Leishmaniaisis affects millions of people worldwide today, resulting in disfiguring lesions and fatal complications. The World Health Organization regards leishmaniasis as a "Neglected Tropical Disease," endemic to poverty-stricken countries and rising in incidence by nearly two million cases annually, but lacking effective treatment or prevention. Its worldwide geographic distribution and its impact on human health and well-being make leishmaniasis one of the most significant of the neglected diseases. Leishmaniasis is not, however, solely a contemporary affliction. The fields of bioarchaeology, ethnohistory and genetics provide evidence to support that leishmaniasis has been a burden to humanity long before recorded history. Nevertheless, the origins and movement of the parasite as it evolved and dispersed throughout the globe remains poorly understood.
This project by doctoral student Kelly Harkins (Arizona State University), under the supervision of Drs. Anne Stone and Jane Buikstra, will use molecular and bioarchaeological techniques to obtain DNA from a sample of poorly understood contemporary Leishmania strains and archaeologically recovered human remains. These data will be used to test hypotheses that address the evolutionary history of the parasite Leishmania and the emergence of human leishmaniasis in the New World, both of which are the source of ongoing debate. Specifically, this project proposes to characterize strains from the earliest known skeletal cases of New World leishmaniasis in the Andes, to explore the relationship of these early strains to modern strains found throughout the globe today, and with additional data from archaeological and ethnohistoric contexts, to address the nature of the disease's movement in prehistoric South American populations. This project will publicly disseminate new genomic sequence data, essential for understanding pathogen biology and also a powerful tool for those fields and researchers working to improve global public health.
The major goals of the project are to investigate the unresolved evolutionary history and the origins of the parasite genus, Leishmania, and the resulting neglected human disease, leishmaniasis. The investigation was designed to incorporate a molecular and bioarchaeological approach by focusing on both the genetics and phylogenetics of the modern parasite as well as what understanding we could be drawn from prehistoric skeletal evidence of the disease in the pre-contact New World. As such the project was separated into a modern and ancient component. Throughout the course of the project we were able to complete a number of objectives. Pilot data informed our modern sampling strategy, which focused on Leishmania species that had few sequence data publicly available. We acquired genomic DNA samples from 21 Leishmania parasite isolates grown in culture in strain banks located at Yale School of Public Health and the University of Sao Paolo. Thirteen of these isolates were chosen for whole genome shotgun (WGS) sequencing on three lanes of a HiSeq2000 at the University of Arizona Genome Core. The large size of our multi-genome dataset, submitted to publicly available sequence databases (BioProject PRJNA267749), led us to collaborate with bioinformaticians at our institution to develop a tool for phylogenomic reconstruction specifically from our WGS data, which, like for many other non-model organisms, do not have suitable reference genomes. This tool is available open-source and has received interest in the research community following dissemination of our preliminary results at international and national conferences. Also of growing interest is the use of this tool to exploit the available diversity within a broad "next-generation" dataset to inform experimental design for targeted approaches. We used this method to design a targeted enrichment experiment (MycroArray MyBaits) to capture over 200,000 variable DNA sites throughout the Leishmania genome as well as 42 known genes, which were then used to test for the presence of leishmaniasis in archaeological remains. The ancient component of the project relied upon previous research that first identified leishmaniasis in human remains from pre-Columbian sites (c. AD 800-1200) in San Pedro de Atacama (SPA), Chile, using paleopathological and molecular methods. In hopes of attaining more information than presence/absence, we sampled additional bone from these individuals and surveyed human remains from six cemetery sites, housed at the RP Gustavo Le Paige Archaeological Museum in SPA. Samples from the original 2009 study and three additional samples were exported to Arizona State University for paleogenetic analyses. Skeletal collections housed at the MAANHP in Lima, Peru, were also evaluated, but no additional samples were discovered. In our dedicated cleanroom at the Stone Molecular Anthropology Lab at ASU, we extracted DNA from eight skeletal remains and tested for the presence of multicopy kinetoplastid DNA via PCR and quantitative PCR. We could not confirm previous analyses that reported the presence of Leishmania kDNA. Sequencing libraries were generated from each sample. Two enrichment experiments were performed with the libraries and sequenced on a Illumina MiSeq 2000. Ultimately, however, we were not able to confirm the previous analyses that found the presence of Leishmania DNA in skeletal remains from Coyo Oriente and Tchecar T?mulo Sur, nor in our newly acquired samples. However, the sequencing libraries produced as a part of this project are a renewable resource and are permanently stored at -80º C at ASU, available for future work and collaboration with other scholars. Additionally, remaining bone is still available and can be used with even newer extraction and library methods that are shown to be successful with very poorly preserved samples by improving retention of the smallest molecules during library preparation. We have provided the broader research community with access to our whole genome shotgun sequencing data on the Sequence Read Archive (released to the public in December 2014). Outreach efforts to middle and high school students focuses on how we can use the past to learn about the present, and how our interdisciplinary research combining archaeology and genetics works to elucidate the evolution of human disease. One such outreach effort can be found at Ask a Biologist educational science resource (askabiologist.asu.edu), called Indiana Jane: Uncovering Mysteries of Ancient Disease. In many ways, we believe this project has prepared the way for future research and informed the public, young and old, as well as interested research communities.
