Molecular detection of Anaplasma spp., Babesia spp. and Theileria spp. in yaks (Bos grunniens) and Tibetan sheep (Ovis aries) on the Qinghai-Tibetan Plateau, China

Anaplasma, Babesia and Theileria are tick-borne pathogens (TBPs) that affect livestock worldwide. However, information on these pathogens in yaks (Bos grunniens) and Tibetan sheep (Ovis aries) on the Qinghai-Tibet Plateau (QTP), China, is limited. In this study, Anaplasma spp., Babesia spp. and Theileria spp. infections were assessed in yaks and Tibetan sheep from Qinghai Province. A total of 734 blood samples were collected from 425 yaks and 309 Tibetan sheep at nine sampling sites. Standard or nested polymerase chain reaction was employed to screen all the blood samples using species- or genus-specific primers. The results showed that 14.1% (60/425) of yaks and 79.9% (247/309) of Tibetan sheep were infected with at least one pathogen. Anaplasma ovis, Anaplasma bovis, Anaplasma capra, Anaplasma phagocytophilum, Babesia bovis and Theileria spp. were detected in this study, with total infection rates for all the assessed animals of 22.1% (162/734), 16.3% (120/734), 23.6% (173/734), 8.2% (60/734), 2.7% (20/734) and 19.3% (142/734), respectively. For yaks, the infection rate of A. bovis was 6.4% (27/425), that of B. bovis was 4.7% (20/425) and that of Theileria spp. was 3.3% (14/425). Moreover, 52.4% (162/309) of the Tibetan sheep samples were infected with A. ovis, 30.1% (93/309) with A. bovis, 56.0% (173/309) with A. capra, 19.4% (60/309) with A. phagocytophilum and 41.4% (128/309) with Theileria spp. This study revealed the prevalence of Anaplasma spp., Babesia spp. and Theileria spp. in yaks and Tibetan sheep in Qinghai Province, China, and provides new data for a better understanding of the epidemiology of TBPs in these animals in this area of the QTP, China.

. Yak and Tibetan sheep are indigenous to the QTP, and provide local herdsmen with milk, meat, fuel (yak dung) and wool [4].
In recent years, tick-borne pathogens (TBPs) have attracted increasing attention due to the economic losses they cause in animal production and the risk they pose to humans. Tibetan sheep and yaks range freely on the high-altitude QTP, a harsh environment with a cold climate and low rainfall, and share pastureland with other animals there [5]. This increases the potential risk of transmission of pathogens, including TBPs, such as Anaplasma, Babesia and Theileria, the respective etiological agents of anaplasmosis, babesiosis and theileriosis in animals [6].
Anaplasma, a genus of the class Alphaproteobacteria, are Gram-negative obligate intracellular pathogens which are transmitted by hard ticks to vertebrate hosts and infect different blood cells of the host [7]. In sheep and cattle, infection with these bacteria is characterized by high fever and fatigue, loss of appetite, a sudden decrease in milk production, miscarriage, stillbirth, low fertility, decreased semen quality and other clinical symptoms [8]. Anaplasma infections have been reported in many areas of China. For example, Anaplasma ovis, Anaplasma bovis and Anaplasma phagocytophilum have been detected in sheep in Qinghai Province and the Xinjiang Uygur Autonomous Region [9][10][11][12][13], while Anaplasma capra, Anaplasma marginale, Anaplasma centrale and Anaplasma platys have been detected in both humans and cattle in Heilongjiang Province and Chongqing City, China [6,14].
The genus Babesia was discovered from the red blood cells of cattle in Romania in the nineteenth century [15]. Bovine babesiosis is caused by Babesia bigemina, Babesia bovis, Babesia divergens, Babesia major and Babesia occultans. In an acute case of bovine babesiosis, the main clinical features include high fever, loss of appetite, anemia, hemoglobinuria and lethargy [16], and the disease in farm animals leads to economic losses for farmers. Babesia spp., including Babesia motasi-like, Babesia sp. BQ1 (Lintan and Ningxian), Babesia sp. Tianzhu and Babesia sp. Hebei subgroups, have been found in and identified from sheep and goats in 16 provinces of China [17]. Investigations have also been undertaken on B. bovis, B. bigemina and B. ovata infections in beef cattle, dairy cattle and yaks in 14 provinces in China [18].
Theileria is an obligate intracellular hemoprotozoan parasite which is transmitted by ixodid ticks and affects a range of domestic and wild animals. Theileriosis leads to a decline in the growth rate and productivity of infected animals, and thus is a limiting factor in the development of animal husbandry [19]. On the eastern Tibetan Plateau in China (Sichuan Province), infections with Theileria sinensis, Theileria luwenshuni and Theileria equi have been detected in yaks, Tibetan sheep and Tibetan horses [20]. Moreover, Theileria orientalis [21], Theileria uilenbergi [13], Theileria ovis and Theileria spp. [22] have also been identified in cattle and yaks in northwestern China.
Epidemiological and molecular information on TBP infections in livestock on the QTP is limited. The data provided herein increase the available knowledge on the epidemiology of TBPs in livestock on the QTP, and provide a theoretical basis for the prevention and treatment of these pathogens in this area of China.

Blood sample collection and DNA extraction
A total of 734 whole blood samples (comprising those from 425 yaks and 309 Tibetan sheep) were randomly collected from animals on different farms in Guoluo Tibetan Autonomous Prefecture (hereafter 'Guoluo') and Yushu Tibetan Autonomous Prefecture (hereafter 'Yushu') in the Sanjiangyuan area (which is sometimes referred to as the "water tower of China") of Qinghai Province ( Fig. 1; Additional file 1: Table S1). Blood samples were taken from the jugular vein and collected in tubes containing ethylenediaminetetraacetic acid. Genomic DNA was extracted using the MagPure Blood DNA KF Kit (Magen, China) according to the manufacturer's manual. The DNA concentration was confirmed using a K5800 ultramicro spectrophotometer (Kaiao Technology, China), and the DNA was stored at − 80 °C until further use.

Sequencing and phylogenetic analyses
The positive PCR products of 30% of each organism were selected randomly and sequenced. The PCR products were purified using the EasyPure Quick Gel Extraction Kit (TransGen, China) and cloned into the pMD19 T vector, which was transformed into competent Escherichia coli DH5α cells using the pMD19 (Simple) T-Vector Cloning Kit (TaKaRa, Japan). At least two positive clones were sequenced at Sangon Biotech (Shanghai). The nucleotide sequence identities were determined by performing GenBank Basic Local Alignment Search Tool nucleotide (BLASTn) analysis (https:// blast. ncbi. nlm. nih. gov/ Blast. cgi). Phylogenetic trees based on the obtained sequences were constructed using MEGA 7.0 [30].

Statistical analysis
The chi-square test was performed to evaluate the difference in prevalence between different parameters. Exposure variables included area (Guoluo and Yushu) and altitude (3000-4000 m and 4000-5000 m). Observed differences were considered to be statistically significant when P < 0.05.

Infection rates of Anaplasma, Babesia and Theileria in yaks and Tibetan sheep
A total of 734 whole blood samples were collected and screened. The pathogens detected in these animals were A. capra (23.6%, n = 173), followed by A. ovis ( Table 2).

Sequencing analysis
The BLASTn analysis showed that the two partial sequences of the msp4 genomic region of A. ovis from Tibetan sheep had 100% identity with the A. ovis In addition, BLASTn analysis of the SBP4 gene showed that the B. bovis sequences obtained in this study shared 99.0-99.8% identity with each other and 99.4-100% similarity with previously published sequences from Benin (KX685399) and Syria (AB617641). Moreover, sequence analysis revealed that the nucleotide sequences of Theileria spp. 18S rRNA in this study shared 96.0-100% identity with each other and 99.1-100% similarity with T. ovis species (MN394810) from China.

Phylogenetic analyses
Phylogenetic analysis of the sequences obtained in this study was based on the neighbor-joining method. The analysis based on the msp4 gene of A. ovis in Tibetan sheep grouped the sequences from the present study into the same clade along with A. ovis isolates from Qinghai, China (Fig. 2). All A. bovis 16S rRNA sequences obtained in this study from Tibetan sheep and yaks were grouped into the same clade as isolates from Russia, Iran, Tunisia, Pakistan, Japan and China in the phylogenetic tree (Fig. 3). All four A. phagocytophilum 16S rRNA sequences obtained in this study were in the same clade as those from Japan, Korea and other provinces of China (Fig. 4). In the B. bovis phylogenetic tree, all the yak-derived sequences in this study were grouped into the same clade as those from cattle from Indonesia, Benin and China (Fig. 5). In addition, phylogenetic analysis of Theileria spp. based on the 18S rRNA gene showed that sequences obtained from the Tibetan sheep and yaks were grouped with the T. ovis clade with sheep, cattle, Tibetan sheep, yak and goat isolates from Iran, Egypt, China and Turkey (Fig. 6). However, in the phylogenetic tree of A. capra, all the obtained sequences from Tibetan sheep in the current study formed a separate branch, while other sequences of different animals from China were clustered together (Fig. 7).

Discussion
We investigated the molecular prevalence and genetic diversity of TBPs in yaks and Tibetan sheep on the QTP to increase the amount of available epidemiological data on these pathogens in this area of China. Anaplasma spp., Babesia spp. and Theileria spp. were detected in the yaks and Tibetan sheep in the locations studied.
A total of four Anaplasma species were detected in blood samples of yaks and Tibetan sheep from Guoluo and Yushu. The infection rates of A. ovis, A. capra and A. phagocytophilum in Tibetan sheep were 52.4%, 56.0% and 19.4%, respectively, but none of these species were detected in yaks. Anaplasma bovis was detected in samples from both types of animals, although the infection rate was higher in Tibetan sheep (30.1%) than in yaks (6.4%), which suggests that the former may be more susceptible to this pathogen than the latter. Anaplasma ovis has not only been reported in many areas of China but also in other countries, at a positivity rate ranging from 16.05 to 83.9% [9,10,12,13,[31][32][33][34]. This suggests that this pathogen, which causes sheep anaplasmosis, is an important infectious agent. Anaplasma bovis has also been detected in animals from different countries, such as cattle from Pakistan [35], cats from Angola [36], sheep and goats from Tunisia [37], goats from China [38] and Korean water deer from Korea [39].
In the present study, positive rates of 19.4% and 56.0% were found for A. phagocytophilum and A. capra respectively, in Tibetan sheep. These two pathogens can infect not only ruminants but also humans [14,40]. The infection rate of A. phagocytophilum in Tibetan sheep in the present study was lower than that in sheep (42.9%) and goats (38.5%) in previous studies carried out in Gansu    [41]. However, the infection rate of A. capra was higher in the present study than in previous investigations [42]. These markedly different results may be due to the fact that A. capra is found in a variety of ticks, including Haemaphysalis qinghaiensis [42], a species of tick unique to the QTP, and grazing is more likely to increase the exposure of animals to ticks. The main pathogens that cause bovine babesiosis, which was first reported in China in 1948, are B. bovis and B. bigemina [43]. Previous studies on the prevalence of B. bovis in China found that this species was widespread in cattle in 14 provinces of the country, with infection rates ranging from 1.0 to 60.0%. Among these, the infection rate of B. bovis in yaks in two cities in Gansu Province, one of which is located east and the other northeast of the QTP, was 13.0%, while this species was not detected in yaks in Qinghai Province [18]. In this study, the infection rate of B. bovis was 4.7%, which is lower than that previously reported [18]. This could be due to differences between the studies in terms of geographic and temporal factors and vector distribution [44].
Ovine theileriosis was reported as early as 1956 in Qinghai, China [45]. This disease was originally thought to be caused by T. ovis [46], but infection with different Theileria species has been detected in different animals in China and in other countries worldwide [6,13,[47][48][49]. Previous studies have reported T. sinensis and T. orientalis infections in cattle and T. luwenshuni and T. uilenbergi infections in sheep from Chongqing City and Xinjiang Province in China, respectively [6,13]. Sequencing analysis performed in the present study showed that only T.   14:613 ovis was present in yaks and Tibetan sheep on the QTP. The infection rates of Theileria in Tibetan sheep were significantly higher than those in yaks, which is a similar finding to that of a previous report [50]. The high prevalence of T. ovis in sheep in China and in other countries indicates that this pathogen cannot be neglected [47][48][49][50][51][52][53][54]. A study by Luo et al. [55] showed that H. qinghaiensis was the main disseminator of T. ovis on the QTP, and this may explain the high infection rate of this pathogen in Tibetan sheep in the present study. The T. ovis sequences obtained in the present study were also in the same clade as the T. ovis sequence detected in Rhipicephalus turanicus from Xinjiang [22], a neighboring province of Qinghai.
The results of this study show that Guoluo and Yushu are significantly impacted by the prevalence of B. bovis (χ 2 = 4.56, df = 1, P = 0.0328) in yaks and the prevalence of A. ovis (χ 2 = 23.77, df = 1, P < 0.0001), A. bovis (χ 2 = 52.14, df = 1, P < 0.0001), A. capra (χ 2 = 46.81, df = 1, P < 0.0001) and T. ovis (χ 2 = 50.75, df = 1, P < 0.0001) in Tibetan sheep. These results may be related to the vegetation type, climate and landform of the two sampling areas. Altitude was shown to have a significant impact on the prevalence of B. bovis (χ 2 = 5.77, df = 1, P = 0.0163) and T. ovis (χ 2 = 6.02, df = 1, P = 0.0141) in yaks, and that of A. phagocytophilum (χ 2 = 23.10, df = 1, P < 0.0001) in Tibetan sheep. Han et al. [56] investigated mixed infections of Anaplasma species in ixodid ticks and sheep, and found high co-infections in the latter. Several Anaplasma species have been detected in H. qinghaiensis [57], which implies that this common tick vector may be responsible for mixed infections with these pathogens. Previous studies detected A. marginale and B. bigemina in sheep and yaks in Xinjiang Province, respectively, and B. motasi-like L/N/T in sheep in Qinghai Province [9,12,18]. However, none of these pathogens, or B. ovis, were detected in any of the animals in the present study, which may be due to differences in species distributions and abundances of tick vectors between the sampling sites. The fact that none of these four pathogens were detected in this study also suggests that they may have low prevalences in Guoluo and Yushu or that they may not be present at all.

Conclusions
This study reports the prevalence of Anaplasma spp., Babesia spp. and Theileria spp. in yaks and Tibetan sheep in Qinghai Province, China. The results of this study add to existing epidemiological information on tick-borne diseases in yaks and Tibetan sheep in Sanjiangyuan, and provide basic data for the development of programs for the prevention and control of TBPs in domestic animals in this area of the QTP hinterland. However, further studies are needed to investigate the relationship between ticks and pathogens in Qinghai Province to provide more information on the epidemiology of TBPs in this area of China.