| Anaplasma phagocytophilum(AP)is an emerging tick-borne zoonosis pathogen,which host spectrum is very extensive,including people,livestock,wild animals.Human granulocytic anaplasmosis(HGA)caused by this pathogen can present clinical manifestations ranging from fever to death.At present,it has become an important vector-borne pathogen which endangers public health worldwide.Yunnan Province is located in the southwestern border of China.Its geographical and climatic conditions are complex.The unique natural conditions make Yunnan Province a high-incidence area of various insect-borne infectious diseases due to the large variety and density of harmful microorganisms and insect vectors,as well as the wide distribution of various host animals.Ticks are widely distributed and varied in Yunnan Province,and there are about identified 47 species of ticks belonging to 7 genera in this area.Therefore tick-borne diseases are prevalent in most areas of Yunnan,which seriously affects human health.However,the study of AP in Yunnan started late,and has not yet been systematically carried out.The distribution and prevalence of AP in human,animal and tick vectors are still unclear.In order to further study the occurrence and prevalence of AP in Yunnan and provide information for the effective prevention and control of HGA,we intend to conduct a systematic survey of the distribution of AP in Yunnan Province and make a comprehensive comparison with similar global studies.At the same time,in order to understand this new tropical infectious disease from different perspectives,we chose MSP2 as a starting point to microscopic research.As the main membrane dominant antigen protein of AP,MSP2 plays an important role in AP evading host immunity in addition to its rich content,and has been the main antigen for the detection and diagnosis of AP infection in various hosts.If recombinant MSP2 protein is obtained by genetic engineering technology,it can lay a foundation for further study on pathogenesis,diagnosis and vaccine of APObjective:To understand systematically the infection and carrying status of AP in population,vector ticks and host animals in Yunnan Province,and to grasp the epidemic characteristics and related epidemic factors of the pathogen.Meanwhile,the recombinant AP membrane surface protein MSP2 lays a foundation for exploring new diagnostic and detection methods suitable for the actual situation of Yunnan,further studying the pathogenesis of AP and preparing related vaccines.Methods:During the period of 2017-2018,blood samples from different populations,animal serum samples and transmission vectors tick samples were collected from selected sites in Yunnan Province.Anti-AP antibodies were detected in human and animal serum samples by ELISA or IF A,and the positive rates of human serum and animal serum in Yunnan Province were obtained by statistical calculation and analysis.DNA was extracted from whole blood samples.of the IgM positive samples in fever patients,and the 16SrRNA,msp2 and groEL genes of AP were amplified by nested PCR.According sequenced from amplified bands,the phylogenetic tree was constructed.DNA extraction from tick specimens and qPCR with specific primers to find molecular evidence of tick infection AP.A meta-analysis of AP seropositive rate in different regions of the world was carried out by R software,and the results were compared with those of AP seropositive rate in Yunnan population.Using genetic engineering technology,msp2-PET-30a plasmid was constructed to induce the expression of rMSP2 protein,and the expressed protein was purified and identified.Results:1.A total of 1430 blood samples were collected from 4 areas of Yunnan Province,including 1185 healthy people and 245 febrile patients.The positive rate of anti-AP IgG was 7.59%in healthy people,and 11 anti-AP IgM antibodies samples were detected in fever patients,with a positive rate of 4.49%.The seroprevalence of male and female were 9.29%and 5.96%respectively,and there was statistical difference between them.AP seroprevalence in 51-60 years old was the highest,reaching18.64%.AP seroprevalence from southern Yunnan was higher than that from north-central and Western Yunnan.DNA was extracted from 11 AP IgM antibody positive samples.All of 11 DNA samples were successfully amplified by nested PCR.Among them,16SrRNA gene belonged to the evolutionary tree constructed by Eh-gs amplification and sequencing.The results showed that the sequence of AP isolates from Hekou and Yanshan of Yunnan had little difference,and had high similarity with that of human isolates from Korea(MH482862.1)and another Czech vector Haemophilus(KY462782.1)group.2.In the AP infections survey of animal and tick in Yunnan,a total of 364 samples of goats,cattle,pigs and ducks and 273 samples of tick samples were collected from Yunnan.By ELISA detecting,the seropositivity of AP in cattle,sheep and pigs was 8.42%,10.34%and 5.56%respectively.There was no statistical difference in infection among different regions.A total of 273 ticks were identified as Rhipicephalus microplus.108 DNA samples were extracted by mixed pool method.No positive amplified samples were detected by qPCR.3.In meta-analysis of AP seroprevalence in global population,71 articles met the criteria and 36809 people were included in the study.The target population included healthy people,patients with fever,high-risk popualtion,people bitten by ticks and patients with tick-borne diseases.The literature included in the study came from 29 countries around the world,focusing mainly on three continents,Asia,Europe and the Americas.The seropositive rate of AP ranged from 0%to 66.3%.Meta-analysis results showed that the combined infection rate of AP was 10.2%(95%CI 8.3%-12.4%)in the global population.According to the results of regional subgroup analysis,the highest combined infection rate of AP in Asia was 14.6%,(95%CI 10.6%-19.2%).AP infection rate in high-risk occupational groups had the highest of 14.1%according to the results of subgroup analysis.Sensitivity analysis showed that the results of this study were stable.Begger test and egger test suggest that there is publication bias in the study.4.The synthetic msp2 gene sequence was successfully constructed on the PET-30a plasmid and induced to express the recombinant rMSP2 protein.The best inducing expression condition was determined to be 370C and IPTG concentration was 1.0 mmol/L for 4 hours.After identification by SDS-PAGE and Western-Blot,the protein was further purified.Finally,the recombinant purified protein was identified as AP MSP2 protein by mass spectrometry.Conclusions:1.Serological and molecular evidence of AP infection in human were found in Yunnan Province.2.At present,there are acute cases of human granulocytic anaplasmosis(HGA)case in Yunnan,which has a high risk of transmission.3.AP sequence of infected people in Yunnan Province is similar,with human-to-animal transmission risk.4.It is necessary to carry out a more systematic and extensive survey of AP infection in animals and ticks in Yunnan Province.5.The seroprevalence of AP in global population was 10.2%.Meta-study shows that heterogeneity may originate from studies in high-risk population,fever population and European region.Seroprevalence of Yunnan healthy population is similar to that of global healthy population.6.The recombinant rMSP2 protein obtained in this experiment can be used as a material basis for further study of AP. |
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