Amphibian cellular immune response to chytridiomycosis at metamorphic climax

The fungal disease chytridiomycosis (caused by Batrachochytrium dendrobatidis [Bd]) is a primary contributor to amphibian declines. The frog metamorphic stages, characterised by extensive physiological reorganisation and energy expenditure, have heightened susceptibility to Bd. However, little is known about how these metamorphic stages respond immunologically to Bd infection. In this study, we examined Bd infection and the cellular immune response of Mixophyes fleayi at Gosner stages 40, 42 and 45, using blood smears and skin and liver histology. Although proportional differences were observed, the impact of Bd exposure appeared negligible prior to Gosner stage 45 (onset of morbidity), with no significant differences observed in absolute leukocyte counts for blood or liver samples between control and Bd-exposed groups at Gosner stages 40 and 42. Animals exhibiting clinical signs at Gosner stage 45 demonstrated significant elevation in liver leukocyte counts, blood neutrophil and monocyte counts and neutrophil-to-lymphocyte ratios. These findings are reminiscent of the amplified inflammatory response characteristic of immunopathology in clinically infected amphibians. Interestingly, a subset of exposed animals that had apparently cleared infections at Gosner stage 45 had similar blood leukocyte counts but reduced liver leukocyte counts compared to naïve controls. This could be a consequence of prior cellular consumption during pathogen removal or effective immune regulation via anti-inflammatory protective feedback mechanisms. We recommend targeted gene expression analyses (e.g. immunomodulatory cytokines) to establish the mechanisms responsible for the varied immune expression and infection outcomes across metamorphosis.