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

Chytridiomycosis is a devastating amphibian fungal disease and methods to understand host pathogen interactions and test novel mitigation strategies are urgently needed. A recently developed in vitro frog cell infection model offers an efficient and ethical approach for chytridiomycosis research, allowing precise manipulation of host and fungal traits. However, reliable assays are needed to quantify disease outcomes. Here, we validate methods to quantify chytridiomycosis severity in an in vitro model by measuring pathogen loads and cytopathic effects on host cells. We assessed the ability of methods to distinguish fungal loads by comparing results of exposure to various Batrachochytrium dendrobatidis (Bd) infectious doses incubated under optimal and suboptimal conditions for fungal growth. Using a genetically modified strain of fluorescent Bd allowed quantification of fungal burden via microscopy, spectrophotometry and flow cytometry. For host cell effects, a DAPI staining protocol quantified cell damage and a modified MTT assay quantified cell viability. Our work provides a toolbox of methods to utilise in vitro cell infection models to investigate the function and effect size of fungal virulence and host resistance factors, enabling diverse research aimed at understanding and mitigating chytridiomycosis.