aDNA Analysis of Pre-aDNA Analysis of Pre-Contact TBContact TB

Frederika Kaestle, Jennifer Raff, Della CookFrederika Kaestle, Jennifer Raff, Della CookIndiana University Departments of Indiana University Departments of

Anthropology and BiologyAnthropology and Biology

Outline of TalkOutline of Talk

Background on TB complex evolutionBackground on TB complex evolution Introduction to tuberculosis in ancient North Introduction to tuberculosis in ancient North

AmericaAmerica Approaches in detecting and analyzing TB Approaches in detecting and analyzing TB

complex DNA from Schild populationcomplex DNA from Schild population Results of ancient TB complex strain Results of ancient TB complex strain

analysisanalysis Implications for evolution of TB complex in Implications for evolution of TB complex in

North AmericaNorth America

How has tuberculosis evolved How has tuberculosis evolved as a human pathogen?as a human pathogen?

Null hypothesis: Null hypothesis: M. M. tuberculosistuberculosis evolved from evolved from M. M. bovisbovis Domestication of Domestication of

cattle allowed jump cattle allowed jump from animal to from animal to human populationshuman populations

How has tuberculosis evolved How has tuberculosis evolved as a human pathogen?as a human pathogen?

Null hypothesis: Null hypothesis: M. M. tuberculosistuberculosis evolved from evolved from M. M. bovisbovis Domestication of Domestication of

cattle allowed jump cattle allowed jump from animal to from animal to human populationshuman populations

From Marmiesse et al., 2004 p 150 (modified from Brosch et al., 2002)

How has tuberculosis evolved How has tuberculosis evolved as a human pathogen?as a human pathogen?

Null hypothesis: Null hypothesis: M. M. tuberculosistuberculosis evolved from evolved from M. M. bovisbovis Domestication of Domestication of

cattle allowed jump cattle allowed jump from animal to from animal to human populationshuman populations

Genomic analysis: Genomic analysis: M. tuberculosisM. tuberculosis older than older than M. bovisM. bovis From Marmiesse et al., 2004 p 150 (modified from Brosch et al., 2002)

How has tuberculosis evolved How has tuberculosis evolved as a human pathogen?as a human pathogen?

Null hypothesis: Null hypothesis: M. M. tuberculosistuberculosis evolved evolved from from M. bovisM. bovis Domestication of cattle Domestication of cattle

allowed jump from allowed jump from animal to human animal to human populationspopulations

Genomic analysis: Genomic analysis: M. M. tuberculosistuberculosis older older than than M. bovisM. bovis Suggests very ancient Suggests very ancient

association between TB association between TB complex, humanscomplex, humans From Marmiesse et al., 2004 p 150 (modified from Brosch et al., 2002)

How has tuberculosis evolved How has tuberculosis evolved in North America?in North America?

Which member(s) of the TB complex Which member(s) of the TB complex infected prehistoric Native infected prehistoric Native Americans?Americans? Was TB brought to the New World in Was TB brought to the New World in

human populations?human populations? M. tuberculosisM. tuberculosis or other predominantly or other predominantly

human infecting specieshuman infecting species Was TB acquired by contact with Was TB acquired by contact with

infected animals?infected animals? M. bovisM. bovis or related species or related species

ApproachApproach

Determine the species of TB complex Determine the species of TB complex present in a pre-contact Native present in a pre-contact Native American population using ancient American population using ancient DNA techniquesDNA techniques Schild: a Late Woodland and Schild: a Late Woodland and

Mississippian burial population from Mississippian burial population from West-Central IllinoisWest-Central Illinois

The Schild site : AD 930-The Schild site : AD 930-13001300

Perino, 1971

Tuberculosis complex infection Tuberculosis complex infection at Schildat Schild

Previous studies (Perino, 1971;Buikstra 1977;Buikstra and Cook, 1978; Cook 1980;Buikstra and Cook 1981; Roberts and Buikstra 2003) have identified pathology consistent with TB complex infection in 11 individuals at Schild.

TB complex infection in individual at right, SB201, confirmed molecularly by Braun et al., 1998

SB201, a 20-25 year old female

MethodsMethods

Extract DNA from Schild skeletons and test Extract DNA from Schild skeletons and test for TB complex infection, using standard for TB complex infection, using standard aDNA precautions.aDNA precautions. Insertion sequence Insertion sequence IS6110IS6110

Amplify and sequence segments of genes Amplify and sequence segments of genes informative to complex evolution and informative to complex evolution and strain discriminationstrain discrimination DNA DNA Gyrase BGyrase B

Extend amplifications in overlapping Extend amplifications in overlapping fragmentsfragments

TB complex infected TB complex infected individualsindividuals

IS6110IS6110 was amplified and was amplified and sequenced from 5.3 % (8) sequenced from 5.3 % (8) of samples tested (n=150).of samples tested (n=150).

7/8 individuals had TB 7/8 individuals had TB lesionslesions

DNA amplified from non-DNA amplified from non-lesion sourceslesion sources

IS6110IS6110 sequence identical sequence identical to TB complex members to TB complex members published in literature. published in literature.

Knoll A: SA41, SA96a, Knoll A: SA41, SA96a, SA141SA141

Knoll B: SB201, SB250, Knoll B: SB201, SB250, SB259a, SB269, SB297SB259a, SB269, SB297

We amplified We amplified GyrBGyrB from four skeletons in from four skeletons in two overlapping two overlapping fragments: SA41, fragments: SA41, SA96a, SB201, SB297SA96a, SB201, SB297 165 base pairs of 165 base pairs of

sequence was sequence was obtained from SA41, obtained from SA41, SB201, SB297SB201, SB297

203 base pairs of 203 base pairs of sequence was sequence was obtained from SA96aobtained from SA96a

GyrB amplification product

SB

297

Neg

. extr

. co

ntr

ol

Neg

. am

plifi

cati

on

co

ntr

ol

Primer-dimer(50bp)

SB

201

GyrB amplification

Neg

. extr

. co

ntr

ol

Neg

. extr

. co

ntr

olS

A96a

Sequence analysisSequence analysis GyrBGyrB sequences showed numerous differences from all sequences showed numerous differences from all

other TB complex members. other TB complex members.

Sequence analysisSequence analysis GyrBGyrB sequences showed numerous differences from all other TB complex members. sequences showed numerous differences from all other TB complex members.

Do not appear more similar to any one species over another: at most 89% similarity to other Do not appear more similar to any one species over another: at most 89% similarity to other MycobacteriaMycobacteria

Samples are distinguished from each other by several polymorphisms, supporting lack of contamination.Samples are distinguished from each other by several polymorphisms, supporting lack of contamination.

Comparison of amino acid sequences of Comparison of amino acid sequences of

GyrB GyrB from ancient and modern from ancient and modern MycobacteriaMycobacteria

--Most changes are synonymous, suggesting purifying selection--Non-synonymous changes (e.g. K to R) conserve amino acid properties, do not alter protein structure significantly at that site.

ConclusionsConclusions A substantial number of individuals from Schild were A substantial number of individuals from Schild were

infected by a member of the TB complex.infected by a member of the TB complex.

Phylogenetic analysis of Phylogenetic analysis of GyrB GyrB sequences recovered from sequences recovered from four infected individuals suggests a long evolutionary four infected individuals suggests a long evolutionary separation from TB complex strains in the Old World.separation from TB complex strains in the Old World.

Cannot distinguish between human or animal origin for TB Cannot distinguish between human or animal origin for TB complex in New World on basis of these resultscomplex in New World on basis of these results

Based upon a survey of the literature, and of GenBank, it Based upon a survey of the literature, and of GenBank, it appears that a novel strain of TB complex was present in appears that a novel strain of TB complex was present in pre-contact North American populationspre-contact North American populations

AcknowledgmentsAcknowledgments

Kaestle LabGraduate StudentsAlison French DoubledayMarisa FennCharla McCormickUndergraduate StudentsJake EnkSam OrrRotation studentsSkye Chang Mike BarnoKate GiestingPK Moua

IUB Faculty Mark Braun Clay Fuqua Armin Moczek Jeff Palmer Rudy Raff Beth Raff Loren Rieseburg Bill Saxton Susan Strome

FundingNSF IGERT Evolution, Development and Genomics Training Grant to JARIUB Anthropology Skomp Research Feasibility Grant to JARIUB Start Up Grant to FAK

CollaboratorsJason EschlemanRipan Malhi