从山东省家犬血液中分离出致病性巴尔通体——文森巴尔通体伯格霍夫亚种

Study on Bartonella vinsonii berkhoffii isolated from blood of native dogs in China

目的调查家犬巴尔通体的带菌状况,分离培养并鉴定菌种。方法将采获的家犬血标本抗凝血接种于含5％去纤维兔血的脑心浸液培养基上,置于37℃含5％CO2培养箱中分离巴尔通体。然后挑选巴尔通体疑似菌落染色镜检,应用聚合酶链反应技术(PCR)在属分类水平鉴定巴尔通体,通过PCR产物限制性片段长度多态性分析(PCR-RFLP)方法在分离菌株及与阳性对照菌株之间鉴别。选择16S rRNA、gltA和16S-23S rRNA ITS的PCR产物测序,将所测核酸序列进行同源性比较及系统发育分析确定巴尔通体种或基因型。结果从山东省采获的71份犬血液中分离培养出 2株巴尔通体疑似菌株,光镜下观察为革兰染色阴性、微弯曲的细小杆菌,3对巴尔通体属特异性引物扩增结果阳性,PCR-RFLP分析2株分离菌株相同,与阳性对照不同,16S rRNA、gltA和16S-23S rRNA ITS序列分析结果表明2株巴尔通体分离株与文森巴尔通体伯格霍夫亚种的同源性分别为 100．0％、99．7％和97．2％。结论山东省家犬中存在巴尔通体感染,分离培养出的菌株经鉴定为文森巴尔通体伯格霍夫亚种,该亚种属于致病性巴尔通体。

Objective To isolate and identify Bartonella strains from native dogs in Shandong province in China. Methods EDTA-anticoagulated blood samples were collected from 71 native dogs in Yanggu county of Shandong province in March 2005. All isolates were grown on brain heart infusion agar plates containing 5 % defibrinated rabbit blood. The agar plates were incubated at 37℃ in a humidified with 5 % CO2 environment for 4 weeks or longer. All Bartonella-like isolates were examined by routine Gram and Gimenez staining and then followed by polymerase chain reaction （PER） and PCR-RFLP analysis for identification and differentiation of the isolates. Sequencing 16S rRNA, citrate synthase（gltA ） gene and 16S 23S rRNA ITS were carried out and sequeneial similarities were calculated using the DNASTAR5 software package. The phylogenetic tree was inferred from each bootstrap sample, using the neighbor- joining methods as executed in the MEGA 3. 1 software. The translation from DNA to protein were determined by DNASlS 2.5. Results The two Bartonella-like oganisms（strains Q52SHD and Q64SHD） were isolated from the blood of 71 dogs. Light microscopic examination of the Gram and Gimenez-stained microorganisms showed small, short and slightly curved pleomorphic Gram-negative bacilli. Amplified products of the three pairs of Bartonella genus-specific primers carried the same size as the predicted of those Bartonella species. Data from PCR RFLP analysis showed that the two strains that having the same profiles were all different from the B. henselae type strain 16S rRNA, gltA and 168-23S rRNA ITS sequences from the two isolates were 100.0% ,99.7% and 97.2% homologous to B. vinsonii berkhoffii. Conclusions Based on these findings, the two isolates Q52SHD and Q64SHD were demonstrated as B. vinsonii berkhoffii, To our knowledge,this was the first report on the presence of Bartonella infection in native dogs from China, which constituted a large reservoir of Bartonella species in this country.