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.

FIGURE 44-1 Yeast and mold forms of fungi.

(Courtesy Ashley E. Harmon.)

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.

FIGURE 44-2 Sporangiospores and conidia, the two main types of asexual spores.

(Courtesy Ashley E. Harmon.)

FIGURE 44-3 Sexual spores (ascospores, basidiospores, and zygospores) are produced through fusion of the protoplasm and nuclei of two cells by meiosis.

(Courtesy Ashley E. Harmon.)

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

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

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.

FIGURE 44-4 Processing scheme for clinical specimens: cutaneous and subcutaneous mycoses.

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