TY - JOUR
AU - Otranto, Domenico
AB - Abstract Malassezia pachydermatis isolates (n=185) from skin sites from dogs (n=30) were characterized genetically and biochemically following in vitro culture. Two regions in the chitin synthase-2 gene (chs-2) and the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA were sequenced, and the phospholipase activity of each isolate was assessed. Three chs-2 (i.e. Ac, Bc and Cc) and eight ITS-1 (i.e. AI1, AI2, AI3, AI4, BI1, CI1, CI2 and CI3) sequence types were defined for all 185 samples. The findings revealed that multiple M. pachydermatis genotypes/subgenotypes could be cultured from healthy dogs or from dogs with single or multiple, generalized skin lesions. Subgenotypes AI1 and BI1 were associated with all skin sites of dogs sampled, whereas subgenotype CI2 was mostly linked to a particular location. Isolates derived from skin lesions showed a significantly higher phospholipase activity compared with those from skin sites with no detectable lesions. Genotype B was mainly cultured from healthy skin; only four isolates (9.3%) had low phospholipase activity, whereas other genotypes/subgenotypes were predominantly associated with skin lesions and had a high phospholipase activity. The results of the present study suggest that the distribution pattern of particular genotypes or subgenotypes of M. pachydermatis on the skin of dogs relates to the affinity of the yeast to the host and to particular skin sites. Malassezia pachydermatis, chitin synthase-2 gene (chs-2), first internal transcribed spacer (ITS-1), phospholipase, single-strand conformation polymorphism (SSCP) analysis Introduction Members of the genus Malassezia are lipophilic, unipolar, budding yeasts characterized by a thick cell wall (Guillot & Bond, 1999; Batra et al., 2005). In recent years, this genus has received considerable attention from dermatologists and clinicians (Batra et al., 2005). Although these yeasts are common commensals of the skin of animals, they may become pathogenic under the influence of particular predisposing factors (Guého et al., 1998; Guillot & Bond, 1999; Batra et al., 2005; Chen & Hill, 2005; Ashbee, 2007). Currently, some Malassezia spp. are considered as emerging pathogens (Batra et al., 2005). Among them, Malassezia pachydermatis, the only member that does not require lipid supplementation for growth in culture, has been considered traditionally to be zoophilic, and is frequently found on wild and domestic carnivores (including dog, fox, cat, ferret and bear). This yeast is usually associated with otitis externa and with different clinical forms of dermatitis in domestic animals, particularly dogs (Guillot & Bond, 1999; Batra et al., 2005). It has also been reported as a commensal on the skin of dog owners (Morris, 2005), and a causative agent of nosocomial infection in humans, being transmitted by human health-care workers from their pet dogs to neonatal patients (Chang et al., 1998; Chryssanthou et al., 2001). Recently, the sequencing of regions in nuclear ribosomal DNA (e.g. large subunit, LSU, and first internal transcribed spacer, ITS-1) and chitin synthase-2 gene (chs-2) of Malassezia species has been used for taxonomic and/or epidemiological studies (Guého et al., 1995; Guillot et al., 1997; Aizawa et al., 2001; Sugita et al., 2005; Cabanes et al., 2007; Cafarchia et al., 2007b, c), allowing Malassezia species to be classified genetically. Other studies have revealed concordance in the genotypic classification of M. pachydermatis from dogs among the loci LSU, chs-2 and ITS-1 (Cafarchia et al., 2007b, c). It has been proposed that phospholipase production by M. pachydermatis is associated with its occurrence in skin lesions and pathogenicity (Cafarchia & Otranto, 2004). Phospholipases are a heterogeneous group of enzymes that hydrolyze one or more ester linkages in glycerophospholipids, and specifically cleave ester bonds (Ghannoum, 2000). They are also produced by fungi known to be pathogenic via damage to host cell membranes (Barret-Bee et al., 1985; Chen et al., 1997). Interestingly, to date, there is no information linking pathogenicity and/or the ability to produce phospholipase to genetic variants within M. pachydermatis. As a first step toward addressing these issues, the aim of this work was to molecularly characterize isolates of M. pachydermatis from different anatomical sites of dogs with and without dermatitis, and to establish their phospholipase activity profiles in vitro. Materials and methods Malassezia isolates and their phenotypic identification Malassezia pachydermatis isolates (n=185), with at least two being derived from seven skin sites (i.e. periorbital, perioral, dorsal area of neck, perianal, inguinal, interdigital and external acustic meatus with pinna), were sampled from 30 dogs with or without skin lesions and/or otitis, and cultured using modified Dixon agar (Guého et al., 1995). Included in the study were privately owned, domestic dogs referred to the Faculty of Veterinary Medicine of the University of Bari, Italy. The dogs were in good general health, with no known history of antibiotic or antifungal treatment in the preceding 5 months. Specifically, isolates were divided into three groups: Group I consisting of 81 isolates collected from 15 healthy dogs, Group II comprising 59 isolates from nine dogs with localized dermatitis characterized by erythema and pruritus at only one site (i.e. two dogs, each presenting periorbital or otitis with pinna hyperkeratosis and two dogs presenting perioral, two interdigital and three inguinal dermatitis), and Group III consisting of 45 isolates collected from six dogs with generalized dermatitis characterized by erythema and/or hyperkeratosis in more than one site and being associated clinically with pruritus (Table 1). Malassezia pachydermatis was identified phenotypically based on morphology (macroscopically and microscopically) and its ability to grow on medium without lipid supplementation (Sabouraud Dextrose Agar, Biolife®-SAB) (Guillot et al., 1996). Isolates were maintained on modified Dixon agar. All isolates were subjected to molecular characterization and evaluated for phospholipase activity. 1 Number of Malassezia pachydermatis genotypes/subgenotypes for chs-2 (Ac–Cc) and ITS-1 genotypes (AI–CI) or subgenotypes (with subscript numbers) from dogs of Group I (healthy dogs), Group II (dogs with localized dermatitis at only one site) and Group III (dogs with generalized dermatitis and hyperkeratosis localized in more than one site), divided according their sites of collection chs-2 genotype  ITS-1 genotype/ subgenotype  Group I  Group II  Group III  Total  α  β  γ  δ  ɛ  ζ  η  α  β  γ  δ  ɛ  ζ  η  α  β  γ  δ  ɛ  ζ  η  Ac  AI1  –  2  –  2  4  –  4  2  –  6  –  4  –  2  5  4  8  –  6  –  8  57  AI2  –  –  –  –  –  –  –  –  2  2  2  2  2  2  –  –  –  2  –  –  –  14  AI3  –  –  –  –  –  –  –  2  –  –  –  2  –  –  –  –  2  –  –  –  –  6  AI4  –  –  –  –  –  –  –  –  –  –  –  2  –  2  –  –  –  –  –  –  –  4  Bc  BI1  17  12  8  2  2  –  2  6  –  –  –  2  2  –  –  –  –  –  –  –  2  55  Cc  CI1  2  –  2  –  2  2  –  –  2  –  –  –  –  2  2  –  –  –  –  –  –  14  CI2  –  2  –  –  –  –  2  2  3  2  –  –  –  2  2  2  –  –  –  –  2  19  CI3  4  4  –  –  2  –  4  2  –  –  –  –  –  –  –  –  –  –  –  –  –  16  Total  23  20  10  4  10  2  12  14  7  10  2  12  4  10  9  6  10  2  6  12  185  chs-2 genotype  ITS-1 genotype/ subgenotype  Group I  Group II  Group III  Total  α  β  γ  δ  ɛ  ζ  η  α  β  γ  δ  ɛ  ζ  η  α  β  γ  δ  ɛ  ζ  η  Ac  AI1  –  2  –  2  4  –  4  2  –  6  –  4  –  2  5  4  8  –  6  –  8  57  AI2  –  –  –  –  –  –  –  –  2  2  2  2  2  2  –  –  –  2  –  –  –  14  AI3  –  –  –  –  –  –  –  2  –  –  –  2  –  –  –  –  2  –  –  –  –  6  AI4  –  –  –  –  –  –  –  –  –  –  –  2  –  2  –  –  –  –  –  –  –  4  Bc  BI1  17  12  8  2  2  –  2  6  –  –  –  2  2  –  –  –  –  –  –  –  2  55  Cc  CI1  2  –  2  –  2  2  –  –  2  –  –  –  –  2  2  –  –  –  –  –  –  14  CI2  –  2  –  –  –  –  2  2  3  2  –  –  –  2  2  2  –  –  –  –  2  19  CI3  4  4  –  –  2  –  4  2  –  –  –  –  –  –  –  –  –  –  –  –  –  16  Total  23  20  10  4  10  2  12  14  7  10  2  12  4  10  9  6  10  2  6  12  185  (i.e. α, perioral; β, periorbital; γ, inguinal; δ, perianal; ɛ, interdigital; ζ, dorsal; η, external acustic meatus). View Large 1 Number of Malassezia pachydermatis genotypes/subgenotypes for chs-2 (Ac–Cc) and ITS-1 genotypes (AI–CI) or subgenotypes (with subscript numbers) from dogs of Group I (healthy dogs), Group II (dogs with localized dermatitis at only one site) and Group III (dogs with generalized dermatitis and hyperkeratosis localized in more than one site), divided according their sites of collection chs-2 genotype  ITS-1 genotype/ subgenotype  Group I  Group II  Group III  Total  α  β  γ  δ  ɛ  ζ  η  α  β  γ  δ  ɛ  ζ  η  α  β  γ  δ  ɛ  ζ  η  Ac  AI1  –  2  –  2  4  –  4  2  –  6  –  4  –  2  5  4  8  –  6  –  8  57  AI2  –  –  –  –  –  –  –  –  2  2  2  2  2  2  –  –  –  2  –  –  –  14  AI3  –  –  –  –  –  –  –  2  –  –  –  2  –  –  –  –  2  –  –  –  –  6  AI4  –  –  –  –  –  –  –  –  –  –  –  2  –  2  –  –  –  –  –  –  –  4  Bc  BI1  17  12  8  2  2  –  2  6  –  –  –  2  2  –  –  –  –  –  –  –  2  55  Cc  CI1  2  –  2  –  2  2  –  –  2  –  –  –  –  2  2  –  –  –  –  –  –  14  CI2  –  2  –  –  –  –  2  2  3  2  –  –  –  2  2  2  –  –  –  –  2  19  CI3  4  4  –  –  2  –  4  2  –  –  –  –  –  –  –  –  –  –  –  –  –  16  Total  23  20  10  4  10  2  12  14  7  10  2  12  4  10  9  6  10  2  6  12  185  chs-2 genotype  ITS-1 genotype/ subgenotype  Group I  Group II  Group III  Total  α  β  γ  δ  ɛ  ζ  η  α  β  γ  δ  ɛ  ζ  η  α  β  γ  δ  ɛ  ζ  η  Ac  AI1  –  2  –  2  4  –  4  2  –  6  –  4  –  2  5  4  8  –  6  –  8  57  AI2  –  –  –  –  –  –  –  –  2  2  2  2  2  2  –  –  –  2  –  –  –  14  AI3  –  –  –  –  –  –  –  2  –  –  –  2  –  –  –  –  2  –  –  –  –  6  AI4  –  –  –  –  –  –  –  –  –  –  –  2  –  2  –  –  –  –  –  –  –  4  Bc  BI1  17  12  8  2  2  –  2  6  –  –  –  2  2  –  –  –  –  –  –  –  2  55  Cc  CI1  2  –  2  –  2  2  –  –  2  –  –  –  –  2  2  –  –  –  –  –  –  14  CI2  –  2  –  –  –  –  2  2  3  2  –  –  –  2  2  2  –  –  –  –  2  19  CI3  4  4  –  –  2  –  4  2  –  –  –  –  –  –  –  –  –  –  –  –  –  16  Total  23  20  10  4  10  2  12  14  7  10  2  12  4  10  9  6  10  2  6  12  185  (i.e. α, perioral; β, periorbital; γ, inguinal; δ, perianal; ɛ, interdigital; ζ, dorsal; η, external acustic meatus). View Large Isolation of genomic DNA and enzymatic amplification Yeasts were cultured in 3 mL of modified Dixon broth at 32 °C for 7 days. Genomic DNA was extracted from 1 mL of culture (containing c. 1–2 × 108 cells), as described previously (Cafarchia et al., 2007c). The chs-2 gene (c. 540 bp) was amplified from genomic DNA by the PCR using the primers CED1 (5′-CTATTCACTCGAACCATGCATGGTGTC-3′) and CED2 (5′-GAGAAGCGCGTGCCACATGGTGCC-3′), as was the ITS-1 region (c. 285 bp) employing the primers 18SF1 (5′-AGGTTTCCGTAGGTGAACCT-3′) and 5.8SR1 (5′-TTCGCTGCGTTCTTCATCGA-3′) (Cafarchia et al., 2007c). Genomic DNA (4 μL) was added to the PCR mix (46 μL) containing 2.5 mM MgCl2, 10 mM Tris-HCl, pH 8.3 and 50 mM KCl, 250 μM of each dNTP, 50 pmol of each primer and 1.25 U of Ampli Taq Gold (Applied Biosystems). PCR was performed in a thermal cycler (2700, Applied Biosystems) at 94 °C for 12 min (polymerase activation), followed by 25–30 cycles of 94 °C for 1 min (chs-2) or 30 s (ITS-1) (denaturation), respectively; 60 °C for 1 min (chs-2) or 15 s (ITS-1) (annealing); 72 °C for 2 min (chs-2) or 15 s (ITS-1) (extension), followed by 7 min at 72 °C (final extension). Agarose gel electrophoresis and sequence analysis Amplicons were examined in 2% w/v agarose (Ambion) gels, stained with ethidium bromide (10 mg mL−1) and then purified using Ultrafree-DA columns (Amicon, Millipore, Bedford) and sequenced directly using the Taq DyeDeoxyTerminator Cycle Sequencing Kit (v.2, Applied Biosystems), employing an automated sequencer (ABI-PRISM 377). Sequences were determined from both strands (using the same primers individually as for the PCR) and the electro-pherograms verified by eye. Sequences were aligned using the clustalx program (Thompson et al., 1997). Pairwise comparisons of sequence differences (D) were made using the formula D=1−(M/L) (Chilton et al., 1995), where M is the number of alignment positions at which the two sequences have a base in common, and L is the total number of alignment positions over which the two sequences are compared. The nucleotide sequences reported in this paper are available in the GenBank, EMBL and DDBJ databases under accession nos. EU158826, EU158827, EU158828 and EU158829. Mutation scanning analysis Amplicons representing the different genetic variants of M. pachydermatis were subjected to nonisotopic single-strand conformation polymorphism analysis, according to protocol B (Gasser et al., 2006b). In brief, after denaturation at 94 °C for 30 min and snap cooling on a freeze block (−20 °C), 15 μL of each sample (i.e. 1–2 μL of amplicon plus 8 μL of water plus 5 μL of loading buffer) were loaded into the wells of precast Gene Mutation Analysis (GMA) gels (S-2 × 13, Elchrom Scientific AG) and subjected to electrophoresis for 19 h at 74 V and 7.4 °C (constant) in a horizontal SEA2000 apparatus (Elchrom Scientific) connected to a MultiTemp III (Pharmacia) cooling system. After electrophoresis, gels were stained for 15 min with SYBR Gold (1 : 10 000 dilution) and photographed using a GelDoc system (Bio-Rad). SSCP profiles were demonstrated to be reproducible on different days using amplicons produced on different days (not shown). Biochemical evaluation of phospholipase production The phospholipase production was assessed using the egg-yolk plate method (Cafarchia & Otranto, 2004), with some modifications in the preparation of the inoculum. Briefly, the isolates were incubated in slants of Dixon agar and suspended in Dixon broth (CFU: 2.5 × 103 mL−1) and incubated again (3 days at 32 °C for both media). Ten microliters of yeast suspension were then transferred to the egg-yolk plates and incubated at 32 °C for 10 days. After this time, the formation of zones of precipitation around the colonies was considered as indicative of enzyme production. The production of phospholipase was expressed as a ratio of colony diameter (a) to total diameter of the colonies plus zone of precipitation (b) (Price et al., 1982). Hence, the higher the phospholipase value, the lower the production of phospholipases. Each strain was tested in duplicate, and the phospholipase recorded as an average of the two phospholipase values for each isolate. Phospholipase activity was expressed as a mean of phospholipase values. Statistical analysis The χ2-test was used to compare the number of isolates with each (chs-2 or ITS-1) sequence type from Groups I, II and III as well as the number of isolates within each group producing phospholipase. The anova, followed by the post hoc test of Tukey, was used to evaluate the differences among the phospholipase mean values of different genotypes/subgenotypes within each Group (I, II or III). A value of P≤0.05 was considered to be statistically significant. Results All 185 M. pachydermatis isolates, taken from at least two different skin sites from each of 30 dogs (clinically unaffected or affected), were cultured using modified Dixon agar. Genomic DNA isolated from each of the cultured samples was then subjected to PCR-coupled sequencing of chs-2 (c. 540 bp) and ITS-1 (c. 280 bp). Three chs-2 and eight ITS-1 sequence types (Fig. 1) were determined for all 185 samples. There was no evidence of multiple types of sequence (via the detection of sequence polymorphism) within any of the samples following in vitro culture. All three chs-2 sequence types matched previously determined sequences (accession nos. DQ915507, DQ915508 and DQ915509;Cafarchia et al., 2007c). Four ITS-1 sequences matched those published previously (accession nos. DQ915503, DQ915504, DQ915505 and DQ915506; Cafarchia et al., 2007c). Four new ITS-1 sequence types (accession nos. EU158826, EU158827, EU158828 and EU158829; Fig. 1) were defined, and did not match any sequences available in current gene databases. Pairwise comparisons among the sequences revealed differences of 1.9–3.4% in chs-2 and 0.4–7.0% in ITS-1 (Table 2). 1 View largeDownload slide Alignment of all ITS-1 sequence types representing Malassezia pachydermatis genotypes or subgenotypes cultured from the skin from dogs (Groups I–III). 1 View largeDownload slide Alignment of all ITS-1 sequence types representing Malassezia pachydermatis genotypes or subgenotypes cultured from the skin from dogs (Groups I–III). 2 Pairwise comparisons (Pwc) of sequence differences (%) in ITS-1 among all Malassezia pachydermatis isolates (n=185) cultured from canine skin Sequence type  Pwc (%)  AI1  AI2  AI3  AI4  BI1  CI1  CI2  CI3  AI1  –  AI2  0.4  –  AI3  0.4  0.77  –  AI4  0.4  0.77  0.77  –  BI1  4.6  4.3  5.4  5  –  CI1  6.2  5.8  6.6  6.6  3.9  –  CI2  6.2  5.8  6.6  6.6  3.5  0.4  –  CI3  6.6  6.2  7  7  3.9  0.77  0.4  –  Sequence type  Pwc (%)  AI1  AI2  AI3  AI4  BI1  CI1  CI2  CI3  AI1  –  AI2  0.4  –  AI3  0.4  0.77  –  AI4  0.4  0.77  0.77  –  BI1  4.6  4.3  5.4  5  –  CI1  6.2  5.8  6.6  6.6  3.9  –  CI2  6.2  5.8  6.6  6.6  3.5  0.4  –  CI3  6.6  6.2  7  7  3.9  0.77  0.4  –  View Large 2 Pairwise comparisons (Pwc) of sequence differences (%) in ITS-1 among all Malassezia pachydermatis isolates (n=185) cultured from canine skin Sequence type  Pwc (%)  AI1  AI2  AI3  AI4  BI1  CI1  CI2  CI3  AI1  –  AI2  0.4  –  AI3  0.4  0.77  –  AI4  0.4  0.77  0.77  –  BI1  4.6  4.3  5.4  5  –  CI1  6.2  5.8  6.6  6.6  3.9  –  CI2  6.2  5.8  6.6  6.6  3.5  0.4  –  CI3  6.6  6.2  7  7  3.9  0.77  0.4  –  Sequence type  Pwc (%)  AI1  AI2  AI3  AI4  BI1  CI1  CI2  CI3  AI1  –  AI2  0.4  –  AI3  0.4  0.77  –  AI4  0.4  0.77  0.77  –  BI1  4.6  4.3  5.4  5  –  CI1  6.2  5.8  6.6  6.6  3.9  –  CI2  6.2  5.8  6.6  6.6  3.5  0.4  –  CI3  6.6  6.2  7  7  3.9  0.77  0.4  –  View Large For chs-2 and ITS-1, three genotypes Ac–Cc and AI–CI (subscript c and I referring to chs-2 and ITS-1, respectively) of M. pachydermatis were defined (Tables 1–3). Thus, based on levels of sequence difference (Table 2; Fig. 1), the genotypic grouping (A–C) were concordant between chs-2 and ITS-1. As ITS-1 was considerably more variable in sequence than the protein coding gene chs-2, subgenotypes (i.e. variants within genotypes AI1–AI4 and CI1–CI3) could be classified (based on the nature of nucleotide alterations and sequence similarity); no variation was detected within genotype BI1. While these genotypic/subgenotypic classifications based on the apparent relatedness of ITS-1 sequence types were practical, phylogenetic analysis (using neighbor joining, maximum likelihood and maximum parsimony methods) did not necessarily provide strong bootstrap support for some subgenotypic groups (data not shown), likely due to the relatively short length of ITS-1. SSCP-based analysis of ITS-1 amplicons allowed the display of sequence variation (0.4–7.0%) among all genotypes/subgenotypes (Fig. 2). 3 Frequency distribution of Malassezia pachydermatis genotypes/subgenotypes (for the loci chs-2 and ITS-1) from dogs in Groups I–III Group  Number of isolates  chs-2 genotypes  ITS-1 genotypes/subgenotypes  Ac  Bc  Cc  AI1  AI2  AI3  AI4  BI1  CI1  CI2  CI3  I  81  12a,b  43a  26b  12ijk  0  0  0  43ilmn  8jl  4kmo  14no  (14.8)  (53.1)  (32.1)  (14.8)  (53.1)  (9.8)  (4.9)  (17.3)  II  59  34c,d  10c  15d  14pqrs  12  4p  4q  10t  4r  9  2st  (57.6)  (16.9)  (25.4)  (23.7)  (20.3)  (6.78)  (6.78)  (16.9)  (6.78)  (15.2)  (3.39)  III  45  35e,f  2e  8f  31uvwxy  2u  2v  0  2w  2x  6s  0  (77.7)  (4.4)  (17.7)  (68.8)  (4.44)  (4.44)  (4.44)  (4.44)  (13.3)  Total  185  81g,h  55g  49h  57zαβδɛ  14zγ  6αζη  4θιλ  55γθημξπ  14βμ  19δζιξ  16ɛλπ  (43.8)  (29.7)  (26.5)  (30.8)  (7.56)  (3.24)  (2.16)  (29.7)  (7.56)  (10.2)  (8.64)  Group  Number of isolates  chs-2 genotypes  ITS-1 genotypes/subgenotypes  Ac  Bc  Cc  AI1  AI2  AI3  AI4  BI1  CI1  CI2  CI3  I  81  12a,b  43a  26b  12ijk  0  0  0  43ilmn  8jl  4kmo  14no  (14.8)  (53.1)  (32.1)  (14.8)  (53.1)  (9.8)  (4.9)  (17.3)  II  59  34c,d  10c  15d  14pqrs  12  4p  4q  10t  4r  9  2st  (57.6)  (16.9)  (25.4)  (23.7)  (20.3)  (6.78)  (6.78)  (16.9)  (6.78)  (15.2)  (3.39)  III  45  35e,f  2e  8f  31uvwxy  2u  2v  0  2w  2x  6s  0  (77.7)  (4.4)  (17.7)  (68.8)  (4.44)  (4.44)  (4.44)  (4.44)  (13.3)  Total  185  81g,h  55g  49h  57zαβδɛ  14zγ  6αζη  4θιλ  55γθημξπ  14βμ  19δζιξ  16ɛλπ  (43.8)  (29.7)  (26.5)  (30.8)  (7.56)  (3.24)  (2.16)  (29.7)  (7.56)  (10.2)  (8.64)  a–z;α–π : χ-2-test – statistically significant differences (P<0.05) were marked with the same letters. View Large 3 Frequency distribution of Malassezia pachydermatis genotypes/subgenotypes (for the loci chs-2 and ITS-1) from dogs in Groups I–III Group  Number of isolates  chs-2 genotypes  ITS-1 genotypes/subgenotypes  Ac  Bc  Cc  AI1  AI2  AI3  AI4  BI1  CI1  CI2  CI3  I  81  12a,b  43a  26b  12ijk  0  0  0  43ilmn  8jl  4kmo  14no  (14.8)  (53.1)  (32.1)  (14.8)  (53.1)  (9.8)  (4.9)  (17.3)  II  59  34c,d  10c  15d  14pqrs  12  4p  4q  10t  4r  9  2st  (57.6)  (16.9)  (25.4)  (23.7)  (20.3)  (6.78)  (6.78)  (16.9)  (6.78)  (15.2)  (3.39)  III  45  35e,f  2e  8f  31uvwxy  2u  2v  0  2w  2x  6s  0  (77.7)  (4.4)  (17.7)  (68.8)  (4.44)  (4.44)  (4.44)  (4.44)  (13.3)  Total  185  81g,h  55g  49h  57zαβδɛ  14zγ  6αζη  4θιλ  55γθημξπ  14βμ  19δζιξ  16ɛλπ  (43.8)  (29.7)  (26.5)  (30.8)  (7.56)  (3.24)  (2.16)  (29.7)  (7.56)  (10.2)  (8.64)  Group  Number of isolates  chs-2 genotypes  ITS-1 genotypes/subgenotypes  Ac  Bc  Cc  AI1  AI2  AI3  AI4  BI1  CI1  CI2  CI3  I  81  12a,b  43a  26b  12ijk  0  0  0  43ilmn  8jl  4kmo  14no  (14.8)  (53.1)  (32.1)  (14.8)  (53.1)  (9.8)  (4.9)  (17.3)  II  59  34c,d  10c  15d  14pqrs  12  4p  4q  10t  4r  9  2st  (57.6)  (16.9)  (25.4)  (23.7)  (20.3)  (6.78)  (6.78)  (16.9)  (6.78)  (15.2)  (3.39)  III  45  35e,f  2e  8f  31uvwxy  2u  2v  0  2w  2x  6s  0  (77.7)  (4.4)  (17.7)  (68.8)  (4.44)  (4.44)  (4.44)  (4.44)  (13.3)  Total  185  81g,h  55g  49h  57zαβδɛ  14zγ  6αζη  4θιλ  55γθημξπ  14βμ  19δζιξ  16ɛλπ  (43.8)  (29.7)  (26.5)  (30.8)  (7.56)  (3.24)  (2.16)  (29.7)  (7.56)  (10.2)  (8.64)  a–z;α–π : χ-2-test – statistically significant differences (P<0.05) were marked with the same letters. View Large 2 View largeDownload slide Image of an SSCP gel displaying sequence variation among the eight different ITS-1 sequence types (i.e. AI1, AI2, AI3, AI4, BI1, CI1, CI2 and CI3) representing Malassezia pachydermatis genotypes or subgenotypes, cultured from the skin from dogs (Groups I–III). 2 View largeDownload slide Image of an SSCP gel displaying sequence variation among the eight different ITS-1 sequence types (i.e. AI1, AI2, AI3, AI4, BI1, CI1, CI2 and CI3) representing Malassezia pachydermatis genotypes or subgenotypes, cultured from the skin from dogs (Groups I–III). Following the classification of samples using each chs-2 or ITS-1, the frequency distributions of individual genotypes/subgenotypes according to skin location and dog group were determined (see Tables 1 and 3). Because there was no evidence of multiple genotypes (inferred using both chs-2 and ITS-1) within the same sample following in vitro culture, one genotype or subgenotype could be inferred for each skin site (Table 1). Because ITS-1 was considerably more variable in sequence among samples, this locus was more informative for an appraisal of genetic substructuring within M. pachydermatis. Therefore, the frequencies of ITS-1 genotypes and subgenotypes were determined for dogs in individual groups (I–III), according to skin sites (Table 1). All eight genotypes/subgenotypes were identified on dogs in Group II (see Table 3), and five and six (i.e. 62% and 75%) in Groups I and III, respectively. Based on the analyses, a single genotype or subgenotype was characterized per anatomical site, whereas in 50% of dogs multiple genotypes were identified at different anatomical sites on the same individual dog (data not shown). In particular, perioral, periorbital and external acustic meatus were the sites from which a greater number (≥3) of different genotypes/subgenotypes were obtained for the three groups (Table 1). Overall, subgenotype AI1 and genotype BI1 were associated with all anatomical sites on the dogs sampled, whereas subgenotype CI2 was usually linked to a particular location (Table 1). Genotype BI1 and subgenotype AI1 were most frequently detected in Groups I and III, respectively (P<0.05; Table 3). No statistically significant differences in frequency among genotypes/subgenotypes AI1, BI1, AI2 and CI2 were found in Group II (Table 3). In order to examine the relationship between genotypes of M. pachydermatis and pathogenicity, cultured samples were tested for their ability to produce phospholipase. All eight M. pachydermatis genotypes/subgenotypes, defined based on ITS-1, produced phospholipase (Table 4). Overall, the number of M. pachydermatis cultured isolates producing phospholipase and the phospholipase activity were statistically lower for Group I than for Groups II and III (Table 4). Genotypes BI1 and CI1 produced phospholipase only if they were from Groups I and III, respectively. 4 Number (positive/total; P/T) and percentage of Malassezia pachydermatis genotypes/subgenotypes producing phospholipase, listed according to their ITS-1 sequence types ITS-1 genotype/ subgenotype  Group I  Group II  Group III  Total  P/T (%)  Mean phospholipase (SD)  P/T (%)  Mean phospholipase (SD)  P/T (%)  Mean phospholipase (SD)  P/T (%)  Mean phospholipase (SD)  AI1  5/12  0.83  8/14  0.80  20/31  0.84  33/57  0.83  (41.6)  (0.21)  (57)  (0.19)  (64.5)  (0.15)  (57.9)  (0.17)fg  AI2  –  –  8/12  0.88  2/2  0.88  10/14  0.88  (66.6)  (0.09)  (100)  (0.00)  (71.4)  (0.08)  AI3  –  –  1/4  0.89  0/2  1  1/6  0.93  (25)  (0.22)  (0)  (0.00)  (16.6)  (0.18)  AI4  –  –  2/4  0.76  –  –  2/4  0.76  (50)  (0.27)  (50)  (0.27)fh  BI1  4/43  0.97  0/10  1  0/2  1  4/55 (7.2)  0.98  (9.3)  (0.10)  (0)  (0.00)  (0.00)  (0.09)ghil  CI1  0/8  1  0/4  1  2/2  0.57  2/14  0.94  (0)a  (0.00)d  (0)  (0.00)  (100)a  (0.00)d  (14.3)  (0.16)  CI2  1/4  0.90  3/9  0.90  3/6  0.90  7/19  0.90  (25)  (0.21)  (33)  (0.22)  (50)  (0.14)  (36.8)  (0.18)i  CI3  7/14  0.83  1/2  0.75  –  –  8/16  0.82  (50)  (0.20)  (50)  (0.35)  (50)  (0.21)l  Total  17/81  0.93  23/59  0.88  27/45  0.85  67/185  0.89  (21)bc  (0.15)e  (38.9)b  (0.18)  (60)c  (0.16)e  (36.2)  (0.16)  ITS-1 genotype/ subgenotype  Group I  Group II  Group III  Total  P/T (%)  Mean phospholipase (SD)  P/T (%)  Mean phospholipase (SD)  P/T (%)  Mean phospholipase (SD)  P/T (%)  Mean phospholipase (SD)  AI1  5/12  0.83  8/14  0.80  20/31  0.84  33/57  0.83  (41.6)  (0.21)  (57)  (0.19)  (64.5)  (0.15)  (57.9)  (0.17)fg  AI2  –  –  8/12  0.88  2/2  0.88  10/14  0.88  (66.6)  (0.09)  (100)  (0.00)  (71.4)  (0.08)  AI3  –  –  1/4  0.89  0/2  1  1/6  0.93  (25)  (0.22)  (0)  (0.00)  (16.6)  (0.18)  AI4  –  –  2/4  0.76  –  –  2/4  0.76  (50)  (0.27)  (50)  (0.27)fh  BI1  4/43  0.97  0/10  1  0/2  1  4/55 (7.2)  0.98  (9.3)  (0.10)  (0)  (0.00)  (0.00)  (0.09)ghil  CI1  0/8  1  0/4  1  2/2  0.57  2/14  0.94  (0)a  (0.00)d  (0)  (0.00)  (100)a  (0.00)d  (14.3)  (0.16)  CI2  1/4  0.90  3/9  0.90  3/6  0.90  7/19  0.90  (25)  (0.21)  (33)  (0.22)  (50)  (0.14)  (36.8)  (0.18)i  CI3  7/14  0.83  1/2  0.75  –  –  8/16  0.82  (50)  (0.20)  (50)  (0.35)  (50)  (0.21)l  Total  17/81  0.93  23/59  0.88  27/45  0.85  67/185  0.89  (21)bc  (0.15)e  (38.9)b  (0.18)  (60)c  (0.16)e  (36.2)  (0.16)  Phospholipase activity (expressed as the mean phospholipase value) and standard deviations (SD) are also given. a–cχ2-test – statistically significant differences (P<0.05) were marked with the same letters. d–lanova (Tukey post hoc test); statistically significant differences (P<0.05) were marked with the same letters. View Large 4 Number (positive/total; P/T) and percentage of Malassezia pachydermatis genotypes/subgenotypes producing phospholipase, listed according to their ITS-1 sequence types ITS-1 genotype/ subgenotype  Group I  Group II  Group III  Total  P/T (%)  Mean phospholipase (SD)  P/T (%)  Mean phospholipase (SD)  P/T (%)  Mean phospholipase (SD)  P/T (%)  Mean phospholipase (SD)  AI1  5/12  0.83  8/14  0.80  20/31  0.84  33/57  0.83  (41.6)  (0.21)  (57)  (0.19)  (64.5)  (0.15)  (57.9)  (0.17)fg  AI2  –  –  8/12  0.88  2/2  0.88  10/14  0.88  (66.6)  (0.09)  (100)  (0.00)  (71.4)  (0.08)  AI3  –  –  1/4  0.89  0/2  1  1/6  0.93  (25)  (0.22)  (0)  (0.00)  (16.6)  (0.18)  AI4  –  –  2/4  0.76  –  –  2/4  0.76  (50)  (0.27)  (50)  (0.27)fh  BI1  4/43  0.97  0/10  1  0/2  1  4/55 (7.2)  0.98  (9.3)  (0.10)  (0)  (0.00)  (0.00)  (0.09)ghil  CI1  0/8  1  0/4  1  2/2  0.57  2/14  0.94  (0)a  (0.00)d  (0)  (0.00)  (100)a  (0.00)d  (14.3)  (0.16)  CI2  1/4  0.90  3/9  0.90  3/6  0.90  7/19  0.90  (25)  (0.21)  (33)  (0.22)  (50)  (0.14)  (36.8)  (0.18)i  CI3  7/14  0.83  1/2  0.75  –  –  8/16  0.82  (50)  (0.20)  (50)  (0.35)  (50)  (0.21)l  Total  17/81  0.93  23/59  0.88  27/45  0.85  67/185  0.89  (21)bc  (0.15)e  (38.9)b  (0.18)  (60)c  (0.16)e  (36.2)  (0.16)  ITS-1 genotype/ subgenotype  Group I  Group II  Group III  Total  P/T (%)  Mean phospholipase (SD)  P/T (%)  Mean phospholipase (SD)  P/T (%)  Mean phospholipase (SD)  P/T (%)  Mean phospholipase (SD)  AI1  5/12  0.83  8/14  0.80  20/31  0.84  33/57  0.83  (41.6)  (0.21)  (57)  (0.19)  (64.5)  (0.15)  (57.9)  (0.17)fg  AI2  –  –  8/12  0.88  2/2  0.88  10/14  0.88  (66.6)  (0.09)  (100)  (0.00)  (71.4)  (0.08)  AI3  –  –  1/4  0.89  0/2  1  1/6  0.93  (25)  (0.22)  (0)  (0.00)  (16.6)  (0.18)  AI4  –  –  2/4  0.76  –  –  2/4  0.76  (50)  (0.27)  (50)  (0.27)fh  BI1  4/43  0.97  0/10  1  0/2  1  4/55 (7.2)  0.98  (9.3)  (0.10)  (0)  (0.00)  (0.00)  (0.09)ghil  CI1  0/8  1  0/4  1  2/2  0.57  2/14  0.94  (0)a  (0.00)d  (0)  (0.00)  (100)a  (0.00)d  (14.3)  (0.16)  CI2  1/4  0.90  3/9  0.90  3/6  0.90  7/19  0.90  (25)  (0.21)  (33)  (0.22)  (50)  (0.14)  (36.8)  (0.18)i  CI3  7/14  0.83  1/2  0.75  –  –  8/16  0.82  (50)  (0.20)  (50)  (0.35)  (50)  (0.21)l  Total  17/81  0.93  23/59  0.88  27/45  0.85  67/185  0.89  (21)bc  (0.15)e  (38.9)b  (0.18)  (60)c  (0.16)e  (36.2)  (0.16)  Phospholipase activity (expressed as the mean phospholipase value) and standard deviations (SD) are also given. a–cχ2-test – statistically significant differences (P<0.05) were marked with the same letters. d–lanova (Tukey post hoc test); statistically significant differences (P<0.05) were marked with the same letters. View Large Discussion Multiple M. pachydermatis genotypes/subgenotypes were cultured from healthy dogs (Group I) or from dogs with single (Group II) or multiple, generalized skin lesions (Group III). Isolates from dogs with skin lesions (Groups II and III) showed a significantly higher phospholipase activity compared with those derived from skin with no detectable lesions (Group I). For both chs-2 and ITS-1, three genotypes (i.e. A–C) of M. pachydermatis were classified. While all three chs-2 sequences detected herein were the same as those from a previous study (Cafarchia et al., 2007c), four new ITS-1 sequence types (i.e. AI2, AI3, AI4 and CI3; accession numbers EU158826, EU158827, EU158828, EU158829, respectively) were defined (see Fig. 2), showing that this rRNA gene region is more variable and suggesting that it evolves at a higher rate, and that it is therefore useful for classifying genotypes/subgenotypes. The magnitude of nucleotide variation among all the ITS-1 sequence types was <7%, thus falling within the range of intraspecific variability reported previously (Makimura et al., 2000; Sugita et al., 2005) and being significantly less than levels of interspecific differences (i.e. >26%) (Makimura et al., 2000; Sugita et al., 2005). A number of previous molecular studies have genetically characterized M. pachydermatis from dogs (Guillot et al., 1997; Aizawa et al., 2001; Castella et al., 2005; Hossain et al., 2007). In particular, the study by Guillot. (1997) indicated that M. pachydermatis (from dogs, cats, “wild animals” and humans) could be divided into seven genetic groups (‘sequevars’ Ia–Ig) based on LSU sequence data. More recently, Hossain. (2007) inferred 28 different M. pachydermatis‘genotypes’ from dogs and cats based on data from a random amplification of polymorphic DNA (RAPD) analysis (Hossain et al., 2007). However, given the technical limitations of the method of RAPD (i.e. poor reproducibility and specificity – see, Ellsworth et al., 1993; MacPherson et al., 1993; Gasser, 2006a) and that the profile data sets generated are phenetic rather than genetic (thus making genetic interpretations inappropriate), the number of phenotypes reported (i.e. 28) may not all represent distinct genotypes. Further investigation would be required to establish the genetic diversity among the isolates from this study (Hossain et al., 2007) using loci, such as chs-2 and/or ITS-1, or by whole genome sequencing. Of the seven M. pachydermatis groups identified by Guillot. (1997), three different LSU sequence types were from dogs with or without dermatitis and/or otitis. In the present study, eight different ITS-1 sequence types were defined for M. pachydermatis cultured from canine skin samples. For both ITS-1 and chs-2, genotypes B and C were most common (53% and 32.1%, respectively) on healthy skin (Table 3). Although 12 isolates cultured from dogs in Groups II and III were assigned genotype BI1, 10 of them originated from healthy skin from dogs in Group II, suggesting a relationship between genotype B and healthy skin. Genotype A (subgenotypes AI2 and AI3) was unique to Groups II and III, indicating an affiliation to affected skin. Genotype AI1 was defined from cultures representing all dog Groups, but predominantly (68%) in those from Group III with multiple, generalized skin lesions. Genotype C occurred in Groups II and III at a lower frequency (25.4% and 17.7%, respectively). Although some degree of caution is warranted in the interpretation of the genetic make-up of yeast populations following in vitro culture, M. pachydermatis isolated from the same skin site of a single dog were genetically identical. However, different genotypes or subgenotypes were cultured from multiple distinct skin sites of individual dogs. These findings contrast those of a previous publication (Castella et al., 2005), most likely due to the small number (n=5) of dogs examined therein. Some ITS-1 subgenotypes (i.e. AI1, CI2) were consistently isolated from the same anatomical site from each of the three groups of dogs, whereas there was no consistent pattern for other subgenotypes (i.e. AI2, AI3, AI4, CI1 and CI3; see Table 1). This information may indicate an affiliation of M. pachydermatis genotypes/subgenotypes with a particular biochemical composition of the skin surface, which may relate to growth linked to specific physiological requirements of this yeast. Indeed, it has been demonstrated that the biochemical composition of canine skin may differ, depending on skin site and its health and integrity (Chen & Hill, 2005). In previous studies, an affiliation between the presence of particular genotypes of Malassezia globosa and Malassezia furfur and site of infection has been demonstrated in humans (Theelen et al., 2001; Sugita et al., 2003; Gupta et al., 2004). However, the specific locations of three to five ITS-1 genotypes/subgenotypes to perioral, periorbital regions and the external acustic meatus of dogs in the three different groups may suggest that particular populations of M. pachydermatis are transmitted specifically from dog to dog via licking and/or scratching. Although the precise mode(s) of reproduction of M. pachydermatis is not yet known, the occurrence of multiple genetic variants of M. pachydermatis on the skin of a host individual (as demonstrated herein) supports the notion that yeasts of the genus Malassezia may have a sexual reproductive phase (Midreuil et al., 1999; Xu et al., 2007). Future investigations into the reproduction of M. pachydermatis are warranted, given that sexual or parasexual reproduction may be linked to virulence in yeasts (Heitman, 2006). Most phospholipase activity was recorded in M. pachydermatis cultured from dogs with skin lesions (Groups II and III) and supports the proposal of an association between phospholipase production and pathological effect (Cafarchia & Otranto, 2004). The fact that only 21% of cultured M. pachydermatis derived from healthy skin (Group I) produced small amounts of phospholipase (Table 4) further supports this proposal. Nonetheless, although phospholipase activity was detectable in M. pachydermatis cultured from dogs with healthy skin (Group I), it is possible that microscopic skin alterations were ‘underway’ in some dogs. That all M. pachydermatis genotypes/subgenotypes produced phospholipase indicates that there is no relationship between genotype/subgenotype and the activity of this enzyme. However, particular genotype/subgenotypes were associated with skin lesions, although the sample size studied may be somewhat too small to make a definitive conclusion in this regard. This latter finding is not surprising, considering that the genetic markers selected (i.e. chs-2 and ITS-1) are not known to be associated with any functional molecules linked to a pathogenic effect by the yeast in skin. However, M. pachydermatis phospholipase production and the occurrence of skin lesions might be linked to β-endorphin (Bigliardi-Qi et al., 2000; Cafarchia et al., 2007a). In humans, a significantly greater amount of β-endorphin has been found in skin and blood of patients with atopic dermatitis (Glinski et al., 1994) because the μ-opiate receptor expression in the epidermis of skin cultured from such patients is downregulated and the proliferation of keratinocyte increased (Bigliardi-Qi et al., 2000, 2005). In conclusion, the distribution pattern of particular genotypes/subgenotypes of M. pachydermatis on the skin of dogs may relate to the affinity of the yeast itself to the host and skin. Again, it is likely that a range of microenvironmental factors in and on the skin (including bacterial flora, pH, salts, immune responses, biochemistry and physiology) play a significant role in the adherence, establishment and growth of Malassezia species. Genotype B appears to be present mainly on healthy skin without producing phospholipase, whereas other genotypes and subgenotypes seem to be predominantly associated with skin lesions and high phospholipase activity. The specific physiological requirements of Malassezia species raises questions about the efficacy of certain treatments, because the lipid composition of different therapeutic agents may selectively affect the growth and survival of Malassezia. 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TI - Genetic variants of Malassezia pachydermatis from canine skin: body distribution and phospholipase activity
JF - FEMS Yeast Research
DO - 10.1111/j.1567-1364.2008.00358.x
DA - 2008-05-01
UR - https://www.deepdyve.com/lp/oxford-university-press/genetic-variants-of-malassezia-pachydermatis-from-canine-skin-body-4usLCEGe8A
SP - 451
EP - 459
VL - 8
IS - 3
DP - DeepDyve
ER -