Eur J Wildl Res (2012) 58:9971000 DOI 10.

1007/s10344-012-0662-y

SHORT COMMUNICATION

Comparative pathological and immunohistochemical features of sarcoptic mange in five sympatric wildlife species in Northern Spain
A. Oleaga & R. Casais & J. M. Prieto & C. Gortzar & A. Balseiro

Received: 11 June 2012 / Revised: 24 August 2012 / Accepted: 27 August 2012 / Published online: 20 September 2012 # Springer-Verlag 2012

Abstract This study collects pathological information concerning sarcoptic mange in sympatric red deer, roe deer, chamois, wolf and red fox from Northern Spain, and discusses reported variations in the response from the different species against Sarcoptes scabiei. Wolf is the only studied species which shows prevalence of the hypersensitivity (alopecic) response to the detriment of the hyperkeratotic form, usually observed in ungulates and fox. The present work shows the use of immunohistochemistry as a valuable tool for the study and diagnosis of sarcoptic mange in those species whose immune response or stage of infestation limits the number of detectable mites, not an unusual difficulty faced when working with sarcoptic mange in wildlife species. Keywords Sarcoptic mange . Sarcoptes scabiei . Comparative histopathology . Wildlife . Immunohistochemistry

Introduction Sarcoptic mange is a common, widespread, highly contagious skin disease of mammals caused by the mite Sarcoptes scabiei. This parasitosis has been reported in many species of wild mammals worldwide (Pence and Ueckerman 2002). In Spain, epizootic sarcoptic mange has been described in Cantabrian chamois and Spanish ibex with high mortality rates and population declines during the first years after appearance (Fernndez-Morn et al. 1997; Len Vizcaino et al. 1999), whereas an enzootic status of the disease has been reported in red foxes (Gortzar et al. 1998) and little information is still available regarding the effects of this parasite on wolf populations (Oleaga et al. 2011). The epidemiology and pathology of sarcoptic mange in wildlife populations seems to differ between different areas of the world and animal species (Pence and Ueckerman 2002), with two pathological forms described: a parakeratotic form, consistent with a type I (immediate) hypersensitive response and an alopecic form, consistent with a type IV (delayed) hypersensitive response (Bates 2003; Skerrat 2003). The aim of this study is to collect pathological information concerning sarcoptic mange lesions in different sympatric wild species and to discuss their possible variation in the response against S. scabiei.

Communicated by C. Gortzar A. Oleaga SERPA, Sociedad de Servicios del Principado de Asturias S.A., 33203 Gijn, Asturias, Spain R. Casais : J. M. Prieto : A. Balseiro (*) SERIDA, Centro de Biotecnologa Animal, Camino de Rioseco, 1225, La Olla, Deva, 33394 Gijn, Asturias, Spain e-mail: abalseiro@serida.org C. Gortzar IREC (CSICUCLMJCCM), Ronda de Toledo s/n., 13071 Ciudad Real, Spain

Material and methods From 2003 to 2010, 22 red deer (Cervus elaphus), 10 chamois (Rupycapra pyrenaica parva), 9 wolves (Canis lupus), 8 red foxes (Vulpes vulpes) and 2 roe deer (Capreolus capreolus) with sarcoptic mange compatible lesions and mite isolation (see Alasaad et al. 2011) were necropsied in the frame of the wildlife disease surveillance programme of Asturias

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(Northern Spain). Animals were collected in population control hunts by wildlife officers or found dead. Macroscopic lesions were recorded and for histopathological studies skin samples from wolves (n 0 9), foxes (n 0 8), red deer (n 0 6), chamois (n 0 3) and roe deer (n 0 2) were collected and submitted to standard histological procedures. Several serial sections, 4 m thick were cut from each sample and stained with hematoxylin and eosin (HE). Immunohistochemical examination was performed by means of the peroxidase antiperoxidase (PAP) method and the sections incubated with a rabbit antiserum against the S. scaibei Ss20B3 antigen (Casais et al. 2007) diluted 1 in 700. Preimmunization rabbit serum was used as negative control. Positive and negative control tissues were also included.

Results Macroscopically, lesions in the ungulate species (red deer, roe deer and chamois) consisted of crusty skin lesions with abundant mites presented during isolation procedures. The location and progression of detected lesions agreed on the whole with those described for another ungulate affected by sarcoptic mange as the Spanish ibex (Len Vizcaino et al. 1999). In wild canids, two different presentations were reported: the most frequently detected in red fox included crusty lesions leading to an extremely thickened and fissured skin, especially in the back, head, rear legs and base of tail. As reported in affected ungulates, red foxes with this kind of lesions showed an extremely poor body condition and a large number of mites in studied skin pieces. The

second sarcoptic mange presentation detected in two red foxes but representing the only form reported in the nine studied wolves, included alopecia as the main lesion, mildly thickened skin often with a slate-grey colour in affected areas and almost complete absence of crusts or fissuring. This alopecic presentation hardly allowed isolation of S. scabiei, while seemed to affect body condition less than the parakeratotic form described above. Thus, while red fox, red deer, roe deer and chamois often succumb to the disease in the study area, mangy wolves have not been found in such poor body condition. Microscopically, the histological study revealed ungulates and foxes exhibiting a crusting dermatitis characterised by hyperkeratosis, acanthosis, severe degeneration of epidermal cells, presence of large number of mites and an inflammatory infiltrate consisting mainly of lymphocytes, macrophages and eosinophils (Table 1, Fig. 1). The predominant alteration in wolves was an alopecic hypersensitive reaction with almost complete lack of mites and the presence of an infiltrate consisting of macrophages, lymphocytes and few neutrophils (Table 1, Fig. 1). In two foxes, both hyperkeratotic and alopecic forms were observed (Table 1, Fig. 1). Sebaceous gland hyperplasia and hair follicles clogged with keratin were observed in all studied species. Positive immunostaining was detected in 5/9 wolves, 8/ 8 foxes, 6/6 red deer, 3/3 chamois and 2/2 roe deer. This reaction was noted in the integument of the epidermis and in the cavities surrounding vital organs of the mites and in the cells forming the inflammatory infiltrate (Table 1, Fig. 1). Interestingly, the positive immunolabelling in ungulates and foxes showing the hyperkeratotic form was predominantly

Table 1 Comparative histopathological and immunohistochemical features in five sympatric wildlife species affected by sarcoptic mange Lesions/features Red deer (Cervus elaphus) Yes Yes Yes Low Numerous Chamois (Rupycapra Roe deer pyrenaica parva) (Capreolus capreolus) Yes Yes Yes Low Numerous Yes Yes Yes Low Numerous Wolf (Canis lupus) Red fox (Vulpes vulpes)

Hyperplasia epidermis Hyperplasia of sebaceous glands Hyperkeratotic form Alopecic form Abundance of mites

Yes Yes No Yes Scarce/abscence

Yes Yes Yes Yes Hyperkeratotic form: Numerous Alopecic form: scarce Hyperkeratotic form: macrophages, lymphocytes and eosinophils Alopecic form: macrophages, lymphocytes and neutrophils Hyperkeratotic form: positivity mainly in mites Alopecic form: positivity in macrophages Yes

Inflammatory infltrate Macrophages, plasma cells, lymphocytes, histiocytes and Macrophages, eosinophils lymphocytes and neutrophils Immunohistochemistry Positivity mainly in mites Positivity in macrophages in 5 wolves Yes No

Mortality

Yes

Yes

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noted in mites, whereas wolves and foxes affected by the hypersensitivity (alopecic) form (virtually without mites in skin sections) showed a strong positive immunolabelling mainly located in macrophages in the dermis (see Fig. 1). This feature seems to reflect the important role that macrophages can play in the apparently more effective alopecic form against S. scabiei developed by wolves in the study area.

Discussion The scarce number of mites detected in wolves and red foxes with the hypersensitivity (alopecic) form contrasts

with the numerous mites observed in ungulates and red fox (hyperkeratotic form). As the hypersensitivity reaction progresses mites decrease in the lesions (Arlian 1996). Numbers of eosinophils in the dermis are correlated with the density of mites, suggesting that recruitment of eosinophils is influenced by mites or their products (Skerrat 2003). In animals showing the alopecic form, neutrophils were observed forming the inflammatory infiltrate. Although neutrophils can be related to secondary infections or skin damage, the oxidation burst of neutrophils may be important in eliminating mites (Arlian 1996). Although not all mangy confirmed wolves offered immunohistochemical positive result, the extremely low number of live mites detected in studied mangy wolves (ranging from 1 to 78 mites, Oleaga

Fig. 1 Comparative analysis of pathological features of the skin from wildlife species using hematoxylineosin stain (HE) and immunohistochemistry (PAP). a Red deer showing sarcoptic mange in neck, legs and ventral surface of the body. b Severe hyperkeratosis and presence of mites. HE; bar 0 100 m. c Inflammatory infiltrate consisted of macrophages, lymphocytes and eosinophils. HE; bar 0 100 m. d Mites in stratum corneous with intense immunolabelling. PAP; bar 0 100 m. e Chamois showing sarcoptic mange. f Hyperkeratosis, acanthosis and numerous mites in the epidermis. HE; bar 0 100 m. g Inflammatory infiltrate in the dermis consisted of macrophages, lymphocytes and eosinophils. HE; bar 0 100 m. h Positive immunolabelling against S. scabiei in mites. PAP; bar 0 100 m. i Roe deer showing sarcoptic mange. j Skin showing hyperplasia in the epidermis, one mite and an inflammatory infiltrate in the dermis. HE; bar 0 100 m. k

Inflammatory infiltrate in the dermis consisted of macrophages, lymphocytes and eosinophils. HE; bar 0 100 m. l Positive immunolabelling in a mite. PAP; bar 0 100 m. m Wolf with sarcoptic mange showing alopecic areas in the body. n Alopecic form of sarcoptic mange. A mite can be seen located in the stratum corneous. HE; bar 0 100 m. o Inflammatory infiltrate in the dermis consisted of macrophages, lymphocytes and neutrophils. HE; bar 0 100 m. p Positive immunolabelling in macrophages in the dermis. PAP; bar 0 100 m. q Red fox showing sarcoptic mange. r Hyperkeratotic form of sarcoptic mange in the fox. HE; bar 0 100 m. s Alopecic form of sarcoptic mange in the fox. HE; bar 0 100 m. t Alopecic form of sarcoptic mange in the fox. Positive immunolabelling in macrophages in the dermis. PAP; bar 0 100 m

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Eur J Wildl Res (2012) 58:9971000 recipient of a Contrato de Investigacin para Doctores from the Instituto Nacional de Investigacin Agraria y Agroalimentaria (INIA). This project is supported by INIA RTA2008-00041-00-00 and is a contribution to the agreement between CSIC and Principado de Asturias. Conflict of interest None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.

et al. 2011) confers immunohistochemistry a special value as survey and diagnostic technique of sarcoptic mange in this species. When the low number of mites prevents their detection in lesions or when samples cannot be immediately submitted for live mites isolation, and histopathology cannot be used as a reliable marker of infestation, immunohistochemistry has proven to be a useful tool in order to discern sarcoptic mange from other pathologies. Both macroscopical and microscopical skin lesions in wolves (at least in the nine wolves surveyed in the present study) suggest a prevalence of the hypersensitivity (alopecic) response to the detriment of the hyperkeratotic form against sarcoptic mange in this wild canid. The coincidence in wolves of this presentation with the apparent higher ability of the species to control the parasite (Oleaga et al. 2011), not reported in the rest of species surveyed in the present work, could indicate that this form of immune response is a more effective defence mechanism against S. scabiei. The intimate immunological mechanisms developed by wolf should be better studied and compared with species taxonomically close like red fox, where the same mite seems to trigger different pathological features and population effects. A correct interpretation of observed pathological findings of sarcoptic mange and an appropriate understanding of the immune response mechanisms and their effectiveness are key pieces in surveillance programmes dealing with wildlife species and also in their populations' management. The present work shows the use of immunohistochemistry as a valuable tool for the study and diagnosis of sarcoptic mange in those species whose immune response or stage of infestation limits the number of detectable mites, not an unusual finding when working with sarcoptic mange in wildlife species.
Acknowledgments The authors thank Cotos de Caza, Veterinary Services of the Seccin de Caza de la Consejera de Medio Ambiente del Principado de Asturias, and our colleagues of IREC Oscar Rodrguez and Mara Surez for helping with the sample collection. Dr. Marta Muoz is thanked for critically reviewing the manuscript. Dr. Ana Balseiro is

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