CHYTRIDIOMYCOSIS AND AMPHIBIAN POPULATION DECLINES

Amphibian population declines have been increasingly recognized since the late 1980s and have generated much public and scientific interest. A recent survey suggests that 32.5% of known amphibian species are globally threatened and 43.2% are experiencing a population decrease.³⁸ Although in some declines a clear anthropogenic cause such as habitat loss or species exploitation may be implicated, in many others the cause is not obvious (“enigmatic decline”). Of the enigmatic-type population declines, chytridiomycosis and climate change are most frequently cited as potential causes.

Population declines attributed to chytridiomycosis are best documented in stream-dwelling species at high elevations in the rain forests of Central America and eastern Australia.^19,41 In both locations, evidence suggests temporal and geographic progression of declines.⁸ The apparent southward progression of disease incidence in Costa Rica and Panama has allowed for prediction of future sites of decline.²⁰ Disease progression may have significant implications for worldwide amphibian species diversity because up to half of all species live in neotropical regions that environmental modeling suggests could be a suitable niche for Bd.³⁷

It is unclear whether Bd is a novel pathogen that has recently been introduced to naive populations or an endemic pathogen that has emerged because of environmental or other cofactors.³⁵ It may be that both circumstances exist, depending on geographic location. Preliminary genetic information obtained from isolates originating from several continents indicates that Bd is a recently emerged clone, possibly consistent with an introduced novel pathogen.²³ Research on the means of introduction of Bd to new locations has focused on international movement of amphibians for food, laboratory research, and the pet trade. In particular, the African clawed frog (Xenopus laevis) and the bullfrog (Rana catesbeiana) are species that have been widely moved or introduced worldwide and are good potential reservoir hosts for Bd because they carry infection without significant clinical signs.^9,40 The occurrence of Bd-associated mortality events at lower temperatures (within preferred temperature ranges for Bd) suggests that environmental cofactors may play a role in mortality events resulting from either recent introduction of Bd to a region or exacerbation of endemic infections.^3,33 Stable endemic Bd infection of populations has been documented after catastrophic declines presumed to have resulted from introduced Bd infection,³⁶ as well as in populations without documented declines.²⁸

PATHOLOGY AND PATHOGENESIS

Lesions of chytridiomycosis are limited to keratinizing epithelium in the skin of postmetamorphic animals and the mouthparts (tooth rows and jaw sheaths) of tadpoles.^2,32,34 Dissemination to deeper portions of the skin or to viscera does not occur. Lesions consist of varying degrees of epidermal hyperplasia and hyperkeratosis, with intralesional thalli characteristic of Bd (Figure 17-1). The keratinized layers (stratum corneum) of amphibian skin are usually very thin, and hyperkeratosis may be overlooked if using criteria established for other species. Associated inflammatory cell infiltrates are an inconsistent finding and, when observed, are usually in association with severe or chronic infections or in cases with secondary bacterial or fungal infection. Secondary infections are common, presumably because environmental bacteria and fungi become trapped in excessive keratin layers or within empty thalli of Bd that have expelled zoospores. The severity and distribution of lesions may range from relatively minimal focal lesions in subclinically infected animals¹³ to severe, multifocally extensive to diffuse lesions considered to be clinically significant.

Fig 17-1 A, Histologic section of normal skin from boreal toad (Bufo boreas boreas). The stratum corneum (SC) is thin and only one or two cell layers thick. B, Histologic section of skin from Wyoming toad (Bufo baxteri) with severe chytridiomycosis. There is moderate epidermal hyperplasia, and the SC is greatly thickened with numerous chytrid thalli (arrowheads). (See Color Plate 17-1.)

Characteristic features of Bd thalli aid in identification in cytologic preparations or histologic section. The spherical thalli, which are intracellular in superficial keratinocytes, range from approximately 7 to 20 μm in diameter, and mature thalli (zoosporangia) may contain discrete, 1- to 2-μm basophilic zoospores. Empty thalli that have discharged their zoospores are common and should be distinguished from cross sections of fungal hyphae or ducts of cutaneous glands. Flask-shaped thalli with prominent discharge papillae (discharge tubes) can usually be found in most heavy infections (Figure 17-2). Colonial thalli have evidence of fine internal septation and are best appreciated in empty thalli or in Gomori’s methenamine silver (GMS)–stained sections. An inconsistent, but sometimes helpful, finding in GMS–stained sections are thin, rootlike extensions from thalli termed rhizoids (Figure 17-2, D).

Fig 17-2 A, Four immature, spherical chytrid thalli in stratum corneum of a boreal toad (Bufo boreas boreas). B, Mature chytrid thallus (B. boreas boreas). Also visible in this section is the discharge tube (arrows), giving the thallus a flasklike shape. C, Numerous empty chytrid thalli that previously discharged zoospores in the keratinized mouthparts of mountain stream tadpole (Ptychohyla sp.). The thallus in the center (arrow) has faintly visible internal septation (colonial thallus). D, Gomori’s methenamine silver (GMS)–stained section showing rootlike extension (rhizoid, arrow) from a colonial thallus in the stratum corneum of a Limon giant glass frog (Centrolenella ilex). (See Color Plate 17-2.)

The mechanism by which Bd causes death of susceptible animals is still unclear. Disruption of normal skin function, especially in regard to water absorption and electrolyte balance, and secretion of a mycotoxin are the most frequently cited hypotheses. Some clinically affected animals show evidence of dehydration and hemoconcentration, which may provide support for disruption of skin function.¹¹ Death in Bufo boreas tadpoles shortly after exposure to cultures of Bd might support the role of a mycotoxin.⁴ There is evidence for both species-related and age-related differences in susceptibility to clinically significant infections.^10,13,18

Transmission of Bd infection is through the motile, flagellated zoospores and may occur by direct contact between animals or by contact with water or substrates used in housing affected animals. Extension of infection from the keratinized mouthparts of tadpoles may occur as the tadpoles metamorphose and develop a keratinized epidermis.^18,34

CLINICAL SIGNS

Clinical signs of chytridiomycosis are variable and range from unexpected death without premonitory signs to animals with evidence of significant skin disease. The most common cutaneous signs of chytridiomycosis are excessive shedding (“sloughing”) of skin, rough or granular changes in skin texture, and brown to red (hyperemia) skin discoloration. Findings are most often distributed on the ventral body and feet of terrestrial animals, but may be diffuse in totally aquatic species. Hyperemia and other features, such as cutaneous ulceration, are more common in animals with secondary bacterial, fungal, or water mold (Oomycetes) infections. In these cases, chytridiomycosis should be distinguished from other potential causes of “red leg” syndrome, including bacterial septicemia and iridovirus infection.

Other clinical signs that may be observed include postural changes, in which animals hold their legs away from the body, apparently to avoid contact with substrate; avoidance of or increased preference for water; anorexia; lethargy; and neurologic signs such as loss of righting reflex.

Tadpoles infected with Bd are usually asymptomatic and may serve as reservoirs of infection for postmetamorphic animals. Detailed gross examination of the keratinized mouthparts may show areas of depigmentation, which should be distinguished from other potential causes of mouthpart abnormality, including chemical exposure and low environmental temperature.³⁴ Reduced body mass of infected tadpoles and deaths of some individuals in acute-exposure studies have been reported for selected species.^4,30