biomolecular archaeology

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Lecture 5
Definitions
Objectives
Applications
Cases
Extra resources
Molecular
Archaeoparasitology
as a novel archaeological
tool

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Topic 1
Parasitology Archaeoparasitoloy
History

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Parasites are organisms which live in close association with another organism, called the
host, in which the parasite benefits from the association, to the detriment of the host.
Many other kinds of associations may exist between two closely allied organisms, such as
commensalism or mutualism . Endoparasites (such as protozoans and helminthes), tend
to be found inside the host, while ectoparasites (such as ticks, lice and fleas) live on the
outside of the host body. Parasite lifecycles often require that different developmental
stages pass sequentially through multiple host species in order to successfully mature and
reproduce. Some parasites are very hostspecific; means that only one or a few species of
hosts are capable of perpetuating their life cycle. Others are not host-specific, since many
different hosts appear to harbor and pass on the infective stages of the parasite.
Parasitology basics
Topic 1| lesson 1

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Archaeoparasitology, a multi-disciplinary field within paleopathology, is the study of
parasites in archaeological contexts. It includes studies of the protozoan and metazoan
parasites of humans in the past, as well as parasites which may have affected past human
societies, such as those infesting domesticated animals. Reinhard suggested that the term
"archaeoparasitology" be applied to "... all parasitological remains excavated from
archaeological contexts ... derived from human activity" and that "the term
Paleoparasitology be applied to studies of nonhuman, paleontological materials.
"Paleoparasitology includes all studies of ancient parasites outside of archaeological
contexts; such as those found in amber and even dinosaur parasites.
Archaeoparasitology
Topic 1 | Lesson 2

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The first archaeoparasitology report described calcified eggs of
Bilharzia haematobia (now Schistosoma haematobium) from the
kidneys of an ancient Egyptian mummy. Since then, many fundamental
archaeological questions have been answered by integrating our
knowledge of the hosts, life cycles and basic biology of parasites, with
the archaeological, anthropological and historical contexts in which
they are found.
Topic 1 | Lesson 3
History

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Most archaeoparasitology reports involve species which are considered to be true parasites of
humans today. However, incidental parasitism (referred to by some authors as
"pseudoparasitism", "false parasitism" or "accidental parasitism") occurs when a parasite which
does not normally utilize a host for the perpetuation of its lifecycle is found in that host
incidentally. One example is finding the eggs of Cryptocotyle lingua (a fish parasite) in the
stomach contents of an Eskimo mummy. It is estimated that 70% of the "parasite" species
reported from present-day humans are actually only incidental parasites. Some incidental
parasites do cause harm to the infested pseudohosts.
Topic 1 | Lesson 3

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Topic 2
Sources Evidences
Evidences

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In archaeological contexts, Endoparasites (or their eggs or cysts) are usually found in
(i)fossilized human or animal dung (coprolites), (ii) the tissues and digestive contents of
mummified corpses, or (iii) soil samples from latrines, cesspits, or middens (dumps for
domestic waste). A cyst of Echinococcus granulosus was even retrieved from cemetery soil in
Poland. Ectoparasites may be found on the skin or scalp, as well as wigs, clothing, or
personal grooming accessories found in archaeological sites. Ectoparasite eggs may be found
attached to individual hairs. The International Ancient Egyptian Mummy Tissue Bank in
Manchester, England, provides tissue samples for a variety of uses, including parasitological
studies.
Sources of material
Topic 2 | Lesson 1

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Since 1910, parasite remains have been found in archaeological
samples from Africa, the Americas, Asia, Europe, the Middle East,
and New Zealand. The age of archaeological sites yielding human
parasite remains ranges from approx. 25,000-30,000 years ago to late
19th-early20th century. Parasite remains have also been found in
domestic animal remains at archaeological sites.
Topic 2 | Lesson 2

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Human skeletal remains may exhibit indirect evidence of parasitism.
For example, hookworm(Ancylostoma duodenale) parasitism may lead
to anemia, and anemia is one factor associated with the skeletal changes
of cribra orbitalia and porotic hyperostosis. Thus, hookworm
parasitism may be a causal factor in observed cribra orbitalia and
porotic hyperostosis, though dietary factors may also lead to anemia.
Evidences
Topic 3| Lesson 1

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Information on the presence of intermediate hosts, required for
life cycle completion by many parasites, is also useful in
determining the likelihood that a parasite may have infected a
particular ancient society. One example is the identification of
molluscan intermediate hosts of schistosomiasis in an Islamic
archaeological context.
Evidences
Topic 3 | Lesson 2

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Artifacts depicting the appearance of individuals may also indicate cases of
parasitism. Examples include the characteristic facial deformities of
leishmaniasis found on pre-Columbian Mochica pottery, and morphological
features of certain ancient Egyptian figurative art. Literary sources also
provide valuable information regarding not only the parasites present in
historic societies. but also the knowledge and attitudes that the people had
towards their parasitic infestations. However, specific parasitological diagnoses
reported in ancient and medieval texts must always be read with some degree
of skepticism.
Topic 3 | Lesson 2

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Topic 3
Methods Techniques

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Parasite remains in archaeological samples are identified by a variety of
techniques. Very durable remains, such as eggs and cysts, may remain intact for
many thousands of years. In some cases, relatively intact soft-bodied adult
helminthes and ectoparasitic arthropods have been found. All of these forms can
be identified to the family, genus or species level by compound or electron
microscopy. Petrographic techniques have been used for eggs of Capillaria
hepatica found in cysts in the corpse of an adolescent from the late Roman
period buried in Amiens (France).
Methods
Topic 3 | Lesson 1

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The authors stated that identification of tissue-dwelling
parasites such as Capillaria hepatica in archaeological remains
is particularly dependent on preservation conditions and
taphonomic changes and should be interpreted with caution due
to morphological similarities with Trichiuri ssp. eggs
Methods
Topic 3 | Lesson 1

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In cases where the intact bodies of parasites are not found,
protein or DNA from the parasite may still be present. Antigenic
and immunological assays (including enzyme-linked
immunoassay -ELISA, and DNA sequencing are used to identify
the source of these chemical remains, often tot he species level.
Techniques
Topic3 | Lesson 2

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Archaeoparasitological studies have provided information on many fundamental
archaeological, historical, and biogeographical questions. These questions may be grouped
into the following broad categories: past dietary and farming practices, animal
domestication, migration patterns, climate change, sanitary practices, cultural contacts,
ethnomedicine, and the overall health of various human societies. Archaeoparasitology
data, combined with our knowledge of present host-parasite associations, also contributes
to our understanding of the co-evolution of human host-parasite interactions. Our
understanding of the geographic origins, evolution and biogeography of the parasites
themselves and human diseases associated with them has also benefitted tremendously from
archaeoparasitological studies.
Applications
Topic 4| Lesson 1

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To aim of identifying genetic signatures which might correlate with the
geographical location or the corresponding historic era required the premise
that such signatures are possible. An initial basic phylogenetic alignment on
sequences available in the GenBank database showed that modern database
sequences of Trichuris trichiura from different parts of the world where the
parasites are still prevalent reveal genetic variation according to geographic
origin, yet these initial findings showed only very weak phylogenetic support,
even suggesting that some of the sequences labelled Trichuris trichiura might
not be from the same parasite species.
Applications
Topic 4| Lesson 1

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The sequence used in this comparison was the ribosomal internal transcribed
spacer 1 (ITS1) located between the 18S and the 5.8S rDNA and commonly used
to determine the species of Trichuris sp. A part of this ITS1 sequence was
subsequently used on the aDNA samples obtained.
Further evidence for geographical grouping was found for the related species T.
muris (parasitizing rodents) from several locations across Europe Since modern
samples can be geographically grouped based upon sequence identity it is likely
that signatures of temporal and/or geographic origin can be found in ancient
samples.
Applications
Topic 4| Lesson 1

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the molecular epidemiology of Ascaris significant genetic
differentiation exits between parasites of different countries or
even villages based on the mitochondrial cytochrome c oxidase I
(CO1) gene
Applications
Topic 4| Lesson 1

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Topic 5
Case 1 Case 2
Case 3

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By analysing the occurrence of helminth parasite eggs within 152 samples that dated
between Neolithic (ca 3600 BCE) and the Post Medieval (seventeenth century) which
provided insight into historical living conditions and cultural practices. Parasite aDNA was
amplified and sequenced to provide species-level diagnosis and, with Trichuris trichiura (the
most frequently detected parasite), epidemiological patterns including genetic diversity
provided a unique, artefact-independent source of historical information. The novel
approaches developed in this work demonstrate the wide applicability and potential for
molecular archaeoparasitology to broadly impact the study of historical events ranging from
health to socio-economic and dietary practices.
CASE 1
UNIT 5| Lesson 1

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A combination of parasitological and molecular approaches was used to
identify changes in eating habits and trading patterns centred on
Lübeck, a dominant medieval port and founding city of the Hanseatic
League. We propose that the integrated application of molecular
parasitology and epidemiology to the study of archaeological sites
represents a novel addition to the repertoire of approaches that can be
used to interrogate historical events.
CASE 1
UNIT 5| Lesson 1

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● (a) Sample handling and preparation workflow
● aDNA handling practices have been outlined in a range of
publications . None of the parasites targeted in this study is endemic
in the UK or any country where the material was received from.
None of the laboratories has ever handled or stored modern samples
containing these parasites,
CASE 1
UNIT 5| Lesson 1

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● (a) Sample handling and preparation workflow
● hence modern contamination is extremely unlikely. The processing pipeline
involved sample preparation and PCR set up in a ‘clean’ laboratory within
dedicated UV hoods using dedicated equipment with all handling of
amplified material undertaken in a separate laboratory. Researchers also
followed a strict unidirectional protocol, never entering the ‘clean area’ or
handling samples after entering any downstream processing laboratory.
CASE 1
UNIT 5| Lesson 1

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● (b) aDNA extraction
● Soil subsamples (5 g) were re-hydrated in 20 ml of PCR-grade water
(Qiagen, Hilden, Germany) as described above. The samples were then
sieved through a series of disposable, single-use nylon mesh sieves with
decreasing aperture size (1 030 µm, 500 µm, 100 µm; Plastok Associates Ltd,
Birkenhead, UK). The eggs present in the filtrate were pelleted by
centrifugation (400g, 10 min) and the pellet homogenized in a BeadBeater
(BSP BioSpec, Bartlesville, USA) using 1 mm glass beads (Hecht, Sondheim,
Germany). aDNA was extracted using a Qiagen Blood&Tissue or Mericon
Food kit (Qiagen). Blank extraction controls were routinely included as
contamination checks.
CASE 1
UNIT 5| Lesson 1

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● (c) Microscopic diagnosis
● Aliquots of the initial subsample were analysed microscopically
using a Nikon Eclipse E400 with 10× and 40× lenses (Nikon UK,
Kingston-Upon-Thames, UK). Photographs were recorded on a
QImaging MP5.0 RTV camera (QImaging, Surrey, Canada).
Parasite egg counts were extrapolated from replicated counts to the
dry weight of the sample.
CASE 1
UNIT 5| Lesson 1

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● (d) PCR amplification
● The PCR was performed in two stages; with the first stage boosting the starting
concentration using a very robust polymerase (AmpliTaq Gold 360, ThermoFisher,
Loughborough, UK) and a second stage to produce sufficient amounts of target DNA
using a high-fidelity polymerase and to attach the barcodes for MiSeq sequencing
(Phusion Hot Start Flex, New England Biolabs, Hitchin, UK). PCR primers were
designed based on published sequences or reported previously (electronic
supplementary material, Methods). Multiple water controls were included with each set
of PCRs, producing either no product or primer concatemers.
CASE 1
UNIT 5| Lesson 1

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● (e) Sequencing
● Second stage PCR products were cleaned up prior to sequencing
(MinElute, Qiagen). Initial Sanger sequence data were generated
using the BigDye Terminator (ThermoFisher).
CASE 1
UNIT 5| Lesson 1

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● (d) PCR amplification
● The PCR was performed in two stages; with the first stage boosting the starting
concentration using a very robust polymerase (AmpliTaq Gold 360, ThermoFisher,
Loughborough, UK) and a second stage to produce sufficient amounts of target DNA
using a high-fidelity polymerase and to attach the barcodes for MiSeq sequencing
(Phusion Hot Start Flex, New England Biolabs, Hitchin, UK). PCR primers were
designed based on published sequences or reported previously (electronic
supplementary material, Methods). Multiple water controls were included with each set
of PCRs, producing either no product or primer concatemers.
CASE 1
UNIT 5| Lesson 1

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● A dog mummy has revealed the first archaeological evidence of bloodsucking parasites
plaguing Fido's ancestors in Egypt during the classical era of Roman rule.
● The preserved parasites discovered in the mummified young dog's right ear and coat
include the common brown tick and louse fly — tiny nuisances that may have carried
diseases leading to the puppy's early demise. French archaeologists found the infested
dog mummy while studying hundreds of mummified dogs at the excavation site of El
Deir in Egypt, during expeditions in 2010 and 2011.
Egyptian dog mummy infested with
bloodsucking parasites
Topic5| Case 2

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Topic5| case 2

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Digging deeper into history
The French archaeologists hope to find answers to a
different set of questions by searching for more
preserved ticks and flies among the mummified dogs
of El Deir. Such archaeological evidence could show
how diseases originated throughout history, provide
clues about the geographical spread of parasites and
reveal more about the relationship between parasites
and both human and animal evolution.
Topic 5| case 2

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Malaria is a disease caused by parasites of the
genus Plasmodium. The infection is transmitted to humans through the
bites of female mosquitoes of the genus Anopheles. Four species
of Plasmodium are pathogenic to humans, and each leads to different
clinical features: P. falciparum causes severe malaria with undulating
high fever (malaria tropica); P. malariae, P. vivax, and P. ovale cause
less severe clinical courses of disease with the manifestations of malaria
quartana (P. malariae) and malaria tertiana (P. vivax and P. ovale).
Topic 5| case 3

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Literary evidence for malaria infection dates back to the early Greek period when
Hippocrates described the typical undulating fever , highly suggestive of plasmodial
infection. Although it is believed that malaria widely affected early pre-Hippocrates
populations, until now only 1 study, which used molecular analysis, clearly identified P.
falciparum in a Roman infant dating back to the 5th century AD .Two other studies
used molecular analysis to identify more recent plasmodial DNA in ancient human
remains, i.e., from 100–400 years ago A substantial number of nonspecific
amplifications in these previous studies raised concerns as to the specificity of current
molecular markers for ancient malaria.
Topic 5| case 3

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2 of the 91 ancient Egyptian samples tested positive for the 134-bp fragment of the pfcrt
region of P. falciparum . The specificity of the amplification was verified by sequencing,
which showed 99% sequence concordance. The result was verified by parallel analysis in 2
independent laboratories; observations were fully concordant. The 2 positive samples
originated from 2 different tomb complexes dating from the New Kingdom until Late
Period (1500–500 BC). Each sample was obtained from adults who had osteopathologic
evidence of chronic anemia. No positive results were found for the earlier samples from the
Predynastic to Early Dynastic or Middle Kingdom periods.
Topic 5| case 3

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Topic 5| case 3

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Previously, immunologic tests have been used to
investigate the presence and incidence of malaria
in ancient Egyptian mummies. Because >40% of
all samples and 92% of samples from persons
with bone lesions suggestive of chronic anemia
tested positive for the P. falciparum histidine-rich
protein-2 antigen, doubts as to the specificity of
those tests have been raised.
Topic 5| case 3

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Our study unambiguously identified P. falciparum aDNA in Egyptian
mummy samples, thereby proving a specific infection by falciparum
malaria in ancient Egypt. With respect to the infection incidence, our
molecular analysis suggests a more realistic frequency than had been
previously suggested by paleoimmunologic methods. Consequently, the
aDNA analysis is superior with respect to the reaction specificity, so
that the latter should not further be used for that purpose.
Topic 5| case 3

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Our study unambiguously identified P. falciparum aDNA in Egyptian
mummy samples, thereby proving a specific infection by falciparum
malaria in ancient Egypt. With respect to the infection incidence, our
molecular analysis suggests a more realistic frequency than had been
previously suggested by paleoimmunologic methods. Consequently, the
aDNA analysis is superior with respect to the reaction specificity, so
that the latter should not further be used for that purpose.
Topic 5| case 3

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This report adds another infectious disease to the spectrum of
paleomicrobiology in ancient Egypt, thereby further explaining the
previously postulated influence of infectious diseases on the low life
expectancy for ancient Egyptian populations. Molecular detection of
pathogen aDNA can be used not only to identify a certain disease, but it
may also provide information on disease frequency, evolutionary
origin, and pathways.
Topic 5| case 3

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Ancient Salt Man.
TOPIC 6

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Extra resources
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4293883/