A rapid technique to detect chytrid infection in adult frogs

Investigations into worldwide population declines in amphibians are under way. A wide range of causes have been identified, e.g., habitat destruction and the introduction of non-native fish. However, a newly discovered chytrid fungus has been consistently found in mass mortality events in wild populations of frogs in Australia, Panama and Spain. This is the first known instance of a keratinophilic fungus pathogenic to amphibians. The chytrid fungi present in all of these events were morphologically and ultrastructurally identical and given the name Batrachochytrium dendrobatidis gen. et sp. nov. Longcore. The disease caused by B. dendrobatidis was called chytridiomycosis. The fungus has been found inside the keratinized cells of the epidermis of adult or newly metamorphosed animals (particularly the ventral body, limbs, and feet areas); it has also been observed to invade the keratinous teeth of tadpoles. Histological sections made from frog toe clippings have revealed the fungus to occur within the inner layers of the epidermis, within thickening or erosions of the epidermis, and occasionally within ulcers. Skin scrapings, histological sections from toe clippings and elsewhere, transmission and scanning electron microscopy, and immunocytochemistry are all methods that have been used to detect the presence of chytrid fungus in frog skin. All of these have associated problems. To address these problems we sought to develop a rapid cost-effective method suitable for non-mycologists to distinguish clearly the chytrid fungus in frogs suspected of being infected with chytridiomycosis. Because the cationic dye Congo red stains cellulose and chitin, and has heen used to reveal fungal hyphae in histological sections of human tissue, we investigated the feasibility of this dye to reveal the chytrid fungus in frog skin scrapings and intact skin.