Development Of Two Detection Methods For Lyme Disease

Lyme disease is an important vector-borne zoonotic disease, caused by Borrelia burgdorferi; its biological vectors are Ixodid ticks. Tick bites can transmit the disease to human beings and animals. It can result in damage to multiple systems and organs, life-long disability or even death in severe cases in human. The disease restrains the development of animal husbandry, and has been considered the globel problem of public health, and was listed by the WHO in 1992 as a key target for prevention and control.Currently, most of studies of Lyme disease focus on human, while studies on animals are relatively rare. Both tick and livestock hosts are important in transmission and reservioer of Borrelia burgdorferi sensu lato in nature. In this study, ELISA and real-time PCR methods for detection B. burgdorferi sensu lato were developed and used to investigate epidemiology in some regions in China.The OspC gene fragments from B. burgdorferi sensu lato SZ, BO23, B31 strain were amplified by PCR, and then cloned into the expression vector pGEX-4T-1. The recombinant OspC protein was expressed and purified using MagneGSTTM Protein Purification System. The protein was detected by WB with positive sera from sheep infected by the three strains of Borrellia. The results showed that the recombinant proteins have good antigenicity and cross-reactivities with the sera from different Borrelia strains, which indicated that the recombinant OspC from any strains could be used as antigen to establish ELISA.With the OspC protein from SZ straiin, an ELISA was developed. Evaluation of the assay with 200 positive sera (sheep) and 214 negative sera (sheep)showed that the sensitivity and specificity of the method were 87.5% and 86.4% respectively. No cross-reaction between recombinant Protein OspC and positive sera of Theileria luwenshuni, T. uilenbergi, Babesia (Lintan, Ningxian, Xinjiang, Hebei, Tianzhu), Anaplasma and Toxophasma was observed. In addition, examination on a total of 502 (2004) and 360 (2010) sheep serum samples collected from Gannan Tibetan Autonomous Region of Gansu Province showed the positive rate were 5.4% (2004) and 9.4% (2010)respectively.Regarding to another method, a real-time PCR method for detection of B. burgdorferi was established using universal primers and probes selected on the basis of the 16S rRNA gene sequence of B. burgdorferi in GenBank?. The results showed that this method could specifically detect the B31 strain (B. burgdorferi sensu stricto), the BO23 strain (B. afzelii) and the SZ strain (B. garinii), without cross-reaction with genome DNA of Theileria, Babesia, Anaplasma, Chlamydia and Mycoplasma. The sensitivity of the assay was 103–104 times greater than that of conventional PCR. The real-time PCR could detect Borrelia from as little as 22.88 fg genomic DNA. DNA from 369 field collected ticks in Shangzhi City, Heilongjiang Province, China was tested by the real-time PCR; as a result, the infection rate was 42.8%. When a total of 332 genomic DNA from blood of 186 yaks and 146 sheep in the Gannan Tibetan Autonomous Prefecture of Gansu Province, China, were tested, the positive rates were 24.19% in yaks and 39.04% in sheep.