Molecular detection of pathogens in ticks and rodents, Northeast and Middle-east China

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However, the types and distribution of pathogens they carry remain poorly understood in China. Methods In this study, we collected a total of 1004 Haemaphysalis longicornis ticks (503 adults and 501 nymphs), divided into 670 groups, from Liaoning Province in northeastern China and Anhui Province in middle-eastern China, along with 42 Spermophilus dauricus from Heilongjiang Province in northeastern China. Morphological analysis and cytochrome oxidase I ( COI ) sequencing were used to identify tick and rodent species. Using nested PCR, all samples were first tested for severe fever with thrombocytopenia syndrome virus (SFTSV) and the bacterial 16S rRNA gene, followed by detection of the corresponding genus-specific genes. Samples were tested for Rickettsia 16S rRNA/ rrs , 17kDa , gltA , ompA , ompB , and sca4 genes; for Coxiella 16S rRNA, groEL , and rpoB genes; and for Legionella groEL and mip genes. Results Spotted fever group Rickettsia spp. (SFGR) were identified in a total of 14 tick groups: Candidatus Rickettsia jingxinensis (12/594) from Liaoning and Rickettsia heilongjiangensis (2/76) from Anhui. Four of the 14 groups were co-infected with Coxiella -like endosymbionts (CLE). Notably, Legionella pneumophila was detected in 2 of 42 S. dauricus from Heilongjiang, with genetic similarity to the OLDA strain from a U.S. patient exceeding 99.8% for 16S rRNA, groEL , and mip genes, confirming the high pathogenic potential of these strains in humans. Conclusions Our findings suggest that local residents should be cautious about potential infections from these two SFGR species, and we recommend further surveillance and investigation of H. longicornis and S. dauricu s as pathogen carriers. Spotted fever group Rickettsia (SFGR) Rickettsia heilongjiangensis Candidatus Rickettsia jingxinensis Haemaphysalis longicornis Coxiella-like endosymbiont (CLE) Tick Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Ticks are obligate blood-feeding ectoparasites and are second only to mosquitoes as vectors of human pathogens [1,2]. H. longicornis is a common species worldwide and serves as an important vector of diseases in both humans and animals [3,4]. This tick species exhibits a complex biogeography: it is native to East Asia (eastern China, Japan, the Russian Far East, and Korea) but has also established invasive populations in Australia, New Zealand, and the eastern United States [3–5]. Retrospective analyses of historical tick specimens revealed its presence in the U.S. as early as 2010. Its dual status as both native and invasive, depending on the region, underscores its ecological adaptability and capacity to transmit tick-borne pathogens [6,7]. H. longicornis is a vector for at least 30 pathogens that infect humans, including seven species of spotted fever group Rickettsia (SFGR), seven species from the Anaplasmataceae family, four genospecies within the Borrelia burgdorferi sensu lato complex, and two Babesia species. Additionally, it can carry Francisella , Bartonella , Coxiella , Toxoplasma , and six viruses [7], one of which is severe fever with thrombocytopenia syndrome virus (SFTSV), which has spread across China, Korea, and Japan [1,8–12]. Beyond pathogens, ticks also harbor a diverse array of commensal and symbiotic microorganisms. Non-pathogenic microbes play key roles in tick fitness, nutritional adaptation, development, and reproduction [13,14]. The massive infestations of H. longicornis may be facilitated by their ability to reproduce parthenogenetically [4,15–17] and to survive a wide range of environmental temperatures (–2°C to 40°C), although they are most suited to warm and moist temperate conditions [18]. Like other Ixodid ticks, H. longicornis is a three-host tick with variable phenology depending on latitude [18,19]. Enzootic tick–vertebrate–tick cycle was influenced by ecological shifts, including climate variability, habitat fragmentation, and anthropogenic activities. Rodents are frequently infested by ticks, providing blood meals necessary for the development of ticks in their immature stages. Rodents are considered competent reservoirs for various tick-borne pathogens, such as tick-borne encephalitis virus, Rickettsia spp ., Ehrlichia spp ., Coxiella burnetii , Francisella tularensis , and Babesia microti [20]. Additionally, rodents are significant reservoir hosts for many zoonotic infections, including Hantavirus hemorrhagic fever with renal syndrome, Hantavirus cardiopulmonary syndrome, plague, borreliosis, salmonellosis, murine typhus, leptospirosis, Lassa fever, rat-bite fever, and campylobacteriosis [21,22]. Climate change has led to an increased risk of tick-borne infectious diseases, as it enhances tick survival rates, egg-laying rates, and overall population growth [23]. Moreover, the general population, not just agricultural workers, is facing a higher risk of tick and rodent exposure due to the growing popularity of outdoor activities such as hiking and camping, as well as the increasing pet population [24]. We focused on H. longicornis due to its dominance in the studied regions, parthenogenetic reproduction (enhancing population growth), and role as a vector for >30 human pathogens [7]. S. dauricus was selected as a key rodent reservoir in Northeast China that supports immature tick development and zoonotic pathogen transmission. Investigating these species helps elucidate local disease risks and informs targeted surveillance. Given this context, to evaluate the potential ability of ticks and rodents to acquire endemic pathogens and the possible exposure risk to humans, we collected ticks from Liaoning Province in northeastern China and Anhui Province in central-eastern China, and rodents from Heilongjiang Province in northeastern China, from 2021 to 2022. Methods Sample Collection and Classification In June 2021, in Helan Town, Liaoyang City, Liaoning Province, and in August 2022, in Hanshan County, Maanshan City, Anhui Province, we surveyed 30 forest and grassland sites for questing ticks. Ticks were collected by dragging a 1-m² corduroy cloth over the grass in woodland areas or along roadsides. The cloth was inspected every 10-20 meters, and all attached ticks were removed. Additionally, in June 2021, mouse traps were placed at 12 field sites in the suburbs of Harbin City, Heilongjiang Province. The traps were set daily at 10:00 a.m. in front of rodent holes, with dirt sprinkled over them to reduce visibility. At 3:00 p.m. the same day, all traps were checked, and the captured rodents were collected and placed in cloth bags. During sampling, background data such as landform, vegetation, and the latitude and longitude of the collection sites were recorded. Ticks and rodents were first identified morphologically and then processed individually for PCR amplification of the mitochondrial cytochrome oxidase I (COI) gene. Sample Processing and Pathogenic Culture Ticks were pooled by developmental stage (adults individually; nymphs in groups of 3) to prioritize detection sensitivity. Sex-based differences in pathogen prevalence are minimal in unfed ticks [25,26], and nymph pooling followed established protocols [26]. Cryopreserved ticks were first washed once with 75% ethanol and three times with sterile phosphate-buffered saline (PBS) to remove surface impurities. Subsequently, 500 μl of PBS was added to a 2-ml sterile glass tube for manual grinding using a glass pestle. For rodents, about 50 mg of liver tissue was aseptically ground following the same method. To eliminate environmental microorganisms, cysteine heart agar blood (CHAB) media containing five antibiotics (colistin, amphotericin, lincomycin, methicillin, and ampicillin) was prepared. From each tick and mouse group, 300 μl of the grinding solution was pipetted and evenly distributed onto freshly prepared CHAB plates with antibiotics, which were then incubated in a 5% CO₂ atmosphere. The CHAB plates were monitored every 24 hours, and when colonies appeared, individual colonies were isolated and inoculated onto new CHAB plates for bacterial proliferation. Freshly grown bacteria were scraped off and suspended in saline for DNA extraction, while another portion was mixed with glycerol preservation solution and stored at -70°C. DNA/RNA Extraction The remaining 200 μl of tick or mouse grinding solution was directly subjected to nucleic acid extraction using the AllPrep DNA/RNA Mini Kit (QIAGEN, Germany) according to the manufacturer’s instructions. The total extracted nucleotides were eluted twice with 60 μl of Elution Buffer. Nucleic acid concentration was measured using a NanoDrop 1000 spectrophotometer (Thermo Scientific, USA), and the samples were stored at -40°C until further use. Molecular Detection of Pathogens All nucleic acid samples were initially tested for SFTSV and the bacterial mitochondrial 16S rRNA gene. SFTSV detection was carried out using one-step RT-PCR (Access RT-PCR System Kit, Promega, USA) with primers specific to the small RNA segment of the virus. Bacterial identification was first performed using the 16S rRNA gene, followed by further amplification with genus-specific primers to confirm the bacterial species. Nested PCR was employed for the amplification of all genes, and detailed information on the primers used for pathogen detection is shown in Table 1. PCR products were electrophoresed through 1.0% agarose gels, and the target amplicons were purified using the QIAquick PCR Purification Kit (QIAGEN, Germany). The purified PCR products were sent to Beijing Tianyihuiyuan Biotechnology Co., Ltd. (Beijing, China) for directional sequencing. All PCR products were sequenced bidirectionally, prioritizing longer fragments (>600 bp) to ensure accuracy. Phylogenetic Analysis and MIR The pathogen sequences and mitochondrial cytochrome oxidase I (COI) gene sequences from ticks or rodents were aligned against those in GenBank using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic trees were constructed using the neighbor-joining method with 1000 bootstrap replicates, implemented in MEGA 11 software (http://www.megasoftware.net). The prevalence of pathogens was calculated as the minimum infection rate (MIR), which for grouped ticks was determined by dividing the number of positive groups by the total number of ticks. Results Sample collection and identification From June 2021 to August 2022, a total of 1004 ticks (503 adults and 501 nymphs) were collected from Helan Town, Liaoning Province (450 adults and 432 nymphs) and Hanshan County, Anhui Province (53 adults and 69 nymphs), as well as 42 rodents from Harbin City, Heilongjiang Province. Morphological and molecular identification confirmed that all ticks were H. longicornis , divided into 594 groups, and all rodents were S. dauricus . BLASTn analysis showed that the COI sequences of H. longicornis and S. dauricus had greater than 99.9% identity to reference sequences in GenBank. The COI sequences of H. longicornis from Liaoning (OR492359) and Anhui (OR492358) showed the highest similarity, clustering on a branch with a node value of 100. Similarly, the COI sequences of S. dauricus from Heilongjiang (OR492360) and the reference sequence from China (KP708706) also clustered on a branch with a node value of 100 (Fig. 1). Detection of Pathogen in Ticks and Rodents In 14 out of 670 groups of H. longicornis , Rickettsia was detected, with an MIR of 1.4% (14/1004). Ca . R. jingxinensis was detected in 12 groups from Liaoning Province, while R. heilongjiangensis was found in two groups from Anhui Province. Coxiella -like endosymbiont (CLE) was detected in 20 groups, with an MIR of 2.0% (20/1004), including 7 groups from Liaoning and 13 from Anhui. Notably, co-infections with CLE were observed in samples hl182 and hl466 from Liaoning and st5 and st6 from Anhui. Unexpectedly, L. pneumophila was detected in 2 out of 42 S. dauricus from Heilongjiang, with an infection rate of 4.8% (2/42). Additionally, various bacteria were detected on the surface of ticks, including Pseudomonas , Staphylococcus , Bacillus , Paenibacillus , Enterococcus , Bacillus parasporus , and Salmonella . Staphylococcus and Enterococcus were also detected in rodents. The distribution of these bacteria is shown in Table 2. In terms of microbial abundance, Pseudomonas and Staphylococcus had the highest composition ratios on ticks from Liaoning, with 28.7% (84/293) and 15.7% (46/293), respectively. Using both pathogen culture and molecular biology methods on the same samples, the results provided mutual validation in some cases. For instance, 56 of 84 Pseudomonas strains isolated from H. longicornis in Liaoning were also detected in the grinding solution of the corresponding samples, and 7 of 21 Paenibacillus strains were similarly detected. However, in Anhui, 1 Pseudomonas strain, 3 Staphylococcus strains, 7 Bacillus strains, and 7 Bacillus parasporus strains were isolated from H. longicornis , and 26 Staphylococcus strains and 13 Enterococcus strains were isolated from S. dauricus in Heilongjiang, none of which were detected in the grinding solution. This suggests that pathogen culture may be more sensitive than molecular methods for detecting these microorganisms on ticks. Additionally, no SFTSV was detected in any of the collected samples. Genetic and Phylogenic Analysis of Rickettsia The 16S rRNA/ rrs (OR477299) and 17 kDa (OR500965) genes of 12 Ca . R. jingxinensis isolates (tick73) from Liaoning clustered on the same branch as tick-XA188 and Xian-HL-21 from Shaanxi Province, with 99.89% identity and 99.77% identity, respectively. The ompA (OR500967) gene clustered with Meixian-HI-242 from Shaanxi, YBHC-T32 from Jilin, and J244 from Shandong, sharing 99.98% homology. The gltA (OR500963), ompB (OR500969), and sca4 (OR500971) genes were clustered with those of Huaian-RM from Jiangsu, China, showing 100% identity (Fig. 2). Genetic analysis revealed that the two Anhui strains of R. heilongjiangensis shared highest homology with strain B8 (CP112971) for five genes (16S rRNA, 17kDa , ompA , gltA , and sca4 ). In contrast, their ompB sequences (OR500968) showed 100% identity with strain 054 (CP002912) from Heilongjiang, differing from B8 at 97.3% of sites (748/769). Genetic and Phylogenic Analysis of CLE In seven CLE strains from Liaoning and 13 CLE strains from Anhui, their 16S rRNA (OR477301 and OR477300), groEL (OR500973 and OR500972), and rpoB (OR500976 and OR500975) genes were clustered together with the 580 strain from Jiangxi Province (Fig. 3), all showing 100% homology. Genetic and Phylogenic Analysis of L. pneumophila For L. pneumophila detected in two S. dauricus from Heilongjiang, the 16S rRNA (OR477302), groEL (OR500974), and mip (OR519871) genes clustered with those of the OLDA strain from a U.S. patient, with 96.9%, 99.81%, and 100% homology, respectively (Fig. 4). Discussion In this study, All PCR products were sequenced bidirectionally, which were divided into 670 groups. Ca . R. jingxinensis was found in 12 of 594 groups of H. longicornis ticks from Helan Town, Liaoning Province, northeastern China, while R. heilongjiangensis was identified in 2 of 76 groups of H. longicornis ticks from Hanshan County, Anhui Province, middle-east China. The MIR of Rickettsia in H. longicornis ticks was low at 1.4%. Typically, in unfed ticks from vegetation, the pathogen carriage rate [25,26] is lower than that in ticks collected from animals, where the rate can be as high as 20–70% [27,28]. Notably, a Legionella species, highly similar to L. pneumophila , was detected in 2 of 42 S. dauricus from Harbin, Heilongjiang Province, northeastern China, which is known to cause disease in humans. The geographic distribution of detected pathogens ( Ca . R. jingxinensis in Liaoning, R. heilongjiangensis in Anhui) may reflect climate-influenced vector-host interactions. Future studies should incorporate climate variables to assess these relationships. Ca . R. jingxinensis was first discovered in Japan [29] and named in 2016 in Jingxin City, Jilin Province, China [30]. It is widely distributed across Asia and has been detected in several provinces in China, including Liaoning, Guangxi, Sichuan, Hebei, and Shaanxi, as well as in other Asian countries such as South Korea, Thailand, and India [31–34]. Studies suggest that Ca. R. jingxinensis may be pathogenic to humans [35]. Of the six genes of Ca. R. jingxinensis detected from Liaoning, 16S rRNA/ rrs , 17kDa , and ompA showed the highest similarity with strains from Shaanxi, while gltA , ompB , and sca4 showed the highest similarity with strains from Jiangsu. Additionally, ompA displayed similar high similarity with strains from Jilin and Shandong. In H. longicornis from Anhui, R. heilongjiangensis was detected, which was first isolated from Dermacentor silvarum ticks in Heilongjiang Province in 1982 [36] and later confirmed as a new Rickettsia species [37]. The epidemic zone of R. heilongjiangensis has been reported primarily in northeastern China [38] and has also been found in ticks from Altay and the Russian Far East [39,40]. In this study, the two isolates of R. heilongjiangensis from Anhui showed the highest homology across six genes, including 16S rRNA, 17kDa , ompA , gltA , and sca4 , with strain B8 [41], which was isolated from a patient in the same province. It is speculated that the source of R. heilongjiangensis in Hanshan may have originated within the province. Interestingly, L. pneumophila was detected in two liver samples (Heb1 and Heb3) from S. dauricus in Heilongjiang. The three genes—16S rRNA, groEL , and mip —showed the highest homology with the OLDA strain, which was isolated from a U.S. case, indicating that S. dauricus may carry a highly pathogenic strain of L. pneumophila . Legionella species were first recognized in the summer of 1976 following an outbreak of pneumonia in Philadelphia, Pennsylvania, USA [42]. Legionella was the first bacterium identified to multiply within protozoan hosts, primarily aquatic amoebae [43]. Human infections typically occur through inhalation of Legionella-containing aerosols generated by contaminated man-made water sources, such as showers [44]. This study may be the first report of rodents carrying L. pneumophila , suggesting that preventive measures should target not only known rodent-borne pathogens but also L. pneumophila infections from rodents. The distribution of SFTS cases in China is expanding, with a total of 27 provinces reporting cases. Recently, some suspected SFTS cases were reported in Hanshan, but H. longicornis was not found to be infected with SFTSV in this study. This could be due to the low detection rate of free-living ticks [26], or it may be a result of the small sample size. Additionally, co-infections were identified in four groups of samples. CLE were detected in samples hl182 and hl466 from Liaoning, which also carried Ca . R. jingxinensis, and in samples st5 and st6 from Anhui, which were infected with R. heilongjiangensis . The natural symbiont Coxiella has been shown to promote feeding behavior and blood intake in H. longicornis [45]. Endosymbiotic bacteria often become essential or even irreplaceable for their hosts’ fitness and reproduction [46]. Furthermore, Pseudomonas , Staphylococcus , Bacillus , Paenibacillus , Enterococcus , Bacillus parasporus , and Salmonella were detected on the surface of the ticks, which is similar to the pathogen spectrum seen in ticks from animals [47]. This suggests the possibility of acquiring conditional pathogens through mechanical transmission during tick exposure, although the human pathogenicity of most of these bacteria remains to be determined. Staphylococcus and Enterococcus were also detected in rodents, adding to our understanding of the pathogens carried by rodents. Conclusion In conclusion, two SFGR species, Ca. R. jingxinensis and R. heilongjiangensis , were detected in H. longicornis from Liaoning and Anhui, with four groups co-infected with CLE. Notably, two S. dauricus from Heilongjiang were found to carry L. pneumophila . These findings expand our understanding of the role ticks and rodents play in the local ecosystem, particularly in pathogen acquisition and transmission. The threat posed to public health and veterinary medicine should not be underestimated, and there is a need for enhanced surveillance and investigation of H. longicornis and S. dauricus as pathogen carriers. Declarations Author Contributions Investigation—Y. S., L. G., S. G., L. M., D. S., Y. G., Y. J., K. Y., X. L., M. W.; investigation and data curation—W. W., R. Z. and G. Y.; resources—T. Q.; conceptualization, investigation, resources, and writing – review & editing—Y. W. Funding This work was funded by the Natural Science Foundation of Beijing Municipality (Grant No. 7242188) and the National Natural Science Foundation of China (Grant No. 81874275). Data Availability Statement All data supporting this study are available within the manuscript. Declaration of Competing Interest The authors declare no conflict of interest. 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Characterization of Rickettsiae in Ticks in Northeastern China. Parasites Vectors, 9, 498. doi:10.1186/s13071-016-1764-2. Bang, M.S., Kim, C.-M., Pyun, S.-H., Kim, D.-M., Yun, N.R., 2021. Molecular Investigation of Tick-Borne Pathogens in Ticks Removed from Tick-Bitten Humans in the Southwestern Region of the Republic of Korea. PLoS ONE, 16, e0252992. doi:10.1371/journal.pone.0252992. Liu, H., Liang, X., Wang, H., Sun, X., Bai, X., Hu, B., Shi, N., Wang, N., Zhang, X., Huang, L., et al. 2020. Molecular Evidence of the Spotted Fever Group Rickettsiae in Ticks from Yunnan Province, Southwest China. Exp Appl Acarol, 80, 339–348. doi:10.1007/s10493-020-00467-5. Takhampunya, R., Korkusol, A., Pongpichit, C., Yodin, K., Rungrojn, A., Chanarat, N., Promsathaporn, S., Monkanna, T., Thaloengsok, S., Tippayachai, B., et al. 2019. Metagenomic Approach to Characterizing Disease Epidemiology in a Disease-Endemic Environment in Northern Thailand. Front. Microbiol., 10, 319. doi:10.3389/fmicb.2019.00319. Wang, Q., Guo, W.B., Pan, Y.-S., Jiang, B.G., Du, C.H., Que, T.C., Zhan, L., Wu, J.H., Yu, M.H., Cui, X.M., Zhao, L., Xu, D.L., Xia, L.Y., Ye, R.Z., Li, J., Li, L.F., Wei, W., Zhou, Y.H., Jiang, J.F., Jia, N., Cao, W.C. 2021. Detection of Novel Spotted Fever Group Rickettsiae (Rickettsiales: Rickettsiaceae) in Ticks (Acari: Ixodidae) in Southwestern China. Journal of Medical Entomology, 58, 1363–1369. doi:10.1093/jme/tjaa294. Lu, M., Tian, J., Zhao, H., Jiang, H., Qin, X., Wang, W., Li, K., 2022. Molecular Survey of Vector-Borne Pathogens in Ticks, Sheep Keds, and Domestic Animals from Ngawa, Southwest China. Pathogens, 11, 606. doi:10.3390/pathogens11050606. Zhang, J.Z., Fan, M.Y., Wu, Y.M., Fournier, P.E., Roux, V., Raoult, D., 2000. Genetic Classification of “Rickettsia Heilongjiangii” and “Rickettsia Hulinii,” Two Chinese Spotted Fever Group Rickettsiae. J Clin Microbiol, 38, 3498–3501. doi:10.1128/JCM.38.9.3498-3501.2000. Fournier, P.E., Dumler, J.S., Greub, G., Zhang, J., Wu, Y., Raoult, D., 2003. Gene Sequence-Based Criteria for Identification of New Rickettsia Isolates and Description of Rickettsia Heilongjiangensis Sp. Nov. J Clin Microbiol, 41, 5456–5465. doi:10.1128/JCM.41.12.5456-5465.2003. Kasama, K., Fujita, H., Yamamoto, S., Ooka, T., Gotoh, Y., Ogura, Y., Ando, S., Hayashi, T., 2019. Genomic Features of Rickettsia Heilongjiangensis Revealed by Intraspecies Comparison and Detailed Comparison With Rickettsia Japonica. Front Microbiol, 10, 2787. doi:10.3389/fmicb.2019.02787. Shpynov, S., Fournier, P.-E., Rudakov, N., Tarasevich, I., Raoult, D., 2006. Detection of Members of the Genera Rickettsia, Anaplasma, and Ehrlichia in Ticks Collected in the Asiatic Part of Russia. Ann N Y Acad Sci, 1078, 378–383. doi:10.1196/annals.1374.075. Mediannikov, O.Y., Sidelnikov, Y., Ivanov, L., Mokretsova, E., Fournier, P.-E., Tarasevich, I., Raoult, D., 2004. Acute Tick-Borne Rickettsiosis Caused by Rickettsia Heilongjiangensis in Russian Far East. Emerg Infect Dis, 10, 810–817. doi:10.3201/eid1005.030437. He, M., Zhang, L., Hu, H., Liu, X., Zhang, C., Xin, Y., Liu, B., Chen, Z., Xu, K., Liu, Y., 2023. Complete Genome Sequencing and Comparative Genomic Analyses of a New Spotted-Fever Rickettsia Heilongjiangensis Strain B8. Emerg Microbes Infect, 12, 2153085. doi:10.1080/22221751.2022.2153085. Abu Kwaik, Y., Gao, L.Y., Stone, B.J., Venkataraman, C., Harb, O.S., 1998. Invasion of Protozoa by Legionella Pneumophila and Its Role in Bacterial Ecology and Pathogenesis. Appl Environ Microbiol,64(9),3127–3133. doi:10.1128/AEM.64.9.3127-3133.1998. Rowbotham, T.J., 1980. Preliminary Report on the Pathogenicity of Legionella Pneumophila for Freshwater and Soil Amoebae. J Clin Pathol, 33, 1179–1183. doi:10.1136/jcp.33.12.1179. Blatt, S.P., Parkinson, M.D., Pace, E., Hoffman, P., Dolan, D., Lauderdale, P., Zajac, R.A., Melcher, G.P., 1993. Nosocomial Legionnaires’ Disease: Aspiration as a Primary Mode of Disease Acquisition. Am J Med, 95, 16–22. doi:10.1016/0002-9343(93)90227-g. Ma, R., Zhao, M., Wang, H., Hou, R., Qin, K., Qian, Y., Zhang, H., Zhou, Y., Wu, W., Gu, J., et al. 2022. Virome of Giant Panda-Infesting Ticks Reveals Novel Bunyaviruses and Other Viruses That Are Genetically Close to Those from Giant Pandas. Microbiol Spectr, 10, e0203422. doi:10.1128/spectrum.02034-22. Tsementzi, D., Castro Gordillo, J., Mahagna, M., Gottlieb, Y., Konstantinidis, K.T., 2018. Comparison of Closely Related, Uncultivated Coxiella Tick Endosymbiont Population Genomes Reveals Clues about the Mechanisms of Symbiosis. Environ Microbiol, 20, 1751–1764. doi:10.1111/1462-2920.14104. Yao, J.L., Chen, Z.G., Gong, H.Y., 2021. Investigation of Tick Harbored Bacteria and Protoza from Several Animal Species. Chin. J. Anim. Infect, 29(03): 107-113. doi:10.19958/j.cnki.cn31-2031/s.2021.03.016. Wen, H.L., Zhao, L., Zhai, S., Chi, Y., Cui, F., Wang, D., Wang, L., Wang, Z., Wang, Q., Zhang, S., et al. 2014. Severe Fever with Thrombocytopenia Syndrome, Shandong Province, China, 2011. Emerg Infect Dis, 20, 1–5. doi:10.3201/eid2001.120532. Lv, J.Z., Wu, S.Q., Zhang,Y.N., Chen, Y., Feng, C.Y.; Yuan, X.F., Jia, G.L., Deng, J.H., Wang, C.X.; Wang, Q., Mei, L., Lin, X.M., 2014. Assessment of four DNA fragments (COI, 16S rDNA, ITS2, 12S rDNA) for species identification of the Ixodida (Acari: Ixodida). Parasit Vectors. ,3:7:93. doi: 10.1186/1756-3305-7-93. Borsoi, A.B.P., Bitencourth, K., De Oliveira, S.V., Amorim, M., Gazêta, G.S., 2019. Human Parasitism by Amblyomma Parkeri Ticks Infected with Candidatus Rickettsia Paranaensis, Brazil. Emerg. Infect.Dis.,25,2339–2341. doi:10.3201/eid2512.190988. Li, H., Zhang, P.-H., Huang, Y., Du, J., Cui, N., Yang, Z.-D., Tang, F., Fu, F.-X., Li, X.-M., Cui, X.-M., et al. 2018. Isolation and Identification of Rickettsia Raoultii in Human Cases: A Surveillance Study in 3 Medical Centers in China. Clinical Infectious Diseases, 66, 1109–1115. doi:10.1093/cid/cix917. Noh, Y., Lee, Y.S., Kim, H.-C., Chong, S.-T., Klein, T.A., Jiang, J., Richards, A.L., Lee, H.K., Kim, S.Y., 2017. Molecular Detection of Rickettsia Species in Ticks Collected from the Southwestern Provinces of the Republic of Korea. Parasites Vectors, 10, 20. doi:10.1186/s13071-016-1955-x. Jiang, J., An, H., Lee, J.S., O’Guinn, M.L., Kim, H.C., Chong, S.T., Zhang, Y., Song, D., Burrus, R.G., Bao, Y., et al. 2018. Molecular Characterization of Haemaphysalis Longicornis-Borne Rickettsiae, Republic of Korea and China. Ticks and Tick-borne Diseases, 9, 1606–1613. doi:10.1016/j.ttbdis.2018.07.013. Duron, O., Noël, V., McCoy, K.D., Bonazzi, M., Sidi-Boumedine, K., Morel, O., Vavre, F., Zenner, L., Jourdain, E., Durand, P., et al. 2015. The Recent Evolution of a Maternally-Inherited Endosymbiont of Ticks Led to the Emergence of the Q Fever Pathogen. Coxiella Burnetii. PLoS Pathog, 11, e1004892. doi:10.1371/journal.ppat.1004892. Pascale, M.R., Salaris, S., Mazzotta, M., Girolamini, L., Fregni Serpini, G., Manni, L., Grottola, A., Cristino, S., 2021. New Insight Regarding Legionella Non- Pneumophila Species Identification: Comparison between the Traditional Mip Gene Classification Scheme and a Newly Proposed Scheme Targeting the rpoB Gene. Microbiol Spectr, 9, e01161-21. doi:10.1128/Spectrum.01161-21. Valeria Gaia, Norman K Fry, Timothy G Harrison, Raffaele Peduzzi. 2003. Sequence-Based Typing of Legionella pneumophila Serogroup 1 Offers the Potential for True Portability in Legionellosis Outbreak Investigation. J Clin Microbiol, 41(7):2932-9. doi: 10.1128/JCM.41.7.2932-2939.2003. Tables Table 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table12025414.docx Table22025414.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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08:55:06","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":24058,"visible":true,"origin":"","legend":"","description":"","filename":"Table12025414.docx","url":"https://assets-eu.researchsquare.com/files/rs-6464024/v1/e68846fa795b9eca3031a003.docx"},{"id":82162524,"identity":"366c3345-64cb-4d74-ade4-ba66cdc0cac8","added_by":"auto","created_at":"2025-05-07 08:47:06","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":20842,"visible":true,"origin":"","legend":"","description":"","filename":"Table22025414.docx","url":"https://assets-eu.researchsquare.com/files/rs-6464024/v1/86558ad84488d85955535e97.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eMolecular detection of pathogens in ticks and rodents, Northeast and Middle-east China\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eTicks are obligate blood-feeding ectoparasites and are second only to mosquitoes as vectors of human pathogens [1,2]. \u003cem\u003eH. longicornis\u003c/em\u003e is a common species worldwide and serves as an important vector of diseases in both humans and animals [3,4]. This tick species exhibits a complex biogeography: it is native to East Asia (eastern China, Japan, the Russian Far East, and Korea) but has also established invasive populations in Australia, New Zealand, and the eastern United States [3\u0026ndash;5]. Retrospective analyses of historical tick specimens revealed its presence in the U.S. as early as 2010. Its dual status as both native and invasive, depending on the region, underscores its ecological adaptability and capacity to transmit tick-borne pathogens [6,7].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eH. longicornis\u003c/em\u003e is a vector for at least 30 pathogens that infect humans, including seven species of spotted fever group Rickettsia (SFGR), seven species from the \u003cem\u003eAnaplasmataceae\u0026nbsp;\u003c/em\u003efamily, four genospecies within the \u003cem\u003eBorrelia burgdorferi\u003c/em\u003e sensu lato complex, and two \u003cem\u003eBabesia\u003c/em\u003e species. Additionally, it can carry \u003cem\u003eFrancisella\u003c/em\u003e, \u003cem\u003eBartonella\u003c/em\u003e, \u003cem\u003eCoxiella\u003c/em\u003e, \u003cem\u003eToxoplasma\u003c/em\u003e, and six viruses [7], one of which is severe fever with thrombocytopenia syndrome virus (SFTSV), which has spread across China, Korea, and Japan [1,8\u0026ndash;12]. Beyond pathogens, ticks also harbor a diverse array of commensal and symbiotic microorganisms. Non-pathogenic microbes play key roles in tick fitness, nutritional adaptation, development, and reproduction [13,14]. The massive infestations of \u003cem\u003eH. longicornis\u003c/em\u003e may be facilitated by their ability to reproduce parthenogenetically [4,15\u0026ndash;17] and to survive a wide range of environmental temperatures (\u0026ndash;2\u0026deg;C to 40\u0026deg;C), although they are most suited to warm and moist temperate conditions [18]. Like other \u003cem\u003eIxodid\u003c/em\u003e ticks, \u003cem\u003eH. longicornis\u003c/em\u003e is a three-host tick with variable phenology depending on latitude [18,19].\u003c/p\u003e\n\u003cp\u003eEnzootic tick\u0026ndash;vertebrate\u0026ndash;tick cycle was influenced by ecological shifts, including climate variability, habitat fragmentation, and anthropogenic activities.\u0026nbsp;Rodents are frequently infested by ticks, providing blood meals necessary for the development of ticks in their immature stages. Rodents are considered competent reservoirs for various tick-borne pathogens, such as tick-borne encephalitis virus, \u003cem\u003eRickettsia spp\u003c/em\u003e., \u003cem\u003eEhrlichia spp\u003c/em\u003e., \u003cem\u003eCoxiella burnetii\u003c/em\u003e, \u003cem\u003eFrancisella tularensis\u003c/em\u003e, and \u003cem\u003eBabesia microti\u003c/em\u003e [20]. Additionally, rodents are significant reservoir hosts for many zoonotic infections, including Hantavirus hemorrhagic fever with renal syndrome, Hantavirus cardiopulmonary syndrome, plague, borreliosis, salmonellosis, murine typhus, leptospirosis, Lassa fever, rat-bite fever, and campylobacteriosis [21,22].\u003c/p\u003e\n\u003cp\u003eClimate change has led to an increased risk of tick-borne infectious diseases, as it enhances tick survival rates, egg-laying rates, and overall population growth [23]. Moreover, the general population, not just agricultural workers, is facing a higher risk of tick and rodent exposure due to the growing popularity of outdoor activities such as hiking and camping, as well as the increasing pet population [24]. We focused on \u003cem\u003eH. longicornis\u003c/em\u003e due to its dominance in the studied regions, parthenogenetic reproduction (enhancing population growth), and role as a vector for \u0026gt;30 human pathogens [7]. \u003cem\u003eS. dauricus\u003c/em\u003e was selected as a key rodent reservoir in Northeast China that supports immature tick development and zoonotic pathogen transmission. Investigating these species helps elucidate local disease risks and informs targeted surveillance. Given this context, to evaluate the potential ability of ticks and rodents to acquire endemic pathogens and the possible exposure risk to humans, we collected ticks from Liaoning Province in northeastern China and Anhui Province in central-eastern China, and rodents from Heilongjiang Province in northeastern China, from 2021 to 2022.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eSample Collection\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and Classification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn June 2021, in Helan Town, Liaoyang City, Liaoning Province, and in August 2022, in Hanshan County, Maanshan City, Anhui Province, we surveyed 30 forest and grassland sites for questing ticks. Ticks were collected by dragging a 1-m\u0026sup2; corduroy cloth over the grass in woodland areas or along roadsides. The cloth was inspected every 10-20 meters, and all attached ticks were removed. Additionally, in June 2021, mouse traps were placed at 12 field sites in the suburbs of Harbin City, Heilongjiang Province. The traps were set daily at 10:00 a.m. in front of rodent holes, with dirt sprinkled over them to reduce visibility. At 3:00 p.m. the same day, all traps were checked, and the captured rodents were collected and placed in cloth bags.\u003c/p\u003e\n\u003cp\u003eDuring sampling, background data such as landform, vegetation, and the latitude and longitude of the collection sites were recorded. Ticks and rodents were first identified morphologically and then processed individually for PCR amplification of the mitochondrial cytochrome oxidase I (COI) gene.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample Processing and Pathogenic Culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTicks were pooled by developmental stage (adults individually; nymphs in groups of 3) to prioritize detection sensitivity. Sex-based differences in pathogen prevalence are minimal in unfed ticks [25,26], and nymph pooling followed established protocols [26]. Cryopreserved ticks were first washed once with 75% ethanol and three times with sterile phosphate-buffered saline (PBS) to remove surface impurities. Subsequently, 500 \u0026mu;l of PBS was added to a 2-ml sterile glass tube for manual grinding using a glass pestle. For rodents, about 50 mg of liver tissue was aseptically ground following the same method. To eliminate environmental microorganisms, cysteine heart agar blood (CHAB) media containing five antibiotics (colistin, amphotericin, lincomycin, methicillin, and ampicillin) was prepared. From each tick and mouse group, 300 \u0026mu;l of the grinding solution was pipetted and evenly distributed onto freshly prepared CHAB plates with antibiotics, which were then incubated in a 5% CO₂ atmosphere. The CHAB plates were monitored every 24 hours, and when colonies appeared, individual colonies were isolated and inoculated onto new CHAB plates for bacterial proliferation. Freshly grown bacteria were scraped off and suspended in saline for DNA extraction, while another portion was mixed with glycerol preservation solution and stored at -70\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA/RNA Extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe remaining 200 \u0026mu;l of tick or mouse grinding solution was directly subjected to nucleic acid extraction using the AllPrep DNA/RNA Mini Kit (QIAGEN, Germany) according to the manufacturer\u0026rsquo;s instructions. The total extracted nucleotides were eluted twice with 60 \u0026mu;l of Elution Buffer. Nucleic acid concentration was measured using a NanoDrop 1000 spectrophotometer (Thermo Scientific, USA), and the samples were stored at -40\u0026deg;C until further use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular Detection of Pathogens\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll nucleic acid samples were initially tested for SFTSV and the bacterial mitochondrial 16S rRNA gene. SFTSV detection was carried out using one-step RT-PCR (Access RT-PCR System Kit, Promega, USA) with primers specific to the small RNA segment of the virus. Bacterial identification was first performed using the 16S rRNA gene, followed by further amplification with genus-specific primers to confirm the bacterial species. Nested PCR was employed for the amplification of all genes, and detailed information on the primers used for pathogen detection is shown in Table 1. PCR products were electrophoresed through 1.0% agarose gels, and the target amplicons were purified using the QIAquick PCR Purification Kit (QIAGEN, Germany). The purified PCR products were sent to Beijing Tianyihuiyuan Biotechnology Co., Ltd. (Beijing, China) for directional sequencing. All PCR products were sequenced bidirectionally, prioritizing longer fragments (\u0026gt;600 bp) to ensure accuracy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic Analysis and MIR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe pathogen sequences and mitochondrial cytochrome oxidase I (COI) gene sequences from ticks or rodents were aligned against those in GenBank using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic trees were constructed using the neighbor-joining method with 1000 bootstrap replicates, implemented in MEGA 11 software (http://www.megasoftware.net). The prevalence of pathogens was calculated as the minimum infection rate (MIR), which for grouped ticks was determined by dividing the number of positive groups by the total number of ticks.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eSample\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;collection and identification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFrom June 2021 to August 2022, a total of 1004 ticks (503 adults and 501 nymphs) were collected from Helan Town, Liaoning Province (450 adults and 432 nymphs) and Hanshan County, Anhui Province (53 adults and 69 nymphs), as well as 42\u0026nbsp;rodents\u0026nbsp;from Harbin City, Heilongjiang Province. Morphological and molecular identification confirmed that all ticks were \u003cem\u003eH. longicornis\u003c/em\u003e, divided into 594 groups, and all\u0026nbsp;rodents\u0026nbsp;were \u003cem\u003eS. dauricus\u003c/em\u003e. BLASTn analysis showed that the COI sequences of \u003cem\u003eH. longicornis\u003c/em\u003e and \u003cem\u003eS. dauricus\u003c/em\u003e had greater than 99.9% identity to reference sequences in GenBank. The COI sequences of \u003cem\u003eH. longicornis\u003c/em\u003e from Liaoning (OR492359) and Anhui (OR492358) showed the highest similarity, clustering on a branch with a node value of 100. Similarly, the COI sequences of \u003cem\u003eS. dauricus\u003c/em\u003e from Heilongjiang (OR492360) and the reference sequence from China (KP708706) also clustered on a branch with a node value of 100 (Fig. 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of Pathogen in Ticks and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eRodents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn 14 out of 670 groups of \u003cem\u003eH. longicornis\u003c/em\u003e, \u003cem\u003eRickettsia\u003c/em\u003e was detected, with an MIR of 1.4% (14/1004). \u003cem\u003eCa\u003c/em\u003e. R. jingxinensis was detected in 12 groups from Liaoning Province, while \u003cem\u003eR. heilongjiangensis\u003c/em\u003e was found in two groups from Anhui Province. \u003cem\u003eCoxiella\u003c/em\u003e-like endosymbiont (CLE) was detected in 20 groups, with an MIR of 2.0% (20/1004), including 7 groups from Liaoning and 13 from Anhui. Notably, co-infections with CLE were observed in samples hl182 and hl466 from Liaoning and st5 and st6 from Anhui. Unexpectedly, \u003cem\u003eL. pneumophila\u003c/em\u003e was detected in 2 out of 42 \u003cem\u003eS. dauricus\u003c/em\u003e from Heilongjiang, with an infection rate of 4.8% (2/42).\u003c/p\u003e\n\u003cp\u003eAdditionally, various bacteria were detected on the surface of ticks, including \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eStaphylococcus\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003ePaenibacillus\u003c/em\u003e, \u003cem\u003eEnterococcus\u003c/em\u003e, \u003cem\u003eBacillus parasporus\u003c/em\u003e, and \u003cem\u003eSalmonella\u003c/em\u003e. \u003cem\u003eStaphylococcus\u0026nbsp;\u003c/em\u003eand \u003cem\u003eEnterococcus\u003c/em\u003e were also detected in rodents. The distribution of these bacteria is shown in Table 2. In terms of microbial abundance, \u003cem\u003ePseudomonas\u003c/em\u003e and \u003cem\u003eStaphylococcus\u003c/em\u003e had the highest composition ratios on ticks from Liaoning, with 28.7% (84/293) and 15.7% (46/293), respectively. Using both pathogen culture and molecular biology methods on the same samples, the results provided mutual validation in some cases. For instance, 56 of 84 \u003cem\u003ePseudomonas\u003c/em\u003e strains isolated from \u003cem\u003eH. longicornis\u003c/em\u003e in Liaoning were also detected in the grinding solution of the corresponding samples, and 7 of 21 \u003cem\u003ePaenibacillus\u003c/em\u003e strains were similarly detected. However, in Anhui, 1 \u003cem\u003ePseudomonas\u003c/em\u003e strain, 3 \u003cem\u003eStaphylococcus\u003c/em\u003e strains, 7 \u003cem\u003eBacillus\u003c/em\u003e strains, and 7 \u003cem\u003eBacillus parasporus\u003c/em\u003e strains were isolated from \u003cem\u003eH. longicornis\u003c/em\u003e, and 26 \u003cem\u003eStaphylococcus\u003c/em\u003e strains and 13 \u003cem\u003eEnterococcus\u003c/em\u003e strains were isolated from \u003cem\u003eS. dauricus\u003c/em\u003e in Heilongjiang, none of which were detected in the grinding solution. This suggests that pathogen culture may be more sensitive than molecular methods for detecting these microorganisms on ticks. Additionally, no SFTSV was detected in any of the collected samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenetic and Phylogenic Analysis of \u003cem\u003eRickettsia\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 16S rRNA/\u003cem\u003errs\u003c/em\u003e (OR477299) and 17 \u003cem\u003ekDa\u0026nbsp;\u003c/em\u003e(OR500965) genes of 12 \u003cem\u003eCa\u003c/em\u003e. R. jingxinensis\u0026nbsp;isolates (tick73) from Liaoning clustered on the same branch as tick-XA188 and Xian-HL-21 from Shaanxi Province, with 99.89%\u0026nbsp;identity\u0026nbsp;and\u0026nbsp;99.77% identity, respectively. The \u003cem\u003eompA\u0026nbsp;\u003c/em\u003e(OR500967) gene clustered with Meixian-HI-242 from Shaanxi, YBHC-T32 from Jilin, and J244 from Shandong, sharing 99.98% homology. The \u003cem\u003egltA\u003c/em\u003e (OR500963), \u003cem\u003eompB\u0026nbsp;\u003c/em\u003e(OR500969), and \u003cem\u003esca4\u003c/em\u003e (OR500971) genes were clustered with those of Huaian-RM from Jiangsu, China, showing 100% identity (Fig.\u0026nbsp;2).\u0026nbsp;Genetic analysis revealed that the two Anhui strains of \u003cem\u003eR. heilongjiangensis\u003c/em\u003e shared highest homology with strain B8 (CP112971) for five genes (16S rRNA, \u003cem\u003e17kDa\u003c/em\u003e, \u003cem\u003eompA\u003c/em\u003e, \u003cem\u003egltA\u003c/em\u003e, and \u003cem\u003esca4\u003c/em\u003e). In contrast, their \u003cem\u003eompB\u003c/em\u003e sequences (OR500968) showed 100% identity with strain 054 (CP002912) from Heilongjiang, differing from B8 at 97.3% of sites (748/769).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenetic and Phylogenic Analysis of CLE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn seven CLE strains from Liaoning and 13 CLE strains from Anhui, their 16S rRNA (OR477301 and OR477300), \u003cem\u003egroEL\u003c/em\u003e (OR500973 and OR500972), and \u003cem\u003erpoB\u003c/em\u003e (OR500976 and OR500975) genes were clustered together with the 580 strain from Jiangxi Province (Fig. 3), all showing 100% homology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenetic and Phylogenic Analysis of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eL. pneumophila\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor \u003cem\u003eL. pneumophila\u003c/em\u003e detected in two \u003cem\u003eS. dauricus\u003c/em\u003e from Heilongjiang, the 16S rRNA (OR477302), \u003cem\u003egroEL\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e(OR500974), and \u003cem\u003emip\u003c/em\u003e (OR519871) genes clustered with those of the OLDA strain from a U.S. patient, with 96.9%, 99.81%, and 100% homology, respectively (Fig. 4).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, All PCR products were sequenced bidirectionally, which were divided into 670 groups. \u003cem\u003eCa\u003c/em\u003e. R. jingxinensis was found in 12 of 594 groups of \u003cem\u003eH. longicornis\u003c/em\u003e ticks from Helan Town, Liaoning Province, northeastern China, while \u003cem\u003eR. heilongjiangensis\u003c/em\u003e was identified in 2 of 76 groups of \u003cem\u003eH. longicornis\u003c/em\u003e ticks from Hanshan County, Anhui Province, middle-east China. The MIR of Rickettsia in \u003cem\u003eH. longicornis\u003c/em\u003e ticks was low at 1.4%. Typically, in unfed ticks from vegetation, the pathogen carriage rate [25,26] is lower than that in ticks collected from animals, where the rate can be as high as 20\u0026ndash;70% [27,28]. Notably, a Legionella species, highly similar to \u003cem\u003eL. pneumophila\u003c/em\u003e, was detected in 2 of 42 \u003cem\u003eS. dauricus\u003c/em\u003e from Harbin, Heilongjiang Province, northeastern China, which is known to cause disease in humans. The geographic distribution of detected pathogens (\u003cem\u003eCa\u003c/em\u003e. R. jingxinensis in Liaoning, \u003cem\u003eR. heilongjiangensis\u003c/em\u003e in Anhui) may reflect climate-influenced vector-host interactions. Future studies should incorporate climate variables to assess these relationships.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCa\u003c/em\u003e. R. jingxinensis was first discovered in Japan [29] and named in 2016 in Jingxin City, Jilin Province, China [30]. It is widely distributed across Asia and has been detected in several provinces in China, including Liaoning, Guangxi, Sichuan, Hebei, and Shaanxi, as well as in other Asian countries such as South Korea, Thailand, and India [31\u0026ndash;34]. Studies suggest that \u003cem\u003eCa.\u003c/em\u003e R. jingxinensis may be pathogenic to humans [35]. Of the six genes of \u003cem\u003eCa.\u003c/em\u003e R. jingxinensis detected from Liaoning, 16S rRNA/\u003cem\u003errs\u003c/em\u003e, \u003cem\u003e17kDa\u003c/em\u003e, and \u003cem\u003eompA\u003c/em\u003e showed the highest similarity with strains from Shaanxi, while \u003cem\u003egltA\u003c/em\u003e, \u003cem\u003eompB\u003c/em\u003e, and \u003cem\u003esca4\u003c/em\u003e showed the highest similarity with strains from Jiangsu. Additionally, \u003cem\u003eompA\u003c/em\u003e displayed similar high similarity with strains from Jilin and Shandong. In \u003cem\u003eH. longicornis\u003c/em\u003e from Anhui, \u003cem\u003eR. heilongjiangensis\u003c/em\u003e was detected, which was first isolated from \u003cem\u003eDermacentor silvarum\u003c/em\u003e ticks in Heilongjiang Province in 1982 [36] and later confirmed as a new Rickettsia species [37]. The epidemic zone of \u003cem\u003eR. heilongjiangensis\u003c/em\u003e has been reported primarily in northeastern China [38] and has also been found in ticks from Altay and the Russian Far East [39,40]. In this study, the two isolates of \u003cem\u003eR. heilongjiangensis\u003c/em\u003e from Anhui showed the highest homology across six genes, including 16S rRNA, \u003cem\u003e17kDa\u003c/em\u003e, \u003cem\u003eompA\u003c/em\u003e, \u003cem\u003egltA\u003c/em\u003e, and \u003cem\u003esca4\u003c/em\u003e, with strain B8 [41], which was isolated from a patient in the same province. It is speculated that the source of \u003cem\u003eR. heilongjiangensis\u003c/em\u003e in Hanshan may have originated within the province.\u003c/p\u003e\n\u003cp\u003eInterestingly, \u003cem\u003eL. pneumophila\u003c/em\u003e was detected in two liver samples (Heb1 and Heb3) from \u003cem\u003eS. dauricus\u003c/em\u003e in Heilongjiang. The three genes\u0026mdash;16S rRNA, \u003cem\u003egroEL\u003c/em\u003e, and \u003cem\u003emip\u003c/em\u003e\u0026mdash;showed the highest homology with the OLDA strain, which was isolated from a U.S. case, indicating that \u003cem\u003eS. dauricus\u003c/em\u003e may carry a highly pathogenic strain of \u003cem\u003eL. pneumophila\u003c/em\u003e. \u003cem\u003eLegionella\u003c/em\u003e species were first recognized in the summer of 1976 following an outbreak of pneumonia in Philadelphia, Pennsylvania, USA [42]. \u003cem\u003eLegionella\u003c/em\u003e was the first bacterium identified to multiply within protozoan hosts, primarily aquatic amoebae [43]. Human infections typically occur through inhalation of Legionella-containing aerosols generated by contaminated man-made water sources, such as showers [44]. This study may be the first report of rodents carrying \u003cem\u003eL. pneumophila\u003c/em\u003e, suggesting that preventive measures should target not only known rodent-borne pathogens but also \u003cem\u003eL. pneumophila\u003c/em\u003e infections from rodents.\u003c/p\u003e\n\u003cp\u003eThe distribution of SFTS cases in China is expanding, with a total of 27 provinces reporting cases. Recently, some suspected SFTS cases were reported in Hanshan, but \u003cem\u003eH. longicornis\u003c/em\u003e was not found to be infected with SFTSV in this study. This could be due to the low detection rate of free-living ticks [26], or it may be a result of the small sample size. Additionally, co-infections were identified in four groups of samples. CLE were detected in samples hl182 and hl466 from Liaoning, which also carried \u003cem\u003eCa\u003c/em\u003e. R. jingxinensis, and in samples st5 and st6 from Anhui, which were infected with \u003cem\u003eR. heilongjiangensis\u003c/em\u003e. The natural symbiont Coxiella has been shown to promote feeding behavior and blood intake in \u003cem\u003eH. longicornis\u003c/em\u003e [45]. Endosymbiotic bacteria often become essential or even irreplaceable for their hosts\u0026rsquo; fitness and reproduction [46]. Furthermore, \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eStaphylococcus\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003ePaenibacillus\u003c/em\u003e, \u003cem\u003eEnterococcus\u003c/em\u003e, \u003cem\u003eBacillus parasporus\u003c/em\u003e, and \u003cem\u003eSalmonella\u003c/em\u003e were detected on the surface of the ticks, which is similar to the pathogen spectrum seen in ticks from animals [47]. This suggests the possibility of acquiring conditional pathogens through mechanical transmission during tick exposure, although the human pathogenicity of most of these bacteria remains to be determined. \u003cem\u003eStaphylococcus\u003c/em\u003e and \u003cem\u003eEnterococcus\u003c/em\u003e were also detected in rodents, adding to our understanding of the pathogens carried by rodents.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, two SFGR species, \u003cem\u003eCa.\u003c/em\u003e R. jingxinensis and \u003cem\u003eR. heilongjiangensis\u003c/em\u003e, were detected in \u003cem\u003eH. longicornis\u003c/em\u003e from Liaoning and Anhui, with four groups co-infected with CLE. Notably, two \u003cem\u003eS. dauricus\u003c/em\u003e from Heilongjiang were found to carry \u003cem\u003eL. pneumophila\u003c/em\u003e. These findings expand our understanding of the role ticks and rodents play in the local ecosystem, particularly in pathogen acquisition and transmission. The threat posed to public health and veterinary medicine should not be underestimated, and there is a need for enhanced surveillance and investigation of \u003cem\u003eH. longicornis\u003c/em\u003e and \u003cem\u003eS. dauricus\u003c/em\u003e as pathogen carriers.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInvestigation\u0026mdash;Y. S., L. G., S. G., L. M., D. S., Y. G., Y. J., K. Y., X. L., M. W.; investigation and data curation\u0026mdash;W. W., R. Z. and G. Y.; resources\u0026mdash;T. Q.; conceptualization, investigation, resources, and writing \u0026ndash; review \u0026amp; editing\u0026mdash;Y. W.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the Natural Science Foundation of Beijing Municipality (Grant No. 7242188) and the National Natural Science Foundation of China (Grant No.\u0026nbsp;81874275).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting this study are available within the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics and Consent to Participate declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eDeclaration of Interest Statement\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJongejan, F., Uilenberg, G., 2004. 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The Recent Evolution of a Maternally-Inherited Endosymbiont of Ticks Led to the Emergence of the Q Fever Pathogen. Coxiella Burnetii. PLoS Pathog, 11, e1004892. doi:10.1371/journal.ppat.1004892.\u003c/li\u003e\n\u003cli\u003ePascale, M.R., Salaris, S., Mazzotta, M., Girolamini, L., Fregni Serpini, G., Manni, L., Grottola, A., Cristino, S., 2021. New Insight Regarding Legionella Non- Pneumophila Species Identification: Comparison between the Traditional Mip Gene Classification Scheme and a Newly Proposed Scheme Targeting the rpoB Gene. Microbiol Spectr, 9, e01161-21. doi:10.1128/Spectrum.01161-21.\u003c/li\u003e\n\u003cli\u003eValeria Gaia, Norman K Fry, Timothy G Harrison, Raffaele Peduzzi. 2003. Sequence-Based Typing of Legionella pneumophila Serogroup 1 Offers the Potential for True Portability in Legionellosis Outbreak Investigation. J Clin Microbiol, 41(7):2932-9. doi: 10.1128/JCM.41.7.2932-2939.2003.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Spotted fever group Rickettsia (SFGR), Rickettsia heilongjiangensis, Candidatus Rickettsia jingxinensis, Haemaphysalis longicornis, Coxiella-like endosymbiont (CLE), Tick","lastPublishedDoi":"10.21203/rs.3.rs-6464024/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6464024/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u003c/strong\u003eIn recent years, tick-borne and rodent-borne pathogens have increasingly threatened humans and domestic animals. However, the types and distribution of pathogens they carry remain poorly understood in China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e In this study, we collected a total of 1004 \u003cem\u003eHaemaphysalis longicornis\u003c/em\u003e ticks (503 adults and 501 nymphs), divided into 670 groups, from Liaoning Province in northeastern China and Anhui Province in middle-eastern China, along with 42 \u003cem\u003eSpermophilus dauricus\u003c/em\u003e from Heilongjiang Province in northeastern China. Morphological analysis and cytochrome oxidase I (\u003cem\u003eCOI\u003c/em\u003e) sequencing were used to identify tick and rodent species. Using nested PCR, all samples were first tested for severe fever with thrombocytopenia syndrome virus (SFTSV) and the bacterial 16S rRNA gene, followed by detection of the corresponding genus-specific genes. Samples were tested for Rickettsia 16S rRNA/\u003cem\u003errs\u003c/em\u003e, \u003cem\u003e17kDa\u003c/em\u003e, \u003cem\u003egltA\u003c/em\u003e, \u003cem\u003eompA\u003c/em\u003e,\u003cem\u003e ompB\u003c/em\u003e, and \u003cem\u003esca4\u003c/em\u003egenes; for Coxiella 16S rRNA, \u003cem\u003egroEL\u003c/em\u003e, and \u003cem\u003erpoB\u003c/em\u003e genes; and for Legionella \u003cem\u003egroEL\u003c/em\u003e and \u003cem\u003emip\u003c/em\u003e genes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e Spotted fever group Rickettsia spp. (SFGR) were identified in a total of 14 tick groups: \u003cem\u003eCandidatus\u003c/em\u003e Rickettsia jingxinensis (12/594) from Liaoning and \u003cem\u003eRickettsia heilongjiangensis\u003c/em\u003e (2/76) from Anhui. Four of the 14 groups were co-infected with \u003cem\u003eCoxiella\u003c/em\u003e-like endosymbionts (CLE). Notably, \u003cem\u003eLegionella pneumophila\u003c/em\u003e was detected in 2 of 42 \u003cem\u003eS. dauricus\u003c/em\u003e from Heilongjiang, with genetic similarity to the OLDA strain from a U.S. patient exceeding 99.8% for 16S rRNA, \u003cem\u003egroEL\u003c/em\u003e, and \u003cem\u003emip \u003c/em\u003egenes, confirming the high pathogenic potential of these strains in humans.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions \u003c/strong\u003eOur findings suggest that local residents should be cautious about potential infections from these two SFGR species, and we recommend further surveillance and investigation of \u003cem\u003eH. longicornis\u003c/em\u003e and \u003cem\u003eS. dauricu\u003c/em\u003es as pathogen carriers.\u003c/p\u003e","manuscriptTitle":"Molecular detection of pathogens in ticks and rodents, Northeast and Middle-east China","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-07 08:47:01","doi":"10.21203/rs.3.rs-6464024/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bd4dcff3-000e-4e70-98ff-faeb022ef6bc","owner":[],"postedDate":"May 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-18T19:53:27+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-07 08:47:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6464024","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6464024","identity":"rs-6464024","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}