Microbiome Analysis of Ancient Feces

Sometimes it can be difficult to distinguish who left a pile of poop.

Humans who live in multi-cat or multi-canine households might run into this problem when trying to discover which diarrhea-stricken pet to haul off to the vet.

Similarly, a deposit of scat on a hiking trail can inspire vivid conversations on the origin of the feces. But an intrigued hiker without a phone or camera who wants to bring a friend back for a viewing may find the excrement dispersed beyond recognition from weather conditions and trampling. Only a DNA test might be able to identify the species of animal that left the deposit if contextual clues, like a nearby moose, aren’t there.

CoproID

The microbial species found within excrement – the fecal microbiome – can reveal a lot about an animal.

Archaeologists analyze coprolites – partially mineralized ancient excrement – for clues to the health and diet of our forebears and the animals they lived with or near. The fecal microbiome can indicate obesity, diabetes, blood conditions, parasites, and symbionts.

Maxime Borry and Christina Warinner, of the Max Planck Institute for the Science of Human History, and their colleagues at Harvard University and the University of Oklahoma, developed “CoproID: a reliable method of inferring sources of paleofeces,” published in the journal PeerJ.

“One unexpected finding of our study is the realization that the archaeological record is full of dog poop,” said Warinner. The researchers hope to use CoproID to look back at changes in human health that occurred as agriculture altered diet.

The archaeologists used a technique colorfully called “shotgun metagenomics” to identify microbes from the guts of those who left specimens. “Shotgun” refers to blasting apart a genome, sequencing the pieces, and overlapping them to derive the entire sequence. “Metagenomics” is a strategy that sequences all of the DNA tidbits in an area, such as the Sargasso Sea, an armpit, or the stomach of an aardvark.

Untangling the Microbiomes of Humans and Dogs

DNA is a more precise identifier than simple description.

“Even well-preserved fecal material degrades over time, changing in size, shape, and color. The combined analysis of host and microbial ancient DNA within paleofeces presents a potential solution to this problem,” the researchers write.

They used CoproID to distinguish similar-appearing human and canine paleofeces from each other and from non-tainted sediments, which is particularly challenging because of our close relationship. For more than 12,000 years, we’ve lived with each other, eating some of the same things. In some societies people ate dogs, and dogs sometimes ingest bits of human feces and bite people. Simple DNA comparisons don’t tell the entire story.

And that’s where machine learning comes in. The researchers trained CoproID on contemporary microbiomes – that is, the genome sequences within excrement from a variety of places. They used fresh samples and already-published DNA sequences, from modern populations and from older ones. The bottom line: the technology can reveal the source of poop based on the species represented in it by telltale DNA sequences.

Specifically, the investigators analyzed modern microbiome DNA from people in the US, the Tunapuco and Matses of Peru, people from India, from Fiji (agrarian villages), from Madagascar, the Yanomami of Brazil, the Hadza of Tanzania, and people from Burkina Faso, plus DNA from a dog and three soil samples.

In addition to the modern samples, the researchers analyzed 20 archaeological samples from 10 sites representing times from 7200 BP to the medieval era. Of the 20 samples, 13 were paleofeces, 4 were from “midden” sediments (garbage piles or dung heaps), and 3 were collected from the surfaces of human pelvic bones. The sources were from Mexico, China, Slovakia, and Europe.

Of the 20 samples, coproID quickly found that 7 didn’t harbor any fecal material. Of the 13 paleofeces samples, 5 were all human and 2 were all dog; the rest were a mixture. Three samples from Mexico, for example, were mostly dog, with a few human microbiome representatives.

One of the canine samples, YRK001, was especially intriguing because it had been discovered in a chamber pot from the UK. It contained evidence of parasites not seen in human feces. Did old English hounds have toileting talents? It’s possible. A raccoon once broke into a cellar window in my house and left a personal deposit in the toilet, the animal’s identity deduced by pawprints in the bathtub. But I’ve seen raccoons scoop up frogs; dogs aren’t that dextrous.

In future work the researchers hope to minimize their “uncertain” findings by including more microbiomes in their analyses. “Identifying human coprolites should be the first step for ancient human microbiome analysis. With additional data about the gut metagenomes of non-Westernized rural dogs, we’ll be better able to classify even more ancient dog feces as in fact being canine, as opposed to ‘uncertain,'” Borry said.

The researchers suggest that coproID be used in forensic investigations, to supplement testing dog or cat DNA from hair or skin. Those tests usually detect mitochondrial DNA from the animal, which is less information than a survey of resident microbial genomes.

Other Applications

Microbiome analysis is already part of human as well as veterinary medicine. For example, it is used to diagnose inflammatory bowel disease and a form of digestive tract lymphoma in cats.

Pet DNA testing also embraces stool samples. The KittyBiome gut health test, for example, is $75, with free shipping. A human can repeatedly monitor shifting microbiomes in the pet over time, such as when a cat is stuck indoors in the winter and reverts to eating chipmunks in the summer. The microbiome should change.

Returning to my original idea, PetPoopID would be a machine-learning-based metagenomic tool that would include telltale DNA sequences of all the feline residents of a home. The owner could then provide a vet with data to figure out not only what’s wrong, but which cat is sick.

Photo credit: Human coprolites from the Xiaosungang archaeological site, Anhui Province, China (Jada Ko, courtesy of the Anhui Provincial Institute of Cultural Relics and Archaeology)