Introduction to the pathogenic fungi

General characteristics of the fungi

Fungi have a eucaryotic cell type and are therefore insensitive to most bacterial antibiotics. They have nuclei with well-defined nuclear membranes, mitochondria and networks of microtubules. They are non-photosynthetic, usually non-motile and possess a cell wall external to the plasma membrane. Although their optimum pH is about 6, they can tolerate more acidic conditions. They are strict aerobes with an optimum temperature for growth of 20–30°C, but the pathogenic fungi causing systemic mycoses can tolerate 37°C. The fungi are comparatively slow-growing on laboratory media, the zygomycetes and Aspergillus species may show growth in two to three days but the incubation time for some of the dermatophytes may be as long as three to five weeks. The fungi can be divided into moulds and yeasts. Moulds are filamentous, growing apically and forming lateral branches. The branching filaments or hyphae are 2–10 µm in diameter. The hyphae in most moulds have cross-walls or septa but the zygomycetes rarely produce septa and are termed non-septate. The branching hyphae grow to form a tangled interlacing mass known as the mycelium. Moulds usually form large fluffy colonies on laboratory media and produce aerial fruiting hyphae that bear asexual spores. Yeasts are oval, spherical or elongated cells, about 3–5 µm in diameter, and form moist colonies that are usually larger than, but not unlike bacterial colonies. The yeasts reproduce by budding or by both budding and spore formation. The terms ‘mould’ and ‘yeast’ have no taxonomic significance and are not mutually exclusive. Some of the fungal pathogens are dimorphic, being yeasts or yeast-like in animal tissues and when grown on enriched media at 37°C but are moulds in their natural environment and when grown on media at 25°C. Yeasts such as Candida albicans can grow in animal tissue as elongated cells, joined together, that resemble septate hyphae and are known as pseudohyphae.

Classification of the fungi

The kingdom Fungi comprises more than 250,000 species of which perhaps a few hundred are of pathogenic importance for animals and humans. The kingdom is divided into six phyla: Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota, Microsporidiomycota and Glomeromycota. Members of Chytridiomycota and Glomeromycota are not known to be of veterinary or medical importance. However, chytrids cause infections of the epidermis of frogs, interfering with their ability to respire across the skin. These infections have been implicated in the dramatic worldwide die-off of amphibians in recent times.

The members of the different phyla are primarily characterized by different methods of sexual reproduction. Traditionally fungi with no known sexual stage have been formally grouped in the phylum Deuteromycota, also referred to as the Fungi Imperfecti. Increasingly, molecular methods are being used to place fungal species, for which only the asexual form is known, in one of the recognized phyla without the need to discover a sexual form. The preferred term for fungi that are not known to have a meiotic stage is mitosporic fungi. For many years a dual system of names has been used, one for the asexual form (the anamorph) and one for the sexual form (the teleomorph). For example, the teleomorph of the yeast Cryptococcus neoformans is Filobasidiella neoformans, one of the few pathogenic fungi in the basidiomycetes. Most of the pathogenic fungi, previously in the Fungi Imperfecti, have been found to be ascomycetes when the sexual state is found. Several pathogenic fungi are better known by their anamorph name because it is often the asexual form that is found in pathological situations. However, the usefulness of this dual naming system is increasingly being questioned and it is expected that eventually a single name, that of the teleomorph, will only be recognized and used. A major re-organization of fungal classification based on molecular phylogenetic analyses has been proposed (Hibbett et al. 2007). Two new phyla have been added, Blastocladiomycota and Neocallimastigomycota, while the subkingdom Dikarya containing Ascomycota and Basidiomycota has been created. The status of the phylum Zygomycota is currently in doubt and may be broken up into a number of new phyla in the future. These proposals have been adopted in the Dictionary of the Fungi (Kirk et al. 2008) and Index Fungorum (www.indexfungorum.org accessed 31 December 2012).

A small number of fungal-like agents such as Pythium insidiosum and Rhinosporidium seeberi are traditionally included in texts on fungal diseases because they produce elements in tissue that resemble fungal elements and may form yeast-like colonies on fungal media. Pythium insidiosum is a member of the kingdom Chromista (also known as Stramenopila or Heterokonta), while Rhinosporidium seeberi belongs to a novel group of aquatic protistan parasites at the boundary between fungi and animals, in the class Mesomycetozoea, kingdom Protozoa (Mendoza et al. 2002). Pneumocystis carinii, an occasional cause of pneumonia in immunosuppressed dogs and horses (P. jiroveci is associated with terminal pneumonia in severely immunocompromised people), was considered a protozoa but has in recent years been shown to belong to the kingdom Fungi, phylum Ascomycota.

General features of fungal infections

A few of the dermatophytes are considered to be obligate parasites but the majority of the pathogenic fungi are found widespread in the environment as saprophytes or present as commensals associated with animals and humans. Most are therefore opportunistic pathogens and predisposing factors often contribute to the establishment of fungal infections. These include an upset in the normal flora of the host by prolonged administration of antibiotics, immunosuppression, concurrent infections, breaks in the skin or mucous membranes, perpetually moist areas of skin or the exposure to a large infective dose, such as with Aspergillus fumigatus spores in brooder pneumonia of chicks. Fungal diseases do not usually assume epidemic proportions except in certain instances such as ringworm. No exotoxins or endotoxins have conclusively been shown to be produced, but mycotoxicoses occur due to animals ingesting preformed toxic metabolic products, termed mycotoxins, produced during fungal growth in animal feedstuffs.

Chronic fungal infections lead to a granulomatous reaction that resembles the host’s response to a foreign body or to an actinomycete infection. Immunity to fungal infections is considered to be more dependent on cell-mediated than antibody-mediated activity. Antibodies are produced in most mycoses, but the antibodies do not appear to be protective. Hypersensitivity can develop to a particular fungus in the infected or exposed host and may lead to skin rashes in humans. Hypersensitivity responses form the basis of various diagnostic skin tests such as the use of histoplasmin for the diagnosis of histoplasmosis. The mycoses are sometimes divided into deep (systemic), subcutaneous and superficial mycoses. Several pathogenic fungi such as Candida albicans and Aspergillus fumigatus are capable of both superficial and deep infections. Systemic mycoses are uncommon and associated with opportunistic infections by saprophytic fungi which have been facilitated by predisposing factors such as host debilitation, immunosuppression, exposure to huge numbers of spores or disruption of normal flora due to prolonged antibiotic therapy. Subcutaneous mycoses are characterized by localized involvement of the dermis and subcutis by a range of fungal species. The term phaeohyphomycosis refers to infection with dematiaceous (pigmented) fungi while the term mycetoma (sometimes referred to as eumycetoma to distinguish it from mycetomas associated with actinomycete infections) indicates a tumour-like granulomatous lesion involving cutaneous and subcutaneous tissues. Superficial mycoses may be grouped into dermatomycoses and dermatophytoses. The former conditions refer to opportunistic infections of the skin or mucocutaneous junctions by fungi such as Candida species or Malassezia pachydermatis. Dermatophytes such as Trichophyton species and Microsporum species invade and destroy keratinized structures and are notable for being both easily transmitted and zoonotic.

General methods for the diagnosis of the mycoses

History, clinical signs, gross pathology, and in a few cases intradermal skin tests, are all of value in the diagnosis of fungal infections backed by a laboratory investigation of clinical specimens. These investigations include direct microscopy, isolation and identification of the pathogen. The identification is based on colonial characteristics, examination of the fruiting heads and spores and on the results of biochemical reactions in the case of the yeasts and certain of the dermatophytes.

Direct Microscopic Examination of Clinical Specimens

Table 37.1 indicates methods used for the examination of fungal elements in clinical specimens and Table 37.2 gives a brief summary of the diagnostic features of some of the fungi found in veterinary diagnostic samples. The specimens suspected of containing fungal pathogens range from hairs and skin scrapings for dermatophytes to exudates, biopsies and tissues. Because many of the potentially pathogenic fungi are ubiquitous it is important to take tissue for histopathology whenever possible so as to demonstrate fungal hyphae or yeast cells actually invading the tissue, often invoking a tissue reaction. If there is a correlation between the fungus that is isolated and the histopathological findings there can be greater confidence in the diagnosis of the disease or condition.

Table 37.1

Summary of the methods employed for the direct microscopic examination of fungi

Technique	Use	Fungi
10–20% KOH wet preparations	Clears specimens to make fungi more visible. Examine under low and high-dry objectives or phase contrast	Fungal elements of most moulds and yeasts. Dimorphic fungi as yeast-like forms in tissue. Arthrospores on affected hairs for dermatophytes
Calcofluor white (0.1%)	Fluorescence of fungal elements under fluorescence microscope. Visualization of fungi made easier	Detection of most fungal elements in wet preparations and in tissue sections
India ink or nigrosin	Wet preparation with cerebrospinal fluid or clear exudates	Cryptococcus neoformans, to demonstrate the characteristically large capsule
Gram or methylene blue stain	Fixed smears of tissues or exudates	Yeast cells such as Candida albicans as well as any bacteria that are present. Cryptococcus neoformans stains poorly by these methods
Fluorescent antibody technique	Frozen sections or fixed smears	Available in specialized laboratories for some of the dimorphic fungi such as Blastomyces dermatitidis
Periodic acid-Schiff (PAS) + counter-stain (haematoxylin)	Frozen or paraffin-embedded histological sections from biopsies or tissues	Most fungal elements can be demonstrated in tissues by this method. The fungi stain pink. Any tissue reaction caused by the fungal invasion can also be observed
Methenamine silver stain + counter-stain	Frozen or paraffin-embedded histological sections from biopsies or tissues	Most fungal elements in tissues will be stained a dark brown by this method and are easy to see. Visualization of any internal structures may be harder than with the PAS-haematoxylin stain
Wright or Giemsa stain	Fixed bone marrow smears or impression smears from biopsies	Demonstration is limited to Histoplasma capsulatum

Table 37.2

Morphological features of pathogenic fungi in diagnostic specimens

Fungus	Techniques	Summary of diagnostic features
Aspergillus fumigatus	KOH, calcofluor white, periodic acid-Schiff (PAS) or silver impregnation stains	Septate hyphae, dichotomous branching at a 45° angle. Hyphae 3–6 µm and rarely up to 12 µm in diameter. Tissue reaction is granulomatous or necrotizing, but may not occur in an immunosuppressed host. May see distorted fruiting heads if fungus spreads into an air space in the body
Zygomycetes: Rhizopus, Mucor, Rhizomucor, Absidia and Mortierella spp.	KOH, calcofluor white, PAS or silver impregnation stains	Large, bulging, non-septate hyphae that can be twisted and fragmented. About 10–20 µm in diameter (range 3–25 µm) with irregular branching. The invading hyphae of Mortierella wolfii tend to be finer (2–12 µm diameter) than the other zygomycetes
Candida albicans	Gram stain, KOH, PAS or silver impregnation stains	Budding cells, oval or round, 3–4 µm diameter. Pseudohyphae may be present in tissue; these have regular points of constriction between individual elongated yeast cells. They must be distinguished from moulds with septate hyphae
Malassezia pachydermatis	Gram stain, methylene blue, KOH or calcofluor white	Bottle-shaped, small yeast (1–2 × 2–4 µm). Unipolar budding and reproduction is by bud-fission in which the bud detaches from the mother cell by a septum
Cryptococcus neoformans	India ink, KOH, PAS or Mayer’s mucicarmine stain	Spherical budding yeast cells, 2–15 µm diameter, usually surrounded by a large capsule. Produces pinched-off buds, sometimes multiple. Cells vary greatly in size in a single preparation. Encapsulated pseudohyphae are very occasionally seen
Blastomyces dermatitidis	KOH, calcofluor white, FA technique, PAS or silver-impregnation stains	Large, budding yeast 8–15 µm (range 2–30 µm) in diameter with very thick walls. Buds are connected by a broad base. Intracytoplasmic contents are usually evident
Histoplasma capsulatum	Wright, Giemsa, PAS or silver impregnation stains	Small, budding yeast, spherical to oval, 2–5 µm, intracellular in monocytic cells. A clear halo can be seen around the darker staining cell. Buds are single with narrow bases. The fungus is difficult to detect in unstained preparations
Coccidioides immitis	PAS and silver-impregnation stains, KOH + calcofluor white	Large spherules present in tissue. When mature, up to 200 µm in diameter and contain numerous non-budding endospores (2–5 µm). Immature spherules vary in size and do not contain endospores
Sporothrix schenckii	Gram stain or KOH on exudates. PAS or silver-impregnation stains on biopsies	Small, cigar-shaped yeasts, 2–6 µm. May exhibit multiple budding. Only a small number are usually present in exudates and they may be hard to see
Dermatophytes: Microsporum and Trichophyton spp.	KOH, KOH + calcofluor white, DMSO + KOH, blue-black ink + KOH	Septate hyphae (2–3 µm diameter) surround affected hairs and fragment into arthrospores. Some hyphae may still be present but more usually a sheath of refractile round arthrospores (2–8 µm diameter) is present. These arthropores must not be confused with fat globules or hair-pigment granules (melanosomes)
Fungi in mycetomas	KOH, calcofluor white, PAS and silver-impregnation stains	Irregular granules, 0.5–3.0 mm and variously coloured, are present in biopsies or scrapings. Within crushed granules are intertwined hyphae (2–5 µm) with swollen cells (15 µm or more) at the periphery
Fungi in chromoblastomycoses	KOH, calcofluor white, PAS and silver-impregnation stains	Single-celled or clustered, spherical (4–12 µm), thick-walled bodies and darkly pigmented (sclerotic) bodies. Hyphae may be present (2–6 µm) and are seen in skin scrapings and aspirates
Pneumocystis carinii	Giemsa stain, immunocytochemistry and methenamine silver stain	Trophic, cystic and spore forms may be found in lung tissue and bronchoalveolar lavage fluid of affected animals