Hunting for Germs in an Ancient Graveyard

Researchers hope the discovery of a mass cholera burial pit will allow them to find and sequence the DNA of the bacteria that killed its inhabitants.

Field School Pozzeveri/The Ohio State University/University of Pisa

The third cholera pandemic in recorded history—and the deadliest—began in India sometime in the mid-1800s, making its way across Asia, Europe, Africa, and the Americas throughout the 1850s. By the time it subsided in 1860, the pandemic had killed more than a million people around the world—which, luckily for Clark Larsen, included the victims buried in the mass grave at Badia Pozzeveri, an ancient church in the small town of Altopascio, Italy.

For the past three years, Larsen, a professor of anthropology at the Ohio State University, has helped to lead a team of archaeologists as they sift through a corner of the cemetery known as “Area 2000,” searching for clues to the evolution of the disease.

For six weeks each summer, the Field School at Badia Pozzeveri, a collaboration between Ohio State and the University of Pisa, gives students a chance to excavate the site’s human remains, which stretch back as far as the bubonic-plague outbreak that devastated Europe in the 14th century. The discovery of the grave—which Larsen estimates contains “a couple hundred” bodies—was a happy accident during a dig in the summer of 2012, as Science magazine reported the following year; old records confirmed that the victims had died of cholera when the epidemic swept through Tuscany in 1855.

It also, the team quickly realized, presented them with a rare opportunity. “The wonderful thing about the cholera victims—not so wonderful for them, of course—is the fact that a number of them were buried encased in liquid lime,” Larsen said. Over the centuries, the lime, once used to mask the smell of the bodies as they decomposed, hardened into a shell than enveloped the skeletons and the soil that surrounded them.

In most cases, bones can provide only limited insight into the diseases that sickened their owners. “The problem with skeletons is that they only display a record of chronic diseases. The person has to be alive long enough for the disease to have an impression on bone,” Larsen explained. “The diseases we can’t look at [in skeletons] are those that kill you right away, like plague and smallpox and cholera.” But the soil around these particular bones, trapped in its lime casing from the beginning, may contain a different kind of information: the genetic material of Vibrio cholerae, the bacterium that causes cholera. If they could find the DNA, the researchers reasoned, they may be able to reconstruct the genome of the 19th-century strain.

“Understanding how pathogens evolve through time gives us a picture of the evolutionary history that we can’t really glean from just modern-day sequence data,” said Hendrick Poinar, a professor of genetics and anthropology at McMaster University and the lead researcher on genetic screening for the project. In addition to scanning soil from the gravesite, Poinar says, he also analyzes the bodies’ teeth for genetic material—altogether, he says, “we look for [the DNA from] over 3,000 different bacterial, viral, and fungal species” in the samples that pass through his lab.

Last year, Poinar and his colleagues published an article in the journal The Lancet Infectious Diseases outlining a similar project, unrelated to Larsen and the Field School, with the remains of bodies from the Aschheim-Bajuwarenring cemetery in Bavaria, Germany. Using DNA taken from the teeth of the victims of the Plague of Justinian, a bubonic-plague epidemic that lasted from the sixth to eighth centuries, the researchers were able to sequence the genome of Y pestis, the bacterium that caused the epidemic, enough to determine that it was a different strain from the outbreaks that would take place centuries later (“the Black Death” in the 1300s-1600s, and a third outbreak in the 1800s and 1900s).

Field School students with a skeleton in the Badia
Pozzeveri cemetery (Field School Pozzeveri/The
Ohio State University/University of Pisa)

“If you have a site that contains remains and that level of continuity over six, seven, 800 years, then there is this distinct possibility to look at population health and pathogen burden over time,” Poinar said. “That’s really contributing tremendously to knowledge of our evolutionary fight with pathogens.”

As of now, the team at Badia Pozzeveri hasn’t yet found cholera DNA, Larsen says, though they have found “human-associated DNA” of gut bacteria and other organisms that make their home in the human body, a fact that makes them optimistic about eventually hitting upon their target—and, in turn, about reaching a better understanding of the disease that still sickens between 3 and 5 million people worldwide each year. (Cholera, which kills by dehydration, is easily treatable with oral rehydration salts; even so, it still causes an estimated 100,000 to 120,000 deaths annually.)

“We know what the genome of cholera looks like today. We don’t know what it looked like in 1855,” Larsen said. “If we can track that organism, we can have a much better sense of how it may evolve in the future. And knowing how it evolves, we can get a better handle on how to control it.”