Assessment of the ability of Malassezia pachydermatis to stimulate proliferation of canine keratinocytes in vitro

OBJECTIVE: To investigate the direct interaction between canine keratinocytes and live Malassezia pachydermatis and thereby determine the role of these organisms in the pathogenesis of epidermal hyperplasia associated with Malassezia dermatitis in dogs. SAMPLE POPULATION: Primary canine keratinocyte cultures established from skin samples obtained from clinically normal dogs. PROCEDURE: The proliferative response of keratinocytes co-cultured with Malassezia organisms for 1, 2, or 3 days was assessed by use of direct manual counting (to determine the number of keratinocytes in both the monolayer and the medium) and immunohistochemical staining techniques involving antibodies against proliferating cell nuclear antigen (PCNA) and another cellular proliferation marker, Ki-67. The potential cytotoxic effect of Malassezia organisms was investigated by use of an apoptosis detection kit to label keratinocytes co-cultured with M. pachydermatis that underwent apoptosis. RESULTS: No stimulatory effect of Malassezia organisms on canine keratinocyte proliferation was detected via cell counting and immunohistochemical techniques. However, there was a significant increase in dead keratinocytes in the medium with increasing numbers of Malassezia organisms in the co-culture. More apoptotic cells were observed in keratinocyte monolayers co-cultured with high numbers of M. pachydermatis than there were in monolayers cultured without Malassezia organisms, and the number increased after prolonged incubation. CONCLUSIONS AND CLINICAL RELEVANCE: M. pachydermatis did not stimulate canine keratinocyte proliferation in vitro. The results suggested that the epidermal hyperplasia observed in dogs with Malassezia dermatitis is unlikely to be caused by a direct effect of the organism on the keratinocyte cell cycle, but is likely to involve other mechanisms.     

Serum antibodies to Malassezia yeasts in canine atopic dermatitis

Significant numbers of humans with atopic dermatitis develop Malassezia-specific IgE. Immediate skin-test reactivity to Malassezia has been demonstrated in atopic dogs. The aim of this study was to compare the serum IgG and IgE response to Malassezia in atopic dogs with and without clinical evidence of Malassezia dermatitis and/or otitis, nonatopic dogs with clinical evidence of Malassezia dermatitis and/or otitis and healthy dogs. Cytology was used to diagnose clinically significant Malassezia dermatitis and otitis. Contact plate cultures confirmed the validity of this technique. Reproducible enzyme-linked immunosorbent assays for Malassezia-specific IgG and IgE in canine serum were established. Atopic dogs had significantly higher serum IgG and IgE levels than either healthy dogs or nonatopic dogs with clinical evidence of Malassezia dermatitis and/or otitis. There was no significant difference in IgG and IgE levels between atopic dogs with and without clinical evidence of Malassezia dermatitis and/or otitis. The implications of these findings in the pathogenesis and management of canine atopic dermatitis are discussed.     

Effects of cyclosporin A and cilomilast on activated canine, murine and human keratinocytes

The calcineurin inhibitor cyclosporin A and the phosphodiesterase 4 inhibitor cilomilast exhibit potent immunomodulatory properties which make them interesting therapeutics for the treatment of skin disorders like canine and human atopic dermatitis. Cyclosporin A and phosphodiesterase 4 inhibitors have already demonstrated clinical efficacy in the therapy of canine and human atopic dermatitis. Their direct impact on keratinocytes, especially canine keratinocytes, is less obvious. Thus, an investigation was carried out to ascertain whether cyclosporin A and cilomilast modulate keratinocyte proliferation and secretion of proinflammatory mediators. Cyclosporin A inhibited canine and murine keratinocyte proliferation, whereas cilomilast had no affect. Cyclosporin A and cilomilast reduced the lipopolysaccharide-induced prostaglandin E2 synthesis in canine and murine keratinocytes. Both immunomodulators also inhibited the production of the CXC chemokine KC and CCL2 in the murine keratinocyte cell line MSC-P5. The two immunomodulators also significantly reduced the interferon-gamma-induced production of interferon-gamma-inducible protein 10 in human keratinocytes (HaCaT cells). Thus, cyclosporin A and cilomilast directly modulate keratinocyte functions which might contribute to the anti-inflammatory and immunomodulatory action of these compounds in the treatment of allergic skin diseases.     

Hereditary nasal parakeratosis in Labrador retrievers: 11 new cases and a retrospective study on the presence of accumulations of serum ('serum lakes') in the epidermis of parakeratotic dermatoses and inflamed nasal plana of dogs

We report 11 new cases of hereditary nasal parakeratosis in Labrador retrievers. The disease was first observed when the dogs were 6 months to 2 years of age, and affected dogs of either sex and all coat colours. Hyperkeratosis and depigmentation were confined to the nasal planum, and affected dogs were otherwise healthy. The principal histological findings in biopsy specimens were marked diffuse parakeratotic hyperkeratosis, multiple intracorneal serum lakes and superficial interstitial-to-interface lymphoplasmacytic dermatitis. Topical applications of propylene glycol in water or white petrolatum were often effective for treatment of the dermatosis. However, continued applications were required to maintain a beneficial response. A retrospective histological study of parakeratotic inflammatory diseases of canine haired skin and inflammatory diseases of the canine nasal planum was performed. The degree of parakeratotic hyperkeratosis and the number and size of intracorneal serum lakes were evaluated. The degree of parakeratotic hyperkeratosis was greater in hereditary nasal parakeratosis specimens than that seen in discoid lupus erythematosus and Malassezia dermatitis. There were more serum lakes in hereditary nasal parakeratosis specimens than in specimens from dogs with discoid lupus erythematosus, Malassezia dermatitis, primary seborrheic dermatitis or zinc-responsive dermatosis. Significant differences in sizes of serum lakes (if present) were not seen.     

Adherence by Staphylococcus intermedius to canine keratinocytes in atopic dermatitis

The adherence of Staphylococcus intermedius to canine keratinocytes in normal dogs was compared to that in dogs suffering from atopic dermatitis, primary seborrhoea and bacterial pyoderma. Statistically significant greater adherence by S. intermedius to keratinocytes occurred in atopic dogs and dogs suffering from pyoderma when compared with the normal group (P < 0.01) and dogs suffering from primary seborrhoea (P < 0.05). This is similar to the results of a study of human atopic dermatitis by Cole and Silverberg (1986) who demonstrated increased adherence by S. aureus to keratinocytes from atopic dermatitis patients when compared with adherence to keratinocytes in a variety of non-atopic dermatoses. This increased adherence by pathogenic staphylococci to keratinocytes may in part explain the high incidence of staphylococcal pyoderma seen in both canine and human patients suffering from atopic dermatitis.     

Humoral measurement of type-1 hypersensitivity reactions to a commercial Malassezia allergen

Malassezia pachydermatis is considered to be a contributing factor to canine atopic dermatitis (AD). The purpose of this study was to investigate the humoral response to a commercially produced M. pachydermatis extract. Fifteen atopic dogs with Malassezia overgrowth on the skin (MD), 16 atopic dogs without MD, three atopic dogs with overgrowth of Malassezia in the ears only (MO), and 12 normal dogs were intradermally tested with M. pachydermatis extract at 50, 100, 250, 500, 1000, 2000 and 4000 PNU mL(-1). All dogs were evaluated cytologically by cutaneous tape strip and bilateral ear exudate sampling to determine presence of MD or MO. Each had serum evaluated for anti-Malassezia IgE using three Malassezia extracts with an ELISA assay. The irritant threshold concentration at which healthy nonatopic dogs ceased to react was 1000 PNU mL(-1). There was a significant difference in intradermal test reactivity between the atopic groups. At this dilution, 93% (14/15) of the atopic MD group, 31% (5/16) of the atopic group without MD or MO, and 100% (3/3) of the atopic MO only group reacted. There were no significant differences in the serum IgE levels as measured by the Greer ELISA assay, between any groups using any of the three extracts. These results support that Greer's M. pachydermatis extract is useful for intradermal testing of dogs with an allergic phenotype, and that atopics with MD are more likely to have a type-1 Malassezia hypersensitivity than those without. The ELISA assay may require further development in order to be useful for the diagnosis of Malassezia hypersensitivity.     

Frequency, body distribution, and population size of Malassezia species in healthy dogs and in dogs with localized cutaneous lesions.

Malassezia species are commensal organisms of human and animal skin that occasionally act as opportunistic pathogens. The lipid-dependent species are associated with human skin disorders, whereas the non-lipid-dependent species (Malassezia pachydermatis) is considered as an opportunistic secondary pathogen affecting the canine skin surface and ear canal. This study evaluated the relationship between Malassezia yeasts, their population size, and the occurrence of skin lesions from healthy and skin-diseased dogs. The efficiency of cytological examination and fungal culture for Malassezia detection was also evaluated. From March 2002 to July 2003, 33 healthy dogs and 54 dogs with pruritic localized skin diseases were examined; skin swabs (1218) were collected from 7 anatomical sites for culture and cytological examination. Malassezia prevalence according to anatomical site and the agreement between cytological results and fungal cultures were statistically analyzed. Differences in mean colony forming unit counts between positive healthy and diseased dogs were evaluated using the Bonferroni test for post hoc pair-wise comparisons. In healthy dogs, Malassezia yeasts were most frequently isolated in the perianal and perioral areas. The frequency of isolation and population size of Malassezia species were higher in dogs with localized dermatitis, especially in affected areas, indicating a role for Malassezia in the occurrence of skin lesions. Malassezia pachydermatis was the species most commonly cultured from the skin and external ear canal of healthy and diseased dogs; isolation of lipid-dependent yeasts from healthy dogs was less frequent. Using fungal culture as the gold standard, cytological examination showed good relative specificity (95%) but very low relative sensitivity (30%).     

A noninferiority clinical trial comparing fluconazole and ketoconazole in combination with cephalexin for the treatment of dogs with malassezia dermatitis

This double-blinded noninferiority clinical trial evaluated the use of oral fluconazole for the treatment of Malassezia dermatitis in dogs by comparing it with use of an accepted therapeutic agent, ketoconazole. Dogs presenting with Malassezia dermatitis were treated with either fluconazole or ketoconazole in addition to cephalexin for concurrent bacterial dermatitis. Statistically significant improvements in cytologic yeast count, clinical signs associated with Malassezia dermatitis, and pruritus were seen with both antifungal treatments. There was no statistical difference between the treatments with regard to the magnitude of reduction in these parameters. These results suggest that fluconazole is at least as effective as ketoconazole for the treatment of dogs with Malassezia dermatitis.     

Genotyping of Malassezia pachydermatis isolates from canine healthy skin and atopic dermatitis by internal spacer 1 (IGS1) region analysis

Isolates of Malassezia pachydermatis from healthy dog skin and from dogs with atopic dermatitis were molecularly characterized using internal spacer 1 (IGS1) region analyses, and their phospholipase A2 activity and pH growth profiles were then characterized in vitro. The percentage of isolates from healthy dogs that had the following IGS1 subtypes (isotype, %) were as follows: 1A, 6%; 1B, 27%; 1C, 11%; 2A, 6%; 2B, 6%; 3A, 11%; 3C, 3%; and 3D, 24%. In contrast, 9% of isolates from dogs with atopic dermatitis were isotype IB and 91% were isotype 3D, indicating that isolates of subtype 3D were the most prevalent in dogs with atopic dermatitis. Production of phospholipase A2 was statistically higher in isolates of subtype 3D than in the other subtypes. The subtype 3D isolates showed enhanced growth on alkaline medium compared with non-3D subtype isolates. The main clinical sign of canine Malassezia dermatitis is waxy exudates on the skin, which predispose the patient to development of a yeast overgrowth of the subtype 3D. Increased phospholipase A2 production may be involved in the inflammatory process associated with Malassezia dermatitis.     

Severe bilaterally symmetrical alopecia in a horse

A 9-year-old Tennessee Walking Horse gelding was presented for diagnosis of the cause of extensive alopecia. Complete hair loss was noted over the head, neck, shoulder, thigh, and proximal limbs, but the trunk, distal limbs, pelvic area, mane, and tail were unaffected. The alopecic areas were visually noninflammatory with no exudate or crust except on the shoulder and along the back, where multifocal patchy areas of alopecia with scales and crust were evident. The horse was slightly pruritic. Microscopically, the hair bulbs, inner and outer root sheaths of inferior segments, and perifollicular regions were infiltrated by small to moderate numbers of small lymphocytes. Similar inflammation was occasionally evident in isthmus follicular walls as well as some apocrine glands. No sebaceous glands were affected. Immunohistochemistry confirmed that the small lymphocytes were CD3(+) T lymphocytes. The epidermis from the skin with scale and crusts along the horse's back exhibited mild to moderate hyperplasia, mild lymphocytic exocytosis, mild eosinophilic dermatitis, and diffuse parakeratosis with numerous budding yeasts, consistent with Malassezia spp. The final disease diagnosis was made as alopecia areata with Malassezia dermatitis. Alopecia areata could be a contributing underlying factor for Malassezia dermatitis.     

Peripheral blood mononuclear cell responses to major and minor Dermatophagoides allergens in canine atopic dermatitis.

Atopic dermatitis is a chronic inflammatory and pruritic skin disease commonly seen in dogs and humans. Most cases involve hypersensitivity to the house dust mites (HDM) Dermatophagoides farinae and Dermatophagoides pteronyssinus. Human atopic dermatitis is associated with the HDM derived allergens Der f 1 and 2, and Der p 1 and 2. Serological data, however, suggest that a 98/104kD protein is the most important allergen in dogs with atopic dermatitis. The aim of this study was to characterise the specificity of circulating T-cells in canine atopic dermatitis for HDM derived allergens. Peripheral blood mononuclear cells (PBMCs) from dogs with atopic dermatitis that were skin test positive for D. farinae and D. pteronyssinus were cultured with crude extracts of D. farinae, D. pteronyssinus and D. microceras, a 98/104kD allergen purified from D. farinae, Der f 1 and Der f 2. There was significantly greater responsiveness of PBMCs to the D. farinae and D. pteronyssinus extracts compared to the D. microceras extract, and similarly to the purified 98/104kD allergen compared to Der f 1 and Der f 2. The close association between serological findings and PBMC proliferation implies that the 98/104kD HDM protein is a major target of immune recognition and that T-cells also participate in the pathogenesis of canine atopic dermatitis by supporting IgE production.     

Malassezia dermatitis in the dog: a retrospective histopathological and immunopathological study of 86 cases (1990–95)

A retrospective histopathological and immunopathological study was conducted on 86 dogs with Malassezia dermatitis. West Highland White terriers, English Setters, Shih Tzus, Basset Hounds, American Cocker Spaniels, spayed females, and castrated males were found to be at increased risk. The histopathological reaction pattern of lymphocytic superficial perivascular to interstitial dermatitis with parakeratotic hyperkeratosis, irregular epidermal hyperplasia, diffuse intercellular oedema and lymphocytic exocytosis was found to be consistent with a diagnosis of Malassezia dermatitis whether yeast were histologically visible (73.3% of the cases) or not (26.7%). Immunopathological studies revealed that 60– > 90% of the inflammatory cells within the epidermis, and 25–75% of those within the dermis were CD3+T lymphocytes, and that the only immunoglobulin-positive cells were dermal plasma cells.     

Comparative efficacies of oral ketoconazole and terbinafine for reducing Malassezia population sizes on the skin of Basset Hounds

The objective of this study was to compare the efficacy of oral ketoconazole and terbinafine for reducing population sizes of Malassezia yeasts on canine skin. Twenty-one Basset Hounds were randomised in three groups of seven according to Malassezia populations. Dogs in the first group were treated by oral administration of ketoconazole (Ketofungol) 200 mg, Janssen-Cilag) at 10 mg x kg-1, every 24 h with food, for 3 weeks. Dogs in the second group were treated by oral administration of terbinafine (Lamisil) 250 mg, Novartis) at 30 mg x kg-1, every 24 h with food, for 3 weeks. The seven remaining dogs were used as controls. Malassezia population sizes were assessed by use of contact plates on four cutaneous sites at days 7, 14 and 21. Both ketoconazole and terbinafine were effective in reducing the baseline levels of Malassezia organisms with no significant difference between the two drugs. In further studies, oral terbinafine should be evaluated for the management of canine cases of Malassezia dermatitis.     

Production of GM-CSF Mediated by Cysteine Protease of Der f in Canine Keratinocytes

House dust mite (HDM) allergens are the most common allergens for induction of IgE-mediated hypersensitivity. Recently, epicutaneous sensitization with HDM allergens has been emphasized in the development of atopic dermatitis (AD) by producing various soluble factors in keratinocytes. Among the soluble factors, GM-CSF is a key molecule that activates Langerhans cells, antigen-presenting cells in the epidermis. In the present study, we investigated the effects of Dermatophagoides farinae (Der f) on GM-CSF production in a canine keratinocyte cell line, CPEK. CPEKs were found to produce GM-CSF upon stimulation by Der f. The GM-CSF production was suppressed by addition of a cysteine protease inhibitor. The present results suggest that cysteine protease-derived Der f may be an initiator of allergic inflammation by inducing the production of GM-CSF in keratinocytes.     

Canine distemper virus associated proliferation of canine footpad keratinocytes in vitro

Infection of canine footpads with canine distemper virus (CDV) can result in so-called hard pad disease characterized by footpad epidermal proliferation and hyperkeratosis. Cultured canine footpad keratinocytes (CFK) were inoculated with a virulent canine distemper virus strain (A75/17-CDV) to study the effects of CDV-infection on keratinocyte proliferation. Infection was analyzed by immunohistochemistry and in situ hybridization for CDV nucleoprotein (N-protein) antigen and mRNA. CDV caused a persistent, non-cytocidal infection with spread from single cells to infection of the confluent cell layer 7 days post infection (p.i.). Absolute cell numbers were significantly higher in infected cultures compared to control cultures from day 4 until day 6 p.i. Infected cultures contained significantly more total DNA on day 5 p.i. compared to controls. Immunohistochemical investigation of proliferation markers Ki67 and BrdU demonstrated a nearly two-fold increase in numbers of positive cells on day 5 p.i. compared to controls. These findings demonstrate that canine distemper virus infection of canine footpad keratinocytes in vitro was associated with proliferation.     

Putative Malassezia dermatitis in six goats

Histopathology submissions from 28 goats with dermatological disease were identified in an archival search of pathology files. Microscopic sections of skin biopsy specimens were examined for the presence of Malassezia spp. organisms. Six cases with many Malassezia yeasts were identified histopathologically. Based on the extent of clinical disease, three cases were regarded as localized and three were generalized infections. Clinical findings included alopecia with dry seborrhoea (four cases), greasy seborrhoea (one case), and no clinical findings specific to localized Malassezia infection when concurrent bacterial infection was present (one case). Mild pruritus was reported in two cases of generalized infection. No breed predilection was apparent. Three cases were male and three were female. Malassezia dermatitis occurred in goats from 10 months to 13 years of age. Three of six cases had concurrent bacterial infection. Skin lesions resolved following topical antifungal therapy in the two goats that were treated. Histopathological findings in all cases were severe follicular and epidermal orthokeratotic hyperkeratosis with minimal epithelial change and mild superficial perivascular to interstitial nonsuppurative inflammation. Numerous budding yeasts were visible within the stratum corneum of all cases; however, Malassezia was not isolated in the three cases in which culture was attempted. Based upon these findings, the authors suggest that the diagnosis Malassezia dermatitis in goats is most likely to be made by cytological examination of skin impressions or by examination of skin biopsy samples.     

The ACVD task force on canine atopic dermatitis (XXII): nonsteroidal anti-inflammatory pharmacotherapy.

The pharmacotherapy of canine atopic dermatitis has relied primarily on the use of glucocorticoids and anti-histamines. During the last decade, other anti-inflammatory drugs have been investigated in clinical trials. This paper will review the studies using misoprostol, cyclosporine, tacrolimus, phosphodiesterase inhibitors, capsaicin, leukotriene inhibitors and serotonin-reuptake inhibitors for treatment of dogs with atopic dermatitis. For each drug the mechanism of action, the rationale for use in atopic dermatitis, the clinical efficacy, reported adverse effects and strength of recommendation for treatment of canine atopic dermatitis are described. At the time of this writing, there is fair evidence to support the recommendation for using cyclosporine, misoprostol and pentoxifylline for treatment of canine atopic dermatitis. This recommendation can be strengthened by the performance of additional blinded randomized controlled trials with larger number of dogs. In contrast, there is insufficient evidence to recommend for or against treatment with tacrolimus, leukotriene inhibitors, serotonin-reuptake antagonists and capsaicin.     

Canine serum immunoreactivity to M. pachydermatis in vitro is influenced by the phase of yeast growth

In western immunoblotting studies of canine sera using Malassezia pachydermatis extracts, the reported patterns of immunoreactivity vary between different laboratories. Because the duration of culture influences the antigenic composition of lipid-dependent Malassezia spp. when probed with human sera, we investigated whether the in vitro growth phase of M. pachydermatis influences immunoreactivity using canine sera. Extracts of M. pachydermatis CBS 1879 grown in Sabouraud's liquid medium at 37 degrees C for 2, 4, 6, 8 and 10 days were prepared by mechanical disruption, centrifugation, dialysis and lyophilization. Yeast growth phase was assessed by sequential colony counts and optical density measurements. Patterns of IgG immunoreactivity in high (n = 3) and low (n = 3) titre sera were compared using extracts prepared at each time point by sodium dodecyl sulphate polyacrylamide gel electrophoresis and western immunoblotting. Protein bands of 62 and 49 kDa were recognized by all sera, and 98 and 68 kDa bands were recognized by five sera. Proteins of 188, 66, 58, 57, 38, 28 and 17 kDa were only recognized by high titre sera. All high titre sera used recognized more bands in exponential phase (d2) extracts when compared with decline phase (d8-d10) extracts, and two of these sera showed most bands in stationary phase (d4-d6) extracts. Bands of 62 and 57 kDa were primarily detected in exponential and early stationary phase extracts. There is variation in antigenic expression in different growth phases of M. pachydermatis, which might explain discrepancies between previous laboratory studies of canine immunity to this yeast.     

Malassezia and Candida infections in bull terriers with lethal acrodermatitis

In 12 cases of lethal acrodermatitis (LAD), four sampling techniques (brush, swab, scrape and adhesive tape strip) were used to study the distribution of yeasts in various body sites and these results were compared with those from five cases of atopic dermatitis and those of 10 normal dogs. Malassezia was frequently isolated from lesional and non-lesional skin and haircoat, footpads, nails and mucous membranes from dogs with either LAD or atopic dermatitis, although, generally, more Malassezia organisms were isolated from LAD cases. In normal dogs, Malassezia was most frequently recovered from the ear canal and the perianal skin. Candida was isolated frequently from dogs with LAD, but only a single isolate of this yeast was found in the other two groups. Fungal hyphae and pseudohyphae, probably Candida albicans, could be detected in samples collected from the nails and footpads of dogs with LAD. Both Malassezia and Candida could be isolated using all four sampling techniques. The MacKenzie (toothbrush) technique and adhesive tape strip cultures proved simple methods for the semiquantitative evaluation of yeasts. The high recovery rate of Malassezia and Candida from dogs with LAD is probably related to immune dysfunction, particularly T-cell dysfunction, known to be present in these dogs. C albicans infection may in part be responsible for the pathogenic changes of the nails and footpads commonly seen in cases of LAD.     

Production of recombinant extracellular domains of canine desmoglein 1 (Dsg1) by baculovirus expression

The aim of this study was to generate a recombinant protein to represent the entire extracellular domain of canine desmoglein 1 (Dsg1), a desmosomal cell-cell adhesion molecule, by the baculovirus expression system. Cotransfection of a baculovirus transfer vector containing the cDNA for the entire extracellular domain of canine Dsg1 with baculovirus DNA into insect cells resulted in the secretion of soluble canine Dsg1 into insect culture supernatants. Immunoreactivity of 11 human pemphigus foliaceus (PF) sera against the cell surface of canine keratinocytes was completely removed when the sera were preincubated with the canine Dsg1 baculoprotein. This recombinant canine Dsg1 produced by baculovirus shares the major epitopes of the authentic canine Dsg1 recognized by human PF sera, and will be useful in studying the molecular pathophysiological mechanisms in PF and impetigo in canine patients.