Health

Ancient Skeletons Might Explain One of Humanity's Worst Epidemics

After stumbling on a mass burial site, "paleopathologists" use ancient DNA to tackle a 400-year-old mystery.

by Shayla Love
Jan 15 2018, 4:00pm

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When the inhabitants of the ancient town of Teposcolula left in 1552, they resigned their home to nature. Its remains are covered with dense vegetation on a mountain ridge in southern Mexico, in the Mixteca Alta region of Oaxaca. The only nearby human life is a small farm on the other side of the mountain, where a few families herd sheep and goats.

In 2004, archaeologists traveled there to excavate administrative buildings in Teposcolula’s former Grand Plaza. When they pulled back the overgrown brush and soil, they found strange oval-shaped holes cut directly into an ornate plaster floor. When they dug into them, they discovered they were filled with human remains.

This type of burial didn’t follow any known rituals for the Mixtecs, the native people who lived at the site at the time. Most holes contained multiple bodies, stacked alternating from head to foot, with their arms folded across the chest or abdomen. The burials were laid out in a grid across the plaza; it was as if they would dig a hole, fill it with as many bodies as they could, and then start another.

What began as an architectural exploration became an excavation of a cemetery believed to hold the bodies from one of the worst epidemics in human history.

Excavated structure at the northern edge of the Grand Plaza at Teposcolula-Yucundaa. Photo by: Christina Warinner. Image courtesy of the Teposcolula-Yucundaa Archaeological Project.

No one has lived in Teposcolula since 1552, not since a mysterious disease plagued the Mixtecs, killing as many as 60 to 90 percent of its locals. Historical documents tell us that a series of horrible epidemics ravaged Mexico during the period of Spanish colonization. One Spanish priest claimed he buried 10,000 people in a single year—perhaps hyperbole, but the account reflects the feeling of loss it caused in Mexico in the 16th century. Yet, parts of the story were unresolved: If these people were dying, they had to be buried somewhere—but where? And what did they die of?

At Teposcolula, a long-standing question in epidemiological history was rearing its head through oval-shaped holes in the ground. And a field well-suited to this investigation showed up to get some answers: paleopathology.

Paleopathology is a field with an enticing name that does exactly what it sounds like: studies the diseases of past populations through ancient human remains.

When Sharon DeWitte, a paleopathologist at the University of South Carolina, was 14, she had surgery to correct her scoliosis. She imagined that in a thousand years, archaeologists would dig up her bones and marvel at the metal rods put into her spine. “I’ve had that narcissistic interest in skeletal biology ever since I was a teenager,” she says.

When I look in the mirror, I see what I consider to be “myself.” My nose, my hair, my cheeks. It’s through this appearance that others infer my gender, my age, and my overall state of health. But when I die, and am buried in the ground, all of those things will be lost. For DeWitte, and other paleopathologists, they use clues from what’s left­­—pieces of bones—to solve complex problems about the past.

What could Dewitte tell about me using only my skeleton? She walks me through the basic paleopathological approach. Bone can only do two things: add more bone, or take bone away, she says. Within these basic parameters, she can uncover facts about nutrition, basic health, age, sex, chronic illnesses, diet, and more.

When faced with a skeleton, she starts with the pelvis and skull. Women have several adaptations in their pelvis to ease childbirth and, through those differences, she can tell if she’s looking at a man or woman. As you grow, several of your bones in the arms and legs fuse together and she can tell how old a person was through those bone fusions. Joint surfaces degenerate over time, and help her determine age too.

She’ll look for stress markers called enamel hypoplasia, lines or grooves across the surface of teeth that represent interruptions of enamel formation during childhood because of disease or malnourishment. She can look at stature: people who are shorter could have experienced stress. Excess growth on the surface of bones could be caused by infection or trauma, and lesions on the top of the skull could be attributed to anemia. Diseases like syphilis leave lesions on the bone, and tuberculosis causes a characteristic hunching of the spine.

“We’re looking at a tissue that’s incredibly hard, so people have this idea of it being static,” Dewitte says. “But it’s so responsive to everything that you do to your body throughout your life. You can test the bones and teeth for the presence of certain isotopes, so you can get an idea of diet. You can learn people’s migration patterns, or if they died in a location different from where they were a child. There’s so much that’s present in the skeleton. We don’t realize how much we’re recording as we’re going through our daily lives.”

Paleopathology is not a new field, it gained recognition in the early 1900s when Sir Marc Armand Ruffer, the director of the British Institute of Preventive Medicine (now the Lister Institute) was researching diphtheria and accidentally infected himself with the bacterial infection. He left England to recover in Egypt, and began to study mummies. Through his work there, the term “paleopathology” became well-known.

Starting in 1921, he published several important paleopathological findings from his mummies: he discovered calcified eggs of Schistosoma haematobium bilharzia, a parasitic infection, in the kidneys of two 20th dynasty Egyptian mummies buried from 1187 to 1064 BC, showing that humans had a history with this illness for the past 3,000 years. He saw arterial lesions, showing that mummies had atheromatous degenerative arterial disease, found evidence of smallpox in another twentieth dynasty mummy, and diagnosed tuberculosis in a mummy whose soft tissue was preserved.

Paleopathology hadn’t changed that much since the days of Ruffer—until about 20 years ago. Dating methods had gotten better, but paleopathology was largely bound by what it could see in the bone.

There was a limit to what DeWitte and other paleopathologists could learn using only their eyes, she says. In the last ten years, a field that deals with the very old has been revolutionized by something very new: next-generation sequencing. A tool has emerged to take a closer look at their samples, and uncover secrets lurking in the bones of the remains in the DNA itself of ancient diseases.

Christina Warinner, a molecular anthropologist who now works at the Max Planck Institute for the Science of Human History, arrived in Mexico in 2006 for her dissertation research, and to help figure out the cause of the epidemic in Teposcolula.

Here’s what she knew: In late 1544, an unidentified epidemic broke out in New Spain. The native peoples and the Spanish had never seen anything like it. “The Spanish, if they knew what the disease was, they would name it,” she says. “They named smallpox, they named measles, they named mumps. They recognized them and they named them. For this one, they didn’t know what it was, and they didn’t have a name for it.”

Instead, it was called pujamiento de sangre (abundant bleeding or full bloodiness) in Spanish or huey cocoliztli (the great pestilence) in Nahuatl, a widely spoken indigenous language. In visual depictions of people suffering from the disease, it was shown with gruesome symptoms, like bleeding from the face and full-body rashes.

When Warinner's group began to carefully excavate the skeletons from the Grand Plaza, she noticed not how sick the skeletons looked, but how healthy they were. “In fact, they were young,” she says. “Most of the people that I looked at were in their late teens or early 20s. That itself is a warning sign. When you find a cemetery full of people in their late teens and 20s, which is typically when people are at their healthiest, it often means something terrible has happened.”

Indigenous depiction of the 1545 cocoliztli epidemic in the Codex en Cruz, a 16th century central Mexican document showing the symptoms of the epidemic: bleeding from the face and a full-body rash. Drawing by Christina Warinner, after the original.

She exhaustively researched historical and archival documents for descriptions of disease, and tried to use polymerase chain reaction (PCR), a technique that amplifies and copies a selected sample of DNA, to find an ancient pathogen. But the PCR techniques were crude. She had to know exactly what she was looking for and if she did find a bacterium, she couldn’t be sure it was ancient.

“I ran into barrier after barrier, not knowing what it was, and then I just kept amplifying soil bacteria that was background,” she says. “The project kind of stalled and I really couldn’t take it further.”

Imagine you’re going fishing. The fish you want to catch are very small, and they’re swimming in a pool of millions of other fish that all look similar. One thing is working in your favor: this type of fish has a uniquely shaped mouth, so if you designed a special bait, it could catch only that fish, like a key fitting into a lock. This is the premise for a technique that isn't new to biology, but is transforming paleopathology, called capture-based molecular enrichment. Kirsten Bos, a molecular paleopathologist, physical anthropologist, and one of the leading experts in finding, recovering, and reconstructing ancient pathogen genomes, guided me through the process.

The ancient pathogens they’re looking for might only make up 0.1 percent of the DNA in the remains, she says, even if at the time of death it was raging throughout the person’s body. After death, bones and teeth become filled with other types of bacteria, like the soil bacteria that Warinner kept finding. Some of these bacteria are also involved in decomposing the body.

The "bait" is made by taking the genome of the pathogen you’re interested in, or a closely related organism, and using a computer to chop up the DNA segments. These pieces are then synthetically made. Additional components that function similar to little magnets are attached to those DNA pieces, and they go fishing. They use this to pull out any DNA fragments in an archeological sample that are a close match to the sequence of the bait. After that, they wash away all the human DNA, the soil DNA, and anything else that didn’t bind, and most of what they’re left with is the DNA of the genome they’re interested in.

“You create a bait, you drop it into your pond, and you pull out the fish that bites onto your bait,” Bos says.

Bos has used this technique recently to solve several other mysteries in archaeology and epidemiology, like definitively determining the cause of the Black Death in medieval Europe. There had still been some debate about what exactly the pathogen was. Most experts thought it was Yersinia pestis, the bacterium that causes plague, but others thought it was a hemorrhagic fever, anthrax, or another unknown virus. The controversy was rooted in how deadly the Black Death was: it traveled all the way across Europe in five years and caused the deaths of an estimated 60 million people. Plague still exists today, and that’s not a pattern we observe in modern outbreaks.

Through collaboration with DeWitte, who mainly studies bones associated with Black Death, and other geneticists and paleopathologists, Bos used baits made from Y. pestis DNA, and was able to find and reconstruct an ancient plague genome. They wondered if it was more virulent in the past, but found instead that the strain circulating during the Black Death is very genetically similar to modern Y. pestis.

Though DeWitte can determine many things from a skeleton, genetic similarities to modern pathogens is out of her reach. “This kind of approach is going to be transformative for this field,” DeWitte says. “These molecular methods are just opening up lines of inquiry that were not possible when we only had the ability to look at the skeletons with the naked eye.”

Using those modern tools, it was time to revisit Teposcolula.

Bos and her collaborators in geneticist Johannes Krause’s group at the Max Planck Institute for the Science of Human History had demonstrated they could isolate and recover ancient DNA, but could they find a pathogen when they didn’t know what they were looking for?

Rather than creating baits from a known genome like Y. pestis, and sequencing what they could fish out, they analyzed everything in ancient DNA extracted from 29 skeletons excavated at Teposcolula–any and all DNA that was present in the archeological sample, be it human, plant, or bacteria. Then, they used a computational tool that matched the DNA fragments they obtained to known bacterial species.

“We got a list of several thousand different bacterial species that were found in the inner tooth chambers of the Teposcolula individuals, and it was a little bit intimidating,” Bos says. “We actually went through each and every sample with a fine-tooth comb.”

In a few teeth they found ancient pieces of Salmonella enterica DNA, the cause of enteric fever. This disease is still a major health threat around the world; it caused an estimated 27 million illnesses in the year 2000 alone. Until now, little was known about its past severity or prevalence. The symptoms of enteric fever (high fever, dehydration, belly pains) were in line with the historical descriptions of the epidemic Warinner uncovered. Four hundred years later, Bos and her collaborators think the bug behind the "great pestilence" has likely been revealed. Their findings were published today in Nature Evolution and Ecology.

Overview of Teposcolula-Yucundaa, showing its location in the Mixteca Alta region of Oaxaca, Mexico (A), and its central administrative area (B), where excavations took place. (C) shows a drawing of individual 35, from which an S. enterica genome was isolated. Image by: Åshild J. Vågene et al. Salmonella enterica genomes from victims of a major 16th century epidemic in Mexico. Nature Ecology & Evolution.

“This was a very exciting example where we could use our advances in computational analysis to find a needle in a giant haystack,” Bos says. “The limits of what we can detect seem to keep changing all the time. We no longer have to have a candidate pathogen in mind; we can now screen skeletal material non-selectively and identify pathogens that were previously hidden from view. There’s a lot of relevant information in the archeological record, and it’s great that we’re now developing techniques to get a better understanding of disease in the past.”

Why should we care that, 400 years ago, a bunch of people died from enteric fever? The reasons are as varied as the many diseases that can kill us.

“One of the things that people don’t really realize is we don’t know how microbes evolve,” Warinner says. “We have such limited understanding of it.”

We’ve only been culturing bacteria for little more than a century, she says. That’s a short time to observe evolution. We know that mutations in mammals arise randomly and at a consistent rate. We can count the mutations between different groups and guess their common ancestries—that’s how we estimate the divergence in species between chimpanzees and humans. But each species has a different so-called molecular clock, and evolves at different paces. For bacteria, it’s extremely hard to determine with short-term experiments done in labs. The only way to learn more is to look at bigger time scales.

“This is what’s so cool,” Warinner says. “With the archaeological record, we’ve gone from only being able to look at bacterial evolution over the last century or so, to now looking at it in any sample that we can radiocarbon date.”

Many of these historic diseases were catastrophic; they changed human history, and collapsed cities and societies. And in some cases, we have no idea what they were. As we start to uncover the pathogens, we can start to ask: are they different or the same as the ones that currently exist? “Once we find them, we often find out that they’re the ancestors of pathogens that still cause disease today,” Warinner says. "The question is, were these bacteria more virulent in the past? How do pathogens evolve? Do they evolve towards less virulence over time?”

Our past with disease is intimately connected to our future. The introduction of antibiotics is largely the reason why there aren’t as devastating outbreaks of enteric fever or TB anymore. But with antibiotic resistance on the rise, and no new antibiotics being discovered, there's a lot of knowledge to be mined from ancient epidemics to try and prevent them from happening again.

“One of the big questions is how does an epidemic take place?” Warinner says. “How does it actually work? How does it actually spread? With things like plague, it came in and hit, went away for a while, and then it came back. Is it the same strain that comes back? Did it just hide out in some other population for which we have no historical records, possibly an animal reservoir? Or is it completely reintroduced from a very distant population again? How do these plagues come about and how do they hide for periods of time, only to reemerge?"

Warinner thinks that Ebola is a modern example we can turn to with similar questions questions, like: Where is Ebola coming from? What’s its reservoir? Why does it keep re-emerging? "We can look at these historical epidemics and start to tie them together," she says.

Archaeological excavations at the site of Teposcolula. Photo by: Christina Warinner. Image courtesy of the Teposcolula-Yucundaa Archaeological Project.

There are a lot of questions, and paleopathology is only going to grow in its ability to answer them. Warinner is currently researching ancient DNA left behind in calcified dental plaque and hopes to uncover information about more common diseases like periodontal disease and the makeup of ancient microbiomes. Bos is looking into the genetic diversity in plague after the Black Death, and the diversity of plague that was present during the Neolithic period.

There seems to be a new, cool ancient DNA finding every week on ancient pathogens. A group reconstructed smallpox virus from a 17th century mummy and bacterial microfossils in a Byzantine skeleton that seem related to Staphylococcus aureus. Another paper isolated a 5,300-year-old Helicobacter pylori genome from a European Copper Age glacier mummy.

But whether it’s through DNA, or what can be seen with human eyes, the field of paleopathology continues to be multidisciplinary in nature. Paleopathology only works if its researchers continue to mine historical archives, collaborate with historians and archaeologists, and get the full historical context of the samples they’re studying.

“Ancient DNA is a tool,” Bos says. “It’s an important tool but it’s really something we have to do in conjunction with museum curators, with archaeologists, with historians, with skeletal biologists, and with computational experts.”

This sentiment reveals another aspect of paleopathology I noticed in my reporting: a deep sense of respect for those who've died.

When I looked at Warinner’s original dissertation on the Mexican gravesite, she told me that the excavators weren't comfortable with me publishing pictures of any of the skeletons from the site. Another paleopathologist I spoke to, when I ask if they have nicknames for their skeletons (thinking of the famous "Lucy" skeleton), tells me that’s not an acceptable practice in the field. “Any skeleton that had a name in life, we don’t assign it another name,” she says.

There’s an intimate recognition from this work that life leaves a trace, whether it be in the shape of our skeletons or remnants of DNA left behind. And with that comes the humbling realization that the history of human life is not isolated, but rather a history of our interaction with microorganisms. The story of ancient man is one of his relationship with disease, and our coevolution with these bacteria and viruses.

Every time DeWitte goes to London, to work with Black Death bodies being held at a museum, she takes the time to visit their original gravesite. It's now topped with modern buildings, down the street from the Tower of London. “There’s still thousands of people who are buried there who were never excavated,” DeWitte says. “They’re still under the streets, under the buildings. I go to the site, but not to actually see the skeletons in place, I go and pay homage.”

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