Genotyping of Borrelia burgdorferi sensu lato isolated from Northeastern Forest Areas of China
Objective To isolate Borrelia burgdorferi sensu lato(B.b.s.l.) from ticks collected from Northeastern Forest Areas of China and to determine their genospecies and distribution. Methods BSKH medium was used to isolate B.b.s.l. from ticks. 5S-23S rRNA intergenic spacer from all the isolates was amplified by polymerase chain reaction(PCR). Restriction fragment length polymorphism(RFLP) method and sequencing analysis based on 5S-23S rRNA intergenic spacer were used for genotyping, identifying genotypes. Results A total of 25 strains were isolated, of which 11,13 and 1 were obtained from Great Xing-an Mountains forest in Inner Mongolia Autonomous Region, Small Xing-an Mountains forest in Heilongjiang province and Chang Bai Mountains in Jilin province, respectively. A target amplicon with 254 bp in size was generated from all 25 isolates. The 25 PCR products were digested using MseⅠ and yielded 4 different RFLP patterns: B, C, D and N. Of the 4 patterns, N seemed to be a new one(a new RFLP pattern). Among them, 21 isolates belonged to B.garinii and were divided into two different patterns: pattern B and C; 3 isolates belonged to B.afzelii and fell into one pattern D; another isolate, named as VH10, show a unique pattern. The phylogenitic tree deduced from the sequences of all the 25 strains showed the isolates belonging to 4 branching groups. One branching group comprised 3 isolates of B.afzelii, the others comprised 22 isolates belonged to B.garinii. All 3 B.afzelii isolates were obtained from Weihe area of Heilongjiang province. Twenty two B.garinii isolates were obtained from Inner Mongolia Autonomous Region, Heilongjiang province and Jilin province respectively. Conclusion In Northeastern Forest Areas of China, there are two genospecies, and B.garinii is dominant one. In order to genotype the isolates accurately, RFLP analysis basing on 5S-23S rRNA intergenic spacer should be combined with sequence analysis after PCR assay.