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.     

Macroscopic,cytological and bacteriological evaluation of anal sac content in normal dogs and in dogs with selected dermatological diseases

Abstract Macroscopic and cytological aspects of anal sac content were evaluated in 40 normal dogs and 10 dogs each with pyoderma, Malassezia dermatitis associated with atopic dermatitis and uncomplicated atopic dermatitis. Bacteria isolated from anal sacs were compared with those from abdominal skin and hair in 20 normal dogs and 10 dogs with pyoderma. There was no difference between the groups in anal sac dimension, or in the colour, consistency or presence of granules in their content. Extracellular bacteria were found in higher numbers in diseased animals, whereas intracellular bacteria were observed in 40% of dogs with pyoderma and in only 2.5% of normal dogs. Malassezia spp. were present in 15.7% of dogs, with no difference between groups. Neutrophils were observed in 12.5% of normal dogs, 30% of dogs with Malassezia dermatitis with underlying atopic dermatitis and in 70 and 80% of dogs with pyoderma and uncomplicated atopic dermatitis, respectively. Seven bacterial species were isolated from anal sacs, with no difference between normal dogs and dogs with pyoderma. In five normal animals and in four dogs with pyoderma the same bacterial strains were isolated from anal sacs and from abdominal skin and hair.     

Response to Malassezia pachydermatis by peripheral blood mononuclear cells from clinically normal and atopic dogs

OBJECTIVE: To investigate the potential cell-mediated immune response of atopic dogs to the yeast Malassezia pachydermatis and to correlate it with the type-1 hypersensitivity (humoral) response of the same population of dogs. ANIMALS: 16 clinically normal dogs, 15 atopic dogs with Malassezia dermatitis, 5 atopic dogs with Malassezia otitis, and 7 atopic control (ie, without Malassezia dermatitis or otitis) dogs. PROCEDURE: A crude extract of M pachydermatis was extracted for use as an intradermal allergy testing reagent and for stimulation of isolated peripheral blood mononuclear cells in vitro. Flow cytometry was also used to assess cell surface antigenic determinants (CD3, CD4, CD8, CD14, CD21, CD45RA, surface immunoglobulin) on peripheral blood mononuclear cells. RESULTS: Atopic dogs with cytologic evidence of Malassezia dermatitis had an increased lymphocyte blastogenic response to crude M pachydermatis extract, compared with clinically normal dogs and dogs with Malassezia otitis. Atopic control dogs did not differ significantly in their responses from atopic dogs with Malassezia dermatitis or otitis. A significant correlation was not found between the lymphocyte blastogenic response and the type-1 hypersensitivity response to M pachydermatis within any of the groups. CONCLUSIONS AND CLINICAL RELEVANCE: Cell-mediated and humoral reactivities to M pachydermatis contribute to the pathogenesis of atopic dermatitis in dogs but are not directly correlated. Modification of the dysregulated immune response toward M pachydermatis may assist in the reduction of pathologic changes associated with an atopic dermatitis phenotype in dogs.     

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.     

Late-phase reactions to intradermal testing with Dermatophagoides farinae in healthy dogs and dogs with house dust mite-induced atopic dermatitis

OBJECTIVE: To determine the prevalence of late-phase reactions to intradermal testing with Dermatophagoides farinae in healthy dogs and dogs with atopic dermatitis and an immediate reaction to D farinae. ANIMALS: 6 healthy dogs and 20 dogs with atopic dermatitis and immediate reactions to D farinae. PROCEDURE: ntradermal tests were performed with D farinae at 1:1,000 wt/vol and 1:50,000 wt/vol concentrations, and skin reactivity was evaluated after 0.25, 6, and 24 hours. Serum D farinae-specific IgE antibodies were assayed. Extent of lesions (atopy index) and pruritus (visual analogue scale) were evaluated in dogs with atopic dermatitis. RESULTS: Late-phase reactions were observed in healthy dogs at 6 hours (n = 2 dogs) and 24 hours (1) with the 1:1,000 wt/vol concentration, and at 6 hours (1) and 24 hours (1) with the 1:50,000 wt/vol concentration of allergen. Late-phase reactions in healthy dogs were only observed in dogs with an immediate reaction to D farinae. Late-phase reactions were observed in 11 of 20 dogs with atopic dermatitis at 6 and 24 hours with the 1:1,000 wt/vol concentration and in 10 of 20 at 6 and 24 hours with the 1:50,000 wt/vol concentration of allergen. There was no difference in mean atopy index, mean visual analogue scale of pruritus, or mean serum D farinae-specific IgE concentration of dogs with a late-phase reaction, compared to dogs without a late-phase reaction. CONCLUSIONS AND CLINICAL RELEVANCE: Late-phase reactions may be observed after an immediate reaction to intradermal skin testing in healthy and allergic dogs but are more commonly observed in dogs with atopic dermatitis.     

The immunoglobulin G response to Malassezia pachydermatis extracts in atopic and non-atopic dogs

IgG immunoreactivity to Malassezia pachydermatis was compared in atopic and non-atopic dogs. Malassezia pachydermatis proteins with a molecular weight of 98 kDa were recognized at a significantly higher frequency in the sera of atopic dogs. Most of the atopic dogs with Malassezia dermatitis had a greater IgG response than did normal dogs.     

Identification of major allergens of Malassezia pachydermatis in dogs with atopic dermatitis and Malassezia overgrowth

Abstract We have previously shown that both atopic and normal dogs generate an IgG response to antigens of Malassezia pachydermatis . The aim of this study was to compare IgE responses to separated proteins of M. pachydermatis in 28 atopic dogs with Malassezia dermatitis and 22 clinically normal dogs using Western immunoblotting. Six different detection systems were evaluated in order to assess sensitivity and eliminate nonspecific binding and cross-reactivity. The protocol yielding the best results utilized a monoclonal mouse antidog IgE, an alkaline phosphatase conjugated goat antimouse IgG which had been passed through a canine IgG column 3 times, a chemiluminescent substrate and a digital imaging system. Proteins of 45, 52, 56 and 63 kDa were recognized by more than 50% of the atopic dog sera and thus represented major allergens. Only a minority of normal dogs showed faint IgE binding to these proteins. The results indicate that the majority of atopic dogs with Malassezia dermatitis have a greater IgE response than normal dogs, suggesting an IgE-mediated immune response may be clinically important in the pathogenesis of the disease.     

Evaluation of serum obtained from atopic dogs with dermatitis attributable to Malassezia pachydermatis for passive transfer of immediate hypersensitivity to that organism

OBJECTIVE: To determine the functionality of canine anti-Malassezia IgE via the passive transfer of immediate hypersensitivity localized to the skin (ie, cutaneous anaphylaxis) from atopic dogs with dermatitis attributable to overgrowth of Malassezia pachydermatis (Malassezia dermatitis [MD]) to healthy recipient dogs by use of the Prausnitz-Küstner (P-K) technique. ANIMALS: 7 clinically normal dogs, 32 atopic dogs with MD, serum from 11 atopic dogs with MD, and 3 healthy dogs without prior sensitization to M pachydermatis. PROCEDURE: Serum from atopic dogs with MD was used for P-K tests in 3 clinically normal recipient dogs. Serial dilutions of untreated, heat-inactivated, IgE-absorbed, and bovine serum albumin (BSA)-absorbed (control) aliquots of serum were injected ID in triplicate for dermal sensitization. Twenty-four, 48, and 72 hours later, a crude extract of M pachydermatis was injected ID into the sites used for sensitization injections, and immediate hypersensitivity reactions were graded on a 4-point scale. RESULTS: Untreated serum caused P-K reactivity beginning 24 hours after passive sensitization and persisting through 72 hours (titers, 1:32 to 1:64). Heat inactivation and IgE-absorption of serum eliminated P-K reactivity, whereas treatment of serum with BSA did not. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis of P-K test results supports the passive transfer of cutaneous anaphylaxis by anti-Malassezia IgE and indicates it is functional in type-1 hypersensitivity reactions of atopic dogs with MD. Reduction or blockade of anti-Malassezia IgE in atopic dogs with MD may provide better clinical control of the disease.     

Peripheral blood mononuclear cell responses to Dermatophagoides farinae in canine atopic dermatitis

Atopic dermatitis is a chronic inflammatory and pruritic skin disease commonly seen in dogs and humans that is characterised by the presence of allergen-specific IgE. Data from skin tests and serological analysis suggest that the house dust mite Dermatophagoides farinae is the most important allergen in dogs with atopic dermatitis. The aim of this study was to determine if D. farinae specific peripheral blood mononuclear cell (PBMC) responses could be detected in dogs with atopic dermatitis. PBMCs were isolated by the density centrifugation from dogs with atopic dermatitis that were skin test positive for D. farinae, dogs with atopic dermatitis that were skin test negative for D. farinae, and healthy dogs. Cells were cultured with increasing concentrations of the D. farinae extract, no antigen, vaccine antigens or concanavalin A (ConA). There was significantly greater responsiveness of PBMCs from the D. farinae positive dogs than from either the D. farinae negative or healthy dogs (ANOVA, P<0.05). In contrast, no significant differences were observed in the control responses between the three groups. This is the first study to demonstrate that D. farinae specific circulating memory cells are involved in the pathogenesis of canine house dust mite hypersensitivity.     

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.     

Malassezia spp. overgrowth in allergic cats

A series of 18 allergic cats with multifocal Malassezia spp. overgrowth is reported: atopic dermatitis was diagnosed in 16, an adverse food reaction in another and one was euthanized 2 months after diagnosis of Malassezia overgrowth. All the cats were otherwise healthy and those tested (16 out of 18) for feline leukaemia or feline immunodeficiency virus infections were all negative. At dermatological examination, multifocal alopecia, erythema, crusting and greasy adherent brownish scales were variably distributed on all cats. Cytological examination revealed Malassezia spp. overgrowth with/without bacterial infection in facial skin (n = 11), ventral neck (n = 6), abdomen (n = 6), ear canal (n = 4), chin (n = 2), ear pinnae (n = 2), interdigital (n = 1) and claw folds skin (n = 1). Moreover, in two cats Malassezia pachydermatis was isolated in fungal cultures from lesional skin. Azoles therapy alone was prescribed in seven, azoles and antibacterial therapy in eight and azoles with both antibacterial and anti-inflammatory therapy in three of the cats. After 3-4 weeks of treatment, substantial reduction of pruritus and skin lesions was observed in all 11 cats treated with a combined therapy and in five of seven treated solely with azoles. Malassezia spp. overgrowth may represent a secondary cutaneous problem in allergic cats particularly in those presented for dermatological examination displaying greasy adherent brownish scales. The favourable response to treatment with antifungal treatments alone suggests that, as in dogs, Malassezia spp. may be partly responsible for both pruritus and cutaneous lesions in allergic cats.     

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.     

Serum immunoglobulin E concentrations in West Highland White Terrier puppies do not predict development of atopic dermatitis

Sera from 154 West Highland White Terrier puppies between 6 and 12 weeks of age were assayed for total IgE using a sandwich ELISA method. Development of clinical signs of atopic dermatitis in these dogs was monitored by use of an annual owner questionnaire, until the dog reached 3 years of age. Of 114 evaluated dogs, skin disease severe enough to warrant veterinary examination was reported in 52 (46%) during the three study years. A diagnosis of atopic dermatitis was made by the attending veterinarian in 28 dogs (25%). Certain litters had an especially high prevalence of apparently atopic dogs, consistent with the genetic predisposition towards atopy in this breed, but clear evidence of consistent heritability was not present. The median IgE concentration in 154 puppies at 6–12 weeks of age was 0.9 units mL⁻¹, with a skewed distribution. Significant ( P < 0.01) variation in serum IgE concentrations was observed between litters, with median serum IgE concentrations for a litter ranging from 0 to 27.7 units mL⁻¹. The median serum IgE concentration in puppies that later developed clinical signs of atopic dermatitis was not significantly different from that of puppies that remained healthy. There were no apparent correlations or significant differences found between serum IgE concentration as a puppy, parental history of skin disease, and subsequent emergence of clinical signs of atopic dermatitis. We conclude that early total serum IgE determinations seem to have little usefulness in predicting the later onset of atopic dermatitis in this breed.     

Subepidermal linear alignment of mast cells in inflammatory dermatoses of the dog

A recent study demonstrated that 47.7% of dogs with Malassezia dermatitis had a subepidermal linear alignment of mast cells (SLAM). A retrospective histopathological study was conducted on 419 canine skin biopsies to determine if a SLAM was present in other inflammatory diseases. Cases examined included dogs with demodicosis, sarcoptic mange, dermatophytosis, pemphigus foliaceus, pemphigus erythematosus, discoid lupus erythematosus, systemic lupus erythematosus, erythema multiforme, dermatomyositis, staphylococcal pyoderma, primary seborrhea, arthropod bites, contact hypersensitivity, flea bite hypersensitivity, atopy, and food hypersensitivity. Three cases (3/419, 0.7%) were identified with SLAM. The diagnoses for these cases were atopy (1/23, 4%) with a secondary bacterial folliculitis (1/136, 0.7%), pemphigus erythematosus (1/18, 6%), and discoid lupus erythematosus (1/16, 6%). Based on this study, SLAM is significantly more common in Malassezia dermatitis than in other inflammatory diseases.     

Occurrence, distribution and population size of Malassezia pachydermatis on skin and mucosae of atopic dogs

Aim of the present study was to determine the distribution and quantification of Malassezia yeasts on a wide number of cutaneous sites in atopic dogs by means of a semiquantitative swab technique. A possible relationship between the presence of clinical signs and the occurrence and population size of yeasts was attempted. Forty-one privately owned atopic dogs of different age and breed were sampled. Results were expressed as colony forming units per swab. Malassezia colonies obtained from each plate were counted, scored and typed. All dogs yielded Malassezia pachydermatis from at least one skin area. Yeast population mean size by site was 6.98 (S.D.=3.47) as compared to other body areas. The frequence of isolation was higher from interdigital areas (70.7%), ears (63.4%), nail folds (35.7%), mouth (33.3%), groin (30.9%), conjunctiva and axillae (23.8%), perineum and anus (19%), perianal glands (9.5%). Ears, anus, interdigital areas, perianal glands and groin yielded the largest mycotic amount. M. pachydermatis was the sole species of yeast to colonize canine skin in examined animals. No statistical correlation between the presence of cutaneous alterations and Malassezia isolation was detected. Highest scores were not exclusively found on affected areas, but also on lesion-free sites, demonstrating that atopic animals can be heavily colonized also in apparently healthy areas.     

In vitro antifungal susceptibility of Malassezia pachydermatis from dogs with and without skin lesions

Canine Malassezia dermatitis is frequently treated with systemic ketoconazole (KTZ) and itraconazole (ITZ). However, no information is available on the antifungal susceptibility to azoles and allilamine of Malassezia pachydermatis isolates from dogs with or without skin lesions. The present study was designed to evaluate the in vitro antifungal susceptibility of M. pachydermatis strains from dogs with or without skin lesions to KTZ, ITZ, miconazole (MICO), fluconazole (FLZ), posaconazole (POS), voriconazole (VOR) and terbinafine (TER) using the Clinical and Laboratory Standards Institute reference Broth Microdilution Method (CLSI M27-A2). The association between the susceptibility to antifungal compounds and the origin of M. pachydermatis, from skin with or without lesions has been also assessed. A total of 62 M. pachydermatis strains from healthy dogs (i.e., Group A=30) or with skin lesions (i.e., Group B=32) were tested. ITZ, KTZ and POS showed the highest activity against M. pachydermatis strains, whereas MICO TER and FLZ the lowest. A higher number of Malassezia resistant strains were registered among isolates from Group B than those from Group A. This study indicates that M. pachydermatis strains were susceptible to ITZ, KTZ, and POS. However, dogs with lesions may harbour strains with low susceptibility to antifungal agents and displaying cross-resistance phenomena to azole. The antifungal therapy in Malassezia infections requires careful appraisal of choice of drugs especially in cases of unresponsiveness to antifungal treatment or recurrent infections.     

Heterogeneity of Staphylococcus pseudintermedius isolates from atopic and healthy dogs

Staphylococcus pseudintermedius is part of the normal canine flora but frequently causes pyoderma in canine atopic dermatitis (AD). This study aimed to determine whether particular S. pseudintermedius strains were associated with AD and/or pyoderma. Ninety‐six S. pseudintermedius isolates from the ear, nares, perineum and lesions of 21 atopic and 16 healthy dogs were lysed with proteinase K and digested with 40 U SmaI. Restriction products were separated using pulsed‐field gel electrophoresis (PFGE) with an Oxford S. aureus control and lambda‐ladder DNA concatomer markers. A dendrogram was constructed by the unweighted pair group method. All isolates showed a ≥56% similarity coefficient. Nine distinct PFGE clusters were identified, as follows: five from both atopic and healthy dogs; three from atopic dogs only; and one from healthy dogs only. Nine clusters were isolated from the nares, eight from the perineum, five from the ears and six from pyoderma lesions. There were no significant differences in the frequency of isolation from atopic or healthy skin, body sites or infected lesions for any of the clusters. Two of six healthy dogs and 18 of 20 atopic dogs with multiple isolates had closely related isolates (less than three band differences) at more than one sampling site. Isolates from pyoderma lesions were closely related to at least one mucosal isolate in 11 of 16 dogs. Staphylococcus pseudintermedius isolates appear to be heterogeneous, and colonization or infection of atopic skin was not associated with any particular strain or cluster of strains.     

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%).