Reconstruction of ancient microbial genomes from the human gut.

Loss of gut microbial diversity1-6 in industrial populations is associated with chronic diseases7, underscoring the importance of studying our ancestral gut microbiome. However, relatively little is known about the composition of pre-industrial gut microbiomes. Here we performed a large-scale de novo assembly of microbial genomes from palaeofaeces. From eight authenticated human palaeofaeces samples (1,000-2,000 years old) with well-preserved DNA from southwestern USA and Mexico, we reconstructed 498 medium- and high-quality microbial genomes. Among the 181 genomes with the strongest evidence of being ancient and of human gut origin, 39% represent previously undescribed species-level genome bins. Tip dating suggests an approximate diversification timeline for the key human symbiont Methanobrevibacter smithii. In comparison to 789 present-day human gut microbiome samples from eight countries, the palaeofaeces samples are more similar to non-industrialized than industrialized human gut microbiomes. Functional profiling of the palaeofaeces samples reveals a markedly lower abundance of antibiotic-resistance and mucin-degrading genes, as well as enrichment of mobile genetic elements relative to industrial gut microbiomes. This study facilitates the discovery and characterization of previously undescribed gut microorganisms from ancient microbiomes and the investigation of the evolutionary history of the human gut microbiota through genome reconstruction from palaeofaeces.

Comparison of extraction methods for recovering ancient microbial DNA from paleofeces.

OBJECTIVES: Paleofeces are valuable to archeologists and evolutionary biologists for their potential to yield health, dietary, and host information. As a rich source of preserved biomolecules from host-associated microorganisms, they can also provide insights into the recent evolution and changing ecology of the gut microbiome. However, there is currently no standard method for DNA extraction from paleofeces, which combine the dual challenges of complex biological composition and degraded DNA. Due to the scarcity and relatively poor preservation of paleofeces when compared with other archeological remains, it is important to use efficient methods that maximize ancient DNA (aDNA) recovery while also minimizing downstream taxonomic biases. METHODS: In this study, we use shotgun metagenomics to systematically compare the performance of five DNA extraction methods on a set of well-preserved human and dog paleofeces from Mexico (~1,300 BP). RESULTS: Our results show that all tested DNA extraction methods yield a consistent microbial taxonomic profile, but that methods optimized for ancient samples recover significantly more DNA. CONCLUSIONS: These results show promise for future studies that seek to explore the evolution of the human gut microbiome by comparing aDNA data with those generated in modern studies.

Pinworm Infection at Salmon Ruins and Aztec Ruins: Relation to Pueblo III Regional Violence.

The study of coprolites has been a theme of archaeology in the American Southwest. A feature of archaeoparasitology on the Colorado Plateau is the ubiquity of pinworm infection. As a crowd parasite, this ubiquity signals varying concentrations of populations. Our recent analysis of coprolite deposits from 2 sites revealed the highest prevalence of infection ever recorded for the region. For Salmon Ruins, the deposits date from AD 1140 to 1280. For Aztec Ruins, the samples can be dated by artifact association between AD 1182-1253. Both sites can be placed in the Ancestral Pueblo III occupation (AD 1100-1300), which included a period of cultural stress associated with warfare. Although neither of these sites show evidence of warfare, they are typical of large, defensible towns that survived this time of threat by virtue of large populations in stonewalled villages with easily accessible water. We hypothesize that the concentration of large numbers of people promoted pinworm infection and, therefore, explains the phenomenal levels of infection at these sites.

Confusing a Pollen Grain with a Parasite Egg: an Appraisal of "Paleoparasitological Evidence of Pinworm (Enterobius Vermicularis) Infection in a Female Adolescent Residing in Ancient Tehran".

There is often the risk of confusing pollen grains with helminth eggs from archaeological sites. Thousands to millions of pollen grains can be recovered from archaeological burial sediments that represent past ritual, medication and environment. Some pollen grain types can be similar to parasite eggs. Such a confusion is represented by the diagnosis of enterobiasis in ancient Iran. The authors of this study confused a joint-pine (Ephedra spp.) pollen grain with a pinworm egg. This paper describes the specific Ephedra pollen morphology that can be confused with pinworm eggs.

Autologous Bone Flap Resorption Years After Subtemporal Craniotomy.

In Spring of 2012, the partially undressed and skeletonized remains of a homeless adult Hispanic male was found in a fairly open wooded area in Nebraska. The remains showed evidence of extensive pathologies, which included healed traumas and surgeries. Examination of the decedent's medical records revealed that he had a history of kidney and liver problems, alcohol abuse, several traumas including a major head injury that necessitated a craniotomy, and radiological features of neurocysticercosis. The autologous bone flap, which was replaced after the craniotomy, had resorbed significantly away from the edges of the injury. Death was variously attributed to craniocerebral injury with hypothermia sequel to blunt force trauma most probably due to a fall. The manner of death was ruled as an accident. This study makes use of the forensic evidence and medical records to examine the possibility that the unique combination of failed cranioplasty, hypothermia, and neurocysticercosis may have contributed to the victim's death. Other potential causes of death are considered.

Evidence for maize (Zea mays) in the Late Archaic (3000-1800 B.C.) in the Norte Chico region of Peru.

For more than 40 y, there has been an active discussion over the presence and economic importance of maize (Zea mays) during the Late Archaic period (3000-1800 B.C.) in ancient Peru. The evidence for Late Archaic maize has been limited, leading to the interpretation that it was present but used primarily for ceremonial purposes. Archaeological testing at a number of sites in the Norte Chico region of the north central coast provides a broad range of empirical data on the production, processing, and consumption of maize. New data drawn from coprolites, pollen records, and stone tool residues, combined with 126 radiocarbon dates, demonstrate that maize was widely grown, intensively processed, and constituted a primary component of the diet throughout the period from 3000 to 1800 B.C.

Prehistoric Pathoecology as Represented by Parasites of a Mummy from the Peruaçu Valley, Brazil.

Paleopathologists have begun exploring the pathoecology of parasitic diseases in relation to diet and environment. We are summarizing the parasitological findings from a mummy in the site of Lapa do Boquete, a Brazilian cave in the state of Minas Gerais. These findings in context of the archaeology of the site provided insights into the pathoecology of disease transmission in cave and rockshelter environments. We are presenting a description of the site followed by the evidence of hookworm, intestinal fluke, and Trypanosoma infection with resulting Chagas disease in the mummy discovered in the cave. These findings are used to reconstruct the transmission ecology of the site.

Assessing the Archaeoparasitological Potential of Quids As a Source Material for Immunodiagnostic Analyses.

In the present study, quids from La Cueva de los Muertos Chiquitos (CMC) were subjected to ELISA tests for 2 protozoan parasites, Toxoplasma gondii (n=45) and Trypanosoma cruzi (n=43). The people who occupied CMC, the Loma San Gabriel, lived throughout much of present-day Durango and Zacatecas in Mexico. The known pathoecology of these people puts them into at-risk categories for the transmission of T. gondii and T. cruzi. Human antibodies created in response to these 2 parasites can be detected in modern saliva using ELISA kits intended for use with human serum. For these reasons, quids were reconstituted and subjected to ELISA testing. All test wells yielded negative results. These results could be a factor of improper methods because there is no precedence for this work in the existing literature. The results could equally be a simple matter of parasite absence among those people who occupied CMC. A final consideration is the taphonomy of human antibodies and whether or not ELISA is a sufficient method for recovering antibodies from archaeological contexts. An additional ELISA test targeting secretory IgA (sIgA) was conducted to further examine the failure to detect parasite-induced antibodies from quids. Herein, the methods used for quid preparation and ELISA procedures are described so that they can be further developed by future researchers. The results are discussed in light of the potential future of quid analysis.

Temporal and Spatial Distribution of Enterobius vermicularis (Nematoda: Oxyuridae) in the Prehistoric Americas.

Investigations of Enterobius sp. infection in prehistory have produced a body of data that can be used to evaluate the geographic distribution of infection through time in the Americas. Regional variations in prevalence are evident. In North America, 119 pinworm positive samples were found in 1,112 samples from 28 sites with a prevalence of 10.7%. Almost all of the positive samples came from agricultural sites. From Brazil, 0 pinworm positive samples were found in 325 samples from 7 sites. For the Andes region, 22 pinworm positive samples were found in 411 samples from 26 sites for a prevalence of 5.3%. Detailed analyses of these data defined several trends. First, preagricultural sites less frequently show evidence of infection compared to agricultural populations. This is especially clear in the data from North America, but is also evident in the data from South America. Second, there is an apparent relationship between the commonality of pinworms in coprolites and the manner of constructing villages. These analyses show that ancient parasitism has substantial value in documenting the range of human behaviors that influence parasitic infections.

Paleoparasitological studies on mummies of the Joseon Dynasty, Korea.

Paleoparasitology is the application of conventional or molecular investigative techniques to archeological samples in order to reveal parasitic infection patterns among past populations. Although pioneering studies already have reported key paleoparasitological findings around the world, the same sorts of studies had not, until very recently, been conducted in sufficient numbers in Korea. Mummified remains of individuals dating to the Korean Joseon Dynasty actually have proved very meaningful to concerned researchers, owing particularly to their superb preservation status, which makes them ideal subjects for paleoparasitological studies. Over the past several years, our study series on Korean mummies has yielded very pertinent data on parasitic infection patterns prevailing among certain Joseon Dynasty populations. In this short review, we summarized the findings and achievements of our recent paleoparasitological examinations of Joseon mummies and discussed about the prospects for future research in this vein.

HEAD LOUSE PALEOEPIDEMIOLOGY IN THE OSMORE RIVER VALLEY, SOUTHERN PERU.

Recent studies of louse ectoparasites from mummies have developed robust data sets that allow a true epidemiological approach to the prehistory of louse parasitism. One epidemiological principle is that the binomial of overdispersion is normally negative, meaning that in a host population, parasites are aggregated in a few individuals. We demonstrate the overdispersion of lice in 3 different prehistoric communities that differ along 3 axes or variables: environmental setting, socioeconomic status, and cultural affiliation. Distinct cultural practices could have been involved in different patterns of louse infestation. Prevalence, intensity, and abundance of infestations exhibit statistically significant differences between the communities. We also find differences in prevalence between subadults and adults that contrasted by cultural affiliation and suggest conditions different from those seen today. We show that overall prevalence was affected primarily by ecological setting, not socioeconomic status nor cultural affiliation. These findings demonstrate that statistical analysis of archaeological data can reveal the states of infestation in past populations with lifestyles not seen in modern people. Our approach paves the way for future comparisons of subpopulations within archaeological communities.

PALEOPARASITOLOGY OF HUMAN ACANTHOCEPHALAN INFECTION: A REVIEW AND NEW CASE FROM BONNEVILLE ESTATES ROCKSHELTER, NEVADA, U.S.A.

This study reports a new case of acanthocephalan (thorny-headed worm) eggs in a coprolite from Bonneville Estates Rockshelter in eastern Nevada and uses archaeological and ethnographic data to better understand long-term relationships between people and acanthocephalans. Acanthocephalans are parasitic worms that use arthropods as intermediate hosts in their multi-host life cycles. Though acanthocephaliasis is rare among humans today, cases have increased in the last decade, and the discovery of acanthocephalan eggs in coprolites from archaeological sites in the Great Basin suggests a deep, shared history. At Bonneville Estates Rockshelter, 9 acanthocephalan eggs were recovered using a modified rehydration-homogenization-micro-sieving protocol on a coprolite that was radiocarbon dated to 6,040 ± 60 14C BP (7,160-6,730 cal BP), pushing back the oldest evidence of human acanthocephalan infection by 3 millennia. Researchers have proposed that the paleoepidemiology of acanthocephalans may relate to subsistence practices due to overlap in locations of infection and areas where insects are part of traditional foodways. This paper considers the paleoepidemiology of acanthocephalan infection through the first combined review of paleoparasitological, ethnographic, and archaeological records in western North America. Ethnographic and archaeological records support the hypothesis that archaeological cases of human acanthocephaliasis may be linked to entomophagy. Additional parasitological analyses are advised to determine whether this distribution is the result of dietary practices, host ecology, taphonomic issues, sampling biases, or a combination of factors.

PALEOEPIDEMIOLOGY OF DIPHYLLOBOTHRIOSIS: CULTURAL AND ENVIRONMENTAL FACTORS AFFECTING ADENOCEPHALUS INTENSITY AND PREVALENCE.

Peruvian and Chilean mummies and coprolites provide a source of population-based parasitological information. This is especially true of the fish tapeworm, Adenocephalus pacificus. Our analysis of Chinchorro and Chiribaya mummies and diversified coprolite samples from Chile and Peru show variation in infection. There is a statistically significant difference in prevalence between Chinchorro hunter-gatherer and Chiribaya mixed-subsistence contexts. Furthermore, the most pronounced differences occur between populations within these groups. Chinchorro differences in cemeteries at the same location can be related to El Niño-Southern Oscillation variations. Pronounced prevalence variations between 3 Chiribaya villages within 7 km of each other relate to fish distribution and preparation variation. As with other recent archaeoparasitology studies, eggs-per-gram data exhibit overdispersion.

Possible influence of the ENSO phenomenon on the pathoecology of diphyllobothriasis and anisakiasis in ancient Chinchorro populations.

Current clinical data show a clear relationship between the zoonosis rates of Diphyllobothrium pacificum and Anisakis caused by the El Niño Southern Oscillations (ENSO) phenomenon along the Chilean coast. These parasites are endemic to the region and have a specific habitat distribution. D. pacificum prefers the warmer waters in the northern coast, while Anisakis prefers the colder waters of Southern Chile. The ENSO phenomenon causes a drastic inversion in the seawater temperatures in this region, modifying both the cool nutrient-rich seawater and the local ecology. This causes a latitudinal shift in marine parasite distribution and prevalence, as well as drastic environmental changes. The abundance of human mummies and archaeological coastal sites in the Atacama Desert provides an excellent model to test the ENSO impact on antiquity. We review the clinical and archaeological literature debating to what extent these parasites affected the health of the Chinchorros, the earliest settlers of this region. We hypothesise the Chinchorro and their descendants were affected by this natural and cyclical ENSO phenomenon and should therefore present fluctuating rates of D. pacificum and Anisakis infestations.

Automontage microscopy and SEM: A combined approach for documenting ancient lice.

Human ectoparasites, including lice, have been recovered from a wide range of archaeological materials. The human head louse, Pediculus humanus capitis, has been identified from mummies and sediments for decades. Louse eggs are the body part most commonly encountered and therefore the most frequently quantified. Typically, several types of microscopy are applied for egg documentation. For studies in which quantification of infestation is a goal, counting is done with the naked eye or with the aid of handheld lenses. For determination and stage classification, stereomicroscopy is commonly used. For more detailed examination of microstructure, light microscopy, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM) can be employed. In most reports, researchers use two or more techniques to accomplish interrelated goals. Automontage microscopy is used to document prehistoric arthropods with good success. Herein, we report the results of a combination of SEM and automontage microscopy to document lice and eggs recovered from South American mummies. This combined approach allows for simultaneous examination of internal and external characteristics. Thirty automontage composite images of 2 adult lice and 16 eggs showed that egg internal morphologies were easily examined showing the within-egg anatomy of emergent nymphs. SEM imaging of 9 lice and 129 eggs was completed. In the case of two adults and several eggs, SEM imaging was accomplish after automontage image capture of the same specimens. This one-to-one image comparison of SEM and automontage shows that transmitted light of automontage reveals egg internal structures and details of the adult lice. SEM allows for high magnification examination of egg, nymph and adult microstructures. We conclude that automontage imaging followed by SEM results in efficient graphic documentation of rare louse specimens.

CoproID predicts the source of coprolites and paleofeces using microbiome composition and host DNA content.

Shotgun metagenomics applied to archaeological feces (paleofeces) can bring new insights into the composition and functions of human and animal gut microbiota from the past. However, paleofeces often undergo physical distortions in archaeological sediments, making their source species difficult to identify on the basis of fecal morphology or microscopic features alone. Here we present a reproducible and scalable pipeline using both host and microbial DNA to infer the host source of fecal material. We apply this pipeline to newly sequenced archaeological specimens and show that we are able to distinguish morphologically similar human and canine paleofeces, as well as non-fecal sediments, from a range of archaeological contexts.

Paleoparasitology of Chagas disease--a review.

One hundred years since the discovery of Chagas disease associated with Trypanosoma cruzi infection, growing attention has focused on understanding the evolution in parasite-human host interaction. This interest has featured studies and results from paleoparasitology, not only the description of lesions in mummified bodies, but also the recovery of genetic material from the parasite and the possibility of analyzing such material over time. The present study reviews the evidence of Chagas disease in organic remains excavated from archeological sites and discusses two findings in greater detail, both with lesions suggestive of chagasic megacolon and confirmed by molecular biology techniques. One of these sites is located in the United States, on the border between Texas and Mexico and the other in state of Minas Gerais, in the Brazilian cerrado (savannah). Dated prior to contact with Europeans, these results confirm that Chagas disease affected prehistoric human groups in other regions outside the Andean altiplanos and other transmission areas on the Pacific Coast, previously considered the origin of T. cruzi infection in the human host.

The Skiles Mummy: Care of a debilitated hunter-gatherer evidenced by coprolite studies and stable isotopic analysis of hair.

The Skiles Mummy (SMM), a naturally mummified adult male from the late archaic period of Lower Pecos Canyonlands of South Texas, represents a unique case of care. SMM is an exceptional mummy within this region due to both the retention of a full head of hair, and having a diagnosed case of megacolon, a complication commonly associated with Chagas disease caused by Trypanosoma cruzi. Stable isotopic analysis of his hair is consistent with a diet incorporating of C4/CAM plants with some C3 plants, freshwater resources, and higher trophic level animals. However, the segments of hair most proximal to the scalp exhibited elevated δ15N values. Data from previous research indicate starvation and malnutrition can cause δ15N values to rise. The presence of large fecal boluses in the digestive tract suggest peristalsis ceased in the last four to five months of life, and this, together with results from coprolite analysis, indicate he would not have been able to adequately absorb protein and nutrients during this time. His condition would have rendered him immobile. Following Tilley's index of care, someone would have had to bring him food resources, as well as attending to his daily needs.

The Prevotella copri Complex Comprises Four Distinct Clades Underrepresented in Westernized Populations.

Prevotella copri is a common human gut microbe that has been both positively and negatively associated with host health. In a cross-continent meta-analysis exploiting >6,500 metagenomes, we obtained >1,000 genomes and explored the genetic and population structure of P. copri. P. copri encompasses four distinct clades (>10% inter-clade genetic divergence) that we propose constitute the P. copri complex, and all clades were confirmed by isolate sequencing. These clades are nearly ubiquitous and co-present in non-Westernized populations. Genomic analysis showed substantial functional diversity in the complex with notable differences in carbohydrate metabolism, suggesting that multi-generational dietary modifications may be driving reduced prevalence in Westernized populations. Analysis of ancient metagenomes highlighted patterns of P. copri presence consistent with modern non-Westernized populations and a clade delineation time pre-dating human migratory waves out of Africa. These findings reveal that P. copri exhibits a high diversity that is underrepresented in Western-lifestyle populations.