Detection of Clostridium sporogenes in a Roman-era cattle mass grave at Vilauba

In the ancient Roman world, cattle played an integral role in daily agricultural tasks, providing the means necessary to plow fields, mill grains, and transport goods. The research presented here deals with the remains of 14 cattle discovered in a mass grave at the Roman villa of Vilauba in Catalonia, Spain. According to the archaeological record, it can be ruled out that the animals were slaughtered for consumption, banqueting, or sacrificial purposes. By investigating the metagenomic sequences generated from the bovine remains, we identified in three individuals a group I Clostridium strain, phylogenetically related to known producers of botulinum neurotoxins – suggesting that the Vilauba strain may have had toxigenic potential. Moreover, we discovered a Mycolicibacterium species phylogenetically related to known opportunistic pathogens. While no definitive conclusions can be drawn about disease, the phylogenetic placement of these taxa and the detection of Clostridium virulence-associated genes suggest a possible role beyond postmortem contamination. Collectively, these findings draw attention to atypical bacterial species, such as C. sporogenes, which are often overlooked in palaeogenomic studies due to their ambiguous status as environmental microbes, commensals, or potential pathogens. Their detection in animal remains highlights that they may represent a blind spot in our current understanding of livestock health. More broadly, this study underscores the current complexity of investigating such taxa and emphasizes the need for novel methods to disentangle the roles of these bacterial species.