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Verlag Dr. Friedrich Pfeil
Verlag Dr. Friedrich Pfeil
•
•
München
München
Yearbook of
Mummy Studies
2
2
Heather Gill-Frerking,
Heather Gill-Frerking,
Wilfried Rosendahl
Wilfried Rosendahl
& Albert Zink
& Albert Zink
(editors)
(editors)
Amelie Alterauge
Amelie Alterauge
(guest editor)
(guest editor)
39
Yearbook of Mummy Studies, Vol. 2, pp. 39-47, 4 figs., 1 tab., March 2014
© 2014 by Verlag Dr. Friedrich Pfeil, München, Germany – ISBN 978-3-89937-163-5
Molecular Confirmation of Schistosoma
and Family Relationship
in two Ancient Egyptian Mummies
Carney D. Matheson1, Rosalie David2, Mark Spigelman3, 4 and Helen D. Donoghue4, 5
Abstract
Egg morphology and immunocytochemistry have identified schistosomiasis in ancient Egypt. Our study aimed
to detect and characterize schistosomal DNA in mummified human tissue. Liver samples from the mummy
Nekht-Ankh (c. 3900 BP) and intestinal samples from Khnum-Nakht, possibly his brother, were analyzed using
PCR primers suitable for fragmented ancient DNA, specific for either Schistosoma mansoni or Schistosoma haema-
tobium. Mitochondrial primers examined any relationship between the supposed brothers. Two independent
laboratories confirmed S. mansoni DNA from the Nekht-Ankh liver. One laboratory detected S. haematobium DNA
in both the Nekht-Ankh liver and intestinal samples from Khnum-Nakht in repeat experiments. We believe
this is the first verified report of S. mansoni in ancient Egypt. Although no S. haematobium DNA sequence was
obtained, the results support earlier histological findings of S. haematobium in ancient Egyptian mummies. These
findings demonstrate that both S. mansoni and S. haematobium were present in Central Egypt during the Middle
Kingdom, around 3900 years ago. From the mitochondrial DNA analysis it appears that these two individuals
were not maternally related, which is consistent with the morphology of the skulls. The lack of genetic related-
ness between these supposed brothers throws light upon the habit of adoption in this society.
Introduction
Schistosomiasis has been extensively documented in Egypt, and Schistosoma eggs were detected in a pre-
Dynastic mummy from 3200 BC (Deelder et al 1990). Today around two billion people are infected in the
world, with 300 million suffering severely from the disease. Egypt continues to be a major affected area
and in 2009 there were 820 442 treated cases reported (WHO 2011). Descriptions of medical conditions
consistent with schistosomiasis have been documented in the Egyptological literature (Adamson 1976,
Jordan 2000, Nunn & Tapp 2000). Direct evidence of schistosomiasis was obtained when a paleopatholo-
gist found the calcified eggs of Schistosoma haematobium, recognized by their morphology, in preserved
kidneys from two mummies of the Twentieth Dynasty (Ruffer 1910). Further morphological evidence of
schistosomiasis was obtained from a 5000 year-old mummy (Deelder et al. 1990), the bladder of a New
Kingdom mummy dating to c. 3450 BP (Horne 2003) and a 65 % prevalence found among mummies
(350-550 CE) from the Wadi Halfa area on the Sudan-Egyptian border (Miller et al. 1992). In the 1990s, a
major study of schistosomiasis in ancient Egyptian mummified remains was undertaken at the University
of Manchester (Contis & David 1996). Immunocytochemistry was pioneered as a diagnostic tool to detect
1 Lakehead University, Department of Anthropology, Canada.
2 KNH Centre for Biomedical Egyptology Faculty of Life Sciences, University of Manchester, UK.
3 Kuvin Centre for the Study of Infectious and Tropical Diseases, The Hebrew University, Hadassah Medical
School, Jerusalem, Israel.
4 Centre for Clinical Microbiology, Royal Free Campus, University College London, London, UK.
5 Centre for the History of Medicine, University College London, London, UK;
E-mail: h.donoghue@ucl.ac.uk (corresponding author).
Matheson et al.: Schistosoma and Family Relationship in Egyptian Mummies
40
Yearbook of Mummy Studies 2 (2014)
the disease (Rutherford in David 2008: 99-115), fol-
lowed by an enzyme-linked immunosorbent assay
(ELISA) and DNA as confirmatory tests (Rutherford
in David 2008: 116-132).
Detection and characterization of Schistosoma
infection in ancient human remains may help us
understand the relationship between the human
host and parasite in the context of activities and
environmental surroundings. PCR may be more
sensitive than immunological methods. Therefore,
the present study was undertaken to determine
whether mummified remains contain schistosomal
DNA and, if so, which species are present.
Until recently, PCR received relatively little at-
tention in clinical diagnosis of schistosomiasis, as
non-molecular methods are more cost-effective in
countries with a high incidence of the disease. The
Schisto soma genome project increased the number
and range of available nucleotide sequences, fa-
cilitating the differentiation of the major human
Schistosoma pathogens by PCR (Hamburger et al.
2001, Kane & Rollinson 1998, Pontes et al. 2002).
Today the total genome sequence of Schistosoma
mansoni is known (Berriman et al. 2009), but that of
Schistosoma haematobium was determined only after
the completion of this study (Young et al. 2012). In
archaeological material often only small fragments
of DNA (< 150 bp) survive, although this varies
greatly according to the local environmental con-
ditions at the particular archaeological site (Pääbo
1989). Therefore, the first aim of the present study
was to design and verify species-specific primers
based on a short target sequence. Thereafter, tissues
were examined for Schistosoma from two individu-
als believed to be brothers, dating from 3900 years
ago (Fig. 1) and located in the Manchester Museum:
Nekht-Ankh (inventory no. 11724) and Khnum-
Ankh (inventory no. 11725). Further details can
be found at: http://www.ancient-egypt.co.uk/
manchester/pages/the%20two%20brothers.htm
The bodies of the “two brothers” have been
subjected to various studies. In 1908 they were un-
wrapped and autopsied together with a multidisci-
plinary team by Dr. Margaret Murray, then curator of the Egyptian collection at the Manchester Museum
(Fig. 4). This pioneering investigation marked a critical stage in the development of paleopathology. There
was no opportunity to examine the bodies using radiological or histological techniques, but, relying on
the use of morbid anatomy, the team made several interesting disease identifications (David 2003). In this
original investigation it was noted that while both bodies were poorly preserved the color and texture
of the residual tissue appeared very different in each case. Therefore, it was concluded that different
preservatives had been used in the mummification process. Since 1973 the now skeletal remains of the
“two brothers” have been the subject of continuing investigations by the Manchester Egyptian Mummy
Research Project; techniques have included radiography, histology, electron microscopy, immunocyto-
chemistry and paleoodontology (David 1979, 2000). Studies on the fragments of lung tissue indicated
the presence of sand pneumoconiosis; when the ancient embalmers eviscerated the thoracic cavity they
also removed part of the wall of the heart, which was attached to the lung. Here the histological sections
Fig. 1. Anthropoid coffins of Nekht-Ankh (right) and
Khnum-Nakht (left) as displayed in the Manchester
Museum, inventory numbers 11724 and 11725, re-
spectively. According to the coffin inscriptions, both
individuals were sons of the same mother, who was
called Khnum-Aa and carried the title “Mistress of
the House”, indicating she was an heiress who owned
property. The inscriptions do not make it clear if both
brothers shared the same father. Nekht-Ankh and
Khnum-Nakht were buried together without wives
or offspring, suggesting that neither man was married
(David 2003).
41
show fibrous tissue obliterating the pericardial and pleural cavities, indicating some time before death
there must have been inflammation in this area, possibly associated with pneumonia. In well-preserved
liver tissue a section containing a group of cells with thickened walls was tentatively identified as the
remains of the liver fluke, Facciola hepatica.
A sample of the liver from the Egyptian mummy Nekht-Ankh and a sample of intestine from Khnum-
Nakht, believed to be his brother, were analyzed by PCR for schistosomal DNA. Their family connection
remains controversial, so to try to resolve the matter, primers for human mitochondrial DNA were used
to determine any maternal relationship.
Materials and methods
Precautions against contamination
The standard strict precautions were taken at all stages of the study to ensure there was no cross-con-
tamination (Donoghue 2008). Negative DNA extraction and PCR controls were always included in the
protocols. All initial work on DNA extraction and PCR optimization was completed more than 12 months
before the ancient DNA investigation and thorough cleaning had been undertaken in the interim.
Material for optimization and verification experiments
The Kuvin Centre, Jerusalem (Laboratory 1) supplied purified DNA for optimization and verification
of experimental PCR primers. Schistosoma haematobium, Schistosoma mansoni and Schistosoma intercalatum
were supplied as adult pairs of worms, preserved in ethanol to Laboratory 2 (UCL) by the Natural His-
tory Museum, London.
Ancient Egyptian mummy tissue
The mummy of Nekht-Ankh was discovered in 1905/1906 at a burial site known as the “Tomb of Two
Brothers” and belonged to Nekht-Ankh and Khnum-Nakht (David 2003). These two mummies, discovered
in a previously undisturbed, provincial tomb, are archaeologically important as they have made a major
contribution to the emergence and development of paleopathology, and studies on them continue. In
addition to the mummies, the tomb contained a limited but well made range of artifacts. However only
Nekht-Ankh was provided with a set of four canopic jars in a canopic chest, to contain his mummified
viscera, removed from the body during the mummification process. The exact date of the burial cannot
be determined since there is no evidence of any royal names to be found at the cemetery. The style and
contents of the tomb, however, places it in the 12th Dynasty (c. 3900 BP). A small sample of liver from
the canopic jar of Nekht-Ankh was examined, together with a sample of intestine and another tissue
fragment from Khnum-Nakht.
DNA extraction: adult worms
A small segment was cut off and placed in 100 μl of demineralization solution (0.5 M EDTA pH 8.0/1 mg−1
proteinase K) in a 1.5 ml tube with 10 glass beads (1.5-2.0 mm). After vortexing, tubes were incubated
overnight at 56 °C. Next the samples were incubated in lysis buffer (Qiagen DNeasy™ kit) for I hour at
70 °C. Thereafter the standard protocol was followed. DNA was captured onto spin columns, washed
and eluted.
DNA extraction: ancient Egyptian mummy tissue
In Laboratory 1 DNA was extracted from the liver and intestine using a modification of the procedure
described by Boom et al. (1990). Tissue was placed in 1.5 ml tubes containing 500 μl guanidium thiocyanate
solution (10 M), vortex mixed for 1 min., incubated at 56 °C and gently agitated for 5 to 8 h. Next, samples
were incubated at 94 °C for 10 min., centrifuged at 12 000 rpm for 3 min. and the supernatant transferred
to another sterile 1.5 ml tube. Guanidium thiocyanate solution (1 ml) and 10 μl of silica bead suspen-
sion were added to the sample. Tubes were vortex mixed for 20 s, placed on ice for 1 hour and agitated
every 15 min. Samples were centrifuged at 12 000 rpm for 30 s and the supernatant discarded. Pellets
were re-suspended in 500 μl of washing buffer (2 mM Tris-HCl (pH 7.5)/10 mM EDTA (pH 8.0)/10 mM
Matheson et al.: Schistosoma and Family Relationship in Egyptian Mummies
42
Yearbook of Mummy Studies 2 (2014)
NaCl in a 50 % (v/v) water/alcohol mixture), centrifuged for 30 s at 12 000 rpm, and the supernatant
discarded. This step was repeated if the silica pellet appeared discolored. The silica was washed with
200 μl absolute ethanol and air-dried. DNA was eluted with 100 μl water, mixed for 20 s, incubated at
56 °C for 1 h and re-suspended in double-distilled water. The extract was centrifuged at 12 000 rpm, for
3 min. and the DNA extract stored at 4 °C. Extraction negatives were processed simultaneously to identify
any contamination during this process.
In Laboratory 2 a small quantity of sample was placed in 200 μl demineralization solution in a 1.5 ml
tube with beads, incubated at 56 °C and mixed in a bead beater daily until dispersed. After mixing well
in a bead beater, samples were split and one 100 μl aliquot was incubated for 1 h at 60 °C with 10 μl
0.1 M N-phenacylthiazolium bromide (PTB), a reagent that cleaves glucose-derived protein cross-links
(Poinar et al. 1998) thereby facilitating DNA release and strand separation. Both sets of samples were
then incubated for 2 h at 56 °C with 200 μl of lysis buffer containing guanidium thiocyanate, EDTA and
Triton X-100 (Boom et al. 1990). Subsequently the DNeasy™ protocol was followed.
PCR amplification
In Laboratory 1 the Schistosoma mansoni 121 bp tandem repeat sequence known as Sm1-7 (Hamburger et
al. 1991) was targeted for amplification using primers described by Hamburger et al. (1998): Primer 1:
5'-GAAAATCGTTGTATCTCCG-3' and Primer 2: 5'-GGTGACCTGCCTAAAAATAC-3'. To examine genetic
relatedness between the two mummified individuals, the following primers were used: mitochondrial DNA
16210: 5'-CCC ATGCTTACAAGCAAG TA-3' (Kolman & Tuross 2000) and 16401: 5'-TGATTTCACGGA
GGATGGTG-3' (Vigilant et al. 1989). Amplification was performed using a 25 μl PCR reaction mix com-
prised of: 20 mM Tris-HCl pH 8.4; 50 mM KCl; 2.0 mM MgCl2; 2.5 mM of each nucleotide (dATP, dCTP,
dGTP and dTTP); 0.25 μM of each primer; and 1.25 units of Platinum Taq (Gibco). DNA extract (10.0 μl)
was added to each tube. PCR and extraction negative controls were included in each PCR. Amplification
was achieved using a program consisting of a 3 min. initial denaturation at 94 °C, followed by 45 cycles
of: 94 °C for 1 min., 55 °C (primers 1 and 2) and 60 °C (primers16210 & 16401) for 1 min., and 72 °C for
2 min.; followed by a final extension of 10 min. at 72 °C. Primers 1 & 2 produce a 46-bp product and a
repetitive 121-bp product of S. mansoni; mitochondrial primers 16210 and 16401 amplify a 230-bp product
of the hyper variable region I.
In Laboratory 2 two sets of nested pairs of primers were devised based on the S. mansoni tandem
repeat unit (Hamburger et al. 1991) and an intergenic spacer region in S. haematobium near the 28S ribo-
somal RNA gene (Kane & Rollinson 1998):
Schistosoma mansoni outer primers SM1: 5'-GATCTGAATCCGACCAACCG-3' and SM2: 5'-CTTGTT-
TTATATTAACGCCC-3' (118 bp); inner primers SM3: 5'-GAATCCGACCAACCGTTC-3' and SM4 5'-ACG-
CCCACGCTCTCGCA-3' (98 bp).
Schistosoma haematobium outer primers SH1: 5'-CGCTAGACTTCGGTCTGGTT-3' and SH2: 5'-CGTCG-
CAAGTATACGATAT-3' (107 bp); inner primers SH3: 5'-CTAGACTTCGGTCTGGTTAAAGC-3' and SH4:
5'-CGATATATGGATATATGTTATGC-3' (92 bp).
PCR was carried out in 50 μl volumes containing Qiagen HotStar® buffer; 10 mM bovine serum al-
bumin; 3.5 mM MgCl2; 0.5 mM of each primer; 0.2 mM of dATP, dCTP, dGTP and dTTP; and 1.25 units
of HotStar® Taq polymerase.
Amplification was achieved using a program consisting of initial enzyme activation and DNA de-
naturation for 15 min. at 95 °C; followed by 10 cycles of touchdown PCR with annealing dropping 1 °C
per cycle from 65 °C to 56 °C; followed by 33-43 cycles of: 94 °C for 40 s, 55 °C for 1 min., and 72 °C for
20 sec + 1 s/cycle; followed by a final extension of 1 min. at 72 °C. Nested PCR when performed used
1 μl of the stage 1 PCR product with 25 cycles of amplification.
Detection of amplified DNA
PCR product was added to loading dye and electrophoresed in a 2.0 % (w/v) NuSieve© (FMC Bioprod-
ucts) agarose gel in 1X TAE at 108 volts for 35 min. (Laboratory 1); or a 3.0 % (w/v) NuSieve© agarose
gel in 1 x TBE or TAE at 8.8 volts cm−1 for 65 min. (Laboratory 2). Amplified DNA was visualized using
ethidium bromide staining exposed to ultraviolet light, and recorded with digital imaging equipment
(Pharmacia Biotech, ImageMaster or LabWorks 3.6). PCR products were excised from the agarose using
sterile implements for direct sequencing.
43
DNA sequencing
In Laboratory 1 the PCR product was directly sequenced using a radioactive manual sequencing kit (Se-
quenase Kit, Amersham). Sequencing PCRs consisted of 32P radio-labeled nucleotides, Sequenase buffer
and 4 μl of liquefied (94 °C) excised agarose PCR product to a total reaction volume of 10 μl. Amersham
recommended thermocycling parameters were used and radio-labeled DNA was run on a 6 % acrylamide
gel and exposed to high resolution X-ray film (Kodak) for 48 to 72 hours before development.
Results
Optimization and verification experiments
The S. mansoni PCR primers were highly specific and no amplicons were detected with S. haematobium
or S. intercalatum DNA extracts. The S. haematobium primers generated some amplified DNA from the
S. intercalatum DNA extract. This could be reduced considerably by touchdown PCR.
1 2 3 4 5 6 7 8 9 10 11
← 118 bp
← 118 bp
1 2 3 4 5 6 7 8
107 bp →
←⎯
⎯
⎯
107 bp
Fig. 2. Gel electrophoresis of Schistosoma mansoni
PCR. Wells were loaded as follows: Top row were
DNA extractions with phenacylthiazolium bromide
(PTB); bottom row were extracted without PTB. Lane
1; unrelated archaeological sample; lane 2: Nekht-
Ankh liver; lane 3: Khnum-Nakht intestine; lanes 4-6:
unrelated archaeological samples; lane 7 (top row): gel
positive control – archival stored PCR product; lane
7 (bottom row): blank; lane 8 (top row): DNA extrac-
tion positive control – archived; lane 8 (bottom row):
DNA extraction negative control; lane 9 (top row):
DNA negative control; lane 9 (bottom row): DNA
extraction positive control; lane 10 (top row): PCR
negative control; lane 10 (bottom row): PCR positive
control – archived DNA extract; lane 11: molecular
markers – PhiX174HaeIII digest. The 118 bp S. mansoni
PCR product can be seen in lanes 7 and 8 (top row) and
lanes 2, 9 and doubtfully 10 (bottom row). Negative
and positive controls are satisfactory and the extract
from the Nekht-Ankh liver is weakly positive.
Fig. 3. Gel electrophoresis of Schistosoma haematobium
PCR. Wells were loaded as follows: Top row: lane
1: PCR negative control; lane 2: DNA extraction
negative control; lane 3: S. haematobium positive con-
trol – 1 : 100 000 dilution of worm extract; lanes 4-6:
unrelated archaeological samples; lane 7: Nekht-Ankh
liver. Bottom row: lanes 1-4: samples from four unre-
lated canopic jars; lanes 5-6: Khnum-Nakht intestinal
samples; lane 7: blank. Lane 8 (both rows): molecular
markers – PhiX174HaeIII digest. Negative and positive
controls are satisfactory and the extracts from the Nekht-
Ankh liver, the Khnum-Nakht intestinal samples,
two of the canopic jar samples and another unrelated
archaeological sample are weakly positive.
Matheson et al.: Schistosoma and Family Relationship in Egyptian Mummies
44
Yearbook of Mummy Studies 2 (2014)
Ancient Egyptian mummy tissue
In Laboratory 1 extraction of DNA from the liver and intestine samples was immediately followed by
PCR amplification of the 121-bp S. mansoni tandem repeat unit. A faint 121-bp product was amplified
from the Egyptian liver sample but subsequent attempts
to repeat amplification were unsuccessful. It was noted
that significant degradation of the DNA extract occurred
during a 5-hour period before a second amplification was
attempted. Due to sample limitations this extraction could
not be replicated. A sequence exhibiting 100 % identity with
S. mansoni (reference sequence NCBI nucleotide accession #:
M61098) was obtained. The HVII mitochondrial DNA was
successfully amplified from both individuals (Table 1). All
extraction and amplification controls were negative.
In Laboratory 2 a faint 118-bp S. mansoni product was
obtained from the Nekht-Ankh liver sample, using the DNA extracted without the facilitating reagent PTB.
No amplicons were obtained with the Khnum-Nakht intestine samples. All extraction and amplification
controls were negative (Fig. 2). However, S. haematobium DNA was detected in both the Nekht-Ankh liver
and two samples from Khnum-Nakht, believed to be intestinal (Fig. 3). There was insufficient material
for further DNA extractions.
Discussion
Results confirmed that the DNA extraction procedures resulted in amplifiable DNA from fixed tissues,
without the need for PTB. The sensitivity of PCR amplification was demonstrated by Pontes et al. (2002)
who, using a similar nested PCR and purified DNA, showed that as little as 1 fg was detectable. The
outer and nested S. mansoni primers were specific. However, the S. haematobium PCR allowed some S. in-
tercalatum amplicon unless stringent conditions were employed. This appears to be a general problem,
as Hamburger et al. (2001) reported a similar finding based on the Dra1 repeat sequence.
S. mansoni DNA was amplified and sequenced from the liver of a 3900 year old Egyptian mummy,
Nekht-Ankh, suggesting that he suffered from chronic schistosomiasis. This was confirmed in two in-
dependent laboratories. The intestinal samples from Khnum-Nakht were positive for S. haematobium but
were negative for S. mansoni. These findings support previous microscopic observations of Schistosoma
spp. eggs and immunocytochemistry (Rutherford 1999, David 2003). Data from Laboratory 2 indicate
that the liver sample from Nekht-Ankh was also positive for S. haematobium. In addition, S. haematobium
DNA was present in material from two of four unrelated Middle Kingdom canopic jars, which confirms
earlier conclusions that this species was widespread in ancient Egypt. Although S. haematobium eggs are
usually found in the ureter and bladder, the early observations by Ruffer (1910) described the calcified ova
of Bilharzia haematobia in the straight tubules of the kidney, whilst Reyman et al. (1977) reported schisto-
some eggs in the kidneys of a teenage boy, Nakht. Both these reports also described cirrhosis of the liver,
and calcified schistosome eggs with terminal spines, typical of S. haematobium, were seen in histological
sections of tissue from the portal region of the Nakht liver (Nunn & Tapp 2000). Therefore, although the
presence of this species is less usual in the liver, our finding is in line with the earlier reports. In addition,
where there are high intensities of S. haematobium and mixed infections with S. mansoni, S. haematobium
can be found in the mesenteric veins and thus their eggs appear in the liver and gastrointestinal tract
(Cunin et al. 2003).
It is believed that schistosomiasis originated in Central Africa and that the disease spread from
Ethiopia down the Nile (Adamson 1976). The snail hosts of S. haematobium, Bulinus spp, are bottom
feeders and are thus relatively unaffected by the rate of flow of the river. However, the snail hosts of
S. mansoni, Biomphalaria spp, are surface feeders, and cannot establish themselves except in waters with
a slow current or none. Therefore, it was believed that S. haematobium was always prevalent in Upper
Egypt, whereas S. mansoni was concentrated in the Nile delta (El-Khoby et al. 2000). Recently Hibbs et
al. (2011) demonstrated S. mansoni in desiccated tissue from Wadi Halfa (350-550 CE) and Kulubnarti
(550-750 CE) – settlements in early Christian Nubia, using a specific enzyme-linked immunosorbent
assay that does not cross-react with S. haematobium. There was a clear association between incidence of
Table 1. Hypervariable region I mitochon-
drial polymorphisms.
Individual 16223
C → T 16261
C → T 16292
C → T
Nekht-Ankh + + +
Khnum-Nakht + – +
45
S. mansoni and local irrigation practices in Wadi Halfa and the authors pointed out the need for further
data on the distribution of S. haematobium in the Nile Valley. The present study confirms the prevalence
of S. haematobium and also provides the first direct evidence of S. mansoni from 3900 years ago. The tomb
of Nekht-Ankh and Khnum-Nakht was in Rifeh, near Assiut in Central Egypt. This may have been an
area where both species of Schistosoma were prevalent, as there almost certainly would have been pools,
wells and irrigation channels in the area that could have provided a source of S. mansoni (Butzer 1976).
The mitochondrial DNA sequences obtained from Nekht-Ankh liver and Khnum-Nakht intestinal
samples (Table 1) exhibit the 16223 T and 16292 T mutations common in the Middle East and Egypt.
Nekht-Ankh also exhibited the 16261 T mutation, suggesting that these two “brothers” were not ma-
ternally related. Exploration of postmortem damage of endogenous DNA has characterized genetic
damage, including oxidative modification and hydrolytic deamination, in ancient human samples as
‘miscoding lesions’ (Gilbert et al. 2003). Although results from the HVI mitochondrial analysis have not
been independently verified, they were replicated and showed no evidence of polymorphic variation, in
either the electropherograms or autoradiographs. In addition, the preservation of proteins and lipids in a
skin sample from Nekht-Ankh has been shown to be good (Petersen et al. 2003), which suggests that the
positive DNA results are likely to be genuine. Therefore it was concluded that authentic polymorphisms
were identified from these Egyptian mummies. The difference in the strength of the reactions from the
schistosomal DNA may reflect the limited and localized distribution of the parasite compared with the
human tissue.
In the inscriptions on the tomb equipment both “brothers” were described as the sons of a local
governor or mayor, and this wealthy family would have enjoyed considerable local status. It is recorded
that they were the sons of a woman named Khnum-Aa who carried the title “mistress of the house”.
However, the inscriptional evidence may indicate that Nekht-Ankh and Khnum-Nakht were half broth-
Fig. 4. Margaret Murray and her team unwrap the mummy of Khnum-Nakht in 1908. On May 6 1908 Dr.
Murray and her scientific team carried out an autopsy on the mummy of Khnum-Nakht in the Chemistry
Lecture Theatre, University of Manchester. There was an invited audience of 500 people from the Manchester
Egyptian Association (David 2003). This was a critical event in the development of paleopathology as a scien-
tific discipline.
Matheson et al.: Schistosoma and Family Relationship in Egyptian Mummies
46
Yearbook of Mummy Studies 2 (2014)
ers, the children of different fathers since in Khnum-Nakht’s case it provides information that both his
father and paternal grandfather were local governors, whereas it only states that Nekht-Ankh’s father
held that position. The inscriptions also record that Khnum-Nakht was a “great wa’ab priest” who served
the ram-headed god Khnum, and it would have been customary for Khnum-Nakht, as the son of the
local governor, to become a priest in the local temple. There is no information regarding the career of
Nekht-Ankh.
The original study in 1908 considered the vexed question of the parentage of the “brothers” as there
are marked differences between the brothers’ skeletons particularly their skulls – Khnum-Nakht’s skull
was prognathous, while that of his brother was orthognathous. Adoption was not uncommon in ancient
Egypt and this was thought to explain their physical dissimilarity. The findings of the present study that
these two individuals are maternally unrelated are consistent with these morphological observations.
Acknowledgements
Victoria Friends of the Hebrew University supported student travel to conduct research. Dr Vaughan South-
gate and his colleagues at the Natural History Museum, London kindly provided samples of adult Schistosoma
worms. Samples from Nekht-Ankh and Khnum-Nakht were provided by the Manchester University Museum
Egyptian Mummy Tissue Bank. Thanks are due to Professor Joseph Hamburger, Kuvin Center for the Study
of Infectious and Tropical Diseases, Hebrew University Hadassah Medical School, Israel for his helpful advice
on Schistosoma gene sequences. The technical assistance at UCL of Michelle Groves, Sahir Khurshid, Jasmine
Patel and Kim Vernon is gratefully acknowledged. Figures 1 and 4 are included by the kind permission of the
Manchester Museum, University of Manchester, UK.
References
Adamson, P. B. (1976): Schistosomiasis in antiquity. – Med. Hist. 20: 176-188.
Berriman, M., B. J. Haas, P. T. LoVerde et al. (2009): The genome of the blood fluke Schistosoma mansoni. – Nature
460: 352-358. (doi:10.1038/nature08160)
Boom, R., C. J. Sol, M. M. Salimans, C. L. Jansen, P. M. Wertheim-van Dillen & J. Van der Noordaa (1990): Rapid
and simple method for purification of nucleic acids. – J. Clin. Microbiol. 28 (3): 495-503.
Butzer, K. W. (1976): Early hydraulic civilization in Egypt: a study in cultural ecology. – Chicago, London
(University of Chicago Press).
Contis, G. & A. R. David (1996): The epidemiology of Bilharzia in ancient Egypt: 5000 years of schistosomiasis.
– Parasitol. Today 12 (7): 253-255.
Cunin, P., L.-A. Tchuem Tchuenté, B. Poste, K. Djibrilla & P. M. V. Martin (2003): Interactions between Schis-
tosoma haematobium and Schistosoma mansoni in humans in north Cameroon. – Trop. Med. Int. Health 8:
1110-1117.
David, A. R. (1979) (ed.): The Manchester Museum Mummy Project: Multidisciplinary Research on Ancient
Egyptian Mummified Remains. – Manchester (The Manchester Museum).
– (2000): 5000 years of schistosomiasis in Egypt. – Chungará, Revista de Antropología Chilena 32: 133-135.
– http://dx.doi.org/10.4067/S0717-73562000000100022 (accessed 01 August 2013)
– (2003): The tomb of two brothers at Rifeh, Egypt: palaeopathological and archaeological studies. – Pp. 169-
173 in Lynnerup, N., C. Andreasen & J. Berglund (eds.): Mummies in a New Millennium: Proceedings of
the 4th World Congress on Mummy Studies. Nuuk, Greenland, September 4th to 10th, 2001. Copenhagen
(Danish Polar Center).
David, R. (ed.) (2008): Egyptian Mummies and Modern Science. – Cambridge (Cambridge University Press).
Deelder, A. M., R. L. Miller, N. de Jonge & F. W. Krijger (1990): Detection of schistosome antigen in mummies.
– Lancet 335: 724-725.
Donoghue, H. D. (2008): Molecular palaeopathology of human infectious disease. – Pp. 147-176 in Pinhasi, R.
& S. Mays (eds.): Advances in human palaeopathology. Chichester, UK (John Wiley & Sons Ltd).
El-Khoby, T., N. Galal, A. Fenwick, R. Barakat, A. El-Hawey, Z. Nooman, M. Habib, F. Abdel-Wahab, N. S. Gabr,
H. M. Hammam, M. H. Hussein, N. N. Mikhail, B. L. Cline & G. T. Strickland (2000): The epidemiology of
schistosomiasis in Egypt: summary findings in nine governorates. – Am. J. Trop. Med. Hyg. 62S: 88-99.
Gilbert, M. T., E. Willerslev, A. J. Hansen, I. Barnes, L. Rudbeck, N. Lynnerup & A. Cooper (2003): Distribution
patterns of postmortem damage in human mitochondrial DNA. – Am. J. Hum. Genet. 72: 32-47. Erratum
in: Am. J. Hum. Genet. 72: 779.
47
Hamburger, J., T. Turetski, I. Kapeller & R. Deresiewicz (1991): Highly repeated short DNA sequences in the
genome of Schistosoma mansoni recognized by a species-specific probe. – Mol. Biochem. Parasitol. 44 (1):
73-80.
Hamburger, J., Y. X. Xu, R. M. Ramzy, J. Jourdane & A. Ruppel (1998): Development and laboratory evalua-
tion of a polymerase chain reaction for monitoring Schistosoma mansoni infestation of water. – Am. J. Trop.
Med. Hyg. 59 (3): 468-473.
Hamburger, J., He-Na, I. Abbasi, R. M. Ramzy, J. Jourdane & A. Ruppel (2001): Polymerase chain reaction assay
based on a highly repeated sequence of Schistosoma haematobium: a potential tool for monitoring schistosome-
infested water. – Am. J. Trop. Med. Hyg. 65 (6): 907-911.
Hibbs, A. C., W. E. Secor, D. Van Gervan & G. Armelagos (2011): Irrigation and infection: The immunoepide-
miology of schistosomiasis in ancient Nubia. – Am. J. Phys. Anthropol. 145 (2): 290-298.
Horne, P. (2003): Disease in mummies from two sites in ancient Egypt. – Pp. 111-112 in Lynnerup, N., C. An-
dreasen & J. Berglund (eds.): Mummies in a New Millennium: Proceedings of the 4th World Congress on
Mummy Studies. Nuuk, Greenland, September 4th to 10th, 2001. Copenhagen (Danish Polar Center).
Jordan, P. (2000): From Katayama to the Dakhla Oasis: the beginning of epidemiology and control of bilharzia.
– Acta Trop. 77 (1): 9-40.
Kane, R. A. & D. Rollinson (1998): Comparison of the intergenic spacers and 3' end regions of the large subunit
(28S) ribosomal RNA gene from three species of Schistosoma. – Parasitology 117: 235-242.
Kolman, C. J. & N. Tuross (2000): Ancient DNA analysis of human populations. – Am. J. Phys. Anthropol.
111 (1): 5-23.
Miller, R. L., G. J. Armelagos, S. Ikram, N. De Jonge, F. W. Krijger & A. M. Deelder (1992): Palaeoepidemiology
of Schistosoma infection in mummies. – B.M.J. 304: 555-556.
Nunn, J. F. & E. Tapp (2000): Tropical diseases in ancient Egypt. – Trans. Roy. Soc. Trop. Med. Hyg. 94 (2):
147-153. Erratum in: Trans. R. Soc. Trop. Med. Hyg. 94 (3): 352.
Pääbo, S. (1989): Ancient DNA: extraction, characterization, molecular cloning, and enzymatic amplification. –
Proc. Natl. Acad. Sci. USA 86 (6): 1939-1943.
Petersen, S., O. F. Nielsen, D. H. Christensen, H. G. M. Edwards, D. W. Farwell, R. David, P. Lambert, M. Gnia-
decka & H. C. Wulf (2003): Near infra-red Fourier transform Ramon spectroscopy of skin samples from the
‘Tomb of the Two Brothers,’ Khnum-Nakht and Nekht-Ankh, XIIth Dynasty Egyptian mummies (ca 2000
BC). – J. Ramon Spectroscopy 34: 375-379. (doi: 10.1002/jrs.1006)
Poinar, H. N., M. Hofreiter, W. G. Spaulding, P. S. Martin, B. A. Stankiewicz, H. Bland, R. P. Evershed, G. Pos-
snert & S. Pääbo (1998): Molecular coproscopy: dung and diet of the extinct ground sloth Nothrotheriops
shastensis. – Science 281: 402-406.
Pontes, L. A., E. Dias-Neto & A. Rabello (2002): Detection by polymerase chain reaction of Schistosoma mansoni
DNA in human serum and feces. – Am. J. Trop. Med. Hyg. 66 (2): 157-162.
Reyman, T. A., M. Y. Zimmerman & P. K. Lewin (1977): Autopsy of an Egyptian mummy. 5. Histopathologic
investigation. – Can. Med. Assoc. J. 117 (5): 470-472.
Ruffer, M. A. (1910): Note on the presence of Bilharzia haematobia in Egyptian mummies of the XXth dynasty
(1250-1000 BC). Cited in: Adamson, P. B. (1976): Schistosomiasis in antiquity. Med. Hist. 20: 176-188.
Rutherford, P. (1999): Immunocytochemistry and the diagnosis of schistosomiasis: ancient and modern. – Para-
sitol. Today 15: 390-391.
Schmitt, J., M. Wuhrer, J. Hamburger, J. Jourdane, R. M. Ramzy, R. Geyer & A. Ruppel (2002): Schistosoma
mansoni and Schistosoma haematobium: identification and characterization of glycoconjugate antigens in the
hemolymph of infected vector snails. – J. Parasitol. 88 (3): 505-513.
Vigilant, L., R. Pennington, H. Harpending, T. D. Kocher & A. C. Wilson (1989): Mitochondrial DNA sequences
in single hairs from a southern African population. – Proc. Natl. Acad. Sci. USA 86: 9350-9354.
WHO (2011): Schistosomiasis, 25 February 2011. – Weekly epidemiological record 86: 73-80. – http://www.
who.int/wer/2011/wer8609.pdf. Accessed 2011, December 29.
Young, N. D., A. R. Jex, B. Li, S. Lie, L. Yang, Z. Xiong, Y. Li, C. Cantacessi, R. S. Hall, X. Xu, F. Chen, X. Wu,
A. Zerlotini, G. Oliveira, A. Hofmann, G. Zhang, X. Fang, Y. Kang, B. E. Campbell, A. Loukas, S. Ranga-
nathan, D. Rollinson, G. Rinaldi, P. J. Brindley, H. Yang, Jun Wang, Jian Wang & R. B. Gasser (2012): Whole
genome sequence of Schistosoma haematobium. – Nature Genet. 44 (2): 221-225. (doi:1038/ng.1065)
Matheson et al.: Schistosoma and Family Relationship in Egyptian Mummies
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Yearbook of Mummy Studies 2 (2014)
ISBN 978
ISBN 978
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3
3
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89937
89937
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163
163
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5 www.pfeil-verlag.de
5 www.pfeil-verlag.de
Bodies with any type of preserved soft tissue are considered mummies. The interna-
Bodies with any type of preserved soft tissue are considered mummies. The interna-
tional multidisciplinary research field of Mummy Studies incorporates research from
tional multidisciplinary research field of Mummy Studies incorporates research from
anthropology, medicine and numerous other fields in order to analyze and interpret
anthropology, medicine and numerous other fields in order to analyze and interpret
human and animal remains with preserved soft tissue. The
human and animal remains with preserved soft tissue. The
“
“
Yearbook of Mummy
Yearbook of Mummy
Studies
Studies
”
”
is the first, and only, academic journal dedicated solely to aspects of the study
is the first, and only, academic journal dedicated solely to aspects of the study
of mummies and aims to present research that explores the methods and theories used
of mummies and aims to present research that explores the methods and theories used
in the interpretation of human and animal mummies.
in the interpretation of human and animal mummies.
Front cover
Front cover
CT-based 3D reconstruction of the Late Period Egyptian mummy of Tadimentet inside the coffin.
CT-based 3D reconstruction of the Late Period Egyptian mummy of Tadimentet inside the coffin.
(Daizo et al., pp. 71
(Daizo et al., pp. 71
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76)
76)
Back cover
Back cover
Stages of the facial reconstruction of a Roman Period Egyptian child mummy
Stages of the facial reconstruction of a Roman Period Egyptian child mummy
made by Raymond Evenhouse (University of Illinois at Chicago) in 1990.
made by Raymond Evenhouse (University of Illinois at Chicago) in 1990.
(Wisseman & Hunt, pp. 87
(Wisseman & Hunt, pp. 87
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94)
94)
