Introduction to Veterinary Mycology

Chapter 44 Introduction to Veterinary Mycology


Fungal diseases of nonhumans were reported nearly two centuries ago, and among the first was infection by Beauveria bassiana, a fungal pathogen of silkworms that nearly destroyed the Chinese silk industry in the early nineteenth century. After many decades of relative neglect, modern veterinary mycology has become part of the mainstream of veterinary microbiology. Frequency of diagnosis of mycoses in domestic animals has increased in concert with the pattern in human medicine. This is partly because of advances in veterinary oncology, in which immunosuppressive treatments predispose to fungal infection. In addition, the increased number of effective antifungal agents for use in animals has increased interest in detection, identification, and characterization of animal-associated fungi.


Fungi are unicellular or multicellular chemoheterotrophic eukaryotes. Their cell walls contain cellulose, chitin, glucan, chitosan, and/or mannan. For practical purposes they may be grouped into yeasts, molds, and fungal-like agents. Yeasts are oval to spherical single cells; they reproduce by budding, a phenomenon in which a progenitor cell pinches off part of itself to produce another identical cell. Molds are multicellular filamentous fungi that consist of masses of threadlike filaments called hyphae that grow by elongation of their tips into an intertwining mat called the mycelium (Figure 44-1). Fungal-like agents, as exemplified by Prototheca and Pythium species, have traditionally been discussed with mycotic agents because they produce elements that resemble fungi in tissue, and some may form yeastlike colonies on mycologic media.



The individual reproductive bodies of fungi are called spores. During the evolutionary process, most fungi relied on a combination of asexual and sexual reproduction for survival. Asexual spores are produced by mitosis. The two main types of asexual spores are sporangiospores and conidia (Figure 44-2). Many of the lower fungi of the class Zygomycetes produce sporangiospores that are contained in a closed structure called the sporangium. Conidia are naked spores borne on hyphae and produced by fungi such as Aspergillus and Penicillium spp. Sexual spores are produced through fusion of the protoplasm and nuclei of two cells by meiosis, and include ascospores, basidiospores, and zygospores (Figure 44-3). Sexual spores and the protective structures that surround them serve as the basis for fungal classification.




Pathogenic fungi are categorized into four groups based on sexual reproduction, even if sexual reproduction has not been observed. These groups correspond to phyla within the kingdom Fungi and comprise the ascomycetes, basidiomycetes, zygomycetes, and deuteromycetes (fungi imperfecti). The first three groups produce sexual spores, but the deuteromycetes are “imperfect,”in that they have no known sexual state. The genera comprising the deuteromycetes are called “form” genera, because they are characterized without the use of sexual structures. Taxonomy of the veterinary pathogenic fungi is presented in Table 44-1.


TABLE 44-1 Taxonomy of the Veterinary Pathogenic Fungi





























































Taxonomic Designation Representative Genera Veterinary Disease
Phylum Zygomycota, Class Zygomycetes
Order Mucorales Absidia, Mucor, Rhizopus Abortion, zygomycosis
Order Entomophthorales Basidiobolus, Conidiobolus Nasal infection, zygomycosis
Phylum Ascomycota, Class Ascomycetes
Order Saccharomycetales Pichia (teleomorphs of some Candida spp.) Numerous mycoses
Order Onygenales Arthroderma (teleomorphs of dermatophytes) Ringworm
Order Eurotiales Teleomorphs of some Aspergillus spp. Aspergillosis
Order Microascales Pseudoallescheria boydii (teleomorph of Scedosporium apiospermum) Mycetoma
Order Pyrenomycetes Gibberella (teleomorph of some Fusarium spp.) Mycotic keratitis
Phylum Basidiomycota, Class Basidiomycetes
Order Tremellales Filobasidiella (teleomorph of Cryptococcus neoformans) Cryptococcosis
Phylum Deuteromycota, Class Deuteromycetes
Order Cryptococcales Candida, Cryptococcus, Malassezia, Trichosporon Numerous mycoses
ORDER MONILIALES
Family Moniciliaceae Aspergillus, Coccidioides, Sporothrix Numerous mycoses
Family Dematiaceae Alternaria, Bipolaris, Cladosporium, Curvularia, Exophiala Chromoblastomycosis, mycetoma, nasal granuloma, phaeohyphomycosis

Mycology is often descriptive, and the importance of morphology in the identification process mitigates in favor of a clear understanding of mycologic terminology (Table 44-2). The mycelial mat is known as the thallus, but this term is specifically reserved for colonial growth that arises from a single spore. Some of the medically significant fungi, such as Histoplasma capsulatum and Blastomyces dermatitidis, are dimorphic, in that they can exist in either the yeast or mold form; yeast forms are present at 37° C and the mold form is present at 25° C. Dematiaceous fungi are dark colored, containing melanin in hyphal or conidial cell walls. Dermatophytes invade hair, nails, and claws.


TABLE 44-2 Glossary of Mycologic Terminology



































































































































































































Term Definition
Aerial hyphae Hyphae that develop above the agar surface
Anamorph Asexual form of a conidial fungus
Annelide Cell that produces conidia; as the conidium is released, a ring of cell wall material is formed
Anthropophilic Fungi that almost exclusively infect humans; examples are Epidermophyton floccosum and Trichophyton rubrum
Arthrospore Asexual spore produced by fragmentation of existing hypha into separate cells; examples are Coccidioides immitis and Geotrichum candidum
Ascospore Sexual spore produced in a saclike structure (ascus), characteristic of true yeasts
Ascus Round saclike structure that contains ascospores
Asexual Reproduction by division or redistribution of nuclei but without nuclear fusion
Basidiospore Sexual spore formed on the basidium
Blastoconidium Blastospore; conidium formed by budding along hypha, pseudohypha, or single cell; examples are Candida albicans and Cryptococcus neoformans
Budding Process of asexual reproduction in which new cells develop as outgrowths of the parent cell; examples are yeasts
Capsule Colorless, mucopolysaccharide sheath on the cell wall; example is Cryptococcus neoformans
Chlamydospore Thick-walled resistant cell formed as a result of enlargement of an existing hyphal cell; examples are Histoplasma capsulatum and Candida albicans
Chromoblastomycosis Chronic fungal infection characterized by nodular, cutaneous lesions; contain dark brown sclerotic bodies when examined microscopically
Clavate Club shaped; examples are macroconidia of Microsporum nanum
Cleistothecium Closed, round structure in which asci and ascospores are held until their release; example is Pseudallescheria boydii
Columella Swollen tip of sporangiospore projecting into sporangium in some Mucorales; examples are Rhizopus and Mucor spp.
Conidiophore Specialized hypha or cell on which conidia are produced; examples are Aspergillus and Penicillium spp.
Conidium Asexual spore; examples are Aspergillus and Penicillium spp.
Dematiaceous Fungi that possess dark hyphae; examples are Sporothrix schenckii and Ochroconis (Dactylaria) gallopava
Dermatophtye Member of genus Microsporum, Trichophyton, or Epidermophtyon; infects hair, skin, or nails
Dichotomous Division of hyphae into two equal branches of same diameter as original hypha; examples are Aspergillus spp.
Dimorphic Fungi that exist in yeast and mycelial forms; examples are Blastomyces dermatitidis and Histoplasma capsulatum
Echinulate Spiny; examples are macroconidia of Microsporum spp.
Ectothrix Arthrospores located on outermost portion of hair shaft; examples are Microsporum spp.
Endothrix Arthrospores located within hair shaft; examples are Trichophyton spp.
Fungus Filamentous or unicellular, achlorophyllic organism with true nucleus enclosed in membrane and containing chitin in cell wall
Fusiform Spindle shaped; examples are macroconidia of Fusarium spp.
Geophilic Fungi that reside in soil; examples are Microsporum gypseum and Coccidioides immitis
Germ tube Tubelike outgrowth produced by germinating cells; develops into hypha; example is Candida albicans
Hyaline Transparent, clear, or colorless, usually in reference to hyphae; examples are Fusarium and Penicillium spp.
Hypha Individual, threadlike filament comprising vegetative fungal growth
Macroconidium Larger of two sizes of conidia, usually multicellular; examples are Microsporum gypseum and Microsporum nanum
Microconidium Smaller of two sizes of conidia, usually unicellular; examples are Trichosporon verrucosum and Trichosporon mentagrophytes
Mold Multicellular filamentous form of fungus; consists of masses of hyphae that grow together into a mycelium; examples are Aspergillus spp. and zygomycetes
Muriform Conidium with longitudinal and transverse walls; examples are Alternaria alternata and Curvularia spp.
Mycelium Mass of branching filaments (hyphae) that makes up vegetative growth of fungus
Mycetoma Fungal tumor of cutaneous and subcutaneous tissues with triad of clinical signs (swelling, draining sinuses, grains, or granules in exudates)
Mycosis Disease caused by a fungus
Phaeohyphomycosis Fungal infections whose tissue form is dark-walled hyphae; usually associated with dematiaceous fungi
Phialide Flasklike structure that produces conidia; examples are Aspergillus and Phialophora spp.
Propagule Unit that is able to give rise to another organism
Pseudohypha Chain of yeast cells; result of budding and elongation without detachment, forming hyphalike filament; examples are Candida albicans and Trichosporon spp.
Rhizoid Short, branching hypha that resembles a root; examples are Rhizopus spp.
Ringworm Superficial skin infection caused by dermatophytes; derived from ringlike lesions once thought to be caused by wormlike organisms
Sclerotic bodies Large, round, brownish, thick-walled cells with vertical and horizontal septa; found in tissue sections from animals affected with chromoblastomycosis
Septate Having cross-walls; examples are Aspergillus spp.
Sessile Direct hyphal attachment; no stalk involved.
Sexual state Part of life cycle during which organism reproduces by fusion of two nuclei; in mycology, also known as the “perfect state”
Spherule Large round structure containing spores; examples are Coccidioides immitis and Rhinosporidium seeberi
Sporangiophore Spore-bearing structure, usually closed, on which sporangium develops; examples are Rhizopus and Mucor spp.
Sporangiospore Asexual spore produced within a sporangiophore
Sporangium Closed, often spherical, structure that contains sporangiospores; examples are zygomycetes
Spore Propagule that develops asexually within a sporangium or by sexual reproduction (ascospore, basidiospore, or zygospore)
Sterigmata Specialized hypha that bears a conidium; examples are Aspergillus spp.
Teleomorph Sexual form of conidial fungus
Thallus Vegetative body of a fungus
Tuberculate Knoblike projections from a cell; examples are macroconidia of Histoplasma capsulatum
Vesicle Swollen or bladderlike cell produced at terminal end of condiophore; examples are Aspergillus spp.
Yeasts Unicellular, budding fungi; examples are Malassezia pachydermatis and Candida albicans
Zoophilic Fungi associated with animals
Zoospore Motile asexual spore
Zygospore Thick-walled sexual spore produced by zygomycetes

Fungal diseases may be grouped in several ways. Opportunistic fungi seldom cause disease without some underlying predisposing factor, such as trauma or immunosuppression. Pathogenic fungi cause disease without a predisposing factor, and may be grouped into the superficial or cutaneous, subcutaneous, and systemic mycoses. Superficial and cutaneous mycoses are associated with hair, nails, and keratinized layers of the skin. Subcutaneous mycoses affect mainly dermis, bone, muscle, and fascia. Systemic mycoses represent a group of fungi that invade internal organs following hematogenous dissemination from the lungs. Each group will be covered in detail in subsequent chapters, as will fungal-like agents such as Rhinosporidium seeberi, Pythium insidiosum, Prototheca, and Pneumocystis carinii.


Mycoses should be included in the differential diagnosis of any chronic condition that has failed to respond to treatment or for which the etiology is unknown. Accurate diagnosis is facilitated by understanding and applying criteria by which one can distinguish among contamination, colonization, and infection. Animals are typically covered with hair or feathers that provide an ideal environment for fungal colonization that can take place in the absence of frank disease. In the initial assessment of a suspected case of fungal infection, the practitioner should determine if the symptoms are compatible, if fungal elements are observed in host tissue or sterile body fluids, if fungi are recovered from the specimens, and if fungi detected microscopically in tissue sections or other clinical specimens are compatible with those detected by culture.



PRACTICAL PROCEDURES FOR DIAGNOSIS OF FUNGAL DISEASES


Given a basic knowledge of mycology, many fungal infections can be presumptively diagnosed in the veterinarian’s office. This requires recognition of basic fungal elements (hyphae, yeasts, or spherules) and minimal equipment and reagents; the practitioner will need a microscope with oil immersion lens, 10% potassium hydroxide (KOH) in glycerol, india ink, a hematologic stain, such as Diff-Quik, glass slides and cover slips, and a book containing an atlas of medical mycology.


Obtaining the proper specimen for diagnosis is of paramount importance. General guidelines are much the same as those for bacterial specimen collection and include aseptic collection techniques, collection from sites representative of the disease process, collection before antifungal therapy, and collection of an adequate volume of material. Procedures are detailed in Table 44-3.


TABLE 44-3 Collection of Clinical Specimens for Diagnosis of Fungal Disease















































Specimen Container Comments
Hairs Paper envelope (dry conditions inhibit overgrowth of bacterial or saprophytic fungal contaminants) Wash and dry affected area with soap and water. With forceps, epilate hairs from the periphery of an active lesion. Pull hairs in the direction of growth to include the root. Look for broken, stubby hairs, which are often infected. Useful for diagnosis of dermatophytosis.
Skin Paper envelope Clean skin with alcohol gauze sponge (cotton leaves too many fibers). Scrape the periphery of an active lesion with a sterile scalpel blade. Also, obtain crusts and scabs. Useful for diagnosis of dermatophytosis.
Nails Paper envelope Proven nail infections in animals are rare. Cleanse affected nail with alcohol gauze. Scrape with a scalpel blade so that fine pieces are collected. Also, collect debris under the nail. Useful for diagnosis of onchyomycosis.
Biopsy Sterile tube in sterile saline or water Normal and affected tissue should be included. Important to prevent specimen desiccation.
Urine Sterile tube Centrifuge and use sediment for direct examination and culture. Useful for diagnosis of histoplasmosis.
Cerebrospinal fluid Sterile tube Useful for diagnosis of cryptococcosis. Make india ink preparation to observe encapsulated yeasts.
Pleural/abdominal fluid Sterile tube If fluid contains flakes or granules, they should be included because these are actual colonies of organisms.
Transtracheal/bronchial washings Sterile tube Centrifuge and use sediment for direct examination and culture. Useful for diagnosis of systemic mycoses.
Nasal flush Sterile tube Centrifuge and use sediment for direct examination and culture. Useful for diagnosis of nasal aspergillosis and guttural pouch mycosis.
Ocular fluid Sterile tube or syringe Examine directly. Inoculate onto plated fungal media immediately after collection. Useful for diagnosis of ocular blastomycosis.

Microscopic examination of clinical materials and isolates obtained by fungal culture is used extensively in fungal identification. Most specimens suspected to contain fungi are initially examined in wet mounts to prevent morphologic distortion of fungal morphology. Wet mounts in KOH are incubated for 30 minutes at room temperature; KOH acts as a clearing agent, digesting the proteinaceous components of host cells and leaving the polysaccharide-containing fungal cell wall intact and more apparent. After digestion, mounts are examined at 100× and 400× magnification for fungal elements. The microscopic appearance of fungi and fungal-like agents in direct examination of clinical material is given in Table 44-4.


TABLE 44-4 Microscopic Appearance of Fungi and Fungal-Like Agents in Clinical Specimens































































Disease Microscopic Examination Method Appearance
Aspergillosis Wet mount (lactophenol cotton blue or KOH) Septate hyphae with dichotomous branching; may see fruiting heads
Blastomycosis Wet mount Thick, double-walled budding yeasts with broad bases of attachment to mother cells
Candidiasis and other yeast infections Wet mount Budding and nonbudding yeasts; pseudohyphae may be present
Coccidioidomycosis Wet mount Spherules with and without endospores
Cryptococcosis India ink Encapsulated yeasts
Dermatophytosis Wet mount Hairs with arthrospores (endothrix or ectothrix), hyphae or sheath of spores around skin and nails
Histoplasmosis Hematologic stain Small yeasts with narrow necks of attachment to mother cells, often within macrophages
Mycetoma Wet mount Dark brown chlamydospores and hyaline hyphae in crushed granules
Phaeohyphomycosis Wet mount Dark hyphae
Pneumocystosis Hematologic stain Cysts and trophozoites
Protothecosis Wet mount Spherical to oval nonbudding, small and large cells containing two or more autospores
Rhinosporidiosis Wet mount Spherules (some large) with and without endospores
Sporotrichosis Hematologic stain Small oval to round to cigar-shaped yeasts
Zygomycosis Wet mount Broad, relatively nonseptate hyphae

KOH, Potassium hydroxide.


Fluorochrome stains, such as calcofluor white, bind to cellulose and chitin in the fungal cell wall and may facilitate detection of fungi. On wet preparations of specimens or on smeared and dried clinical materials, these nondifferential stains are examined with ultraviolet (UV) illumination. Fungi and Pneumocystis cysts appear bright blue or green, depending on the type of UV filter used. Hematologic stains can be applied to exudates, and allow examination of fungi without distortion. They are especially useful for demonstration of yeasts. Gram stains are generally not used because they distort fungal morphology.


Fungal specimens should be inoculated onto media that ensure adequate fungal growth and preclude the growth of bacterial contaminants. Traditional media for primary isolation include Sabouraud dextrose and potato dextrose agars. These have a high sugar content and acidic pH that inhibit most bacteria but are supportive for fungi. Media may be made more selective by addition of antibiotics such as chloramphenicol, gentamicin, penicillin, or streptomycin to further inhibit bacterial growth. Cycloheximide, an antifungal agent, may be added to potato dextrose or Sabouraud dextrose agar to prevent overgrowth of saprophytic fungi. Mycosel (or Mycobiotic) are two commercial formulations containing cycloheximide. Because this agent can be inhibitory to some pathogenic fungi (e.g., most zygomycetes, Cryptococcus neoformans, and Aspergillus fumigatus), it is advisable to use media with and without cycloheximide for fungal specimen processing. Plates are routinely incubated at 25° to 30° C for up to 6 weeks. If dimorphic fungi are suspected, enriched media such as brain-heart infusion agar with 5% blood are incorporated. Blood enrichment and 37° C incubation temperatures promote the growth of the yeast phase. For isolation of the ringworm fungi, a partially differential and selective medium is used. Dermatophyte test medium contains antibiotics, cycloheximide, and an indicator to demonstrate a rise in pH, consistent with the growth of dermatophytes. Processing schemes for clinical materials appear in Figures 44-4 and 44-5.


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Jul 18, 2016 | Posted by in PHARMACOLOGY, TOXICOLOGY & THERAPEUTICS | Comments Off on Introduction to Veterinary Mycology
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