Van Rooij P et al. (2012), Germ tube mediated invasion of Batrachochytrium...

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PLoS One 2012 Jan 01;77:e41481. doi: 10.1371/journal.pone.0041481.

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

Van Rooij P , Martel A , D'Herde K , Brutyn M , Croubels S , Ducatelle R , Haesebrouck F , Pasmans F .

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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.

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	Figure 1. Light microscopical overview of the development of Bd in skin explants of Xenopus laevis.(A) 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) at 1 dpi Bd 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; (C) at 2 dpi the infected host cells have lost their cytoplasm (arrow) subsequent to invasion by Bd, only the cell membrane remains; HE stain; scale bar = 20 µm; (D) 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.
	Figure 2. TEM overview of the development of Bd in skin explants of Xenopus laevis.(A) 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) initiation of germ tube development (arrow); note the polarisation of the cell cytoplasm (); scale bar = 2 µm; (C) germ tube (GT) elongating upon the epidermis of X. laevis, with the presence of numerous lipid globules (LG) in the germ tube; scale bar = 1 µm; (D) a growing germ tube protruding the stratum corneum; scale bar = 2 µm; (E) 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; (F) 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.
	Figure 3. Light microscopical overview of the development of Bd in skin explants of Alytes muletensis and Litoria caerulea.(A) at 1 day post infection (dpi) germlings have developed germ tubes (arrow) that invade the epidermis of A. muletensis; Gomori methenamine silver (GMS) stain; scale bar = 10 µm; (B) at 1 dpi both Bd germlings (black arrow) attached upon the epidermal surface as intracellular chytrid thalli (white arrow) in the stratum corneum of L. caerulea are observed; haematoxylin and eosin stain; scale bar = 10 µm.
	Figure 4. TEM overview of the development of Bd in skin explants of Alytes muletesis and Litoria caerulea.(A) infected epidermis of A. mulentensis 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) infected epidermis of L. caerulea 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.
	Figure 5. Skin sections of Alytes muletensis and Litoria caerulea experimentally infected with Bd at 14 days post infection.Exclusively intracellular chytrid thalli (arrows) are observed by light microscopy in the stratum corneum of A. muletensis (A) and L.caerulea (B); haematoxylin and eosin stain; scale bar = 10 µm.
	Figure 6. Intracellular colonization of Litoria caerulea skin by Bd.(A–E): in vitro, (F): in vivo. (A) invasion of the stratum corneum by a germ tube (white arrow) at 2 hour post infection (hpi); (B) strong elongation of the germ tube (white arrow) into the stratum spinosum at 8 hpi; (C) 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; (D) 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); (E) 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); (F) 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.
	Figure 7. Schematic summary of the intracellular colonization process by Bd in amphibian skin.(A) Germination of a zoospore cyst or germling is followed by the development of a germ tube that invades an epidermal cell; (B) at the end of the germ tube a swelling is formed, that gives rise to a new thallus; (C) cell contents of the germling migrate into the newly formed thallus; (D) the emptied germling evanesces; (E) the new intracellular thallus forms a rhizoid-like structure that extends to a deeper epidermal layer and develops a swelling at its end; (F) a new intracellular thallus is formed.

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