Pathogen shedding varies over time in confiscated eastern box turtles, making it unlikely that a sick turtle will always test positive. This can create challenges for reintroducing turtles into the wild.
“That was a shocker,” said Maris Daleo, a TWS member and veterinary student at the University of Illinois Urbana-Champaign. “We thought that if a turtle tested positive for a pathogen, it would continue to test positive.”
But a new study shows how many negative tests you need to receive to be sure a turtle is actually healthy.
For her doctoral research, Daleo compared the pathogens carried by confiscated turtles with local free-ranging populations to determine protocols for safe reintroductions into the wild. Officials confiscate upwards of 2,000 eastern box turtles (Terrapene carolina carolina) at ports of entry each year, some of which they send to zoos for long-term care. But that’s not a sustainable solution.
Illegal wildlife trade is especially damaging to declining species that take decades to reach reproductive maturity, like eastern box turtles, which are listed as vulnerable by the International Union for the Conservation of Nature but not listed by the U.S. Fish and Wildlife Service. “Pulling any animals from the wild, especially adults, is incredibly detrimental to population health,” said Matt Allender, director of the Wildlife Epidemiology Laboratory at the University of Illinois and Daleo’s thesis advisor, “and destines them to long-term extirpation.”
And it’s not just box turtles—according to the U.S. Immigration and Customs Enforcement, illegal wildlife trafficking is estimated to be valued between $7.8 billion and $10 billion per year. “It’s a massive problem,” Allender said. Many smuggled turtles are never discovered by law enforcement officers. But dealing with the ones they do confiscate isn’t easy, either.
“When we’re talking about 2,000 turtles, zoos don’t have the capacity for that,” Allender said. “The goal was, and still remains, finding a way to get turtles back into the wild that have been taken illegally.” But disease remains a significant barrier to reintroduction efforts—the last thing conservationists want to do is infect wild populations with pathogens they picked up in captivity from other species or populations. Testing for disease isn’t as straightforward as it may seem, though.
Many pathogens, like viruses and bacteria, multiply within their hosts and then shed into the environment so they can infect new individuals. But shedding isn’t constant through time with some diseases.
In a new study published in the Journal of Wildlife Management, Daleo and her collaborators studied the pathogen shedding of box turtles confiscated in the fall of 2022.
After confiscation, the U.S. Fish and Wildlife Service tested the box turtles for ranavirus, a disease that can cause mass die-offs in amphibians and turtles that Allender describes as “the Ebola of turtles.” The authorities sent 17 turtles that had been exposed to ranavirus to the Wildlife Epidemiology Lab in Illinois because zoos couldn’t accommodate them without endangering their own collections. Seven of the turtles tested positive for ranavirus.
They placed the reptiles in individual buckets in two rooms, seven in a ranavirus-positive room and 10 in a ranavirus-negative room. To see if the turtles’ pathogen shedding varied over time, they ran an experiment. For one year, Daleo tested each turtle every month for ranavirus and three other well-known box turtle diseases to see how their levels of pathogen shedding changed throughout time: a herpes virus specific to box turtles, an adenovirus specific to box turtles and a bacterial infection. Daleo also took monthly blood samples to see if immune system markers, like white blood cell count, correlated with pathogen shedding.
Two turtles died throughout the study period, neither of which were directly linked to an infection. Also, none of the turtles tested positive for ranavirus after the initial testing done when the turtles were initially confiscated.
The problem of false negatives
Daleo analyzed the swabs with a technique that reports pathogen load, even when shedding is low. She found that the probability that a test would pick up an infection in a sick turtle was relatively low. For example, each turtle tested positive for the herpes virus at least once. Similar to humans, once a turtle gets herpes, it has the virus for life. But the herpes tests only came out positive 32% of the time in a population that was 100% infected.
And it wasn’t the fault of the tests, which are highly accurate. “We think it’s because pathogen shedding patterns vary throughout time and between individuals,” Daleo said. To make things more complicated, some pathogens showed seasonal shedding differences; herpesvirus, for example, had higher shedding rates in the spring. “That’s concerning for the institutions, like zoos, that are taking in these confiscated turtles,” she said. “How confident can you be that these individuals are truly negative?”
To answer that question, she collaborated with her epidemiology professor, Becky Smith, who wrote a paper that answered a similar question for COVID-19. “With all this information, we were able to determine how many swabs you need—and at what frequency—to get a positive detection of a pathogen,” Daleo said.
Daleo determined that the optimal sampling strategy was monthly testing for two months. But many zoos or sanctuaries have limited resources and don’t have the capacity to house turtles in quarantine for long periods of time or conduct multiple rounds of testing, which can get expensive.
If the turtles could be kept longer, and monthly sampling wasn’t an option, two swabs every third month was the ideal strategy.
The future for confiscated turtles
Despite improvements in disease detection, reintroduction of confiscated animals remains a challenge.
There are additional risks to repatriating confiscated animals. Trafficked turtles can be collected from different geographic areas and kept in small enclosures, exposing animals to new diseases. “Pathogen dynamics within groups of confiscated turtles are really complex,” Daleo said. Within a previous group of confiscated turtles the lab received, they detected an adenovirus that was only found in Europe—presumably picked up from other species while held in captivity. Without proper testing, releasing turtles back into the wild could introduce new, potentially foreign pathogens to local populations.
“We are already strapped, even in the best of times, to identify all the confiscations and save turtles, and any reduction in these efforts is going to compound the problem.” He also noted the importance of long-term monitoring efforts for reintroductions, both in monitoring the status of reintroduced individuals and populations as well as making sure that habitats are safe and healthy for reintroduction.
Daleo completed her doctoral research studying confiscated turtles this spring and will finish her veterinary degree within the next three years. “When you’re working with confiscated turtles, it’s emotionally taxing,” she said.
But Daleo and Allender continue with their work because of how critical it is for the future of the species. “It’s a collaborative work on many different fronts, and it’s going to take the continued collaboration and coordination among multiple different stakeholders to save the world,” Allender said. “In this case, one box turtle at a time.”
