The pathogenic yeasts

The pathogenic yeasts

Yeasts are common in the environment, often found on plants or associated with plant materials. They are also found as commensals of the skin and mucous membranes of animals. Some species cause opportunistic infections, facilitated by factors such as immunosuppression or antimicrobial therapy which upsets the resident flora on mucosal surfaces. Asexual reproduction is characterized by the production of blastoconidia, also known as buds or daughter cells. A number of species are known to produce a range of forms while causing infection. Candida albicans tends to a filamentous growth pattern when grown on media with low concentrations of glucose in an atmosphere of elevated CO₂ concentration. Lesions caused by C. albicans may contain budding yeasts, pseudohyphae and hyphae.

Candida albicans

There are more than 200 species of Candida but only C. albicans is commonly associated with disease in animals. Candida albicans grows as a budding yeast cell, oval and 3.5–6.0 × 6.0–10.0 µm in size, on agar cultures and in animal tissues. Pseudohyphae are also produced in animal tissue by elongation of individual yeast cells that fail to separate. These can be mistaken for septate hyphae of moulds. Thick-walled resting cells, known as chlamydospores, are produced in vitro on certain media such as cornmeal Tween 80 agar. Candida albicans will grow on ordinary media over a wide range of pH and temperatures. At both 25°C and 37°C it produces white, shiny, high-convex colonies in 24–48 hours.

Natural Habitat

Candida albicans is a commensal of mucocutaneous areas, particularly of the intestinal and genital tracts of many animal species and humans. Most infections are endogenous in origin and predisposing causes, such as immunosuppression, prolonged antibiotic therapy, intercurrent infection and malnutrition, can initiate infections. In cattle the yeast is often introduced into the udder on the nozzle of tubes of intramammary antibiotics. Candida albicans is worldwide in distribution.

Pathogenesis

Neuraminidase and proteases may play a part in virulence and cell wall glycoproteins have an endotoxin-like activity. Candidal surface proteins facilitate adhesion to matrix proteins. The production of extracellular, cytotoxic phospholipases and proteases is thought to aid tissue invasion and correlate with virulence. Phospholipase activity is concentrated at the growing tips of hyphae. Infections caused by C. albicans frequently involve overgrowth of resident C. albicans on mucous membranes. The yeast form is thought of as the form responsible for colonization of epithelial surfaces whereas tissue penetration and invasion is mediated by transition to the hyphal form. Granulomatous lesions are rare and inflammatory responses are predominantly neutrophilic. In severe, chronic infections, such as infection of the crop in chickens, the wall of the crop becomes thickened and covered by a corrugated pseudomembrane of yellowish-grey necrotic material giving it the characteristic ‘terry-towelling’ effect. Infections due to C. albicans have been given several names including moniliasis, candidosis and candidiasis. Table 40.1 lists the diseases caused by C. albicans in animals. There have been reports of mastitis in cattle being caused by other Candida species including C. tropicalis, C. pseudotropicalis, C. parapsilosis, C. guilliermondii, C. krusei and C. rugosa.

Table 40.1

Diseases and main hosts of Candida albicans

Hosts	Diseases
Birds	Crop mycosis (‘thrush’) may affect the crop, mouth or oesophagus causing stunting and high mortality in young birds
Horses	Gastric ulceration in foals. Genital infections have been described in adults
Cattle	Pneumonic, enteric (gastro-oesophageal ulcers, rumenitis) and generalized candidiasis. Infection seen in calves following prolonged antibiotic therapy. A mild and self-limiting mastitis may occur in cows. Abortion has also been reported
Pigs	Gastro-oesophageal ulcers
Dogs	Mycotic stomatitis occurs in pups. Genital tract infections in bitches. Cutaneous candidiasis has been described. Generalized infections occur rarely
Cats	Mycotic stomatitis in kittens. Pyothorax, rare cases due to C. albicans

Laboratory Diagnosis

Specimens

These may include scrapings from lesions, centrifuged milk samples and biopsy or tissue samples in 10% formalin for histopathology.

Direct microscopy

Candida albicans can be demonstrated in specimens by Gram-stained smears (Fig. 40.1), 10% KOH preparations, or in tissue sections stained by PAS-haematoxylin or methenamine silver stains. Candida albicans stains purple-blue with the Gram-stain. In tissue sections it appears as thin-walled, oval, budding yeast cells, hyphae and/or pseudohyphae (Fig. 40.2).

Figure 40.1 Candidia albicans in a faecal smear from a calf. (Gram stain, ×1000)

Figure 40.2 Candidia albicans: A hypha; B pseudohypha; C yeast cells budding.

Isolation

Candida albicans grows well on blood agar or on Sabouraud dextrose agar with and without inhibitors. However, some of the other Candida species may be inhibited by cycloheximide. The plates are streaked with a small inoculum as for bacteria. The cultures are incubated at 37°C, aerobically, for up to five days. Isolation alone is not sufficient for a diagnosis of candidiasis. Presence of the organism, particularly psuedohyphae, in direct smears from lesion material is usually indicative.

Identification

Colonial appearance

Colonies of C. albicans usually appear in one to three days. They are white to cream, shiny, high-convex and have a pleasant ‘beery smell’. They can attain a diameter of 4–5 mm (Fig. 40.3).

Figure 40.3 Candidia albicans on Sabouraud dextrose agar, five days.

Microscopic appearance

A small amount of growth can be placed in lactophenol cotton blue as a wet preparation, or a Gram or methylene blue stain can be used on fixed smears from the colonies. Candida albicans produces thin-walled, budding yeast cells on Sabouraud dextrose agar or blood agar.

Demonstration of germ tubes

A small inoculum from an isolated colony is suspended in 0.5 mL of sheep, bovine, rabbit or human serum and is incubated at 37°C for two to three hours. A drop of the preparation is examined under phase contrast or the high-dry objective of the light microscope (with the condenser slightly lowered). Small tubes will be seen projecting from some of the yeast cells (Fig. 40.4). This is characteristic of C. albicans (Fig. 40.5). Some strains of C. tropicalis can occasionally produce pseudo-germ-tubes but only after three hours’ incubation or more.

Figure 40.4 Candidia albicans forming germ tubes following two hours’ incubation at 37°C in serum. (Unstained, ×400)

Figure 40.5 Candidia albicans producing germ tubes.

Chlamydospore production

A plate of cornmeal Tween 80 or chlamydospore agar is inoculated by making three parallel cuts in the medium 1.0 cm apart. The cuts are made at 45° to the surface to facilitate later microscopic examination. Subsurface inoculation is made as chlamydospore production is enhanced by lowered oxygen tension. The inoculated plates are incubated at 30°C for two to four days. A thin cover slip is placed on the surface of the agar and the preparation examined under the low and high-dry objectives for the thick-walled chlamydospores (8–12 µm) borne on the tips of pseudohyphae (Fig. 40.6). Clusters of smaller blastospores may also be present (Fig. 40.7).

Figure 40.6 Candidia albicans: thick-walled terminal chlamydospores, pseudohyphae and two clusters of smaller blastospores. (Unstained, ×400)

Figure 40.7 Thick-walled chlamydospores of Candidia albicans, blastospores also illustrated.

Biochemical tests

Positive tests with the production of germ tubes and chlamydospores are sufficient for a good presumptive identification of C. albicans. For a definite identification of C. albicans and some of the other Candida species either conventional biochemical tests can be used or commercial systems such as API 20C, API-yeast-Ident (bioMérieux) and Uni-Yeast-Tek system (Remel). CHROMagar™ Candida is a commercial agar designed for the differentiation of C. albicans, C. tropicalis and C. krusei.

BiGGY agar

Bismuth sulphite glucose glycine yeast (BiGGY) agar can be used for the isolation and/or presumptive identification of Candida species. Most bacterial contaminants are inhibited by the bismuth sulphite. Candida albicans, C. kruseri and C. tropicalis strongly reduce the bismuth sulphite to bismuth sulphide. Candida albicans gives smooth, circular, brownish colonies with a slight white fringe and no colour diffusion into the surrounding medium (Fig. 40.8). The colonies of C. tropicalis are similar but there is diffuse blackening of the medium after 72 hours. Candida krusei gives large, flat, wrinkled, silvery, brown-black colonies with a brown periphery and yellow diffusion into the surrounding medium.

Figure 40.8 Candidia albicans on ‘BiGGY’ agar, seven days.

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Tags: Clinical Veterinary Microbiology

Jul 18, 2016 | Posted by admin in PHARMACOLOGY, TOXICOLOGY & THERAPEUTICS | Comments Off

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