The object of this study was Borrelia burgdorferi sensu lato bacteria, which cause Lyme borreliosis in human organism. The detection of B. burgdorferi s. l. is still complicated, there is not only reliable method to diagnose the disease. To elucidate the relevance of a few methods, the purpose was formed: to detect and identify B. burgdorferi s. l. genotypes in several organisms using different molecular markers and methods.
The material of the study was collected during summer and autumn of 2010 in southern Norway (580 2' 3" N, 70 28' 43" E). 336 Ixodes ricinus larvae collected from rodents, 169 nymphs collected from vegetation and 18 DNA of rodents were used for molecular survey.
Three different methods of DNA extraction, five different molecular markers (16S rRNR, ospA, hbb, rpoB genes and 16S (rrsA) – 23S (rrlA) intergenic spacer) and five different methods (standard, multiplex, nested, real time PCR and sequencing) were used in this study.
Ticks collected from vegetation at first were screened by real time PCR based on 16S rRNR gene. Infection rate of B. burgdorferi s. l. pathogens in ticks collected from vegetation was 11 % (36 from 336). To compare a relevance of other method, all the positive ticks after screening were tested by standard PCR based on ospA gene. It was proven a tenth of infected ticks (11 %: 4 from 36). Infection rate by the same method in ticks from vegetation was 9 % (15 from 169), in rodents 40 % (7 from 18).
The real time PCR based on hbb gene was not sufficient to distinct B. burgdorferi s. l. species. Using a multiplex PCR with ospA gene – target, B. afzelii, B. garinii and B. burgdorferi s. s. species were determined of 36 % samples (25 from 69). Genotyping of undetermined samples was done by direct sequencing of the 16S (rrsA) – 23S (rrlA) intergenic spacer. B. miyamotoi and two different strains of B. afzelii were identified.
There is a discussion about possibilities using methods and molecular markers, which were used in this study.
Raktažodžiai: Lyme borreliosis, Borrelia burgdorferi s. l., genotyping, 16S rRNR, IGS