Molecular Detection of Tick-Borne Pathogens in Ticks Collected From Hainan Island, China

Pathogens like bacteria and protozoa, which affect human and animal health worldwide, can be transmitted by vectors like ticks. To investigate the epidemiology and genetic diversity of bacteria and protozoans carried by ticks in Chengmai county of Hainan province, China, 285 adult hard ticks belonging to two species [ Rhipicephalus sanguineus ( sensu lato): 183, 64.21% and Rhipicephalus microplus: 102, 35.79%] from dogs, cattle, and goats were collected. Microbial families were identified in these ticks by amplifying the 18S rRNA, 16S rRNA ( rrs), citrate synthase ( gltA), and heat shock protein ( groEL) genes. Our data revealed the presence of four recognized species and two Candidatus spp. of Anaplasmataceae and Coxiellaceae. In sum, these data reveal an extensive diversity of Anaplasmataceae bacteria, Coxiellaceae bacteria, Babesiidae, and Hepatozoidae in ticks from Hainan Island, highlighting the need to understand the tick-borne pathogen infection in local animals and humans.


Introduction
Ticks are vectors for a variety of etiological agents of zoonotic disease including viruses, bacteria and protozoa [1]. Tick-borne diseases such as rickettsiosis, anaplasmosis and babesiosis are of substantial concern for both humans and animals all over the world. In China, tick-borne pathogens have posed great threat to residents, especially those in rural and forest [2]. However, most of these pathogens such as rickettsiosis, anaplasmosis, and babesiosis are still underestimated, because the epidemiological and clinical information about these pathogens is quite limited. In addition, misdiagnosis is very often due to their clinical manifestation's similarity to other syndromes such as hemorrhagic fever with renal syndrome (HFRS) [2]. Furthermore, ticks also act as vectors for a variety of pathogens causing diseases to companion animals and livestock [3,4]. These include bovine anaplasmosis (Anaplasma marginale and A. bovis), bovine babesiosis (Babesia bigemina and B. bovis), canine babesiosis (B. canis), canine ehrlichiosis (Ehrlichia canis), caprine anaplasmosis (A. capra), etc [5,[6][7][8]. These pathogens have signi cant economic impacts on livestock production and cause remarkable economic losses each year.
On the other hand, infection in these animals also increased the risk of developing tick-borne disease in their owners.
Hainan Island, the second largest island of China, locates in the Southern China Sea. It has a typical tropical climate and plentiful wildlife. Until the year 2018, it has an area 33, 900 square kilometers and a population of 9.34 million. In the recent decades, it is becoming a tourist attraction because of its beautiful scenery and geographical position. Previous study indicated that Hainan Island is the natural epidemic focus of North Asia Tick-Borne Spotted Fever (NASF), which is caused by Rickettsia sibirica belonged to Spotted Fever Group Rickettsiae (SFGR) [9]. The antibodies investigate of NASF in locality revealed the positive incidence are 38.3% and 53% in local human and mouse, separately [10]. However, the reports on other tick-borne pathogens in Hainan Island have been very few. In this study, to evaluate the potential risk of tick-borne pathogens to local residents, tourists and domestic animals on Hainan Island, we performed a survey of the occurrence and prevalence of bacteria and protozoans in ticks collected from Hainan Island, China.

Samples collection and DNA isolation
Tick samples were collected from stray dogs, goats and cattle in Chengmai county locating in the northwest of Hainan Island from September to October, 2019. Only adult ticks were included in this study.
Ticks were brought to China CDC alive and stored individually at -80 °C before DNA extraction. The tick species was determined by morphological identi cation, and then con rmed by PCR and sequencing.
Before DNA extraction, the ticks were washed with 75% ethyl alcohol followed by 0.01M phosphate buffer solution (PBS) and dried. They were divided into pools (3 ticks each) and homogenized in PBS. Genomic DNA was extracted using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the instructions of the manufacturer. All DNA extracts were stored at -20 °C.

Detection of tick-borne pathogens
All the tick samples were tested for the presence of Rickettsiales bacteria, Coxiellaceae bacteria, Babesiidae, and Hepatozoidae. PCR detection of Babesiidae and Hepatozoidae species targeting the 18S rRNA gene was performed using primers as described previously [11]. The rrs gene of Rickettsiales bacteria and Coxiellaceae bacteria species were ampli ed by nested PCR using primers as described [12,13]. For further exploration of phylogenetic positioning of the bacterial strains, the 760-bp fragment of the gltA gene encoding citrate synthase and the 614-bp fragment of the groEL gene encoding the heat shock protein were ampli ed for samples that were positive for these bacteria. Both negative and positive controls were added. PCR amplicons were analyzed by electrophoresis in 1.0% agarose gels. The amplicons shorter than 600 bp were directly subjected to Sanger sequencing by Sangon Biotechnology Company (Shanghai, China). The PCR products longer than 600 bp were cloned into pMD19-T cloning vector (TaKaRa, China), transformed into E. coli and plated onto culture dish. Then the obtained clones were picked and sent for Bi-directional sequencing.

Phylogenetic analysis
Nucleotide sequence similarities between the obtained 16S/18S rRNA, groEL and gltA sequences and those from GenBank were calculated by DNAStar (ver. 7.0). For phylogenetic analysis, the sequences were aligned with reference sequences using ClustalW (default parameters) within MEGA 6.0 [14]. Phylogenetic trees were then estimated using the Maximum Likelihood (ML) method implemented in PhyML, version 3 [15]. Totally, 1000 bootstrap replicates were used under the same procedure to estimate the support for each node. All trees were mid-point rooted.

Tick Collections
The ticks were collected in Fushan town, Chengmai county locating in Northern Hainan Island, China ( Fig. 1). In total, twelve dogs, ten goats and fteen cattle were inspected and 285 ticks were collected during September to October. These ticks were identi ed by morphological observation and sequence analysis of their 12S rRNA gene, and they were assigned to two tick species, Rhipicephalus sanguineus (183, 64.21%) and R. microplus (102, 35.79%). All the 132 ticks from dogs and 51 ticks from goats are R. sanguineus. They were equally divided into 44 pools and 17 pools, with 3 ticks for each pool. The 102 ticks from cattle are all R. microplus and they are divided into 34 pools (Table 1).

Detection of Babesia/Theileria and Hepatozoon
Genetic analysis of the recovered 18S rRNA gene sequences using the blastn with Nucleotide collection nr/nt revealed that they were most closely related to those of Babesia canis vogeli (100.0%) and Hepatozoon canis (99.54%~99.77%), respectively ( Table 2). Phylogenetic analysis of 18S RNA gene sequences revealed the co-circulation of B. canis and H. canis in the 1 and 3 R. sanguineus pools from dogs, respectively (Fig. 2, Table 3). The sequences of B. canis vogeli HNRS/dog/B1 exhibited a close relationship to those of the known B. canis vogeli found in R. sanguineus from domestic and foreign [7,[16][17][18][19] in the 18S RNA tree ( Fig. 2A)

Detection of bacterial pathogens
Genetic analysis of the rrs, groEL, and gltA gene sequences from ticks revealed that they were most closely related to those of Coxiellaceae, and Anaplasmataceae bacteria. Brie y, the rrs sequences recovered from the ticks pools sampled from Hainan Island exhibited high sequence similarities to those from species of Coxiellaceae bacterium (100%), C. burnetii (95.7%), A. marginale (100%), A. platys (99.87%), and Ehrlichia sp. (100%), respectively ( Table 2). Whereas the similarities between the sequences recovered from this study and known reference sequences from GenBank varied from 81.96-99.86% for the gltA gene sequences, and from 91.56-100.0% for the groEL gene sequences (Table 2). Hence, these data revealed the co-circulation of Ehrlichia, Anaplasma, and (the proposed) Candidatus Coxiellaceae bacterium in ticks collected from Hainan Island (Fig. 3, Fig. 4, Table 2).
Besides, Coxiella like bacterial DNA was mainly found in 3 R. sanguineus tick pools (Table 3). In the rrs gene tree (Fig. 4)

Discussion
The previous studies revealed the dominant species are R. sanguineus and R. microplus in Hainan Island, China [29], as well as in our study. Hainan Island is the natural epidemic focus of North Asia Tick-Borne Spotted Fever (NASF) [9]. The antibodies investigate of NASF in local human and mouse indicated the positive incidence are high [10]. In the current study we evaluated the presence of several pathogens as Rickettsiales bacteria, Coxiellaceae bacteria, Babesiidae, and Hepatozoidae in these two ticks. The results revealed the presence of four recognized species and two Candidatus sp. of Anaplasmataceae and Coxiellaceae. Importantly, A. marginale, A. platys, H. canis, B. canis, and Coxiella burnetii are known to be animal and/or human pathogens [28,30]. Hence, the data clearly indicate that multiple bacteria and protozoa co-circulate in hard ticks in Hainan Island. As more species of ticks are present in Hainan Island, it is likely that more additional tick-associated pathogens will be discovered.
Many members in the family Anaplasmataceae are causative agents of tick-borne diseases (i.e. Anaplasmosis and Ehrlichiosis) with a remarkable impact on human and animal health [31]. Several species have been identi ed in family Anaplasmataceae and some are considered as human or animal pathogens. For example, A. phagocytophilum, A. marginale, and A. platys are the important diseaseproducing pathogens in the genus Anaplasma [5,32]. A. marginale is the common ruminant pathogens infecting buffalo and cattle which is distributed on six continents, especially in tropical and subtropical regions [30,33,34]. While A. platys is the agent of cyclic thrombocytopenia in dogs and is the only classi ed Rickettsiales species known to infect platelets [17]. Besides infecting dogs, the infection of this pathogen has been reported in cats, foxes (Vulpes vulpes), cattle, goats, camels, red deer, and humans [8,25,[35][36][37][38][39][40][41][42]. In our study, two documented bacteria A. marginale and A. platys were identi ed in R. microplus and R. sanguineus ticks separately. The cattle, as the major hosts of R. microplus, possessed the high prevalence of A. marginale, and these results suggests that more surveillance in cattle in Hainan Island needs to be performed. R. sanguineus is considered the primary vector of infection to A. platys, which is all founded in dogs in this study. However, the high infection rate of A. platys in local dog ticks indicated high risk of cyclic thrombocytopenia to other animals and humans, due to the close exposure between the dogs and others. Therefore, more surveillance and researches in dogs and its pathogens in Hainan Province needs to be performed.
Similar to the Anaplasma, several novel Ehrlichia bacteria has been discovered in the past decade [17,31,33]. Here, a tentative species was identi ed in R. microplus ticks from cattle, which is mainly founded in R. microplus ticks from several provinces and countries [12,26,27,43]. Otherwise, the sequence of this candidatus species are close to that of E. chaffeensis which is human pathogen bacteria, so more attentions would be paid to this potential novel species.
Coxiella burnetii is the causative agent of Q fever which is a worldwide zoonosis. In common, the infection is mostly persistent in animals, whereas it is often asymptomatic in humans, which can manifest as an acute disease like a u-like illness, or pneumonia, or as a chronic form like endocarditis, et al [15]. There are not typical symptoms when infection occurred in animals and humans except during pregnancy. The most primary reservoirs of C. burnetii are goats, sheep, and cattle [44]. In nature, the ticks, as the main vector and reservoir, play important part in the bacteria maintaining and transmitting, otherwise the cattle and goats play the most important roles in human infections. In this study, Coxiella like bacteria were identi ed in 3 R. sanguineus ticks sampled from Hainan. It would be paid more attentions to due to its closer relations with the known Coxiella burnetii.
Canine hepatozoonosis is a tick-borne disease distributed worldwide, and H. canis is the etiology of hepatozoonosis in dogs [45]. Canine babesiosis is a vector-borne disease caused by several Babesia spp. like B. canis vogeli et al [46]. In our study, H. canis and B. canis vogeli were identi ed in 3 and 1 R. sanguineus ticks collected from Hainan Island, separately. These data revealed that the two agents are co-circulating in arthropods, and the infection risk is higher for dogs, as well as other animals in certain regions.

Conclusions
In conclusion, two species (R. microplus and R. sanguineus) of hard ticks were sampled from dogs, cattle and goats in Hainan province, China. More importantly, four recognized species and two Candidatus spp. of Anaplasmataceae and Coxiellaceae were discovered from these two hard ticks, indicating that this certain geographic region harbors a considerable diversity of Anaplasmataceae bacteria, Coxiellaceae bacteria, Babesiidae, and Hepatozoidae in nature. The data from this study highlight the need for stated surveillance of local arthropods, mammal, and humans, and this can provide the evidence for several bacteria, and protozoa infection.