Molecular Detection of Theileria ovis, Anaplasma ovis, and Rickettsia spp. in Rhipicephalus turanicus and Hyalomma anatolicum Collected from Sheep in Southern Xinjiang, China

The Xinjiang Uygur Autonomous Region (Xinjiang) borders eight countries and has a complex geographic environment. There are almost 45.696 million herded sheep in Xinjiang, which occupies 13.80% of China’s sheep farming industry. However, there is a scarcity of reports investigating the role of sheep or ticks in Xinjiang in transmitting tick-borne diseases (TBDs). A total of 894 ticks (298 tick pools) were collected from sheep in southern Xinjiang. Out of the 298 tick pools investigated in this study, Rhipicephalus turanicus (Rh. turanicus) and Hyalomma anatolicum (H. anatolicum) were identified through morphological and molecular sequencing. In the southern part of Xinjiang, 142 (47.65%), 86 (28.86%), and 60 (20.13%) tick pools were positive for Rickettsia spp., Theileria spp., and Anaplasma spp., respectively. Interestingly, the infection rate of Rickettsia spp. (73%, 35.10%, and 28.56–41.64%) was higher in Rh. turanicus pools than in H. anatolicum pools (4%, 4.44%, and 0.10–8.79%) in this study. Fifty-one tick pools were found to harbor two pathogens, while nineteen tick pools were detected to have the three pathogens. Our findings indicate the presence of Rickettsia spp., Theileria spp., and Anaplasma spp. potentially transmitted by H. anatolicum and Rh. turanicus in sheep in southern Xinjiang, China.


Introduction
Ticks and tick-borne diseases (TBDs) are a growing global problem that can have a detrimental impact on both human and animal health.As ticks are the second most common vectors worldwide, the biosurveillance of TBDs, such as piroplasmosis (babesiosis and theileriosis), anaplasmosis, and rickettsiosis, is crucial for protecting public and animal health.Ticks and TBDs negatively affect animal production, leading to pruritus, weight loss, reduced immunity, fever, anemia, and even death [1].
In Asia, tick-borne pathogens have become a concern in many countries in recent years, including China, Mongolia, India, Tajikistan, and Pakistan [2,3].The identification and control of several tick-borne diseases are also a concern for sheep.Sheep can be affected by TBDs, such as Theileria spp., Anaplasma spp., and Rickettsia spp.Additionally, these pathogens were detected near the border of the Xinjiang Uygur Autonomous Region (Xinjiang).For example, the Theileria ovis (T.ovis), Anaplasma ovis (A.ovis), and Rickettsia species have been reported in Pakistan and Tajikistan [4].
Importantly, Xinjiang shares its borders with eight other countries and has a complex geographic environment that includes diverse features, such as mountains, plateaus, deserts, and basins.This region is typically divided into two distinct climatic zones: the north, with a temperate continental arid and semi-arid climate, and the south with a warm temperate continental arid climate.These two zones are separated by the Tianshan Mountains [5].According to the Xinjiang Statistics Bureau (https://tjj.xinjiang.gov.cn/tjj/tjgn/202203/7ab304445f174a7eb1f5165be4f94041.shtml,accessed on 25 December 2023) [6], Xinjiang has approximately 45.696 million sheep, comprising 13.8% of China's total sheep farming industry.However, despite the importance of the sheep industry in Xinjiang and China as a whole, there are few studies on ticks in sheep, which are known to transmit tick-borne diseases (TBDs) or tick-borne pathogens (TBPs).
A previous study identified 12 tick species on five animal species, including sheep, in northern Xinjiang counties [7].However, information about the pathogens carried or transmitted by ticks in southern Xinjiang remains limited.Therefore, this study was conducted to identify and characterize the pathogens harbored or transmitted by tick species collected from sheep in southern Xinjiang.

Tick Sample Collection
A total of 894 partially engorged adult ticks (female and male) were randomly collected from three districts (Aksu, Kashgar, and Kizilsu Kirgizil) and seven locations in southern Xinjiang, China.Farms A, B, and C are located in Aksu-Aksu, while Farms D and E are in Aksu-Wensu.Additionally, ticks were also collected from Kashgar-Shufu and Kizilsu Kirgizil-Akto (Figure 1).These ticks were collected from the body surfaces of 128 asymptomatic sheep across four regions: Aksu (48 sheep), Wensu (40 sheep), Shufu (20 sheep), and Akto County (20 sheep).Briefly, the ticks were collected from the surface of the sheep, including the ear, perineum, perianal areas, and the base of the tail [7].The ticks were initially washed with phosphate-buffered saline (PBS, pH 7.2) and subsequently sonicated using ultrasonic waves (80 kHz) for 30 min.All ticks were immersed in boiling water and individually preserved in labeled vials containing 70% ethanol.Before DNA extraction, the ticks were again washed with 70% ethanol and air-dried.The protocol for this study was approved by the Committee on the Ethics of Animal Experiments at the Obihiro University of Agriculture and Veterinary Medicine, Japan (Permit numbers: animal experiment, 230244; DNA experiment, AP0001299621; Pathogen, AP0001299622; Ethical approval numbers, 22-23 and 23-17).

Tick Identification
The collected ticks were identified morphologically using published identification keys [8,9].Specifically, the genera of the ticks were classified based on features such as the basis capituli, scutum, and spiracular plates [8,9].

DNA Extraction
In each sampling county, tick specimens morphologically identified as the same tick species were randomly selected for pathogen detection.Three ticks from the same region and farm, morphologically identified as the same species, were pooled.This pool was washed three times with 1× PBS, ground in liquid nitrogen using a mortar and pestle, and resuspended in 200 µL of 1× PBS.Genomic DNA was extracted from this suspension using a resin-based DNA extraction kit (TIANGEN, Beijing, China) and stored at −20 • C according to the manufacturer's instructions.

Tick Identification
The collected ticks were identified morphologically using published identification keys [8,9].Specifically, the genera of the ticks were classified based on features such as the basis capituli, scutum, and spiracular plates [8,9].

DNA Extraction
In each sampling county, tick specimens morphologically identified as the same tick species were randomly selected for pathogen detection.Three ticks from the same region and farm, morphologically identified as the same species, were pooled.This pool was washed three times with 1× PBS, ground in liquid nitrogen using a mortar and pestle, and resuspended in 200 µL of 1× PBS.Genomic DNA was extracted from this suspension using a resin-based DNA extraction kit (TIANGEN, Beijing, China) and stored at −20 °C according to the manufacturer's instructions.

Molecular Characterization of the Tick Species and Characterization of Tick-Borne Pathogens
The molecular identification of the most common tick species was carried out using PCR assays.Target primers for partial sequences of Babesia spp.18S rRNA, Theileria spp.18S rRNA, Rickettsia spp.outer membrane protein (OmpA), and Anaplasma spp.major surface protein (Msp4) were employed [7,[10][11][12].In brief, all adult ticks (894) were screened via PCR using the primers listed in Table A1.PCR products were visualized on a 1.0% agarose gel stained with ethidium bromide under UV light [7].
All amplicons were extracted from the gels using a TIANquick Midi Purification Kit (TIANGEN, Beijing, China) and sequenced.The purified PCR products (pathogen DNA amplicons) were cloned using a pGEM-T Easy Vector (Promega, Madison, WI, USA).The

Molecular Characterization of the Tick Species and Characterization of Tick-Borne Pathogens
The molecular identification of the most common tick species was carried out using PCR assays.Target primers for partial sequences of Babesia spp.18S rRNA, Theileria spp.18S rRNA, Rickettsia spp.outer membrane protein (OmpA), and Anaplasma spp.major surface protein (Msp4) were employed [7,[10][11][12].In brief, all adult ticks (894) were screened via PCR using the primers listed in Table A1.PCR products were visualized on a 1.0% agarose gel stained with ethidium bromide under UV light [7].
All amplicons were extracted from the gels using a TIANquick Midi Purification Kit (TIANGEN, Beijing, China) and sequenced.The purified PCR products (pathogen DNA amplicons) were cloned using a pGEM-T Easy Vector (Promega, Madison, WI, USA).The plasmids were sent to Shanghai Sangon Company for sequencing [7].
The identities and similarities of the sequenced isolates were assessed using the BLASTn tool against the NCBI GenBank database and the Clustal X program.Phylogenetic trees were constructed using the maximum likelihood (ML) method with the Kimura 2-parameter model in MEGA version 7.0.Bootstrap support values (1000 replicates) were indicated at each node.Confidence intervals (95% CI) and one-way ANOVA analyses were conducted using GraphPad Prism 6.0.

Analyses of the Three Pathogens' DNA Sequences
PCR amplicons from positive samples were cloned and sequenced.(GenBank Accession no.PP065759, PP065760) from Xinjiang shared a identity with T. ovis (MN712508).Interestingly, the T. ovis 18S rRNA se distinct cluster and illustrated a close relationship with the sequ (MT732332) and Thailand (OM802548) (Figure 2).Two A. ovis Msp4 sequ this study exhibited a 99.62% identity with A. ovis (MN198191).Two i Accession no.PP104810-PP104811) shared a 99.62% nucleotide sequenc ovis obtained from goats (FJ460454) in Cyprus and sheep (MF002530) i Rickettsia spp.OmpA (GenBank Accession no.PP104812) from Xinjiang s 99.68% sequence identity to Rickettsia massiliae (KR401143) and R. raoult spectively.The phylogenetic analysis revealed two distinct Rickettsia clade included the Xinjiang isolate and sequences from Spain (KR401146 jiang, MF098409).The other clade showed a close relationship between datus Rickettsia barbariae) and an isolate from Israel (GU212860) (

Discussion
Xinjiang, a region in China with a complex geographic environment, has experienced a surge in the ruminant trade since the launch of the "Silk Road Economic Belt" initiative.Xinjiang shares extensive borders with eight countries, including India, Pakistan, Afghanistan, Tajikistan, Kyrgyzstan, Kazakhstan, Russia, and Mongolia.Due to this proximity, animal transport across these borders may also increase the likelihood of disease introduction into China.The risk of tick-borne pathogens (TBPs) entering China through animal transport is also increased.Such introductions pose a significant threat to both Xinjiang's livestock industry and the nation as a whole, as animals carrying disease and tick vectors can be transported throughout China.Despite the importance of understanding TBPs in sheep from southern Xinjiang, knowledge gaps persist regarding their genetic diversity and transmission.This study aimed to identify and characterize the pathogens harbored by two tick species (Rh.turanicus and H. anatolicum) collected from sheep in southern Xinjiang.
Given the four outbreaks of theileriosis in small ruminants from 2020 to 2022 in other countries, with morbidity rates of 27.95% and mortality rates 17.46% lower than the 62.5% case fatality rate observed in India [13], theileriosis in sheep warrants more attention in Xinjiang.T. ovis and T. lestoquardi were identified and analyzed in these four outbreaks [13].The first identified case of Theileria in sheep and goats from China dates back to 2007 [14], subsequently identified as T. ovis.In the present study, T. ovis identified in both Rh.turanicus and H. anatolicum were found on sheep from southern Xinjiang, China.Additionally, H. anatolicum was detected on one of the deceased sheep with a history of contact with the malignant ovine theileriosis outbreak, as reported by Moudgil et al. (2023) [15].Further investigations in India reported T. ovis (29.1%), T. lestoquardi (12.69%), and T. luwenshuni (5.97%) in sheep [16].An epidemiological investigation of T. lestoquardi was also conducted in Pakistan [3].
The Aksu (Aksu and Wensu) area in the southern part of Xinjiang, near Kazakhstan and Kyrgyzstan, shows the presence of T. ovis, which is aligned with findings by Sang (2021) [17] and Sultankulova (2022) [18].This situation was similar to those in Kizilsu Kirgizil (Akto) and Kashgar (Shufu) regions, near Afghanistan and Pakistan.T. ovis has been reported only in Pakistan, where Rhipicephalus turanicus has also been identified [4,19].Studies have also documented the molecular detection of Theileria spp. in ticks and ruminants in Kazakhstan, with Rh. turanicus identified as a potential vector of T. ovis in the southern part of Kazakhstan [17,18].Our findings support this hypothesis, suggesting Rh. turanicus as potential vector for T. ovis transmission between Xinjiang, China, and Kazakhstan.Comparable infection rates were observed for A. marginale (11%), A. ovis (28%), and T. ovis (3%) in goats and sheep from Pakistan [4].Additionally, Rh. turanicus and H. anatolicum were among over 15 tick species, including Hyalomma, Rhipicephalus, Dermacentor, and Amblyomma, identified as carrying these pathogens in Pakistan [20].
To date, limited research has been conducted on Anaplasma spp.While economic development has increased in Kazakhstan, Tajikistan, Kyrgyzstan, and Pakistan, studies on Anaplasma in these regions remain limited.An overall prevalence of 69.4% (59/85) and 80.5% (70/85) was reported in Mongolia for A. ovis in goats and sheep, respectively [21]. A. ovis prevalence in goats was 15% (185/1200), including cases reported in Punjab, Pakistan [22].Although Rh. turanicus was not identified in Mongolia, according to Enkhtaivan et al. (2019) [23], H. asiaticum was the main vector in Mongolia; therefore, the outcome of their research was similar to that of our study.Our observed A. ovis infection rate of 12.5% and the phylogenetic analysis showed resemblance to the sequences found in China [24].A comparison with studies from India revealed higher T. ovis (37.2%) but similar A. ovis (15.4%) infection rates in sheep [25].Moudgil et al. also implicated that T. lestoquardi, which causes malignant ovine theileriosis, may also be transmitted by ticks.Moreover, a previous study detected R. massiliae and Candidatus R. barbariae in Rh. turanicus from dogs in Xinjiang, China [8].

Conclusions
While this study provides valuable insights into the population structures and epidemiological distribution of TBPs in ticks from sheep in southern Xinjiang, China, it did not determine the infection in sheep and is limited by its focus on the tick vector.Future research should investigate the prevalence of the identified pathogens in the blood DNA samples of local ruminants in China and its surroundings to assess the true impact on animal health.Additionally, identifying risk factors for TBDs in sheep at both the individual animal and farm levels is essential for effective disease control and prevention.
In conclusion, Rh. turanicus and H. anatolicum were the two identified tick species from sheep in southern Xinjiang.Rickettsia spp. was the most frequently detected TBP in both species, followed by Theileria spp., and Anaplasma spp.Co-infections with two or three TBPs were common among tick species in sheep from southern Xinjiang.The sequencing analysis showed that 99-100% of A. ovis Msp4 and Rickettsia spp.were present in T. ovis OmpA from southern Xinjiang.When compared to known isolates from other countries, the OmpA sequences displayed 99-100% identity.Our research indicates that Rickettsia, Theileria, and Anaplasma species are prevalent among H. anatolicum and Rh.turanicus ticks in sheep in southern Xinjiang.This study highlights several important aspects of TBP status in sheep in southern Xinjiang, China, which have serious economic implications for the sheep industry in Asian countries.These aspects should help in the development and implementation of effective prevention and control measures for veterinary practitioners and animal owners.

Figure 2 .
Figure 2. The maximum likelihood method based on the Kimura 2-parameter investigate Theileria spp.Phylogenetic analysis of the 18S rRNA gene sequence w number marked at each node represents the percentage occurring in 1000 bootst sequences in this study are marked with black triangles.

Figure 2 . 1 Figure 3 .
Figure 2. The maximum likelihood method based on the Kimura 2-parameter model was used to investigate Theileria spp.Phylogenetic analysis of the 18S rRNA gene sequence was performed.The number marked at each node represents the percentage occurring in 1000 bootstrap repetitions.The sequences in this study are marked with black triangles.

Figure 3 . 11 Figure 3 .
Figure 3. Phylogenetic analysis of Anaplasma ovis Msp4 gene sequences was performed using the maximum likelihood method based on the Kimura 2-parameter model.The numbers at the nodes indicate the percentage support derived from 1000 bootstrap replicates.Sequences from this study are denoted by black triangles.

Figure 4 .
Figure 4. Phylogenetic analysis of Rickettsia spp.OmpA gene sequences was conducted using the maximum likelihood method based on the Kimura 2-parameter model.The values at the nodes represent the percentage support from 1000 bootstrap replicates.Sequences from this study are indicated by black triangles.

Author
Contributions: Conceptualization, X.X. and Q.G.; methodology, Y.L. and J.L.; software, Y.L. and G.X.; validation, L.G. and J.R.; formal analysis, U.K.M. and S.A.E.-S.E.-S.; investigation, G.X., J.L. and J.R.; resources, G.X. and Q.G.; data curation, G.X. and J.L.; writing-original draft preparation, Y.L. and J.L.; writing-review and editing, M.A.R., A.M.C.M., B.C. and U.K.M.; visualization, Y.L.; supervision, X.X. and Q.G.; project administration, B.C., X.X. and Q.G.; funding acquisition, B.C., X.X. and Q.G.All authors have read and agreed to the published version of the manuscript.Funding: This research was funded by the following projects Innovative research team on biological vectors and transmission of vector-borne zoonotic diseases in the Xinjiang Uygur Autonomous Region (No. 2023TSYCTD0008), National Key Research and Development Program of China (No. 2021YFD1600702-3), Shanghai Cooperation Organization of Xinjiang Uygur Autonomous Region (No. 2021E01001), Postdoctoral program of Xinjiang Agricultural University, Xinjiang Uygur Autonomous Region, the AMED project (Grant No. JP23WM02250317), and the Strategic International Collaborative Research Project (JP008837) promoted by the Ministry of Agriculture, Forestry, and Fisheries of Japan.Institutional Review Board Statement: All protocols were carried out according to the ethical guidelines approved by the Obihiro University of Agriculture and Veterinary Medicine (Permit for the animal experiment: 18-40).Informed Consent Statement: Not applicable.

Table 1 .
The tick-borne pathogens detected in the adult tick pools of Rh. turanicus and H. anatolicum.
"-" means not detected; "No." means the number of ticks tested per animal species.