Microbial Diversity of Ticks And A Novel Tyhpus Group Rickettsia Species ( R. bacterium Ac37b) Detected In Inner Mongolia, China

Background: Ticks are arthropods that can carry multiple pathogens and parasitize on livestock and mammals as well as on humans. Animal husbandry in Inner Mongolia, China, provides a suitable tick habitat. In this study, PacBio full-length 16S rDNA third-generation sequencing was used to analyze the diversity of microbial communities carried by ticks in different regions of Inner Mongolia. The aim of the study is to characterize the microbiome carried by ticks in different geographical locations and to provide theoretical support for regional prevention and control of pathogen populations in the future. Methods: In this study, a total of 905 Dermacentor nuttalli and 36 Ixodes persuleatus were collected from the surface of sheep in four main pasture areas in Inner Mongolia. Pooled DNA samples were prepared from three samples from each region and from each tick species. In total the microbial diversity of 12 samples was analyzed by PacBio full-length 16S rRNA third-generation sequencing, and the α and β diversity were determined. Results: The main bacterial genera we found were Rickettsia (35.27%), Ac37b (19.33%), Arsenophonus (11.21%), Candidatus Lariskella (10.84%), and Acinetobacter (7.17%). There were signicant differences in the microbial composition of ticks from different regions and in different tick species. Rickettsia bellii was found in the I. persuleatus group. In addition, Anaplasma and a novel tyhpus group Rickettsia species (R. bacterium Ac37b) were found in the sample group of D. nuttalli in the city of Ordos. Conclusions: In this study, Rickettsia bellii was rst found in I. persuleatus in Inner Mongolia, and a novel tyhpus group Rickettsia species (R. bacterium Ac37b) was found in D. nuttalli from the city of Ordos. Our study provides a basis for the prevention and control of tick-borne diseases through the analysis of tick microbial diversity in different regions of Inner Mongolia. Furthermore, we were able to detect a new tickborne pathogen in D. nutalli.


Background
Ticks are a kind of arachnids (Arachnida: Acari: Ixodida) parasitic on animals or humans. The larvae, nymphs, and male and female adults of ticks are all bloodsucking. The hosts include terrestrial mammals, birds, reptiles, and amphibians. When ticks bite the host, they can not only cause irritation, anemia, local or systemic hypersensitivity but also become the vector for the transmission of various pathogens [1]. The pathogens transmitted by ticks are even more diverse than in mosquitoes [2,3], including forest encephalitis virus, Rickettsia, Coxiella, Borrelia burgdorferi, Anaplasma, and Babesia [4,5,6,7,8]. Depending on the pathogen the severity of tick-borne disease (TBD) can even be life-threatening.
As human populations have grown and their interactions with the wild have increased, also human exposure to pathogens carrying ticks has greatly increased [9]. The transmission process of TBD is in uenced by many factors, including pathogens, vectors, potential hosts, the environment, and human behavior. In addition, TBD often bene ts from human population mobility, animal migration, and global logistics. Especially the global logistics increases the possibility of tick-borne pathogens spreading across borders [10]. Worldwide, tick-borne diseases (TBD) such as tick-borne encephalitis, Crimean-Congolese hemorrhagic fever, and Rocky Mountain spot fever have posed new threats to public health around the world, and the incidence of TBD is increasing at an alarming rate [11][12][13]. The United States reported nearly 650,000 cases of vector-borne diseases from 2004 to 2016. More than 75% of these cases were tick-borne diseases. From 2012 to 2017 the United States reported nearly 288,000 cases of seven tick-borne diseases to the Centers for Disease Control and Prevention (CDC). Due to underreporting, the true number of cases may be higher [14]. At the same time, the rate of introduction or late identi cation of new or unknown tick-borne pathogens is also accelerating [15].
China has a vast territory, complex geography, diverse climate, and a wide variety of ticks. So tick-borne diseases are prevalent in most areas of China, seriously affecting human health [16,17]. Zhao et al. [18] found that D. nuttalli is one of the tick species carrying many different tick-borne pathogens in China. According to the model prediction [18], the habitat suitable for 19 tick species was 14 -476% larger than the geographical area where these species are currently found, indicating that there are still serious de ciencies in our knowledge of tick distribution. Due to large pasture areas and extensive animal husbandry, Inner Mongolia provides excellent habitat for ticks and the zoonosis incidence is often very high [19]. In 2005 Jia N et al. [20] reported human cases of Rickettsia raoultii in northeast China.
Ticks and tick-borne diseases have a substantial impact on the economy and human life in Inner Mongolia. Nevertheless, the community structure and diversity of microbial communities on ticks parasitizing sheep in various regions of Inner Mongolia have not been thoroughly studied so far. The microbial communities of ticks are in uenced by many factors, including geographical area, feeding status, blood meal source, and developmental stage [21].
Current techniques for 16S ribosomal RNA (rRNA) gene sequence analysis, based on typical clustering thresholds of operational taxa (OTUs), are insu cient for accurate taxonomic allocation and for addressing the phylogenetic relationships at the species level when only a few hypervariable regions are ampli ed. Therefore, 16S rRNA gene ampli cation sequence data using the v3-v4 region can only be explored at the genus level [22]. PacBio full-length 16S rDNA third-generation sequencing technology, however, is more accurate and can be used for precise species identi cation [23]. Therefore, we collected ticks from the surface of sheep in four main pastoral areas of Inner Mongolia, and analyzed their microbial diversity using PacBio full-length 16S rDNA third-generation sequencing technology. The aim of our investigation was to assess the distribution characteristics of tick microbial communities in different geographical locations in Inner Mongolia, providing information for better environmental management.

DNA extraction
The morphologically identi ed tick samples were disinfected with 75 % ethanol, dried on lter paper, washed three times with PBS, and nally dried on lter paper.A total of 941 tick samples were divided into 206 sample pools. In each sample pool, the same area with the genus 5 ~ 20 not only full blood or 1 ~ 2 only full blood tick were taken and put into a sterile grinding tube, add 5 mm magnetic bead 1 and 3 mm magnetic beads 2, grinding tube into the adapter (ahead -20 ℃ refrigerator 30 min), install the adapter into the grinding apparatus, set the parameter to 70Hz and 180S.Finally, according to TIANGEN blood / cell / tissue genomic DNA extraction kit instructions to extract DNA, all extracted DNA stored at-20°C standby. Three DNA samples ( Table 2 ) were randomly selected from each area, and 10 µL of each sample was prepared for microbial diversity sequencing.

16S rRNA Amplicon Sequencing and Data Analysis
The V1-V9 region of the bacteria 16S ribosomal RNA gene were ampli ed by PCR (95°C for 2 min, followed by 27 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 60 s and a nal extension at 72°C for 5 min) using primers PacBio raw reads were processed using the SMRT Link Analysis software version 6.0 to obtain demultiplexed circular consensus sequence (CCS) reads with the following settings: minimum number of passes = 3, minimum predicted accuracy = 0.99. Raw reads were processed through SMRT Portal to lter sequences for length (<800 or >2500 bp) and quality. Sequences were further ltered by removing barcode, primer sequences, chirmas and sequences if they contained 10 consecutive identical bases. OTUs were clustered with 97% similarity cutoff using UPARSE(version 7.1 http://drive5.com/uparse/) and chimeric sequences were identi ed and removed using UCHIME. The phylogenetic a liation of each 16S rRNA gene sequence was analyzed by RDP Classi er (http://rdp.cme.msu.edu/) against the silva (SSU132)16S rRNA database using con dence threshold of 70% [24].Subsequently Alpha and Betadiversity Analyses.The rarefaction analysisbased on Mothur v.1.21.1 [25] was conducted to reveal the diversity indices, including the Chao, ACE, and Shannon diversity indices. The beta diversity analysis was performed using UniFrac [26] to compare the results of the principal component analysis (PCA) using the community ecology package, R-forge (Vegan 2.0 package was used to generate a PCA gure).One way analysis of variance (ANOVA) tests were performed to assess the statistically signi cant difference of diversity indices between samples. Differences were considered signi cant at p < 0.05. Venn diagrams were drawn using online tool "Draw Venn Diagram" (http://bioinformatics.psb.ugent.be/webtools/Venn) to analyze overlapped and unique OTUs during the treatment processes.
For identi cation of biomarkers for highly dimensional colonic bacteria, LEfSe (linear discriminant analysis effect size) analysis was done [27]. Kruskal-Wallis sum-rank test was performed to examine the changes and dissimilarities among classes followed by LDA analysis to determine the size effect of each distinctively abundant taxa [28].

Results
PacBio sequencing data A total of 12 samples were sequenced ( Table 2). After data screening and deletion, a total of 20,1159 reads were generated and classi ed. The reads of each sample ranged from 11,550 to 21996. The dilution curve of the Shannon index at OTU level showed a suitable range for sequencing, and the observed Shannon index accumulation curve also reached a plateau (Fig.3).

Differences Between D. nuttalli and I. persuleatus
North of the Arshan region is adjacent to the New Barag Left Banner and to the Ewenki Autonomous Banner of the Hulun Buir City, close to the Hulun Buir tick collection point. All I. persuleatus came from the Arshan region. Whereas in the Hulun Buir area only D. nuttalli was found. A total of 123 microbial OTUs were found in ticks, and α diversity indicated that specimens of D. nuttalli contained greater microbial diversity than specimens of I. persuleatus (Fig. 5) In general, D. nuttalli in Hulun Buir area showed the highest microbial diversity. We selected pathogenic bacteria with high abundance and obvious harm to humans at the species level (Rickettsia raoultii, Anaplasma, Rickettsia bellii, Coxiella) for further analysis. We found that Ac37b, a new type of Rickettsia, was predominant in the Ordos region, and Anaplasma was much more abundant in the Ordos region than in other regions. The abundance of Rickettsia was very low in Ordos, but it was higher in D. nuttalli from Hulun Buir and Chifeng. Rickettsia bellii was detected for the rst time in Inner Mongolia. It only appeared in the I. persuleatus group and there were many unclassi ed Rickettsia. Arsenophonus unclassi ed was also an important component of its microorganisms in Hulun Buir (Fig.7).

Discussion
D. nuttalli is a tick species widely distributed in Inner Mongolia, eastern Siberia, and China. It is parasitic on livestock and can also cause damage to humans [29]. D.nuttalli can carry different pathogens including Babesia, Anaplasma vois, Rickettsia, and Coxellia [30,31,32]. I. persuleatus is the dominant tick species in northeast China. Inner Mongolia has a large east-west span, so there are I. persuleatus populations at the border with northeast China. This often is associated with the transmission of tickborne encephalitis virus and can pose a serious threat to human life and safety [33,34].
D. nuttalli is the dominant tick species in Inner Mongolia, with signi cant in uence on the economy and health of the local human population. Jiao et al. [35] carried out a simple microbial diversity analysis of ticks on cattle in the Hulun Buir area of Inner Mongolia. Beyond this, the microbial community composition of ticks in other areas of Inner Mongolia was not further investigated. The bacterial diversity on different tick species must be analyzed to further understand the relationships between ticks and microorganisms. Samples collected from multiple regions are more likely to nd new pathogens in microbial diversity.
In our investigation, we applied PacBio full-length 16S rRNA third-generation sequencing to the V1-V9 region of the 16S rRNA. In their study on oral microorganisms, Zhang et al. [36] found that OTU sequences generated by PacBio were much larger than those generated on the MiSeq platform. Therefore, we adopted PacBio full-length 16S rRNA third-generation sequencing for the microbial diversity analysis of ticks in Inner Mongolia. According to our analysis, the microbial diversity on D. nuttalli samples from different regions of Inner Mongolia was signi cantly different. The highest microbial diversity was found in D. nuttalli samples from Hulun Buir, and Rickettsia (35.87%) and Arsenophonus (41.51%) were the main microorganisms. Rickettsia is an arthropod-associated obligate intracellular gram-negative bacterium that can cause mild to severe disease in humans [37]. Arsenophonus is an intracellular symbiotic bacterium of insects with a wide host range and rich biodiversity. An androidal effect on ticks has not been reported so far, and its other biological functions have not yet been identi ed [38].
Linear discriminant analysis effect size (LEfSe) analysis showed that Rickettsia raoultii, Peptostreptococcaceae and Clostridia played an important role in the Chifeng formation. Characteristic of the Ordos formation is Anaplasma. Enterobacterale and Xanthomonadaceae belong to Hulun Buir (Fig. 8). Rickettsia raoultii is the pathogene of the spotted fever group, which is transmitted vertically in arthropods as a symbiotic bacterium and in vertebrates as a pathogenic bacterium and is a pathogen of human diseases [39]. Anaplasma is a gram-negative intracellular obligate parasite, and its pathogenicity poses an important threat to several animals, and even public health security [40]. Currently, there are six species of Anaplasma recognized worldwide, including A. phagocytophilum, A. ovis, A. capra, A. bovis, A. marginale and A. platys [41]. In addition to A. phagocytophilum, A. bovis and A. capra have been reported to infect humans [42,43]. A novel tyhpus group Rickettsia species (R. bacterium Ac37b) was found in the Ordos region [44]. The Rickettsia typhus group is mainly composed of R. przewskii and R. morzewskii. In Australia, three types of typhus, epidemic typhus, mouse typhus, and tsutsugamushi disease, have been found successively. These are closely related to native wild animals and ticks in Australia [45]. In China, typhus has also been an important cause of human morbidity and mortality in the past decade. The disease was initially identi ed only in southern China, but now cases of typhus have been reported in northern China, with a wide geographical distribution [46].
R. bellii is the only known species in a third group that differentiated before the spotted fever group and the typhus group separated. R. bellii is the most common Rickettsia in American ticks and is found in all species of Ixodes. Including Dermacentor and Amblyomma, it is also the only Rickettsia that has been found in both Ixodes, showing the largest arthropod host range among known Rickettsia: It is pathogenic to mammals [47]. But the pathogenic potential for humans is still unknown and should be closely monitored [48].
We can further explore the relationship between Rickettsia bellii and other Rickettsia. In the Ordos area, there were pathogenic bacteria that are more threatening to humans, and a new classi cation of Rickettsia has emerged. The pathogen prevention and control in this area needs to focus on monitoring and strengthening the popular knowledge of methods for personal protection. The newly discovered pathogens in Inner Mongolia need to be isolated and sequenced in future studies, and the pathogenicity of the organisms should be tested through subsequent animal experiments. At the same time, the sampling sites can be expanded in future investigations, and ticks should be collected from different hosts, such as cattle, camels, and other mammals.

Conclusions
In this study, we analyzed the microbial diversity of two ticks in four regions of Inner Mongolia. A novel rickettsia species (R.bacterium Ac37b ) was found in Inner Mongolia for the rst time, and Rickettsia Bellii was found in I.persuleatus. Ticks carried more potential pathogens in Ordos area,and there were coinfections of Rickettsia and Anaplasma, which may be related to its geographical environment. The prevention and control of tick-borne diseases in this region should be strengthened. In the future research, sampling in the western region of Inner Mongolia can be focused on.