The Diversity Study Of The Bartonella Infection In Rodents In Three Provinces In The Midwest China

Objectives: The objectives of the study were to investigate the infection status of Bartonella in wild rodents from the Ningxia Hui Autonomous Region,Nei Monggol Autonomous Region and the Province of Shanxi in China,and to obtain the composition of the host animal and to characterize the Chinese strains by molecular techniques and to provide evidence for the prevention and control of disease.Methods: Samples were selected from 17 sampling points in 8 cities of 3 Provinces with elevational ranges from 800 meters to 2400 meters at east longitude 105 °~116 °,north latitude 35 °~45 °,and annual average temperature ranges from 0.7 degree centigrade to 19 degree centigrade.The night trapping method was employed to capture wild rodents,using DNA barcode technology to identify the types of rat,and to record the sex,the value of longitude and latitude,altitude,and the other information such as habitat,and the livers tissue were extracted using aseptic technic.The liver tissue specimens of rat was inoculated in containing 5% defiber sheep blood on the TSA to train,and identification of the morphological aspects,including the gram stain and microscopic examination.Nucleic acids were crudely extracted from colonies,and to choose Bartonella specific primers to applied gltA gene,and to assay of electrophoresis strip.The PCR amplification products were sequenced by sequencing company.Bartonella sequences obtained from 219 isolates were compared with those of known Bartonella species deposited in NCBI Genebank.According to kit operating steps,Bartonella Nucleic acids from positive strains were extracted.Then the ftsZ gene,rpoB gene,and 16 SrRNA gene of the positive sample were applied.The PCR products were sequenced.A phylogenetic tree was drawn based on the sequences of gltA,rpoB and 16 SrRNA,using the neighbor-joining method with Kimura'S two-parameter distance method in MEGA 6.06.Bootstrap analysis was carried out with 1,000 resamplings.The haplotype diversity and nucleic acid diversity of the strains were calculated.Strain is adopt the method of continuous subculture and freezing preservation.Statistical analyses were performed using the SPSS 19.0 statistical software package.The infection rate among the different groups was tested using Chi-square test.The software of Chromas is used to compare the DNA sequences of peak figure.Sequencing results are homologous comparison with NCBI blast.A phylogenetic tree was drawn to analyze,using the neighbor-joining method in MEGA 6.06.ArcGIS software is used for construction of sampling in rats captured and Bartonella component strain genotype distribution.DnaSPv5 is used to calculate the haploid type diversity and nucleotide diversity.The results were as follows:1.The natural infection of Bartonella in wild rodents.A total number of 787 rodents were collected from the Ningxia Hui Autonomous Region,Nei Monggol Autonomous Region and the province of Shanxi in China during 2013~2015 and 26 species of 13 genera of rodents were captured.219 Bartonella isolates were obtained from 21 species of 12 genera of rodents and the overall were obtain from Spermophilus alaschanicus?Spermophilus dauricus?Citellus erythrogenys?Apodemus agrarprevalence of Bartonella in those rodents was 27.83%.Among them,Prevalence was highest in Nei Monggol Autonomous Region(41.50%).Prevalence was 21.98% in Ningxia Hui Autonomous Region and 20.40% in Shanxi Province.Bartonella isolates from Apodemus peninsulae?Apodemus peninsulae?Apodemus draco ? Cricetulus longicaudatus ? Cricetulus eversmanni ? Cricetulus Barabensis?Cricetulus triton?Mus musculus?Dipus sagittal?Allactaga sibirica?Rattus niviventer?Meriones meridianus?Meriones unguiculatus?Eothenomys eva?Cricetidae?Lasiopodomys brandtii and Phodopus sungorus.The result of statistical analysis show that a significant difference exists in different areas of three provinces(P<0.01).The result of statistical analysis also show that a significant difference exists in different altitudes?annual average temperature?habitats and the wild rodent species.2.Genetic evolution analysis.Sequences were analyzed that Bartonella isolates from rallus were mainly clustered in six known genotypes(B.grahamii,B.elizabethae,B.Jaculi,B.Japonica,B.Taylorii and B.washoensis)and one unknown genotype.To characterize the Bartonella obtained in this study,sequence of gltA,16 SrRNA,ftsZ,rpoB were analyzed,there is great difference in nucleotide sequence of Bartonella in rodents in in the Ningxia Hui Autonomous Region,Nei Monggol Autonomous Region and the province of Shanxi in China.There are many sequence polymorphism loci.Haploid type variety and diversity of nucleic acid value is very high.The evolution of the Bartonella by neutrality test was the negative selection.In conclusion,219 Bartonella isolates were obtained from three provinces in the study.It contains six known genotype and one new genotype and there are at least four kinds of pathogenic genes to people.The results illustrate the prevalence of Bartonella bacteremia was affected in different habitats and under various factors of wild rodents.The evolution of the Bartonella is negative selection.