Intestinal helminths as a biomolecular complex in archaeological research

Abstract

Enteric helminths are common parasites in many parts of the world and in the past were much more widespread both geographically and socially. Many enteric helminths are relatively long-lived in the human host, often benign or of low pathogenicity while producing large numbers of environmentally resistant eggs voided in the faeces or found associated with individual remains (skeletons and mummies). The combination of helminth characters offers opportunities to the field of historical pathogen research that are quite different to that of some of the more intensively studied high impact pathogens. Historically, a wealth of studies has employed microscopic techniques to diagnose infection using the morphology of the helminth eggs. More recently, various ancient DNA (aDNA) approaches have been applied in the archaeoparasitological context and these are revolutionizing the field, allowing much more specific diagnosis as well as interrogating the epidemiology of helminths. These advances have enhanced the potential for the field to provide unique information on past populations including using diseases to consider many aspects of life (e.g. sanitation, hygiene, diet, culinary practices and other aspects of society). Here, we consider the impact of helminth archaeoparasitology and more specifically the impact and potential for application of aDNA technologies as a part of the archaeologists' toolkit.

This article is part of the theme issue ‘Insights into health and disease from ancient biomolecules’.

1. Introduction

A wide range of enteric parasites have afflicted mankind over the course of history. In this paper, we consider the enteric parasites of vertebrates as biomolecular tools to study historical events. We will explore how these parasites represent biomarkers of human activities and events, with a focus on how molecular approaches enhance and provide added value. For a detailed background on the detection of parasites in archaeological contexts, the reader is directed towards some excellent reviews [1–3]. Although all infections are parasitic on their hosts, the term is more generally applied to the eukaryotic infectious organisms that range from single-celled protozoa through to much larger helminths (worms) and ectoparasitic arthropods. From an archaeological perspective, some infections are more informative than others, chiefly due to the environmental resilience of some parasite-associated structures (e.g. the cysts, oocysts and eggs of enteric parasites). In some circumstances where the preservation state of human remains is very high (e.g. mummified bodies), it is possible to detect the presence of systemic parasites including the malaria-causing Plasmodium spp. parasites or trypanosomes using ancient DNA (aDNA) or antibody-based methods [4–8]. However, these soft tissue resident parasites represent special cases and appropriate material is often not available. We will focus on parasites that infect the gut of hosts which transmit using the environmentally resistant stages (e.g. eggs) that are widely found in archaeological contexts.

Pathogens vary in their effects on the human population; some cause devastating disease whereas others exhibit much lower levels of pathogenicity. Many of the ‘high impact pathogens’ that kill or disfigure the infected individuals have received considerable attention (e.g. the causal agents of plague, tuberculosis, leprosy or typhoid) [9–15]. By contrast, the enteric helminths may not have caused acute disease but were common infections in many historical populations. Indeed, in the past, many intestinal helminths had a much broader geographical distribution than in modern times [1–3,16–18]. Various aspects of helminth biology make them attractive sources of information for archaeologists providing insights into the life of past populations. Helminth eggs are environmentally resilient [19], being readily detectable using light microscopy and retaining considerable diagnostic structure in a wide array of archaeological contexts (e.g. [20–23]). Helminths also have complex life histories that can involve different host species, with transmission to humans often occurring via the faecal–oral route or by consumption of particular contaminated food types. Indeed, the helminth egg does more than indicate infection; it can be considered a biomolecular marker of socioeconomic, behavioural and environmental conditions. The presence of eggs and an estimation of their preservation state can also be used to pre-select samples for other analyses including those involving aDNA. The following sections will elaborate on these characteristics and discuss how molecular approaches substantially enhance the potential for parasitological research within the archaeologist's toolbox.

2. The helminths with intestinal associations: a brief guide to the main groups detected in archaeological contexts

The intestinal helminths fall into three broad groups: the roundworms (nematodes), flatworms (trematodes) and tapeworms (cestodes). Members of these groups parasitize most vertebrates, and in the case of humans, some of these parasites represent major burdens on modern populations, principally those in less well-developed countries [24]. The life cycles of parasites that infect the human gut are often complex, involving many stages and sometimes multiple hosts, and the reader is directed towards the many medical parasitology texts for comprehensive coverage of this topic [25]. The presence of enteric helminths is obviously a health issue for the individual but careful consideration of the life cycle, in particular, the process of transmission to humans, allows much wider commentary on the human relationship with their environment. For example, those parasites that are faecal–oral transmitted between humans are found at much higher prevalence in areas with less effective hygiene and sanitation. Some parasitic infections represent an ‘accidental’ zoonotic transfer from other animals and may indicate close proximity to particular groups of source animals. The third group of helminths enter the human via food and are, therefore, influenced by dietary and culinary practices.

In brief, the key infections that are readily reported in archaeological contexts include the soil-transmitted helminths (STH)—principally the human roundworm (Ascaris spp.), the whipworm (Trichuris spp.) and the hookworms (e.g. Necator americanus) [1–3]. Of these the most common in archaeological deposits are the nematodes Ascaris lumbricoides and Trichuris trichiura (e.g. [20,21,23,26–30], figure 1). These nematodes are also prevalent in some parts of the world today, particularly in tropical and sub-tropical low or middle income countries [24]. The human pinworm, Enterobius vermicularis, has also been detected in some archaeological contexts [31] and is a common childhood infection throughout the world [32]. E. vermicularis is less commonly identified in archaeological deposits (compared with STH). This may be due to a less robust egg structure or that eggs are sticky and deposited in the perianal area by the gravid female pinworm. Indeed, with modern samples, faecal examination only detects 5–15% of infected cases [32]. In the prehistoric Americas, wide variation in the rates of detection of E. vermicularis eggs was noted, with increased levels generally associated with agricultural communities [33]. Further studies on the detection and epidemiology of E. vermicularis in different historical contexts may be useful, as this parasite is best transmitted by close human–human interactions rather than the deposit-and-ingest transmission associated with Ascaris and Trichuris.

Figure 1.

The eggs of the most common enteric nematodes found in archaeological deposits. Photomicrographs depict eggs of Ascaris sp. (a) and Trichuris sp. (b) from a medieval latrine in Lübeck. Scale bar: 20 µm.

The trematodes include Fasciola hepatica (the liver fluke), various Schistosoma spp. (the blood flukes) and Clonorchis sinensis (Chinese liver fluke) [31,34]. Some of these parasites are geographically localized and found only in certain regions of the world, such as C. sinensis in Asia [35]. Clonorchis sinensis has been identified in Korean archaeological deposits [36] and at a Silk Road relay station in China [34]. The schistosomes that infect humans also have a restricted geographical range related to the distribution of their freshwater snail intermediate hosts. Although adult schistosomes do not reside in the intestine, the eggs are voided in faeces or urine and may be found in archaeological deposits. Schistosoma eggs have been reported in mummified remains from ancient Egypt [37]. Further, some intestinal-resident helminths are transferred into humans via uncooked or undercooked meat or fish; these include cestodes (Taenia spp. and Diphyllobothrium latum [syn. Dibothriocephalus latus]), nematodes such as Anisakis sp., as well as trematodes such as C. sinensis.

3. What makes helminths so informative in an archaeological context?

Helminth eggs are environmentally resistant and can readily be detected in a wide variety of archaeological contexts. Most helminths are also highly fecund, producing large numbers of eggs over prolonged periods of time thereby providing a consistent source of material for analysis by archaeoparasitologists. Helminth eggs are relatively large (approx. 30–90 µm, depending on the species) and easily observed with a light microscope at moderate magnification [25]. In many cases, the shape and structure of the egg can be used for diagnosis and enumeration of parasites present in a sample. The eggs of intestinal helminths are typically shed in faeces and can be found in various communal deposits (e.g. latrines or waste pits) or associated with the pelvic (typically sacral) region of skeletal or mummified remains. Indeed, microscopy has been employed to detect helminth eggs for many years including the pioneering work performed on Egyptian mummies [37] and bog bodies [38]. In addition to providing a diagnostic structure, the resilience of helminth eggs protects the material within them, including aDNA (discussed below).

A second key character of helminth infections is that they are almost universally less pathogenic than many of the systemic infections studied in archaeological contexts (e.g. plague, typhoid or tuberculosis). Indeed, individuals are often infected with helminth parasites for many years and in the absence of severe clinical symptoms are able to maintain their daily routine. Hence, studying helminth infections provides a very different perspective on the life of past populations than with those pathogens that caused debilitating or life-threatening disease.

Helminth infections are endemic and prevalent, with infection rates often greater than 20–40% for STH in less well-developed countries [16] and this was also true of people in the past [39]. Many helminths were also more widespread in past populations compared with their modern distribution. For example, helminth eggs can be found in historical deposits throughout Europe and North America where they are no longer endemic (reviewed in [1–3]). Indeed, most modern helminth infections detected within Europe can be traced to travel-related acquisition events [25,40].

In an archaeological context, parasite eggs are often found in deposits that contain faecal material (including infills of latrines, cesspits, waste deposits or from the abdominal region of burials). This material, where collected, is incredibly valuable to the helminth researcher and relatively accessible; in particular, there are fewer restrictions or sensitivities in terms of destructive sampling compared with skeletal remains. The presence of human parasites can be used to determine whether any organic-rich deposit contains human waste [41], although this can be limited by the fact that formal differentiation of helminth eggs from humans versus animals by microscopy can be difficult. The application of molecular methods to detect and sequence parasite aDNA can help resolve some of the more subtle identification issues (see below).

4. Helminth applications in archaeological research

Since the versatility of parasites for archaeological research was proposal by Pike in 1968 [41] there have been a multitude of reports of helminth eggs in archaeological contexts (e.g. [20,21,23,27–30,42]). Reports include detection of eggs in the context of communal deposits (e.g. latrines or waste pits) and single individuals (associated with skeletal remains or mummies) as well as implements used for hygiene purposes [2,34,43]. Identifying eggs in each of these circumstances provides a variety of information that relate to general health, levels of sanitation/hygiene, dietary/culinary practices and travel. The most common infections reported in archaeological contexts are those considered soil-transmitted helminths (STH) including the nematodes, Ascaris spp. and T. trichiura. Heavy infestations can cause clinical disease, generally related to intestinal issues, but in children that often harbour higher intensities of infection, there may be associated growth and developmental effects [44]. However, in many cases, particularly in adults, these infections persist for many months or years without causing acute disease. Since Ascaris and Trichuris are transmitted via the faecal–oral route directly between humans the numbers of eggs detected represents a good indicator of the levels of hygiene and sanitation available to the population. The prevalence of STH may also have been supported by the use of human waste (night soil) to fertilize arable fields. These infections are very rare and not endemic in most of modern Europe and North America but were readily detected in past populations. It is likely that improvements in sewage and clean water systems at the end of the nineteenth and early twentieth century led to reduced STH infection. There are reports of helminth infection within Europe in periods around the two world wars (e.g. [45,46]), however it is unclear whether these reflect conflict-based increases in infection rates or are representative of the broader circumstance. Indeed, conflict may have reduced the levels of sanitation especially associated with the most affected areas (e.g. WWI trenches) but may also have led to the importation of infection with the large groups of soldiers entering conflict zones. It is noteworthy that many of the reductions in STH infections occurred prior to the introduction of modern anthelminthic drugs that are considered the cornerstone of modern eradication/control efforts [24,47]. The timing and factors that led to the decline in STH infections in Europe and North America are areas that deserve more attention, and these could have implications for modern control efforts in STH endemic countries.

In modern endemic populations, STH infections are associated with poverty, being considered an indicator of insufficient access to sanitation and clean drinking water [48]. However, in past populations, these parasites infected humans in all sectors of society, including the wealthy and privileged [20,49,50]. The variety of reports of parasite eggs in archaeological contexts as well as the relative ease by which they are detected allows us to speculate that these infections were at least as common throughout history as they are in modern endemic regions. Studies examining large groups of individual-based samples will help to identify the prevalence of these infections in historical populations, and whether there were any changes in prevalence over time or space.

The presence of eggs from helminths that are transferred to humans via fish or red meat can be interpreted in terms of the diet and culinary practices of the local population. For example, the presence of Taenia spp. indicates the consumption of uncooked or undercooked pork or beef, depending upon whether the eggs are from Taenia saginata (beef), T. solium (pork) or T. asiatica (pork, in Asia only). Unfortunately, egg morphology does not allow for differentiation between these Taenia spp., although molecular methods have proven useful in this context (see below, [20]). The presence of eggs from the cestode D. latum in human samples indicates the consumption of uncooked or undercooked freshwater fish. These parasites have been identified in a range of studies (e.g. [51–54]) but the numbers found in latrine samples from the medieval city of Lübeck deserve a mention. Lübeck played a key role in the operation of the Hanseatic League, a major medieval trading organization focussed on the Baltic and North Sea regions [55]. Excavation of a large set of latrines in a merchant's quarter of Lübeck revealed that in addition to the large numbers of STH nematodes, there were also large numbers of the food-transmitted cestodes Taenia spp. and D. latum in the deposits. The large numbers of cestode eggs suggested unusual culinary practices with both red meat and freshwater fish [20]. Moreover, there were dramatic changes in the numbers of eggs from each of these cestodes over time, with D. latum more highly represented in samples that dated from earlier periods and T. saginata in samples from later periods. Indeed, the change from high to low levels of D. latum at around 1300 CE could be interpreted in a number of ways. The simplistic explanation that less freshwater fish was consumed after 1300 CE was not supported by analyses of vertebrate DNA signatures in the deposits [20]. Other events may have either led to a change in supply of contaminated fish, or some other factors breaking the parasite life cycle. Careful consideration of the life history of the parasite offered an alternative explanation. The D. latum life cycle involves at least three hosts: humans, copepods and fish. After the egg exits the human and enters a body of water it matures, hatches and the coracidium stage is ingested by small permissive crustaceans (e.g. copepods) before developing into a larval form that can infect fish, where it further develops. At this stage, it can infect humans but can also be passed between fish by predation. Once in the human gut the mature tapeworm develops in the intestine. It is noteworthy, that copepods and other small crustaceans are sensitive to levels of pollutants that may not kill fish [56]. In Lübeck, there was an expansion in industry along the river (principally butcheries and tanneries) at around the time of the reduction in D. latum eggs, and it is possible that the resultant pollution reduced the availability of the first intermediate host, thereby breaking the D. latum life cycle.

The presence of parasites more commonly found in livestock or other animals can be used as a potential indicator of close proximity and zoonotic infection of humans. Some parasites are highly species-specific and can be used to identify deposits which are contaminated with both human and animal faeces. Identifying and disentangling these events is a tough proposition and difficult (or even impossible) to achieve by microscopic examination of egg morphology. For example, there are multiple species of the genus Trichuris that primarily infect different hosts (e.g. T. muris, T. suis and T. ovis), but some that primarily infect domesticated animals can also infect humans (e.g. the dog parasite, T. vulpis). The development of molecular methods to identify helminths at greater resolution has begun to open this topic to greater scrutiny (see below).

Some helminths are localized to particular geographical areas, yet on occasion eggs are detected far from the location where infection would have been transmitted. The reasons for this are that many helminths can persist in, and travel with, their human hosts for prolonged periods without overt clinical symptoms. These eggs can then be deposited at a site where the conditions would not maintain a transmission cycle for the parasite in question. Examples of this may be the detection of D. latum in Acre and Jerusalem, a parasite much more common in more northerly European regions [57,58]. Similarly, the detection of C. sinensis at the Silk Road relay station at Xuanquanzhi (111 BCE–109 CE), at the eastern margin of the Tarim Basin in north-western China [34], is over 1000 km from any wetland habitat which would support Clonorchis transmission.

5. The impact of molecular approaches in the context of ancient helminths

The growing field of aDNA research has revolutionized the study of many systemic infections in archaeology (e.g. [10,59,60]). This expanding body of work has led to a wealth of information, including complete ancient genome sequences of a number of human pathogens such as Y. pestis [9], M. tuberculosis [61,62] or M. leprae [63]. These studies are considered in detail elsewhere, but indicate the potential of aDNA in the study of past infections. In terms of enteric helminths, molecular approaches have begun to be exploited and the potential for these approaches is immense. The ability to differentiate between helminths with greater specificity allows robust identification of human- versus animal-derived parasites, and within the human parasites, better diagnosis can be key in identifying the source of infection (e.g. with Taenia spp.). Molecular methods are also beginning to provide insights into the historical epidemiology, molecular diversity and evolution of the helminths [20,51,64]. These areas will be discussed after considering the practical issue of how the helminth egg affects the acquisition of parasite aDNA.

The enteric parasites are not represented in aDNA from bones or teeth simply because the pathogens detected in these types of sample are generally those that reach high levels of systemic infection, particularly those present at high frequency in the blood of the affected individuals at the time of death. With the enteric parasites, transmission-associated structures (i.e. eggs of helminths or cysts/oocysts of protozoa) protect and isolate aDNA from the environment. Enrichment techniques have included filtration through sieves of different pore sizes, flotation on density gradients and/or differential centrifugation (e.g. [20,28,45,52]). The approach selected for a specific application has to be carefully considered, as each of these approaches has advantages in terms of purity of material versus potential disadvantages such as the loss of parasite material. Sampling strategies and decisions on whether to employ purification or enrichment strategies are key to any archaeoparasitological analyses and can differ according to the material under examination. Reinhard [65] has recently considered many of these points and we would also reinforce the need for rigour in establishing and reporting methods. Helminth eggs are structurally complex and resilient to the effects of a wide range of environmental conditions [19] which protects the aDNA, but also necessitates the use of effective disruptive procedures to release parasite aDNA. These procedures include sonication, freeze–thaw cycles and/or disruption using small glass beads with high energy mechanical devices. It is important to note that the disruption approaches also differ in their capacity to disrupt eggs of different helminths, and in our hands the eggs of some (e.g. Taenia) are more difficult to disrupt than others (e.g. Ascaris and Trichuris) (PG Flammer and AL Smith 2011, unpublished data). In some circumstances, a molecular signature of helminth presence can be detected despite a negative microscopic diagnosis, probably due to the high sensitivity of PCR [51]. Where small amounts of target DNA are being detected by PCR, it is best to report any result as presence/absence rather than as a quantitative measure. Most helminth aDNA signal in latrine samples derives from intact eggs, and the proportion of intact eggs varies considerably in all types of deposit [66]. Hence, it is important to maintain microscopy-based enumeration alongside any quantitative or semi-quantitative molecular methods (as also indicated by [2,51,67]).

The first aDNA studies on helminths reported obtaining aDNA sequences (fragments of 18S rRNA and cytochrome b) from Ascaris eggs isolated from human coprolites excavated from seventeenth century coprolites in Belgium [26]. Similar studies have reported the use of PCR-based approaches to amplify Ascaris-derived aDNA sequences from latrines, mummified bodies and communal deposits excavated from Asian, South American and other European sites [26,52,68–73]. In the last few years, a much wider array of PCR protocols have been developed to target helminths in archaeological contexts including the other commonly identified nematodes (T. trichiura, Ascaris spp. and E. vermicularis), the cestodes (Taenia spp., Echinoccocus spp. and D. latum) and trematodes (F. hepatica, C. sinensis, Dicrocoelium spp. and Schistosoma spp.) [20,26,51,73]. In most cases, the sequence data has been used to confirm helminth identity within samples, and a range of parasite target genes have been applied to archaeological samples (reviewed in [2]).

There are some circumstances where molecular diagnosis helps to resolve issues that would not be convincingly dealt with using microscopic approaches. For example, aDNA can be used to confirm that Trichuris eggs are derived from the human infecting specialist, T. trichiura, rather than other trichurids that either do not infect humans (e.g. T. muris) or those that occasionally infect humans, but are considered specialist for other hosts (e.g. T. suis in pigs or T. vulpis in dogs). Where significant non-human specialist Trichuris spp. aDNA is found in a sample, it may be important to consider whether the sample includes faeces from non-human sources.

A second example is the differentiation of different species of human-targeting Taenia spp. (i.e. T. solium, T. saginata or T. asiatica). Molecular approaches are necessary to differentiate between the Taenia spp., even in modern samples [74]. PCR-based approaches have also been applied to differentiate Taenia spp. in archaeological samples [20,51], providing important information on the source of infection. T. solium and T. asiatica are both acquired from raw or undercooked pork, whereas T. saginata is acquired from raw or undercooked beef. As the name suggests T. asiatica is mostly transmitted in Asia, whereas T. solium and T. saginata are geographically widespread. Phylogenetic analyses of Taenia spp. reveal a fascinating history of association with humans, with at least two introductions into early hominids (approx. 1 Mya) through hunting and scavenging activities [75,76]. Subsequently, these parasites were influenced by human management and domestication of porcine and bovine intermediate hosts and dispersed during the major migrations out of Africa [75,77]. This is an area where molecular archaeoparasitology approaches will contribute to our understanding of the influence of humans on these parasites. Moreover, the identification of which Taenia spp. are present within a sample immediately has implications for the lifestyle of the population under study, in particular dietary and culinary practices.

It is well established that aDNA is highly fragmented and damaged [59,78–81], which also holds true for the aDNA associated with helminth eggs, and it is important to consider those aspects in the design of suitable primers for PCR amplification. Where aDNA is targeted for purely diagnostic purposes, the PCR amplicon should be very short encompassing at least one, but preferably multiple diagnostic single nucleotide polymorphisms (SNPs) to differentiate between the parasites of interest.

Genomic resources for helminths have improved significantly in recent years [82]. These have been largely focused on human parasites or those used as biomedical models in rodents. There is also increasing interest in molecular identification of parasites and the use of sequence variation in modern epidemiology. The greater breadth and depth of resources will provide new opportunities for contextualizing data obtained from archaeological studies. Since sequence databases may not identify all of the potential products that might be generated with environmental samples (including those used for parasite analysis), it is important that the products are verified. The simplest approach, used by many of the early studies, employed TA-cloning, colony picking and Sanger sequencing (e.g. [26]), but this is a laborious approach and only small numbers of samples can be processed. One alternative higher-throughput approach employs a hybridization-based method whereby PCR products are tested for the ability to hybridize to a known target [83]. This approach is attractive where larger groups of samples are being considered; however, it can become laborious (depending on the number of target parasites) and does not provide sequence information. Hence, the approach more widely used in recent studies involves parallel sequencing bar-coded PCR products (e.g. [20,51]).

In most archaeological contexts, more than one helminth species have been identified, even where samples are derived from single burials [2,38,84]. Therefore, a technique to detect an array of parasite species within a single sample is very attractive, especially when it allows the analysis of a considerable number of samples in parallel. The method published by Côté et al. [51] employs a multiplex PCR-based approach combined with high-throughput sequencing to detect multiple helminths (Taenia, Echinococcus, Diphyllobothrium, Ascaris, T. trichiura, Dicrocoelium, Fasciola and E. vermicularis). With this method, the focus was placed upon detection using very short amplicon lengths (53–113 bp) comparing reasonably well with the microscopic detection of eggs. It is important to remind the reader that this technique should be combined with microscopic evaluation if the numbers of eggs are to be considered.

For some helminth targets, it is possible to routinely generate slightly longer amplicons (up to 200 bp) which may be less sensitive for diagnosis but can provide more sequence data for considering population diversity. This approach was used with a T. trichiura ITS-1 target to differentiate between two groups of T. trichiura [20], one of which was more common in some sites (e.g. medieval Lübeck) than others (e.g. medieval Bristol). Moreover, the slightly longer sequences allowed the observation that some sites contained greater T. trichiura ITS-1 diversity than others. Although many more sites and samples need to be added, the preliminary dataset suggests that genetic diversity of helminths may be able to differentiate more connected sites (e.g. ports) from more isolated sites and may also be able to link the parasite populations between sites [20]. Where PCR-based parallel sequencing approaches are selected, it will be most useful to employ multiple targets with a view to obtaining robust results. It is also important that we standardize protocols and aDNA targets to integrate data more efficiently thereby adding to the power of any individual analysis.

High-throughput shotgun sequencing has proven very powerful in the field of aDNA research; indeed, the genomes of a range of systemic pathogens have been identified as a consequence of studies on human bones and teeth [9,11,15,85]. The shotgun sequencing approach involves random sequencing any aDNA in the sample retaining aDNA damage signatures and providing increased coverage of pathogen DNA. This approach has been successfully applied to samples of communal deposits containing intestinal helminths [52] and allowed the reconstruction of the mitochondrial genomes from three nematodes, the roundworm (Ascaris spp.), as well as the human and rodent whipworms (T. trichiura and T. muris). The sites analysed included three in Denmark, two in the Netherlands and one in each of Lithuania, Jordan and Bahrain, with both Trichuris and Ascaris being detected at significant levels in almost all sites by both microscopic analysis and shotgun sequencing. aDNA from a number of other helminths was detected including parasites where eggs would be derived from humans, livestock and companion animals indicating that some of the samples contained faeces from a range of mammals.

In addition, the full mitochondrial genome sequences of T. trichiura and Ascaris allowed an extensive analysis of potential SNPs, the number of which varied across sites as did the number of inferred dominant haplotypes [52]. These data support the premise that genetic diversity will influence future epidemiological studies and reveal new targets that will be useful alongside the T. trichiura ITS-1 fragment [20]. Interestingly, both shotgun and targeted PCR approaches suggest that the dominant T. trichiura strains found in historic Europe were distinct from Asian strains and perhaps more closely related to those derived from modern Uganda [20,52,64]. In a separate shotgun study of kitchen middens in Greenland, aDNA from the tapeworms (Taenia hydatigena, T. multiceps and Echinococcus spp.) were detected at low levels [86]. With all three of these tapeworms, canids are the host of the mature tapeworm (i.e. definitive host) and the presence of these tapeworms can be interpreted as the samples containing domesticated dog or wild canid (e.g. fox) faeces.

While shotgun sequencing is very powerful it requires large amounts of starting material and can be expensive on a per sample basis. In terms of the physical sample size, the Soe et al. [52] study reported using 75–503 g (mean 183 g) of starting material with communal deposits that contained large numbers of eggs. This compares with much lower amounts of starting material required for PCR-sequencing approaches (less than 5 g). In many circumstances it may not be possible to obtain sufficient material for effective shotgun approaches, for example with single individual derived remains or those with low numbers of eggs. The smaller sample requirements for PCR-sequencing approaches facilitate higher-resolution spatial and temporal (e.g. stratigraphic) sampling of communal features. Integrated approaches could be relevant to many studies and the use of baiting-based approaches represents a logical next step within the field. The use of synthetic baits to enrich for target DNA has been a key development in the study of various pathogens [87], focusing sequencing efforts on the target pathogen. This approach will clearly be important in enriching for helminth DNA; however, due to the very large size of helminth genomes (75–273 Mbp [88–91]), it would be impractical to generate whole-genome enrichment strategies. Nonetheless, a more targeted approach including full mitochondrial sequences supplemented by selected regions of the nuclear genome would represent a powerful approach.

6. Concluding remarks

The use of helminths in archaeoparasitology has a long history, and it is clear that many enteric helminths were much more widespread in past communities. Traditional microscopic methods remain critical, especially for the enumeration and identification of eggs that may have been broken and lost any associated aDNA. However, aDNA studies have demonstrated their potential to enhance archaeoparasitological studies and will have an impact on understanding helminth infections in the past and exploiting them to interrogate the activities and habits of past populations. aDNA enables the diagnosis of infections with much greater certainty, and can provide significant molecular resources to examine the evolution and molecular epidemiology of parasites in the past. Some of the key features of intestinal parasites, including the environmentally resilient transmission stages and the persistence of infection with low clinical pathology, support both traditional and molecular analyses. The relative lack of modern molecular epidemiology with most helminths remains a problem in terms of contextualising data obtained from archaeological contexts, but as both modern and ancient data sets become larger these problems will be reduced.

Although in this article we have focused on enteric helminths and their use in archaeological contexts, it is appropriate to very briefly consider the broader impact of infection with intestinal helminths on general health. Enteric helminth infections typically cause relatively mild clinical symptoms (unless high numbers of worms are present), but their presence may affect other aspects of health. The immune system responds to helminths in a particular way and the helminth-induced response may affect the capacity of the host to appropriately respond to other infections (reviewed in [92]). Mechanistically this is quite complex (and beyond the scope of the current article), however, increased susceptibility to co-infection could have influenced the outcomes of some of the most devastating diseases in history including plague, smallpox, typhoid and tuberculosis. Interestingly, populations challenged with a high diversity of pathogens tend to experience reduced incidence of allergy [93] and autoimmunity [94], a phenomenon originally defined as the ‘hygiene hypothesis’ [93]. The mechanisms are complex (and at times controversial), but is clear that helminth infections can reduce the development of allergic diseases probably by establishing regulatory networks in the immune system [95–97]. In short, allergic and autoimmune diseases that are common in modern ‘pathogen-deprived’ environments would have been much rarer in historic populations and intestinal helminths would have been a key contributing factor.

Technically, we support the use of multiple approaches but would always include microscopic analyses in any study. The choice of molecular approach will be defined by the questions that are being addressed in the study and the available resources (sample size and finances). Many approaches are complimentary and could be used together to provide high depth resolution of specific targets alongside shotgun and baiting strategies that offer broader acquisition of parasite aDNA albeit generally with lower depth in specific targets than PCR-parallel sequencing approaches. As with all aspects of using molecular archaeology to study infectious disease in the past the value of any sample is dependent upon the contextual data provided by archaeologists and historians. That said, we are convinced that the study of enteric parasites has reached a new level with the application of aDNA methodologies, representing a powerful addition to the archaeological toolbox. We predict that the future is strong and that in the coming years there will be key advances related to understanding the evolution and historical epidemiology of the parasites as well as contributions from this field to understanding the life of people and animals in the past.

Data accessibility

This article has no additional data.

Authors' contributions

P.G.F. and A.L.S. completed this work together both drafting and revising the manuscript.

Competing interests

We declare we have no competing interests.

Funding

We have received support from The John Fell Fund (Oxford), the Poesshl Foundation, the Swiss National Science Foundation (PBSKP3-140149/PBSKP3-145846) as well as the BBSRC (BB/K004468/1 and BB/K001388/1).