Epidemiological Investigation Of Ehrlichiosis And Immunogenicity Study Of E.canis Chimeric Virus-like Particles

Ehrlichiosis,transmitted through ticks’biting,is a natural focal disease caused by ehrlichiae.It attracted the attention of scholars around the world with the increase of pathogen types,emergence of new disease types and human infection.At present,there are three urgent scientific problems to be solved:(1)there is no systematic epidemiological investigation of ehrlichiosis in China.As the only vector of the disease,it is of great economic and public health significance to investigate the prevalence of ehrlichiae in ticks.(2)Serological diagnosis of ehrlichiosis disease has not been reported in China,and the clinical symptoms of this disease are not typical and similar to some viral diseases.Infected animals cannot be diagnosed in time for medical treatment and often miss the best time for treatment because of the lack of effective diagnostic methods.(3)No commercial vaccine is currently available.Now,the research and development of ehrlichiae vaccine mainly focused on live-attenuated vaccine,subunit vaccine and nucleic acid vaccine.However,single antigen or single virus strain cannot produce complete protection with the increase of genotypes.To solve the above problems,this study conducted a molecular epidemiological survey on tick carrying ehrlichiae in some areas of China to provide data support for the distribution of the pathogen;An indirect ELISA method for E.canis antibody detection was established to provide a rapid and effective clinical diagnosis method;The E.canis gp36 sequence was used to replace the extracellular domain of NDV HN protein and form cVLPs-HNcgp36 with NDV M protein.And then the immunogenicity of cVLPs-HNcgp36 was analyzed,which provides a new strategy for the development of E.canis vaccine.The main research contents are as follows:1.Molecular epidemiological investigation of ehrlichiae carried by ticks in some areas of ChinaIn this study,1142 tick samples were collected from the body surface of cattle,sheep,dogs and ground squirrels in Inner Mongolia,Jilin,Heilongjiang,Zhejiang and Hainan province during 2016-2018.PCR identification of tick genomic DNA using Ehrlichia spp.16S rRNA,dsb and gp36 related primers found 10 groups of positive samples,and phylogenetic analysis indicated that they belong to 3 species:E.ruminantium(2 groups),E.muris(2 groups)and E.canis(6 groups).The positive rate was 0%(0/54)groups in Inner Mongolia,0.7%(1/144)in Heilongjiang province,4.1%(3/74)in Jilin province,0%(0/8)in Zhejiang province and 28.6%(6/21)in Hainan province.2.Establishment and preliminary application of indirect ELISA method for E.canis antibody detectionIn this study,E.canis P30 protein was expressed by E.coli and used as the cladding plate antigen to establish an indirect ELISA method for detection of E.canis antibody.Then the indirect ELISA method was used to detect 172 canine serum samples from Beijing city,Shanghai city,Jilin,Liaoning and Jiangxi provinces and the average infection rate of serum samples from 5 provinces and cities was 26.74%.33 positive serum samples and 37 negative serum samples detected by indirect ELISA method established in this study were detected again by SNAP*4Dx*PLUS,a commercial kit developed by IDEXX company in the United States,and the coincidence degree of the two methods is 92.86%(κ=0.856).Therefore,the indirect ELISA method for E.canis antibody detection established in this study has a high coincidence with commercial kits and can be used for the detection of clinical samples which provides technical support for the diagnosis of ehrlichiosis in dogs.3.Construction and immunogenicity study of E.canis cVLPsIn this study,the insect-baculovirus expression system was used to form cVLPs-HNcgp36 with NDV M protein by using the strategy of E.canis gp36full-length replacement of NDV HN protein in the extracellular domain.Results of western blot showed that the protein was expressed correctly.Under transmission electron microscope,viroid particles with a diameter of 80-200nm can be seen.C57BL/6 mice were immunized by intraperitoneal injection,and then the immunogenicity was analyzed by detecting the humoral and cellular immune levels of the mice.The results showed that the cVLPs-HNcgp36 group could induce higher antibody titers than the soluble protein group in humoral immunity.In terms of cellular immunity,the proportions of CD3~+CD4~+and CD3~+CD8~+T lymphocytes in cVLPs-HNcgp36 group were higher than those in the other three groups.In conclusion,cVLPs-HNcgp36 constructed in this study has better immunogenicity than soluble protein,which provides a new strategy for the development of E.canis new vaccine.