Introduction

Ticks are hematophagous parasites that transmit a wide range of pathogens—bacteria, viruses, and protozoa—causing diseases in humans and animals. One of the most prevalent tick-borne pathogens, especially in the northern latitudes, are spirochetes from Borreliaceae family. The Borreliaceae family includes two main genera: Borreliella, with the causative agents of Lyme borreliosis, transmitted by hard (ixodid) ticks and Borrelia, containing the causative agents of relapsing fever (RF). The RF borreliae can be divided into three groups depending on the vector: soft (argasid) ticks relapsing fever (STRF), hard ticks relapsing fever (HTRF), and louse-borne relapsing fever borreliae1.

Borrelia miyamotoi is an emerging human tick-borne pathogen causing hard tick relapsing fever (Borrelia miyamotoi disease - BMD), detected for the first time from tissues of Ixodes perculcatus ticks in Japan in 19922. Further studies confirmed that the spirochaete was prevalent in Ixodes ticks in the northern hemisphere. In Europe, B. miyamotoi is transmitted by I. ricinus, in North America by I. scapularis and I. pacificus, and in Asia by I. persulcatus and I. pavlovskyi. All three genotypes currently described, European, American, and Asian, are pathogenic to humans3,4. Borreliella burgdorferi and B. miyamotoi share tick vectors, except that the prevalence of Lyme spirochaetes is higher compared to B. miyamotoi. The meta-analysis by Hoornstra et al. (2022) showed the overall incidence of B. miyamotoi in questing Ixodes ticks (adults, nymphs, and larvae) worldwide at 1.1%, with the highest prevalence of spirochaete in I. persulcatus (2.8%), followed by I. scapularis (1.1%), I. ricinus (1.0%), and the lowest in I. pacificus (0.7%)5. For comparison, the frequency of occurrence of Borreliella species in I. ricinus and I. scapularis was 12% and over 20%, respectively6,7,8,9,10,11.

In Europe, several cases of B. miyamotoi infection in humans were described, including the Netherlands12, Germany13, Sweden14, and France15. The disease was described both in immunocompromised patients and in healthy individuals. Symptoms in healthy people are mostly non-specific and flu-like, including fever, headaches, muscle and joint pain, weight loss16. In immunocompromised patients, B. miyamotoi infection may lead to meningoencephalitis, which hardly ever affects immunocompetent people14. In Poland, only one case of B. miyamotoi infection has been described so far17.

Limited access to diagnostic tools and nonspecific clinical symptoms of the disease, make it difficult to provide full insights into disease epidemiology.

This study aimed to determine the prevalence of B. miyamotoi in ticks parasitizing humans over a three-year period (2022–2024) in Poland.

Materials and methods

Tick collection and identification

Human-attached ticks removed from skin were collected over a three-year period, from 2022 to 2024 in all voivodeships of Poland (Fig. 1). The research was carried out as part of a National Health Programme for 2021–2025 financed by the Minister of Health (Poland) and participation in the research was voluntary. Tick specimens were delivered from patients with tick bites in person or sent by post to the Department of Health Biohazards and Parasitology at the Institute of Rural Health in Lublin, Poland. All participants (in the case of minors, guardians) were asked to complete and sign the Project Participant Declaration and questionnaire. We collected information on gender, age, occupation, and some information regarding tick bite history: geographic origin and date of exposure, habitat types, activity performed during exposure, time of tick attachment, part of the body that was bitten, skin lesions, tick removal method. Engorgement status (not engorged, semi-engorged, engorged) was assessed by a scientific staff member. Ticks without visible morphological changes from feeding (no evidence of blood intake) were considered unengorged. Specimens were classified as semi-engorged when moderate changes in appearance (size, shape, colour) were observed, and when visual assessment confirmed the presence of a blood meal in the tick’s visceral organs after mechanical disintegration in a sterile transparent plastic bag, performed as a preliminary step for nucleic acid isolation. Ticks with a markedly enlarged, rounded shape, a change in colour, and a large amount of blood released during crushing were classified as fully engorged. Ticks were identified morphologically to the species and developmental stage using taxonomic keys18,19. Identification, homogenization, and first steps of isolation were performed on the same day the tick was delivered. Molecular identification of randomly selected samples was performed using polymerase chain reaction (PCR), based on the fragment of the mitochondrial 16 S rRNA gene (rDNA) and sequencing (Supplementary Table S1)20. Specimens that could not be identified due to extensive damage caused during removal of the tick from the skin were excluded from the analysis, as well as ticks collected from animals (Fig. 2).

Fig. 1
Fig. 1
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Voivodeships from which ticks were with the number of delivered ticks (in brackets the number of Borrelia miyamotoi-infected ticks).

Fig. 2
Fig. 2
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Flowchart with ticks included and excluded from the study.

DNA extraction

Ticks were individually homogenized, and a genomic DNA was isolated with QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol for tissues with some modifications. The ticks were placed individually in disposable transparent bags and crushed mechanically with a pestle. We mixed homogenized tick’s tissue with: (1) 180 µl ATL (animal tissue lysis solution) buffer and 20 µl proteinase K for adults or (2) 90 µl ATL and 10 µl proteinase K for nymphs and larvae, and incubated at 56 °C overnight for lysis. For adults, further treatment followed the tissue protocol, while for nymphs, half the volumes of the remaining reagents were used. The DNA samples were eluted with 100 µl elution buffer for adult ticks and 50 µl for nymphs and larvae, and stored at -20 °C for further study. Extraction was performed separately from PCR, in different rooms and using dedicated workstations21. DNA concentration was measured using the QIAxpert spectrophotomerer (Qiagen, Hilden, Germany).

Borrelia miyamotoi DNA detection

The presence of Borrelia miyamotoi was detected by amplification of the p66 and the gplQ gene fragments, specific for B. miyamotoi, according to the method described by Geller et al. and Fomenko et al.21,22 (Supplementary Table S1). Nuclease-free water was used as negative control and B. miyamotoi DNA isolated from I. ricinus (confirmed by sequencing in an earlier study, Acc. No. MK977951) as positive control23. PCR products were electrophoresed on 2% agarose gels stained in ethidium bromide (2 µg/ml), visualised and photographed under ultraviolet radiation.

Borreliella DNA detection

To estimate the prevalence of B. miyamotoi and Lyme spirochaetes infections, in samples positive for B. miyamotoi, Borreliella genus and three species frequently detected in Europe (Bl. afzelii, Bl. burgdorferi and Bl. garinii) were detected by amplifying fragment of the flaB gene as described previously by Wójcik-Fatla et al.24 (Supplementary Table S1).

Sequencing

Sequencing was performed with inner forward and reverse primers used for PCR amplification (Supplementary Table S1). It was carried out for 23 randomly selected p66-positive or glpQ-positive samples from I. ricinus ticks and for all four p66- and glpQ-positive samples from D. reticulatus ticks. Sequencing was performed with the ABI PRISM 310 Genetic Analyzer (Applied Biosystems, USA) using a BigDye® Terminator v3.1 Cycle Sequencing Kit and Big Dye XTerminator Purification Kit (Applied Biosystems, USA), or by a private company (Genomed S.A., Warsaw, Poland). The nucleotide sequences were compared with data stored in GenBank using the Basic Local Alignment Search Tool (BLAST).

Ethics statement

Ethical approval for the examination of ticks removed from the skin of bitten people tested for Borrelia presence was received by the Ethics Committee of the Institute of Rural Health in Lublin (Approval no. 1/2022) as a part of the National Health Programme for the years 2021–2025, financed by the Minister of Health in Poland, under the name: “Taking initiatives to prevent occupational and work-related diseases, including those related to the service of professional soldiers and officers, and strengthening the health of workers, Operational Objective No. 4: Environmental health and infectious diseases”.

The participants then signed an informed consent to participate in the programme. The processing of personal data in the laboratory in order to ensure confidentiality was carried out in accordance with the provisions of European law (Regulation (EU) 2016/679 of the European Parliament and of the Council of 27 April 2016 on the protection of natural persons with regard to the processing of per-sonal data and on the free movement of such data, and repealing Directive 95/46/EC (General Data Protection Regulation) (Text with EEA relevance). Each participant read and signed the Information Clause on the Processing of Personal Data.

Statistical analysis

Statistical analysis was performed using the StatSoft, Inc. (2011) STATISTICA (data analysis software system), version 10. Relationships between variables were tested using contingency tables and Pearson’s chi-square test. Associations with a p-value < 0.05 were considered statistically significant.

Results

Tick material description

A total of 2,263 tick specimens collected from humans over the three years were submitted for further study, of which 699 (30.9%), 721 (31.9%), and 843 (37.2%) were collected in 2022, 2023, and 2024, respectively. Over 80% of the entire collection came from eastern Poland (Fig. 1; Table 1). The most frequently identified tick species was I. ricinus (2,123 ticks, 93.81%), followed by D. reticulatus (137 ticks, 6.1%). Additionally, two Ixodes hexagonus and one Argas reflexus species were identified (Fig. 2; Table 1).

Table 1 Borrelia miyamotoi prevalence in Ixodes ricinus and Dermacentor reticulatus ticks feeding on humans between 2022 and 2024.
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Borrelia miyamotoi prevalence

The DNA concentration varied depending on the tick stage, engorgement status, and condition of the individual specimen (dried, alive, or dead): in larvae it ranged from 0.5 to 20 ng/µl, in nymphs from 1 to 70 ng/µl, and in adults from 20 to 250 ng/µl. In total, the B. miyamotoi prevalence in human-derived ticks was 3% (67/2,263; CI: 2.3–3.7). Among I. ricinus, 63 ticks carried the pathogen (3%; 95% CI: 2.3–3.8) (Table 1). Borrelia miyamotoi was detected only in females and nymphs (3.5% and 2.9%, respectively), with no positive results among males (n = 31) and larvae (n = 72). B. miyamotoi spirochetes were not detected in larvae, which also yielded the lowest average amounts of extracted DNA, although the present data do not allow for assessment of a causal relationship between DNA concentration and detection sensitivity. There was no significant difference in the infection rate between females and nymphs (p = 0.2712). Dermacentor reticulatus ticks were infected with B. miyamotoi with a prevalence of 2.9% (4/137; CI: 0.8–7.3). All ticks carrying the pathogen were females, and no statistical significance was observed between females and males (p = 0.1377). There was no significant relationship between B. miyamotoi infection and species (I. ricinus vs. D. reticulatus; p = 0.9745). None of I. hexagonus and (A) reflexus ticks were infected with (B) miyamotoi.

Sequencing results

The BLAST-NCBI sequence analysis of the p66-positive samples isolated from I. ricinus showed a 99.8–100% similarity to European B. miyamotoi strains isolated from I. ricinus ticks (KJ425366). The generated sequences were deposited in the GenBank database under the accession numbers PQ492203-PQ492208 for the p66 gene. These sequences were 100% identical to those obtained from I. ricinus collected from vegetation in the previous study (MK977951)23.

In the case of D. reticulatus ticks, sequence analysis of the p66 gene fragment showed a similarity of 99.68% to European B. miyamotoi strains (CP114720). The additional sequence analysis of the glpQ gene fragment showed 100% similarity to B. miyamotoi strains (MG136725). The generated sequences were deposited in the GenBank database under the accession numbers PV256434-PV256437.

The dependence of Borrelia miyamotoi infection on the month of tick feeding

Infected I. ricinus ticks were collected primarily in late spring and early summer, in May (n = 18) and June (n = 29) (Fig. 3). Ticks were most frequently examined during these months, which coincide with the spring peak of I. ricinus activity. Study participants found infected D. reticulatus ticks in February (n = 1), April (n = 2), and May (n = 1). In the months April–October, a significant decrease in the number of infected ticks was confirmed from July (p = 0.00302). The detection of B. miyamotoi infection in the studied tick population was significantly lower in 2023 compared to other years (p = 0.00031) (Table 1).

Fig. 3
Fig. 3
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Number of Ixodes ricinus and Dermacentor reticulatus ticks delivered in each month with Borrelia-miyamotoi-infected ticks marked in orange squares.

The dependence of Borrelia miyamotoi infection on tick habitat

Most ticks originated from rural environments, but a higher proportion of infected ticks originated from urban areas (2.5% vs. 3.1%). However, there was no statistically significant difference in the prevalence of tick infestations between rural and urban areas for either I. ricinus (p = 0.4576) or D. reticulatus (p = 0.1038). Study participants frequently spent time in forests, homesteads, and meadows, but analysis revealed no significant differences in the prevalence of I. ricinus tick infections between different habitats (p = 0.9665). D. reticulatus ticks infected with B. miyamotoi were more likely to originate from other locations than from meadows (p = 0.04882). Tick bites most often occurred during recreation and residential activities (Table 2).

Table 2 Borrelia miyamotoi prevalence in Ixodes ricinus ticks collected from humans depending on the environment and type of activity.
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Borrelia miyamotoi-positive ticks and their human hosts

Infected ticks were most frequently removed from the skin of adults (n = 51), less frequently from children (n = 16). These ticks were removed from the legs (n = 19), torso (n = 19), head/neck (n = 11), arms (n = 7), and genital areas (n = 7). In survey studies, 37 participants bitten by B. miyamotoi-infected ticks reported skin lesions after tick removal. Most patients experienced only minor skin lesions around the tick bite, such as redness (n = 33), bruising and swelling (n = 1), and induration and swelling (n = 1). Only one participant reported a significant skin lesion – a 5 cm diameter redness. Patients from whom B. miyamotoi-uninfected ticks were collected reported the presence of a skin lesion with the similar frequency (60.4%). No statistically significant association was found between the presence of a skin lesion and B. miyamotoi infection (p = 0.4212). The majority of infected, attached I. ricinus ticks were classified as semi-engorged (n = 43). There was no statistically significant association between B. miyamotoi infection in I. ricinus and the degree of tick engorged (p = 0.3901). For D. reticulatus, two infected ticks were partially engorged, one engorged, and one unfed (Table 3).

Table 3 Number of single infections with Borrelia miyamotoi and co-infections with Borreliella burgdorferi in Ixodes ricinus ticks collected from human skin depending on engorged status.
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Co-infections with Borreliella Speceis

Among I. ricinus ticks tested positive for B. miyamotoi, co-occurrence with Bl. burgdorferi was detected in 15 cases (23.8% of B. miyamotoi-positive samples). The most common co-infection was Bl. afzelii (n = 10), followed by two infections with Bl. burgdorferi s.s. and Bl. afzelii, one co-infection with Bl. burgdorferi s.s., and single co-infections with two genospecies (Bl. burgdorferi s.s. with Bl. garinii, and Bl. afzelii with Bl. garinii). Borrelia miyamotoi was not found to co-occur with Bl. burgdorferi in D. reticulatus ticks. Patients bitten by co-infecting ticks reported only minor skin lesions around the bite site or no lesions at all (Table 3).

Discussion

According to the conducted studies, the percentage of B. miyamotoi-infected I. ricinus ticks removed from human skin is low (ranging from 2.3 to 3.8%) compared to values at least twice as high in the case of Bl. burgdorferi infections25. The obtained average value of 3% corresponds to the value obtained for ticks collected from vegetation in previous own studies23. Similarly in Europe, the relapsing-fever spirochaete B. miyamotoi was detected in human-biting I. ricinus ticks, including Belgium, the Netherlands, Italy, Germany, and Poland, with prevalence ranging from 0.6 to 2.9%25,26,27,28,29. Although information on the geographical origin of ticks was collected, the sample distribution was highly uneven, with 75.5% of ticks originating from the Lublin Voivodeship. The remaining samples were distributed across other regions of Poland. Due to this imbalance, a regional comparison of B. miyamotoi prevalence was not performed, as this could lead to biased or unreliable conclusions. Therefore, the analysis was conducted at the national level to obtain an overall picture of the risk associated with tick bites in Poland.

The presence of RF Borrelia bacteria was only demonstrated in I. ricinus females and nymphs, which makes it impossible to confirm transovarial transmission of the pathogen in this study. The efficiency of pathogen transmission and the frequency of offspring infection depend largely on the concentration of B. miyamotoi in females30. This may explain the negative results among the larvae tested, as well as the small larval sample size (n = 72) obtained for the study. Moreover, the absence of Borrelia detection in larvae coincided with the lowest DNA concentrations obtained from this developmental stage. Nevertheless, transovarial transmission of B. miyamotoi was confirmed in Ixodid ticks31, e.g., for Ixodes scapularis, with estimated filial infection rates ranging from 3.3 to 100%30. A small group of males obtained for research (n = 31) confirms the fact that adult male ticks are collected from humans sporadically32,33.

Compared to other Borrelia and Borreliella species, it is still unknown whether, and if, to what extent, wild animals play a role in maintaining these spirochaetes in the environment. Initially, it was suggested that B. miyamotoi might share an animal reservoir with Bl. burgdorferi. It has been even said that the white-footed mouse is a competent reservoir host of B. miyamotoi in the USA34. In Europe, numerous field studies have reported B. miyamotoi presence in various species of small mammals, including Apodemus sylvaticus, Microtus arvalis, Myodes glareolus, Apodemus flavicollis, Apodemus agrarius, and Sorex araneus35,36,37,38,39. Burri et al.40 performed an experiment which results suggested that xenodiagnostic larvae can acquire the pathogen from rodents and produce infected molted nymphs. However, more recent reports indicate minimal horizontal transmission and question the reservoir role of small mammals, suggesting that the circulation of spirochaetes in nature depends primarily on successful transovarial transmission30,41. Further research on the transmission of B. miyamotoi in mammalian hosts is therefore necessary.

So far, only a few studies have shown the presence of B. miyamotoi in single specimens of D. reticulatus collected from vegetation, indicating the need for research to assess the level of infection among the meadow tick population42,43. In this study, the presence of the pathogen was demonstrated for the first time in four D. reticulatus ticks removed from human skin. D. reticulatus rarely fed on humans (137 cases over three years), so it was difficult to assess the role of this species as a competent reservoir of B. miyamotoi. To the best of our knowledge, no experimental studies on the transmission of B. miyamotoi by D. reticulatus ticks have been reported to date. Available data indicate that the ticks of the genus Dermacentor do not play a role in the transmission of Bl. burgdorferi species. Dermacentor variabilis ticks usually acquire Lyme Borreliella burgdorferi with low efficiency, are unable to maintain the bacterium, and do not transmit spirochaetes between animals44,45. However, it cannot be ruled out that Dermacentor ticks may serve as vectors of RF Borrelia, although further research is needed.

In the current study tick bites occurred more frequently in rural areas, but more than a quarter of them originated in urban areas. Some studies have shown that despite the larger tick population in natural areas compared to urban areas, the incidence of tick-borne pathogens may be comparable or even higher, in cities46,47.

It has been experimentally demonstrated that spirochaete transmission may occur within the first 24 h of attachment of a B. miyamotoi-infected I. scapularis nymph to mice48. Most participants in the current study (n = 46) with Borrelia-positive ticks indicated in the survey that the duration of tick feeding was less than 24 h, and except one case, participants indicated no or mild skin lesions. In contrast to Bl. burgdorferi, infections caused by B. miyamotoi rarely associated with skin symptoms4. Thus, results concerning skin symptoms should be treated with caution, as they may be related to the tick bite itself or another tick-borne pathogen, e.g., Lyme Borreliella. We have found that co-infection of B. miyamotoi and Bl. burgdorferi affected 0.7% of all delivered I. ricinus ticks. It is not known whether co-transmission of Bl. burgdorferi and B. miyamotoi through a single tick bite is possible. Borrelia miyamotoi co-infection with Bl. burgdorferi has been detected in patients4, although this may be the result of multiple bites by infected ticks. The impact of co-infection on the course of infection in patients is unknown, but no exacerbation of symptoms during infection has been observed49.

Conclusion

Based on studies of over 2,200 ticks of the species I. ricinus and D. reticulatus removed from human skin, the prevalence of B. miyamotoi infection in humans was considered low (3% positive results). However, the risk of human infection depends primarily on the degree of exposure to ticks, and not only on the prevalence of the pathogen in the tick population. The environment is constantly changing and is influenced by ecological and climatic factors, which influence tick vectors, animal reservoirs, and the pathogen’s distribution. Therefore, further monitoring of B. miyamotoi among tick populations should be considered. As this study demonstrates, the novel tick-borne pathogen B. miyamotoi can occur in ticks of various species that bite humans, posing a risk of transmission to humans.