SL1 RNA gene recovery from Enterobius vermicularis ancient DNA in pre-Columbian human coprolites

Enterobius vermicularis, pinworm, is one of the most common helminths worldwide, infecting nearly a billion people at all socio-economic levels. In prehistoric populations the paleoparasitological findings show a pinworm homogeneous distribution among hunter-gatherers in North America, intensified with the advent of agriculture. This same increase also occurred in the transition from nomad hunter-gatherers to sedentary farmers in South America, although E. vermicularis infection encompasses only the ancient Andean peoples, with no record among the pre-Colombian populations in the South American lowlands. However, the outline of pinworm paleoepidemiology has been supported by microscopic finding of eggs recovered from coprolites. Since molecular techniques are precise and sensitive in detecting pathogen ancient DNA (aDNA), and also could provide insights into the parasite evolutionary history, in this work we have performed a molecular paleoparasitological study of E. vermicularis. aDNA was recovered and pinworm 5S rRNA spacer sequences were determined from pre-Columbian coprolites (4110 BC-AD 900) from four different North and South American archaeological sites. The sequence analysis confirmed E. vermicularis identity and revealed a similarity among ancient and modern sequences. Moreover, polymorphisms were identified at the relative positions 160, 173 and 180, in independent coprolite samples from Tulán, San Pedro de Atacama, Chile (1080-950 BC). We also verified the presence of peculiarities (Splicing leader (SL1) RNA sequence, spliced donor site, the Sm antigen biding site, and RNA secondary structure) which characterise the SL1 RNA gene. The analysis shows that the SL1 RNA gene of contemporary pinworms was present in pre-Columbian E. vermicularis by 6110 years ago. We were successful in detecting E. vermicularis aDNA even in coprolites without direct microscopic evidence of the eggs, improving the diagnosis of helminth infections in the past and further pinworm paleoepidemiological studies.     

Human intestinal parasites in the past: new findings and a review

Almost all known human specific parasites have been found in ancient feces. A review of the paleoparasitological helminth and intestinal protozoa findings available in the literature is presented. We also report the new paleoparasitologic findings from the examination performed in samples collected in New and Old World archaeological sites. New finds of ancylostomid, Ascaris lumbricoides, Trichuris trichiura, Enterobius vermicularis, Trichostrongylus spp., Diphyllobothrium latum, Hymenolepis nana and Acantocephalan eggs are reported. According to the findings, it is probable that A. lumbricoides was originally a human parasite. Human ancylostomids, A. lumbricoides and T. trichiura, found in the New World in pre-Columbian times, have not been introduced into the Americas by land via Beringia. These parasites could not supported the cold climate of the region. Nomadic prehistoric humans that have crossed the Bering Land Bridge from Asia to the Americas in the last glaciation, probably during generations, would have lost these parasites, which life cycles need warm temperatures in the soil to be transmitted from host to host. Alternative routes are discussed for human parasite introduction into the Americas.     

Parasite remains in archaeological sites

Organic remains can be found in many different environments. They are the most significant source for paleoparasitological studies as well as for other paleoecological reconstruction. Preserved paleoparasitological remains are found from the driest to the moistest conditions. They help us to understand past and present diseases and therefore contribute to understanding the evolution of present human sociality, biology, and behavior. In this paper, the scope of the surviving evidence will be briefy surveyed, and the great variety of ways it has been preserved in different environments will be discussed. This is done to develop to the most appropriated techniques to recover remaining parasites. Different techniques applied to the study of paleoparasitological remains, preserved in different environments, are presented. The most common materials used to analyze prehistoric human groups are reviewed, and their potential for reconstructing ancient environment and disease are emphasized. This paper also urges increased cooperation among archaeologists, paleontologists, and paleoparasitologists.     

Old parasites for a new world: the future of paleoparasitological research. a review

Paleoparasitological research has made important contributions to our understanding of parasite evolution and ecology since the 1960s. Since then, most studies have focused on paleoparasitological evidence from single sites or samples. With the development of high throughput sequencing techniques, new avenues of investigation for paleoparasitological material are opening up. Here, I provide an overview of recent developments and highlight how these results will broaden the scope of the field, placing paleoparasitology at the interface of a wide array of studies, including parasitology, climate change, human evolution, and evolutionary processes.     

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.     

Molecular Paleoparasitological Hybridization Approach as Effective Tool for Diagnosing Human Intestinal Parasites from Scarce Archaeological Remains

Paleoparasitology is the science that uses parasitological techniques for diagnosing parasitic diseases in the past. Advances in molecular biology brought new insights into this field allowing the study of archaeological material. However, due to technical limitations a proper diagnosis and confirmation of the presence of parasites is not always possible, especially in scarce and degraded archaeological remains. In this study, we developed a Molecular Paleoparasitological Hybridization (MPH) approach using ancient DNA (aDNA) hybridization to confirm and complement paleoparasitological diagnosis. Eight molecular targets from four helminth parasites were included: Ascaris sp., Trichuris trichiura, Enterobius vermicularis, and Strongyloides stercoralis. The MPH analysis using 18^th century human remains from Praça XV cemetery (CPXV), Rio de Janeiro, Brazil, revealed for the first time the presence E. vermicularis aDNA (50%) in archaeological sites of Brazil. Besides, the results confirmed T. trichiura and Ascaris sp. infections. The prevalence of infection by Ascaris sp. and E. vermicularis increased considerably when MPH was applied. However, a lower aDNA detection of T. trichiura (40%) was observed when compared to the diagnosis by paleoparasitological analysis (70%). Therefore, based on these data, we suggest a combination of Paleoparasitological and MPH approaches to verify the real panorama of intestinal parasite infection in human archeological samples.     

Paleoparasitological analysis of a raptor pellet from southern Patagonia

Organic remains attributable to one regurgitated pellet were examined. The pellet, belonging to a bird of prey and collected from a cave of Southern Patagonia, was dated at 6,540 +/- 110 yr. With standard paleoparasitological procedures, eggs of Capillaria sp. and a mite, Demodex sp., were found. The parasites found in the pellet belong to a rodent ingested by the bird. The present report constitutes the first paleoparasitological study of a regurgitated pellet.     

Prehistoric parasitism in Tennessee: evidence from the analysis of desiccated fecal material collected from Big Bone Cave, Van Buren County, Tennessee

Eight samples of desiccated human feces collected from Big Bone Cave (40VB103), Van Buren County, Tennessee, were analyzed to determine the presence of ecto- and endoparasitic infection among the prehistoric population using the cave. Radiocarbon-dated torch material from the cave indicated that it was a locus of human activity 2,177 +/- 145 yr ago. Parasitic species identified were: Ascaris lumbricoides, Enterobius vermicularis, fleas of the tribe Phalacropsyllini, and protozoan cysts. The cysts were identified as Giardia using an indirect immunofluorescent antibody test. The only report of Giardia in a prehistoric context is the identification of cysts in 2 1,800-yr-old paleofecal specimens from a cave in Israel. This is the first report of Giardia from paleofeces in the New World.     

Purification and characterization of an arylamine N-acetyltransferase in the nematode Enterobius vermicularis.

N-acetyltransferase (NAT) activities were determined by incubation of Enterobius vermicularis cytosols with 2-aminofluorene (2-AF) as the substrate followed by high pressure liquid chromatography assays. The NAT activity from E. vermicularis was found to be 0.41 +/- 0.08 nmol/min/mg protein for 2-AF. The apparent K(m) and Vmax values obtained were 0.81 +/- 0.11 mM and 2.25 +/- 0.22 nmol/min/mg protein respectively, for 2-AF. The optimal pH value for the enzyme activity was 7.5 for 2-AF. The optimal temperature for enzyme activity was 37 degrees C for the 2-AF substrate. The molecular weight of NAT from E. vermicularis was 44.9 kD. Among a series of divalent cations and salts, Zn2+, Ca2+, and Fe2+ were the most potent inhibitors. Of the protease inhibitors, only ethylenediaminetetraacetic acid significantly protected the NAT. Iodoacetate, in contrast to other agents, markedly inhibited NAT activity. This report is the first demonstration of acetyl coenzyme A-dependent arylamine NAT activity in E. vermicularis and extends the number of phyla in which this activity has been found.     

Paleoparasitological analysis of coprolites from K2, an Iron Age archaeological site in South Africa: the first finding of Dicrocoelium sp. eggs

Until now, Dicrocoelium sp. eggs have only been recorded from European and 1 North American archaeological sites. We present evidence for the first record of Dicrocoelium sp. from an African archaeological site. A paleoparasitological study was conducted on 7 coprolite samples from K2, a Late Iron Age site on the farm Greefswald, in the Northern Province of South Africa. Standard parasitological analysis revealed the presence of Dicrocoelium sp. and Trichuris sp. eggs. Today, the parasite does not occur in this region. Trichurid eggs are a relatively common find in paleoparasitological analysis. The presence of Dicrocoelium sp. provides new clues about the antiquity of this parasite, as well as aspects of ancient environment, climate, and interactions among humans, animals, and parasites.     

First study of fossil rodent middens as source of paleoparasitological evidences (northwestern Patagonia,Argentina)

The present paper reports the first paleoparasitological results obtained from coprolites of fossil rodent middens and demonstrates the potential of rodent middens as a source of paleoparasitological evidences in South America. Ten fossil rodent middens from northwestern Patagonia, Argentina, were studied. Five coprolites of each midden were fully processed, rehydrated, homogenized, subjected to spontaneous sedimentation, and examined through light microscopy. Eight of the 10 examined rodent middens contained parasite eggs. The eggs of parasites were assigned to Heteroxynema (Cavioxyura) viscaciae Sutton & Hugot, 1989 and Helminthoxys sp. (Nematoda: Oxyuridae), Trichuris sp. (Nematoda: Trichuridae) and one unidentified nematode. Fossil rodent middens were assigned to Lagidium viscacia (Caviomorph: Chinchillidae). The excellent preservation of parasite remains in coprolites from fossil rodent middens provided an opportunity to perform paleoparasitological inferences. The results of this papers demonstrates that fossil rodent middens offer an excellent opportunity for the recovery of parasite remains for future paleoparasitological studies in the southwest of South America.     

Enterobius vermicularis egg positive rates in primary school children in Gangwon-do (province), Korea.

A survey of the infection rate of Enterobius vermicularis among students in 4 primary schools located in Gangwon-do (Province) was done from May to June 2001. Among the 398 examinees, 39 (9.8%) were infected with E. vermicularis demonstrated by the adhesive cellotape anal swab method. The infection rates ranged from 8.3% to 11.8% among the four schools. The infection rate of males and females was 10.7% and 7.7% respectively. The first grade students showed the highest infection rate, 28.7%. The confirmed cases were treated with albendazole three times at an interval of 15 days. We were able to confirm that E. vermicularis infection is still prevalent among students in Gangwon-do, Korea.     

Trichuris sp. from 1,040 +/- 50-year-old Cervidae coprolites from the archaeological site Furna do Estrago, Pernambuco, Brazil

We present results of the paleoparasitological analysis of Cervidae coprolites that were recovered from the archaeological site Furna do Estrago, Pernambuco, Brazil. Trichuris sp. eggs were recovered from the coprolite samples dated 1,040 ± 50 years before present. This is the first record of Trichuris sp. in semiarid Cervidae, unexpectedly recorded in archaeological material.     

Paleoparasitologic, paleogenetic and paleobotanic analysis of XVIII century coprolites from the church La Concepción in Santa Cruz de Tenerife, Canary Islands, Spain

We present the results of a paleoparasitologic, paleogenetic and paleobotanic analysis of coprolites recovered during the excavation of the church La Concepción in Santa Cruz de Tenerife. Coprolites (n = 4) were rehydrated and a multidisciplinary analysis was conducted. The paleobotanic analysis showed numerous silicates, seeds and fruits of the family Moraceae. In the paleoparasitologic study, Ascaris sp. eggs (n = 344) were identified. The paleogenetic results confirmed the Ascaris sp. infection as well as the European origin of human remains. These findings contribute to our knowledge of ancient helminthes infections and are the first paleoparasitological record of Ascaris sp. infection in Spain.     

The finding of Enterobius vermicularis eggs in pre-Columbian human coprolites

Enterobius vermicularis eggs were found in human coprolites collected in the archaeological site of Caserones, Tarapaca Valley, Chile, dating from 400 BC to 800 AD. The human pinworm had already been found in other pre-historic archaeological sites in America, and its introduction in this continent is discussed.     

The mitochondrial genome sequence of Enterobius vermicularis (Nematoda: Oxyurida)--an idiosyncratic gene order and phylogenetic information for chromadorean nematodes

The complete mitochondrial genome sequence was determined for the human pinworm Enterobius vermicularis (Oxyurida: Nematoda) and used to infer its phylogenetic relationship to other major groups of chromadorean nematodes. The E. vermicularis genome is a 14,010-bp circular DNA molecule that encodes 36 genes (12 proteins, 22 tRNAs, and 2 rRNAs). This mtDNA genome lacks atp8, as reported for almost all other nematode species investigated. Phylogenetic analyses (maximum parsimony, maximum likelihood, neighbor joining, and Bayesian inference) of nucleotide sequences for the 12 protein-coding genes of 25 nematode species placed E. vermicularis, a representative of the order Oxyurida, as sister to the main Ascaridida+Rhabditida group. Tree topology comparisons using statistical tests rejected an alternative hypothesis favoring a closer relationship among Ascaridida, Spirurida, and Oxyurida, which has been supported from most studies based on nuclear ribosomal DNA sequences. Unlike the relatively conserved gene arrangement found for most chromadorean taxa, E. vermicularis mtDNA gene order is very unique, not sharing similarity to any other nematode species reported to date. This lack of gene order similarity may represent idiosyncratic gene rearrangements unique to this specific lineage of the oxyurids. To more fully understand the extent of gene rearrangement and its evolutionary significance within the nematode phylogenetic framework, additional mitochondrial genomes representing a greater evolutionary diversity of species must be characterized.     

Egg positive rate of Enterobius vermicularis and Taenia spp. by cellophane tape method in primary school children in Sivas, Turkey

The aim of the present study was to find out the number of students with enterobiasis and/or taeniasis in primary schools of Sivas. Among the 2,029 students in 6 primary schools, 316 (15.6%) were positive to Enterobius vermicularis eggs and 32 (1.6%) were positive to Taenia spp. eggs by the cellophane tape method. The egg positive rates of E. vermicularis and Taenia spp. ranged from 9.4% to 27.2% and from 0.8% to 2.6% respectively among six schools. The egg positive rate of E. vermicularis was found to be significantly different among these schools (chi2 = 31.96, P < 0.05), whereas there was no significant difference between the schools for Taenia spp. (chi2 = 4.37; P > 0.05). The rate (18.7%) of E. vermicularis in the urban slum regions was higher than the rate (11.5%) in the urban central regions (chi2 = 19.20; P < 0.05). Above results demonstrate that the egg positive rate of E. vermicularis and Taenia spp. was still prevalent among primary school children.     

Paleoparasitological report on the stool from a Medieval child mummy in Yangju, Korea

Previous studies have successfully shown evidence for parasitic infections in human remains from various archaeological sites. However, in the case of Korea, since there have been very few paleoparasitological reports published, pre-20th century parasitic infection patterns remain obscure. Therefore, in order to partly fill this gap, we are reporting on a case of paleoparasitic infection from the feces of a 15th century child mummy from Yangju, Korea. In the course of the present study, we found the eggs of Clonorchis sinensis, Ascaris lumbricoides, and Trichuris trichiura in the feces of the mummy. Trichuris trichiura eggs were found in far greater numbers than other parasite eggs; in fact, intact bipolar plugs were clearly observed and even the larvae were still visible in some eggs. The eggs of C. sinensis and A. lumbricoides were also well preserved, though not in as great a number. Since we could find a number of well-preserved larvae-containing eggs, we are encouraged that successful extraction, amplification, and sequence determination of ancient DNA from the paleoparasite eggs might be possible in future studies. With additional paleoparasitological investigation using feces from Korean mummies, we hope that a history of parasite infection in Korea will be reconstructed.     

Detection of Toxoplasma gondii DNA by polymerase chain reaction in experimentally desiccated tissues

Despite toxoplasmosis being a common infection among human and other warm-blooded animals worldwide, there are no findings about Toxoplasma gondii evolutionary forms in ancient populations. The molecular techniques used for amplification of genetic material have allowed recovery of ancient DNA (aDNA) from parasites contained in mummified tissues. The application of polymerase chain reaction (PCR) to paleoparasitological toxoplasmosis research becomes a promising option, since it might allow diagnosis, acquisition of paleoepidemiological data, access to toxoplasmosis information related origin, evolution, and distribution among the ancient populations. Furthermore, it makes possible the analysis of parasite aDNA aiming at phylogenetic studies. To standardize and evaluate PCR applicability to toxoplasmosis paleodiagnostic, an experimental mummification protocol was tested using desiccated tissues from mice infected with the ME49 strain cysts, the chronic infection group (CIG), or infected with tachyzoites (RH strain), the acute infection group (AIG). Tissues were subjected to DNA extraction followed by PCR amplification of T. gondii B1 gene. PCR recovered T. gondii DNA in thigh muscle, encephalon, heart, and lung samples. AIG presented PCR positivity in encephalon, lungs, hearts, and livers. Based on this results, we propose this molecular approach for toxoplasmosis research in past populations.     

Enterobius vermicularis egg positive rate in a primary school in Chungchongnam-do (province) in Korea

The egg positive rate of Enterobius vermicularis was investigated among students of a primary school and a kindergarten located in the rural area of Tangjin-gun, Chungchongnam-do in December, 1998. Of the 189 examinees, 28 (14.8%) were found to be infected with E. vermicularis by the adhesive cellotape anal swab method. The infection rates ranged from 4.2% to 26.1% among school children, and the highest rate was observed in children attending kindergarten. Three months after treatment with albendazole, four (14.3%) out of 28 infected children still remained infected with E. vermicularis. Through this survey, we were able to determine that E. vermicularis infection is still prevalent among children in rural areas of Korea.