鹿出血性肠炎病原菌的分离和鉴定

Isolation and Characterization of the Pathogens within Deers Died from Hemorrhagic Enteritis

通过对死于出血性肠炎的圈养鹿的病原菌进行分离鉴定,为研制产气荚膜梭菌β-毒素单价和多价疫苗奠定基础。采集山西省内不同地区鹿场因出血性肠炎而死亡鹿的病料32例,经病原微生物分离培养、生化试验和血清型鉴定,分离得到C型产气荚膜梭菌,并测定分离菌所产毒素对小鼠的最小致死量。PCR扩增C型产气荚膜梭菌β-毒素基因,构建重组质粒p MD18-T-J28-C,进行酶切鉴定和核苷酸序列分析。结果 32株分离菌中有6株是C型产气荚膜梭菌,占18.7%;其余均为A型,占81.3%。筛选出毒力最强的菌株J28-C,最小致死量(MLD)为5.0×105CFU/m L。PCR扩增和核苷酸序列分析表明,经PCR得到了特异性的β毒素基因片段。表明造成山西省鹿出血性肠炎的病原菌为A型和C型产气荚膜梭菌,以A型为主。

Isolating and identifying the pathogens within deer died from hemorrhagic enteritis,and providing information for developing beta toxin gene engineering subunit vaccine and bacterial toxin polyvalent vaccine.Samples were collected from 32 cases of cervus hemorrhagic enteritis in Shanxi province,and the major microbial pathogen were isolated and examined microscopically,and then identified through serum reaction.A Beta-toxin gene was amplified by PCR and The recombinant plasmid p MD18-TJ28-C was constructed.The recombinant plasmid p MD18-T-J28-C was studied in detail by restriction endonucleases analysis and nucleotide sequencing.The results showed that 6 of 32 isolated were Clostridium perfringens type C,accounting for 18.7%,and the rest were type A,about 81.3%.The most virulent strain J28-C was selected and its minimum lethal dose（MLD）was 5.0×105CFU.Further,PCR and sequence analysis showed that the recombinant p MD18-T-J28-C possessed a positive beta-toxin gene sequences,and a reading frame.Hemorrhagic enteritis in Shanxi province is caused by Clostridium perfringens of type A and C,and type A is the major pathogen.