巴尔通体分离培养及生物学特性研究

Culture and isolation of Bartonella and analysis of its biological characteristics

目的研究巴尔通体的分离培养及生物学特性。方法采用含5%去纤维兔血脑心浸液琼脂培基对收集自云南省的16个县(市)的913份鼠类血液进行巴尔通体分离培养,对疑似菌落用巴尔通体属特异性引物进行聚合酶链反应(PCR)及进行枸橼酸合酶基因(gltA)的379bp片断扩增,从电泳图中出现目标带的阳性菌中随机选出菌株进行电镜检测,同时将其gltA扩增产物行已序列测定并与已知菌株进行比较。结果从913份鼠类血液中分离到巴尔通体251份,分离率为27.5%(251/913)。阳性菌落长出时间最早为3d,菌落形态随培养时间延长而改变且特点多样;电镜扫描见多个菌缠绕成团,菌体密集,透射见菌细胞呈短杆状、菌壁较厚。核苷酸序列及相似性比较发现家鼠中巴尔通体有3个基因型。结论分离的巴尔通体种类多、生长特性不一、形态多样,呈高度流行及基因型的多样性,一些不明原因的疾病可能与之感染有关。

Objective To culture and isolate of Bartonella and analysis of its biological characteristics. Methods Bartonella strains were isolated from 913 rat blood samples collected from 16 counties in Yunnan Province and inoculated into macconkey agar with 5 % non - fiber rabbit blood brain heart infusion broth, then cultured and isolated at 35 ℃ in 5 % CO2 chamber, stained with Gram staininig and examined under microscope to observed their characteristics. The suspected bacteria colonies were amplified by PCR with specific Bartonella introducer to amplify specific gene fragment（379 bp of glt A gene）, if the objective band appears on electrophoresis chart it can be judged that the bacteria were the positive strains. The ultrastructure of Bartonella from any positive strain was observed with electron micmscopy, and the 379 bp fragment of gltA gene was sequenced and compared with other Bartonella strains. Results 251 Bartonella strains were isolated from 913 rat blood samples with the isolation rate of 27.5 %. The earliest growing period of positive colonies was 3 days. The morphological changes of positive colony were observed along with the elongation of culturing period with diversity of characteristics. Groups of Bartonella were seen twining round through scanning electron microscopy. Small coccobacillus and thicker membrane of Bartonella were noticed through transmission electron microscopy. The three genotypes were found in the Bartonella in Yunnan by sequencial analysis. Conclusion Bartonella strains are diverse, and its morphology may metamorphose associated with its broad pathogenesis to human. These findings demonstrate that Bartonella spp is highly pprevalent and its genotype is highly diverse. The findings also indicate further investigations be carried out on elucidate its role in the transmission of unknown infection.