Accepted Manuscript

Title: Isolation and characterization of Malassezia spp. inhealthy swine of different breeds

Authors: S. Nardoni, V. Merildi, S. Frangioni, G. Ariti, R.Verin, P. Vannucci, F. Mancianti

PII: S0378-1135(09)00355-1DOI: doi:10.1016/j.vetmic.2009.07.033Reference: VETMIC 4525

To appear in: VETMIC

Received date: 2-4-2009Revised date: 8-7-2009Accepted date: 31-7-2009

Please cite this article as: Nardoni, S., Merildi, V., Frangioni, S., Ariti, G.,Verin, R., Vannucci, P., Mancianti, F., Isolation and characterization of Malasseziaspp. in healthy swine of different breeds, Veterinary Microbiology (2008),doi:10.1016/j.vetmic.2009.07.033

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Isolation and characterization of Malassezia spp. in healthy swine of 1

different breeds2

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Nardoni S., Merildi V., Frangioni S., Ariti G., Verin R., Vannucci P*, Mancianti F.4

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Dipartimento di Patologia Animale, Profilassi ed Igiene degli Alimenti Università di Pisa8

*Practitioner, Firenze, Italy9

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Corresponding Author:13

Dr Simona Nardoni14

Dipartimento di Patologia Animale, Profilassi ed Igiene degli Alimenti 15

Viale delle Piagge, 216

56100 Pisa 17

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Telephone number: 0039050 2216952; 19

Fax number: 0039050 221694120

E-mail address: snardoni@vet.unipi.it21

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Manuscript

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Abstract 23

Malassezia spp. genus is represented by several lipophilic yeasts, normally present on the skin 24

of many warm-blooded vertebrates, including man. Swine are one of the less investigated 25

animal species. The aim of the present work was to study the occurrence of Malassezia spp. in 26

the external ear canal of 408 healthy swine of different breeds, under different breeding 27

conditions. For this purpose N. 185 free ranging wild boars, N. 107 large size pigs and 116 28

Cinta Senese breed were selected. Animals were of both genders, with age ranging from 8 29

months to 4 years. The subjects were culturally and molecularly checked for Malassezia spp. 30

Ninety-two out of 408 animals scored positive for Malassezia yeasts (22.5%). M. 31

pachydermatis, M. sympodialis and M. furfur were recognized. M. pachydermatis was the sole 32

species isolated from wild boars (12.9%), Cinta Senese (20.7%) and juvenile large size pigs 33

(13.6%); 88% of large size breeds adult subjects scored positive for M. sympodialis (63.6%) 34

and M. furfur (22.7%), respectively. The study focus on scarcely investigated 35

epidemiological aspects of Malassezia spp. in this animal species. 36

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Keywords: Malassezia spp.; swine; PCR; culture; external ear canal.39

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Introduction48

Malassezia genus encounters lipophilic yeasts, normally present on the skin of many warm-49

blooded vertebrates. Based on morphological, ultrastructural, physiological, and genetic 50

features, 7 species were assigned to the genus in the 90ties (Guého et al., 1996; Guillot and 51

Guého, 1995), Malassezia pachydermatis, the only non lipid-dependent species, and the six 52

lipid-dependent species Malassezia furfur, Malassezia globosa, Malassezia obtusa, 53

Malassezia restricta, Malassezia slooffiae and Malassezia sympodialis. Further lipid-54

dependent species, namely Malassezia dermatis (Sugita et al., 2002) Malassezia japonica55

(Sugita et al., 2003), Malassezia nana (Hirai et al., 2004), Malassezia yamatoensis (Sugita et 56

al., 2004), Malassezia equina and Malassezia caprae (Cabañes et al., 2007) were then 57

described. These fungi have a role in otitis externa and seborrhoeic dermatitis of animals, 58

mainly carnivores, as well as in pityriasis versicolor, seborrhoeic dermatitis, atopic dermatitis 59

and folliculitis of man. In humans, M. pachydermatis was the responsible agent of infection 60

both in immunocompetent (Ming Fan et al., 2006), and in immunocompromised subjects 61

(Midgley, 2000); lipid-dependent species alone or variously associated among them or with62

M. pachydermatis are described in domestic animals (Crespo et al., 2002a; Crespo et al., 63

2002b; Nardoni et al., 2004). 64

Swine seem to be one of the less investigated animal species. Some reports prior to the 65

description of the new species demonstrated the occurrence of Malassezia spp. on swine skin 66

(Gustafson, 1959; Gustafson, 1960; Dufait, 1985; Kuttin and Glas, 1985; Guillot et al., 1994) 67

with a marked predominance of lipid-dependent yeasts. Garau et al. (2005), identified M. 68

sympodialis and M. slooffiae from 73% of ear canals in dermatologically healthy animals. To 69

the best of our knowledge, there are no reports about the presence of Malassezia spp., related 70

to breed and management in this animal species. Aim of the present work was to investigate 71

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the occurrence of Malassezia spp. in the external ear canal of different swine breeds, under 72

different breeding conditions.73

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Materials and methods 75

External ear canals from 408 healthy swine were culturally and molecularly checked for 76

Malassezia spp. The animals were of both genders, with age ranging from 8 months to 4 77

years. Animals aged more than one year were classified as adults. On the basis of breed, the 78

animals were divided into 3 groups: free-ranging wild boars (N. 185), large size swine, such 79

as Landrace, Large White and their crosses (N. 107) and Cinta Senese breed (N. 116). The 80

first group consisted of 68 juvenile and 117 adult wild boars, the second comprehended 64 81

juvenile and 43 adults, while the last one 42 juvenile and 74 adult subjects. Large size pigs 82

were intensively raised in 3 farms (No. 1, 2, 3), as well as Cinta Senese subjects were 83

managed with extensive/outdoor production system in 3 different farms (No. 4, 5, 6).84

Samples were collected by means of sterile cotton tip swabs immediately after killing during 85

wild board hunting season, and at the swine farm in the others. 86

After collection, the specimens were promptly seeded onto Sabouraud Dextrose Agar added 87

with 0.5% of chloramphenicol and cycloheximide (Mycobiotic Agar®, DID, Milano, Italy) 88

and mDixon Agar (3.6% malt extract, 0.6% peptone, 2% desiccated ox-bile, 1% Tween 40, 89

0.2% glycerol, 0.2% oleic acid, 1.2% agar, 0.5% chloramphenicol and 0.5% cycloheximide). 90

The plates were incubated at 30 ° C for 7 days and processed as previously reported (Nardoni 91

et al., 2004). Briefly, lipid-dependent species were morphologically and biochemically 92

identified, as described by Guého et al. (1996). The presence of M. furfur, M. sympodialis and 93

M. slooffiae was proven by the Tween diffusion test, on the basis of their ability to assimilate 94

various polyoxyethylene sorbitan esters. Bond and Anthony (1995) demonstrated the possible 95

lipid dependence of some isolates of M. pachydermatis. Also, the presence of some lipids in 96

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the primary culture from the specimen can allow the growth of M. furfur in lipid-free medium. 97

For these reasons, subsequent transfers were performed to clearly distinguish these two 98

species. The identification of M. furfur was confirmed by using the Cremophor EL (Sigma, St 99

Louis, MO, USA) assimilation test, as reported by Raabe et al. (1998). The splitting of esculin 100

was also performed as additional key to identify both M. furfur and M. sympodialis (Mayser et 101

al., 1997). The lack of catalase activity, which is a specific feature of M. restricta, proved the 102

presence of this species. 103

Morphological and biochemical identification was confirmed by means of a PCR-based 104

technique using restriction enzyme digestion, specific for the discrimination of 11 Malassezia105

species, as described by Mirhendi et al. (2005). In order to achieve pure cultures, for each 106

positive sample 5 colonies were subcultured onto mDixon Agar and stored at -20°C until 107

analysis. The cell walls were mechanically disrupted by freeze-thawing and genomic DNA 108

was extracted and purified according to the DNeasyTM protocol for animal tissue (QIAGEN 109

Inc., Valencia, Ca, USA). The primers selected for this protocol amplify the target part of 26S 110

rDNA, providing a single PCR product of an expected size of 580 bp. The PCR products were 111

subjected to REA using CfoI and BstF51, separately, according to the manufacturer’s 112

instructions (Fermentas International Inc. Burlington, Ontario, Canada). Digested fragments 113

were analyzed by electrophoresis in 2% agarose gel stained with ethidium bromide. 114

Yeast strains with morphological, physiological and molecular characters consistent with M. 115

sympodialis were discriminated from the newly described species M. caprae, and M. equina, 116

amplifying by PCR the variable D1 and D2 regions of the 26S rRNA gene, and the ITS and 117

5.8S rRNA gene. Isolation of DNA, PCR amplification process and DNA sequencing of 118

rRNA genes of the isolates were performed as described by Cabañes et al., (2007). Statistic 119

analysis to evaluate significant differences among breeds and age of pigs was performed by 120

chi square test (P<0.01).121

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Results122

Ninety-two out of 408 animals scored positive for Malassezia spp. (22.5%). Three different 123

species were recognized, namely M. pachydermatis, M. sympodialis and M. furfur. All M. 124

pachydermatis isolates grew without lipid supplementation. Some of the lipid-dependent 125

isolates grew scarcely, making difficult conventional identification. However most of the 126

lipid-dependent species showed a strong catalase reaction, scored positive for assimilation of 127

Cremophor EL (M. furfur isolates) and the splitting of esculin (M. sympodialis isolates) and 128

were inhibited from Tween 20 only (M. sympodialis). PCR-RFLP results agreed with cultural 129

findings, and resulted discriminating for isolates with a reduced growing rate, generating 130

fragments identificative for M. pachydermatis, M. sympodialis and M. furfur, respectively. 131

The sequences of the isolates identified as M. sympodialis on a preliminary count, resulted 132

completely identical to M. sympodialis CBS 7222.133

M. pachydermatis was the sole species isolated from both wild boars and Cinta Senese breed, 134

being cultured from 24 animals for each group (12.9% and 20.7%, respectively). The 135

prevalence of recovery for Malassezia spp. in large size animals was 44.1%; M. sympodialis 136

was cultured from 28 subjects (63.6%), M. furfur from 10 (22.7%) and M. pachydermatis137

from 6 (13.6%). All yeasts species were obtained as a pure culture. Table shows the 138

prevalences of Malassezia spp. recovery from different swine breeds, based on farm and age 139

of animals.140

Animals harboring Malassezia species were present in 2 out of 3 intensive breeding farms, 141

while all farms in which Cinta Senese were raised yielded yeasts, with different prevalences. 142

Lipid-dependent species among large size breeds were limited to one farm only, where 88.3% 143

of adults scored positive. Statistical analysis showed significant differences among overall144

prevalence values obtained from wild boars and Cinta Senese on one hand, and large size 145

swine, on the other hand (P> 0.01). 146

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Among juvenile wild boars, M. pachydermatis was isolated from 3 subjects only (4.4%), 147

while this same species was recovered from 23/117 adults (19.6%). Thirty-eight out of 43 148

(88%) large size adult animals scored positive for lipid-dependent Malassezia species, while 149

M. pachydermatis was cultured from 6 juvenile subjects only (9.4%). This difference was 150

statistically significant (P<0.01). Sixteen out of 74 Cinta Senese adults (21.6%) were positive 151

for M. pachydermatis, and 8/42 juvenile pigs (19%) harbored this fungal species. For animals 152

belonging to this last breed, different prevalences were not statistically significant. 153

154

Discussion155

Epidemiology of Malassezia species in swine has been scarcely investigated. In literature 156

there is an unique paper dealing with this topic published since the taxonomic revision in 157

1996, while previous reports concern the presence of both lipid-dependent species and M. 158

pachydermatis (formerly Pityrosporum pachydermatis) in the external ear canal of healthy 159

animals (Garau et al., 2005). These authors recovered M. sympodialis and M. slooffiae from 160

73 out of 100 swine ears. In our paper 408 pigs were investigated, with an overall prevalence 161

of 22.5%. The occurrence of Malassezia yeasts appeared to be significantly different 162

depending on the productive aptitude of the animals and, in group of large size and in wild 163

boars on the age, also. To the best of our knowledge there are no reports about the occurrence 164

of Malassezia species in different breeds of Sus scropha.165

The most striking feature was represented by the fact that lipid-dependent fungal species were 166

cultured from adults belonging to large size breeds, only. Among these species, M. 167

sympodialis and M. furfur were identified, in partial contrast to the data published by Garau et 168

al. (2005), who isolated M. sympodialis and M. slooffiae. The presence of lipid dependent 169

Malassezia organisms in this group of animals could suggest that breed and lines used in 170

commercial production as well as diet may represent sources of variation of fat quality not 171

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only on fatty acid profiles of muscle and adipose tissue (Guo et al., 2006), but also on skin 172

composition traits. M. pachydermatis was present in all the 3 groups of investigated animals, 173

but the occurrence of isolation showed remarkable differences. It was the unique species 174

recovered both from wild boars and Cinta Senese breed animals, although a significant higher 175

fungal burden was present in adult wild boars, only. The presence of this yeast species in 176

juvenile large size pigs only was striking, considering that a simultaneous recovery of mixed 177

lipid-dependent and M. pachydermatis was not recorded neither by Garau et al. (2005) nor in 178

the present study. The colonies isolated from each positive animal were representative of an 179

unique Malassezia species as reported by the above cited Authors.180

The number animals checked for each group within different farms didn’t allow to draw 181

general conclusions from a statistic point of view. Nevertheless, different prevalence values 182

were observed among different farms, harbouring animals with similar management systems, 183

suggesting a circulation of fungal organisms among animals of the same piggery, whereas184

they are present.185

PCR-RFLP performed in the present study contextually with phenotypic identification 186

methods was chosen for its ability to discriminate among 11 yeasts species by using 2 187

restriction enzymes (Mihrendi et al., 2005). This identification approach, applied to cultured 188

isolates, appeared to be relatively fast and reliable, showing both a good discriminatory 189

power, and an unambiguous capability to differentiate newly identified species, with the 190

exception of M. sympodialis. To distinguish this last species from related ones, the sequencing 191

and analysis of the D1/D2 26S rRNA gene, and ITS-5.8S rRNA gene is necessary, as 192

demonstrated by Cabañes et al., (2007). 193

Lipid-dependent Malassezia species have been cultured from healthy subjects as stressed by 194

Garau et al. (2005), who appreciate studies dealing with yeast prevalence in skin/ear diseased 195

swine. It is remarkable to considerate that swine is a strictly zootechnic species, characterized 196

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by a relatively short productive life cycle, so a possible occurrence of otitis externa is often 197

neglected by breeders.198

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References199

Bond, R., Anthony, R.,M., 1995. Characterization of markedly lipid-dependent Malassezia 200

pachydermatis isolates from healthy dogs. J Appl Bacteriol. 78: 537-542.201

Cabañes, F. J., Theelen, B., Castellà, G., Boekhout, T., 2007. Two new lipid-dependent 202

Malassezia species from domestic animals. FEMS Yeast Res 7: 1064-1076.203

Crespo, M.,J., Abarca, M.,L., Cabañes, F.,J., 2002a. Occurrence of Malassezia spp. in the 204

external ear canals of dogs and cats with and without otitis externa. Med Mycol. 40: 115-121.205

Crespo, M.,J., Abarca, M.,L., Cabañes, F.,J., 2002b. Occurrence of Malassezia spp. in horses 206

and domestic ruminants. Mycoses. 45: 333–337.207

Dufait, R., 1985, Présence de Malassezia pachydermatis (syn. Pityrosporum canis) sur les 208

poils et les plumes des animaux domestiques. Bull Soc Fr Mycol Méd. 14: 19–22.209

Garau, M., Del Palacio, A., García, J., 2005. Prevalence of Malassezia spp. in healthy pigs. 210

Mycoses. 48: 17-20.211

Guého, E., Midgley, G., Guillot, J., 1996. The genus Malassezia with description of four new 212

species. Antoine van Leeuwenhoek. 69: 337-355.213

Guillot, J., Chermette, R., Guého, E., 1994. Prévalence du genre Malassezia chez les 214

mammifères. J Mycol Med. 4: 72–79.215

Guillot, J., Guého, E., 1995. The diversity of Malassezia yeasts confirmed by rRNA sequence 216

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Guo, Q., Richert, B., T., Burgess, J., R., Webel, D., M., Orr, D., E., Blair, M., Fitzner, G., E., 218

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different kinds of animals. Acta Path Microbiol Scand. 48: 51–55.225

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H., Hasegawa, A., 2004. Malassezia nana sp. nov., a novel lipid-dependent yeast species 227

isolated from animals. Int J Syst Evol Microbiol. 54: 623-627.228

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acid for M. furfur. Br J Dermatol. 137: 208-213.232

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Dermatol. 142: 1181-1184. 236

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Farm No. N. of animals juvenile adults positive juvenile animals (%) M. pachydermatis positive adult animals (%) M. furfur M. pachydermatis M. sympodialis1 29 29 0 4/29 (13.8%) recovered 0/0 (0%) 0 0 02 54 11 43 2/11 (18.2%) recovered 38/43 (88.4%) 10 0 283 24 24 0 0/24 (0%) not recovered 0/0 (0%) 0 0 0

total 107 64 43 6/64 (9.4%)* 38/43 (88.4%)* 10 0 284 36 4 32 4/4 (100%) recovered 8/32 (25%) 0 8 05 36 2 34 0/2 (0%) not recovered 8/34 (23.5%) 0 8 06 44 36 8 4/36 (11.1%) recovered 0/8 (0%) 0 0 0

total 116 42 74 8/42 (19%) 16/74 (21.6%) 0 16 0

185 68 117 3/68 (4.4%) recovered 23/117 (19.6%) 0 23 0

larg

e br

eeds

§§Ci

nta

Sene

se §

Wild boars§

§ statistically not significant values§§; * statistically significant values

Table - Prevalence and characterization of Malassezia species in function of farms, breed and age of examined animals.

Table