Prior infection of harlequin toads by Batrachochytrium dendrobatidis alters their immune-microbial state and increases mortality upon re-infection

Strategies to improve survival of wildlife impacted by emerging infectious disease are needed for species of conservation concern. We investigated how prior infection by the skin fungal pathogen, Batrachochytrium dendrobatidis (Bd), affected subsequent disease outcomes in a highly susceptible and critically endangered toad, Atelopus glyphus. Our experimental design consisted of: (1) Bd infection followed by itraconazole clearance and Bd re-infection (Bd-Bd n = 20), (2) mock infection followed by itraconazole treatment and Bd infection (mock-Bd n = 20), (3) Bd infection followed by itraconazole clearance and mock infection (Bd-mock n = 10), (4) mock infection followed by itraconazole treatment and mock infection (mock-mock n = 10), (5) untreated animals (untreated n = 10). Prior infection shifted the skin bacterial and fungal communities to a new state enriched in putatively anti-Bd bacteria. However, prior infection worsened disease outcomes, accelerating Bd infection and mortality in prior infected animals compared to naïve animals. Prior infection animals not re-infected (Bd-mock) showed immune gene downregulation compared to re-infected Bd-Bd animals suggesting that even after Bd clearance, Bd caused sustained immunosuppression in genes that respond to Bd; this likely led to lowered immune efficacies when re-infected. Upon re-infection, as Bd load increased, Bd became the primary driver of both immune expression and bacterial composition changes regardless of infection history. Observed bacterial community differences were closely tied to host immune gene expression. The fungal community did not shift in response to re-infection, suggesting that the initial shift was due to itraconazole treatment. Prior infected Bd-Bd animals did not show any novel shifts in immune gene expression, rather they had similar expression patterns that were just more pronounced compared to naïve mock-Bd animal. This suggests that the infection-clearance-infection treatment was ineffective in improving animal survival because there was limited long-lasting effects on immune genes once the pathogen was re-encountered. Unlike previous studies demonstrating protective effects following Bd clearance in other species, this highly susceptible amphibian showed no evidence of acquired resistance. Our results underscore the species-specific nature of disease outcomes and the value of integrative approaches for examining strategies to reduce pathogen susceptibility in endangered wildlife.