Crisis biology: Can bacteria save bats and frogs from deadly diseases?
by Emily Guerin
In 2007, Valerie McKenzie volunteered for a large study of human body bacteria. It was the dawn of the golden age of the microbe. Researchers were just beginning to understand how bacteria and other microbes in human intestines influence everything from obesity to allergies and infections. McKenzie, a University of Colorado-Boulder biologist, was mildly curious about her "microbiome." But she was more interested in the bacteria living on the skin of frogs and toads.
Amphibian populations worldwide are plummeting, and entire species are going extinct. The West's struggling species include boreal toads and mountain yellow-legged frogs. Invasive species and habitat degradation play a major role, but amphibians are dying even in places with good habitat. Batrachochytrium dendrobatidis, an aggressive fungus commonly known as chytrid, is often to blame.
McKenzie, who was studying the role of farmland conversion and suburbanization in the decline of leopard frogs in Colorado, suspected chytrid was also a factor. When she read a paper about a strain of bacteria found on red-backed salamanders that inhibited chytrid's growth, she began to wonder: What microbes lived on the skin of her frogs and toads? And could any of them fight chytrid?
She captured boreal toads and leopard frogs and swabbed their legs, feet and bellies at research sites near Boulder and Meeker, Colo. She took the samples to Rob Knight, a fellow CU-Boulder biologist who studies the human microbiome. In exchange for DNA analyses of the bacteria on her amphibians, McKenzie volunteered for Knight's study, handing over fecal samples and forehead, armpit and hand swabs. "I was like, 'Whatever you want, can you just run some of my frog samples?' " she laughs. "That's how I got my first data set."
McKenzie and others now hope to harness the breakthroughs of the human-microbe revolution to slow two of wildlife's most prolific – and seemingly unstoppable – modern killers: chytrid, "the worst infectious disease ever recorded among vertebrates," according to the International Union for Conservation of Nature, and white-nose syndrome, a fungal disease that has killed more than 5.7 million bats in North America since 2006.
"When you see almost 99 percent of your population perished on the (cave) floor, it's incredibly alarming," says Tina Cheng, a University of California-Santa Cruz Ph.D. student who studies white-nose syndrome. "People are desperate for solutions."
Chytrid is a cruel and efficient killer. It spreads through water and skin-to-skin contact – during mating, for instance – infecting amphibians' skin, reducing their ability to absorb water and disrupting electrolyte levels enough to cause major organ failure. In weeks, it can obliterate populations.
Scientists suspect the disease is so devastating because most amphibians have never encountered this strain of the fungus before. Humans likely transported it around the globe in the pet trade, on American bullfrogs, whose legs are a delicacy, or on African clawed frogs, which doctors used for pregnancy tests until the 1970s. (The frog laid eggs when injected with a pregnant woman's urine.) Scientists first linked mass mortalities to chytrid in Australia and Central America in the late 1990s. It's since driven more than 200 species to collapse or extinction and colonized every continent except Antarctica.
For years, Vance Vredenburg, a blond, Chaco-wearing frog biologist at San Francisco State University, watched miserably as his favorite animals, mountain yellow-legged frogs, fell victim to chytrid. "These frogs were incredibly abundant for a very long time," he says. "Now they've become so scarce that most people don't know they live up there."
Vredenburg has monitored chytrid's march through the Sierra Nevada since the early 2000s, often hiking 15 miles over 12,000-foot passes to check alpine lakes for the disease. He knew chytrid had arrived when mountain yellow-legged frogs disappeared from lakes or floated belly-up near shore. He watched some frogs, too sick to swim, drown. "It was one of the worst experiences of my life," he says.
On a 2002 trip to Sixty Lake Basin in Kings Canyon National Park, he found the usual carnage. But at a remote lake in Yosemite National Park that same year, a few populations of mountain yellow-legged frogs appeared to be living with the usually fatal disease. What was the difference?
He found the first clues in 2005, during a talk by Reid Harris, the biologist who discovered the chytrid-inhibiting bacterium that would also inspire McKenzie's research. Just like McKenzie, Vredenburg began to suspect that microbes helped determine which frogs lived or died. To test the theory, he and one of Harris' post-docs hiked back into the Sierras. They caught and swabbed mountain yellow-legged frogs in a Kings Canyon lake that was still chytrid-free, and returned to sample the surviving Yosemite frogs.
Their results affirmed Vredenburg's hypothesis. A significantly higher proportion of the Yosemite frogs had Janthinobacterium lividum on their skin – the same anti-fungal species Harris found on salamanders. Just one year later, the Kings Canyon population went extinct. Harris, Vrendenburg and others then exposed one group of mountain yellow-legged frogs to chytrid, and one to J. lividum and chytrid. The results were dramatic: Every frog in the first group died. Every frog in the second group lived.
The experiment changed Vredenburg's career. "It forced me to become a much bigger-picture biologist," he says. Instead of doing field research on "my favorite little frog that nobody else in the world cares about," he now works in a lab with bacteria and has graduate students studying the skin microbes of amphibians around the world. He hopes to discover a way to fight chytrid by treating frogs with higher concentrations of anti-fungal bacteria than already live on their skin.
One obvious way to do that is to dose them all with J. lividum. But scientists are hesitant to spread the microbe around for fear of inadvertently spawning a new biological threat, as happened with the chytrid fungus. When animals are exposed to microbes their immune systems have never encountered, "it's a huge concern," explains McKenzie. Plus, J. lividum may not take to every amphibian, she says. "It's not one-size-fits-all."
Scientists prefer to identify and culture anti-fungal bacteria already familiar to the frogs. But it's hard to do. Even though DNA analysis revealed local strains of J. lividum growing on McKenzie's endangered boreal toads, she has failed to grow any of them in a petri dish. That means she can't test how reliably the bacteria can protect toads from chytrid in the lab. And so her ultimate goal – to increase the survival rate of captive toads released into the wild to boost populations by bathing them in beneficial bacteria – remains out of reach.
Scientists worry that because research takes so long and chytrid kills so quickly, amphibians will continue to go extinct before they find the bacteria that could save them. To get ahead of the fungus, Harris and a grad student have set their sights on Madagascar, one of the world's last chytrid-free areas. They hope to find and stockpile native fungus-fighters before chytrid appears. That way, if the disease arrives, they can respond quickly by releasing local strains of bacteria into the environment and letting them spread from frog to frog. "I like to call it crisis biology," Vredenburg says. "We don't have time to figure out the nuances. We have to do something now before these frogs go extinct."
Unlike chytrid, white-nose syndrome has yet to wipe out an entire bat species, and there are still many places in North America unaffected by it. That gives researchers like Tina Cheng hope. If they can identify an anti-white-nose strain of bacteria before the disease hits Western North America, they may be able to blunt its impact.
White-nose syndrome is caused by a cold-loving fungus called Pseudogymnoascus destructans, which grows on bat's wings, faces and tails during hibernation. Irritated, the bats wake from their torpor too often, flying around and expending energy when they should be resting. Eventually, they lose body fat and starve. The disease spreads easily, likely through bat-to-bat transmission, and can kill more than 90 percent of the individuals in a cave.
Thought to have originated in Europe, white-nose was first discovered on American soil in New York in 2006. The disease has spread to 22 states in the Eastern U.S. and five Canadian provinces. It's not clear when white-nose will reach the West, and it will likely act differently when it does, given the region's low humidity and the propensity of some Western bats to hibernate in smaller, more dispersed groups.
Cheng, who was one of Vredenburg's students, originally studied frogs but switched to bats for her Ph.D. "I thought it would be really important to address white-nose syndrome," she says. "Unfortunately for chytrid, it was too late for many species."
She has already identified and cultured a strain of bacteria that seems to slow the growth of P. destructans. Now, she's replicating Vredenburg's experiment on mountain yellow-legged frogs. In Winnipeg last fall, Cheng wiped the bacterium and the fungus on little brown bats collected from a northern Manitoba cave at the start of their hibernation. She then released them into a refrigerator that mimics the cave environment. The bats settled back into sleep and Cheng flew home to Santa Cruz, where she spends hours looking for signs of infection through a camera feed. Sick bats typically live two to three months before dying, but when really ill they will sit alone on the cave floor, shaking. In early February, Cheng said all the bats were calmly hibernating; it was too soon to tell if any were infected.
Eventually, she hopes to develop a bacterial soup that could be misted onto bats as they begin hibernation. "My interest is in prevention for those populations that have not been hit by white-nose," like those in the West, she says. But she's also interested in helping species that have already been affected. "We could prevent species from going extinct."
Research on white-nose has progressed much faster than on chytrid. Only two years after the first New York bats died, scientists had identified P. destructans, linked it to the die-offs, and started looking for ways to combat it. It took years after chytrid was first described for most amphibian biologists to agree that widespread declines were happening and to acknowledge the fungus' contribution. "By the time white-nose came along, people were familiar with a fungal disease affecting wildlife. With chytrid, there was no reference," says Katie Gillies of Bat Conservation International. Still, bat scientists face their own challenges. Because white-nose affects bats at their most vulnerable – while they hibernate – Cheng's proposed spray-bottle application could be as disruptive to bats as the fungus itself.
This disturbance is what Christopher Cornelison, a post-doctoral researcher at Georgia State University, is trying to avoid. Trained as a microbiologist with a specialty in fungi, he isn't interested in the outdoor fieldwork many scientists live for. "The bat people, they always joke that they're going to drag me into a cave and make me do some sampling," he says. He prefers white lab coats, gloves and tidy laboratories, where he's working with the U.S. Forest Service to turn Rhodococcus rhodochrous, a common soil bacterium, into an anti-white-nose weapon – without touching bats.
Cornelison discovered R. rhodochrous through a research group studying how it delayed the ripening of peaches. They noticed that less fungus grew on fruit that had been exposed to R. rhodochrous. Cornelison, who had just begun researching white-nose as a possible dissertation topic, realized the fungus might do for bats what it did for peaches.
R. rhodochrous not only slows the growth of P. destructans, he found, it annihilates it. The bacterium produces volatile organic compounds so potent they can permanently stop the fungus' spores from germinating just by being enclosed in the same chamber together for 12 hours. Now, Cornelison is designing ways for land managers to deploy the bacterium. He's come up with a bacterial paste that can be spread on a plastic sheet, covered with a semi-permeable membrane, and hung in a cave during winter, allowing R. rhodochrous VOCs to escape and kill P. destructans. In the spring, when bats begin leaving caves by day and are at lower risk of infection, the sheet could be removed. Then, says Cornelison, "you no longer have the potential collateral impacts that everybody's concerned about," such as bats dying after being disturbed by well-intentioned humans or the disruption of the cave's microbial community.
Few of these technologies have been field-tested. Still, bat and amphibian researchers feel a sense of urgency that inspires them to work faster, collaborate more, and break down the traditional barriers between microbiology, medicine and field biology. "We have no time to waste," says Vredenburg. With humans spreading germs around the globe at unprecedented rates, "(we need) to figure out what we can do to ameliorate some of the terrible consequences of (an increasingly interconnected) biosphere."© High Country News