Detection And Isolation Of Bartonella Spp. In Rodents In Helongjiang Province Of China

ObjectivesThe objectives of the study were to investigate the infection status of Bartonella in wild rodents from Helongjiang province of China, and to obtain isolated strains of of Bartonella and to characterize the Chinese strains by molecular techniques.MethodsThe region where the rodents were captured was taken as the points of Surveillance of Tick-borne disease, including tongjiang, suifenghe, dongning and mudanjing area. The study was taken by the State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, The sample size was determined by the method of estimation of sample size being used in the cross-sectional study.Wild rodents were captured. According to kit operating steps, Bartonella DNA from spleen samples was extracted. Specific primers of Bartonella were used to amplify the gltA gene fragment. Specimens of gltA gene positive were defined as positive judgement standard. Then the16S/23SrRNAITS gene of the positive sample was applied. The PCR products were sequenced. Bartonella sequences obtained in this study were compared with those of known Bartonella species deposited in the GenBank. A phylogenetic tree was drawn based on the sequences of gltA and16S/23SrRNAIT, using the neighbor-joining method with Kimura’s two-parameter distance method in MEGA5.0. Bootstrap analysis was carried out with1,000resamplings. The spleen issues of the positive samples were inoculated on chocolate agar plate. The gltA of suspected colonies on chocolate agar plate were detected, the Bartonella isolates obtained were confirmed further as follows:①6S/23SrRNAITS gene,16SrRNA gene, FtsZ gene was amplified as well.②Identification of the morphological aspects, including the gram stain, Giemsastaining, optical microscope, electron microscope.③The Successfully isolated Bartonella were preservation by continuous passage culture and Cryopreservation.④the products of the Bartonella isolates were sequenced, and then compared with the sequence obtained from the spleen issues.All statistical analyses were performed with the SPSS16.0statistical software package, x2-test, Fisher’s exact test and Binary Logistic method were used.Results1. The natural infection of Bartonella in wild rodents.A total number of514rodents were collected from Heilongjiang province during2009-2011and11types of Species were included. The overall prevalence of Bartonella in those rodents was13.81%(95%CI:11.09%-17.06%). The result of statistical analysis show that there is no significant difference exists in different species of rodents (P=0.098). The result of statistical analysis show that a significant difference exists in different areas of Heilongjiang province (P=0.003). Prevalence was highest in Mudanjing area (18.15%).2. The isolation and identification of Bartonella.By inoculation of chocolate agar plate and Columbia blood agar plate,6strains of Bartonella were successfully isolated,3strains derived from Apodemus agrarius, the other3strains were isolated from the Clethrionomys rufocanus, Tscherskia triton Rattus norvegicus respectively. The Successfully isolated Bartonella were confirmed by PCR, gram stain, Giemsa staining, optical microscope and electron microscope.3. Sequence analysis of the Bartonella.To characterize the Bartonella obtained in this study, sequence of gltA,16S rRNA were analyzed, there is great difference in nucleotide sequence of Bartonella in rodents in in Heilongjiang proviceConclusionThe study is the first report of detection by PCR amplification of Bartonella in rodents in Heilongjiang provice and seven strains of Bartonella were isolated. Sequence analysis revealed the genotypes diversity of Bartonella. This study promoted monitoring of Bartonellosis in Heilongjiang provice and laid a foundation.for the further research of Bartonellosis in the north region.