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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 4872 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 (4.70.6-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.

  Aucun commentaire | Ecrire un nouveau commentaire Posté le 05-06-2017 à 08h46

 Our study shows that Bd is able to infect Alerter l'administrateur Recommander à un ami Lien de l'article 

Figure 4: Intracellular colonization by Bd in zebrafish larvae.
Figure 4
(a) Zebrafish larvae bath water was inoculated with high (>200 zsp per μl) dose Bd zoospores and incubated for 72 h.p.i., then fixed and labelled with Hoechst (for DNA; blue) and mAb 5C4 (for Bd; green) for visualization by confocal microscopy. Images taken at 63 X, maximum intensity projection of Z-stack shown here. Cartoon depicts imaged region. Representative images with arrows highlight colocalization between Bd and blisters on larvae skin. Scale bar, 50 μm. (b) Zebrafish larvae bath water was inoculated with high dose Bd zoospores and incubated for 72 h.p.i., then fixed and labelled with Hoechst (for DNA; blue), mAb 5C4 (for Bd; green) and phalloidin (for F-Actin; red) for visualization by confocal microscopy. Images taken at 63, maximum intensity projection of Z-stack shown here. Cartoon depicts imaged region. Representative images with insets highlight Bd adjacent to host cell actin rearrangements. Scale bar, 50 μm. See also Supplementary Fig. 4c for host cell actin in control treated larvae. (c) Zebrafish larvae bath water was treated as in a. Images taken at 40 or 63, maximum intensity projection of Z-stack shown here. Images showing different stages of Bd invasion and infection on larvae, also depicted using cartoons in the right column. 1, rhizoid-like germ tube attached to encysting zoospore, 2, chytrid thallus growth on zebrafish larvae skin, 3, encysted sporangium amongst hyperplasic epithelial cells. Scale bars, 10 μm.
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Discussion
Our study shows that Bd is able to infect and multiply on zebrafish larvae treated with antibiotics in a dose dependent manner that mimics the process of infection seen in amphibians. This demonstration of a non-amphibian vertebrate host being infected by Bd widens the host range previously known to be exploited by this hypervirulent chytrid lineage. Using a Bd monoclonal antibody (mAb 5C4), we were able to image the different stages of Bd infection with unprecedented resolution in vivo. Collectively, these results validate zebrafish larvae as a powerful aquatic model system within which these host-Bd interactions can be more fully explored. Furthermore, our observations that treating zebrafish with antibiotics results in higher cable accessories burdens of infection highlight the use of probiotic bacteria to combat Bd infection34.

Although the specific host factors necessary for Bd infection remain to be discovered, Bd is commonly found to parasitize the keratinized tissue of both amphibian and non-amphibian hosts8,9,35,36. Consistent with these observations, we found Bd parasitizing zebrafish larvae structures known to express high levels of keratin, such as the edges of the caudal fin32, where we observed fin erosion, tissue damage and apoptotic cells. These observations are in agreement with studies showing widespread apoptosis of amphibian skin cells in response to Bd infection6,31. However, in common with many macroparasite disease systems37, not all larvae became infected and mortality is heterogeneous among experiments (Supplementary Table 1), suggesting that there are unknown factors underlying the susceptibility of zebrafish larvae to Bd.

  Aucun commentaire | Ecrire un nouveau commentaire Posté le 05-06-2017 à 08h48


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  Blog créé le 16-05-2017 à 08h36 | Mis à jour le 11-08-2017 à 03h24 | Note : Pas de note