| Anaplasma capra belongs to the genus of Anaplasma,which is a new Anplasma sp.that can be transmitted by ticks and can infect the erythrocytes of human and animals.In 2015,it was first reported human infected cases.The clinical features include acute fever,headache and discomfort,which is difficult to distinguish from other acute febrile diseases clinically.Hence,it is easy to cause missed diagnosis or misdiagnosis,causing a serious impact on human and animal health.However,the prevalence of A.capra in animals in China,its parasitic sites in animals and its biological characteristics such as cell infection are still unclear.We introduce a novel multiplex PCR for the simultaneous detection of A.capra,A.bovis,A.ovis and A.phagocytophilum,based on species-specific primers against the groEL（A.capra and A.bovis）,msp4（A.ovis）and 16S rRNA（A.phagocytophilum）genes.To verify the specificity of the PCR reactions,we evaluated four sets of primers to analyze samples containing different blood pathogens.The sensitivity of the multiplex PCR was evaluated by amplifying 10-fold dilutions of total genomic DNA extracted from sheep blood infected with A.capra,A.bovis,A.ovis or A.phagocytophilum.The reproducibility of the assay was evaluated by testing 10-fold dilutions of total genomic DNA extracted from sheep blood infected with these pathogens from 100-10-3 ng/μL per reaction in triplicate on three different days.A total of 175 field blood DNA samples were used to evaluate the reproducibility of multiplex PCR compared with the simplex PCRs.The lower limit of detection of the multiplex PCR with good repeatability enabled the detection of A.capra,A.bovis,A.ovis and A.phagocytophilum at concentrations of 3×10-5,5×10-7,2×10-5,and 7×10-7 ng/μL,respectively.There was no significant difference between conventional and multiplex PCR protocols used to detect the four Anaplasma species（P>0.05）.Our study provides an effective,sensitive,specific and accurate tool for the rapid differential clinical diagnosis and epidemiological surveillance of Anaplasma pathogens in sheep and goats.This study was conducted to determine A.capra in blood samples of sheep and goats in China.Using nested polymerase chain reaction（nPCR）targeting the citrate synthase（gltA）gene and conventional PCR targeting the heat shock protein（groEL）and the major surface protein4 gene（msp4）,A.capra was detected from 129（8.9%）of 1453 sheep and goat blood samples.The positive rate was higher in goats（9.4%,89/943）than in sheep（7.8%,40/510）.For sheep,A.capra was found in 17 sites from 2 Provinces.The prevalence was 28.6%in sheep from Liaoning Province,which was higher than in Henan Province（7.3%）.For goats,A.capra was detected in 35 sites from 7 Provinces.The prevalence varied from 0%to 19.4%in the goat sites examined.The prevalences were 19.4%,19.3%,10%,8.8%,6.8%,1.8%and 0%in goats from Guizhou Province,Henan Province,Inner Mongolia Autonomous Region,Shanxi Province,Xinjiang Uygur Autonomous Region,Yunnan Province and Gansu Province respectively.Based on the analysis of the A.capra gltA gene,two variants were identified.Variant I showed high sequence similarity to those A.capra,previously reported in sheep,goats,Ixodes persulcatus,Haemaphysalis longicornis,H.qinghaiensis,Rhipicephalus microplus and human.Variant II was only found in Luoyang,Anyang,and Sanmengxia,of Henan Province.To our knowledge,this is the first detection of this variant of A.capra in sheep and goat blood in China.Phylogenetic analysis based on groEL and msp4 genes showed that the Anaplasma sp.sequences clustered independently from A.capra and other Anaplasma species with high bootstrap values.We found A.capra DNA in sheep and goats in China,providing evidence that sheep and goats can be infected by A.capra.We also found that this zoonotic pathogen is widely distributed in China.This study provides information for assessing the public health risks for human anaplasmosis.In order to study the host cell types and pathogen morphological characteristics of A.capra,the A.capra-positive blood samples of goats were collected and isolated in this study.The DNA of whole blood cells,erythrocytes and leukocytes were extracted respectively.They were detected by multiplex PCR,phylogenetic analysis based on groEL,16S rRNA,gltA and msp4 genes of A.capra were performed.Scanning electron microscopy（SEM）,transmission electron microscopy（TEM）,Giemsa staining,in situ hybridization,immunocytochemistry and indirect immunofluorescence were used to characterise the parasitic location and morphological characteristics of A.capra.The results of multiplex PCR showed that all the erythrocyte DNA samples were positive-A.capra,while the leukocyte DNA samples were negative-A.capra.The phylogenetic analysis showed that the A.capra identified in this study belonged to the same genotype as the human isolates previously reported.Giemsa staining,in situ hybridization,immunocytochemistry,and indirect immunofluorescence were further confirmed by SEM observations of A.capra morulae inner or outer surface of erythrocytes.This study first proved that the goat erythrocyte is the host cell of A.capra.According to its molecular-characteristics,morphological characteristics and host cell type,we suggest that the genus.Anaplasma should be revised,and the newly discovered A.capra should be formally classified into the genus of Anaplasma.In order to verify the ability of goat-derived A.capra to infect human cells,this study will be separated and derived A.capra,and inoculated to human erythrocytes,HL-60 and TF-1 cell lines in vitro culture.In the Giemsa staining,many dark colored corpuscles or purple granules were seen in the inoculated erythrocytes,HL-60,and TF-1 cells.The results of chromogenic in situ hybridization show that there were brown precipitates on the surface of most erythrocytes.Immunocytochemistry results show many dark brown vacuolar structures or corpuscles in the cytoplasm of erythrocytes,HL-60,and TF-1 cell lines.The A,capra morulae were seen in the cytoplasm of both HL-60 and TF-1 in TEM,and their diameter was about 295-518 nm.Both dense-cored（DC）and reticulate cell（RC）form morulae could be seen.This study confirmed the ability of A.capra to infect human erythrocytes,HL-60,and TF-1.This study is of profound significance in further verifying the zoonotic characteristics of the pathogen and for establishing an in vitro cultivation model.Above all,the A.capra as a kind of new discovered Anaplasma sp.,the research results show that the zoonotic genotype of this pathogen is widely distributed in the goats and sheep in our country,and first confirmed that the host cell types for A.capra was erythrocytes.The human erythrocytes,HL-60 and TF-1 cell lines have infection ability,so the zoonotic characteristics of A.capra cannot be ignored;effective prevention and control measures should be taken to prevent the spread of the disease. |
