Molecular detection and identification of tick-borne bacteria and protozoans in goats and wild Siberian roe deer (Capreolus pygargus) from Heilongjiang Province, northeastern China

Background Small ruminants are important hosts for various tick species and tick-associated organisms, many of which are zoonotic. The aim of the present study was to determine the presence of tick-borne protozoans and bacteria of public health and veterinary significance in goats and wild Siberian roe deer (Capreolus pygargus) from Heilongjiang Province, northeastern China. Methods The occurrence of piroplasms, Anaplasma phagocytophilum, A. bovis, A. marginale, A. capra, A. ovis, Ehrlichia spp. and spotted fever group rickettsiae was molecularly investigated and analyzed in 134 goats and 9 free ranging C. pygargus living in close proximity. Results Piroplasm DNA was detected in 16 (11.9%) goats and 5 C. pygargus. Sequence analysis of 18S rRNA sequences identified 3 Theileria species (T. luwenshuni, T. capreoli and T. cervi). Four Anaplasma species (A. ovis, A. phagocytophilum, A. bovis and A. capra) were identified in goats and C. pygargus. Anaplasma ovis and A. bovis were detected in 11 (8.2%) and 6 (4.5%) goats, respectively; A. phagocytophilum, A. bovis and A. capra were found in 3, 7 and 3 C. pygargus, respectively. Sequence analysis of 16S rRNA sequences revealed the presence of 5 different genetic variants of A. bovis in goats and C. pygargus, while the analysis of 16S rRNA and gltA sequence data showed that A. capra isolates identified from C. pygargus were closely related to the genotype identified from sheep and Haemaphysalis qinghaiensis, but differed with the genotype from humans. Anaplasma/Theileria mixed infection was observed in 2 (1.5%) goats and 5 C. pygargus, and co-existence involving potential zoonotic organisms (A. phagocytophilum and A. capra) was found in 2 C. pygargus. All samples were negative for A. marginale, Ehrlichia spp. and SFG rickettsiae. Conclusions These findings report the tick-borne pathogens in goats and C. pygargus, and a greater diversity of these pathogens were observed in wild animals. Three Theileria (T. luwenshuni, T. capreoli and T. cervi) and four Anaplasma species (A. ovis, A. phagocytophilum, A. bovis and A. capra) with veterinary and medical significance were identified in small domestic and wild ruminants. The contact between wild and domestic animals may increase the potential risk of spread and transmission of tick-borne diseases.


Background
Small ruminants are known to harbor various ticks that act as vectors and reservoirs of tick-borne pathogens of veterinary and/or medical importance. They play an important role, not only in the life-cycle of ticks, but also in the natural maintenance and transmission of these pathogens [1]. Tick-borne diseases affect domestic and wild ruminants, especially sheep, goats, cattle and deer [2]. In the past two decades, an increasing number of tick-borne pathogens have been identified in domestic animals and a variety of wild cervids, some of which have been confirmed as causes of human infection, such as Anaplasma phagocytophilum, Anaplasma capra, Babesia divergens, Babesia venatorum, Ehrlichia canis, etc. [3][4][5][6]. This fact is of great concern in terms of tick-borne disease control, since animals are usually asymptomatic carriers that may serve as reservoirs of the infection.
With advances in molecular techniques, a number of well-known and potential novel tick-borne bacteria and protozoans have been identified in unexpected hosts and geographical locations [7]. In China, Theileria annulata is the causative agent of bovine theileriosis and has been identified in sika deer [8]; Theileria uilenbergi causes ovine theileriosis and has been detected in red deer and sika deer [9]; and Babesia motasi infects sheep and goats and has been reported in sika deer [8]. This information warrants further investigation of tick-borne pathogens in both domestic and wild animals simultaneously. Siberian roe deer (Capreolus pygargus) is a commonly encountered wild animal and distributed mainly in Xinjiang and northeastern China [10]. The aim of this study was to determine the occurrence of the tick-borne bacteria and protozoans in goats and free-ranging C. pygargus from Heilongjiang Province, northeastern China.

Study sites and collection of specimens
In the present study, EDTA-anticoagulated blood samples were collected from 134 asymptomatic goats and 9 free-ranging C. pygargus from September 2017 to August 2018 in forest farms in Hebei Forestry Bureau, Mudanjiang city and Chaihe Forestry Bureau from Heilongjiang Province, northeastern China. The forest farms are local administrative units which have changed their role from timber production to forest and wildlife conservation over the last two decades. They never serve as large scale husbandry units, while backyard livestock breeding by its employees is common. Those domestic animals graze freely and share a common habitat with wild ungulates, which can frequently be found feeding very close to the local settlements. The samples were collected from rescued C. pygargus during the course of daily routine patrol by local wildlife disease monitoring stations. The goats were sampled in the herds close to the sites where C. pygargus included in this study were found. Sample collection and animal handling complied with the Animal Ethics Procedures and Guidelines and was approved by the Animal Ethics Committee of Northeast Forestry University. Genomic DNA was extracted from 200 μl of whole blood using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the instructions of the manufacturer.

PCR reactions
The extracted DNA was screened for the presence of piroplasms, Anaplasma, Ehrlichia and spotted fever group (SFG) rickettsiae by PCR. The PCR primers and cycling conditions used in this study are listed in Table 1. Briefly, nested PCRs were employed for the detection of piroplasms, A. phagocytophilum, A. bovis, and A. capra based on 18S rRNA, 16S rRNA and gltA genes, respectively. Anaplasma ovis, A. marginale, Ehrlichia spp. and SFG rickettsiae were detected by conventional PCR based on msp4 gene, 16S rRNA and ompA genes, respectively. PCR reactions were conducted in an automatic thermocycler (Bio-Rad, Hercules, CA, USA) in a total volume of 25 μl, including 2 μl of DNA sample as previously described [11]. The DNAs extracted from the whole blood of animals infected with T. annulata, A. phagocytophilum, A. bovis, A. marginale and A. ovis, and the DNAs from ticks positive for E. chaffeensis and SFG rickettsiae that had been verified by sequencing, were used as the positive control for corresponding PCR reactions; sterile water was used as the blank control for each run. Amplified fragments were electrophoresed on a 1.0% agarose gel containing 10 μl of GoldView (SolarBio, Beijing, China) and visualized under UV transillumination.

DNA sequencing and phylogenetic analysis
The DNA fragments were purified with a AxyPrepTM DNA Gel Extraction Kit (Axygen, Union City, CA, USA), cloned into pGEM-T Easy vector (Promega, Madison, WI, USA) and transformed for sequencing using Big-Dye Terminator Mix (GenScript, Nanjing, China). The nucleotide sequences obtained in this study were compared with previously published sequences deposited in GenBank by a BLASTn search or by using the ClustalW multiple alignment algorithm in the MegAlign program of the Lasergene 7.1 software package (DNAStar, Madison, WI, USA). The phylogenetic trees were inferred by using the neighbor-joining (NJ) method with the Kimura two-parameter model, and the bootstrap test was replicated 1000 times [12].

Nucleotide sequence accession numbers
The representative sequences of the identified pathogens in this study were deposited in the GenBank database and assigned accession numbers as follows: MH085202, MH085203 and MK271372 for 18S rRNA gene sequences of T. capreoli, T. cervi and T. luwenshuni, respectively; MH085195-MH085196 and MK271373-MK271375 for 16S rRNA gene sequences of A. bovis; MH085197 and MH085198 for 16S rRNA gene sequences of A. phagocytophilum and A. capra, respectively; MK271379 for the msp4 gene sequence of A. ovis; and MH094751 for the gltA gene sequence of A. capra.

Results
Sixteen (11.9%) of 134 goats and 5 (55.6%) of 9 free-ranging C. pygargus tested positive for piroplasms by nested PCR, which amplifies an approximately 430 bp band of the 18S rRNA gene of Theileria/Babesia spp. All amplicons were sequenced, and BLAST analysis revealed that the obtained sequences belonged to three different Theileria species; Babesia was not identified in any of these amplicon sequences. Sequence analysis revealed that the 18S rRNA sequences detected from goats were 100% identical to each other and to the isolates GNhl4 (MG799814), SX01 (MG930123) and PZG5 (LC326009) of T. luwenshuni identified in Haemaphysalis longicornis and goats from China and Myanmar. The 18S rRNA sequences of Theileria detected from C. pygargus were classified into two groups. Two 18S rRNA sequences were 100% identical to the Theileria sp. 3185/02 (Gen-Bank: DQ866842) identified in roe deer (Capreolus capreolus) from Spain and the isolate TCCRO1 of T. capreoli (GenBank: KY359359) identified in grey wolf (Canis lupus) from Croatia [13]. Three sequences were 100% identical to the isolates Am4 (GenBank: MG041373) and 13WYs1a (GenBank: KP407020) of T. cervi isolated from Ixodes persulcatus and sika deer (Cervus nippon) in Russia and China, respectively. To further characterize these three Theileria species, the first-round PCR amplicons from positive samples (~ 1410 bp) were  (Fig. 2). However, based on the gltA gene, the isolate was closely related to A. capra strains from sheep and H. qinghaiensis, but separated clearly from the human isolate HLJ-14 (Fig. 3).
Co-infection with Anaplasma and Theileria was observed in 2 goats and 5 C. pygargus. Co-infection that involved potential zoonotic organisms (A. phagocytophilum and A. capra) was found in 2 C. pygargus. Moreover, all animals included in this study tested negative for A. marginale, Ehrlichia spp. and SFG rickettsiae.

Discussion
The occurrence of tick-borne pathogens has been well documented in various domestic and wild ruminants in many countries. In addition to their veterinary importance, many tick-borne pathogens are known to have zoonotic potential. In the present study, the presence  Protozoan parasites of the genus Theileria are obligatory intracellular parasites that infect leukocytes and erythrocytes of both wild and domestic animals. Aside from highly pathogenic Theileria species, such as T. parva and T. annulata, others are described as low pathogenic [13]. Asymptomatic infections by different Theileria species have so far been identified in a variety of wild and domestic animals [1,[14][15][16][17]. In this study, T. luwenshuni was identified in goats, and T. capreoli and T. cervi in C. pygargus in Heilongjiang Province, northeastern China. Theileria luwenshuni is a new Theileria species highly pathogenic for small domestic ruminants (goats and sheep); it is widely distributed in China and causes substantial economic losses for the livestock industry [18]. Theileria capreoli and T. cervi have been frequently reported in cervid species. Theileria capreoli was first described in roe deer, and recorded subsequently in red deer, fallow deer, roe deer and Chinese water deer [16,19]. Theileria cervi is a non-pathogenic species that has been reported in brown brocket deer, white-tailed deer, sika deer, axis deer, marsh deer, elk, pampas deer and mule deer [14,15,20]. Our findings suggest that C. pygargus may serve as a reservoir of T. capreoli and T. cervi in northeastern China.
The genus Anaplasma encompasses a group of obligate intracellular bacteria that are causative agents of anaplasmosis with veterinary and public health significance [5]. Apart from those well-known Anaplasma species, a novel Anaplasma species named A. capra has been recently described in goats and identified subsequently as a human pathogen in Heilongjiang Province in northeastern China [6]. The molecular investigations of A. capra in small domestic ruminants and several tick species revealed that this pathogen is widely distributed in China, subdivided into two genotypes [11,21,22]. In this study, A. capra was identified in C. pygargus in Heilongjiang Province, where the human isolate HLJ-14 was described [6]. Phylogenetic analysis showed that the isolates from C. pygargus were closely related to A. capra genotype identified from sheep and H. qinghaiensis, but differ with the genotype from human, suggesting the high degree of genetic diversity of this agent. Moreover, since 16S rRNA sequences of A. capra have been previously detected in Japanese serows (AB509223) and deer (Anaplasma sp. NS104, AB454075) from Japan [23], the finding of this agent in C. pygargus was not surprising. These findings, together with previous reports, suggest that A. capra could be efficiently maintained in nature through enzootic cycles between ticks and wild cervids, and the A. capra genotype identified in C. pygargus is distinct from the human genotype.
In summary, several tick-borne pathogens were identified in small domestic and wild ruminants from northeastern China; three (T. luwenshuni, A. ovis and A. bovis) were identified in goats and five (T. capreoli, T. cervi, A. phagocytophilum, A. bovis and A. capra) in free-ranging C. pygargus. These findings suggest a greater diversity of tick-borne pathogens in wild animals than that in domestic animals. Furthermore, the infection of A. marginale, Ehrlichia spp. and SFG rickettsiae was not found in those animals included this study. Goats and C. pygargus serve as reservoirs for tick-borne protozoans and bacteria with a broad host range, such as A. phagocytophilum, A. bovis and A. capra, posing a potential threat to domestic and other wild animals as well as humans.

Conclusions
This study describes the occurrence of tick-borne bacteria and protozoans in goats and C. pygargus from northeastern China. Three Theileria (T. luwenshuni, T. capreoli and T. cervi) and four Anaplasma species (A. ovis, A. phagocytophilum, A. bovis and A. capra) with veterinary and medical significance were identified in small domestic and wild ruminants. The identification of these causative agents in domestic and wild animals provides useful information for the control and management of tick-borne diseases.