From modern-day parasitology to paleoparasitology: the elusive past record and evolution of Cryptosporidium.

Recent efforts have been made to review the state of the art on a variety of questions and targets in paleoparasitology, including protozoan taxa. Meanwhile, these efforts seemed to let aside Cryptosporidium, and we then intended to review its paleoparasitological record to assess its past distribution and favored detection methods, and eventually highlight needed research trajectories. This review shows that contrary to other parasites, most of the positive results came from South-American sites and coprolites rather than sediment samples, highlighting the need to test this kind of material, notably in Europe where many negative results were reported in the published literature from sediment samples. Moreover, aDNA-based detections are nearly absent from the paleoparasitological record of this parasite, though punctually shown successful. With their potential to address the evolutionary history of Cryptosporidium species, notably through their 18S rRNA tree, aDNA-based approaches should be encouraged in the future. In sum, and though the limits of currently used methods and materials remain unclear, this review highlights the potential role of coprolites and aDNA for the study of Cryptosporidium species in the past and how this history shaped their current diversity and distribution, notably among human populations but also farm animals.

Implications of the prevalence of Ascaris sp. in the funerary context of a Late Antique population (5th-7th c.) in Granada (Spain).

OBJECTIVE: To evaluate the prevalence of gastro-intestinal parasites in human remains from Late Antiquity (5th - 7th c.) Granada (Spain). MATERIALS: The study included pelvic and cranial control samples from 17 skeletons from the archaeological sites of Los Mondragones (n = 13) and Rafael Guillén (n = 4). METHODS: In the paleoparasitological study, soil samples from pelvic area and cranium were analyzed using the rehydration, homogenization, and micro-sieving method and visualization under brightfield microscopy. RESULTS: Ascaris sp. eggs were detected in pelvic samples from seven individuals. CONCLUSIONS: These findings may indicate that this parasite was endemic. Its detection frequency is one of the highest reported at group level in an osteological series from Late Antiquity. SIGNIFICANCE: The prevalence of Ascaris sp. associated with skeletal remains has implications for assessing the lifestyle and health of populations in southern Spain during the Late Antique period. LIMITATIONS: The number of individuals is small and taphonomic processes could have limited paleoparasitological findings SUGGESTIONS FOR FURTHER RESEARCH: Future interdisciplinary studies of this type are warranted in larger osteological series to improve knowledge of parasitosis in the past.

Multi-proxy analyses of a mid-15th century Middle Iron Age Bantu-speaker palaeo-faecal specimen elucidates the configuration of the 'ancestral' sub-Saharan African intestinal microbiome.

BACKGROUND: The archaeological incidence of ancient human faecal material provides a rare opportunity to explore the taxonomic composition and metabolic capacity of the ancestral human intestinal microbiome (IM). Here, we report the results of the shotgun metagenomic analyses of an ancient South African palaeo-faecal specimen. METHODS: Following the recovery of a single desiccated palaeo-faecal specimen from Bushman Rock Shelter in Limpopo Province, South Africa, we applied a multi-proxy analytical protocol to the sample. The extraction of ancient DNA from the specimen and its subsequent shotgun metagenomic sequencing facilitated the taxonomic and metabolic characterisation of this ancient human IM. RESULTS: Our results indicate that the distal IM of the Neolithic 'Middle Iron Age' (c. AD 1460) Bantu-speaking individual exhibits features indicative of a largely mixed forager-agro-pastoralist diet. Subsequent comparison with the IMs of the Tyrolean Iceman (Ötzi) and contemporary Hadza hunter-gatherers, Malawian agro-pastoralists and Italians reveals that this IM precedes recent adaptation to 'Western' diets, including the consumption of coffee, tea, chocolate, citrus and soy, and the use of antibiotics, analgesics and also exposure to various toxic environmental pollutants. CONCLUSIONS: Our analyses reveal some of the causes and means by which current human IMs are likely to have responded to recent dietary changes, prescription medications and environmental pollutants, providing rare insight into human IM evolution following the advent of the Neolithic c. 12,000 years ago. Video Abtract.

Worldwide paleodistribution of capillariid parasites: Paleoparasitology, current status of phylogeny and taxonomic perspectives.

INTRODUCTION: Paleoparasitology, the study of parasites in the past, brings the knowledge of where and when they occurred in preterit populations. Some groups of parasites, as capillariids, have a complex and controversial systematic, hindering the paleoparasitological diagnosis. In this article, we synthesized the occurrence of capillariids in both the New and the Old World in ancient times, and discussed the difficulty of the diagnosis of species and the strategies for identification. The present review also shows the current status of the phylogeny in capillariids and indicates the necessity to try new approaches for a better understanding of capillariid paleodistribution. METHODS: For the systematic review, a predefined guideline defined by PRISMA was used. The articles collected were identified, screened, and included in the review following criteria for eligibility. The current status of the phylogeny of capillariids was accessed using MUSCLE, Bioedit v.7.0.5 and MEGA v. 7.0.21 programs. RESULTS: The review discussed 38 articles that presented information about capillariids in past populations. Most of capillariid eggs found in the New and Old World were not identified. However, Calodium hepaticum eggs were the most identified, as some from Eucoleus genus. It was observed that sites from the New World had a better chance for capillariid egg identification, due to previous knowledge of its host, when compared to the Old World. In the 18S rDNA phylogenetic analyses, two datasets were constructed, one including sequences from 7 Moravec's genera, where 3 genus-specific clusters, with high bootstrap values, could be observed for Capillaria (ML = 99%, NJ = 96%), Eucoleus (ML / NJ = 100%) and Paratrichosoma (ML / NJ = 100%). A fourth cluster of 18S rDNA dataset I revealed lack of definition of Pearsonema and Aonchotheca genera. The 18S rDNA dataset II comprised 8 Moravec's genera and defined 3 clusters, 2 genus-specific for Eucoleus (ML = 99%, NJ = 100%) and Capillaria (ML / NJ = 98%). The third 18S rDNA dataset II cluster included 6 genera and exhibited, once again, Pearsonema and Aonchotheca poor discrimination. The cox1 gene data consist of 4 Moravec's genera, and in spite of grouping some species-specific clusters, did not show genera-specific definition. CONCLUSIONS: Despite the numerous archaeological findings, both in the New and the Old Worlds, the identification of capillariid species based on the morphology and morphometry of eggs remains imprecise, often resulting in a generic diagnosis of a group or morphotype of capillariid. Capillariid is one of the most diverse group of helminths recovered in archaeological sites. The phylogenetic trees produced in this study showed limited genetic information available, unresolved genera and incongruence with the classical taxonomy. The elucidation of the paleodistribution of capillariids can give insights of the ancient host-parasite associations but also in modern sceneries.

Assessing the Parasitic Burden in a Late Antique Florentine Emergency Burial Site.

Excavation (2008-2014) carried out under the Uffizi Gallery (Florence, Italy) led to the discovery of 75 individuals, mostly buried in multiple graves. Based on Roman minted coins, the graves were preliminarily dated between the second half of the 4th and the beginning of the 5th centuries CE. Taphonomy showed that this was an emergency burial site associated with a catastrophic event, possibly an epidemic of unknown etiology with high mortality rates. In this perspective, paleoparasitological investigations were performed on 18 individuals exhumed from 9 multiple graves to assess the burden of gastrointestinal parasitism. Five out of eighteen individuals (27.7%) tested positive for ascarid-type remains; these are considered as "decorticated" Ascaris eggs, which have lost their outer mammillated coat. Roundworms (genus Ascaris) commonly infest human populations under dire sanitary conditions. Archaeological and historical evidence indicates that Florentia suffered a period of economic crisis between the end of 4th and the beginning of the 5th centuries CE, and that the aqueduct was severely damaged at the beginning of the 4th century CE, possibly during the siege of the Goths (406 CE). It is more than plausible that the epidemic, possibly coupled with the disruption of the aqueduct, deeply affected the living conditions of these individuals. A 27.7% frequency suggests that ascariasis was widespread in this population. This investigation exemplifies how paleoparasitological information can be retrieved from the analysis of sediments sampled in cemeteries, thus allowing a better assessment of the varying frequency of parasitic infections among ancient populations.

Paleoparasitology of Merovingian Corpses Buried in Stone Sarcophagi in the Saint-Martin-au-Val Church (Chartres, France).

Paleoparasitological analysis was carried on 4 Merovingian skeletons, dated from the late-5th to the late-9th centuries, and recovered in the church of Saint-Martin-au-Val in Chartres (Center region, France). The corpses were buried in stone sarcophagi, which were still sealed at the time of excavation. Parasite marker extraction was conducted on sediment samples taken from the abdominal and pelvic regions, but also on samples taken from under the head and the feet as control samples. Microscopic observation revealed the presence of 3 gastrointestinal parasites, namely the roundworm (Ascaris lumbricoides), the whipworm (Trichuris trichiura) and the fish tapeworm (genus Diphyllobothrium). This analysis contributes to a better knowledge of the health status and the lifestyle of ancient medieval populations during the Merovingian period, for which very few paleoparasitological data were available, up until now. It demonstrates the presence of the fish tapeworm for the first time during this period.

Palaeoparasitology and palaeogenetics: review and perspectives for the study of ancient human parasites.

While some species of parasites can be identified to species level from archaeological remains using microscopy (i.e. Enterobius vermicularis, Clonorchis sinensis), others can only be identified to family or genus level as different species produce eggs with similar morphology (i.e. Tænia sp. and Echinococcus sp.). Molecular and immunological approaches offer the possibility to provide more precise determination at the species level. They can also identify taxa when classic parasite markers such as eggs or cysts have been destroyed over time. However, biomolecules can be poorly preserved and modern reference DNA is available only for a limited number of species of parasites, leading to the conclusion that classic microscopic observation should be combined with molecular analyses. Here we present a review of the molecular approaches used over the past two decades to identify human pathogenic helminths (Ascaris sp., Trichuris sp., E. vermicularis, Fasciola sp. etc.) or protists (Giardia sp., Trypanosoma sp., Leishmania sp. etc.). We also discuss the prospects for studying the evolution of parasites with genetics and genomics.

A First Case of Human Trichuriasis from a Roman Lead Coffin in France.

A paleoparasitological study was carried out on 2 lead coffins recovered from the Roman site of Jaunay-Clan (near Poitiers, France). For the first time, this particular type of burial gave positive parasitological results, and eggs of the whipworm Trichuris trichiura were identified in 1 individual. In the present case, thanatomorphose associated with funerary practices may explain the scarcity of the recovered eggs. However, human whipworm has now been observed in 9 individuals dated to the Roman period. The very high frequency of Trichuris sp. eggs in Roman archaeological sites (up to 80%) suggests that fecal peril, hygiene, and waste management were problematic during this period. Finally, due to the fact that very few analyses have been conducted on human bodies dated to the Roman period, more analyses must be performed in the future to provide further information about diseases in the Roman world.

Human Coprolites as a Source for Paleomicrobiology.

The paleomicrobiology of coprolites, which are fossilized fecal materials, has already yielded data about various organisms, including micro-eukaryotes, bacteria, and archaea, thus expanding our comprehension of ancient human dietary habits, gut microbiota, and intestinal and systemic infections. This mini-review briefly describes previous works and summarizes the main techniques used in handling coprolites and the findings obtained about ancient gut microbiota. Past intestinal and systemic infections are outlined.

Past Intestinal Parasites.

This chapter aims to provide some key points for researchers interested in the study of ancient gastrointestinal parasites. These few pages are dedicated to my colleague and friend, Prof. Adauto Araújo (1951-2015), who participated in the writing of this chapter. His huge efforts in paleoparasitology contributed to the development and promotion of the discipline during more than 30 years.

Archaeological occurrences and historical review of the human amoeba, Entamoeba histolytica, over the past 6000years.

Understanding parasite history and the evolution of host/parasite relationships is one of the most important aspects of paleoparasitology. Within the framework of this research topic, this paper focuses on the human pathogenic amoeba, Entamoeba histolytica. The compilation of all the available archaeological data concerning this parasite leads to a first glimpse of the history of this parasite of current medical importance. Paleoparasitological investigation into this parasite uses immunological techniques and shows that the modern strain of E. histolytica has been present in Western Europe since at least the Neolithic period (3700yearsBCE), and could have originated in the Old World. The appearance of the modern amoeba strain in the pre-Columbian Americas and the Middle East around the 12th century CE gives rise to hypotheses as to how human migrations (Atlantic or Pacific routes) contributed to the diffusion of this pathogen, resulting in its current distribution. This compilation proves that parasites are valuable proxies for studying past human and animal migrations, and should be given more consideration in the future.

A New High-Throughput Approach to Genotype Ancient Human Gastrointestinal Parasites.

Human gastrointestinal parasites are good indicators for hygienic conditions and health status of past and present individuals and communities. While microscopic analysis of eggs in sediments of archeological sites often allows their taxonomic identification, this method is rarely effective at the species level, and requires both the survival of intact eggs and their proper identification. Genotyping via PCR-based approaches has the potential to achieve a precise species-level taxonomic determination. However, so far it has mostly been applied to individual eggs isolated from archeological samples. To increase the throughput and taxonomic accuracy, as well as reduce costs of genotyping methods, we adapted a PCR-based approach coupled with next-generation sequencing to perform precise taxonomic identification of parasitic helminths directly from archeological sediments. Our study of twenty-five 100 to 7,200 year-old archeological samples proved this to be a powerful, reliable and efficient approach for species determination even in the absence of preserved eggs, either as a stand-alone method or as a complement to microscopic studies.

The horse pinworm (Oxyuris equi) in archaeology during the Holocene: Review of past records and new data.

This paper focuses on the horse pinworm, Oxyuris equi, in archaeology during the Holocene period, and presents an overview of past published occurrences, early mentions in texts, and new data from our paleoparasitology research. This original compilation shows that the most ancient record of the horse pinworm dates to ca. 2500 years before present (ybp) in Central Asia and to ca. 2020 ybp in Western Europe. It also shows that the parasite is not detected on the American continent until contemporary periods. The role of European migrations from 1492 (Christopher Columbus) is discussed to explain the transfer of the horse pinworm from the Old World to the Americas. The absence of any record of this parasite before ca. 2500 ybp in Eurasia could be explained by parasite ecology, unfavorable sampling and scarcity of horse archeological remains. For the Americas, the absence of horse for long periods can be an additional explanation for the absence of the parasite.

Intestinal parasites in First World War German soldiers from "Kilianstollen", Carspach, France.

Paleoparasitological investigations revealed the presence of intestinal helminths in samples taken from the abdominal cavities of two German soldiers, recovered in the First World War site named "Kilianstollen" in Carspach, France. Eggs from roundworm, whipworm, tapeworm and capillariids were identified. The morphological and morphometrical comparison, followed by statistical analyses, showed that the Carspach capillariid eggs are similar to rodent parasites. Poor sanitary conditions in the trenches, the lack of knowledge of parasites, and the widespread presence of commensal animals, can explain the occurrence of such parasites in human intestines. This study is the second dealing with 20th century human samples. It confirms the presence of intestinal worms in First World War German soldiers. In this case study, the application of statistics to precise measurements facilitated the diagnosis of ancient helminth eggs and completed the microscopic approach.

Polyphasic analysis of a middle ages coprolite microbiota, Belgium.

Paleomicrobiological investigations of a 14(th)-century coprolite found inside a barrel in Namur, Belgium were done using microscopy, a culture-dependent approach and metagenomics. Results were confirmed by ad hoc PCR--sequencing. Investigations yielded evidence for flora from ancient environment preserved inside the coprolite, indicated by microscopic observation of amoebal cysts, plant fibers, seeds, pollens and mold remains. Seventeen different bacterial species were cultured from the coprolite, mixing organisms known to originate from the environment and organisms known to be gut inhabitants. Metagenomic analyses yielded 107,470 reads, of which known sequences (31.9%) comprised 98.98% bacterial, 0.52% eukaryotic, 0.44% archaeal and 0.06% viral assigned reads. Most abundant bacterial phyla were Proteobacteria, Gemmatimonadetes, Actinobacteria and Bacteroidetes. The 16 S rRNA gene dataset yielded 132,000 trimmed reads and 673 Operational Taxonomic Units. Most abundant bacterial phyla observed in the 16 S rRNA gene dataset belonged to Proteobacteria, Firmicutes, Actinobacteria and Chlamydia. The Namur coprolite yielded typical gut microbiota inhabitants, intestinal parasites Trichuris and Ascaris and systemic pathogens Bartonella and Bordetella. This study adds knowledge to gut microbiota in medieval times.

Viruses in a 14th-century coprolite.

Coprolites are fossilized fecal material that can reveal information about ancient intestinal and environmental microbiota. Viral metagenomics has allowed systematic characterization of viral diversity in environmental and human-associated specimens, but little is known about the viral diversity in fossil remains. Here, we analyzed the viral community of a 14th-century coprolite from a closed barrel in a Middle Ages site in Belgium using electron microscopy and metagenomics. Viruses that infect eukaryotes, bacteria, and archaea were detected, and we confirmed the presence of some of them by ad hoc suicide PCR. The coprolite DNA viral metagenome was dominated by sequences showing homologies to phages commonly found in modern stools and soil. Although their phylogenetic compositions differed, the metabolic functions of the viral communities have remained conserved across centuries. Antibiotic resistance was one of the reconstructed metabolic functions detected.

Diphyllobothrium in the past: Review and new records.

This paper presents a paleoparasitological review of the fish tapeworm genus Diphyllobothrium. Comprehensive data from published articles and original results from the Reims paleoparasitology laboratory show that the fish tapeworm has co-evolved with humans for several thousand years, influenced by human dietary habits, cultural differences, and sometimes environmental changes. This taxon has been present in the Old World for at least 9500 years (early Neolithic period) and in the New World for 10,000 years. Unlike the current geographical distribution of other parasite species, the human history of the fish tapeworm has still not been elucidated. Future studies and advances in paleoparasitological research may help to resolve the question of the evolution of this parasite.

Testing new parasite egg extraction methods in paleoparasitology and an attempt at quantification.

In order to improve paleoparasitological analyses, we tested different acid (hydrochloric and hydrofluoric acids) and base (sodium hydroxide) combinations for parasite egg extraction in archaeological sediments. We used a method of egg counting to compare these results to those obtained with the standard paleo-parasitological RHM extraction protocol (rehydration-homogenization-micro-sieving). Tests show that the use of hydrochloric acid results in a concentration of some taxa like Ascaris sp. or Trichuris sp. and an appreciable decrease in vegetal and mineral remains. However, tests also show that acid use systematically decreases parasite species identified, as compared to the standard RHM protocol. Base use yielded even more negative results with systematically lower biodiversity than with the standard protocol, probably due to chemical processes on chitin contained in the eggshell. These results suggest acids and sodium hydroxide should be used as little as possible during extraction due to the damages they cause to the eggs of some parasite species. The counting method was an efficient technique with which to demonstrate our results and could be used in future paleoparasitology studies.

Ancient dicrocoeliosis: Occurrence, distribution and migration.

This paper focuses on the lancet fluke, Dicrocoelium sp., and includes a complete review of the literature as well as numerous new research results. The compilation of all these data led to a global overview of ancient dicrocoeliosis history. The presence of the lancet fluke in Western Europe was attested from 550,000 years BP to the 16th century AD Moreover, the parasite was identified in the New World around the 17th century AD following the colonization of Canada by Europeans. The role of human and animal migrations is discussed.