Investigation And Whole-Genome Sequencing And Analysis Of Bartonella In Rodents In Guangdong Province

Objective1.To investigate the distribution and prevalence and genetic evolution of Bartonella spp.in rodents in Guangdong Province and provide evidence for the prevention and control of Bartonellosis.2.Bartonella strains were sequenced by high-throughput sequencing technology to obtain its genome sequence information and to analyse and discuss its functional genes,virulence genes and pathogenic mechanisms.Methods1.We selected seven cities,distributed in the province on the basis of the geographic division of Guangdong Province.The rodents were captured and total nucleic acids were extracted from liver specimens.2.Bartonella nucleic acid was detected using polymerase chain reaction(PCR).Bartonella non-coding RNA(ssrA)gene was detected by real-time PCR method.For positive samples,the bacterial isolation was performed,and further amplified the citrate synthase gene(gltA),about 379 bp in length,and the RNA polymerase subunit gene(rpoB)with the length of 866 bp by ordinary PCR.The amplified products were sequenced in biotech company.3.The sequences of ssrA,gltA,and rpoB gene were homologously compared with sequences deposited in NCBI,and the optimal model for topology construction was tested using Mega 6.0 software.The phylogenetic trees were constructed by neighboring joining method and Maximum Likelihood method.The statistical analysis were performed using SPSS 19.0 and comparison of infection rates among different groups was performed using the Chi-square test and Fisher's exact test.4.The whole genome sequence of two Bartonella strains were sequenced using the IonTorrent PGM platform and genome was annotated through the RAST platform to obtain genome-wide maps,and to compare and analyse functional and virulence genes with different bartonella isolates.Results1.In this study,495 liver tissue samples were collected from 7 cities,Huizhou,Foshan,Maoming,Yunfu,Chaozhou,Jieyang and Heyuan in Guangdong Province,the bartonella-infected animals belonging to 2 orders,3 families,6 genera and 8 species.The total infection rate of Bartonella in rodents was 12.52%and infected species included Rattus norvegicus,Rattus flavipectus,Rattus rattus,Rattus losea,Bandicota indica,and Suncus murinus.2.There were statistical differences in the infection rates among different regions(P=0.008)and the Bartonella genotypes of these areas were different.Except for Foshan,there were more than two Bartonella genotypes in other regions.The differences of infection rates in rodents among different species(P=0.014)had statistical significance,but there were no differences between different gender(P=0.826)and habitat(P=0.512).3.According to the homology and phylogenetic analysis,7 genotypes of Bartonella were detected in rodents,which were B.elizabethae?B.henselae?B.rochalimae?B.tribocorum?B.coopersplainsensis?B.queenslandensis?B.phoceensis.4.The genotype of B1601 strain was B.elizabethae,and whole genome size was 1929474 bp with GC content of 39.91%.B.1607 was B.queenslandensis with a total genome length of 2173468 bp and GC content of 39.78%.There were prophage genes and flagellar genes in the genome of both strains,and the their virulence genes were also different,which provided some clues to explore the pathogenicity and mechanism of pathogenesis for Bartonella study.ConclusionsIn Guangdong province,Bartonella is widespread in rodents with obvious diverse genotypes.There were four kinds of pathogenic genotypes,which poses a potential threat to human health.In our study,both Bartonella strains had functional genes associated with cell adhesion and invasion,and the expression of some genes were different compared with other strains,but whether these genes were associated with pathogenicity and pathogenical mechanism remained to be further explored.Considering rodents closely contacted with other animals and humans,the infection of Bartonella in rodents may spread to other hosts,and cause human Bartonellosis which may present as latent infection or obvious clinical disease.So it is necessary to strengthen surveillance of prevalence of Bartonella in animals and investigation of suspicious clinical cases.