Molecular study of Rickettsia species in ticks and blood collected from hedgehogs (Erinaceus europaeus) and tortoises (Testudo graeca) in Iran

TL;DR

This study investigates Rickettsia in ticks and blood from hedgehogs and tortoises in Iran, finding infected ticks but no Rickettsia in the blood.

Contribution

The study provides new molecular evidence of Rickettsia in ticks from hedgehogs and tortoises in Iran.

Findings

Rickettsia DNA was detected in ticks, with the highest prevalence in R. turanicus.

No Rickettsia was found in the blood samples from hedgehogs or tortoises.

Hedgehogs and tortoises are confirmed as hosts for Rickettsia-infected ticks.

Abstract

The family Rickettsiaceae, especially the genus Rickettsia, refers to obligate intracellular pathogens responsible for zoonotic diseases such as rickettsiosis. Primarily transmitted through arthropod vectors such as ticks, fleas, and lice, they pose serious concerns for both public health and veterinary fields.

This study is conducted in northwestern Iran to investigate ticks collected from European hedgehogs (Erinaceus europaeus) and spur-thighed tortoises (Testudo graeca), with specific focus on the molecular detection of Rickettsia spp. In Hyalomma and Rhipicephalus tick species. Due to their ecological distribution and proximity to human habitats, hedgehogs and tortoises are important hosts for the maintenance and spread of these pathogens. A total of 106 ticks and 31 blood samples were collected from hedgehogs, while 234 ticks and 87 blood samples were collected from tortoises. Hedgehog blood sampling was performed directly from the heart under deep anesthesia to minimize stress, ensuring full recovery before release; tortoise blood was collected randomly under sterile conditions. All specimens were transferred to the Faculty of Veterinary Medicine at the University of Urmia. Overall, 339 tick samples and 118 blood samples were tested. Rickettsia DNA was detected using Real-time PCR (16S rRNA gene) and conventional PCR (gltA gene) in tick species including H. aegyptium, R. sanguineus, R. turanicus, and R. bursa. The highest prevalence was observed in R. turanicus. None of the blood samples were positive for Rickettsia.

These results confirm the role of hedgehogs and tortoises as hosts for Rickettsia-infected ticks and underscore the need for further molecular studies and expanded wildlife surveillance for better assessment of the zoonotic risk.

Figures

Pictures of hedgehogs and tortoises with ticks on their bodies.Fig. 1Table 1Number of ticks collected from different body parts of specified hosts.Table 1Host AnimalsBody PartsAnimal typeExaminedBellyHead and neck regionAnal regionLegsBetween the quillsTotal collectionHedgehogs3195810029106Turtles15010758960–234

Geographic map of the study locations in West Azerbaijan, Iran.Fig. 2

Agarose gel image PCR test on hedgehog ticks to identify *Rickettsia* sp., with *gltA* gene (834 bp), which Lane 1-marker 100 bp DNA (Smobio Technology Inc., Taiwan), Lanes 1–5 positive samples,6 Negative sample and 8 Lane of negative control).Fig. 3

A phylogenetic tree was constructed based on partial sequences (835 base pairs) of the *gltA* gene from *Rickettsia* species, incorporating both sequences obtained in this study and reference sequences retrieved from the GenBank database. The sequence generated in this study is distinctly highlighted in the tree using two unique symbols: two red bold Circle and two Blue bold lozenge, to facilitate differentiation from the reference sequences and to illustrate its phylogenetic position.Fig. 4

Full text

Introduction

1

As the second most important vectors of pathogens after mosquitoes, ticks transmit viruses, bacteria, and protozoa that may cause disease in humans and animals, leading to economic losses in livestock [1]. Among them, Rickettsia spp. obligates intracellular Gram-negative bacteria are globally distributed, with the spotted fever group (SFG) being the largest and mainly transmitted by hard ticks [2], [3]. Key species include R. conorii and R. massiliae.

European hedgehogs (Erinaceus europaeus) are widespread in Europe, often living near humans and carrying ectoparasites such as Ixodes hexagonus, I. ricinus, Rhipicephalus sanguineus, Hyalomma spp., and the flea Archaeopsylla erinaceid [4]. These vectors transmit Rickettsia spp., Borrelia burgdorferi, Anaplasma phagocytophilum, and tick-borne encephalitis virus. Hedgehogs can also host R. felis-like organisms with uncertain pathogenicity. Studies in Europe and Asia confirm their role in maintenance and spread of the Rickettsial pathogens [5], [6].

Tortoises host Hyalomma aegyptium, whose larvae and nymphs feed on multiple hosts, including humans, enabling transmission of zoonotic pathogens such as R. africae, R. aeschlimannii, and Coxiella burnetiid [7], [8]. International reptile trade risks introducing exotic ticks and pathogens, emphasizing the need for surveillance [9]. Tortoise ticks also harbor endosymbionts that may influence pathogen transmission [10].

Ticks serve as both vectors and reservoirs for Rickettsiae through transstadial, transovarial, and other transmission routes. Hedgehogs and tortoises, through their host–tick relationships, play important roles in the ecology of these pathogens. However, genomic diversity and host roles remain poorly understood in certain regions, including Iran [11].

In this regard, the present study investigates the genomic presence of Rickettsia in ticks from hedgehogs and tortoises particularly Hyalomma and Rhipicephalus species to identify circulating pathogens, assess their potential risk, and improve understanding on disease prevention and control.

Materials and methods

2

Blood and tick sampling

2.1

A descriptive cross-sectional study was conducted in spring and summer 2024 to collect tick and blood samples from hedgehogs (Erinaceus europaeus) and tortoises (Testudo graeca) in northwestern Iran (Fig.1). Due to the small size of the animals and the difficulty in accessing peripheral veins, thirty-one blood samples were aseptically collected directly from the heart under deep anesthesia using ketamine and xylazine (ketamine at a dose of 10 mg/kg and xylazine at a dose of 0.1 mg/kg) to minimize pain and distress. The samples were immediately placed in EDTA-containing tubes and stored in specialized containers on ice. After full recovery from anesthesia, all animals were released back into their natural habitat. Additionally, 106 ticks were collected from various body surfaces (abdomen, head and neck, anal region, legs, and between spines) of hedgehogs (Table 1) and preserved in 96% alcohol. Concurrently, from 150 spur-thighed tortoises examined in West Azerbaijan, 87 blood samples were randomly collected and immediately frozen on ice. Furthermore, 234 ticks were randomly collected from different body parts of the tortoises and stored in sterile glass containers with 95% ethanol [12]. Tick samples were first sent to the parasitology laboratory at the same faculty for species identification and subsequently transferred to the microbiology laboratory for further analyses. Additionally, as part of this study, four blood samples were collected in total: one from a Persian fallow deer (Dama mesopotamica), one from a red deer (Cervus elaphus), and two from wild rams. The samples were obtained aseptically by trained wildlife expert during authorized animal translocation procedures. All blood specimens were transported to the Microbiology Laboratory at the Faculty of Veterinary Medicine, University of Urmia, for species identification and molecular analyses.Fig. 1. Pictures of hedgehogs and tortoises with ticks on their bodies.Fig. 1. Table 1Number of ticks collected from different body parts of specified hosts.Table 1. Host AnimalsBody PartsAnimal typeExaminedBellyHead and neck regionAnal regionLegsBetween the quillsTotal collectionHedgehogs3195810029106Turtles15010758960–234

Study area

2.2

West Azerbaijan Province possesses exceptional biodiversity, with diverse habitats from wetlands and mountain forests to open plains supporting a wide range of native wildlife. Key species include the European hedgehog (Erinaceus europaeus) and the Greek tortoise (Testudo graeca), both of crucial ecological importance. Hedgehogs help regulate insect and rodent populations, while tortoises promote seed dispersal and improve soil structure. Additionally, both species are natural hosts for ticks, making them key players in the cycle of vector-borne pathogens and valuable indicators of ecosystem health (Fig.2).Fig. 2. Geographic map of the study locations in West Azerbaijan, Iran.Fig. 2

DNA extraction from blood and ticks

2.3

Genomic DNA was extracted from blood samples using the FavorPrep™ Blood Genomic DNA Extraction Mini Kit (Favorgen, Taiwan), following the manufacturer's protocol. In a typical procedure, ticks were washed twice with phosphate-buffered saline (PBS), air-dried, and rapidly frozen in liquid nitrogen. After freezing, they were pooled in groups of five, crushed with a scalpel blade, and transferred to 2 mL microcentrifuge tubes. DNA extraction was performed using the RXNS Tissue and Blood DNA Extraction Kit (Iran Biotechnology Company). The quality and concentration of extracted DNA were evaluated using a NanoDrop 2000c spectrophotometer (Thermo Scientific, USA). Absorbance of the samples was measured at 260 nm, and the 260/280 nm ratio was used to evaluate the DNA purity [13], [14].

Molecular study based on nested-PCR method

2.4

To detect the genus Rickettsia sp., Real-Time PCR with SYBR Green was performed targeting the 16SrRNA gene using the forward (F: 5’-CGCAACCCTCATTCTTATTTGC-3′) and reverse (R: 5’-CGCAACCCTTATTCTTATTTGC-3′) primers, yielding a 149 bp amplicon. The thermal cycling conditions involved an initial denaturation at 94 °C for 5 min, followed by 36 sixty-second cycles at 94 °C, 56 °C, and 72 °C. Species identification of Rickettsia involved conventional PCR targeting of the gltA gene, producing an 834 bp amplicon using degenerate primers: forward (F: 5’-GCTCTTCTCATCCTATGGCTATTA-3′), reverse 1 (R1: 5’-CAGGGTCTTCRTGCATTTCTT-3′), and reverse 2 (R2: 5’-CAGGGTCTTCATGCATTTCTT-3′). The thermal profile consisted of one cycle at 95 °C for 4 min, followed by 32 ninety-second cycles at 95 °C, 58 °C, and 72 °C, with a final extension at 72 °C for 7 min.

The PCR was performed in a 25 μL reaction mixture containing 4 μL of template DNA, 1 μL of each primer, and 12.5 μL of master mix, with the remaining volume adjusted with sterile distilled water. A touchdown PCR program was employed to prevent non-specific amplification. The specific touchdown thermal program, along with the PCR conditions outlined in Table 1, was set up on a thermocycler (Quanta Biotech, UK). For nested PCR, 2.5 μL of the standard PCR product was used as the template, with other components added as in the initial stage. PCR products were then electrophoresed on a 1.5% agarose gel and stained with a safe dye (Labnet, ENDURO, USA). The results were visualized by the Genius gel documentation system developed by Syngene Bio-Imaging [15], as shown in Fig. 2, Fig. 3.Fig. 3. Agarose gel image PCR test on hedgehog ticks to identify Rickettsia sp., with gltA gene (834 bp), which Lane 1-marker 100 bp DNA (Smobio Technology Inc., Taiwan), Lanes 1–5 positive samples,6 Negative sample and 8 Lane of negative control).Fig. 3

Sequences and phylogenetic analysis

2.5

The obtained sequences were edited using Geneious Prime software (version 2022.01, Biomatters). Consensus sequences were compared with reference sequences in the GenBank database using the BLASTn algorithm. Multiple sequence alignment was performed using CLUSTAL X [16]. Phylogenetic trees were constructed using MEGA version 11.0, employing the Neighbor-Joining (NJ) method (Fig. 4). To evaluate the reliability of the branching patterns, a consensus tree reflecting the evolutionary lineage was generated from 1000 replicates through bootstrap analysis [17], and values greater than 70% were considered significant. The tree was rooted using Brucella abortus (Accession No. CP044339) as the outgroup. All sequences generated in this study have been deposited in GenBank under accession numbers PV684243, PV685004, PV692319, and PV692320.Fig. 4A phylogenetic tree was constructed based on partial sequences (835 base pairs) of the gltA gene from Rickettsia species, incorporating both sequences obtained in this study and reference sequences retrieved from the GenBank database. The sequence generated in this study is distinctly highlighted in the tree using two unique symbols: two red bold Circle and two Blue bold lozenge, to facilitate differentiation from the reference sequences and to illustrate its phylogenetic position.Fig. 4

Results

3

In this study, a total of 339 tick samples and 118 blood samples were collected from spur-thighed tortoises (Testudo graeca) and hedgehogs (Erinaceus europaeus), comprising 234 tick samples from tortoises and 106 from hedgehogs, as well as 87 blood samples from tortoises and 31 from hedgehogs. To detect bacterial presence, the 16SrRNA gene was analyzed using real-time PCR, while the gltA gene was utilized to identify the genus Rickettsia. Ticks of the genera Rhipicephalus and Hyalomma were collected from both animal species. Analysis of the 16SrRNA gene in ticks from tortoises revealed 5 positive samples (n = 234; 2.14%; 95% CI: 0.92%–4.91%), all belonging to the species Hyalomma aegyptium. The tick species were predominantly collected from tortoises of the region. In ticks collected from hedgehogs, samples tested positive for Rickettsia. Notably, the tick species isolated from hedgehogs included Rhipicephalus sanguineus, Hyalomma aegyptium, Rhipicephalus turanicus, and Rhipicephalus bursa, with the highest prevalence of infection observed in R. turanicus and the lowest in H. aegyptium and R. bursa (Table 2). In this study, blood samples from one Persian fallow deer (Dama mesopotamica), one red deer (Cervus elaphus), and two wild rams (Ovis orientalis) were analyzed using PCR and Real Time PCR to detect the presence of Rickettsia spp. DNA. None of these blood samples tested positive for Rickettsia spp., suggesting the absence of active bacteremia in these individuals indicating the limited role of these ungulate species as systemic reservoirs for Rickettsia in the studied region. Due to the high specificity and sensitivity of real-time PCR, a greater number of Rickettsia-infected samples were detected by this method compared to conventional PCR. However, conventional PCR targeting the Rickettsia-specific gltA gene was employed to determine the sequence, construct a phylogenetic tree, and accurately identify the Rickettsia species isolated from ticks of tortoises and hedgehogs (Fig. 3). None of the blood samples collected from tortoises or hedgehogs were positive for either the 16SrRNA or gltA genes (Table 2 and 3). As a part of this study, the gene sequences obtained from Rickettsia-positive tick samples collected from hedgehogs and turtles were submitted and deposited in the National Center for Biotechnology Information (NCBI) database. Specifically, the sequences from hedgehog-associated ticks are assigned to the accession numbers PV684243 and PV685004, while the sequences from turtle-associated ticks are available under the accession numbers PV692319 and PV692320 in the GenBank repository (Fig. 4)Table 2. The results obtained pertain to the contamination level of blood and ticks gathered from hedgehogs.Table 2. AnimalSamples of ticks and bloodsNumber of ticks and bloodsSex (Female and male)Number of ticks (Female and male)Number of positive (%) Rickettsia sp. For 16SrRNA gene by Real Time PCRNumber of positive (%) Rickettsia For gltA gene by PCRHedgehogRhipicephalus sanguineus31Female91(11.11%)1/1Male222(9.09%)0Hyalomma aegyptium40Female1300Male271/27(3.70)0Rhipicephalus turanicus26Female71(14.28%)1/1Male192(10.52%)1/3Rhipicephalus bursa9Female100Male800Blood31––00TurtleHyalomma aegyptium234Female864 (4.65%)2/4Male1481 (0.67%)0Blood87––00Table 3The obtained results are related to the amount of blood and tick contamination according to the lower and upper limit of 95% confidence.Table 3. AnimalsSamplesNO.Rickettsia sp. For 16SrRNA gene by Real Time PCRRickettsia For gltA gene by PCRHedgehogTicks1067 (n = 106; 6.6%; 95%Cl: 3.23%–13%)3 (n = 7; 42.86%; 95%Cl: 15.82%–74.96%)Blood310 (n = 31; 0%; 95%Cl: 0%–11.03%)0 (n = 31; 0%; 95%Cl: 0%–11.03%)TurtleTicks2345 (n = 234; 2.14%; 95%Cl: 0.92%–4.91%)2 (n = 234; 0.85%; 95%Cl: 0.23%–3.05%)Blood870 (n = 87; 0%; 95%Cl: 0%–4.23%)0 (n = 87; 0%; 95%Cl: 0%–4.23%)

Discussion

4

This study provides the first evidence on the genomic presence of Rickettsia bacteria in ticks collected from European hedgehogs (Erinaceus europaeus) and spur-thighed tortoises (Testudo graeca) in West Azerbaijan Province, Iran. Of the 339 ticks examined, 10 (2.95%) were positive for Rickettsia spp., including 5 (2.14%; 95% CI: 0.92–4.91) from tortoise-associated ticks and 7 (6.6%; 95% CI: 3.23–13.00) from hedgehog-associated ticks.

All infected ticks belonged to the genera Hyalomma and Rhipicephalus. Hyalomma aegyptium was the predominant species in tortoises with a prevalence of 2.13%, identified as the sole infected vector in this host. In contrast, ticks collected from hedgehogs comprised Rhipicephalus sanguineus, H. aegyptium, R. turanicus, and R. bursa, with the highest infection rate in R. turanicus and the lowest in H. aegyptium and R. bursa.

Despite the detection of Rickettsia in ticks, none of the 118 blood samples from tortoises (n = 87) and hedgehogs (n = 31) contained detectable Rickettsia DNA. Given the high sensitivity and specificity of Real-Time PCR, although no DNA was detected in blood, it is important to note that bacteremia can be transient. Thus, while active infection was not found at the time of sampling, the high prevalence of infected ticks suggests these animals play a significant role in the maintenance of the pathogen in the environment.

Similarly, none of the blood samples from three wild herbivore species the Persian fallow deer (Dama mesopotamica), red deer (Cervus elaphus), and wild sheep (Ovis orientalis) were positive, indicating no active bacteremia at the time of sampling, therefore, reducing the role of these species as natural reservoirs.

Overall, the findings underscore the significant role of wildlife-associated ticks in the ecosystem of the region and their potential to transmit zoonotic pathogens such as Rickettsia. Given the close association of certain hosts, particularly hedgehogs, with human environments, the results highlight the need for increased tick surveillance and monitoring of potential natural reservoirs in public health and epidemiological programs.

This research is the first documented report to simultaneously investigate the genomic presence of Rickettsia in ticks and blood from two key small vertebrates spur-thighed tortoises (T. graeca) and European hedgehogs (E. europaeus) in Iran. Previous Rickettsia-related studies in Iran largely focused on domestic livestock or specific tick species, with little information on the potential role of these two wildlife species as hosts or vectors. The novelty of this work lies not only in the choice of hosts but also in the combined application of highly sensitive molecular methods, including both conventional PCR and Real-Time PCR, for detection and phylogenetic characterization of the bacteria, further enhancing the importance of the study.

The results of this research can serve as a milestone in assessing the ecological role of wildlife species in the transmission cycles of tick-borne diseases in Iran, paving the way for future studies on epidemiology, public health, and the management of zoonotic pathogens.

Notable differences emerge when comparing these findings with a similar study conducted in Xuyi, southeastern China, where 114 ticks were collected from 45 hedgehogs. In the mentioned study, 78.1% of ticks were infected with spotted fever group Rickettsia (SFGR) species, and 17.8% of hedgehogs harbored bacterial DNA in their internal tissues. Sequencing of multiple genes (rrs, gltA, ompA, ompB, sca4) identified Rickettsia heilongjiangensis and a novel species, Candidatus Rickettsia xuyiensis XY-2. These results suggested the possible role of hedgehogs as active hosts in the natural transmission cycle of Rickettsia in China [18].

Although our study detected Rickettsia in ticks from hedgehogs, no bacterial DNA was found in hedgehog blood samples, reflecting ecological and epidemiological differences between the two regions. One possible explanation is variation in tick species composition; in the Chinese study, most ticks belonged to the genus Haemaphysalis (particularly H. flava), whereas in our research, ticks were predominantly Rhipicephalus and Hyalomma, which may differ in feeding behavior, vector competence, and host preference [19].

Furthermore, the higher infection prevalence in China could be influenced by factors such as tick density, season of sampling, genetic differences in hedgehog populations, or host immune characteristics. Another possibility is early or inactive stage of Rickettsia infection in Iran, rendering bacterial DNA undetectable in blood but still present in ticks [20], [21].

While hedgehogs are confirmed hosts of Rickettsia-infected ticks in both regions, their active participation in the life cycle of pathogens may be strongly influenced by environmental conditions, tick species, bacterial strain, and local ecological factors. Further research involving larger sample size, tissue analysis, and serological testing is required to clarify the precise epidemiological role of these small mammals.

A separate study in Portugal sampled 33 rescued hedgehogs from northern and central regions, collecting 1892 ticks and 213 fleas. Rhipicephalus sanguineus was the most abundant tick (91%), followed by Ixodes hexagonus (9%), while all fleas were identified as Archaeopsylla erinacei. Molecular analysis detected Rickettsia massiliae DNA in 22 R. sanguineus ticks and R. asembonensis DNA in 55 A. erinacei fleas, with no infection detected in I. hexagonus. Noteworthy, this was the first report of R. asembonensis in hedgehog fleas in Portugal [7], whereas our study in Iran reports the first genomic detection of Rickettsia sibirica in H. aegyptium ticks from tortoises. Such differences in Rickettsia species and vectors may reflect environmental, host, and ecological variation across regions.

Collectively, findings from Iran, China, Portugal, and other countries highlight the increasing importance of hedgehogs in the epidemiology of zoonotic bacteria such as Rickettsia. Given their proximity to human settlements and ability to host diverse tick species, these animals may play a critical role in sustaining the ecological cycle of these pathogens.

A large-scale study in North Africa and Anatolia, involving 131 adult H. aegyptium ticks collected from wild T. graeca tortoises, exhibited a much higher diversity of microorganisms, including Hemolivia mauritanica (22.9%), Midichloria mitochondrii (11.4%), Borrelia (8.4%), Ehrlichia (7.6%), and Rickettsia spp. (3.4%). Identified Rickettsia species included R. sibirica mongolitimonae (Algeria), R. aeschlimannii (Turkey), and R. africae (Morocco), demonstrating a wide geographic distribution across the Mediterranean region [8]. Our detection of R. sibirica in H. aegyptium from Iran aligns with these reports and constitutes the first such documentation from the country.

The lower prevalence of Rickettsia in the current study compared to North Africa could be related to differences in climate, vegetation cover, host diversity, or genetic variation in tick populations. Given that T. graeca is widely transported in the pet trade (both legally and illegally), monitoring the role of this reptile and its ticks in transmitting tick-borne microorganisms to humans and other animals is of particular public health relevance.

Conclusion

5

According to the results of this study, only ticks, not the blood of hedgehogs and turtles, tested positive for Rickettsia spp., suggesting these animals are not reservoirs but may serve as transporting hosts for infected ticks. This highlights their indirect role in pathogen spread and underscores the importance of monitoring both wildlife and their ectoparasites. Given the zoonotic risks of Rickettsia, enhanced tick surveillance and further molecular studies are essential for deeper understanding of transmission dynamics and selecting more effective public and veterinary health strategies.

CRediT authorship contribution statement

Abdolghaffar Ownagh: Writing – review & editing, Methodology. Ahmad Enferadi: Writing – original draft, Software. Mahdi Rezaverdinejad: Writing – original draft, Software, Methodology.

Consent to participate

Not applicable.

Ethics approval

The study protocol was approved by the Animal Ethics Committee of Urmia University (Code: IR-UU-AEC-3/16). In addition, wildlife sampling was conducted under the official authorization of the Department of Environment (Permit No. 19110). All field procedures, including animal handling and biological sampling, were carried out in strict compliance with national wildlife and environmental regulations.

Authors' statement

Abdolghaffar Ownagh: Supervision, Project administration, Resources, Writing – review & editing. Ahmad Enferadi: Conceptualization, Methodology, Software, Formal analysis, Investigation, Writing – original draft. Mahdi Rezaverdinejad: Investigation, Data curation, Validation. All authors have read and agreed to the published version of the manuscript.

Code availability

Not applicable.

Funding

Not applicable.

Declaration of competing interest

This manuscript has not been published and is not under consideration for publication elsewhere. We have no conflicts of interest to disclose.