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Nuvee Prapasarakul (D.V.M.,Ph.D)Depart. of Vet. Micro. Faculty of Vet. Sci. Chulalongkorn University

cell wall

Ultrastructure of yeast and moldTargeted immune modulation,TM

Norwegian Beta Glucan (NBG)

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1. Hypersensitivity - an allergic reaction to molds and spores.

2. Mycotoxicoses - poisoning of man and animals by food products contaminated by fungi

3. Mycetismus- the ingestion of preformed toxin

4. Infection

Organ involvements • Phaeohyphomycosis• Hyalohyphomycosis

D t h t i

• Mycetoma• Chromoblastomycosis• Lobomycosis

• Histoplasmosis• Coccidioidomycosis• Blastomycosis• Dermatophytosis

• Dermatomycosis

Cutaneousmycoses

• Lobomycosis

Subcutaneous mycoses

• Blastomycosis• Invasive candidiasis• Zygomycosis

Systemic mycoses

Species of Fungi : DeuteromycotaCandidiasis

CryptococcosisHistoplasmosis

Sporothricosis

PythiosisSaprolegniasis

Chytridiomycosis

yp g

chytrid Batrachochytrium dendrobatidis

Pathogenic fungi• Blastomycosis,

Opportunistic fungi• Mycetoma, otomycosis, mycotic

k y

Coccidiomycosis, Paracoccidomycosis, Histoplasmosis

keratitis, meningitis, mycoticabscess, pulmonary aspergillosis, onychomycosis, dermatophytosis

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Fungi are divided into 2 broad groups

• Simple yeasts• Simple yeasts

• Mold

Fungal diseasesFungal diseases•• DermatophytosisDermatophytosis•• Subcutaneous mycosesSubcutaneous mycosesyy

–– ChromomycosisChromomycosis–– SporothrichosisSporothrichosis

•• Systemic mycosesSystemic mycoses–– AspergillosisAspergillosis–– Pathogenic yeastsPathogenic yeasts

Di hi f gi Di hi f gi –– Dimorphic fungi Dimorphic fungi –– PhycomycosesPhycomycoses

•• MucomycosesMucomycoses•• EntomophthomycosesEntomophthomycoses

•• MycotoxicosisMycotoxicosis

Mycoses in domestic animalsMycoses in domestic animalsAspergillosis, Dermatophytoses, OtitisDermatophytoses, Cryptococcosis

Fungal abortion caused by Aspergillus CandidaFungal abortion caused by Aspergillus, Candida, Zygomycetes

Dermatophytoses Cryptococcosis

•Pnuemonic cryptococcosis

•Aspergillosis

• Pnuemocystic carinii and arthritis

•Aspergillosis

Blanco, 2006

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Sporotrichosis Mycetoma Rhinosporidiosis Chromoblastomycosis

Subcutaneous zygomycosis

PhaeohyphomycosisUlocladium sp.

Hyalohyphomycosis

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locali ed lesionlocalized lesion

Cat: nonpruritic swelling in nasal planum

Dog: ventral abdominal lesion

sclerotic bodies: 5-12 mm

Sheep Conidiobolomycosis in Brazil, Silva et al., 2007.

การตรวจทางห้องปฏบิตักิาร

Tissue sample Splendore hoeppli material observed by H&E

KOH-preparation Histopathology

eosinophilic material coats the grains that are characteristic of the exudate in lesions of mycosis

Large hyphae8-20 μm

Dematiaceous fungus within the keratin of follicular infundibulum.

Epithelioid-like macrophages, multinucleated giant cells, and lymphocytes

Pyogranulomatous perifolliculitis.

Intrafollicular fungi stained black.

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Cutaneous dog pythiosisAtypical case of equine pythiosis. The infection began in the lower extremity and spread, trough lymphatic vessels, to the horse's chest.

Typical cutaneous pythiosis in an equine. Small white masses, known as "kunkers", are observed in the center of the lesion.

Granulomatous coagula consist of necrotic Mc, Eo and hyphae• Amputated section of a patient leg that had pythiosis.The

picture shows the aneurysm in the femoral artery caused by P. insidiosum from a Thai human patient.

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• Disseminated cryptococcosisMultiple skin-colored 2 to 10 mm nodules on the face, resembling mollusca contagiosa. Identical lesions are also seen in disseminated histoplasmosis and disseminated coccidoidomycosis.

• Aspergillus fumigatus **• Aspergillus niger

Clinical sign• Chronic nasal dischargep g g

• Aspergillus nidulans• Aspergillus flavus• Aspergillus terreus*

• Involve brain, heart, kidneys, spleen, lymph nodes and bones

• Aplastic anemia• Mortal

Histoplasmosis Paracoccidioidomycosis

Coccidioidomycosis Blastomycosis

H. capsulatumvar. capsulatum :Common histoplasmosis

H capsulatumH. capsulatumvar. duboisii : African type

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Fungal isolation

• fine-needle aspirate• tissue biopsy• blood• body cavity fluid

• fine-needle aspirate• tissue biopsy• blood• body cavity fluid

Caution: Fungal isolation is not recommended in routine veterinary practice because the organism is highly

pathogenic when grown in culture.

Gross pathology specimen of lung showing cut surface of fibrocaseous nodule due to Histoplasma capsulatum.

"Molluscum contagiosum" lesions

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Histopathology of coccidioidomycosis of lung. Mature spherulewith endospores of Coccidioides immitis, intense infiltrate of neutrophils.

HSP90

Glucuronylxylomannan (GXN)

Histone 2B

Phosphomannoprotein

Laminarin (1 3 β glucan)

Yeast and Mold Antigens

Laminarin (1,3 β glucan)

Adhesin

Special structures: conidia, hyphae, spherules.

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Defects in host defences that predispose patients to infections with specific fungi.

Fungal pathogen Host factor

• Candida (mucosal) Impaired cell mediated immunityC did (di i d) i d i• Candida (disseminated) Impaired mucosa or integument,

neutropenia• Aspergillus Neutropenia, high-dose corticosteroids• Cryptococcus Impaired cell mediated immunity,

corticosteroids• Zygomycetes Neutropenia, deferoxamine treatment,

corticosteroids, diabetic ketoacidosis• Fusarium Neutropenia, impaired integument,

corticosteroidscorticosteroids• Scedosporium Neutropenia• Trichosporon Neutropenia, impaired integument

How the pathogen elude and subvert the host defense.

Neutrophil, Macrophage, Monocyte, Complement, antimicrobial peptides.

VS

Immune response to specific Immune response to specific fungal antigensfungal antigens

eg. Cutaneous CandidiasisDifference of CMI and HMI response to pathogen

Yeast Germinating

Ab in normal Ab in patient

Cause of CMI deficiency has still not elucidated in chronic form.

Transformation of yeast: escape recognition

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Non specific host defence

Primary line of defense against invasion by fungi include:

- intact skin and mucus membrane

- inflammatory response and recruitment of

PMN neutrophil and macrophage

Host Physiology Defects

-Pregnancy, alcohol, drug abuse

-Prolong antibiotic administration

-Malnutrition Diabetic ketoacidosis

Patient Risk-Neutropenia or neutrophil dysfunction

-Cytotoxic chemotherapy for cancer

Corticosteroid:

Depress neutrophil and macrophage function.

Inhibit reactive oxidant intermediates and

-Immunosuppressant for tissue graft

Where do fungi from?

intermediates and killing of

phagocytosed fungi.

gYeasts normal flora

Mold environmental contamination

Host response to fungal infections

Extracellular and Combinative response

Intracellular antigens

Animal models is just an individual response, that do not make a complete explanation.

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Role Of Immune System VS Fungus

Most of fungi are too large to be digested by phygocytic neutrophil. However, these organism invoke HMI and p , gCMI.

HMI: Antibody does not appear to play the protective role but it is an important defense against yeast and mold…How?

Humeral antibody or complement fragments stimulate phagocytic cell to produce enzyme that can digest these fungican digest these fungi extracellulary

Common infection associated with immunodeficiency

Common infection associated with immunodeficiency

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Which cell is a principle mediator of antifungal mechanisms of phagocytic cell ?A Neutrophil D NK cellA. Neutrophil D. NK cellB. Basophile E. CTL C. Macrophage F. Eosinophil

Major mediator by Neutrophil Minor action by Macrophage

• Neutrophil: - Microbicidal oxidant; H2O2, hypochlorous acid,

monochloramine- non-oxidative substance; lysozyme, defensin,

lactoferrin, elastase, azuricidine, - Chemotactic responses - Calprotectin

Macrophagep g

- nitric oxide- lysozyme

Toll-like receptor (TLR) leukocyte expression patterns and PAMP specificities. Each of the TLRs is expressed on a different subset of leukocytes and each of the TLRs detects different subsets of pathogens allowing vigilant surveillance by the

immune system.

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Source of type 1 hypersensitivity

Source Of type 4 hypersensitivity

Principle of antigen to activated systemic immunity

Th1 and Th2

Promote killing of the intracellular microbes.

Skin : erythema, wheal, flare, urticaria

Lung : asthma

Intestine : diarrhea

Invasive Aspergillosis

Fungus triggers immune phenomenaAllergic rhinitis, asthma, hypersensitivity

pneumonitis, allergic bronchopulmonary aspergillosis, aspergilloma.

Normal Host Susceptible hostNormal Host Susceptible host

Maintain only conidia form Switch to hyphae form

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Toll like receptors identify microbial productsToll like receptors identify microbial products

• To conidia l i h

• To hyphaeAlternative pathway

TLR4 Dc and Mc : TNFα, IL1, MIPα

Classical pathwayTLR2 Dc and Mc: IL10

Th2Corticosteroid Th1, Mc, NeuCorticosteroid

Oxidative burst and superoxide anion of Neutrophils

Specific immune response to fungi

Major defense mechanism esp for intracellular fungiMajor defense mechanism esp. for intracellular fungi.

CD4+ and CD8+ T cell limit dissimilation of fungi to other organs.Th1 Th2

Phagocytic dependence Non-phagocytic dependence

Phagocytosis, Tissue injury, inflammationIgG production granulomatous, IgE

Host resistance Host susceptible

GM-CSF, IFN, TNF, IL1, IL6, IL12, IL18, MIP1, MCP

IL4, IL10

Macrophage and T-cell Interactions in Aspergillosis

Macrophages secrete chemokines that attract leukocytes and produce IFN-gamma and TNFα, thereby activating CD4 + T lymphocytes.

These lymphocytes produce cytokines, IL-2 and IFN-gammaand IFN-gamma. Cytolytic T cells and macrophage-produced,

lysosomal, reactive nitrogen and oxygen intermediates.In mice, the presence of TNF-alfa and IFN-

gamma: granuloma formation and control of infection.TGF-β, cause tissue destruction and

fibrosis.

In the process of macrophage activation, p p g ,epithelioid, loses its phagocytic ability.

Hyphae damage is achieved via extracellular means.

TNFα recruits neutrophils to damage hyphae via oxidative mechanism.GM-CSF, IFN, IL15, IL8

C did lbiCandida albicans

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Commensal Candida: Protective immunity in absence of immuneopathology

Commensal mucocutaneous systemic dissimilation

• Antigens–Adhesin

–Mannan

–Unicellulaer yeast

• Host defense–Salivary proteins

–CMI

–Complement

OPC, CMC CDC

–Pseudohyphae

–Hyphae

• ±HMI

Specific immune response to Candida

Major defense mechanism IFN, TNF, IL1, IL6, IL12

DC presents antigens via TLR/MyD88 signaling TLR superfamily/IL1

CD4+T cell antifungal Th1response

Induce proinflammatory cytokines

IL12,

NK cell, Mc Nu limit dissimilation of fungi

Host resistance

Host susceptible

IL4, IL10

Predominance of Th2 response progression of infection

Cryptococcus neoformansCryptococcus neoformans

• 90% associate with AIDS• Virulent factorsVirulent factors

– Polysaccharide antigens– Mannitol, Superoxide dismutase and

Melanin

•• glucuronoxylomannanglucuronoxylomannan

Capsular Capsular polysaccharidepolysaccharide

glucuronoxylomannanglucuronoxylomannan•• Ab unresponsive to AgAb unresponsive to Ag•• Inhibit leukocyte migrationInhibit leukocyte migration•• Down regulate TNFDown regulate TNFαα•• Up regulate ILUp regulate IL10 10 and Thand Th22•• Dysregulation of cytoineDysregulation of cytoine•• Inhibit phagocytosisInhibit phagocytosis

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• Intracellular killing

–Lysosomal fusion

–Phagosomal acidification

–Iron competition

–Enzymatic degradation

• Extracellular killing–Antifungal peptides

–Nitric oxide

–Oxygen intermediate

Host defense against Cryptococcosis

• CMI: Mc, Dc, NK, CD4+ and CD8+ infiltrate in infective sites.

• Complement: C3, C5a, C3a

• HMI: anticapsular antibody

ADCC enhances fungal killing.

Dermatomycosis (ringworm) of hair follicles

DermatophytosisDermatophytosis

• Major antigenic substance: chitin, glucan, glycopeptide, CHO and keratinase DTH

IgG• Broadly cross-reactive to other fungi

– Immediate hypersensitivity to air-born fungi and penicillin.

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Sublayer in Epidermis

Cells with immunologic potential in the skin

• Epidermal langerhans cells: MHC class I and II

• Dermal dendritic cells: MHC class II

• Epidermal T lymphocytes: 60% αβ heterodimer / 29 % δγ heterodimer

• Dermis T lymphocytes:80% αβ heterodimer / 7 % δγ heterodimer

• Keratinocytes: structural role, cell proliferationMHC class I CTL contact hypersensitivity, graft-versus-host yp y, g

defense, lichenoid infiltrate MHC class II induce IFN γ

• Microvascular endothelial cells: wound healing, angiogenesis

Produce cytokine and chemotactic factor esp. macrophage

Epidermal langerhans cells

• 2-5% esp at head, face trunk, limbs2 5% esp at head, face trunk, limbs > genital region, plam.

• Surface makers: CD1a, CD32, CD23, CD11b-CD18, CD25, CD40, CD54, CD58, MHC I and MHC II

• Adhesion molecules: LFA-3, ICAM-1, B7

C t ki• Cytokines: IL-1β, IL-6, TNFα

Dermal dendritic cell

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The Inflammatory Response

Relationship between inflammation and chronicityRelationship between inflammation and chronicity

Anthropophilic Zoophilic M. furfur, E. floccosum T. verrucosum

T. rubrum

Chronic response Acute response

Dermatophytosis

• Major antigenic substance: chitin• Major antigenic substance: chitin, glucan, glycopeptide, CHO and keratinase DTH

IgG• Broadly cross-reactive to other fungiy g

– Immediate hypersensitivity to air-born fungi and penicillin.

Pathogenesis Pathogenesis and pathogenicityPathogenesis and pathogenicity

• Human: response in Type 1 > Type 4 to trichophytin (IgE inhibit development of CMI??)

Acute Chronic+ve for DTH ± ve CMI response testingp g

• Guinea pig: Type 1 and 4 erythema • Cattle: Type 4 desquamation of St corneum Hyperkeratinization and hyperpigmentation at

anal region and tail caused by M. pachydermatis

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Exudate stained with methylene blue observed bottle-shaped cells.

Biological properties of Malassezia

• Over reaction of yeast on epidermis by y p yproteolytic enzyme

• Lipid is compensated by cerunimous gland• Gamma lactone and decarboxylic acid

products• Lipase, phospholipase, hydrolase

C l lik l ll ti

Azelaic acid

• Capsular-like or lamellar antigen• Tyrosinase: key of melanogenesis• Malassezin: apoptosis of melanocytes

Malassezia associated skin disease

• Increased Type I hypersensitivity• Increased Malassezia sp IgE• Malassezia sp Th2 response• Induction eczematous lesion when

exposed to Malassezia

Type 4/Type 1 hypersensitivity

Granulomatous response

Immunocompromised

Fungal species