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The mystery of the sick turtles: Q&A with animal disease detectives Chrissy Cabay and Daniel Woodburn

  • A puzzling shell disease is affecting western pond turtles, a species listed as vulnerable by the IUCN, in the state of Washington in the U.S.
  • Researchers from Chicago’s Shedd Aquarium are investigating if disinfection practices that keep zoos clean could actually be removing good microbes that are beneficial for turtles, making them vulnerable to shell disease when they are released into the wild.
  • Other experts, from the University of Illinois’s Zoological Pathology Program, are trying to understand what causes the disease and how it spreads. So far, they have discovered a new species of fungus that appears to be associated with the lesions that are characteristic of the shell disease.
  • Mongabay spoke with Chrissy Cabay of Shedd Aquarium and Daniel B. Woodburn of the University of Illinois to find out more about their work.

A puzzling disease lurks among western pond turtles, one of only two native freshwater turtle species that live in the state of Washington in the U.S.

The disease produces deep lesions on the turtles’ shells, exposing the underlying bone and sometimes causing paralysis and even death. But researchers are yet to figure out what causes this shell disease or how it spreads and affects the turtles themselves. One thing is clear, though: the shell disease threatens to undo decades of recovery efforts that have been aimed at protecting the species, which is listed as vulnerable by the IUCN.

In the early 1990s, only about 150 western pond turtles (Actinemys marmorata) remained in Washington, their decline prompted by a combination of threats including the loss of their preferred wetland habitats, commercial exploitation for food, and introduction of non-native predators like the American bullfrog (Lithobates catesbeianus) that preyed on young turtle hatchlings.

Researchers scrambled to reverse the turtle’s decline. They tracked turtle nests in the wild, retrieved hatchlings and eggs from those nests, and raised them in warm, safe places within labs at the Woodland Park Zoo in Washington and Oregon Zoo until the hatchlings were big enough to be safely released into the wild. The researchers hoped the head start would give these individuals a better fighting chance in their native habitats.

Some of them did. Today, more than 1,000 of the turtles are known to live across six locations in Washington. But shell disease is slowly creeping into these populations. A survey from 2013-2014 found that 29 to 49 percent of all examined turtles in these populations showed signs of the disease.

Now, Chicago’s Shedd Aquarium and the University of Illinois at Urbana-Champaign Zoological Pathology Program have joined forces with Woodland Park Zoo, Oregon Zoo and the Washington Department of Fish & Wildlife (WDFW) to unravel the mystery of the shell disease.

The researchers at Shedd Aquarium are exploring a hypothesis: they think that disinfection practices that keep zoos clean could actually be removing good microbes that are beneficial for the turtles, and reducing their immune fitness in the wild. This could be making the western pond turtles vulnerable to the shell disease.

Researchers from Shedd Aquarium sampling western pond turtles. Image © Shedd Aquarium/Inga Mesker.

To investigate their hypothesis, the researchers are looking at the microbial communities within the “head-start” habitats (the environment in which the hatchlings are raised in captivity before being released into the wild) and comparing them to those in the turtle’s native wild habitats. So far, the team has collected more than 600 samples from the head-start environments, including samples from 200 individual turtles, according to Chrissy Cabay, program director for Shedd Aquarium’s Microbiome Project. Researchers will be heading out to collect more samples over the next few weeks, she told Mongabay.

“Our question is really what can we do in the head-start environment to shift the microbiome closer to that of the native, and see if that has a positive impact on outcomes for the turtles when they’re released,” Cabay said.

At the same time, researchers at the University of Illinois’s Zoological Pathology Program are trying to understand what causes the disease and how it spreads. So far, the team has discovered a new species of fungus that appears to be associated with the lesions of shell disease.

“We are now working to fully characterize the fungus and discover its role in the development of shell lesions,” said Daniel B. Woodburn, a pathologist and doctoral candidate at the Zoological Pathology Program. “By learning about this new fungus, we can better advise caretakers, veterinarians and wildlife biologists on how to best protect head-start turtles from this disease and how to care for affected populations in the wild.”

Mongabay spoke with Cabay and Woodburn to find out more about their research.

Mongabay: When did the new shell disease first start appearing in the Western pond turtles? And how does it affect the turtles?

Daniel B. Woodburn: Shell disease has been a concern in western pond turtles since at least 2013, when major research and surveillance efforts began with populations in Washington. However, by looking at historical records and pictures, researchers at the Washington Department of Fish & Wildlife (WDFW) have identified similar shell lesions in turtles as far back as 2003. From continued monitoring of these early turtles, we know the disease is progressive and slowly eats away at the shell, but the full long-term effects are unknown. A variety of projects are currently underway to study these effects, particularly in adult females where disease may result in a reduced number of eggs and lower survival rates for hatchlings.

Chrissy Cabay: It took multiple years to really figure out what was going on, and we’re definitely still learning more about what the disease is — is it one pathogen, is it several, what is its etiology, how it affects the turtle.

We are also still learning the full extent to which the disease affects the turtles. It’s first noticed visually by lesions on the surface of the shell. But for a proper diagnosis you need a CT scan. The disease causes these necrotic lesions inside the body cavity of the animal, which are first spotted typically by a CT. So the disease will typically show up on CT before it shows up on the surface of the shell.

Dr. Daniel Woodburn of the University of Illinois discovered a new fungus that appears to be associated with shell disease. Image courtesy of the University of Illinois College of Veterinary Medicine.

Does it affect wild turtles? Or only captive ones?

Woodburn: So far, this new shell disease has only been confirmed in turtles that have spent at least part of their lives under human care. This includes turtles currently living in zoos and aquariums, as well as turtles that were raised in head-start programs and released into the wild. While there are reports of completely wild turtles with suspicious-looking shell defects, we haven’t yet been able to confirm infection.

Cabay: We’ve seen the disease so far only in head-started animals. But there are just so few truly wild turtles left that it’s impossible to do a proper scientific comparison and really say whether head-starting is the cause.

Your research found a newly described fungus associated with lesions that are characteristic of the shell disease. What do you know about the fungus’s role in the disease so far?

Woodburn: This new shell disease is characterized by defects and changes in the shell bone that are microscopically very different from the lesions we see more commonly with other shell infections. Whenever we’ve seen these unique shell lesions in a turtle, we’ve also found this new fungus, so we know the two are associated with each other. Further research is needed to determine whether the fungus itself is causing these lesions on its own, needs another contributing factor, or is taking advantage of damage due to some other cause. However, as this new fungus is related to other more well-known fungi that infect other types of reptiles, we suspect it may be a primary pathogen.

A severely affected turtle with fungus on its shell. Image courtesy of Daniel Woodburn.

How is the Shedd Aquarium involved?

Cabay: We are the home of the microbiome project, which is a relatively recent program that started in 2015. It was based on generating some data to help inform the regulations that we’re subject to. We intensively treat the water in many of our habitats, for example, and the thinking for years was to make it as clean as possible through filtration, disinfection. We’ve since learned with the advent of the human hygiene hypothesis that there can be some health advantages to microbial diversity in the environment. We have some questions for what that means for our animals. We know what temperature to maintain them at. We know what salinity to keep them at. We know a lot about the environmental parameters that are best for their health. What we don’t know is what kind of microbial community should we be promoting in their habitats to provide them the best possible care. It’s a new field and there’s still much to be learned.

But our original core question was “how clean is too clean?” for animal habitats. We are sort of unique in the zoo and aquarium world in that we have a research team whose specialty is microbial ecology and we have an onsite laboratory with this expertise. Because of our reputation, we’ve sort of become the go-to for our peers that are interested in microbial questions of their own. We were contacted by the Washington Department of Fish & Wildlife who asked for our help with the analysis of a separate study, but we began a partnership with them and learned about challenges that these turtles faced. And that’s when they asked us to contribute our expertise and our unique resources to what has been for them a serious challenge.

How did you arrive at your hypothesis?

Cabay: It really stems from some recent work being done in human health that shows that exposure to a diverse array of microbes in the environment, particularly early in life, can have important implications for an animal’s development. It’s been shown in people that exposure can have an immuno-protective effect on individuals. We know that the head-started animals are maintained in environments that are pretty different from the native environment especially when it comes to the microbial load. So we are curious to know whether there is any change we could make in the head-start environment that might induce microbial diversity that the animals could be exposed to. That might benefit them in terms of being able to resist this disease down the road when they’re released. And there’s still a lot we don’t know. Immune parameters in reptiles are not where they are for people, so we’re starting with the baseline. At the moment we’re in the better part of year one of a two-year study that is examining the native environment of the turtle and the head-start environment, and simply comparing to see how the microbiome differs.

Our question is really what can we do in the head-start environment to shift the microbiome closer to that of the native, and see if that has a positive impact on outcomes for the turtles when they’re released.

Western pond turtle. Image © Shedd Aquarium/Inga Mesker.

Can you tell us a little about your sampling process?

Cabay: Our strategy is to sample places that are analogous between the head-start and the native environments. That would be water or basking structures — basking is common with any ectothermic animals like these turtles, so they are likely to interact with the biofilm that tends to accumulates on the surface of the basking structures. In the native environment that might be a log. In the head-start environment that might be a floating mat or rock that has been provided. We’re also sampling the animals themselves; their shell, and their cloaca or anal opening. We’re interested in this opening because it’s one route of potential transmission of the disease because it comes in contact with water. It’s also an opportunity to look at the gut microbiota of the turtles. In other species, gut microbiome has also been associated significantly with health.

Once we collect those samples from the environment or from the turtle shells, we store them at -80 degrees Celsius [-112 degrees Fahrenheit], unless they are ready to be processed. At that point, we extract DNA from the samples, and we do what is called 16s rRNA gene profiling. We use a special marker gene, sort of a biomarker, that is known to have utility as an identifier for microbes. It’s highly conserved among bacteria and archaea [microbes] but has enough variability where its sequence can be associated back to a particular taxonomic group. And typically that gets us to the genus level for bacteria and archaea.

What do you hope to achieve at the end of the microbiome project?

Woodburn: By looking at microbiome data from turtles with shell disease and comparing to healthy turtles, we hope to identify any other infectious organisms (such as bacteria, viruses or even other fungi) that are involved in the development or progression of shell disease. This information will help guide future studies on disease transmission and may reveal new options for treating and preventing shell disease in western pond turtles.

What have been some of the challenges in your research?

Woodburn: Since this disease involves a newly recognized fungus, no specific diagnostic tests were available when we started this project. Therefore, we’ve had to develop our own set of tests which look for fungal DNA in shell samples. This means a lot of trial and error, but having a rapid and accurate test for this shell disease is necessary to help identify sick turtles, monitor treatment successes, and guide future research efforts.

Cabay: I would say that the biggest challenge has been the gaps in knowledge in what we know about the turtles compared to what’s known about health in mammals and in particular in humans. How do you know if a turtle’s healthy? How do you know if a turtle is immunologically robust? The same parameters that have been measured in humans have not been identified in turtles. It may be that we may not know for years whether these animals have benefited from our manipulation or not. We have to accept the reality that there is some inherent gap in research on these animals.

Is there anything else that you would like to add?

Woodburn: By tapping into the expertise from multiple collaborators, including WDFW and the Shedd Aquarium Microbiome Project, we hope to gain a fuller understanding of this disease so that we can minimize its effects on western pond turtle recovery efforts. This project would not be possible without the hard work, knowledge and support of everyone involved, from the field into the laboratory.

Cabay: Many partners and organizations are involved in trying to solve this mystery when it comes to shell disease. We’re just one piece of that. But with these large complex questions particularly in the conservation sphere, it’s an all-hands-on-deck approach. It takes many different approaches to science to help inform strategies for managers.

Western pond turtles have been suffering from a mystery shell disease. Image courtesy of the University of Illinois College of Veterinary Medicine.



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