Ancient DNA from Ascaris: extraction amplification and sequences from eggs collected in coprolites

doi:10.1016/S0020-7519(01)00214-4

International Journal for Parasitology

Volume 31, Issue 10, August 2001, Pages 1101-1106

https://doi.org/10.1016/S0020-7519(01)00214-4 Get rights and content

Abstract

On the Middle-Age site of Namur (Belgium) the analysis of coprolites revealed the presence of many well-preserved Ascaris eggs. Following rehydratation of the coprolite samples, 104 eggs were collected and extracted with an ultrasonication and phenol-chloroform based method. Three overlapping fragments of the 18S rRNA gene and one fragment of the cytochrome b gene have been reproducibly amplified, cloned and sequenced. The analysis of these sequences confirms the identification of the eggs as coming from Ascaris. Our study reveals that coprolites can be an interesting source of parasites that can be readily identified using molecular approaches. The study of ancient DNA from helminth parasites is of interest as it may answer long-standing questions in the history of infectious diseases and gives a possibility to compare these ancient sequences with those of modern populations.

Section snippets

Acknowledgments

We thank CNRS, UCBL, MENRT and FRM for support. O.L. was supported by a fellowship from Fondation Les Treilles. We are grateful to C. Donne-Goussé and T. Delefosse for help in the laboratory, V. Laudet and M. Robinson-Rechavi for critical reading of the manuscript as well as L. Orlando, B. Herrmann, S. Hummel, B. Bramanti and T.J.C. Anderson for their comments on this work. We are grateful to G.C. Barker and S. Morand for providing us tissues of A. lumbricoides and A. suum, respectively. We

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Coprolites are promising tools for the understanding of paleoecological and paleoenvironmental aspects. Through its chemical composition and analysis of micro- and macrostructures, it is possible to obtain information about the diet of ancient animals and insights into the climate and vegetation of paleoenvironments. Paleoparasitological analysis of these trace fossils help to understand the parasite-host relationship and how parasitic infections were processed in the past. The present study aims to identify helminths in coprolites of the Tremembé Formation (Oligocene of the Taubaté Basin, São Paulo). Furthermore, we included the descriptive association of the paleoparasitological data with the paleoenvironmental information present in the literature for this geological formation. Eggs were identified through linear morphometry and morphology. The paleoparasitological analysis revealed the presence of Nematode eggs of Ascaridina, Spirurina, and Trichocephalida, with a prevalence of 5% for each group. The complex life cycle of some nematodes would be made possible by the wide faunal diversity of birds, fish, and arthropods that inhabited the Tremembé paleolake. The low prevalence of helminths in the samples may be related to taphonomic processes. This study represents the first paleoparasitological analysis of avian coprolites from the Tremembé Formation. In addition, the finding of these helminth records creates new perspectives for the studies of this geological unit through the epidemiological point of view associated with the local paleofauna and also provides new subsidies for the further development of evolutionary and biogeographic helminth studies.
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Ascarid infections constitute a major concern for both human and animal health risk assessment. Although being effectively transmitted by soil, water and contaminated food, reliable detection of ascarid eggs in environmental media often remains challenging. However, contamination of the environment with ascarid ova has gained more attention as a decisive part of proper risk assessment in recent years. Due to various factors, such as sample matrices, dissociation detergents and flotation solutions, defined and standardised protocols for the isolation of eggs from complex environmental matrices are difficult to establish and therefore limited. Thus, this study reviews common techniques used for the recovery of ascarid eggs from environmental media with special emphasis on sampling strategies, purification procedures and microscopic as well as molecular detection of egg contamination. Despite various advancements, mainly in the field of molecular methods leading to more reliable and sensitive detection, it can be concluded that there is still a need for unified guidelines for sampling and recovery of ascarid eggs derived from complex environmental matrices.
Parasites in the Atacama Desert: New insights into the lifestyles of ancient human populations (3000–500 BP)
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Citation Excerpt :
Extraction blanks, containing ultrapure water instead of sample, were included in every six archaeological samples. PCR reactions were performed with specific primers to amplify the cyt b, SSUrRNA, and 5S rRNA targets of Ascaris lumbricoides, Trichuris trichiura, and Enterobius vermicularis, respectively (Iñiguez et al., 2003; Loreille et al., 2001; Oh et al., 2010). Amplification reactions for each sample contained 1 U/rxn Platinum Taq DNA Polymerase High Fidelity (Invitrogen), 1X High Fidelity PCR Buffer, 0.2 mM dNTPs mix, 0.2 μM of each primer, 2 mM MgSO4, 0.1 pmol/µL BSA, 5–10 µL of aDNA extracts or dilutions, and ultrapure water to a final volume of 50 µL.
Paleoparasitological studies have provided insights into ancient parasite occurrence and its relationship to the health, lifestyle, and cultural practices of past populations. We analyzed 26 human coprolites and digestive tract contents, most of them contained in mummified human bodies, from coastal and inland archaeological sites in the Atacama Desert (3000–500 BP), to test for gastrointestinal parasites. We rehydrated and sedimented the samples to concentrate the micro-remains and then conducted light microscopy examination and paleogenetic analysis. The microscopy analysis allowed us to recover helminth eggs of five different taxa in seven samples, attributed to Diphyllobotrium sp., Dipylidium caninum, Enterobius vermicularis, Trichuris sp., and possible strongylid. PCR amplification yielded negative results in all cases. Results suggest a low parasite burden relating to infection with pinworms, whipworms, and possible strongylids, as well as an absence of infection in most of the individuals. The presence of Diphyllobotrium sp. in coastal sites is associated with the consumption of insufficiently cooked fish resources. The occurrence of D. caninum in an inland site may indicate cohabitation with dogs and/or wild canids. These findings contribute to knowledge of parasite occurrence in human groups from the southern Andes and its relationship with the environment and the cultural practices involved.
Molecular identification of parasites in an intestinal coprolite from a mummified religious dignitary of the Piraino Mother Church crypt, Sicily
2021, Journal of Archaeological Science: Reports
Intestinal contents were sampled from a spontaneously enhanced mummy from the Sepulcher of the Priests of the Piraino Mother Church in the Province of Messina, Sicily. This adult male mummy, Piraino 1, is an unidentified religious dignitary dating from the late-18th to mid-19th centuries. Immunological and molecular diagnostics were used to test for common and clinically significant parasites. A morphological diagnosis of Trichuris trichiura (whipworm) was confirmed genetically. A previously undetected Enterobius vermicularis (pinworm) infection was also identified genetically. These data indicate that the Piraino 1 individual was simultaneously infected with multiple intestinal parasite species indicative of poor hygiene. This study also emphasizes the importance of utilizing multiple diagnostic techniques to detect pathogens from archaeological contexts.
Gastrointestinal parasite burden in 4th-5th c. CE Florence highlighted by microscopy and paleogenetics
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The study of ancient parasites, named paleoparasitology, traditionally focused on microscopic eggs disseminated in past environments and archaeological structures by humans and other animals infested by gastrointestinal parasites. Since the development of paleogenetics in the early 1980s, few paleoparasitological studies have been based on the ancient DNA (aDNA) of parasites, although such studies have clearly proven their utility and reliability. In this paper, we describe our integrative approach for the paleoparasitological study of an ancient population from Florence in Italy, dated to the 4th-5th c. CE. The first stage consisted in the study of sediment samples from the pelvic area of 18 individuals under light microscopy. This allowed us to detect Ascarid-type eggs belonging very probably to the human-infesting roundworm Ascaris lumbricoides. Ten subsamples were selected corresponding to five individuals, and we extracted their whole DNA following sediment aDNA protocols. A targeted approach allowed us to detect two nematodes and one trematode aDNA fragments, namely Ascaris sp., Trichuris trichiura, and Dicrocoelium dendriticum. Among the five individuals tested for microscopic eggs and aDNA, three of them showed the remains of eggs (only Ascarid-type), but all of them tested positive to the presence of at least one parasite aDNA. Microscopic diagnosis first guided our research for the selection of promising samples while the targeted aDNA approach significantly improved our knowledge in terms of parasitic diversity and frequency in this population subgroup. These results enabled us to discuss the possible impact of latent parasitism in this past population at the time of an epidemic, as suggested in Florence. In particular, the singular case of D. dendriticum detection is discussed in light of the present-day scarcity of genuine human infections. Nevertheless, actual infections are known in the paleoparasitological record, and food habits may have led to false parasitism in this historical context. aDNA leaching from overlying strata may also explain this detection. This study strongly pleads for a systematic integrative approach combining microscopy and aDNA in paleoparasitology.
African helminth infection out of Africa: Paleoparasitological and paleogenetic investigations in Pretos Novos cemetery, Rio de Janeiro, Brazil (1769–1830)
2020, Acta Tropica
Pretos Novos cemetery (PNC), Rio de Janeiro, Brazil (1769–1830) was created exclusively to bury enslaved Africans who died upon arrival at the city or before being sold in the slave market. The PNC site may be unique in the Americas in allowing the study of African parasite infections acquired in Africa. We aimed to identify parasites infecting PNC individuals through paleoparasitological and paleogenetic analyses. The bodies had been dismembered, placed in mass graves, and burned, and most human remains collected from the site are highly fragmented and show extensive degradation. Sacrum and pelvic sediments were collected from five individuals along with seven samples of sediment from other areas of the body, as controls. Samples were submitted to three parasitological techniques and, in paleogenetic analysis, to four molecular targets. Larvae, mites, pollen grains, and structure suggestive of plants and fungus were observed, but we found no evidence of helminth infection. Ascaris sp. cytb sequence was recovered in one individual. We emphasize that, even with the extensive degradation of PNC human remains and the process of curation of samples, it was possible to recover helminth aDNA. The origin of PNC individuals confirms that these infections were brought to Brazil from western and central Africa during the colonial era.

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¹: Present address: Institut für Zoologie und Anthropologie, Georg August Universitaet Goettingen, Buergerstr. 50, 37073 Goettingen, Germany.

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International Journal for Parasitology

Abstract

Section snippets

Acknowledgments

References (27)

Value of the specific distinction between Ascaris lumbricoides Linné 1758 and Ascaris suum Goeze 1782

Int. J. Parasitol.

Paleoparasitology of Chagas disease revealed by infected tissues from Chilean mummies

Acta Tropica

Ancient DNA analysis reveals divergence of the cave bear Ursus spelaeus, and brown bear, Ursus arctos, lineages

Curr. Biol.

Molecular genetic approaches to parasite identification: their value in diagnostic parasitology and systematics

Int. J. Parasitol.

Ribosomal DNA and phylogeny of the Ascaridoidea (Nemata: Secernentea): Implications for morphological evolution and classification

Mol. Phylogenet. Evol.

Ascaris infections in human from North America: molecular evidence for cross-infection

Parasitology

Host specificity, evolutionary relationships and macrogeographic differentiation among Ascaris populations from humans and pigs

Parasitology

Recovery of helminth eggs from archaeological excavations of the Grand Louvre (Paris, France)

J. Parasitol.

Paléoparasitologie en contexte Pléistocène: première observation à la Grande Grotte d'Arcy sur Cure (Yonne, France)

C. R. Acad. Sci. Paris, III

Parasitological investigation of the ancient corpse of the Western Han dynasty unearthed from tomb No 168 on Phoenix Hill in Jiangling country

Acta Acad. Med. Wuhan

Nematode larvae in fossilized animal coprolites from lower and middle Pleistocene sites, central Italy

J. Parasitol.

Ancient bone DNA amplified

Nature

Amplification de fragments d'ADN mitochondrial partir de dents et d'os humains anciens

C. R. Acad. Sci. Paris 310 Ser

Cited by (99)

Paleoparasitological study of avian trace fossils from the Tremembé Formation (Oligocene of the Taubaté Basin), São Paulo, Brazil

Potentials and challenges in the isolation and detection of ascarid eggs in complex environmental matrices

Parasites in the Atacama Desert: New insights into the lifestyles of ancient human populations (3000–500 BP)

Molecular identification of parasites in an intestinal coprolite from a mummified religious dignitary of the Piraino Mother Church crypt, Sicily

Gastrointestinal parasite burden in 4th-5th c. CE Florence highlighted by microscopy and paleogenetics

African helminth infection out of Africa: Paleoparasitological and paleogenetic investigations in Pretos Novos cemetery, Rio de Janeiro, Brazil (1769–1830)

Research noteAncient DNA from Ascaris: extraction amplification and sequences from eggs collected in coprolites

Abstract

Section snippets

Acknowledgments

Int. J. Parasitol.

Acta Tropica

Curr. Biol.

Int. J. Parasitol.

Mol. Phylogenet. Evol.

Ascaris infections in human from North America: molecular evidence for cross-infection

Parasitology

Host specificity, evolutionary relationships and macrogeographic differentiation among Ascaris populations from humans and pigs

Parasitology

Recovery of helminth eggs from archaeological excavations of the Grand Louvre (Paris, France)

J. Parasitol.

Paléoparasitologie en contexte Pléistocène: première observation à la Grande Grotte d'Arcy sur Cure (Yonne, France)

C. R. Acad. Sci. Paris, III

Parasitological investigation of the ancient corpse of the Western Han dynasty unearthed from tomb No 168 on Phoenix Hill in Jiangling country

Acta Acad. Med. Wuhan

Nematode larvae in fossilized animal coprolites from lower and middle Pleistocene sites, central Italy

J. Parasitol.

Ancient bone DNA amplified

Nature

Amplification de fragments d'ADN mitochondrial partir de dents et d'os humains anciens

C. R. Acad. Sci. Paris 310 Ser

Research note
Ancient DNA from Ascaris: extraction amplification and sequences from eggs collected in coprolites