By Kathleen Wong, UC Natural Reserve System
A deadly disease has been burning through amphibians around the world. What some call the most devastating pathogen of all time has played a role in over 90 extinctions, and caused over 500 more species to decline.
Research associated with the first outbreaks of chytrid disease, caused by the fungus Batrachochytrium dendrobatidis (Bd), was published in late 1990s. Since then, Bd has proved to be a formidable foe. The pathogen has decimated populations in seemingly pristine habitats, yet avoided its own demise by jumping from species to species.
A range of interventions have been tested to halt the carnage, from injecting vulnerable frogs one by one to repopulating habitats with animals that have evolved resistance to the pathogen.
Now scientists have brought the fight to Blue Oak Ranch Reserve, one of 41 reserves in the UC Natural Reserve System. This spring, the reserve will be the site of a promising new method to protect frogs against Bd infection. If successful, the prophylactic treatment—akin to a vaccination—could be used to keep amphibians healthy even in areas rife with Bd.
“This is the first time this treatment has been tried in the field,” says KM Barnett, who is leading the effort at the reserve. “It’s such a daunting task to apply vaccination to wildlife, but there is precedent for success,” says the Emory University PhD candidate, citing campaigns to reduce rabies in foxes and raccoons, and sylvatic plague among prairie dogs.
An immune response eureka
For a long time, scientists have been unsure whether Bd could be tackled with a vaccine. By definition, a vaccine works by raising an immune response involving antibodies against a target pathogen. Yet suppression of the immune system is one of the hallmarks of chytrid disease, which could make vaccination success more elusive.
In 2014, a team of researchers including Taegan McMahon of Connecticut College, Jason Rohr of University of Notre Dame, and Barnett’s advisor Dave Civitello at Emory found that frogs surviving Bd infection and those exposed to killed Bd were better at warding off new exposure to the fungus. This was a game changer. It demonstrated frogs could raise an anti-Bd immune response given prior infection or exposure, signaling that vaccination could be possible. Now the researchers could focus on triggering that response artificially.
Flushing out a vaccine
Vaccines are generally made from actual viruses or bacteria that have been weakened or killed. Sometimes, as with the COVID vaccines, molecules associated with a pathogen are enough.
To obtain material for a vaccine, McMahon cultured Bd on Petri dishes in her lab. Spores of the fungus mature into zoosporangia, structures resembling tiny volcanoes, just as they do on frogs. These zoosporangia incubate the next generation of spores. Spores typically erupt from zoosporangia when amphibian skin cells die and dissolve. The scientists coaxed the spores out manually, by rinsing the cultures with water. The spores, in turn, produce chemicals (i.e. metabolites) which are thought to aid in helping establish infections. The scientists pass the Bd in water solution through a fine filter which removes Bd spores and zoosporangia from the mixture, leaving a solution containing only these byproduct metabolites. This metabolite-containing liquid is used as the prophylactic treatment and contains no live or transmissible components of Bd.
Frogs were then bathed in this prophylactic treatment, and were subsequently exposed to the fungus. Those who received the prophylaxis bath were more able to fight off infection. The prophylaxis didn’t necessarily stop frogs from getting infected, but did help reduce infection intensity. This meant the frogs remained healthier, with less fungus to transmit the disease to others.
Mysterious metabolites
The magic in the prophylaxis, scientists suspect, comes from these fungal metabolites—proteins and other chemicals that help Bd gain a foothold on amphibian skin. For an amphibian, which relies on its skin to breathe and drink, Bd infection can be devastating. As the pathogen tears more and more holes into an animal’s epidermis, “over time it causes an electrolyte imbalance, and then the frog can die by heart attack,” Barnett says.
How exposure to Bd metabolites protects frogs, however, is unknown. “It could be changing the microbiome of the skin, or stimulating the immune system to produce protective skin peptides,” Barnett says. But amid an amphibian pandemic, understanding why the treatment works seems less critical than harnessing its powers to aid frogs.
Several features of the metabolite liquid make it a great vaccine candidate. For example, it works on contact with skin, as opposed to requiring an injection. And a lack of Bd spores in the formulation makes it safer, as it can’t spread infection.
The treatment also appears nontoxic to other species. McMahon has already exposed a variety of species to it in her lab. Frog tadpoles exposed to Bd metabolites mature faster and are more robust in size, but otherwise develop normally. The only invertebrates that suffered side effects were crayfish, and only when metabolite concentrations were orders of magnitude higher than used to treat ponds.
A Bay Area field trial
At Blue Oak Ranch Reserve, Barnett is working with Pieter Johnson of the University of Colorado Boulder to test the treatment on tadpoles of the Pacific chorus frog, Pseudacris regilla. “Pseudacris can be infected with Bd but are not too susceptible to the disease; their populations are very sturdy. And they’re very abundant, found in almost every pond. That’s why we’re comfortable studying this species, because they’re doing great,” Barnett says.
Ground zero for the treatment will be four ponds on the reserve, and 11 ponds in adjacent Joseph D. Grant County Park. A long record of monitoring data exists for all of the ponds, thanks to study collaborator Johnson. Johnson has been using Blue Oak Ranch as his California headquarters for more than a decade during his studies of these and other frog ponds around Mount Hamilton.
This treasure trove of pond information is a major reason why Barnett came to Blue Oak Ranch to run her experiments. “Being able to go to a pond and knowing because of 14 years of data what size they are, what amphibian species will or won’t be there, and even Bd prevalence was really helpful,” she says.
The reserve’s comfortable accommodations, combined with the thriving intellectual company of many other frog researchers, means she looks forward to every visit. “I haven’t gotten to go to any in-person conferences during my PhD because of COVID. But getting to live and work with people from other institutions who are studying similar things—it’s almost like being at a scientific meeting.”
Treating wild frogs
Barnett will treat her selected ponds with the vaccine material this spring. The treatment window is after adult frogs have left the pond and their eggs have hatched, but before the tadpoles have grown legs.
She’ll use a watering can to sprinkle the treatment around edges of each pond, where tadpoles tend to congregate. “It looks like I’m watering water,” Barnett says.
A few weeks later, Barnett will return to swab metamorphosing tadpoles. Analyses of the swabs will reveal whether the treatment affected their levels of Bd. She’ll also survey pond invertebrates to determine if the treatment affected their populations.
“This type of field experiment is a long shot,” Barnett admits. She and her collaborators are still conducting trials to dial in the optimal dose, treatment frequency, and timing.
Even so, when tadpoles at the reserve get so numerous that they resemble clouds of smoke in the water, Barnett can’t help but have faith in the future. “It just makes me feel so hopeful to see all of that wildlife, and to realize that nature can be resilient.”
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