Chytridiomycosis related mortality in a midwife toad (Alytes obstetricans) in Belgium

Chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis, contributes to amphibian declines worldwide. Recently, the fungus has shown to be widely distributed in Belgium and the Netherlands, although no clinical cases of the disease have been diagnosed yet. This case report describes the first case of mortality due to chytridiomycosis in Belgium in a wild population of midwife toads (Alytes obstetricans). The presence of clinical chytridiomycosis, combined with the relatively high prevalence of the fungus in Belgium, emphasizes the urgent need for a thorough study on the impact of infection on the native amphibian populations in Belgium

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 (TM); 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

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

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Germ Tube Mediated Invasion of <em>Batrachochytrium dendrobatidis</em> in Amphibian Skin Is Host Dependent

<div><p><em>Batrachochytrium dendrobatidis</em> (<em>Bd</em>) is the causative agent of chytridiomycosis, a fungal skin disease in amphibians and driver of worldwide amphibian declines.</p> <p>We focussed on the early stages of infection by <em>Bd</em> in 3 amphibian species with a differential susceptibility to chytridiomycosis. Skin explants of <em>Alytes muletensis</em>, <em>Litoria caerulea</em> and <em>Xenopus leavis</em> were exposed to <em>Bd</em> in an Ussing chamber for 3 to 5 days. Early interactions of <em>Bd</em> with amphibian skin were observed using light microscopy and transmission electron microscopy. To validate the observations <em>in vitro</em>, comparison was made with skin from experimentally infected frogs. Additional <em>in vitro</em> experiments were performed to elucidate the process of intracellular colonization in <em>L. caerulea</em>.</p> <p>Early interactions of <em>Bd</em> 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 <em>A. muletensis</em> and <em>L. caerulea</em> 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 <em>Bd</em> 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.</p> <p>In <em>X. laevis</em>, interaction of <em>Bd</em> 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 <em>in vitro</em> data agreed with the results obtained after experimental infection of the studied frog species. These data suggest that the colonization strategy of <em>B. dendrobatidis</em> is host dependent, with the extent of colonization most likely determined by inherent characteristics of the host epidermis.</p> </div

TEM overview of the development of <i>Bd</i> in skin explants of <i>Alytes muletesis and Litoria caerulea</i>.

<p>(<b>A</b>) infected epidermis of <i>A. mulentensis</i> at 1 dpi, with loss of the host cell cytoplasma and the presence of germ tube fragments inside the infected cell in cross and longitudinal section (arrow); scale bar = 2 µm; (<b>B</b>) infected epidermis of <i>L. caerulea</i> at 2 dpi showing colonization of the stratum corneum, loss of the host cell cytoplasm and the presence of germ tube fragments (arrow); intracellular chytrid sporangia are observed in the stratum spinosum; scale bar = 2 µm; GT; germ tube, SC: stratum corneum, SP: sporangium, SS: stratum corneum, ZS: encysted zoospore.</p

Light microscopical overview of the development of <i>Bd</i> in skin explants of <i>Alytes muletensis</i> and <i>Litoria caerulea</i>.

<p>(<b>A</b>) at 1 day post infection (dpi) germlings have developed germ tubes (arrow) that invade the epidermis of <i>A. muletensis</i>; Gomori methenamine silver (GMS) stain; scale bar = 10 µm; (<b>B</b>) at 1 dpi both <i>Bd</i> germlings (black arrow) attached upon the epidermal surface as intracellular chytrid thalli (white arrow) in the stratum corneum of <i>L. caerulea</i> are observed; haematoxylin and eosin stain; scale bar = 10 µm.</p

Light microscopical overview of the development of <i>Bd</i> in skin explants of <i>Xenopus laevis</i>.

<p>(<b>A</b>) adhesion of encysted zoospores (arrow) to the host epidermis at 1 dpi; (1) stratum corneum, (2) stratum spinosum; haematoxylin and eosin (HE) stain; scale bar = 20 µm; (<b>B</b>) at 1 dpi <i>Bd</i> germlings have developed germ tubes, that penetrate the stratum corneum and develop into a branched mesh work of rhizoids (arrow) in heavily infected epidermis; Gomori methenamine silver stain; scale bar = 10 µm; (<b>C</b>) at 2 dpi the infected host cells have lost their cytoplasm (arrow) subsequent to invasion by <i>Bd</i>, only the cell membrane remains; HE stain; scale bar = 20 µm; (<b>D</b>) at 4 dpi germlings have developed into mature zoosporangia (arrow), the upper layer of the stratum corneum is shed; HE stain; scale bar = 20 µm.</p

TEM overview of the development of <i>Bd</i> in skin explants of <i>Xenopus laevis</i>.

<p>(<b>A</b>) adhesion of an encysted zoospore (ZS) to the superficial mucus layer (M) on top of the stratum corneum (SC); at the site where adhesion occurs the cell wall of the encysted zoospore is remarkably thickened (arrow); scale bar = 500 nm; (<b>B</b>) initiation of germ tube development (arrow); note the polarisation of the cell cytoplasm (*); scale bar = 2 µm; (<b>C</b>) germ tube (GT) elongating upon the epidermis of <i>X. laevis</i>, with the presence of numerous lipid globules (LG) in the germ tube; scale bar = 1 µm; (<b>D</b>) a growing germ tube protruding the stratum corneum; scale bar = 2 µm; (<b>E</b>) invasion of a host cell resulting in the loss of cell cytoplasm; remnants of the host cell cytoplasm (arrow) are seen at the tip of a protruded germ tube; note the presence of a collapsed sporangium (ZS) due to cell polarisation (*); (SS): stratum spinosum; scale bar = 2 µm; (<b>F</b>) infected epidermal cell with digested cell content (*) alternated by an uninfected normal epidermal cell; note the presence of lipid globules in the infected host cell; scale bar = 1 µm.</p

Intracellular colonization of <i>Litoria caerulea</i> skin by <i>Bd</i>.

<p>(<b>A–E</b>): <i>in vitro</i>, (<b>F</b>): <i>in vivo</i>. (<b>A</b>) invasion of the stratum corneum by a germ tube (white arrow) at 2 hour post infection (hpi); (<b>B</b>) strong elongation of the germ tube (white arrow) into the stratum spinosum at 8 hpi; (<b>C</b>) development of intracellular chytrid thalli (white arrow) at the end of a germ tube at 24 hpi; rhizoid-like structures (black arrow) arise from newly developed chytrid thalli; (<b>D</b>) development of a new chytrid thallus at 24 hpi; a swelling is formed at the end of a rhizoid-like structure, a thin cell wall is formed and the cell content of the mother thallus (white arrow) is transferred into the new daughter thallus (white circle); a new thallus in a later developmental stage (black circle); (<b>E</b>) thalli connected by a rhizoid-like structure (white arrow); remnants of a germling, after having injected its cell content into a new intracellular thallus (black arrow); (<b>F</b>) mother thallus connected to a newly formed daughter thallus by a rhizoid-like structure (white arrow) at 14 days post infection. Gomori methenamine silver stain, scale bar = 10 µm.</p