The amphibian chytrid fungus (Batrachochytrium dendrobatidis, or “Bd”) has been linked to the decline or disappearance of roughly 200 amphibian species, and nowhere has the damage been worse than among harlequin toads of the genus Atelopus. Today, 93% of harlequin toad species are threatened with extinction, largely because of this pathogen. One strategy that has helped some amphibians survive Bd is borrowed from vaccine science: expose an animal to the fungus, clear the infection with an antifungal drug, and see whether that prior exposure primes the immune response to prepare the animal for its next encounter. We tested this approach in the Pirre harlequin toad (Atelopus glyphus), one of the most imperiled species in our care. The results, published this month in BMC Genomics found that prior infection did not protect frogs from subsequent infections.

The experiment

We infected captive-bred A. glyphus with live Bd, cleared the infection with a 10-day itraconazole treatment, then waited 90 days before re-exposing them to Bd. We compared these “prior-infected” toads to naïve toads meeting Bd for the first time, tracking disease severity, immune gene expression (via non-lethal toe-clip sampling), and skin microbiome changes throughout. All Bd-exposed toads died, but previously infected toads died faster and developed heavier infections more quickly than toads facing Bd for the first time. Prior infection was a significant, independent predictor of earlier death — the opposite of what a “vaccination” strategy is supposed to achieve.

The immune system never fully recovered, and a shifting microbiome did not protect them

Ninety days after clearance, with no fungus detectable, previously infected toads still showed substantially reduced expression of core adaptive immune genes — the machinery behind immune signaling and lymphocyte responses. Their immune systems hadn’t returned to baseline; they were left in a depressed state. Upon re-infection, these toads didn’t mount a faster, targeted defense. Instead, they showed the same patterns as naïve toads, just more extreme, layered with markers of active immune suppression. Their immune systems appear to remember the infection and mounted a more vigorous immune response, but it was ineffective. The Bd itself may have also had a role in further suppressing some immune responses. Skin bacteria known to fight Bd increased on the skin following the first infection, likely driven by host immune responses. But that shift reversed during re-infection, dropping as disease worsened, and did not provide any protection during reinfection.

These findings indicate that prior exposure as a strategy may not work in some highly susceptible species. This study gives us unique insight into the skin-microbial interface in a highly susceptible species, however, a better understanding of why some individuals seem to have evolved more innate resilience to the pathogen remains a high research priority for harlequin toads as it may allow us to breed more resilient frogs.

Read the full open-access paper in BMC Genomics. Gratwicke B, Osborne OG, Hartmann A, Ávila N, Baitchman E, Togna GD, Figueroa Y, Guerrel J, Illueca E, Linhoff L, Madison JD, Ellison A, Grayfer L, Ibáñez, R, Muletz-Wolz C.R. Prior infection of harlequin toads by Batrachochytrium dendrobatidis alters their immune-microbial state and increases mortality upon re-infection. BMC Genomics. 2026 Sep 2.