Centuries ago, between approximately 725 A.D. and 920 A.D., numerous individuals defecated within a cave situated north of Durango, Mexico. The cave’s arid environment preserved these ancient fecal samples remarkably well, allowing archaeologists excavating in the 1950s to discover them in excellent condition. These weathered, dry, and fibrous stool samples have since provided scientists with crucial insights into the diets of these ancient populations and the organisms residing within their digestive systems.
These cave deposits, often referred to as paleofeces, have since been extensively circulated among different research laboratories for analysis. A global collaborative team in 2021 specifically investigated these samples to identify the microbial composition of the individuals’ gut microbiomes.
More recently, in a study published in PLoS One, a distinct group of researchers re-examined DNA extracted from 10 of these ancient fecal specimens. Their findings largely corroborated a previous discovery: the individuals who produced these samples harbored a diverse array of parasites.
Hosting Parasitic Organisms
Drew Capone, the study’s lead author and an environmental microbiologist at Indiana University, typically examines much more recent fecal matter. His research focuses on the health implications of sanitation. Capone elaborates: ‘Our studies investigate how feces enter the environment, its distribution, how infrastructure can prevent its environmental presence, and its subsequent pediatric health effects.’
Capone and his team sought to apply modern fecal pathogen detection techniques to these ancient samples. These methodologies involve screening sample DNA for specific genetic markers indicative of parasites, such as pinworms, and various bacterial pathogens.
Extracting DNA from the paleofeces proved more challenging than anticipated. Capone explained, ‘We were unable to simply break off pieces; we had to grind these ancient fecal samples into a powder.’ The team proceeded with the analysis for DNA matches, yielding results that indicated the presence of multiple pathogens, including pinworms, the protozoan parasite Giardia, and various pathogenic bacteria.
Numerous samples tested positive for multiple organisms. Capone notes that encountering such a high pathogen load is typical in areas with inadequate sanitation, leading him to infer that the ancient populations responsible for these deposits likely faced similar conditions.
Significance of Methodological Choices
Conversely, Kirsten Bos and Alexander Hubener, ancient DNA specialists at the Max Planck Institute for Evolutionary Anthropology, highlight why most labs now avoid these older procedures. DNA degrades over millennia, becoming frayed and fragmented. The technique employed in the PLoS ONE paper is less rigorous in verifying the antiquity of DNA, making it difficult to differentiate true ancient DNA from incidental modern contamination. Specialized ancient DNA laboratories, in contrast, utilize high-tech clean rooms to prevent contamination and employ next-generation sequencing methods specifically optimized for such delicate material.
Furthermore, most labs confirm the age of DNA fragments by examining their ends for characteristic degradation patterns. Bos explains that the PLoS ONE paper’s technique ‘doesn’t readily allow for determining if the chemical modifications typical of ancient DNA have occurred.’
Capone counters that many of the target organisms have limited survival outside the human gut, suggesting a low probability of false positives from modern DNA contamination acquired during the samples’ transport. He also notes that specialized ancient DNA analysis is expensive, making the older technique more broadly accessible.
Hubener, a member of the team responsible for the 2021 analysis of these cave samples, expresses skepticism regarding the bacterial matches, as these are notoriously difficult to ascertain in ancient specimens using this methodology. However, based on his team’s earlier findings and established parasite biology, he considers the results concerning larger parasites, such as worms, to be more credible. ‘That, for me, is plausible,’ Hubener stated.
Bos suggests that a particularly valuable approach would have been to apply both the older and newer techniques to the same samples. This comparative analysis would clarify which findings from the older methods are consistently replicated by the more rigorous, modern procedures.
She concludes, ‘That would have represented a very effective path forward.’