Molecular detection of spotted fever group rickettsiae in hedgehogs (Erinaceus amurensis) and hedgehog-attached ticks in Xuyi County, Southeast China

Tick-borne diseases like Rickettsia, Anaplasma and Ehrlichia are widespread infectious zoonoses that threaten the health of both humans and animals worldwide. Ticks and their hosts, such as hedgehogs, can play a crucial role in transmitting tick-borne diseases and the cycle of Rickettsia. To investigate the presence and identity of Rickettsia in hedgehogs and hedgehog-attached ticks in Xuyi County, Southeast China, 114 ticks were collected from 45 hedgehogs captured totally. Via morphological and molecular methods, all these ticks were identified as two species: Haemaphysalis flava (110/114, 96.5%) and Haemaphysalis longicornis (4/114, 3.5%). Rickettsia spp. were genotypically characterized by PCR targeting rrs, gltA, ompA, ompB, and sca4 gene fragments. The prevalence of spotted fever group rickettsiae (SFGR) infection found in hedgehogs and ticks was 17.8% (8/45) and 78.1% (89/114), respectively. Phylogenetic analyses demonstrated that those Rickettsia spp. belong to two species: Rickettsia heilongjiangensis (R. heilongjiangensis XY-1) and a potential new species, Candidatus Rickettsia xuyiensis XY-2. The present study gave the first evidence of R. heilongjiangensis and Candidatus R. xuyiensis in ticks and hedgehogs of Southeast China. Our findings suggest that hedgehogs might be involved in the natural transmission cycle of Rickettsia species. Supplementary Information The online version contains supplementary material available at 10.1007/s10493-022-00721-y.

Ticks are the primary vector and reservoir of Rickettsia. The spotted fever group rickettsiae (SFGR), including pathogenic and nonpathogenic species found worldwide, are transmitted mainly by hard ticks (Ixodidae) to vertebrate hosts (Parola et al. 2000;Socolovschi et al. 2009). In China, > 110 species of hard ticks have been identified, of which Haemaphysalis longicornis and H. flava are the most common species throughout China (Zhang et al. 2019). A variety of Rickettsia species-including R. japonica, R. heilongjiangensis, R. raoultii, Candidatus Rickettsia tarasevichiae and Candidatus Rickettsia principis-have been screened out from H. longicornis and H. flava (Fang et al. 2021;Jiang et al. 2018;Liu et al. 2016).
Hedgehogs mainly inhabit natural open and green spaces as well as artificial, rural and urban areas, including farmlands, parks, gardens, scrubby habitats at the edge of forests, and shrubby vegetation. They feed on a broad spectrum, including caterpillars, earthworms, small vertebrates, bird eggs, and berries and fruits (Reuter et al. 2019). Hedgehogs are crucial wild animal hosts for various ticks, including Ixodes hexagonus, H. flava, H. longicornis, H. erinacei, H. aegyptium, H. marginatum and Rhipicephalus sanguineus (Jahfari et al. 2017;Khaldi et al. 2012;Marié et al. 2012;Szekeres et al. 2019;Orkun et al. 2019; Barradas et al. 2021). Hedgehogs' ecological and feeding habits, along with high population densities, resulting in their frequent contact with either human or domestic and wild animals, implicates the possibility of tick-borne diseases (Delogu et al. 2020). Therefore, hedgehogs may be involved in the ecology of several potential emerging pathogens.
A wide range of tick-borne bacteria has been reported in hedgehogs and their attached ticks (Skuballa et al. 2010;Szekeres et al. 2019;Bolanos-Rivero et al. 2017;Gong et al. 2020). Therefore, from a public health perspective it is of great importance to understand the local tick species, tick hosts, and SFG rickettsiae carried by them. Despite the previous extensive efforts of clarifying this problem, the knowledge about the circulation of SFG rickettsiae in areas of Southeast China, such as Xuyi County, Jiangsu Province, is still unclear (Jiang et al. 2010;Tan et al. 2012;Li et al. 2018a, b). Therefore, to evaluate the prevalence of SFG rickettsiae within the Southeast China region, the present study collected free-ranging hedgehogs and ticks from Xuyi County, Southeast China, and investigated their diversity and related SFG rickettsiae, in order to provide a scientific basis for the prevention and control of SFGR.

Ethical approval
All procedures and protocols for sample collection and processing were approved by the Administrative Committee on Animal Welfare of the Institute of Jiangsu CDC Veterinary and the Ethics Committee of the CDC of Eastern Theater (approval nrs. 2017011 and 2018012; approval dates 26-10-2017 and 15-08-2018).

Ticks and animal collection
Between November 2017 and April 2019, 45 hedgehogs were captured from forest sites near Tieshan Temple in Xuyi County, Jiangsu Province, China (Fig. 1). After careful examination, ectoparasitic ticks were removed from the hedgehogs using fine forceps and placed individually into 1.5-mL tubes with 70% cleaning ethanol. After cleaning, all ticks were marked with the collection date and stored at −80 °C. The tick species were identified based on morphological criteria (Deng and Jiang 1991) and molecular biology tools (Liu et al. 2016). After being anesthetized with diethyl ether, all hedgehogs were sacrificed to collect muscle tissue, hearts, livers, spleens, lungs, kidneys, brains, and intestines, which were all stored at −80 °C.

DNA extraction
Ticks and hedgehog tissues were homogenized with a stroke-physiological saline solution individually. Homogenates were centrifuged for 10 min at 1000×g and 4 °C, and pellets were collected for DNA extraction. Genomic DNA was extracted from each specimen by using the MiniBEST Universal Genomic DNA Extraction Kit (Takara, Beijing, China) according to the manufacturer's instructions and subsequently stored at −20 °C before use.

PCR amplification and sequencing
To identify the species of each hedgehog, a partial sequence of the mitochondrial 16S rRNA gene-approximately 201-211 nucleotides (nt) in length-was PCR-amplified using genomic DNA from hedgehog muscle tissues, based on primers (HedF and HedR) as described by Sarri et al. (2014). To identify the species of each tick, the mitochondrial 16S rRNA gene from the genomic DNA of each tick was PCR-amplified using the forward and reverse primers TickHF and TickHR (Liu et al. 2016).
The rickettsial citrate synthase (gltA) gene was chosen as the target for its genus specificity and conservativeness (Mediannikov et al. 2004). All samples were screened for the presence of gltA by nested PCR using two sets of primers, RpCS877F and RpCS1258R, and approximately a 380 bp fragment of the gltA gene was amplified. The second PCR round will be performed if no product was visible by agarose gel electrophoresis. The full-length of gltA gene was amplified in 22 tick samples using primers CS2d and CSEndr. To further characterize SFGR strains, each positive sample for the gltA gene was tested for four other genes: the 16S ribosomal RNA gene (16S rRNA), outer membrane protein A gene (ompA), outer membrane protein B gene (ompB), and surface cell antigen-4 gene (sca4). The primer sets used in each of these assays are listed in Table 1. Sterile distilled water and a previously determined rickettsial-positive tick sample were used as negative and positive controls in each run, respectively. All positive amplicons were purified with PCR Clean-Up Kit (Beyotime, Shanghai, China). Sanger dideoxy DNA sequencing was performed using the BigDye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and an ABI Prism 3130 × genetic analyzer.

Phylogenetic analysis
Partial nucleotide sequences of rrs, gltA, ompA, ompB, and sca4 obtained from ticks, and hedgehog organs were compared to known sequences using the BLAST program from the NCBI website (https:// blast. ncbi. nlm. nih. gov/ Blast. cgi). The best-fit evolution model for each gene was calculated via MrModeltest v.2.3 in cooperation with BEAST v.1.10.4 using the Bayesian information criterion (BIC). The K81u (K3Pu), TVM, TPM3u, TN(TN93) and K81u (K3Pu) models were selected for the gltA, ompA, ompB, rrs, and sca4 gene, respectively. Substitution rates at polymorphic sites in both genes followed a gamma distribution with a large proportion of invariable sites. Maximum Likelihood (ML) methods of phylogeny inference on the individual were conducted in BEAST in the 'Ultrafast' bootstrap model with the 5000 bootstrap samples, maximum interaction value 1000, and minimum correlation coefficient of 0.90. Nodal support was evaluated by bootstrap resampling for the ML trees from posterior probabilities (PP) for Bayesian inferences. Bootstrap values of 70% or more and Bayesian support values of 0.95 and higher were considered significant nodal support.

Results
The amplified partial sequences (203-215 nt) of the hosts' 16S rRNA gene (GenBank acc. nr. of the hedgehogs: OM865773) showed the highest nucleotide similarity (96.0-97.0%) to those of Erinaceus amurensis from the GenBank database (KX9646061). Therefore, all hedgehogs (n = 45) were identified as E. amurensis. 17.8% (8/45) of the hedgehogs were PCR positive for rickettsial gltA. The highest infection rate of SFGR in the eight positive hedgehogs was seen in the brain, whereas other organs varied in their presence of rickettsial gltA (Table 2). A total of 114 adult ticks were collected from 45 hedgehogs and identified as H. flava and H. longicornis through their morphological characteristics and partial 16S rRNA gene (  Fig. 2A-E, respectively), inferred from these genes, also showed one isolated strain formed a distinct cluster with R. heilongjiangensis in all trees, which also confirmed the identification of R. heilongjiangensis (Rickettsia heilongjiangensis XY-1).

Discussion
This study reports Rickettsia sp.'s finding in hedgehogs and ticks from Jiangsu Province, Southeast China. In addition, this is the first report of R. heilongjiangensis and a novel potential species of Rickettsia (Candidatus R. xuyiensis XY-2) in H. flava and hedgehogs of Xuyi, Southeast China. SFGRs are widely distributed throughout China and tend to have regional characteristics. Previous studies have revealed the extensive diversity of rickettsiae among tick species and geographic areas (El-Mahallawy et al. 2015;Fang et al. 2021). There are many hills and low mountains in the Xuyi area, and it has a developed presence of animal husbandry. Many tick bites have been reported among fever cases with thrombocytopenia syndrome in Jiangsu Province (Li et al. 2017). Therefore, improving the knowledge on the prevalence of Rickettsia in ticks and hosts from this region can identify potential rickettsioses in the population and reduce the risk for tick-borne Rickettsia transmission.
Small mammals and ticks are intermediate hosts or vectors of many zoonoses. Hedgehogs, one of the most important hosts of ticks, can play an essential role in the natural foci of tick-borne pathogens (Orkun et al. 2019). Our results demonstrate that the dominant tick species carried by hedgehogs in the Xuyi area is H. flava, followed by H. longicornis, which agrees with the findings of both Sun et al. (2009) andLan et al. (2016). The number of ticks carried by each hedgehog in this study may vary significantly due to the sampling season or the activity tracking of hedgehogs. Additionally, an initial screening test using gltA nested PCR revealed that 78.1% of the ticks and 17.8% of the hedgehogs were infected with SFG rickettsiae. This percentage was significantly higher than previous work from the Sichuan (33.5%), Yunnan (12.1%), and Zhejiang (7.5%) provinces Sun et al. 2015;Zhang et al. 2018). We found that the prevalence of SFGR infection of ticks in eight positive hedgehogs was 76.9 ~ 100%, and most SFGR infection of ticks collected from negative hedgehogs was 0-100%. Of 89 ticks infected with SFG rickettsiae, 63 positive ticks were carried by eight positive hedgehogs ( Table 2). As the ticks may suck the blood of these hedgehogs, they had a high positive rate of spotted fever.
Furthermore, we determined partial sequences of the gltA gene of SFG rickettsiae by conventional PCR in 45 hedgehogs organs (heart, brain, intestine, spleen, lung, liver, kidney). Based on the sequences of the gltA gene obtained from 45 hedgehogs' organs, the highest infection rate of SFG rickettsiae was detected in the brain (5/45); other organs varied in their presence of rickettsial gltA (Table 2). Therefore, the brain of the hedgehog may be particularly susceptible to Rickettsia. Our findings also indicate that hedgehogs and their carrying ticks can serve as the animal host and vector for SFG rickettsiae.
For the molecular classification of SFG rickettsiae that were obtained in the study, partial sequences of rrs, gltA, ompA, ompB, and sca4 were analyzed. Phylogenetic trees inferred from rrs, gltA, ompA, ompB, and sca4 analysis are shown in Fig. 2. One isolated strain formed a distinct cluster with R. heilongjiangensis in all trees (Fig. 2) and thus were identified as R. heilongjiangensis (XY-1). Nucleotide sequence analysis of five genes of R. heilongjiangensis XY-1 showed 97-100% similarity with R. heilongjiangensis isolate Xinxian-HL9 and R. japonica YH_M. The isolated strain could not be classified into specific species due to a lack of consensus between the phylogenetic trees. It shares a branch with the previously reported Candidatus R. principis isolate Kh-81, uncultured Rickettsia sp. Hme_2021, uncultured Rickettsia sp. clone 7-17, whereas it forms a separate branch in the ompB and sca4 phylogenetic tree (Fig. 2). Moreover, in the isolated strain, the sequence nucleotide identity to recognized Rickettsia species was < 99.8, 99.9, 98.8, 99.2 and 99.3% for rrs, gltA, ompA, ompB, and sca4, respectively, which suggests that this agent is novel potential SFG Rickettsia according to Fournier et al. (2003). Therefore, this species was provisionally named Candidatus R. xuyiensis-XY2, concerning the location where it was found. Our findings indicate that hedgehogs and H. flava collected from hedgehogs in Southeast China were infected with R. heilongjiangensis and Candidatus R. xuyiensis. The diseases caused by these pathogens should therefore be monitored in Southeast China. Further isolation and identification are needed to obtain morphological characteristics and the entire genome of these species. Previous studies have reported that H. longicornis is an essential vector of R. heilongjangensis (Jiang et al. 2019;Liu et al. 2020;Zhuang et al. 2018). However, no rickettsiae were detected in any of the H. longicornis ticks collected in our study, which may be due to the limited sample size of H. longicornis ticks.
There are some limitations of this study worth noting. Firstly, our investigation is biased because the infection rates were calculated using ticks collected from the infected hedgehogs, where we collected fewer ticks from uninfected hedgehogs. Therefore, the actual infection rates might be lower than those determined by this research. Secondly, we mainly focused on the infection rates and tick species collected from hedgehogs, and we did not identify ticks carried by other small mammals in the Xuyi area. Thus, it is crucial to find Rickettsiales infection among other local animals and humans in a subsequent study.