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TRANSCRIPT
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The International Ancient Egyptian Mummy Tissue Bank at the Manchester Museum as a
Resource for the Palaeoepidemiological Study of Schistosomiasis
Author(s): Patricia I. Lambert-Zazulak, Patricia Rutherford, A. Rosalie David
Source: World Archaeology, Vol. 35, No. 2, Archaeology of Epidemic and Infectious Disease,
(Oct., 2003), pp. 223-240
Published by: Taylor & Francis, Ltd.
Stable URL: http://www.jstor.org/stable/3560224
Accessed: 01/07/2008 02:53
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h e nternational
n c i e n t
gypt ian
Mummy
T i s s u e
a n k t t h Manchester
Mu s e um
s
resource
o r
t h
p l eoepdemologic l
t u d y
o f
schistosomi sis
Patricia
I.
Lambert-Zazulak,
Patricia
Rutherford
and
A.
Rosalie
David
Abstract
This
paper
outlines the
setting up
of
the
Mummy
Tissue Bank at
the Manchester
Museum
as
an
international research
resource,
conceived
in
the
context
of
the
worldwide Schistosomiasis
Research
Project
n
1996. It considers
he
value
of
such a
resourcefor both
ancient
and modern
studies,
with
particular
reference
to the work
done
at Manchesteron
ancient schistosomiasis.
Immunocytochemicalechniques
have been
developed
to be
applied
o
mummy
issue
for the first
time,and the resultsmaybe comparedwithdata from the modernEgyptianpopulation.The Tissue
Bank facilitatesvarious
research
projects,
and it is
hoped
that the
work on
schistosomiasis
will
provide
a model for
future
palaeoepidemiological
esearch nto other
diseases.
Keywords
Egypt;
mummy;
issue
bank; chistosomiasis;
alaeopathology;
Manchester.
Introduction
Because the environmental conditions
in
Egypt
have ensured
that a wealth
of evidence
has survived
from
antiquity,
it
is
possible
to
gain
information
from
a wide
range
of
material about
disease in the ancient
population.
The
sources
include
tomb
wall
scenes,
medical
papyri
and
human
remains.
The
Manchester
Egyptian
Mummy
Research
Project
was established
in 1973 at
the
University
of
Manchester
(UK),
with
the aim
of
developing
an
interdisciplinary,
scientific
study
of
human
and
animal mummified remains
(David 1979).
Initially,
the
project
g
Routledge
World
Archaeology
Vol.
35(2):
223-240
Epidemic
nd
Infectious
isease
Taylor&FrancisGroup
?
2003
Taylor
&
FrancisLtdISSN
0043-8243
print/1470-1375
nline
DOI:10.1080/0043824032000111399
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International Ancient
Egyptian
Mummy
Tissue Bank
225
Ancient evidence for the
disease
In order to
compare
the
100,000
case studies identified
by
the SRP
in
modern
Egypt
with
examples
from
antiquity,
it was
necessary
to examine evidence
provided
by
the ancient
literary
sources
and
the
mummified remains.
Written information relating to disease in ancient Egypt is preserved in ten major
documents known as
medical
papyri
(Leake 1952).
Most of
these
can be
dated
to c.1550
BC,
although
some are
almost
certainly
copies
of earlier works. These texts show little
uniformity:
some were
probably
handbooks for
surgeons' regular
use,
while others
were
perhaps
the
outlines of
medical
lectures,
or
the
lecture
notes and
clinical notebooks
that
once
belonged
to
students.
However,
many
of
them
present
individual case
studies,
which
each
include
lists of
symptoms,
a
diagnosis
and recommended treatment.
One
condition,
referred
to as the
aaa-disease,
is mentioned
many
times in the
papyri.
In
Egyptian hieroglyphs,
the
symbol
drawn at the end of a word
(known
as a
determinative)
was
used
to
convey
its
meaning.
In the
medical
texts,
the determinative which
appears
at
the
end of
aaa
represents
a
discharging phallus,
and thus
the
translator of
one medical
papyrus
(Ebbell 1937)
has
identified aaa as haematuria
(blood
in the
urine).
Since schisto-
somiasis is the
commonest cause of haematuria in
Egypt,
some
Egyptologists
have
claimed that these
references to the
aaa-disease
prove
that the
ancient
Egyptians
specifi-
cally diagnosed,
identified
and recorded this disease
(Jonckheere
1944).
However,
there
are
alternative
interpretations
which
identify
aaa not
as
schistosomiasis
but as a toxic and
poisonous
substance,
introduced into the
patient
by
means of
magic,
which
caused
a
variety
of
diseases
(Nunn
1996:
63).
One
medicinal
remedy (Papyrus
Ebers
No.62),
intended for the treatment
of
a
patient
who
has hrrwt-worms
in his
belly,
states
that the
hrrwt-worms have been created
by
the
aaa.
Although
it has
been
suggested
that the
hrrwt-worm was the adult
parasitic
worm
(schistosome)
that causes schistosomiasis, the hrrwt-worm has never been conclusively
identified. Some
translators claim that
this
passage proves
that the aaa-disease
should be
identified as
schistosomiasis,
but others
dispute
this,
stating
that,
although
the medical
papyri
contain
many
references to intestinal infestations
by parasitic
worms,
exact identi-
fication of
some
of
these
worms,
including
the
schistosome,
would
have
been
impossible
in
antiquity.
It is
argued
that the
Egyptians
almost
certainly
would not have
performed
autopsies
within
a
short
enough
time-frame and
in sufficient
detail
to discover
the
presence
of
this
parasite (Nunn
1996:
68-9)
and that even
the
adult male
worm,
ranging
from 0.6 to
2.5cm
in
length
(Neva
and
Brown 1994:
245),
would
probably
not
have
been
visible to the naked
eye.
The literary evidence is therefore inconclusive with regard to the identification and
diagnosis
of
schistosomiasis,
but confirmation of its existence
in
antiquity
has
been
provided by
examination
of
the
mummified remains. The earliest
of
these
reports
(Ruffer
1910)
described
the
discovery
of calcified
bilharzia
haematobia
(schistosoma)
ova in
the
kidneys
of
two
mummies of the Twentieth
Dynasty.
Subsequent
radiological,
histological
and
immunological
studies have revealed
the
presence
of
schistosomiasis
in several other
mummies.
Although
the
disease had been
identified
in
these
few
examples,
it was decided
that,
for
the
Manchester
epidemiological study,
it would be
necessary
to include as
large
a
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226
Patricia
I.
Lambert-Zazulak
et
al
population sample
as
possible,
and also to
develop
a
technique
that could be used as an
effective and
relatively
cheap
diagnostic
tool for
large
numbers
of
fragmentary
ancient
tissue
samples.
Consequently,
the
International Ancient
Egyptian Mummy
Tissue Bank
was
established at
Manchester,
and
immunocytochemistry
was selected as an
appropriate
diagnostic
technique.
Preservation of tissues
from ancient
Egypt
The Schistosomiasis
Research
Project proposed
to
study
the
palaeoepidemiology
of this
disease for
the first
time,
as
part
of
the
largest-scale epidemiological
study
carried out to
date
for
any
disease,
and
covering
a
longer
time
period
than
is
usually possible.
The
evidence for
the
presence
of ancient
schistosomiasis
can be found
preserved
in
small
samples
of the
soft tissues of the human
body, especially
the
bladder, liver,
intestine
and
kidneys
(Ruffer 1910),
although
other tissue
types
can also reveal
positive
results.
The
preservation
of
such tissues from ancient
Egypt
occurred due to two
main factors
-
the
physical
environment and
the ancient
religious
beliefs. A shallow
grave
made
in the
desert
sand allowed the
hot,
dry
and well-drained
environment to transform
a
corpse
naturally
into a
stable,
desiccated
mummy
in
just
a few weeks. Some bodies
found
in
this
condition
date from
Predynastic
times
(up
to 3050
BC),
some
represent
the
poorer
Egyptian
classes
throughout Dynastic history (Murnane
1983:
351-6),
some
are
found
among
later
Christian burials after
religious
beliefs had
changed
(Van
Gerven
1981;
Dzierzykray-Rogalski
1986),
and
a
few
may
continue
to occur in modern times. Some
'fake'
ancient
Egyptian
mummies were also
produced
in
this
way, using
the
bodies
of
executed criminals
(Budge
1890:
13),
from medieval times to
the nineteenth
century,
to
satisfy
the needs of the
trade in mummies and
mummy parts
for
making
the
drug
'mumia'
(Dawson 1926-7) and for the antiquities and souvenir markets.This category of Egyptian
mummy
is often referred
to as 'natural' or
'unintentional',
as environmental
factors
alone
account
for the
preservation
and stabilization of the
corpse.
Natural
mummies are
also
free of
the
embalming
materials,
such as
resins,
which can hinder tissue
preparation
in
the
laboratory.
Many
of
even the most
delicate structures such as fine membranes
and blood
vessels
may
be found
preserved
in
mummified
individuals,
and the fact that such structures
are
demonstrable in mummies
suggests
the
possibility
that
parasites
and their
eggs may
also
be
preserved.
By
far
the
major category
of
Egyptian
mummy
available for
study today
is the
inten-
tionally preserved and wrapped body. This was produced using contemporaneous
anatomical
and medical
knowledge,
specialized
tools and
materials,
and treated
in accor-
dance with
elaborate
religious
beliefs. These beliefs
may
have had some
of their
origins
in
the
observation
of
the
phenomenon
of natural
mummification
(Breasted 1912:49).
Herodotus
(De
Selincourt 1972:
160-2)
visited
Egypt
in the
fifth
century
BC and was
told
by
the
priests
of
three methods of
preparing
mummies,
related
to cost. The
first and
most
elaborate method
caused the
mummy
to become
most like
the revivified
god
of the
dead,
Osiris,
the first and
archetypal Egyptian
mummy,
with
whom the
Egyptians
sought
to
identify
after
death
(Watterson 1984:72-88).
This first-class method
of mummification
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International Ancient
Egyptian
Mummy
Tissue Bank 227
involved the removal of the brain
using
a
metal instrument
forced
through
the cribriform
plate
of the ethmoid
bone into the cranial
cavity,
which
was then rinsed out.
The
major
viscera were removed
(except
for
the heart and
kidneys),
via an
incision,
and
were
then
placed
in
canopic packages
under the
protection
of the Four Sons
of Horus.
The
human-headed Imset
protected
the
liver,
baboon-headed
Hapy
the
lungs,
jackal-headed
Duamutef the stomach and hawk-headed Qebehsenuf the intestines (Smith 1906; Edit-
orial
1907).
The
packages
were
placed
either
in
canopic jars
or within or
upon
the
mummy
before it was
wrapped.
Desiccation
of
the
body
was achieved
with natron salt
(Garner
1979)
and the
mummy
was washed
and anointed
with
a
number of substances
(Lucas
1962:
270-326),
including palm
wine, resins, oils,
unguents
and
possibly
sometimes
bitumen
(Spielmann 1932)
before
being wrapped.
This
type
of
mummy
often contains
body cavity-packing
materials
including
linen, resin,
spices
and
sawdust,
and sometimes
subcutaneous
packing
materials such as
mud,
sand and butter. So-called embalmers'
dumps
are also associated with the
higher-class
burials,
and contain the
waste materials
of
embalming,
ritually
buried near
a
tomb,
as
they may
also
have included
fragments
of
body
tissue
(Winlock
1941).
The second method of
mummification described
by
Herodotus
involved evisceration
by
injecting
cedar oil
through
the
anus,
which was then
stopped
up
and the
body
desiccated
in
natron. When the oil was
drained
off,
it carried with it all
the dissolved internal
organs,
and
Herodotus wrote of this
type
of
mummy, 'nothing
of the
body
is
left
but the bones
and
skin'
(De
Selincourt 1972:
161).
The
third
method,
used
only
for the
poor,
was to
clean out
the intestines with a
purge,
and desiccate the
body
in natron.
Ancient
Egypt
has
clearly
left a
variety
of
types
of desiccated
mummies
available
for
modern-day study
(Smith
and Dawson 1991
[1924];
Cockburn et al.
1983;
Millet
et al.
1983;
Reyman
and Peck
1983;
Editorial
1912),
depending
on their
original
environmental condi-
tions
and
preparation.
Intentional,
ritual
preparation
of
each
part
of the
body
was carried
out in accordancewith a highly elaborate magico-religious system
(Lambert-Zazulak
1996)
and this
profoundly
affects the
range
of
tissue
types
available
today,
their
differential
levels
of
preservation,
the
presence
of
embalming
or other materials
(sand
is almost
universally
present),
the
accessibility
of
tissues for
sampling
and the
techniques by
which
they
can
be
prepared
for
study.
The work of earlier
investigators
and conservators
upon
the
mummy
is
another factor which
impacts
on the tissue
available
for
study today,
and
its
condition,
and
clearly
demonstrates
the
importance
of accurate record
keeping
(Granville
1825;
Osburn
1828;
Murray
1910;
Tapp
1979;
Dawson
et
al.
2002).
The Tissue Bank
The
objective
of the
International Ancient
Egyptian
Mummy
Tissue
Bank is to
obtain
and
store small
(up
to
2g) samples
of as
many
tissue
types
as
possible,
from mummies
representing
the
span
of
Egyptian history (3100
BC to AD
641)
and
many geographical
regions
of the
country.
These
samples
are obtained from mummies now
outside
Egypt,
and
their
collection involves a
range
of non-destructive
methods,
designed
to
maintain
the
conservation
of
the
body.
Potential sources of tissue include
whole
mummies
(wrapped
or
unwrapped),
mummy parts, canopic
packages
and
samples
of the
'drug'
mumia.
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228 Patricia
I
Lambert-Zazulak
et
al.
Locating
and
documenting potential
sources of
mummy
tissue is itself a
major
research
project,
which
began
in
1997.
The
data obtained
by
the
project
can be included
in the
International
Mummy
Database
(Pettitt
and Fildes
1984,
1986),
which
was set
up
in
Manchester in
1979.
The search
conducted for the Tissue Bank
project
represents perhaps
the
largest survey
of the
locations of ancient
Egyptian
human remains worldwide
outside
Egypt ever undertaken. This research was initiated by the compilation of a database
containing
some
8,000
entries
of names and
addresses,
mainly
obtained
from
specialized
directories,
of
the
potential
locations of ancient
Egyptian
human remains.
They
each
received a
letter
introducing
the Tissue Bank
project,
translated into various
languages,
along
with a
reply
form.
The
response
to
the
shot
was extensive and
very encouraging, resulting
in remains
being
located in
many places
not
previously centrally
recorded. Places where remains
have been
located cover museums
(including
those
specializing
in the
history
of
medicine,
pharmacy,
Bible
studies,
circuses
and the
military),
universities,
research
institutes,
learned
societies,
hospitals,
medical
schools,
stately
homes,
public
schools,
libraries
and
monasteries
(Lambert-Zazulak
2000).
This has reflected the
history
of the
collecting
of
Egyptian
human
remains
by
institutions and
individuals,
and the uses and trades to
which
mummies have
been
subjected
in
the
long history
of their
departure
from
Egypt
and
subsequent
movements
(Germer
1997:
95-115).
Respondents
were
requested
to
provide
any
historical and medical
data available
on
their
ancient
Egyptian
human
remains for the
central records held at
Manchester,
along
with
an
indication of their
interest in
depositing
tissue with the Tissue
Bank,
and a note of
any
other
locations
of
Egyptian
human
remains in their
area,
which
may
not otherwise
have been
included
in
the
mail shot. The
records
compiled
from this
survey, along
with the
Interna-
tional
Mummy
Database,
will
themselves form a
large
centralized research
resource,
as
they
contain
information
on
all
the
remains
located,
whether or not
they
are also contributors
of
tissue to the Bank. For example, reports on a mummy's archaeological context may include
descriptions
of
artwork,
nscriptions
and artefacts which
suggest
the
person's age,
sex,
family
relationships, ethnicity,
occupation,
social
status,
locality
and historical
period.
When
avail-
able,
this
information
may
help
to
corroborate examinations
of
the
body
and contribute
important
demographic
data for
various
types
of studies.
The
survey
has
revealed
some
fascinating insights
into how mummies came
to be
collected from
Egypt
at
different
times,
up
until the late twentieth
century,
and reflects
the
interests and
motives
of
the
collectors and also
the
changing
popular
perceptions
of
mummies
(Brier
1996:
299-322).
Unfortunately,
however,
this
history
has often
resulted
in the
provenance
of the
remains
being
entirely
lost.
Once the governing body of the depositing institution has granted permission to sample
human
remains for the
Tissue
Bank,
the
application
of non-destructive
techniques
ensures
the
preservation
of
the
integrity
of the
specimen. Techniques
include dissection
from
damaged
areas of a
mummy,
especially
the severed areas of a
separated
head
or
limb,
and also
the
application
of
specially adapted endoscopes (Tapp
et al.
1984),
which
are
capable
of
retrieving
small
samples
of material
from
within
body
cavities,
after
prior
radiographic
examination.
This
is
especially
useful for
pinpointing
areas
from
which
diseased tissue
may
be obtained
by endoscopy,
after
patterns
of calcification
characteristic
of
schistosomiasis
have been
demonstrated
radiographically.
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International
Ancient
Egyptian Mummy
Tissue
Bank 229
The Tissue Bank's
procedures
ensure that
mummies and
the
samples
obtained
from
them are handled
ethically
and with a
sense of
dignity
and
respect
for
human
remains at
all
stages
of the
process
of tissue collection
and
subsequent
study.
Samples
remain
the
property
of their
depositors,
and are
allocated to the
Bank for a
renewable
period
of ten
years.
The
samples
are
labelled,
recorded,
checked and
stored in a
fireproof
safe in
an
environmentally
controlled secure
area
(Plate 1). Routinely,
tissue is
allocated from the
Bank for
use
in
scientific research
projects by
the
Tissue Bank
Supervisor
(currently
Professor Rosalie
David),
but reference
may
also
be made
to the
Bank's
multidisciplinary
advisory panel.
The
panel
includes
Egyptologists
and scientists
of
several
nationalities
(Lambert-Zazulak
2000).
The
Tissue Bank's
procedures
and
legal
documentation
ensure
that the results of research are
reported
in
due course
for the
Tissue
Bank's
central
records,
and that
any remaining
tissue is
returned to the
Bank,
including permanent
preparations
such as
histological
blocks
and
microscope
slides.
Results of
any
investiga-
tions are
reported
to the
depositing
institutions,
and
any publications
arising
from
the
research will include an
acknowledgement
of the
depositors
of
the
tissue
studied.
While the Tissue Bank was
originally
initiated in
the context
of the
Schistosomiasis
Research
Project,
it can also facilitate a number of other,
carefully
selected studies
Plate
1
The
Tissue Bank:
ancient
tissue
samples
are
stored
in
a
secure
cabinet in an
environmentally
controlled
area.
?
The
Manchester
Museum,
The
University
of
Manchester.
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230
Patricia I.
Lambert-Zazulak
et
al.
requiring
small
samples
of
mummy
tissue.
The
particular
strength
of the
Tissue Bank as a
research
facility
is that it is able to collect
large
numbers
of
samples,
which
may
then
be
made
available for
large-scale palaeoepidemiological
studies,
thereby
increasing
their
statistical value.
Importantly,
tissue collection
procedures
have
been
designed
to minimize
destruction,
by directing investigators
to
samples
obtained with
the issue of the conserva-
tion of human remains in mind.
The
antiquity
of
schistosomiasis
in
Egypt
is an
appropriate
subject
to
study
using
material collected
for
the Tissue
Bank
facility
because
of the
disease's
long-standing
historical
presence
in
Egypt
and its
continued
impact
on the
modern
population
(Pain
2001).
Its
diagnosis
in
ancient tissues
at Manchester
required
the
development
of
special-
ized methods of
preparation
and
detection,
applied
to
mummy
tissue for the
first time.
Schistosomiasis: the disease
Currently
more
than
300 million
people
are infected
by
schistosomes.
The
resulting
disease
is
widespread
in the
tropics, infecting
between
75
and
95
per
cent of the
popula-
tion
in certain areas
(Neva
and
Brown 1994:
251),
and
it
is
thought
that
20
per
cent of
the
Egyptian
population today
are
infected,
with a
prevalence
of 85
per
cent
in
some
of
the
small
villages (Contis
and
David
1996).
Schistosoma
mansoni
is the
most common
species,
and lives
within the
veins
of the
hepatic portal
system,
which
drains the
intestines.
The
Schistosoma
japonicum
species
also lives in the
hepatic portal
system,
in contrast
to
Schistosoma
haematobium,
which
lives in the veins
draining
the bladder.
Although
prevalent
today,
schistosomes were
evident
in
ancient
Egyptian
times;
tissues
dating
as far back as
5,000
years
have been
positively
diagnosed
with
the disease
(Deelder
et
al.
1990).
Records
of its
comparable
antiquity
have also been
found
in China
(Strick-
land 1991:781). The schistosoma belong to the platyhelminth phylum, a blood fluke that
lives and
feeds
upon
the
cells, blood,
mucus and
tissue fluids
of its
primary
host. To
date,
eighteen
different
species
of schistosoma have been
found,
the
majority
of which
infect
animals;
although
the cercaria can
penetrate
man's
skin,
most
species
die without
migrating,
and
produce
no more
than dermatitis.
However,
humans
can
be
successfully
infected,
the three
main
species
responsible being
Schistosoma
mansoni,
haematobium
and
japonicum.
Schistosoma
worms
can
successfully
live in
man,
sometimes
for
over
twenty
years,
continually breeding
and
producing
thousands of
eggs,
half of
which are released
back
into the
water via
faeces
or
urine,
depending
on
their
species
(Cheever
1969),
while
the
other half remain in the body, causing continuous damage by both mechanical and
immunological
means. After
mating
has taken
place,
between 300
(S. mansoni)
and
3,000
(S.
japonicum)
eggs
are
laid
each
day.
Within
each
egg
is a
fully
developed
larva known
as
a miracidium. This secretes
lytic enzymes
that
diffuse
through
the
micropores
of the
egg's
shell
thus
assisting
the
spines
readily
to break
through
the
walls of
the veins
and
surrounding
tissues,
causing
considerable
damage
to
lung,
renal
and neural
tissues.
It is
the
reactions
towards
the
eggs
that
produce
inflammation,
fibrosis,
cirrhosis of the
liver,
diarrhoea and
abdominal
distension,
enlargement
of the
liver
and
spleen
and
even
haem-
orrhaging.
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International Ancient
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231
The miracidium
penetrates
species-specific
water
snails,
where
two
generations
of
sporocytes
follow. This results in
cercariae
larvae
burrowing
out of
the snail's
body
into
the
water,
where
they readily
infect
people by penetrating
the skin. Each snail
can release
300 to
3,000
larvae each
day. Upon penetration
the cercaria
sheds its
tail,
becoming
a
shistosomulum. Now
tolerant of a saline
environment,
it
migrates through
the host's
subcutaneous tissue into the blood vessels. It is then passively taken in the blood stream
to the
lungs
and heart
where,
via the
systemic
circulatory system,
it
reaches
the
liver. Once
in the liver it
feeds,
grows
and
pairs
with the
opposite
sex:
they
then move
against
the
blood flow in
the
hepatic portal
vein to their
primary
site
of infection
(which
depends
on
the
species),
where
they begin
to breed
(Fig. 1).
Upon
maturity
the adult worms are
totally
beyond
eradication
as
they begin
to
decrease
their
expression
of
antigens.
The worm encloses itself
in both
glycolipids
and
proteins
derived from the host
by inserting
them into the outer
lipid layer,
thus
masking
its own
antigens
and
creating
a
pseudo-identity
(Clegg
et al.
1970).
Circulating
anodic
and cathodic
antigens
(CAA
and
CCA)
are
regurgitated
from
the
gut
of the
worm into the blood
stream,
possibly
to divert immune reactions
away
from
the
worm itself.
The worms therefore have
very
little
pathogenic
effect within the
body,
unlike
the
spiny eggs
that remain
lodged
within
the vessel
walls. It
is the immune reaction
to
these
eggs
that
causes the disease state schistosomiasis.
People living
in
village
communities
by
the rivers
constantly
swim,
fish
and
wash
there,
and this
lifestyle,
combined with
increased
irrigation
and bad
sanitation
habits,
makes
them
vulnerable to the
free-swimming
cercariae.
Schistosomiasis
is therefore still endemic
in
certain areas of the world
today.
Evidence of ancient
schistosomiasis
Although
first described in
depth
in
the
early
1900s
by
Bilharz
(1852,
S.
haematobium),
Sambon
(1907,
S.
mansoni)
and
Logan
(1905
S.
japonicum),
this
is not a
new
disease.
As
previously
described,
it was
evident
in
ancient
Egyptian
times,
possibly
in
literary
sources.
Archeological
evidence,
such as wall
reliefs,
hieroglyphs
and
papyri,
confirms
that the
people's
lifestyle encompassed
activities such as
bathing, fishing
and
playing
in
the
Nile.
Combined with bad sanitation
habits,
this would make almost
everyone
susceptible
to this
infection.
Scientific evidence
also
supports
the
presence
of
ancient
schistosomiasis.
Schistosome
eggs
have been
positively
identified
in
ancient
tissues,
by histological
investigations
done
by Ruffer (1910) and studies by Millet et al. (1983) on gut, kidney and liver samples taken
from a
young
weaver known as Nakht
(ROM1),
held at the
Royal
Ontario Museum.
More
recently,
work
carried out
by
Deelder et al.
(1990)
has
confirmed the
presence
of circu-
lating
anodic
antigens
(CAA)
in
tissue
samples
investigated
at the
British
Museum.
The
oldest
diagnosis
of
schistosomiasis to date has been made
in tissue taken
from
the
shin
of
a
5,000-year-old Predynastic mummy
(BM32753).
This
was achieved
by
means
of an
enzyme-linked
immunosorbent
assay
(ELISA).
Not
only
did the ELISA
confirm the
presence
of
CAA
but
also that the worm was still alive
upon
the
death
of the host.
This
was
established as the CAA can be
detected
in
the
host's serum
only
if the worm
is
alive,
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232
Patricia
L
Lambert-Zazulak
et al
\^B~^~. ~~~~~~~~~~Hepatic
ortal
veins
Pairedmature
lukes
Primary
site
of
infection
Liver
S.
haematobium
Eggs
in veins of bladder a.Retainedn tissue
Arteries
/_::^/^ ~~causing
damage
-
S. minansoni
a
rt
S.jaonhum
^,
b.
Excreted
nto water.
Heart
in nesenteric
Cr
(yclv--ce
begins
again
veinsof bowel
Luligs
'
cercariae
'
penetrate
Bulinus
p
skin
S.
haematobiut
i
---
--
-
-- ----
Biomphalaria sp
S.
nmansoni
.
.
g , ,,
.
,/ , /
.
-\
) )
-l
Oncomelania
sp
S.
japonimcum
i X- t-
-
ovum
miracidium
Snail
cercaria
Figure1 The
life
cycle
of
Schistosoma
mansoni,
haematobium
nd
japonicum. Drawingby
Jane
Sherry
after a
drawing by
G. Barnish in
Jordan et al.
1993.)
as its levels
reduce
rapidly
to
nothing
in the host's serum
within ten
days
of the
worm's
death. The same ELISA tests have also been carried out successfully on cheek samples
taken from
ROM1. Serum
levels found in the ROM1 cheek tissue
correspond
with those
of modern
patients
(De
Jonge
et al.
1989).
The
aforementioned work
inspired
Miller et al.
(1993)
to
conclude
that the
identifica-
tion of CAA
in
both these
mummies confirms that
complex polysaccharides
can survive
over thousands
of
years
as
they
are more stable
than
proteins;
and such
carbohydrate
survival is
now
comparable
to
the survival of short
DNA
fragments
found
in the same
Predynastic
mummy
(BM
32753)
and others
by
Paabo
(1985,1989).
Radiology
has
also
been
successfully
applied
to
studying
ancient
remains,
usually
with
intact mummies
that
are
often covered
by
a
cartonnage
case.
An
excellent
example
of this
was carried out by Isherwood et al. (1979), who demonstrated the presence of calcification
of
the
bladder,
a classic
symptom
of
S.
haematobium,
in
two
mummies;
one
of
these,
Manchester
Museum
registration
number
1766,
is
shown in Plate
2.
Thus,
bladder
tissue
from this
mummy
has
proved
to
be
an
invaluable source
of material
when
investigating
schistosomiasis in
ancient
tissues. More
recently,
when Adams
(2000)
X-rayed
other
mummies from
the Leicester
and
Manchester
Museum
collections,
calcification
of
the
bladder was
seen in
several of
these
mummies,
including
Asru
(Manchester
Museum
registration
number
1777).
Endoscopy
was used to
obtain
samples
of
Asru's
bladder
tissue
for
testing. Although
some
of
the modern
methods are
not
appropriate
for
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International Ancient
Egyptian Mummy
Tissue Bank
233
Plate
2 Manchester
Mummy
1766 is
carefullyendoscoped
n
order to obtain tissue
samples
for
testing.
?
The Manchester
Museum,
The
University
of
Manchester.
diagnosing
schistosomiasis
in ancient tissues
(such
as the
detection of schistosoma
ova
in
urine,
stools or rectal
mucosa,
or the use of
reagent strips
that can detect blood in
both
stools and
urine),
other methods
such
as
histology (Ruffer
1910;
Millet et al.
1983),
radiology
(Isherwood
et al.
1979;
Adams
2000)
and
ELISA
(Deelder
et al.
1990;
Miller et
al. 1992), can be readily adapted for this purpose.
Immunocytochemistry
as a
diagnostic
tool
The
establishment of the Tissue
Bank at
Manchester Museum to facilitate the
epidemio-
logical study
of schistosomiasis has
generated
the need for
reasonably cheap,
robust,
reproducible
tests that can be
applied
to such a
large-scale study.
Some
of
the
diagnostic
tools
already
mentioned can
be
impractical
as
they may
be
very expensive
(X-rays
and
ELISA),
or
low in
sensitivity (histology).
To overcome such problems immunocytochemistry has been used, and successfully
applied
to both modern and ancient tissues
(Rutherford
1997).
The use of
immuno-
cytochemistry
to
diagnose
disease in ancient tissues is
not common
practice,
although
applying
it to
tissues embedded in
wax,
to show cellular
components
and
neurotransmitters,
has been
reported
(Fulcheri
et al.
1992).
Prior to the current
project,
immunocytochemistry
had not been
successfully applied
to
diagnose
schistosomiasis
in
either modern or ancient
tissues,
as
sectioning
modern
tissue often obscures the distinctive
egg shapes.
Using
antisera
to
both S.
mansoni and
S.
haematobium worm
and
egg antigens,
visual-
ization of S.
mansoni and
S.
haematobium
antigens
in modern
mouse and hamster tissues
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234
Patricia
I
Lambert-Zazulak
et
al
was
achieved
respectively.
Positive
staining
was also achieved
upon
a human
bladder
sample
infected with
S. haematobium some
fifty years
old,
from an
Egyptian
cadaver.
Positive
immunostaining upon
ancient
Egyptian
tissues has also been
achieved,
suggesting
that schistosoma
antigens
may
still be
present
after thousands
of
years.
Twenty-five
per
cent of all
samples
tested
using immunocytochemistry
showed
positive
results. In particular the bladder of mummy 1766 displayed positive immunostaining of
eggs
and also
revealed the
presence
of
S.
haematobium worms.
Researchers
from
the
VACSERA
laboratory, Egypt,
confirmed the identification of these worms
after
comparing
them
with
contemporary samples.
However,
positive
staining
of
the
often-distorted
egg shapes
meant
that
species
identification
was
not
always
possible.
Such a test
was
predominantly
dependent upon
the successful
sectioning
of
both the
modern
and
ancient
tissues,
the
majority
of which
were
embedded
in immunoresin for
higher
sensitivity (Heryet
and
Gatter
1992).
It was found
that,
although
there
were no
problems
sectioning
the modern
tissue,
the
presence
of
resins used
in the
mummification
process
and
silica
particles
enmeshed within
the
mummified tissues made
sectioning
of the
ancient
tissues
difficult. In order to
overcome
this,
a diamond knife was often used
to cut
the
sections.
Alternatively, soaking
the
gritty samples
in
a
very
weak solution of
hydrofluoric
acid for several
weeks dissolved the
silica without
damaging
the
antigens
of interest.
Other
diagnostic
tests
The
application
of
immunocytochemistry
to ancient
tissues has
proven
to be
relatively
cheap
to
perform
and is a
prelude
to other more
complicated
tests.
However,
limitations
have been
imposed
by
the
unavailability
of tissue
samples
such as liver and bladder
that
harbour
the
infecting
schistosomes and their
eggs.
Several alternative tests have
been
incorporated into the study (Rutherford 2002). For example, the ELISA test performed
by
Deelder et
al.
(1990)
was
used to
diagnose
the disease
in
other tissue
samples
such
as
skin
and brain.
This has allowed
fifty
mummies to be
tested to
date,
of which 36
per
cent
were
found
to be
positive.
However,
the
ELISA test
does have
limitations.
Al
Sherbiny
et al.
(1999)
reported
that,
even when
testing
modern
patients
known to be
infected,
the
low
sensitivity
of
the test
cannot detect
positive
infections if
the
patient
is
suffering
from
only
a
light
infection.
Such
a
test is also
appropriate
for
showing
only
active infections.
This,
combined with
the
high
cost of
using
monoclonal
antibodies to
perform
the
ELISA,
has been taken into
account;
and it has
been
concluded
that,
as an
independent
test on such a
large
number
of
samples,
it would prove to be very expensive and insensitive. The ELISA test is therefore most
suitable for
reinforcing
immunocytochemistry
results.
A
small
number of
ancient
samples
that were
positively
immunostained
were also
tested
for the
presence
of the S.
haematobium GP23
(HAMA)
and the S. mansoni GP30
(MAMA)
antibodies
towards
microsomal
antigens.
This was carried out
using
the
enzyme-linked
immunoelectro transfer
blot
(EITB).
This entails
antibodies
reacting
with
specific
antigens
that have been
absorbed onto
paper,
which is then cut
into
strips.
These
were
kindly
supplied by
the VACSERA
laboratory
in
Egypt,
where the
strips
are
used to
diagnose
the
disease
in
modern
patients (Al Sherbiny
et al.
1999).
A
positive
result
for the
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International Ancient
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235
S. haematobium
GP23
(HAMA) antibody
has been
achieved
upon
bladder
tissue
extracted
from
mummy
1766,
thus
reinforcing
the
positive
immunostaining
results.
Only
one other
sample,
mummy
1777
bladder,
has
proved
to
be
positive.
The
presence
of
antibodies
shown
in
mummy
1766
and
mummy
1777
bladder confirm
that,
although antigens
are
larger
and
thought
to survive
for
longer periods
of
time,
antibodies can also survive for over two thousand years. However, 1766 is an intact
mummy
covered in
cartonnage,
and
therefore
the
endoscoped
bladder
will have
been
protected
from the outside elements
and remained
in a
static,
stable
environment.
Whether the
antibodies would
still
be
present
in
samples
from
detached
body parts
that
do not have
such
protection
is
unknown,
but
they may
be
considerably degraded.
The
very
pale
reaction
achieved for
GP23
S.
haematobium
antibodies
from
the
unwrapped body
of
mummy
1777
supports
this
theory.
The
drawback
of
testing
for antibodies is that
their
presence
does not
necessarily
indicate the
presence
of an active infection. This is because antibodies to
parasite antigens
can
persist
in
the bloodstream
after
elimination of
the
parasite (Neva
and Brown 1994:
330).
Therefore it can be concluded
only
that
the
subject
did suffer
from an infection but
it
may
have
been
in the
past.
In
modern
patients
this is not
the case as other
symptoms
manifest at
the time of
testing.
Unfortunately,
these
symptoms
do
not manifest
in the
mummies.
Samples
that
produced positive immunostaining
results were
also
analysed
for
the
presence
of
ancient
schistosome
DNA.
Ancient
DNA
has
been
successfully
extracted
from
several ancient
samples.
A
small
region
of the human
haemoglobin gene
and
236
base
pairs
of the
schistosome
cytochrome
oxidase C
gene (COI)
have been
successfully
amplified.
Sequencing
of the ancient
S. haematobium
COI DNA
has also been
successful,
producing
a
97
per
cent
match
to
the modern
sequence
found
today.
Analysing
ancient
samples
for the
presence
of
ancient DNA involves destruction
of the
sample, although it is small. Therefore, only samples that displayed positive immun-
ostaining
results have
been
investigated
at this level. Total
destruction of tissue
is also
required
to
carry
out
the
ELISA
and
EITB
tests,
as
it
involves
incubation
of
the
super-
natant
produced
from
homogenizing
the tissue
sample.
Therefore
again,
only
positive
immunostained
samples
have been
tested.
Clearly
there are limitations to
each
type
of
test,
but,
by
combining
results
from
several
tests,
they
can
provide
an overall
picture
of the state
of health
in the case of each
mummy.
An
excellent
example
of
this is
the overall results
achieved with the
bladder
samples
taken
from
mummy
1766.
Immunocytochemistry
has shown
positive
immunostaining
of
eggs
and
the
presence
of the S.
haematobium
worm.
Also,
tests for
the
presence
of S.
haematobium GP23 antibodies and the CAA were both positive. Calcification of the
bladder
was
also
clearly
evident,
suggesting
that chronic
schistosomiasis
may
have been
present
for
several
years.
Although combining
the results of several tests
produced
a
higher percentage
of
positive
results,
a minimum of
750mg
to
Ig
of tissue
is
required
to do the
full
range
of
tests,
and this
amount
may
not
always
be available. The use of
immunocytochemistry
entails
blocking only
a small amount of
tissue,
upon
which
other
tests,
such as
histology,
DNA
staining
and even in situ
hybridization,
can
be carried out
at
a later
date.
Very
few of
the
fifty
samples
tested for
schistosomiasis
to date have been
subjected
to all
the
available
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236 Patricia I.
Lambert-Zazulak
et
al
tests.
However,
all
samples
have been
subject
to
immunocytochemistry,
and the
use of
immunocytochemistry
is
now a
prelude
to all other tests.
Science and
Egyptology
Combining
science and
Egyptology
can
give
an overall
picture
of a disease.
However,
because of the nature of the
ancient tissues
studied,
several factors
have dictated
the
design
of the
experiments.
The
ethics
of
destroying
finite
mummy
tissue
is
always
an
important
factor and
therefore the use of destructive
methods must be carried
out
conservatively.
The
availability
of
optimum samples
(i.e.
liver and
bladder)
is
often limited
and
therefore,
in
this
project,
other
samples
have had to be used.
In order to
study
the
distribution
patterns
of
infection,
provenance
of the
mummies is
needed, and,
especially
for
detached
body
parts,
this is
not
usually
known.
Obviously,
for a
large-scale
epidemi-
ology study, samples
with a
provenance
are most desirable.
In
continuing
work,
medieval
samples
collected from Sudanese Nubia
dating
to
circa
AD
500 are now
being
tested.
Immunostaining
results
will dictate which
samples
are
investigated
at the DNA
level. All the data obtained from the tested
samples
are recorded
in
the
Tissue Bank
database,
and
ultimately,
after
many provenanced
samples
have
been
tested,
a
distribution
pattern
should
emerge.
The aim
of the initial
project
was to establish
working
methods which could be
applied
to a
large-scale
study
and several have now been established
for future work. For
example,
immunoresin
(GMA)
provided
a better medium than
wax
when
working
with
mummy
tissue.
It
not
only provides
support,
but also can be
prepared
and
kept
in the
cold,
thus
preserving
epitopes
of
interest.
Also,
thinner sections can be
cut,
which increases
the
sensitivity
of
immunostaining
tests.
However,
the
delicate
tissue
may
not
always
stand
up
to the excessive soaking and may disintegrate before it can be blocked.
Tissue
samples
containing
less resins
(used
in
mummification)
and sand were easier
to
process. Early
Dynastic
or
naturally
preserved
mummy
tissue is therefore
the
optimum
tissue
to
obtain. If
expensive
knives,
such as
diamond,
are not
available,
a
very
weak
solution
of
hydrofluoric
acid at an
optimum
concentration
of
2.5
per
cent
will dissolve
silica
particles
without
destroying
epitopes.
Not all
tissue
samples,
such as
bladder,
are available for
testing,
as mummies are often
covered in
cartonnage
or
bandages.
The ethics
of
causing
destruction of
any
kind to the
mummy
must
always
be
addressed.
Therefore,
alternative tests
may
have
to be
tried,
e.g.
ELISA for
CAA,
using
skin and brain from the detached feet and heads found
in
many
museum collections.
Conclusion
Diagnosis
of
schistosomiasis in modern
individuals
can be achieved
by using
a wide
range
of
diagnostic
tools,
such as
radiology,
histology
and the
enzyme
linked immuno-
sorbent
assay
(ELISA).
However,
it is often difficult to utilize
diagnostic
tools
to their
optimum
in
modern
tissues,
but even harder to achieve
any
results
on
ancient
tissues
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International
Ancient
Egyptian Mummy
Tissue
Bank
237
successfully. Although
most
reports
today
generally
highlight
work carried out
with the
ancient DNA
molecule,
especially
the
retrieval
of
small
DNA
fragments
that
may help
with
migration patterns,
there
has also
been some success
regarding
the
diagnosis
of
diseases.
Miller et al.
(1994)
reported
positive
results for the malaria
antigen
PFhrh-2 in
several
ancient
Egyptian
and Nubian
mummies. The
group
used a
rapid
manual Para-
sight tm-f test. This entailed absorbing homogenized tissue onto a flattened fibre wick to
which
antibody
conjugated
liposomes
containing
a
dye
were
applied,
and
if
positive
a
red line
was seen on
the wick.
Chagas'
disease has been found in Inca
mummy
tissue
(Fornaciara
et al.
1992)
and
even
syphilis
and
smallpox
have
been
found in sixteenth
century
remains
(Fornaciari
et
al.
1989).
These
reports
all reinforce other evidence that
antigens
can survive
the
rigours
of
time,
even
as
long
as
5,000
years
(Deelder
et
al.
1990).
However,
since
many
diagnostic
tools are
very
expensive,
low
in
sensitivity,
and
often need
body
fluids to
work,
they
are
impractical
when
working
with
ancient,
dehydrated
tissues.
This
current
study
has
produced
answers to several
problems regarding
how to
approach
tissue
collection,
its
preparation,
and what
tests
are
practical.
The
ethics of
working
with ancient tissue
and the
use of destructive
techniques
have also
been
addressed.
Some tests have
proved
to
be
impractical
and insensitive whereas
others have
been
identified as
suitable for ancient
tissues.
The
findings
of this
study
will assist other
investigators,
regardless
of which
disease is
being
studied;
also,
they
demonstrate
how the
destruction
of finite ancient
tissues can be
minimized.
Acknowledgements
The
authors would
like to
acknowledge
the
contributions
of the
following:
the
Lever-
hulme
Trust;
the
Kay
Hinckley
Charitable
Trust;
the
North
West Museum
Service;
Mr
Tristram
Besterman,
Director of
the
Manchester
Museum;
Professor Mark J.
Ferguson,
School
of
Biological
Sciences,
University
of
Manchester;
the
Egyptian
Reference
Diag-
nostic
Centre of the
Egyptian
Organization
for
Biological
and Vaccine
Production
(VACSERA);
Dr
G.
Contis,
President of
Medical Service
Corporation
International;
Professor M.
Doenhoff,
University
of
Bangor,
Wales;
Jane
Sherry,
Manchester
Museum,
for
Figure
1.
University of
Manchester
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