Veterinary Parasitology 181 (2011) 255266

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Veterinary Parasitology
journal homepage: www.elsevier.com/locate/vetpar

New techniques for an old disease: Sarcoptic mange in the Iberian wolf
lvaro Oleaga a, , Rosa Casais b , Ana Balseiro b , Alberto Esp b , Luis Llaneza c , Alfonso Hartasnchez d , Christian Gortzar a
a b c d

Instituto de Investigacin en Recursos Cinegticos IREC (CSIC-UCLM-JCCM), Ronda de Toledo, s/n, 13071, Ciudad Real, Spain Centro de Biotecnologa Animal SERIDA, Camino de Rioseco 1225, La Olla-Deva, 33394 Gijn-Asturias, Spain Asesores en Recursos Naturales ARENA S.L., c/Perpetuo Socorro, n 12 Entlo, 2B, 27003 Lugo, Spain Fondo para la Proteccin de los Animales Salvajes FAPAS, Carretera AS-228, Km 8.9, 33115 Tu nn/Sto, Adriano-Asturias, Spain

a r t i c l e

i n f o

a b s t r a c t
Sarcoptic mange, a parasitic skin infection caused by the burrowing mite Sarcoptes scabiei, has been reported in over 100 mammals, including humans. In endangered species, mange causes conservation concerns because it may decimate isolated populations and contribute to extinction. The Iberian Peninsula still maintains one of the largest wolf (Canis lupus) populations in Europe. In Iberia, sarcoptic mange is endemic in red foxes (Vulpes vulpes) and the rst conrmed wolf mange cases were recently reported. However, knowledge on S. scabiei in wolves is scarce because of the sampling difculties inherent to research on scarce species. In order to describe wolf mange epidemiology and to infer conservation implications, this study combined traditional laboratory techniques with the revision of wolf carcass pictures taken by eld biologists and original information obtained by camera trapping. A total of 125 necropsies and 8783 camera-trap days allowed insights into wolf mange epidemiology between 2003 and 2010. Living Sarcoptes mites were detected in 19% of the fresh carcasses. Alopecic (delayed) type IV hypersensitive response reactions were observed, while parakeratotic lesions were infrequent. The number of mites isolated per wolf ranged from 1 to 78, and had a negative correlation with the percentage of alopecic skin. No effect by sex on mange prevalence was found. Yearlings showed a lower probability to present mange-compatible lesions than pups or adults. Wolves with mange-compatible lesions had a lower kidney fat index than apparently healthy ones. ELISA testing of 88 sera yielded an antibody prevalence of 20%. Photo-trapping recorded mange-compatible lesions since 2003 with a peak in 2008. The percentage of wolves with mange-compatible lesions registered in camera-traps during 1 year correlated with the percentage of red foxes with lesions in the previous year. This is the rst large survey on sarcoptic mange in the Iberian wolf. Necropsy data, with alopecia as the main feature and a slight effect on body condition, and trends derived from camera trapping coincided in showing a rather low prevalence and an apparently stable situation of the disease and its host, suggesting that this parasite is currently not a major threat for this wolf population. However, more information is needed in order to assess the effect of mange on aspects such as pup survival. 2011 Elsevier B.V. All rights reserved.

Article history: Received 15 February 2011 Received in revised form 11 April 2011 Accepted 20 April 2011 Keywords: Iberian wolf Mange Sarcoptes scabiei Epidemiology Histopathology Camera trapping

1. Introduction Sarcoptic mange is a parasitic skin infection caused by the burrowing mite Sarcoptes scabiei, whose antigenic material deposited in tunnels dug in the epidermis causes intense irritation and an inammatory response producing

Corresponding author. Tel.: +34 926 295450; fax: +34 926 295451. E-mail addresses: alvaro.oleaga@uclm.es, alvaroleaga@yahoo.es (. Oleaga). 0304-4017/$ see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.04.036

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characteristic skin lesions. Such lesions are distinguished by dermal inammation, alopecia, hyperkeratosis and a characteristic thickened, wrinkled and slate grey-coloured skin, leading in some cases to dehydration, emaciation and eventually death of the animal (Mrner and Christensson, 1984). The disease has been reported for more than 100 mammal species, including humans and domestic and wild mammals (Bornstein et al., 2001). It has been proposed that the mite S. scabiei is a single highly variable species with different strains manifesting physiological specicity in different hosts (Pence et al., 1975; Arlian et al., 1989, 1996a), with this specicity as a subject of ongoing debate (Alasaad et al., 2011). Molecular studies carried out in recent years support this hypothesis (Walton et al., 2004; Rasero et al., 2010). Apart from the importance as a zoonotic disease and its economical relevance in domestic animals, sarcoptic mange may be an important problem for isolated or small size populations (Henriksen et al., 1993; Martin et al., 1998), and reaches epizootic proportions in certain wildlife populations (Fernndez-Moran et al., 1997; Leon Vizcaino et al., 1999; Pence and Ueckerman, 2002; Gonzlez-Candela et al., 2004). The family Canidae (Order Carnivora) includes at least 12 different species from all over the world described as affected by sarcoptic mange (Bornstein et al., 2001; Deem et al., 2002). The epidemiology of the process seems to vary between different areas and host populations, and both endemic and epidemic situations have been reported in the scientic literature concerning sarcoptic mange and wild canids (Todd et al., 1981; Pence et al., 1983; Mrner, 1992; Pence and Windberg, 1994; Gortzar et al., 1998; Bates, 2003; Davidson et al., 2008). This parasitic disease has been widely described in American coyotes Canis latrans and red foxes Vulpes vulpes (Todd et al., 1981; Gosselink et al., 2007) and in European red foxes (Lindstrm et al., 1994). In the coyote and wolf (Canis lupus), mortality due to this parasite has usually been considered a compensatory cause of death, usually related to lack of hair and cold temperatures (Pence et al., 1983; Pence and Windberg, 1994). However, knowledge focussed on S. scabiei in grey wolves is scarce (Todd et al., 1981; Jimenez et al., 2010), with most references mentioning a small number of individuals presenting dermal lesions, often without detailed clinical descriptions of the process et al., 1996; Shelley and (Wobeser, 1992; Jedrzejewska Gehring, 2002; Lovari et al., 2007). Recently, Domnguez et al. (2008) reported the rst conrmed mange cases in Iberian wolves. Sarcoptic mange can present two different pathological forms: the parakeratotic form, consistent with a type I (immediate) hypersensitive response; and the alopecic form, consistent with a type IV (delayed) hypersensitive response (Skerratt, 2003). Both forms of sarcoptic mange have been recorded in red foxes in England (Bates, 2003). Clinical manifestations of sarcoptic mange vary depending on individual variations in the duration and intensity of the hypersensitivity reaction and on the capacity of each individual host to limit parasite multiplication (Yager and Scott, 1993).

The grey wolf distribution area includes Europe, Asia and North America, with many populations fragmented and reduced as a result of human persecution and habitat alteration (Boitani, 2003; Fernndez and Ruiz de Azua, 2009). The largest population in Western Europe survives in the Iberian Peninsula, where after the recovery experienced since 1970 the total wolf population was estimated in more than 2000 wolves in 2008, with 322 packs occupying a continuous area of 120,000 km2 in the northwest and a small isolated population in the south of Spain (lvares et al., 2005; Blanco et al., 2008). The conrmation of sarcoptic mange by mite isolation carried out by authors in 6 wolves submitted for necropsy in 2008 as part of the wildlife disease surveillance program of Asturias (North Spain) suggested the need of a specic study to evaluate the importance of a possible threat of the disease for wolf population. Serological, histopathological and necropsy data were complemented with the retrospective revision of wolf carcass pictures and epidemiological information obtained by camera trapping. The objective in this study is to present the collected information concerning sarcoptic mange and wolves in Asturias, analyse data reported in epidemiological surveillance programs carried out since 2003, evaluate the usefulness of different study methods and discuss the possible causes and management or conservation implications of mange in Iberian wolves. 2. Materials and methods 2.1. Study area The Principality of Asturias (Spain) is a 10,603 km2 autonomous region located on the North coast of the Iberian Peninsula, with an abrupt orography that includes the central part of the Cantabrian mountain chain and altitudes ranging from sea level to 2640 m within only 40 km. This region is included in the Eurosiberian climatic dominion of Atlantic type climate, with cold winters (minimum of 6 months of potential frosts in non-coastal areas and temperatures ranging from 3 to 8 C to 4 to 0 C in the coldest months) and abundant precipitations (14002100 L/m2 per year), with frequent snowfalls in the winter season in the highest areas (Lines Escard, 1970). The predominant landscape is a mixture of deciduous and mixed forests with pastures and meadows for cattle feeding that share this territory with different wild ungulate species such as Southern chamois (Rupicapra pyrenaica parva), roe deer (Capreolus capreolus), red deer (Cervus elaphus), fallow deer (Dama dama) and wild boar (Sus scrofa), and also with other wildlife species such as the red fox, beech marten (Martes martes), stone marten (Martes foina), badger (Meles meles), brown bear (Ursus arctos) and wolf, all of them listed among the hosts of S. scabiei in Europe (Bornstein et al., 2001), in some cases even being diagnosed as suffering sarcoptic mange in the study area (Fernndez-Moran et al., 1997; Gortzar et al., 1998; Oleaga et al., 2008a,b). The distribution area of the wolf in Asturias includes almost the whole territory except the most populated Central region (Blanco et al., 2008) (Fig. 1). The wolf population showed a gradual increase in Asturias from the late nineties

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Fig. 1. Distribution in the three different geographic areas considered in Asturias for this work (Western, Central and Eastern) of wolves submitted and 2010: . Icon colours represent for necropsy in 2008: , 2009: mangy-conrmed wolves: , skin-injured wolves without mite isolation: and wolves without lesions or mite isolation: . The icon represents the only wolf without skin lesions that allowed mite isolation. Grey areas correspond to the three council districts (Proaza, Belmonte and Somiedo) where the long-term camera-trapping study was carried out.

Fig. 2. Body scheme indicating the number of conrmed mangy wolves showing alopecia and mite isolation in the 8 different body regions examined during the mite searching protocol, and the percentage of total mites isolated in each studied body region.

up to 2004, with an apparent maintenance of population values in recent years with an estimation of average density of 3.94.4 wolves/100 km2 in 2004 (Llaneza et al., 2004). For the work presented here, Asturias was divided into three different geographical areas: Western, Central and Eastern Asturias, in order to group samples and study animals for analysis. 2.2. Studied animals A total number of 125 wolves were submitted for necropsy between 2003 and March 2010 (Table 1), which included animals collected in population control hunts carried out by wildlife ofcers (n = 95; the wolf is not a hunted species in Asturias) and those found dead (vehicle collision n = 12, illegal kill n = 5, poisoning n = 2 and infectious disease n = 1). In 10 cases it was not possible to determine the cause of death. Animals came from almost the whole distribution area of wolves in Asturias, but they were not homogeneously distributed due to larger hunting effort carried out by ofcers in some areas according to the established Wolf Management Plan followed in the region (Fig. 1). Based on tooth wear (Gipson et al., 2000) three age classes were determined for this work: pups (<1 year), yearlings (between 1 and 2 years) and adults (>2 years). The studied group was composed of 58 males (11 pups, 10 yearlings and 37 adults) and 61 females (18 pups, 22 yearlings and 21 adults). In 6 cases it was not possible to register complete data regarding sex and age due to bad preservation. 2.3. Samples and pathological study From January 2003 to December 2007, 78 necropsies were carried out in SERIDA (Regional Government Animal Health Laboratory, located in Gijn-Asturias). During necropsies photographs of the general aspect and particular features of the animals studied were taken, sex, age, weight and different body measurements were registered, and whenever possible blood (taken by cardiac puncture for serology), parasites and tissue samples were collected.

The above-described necropsy procedure was improved in order to get more data about the aetiology and characteristics of skin lesions observed since January 2008 (Table 1). Thus, in the 47 animals submitted for necropsy from January 2008 to March 2010 location, extension and characteristics of dermal and/or internal lesions were recorded; KFI (kidney fat index) and adrenal glands weight were noted (as body condition and stress degree indicators, Salak-Johnson and McGlone, 2007); eight 5 cm 5 cm skin samples (from ears, head, neck, back, foreleg, rear leg, abdomen and tail, Fig. 2) were resected with a scalpel and additional skin sections, lymph node pieces and any lesion detected in other tissues were xed in 10% buffered formalin, embedded in parafn and sectioned at 4 m for histopathological studies whenever possible. 2.4. Laboratory techniques 2.4.1. Mite isolation/identication Between January 2008 and March 2010, the eight skin samples taken from every wolf that arrived for necropsy less than 12 h after death (n = 32, including 12 wolves with mange-compatible lesions and 20 apparently healthy animals, Table 1) were submitted to an established ectoparasites search protocol. Briey, the skin pieces were placed on Petri dishes and incubated at 37 C for 24 h in order to stimulate mites to leave the skin. Both Petri dishes and skin samples were then meticulously examined for the presence and identication of ectoparasites using an Olympus SZX9 (1057) magnier. Detected mites were identied according to Wall and Shearer (1997), collected and preserved in 70% ethanol. 2.4.2. Histopathological study Skin samples and prescapular lymph nodes from the nine wolves with sarcoptic mange-compatible lesions and S. scabiei isolation were collected, xed in 10% neutralbuffered formalin, and dehydrated through graded alcohols and xylol before being embedded in parafn wax. Several serial sections, 4 m thick, were cut from each sample and

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Table 1 Wolves subjected to necropsy during the study period. Year Wolves studied at necropsy 78 21 10 16 125 Wolves with mange-compatible lesions at necropsy 0 9 3 3 15 Wolves submitted to mites searching protocol 0 12 5 15 32 Wolves with mange-compatible lesions submitted to mites searching protocol 0 7 2 3 12 Wolves with mange-compatible lesions and mites isolation 0 6 1 2 9

2003 2007 2008 2009 2010 Total

Number of wolves subjected to necropsy during the study period, including wolves with mange-compatible lesions, those subjected to the established mite searching protocol and those with skin lesions and conrmed sarcoptic mange by mite isolation. Data from 2003 to 2007 have been grouped due to the absence of mange-compatible lesions detected at necropsy before 2008 and the lack up to that year of a mite searching protocol.

stained with hematoxylin and eosin (HE) for histological characterization of detected lesions. From January 2008 to January 2009, 16 wolves (including 7 conrmed mangy animals, 4 skin-injured without mite isolation and 5 apparently healthy animals) were subjected to immunohistochemical examination by means of the peroxidase anti-peroxidase (PAP) method (Sternberger et al., 1970). The sections were incubated with a rabbit antiserum against the S. scabiei Ss20 B3 antigen (Casais et al., 2007) diluted 1 in 700. Preimmunization rabbit serum was used as negative control. 2.4.3. Serum antibody survey (ELISA test) The antibody levels against S. scabiei present in blood sera taken from 88 wolves submitted for necropsy between 2004 and 2010 (annual average of 12.6 samples, range 719) were assessed using an in-house ELISA. This ELISA uses as antigen the recombinant protein Ss20 B3 and has been previously used successfully for diagnosis of sarcoptic mange in other wild species (Casais et al., 2007; Oleaga et al., 2008a; Falconi et al., 2010). The absorbance at 450 nm was measured in a microplate reader (Sigma, model 680). Results were expressed as a percentage of the relative optical density (% relative OD450 nm), which was calculated according to the following formula: OD = ODunknown serum ODnegative serum 100 ODpositive serum ODnegative serum

pictures obtained from dead animals before that year was used as a retrospective method for studying the presence of mange-compatible lesions and to compare the obtained data with those coming from other study methods. A total of 1195 photographs (958 from 2003 to 2007 and 237 from 2008) were taken and provided by ARENA S.L. biologists in order to evaluate the presence of skin lesions in dead wolves. The rst 958 pictures revised included 71 of the 78 animals submitted for necropsy from 2003 to 2007 (Table 1) and 10 additional wolves dead before 2008 where necropsy was not possible but the external general aspect was recorded (average value = 16.2 animals/year). The animals studied were 43 males and 50 females, including 17 pups, 31 yearlings and 45 adult wolves. The abovementioned pictures and results obtained in their revision were compared with 237 photographs and subsequent necropsy and laboratory data of 15 animals studied in 2008 (when the improved necropsy protocol for the diagnosis of sarcoptic mange had already been established). This comparison allowed us to evaluate the suitability of the method to detect skin lesions and measure its success rate in correctly detecting sarcoptic mange. Pictures were obtained using both analogical and digital cameras, recording external general aspect, particular features of skin and different parts of the body and paying special attention to the presence of any kind of lesion or anomaly. 2.6. Camera-trapping data The evaluation of camera trapping as a potential tool for the epidemiological study of sarcoptic mange in the Iberian wolf was carried out using two different approaches: (a) A long-term evaluation of the detection frequencies of wild canids and the presence of mange-compatible lesions in their skin was carried out taking advantage of a camera-trapping monitoring program established by FAPAS (fund for the protection of wild animals) for the study of free-ranging brown bear populations in Central Asturias. Pictures from wolves and red foxes obtained by the eight camera-traps (4 in Somiedo, 2 in Belmonte and 2 in Proazas council district, Central Asturias Fig. 1) maintained in the same location for at least 5 consecutive years during the period 20032009 were carefully revised in order to: (i) detect presence and

Serum samples were tested in duplicated, with control sera being included in every series. The sera from a S. scabiei-infected wolf and a wolf without mangecompatible lesions or mite isolation were used as positive and negative controls, respectively. The mean value from the % relative OD450 nm of seven scabies-free animals without skin lesions plus three times the standard deviation (Bornstein and Wallgren, 1997; Hollanders et al., 1997) was dened as the cut-off between positive and negative serum samples, offering for this species a cut-off value of 8.9%, indicating that animals with a relative OD450 nm percentage of 8.9% or above were considered positive. 2.5. Revision of wolf carcass pictures Due to the execution of necropsies by different technicians (belonging to ARENA S.L., SERIDA and IREC) during the study period and the lack of information concerning sarcoptic mange in Spanish wolves before 2008, revision of

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characterize mange-compatible lesions in free-ranging wolves and red foxes; (ii) signal possible trends in the frequency of the appearance of these lesions in nature through time and compare these trends with those reported at necropsy during the same period; and (iii) detect possible temporal and spatial agreements between the presence and frequency (as number of wild canid contacts per 100 camera-trap days of work) of both apparently healthy and skin-injured red foxes and wolves. The eight camera traps used in this long-term evaluation were analogical Canon camera systems activated by a heat and movement sensor (external Scanfer sensor connected from 2003 to 2006, and a Trail scan Canon system with incorporated sensor since 2007, in all cases using ash as articial light source). These cameras were placed along natural animal paths through beech woods (n = 3), oak woods (n = 1), grazing land (n = 1) and chestnut tree woods (n = 3), with an average altitude of 845 m above sea level (ranging from 480 to 1520 m a.s.l.). Distances from the closest camera and to the nearest village were 3.49 (ranging from 1.6 to 5.6 km) and 1.41 (ranging from 0.4 to 3.1) km average values, respectively. Cameras were usually operative from March to November (average value of 179.25 days working/year throughout the study period for the 8 cameras) due to the bad meteorological conditions existing during the winter season and the lower bear activity during that period. Revision of cameras and picture collection was done with a periodicity of 1530 days. The analysis of obtained pictures was carried out for each camera, district and the whole study area. All pictures of a single animal obtained during a 15-min period were considered and analysed as a single contact. (b) In order to get all the information available regarding epidemiology of the process in particular wolf packs, the careful revision of all pictures obtained in the camera-trapping long-term study signalled above was completed with photographs obtained close to carrions or temporal locations since 2003 by FAPAS and those got by 6 additional digital camera traps (passive systems Leaf River IR-3BU Leaf River Outdoor Products, Taylorsville, Mississippi, USA, with an infrared light source and an infrared movement and temperature sensor) set up since January 2008 nearby to suspicious sarcoptic mange-affected pack reproduction areas. 2.7. Statistics Statistical analysis was performed using the Epi Info 3.5.1 and SPSS 15.0 software. 3. Results 3.1. Sarcoptic mange conrmation and distribution Different degrees of alopecia or dermal lesion compatible with sarcoptic mange were detected in 15 out of the 47 wolves submitted for necropsy from January

2008 to March 2010. From the 32 fresh submitted wolves during that period, S. scabiei was isolated from 9 out of 12 animals showing skin lesions and 1 out of 20 without skin alterations (Table 1 and Fig. 1). The presence of mange-compatible lesions offered an apparent average prevalence value of 31.91% (19.5247.25%, 95% condence interval C.I.) for the period 20082010, reaching a peak value of 42.86% (22.5965.56%, 95% C.I.) in 2008. The real prevalence (wolves with lesions and conrmed S. scabiei presence) average value was 19.15% (9.6533.73%, 95% C.I.) from 2008 to 2010, and 28.57% (12.1952.31%, 95% C.I.) in 2008. No data regarding this kind of skin alterations were signalled in necropsies of animals carried out before 2008 (n = 78). Sarcoptic mange was conrmed in the three considered geographical areas of Asturias Eastern, Central and Western, with 3 (out of 13), 5 (n = 23) and 1 (n = 11) wolves with conrmed mange by isolation of mites, respectively, since January 2008 (Fig. 1). The Chi square test revealed no sex-related difference in the probability to show mange-compatible lesions or even harbour S. scabiei. Nevertheless, yearling wolves studied according to the necropsy protocol established in 2008 (n = 11) showed a statistically signicant lower probability to present mangecompatible lesions than pups (n = 11) or adults (n = 21) ( 2 = 8.353, p = 0.015), offering apparent prevalence values of 0%, 54.54% and 42.86%, respectively. While months like April, May and November had 2 mangy-conrmed wolves (this happened at least in one of the study years), none of the 15 wolves with sarcoptic mange or compatible lesions recorded at necropsy was collected in March, August, September or October. 3.2. Clinical characterization The number of mites isolated in conrmed parasitized wolves ranged from 1 to 78 per animal. With the exception of two wolves (with only 1 and 2 detected mites), all animals with lesions and S. scabiei isolation showed mite presence in at least 2 of the 8 studied skin areas (ears, head, neck, back, foreleg, rear leg, abdomen and tail), with a maximum of 7 of the 8 body areas tested with acari detection in one single animal. Skin pieces from tail, ears and rear legs showed an overall higher number of live mites detected (55, 57 and 47, respectively), and were also the body areas where more wolves (4, 6 and 6, respectively) allowed S. scabiei identication during examination (Fig. 2). 28.35% of detected mites were isolated in areas without dermal lesions. The most frequent and conspicuous detected macroscopical skin alteration was alopecia, present in the 9 mangy-conrmed animals in different degrees (from 10% to 90% injured skin surface); rear leg, abdomen, ears and foreleg were the most frequently alopecic areas (Figs. 2 and 3a, b and d). The relationship between the percentage of alopecic skin and the number of isolated mites for each conrmed mangy wolf presented a linear distribution (Fig. 4), with a statistically signicant decrease in the number of alive mites as the percentage of affected skin increases (r = 0.7818; p < 0.05).

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Fig. 3. Macroscopical and microscopical pictures showing characteristic lesions detected in conrmed mangy wolves at necropsy. The most frequently affected skin areas were abdomen (a), rear legs (b), base of tail (c) and ears (d). Cracking and thickened skin was infrequent, very localized when present (b and c). (e) The most frequently reported microscopical alterations were focal hyperkeratosis (A), hyperplasic epidermis (B) and inammatory inltrate in the dermis (C). Hypertrophy of the sebaceous glands (D) and hair follicles clogged with keratin (E) can be also observed (HE = 400, bar = 200 m). (f) Skin. Positive immunostaining in macrophages (arrows) located in the dermis (PAP = 200, bar = 100 m). (g) Prescapular lymph node. Positive immunostaining in macrophages (arrows) (PAP = 400, bar = 50 m).

Both hyperpigmentation and hyperkeratosis were detected in 4 conrmed mangy wolves, although especially in the case of hyperkeratosis reported alterations were of a small size and thickness (Fig. 3b and c). Three of the four animals showing hyperkeratosis were those with the highest percentage of affected skin surface (90, 80 and 70%, respectively). No recent ulcers or wounds were detected. Histologically, an alopecic (delayed) type IV hypersensitive response reaction was observed in the nine wolves with conrmed sarcoptic mange. Skin samples from the alopecic areas demonstrated an inammatory inltrate consistent with lymphocytes, macrophages and few neutrophils in the dermis (Fig. 3e and f). Occasionally, the

Fig. 4. Regression analysis carried out considering the percentage of affected skin versus the number of isolated mites in the nine conrmed mangy wolves (r2 = 0.6112; r = 0.7818, p = 0.0128; y = 55.5391933 0.765068493x).

stratum corneum showed focal parakeratosis and hyperkeratosis, and the epidermis was hyperplasic. The sebaceous glands were hypertrophied and some of the hair follicles clogged with keratin (Fig. 3e). Scab formation was not observed and mites could only be detected in one case. Lymphocytes and macrophages, located both in the dermis (Fig. 3f) and in the prescapular lymph nodes (Fig. 3g), showed positive immunostaining against S. scabiei antigen in 4 of the 7 wolves with conrmed sarcoptic mange subjected to immunohistochemical examination. There was no statistical relationship between the appearance of immunostaining and the percentage of affected skin or the number of isolated mites in these animals. None of the 4 wolves with mange-compatible lesions but without mite isolation showed positive immunostaining and only 1 of the 5 apparently healthy animals showed positive immunostaining, located only in the prescapular lymph node. The preimmune serum controls showed no staining. With the exception of one wolf with mange-compatible lesions and S. scabiei isolation where histological analysis suggested co-infection by a virus, no other pathological alteration was detected in studied animals besides those attributed to sarcoptic mange. The KFI gave a statistically signicant (t = 3.035, p < 0.005, n = 39) lower value for wolves with mangecompatible lesions, but showed no signicant relationship (t = 1.637, p = 0.110) with the isolation of mites in suspicious wolves. The KFI average values were 0.407 0.04, 0.228 0.024 and 0.264 0.04 for apparently healthy wolves (n = 26), animals with injured skin (n = 13), and conrmed mangy wolves (n = 9), respectively. No statistical relationship was detected regarding adrenal mass and the presence of mange-compatible lesions or conrmed sarcoptic mange.

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Fig. 5. Temporal evolution during the 7 years of the study period of: (i) seroprevalence, (ii) percentage of wolves with mange-compatible lesions at necropsy, (iii) percentage of wolves with isolated mites conrming sarcoptic mange at necropsy, (iv) percentage of dead wolves with mange-compatible lesions in pictures and (v) percentage of wolves with mange-compatible lesions in camera-trapping pictures. (ii), (iii), (iv) and (v) percentages are in agreement with the apparent morbidity increase reported in Asturian wolves during 2008.

3.3. Serology Considering the 8.9% (percentage of relative OD450 nm) calculated cut off level, the ELISA test was characterized by 75.0% sensitivity and 87.5% specicity. The analysis of 88 sera samples obtained from 2004 to 2008 allowed the detection of 18 seropositive wolves, offering an average seroprevalence value of 20.45% (12.5930.39%, 95% C.I.). The percentages of OD450 nm of the ELISA positive wolves were below 27.3%, indicating a weak antibody response. The rst seropositive animal was detected in 2004 (rst year with collected blood samples), with seropositive wolves detected during the whole study period (Fig. 5). ELISA positive animals appeared equally distributed between both sexes, with 20.93% (10.0436.05, 95% C.I.) and 21.43% (10.2936.82, 95% C.I.) seropositive males and females, respectively. Regarding age groups, pups showed a slightly lower percentage of seropositivity (14.28% seroprevalence average value, 95% C.I. = 3.0436.35%) than adults (22.5% average value, 95% C.I. = 10.8338.46%) and yearlings (25% average value, 95% C.I. = 9.7746.72%). The effect of sex and age on seroprevalence was not statistically signicant. The Western area, with a 35% seropositivity (15.3959.22%, 95% C.I.), showed the highest seroprevalence average value, whereas similar data of 17.2% (5.8435.78%, 95% C.I.) and 16.7% (6.3732.82%, 95% C.I.) were reported for the Central and Eastern regions, respectively. Differences were not statistically signicant. The rst half of the year showed a statistically signicant ( 2 = 10.159, p = 0.017) higher seroprevalence value, with 16 out of 18 seropositive wolves sampled between January and June. 3.4. Photographic studies The revision of pictures of dead wolves obtained from 2003 to 2007 (n = 958 pictures), focussed on the detection of skin lesions and alterations similar to those described for conrmed mangy wolves since 2008, permitted the detection of six animals showing mange-compatible lesions. The

rst suspicious animal was detected in 2003, and since then, wolves showing mange-compatible skin alterations were detected in pictures taken in 2005, 2006 and 2007 (with 1, 1 and 3 skin-injured animals, respectively, Fig. 5). The average global percentage of animals showing mangecompatible lesions was 7.40% between 2003 and 2007 (Fig. 5). Distribution of these animals included Western (1), Central (2) and Eastern (3) areas. The apparently affected animals were 2 males and 4 females, including 1 pup, 2 yearlings and 3 adults. From the 15 animals photographed by ARENA S.L. biologists in 2008 whose pictures were revised as an evaluation of this study method, mange-compatible lesions were detected in 6 (40%) wolves, from which sarcoptic mange was conrmed by the subsequent mite searching protocol in 4 cases, indicating the suitability of the evaluation method used. Since the beginning of the camera-trapping long-term study (2003), an 8783 camera-trap/day monitoring effort was carried out. A total of 832 and 184 contacts with red foxes and wolves, respectively, were registered, including 20 contacts with red foxes and 22 with wolves showing mange-compatible lesions. Both canid species were registered in all 8 camera traps used in this study, with average values of 16.50 (ranging from 0.29 to 48.1) and 3.69 (from 0.14 to 8.8) contacts/year/camera for red foxes and wolves, respectively. The rst pictures showing mange-compatible lesions in red fox and wolf were obtained in 2003 in Belmonte and Somiedo, respectively. Since then, both wolves and red foxes with cutaneous alterations were photographed in 2006, 2007 and 2008 (Figs. 6 and 7). Mange-compatible lesions were detected in the three camera-trapping studied areas in both species with the exception of Belmonte (Fig. 1), where no apparently affected wolves were registered (in this area a wolf with dermal lesions was photographed in a camera trap placed close to a carrion, thus not being included in this monitoring program). In one single camera-trap (from Somiedo) no canid with mangecompatible lesions was photographed. The analysis of recorded data both for each individual camera trap and for the group of cameras allocated in each area (Somiedo n = 4, Belmonte n = 2 and Proaza n = 2) showed a different evolution for each camera and even area, with a great annual variation in detection of red foxes not observed for wolves (expressed as the number of contacts registered in 100 camera trap days), and often single or very scarce data regarding the detection of mange-compatible lesions, preventing trend descriptions and statistical analysis on this scale. Nevertheless, the study of camera-trapping collected data from the 8 cameras grouped as a whole provided data enough to attempt analysis, which agreed to a large extent with results obtained by the other study techniques used in this work (Figs. 5 and 7). A gradual increase in the percentage of skin-injured red foxes and consecutive decreases in the number of red fox-contacts/100 camera-trap days were detected, reaching the lowest detection rate and the highest percentage of red foxes with mange-compatible lesions in 2007, suggesting a possible effect of the disease on the presence or density of this species in the studied area.

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Fig. 6. Example of pictures obtained by the camera-trapping technique in the present work showing different degrees of mange-compatible lesions in wild wolves. The last picture shows a skin-injured animal next to another member of its wolf pack without detectable skin lesions.

Regarding the wolf, an important increase (reaching 27.7% of registered wolves) in the percentage of wolves photographed with mange-compatible lesions was observed in 2008, whereas the number of wolf-contacts detected for 100 camera-trap days as a measure of detection showed very small variations during the study period (Fig. 7). The statistical analysis of obtained pictures, although carried out on a limited number of years and contacts, showed a close statistically signicant relationship between the percentage of skin-injured wolves registered in camera traps during 1 year and the percentage of red foxes with mange-compatible lesions registered in the previous year (Rs = 0.9856, p < 0.0005, n = 6; Fig. 7).

The examination of all pictures obtained by the 8 camera traps used in the long-term study plus those placed near carrions or added since 2008 provided valuable information regarding clinical and epidemiological aspects of sarcoptic mange in wolves: (i) showing the similarity of lesions reported in the eld with those conrmed as a consequence of sarcoptic mange at necropsy; (ii) allowing the detection of wolf packs with members showing mangecompatible lesions for two consecutive years or in areas where these skin alterations had not been reported before; (iii) reporting the presence of skin-injured and apparently healthy pups from one same pack and reproductive season and (iv) showing the apparently slow extension of skin lesions and limited body condition worsening in individually recognizable wolves (especially compared to the quicker, often lethal, clinical evolution of the process in red foxes from the same area unpublished data). A single wolf with mange-compatible lesions recognizable in pictures obtained during a long enough time period (from 14/08/2008 to 31/10/2008 in the Proaza area) showed the maintenance of a good body condition and even the apparent recovery of hair and skin condition through the series of pictures, as signalled in particular cases from other affected wolf populations (Jimenez et al., 2010). 4. Discussion

Fig. 7. Comparison between year-on-year variations undergone by the number of contacts/100 camera-trap days and the percentage of contacts showing mange-compatible lesions on pictures obtained by camera trapping for both red foxes and wolves from the study area.

This is the rst large survey on sarcoptic mange in the Iberian wolf. Although the number of samples and obtained data available were limited due to the species

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status, data compiled herein by a combination of pathological and retrospective studies (including serological and photographic techniques) allowed valuable insights into the epidemiology of this parasitehost relationship. Mange was conrmed by isolation of S. scabiei mites from wolves necropsied between 2008 and 2010. Moreover, visible lesions recorded on photographs and serum antibody detection by ELISA coincide, suggesting that the disease was already present at least since 2003. Lesion distribution was similar to those described in previous reports in wolves (Todd et al., 1981; Domnguez et al., 2008), and in other canids such as the red fox and coyote (Trainer and Hale, 1969; Pence et al., 1983; Mrner, 1992). However, parakeratotic lesions were infrequently reported in this study. Regarding the ELISA, the low number of mites usually detected on infected wolves may have limited the sensitivity of this antibody test. The mean antibody prevalence was similar to the 21% reported in a Scandinavian study, where 86 wolves collected from 1998 to 2002 included 6 individuals that died of sarcoptic mange (Olsen, 2003). The lack of sex, age or site differences in antibody prevalence suggests a widespread contact with the parasite. Visualizing the mite by isolation or histology is the only denitive diagnosis of sarcoptic mange (Bornstein et al., 1996; Morris and Dunstan, 1996; Davidson et al., 2008). In the present study, mites were detected in only one of 20 apparently healthy wolves (with no visible lesions), whereas they were isolated in 9 of 12 animals with dermal lesions and were also immunohistochemically detected in 4 of the 7 mangy animals studied at this level. This, along with an apparent sarcoptic mange/viral co-infection in one male as the only pathological process different of sarcoptic mange detected by histology or other applied techniques in the frame of the wildlife disease surveillance program in studied wolves, signals mange as the cause of the lesions observed in these animals. Live mites were detected in only 75% of the properly tested suspect wolves, and usually in a low number. This agrees with data on coyote and other studies on sarcoptic mange in wolves (Todd et al., 1981; Domnguez et al., 2008), and coincides also with low isolation rates obtained by skin scraping in mange-suspect dogs (Canis familiaris) (Bornstein, 1991; Hill and Steinberg, 1993; Grifn, 1993; Carlotti and Bensignor, 1997; Beck and Hiepe, 1998; Davidson et al., 2008). These isolation numbers contrast with those reported for the red fox (up to 5000 mites per cm2 , Little et al., 1998a). Similarly, the inverse relationship observed between the number of mites and the size of the affected skin (Fig. 4) contrasts with ndings in foxes (and wild ruminants, data not shown), suggesting a different evolution of the process and an apparent greater ability of wolves to control the number of alive mites as compared to most of the other susceptible natural S. scabiei hosts in the study area (different immune responses and efcacy against the mite have already been signalled for dog and red fox: Arlian et al., 1996b; Little et al., 1998a). However, caution is required in using the number of isolated mites as a proxy for parasite burden (Alasaad et al., 2009). Most wolf lesions were histologically conrmed as alopecic, opposed to the frequent parakeratotic form (type

I immediate hypersensitivity response) observed in red foxes from the study area by the authors (data not shown) and most frequently reported in other red fox populations (Little et al., 1998b; Newman et al., 2002). This alopecic form reported in Asturian wolves, a type IV hypersensitivity response, is also the most common presentation of sarcoptic mange in dogs (Paterson et al., 1995), but rarely reported in the fox, where this presentation has been described as characterized by low mite burdens, few (if any) crusty lesions and a limited effect on body condition (Bates, 2003). Experimental studies have shown no signicant KFI value differences between mangy and healthy dogs (Arlian et al., 1995; Little et al., 1998a), whereas coyotes and wolves tend to show limited effects of mange on body condition (Todd et al., 1981; Pence et al., 1983; Pence and Windberg, 1994), and this effect is more severe in red foxes (Gortzar et al., 1998; Little et al., 1998b; Newman et al., 2002; Davidson et al., 2008). In this study wolves with mange-compatible lesions showed a 50% lower KFI value than those without visible lesions, but never appeared emaciated or with an extremely poor body condition at necropsy. These ndings coincide with those signalled in the few cases of red foxes reported by Bates (2003) with the alopecic presentation of sarcoptic mange, and are lower than the 85% difference observed for foxes with parakeratotic mange lesions (Little et al., 1998a; Newman et al., 2002). Nevertheless, the detection of both parakeratotic and alopecic forms in a red fox population with different effects on body condition (Bates, 2003) conrms the existence of individual differences in the response to the mite, warning us about the possible presence of different disease presentation forms in Asturian wolves not yet reported at necropsy. In this sense, the authors were able to observe in the eld in 2010 one wolf with extremely poor body condition, abnormal behaviour and severe parakeratoticmange-compatible lesions, frequent in red foxes, but not registered in Asturian wolves at necropsy up to date. Regarding the time trends, both the photographic records and the necropsy data showed an increased morbidity in 2008. Possible explanations for this increase include changes in mite pathogenicity (Pence and Windberg, 1994), density dependence in transmission rates (Pence et al., 1983; Pence and Windberg, 1994), coinfections or other debilitating factors (Lloyd, 1980) and a higher mange circulation among sympatric carnivores (Trainer and Hale, 1969; Todd et al., 1981; Clayton, 2003; Mrner et al., 2005; Soulsbury et al., 2007; Domnguez et al., 2008). Mite pathogenicity variation is difcult to test in the eld. On the subject of density dependence, there are no indications of an increase in wolf numbers during the study period in Asturias: after a moderate increase observed from 1998 to 2004, an apparent stabilization in wolf density was reported from 2005 to 2008 (Llaneza et al., 2004; Garca and Llaneza, 2009). Regarding co-infections, it is known that Iberian wolves are often exposed to disease agents including canine distemper virus, canine parvovirus and Leishmania infantum (Sobrino et al., 2008a,b); the histopathological detection of a wolf apparently co-infected by S. scabiei and a viral agent emphasizes the need of further studies about the role that concomitant agents can play in the development of

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sarcoptic mange and skin lesions, with ongoing research in the studied population by the authors. Finally, the statistically signicant relationship signalled between the trends in mange-compatible lesions detection for red fox and wolf with 1-year delay (Fig. 7) strongly suggests sarcoptic mange in the red fox as the most likely origin of the observed time trend in wolf disease. Interspecic transmission of S. scabiei has been observed between the red fox and coyote (Trainer and Hale, 1969), the red fox and dog (Soulsbury et al., 2007; Clayton, 2003) and coyote and wolf (Todd et al., 1981; Jimenez et al., 2010), also with 1-year difference in the morbidity peak of mange reported between the coyote and wolf by Todd et al. (1981). Mrner et al. (2005) and Domnguez et al. (2008) indicate that the red fox is the most probable origin of sarcoptic mange in wolf. The possible role of sympatric ungulates conrmed as S. scabiei hosts in Asturias (Fernndez-Moran et al., 1997; Oleaga et al., 2008a,b) in the epidemiology of sarcoptic mange in Iberian wolf is still unclear. The high percentage of macroscopically affected skin in conrmed mangy wolves at necropsy suggested the usefulness of photographic techniques for the detection of sarcoptic mange lesions in the Iberian wolf, which was conrmed by data on pictures taken by technicians in 2008 from dead wolves subsequently studied at necropsy. Remote photography methods have been increasingly used in recent years to address a great variety of questions in wildlife biology (Karath and Nichols, 1998; Cutler and Swann, 1999; Carbone et al., 2001; Grassman et al., 2006; Lucherini et al., 2009), whereas their use in wildlife sanitary studies is infrequent, usually related to animal behaviour or biological estimations with epidemiological interest (Hegglin et al., 2004; VerCauteren et al., 2007; Jennelle et al., 2009). Even with the limited number of cameras available and contacts obtained (that prevented weighty conclusions), this camera-trapping technique has been shown to be useful for the detection of animals with mange-compatible lesions in the eld, providing interesting information about the presence of injured animals at different times of year and in different areas and interesting data regarding population trends and the epidemiology of the disease (conrming in the eld the apparent increase in morbidity reported for sarcoptic mange at necropsy during 2008, Figs. 5 and 7). To the best of our knowledge, this is the rst time photo-trapping has been used to study disease-compatible lesions and time trends in a wildlife disease. The combination of these eld techniques with the necropsies and laboratory results allowed interesting insights of mange epidemiology in Iberian wolves. Necropsy data (with skin alteration as the only detected lesion related to S. scabiei and a limited effect on body condition) and reported trends (showing a rather low prevalence and an apparently stable distribution of the disease and its host) suggest that this parasite is not a major threat for wolf populations in Asturias. However, the possible lack of data and incomplete overview of the disease in such a scarce species emphasize the need of more information in order to evaluate its possible effect on fertility and pup survival rates, where the effect of the parasite seems to be more important (Todd

et al., 1981; Pence and Windberg, 1994; Soulsbury et al., 2007; Jimenez et al., 2010), and even the long-term effect on the health of adult wolves. Hence, surveillance should be maintained and, eventually, more information gathered through larger surveys (increasing the number of available cameras; including wolf tagging with epidemiological surveillance purposes) in order to better understand and characterize sarcoptic mange in the Iberian wolf and assess the epidemiology of this parasitic disease and its effect on wild canids population trends and wolf conservation.

Acknowledgements This is a contribution to the agreement between CSIC and Principado de Asturias. We thank the rangers of the game preserves (specially Jaime Marcos Beltrn, Francisco Alonso Mier, Fernando Rodrguez Prez, Pedro Gonzlez for their help in carcasses submission and Angel Nuno) and camera-trapping, our colleagues from ARENA S.L. and IREC (Emilio Jos Garca, scar Rodrguez and Mara Surez) for their assistance in eld, necropsy and laboratory work, and other workmates (Jose Luis Garca Daz and Rafael Alba Zarabozo) for their advice and eld collaboration in camera-trapping work. We wish to thank also Kevin Dalton for revising the English of the manuscript, Pelayo Acevedo for his contribution in statistical and geographical analysis and Joaqun Vicente for his advice in the study design and camera trapping procedures. Projects RTA 2009-00114-00-00 (INIA) and CIT-060000-2009-0034 (MICINN) contributed to this study.

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