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Veterinary Parasitology 181 (2011) 255266

Contents lists available at ScienceDirect

Veterinary Parasitology

j ourna l homepage: www.e lsev ier .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 Espb, Luis Llanezac,Alfonso Hartasnchez d, Christian Gortzar a

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

a r t i c l e i n f o

Article history:Received 15 February 2011Received in revised form 11 April 2011Accepted 20 April 2011

Keywords:Iberian wolfMangeSarcoptes scabieiEpidemiologyHistopathologyCamera trapping

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, mangecauses conservationconcerns because it may decimate isolated populations and contributeto extinction. The Iberian Peninsula still maintains one ofthe largest wolf (Canis lupus)populations in Europe. In Iberia, sarcoptic mange is endemic in red foxes (Vulpes vulpes)and the first confirmed wolfmange cases were recently reported. However, knowledge onS. scabiei in wolves is scarce because ofthe sampling difficulties inherent to research onscarce species. In order to describe wolf mange epidemiology and to infer conservationimplications, this study combined traditional laboratory techniques with the revision ofwolf carcass pictures taken by field biologists and original information obtained by cameratrapping. A total of125 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 wereobserved, while parakeratotic lesions were infrequent. The number ofmites isolated perwolfranged from 1 to 78, and had a negative correlation with the percentage ofalopecicskin. No effect bysex onmange prevalencewas found. Yearlingsshowed alower probabilityto present mange-compatible lesions than pups or adults. Wolves with mange-compatiblelesions had a lower kidney fat index than apparently healthy ones. ELISA testing of88 serayielded an antibody prevalenceof20%. Photo-trappingrecorded mange-compatiblelesionssince 2003 with a peak in 2008. The percentage ofwolves with mange-compatible lesionsregistered in camera-traps during 1 year correlated with the percentage ofred foxes withlesions in the previous year. This is the first large survey on sarcoptic mange in the Iberianwolf. Necropsy data, with alopecia as the main feature and a slighteffect on body condition,and trends derived from camera trapping coincidedin showinga ratherlow prevalenceandan apparently stable situation ofthe disease and its host, suggesting that this parasite is

currently not a major threat for this wolfpopulation. However, more informationis neededin order to assess the effect ofmange on aspects such as pup survival.

2011 Elsevier B.V. All rights reserved.

Corresponding author. Tel.: +34 926 295450; fax: +34 926 295451.E-mail addresses: [email protected], [email protected]

(. Oleaga).

1. Introduction

Sarcoptic mange is a parasitic skin infection causedby the burrowing mite Sarcoptes scabiei, whose antigenicmaterial deposited in tunnels dug in the epidermis causesintense irritation and an inflammatory response producing

0304-4017/$ see front matter 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.vetpar.2011.04.036
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256 . Oleaga et al. / Veterinary Parasitology 181 (2011) 255266

characteristic skin lesions. Such lesions are distinguishedby dermal inflammation, alopecia, hyperkeratosis and acharacteristic thickened, wrinkled and slate grey-colouredskin, leading in some cases to dehydration, emaciation andeventually death of the animal (Mrner and Christensson,1984).

The disease has been reported for more than 100 mam-mal species, including humans and domestic and wildmammals (Bornstein et al., 2001). It has been proposedthat the mite S. scabiei is a single highly variable specieswith different strains manifesting physiological specificityin different hosts (Pence et al., 1975; Arlian et al. , 1989,1996a), with this specificity as a subject of ongoing debate(Alasaad et al., 2011). Molecular studies carried out inrecent years support this hypothesis (Walton et al., 2004;Rasero et al., 2010).

Apart from the importance as a zoonotic disease andits economical relevance in domestic animals, sarcop-tic mange may be an important problem for isolated orsmall size populations (Henriksen et al. , 1993; Martinet al., 1998), and reaches epizootic proportions in cer-tain 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 least12 different species from all over the world described asaffected by sarcoptic mange (Bornstein et al., 2001; Deemet al., 2002). The epidemiology of the process seems to varybetween different areas and host populations, and bothendemic and epidemic situations have been reported inthe scientific literature concerning sarcoptic mange andwild 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 dis-ease 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 (Canislupus), mortality due to this parasite has usually been con-sidered a compensatory cause of death, usually relatedto lack of hair and cold temperatures (Pence et al., 1983;Pence andWindberg,1994). However, knowledge focussedon S. scabiei in grey wolves is scarce (Todd et al., 1981;

Jimenez et al., 2010), with most references mentioninga small number of individuals presenting dermal lesions,often without detailed clinical descriptions of the process(Wobeser, 1992; Jedrzejewska et al., 1996; Shelley andGehring, 2002; Lovari et al., 2007). Recently, Domnguezet al. (2008) reported the first confirmed mange cases inIberian wolves.

Sarcoptic mange can present two different pathologi-cal forms: the parakeratotic form, consistent with a typeI (immediate) hypersensitive response; and the alopecicform, consistent with a type IV (delayed) hypersensitiveresponse (Skerratt, 2003). Both forms of sarcoptic mangehave been recorded in red foxes in England (Bates, 2003).Clinical manifestations of sarcoptic mange vary dependingon individual variations in the duration and intensity ofthe hypersensitivity reaction and on the capacity of eachindividual host to limit parasite multiplication (Yager andScott, 1993).

The grey wolf distribution area includes Europe, Asiaand North America, with many populations fragmentedand reduced as a result of human persecution and habi-tat alteration (Boitani, 2003; Fernndez and Ruiz de Azua,2009). The largest population in Western Europe survivesin the Iberian Peninsula, where after the recovery experi-enced since 1970 the total wolf population was estimatedin more than 2000 wolves in 2008, with 322 packs occupy-ing a continuous area of 120,000 km2 in the northwest anda small isolated population in the south of Spain (lvareset al., 2005; Blanco et al., 2008).

The confirmation of sarcoptic mange by mite isolationcarried out by authors in 6 wolves submitted for necropsyin 2008 as part of the wildlife disease surveillance programof Asturias (North Spain) suggested the need of a specificstudy to evaluate the importance of a possible threat of thedisease for wolf population. Serological, histopathologicalandnecropsydatawerecomplementedwiththeretrospec-tive revision of wolf carcass pictures and epidemiologicalinformation obtained by camera trapping.

The objective in this study is to present the collectedinformation concerning sarcoptic mange and wolves inAsturias, analyse data reported in epidemiological surveil-lance programs carried out since 2003, evaluate theusefulness of different study methods and discuss the pos-sible causes and managementor conservation implicationsof mange in Iberian wolves.

2. Materials and methods

2.1. Study area

The Principality of Asturias (Spain) is a 10,603km2

autonomous region located on the North coast of theIberian Peninsula, with an abrupt orography that includesthe central part of the Cantabrian mountain chain and alti-tudes ranging from sea level to 2640m within only 40km.This region is included in the Eurosiberian climatic domin-ionof Atlantic type climate, with cold winters (minimum of6 months of potential frosts in non-coastal areas and tem-peratures ranging from 3 to 8 C to4 t o 0 C in the coldestmonths)andabundantprecipitations(14002100L/m2 peryear), with frequent snowfalls in the winter season in thehighest areas (LinesEscard, 1970). The predominant land-scape is a mixture of deciduous and mixed forests withpastures andmeadows for cattlefeeding that share this ter-ritorywithdifferentwildungulatespeciessuchasSouthernchamois (Rupicapra pyrenaica parva), roe deer (Capreoluscapreolus), red deer (Cervus elaphus), fallow deer (Damadama) and wild boar (Sus scrofa), and also with otherwildlife species such as the red fox, beech marten (Martesmartes), stone marten (Martes foina), badger (Meles meles),brownbear(Ursus arctos) andwolf,allofthemlistedamongthe hosts ofS. scabiei in Europe (Bornstein et al., 2001),in some cases even being diagnosed as suffering sarcop-tic 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 includesalmost the whole territory except the most populated Cen-tral region (Blanco et al., 2008) (Fig.1). The wolf populationshowedagradualincreaseinAsturiasfromthelatenineties


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. Oleaga et al. / Veterinary Parasitology 181 (2011) 255266 257

Fig. 1. Distribution in the three different geographic areas considered inAsturiasfor thiswork (Western, Central and Eastern)of wolves submittedfor necropsy in 2008: , 2009: and 2010: . Icon colours representmangy-confirmed wolves:, skin-injured wolves without miteisolation:

and wolves without lesionsor miteisolation:. Theicon representstheonly wolf without skin lesions that allowed mite isolation. Grey areascorrespond to the three council districts (Proaza, Belmonte and Somiedo)where the long-term camera-trapping study was carried out.

up to 2004, with an apparent maintenance of population

valuesinrecentyearswithanestimationofaveragedensityof 3.94.4 wolves/100 km2 in 2004 (Llaneza et al., 2004).

For the work presented here, Asturias was divided intothree different geographical areas: Western, Central andEastern Asturias, in order to group samples and study ani-mals for analysis.

2.2. Studied animals

A total number of 125 wolves were submitted fornecropsy between 2003 and March 2010 (Table 1), whichincluded animals collected in population control huntscar-ried out by wildlife officers (n = 95; the wolf isnot a huntedspecies in Asturias) and those found dead (vehicle collisionn = 12, illegal kill n = 5, poisoning n = 2 and infectious dis-ease n = 1). In 10 cases it was not possible to determine thecause of death.

Animals came from almost the whole distribution areaof wolves in Asturias, but they were not homogeneouslydistributed due to larger hunting effort carried out byofficers in some areas according to the established WolfManagement Plan followed in the region (Fig. 1). Basedon tooth wear (Gipson et al., 2000) three age classeswere determined for this work: pups (2 years). The studiedgroup was composed of 58 males (11 pups, 10 yearlingsand 37 adults) and 61 females (18 pups, 22 yearlings and21adults).In6casesitwasnotpossibletoregistercompletedata regarding sex and age due to bad preservation.

2.3. Samples and pathological study

From January 2003 to December 2007, 78 necropsieswere carried out in SERIDA (Regional Government Ani-mal Health Laboratory, located in Gijn-Asturias). Duringnecropsies photographs of the general aspect and partic-ular features of the animals studied were taken, sex, age,weight and different body measurements were registered,and whenever possible blood (taken by cardiac puncturefor serology), parasites and tissue samples were collected.

Fig. 2. Body scheme indicating the number of confirmed mangy wolvesshowing alopecia and miteisolation in the 8 different bodyregionsexam-inedduring the mitesearching protocol, and the percentage of total mitesisolated in each studied body region.

The above-describednecropsy procedure was improvedin order to get more data about the aetiology and char-acteristics of skin lesions observed since January 2008(Table 1). Thus, in the 47 animals submitted for necropsyfrom January 2008 to March 2010 location, extensionand characteristics of dermal and/or internal lesions wererecorded; KFI (kidney fat index) and adrenal glands weightwere noted (as body condition and stress degree indica-tors, Salak-Johnson and McGlone, 2007); eight 5 cm5 cmskin samples (from ears, head, neck, back, foreleg, rear leg,abdomen and tail, Fig. 2) were resected with a scalpel andadditional skin sections, lymph node pieces and any lesiondetected in other tissues were fixed in 10% buffered for-

malin, embedded in paraffin and sectioned at 4m forhistopathological studies whenever possible.

2.4. Laboratory techniques

2.4.1. Mite isolation/identificationBetween January 2008 and March 2010, the eight skin

samples taken from every wolf that arrived for necropsyless than 12 h after death (n =32, including 12 wolveswith mange-compatible lesions and 20 apparently healthyanimals, Table1) weresubmittedtoanestablishedectopar-asites search protocol. Briefly, the skin pieces were placedon Petri dishes and incubated at 37C for 24h in order tostimulate mites to leave the skin. Both Petri dishes and skinsamples were then meticulously examined for the pres-ence and identification of ectoparasites using an OlympusSZX9 (1057) magnifier. Detected mites were identifiedaccording to Wall and Shearer (1997), collected and pre-served in 70% ethanol.

2.4.2. Histopathological studySkin samples and prescapular lymph nodes from the

nine wolves with sarcoptic mange-compatible lesions andS. scabiei isolation were collected, fixed in 10% neutral-buffered formalin,anddehydratedthrough graded alcoholsand xylol before being embedded in paraffin wax. Severalserial sections, 4m thick, were cut from each sample and


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Table 1

Wolves subjected to necropsy during the study period.

Year Wolves studiedat necropsy

Wolves withmange-compatiblelesions at necropsy

Wolves submittedto mites searchingprotocol

Wolves with mange-compatiblelesions submitted to mitessearching protocol

Wolves withmange-compatible lesionsand mites isolation

20032007 78 0 0 0 02008 21 9 12 7 62009 10 3 5 2 1

2010 16 3 15 3 2Total 125 15 32 12 9

Number of wolves subjected to necropsy during the study period, including wolves with mange-compatible lesions, those subjected to the establishedmite searching protocol and those with skin lesions and confirmed sarcoptic mange by mite isolation. Data from 2003 to 2007 have been grouped due tothe 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 histologicalcharacterization of detected lesions.

From January 2008 to January 2009, 16 wolves (includ-ing 7 confirmed mangy animals, 4 skin-injured withoutmite isolation and 5 apparently healthy animals) weresub-

jected to immunohistochemical examination by means ofthe peroxidase anti-peroxidase (PAP) method (Sternberger

etal.,1970). Thesectionswere incubatedwith a rabbitanti-serumagainst the S. scabiei Ss20B3 antigen (Casaiset al.,2007) diluted 1 in 700. Preimmunization rabbit serum wasused 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 fornecropsy between2004 and 2010 (annual average of 12.6 samples, range719) were assessed using an in-house ELISA. This ELISAuses as antigen therecombinantprotein Ss20B3 andhasbeen previously used successfully for diagnosis of sarcop-tic mange in other wild species (Casais et al., 2007; Oleagaet al., 2008a;Falconi et al., 2010). Theabsorbanceat450nmwas measured in a microplate reader (Sigma, model 680).Results were expressed as a percentage of the relative opti-cal density (% relative OD450 nm), which was calculatedaccording to the following formula:

OD =ODunknown serum ODnegative serumODpositive serumODnegative serum

100

Serum samples were tested in duplicated, with con-trol sera being included in every series. The sera froma S. scabiei-infected wolf and a wolf without mange-compatible lesions or mite isolation were used as positiveand negative controls, respectively. The mean value fromthe % relative OD450nm of seven scabies-free animalswithout skin lesions plus three times the standard devia-tion(Bornstein andWallgren, 1997; Hollanders et al., 1997)was defined as the cut-off between positive and negativeserum samples, offering for this species a cut-off value of8.9%, indicating that animals with a relative OD450 nm per-centage of 8.9% or above were considered positive.

2.5. Revision of wolf carcass pictures

Due to the execution of necropsies by different techni-cians (belonging to ARENA S.L., SERIDA and IREC) duringthe study period and the lack of information concerningsarcoptic mange in Spanish wolves before 2008, revision of

pictures obtained from dead animals before that year wasused as a retrospective method for studying the presenceof mange-compatible lesions and to compare the obtaineddata with those coming from other study methods.

A total of 1195 photographs (958from2003to 2007 and237 from 2008) were taken and provided by ARENA S.L.biologists in order to evaluate the presence of skin lesions

indeadwolves.Thefirst958picturesrevisedincluded71ofthe 78 animals submitted for necropsy from 2003 to 2007(Table 1) and 10 additional wolves dead before 2008 wherenecropsy was not possible but the external general aspectwas recorded (average value = 16.2 animals/year). The ani-mals studied were 43 males and 50 females, including17 pups, 31 yearlings and 45 adult wolves. The above-mentioned pictures and results obtained in their revisionwere compared with 237 photographs and subsequentnecropsy and laboratory data of 15 animals studied in 2008(when the improved necropsy protocol for the diagnosis ofsarcoptic mange had already been established). This com-parison allowed us to evaluate thesuitabilityof themethod

to detect skin lesions and measure its success rate in cor-rectly detecting sarcoptic mange.

Pictures were obtained using both analogical and dig-ital cameras, recording external general aspect, particularfeatures of skin and different parts of the body and payingspecial attention to the presence of any kind of lesion oranomaly.

2.6. Camera-trapping data

The evaluation of camera trapping as a potential tool fortheepidemiological study of sarcoptic mange in the Iberianwolf was carried out using two different approaches:

(a) A long-term evaluation of the detection frequenciesof wild canids and the presence of mange-compatiblelesions in their skin was carried out taking advantageof a camera-trapping monitoring program establishedby FAPAS (fund for the protection of wild animals) forthe study of free-ranging brown bear populations inCentral Asturias.

Pictures from wolves and red foxes obtained bythe eight camera-traps (4 in Somiedo, 2 in Belmonteand 2 in Proazas council district, Central Asturias Fig. 1) maintained in the same location for at least 5consecutive years during the period 20032009 werecarefully revised in order to: (i) detect presence and


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characterize mange-compatible lesions in free-rangingwolves and red foxes; (ii) signal possible trends inthe frequency of the appearance of these lesions innature through time and compare these trends withthose reported at necropsy duringthe same period;and(iii) detect possible temporal and spatial agreementsbetween the presence and frequency (as number ofwild canid contacts per 100 camera-trap days of work)of both apparently healthy and skin-injured red foxesand wolves.

The eight camera traps used in this long-term evalua-tion were analogical Canon camera systems activatedby a heat and movement sensor (external Scanfer sen-sorconnectedfrom2003to2006,andaTrailscanCanonsystemwith incorporatedsensor since 2007, in allcasesusing flash as artificial light source). These cameraswere placed along natural animal paths through beechwoods(n =3),oakwoods(n =1),grazingland(n =1)andchestnut tree woods (n = 3), with an average altitudeof 845 m above sea level (ranging from 480 to 1520 ma.s.l.). Distances from the closest camera and to thenearest 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 toNovember (average value of 179.25 days working/yearthroughout the study period for the 8 cameras) due tothe bad meteorological conditions existing during thewinter season and the lower bear activity during thatperiod. Revision of cameras and picture collection wasdone with a periodicity of 1530 days.

The analysis of obtained pictures was carried outfor each camera, district and the whole study area. Allpictures of a single animal obtained during a 15-minperiod were considered and analysed as a single con-tact.

(b) In order to get all the information available regardingepidemiology of the process in particular wolf packs,the careful revision of all pictures obtained in thecamera-trapping long-term study signalled above wascompleted with photographsobtained close to carrionsor temporal locations since 2003 by FAPAS and thosegot by 6 additional digital camera traps (passive sys-tems Leaf River IR-3BU Leaf River Outdoor Products,Taylorsville, Mississippi, USA, with an infrared lightsource and an infrared movement and temperaturesensor) set up since January 2008 nearby to suspicioussarcoptic mange-affected pack reproduction areas.

2.7. Statistics

Statistical analysis was performed using the Epi Info3.5.1 and SPSS 15.0 software.

3. Results

3.1. Sarcoptic mange confirmation and distribution

Different degrees of alopecia or dermal lesion com-patible with sarcoptic mange were detected in 15 outof the 47 wolves submitted for necropsy from January

2008 to March 2010. From the 32 fresh submitted wolvesduring that period, S. scabiei was isolated from 9 out of 12 animals showing skin lesions and 1 out of 20 with-out skin alterations (Table 1 and Fig. 1). The presence ofmange-compatible lesions offered an apparent averageprevalence value of 31.91% (19.5247.25%, 95% confidenceinterval C.I.) for the period 20082010, reaching a peakvalue of 42.86% (22.5965.56%, 95% C.I.) in 2008. The realprevalence (wolves with lesions and confirmed S. scabieipresence)average value was 19.15% (9.6533.73%, 95% C.I.)from 2008 to 2010, and 28.57% (12.1952.31%, 95% C.I.) in2008. No data regarding this kind of skin alterations weresignalled in necropsies of animals carried out before 2008(n =78).

Sarcoptic mange was confirmed in the three consid-ered geographical areas of Asturias Eastern, Central andWestern, with 3 (out of 13), 5 (n = 23) and 1 (n =11)wolveswith confirmed mange by isolationof mites,respec-tively, since January 2008 (Fig. 1). The Chi square testrevealed no sex-related difference in the probability toshow mange-compatible lesions or even harbour S. sca-biei. Nevertheless, yearling wolves studied accordingto thenecropsy protocol established in 2008 (n =11) showed astatisticallysignificant lowerprobability to present mange-compatible lesions than pups (n = 11) or adults (n =21)(2 =8.353,p = 0.015), offering apparent prevalence val-ues of 0%, 54.54% and 42.86%, respectively. While monthslike April, May and November had 2 mangy-confirmedwolves (this happened at least in one of the study years),none of the 15 wolves with sarcoptic mange or compati-ble lesions recorded at necropsy was collected in March,August, September or October.

3.2. Clinical characterization

The number of mites isolated in confirmed parasitizedwolves ranged from 1 to 78 per animal. With the excep-tion of two wolves (with only 1 and 2 detected mites), allanimals with lesions and S. scabiei isolation showed mitepresence in at least 2 of the 8 studied skin areas (ears, head,neck, back, foreleg, rear leg, abdomen andtail), with a max-imum of 7 of the 8 body areas tested with acari detection inone single animal. Skin pieces from tail, ears and rear legsshowed an overall higher number of live mites detected(55, 57 and 47, respectively), and were also the body areaswhere more wolves (4, 6 and 6, respectively) allowed S.scabiei identification during examination (Fig. 2). 28.35%of detected mites were isolated in areas without dermallesions.

The most frequent and conspicuous detected macro-scopical skin alteration was alopecia, present in the 9mangy-confirmed animals in different degrees (from 10%to 90% injured skin surface); rear leg, abdomen, ears andforeleg werethe most frequently alopecic areas (Figs. 2 and3a, b and d).

The relationship between the percentage of alopecicskin and the number of isolated mites for each con-firmed mangy wolf presented a linear distribution (Fig. 4),with a statistically significant decrease in the number ofalive mites as the percentage of affected skin increases(r=0.7818;p


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Fig. 3. Macroscopical and microscopical pictures showing characteristic lesions detected in confirmed mangy wolves at necropsy. The most frequently

affected skin areas were abdomen (a), rear legs (b), base of tail (c)and ears (d). Cracking andthickened skin wasinfrequent, very localized when present (band c). (e) The most frequently reported microscopical alterations were focal hyperkeratosis (A), hyperplasic epidermis (B) and inflammatory infiltrate inthe dermis(C). Hypertrophyof the sebaceousglands (D)and hair follicles clogged with keratin (E)can be also observed (HE= 400, bar= 200m). (f) Skin.Positive immunostaining in macrophages (arrows)located in the dermis (PAP= 200, bar= 100m). (g) Prescapular lymph node. Positive immunostainingin macrophages (arrows) (PAP= 400, bar=50m).

Both hyperpigmentation and hyperkeratosis weredetected in 4 confirmed mangy wolves, although especiallyin the case of hyperkeratosis reported alterations were ofa small size and thickness (Fig. 3b and c). Three of the fouranimals showing hyperkeratosis were those with the high-est percentage of affected skin surface (90, 80 and 70%,respectively). No recent ulcers or wounds were detected.

Histologically, an alopecic (delayed) type IV hypersen-sitive response reaction was observed in the nine wolveswith confirmed sarcoptic mange. Skin samples from thealopecic areas demonstrated an inflammatory infiltrateconsistent with lymphocytes, macrophages and few neu-trophils in the dermis (Fig. 3e and f). Occasionally, the

Fig. 4. Regression analysis carried out considering the percent-age of affected skin versus the number of isolated mites in the

nine confirmed mangy wolves (r2

=0.6112; r=0.7818, p =0.0128;y =55.53919330.765068493x).

stratum corneum showed focal parakeratosis and hyperk-eratosis,and the epidermiswas hyperplasic. The sebaceousglands were hypertrophied and some of the hair folliclesclogged with keratin (Fig. 3e). Scab formation was notobserved and mites could only be detected in one case.

Lymphocytes andmacrophages, located both in theder-mis (Fig. 3f) and in the prescapular lymph nodes (Fig. 3g),

showed positive immunostaining against S. scabiei anti-gen in 4 of the 7 wolves with confirmed sarcoptic mangesubjected to immunohistochemical examination. Therewas no statistical relationship between the appearance ofimmunostaining and the percentage of affected skin orthe number of isolated mites in these animals. None ofthe 4 wolves with mange-compatible lesions but withoutmite isolation showed positive immunostaining and only1 of the 5 apparently healthy animals showed positiveimmunostaining, located only in the prescapular lymphnode. The preimmune serum controls showed no staining.

With the exception of one wolf with mange-compatiblelesions and S. scabiei isolation where histological analysis

suggested co-infection by a virus, no other pathologicalalteration was detected in studied animals besides thoseattributed to sarcoptic mange.

The KFI gave a statistically significant (t=3.035,p


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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-compatiblelesions at necropsy, (iii) percentage of wolves with isolated mites con-firming sarcoptic mange at necropsy, (iv)percentage of dead wolves withmange-compatible lesions in pictures and (v) percentage of wolves withmange-compatible lesions in camera-trapping pictures. (ii), (iii), (iv) and(v) percentages are in agreement with the apparent morbidity increasereported 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 by75.0% sensitivity and 87.5% specificity.

The analysis of 88 sera samples obtained from 2004to 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 OD450nm ofthe ELISA positive wolves were below 27.3%, indicating aweak antibody response.

The first seropositive animal was detected in 2004(first year with collected blood samples), with seropos-itive wolves detected during the whole study period

(Fig. 5). ELISA positive animals appeared equally dis-tributed between both sexes, with 20.93% (10.0436.05,95% C.I.) and 21.43% (10.2936.82, 95% C.I.) seropos-itive males and females, respectively. Regarding agegroups, 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 seropreva-lence was not statistically significant.

The Western area, with a 35% seropositivity(15.3959.22%, 95% C.I.), showed the highest sero-prevalence 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 significant.The first half of the year showed a statistically significant(2 =10.159, p = 0.017) higher seroprevalence value, with16 out of 18 seropositive wolves sampled between Januaryand June.

3.4. Photographic studies

The revision of pictures of dead wolves obtained from2003 to 2007 (n = 958 pictures), focussed on the detectionof skin lesions andalterationssimilar to those described forconfirmed mangy wolves since 2008, permitted the detec-tion of six animals showing mange-compatible lesions. The

first suspicious animal was detected in 2003, and sincethen, wolves showing mange-compatible skin alterationswere 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 mange-compatible 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 affectedanimals were 2 males and 4 females, including 1 pup, 2yearlings and 3 adults.

From the 15 animals photographed by ARENA S.L.biolo-gists in 2008 whose pictures were revised as an evaluationof this study method, mange-compatible lesions weredetected in 6 (40%) wolves, from which sarcoptic mangewas confirmed by the subsequent mite searching proto-col in 4 cases, indicating the suitability of the evaluationmethod used.

Since the beginning of the camera-trapping long-termstudy (2003), an 8783 camera-trap/day monitoring effortwas carried out. A total of 832 and 184 contacts with redfoxes and wolves, respectively, were registered, including20 contacts with red foxes and 22 with wolves showingmange-compatible lesions. Both canid species were regis-tered in all 8 camera traps used in this study, with averagevalues of 16.50 (ranging from 0.29 to 48.1) and 3.69 (from0.14 to 8.8) contacts/year/camera for red foxes and wolves,respectively.

The first pictures showing mange-compatible lesionsin red fox and wolf were obtained in 2003 in Belmonteand Somiedo, respectively. Since then, both wolves andred foxes with cutaneous alterations were photographedin 2006, 2007 and 2008 (Figs. 6 and 7). Mange-compatiblelesions were detected in the three camera-trapping stud-ied areas in both species with the exception of Belmonte(Fig. 1), where no apparently affected wolves were reg-istered (in this area a wolf with dermal lesions wasphotographed in a camera trap placed close to a carrion,thus notbeingincluded in this monitoring program). In onesingle camera-trap (from Somiedo) no canid with mange-compatible lesions was photographed.

The analysis of recorded data both for each individ-ual camera trap and for the group of cameras allocatedin each area (Somiedo n = 4, Belmonte n =2 and Proazan =2) showed a different evolution for each camera andeven area, with a great annual variation in detection ofred foxes not observed for wolves (expressed as the num-ber of contacts registered in 100 camera trap days), andoften single or very scarce data regarding the detection ofmange-compatible lesions, preventing trend descriptionsand statistical analysis on this scale.

Nevertheless, the study of camera-trapping collecteddata from the 8 cameras grouped as a whole provided dataenough to attempt analysis, which agreed to a large extentwith results obtained by the other study techniquesused inthis work (Figs. 5 and 7). A gradual increase in the percent-age of skin-injured red foxes and consecutive decreases inthenumber of red fox-contacts/100 camera-trap days weredetected, reaching the lowest detection rate and the high-est percentage of red foxes with mange-compatible lesionsin 2007, suggesting a possible effect of the disease on thepresence or density of this species in the studied area.


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Fig. 6. Example of pictures obtainedby the camera-trapping technique in the present workshowingdifferent degrees of mange-compatible lesions in wildwolves. 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 (reach-ing 27.7% of registered wolves) in the percentage of wolves photographed with mange-compatible lesions wasobserved in 2008, whereas the number of wolf-contactsdetected for 100 camera-trap days as a measure of detec-tion showed very small variations during the study period(Fig. 7).

The statistical analysis of obtained pictures, althoughcarried out on a limited number of years and con-tacts, showed a close statistically significant relationshipbetween the percentage of skin-injured wolves registeredin camera traps during 1 year and the percentage of redfoxes with mange-compatible lesions registered in the pre-vious year (Rs= 0.9856,p


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status, data compiled herein by a combination of patho-logical and retrospective studies (including serologicaland photographic techniques) allowed valuable insightsinto the epidemiology of this parasitehost relationship.Mange was confirmed by isolation ofS. scabiei mites fromwolves necropsied between 2008 and 2010. Moreover, vis-ible lesions recorded on photographs and serum antibodydetection by ELISA coincide, suggesting that the diseasewas already present at least since 2003. Lesion distribu-tion was similar to those described in previous reports inwolves (Todd et al., 1981; Domnguez et al., 2008), and inother canids such as the red fox and coyote (Trainer andHale, 1969; Pence et al., 1983; Mrner, 1992). However,parakeratotic lesions were infrequently reported in thisstudy.

Regarding the ELISA, the low number of mites usuallydetected on infected wolves may have limited the sensi-tivity of this antibody test. The mean antibody prevalencewas similar to the 21% reported in a Scandinavian study,where 86 wolves collected from 1998 to 2002 included 6individuals that died of sarcoptic mange (Olsen, 2003). Thelack of sex, age or site differences in antibody prevalencesuggests a widespread contact with the parasite.

Visualizing the mite by isolation or histology is the onlydefinitive 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 of20 apparently healthy wolves (with no visible lesions),whereas they were isolated in 9 of 12 animals with dermallesions and were also immunohistochemically detected in4 of the 7 mangy animals studied at this level. This, alongwith an apparent sarcoptic mange/viral co-infection in onemale as the only pathological process different of sarcopticmange detected by histology or other applied techniquesin the frame of the wildlife disease surveillance program instudied wolves, signals mange as the cause of the lesionsobserved in these animals.

Live mites were detected in only 75% of the properlytested suspect wolves, and usually in a low number. Thisagrees with data on coyote and other studies on sarcop-tic mange in wolves (Todd et al., 1981; Domnguez et al.,2008), and coincides also with low isolation rates obtainedby skin scraping in mange-suspect dogs (Canis famil-iaris) (Bornstein, 1991; Hill and Steinberg, 1993; Griffin,1993; Carlotti and Bensignor, 1997; Beck and Hiepe, 1998;Davidson et al., 2008). These isolation numbers contrastwith those reported for the red fox (up to 5000 mites percm2, Little et al., 1998a). Similarly, the inverse relationshipobserved between the number of mites and the size of theaffected skin (Fig. 4) contrasts with findings in foxes (andwild ruminants, data not shown), suggesting a differentevolution of the process and an apparent greater ability ofwolves to control the number of alive mites as comparedto most of the other susceptible natural S. scabiei hosts inthe study area (different immune responses and efficacyagainst the mite have already been signalled for dog andred fox: Arlian et al., 1996b; Little et al., 1998a). However,caution is required in using the number of isolated mitesas a proxy for parasite burden (Alasaad et al., 2009).

Most wolf lesions were histologically confirmed asalopecic, opposed to the frequent parakeratotic form (type

I immediate hypersensitivity response) observed in redfoxes 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 alopecicformreportedinAsturianwolves,atypeIVhypersensitivityresponse, is also the most common presentation of sarcop-ticmangeindogs(Patersonetal.,1995), butrarelyreportedin the fox, where this presentation has been describedas characterized by low mite burdens, few (if any) crustylesions anda limited effecton body condition(Bates,2003).

Experimental studies have shown no significant KFIvalue differences between mangy and healthy dogs (Arlianet al., 1995; Little et al., 1998a), whereas coyotes andwolves tend to show limited effects of mange on bodycondition (Todd et al., 1981; Pence et al., 1983; Pence andWindberg, 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 withmange-compatible lesions showed a 50% lower KFI valuethan those without visible lesions, but never appearedemaciated or with an extremely poor body condition atnecropsy. These findings coincide with those signalled inthe few cases of red foxes reported by Bates (2003) withthealopecicpresentation of sarcopticmange, andare lowerthan the 85% difference observed for foxes with paraker-atotic mange lesions (Little et al., 1998a; Newman et al.,2002). Nevertheless, the detection of both parakeratoticand alopecic forms in a red fox population with differenteffects on body condition (Bates, 2003) confirms the exis-tence of individual differences in the response to the mite,warning us about the possible presence of different diseasepresentation forms in Asturian wolves not yet reported atnecropsy. In this sense, the authors were able to observein the field in 2010 one wolf with extremely poor bodycondition, abnormal behaviour and severe parakeratotic-mange-compatible lesions, frequent in red foxes, but notregistered in Asturian wolves at necropsy up to date.

Regarding the time trends, both the photographicrecords and the necropsy data showed an increased mor-bidity in 2008. Possible explanations for this increaseinclude changes in mite pathogenicity (Pence andWindberg, 1994), density dependence in transmissionrates (Pence et al., 1983; Pence and Windberg, 1994), co-infections or other debilitating factors (Lloyd, 1980) anda higher mange circulation among sympatric carnivores(Trainer and Hale, 1969; Todd et al., 1981; Clayton, 2003;Mrner et al., 2005; Soulsbury et al., 2007; Domnguezet al., 2008). Mite pathogenicity variation is difficult totest in the field. On the subject of density dependence,there are no indications of an increase in wolf numbersduring the study period in Asturias: after a moderateincrease observed from 1998 to 2004, an apparent sta-bilization in wolf density was reported from 2005 to2008 (Llaneza et al., 2004; Garca and Llaneza, 2009).Regarding co-infections, it is known that Iberian wolvesare often exposed to disease agents including canine dis-temper virus, canine parvovirus and Leishmania infantum(Sobrino et al., 2008a,b); the histopathological detectionof a wolf apparently co-infected by S. scabiei and a viralagent emphasizes the need of further studies about the rolethat concomitant agents can play in the development of


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sarcoptic mange and skin lesions, with ongoing research inthe studied population by the authors. Finally, the statisti-cally significant relationship signalled between the trendsin mange-compatible lesions detectionfor red fox and wolfwith1-yeardelay(Fig.7) stronglysuggestssarcopticmangein the red fox as the most likely origin of the observed timetrend in wolf disease. Interspecific transmission ofS. sca-biei has been observed between the red fox and coyote(Trainer and Hale, 1969), the red fox and dog (Soulsburyet al., 2007; Clayton, 2003) andcoyoteandwolf(Toddetal.,1981; Jimenez et al., 2010), also with 1-year difference inthe morbidity peak of mange reported between the coy-ote and wolf by Todd et al. (1981). Mrner et al. (2005)and Domnguez et al. (2008) indicate that the red fox isthe most probable origin of sarcoptic mange in wolf. Thepossible role of sympatric ungulates confirmed as S. sca-biei hosts in Asturias (Fernndez-Moran et al., 1997;Oleagaet al., 2008a,b) in the epidemiology of sarcoptic mange inIberian wolf is still unclear.

The high percentage of macroscopically affected skin inconfirmed mangy wolves at necropsy suggested the use-fulness of photographic techniques for the detection ofsarcopticmange lesions in theIberian wolf, which wascon-firmed by data on pictures taken by technicians in 2008from dead wolves subsequently studied at necropsy.

Remote photography methods have been increasinglyused in recent years to address a great variety of ques-tions in wildlife biology (Karath and Nichols, 1998; Cutlerand Swann, 1999; Carbone et al., 2001; Grassman et al.,2006; Lucherini et al., 2009), whereas their use in wildlifesanitary studies is infrequent, usually related to animalbehaviour or biological estimations with epidemiologicalinterest (Hegglin et al., 2004; VerCauteren et al., 2007;

Jennelle et al., 2009). Even with the limited number ofcameras available and contacts obtained (that preventedweighty conclusions), this camera-trapping technique hasbeen shown to be useful for the detection of animals withmange-compatible lesions in the field, providing interest-ing information about the presence of injured animals atdifferent times of year and in different areas and interest-ingdata regarding population trendsand the epidemiologyof thedisease (confirming in thefield the apparent increasein morbidityreported for sarcoptic mange at necropsy dur-ing 2008, Figs. 5 and 7). To the best of our knowledge, thisis the first time photo-trapping has been used to studydisease-compatible lesions and time trends in a wildlifedisease.

The combination of these field techniques withthe necropsies and laboratory results allowed interest-ing insights of mange epidemiology in Iberian wolves.Necropsy data (with skin alteration as the only detectedlesion related to S. scabiei and a limited effect on bodycondition) and reported trends (showing a rather lowprevalence and an apparently stable distribution of the dis-ease and its host) suggest that this parasite is not a majorthreat for wolf populations in Asturias.

However, the possible lack of data and incompleteoverview of the disease in such a scarce species emphasizethe need of more information in order to evaluate its pos-sible effect on fertility and pup survival rates, where theeffect 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 effecton the health of adult wolves. Hence, surveillance shouldbe maintained and, eventually, more information gatheredthrough larger surveys (increasing the number of avail-able cameras; including wolf tagging with epidemiologicalsurveillance purposes) in order to better understand andcharacterize sarcoptic mange in the Iberian wolf and assessthe epidemiology of this parasitic disease and its effect onwild canids population trends and wolf conservation.

Acknowledgements

This is a contribution to the agreement between CSICand Principado de Asturias. We thank the rangers of thegame preserves (specially Jaime Marcos Beltrn, FranciscoAlonso Mier, Fernando Rodrguez Prez, Pedro Gonzlezand Angel Nuno) for their help in carcasses submissionand camera-trapping, our colleagues from ARENA S.L. andIREC (Emilio Jos Garca, scar Rodrguezand MaraSurez)for their assistance in field, necropsy and laboratory work,and other workmates (Jose Luis Garca Daz and RafaelAlba Zarabozo) for their advice and field collaborationin camera-trapping work. We wish to thank also KevinDalton for revising the English of the manuscript, PelayoAcevedo for his contribution in statistical and geograph-ical analysis and Joaqun Vicente for his advice in thestudy design and camera trapping procedures. ProjectsRTA 2009-00114-00-00 (INIA) and CIT-060000-2009-0034(MICINN) contributed to this study.

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