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 Intracellular colonization by Bd in zebrafish larvae Alerter l'administrateur Recommander à un ami Lien de l'article 

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A hallmark of chytrid infection in amphibians includes the presence of zoosporangia and rhizoid-like structures in the keratinized epidermal tissue layers of host skin28,30. To investigate symptoms of zebrafish larvae infection, Bd-infected larvae were labelled with calcofluor white (CFW). We first observed that CFW clearly labelled the chitinous cell wall of Bd sporangia in broth culture (Supplementary Fig. 2d). At 72 h.p.i., infected larvae showed fluorescent punctae (bright round spots) throughout the entire body and fin (Fig. 2c). Time-course imaging experiments showed that these phenotypes appeared within 48–72 h.p.i. (Fig. 2c), consistent with the appearance of fin erosion (Supplementary Fig. 2c). We quantified the number cable accessories of CFW-positive punctae on each larva and observed that larvae infected with a high dose of Bd zoospores show significantly more (4.7±0.6-fold) CFW-positive punctae compared to controls (Fig. 2d, Supplementary Fig. 2e). Larvae infected with a low dose of Bd zoospores show similar values to controls, highlighting the dose dependent nature of infection. Moreover, CFW-positive punctae colocalized with blister-like structures on eroded muscle of infected larvae (Fig. 2e), suggesting that both CFW-positive punctae and blistering of skin are symptoms of infection by Bd.

Consequence of Bd infection on zebrafish larvae host tissue
We used confocal microscopy to investigate the consequence of Bd infection on zebrafish larvae. Bd-infected larvae were fluorescently labelled with CFW and Evans Blue (EB) to detect tissue damage. CFW-positive punctae were observed on the fins and muscle of low dose infected larvae, which colocalize with EB-positive tissue damage (Fig. 3a, Supplementary Fig. 3a). Moreover, both CFW-positive punctae and EB-positive tissue damage were found to colocalize with fin erosion and blister-like structures on Bd-infected larvae (Supplementary Fig. 3b).

 

Intracellular colonization by Bd in zebrafish larvae
To determine how symptoms of infection in zebrafish larvae are related to the colonization of Bd on larvae skin, we labelled infected larvae at 72 h.p.i. with a novel Bd-specific monoclonal antibody, mAb 5C4. This antibody binds to a carbohydrate epitope on an extracellular antigen produced by Bd33, labelling both Bd zoospores and zoosporangia with minimal background labelling in vivo (Supplementary Fig. 4a,b). Infected larvae present areas of skin harbouring the Bd secreted antigen (Fig. 4a). Antibody labelling also colocalizes with blisters and actin reorganization on infected larvae, showing disruption of host tissue in response to Bd infection (Fig. 4a,b, Supplementary Fig. 4c). These results are consistent with the pattern of CFW-labelled punctae described above, indicating that mAb 5C4 is suitable for highly specific, in-depth analysis of the Bd infection process. Indeed, various stages of Bd infection can be visualized in infected larvae by 72 h.p.i. (Fig. 4c), including germ-tubes invading epidermal cells, encysting zoospores and intracellular zoosporangia amongst hyperplastic epithelial cell build-ups. These results show that zebrafish larvae undergo an infection process similar to that of amphibians, and that Bd infection of zebrafish can be powerful model system to study the invasion and proliferation of Bd in vivo.

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