海南省小型兽类巴尔通体的分离培养和序列分析

Isolation and sequence analysis of Bartonella in small mammals in Hainan province

目的调查海南省小型兽类巴尔通体感染状况以及携带巴尔通体的种类,为巴尔通体感染的预防控制提供科学依据。方法用鼠笼法捕获小型兽类,取心脏血,抗凝,取100μl抗凝血用胰酶大豆肉汤按1∶4稀释后接种于含5%去纤维羊血的胰酶大豆琼脂培养基上,置含5%CO2的培养箱中37℃培养45d。挑选疑似菌落涂片,革兰染色、Gimanez染色后做镜检进行初筛,将革兰阴性杆菌用巴尔通体属特异性引物进行聚合酶链反应(PCR)。然后对其PCR产物测序,将所测核酸序列提交到GenBank,做相似性比较及序列分析。结果从65份样本中分离培养出疑似菌落6株,革兰染色镜检均见阴性小杆菌,Gimanez染色为红色杆菌,经PCR证实6株均为巴尔通体,其中分离自黄毛鼠和屋顶鼠各2株,针毛鼠和臭鼩鼱各1株。经过序列分析证实,所分离到的巴尔通体与Bartonella rattimassiliensis、B.tribocorum和B.queenslandensis3种巴尔通体相似度最高。结论海南省小型兽类中有巴尔通体感染,存在人群感染的风险。

Objective To determine the current situation of Bartonella infection in and types of Bartonella carrying small mammals in Hainan province,providing evidence for prevention and control of Bartonella infection. Methods The rat-cage method was employed to capture small mammals,of which heart blood was collected and treated with anticoagulants. A 1∶4 dilution of 100 μl anti-coagulation blood and trypsin soy broth was inoculated onto the trypsin soybean agar culture media containing 5% de-fiber sheep blood and cultured in a 5% CO2 incubator at 37 ℃ for 45 d. Suspected colonies were smeared,Gram-and Gimanez-stained and screened under microscopy. The polymerase chain reaction （PCR） was performed for the Gram-negative bacilli with Bartonella genus-specific primers,followed by sequencing of the PCR products. The nucleic acid sequences were submitted to GenBank for similarity comparison and sequence analysis. Results A total of 6 suspected colonies were isolated from 65 samples,all being Gram-negative small bacilli,and red for Gimanez staining. The PCR results confirmed that the six strains were all Bartonella,two isolated from Rattus losea and Rattus brunneusculue each,and one from Niniventer fulvescens and Suncus murinus each. The sequence analysis suggests that the isolated Bartonella had the highest similarity with Bartonella rattimassiliensis,B. tribocorum and B. queenslandensis. Conclusion Bartonella infection was found among small mammals in Hainan province,indicating risks of infection among human beings.