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REVIEWS Unité des Rickettsies, CNRS UMR 6020, IFR 48, Faculté de Médecine, Université de la Méditerranée, 27 Bd Jean Moulin, 13385 Marseille Cedex 05, France. Correspondence to D.R. e-mail: Didier.Raoult@ medecine.univ-mrs.fr doi:10.1038/nrmicro1063 NATURE REVIEWS | MICROBIOLOGY VOLUME 3 | JANUARY 2005 | 23 Palaeomicrobiology is an emerging field of research in microbiology that is devoted to the detection, identifica- tion and characterization of microorganisms (bacteria, viruses and parasites) in ancient specimens that can date from centuries to thousands of years in age. Although there is no strict definition to characterize the antiquity of specimens that are considered under the term ‘palaeomicrobiology’, in this article, we will consider specimens that date from the era before the emergence of the concept of microorganisms (~100 years ago) as ancient. Therefore, twentieth-century specimens are regarded as forensic specimens, and research dealing with such specimens, for example, DNA analyses to identify the cause of the 1979 out- break of anthrax in Sverdlovsk, in the former Soviet Union 1 , is presented in a separate box (BOX 1). As a discipline, palaeomicrobiology began in 1993 with the molecular detection of Mycobacterium tubercu- losis DNA in an ancient human skeleton 2 . The objectives of palaeomicrobiological studies include the diagnosis of past infectious diseases through the detection of specific microorganisms in ancient remains, the elucidation of the epidemiology of past infectious diseases by reconstituting the temporal and geographical distributions of infected individuals, reservoirs and vectors, and the tracing of the genetic evolution of the microorganisms themselves. Data from such studies benefit modern microbiology and studies of host– pathogen relationships, particularly in the context of the emergence of infectious diseases. Refinements in molecular typing now allow researchers to study the genetic evolution of microorganisms and the timing of their introduction into human populations. Palaeomicrobiological investigations can provide new information about the source of a particular epidemic, as well as details of its epidemiology. Also, such data can benefit social sciences such as archaeology, history and anthropology by providing a proven cause of death instead of the putative hypotheses that can be drawn from archaeological or historical sources alone. As laboratory techniques have advanced, the materials that can be used to search for ancient microorganisms have changed. Therefore, initial palaeomicrobiological studies used bone tissue, whereas later studies have progressed to use mummified tissues and dental pulp for analysis 3–5 . Furthermore, experimental standards for PALAEOMICROBIOLOGY: CURRENT ISSUES AND PERSPECTIVES Michel Drancourt and Didier Raoult Abstract | Palaeomicrobiology is an emerging field that is devoted to the detection, identification and characterization of microorganisms in ancient remains. Data indicate that host-associated microbial DNA can survive for almost 20,000 years, and environmental bacterial DNA preserved in permafrost samples has been dated to 400,000–600,000 years. In addition to frozen and mummified soft tissues, bone and dental pulp can also be used to search for microbial pathogens. Various techniques, including microscopy and immunodetection, can be used in palaeomicrobiology, but most data have been obtained using PCR-based molecular techniques. Infections caused by bacteria, viruses and parasites have all been diagnosed using palaeomicrobiological techniques. Additionally, molecular typing of ancient pathogens could help to reconstruct the epidemiology of past epidemics and could feed into current models of emerging infections, therefore contributing to the development of appropriate preventative measures.

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Page 1: Palaeomicrobiology: current issues and perspectives

R E V I E W S

Unité des Rickettsies,CNRS UMR 6020, IFR 48,Faculté de Médecine,Université de laMéditerranée, 27 Bd JeanMoulin, 13385 MarseilleCedex 05, France.Correspondence to D.R.e-mail: [email protected]:10.1038/nrmicro1063

NATURE REVIEWS | MICROBIOLOGY VOLUME 3 | JANUARY 2005 | 23

Palaeomicrobiology is an emerging field of research inmicrobiology that is devoted to the detection, identifica-tion and characterization of microorganisms (bacteria,viruses and parasites) in ancient specimens that candate from centuries to thousands of years in age.Although there is no strict definition to characterizethe antiquity of specimens that are considered underthe term ‘palaeomicrobiology’, in this article, we willconsider specimens that date from the era before theemergence of the concept of microorganisms (~100years ago) as ancient. Therefore, twentieth-centuryspecimens are regarded as forensic specimens, andresearch dealing with such specimens, for example,DNA analyses to identify the cause of the 1979 out-break of anthrax in Sverdlovsk, in the former SovietUnion1, is presented in a separate box (BOX 1).

As a discipline, palaeomicrobiology began in 1993with the molecular detection of Mycobacterium tubercu-losis DNA in an ancient human skeleton2. The objectivesof palaeomicrobiological studies include the diagnosisof past infectious diseases through the detection ofspecific microorganisms in ancient remains, theelucidation of the epidemiology of past infectious

diseases by reconstituting the temporal and geographicaldistributions of infected individuals, reservoirs andvectors, and the tracing of the genetic evolution of themicroorganisms themselves. Data from such studiesbenefit modern microbiology and studies of host–pathogen relationships, particularly in the context ofthe emergence of infectious diseases. Refinements inmolecular typing now allow researchers to study thegenetic evolution of microorganisms and the timingof their introduction into human populations.Palaeomicrobiological investigations can provide newinformation about the source of a particular epidemic,as well as details of its epidemiology.Also, such data canbenefit social sciences such as archaeology, history andanthropology by providing a proven cause of deathinstead of the putative hypotheses that can be drawnfrom archaeological or historical sources alone.

As laboratory techniques have advanced, the materialsthat can be used to search for ancient microorganismshave changed. Therefore, initial palaeomicrobiologicalstudies used bone tissue, whereas later studies haveprogressed to use mummified tissues and dental pulpfor analysis3–5. Furthermore, experimental standards for

PALAEOMICROBIOLOGY:CURRENT ISSUES AND PERSPECTIVESMichel Drancourt and Didier Raoult

Abstract | Palaeomicrobiology is an emerging field that is devoted to the detection,identification and characterization of microorganisms in ancient remains. Data indicate thathost-associated microbial DNA can survive for almost 20,000 years, and environmentalbacterial DNA preserved in permafrost samples has been dated to 400,000–600,000 years.In addition to frozen and mummified soft tissues, bone and dental pulp can also be used tosearch for microbial pathogens. Various techniques, including microscopy andimmunodetection, can be used in palaeomicrobiology, but most data have been obtainedusing PCR-based molecular techniques. Infections caused by bacteria, viruses andparasites have all been diagnosed using palaeomicrobiological techniques. Additionally,molecular typing of ancient pathogens could help to reconstruct the epidemiology of pastepidemics and could feed into current models of emerging infections, therefore contributingto the development of appropriate preventative measures.

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Search strategy, selection criteria and analysisWe searched the following databases: MEDLINE(from 1 Jan 1993 to 31 Aug 2004), Current Contents(from 1 Jan 1993 to 31 Aug 2004) and PubMed (from1 Jan 1993 to 31 Aug 2004). The following keywordswere used in combination: ancient, past, DNA, microbe,bacteria, virus, parasite, infection, DNA, tuberculosis,leprosy, syphilis, plague, bartonellosis, mycobacteria,Mycobacterium tuberculosis, Mycobacterium leprae,Treponema pallidum, Bartonella, Rickettsia. Referencesfrom a recent review6,7 as well as references fromselected papers were also reviewed. The criteria for astudy to be included in our review were that it had tobe published in English, in a peer-reviewed journalwith an impact factor >0.5 (2003 value), and providesufficient information about the materials and methodsto allow the authenticity of the data to be measured.Therefore, non-English-language studies, non-peer-reviewed monographs and ‘letters to the Editor’ wereexcluded.

We critically reviewed selected papers using astrength and quality of evidence system derived fromone that had been established for evidence-basedmedicine8 (BOX 2). With regard to the strength ofevidence of authenticity, level A includes reports withgood evidence of authenticity, that is, detection of anoriginal identifying sequence, detection of two,unrelated identifying sequences, or detection of anidentifying sequence and another unrelated biologi-cal molecule such as an antigen or a mycolic acid,all in the presence of negative controls. Level Bincludes reports with moderate evidence of authen-ticity, that is, detection of one identifying sequence orthe detection of one specific biological molecule.Level C includes reports with low evidence of authen-ticity, that is, the detection of one sequence or biolog-ical molecule of low specificity. With regard to thequality of evidence of authenticity, level I includesdata reproduced from the same collection of speci-mens in two independent laboratories using agreedprotocols; level II includes data produced by one laboratory using agreed protocols; and level IIIincludes data produced by one laboratory using non-agreed protocols. Agreed protocols in palaeo-microbiology include: pre-analysis manipulation of ancient material in a building that is free of thebacterial target; extraction of DNA using standardprotocols; use of several negative controls that are assimilar as possible to the experimental material;omission of a positive control in parallel with thetested material; analysis of several molecular targets(either DNA or antigenic) and sequencing of anyamplicons. This system of categorization allows readers to differentiate between published studieswith conclusive results (levels AI and AII) and studies with inconclusive results that might merely bethe result of contamination. In this review, we havecited ancient DNA studies that have reported positiveresults (TABLES 1,2), but we recommend that readersconsult BOX 2 to determine whether these results areconclusive.

palaeomicrobiology have emerged to deal with theproblems of contamination and the authenticity ofdata. Most of the data regarding ancient pathogenshave been obtained from molecular studies. However,some results have been obtained using microscopy,immunological detection techniques, and the isolationand culture of microorganisms from ancient samples.

Box 1 | Analysis of twentieth century specimens

The molecular biology of Spanish influenzaThe genome of the strain of influenza virus that was responsible for the ‘Spanish’influenza pandemic has been extensively studied49. Between 1918 and 1919, this viruskilled ~40 million people worldwide, and it is the worst recorded infectious pandemicin history. In initial work, viral RNA was extracted from formalin-fixed, paraffin-embedded lung tissue samples and nine fragments of viral RNA that comprised thecoding regions of haemagglutinin, neuraminidase, nucleoprotein, matrix protein-1 andmatrix protein-2 were sequenced49. The sequences were consistent with a novel H1N1influenza-A virus. The complete sequence of the haemagglutinin and neuraminidasegenes were further determined from a formalin-fixed, paraffin-embedded lung sampleand from a lung biopsy from a frozen corpse41,42. Recently, the structure of the ‘Spanish’influenza virus H1 haemagglutinin was determined to be of avian type120. These resultsallowed researchers to determine that the ‘Spanish’ influenza virus belonged to asubgroup that was an intermediate between the avian and mammal subgroups, andwas introduced into mammals just before the 1918 pandemic41. Also, these works pavedthe way for the complete sequencing of this virus and allowed antiviral drugs andvaccines to be tested on recombinant viruses42,44.

Molecular analysis of HIV in archived specimensElegant phylogenetic work based on the sequence analysis of the HIV-2 env, pol and gaggenes concluded that a zoonotic transfer of HIV-2 occurred during the first half of thetwentieth century; however, no experimental data have been obtained from archivedsamples to confirm this hypothesis121. HIV-1 RNA has been amplified from plasma thatwas collected in 1959 from an adult African male in Kinshasa, Congo, thereforedemonstrating that HIV infection was already present in Africa in the 1950s (REF. 122).This molecular study placed this HIV-1 strain sequence near the ancestral node ofsubtypes B and D and indicated that all major-group viruses could have evolved from asingle introduction into the African population shortly before 1959. Other molecularevidence of HIV-1 infection before 1970 was obtained from a Norwegian family123.

Box 2 | System used in this article to assess authenticity of data

Selection of papers:

• Published in English

• Peer-reviewed journals with impact factor >0.5 (2003 value)

Strength of evidence:A. Good evidence of authenticity:

• Detection of one or more original identifying sequences

• Detection of two unrelated identifying sequences

• Detection of one identifying sequence and one unrelated biological molecule

B. Moderate evidence of authenticity:

• Detection of one identifying sequence

• Detection of one specific biological molecule

C. Low evidence of authenticity:

• Detection of one sequence or biological molecule of low specificity

Quality of evidence:

I. Evidence from two independent teams using agreed protocols

II. Evidence from one laboratory using agreed protocols

III. Evidence from one laboratory using non-agreed protocols

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Table 1 | Current data in palaeomicrobiology for bacteria

Source Specimen, Conservation Datation Method PCR target Strength/ Refsbody site quality of

evidence

Mycobacterium tuberculosis

Bison Metacarpal Buried 17,000 yrs BP Molecular biology, S12-protein gene, IS6110, A/I 19spoligotyping DR locus

Human Lung, lymph node Mummified 1,000 yrs BP Molecular biology IS6110 B/III 3

Human Bone Mummified 5,400 yrs BP Molecular biology, 65-kDa-antigen gene, IS6110 A/III 51spoligotyping

Human Metacarpal, Buried Medieval Molecular biology, IS6110, rpoB gene, Mtp40 B/III 52lumbar vertebrae spoligotyping genome fragment, oxyR

pseudogene

Human Rib Buried Medieval Molecular biology IS6110, oxyR pseudogene B/III 53

Human Vertebrae Buried 1,000 yrs BP Molecular biology IS6110 B/III 54

Human Mandible Buried AD 1400–1800 Molecular biology IS6110 B/III 55

Human Vertebrae, femur, Buried 7th–8th century, Molecular biology IS6110, hsp65 gene B/III 76ankle, rib, pleura 17th century

Human Lung, pleura Buried AD 600 Chromatography, IS6110 B/III 31molecular biology

Human Bone Buried 1,000 yrs BP Chromatography, IS6110 B/III 12molecular biology

Human Vertebrae, rib Buried 400–230 BC Molecular biology IS6110, oxyR pseudogene, B/II 56RD7, gyrA, katG

Human Bone, soft tissues Mummified 2050–500 BC Molecular biology, IS6110 A/I 32spoligotyping

Human Bone Buried Molecular biology C/III 2

Human Lungs, pleura, Mummified 18th–19th century Molecular biology IS6110, gyrA, katG, dnaA- A/I 47abdomen, ribs, dnaN, 19-kDa-antigen gene,hair, teeth MPB70-antigen gene

Human Wrist, lumbar vertebrae Buried 14th–16th century Molecular biology IS6110 C/III 117

Mycobacterium leprae

Human Foot bones Buried 12th century Molecular biology RLEP B/II 18

Human Metacarpals Buried AD 300–600 Molecular biology RLEP, 18-kDa gene B/II 77

Human Nasal bony tissue Buried 1,100 yrs BP Molecular biology RLEP, 18-kDa gene B/II 57

Human Skulls Buried AD 1400–1800 Molecular biology RLEP1, RLEP3 B/II 57

Human Hard palate, skull Buried AD 1400–1800, 10th century Molecular biology RLEP1, RLEP3 B/II 55

Enteric bacteria

Mastodon Bowel Frozen 12,000 yrs BP Culture C/II 94

Human Metatarsal Mummified 1400 BC Molecular biology 16S rRNA gene B/II 10

Human Upper gut content Bogged 300 BC Molecular biology uidA gene, lacZ gene C/III 124

Treponema pallidum

Human Bone Buried 240 yrs BP Immunodetection, TPP15 gene, T. pallidum B/II 30molecular biology whole antigen

Borrelia burgdorferi

Ticks Dry AD 1884 Molecular biology ospA gene A/III 87

Rodents Dry 19th century Molecular biology ospA gene B/II 86

Other spirochaetes

Termite Intestinal tissue Amber Miocene Microscopy A/II 91

Bartonella quintana

Human Dental pulp Buried 4,000 yrs BP Molecular biology groEL gene, hbpE gene A/II 92

Bartonella henselae

Cat Dental pulp Buried 13th–18th century Molecular biology groEL gene, pap31 gene A/II 93

Yersinia pestis

Human Dental pulp Buried 5th–14th century Molecular biology Intergenic spacers A/II 26

Human Dental pulp Buried AD 1590–1722 Molecular biology pla gene, rpoB gene B/III 4

Human Dental pulp Buried AD 1348 Molecular biology pla gene A/II 5

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16S rRNA gene-based molecular diagnostic techniqueand we have observed a 5–7% rate of contamination. Inour experience, and in the experience reported by othergroups, contamination is always caused by bacterialspecies that have been previously amplified in the labo-ratory or that are present in the water and reagents usedfor DNA extraction, and never from the bacterial targetsof the experiment or from bacteria isolated in a neigh-bouring laboratory14,15. Furthermore, we have neverobtained DNA sequences from previously unknownenvironmental microorganisms, however this conta-mination problem has been reported by other groups9.

Several protocols can be used to limit the risk ofcontamination in the laboratory (BOX 3). The externalcleansing of bone using filtered compressed air andsterile distilled water, scraping the external surfaceand irradiation with 254-nm ultraviolet (UV) lighthave been advocated16.For the manipulation of ancientteeth, encasing the specimen in sterile resin has beenproposed17. As previous amplicons are a highly probablesource of contamination, all PCR-based experimentsshould be carried out in designated one-way PCR suiteswith appropriate ventilation. Primer optimization forPCR should be carried out in a separate building fromthe one in which the ancient material is handled, andPCR and post-PCR experiments should be performed

Prevention of contamination The sources of contamination in palaeomicrobiologicalstudies include microorganisms from the burial site,which can contaminate the specimens before laboratoryanalyses, and microorganisms and their DNA fromthe laboratory, which can contaminate the specimensduring laboratory analyses (FIG. 1). Environmentalbacteria can also contaminate specimens and thereforetheir nucleic acids can contaminate PCR-based detectionof specific pathogens in these samples. This threat isparticularly great when using a universal approachsuch as 16S rRNA gene-based PCR9,10. Specific moleculartargets carry a smaller risk, but there is still the possibilitythat these targets are shared by as yet unknown, envi-ronmental bacteria, which might result in misleadinginterpretation of sequences. The specificity of detec-tion has been shown by analysis of environmentalsamples in parallel with buried specimens11,12. The useof naturally protected specimens, such as dental pulp,might also limit the risk of external contamination.Contamination can occur in the laboratory and resultsfrom water and laboratory reagents, previous ampli-cons in the laboratory and cross-contamination fromone positive sample to another (carry-over)13.

In our laboratory, we have processed more than6,000 modern specimens in 5 years using a home-made

Table 2 | Current data in palaeomicrobiology for parasites

Source Specimen, Conservation Datation Method PCR target Strength/ Refsbody site quality of

evidence

Plasmodium falciparum

Human Bone Buried 1,500 yrs BP Molecular biology 18S rRNA gene A/II 108

Trypanosoma cruzi

Human Human visceral tissue Mummified 4,000 yrs BP Molecular biology Minicircle DNA C/III 60

Human Heart, lung, liver, Mummified 9,000 yrs BP Molecular biology Kinetoplast regions B/III 106kidney, ileum, colon,muscle, brain

Schistosoma species

Human Human visceral tissue Mummified 1,000 yrs BP Microscopy B/II 102

Toxocara canis

Hyena Coprolites Buried 300,000—500,000 yrs BP Microscopy B/II 125

Taenia saginata

Human Embalming rejects Buried 2,700 yrs BP Microscopy B/II 104

Ascaris lumbricoides

Human Embalming rejects Buried 2,700 yrs BP Microscopy B/II 104

Enterobius vermicularis

Human Coprolites Buried Molecular biology 5S rRNA spacer B/III 126

Pediculus humanus humanus

Textile Buried AD 76–77 Microscopy B/II 100

Human Scalp Mummified Prehistoric Microscopy B/II 96

Human Scalp Mummified 9,000 yrs BP Microscopy B/II 95

Human Scalp Mummified Prehistoric Microscopy B/II 98

Human Scalp Mummified AD 1000–1250 Microscopy B/II 99

Hair comb Buried 2,000 yrs BP Microscopy B/II 97

Pthirus pubis

Human Pubic hairs Mummified 1,000–2,000 yrs BP Microscopy B/II 101

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new genomic region by using a new PCR primer pair inevery new experiment, to prevent vertical contaminationfrom previous amplifications5. However, this protocoldoes not prevent pre-PCR contamination of theextracted specimen.

As carry-over can be a significant source of cross-contamination, it is important to strictly enforce basicrules such as the changing of cotton-filtered tips betweenevery specimen. The introduction of numerous negativecontrols also helps to monitor the latter source of conta-mination. In our laboratory, we use one negative controlthat comprises both negative tissue and uninoculated

in a separate room using disposable equipment andfreshly prepared reagents that have been irradiatedwith UV light5. Bench workers should wear disposablecaps, gloves and clothes. It is also recommended thatresearchers should perform experiments with ancientmaterials in a laboratory in which the modern pathogenhas not been amplified previously or produced in largeamounts. We (and others) also advocate performingancient DNA experiments without using a positivecontrol (the presence of a positive control is useful onlyfor the validity of negative results)5,18. Furthermore, inour group, we use ‘suicide PCR’ reactions, which target a

• Wearing gloves

• Scraping external surface

• Cleansing with filtered compressed air

• Cleansing with sterile water

• Ultraviolet irradiation

• Using dedicated, controlled room

• Wearing gloves

• Using primers only once (suicide PCR)

• Avoiding positive control

• Running 1 negative control for 3 samples

• Sequencing all the amplicons

DNA extraction PCR

Ris

kP

roce

dur

e

• Environmental flora• Positive control• Previous amplicons

• Environmental flora• Hand-borne flora

• Cross-contamination(carry-over)

Pre

vent

ativ

e m

easu

res

Sample collection

1 2 3

4 5 6

7 8 9

* 0 #~ ~ ~ ~

Figure 1 | Prevention of contamination in palaeomicrobiology. Preventing contamination is an issue of crucial importance forpalaeomicrobiological studies. Sources of contamination include microbial flora from the burial site and from the laboratory wherethe analysis is taking place. There is now general consensus on the preventative measures that should be taken bypalaeomicrobiological researchers.

Box 3 | Criteria for the authentication of molecular data in palaeomicrobiology

Absence of a positive control

• The positive control should be removed from the laboratory in which ancient specimens are processed.

Negativity of negative controls

• Several negative controls should be analysed in parallel with the specimens being processed.

• Negative controls should be as similar as possible to the ancient specimens.

• Negative controls should remain free of amplicons.

Sequencing of PCR amplicons

• PCR alone does not ensure the specificity of the diagnosis, and amplicons have to be sequenced to identify ancientmicroorganisms.

Targeting a new sequence in the laboratory

• PCR should target a specific sequence that has not previously been amplified in the laboratory.

Amplification and sequencing of a second target

• A positive result must be confirmed by amplification and sequencing of a second specific molecular target.

Originality of the ancient sequence

• The acquisition of an original sequence that differs from modern homologues by mutation or deletion excludescontamination.

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preservation of biological molecules in ancientremains29. An active search for variable sequences can behelpful in this regard. Where available, bioinformaticcomparisons of complete genome sequences of thepathogen of interest can identify the most variablegenomic regions to target in PCR-sequencing experi-ments. This strategy maximizes the probability ofrecovering original microbial sequences from ancientmaterials, thereby ensuring its authenticity.

In our laboratory, we have successfully applied thisstrategy to search for Y. pestis DNA in individuals thatwere suspected to have died from the Justinian plague26.After comparison of the two Y. pestis genome sequencesavailable in GenBank, we found that some intergenicspacer sequences were highly variable, and we amplifiedsix of these sequences from the ancient specimens.Sequence analyses showed that the sequences obtainedwere original sequences owing to the presence of pointmutations. These mutations were consistently found inseveral clones, therefore ensuring that they were notmerely caused by Taq polymerase misincorporation ofnucleotides. Also, the demonstration of two unrelatedsequences that identified the same pathogen in thesame specimen further increases the specificity ofthe identification. For example, we have detected boththe plasmid-encoded pla gene and the chromosome-encoded rpoB gene of Y. pestis in the skeletons of peoplethought to have died of plague4. The demonstration ofa specific, identifying sequence and another unrelatedbiological molecule, such as an antigen or a mycolicacid, has the same value12,30,31. Obtaining parallel repli-cates of these results in two geographically separatelaboratories has been advocated as a method to addconviction to these results9,19,32. However, we wouldargue that replicates do not prevent contaminationbefore analysis and that they are not warranted if anoriginal sequence is obtained.

Identifying suitable sources of materialBuried human skeletons — which can be preserved inindividual coffins, as collections of a few skeletonsburied in the same area or as large collections of skele-tons in cemeteries or mass graves — are the mainsource of material in the search for ancient pathogens.Catastrophic graves comprising numerous corpses thatare buried together, often without shrouds or coffins, aregenerally located outside cemeteries and traditionalburial sites, and are often discovered during urbaniza-tion works. They can be indicative of a demographiccrisis, typically owing to acts of violence, war, famine,cold or epidemics. Discriminating between these differ-ent hypotheses relies on historical data obtained fromancient texts and pictoral sources, such as paintings andpotteries, as well as anthropological analysis of the gravestructure, the sex ratio, age distribution and thepalaeopathological examination of the skeletons. Ifinfection is proposed as the cause of death solely on thebasis of historical and anthropological data, such ahypothesis remains presumptive and not demonstrative.For example, the death of Alexander the Great has beenattributed to West Nile virus because a historical

reaction mix for every three specimens. In the case ofpathogens with focal lesions, such as mycobacteria,material from non-lesional tissue has been included,but this might not be an appropriate negative controlbecause of the haematogenous spread of M. tuberculosis11.Material collected from unaffected individuals or un-affected species that are closely related to the specimensof interest are also of value; for example, lesion-freebones collected from fossilized Canis and Equus specieshave been used as controls for the molecular detectionof M. tuberculosis DNA in extinct bison19.

The interpretation and authenticity of dataThe interpretation of palaeomicrobiological dataincludes the verification of authenticity and the furtherinterpretation of authentic data. Establishing theauthenticity of data in palaeomicrobiology is easierfor ancient microbial DNA experiments than forancient human DNA experiments because microbialcontaminants in the laboratory are well-known andresults are therefore easier to interpret. Any microbialsequence or biological molecule that was present inthe specimen before the experiment began, regardlessof whether it was originally present or was introducedthereafter, is authentic20. Indeed, as yet unknown as wellas known environmental bacteria can contaminate spec-imens before the commencement of laboratory benchwork. For example, Gilbert et al. amplified previouslyunknown sequences when using PCR primers that tar-geted the Yersinia pestis plasminogen-activator (pla) geneand the rpoB gene, which encodes the α-subunit of theRNA polymerase rpoB genes9. The authenticity criteriathat have been proposed for ancient human DNAshould be adapted to palaeomicrobiology21,22.

Strict adherence to the rules for the prevention ofcontamination is a first step towards ensuring theauthenticity of ancient microbial isolates. Phylogeneticanalyses of the gene sequence from the ancient micro-organism can confirm its antiquity; for example, thephylogenetic analyses of a Bacillus sample that wasclaimed to be 250 million years old showed that it was,in fact, modern23,24. Similar to general molecular biologystudies, the authenticity criteria include the absence of adetectable amplicon in the negative controls. The repro-ducibility of results using different specimens that havebeen collected from the same individual is a secondcriterion. Some studies have been criticized becauseinvestigators found ancient DNA that containedsequences that were identical to those of modernDNA25. The recovery of an original sequence indicatesthat laboratory contamination has not occurred and isgood evidence for authenticity 5,19,26. The originalsequence must be shown in several clones. Chemicalmodifications of ancient DNA can result in ‘jumpingPCR’ — template switching during the PCR and C→Tand G→A substitutions. The sequencing of multipleclones that are derived from more than one indepen-dent amplification has been advocated to reduce therisk of obtaining incorrect DNA sequences20,22,27,28.Several methods, including pyrolysis coupled withmass spectrometry, have been proposed to evaluate the

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suspected to be the cause of death. In the UnitedKingdom, the Health and Safety Executive temporarilyhalted archaeological work on the site of Christchurch,Spitalfields, London, when a corpse with the externalclinical signs of smallpox was uncovered. Indeed, reportshave indicated that smallpox inactivation might take 25 years under optimal conditions, and extrapolatingthe initial viable inoculum to the whole of a burial siteled to the calculation of a period of >100 years38.Vaccination against smallpox has been advocated forarchaeologists who might disturb the corpses of possi-ble smallpox victims, but this recommendation remainscontroversial39.

The materials used to detect ancient pathogensHuman remains that have been investigated inpalaeomicrobiological studies include frozen tissues,which are rare40–45, mummified corpses3,46–48, fixed tissuesfrom pathology laboratory collections41,42,49,50 andburied skeletons2,12,19,30,51–57. In the case of frozen tissues,ancient nucleic acids can be extracted using standardprocedures, but mummified tissues must first berehydrated58,59. Also, contamination of frozen andmummified human corpses can hamper the correctinterpretation of data: contamination of a Tyroleanfrozen corpse and of Andean mummies was shownby PCR-sequencing, and Mycobacterium flavescens wasidentified in later samples40,60. Formalin-fixed paraffin-embedded tissues have been used for the immunodetec-tion of pathogens and require specific pre-extractiontreatment to remove traces of the fixative for molecularstudies49. Bone is the most widely used material in thedetection of human pathogens, although it can be con-taminated by soil flora and the organic content can bewashed out. Gross and histological preservation ofbone predicts DNA survival, indicating that it isworthwhile to preferentially select morphologicallywell-preserved bones for ancient microbial DNA stud-ies61. Bone samples require mechanical grinding andchemical decalcification by incubation in EDTA beforenucleic-acid extraction30. DNA has also been successfullyextracted from non-decalcified bone samples usingguanidinium thiocyanate and silica19. Bone is nowregarded as a suitable tissue for the molecular detectionof pathogens that cause focal bone infections such as M. tuberculosis and M. leprae.

Teeth are another important source of DNA becausethey are the longest-lasting human tissue. Varioussamples can be prepared from teeth: whole teeth canbe ground, a mixture of dentine and dental pulp can bedrilled out of the tooth9, or the dental pulp alone canbe extracted and the DNA extracted from the pulp4,9.These three different protocols result in three differentdental samples that can have different applications.Whole ground teeth have been used for PCR amplifica-tion of ancient animal and human DNA21. In theseexperiments, whole-tooth material and dentine wereused. The whole tooth, however, is subject to externalcontamination and DNA extraction requires a specificprotocol. Using whole teeth therefore offers no advan-tage over bone tissue, and contamination has been

description of a flock of dying ravens around the timeAlexander entered Babylon is reminiscent of modernepisodes of the disease33. Combining historical andanthropological data allows researchers to select thosemass graves where an epidemic is suspected and there-fore might warrant further microbiological investiga-tion. The dating of ancient corpses is crucial for theaccurate interpretation of any associated microbialdata. This is based on the analysis of funeral coins,furniture, textiles and inscriptions and, for specimensdating >600 years, the 14C isotope34. Analysis of fauna,flora and entomological remains in the sediments andstratification data can be used to estimate dates on thescale of several centuries — but millennia can be impor-tant in dating ancient corpses35. A putative identificationcan be made using historical and anthropological data,including individual anatomical characteristics reportedin historical sources, but this putative identity can onlybe confirmed by DNA analysis36.

There can be ethical issues associated with the scientificexploitation of ancient corpses as they are generallyregarded as the property of the country of burial. Thecase of a Late Neolithic Tyrolean ice man illustrates adispute between Austria and Italy regarding the scien-tific exploitation of remains37. The question of whethera past infection is still contagious was addressed after thediscovery of ancient corpses in whom smallpox was

a b

c d

Figure 2 | Tooth processing for the collection of dental pulp. The entire (a), radiographed (b),opened (c) and reconstituted tooth (d) are shown. As the longest-lasting tissues, teeth are animportant source of DNA. Our laboratory advocates the use of dental pulp in palaeomicrobiologystudies, however, a more universal protocol that can be used by other laboratories is required.

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inhibitors. The introduction of a mimic might be asuitable alternative, although it prevents the interpreta-tion of the original sequence that has been derivedfrom ancient material3,72. Any inhibitors that aredetected can be partially removed by diluting theancient DNA, isopropanol washing and precipitation55,71.Ancient microbial DNA is usually fragmented to lessthan 300 bp4,5 and an inverse relationship between PCRpositivity and target size was observed in tuberculousspecimens47. In these specimens, an inverse relationshipwas also observed between copy number and the targetsize47. It is possible that this limitation could be over-come by amplifying overlapping fragments and byDNA repair using DNA polymerase I and T4 ligase,although this technique has not yet been applied toancient microbial DNA73,74. Also, the quantity ofDNA/RNA that is extracted from ancient specimens issmall, in the order of nanograms, therefore limitingthe number of experiments that can be carried out4.

The diagnosis of past infectionsSee TABLES 1 and 2 for a summary of the availablepalaeomicrobiological data, and FIG. 3 for a summary ofthe techniques that are used for the detection of ancientpathogens.

Bacterial infections: mycobacterial infections. Tuberculosis,which is caused by bacteria of the M. tuberculosis com-plex, is primarily a disease of soft tissues but it canoccasionally affect the skeleton. Evidence from skeletalremains attests to the presence of tuberculosis in theearliest urban societies. However, several studies found alack of correlation between rib lesions and documentedtuberculosis in medieval skeletons32,53. Numerouspapers have been published on the molecular detectionof M. tuberculosis DNA, but some included positive con-trols or did not sequence all amplicons, and thereforelack the standards proposed in this review. These papershave been classified in group III in BOX 2.

The insertion sequence IS6110, which is specific fortubercle bacilli, was the molecular target used in all ofthese works.Verification was performed by PCR targetingof the mycobacterial hsp65 gene, oxyR pseudogene, rpoBgene, gyrA, katG, dnaA-dnaN region, 19-kDa-antigengene, MPB70-antigen gene, mtp40 genome fragment,surface-antigen gene and ribosomal-protein-S12gene11,19,46,47,52,53,55. Similar to the detection of IS6110,most studies used nested PCR when conventional PCRwas used for the detection of other molecular targets12,31.DNA sequences from the M. tuberculosis complex havebeen obtained from 5,400-year-old diseased bones andmummified soft tissues that originated from Egyptianmummified corpses and medieval buried skeletons3,46,51,52,and also from bone lesions from a 17,000-year-oldbison19. Two reports of M. tuberculosis-specific mycolicacids have been published12,31. In these reports, a stan-dard mycolic-acid extraction and derivatization protocoland chromatography were used to show the presence ofspecific biological molecules. In both reports, theIS6110 sequence was detected in parallel by nestedPCR, although a positive control was used12,31.

observed21. Dental samples were used in the search for Y. pestis DNA: whole ground teeth were used in 18 of 108samples of North European individuals from the MiddleAges, in parallel with mixed dentine and dental pulpdrilled from 75 of 108 teeth, and the dental pulp alonerecovered from 28 of 108 teeth9. In this report, theauthors observed no amplification of the Y. pestis target.

We have proposed the use of dental pulp in thedetection of ancient bacterial or viral DNA or RNA.Dental pulp is a well-vascularized soft tissue, in con-trast to bone or dentine4. We have shown that dentalpulp is the equivalent of a small blood specimen thatis sterile in undiseased mammals with definite dentalgrowth, including humans62. In our laboratory, wehave established an experimental model of Coxiellaburnetii bacteraemia in guinea-pigs and have recov-ered C. burnetii-specific DNA from the dental pulp ofthese animals63. We also showed that C. burnetii couldbe isolated and cultured from dental pulp in these ani-mals64. Similarly, we detected Bartonella henselae DNAin the dental pulp of buried stray cats65. HIV-specificRNA sequences have also been recovered from thedental pulp of HIV-infected patients, and Bartonellaquintana has been recovered from the dental pulp ofone homeless patient66–68. Recovery of dental pulp isdestructive, but it is possible to reconstitute the toothto preserve the appearance of collection specimens andto allow for further morphological studies (FIG. 2; seeonline supplementary information S1 (movie)).

The dental pulp is better protected from externalcontamination in teeth with no trauma and inunerupted teeth; however, the dental tubules connectthe dental-pulp chamber to the outside at the root and,using broad-range 16S rRNA gene primers, Gilbert et al.found molecular contamination of dental pulp withboth previously characterized environmental sequencesand unknown sequences, when using pla and rpoBgene-based PCR primers9. Therefore, in its presentform, the examination of dental pulp does not produceresults that are reproducible in all laboratories, and amore universal protocol will be required to promote theuse of dental pulp in palaeomicrobiology.

Regardless of the source of material, the presence ofPCR inhibitors, chemical modifications that occur inancient DNA and contamination can all limit PCR-based detection of ancient pathogens69. However, reportsindicate that bacterial DNA can survive for long periods,as it has been detected in 20,000-year-old specimens19

and environmental bacterial DNA preserved in perma-frost samples has been dated to 400,000–600,000 years70.Particular conditions of specimen preservation, includinglow temperature and dryness, can increase the survivaltime for microbial DNA19. Also, the encapsulation ofDNA into a bacterial spore, in the case of Bacillusspecies, or a strong mycolic-acid-containing wall, in thecase of mycobacteria, might extend the survival time ofDNA. It has been suggested that the brown colour ofextracted DNA indicates that inhibitors are present,however this has not been confirmed5,71. Some authorspropose that host DNA should be amplified in parallelwith the microbial DNA to monitor the presence of

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sequences were detected among 108 teeth collectedfrom 5 putative Black-Death sites in 3 Europeancountries9,83. In French specimens, we used dentalpulp for the molecular detection of the Y. pestis plaand rpoB genes4,5. The first study did not use agreedprotocols, as it incorporated a positive control, andthe second study used a ‘suicide PCR’ protocol. Werecently reported a study that used highly variablespacers, which confirmed these results. Moreover, wealso found evidence that Y. pestis was involved in theJustinian plague26. The authenticity of any ampliconswas ensured as no amplicons were present in the neg-ative controls and original sequences were detected.So far, we have detected specific Y. pestis sequences inthree individuals from the fifth–sixth century, sevenindividuals from the twelfth–fourteenth century, twoadults dated from AD 1590 and three individuals fromAD 1722 (REFS 5,26). Although it is difficult to interpretnegative results, discrepancies between these studiescould be caused by artefactual selection in sourcesand materials — differences in taphonomic preserva-tion (the conditions and processes by which organismsbecome fossilized), the use of mixed dentine and den-tal pulp versus dental pulp alone — or by the fact thatindividuals in Northern European countries did notsuffer from Y. pestis infection. The teeth that we havetested in parallel with Gilbert et al. were negative in bothlaboratories84.

Spirochaetes. The survival of immunoglobulin G (IgG)was first shown using a quantitative dot-blot techniqueafter extraction of IgG from a 0.12-million-year-oldequine fossil bone and from 1.6-million-year-old equineand hominid fossil bones85. Kolman et al. purifiedhuman IgG from a 200-year-old femoral bone of anindividual with syphilitic lesions. The IgG reactedstrongly with commercially available T. pallidum anti-gen, but as the nature and the specificity of the antigenwere not determined, the specificity of the detectioncannot be evaluated30. However, the diagnosis of syphiliswas further confirmed by sequencing the 15-kDa-lipoprotein gene of T. pallidum, which showed a singlebase mutation that distinguished T. pallidum subspeciespallidum from closely related spirochaetes30. AlthoughBorrelia burgdorferi sensu lato has not yet been detectedin ancient human material, its three genotypes,B. burgdorferi sensu stricto, Borrelia afzelii and Borreliagarinii have been documented in European tick vectorsarchived since 1884, whereas only B. burgdorferi sensustricto has been detected in New World vectors, whichinclude archived ticks and rodents86–88. Further studiesthat incorporated more isolates and new moleculartools showed a larger diversity within North-AmericanB. burgdorferi sensu lato isolates, indicating that B. burgdorferi sensu stricto originated from the OldWorld and was imported into the New World aroundthe time of Columbus, 500 years ago, or by migratingbirds89,90. Also, unidentified spirochaetes were clearlyobserved by light and transmission electronmicroscopy in the intestinal tissue of a Miocene termitethat was preserved in amber91.

Lepromatous lesions that caused rhinomaxillarychanges with subsequent local bone alterations,widening of the nasal aperture, loss of the anteriornasal spine and thinning of the hard palate wereobserved on the skeleton of the German king HenriVII, who died in AD 1242 (REF. 75). Indeed, M. lepraeRLEP fragments were detected by PCR from tenthcentury hard palate and AD 1400–1800 skull speci-mens57,76. Also, leprosy was retrospectively diagnosedby the molecular detection of dispersed repetitivesequences that were specific for M. leprae in diseasedmetacarpal bones from twelfth century individuals inSeville, Spain18. The identification was confirmed byrestriction enzyme analysis and sequencing of oneamplicon that was obtained after nested PCR. Leprosywas identified by sequencing specific RLEP and 18-kDa-antigen gene amplicons obtained by nestedPCR from an individual dated AD 300–600 in Israel77.This approach was applied to other individuals57.

Plague. Historical sources describe three devastatingplague pandemics in the Old World: the Justinianplague that occurred between AD 541 and AD 750; themedieval Black Death, which started in Europe in AD 1347; and the modern and current pandemic, whichprobably started in AD 1855 in the Chinese province ofYünnan78. Although the historical descriptions ofbuboes and the characteristics of these epidemics arehighly suggestive of plague, their aetiology is disputedand alternative aetiologies have been proposed79–82.Also, the aetiology of the Black Death in NorthernEurope needs to be confirmed, as no Y. pestis DNA

Electron microscope

• Hairs• Preserved insects• Textile fragments

Isolation and culture

• Frozen specimens

Immunodetection

• Fixed specimens

Late serology

• Bone

Optic microscope

• Coprolites• Hairs

PCR-based detectionand sequencing

• Bone• Mummified tissues• Frozen tissues• Dental pulp

Sample

Figure 3 | Techniques used for the detection of ancient pathogens. A variety of techniqueshave been used in palaeomicrobiolgical studies over the years, including microscopy andimmunodetection. However, most data have been obtained and most different materials can beexamined using PCR-based molecular techniques.

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was detected in Egyptian mummies using a doubleantibody immunoassay, but this detection was notconfirmed by P. falciparum DNA detection in the samemummy107. P. falciparum DNA has been retrievedfrom the bones of a baby that were excavated from afifth-century cemetery outside Rome, Italy108.

Viruses. Human T-cell lymphotropic virus type-1(HTLV-1) DNA has been detected in the bone marrowof an ~1,500-year-old Andean mummy, and both theHTLV-1-pX gene and the HTLV-1-LTR gene wereamplified and sequenced109. Further sequence analyses,however, concluded that the reported sequences wereof modern origin and therefore this report remainsdoubtful110,111.

Genotyping ancient bacteriaThe adoption of experimental standards in palaeo-microbiology, as well as the availability of genome-based genotyping systems, has facilitated pioneeringwork in the genotyping of some ancient bacteria. TheM. tuberculosis complex includes the related species M. tuberculosis, Mycobacterium bovis, Mycobacteriumafricanum and Mycobacterium microti. Ancientmycobacteria were genotyped by sequencing the phos-pholipase-C mtp40 gene, an M. tuberculosis-specificregion, another M. bovis-specific fragment and the oxyRpseudogene52. This work demonstrated that medievalmycobacteria were more closely related to modern M. tuberculosis than to M. bovis. Spoligotyping of thesame specimens indicated a close relationship betweenmycobacteria in two separate burial sites52. Similar con-clusions were obtained from a spoligotyping analysis of12 M. tuberculosis strains that were characterized amongEgyptian mummies dating from 2050–500 BC (REF. 48).This spoligotyping analysis indicated that these strainswere more closely related to the M. tuberculosis/M. africanum group than to M. bovis. Similarly, princi-pal-component analysis of spoligotypes obtained frommycobacterial DNA from an extinct bison demonstratedthat it was more closely related to the M. tuberculosis/M. africanum group than it was to M. bovis19. Takentogether, these results indicate that the speculation thatM. tuberculosis evolved from M. bovis by specific adap-tation of an animal pathogen to the human host mightnot be correct112. Recent complete-genome-basedanalysis also concluded that the common ancestor ofmodern tubercle bacilli resembled M. tuberculosis andMycobacterium canettii, and that it might already havebeen a human pathogen113. Detection of specific junctionsequences in ancient specimens might help to furtherresolve the timescale in which members of the M. tuberculosis complex have evolved. Genotyping M. tuberculosis using five molecular targets also allowedthe study of the distribution of tubercle bacilli amongfamily members in Hungarian mummies dating fromAD 1731–1838 (REF. 47). Present data also indicate that M. tuberculosis was widespread during the Pleistocene.

Multiple spacer typing (MST) has been developedfor the genotyping of past Y. pestis26. This method isbased on complete genome analysis. Y. pestis has been

Bartonellae. Microorganisms that were morphologicallycompatible with Bartonella species have been observedin verruga peruana (a skin eruption characteristic ofbartonellosis) in skin specimens from the tenthcentury45. We recently used dental pulp to detect twospecific B. quintana sequences in a prehistoric humantooth dated from 4,000 years BP (REF. 92). Similarly, werecently detected flea-borne B. henselae DNA in dentalpulp collected from three domestic cats dated from thethirteenth, fourteenth and sixteenth century and con-firmed that these animals were infected by the Houstongenotype of the bacterium93. This species causes humancat-scratch disease and these data showed that medievalpeople were exposed to this disease. Such palaeo-microbiological investigations of bartonelloses haveassisted our understanding of the molecular evolutionof the bartonellae and the co-evolution of these uniquemicroorganisms with their mammalian host.

Miscellaneous bacteria. Microbiological investigationsof frozen materials include 750,000-year-old glacial iceand the intestinal contents of a 12,000-year-oldmastodon, which yielded 295 bacterial isolates94, 41% ofwhich were Enterobacteriaceae, with Enterobacter cloacaebeing the most frequently isolated taxon. Whether thesemicroorganisms were direct descendants of the originalintestinal microbiota could not be established.

Parasites. Microscopy has contributed to the detectionof microorganisms in mummified individuals and tis-sues, but this approach lacks specificity for the accurateidentification of pathogens. It has been most useful inthe detection of parasites. The oldest trace of human licewas detected by stereo-microscopic observation of thehair of an individual who lived about 9,000 years ago inthe Judean desert95. Other studies include the detectionof head-lice eggs on hair from human mummies —including preColumbian Peruvian mummies, 20 pre-historic American-Indian mummies and 1 Egyptianmummy from the fourth century AD — from 2,000-year-old hair combs that were recovered in excavations inthe Judean and Negev deserts and from pre-historicAmerican-Indians96–99. Most of these specimens had nitsand one prehistoric mummy had dead lice at all stagesof development. Also, the leg from the third nymphalstage of the body louse Pediculus humanus humanus wasidentified in textile fragments excavated in Massada,Israel, and dated to AD 73–74 (REF. 100). Pthirus pubiswas also found in ancient populations, as illustratedby its detection by microscopy in the pubic hairs of a2,000-year-old Chilean mummy101. Toxacara canis,Ascaris lumbricoides and Taenia saginata eggs, and schis-tosomes have also been observed microscopically inancient specimens102–104. Enterobius vermicularis DNAwas amplified from human coprolites from Chile andthe USA105.

DNA from the causative agent of Chagas disease,Trypanosoma cruzi, was detected in 41% of tissueextracts from naturally dessicated human mummiesdating from 9,000 years ago in northern Chile106.Plasmodium falciparum histidine-rich-protein-2 antigen

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The application of the universal 16S rRNA gene-based detection and identification of bacteria topalaeomicrobiology has been limited by the contami-nation of ancient material. However, this powerfulmolecular tool would be invaluable to study thenature and epidemiology of unpredicted pathogens.So far, palaeomicrobiological techniques have con-firmed the suspected presence of one particularpathogen in ancient remains. However, the aetiologyof numerous past epidemics remains unknown,despite testing for the presence of one or more bacter-ial pathogens. Tracing any bacterial pathogen withinthe remains of this past population could help toresolve the question of the aetiology of some mysteri-ous epidemics. As there are few valuable materials(such as dental pulp) available in the majority of thesecases, testing for all bacterial pathogens at the sametime would be helpful. Studies must be performed todevelop such a protocol of universal amplification andsequencing that is adapted to ancient bacterial DNA.

Genotyping will create the necessary bridgebetween the detection of microbial DNA in ancientenvironmental and human specimens and in modernmicrobiology. The lack of a universal molecular geno-typing method that is adaptable to altered ancientmicrobial DNA has limited the genotyping of ancientmicroorganisms to M. tuberculosis117. The availabilityof a large database of complete microbial genomesequences has already prompted the establishment ofnew genotyping methods for modern microorgan-isms, including microarray-based genotyping ofFrancisella tularensis118 and SNP-based genotyping ofM. tuberculosis119. These new typing methods shouldbe applied to ancient specimens.

The transfer of the techniques specific topalaeomicrobiology to more laboratories around theworld, the sharing of environmental and humanmaterials of interest between such laboratories, andthe bridging of gaps that still exist between palaeomi-crobiology, anthropology and history will hopefullyallow us not only to resolve historical mysteries, butalso to understand the mechanisms of the emergenceof infectious diseases and to design predictive modelsof this emergence.

subdivided into three biovars on the basis of its ability toferment glycerol and to reduce nitrate. On the basis oftheir current geographical niche and on historicalrecords that indicate the geographical origin of thepandemics, it was speculated that each biovar caused aspecific pandemic114. We used MST to test this hypoth-esis and found that the genotype involved in all threepandemics was associated with the Orientalis biovar.

Concluding remarksPalaeomicrobiology is a young discipline, and some ofthe early works in this field did not adhere to the nowgenerally accepted standards. The enforcement of thesestandards in laboratories that are devoted to palaeo-microbiology will help to avoid contamination and willaid data authentication. In our opinion, therefore,palaeomicrobiology has the potential to contribute toour understanding of the emergence and re-emergenceof infectious diseases. The emergence of infectious dis-eases in humans requires both the introduction of apathogen from an environmental or animal reservoirinto the human population, and its subsequent spreadand maintenance in this population. The role of envi-ronmental factors that influence both of these elements,including human behaviour, can be addressed byanthropological and historical studies. The importanceof evolutionary factors in the emergence of infectiousdiseases has recently been emphasized115 and thesecould be studied by palaeomicrobiology. The detectionof pathogens in their ancient reservoirs and of vectorswill be a key step towards achieving this goal. Suchdetection will benefit from improved collaborationbetween palaeozoologists, specialists in ancient ecto-parasites and palaeomicrobiologists. Specific issuesinclude the correct collection and identification ofburied animals and ectoparasites by using appropriatemethods of collection for small animal debris andectoparasites, and molecular methods, such as 18SrRNA gene sequencing, for the identification of ecto-parasites116. With regards to human remains and theremains of other mammals, in our opinion, the broaduse of dental pulp could help to resolve the aetiologyof ancient bloodborne infections, although universalprotocols are still required4,5.

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AcknowledgementsThe authors acknowledge G. Aboudharam, L. V. Dang and L. Tran-Hung for expert help in the preparation of teeth picturesand P. Kelly for reviewing the manuscript.

Competing interests statementThe authors declare no competing financial interests.

Online links

DATABASESThe following terms in this article are linked online to:Entrez: http://www.ncbi.nlm.nih.gov/entrezBartonella henselae | Coxiella burnetti | Mycobacterium leprae |Mycobacterium tuberculosis | Treponema pallidum | Yersinia pestis

FURTHER INFORMATIONDidier Raoult’s laboratory: http://www.mediterranee.univ-mrs.fr/recherche/lab.asp?lng=gb&view=unit&id=50Encyclopedia of Life Sciences: http://www.els.net/els/Molecular Palaeontology

SUPPLEMENTARY INFORMATIONSee online article: S1 (movie)Access to this links box is available online.