Mycology: Introduction

28 Mycology: Introduction G. R. Carter

Mycology has been a neglected field in many veterinary diagnostic laboratories. A number of important pathogenic fungi can be identified without difficulty. Those that cannot should be forwarded to a reference mycology laboratory. A duplicate culture of the strain submitted may be kept for study after an identification has been made. By this means, a collection of the more common pathogenic and nonpathogenic fungi can be acquired. The outline that follows is provided as an aid and is not meant to be a substitute for standard texts. A number of useful texts are listed as supplementary readings. The older ones, although useful, do not include the more recent changes in the names of some fungi. It should be remembered that a number of fungi producing disease in animals are transmissible to humans. Special care should be taken to prevent laboratory infections.

Significance of Fungous Isolations The great majority of fungi live in the soil or water, or as harmless commensals associated with humans and animals; for example, Histoplasma capsulatum and Coccidioides immitis occur in soil (geophilic), while Candida albicans lives in the alimentary canal. Many of the fungi, such as Aspergillus spp., Rhizopus spp., and Geotrichum are widespread in nature and thus are frequently found in clinical materials. The significance of the isolation will depend on such considerations as (1) demonstration of fungi in tissue sections, (2) presence of clinical disease, (3) presence of pathologic lesions, and (4) repeated isolation of the same fungus. The widespread saprophytic fungi that Diagnostic Procedures in Veterinary Bacteriology and Mycology, Fifth Edition Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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occasionally cause disease, such as Mucor spp., Aspergillus spp., or Candida albicans, are frequently referred to as "opportunistic fungi." Production of disease by these fungi is thought to be related to such factors as "impaired resistance," prolonged steroid or antibiotic therapy, and various stresses, including terminal diseases and metabolic disturbances.

Mycological Examinations Procedures for a Mycological Examination 1. Direct examination of the clinical material: A small amount of material is added to a drop of 10% potassium hydroxide or lactophenol, and a coverslip is applied. The slide is gently heated to remove air bubbles and promote clarification. Some laboratories prefer 20% potassium hydroxide with glycerol (see Appendix C). Gram (special procedure for clinical material), Giemsa, and Wright's stains are also frequently of value. Direct examinations are almost always negative in histoplasmosis and sporotrichosis. Tissue should be set aside in buffered formalin for histopathologic study, if this was not already done. 2. If there is evidence of a fungous infection, appropriate media are inoculated. 3. Examination of the growth both macroscopically and microscopically should be conducted. 4. Those cultures that cannot be identified with certainty are forwarded to a mycology laboratory.

Material Required for Mycological Procedures Almost all of the equipment required can be found in the diagnostic bacteriology laboratory. Straight dissecting needles are useful for breaking up colonies of fungi for examination. The inocula used to seed media are generally larger than those used in bacteriological work. Forceps and scalpels with small, sharp blades are especially useful. Bacterial contamination can be reduced by aseptic operations. The reagents, stains, culture media, and other items required especially for mycological work are described in Appendix C.

Culture Media The media recommended for isolation of the pathogenic fungi along with the incubation temperatures and duration of incubation are summarized in Table 28-1.

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Table 28-1 Media for Isolation/ Incubation Temperature/ and Usual Length of Incubation for the Pathogenic Fungi Isolation media Disease

37°C

25°C

Zygomycosis (Mucormycosis)

Sabouraud dextrose agar; chloramphenicol (not cycloheximide) can be used (1-3 days)

Aspergillosis

Same as for zygomycosis (1-3 days)

Candidiasis

Sabouraud dextrose agar, Sabouraud C and C agar (1-3 days)

0

Dermatophytosis (Ringworm)

Sabouraud C and C agar (2-3 weeks)

Blastomycosis

Sabouraud C and C agar (2-3 weeks)

Cryptococcosis

Sabouraud dextrose agar; chloramphenicol (not cycloheximide) can be used (1-2 weeks)

Histoplasmosis

Sabouraud C and C agar (2-4 weeks)

Coccidioidomycosis

Sabouraud C and C agar (1-2 weeks)

Sporotrichosis

Sabouraud C and C agar (7-10 days)

Brain—heart infusion agar (1% blood) or blood agar (7-10 days)

Epizootic lymphangitis

Sabouraud C and C agar (2-8 weeks)

Horse blood or serum agar (2-8 weeks)

Geotrichosis

Sabouraud C and C agar (1-2 weeks)

Chromomycosis

Sabouraud C and C agar (2-3 weeks)

Maduromycosis

Sabouraud dextrose agar (2-3 weeks)

Brain-heart infusion agar or blood agar (3-7 days)

Brain-heart infusion agar or blood agar (2-4 weeks)

hi some instances, depending on source of the specimen and history, it will be advisable to inoculate both kinds of Sabouraud media as well as blood agar. Usual incubation period. Cycloheximide and chloramphenicol added (Mycosel, Mycobiotic, etc.). Some Trichophyton spp. require special media (see Chapter 29).

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b c

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Blood agar, Sabouraud dextrose agar, and Sabouraud C and C agar (Mycosel, Mycobiotic, etc.) are used routinely for primary fungous cultivation. The last-mentioned medium is basically the same as the second except that it contains cycloheximide and chloramphenicol for the suppression of some saprophytic fungi and bacteria, respectively. Brain-heart infusion agar can usually be used instead of blood agar. Media are most useful in Petri dishes; the medium should be thicker than usual, 25-35 ml per plate. It should be kept in mind that media containing cycloheximide should be incubated at room temperature (25-28°C) only. If one is uncertain as to the probable fungus involved, it is advisable to inoculate each specimen onto both kinds of Sabouraud media, and onto blood and brain-heart infusion agar with 1% blood. The latter two media are incubated at 37°C. The media for isolation, the incubator temperature, and the usual time required for growth are listed in Table 28-1. It is a good practice to incubate all plates for fungous isolation for at least 4 weeks.

Examination of Cultures After gross characteristics of the cultures are observed, a portion of the colony is teased apart with needles, then transferred to a drop of lactophenol cotton blue. A coverslip is then added. An alterative useful procedure is to prepare a tape mount. This procedure is described in detail in Chapter 29. For verification of the morphologic observations, a slide culture may be prepared. The technique is described by Campbell and associates (1) and in Appendix C. Difficulty may be experienced in obtaining both growth phases of the dimorphic fungi. Methods are described (1) for the conversion from one phase to another. Many of the saprophytic fungi can be identified by their characteristic fruiting bodies. Figure 28-1 has been provided to aid in the identification of some of the more common contaminants. Unfortunately, fruiting bodies are not always present.

Classification of Fungi The classification that follows is based upon that of Copper (2), which was patterned after Alexopoulos and Mims (3). Fungi are placed in one of the subdivisions listed below (excepting subdivision Deuteromycotina) on the basis of their sexual spores. The sexual state of a fungus is called the teleomorph. The sexual state of

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fungi in the subclass Deuteromycetes has not yet been discovered. The term anamorph is used to denote the asexual reproductive state. Kingdom Mycetae (Fungi) Division Amastigomycota Subdivision Zygomycotina Class Zygomycetes Order Mucorales Representative genera: Absidia, Mucor, Rhizopus, Cunningham ell a Order Entomophthorales Representative genera: Basidiobolus, Conidiobolus Subdivision Ascomycotina Class Ascomycetes Subclass Hemiascomycetidae Representative genera: Pichia, Saccharomyces Rarely cause disease. Some Candida spp. are related to this subclass. Subclass Plectomycetidae Order Onygenales Family Gymnoascaceae Representative genera: Nannizzia, teleomorph of Microsporum; Arthroderma, teleomorph of Trichophyton; Emmonsiella, teleomorph of Histoplasma; Ajellomyces, teleomorph of Blastomyces Order Eurotiales Family Eurotiaceae Includes teleomorphs of some Penicillium and Aspergillus spp. Subdivision Basidiomycotina Class Basidiomycetes Subclass Holobasidiomycetidae Order Agaricales Includes poisonous and edible mushrooms Subclass Teliomycetidae Order Ustilagenales Family Ustilagenaceae Includes Filobasidiella, the teleomorph of Cryptococcus Subdivision Deuteromycotina Form class Deuteromycetes (Imperfect fungi; sexual state not discovered) Form subclass Blastomycetidae: Imperfect yeasts Representative genera: Cryptococcus, Candida Form subclass Hyphomycetidae

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Form family Moniliaceae Representative genera: Coccidioides, Epidermophyton, Sporothrix, Paracoccidioides Form family Dermatiaceae Representative genera: Phialophora, Exophiala, Fonsecaea, Cladosporium, Wangiella. These are molds with darkly pigmented hyphae.

Glossary of Mycological Terms Arthrospore: An asexual spore formed by the disarticulation of the mycelium. Ascospore: A sexual spore characteristic of the true yeasts and ascomycetes. They are produced in a saclike structure called an ascus. The ascospore results from the fusion of two nuclei. Ascus: The specialized saclike structure characteristic of the true yeasts in which ascopores (usually eight) are produced. Blastospore: A spore produced as a result of a budding process along the mycelium or from a single spore. Chlamydospores: Thick-walled, resistant spores formed by the direct differentiation of hyphae. Clavate: Club-shaped. Columella: The persisting, dome-shaped upper portion of the sporangiophore. Conidium: An asexual spore formed from hyphae by abstriction, budding, or septal division. Conidiophore: A stalklike branch of the mycelium on which conidia develop either singly or in numbers. Dematiaceous: Used to denote the dark brown or black fungi. Dimorphic: Having a yeast form and mycelial form. Echinulate: This refers to the spiny walls of conidia and conidiophores. Endogenous: Originating or produced from within. Endothrix: Arthrospores appear within the hair shaft. Exogenous: Originating from without. Favic Chandeliers: These are spherical hyphae that branch with curved and irregular ends, which give an appearance of antlerlike branches. Geophilic: Denotes fungi whose natural habitat is the soil. Germ Tube: Tubelike structure produced by germinating spores. They develop into hyphae. Glabrous: The smooth form. Hyphae: The filaments that compose the body of the thallus of a fungus. Macroaleuriospore: This is the larger of the two kinds of conidia that break from the attachment to hyphae by rupture through the cell wall. It is also called a macroconidium. Macroconidium: A large, sometimes multicellular spore. It is the larger of two types of conidia produced in the same manner by the same fungus. Microaleuriospore: This is the smaller of the two kinds of conidia that break from the attachment to hyphae by rupture through the cell wall. It is also called a microconidium. Microconidium: A small, single-celled conidium borne laterally on hyphae. Mycelium: The mat made up of the intertwining, threadlike hyphae.

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Nodes: The points on the stolons from which the rhizoids arise. Obovate: Inversely ovate, that is, ovate with the narrow end at the base. Obovoid: Inversely ovoid, that is, ovoid with the narrow end at the base. Ovate: Egg-shaped. Phialide: This is a tip cell of a conidiophore (phialophore), which is usually flask-shaped, and from which conidia (phialospores] arise. Phialophore: A conidiophore which bears phialides. Pseudohyphae: Filaments constituted by elongated budding cells that have failed to detach. Pyriform: Pear-shaped. Racquet Hyphae: Hyphae with terminal swelling of segments giving a shape resembling that of a tennis racquet. Rhizoid: Rootlike, branched hyphae extending into the medium. Septate: Having cross walls or septa in the hyphae. Sporangium: The closed, often spherical structure in which are produced asexual spores by cleavage. Sterigmata: Specialized structures, short or elongated, borne on a vesicle and producing conidia. Stolon: A horizontal hypha or runner that sprouts where it touches the substrate. It forms rhizoids in the substrate. Yeasts: Unicellular fungi that reproduce by asexual budding or by sexually produced ascospores. Zygospore: A thick-walled, sexual spore of the true fungi that results from the fusion of two similar gametangia.

Serodiagnosis of Fungal Diseases This area of laboratory diagnosis has received little attention in fungal diseases of animals. This is partly because these diseases are relatively infrequent and the required reagents have not usually been readily available to the veterinary microbiologist. The expense of the reagents has also been a deterrent. Some hospital and public health laboratories will make their serodiagnostic capability available to veterinarians, and some fungal diagnostic reagents are available commercially. Readers are referred to Kaufman (4) for details of procedures and some sources of reagents. The results of these human tests when used in animals may require different interpretations. Attleberger (5) has discussed the use of serologic procedures in the diagnosis of fungal diseases of dogs and cats.

Safety All fungous cultures and specimens thought to harbor fungi should be considered potentially dangerous. Work with these materials should be carried out in an approved biological safety hood (see Chapter 1).

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References 1. Campbell, Β. Α., Stewart, J. L., and Larsh, H. W.: How it is done: Clinical laboratory methods. The Medical Mycology Handbook. New York, Wiley, 1980. 2. Cooper, Β. H. (Sect. Ed.): Section VI. Fungi. In Lennette, Ε. H. (Ed.-in-chief): Manual of Clinical Microbiology, 4th ed. Washington, D.C., American Society for Microbiology, 1985. 3. Alexopoulos, C. J., and Mims, C. W.: Introductory Mycology, 3rd ed. New York, Wi­ ley, 1979. 4. Kaufman, L., and Reiss, E.: Serodiagnosis of fungal diseases. In Lennette, Ε. H. (Ed.in-chief): Manual of Clinical Microbiology, 4th ed. Washington, D.C., American Soci­ ety for Microbiology, 1985. 5. Attleberger, Μ. H.: Laboratory diagnosis of fungal and achloric algal infections. In Greene, C. E. (Ed.): Clinical Microbiology and Infectious Diseases of the Dog and Cat. Philadelphia, Saunders, 1984.

Supplementary Readings Ainsworth, G. C , and Austwick, P. K. C : Fungal Diseases of Animals, 2nd ed. Farnham Royal, Slough, England, Commonwealth Agricultural Bureaux, 1973. Beneke, E. S., and Rogers, A. L.: Medical Mycology Manual, 4th ed. Minneapolis, Bur­ gess, 1980. Conant, N. F., Smith, D. T., Baker, R. D., and Calloway, J. L.: Manual of Clinical Micro­ biology, 3rd ed. Philadelphia, Saunders, 1971. Haley, L. D., Trandel, J., and Coyle, Μ. B.: Practical Methods for Culture and Identifica­ tion of Fungi in the Clinical Microbiology Laboratory, Cumitech 11. Washington, D.C., American Society for Microbiology, 1980. Jungerman, P. F., and Schwartzman, R. M.: Veterinary Mycology. Philadelphia, Lea & Febiger, 1972. Rippon, J. W.: Medical Mycology, 3rd ed. Philadelphia, Saunders, 1988.