First report of Theileria and Anaplasma in the Mongolian gazelle, Procapra gutturosa

Background Theileria and Anaplasma are especially important emerging tick-borne pathogens of animals and humans. Molecular surveys and identification of the infectious agents in Mongolian gazelle, Procapra gutturosa are not only crucial for the species’ preservation, but also provide valuable information on parasite and bacterial epidemiology. Findings A molecular surveillance study was undertaken to assess the prevalence of Theileria spp. and Anaplasma spp. in P. gutturosa by PCR in China. Theileria luwenshuni, A. bovis, A. phagocytophilum, and A. ovis were frequently found in P. gutturosa in China, at a prevalence of 97.8%, 78.3%, 65.2%, and 52.2%, respectively. The prevalence of each pathogens in the tick Haemaphysalis longicornis was 80.0%, 66.7%, 76.7%, and 0%, respectively, and in the tick Dermacentor niveus was 88.2%, 35.3%, 88.2%, and 58.5%, respectively. No other Theileria or Anaplasma species was found in these samples. Rickettsia raoultii was detected for the first time in P. gutturosa in China. Conclusions Our results extend our understanding of the epidemiology of theileriosis and anaplasmosis in P. gutturosa, and will facilitate the implementation of measures to control these tick-borne diseases in China.


Background
Theileria is mainly transmitted by tick vectors and cause heavy economic losses to the livestock industry. The family Anaplasmataceae in the order Rickettsiales was reclassified in 2001, and includes several genera, including Anaplasma, Ehrlichia, Neorickettsia, and Wolbachia. Of them, the genera Anaplasma and Ehrlichia are especially important as emerging tick-borne pathogens in both humans and animals [1]. Anaplasma phagocytophilum is the causative agent of human granulocytic anaplasmosis, an extremely dangerous disease associated with high mortality rates in humans [2][3][4]. Other Anaplasma spp., such as A. bovis, A. ovis, A. marginale, and A. centrale, infect the erythrocytes and other cells of ruminants [3,4]. Anaplasmosis is endemic in tropical and subtropical areas, but is also frequently reported in temperate regions. Six or seven Anaplasma species have been reported in North America, Europe, Africa, and Asia [5][6][7][8][9][10][11], and some have also been reported in sheep, goats, and cattle throughout China [9,12,13].
The detection and isolation of Theileria and Anaplasma require specialized laboratories staffed by technicians with a high degree of expertise, primarily because the species' life cycles are intracellular. Several sensitive molecular tools, such as PCR, have been used to detect and identify Theileria and Anaplasma species in both hosts and vectors [10][11][12][13][14][15][16][17].
The Mongolian gazelle, an endemic ungulate species designated a threatened species by the World Conservation Union, is facing human and livestock disturbances of varying intensity in northern China. Although several studies have demonstrated that various Theileria, Babesia, Ehrlichia, and Anaplasma species circulate among sheep, goats, cattle, cervids, and humans in China, almost no data are available on the possible role of P. gutturosa as a host organism. The aim of this study was to detect and identify Theileria and Anaplasma spp. in P. gutturosa, a potential natural host of animal theileriosis and anaplasmosis in China.

Sample collection
The region investigated in China is located at latitudes 35°03′-35°55′ north and longitudes 105°37′-108°08′ east. The study was performed in April 2014. A total of 92 blood samples were collected randomly from P. gutturosa, and 242 ticks were collected from both P. gutturosa and grass in its environment. Of them, 30 unfed adult ticks were collected directly from grass in the gazelles' environment; 212 engorged nymph ticks collected from P. gutturosa were kept at 28°C and 80-90% relative humidity during molt, until nymph ticks were molted into adult ticks. All of adults were identified with Teng's methods [18]. Blood smears were prepared from the ear blood of every P. gutturosa individual. During the blood collection process, cases of suspected theileriosis or anaplasmosis were investigated. Theileriosis and/or anaplasmosis should be suspected in tick-infested animals with fever, enlarged lymph nodes (theileriosis only), anemia, and jaundice.

Microscopic analysis of blood smears
The blood smears were air-dried, fixed in methanol, stained with a 10% solution of Giemsa in phosphatebuffered saline (pH 7.2), and then analyzed microscopically and photographed ( Figure 1).

DNA extraction
Genomic DNA was extracted from the 92 whole blood samples and 222 tick samples using a genomic DNA extraction kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. The DNA yields were determined with a NanoDrop 2000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA).

Molecular detection of Theileria and
Anaplasma using species-specific primers PCR was used to detect and identify Theileria and Anaplasma spp. in P. gutturosa with the species-specific primers shown in Table 1 [10,11,[14][15][16][17]. The PCR reactions were performed in an automatic DNA thermocycler (Bio-Rad, Hercules, CA, USA) and the PCR products were used to assess the presence of specific bands indicative of Theileria and Anaplasma.
The DNA fragments were sequenced by the GenScript Corporation (Piscataway, NJ, USA). Representative sequences of the 18S rDNA/16S rDNA (or msp4) genes of the Theileria and Anaplasma spp. newly identified in this study were deposited in the GenBank database of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/genbank/).

Sequence alignments and phylogenetic analyses
The MegAlign component of the Lasergene® program version 4.01 (DNASTAR) was used to generate multiple sequence alignments with the ClustalW algorithm (www. clustal.org/) and for the phylogenetic analyses using the neighbor-joining method. A phylogenetic tree was constructed ( Figure 2

Tick identification
In this study, all 242 ticks were collected from P. gutturosa or grass in its environment in north-western China. The identification result showed that the adult ticks were either Haemaphysalis longicornis (n = 130: 86 female; 44 male) or Dermacentor niveus (n = 112: 78 female; 34 male). The whole DNA of 120 H. longicornis ticks and 102 D. niveus ticks was extracted.

Sequence alignments and phylogenetic analyses
The phylogenetic tree based on the Theileria and Babesia 18S rDNA sequences showed that only one pathogen was detected, which was placed in the T. luwenshuni cluster (Figure 2). The phylogenetic tree based on the 16S rDNA sequences of Anaplasma and Ehrlichia revealed four pathogens existed and they were A. bovis, A. phagocytophilum, A. ovis, and Anaplasma sp., respectively, in the blood samples from P. gutturosa roaming northern China (Figure 3).

Discussion
To the best of our knowledge, this study is the first to report the prevalence of theileriosis and anaplasmosis in P. gutturosa in China.  [9]. A. bovis and A. ovis have also been reported in red deer, sika deer, and D. everestianus in northwestern China [12]. In Japan, A. bovis and A. centrale have been detected in wild deer and H. longicornis ticks on Honshu Island, Japan [10]; A. bovis and A. phagocytophilum were initially detected in cattle on Yonaguni Island, Okinawa, Japan [19]. Therefore, H. megaspinosa is considered a dominant vector tick species for both these species in cattle in Japan [20]. In this study, four Anaplasma spp. (A. bovis, A. ovis, A. phagocytophilum, and an Anaplasma sp.) were detected in P. gutturosa. Rickettsia raoultii was also detected for the first time in P. gutturosa in China.
In this study, all 242 ticks were collected from gazelle or from their environment in the investigated area. They consisted of H. longicornis and D. vineus. Theileria luwenshuni and Anaplasma spp. (including A. bovis, A. phagocytophilum, A. ovis, and Anaplasma sp.) were detected and identified by PCR. Therefore, we speculate that these ticks play an important role as natural vectors of Anaplasma spp. in northern China. Theileria luwenshuni were first reported in sheep and goats, and widely distributed in north-western China [21]; recently, it was also reported in sheep and goats in central and southern China [22][23][24]. T. luwenshuni can be transmitted by H. qinghaiensis and H. longicornis in north-western China [25], but only H. longicornis and D. niveus were found in this study. Therefore, H. longicornis must play an important role as a natural vector of T. luwenshuni in P. gutturosa in northern China. However, whether T. luwenshuni can be transmitted by D. niveus remains to be determined.

Conclusion
Our results provide important data that extend our understanding of the epidemiology of theileriosis and anaplasmosis, and should facilitate the implementation of measures to control the transmission of Theileria and Anaplasma among P. gutturosa and other relative ruminants in China. Clarification of the role of P. gutturosa as a reservoir host for some Theileria and Anaplasma species is critical in determining whether P. gutturosa contributes to the spread of ruminant theileriosis and anaplasmosis in China.