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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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8/11/2019 213 no già


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

8/11/2019 213 no già


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


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

8/11/2019 213 no già



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


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.

8/11/2019 213 no già


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

mail

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.

8/11/2019 213 no già


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.

8/11/2019 213 no già


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.

8/11/2019 213 no già



Egyptian

Mummy

Tissue Bank

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,

8/11/2019 213 no già


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

8/11/2019 213 no già



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

8/11/2019 213 no già


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

8/11/2019 213 no già



Egyptian Mummy

Tissue

Bank

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

8/11/2019 213 no già


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

8/11/2019 213 no già


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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