Molecular Detection and Identification of Babesia spp., Theileria spp., and Anaplasma spp. in Sheep From Border Regions, Northwestern China

Babesia, Theileria, and Anaplasma are important causative agents of tick-borne diseases that severely affect sheep. However, there is paucity in the occurrence genetic diversity of the infections of tick-borne diseases in sheep in border regions, northwestern China. In this study, nested polymerase chain reaction (nPCR) assays and gene sequencing were used to identify tick-borne Babesia spp., Theileria spp., and Anaplasma spp. infections in border regions, northwestern China. Out of 323 samples tested in this study, 225 (69.7%) sheep were infected with Babesia spp., Theileria spp., and Anaplasma spp. Two hundred six (63.8%), 60 (18.6%), 54 (16.7%), 51 (15.8%), 32 (9.9%), 19 (5.9%), and 16 (5.0%) were positive for A. ovis, B. motasi-like, A. bovis, T. uilenbergi, A. phagocytophilum, T. luwenshuni, and B. motasi-like Xinjiang, respectively. The most common dual infection was with A. ovis and B. motasi-like while the most frequent triple coinfection was A. ovis, B. motasi-like, and T. uilenbergi with coinfection rates of 17.0% (55/323) and 5.0% (16/323), respectively. Sequencing analysis indicated that A. ovis MSP4, A. phagocytophilum epank1, A. bovis 16S rRNA, B. motasi-like rap1-b, B. motasi-like Xinjiang rap1-a, T. luwenshuni 18S rRNA, and T. uilenbergi 18S rRNA from border regions, northwestern China, showed 99–100% identity with documented isolates from other countries. To the best of the authors' knowledge, this is the first report of T. uilenbergi and T. luwenshuni infections of sheep in border regions, northwestern China. Furthermore, these findings provide important data for understanding the distribution of Babesia, Theileria, and Anaplasma in sheep between border countries and China.

Xinjiang Uygur Autonomous Region (XUAR), an area surrounded by multiple land forms including Gobi desert, valley, mountain, grassland, and plates, occupies one-sixth of China's land area and borders eight countries including Russia, Mongolia, Kazakhstan, Kyrgyzstan, Tajikistan, Afghanistan, Pakistan, and India (13). According to Song et al. (14), in 11 border counties of XUAR, five Babesia species, two Theileria species and A. ovis were identified in Haemaphysalis punctata, Hyalomma asiaticum, Dermacentor nuttalli, D. marginatus, Rhipicephalus turanicus. Furthermore, in 2010, there were more than 30 million sheep and goats in XUAR (15), but information about ovine tick-borne pathogens is still lacking in border regions, northwest China. Subsequently, this study was conducted to help in full filling the information gap regarding the occurrence and genetic diversity of Babesia spp., Theileria spp., and Anaplasma spp. in sheep from northern and southern XUAR.
All samples were screened with species-specific primers for Theileria spp., Babesia spp., and Anaplasma spp. by PCR or nested PCR (nPCR) ( Table S1). Based on fragments of major surface protein 4 (MSP4) gene of A. ovis and A. marginale, epank1 gene of A. phagocytophilum, 16S rRNA gene of A. bovis and A. capra, 18 ssu rRNA gene of B. ovis and T. ovis, rhoptry-associated protein (rap)1-b gene of B. motasilike, rap1-a gene of B. motasi-like Xinjiang, 18S rRNA of T. luwenshuni and T. uilenbergi, and major piroplasm surface protein (MPSP) of T. orientalis. The PCR reaction mixture was composed of the following Takara super mix (Takara, Japan), PCR products were checked by electrophoresis in 1.5% agarose gels. Five positive samples were randomly selected from the six sampling areas, and at least three clones were sequenced. When the sequences obtained from different amplicons were identical, only one amplicon was retained for sequence analysis. Representative amplicons were cloned in pGEM-T Easy Vector. In brief, after extracting the amplicons from the agarose using QIAquick Gel Extraction Kit (Qiagen, Germany), pGEM-T Easy Vector (Promega, USA), and NucleoSpin R Plasmid QuickPure (Macherey-Nagel, Germany). BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, USA) and 3100 Genetic Analyzer (Applied Biosystems, USA) were used for sequencing (13). The nucleotide sequence identities were determined by performing GenBank BLASTn analysis on the NCBI database. The confidence interval (95% CI) analyses were conducted using GraphPad Prismer 6.0.
All A. ovis, A. bovis, A. phagocytophilum, B. motasi-like, B. motasi-like Xinjiang, T. luwenshuni, and T. uilenbergi sequences in this study were the expected sizes of 347, 551, 444, 536, 507, 389, and 388 bp, respectively. The MSP4 gene sequences of A. ovis (GenBank accession numbers MN946542) were 97.9% identical to other and to an isolate from Sudan (KU497712). A. bovis 16S rRNA gene sequences (GenBank Accession no. MN947620) was 99% identical to an isolate obtained from cattle in China (MK345480). A. phagocytophilum epank1 gene sequences (GenBank Accession no. MN946539) were 98% identical to an isolate obtained from sheep in Germany (GU236795). Sequences of B. motasi-like rap1-b sequences (GenBank Accession nos. MN946540) showed 99% identity to a previous isolate from XUAR sheep (KU510048). The sequence identity of B. motasilike Xinjiang rap1-a (GenBank Accession no. MN946541) with an isolate from Haemaphysalis longicornis in Gansu, China (KX708614), was 99%. The accession numbers for 18S rRNA of T. luwenshuni and T. uilenbergi are MN944535 and MN944557, respectively. The complete list of GenBank accession numbers of sequences from this study is shown in Table S3. Tick-borne pathogens (TBPs) of ovines represent a serious threat to veterinary and public health worldwide (16)(17)(18). In this study, three species of Anaplasma and two species of Babesia and Theileria were molecularly detected from sheep. The result also revealed that 69.7% of the samples were infected with at least one pathogen. Generally, the relatively high incidence of TBPs could be attributed to the abundance of vectors in the study area. In XUAR, 45 species from 6 genera of ticks, namely, Hyalomma, Dermacentor, Haemaphysalis, and Rhipicephalus are reported (19). In addition, Rhipicephalus turanicus, Dermacentor niveus, Hyalomma asiaticum, and Dermacentor marginatus are the most frequent tick species in domestic animals from thirtyfive counties (cities) in XUAR during 2011-2017 (20).
A number of Anaplasma species including A. ovis, A. bovis, and A. phagocytophilum, have been documented to cause anaplasmosis in ruminants (21). In this study, we identified A. ovis (63.8%), A. bovis (16.7%), and A. phagocytophilum (9.9%). Previously, (11) found A. ovis and A. phagocytophilum in sheep in XUAR, while A. ovis was identified in ticks in border regions (14). In addition, A. ovis and A. bovis were identified in Fuhai and Qinghe while A. phagocytophilum was detected in Fuhai, which both border Mongolia. A. ovis was identified in sheep and Dermacentor nuttalli tick (22) and A. phagocytophilum in I. persulcatus in areas surrounding Mongolia (23). We also detected A. ovis, A. bovis, and A. phagocytophilum in Bole and A. ovis and A. phagocytophilum in Jimunai which both areas are adjoined to Kazakhstan. According to Shpynov et al. (24), A. phagocytophilum DNA was identified in I. persulcatus ticks in the Altai and Primorye territories, which is also borders Kazakhstan. Furthermore, A. ovis (79.0%), A. bovis (24.7%), and A. phagocytophilum (28.4%) in Yecheng sheep in current study, while A. ovis (1.5%), A. marginale (5.7%), and A. centrale (2.7%) were identified in ticks from Pakistan, the country that Yecheng borders (25). Other border countries of XUAR also have reports about Anaplasma infection (A. bovis, A. centrale, A. phagocytophilum, and A. marginale) in ruminants or ticks including Russia (26), Philippines (27), and south India (28). Notably, A. capra, a newly discovered emerging zoonotic species  of Anaplasma, was not identified in the current study, although it has been reported in both XUAR (only four positive samples) and Gansu of China (29,30). Such discrepant is attributed to the difference in the area where the samples were collected in our study and in the previous one in XUAR (29). Several Babesia species (B. motasi-like and B. ovis) are widely spread among sheep and goat in China, specifically in Gansu and Qinghai (31,32), consistent with the distribution of the tick vectors (33). The total infection rates were 0.4% for B. motasi in sheep from Gansu according to (34). Song et al. (14) reported that in XUAR, five Babesia were identified in H. asiaticum, H. punctata, D. nuttalli, and R. turanicus ticks which were collected from sheep, including B. occultans, B. motasi-like, and B. major. Meanwhile, none of the samples was positive for B. motasi-like in ticks which were collected from Fuhai, Jimunai, Qinghe, and Yecheng (14). However, except in Yecheng, we found B. motasi-like infection in sheep in Fuhai (31.0%), Jimunai (18.2), and Qinghe (25.9%). According to Niu et al. (31), the recorded infection rate for B. motasi-like was 21.87%, while Song et al. (14) also reveal that B. motasi-like was identified in H. punctata ticks in Yili district. In countries nearby XUAR, such as Pakistan, B. ovis (16%) and T. ovis (23%) were also detected from sheep (35).
The first infection by Theileria spp. in small ruminants imported into Sichuan province of China was reported in 1958 (36). Infection with other Theileria species, such as T. uilenbergi, T. luwenshuni, and T. ovis, infective to small ruminants, has been documented since the first report (34). Furthermore, T. luwenshuni and T. uilenbergi were found in Gansu and Sichuan, Heilongjiang, Qinghai, Hubei, and Hainan, China (12,16,32). In this study, pathogenic T. luwenshuni was detected in sheep from five counties (Fuhai, Bole, Qinghe, Yecheng, Tashikurgan), with detection rates of 24.1, 8.8, 6.2, 3.7, and 1.3%, respectively. Although T. ovis (7.25%) was previously detected in R. turanicus ticks infesting sheep in Yecheng (14), we only identified T. luwenshuni and T. uilenbergi in sheep from Yecheng in this study. T. lestoquardi was also found infecting apparently healthy sheep and goats from two districts of Pakistan, which was near Yecheng county (37). In addition, T. luwenshuni infection in ticks from dogs and cattle was identified by reverse line blot hybridization (34). Aktaş et al. (38) revealed the presence of T. orientalis and T. annulata from cattle in Kyrgyzstan, which is near Bole. In south India, which is near Yecheng and Tashikurgan, researchers also identified T. orientalis in ticks (29). In previous studies, T. ovis were found in ticks (39) and goats (40) from XUAR, but in the present study, all sheep DNA samples showed a negative result for T. ovis and T. orientalis. The reasonable explanation for such obtained finding might be attributed to the difference between collection sample areas and tick species with previous study in XUAR.
The distribution and diversity of pathogens in sheep may be attributed to the difference in macroclimate, tick species, tickdwelling habitat, and landscape between districts. Particularly, questing ticks can be found in habitats like herb layer and vegetation (41,42). In XUAR, cattle, horses, and sheep often co-feed in the same district, and there are also many small mammals in areas with high vegetation (43). These expand the possibility of ticks finding an animal as its host, but reduce the possibility of ticks encountering the same animal (44). Furthermore, the districts containing alpine and meadow climates have humid soils rich in organic matter resulting in abundant vegetation which favors the tick population (42,45). Those aspects might explain why a lot of TBPs and different tick species were identified in XUAR. Although this study screened several TBPs in sheep reared in the southern and northern parts of XUAR, China, this survey did not include ticks from this area. Therefore, other future studies are required to detect those pathogens in tick spp. collected from different parts of XUAR. Furthermore, the present study did not determine the risk factors associated with screened pathogens on either animal or farm level. Subsequently, more studies are warranted in the future to avoid the risk factors correlated with TBPs in XUAR.
This study revealed the presence of A. ovis, A. bovis, A. phagocytophilum B. motasi-like, B. motasi-like Xinjiang, T. uilenbergi, and T. luwenshuni in sheep from six border counties (Qinghe, Fuhai, Jimunai, Bole, Tashikuergan, Yecheng) of XUAR, China. The current results present valuable information about ovine tick-borne infections in XUAR, which might suggest the possible emergence of tick-borne diseases in small ruminants in border regions of northwest China. Moreover, information generated from this study is expected to improve the approach for prevention and control of tick-borne diseases in China.

DATA AVAILABILITY STATEMENT
Publicly available datasets were analyzed in this study. This data can be found in the NCBI: MN946542, MN947620, MN946539, MN946540, MN946541, MN944535, and MN944557.

ETHICS STATEMENT
The animal study was reviewed and approved by the owners of the selected farms were informed of the study and provided their written approval for sampling of their sheep. All procedures were carried out according to the ethical guidelines for the use of animal samples permitted by Obihiro University of Agriculture and Veterinary Medicine (Approval ID: . Written informed consent was obtained from the owners for the participation of their animals in this study.

AUTHOR CONTRIBUTIONS
YL: methodology, validation, statistical analysis of the results, writing-original draft, and writing-review & editing.
EG: validation, statistical analysis the results, and writingreview & editing. QG, MR, and PM: investigation and writing-review & editing. ML, JL, and SJ: Investigation and recorded samples' data. BC: investigation, recorded samples' data, and funding acquisition. XX: conceptualization, writing-review & editing, and funding acquisition. All authors contributed to the article and approved the submitted version.

FUNDING
This study was supported by a grant from Japan Society for the Promotion of Science (JSPS) Core to Core Program and National Natural Science Foundation of China (31660711).

ACKNOWLEDGMENTS
We are grateful to the farmers for their cooperation and the state veterinarians for helping in the blood sample collection in the field.