Molecular Survey of Vector-Borne Pathogens in Ticks, Sheep Keds, and Domestic Animals from Ngawa, Southwest China

Vector-borne pathogens are mainly transmitted by blood-feeding arthropods such as ticks, mosquitoes, fleas, lice, mites, etc. They pose a significant threat to animal and human health due to their worldwide distribution. Although much work has been performed on these pathogens, some neglected areas and undiscovered pathogens are still to be further researched. In this study, ticks (Haemaphysalis qinghaiensis), sheep keds (Melophagus ovinus), and blood samples from yaks and goats were collected in Ngawa Tibetan and Qiang Autonomous Prefecture located on the eastern edge of the Qinghai–Tibet Plateau, Southwest China. Several vector-borne bacterial pathogens were screened and studied. Anaplasma bovis strains representing novel genotypes were detected in ticks (8.83%, 37/419), yak blood samples (45.71%, 64/140), and goat blood samples (58.93%, 33/56). Two spotted fever group (SFG) Rickettsiae, Candidatus Rickettsia jingxinensis, and a novel Rickettsia species named Candidatus Rickettsia hongyuanensis were identified in ticks. Another Rickettsia species closely related to the Rickettsia endosymbiont of Polydesmus complanatus was also detected in ticks. Furthermore, a Coxiella species was detected in ticks (3.34%, 14/419), keds (1.89%, 2/106), and yak blood (0.71%, 1/140). Interestingly, another Coxiella species and a Coxiella-like bacterium were detected in a tick and a goat blood sample, respectively. These results indicate the remarkable diversity of vector-borne pathogens circulating in this area. Further investigations on their pathogenicity to humans and domestic animals are still needed.

Notably, domestic ungulates are often infected with Anaplasma spp., causing weight loss, abortion, reduced milk production, or even death, leading to numerous economic losses in the livestock industry annually [9]. Meanwhile, many infected domestic ungulates are permanently and asymptomatically infected. They may act as reservoirs and sources of pathogens for vectors in their life cycles. Meanwhile, Coxiella burnetii, the etiologic agent of Q fever, is the most notorious pathogen of the genus Coxiella [10]. As a worldwide distributed pathogen, it has been detected in multiple animal species, including goats, cattle, horses, deer, rodents, and even birds [11][12][13]. Coxiella burnetii infection in domestic animals is usually asymptomatic but may lead to infertility, abortion, and neonatal death [14], thus resulting in tremendous economic losses. Furthermore, human Q fever caused by C. burnetii is a worldwide zoonosis that has long been under-reported and underdiagnosed due to its non-specific symptoms.
Ngawa Tibetan and Qiang Autonomous Prefecture has an area of 84,242 km 2 . It is located in the northwest of Sichuan Province and on the eastern edge of the Qinghai-Tibet Plateau, China. The high altitude (982-6250 m, mostly 2500-4000 m) and the abundant grassland resources of Ngawa make animal husbandry the major income source for rural households. Yaks, goats, horses, and pigs are the common livestock animals in Ngawa. The frequent contact between herdsmen and livestock animals makes it possible for zoonotic and vector-borne pathogens to be easily transmitted from animals or their ectoparasites to humans. In recent years, some studies on vector-borne pathogens have been performed in some areas of the eastern Qinghai-Tibet Plateau [15][16][17]. In 2016, Yang et al. reported a novel Anaplasma species closely related to A. capra in H. qinghaiensis ticks from Gannan Tibetan Autonomous Prefecture located in the east Qinghai-Tibet Plateau [15]. In 2020, diverse Bartonella species were detected in H. qinghaiensis and Dermacentor everestianus ticks collected in Shiqu County, located on the Qinghai-Tibetan Plateau in Ganze Prefecture [16]. However, except for one study on Theileria detected in H. qinghaiensis, D. everestianus, Rhipicephalus microplus, and Melophagus ovinus reported in 2020 [17], vector-borne pathogens in Ngawa remain understudied.
The objective of our study was to update the epidemiological profile of circulating bacterial VBDs in Ngawa. In this study, we collected ticks (H. qinghaiensis), sheep keds (Melophagus ovinus), and the blood of yaks and goats in Hongyuan County of Ngawa. The presence, prevalence, and genetic characteristics of several vector-borne bacterial pathogens were studied.

Sample Collection
From February to March 2021, a total of 419 ticks (adults and nymphs) were collected from yaks (Bos mutus), and 106 keds were collected from goats (Capra hircus) in Hongyuan County of Ngawa, China ( Figure 1). All ticks were morphologically determined to be H. qinghaiensis and further confirmed by analysis of the COI gene (accession numbers: ON358162-ON358175). All keds were morphologically identified as M. ovinus and molecularly confirmed (accession numbers: ON358236-ON358247). From March to May 2021, 140 and 56 blood samples were collected from apparently healthy yaks and goats, respectively.

Discussion
In this study, most pathogens were detected in H. qinghaiensis ticks. Meanwhile, some pathogens detected in ticks were also detected in goats, yaks, and keds, indicating the possible role of H. qinghaiensis in the maintenance and transmission of these pathogens in this area. Haemaphysalis qinghaiensis is an endemic tick species widely distributed in the western plateau of China [15]. As a three-host tick, it often infests domestic animals, including goats, cattle, yaks, horses, etc. Occasionally, it also bites humans [15]. In previous studies, it has been demonstrated that H. qinghaiensis transmits a lot of human and animal pathogens, such as Theileria sp., Babesia sp., Borrelia burgdorferi, and A. phagocytophilum [21]. The potential role of H. qinghaiensis ticks in the transmission of human and animal diseases in this area should be further considered.
Anaplasma bovis has been well recognized as a tick-borne pathogen infecting cattle, buffalos, yaks, goats, and rodents, as well as humans [4,[30][31][32]. It is one of the etiologic agents of bovine anaplasmosis, characterized by fluctuating fever, depression, and death [33]. Occasionally, it also infects humans and causes fever, headache, myalgia, rash, eschar, etc. [4]. In this study, we observed remarkably high positivity rates of A. bovis in apparently healthy goats (58.93%) and yaks (45.71%), indicating that A. bovis is circulating in domestic animals in this region. Genetic and phylogenetic analyses both indicate that these strains are different from previously reported A. bovis strains, and they may represent more than one novel genotype. Phylogenetic analysis based on groEL and gltA genes indicated that they are divided into two clusters, and genetic recombination between the two clusters may exist, suggesting the long-history evolution of A. bovis in this area. The pathogenicity of these strains to humans is still to be determined. Ticks also tested positive for this A. bovis strain, with a positivity rate of 8.83%. As reported previously, the detection rate of Anaplasma in ticks was likely skewed due to Anaplasma infection in livestock [33]. Although the possibility cannot be ruled out that the A. bovis DNA may come from the blood meal of ticks, we suspect that H. qinghaiensis ticks might play a role in the transmission of A. bovis in domestic animals.
Three Rickettsia species, including Rickettsia sp. strain tick14, Candidatus R. jingxinensis, and a novel SFG Rickettsia species named Candidatus R. hongyuanensis, were identified in ticks. Candidatus R. jingxinensis, belonging to SFG Rickettsia, was first identified in Ha. longicornis from northeast China in 2016 [34]. In recent years, it has been detected in Rhipicephalus microplus, Ha. turturis, and Ixodes persulcatus from multiple locations in China, Korea, Thailand, and India, indicating that it is a widely distributed Rickettsia circulating in Asia [35][36][37][38][39]. Notably, a gltA sequence (KU853023) from a patient in China was submitted to GenBank (unpublished), which shows 99.91% nucleotide identity to Ca. R. jingxinensis, suggesting its potential human pathogenicity. Furthermore, a novel SFG Rickettsia named Candidatus R. hongyuanensis was identified in H. qinghaiensis ticks. Its 16S, gltA, groEL, ompA, and ompB genes have the highest identities of 99.58%, 98.81%, 98.77%, 81.87%, and 96.81% to validated species, supporting that it represents a novel Rickettsia species based on criteria established by Fournier et al. [27]. Genetic analyses indicate that it is closely related to human pathogenic R. raoultii and R. conorii. Due to the fact that H. qinghaiensis is a three-host tick, it can easily transmit pathogens between different hosts during its life cycle [15]. Therefore, the role of H. qinghaiensis in the transmission of SFG Rickettsia should be further considered, and investigations on the human pathogenicity of these Rickettsia species are still needed. Furthermore, Rickettsia sp. strain tick14, closely related to the Rickettsia endosymbiont of Polydesmus complanatus Bel-53 [28], was identified, and it is located in a basic position of the phylogenetic trees based on 16S and groEL sequences.
Similar to Rickettsiales bacteria, the genus Coxiella, belonging to Coxiellaceae, is a group of intracellular bacteria [40]. This genus includes two recognized species (C. burnetii and C. cheraxi), one Candidatus species (Candidatus Coxiella mudrowiae), and some unclassified Coxiella symbionts [41,42]. In this study, three Coxiella species were detected, with two of them infecting domestic animals. This may be one of the few recent studies in which Coxiella species were found to infect mammals. One Coxiella species (strain yak17, strain tick103, and strain tick166) was detected in ticks (3.34%), sheep keds (1.89%), and one yak blood sample (0.71%). Interestingly, sheep keds also tested positive for this Coxiella. There might be three possibilities: (1). Sheep keds are infected, and they act as the vector of this Coxiella. (2). The Coxiella DNA is from the blood meal of sheep keds. That is, goats may be infected by this Coxiella. (3). The Coxiella strains could be just Coxiella-like endosymbionts that co-exist with hosts/vectors. Each possibility is intriguing, and further studies are still needed. Furthermore, it is noteworthy that a Coxiella-like bacterium was identified in a goat blood sample. Its 16S gene shared the highest (96.77%) identity with the human and animal pathogen C. burnetii strain CB_25 (LC464975.1), strain CB_30 (LC464974.1), etc. (unpublished). However, sequences of other key genes are not available for this strain, possibly due to the low identity to known strains.
In summary, we identified seven bacterial species in arthropods and domestic animals in Ngawa. Although the human pathogenicity for most of these bacteria is still to be determined, more attention should be paid to the risk of human infection and the possible circulation of these pathogens in local people.

Sample Collection and Processing
Ngawa Tibetan and Qiang Autonomous Prefecture is located in the northwest of Sichuan Province and on the eastern edge of Qinghai-Tibet Plateau, China. From February to March 2021, 419 ticks and 106 sheep keds were respectively collected from yaks and goats in Sedi town (102.98 • E, 30.01 • N, with an altitude of 3528 m), Hongyuan County of Ngawa, Sichuan Province, China. The ticks and keds were stored at −40 • C before DNA extraction. From March to May, blood samples were collected from asymptomatic domestic animals, including yaks and goats, in Hongyuan County. Whole blood samples were stored in tubes containing EDTA as anticoagulant and then stored at −40 • C before DNA extraction.

DNA Extraction
The cryopreserved tick and sheep ked samples were washed three times with phosphatebuffered saline (PBS) and then were individually placed into 2 mL sterile tubes with 0.5 mL of PBS solution. After homogenization using Mixer Mill MM 400 (Retsch, Hann, Germany), the arthropod suspension was directly subjected to DNA extraction with a Mollusc DNA Extraction Kit (Omega Bio-Tek, Norcross, GA, USA). The kit was operated according to the manufacturer's instructions, and the genomic DNA was extracted and eluted with 60 µL of Elution Buffer twice.
According to the protocol of Blood & Tissue DNA Kit (Omega, Norcross, GA, USA), DNA was extracted from 200 µL of the animal blood samples. After extraction, the DNA samples were diluted into 60 µL of Elution Buffer repeatedly.

Species Identification of Ticks and Keds
Ticks and keds were morphologically identified by an arthropod taxonomist using microscopy and then stored at −40 • C until DNA isolation. The tick species was morphologically determined by observing the basis scapituli and palp, while the ked species was determined by checking the mouthparts, bristles on the body surface, and legs tipped with pointed claws [43]. Tick and ked species were further confirmed by PCR amplification and analysis of the COI gene (cytochrome C oxidase I) (primers shown in Table S5). PCR amplification was performed using a Sensoquest PCR System LabCycler (Göttingen, Germany). The cycling conditions were: 94 • C for 3 min, 35 cycles of 94 • C for 30 s, 55 • C for 1 min, 72 • C for 1 min, and a final extension of 72 • C for 5 min [44]. After electrophoresis, the amplicons were subjected to sequencing and analyzed by BLASTN alignment.

PCR Detection and Amplification of Key Genes
To determine the presence and prevalence of Rickettsia spp., Anaplasma spp., and Coxiella spp., semi-nested or nested PCR targeting a conserved domain of 16S rDNA gene was performed on all DNA samples. The primers are shown in Table S5 and previous reports [43,45]. PCR amplification was performed using a Sensoquest PCR System LabCycler (Germany). For further analysis of the molecular characteristics and phylogenetic positions of these bacterial strains, the sequences of two other genes (the gltA gene encoding citrate synthase and the groEL gene encoding the heat shock protein) were obtained for Rickettsia (gltA: 1006 bp; groEL: 656-1053 bp) and Anaplasma (gltA: 826 bp; groEL: 769 bp) positive samples (primers and details shown in Table S5). Additionally, a longer 16S fragment (1201 bp), ompA (705 bp), ompB (376 bp), and htrA (411 bp) genes were recovered from Rickettsia strains. The primers used were previously described [43,46] (Table S5). For Coxiella strains, the sequences of groEL and rpoB genes were obtained using the primers shown in Table S5 [45]. The details of amplification are provided in Table S5. After electrophoresis in 1.0% agarose gel, the PCR amplicons were subjected to sequencing by Sangon Biotech Company (Shanghai, China), and the sequencing results were analyzed by BLASTN alignment.

Genetic and Phylogenetic Analyses
Protein-coding sequences from Rickettsia spp., Anaplasma spp., and Coxiella spp. obtained in this study, as well as the reference sequences retrieved from GenBank, were aligned using ClustalW ("protein-coding genes" strategy) in the MEGA software, version 7.0 [47]. Nucleotide sequence identities were calculated by MegAlign program in the DNASTAR Lasergene package (DNASTAR, Inc., Madison, WI, USA). Phylogenetic trees of the 16S and other key genes based on the maximum likelihood method were reconstructed by PhyML v3.2 [48]. The substitution model test was performed to determine the bestfit phylogenetic model. The confidence values for individual branches of the tree were determined by bootstrap analysis with 100 repetitions.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/pathogens11050606/s1, Table S1. Nucleotide identity of 16S, gltA, and groEL genes of Anaplasma bovis strains to reported strains in the Genbank database. Table S2. Nucleotide identity of 16S, gltA, groEL, ompA, ompB, and htrA genes of Rickettsia strains to reported strains in the Genbank database. Table S3. Nucleotide identity of 16S, groEL, and rpoB genes of Coxiella strains to reported strains in the Genbank database. Table S4. Genbank numbers of Rickettsia spp., Anaplasma spp., and Coxiella spp. sequences obtained in this study. Table S5. The primers used for amplification of 16S, gltA, groEL, htrA, ompA, ompB, and rpoB genes from Rickettsia spp., Anaplasma spp., and Coxiella spp. by nested PCR or semi-nested PCR. Supporting Data. Nucleotide sequences of Candidatus Rickettsia hongyuanensis.

Informed Consent Statement: Not applicable.
Data Availability Statement: All sequence files are available from the NCBI database (shown in Table S4).