Single and Co-Infections by Tick-Borne Pathogens in Synanthropic European Hedgehogs (Erinaceus europaeus) in Northwestern Italy

TL;DR

European hedgehogs in urban areas can carry tick- and flea-borne pathogens, posing a zoonotic disease risk to humans and pets.

Contribution

First report of a flea-borne Rickettsia related to R. asembonensis in European hedgehogs and evidence of skin as a key site for pathogen detection.

Findings

Anaplasma phagocytophilum, Borrelia burgdorferi s.l., and Rickettsia spp. were detected in European hedgehog skin and ticks.

A flea-borne Rickettsia closely related to R. asembonensis was identified for the first time in European hedgehogs.

Pathogens showed skin tropism and interactions, with A. phagocytophilum and B. burgdorferi s.l. excluding Rickettsia spp.

Abstract

The rapid increase in wildlife species adapted to urban environments may contribute to the maintenance of zoonotic pathogens in cities, thereby increasing infection risk for urban residents and domestic animals. The European hedgehog (Erinaceous europaeus) is one of these widespread synantropic mammals and is frequently infested with ticks and fleas that act as vectors for zoonotic pathogens. We examined fleas, ticks, skin and spleen samples from 129 European hedgehogs admitted to two wildlife rescue centers in northwestern Italy to assess their role in maintaining pathogenic bacteria. We detected Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, B. myiamotoi and a flea-borne Rickettsia closely related to the zoonotic R. asembonensis, reported here for the first time in European hedgehogs. All bacteria were found more often in skin than in spleen, where A. phagocytophilum and B. burgdorferi s.l. tended to occur together while both appeared to exclude the presence of Rickettsia spp. These results indicate that hedgehogs can harbor vector-borne pathogens and highlight the importance of considering urban wildlife in surveillance and control programs for diseases transmitted by ticks and fleas.

Tick-borne pathogens (TBPs) are increasingly recorded in urban areas, where synanthropic wildlife may sustain pathogen transmission cycles. The European hedgehog (Erinaceus europaeus), frequently infested with ectoparasites, may serve as an urban reservoir of zoonotic TBPs. We investigated TBPs in host tissues and associated ectoparasites (Ixodes spp. and Archaeopsylla erinacei) from 129 hedgehogs in northwestern Italy. Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato and Rickettsia spp. were detected in skin, spleen and ectoparasites (Ixodes spp. and Archaeopsylla erinacei). One spleen sample was positive for B. miyamotoi. Molecular identification revealed A. phagocytophilum ecotype 1, B. afzelii and B. bavariensis. A flea-borne Rickettsia closely related to the zoonotic Rickettsia asembonensis was identified for the first time in European hedgehogs. All pathogens were more prevalent in skin than in spleen. In skin, A. phagocytophilum and B. burgdorferi s.l. showed a positive interaction, whereas both were negatively associated with Rickettsia spp. These findings highlight hedgehogs as potential urban reservoirs of zoonotic TBPs, posing a potential risk for humans and domestic animals. The marked skin tropism of these pathogens supports the use of skin for TBP surveillance and underlines its role as a key interface for vector-borne transmission.

Figures

Full text

1. Introduction

Infections by tick-borne pathogens (TBPs) have markedly increased worldwide over the past decades, causing substantial medical and economic burdens [1,2]. Once mostly prevalent in rural and natural environments, they are increasingly detected in urban areas, where synanthropic wild species (e.g., wildlife living in close association with humans but remaining free-living and not under direct human control) contribute to the survival and transmission of blood-feeding ectoparasites and hard ticks [3,4,5]. However, the role of urban wildlife in the enzootic cycle of TBPs and their potential transmission to humans and domestic animals remains poorly understood [6]. Elucidating transmission dynamics in urban and periurban environments is therefore essential for the development of effective monitoring and management strategies.

Among urban-adapted wildlife species, European hedgehogs (Erinaceus europaeus) represent key hosts for hard ticks, including Ixodes ricinus and Ixodes hexagonus, which are competent vectors for several TBPs [1,4]. Hedgehogs have successfully colonized urban areas, often reaching higher population densities than in rural settings [7]. This close proximity to human settlements increases the likelihood of ectoparasite contact and pathogen transmission to humans and domestic animals [8,9]. Accordingly, hedgehogs are recognized as reservoirs for multiple TBPs [1], including Borrelia burgdorferi sensu lato (s.l.), Anaplasma phagocytophilum, and Rickettsia spp., and may facilitate their maintenance and circulation in urban environments [1,10,11,12].

Borrelia burgdorferi s.l., the causative agent of Lyme disease [13], represents the most prevalent human tick-borne pathogen in the Northern hemisphere [11,14,15]. This spirochete is associated with a wide spectrum of clinical manifestations, ranging from localized skin lesions (erythema migrans) to severe systemic complications involving the nervous system, joints, and heart [16]. Distinct genospecies within this complex display differences in reservoir hosts and disease outcomes. For instance, Borrelia afzelii primarily infects rodents and small mammals [17,18,19], whereas Borrelia bavariensis is associated with both rodents and hedgehogs [11,20] and has been linked to human neuroborreliosis [21]. Likewise, Borrelia miyamotoi, a relapsing-fever spirochete [22], infects hedgehogs and other vertebrate hosts [1,23], causing febrile viral-like symptoms in humans [24].

Other relevant TBPs include members of the order Rickettsiales, such as A. phagocytophilum, a Gram-negative intracellular bacterium responsible for human granulocytic anaplasmosis [25,26], and Neoehrlichia mikurensis, an emerging pathogen associated with severe thromboembolic complications, particularly in immunocompromised individuals [27]. Bacteria of the genus Rickettsia, classified into the Spotted Fever Group (SFG) and the Typhus Group (TG) [28], are primarily maintained in nature by arthropod vectors but can also infect wild and domestic vertebrates. These bacteria pose significant health risks, causing febrile illnesses in humans [29,30].

Previous studies on European hedgehogs and other Erinaceinae species (e.g., Erinaceus roumanicus, Erinaceus amurensis, Atelerix algirus) have largely focused on ectoparasite prevalence [12,31,32] or pathogen detection in selected tissues [5,33,34]. However, information on the most appropriate tissues for pathogen detection remains limited [6], as does knowledge regarding co-infections within these hosts. Co-infections—defined as the simultaneous presence of two or more genetically distinct microorganisms within the same host—are common in both vertebrates and vectors [35,36,37]. Such interactions may be synergistic, antagonistic, or neutral, thereby influencing host susceptibility, disease progression, and pathogen transmission dynamics [35,38,39,40]. Despite their relevance, co-infections are frequently overlooked, limiting our understanding of enzootic cycles and pathogen interactions [39]. This gap is particularly evident in studies on European hedgehogs, which have predominantly addressed single-pathogen prevalence.

The present study aims to investigate the role of European hedgehogs in the enzootic cycles of TBPs in urban and suburban environments within the Piedmont region, northwestern Italy. To date, hedgehogs have been only marginally investigated in Italy, particularly with respect to their role as carriers of TBPs. We therefore focused on an area characterized by high levels of urbanization and frequent human–wildlife interactions, where increasing trends in tick abundance and human TBP reports have been documented in recent years [41,42]. By identifying the presence of key TBPs, their tissue distribution in vertebrate hosts, and the occurrence of co-infections, this study seeks to improve current understanding of pathogen transmission mechanisms and interactions, providing novel insights into tick-borne diseases in urban ecosystems.

2. Materials and Methods

2.1. Tested Animals and Sample Collection

Hedgehogs included in this study (n = 129) were admitted between 2022 and 2023 to two wildlife rescue centers (WRCs) in the Piedmont region, northwestern Italy: ‘Centro Animali Non Convenzionali’ (C.A.N.C.) of the University of Turin and ‘Centro Recupero Ricci “La Ninna”’ in the province of Cuneo. A graphical representation of the animal sampling sites is provided in Figure 1. Anamnestic data, including age (determined based on morphological characteristics [43,44,45] and classified as juvenile or adult), sex and Body Condition Score (BCS, recorded on a scale of 1 to 5, with 1 indicating cachexia and 5 obesity), were collected at admission.

In cases of spontaneous death or euthanasia, full post-mortem examinations were performed as part of a separate research study [46]. Euthanasia, carried out in cases of clinical deterioration and poor prognosis, was performed by inducing general anesthesia followed by intracardiac administration of a preparation containing mebezonium iodide, embutramide, and tetracaine. During necropsy, spleen and skin samples (collected from the auricular and perineal regions) were obtained and stored individually at −80 °C. Two different skin sites were sampled to evaluate potential differences in pathogen detection. The auricular region is generally more heavily infested by ticks, and a higher pathogen prevalence was therefore expected. During the clinical examination and during necropsy, ticks and fleas (up to ten per animal) were collected and stored separately at −20 °C. Ectoparasites were identified under a stereomicroscope using identification keys [47,48,49].

2.2. DNA Extraction and PCR Analysis

DNA extraction from tissues was performed using DNeasy Blood and Tissue Kit (Qiagen, Venlo, Limburg, The Netherlands) following the manufacturer’s instructions. A negative control was included at each step to monitor potential contamination. For skin samples, the auricular and perianal regions were extracted and analyzed separately, and their results were subsequently combined; animals were considered positive if they tested positive in either or both regions.

Due to economic constraints, ectoparasite specimens were analyzed in pools rather than individually. A maximum of six ticks per pool was selected to remain within the DNA input limits recommended by the extraction kit and to ensure a sufficient target DNA concentration for reliable PCR amplification. Flea pools (maximum of six specimens per animal per pool) were also analyzed. DNA from ectoparasites was extracted using the same protocol described above. Multiplex real-time qPCRs assays were used to detect the presence of Anaplasma spp. (gene Msp2) [50], B. burgdorferi s.l. (genes FlaB and OspA) [51], B. miyamotoi (gene FlaB) [52], Neoehrlichia spp. (gene GroEL) [53] and Rickettsia spp. (gene gltA) [54] in skin, spleen and ectoparasite pools. Fleas were tested only for Rickettsia spp., as the other pathogens are transmitted exclusively by ticks. Anaplasma spp. positive samples were further analyzed using real-time qPCR targeting the GroEL gene to identify A. phagocytophilum Ecotype 1 and 2 [55]. All qPCR assays were performed using iQ Multiplex Powermix (Bio-Rad Laboratories Inc., Hercules, CA, USA). Positive controls and negative water controls were included in all molecular assays.

2.3. Amplicon Purification and Sequencing

DNA from qPCR-positive samples was amplified using conventional PCR for the detection of the A. phagocytophilum GroEL gene [56], B. burgdorferi s.l. igs gene [57], and the Rickettsia spp. genes gltA [58], OmpA (hemi-nested technique [59,60]), and OmpB [61]. The MyTaq™ HS Red DNA Polymerase (Bioline Reagents Ltd., London, UK) was used for the detection of A. phagocytophilum and Rickettsia spp., whereas Platinum™ Taq DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA) was used for the detection of B. burgdorferi s.l. Further details regarding the PCR and qPCR assays used for the detection of the selected TBPs are provided in Supplementary Table S1.

Amplicons from positive samples were purified using the ExoSAP-IT™ PCR Product Clean-up Kit (GE Healthcare Limited, Chalfont, UK) and sent to external laboratories (BMR Genomics, Padua, Italy and BaseClear, Leiden, The Netherlands) for Sanger sequencing. Nucleotide sequences were analyzed using BioEdit [62], and the sequences obtained were compared with those available in the National Centre for Biotechnology Information GenBank^®^ database using the BLASTn tool, version 2.17.0 [63].

2.4. Statistical Analysis

All statistical analyses were performed using R Studio (version 4.3.2) [64].

The probabilities of detecting Anaplasma spp., B. burgdorferi s.l., and Rickettsia spp. in skin, spleen and ectoparasites were described using the mean and standard deviation. The prevalence and 95% exact binomial confidence intervals (CI) of TBP infection were calculated using a binomial exact test. Differences in pool positivity for Rickettsia spp. between infected and non-infected animals were evaluated using Fisher’s exact test, with the statistical significance level set at 0.05.

In hedgehogs, differences in detection probability between skin and spleen were assessed using a generalized linear mixed model (GLMM) with maximum likelihood estimation (Laplace Approximation) implemented in the package lme4 version 1.1-38 [65]. The null hypothesis assumed equal detection probabilities for the three pathogens in both organs, with individual hedgehogs included as random effects.

Co-occurrence patterns among detected microorganisms were analyzed to determine whether Anaplasma spp., B. burgdorferi s.l., and Rickettsia spp. were positively, negatively, or randomly associated in skin samples. Pairwise co-occurrence analyses were performed [66,67] using the Cooccur package version 1.3 [67,68]. Data were organized into a presence-absence matrix, in which rows represented pathogens and columns represented sampled hedgehogs. In the matrix, “1” indicated pathogen detection, whereas “0” indicated absence. The expected number of co-occurrences between pathogen pairs was calculated by randomizing pathogen presence across sampled animals [67].

3. Results

A total of 129 animals were included in the study, with 74 (57.4%) originating from C.A.N.C. and 55 (42.6%) from “La Ninna” WRC. An overview of the population characteristics is provided in Table 1.

Overall, 70 animals (54.3%, 95% CI: [45.3–63.1]) were infested by ectoparasites. Ixodid ticks were collected from 62 animals and were identified as I. hexagonus, I. ricinus, and Ixodes spp. Of the 228 ticks identified as I. hexagonus, 149 were females, 21 males, and 58 nymphs. A further 13 ticks were identified as I. ricinus, including four females and nine nymphs. An additional 78 ticks were too damaged to allow species-level identification and were therefore classified as Ixodes spp.

I. hexagonus was detected in 41 hedgehogs (31.8%, 95% CI: [23.9–40.6]), whereas I. ricinus was found in five animals (3.9%, 95% CI: [1.3–8.8]). Ticks identified as Ixodes spp. were detected in 29 animals (22.5%, 95% CI: [15.6–30.7]), of which 13 were co-infested with I. hexagonus and/or I. ricinus, whereas the remaining 16 harbored Ixodes spp. alone.

Fleas were collected from 17 hedgehogs (13.2%, 95% CI: [7.9–20.3]), all identified as Archaeopsylla erinacei. Nine animals simultaneously hosted both fleas and ticks (7.0%, 95% CI: [3.2–12.8]).

The number of hedgehogs, categorized by BCS classification, hosting each ectoparasite species and stage is provided in Supplementary Table S2.

The prevalence of infection by the target pathogens in spleen, skin and ectoparasite pools is presented in Table 2.

Results from the auricular and perianal skin regions were initially analyzed separately; however, no significant differences in pathogen prevalence were observed. Accordingly, data from both regions were pooled for subsequent analyses. Anaplasma phagocytophilum was detected in 13 animals (10.1%, 95%CI: 5.5–16.6), with four animals testing positive in both skin and spleen. Borrelia burgdorferi s.l. was identified in 14 animals (10.9%, 95%CI: 6.1–17.5) in either skin or spleen samples. Rickettsia spp. was detected in the skin of 88 animals (68.2%, 95%CI: 59.4–76.1), with five animals also testing positive in the spleen. One spleen sample (0.8%, 95%CI: 0.0–4.2) was positive for B. myiamotoi, while N. mikurensis was not detected in any animal.

Two tick pools tested positive for A. phagocytophilum (n = 2, 95% CI: 0.2–1.0), and both originated from animals with Anaplasma-positive tissues. Borrelia burgdorferi s.l. was detected from one tick pool derived from a B. burgdorferi s.l.-positive hedgehog. Ten tick pools and ten flea pools tested positive for Rickettsia spp. Thirty percent (n = 3, 95% CI: 0.1–0.7) of the positive tick pools originated from Rickettsia-positive animals, while 90% (n = 9, 95% CI: 0.6–1.0) of positive fleas were collected from Rickettsia-positive hedgehogs. No animals simultaneously hosted ticks and fleas that were both positive for Rickettsia spp. Parasite pool positivity did not differ significantly between positive and negative animals (p > 0.05). Neither B. miyamotoi nor N. mikurensis were detected in any tick pools.

All A. phagocytophilum positive samples were identified as Ecotype 1; one representative sequence was deposited in GenBank (accession no. PX597552). All B. burgdorferi s.l. positive samples, except one, were identified as B. afzelii (Genbank accession no. PX597535-7) and showed 98.9–100% identity with GenBank sequences PP874234 and PP874245 isolated from I. ricinus in Belgium. One skin sample was positive for *B. bavariensis *(Genbank accession no. PX597538) and showed 100% identity with sequence CP117799 from a human isolate in Germany. Regarding Rickettsia spp., the gltA fragment detected in hedgehog tissues and ectoparasites (ticks and fleas; Genbank accession no. PX597539-44) showed 98.4–100% identity with R. asembonensis identified in fleas collected from wildlife in Turkey, including A. erinacei collected from Erinaceus concolor (OR187501). The OmpA fragments (GenBank no. PX597545-9) showed the highest similarity (89.5–91.4%) with R. asembonensis strains detected in Ctenocephalides fleas from Peru (e.g., MK923722). The OmpB gene could not be amplified.

The detection probabilities of A. phagocytophilum, B. burgdorferi s.l., and Rickettsia spp. in skin and spleen samples are shown in Figure 2. The GLMM revealed significantly higher detection probabilities for each pathogen in skin compared to the spleen (A. phagocytophilum Estimate = −4.078, SE = 1.965, p = 0.0379; B. burgdorferi s.l. Estimate = −1.8738, SE = 0.7744, p = 0.0155; Rickettsia spp. Estimate = −23.657, SE = 3.133, p = 4.29 × 10^−14^).

Co-occurrence patterns of A. phagocytophilum, B. burgdorferi s.l., and Rickettsia spp. in skin samples indicated a positive association between B. burgdorferi s.l. and A. phagocytophilum, whereas both pathogens were negatively associated with Rickettsia spp. Table 3 summarizes the interactions among the studied pathogens. No co-occurrence analysis was performed for pathogens detected in hedgehog ectoparasites due to the limited number of positive samples for A. phagocytophilum and B. burgdorferi s.l

4. Discussion

Our study highlights the presence of multiple TBPs in tissues and ectoparasites of European hedgehogs.

Anaplasma phagocytophilum was detected in 10.1% of tissue samples; this prevalence is lower than that reported in European hedgehogs from central Europe, where values ranged between 25.8% and 97.6% [5,10,69,70]. Regarding ticks, the prevalence of A. phagocytophilum observed in this study (3.2%) was also lower than that reported for hedgehog-associated ticks in the Netherlands, where prevalences of 27% in I. hexagonus and 24% in I. ricinus were described [12]. Infection rates in questing I. ricinus ticks across Europe vary considerably, ranging from 0.4% to 20.0% [71]. In the Piedmont region, for example, a prevalence of 1.9% has been reported in questing I. ricinus [42]. Similarly, A. phagocytophilum was detected in 4.2% of Rhipicephalus turanicus ticks collected from European hedgehogs in southern Italy [32]. Taken together, the low detection rate observed in our study and in that by Bezerra-Santos et al. may reflect the limited circulation of the pathogen in Italy.

Anaplasma phagocytophilum is classified into nine genotypes based on the groEL gene, which are further grouped into four ecotypes [69]. Sequencing results from both ectoparasites and tissue samples identified all positive samples as belonging to ecotype 1, which is zoonotic and is also responsible for canine granulocytic anaplasmosis [72]. This ecotype has been isolated from a wide range of vertebrate hosts, including European hedgehogs, which may therefore act as reservoirs of the bacterium in urban environments [69]. In hedgehog ectoparasites, A. phagocytophilum ecotype 1 has been detected in both I. ricinus and I. hexagonus ticks [1].

Borrelia burgdorferi s.l. was detected in 10.9% of the examined hedgehogs, a prevalence consistent with previously reported European values (ranging from 9.8% to 90%) [5,6,11]. Regarding Borrelia infection in ticks, our findings (1.6%) are comparable to those reported for I. hexagonus collected from rescued hedgehogs in Germany and Great Britain, where infection rates ranged from 1.1% for nymphs to 1.5% for females [11]. However, the prevalence of B. burgdorferi s.l. in ticks varies widely across Europe, with rates of 14% in I. hexagonus and 28% in I. ricinus reported from hedgehogs in the Netherlands [12], increasing to 60% in engorged I. hexagonus females collected from Romanian urban wildlife, including hedgehogs (E. roumanicus) [13].

All Borrelia-positive samples in this study were identified as B. afzelii, except for one skin sample corresponding to B. bavariensis. These species primarily utilize small mammals (including hedgehogs) and rodents as reservoir hosts [1,11] and represent two of the main pathogenic Borrelia species affecting humans [15]: B. afzelii is typically associated with dermal manifestations, whereas B. bavariensis is linked to human neuroborreliosis [21]. Both species are mainly transmitted by I. ricinus ticks; however, I. hexagonus also appears to play a relevant role in the epidemiology of B. bavariensis [11]. Borrelia afzelii is the predominant genospecies identified in European hedgehogs and their associated ticks across multiple regions [6,11,12,13], and is also the most prevalent circulating genospecies in the study area [42]. Accordingly, its predominance in hedgehogs may reflect both its widespread circulation and a degree of host specificity. To our knowledge, B. bavariensis has not been previously reported in the region.

Rickettsia spp. was the most prevalent pathogen detected in this study, occurring in 68.2% of skin samples, with five animals also testing positive in the spleen. Reports of Rickettsia spp. infection in hedgehogs vary widely, ranging from no detection in liver and lung samples in Portugal [31] to 10% prevalence in internal organs of A. algirus in Tunisia [73] and 65.2% positivity in skin and internal organs in Hungary [5]. Several Rickettsia species have been identified in ticks collected from hedgehogs, including R. massiliae and R. conorii [74], with detection rates ranging from no positivity in I. hexagonus in Portugal [31] to 91.7% in Rhipicephalus sanguineus ticks [75]. Likewise, A. erinacei fleas collected from European hedgehogs have tested positive for R. felis, R. typhi, and Rickettsia felis-like organisms (RFLOs) [74]. In the present study, Rickettsia spp. strains detected in tissues, ticks, and fleas showed the highest sequence identity with R. asembonensis. The gltA gene fragments were identical to those of R. asembonensis; however, OmpA sequence similarity was below the 98.8% cut-off required for species-level classification based on this gene [76]. Therefore, further molecular analyses are needed to confirm the precise taxonomic status of these strains. Rickettsia asembonensis is classified as an RFLO within the Spotted Fever Group and has been detected in fleas and other arthropods worldwide [77]. Although its pathogenicity remains poorly defined, it has been associated with non-specific acute febrile illness in humans in Peru [78]. In hedgehogs, R. asembonensis has previously been detected in A. erinacei fleas in Portugal (47%) [31], southern Italy (93.3%) [32], and Tunisia (82.4%, in A. algirus) [73], with prevalences comparable to the 58.8% observed in fleas in the present study. Notably, we also detected R. asembonensis in engorging ticks and hedgehog tissues, which tested negative in the Portuguese study by Barradas and colleagues [31]. To our knowledge, this represents the first report of R. asembonensis in hedgehog tissues. The detection of a Rickettsia species closely related to R. asembonensis in fleas, engorged ticks, and tissues may support a role for hedgehogs in the transmission and maintenance of this bacterium in natural environments.

Borrelia miyamotoi was detected in only one spleen sample, whereas Neoehrlichia spp. was not detected in either tissues or arthropods. These findings are consistent with those of Szekeres et al. [5], who reported no detection of B. miyamotoi or Neoehrlichia spp. in hedgehog tissues or ectoparasites. However, Majerová et al. reported a 5.0% prevalence of B. miyamotoi in hedgehog tissues [6], and Földvári et al. identified N. mikurensis in 2.3% of ear skin samples from northern white-breasted hedgehogs [79].

All TBPs detected in the present study are zoonotic and may pose significant risks to human health [80]. The overall high prevalence observed suggests that hedgehogs may contribute to the maintenance of these pathogens in natural environments, acting as reservoir hosts that facilitate pathogen persistence and transmission [81]. This role is of particular concern given the increasing presence of hedgehogs in urban and suburban areas, where their interactions with humans, pets, and other wildlife may promote both direct and indirect transmission of zoonotic agents [74,82]. However, not all TBPs appear to follow this pattern: N. mikurensis does not seem to rely on European hedgehogs or their ectoparasites for maintenance, and hedgehogs may also play a limited role in the circulation of B. miyamotoi.

In our study, complete removal of all arthropods infesting the animals was not performed, and no more than ten ectoparasites were collected from each hedgehog. This approach was adopted because the primary focus of the study was on hedgehogs, while ectoparasites were sampled only to assess the TBPs they carried and could potentially transmit. Consequently, tick infestation burden could not be evaluated. In addition, species-level identification was possible only for a subset of the collected specimens, which were identified as I. hexagonus and I. ricinus. The remaining specimens were too damaged or engorged to allow reliable identification and were therefore classified as Ixodes spp. only. This limitation precluded assessment of complete species-specific infestation patterns and pathogen prevalence.

The pathogens most frequently detected in this study—A. phagocytophilum, B. burgdorferi s.l., and Rickettsia spp.—exhibited higher prevalence in skin samples than in spleen samples. This difference was particularly pronounced for Rickettsia spp., whose prevalence in skin was 17.5 times higher than in spleen (68.2% vs. 3.9%). This finding may explain the wide variability in detection rates reported in the literature, with lower prevalence in internal organs [31,73] and higher rates when skin samples are included [5]. Although these pathogens can disseminate from the inoculation site to internal organs such as the spleen, all three displayed a clear tropism for the skin, which represents the first physical and immunological barrier encountered following a tick bite [83]. Tropism refers to the ability of a microorganism to persist in specific host tissues and plays a crucial role in pathogen survival, proliferation, and dissemination, thereby shaping clinical manifestations and transmission dynamics [83]. Assessing tissue tropism in vertebrate hosts therefore provides valuable insights into transmission efficiency and reservoir competence [84]. Previous studies have demonstrated the effectiveness of skin biopsies for detecting A. phagocytophilum [69], B. burgdorferi s.l. [6,15,83,84,85,86] and Rickettsia spp. [83,87]. As the skin represents the primary interface between ectoparasites and vertebrate hosts, these findings further support its value in evaluating host contributions to TBP maintenance in natural environments [83]. The suitability of skin as a diagnostic matrix is additionally reinforced by the relative ease of collection from live animals. The frequent detection of A. phagocytophilum, B. burgdorferi s.l., and Rickettsia spp. in hedgehog skin samples strengthens the hypothesis that hedgehogs contribute to the maintenance of these TBPs and facilitate their transmission to other hematophagous vectors.

In the present study, A. phagocytophilum and B. burgdorferi s.l. showed a positive association, whereas both were negatively associated with Rickettsia spp. Co-infections are common in wildlife and play a key role in shaping immune responses, host susceptibility, pathogen replication, transmission, and disease dynamics [13,39,40,88]. Positive co-occurrence is often attributed to host co-exposure to pathogens that do not elicit cross-immunity or competitive exclusion within host tissues [89]. Previous studies on I. hexagonus ticks collected from hedgehogs have reported co-infections involving A. phagocytophilum, B. burgdorferi s.l. and R. helvetica [1]. Positive interactions between A. phagocytophilum and B. burgdorferi s.l. have also been documented in both feeding and questing ticks [13,90], with A. phagocytophilum potentially facilitating the dissemination of B. burgdorferi s.l. [91]. Co-infection with A. phagocytophilum and B. burgdorferi s.l. is frequently reported in human clinical cases [92]. Experimental studies have shown that Anaplasma-infected neutrophils may promote the translocation of B. burgdorferi across the blood–brain barrier [93], potentially leading to increased bacterial replication, disease severity, and more complex clinical manifestations [94,95]. Co-occurring TBPs may therefore increase host susceptibility to subsequent infections, as illustrated in wildlife systems such as African lions, where feline immunodeficiency virus facilitates co-infection by gastrointestinal parasites and tick-borne hemoparasites [96].

In contrast to our findings, other studies have reported positive associations between B. burgdorferi s.l. and Rickettsia spp. [22], as well as between A. phagocytophilum and R. helvetica [1] or R. amblyommatis [40]. These discrepancies may reflect differences in host immune systems, vector species composition [40], pathogen genospecies, geographical settings, or sampled tissues [97]. The occurrence of simultaneous co-infections in hedgehog tissues, particularly in the skin—the primary interface with ectoparasites—suggests that ectoparasites may acquire multiple pathogens during a single blood meal, further supporting a role for hedgehog in the maintenance and circulation of TBPs in both natural and urban environments.

5. Conclusions

This study reinforces the role of European hedgehogs in the maintenance and transmission of TBPs. The higher prevalence of A. phagocytophilum, B. burgdorferi s.l. and Rickettsia spp. in skin samples compared with spleen samples underscores the importance of skin in the tropism of these TBPs and highlights its value as a diagnostic tissue, with the additional advantage that it can also be collected from living animals. Nonetheless, sampling multiple tissue types remains useful for improving pathogen detection rates. Furthermore, the constant presence of hedgehogs in urban environments facilitates the persistence of hematophagous ectoparasites and increases the risk of TBP transmission to humans and other animal species. Further research should focus on elucidating the mechanisms underlying pathogen interactions, assessing their public health implications, and developing effective strategies to mitigate the risks associated with wildlife reservoirs in urban ecosystems.