Molecular Detection of Tick-Borne Bacterial and Protozoan Pathogens in Haemaphysalis longicornis (Acari: Ixodidae) Ticks from Free-Ranging Domestic Sheep in Hebei Province, China

Ticks and tick-borne pathogens significantly threaten human and animal health worldwide. Haemaphysalis longicornis is one of the dominant tick species in East Asia, including China. In the present study, 646 Ha. longicornis ticks were collected from free-ranging domestic sheep in the southern region of Hebei Province, China. Tick-borne pathogens of zoonotic and veterinary importance (i.e., Rickettsia, Anaplasma, Ehrlichia, Borrelia, Theileria, and Hepatozoon spp.) were detected in the ticks using PCR assays and sequence analysis. The prevalence rates of these pathogens were 5.1% (33/646), 15.9% (103/646), 1.2% (8/646), 17.0% (110/646), 0.15% (1/646), and 0.15% (1/646), respectively. For Rickettsia spp., R. japonica (n = 13), R. raoultii (n = 6), and Candidatus R. jingxinensis (n = 14) were detected for the first time in the province, while several Anaplasma spp. were also detected in the ticks, including A. bovis (n = 52), A. ovis (n = 31), A. phagocytophilum (n = 10), and A. capra (n = 10). A putative novel Ehrlichia spp. was also found with a prevalence of 1.2% in the area. The present study provides important data for effectively controlling ticks and tick-borne diseases in the Hebei Province region of China.


Introduction
Ticks are small, blood-sucking arachnids that are found throughout the world [1]. They feed on the blood of mammals, birds, reptiles, and other hosts, and in the process, can transmit various pathogens that cause disease in humans and animals [2][3][4]. There are approximately 124 species of tick found in China. Among them, Ha. longicornis is distributed in the northeastern, central, southern, and western regions of China [5]. Ticks are considered to be one of the most competent vectors, as they can transmit at least 15 bacterial, parasitic, and viral pathogens of zoonotic and veterinary importance [6,7]. They have become a significant public health concern due to the number of diseases they transmit, including Lyme disease, spotted fever, ehrlichiosis, and anaplasmosis [8].
Ticks are the main vector for numerous protozoan pathogens belonging to the phylum Apicomplexa, including Babesia, Theileria, and Hepatozoon [26]. They can infect a variety of animal hosts, including mammals and birds [27]. The parasites are transmitted through the bite of infected arthropods (especially ticks), causing significant illness in hosts. Ha. longicornis ticks have been implicated in the transmission of Theileria, Hepatozoon, and Babesia [28,29]. Though Theileria and Hepatozoon agents have not been associated with human infection in China, they inflict damage to animal husbandry production and wildlife. Babesia agents are significant emerging threats to animal and human health. Ha. longicornis ticks can act as vectors of several Babesia species, including human babesiosis agents, such as Ba. microti and Ba. divergens [30].
Hebei Province is located in northern China and is adjacent to Beijing and Tianjin cities. The province presents varied landscapes, including rolling hills, forests, and plains. There are several tick-borne pathogen surveillance projects in the province, mainly concentrated in the northern regions. Based on the surveillance project for tick and tick-borne pathogens of the National Institute for Communicable Disease Control and Prevention (ICDC), we found that Ha. longicornis is the dominant tick species in the province, especially the ticks that are parasitic on sheep. The present study aimed to investigate the prevalence and genetic diversity of the bacterial and protozoan pathogens in Ha. longicornis ticks on free-range sheep in Shijiazhuang City, Hebei Province, China.

Study Area and Tick Sampling Protocol
This study was conducted in Shijiazhuang City in the south of Hebei Province to the east of Taihang Mountains and presents the stepped landform features, including mountains, hills, plains, and wetlands. We sampled ticks from the ears, periocular, axillary, and neck of free-ranging sheep ( Figure 1) in Pingshan, Luquan, Jingxing, Jingxing Mining District, Yuanshi, Lingshou, Xingtang, and Zanhuang counties in the spring and autumn of 2022. Tick species were first identified morphologically using taxonomic keys and then confirmed by nested PCR amplification and sequencing of the CO1 genes (Table S1) [31,32]. locytic anaplasmosis (HGA), and human monocytic ehrlichiosis (HME), respectively. In addition, A. bovis, A. capra, A. ovis, E. ewingii, and E. muris have been reported to cause human infection [21][22][23][24]. Haemaphysalis spp. are also an important part of the natural transmission cycle of B. burgdorferi, a causative agent of Lyme disease [25].
Ticks are the main vector for numerous protozoan pathogens belonging to the phylum Apicomplexa, including Babesia, Theileria, and Hepatozoon [26]. They can infect a variety of animal hosts, including mammals and birds [27]. The parasites are transmitted through the bite of infected arthropods (especially ticks), causing significant illness in hosts. Ha. longicornis ticks have been implicated in the transmission of Theileria, Hepatozoon, and Babesia [28,29]. Though Theileria and Hepatozoon agents have not been associated with human infection in China, they inflict damage to animal husbandry production and wildlife. Babesia agents are significant emerging threats to animal and human health. Ha. longicornis ticks can act as vectors of several Babesia species, including human babesiosis agents, such as Ba. microti and Ba. divergens [30].
Hebei Province is located in northern China and is adjacent to Beijing and Tianjin cities. The province presents varied landscapes, including rolling hills, forests, and plains. There are several tick-borne pathogen surveillance projects in the province, mainly concentrated in the northern regions. Based on the surveillance project for tick and tick-borne pathogens of the National Institute for Communicable Disease Control and Prevention (ICDC), we found that Ha. longicornis is the dominant tick species in the province, especially the ticks that are parasitic on sheep. The present study aimed to investigate the prevalence and genetic diversity of the bacterial and protozoan pathogens in Ha. longicornis ticks on free-range sheep in Shijiazhuang City, Hebei Province, China.

Study Area and Tick Sampling Protocol
This study was conducted in Shijiazhuang City in the south of Hebei Province to the east of Taihang Mountains and presents the stepped landform features, including mountains, hills, plains, and wetlands. We sampled ticks from the ears, periocular, axillary, and neck of free-ranging sheep ( Figure 1) in Pingshan, Luquan, Jingxing, Jingxing Mining District, Yuanshi, Lingshou, Xingtang, and Zanhuang counties in the spring and autumn of 2022. Tick species were first identified morphologically using taxonomic keys and then confirmed by nested PCR amplification and sequencing of the CO1 genes (Table S1) [31,32].

DNA Extraction
All the ticks were washed with bromogeramine (5%), alcohol, and water, respectively, for 15 min each. After air-drying, the ticks were individually homogenized, and then the DNA was extracted following the protocol of the QIAamp DNA Mini Kit (Qiagen, Germany).

Detection of Bacteria and Parasites in Ticks
Bacterial pathogens, including Rickettsia spp., Anaplasma spp., Ehrlichia spp., Bartonella spp., Borrelia spp., C. burnetii, and F. tularensis, were screened using real-time PCR (qPCR) with the corresponding primers described in Table S1. The tick DNA samples positively detected in the rickettsia-specific qPCR test (CT value < 38) were further tested using nested PCR targeting an 1100 bp region of the gltA gene, a 440 bp region of the 17 kD gene, a 530 bp sequence of the ompA gene, and a 1200 bp region of the rrs gene of SFGR. The tick DNA samples testing positive for Anaplasmataceae in qPCR (CT value of <38) were confirmed and preliminarily typed using nested PCR targeting a 500 bp region of the rrs gene that could amplify both Anaplasma spp. and Ehrlichia spp. A set of genus-specific or species-specific primers of Anaplasma and Ehrlichia targeting the rrs, gltA, and groEL genes (heat shock protein) were used for the identification of bacterial species and phylogenetic analysis. For the putative novel Ehrlichia strains, sequences of ftsZ (cell division protein gene), conP28 (P28 major membrane protein gene), and dsb (disulfide oxidoreductase) genes were also obtained with amplification and sequencing. Borrelia-positive samples were reevaluated by nested PCR targeting the 350 bp ospA (outer surface protein A) gene.
For the detection and characterization of tick-borne protozoan pathogens, nested PCR was performed using a universal primer set targeting the 18S rRNA gene of Babesia-Theileria-Hepatozoon [33].
The target amplicons were isolated with the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and then sent to Beijing De'aoping Biotechnology Co., Ltd. (Beijing, China), for sequencing. The PCR primers are shown in Table S1. The agarose gel electrophoresis images of representative isolates in each pathogen are provided in Supplementary Data.

Phylogenetic Data Analysis
For the rrs genes of Rickettsia, Anaplasma spp., and Ehrlichia spp., two overlapping fragments were first edited and assembled using SeqMan software (DNASTAR, Madison, WI, USA) to obtain the almost complete gene sequences. Qualified and trimmed sequences were identified by comparison with the sequences available in GenBank with the Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov/Blast.cgi accessed on 28 March 2023). Phylogenetic and molecular evolutionary analysis was performed using the neighbor-joining method with 1000 replicates for bootstrap analysis in MEGA 7.0 (https://www.megasoftware.net accessed on 28 March 2023).

Tick Sampling and Identification
A total of 646 adults ticks (29 fully engorged ticks, 131 partially engorged, and 486 unfed ticks) were collected from free-ranging sheep in the Shijiazhuang City in the south of Hebei Province ( Figure 1). All collected ticks were identified as Ha. longicornis based on morphology. CO1 gene sequences of the collected ticks were acquired by amplifying and sequencing (Table S1) those sharing 99-100% identity with the sequences of Ha. longicornis from GenBank (MK450606).
Co-infection with two or three tick-borne pathogens within an individual tick was detected in 42 (6.5%) of the ticks tested. Two (0.3%) ticks were co-infected with Anaplasma spp. and Rickettsia spp., seven (1.1%) ticks were co-infected with Rickettsia spp. and Theileria spp.), twenty-seven (4.2%) ticks were co-infected with Anaplasma spp. and Theileria spp., and only one tick was co-infectedwith Ehrlichia spp. and Hepatozoon spp.

Phylogenic Analysis of Different Tick-Borne Pathogens
Rickettsia: Phylogenetic analysis based on the rrs, gltA, ompA, and 17 kD genes showed that three Rickettsia spp. identified in the ticks clustered together with R. japonica, R. raoultii, and Ca. R. jingxinensis ( Figure 2). The sequences of rrs, gltA, ompA, and 17 kD genes for R. japonica and rrs, gltA, and ompA for R. raoultii were identical to R. japonica (CP047359) and R. raoultii (CP019435). The 17kD gene of the obtained R. raoultii strain was 99.42% similar to R. raoultii (CP019435). For the samples HBSJZJX40, HBSJZJK16, and HBSJZPS105, which clustered closely with Ca. R. jingxinensis, the rrs, ompA, and gltA gene sequences obtained from the ticks showed 100% identity, and the 17 kD gene sequence showed 99.28-99.76% identity to Ca. R. jingxinensis (MH932038) or Ca. R. longicornii (KY617773).  Anaplasma: Four Anaplasma species (A. bovis, A. ovis, A. phagocytophilum, and A. capra) were identified in the ticks. A. bovis detected in the current study were classified into three genotypes in the phylogenetic tree based on the rrs, gltA, and groEL genes. The sequences of the three genes shared 99.77-100% identity with A. bovis strains from other provinces of China. All rrs gene sequences of A. ovis obtained in this study were identical to each other, and the isolates were closely related to A. ovis isolates from goats (MG869525) and sheep (KX579073) in China. Sequences of the gltA and groEL genes for the three A. ovis isolates showed 99.97% and 99.51 to 100% intersequence identities, since they are still closely related to A. ovis strains in Shannxi Province (gltA: MG869310 and MG869296; groEL: MG869402 and OM648130). The partial rrs gene sequences of A. phagocytophilum identified in sheep were 99.83 to 99.91% identical to the isolates derived from Ha. longicornis (KF569915) and goat (KR002115). The gltA and groEL sequences were closely related to A. phagocytophilum strains reported in China (KP076361 and KX388358), with similarities of 99.12% and 99.28%. For the two isolates of A. capra, the rrs, gltA, and groEL sequences had 99.91 to 99.93%, 100%, and 99.86% identity with those of reported A. capra strains (MH762076, KX987362, and MG869399), respectively (Figure 3).   Ehrlichia: PCR detection indicated that eight ticks were infected with Ehrlichia spp. The sequences of nearly complete rrs genes showed 100% identity to E. sp. NS101 (AB454074) or E. chaffeensis isolate X1 (KX505292) and 99.60% identity to other strains of E. chaffeensis (query cover: 100%; E-value: 0.0). However, the partial gltA and groEL sequences of the Ehrlichia spp. share 86.17 to 86.28% and 94.33% identity with the E. chaffeensis strains (Query cover: 100%, E-value: 0.0). The obtained groEL sequences were 98.26% similar to E. sp. NS101, but the gltA gene records were absent for the NS101 strain. Although these strains were closest to the Candidatus Ehrlichia zunyiensis found in Guizhou Province in the phylogenetic trees of the gltA and groEL genes, the identity similarity was only 97.33% and 96.47% for the two single genes. The sequences of partial ftsZ, conP28, and dsb genes of the Ehrlichia spp. were also obtained and deposited in Genbank. The dsb and ftsZ genes of the Ehrlichia strains detected in the present study had 85 to 85.33% (query cover: 100%; E-value: 0.0) and 88.31 to 88.57% (query cover: 99%; E-value: 0.0) identity to E. chaffeensis (Figure 4). We therefore propose that they represent a novel species, and we name it "Candidatus Ehrlichia luquansis" according to the site where they were detected.

Discussion
Ha. longicornis, also named the Asian longhorned tick, is a human-biting Ixodid species native to East Asia, especially in eastern China, Japan, and Korea. Howev distribution regions of the ticks have expanded to Australia, New Zealand, and th [7,34]. In China, Ha. longicornis is the most prevalent tick species, distributed thro least 17 provinces [31], and it is regarded as an important vector of infectious di Protozoan: Two sequences of the 18S rRNA gene exhibiting 99.98% intersequence identities were identified as belonging to Theileria strains in the ticks. As shown in Figure 6, the 18S rRNA gene sequences of the Theileria strains detected in this study showed 99.3% to 100% identity to T. luwenshuni (MH208630) that was detected in the Rh. microplus ticks in China and had 99.79 to 99.86% identity to T. luwenshuni (JX469515) from small ruminants in China. Sequencing and BLAST analysis revealed that a sequence of the 18S rRNA gene was highly similar to the gene of H. felis isolated from wildcats (Felis silvestris) in Hungary (OM256568 and OM256569) and from an Asiatic lion in India (ON075470 and KX017290) with 98.68% and 99.85% identities, respectively.

Discussion
Ha. longicornis, also named the Asian longhorned tick, is a human-biting Ixodidae tick species native to East Asia, especially in eastern China, Japan, and Korea. However, the distribution regions of the ticks have expanded to Australia, New Zealand, and the USA

Discussion
Ha. longicornis, also named the Asian longhorned tick, is a human-biting Ixodidae tick species native to East Asia, especially in eastern China, Japan, and Korea. However, the distribution regions of the ticks have expanded to Australia, New Zealand, and the USA [7,34]. In China, Ha. longicornis is the most prevalent tick species, distributed through at least 17 provinces [31], and it is regarded as an important vector of infectious diseases threatening human and animal health, due to its broad host range, diverse vegetation habitats, and multiple pathogens associated with a wide spectrum of human and animal diseases [35]. In this study, SFG Rickettsia (R. japonica, R. raoultii, and Ca. R. jingxinensis), Anaplasma spp. (A. phagocytophilum, A. bovis, A. ovis, and A. capra), Ehrlichia spp., B. burgdorferi, T. luwenshunni, and H. felis were identified in Ha. longicornis ticks which were collected from free-ranging sheep in eight counties along the eastern side of the Taihang Mountains in southern Hebei Province.
Ha. Longicornis were reported as vectors of several Rickettsia spp. (R. raoultii, R. japonica, R. heilongjiangensis, Ca. R. tarasevichiae, Ca. R. jingxinensis, Ca. R. jiaonani, and Ca. R. hebeiii) in China [6]. In the present work, R. japonica, R. raoultii and Ca. R. jingxinensis were first detected in the ticks of Hebei Province in the present surveillance. R. japonica was the causative agent of Japanese spotted fever (JSF). The pathogen was first described in Japan and human cases were found in Japan, South Korea, the Philippines, Thailand, and China [15,36,37]. In recent years, human JSF cases have been found in Zhejiang, Anhui, Hubei, and Henan provinces of China [38][39][40], and two fatal cases occurred in Hubei province [15]. R. japonica has been detected in ticks from many provinces in northern, eastern, central, and northeastern China [6,41]. R. raoultii, a causative agent of tick-borne lymphadenopathy in humans was first described in the D. nuttalli ticks in Siberia and the Rhipicephalus pumilio ticks in the Astrakhan region in 1999 [42]. Human infections with R. raoultii were first confirmed in Spain and have since been reported in several provinces of China [43,44]. Although Dermacentor ticks were considered to be the dominant vectors of R. raoultii, it was also detected in other ticks, including Ha. erinaceid, Ha. concinna, Ha. qinghaiensis, and Ha. longicornis [45,46]. Ca. R. jingxinensis is a novel Rickettsia species with potential pathogenicity that has been reported to be widespread in China and co-circulates in various ticks [47]. To date, no human cases of SFGR infection have been reported in Hebei Province, but the presence of R. raoultii and R. japonica suggests a risk of Rickettsial infection in local residents. A putative novel Rickettsia spp., named Ca. R. hebeiii was previously reported in ticks with a minimum prevalence of 0.7% in the ticks in the province, but it was not detected in the present study [48]. Ticks and the pathogens that they carry can exhibit temporal variations, with changes in their distribution and prevalence occurring over time.
A high diversity of Anaplasma spp. were found in the Ha. longicornis ticks in this study. The presence of A. phagocytophilum, A. bovis, A. ovis, and A. capra was detected in the ticks, and the infection rate of A. bovis (8.0%) and A. ovis (4.8%) were the highest, suggesting that A. bovis and A. ovis were the dominant species of the genus Anaplasma, which is prevalent in Ha. longicornis ticks in Hebei Province. Interestingly, most A. ovis strains were detected in ticks collected in Pingshan county, indicating the potential differences in the geographical distribution of A. ovis, as it was not detected in the ticks from the adjacent counties, even though the adjacent regions have similar ecological environments. A. bovis was initially found as a pathogen of cattle but has been reported to be present across a broad host range [21]. Three genotypes of A. bovis were found in the present study, demonstrating its diversity in the ticks of Hebei province. A. ovis is widely distributed in North America, Asia, Africa, and Europe [49]. Sheep and goats are the main hosts, and livestock infection can lead to the loss of the local pasturage economy. A. capra, a zoonotic pathogen, was also detected in the present study, with a minimum infection rate of 1.5%. In addition, A. bovis and A. ovis can also cause human infection. Though the prevalence of A. phagocytophilum was 1.5% in the ticks, lower than that of others, it is a well-known tick-borne pathogen causing HGA. HGA cases and a high prevalence of antibodies to A. phagocytophilum in local residents were found in different regions of China [50][51][52]. Attention should be paid to the risks of HGA for local residents in Hebei Province.
A putative novel species of the genus Ehrlichia was detected in the ticks in the present study. The highest degree of identities of rrs, gltA, groEL, dsb, and ftsZ amplified from the novel species in the ticks were 100%, 96.47%, 98.26%, 85.33%, and 88.57%, respectively, compared with those from known Ehrlichia species. The species was close to Ca. E. zunyiensis which was detected in Berylmys bowersi in Guizhou Province, China, and Ehrlichia sp. NS101, which was identified in deer in Japan. Similarly, the rrs genes of these species were most closely related to that of E. chaffeensis, but other test genes were not. There may be a cluster of Ehrlichia spp. with similar rrs genes, but which are diverse in genomes widely distributed in East Asia. This result suggests that multiple genes should be analyzed in the genotyping of the Ehrlichia species. The potential pathogenicity of the Ehrlichia species needs to be further studied.
B. burgdorferi was also detected in the Ha. longicornis ticks with a low prevalence (0.2%) in the present study. Ixodes persulcatus and Ha. japonica ticks are recognized as the primary vector of B. burgdorferi in northern China [25]. In a previous study, 17.14% of I. persulcatus and 10% of Ha. japonica ticks were positively detected in PCR targeting the B. burgdorferi gene, but all of the Ha. longicornis ticks were negative [53]. However, B. burgdorferi strains were isolated from Ha. longicornis ticks in Beijing, which is surrounded by Hebei [54]. Our results indicated that the Ha. longicornis ticks in the investigated sites can serve as a vector of B. burgdorferi.
T. luwenshuni and H. felis were detected in the Ha. longicornis ticks using PCR targeting Babesia, Theileria, and Hepatozoon 18sRNA, with a 17.9% and 0.15% prevalence, respectively. T. luwenshuni can be transmitted by Ha. qinghaiensis and Ha. longicornis ticks, which are mainly reported in northwestern regions of China. Our results agree and suggest that Ha. longicornis acts as a vector of T. luwenshuni. The pathogen can cause theileriosis that affects domestic and wild ruminants, including sheep, goats, cattle, and deer. T. luwenshuni is transmitted to animals through the bite of infected ticks, causing a range of symptoms, including fever, anemia, and weight loss in livestock, especially goats and sheep, and even causing death in serious cases. An Ha. longicornis tick was shown to be positive for H. felis, a pathogen to felids. It can infect hosts via the bite of ticks or infected prey. Our study provides evidence that Ha. longicornis may be a biological vector of H. felis in Hebei Province and poses threats to wild felids and domestic cats with field contact.
The present study does have limitations. The PCR-positive detection of pathogens in the collected parasitic ticks from sheep cannot differentiate whether the pathogens' DNA templates were from infected ticks or sheep blood that was degraded in tick guts. The investigation of tick-borne pathogens in local sheep and free ticks should be carried out in further studies.

Conclusions
In summary, we identified numerous bacterial and protozoan pathogens in Ha. longicornis ticks from free-ranging domestic sheep in Hebei Province. R. japonica and R. raoultti, the agents of spotted fever, were first detected in the province. A high diversity of pathogens belonging to Anapasmatacae, including a putative novel Candidatus Ehrlichia spp., were found harboring in Ha. longicornis ticks. In addition, it was determined that protozoan pathogens that can infect wild and domestic animals were found with a high prevalence. The results indicate that tick-borne diseases are a threat to public health and animal husbandry in the region. Due to the constantly changing climate, environment, and human activities affecting the prevalence of ticks and their vector pathogens, surveillance of tick-borne pathogens is required for developing new control strategies.
Author Contributions: Conceptualization, T.Q., B.X. and Z.T.; methodology, Z.T., N.Z. and Y.S.; software, N.Z.; validation, Z.T. and Y.S.; formal analysis, N.Z. and X.J.; investigation, N.Z., X.Z. and J.H.; resources, Y.S.; data curation, N.Z.; writing-original draft preparation, Z.T.; writing-review and editing, Z.T., N.Z., Y.S., B.X. and T.Q.; visualization, N.Z.; supervision, T.Q. and B.X.; project administration, T.Q. and B.X.; funding acquisition, T.Q. and Z.T. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki. Experimental protocols were approved by the Ethical Review Committee of the ICDC and the Chinese Center for Disease Control and Prevention (China CDC).

Informed Consent Statement: Not applicable.
Data Availability Statement: All relevant data are within the paper and its supporting information files.

Conflicts of Interest:
The authors declare no conflict of interest.