Ecological preferences of exophilic and endophilic ticks (Acari: Ixodidae) parasitizing wild carnivores in the Iberian Peninsula

Veterinary Parasitology 184 (2012) 248–257

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Ecological preferences of exophilic and endophilic ticks (Acari: Ixodidae) parasitizing wild carnivores in the Iberian Peninsula Raquel Sobrino a , Javier Millán b , Álvaro Oleaga c , Christian Gortázar c , José de la Fuente c,d , Francisco Ruiz-Fons c,∗ a Università degli Studio di Torino, Facoltà di Medicina Veterinária, Dipartimento di Produzioni Animali, Epidemiologia ed Ecologia, Vía L. Da Vinci, 44, Grugliasco, Torino, Italy b Servei d‘Ecopatologia de Fauna Salvatge (SEFaS) (Wildlife Diseases Research Group), Departament de Medicina i Cirurgia Animals, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain c Instituto de Investigación en Recursos Cinegéticos IREC (CSIC-UCLM-JCCM), Animal Health Dept., Ronda de Toledo s/n, 13005 Ciudad Real, Spain d Dept. of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078, USA

a r t i c l e

i n f o

Article history: Received 30 June 2011 Received in revised form 26 August 2011 Accepted 5 September 2011 Keywords: Behaviour Climate Ecology Endophilic Exophilic Mediterranean Tick

a b s t r a c t Ticks parasitizing wild carnivores and the tick-borne pathogens (TBPs) that they transmit may affect domestic carnivores and humans. Thus, investigating the role of wild carnivores as tick hosts is of relevance for understanding the life cycle of ticks in natural foci and the epidemiology of TBPs shared with domestic animals and humans. Therefore, the main objective of this study was to determine the ixodid tick fauna of wild carnivores in Peninsular Spain and the environmental factors driving the risk of wild carnivores to be parasitized by ixodid ticks. We hypothesized that the adaptation of tick species to differing climatic conditions may be reflected in a similar parasitization risk of wild carnivores by ticks between bioclimatic regions in our study area. To test this, we surveyed ixodid ticks in wild carnivores in oceanic, continental-Mediterranean, and thermo-Mediterranean bioclimatic regions of Peninsular Spain. We analyzed the influence of environmental factors on the risk of wild carnivores to be parasitized by ticks by performing logistic regression models. Models were separately performed for exophilic and endophilic ticks under the expected differing influence of environmental conditions on their life cycle. We found differences in the composition of the tick community parasitizing wild carnivores from different bioclimatic regions. Modelling results partially confirmed our null hypothesis because bioclimatic region was not a relevant factor influencing the risk of wild carnivores to be parasitized by exophilic ticks. Bioclimatic region was however a factor driving the risk of wild carnivores to be parasitized by endophilic ticks. Spanish wild carnivores are hosts to a relevant number of tick species, some of them being potential vectors of pathogens causing serious animal and human diseases. Information provided herein can be of help to understand tick ecology in Spanish wildlife, the epidemiology of tick-borne diseases, and to prevent the risks of TBPs for wildlife, domestic animals, and humans. © 2011 Elsevier B.V. All rights reserved.

1. Introduction

∗ Corresponding author. Tel.: +34 926295450; fax: +34 926295451. E-mail addresses: [email protected], [email protected] (F. Ruiz-Fons). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.09.003

Ticks are obligate blood-sucking arthropods that parasitize vertebrates worldwide (Sonenshine et al., 2002). As a consequence of their blood-feeding lifestyle, ticks are able to get infected, replicate, and maintain active

R. Sobrino et al. / Veterinary Parasitology 184 (2012) 248–257

pathogens that can be later transmitted to susceptible vertebrate hosts. Ticks are vectors of a wide range of pathogens such as viruses, bacteria, and protozoa that cause diseases to humans and animals (Sonenshine et al., 2002; Jongejan and Uilenberg, 2004). Tick-borne pathogens (TBPs) life cycle is highly dependent on tick ecology which is one of the most important factors driving the epidemiology of tick-borne diseases (Randolph, 2009). Most of the emergent tick-borne diseases in the world emerge from the wild reservoir (Daszak et al., 2001). Thus, preventing emergence of tick-borne diseases includes determining the role of wildlife as tick hosts and their role in the life cycle of TBPs. Some tick species may be generalists and may feed on different vertebrate species depending on their availability and abundance (Wilson et al., 1984) whereas other species may be more specific and use a narrow host range (Sonenshine et al., 2002). Many ticks parasitize domestic animals but few ticks feed exclusively on them and most tick species may also parasitize wild animals. Wild and domestic cycles are often complementary. Immature tick stages that parasitize wild and peridomestic animals can feed later as adults on domestic animals. Thus, investigating the role of wildlife species as tick hosts is of great relevance for understanding the life cycle of ticks and the epidemiology of tick-borne diseases shared with domestic animals and humans (Ruiz-Fons et al., 2006; Ruiz-Fons and Gilbert, 2010). Among the wide range of wildlife hosts of ticks, wild carnivores are important hosts to endophilic ixodid ticks because they spend many daily hours in burrows, nests or caves where ticks remain active. Wild carnivores are also hosts for some instars of exophilic tick species such as larvae and nymphs of Ixodes ricinus (Santos-Silva et al., 2011). I. ricinus is the main European vector of Borrelia burgdorferi sensu lato – the causal agent of Lyme disease – for which wild carnivores are good sentinels (Sobrino and Gortázar, 2008). Another important group of pathogens transmitted by ticks hosted by carnivores is the order Rickettsiales. In Spain, Rickettsia monacensis, Rickettsia helvetica, and Rickettsia massiliae have been recently detected in wild carnivores (Márquez and Millán, 2009), all of them with demonstrated zoonotic character (Parola et al., 2005). Anaplasma phagocytophilum and Ehrlichia spp. have also been detected by molecular and serological techniques in European wild carnivores (Petrovec et al., 2002; Ryser-Degiorgis et al., 2005; Millán et al., 2009). Nonetheless, the role of wild carnivores in the epidemiology of Rickettsia spp., A. phagocytophilum, and other TBPs such as Ehrlichia canis and A. platys is poorly understood. Across their evolutionary history, ticks have diversified from their ancestors allowing them to colonize very different biotopes, adapting their life cycle to the environment (Black and Kondratieff, 2004). Ticks have developed two different feeding strategies, an endophilic strategy in which ticks adopt a nidicolous lifestyle remaining in burrows, nests, hollows or craks near resting or breeding places of hosts, and an exophilic strategy in which ticks must search for their hosts. Ticks in the family Argasidae follow an endophilic strategy whereas ticks

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in the family Ixodidae – those of interest in this work – may follow both strategies. Many ixodid ticks follow only exophilic or endophilic strategies across their complete life-cycle, but some exophilic species may follow a pseudo-endophilic strategy because particular instars of the species remain on the host for more than one developmental stage (Klompen, 2005). As an example, some species in the genera Hyalomma, Rhipicephalus, and Dermacentor quest as larvae and adults in the vegetation looking for hosts while nymphs remain in the host (two-host cycle). As a consequence, endophilic and exophilic ticks differ in their exposure to environmental conditions due to their differing host-seeking behaviour. While endophilic ticks remain better protected from environmental changes and hosts are easily accessible, exophilic ticks are highly exposed to environmental conditions and the accessibility to the host is also dependent on host ecology and population dynamics (Wilson et al., 1990; Ruiz-Fons and Gilbert, 2010). Additionally, the ecology of certain tick species is influenced by climatic and meteorological conditions (Sonenshine, 1993) and by host population and host individual factors such as host abundance, host community composition, sex, age, and physical condition as well (LoGiudice et al., 2008; Alzaga et al., 2009; Vor et al., 2010). Most wild carnivores are widely distributed in Spain (Palomo et al., 2007) and they occupy from natural to humanized biotopes (Palomo and Gisbert, 2002). Wild and domestic carnivores may be parasitized by the same tick species and wild species such as the red fox (Vulpes vulpes) are peridomestic animals (Palomo and Gisbert, 2002) increasing the risk of indirect contacts with domestic animals through ticks. This wildlife–domestic connection may have consequences for human health as many pathogens shared by foxes and dogs may be transmitted to humans (Márquez and Millán, 2009). Information on the ticks parasitizing wild carnivores in Spain is scarce and most of the Spanish mainland has not been surveyed before, especially most of the Atlantic regions and continental inner areas of mainland Spain. Wild carnivores in Spain have been found to host different endophilic species such as Ixodes hexagonus, Ixodes canisuga, Ixodes trianguliceps, Ixodes ventalloi, Riphicephalus sanguineus, Riphicephalus pusillus, Haemaphysalis hispanica and exophilic ticks such as I. ricinus, Haemaphysalis punctata, Haemaphysalis concinna, Riphicephalus turanicus, Hyalomma spp., and adult Dermacentor reticulatus (Encinas Grandes, 1986; Domínguez, 2004; MartínezCarrasco et al., 2007; Millán et al., 2007; Gerrikagoitia, 2010). The important gap in the knowledge about ticks parasitizing wild carnivores in some areas of their distribution in Spain led us to this study. We hypothesize that in spite of big climatic variations across different bioclimatic areas in mainland Spain, the risk of animals to be parasitized by ticks would remain constant due to adaptations of the different tick species present in these areas to the predominant environmental conditions. This study adds information on the distribution, host range, and life-cycle of ixodid ticks in the Iberian Peninsula.

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Fig. 1. Map of Peninsular Spain showing the bioclimatic areas where the wild carnivore survey for ticks was carried out (A: Atlantic bioclimatic area; C: continental-Mediterranean bioclimatic area, and T: thermoMediterranean bioclimatic area).

2. Material and methods 2.1. Sampling wild carnivores and ticks Two hundred and four wild carnivores were surveyed for ticks from 2003 to 2010 in different geographic areas in mainland Spain (Fig. 1). Our survey approach was based on the collection of ticks on experimentally live-trapped wild carnivores and the collection of wild carnivore carcasses, both animals shot (red fox (V. vulpes) by hunters and Iberian wolf (Canis lupus signatus) by gamekeepers) or found dead. A total of 88 red foxes, 14 Iberian wolves, 15 stone martens (Martes foina), eight pine martens (Martes martes), 17 common genets (Genetta genetta), 26 Eurasian badgers (Meles meles), 23 Egyptian mongooses (Herpestes ichneumon), seven polecats (Mustela putorius), one stoat (Mustela erminea), one weasel (Mustela nivalis) and four wildcats (Felis silvestris) were surveyed for ticks. Live animals were anesthetized before inspection according to the protocol reported elsewhere (Millán et al., 2007). Live carnivores were visually inspected for tick detection and ticks were collected in tubes containing 70% ethanol. Shot carnivores were preserved in sealed plastic bags and transported to the laboratory. Sealed plastic bags impeded ticks detaching from the carcass to escape. Animals found dead were also transported to the laboratory in sealed plastic bags. Dead animals and container plastic bags were thoroughly inspected in the laboratory for ticks. Sampled animals were sexed and age was estimated by dentition and size (Saénz de Buruaga et al., 1991). Date of collection was recorded from several animals although it was not available for most of them (64.7%). Every tick collected from dead animals was stored at −20 ◦ C until identification (Gil Collado et al., 1979; Manilla, 1998; ˜ et al., 2004b). Some immature ticks could not Estrada-Pena be identified to species level due to the damage suffered during collection. Description of ticks collected in wild carnivores from southern Spain has been formerly reported

(Millán et al., 2007) and these data were herein used only to test the influence of environmental factors on tick prevalence. Each tick was classified according to its behavioural pattern in two classes: exophilic and endophilic. As different stages of the same tick species may show variations in their feeding strategy, the classification of ticks as exophilic or endophilic was done at the developmental stage level. By doing this, those stages of exophilic ticks that remained on the host after feeding and molting were considered as endophilic. We classified as exophilic every stage in the species I. ricinus and adult Hyalomma marginatum, Riphicephalus bursa, R. turanicus, and D. reticulatus. Every stage of I. ricinus is known to quest in the vegetation (e.g., RuizFons and Gilbert, 2010) and adult Hyalomma marginatum and R. turanicus are usually found parasitizing ungulates on which no immature stages are found (Pegram et al., 1987; Ruiz-Fons et al., 2006). Adult D. reticulatus and R. bursa ticks are frequently collected from the vegetation (Bullová et al., 2009; the authors, unpublished). Every stage in the species R. sanguineus, R. pusillus, I. hexagonus, I. canisuga, and I. ventalloi were considered as endophilic as they are not commonly found questing in the vegetation in our study areas (unpublished results). R. sanguineus can be occasionally collected outside houses, cracks or crevices (Barandika et al., 2006; Ruiz-Fons et al., 2006) but it is considered a true endophilic species (Danta-Torres, 2010). In contrast, R. turanicus frequently parasitizes ungulates ˜ et al., 2004a), which suggests an endophilic (Estrada-Pena behaviour.

2.2. Study areas Due to the opportunistic nature of the data collected due to difficulties in sampling wild carnivores in such a wide land surface (Sobrino and Gortázar, 2008), sampling sites were grouped into three main biogeographic areas (Fig. 1) according to gross climatic differences: (i) an Atlantic area in the northern third of mainland Spain; (ii) a continental-Mediterranean area in inland Spain; and (iii) a thermo-Mediterranean area in the south-west coast of Spain. The Atlantic area is dominated by an oceanic climate with year-round persistent rains (above 1000 mm), mild winters and slightly warm summers. The continental area is under the Mediterranean influence but characterized by extreme weather conditions with cold winters and very hot summers. Rains range 300–800 mm annually and are under a Mediterranean influence and thus highly seasonal – concentrated mainly in autumn and spring. The thermo-Mediterranean area is characterized by mild winters and hot summers with also a Mediterranean influence on rains (ranging 700–1000 mm in a year). Red foxes, genets, polecats, and European badgers were surveyed in the three bioclimatic areas. Iberian wolves, pine martens, the stoat, and the weasel were collected only in Atlantic areas. Finally, stone martens and wild cats were collected only from the Atlantic and continentalMediterranean regions meanwhile Egyptian mongooses were only surveyed in the continental-Mediterranean and thermo-Mediterranean regions.

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2.3. Statistical analyses We wanted to test the hypothesis that the risk of wild carnivores to be parasitized by ticks would remain constant across bioclimatic area in spite of the differences in the composition of the community of ticks in these areas. For this purpose, every sampled animal was categorized according to the presence/absence (0 and 1, respectively) of exophilic and endophilic ticks. Using sampled wild carnivores in which every data of interest in the statistical approach was collected (n = 138), we performed logistic binomial regression (binomial distribution and logistic link function) considering the presence/absence of ticks as the response variable and sampling area (categorical, three classes; Fig. 1), season (categorical, four classes) and the interactions between taxonomic family and area and taxonomic family and season as environmental predictor variables. Sampling area was considered as a proxy of the predominant macroclimatic conditions, season as a proxy of tick activity and interactions of taxonomic family with area and season as a proxy of eco-environmental features of individuals influencing exposure to ticks. We measured the proportion of individuals sampled after being trapped or surveyed immediately after being hunted and the proportion of individuals found dead in each taxonomic family. We found statistically significant differences between taxonomic families (Chi-square = 27.3, df = 3, p < 0.001) and this was the reason for not considering this variable as a pure effect in the models. We assumed the influence of environmental factors on the prevalence of ticks in wild carnivores would vary according to their host-seeking behaviour, being exophilic ticks more exposed to the environment than endophilic ones. Therefore logistic models were performed for exophilic and endophilic ticks in separate. For details about the modelling approach see Ruiz-Fons et al. (2008). We studied the particular case of the red fox separately from the rest of wild carnivores because red fox is the most abundant wild carnivore in Spain and thus accounted for the majority of our samples (Palomo et al., 2007). We aimed to test in foxes the same hypothesis tested

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for the entire carnivore taxa surveyed. Therefore, logistic binomial regression was performed with bioclimatic area, season and two additional host individual variables, sex and age. The interaction between sex and age was also considered in the models. Model performance followed the same approach described above. Statistical uncertainty of the calculated tick prevalences was assessed by calculating the 95% confidence interval for each of the proportions according to the expression C.I.95% = 1.96[p(1 − p)/n]1/2 . 3. Results A total of 475 ixodid ticks were collected during the sampled period (393 adults and 82 immatures) in the three bioclimatic areas. No ticks were collected from the stoat, weasel, polecats, and pine martens surveyed. Forty-five of 88 red foxes (51.1 ± 10.4%), 13 of 14 Iberian wolves (92.9 ± 13.5%), 9 of 26 European badgers (34.6 ± 18.3%), 12 of 23 Egyptian mongooses (52.2 ± 13.4%), 3 of 15 stone martens (20 ± 20.2%), 1 of 4 wild cats (25 ± 42.4%), and 3 of 17 genets (17.7 ± 18.1%) were parasitized by ticks. Species of ticks identified in the different bioclimatic areas by carnivore species are shown in Table 1. I. ricinus was the predominant species in wild carnivores from Atlantic areas, where it was detected in almost every sampling site. I. ricinus was rarely found in Mediterranean areas where it was present only in a reduced number of sites. I. canisuga and D. reticulatus were found only in Atlantic areas. I. hexagonus was together with I. ricinus the most distributed tick species in the different bioclimatic areas (Table 1). We collected mainly adult stages of I. hexagonus and a small number of nymphs. In contrast, R. pusillus, R. sanguineus, R. turanicus, and R. bursa were found in carnivores from Mediterranean areas but not in Atlantic areas (Table 1). I. ventalloi was collected only in wild carnivores from thermo-Mediterranean areas. Ticks of the genus Hyalomma were collected in Mediterranean areas in low numbers while they were not collected in Atlantic areas. The prevalence of exophilic and endophilic ticks in the examined wild carnivores was 19.1 ± 5.2% and 29.4 ± 6.1% respectively. Twenty-six (29.5 ± 9.2%) and 29 (32.9 ± 9.8%)

Table 1 Tick species and developmental stages parasitizing wild carnivores through host species and bioclimatic area. Host

Bioclimatic area (No. of parasitized)

Tick species and stages

Iberian wolf (Canis lupus signatus)

Atlantic (13) Atlantic (6) Continental (11) Thermo-Mediterranean (19)a Continental (2) Atlantic (3) Continental (5) Thermo-Mediterranean (1)a Continental (1) Thermo-Mediterranean (2)a Continental (1) Continental (1) Thermo-Mediterranean (10)a

Ir (A), I. spp. (A, N), Dr (A) Ir (A), Ih (A, N), Ic (A), I. spp. (A, N), Dr (A) Ir (A, N, L), Ih (A), I. spp. (A, N), Hm (A), Hy. spp. (N), Rs (A), Rp (A) Ir (A), Iv (A), Rp (A), Rt (A), R. spp. (A) Ih (N), R. spp. (A) Ir (A), Ih (A), Dr (A) Ih (A), Ic (A), I. spp. (N), Rs (A), Rb (A) Rp (A) I. spp. (L) I. spp. (N), Rp (A) Rs (A), Rp (A) Ih (A) Ih (A), Iv (A), I. spp. (A, N), Hy. spp. (A), Rp (A), Rt (A), R. spp. (A)

Red fox (Vulpes vulpes) Stone marten (Martes foina) European badger (Meles meles) Genet (Genetta genetta) Wild cat (Felis silvestris) Egyptian mongoose (Herpestes ichneumon)

Abbreviations: Ir: Ixodes ricinus; Ih: I. hexagonus; Ic: I. canisuga; Iv: I. ventalloi; I. spp.: Ixodes genus; Hm: Hyalomma marginatum; Hy. spp.: Hyalomma genus; Rb: Rhipicephalus bursa; Rs: R. sanguineus; Rp: R. pusillus; Rt: R. turanicus; R. spp.: Rhipicephalus genus; Dr: Dermacentor reticulatus; A: adult; N: nymph; L: larva. a Information reported by Millán et al. (2007).

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of 88 red foxes, 6 (42.8 ± 25.4%) and 8 (57.1 ± 25.4) of 14 Iberian wolves, 3 (11.5 ± 12.1) and 7 (26.9 ± 16.8%) of 26 European badgers, 4 (17.3 ± 15.2%) and 10 (43.4 ± 19.6%) of 23 Egyptian mongooses, 0 and 3 (17.6 ± 17.4%) of 17 genets, 0 and 2 (13.3 ± 17.1%) of 15 stone martens and 0 and 1 (25 ± 41.9%) of 4 wild cats were parasitized by exophilic and endophilic ticks, respectively. The best model showed that none of the variables had a statistically significant influence on the risk of wild carnivores to be parasitized by exophilic ticks (Table 2). Nonetheless, we observed that the risk of wild carnivores to be parasitized by exophilic ticks in thermo-Mediterranean areas was three times higher (31.3 ± 11.1%, n = 67) than in the rest of the areas (11.4 ± 9.4%, n = 44; and 7.4 ± 9.8%, n = 27 for Atlantic and continental areas, respectively). Prevalence of exophilic ticks peaked in summer in Atlantic and continental areas while the peak in the thermoMediterranean area took place in spring (Table 3). The final model for endophilic ticks showed a statistically significant influence of bioclimatic area and season on the risk of wild carnivores to be parasitized (Table 2). The prevalence in continental areas was higher than that found in Atlantic and thermo-Mediterranean areas (Table 3). Prevalence was observed to peak in spring (44.2 ± 13.4%, n = 52) meanwhile lower prevalence values were observed in summer (18.2 ± 16.1%, n = 22), autumn (19.0 ± 16.7%, n = 21) and winter (18.6 ± 11.6%, n = 43). Prevalence peaked in spring in the three bioclimatic areas (Table 3). None of the factors considered was statistically significant in the final models for endophilic and exophilic ticks in foxes. However, we observed differences in the prevalence in the thermo-Mediterranean area (51.5 ± 17.1%, n = 33) and in the Atlantic area (7.1 ± 13.4%, n = 14). The prevalence peaked in spring in thermo-Mediterranean areas while it peaked in summer in Atlantic areas. In the case of endophilic ticks, the final model also showed that none of the considered factors influenced the risk of foxes to be parasitized. However, the prevalence was also higher in thermo-Mediterranean (27.3 ± 15.2%, n = 33) than in Atlantic (14.3 ± 18.3%, n = 14) areas and the prevalence peaked in spring in both thermo-Mediterranean and Atlantic areas. 4. Discussion To the best of our knowledge this is the first study addressing the ecological determinants of ticks parasitizing wild carnivores in Spain according to their different

life cycles, contributing to our knowledge on the ecology of ticks in wildlife in the Iberian Peninsula. Our hypothesis was partially confirmed as no statistically significant differences in the prevalence throughout bioclimatic areas were evidenced for exophilic ticks. However, the prevalence of endophilic ticks statistically differed between bioclimatic areas, suggesting the influence of macroclimatic conditions on the risk of wild carnivores to be parasitized by endophilic ticks. This finding may nonetheless be modulated by tick host population dynamics that may vary between the considered bioclimatic areas. Thus, confirming the effect of climate would need further research. Our results are a preliminary approach to understand the epidemiology of tick-borne pathogens transmitted by the tick species that parasitize wild carnivores. 4.1. Methodological considerations Ticks are expected to detach from animals after death in a variable time lapse. Shot animals were collected immediately after death and ticks had no chance to escape from the sealed plastic bag in which they were placed. The rest of carnivore carcasses surveyed were collected after being found death. Hence, we ignore the time between death and sampling. We only selected those animals whose condition was good enough to be necropsied, normally before 24 h after death. Thus, in order to avoid any confusing effect of the sampling approach on our findings, we estimated the percentage of animals properly sampled for ticks and those found dead in any of the surveyed regions, season, and families. There were no statistically significant (Chi-square = 1.4, df = 2, p > 0.05) differences in the proportion of animals sampled alive or immediately after death between bioclimatic areas (27.3%, 29.6%, and 37.3% in Atlantic, continental-Mediterranean, and thermoMediterranean bioclimatic areas, respectively) and this pattern was similar for the different seasons (data not shown). We expected a lower influence of climatic conditions on endophilic than on exophilic ticks due to the buffering microclimate provided by nests, burrows, caves or crevices which endophilic ticks inhabit. However, endophilic tick activity is influenced by the predominant climatic conditions such as seasonal changes in temperature and ˜ et al., 1992) and season may hence humidity (Estrada-Pena influence its prevalence on hosts as well. This gross classification does not consider particular differences within endophilic or exophilic tick species. A good example of

Table 2 Model statistics (Wald Chi-square statistic value, degrees of freedom (df) and the P-value) for the final binomial regression models (binomial distribution and a logistic link function) of endophilic and exophilic ticks. Models were carried in a separate way for of all of the carnivores surveyed and for red foxes only. Host

Tick class Exophilic

Wild carnivores Endophilic

Red fox

Exophilic Endophilic

Variable

Wald

Intercept Family × Season Intercept Area Season

0.000 13.59 18.77 17.89 9.89

Intercept Intercept

3.81 12.12

df 1 16 1 2 3 1 1

P-value 0.999 0.629 <0.0001 <0.0001 <0.05 0.051 <0.001

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Table 3 Prevalence (Prev.) of exophilic and endophilic ticks found on different wild carnivore species by climatic region and season. Area

Season

Carnivore species (Family)

Spring

Canis lupus signatus (Canidae) Vulpes vulpes (Canidae) Martes foina (Mustelidae) Martes martes (Mustelidae)

Subtotal spring

Summer

Canis lupus signatus (Canidae) Vulpes vulpes (Canidae) Martes foina (Mustelidae) Meles meles (Mustelidae) Martes martes (Mustelidae)

Subtotal summer Atlantic Autumn

Canis lupus signatus (Canidae) Vulpes vulpes (Canidae) Mustela erminea (Mustelidae) Mustela nivalis (Mustelidae) Martes foina (Mustelidae) Meles meles (Mustelidae) Felis silvestris (Felidae)

Subtotal autumn

Winter

Vulpes vulpes (Canidae) Martes foina (Mustelidae) Meles meles (Mustelidae) Mustela putorious (Mustelidae) Genetta genetta (Viverridae)

Subtotal winter Subtotal Atlantic area

Spring

Vulpes vulpes (Canidae) Martes foina (Mustelidae) Meles meles (Mustelidae)

Subtotal spring Summer

Vulpes vulpes (Canidae) Martes foina (Mustelidae) Meles meles (Mustelidae)

Subtotal summer Continental-Mediterranean Autumn

Vulpes vulpes (Canidae) Genetta genetta (Viverridae) Herpestes ichneumon (Herpestidae)

Subtotal autumn Winter

Vulpes vulpes (Canidae) Mustela putorious (Mustelidae) Felis silvestris (Felidae) Genetta genetta (Viverridae)

Subtotal winter Subtotal continental-Mediterranean area Spring

Vulpes vulpes (Canidae) Genetta genetta (Viverridae) Herpestes ichneumon (Herpestidae)

Subtotal spring Summer

Vulpes vulpes (Canidae) Meles meles (Mustelidae) Mustela putorious (Mustelidae) Genetta genetta (Viverridae)

Subtotal summer Autumn Subtotal autumn

Vulpes vulpes (Canidae)

Winter

Vulpes vulpes (Canidae) Genetta genetta (Viverridae) Herpestes ichneumon (Herpestidae)

Thermo-Mediterranean

Subtotal winter Subtotal thermo-Mediterranean area P: number of parasitized animals; S: number of sampled animals.

Prev. exophilic (P/S)

Prev. endophilic (P/S)

100 (2/2) 0 (0/3) 0 (0/1) 0 (0/3) 22 (2/9) 100 (1/1) 25 (1/4) 0 (0/1) 50 (1/2) 0 (0/4) 25 (3/12) 0 (0/1) 0 (0/6) 0 (0/1) 0 (0/1) 0 (0/1) 0 (0/3) 0 (0/1) 0 (0/14) 0 (0/2) 0 (0/2) 0 (0/2) 0 (0/1) 0 (0/2) 0 (0/9)

0 (0/2) 33 (1/3) 0 (0/1) 0 (0/3) 11 (1/9) 0 (0/1) 25 (1/4) 0 (0/1) 0 (0/2) 0 (0/4) 8 (1/12) 0 (0/1) 0 (0/6) 0 (0/1) 0 (0/1) 0 (0/1) 0 (0/3) 0 (0/1) 0 (0/14) 0 (0/2) 0 (0/2) 50 (1/2) 0 (0/1) 0 (0/2) 11 (1/9)

11.4 (5/44)

6.8 (3/44)

0 (0/1) 0 (0/2) 0 (0/3) 0 (0/6) 50 (1/2) 0 (0/3) 0 (0/1) 17 (1/6) 0 (0/3) 0 (0/2) 0 (0/1) 0 (0/6) 25 (1/4) 0 (0/2) 0 (0/1) 0 (0/2) 11 (1/9)

100 (1/1) 100 (2/2) 100 (3/3) 100 (6/6) 50 (1/2) 0 (0/3) 100 (1/1) 33 (2/6) 66 (2/3) 50 (1/2) 100 (1/1) 67 (4/6) 50 (2/4) 0 (0/2) 100 (1/1) 0 (0/2) 33 (3/9)

7.4 (2/27)

55.6 (15/27)

69 (11/16) 0 (0/5) 19 (3/16) 38 (14/37) 100 (1/1) 0 (0/1) 0 (0/1) 0 (0/1) 25 (1/4) 0 (0/1) 0 (0/1) 33 (5/15) 0 (0/4) 17 (1/6) 24 (6/25)

44 (7/16) 40 (2/5) 44 (7/16) 43 (16/37) 0 (0/1) 100 (1/1) 0 (0/1) 0 (0/1) 25 (1/4) 0 (0/1) 0 (0/1) 13 (2/15) 0 (0/4) 34 (2/6) 16 (4/25)

31.3 (21/67)

31.3 (21/67)

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particular differences within endophilic ticks is that between R. pusillus and H. hispanica, two wild rabbit (Oryctolagus cuniculus) ticks. While R. pusillus can be occasionally found outside rabbit burrows and can consequently parasitize other hosts (Ruiz-Fons et al., 2006), H. hispanica is a true endophilic tick that can be only collected inside bur˜ et al., 1992). rows (Estrada-Pena

4.2. Tick distribution The tick I. ricinus is predicted to be present at different ˜ et al., 2006), latitudes in Peninsular Spain (Estrada-Pena although its populations in Mediterranean climates are predicted to be scarce. However, I. ricinus was collected in wild carnivores from Mediterranean areas. The finding of every developmental stage of I. ricinus on Mediterranean wild carnivores suggests the existence of stable populations of I. ricinus in these areas. These stable populations would be perhaps restricted to particular humid microclimates within a sub-humid to dry Mediterranean macro-climate. The epidemiological relevance of these Mediterranean populations in I. ricinus-borne pathogens remains unknown. I. hexagonus is a widely distributed species due to its nidicolous habits that offer it a buffering microclimate inside host nests, burrows or caves (Piesman and Gern, 2008). We found I. hexagonus in several wild carnivore species in different bioclimatic areas. This agrees with the buffering effect burrows, nests of caves of hosts offer I. hexagonus against adverse climatic conditions. The predominant collection of adult I. hexagonus in relation to immature stages suggests that other animals host the first stages of this tick species. This contrasts with findings in Portugal where wild carnivores are important hosts of immature stages (Santos-Silva et al., 2011). I. hexagonus is a competent vector of B. burgdorferi sensu lato (Piesman and Gern, 2008). This pathogen has also been detected in Hyalomma spp. ticks in areas of the Iberian Peninsula apparently unsuitable for I. ricinus (Toledo et al., 2009) where I. hexagonus is expected to be present according to our findings. We suggest that endophilic cycles of B. burgdorferi may exist in the absence of its main vector – I. ricinus – linked to I. hexagonus and its hosts. The impact of B. burgdorferi in wild carnivore health and conservation is unknown (Sobrino and Gortázar, 2008). In areas where exophilic cycles of B. burgdorferi exist, e.g., the northern third of Peninsular Spain, a link between endophilic and exophilic cycles may influence the maintenance of this pathogen and consequently interfere in Lyme disease control measures if these target I. ricinus only. I. canisuga was collected from Atlantic and continental areas but only occasionally. I. canisuga was previously collected on foxes in winter in dry areas of north-eastern ˜ et al., 1992). It was surprising that, Spain (Estrada-Pena according to our results, the only capture of I. canisuga in continental areas was in May while it was absent in carnivores sampled in winter (data not shown). Our findings contrast with observations in Portugal where I. canisuga is distributed in southern regions where it parasitizes mainly wild carnivores (Santos-Silva et al., 2011).

The endophilic behaviour of I. ventalloi suggests it may be more widely distributed than we found. I. ventalloi parasitizes mainly wild rabbits, which had experienced increasing population changes in our thermoMediterranean study areas although linked to game management (Delibes-Mateos et al., 2008). The absence of I. ventalloi in continental areas could be due to our survey approach that was partially biased to big game hunting areas. Big game management for hunting purposes may negatively influence rabbit population abundance and consequently its associated ticks. In fact, I. ventalloi has been reported in wild lagomorphs from continental areas of Spain (Rodríguez Rodríguez et al., 1981). Its absence from Atlantic areas where rabbits are testimonial (Villafuerte et al., 1998) suggests an accidental role of wild carnivores as hosts for I. ventalloi that may be caused by predation on rabbits. Independently of their role, wild carnivores may be considered when studying the epidemiology of I. ventalloiborne pathogens. It was surprising not to find wild carnivores parasitized by exophilic ticks of the genus Haemaphysalis such as H. concinna and H. punctata that are among the most abundant exophilic tick species in Atlantic areas (the authors, unpublished). Gerrikagoitia (2010) found both H. concinna and H. punctata parasitizing red foxes in the Basque Country although at very low intensities. Our results suggest wild carnivores in Atlantic areas are not among the preferred hosts for Haemaphysalis spp. and may only serve as accidental hosts. This may have important implications in Haemaphysalis-borne pathogen epidemiology. The species D. reticulatus was found to be restricted to its known geographic distribution in Atlantic bioclimatic areas ˜ et al., 2004a,b). However, of Peninsular Spain (Estrada-Pena it is surprising that we found no wild carnivore parasitized by Dermacentor marginatus in Mediterranean areas where ˜ et al., 1992, 2004a,b; it is a common species (Estrada-Pena Ruiz-Fons et al., 2006). D. marginatus larvae and adults behave as exophilic ticks and are usually found questing in the vegetation (de la Fuente et al., 2004; the authors, unpublished). D. marginatus immature stages are known to parasitize rabbits in Spain but at low intensities (Rodríguez Rodríguez et al., 1981). In Portugal, D. marginatus larvae and nymphs were found parasitizing small rodents and rabbits (Santos-Silva et al., 2011). Therefore, we expected wild carnivores to be relevant as hosts for immature stages of this species but our results suggest that D. marginatus does not select wild carnivores as preferred hosts. However, adult D. marginatus were collected from Iberian wolf, red fox, wildcat, and stone marten in Portugal (Santos-Silva et al., 2011) while in Spain this species has been found mainly on wild ˜ et al., 2004a; Ruizand domestic ungulates (Estrada-Pena Fons et al., 2006; Toledo et al., 2009). This is important since D. marginatus is one of the main reservoirs and vectors for spotted-fever group Rickettsia (de la Fuente et al., 2004). An important finding was the scarcity of Hyalomma spp. ticks. Hyalomma is the most abundant tick genus in the southern half of Peninsular Spain (Ruiz-Fons et al., 2006). This finding suggests the limited role of wild carnivores in Hyalomma-borne pathogens. This information is especially relevant due to the current concern about the emergence of Crimean-Congo hemorrhagic fever virus – a virus

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transmitted by Hyalomma spp. – in the Mediterranean (Maltezou and Papa, 2010). Species in the R. sanguineus group – R. sanguineus, R. turanicus and R. pusillus – showed a Mediterranean climate preference because of their absence from Atlantic bioclimatic areas. Ticks in the genus Rhipicephalus are the main reservoirs of Ehrlichia spp. and Rickettsia spp. in nature (de la Fuente et al., 2004; Toledo et al., 2009). Thus, the role of wild carnivores in the life cycle of these species must be borne in mind when studying their epidemiology. Our findings on the parasitization of wild carnivores by ticks whose main hosts are deemed to be wild rabbits, i.e., I. ventalloi and R. pusillus, suggest that I. ventalloi is a specialist tick of rabbits that accidentally parasitizes other animals in sympatry with rabbits while R. pusillus may establish stable populations independently of rabbits. This is suggested because I. ventalloi was not collected in areas where rabbits might not be abundant while R. pusillus was collected. Nonetheless, further studies on the ecology of these tick species under different host community composition assemblages may confirm or reject this hypothesis. The finding of R. bursa may be caused as well by our sampling bias towards big game hunting areas where deer – main wild hosts for adult and immature R. bursa (Ruiz-Fons et al., 2006) – are abundant (Acevedo et al., 2008). R. bursa is a monotropic tick ˜ et al., 2004a), which parasitizes ruminants (Estrada-Pena so this finding is thought to be accidental. 4.3. Ticks parasitizing wild carnivores The highest diversity of ticks was observed in wild carnivores collected from continental and thermoMediterranean areas (Table 1). The parasitization of Iberian wolves by I. ricinus and D. reticulatus was not surprising since these tick species were already reported in wolves from the nearby province of Burgos and from the Basque Country (Domínguez, 2004; Gerrikagoitia, 2010). However, wolves in Atlantic areas were not parasitized by R. pusillus in contrast to those from Burgos, supporting the restriction of this species to Mediterranean climates with hot ˜ et al., and dry summers and cold winters (Estrada-Pena 1992). Tick species collected on badgers and foxes from Atlantic areas had been previously reported (Cordero Del Campillo et al., 1994; Domínguez, 2004; Astobiza et al., 2010; Gerrikagoitia, 2010). The limited role of wild carnivores as hosts for Hyalomma spp. in Mediterranean areas is in line with previous observations that suggest that other animals such as lagomorphs (Rodríguez Rodríguez et al., 1981), small mammals and birds, may act as hosts for immature stages of Hyalomma spp. We expected to find ticks of the R. sanguineus group parasitizing wild carnivores since carnivores are deemed as main hosts for R. sanguineus, R. turanicus, and R. pusillus (Santos-Silva et al., 2011). Almost every parasitized wild carnivore species from Mediterranean areas hosted ticks of the R. sanguineus group, thus showing the high plasticity of these tick species when looking for hosts. To the best of our knowledge, this is the first description of R. sanguineus in wildcat from Spain. However, R. sanguineus was identified in freeranging domestic cats (Millán et al., 2007). This is the first description of ticks in Eurasian badger from continental

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areas, where I. hexagonus and R. sanguineus were found and contrast with ticks collected from badgers in thermoMediterranean areas (Millán et al., 2007). For the first time R. bursa is described in Eurasian badger in Spain, although it was previously reported in red fox (Cordero Del Campillo et al., 1994). It is remarkable that ticks found in Atlantic and continental areas are also found in domestic dogs (Camacho et al., 2003; Herrero et al., 1992; Millán et al., 2007). R. sanguineus is vector of E. canis – the causal agent of canine monocytic ehrlichiosis – and spotted fever group Rickettsia (Herrero et al., 1992) while I. ricinus is the main vector of B. burgdorferi which has been detected in wild carnivores from Spain (Sobrino and Gortázar, 2008). Dogs and wild carnivores share ticks and tick-borne pathogens which poses concerns for pet and human health. The prevalence of ticks we found in Iberian wolf was similar to that reported in wolves from the province of Burgos (50%; Domínguez, 2004). The prevalence in red fox was similar to the prevalence reported in foxes in south-eastern Spain (55%; Martínez-Carrasco et al., 2007) and the Basque Country (50%; Gerrikagoitia, 2010) but lower than that reported in Burgos (73%; Domínguez, 2004). The prevalence in Eurasian badgers was substantially lower than that found in Burgos (71%; Domínguez, 2004) and the Basque Country (53%; Gerrikagoitia, 2010). Finally, the prevalence in stone marten was slightly lower to that reported in Burgos (33%; Domínguez, 2004) and similar to what was reported in the Basque Country (23%; Gerrikagoitia, 2010). 4.4. Environmental determinants of tick prevalence The lack of influence of the bioclimatic area on the risk of wild carnivores to be parasitized by exophilic ticks together with the diversity of tick species observed in wild carnivores from different bioclimatic areas confirmed our hypothesis. Different tick species have evolved to adapt to different environmental conditions while some wild carnivores are tolerant to changing environmental conditions. This would make the risk of wild carnivores to be parasitized by ticks independent of the bioclimatic area, at least at our study scale. A similar finding was expected across seasons when considering wild carnivores from different bioclimatic areas together. This would be caused by differences in activity periods among different tick species (Pfäffle et al., 2011) that would make the risk of wild carnivores to be parasitized by exophilic ticks almost constant across the year. Our observations suggest that the risk of wild carnivores to be parasitized may be influenced by season due to the particular activity of ticks in these areas (Table 3). Our sample size did not allow for comparisons within each bioclimatic area studied. Differences observed in red foxes are worth to be further studied since behavioural differences due to the variable availability of prey between bioclimatic areas may significantly condition the risk of being parasitized by exophilic ticks. Additionally, age and sex may also influence this risk, which may be caused by host individual traits (Alzaga et al., 2009). The higher prevalence of endophilic ticks observed in Mediterranean versus Atlantic areas is surprising and may be associated with the more extreme climatic

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conditions influencing microclimate in burrows, nests and caves to which endophilic ticks are exposed in Mediterranean ecosystems in comparison to their Atlantic counterparts. Host community composition is a highly relevant factor determining exophilic tick ecology (LoGiudice et al., 2008) and may also influence endophilic tick ecology. Seasonal differences in prevalence showed that endophilic ticks may be adapted to host reproduction as the higher prevalence was observed in spring when reproduction occurs. Nonetheless, ticks may adapt their activity to climatic conditions as well in parallel to what their hosts do as happens with the argasid Ornithodorus erraticus (OleagaPérez et al., 1990).

5. Conclusion In conclusion, our study indicates that Spanish wild carnivores are hosts to a relevant number of tick species. Some of them are potential vectors of pathogens causing serious animal and human diseases. We show that abiotic environmental conditions do not modulate the risk of wild carnivores to be parasitized by exophilic ticks. This is suggested to be caused by adaptation of ticks to different climatic conditions. Meanwhile, abiotic environmental conditions may drive the risk of wild carnivores to be parasitized by endophilic ticks. Information provided herein can be of help to understand tick ecology in Spanish wildlife, the epidemiology of tick-borne diseases, and to prevent the risks for wildlife, domestic animals, and humans posed by tick-borne pathogens.

Conflict of interest statement The authors declare they have no conflict of interest.

Acknowledgements We are grateful to those who contributed to the wild carnivore survey: gamekeepers of preserves at the “Principado de Asturias” region and “Organismo Autónomo de Parques Nacionales”, the Animal Health Service of the “Xunta de Galicia”, Joaquín Vicente, Pelayo Acevedo, Óscar Rodríguez, Pablo Ferreras, Diego Villanúa and Vanesa Alzaga. We acknowledge the contribution of two anonymous reviewers in improving the manuscript. This work is a contribution to project POII09-0141-8176 by “Junta de Comunidades de Castilla – La Mancha – JCCM” and to the agreement between the Spanish National Research Council (CSIC) and “Principado de Asturias”. Raquel Sobrino is supported by a post-doctoral fellowship awarded by JCCM and the EU. Álvaro Oleaga and F. Ruiz-Fons are supported by CSIC. Javier Millán holds a “Ramón y Cajal” contract awarded by the Spanish Ministry for Science and Innovation and the European Social Fund. No sponsor of this work participated in the study design, in the collection, analysis and interpretation of data, in the writing of the manuscript and in the decision to submit the manuscript for publication.

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