Urine and fecal residue encrusted on the inside of ancient Roman chamber pots unearthed in Bulgaria has revealed the world's oldest known evidence of humans infected with the Cryptosporidium parasite, which causes acute gastrointestinal distress.

In the first century, the Romans established a province called Moesia Inferior in the Balkan Peninsula, which includes the modern country of Bulgaria. Roman legions were tasked with defending the imperial border from the Goths, primarily from a fortress called Novae (near present-day Svishtov) and a town known as Marcianopolis (modern-day Devnya). While excavating at Novae and Marcianopolis in Bulgaria, archaeologists recovered four chamber pots, whose long-dried contents have revealed new information about health and disease in the Roman Empire.

Using ELISA (short for "enzyme-linked immunosorbent assay") ‪—‬ a laboratory test that can detect bacteria, parasites and viruses in a sample of bodily fluid ‪—‬ the researchers identified three pathogens in the chamber pot samples: the protozoan Entamoeba histolytica, the parasite Cryptosporidium parvum, and the tapeworm Taenia. All three pathogens infect the human gut and can cause gastrointestinal distress, including diarrhea and stomach pain.

Previous ancient-parasite studies have shown that Roman soldiers on the frontiers of the empire dealt with intestinal worms and the parasite Giardia, as did people living in the city of Rome. However, the new study has identified Cryptosporidium for the first time in an ancient Roman context, and it is the earliest known evidence of human Cryptosporidium infection in the world.

"The evidence of Cryptosporidium comes from two separate chamber pots from Novae," study first author Elena Klenina, a historian at Adam Mickiewicz University in Poland, told Live Science in an email. "The parasite's presence across distinct contexts suggests the infection may have been relatively widespread within that community."

C. parvum (often dubbed Crypto, for short) is a parasite that infects dozens of species of domesticated and wild animals, the researchers wrote in the study. When those infected animals contaminate soil or water, the infection can spread to humans. But because Crypto generally causes mild symptoms, the first human cases of the disease were not identified until 1976. Since then, researchers have been investigating the origin of this organism.

Archaeologists in Mexico found Crypto in preserved human feces dated to around A.D. 700, which led some experts to propose that the parasite originated in the Americas. But because the remains of a 5,000-year-old goat (Myotragus balearicus) from the western Mediterranean tested positive for Crypto, Klenina and colleagues suspect that it may have first started infecting humans in Europe rather than in the Americas.

It's unclear how the Romans living at Novae picked up the parasite, but the researchers think the infected individuals might have been drinking contaminated water supplied by an aqueduct in the rural countryside. When infected with any of the three pathogens the researchers identified, the ancient Romans may have dealt with severe diarrhea and thus had to use chamber pots in the middle of the night rather than the more sanitary public latrines and bathhouses that were open during the day.

Analyzing ancient bodily fluids is important for understanding health and disease in the Roman Empire, the researchers noted in the study, and chamber pots present a wealth of information on these topics.

"This type of research poses no significant health risk to archaeologists," Klenina said. "The biological material we analyze is extremely old and typically no longer viable or infectious."

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