Malassezia Dermatitis:Malassezia Dermatitis:The Host-Pathogen The Host-Pathogen

RelationshipRelationship

Daniel O. Morris, DVMDaniel O. Morris, DVMDiplomate, ACVDDiplomate, ACVD

University of PennsylvaniaUniversity of Pennsylvania

Philadelphia, PA USAPhiladelphia, PA USA

D Morris ACVD Resident Review

2003

The genus MalasseziaThe genus Malassezia: Background

Lipophilic:Lipophilic: Able to utilize lipids as a source of carbonAble to utilize lipids as a source of carbon Lipid-dependent species lack the ability to Lipid-dependent species lack the ability to

synthesize long-chain (C12-C24 series) synthesize long-chain (C12-C24 series) fatty acids- hence their requirement for fatty acids- hence their requirement for an exogenous lipid sourcean exogenous lipid source

Only M. pachydermatis is Only M. pachydermatis is trulytruly non-lipid non-lipid dependent, but its growth is enhanced by dependent, but its growth is enhanced by exogenous lipidexogenous lipid

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Ecology

Opportunistic pathogensOpportunistic pathogens

Commensal microflora in nearly all Commensal microflora in nearly all mammals (and some birds) studiedmammals (and some birds) studied

KeratinophilicKeratinophilic Dwell primarily within the stratum Dwell primarily within the stratum

corneumcorneum Isolated from the canine distal hair shaftIsolated from the canine distal hair shaft Rarely found in the follicular infundibulumRarely found in the follicular infundibulum

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Species classification

Lipid-dependent speciesLipid-dependent species M. furfur (the primary human pathogen)M. furfur (the primary human pathogen) M. sympodialisM. sympodialis M. globosaM. globosa M. obtusaM. obtusa M. restrictaM. restricta M. slooffiaeM. slooffiae M. equiM. equi

Non-lipid dependent speciesNon-lipid dependent species M. pachydermatis (the primary animal M. pachydermatis (the primary animal

pathogen)pathogen)

M. pachydermatis

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M. globosa

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M. restricta

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M. sympodialis

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-Occur in yeast form in-vivo (non--Occur in yeast form in-vivo (non-mycelial), but hyphae may occur in mycelial), but hyphae may occur in

culturecultureM. furfur

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-Thick, multilayered cell wall-Thick, multilayered cell wall-Repetitive uni-polar (sympodial) budding -Repetitive uni-polar (sympodial) budding

to produce blastoconidiato produce blastoconidia

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Microbiology of M. pachydermatis

Optimum growth at 37oC (range 32-40)Lipid-enhanced SDA or Dixon’s agarMicroaerophilic conditions (5% CO2)

Colonies mature at 72 hours

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Candida species

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Microbiology: Isolation and I.D.

CytologyCytology Quantitative: direct impression Quantitative: direct impression

smear, tape strippingsmear, tape stripping Non-quantitative: cotton-tip swab, Non-quantitative: cotton-tip swab,

dry skin scrapingdry skin scraping

Culture Culture Quantitative methods: tape-stripping, Quantitative methods: tape-stripping,

detergent scrub, contact platedetergent scrub, contact plate Non-quantitative: sterile swabNon-quantitative: sterile swab

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Microbiology: Isolation and I.D.

Molecular techniquesMolecular techniques Nested PCR: Species-specific primers Nested PCR: Species-specific primers

for ribosomal RNAfor ribosomal RNA DNA fingerprinting by PFGE: Allows DNA fingerprinting by PFGE: Allows

testing for concordance of identity testing for concordance of identity between strains (allows tracking of between strains (allows tracking of epizootics)epizootics)

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Correlation with pathogenic effect

From the literature:From the literature: Quantitative cytologyQuantitative cytology

>10 organisms per ½ inch>10 organisms per ½ inch2 2 (evidence-based)*(evidence-based)* >1 organism/oil-immersion field (non-evidence based)>1 organism/oil-immersion field (non-evidence based) Ear exudate: >5 organisms/oil-immersion fieldEar exudate: >5 organisms/oil-immersion field

Quantitative cultureQuantitative culture Colony counts from contact-plate cultures significantly Colony counts from contact-plate cultures significantly

correlate with cytological (tape-strip) countscorrelate with cytological (tape-strip) counts††

*Morris, et al. Type-1 hypersensitivity reactions to M. *Morris, et al. Type-1 hypersensitivity reactions to M. pachydermatis extracts in atopic dogs. AJVR 1998.pachydermatis extracts in atopic dogs. AJVR 1998.

††Nuttal, et al. Serum antibodies to Malassezia yeasts in canine Nuttal, et al. Serum antibodies to Malassezia yeasts in canine atopic dermatitis. Vet Dermatol 2001atopic dermatitis. Vet Dermatol 2001

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2003

Clinical manifestations

Primary MD is rare: chronic wetting/ high humidity?

Primary Malassezia otitis: much more common - “swimmer’s ear”

Secondary MD: Atopy (and other allergic diseases) Seborrheic disorders Endocrine/metabolic diseases Systemic neoplasia (cats)

Virulence differences between strains??

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Non-atopic disease associations

Parakeratotic diseases Zinc-responsive dermatosis Generic dog food dermatosis Superficial necrolytic dermatitis

Cutaneous T-cell lymphomaCanine Cushing’s disease

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Occurrence in the oral cavity

Considered to be part of the normal oral microflora of dogsImplicated in a case of severe, ulcerative stomatitis/pharyngitis/ tonsillitis

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Malassezia dermatitis - feline

Feline paraneoplastic Feline paraneoplastic alopeciaalopecia

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Malassezia in avian species??

Role in feather picking?Role in feather picking?

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Malassezia in domestic farm animals

Isolated from normal ruminants, horses, and swine. Role in otic or cutaneous diseases remains to be determined.

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Pathogen-dependent immunological factors (M. pachydermatis/M. furfur)

Production of allergens relevant to dogs and human beings, respectivelyFixation of complement (alternative pathway) anaphylatoxin production (C3a, C4a, C5a) inflammation Does not require sensitization or

allergen-specific recognition Role in non-atopic inflammatory

diseases?

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Immunological factors, cont…

Directed effect to human keratinocytes leading to cytokine production; net effect is pro-inflammatory This effect varies according to yeast

species M. furfur does not induce cytokine

release in human keratinocytes M. pachydermatis does

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Pathogen virulence factors

Adherence to corneocytesAdherence to corneocytes Increased at higher temperaturesIncreased at higher temperatures No influence by concurrent Staph. No influence by concurrent Staph.

colonizationcolonization Mediated by trypsin-sensitive Mediated by trypsin-sensitive

proteins/glyco-proteins and mannosyl-proteins/glyco-proteins and mannosyl-bearing carbohydrate ligands on the yeast bearing carbohydrate ligands on the yeast cell wall, or by lipid ligands in otic infectionscell wall, or by lipid ligands in otic infections

Marked variability amongst strainsMarked variability amongst strains Implication: identification of an universal Implication: identification of an universal

ligand inhibitor is unlikely ligand inhibitor is unlikely

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Pathogen virulence factors II

Lipid hydrolysis Lipid hydrolysis FFA’s FFA’s inhibition inhibition of competing microorganismsof competing microorganismsProduction of catabolic enzymes (role Production of catabolic enzymes (role in pruritus?)in pruritus?) Proteinase – role in keratin penetration?Proteinase – role in keratin penetration? Chondroiton-sulphataseChondroiton-sulphatase PhospholipasePhospholipase HyaluronidaseHyaluronidase

Epidermal hyperplasia – relevance?Epidermal hyperplasia – relevance?

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Host factors: colonization of skin surface

Dogs, cats, human beings: colonization occurs Dogs, cats, human beings: colonization occurs within hours of birthwithin hours of birthSparsely haired skinSparsely haired skin Peri-orificial, periungualPeri-orificial, periungual Seborrheic “zones” (head and neck in human Seborrheic “zones” (head and neck in human

beings)beings) External ear canals in dogs and catsExternal ear canals in dogs and cats

MucosaeMucosae Mucosal disease due to Malassezia has been Mucosal disease due to Malassezia has been

reported but appears to be reported but appears to be rare rare as opposed to as opposed to Candidiasis. Anal sacs a reservoir for Candidiasis. Anal sacs a reservoir for inoculation?inoculation?

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Host factors: Microclimate

Areas with increased moisture and/or Areas with increased moisture and/or sebum are favoredsebum are favored Ear canals express a higher temp. and Ear canals express a higher temp. and

humidity than the skin surfacehumidity than the skin surface Pinnal shape is unrelated to occurrence rates of MOPinnal shape is unrelated to occurrence rates of MO

Sterile saline added to the ear canal is Sterile saline added to the ear canal is sufficient to induce Malassezia otitissufficient to induce Malassezia otitis

Anecdotal reports of increased rate of Anecdotal reports of increased rate of primary Malassezia otitis and pododermatitis primary Malassezia otitis and pododermatitis in humid climatesin humid climates

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Host factors II: Immunological Immunological responsesresponses

Type-1 hypersensitivityType-1 hypersensitivity IgE-mediated: IgE-mediated:

Documented via IDAT, SPT, ELISA, RAST, and APT in Documented via IDAT, SPT, ELISA, RAST, and APT in atopic human beings. Major allergens characterized.atopic human beings. Major allergens characterized.

Documented via IDAT, ELISA, and PCA testing in Documented via IDAT, ELISA, and PCA testing in dogs with atopic dermatitis. Major allergens dogs with atopic dermatitis. Major allergens characterized.characterized.

Cell-mediated response:Cell-mediated response: Lymphocyte-mediated (ThLymphocyte-mediated (Th22))

Documented via Documented via in-vivoin-vivo and and in-vitroin-vitro LT testing and LT testing and cytokine profiling in human beings with ADcytokine profiling in human beings with AD

Documented via Documented via in-vitroin-vitro LT testing in dogs with AD LT testing in dogs with AD

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Evidence for Type-1 hypersensitivity in dogs

Intradermal testing:Intradermal testing: Dogs with increased M. pachydermatis Dogs with increased M. pachydermatis

colonization, in association with atopic colonization, in association with atopic dermatitis, have stronger IDAT reactions to dermatitis, have stronger IDAT reactions to crude extracts of M. pachydermatis than do crude extracts of M. pachydermatis than do atopic dogs without MD. atopic dogs without MD.

Normal dogs do not react to the extract.Normal dogs do not react to the extract.

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Evidence for Type-1 hypersensitivity in dogs, II

Testing for passive cutaneous anaphylaxis (P-Testing for passive cutaneous anaphylaxis (P-K)K) Pooled serum from atopic dogs with MD that are Pooled serum from atopic dogs with MD that are

IDAT positive for M. pachydermatis is injected ID in IDAT positive for M. pachydermatis is injected ID in serial dilutions into normal dogsserial dilutions into normal dogs

At 24, 48 and 72 hours, the M. pachydermatis extract At 24, 48 and 72 hours, the M. pachydermatis extract is injected ID at the former serum injection sitesis injected ID at the former serum injection sites

MS

H1:2 1:4 1:8 1:16 1:32 1:64

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P-K testing, cont….

Formation of a wheal/flare reaction indicates Formation of a wheal/flare reaction indicates transfer of anti-Malassezia antibody from the transfer of anti-Malassezia antibody from the donor serum – persistence to 72 hours donor serum – persistence to 72 hours indicates IgE is present (vs. IgG)indicates IgE is present (vs. IgG)

Heating (56Heating (56ooc for 4 hours) and adsorption with c for 4 hours) and adsorption with anti-canine IgE removes the IgEanti-canine IgE removes the IgE

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Evidence for Type-1 hypersensitivity in dogs, III

ELISA testing:ELISA testing: Atopic dogs (both with or without MD) have Atopic dogs (both with or without MD) have

significantly higher levels of anti-Malassezia significantly higher levels of anti-Malassezia IgE than normal dogs, or non-atopic dogs IgE than normal dogs, or non-atopic dogs with Malassezia overgrowth.with Malassezia overgrowth.

Normal dogs do not have high levels of Normal dogs do not have high levels of circulating antibody toward the normal circulating antibody toward the normal (commensal) population of yeast(commensal) population of yeast

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Evidence for Type-1 hypersensitivity in dogs, IV

Clinical evidenceClinical evidence Specific antifungal therapy of atopic dogs Specific antifungal therapy of atopic dogs

with MD will often ameliorate the majority of with MD will often ameliorate the majority of pruritus within several days. Rapid relapse pruritus within several days. Rapid relapse of pruritus occurs when yeast counts rise of pruritus occurs when yeast counts rise againagain..

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Evidence for T-cell Dysregulation in atopic dogs with MD

Lymphocyte blastogenesis testing (LT) Lymphocyte blastogenesis testing (LT) methods:methods: Peripheral blood mononuclear cells isolated Peripheral blood mononuclear cells isolated

by hypaque-ficoll density gradientby hypaque-ficoll density gradient PBMC incubated for 7 days with M. PBMC incubated for 7 days with M.

pachydermatis extract at serial pachydermatis extract at serial concentrationsconcentrations

PHA used as pos. control; untreated media PHA used as pos. control; untreated media used as neg. controlused as neg. control

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T-cell responses II

Results: Results: Atopic dogs with MD have increased LT Atopic dogs with MD have increased LT

response to M. pachydermatis compared to response to M. pachydermatis compared to normal dogs and atopic dogs with Malassezia normal dogs and atopic dogs with Malassezia otitis. Atopic dogs without MD or MO are otitis. Atopic dogs without MD or MO are intermediateintermediate

[Bond, et al: Basset hounds with idiopathic [Bond, et al: Basset hounds with idiopathic seborrhea have a blunted LT response to M. seborrhea have a blunted LT response to M. pachydermatis when compared to normal pachydermatis when compared to normal Bassets]Bassets]

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T-cell responses III

Conclusions:Conclusions: T-cell dysregulation contributes to the T-cell dysregulation contributes to the

pathogenesis of MD (as in human beings pathogenesis of MD (as in human beings with AD)with AD)

Like human beings with Seb Derm, Basset Like human beings with Seb Derm, Basset hounds with seborrhea do not have a hounds with seborrhea do not have a normal (inadequate?) T-cell response normal (inadequate?) T-cell response

Cerumen within the ear canal may be Cerumen within the ear canal may be protective against penetration by (and protective against penetration by (and recognition of) Malassezia allergensrecognition of) Malassezia allergens

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A role for superantigens?

What are superantigens?What are superantigens? Microbial products that bind directly to the VMicrobial products that bind directly to the V region of region of

the TCR without regard for antigen specificitythe TCR without regard for antigen specificity Result: activation of large “families” of T-cells that Result: activation of large “families” of T-cells that

express a common Vexpress a common V region region Activation of 5-30% of the T-cell population Activation of 5-30% of the T-cell population

simultaneously with production of inflammatory simultaneously with production of inflammatory cytokines and binding to “innocent” antigens = cytokines and binding to “innocent” antigens = “innocent bystander” damage“innocent bystander” damage

Role in Malassezia dermatitis:Role in Malassezia dermatitis: Ruled out in human beingsRuled out in human beings Studies in atopic dogs planned with newly developed Studies in atopic dogs planned with newly developed

monoclonal antibodies for Vmonoclonal antibodies for V--region gene products of region gene products of TCRTCR

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Future therapies for Malassezia dermatitis

Currently, treatment continues to depend Currently, treatment continues to depend upon topical and systemic anti-fungal upon topical and systemic anti-fungal agentsagents

Allergen immunotherapy?Allergen immunotherapy? Crude extracts vs. recombinant allergens?Crude extracts vs. recombinant allergens? Double-masked, placebo-controlled trials will Double-masked, placebo-controlled trials will

be requiredbe required 12 to 18 months in duration12 to 18 months in duration

Anti-IgE therapiesAnti-IgE therapies Trials on-going in human beingsTrials on-going in human beings

D Morris ACVD Resident Review 2003