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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.
La réponse en immunoglobulines G à des extraits de Malassezia pachydermatis chez les chiens atopiques et non atopiques. L’immunoréactivité des IgG envers Malassezia pachydermatis a été comparée chez des chiens atopiques et non atopiques. Des protéines de Malassezia pachydermatis avec un poids moléculaire de 98 kDa ont été reconnues à une fréquence significativement supérieure dans le sérum des chiens atopiques. La plupart des chiens atopiques avec une dermatite à Malassezia présentaient une réponse en IgG supérieure à celle des chiens normaux.
(Traduit par Isabelle Vallières)
Malassezia pachydermatis is frequently isolated from the skin and mucosal sites of healthy dogs, but some dogs with dermatitis have remarkably increased populations of the yeasts, suggesting that the organism is an opportunistic pathogen (1). Previous studies have suggested that Malassezia species induce both humoral and cell-mediated immune responses in humans and dogs (2,3). Elevated Malassezia-specific serum IgG and IgE have been identified in atopic dogs (4). Atopic dogs also have elevated serum IgE titers to M. pachydermatis, which frequently show immunoreactivity to antigens of 45, 52, 56, and 65 kDa (5), and dogs with M. pachydermatis have a greater IgG response to a clinically important 25 kDa antigen when compared with control animals (6). Furthermore, anti-M. pachydermatis IgG immunoreactivity towards proteins with molecular weights of 219, 110, 71, and 42 kDa has been observed in affected dogs (7). The results of many previous studies designed to identify antigenic proteins of M. pachydermatis and the role of immunoglobulins have been so variable that the pathogenesis of M. pachydermatis is still unclear.
The purpose of this study was to compare the IgG immune responses to M. pachydermatis in atopic and non-atopic dogs and to identify antigenic proteins of the yeast.
Serum samples were collected from 14 atopic dogs and 14 non-atopic dogs presented to the Konkuk University Veterinary Teaching Hospital.
Identification of atopic dermatitis (AD) was based on a combination of consistent history and clinical signs, and exclusion of other causes of pruritic skin diseases (8). We performed skin scraping to exclude ectoparasites. In addition, antiparasitic control included monthly topical application of Fipronil (Frontline; Mérial, Lyon, France), systemic Ivermectin (Virbamec; Virbac Laboratorios, Guadalajera, Mexico), 0.5 mg/kg, SC, weekly for 1 mo, and bathing with amitraz (Greentix; Green Crtoss Veterinary Products, Young-In, Korea) once a week for 2 wk.
The atopic dogs were fed an 8-week trial diet with a commercial hypoallergenic food to rule out food allergy. No anti-inflammatory medication was given for at least 3 wk prior to examination (5). Four of the dogs were clipped, sedated (medetomidine, Pfizer Animal Health, New York, New York, USA), 20 μg/kg, IV, and intradermal skin tests were performed with 40 allergens (Greer laboratories, Lenoir, North Carolina, USA) using 0.05 mL of each allergen extract on the lateral flank. Histamine (1:100 000 w/v) and a buffered-saline diluent were also injected as positive and negative controls, respectively. Test sites were assessed after 15 min and scored from 0 to + 4 by comparison with the controls. Reactions ≥ 2 were considered to be positive (9).
Firm cytological criteria for Malassezia overgrowth have not been established, and there are important breed and site differences in yeast numbers in healthy dogs (10). Malassezia overgrowth was diagnosed by microscopic observation of Diff-Quik (Sysmex, Kobe, Japan) stained tape strips. Samples were obtained from the groin, axilla, and interdigital areas. For this study, Malassezia overgrowth was defined as an average of 5 or more Malassezia organisms per 400 × field (5,10).
Non-atopic dogs had no history or clinical signs of skin disease and had no lesions upon dermatological examination. Most of the non-atopic dogs had been presented for other diseases.
An isolate of M. pachydermatis was obtained from the skin of an atopic dog with Malassezia overgrowth. The sample was cultured for 48 h at 37°C on Sabouraud dextrose agar (Becton Dickinson, Le Pont de Claix-Cedex, France) containing 20 mg/mL chlor-amphenicol (Helocetin; Chong Gun Dang Pharm, Korea), and the colonies were identified as M. pachydermatis by microscopic examination. The colonies were subcultured onto a large number of plates to obtain an adequate number of organisms for the subsequent studies (5).
Malassezia colonies were harvested, suspended in phosphate-buffered saline (PBS) (pH 7.4), and washed by 3 cycles of centrifugation at 500 × g for 5 min followed by removal of the supernatant and resuspension of the pellet in PBS. After the last washing cycle, the cells were resuspended in an extraction buffer (pH 7.4) containing 125 mM NH4HCO3 (Sigma, St. Louis, Missouri, USA) and protease inhibitors (20 mM -aminocaproic acid, 5 mM ethylenediaminetetra-acetic acid, and 1 mM phenylmethylsulfonyl fluoride; Sigma) (11,12). The Malassezia colonies in the extraction buffer were mixed vigorously on a vortex mixer for 10 min with an equal volume of glass beads (0.4 mm; Sigma) to mechanically disrupt the cell membranes. After extraction, the cell suspensions were stored at 4°C overnight, centrifuged at 6000 × g for 5 min and the supernatants were collected. The amount of protein obtained was measured by the protein A 280 method (13).
Gel electrophoresis was performed according to the method of Laemmli (13) using a mini-protean 3 system (Bio-Rad, USA) in a discontinuous buffer system containing 0.025 M Tris (Bio-Rad), 0.192 M Glycine (Bio-Rad), and 0.1% SDS (Bio-Rad), pH 8.3. The gel was comprised of a 10% separating and a 4% stacking gel. The extracts (400 μg) were diluted 2:1 with reducing sample buffer (containing 5% β-mercaptoethanol, Bio-Rad), heated at 95°C for 5 min and then loaded onto gels next to a lane loaded with molecular markers (Bio-Rad). The electrophoresis was run at 130 V for 60 min and the gel stained with Coomassie Brilliant Blue R-250 (Bio-Rad).
The separated proteins and molecular weight standards were transferred from the gel to a nitrocellulose membrane in a Bio-Rad Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell according to the manufacturer’s instructions. The transfer buffer contained 48 mM Tris (Sigma, UK), 39 mM glycine, 0.0375% SDS (Bio-Rad), and 20% methanol (Duksan Chemical, Pusan, South Korea), pH 8.9. The transfer was run for 20 min at 15 V.
The individual serum samples were analyzed under the following conditions. The nitrocellulose membranes were removed and washed in Tris-buffered saline pH 7.5 (TBS) for 30 min with gentle rocking. The residual binding sites were blocked by incubating the membranes in TBS containing 1% skimmed milk for 30 min at room temperature with gentle rocking. The membranes were washed with PBS containing 0.05% Tween 20 (Sigma, USA) (PBST) for 30 min (three, 10-minute rinses), cut into vertical strips (5-mm wide), and then each strip was placed into a small petri dish. A strip was incubated with each serum sample at 1/100 in PBST for 2 h at room temperature. The membranes were washed with PBST for 30 min (three, 10-minute rinses), and then incubated for 2 h at room temperature with Horseradish Peroxidase (HRP)-conjugated rabbit anti-dog IgG (Sigma, USA) at 1:2000 in PBST. After washing with PBST for 30 min (three, 10-minute rinses), the strips were developed for 25 min in a substrate solution containing 4-chloro-1-naphthol (30 mg/mL in methanol). The molecular weights of the visualized bands were calculated by their relative mobility from the regression line of the log10 of the molecular weight of the standard proteins plotted against their relative mobility. The gel was analyzed using image analysis software (ImageJ 1.40g, National Institutes of Health, USA).
The bands were classified into negative, weak, and strong reaction based on their intensity. The intensities of zero to half of the maximum intensity, half to the maximum intensity, were defined as weak and strong, respectively. Fisher’s exact test was used to assess the differences between the percentages of atopic and non-atopic dogs showing strong IgG binding to individual proteins in the M. pachydermatis extracts.
Age, sex, and breed of the study animals and the diseases of the non-atopic dogs are detailed in Table 1. The mean ages of atopic and non-atopic dogs were 6.0 y and 3.4 y, respectively. Of the 28 dogs in the study, 15 were male and 13 were female. Only 1 dog was healthy; the others had various systemic diseases.
The extracts of M. pachydermatis were separated in SDS-PAGE. The IgG immunoblots of dog sera from atopic dogs and non-atopic dogs are shown in Figure 1. The IgG binding proteins with molecular weights of 73 (50% of dogs), 74 (50%), 86 (71%), 88 (71%), 98 (50%) kDa were recognized in > 50% of the atopic dogs. Most of the non-atopic dogs showed weak binding to those proteins, and strong binding was detected in 28.5%, 21.4%, 50%, 42.8%, and 7.1%, respectively. The differences between atopic and non-atopic dogs in IgG binding to the main proteins of M. pachydermatis, however, were not statistically significant except for the 98 kDa protein (P < 0.05) (Figure 1).
Malassezia pachydermatis is a resident organism in the canine stratum corneum. Previous studies (5,6) have shown that both healthy dogs and dogs with M. pachydermatis-related skin disease had serum IgG antibodies to the yeast. Cutaneous hypersensitivity, including AD, is a common underlying cause of alterations to the cutaneous microclimate or to the host defense mechanisms allowing M. pachydermatis to multiply and cause disease. Atopic dogs, therefore, may have a greater sensitivity to the variety of immune responses induced by M. pachydermatis.
Previous studies have reported that atopic dogs, with clinical evidence of Malassezia dermatitis and otitis, had significantly higher serum IgG and IgE levels than either healthy dogs or non-atopic dogs (4,5). Another study showed that atopic working dogs had a significantly higher concentration of serum total IgG than non-atopic working dogs (14). These results suggest that immunoreactivity to M. pachydermatis in atopic dogs could be associated with an increased concentration of serum IgG or IgE.
In our study, the intensity of IgG binding seen in atopic dogs was higher than that in the non-atopic dogs for most of the identified M. pachydermatis proteins, although the differences were statistically significant for only the 98 kDa protein. Our study showed that atopic dogs had a high IgG immunoreactivity to the M. pachydermatis proteins with molecular weights of 73, 74, 86, 88, 98 kDa, suggesting that these specific yeast proteins may be relevant in atopic dogs. These observations do not agree with the results of previous studies in which proteins of different molecular weights were identified (5,6). The differences in immunoreactivity to M. pachydermatis between different studies could be related to differences in the phase of yeast growth, individual host responses, strain variability, and the different extraction processes affecting antigen release and stability (15). In addition, the different results could be related to differences in immunoreactivity between IgG and IgE in various studies.
According to a previous report (6), the IgG may act as an opsonin to facilitate phagocytosis of Malassezia antigens. This could have a protective function but it could also activate the complement system and exacerbate the underlying inflammation. It has been shown that Malassezia is capable of activating both the classical and alternative complement pathways (16).
In our study, an antigen of 98 kDa and of unknown function and identity was recognized by 50% of the atopic dogs and only 7.1% of the non-atopic dogs. Protein purification and N-terminal amino acid sequencing may assist in positively identifying this antigen and the other relevant antigens of M. pachydermatis with molecular weights of 73, 74, 86, and 88 kDa.
In conclusion, this study demonstrates that total serum IgG immunoreactivity to M. pachydermatis antigens was higher in atopic dogs than non-atopic dogs, particularly with a 98 kDa antigen.
This study was supported in part by a grant from the Brain Korea 21 Project and the Research Institute for Veterinary Science, College of Veterinary Medicine, Konkuk University, Korea. CVJ
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