Malassezia Dermatitis: The Host-Pathogen Relationship

1. Malassezia Dermatitis:The Host-Pathogen Relationship Daniel O. Morris, DVM Diplomate, ACVD University of Pennsylvania Philadelphia, PA USA

2. The genus Malassezia: Background • Lipophilic: • Able to utilize lipids as a source of carbon • Lipid-dependent species lack the ability to synthesize long-chain (C12-C24 series) fatty acids- hence their requirement for an exogenous lipid source • Only M. pachydermatis is truly non-lipid dependent, but its growth is enhanced by exogenous lipid D Morris ACVD Resident Review 2003

3. Ecology • Opportunistic pathogens • Commensal microflora in nearly all mammals (and some birds) studied • Keratinophilic • Dwell primarily within the stratum corneum • Isolated from the canine distal hair shaft • Rarely found in the follicular infundibulum D Morris ACVD Resident Review 2003

4. Species classification • Lipid-dependent species • M. furfur (the primary human pathogen) • M. sympodialis • M. globosa • M. obtusa • M. restricta • M. slooffiae • M. equi • Non-lipid dependent species • M. pachydermatis (the primary animal pathogen) D Morris ACVD Resident Review 2003

5. M. pachydermatis

6. M. globosa

7. M. restricta

8. M. sympodialis

9. -Occur in yeast form in-vivo (non-mycelial), but hyphae may occur in culture M. furfur

11. -Thick, multilayered cell wall-Repetitive uni-polar (sympodial) budding to produce blastoconidia

12. Microbiology of M. pachydermatis • Optimum growth at 37oC (range 32-40) • Lipid-enhanced SDA or Dixon’s agar • Microaerophilic conditions (5% CO2) • Colonies mature at 72 hours D Morris ACVD Resident Review 2003

15. Candida species

16. Microbiology: Isolation and I.D. • Cytology • Quantitative: direct impression smear, tape stripping • Non-quantitative: cotton-tip swab, dry skin scraping • Culture • Quantitative methods: tape-stripping, detergent scrub, contact plate • Non-quantitative: sterile swab D Morris ACVD Resident Review 2003

17. Microbiology: Isolation and I.D. • Molecular techniques • Nested PCR: Species-specific primers for ribosomal RNA • DNA fingerprinting by PFGE: Allows testing for concordance of identity between strains (allows tracking of epizootics) D Morris ACVD Resident Review 2003

18. Correlation with pathogenic effect • From the literature: • Quantitative cytology • >10 organisms per ½ inch2 (evidence-based)* • >1 organism/oil-immersion field (non-evidence based) • Ear exudate: >5 organisms/oil-immersion field • Quantitative culture • Colony counts from contact-plate cultures significantly correlate with cytological (tape-strip) counts† *Morris, et al. Type-1 hypersensitivity reactions to M. pachydermatis extracts in atopic dogs. AJVR 1998. †Nuttal, et al. Serum antibodies to Malassezia yeasts in canine atopic dermatitis. Vet Dermatol 2001 D Morris ACVD Resident Review 2003

19. 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?? D Morris ACVD Resident Review 2003

23. Non-atopic disease associations • Parakeratotic diseases • Zinc-responsive dermatosis • Generic dog food dermatosis • Superficial necrolytic dermatitis • Cutaneous T-cell lymphoma • Canine Cushing’s disease D Morris ACVD Resident Review 2003

26. Occurrence in the oral cavity • Considered to be part of the normal oral microflora of dogs • Implicated in a case of severe, ulcerative stomatitis/pharyngitis/ tonsillitis D Morris ACVD Resident Review 2003

27. Malassezia dermatitis - feline Feline paraneoplastic alopecia D Morris ACVD Resident Review 2003

28. Malassezia in avian species?? Role in feather picking? D Morris ACVD Resident Review 2003

30. Malassezia in domestic farm animals • Isolated from normal ruminants, horses, and swine. • Role in otic or cutaneous diseases remains to be determined. D Morris ACVD Resident Review 2003

32. Pathogen-dependent immunological factors (M. pachydermatis/M. furfur) • Production of allergens relevant to dogs and human beings, respectively • Fixation of complement (alternative pathway)  anaphylatoxin production (C3a, C4a, C5a) inflammation • Does not require sensitization or allergen-specific recognition • Role in non-atopic inflammatory diseases? D Morris ACVD Resident Review 2003

33. 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 D Morris ACVD Resident Review 2003

34. Pathogen virulence factors • Adherence to corneocytes • Increased at higher temperatures • No influence by concurrent Staph. colonization • Mediated by trypsin-sensitive proteins/glyco-proteins and mannosyl-bearing carbohydrate ligands on the yeast cell wall, or by lipid ligands in otic infections • Marked variability amongst strains • Implication: identification of an universal ligand inhibitor is unlikely D Morris ACVD Resident Review 2003

35. Pathogen virulence factors II • Lipid hydrolysis  FFA’s  inhibition of competing microorganisms • Production of catabolic enzymes (role in pruritus?) • Proteinase – role in keratin penetration? • Chondroiton-sulphatase • Phospholipase • Hyaluronidase D Morris ACVD Resident Review 2003

36. Epidermal hyperplasia – relevance?

37. Host factors: colonization of skin surface • Dogs, cats, human beings: colonization occurs within hours of birth • Sparsely haired skin • Peri-orificial, periungual • Seborrheic “zones” (head and neck in human beings) • External ear canals in dogs and cats • Mucosae • Mucosal disease due to Malassezia has been reported but appears to be rare as opposed to Candidiasis. Anal sacs a reservoir for inoculation? D Morris ACVD Resident Review 2003

38. Host factors: Microclimate • Areas with increased moisture and/or sebum are favored • Ear canals express a higher temp. and humidity than the skin surface • Pinnal shape is unrelated to occurrence rates of MO • Sterile saline added to the ear canal is sufficient to induce Malassezia otitis • Anecdotal reports of increased rate of primary Malassezia otitis and pododermatitis in humid climates D Morris ACVD Resident Review 2003

39. Host factors II: Immunological responses • Type-1 hypersensitivity • IgE-mediated: • Documented via IDAT, SPT, ELISA, RAST, and APT in atopic human beings. Major allergens characterized. • Documented via IDAT, ELISA, and PCA testing in dogs with atopic dermatitis. Major allergens characterized. • Cell-mediated response: • Lymphocyte-mediated (Th2) • Documented via in-vivo and in-vitro LT testing and cytokine profiling in human beings with AD • Documented via in-vitro LT testing in dogs with AD D Morris ACVD Resident Review 2003

40. Evidence for Type-1 hypersensitivity in dogs • Intradermal testing: • Dogs with increased M. pachydermatis colonization, in association with atopic dermatitis, have stronger IDAT reactions to crude extracts of M. pachydermatis than do atopic dogs without MD. • Normal dogs do not react to the extract. D Morris ACVD Resident Review 2003

41. Evidence for Type-1 hypersensitivity in dogs, II • Testing for passive cutaneous anaphylaxis (P-K) • Pooled serum from atopic dogs with MD that are IDAT positive for M. pachydermatis is injected ID in serial dilutions into normal dogs • At 24, 48 and 72 hours, the M. pachydermatis extract is injected ID at the former serum injection sites S 1:8 1:16 1:32 1:64 1:4 M 1:2 H D Morris ACVD Resident Review 2003

42. P-K testing, cont…. • Formation of a wheal/flare reaction indicates transfer of anti-Malassezia antibody from the donor serum – persistence to 72 hours indicates IgE is present (vs. IgG) • Heating (56oc for 4 hours) and adsorption with anti-canine IgE removes the IgE D Morris ACVD Resident Review 2003

43. Evidence for Type-1 hypersensitivity in dogs, III • ELISA testing: • Atopic dogs (both with or without MD) have significantly higher levels of anti-Malassezia IgE than normal dogs, or non-atopic dogs with Malassezia overgrowth. • Normal dogs do not have high levels of circulating antibody toward the normal (commensal) population of yeast D Morris ACVD Resident Review 2003

46. Evidence for Type-1 hypersensitivity in dogs, IV • Clinical evidence • Specific antifungal therapy of atopic dogs with MD will often ameliorate the majority of pruritus within several days. Rapid relapse of pruritus occurs when yeast counts rise again. D Morris ACVD Resident Review 2003

47. Evidence for T-cell Dysregulation in atopic dogs with MD • Lymphocyte blastogenesis testing (LT) methods: • Peripheral blood mononuclear cells isolated by hypaque-ficoll density gradient • PBMC incubated for 7 days with M. pachydermatis extract at serial concentrations • PHA used as pos. control; untreated media used as neg. control D Morris ACVD Resident Review 2003

48. T-cell responses II • Results: • Atopic dogs with MD have increased LT response to M. pachydermatis compared to normal dogs and atopic dogs with Malassezia otitis. Atopic dogs without MD or MO are intermediate • [Bond, et al: Basset hounds with idiopathic seborrhea have a blunted LT response to M. pachydermatis when compared to normal Bassets] D Morris ACVD Resident Review 2003

49. T-cell responses III • Conclusions: • T-cell dysregulation contributes to the pathogenesis of MD (as in human beings with AD) • Like human beings with Seb Derm, Basset hounds with seborrhea do not have a normal (inadequate?) T-cell response • Cerumen within the ear canal may be protective against penetration by (and recognition of) Malassezia allergens D Morris ACVD Resident Review 2003

50. A role for superantigens? • What are superantigens? • Microbial products that bind directly to the V region of the TCR without regard for antigen specificity • Result: activation of large “families” of T-cells that express a common V region • Activation of 5-30% of the T-cell population simultaneously with production of inflammatory cytokines and binding to “innocent” antigens = “innocent bystander” damage • Role in Malassezia dermatitis: • Ruled out in human beings • Studies in atopic dogs planned with newly developed monoclonal antibodies for V-region gene products of TCR D Morris ACVD Resident Review 2003