title: Development of in vitro models for a better understanding of the early pathogenesis of Batrachochytrium dendrobatidis infections in amphibians.
abstract: 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.
authors.affiliation: Laboratory of Bacteriology and Mycology, Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium. pascale.vanrooij@ugent.be
authors.forename: Pascale
authors.initials: P
authors.lastname: Van Rooij
authors.forename: An
authors.initials: A
authors.lastname: Martel
authors.forename: Melanie
authors.initials: M
authors.lastname: Brutyn
authors.forename: Sofie
authors.initials: S
authors.lastname: Maes
authors.forename: Koen
authors.initials: K
authors.lastname: Chiers
authors.forename: Lieven
authors.initials: L
authors.lastname: Van Waeyenberghe
authors.forename: Siska
authors.initials: S
authors.lastname: Croubels
authors.forename: Freddy
authors.initials: F
authors.lastname: Haesebrouck
authors.forename: Frank
authors.initials: F
authors.lastname: Pasmans
mesh_terms: D023401:Animal Use Alternatives; D000818:Animals; D008411:Chytridiomycota / Q000472:pathogenicity* / Q000502:physiology / Q000648:ultrastructure; D004271:DNA, Fungal / Q000235:genetics; D003881:Dermatomycoses / Q000382:microbiology / Q000473:pathology / Q000662:veterinary*; D054884:Host-Pathogen Interactions / Q000502:physiology; D015603:Keratinocytes / Q000382:microbiology / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission / Q000662:veterinary; D016133:Polymerase Chain Reaction / Q000662:veterinary; D046509:Tissue Culture Techniques / Q000662:veterinary*; D014981:Xenopus / Q000382:microbiology*
chemical_list: D004271:DNA, Fungal