Co-infection of tick-borne bacterial pathogens in ticks in Inner Mongolia, China

Tick-borne infectious diseases pose a serious health threat in certain regions of the world. Emerging infectious diseases caused by novel tick-borne pathogens have been reported that are causing particular concern. Several tick-borne diseases often coexist in the same foci, and a single vector tick can transmit two or more pathogens at the same time, which greatly increases the probability of co-infection in host animals and humans and can lead to an epidemic of tick-borne disease. The lack of epidemiological data and information on the specific clinical symptoms related to co-infection with tick-borne pathogens means that it is not currently possible to accurately and rapidly distinguish between a single pathogen infection and co-infection with multiple pathogens, which can have serious consequences. Inner Mongolia in the north of China is endemic for tick-borne infectious diseases, especially in the eastern forest region. Previous studies have found that more than 10% of co-infections were in host-seeking ticks. However, the lack of data on the specific types of co-infection with pathogens makes clinical treatment difficult. In our study, we present data on the co-infection types and the differences in co-infection among different ecological regions through genetic analysis of tick samples collected throughout Inner Mongolia. Our findings may aid clinicians in the diagnosis of concomitant tick-borne infectious diseases.

Introduction Tick-borne pathogens are transmitted via hematophagous blood-sucking ticks to hosts (including humans), in which they may cause infectious disease. In some cases, ticks harbor multiple pathogens, which can result in co-infection. The two forms of co-infection are interspecific infection and intraspecific infection with different genospecies, and regional differences between these two types of infection have been reported [1][2][3]. Distinct environmental conditions provide the habitat for specific tick species and several tick-borne diseases often coexist in the same foci, which defines their geographical distribution and, consequently, the areas of risk for human tick-borne infections [4][5][6]. In these areas, the probability of co-infection of host animals and humans is greatly increased, leading to an epidemic of tick-borne disease.
During the 20th century, Mitchell and colleagues proposed serological evidence of co-infection with Borrelia burgdorferi, Babesia, and human granulocytic Ehrlichia species in residents of Wisconsin and Minnesota in the USA [7]. In a 4-year prospective study conducted in Germany and Latvia, 75 of 192 patients (39%) were co-infected with tick-borne pathogens, and 61 of the 75 patients were co-infected with B. burgdorferi and Babesia, with a co-infection rate of 81% in Ixodes ricinus ticks [8]. Dibernardo and colleagues reported co-infection of B. burgdorferi and Anaplasma phagocytophilum in Ixodes scapularis ticks collected in Canada [9]. These studies suggested that co-infection of B. burgdorferi with Babesia is common in both patients and tick samples. Lu and coworkers also found Candidatus R. tarasevichiae infection in patients with severe fever that were also infected with thrombocytopenia syndrome virus, with a co-infection rate of 9.4% (77/823) in China [10]. The results of laboratory examination and clinical manifestations suggested that the co-infection group included more severe cases than the single infection group. Furthermore, the course of disease was longer, the recovery of laboratory indicators was slower, and fatalities were reported among the co-infection group [10,11].
Located on the border of China and Russia, the Greater Hinggan Mountains in the eastern part of Inner Mongolia are rich in wildlife and have a diverse ecosystem. This region is one of the major epidemic areas of tick-borne infectious diseases in China because its unique geographical and ecological features make it an ideal habitat for ticks [3,[12][13][14]. In Inner Mongolia, and elsewhere in China and the rest of the world, limited research has been carried out on the occurrence of different genospecies in co-infections in host-questing ticks, despite progress on tick-borne infections. Indeed, most studies have focused on the identification of diversity in a single or a few pathogens, or on the prevalence of pathogen species [15,16]. Our present study aimed to detect the co-infection rates and co-infection diversity of tick-borne pathogens in questing ticks collected from three different ecological sites in Inner Mongolia.

Ethics statement
The collection of ticks from the body surface of cattle, goats, and horses in this study was verbally approved by the animal owners and performed in strict accordance with the National Guidelines for Experimental Animal Welfare of China . In addition, it has been applied and reviewed by the Animal Experimental Ethics Committee of the Medical Department of Hetao College, and it has been put on record in accordance with the "Animal Experimental Ethics Review Measures of Hetao College" ([2022] No. 112).

Study area and tick sampling sites
Inner Mongolia Autonomous Region is located in the northern frontier of China (97˚E-126˚E; 37˚N-53˚N), bordering Mongolia and Russia 4200 km to the north. The natural grassland in Inner Mongolia is vast and broad, with the total area ranking highest among the five grasslands in China, and it is an important region for livestock production. The topography of Inner Mongolia comprises mainly the Mongolian plateau, which is complex and diverse in form, with an average elevation of about 1000 m and a temperature that changes greatly between winter and summer. The sampling sites for this study were distributed across 103 counties (banners) of 12 league sites, covering three ecologically and geographically distinct areas of Inner Mongolia (Fig 1).
From March to September of each year from 2015 to 2019, ticks were collected from vegetation in the forest by the lab-cloth flagging method from area 1, and exoparasitic ticks were collected from cattle, goat, sheep, camel and horse by the animal physical examination method in area 2 and 3. Each area represented a distinct habitat. In sampling area 1, a forested area in the north-eastern part of Inner Mongolia, the main habitat was primeval forest at an altitude of 250-1745 m, with an average annual temperature of ˗3.5˚C, and annual precipitation of 300-450 mm. In sampling area 2, a grassland area in central Inner Mongolia, the habitat is considered a frigid temperate zone of semi damp grassland with monsoonal conditions, at an altitude of 800-1200 m, and with annual precipitation of 150-400 mm. In sampling area 3, an area encompassing the Gobi Desert and the semi-desertification steppe of the western part of Inner Mongolia, the land is arid, at an altitude of 800-1500 m, and with annual precipitation of 40-240 mm (Fig 1).
Land cover data of Inner Mongolia were obtained free from the National Earth System Science Data Sharing Infrastructure (http://www.geodata.cn). ArcGIS 10.2 software was used for visualization.

Tick species identification, DNA extraction, and detection
Ticks were identified by morphological characteristics combined with tick mt-rrs gene identification method [17]. The ticks were soaked with sodium hypophosphite, 75% ethanol, and iodophor for 5 min, then washed with sterile water, dried naturally, and DNA was extracted using a genomic extraction kit (Qiagen, Hilden, Germany). Extracted DNA was stored at ˗20˚C before use.

Detection of co-infections with other tick-borne pathogens
PCR was used for detection of the citrate synthase gene (gltA) in spotted fever group rickettsiae (SFGR). The gltA-positive samples were classified, and representative samples were sequenced according to the tick species and regional distribution. The Rickettsia outer membrane protein A gene (rOmpA) was also amplified for confirmation of the gltA PCR [18]. The outer membrane protein-1 gene (p28/omp-1) of Ehrlichia and the major surface protein-2 gene (p44/ msp2) of Anaplasma were detected by nested PCR [12,19]. Targeting the 16S rRNA gene for borreliae, DNA primers and Taqman probes were designed from conserved sequences. Specific DNA probes were labeled with two types of fluorescent dyes, 6-carboxyfluorescein (FAM) and 4,7,2 0 -trichloro-7 0 -phenyl-6-carboxyfluorescein (VIC), and were conjugated with the nonfluorescent quencher (NFQ) and minor groove-binder architectural protein (MGB) according to a report by Barbour and colleagues (Table 1) [20]. Multiplex PCR was performed by realtime PCR according to a previously described protocol [17]. The 16S rRNA PCR-positive samples were classified, and conventional PCRs based on the borrelial flagellin (flaB) gene or the glycerophosphodiester phosphodiesterase (glpQ) gene were performed for confirmation of the real-time PCR results [21]. The PCR-specific primers and reaction conditions were derived from our previous studies [12,[18][19][20][21], and the primers used in these experiments were synthesized by Nanjing Kingsley Biotechnology Company (Nanjing, China).

PCR product purification and sequence analysis
The obtained PCR products of Anaplasma and Ehrlichia were gel-purified and cloned into a pCR2.1 vector using a TA Cloning kit (Thermo Fisher Scientific, Waltham, MA, USA). Escherichia coli DH5α (TOYOBO, Osaka, Japan) was transformed with the recombinant plasmids. Ten clones were selected randomly for each PCR product and the insert DNA of each clone was sequenced. Other PCR products were purified and sequenced directly. All obtained sequences were assembled and translated into protein sequences using the Sequencher program (Gene Codes Corp., Ann Arbor, MI, USA). Homology searches and species identification were performed using blastn or blastp (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic analysis of the flaB, glpQ, gltA, p28/omp-1, and p44/msp2 sequences were performed using MEGA 7 with 1000 bootstrap replicates [22].

Statistical analysis
Excel software was used to establish the database and IBM SPSS Statistics version 19 (IBM R Corporation, Chicago, IL, USA) was used for data analysis. Data counts were described by the number of cases (percentage), and the chi-square test (χ 2 ) was performed on data relating to the tick infection ratio of geographic groups. A P-value was determined to be statistically significant when P < 0.05.

Co-infections
Among the 6456 collected ticks, the overall prevalence of tick-borne pathogens was 61.4% (3966/6456), and the co-infection rate of the 3 regions was significantly different.

PLOS NEGLECTED TROPICAL DISEASES
Co-infection of tick-borne bacterial pathogens in ticks in Inner Mongolia, China
In sampling area 1, 25 ticks were detected to be co-infected with four different pathogens, eight of which were infected with C. R. tarasevichiae, E. muris, the B. garinii-complex, and B. miyamotoi, and eight were infected with C. R. tarasevichiae, A. phagocytophilum, the B. garinii-complex, and B. miyamotoi. In sampling areas 2 and 3, no ticks were found to be coinfected with four different pathogens (Table 4).
Only in sampling area 1, was one tick found to be co-infected with five different pathogens, namely C. R. tarasevichiae, E. muris, A. phagocytophilum, the B. garinii-complex, and B. miyamotoi (Table 4).

Discussion
Co-infection with tick-borne pathogens has been suggested to reflect the fact that ticks can carry and transmit multiple pathogens and the need for ticks to switch to different hosts to complete their entire growth process, thereby increasing the likelihood of acquiring different pathogens from different hosts [23,24]. Co-infection can occur when a livestock tick carrying multiple pathogens or multiple ticks carrying multiple pathogens bite a person in succession [25].
In this study, a comprehensive investigation of the epidemiological status of 12 tick-borne bacterial pathogens of four genera was performed in co-infected ticks isolated in northern China. The important findings of our study were as follows. (1) The identification of a variety of tick-borne bacterial pathogens, with an overall high prevalence rate (61.4% of ticks infected). (2) The frequency of co-infection. Among infected ticks, 24.2% were co-infected, with co-infection of C. R. tarasevichiae and the B. garinii-complex being the most common.
(3) The unexpected high infection and co-infection rates of ticks collected from the forest region of eastern Inner Mongolia (sample area 1). (4) The significant changes in the ecological and geographical distribution of the main dominant tick species, and the corresponding increase in pathogen diversity between the Gobi Desert and the semi-desertification steppe, and the grasslands and forest. Together, these results indicate the significant potential threat to public health of tick-borne pathogens.
In this study, we detected the B. garinii-complex in ticks. In a previous study, B. garinii, comprising of the formerly designated B. garinii and B. bavariensis, was reported to be distributed across China [26]. Although these two Borrelia spp. are distinguishable by multi-locus sequence typing [27], they cannot be distinguished by flaB-sequencing because of its low resolution. Therefore, it is highly probable that both the former B. garinii and B. bavariensis have previously been designated as B. garinii.
Inner Mongolia covers a wide geographic area from east to west, and the ecological environments across this region are therefore quite distinct. The vegetation that constitutes the tick habitat changes from east to west, from forests to grasslands to semi-desert grasslands to deserts. The distribution of tick species is closely related to host species and the ecological environment and, consequently, the risk of human tick-borne infection varies from region to region [4][5][6]. I. persulcatus was the dominant tick species in the eastern forest region (sample area 1). I. persulcatus is a typical forest tick species, which is dominant in conifer and broadleaved mixed forest, and its host range is wide, including domestic or wild medium and small mammals. The distribution of I. persulcatus covers Inner Mongolia [13,14], mainland China [28], and more specifically the southwest and northeast of China [29][30][31][32], among other places. D. nuttalli was the dominant tick species in the central and western grasslands (sample areas 2 and 3). D. nuttalli inhabits the arid semi-desert steppe regions, mainly parasitizing livestock and humans. The distribution of D. nuttalli covers Inner Mongolia [12,14], Gansu [33], and the southwest and northeast of China [29,31,32], among other places. In sample area 1, C. R. tarasevichiae was found to reside with other pathogens in 23 coinfection patterns, accounting for 81.7% of co-infections. Among them, the co-infection of C. R. tarasevichiae and the B. garinii-complex was most common, followed by C. R. tarasevichiae and A. phagocytophilum. C. R. tarasevichiae was first identified in I. persulcatus ticks from various sites in Russia in 2001 [34]. Human infection with C. R. tarasevichiae was first reported in northeastern China in 2012 [35] and is widely distributed in eastern and northeastern China [11,36,37]. The forest region in northeast China is close to Russia in terms of geography. With the development of tourism, animal husbandry, and logging, human activities have increased the opportunity for humans, livestock, and ticks to come into contact, possibly creating conditions for the spread of C. R. tarasevichiae. A previous study found that B. burgdorferi sensu lato infection in ticks and mice in the Greater Khingan Mountains forest region of Inner Mongolia is mainly caused by B. garinii [38]. Pan and colleagues [39] also found that the co-infection rate of C. R. tarasevichiae and B. burgdorferi sensu lato was high (20%) in I. persulcatus in Heilongjiang Province. Co-infection with C. R. tarasevichiae has been reported to aggravate disease symptoms and has been linked with mortality [11]. Our results suggest that C. R. tarasevichiae has a high rate of co-infection with other pathogens, which highlights the importance of considering C. R. tarasevichiae in the differential diagnosis of other tick-borne pathogens in endemic regions. In sample areas 2 and 3, the most frequent genospecies association was between R. raoultii and B. garinii. Co-infection with B. garinii is relatively common [1,40]. R. raoultii is widely distributed in the steppe of central and western Inner Mongolia, and D. nuttalli is the main vector and host. In central and western Inner Mongolia, the grassland is arid and the vegetation coverage rate is low, but the parasitism rate of D. nuttalli remains high in spring and summer, which seriously affects local livestock production. The infection and co-infection rates in ticks from sample area 1 (deciduous and mixed forest vegetation) were the most serious, thus highlighting a significant disease risk in this area, a heavily frequented recreational area and tourist hotspot. In addition, the detection rate of Rickettsia and Borrelia was high in ticks in this study, indicating an increased probability of their co-infection with other pathogens.
In this study, the predominant host among co-infection cases was I. persulcatus, accounting for more than 85% of co-infections. It has been confirmed that I. persulcatus can be naturally

PLOS NEGLECTED TROPICAL DISEASES
Co-infection of tick-borne bacterial pathogens in ticks in Inner Mongolia, China infected with a variety of pathogens. Fu and colleagues [41] found that at least 40% of I. persulcatus individuals were co-infected, including both double and triple infections. Co-infections might have consequences in terms of pathogen co-transmission [24], and the high co-infection rate among ticks poses a health threat to humans and livestock [3,42]. The clinical presentation of tick-borne-associated bacterial infections is similar, therefore, diagnosis is challenging and co-infection can be easily missed. The co-infecting pathogens might play different roles within their respective host, thus modulating disease severity [43,44].
Ecological changes and social development may have contributed to the emergence of the tick-borne diseases by placing people in increasing contact with ticks and potential animal reservoirs. Therefore, medical personnel should be trained in identified tick-borne disease hotspots (sample area 1), to improve the detection and identification of TBRD and treatment strategies to reduce the fatality rate linked to co-infection. Disease control and prevention personnel should also be trained to conduct epidemiological investigations and to control the spread and prevalence of outbreaks.
Our findings highlight the severity of tick-borne pathogen infections in the eastern forest region through the collection of field data across all regions of Inner Mongolia from 2015 to 2019. In response, it is hoped that relevant departments can pay increased attention to the coinfection of tick-borne pathogens, and conduct timely screening and clinical treatment for common co-infection patterns to avoid the occurrence of more serious complications.
In this study, sequencing was performed on some samples and R. raoultii was detected from D. nuttalli and C. R. tarasevichiae from I. persulcatus. Data from neighboring countries showed the presence of Rickettsia helvetica [45], but we failed to detect it within the scope of this investigation. Therefore, the genotypes of rickettsiae may be incomplete, and we will continue to expand the sample size to be sequenced for verification.