review tuberculosis and ancient dna - clas...

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

During the past 10 years palaeomicrobiology, a newscientific discipline, has developed. The study of ancientpathogens by direct detection of their DNA has answeredseveral historical questions and shown changes topathogens over time. However, ancient DNA (aDNA)continues to be controversial and great care is needed toprovide valid data. Here we review the most successfulapplication of the technology, which is the study oftuberculosis. This has provided direct support for the currenttheory of Mycobacterium tuberculosis evolution, andsuggests areas of investigation for the interaction ofM tuberculosis with its host.

Lancet Infect Dis 2004; 4: 584–92

Ancient DNA (aDNA) has been controversial since it was retrieved from an extinct animal, the quagga, in 1984.1

Originally, cloning was used to obtain the DNA sequencebut the preferred method has become DNA amplificationby PCR.2 There are many anthropological and epide-miological reasons for the appeal of aDNA analysis—eg, 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 about early social and agricultural practices, and coprolites(ancient faeces) give information about health status and diet. Nuclear DNA from human beings can reveal their sex, which may not be determined from skeletalmorphometrics or inferences from grave goods. Analysis of mitochondrial DNA provides information about familial or kinship relatedness and the data to testhypotheses 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, dryenvironments 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 ancientDNA—from prehistoric human remains—led 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 preservationand specific DNA sequences are detected, cloning is rarelyneeded.

Review Tuberculosis and ancient DNA

HDD and MS are at the Centre for Infectious Diseases andInternational Health, University College London, London, UK; CLG,GLM, MS, and KV are at the Kuvin Centre for the Study of Infectiousand Tropical Diseases, The Hebrew University, Hadassah MedicalSchool, Jerusalem, Israel; GKBG is at the Laboratory of GenomicDiversity, National Cancer Institute, Frederick, MD, USA; CM is atthe Paleo-DNA Laboratory, Faculty of Science and EnvironmentalStudies, Lakehead University, Thunder Bay, ON, Canada; KV is atthe Department of Zoology, The University of Queensland, Brisbane,Queensland, Australia; AGN and ARZ are at the Division ofPalaeopathology, Institute of Pathology, Academic TeachingHospital München-Bogenhausen, Munich, Germany.

Correspondence: Dr Helen D Donoghue, Centre for InfectiousDiseases 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, asrevealed by ancient DNA

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


Microbial pathogens and ancient DNAThe success in obtaining mammalian aDNA soon led to thesearch 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 the M 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 tuberculosis complex.

11 specimens were used in the first report ofarchaeological M tuberculosis DNA26 and four of these, bonesranging in age from 300–1400 years, tested positive. Thebones came from Europe, Turkey, and Borneo (beforeEuropean 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.30–40 This method continues to be themost sensitive for the detection of the M tuberculosiscomplex. Other specific sequences—in addition to theIS6110 region—have been targeted.10,41–48

Verification of findings of ancientM tuberculosis DNAAt 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 biomolecules—amplification of the DNA anddirect detection of the cell wall mycolic with highperformance liquid chromatography—is 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 tuberculosis DNA 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 fromVàc who had tuberculosis.

Panel 1. Advantages of studying aDNA of microbialpathogens

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


by the authors). The reason is likely to be the differentialpreservation of M tuberculosis DNA.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 tointerpretation 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 UniversityCollege 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 wellbecause they were in a sealed church crypt in Vàc, 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 tocharacterise M tuberculosis aDNAIn addition to the IS6110 region, other sites on theM tuberculosis genome are being assessed to validateM tuberculosis and to characterise the organisms that causedtuberculosis in the past. Initially this work aimed at findingwhich species of the M tuberculosis complex was present. InM tuberculosis a 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 inthe M tuberculosis genome,55 which can be used todifferentiate the members of the M tuberculosis complex anddistinguish between strains.56,57 It seems that deletions are



Figure 3. Microscopic appearance of chest material from a 36-year-oldman who haemorrhaged from the mouth and died in Vàc in 1808.Samples were strongly positive for M tuberculosis DNA with Ziehl-Neelsen stain for acid-fast bacilli.

Figure 4. Mummified body 116 from Vàc. DNA from this woman’s chestwas M tuberculosis positive. She died aged 26 years after a caesareanoperation; her child lived for 1 day.


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

Initial findings from ancient M tuberculosisstudiesEven the earliest experiments in this field answeredquestions that have interested palaeopathologists for a longtime—such 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 findingthat is consistent with the course of tuberculosis infection isthat although M tuberculosis can be detected in samplesshowing typical pathology—such 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.10,41,46,59–61 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 tuberculosis rather than M bovis in a subculture (1901) of the original Koch’s bacillus, increasing our knowledge of tuberculosis during past times.62 This work was based on 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 of M tuberculosis commonly show deletions,which form the basis of an international database that isused 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 of M tuberculosis in archaeological material since it ispossible to obtain useful data even when the DNA is highlyfragmented. The distance between adjacent DR loci is only35–41 bp and each DR locus is 36 bp,63 so a fragment of55–60 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 of M tuberculosis DNA 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 thewell-preserved remains of 12 different people; results werereproducible on different occasions and in separatelaboratories.

Spoligotyping was also central to Zink et al’s work46 andwas done on all of the samples obtained by theJerusalem–UCL 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 Jerusalem–UCLstudy, 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 andthe 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 with M africanum?Although M africanum is recognised as a separate specieswithin the M tuberculosis complex, some researchers dispute



Panel 2. Questions answered by aDNA studies ofM 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

Koch’s bacillus was M tuberculosis not M bovis

General absence of M bovis in human remains

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

Indication that M tuberculosis in North America was spread from Asia byungulates that crossed the Bering land bridge

Confirmation of latest theory of M tuberculosis evolution

M africanum and M tuberculosis existed over 4000 years ago

Figure 5. Lesion on a rib bone with M tuberculosis DNA.39


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 Jerusalem–UCLstudies found with spoligotyping that some samples weremissing single spacers in numbers 38–43; the gradualdeletion of these spacers may suggest a tendency towards theM africanum and M bovis genotypes.66,67 Several spacers(33–36) were under-represented in Zink et al46 (figure 8) andthis also happened in the Jerusalem–UCL 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(2050–1650 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 tuberculosis or M bovis. Therefore these data indicate thatthis group had become differentiated from the ancestralform because of the loss of the RD9 region by this time.

Absence of M bovis in archaeological humanremainsAlmost all archaeological M tuberculosis complex aDNAobtained from human remains and assessed withspoligotyping consistently shows at least some of the spacers38–43. 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 indicatethat skeletal lesions were probably attributable to infectionwith M bovis. Climate could have been a factor in preservationof the M bovis DNA (GM Taylor, Imperial College London,

UK, personal communication). The apparent absence ofM bovis in 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 controlprogrammes 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 10 000–15 000 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 40 000 bone samples accumulated over100 000 years. These samples lay in the bottom of the pit attemperatures of about 4°C, 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 wasmore closely aligned to the M tuberculosis group than to theM africanum and M bovis groups. Among other animals thatfell into the pit, tuberculosis-like pathology has been found



Figure 6. A mummy torso of a 6–8 year old girl from a tomb in Thebes-West Dra Abu El-Naga.15 The tomb was built in the New Kingdom but used untilthe Late Period; it is dated about 1500–500 BC. The arrow shows major connective tissue adhesions that extend to the left body wall, suggestingchronic pleural infection.


in the metacarpal–phalangeal 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 indetermining how and when M bovis and M tuberculosis arosefrom a progenitor species. Further work in this area isneeded.

Ancient M 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 of katG463and gyrA95 and discovery of genotypes 2 and 3 by Fletcher etal48 was used by Brosch et al65 as evidence that the TbD1deletion of M tuberculosis took 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 tuberculosis strains 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 N3

Younger daughter L1

Younger daughter L2Younger 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 Vàc.48 (Spacers 28, 29, 30, and 32 are present butdifficult to see.) (B) Schematic representation of spoligotyping results from London and Netherlands. L1=extract 1 typed in London, L2=extract 2 typedin London, and N3=extract 3 typed in Netherlands. Reproduced with permission from the Society of General Microbiology.

5 10 15 20 25 30 35 40MK TT196-44MK TT196-M5MK TT196-MW7MK TT196-MW18NK TT453-PC14NK TT453-PC9NK TT183-8NK TT95-PC40NK TT95-PC122NK TT95-PC169NK DAN 93·11MK DAN 95·1-1M tuberculosis H37RvM bovis BCG P3M africanum I 25420M africanum II 1567/99

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=2050–1650 BC; DAN 95·1-1=2050–500 BC; DAN 93·11=1550–500 BC; TT 95=1450–500 BC; TT 453=1450–500 BC; TT183=1250–500 BC. Spoligotypes of M africanum: M africanum subgroup I isolate ATCC 25420; M africanum subgroup II isolate 1567/99.67


modern M tuberculosis epidemic strains, which aredescended from a single clone. Therefore, modernM tuberculosis strains—which account for the vast majorityof today’s tuberculosis cases—are 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-scaleinternational travel and antimicrobial therapy has a specificrole in the understanding of M 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 Vác Hungarian mummiesand the archives available for many of them should enablethe 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 of M tuberculosis DNA 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, mycobacterialcell 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 ancient M tuberculosis DNAresearchThe genomes of both M tuberculosis and M bovis have 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 ancestorof the turbercle bacilli RD 9

RD 7

RDmic

RDseal

RD 8RD 10

TbD 1

katG463CTG CGG

mmplL6561AAC AAG

oxyR296G A

pncA57CAC GAC

gyrA95AGC ACC

Numerous sequencepolymorphisms

RDcan M canettii

M africanum

M microti

M bovis

Seal isolates

gr1, Ancestral M tuberculosis

ModernM 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 13RD 12

RD 4

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


“ancient M tuberculosis genome project” could beconceived; genomics and proteomics of strains ofM 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 theM tuberculosis complex and has the potential to aid ourunderstanding of the development of the relation betweenhost and pathogen.

AcknowledgmentsWe 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, Antónia Marscik, Ildicó Pap, Bruce Rothschild, DouglasUbelaker, and Joe Zias provided. Hillel Bercovier’s 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 interestWe have no conflicts of interest.



Search strategy and selection criteriaMost references were obtained from the authors’ extensivefiles; relevant articles referenced in these were also identified.Medline searches were done with the search term “ancientDNA” together with the search terms “tuberculosis” and“PCR”. Selection criteria were historical importance, scientificrelevance, and illustrations of the breadth of the field.

References1 Higuchi R, Bowman B, Freiberger M, Ryder OA,

Wilson AC. DNA sequences from the quagga, anextinct member of the horse family. Nature 1984;312: 282–84.

2 Pääbo S. Ancient DNA: extraction, characterization,molecular cloning, and enzymatic amplification.Proc Natl Acad Sci USA 1989; 86: 1939–43.

3 Marota I, Rollo F. Molecular paleontology.Cell Mol Life Sci 2002; 59: 97–111.

4 Kaestle FA, Horsburgh KA. Ancient DNA inanthropology: methods, applications and ethics.Am J Phys Anthropol 2002; Suppl 35: 92–130.

5 O’Rourke DH, Hayes MG, Carlyle SW. AncientDNA studies in physical anthropology.Annu Rev Anthropol 2000; 29: 217–42.

6 Höss M, Jaruga P, Zastawny TH, Dizdaroglu M,Pääbo S. DNA damage and DNA sequence retrievalfrom ancient tissues. Nucleic Acids Res 1996; 24:1304–07.

7 Höss M. Neanderthal population genetics.Nature 2000; 404: 453–54.

8 Smith CI, Chamberlain AT, Riley MS, Cooper A,Stringer CB, Collins MJ. Neanderthal DNA. Not justold but old and cold? Nature 2001; 410: 771–72.

9 Pääbo S, Gifford JA, Wilson AC. MitochondrialDNA sequences from a 7000-year old brain.Nucleic Acids Res 1988; 16: 9775–87.

10 Zink A, Haas CJ, Reischl U, Szeimies U, Nerlich AG.Molecular analysis of skeletal tuberculosis in anancient Egyptian population. J Med Microbiol 2001;50: 355–66.

11 Lindahl T. Facts and artefacts of ancient DNA.Cell 1997; 90: 1–3.

12 Cooper A, Poinar HN. Ancient DNA: do it right ornot at all. Science 2000; 289: 1139

13 Reid AH, Fanning TG, Hultin JV, Taubenberger JK.Origin and evolution of the 1918 “Spanish”influenza virus hemagglutinin gene.Proc Natl Acad Sci USA 1999; 96: 1651–56.

14 Reid AH, Taubenberger JK, Fanning TG. The 1918Spanish influenza: integrating history and biology.Microbes Infect 2001; 3: 81–87.

15 Zink AR, Reischl U, Wolf H, Nerlich AG. Molecularanalysis of ancient microbial infections.FEMS Microbiol Lett 2002; 213: 141–47.

16 Herrmann B, Hummel S. Ancient DNA can identifydisease elements. In: Greenblatt C, Spigelman M,eds. Emerging pathogens: archaeology, ecology andevolution of infectious disease. Oxford:Oxford University Press, 2003: 143–49.

17 Spigelman M, Donoghue HD. Paleobacteriologywith special reference to pathogenic mycobacteria.In: Greenblatt C, Spigelman M, eds. Emergingpathogens: archaeology, ecology and evolution ofinfectious disease. Oxford: Oxford University Press,2003: 175–88.

18 Cano RJ, Tiefenbrunner F, Ubaldi M, et al. Sequenceanalysis of bacterial DNA in the colon and stomachof the Tyrolean Iceman. Am J Phys Anthropol 2000;112: 297–309.

19 World Health Organization. Tuberculosis: a globalemergency. World Health Forum 1993; 14: 438.

20 Kumaresan J, Heitkamp P, Smith I, Billo N. Globalpartnership to stop TB: a model of an effectivepublic health partnership. Int J Tuberc Lung Dis2004; 8: 120–29.

21 Daffé M, Draper P. The envelope layers ofmycobacteria with reference to their pathogenicity.Adv Microb Physiol 1998; 39: 131–203.

22 Lambert PA. Cellular impermeability and uptake ofbiocides and antibiotics in Gram-positive bacteriaand mycobacteria. J Appl Microbiol Symp 2002; 92(suppl): 46S–54S.

23 Gerston KF, Blumberg L, Gafoor H. Viability ofmycobacteria in formalin-fixed tissues.Int J Tuberc Lung Dis 1998; 2: 521–23.

24 Weed LA, Baggenstoss AH. The isolation ofpathogens from tissues of embalmed human bodies.Am J Clin Pathol 1951; 21: 1114–20.

25 Sterling TR, Pope DS, Bishai WR, Harrington S,Gershon RR, Chaisson RE. Transmission ofMycobacterium tuberculosis from a cadaver to anembalmer. N Engl J Med 2000; 342: 246–48.

26 Spigelman M, Lemma E. The use of the polymerasechain reaction (PCR) to detect Mycobacteriumtuberculosis in ancient skeletons. Int J Osteoarchaeol1993; 3: 137–43.

27 Eisenach KD, Cave MD, Bates JH, Crawford JT.Polymerase chain reaction amplification of arepetitive DNA sequence specific for Mycobacteriumtuberculosis. J Infect Dis 1990; 161: 977–81.

28 Spigelman M, Matheson C, Lev G, Greenblatt C,Donoghue HD. Confirmation of the presence ofMycobacterium tuberculosis complex-specific DNA inthree archaeological specimens. Int J Osteoarchaeol2002; 12: 393–401.

29 Salo WL, Aufderheide AC, Buikstra J, Holcomb TA.Identification of Mycobacterium tuberculosis DNA ina pre-Columbian Peruvian mummy.Proc Natl Acad Sci USA 1994; 91: 2091–94.

30 Arrieza BT, Salo W, Aufderheide AC, Holcomb TA.Pre-Columbian tuberculosis in northern Chile:molecular and skeletal evidence.Am J Phys Anthropol 1995; 98: 37–45.

31 Baron H, Hummel S, Herrmann B. Mycobacteriumtuberculosis complex DNA in ancient human bones.J Archaeol Sci 1996; 23: 667–71.

32 Taylor G, Crossey M, Saldanha J, Waldron T. DNAfrom Mycobacterium tuberculosis identified inmediaeval human skeletal remains using polymerasechain reaction. J Archaeol Sci 1996; 23: 789–98.

33 Nerlich AG, Haas CJ, Zink A, Szeimies U,Hagedorn HG. Molecular evidence for tuberculosis inan ancient Egyptian mummy. Lancet 1997; 350: 1404.

34 Braun M, Cook DC, Pfeiffer S. DNA fromMycobacterium tuberculosis complex identified inNorth American, pre-Columbian skeletal remains.J Archaeol Sci 1998; 25: 271–77.

35 Crubézy É, Ludes B, Poveda JD, Clayton J,Crouau-Roy B, Montagnon D. Identification ofMycobacterium DNA in an Egyptian Pott’s disease of5,400 years old. CR Acad Sci Paris 1998; 321: 941–51.

36 Donoghue HD, Spigelman M, Zias J,Gernaey-Child AM, Minnikin DE. Mycobacteriumtuberculosis complex DNA in calcified pleura fromremains 1400 years old. Lett Appl Microbiol 1998;27: 265–69.

37 Nuorala E. Tuberculosis on the 17th century man-of-war Kronan. Int J Osteoarchaeol 1999; 9: 344–48.

38 Haas CJ, Zink A, Molnar E. Molecular evidence fordifferent stages of tuberculosis in ancient bonesamples from Hungary. Am J Phys Anthropol 2000113: 293–304.

39 Ubelaker DH, Jones EB, Donoghue HD,Spigelman M. Skeletal and molecular evidence fortuberculosis in a forensic case. Anthropologie 2000;38: 193–200.

40 Mays S, Taylor GM. A first prehistoric case oftuberculosis from Britain. Int J Osteoarchaeol 2003;13: 189–196.

41 Faerman M, Jankauskas R, Gorski A, Bercovier H,Greenblatt CL. Prevalence of human tuberculosis ina medieval population of Lithuania studied byancient DNA analysis. Ancient Biomolecules 1997;1: 205–14.

42 Taylor GM, Goyal M, Legge AJ, Shaw RJ, Young D.Genotypic analysis of Mycobacterium tuberculosisfrom medieval human remains. Microbiology 1999;145: 899–904.

43 Faerman M, Jankauskas R. Paleopathological andmolecular evidence of human bone tuberculosis inIron Age Lithuania. Anthropol Anz 2000; 58: 57–62.

44 Mays S, Taylor GM, Legge AJ, Young DB,Turner-Walker G. Paleopathological andbiomolecular study of tuberculosis in a medievalskeletal collection from England. Am J PhysAnthropol 2001; 114: 298–311.

45 Rothschild BM, Martin LD, Lev G, et al.Mycobacterium tuberculosis complex DNA from anextinct bison dated 17,000 years before the present.Clin Infect Dis 2001; 33: 305–11.

46 Zink AR, Sola C, Reischl U, et al. Characterization ofMycobacterium tuberculosis complex DNAs fromEgyptian mummies by spoligotyping.J Clin Microbiol 2003; 41: 359–67.

47 Fletcher HA, Donoghue HD, Holton J, Pap I,Spigelman M. Widespread occurrence ofMycobacterium tuberculosis DNA from 18th–19thcentury Hungarians. Am J Phys Anthropol 2003;120: 144–52.

48 Fletcher HA, Donoghue HD, Taylor GM,van der Zanden AG, Spigelman M. Molecularanalysis of Mycobacterium tuberculosis DNA from afamily of 18th century Hungarians.Microbiology 2003; 149: 143–51.

49 Gernaey AM, Minnikin DE, Copley MS, Dixon RA, Middleton JC, Roberts CA. Mycolic acids and ancient DNA confirm an osteologicaldiagnosis of tuberculosis. Tuberculosis 2001; 81:259–65.

50 Gilbert MT, Hansen AJ, Willersley E, et al.Characterization of genetic miscoding lesions causedby post-mortem damage. Am J Hum Genet 2003; 72:48–61.


51 Sreevatsan S, Pan X, Stockbauer KE, et al. Restrictedstructural gene polymorphism in the Mycobacteriumtuberculosis complex indicates evolutionarily recentglobal dissemination. Proc Natl Acad Sci USA 1997;94: 9869–74.

52 Lev-Maor G, Blondiaux J, Spigelman M, et al.Ancient tuberculosis: problems of diagnosis andcharacterization. Ancient Biomolecules 2002; 4: 142.

53 Frothingham R. Evolutionary bottlenecks in theagents of tuberculosis, leprosy and paratuberculosis.Med Hypotheses 1999; 52: 95–99.

54 Taylor GM, Mays S, Legge AJ, Ho TBL, Young DB.Genetic analysis of tuberculosis in human remains.Ancient Biomolecules 2001; 3: 267–80.

55 Ho TB, Robertson BD, Taylor GM, Shaw RJ,Young DB. Comparison of Mycobacteriumtuberculosis genomes reveals frequent deletions in a20-kb variable region in clinical isolates. Yeast 2000;17: 272–82.

56 Parsons LM, Brosch R, Cole ST, et al. Rapid andsimple approach for identification of Mycobacteriumtuberculosis complex isolates by PCR-based genomicdeletion analysis. J Clin Microbiol 2002; 40: 2339–45.

57 Huard RC, de Oliveira Lazzarini LC, Butler WR,van Soolingen D, Ho JL. PCR-based method todifferentiate the subspecies of the Mycobacteriumtuberculosis complex on the basis of genomicdeletions. J Clin Microbiol 2003; 41: 1637–50.

58 Kato-Maeda M, Rhee JT, Gingeras TR, et al.Comparing genomes within the speciesMycobacterium tuberculosis. Genome Res 2001;11: 547–54.

59 Mays S, Fysh E, Taylor GM. Investigation of the linkbetween visceral surface rib lesions and tuberculosisin a medieval skeletal series from England usingancient DNA. Am J Phys Anthropol 2002; 119: 27–36.

60 Zink AR, Grabner W, Reischl U, Wolf H, Nerlich AG.Molecular study on human tuberculosis in threegeographically distinct and time delineatedpopulations from ancient Egypt. Epidemiol Infect2003; 130: 239–49.

61 Fusegawa H, Wang BH, Sakurai K, Nagasawa K,Okauchi M, Nagakura K. Outbreak of tuberculosis ina 2000-year-old Chinese population. KansenshogakuZasshi 2003; 77: 146–49.

62 Taylor GM, Stewart GR, Cooke M, et al. Koch’sBacillus—a look at the first isolate of Mycobacterium

tuberculosis from a modern perspective.Microbiology 2003; 149: 3213–20.

63 Kamerbeek J, Schouls L, Kolk A, et al. Simultaneousdetection and strain differentiation ofMycobacterium tuberculosis for diagnosis andepidemiology. J Clin Microbiol 1997; 35: 907–14.

64 Parwati I, van Crevel R, van Soolingen D, van derZanden A. Application of spoligotyping tononcultured Mycobacterium tuberculosis bacteriarequires an optimized approach.J Clin Microbiol 2003; 41: 5350–51.

65 Brosch R, Gordon V, Marmiesse M, et al. A newevolutionary scenario for the Mycobacteriumtuberculosis complex. Proc Natl Acad Sci USA 2002;99: 3684–89.

66 Sales MPU, Taylor GM, Hughes S, et al. Geneticdiversity among Mycobacterium bovis isolates: apreliminary study of strains from animal and humansources. J Clin Microbiol 2001; 39: 4558–62.

67 Niemann S, Rüsch-Gerdes S, Joloba ML, et al.Mycobacterium africanum subtype II is associatedwith two distinct genotypes and is a major cause ofhuman tuberculosis in Kampala, Uganda.J Clin Microbiol 2002; 40: 3398–3405.

68 Frothingham R, Strickland PL, Bretzel G,Ramaswamy S, Musser JM, Williams DL. Phenotypicand genotypic characterization of Mycobacteriumafricanum isolates from West Africa.J Clin Microbiol 1999; 37: 1921–26.

69 Viana-Niero C, Gutierrez C, Sola C, et al. Geneticdiversity of Mycobacterium africanum clinical isolatesbased on IS6110-restriction fragment lengthpolymorphism analysis, spoligotyping, and variablenumber of tandem DNA repeats.J Clin Microbiol 2001; 39: 57–65.

70 Grange JM. Human aspects of Mycobacterium bovisinfection. In: Thoen CO, Steele JH, eds.Mycobacterium bovis infection in animals andhumans. Iowa: Iowa State University Press, 1995:29–46.

71 Haas F, Haas SS. The origins of Mycobacteriumtuberculosis and the notion of its contagiousness. In:Rom WN, Garay SM, eds. Tuberculosis, 1st edn.Boston MA: Little Brown and Company, 1996: 3–19.

72 Cole ST. Comparative and functional genomics ofthe Mycobacterium tuberculosis complex.Microbiology 2002; 148: 2919–28.

73 Mostowy S, Cousins D, Brinkman J, Aranaz A,Behr MA. Genomic deletions suggest a phylogeny forthe Mycobacterium tuberculosis complex.J Infect Dis 2002; 186: 74–80.

74 Rothschild BM, Martin LD. Frequency of pathologyin a large natural sample from Natural Trap Cavewith special remarks on erosive disease in thePleistocene. Reumatismo 2003; 55: 58–65.

75 Kapur V, Whittam TS, Musser JM. IsMycobacterium tuberculosis 15,000 years old? J Inf Dis 1994; 170: 1348–49.

76 Hill AVS. The genomics and genetics of humaninfectious disease susceptibility.Annu Rev Genom Hum Genet 2001; 2: 373–400.

77 Bellamy R. Susceptibility to mycobacterialinfections: the importance of host genetics. GenImmun 2003; 4: 4–11.

78 Matheson C, Donoghue HD, Fletcher H, et al.Tuberculosis in ancient populations: a linkage study.Ancient Biomolecules 2001; 3: 295.

79 Lopez B, Aguilar D, Orozco H, et al. A markeddifference in pathogenesis and immune responseinduced by different Mycobacterium tuberculosisgenotypes. Clin Exp Immunol 2003; 133: 30–37.

80 Khoo KH, Tang JB, Chatterjee D. Variation inmannose-capped terminal arabinan motifs oflipoarabinomannans from clinical isolates ofMycobacterium tuberculosis and Mycobacteriumavium complex. J Biol Chem 2000; 276: 3863–71.

81 Appelmelk BJ, van Die I, van Vliet SJ,Vandenbroucke-Grauls CMJE, Geijtenbeek TBH,van Kooyk Y. Cutting edge: carbohydrate profiling identifies new pathogens that interact withdendritic cell-specific ICAM-3-grabbingnonintegrin on dendritic cells. J Immunol 2003; 170:1635–39.

82 Alexander DC, Jones JR, Tan T, Chen JM, Liu J.PimF, a mannosyltransferase of mycobacteria, isinvolved in the biosynthesis of phosphatidylinositolmannosides and lipoarabinomannan.J Biol Chem 2004; 279: 18824–33.

83 Cole ST, Brosch R, Parkhill J, et al. Deciphering thebiology of Mycobacterium tuberculosis from thecomplete genome sequence. Nature 2001; 393: 537–44.

84 Garnier T, Eiglmeier K, Camus J-C, et al. Thecomplete genome sequence of Mycobacterium bovis.Proc Natl Acad Sci USA 2003; 100: 7877–82.



review tuberculosis and ancient dna - clas...

Documents