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1 Department of Basic Veterinary Sciences, Faculty of Veterinary Medicine, PO Box 66, FIN-00014 University of Helsinki, Finland;
2 Department of Pathology, National Veterinary and Food Research Institute, PO Box 45, FIN-00580 Helsinki, Finland;
4 Kuopio Department, National Veterinary and Food Research Institute, Neulamäentie 4, FIN-70210 Kuopio, Finland;
5 Oulu Regional Unit, National Veterinary and Food Research Institute, PO Box 517, FIN-90101 Oulu, Finland
6 Corresponding author (email: seppo.saari{at}helsinki.fi)
ABSTRACT:
Contagious mucocutaneous dermatitis is a frequently encountered disease of mountain hares (Lepidus timidus) in Finland. We describe the histopathologic changes and propose an etiologic cause for this disorder. Fifty-three cases collected during 1982–2000 were examined histologically. Transmission electron microscopy was performed in one case. In fully developed lesions, keratinocytes in epidermis and follicular infundibula were swollen and contained large eosinophilic intracytoplasmic inclusion bodies with marked reticular and ballooning degeneration. In later stages, there was marked necrosis and ulceration with severe pyogranulomatous and suppurative inflammation. At this stage, no viral inclusions were detectable, but secondary Staphylococcus warnerii infection was present in most cases. In late lesions, there was dermal fibrosis with epidermal hyperplasia. No spiral-shaped bacteria suggesting treponematosis were detected at any stage. Ultrastructurally, swollen epidermal and follicle infundibular cells contained round intracytoplasmic inclusion bodies with a myriad of virions typical of poxvirus with a biconcave nucleocapsid core, two lateral bodies, and a clearly discernible outer lipoprotein capsule. The findings suggest that contagious mucocutaneous dermatitis in mountain hares is a viral disease caused by a poxvirus. The disease is often complicated by secondary bacterial infection, most commonly S. warneri.
Key words: Mountain hare, mucocutaneous dermatitis, poxvirus, Staphylococcus warnerii, ulcerative dermatitis.
Contagious mucocutaneous dermatitis of mountain hares (Lepidus timidus) in Finland is characterized by proliferative lesions with marked crust formation at mucocutaneous junctions. The lesions are most often present on the lips and around the nose (Fig. 1
). Prepuce, vulva, conjunctiva, and sometimes skin of extremities may be affected as well. Diagnosis has been based on the presence of typical macroscopic lesions but etiology has remained unsolved. Viruses, bacteria, and even parasites have been suggested as the causative agent. Clinically, similar lesions have been reported in rabbits (Oryctolagus cuniculus) and European brown hares (Lepus europeaus) with bacterial syphilis, caused by Treponema paraluiscuniculi and T. paraluisleporis. Based on these reports, wildlife pathologists have suggested that the disease in mountain hares is also caused by Treponema spp. In this retrospective study, we evaluated 53 cases of contagious mucocutaneous dermatitis in mountain hares examined during 1982–2000 in the laboratories of National Veterinary and Food Research Institute in Helsinki, Kuopio, and Oulu, Finland. We describe the histopathologic changes and bacteriologic findings and propose an etiologic agent and a new name for this disease.
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Transmission electron microscopy was used in one case. Several small samples from formalin-fixed affected skin were trimmed into 1 mm3 pieces, fixed for 2 hr in 2.5% glutaraldehyde in 0.1 M Sörensen phosphate buffer (pH 7.3). Samples were postfixed in 1% osmium tetroxide, dehydrated in acetone, embedded in epoxy resin, and polymerized. Ultrathin sections (0.06 µm) were stained with uranyl acetate and lead citrate. Sections were viewed with a JEM 100 S transmission electron microscope (Jeol, Tokyo, Japan).
During 1990–2000, skin samples from 32 hares were cultured. The crust covering the skin was removed and the underlying inflamed dermis scraped with a blade. Removed material was stained with a Gram stain and culture was attempted on tryptose-soy-agar (TSA; supplemented with 5% bovine blood and incubated at 37 C for 2 days) or Saborraud agar (incubated at 20 C and 37 C). Nineteen samples were cultivated microaerophilically and anaerobically on Fastidious Anaerobe Agar (FAA) plates (Lab M, Bury, UK).
Bacteria were identified using standard biochemical methods. Appropriate api-methods (Bio Merieux: api STAPH, api 20 E, api 20 NE) were selected. Coagulase test and Staphytect Plus (Oxoid) test were used to identify staphylococci spp. Gram stain, api 20 C AUX, and germ tests were used to identify yeast.
Because samples were from natural cases, they varied in stage of disease. In the fully developed acute lesions, multifocal clusters of swollen keratinocytes were present in hyperplastic epidermis. Changes extended to follicular infundibula. As a result of severe hydropic swelling, keratinocytes were large and pale. Numerous eosinophilic, often floccular, intracytoplasmic inclusion bodies were present, in particular, in cells with ballooning degeneration (Figs. 2, 3). In 12 cases (28%), myriads of inclusion bodies were present. Dermal lesions were sparse, consisting mainly of edema and mild superficial perivascular lymphocytic infiltration.
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In chronic advanced lesions, there was variable dermal fibrosis and epidermal hyperplasia with mild to minimal infiltration of plasma cells with lesser numbers of lymphocytes and neutrophils. Dermal fibrosis was marked in three (7%) cases.
Ultrastructurally, the swollen epidermal and follicle infundibular keratinocytes contained round intracytoplasmic inclusion bodies with numerous virions (Fig. 4). The virions were large, ovoid, 300 nmx130–150 nm, and typical for poxviruses in terms of size and shape, biconcave nucleocapsid core, two lateral bodies, and clearly discernible outer lipoprotein capsule (Fig. 5).
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Staphylococcus warneri colonies were gray-white and clearly ß-hemolytic after 1 day of culture and became yellowish after a longer incubation period. Bacteria were catalase-positive and oxidase-negative, did not coagulate rabbit plasma, and were negative in the Staphytect Plus test indicating that clumping factors were not present. They also grew in anaerobic conditions and were typical in api STAPH; however, the number of nitrate-reducing, phosphatase-positive strains was greater than described in the standard.
The microscopic lesions described in this study represented a continuum of changes from an acute viral dermatitis to secondary pyoderma and finally to cutaneous scarring and substantiate that contagious mucocutaneous dermatitis of mountain hares in Finland is a viral disease. Numerous viral inclusions were present in the acute fully developed stage of the disease, which represented one third of the cases. This suggests that the period of virus replication is fairly short and is rapidly followed by epidermal necrosis and secondary pyoderma. Samples are often taken at the later stages of the disease when the secondary or chronic lesions predominate and no viral inclusions are present. In such cases, an etiologic diagnosis cannot be made. The secondary lesions described here are typical for many epitheliotrophic viral diseases. In our opinion, they have been given too much emphasis in the past; thus, the true nature of the disease has remained unsolved. The macroscopic and microscopic lesions were typical for a poxvirus infection. The presence of poxvirus in the inclusions was confirmed by electron microscopy. Proportions and dimensions of virions were suggestive of parapox virus (Quinn et al., 2002). However, molecular studies would be needed to characterize the virus more thoroughly.
Viruses classified as Poxviridae are responsible for many types of infections in lagomorphs (Robinson and Kerr, 2002). These infections differ in many respects from our cases. Myxomatosis, caused by Myxoma virus (Leporipoxvirus) infection, occurs in wild and domestic rabbits. The outcome and clinical disease largely depend on the virus strain. Although initial lesions may be present at the same sites (face, mucocutaneous junctions) as in contagious mucocutaneous dermatitis of the mountain hare, the histopathologic findings are very different. Epithelial swelling and intracytoplasmic inclusions may be present; however, the characteristic findings in myxomatosis are proliferation of myxomatous tissue around blood vessels and necrotizing vasculitis. The disease may become generalized with high mortality (Robinson and Kerr, 2002). Myxomatosis is very rare in hares. Only a few mountain hares develop clinical signs after experimental infection, but myxomatosis after natural infection has been reported (Fenner and Ratcliffe, 1965). Rabbit fibroma virus and Hare fibroma virus are also included in the genus Leporipoxvirus. The disease caused by these viruses is manifested by multiple firm cutaneous tumor-like growths, most commonly seen in the extremities. Histopathologically, marked irregular epidermal hyperplasia extends down into the dermal tumor mass consisting of a proliferation of fibroblasts. Large eosinophilic inclusions may be observed in the cytoplasm of keratinocytes and to a lesser extent in fibroblasts. The disease is reported in brown hares (Grilli et al., 2003) but, to our knowledge, not in mountain hares. Another poxvirus disease occurring in rabbits is rabbitpox, caused by a strain of vaccinia virus in the genus Orthopoxvirus. Typically, there are numerous small cutaneous nodular lesions that are histologically characterized by central necrosis and mononuclear cell infiltration. Inclusion bodies are absent. In addition, this infection may become generalized, with necrotizing lesions in oral mucosa, lungs, liver, adrenal glands, lymph nodes, and testicles (Mare, 1974). The disease has not been reported in wild lagomorphs (Robinson and Kerr, 2002).
A disease similar to what has been described here was reported in Sweden. Eleven cases (4%) of 292 mountain hares and two cases (1%) of 551 brown hares examined during 1986–89 in the National Veterinary Institute of Sweden were diagnosed as "elakartat noseksem" (malignant nasal eczema). Treponema-type bacteria were suggested as the etiologic agent (Mörner, 1992).
Treponemal infection described in wild European brown hares is characterized by a mucocutaneous dermatitis of the lips and prepuce with numerous spirochetes. The infection can be confirmed by special stains and by serology (Lumeij et al., 1994; Lumeij, 1996). In our study, there was no indication of infection with spiral-shaped bacteria, although bacteria, predominantly coccoid bacteria, were a constant finding in ulcerated lesions.
Bacteria of the staphylococcus family mainly reside on mucous membranes and in smaller numbers on the skin surface in a benevolent symbiosis with their host. They may multiply on the skin if the protective skin barrier changes (e.g., after an abrasion or necrosis of the epithelium). The staphylococcal strains colonizing the skin appear to depend on the host species. For example, the Staphylococcus sp. typical for human skin is S. aureus, in dogs it is Staphylococcus intermedius, and in piglets Staphylococcus hyicus predominates. According to our study, S. warneri appears to be typical for hares. Staphylococcus warneri has previously been considered more likely to infect humans than animals (Kloos and Schleifer, 1986).
In our study, we have shown that contagious mucocutaneous dermatitis in mountain hares is caused by a poxvirus. The disease is often complicated by secondary bacterial infection, most commonly by S. warneri. We propose this disease be called "mucocutaneous poxviral dermatitis of the mountain hare."
Our sincere thanks to Pirkko Niemelä from the Department of Basic Veterinary Sciences, The University of Helsinki, for her technical support with processing the samples for the transmission electron microscopy.
3 Present address: Helsinki Zoo, PO Box 4600, FIN-00099 Helsinki, Finland 
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Received for publication 10 April 2003.
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