Molecular detection of Anaplasma infections in ixodid ticks from the Qinghai-Tibet Plateau

Anaplasma species are tick-transmitted obligate intracellular bacteria that infect many wild and domestic animals and humans. The prevalence of Anaplasma spp. in ixodid ticks of Qinghai Province is poorly understood. In this study, a total of 1104 questing adult ticks were investigated for the infection of Anaplasma species. As a result, we demonstrated the total infection rates of 3.1, 11.1, 5.6, and 4.5% for A. phagocytophilum, A. bovis, A. ovis and A. capra, respectively. All of the tick samples were negative for A. marginale. The positive rates of A. phagocytophilum, A. ovis and A. capra in different tick species were significantly different. The positive rates of A. capra and A. bovis in the male ticks were significantly higher than that in the female ticks. Sequence analysis of A. ovis showed 99.5–100% identity to the previous reported isolates. The sequences of A. phagocytophilum had 100% identity to strains Ap-SHX21, JC3–3 and ZAM dog-181 from sheep, Mongolian gazelles, and dogs. Two genotypes of A. capra were found based on 16S rRNA, citrate synthase (gltA) gene and heat shock protein (groEL) gene analysis. In conclusion, A. bovis, A. ovis, A. phagocytophilum, and A. capra were present in the ticks in Qinghai Province. Anaplasma infection is associated with tick species, gender and distribution. These data will be helpful for understanding prevalence status of Anaplasma infections in ticks in Qinghai-Tibet Plateau. Electronic supplementary material The online version of this article (10.1186/s40249-019-0522-z) contains supplementary material, which is available to authorized users.


Background
Ticks are important vectors of many viral, bacterial, and protozoal pathogens that infect to humans and animals, and tick species are widely distributed all over the world. Among tick-borne pathogens, the genus Anaplasma (order Rickettsiales, family Anaplasmataceae) is composed of tick-transmitted obligate intracellular bacteria, which include A. ovis, A. bovis, A. marginale, A. phagocytophilum, A. platys, A. centrale and A. capra [1,2]. A. ovis is an obligate intra-erythrocytic organism of small ruminants. A. centrale has relatively mild virulence and it has been used as a live vaccine against A. marginale infection in several countries [3]. A. bovis infects monocytes of small mammals and ruminants [4,5]. A. phagocytophilum infects neutrophils of many wild and domestic animals and humans, is the etiological agent of human granulocytic anaplasmosis and tick-borne fever [6]. A. platys is unique in infecting the platelets of dogs and it is the etiological agent of the infectious canine cyclic thrombocytopenia [7]. A. capra has been identified in China as a novel tick-transmitted zoonotic pathogen but its vectors and infected cell types are unclear [1]. Ixodid ticks play a critical role in the transmission and maintenance of Anaplasma species [8]. Dermacentor nuttalli, Hyalomma asiaticum and Rhipicephalus pumilio are the main vectors of A. ovis in China [9]. Although ixodid tick infestation of livestock is common, little is known about the Anaplasma infection in the ticks in Qinghai Province.
Qinghai Province is located in the northeastern part of Qinghai-Tibet Plateau in western China. Qinghai has an average attitude of more than 3000 m with 54% of the total area being between 4000 m and 5000 m. The provincial climate is characterized by being relatively arid, windy, and cold. Qinghai contains significant amounts of pastures and is an important region for animal production. Qinghai has 33.45 million ha of grassland. The grassland meadows are classified as alpine, swamp, Gobi, forest, and prairie. Yaks, Tibetan sheep, sheep and goats are adapted for survival and growth on these grasslands. Ixodid ticks infestation of livestock is often found in Qinghai Province, including 54.5, 24.0, 36.1% infection rates of A. ovis in sheep [10], Babesia spp. in wild yaks [11], and Theileria spp. in yaks [12], respectively. However, very little is known about the Anaplasma infection in animals and ticks. In this study, we identified and analyzed the infections of A. phagocytophilum, A. bovis, A. ovis, A. marginale and A. capra in ticks. The data provide an overview of Anaplasma infections and the potential threats to both livestock and humans in the study areas.

Sampling sites and tick collection
Samples were collected in the Qinghai Province, the Qinghai-Tibetan Plateau at an average altitude of > 3000 m. From February to October in 2015-2017, a total of 1104 questing adult ticks were collected from vegetation on 22 counties of Qinghai by using the flagging method. All of the tick specimens were identified according to morphological criteria [13] and a few were confirmed by sequence analysis of a partial fragment of the 16S rRNA gene.
DNA extraction, PCR amplification and sequencing DNA extraction of each individual ticks was conducted as described previously [2]. DNA samples were detected for the presence of the agents in the genus Anaplasma by PCR targeting the msp4 gene for A. ovis and A. marginale, the 16S rRNA gene for A. phagocytophilum and A. bovis, and the citrate synthase (gltA) gene for A. capra, respectively. For further confirmation of the A. capra, the 16S rRNA gene and the heat-shock protein gene (groEL) were amplified from A. capra positive samples. The 16S rRNA gene was amplified for the molecular identification of the tick species. The PCR was carried out by using an automatic thermocycler (Bio-Rad, Hercules, USA). The reaction system for the PCRs was the same as described in our previous study [14] and the PCR primers and cycling conditions were shown in Table 1. The DNAs extracted from the animals infected with A. ovis, A. marginale, A. phagocytophilum, A. bovis and A. capra were used as positive controls, and double distilled water was used as a negative controls. The PCR products were visualized under UV illumination in a 1.2% agarose gel followed by electrophoresis and treated with GoldView I (Solarbio, Beijing, China). The PCR products were purified with the TaKaRa Agarose Gel DNA Purification Kit Ver.2.0 (TaKaRa, Dalian, China). Purified PCR products were cloned into a pGEM-T Easy vector (Promega, Madison, WI, USA), and then transformed into Escherichia coli JM109 competent cells (TaKaRa, Dalian, China). Three positive colonies from each sample were subjected to sequencing. The obtained sequences were used to conduct BLAST search in GenBank® of the National Center for Biotechnology Information (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Data analysis
The data were grouped into three variables in terms of tick species, tick gender and the altitude of the sampling sites, respectively. Differences in each group were statistically calculated using a Chi-square test in Predictive for Analytics Software Statistics 18 (PASW, SPSS Inc.,Chicago, IL, USA). A P-value of < 0.05 was considered significant.

Detection of the Anaplasma spp. in ticks
Five Anaplasma species were investigated in the ticks. Of the 1104 samples tested, the average infection rates were 3.1, 11.1, 5.6, and 4.5% for A. phagocytophilum, A. bovis, A. ovis, and A. capra, respectively. All of the samples were negative for A. marginale. A. phagocytophilum was detected in four tick species from ten sampling sites, and it was detected for the first time in D. abaensis, D. nuttalli, and H. danieli. A. bovis was detected in five tick species from 14 sampling sites, whereas A. ovis was detected in three tick species from nine sampling sites. Three tick species including H. qinghaiensis, D. abaensis  Table 2.
Molecular characterization was based on the partial sequences of 16S rRNA gene (642 and 551 bp) for A. phagocytophilum and A. bovis, msp4 gene (869 bp) for A. ovis, 16S rRNA, gltA and groEL genes (1261 bp, 594 bp and 874 bp) for A. capra. These sequences were generated from positive samples representing the different sampling sites. As listed in Table 3, A. ovis were grouped into four genotypes. A. phagocytophilum were classified into three genotypes, and they were 100% identical to sequences of strains Ap-SHX21, JC3-3, and ZAM dog-181 from sheep, Mongolian gazelles, and dogs, respectively. A.
bovis were classified into five genotypes. The 16S rRNA gene sequences of A. capra showed 99.8-100% similarity to strain S62b from sheep and strain 9-13a from goat, and the groEL gene sequences were identical with strain tick102/China/2013 and M141a, respectively. These sequences showed a close relation to the sequences of strain HLJ-14 from a patient. In addition, two genotypes of gltA gene sequences of A. capra were obtained in this study.

Risk factors for Anaplasma infection to in the tick species
Risk factors, including tick species, gender, and altitude of sampling sites, were used as variables for statistical analysis of the infection patterns of Anaplasma spp. (Table 4). As a result, tick species was positively associated with the bovis and A. capra infections. Ticks collected below 3000 m areas had a higher risk for being infected by A. phagocytophilum and A. capra than in the ticks collected at elevations greater than 3000 m. A. bovis infection rates in ticks collected above 4000 m were higher than in the ticks collected below 4000 m.

Discussion
Qinghai is one of the five largest animal grazing regions in China. Grazing animal production is a supporting industry in this region. The Qinghai ecosystem is very suitable for ixodid tick infestation and 25 tick species in six genera has been reported [15]. In this study we collected seven tick species from three genera.   Aanaplasma prevalence in ticks demonstrated a wide distribution of A. phagocytophilum, A. bovis, A. ovis and A. capra. Among the Anaplasma species, A. phagocytophilum is an emerging tick-borne zoonotic pathogen of public health significance [16], and it has been detected in many tick species, including H. qinghaiensis, H. concinna, H. longicornis, I. persulcatus, and D. silvarum in China [17][18][19][20]. We detected A. phagocytophilum in H. qinghaiensis, and, for the first time, found it in D. abaensis, D. nuttalli, and H. danieli. The 16S rRNA gene sequences represented three genotypes, which showed high identities to the sequences found in goats from Central and Southern China [21], these genotypes were different from the genotype identified from human samples. Therefore, the significance of these genotypes to public health needs further investigation. A. bovis was initially found as a pathogen of cattle but has also been reported in sheep, goats, wild deer, and dogs [5,22,23], indicating this agent has a broad host range. We detected A. bovis in five tick species (H. qinghaiensis, D. abaensis, D. nuttalli, I. crenulatus, and H. danieli) from 14 sampling sites and it has the highest infection rate when compared with A. phagocytophilum, A. ovis and A. capra. Five genotypes of A. bovis were found, demonstrating its diversity in the ticks of Qinghai. A. bovis can be found in many tick species, such as H. longicornis in China [24], Korea [25] and Japan [26]. A. bovis was also found in H. lagrangei in Thailand [27], H.concinna in Russia [28], H. megaspinosa in Japan [29]; Amblyomma variegatum and R. appendiculatus in Africa [30], Rhipicephalus evertsi in South Africa [31], and R. turanicus in Israel [32]. We found A. bovis in H. qinghaiensis, D. abaensis, D. nuttalli, I. crenulatus, and H. danieli ticks. Statistics analysis indicated that A. bovis was more likely to infect male ticks and ticks at altitude above 4000 m. This result may be related to the distribution of its mammal hosts, since the majority of the yak population lives at altitudes more than 4000 m.
A. ovis is widely distributed in Asia, Europe, Africa and North American. Several msp4 gene variants of A. ovis have been identified in sheep and goats in northwest regions of China [14,33,34]. D. nuttalli, Hyalomma asiaticum and Rhipicephalus pumilio are vectors of A. ovis in China [9]. We detected A. ovis in D. abaensis, D. nuttalli, H. tibetensis, and four msp4 gene variants were identified in ticks. These variants showed high similarities to those from Chinese and Spanish strains, indicating diversity of A. ovis in the study ticks.
A. capra was initially identified in goats, and was subsequently considered to be an emerging human pathogen [1]. A. capra was previously identified in H. qinghaiensis in Gansu Province, in H. longicornis in Shandong Province, and in I. persulcatus in Heilongjiang Province [35,36]. We detected A. capra in H. qinghaiensis, D. abaensis, and D. nuttalli, and two genotypes were identified on the basis of gltA, 16S rRNA, groEL gene analysis. One genotype showed high sequence identity to the A. capra HLJ-14 strain, which had been reported in both goats and humans in China [1]. Another genotype showed low sequence identity to the strain HLJ-14 of A. capra, but high identity to an A. capra-like bacteria from H. qinghaiensis ticks [35]. Additionally, H. qinghaiensis is the dominant tick species for the infection of A. capra, and high prevalence occurs in the ticks found at altitudes less than 3000 m.
Although the present study has revealed the current status of ixodid tick infestation with Anaplasma spp. in the investigated areas, the specific biological vector for the individual Anaplasma species need to be further studied by transmission experiments. In addition, the infections of Anaplasma species in animals or humans should be investigated to understand the true impact of anaplasmosis in Qinghai Province.

Conclusions
We demonstrated the prevalence of A. bovis, A. ovis, A. phagocytophilum, and A. capra in ticks from 22 counties of Qinghai Province. Anaplasma infection in ticks is associated with the species, gender and distribution of the ticks. The prevalence of A. capra in ticks may be a threat to public health in Qinghai Province.

Additional file
Additional file 1: Multilingual abstracts in the five official working languages of the United Nations. (PDF 726 kb) Abbreviations gltA: Citrate synthase; groEL: Heat shock protein Acknowledgements Qinghai Provincial Center for Animal Disease Control and Prevention assisted with tick collection in Qinghai province. We thank them for their help and constructive comments.

Funding
This study was financially supported by the National Key Research and Development Program of China (2016YFC1202000,2017YFD0501200);and the Jiangsu Co-Innovation Center Program for the Prevention and Control of Important Animal Infectious Diseases and Zoonoses, State Key Laboratory of Veterinary Etiological Biology Project.

Availability of data and materials
The datasets used or analyzed for this study are available from the corresponding author.
Authors' contributions HY and Z-JL designed this study and critically revised the manuscript. RH participated in study design, coordination, and manuscript revision. RH, Q-LN, and YQ-L participated in sample collection. RH, YJ, M-UM, ZC,Q-LN, and G-YL performed the experiments, data analysis, and drafted the manuscript. All of the authors read and approved the final manuscript.
Ethics approval and consent to participate This study was approved by the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

Consent for publication
All of the authors of this manuscript declare that we have seen and approved the submitted version of this manuscript. Not applicable any individual persons data.

Competing interests
The authors declare that they have no competing interests.