1. aDNA Analysis of Pre-Contact TB Frederika Kaestle, Jennifer Raff, Della Cook Indiana University Departments of Anthropology and Biology

2. Outline of Talk Background on TB complex evolution Background on TB complex evolution Introduction to tuberculosis in ancient North America Introduction to tuberculosis in ancient North America Approaches in detecting and analyzing TB complex DNA from Schild population Approaches in detecting and analyzing TB complex DNA from Schild population Results of ancient TB complex strain analysis Results of ancient TB complex strain analysis Implications for evolution of TB complex in North America Implications for evolution of TB complex in North America

3. How has tuberculosis evolved as a human pathogen? Null hypothesis: M. tuberculosis evolved from M. bovis Null hypothesis: M. tuberculosis evolved from M. bovis Domestication of cattle allowed jump from animal to human populations Domestication of cattle allowed jump from animal to human populations

4. How has tuberculosis evolved as a human pathogen? Null hypothesis: M. tuberculosis evolved from M. bovis Null hypothesis: M. tuberculosis evolved from M. bovis Domestication of cattle allowed jump from animal to human populations Domestication of cattle allowed jump from animal to human populations From Marmiesse et al., 2004 p 150 (modified from Brosch et al., 2002)

5. How has tuberculosis evolved as a human pathogen? Null hypothesis: M. tuberculosis evolved from M. bovis Null hypothesis: M. tuberculosis evolved from M. bovis Domestication of cattle allowed jump from animal to human populations Domestication of cattle allowed jump from animal to human populations Genomic analysis: M. tuberculosis older than M. bovis Genomic analysis: M. tuberculosis older than M. bovis From Marmiesse et al., 2004 p 150 (modified from Brosch et al., 2002)

6. How has tuberculosis evolved as a human pathogen? Null hypothesis: M. tuberculosis evolved from M. bovis Null hypothesis: M. tuberculosis evolved from M. bovis Domestication of cattle allowed jump from animal to human populations Domestication of cattle allowed jump from animal to human populations Genomic analysis: M. tuberculosis older than M. bovis Genomic analysis: M. tuberculosis older than M. bovis Suggests very ancient association between TB complex, humans Suggests very ancient association between TB complex, humans From Marmiesse et al., 2004 p 150 (modified from Brosch et al., 2002)

7. How has tuberculosis evolved in North America? Which member(s) of the TB complex infected prehistoric Native Americans? Which member(s) of the TB complex infected prehistoric Native Americans? Was TB brought to the New World in human populations? Was TB brought to the New World in human populations? M. tuberculosis or other predominantly human infecting species M. tuberculosis or other predominantly human infecting species Was TB acquired by contact with infected animals? Was TB acquired by contact with infected animals? M. bovis or related species M. bovis or related species

8. Approach Determine the species of TB complex present in a pre-contact Native American population using ancient DNA techniques Determine the species of TB complex present in a pre-contact Native American population using ancient DNA techniques Schild: a Late Woodland and Mississippian burial population from West-Central Illinois Schild: a Late Woodland and Mississippian burial population from West-Central Illinois

9. The Schild site : AD 930-1300 Perino, 1971

10. Tuberculosis complex infection at 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

11. Methods Extract DNA from Schild skeletons and test for TB complex infection, using standard aDNA precautions. Extract DNA from Schild skeletons and test for TB complex infection, using standard aDNA precautions. Insertion sequence IS6110 Insertion sequence IS6110 Amplify and sequence segments of genes informative to complex evolution and strain discrimination Amplify and sequence segments of genes informative to complex evolution and strain discrimination DNA Gyrase B DNA Gyrase B Extend amplifications in overlapping fragments Extend amplifications in overlapping fragments

12. TB complex infected individuals IS6110 was amplified and sequenced from 5.3 % (8) of samples tested (n=150). IS6110 was amplified and sequenced from 5.3 % (8) of samples tested (n=150). 7/8 individuals had TB lesions 7/8 individuals had TB lesions DNA amplified from non- lesion sources DNA amplified from non- lesion sources IS6110 sequence identical to TB complex members published in literature. IS6110 sequence identical to TB complex members published in literature. Knoll A: SA41, SA96a, SA141 Knoll A: SA41, SA96a, SA141 Knoll B: SB201, SB250, SB259a, SB269, SB297 Knoll B: SB201, SB250, SB259a, SB269, SB297

13. We amplified GyrB from four skeletons in two overlapping fragments: SA41, SA96a, SB201, SB297 We amplified GyrB from four skeletons in two overlapping fragments: SA41, SA96a, SB201, SB297 165 base pairs of sequence was obtained from SA41, SB201, SB297 165 base pairs of sequence was obtained from SA41, SB201, SB297 203 base pairs of sequence was obtained from SA96a 203 base pairs of sequence was obtained from SA96a GyrB amplification product SB297 Neg. extr. controlNeg. amplification control Primer-dimer (50bp) SB201 GyrB amplification Neg. extr. control SA96a

14. Sequence analysis GyrB sequences showed numerous differences from all other TB complex members. GyrB sequences showed numerous differences from all other TB complex members.

15. Sequence analysis GyrB sequences showed numerous differences from all other TB complex members. GyrB 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 Mycobacteria 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. Samples are distinguished from each other by several polymorphisms, supporting lack of contamination.

17. 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.

18. Conclusions A substantial number of individuals from Schild were infected by a member of the TB complex. 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. Phylogenetic analysis of GyrB sequences recovered from four infected individuals suggests a long evolutionary separation from TB complex strains in the Old World. Cannot distinguish between human or animal origin for TB complex in New World on basis of these results 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 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

19. 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 NSF IGERT Evolution, Development and Genomics Training Grant to JAR IUB Anthropology Skomp Research Feasibility Grant to JAR IUB Start Up Grant to FAK Collaborators Jason Eschleman Ripan Malhi