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All issues Volume 31 (2024) Parasite, 31 (2024) 21 Full HTML
Open Access
Issue
Parasite
Volume 31, 2024
Article Number 21
Number of page(s) 12
DOI https://doi.org/10.1051/parasite/2024011
Published online 10 April 2024

© J. Habib et al., published by EDP Sciences, 2024

Licence Creative CommonsThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction

Ticks (order Ixodida) are the most important vectors of infectious diseases for animals and the second for humans, after mosquitoes [22, 65]. They can transmit a variety of bacteria (e.g., Borrelia burgdorferi s.l. [= Borreliella burgdorferi s. l.],, Anaplasma spp. and Rickettsia spp.), viruses (e.g., tick-borne encephalitis virus) and parasites (e.g., Babesia spp. and Theileria spp.) [38, 79]. The diseases caused by these pathogens represent major threats to public and animal health [15]. In the context of global climate and environmental change, concerns about ticks and tick-borne diseases are growing, as these changes over the last 30 years have led to modified phenology and geographical expansion of ticks [38, 63, 91].

Among these changes, those involving temperature and humidity have been accompanied by modifications in phenology, survival and tick development [21, 31, 71], which have favoured an increase in tick abundances, the seasonality of tick activity and/or their geographical expansion in some areas [31, 59, 71]. During their life cycle, ticks spend most of their time in the environment and therefore their life history traits and fitness are highly dependent on environmental conditions, such as the weather, the presence of potential hosts, and predation risk. For example, a decrease in hatching and moulting time has been demonstrated, and also of the proportion of questing ticks, as the ambient temperature increases [30, 69, 87]. Milder winter weather conditions may be observed in some areas, potentially allowing ticks to remain active and seek hosts during this time of the year in these areas [16]. Hence, in areas where ticks were already present, global warming will probably result in an extended period of tick activity.

Considering the spatial distribution of ticks, various tick species are also invading new areas at higher latitudes and/or altitudes. This is the case in Africa for ticks of the genera Amblyomma [23] and Rhipicephalus [18, 72], and for the tick Ixodes ricinus in Europe [63]. Land use changes, e.g., in forest management, and their effects on host communities have contributed to increased geographical distribution [90]. It is considered that the spatial expansion of I. ricinus is mainly explained by the opening of new habitats favourable to its installation, rather than by its adaptation [90]. This colonisation of new areas is partially facilitated by more mobile hosts such as migratory bird species [70] and some mammals, such as roe deer (Capreolus capreolus) [46]. However, although this is not the only factor, the expansion of I. ricinus seems to be explained in the first place by global warming [63]. In fact, local weather conditions in some areas did not allow for the activity, development and survival of ticks a few decades ago. Due to long-term changes in temperature and humidity, some areas now have weather conditions suitable for certain tick species, allowing their local maintenance. This phenomenon partially explains the northward (e.g., Sweden, Norway, European Russia [46, 48, 99]) and altitudinal (e.g., Czechia, Bosnia-Herzegovina [61, 73]), expansion of ticks observed during the past few decades. This results in higher exposure of either humans or domestic and wild animals to ticks and to pathogen transmission at higher latitudes and altitudes [22, 45].

Wild ungulates play a major role as a tick abundance amplifier and/or in the epidemiology of some tick-borne diseases of veterinary and/or public health importance [26, 52, 79, 80]. In Hungary, during the summer months, red deer (Cervus elaphus) and roe deer harbour many more ticks (I. ricinus and Haemaphysalis concinna) than domestic goats and sheep [41]. Because of their frequentation of tick-tolerant woodland habitats, red deer, roe deer and Mediterranean mouflon (Ovis gmelini musimon × Ovis sp.) have been shown to be important hosts of adults and immature stages of the tick I. ricinus [41, 47]. In Corsica, D. marginatus, Rh. bursa and I. ricinus were the most prevalent tick species collected from wild boars, Corsican mouflon (Ovis gmelini musimon) and red deer, respectively [32]. However, despite the geographical expansion of ticks in mountainous areas, knowledge on ticks in mountain ungulates and the associated vector-borne disease risks in these areas remains low [52, 92], especially in France [17, 33]. Yet, the recent discovery of Rickettsia monacensis in questing I. ricinus ticks in a French Pyrenees area inhabited by Pyrenean chamois (Rupicapra pyrenaica) [1] supports the need for a better understanding of the presence of tick species and of tick-borne pathogens in mountainous areas.

We aimed to study here the diversity of tick species infecting wild ungulates and the presence of major tick-borne pathogens for both animals and humans in mountainous areas of France. We first collected and identified ticks from different species of wild mountain ungulates (Corsican and Mediterranean mouflon, chamois Rupicapra rupicapra and Pyrenean chamois), in 4 mountainous areas in France, and then investigated the presence of pathogens in these ticks.

Materials and methods

Study regions, tick collection and identification

The study was conducted in 4 mountainous areas in France: (1) Bauges (French Alps), (2) Caroux-Espinouse (Massif Central), (3) Cinto (Corsica) and (4) Bazès (French Pyrenees) (see Table 1 for further details on the location and study areas).

Table 1

Characteristics of the study areas.

Wild ungulates are captured annually in spring-summer (Bauges, Bazès and Caroux-Espinouse) or winter (Cinto) by the Office Français de la Biodiversité (formerly Office National de la Chasse et de la Faune Sauvage) in each of the 4 study areas (Table 1). Animals are captured with traps (Caroux-Espinouse and Cinto), falling net (Bauges), or leg-hold snares (Bazès), and are then manually immobilised. Information on the species, sex, and age of each animal are noted. A meticulous examination of the fur on the head, in the armpit and in the inguinal areas is also performed to detect attached ticks. All or a random sample of ticks were collected from the examined parts of the trapped animals. Collected ticks were placed in small tubes with 70% ethanol and stored at room temperature before their transport to the laboratory of parasitology at VetAgro Sup (Lyon, France). Using a binocular microscope, species, stage and sex were identified using morphological criteria following standard taxonomic keys [24]. Ticks were then stored individually in 1.5 mL plastic tubes with 70% ethanol and stored at −20 °C. For a few ticks, morphological identification was confirmed by molecular analyses.

Tick selection and DNA extraction

Among the collected ticks, we randomly selected adult male and female ticks in each study area among host species, tick species and years. Prior to DNA extraction, ticks were individually washed twice for 10 min in 800 μL of 70% ethanol then in sterile phosphate buffered saline (PBS), after which ticks were transferred into new 1.5 mL tubes and washed a final time with PBS. The tubes were vigorously vortexed after each bath and finally, PBS was removed to allow ticks to dry. Each tick was incised into small pieces while in the tube with a disposable scalpel blade. DNA was then extracted from each tick using NucleoSpin® Tissue kits (Macherey-Nagel, Düren, Germany). At the final extraction step, DNA was eluted in 80 μL of kit solution. Extracted DNA was stored at −20 °C prior to molecular analysis.

Molecular analyses

All molecular analyses were processed on individual samples (no pool). The quality of the extracted DNA was verified by PCR amplification of a 320 bp region of the mitochondrial 16S rDNA specific to ticks using the primers TQ 16S+1F and TQ 16S-2R (Supplementary material) [3]. PCR products were then examined by gel electrophoresis (1.5% agarose gel, Standard Agarose, Eurobio, France), stained with bromophenol blue stain and detected using ultraviolet light (Kodak EDAS 290, New York, NY, USA). For a few ticks, PCR product was sequenced to confirm morphological identification.

Successfully extracted DNA from ticks was used to screen for the presence of DNA of Borrelia burgdorferi s.l., Anaplasmataceae, Babesia/Theileria spp. and Rickettsia spp. with individual PCR assays. The samples positive in the Anaplasmataceae PCR assay were further analysed with (1) a nested PCR assay using ge3a/ge10r as the first primer couple and ge9f/ge2r as the second, amplifying a part of the 16S rDNA specific to Anaplasma phagocytophilum and with PCR assays using the primers (2) AovisMSP4Fw/AovisMSP4Rev and (3) A. marginale F/A. marginale R amplifying the 16s rDNA specific to A. ovis and A. marginale, respectively. Similarly, for SFG Rickettsia, only samples that were positive in the Rickettsia spp. PCR assay were further analysed using the primer couple Rr190.70p/Rr190.602n amplifying the ompA gene specific for SFG Rickettsia. Positive and negative controls were included. All primers and PCR conditions are presented in the Supplementary material. All amplified products were examined by gel electrophoresis, as described previously.

Sequence analysis

Positive samples for each tested pathogen showing clear positive strands on gel electrophoresis were randomly selected for sequence analysis. Samples were sent to Biofidal Laboratory (Villeurbanne, France) for sequencing in both directions using the same primers as those used in the PCR assays. We used CLC Main Workbench 8 (QIAGEN, Hilden, Germany) to analyse the quality of the sequences and create consensus sequences. Consensus sequences, excluding primers, were compared with sequences available from the GenBank® database with the BLAST tool of the CLC Main Workbench.

Statistical analysis

We used the association screening approach [101] to test for statistically significant associations among pathogens. Briefly, it compares a simulated theoretical distribution of all possible combinations of pathogens under the null hypothesis H0 (i.e., random association of pathogens) and the observed counts.

Results

Ticks collected

A total of 2,081 ticks were collected and identified from wild ungulates during the study (Figure 1). We morphologically identified 6 tick species: Ixodes ricinus, Rhipicephalus bursa, Rhipicephalus sanguineus s.l., Haemaphysalis punctata, Haemaphysalis sulcata and Dermacentor marginatus.

thumbnail Figure 1

Location of the 4 study areas in France (top left), and for each area, description of the species and number of ungulates on which ticks were collected (top right). For each tick species, description of the total number (middle) and the detailed (i.e., value for each stage; bottom) number of ticks collected and identified. *When collected females were mating with a male, both the female and male were counted and considered in the table.

Only I. ricinus ticks were identified in the French Alps (Bauges) from the 273 ticks collected from 12 Mediterranean mouflon and 71 chamois, and in Bazès from the 254 ticks collected from 27 Pyrenean chamois, where a typical mountain climate occurs (Table 1). In the Caroux-Espinouse massif, with Mediterranean, oceanic and mountain climatic influences, different tick species were collected on 110 Mediterranean mouflon. Among the 1,059 collected ticks, the most abundant species were I. ricinus (54.0%) and H. punctata (39.1%), whereas Rh. sanguineus s.l. and D. marginatus were more rare (6.7% and 0.2%, respectively). In Cinto, a study site with a Mediterranean mountain climate, no I. ricinus ticks were identified in the 495 ticks collected from 86 Corsican mouflon. Rhipicephalus bursa was highly predominant in this area (96.0%), and only 16 ticks were identified as H. sulcata (3.2%) and 4 as H. punctata (0.8%). Most of the collected ticks were females (n = 1,125; 54.1%), followed by nymphs (n = 474; 22.8%), males (n = 472; 22.7%) and larvae (n = 10; 0.5%; Figure 1).

DNA extraction and PCR assays for the detection of vector-borne pathogens and validation of morphological identification of a few ticks were conducted on 791 adult ticks randomly selected: 155 I. ricinus in Bauges, 202 I. ricinus in Bazès, 93 I. ricinus, 55 Rh. sanguineus s.l. and 66 H. punctata in Caroux-Espinouse and 206 Rh. bursa and 14 H. sulcata in Cinto.

Detection of Babesia/Theileria spp.

Among the 791 tested ticks, only 7 (0.88%) were positive for Babesia/Theileria spp. One I. ricinus (0.5%; 1/202) in Bazès, 1 I. ricinus (1.1%; 1/93) in Caroux-Espinouse and 5 Rh. bursa (2.4%; 5/206) ticks, all collected in 2015, in Cinto, were positive for this pathogen (Table 2). All seven positive samples were submitted for sequence analysis.

Table 2

Results of PCR assays for the different tested pathogens (number of positive samples/number of tested samples, with the corresponding percentages in brackets), for each tick species from the 4 study areas in France.

In Cinto, the sequences from the five Rh. bursa collected were all identical (578 bp amplicon; GenBank accession number (AN): OR420709) and showed 100% similarity with Theileria ovis previously isolated from small ruminants mostly in the Middle East, other Mediterranean countries and China (GenBank AN: e.g., MN625886.1, MN493111.1). The 2 samples isolated in I. ricinus from Bazès (518 bp [incomplete sequence]) and Caroux-Espinouse (560 bp; GenBank accession number (AN): OR420710) (100% similarity) showed 100% similarity with Babesia venatorum (a.k.a. Babesia sp. EU1) detected in ticks, roe deer and also humans in several countries (e.g., European countries, Russia, China, Mongolia; GenBank AN: e.g., MG344777.1, MG052939.1).

Detection of Borrelia burgdorferi s.l.

PCR screening for the presence of B. burgdorferi s.l. in I. ricinus revealed an overall prevalence of infection of 2.0% (9/450) in tested ticks. Among the positive samples, eight females I. ricinus (8.6%; 8/93) were collected from Caroux-Espinouse, and one female I. ricinus (0.6%; 1/155) was collected from Bauges (Table 2).

Analysis of the sequence isolated from the positive sample from Bauges (351 bp; GenBank accession number (AN): OR421277) showed 100% similarity with B. afzelii previously isolated from Ixodes sp. ticks in e.g., Europe, Russia, and China (GenBank AN: e.g., CP018262.1, KX622580.1). Two samples out of the 8 positives from Caroux-Espinouse were submitted for sequence analysis. The two sequences were identical (351 bp; GenBank AN: OR421278) and showed 100% similarity with B. garinii isolated from Ixodes spp. ticks or mammal hosts in e.g., Europe, Russia or the USA (GenBank AN: e.g., CP028861.1, KY346892.1).

Detection of anaplasmataceae and species identification of Anaplasma

A 345 bp fragment of the 16s RNA gene of Anaplasmataceae species was detected in 36.4% (288/791) of the collected ticks (Table 2). Additional PCRs were performed on all these positive samples to deepen the identification of Anaplasmataceae. No ticks were positive for A. marginale.

Anaplasma phagocytophilum

Anaplasma phagocytophilum was detected in 4.3% (34/791) of all the tested ticks (11.8% [34/288] of ticks positive for Anaplasmataceae) (Table 2). It was detected only in I. ricinus ticks (7.6% [34/450]) and at all study sites where this tick species was collected (i.e., not in Cinto). More precisely, 11.6% (18/155) of I. ricinus in Bauges, 1.0% (2/202) in Bazès, and 15.1% (14/93) in Caroux-Espinouse were positive for A. phagocytophilum (Table 2).

We further analysed by sequencing 13 of the 48 Anaplasma phagocytophilum positive samples (n = 6, 5 and 2 from Caroux-Espinouse, Bauges and Bazès, respectively) and identified four different sequences of A. phagocytophilum (546 bp amplicon; 99.4–99.8% similarity between sequences). All the sequences from Bauges and Bazès and two from Caroux-Espinouse were identical (GenBank AN: OR426540) and had 100% similarity with several sequences isolated in domestic and wild ungulate species and in Ixodes sp. ticks in various countries (e.g., Germany, Norway, USA, Turkey, Russia; GenBank AN: e.g., KU705198.1, KP276588.1). The other three sequences were from Caroux-Espinouse (GenBank AN: OR426541–OR426543) and had 99.8–100% similarity with several sequences isolated from various species and countries (GenBank AN: e.g., KU705203.1, KU705198.1)

Anaplasma ovis

Tests were performed on 784/791 ticks as we did not have sufficient DNA for 7 ticks to test for A. ovis. Anaplasma ovis was detected in 0.8% (6/784) of all the tested ticks (2.1% [6/281] of the ticks positive for Anaplasmataceae) (Table 2). All the positive ticks were collected in Caroux-Espinouse, including 5 Rh. sanguineus s.l. (9.3%; 5/54) and 1 I. ricinus (1.1%; 1/88).

All positive samples were submitted for sequence analysis and 2 different sequences were identified (347 bp amplicon; 99.7% similarity between sequences; GenBank AN: OR501022; OR501023) that showed 99.7% and 100% similarity with several sequences of Anaplasma ovis (e.g., GenBank AN: LC553537.1, MT344082.1) isolated from goats, sheep, cattle, dromedaries and ticks from various countries (e.g., Malawi, Turkey, China, Portugal and Tunisia). Both sequences were detected in Rh. sanguineus s.l.

Detection of Rickettsia spp.

Of the 791 tested ticks, DNA of Rickettsia spp. was found in 13.65% (108/791) of collected ticks (Table 2). An additional PCR assay was conducted on all positive samples with a probe amplifying the ompA gene specific to SFG Rickettsia. SFG Rickettsia was detected in 2.2% (18/791) of all the tested ticks (16.6% [18/108] of Rickettsia spp.-positive ticks). More precisely, 5.5% (3/55) of Rh. sanguineus s.l. were positive for Rickettsia spp. in Caroux-Espinouse and 1.0% (2/206) of Rh. bursa in Cinto. Among all the tested I. ricinus, SFG Rickettsia was detected in 2.9% (13/450), with 6.5% (10/155) in Bauges, 1.0% (2/202) in Bazès and 1.1% (1/93) in Caroux-Espinouse (Table 2).

We sequenced 7 samples randomly selected among the samples positive for the gltA gene (i.e., Rickettsia spp.) but negative for SFG Rickettsia (n = 1, 2, 2 and 2 from Bauges, Bazès, Caroux-Espinouse and Cinto, respectively). One sequence of the gltA gene in I. ricinus from the Caroux-Espinouse (382 bp; GenBank AN: OR501024) showed 100% similarity with R. monacensis. The other sequences detected in I. ricinus from Caroux-Espinouse (n = 1), Bauges (n = 1) and Bazès (n = 2) were identical (382 bp; GenBank AN: OR501025) and had 100% similarity with R. helvetica. These species were previously isolated in European countries (GenBank AN: e.g., MH618388.1, MH589996.1 and MH618386.1, MN226407.1, respectively). The two sequences of the gltA gene in Rh. bursa from Cinto were identical (382 bp; GenBank AN: OR501026) and showed 100% similarity with R. hoogstraalii described in e.g., Turkey, Cyprus, Japan or South Africa (GenBank AN: e.g., MK929389.1, AB795196.1).

Twelve positive samples for SFG Rickettsia out of 18 (n = 4, 2, 4 and 2 from Bauges, Bazès, Caroux-Espinouse and Cinto, respectively) were further analysed by sequencing. The two positive Rh. bursa from Cinto were identical (530 bp; GenBank AN: OR501027) and showed 100% similarity with Candidatus Rickettsia barbariae in Rhipicephalus sp. ticks from various countries (e.g., Algeria, China, Turkey, Cyprus; GenBank AN: e.g., MK028340.1, MF002506.1). Three positive Rh. sanguineus s.l. from Caroux-Espinouse had identical sequence (533 bp; GenBank AN: OR501030) with 100% similarity to the ompA gene of Rickettsia massiliae in Rh. sanguineus s.l. ticks isolated in e.g., southern European countries (Italy, Spain, Portugal, Greece and France) and China (GenBank AN: e.g., MH532237.1, MF098409.1). Two different sequences were identified (530 bp; 99% similarity) in I. ricinus ticks with 99.0–100% similarity to the ompA gene of Rickettsia monacensis isolated in ticks collected in e.g., Turkey, Italy and Estonia (GenBank AN: e.g., MK211314.1, MG432690.1). The first sequence (GenBank AN: OR501028) was detected in all tested samples from Bauges (n = 4), one from Bazès, and one from Caroux-Espinouse, and the second sequence (GenBank AN: OR501029) was detected in the other sample from Bazès.

Co-infections

For Rickettsia, we present only the results of co-infections with positive results for ompA (SFG Rickettsia) and results positive for gltA but negative for ompA that were sequenced (Rickettsia spp.). Co-infections were found in 0.8% (6/791) of all the tested ticks, but only in I. ricinus (1.3%; 6/451) in Bauges (n = 2) and Caroux-Espinouse (n = 4). The co-infections in Caroux-Espinouse involved A. phagocytophilum with B. burgdorferi s.l. [sequencing result: B. garinii] (n = 1), SFG Rickettsia [R. monacensis] (n = 1) and Babesia venatorum (n = 1) and three pathogens in one tick (A. phagocytophilum × B. burgdorferi s.l. [not sequenced] × Rickettsia spp. [R. monacensis]). In Bauges, we detected 1 tick with A. phagocytophilum × Rickettsia spp. [R. helvetica] co-infection and 3 pathogens in one tick (A. phagocytophilum × B. burgdorferi s.l. [B. afzelii] × SFG Rickettsia [R. monacensis]). Due to the limited sequence availability for Rickettsia spp., positive results for this bacteria were not used to test for parasite associations as they may contain non-pathogenic strains. With the remaining data, the association screening approach did not reveal any specific parasite associations.

Discussion

Based on monitoring of wild ungulate populations inhabiting four mountainous regions of France, we described the diversity of tick species and tick-borne pathogens in these areas. We observed that wild living ungulates were parasitised by a range of five species of ticks: I. ricinus, Rh. bursa, Rh. sanguineus s.l., H. sulcata and H. punctata. Ixodes ricinus was the sole or predominant species in all areas, except in Corsica (Cinto) where it was not detected, and where Rh. bursa was dominant. This study also allowed the simultaneous detection and identification of various major tick-borne pathogens including Babesia/Theileria spp., B. burgdorferi s.l., Anaplasma phagocytophilum, A. ovis and SFG Rickettsia in collected ticks, with variation in prevalence among tick species and study areas. We also reported for the first time to our knowledge, the presence of Rickettsia hoogstraalii in Rh. bursa ticks in mainland France and Theileria ovis in Corsica. These results highlight the heterogeneous potential risks of pathogen transmission for animals and humans in these mountain areas.

Strong variations in tick species were observed among study sites, which can be explained by the differences in environmental conditions among sites, including e.g., climate and microclimate, habitats, and abundance and diversity of hosts species. The tick Ixodes ricinus was the most frequent species (52.8%; 1099/2081) and the only species detected in Bauges and Bazès, while additional and/or distinct species were detected in Caroux-Espinouse and Cinto. Ixodes ricinus is a widespread species in temperate countries and covers most of France, except for warm and dry areas (e.g., with a Mediterranean climate) and high elevations [81]. It is often the only species observed in mountainous areas due to its ability to develop under temperate climatic conditions and its requirements for habitats with high humidity, compared to the other species of ticks present in Europe. In accordance with these findings, we only detected I. ricinus in Bauges (French Alps) and Bazès (Pyrenees), where mountain climate occurs. This tick is also observed in Caroux-Espinouse in addition to other tick species. Ixodes ricinus is predominant in this area, but the presence of various climatic influences (Mediterranean, oceanic and mountain climate; Table 1) enables the development of more thermophilic tick species, such as Rh. sanguineus s.l. and H. punctata. In fact, Rh. sanguineus s.l. is present in the Mediterranean zone and the tropical and subtropical zones, but also in temperate regions. H. punctata inhabits pastures, forest margins, forest steppes, brush areas, limestone pastures, artificial conifer forests, oak forests with scarce undercover and, rarely, even evergreen oak forests [24]. In other studies in Sardinia (Italy), they were the most frequent tick species detected in mouflon [6, 7]. No I. ricinus were detected in Cinto, but only Rh. bursa and H. sulcata. This area has a strong Mediterranean influence, especially at low altitudes of the range used by mouflon (Table 1), explaining the absence of I. ricinus more adapted to a temperate climate, but which favours the development of more thermophilic tick species, such as Rh. bursa and H. sulcata. Rhipicephalus bursa is typically found in coastal and mountainous areas in the Mediterranean area and it prefers grassy slopes and low to medium altitude mountain slopes [86]. Haemaphysalis sulcata is widespread mostly in wormwood foothills, mountain steppe, dry steppe and semi-desert habitats [24]. They were also the most prevalent species collected on the same population of Corsican mouflon [32].

In addition to the local influence of climatic conditions specific to each study site, the season and date of capture of ungulates could influence the species and stage of ticks collected. In fact, the phenology of questing activity of ticks is highly seasonal and dependent of local meteorological conditions, and especially of the habitat and microclimate, such as temperature and humidity [94]. In our study, the ticks were mostly collected in spring–summer, but in winter only in Cinto. While winter is often considered as a season of no or low questing activity of ticks in northern countries and mountain areas, the winter weather conditions in Cinto seem to be favourable to the questing activity of some tick species, notably Rh. bursa. Better knowledge of the tick species and their phenology at the different study sites would require sampling of ticks in the environment and on different host species at different periods of the year.

We mostly collected adult ticks (76.7%, n = 1,597/2 081), and especially females (n = 1,125), but only 10 larvae (0.5 %). While differences in the relative proportion of the different stages of ticks can be partly explained by a lag in their phenology [94], the size of the ticks according to the stage, sex and engorgement level can create a sampling bias. In fact, even though we tried to randomly sample ticks on animals, larvae are tiny and therefore highly difficult to detect in the fur and to detach from animals, especially considering that ticks were collected from conscious wild individuals in a limited amount of time. The higher proportion of females compared to males can partly be explained by the biology of ticks. For instance, males Ixodes ricinus do not generally blood feed and part of the males we collected were copulating with a female. Regarding tick species, ticks were collected at the same period in the same population of mouflon in a previous study in Cinto [32]. In this latter study, other tick species not detected here (Rh. sanguineus s.l. and D. marginatus) were found, but in very low numbers (0.7% each). This can be explained by sampling bias, such as the number and the “random” selection of the ticks to collect.

During recent decades, changes in tick distribution and species composition have been observed in different parts of the world. For instance, I. ricinus has been expanding both northwards as well as in altitude in mountainous areas as a consequence of climate change, and host movements and abundance [24, 46, 61]. Based on GPS data from collared wild ungulates at each study sites (Table 1), we can see that they use a wide range of elevation (500–1,100 m). They can therefore transport ticks from low to higher elevation, where ticks can further develop if they meet favourable climatic conditions and hosts for their development. In addition, domestic ungulates are moved to the mountain pastures during summer months, especially in Bauges and Bazès. These animals can carry ticks and vector-borne diseases, and hence contaminate the environment of wild species. However, the relative contribution of wild and domestic species in the dynamic of ticks and vector-borne diseases in mountainous areas remains to be determined.

We detected and identified DNA of various major tick-borne pathogens in collected ticks, with variation in prevalence among tick species and study areas. While positive ticks could have been infected by the pathogen before feeding on the sampled wild ruminant (i.e., infected during a blood meal at a previous stage or trans-ovarial transmission), we cannot exclude a contamination from the blood of the wild ruminant because of an ongoing infection and ticks were attached on it and partially fed. In addition, when 2 ticks or more were collected and analysed from a same animal (number of ticks per animal analysed for pathogens: median = 2; 95%IQR [1; 10]), co-feeding transmission of pathogens among ticks is possible. Although most of the time the pathogen was not detected in all the ticks analysed from the same animal, which is not in favour of contamination of ticks from a contaminated wild ruminant, we should remain cautious regarding the observed prevalence of pathogens in feeding ticks, which can be overestimated (see also our comment on the detection of B. burgdorferi s.l. below).

We detected the pathogens Babesia venatorum and Theileria ovis with low prevalence (0.4% in I. ricinus and 2.4% in Rh. bursa, respectively; Table 2). Several species of wild ungulates are considered to be reservoirs for babesiosis [103]. Although wild ungulate positive antibodies or DNA from Babesia sp. were detected, reports of clinical cases of piroplasmosis in the wild are rare [40]. For instance, cases of fatal babesiosis were recorded in Alpine chamois infected with Babesia capreoli alone in Switzerland [39]. We previously detected Babesia venatorum in Bazès from Ixodes ricinus collected by dragging technique [1], as well as from six Pyrenean chamois in healthy conditions in 2008 [29]. This protozoan species was also detected in wild ungulate species in other countries (e.g., roe deer and mouflon in Germany [50]; Alpine chamois and ibex Capra ibex ibex in Switzerland [64]; Alpine chamois in Austria [93]) and has zoonotic potential (e.g., B. venatorum was first reported in two splenectomised patients from Italy and Austria) [37].

We detected Theileria ovis in Rh. bursa ticks collected from mouflon in Cinto. In a recent study in Corsica on ticks collected on domestic and wild hosts [33], this pathogen was not detected. This is, to our knowledge, the first identification of Theileria ovis in Corsica and in ticks collected on wildlife in France (DNA from T. ovis was detected in blood sample from a sheep in France [12]). This pathogen was reported in surrounding countries (e.g., Spain and Italy) [5, 27] and is known to cause a benign type of theileriosis in small ruminants. However, knowledge on the consequences of this parasite on wild ungulates is lacking.

The overall prevalence of B. burgdorferi s.l. in I. ricinus ticks in our study was variable among study sites (0–8.6%). These values are comparable with those observed in previous studies in France (e.g., 3.3% [36] and 8.4% [1]) and other European countries, such as southern Norway (0%) [53, 67], Northwest Italy (2.2%) [82], Slovakia (1.7%) [52], and the Netherlands (0.7%) [75]. Sequence analysis showed 100% similarity to two species of the B. burgdorferi s.l. complex: B. garinii and B. afzelii. Borrelia afzelii is less responsible for disseminated clinical manifestations in humans than B. burgdorferi s.s. [49]. Borrelia afzelii and B. miyamotoi were recently detected in I. ricinus ticks from cattle in Corsica [33], but we did not collect this tick species on captured mouflon in Cinto. In Bazès, we did not detect B. burgdorferi s.l. in ticks collected from Pyrenean chamois, while Akl et al. [1] reported a prevalence of 8.4% for B. burgdorferi s.l. in I. ricinus collected in the same area by dragging technique. The significant difference in infection prevalence between feeding and questing ticks can be explained by sampling bias, but it also supports the perception of the incompetency of ungulates as a reservoir for B. burgdorferi s.l. [75, 89]. Ticks infected by this pathogen appear to lose their infection when feeding on wild ungulates due to borreliacidal effects of host derived molecules [57, 68, 75]. Further investigations should be performed to confirm this hypothesis of a borreliacidal effect.

Most Anaplasma sp. infecting domestic ruminants have also been detected in wild ruminants [25, 97]. Among Anaplasma species, A. phagocytophilum is an intracellular bacterium infecting neutrophils that can cause Tick-Borne Fever in domestic animals and is also a zoonotic bacterium responsible for granulocytic anaplasmosis in humans [102]. It may cause anaemia due to erythrophagocytosis, mottled liver and enlarged spleen in humans and animals. Anaplasma phagocytophilum was detected only in I. ricinus ticks, the main vector for this pathogen, and in all our study areas where this tick was present (i.e., not in Cinto). Prevalence in I. ricinus ticks was heterogeneous among study sites (1.0–15.1%). Prevalence values are comparable to those from previous studies on ticks collected in France (10.7–22.4%) [13, 33–35], except for Bazès where the prevalence was relatively low (1.0%). We observed a similar prevalence in I. ricinus collected by dragging method in Bazès in a previous study (2.3% [16/696]) [1]. The presence of this bacterium in tissue or ticks collected from wild ungulates (mostly deer species but also some mountain ungulates such as mouflon, chamois and Alpine ibex) has been reported from several countries with highly variable prevalence values among species and sites [47, 54, 97].

We detected A. ovis in Rh. sanguineus s.l. and I. ricinus in Caroux-Espinouse. Anaplasma ovis can cause ovine anaplasmosis, a subclinical disease related to haemolytic anaemia in goats and sheep [100]. This bacterium was also reported in a young woman in Cyprus with thrombocytopaenia and hyperthermia [9]. It has been detected in wild ungulates such as red deer in Portugal [80] and mouflon in Cyprus [42]. Anaplasma ovis was previously detected at high prevalence in its main vector, Rh. bursa, and in blood collected from goat and sheep herds, in Corsica [4, 14]. However, we did not detect this pathogen in ticks sampled in Corsica, as in a recent study on ticks collected on both domestic and wild ungulates in Corsica [33].

We detected five rickettsial species (Rickettsia monacensis, R. helvetica, R. massiliae, R. hoogstraalii and Candidatus R. barbariae) in three tick species (I. ricinus, Rh. sanguineus s.l. and Rh. bursa). Rickettsia helvetica is a bacterium distributed across Europe and transmitted by the tick Ixodes ricinus [77, 96]. Few human infections have been reported in e.g., France, Switzerland and Italy, with mild and self-limiting illness [77]. We detected this Rickettsia only in I. ricinus collected in the Caroux-Espinouse, but it was previously isolated in Bazès [1] and in Corsica [33].

Rickettsia massiliae is an emerging pathogen causing spotted fever in humans [77]. It has been detected previously in different species of the genus Rhipicephalus and I. ricinus collected from domestic and wild animals in several European countries [77], including France (South-East of France and Corsica) [2, 11, 62]. We detected R. massiliae in Rh. sanguineus s.l. collected from mouflon in Caroux-Espinouse, but not in Rh. bursa from Cinto, Corsica.

We detected Candidatus Rickettsia barbariae only in Cinto in Rh. bursa. This pathogen was previously detected in Corsica in Rh. bursa and H. marginatum ticks collected from sheep and cattle [10]. It is generally associated with Rhipicephalus ticks (Rh. bursa, Rh. sanguineus s.l., Rh. turanicus, Rh. annulatus) and was also detected in H. marginatum ticks [8, 10, 79, 98]. The pathogenic potential in animals of Candidatus R. barbariae remains unknown.

Rickettsia monacensis has been detected in I. ricinus collected in numerous European countries [77] and in lizard tissue on Madeira Island, Portugal [95]. This SFG Rickettsiae was isolated in patients in Spain and Italy (Sardinia) and identified as a human pathogen [44, 60]. Rickettsia monacensis was reported for the first time in France in a study focusing on I. ricinus collected in Bazès [1]. Our results confirm the presence of this Rickettsia in Bazès, but also in I. ricinus from two other areas: Caroux-Espinouse and Bauges.

We also identified two DNA sequences of Rickettsia spp. isolated in one male and one female of Rh. bursa from Cinto, as Rickettsia hoogstraalii. After its first detection in Croatia in 2006 [20], this Rickettsia was detected in several countries across Europe (e.g., Italy, Spain, Cyprus, Greece, Romania, Georgia and Turkey) in H. sulcata, H. punctata, H. parva, D. marginatus, and Rh. rossicus [8, 19, 43, 66, 74, 78, 84, 98], and in other parts of the world (e.g., Iran, Zambia, Ethiopia, Namibia, USA and Japan), mostly in soft-bodied ticks [51, 55, 58, 76, 85, 88]. In addition, R. hoogstraalii has been detected in H. sulcata and H. punctata ticks collected from a mouflon in Sardinia, Italy, an island close to Corsica [7]. The pathogenicity of R. hoogstraalii in vertebrates is currently unknown.

Conclusion

Our results show that ticks collected on wild ungulates in mountainous areas of France carry several pathogens. Here we report for the first time the detection of Rickettsia hoogstraalii in Rh. bursa ticks in Corsica, France. The presence of major pathogens and the increased tick risk in mountainous areas associated with climate change highlight that tick-borne diseases in middle- to high elevation areas should not be neglected. More epidemiological data are required to better understand the epidemiology of tick-borne pathogens in these areas and their potential disease threats for both human and wild animal populations.

Acknowledgments

We are grateful for data collection efforts of all professionals from the Office Français de la Biodiversité, the Regional Natural Park of Corsica, as well as all the trainees and volunteers.

Conflict of interests

The authors declare that they have no competing interests.

Ethics approval

All captures, handling and sampling were conducted according to the appropriate national laws for animal welfare, following the ethical conditions detailed in the specific accreditations delivered by the Préfecture de Paris (prefectorial decree n°2009e014) in agreement with the French environmental code (Art. R421-15 to 421e31 and R422-92 to 422e94-1).

Supplementary materials

Supplementary File: Primers and PCR conditions used in the PCR assays conducted in this study with their respective references. Access here

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Cite this article as: Habib J, Zenner L, Garel M, Mercier A, Poirel M-T, Itty C, Appolinaire J, Amblard T, Benedetti P, Sanchis F, Benabed S, Abi Rizk G, Gibert P & Bourgoin G. 2024. Prevalence of tick-borne pathogens in ticks collected from the wild mountain ungulates mouflon and chamois in 4 regions of France. Parasite 31, 21.

All Tables

Table 1

Characteristics of the study areas.

In the text
Table 2

Results of PCR assays for the different tested pathogens (number of positive samples/number of tested samples, with the corresponding percentages in brackets), for each tick species from the 4 study areas in France.

In the text

All Figures

thumbnail Figure 1

Location of the 4 study areas in France (top left), and for each area, description of the species and number of ungulates on which ticks were collected (top right). For each tick species, description of the total number (middle) and the detailed (i.e., value for each stage; bottom) number of ticks collected and identified. *When collected females were mating with a male, both the female and male were counted and considered in the table.
In the text

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