Molecular identification of Theileria parasites of northwestern Chinese Cervidae

Background Theileria and Babesia protozoan parasites are transmitted mainly by tick vectors. These parasites cause heavy economic losses to the live-stock industry, as well as affecting the health of wild animals in parasite-endemic areas. Identification of infectious agents in wild animals is not only crucial for species preservation, but also provides valuable information on parasite epidemiology. Here, we conducted a molecular surveillance study in Northwestern China to assess the prevalence of blood pathogens in cervids. Methods PCR analysis and microscopic evaluation of blood smears to detect Theileria- and Babesia-related diseases in Cervidae were conducted, in which 22 blood samples from red deer (n = 22) in Qilian Mountain and 20 from sika deer (n = 20) in Long Mountain were collected and tested for the presence of Theileria and Babesia. The 18S rRNA gene was amplified, and selected polymerase chain reaction (PCR)-positive samples were sequenced for species identification. Results PCR revealed that 9.1% of the Qilian Mountain samples and 20% of the Long Mountain samples were positive for Theileria uilenbergi; 90.09% of the Qilian Mountain samples (n = 22) were positive for T. capreoli, but all of the Long Mountain samples (n = 20) were negative for T. capreoli; no other Theileria or Babesia species were found. PCR showed that T. uilenbergi and T. capreoli were present in red deer in Qilian Mountain, while only T. uilenbergi was found in sika Deer in Long Mountain. The 18S rRNA gene sequences were aligned against the corresponding GenBank sequences of known isolates of Theileria and Babesia and subjected to phylogenetic analysis. The phylogenetic tree showed that the newly isolated Theileria spp. could be classified as belonging to two clades: one group belonged to the same clade as T. uilenbergi, the other to a clade containing T. capreoli. Conclusions Our results provide important data to increase understanding of the epidemiology of Cervidae theileriosis, and will assist with the implementation of measures to control theileriosis transmission to Cervidae and small ruminants in central China.


Background
Theileria and Babesia, parasites that are mainly transmitted by tick vectors, cause heavy economic losses to live-stock and affect the health of wild animals where ever such parasites are endemic. Theileria and the closely related genus, Babesia, exhibit complex life cycles that involve mammalian intermediate hosts and hard ticks as the definite hosts [1]. Theileria is an obligatory intracellular parasite that infects leukocytes and erythrocytes in the intermediate host, unlike Babesia, which only infects erythrocytes. Among the Theileria and Babesia parasites that infect cervids, several Theileria spp. (e.g. T. cervi, T. capreoli, Theileria sp. OT3, Theileria sp. ZS OT4, and T. ovis), and Babesia spp. (e.g. B. bigemina, B. bovis, B. capreoli, B. divergens, and B. odocoilei) are described as being moderately pathogenic or benign. Theileria and Babesia are cosmopolitan parasites [2] that have been detected in wild ruminants in many countries including Japan [3][4][5], South Korea [6], Brazil [7,8], the United States [9][10][11][12], Italy [13], eastern Austria [14], northern and central Spain [15][16][17], and the United Kingdom [18]. In China, He et al. [19] first reported the existence of Theileria infection in sika deer in the Hubei province of central China.
Although there are no published epidemiological data on the Cervidae of northwestern China, no suspected cases of theileriosis in small ruminants have been observed here. In the current study, we performed an epidemiologic survey of Theileria and Babesia infections of Cervidae in northwestern China. Polymerase chain reaction (PCR) analysis and microscopic examination of blood smears were used for species identification and to elucidate the evolutionary relationships among the newly identified hemoparasite species based on the 18S rRNA gene sequences of known Theileria and Babesia species.

Sample collection
The two regions investigated in northwestern China are located at latitudes 34°44′ to 73°28′ north and longitudes 97°20′ to 106°35′ east. The study period was August 2013. Twenty-two blood samples (n = 22) were collected randomly from wild red deer in Qilian Mountain, and 20 blood samples (n = 20) were collected randomly from domesticated sika deer at a ranch in Long Mountain, northwestern China. Blood smears were prepared for both groups (Table 1). During the blood collection process, cases of suspected theileriosis and babesiosis were investigated. Theileriosis and/or babesiosis should be suspected in tickinfested animals with a fever, enlarged lymph nodes (only for theileriosis), anemia and jaundice, or hemoglobinuria (only for babesiosis).

Microscopy of blood smears
Blood smears were air-dried, fixed in methanol, stained with a 10% solution of Giemsa in phosphate-buffered saline (pH 7.2), and then subjected to microscopic analysis and photography.

DNA extraction
Genomic DNA from 42 whole blood samples was extracted using a genomic DNA extraction kit (Qiagen, Shayan et al., [33] Hilden, Germany) according to the manufacturer's instructions. The DNA yields were determined using a NanoDrop ND-2000 Spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA).
PCR amplification of the 18S rRNA gene of Theileria PCR was used to amplify protozoan 18S rRNA gene sequences with primers A/B as described by Medlin et al. [20]. The DNA fragments generated were ligated into pGEM T easy vectors (Invitrogen, Carlsbad, CA, USA) and transformed into competent JM109 cells (Takara Bio Inc., Shiga, Japan). The transformed bacteria were plated onto selective LB medium, and at least three positive clones identified by the 989/990 primers specific to Theileria spp. [21] were sequenced by the GenScript Corporation (Piscataway, NJ, USA). Representative sequences of the newly identified Theileria and Babesia 18S rRNA genes from this study were deposited in the GenBank database of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/genbank/).

Ethical approval and consent
This molecular approach was then employed to identify Theileria parasites of northwestern Chinese cervidae at Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Science. Use of these clinical samples was approved by the National Review Board (China).

Microscopic examination of thin blood smears
Piroplasm infections in the blood smears were observed microscopically in samples from Qilian Mountain (72.7%, 16/22) and Long Mountain (10%, 2/20); all infections exhibited low parasitemia levels (0.01-2.5%) ( Table 2). The piroplasms were polymorphous, and most presented as rod-like, needle-like, pear-shaped, or spherical forms ( Figure 1). Cases of theileriosis and babesiosis were not seen in the Qilian Mountain and Long Mountain regions of northwestern China.
PCR detection of Theileria and Babesia using speciesspecific primer sets All 42 blood samples were negative for Babesia spp.
using Babesia genus and Babesia species-specific primer sets; 24 of them (n = 42) were positive for Theileria spp.
using Theileria genus and Theileria species-specific primer sets. PCR using twenty sets of primers revealed that only two Theileria species, T. capreoli and T. uilenbergi, were detectable in the blood samples. The PCR-positive rates for T. uilenbergi in the blood samples from red deer in Qilian Mountain and sika deer in Long Mountain were 9.1% (2/22), and 20% (4/20), respectively. While the PCR-positive rates for T. capreoli in blood  (Table 2). These results confirm that all of the samples that were blood smear-positive were also PCR-positive for Theileria parasites. Of the 24 samples for which no piroplasms were observed by microscopy, we found that one was T. capreoli-and T. uilenbergi-positive, three were T. capreoli-positive, and two were T. uilenbergi-positive by PCR. Compared with microscopy of blood smears, the PCR method had higher sensitivity and was recommended to be utilized in the diagnosis of piroplasmosis.

18S rRNA amplification
The 18S rRNA gene sequences were determined for the

Discussion
To the best of our knowledge, this study is the first to report on cervine theileriosis in northwestern China. Molecular screening for Theileria and Babesia in red deer and sika deer in northwestern China showed that the most prevalent species of Theileria in the deer were T. capreoli and T. uilernbergi; however, no Babesia parasites were found. The prevalence of T. capreoli was very T. annulata, and T. sinensis) were detected using genusand species-specific primers. It has been speculated that in areas with high infection rates, there is an inverse relationship between age and resistance to infection, where fawns gradually acquire immunity without showing clinical symptoms, and immunity is maintained by repeated challenges with the parasites. Consequently, a persistent parasite reservoir is established in the wild ruminants [16]. However, in these hosts, stressors like high parasitemia, poor nutrition, high population density, harsh weather conditions, or handling (e.g. translocation) can lead to symptomatic piroplasmosis, a cause of severe disease and death [15]. The diagnosis of parasitic diseases and detection of parasite species is often difficult at the carrier stage and in animals with mixed infections. Nevertheless, molecular diagnostic methods can enable the direct, specific, and sensitive detection of parasite species [27,34]. PCR-based molecular techniques allow the detection and discrimination of these parasites at low parasitemias and in animals with mixed infections [33,34]. The PCR methods used in this survey can detect cervine Theileria and Babesia directly in the same samples based on the 18S rRNA gene sequence using Theileria and Babesia specific primer sets. In the present study, the prevalence of Theileria infections in red deer and silk deer, which were detected by PCR, was statistically higher than that observed via microscopic examination. These results were expected because carrier animals often have low parasitemias, which are difficult to detect microscopically.
The maximum parsimony tree of the 18S rRNA genes indicated that the cervid infections detected in this study could be divided into two groups within the Theileria species clade. Group 1 sequences fell within the low-pathogenicity clade containing T. capreoli BAB1158 (AY7266011: isolated from European roe deer in Spain), Theileria spp. 3185/02 (AY421708: isolated from red deer in Spain), and Theileria spp. 3185/02 (DQ866842: isolated from roe deer from the Basque Country of Spain), and were also present in roe deer and red deer from northern Spain [16], and in fallow deer from Italy [13], but absent from chamois. In contrast, group 2 Theileria spp. fell into the same clade as the high-pathogenicity T. uilenbergi Longde isolate (AY262120), as did the T. uilenbergi Lintan isolate (AY262116) and the T. uilenbergi Yongjing isolate (JF719835), which are all widely present in sheep and goats and transmitted by Haemaphysalis qinghaiensis and H. longicornis in northern and northwestern China; their sequences were almost identical to each other with high bootstrap values (Figure 1). Clustering of the 18S rRNA sequences into two groups indicates the heterogeneity of the 18S rRNA genes of cervine Theileria spp. in China. Therefore, the two Theileria spp. newly identified in Cervidae in northwestern China should be classified as T. capreoli and T. uilenbergi. The 18S rRNA gene sequence of T. capreoli was most closely related to Theileria CYN3B of Japan and T. ovis, which belong to an evolutionary distinct lineage of non-lymphoproliferative Theileria species. This species was clearly divergent from Babesia and a second lineage of lymphoproliferative Theileria species that included T. annulata and T. parva.
Ideally, tick transmission studies on ticks that are associated with infected herds should be conducted to identify the species involved in transmitting theileriosis to Cervidae. In this study, no ticks were collected from the deer or the surrounding environments of the two areas investigated because the sampling was carried out in August, a time of year when the ticks are inactive. Yin et al. [35] and Li et al. [36] identified H. qinghaiensis and H. longicornis as vectors for T. uilenbergi in China. H. qinghaiensis is a triphasic tick common in Northwestern China and H. longicornis is a ubiquitous triphasic tick found in most parts of China. Therefore, it can be speculated that H. qinghaiensis and H. longicornis may play an important role as natural vectors of T. uilenbergi in northwestern China. Galuppi et al. [13] detected T. capreoli DNA in Ixodes ricinus, but whether other vectors transmit T. capreoli remains unclear. At present, identification of the full range of tick species that can transmit T. capreoli to Cervidae awaits further investigation.

Conclusions
Our results provide important data that should increase understanding of the epidemiology of cervine theileriosis, and assist with the implementation of measures to control theileriosis transmission to Cervidae and small ruminants in northwestern China. Clarification of the role that Cervidae might have as reservoir hosts for maintaining T. capreoli and T. uilenbergi is critical to understanding whether Cervidae contribute to the spread of ruminant theileriosis in China.