结核棒状杆菌，命名为FJ-PN。小鼠攻毒试验结果显示，小鼠腹腔与皮下注射0.3 mL含量为4×105CFU·mL-1的分离菌，在40 h内全部死亡；山羊攻毒试验结果显示，山羊经皮下注射2 mL、鼻腔接种1 mL含量为4×105CFU·mL-1的分离菌3 d后死亡。药敏试验结果显示该菌对克拉霉素、环丙沙星、青霉素、四环素、左氟沙星、氧氟沙星、庆大霉素高度敏感，对红霉素、氯霉素、氨苄西林、头孢噻肟、头孢西丁、利福平低度敏感，对链霉素、甲氧苄啶表现耐药。
      结论  本研究分离到的1株细菌为山羊伪结核棒状杆菌，具有较强的致病性，且对抗生素表现出一定的耐药性，为该病的诊断和合理用药提供了理论支持。

     

    Abstract:
      Objective  To identify the pathogen that caused the death of goats in a farm in Fujian for prevention and treatment of Pseudotuberculosis.
      Method   Aseptically collected specimens of lung tissues from the dead goats were used to isolate the microbes for morphological observation and strain identification. DNA of the isolates were extracted, and the specific and 16S rRNA universal primers selected for PCR. The 16S rRNA amplified fragment was cloned, sequenced and analyzed. Biochemical, drug sensitivity, and animal challenge tests were performed on the isolate for taxonomic identification.
      Results  The isolated strain was cultured on an agar medium containing 10% defibrated sheep blood to grow into a colony with a diameter of about 1 mm, a central bulge, a milky white color, an irregular edge, and a beta hemolysis ring. It did not grow well on a nutrient agar nor a MacConkey agar medium. The 16S rRNA sequencing showed an up to 100% homology with Corynebacterium pseudotuberculosis on GenBank by the NCBI online software Blast. Like the specific PCR of C.pseudotuberculosis, that of the isolate also had the same amplified targeted bands. In addition, the biochemical test on the isolate duplicated the characteristics of C. pseudotuberculosis shown in the Berger's Bacterial Identification Manual. Hence, the isolated pathogen named as FJ-PN was verified to be C. pseudotuberculosis. A challenge test on mice with an intraperitoneal or subcutaneous injection of 0.3 mL of 4×105 CFU·mL-1 of the isolate died within 40 h. Furthermore, two goats injected subcutaneously with 2 mL of 4×105 CFU·mL-1 and intranasal inoculated simultaneously with 1 mL of 4×105 CFU·mL-1 of the isolate died within 72 h. The drug sensitivity test showed that the strain was highly sensitive to clarithromycin, ciprofloxacin, penicillin, tetracycline, levofloxacin, ofloxacin, and gentamicin, slightly sensitive to erythromycin, chloramphenicol, ampicillin, cefotaxime, cefoxitin, and rifampicin, and resistant to streptomycin or trimethoprim.
      Conclusion  The pathogenic bacteria isolated from the diseased goats was identified as C. pseudotuberculosis. It had a strong pathogenicity with varying degrees of drug resistance to antibiotics.

     

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