Development Of Three Detection Techniques For Borrelia Burgdorferi Sensu Lato

Lyme disease caused by Borrelia burgdorferi sensu lato and transmitted by ticks, was a natural focal zoonoses. Infection with B. burgdorferi s.l. can result in many organs'disorder. Human or animals with B. burgdorferi s.l. infection may experience one or more clinical syndromes of early or late LB. As one of the most common vector-borne diseases, Lyme disease is widely distributed in the worldwide. It is considered as a challenge for public health and has been paid more and more attention in recent years.Molecular and serology diagnostic techniques was developed for diagnosis of Lyme disease based on the difference and characteristic of Borrelia burgdorferi sensu lato isolates in different country due to there are scarce of research in China.Oligonucleotide primers specific for the Borrelia burgdorferi s. l. were designed corresponding to the region of 16S rRNA gene according to sequences submitted to GenBank. A loop-mediated isothermal amplification (LAMP) was developed and compared with a referred PCR method for detection of Borrelia burgdorferi s. l.. No cross-reaction was observed with other pathogens, including Chlamydia, Mycoplasma, Anaplasma, Babesia, Theileria. The LAMP assay detected B. burgdorferi s. l. with high sensitivity comparing with PCR. The PCR could detect 1pg, 1pg and 0.1pg of genomic DNA of B. burgdorferi s. l., B. afzelii and B. garinii, respectively, but those of LAMP were 0.2pg, 0.2pg and 0.02pg, respectively. Of 110 tick DNAs were detected by LAMP and PCR simultaneously, the positive rate was 15.5% by PCR and 32.7% by LAMP. A total of 1052 ticks from 4 genus, collected from 8 provinces in China were examined for Borrelia burgdorferi s. l. by the LAMP targeting the 16S rRNA gene. The result showed that the infection rate of B. burgdorferi s. l. was 28.6% in Dermacentors, 36.5% in Haemaphysalis, 61% in Boophilus microplus and 54.9% in Rhipicephalus.A loop-mediated isothermal amplification (LAMP) rapid assay was developed based on outer surface protein A (OspA) gene for genotyping Borrelia borgdorferi s. l. including B. burgdorferi sensu stricto, B. afzelii and B. garinii that are the main genotype strains in China. Three sets of primers were specific for B. burgdorferi sensu stricto, B. afzelii and B. garinii and no cross-reaction was observed with other pathogens, including the Chlamydia, Mycoplasma and Anaplasma, Babesia, Theileria. The sensitivity of this assay for detection of B. burgdorferi sensu stricto, B. afzelii and B. garinii was 2ng, 0.2ng, 0.02ng, respectively. The result revealed that this LAMP assay could be used to genotype for B. burgdorferi s.s., B. afzelii and B. garinii. However, due to its low sensitivity, it should not be recomanded in epidemiological surveillance.Finally, an indirect ELISA for diagnosis of sheep Lyme disease was developed with whole-cell antigen prepared with in vitro B. burgdorferi s.s. B31. Positive coincidence of given sera from infected sheep was 90.1% and that of from neative sheep was 90% with the ELISA. There was no cross-reaction with positive sera of ovine Mycoplasma, Brucella, Toxoplasma, Anaplasma, Babesia, Theileria, respectively. A total of 450 sheep sera collected from Gannan Tibet autonomous region in Gansu province were examined. The result indicated that the positive rates of samples from Xiahe, Lintan and Zhuoni counties were 3.3%, 6.1% and 4.1%, respectively. The average positive rate was 5.1%. It is supposed that this region should be a natural focus of Lyme disease.