Establishment And Application Of Diagnosis Method For Canine Lyme Disease

Lyme disease is a natural epidemic disease caused by Borrelia burgdorferi infection transmitted by vector ticks.It is a zoonosis transmitted by vector vectors.The occurrence and prevalence of Lyme disease is the same as the active time of ticks.Lyme disease poses a serious hazard to the health of humans and animals.It mainly affects the healthy breeding of wild animals and pets and has become a serious public health problem in the world.In 1986,since Ai Chengxu first discovered the disease in Hailin County of Heilongjiang Province,there are currently 30 provinces(cities and districts)in China that have been identified as natural sources of Lyme disease.Lyme disease is mainly distributed in north,northwest and northeast forest regions in China.The distribution shows a tendency of regional aggregation.The occurrence of the disease depends on the media and the distribution of host animals.However,because the locust is not painful when bitten,and the early symptoms of the disease are not obvious,often confused with rheumatoid arthritis and other diseases,fever,etc.,leading to missed the best treatment time.As early as 1993,the Lyme disease rate was between 6.5%and 85.2%in 1446 canine blood samples collected from 25 cities and towns in New York.This data shows that due to the close relationship between humans and pet dogs,the chance of people being infected with Lyme disease increases.With the rapid economic development of our country and the improvement of people's living standards,pets have gradually become an integral part of people's lives.However,the serological detection methods for canine Lyme disease in China have not been reported.Therefore,it is urgent to establish a rapid and accurate diagnosis method for Lyme disease in canines.This research was supported by the National Thirteenth Five-Year Key Research and Development Project“Studies on New Technologies for the Diagnosis,Treatment,Prevention,and Control of Pet Bacterial and Fungal Infections”(Project Number:2016YFD0501005).The establishment of SYBR Green I fluorescence quantitative PCR method and canine Lyme disease indirect ELISA diagnosis method provide technical support and protection for pets and human health and safety.The main research content is as follows:I.Establishment of SYBR Green I Real-time PCR Assay for Detection of Borrelia burgdorferiThis study was based on the three genotypes of the predominant strains of Borrelia burgdorferi at home and abroad:the 16S rRNA gene fragment in Borrelia garinii,Borrelia afzelii,Borrelia burgdorferi sensu stricto as a template,which is conserved against Borrelia burgdorferi.Regional design of Real-time PCR specific primers,established SYBR Green I fluorescence quantification method.The relative coefficient R2 of the fluorescence quantitative standard curve is 0.99,and the amplification efficiency is 98.217%.The specific test results showed that the established method only detected B.burgdorferi specifically,but did not cross-react with other pathogens.Sensitivity test results show that this method is 10~4 times more sensitive than ordinary PCR,and the minimum copy number that can be detected is4.72 copies/?L.This method was applied to the detection of 25 tick samples collected from Siping City,Jilin Province.The positive rate of samples was 40.00%.II.Cloning and expression of OspC from Borrelia burgdorferi and antigenicity analysisOspC of Borrelia burgdorferi was selected.After bioinformatics analysis,the OspC gene of B31 strain was expressed,purified and Western-blot analyzed using prokaryotic expression vector,indicating that the protein had good immunogenicity.III.Establishment and application of indirect ELISA for diagnosis of canine Lyme diseaseThis study established an ELISA method for diagnosis of canine Lyme disease and serum prevalence investigations.OspC protein was used as coating antigen to establish an indirect ELISA for detection of Lyme disease in canines.This method was applied to the clinical samples of 309 canine serum samples collected.The average antibody positive rate was 20.71%.The results show that the ELISA method established in this study can be used for the detection of clinical serum samples.The test results show that the infection rate of Lyme disease is high in the canine population in China.