# Ticks and tick-borne pathogens on Belle-Île-en-Mer: An exploratory study in a western French island environment

Ali Haidar-Ahmad, Cathy Barthel, Pierre H. Boyer, Guy Joncour, Brigitte

Degeilh, Nathalie Boulanger

► To cite this version:

Ali Haidar-Ahmad, Cathy Barthel, Pierre H. Boyer, Guy Joncour, Brigitte Degeilh, et al.. Ticks and tick-borne pathogens on Belle-Île-en-Mer: An exploratory study in a western French island environment. Ticks and Tick-borne Diseases, 2025, 16 (1), pp.102399. 10.1016/j.ttbdis.2024.102399. hal-04954140

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Short communication

# Ticks and tick-borne pathogens on Belle-Île-en-Mer: An exploratory study in a western French island environment

A. Haidar-Ahmad<sup>a</sup>, C. Barthel<sup>a</sup>, P. Boyer<sup>a,b</sup>, G. Joncour<sup>c</sup>, B. Degeilh<sup>d</sup>, N. Boulanger<sup>a,b,*</sup>

<figure><img src="image_3.png" /></figure>

## ARTICLE INFO

**Keywords:**

Island

Ecosystem

Ticks and tick-borne pathogens

## ABSTRACT

The islands of Brittany provide unique ecosystems for ticks and tick-borne diseases owing to their oceanic climate, influencing interactions among ticks, hosts, and pathogens. We conducted a preliminary investigation on Belle-Île-en-Mer, an island off the Atlantic coast of Brittany in western France, to assess the prevalence of questing adult ticks and associated human pathogenic bacteria. Dermacentor spp. were found to dominate the tick population (61 %): 23 % Dermacentor reticulatus and 77 % *D. marginatus*. Haemaphysalis punctata (27 %) was also prevalent on the island, and *Ixodes ricinus* (12 %) was detected for the first time on Belle-Île-en-Mer. Both Dermacentor species harbored either Rickettsia slovaca (24 %) or Rickettsia raoultii (20 %), whereas *I. ricinus* carried Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum but not Borrelia miyamotoi or Neohrichia mikurensis. Detection of two potentially pathogenic species in the *B. burgdorferi* sensu lato complex, *B. afzelii* and *B. lusitaniae*, along with *A. phagocytophilum* underscores the current risk of Lyme borreliosis and anaplasmosis. The high prevalence of Rickettsia infection in Dermacentor indicates an additional risk of human rickettsioses.

## 1. Introduction

The islands off the cost of Brittany constitute unique ecosystems for ticks and tick-borne pathogens owing to their closed environment and oceanic climate. In this study, we selected Belle-Île-en-Mer, an island off western France, to explore current tick populations and associated pathogens, especially those affecting humans, and to compare our findings with previous research in the region (Michelet et al., 2016). The 2016 study identified key populations of Dermacentor and Haemaphysalis ticks as well as the presence of potentially pathogenic microorganisms of veterinary interest in domestic animals, including Rickettsia and Babesia (Michelet et al., 2016). We focused on surveying collected ticks and medically important tick-borne pathogens. Although human cases of tick-borne lymphadenopathy and Lyme borreliosis have been reported by physicians of the island, they are poorly documented. In this study, tick collections were made from vegetation across three sites, including both anthropized and nonanthropized ecosystems. Molecular analysis was used to identify potential pathogens. Regarding tick species, *Ixodes ricinus*, *Haemaphysalis punctata*, Dermacentor reticulatus, and Dermacentor marginatus were identified, with *I. ricinus* and its associated bacteria,

including three species of *Borrelia burgdorferi* sensu lato alongside *Anaplasma phagocytophilum*, identified for the first time. Furthermore, the presence of *Rickettsia raoultii* and *Rickettsia slovaca* in Dermacentor spp. was confirmed on the island.

## 2. Materials and methods

### 2.1. Sites and tick collection

Belle-Île is a touristic island off the Atlantic coast of Brittany, situated 14 km from the Quiberon peninsula, covering approximately 84 km² with an average altitude of 40 m above sea level. Designated a Natura 2000 site since 2014 (Office française de la biodiversité, 2024), the island, being relatively close to continental land, hosts diverse fauna, including batrachians, reptiles (e.g., *Podarcis muralis*, *Lacerta viridis*, and *Natrix natrix*), and 189 native bird species (mainly seabirds), with 3 classified as game birds (*Perdrix perdrix*, *Alectoris rufa*, and *Phasianus colchicus* introduced in 1966). The island's mammals included small mammals and lagomorphs, with deer and wild boar being absent. Notably, pheasants and lagomorphs are especially abundant (Gélinaud,

<footnote><sup>a</sup> UR3073: PHAVI: Groupe Borrelia, Institut de Bactériologie, FMTS, University of Strasbourg, 67000 Strasbourg, France</footnote>

<footnote><sup>b</sup> French National Reference Center for Borrelia, Centre Hospitalier Régional Universitaire, 67000 Strasbourg, France</footnote>

<footnote><sup>c</sup> GTV Bretagne. 22160 Callac, France</footnote>

<footnote><sup>d</sup> Laboratoire de Parasitologie, Mycologie CHU Rennes rue Henri Le Guilloux 35033 Rennes, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: nboulanger@unistra.fr (N. Boulanger).</footnote>
2015). Domestic animals primarily comprise farm livestock (e.g., horses, cows, and sheep). Agriculture practices focus on corn cultivation and artificial meadows, with natural meadows, wet valleys, coastal meadows, and moors having been abandoned. In recent years, coniferous woodlands and hedgerows have been extensively planted. Human activities are considered to have markedly impacted the island (Brien, 2015).

Three sites were surveyed (Fig. 1 and Table 1). Site 1, Donnant, a protected natural area owned by the Conservatoire du Littoral, is characterized by expansive sandy dunes populated primarily by seabirds. Site 2, Saint Marc, a coastal heath, is dominated by European gorse (Ulex europaeus) and bramble (Rubus fruticosus)-associated bushes and harbors grazing animals, such as goats, horses, and cattle. Site 3, Port Guen, an anthropized valley, is an ecosystem of shrub vegetation with a stream and high humidity. Over the year, the average temperature in Belle-Île-en-Mer is 12.5 °C and rainfall averages 600.1 mm. Adult ticks were collected in April 2022 at all three sites using the flagging method to sweep vegetation on a distance of 100 m. Ticks were subsequently preserved in ethanol.

## 2.2. Tick identification

Ticks were morphologically identified at the genus level (Table 1). All ticks were removed from ethanol and individually dissected, separating the four legs from the idiosoma. Each part was placed in a tube, and the remaining ethanol was evaporated overnight at 37 °C. From 2 to 5 morphologically distinct specimens were selected for molecular identification using the COI gene for Dermacentor spp. and *I. ricinus*, or the 16S rDNA gene for *H. punctata*. The extraction protocol, primers, PCR conditions, and PCR revelation are described previously (Boyer et al., 2019). Sanger sequencing was performed by the Eurofins Genomics company (Basel, Switzerland). The quality of the sequences was assessed by inspecting the chromatograms with SeqTrace, then the forward and reverse sequences were assembled and converted into finished DNA sequences using the SeqTrace software (PMID: 22942788). The sequences were compared with sequences from GenBank.

The four legs of all specimens were submitted to Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS), using the same protocol and settings as described previously. MALDI-TOF MS Spectra issuing from specimens identified by molecular biology were added to our in-house database (Boyer et al., 2019). The remaining specimens were identified using the upgraded database. A log score value (LSV) of at least 1.8 was required for species

<table><caption>**Table 1**

Description of the different sites for tick collections in April 2022.</caption><thead><tr><th>Site</th><th>Collected adult ticks</th><th>Total</th><th>Site description</th><th>GPS</th></tr></thead><tbody><tr><td rowspan="2">Donnant</td><td><em>Dermacentor</em></td><td>36</td><td>Protected natural site</td><td rowspan="2">47.32N,3.24W</td></tr><tr><td><em>Haemaphysalis</em></td><td>12</td><td>with dunes.</td></tr><tr><td rowspan="2">Saint Marc</td><td><em>Dermacentor</em></td><td>40</td><td>Coastal heath with gorse</td><td rowspan="2">47.29N,3.14W</td></tr><tr><td><em>Haemaphysalis</em></td><td>29</td><td>and bushes associated with bramble</td></tr><tr><td>Port Guen</td><td><em>Ixodes</em></td><td>18</td><td>Anthropized valley with stream, ecosystem with high humidity.</td><td>47.33N,3.15W</td></tr><tr><td></td><td><em>Dermacentor</em></td><td>18</td><td></td><td></td></tr></tbody></table>

identification to be considered reliable. Additionally, a minimum difference of 0.2 between the score of the best species match and the second species match score was required (Boyer et al., 2019).

## 2.3. Molecular detection of microorganisms

DNA extraction from idiosomes was performed using ammonium hydroxide following established procedures (Guy and Stanek, 1991). In *I. ricinus*, the investigation focused on human pathogens, particularly *B. burgdorferi* sensu lato and *A. phagocytophilum* (Boyer et al., 2022). Briefly, for *B. burgdorferi* sensu lato, initial real-time polymerase chain reaction (RT-PCR) targeted the *flagellin* gene, followed by species-specific RT-PCR targeting variable regions of the *flagellin* gene for each *Borrelia* species. Regarding *A. phagocytophilum*, the major surface protein 4 gene was targeted, whereas for *Borrelia miyamotoi*, the relapsing fever *flagellin* gene was targeted (Hovius et al., 2013). For *Neoehrlichia mikurensis*, a RT-PCR assay targeting the chaperonin 60 kDa subunit gene was adapted from Jahfari et al. (2012). All PCR assays were performed using the Biorad Opus 384 system.

Detection of *Rickettsia* spp. in *Dermacentor* adults was performed using a RT-PCR assay initially targeting the *citrate synthase* gene, followed by nested end-point PCR targeting the *outer membrane protein B (ompB)* gene specific to the spotted fever group *Rickettsia*, with subsequent sequencing (GATC Eurofins Genomics, Basel, Switzerland) achieved using inner primers via the Sanger method (Boyer et al., 2022).

# 3. Results

## 3.1. Tick identification

In the tick collection performed during April 2022, 153 adult ticks

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Geographic localisation of Belle-Île en Mer with the 3 investigated sites for ticks and tick-borne pathogens: 1: Donnant, 2: Saint Marc, 3: Port Guen.</figcaption></figure>
were collected: 41 *Haemaphysalis* (27 %), 94 *Dermacentor* spp. (61 %), and 18 *Ixodes* (12 %) (Table 1). To verify morphological identification, 15 specimens were randomly chosen for molecular analysis: 5 *D. marginatus*, 2 *D. reticulatus*, 3 *H. punctata*, and 5 *I. ricinus*. A query on GenBank showed that the COI gene sequences were available for *Dermacentor* spp. and *I. ricinus*, whereas only the 16S rDNA sequences were found for *H. punctata*. Sequencing and comparing of the 16S rDNA and *COI* gene sequences with the GenBank database using BLAST revealed reliable and consistent tick species identification, which matched the morphological data (Supplementary file 1). The protein MS profiles from the MALDI-TOF analysis reached high-intensity peaks in the range of 2–20 kDa. Of the remaining 138 ticks, all were identified with an LSV > 1.8 as follows: 13 as *I. ricinus*, 20 as *D. reticulatus*, 67 as *D. marginatus*, and 38 as *H. punctata*.

In total, among *Dermacentor* spp., 22 specimens (23 %) were *D. reticulatus* and 72 (77 %) were *D. marginatus*. Notably, *I. ricinus* was exclusively found at one site, Port Guen, an anthropized environment with an ecosystem favorable for *Ixodes*. *Dermacentor* was observed across all study sites, whereas *H. punctata* was absent at Port Guen. *Dermacentor* spp. were predominantly detected on the coast, whereas *H. punctata* was found both on the coast and inland. Table 2 details the tick species collected according to the collection sites.

## 3.2. Detection of microorganisms

This study focused on bacterial microorganisms potentially transmissible to humans. Specifically, 22 % of the collected *I. ricinus* were positive for *B. burgdorferi* sensu lato, with two cases of *B. afzelii*, one of *B. valaisiana*, and one of *B. lusitaniae*. Additionally, *A. phagocytophilum* was identified in one *I. ricinus* specimen. A high percentage of *Dermacentor* ticks (73 %) were positive for *Rickettsia* spp., comprising 28 % for *R. raoultii*, and 33 % for *R. slovaca*. For 39 % of these positive ticks, the precise identification of the bacterial species could not be achieved using PCR for *OmpB* gene sequencing. Table 2 shows the number of positive ticks and the type of microorganisms identified by tick species and collection site.

## 4. Discussion

In this study, three sites on Belle-Île were investigated compared with five sites in a 2016 study (Michelet et al., 2016), with one site being consistent between the studies (Saint Marc). The predominant tick

genera identified were *Haemaphysalis* and *Dermacentor*, with *D. marginatus* being the most prevalent within the *Dermacentor* genus. All ticks collected were only adults due to the type of vegetation. France hosts four species of *Haemaphysalis*: *H. concinna*, *H. hispanica*, *H. inermis*, and *H. punctata* (Perez-Eid, 2007). Only *H. punctata* was found on Belle-Île Island. This species is a three-host and exophilic tick, mainly inhabiting vegetation in forested area or pastures in bocage zones, and its medical importance in France is limited (Perez-Eid, 2007). We did not screen for specific bacteria in *H. punctata*, as no bacterial infections associated with this tick have been reported in Europe (Del Cerro et al., 2022) and it infrequently bites humans (Jumpertz et al., 2023). On Belle-Île, *Theileria buffeli* has been identified in *H. punctata* collected from cattle, contributing to mortality. These cattle were primarily imported from mainland France (Michelet, 2015). This tick species commonly coexists with the xerophilic tick *D. marginatus* in similar ecosystems. As noted by Michelet et al. (2016), *D. reticulatus* and *D. marginatus* are present on the island. Specifically, *D. marginatus* favors meadows and pastures, whereas *D. reticulatus* thrives in highly humid areas and exhibits greater cold tolerance (Estrada-Pena et al. 2017). Furthermore, *D. reticulatus* is undergoing expansion in Europe (Bilbija et al., 2023; Földvári et al., 2016). Although the presence of *I. ricinus* was suspected on Belle-Île, it was never previously confirmed. This tick species is widespread on mainland France (Perez et al., 2023) but surprisingly unreported on Belle-Île Island. In the present study, *I. ricinus* was collected solely at the Port Guen site, characterized by an anthropized valley with a small stream, a site not investigated by Michelet et al. (2016).

The distribution of ticks across study sites indicated a potential risk of human exposure to tick bites on the island. All life stages of *I. ricinus* can bite humans, with nymphs showing a preference (Lernout et al., 2019). This tick species requires high humidity levels, found in oceanic climates not only in forests but also in pastures, grazing areas, and hillsides (Kahl and Gray 2023); such conditions are present at Port Guen. *Haemaphysalis punctata* adults typically infest Bovidae and Cervidae, whereas immature stages feed on small mammals, birds, and reptiles. Both male and female *Dermacentor* spp. are commonly found on Bovidae, Suidae, and Equidae and can bite humans (Jumpertz et al., 2023). Immature stages are mainly endophilic, feeding on rodents, Erinaceidae, and lagomorphs (Guglielmone and Robbins, 2018). Nymphs can also bite larger mammals (deer, dog, goat) and occasionally human (Földvári et al., 2016).

Tick-borne diseases affecting humans have been reported on Belle-Île.

<table><caption>**Table 2**
Infection rate of human tick-borne pathogens identified in different adult ticks collected on Belle-Île en Mer.</caption><thead><tr><th>Collection site</th><th>Identified adult ticks</th><th>Total</th><th>B. burgdorferi s.l.</th><th>A. phagocytophilum</th><th>B. miyamotoi</th><th>Neoehrlichia mikurensis</th><th>Rickettsia spp.</th></tr></thead><tbody><tr><td rowspan="3">Port Guen Anthropized valley</td><td rowspan="3">Ixodes ricinus</td><td>18</td><td>B. afzelii<br/>2/18<br/>B. valaisiana 1/18<br/>B. lusitaniae 1/18</td><td>1/18</td><td>0/18</td><td>0/18</td><td></td></tr><tr><td>18</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>6</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="3">Donnant Sandy dunes</td><td rowspan="3">D. reticulatus<br/>2<br/>D. marginatus: 34</td><td rowspan="3">36</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="5">Saint Marc Coastal heath</td><td>H. punctata</td><td>12</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>D. reticulatus<br/>14</td><td>40</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>D. marginatus<br/>26</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>H. punctata</td><td>29</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

1 Rickettsia spp.

1 R. slovaca

2 R. raoultii

2 Rickettsia spp.

1 R. slovaca

1 R. raoultii

0

11 Rickettsia spp.

12 R. slovaca

2 R. raoultii

1 Rickettsia spp.

5 R. slovaca

5 R. raoultii

12 Rickettsia spp.

4 R. slovaca

9 R. raoultii

-
île; however, they have not been conclusively linked to local tick populations and their associated pathogens. Lyme borreliosis is highly endemic on the French mainland (Figoni et al., 2019), and local island physicians report three to four erythema migrans cases annually (Dr. E. de Poix, personal communication). Among the relatively few *I. ricinus* ticks collected (18 in total), a notable proportion was infected with *B. burgdorferi* sensu lato, including two species potentially responsible for Lyme borreliosis in humans: *B. afzelii* and *B. lusitaniae* (Stanek et al., 2012). Notably, *B. afzelii* is the most commonly identified species in human infections across Europe, whereas *B. lusitaniae* is less prevalent in *I. ricinus* populations, except in specific ecosystems (Boyer et al., 2022). The three *Borrelia* species, *B. afzelii*, *B. valaisiana*, and *B. lusitaniae*, are mainly associated with rodents, birds, and lizards, respectively (Cirkovic et al., 2024; Wolcott et al., 2021). Interestingly, one tick tested positive for *A. phagocytophilum*, although the precise ecotype was not investigated, leaving uncertainty regarding its potential pathogenicity for humans (Jahfari et al., 2014). No ticks were found to be infected with *B. miyamotoi* or *N. mikurensis*. On the French mainland, *B. miyamotoi* and *N. mikurensis* are regularly detected in ticks collected in Alsace but are rare in Brittany (Boyer et al., 2022, 2021). Considered an emerging bacterium, *N. mikurensis* belongs to the order Rickettsiales and is closely related to *A. phagocytophilum* (Li et al., 2012; Portillo et al., 2018). It is capable of causing severe febrile syndromes and fever in immunocompromised and immunocompetent individuals, respectively (Boyer et al., 2021; Grankvist et al., 2015). Regarding *Dermacentor* spp., *Rickettsia* spp. infections are frequently reported in Europe (Garcia-Vozmediano et al., 2020; Parola et al., 2009; Portillo et al., 2015; Santibáñez et al., 2022). A confirmed case was clinically documented at a hospital in Rennes in April 2008, involving a patient from Belle-Île (Rennes-Brittany, France; Dr. Degeilh, personal communication). The present study revealed a high prevalence of *Dermacentor* ticks with high infection rates of either *R. raoultii* or *R. slovaca* (28 % and 33 %, respectively).

Belle-Île-en-Mer hosts a diverse community of domestic animals (e. g., cattle and sheep) and wildlife (e.g., pheasants and hares). The island's oceanic climate supports year-round tick activity. Additionally, domestic animals accompanying tourists, particularly dogs, may introduce *D. reticulatus* ticks from the mainland. Sea birds can also transport ticks from the continent (Wilhelmsson et al., 2020). These factors collectively create a conducive ecosystem for ticks on Belle-Île en Mer, underscoring the importance of raising public and physician awareness regarding ticks and potential tick-borne diseases on the island.

### CRediT authorship contribution statement

**A. Haidar-Ahmad:** Investigation. **C. Barthel:** Methodology, Investigation, Formal analysis. **P. Boyer:** Methodology, Investigation, Formal analysis. **G. Joncour:** Resources, Investigation. **B. Degeilh:** Resources, Investigation. **N. Boulanger:** Writing – review & editing, Writing – original draft, Validation, Supervision, Funding acquisition, Formal analysis.

### Declaration of competing interest

The authors declare no conflict of interest.

### Data availability

Data will be made available on request.

### Acknowledgements

We are grateful to Dr E. de Poix, physician on Belle-Île since 2013 for sharing his information on Lyme borreliosis cases. We thank Santé Publique France for its financial support.

### Funding

University of Strasbourg, France.

### Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.tttdis.2024.102399.

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