Once upon a time, in a cave just north of Durango, Mexico, someone pooped. In fact, it involved a number of people, and these events took place over a period of time – from around 725 AD to 920 AD, according to the researchers. Thanks to the cave’s arid conditions, when archaeologists excavated the place in the 1950s, the droppings were in fairly good condition. Weathered, dry and full of fiber, these stool samples gave scientists valuable insight into the type of food ancient people ate and what lived in their intestines.
The deposits in the cave are now widely used and have been taken to various laboratories interested in studying them. In 2021, a global team of collaborators analyzed the DNA in old poop – or paleofeces, as it’s delicately called – to see if they could identify the microbes present in poop’s gut microbiomes.
Now, in a new article published in PLoS Oneanother group of researchers took a new look at DNA collected from 10 of the droppings. Their results largely confirm an earlier finding: that the people who made these droppings were harboring a menagerie of parasites.
Welcoming worms
Usually, the droppings that Drew Capone, the lead author of the study, works with are much fresher. An environmental microbiologist at Indiana University, Capone studies the impact of sanitation on health. “Our job is to figure out, ‘How is feces getting into the environment? Where are feces found in the environment? How does infrastructure prevent excrement from entering the environment? And then, what are the impacts of excrement on pediatric health?” he says.
Capone and his colleagues wanted to use techniques to detect pathogens in modern stools on ancient stools. These methods sort the DNA of a sample looking for specific genes that are signatures of parasites like pinworms, as well as pathogenic bacteria.
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To extract this DNA, the researchers had to take samples of paleofeces from the cave. It was more difficult than expected: “We had to grind this ancient excrement into powder. We couldn’t really break it into pieces,” says Capone. They performed the procedure to look for DNA matches and got results suggesting that a number of different pathogens were in the feces, including pinworms, the protozoan parasite Giardia, and various disease-causing bacteria.
Many stools tested positive for multiple organisms. In Capone’s experience, such large numbers of pathogens are not uncommon in places with poor sanitation, making him suspect that the people who deposited these droppings so many centuries ago were in a similar situation.
Why the choice of technique is important
However, there are reasons why most labs working with ancient DNA no longer use these procedures, say Kirsten Bos and Alexander Hubener, both ancient DNA specialists at the Max Planck Institute for Evolutionary Anthropology. DNA tends to disintegrate over time, becoming frayed and fragmented. The ancient technique used in PLoS ONE paper favors longer pieces of DNA, which means it’s hard to be sure that what you’re seeing is actually ancient DNA and not modern DNA that slipped in by accident. Laboratories specializing in ancient DNA have high-tech clean rooms to minimize contamination. They also use next-generation sequencing optimized for such a fragile substance.
Additionally, most labs check the ends of DNA fragments, where distinctive fraying occurs, to confirm that what they’re looking at is truly old. With the technique in the PLoS ONE In his paper, “it’s difficult to know whether these chemical changes that occur in ancient DNA ever happened,” Bos says.
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Capone says many of the organisms tested are not capable of living long outside the human gut, so the risk of getting a false positive from modern DNA detected during feces travels could be quite low. Additionally, specialized laboratory work on ancient DNA can be expensive, and this older technique is more accessible.
Hubener, who was part of the team behind the 2021 paper analyzing poop samples from the cave, says he is skeptical of matches to bacteria — these can be particularly difficult to identify in ancient samples with this technique. However, given his team’s findings and what we know about parasite biology, he says the findings about larger parasites like worms are on somewhat firmer footing. “It’s credible to me,” says Hubener.
What would have been particularly interesting would have been to use both the old techniques and the new ones on the same samples, says Bos. This would make it clear what older techniques can reliably detect that also shows up with the newer, more stringent procedures.
“That would have been a really good way to move forward,” she said.
