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.

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.

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.

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.

Palynological Investigation of Mummified Human Remains.

Pollen analysis was applied to a mummified homicide victim in Nebraska, U.S.A., to determine the location of death. A control sample showed the normal ambient pollen in the garage crime scene. Ambient windborne types, common in the air of the region, dominated the control. Internal samples were analyzed from the sacrum, intestine, and diaphragm. Microfossils were recovered from the rehydrated intestine lumen. The intestinal sample was dominated by Brassica (broccoli). The sacrum sample was high in dietary types but with a showing of ambient types. The pollen from the diaphragm was dominated by ambient pollen similar to the control samples. The discovery of diverse pollen spectra from within a single mummy was unexpected. They show that ingested and inhaled pollen mixed in the corpse. The data linked the decedent to a specific crime scene in her Nebraska home in the southern tier of eastern counties on the border with Kansas.

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.

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.

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.

Cleaning Puparia for Forensic Analysis.

We tested procedures for removing adipocere from insect samples to allow identification. An acceptable procedure was determined: (i) Samples were sorted in petri dishes with 75% alcohol to remove any larvae, adult insects, or other soft-bodied material. (ii) Samples of up to 24 puparia were placed in a vial with 15 mL of 95% acetone, capped, and vortexed for a total of 30-90 sec in 10- to 15-sec bursts. This step removed large masses of adipocere or soil from specimen. (iii) Specimens were removed from acetone and placed in a vial of 15 mL of 2% potassium hydroxide (KOH) and vortexed in 10- to 15-sec bursts until all puparia appeared clean (with our samples this required a total of 60-120 sec). (iv) Specimens were removed from the 2% KOH, placed in 75% ethanol, and examined microscopically. (v) Material was stored in 75% ethanol for identification and long-term preservation.

Cryptosporidium parvum Among Coprolites from La Cueva de los Muertos Chiquitos (600-800 CE), Rio Zape Valley, Durango, Mexico.

: In the present study, 90 coprolites from La Cueva de los Muertos Chiquitos (CMC) were subjected to enzyme-linked immunosorbent assay (ELISA) tests for 3 diarrhea-inducing protozoan parasites, Entamoeba histolytica , Giardia duodenalis , and Cryptosporidium parvum , to determine whether these parasites were present among the people who utilized this cave 1,200-1,400 yr ago. These people, the Loma San Gabriel, developed as a culture out of the Archaic Los Caracoles population and lived throughout much of present-day Durango and Zacatecas in Mexico. The Loma San Gabriel persisted through a mixed subsistence strategy of hunting-gathering and agricultural production. The results of ELISA testing were negative for both E. histolytica and G. duodenalis across all coprolites. A total of 66/90 (∼73% prevalence) coprolites tested positive or likely positive for C. parvum . The high prevalence of C. parvum among CMC coprolites contributes to our growing knowledge of the pathoecology among the Loma San Gabriel who utilized CMC. Herein, we report the successful recovery of C. parvum coproantigens from prehistoric coprolites. The recovery of these coproantigens demonstrates the existence of C. parvum in Mesoamerica before European contact in the 1400s.

Taphonomic considerations for the analysis of parasites in archaeological materials.

Archaeoparasitological analyses of human remains can present interpretative challenges arising from diverse preservation environments. Three archaeoparasitological studies are used to demonstrate the impacts of five major types of taphonomic factors on parasite egg preservation. In the first case, an analysis of a historic Lithuanian mummy revealed infections with Trichuris trichiura and Ascaris lumbricoides and illustrates taphonomic issues unique to mummies. The second case involved the analysis of coprolites collected from medieval burials in Nivelles, Belgium. One burial demonstrated a high concentration of T. trichiura eggs (approximately 1,577,679 total eggs) and A. lumbricoides eggs (approximately 202,350 total eggs). Preservation was affected mostly by water percolation with differential preservation of eggs based on morphological characteristics. The third case is based on material from embalming jars of the Medici family. No parasite eggs were recovered; however, an abundance of mites and dipteran puparia were encountered, suggesting that arthropods may play a larger role in parasite egg preservation than previously supposed. Differential parasite egg preservation is discussed in light of variances in five major types of taphonomic factors: abiotic, contextual, anthropogenic, organismal, and ecological. Accounting for these factors is a vital component in the interpretation of archaeoparasitological data and should be included in future archaeoparasitological reports.

Curatorial implications of Ophyra capensis (Order Diptera, Family Muscidae) puparia recovered from the body of the Blessed Antonio Patrizi, Monticiano, Italy (Middle Ages).

The discovery of dipteran remains on mummified individuals can lead to either cause for curatorial concern or to a better understanding of the individual's post-mortem environment. The present study analyzed insect remains associated with the body of a unique medieval mummy of religious significance, that of the Blessed Antonio Patrizi da Monticiano. A total of 79 puparia were examined and all were identified as Ophyra capensis (Diptera: Muscidae). Additionally, a desiccated moth (Lepidoptera: Tineidae) was encountered. Puparia of O. capensis would be associated with normal decomposition shortly after the death of the mummified individual, and not an infestation beginning during more recent years. Similarly, the tineid moth found would likely be related with decomposition of cloth associated with the remains. These findings illustrate how collection and identification of insects associated with human remains can distinguish between historical decomposition versus issues of modern curatorial concern.

Agave Chewing and Dental Wear: Evidence from Quids.

Agave quid chewing is examined as a potential contributing behavior to hunter-gatherer dental wear. It has previously been hypothesized that the contribution of Agave quid chewing to dental wear would be observed in communities wherever phytolith-rich desert succulents were part of subsistence. Previous analysis of coprolites from a prehistoric agricultural site, La Cueva de los Muertos Chiquitos in Durango, Mexico, showed that Agave was a consistent part of a diverse diet. Therefore, quids recovered at this site ought to be useful materials to test the hypothesis that dental wear was related to desert succulent consumption. The quids recovered from the site were found to be largely derived from chewing Agave. In this study, the quids were found to be especially rich in phytoliths, and analysis of dental casts made from impressions left in the quids revealed flat wear and dental attrition similar to that of Agave-reliant hunter-gatherers. Based on evidence obtained from the analysis of quids, taken in combination with results from previous studies, it is determined that Agave quid chewing was a likely contributing factor to dental wear in this population. As such, our method provides an additional avenue of dental research in areas where quids are present.

Establishing tobacco origin from pollen identification: an approach to resolving the debate.

Previous research into pollen content of tobacco resulted in a debate. We address this debate and determine that pollen analysis may be able to assist with identifying geographical origin of tobacco. However, the value of any results should be assessed on a case-by-case regional basis until sufficient database information is available for an objective interpretation to be undertaken on a global basis. As a first step toward developing comparative data for South America, we analyzed a tobacco sample from Brazil in an effort to identify signature taxa from the state of Minas Gerais. We also assessed the role of honey additives to tobacco to assess this issue. Comparing the data to previously published data, we conclude that pollen signatures can distinguish broad geographic areas. We conclude that this forensic interpretation framework needs to be developed in context of the National Academy of Sciences recommendations for tightening methods in forensic science.

A retrospective examination of paleoparasitology and its establishment in the Journal of Parasitology.

Volume 95 (2009) of the Journal of Parasitology represented a significant benchmark in the history of paleoparasitology when it received on the cover formal recognition as a topical area for publication. This retrospective examination chronicles the emergence of paleoparasitology, from its origins as an adjunct contribution to the study of prehistoric human populations to its modern expression as a sub-disciplinary interest. The aim of paleoparasitology is to elucidate the temporal and spatial dimensions of parasitism from the fossil record of human and non-human host populations.

Taphonomic considerations of a whipworm infection in a mummy from the Dominican Church of the Holy Spirit, Vilnius, Lithuania.

In the present study, the abdominal contents of 10 mummies from beneath the Dominican Church of the Holy Spirit in Vilnius, Lithuania, were examined for the presence of helminth parasites using standard archaeoparasitological techniques. Of the mummies examined, only one individual presented with evidence of parasitism. This individual was infected with both Trichuris trichiura and Ascaris lumbricoides (5,222 parasite eggs/gram). The conditions of many of the T. trichiura eggs suggest that a fortuitously embedded female whipworm decomposed within the individual's gut to release the eggs, as opposed to the eggs actually being passed by the adult helminth. This study highlights a taphonomic issue unique to mummies by demonstrating the differential preservation of parasite eggs existing in various stages of development. Whenever one is not dealing with parasite eggs that have already been passed by the host, as is the case when analyzing intestinal tissues, one must understand that some types of parasite eggs may not be fully formed. It is imperative, as demonstrated by our findings, that researchers have the knowledge to recognize under-developed intestinal helminth eggs in addition to fully formed intestinal helminth eggs from mummy source materials. Together, these findings demonstrate the persistence of these helminth parasites in Europe during the 18th and 19th centuries and represent the first archaeoparasitological evidence from mummies in Vilnius, Lithuania.

Clarifying Prehistoric Parasitism from a Complementary Morphological and Molecular Approach.

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.

Applying forensic anthropological data in homicide investigation to the depravity standard.

Forensic anthropology can provide detailed information regarding the perpetrator's treatment of a homicide victim. This data may inform The Depravity Standard (DS), a forensic science inventory used to assess the severity of a homicide's intent, actions, victimology, and attitudes. Skeletal data enabled the reconstruction of a homicide case involving mutilation and possible torture. Using The Depravity Standard (DS) the skeletal data underwent evaluation in order to provide evidence of depravity. The osteological data alone offered sufficient evidence for a number of criteria of depravity, demonstrating the importance and application of osteology in resolving specific questions about the depravity of a homicide.

Parasitism of the Zweeloo Woman: Dicrocoeliasis evidenced in a Roman period bog mummy.

We undertook the analysis of Zweeloo Woman, a bog mummy from the Netherlands, to assess her parasitic state. Evidence of infection came from two areas: (1) liver paraffin sections and (2) microfossils washed from an intestinal section. Although the liver had shrunken considerably, objects consistent with operculated trematode eggs were found. After evaluating the range of trematode species that produce eggs in liver tissue, we arrived at the diagnosis of Dicrocoelium dendriticum. Although only 0.1ml of sediment was recovered from an intestinal section, eggs of Ascaris lumbricoides and Trichuris trichiura were also identified. No eggs of D. dendriticum were revealed by the intestinal wash although they were observed in the liver. The lancet fluke, D. dendriticum, is a zoonosis that usually infects ruminants such as cattle. Eggs of D. dendriticum may be found in human coprolites if infected cow liver, for example, was eaten. This is false parasitism. Since eggs of D. dendriticum were found in the liver of Zweeloo Woman, we are assured this was a true infection. This find is especially significant because it is the oldest known, patent infection of D. dendriticum in humans.