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ABSTRACT An emerging infectious disease caused by " Anaplasma capra " was reported in a 2015 survey of 477 hospital patients with a tick-bite history in China. However, the morphological characteristics and parasitic location of this pathogen are still unclear, and the pathogen has not been officially classified as a member of the genus Anaplasma . Anaplasma capra -positive blood samples were collected, blood cells separated, and DNA of whole blood cells, erythrocytes, and leukocytes extracted. Multiplex PCR detection assay was used to detect whole blood cell, erythrocytes and leukocytes, DNA samples, and PCR identification, nucleic acid sequencing, and phylogenetic analyses based on A. capra groEL , 16S rRNA, gltA, and msp4 genes. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), Wright-Giemsa staining, chromogenic in situ hybridization (CISH), immunocytochemistry, and indirect immunofluorescence assay (IFA) were used to identify the location and morphological characteristics of A. capra . Multiple gene loci results demonstrated that erythrocyte DNA samples were A. capra -positive, while leukocyte DNA samples were A. capra -negative. Phylogenetic analysis showed that A. capra is in the same clade with the A. capra sequence reported previously. SEM and TEM showed one or more pathogens internally or on the outer surface of erythrocytes. Giemsa staining, CISH, immunocytochemistry, and IFA indicated that erythrocytes were A. capra -positive. This study is the first to identify the novel zoonotic tick-borne Anaplasma sp., " Anaplasma capra ," in host erythrocytes. Based on our results, we suggest revision of Genus Anaplasma and formally name " A. capra " as Anaplasma capra sp. nov.

Anaplasma species, which are distributed worldwide, are gram-negative obligate intracellular tick-borne bacteria that pose a threat to human and animal health. Haemaphysalis longicornis ticks play a vital role as vectors in the transmission of Anaplasma pathogens. However, the Anaplasma species carried by H. longicornis in China are yet to be characterized. In this study, 1074 H. longicornis specimens were collected from goats in four provinces of China from 2018 to 2019 and divided into 371 sample pools. All tick sample pools were examined for the presence of Anaplasma species via nested PCR amplification of 16S ribosomal RNA, major surface protein 4 (msp4), or citric acid synthase (gltA) genes, which were sequenced to determine the molecular and phylogenetic characteristics of the isolates. The overall Anaplasma spp-positive rate of H. longicornis was determined to be 26.68% (99/371). The percentage prevalence of A. phagocytophilum-like1, A. bovis, A. ovis, A. marginale, and A. capra were 1.08% (4/371), 13.21% (49/371), 13.21% (49/371), 1.35% (5/371), and 10.24% (38/371), respectively, and the co-infection rate of two or more types of Anaplasma was 6.47% (24/371). Phylogenetic analyses led to the classification of A. phagocytophilum into an A. phagocytophilum-like1 (Anaplasma sp. Japan) group. Anaplasma bovis sequences obtained in this study were 99.8-100% identical to those of an earlier strain isolated from a Chinese tick (GenBank accession no. KP314251). Anaplasma ovis sequences showed 99.3-99.6% identity to an A. ovis human strain identified from a Cypriot patient (GenBank accession no. FJ460443). Only one msp4 sequence of A. marginale was detected and was grouped with those of other A. marginale isolates, and these A. capra isolates obtained in this present study may be zoonotic. The detection and characterization of four Anaplasma species in H. longicornis in this study have added to the current knowledge of the parasite and provided data on multiple Anaplasma species with veterinary and medical significance from four provinces of China.

Anaplasma are tick-borne obligate intracellular bacteria that can endanger human and animal health, and until now, there have been few reports on the seasonal dynamics of Anaplasma species in China. In this study, a total of 491 goat blood samples were collected in spring (n = 124), summer (n = 135), autumn (n = 110), and winter (n = 122) from Shaanxi provinces. Single and mixed infections of Anaplasma spp. from warm-temperate regions of China were analyzed according to seasons using a nested PCR method. Positive samples were sequenced to observe the molecular and phylogenetic characteristics of the Anaplasma species, and we determined the co-infection rates of Anaplasma spp. for each season. A molecular survey of Anaplasma phagocytophilum, A. bovis, A. ovis, and A. capra in goats showed average prevalences of 71.6 % (maximum 86.7 % in summer and minimum 48.4 % in winter), 62.2 % (minimum 38.7 % in spring and maximum 94.1 % in summer), 25.5 % (minimum 0% in summer and maximum 51.6 % in spring), and 26.6 % (minimum 8.2 % in winter and maximum 55.6 % in summer), respectively. In the phylogenetic analysis, A. phagocytophilum and A. capra occupied two separate groups, Chinese A. bovis and foreign isolates appeared to be geographically isolated, and all A. ovis isolates were in the same branch as the previously described sequences. The survey indicated that goats in warm-temperate regions of China are frequently exposed to Anaplasma spp. all year round, and thus prevention and treatment efforts for anaplasmosis in the region should be strengthened., (Copyright © 2021 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Anaplasma capra is an emerging pathogen, which can infect ruminants and humans. This study was conducted to determine the occurrence of A. capra in the blood samples of sheep and goats in China. Using nested polymerase chain reaction (nested-PCR) targeting the gltA gene and conventional PCR targeting the heat shock protein ( groEL ) gene and the major surface protein4 gene ( msp4 ), A. capra was detected in 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) (χ 2 = 1.04, p > 0.05, df = 1). 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 prevalence rates 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 citrate synthase gene (gltA ), two variants were identified. Variant I showed a high sequence similarity to the A. capra , which were previously reported in sheep, goats, Ixodes persulcatus, Haemaphysalis longicornis, Haemaphysalis qinghaiensis , and humans. 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.

A loop-mediated isothermal amplification (LAMP) technique has been widely used in detecting the nucleic acid of various pathogenic bacteria. In this study, a set of four LAMP primers was designed to specifically test Anaplasma bovis. The LAMP assay was performed at 62°C for 60min in a water bath. The specificity was confirmed by amplifying A. bovis isolate, while no cross reaction was observed with other five pathogens (Anaplasma bovis, Anaplasma phagocytophilum, Theileria luwenshuni, Babesia motasi and Schistosoma japonicum). The sensitivity of LAMP was 5×10 0 copies/μL, 100 times more than that of conventional PCR (5×10 2 copies/μL). Of 120 blood DNA extracted from sheep and goats field samples, 81 (67.5%), 22 (18.3%) and 43 (35.8%) were positively detected by LAMP, conventional PCR and nested PCR, respectively. The findings indicated that the developed LAMP assay is a new convenient tool for rapid and cost-effective detection of A. bovis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In recent years, tick-borne diseases like anaplasmosis have become widespread worldwide threatening the health of both human and animals. Dogs play an important role in the epidemiology of several zoonotic tick-borne pathogens by acting as reservoirs. In this study, the status of Anaplasma phagocytophilum, A. platys, A. bovis and A. ovis infection were assessed in dogs in Henan, China, with PCR and phylogenetic analyses. Nested PCRs on 243 blood samples collected from dogs from different sampling sites revealed that thirty-three (13.6%) dogs were positive for one or more pathogens. The prevalence of Anaplasma spp. in stray dogs was 40.7% (24/59), which was much higher than that of pet dogs (4.0%, 7/175). The prevalence for A. ovis, A. bovis and A. phagocytophilum was 6.2%, 4.1% and 0.4%, respectively and mixed-infection of these three pathogens was found in only one stray dog (prevalence, 0.4%). None of the dogs was positive for A. platys. Phylogenetic analyses classified A. phagocytophilum into two distinct groups (East Asia and south Africa group, Europe and America group), whereas A. ovis and A. bovis showed a general classification into two groups (cluster 1 and cluster 2), respectively. The isolate (KX190783) of A. ovis from a stray dog fell in a clade with a human isolate from Cyprus (FJ460443) and shared 99.8% similarity with it. To the best of our knowledge, this study is the first report to identify A. bovis and A. ovis DNA in dogs in China and the mixed-infection of the three Anaplasma spp. (A. phagocytophilum, A. bovis and A. ovis) in dogs., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Members of the genus Anaplasma are important emerging tick-borne pathogens in both humans and animals in tropical and subtropical areas. Here, we investigated the presence of Anaplasma spp. in 621 sheep and 710 goats from six provinces of China. Polymerase chain reaction (PCR) and DNA sequencing were conducted to determine the prevalence of Anaplasma (A.) phagocytophilum, A. ovis and A. bovis targeting the 16S ribosomal RNA or the major surface protein 4 gene. PCR revealed Anaplasma in 39.0% (240/621) of sheep and 45.5% (323/710) of goats. The most frequently detected species was A. ovis (88/621, 14.2% for sheep; 129/710, 18.2% for goats), followed by A. bovis (60/621, 9.7% for sheep; 74/710, 10.4% for goats) and A. phagocytophilum (33/621, 5.3% for sheep; 15/710, 2.1% for goats). Additionally, eight sheep and 20 goats were found to be infected with three pathogens simultaneously. DNA sequencing confirmed the presence of these three Anaplasma species in the investigated areas, and phylogenetic analysis indicated that there was geographic segregation to a certain extent, as well as a relationship between the host and cluster of A. ovis. The results of the present study provide valuable data that helps understand the epidemiology of anaplasmosis in ruminants from China., Competing Interests: There is no conflict of interest.

Organisms of the genera Anaplasma and Theileria are important intracellular bacteria and parasites that cause various tick-borne diseases, threatening the health of numerous animals as well as human beings. In the present study, a 12-month-old male wild South African giraffe (Giraffa camelopardalis giraffa) originating from South Africa, and living in Zhengzhou Zoo (located in the urban district of Zhengzhou in the provincial capital of Henan), suddenly developed an unknown fatal disease and died 1day after the onset of the clinical signs. By microscopic examination of Giemsa-stained blood smears combined with nested PCR and DNA sequence analysis, Anaplasma phagocytophilum, Anaplasma bovis and a novel Theileria spp. were found in the blood of this giraffe. The six other Cervidae animals in the zoo and three ruminants living in the same colony house with them were found to be negative for both Anaplasma and Theileria in their blood specimens. We report on the first case of an A. phagocytophilum infection and the occurrence of a novel Theileria spp. in the blood of a giraffe. This is the first reported case of a multi-infection of A. bovis, A. phagocytophilum and Theileria spp. in a giraffe, as revealed by microscopic examination of blood smears and the results of nested PCR and DNA sequencing., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Anaplasmosis, a disease caused by bacteria in the genus of Anaplasma, imposes economic constraints on animal breeders and also threatens human health. In the present study, we investigated the presence of Anaplasma spp. DNA in milk collected from sheep and goats in China. A total of 120 milk samples and 414 field-sampled blood specimens from sheep and goats were analyzed by PCR and DNA sequencing. Anaplasma ovis was detected in 12 milk samples (three from sheep and nine from goats). The blood specimens corresponding to the A. ovis DNA-positive milk were analyzed for Anaplasma DNA presence, and A. ovis DNA was identified in 10 out of the 12 blood specimens. One goat's milk sample was Anaplasma bovis DNA-positive, as was the corresponding blood sample. Anaplasma phagocytophilum was found in a milk sample and blood sample from one goat. One milk sample from Xinmi in Henan province was simultaneously positive for A. bovis and A. phagocytophilum; however, the corresponding blood was negative for both species. DNA sequencing of the PCR products and phylogenetic analysis confirmed that the sequences from the milk samples matched those of the corresponding blood samples. This is the first report to detect the Anaplasma DNA in milk samples under natural condition, and represents the first evidence of the presence of A. ovis, A. bovis and A. phagocytophilum DNA in milk from sheep and goats.

Anaplasma capra, a species of the family Anaplasmataceae, is zoonotic tick-borne obligate intracellular bacteria. There have been no reports of human infection with this pathogen since 2015. Therefore, the zoonotic characteristics of A. capra need to be further studied. To verify the ability of A. capra to infect human cells, A. capra were inoculated in human erythrocytes, HL-60, and TF-1 cell lines in vitro. Cell smears were taken after inoculation, using Giemsa staining, transmission electron microscope (TEM), chromogenic in situ hybridization and immunocytochemistry for detection. 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. [ABSTRACT FROM AUTHOR]