Spotted fever group Rickettsia, Anaplasma and Coxiella-like endosymbiont in Haemaphysalis ticks from mammals in Thailand

Ticks are ectoparasites of vertebrates and vectors of various pathogenic microorganisms. In this study, the presence of bacteria and protozoa was evaluated by PCR and DNA sequencing in 233 mammal ticks collected from 8 provinces in Thailand. Sequence and phylogenetic analyses of partial rickettsial ompA, ompB, sca4 and partial Coxiella 16S rRNA, GroEL, rpoB genes clearly revealed, for the first time, a co-infection of SFG Rickettsia belonging to R. massiliae subgroup and Coxiella-like endosymbiont (CLE), Cox-hein, in a male of Haemaphysalis heinrichi tick infesting Burmese ferret-badger in Loei province. Moreover, a male of H. hystricis tick infesting the same host was infected with another CLE, Cox-hys. Based on the 16S rRNA gene sequence, Anaplasma sp., closely related to Anaplasma bovis was also detected in a male of H. heinrichi infesting the same Burmese ferret-badger. In addition, the third CLE, Cox-asia, found in H. asiatica collected from Asian palm civet in Chiang Rai province, was different from both Cox-hein and Cox-hys. This study provided important data and broadened our knowledge on tick-borne pathogens and endosymbionts in Thailand and Southeast Asia.


Introduction
Ticks are important vectors of infectious pathogens including bacteria, protozoa, and viruses. The obligate intracellular bacteria, Rickettsia, is notorious for causing infection and mild to severe diseases in humans and other mammals (Raoult and Roux 1997). Based on phylogenomic analyses, members of the genus Rickettsia are classified into four groups, namely, spotted fever group (SFG), typhus group (TG), transitional group (TRG), and ancestral group (AG) (Gillespie et al. 2007).
Coxiella burnetii is the etiological agent of a worldwide zoonotic disease called Q fever. In Thailand, infective endocarditis caused by C. burnetii has been reported (Pachirat et al. 2012). Both animals and humans can be infected by this bacterium, usually through inhalation of contaminated aerosols. Its reservoir hosts comprise mammals, birds, and arthropods, particularly ticks (Raoult and Marrie 1995). However, the role of ticks as the vector of C. burnetii remains controversial. Many Ixodidae and Argasidae ticks, including Amblyomma, Dermacentor, Haemaphysalis, Ixodes, Rhipicephalus, Argas, and Ornithodoros also harbor Coxiella-like endosymbionts (CLE) (Duron et al. 2015). Thus far, there is no evidence of the transmission of CLE by ticks to vertebrates. In addition to CLE, another endosymbiont commonly found in many arthropods and filarial nematodes is Wolbachia bacteria. In arthropods, Wolbachia has been reported to manipulate host reproduction including cytoplasmic incompatibility, parthenogenesis, feminization of genetic males, and male killing. Therefore, current research focuses on Wolbachia application to protect humans from vector-borne diseases (Werren et al. 2008).
Anaplasma and Ehrlichia are tick-transmitted bacteria in the family Anaplasmataceae, order Rickettsiales. Anaplasma is an obligate intracellular bacterium living in mammal blood cells. Members of this genus include tick-borne pathogens causing fatal infectious diseases in humans and catastrophic diseases in animals (Dumler et al. 2001). Anaplasmosis in humans is principally caused by Anaplasma phagocytophilum. Anaplasma has also been found in ticks of the family Ixodidae, including the genera Amblyomma, Dermacentor, Ixodes, and Rhipicephalus (Dumler et al. 2001). Ehrlichia spp. are obligatory intracellular bacteria that cause ehrlichiosis in animals and humans. In Thailand, several Ehrlichia spp. that are the etiologic agents of ehrlichiosis including E. canis, E. chaffeensis, and E. platys, have been reported (Parola et al. 2003;Pinyoowong et al. 2008).
Lyme disease is a tick-borne disease caused by Borrelia burgdorferi, whose main vectors are Ixodes ticks, i.e., Ixodes ricinus and Ixodes scapularis. This bacterial pathogen has also been reported to be carried by other tick genera, namely Amblyomma, Dermacentor, Haemaphysalis, Hyalomma, and Rhipicephalus. Moreover, other members of the genus Borrelia are known to be the cause of human diseases, for example, B. afzelii and B. garinii (Margos et al. 2009).
The study of pathogens that are transmitted by ticks will be useful for understanding the role of ticks as vectors of human and other animal disease agents. This information is crucial for effective monitoring and control of tick-borne diseases. Thus, the aim of this study was to investigate the presence of bacteria and protozoa in mammal ticks collected from several regions in Thailand using molecular approaches.

Tick collection and identification
A total of 233 ticks were collected from domestic and roadkilled mammals (23 hosts belonging to 7 species) in 8 provinces of Thailand during 2015-2019 (Table 2). All tick samples were kept in labeled collection vials individualized per host, containing 70% ethanol. They were identified morphologically by using a stereomicroscope following previously published taxonomic keys (Cooley 1946;Wassef and Hoogstraal 1984;Tanskul and Inlao 1989). Species of ticks were molecularly identified by partial sequencing of 16S rRNA gene (Black and Piesman 1994) to confirm their morphological identifications.

DNA extraction and PCR amplification
Each individual tick was rinsed in 10% sodium hypochlorite, 70% ethanol, and sterile distilled water three times (1 min each). Genomic DNA was extracted from individual adults, nymphs, pooled nymphs, and pooled larvae using the QIAamp DNA Extraction Kit for Tissue (QIAGEN) according to the manufacturer's protocol. The presence of bacteria and protozoa, including Anaplasma, Borrelia, Coxiella, Ehrlichia, Rickettsia, Wolbachia, Babesia, and Hepatozoon in tick samples was initially screened by Polymerase Chain Reaction (PCR) using primers as shown in Table 1. For Rickettsia characterization, the primers Rr17.61p/Rr17.492n were initially used to amplify a 434 bp fragment of the 17-kDa antigen gene. Subsequently, the 17-kDa-positive samples were amplified and sequenced with primers specific to gltA, ompA, ompB, and sca4 genes (Webb et al. 1990;Regnery et al. 1991;Roux and Raoult 2000;Jiang et al. 2005). For Coxiella, the 16S rRNA positive samples were further amplified with primers targeting rpoB (DNA directed RNA polymerase beta) and GroEL (60 kDa chaperone heat shock protein B) genes (Duron et al. 2014(Duron et al. , 2015. Additionally, the positive sample tested with EHR16SD/EHR16SR primers was amplified and sequenced with Anaplasma 16S rRNA primers as previously described by Zobba et al. (2014) (Table 1).

DNA sequencing and phylogenetic analysis
All positive amplicons were purified using the GF-1 Ambi Clean kit (Vivantis) according to the manufacturer's instructions and sequenced in both directions on an ABI 3730xl DNA analyzer (Applied Biosystems). All obtained DNA sequences were assembled and edited using BioEdit (Alzohairy 2011). Edited sequences were assembled into a contig using SeqMan software (DNASTAR, Lasergene), and thus were subjected to BLASTn analysis (http:// blast. ncbi. nlm. nih. gov/ Blast. cgi) to find sequence similarity to known sequences. Phylogenetic analyses were performed using the maximum parsimony method (PAUP v. 4.0b1) and bootstrap analysis was calculated with 1,000 replicates.

Identification of ticks
A total of 23 mammals from 8 provinces of Thailand were examined for tick infestation. These mammals belonged Haemaphysalis bispinosa (2)

Detection of tick-borne bacteria and protozoa in tick samples
A total of 233 tick specimens were molecularly detected for bacterial and protozoal microorganisms by PCR technique. All tick samples collected from domestic mammals showed PCR negative results while some of the ticks collected from Burmese ferret-badger in Loei province and from Asian palm civet in Chiang Rai province were positive for Rickettsia (0.4%; 1/233) and Coxiella (3.4%; 8/233) ( Table 2). Rickettsial DNA was detected in a male of H. heinrichi. Coxiella-like endosymbionts were detected in a male of H. hystricis (Cox-hys), six males of H. asiatica (Cox-asia), and a male of H. heinrichi (Cox-hein) which was co-infected with SFG Rickettsia sp. Interestingly, Anaplasma DNA was detected in another male of H. heinrichi infesting Burmese ferret-badger from Loei province ( Table 2). None of the 233 mammal ticks gave specific PCR products of Borrelia, Ehrlichia, Wolbachia, Babesia, and Hepatozoon.
Based on three partial sequences of 16S rRNA, GroEL, and rpoB, all Coxiella spp. amplified from three Haemaphysalis tick species were CLE. In contrast, C. burnetii was not detected in this study. Nucleotide sequences of the three CLE (Cox-hein, Cox-hys, and Cox-asia) were different from each other. The BLAST results of these Coxiella partial 16S rRNA sequences indicated that their nucleotide sequences were highly similar to other CLE of several Haemaphysalis tick species represented in GenBank (98.52-100%). However, BLAST results of partial GroEL and rpoB sequences showed lower level of nucleotide sequence similarity to other CLE available in GenBank than that found with 16S rRNA. The nucleotide sequence similarity of CLE detected in this study was 89.42-93.64% for GroEL and 91.04-100% for rpoB. Phylogenetic analysis of partial 16S rRNA revealed that Cox-hein and Cox-hys in ticks removed from Burmese ferret-badger were grouped in the same subclade of clade D, while Cox-asia in ticks removed from Asian palm civet was placed in another subclade (Fig. 2). In contrast, the phylogenetic trees generated from the partial sequences of GroEL and rpoB, Cox-hein were more closely related to Cox-asia than to Cox-hys (Fig. 2). However, all three CLE detected in this study were grouped in clade D with other CLE of Haemaphysalis ticks from previous reports.
Anaplasma sp. was found only in a male of H. heinrichi using PCR with 16S rRNA primers. BLAST results of the partial sequences of 16S rRNA illustrated a remarkably high similarity (99.73%; 730/732 bp) to several strains of A. bovis, for example, Zhengxiaocon-goat-48 (MH255939), ZJ69 (KP062958), sika35 (LC060988), NR07 (AB196475), and many other strains. Based on phylogenetic analysis of partial 16S rRNA gene sequences, Anaplasma sp. detected in this study manifestly belonged to the same clade with A. bovis with strong bootstrap support (100%) (Fig. 3). The results of BLAST and phylogenetic analysis thus explicitly demonstrated that a male of H. heinrichi was infected with Anaplasma sp. closely related to A. bovis.

Discussion
Ticks serve as vectors and/or reservoirs of several pathogens such as Anaplasma, Coxiella, Ehrlichia, and Rickettsia (Walker and Yu 2012). To better understand tick-associated microorganisms among various mammal ticks in Thailand, we conducted specific molecular screening for the presence of bacterial and protozoal microorganisms. Previous studies showed that Rickettsia spp. were widely distributed throughout Thailand in various locations, hosts, and in tick species, including H. ornithophila, Amblyomma testudinarium, H. shimoga, H. lagrangei, A. helvolum, and A. varanense (Hirunkanokpun et al. 2003;Ahantarig et al. 2011;Sumrandee et al. 2014;Malaisri et al. 2015;Nooroong et al. 2018). In this study, a SFG Rickettsia sp. was detected in a male of H. heinrichi infesting Burmese ferret-badger in Loei province, northeastern Thailand. The members of SFG rickettsiae have been categorized into four subgroups: R. rickettsii, R. massiliae, R. helvetica, and R. akari (Merhej and Raoult 2011). Our results revealed five partial rickettsial sequences of 17-kDa, gltA, ompA, ompB, and sca4 genes that were amplified and sequenced from a H. heinrichi tick. The analysis of partial 17-kDa and gltA sequences indicated that the Rickettsia sp. was a member of SFG rickettsiae. However, these two genes could not differentiate whether it belonged to the R. massiliae or R. rickettsii subgroups. Since 17-kDa and gltA genes are highly conserved among SFG rickettsiae, previous studies have suggested that less conservative rickettsial genes such as ompA, ompB, and sca4 should be more suitable for the comparisons of closely related SFG species (Robinson et al. 2019). Therefore, these gene sequences were used for characterization of Rickettsia sp. in this study. BLAST and phylogenetic analyses of the partial ompA, ompB, and sca4 genes showed that the detected SFG Rickettsia sp. found in this study was a member of R. massiliae   The Anaplasma sp. sequence obtained in present study is indicated in bold subgroup and was closely related to both R. rhipicephali and R. massiliae. However, it remains uncertain whether this Rickettsia sp. is pathogenic R. massiliae or non-pathogenic R. rhipicephali. Further characterizations by amplification and sequencing of additional genes, such as sca2 and GroEL, are required to clarify this point. In Thailand, a Rickettsia sp. closely related to members of the R. massiliae subgroup has been reported in H. lagrangei collected from sambar deer (Sumrandee et al. 2016). We reported herein for the first time SFG Rickettsia sp. belonging to the R. massiliae subgroup in H. heinrichi infesting a wild Burmese ferret-badger.
The distribution and diversity of tick species may be crucial to the investigation of tick-borne diseases. Cornet et al. (2009) indicated that Haemaphysalis was one of the most common genera widely distributed throughout Thailand and was capable of transmitting tick-borne diseases to humans. Humans bitten by infected Haemaphysalis ticks are consequently exposed to a high risk of bacterial infection. Haemaphysalis heinrichi was previously found infesting four species of mammal hosts: Arctonyx collaris, Bos domesticus, Canis familiaris, and Melogale personata in Chiang Mai, Chiang Rai, Khon Kaen, Bangkok, Nakhon Ratchasima, Prachinburi, and Ubon Ratchathani (Tanskul et al. 1983). Our findings have provided additional evidence of H. heinrichi infesting Burmese ferret-badger in Loei province, northeastern Thailand. Further study of the abundance and distribution of H. heinrichi and related tick species and the prevalence of bacterial infection will provide a better understanding of the epidemiology of rickettsioses and other tick-borne diseases in Thailand.
Coxiella-like endosymbionts have frequently been found in ticks (Ahantarig et al. 2011;Almeida et al. 2012;Arthan et al. 2015;Duron et al. 2015). Multilocus sequence analysis of five Coxiella housekeeping genes indicated that Coxiella endosymbiont in ticks served as the common ancestor of C. burnetii (Duron et al. 2015). The endosymbiont may play a vital role in providing vitamin and cofactor biosynthesis pathways and in defining the reproductive fitness of tick hosts (Smith et al. 2015). Consequently, understanding its role in sustaining growth and survival of ticks may provide new methods for control and management of tick populations. Q fever and seroprevalence of C. burnetii have been reported in rural areas of Malaysia and Thailand (Suputtamongkol et al. 2003;Bina Rai et al. 2011). In Malaysia, both C. burnetii and Coxiella endosymbiont were reported in H. hystricis ticks collected from the same wild boar. In this study, C. burnetii was not detected in all tick samples and CLE were detected solely in one (Haemaphysalis) of three analyzed tick genera. We identified three different types of CLE, namely Cox-hein in H. heinrichi, Cox-hys in H. hystricis, and Cox-asia in H. asiatica. Phylogenetic analysis with five concatenated gene sequences by Duron et al. (2015) showed that the genus Coxiella was divided into four clades (A-to-D) corresponding to the host genera. In this study, the phylogenetic trees generated from partial 16S rRNA, GroEL, and rpoB gene sequences demonstrated that the three CLE were clustered with those of Haemaphysalis ticks and were placed in clade D. The result of CLE partial 16S rRNA sequences obtained in this study was in accordance with previous authors who suggested that CLE detected from the same host genera were clustered within the same clade (Khoo et al. 2016;Trinachartvanit et al. 2018). However, GroEL and rpoB gene sequences did not cluster together with those previously reported in the same host genera (Khoo et al. 2016;Trinachartvanit et al. 2018). In Thailand, co-infections of Rickettsia and Coxiella have been reported in H. lagrangei and A. testudinarium (Nooroong et al. 2018;Sumrandee et al. 2016). Our results have also demonstrated the first case of coinfection of CLE and SFG Rickettsia sp. closely related to R. massiliae subgroup in H. heinrichi infesting Burmese ferret-badger. Interestingly, many wild animals have been reported as reservoirs of pathogenic bacterial agents of humans, including A. phagocytophilum, C. burnetii, and Rickettsia spp. (Meerburg and Reusken 2011;Silaghi et al. 2014). Further investigation in this field of research will provide more information on the prevalence of tick-borne pathogens in various tick species infesting wild mammals in Thailand before any conclusion can be made.
Anaplasmosis is an infectious hemotropic disease of cattle, sheep, goats, and other ruminants worldwide. Several Anaplasma species are causative agents of anaplasmosis in ruminants, e.g., A. phagocytophilum, A. marginale, and A. bovis (Inokuma 2007). In Thailand, Anaplasma spp. have been reported in both domestic and wild mammals and their associated ticks, including A. platys which have been detected from blood of mammal hosts i.e., cats, dogs, rodents, sambar deer, and wild boar, or from their parasitic ticks (Parola et al. 2003;Pinyoowong et al. 2008;Foongladda et al. 2011;Salakij et al. 2012;Sumrandee et al. 2016). Anaplasma marginale and A. phagocytophilum were found in water buffalo and ticks collected from vegetation, respectively (Nguyen et al. 2020;Nooroong et al. 2018); A. bovis has been reported in ticks infesting bear, sambar deer, rodents, and ticks collected from vegetation (Parola et al. 2003;Malaisri et al. 2015;Sumrandee et al. 2016;Takhampunya et al. 2019). In this study, an Anaplasma sp. closely related to A. bovis was detected in H. heinrichi removed from Burmese ferret-badger. Thus, the results of the present and previous studies in Thailand demonstrated that A. bovis were commonly found in ticks parasitizing domestic and wild mammals.
Ixodid ticks of the genera Ixodes, Dermacentor, Rhipicephalus, and Amblyomma are the main vectors of Anaplasma bacteria (Dumler et al. 2001). However, this bacterium has also been found in Haemaphysalis ticks in Asia and North America (Goethert and Telford 2003;Kim et al. 2003;Qin et al. 2018;Fukui and Inokuma 2019). In Thailand, Anaplasma spp. have been detected in ticks such as A. platys in D. auratus (Parola et al. 2003) and R. sanguineus (Foongladda et al. 2011); A. phagocytophilum in D. auratus (Nooroong et al. 2018); A. bovis in H. lagrangei (Parola et al. 2003;Sumrandee et al. 2016), H. shimoga (Malaisri et al. 2015), H. obesa (Sumrandee et al. 2016), and H. bandicota (Takhampunya et al. 2019). In this study, the presence of Anaplasma sp. closely related to A. bovis in H. heinrichi has been demonstrated by sequence and phylogenetic analysis of partial Anaplasma 16S rRNA. Our findings support previous studies in Thailand and other countries in Asia in which A. bovis is mostly found in Haemaphysalis, and this tick genus also associates with the transmission of Anaplasma (Goethert and Telford 2003;Parola et al. 2003;Lee and Chae 2010;Yoshimoto et al. 2010;Malaisri et al. 2015;Sumrandee et al. 2016). To our knowledge, this is the first report of Anaplasma sp. closely related to A. bovis in H. heinrichi tick-infested Burmese ferret-badger.
In summary, we identified tick-borne bacteria among various mammal ticks collected from 8 provinces of Thailand. Based on DNA sequencing and phylogenetic analyses, we found SFG Rickettsia sp. in the R. massiliae subgroup in H. heinrichi and three CLE, namely Cox-hein, Cox-hys in H. heinrichi and H. hystricis ticks infesting a Burmese ferret-badger, and Cox-asia in H. asiatica. Co-infection of SFG Rickettsia sp. and CLE (Cox-hein) was detected in H. heinrichi. This study also provided the first evidence for the presence of an Anaplasma sp. closely related to A. bovis in a H. heinrichi tick. Our results extended the knowledge of geographic distribution of ticks parasitizing different species of mammals and vector-borne bacteria in Southeast Asia.