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aDNA Analysis of Pre-Contact TB
Frederika Kaestle, Jennifer Raff, Della Cook
Indiana University Departments of Anthropology
and Biology
Outline of Talk
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Background on TB complex evolution
Introduction to tuberculosis in ancient North
America
Approaches in detecting and analyzing TB
complex DNA from Schild population
Results of ancient TB complex strain analysis
Implications for evolution of TB complex in
North America
How has tuberculosis evolved as a
human pathogen?
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Null hypothesis: M.
tuberculosis evolved
from M. bovis
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Domestication of cattle
allowed jump from
animal to human
populations
How has tuberculosis evolved as a
human pathogen?

Null hypothesis: M.
tuberculosis evolved
from M. bovis

Domestication of cattle
allowed jump from
animal to human
populations
From Marmiesse et al., 2004 p 150 (modified from Brosch et al., 2002)
How has tuberculosis evolved as a
human pathogen?

Null hypothesis: M.
tuberculosis evolved
from M. bovis
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Domestication of cattle
allowed jump from
animal to human
populations
Genomic analysis: M.
tuberculosis older
than M. bovis
From Marmiesse et al., 2004 p 150 (modified from Brosch et al., 2002)
How has tuberculosis evolved as a
human pathogen?

Null hypothesis: M.
tuberculosis evolved from
M. bovis
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
Domestication of cattle
allowed jump from animal
to human populations
Genomic analysis: M.
tuberculosis older than M.
bovis
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Suggests very ancient
association between TB
complex, humans
From Marmiesse et al., 2004 p 150 (modified from Brosch et al., 2002)
How has tuberculosis evolved in
North America?
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Which member(s) of the TB complex
infected prehistoric Native Americans?
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Was TB brought to the New World in human
populations?
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M. tuberculosis or other predominantly human
infecting species
Was TB acquired by contact with infected
animals?
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M. bovis or related species
Approach
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Determine the species of TB complex
present in a pre-contact Native American
population using ancient DNA techniques
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Schild: a Late Woodland and Mississippian
burial population from West-Central Illinois
The Schild site : AD 930-1300
Perino, 1971
Tuberculosis complex infection at
Schild SB201, a 20-25 year old female
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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
Methods
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Extract DNA from Schild skeletons and test for
TB complex infection, using standard aDNA
precautions.
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Amplify and sequence segments of genes
informative to complex evolution and strain
discrimination
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Insertion sequence IS6110
DNA Gyrase B
Extend amplifications in overlapping fragments
TB complex infected individuals
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IS6110 was amplified and
sequenced from 5.3 % (8) of
samples tested (n=150).
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7/8 individuals had TB
lesions
DNA amplified from nonlesion sources
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IS6110 sequence identical to
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Knoll A: SA41, SA96a, SA141
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Knoll B: SB201, SB250,
SB259a, SB269, SB297
TB complex members
published in literature.
GyrB amplification
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GyrB
amplification
product
We amplified GyrB from
four skeletons in two
overlapping fragments:
SA41, SA96a, SB201,
SB297
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Primer-dimer
(50bp)
Neg. amplification
control
Neg. extr. control
Neg. extr. control
SA96a
SB297
Neg. extr. control
SB201
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165 base pairs of
sequence was
obtained from SA41,
SB201, SB297
203 base pairs of
sequence was
obtained from SA96a
Sequence analysis
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GyrB sequences showed numerous differences from all other
TB complex members.
Sequence analysis
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GyrB sequences showed numerous differences from
all other TB complex members.
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Do not appear more similar to any one species over
another: at most 89% similarity to other Mycobacteria
Samples are distinguished from each other by several
polymorphisms, supporting lack of contamination.
Comparison of amino acid sequences of GyrB from
ancient and modern Mycobacteria
--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.
Conclusions
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A substantial number of individuals from Schild were infected by a
member of the TB complex.
Phylogenetic analysis of GyrB sequences recovered from four
infected individuals suggests a long evolutionary separation from TB
complex strains in the Old World.
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Cannot distinguish between human or animal origin for TB complex in
New World on basis of these results
Based upon a survey of the literature, and of GenBank, it appears
that a novel strain of TB complex was present in pre-contact North
American populations
Acknowledgments
Kaestle Lab
Graduate Students
Alison French Doubleday
Marisa Fenn
Charla McCormick
Undergraduate Students
Jake Enk
Sam Orr
Rotation students
Skye Chang
Mike Barno
Kate Giesting
PK Moua
IUB Faculty
Mark Braun
Clay Fuqua
Armin Moczek
Jeff Palmer
Rudy Raff
Beth Raff
Loren Rieseburg
Bill Saxton
Susan Strome
Funding
Collaborators
Jason Eschleman
Ripan Malhi
NSF IGERT Evolution, Development and Genomics Training Grant to JAR
IUB Anthropology Skomp Research Feasibility Grant to JAR
IUB Start Up Grant to FAK