羊源A型产气荚膜梭菌的分离鉴定与进化树分析
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摘要
无菌采取内蒙古通辽市某羊场病死羊肠道内容物、肝脏和肺脏,进行细菌的分离培养。将从十二指肠内分离到的1株疑似致病菌株进行生化试验、小鼠致病性试验,再将其通过魏氏梭菌多重PCR试验、魏氏梭菌ELISA试验及16S rRNA PCR试验进行鉴定。将PCR产物进行测序并进行了16S rRNA基因的进化树分析。结果显示,经细菌生化试验、多重PCR试验、ELISA试验和16S rRNA试验均证实此分离株为A型产气荚膜梭菌;进化树分析显示该菌与序列号为HQ808749.1(美国)的A型产气荚膜梭菌遗传距离最近。结果表明,该羊病例所分离的致病菌为A型产气荚膜梭菌。
To identify a suspected pathogen isolated from the duodenum of a sheep in a sheep farm in Tongliao City,Inner Mongolia.Intestinal contents,liver and lung were taken aseptically,and bacteria were isolated and cultured.The strains isolated were used for biochemical tests,mouse pathogenicity tests,multiplex PCR experiment,ELISA test and 16 S rRNA PCR test.The PCR product was sequenced and a phylogenetic tree analysis of the 16 S rRNA gene was performed.Clostridium perfringens type A was confirmed by bacterial biochemical test,multiplex PCR test,ELISA test and 16 S rRNA test.Phylogenetic tree analysis showed that the strain had the closest genetic distance to Clostridium perfringens type A with the serial number HQ808749.1(USA).The pathogenic bacteria from diseased sheep is Clostridium perfringens type A.
To identify a suspected pathogen isolated from the duodenum of a sheep in a sheep farm in Tongliao City,Inner Mongolia.Intestinal contents,liver and lung were taken aseptically,and bacteria were isolated and cultured.The strains isolated were used for biochemical tests,mouse pathogenicity tests,multiplex PCR experiment,ELISA test and 16 S rRNA PCR test.The PCR product was sequenced and a phylogenetic tree analysis of the 16 S rRNA gene was performed.Clostridium perfringens type A was confirmed by bacterial biochemical test,multiplex PCR test,ELISA test and 16 S rRNA test.Phylogenetic tree analysis showed that the strain had the closest genetic distance to Clostridium perfringens type A with the serial number HQ808749.1(USA).The pathogenic bacteria from diseased sheep is Clostridium perfringens type A.
引文
[1] 吕长辉,孙佳芝,刘晶晶,等.青岛地区兔源产气荚膜梭菌的分离鉴定及遗传多样性分析[J].中国兽医学报,2013,33(12):1822-1827.
[2] 姜艳芬,王清爱.零售肉品中产气荚膜梭菌的检测与定型:以陕西关中地区为例[J].西北农林科技大学学报(自然科学版),2016(11):55-60.
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[7] 叶宝宏,冯平,许信刚,等.羊源C型产气荚膜梭菌的分离鉴定、药敏试验及免疫治疗[J].西北农业学报,2016,25(7):960-965.
[8] 文其乙,刘秀梵.多重PCR方法快速检测粪样中产气荚膜梭菌[J].中国预防兽医学报,2002,24(1):50-53.
[9] FINEGOLD S M,SUMMANEN P H,DOWNES J,et al.Detection of Clostridium perfringens toxin genes in the gut microbiota of autistic children[J].Anaerobe,2017,45:133-137.
[10] WATTS T D,JOHANESEN P A,LYRAS D,et al.Evidence that compatibility of closely related replicons in Clostridium perfringens depends on linkage to parMRC-like partitioning systems of different subfamilies[J].Plasmid,2017,91:68-75.
[11] OSTERBAUER K J,KING A M,SEMAN D L,et al.Effects of nitrite and erythorbate on Clostridium perfringens growth during extended cooling of cured ham[J].J Food Prot,2017,80(10):1697-1704.
[12] PARK M,RAFII F.Exposure to β-lactams results in the alteration of penicillin-binding proteins in Clostridium perfringens[J].Anaerobe,2017,45:78-85.
[2] 姜艳芬,王清爱.零售肉品中产气荚膜梭菌的检测与定型:以陕西关中地区为例[J].西北农林科技大学学报(自然科学版),2016(11):55-60.
[3] 吕长辉,王方山,孙佳芝,等.青岛地区规模化兔场产气荚膜梭菌流行株毒素型、遗传多样性和耐药性研究[J].中国预防兽医学报,2014(2):102-106.
[4] 孙雨,翟新验,董浩,等.产气荚膜梭菌多毒素融合蛋白的表达与纯化及基因工程亚单位多价疫苗的制备[J].中国兽医学报,2017,37(12):2249-2255.
[5] OSOEGAWA K,YAMAGUCHI K,KISHIKAWA K,et al.An autopsy case of severe acute pancreatitis caused by Clostridium perfringens infection accompanied by pneumoretroperitoneum[J].Jpn J Med Technol,2017,66(6):15-17.
[6] DALIA H,SOHAD D,ASHRAF H,et al.Toxinotyping and antimicrobial resistance of Clostridium perfringens isolated from processed chicken meat products[J].J Vet Res,2017,61(1):53-58.
[7] 叶宝宏,冯平,许信刚,等.羊源C型产气荚膜梭菌的分离鉴定、药敏试验及免疫治疗[J].西北农业学报,2016,25(7):960-965.
[8] 文其乙,刘秀梵.多重PCR方法快速检测粪样中产气荚膜梭菌[J].中国预防兽医学报,2002,24(1):50-53.
[9] FINEGOLD S M,SUMMANEN P H,DOWNES J,et al.Detection of Clostridium perfringens toxin genes in the gut microbiota of autistic children[J].Anaerobe,2017,45:133-137.
[10] WATTS T D,JOHANESEN P A,LYRAS D,et al.Evidence that compatibility of closely related replicons in Clostridium perfringens depends on linkage to parMRC-like partitioning systems of different subfamilies[J].Plasmid,2017,91:68-75.
[11] OSTERBAUER K J,KING A M,SEMAN D L,et al.Effects of nitrite and erythorbate on Clostridium perfringens growth during extended cooling of cured ham[J].J Food Prot,2017,80(10):1697-1704.
[12] PARK M,RAFII F.Exposure to β-lactams results in the alteration of penicillin-binding proteins in Clostridium perfringens[J].Anaerobe,2017,45:78-85.