Study On The Genetic Diversity Of Borrelia Burgdorferi Sensu Lato In China

Lyme disease (Lyme borreliosis) is a tick-borne and multisystemic enzootic disease. Its etiologic agent, Borrelia burgdorferi sensu lato (B. burgdorferi, also called Lyme spirochete), was originally thought to be homogeneous. However, many studies have demonstrated that B. burgdorferi is phenotypically and genotypically heterogeneous. Till date, at least 14 species of B. burgdorferi have been described:B. burgdorferi sensu stricto, B. garinii, B. afzelii, B. japonica, B. valaisiana, B. lusitaniae, B. andersonii, B. tanukii, B. turdi, B. bissettii, B. sinica, B. spielmani, B. californiensis, and B. carolinensis sp. nov. And new species are emerging. Distribution of Lyme spirochete genotypes is closely associated with geographical environment, vector and reservoirs. The main clinical features caused by different species of B. burgdorferi are different. So it has great significance to clearly understand the genetic diversity, genotype species and distribution of B. burgdorferi in China for epidemiological study and prevention and treatment of Lyme disease.Multilocus sequence analysis (MLSA) was used in our research. Seven locus(rrf-rrl spacer, rrs, hbb, groEL, recA, fla and ospA) of 102 Chinese isolates from ticks, animals and patients in 12 provinces (municipality, autonomous region) were amplified by PCR and DNA sequencing. All the sequences of Chinese strains as well as 8 foreign reference isolates were analysed by MEGA4.0 software. Combining the results of 5S-23S rRNA spacer PCR-RFLP, we investigated the genetic diversity, genotype species and distribution of B. burgdorferi in China as well as the relationship between the pathogen and vector, the pathogen and animal reservoirs.The results showed that 102 isolates were classified as four genotypes: B. burgdorferi sensu stricto, B. garinii, B. afzelii, and B. valaisiana.67 strains (65.69%) were belonged to B. garinii, which were distributed in Heilongjiang, Jilin, Liaoning, Inner Mongolia, Xinjiang Uygur Autonomous Region, Hebei and Guangdong; 20 strains (19.61%) were belonged to B. afzelii, which were distributed in Heilongjiang, Jilin, Liaoning, Xinjiang Uygur Autonomous Region, Shandong, Beijing, Chongqing, Guangdong and Guizhou; 14 strains (13.73%) were belonged to B. valaisiana ,which were distributed in Guizhou province; Additionally, one B. burgdorferi sensu stricto strain was isolated from Hunan province. According our research B. garinii and B. afzelii are the main pathogenetic genotypes in China, B. burgdorferi sensu stricto is rare. And also we found there were two genotypes in some provinces:B. garinii and B. afzelii in Heilongjiang, Jilin, Liaoning, Xinjiang Uygur Autonomous Region and Guangdong; B. valaisiana and B. afzelii in Guizhou. This shows that there is obvious genetic diversity of Chinese Lyme disease spirchete and B. burgdorferi is great heterogeneous in many provinces of China.The study also shows Ixodes persulcatus, which mainly distributed in northern China, was the main vector of B. garinii and B. afzelii; Ixodes granulatus was the main vector of B. valaisiana in Guizhou province; Haemaphsalis longicornis and Haemaphsalis bispinosa were the main vectors of B. afzelii. And we also found that Apodemus agrarius was the important host animal, and could carry B. garinii, B. afzelii, and B. valaisiana. At present it shows that Borrelia burgdorferi sensu lato (B. burgdorferi) is a genetic diversity complex, in which there are at least 14 genospecies. But only B. burgdorferi sensu stricto (B.b.s.s), B. garinii (B.g) and B. afzelii(B.a) of this complex cause the multisystem disorder (Lyme disease). It has confirmed that there are at least four species of B. burgdorferi in China by 5S-23S rRNA intergenic PCR-RFLP and Multilocus Sequence Analysis (MLSA), including B.b.s.s, B. garinii, B. afzelii and B. valaisiana。To gain the genomic information of Chinese B. burgdorferi strains, reveal the differences between species and analyse the evolution law as well as find clues for pathogenesis, we conducted a whole genome sequencing of five Chinese strains, including CS4 of B.b.s.s, PD91 of B. garinii, FP1 and R9 of B. afzelii, QX-S13 of B. valaisiana. And also analysed the sequences preliminarily.The results show that in Chinese strains the chromosome length is between 946,464 bp and 1,024,390 bp, G+C content is between 27.66% and 28.3%, the functional annotation and distribution of metabolic modules of chromosome for the five strains are almost the same:mainly for cell growth maintaining, reproduction, cell motility, defense and other ingredients for cell survival. And also there are some unknown genes.The chromosome sequences of the five strains were compared with the corresponding reference strains respectively. The results show that genetic differences between the two strains of B.B.s.s genotype are minimal, no PA genes, and only 70 SNPs. However there are great genetic differences between chromosome sequences of the two strains of B.g genotype.The number of SNPs was 5917,85 times over the SNP number of B.b.s.s strains. There are more PA genes in B.v strains.Sequence analysis shows that the plasmids of 5 strains are quite different. But plasmids cp26 and 1p54 or their homologous plasmids exist in all five strains.