Survey On The Prevalence And Genotyping Of Borrelia Burgdorferi Sensu Lato In Vectors And Hosts

The spirochetes of the Borrelia burgdorferi sensu lato complex, the causative agents of Lyme borreliosis, are maintained in natural foci by circulation between the vector ticks in the Ixodes ricinus complex and animal hosts in various vertebrate taxa. This complex comprises at least 13 species, among which 4 species have been believed to be pathogenic to humans, including Borrelia burgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii and Borrelia spielmanii. Some of species have a wide and overlapping distribution while other species are distributed in different geographical regions. Different species are associated with distinct ecologic features, levels of pathogenicity, and clinical symptoms in patients.In order to find out underlying risk of Borrelia infection in the different regions of mainland China, and to infer the role of hosts that shape the evolution and epidemiology of B. garnii and B. afzelii, which were widely distributed in Eurasia, surveys were conducted in endemic foci (i.e. Heilongjiang province, Inner Mongolia autonomous region, Zhejiang province, Guizhou province, Xinjiang autonomous region) and potential endemic foci (i.e. Yunan province and Tibet autonomous region) in peak seasons of ticks during 2004 to 2008. Samples were collected, and Borrelia strains were isolated, and further PCR-RFLP analysis of 5S-23S rRNA intergenic spacer was conducted to investigate the prevalence and genospecies of B. burgdorferi s.l. in vectors and hosts. For samples which genospecies were not be clearly identified by using PCR-RFLP analysis, phylogenetic analyses of single gene (i.e. 16S rRNA,fla) and multilocus sequence analysis (MLSA) were performed to clarify their taxonomic status. Moreover, three B. valaisiana-like strains were inoculated into C3H/HeN mice in order to examine their pathogenic potential to humans. Besides, we analyzed the phylogeographic population structure of B. burgdorferi sensu lato in Eurasia by using a recently developed multilocus sequence typing (MLST) scheme based on eight chromosomally located housekeeping genes.A total of 1632 unfed adult ticks (i.e. Ixodes persulcatus, Dermacento silvarum, Haemaphysalis concinna and Haemaphysalis tibetensis) and 929 small animals (i.e. rodent, hare and animals of insectivora) and 916 Melophagus ovinus were collected and detected by nested PCR targeting 5S-23S rRNA intergenic spacer and RFLP analysis, with additional sequence analysis when necessary. B. burgdorferi s.l. was found in other six study areas except for Xinjiang autonomous region. This finding suggested that the endemic foci of Lyme disease might be still maintained in most of survery areas in mainland.Among 38 rodent species detected, 11 species were positive. Significant difference in prevalence of B. burgdorferi s.l. in rodents was observed among various regions (χ2=21.255,df = 5, P=0.001), with the highest in southern areas, which might be caused by the difference of climate. This finding suggested that the transmission cycle of B. burgdorferi s.l. might be easily maintained in the region with the warmer climate than that with the colder climate. In addition, B. burgdorferi s.l. was also detected in 3 Chinese hares (Lepus sinensis Gray) and 1 Hodgson's brown-toothed shrew (Soriculus caudatus), which suggested that there were much species of animals serving as potential reservoir host of B. burgdorferi s.l. in mainland China.In Inner Mongolia autonomous region, the infection rate of I. persulcatus was significantly higher than that of D. silvarum, and H. concinna was free of B. burgdorferi s.l. (χ2=62.965,df = 2, P=0.000). I. persulcatus is the principal species of ticks in this forest area. These results indicated that I. persulcatus was still the major vector of B. burgdorferi s.l. in this area. However, D. silvarum and H. concinna usually inhabit grassland. This suggested that the threat of Lyme disease to humans might be less in grassland than that in forested area. Additionally, 3.6% (33/916) of M. ovinus samples was positive in Tibet, which indicated that some sheep in this pasture might be infected with B. burgdorferi s.l. or in risk of Lyme disease.Three genospecies (or group) were identified as B. garinii, B. afzelii and B. valaisiana-like group according to PCR-RFLP analysis. B. garinii being pathogenic to humans was the most prevalent genospecies and widely distributed in five regions except for Xinjiang, followed by B. afzelii. This finding suggested that the risk of Lyme disease to humans and livestock existed far and wide in mainland China. Multiple infections of different genospecies were found in single tick from Inner Mongolia and in single M. ovinus sample from Tibet. Multiple infections of different genospecies might also occur in humans by the tick bite. Since different genospecies of B. burgdorferi s.l. are related with distinct clinical manifestation of human Lyme disease, clinical symptom of patient being multiply infected with different Borrelia genospecies may be untypical and even complex, which should be noticed in diagnosis. B. valaisiana-like group was only found in Zhejiang, Guizhou, Yunnan and Tibet of south China, and its taxonomic status and pathogenic potential to humans remain unclear.Phylogenetic analyses of 16S rRNA gene and fla gene indicated that B. valaisiana-like strains were closely related to B. valaisiana. Meanwhile, difference was observed between two species. Further study based on multilocus sequence analysis (MLSA) indicated that the concatenated sequences of B. valaisiana-like strains used in this study were 98.4-99.8% identical with each other, whereas they were 96.4-96.9% related to those of B. valaisiana strains and only 92.5-94.9% related to those of other B. burgdorferi s.l. on the basis of the sequence identity matrix. According to MLSA sequence identity cut-off of 97.9% to differentiate B. burgdorferi s.l. species, B. valaisiana-like strains deserve a species status and are proposed to be named Borrelia yangtze sp., because of its distribution usually in the valley of Yangtze River. The finding explained the differences in transmission cycle and phenotype between B. valaisiana strains from Europe and B. valaisiana-like strains from Eastern Asia.Three low-passage B. valaisiana-like strains as well as positive control B. garinii isolate JW1 were inoculated into C3H/HeN mice and disease was monitored by histopathology at 4 weeks, 8 weeks and 12 weeks after spirochaete challenge. Joint, heart, kidney, spleen, brain and liver were examined. B. valaisiana-like strains induced similar or more serious lesions in the joints, hearts, kidneys, spleens, brains of infected mice with B. garinii isolate JW1. No lesions were noted in the livers with inoculation of with B. valaisiana-like strains or B. garinii isolate JW1. But the pathogeneicity of these B. valaisiana-like strains to humans remains to be further clarified by the data of the infected patients.Based on sequence analyses of multiple housekeeping genes (i.e. clpA, clpX, nifS, pepX, pyrG, recG and rplB), B. garinii represented the most diverse species in Eurasia, followed by B. burgdorferi sensu stricto, B. afzelii and B. valaisiana. B. garinii OspA serotype 4 strains, which were seriously pathogenic to humans and proposed to be classified as a new species Borrelia bavariensis, were also widely distributed in Eurasia. Phylogenetic analyses on basis of the concatenated sequence analyses of 8 housekeeping genes showed that B. bavariensis strains from Europe fell into a cluster, which was separated from that of Asian B. bavariensis strains. B. afzelii strains from Europe branched into one cluster with one strains XY1 from Northwest China, which was separated from the cluster of B. afzelii strains from Northeast China. The form of geographical cluster of Borrelia strains might be caused by the highly parapatric distribution of their reservoir host of rodents. Moreover, B. garinii strains from Europe also clustered with those from Northeast China. Unfortunately, B. garinii strains from Northwest China was absent in this study. Further study is needed to clarify whether the high genetic similarity of B. garinii strains from Europe and Northeast China was associated with the migration of their reservoir host of birds. Above all, this study captured the genetic diversity and phylogeographic population structure of B. garinii and B. afzelii in Eurasia, which were related to the species and distribution of their reservoir host.