Donoghue Etal LancetInfDisease 2004 TB

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For personal use. Only reproduce with permission from Elsevier Ltd.584

During the past 10 years palaeomicrobiology, a new

scientific discipline, has developed. The study of ancient

pathogens by direct detection of their DNA has answered

several historical questions and shown changes to

pathogens over time. However, ancient DNA (aDNA)

continues to be controversial and great care is needed toprovide valid data. Here we review the most successful

application of the technology, which is the study of

tuberculosis. This has provided direct support for the current

theory of Mycobacterium tuberculosis evolution, and

suggests areas of investigation for the interaction of

M tuberculosis with its host.

Lancet Infect Dis 2004; 4: 58492

Ancient DNA (aDNA) has been controversial since itwas retrieved from an extinct animal, the quagga, in 1984. 1

Originally, cloning was used to obtain the DNA sequence

but the preferred method has become DNA amplificationby PCR.2 There are many anthropological and epide-miological reasons for the appeal of aDNA analysiseg, itprovides information on sequence changes in real time,rather than relying on calculations based on a molecularclock. In addition to phylogenetics and populationgenetics, animal and plant remains can tell us aboutearly social and agricultural practices, and coprolites(ancient faeces) give information about health statusand diet. Nuclear DNA from human beings can revealtheir sex, which may not be determined from skeletalmorphometrics or inferences from grave goods. Analysisof mitochondrial DNA provides information aboutfamilial or kinship relatedness and the data to test

hypotheses on population migrations in prehistory. Somereviews of the use of aDNA in palaeontology3 andanthropology4 include comprehensive overviews of itsapplications.

DNA is not a stable molecule, and oxidation andhydrolysis damage DNA over time.5 As a result, the DNAbecomes fragmented, especially during the strand separationstage of PCR. Therefore, the best amplifications are obtainedusing a small DNA target size, preferably below 200 bp. It isnot the age of the DNA but the environmental conditionswhich are critical in preservation.6,7 A stable environment isbest for DNA preservation. Cool temperatures have allowedthe recovery of Neanderthal DNA,7,8 but hot, dry

environments such as that of Egypt9,10

have also yieldedaDNA (figure 1). The upper limit for recovery of amplifiableDNA is believed to be around 100 000 years.7

Unfortunately, some of the earliest work on ancient

DNAfrom prehistoric human remainsled to concernsabout contamination from modern DNA, including that ofthe investigators.2,11 This problem resulted in a series ofrecommendations for good practice: physical separation ofactivities before and after PCR, strict protocols to preventand monitor the introduction of modern DNA, the use ofnegative controls, observation of an inverse relationshipbetween target sequence size and PCR efficiency, replicationof samples (preferably) in different laboratories to confirmresults, and assessment of sequence data to checkphylogenetics.5,12 Cloning has been used for aDNA analyses,especially of human remains, since it can identify DNAdamage, PCR error, or the presence of mixtures in thesample. However, where there is good DNA preservation

and specific DNA sequences are detected, cloning is rarelyneeded.

Review Tuberculosis and ancient DNA

HDD and MS are at the Centre for Infectious Diseases and

International Health, University College London, London, UK; CLG,

GLM, MS, and KV are at the Kuvin Centre for the Study of Infectious

and Tropical Diseases, The Hebrew University, Hadassah Medical

School, Jerusalem, Israel; GKBG is at the Laboratory of Genomic

Diversity, National Cancer Institute, Frederick, MD, USA; CM is at

the Paleo-DNA Laboratory, Faculty of Science and Environmental

Studies, Lakehead University, Thunder Bay, ON, Canada; KV is at

the Department of Zoology, The University of Queensland, Brisbane,

Queensland, Australia; AGN and ARZ are at the Division of

Palaeopathology, Institute of Pathology, Academic Teaching

Hospital Mnchen-Bogenhausen, Munich, Germany.

Correspondence: Dr Helen D Donoghue, Centre for Infectious

Diseases and International Health, University College London, 46Cleveland Street, London W1T 4JF, UK. Tel +44 207 679 9153;

fax +44 207 636 8175; email [email protected]

Tuberculosis: from prehistory to Robert Koch, as

revealed by ancient DNA

Helen D Donoghue, Mark Spigelman, Charles L Greenblatt, Galit Lev-Maor, Gila Kahila Bar-Gal, Carney

Matheson, Kim Vernon, Andreas G Nerlich, and Albert R Zink

Infectious Diseases Vol 4 September 2004 http://infection.thelancet.com

Figure 1. Use of endoscopy to obtain samples from an ancient Egyptian

mummy.


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Microbial pathogens and ancient DNAThe success in obtaining mammalian aDNA soon led to the

search for microbial pathogens. Initially, interest focused onretrospective disease diagnosis and confirmation ofdiagnoses that had been inferred from pathological skeletalchanges. However, it became clear that phylogenetic studiesmight be possible. The clearest example of this technology inthe field of acute infections is the characterisation of thestrain of influenza virus13,14 that was responsible for the 1918pandemic, which killed 40 million people worldwide. Thiswork relied on remains preserved in the Arctic permafrostand archival pathology samples. It is hoped that the study ofchronic diseases will clarify the interaction between host andpathogen. Ancient pathogen DNA research has beenreviewed15,16 and Spigelman and Donoghue17 have reviewedmycobacterial diseases.

Two strategies have been adopted to detect specificpathogenic microbes. The most successful strategy usesspecific detection techniques that were developed in clinicaldiagnostic microbiology; these techniques are applied toancient material. The other strategy uses non-specificmethods to amplify aDNA, which is then separated andsequenced.18 In theory, mycobacteria are the ideal micro-organisms for studying the aDNA of pathogens and were theearliest to be sought. This group includes the organisms thatcause tuberculosis and leprosy, and these pathogens arefound only in an infected host. Tuberculosis was widespreadin the past and is a re-emerging global threat.19,20 Thepathology of tuberculosis is characteristic (figure 2);

localised lesions tend to contain residual microbial DNA.Mycobacteria have DNA with a high proportion of guanineand cytosine; this increases DNA stability and may aid itssurvival. In addition, the thick cell walls of thesemycobacteria are lipid-rich,21,22 protecting DNA from theattack of lytic enzymes after autolysis and necrosis of thehost cells, the other microflora and fauna of the host upondeath, and the first-stage decomposers that invade once aperson dies. M tuberculosis has survived after fixing informalin and staining,23 the death of the host,24 and has evenbeen transmitted from the corpse to a new host 1 year afterburial.25

Ancient DNA from theM tuberculosis complexIn 1993, Spigelman and Lemma26 published the first findingof aDNA in a human microbial pathogen (panel 1).

Archaeological specimens from human beings that had beenmorphologically diagnosed with tuberculosis were used. Theinsertion sequence IS6110 was found with PCR primersspecific to the M tuberculosis complex, which includesM tuberculosis, M bovis, and M aftricanum.27 This sequence ishighly conserved and is typically present in multiple copiesin M tuberculosis, which increases detection sensitivity.These primers also detect IS6110 in the other members ofthe M tuberculosiscomplex.

11 specimens were used in the first report ofarchaeological M tuberculosisDNA26 and four of these, bonesranging in age from 3001400 years, tested positive. Thebones came from Europe, Turkey, and Borneo (before

European contact) and M tuberculosis in these samples hassince been confirmed with sequence data.28 Subsequently,Salo et al29 used identical primers to clone and sequenceamplicons from the lung tissue of a 1000-year-old Peruvianmummy. Shortly after these publications a number oflaboratories published similar studies, the great majority ofwhich used the IS6110 target site for the detection ofM tuberculosis DNA.3040 This method continues to be themost sensitive for the detection of the M tuberculosiscomplex. Other specific sequencesin addition to theIS6110regionhave been targeted.10,4148

Verification of findings of ancientM tuberculosis DNA

At least two reports provide biomolecular evidence for thepreservation of the tubercle bacillus in ancient specimens:M tuberculosis-specific mycolic acids were found togetherwith M tuberculosis-specific IS6110 DNA. The presence ofboth types of biomoleculesamplification of the DNA anddirect detection of the cell wall mycolic with highperformance liquid chromatographyis one method ofverifying the diagnosis of tuberculosis.36,49

Pathogenic microbial DNA in a specimen has beenvalidated with the corresponding host DNA. For example,using material from ancient Egyptians, Zink et al46

demonstrated host DNA by amplification of a 202 bpsegment of the human beta-actin gene. In further work,

Zink and Nerlich found a few samples in whichM tuberculosisDNA could be amplified but the human beta-actin gene could not be amplified (unpublished observations

ReviewTuberculosis and ancient DNA


Figure 2. Gross pathology of the spine in an 18th century Hungarian from

Vc who had tuberculosis.

Panel 1. Advantages of studying aDNA of microbial

pathogens

Confirmation of palaeopathology

To answer historical questions

Epidemiology of the disease

Geographic range in the past

Population studies

Comparison of microbial pathogens from the past with those of today

Molecular evolution

Changes in virulence

Development of the relationship between host and pathogen relation


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by the authors). The reason is likely to be the differentialpreservation ofM tuberculosisDNA.17 Matheson and Vernonhave also observed that mycobacterial DNA can be detectedwhen genomic and mitochondrial host DNA is severelydegraded (unpublished observations by the authors). Insome cases the tubercle bacilli have persisted (figure 3).Taphonomy of mammalian DNA after death commonlydamages mitochondrial DNA50 and this can lead to

interpretation errors in ancient human population genetics.By contrast, point mutations are relatively rare inmycobacteria51 and molecular analysis mainly uses genomicdeletions. Therefore, it is not surprising that mycobacterialDNA seems better than human DNA in preservation andsequence fidelity.

Independent verificationIndependent verification by another laboratory, and thedetection of several different target sequences in theM tuberculosis genome, are important strategies forverification of aDNA data. An indication of the reliability ofdata obtained from different laboratories is provided bysome research conducted at Jerusalem and University

College London (UCL). Lev-Maor et al52 used PCR toanalyse 30 suspected tuberculosis samples obtained across awide range of times and locations. 15 specimens testedpositive and replication in a second laboratory confirmedthis result in 12 specimens. Different quantities of bone-powder sample were assessed in the two laboratories, whichmay explain the discrepancy. Also, it should be noted that bycontrast with mitochondrial or nuclear mammalian DNA,pathogenic prokaryotic DNA is not likely to be distributedevenly within a sample and this will lead to variable successin its detection.

Fletcher et al47 studied the remains of 263 individualsfrom the 18th century; the remains were preserved well

because they were in a sealed church crypt in Vc, Hungary,where they naturally mummified (figure 4). In a study of thereproducibility of their findings, one to four independent

laboratories examined 27 samples from 26 individuals withvarious target sequences for M tuberculosis. Identical resultswere obtained in 23 of the samples and in the four remainingsamples the results were compatible.

The use of different target sequences tocharacteriseM tuberculosis aDNAIn addition to the IS6110 region, other sites on theM tuberculosis genome are being assessed to validateM tuberculosisand to characterise the organisms that causedtuberculosis in the past. Initially this work aimed at finding

which species of the M tuberculosiscomplex was present. InM tuberculosisa few single nucleotide polymorphisms in thekatG463 and gyrA95 sites have been used to genotypestrains.51,53 Strains can be identified as M tuberculosis orM bovis from genotyping, amplification of the oxyR andmtp40 loci, and spoligotyping based on the direct repeat(DR) region spacers.48,54 Amplification of these single-copychromosomal loci are highly dependent on the state ofpreservation of the material and, commonly only the IS6110PCR yields data.40

More recently, investigators have begun to use deletionanalysis to characterise the M tuberculosis DNA that ispresent in ancient remains. There are deletion hotspots in

the M tuberculosis genome,55

which can be used todifferentiate the members of the M tuberculosiscomplex anddistinguish between strains.56,57 It seems that deletions are



Figure 3. Microscopic appearance of chest material from a 36-year-old

man who haemorrhaged from the mouth and died in Vc in 1808.

Samples were strongly positive forM tuberculosis DNA with Ziehl-

Neelsen stain for acid-fast bacilli.

Figure 4. Mummified body 116 from Vc. DNA from this womans chestwas M tuberculosispositive. She died aged 26 years after a caesarean

operation; her child lived for 1 day.


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associated with some loss of virulence,58 which suggests thatdeletion analysis may also throw some light on thehostpathogen relationship.

Initial findings from ancientM tuberculosisstudiesEven the earliest experiments in this field answeredquestions that have interested palaeopathologists for a longtimesuch as proving that human tuberculosis existedbefore European contact in both Borneo and the New World(panel 2). It soon became clear that tuberculosis is an ancientdisease with a wide geographic distribution. Another finding

that is consistent with the course of tuberculosis infection isthat although M tuberculosis can be detected in samplesshowing typical pathologysuch as vertebral lesions (figure2), rib lesions (figure 5), or pleural adhesions (figure 6)itcan also be detected at a lower frequency in bones with nopathological changes.10,41,46,5961 The spread of tubercle bacillithroughout the body, via the bloodstream in severe cases ofmilliary tuberculosis and during bacteraemia after death, canexplain this finding.

aDNA techniques have identified a strain ofM tuberculosisrather than M bovis in a subculture (1901)of the original Kochs bacillus, increasing our knowledgeof tuberculosis during past times.62 This work was basedon a museum specimen that had probably been treated

with formalin; further molecular characterisation wasunsuccessful.

The use of spoligotyping to characterise theM tuberculosis complexSpoligotyping is a form of molecular typing. PCR of spacersequences in the DR region63 are seen by way of dot-blothybridisation on a membrane, giving a molecularfingerprint. The primers are in the DR region and amplifyup to 43 unique spacer regions, which are between each DRsequence. The function of this region is unknown anddifferent strains ofM tuberculosiscommonly show deletions,which form the basis of an international database that is

used for epidemiological investigations. The database hasbeen used for evolutionary studies because loss of spacers isunidirectional; care is needed in interpretation since

particular patterns can result from an accumulation ofseveral independent events or mixed infections.

Spoligotyping is an especially valuable technique for thestudy ofM tuberculosisin archaeological material since it ispossible to obtain useful data even when the DNA is highlyfragmented. The distance between adjacent DR loci is only3541 bp and each DR locus is 36 bp,63 so a fragment of5560 bp is sufficient to provide a result.

There are increasing numbers of reports of theapplication of spoligotyping of aDNA for M tuberculosis. Forexample, with methods described by Parwati et al,64 Fletcheret al48 did spoligotyping ofM tuberculosisDNA samples from18th century Hungary in two different laboratories. Thetechnique distinguished different genotypes ofM tuberculosis in three members of the same family (figure7). Almost complete spoligotypes were obtained from the

well-preserved remains of 12 different people; results werereproducible on different occasions and in separatelaboratories.

Spoligotyping was also central to Zink et als work 46 andwas done on all of the samples obtained by theJerusalemUCL group.64 Of 25 positive Egyptian specimens,12 had acceptable spoligotyping patterns. This was definedas three separate PCR amplifications summarised as aconsensus hybridisation pattern.46 In the JerusalemUCLstudy, 21 out of 24 positive samples had a partialhybridisation pattern. Other studies that have presentedspoligotyping patterns of ancient samples include Taylor etal42 who examined three mediaeval English specimens and

the Koch bacillus from the museum specimen.62

Rothschildet al45 used spoligotyping to examine a sample of bone fromancient bison. However, the interpretation of spoligotypes,which are obtained by repeated typing, and theaccumulation of a consensus pattern remains controversial.Taylor et al42 note that conclusions from spoligotypes ofarchaeological material must be made cautiously; somespacer regions may be lost due to differences in preservation.

Were ancient Egyptians from the MiddleKingdom infected withM africanum?Although M africanum is recognised as a separate specieswithin the M tuberculosiscomplex, some researchers dispute



Panel 2. Questions answered by aDNA studies of

M tuberculosis

Confirmation of diagnosis from palaeopathology

M tuberculosis DNA is in bones with no pathology

Tuberculosis existed in Borneo before European contact

Tuberculosis existed in America before Columbus

Tuberculosis was widespread in ancient Egypt and Rome

Kochs bacillus was M tuberculosis not M bovis

General absence ofM bovis in human remains

M tuberculosis DNA in North American bison during Pleistocene (17 500

years BP)

Indication that M tuberculosis in North America was spread from Asia by

ungulates that crossed the Bering land bridge

Confirmation of latest theory ofM tuberculosis evolution

M africanum and M tuberculosis existed over 4000 years ago

Figure 5. Lesion on a rib bone withM tuberculosis DNA.39


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this. However, the RD9 region is absent from bothM africanum types I and II,65 so they can be distinguishedfrom M tuberculosis. Both the German and JerusalemUCLstudies found with spoligotyping that some samples weremissing single spacers in numbers 3843; the gradualdeletion of these spacers may suggest a tendency towards the

M africanum and M bovis genotypes.66,67

Several spacers(3336) were under-represented in Zink et al46 (figure 8) andthis also happened in the JerusalemUCL study,52 except forspacer 33. The absence of these spacers is characteristic ofM tuberculosis and M africanum subtype II.67,68 Thediscrepancy between the two studies may be because theformer used exclusively African samples. An excitingpreliminary result from Zink et al46 is the indication thatthree spoligotypes obtained from the Middle Kingdom(20501650 BC) were missing spacer 39, a characteristic ofM africanum subtype I.67,69 M africanum is believed to becloser in evolutionary terms to their common ancestor thanM tuberculosisor M bovis. Therefore these data indicate thatthis group had become differentiated from the ancestral

form because of the loss of the RD9 region by this time.

Absence ofM bovis in archaeological humanremainsAlmost all archaeological M tuberculosis complex aDNAobtained from human remains and assessed withspoligotyping consistently shows at least some of the spacers3843. The presence of these spacers indicates that theorganisms are not M bovis. This is consistent with the failureto identify M bovis from archaeological material with othergenetic markers that distinguish between the twospecies.44,47,48,54 However, preliminary data from a study of fourarchaeological samples from a single site in Siberia indicate

that skeletal lesions were probably attributable to infectionwith M bovis. Climate could have been a factor in preservationof the M bovisDNA (GM Taylor, Imperial College London,

UK, personal communication). The apparent absence ofM bovisin an overwhelming number of human archaeologicalspecimens suggests that either the organism was absent or thatit was present at such a low level that far more samples need tobe examined to gain a robust assessment of its distribution.Before the introduction of any effective M bovis control

programmes in Great Britain, it is estimated that it caused 6%of human deaths due to tuberculosis.70 Unfortunately, veryfew studies have been completed on animal remains so it isnot known whether M bovis was present in wild ordomesticated animals that could have been a reservoir ofinfection.

Tuberculosis in prehistoryPreviously it was believed that the original source ofM tuberculosis in human beings was newly domesticatedcattle about 10 00015 000 years ago.71,72 However, recentwork45,65,73 suggests that the relationship between bovine andhuman tuberculosis may have involved co-evolution ratherthan a direct transmission from bovines to human beings.

Bone samples with lesions suggestive of a tuberculosis-likeinfection can now be traced back to the Pleistocene. 45,74

Material came from the Natural Trap Cave in Wyoming,USA. This is a vertical pit in the middle of a game trail inwhich more than 40000 bone samples accumulated over100 000 years. These samples lay in the bottom of the pit attemperatures of about 4C, which helped preservation. Onebison metacarpal showed tubercular-like infection and datedto about 17 500 years BP (according to stratigraphy and C14

dating), a period when the bison was unlikely to have beendomesticated. Spoligotyping of this ancient material wasdifficult and only partial patterns were obtained. However,discriminant analysis showed that the bison pattern was

more closely aligned to the M tuberculosisgroup than to theM africanum and M bovisgroups. Among other animals thatfell into the pit, tuberculosis-like pathology has been found



Figure 6. A mummy torso of a 68 year old girl from a tomb in Thebes-West Dra Abu El-Naga.15 The tomb was built in the New Kingdom but used until

the Late Period; it is dated about 1500500 BC. The arrow shows major connective tissue adhesions that extend to the left body wall, suggesting

chronic pleural infection.


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in the metacarpalphalangeal joints of sheep, bison, andmusk oxen. These are all species believed to have migrated toAmerica from Asia across the land bridge before theformation of the Bering Strait during the late Pleistocene. 74

Spoligotyping of this ancient material may assist in

determining how and when M bovisand M tuberculosisarosefrom a progenitor species. Further work in this area isneeded.

AncientM tuberculosis DNA and pathogenevolutionIt has been suggested that genotype 1 strains are older inevolutionary terms than genotypes 2 and 3.51,53 Brosch et al65

used a comparative study of members of the M tuberculosis

complex to propose a phylogeny on the premise that thisgroup of bacteria has evolved through the unidirectionalaccumulation of deletions (figure 9). The analysis ofkatG463and gyrA95 and discovery of genotypes 2 and 3 by Fletcher etal48 was used by Brosch et al65 as evidence that the TbD1

deletion ofM tuberculosistook place before the 18th century.Mostowy et al73 supported this phylogeny, suggesting thatgenomic deletions should be an appealing method ofstudying mycobacterial evolution. The conclusion is thatincreased cases of tuberculosis in the industrial revolutionresulted from modern M tuberculosisstrains and not M bovisor ancestral strains of M tuberculosis. It is proposed thatancestral tubercle bacilli may have originated from endemicfoci, whereas strains with the TbD1 deletion represent



Mother L1

Mother L2

Mother N3

Older daughter L1

Older daughter L2

Older daughter N3Younger daughter L1

Younger daughter L2

Younger daughter N3

H37Rv

PCR control

Mother

Older daughter

Younger daughter

10 15 20 25 30 35 405

5 10 15 20 25 30 35 40

A

B

Figure 7. (A) Spoligotyping of DNA from a mother and two daughters from the 18th century in Vc.48 (Spacers 28, 29, 30, and 32 are present but

difficult to see.) (B) Schematic representation of spoligotyping results from London and Netherlands. L1=extract 1 typed in London, L2=extract 2 typed

in London, and N3=extract 3 typed in Netherlands. Reproduced with permission from the Society of General Microbiology.

5 10 15 20 25 30 35 40

MK TT196-44

MK TT196-M5

MK TT196-MW7

MK TT196-MW18

NK TT453-PC14

NK TT453-PC9

NK TT183-8

NK TT95-PC40

NK TT95-PC122

NK TT95-PC169

NK DAN 9311

MK DAN 951-1

M tuberculosis H37Rv

M bovis BCG P3

M africanum I25420

M africanum II1567/99

Figure 8. Spoligotypes of samples from the Middle Kingdom (MK) and New Kingdom (NK).46 Tomb complexes are estimated to have been in use atthese times: TT 196=20501650 BC; DAN 951-1=2050500 BC; DAN 9311=1550500 BC; TT 95=1450500 BC; TT 453=1450500 BC; TT

183=1250500 BC. Spoligotypes ofM africanum: M africanumsubgroup I isolate ATCC 25420; M africanumsubgroup II isolate 1567/99.67


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modern M tuberculosis epidemic strains, which aredescended from a single clone. Therefore, modernM tuberculosisstrainswhich account for the vast majorityof todays tuberculosis casesare believed to haveundergone a genetic evolutionary bottleneck some 15 000 to20 000 years ago,51,75 before global dissemination.

It is not yet known whether the strains of M tuberculosisfound in pre-Columbian America and other parts of theworld differ from the virulent epidemic strains associated with18th century western Europe. The origin of these virulentstrains is of interest because the location, mechanisms, andtime-scale of their development and spread must beunderstood. Archaeological material that pre-dates large-scale

international travel and antimicrobial therapy has a specificrole in the understanding ofM tuberculosis.

Host resistance and susceptibility genesWhile characterising the phylogeny of ancient pathogens,any pathogen interactions with the human host can also beaddressed. Many research projects have focused on theidentification of genetic markers for host resistance orsusceptibility to M tuberculosis infection, which includepolymorphisms in CD receptors, cytokines and theirreceptors, various classes of leucocytes, and MHC genes.76,77

The excellent preservation of the Vc Hungarian mummiesand the archives available for many of them should enable

the family group and state of health to be established. Inaddition, it has proved feasible to distinguish individualswith active and latent M tuberculosis disease at the time of

their death.47 These data provide an unprecedentedopportunity to analyse host susceptibility to disease. Atpresent there is evidence ofM tuberculosisDNA in the tissuesof 62% of the samples, suggesting the possibility ofaddressing past epidemiology. Preliminary research on thecorrelation of NRAMP1 (SLC11A1) alleles with Hungarianmummies that show active and latent tuberculosis hasconfirmed that authentic sequences can be detected78 and dohave a susceptibility and resistance correlation.

A promising direction of research seems to be theassessment of the M tuberculosis DNA, because differentgenotypes are known to produce different immunologicaland pathological host responses.79 For example, mycobacterial

cell walls contain mannose-capped lipoarabinomannans thatare key molecules in the virulence and immunopathogenesisof tuberculosis.80,81 One of the genes involved has just beenidentified82 and should be a fruitful target for studies of thedevelopment of the host and pathogen relationship. Theexploration of genetic markers for mycobacterial virulence,from an era before any selection pressures brought about byantimicrobial therapy, is an exciting prospect.

The future of ancientM tuberculosis DNAresearchThe genomes of both M tuberculosisand M bovishave beensequenced83,84 and this has improved the understanding

of the evolution of these organisms and their interactionwith the host. Several target sequences can be amplifiedfrom very old material, and as techniques improve an



Last common ancestor

of the turbercle bacilli RD 9

RD 7

RDmic

RDseal

RD 8

RD 10

TbD 1

katG463CTG CGG

mmplL6561AAC AAG

oxyR296G A

pncA57CAC GAC

gyrA95AGC ACC

Numerous sequence

polymorphisms

RDcan M canettii

M africanum

M microti

M bovis

Seal isolates

gr1, Ancestral M tuberculosis

Modern

M tuberculosis

gr1, eg, Beijing

gr2, eg, CDC1551

gr3, eg, H37Rv

Oryx isolates

Goat isolates

Classic

BCG Tokyo

BCG Pasteur

RD 14

RD 2

RD 1

RD 13

RD 12

RD 4

Figure 9. Diagram of the evolution of the M tuberculosis complex, from reference 65. Reproduced with permission from the National Academy of

Sciences, USA.


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ancient M tuberculosis genome project could beconceived; genomics and proteomics of strains of

M tuberculosis from the past could be compared withmodern isolates. Already we have discovered thepreponderance of the human strain of the tubercle bacillusand have verified the hypothesis made by evolutionarymicrobial geneticists. The improvement in methodologyand widespread acceptance of the need for independentverification in the study of ancient pathogens by DNAtechniques has resulted in data that can withstand externalscrutiny. Technologies identify the agents and also allowfurther study of their genetics and evolution; they are nowbeing applied to ancient material. The study of tuberculosisprovides a reliable basis for the establishment of molecularpaleomicrobiology. Conversely, the paleomicrobiology oftuberculosis is informing the debate on the evolution of the

M tuberculosis complex and has the potential to aid ourunderstanding of the development of the relation betweenhost and pathogen.

Acknowledgments

We are grateful to the Center for the Study of Emerging Diseases fortheir generous support of this research. This work would not havebeen possible without the specimens that Joel Blondiaux, BerndHerrmann, Antnia Marscik, Ildic Pap, Bruce Rothschild, DouglasUbelaker, and Joe Zias provided. Hillel Bercoviers enthusiasm andsupport is thankfully acknowledged. Authors GKBG and CMpreviously worked at the Kuvin Centre for the Study of Infectious andTropical Diseases, The Hebrew University, Hadassah Medical School,Jerusalem, Israel.

Conflicts of interest

We have no conflicts of interest.



Search strategy and selection criteria

Most references were obtained from the authors extensivefiles; relevant articles referenced in these were also identified.

Medline searches were done with the search term ancient

DNA together with the search terms tuberculosis and

PCR. Selection criteria were historical importance, scientific

relevance, and illustrations of the breadth of the field.

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