# Dermacentor ticks and their human pathogens in various ecosystems of eastern France

C. Barthel, J. Stynen, M. Grau, Marie-Lazarine Poulle, P. Bauda, N.

Boulanger

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## To cite this version:

C. Barthel, J. Stynen, M. Grau, Marie-Lazarine Poulle, P. Bauda, et al., Dermacentor ticks and their human pathogens in various ecosystems of eastern France. Ticks and Tick-borne Diseases, 2025, 16 (4), pp.102520. 10.1016/j.ttbdis.2025.102520. hal-05191572

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Original article

# Dermacentor ticks and their human pathogens in various ecosystems of eastern France

C. Barthel<sup>a</sup>, J. Stynen<sup>a</sup>, M. Grau<sup>b, d</sup>, M-L. Poulle<sup>c, d</sup>, P. Bauda<sup>e, d</sup>, N. Boulanger<sup>a, f, *</sup>

## ARTICLE INFO

### Keywords:

Dermacentor reticulatus

Dermacentor marginatus

Rickettsia raoultii

Rickettsia slovaca

Wetlands

Ecosystem

Acarological risk

## ABSTRACT

Dermacentor is Europe's second most important tick genus. It comprises two species: *Dermacentor reticulatus*, which has a more northerly range, and *Dermacentor marginatus*, which is typically found in Mediterranean regions. In France, especially in the east, the presence of *Dermacentor* remains understudied. Leveraging projects on *Ixodes ricinus*, we identified high-risk ecosystems for *Dermacentor* ticks. Both species were present, but *D. reticulatus* was dominant (96.3 %), always coexisting with *Ixodes*, at densities reaching 30.3 questing adults/ 100 m² in Bas-Rhin. This species was found to thrive in wet meadows, alluvial forests, and lakes, where wild boars (*Sus scrofa*) are common. Among domestic animals, dogs and horses were the preferred hosts. Regarding human pathogens, we focused on the circulation of rickettsiae responsible for tick-borne lymphadenopathy. Acarological risk varied by region, with *Rickettsia raoultii* being the most prevalent bacterial species in all the departments, whereas *Rickettsia slovaca* was rarer and present only in 2 Bas-Rhin nature reserves. At the peak of tick activity, up to 18.7 % of *Dermacentor* adult ticks carried *R. raoultii*. In the studied region, *D. reticulatus* is the only known vector of these potentially pathogenic microorganisms.

## 1. Introduction

The two most common tick genera in Europe are *Ixodes* and *Dermacentor*. *Ixodes ricinus* is the most widespread, primarily inhabiting forest ecosystems with dense vegetation and leaf litter, requiring 80 % relative humidity (Kahl and Gray, 2023). *Dermacentor* often coexists with *Ixodes*, but its habitat varies by species: *Dermacentor marginatus* is found mainly in the xerophilic ecosystems of southern Europe, whereas *Dermacentor reticulatus* favors open, humid environments in northern regions, often in fallow land (Földvári et al., 2016; Rubel et al., 2016). The tick life cycle takes 1–2 years. Adult ticks feed on wild hosts, such as deer, hares, and wild boars, as well as domestic animals, including sheep, dogs, cattle, and horses, whereas larvae and nymphs parasitize small mammals (Rubel et al., 2016). In recent years, the presence of ticks has increased due to human-driven ecosystem changes, including vegetation management for enhanced biodiversity (Kilpatrick and

Randolph, 2012; Köhler et al., 2023; Mysterud et al., 2016), and the rise in wild ungulate populations (Gilbert et al., 2012; Kilpatrick et al., 2014). Increased temperatures (milder winters and less snowfall) are also conducive to the expansion of *D. reticulatus* populations (Probst et al., 2023). These socioeconomic and environmental changes have created more favorable conditions for tick proliferation, particularly benefiting *D. reticulatus* (Karbowiak, 2014).

In France, *I. ricinus* has been extensively studied owing to its role in Lyme borreliosis and public health impact (Perez et al., 2023; Septfons et al., 2019). However, fewer studies have examined *Dermacentor* habitats (Bonnet et al., 2013; Michelet et al., 2013) and the genus' potential human health risks. Leveraging various *I. ricinus* research projects in the Grand Est region, we investigated ecosystems where *Dermacentor* ticks are present. Some projects were conducted under Centre National de la Recherche Scientifique (CNRS; a French agency) programs, encouraging collaboration with local stakeholders using a multidisciplinary "one

<footnote><sup>a</sup> UR3073: PHAVI: Pathogen-Host-Arthropod Vector Interactions: ticks and tick-borne diseases group, Université de Strasbourg, France</footnote>

<footnote><sup>b</sup> Centre d'épidémiologie et de santé publique des armées (CESPA), Marseille, France</footnote>

<footnote><sup>c</sup> UR ESCAPE, Université de Reims, France</footnote>

<footnote><sup>d</sup> Zone Atelier Argonne, Institut de Chimie Moléculaire de Reims, UMR CNRS 7312, Case postale 44, UFR Sciences Exactes et Naturelles, BP 1039, 51687 Reims Cedex 2, France</footnote>

<footnote><sup>e</sup> LIEC, CNRS, Université de Lorraine, 57 000 Metz, France</footnote>

<footnote><sup>f</sup> Centre National de Référence Borrelia, Hôpitaux Universitaires de Strasbourg, France</footnote>

<footnote>* Corresponding author: Boulanger N.</footnote>

<footnote>E-mail address: nboulanger@unistra.fr (N. Boulanger).</footnote>
health” approach. We worked with hunters, forestry workers, and biodiversity organizations and we were consulted by clinicians at the Grand Est reference center for tick-borne diseases to identify ticks in several tick-borne lymphadenopathy (TIBOLA) cases (Santibánez et al., 2022). This epidemiological context led us to investigate not only risk ecosystems for Dermacentor exposure in the Grand Est region but also its infection rates with human pathogens, such as the bacteria Rickettsia, particularly Rickettsia raoultii and Rickettsia slovaca, both linked to TIBOLA (Parola et al., 2009). This syndrome, also known as scalp eschar neck lymphadenopathy or Dermacentor-borne necrosis erythema and lymphadenopathy, has been well documented (Portillo et al., 2015). Additionally, D. reticulatus is a secondary vector of tick-borne encephalitis virus (Chitimia-Dobler et al., 2019; Sidorenko et al., 2021).

In this study, we collected ticks in different ecosystems across the Grand Est region between 2021 and 2024. Of 59 sites sampled, only 18 contained Dermacentor ticks. Adult ticks were identified as well as Rickettsia species with potential human pathogenicity. Moreover, the key vertebrate hosts and most suitable ecosystems for Dermacentor ticks were determined.

## 2. Materials and methods

### 2.1. Tick collection sites

During four projects (*I. ricinus* studies: OHM, ZARG, ZAEU1, and ZAEU2; refer to the Supplementary data) conducted in the Grand Est region, we also collected *Dermacentor* adults (**Fig. 1**). In 2021, ticks were collected at 40 sites in Achen and Eguelhardt (OHM project: Moselle department) and 7 sites (ZARG project: Ardennes, Marne, and Meuse departments). OHM sites have been described previously (Boulanger et al., 2024), and ZARG sites are detailed in the Supplementary data (Table S1). In the Bas-Rhin department, 4 rural sites around Strasbourg

were sampled for 12 months (2022–2023; ZAEU1 project), and 8 urban and peri-urban sites in Strasbourg were sampled in 2024 (ZAEU2 project). Site characteristics of Bas-Rhin are provided in the Supplementary data (Tables S2 and S3). Ticks were collected on vegetation once a month mainly during the peak of *Ixodes* activity (March to June), except for the ZAEU1 project where a one year-study was conducted.

Ticks (*I. ricinus* and *Dermacentor* spp.) were collected by dragging a 1-m² white cloth over a 300-m² transect (200-m² transect for OHM). Ticks were transferred to tubes with tweezers and kept alive while being transported to the laboratory, where they were stored at -20 °C for DNA extraction.

For tick collections removed from horses, all adult ticks were manually removed from the necks of three horses at a farm once a month from March 2022 to March 2023 (ZARG project: Meuse department, Ferme Welfarm “La Hardonnerie” -55,270 Vauquois). Ticks were preserved in ethanol in tubes, mailed to the laboratory, and stored at room temperature. The farm is located in wet meadows surrounded by small hills.

### 2.2. Tick identification via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or molecular analysis

Each tick's four legs were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), following the protocol described by Boyer et al. (2019) and Hai-dar-Ahmad et al. (2025). Tick protein mass spectra were identified using the Microflex LT MALDI-TOF instrument (Brucker) and compared with an in-house MALDI-TOF MS database. A log score value of ≥1.8 was required for reliable species identification. Ticks collected from horses were identified via molecular analysis, as ethanol preservation at room temperature led to protein degradation, making MALDI-TOF MS identification unfeasible.

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** The five investigated departments are presented. Project 1: OHM, Moselle department, 40 investigated sites (5 positive sites presented for *Dermacentor*: #1 to 5); Project 2: ZARG, Ardennes, Meuse and Marne departments, 7 investigated sites (sites #6 to 12); Project 3: ZAEU 1: Bas-Rhin department, 4 investigated sites (sites #13 to 16); Project 4: ZAEU 2: Bas-Rhin department, 8 investigated sites (3 positive sites presented for *Dermacentor* #17 to 20). ▲: site investigated for ticks from horses.</figcaption></figure>
## 2.3. DNA extraction and polymerase chain reaction-based detection of rickettsia

Ticks were analyzed individually. *Dermacentor* DNA was extracted either using the MagNA Pure 96 (Roche Diagnostics) with the MagNA PURE 96 DNA and Viral NA Small Volume Kit (Roche Diagnostics) from 200 µL of tick lysate or via ammonium hydroxide (Guy and Stanek, 1991; Rijpkema et al., 1996). DNA from unfed ticks was extracted with 100 µl ammonium hydroxide, and if fed ticks were too large, they were cut in half and DNA extracted with 200 µl ammonium hydroxide. DNA extracts were stored at -80 °C for further analysis.

Detection of *Rickettsia* spp. in adult *Dermacentor* was performed using real-time quantitative polymerase chain reaction (RT-qPCR) targeting the *gltA* gene on the CFX Opus 96 instrument (Bio-Rad). Species identification was conducted via a nested end-point PCR assay targeting the *rompB* (rickettsial outer membrane protein B) gene of spotted fever group *Rickettsia* (Choi et al., 2005). PCR amplicons were sequenced using the Sanger method (GATC Eurofins Genomics). *Dermacentor* ticks removed from horses were identified by sequencing the 16S rDNA gene (Boyer et al., 2019).

## 2.4. Statistical analyses

To assess rickettsiosis risk, we determined the density of questing adult ticks per 100 m² (DOT), tick infection prevalence (TIP), and the density of infected ticks per 100 m² (DIT) by multiplying DOT by TIP to estimate acarological risk (Hartemink et al., 2024). DOT was modeled using classification and regression tree (CART) analysis with the rpart package (Therneau and Atkinson, 2025) in R version 4.4.2. Recursive partitioning was used to divide the dataset into homogeneous response-value groups (Strobl et al., 2009). DOT analysis included collection month (four levels: March, April, May, and June), collection year (three levels: 2021, 2022, and 2024), department (five levels: Moselle, Ardennes, Bas-Rhin, Marne, and Meuse), altitude (continuous numerical variable), and ecosystem type (six levels: alluvial forest, botanical garden, city park, deciduous forest, wetland, and meadow). Variables contributing ≥20 % weight in CART were retained.

# 3. Results

## 3.1. Dermacentor density during peak tick activity: global analysis

As part of four projects (*I. ricinus* studies: OHM, ZARG, ZAEU1, and ZAEU2), *Dermacentor* spp. and *I. ricinus* were collected from vegetation at 59 sites in the Grand Est region mainly during peak of tick activity from March to June (Fig. 2 and Table 1). *Dermacentor* spp. were found at 18 sites, with densities ranging from 0.3 to 9.5 ticks/100 m² (Table 1). Sites spanned diverse ecosystems, including forests, meadows, wetlands, rural areas, peri-urban areas, and urban areas, as well as low- and high-altitude regions (up to 500 m at Site 14) and natural protected areas distributed across five French departments (Ardennes, Bas-Rhin, Marne, Meuse, and Moselle; Supplementary data).

The highest *D. reticulatus* density (9.5 ticks/100 m²) was observed at Site 4 (A13, Moselle), a relatively new deciduous forest with wild boars (*Sus scrofa*), surrounded by agricultural land (Boulanger et al., 2024). Three additional sites in Bas-Rhin (Sites 15, 16, and 19) also had high *D. reticulatus* densities and were either nature reserves (Sites 15 and 16) or alluvial forest near peri-urban Strasbourg (Site 19) (Table 1 and Fig. 2). Wetland sites (Sites 8 and 9) were also positive for *Dermacentor*. In contrast, all negative sites were either large-forest areas with tall trees (Site 6, ZARG; 35 sites. OHM; Site 18, ZAEU2) or urban parks and botanical garden in Strasbourg (Sites 21–24, ZAEU2). Peak activity was recorded in April. Most of *Dermacentor* specimens collected during this period (March to June) were identified as *D. reticulatus* (228–97 %); only few *D. marginatus* (7–3 %) were collected and in only two sites (13-Urlosenholz; 15-Bernarswiller) (Table 2).

## 3.2. One-year study of dermacentor populations in bas-rhin

A one-year follow-up (2022–2023) was conducted at the four rural Bas-Rhin sites with high *Dermacentor* densities to assess seasonal variation in density and *Rickettsia* infection rates. Three sites showed high *Dermacentor* presence, with densities ranging from 0 in June to 30.33 in May 2023 (Fig. 3). These sites, wetlands located in the Rhine Valley, included two nature reserves (Bernardswiller and Herrenwald). The fourth site (Site 14, Niedermunster) served as a high-altitude reference (~500 m) with rare *Dermacentor* presence.

As in the global analysis, peak activity occurred in April, with annual variation observed (adult *D. reticulatus* questing density was lower in 2022 than in 2023 likely due to a milder climate in winter and spring

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Density of *Dermacentor* ticks/100 m² per site. From the 4 projects (OHM, ZARG, ZAEU1 and ZAEU2), we selected 24 sites among the 59 investigated sites to represent the different ecosystems where *Dermacentor* ticks are potentially present in the different departments of Eastern France (Moselle, Meuse, Marne, Ardennes, Bas-Rhin). Study performed during the peak of tick activity. The number of the different sites correspond to the number of each site listed in Table 1. F: forest; WL: Wetland; HP: Hunting park; NR: Nature reserve; AF: Alluvial forest; P: park.</figcaption></figure>
<caption>**Table 1**</caption>

Density of ticks (DOT)/100 m², infection prevalence and density of infected ticks (DIT)/100 m² for 20 sites illustrating the diverse *Dermacentor* ecosystems from March to June 2021 to 2024. Sites #21 to 24 are not included, they were negative for all the collection times.

<table><thead><tr><th rowspan="2">Positive Sites for Dermacentor spp.</th><th rowspan="2">Month</th><th rowspan="2"># of collected Dermacentor ticks</th><th rowspan="2">Density of tick/ 100 m²</th><th colspan="4">Number of positive ticks</th><th rowspan="2">Tick infection prevalence (%)</th><th rowspan="2">Density of infected ticks / 100 m² (Rickettsia)</th></tr><tr><th>1st PCR Rickettsia</th><th>R. raoultii</th><th>R. slovaca</th><th>Rickettsia spp.</th></tr></thead><tbody><tr><td colspan="9"><strong>OHM project - 2021 - 40 sites -Moselle</strong></td></tr><tr><td rowspan="3">1-Achen 3</td><td>April</td><td>3</td><td>1.5</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>May</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>June</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td rowspan="3">2-Achen 7</td><td>April</td><td>3</td><td>1.5</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>May</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>June</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td rowspan="3">3-Achen 12</td><td>April</td><td>4</td><td>2</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>May</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>June</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td rowspan="3">4-Achen 13</td><td>April</td><td>19</td><td>9.5</td><td>4</td><td>1</td><td>0</td><td>3</td><td>21.1</td><td>2</td></tr><tr><td>May</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>June</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td rowspan="3">5-Achen 17</td><td>April</td><td>2</td><td>1</td><td>1</td><td>0</td><td>0</td><td>1</td><td>50</td><td>0.5</td></tr><tr><td>May</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>June</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td colspan="9"><strong>ZARG Project - 7 investigated sites - 2021 - Ardennes, Marne, Meuse</strong></td></tr><tr><td rowspan="4">6-Dannevoux forest</td><td>March</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>April</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>May</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>June</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td rowspan="4">7-Belval/Argonne-forest</td><td>March</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td></tr><tr><td>April</td><td>7</td><td>2.3</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td></tr><tr><td>May</td><td>1</td><td>0.3</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td></tr><tr><td>June</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td rowspan="4">8-Belval/Argonne-lake</td><td>March</td><td>2</td><td>0.7</td><td>1</td><td>1</td><td>0</td><td>0</td><td>50</td><td>0.4</td></tr><tr><td>April</td><td>10</td><td>3.3</td><td>4</td><td>2</td><td>0</td><td>2</td><td>66</td><td>1.1</td></tr><tr><td>May</td><td>1</td><td>0.3</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td></tr><tr><td>June</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td rowspan="4">9-Germont-swamp</td><td>March</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>April</td><td>6</td><td>2</td><td>5</td><td>5</td><td>0</td><td>0</td><td>83.3</td><td>1.7</td></tr><tr><td>May</td><td>4</td><td>1.3</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>June</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td rowspan="4">10-Belval Park-Cuve</td><td>March</td><td>2</td><td>0.7</td><td>2</td><td>1</td><td>0</td><td>1</td><td>100</td><td>0.7</td></tr><tr><td>April</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>May</td><td>2</td><td>0.7</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td></tr><tr><td>June</td><td>1</td><td>0.3</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td></tr><tr><td rowspan="4">11-Belval Park-red deer</td><td>March</td><td>5</td><td>1.7</td><td>2</td><td>1</td><td>0</td><td>1</td><td>40</td><td>0.7</td></tr><tr><td>April</td><td>3</td><td>1</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td></tr><tr><td>May</td><td>1</td><td>0.3</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td></tr><tr><td>June</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td rowspan="4">12-Belval Park-roe deer</td><td>March</td><td>2</td><td>0.7</td><td>1</td><td>0</td><td>0</td><td>1</td><td>100</td><td>0.7</td></tr><tr><td>April</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>May</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>June</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td colspan="9"><strong>ZAEU 1 - Project rural sites around Strasbourg - 4 sites - 2022 - Bas-Rhin</strong></td></tr><tr><td rowspan="4">13-Urlosenholz</td><td>March</td><td>10</td><td>3.3</td><td>4</td><td>3</td><td>0</td><td>1</td><td>40</td><td>1.3</td></tr><tr><td>April</td><td>15</td><td>5</td><td>10</td><td>7</td><td>0</td><td>3</td><td>66.7</td><td>3.3</td></tr><tr><td>May</td><td>5</td><td>1.7</td><td>1</td><td>1</td><td>0</td><td>0</td><td>20</td><td>0.3</td></tr><tr><td>June</td><td>3</td><td>1</td><td>1</td><td>1</td><td>0</td><td>0</td><td>33.3</td><td>0.3</td></tr><tr><td rowspan="4">14-Niedermunster</td><td>March</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>April</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>May</td><td>1</td><td>0.3</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>June</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td rowspan="4">15-Bernardswiller</td><td>March</td><td>5</td><td>1.7</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>April</td><td>22</td><td>7.3</td><td>5</td><td>4</td><td>0</td><td>1</td><td>22.7</td><td>1.7</td></tr><tr><td>May</td><td>2</td><td>0.7</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>June</td><td>2</td><td>0.7</td><td>1</td><td>1</td><td>0</td><td>0</td><td>50</td><td>0</td></tr><tr><td rowspan="4">16-Herrenwald</td><td>March</td><td>6</td><td>2</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>April</td><td>25</td><td>8.3</td><td>10</td><td>8</td><td>0</td><td>2</td><td>40</td><td>3.3</td></tr><tr><td>May</td><td>3</td><td>1</td><td>3</td><td>1</td><td>0</td><td>2</td><td>100</td><td>1</td></tr><tr><td>June</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td colspan="9"><strong>ZAEU 2 - Project sub-urban and urban - 8 sites - 2024 - Bas-Rhin (4 negative parks not included: 21 to 24)</strong></td></tr><tr><td rowspan="4">17-Rohrschollen island</td><td>March</td><td>8</td><td>2.7</td><td>3</td><td>2</td><td>0</td><td>1</td><td>37.5</td><td>1</td></tr><tr><td>April</td><td>11</td><td>3.7</td><td>8</td><td>3</td><td>0</td><td>5</td><td>72.7</td><td>2.7</td></tr><tr><td>May</td><td>6</td><td>2</td><td>3</td><td>1</td><td>0</td><td>2</td><td>50</td><td>1</td></tr><tr><td>June</td><td>2</td><td>0.7</td><td>1</td><td>1</td><td>0</td><td>0</td><td>50</td><td>0.3</td></tr><tr><td rowspan="4">18-Neudorf forest</td><td>March</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>April</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>May</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>June</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 1 (continued)**</caption><thead><tr><th rowspan="2">Positive Sites for Dermacentor spp.</th><th rowspan="2">Month</th><th rowspan="2"># of collected Dermacentor ticks</th><th rowspan="2">Density of tick/ 100 m²</th><th colspan="4">Number of positive ticks</th><th rowspan="2">Tick infection prevalence (%)</th><th rowspan="2">Density of infected ticks / 100 m² (Rickettsia)</th></tr><tr><th>1st PCR Rickettsia</th><th>R. raoultii</th><th>R. slovaca</th><th>Rickettsia spp.</th></tr></thead><tbody><tr><td rowspan="5">19-Robertsau</td><td>March</td><td>20</td><td>6.7</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td></tr><tr><td>April</td><td>20</td><td>6.7</td><td>2</td><td>0</td><td>0</td><td>2</td><td>10</td><td>0.7</td></tr><tr><td>May</td><td>10</td><td>3.3</td><td>1</td><td>0</td><td>0</td><td>1</td><td>10</td><td>0.3</td></tr><tr><td>June</td><td>1</td><td>0.3</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>July</td><td>2</td><td>0.7</td><td>*</td><td>*</td><td>*</td><td>*</td><td>0</td><td>0</td></tr><tr><td rowspan="5">20-Pourtalès park</td><td>March</td><td>2</td><td>3.7</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>April</td><td>11</td><td>1.7</td><td>3</td><td>0</td><td>0</td><td>3</td><td>60</td><td>1</td></tr><tr><td>May</td><td>5</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>June</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>TOTAL</td><td>273</td><td>17</td><td>76</td><td>44</td><td>0</td><td>32</td><td></td><td></td></tr></tbody></table>

<caption>**Table 2**</caption>

Summary of the prevalence of *Rickettsia* spp. in ticks and *Dermacentor* species identification in the different departments.

<table><thead><tr><th>Collected ticks</th><th>Tested ticks</th><th>Rickettsia raoultii</th><th>Rickettsia slovaca</th><th>Rickettsia spp.</th><th>Dermacentor reticulatus</th><th>Dermacentor marginatus</th></tr></thead><tbody><tr><td colspan="7">(A) Rickettsia prevalence during the peak of tick activity – March to June- All projects/ all departments</td></tr><tr><td>273</td><td>235</td><td>18.7 %<br>(44)</td><td>0 %</td><td>13.6 %<br>(32)</td><td>97 %<br>(228)</td><td>3 %<br>(7)</td></tr><tr><td colspan="7">(B) Rickettsia prevalence in the one-year study in Bas-Rhin – rural area</td></tr><tr><td>292</td><td>237</td><td>22.8 %<br>(54)</td><td>1.7 %<br>(4)</td><td>7.2 %<br>(17)</td><td>94.1 %<br>(223)</td><td>5.9 %<br>(14)</td></tr><tr><td colspan="7">(C) Rickettsia prevalence across all study sites – Grand Est region</td></tr><tr><td>462</td><td>372</td><td>19.9 %<br>(74)</td><td>1.1 %<br>(4)</td><td>9.4 %<br>(35)</td><td>96.3 %<br>(358)</td><td>3.7 %<br>(14)</td></tr></tbody></table>

Numbers in parentheses correspond to total number of ticks.

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Density of *Dermacentor* ticks/100 m² collected for a year in 4 rural sites in the region of Strasbourg in 2022–2023. F: Forest, NR: Nature reserve.</figcaption></figure>

2023). A smaller activity peak was detected in fall. Compared with *I. ricinus*, *Dermacentor* activity began earlier in spring, with some individuals collected as early as February. Most collected ticks (94.1 %) were *D. reticulatus* (Table 2). *Dermacentor marginatus* ticks (5.9 %) were only detected in two sites in Bas-Rhin, one a nature reserve (Site15–14.4 %) and one in an old forest with a small wet meadow, regularly frequented by wild boar (site 13–5.6 %) (Fig. 4). The two sites are located very close to each other.

Regression tree analysis (Fig. 5) based on 91 observations confirmed the impact of collection month, altitude, and ecosystem type on tick

density. Year and department were not retained, given their negligible impact in CART analysis (<20 %). Collection during April, particularly in alluvial or wetland environments, led to the highest predicted DOT (3.8 ticks/100 m² in 9 % of cases). In other months, DOT declined at altitudes above 140 m (Fig. 6).

### 3.3. Presence of *rickettsia* spp. across all study sites

During the peak of *Dermacentor* activity from March to June, among the 235 analyzed 76 were positive for *Rickettsia* spp. (32.3 % infection
<figure><img src="image_7.png" /><figcaption>Fig. 4. Presence of *Dermacentor* species at the collection sites in the rural region of Strasbourg (project ZAEU 1). F: Forest; NR: Nature reserve.</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>Fig. 5. Ecosystems the most at risk for *Dermacentor*. The classification tree is based on variation of density of ticks (DOT) depending on the (1) month, (2) ecosystem and altitude. Lecture is from the top to the bottom. (3) At the bottom, the first number is the predicted DOT (higher in dark blue and lower with light blue), then the percentage of sites in the database which belong to this classification.</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>Fig. 6. Infection rate of *Dermacentor reticulatus* with *Rickettsia* spp. in rural sites of Bas-Rhin. F: Forest; NR: Nature reserve.</figcaption></figure>

rate; Tables 1 and 2). Nested PCR and sequencing identified *R. raoultii* in 44 ticks (18.7 %), whereas *R. slovaca* was not detected. The *Rickettsia* species of 32 ticks (13.6 %) could not be determined. The DOT was

highest in April, coinciding with peak *Dermacentor* activity. The highest risk areas for *R. raoultii*, the most pathogenic *Rickettsia* species in humans, were rural sites (#13 and 16) in Bas-Rhin with a density of 3.3
infected ticks/100 m² (Table 1).

The one-year study in rural areas of Bas-Rhin (ZAEU1) confirmed a high prevalence of *R. raoultii* (22.8 %) while detecting a low proportion of *R. slovaca* (1.7 %) in 2023 (Table 2). No *Rickettsia* spp. were detected in *D. marginatus* (data not shown).

Among the 462 *Dermacentor* ticks collected on vegetation in all study sites, a total of 372 was tested for *Rickettsia* spp. (Table 2). *Rickettsia raoultii* was the most detected bacterium with an average of 19.9 % infection prevalence in *D. reticulatus*; *R. slovaca* had an infection prevalence of 1.1 % in *D. reticulatus* and was present only in Bas-Rhin department (two rural sites).

### 3.4. One-year study of tick infestation in horses

A farmer in the ZARG project reported persistent tick infestations on horses. In response, ticks were collected monthly from three horses for one year. Of all collected ticks, only one was identified as *I. ricinus*; the remainder were *Dermacentor* spp. (both males and females). Ticks were observed year-round, firmly attached to the horses (Fig. 7), with major cement deposits at bite sites (data not shown). Owing to poor preservation in ethanol, MALDI-TOF MS identification was unsuccessful; however molecular analysis confirmed all ticks as *D. reticulatus*. Seasonal variation in infestation was noted: the highest tick burdens occurred in winter and in spring, with reduced numbers in summer. Tick parasitism varied markedly among the three horses.

## 4. Discussion

*Dermacentor reticulatus* has been expanding across Europe, as reported in multiple studies (Bilbija et al., 2023; Földvári et al., 2016; Rubel et al., 2016; Springer et al., 2022). This expansion is attributed to several factors, including land use changes and shifts in host distribution (Drehmann et al., 2020; Földvári et al., 2016). In France, *Dermacentor* ticks have been studied sporadically, likely owing to their historically low presence. *Dermacentor reticulatus* has been identified in different ecosystems, predominantly in southern France on dogs and cattle (Gilot and Perez-Eid, 1998). A previous study exploring sites in southern and northwestern France found *D. marginatus* to be the dominant *Dermacentor* species, with a focus on veterinary-associated pathogens (Bonnet et al., 2013). In a study conducted on Belle-Ile-en-mer, an island in western France, *D. marginatus* was again the predominant species, likely due to its preference for oceanic and xerophilic climates (Haidar-Ahmad et al., 2025). Both *R. slovaca* and *R. raoultii* were detected in *D. marginatus* and *D. reticulatus* collected from this island.

In the present study, we focused on eastern France, confirming that

wetlands and alluvial forests are high-risk environments, particularly in rural areas around Strasbourg (Bas-Rhin), and in agricultural landscapes, such as those in Moselle (OHM), or wetlands, such as those in Ardennes (ZARG). In contrast, *Dermacentor* ticks were rare in high-altitude forested areas and urban settings, including parks, likely due to the absence of key hosts. We observed *D. reticulatus* activity in winter, even on frozen ground during sunny days, as previously described (Drehmann et al., 2020). *Dermacentor reticulatus* was found to be markedly more abundant than *D. marginatus*, which was restricted to the Rhine Valley (Bas-Rhin). For the statistical analyses of these data, the choice was made to use the random forest method to process the data (Boulesteix et al., 2012). The decision tree used enables a non-parametric and robust analysis, particularly with regard to outliers, adapted to our dataset. This method automatically selects relevant variables. Rendering the results in the form of a decision tree enables simple, comprehensible visualization of the data. This type of analysis is regularly used in environmental or sociological studies.

The role of *Dermacentor* ticks in human tick-borne infections cannot be underestimated. In the agricultural area of Moselle (site#4) and rural sites around Strasbourg (sites #15 and 16), *Rickettsia* infection rates were high, with *R. raoultii* being the predominant species found in *D. reticulatus*. Notably, *D. marginatus* was present in two nature reserves in Bas-Rhin but was negative for *R. slovaca* and *R. raoultii*. Although *D. reticulatus* was positive for these *Rickettsia* species, infection levels were low. In eastern France, *R. raoultii* appears more frequently in ticks compared with *R. slovaca* (Table 2). However, given its potential pathogenicity, *R. slovaca* should not be overlooked (Parola et al., 2009).

A study in Marseille (southern France) found *Dermacentor* ticks to be the second most commonly removed tick genus from bitten patients (136 cases, 33 %), following *Ixodes* (197 cases, 47 %) (Jumpertz et al., 2023). *Dermacentor marginatus* was removed more frequently (113 cases) from patients compared with *D. reticulatus* (5 cases), aligning with its endemic presence in southern France. Among patients bitten by *Dermacentor*, 14 cases of TIBOLA were diagnosed, associated with *R. raoultii* or *R. slovaca* (Jumpertz et al., 2023). Similarly, (Földvári et al., 2013) noted the presence of *R. raoultii* in both *Dermacentor* species, whereas *R. slovaca* was only detected in *D. marginatus* retrieved from patients. In Spain, *D. marginatus* was the only *Dermacentor* species found on patients (88.73 % females; 11.27 % males), with infections caused by *Candidatus Rickettsia rioja* (91 cases), *R. slovaca* (66 cases), and *R. raoultii* (4 cases) (Santibáñez et al., 2022). In eastern France, clinicians are increasingly reporting *Dermacentor*-associated rickettsiosis (Hansmann and Baux, Centre Référence Maladies Vectorielles à Tiques, Grand Est- personal communication).

Both tick *Dermacentor* species are proven vectors of *Babesia caballi*

<figure><img src="image_10.png" /><figcaption>**Fig. 7.** Evolution of *Dermacentor reticulatus* parasitism in 3 horses maintained in the same pasture in Meuse department (project ZARG).</figcaption></figure>
and *Theileria equi* (Estrada-Pena et al., 2017), and *D. reticulatus* is a vector of Anaplasma marinale and *Babesia canis* (Estrada-Pena et al., 2017; Gray et al., 2019). In the present study, we did not test for specific pathogens in *Dermacentor* collected from horses but identified all specimens as *D. reticulatus*. Male and female ticks were present on horses year-round, as indicated by the presence of cement around bite sites (Suppan et al., 2018). A German study showed that *D. marginatus* (35 %) was more prevalent than *D. reticulatus* (16.7 %) on horses. In contrast, *D. reticulatus* was mainly found on dogs, 47 % versus 3 % of *D. marginatus* (Drehmann et al., 2020).

In conclusion, this study confirms that *D. reticulatus* is the most prevalent *Dermacentor* species in eastern France, with the highest densities in rural and agricultural lowland areas, especially wetlands and nature reserves (supplementary data S4-Figure1). In suburban environments, alluvial forests were particularly suitable for these ticks. However, they were absent in urban areas, likely due to dry conditions and a lack of suitable hosts. The majority of collected *Dermacentor* ticks were infected with *R. raoultii*, with fewer cases of *R. slovaca*. Peak activity was in April, although ticks were already present as early as February during warm spells. Interestingly, *D. reticulatus* was found on horses throughout the year, with some animals being more heavily parasitized than others. These findings support previous observations (Földvári et al., 2016). Given the increasing presence of *Dermacentor* ticks across various European ecosystems, particularly in low-altitude rural and suburban wetlands, greater awareness among the public and clinicians is necessary to mitigate associated health risks.

## Funding

We are grateful to the CNRS for funding various projects: Observatoire Homme Milieu (OHM), Pays de Bitche, and Zone Atelier Environnementale Rurale Argonne (ZARG), part of the French Réseau des Zones Ateliers rZA, a member of LTER Europe and ILTER. We thank the Agence Régionale de Santé (ARS) Grand Est and the Starsbourg Eurometropole for their financial support for the project in the urban and suburban regions of Strasbourg (Bas-Rhin).

## CRediT authorship contribution statement

**C. Barthel:** Formal analysis, Data curation. **J. Stynen:** Methodology, Investigation, Formal analysis, Data curation. **M. Grau:** Methodology, Formal analysis, Data curation. **M.-L. Poulle:** Resources, Methodology, Investigation. **P. Bauda:** Funding acquisition, Formal analysis, Data curation, Conceptualization. **N. Boulanger:** Writing – review & editing, Writing – original draft, Validation, Supervision, Investigation, Funding acquisition, Conceptualization.

## Declaration of competing interest

The authors declare no conflict of interest.

## Acknowledgements

We thank L. Placon from WELFARM association, La Hardonnerie farm, 55270 Vauquois, France, for collecting ticks from horses; the Eurométropole of Strasbourg for permission to access the protected aluvial forests of Robertsau, Neudorf, and Rohrschollen (Bas-Rhin); Ph. Vauchelet, curator of the Germont marsh, Conservatoire d'espaces naturels de Champagne-Ardennes; D. Pierrard, director of the Parc de Belval Bois des Dames, Fondation François Sommer, Ardennes; F. Baudet and A. Derrez of the Ardennes and Bas-Rhin Hunter Federation respectively; and E. Wilhem from the National Forest Office, Réserve Naturelle Régionale des Etangs de Belval en Argonne (Marne, 51), Conservatoire des Espaces Naturels Champagne-Ardennes.

## Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.ttbdis.2025.102520.

## Data availability

No data was used for the research described in the article.

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