犬种布鲁菌病病原学和遗传特征分析

摘要

Objective To analyze the etiology and genetic characteristics of canine brucellosis in a relatively closed population.Methods Canine brucellosis was detected in an experimental animal breeding base.The research methods consisted of serological testing [tube agglutination test (TAT)],strain identification (biochemical and phage testing),histopathological examination of tissue (liver,spleen and testis) specimens of dog,genetic characteristics analyzing of the strains [16S rRNA gene sequencing,real-time PCR (RT-PCR),multilocus sequence typing (MLST),pulse field gel electrophoresis analysis (PFGE),and multilocus variable number tandem repeat analysis (MLVA) and IS711 southern hybridization].Results Totally 51.25％ (41/80) of serum specimen from Beagle dogs was positive with antibody titer above 1:50.The Brucella DNA of 80 blood specimen was positive by RT-PCR detection.The lesion tissues and pathogenic bacteria like cells were found in the liver,spleen and testis.Totally 10 isolates,which were cultured from blood specimen were confirmed as Brucella canis by biochemistry and phage testing.Sequence analysis of 16S rRNA gene for the 10 isolates was consistent with that of Brucella in the database.The results of the three genotyping methods for the 10 strains were identical by MLST,PFGE and IS711 southern hybridization.The 10 isolates were clustered into two different MLVA genotypes.Conclusions It is identified as a canine brucellosis outbreak based on the databases of serology,bacteriology,16S rRNA gene sequence analysis,histopathology and genotyping by PFGE,MLST and MLVA.The ten Brucella canis isolates seem to origin from a same clone,and evolve into two stable and heritable MLVA genotypes in the natural environment.%目的 分析相对封闭群体中犬种布鲁菌病的病原学和遗传特征.方法 对北京一个实验动物基地饲养的犬进行布鲁菌病检测,包括血清学实验[试管凝集法(TAT)]、菌株鉴定(噬菌体实验和生化检测方法).采集犬组织(肝、脾和睾丸)标本进行病理组织学检查,对菌株进行基因特征分析[16S rRNA基因测序、荧光定量PCR(RT-PCR)检测血液标本核酸DNA、基因组DNA多位点序列分型(MLST)、脉冲场凝胶电泳(PFGE)分析、多位点可变数目串联重复序列(MLVA)分析和IS711插入序列Southern杂交分析].结果 51.25％(41/80)的比格犬血清中存在高滴度(≥1:50)的犬种布鲁菌抗体.80份血液样本的RT-PCR核酸检测全部阳性.病犬肝、脾和睾丸组织中发现有明显的病变和病菌样细胞.血液组织分离出10株细菌,经生化检测和噬菌体实验证实为犬种布鲁菌.对菌株16S rRNA基因测序分析,结果与数据库中犬种布鲁菌完全吻合.MLST、PFGE和IS711插入序列Southern杂交分析10株菌株,基因型完全相同.MLVA分析发现,10株菌株分为2个不同的基因型.结论 通过血清学、细菌学、组织学、16S rRNA基因测序、MLST、PFGE、MLVA和IS711插入序列Southern杂交全面分析一起犬种布鲁菌病爆发疫情,经MLVA分析发现,犬种布鲁菌在自然环境中,进化为两个相对稳定的MLVA基因型.