V Cicculli

Capai
email: capai@univ-corse.fr

Fernández-Alvareza

Ea7310

Fernandez-Alvarez
email: angela.fdez.alvarez@gmail.com

Falchi A : Ea7310

Alessandra Falchi
email: falchi@univ-corse.fr

Molecular investigation of tick-borne pathogens in ixodid ticks infesting domestic animals (cattle and sheep) and small rodents (black rats) of Corsica, France

Keywords: Quantitative real-time polymerase chain reaction, qPCR, Crimean-Congo hemorrhagic fever virus, CCHFV Ticks, Animals, Anaplasmataceae, Rickettsia spp., Borrelia burgdorferi sl, Candidatus Ri. barbariae and Corsica Quantitative real-time polymerase chain reaction, qPCR, Crimean-Congo hemorrhagic fever virus, CCHFV

Although livestock farming (sheep, goats, pigs, and cattle) is an important economic activity in Corsica, a French Mediterranean island, knowledge about the tick fauna and microorganisms carried by them remains scarce. This study aimed to investigate the presence and perform molecular characterization of Anaplasmataceae, Rickettsia spp., and Borrelia burgdorferi sensu lato (sl) in tick species collected in Corsica. Ticks from cattle (Bos taurus), sheep (Ovis aries), and rodents (Rattus rattus) were collected from May to September 2016. DNA was purified from ticks, submitted to quantitative real-time polymerase chain reaction (qPCR) and sequenced for phylogenetic analysis. In total, 660 ticks were collected from 111 animals during the study. The most abundant collected tick species from cattle was Rhipicephalus bursa (n = 495; 84.5%), followed by Hyalomma marginatum (n = 91; 15.5%). Rhipicephalus bursa and Ixodes ricinus were the only tick species collected from sheep and rodents, respectively. Overall, Rickettsia was the most common pathogen group (n = 48; 24%) detected in ticks. Sequence analysis of partial gltA and ompA genes revealed the presence of Ri. aeschlimannii and Candidatus Ri. barbariae. Anaplasmataceae DNA was detected in eight (6%) of the 127 cattle pools and in one (2%) of the 61 R. bursa specimens collected from sheep. Sequence analysis of the rpoB gene revealed the presence of one Anaplasma species, A. marginale. Borrelia burgdorferi sl DNA was detected in one pool of H. marginatum collected from cattle and in two (15%) of the 13 I. ricinus pools collected from nine black rats. To our knowledge, this is the first report of the occurrence and molecular characterization of Candidatus Ri. barbariae, an emerging member of the Rickettsia group causing spotted fever, in Corsica. The detection of B. burgdorferi sl DNA, which was previously believed to be rare in Corsica, confirms the presence of this agent on the island.

Introduction

Ticks, ubiquitous ectoparasitic arthropods that belong to the subclass Acari, order Ixodida, are considered to be the most common vectors of disease-causing pathogens in animals (domestic and wild), and are the second most common vector of human pathogens worldwide after mosquitoes [START_REF] Colwell | Vector-borne parasitic zoonoses: emerging scenarios and new perspectives[END_REF][START_REF] Rizzoli | Ixodes ricinus and Its Transmitted Pathogens in Urban and Peri-Urban Areas in Europe: New Hazards and Relevance for Public Health[END_REF]. Arthropod-borne microorganisms, such as Toscana virus in dogs [START_REF] Dahmani | Seroprevalence of Toscana virus in dogs from Corsica, France[END_REF] and West Nile virus in domestic animals have been reported in Corsica [START_REF] Maquart | First Serological Evidence of West Nile Virus in Horses and Dogs from Corsica Island, France[END_REF]. Corsica, a French Mediterranean island, is characterized by a mild Mediterranean climate and a high variability of microclimates because of its specific geographical situation [START_REF] Grech-Angelini | Ticks (Acari: Ixodidae) infesting cattle and some other domestic and wild hosts on the French Mediterranean island of Corsica[END_REF]. Corsican livestock farming (sheep, goats, pigs, and cattle) is extensive, so important interactions between livestock, wildlife, and human populations could favor the circulation of tick-borne diseases. A systematic survey of tick fauna on Corsican livestock reported the dominance of the typical Mediterranean species Rhipicephalus bursa and Hyalomma marginatum [START_REF] Grech-Angelini | Ticks (Acari: Ixodidae) infesting cattle and some other domestic and wild hosts on the French Mediterranean island of Corsica[END_REF]. Hyalomma marginatum is one of the main vectors of the zoonotic Crimean-Congo hemorrhagic fever virus (CCHFV) in the Mediterranean Basin and the Afrotropical region [START_REF] Estrada-Pena | Unraveling the ecological complexities of tick-associated Crimean-Congo hemorrhagic fever virus transmission: a gap analysis for the western Palearctic[END_REF]. Rhipicephalus bursa is recognized as a primary vector of Babesia ovis [START_REF] Moltmann | Electron microscopic study on the development of Babesia ovis (Piroplasmia) in the salivary glands of the vector tick Rhipicephalus bursa[END_REF], but it transmits other pathogens such as Rickettsia spp. and Anaplasma spp. [START_REF] Dahmani | Molecular investigation and phylogeny of Anaplasmataceae species infecting domestic animals and ticks in Corsica, France[END_REF][START_REF] Raele | Coxiella-like endosymbiont associated to the "Anatolian brown tick" Rhipicephalus bursa in Southern Italy[END_REF]. An epidemiological survey on Anaplasmataceae species infecting domestic animals and ticks in Corsica described the presence of six species belonging to the genus Anaplasma from blood samples taken from ruminants and from ticks infecting cattle [START_REF] Dahmani | Molecular investigation and phylogeny of Anaplasmataceae species infecting domestic animals and ticks in Corsica, France[END_REF]. Ehrlichia canis was detected once in Corsica in a nonengorged R. bursa tick collected from a cow [START_REF] Dahmani | Molecular investigation and phylogeny of Anaplasmataceae species infecting domestic animals and ticks in Corsica, France[END_REF]. Anaplasma spp. and Ehrlichia spp. are transmitted by ticks, and both genera contain obligate intracellular Gram-negative bacterial parasites. In the vertebrate host, these bacteria infect hematopoietic cells. There are four pathogens Abbreviations: Quantitative real-time polymerase chain reaction, qPCR; Crimean-Congo hemorrhagic fever virus, CCHFV.

of ruminants in the genus Anaplasma: A. marginale, A. centrale, A. bovis, and A. ovis; in addition, there is A. phagocytophilum, which infects a variety of hosts, including humans and other animals, and A. platys, which infects dogs [START_REF] Ndip | Predominance of Ehrlichia chaffeensis in Rhipicephalus sanguineus ticks from kennel-confined dogs in Limbe, Cameroon[END_REF]. Rickettsia aeschlimannii was detected and isolated in H. marginatum in Corsica [START_REF] Matsumoto | Rickettsia aeschlimannii in Hyalomma ticks from Corsica[END_REF].

This study aimed to investigate the presence of Anaplasmataceae, Rickettsia spp. and B. burgdorferi sensu lato (sl) in tick species collected from cattle, sheep, and black rats in Corsica, and to characterize them using molecular approaches.

Materials and methods

Tick collection and morphological identification

Ticks from cattle (Bos taurus), sheep (Ovis aries), and rodents (Rattus rattus) were collected from May to September 2016 in northern Corsica, France (Figure 1). Ticks were collected from June to July 2016 in the Ponte-Leccia slaughterhouse in Haute Corse. During each visit, the whole skin of slaughtered animals was inspected, and ticks were collected manually. The national cattle identification system, which uses ear tags, allowed the origin of animals to be tracked and identified three farm owners. Cattle sampled in the Ponte-Leccia slaughterhouse come from Haute Corse municipalities. Ticks were also collected from sheep (n = 60) belonging to one farm in Corte on May 2016 and from rodents (n = 9) trapped on September 2016 in Lozari, Urbino, and Terrenzana (Figure 1). Ticks were identified at species level using a pictorial guide [START_REF] Estrada-Pena | Ticks of veterinary and medical importance: the Mediterranean basin. A guide of identification of species[END_REF].

Ixodes ticks were checked for being I. inopinatus [START_REF] Estrada-Pena | Description of all the stages of Ixodes inopinatus n. sp. (Acari: Ixodidae)[END_REF].

DNA extraction

Ticks were washed once in 70% ethanol for 5 min and then twice in distilled water for 5 min.

Those collected from sheep and black rats were analyzed individually, whereas those collected from cattle were analyzed after monospecific pools consisting of 2-6 ticks were collected from the same animal.

Individual ticks or pools of ticks were crushed using the TissueLyser II (Qiagen, Hilden, Germany) in phosphate-buffered saline at 5500 rpm for 20 s. DNA extraction was performed on a QIAcube HT (Qiagen) using a QIAamp cador Pathogen Mini kit according to the manufacturer's instructions. DNA was eluted in 150 µl of buffer and stored at -20 °C. For each PCR reaction, the template DNA had a final concentration <200 ng.

Molecular analysis of ticks and bacteria

Morphological identification of ticks was confirmed by polymerase chain reaction (PCR) amplification and sequencing mitochondrial 16S rDNA (Table 1) [START_REF] Black | Phylogeny of hard-and soft-tick taxa (Acari: Ixodida) based on mitochondrial 16S rDNA sequences[END_REF]. At least two specimens from each species were randomly selected and subjected to molecular identification. The PCR assays for Anaplasmataceae [START_REF] Dahmani | Molecular investigation and phylogeny of Anaplasmataceae species infecting domestic animals and ticks in Corsica, France[END_REF], Rickettsia spp. [START_REF] Labruna | Rickettsia species infecting Amblyomma cooperi ticks from an area in the state of Sao Paulo, Brazil, where Brazilian spotted fever is endemic[END_REF], B. burgdorferi sl [START_REF] Courtney | Multiplex real-time PCR for detection of Anaplasma phagocytophilum and Borrelia burgdorferi[END_REF] and B. miyamotoi [START_REF] Diaz | Development of a novel genusspecific real-time PCR assay for detection and differentiation of Bartonella species and genotypes[END_REF] were performed using the primers and probes listed in Table 1. Reactions were performed on a 96well Applied Biosystems™ QuantStudio™ 3 Real-Time PCR System using QuantiFast Pathogen + Internal Control Kits (Qiagen). Internal and negative controls were included in each run. Each run was repeated three times. Samples that were positive for Anaplasmataceae were tested by conventional PCR using Anaplasma genus-specific primers targeting the 525-bp fragment of the RNA polymerase subunit beta (rpoB) gene [START_REF] Dahmani | Molecular investigation and phylogeny of Anaplasmataceae species infecting domestic animals and ticks in Corsica, France[END_REF] Rickettsia spp. were analyzed using primers that amplified the 850-bp fragment of the gltA gene encoding citrate synthase (Table 1) [START_REF] Mediannikov | Acute tick-borne rickettsiosis caused by Rickettsia heilongjiangensis in Russian Far East[END_REF][START_REF] Roux | Citrate synthase gene comparison, a new tool for phylogenetic analysis, and its application for the rickettsiae[END_REF] and primers to amplify the 532-bp fragment of the 190-kDa outer membrane protein (ompA) gene [START_REF] Regnery | Genotypic identification of rickettsiae and estimation of intraspecies sequence divergence for portions of two rickettsial genes[END_REF]. The reactions were carried out using Applied Biosystems GeneAmp PCR System 9700 (Courtabouef, France). Negative and positive controls were included. The PCR products were visualized in 2% agarose gels in Tris-Acetate-EDTA (TAE Buffer) and were visualized under ultraviolet light after staining with ethidium bromide. A 100-bp DNA ladder was used as a standard marker.

The pathogens detected in pools were expressed as the percentage and minimum infection rate based on the assumption that each PCR-positive pool contained at least one positive tick [START_REF] Sosa-Gutierrez | Tick-borne rickettsial pathogens in questing ticks, removed from humans and animals in Mexico[END_REF]. Detection rate of DNA bacteria were compared by using Fisher exact test (p<0.05). The analysis was conducted using the R statistical platform (version 3.1.2) (R, 2015).

Sequencing and phylogenetic analysis

A selected number of positive samples for Rickettsia and Anaplasmataceae were purified and directly sequenced using an Applied Biosystems model 3730XL (Fisher Scientific S. A. S., Illkirch-Graffenstaden, France). Sequences of ticks, Anaplamataceae, or Rickettsia spp. were aligned using MEGA X [START_REF] Kumar | MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms[END_REF]. All sequences were assembled and compared with similar sequences retrieved from the GenBank nucleotide database using BLASTn [START_REF] Altschul | Gapped BLAST and PSI-BLAST: a new generation of protein database search programs[END_REF].

Phylogenetic analyses were inferred using the maximum-likelihood method implemented on Mega X.

Abbreviations: Quantitative real-time polymerase chain reaction, qPCR; Crimean-Congo hemorrhagic fever virus, CCHFV.

Results

Tick identification

In total, 660 ticks were collected during the study (Table 2). Forty-two cattle were inspected in the Ponte-Leccia slaughterhouse from June to July 2016 and 586 ticks were collected. Among the 42 infested cows, 57% were infested with less than 10 ticks, 28% with 10-30 ticks, and 15% with more of 30 ticks. Two tick species were identified morphologically on cattle (Table 2). The most abundant species was R. bursa (n = 495; 84.5%), followed by H. marginatum (n = 91; 15.5%).

Among the 586 ticks collected from cattle, 541 (94.1%) were adults (38.6% females and 55.5% males) and 34 (5.9%) were nymphs. Sex and life stage were not determined for 11 ticks. Sixty-one ticks were collected from 60 sheep in one farm on May 2016 (Corte, Haute-Corse) (Table 2). All these were R. bursa; 51 (83.6%) were adults (33% males and 51% females). Thirteen I. ricinus ticks (all adult females) were collected from nine rodents (black rats) captured in Lozari, Urbino, and Terrenzana (Haute-Corse) in September 2016. Among them, six were infested by one tick, two by two ticks, and one by three ticks. The sequences of mitochondrial 16S rDNA fragments of 16 ticks selected in this study after blast analysis were confirmed to be R. bursa (n = 7), H. marginatum (n = 7), and I. ricinus (n = 2) (GenBank accession numbers MH663984-90 for R. bursa, MH663977-83 for H. marginatum and MH663991-92 for I. ricinus). The seven 16S rDNA sequences of H marginatum had 98-100% identity with each other and 99-100% identity with H. marginatum from Sardinia (KT931964), Israel (KT391060), Turkey (KR870973), and Algeria (KP776645) (Figure 2).

The seven (MH663984-90) 16S rDNA sequences of R. bursa were identical to each other and showed 100% identity with R. bursa from the Iberian Peninsula (AJ002956), Italy (KJ814007-KJ814010), and Turkey (KR870983.1-KU664350.1) (Figure 2). The two (MH663991-92) 16S Abbreviations: Quantitative real-time polymerase chain reaction, qPCR; Crimean-Congo hemorrhagic fever virus, CCHFV.

rDNA sequences of I. ricinus were identical to each other and showed 100% identity with I. ricinus from Germany (JF928526), Italy (KF197116), Sweden (KX384811), Belgium (KJ414457), and Turkey (KR870982). They had 98% identity with 16S rDNA sequences from Morocco (GU074602) and France (GU074612) (Figure 2). Seventy-four individual ticks (61 collected from sheep and 13 from rodents) and 127 pools prepared with cattle ticks (100 pools of R. bursa and 27 pools of H. marginatum in cattle) were analyzed (Tables 2 and3).

Detection of Rickettsia spp.

In ticks collected from cattle, Rickettsia spp. DNA was detected in 37 pools (29%), with the highest detection rate in H. marginatum (59%) compared with R. bursa (21%) (p = 0.00; Table 3).

Similar Rickettsia spp. detection rates were observed in R. bursa collected from cattle and sheep (21% and 18%, respectively; p = 0.68). The 13 I. ricinus specimens collected from rodents were all negative for Rickettsia spp. Nine pools of ticks (three R. bursa and six H. marginatum) and seven individual ticks (R. bursa) collected from cattle and sheep, respectively, were screened for the presence of the gltA gene (GenBank accession numbers MH675633-MH675648). Sequence analysis of the gltA sequences revealed the presence of two Rickettsia species: Ri. aeschlimannii and Candidatus Ri. barbariae. The seven gltA sequences derived from five pools of H. marginatum and two pools of R. bursa collected from cattle were 100% identical to each other and clustered together with Ri. aeschlimannii sequences (KU961540, DQ235776, and HM50282) (Figure 3).

Nine gltA sequences derived from eight R. bursa specimens collected from sheep and one pool of H. marginatum ticks collected from cattle showed 99-100% identity with each other and 99% identity with Candidatus Ri. barbariae collected in Sardinia (EU272185) (Figure 3). The analyses of the ompA gene (GenBank accession numbers MH797764-MH79777) confirmed these results (99% Abbreviations: Quantitative real-time polymerase chain reaction, qPCR; Crimean-Congo hemorrhagic fever virus, CCHFV.

of identity with Candidatus Ri. barbariae with KY233248-49 from Lebanon).

Detection of Anaplasmataceae

Anaplasmataceae DNA was detected in eight (6%) of the 127 tick cattle pools. Among them, DNA was detected in five (18%) of the 27 H. marginatum pools and in three (3%) of the 100 R. bursa pools (Table 3). Anaplamataceae DNA was detected in one (2%) of the 61 R. bursa ticks collected from sheep and analyzed individually. Anaplamataceae DNA was not detected in I.

ricinus. Sequence analysis of the rpoB gene sequencing of three pools (one R. bursa and two H. marginatum) revealed the presence of one Anaplasma species, A. marginale (GenBank accession numbers MH651041-MH651043). The three sequences of A. marginale had 99-100% identity with each other and showed 99-100% identity with an A. marginale strain reported in Corsica (KY498343-KY498345) (Figure 4).

Detection of B. burgdorferi (sl)

Borrelia burgdorferi sl DNA was detected in two (15%) out of the 13 I. ricinus ticks collected from nine rodents and in H. marginatum one (4%) cattle pool. B. miyamotoi DNA was not detected in any of the 13 I. ricinus analyzed.

Discussion

Here we report the detection rate of Rickettsia spp., Anaplasmataceae, and B. burgdorferi sl in ticks collected from cattle, sheep, and black rats in Corsica, France. Although R. bursa was the most-represented tick species, H. marginatum ticks showed the highest detection rate for Rickettsia from sheep and cattle examined. To our knowledge, we report here for the first time the detection of Candidatus Ri. barbariae and B. burgdorferi sl DNA in ticks collected from ruminants and black rats in Corsica, respectively.

Rhipicephalus bursa was the most abundant tick species collected from cattle (>80%) and the only tick species collected from small ruminants. This was in line with previous findings from Corsica [START_REF] Dahmani | Molecular investigation and phylogeny of Anaplasmataceae species infecting domestic animals and ticks in Corsica, France[END_REF][START_REF] Grech-Angelini | Ticks (Acari: Ixodidae) infesting cattle and some other domestic and wild hosts on the French Mediterranean island of Corsica[END_REF]. In the Mediterranean basin, this species is considered as the major ectoparasite of sheep [START_REF] Yeruham | The life cycle of Rhipicephalus bursa Canestrini and Fanzago, 1877 (Acarina: ixodidae) under laboratory conditions[END_REF]. The majority of R. bursa specimens collected in this study were adults, as they were collected during the summer period [START_REF] Ferrolho | Detection and phylogenetic characterization of Theileria spp. and Anaplasma marginale in Rhipicephalus bursa in Portugal[END_REF]. Majority of R. bursa ticks analyzed were positive for Rickettsia spp., with a similar detection rate between cattle and sheep. Sequence analysis revealed the detection of Ri. aeschlimannii and Candidatus Ri. barbariae in R. bursa. Rickettsia spp., such as Ri. aeschlimannii and Ri. massiliae, had previously been detected in R. bursa in several Mediterranean countries in a wide range of animal hosts [START_REF] Parola | Update on tick-borne rickettsioses around the world: a geographic approach[END_REF]; however, to the best of our knowledge, this is the first detection report of Candidatus Ri. barbariae in R. bursa ticks collected from domestic animals in Corsica. These species were described previously in several tick species in Portugal (R. bursa), Cyprus (R. turanicus), Sardinia (R. turanicus), and China in the flea (Vermipsylla alakurt) [START_REF] Zhao | First detection of Candidatus Rickettsia barbariae in the flea Vermipsylla alakurt from north-western China[END_REF]. Here, 3% of R. bursa tick cattle pools were positive for Anaplasmataceae DNA. We found a detection rate of Anaplamataceae DNA of 2% among the 61 R. bursa collected from sheep, which is similar to the detection rate reported in other Mediterranean regions [START_REF] Satta | Pathogens and symbionts in ticks: a survey on tick species distribution and presence of tick-transmitted micro-organisms in Sardinia, Italy[END_REF][START_REF] Torina | Prevalence of tickborne pathogens in ticks in Sicily[END_REF]. We also detected the DNA of A. marginale. This is consistent with a previous study reporting that the DNA of A. marginale was detected in two engorged female ticks removed from cattle (2%) and from all (12/12) cattle blood samples in Corsica [START_REF] Dahmani | Molecular investigation and phylogeny of Anaplasmataceae species infecting domestic animals and ticks in Corsica, France[END_REF].

Hyalomma marginatum was the second most abundant tick species collected from cattle (15.5%), as described previously [START_REF] Grech-Angelini | Ticks (Acari: Ixodidae) infesting cattle and some other domestic and wild hosts on the French Mediterranean island of Corsica[END_REF]. Hyalomma marginatum, which is Abbreviations: Quantitative real-time polymerase chain reaction, qPCR; Crimean-Congo hemorrhagic fever virus, CCHFV.

the vector of CCHFV, is present in southeastern continental France [START_REF] Vial | Strong evidence for the presence of the tick Hyalomma marginatum Koch, 1844 in southern continental France[END_REF], but also in Mediterranean islands such as Sicily (Italy) and Minorca (Spain) [START_REF] Castella | A survey of ticks (Acari: Ixodidae) on dairy cattle on the island of Menorca in Spain[END_REF]. More than 50% of H. marginatum pools were positive for DNA of Ri. aeschlimannii and Candidatus Ri. barbariae. Such a high infection rate is in agreement with Ri. aeschlimannii infection rate of Hyalomma (>70%) previously reported in Corsica [START_REF] Matsumoto | Rickettsia aeschlimannii in Hyalomma ticks from Corsica[END_REF] and Croatia (64%) [START_REF] Punda-Polic | Detection and identification of spotted fever group rickettsiae in ticks collected in southern Croatia[END_REF]. Lower rates of Rickettsia spp. were observed in Sicily (4%) [START_REF] Torina | Prevalence of tickborne pathogens in ticks in Sicily[END_REF] and Turkey (6.5%) [START_REF] Keskin | Molecular detection of spotted fever group rickettsiae in ticks removed from humans in Turkey[END_REF]. These differences in results could be related to differences in environmental factors, animal species, the numbers of ticks collected and the analysis methods. In our study, the detection of Candidatus Ri. barbariae in a pool of H. marginatum, not a recognized vector for this Rickettsia species [START_REF] Parola | Update on tick-borne rickettsioses around the world: a geographic approach[END_REF], could also be explained by the presence of this bacterium in the blood meal of the ticks.

Borrelia burgdorferi sl is the causative agent of Lyme borreliosis, which is the most prevalent tick-borne disease in Europe and North America [START_REF] Raileanu | Borrelia Diversity and Co-infection with Other Tick Borne Pathogens in Ticks[END_REF]. The French Sentinelles General Practitioner Network observed that the incidence of Lyme borreliosis is much lower in Corsica (incidence rate reported, 20-50 cases per 100,000 inhabitants) compared with endemic regions of France (>150 per 100,000 inhabitants) [START_REF] Vandenesch | Incidence and hospitalisation rates of Lyme borreliosis, France, 2004 to 2012[END_REF]. Here we report for the first time the detection of B. burgdorferi sl DNA in two I. ricinus ticks collected from rodents (15%). The detection rate of B. burgdorferi sl reported here was similar to that reported for I. ricinus collected from rodents in Italy (14.7%) [START_REF] Pascucci | Detection of Lyme Disease and Q Fever Agents in Wild Rodents in Central Italy[END_REF] and was in the range of previous studies from France, Ireland, and Austria, which found a detection rate of B. burgdorferi sl in small mammal species ranging from 2.3% to 24% [START_REF] Gray | Borrelia burgdorferi sensu lato in Ixodes ricinus ticks and rodents in a recreational park in south-western Ireland[END_REF][START_REF] Khanakah | Seasonal variations in detecting Borrelia burgdorferi sensu lato in rodents from north eastern Austria[END_REF][START_REF] Marsot | Introduced Siberian chipmunks (Tamias sibiricus barberi) harbor more-diverse Borrelia burgdorferi sensu lato genospecies than native bank voles (Myodes glareolus)[END_REF]. Although cattle are not a reservoir host, we detected B. burgdorferi sl DNA in one H. marginatum cattle pool. This detection could be explained by the presence of reservoir hosts of B. burgdorferi in the same location [START_REF] Gern | Life cycle of Borrelia burgdorferi sensu lato and transmission to humans[END_REF].

Abbreviations: Quantitative real-time polymerase chain reaction, qPCR; Crimean-Congo hemorrhagic fever virus, CCHFV.

The limitations of our study were that first, the ticks were collected mostly in a municipal slaughterhouse in northern Corsica. We have no data about the southern part of the island. Second, ticks were analyzed mostly in pooled samples and not individually. The screening of pooled samples is complicated because it is impossible to determine whether a positive result is caused by one or more infected ticks. Third, the ticks investigated here were removed from hosts. The presence of bacterial DNA in an engorged tick could be caused by its presence in the blood meal.

These results contribute to the knowledge of tick-borne disease in Corsica and provide a useful contribution to understanding the epidemiology. Further research should be carried out to investigate the eco-epidemiological cycle of the pathogenic agents detected here.

Declarations

Abbreviations: Quantitative real-time polymerase chain reaction, qPCR; Crimean-Congo hemorrhagic fever virus, CCHFV.

Abbreviations:

  Abbreviations:Quantitative real-time polymerase chain reaction, qPCR; Crimean-Congo hemorrhagic fever virus, CCHFV.

  Abbreviations:Quantitative real-time polymerase chain reaction, qPCR; Crimean-Congo hemorrhagic fever virus, CCHFV.

Figure 2 .

 2 Figure 2. Phylogenetic tree showing the position of Rhipicephalus bursa (green circles), Hyalomma marginatum (red circles) and Ixodes ricinus (blue squares) compared to other tick species based on 16S rDNA sequences. The evolutionary history was inferred by using the Maximum Likelihood method. The analysis involved 50 nucleotide sequences. There were a total of 291 positions in the final dataset.

Figure 3 .

 3 Figure 3. Phylogenetic analysis of Rickettsia spp. identified in Corsica. The evolutionary history was inferred by using the Maximum Likelihood method based on Rickettsia gltA sequences. The analysis involved 30 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 331 positions in the final dataset.

Figure 4 .

 4 Figure 4. Phylogenetic analysis of Anaplasmatacea identified in Corsica. The evolutionary history was inferred by using the Maximum Likelihood method based on Anaplasmatacea rpob gene sequences. The analysis involved 30 nucleotide sequences. There were a total of 331 positions in the final dataset.

  (Table1). Positive samples for

Abbreviations:

Quantitative real-time polymerase chain reaction, qPCR; Crimean-Congo hemorrhagic fever virus, CCHFV.

Table 2 .

 2 Total tick species collected from hosts sampled. Infection rate of bacteria in ticks collected from cattle.

	Host (n) Cattle (n = 42) Sheep (n = 60) Rodents (n = 9) Total (n = 111) *Sex and life were not determined for 11 ticks Tick species R. bursa H. marginatum Total* R. bursa I. ricinus Total tick species (n) Number of pools H. marginatum Number of pools R. bursa Tableau 3. Number of ticks per pool with n ticks with n ticks	n ticks (%) 495 (84.5) 91 (15.5) 586 61 (100) 13 (100) 660 Positive Rickettsia spp. n (%)	Male n (%) 251 (51.1) 68 (81.0) 319 (55.5) 20 (33.0) 0 (0) 339 (52.2)	Female n (%) 207 (42.2) 15 (18.0) 222 (38.6) 31 (51.0) 13 (100) 266 (41.0) Anaplasmataceae n (%) Positive	Nymph n (%) 33 (6.7) 1 (1.0) 34 (5.9) 10 (16.0) 0 (0) 34 (6.8)	Positive Borrelia burgdorferi (sl) n (%)

Abbreviations:Quantitative real-time polymerase chain reaction, qPCR; Crimean-Congo hemorrhagic fever virus, CCHFV.

Acknowledgements

We are grateful to the staff of the slaughterhouse of Ponte-Leccia for their help in collecting ticks.

Funding

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Abbreviations: Quantitative real-time polymerase chain reaction, qPCR; Crimean-Congo hemorrhagic fever virus, CCHFV.

Ethics approval and consent to participate

The inspected cattle were slaughtered for human consumption. Living sheep were examined with the assistance of their owner. 25) 0 (0) 2 ( 22) 1( 12) 0 (0) 1 ( 11) 0(0) 0 (0) (0) 2 5 7 4 (80) 1 ( 14) 5 ( 42) 0 (0) 0 (0) 0 (0) 1 ( 20) 0 (0) (8) 3 2 9 1 (50) 1 ( 11) 2 ( 18) 2 (100) 0 (0) 2 ( 18) 0 (0) 0 (0) (0) 4 3 15 2 (66) 3 ( 20) 5( 28) 0 (0) 1 ( 33) 1 ( 5) 0 (0) 0 (0) (0) 5 3 12 2 (66) 2 ( 17) 4 ( 27) 1 ( 33) 0 (0) 1 ( 7) 0 (0) 0 (0) (0) 6 6 16 5 (83) 14 ( 87) 19 ( 86) 1 ( 17) 2 ( 12) 3 ( 14) 0 (0) 0 (0) (0)

Total pools 27 100 16 ( 59) 21 ( 21) 37 ( 29) 5 ( 18) 3 (3) 8 ( 6) 1 ( 4) 0 (0) (1)