Clarifying prehistoric parasitism from a complementary morphological and molecular approach

Abstract

This paper reports an approach to the identification of prehistoric parasitic infection, which integrates traditional morphological methods with molecular methods. The approach includes the strengths of each method while mitigating the limitations. Demonstrating the efficacy of this approach, we provide a case study from a 1,400 year old desiccated fecal sample from La Cueva de los Muertos Chiquitos, archaeological site, near Rio Zape, Durango, Mexico. Traditionally prepared microscope slides were processed via microscopy and tentative ascarids were identified. Information regarding the parasites’ developmental stage was recorded. DNA was then extracted directly from the slide material. From this DNA extract, a small segment of the 18S ribosomal RNA gene variant that is specific to Ascaris, and its phylogenetically close relatives, was targeted for PCR amplification and sequencing. Phylogenetic analysis of the DNA sequence best matched a member of physalopterids, rather than ascarids, with a single exception of a match to Contracaecum spiculigerum. Subsequent extractions, amplifications and sequencing of the original rehydrated coprolite material confirmed these results. The C. spiculigerum sequence represented a phylogenetic anomaly and subsequent analysis determined the sequence was an error in the BLAST database, likely attributable to misidentification of juvenile specimens prior to sequencing and submission. Physaloptera are a difficult genus to identify morphologically and can carry major health burdens. They may be underreported in humans, in part, because of morphological similarities to the more common human parasites belonging to ascarids. We conclude that integrating traditional morphological methods with molecular methods can help resolve this issue, in both contemporary and prehistoric populations.

DNA Typing of Ancient Parasite Eggs from Environmental Samples Identifies Human and Animal Worm Infections in Viking-Age Settlement

“…eggs were morphologically observed in 3 contexts but only positively identified in the K283 sample using PCR and sequencing data. Combining morphological and molecular methods may therefore reduce the risk of misclassification (Cleeland et al, 2013). The K283 sample originates from a field that is separated from the latrine by a wooden fence.…”

“…Loreille et al (2001) were the first to report on ancient DNA (aDNA) isolated from parasite egg remains. Since then, aDNA analysis has increasingly been applied to paleoparasitological studies (I˜niguez et al, 2003;Leles et al, 2008Leles et al, , 2014Botella et al, 2010;Oh, Seo, Chai et al, 2010;Oh, Seo, Lim et al, 2010;Cleeland et al, 2013;Shin et al, 2013;My skova´et al, 2014). aDNA analysis has, for example, been used to reclassify an egg finding, as shown by Cleeland et al (2013); they identified Physaloptera sp.…”

Food, parasites, and epidemiological transitions: A broad perspective

“…Zoonotic parasites normally exist in animals but can be transmitted to humans. New studies that compare parasite diversity show that zoonotic parasites were taxonomically more diverse and with highly varied life cycles compared to human specific parasites in New World prehistoric agriculturalists (Jiménez et al, 2012;Cleeland et al, 2013). In other words, Native Americans exposed themselves to a greater variety of zoonotic parasites with a greater variety of life cycles than human-specific parasites.…”

A Retrospective Examination of Paleoparasitology and Its Establishment in the Journal of Parasitology

“…Recently published papers by Wood et al (2013) and Cleeland et al (2013) illustrated the complementary nature of combining molecular analysis of aDNA with the morphologic study of diagnostic parasite products in preserved fecal samples. Wood et al (2013) examined feces of an extinct Moa (Aves: Dinorthiformes) from New Zealand.…”

“…Parasite aDNA sequences of Cryptosporidium and Eimeriorina indicated the presence of infection in the absence of morphologic evidence. Cleeland et al (2013) used aDNA sequencing in an effort to confirm the putative identification of ascarid eggs in a prehistoric canine fecal sample. The resulting analysis demonstrated that the eggs were likely Physaloptera sp., a spiruid nematode parasite that is relatively common, although difficult to diagnose, in canine hosts.…”