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Epidemiological Investigation of Tick-Borne Bacterial Pathogens in Domestic Animals from the Qinghai-Tibetan Plateau Area, China

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18 December 2023

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Abstract
The Qinghai-Tibetan Plateau area (QTPA) features a unique environment that has witnessed the selective breeding of diverse breeds of domestic livestock, exhibiting remarkable adaptability. Nevertheless, Anaplasma spp., Rickettsia spp., Coxiella spp., and Borrelia spp. represent tick-borne bacterial pathogens that pose a global threat and have substantial impacts on both human and animal health, as well as on the economy of animal husbandry within the Qinghai-Tibetan plateau area. In this study, a total of 428 samples were systematically collected from 20 distinct areas within the Qinghai plateau. The samples included 62 ticks and 366 blood samples obtained from diverse animal species, to detect the presence of Anaplasma spp., Rickettsia spp., Coxiella spp., and Borrelia spp. The prevalence of infection in this study was determined as follows: Anaplasma bovis accounted for 16.4% (70/428), A. capra for 4.7% (20/428), A. ovis for 5.8% (25/428), Borrelia burgdorferi sensu lato for 6.3% (27/428), Coxiella burnetii for 0.7% (3/428), and Rickettsia spp. for 0.5% (2/428). Notably, no cases of A. marginale and A. phagocytophilum infections were observed in this study. The findings revealed an elevated presence of these pathogens in Tibetan sheep and goats, with no infections detected in yaks, Bactrian camels, donkeys, and horses. To the best of our knowledge, this study represents the first investigation of tick-borne bacterial pathogens infecting goats, cattle, horses, and donkeys within the Qinghai plateau of the Qinghai-Tibetan Plateau Area. Consequently, our findings contribute valuable insights into the distribution and genetic diversity of Anaplasma spp., Rickettsia spp., Coxiella spp., and Borrelia spp. within China.
Keywords: 
Subject: Biology and Life Sciences  -   Parasitology

1. Introduction

The Qinghai-Tibetan plateau area (QTPA), boasting the highest average elevation globally, harbors a diverse range of ungulate species, comprising approximately 42% of the total ungulate species in China [1]. The significant variations in altitude and the diverse climatic conditions of the plateau have fostered a multitude of ecological environments, which, in turn, have created ecological niches for a wide range of high-altitude species, including different kinds of domestic animals [2]. Nonetheless, the sensitivity of the Qinghai-Tibetan plateau to the effects of global climate change has been noted [3].
The QTPA is home to a diverse range of livestock species that have adapted to survive in the high altitude and cold climate conditions [4]. Among these unique species are Tibetan sheep (Ovis aries), goats (Capra hircus), yaks (Bos grunniens), cattle (Bos taurus domestica), horses (Equus ferus caballus), camels (Camelus bactrianus), and donkeys (Equus asinus) [5]. Within the QTPA, particular livestock species coexist alongside thriving ticks that exhibit adaptability to various environmental niches, characterized by their sensitivity to temperature and humidity conditions [6]. Ticks serve as primary vectors for various pathogens worldwide, impacting both wildlife and domestic animals. While the majority of these pathogens are specific to animals, certain tick-borne diseases are zoonotic, posing significant health risks to humans and, in some cases, leading to severe or fatal consequences [7]. Previous studies have identified the presence of 2 families, 6 genera, and 31 distinct species of ticks in Qinghai plateau of the QTPA. Notably, Haemaphysalis qinghaiensis and Dermacentor nuttalli are the predominant tick species found in Qinghai province and are widespread across most regions [8]. Consequently, the presence of tick-borne bacterial pathogens including Anaplasma, Borrelia, Rickettsia, Coxiella poses a significant concern in this region.
Anaplasmosis is caused by a group of important tick-borne bacteria in the genus Anaplasma. In China, anaplasmosis is generally found in livestock. A. platys was detected in Bactrian camels in China [9]. Furthermore, the occurrence of A. bovis in sheep, goats and yaks, A. ovis in sheep and goats, A. phagocytophilum in yaks, as well as A. marginale in cattle, has been reported within China [10,11,12,13]. Notably, A. bovis and A.ovis can cause anaplasmosis in cattle and sheep, with a high infection rate among livestock; the infection rate of sheep in Haixi and Haibei reached 58% [14].
Lyme disease is a global natural zoonotic disease caused by Borrelia burgdorferi [15]. In China, at least 14 species of B. burgdorferi sensu lato have been indentified in ticks, animals, and humans, providing evidence of the wide distribution and diversity of B. burgdorferi s.l. across China [16]. Results of an epidemiological survey conducted in the forested region of Qinghai plateau revealed a higher prevalence of Lyme disease in the agricultural area as compared to the pastoral area [17].
Earlier study has indicated the presence of B. burgdorferi s.l. in tick hosts, including humans, yaks, cattle, mice, and Tibetan sheep. [18].
Rickettsia spp., categorized under the spotted fever group (SFG), have been identified as causative agents of zoonotic diseases affecting humans, domestic animals, and wildlife, thus emerging as a significnt global health concern [19]. In China, several species including R. heilongjiangensis, R. sibirica subsp. sibirica BJ-90, R. raoultii, R. japonica, Rickettsia sp. XY99, and Candidatus Rickettsia tarasevichiae have been reported [20,21,22,23,24,25,26]. While Rickettsia is predominantly present in ticks within Qinghai plateau [19,27,28,29], there is limited information regarding its incidence in domestic animals.
Coxiella burnetii, the causative agent of Q fever, is a significant zoonotic pathogen with a wide distribution [30]. Infection of C. burnetii in goats has been identified as a major reservoir for human infection. Furthermore, infections in cattle have been associated with cases of abortion and have shown higher prevalence rates in older animals [31]. A study conducted in the Qinghai plateau region reported the detection of C. burnetii within ticks [14].
Previous research has primarily focused on tick-borne pathogens that cause diseases in ticks. However, limited information is available regarding the incidence of these pathogens in domestic animals, particularly goats, cattle, horses, and donkeys in the QTPA. In order to gain a comprehensive understanding of bacterial microorganisms associated with ticks in various domestic animals in Qinghai-Tibetan plateau area, China, we performed targeted molecular screening to identify the presence of bacterial pathogens. Therefore, the current study aims to investigate the incidence of tick-borne bacterial pathogens that infect both ticks and seven distinct types of livestock within the Qinghai-Tibetan Plateau area, providing a comprehensive understanding of the prevalence and distribution of pathogens within the study area.

2. Materials and Methods

2.1. Blood sample collection

A comprehensive collection of 366 randomly obtained blood samples was assembled, representing a diverse range of livestock species, including 52 Tibetan sheep (Ovis aries), 67 goats (Capra hircus), 43 yaks (Bos grunniens), 49 cattle (Bos taurus domestica), 40 donkeys (Equus ferus caballus), 50 camels (Camelus bactrianus), and 65 horses (Equus asinus). These samples were gathered from 20 distinct locations situated within the Qinghai plateau of QTPA (Figure 1). Prior to including livestock in this study, the farm owner was asked for their willingness to participate in the research activity and verbal consent was obtained.

2.2. Morphological and molecular identification of ticks

A total of 62 tick samples were collected from three distinct locations within the Qinghai plateau of the Qinghai-Tibetan Plateau Area (QTPA), specifically Jinchangou in Huzhu County, Lijiatai Village in Ledu County, and Songshu Village in Minhe County (Figure 1). Tick samples were obtained from the field and external surfaces of domestic animals. All tick specimens were carefully preserved in sampling vials and promptly transported to the laboratory for detailed analyses. The identification of each tick specimen was conducted based on morphological criteria, following the methodology outlined by Chen et al. [32]. Additionally, the identification was further confirmed through sequence analyses of a partial fragment of the cytochrome c oxidase subunit I (coxI) gene. A fragment of approximately 850 base pairs (bp) within the coxI gene was amplified through polymerase chain reaction (PCR) using the primers coxI F (5'-GGAACAATATATTTAATTTTTGG-3') and coxI R (5'-ATCTATCCCTACTGTAAATATATG-3') [33].

2.3. DNA extraction

The genomic DNA was extracted from whole blood, which included an anticoagulant (EDTA), utilizing the TIANamp Genomic DNA Kit (TIANGEN, China) following the guidelines provided by the manufacturer. The concentration of DNA was determined using a Biochrom WPA Biowave DNA Life Science Spectrophotometer (Biochrom, UK), and the DNA samples were subsequently stored at -20℃ until their future utilization.

2.4. Molecular identification of tick-borne bacterial pathogens

Tick-borne bacterial pathogens were detected and charaterized by PCR in Tibetan sheep, goats, yaks, cattle, donkeys, horses, Bactrian camels, and ticks from the 20 sites of Qinghai plateau, using the primers listed in Table 1. The PCR reaction was performed with a total volume of 10 μL, containing 1 μL of the DNA template, 0.5 μL each of the forward and reverse primer (100 μM), 0.2 μL of deoxyribonucleotide triphosphate (200 μM; New England BioLab, USA), 1 μL of 10×ThermoPol Reaction Buffer (New England BioLab, USA), 0.1 μL of Taq polymerase (0.5 U; New England BioLab, USA), and double-distilled water up to 10 μL [34]. A negative control was included, using double-distilled water instead of DNA template. The PCR products were visualized by UV transillumination in a 1.5% agarose gel following electrophoresis and staining with ethidium bromide.

2.5. Sequencing analyses of detected tick-borne bacterial pathogens

For the detection of tick-borne bacterial pathogens, at least one positive sample per animal species within each sampling area was selected for sequencing and subsequent molecular characterization. The PCR product obtained from the positive sample was purified using the EasyPure Quick Gel Extraction Kit (TransGen Biotech, China) and subsequently cloned into E. coli DH5É‘ via the pMDâ„¢18-T Vector Cloning Kit (Takara, Japan). At a minimum, three positive clones were sent for sequencing services provided by Genewiz (USA) company.

2.6. Sequencing analyses of detected tick-borne bacterial pathogens

The acquired sequences were validated through a BLASTn search in GenBank (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Subsequently, phylogenetic trees were constructed using the maximum likelihood statistical method and 1000 replications of bootstrap analyses through MEGA X: Molecular Evolutionary Genetics Analysis version X, designed for larger datasets [45].

2.7. Statistical analyses

The proportion of samples tested positive between different regions and different animals was compared by Epitools online (https://epitools.ausvet.com.au). The chi-squared test for contingency table was applied to the original data to quantify the prevalence of each pathogen's infection rate, along with a corresponding 95% confidence interval (CI). Observed differences were considered statistically significant when the resulting p-values were lower than 0.05.

3. Results

3.1. Morphological and molecular identification of ticks

A light microscope was utilized to facilitate the observation of tick species. The results of the morphological identification of ticks revealed the detection of two primary species within the Qinghai plateau, namely H. qinghaiensis and D. nuttalli (Figure 2). A total 23 of ticks were submitted to the Genebank. After conducting a rigorous sequence alignment analyses, our investigation has provided evidence of the presence of two distinct tick species, H. qinghaiensis and D. nuttalli, in the Qinghai plateau region, with nucleotide consistency levels ranging from 97.53% -100% (Table 2). The results of the phylogenetic analyses indicate that based on the coxI gene, the tick species obtained in this study can be mainly classified into two clades: One clade is closely related to H. qinghaiensis, while the other is closely related to D. nuttalli from China (Figure 3). In this investigation, we have observed that OQ816756 exhibits notable dissimilarity when compared to the D. nuttalli and D. silvarum clade. It is suggested that OQ816756 could potentially represent novel variants within the Dermacentor genus.

3.2. Identification of tick-borne bacterial pathogens

A. bovis, A. capra, A. marginale, A. ovis, A. phagocytophilum, B. burgdorferi s.l., C. burnetii, and Rickettsia spp. transmitted by ticks were investigated. As presented in Table 3, the prevalence of infection was found to be 16.4% (70/428) for A. bovis, 4.7% (20/428) for A. capra, 5.8% (25/428) for A. ovis, 6.3% (27/428) for B. burgdorferi s.l., 0.7% (3/428) for C. burnetii, and 0.5% (2/428) for Rickettsia spp. No cases of infections caused by A. marginale and A. phagocytophilum were detected. Additionally, no instances of tick-borne bacterial pathogen infections were found in yaks, Bactrian camels, donkeys, and horses among the livestock examined. The prevalence of tick-borne bacterial pathogens in the dominant animals showed no statistically significant difference (chi-squared test, p >0.05).

3.3. Sequencing and Comparative Analyses

The sequences obtained in the present investigation were analyzed using the the Basic Local Alignment Search Tool (BLAST) at the National Center for Biotechnology Information (NCBI). Thirteen sequences of Anaplasma bovis, two of A. ovis, eight of A. capra, two of Rickettsia spp., two of B. burgdorferi s.l., and two sequences of C. burnetii were submitted to the Genebank. The results presented in this study include 24 distinctive sequences of tick-borne bacterial pathogens (Table 4).
The investigation into tick-borne bacterial pathogens has revealed a high degree of sequence similarity among the identified species of Anaplasma, ranging from 99.27% to 100% for A. bovis, 99.71% to 100% for A. ovis, and 97.58% to 99.62% for A. capra. For the identified species of Rickettsia spp. (R. raoultii), the sequence similarity was 100%. The identified species of Borrelia (KY284014) exhibited sequence similarity ranging from 99.52% to 99.84%, and the identified species of C. burnetii (MK416231) showed a sequence similarity of 99.69%.

3.3. Phylogenetic analyses

Based on our analyses of the 16S rRNA gene, the isolates of A. bovis obtained from goats, cattle, and ticks exhibited a close genetic relationship with previously reported A. bovis isolates. However, it is noteworthy that the isolate OQ826705 displayed distinct dissimilarity compared to A. bovis, suggesting its potential classification as a novel variant within the species A. bovis (Figure 4a). Additionally, our phylogenetic analyses based on partial sequences of the citrate synthase gene (gltA) revealed interesting findings. They showed that A. capra isolates obtained from goats in our study are genetically related to A. capra isolates previously detected from ticks in China [46]. Moreover, the analyses demonstrated that A. capra isolates derived from Tibetan sheep and ticks are positioned within the same clade as Tibetan sheep isolates obtained from Qinghai, as reported by He et al. [47]. These results suggest a genetic similarity and potential association between A. capra variants found in Tibetan sheep, ticks, and the Tibetan sheep population from the Qinghai region (Figure 4b). Moreover, our phylogenetic analyses of A. ovis, based on the Major surface protein 4 (msp4) partial sequences obtained from Tibetan sheep and goats, revealed the presence of a new clade, distinct from those previously identified in a study conducted by de la Fuente et al. [48] (Figure 4c).
The 16S rRNA sequences of B. burgdorferi s.l. obtained from Tibetan sheep and goats in this study were found to cluster with homologous sequences from camels in China (Figure 5).
Furthermore, the phylogenetic analyses of Rickettsia spp. based on the gltA gene revealed that the tick isolates obtained in this study belong to the R. raoultii clade along with ticks from China and Russia (Figure 6).
Moreover, the C. burnetii based on heat shock protein B (htpB) was found to be in the same clade as those from ticks and humans in other countries (Figure 7). The detection of similar clade membership across diverse geographic locations underscores the potential for transnational transmission.

4. Discussion

The Qinghai-Tibetan plateau region is characterized by vast grasslands, towering mountains, and serene lakes, constituting a unique geographical environment. The landscape of this region is intricately intertwined with diverse livestock species that rely on the grasslands for grazing, establishing a symbiotic relationship with the natural surroundings. Moreover, ticks, known to inhabit grasslands, forests, and shrubbery, are also prevalent in this ecosystem. Ticks play a crucial role as carriers of human and animal pathogens globally. Notable examples of these tick-borne bacterial pathogens, with implications for both veterinary and public health, include Rickettsia spp., Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato (s.l.), Coxiella burnetii. These pathogens have the capacity to infect a diverse array of hosts, encompassing humans as well as wild and domestic animals [49]. While various studies have investigated the prevalence of tick-borne pathogens in different regions [50,51,52,53], limited research has been conducted specifically focusing on the Qinghai-Tibetan plateau [14,18,19]. To date, this study represents the first comprehensive investigation into the presence of tick-borne bacterial pathogens in this unique geographical region.
The investigation of tick-borne diseases may depend significantly on the distribution and diversity of tick species. Individuals bitten by Haemaphysalis ticks carrying bacterial infections face increased risk of bacterial infection [54]. Some tick species have successfully adapted to the dry and cold climate of the plateau, such as H. qinghaiensis [55], which is the predominant tick species in Qinghai province, China. Our study confirmed the presence of H. qinghaiensis and D. nuttalli, aligning with previous research findings [8]. To date, there has not been a comprehensive and organized investigation, leading to a lack of knowledge about various pathogens in Qinghai province. This study employed molecular detection methods to investigate the prevalence of these pathogens among major livestock species in Qinghai province. The primary objective of this study was to ascertain the presence of tick-borne pathogens and acquire a more profound comprehension of their biological characteristics and classification within the province. Furthermore, it facilitates research into the biological traits of potential novel tick-borne pathogens.
In the context of Anaplasma spp., this investigation has provided valuable insights into the geographical distribution of diverse tick-borne bacterial pathogens in China. Importantly, it marks the first identification of A. bovis, A. capra, and A. ovis in goats, as well as the detection of A. bovis in cattle within the Qinghai plateau. Notably, Tibetan sheep and goats displayed a high susceptibility to Anaplasma infections, with a remarkable 65.7% of goats found to be infected with A. bovis. Among the five different Anaplasma species tested, the highest infection rate was observed for A. bovis, with 16.4% of the samples testing positive. The presence of A. phagocytophilum has been previously documented in Gansu province [56], while A. marginale has been detected in both Sichuan province [57] and the Tibet Autonomous Region [58], which shares a border with the Qinghai plateau. Additionally, ticks from Qinghai have also tested positive for both A. marginale and A. phagocytophilum [46,61]. However, contrary to these findings, the present study observed the absence of these pathogens.
Based on the results of this study, only two instances of Rickettsia spp. infection were detected in the ticks. Detailed analyses of the genetic sequences unveiled 100% similarity between the positive sequences and the previously identified R. raoultii isolates originating from Russia and China. It is worth noting that earlier studies had already established the presence of R. raoultii in ticks from the Qinghai province [19,27,28], further confirming our findings.
The findings regarding to C. burnetii reveal that Tibetan sheep and ticks in Qinghai demonstrate a seropositivity rate of 0.5% (2/428). It is noteworthy that the sequencing analyses revealed a remarkable 99.69% similarity to the tick sample obtained from Tunisia (MK416231). This finding differs from the strain of C. burnetii previously detected in ticks from the Qilian area of Qinghai province [13]. Moreover, phylogenetic tree analyses further establish that these organisms belong to the same clade as those found in foreign nations, strongly suggesting the possibility of pathogen importation from other countries. The identification of C. burnetii in neighboring provinces [62,63,64] and the Qinghai plateau implies the widespread presence of this bacterium in diverse geographical regions, indicating its potential for transmission to both animals and humans across a broader geographic scope.
In this study, the presence of B. burgdorferi s.l. was detected in Tibetan sheep and goats. It is worth mentioning that while previous studies have reported various animal species being infected with B. burgdorferi s.l. [18], this study represents the first documented instance of its detection in goats within the Qinghai region.
In summary, this study represents the inaugural exploration of tick-borne pathogens in goats, horses, and donkeys within the QTPA. It offers crucial insights into the prevalence and genetic diversity of Anaplasma spp., Rickettsia spp., Coxiella spp., and Borrelia spp. throughout China. The significance of these pathogens lies in their zoonotic potential, indicating the risk of transmission from animals to humans. The intricate interplay between animal and human ecosystems underscores the imperative for robust surveillance and prevention strategies to alleviate the impact of these zoonotic tick-borne bacteria on health and productivity. This study reveals the zoonotic risk of tick-borne pathogens in the Qinghai-Tibetan plateau, emphasizing the need for public health awareness, veterinary vigilance, and conservation efforts. The findings underscore international health security concerns and advocate for a comprehensive One Health approach, urging further research and collaborative strategies. Nevertheless, it is essential to acknowledge certain limitations inherent in this study, including its focused scope, potential diagnostic biases, and the absence of long-term monitoring capabilities, which may impact its generalizability or applicability beyond specific contexts or regions. Consequently, future research initiatives should conscientiously address these constraints to foster a more comprehensive understanding of tick-borne infections, not only within the QTPA but potentially extending to broader geographical contexts as well.

Author Contributions

Conceptualization, X.X. and L.M.; methodology, Y.M.; software, Y.M. and Y.J.; validation, Y.J. and G.W.; formal analysis, Y.J.; investigation, X.L., G.W., Y.H.; resources, L.M.; data curation, X.L.; writing—original draft preparation, Y.M.; writing—review and editing, J.L., I.Z., H.L., M.L., and N.Y.; visualization, Y.M.; supervision, L.M.; project administration, X.X.; funding acquisition, X.X. and L.M. All authors have read and agreed to the published version of the manuscript.

Funding

This study received generous support from several esteemed funding sources in China, including the Transformation of Scientific and Technological Achievements of Qinghai province (Grant No. 2021-SF-146), the China Agriculture Research System (Grant No. CARS-39-16), and the National Foreign Experts Program of China (Grant No. G2022043003L). Additionally, we would like to express our sincere appreciation for the support received from Japan, including the AMED project (Grant No. JP23WM02250317), the JSPS Core-to-Core program, and a grant from the Strategic International Collaborative Research Project (JPJ008837) promoted by the Ministry of Agriculture, Forestry, and Fisheries of Japan. Their financial support has played a crucial role in facilitating the successful execution of this research endeavor.

Institutional Review Board Statement

All protocols were carried out according to the ethical guidelines approved by the Obihiro University of Agriculture and Veterinary Medicine (Permit for the animal experiment: 22-23).

Data Availability Statement

All data are disclosed in the paper.

Acknowledgments

The authors would like to express their heartfelt gratitude to all individuals who played a crucial role in the successful completion of this project. In particular, we extend our sincere appreciation to the dedicated technical staff at the Animal Husbandry and Veterinary Station for their invaluable assistance in collecting blood and tick samples from various regions, including Minhe, Ledu, Huzhu, Menyuan, Huangyuan, Datong, and other counties of Qinghai province, China. Their unwavering commitment and expertise have significantly contributed to the achievements of this study.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. A map of the Qinghai plateau, highlighting the various sampling sites and animals included. The figure was created and adjusted using map data in Excel.
Figure 1. A map of the Qinghai plateau, highlighting the various sampling sites and animals included. The figure was created and adjusted using map data in Excel.
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Figure 2. Morphological identification of ticks in QTPA. (a) H. qinghaiensis detected in QTPA; (b) D. nuttalli from QTPA in this study.
Figure 2. Morphological identification of ticks in QTPA. (a) H. qinghaiensis detected in QTPA; (b) D. nuttalli from QTPA in this study.
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Figure 3. Phylogenetic tree of ticks using Maximum Likelihood method. The numbers indicated at the nodes represent the percentage of occurrence of clades based on 1000 bootstrap replications of the data. The sequences of isolates obtained in this study, along with their corresponding accession numbers, are highlighted in red.
Figure 3. Phylogenetic tree of ticks using Maximum Likelihood method. The numbers indicated at the nodes represent the percentage of occurrence of clades based on 1000 bootstrap replications of the data. The sequences of isolates obtained in this study, along with their corresponding accession numbers, are highlighted in red.
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Figure 4. Phylogenetic trees of Anaplasma spp. constructed using Maximum Likelihood method in MEGA X, employing the Kimura 2-parameter model. (a) The phylogenetic tree of A. bovis based on 16s rRNA gene. (b) The phylogenetic tree of A. capra based on gltA genes. (c) The phylogenetic of A. ovis based on msp4 gene. The numbers assigned to the nodes represent the percentage of occurrence of clades, determined through 1000 bootstrap replications of the data. Isolates from this study, along with their corresponding accession numbers, are highlighted in red. .
Figure 4. Phylogenetic trees of Anaplasma spp. constructed using Maximum Likelihood method in MEGA X, employing the Kimura 2-parameter model. (a) The phylogenetic tree of A. bovis based on 16s rRNA gene. (b) The phylogenetic tree of A. capra based on gltA genes. (c) The phylogenetic of A. ovis based on msp4 gene. The numbers assigned to the nodes represent the percentage of occurrence of clades, determined through 1000 bootstrap replications of the data. Isolates from this study, along with their corresponding accession numbers, are highlighted in red. .
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Figure 5. The phylogenetic tree of B. burgdorferi s.l., based on 16s rRNA partial sequences obtained from Tibetan sheep and goats in this study, as well as sequences retrieved from the GenBank database, was constructed using the Maximum Likelihood method in MEGA X and Kimura 2-parameter model. The numbers assigned to the nodes indicate the percentage of occurrence of clades, determined through 1000 bootstrap replications of the data. Isolates from this study along with their corresponding accession numbers, are highlighted in red.
Figure 5. The phylogenetic tree of B. burgdorferi s.l., based on 16s rRNA partial sequences obtained from Tibetan sheep and goats in this study, as well as sequences retrieved from the GenBank database, was constructed using the Maximum Likelihood method in MEGA X and Kimura 2-parameter model. The numbers assigned to the nodes indicate the percentage of occurrence of clades, determined through 1000 bootstrap replications of the data. Isolates from this study along with their corresponding accession numbers, are highlighted in red.
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Figure 6. The phylogenetic tree of Rickettsia spp., constructed using the Maximum Likelihood method in MEGA X and employing the Tamura 3-parameter model, includes numbers assigned to the nodes representing the percentage of occurrence of clades, determined through 1000 bootstrap replications of the data. Isolates from this study, along with their corresponding accession numbers, are highlighted in red.
Figure 6. The phylogenetic tree of Rickettsia spp., constructed using the Maximum Likelihood method in MEGA X and employing the Tamura 3-parameter model, includes numbers assigned to the nodes representing the percentage of occurrence of clades, determined through 1000 bootstrap replications of the data. Isolates from this study, along with their corresponding accession numbers, are highlighted in red.
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Figure 7. The phylogenetic tree of C. burnetii constructed using the Maximum Likelihood method in MEGA X and employing the Kimura 2-parameter model, includes numbers assigned to the nodes representing the percentage of occurrence of clades, determined through 1000 bootstrap replications of the data. Isolates from this study, along with their corresponding accession numbers, are highlighted in red. .
Figure 7. The phylogenetic tree of C. burnetii constructed using the Maximum Likelihood method in MEGA X and employing the Kimura 2-parameter model, includes numbers assigned to the nodes representing the percentage of occurrence of clades, determined through 1000 bootstrap replications of the data. Isolates from this study, along with their corresponding accession numbers, are highlighted in red. .
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Table 1. The list contains the sequences of the PCR primers used in tick-borne bacterial pathogens.
Table 1. The list contains the sequences of the PCR primers used in tick-borne bacterial pathogens.
Species Target gene Method Primer sequence Annealing temperature
(℃) 		Amplicon size
(bp) 		Reference
A. bovis 16S rRNA PCR F TCCTGGCTCAGAACGAACGCTGGCGGC 55 1433 [35]
R AGTCACTGACCCAACCTTAAATGGCTG
nPCR nF CTCGTAGCTTGCTATGAGAAC 55 551 [36]
nR TCTCCCGGACTCCAGTCTG
A. capra gltA PCR F GCGATTTTAGAGTGYGGAGATTG 55 1031 [9]
R TACAATACCGGAGTAAAAGTCAA
nPCR nF GGGTTCMTGTCYACTGCTGCGTG 55 793
nR TTGGATCGTARTTCTTGTAGACC
A. marginale msp4 PCR F CTGAAGGGGGAGTAATGGG 60 344 [37]
R GGTAATAGCTGCCAGAGATTCC
A. ovis msp4 PCR F TGAAGGGAGCGGGGTCATGGG 62 347 [37]
R GAGTAATTGCAGCCAGGCACTCT
A. phagocytophilum 16S rRNA PCR F CACATGCAAGTCGAACGGATTATTC 55 932 [38]
R TTCCGTTAAGAAGGATCTAATCTCC
nPCR nF AACGGATTATTCTTTATAGCTTGCT 55 546/565
nR GGCAGTATTAAAAGCAGCTCCAGG
B. burgdorferi s.l. 23S rRNA PCR F GCGAACGGGTGAGTAACG 50 1360 [39]
R CCTCCCTTACGGGTTAGAA
16S rRNA PCR F GAGGCGAAGGCGAACTTCTG 60.2 622 [40]
R CTAGCGATTCCAACTTCATGAAG
C. burnetii htpB PCR F GCGGGTGATGGTACCACAACA 57 501 [41]
R GGCAATCACCAATAAGGGCCG
nPCR nF TTGCTGGAATGAACCCCA 52 325
nR TCAAGCTCCGCACTCATG
Rickettsia spp. ompB PCR F AAACAATAATCAAGGTACTGT 55 212/209 [42]
R TACTTCCGGTTACAGCAAAGT
ompA nPCR nF GCTTTATTCACCACCTCAAC 811 [43]
nR TR(g/a)ATCACCACCGTAAGTAAAT
gltA nPCR nF GCAAGTATCGGTGAGGATGTAAT 401 [44]
nR GCTTCCTTAAAATTCAATAAATCAGGAT
Table 2. Accession numbers assigned to the isolates of ticks in Qinghai plateau of QTPA.
Table 2. Accession numbers assigned to the isolates of ticks in Qinghai plateau of QTPA.
Tick species Target gene GenBank accession number Length (bp) Identity (%) Accession number (host, country)
D. nuttalli coxI OQ816756 849 97.76% OM368307 cattle China
H. qinghaiensis coxI OQ816757 849 97.76% NC_062067 yak China
D. nuttalli coxI OQ816758 849 100% MK028675 sheep China
H. qinghaiensis coxI OQ816759 849 99.18% NC_062067 yak China
D. nuttalli coxI OQ816760 849 100% MK028675 sheep China
D. nuttalli coxI OQ816761 849 100% MK028675 sheep China
D. nuttalli coxI OQ816762 849 100% MK028675 sheep China
D. nuttalli coxI OQ816763 849 100% MK028675 sheep China
D. nuttalli coxI OQ816764 849 99.88% MK028675 sheep China
D. nuttalli coxI OQ816765 849 100% MK028675 sheep China
D. nuttalli coxI OQ816766 849 100% MK028675 sheep China
D. nuttalli coxI OQ816767 849 100% MK028675 sheep China
D. nuttalli coxI OQ816768 849 100% MK028675 sheep China
D. nuttalli coxI OQ816769 849 100% MK028675 sheep China
D. nuttalli coxI OQ816770 849 100% MK028675 sheep China
D. nuttalli coxI OQ816771 849 99.88% MK028675 sheep China
D. nuttalli coxI OQ816772 849 100% MK028675 sheep China
H. qinghaiensis coxI OQ816773 849 98.12% OK094412 goat China
D. nuttalli coxI OQ816774 849 98.59% OM368307 cattle China
H. qinghaiensis coxI OQ816775 849 98.23% OK094412 goat China
H. qinghaiensis coxI OQ816776 849 98.12% OK094412 goat China
H. qinghaiensis coxI OQ816777 849 98.82% OK094412 goat China
D. nuttalli coxI OQ816778 849 97.53% OM368307 cattle China
Table 3. The prevalence of tick-borne bacterial pathogens in livestock from Qinghai plateau.
Table 3. The prevalence of tick-borne bacterial pathogens in livestock from Qinghai plateau.
TBPs Livestocks Sheep goat Cattle Yak Camel Donkey Horse Tick p-value
n 52 67 49 43 50 40 65 62
A. bovis No. of positives (%) n. d.1 44 (66) 1 (2) n. d. n. d. n. d. n. d. 3 (5) 0.2931
A. capra No. of positives (%) 8 (15) 10 (15) n. d. n. d. n. d. n. d. n. d. 2 (3) 0.2931
A. marginale No. of positives (%) n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. -
A. ovis No. of positives (%) 24 (46) 1 (2) n. d. n. d. n. d. n. d. n. d. n. d. 0.3134
A. phagocytophilum No. of positives (%) n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. -
B. burgdorferi s.l. No. of positives (%) 14 (27) 13 (19) n. d. n. d. n. d. n. d. n. d. n. d. 0.3134
C. burnetii No. of positives (%) 2 (4) n. d. n. d. n. d. n. d. n. d. n. d. 1 (2) 0.3134
Rickettsia spp. No. of positives (%) n. d. n. d. n. d. n. d. n. d. n. d. n. d. 2 (3) 0.3326
1 n.d. = not detected.
Table 4. Accession numbers for sequences of tick-borne bacterial pathogens deposited in GenBank.
Table 4. Accession numbers for sequences of tick-borne bacterial pathogens deposited in GenBank.
Obtained sequences The closest BLASTn match
Pathogen Animal Target gene GenBank accession number Length (bp) Identity (%) Pathogen isolate Accession number (host, country)
A. bovis cattle 16s rRNA OQ826693 551 99.64% Uncultured Anaplasma sp. KM186933 dzeren China
goat 16s rRNA OQ826694 551 99.82% Uncultured Anaplasma sp. KM186933 dzeren China
goat 16s rRNA OQ826695 551 99.64% Uncultured Anaplasma sp. KM186933 dzeren China
goat 16s rRNA OQ826696 548 100% A. bovis MN213735 giraffe Pakistan
goat 16s rRNA OQ826697 551 99.82% Uncultured Anaplasma sp. MZ069112 yak China
goat 16s rRNA OQ826698 551 99.82% Uncultured Anaplasma sp. KM186933 dzeren China
goat 16s rRNA OQ826699 551 99.82% Uncultured Anaplasma sp. KM186933 dzeren China
goat 16s rRNA OQ826700 551 100% A. bovis MN213735 giraffe Pakistan
goat 16s rRNA OQ826701 551 100% A. bovis MN213735 giraffe Pakistan
goat 16s rRNA OQ826702 551 99.82% Uncultured Anaplasma sp. MZ069112 yak China
tick 16s rRNA OQ826703 551 100% Uncultured Anaplasma sp. MZ069112 yak China
tick 16s rRNA OQ826704 551 99.27% Uncultured Anaplasma sp. KM186933 dzeren China
tick 16s rRNA OQ826705 551 99.46% A. bovis MN213735 giraffe Pakistan
A. ovis goat msp4 OQ847093 347 100% A. ovis MN394791 Tibetan sheep China
Tibetan sheep msp4 OQ847094 347 99.71% A. ovis MN394791 Tibetan sheep China
A. capra tick gltA OQ847092 793 99.37% A. capra MZ130266 Tibetan sheep China
goat gltA OQ847085 793 98.63% A. capra MW930535 goat France
goat gltA OQ847086 793 98.49% A. capra MW930535 goat France
Tibetan sheep gltA OQ847088 793 99.75% A. capra MZ130266 Tibetan sheep China
Tibetan sheep gltA OQ847089 793 99.62% A. capra MZ130266 Tibetan sheep China
Tibetan sheep gltA OQ847090 793 99.62% A. capra MZ130264 Tibetan sheep China
Tibetan sheep gltA OQ847091 793 97.86% A. capra MZ130266 Tibetan sheep China
Rickettsia spp. tick gltA OQ872771 400 100% R. raoultii MK304547 tick Russia
tick gltA OQ872772 400 100% R. raoultii MT178338 tick China
Borrelia spp. goat 16s rRNA OQ875206 623 99.52% Borrelia sp. KY284014 camel China
Tibetan sheep 16s rRNA OQ875207 623 99.84% Borrelia sp. KY284014 camel China
C. burnetii tick htpB OQ872773 325 99.69% C. burnetii MK416231 tick Tunisia
Tibetan sheep htpB OQ872774 325 99.69% C. burnetii MK416231 tick Tunisia
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