103 Veterinaria Italiana 2022, 58 (1), 103-109. doi: 10.12834/VetIt.2124.12936.1 Accepted: 10.09.2020 | Available on line: 16.11.2022 1University of Padova, Via 8 Febbraio 2, 35122 Padova, Italy. 2Istituto Zooprofilattico Sperimentale delle Venezie, Viale dell’Università 10, 35020 Legnaro (PD), Italy. *Corresponding author at: University of Padova, Via 8 Febbraio 2, 35122 Padova, Italy. E‑mail: michele.berlanda@unipd.it. Michele Berlanda1, Carlotta Valente1, Carlo Guglielmini1, Patrizia Danesi2, Barbara Contiero1 and Helen Poser1 Keywords Dermatology, Dog, Malassezia, Yeast count, Skin disease. Summary The present study describes Malassezia populations in clinically healthy dogs (HD) and dogs with Malassezia overgrowth (MO), and evaluates the correlation with clinical signs and previous treatments. Thirteen clinically HD and 84 dogs with MO were enrolled. Clinical history and previous treatments were recorded. After a complete physical and dermatological examination, Canine Atopic Dermatitis Extent and Severity Index_03 scores were calculated. Samples for cytology and mycological cultures were obtained from four body regions and from skin lesions. Malassezia overgrowth was diagnosed by cytology. A global score (GS) for quantitative evaluation of the population of Malassezia was calculated. In dogs with MO, the highest frequency of yeast detection was found in skin lesions (82%, P < 0.001). Sum of GS (GSs) obtained from dogs with MO (68, 0-621) was significantly higher compared to those of HD (3, 0-48, P < 0.001). GSs in dogs previously treated with antibiotics (312.5, 30-975) was significantly higher compared to those of dogs that not have received antibiotics (80, 0-975, P = 0.015). No difference was found between dogs treated and those not treated with steroids. Malassezia overgrowth in dogs in Northern Italy: frequency, body distribution, clinical signs and effects of pharmacologic treatments treatment (Bond and Loyd 1997). Pruritus is a major clinical sign of Malassezia dermatitis. Other symptoms include erythema, alopecia, greasy exudation, and scaling. Hyperpigmentation and lichenification are usually observed in chronic cases. Frequently, affected body regions include lips, ear canals, ventral neck, axillae, groin, interdigital webs, perianal area, and intertriginous regions (Miller et al. 2012). The exact pathogenesis of skin inflammation caused by M.  pachydermatis is still unclear. A key role seems to be played by phospholipase production (Coutinho and Paula 2000, Teramoto et al. 2015) and by hypersensitivity against yeast antigens (Bond et  al. 2002, Bond et  al. 2006a, Bond et  al. 2006b, Khosravi et  al. 2007, Layne and Deboer 2016). Many skin disorders, especially atopic dermatitis, keratinization defects, recurrent bacterial pyoderma, and endocrine diseases alter sebum production and cause a disruption of the epidermal barrier, favouring yeast proliferation (Bond and Ferguson 1996). It has also been proposed by some authors that long-term Introduction Malassezia sp. yeasts are normal inhabitants of the mammalian skin surface and are usually considered opportunistic pathogens (Guillot and Bond 1999). The Taxonomic classification divided the genus Malassezia into 18 species (Gupta et  al. 2004, Batra et  al. 2005, Cabañes et  al. 2007, Castellà et  al. 2014, Theelen et  al. 2018, Lorch et  al. 2018). All species within the genus are non-mycelial, unipolar, and budding. Furthermore, all species except Malassezia pachydermatis are lipid-dependent due to their inability to synthesize some fatty acids and their requirements for lipid supplementation for in vitro growth. M.  pachydermatis is the most common species of this genus in dogs and is frequently isolated from ear canals, skin, and mucosal surfaces (oral and anal), and less commonly from the anal sacs and vagina (Guillot and Bond 1999, Bond et al. 1995). Malassezia dermatitis is a term used to describe skin diseases associated with Malassezia overgrowth (MO) in affected regions that show a good clinical and cytological response to appropriate antifungal 104 Veterinaria Italiana 2022, 58 (1), 103-109. doi: 10.12834/VetIt.2124.12936.1 Malassezia overgrowth in dogs Berlanda et al. in sterile saline (0.9%) solution on a skin area of approximately 1 cm2 in all the above-mentioned cutaneous sites. Cytology Each tape-strip was stained with modified Diff-Quick® method skipping the first step of alcohol fixation, and examined microscopically at 40x magnification. Ear samples were fixed and stained with the Diff-Quick® method and examined as skin specimens. Yeasts were identified according to their typical morphology (cell shape, size, and budding pattern). The number of yeasts was assessed in 5  random fields at 40x magnification (Cafarchia et  al. 2005), and number of yeasts per field was graded as follows: 0, no yeasts found; A, 1-5 yeasts; B, 6-10  yeasts; C, 11-50 yeasts; D, 51-100 yeasts; and E,  >  100 yeasts. Malassezia overgrowth was diagnosed when more than 2 and 10 yeasts were counted in 5 random fields at 40x magnification in skin and ear cytology, respectively (Mauldin et  al. 1997, Bond et al. 1993). Mycological culture Swabs were preserved not more than 24 hours at 4  °C before mycological cultures were performed. Swabs were plated directly onto Petri dishes containing Sabouraud Dextrose Agar (SDA) and modified Dixon’s Agar (DA) medium and incubated at 32 °C for 15 days. The positivity threshold was set at 70 UFC. Identification Colonies were identified as Malassezia based on microscopic and macroscopic morphology and were suspected to belong to the non-lipid dependent M.  pachydermatis species when grown on the SDA medium. Identification was confirmed by PCR and sequencing of the large-subunit (26S) ribosomal DNA gene as previously reported (Kurtzman and Robnett 1997). Statistical analysis The statistical analyses were carried out using the Statistical Analysis System version 9.0 (SAS Inst. Inc., Cary, NC, USA). Distribution of data was assessed by use of the Shapiro-Wilk’s normality test. Normally and non-normally distributed data were reported as mean ± SD and median (range), respectively. Frequencies of the detection from the considered body regions were reported as a percentage and compared using the chi-square test. Due to the non-normal distribution of the yeast population sizes, a Global Score (GS) was calculated by summing administration of glucocorticoids or antibiotics may increase Malassezia populations (Bond and Ferguson 1996, Ihrke et  al. 1993, Mauldin et  al. 1997). A significant effect from prednisone and cyclosporine on cutaneous Malassezia populations in dogs with atopic dermatitis has recently been excluded (Widmer et  al. 2018). Lastly, studies providing evidence on the effect of antibiotic treatments on the Malassezia population are lacking in dogs. The present study describes the frequency of detection and body distribution of Malassezia yeasts in dogs in Italy. We also examined the clinical signs, grade of lesions, and predisposing factors of Malassezia dermatitis with particular attention on the effects of previous pharmacological treatments. Materials and methods Study design Dogs with skin and ear diseases and cytological evidence of MO were prospectively enrolled in a veterinary teaching hospital (VTH) in northern Italy for a period of three years. A control group of healthy dogs (HD) was also recruited, including dogs in good general health that were presented for routine visits or vaccinations. These animals were free of clinical signs and had no history of skin and ear diseases. Moreover, these dogs had not received any medication during the previous three months. Clinical examination Clinical history was collected from all dogs with a special focus on therapies in the previous three months. In addition, to complete physical and dermatological examination, the Canine Atopic Dermatitis Extent and Severity Index-03 score (CADESI-03) was calculated in dogs with MO (Olivry et  al. 2007, Machado et  al. 2011). Client’s informed consent was obtained for each dog before examination. Sampling procedures Samples were obtained from all dogs from axilla, ventral interdigital webs of the forelimb, ear pinna, and ear canal of the right side. Furthermore, in animals with dermatitis or otitis, additional samples were collected from specific lesion. Samples for cytologic examination were collected from the skin with a tape-strip technique (Bond et  al. 1994) and from the ear canal using cotton swabs rolled on a glass slide. Samples for mycological cultures were collected by rubbing a sterile swab moistened 105Veterinaria Italiana 2022, 58 (1), 103-109. doi: 10.12834/VetIt.2124.12936.1 Berlanda et al. Malassezia overgrowth in dogs and in five sampling sites in dogs with MO (n = 84). In the HD group, Malassezia yeasts were detected by cytology in 8/13 dogs (62%) without any significant difference (chi-square  =  4.42, P  =  0.22) in the frequency of yeasts among the four anatomical areas. In dogs with MO, the greatest frequency of detection of Malassezia spp. was recorded from the cutaneous lesions (82%, P < 0.001). The frequency of yeasts was significantly higher on the ear canal compared to the axilla (43% vs 23%, P < 0.001), while interdigital webs and ear pinna showed intermediate values (33% and 26%, respectively; P < 0.001). The comparison of the frequencies of cytological detection of Malassezia species in different affected regions among MO and HD showed a tendency for significant differences regarding ear canal and ear pinna (43% vs 15%, chi-square = 3.6 P = 0.05; and 26% vs 54% chi-square = 4.1 P = 0.05, respectively) (Table II). Global score (GS) Although the GS was not available in 53.6% (45/84) of dogs with MO, the GSs obtained from four sampled anatomical regions in HD (3, 0-48) were significantly lower compared to those of 39/84 dogs with MO (68, 0-612, P < 0.001) (Table III). In dogs with MO, the GS (312.5, 30-975) of the 12 dogs treated with systemic antibiotics in the previous three months was significantly higher compared to that of the 27 not treated dogs (80, 0-975, P = 0.015) (Figure 1). The GS was not significantly affected by treatment with systemic or topical steroids (P = 0.35) (Figure 2). the corresponding scores of various sampled regions. In particular, the score of the sampled regions was calculated using the following formula: Score = 3*number of microscopic fields A + 8* number of microscopic fields B + 30*number of microscopic fields C + 75*n° microscopic fields D + 100*number of microscopic fields E. The coefficients were determined considering the median of the number of yeasts per field per each grade, except for the E grade, as all fields were considered having 100 yeasts per field. The association between the the sum of GS (GSs) of Malassezia and the severity of skin lesions (CADESI-03 scores) among the dogs with MO was also evaluated by use of the Spearman’s correlation index. The effect of pharmacological therapies (systemic antibiotics and steroids in the previous three months of examination) on non-normally distributed data was tested with the Mann-Whitney Test. Normally distributed data were analysed using an analysis of variance (ANOVA). The statistical models included the fixed effects of group of dogs (healthy dogs vs dogs with MO), sex, clinical problem (pruritus, multifocal alopecia, diffuse alopecia, and otitis externa), and treatments (topical and systemic corticosteroid, topical, and systemic antibiotic). A value of P ≤ 0.05 was considered to be statistically significant. Results Population and clinical data A total number of 240 dogs with skin problems was presented at the VTH in the enrolment period. Among these, 97 dogs fitted the inclusion criteria and were enrolled in the study. They were grouped as follows. 1. Healthy dogs (HD) (n = 13). 2. Dogs with MO (n = 84). The population included 52 females (2 HD and 50 with MO) and 45 males (11 HD and 34 with MO). All animals lived in Northern Italy. The median age at first examination of HD and dogs with MO was 58 months (3-132 months) and 71 months (4-192 months), respectively. In dogs with MO, 31 received systemic antibiotics (11 cefalexin, 8  amoxicillin + clavulanic acid, 3 benzylpenicillin + dihydrostreptomycin, 1 enrofloxacin, 1 clindamycin and 1 enrofloxacin + metronidazole, 6 not specified) in the previous three months. Table I summarizes the list of presenting complaint in dogs with MO. Frequency of cytological detection Table II summarizes the results of detection of Malassezia spp. in four sampling sites in HD (n = 13) Table I. Main presenting complaints (%) in 84 dogs with Malassezia overgrowth. Problem % of cases* Pruritus 73%a Otitis externa 18%b Diffuse alopecia 5%b Multifocal alopecia 5%b *Values with different letters along column are significantly different (Chi square = 80.0; P < 0.001). Table II. Cytological detection of Malassezia spp. yeasts in four and five sampling sites in 13 clinically healthy dogs (HD) and 84 dogs with Malassezia overgrowth (MO), respectively. Sampling site HD° Dogs with MO* P Ear canal (%) 15% 43%b 0.05 Interdigital webs (%) 38% 33%bc 0.72 Ear pinna (%) 54% 26%bc 0.05 Axilla (%) 31% 23%c 0.52 Skin lesions (%) - 82%a °Chi square = 4.42, P = 0.22; *Values with different letters along column are significantly different (Chi square = 80.0; P < 0.001). 106 Veterinaria Italiana 2022, 58 (1), 103-109. doi: 10.12834/VetIt.2124.12936.1 Malassezia overgrowth in dogs Berlanda et al. Lesion scores In 39/84 dogs with MO, the CADESI-03 score was 18 (0-43). No significant difference was found in CADESI-03 score between male and female (P  =  0.163). Neither antibiotics nor corticosteroids significantly influenced the CADESI-03 score. No correlation (R = 0.48; P = 0.0021) was found between CADESI-03 scores and modified GSs. Since the CADESI-03 score does not consider ear canals, the GS of this area was not considered in this single analysis. Culture and identification of the Malassezia yeasts From 142 swabs taken from 52 animals (39 with MO and 13 HD), 26 samples (18.3%) were considered positive. All isolates were able to grow on SDA (at 32  °C) and were identified as non-lipid dependent Malassezia yeasts. Sequencing of the 26S rDNA gene confirmed Malassezia pachydermatis in all isolates. 1000 900 800 700 600 500 400 300 No antibiotics Antibiotics 200 100 0 -100 G Ss Figure 1. Box plot showing the sum of global score (GSs) of 12 and 27 dogs that received (Antibiotics) and did not receive (No antibiotics) systemic antibiotic therapy in the previous three months before examination. Boxes represent the interquartile range (25th to 75th percentile). The horizontal line in each box represents the median. Whiskers represent the 5th and 95th percentiles. Outliers are plotted separately as circles. Values are significantly different, P = 0.015. Table III. Global scores (GS) of the four and five sampling sites in 13 clinically healthy dogs (HD) and 39 dogs with Malassezia overgrowth (MO), respectively. Data are reported as Median (range). Sampling site HD MO P Ear canal 0 (0-30) 0 (0-475) 0.132 Interdigital webs 0 (0-3) 0 (0-500) 0.196 Ear pinna 0 (0-39) 0 (0-500) 0.748 Axilla 0 (0-39) 0 (0-35) 0.525 Skin lesions - 12 (0-475) - Sum of GS* 3 (0-48) 68 (0-621) < 0.001 *Without GS of lesions. GenBank® (http://www.ncbi.nlm.nih.gov/genbank/) accession numbers were MN198166, MN198167, MN198171, MN198176, MN198172, MN198173, MN198174, MN198175, MN198177, MN198179, MN198170, MN198168, MN198178 and MN198169. Discussion The main result of the present study is the observed increased number of Malassezia yeast in a canine population treated with systemic antibiotic therapy. Therefore, this is the first time that the effect of antibiotic treatments on the number of cutaneous Malassezia yeasts is evidenced by a clinical study. The GS and then the number of Malassezia yeasts in dogs that had received antibiotics were indeed significantly higher compared to those of non-treated dogs. In fact, the association between Malassezia dermatitis and previous antibiotic therapy has been reported in the canine literature without clinical evidence (Miller et  al. 2012). A recent study evidenced that the skin microbiota can be influenced by topical antimicrobial therapy, but the effect of systemic antibiotic treatment was not considered (Chermaprai et  al. 2019). There are at least two possible explanations for our results. First, the changes in the skin microbiota following antibiotic treatment may predispose it to Malassezia overgrowth giving rise to the thought that Staphylococcus and Malassezia species might have a symbiotic relationship. Second, there may be some common predisposing factors that can favour both the secondary bacterial 1000 900 800 700 600 500 400 300 Steroids No steroids 200 100 0 -100 G Ss Figure 2. Box plot shows the sum of global score (GSs) of 10 and 27 dogs that receive (Steroids) and did not receive (No steroids) systemic steroid therapy during the previous three months before examination. The horizontal line in each box represents the median. Boxes represent the interquartile range (25th to 75th percentile). Whiskers represent the 5th and 95th percentiles. Outliers are plotted separately as circles. Values are not significantly different, P = 0.35. 107Veterinaria Italiana 2022, 58 (1), 103-109. doi: 10.12834/VetIt.2124.12936.1 Berlanda et al. Malassezia overgrowth in dogs differences or correlations were found in the evaluation of CADESI-03 scores (Olivry et  al. 2007, Olivry et  al. 2008). However, it has to be said that CADESI-03 scores were developed and validated only for canine atopic dermatitis and may not be valid for Malassezia dermatitis. GS also has not been validated. This could represent a weakness of the present study. It is generally accepted that MO is secondary to hypersensitivity disorders, endocrine diseases, and defects of keratinization (Miller et al. 2012). Therefore, in the present study only a part of the dogs could have had hypersensitivity disorder and, in particular, atopic dermatitis. This could also represent a study limitation, as already highlighted in a previously published study that uses the CADESI-03 scores for evaluating the difference between dogs with Malassezia dermatitis and dogs with cutaneous lesions but without evidence of Malassezia dermatitis (Machado et  al. 2011). Moreover, the present study is based on the count of yeasts, which is probably a relevant factor, but does not consider that some strains could express different virulence factors such as phospholipase productions (Coutinho et al. 2000, Teramoto et al. 2015, Buommino et al. 2016, Cafarchia and Otranto 2004), or that the susceptibility to this organism could differ between hosts (e.g. Malassezia hypersensitivity) (Bond et al. 2006a, Bond et al. 2006b). In both cases, clinical signs of Malassezia dermatitis would likely develop also with low numbers of yeasts. Finally, secondary bacterial infections that may have influenced the CADESI score should have been considered. This represents, as mentioned before, another study limitation. In conclusion, the present study provides helpful insights into the frequency of detection and body distribution of Malassezia in healthy dogs and in dogs with skin disease. In particular, our results highlight the importance of the number of yeasts resulting from the skin cytology for the diagnosis of MO, even though the different pathogenicity of the involved yeast strains needs also to be considered. Previous antibiotic treatments may represent a predisposing factor for the increased number of Malassezia yeast supporting what has previously been assumed but without clinical evidence. However, further studies are necessary to confirm this hypothesis, also considering the potential effect of other factors not evaluated in the present study such as the concurrent predisposition to both MO and bacterial infections. infections, which justify the previous antibiotic treatments, and the overgrowth of Malassezia. Some changes in the skin barrier and immunological status have demonstrated a predisposition to bacterial and yeast overgrowth in dogs with atopic dermatitis (Santoro et al. 2015). In the present study, 61 (73%) dogs with Malassezia overgrowth were examined for pruritus with a presumptive diagnosis of allergic dermatitis, confirming the findings of other investigations concerning this association (Machado et  al. 2011). Therefore, these subjects could have been predisposed to skin bacterial secondary infections and to Malassezia overgrowth. Many patients of the present study were referred by other practices and so the lack of information about prior bacterial infections was consequently a limitation of the present study. Further studies are necessary to understand whether there may be an altered competition with the resident microbial flora or if other mechanisms, not considered in the present study, can explain the presence of a greater number of Malassezia in dogs treated with antibiotics (Plant et  al. 1992, Bond and Ferguson 1996). In contrast to the effect of antibiotic therapy, glucocorticoids treatment showed no significant influence on GS. Recently, Widmer and colleagues (Widmer et  al. 2018) have shown no significant impact of prednisone on canine cutaneous microbiota. Thus, previous steroid therapy does not seem to affect the composition of the canine skin flora. The cytological examination revealed that the number of yeasts on five random fields can differ considerably in areas within the same slide. For this reason, we studied the Global Score (GS), which permits to overtake the effect of this variability and enhance the importance of fields with a high number of yeasts. As expected and reported in previous studies, the yeast population size (expressed by the GS) differed significantly between the healthy and diseased animals (Crespo et al. 2002, Nardoni et  al. 2004, Yurayart et  al. 2011, Cafarchia et  al. 2005). Furthermore, in the present study, the frequency of yeast detection from dogs with skin disease was significantly higher when compared to that of clinically HD; this was also evident from areas not interested by lesions, although the frequency of yeast detection was higher in areas with skin lesions, as previously reported (Bond and Lloyd 1997, Maachado et  al. 2011, Nardoni et  al. 2004, Yurayart et  al. 2011). Despite the differences in GS and in the frequency of yeast detection, no significant 108 Veterinaria Italiana 2022, 58 (1), 103-109. doi: 10.12834/VetIt.2124.12936.1 Malassezia overgrowth in dogs Berlanda et al. Batra R., Boekhout T., Guého E., Cabañes F.J., Dawson T.L. Jr & Gupta A.K. 2005. Malassezia Baillon, emerging clinical yeasts. FEMS Yeast Res, 5, 1101-1113. Bond R. & Ferguson E. 1996. Factors associated with elevated cutaneous Malassezia pachydermatis populations in dogs with pruritic skin. J Small Anim Pract, 37, 103-107. Bond R. & Lloyd D.H. 1997. Skin and mucosal populations of Malassezia pachydermatis in healthy and seborrhoeic Basset Hounds. Vet Dermatol, 8, 101-106. Bond R. & Sant R.E. 1993. The recovery of Malassezia pachydermatis from canine skin. Vet Dermatol Newsletter, 15, 25. Bond R., Collin N. & Lloyd D. 1994. Use of contact plates for the quantitative culture of Malassezia pachydermatis from canine skin. J Small Anim Pract, 35, 68-72. Bond R., Curtis C.F., Hendricks A., Ferguson E.A. & Lloyd D.H. 2002. Intradermal test reactivity to Malassezia pachydermatis in atopic dogs. Vet Rec, 150, 448-449. Bond R., Habibah A., Patterson-Kane J.C. & Lloyd D.H. 2006a. Patch test responses to Malassezia pachydermatis in healthy dogs. Med Mycol, 44, 175-184. Bond R., Patterson-Kane J.C., Perrins N. & Lloyd D.H. 2006b. Patch test responses to Malassezia pachydermatis in healthy basset hounds and in basset hounds with Malassezia dermatitis. Med Mycol, 44, 419-427. Bond R., Lloyd D.H. & Plummer J.M. 1995. Evaluation of a detergent scrub technique for the quantitative culture of Malassezia pachydermatis from canine skin. Res Vet Sci, 58, 133-137. Buommino E., Nocera F.P., Parisi A., Rizzo A., Donnarumma G., Mallardo K., Fiorito F., Baroni A. & De Martino L. 2016. Correlation between genetic variability and virulence factors in clinical strains of Malassezia pachydermatis of animal origin. New Microbiol, 39, 216-223. Cabañes F.J., Theelen B., Castellá G. & Boekhout T. 2007. Two new lipid-dependent Malassezia species from domestic animals. FEMS Yeast Res, 7, 1064-1076. Cafarchia C. & Otranto D. 2004. Association between phospholipase production by Malassezia pachydermatis and skin lesions. J Clin Microbiol, 42, 4868-4869. Cafarchia C., Gallo S., Romito D., Capelli G., Chermette R., Guillot J. & Otranto D. 2005. Frequency, body distribution, and population size of Malassezia species in healthy dogs and in dogs with localized cutaneous lesions. J Vet Diagn Invest, 17, 316-322. Castellá G., Coutinho S.D.A. & Cabañes F.J. 2014. Phylogenetic relationships of Malassezia species based on multilocus sequence analysis. Med Mycol, 52, 99-105. Chermprapai S., Ederveen T.H.A., Broere F., Broens E.M., Schlotter Y.M., van Schalkwijk S., Boekhorst J., van Hijum S.A.F.T. & Rutten V.P.M.G. 2019. The bacterial and fungal microbiome of the skin of healthy dogs and dogs with atopic dermatitis and the impact of topical antimicrobial therapy, an exploratory study. Vet Microbiol, 229, 90-99. References Coutinho S. & Paula C. 2000. Proteinase, phospholipase, hyaluronidase and chondroitin-sulphatase production by Malassezia pachydermatis. Med Mycol, 38, 73-76. Crespo M.J., Abarca M.L. & Caban F.J. 2002. Occurrence of Malassezia spp. in horses and domestic ruminants. Mycoses, 337, 333-337. Guillot J. & Bond R. 1999. Malassezia pachydermatys: a review. Med Mycol, 37, 295-306. Gupta A.K., Boekhout T., Theelen B., Summerbell R. & Batra R. 2004. Identification and typing of Malassezia species by amplified fragment length polymorphism and sequence analyses of the internal transcribed spacer and large-subunit regions of ribosomal DNA. J Clin Microbiol, 42, 4253-4260. Ihrke P.J., Mason I. & White S.D. 1993. Advances in veterinary dermatology. Vol 2: Proceedings of the 2nd World Congress, Montreal, Canada, May 13th-16th 1992, World Congress of Veterinary Dermatology. Khosravi A.R., Hedayati M.T., Mansouri P., Shokri H. & Moazzeni M. 2007. Immediate hypersensitivity to Malassezia furfur in patients with atopic dermatitis. Mycoses, 50, 297-301. Kurtzman C.P. & Robnett C.J. 1997. Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA gene. J Clin Microbiol, 35, 1216-1223. Layne E.A. & Deboer D.J. 2016. Veterinary immunology and immunopathology serum Malassezia -specific IgE in dogs with recurrent Malassezia otitis externa without concurrent skin disease. Vet Immunol Immunopathol, 176, 1-4. Machado M.L.S., Ferreiro L., Ferreira R.R., Corbellini L.G. & Deville M., Berthelemy M., Guillot J. 2011. Malassezia dermatitis in dogs in Brazil: diagnosis, evaluation of clinical signs and molecular identification. Vet Dermatol, 22, 46-52. Mauldin E.A., Scott D.W., Miller W.H. & Smith C. 1997. Malassezia dermatitis in the dog: a retrospective histopathological and immunopathological study of 86 cases (1990-1995). Vet Dermatol, 8, 191-202. Miller W.H., Griffin C. & Campbell K. 2012. Muller & Kirk’s small animal dermatology, 7th ed. St Louis, MO, USA Elsevier, 243-249. Nardoni S., Mancianti F., Corazza M. & Rum A. 2004. Occurrence of Malassezia species in healthy and dermatologically diseased dogs. Mycopathologia, 157, 383-388. Olivry T., Marsella R., Iwasaki T. & Mueller R. 2007. Validation of CADESI-03, a severity scale for clinical trials enrolling dogs with atopic dermatitis. Vet Dermatol, 18, 78-86. Olivry T., Mueller R., Nuttall T., Favrot C., Prélaud P. & International Task Force on Canine Atopic Dermatitis. 2008. Determination of CADESI-03 thresholds for increasing severity levels of canine atopic dermatitis. Vet Dermatol, 19, 115-119. Plant J., Rosenkrantz W. & Griffin C. 1992. Factors associated 109Veterinaria Italiana 2022, 58 (1), 103-109. doi: 10.12834/VetIt.2124.12936.1 Berlanda et al. Malassezia overgrowth in dogs Theelen B., Cafarchia C., Gaitanis G., Bassukas I.D., Boekhout T. & Dawson T.L. Jr. 2018. Malassezia ecology, pathophysiology, and treatment. Med Mycol, 56, S10-S25. Widmer G., Ferrer L., Favrot C., Paps J., Huynh K. & Olivry T. 2018. Glucocorticosteroids and ciclosporin do not significantly impact canine cutaneous microbiota. BMC Vet Res, 14, 51. Yurayart C., Chindamporn A., Suradhat S., Tummaruk P., Kajiwara S. & Prapasarakul N. 2011. Comparative analysis of the frequency, distribution and population sizes of yeasts associated with canine seborrheic dermatitis and healthy skin. Vet Microbiol, 148, 356-362. with and prevalence of high Malassezia pachydermatis numbers on dog skin. J Am Vet Med Assoc, 201, 879-882. Santoro D., Marsella R., Pucheu-Haston C.M., Eisenschenk M.N., Nuttall T. & Bizikova P. 2015. Review: Pathogenesis of canine atopic dermatitis: skin barrier and host-micro-organism interaction. Vet Dermatol, 26, 84-e25. Teramoto H., Kumeda Y., Yokoigawa K., Hosomi K., Kozaki S., Mukamoto M. & Kohda T. 2015. Genotyping and characterisation of the secretory lipolytic enzymes of Malassezia pachydermatis isolates collected from dogs. Vet Rec Open, 21, 1-8.