The paper in Nature Ecology & Evolution is here: http://go.nature.com/2mENZSa

After the arrival of Europeans in the New World dozens of epidemics devastated its indigenous inhabitants. One such epidemic outbreak was the 1545-1550 ‘cocoliztli’ epidemic (cocoliztli means 'pestilence' in Nahuatl) that heavily affected Mexico and Guatemala, the pathogenic cause of which has been debated for over a century.

Teposcolula-Yucundaa, an ancient Mixtec town situated in the Oaxaca region of southern Mexico, contains the only known cemetery linked to this epidemic. Archaeologists estimate that more than 800 individuals are buried here, in what was the town’s main square or “Grand Plaza”, many of whom were victims of the cocoliztli. Shortly after the epidemic, Teposcolula-Yucundaa was abandoned and the town was relocated. The epidemic cemetery was left untouched until a team of archaeologists, led by Nelly Robles García of the Mexican National Institute of Anthropology and History (INAH), excavated the site between 2004 and 2010. Also located at this site is a second cemetery in the town’s churchyard that contains individuals who died prior to European contact in the region.

In 2013 we formed a collaborative team comprising researchers from the Max Planck Institute for the Science of Human History, Harvard University, INAH, and the University of Tübingen with the aim of using ancient DNA to shed light on the question:

Can ancient DNA help us to determine the pathogenic cause of the 1545-1550 cocoliztli epidemic at Teposcolula-Yucundaa? 

Visual clues as to what caused this epidemic were absent from the excavated skeletons. This was perhaps not surprising, as only a handful of infectious diseases leave visibly distinctive lesions on bone – the most common biological material to survive in the archaeological record. Although historical documents by both indigenous and European authors describe first-hand accounts of the disease symptoms, historians have not been able to reach a consensus on its pathogenic cause. The historical evidence alone suggests that the cause of the epidemic was some sort of malady that produced a body rash and could lead to bleeding from facial orifices. However, these symptoms are not specific to one particular disease. Over the years suggestions have accumulated, including: typhus, measles, enteric (typhoid) fever, viral haemorrhagic fever and pneumonic plague, to name a few. 

We collected teeth from 24 individuals from the epidemic Grand Plaza cemetery and 5 from the churchyard cemetery and brought them to the University of Tübingen, where the tooth pulp-chambers were sampled for DNA extraction. Based on previous experience we knew that our extracts would contain DNA from many different sources, including from the bacterial, fungal, viral, plant, insect and animal communities present in the soil in which the individuals were buried. This complex environmental background greatly confounds our attempts to find ancient pathogen DNA, which is usually preserved in very low abundance. However, the interiors of teeth are shielded to some degree from the burial environment by their hard enamel surface. Thus, we determined that teeth would be our best bet to detect traces of DNA from any blood-borne pathogens infecting the individuals at their time of death.

We needed a strategy that would handle two issues: the fact that we would be dealing with DNA from a large number of environmental sources that could hinder our ability to find the pathogen, and the fact that we did not know which pathogen we were looking for. Therefore, approaches targeting one or a selected set of pathogens were out of the question. We needed a broad scale screening approach – the more unbiased and broader, the better.   Like others before us who have faced similar predicaments, we chose a metagenomic approach and made use of a novel computational tool, termed MALT (MEGAN ALignment Tool), developed by Daniel Huson at the University of Tübingen and adapted for our screening purposes by Alexander Herbig (available to download here). MALT works by placing all sequenced DNA fragments at the taxonomic position of best fit by comparing every read to every possible position in every genome included in your database of choice. Ours contained all complete bacterial genomes available through NCBI RefSeq, providing us with a screening approach for bacterial pathogens that was both of limited bias and would also categorize and filter out DNA from soil-dwelling species that had made it into our data.

Listed in our results, amongst hundreds of environmental bacterial species, we discovered Salmonella enterica subsp. enterica serovar Paratyphi C, a cause of enteric fever, as a potential candidate pathogen in ten of our individuals from the epidemic cemetery. This was unexpected as S. Paratyphi C rarely causes human infection today. However, further experiments and analyses confirmed our S. Paratyphi C finding in all ten individuals from the epidemic cemetery. For five individuals, we were able to reconstruct complete S. Paratyphi C genomes. The pre-contact samples, and a soil sample included as a measure of the environmental background, did not test positive for S. Paratyphi C, indicating that S. Paratyphi C was not an environmental contaminant, and was not present in the pre-contact community.

Now we are able to say, through direct ancient DNA evidence, that S. Paratyphi C was at least one of the pathogens circulating in the indigenous population at Teposcolula-Yucundaa during the 1545-1550 cocoliztli epidemic. Whether enteric fever affected people in other parts of Mexico or Guatemala during this epidemic remains to be seen. Being able to contribute to a longstanding historical epidemic mystery via ancient DNA, as well as the proof of principal that we can indeed look for ancient pathogens without having to specify a target organism, has made this an extremely exciting project. We hope others will use these methods to tackle similar ancient and modern mysteries in the future.