A survey for Batrachochytrium dendrobatidis in endangered and highly susceptible Vietnamese salamanders (Tylototriton spp.).

Until now, Asian amphibians appear to have largely escaped declines driven by chytridiomycosis. Vietnamese salamanders that belong to the genus Tylototriton are rare and have a patchy distribution in mountainous areas, falling within the proposed environmental envelope of chytrid infections, surrounded by Batrachochytrium dendrobatidis infected regions. If these salamanders are susceptible to chytridiomycosis, then their populations could be highly vulnerable after the introduction of B. dendrobatidis. Examination for the presence of the chytrid fungus in skin swabs from 19 Tylototriton asperrimus and 104 Tylototriton vietnamensis by using quantitative polymerase chain reaction was performed. Susceptibility of T. asperrimus to experimental infection by using the global panzootic lineage (BdGPL) strain of B. dendrobatidis was examined. The fungus was absent in all samples from all wild salamanders examined. Inoculation with the BdGPL strain resulted in mortality of all five inoculated salamanders within 3 weeks after inoculation with infected animals that manifested severe orthokeratotic hyperkeratosis, epidermal hyperplasia, and spongiosis. Although infection by B. dendrobatidis currently appears absent in Vietnamese Tylototriton populations, the rarity of these animals, their pronounced susceptibility to chytridiomycosis, an apparently suitable environmental context and increasing likelihood of the pathogen being introduced, together suggest the need of urgent measures to avoid future scenarios of extinction as witnessed in Central America and Australia.

Batrachochytrium dendrobatidis zoospore secretions rapidly disturb intercellular junctions in frog skin.

Global amphibian declines are in part driven by the chytrid fungus Batrachochytrium dendrobatidis, causing superficial dermatomycosis with epidermal hyperplasia and hyperkeratosis in infected amphibians. The susceptibility to chytridiomycosis and the severity of epidermal lesions in amphibians with chytridiomycosis are not consistent across species or even among individuals. Severe infections cause death of the animal most likely through disturbance of ion homeostasis. The mechanism by which this superficial skin infection results in epidermal lesions has so far eluded precise definition. It was the aim of this study to unravel how B. dendrobatidis causes alterations that affect skin integrity. Exposure of Xenopus laevis skin to B. dendrobatidis zoospore supernatant using skin explants and Ussing chambers caused rapid disruption of intercellular junctions, demonstrated using histology and transmission electron microscopy. The loss of intercellular junctions led to detachment-induced cell apoptosis, or anoikis. The zoospore supernatant induced neither apoptosis nor necrosis in isolated primary keratinocytes of X. laevis. This supports the idea that the loss of cell contacts triggered apoptosis in the skin explants. Mass spectrometric analysis of the protein composition of the supernatant revealed a complex mixture, including several new virulence associated proteins, such as proteases, biofilm-associated proteins and a carotenoid ester lipase. Protease and lipase activity of the supernatant was confirmed with a protease and lipase assay. In conclusion, B. dendrobatidis zoospores produce a complex mixture of proteins that quickly disturbs epidermal intercellular junctions leading to anoikis in the anuran skin. The role of the identified proteins in this process remains to be determined.

Development of in vitro models for a better understanding of the early pathogenesis of Batrachochytrium dendrobatidis infections in amphibians.

Batrachochytrium dendrobatidis, the causal agent of chytridiomycosis, is implicated in the global decline of amphibians. This chytrid fungus invades keratinised epithelial cells, and infection is mainly associated with epidermal hyperplasia and hyperkeratosis. Since little is known about the pathogenesis of chytridiomycosis, this study was designed to optimise the conditions under which primary keratinocytes and epidermal explants of amphibian skin could be maintained ex vivo for several days. The usefulness of the following set-ups for pathogenesis studies was investigated: a) cultures of primary keratinocytes; b) stripped epidermal (SE) explants; c) full-thickness epidermal (FTE) explants on Matrigel™; d) FTE explants in cell culture inserts; and e) FTE explants in Ussing chambers. SE explants proved most suitable for short-term studies, since adherence of fluorescently-labelled zoospores to the superficial epidermis could be observed within one hour of infection. FTE explants in an Ussing chamber set-up are most suitable for the study of the later developmental stages of B. dendrobatidis in amphibian skin up to five days post-infection. These models provide a good alternative for in vivo experiments, and reduce the number of experimental animals needed.

Germ tube mediated invasion of Batrachochytrium dendrobatidis in amphibian skin is host dependent.

Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis, a fungal skin disease in amphibians and driver of worldwide amphibian declines.We focussed on the early stages of infection by Bd in 3 amphibian species with a differential susceptibility to chytridiomycosis. Skin explants of Alytes muletensis, Litoria caerulea and Xenopus leavis were exposed to Bd in an Ussing chamber for 3 to 5 days. Early interactions of Bd with amphibian skin were observed using light microscopy and transmission electron microscopy. To validate the observations in vitro, comparison was made with skin from experimentally infected frogs. Additional in vitro experiments were performed to elucidate the process of intracellular colonization in L. caerulea. Early interactions of Bd with amphibian skin are: attachment of zoospores to host skin, zoospore germination, germ tube development, penetration into skin cells, invasive growth in the host skin, resulting in the loss of host cell cytoplasm. Inoculation of A. muletensis and L. caerulea skin was followed within 24 h by endobiotic development, with sporangia located intracellularly in the skin. Evidence is provided of how intracellular colonization is established and how colonization by Bd proceeds to deeper skin layers. Older thalli develop rhizoid-like structures that spread to deeper skin layers, form a swelling inside the host cell to finally give rise to a new thallus. In X. laevis, interaction of Bd with skin was limited to an epibiotic state, with sporangia developing upon the skin. Only the superficial epidermis was affected. Epidermal cells seemed to be used as a nutrient source without development of intracellular thalli. The in vitro data agreed with the results obtained after experimental infection of the studied frog species. These data suggest that the colonization strategy of B. dendrobatidis is host dependent, with the extent of colonization most likely determined by inherent characteristics of the host epidermis.