Using fluorescent in vitro amphibian cell infection models to quantify pathogenicity of Batrachochytrium...

Area of Science:

• Amphibian pathology
• Mycology
• Infectious disease research

Background:

• Chytridiomycosis, a fungal disease, devastates amphibian populations globally.
• Understanding host-pathogen interactions is crucial for developing mitigation strategies.
• Existing research methods for chytridiomycosis are limited in efficiency and ethical considerations.

Purpose of the Study:

• To validate reliable assays for quantifying chytridiomycosis severity in an in vitro frog cell infection model.
• To establish methods for measuring pathogen loads and host cell damage.
• To provide a toolbox for investigating fungal virulence and host resistance factors.

Main Methods:

• Utilized a genetically modified fluorescent strain of Batrachochytrium dendrobatidis (Bd) for fungal quantification.
• Employed microscopy, spectrophotometry, and flow cytometry to measure fungal burden.
• Assessed host cell damage using DAPI staining and cell viability with a modified MTT assay.
• Compared results under optimal and suboptimal fungal growth conditions to validate assay sensitivity.

Main Results:

• Validated methods accurately quantified fungal loads (pathogen burden) in vitro.
• Successfully measured cytopathic effects, including cell damage and viability, on host cells.
• Demonstrated the ability of assays to distinguish varying levels of fungal exposure and growth conditions.
• Established a reliable suite of assays for in vitro chytridiomycosis research.

Conclusions:

• The validated toolbox of methods enables precise quantification of chytridiomycosis severity in cell models.
• These assays facilitate research into the functional impact of fungal virulence and host resistance.
• The developed methods support ethical and efficient investigation of amphibian disease, aiding mitigation efforts.