Mycobacterium tuberculosis Complex Lipid Virulence Factors Preserved in the 17,000-Year-Old Skeleton of an Extinct Bison, Bison antiquus
Identifieur interne : 000361 ( Pmc/Corpus ); précédent : 000360; suivant : 000362Mycobacterium tuberculosis Complex Lipid Virulence Factors Preserved in the 17,000-Year-Old Skeleton of an Extinct Bison, Bison antiquus
Auteurs : Oona Y-C. Lee ; Houdini H. T. Wu ; Helen D. Donoghue ; Mark Spigelman ; Charles L. Greenblatt ; Ian D. Bull ; Bruce M. Rothschild ; Larry D. Martin ; David E. Minnikin ; Gurdyal S. BesraSource :
- PLoS ONE [ 1932-6203 ] ; 2012.
Abstract
Tracing the evolution of ancient diseases depends on the availability and accessibility of suitable biomarkers in archaeological specimens. DNA is potentially information-rich but it depends on a favourable environment for preservation. In the case of the major mycobacterial pathogens,
Url:
DOI: 10.1371/journal.pone.0041923
PubMed: 22860031
PubMed Central: 3408397
Links to Exploration step
PMC:3408397Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en"><italic>Mycobacterium tuberculosis</italic>
Complex Lipid Virulence Factors Preserved in the 17,000-Year-Old Skeleton of an Extinct Bison, <italic>Bison antiquus</italic>
</title>
<author><name sortKey="Lee, Oona Y C" sort="Lee, Oona Y C" uniqKey="Lee O" first="Oona Y-C." last="Lee">Oona Y-C. Lee</name>
<affiliation><nlm:aff id="aff1"><addr-line>School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Wu, Houdini H T" sort="Wu, Houdini H T" uniqKey="Wu H" first="Houdini H. T." last="Wu">Houdini H. T. Wu</name>
<affiliation><nlm:aff id="aff1"><addr-line>School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Donoghue, Helen D" sort="Donoghue, Helen D" uniqKey="Donoghue H" first="Helen D." last="Donoghue">Helen D. Donoghue</name>
<affiliation><nlm:aff id="aff2"><addr-line>Centre for Clinical Microbiology (M9), Royal Free Campus, University College London, London, United Kingdom</addr-line>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff3"><addr-line>Centre for the History of Medicine, University College London, London, United Kingdom</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Spigelman, Mark" sort="Spigelman, Mark" uniqKey="Spigelman M" first="Mark" last="Spigelman">Mark Spigelman</name>
<affiliation><nlm:aff id="aff2"><addr-line>Centre for Clinical Microbiology (M9), Royal Free Campus, University College London, London, United Kingdom</addr-line>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff4"><addr-line>Kuvin Center for the Study of Infectious and Tropical Diseases and Ancient DNA, Hadassah Medical School, Hebrew University, Jerusalem, Israel</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Greenblatt, Charles L" sort="Greenblatt, Charles L" uniqKey="Greenblatt C" first="Charles L." last="Greenblatt">Charles L. Greenblatt</name>
<affiliation><nlm:aff id="aff4"><addr-line>Kuvin Center for the Study of Infectious and Tropical Diseases and Ancient DNA, Hadassah Medical School, Hebrew University, Jerusalem, Israel</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Bull, Ian D" sort="Bull, Ian D" uniqKey="Bull I" first="Ian D." last="Bull">Ian D. Bull</name>
<affiliation><nlm:aff id="aff5"><addr-line>Organic Geochemistry Unit, School of Chemistry, University of Bristol, Bristol, United Kingdom</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Rothschild, Bruce M" sort="Rothschild, Bruce M" uniqKey="Rothschild B" first="Bruce M." last="Rothschild">Bruce M. Rothschild</name>
<affiliation><nlm:aff id="aff6"><addr-line>Department of Medicine, Northeast Ohio Medical University, Rootstown, Ohio, United States of America</addr-line>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff7"><addr-line>Biodiversity Institute, University of Kansas, Lawrence, Kansas, United States of America</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Martin, Larry D" sort="Martin, Larry D" uniqKey="Martin L" first="Larry D." last="Martin">Larry D. Martin</name>
<affiliation><nlm:aff id="aff7"><addr-line>Biodiversity Institute, University of Kansas, Lawrence, Kansas, United States of America</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Minnikin, David E" sort="Minnikin, David E" uniqKey="Minnikin D" first="David E." last="Minnikin">David E. Minnikin</name>
<affiliation><nlm:aff id="aff1"><addr-line>School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Besra, Gurdyal S" sort="Besra, Gurdyal S" uniqKey="Besra G" first="Gurdyal S." last="Besra">Gurdyal S. Besra</name>
<affiliation><nlm:aff id="aff1"><addr-line>School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom</addr-line>
</nlm:aff>
</affiliation>
</author>
</titleStmt>
<publicationStmt><idno type="wicri:source">PMC</idno>
<idno type="pmid">22860031</idno>
<idno type="pmc">3408397</idno>
<idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3408397</idno>
<idno type="RBID">PMC:3408397</idno>
<idno type="doi">10.1371/journal.pone.0041923</idno>
<date when="2012">2012</date>
<idno type="wicri:Area/Pmc/Corpus">000361</idno>
<idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000361</idno>
</publicationStmt>
<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main"><italic>Mycobacterium tuberculosis</italic>
Complex Lipid Virulence Factors Preserved in the 17,000-Year-Old Skeleton of an Extinct Bison, <italic>Bison antiquus</italic>
</title>
<author><name sortKey="Lee, Oona Y C" sort="Lee, Oona Y C" uniqKey="Lee O" first="Oona Y-C." last="Lee">Oona Y-C. Lee</name>
<affiliation><nlm:aff id="aff1"><addr-line>School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Wu, Houdini H T" sort="Wu, Houdini H T" uniqKey="Wu H" first="Houdini H. T." last="Wu">Houdini H. T. Wu</name>
<affiliation><nlm:aff id="aff1"><addr-line>School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Donoghue, Helen D" sort="Donoghue, Helen D" uniqKey="Donoghue H" first="Helen D." last="Donoghue">Helen D. Donoghue</name>
<affiliation><nlm:aff id="aff2"><addr-line>Centre for Clinical Microbiology (M9), Royal Free Campus, University College London, London, United Kingdom</addr-line>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff3"><addr-line>Centre for the History of Medicine, University College London, London, United Kingdom</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Spigelman, Mark" sort="Spigelman, Mark" uniqKey="Spigelman M" first="Mark" last="Spigelman">Mark Spigelman</name>
<affiliation><nlm:aff id="aff2"><addr-line>Centre for Clinical Microbiology (M9), Royal Free Campus, University College London, London, United Kingdom</addr-line>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff4"><addr-line>Kuvin Center for the Study of Infectious and Tropical Diseases and Ancient DNA, Hadassah Medical School, Hebrew University, Jerusalem, Israel</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Greenblatt, Charles L" sort="Greenblatt, Charles L" uniqKey="Greenblatt C" first="Charles L." last="Greenblatt">Charles L. Greenblatt</name>
<affiliation><nlm:aff id="aff4"><addr-line>Kuvin Center for the Study of Infectious and Tropical Diseases and Ancient DNA, Hadassah Medical School, Hebrew University, Jerusalem, Israel</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Bull, Ian D" sort="Bull, Ian D" uniqKey="Bull I" first="Ian D." last="Bull">Ian D. Bull</name>
<affiliation><nlm:aff id="aff5"><addr-line>Organic Geochemistry Unit, School of Chemistry, University of Bristol, Bristol, United Kingdom</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Rothschild, Bruce M" sort="Rothschild, Bruce M" uniqKey="Rothschild B" first="Bruce M." last="Rothschild">Bruce M. Rothschild</name>
<affiliation><nlm:aff id="aff6"><addr-line>Department of Medicine, Northeast Ohio Medical University, Rootstown, Ohio, United States of America</addr-line>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff7"><addr-line>Biodiversity Institute, University of Kansas, Lawrence, Kansas, United States of America</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Martin, Larry D" sort="Martin, Larry D" uniqKey="Martin L" first="Larry D." last="Martin">Larry D. Martin</name>
<affiliation><nlm:aff id="aff7"><addr-line>Biodiversity Institute, University of Kansas, Lawrence, Kansas, United States of America</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Minnikin, David E" sort="Minnikin, David E" uniqKey="Minnikin D" first="David E." last="Minnikin">David E. Minnikin</name>
<affiliation><nlm:aff id="aff1"><addr-line>School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Besra, Gurdyal S" sort="Besra, Gurdyal S" uniqKey="Besra G" first="Gurdyal S." last="Besra">Gurdyal S. Besra</name>
<affiliation><nlm:aff id="aff1"><addr-line>School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom</addr-line>
</nlm:aff>
</affiliation>
</author>
</analytic>
<series><title level="j">PLoS ONE</title>
<idno type="eISSN">1932-6203</idno>
<imprint><date when="2012">2012</date>
</imprint>
</series>
</biblStruct>
</sourceDesc>
</fileDesc>
<profileDesc><textClass></textClass>
</profileDesc>
</teiHeader>
<front><div type="abstract" xml:lang="en"><p>Tracing the evolution of ancient diseases depends on the availability and accessibility of suitable biomarkers in archaeological specimens. DNA is potentially information-rich but it depends on a favourable environment for preservation. In the case of the major mycobacterial pathogens, <italic>Mycobacterium tuberculosis</italic>
and <italic>Mycobacterium leprae</italic>
, robust lipid biomarkers are established as alternatives or complements to DNA analyses. A DNA report, a decade ago, suggested that a 17,000-year-old skeleton of extinct <italic>Bison antiquus</italic>
, from Natural Trap Cave, Wyoming, was the oldest known case of tuberculosis. In the current study, key mycobacterial lipid virulence factor biomarkers were detected in the same two samples from this bison. Fluorescence high-performance liquid chromatography (HPLC) indicated the presence of mycolic acids of the mycobacterial type, but they were degraded and could not be precisely correlated with tuberculosis. However, pristine profiles of C<sub>29</sub>
, C<sub>30</sub>
and C<sub>32</sub>
mycocerosates and C<sub>27</sub>
mycolipenates, typical of the <italic>Mycobacterium tuberculosis</italic>
complex, were recorded by negative ion chemical ionization gas chromatography mass spectrometry of pentafluorobenzyl ester derivatives. These findings were supported by the detection of C<sub>34</sub>
and C<sub>36</sub>
phthiocerols, which are usually esterified to the mycocerosates. The existence of Pleistocene tuberculosis in the Americas is confirmed and there are many even older animal bones with well-characterised tuberculous lesions similar to those on the analysed sample. In the absence of any evidence of tuberculosis in human skeletons older than 9,000 years BP, the hypothesis that this disease evolved as a zoonosis, before transfer to humans, is given detailed consideration and discussion.</p>
</div>
</front>
<back><div1 type="bibliography"><listBibl><biblStruct></biblStruct>
<biblStruct><analytic><author><name sortKey="Machowiak, Pa" uniqKey="Machowiak P">PA Machowiak</name>
</author>
<author><name sortKey="Blos, Vt" uniqKey="Blos V">VT Blos</name>
</author>
<author><name sortKey="Aguilar, M" uniqKey="Aguilar M">M Aguilar</name>
</author>
<author><name sortKey="Buikstra, Je" uniqKey="Buikstra J">JE Buikstra</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Rothschild, Bm" uniqKey="Rothschild B">BM Rothschild</name>
</author>
<author><name sortKey="Martin, Ld" uniqKey="Martin L">LD Martin</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Rothschild, Bm" uniqKey="Rothschild B">BM Rothschild</name>
</author>
<author><name sortKey="Laub, R" uniqKey="Laub R">R Laub</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Martin, Ld" uniqKey="Martin L">LD Martin</name>
</author>
<author><name sortKey="Rothschild, Bm" uniqKey="Rothschild B">BM Rothschild</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Rothschild, Bm" uniqKey="Rothschild B">BM Rothschild</name>
</author>
<author><name sortKey="Martin, Ld" uniqKey="Martin L">LD Martin</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Rothschild, Bm" uniqKey="Rothschild B">BM Rothschild</name>
</author>
<author><name sortKey="Martin, Ld" uniqKey="Martin L">LD Martin</name>
</author>
<author><name sortKey="Lev, G" uniqKey="Lev G">G Lev</name>
</author>
<author><name sortKey="Bercovier, H" uniqKey="Bercovier H">H Bercovier</name>
</author>
<author><name sortKey="Bar Gal, Gk" uniqKey="Bar Gal G">GK Bar-Gal</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Tran, T N N" uniqKey="Tran T">T-N-N Tran</name>
</author>
<author><name sortKey="Aboudharam, G" uniqKey="Aboudharam G">G Aboudharam</name>
</author>
<author><name sortKey="Raoult, D" uniqKey="Raoult D">D Raoult</name>
</author>
<author><name sortKey="Drancourt, M" uniqKey="Drancourt M">M Drancourt</name>
</author>
</analytic>
</biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct><analytic><author><name sortKey="Minnikin, De" uniqKey="Minnikin D">DE Minnikin</name>
</author>
<author><name sortKey="Kremer, L" uniqKey="Kremer L">L Kremer</name>
</author>
<author><name sortKey="Dover, Lg" uniqKey="Dover L">LG Dover</name>
</author>
<author><name sortKey="Besra, Gs" uniqKey="Besra G">GS Besra</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Hershkovitz, I" uniqKey="Hershkovitz I">I Hershkovitz</name>
</author>
<author><name sortKey="Donoghue, Hd" uniqKey="Donoghue H">HD Donoghue</name>
</author>
<author><name sortKey="Minnikin, De" uniqKey="Minnikin D">DE Minnikin</name>
</author>
<author><name sortKey="Besra, Gs" uniqKey="Besra G">GS Besra</name>
</author>
<author><name sortKey="Lee, Oy C" uniqKey="Lee O">OY-C Lee</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Redman, Je" uniqKey="Redman J">JE Redman</name>
</author>
<author><name sortKey="Shaw, Mj" uniqKey="Shaw M">MJ Shaw</name>
</author>
<author><name sortKey="Mallet, Ai" uniqKey="Mallet A">AI Mallet</name>
</author>
<author><name sortKey="Santos, Al" uniqKey="Santos A">AL Santos</name>
</author>
<author><name sortKey="Roberts, Ca" uniqKey="Roberts C">CA Roberts</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Minnikin, De" uniqKey="Minnikin D">DE Minnikin</name>
</author>
<author><name sortKey="Dobson, G" uniqKey="Dobson G">G Dobson</name>
</author>
<author><name sortKey="Goodfellow, M" uniqKey="Goodfellow M">M Goodfellow</name>
</author>
<author><name sortKey="Magnusson, M" uniqKey="Magnusson M">M Magnusson</name>
</author>
<author><name sortKey="Ridell, M" uniqKey="Ridell M">M Ridell</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Minnikin, De" uniqKey="Minnikin D">DE Minnikin</name>
</author>
<author><name sortKey="Dobson, G" uniqKey="Dobson G">G Dobson</name>
</author>
<author><name sortKey="Hutchinson, Ig" uniqKey="Hutchinson I">IG Hutchinson</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Minnikin, De" uniqKey="Minnikin D">DE Minnikin</name>
</author>
<author><name sortKey="Dobson, G" uniqKey="Dobson G">G Dobson</name>
</author>
<author><name sortKey="Sesardic, D" uniqKey="Sesardic D">D Sesardic</name>
</author>
<author><name sortKey="Ridell, M" uniqKey="Ridell M">M Ridell</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Daffe, M" uniqKey="Daffe M">M Daffé</name>
</author>
<author><name sortKey="Lacave, C" uniqKey="Lacave C">C Lacave</name>
</author>
<author><name sortKey="Laneelle, M A" uniqKey="Laneelle M">M-A Lanéelle</name>
</author>
<author><name sortKey="Gillois, M" uniqKey="Gillois M">M Gillois</name>
</author>
<author><name sortKey="Laneelle, G" uniqKey="Laneelle G">G Lanéelle</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Besra, Gs" uniqKey="Besra G">GS Besra</name>
</author>
<author><name sortKey="Bolton, Rc" uniqKey="Bolton R">RC Bolton</name>
</author>
<author><name sortKey="Mcneil, Mr" uniqKey="Mcneil M">MR McNeil</name>
</author>
<author><name sortKey="Ridell, M" uniqKey="Ridell M">M Ridell</name>
</author>
<author><name sortKey="Simpson, Ke" uniqKey="Simpson K">KE Simpson</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Onwueme, Kc" uniqKey="Onwueme K">KC Onwueme</name>
</author>
<author><name sortKey="Vos, Cj" uniqKey="Vos C">CJ Vos</name>
</author>
<author><name sortKey="Zurita, J" uniqKey="Zurita J">J Zurita</name>
</author>
<author><name sortKey="Ferreras, Ja" uniqKey="Ferreras J">JA Ferreras</name>
</author>
<author><name sortKey="Quadri, Len" uniqKey="Quadri L">LEN Quadri</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Jackson, M" uniqKey="Jackson M">M Jackson</name>
</author>
<author><name sortKey="Stadhagen, G" uniqKey="Stadhagen G">G Stadhagen</name>
</author>
<author><name sortKey="Gicquel, B" uniqKey="Gicquel B">B Gicquel</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Neyrolles, O" uniqKey="Neyrolles O">O Neyrolles</name>
</author>
<author><name sortKey="Guilhot, C" uniqKey="Guilhot C">C Guilhot</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Korf, J" uniqKey="Korf J">J Korf</name>
</author>
<author><name sortKey="Stoltz, A" uniqKey="Stoltz A">A Stoltz</name>
</author>
<author><name sortKey="Verschoor, J" uniqKey="Verschoor J">J Verschoor</name>
</author>
<author><name sortKey="De Baetselier, P" uniqKey="De Baetselier P">P De Baetselier</name>
</author>
<author><name sortKey="Grooten, J" uniqKey="Grooten J">J Grooten</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Peyron, P" uniqKey="Peyron P">P Peyron</name>
</author>
<author><name sortKey="Vaubourgeix, J" uniqKey="Vaubourgeix J">J Vaubourgeix</name>
</author>
<author><name sortKey="Poquet, Y" uniqKey="Poquet Y">Y Poquet</name>
</author>
<author><name sortKey="Levillain, F" uniqKey="Levillain F">F Levillain</name>
</author>
<author><name sortKey="Botanch, C" uniqKey="Botanch C">C Botanch</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Domenech, P" uniqKey="Domenech P">P Domenech</name>
</author>
<author><name sortKey="Reed, Mb" uniqKey="Reed M">MB Reed</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Astarie Dequeker, C" uniqKey="Astarie Dequeker C">C Astarie-Dequeker</name>
</author>
<author><name sortKey="Le Guyader, L" uniqKey="Le Guyader L">L Le Guyader</name>
</author>
<author><name sortKey="Malaga, W" uniqKey="Malaga W">W Malaga</name>
</author>
<author><name sortKey="Seaphanh, F K" uniqKey="Seaphanh F">F-K Seaphanh</name>
</author>
<author><name sortKey="Chalut, C" uniqKey="Chalut C">C Chalut</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Mostowy, S" uniqKey="Mostowy S">S Mostowy</name>
</author>
<author><name sortKey="Cousins, D" uniqKey="Cousins D">D Cousins</name>
</author>
<author><name sortKey="Brinkman, J" uniqKey="Brinkman J">J Brinkman</name>
</author>
<author><name sortKey="Aranaz, A" uniqKey="Aranaz A">A Aranaz</name>
</author>
<author><name sortKey="Behr, Ma" uniqKey="Behr M">MA Behr</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Brosch, R" uniqKey="Brosch R">R Brosch</name>
</author>
<author><name sortKey="Gordon, Sv" uniqKey="Gordon S">SV Gordon</name>
</author>
<author><name sortKey="Marmiesse, M" uniqKey="Marmiesse M">M Marmiesse</name>
</author>
<author><name sortKey="Brodin, P" uniqKey="Brodin P">P Brodin</name>
</author>
<author><name sortKey="Buchrieser, C" uniqKey="Buchrieser C">C Buchrieser</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Gutierrez, Mc" uniqKey="Gutierrez M">MC Gutierrez</name>
</author>
<author><name sortKey="Brisse, S" uniqKey="Brisse S">S Brisse</name>
</author>
<author><name sortKey="Brosch, R" uniqKey="Brosch R">R Brosch</name>
</author>
<author><name sortKey="Fabre, M" uniqKey="Fabre M">M Fabre</name>
</author>
<author><name sortKey="Omais, B" uniqKey="Omais B">B Omaïs</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Arnold, C" uniqKey="Arnold C">C Arnold</name>
</author>
<author><name sortKey="Thorne, N" uniqKey="Thorne N">N Thorne</name>
</author>
<author><name sortKey="Underwood, A" uniqKey="Underwood A">A Underwood</name>
</author>
<author><name sortKey="Baster, K" uniqKey="Baster K">K Baster</name>
</author>
<author><name sortKey="Gharbia, S" uniqKey="Gharbia S">S Gharbia</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Wirth, T" uniqKey="Wirth T">T Wirth</name>
</author>
<author><name sortKey="Hildebrand, F" uniqKey="Hildebrand F">F Hildebrand</name>
</author>
<author><name sortKey="Allix Beguec, C" uniqKey="Allix Beguec C">C Allix-Béguec</name>
</author>
<author><name sortKey="Wolbeling, F" uniqKey="Wolbeling F">F Wölbeling</name>
</author>
<author><name sortKey="Kubica, T" uniqKey="Kubica T">T Kubica</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Hershberg, R" uniqKey="Hershberg R">R Hershberg</name>
</author>
<author><name sortKey="Lipatov, M" uniqKey="Lipatov M">M Lipatov</name>
</author>
<author><name sortKey="Small, Pm" uniqKey="Small P">PM Small</name>
</author>
<author><name sortKey="Sheffer, H" uniqKey="Sheffer H">H Sheffer</name>
</author>
<author><name sortKey="Niemann, S" uniqKey="Niemann S">S Niemann</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Smith, Nh" uniqKey="Smith N">NH Smith</name>
</author>
<author><name sortKey="Hewinson, Rg" uniqKey="Hewinson R">RG Hewinson</name>
</author>
<author><name sortKey="Kremer, K" uniqKey="Kremer K">K Kremer</name>
</author>
<author><name sortKey="Brosch, R" uniqKey="Brosch R">R Brosch</name>
</author>
<author><name sortKey="Gordon, Sv" uniqKey="Gordon S">SV Gordon</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Gordon, Sv" uniqKey="Gordon S">SV Gordon</name>
</author>
<author><name sortKey="Bottai, D" uniqKey="Bottai D">D Bottai</name>
</author>
<author><name sortKey="Simeone, R" uniqKey="Simeone R">R Simeone</name>
</author>
<author><name sortKey="Stinear, Tp" uniqKey="Stinear T">TP Stinear</name>
</author>
<author><name sortKey="Brosch, R" uniqKey="Brosch R">R Brosch</name>
</author>
</analytic>
</biblStruct>
<biblStruct></biblStruct>
<biblStruct><analytic><author><name sortKey="Djelouadji, Z" uniqKey="Djelouadji Z">Z Djelouadji</name>
</author>
<author><name sortKey="Raoult, D" uniqKey="Raoult D">D Raoult</name>
</author>
<author><name sortKey="Drancourt, M" uniqKey="Drancourt M">M Drancourt</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Gagneux, S" uniqKey="Gagneux S">S Gagneux</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Sreevatsan, S" uniqKey="Sreevatsan S">S Sreevatsan</name>
</author>
<author><name sortKey="Pan, X" uniqKey="Pan X">X Pan</name>
</author>
<author><name sortKey="Stockbauer, Ke" uniqKey="Stockbauer K">KE Stockbauer</name>
</author>
<author><name sortKey="Connell, Nd" uniqKey="Connell N">ND Connell</name>
</author>
<author><name sortKey="Kreiswirth, Bn" uniqKey="Kreiswirth B">BN Kreiswirth</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Smith, Nh" uniqKey="Smith N">NH Smith</name>
</author>
<author><name sortKey="Gordon, Sv" uniqKey="Gordon S">SV Gordon</name>
</author>
<author><name sortKey="De La Rua Domenech, R" uniqKey="De La Rua Domenech R">R de la Rua-Domenech</name>
</author>
<author><name sortKey="Clifton Hadley, Rs" uniqKey="Clifton Hadley R">RS Clifton-Hadley</name>
</author>
<author><name sortKey="Hewinson, Rg" uniqKey="Hewinson R">RG Hewinson</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Rosas Magallanes, V" uniqKey="Rosas Magallanes V">V Rosas-Magallanes</name>
</author>
<author><name sortKey="Deschavanne, P" uniqKey="Deschavanne P">P Deschavanne</name>
</author>
<author><name sortKey="Quintana Murci, L" uniqKey="Quintana Murci L">L Quintana-Murci</name>
</author>
<author><name sortKey="Brosch, R" uniqKey="Brosch R">R Brosch</name>
</author>
<author><name sortKey="Gicquel, B" uniqKey="Gicquel B">B Gicquel</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Becq, J" uniqKey="Becq J">J Becq</name>
</author>
<author><name sortKey="Gutierrez, Mc" uniqKey="Gutierrez M">MC Gutierrez</name>
</author>
<author><name sortKey="Rosas Magallanes, V" uniqKey="Rosas Magallanes V">V Rosas-Magallanes</name>
</author>
<author><name sortKey="Rauzier, J" uniqKey="Rauzier J">J Rauzier</name>
</author>
<author><name sortKey="Gicquel, B" uniqKey="Gicquel B">B Gicquel</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Jang, J" uniqKey="Jang J">J Jang</name>
</author>
<author><name sortKey="Becq, J" uniqKey="Becq J">J Becq</name>
</author>
<author><name sortKey="Gicquel, B" uniqKey="Gicquel B">B Gicquel</name>
</author>
<author><name sortKey="Deschavanne, P" uniqKey="Deschavanne P">P Deschavanne</name>
</author>
<author><name sortKey="Neyrolles, O" uniqKey="Neyrolles O">O Neyrolles</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Smith, Nh" uniqKey="Smith N">NH Smith</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Fabre, M" uniqKey="Fabre M">M Fabre</name>
</author>
<author><name sortKey="Hauck, Y" uniqKey="Hauck Y">Y Hauck</name>
</author>
<author><name sortKey="Soler, C" uniqKey="Soler C">C Soler</name>
</author>
<author><name sortKey="Koeck, J L" uniqKey="Koeck J">J-L Koeck</name>
</author>
<author><name sortKey="Van Ingen, J" uniqKey="Van Ingen J">J van Ingen</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Koeck, J L" uniqKey="Koeck J">J-L Koeck</name>
</author>
<author><name sortKey="Fabre, M" uniqKey="Fabre M">M Fabre</name>
</author>
<author><name sortKey="Simon, F" uniqKey="Simon F">F Simon</name>
</author>
<author><name sortKey="Daffe, M" uniqKey="Daffe M">M Daffé</name>
</author>
<author><name sortKey="Garnotel, E" uniqKey="Garnotel E">E Garnotel</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Klaus, Hd" uniqKey="Klaus H">HD Klaus</name>
</author>
<author><name sortKey="Wilbur, Ak" uniqKey="Wilbur A">AK Wilbur</name>
</author>
<author><name sortKey="Temple, Dh" uniqKey="Temple D">DH Temple</name>
</author>
<author><name sortKey="Buikstra, Je" uniqKey="Buikstra J">JE Buikstra</name>
</author>
<author><name sortKey="Stone, Ac" uniqKey="Stone A">AC Stone</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Kappelman, J" uniqKey="Kappelman J">J Kappelman</name>
</author>
<author><name sortKey="Alcicek, Mc" uniqKey="Alcicek M">MC Alçiçek</name>
</author>
<author><name sortKey="Kazanci, N" uniqKey="Kazanci N">N Kazanci</name>
</author>
<author><name sortKey="Schultz, M" uniqKey="Schultz M">M Schultz</name>
</author>
<author><name sortKey="Ozkul, M" uniqKey="Ozkul M">M Özkul</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Roberts, Ca" uniqKey="Roberts C">CA Roberts</name>
</author>
<author><name sortKey="Pfister, L A" uniqKey="Pfister L">L-A Pfister</name>
</author>
<author><name sortKey="Mays, S" uniqKey="Mays S">S Mays</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Donoghue, Hd" uniqKey="Donoghue H">HD Donoghue</name>
</author>
<author><name sortKey="Lee, Oy C" uniqKey="Lee O">OY-C Lee</name>
</author>
<author><name sortKey="Minnikin, De" uniqKey="Minnikin D">DE Minnikin</name>
</author>
<author><name sortKey="Besra, Gs" uniqKey="Besra G">GS Besra</name>
</author>
<author><name sortKey="Taylor, Jh" uniqKey="Taylor J">JH Taylor</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Taylor, Gm" uniqKey="Taylor G">GM Taylor</name>
</author>
<author><name sortKey="Blau, S" uniqKey="Blau S">S Blau</name>
</author>
<author><name sortKey="Mays, S" uniqKey="Mays S">S Mays</name>
</author>
<author><name sortKey="Monot, M" uniqKey="Monot M">M Monot</name>
</author>
<author><name sortKey="Lee, Oy C" uniqKey="Lee O">OY-C Lee</name>
</author>
</analytic>
</biblStruct>
</listBibl>
</div1>
</back>
</TEI>
<pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">PLoS One</journal-id>
<journal-id journal-id-type="iso-abbrev">PLoS ONE</journal-id>
<journal-id journal-id-type="publisher-id">plos</journal-id>
<journal-id journal-id-type="pmc">plosone</journal-id>
<journal-title-group><journal-title>PLoS ONE</journal-title>
</journal-title-group>
<issn pub-type="epub">1932-6203</issn>
<publisher><publisher-name>Public Library of Science</publisher-name>
<publisher-loc>San Francisco, USA</publisher-loc>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">22860031</article-id>
<article-id pub-id-type="pmc">3408397</article-id>
<article-id pub-id-type="publisher-id">PONE-D-12-11889</article-id>
<article-id pub-id-type="doi">10.1371/journal.pone.0041923</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject>
</subj-group>
<subj-group subj-group-type="Discipline-v2"><subject>Biology</subject>
<subj-group><subject>Evolutionary Biology</subject>
<subj-group><subject>Comparative Genomics</subject>
<subject>Evolutionary Ecology</subject>
<subject>Evolutionary Processes</subject>
<subject>Genomic Evolution</subject>
<subject>Organismal Evolution</subject>
<subject>Paleontology</subject>
</subj-group>
</subj-group>
<subj-group><subject>Microbiology</subject>
<subj-group><subject>Bacterial Pathogens</subject>
<subject>Host-Pathogen Interaction</subject>
<subject>Microbial Evolution</subject>
<subject>Microbial Pathogens</subject>
</subj-group>
</subj-group>
<subj-group><subject>Paleontology</subject>
<subj-group><subject>Biogeography</subject>
<subject>Paleobiology</subject>
<subject>Paleoecology</subject>
</subj-group>
</subj-group>
</subj-group>
<subj-group subj-group-type="Discipline-v2"><subject>Chemistry</subject>
<subj-group><subject>Chemical Biology</subject>
</subj-group>
<subj-group><subject>Chromatography</subject>
<subj-group><subject>Gas Chromatography</subject>
<subject>Liquid Chromatography</subject>
</subj-group>
</subj-group>
</subj-group>
<subj-group subj-group-type="Discipline-v2"><subject>Veterinary Science</subject>
<subj-group><subject>Veterinary Diseases</subject>
<subj-group><subject>Veterinary Bacteriology</subject>
<subject>Zoonotic Diseases</subject>
</subj-group>
</subj-group>
</subj-group>
</article-categories>
<title-group><article-title><italic>Mycobacterium tuberculosis</italic>
Complex Lipid Virulence Factors Preserved in the 17,000-Year-Old Skeleton of an Extinct Bison, <italic>Bison antiquus</italic>
</article-title>
<alt-title alt-title-type="running-head">Lipid Biomarkers for Pleistocene <italic>Tuberculosis</italic>
</alt-title>
</title-group>
<contrib-group><contrib contrib-type="author"><name><surname>Lee</surname>
<given-names>Oona Y-C.</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Wu</surname>
<given-names>Houdini H. T.</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Donoghue</surname>
<given-names>Helen D.</given-names>
</name>
<xref ref-type="aff" rid="aff2"><sup>2</sup>
</xref>
<xref ref-type="aff" rid="aff3"><sup>3</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Spigelman</surname>
<given-names>Mark</given-names>
</name>
<xref ref-type="aff" rid="aff2"><sup>2</sup>
</xref>
<xref ref-type="aff" rid="aff4"><sup>4</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Greenblatt</surname>
<given-names>Charles L.</given-names>
</name>
<xref ref-type="aff" rid="aff4"><sup>4</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Bull</surname>
<given-names>Ian D.</given-names>
</name>
<xref ref-type="aff" rid="aff5"><sup>5</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Rothschild</surname>
<given-names>Bruce M.</given-names>
</name>
<xref ref-type="aff" rid="aff6"><sup>6</sup>
</xref>
<xref ref-type="aff" rid="aff7"><sup>7</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Martin</surname>
<given-names>Larry D.</given-names>
</name>
<xref ref-type="aff" rid="aff7"><sup>7</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Minnikin</surname>
<given-names>David E.</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Besra</surname>
<given-names>Gurdyal S.</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>1</sup>
</xref>
<xref ref-type="corresp" rid="cor1"><sup>*</sup>
</xref>
</contrib>
</contrib-group>
<aff id="aff1"><label>1</label>
<addr-line>School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom</addr-line>
</aff>
<aff id="aff2"><label>2</label>
<addr-line>Centre for Clinical Microbiology (M9), Royal Free Campus, University College London, London, United Kingdom</addr-line>
</aff>
<aff id="aff3"><label>3</label>
<addr-line>Centre for the History of Medicine, University College London, London, United Kingdom</addr-line>
</aff>
<aff id="aff4"><label>4</label>
<addr-line>Kuvin Center for the Study of Infectious and Tropical Diseases and Ancient DNA, Hadassah Medical School, Hebrew University, Jerusalem, Israel</addr-line>
</aff>
<aff id="aff5"><label>5</label>
<addr-line>Organic Geochemistry Unit, School of Chemistry, University of Bristol, Bristol, United Kingdom</addr-line>
</aff>
<aff id="aff6"><label>6</label>
<addr-line>Department of Medicine, Northeast Ohio Medical University, Rootstown, Ohio, United States of America</addr-line>
</aff>
<aff id="aff7"><label>7</label>
<addr-line>Biodiversity Institute, University of Kansas, Lawrence, Kansas, United States of America</addr-line>
</aff>
<contrib-group><contrib contrib-type="editor"><name><surname>Karakousis</surname>
<given-names>Petros C.</given-names>
</name>
<role>Editor</role>
<xref ref-type="aff" rid="edit1"></xref>
</contrib>
</contrib-group>
<aff id="edit1"><addr-line>Johns Hopkins University School of Medicine, United States of America</addr-line>
</aff>
<author-notes><corresp id="cor1">* E-mail: <email>g.besra@bham.ac.uk</email>
</corresp>
<fn fn-type="conflict"><p><bold>Competing Interests: </bold>
The authors have declared that no competing interests exist.</p>
</fn>
<fn fn-type="con"><p>Conceived and designed the experiments: OY-CL HDD MS CLG BMR LDM DEM GSB. Performed the experiments: OY-CL HHTW IDB. Analyzed the data: OY-CL HHTW IDB DEM GSB. Wrote the paper: OY-CL HHTW HDD BMR DEM GSB.</p>
</fn>
</author-notes>
<pub-date pub-type="collection"><year>2012</year>
</pub-date>
<pub-date pub-type="epub"><day>30</day>
<month>7</month>
<year>2012</year>
</pub-date>
<volume>7</volume>
<issue>7</issue>
<elocation-id>e41923</elocation-id>
<history><date date-type="received"><day>24</day>
<month>4</month>
<year>2012</year>
</date>
<date date-type="accepted"><day>29</day>
<month>6</month>
<year>2012</year>
</date>
</history>
<permissions><copyright-year>2012</copyright-year>
<copyright-holder>Lee et al</copyright-holder>
<license><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p>
</license>
</permissions>
<abstract><p>Tracing the evolution of ancient diseases depends on the availability and accessibility of suitable biomarkers in archaeological specimens. DNA is potentially information-rich but it depends on a favourable environment for preservation. In the case of the major mycobacterial pathogens, <italic>Mycobacterium tuberculosis</italic>
and <italic>Mycobacterium leprae</italic>
, robust lipid biomarkers are established as alternatives or complements to DNA analyses. A DNA report, a decade ago, suggested that a 17,000-year-old skeleton of extinct <italic>Bison antiquus</italic>
, from Natural Trap Cave, Wyoming, was the oldest known case of tuberculosis. In the current study, key mycobacterial lipid virulence factor biomarkers were detected in the same two samples from this bison. Fluorescence high-performance liquid chromatography (HPLC) indicated the presence of mycolic acids of the mycobacterial type, but they were degraded and could not be precisely correlated with tuberculosis. However, pristine profiles of C<sub>29</sub>
, C<sub>30</sub>
and C<sub>32</sub>
mycocerosates and C<sub>27</sub>
mycolipenates, typical of the <italic>Mycobacterium tuberculosis</italic>
complex, were recorded by negative ion chemical ionization gas chromatography mass spectrometry of pentafluorobenzyl ester derivatives. These findings were supported by the detection of C<sub>34</sub>
and C<sub>36</sub>
phthiocerols, which are usually esterified to the mycocerosates. The existence of Pleistocene tuberculosis in the Americas is confirmed and there are many even older animal bones with well-characterised tuberculous lesions similar to those on the analysed sample. In the absence of any evidence of tuberculosis in human skeletons older than 9,000 years BP, the hypothesis that this disease evolved as a zoonosis, before transfer to humans, is given detailed consideration and discussion.</p>
</abstract>
<funding-group><funding-statement>The study was supported by Leverhulme Trust Project Grant F/00 094/BL (GSB, DEM, OY-CL) and Wellcome Trust Grant 080988/Z/06/Z (GSB, OY-CL). A grant from the UK Natural and Environmental Research Council (DEM, GSB) provided access to the Mass Spectrometry Unit at the University of Bristol, UK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.</funding-statement>
</funding-group>
<counts><page-count count="9"></page-count>
</counts>
</article-meta>
</front>
<body><sec id="s1"><title>Introduction</title>
<p>Tuberculosis was present from an early date in North America, but clear evidence for its distribution and origins is by no means complete <xref ref-type="bibr" rid="pone.0041923-Wilbur1">[1]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Machowiak1">[2]</xref>
. An intriguing aspect of ancient North American tuberculosis is its apparent prevalence in ice-age bovids and mastodons who may have nurtured the disease without being rapidly killed <xref ref-type="bibr" rid="pone.0041923-Rothschild1">[3]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Rothschild2">[4]</xref>
. Bone pathology of skeletons, collected in Natural Trap Cave (Wyoming), indicates the presence of such an ancient animal tuberculosis reservoir <xref ref-type="bibr" rid="pone.0041923-Martin1">[5]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Rothschild3">[6]</xref>
. Lesions suggestive of tuberculosis were seen in skeletons of bighorn sheep, musk ox and <italic>Bison antiquus</italic>
<xref ref-type="bibr" rid="pone.0041923-Rothschild3">[6]</xref>
. In this bison, dated to 17,870±230 BP, it was possible to demonstrate ancient DNA characteristic of the <italic>Mycobacterium tuberculosis</italic>
complex, confirming the oldest proven case of tuberculosis <xref ref-type="bibr" rid="pone.0041923-Rothschild4">[7]</xref>
.</p>
<p>The use of DNA can be complemented by other biomarkers, as reviewed recently <xref ref-type="bibr" rid="pone.0041923-Tran1">[8]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Minnikin1">[9]</xref>
. Mycolic acids (MAs) (<xref ref-type="fig" rid="pone-0041923-g001">Figure 1A</xref>
) and phthiocerol dimycocerosate (PDIM) waxes (<xref ref-type="fig" rid="pone-0041923-g001">Figure 1B, C</xref>
) are characteristic major components of the cell envelopes of <italic>M. tuberculosis</italic>
<xref ref-type="bibr" rid="pone.0041923-Minnikin2">[10]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Minnikin3">[11]</xref>
. Using a combination of DNA and MA analysis, the oldest case of human tuberculosis infection was established in skeletons of a woman and adjacent child from Atlit-Yam (Israel) <xref ref-type="bibr" rid="pone.0041923-Hershkovitz1">[12]</xref>
. Analysis of the mycocerosate components (<xref ref-type="fig" rid="pone-0041923-g001">Figure 1B</xref>
) of PDIM waxes was established in the investigation of a skeletal collection from Coimbra (Portugal) <xref ref-type="bibr" rid="pone.0041923-Redman1">[13]</xref>
. In the same study <xref ref-type="bibr" rid="pone.0041923-Redman1">[13]</xref>
, another lipid biomarker, C<sub>27</sub>
mycolipenic acid (<xref ref-type="fig" rid="pone-0041923-g001">Figure 1B</xref>
), was encountered in a minority of samples.</p>
<fig id="pone-0041923-g001" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0041923.g001</object-id>
<label>Figure 1</label>
<caption><title>Lipid biomarkers for <italic>M. tuberculosis</italic>
.</title>
<p><bold>A.</bold>
Generalized structures of the α-, methoxy- and ketomycolates; the main components are in brackets. <bold>B.</bold>
Structures of mycolipenate and mycocerosates, showing ions used for selected ion monitoring on NI-CI GC-MS analysis. <bold>C.</bold>
Structures of members of the phthiocerol family.</p>
</caption>
<graphic xlink:href="pone.0041923.g001"></graphic>
</fig>
<p>The present study investigated the same bone samples used previously <xref ref-type="bibr" rid="pone.0041923-Rothschild4">[7]</xref>
, and recorded a weak mycolic acid profile. However, a clear profile of mycocerosic acids was obtained, supported by the identification of phthiocerol components. In addition, a strong presence of mycolipenic acids was recorded. These findings independently document the presence of tuberculosis in this ancient extinct bison skeleton and demonstrate the long-term stability of these lipid virulence factors. The confirmation of tuberculosis in an animal bone much older than any positive human skeletons raises the possibility that the ancient evolution of the disease was as a zoonosis.</p>
</sec>
<sec id="s2"><title>Results and Discussion</title>
<sec id="s2a"><title>Bone Samples and Extraction of Lipids</title>
<p>The two bone samples analysed were from an extinct bison (<italic>Bison antiquus</italic>
) buried in sediments, dated to 17,870±230 years BP, in Natural Trap Cave (Wyoming) <xref ref-type="bibr" rid="pone.0041923-Rothschild4">[7]</xref>
. The first sample (“Bison 1”) originated from the undermined articular surface of a metacarpal but the second sample (“Bison 2”) was from a site on the same bone remote from this lesion <xref ref-type="bibr" rid="pone.0041923-Rothschild4">[7]</xref>
. The material was exactly the same as that used for the previous ancient tuberculosis DNA analyses <xref ref-type="bibr" rid="pone.0041923-Rothschild4">[7]</xref>
.</p>
<p>Bone samples were hydrolysed by a protocol designed to release all the long-chain lipid components <xref ref-type="bibr" rid="pone.0041923-Minnikin1">[9]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Hershkovitz1">[12]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Redman1">[13]</xref>
and the acidic components were converted to pentafluorobenzyl (PFB) esters <xref ref-type="bibr" rid="pone.0041923-Hershkovitz1">[12]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Redman1">[13]</xref>
. The extract was fractionated into three distinct lipid classes, containing non-hydroxylated fatty acid PFB esters, mycolic acid PFB esters and underivatised phthiocerols <xref ref-type="bibr" rid="pone.0041923-Hershkovitz1">[12]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Redman1">[13]</xref>
. The latter two classes were allowed to react with pyrenebutyric acid (PBA) to produce PBA-PFB mycolates and di-PBA derivatives of the phthiocerol family (<xref ref-type="fig" rid="pone-0041923-g001">Figure 1</xref>
). These fluorescent derivatives were analysed by sequential combinations of reverse and normal phase high performance liquid chromatography (HPLC) <xref ref-type="bibr" rid="pone.0041923-Hershkovitz1">[12]</xref>
. The non-hydroxylated fatty acid PFB esters were fractionated further to produce material enriched in PFB esters of mycocerosic and mycolipenic acids, which were analysed by negative ion chemical ionization gas chromatography mass spectrometry (NICI-GCMS) and selected ion monitoring (SIM) <xref ref-type="bibr" rid="pone.0041923-Redman1">[13]</xref>
.</p>
</sec>
<sec id="s2b"><title>Detection of Mycolic Acids</title>
<p>Reverse phase HPLC of the PBA-PFB mycolate fractions indicated the presence of long-chain mycolates in sample Bison 1, but the profile for Bison 2 was very weak (<xref ref-type="fig" rid="pone-0041923-g002">Figure 2A</xref>
). The total components in the region corresponding to mycolates, from the reverse phase HPLC of Bison 1 and 2 extracts, were collected and analysed by normal phase HPLC (<xref ref-type="fig" rid="pone-0041923-g002">Figure 2B</xref>
). A recognizable profile was recorded for Bison 1, with the α-mycolates being the most abundant. However, the signal was not clean, with minor additional peaks eluting before and after the main component (<xref ref-type="fig" rid="pone-0041923-g002">Figure 2B</xref>
). Methoxymycolates were apparently present in low amounts, but there was only a weak indication for ketomycolates. In the normal phase HPLC profile of the total mycolates from Bison 2 no clear peaks could be discerned. The fractions, corresponding to the positions of α-mycolates, methoxymycolates and ketomycolates (<xref ref-type="fig" rid="pone-0041923-g002">Figure 2B</xref>
), were collected and re-analyzed by reverse phase HPLC (<xref ref-type="fig" rid="pone-0041923-g003">Figure 3</xref>
). An informative profile was obtained for the α-mycolates from Bison 1 but nothing was observed for Bison 2 (<xref ref-type="fig" rid="pone-0041923-g003">Figure 3A</xref>
). Weak indications of methoxymycolates and ketomycolates were only discernible in Bison 1 (<xref ref-type="fig" rid="pone-0041923-g003">Figure 3B, C</xref>
).</p>
<fig id="pone-0041923-g002" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0041923.g002</object-id>
<label>Figure 2</label>
<caption><title>Fluorescence HPLC of pyrenebutyric acid derivatives of pentafluorobenzyl esters of total mycolic acids.</title>
<p>The derivatives analysed were from Bison 1 and Bison 2 bones and standard <italic>M. tuberculosis</italic>
. <bold>A.</bold>
Reverse phase HPLC of total mycolates. <bold>B.</bold>
Normal phase HPLC of total mycolates collected from reverse phase separations.</p>
</caption>
<graphic xlink:href="pone.0041923.g002"></graphic>
</fig>
<fig id="pone-0041923-g003" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0041923.g003</object-id>
<label>Figure 3</label>
<caption><title>Reverse phase fluorescence HPLC of pyrenebutyric acid derivatives of pentafluorobenzyl esters of mycolic acid classes.</title>
<p>The derivatives analysed were from Bison 1 and Bison 2 bones and standard <italic>M. tuberculosis</italic>
. The analyzed fractions were collected from the normal phase separation (<xref ref-type="fig" rid="pone-0041923-g002">Figure 2B</xref>
). <bold>A.</bold>
α-Mycolates. <bold>B.</bold>
Methoxymycolates. <bold>C.</bold>
Ketomycolates.</p>
</caption>
<graphic xlink:href="pone.0041923.g003"></graphic>
</fig>
</sec>
<sec id="s2c"><title>Detection of Mycocerosic and Mycolipenic Acids</title>
<p>Positive profiles of C<sub>29</sub>
, C<sub>30</sub>
and C<sub>32</sub>
mycocerosates were recorded by SIM NICI-GCMS for both Bison 1 and 2 (<xref ref-type="fig" rid="pone-0041923-g004">Figure 4</xref>
). Additionally, lesser proportions of C<sub>27</sub>
mycocerosates were distinguishable, but no significant proportions of C<sub>33</sub>
and C<sub>34</sub>
mycocerosates were encountered (<bold><xref ref-type="supplementary-material" rid="pone.0041923.s001">Figure S1</xref>
</bold>
). The mycocerosates were observed as characteristic double peaks, due to partial racemization during the alkaline hydrolysis <xref ref-type="bibr" rid="pone.0041923-Redman1">[13]</xref>
. Positive recognition of the <italic>M. tuberculosis</italic>
complex mycocerosate pattern was confirmed by the GC retention time values, particularly the overlapping of the C<sub>29</sub>
and C<sub>30</sub>
components (<xref ref-type="fig" rid="pone-0041923-g004">Figure 4</xref>
). This highly diagnostic chromatographic behaviour is a result of the tetramethyl-branched C<sub>30</sub>
mycocerosate being relatively more volatile in comparison with the trimethyl-branched C<sub>29</sub>
ester <xref ref-type="bibr" rid="pone.0041923-Minnikin1">[9]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Redman1">[13]</xref>
. In both Bison 1 and 2, a substantial proportion of C<sub>27</sub>
mycolipenate was observed (<xref ref-type="fig" rid="pone-0041923-g004">Figure 4</xref>
) as a single peak, racemization not being possible <xref ref-type="bibr" rid="pone.0041923-Minnikin1">[9]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Redman1">[13]</xref>
. The identification of C<sub>27</sub>
mycolipenate was also confirmed by comparison of the GC retention time with an authentic standard (<xref ref-type="fig" rid="pone-0041923-g004">Figure 4</xref>
) and correlation with a previous detailed study <xref ref-type="bibr" rid="pone.0041923-Redman1">[13]</xref>
. The full range of positive and negative NICI-GCMS mycocerosate and mycolipenate profiles is shown in <bold><xref ref-type="supplementary-material" rid="pone.0041923.s001">Figure S1</xref>
.</bold>
</p>
<fig id="pone-0041923-g004" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0041923.g004</object-id>
<label>Figure 4</label>
<caption><title>Selected ion monitoring NI-CI GC-MS of mycolipenic and mycocerosic acid pentafluorobenzyl fractions.</title>
<p><bold>A.</bold>
Bison 1. <bold>B.</bold>
Bison 2. <bold>C.</bold>
Standard <italic>M. tuberculosis.</italic>
The <italic>m/z</italic>
407 (mycolipenate), 409, 437, 451 and 479 (mycocerosates) ions correspond to the components shown in <xref ref-type="fig" rid="pone-0041923-g001">Figure 1B</xref>
. The intensities of the mycocerosate and mycolipenate peaks, in square brackets, are normalized to that [100] of the major C<sub>32</sub>
mycocerosate. It was not possible to record all three profiles on the same occasion, so the retention times for the standard <italic>M. tuberculosis</italic>
extract do not correspond exactly. In profiles <bold>A</bold>
and <bold>B</bold>
, the C<sub>27</sub>
<italic>m/z</italic>
409 peaks at 21.81 and 21.80, respectively, correspond to straight-chain heptacosanoate.</p>
</caption>
<graphic xlink:href="pone.0041923.g004"></graphic>
</fig>
</sec>
<sec id="s2d"><title>Detection of Phthiocerol Family</title>
<p>Reverse phase HPLC of the PBA phthiocerol fraction from Bison 1 and 2 both showed components corresponding to C<sub>34</sub>
and C<sub>36</sub>
phthiocerol A and C<sub>35</sub>
phthiodiolone (<xref ref-type="fig" rid="pone-0041923-g005">Figure 5A</xref>
). The collected total PBA phthiocerol family fractions were analyzed by normal phase HPLC (<xref ref-type="fig" rid="pone-0041923-g005">Figure 5B</xref>
), providing profiles with components corresponding to phthiocerols A and B and phthiodiolone. The separated fractions from the normal phase analysis (<xref ref-type="fig" rid="pone-0041923-g005">Figure 5B</xref>
) were subjected to reverse phase HPLC, but there was insufficient material for profiles to be registered.</p>
<fig id="pone-0041923-g005" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0041923.g005</object-id>
<label>Figure 5</label>
<caption><title>Fluorescence HPLC of di-pyrenebutyric acid derivatives of members of the phthiocerol family.</title>
<p>The derivatives analysed were from Bison 1 and Bison 2 bones and standard <italic>M. tuberculosis</italic>
. <bold>A.</bold>
Reverse phase HPLC of total phthiocerol family fraction. <bold>B.</bold>
Normal phase HPLC of phthiocerol family derivatives collected from reverse phase separations. Abbreviations: <bold>PA</bold>
, phthiocerol A; <bold>PB</bold>
, phthiocerol B; <bold>PO</bold>
, phthiodiolone.</p>
</caption>
<graphic xlink:href="pone.0041923.g005"></graphic>
</fig>
</sec>
<sec id="s2e"><title>Significance of the Presence of Lipid Biomarkers</title>
<p>The presence of mycolic acids, having an overall size similar to those found in <italic>M. tuberculosis</italic>
(<xref ref-type="fig" rid="pone-0041923-g002">Figure 2A</xref>
), is highly significant, providing confirmation of the previous recovery of DNA characteristic of the <italic>M. tuberculosis</italic>
complex <xref ref-type="bibr" rid="pone.0041923-Rothschild4">[7]</xref>
. In that study <xref ref-type="bibr" rid="pone.0041923-Rothschild4">[7]</xref>
, spoligotyping indicated that the infection was not due to <italic>M. bovis</italic>
, but <italic>M. tuberculosis</italic>
or <italic>M. africanum</italic>
were possibilities. Not surprisingly, the bone sample taken from near the observed lesion (Bison 1) gave the best profile, but there was a very faint response for Bison 2 (<xref ref-type="fig" rid="pone-0041923-g002">Figure 2A</xref>
). The normal phase HPLC examination of the collected mycolates (<xref ref-type="fig" rid="pone-0041923-g002">Figure 2B</xref>
) gave confirmatory evidence for α-mycolates in Bison 1, with one good sharp peak accompanied by some possibly degraded components (<xref ref-type="fig" rid="pone-0041923-g002">Figure 2B</xref>
). The Bison 1 extract also had a broad peak, possibly representing methoxymycolates, and a slight elevation of the baseline might be interpreted as a suggestion of ketomycolates (<xref ref-type="fig" rid="pone-0041923-g002">Figure 2B</xref>
). If diagenetic modification of mycolates takes place, it is probable that the more hydrophilic and chemically reactive ketomycolates are more susceptible, followed by the intermediate methoxymycolates and the relatively hydrophobic α-mycolates. For Bison 2, the normal phase HPLC analysis of total mycolates was negative (<xref ref-type="fig" rid="pone-0041923-g002">Figure 2B</xref>
). On subjecting total α-mycolates, collected from the normal phase HPLC analyses (<xref ref-type="fig" rid="pone-0041923-g002">Figure 2B</xref>
), to reverse phase HPLC an acceptable profile was recorded for Bison 1 (<xref ref-type="fig" rid="pone-0041923-g003">Figure 3A</xref>
). This profile (<xref ref-type="fig" rid="pone-0041923-g003">Figure 3A</xref>
) corresponds to the standard, but the peaks are broadened by possible degradation. It is also encouraging that a very weak series of peaks can be discerned for methoxymycolates and ketomycolates in Bison 1 (<xref ref-type="fig" rid="pone-0041923-g003">Figure 3B, C</xref>
). The above evidence points to the presence of mycobacterial mycolic acids, but a clear diagnosis of <italic>M. tuberculosis</italic>
complex infection cannot be made solely on these data.</p>
<p>Of greater significance is the discovery of pristine C<sub>29</sub>
, C<sub>30</sub>
and C<sub>32</sub>
mycocerosates and C<sub>27</sub>
mycolipenate (<xref ref-type="fig" rid="pone-0041923-g004">Figure 4</xref>
) and the C<sub>34</sub>
and C<sub>36</sub>
phthiocerols A (<xref ref-type="fig" rid="pone-0041923-g005">Figure 5</xref>
). This combination of mycocerosates is typical of <italic>M. tuberculosis sensu stricto</italic>
<xref ref-type="bibr" rid="pone.0041923-Minnikin3">[11]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Redman1">[13]</xref>
, as is the presence of comparable amounts of the C<sub>34</sub>
and C<sub>36</sub>
phthiocerols. A limited study <xref ref-type="bibr" rid="pone.0041923-Minnikin4">[14]</xref>
indicated that <italic>M. bovis</italic>
usually has an enhanced proportion of C<sub>27</sub>
mycocerosate and a preponderance of C<sub>34</sub>
over C<sub>36</sub>
phthiocerols, when compared with <italic>M. tuberculosis sensu stricto</italic>
. It is interesting that the proportion of phthiodiolone in Bison 1 is comparable to that of phthiocerol A. For Bison 2, however, phthiodiolone is reduced in proportion; this indicates that this “keto” component may have experienced some selective degradation as for ketomycolates (<xref ref-type="fig" rid="pone-0041923-g002">Figure 2B</xref>
). The normal phase HPLC peaks (<xref ref-type="fig" rid="pone-0041923-g005">Figure 5B</xref>
) for phthiocerol A appeared to be doublets, suggesting some diagenetic racemization. The presence of both mycocerosates and phthiocerols in the same extract suggests that intact PDIM waxes were present in these ancient bones. These PDIM waxes are particularly robust, surviving acid methanolysis and aqueous alkaline hydrolysis <xref ref-type="bibr" rid="pone.0041923-Minnikin4">[14]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Minnikin5">[15]</xref>
. It is not surprising, therefore, that they are apparently recovered intact from such an ancient source, when mycolic acids are diminished in abundance (<xref ref-type="fig" rid="pone-0041923-g002">Figures 2</xref>
, <xref ref-type="fig" rid="pone-0041923-g003">3</xref>
, <xref ref-type="fig" rid="pone-0041923-g004">4</xref>
, <xref ref-type="fig" rid="pone-0041923-g005">5</xref>
). If PDIMs can survive intact for ∼17,000 years, it appears possible that, in favourable circumstances, they might be found in archaeological material way back into antiquity.</p>
<p>It is more surprising, however, that clear evidence for mycolipenic acid was observed in both samples. Mycolipenic acid <xref ref-type="bibr" rid="pone.0041923-Minnikin3">[11]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Redman1">[13]</xref>
is a constituent of characteristic glycolipids <xref ref-type="bibr" rid="pone.0041923-Minnikin6">[16]</xref>
, the penta-acyl trehaloses (PATs) <xref ref-type="bibr" rid="pone.0041923-Minnikin6">[16]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Daff1">[17]</xref>
. Mycolipenates appear to be limited in distribution to virulent strains of members of the <italic>M. tuberculosis</italic>
complex, such as <italic>M. tuberculosis, M. bovis</italic>
and <italic>M. africanum</italic>
<xref ref-type="bibr" rid="pone.0041923-Daff1">[17]</xref>
. It might be expected that the trehalose unit would render PATs susceptible to degradation compared to the non-polar PDIM waxes. However, the PATs are also relatively non-polar <xref ref-type="bibr" rid="pone.0041923-Minnikin6">[16]</xref>
and hydrophobic, due to the presence of long-chain fatty acid substituents on five out of the eight trehalose sites <xref ref-type="bibr" rid="pone.0041923-Daff1">[17]</xref>
. Mycolipenates may survive in this protected hydrophobic environment, although there is currently no evidence for the survival of intact PAT glycolipids. Another possibility is that the conjugated αβ-unsaturated unit in the mycolipenate [<xref ref-type="fig" rid="pone-0041923-g001">Figure 1B</xref>
] confers additional resistance to diagenesis.</p>
<p><italic>M. tuberculosis</italic>
produces a class of relatively polar diacylated trehalose (DAT) glycolipids, with mycolipanolic and mycosanoic (2,4-dimethyldocosanoic) acid components <xref ref-type="bibr" rid="pone.0041923-Minnikin3">[11]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Minnikin6">[16]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Besra1">[18]</xref>
. Mycolipanolate was not investigated, but no evidence was obtained by NICI-GCMS for an ion at <italic>m/z</italic>
367, corresponding to C<sub>24</sub>
mycosanoate (<bold><xref ref-type="supplementary-material" rid="pone.0041923.s001">Figure S1</xref>
)</bold>
. It appears, therefore, that mycolipenic acid (<xref ref-type="fig" rid="pone-0041923-g001">Figure 1B</xref>
) is an informative biomarker for ancient tuberculosis. Mycolipenate was found in low abundance in only 9 out of 37 mycocerosate-positive skeletons from the more-recent Coimbra collection <xref ref-type="bibr" rid="pone.0041923-Redman1">[13]</xref>
. Mycolipenates also have value in distinguishing ancient tuberculosis and leprosy, as they are not synthesized by <italic>Mycobacterium leprae</italic>
<xref ref-type="bibr" rid="pone.0041923-Minnikin3">[11]</xref>
.</p>
<p>The previous aDNA results <xref ref-type="bibr" rid="pone.0041923-Rothschild4">[7]</xref>
indicated that the infection did not correlate with bovine tuberculosis, but could be due to <italic>M. tuberculosis</italic>
or <italic>M. africanum.</italic>
The present lipid results do not contradict this conclusion. In particular, the mycocerosate profiles are indicative of <italic>M. tuberculosis</italic>
, as only small proportions of C<sub>27</sub>
-mycocerosate were recorded (<xref ref-type="fig" rid="pone-0041923-g004">Figure 4</xref>
). In previous studies, it was shown that extracts of <italic>M. bovis</italic>
had relatively enhanced amounts of this component <xref ref-type="bibr" rid="pone.0041923-Minnikin4">[14]</xref>
. The profiles for members of the phthiocerol family (<xref ref-type="fig" rid="pone-0041923-g004">Figure 4</xref>
) also resemble those for <italic>M. tuberculosis</italic>
more closely than those for <italic>M. bovis,</italic>
with a predominance of C<sub>36</sub>
phthiocerol A over the C<sub>34</sub>
component <xref ref-type="bibr" rid="pone.0041923-Minnikin4">[14]</xref>
. The degraded mycolate profiles (<xref ref-type="fig" rid="pone-0041923-g002">Figs. 2</xref>
and <xref ref-type="fig" rid="pone-0041923-g003">3</xref>
) are not particularly informative, but the size of the α-mycolates is compatible with those from members of the <italic>M. tuberculosis</italic>
complex. The presence of C<sub>27</sub>
-mycolipenate is not diagnostic, as this component is found in various virulent members of the <italic>M. tuberculosis</italic>
complex <xref ref-type="bibr" rid="pone.0041923-Daff1">[17]</xref>
. The overall conclusion is that the infection was by a member of the <italic>M. tuberculosis</italic>
complex, with <italic>M. bovis</italic>
being an unlikely candidate.</p>
<p>Looking beyond the obvious paleopathological significance of the present results, it should be noted that, to our knowledge, these MAs and PDIMs are the oldest recognizable virulence factors ever recorded for tuberculosis, or perhaps any other infectious disease. The importance in virulence of these two lipid classes can be attributed to at least two features of these compounds. Firstly, both the MAs and PDIMs are considered to be integral components of the cell envelope architecture of <italic>M. tuberculosis</italic>
<xref ref-type="bibr" rid="pone.0041923-Minnikin2">[10]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Minnikin3">[11]</xref>
. In <italic>M. tuberculosis,</italic>
the intense hydrophobicity of the outer membrane may contribute to the defences of the pathogen in resisting attack by infected host cells. A wide range of biological activities have been associated with the lipids identified in this study and these have been comprehensively reviewed <xref ref-type="bibr" rid="pone.0041923-Onwueme1">[19]</xref>
–<xref ref-type="bibr" rid="pone.0041923-Neyrolles1">[21]</xref>
. In particular, there is evidence that both MAs and PDIMs are actively exported with a direct influence in generating foamy macrophages and granulomas, contributing to the disease process <xref ref-type="bibr" rid="pone.0041923-Korf1">[22]</xref>
–<xref ref-type="bibr" rid="pone.0041923-AstarieDequeker1">[25]</xref>
. These exported lipids may accumulate in bone matrices, thereby enhancing their preservation and eventual detection. Although the virulence factor activity of these lipids has been demonstrated for modern human tuberculosis, it is likely that the lipids would have a similar role in ancient animal disease.</p>
</sec>
<sec id="s2f"><title>Implications for the Evolution of Tuberculosis</title>
<p>Great strides are being made in the paleogenomics of tuberculosis <xref ref-type="bibr" rid="pone.0041923-Mostowy1">[26]</xref>
–<xref ref-type="bibr" rid="pone.0041923-Gagneux1">[36]</xref>
, particularly in unravelling the complexities of the interrelationships between relatively modern variants responsible for current disease <xref ref-type="bibr" rid="pone.0041923-Wirth1">[30]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Hershberg1">[31]</xref>
. However, the early evolutionary pathways, defining exactly from where and how tuberculosis originated, remain indistinct. The challenge is to chart a pathway from ancestral environmental freely-circulating mycobacterial species to <italic>M. tuberculosis sensu stricto</italic>
, which is an obligate pathogen with no environmental niche. Currently favoured hypotheses all point to an evolutionary bottle-neck, estimated to have been around 35,000 BP <xref ref-type="bibr" rid="pone.0041923-Brosch1">[27]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Gutierrez1">[28]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Djelouadji1">[35]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Sreevatsan1">[37]</xref>
. Subsequent to this time period, the evolution of a range of particular clades follows an almost linear clonar evolutionary pattern, with key deletions leading to the well-defined modern <italic>M. tuberculosis</italic>
complex causing tuberculosis in humans and various animals <xref ref-type="bibr" rid="pone.0041923-Mostowy1">[26]</xref>
–<xref ref-type="bibr" rid="pone.0041923-Gagneux1">[36]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Smith2">[38]</xref>
.</p>
<p>There is increasing evidence that, before reaching the discontinuity of the bottle-neck, extensive horizontal gene transfer (HGT) was taking place in ancestral tuberculosis strains <xref ref-type="bibr" rid="pone.0041923-Brosch1">[27]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Smith2">[38]</xref>
–<xref ref-type="bibr" rid="pone.0041923-Jang1">[41]</xref>
. These pre-bottle-neck ancestral strains, sometimes termed <italic>“M. prototuberculosis”</italic>
<xref ref-type="bibr" rid="pone.0041923-Gutierrez1">[28]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Smith3">[42]</xref>
, have been associated with the “smooth” colony-forming Canetti variants of <italic>M. tuberculosis</italic>
<xref ref-type="bibr" rid="pone.0041923-Gutierrez1">[28]</xref>
, but “<italic>Mycobacterium canettii</italic>
” isolates are not necessarily considered to be living representatives of the progenitor of the <italic>M. tuberculosis</italic>
complex <xref ref-type="bibr" rid="pone.0041923-Smith1">[32]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Smith3">[42]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Fabre1">[43]</xref>
. “<italic>M. canettii</italic>
” strains can be regarded as a heterogeneous “out-group” whose evolution is distinct from other members of the <italic>M. tuberculosis</italic>
complex <xref ref-type="bibr" rid="pone.0041923-Wirth1">[30]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Hershberg1">[31]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Gordon1">[33]</xref>
. “<italic>M. canettii</italic>
” smooth strains continue to be encountered in isolated cases of tuberculosis, but they are usually confined to certain locations in the Horn of Africa <xref ref-type="bibr" rid="pone.0041923-Fabre1">[43]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Koeck1">[44]</xref>
. Inter human transfer is not known, but children and expatriates are more susceptible to infection, suggesting that the indigenous population has acquired significant immunity <xref ref-type="bibr" rid="pone.0041923-Koeck1">[44]</xref>
. The geographical restriction, genetic diversity and specialised nutritional requirements of <italic>“M. canettii”</italic>
isolates strongly favour an environmental reservoir <xref ref-type="bibr" rid="pone.0041923-Koeck1">[44]</xref>
. It is not known if these bacteria can also be part of an animal reservoir in the Horn of Africa.</p>
<p>The positive identification of tuberculosis in this ancient bison skeleton establishes a clear beacon point in the historical record around which to explore an evolutionary scenario for tuberculosis in North America and elsewhere. Clearly recognisable human tuberculosis has not been recorded before 9,000 BP in Eurasia/North Africa <xref ref-type="bibr" rid="pone.0041923-Hershkovitz1">[12]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Nerlich1">[34]</xref>
and 2,100–1,900 BP in the Americas <xref ref-type="bibr" rid="pone.0041923-Wilbur1">[1]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Machowiak1">[2]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Klaus1">[45]</xref>
. In an interesting, but isolated, report, tuberculosis was suggested to be a cause of possible endocranial paleopathology in a fossilized <italic>Homo erectus</italic>
skeleton, dated 500,000 BP <xref ref-type="bibr" rid="pone.0041923-Kappelman1">[46]</xref>
; however, alternative interpretations have been suggested <xref ref-type="bibr" rid="pone.0041923-Roberts1">[47]</xref>
. Apart from this single unconfirmed case <xref ref-type="bibr" rid="pone.0041923-Kappelman1">[46]</xref>
, any clear indications for the presence of tuberculosis in very ancient human remains have not been reported. However, in the animal kingdom there are indications of widespread tuberculosis. In addition to the bison metacarpal, analysed in this study, 19% of 1,002 bovid specimens <xref ref-type="bibr" rid="pone.0041923-Rothschild1">[3]</xref>
and 52% of 113 mastodon bones <xref ref-type="bibr" rid="pone.0041923-Rothschild2">[4]</xref>
had similar lesions indicative of tuberculosis. The age range for the bovids is 125,000 to 8,000 BP <xref ref-type="bibr" rid="pone.0041923-Rothschild1">[3]</xref>
and the mastodon skeletons cover a range from 38,000 to 10,000 BP <xref ref-type="bibr" rid="pone.0041923-Rothschild2">[4]</xref>
. Bone lesions cannot be considered as complete proof of tuberculosis diagnosis, but the dearth of human bones with comparable lesions over the same time period of at least 100,000 years is very striking. This could be a consequence of the hunter-gatherer human population being thinly spread, whereas it may be easier to locate bones from large animal herds. A solution of this conundrum could simply be that <italic>M. tuberculosis</italic>
was principally an animal disease during its early evolution, with transmission to humans occurring later. It has been noted previously <xref ref-type="bibr" rid="pone.0041923-Nerlich1">[34]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Djelouadji1">[35]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Smith3">[42]</xref>
that such a scenario should not be dismissed.</p>
</sec>
<sec id="s2g"><title>Conclusions</title>
<p>The highly sensitive analytical protocols employed have detected key tuberculosis lipid biomarkers in two samples from an extinct bison, lending solid support to previous aDNA conclusions <xref ref-type="bibr" rid="pone.0041923-Rothschild4">[7]</xref>
. Mycolic acids (<xref ref-type="fig" rid="pone-0041923-g001">Figure 1A</xref>
) were indicated (<xref ref-type="fig" rid="pone-0041923-g002">Figs. 2</xref>
and <xref ref-type="fig" rid="pone-0041923-g003">3</xref>
), but the profiles demonstrated degradation and a positive diagnosis of tuberculosis could not be given. This contrasts with the very strong profiles (<xref ref-type="fig" rid="pone-0041923-g004">Figure 4</xref>
) recorded for the mycocerosic acid components (<xref ref-type="fig" rid="pone-0041923-g001">Figure 1B</xref>
) of the phthiocerol dimycocerosate (PDIM) waxes and, remarkably, for the mycolipenic acid (<xref ref-type="fig" rid="pone-0041923-g001">Figure 1B</xref>
) component of pentaacyl trehaloses (PATs). Profiles (<xref ref-type="fig" rid="pone-0041923-g005">Figure 5</xref>
) for components of the phthiocerol family (<xref ref-type="fig" rid="pone-0041923-g001">Figure 1C</xref>
) suggested that intact PDIMs may have been present.</p>
<p>Conclusive evidence has been provided for the presence of tuberculosis in this ancient bison by detection of these lipid virulence factors. It has also been confirmed that tuberculosis may be identified in absence of macroscopically recognizable bone lesions and defects. Analysis of ancient DNA provides the most informative way to trace the evolution of tuberculosis, but the developing portfolio of diagnostic lipids, illustrated here, offers alternative routes to chart these evolutionary processes. The confirmation of tuberculosis in this exceptionally old 17,000 BP extinct bison and the current absence of any proven human tuberculosis older than 9,000 BP demands exploration of a hypothesis that tuberculosis may have originated and become established as a widespread zoonosis. Many, many more samples of potentially tuberculosis infected human and animal bones are urgently needed for analysis to support or disprove this or any other viable hypothesis.</p>
</sec>
</sec>
<sec sec-type="materials|methods" id="s3"><title>Materials and Methods</title>
<sec id="s3a"><title>Samples</title>
<p>The material, as used for the previous ancient tuberculosis DNA analyses <xref ref-type="bibr" rid="pone.0041923-Rothschild4">[7]</xref>
, was from an extinct bison (<italic>Bison antiquus</italic>
) buried in sediments, dated to 17,870±230 years BP, in Natural Trap Cave (Wyoming). The first sample (“Bison 1”; 13.5 mg) originated from the undermined articular surface of a metacarpal but the second sample (“Bison 2”; 13.0 mg) was from a site on the same bone remote from this lesion <xref ref-type="bibr" rid="pone.0041923-Rothschild4">[7]</xref>
. The metacarpal bone was stored in a dry sterile environment to avoid any possibility of external contamination. In the prevailing conditions of Natural Trap Cave, the possibility of external <italic>post mortem</italic>
infection with <italic>M. tuberculosis</italic>
is most unlikely. <italic>M. tuberculosis</italic>
H37Rv was used to prepare standard profiles. Stringent precautions were taken against sample carry-over during the analyses. Essentially, this amounted to using new disposables for every analysis and running blanks between samples.</p>
</sec>
<sec id="s3b"><title>Lipid Extraction</title>
<p>Samples were hydrolysed by heating with 30% potassium hydroxide in methanol (2 ml) and toluene (1 ml) at 100°C overnight <xref ref-type="bibr" rid="pone.0041923-Hershkovitz1">[12]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Redman1">[13]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Donoghue1">[48]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Taylor1">[49]</xref>
. Long-chain compounds were extracted by a modification of a published method <xref ref-type="bibr" rid="pone.0041923-Redman1">[13]</xref>
, substituting dichloromethane with toluene <xref ref-type="bibr" rid="pone.0041923-Donoghue1">[48]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Taylor1">[49]</xref>
to ensure efficient extraction of the phthiocerols. The extract was treated with pentafluorobenzyl bromide, under phase-transfer conditions <xref ref-type="bibr" rid="pone.0041923-Hershkovitz1">[12]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Redman1">[13]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Donoghue1">[48]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Taylor1">[49]</xref>
, to convert acidic components into pentafluorobenzyl (PFB) esters. Subsequent fractionation on an Alltech 209250 (500 mg) normal phase silica gel cartridge <xref ref-type="bibr" rid="pone.0041923-Hershkovitz1">[12]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Redman1">[13]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Donoghue1">[48]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Taylor1">[49]</xref>
gave fractions containing non-hydroxylated PFB esters, MA PFB esters and underivatized phthiocerols.</p>
</sec>
<sec id="s3c"><title>Mycolic Acid Analysis</title>
<p>The MA PFB esters were reacted with pyrenebutyric acid (PBA) to produce PBA-PFB MA derivatives, which were purified on an Alltech 205250 (500 mg) C<sub>18</sub>
reverse phase cartridge <xref ref-type="bibr" rid="pone.0041923-Hershkovitz1">[12]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Donoghue1">[48]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Taylor1">[49]</xref>
. The PBA-PFB mycolates were analysed by sequential reverse and normal phase HPLC, as described previously <xref ref-type="bibr" rid="pone.0041923-Hershkovitz1">[12]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Donoghue1">[48]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Taylor1">[49]</xref>
.</p>
</sec>
<sec id="s3d"><title>Mycocerosic and Mycolipenic Acid Analysis</title>
<p>In a simplification of a previous protocol, which involved normal phase HPLC pre-purification <xref ref-type="bibr" rid="pone.0041923-Redman1">[13]</xref>
, the non-hydroxylated PFB ester fraction was fractionated on an Alltech 205250 (500 mg) reverse phase silica gel cartridge, using a water-methanol/methanol/methanol-toluene elution sequence (<bold><xref ref-type="supplementary-material" rid="pone.0041923.s002">Figure S2</xref>
</bold>
). A fraction enriched in mycocerosic acid and other longer chain (>C<sub>20</sub>
) PFB esters was eluted by 100% methanol with the more usual C<sub>12</sub>
to C<sub>20</sub>
esters eluting in the earlier water/methanol fractions. The fraction containing possible mycocerosates and mycolipenates, was analysed by negative ion chemical ionization gas chromatography mass spectrometry (NICI-GCMS), as previously described <xref ref-type="bibr" rid="pone.0041923-Redman1">[13]</xref>
(<bold><xref ref-type="supplementary-material" rid="pone.0041923.s001">Figure S1</xref>
</bold>
). PFB esters, on NICI-GCMS, fragment to produce negative carboxylate [M – H]<sup>-</sup>
ions, which can be detected at high sensitivity. Selected ion monitoring (SIM) was used to search for mycocerosate carboxylate ions at <italic>m/z</italic>
367.6311 (C<sub>24</sub>
), 395.6844 (C<sub>26</sub>
), 409.7111 (C<sub>27</sub>
), 437.7645 (C<sub>29</sub>
), 451.7911 (C<sub>30</sub>
), 479.8445 (C<sub>32</sub>
), 493.8712 (C<sub>33</sub>
) and 507.8978 (C<sub>34</sub>
). Additionally, <italic>m/z</italic>
407.6952 was monitored for the presence of the C<sub>27</sub>
mycolipenate carboxylate ion. Partial racemisation of mycocerosates during the alkaline hydrolysis leads to the formation of diasteroisomers, which resolve on gas chromatography to give characteristic doublets; in contrast, mycolipenates are singlets as they cannot racemise <xref ref-type="bibr" rid="pone.0041923-Redman1">[13]</xref>
.</p>
</sec>
<sec id="s3e"><title>Phthiocerol Family Analysis</title>
<p>In a new procedure, the phthiocerol fraction was converted to PBA esters by reaction with pyrenebutyric acid, under the same conditions used to derivatize MA PFB esters <xref ref-type="bibr" rid="pone.0041923-Hershkovitz1">[12]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Donoghue1">[48]</xref>
, <xref ref-type="bibr" rid="pone.0041923-Taylor1">[49]</xref>
. The crude phthiocerol di-PBA esters were purified by an Alltech 205250 (500 mg) C<sub>18</sub>
reverse phase cartridge, utilizing combinations of water, acetonitrile and dichloromethane; the PBA phthiocerols eluted in 100% acetonitrile and acetonitrile/dichloromethane 54∶6 and 48∶12 fractions (<bold><xref ref-type="supplementary-material" rid="pone.0041923.s003">Figure S3</xref>
</bold>
). Reverse phase HPLC was performed on an Alltech 81412 Alltima C<sub>18</sub>
column (3 µ, 4.6 mm×50 mm) column in a VWR Hitachi Elite LaChrom HPLC linked to an L-2480 fluorescence detector, utilizing a gradient of acetonitrile/tetrahydrofuran, from 85∶15 to 60∶40 in 30 min (<bold><xref ref-type="supplementary-material" rid="pone.0041923.s004">Figure S4</xref>
</bold>
). The fraction, corresponding to derivatives of members of the phthiocerol family, was collected and analyzed by HPLC on normal phase columns (Alltech 81414 Alltima Silica, 3 µm 50×4.6 mm). Eluting with heptane/ethyl acetate 99∶1 for 1 min, was followed by gradient of heptane/ethyl acetate 99∶1 to 91∶9 over 30 mins (<bold><xref ref-type="supplementary-material" rid="pone.0041923.s004">Figure S4</xref>
</bold>
).</p>
</sec>
</sec>
<sec sec-type="supplementary-material" id="s4"><title>Supporting Information</title>
<supplementary-material content-type="local-data" id="pone.0041923.s001"><label>Figure S1</label>
<caption><p><bold>Complete results of selected ion monitoring (SIM) negative ion-chemical ionisation gas chromatography-mass spectrometry (NICI-GCMS) analysis of pentafluorobenzyl (PFB) ester fractions, corresponding to mycocerosates and mycolipenates.</bold>
</p>
<p>(DOC)</p>
</caption>
<media xlink:href="pone.0041923.s001.doc" mimetype="application" mime-subtype="msword"><caption><p>Click here for additional data file.</p>
</caption>
</media>
</supplementary-material>
<supplementary-material content-type="local-data" id="pone.0041923.s002"><label>Figure S2</label>
<caption><p><bold>Solvent system for the purification of pentafluorobenzyl (PFB) mycocerosates on C<sub>18</sub>
reverse phase cartridges.</bold>
</p>
<p>(DOC)</p>
</caption>
<media xlink:href="pone.0041923.s002.doc" mimetype="application" mime-subtype="msword"><caption><p>Click here for additional data file.</p>
</caption>
</media>
</supplementary-material>
<supplementary-material content-type="local-data" id="pone.0041923.s003"><label>Figure S3</label>
<caption><p><bold>Solvent system for the purification of pyrenebutyric acid (PBA) derivatives of members of the phthiocerol family on C<sub>18</sub>
reverse phase cartridges.</bold>
</p>
<p>(DOC)</p>
</caption>
<media xlink:href="pone.0041923.s003.doc" mimetype="application" mime-subtype="msword"><caption><p>Click here for additional data file.</p>
</caption>
</media>
</supplementary-material>
<supplementary-material content-type="local-data" id="pone.0041923.s004"><label>Figure S4</label>
<caption><p><bold>HPLC conditions for analysis of pyrenebutyric acid (PBA) derivatives of members of the phthiocerol family.</bold>
</p>
<p>(DOC)</p>
</caption>
<media xlink:href="pone.0041923.s004.doc" mimetype="application" mime-subtype="msword"><caption><p>Click here for additional data file.</p>
</caption>
</media>
</supplementary-material>
</sec>
</body>
<back><ack><p>A Leverhulme Trust Emeritus Fellowship to DEM is acknowledged. GSB has a James Bardrick Personal Research Chair and a Royal Society Wolfson Research Merit Award.</p>
</ack>
<ref-list><title>References</title>
<ref id="pone.0041923-Wilbur1"><label>1</label>
<mixed-citation publication-type="other">Wilbur AK, Buikstra JE (2006) Patterns of tuberculosis in the Americas – How can modern biomedicine inform the ancient past? Mem Inst Oswaldo Cruz Rio de Janeiro 101(Suppl. II). 59–66.</mixed-citation>
</ref>
<ref id="pone.0041923-Machowiak1"><label>2</label>
<mixed-citation publication-type="journal"><name><surname>Machowiak</surname>
<given-names>PA</given-names>
</name>
, <name><surname>Blos</surname>
<given-names>VT</given-names>
</name>
, <name><surname>Aguilar</surname>
<given-names>M</given-names>
</name>
, <name><surname>Buikstra</surname>
<given-names>JE</given-names>
</name>
(<year>2005</year>
) <article-title>On the origin of American tuberculosis</article-title>
. <source>Clin Infect Dis</source>
<volume>41</volume>
: <fpage>515</fpage>
–<lpage>518</lpage>
<pub-id pub-id-type="pmid">16028161</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Rothschild1"><label>3</label>
<mixed-citation publication-type="journal"><name><surname>Rothschild</surname>
<given-names>BM</given-names>
</name>
, <name><surname>Martin</surname>
<given-names>LD</given-names>
</name>
(<year>2006</year>
) <article-title>Did ice-age bovids spread tuberculosis</article-title>
? <source>Naturwissenschaften</source>
<volume>93</volume>
: <fpage>565</fpage>
–<lpage>569</lpage>
<pub-id pub-id-type="pmid">16896974</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Rothschild2"><label>4</label>
<mixed-citation publication-type="journal"><name><surname>Rothschild</surname>
<given-names>BM</given-names>
</name>
, <name><surname>Laub</surname>
<given-names>R</given-names>
</name>
(<year>2006</year>
) <article-title>Hyperdisease in the late Pleistocene: validation of an early 20th century hypothesis</article-title>
. <source>Naturwissenschaften</source>
<volume>93</volume>
: <fpage>557</fpage>
–<lpage>564</lpage>
<pub-id pub-id-type="pmid">16953418</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Martin1"><label>5</label>
<mixed-citation publication-type="journal"><name><surname>Martin</surname>
<given-names>LD</given-names>
</name>
, <name><surname>Rothschild</surname>
<given-names>BM</given-names>
</name>
(<year>1989</year>
) <article-title>Frequency of pathology in a large natural sample from Natural Trap Cave (late Pleistocene)</article-title>
. <source>J Vertebr Paleontol</source>
<volume>9</volume>
: <fpage>31A</fpage>
</mixed-citation>
</ref>
<ref id="pone.0041923-Rothschild3"><label>6</label>
<mixed-citation publication-type="journal"><name><surname>Rothschild</surname>
<given-names>BM</given-names>
</name>
, <name><surname>Martin</surname>
<given-names>LD</given-names>
</name>
(<year>2003</year>
) <article-title>Frequency of pathology in a large natural sample from Natural Trap Cave with special remarks on erosive disease in the Pleistocene</article-title>
. <source>Rheumatismo</source>
<volume>55</volume>
: <fpage>58</fpage>
–<lpage>65</lpage>
</mixed-citation>
</ref>
<ref id="pone.0041923-Rothschild4"><label>7</label>
<mixed-citation publication-type="journal"><name><surname>Rothschild</surname>
<given-names>BM</given-names>
</name>
, <name><surname>Martin</surname>
<given-names>LD</given-names>
</name>
, <name><surname>Lev</surname>
<given-names>G</given-names>
</name>
, <name><surname>Bercovier</surname>
<given-names>H</given-names>
</name>
, <name><surname>Bar-Gal</surname>
<given-names>GK</given-names>
</name>
, <etal>et al</etal>
(<year>2001</year>
) <article-title><italic>Mycobacterium tuberculosis</italic>
-complex DNA from an extinct bison dated 17,000 years</article-title>
. <source>Clin Infect Dis</source>
<volume>33</volume>
: <fpage>305</fpage>
–<lpage>311</lpage>
<pub-id pub-id-type="pmid">11438894</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Tran1"><label>8</label>
<mixed-citation publication-type="journal"><name><surname>Tran</surname>
<given-names>T-N-N</given-names>
</name>
, <name><surname>Aboudharam</surname>
<given-names>G</given-names>
</name>
, <name><surname>Raoult</surname>
<given-names>D</given-names>
</name>
, <name><surname>Drancourt</surname>
<given-names>M</given-names>
</name>
(<year>2011</year>
) <article-title>Beyond ancient microbial DNA: nonnucleotidic biomolecules for paleomicrobiology</article-title>
. <source>BioTechniques</source>
<volume>50</volume>
: <fpage>370</fpage>
–<lpage>380</lpage>
<pub-id pub-id-type="pmid">21781037</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Minnikin1"><label>9</label>
<mixed-citation publication-type="other">Minnikin DE, Lee OY-C, Wu HHT, Besra GS, Donoghue HD (2012) Molecular biomarkers for ancient tuberculosis. In: Cardona P-J, editor. Understanding Tuberculosis – Deciphering the Secret Life of the Bacilli. Rijeka, Croatia: InTech - Open Access Publisher. 1–36. http://www.intechopen.com/books/understanding-tuberculosis-deciphering-the-secret-life-of-the-bacilli ISBN-13: 978–953–307–946–2.</mixed-citation>
</ref>
<ref id="pone.0041923-Minnikin2"><label>10</label>
<mixed-citation publication-type="book">Minnikin DE (1982) Lipids: Complex lipids, their chemistry, biosynthesis and role. In: Ratledge C, Stanford J, editors. The Biology of the Mycobacteria. London: Academic Press. 95–184.</mixed-citation>
</ref>
<ref id="pone.0041923-Minnikin3"><label>11</label>
<mixed-citation publication-type="journal"><name><surname>Minnikin</surname>
<given-names>DE</given-names>
</name>
, <name><surname>Kremer</surname>
<given-names>L</given-names>
</name>
, <name><surname>Dover</surname>
<given-names>LG</given-names>
</name>
, <name><surname>Besra</surname>
<given-names>GS</given-names>
</name>
(<year>2002</year>
) <article-title>The methyl-branched fortifications of <italic>Mycobacterium tuberculosis.</italic>
</article-title>
. <source>Chem Biol</source>
<volume>9</volume>
: <fpage>545</fpage>
–<lpage>553</lpage>
<pub-id pub-id-type="pmid">12031661</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Hershkovitz1"><label>12</label>
<mixed-citation publication-type="journal"><name><surname>Hershkovitz</surname>
<given-names>I</given-names>
</name>
, <name><surname>Donoghue</surname>
<given-names>HD</given-names>
</name>
, <name><surname>Minnikin</surname>
<given-names>DE</given-names>
</name>
, <name><surname>Besra</surname>
<given-names>GS</given-names>
</name>
, <name><surname>Lee</surname>
<given-names>OY-C</given-names>
</name>
, <etal>et al</etal>
(<year>2008</year>
) <article-title>Detection and molecular characterization of 9000-year-old <italic>Mycobacterium tuberculosis</italic>
from a neolithic settlement in the eastern Mediterranean</article-title>
. <source>PLoS ONE</source>
<volume>3</volume>
: <fpage>e3426</fpage>
<pub-id pub-id-type="pmid">18923677</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Redman1"><label>13</label>
<mixed-citation publication-type="journal"><name><surname>Redman</surname>
<given-names>JE</given-names>
</name>
, <name><surname>Shaw</surname>
<given-names>MJ</given-names>
</name>
, <name><surname>Mallet</surname>
<given-names>AI</given-names>
</name>
, <name><surname>Santos</surname>
<given-names>AL</given-names>
</name>
, <name><surname>Roberts</surname>
<given-names>CA</given-names>
</name>
, <etal>et al</etal>
(<year>2009</year>
) <article-title>Mycocerosic acid biomarkers for the diagnosis of tuberculosis in the Coimbra Skeletal Collection</article-title>
. <source>Tuberculosis</source>
<volume>89</volume>
: <fpage>267</fpage>
–<lpage>277</lpage>
<pub-id pub-id-type="pmid">19493698</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Minnikin4"><label>14</label>
<mixed-citation publication-type="journal"><name><surname>Minnikin</surname>
<given-names>DE</given-names>
</name>
, <name><surname>Dobson</surname>
<given-names>G</given-names>
</name>
, <name><surname>Goodfellow</surname>
<given-names>M</given-names>
</name>
, <name><surname>Magnusson</surname>
<given-names>M</given-names>
</name>
, <name><surname>Ridell</surname>
<given-names>M</given-names>
</name>
(<year>1985</year>
) <article-title>Distribution of some mycobacterial waxes based on the phthiocerol family</article-title>
. <source>J Gen Microbiol</source>
<volume>131</volume>
: <fpage>1375</fpage>
–<lpage>1381</lpage>
<pub-id pub-id-type="pmid">3900276</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Minnikin5"><label>15</label>
<mixed-citation publication-type="journal"><name><surname>Minnikin</surname>
<given-names>DE</given-names>
</name>
, <name><surname>Dobson</surname>
<given-names>G</given-names>
</name>
, <name><surname>Hutchinson</surname>
<given-names>IG</given-names>
</name>
(<year>1983</year>
) <article-title>Characterization of phthiocerol dimycocerosates from <italic>Mycobacterium tuberculosis.</italic>
</article-title>
. <source>Biochim Biophys Acta</source>
<volume>753</volume>
: <fpage>445</fpage>
–<lpage>449</lpage>
</mixed-citation>
</ref>
<ref id="pone.0041923-Minnikin6"><label>16</label>
<mixed-citation publication-type="journal"><name><surname>Minnikin</surname>
<given-names>DE</given-names>
</name>
, <name><surname>Dobson</surname>
<given-names>G</given-names>
</name>
, <name><surname>Sesardic</surname>
<given-names>D</given-names>
</name>
, <name><surname>Ridell</surname>
<given-names>M</given-names>
</name>
(<year>1985</year>
) <article-title>Mycolipenates and mycolipanolates of trehalose from <italic>Mycobacterium tuberculosis.</italic>
</article-title>
. <source>J Gen Microbiol</source>
<volume>131</volume>
: <fpage>1369</fpage>
–<lpage>1374</lpage>
<pub-id pub-id-type="pmid">3930656</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Daff1"><label>17</label>
<mixed-citation publication-type="journal"><name><surname>Daffé</surname>
<given-names>M</given-names>
</name>
, <name><surname>Lacave</surname>
<given-names>C</given-names>
</name>
, <name><surname>Lanéelle</surname>
<given-names>M-A</given-names>
</name>
, <name><surname>Gillois</surname>
<given-names>M</given-names>
</name>
, <name><surname>Lanéelle</surname>
<given-names>G</given-names>
</name>
(<year>1988</year>
) <article-title>Polyphthienoyl trehalose, glycolipids specific for virulent strains of the tubercle bacillus</article-title>
. <source>Eur J Biochem</source>
<volume>172</volume>
: <fpage>579</fpage>
–<lpage>584</lpage>
<pub-id pub-id-type="pmid">3127210</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Besra1"><label>18</label>
<mixed-citation publication-type="journal"><name><surname>Besra</surname>
<given-names>GS</given-names>
</name>
, <name><surname>Bolton</surname>
<given-names>RC</given-names>
</name>
, <name><surname>McNeil</surname>
<given-names>MR</given-names>
</name>
, <name><surname>Ridell</surname>
<given-names>M</given-names>
</name>
, <name><surname>Simpson</surname>
<given-names>KE</given-names>
</name>
, <etal>et al</etal>
(<year>1992</year>
) <article-title>Structural elucidation of a novel family of acyltrehaloses from <italic>Mycobacterium tuberculosis.</italic>
</article-title>
. <source>Biochemistry</source>
<volume>31</volume>
: <fpage>9832</fpage>
–<lpage>9837</lpage>
<pub-id pub-id-type="pmid">1390757</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Onwueme1"><label>19</label>
<mixed-citation publication-type="journal"><name><surname>Onwueme</surname>
<given-names>KC</given-names>
</name>
, <name><surname>Vos</surname>
<given-names>CJ</given-names>
</name>
, <name><surname>Zurita</surname>
<given-names>J</given-names>
</name>
, <name><surname>Ferreras</surname>
<given-names>JA</given-names>
</name>
, <name><surname>Quadri</surname>
<given-names>LEN</given-names>
</name>
(<year>2005</year>
) <article-title>The dimycocerosate ester polyketide virulence factors of mycobacteria</article-title>
. <source>Prog Lipid Res</source>
<volume>44</volume>
: <fpage>259</fpage>
–<lpage>302</lpage>
<pub-id pub-id-type="pmid">16115688</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Jackson1"><label>20</label>
<mixed-citation publication-type="journal"><name><surname>Jackson</surname>
<given-names>M</given-names>
</name>
, <name><surname>Stadhagen</surname>
<given-names>G</given-names>
</name>
, <name><surname>Gicquel</surname>
<given-names>B</given-names>
</name>
(<year>2007</year>
) <article-title>Long-chain multiple methyl-branched fatty acid-containing lipids of <italic>Mycobacterium tuberculosis</italic>
: Biosynthesis, transport, regulation and biological activities</article-title>
. <source>Tuberculosis</source>
<volume>87</volume>
: <fpage>78</fpage>
–<lpage>86</lpage>
<pub-id pub-id-type="pmid">17030019</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Neyrolles1"><label>21</label>
<mixed-citation publication-type="journal"><name><surname>Neyrolles</surname>
<given-names>O</given-names>
</name>
, <name><surname>Guilhot</surname>
<given-names>C</given-names>
</name>
(<year>2011</year>
) <article-title>Recent advances in deciphering the contribution of <italic>Mycobacterium tuberculosis</italic>
lipids to pathogenesis</article-title>
. <source>Tuberculosis</source>
<volume>91</volume>
: <fpage>187</fpage>
–<lpage>195</lpage>
<pub-id pub-id-type="pmid">21330212</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Korf1"><label>22</label>
<mixed-citation publication-type="journal"><name><surname>Korf</surname>
<given-names>J</given-names>
</name>
, <name><surname>Stoltz</surname>
<given-names>A</given-names>
</name>
, <name><surname>Verschoor</surname>
<given-names>J</given-names>
</name>
, <name><surname>De Baetselier</surname>
<given-names>P</given-names>
</name>
, <name><surname>Grooten</surname>
<given-names>J</given-names>
</name>
(<year>2005</year>
) <article-title>The <italic>Mycobacterium tuberculosis</italic>
cell wall component mycolic acid elicits pathogen-associated host innate immune responses</article-title>
. <source>Eur J Immunol</source>
<volume>35</volume>
: <fpage>890</fpage>
–<lpage>900</lpage>
<pub-id pub-id-type="pmid">15724242</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Peyron1"><label>23</label>
<mixed-citation publication-type="journal"><name><surname>Peyron</surname>
<given-names>P</given-names>
</name>
, <name><surname>Vaubourgeix</surname>
<given-names>J</given-names>
</name>
, <name><surname>Poquet</surname>
<given-names>Y</given-names>
</name>
, <name><surname>Levillain</surname>
<given-names>F</given-names>
</name>
, <name><surname>Botanch</surname>
<given-names>C</given-names>
</name>
, <etal>et al</etal>
(<year>2008</year>
) <article-title>Foamy macrophages from tuberculous patients’ granulomas constitute a nutrient-rich reservoir for <italic>M. tuberculosis</italic>
persistence</article-title>
. <source>PLoS Pathog</source>
<volume>4</volume>
: <fpage>e1000204</fpage>
<pub-id pub-id-type="pmid">19002241</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Domenech1"><label>24</label>
<mixed-citation publication-type="journal"><name><surname>Domenech</surname>
<given-names>P</given-names>
</name>
, <name><surname>Reed</surname>
<given-names>MB</given-names>
</name>
(<year>2009</year>
) <article-title>Rapid and spontaneous loss of phthiocerol dimycocerosate (PDIM) from <italic>Mycobacterium tuberculosis</italic>
grown <italic>in vitro</italic>
: implications for virulence studies</article-title>
. <source>Microbiology</source>
<volume>155</volume>
: <fpage>3532</fpage>
–<lpage>3543</lpage>
<pub-id pub-id-type="pmid">19661177</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-AstarieDequeker1"><label>25</label>
<mixed-citation publication-type="journal"><name><surname>Astarie-Dequeker</surname>
<given-names>C</given-names>
</name>
, <name><surname>Le Guyader</surname>
<given-names>L</given-names>
</name>
, <name><surname>Malaga</surname>
<given-names>W</given-names>
</name>
, <name><surname>Seaphanh</surname>
<given-names>F-K</given-names>
</name>
, <name><surname>Chalut</surname>
<given-names>C</given-names>
</name>
, <etal>et al</etal>
(<year>2009</year>
) <article-title>Phthiocerol dimycocerosates of <italic>M. tuberculosis</italic>
participate in macrophage invasion by inducing changes in the organization of plasma membrane lipids</article-title>
. <source>PLoS Pathog</source>
<volume>5</volume>
: <fpage>e1000289</fpage>
<pub-id pub-id-type="pmid">19197369</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Mostowy1"><label>26</label>
<mixed-citation publication-type="journal"><name><surname>Mostowy</surname>
<given-names>S</given-names>
</name>
, <name><surname>Cousins</surname>
<given-names>D</given-names>
</name>
, <name><surname>Brinkman</surname>
<given-names>J</given-names>
</name>
, <name><surname>Aranaz</surname>
<given-names>A</given-names>
</name>
, <name><surname>Behr</surname>
<given-names>MA</given-names>
</name>
(<year>2002</year>
) <article-title>Genomic deletions suggest a phylogeny for the <italic>Mycobacterium tuberculosis</italic>
complex</article-title>
. <source>J Infect Dis</source>
<volume>186</volume>
: <fpage>74</fpage>
–<lpage>80</lpage>
<pub-id pub-id-type="pmid">12089664</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Brosch1"><label>27</label>
<mixed-citation publication-type="journal"><name><surname>Brosch</surname>
<given-names>R</given-names>
</name>
, <name><surname>Gordon</surname>
<given-names>SV</given-names>
</name>
, <name><surname>Marmiesse</surname>
<given-names>M</given-names>
</name>
, <name><surname>Brodin</surname>
<given-names>P</given-names>
</name>
, <name><surname>Buchrieser</surname>
<given-names>C</given-names>
</name>
, <etal>et al</etal>
(<year>2002</year>
) <article-title>A new evolutionary scenario for the <italic>Mycobacterium tuberculosis</italic>
complex</article-title>
. <source>Proc Natl Acad Sci USA</source>
<volume>99</volume>
: <fpage>3684</fpage>
–<lpage>3689</lpage>
<pub-id pub-id-type="pmid">11891304</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Gutierrez1"><label>28</label>
<mixed-citation publication-type="journal"><name><surname>Gutierrez</surname>
<given-names>MC</given-names>
</name>
, <name><surname>Brisse</surname>
<given-names>S</given-names>
</name>
, <name><surname>Brosch</surname>
<given-names>R</given-names>
</name>
, <name><surname>Fabre</surname>
<given-names>M</given-names>
</name>
, <name><surname>Omaïs</surname>
<given-names>B</given-names>
</name>
, <etal>et al</etal>
(<year>2005</year>
) <article-title>Ancient origin and gene mosaicism of the progenitor of <italic>Mycobacterium tuberculosis</italic>
</article-title>
. <source>PLoS Pathog</source>
<volume>1</volume>
: <fpage>e5</fpage>
<pub-id pub-id-type="pmid">16201017</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Arnold1"><label>29</label>
<mixed-citation publication-type="journal"><name><surname>Arnold</surname>
<given-names>C</given-names>
</name>
, <name><surname>Thorne</surname>
<given-names>N</given-names>
</name>
, <name><surname>Underwood</surname>
<given-names>A</given-names>
</name>
, <name><surname>Baster</surname>
<given-names>K</given-names>
</name>
, <name><surname>Gharbia</surname>
<given-names>S</given-names>
</name>
(<year>2006</year>
) <article-title>Evolution of short sequence repeats in <italic>Mycobacterium tuberculosis.</italic>
</article-title>
. <source>FEMS Microbiol Lett</source>
<volume>256</volume>
: <fpage>340</fpage>
–<lpage>346</lpage>
<pub-id pub-id-type="pmid">16499626</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Wirth1"><label>30</label>
<mixed-citation publication-type="journal"><name><surname>Wirth</surname>
<given-names>T</given-names>
</name>
, <name><surname>Hildebrand</surname>
<given-names>F</given-names>
</name>
, <name><surname>Allix-Béguec</surname>
<given-names>C</given-names>
</name>
, <name><surname>Wölbeling</surname>
<given-names>F</given-names>
</name>
, <name><surname>Kubica</surname>
<given-names>T</given-names>
</name>
, <etal>et al</etal>
(<year>2008</year>
) <article-title>Origin, spread and demography of the <italic>Mycobacterium tuberculosis</italic>
complex</article-title>
. <source>PLoS Pathog</source>
<volume>4</volume>
: <fpage>e1000160</fpage>
<pub-id pub-id-type="pmid">18802459</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Hershberg1"><label>31</label>
<mixed-citation publication-type="journal"><name><surname>Hershberg</surname>
<given-names>R</given-names>
</name>
, <name><surname>Lipatov</surname>
<given-names>M</given-names>
</name>
, <name><surname>Small</surname>
<given-names>PM</given-names>
</name>
, <name><surname>Sheffer</surname>
<given-names>H</given-names>
</name>
, <name><surname>Niemann</surname>
<given-names>S</given-names>
</name>
, <etal>et al</etal>
(<year>2008</year>
) <article-title>High functional diversity in <italic>Mycobacterium tuberculosis</italic>
driven by genetic drift and human demography</article-title>
. <source>PLoS Biol</source>
<volume>6</volume>
: <fpage>e311</fpage>
<pub-id pub-id-type="pmid">19090620</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Smith1"><label>32</label>
<mixed-citation publication-type="journal"><name><surname>Smith</surname>
<given-names>NH</given-names>
</name>
, <name><surname>Hewinson</surname>
<given-names>RG</given-names>
</name>
, <name><surname>Kremer</surname>
<given-names>K</given-names>
</name>
, <name><surname>Brosch</surname>
<given-names>R</given-names>
</name>
, <name><surname>Gordon</surname>
<given-names>SV</given-names>
</name>
(<year>2009</year>
) <article-title>Myths and misconceptions: the origin and evolution of <italic>Mycobacterium tuberculosis</italic>
</article-title>
. <source>Nat Rev Microbiol</source>
<volume>7</volume>
: <fpage>537</fpage>
–<lpage>544</lpage>
<pub-id pub-id-type="pmid">19483712</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Gordon1"><label>33</label>
<mixed-citation publication-type="journal"><name><surname>Gordon</surname>
<given-names>SV</given-names>
</name>
, <name><surname>Bottai</surname>
<given-names>D</given-names>
</name>
, <name><surname>Simeone</surname>
<given-names>R</given-names>
</name>
, <name><surname>Stinear</surname>
<given-names>TP</given-names>
</name>
, <name><surname>Brosch</surname>
<given-names>R</given-names>
</name>
(<year>2009</year>
) <article-title>Pathogenicity in the tubercle bacillus: molecular and evolutionary determinants</article-title>
. <source>BioEssays</source>
<volume>31</volume>
: <fpage>378</fpage>
–<lpage>388</lpage>
<pub-id pub-id-type="pmid">19274661</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Nerlich1"><label>34</label>
<mixed-citation publication-type="other">Nerlich AG, Lösch S (2009) Paleopathology of human tuberculosis and the potential role of climate. Interdisc Perspect Infect Dis Volume 2009, Article ID 437187. doi:10.1155/2009/437187.</mixed-citation>
</ref>
<ref id="pone.0041923-Djelouadji1"><label>35</label>
<mixed-citation publication-type="journal"><name><surname>Djelouadji</surname>
<given-names>Z</given-names>
</name>
, <name><surname>Raoult</surname>
<given-names>D</given-names>
</name>
, <name><surname>Drancourt</surname>
<given-names>M</given-names>
</name>
(<year>2011</year>
) <article-title>Palaeogenomics of <italic>Mycobacterium tuberculosis</italic>
: epidemic bursts with a degrading genome</article-title>
. <source>Lancet Infect Dis</source>
<volume>11</volume>
: <fpage>641</fpage>
–<lpage>650</lpage>
<pub-id pub-id-type="pmid">21672667</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Gagneux1"><label>36</label>
<mixed-citation publication-type="journal"><name><surname>Gagneux</surname>
<given-names>S</given-names>
</name>
(<year>2012</year>
) <article-title>Host-pathogen coevolution in human tuberculosis</article-title>
. <source>Phil Trans Roy Soc B</source>
<volume>367</volume>
: <fpage>850</fpage>
–<lpage>859</lpage>
<pub-id pub-id-type="pmid">22312052</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Sreevatsan1"><label>37</label>
<mixed-citation publication-type="journal"><name><surname>Sreevatsan</surname>
<given-names>S</given-names>
</name>
, <name><surname>Pan</surname>
<given-names>X</given-names>
</name>
, <name><surname>Stockbauer</surname>
<given-names>KE</given-names>
</name>
, <name><surname>Connell</surname>
<given-names>ND</given-names>
</name>
, <name><surname>Kreiswirth</surname>
<given-names>BN</given-names>
</name>
, <etal>et al</etal>
(<year>1997</year>
) <article-title>Restricted structural gene polymorphism in the <italic>Mycobacterium tuberculosis</italic>
complex indicates evolutionarily recent global dissemination</article-title>
. <source>Proc Natl Acad Sci USA</source>
<volume>94</volume>
: <fpage>9869</fpage>
–<lpage>9874</lpage>
<pub-id pub-id-type="pmid">9275218</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Smith2"><label>38</label>
<mixed-citation publication-type="journal"><name><surname>Smith</surname>
<given-names>NH</given-names>
</name>
, <name><surname>Gordon</surname>
<given-names>SV</given-names>
</name>
, <name><surname>de la Rua-Domenech</surname>
<given-names>R</given-names>
</name>
, <name><surname>Clifton-Hadley</surname>
<given-names>RS</given-names>
</name>
, <name><surname>Hewinson</surname>
<given-names>RG</given-names>
</name>
(<year>2006</year>
) <article-title>Bottlenecks and broomsticks: the molecular evolution of <italic>Mycobacterium bovis</italic>
</article-title>
. <source>Nat Rev Microbiol</source>
<volume>4</volume>
: <fpage>670</fpage>
–<lpage>681</lpage>
<pub-id pub-id-type="pmid">16912712</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-RosasMagallanes1"><label>39</label>
<mixed-citation publication-type="journal"><name><surname>Rosas-Magallanes</surname>
<given-names>V</given-names>
</name>
, <name><surname>Deschavanne</surname>
<given-names>P</given-names>
</name>
, <name><surname>Quintana-Murci</surname>
<given-names>L</given-names>
</name>
, <name><surname>Brosch</surname>
<given-names>R</given-names>
</name>
, <name><surname>Gicquel</surname>
<given-names>B</given-names>
</name>
, <etal>et al</etal>
(<year>2006</year>
) <article-title>Horizontal transfer of a virulence operon to the ancestor of <italic>Mycobacterium tuberculosis.</italic>
</article-title>
. <source>Mol Biol Evol</source>
<volume>23</volume>
: <fpage>1129</fpage>
–<lpage>1135</lpage>
<pub-id pub-id-type="pmid">16520338</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Becq1"><label>40</label>
<mixed-citation publication-type="journal"><name><surname>Becq</surname>
<given-names>J</given-names>
</name>
, <name><surname>Gutierrez</surname>
<given-names>MC</given-names>
</name>
, <name><surname>Rosas-Magallanes</surname>
<given-names>V</given-names>
</name>
, <name><surname>Rauzier</surname>
<given-names>J</given-names>
</name>
, <name><surname>Gicquel</surname>
<given-names>B</given-names>
</name>
, <etal>et al</etal>
(<year>2007</year>
) <article-title>Contribution of horizontally acquired genomic islands to the evolution of the tubercle bacilli</article-title>
. <source>Mol Biol Evol</source>
<volume>24</volume>
: <fpage>1861</fpage>
–<lpage>1871</lpage>
<pub-id pub-id-type="pmid">17545187</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Jang1"><label>41</label>
<mixed-citation publication-type="journal"><name><surname>Jang</surname>
<given-names>J</given-names>
</name>
, <name><surname>Becq</surname>
<given-names>J</given-names>
</name>
, <name><surname>Gicquel</surname>
<given-names>B</given-names>
</name>
, <name><surname>Deschavanne</surname>
<given-names>P</given-names>
</name>
, <name><surname>Neyrolles</surname>
<given-names>O</given-names>
</name>
(<year>2008</year>
) <article-title>Horizontally acquired genomic islands in the tubercle bacilli</article-title>
. <source>Trends Microbiol</source>
<volume>16</volume>
: <fpage>303</fpage>
–<lpage>308</lpage>
<pub-id pub-id-type="pmid">18515114</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Smith3"><label>42</label>
<mixed-citation publication-type="journal"><name><surname>Smith</surname>
<given-names>NH</given-names>
</name>
(<year>2006</year>
) <article-title>A re-evaluation of <italic>M. prototuberculosis</italic>
</article-title>
. <source>PLoS Pathog</source>
<volume>2</volume>
: <fpage>e98</fpage>
<pub-id pub-id-type="pmid">17009867</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Fabre1"><label>43</label>
<mixed-citation publication-type="journal"><name><surname>Fabre</surname>
<given-names>M</given-names>
</name>
, <name><surname>Hauck</surname>
<given-names>Y</given-names>
</name>
, <name><surname>Soler</surname>
<given-names>C</given-names>
</name>
, <name><surname>Koeck</surname>
<given-names>J-L</given-names>
</name>
, <name><surname>van Ingen</surname>
<given-names>J</given-names>
</name>
, <etal>et al</etal>
(<year>2010</year>
) <article-title>Molecular characteristics of “<italic>Mycobacterium canettii</italic>
” the smooth <italic>Mycobacterium tuberculosis</italic>
bacilli</article-title>
. <source>Infect Genet Evol</source>
<volume>10</volume>
: <fpage>1165</fpage>
–<lpage>1173</lpage>
<pub-id pub-id-type="pmid">20692377</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Koeck1"><label>44</label>
<mixed-citation publication-type="journal"><name><surname>Koeck</surname>
<given-names>J-L</given-names>
</name>
, <name><surname>Fabre</surname>
<given-names>M</given-names>
</name>
, <name><surname>Simon</surname>
<given-names>F</given-names>
</name>
, <name><surname>Daffé</surname>
<given-names>M</given-names>
</name>
, <name><surname>Garnotel</surname>
<given-names>E</given-names>
</name>
, <etal>et al</etal>
(<year>2011</year>
) <article-title>Clinical characteristics of the smooth tubercle bacilli ‘<italic>Mycobacterium canettii</italic>
’ infection suggest the existence of an environmental reservoir</article-title>
. <source>Clin Microbiol Infect</source>
<volume>17</volume>
: <fpage>1013</fpage>
–<lpage>1019</lpage>
<pub-id pub-id-type="pmid">20831613</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Klaus1"><label>45</label>
<mixed-citation publication-type="journal"><name><surname>Klaus</surname>
<given-names>HD</given-names>
</name>
, <name><surname>Wilbur</surname>
<given-names>AK</given-names>
</name>
, <name><surname>Temple</surname>
<given-names>DH</given-names>
</name>
, <name><surname>Buikstra</surname>
<given-names>JE</given-names>
</name>
, <name><surname>Stone</surname>
<given-names>AC</given-names>
</name>
, <etal>et al</etal>
(<year>2010</year>
) <article-title>Tuberculosis on the north coast of Peru: skeletal and molecular paleopathology of late pre-Hispanic and post-contact mycobacterial disease</article-title>
. <source>J Archaeol Sci</source>
<volume>37</volume>
: <fpage>2587</fpage>
–<lpage>2597</lpage>
</mixed-citation>
</ref>
<ref id="pone.0041923-Kappelman1"><label>46</label>
<mixed-citation publication-type="journal"><name><surname>Kappelman</surname>
<given-names>J</given-names>
</name>
, <name><surname>Alçiçek</surname>
<given-names>MC</given-names>
</name>
, <name><surname>Kazanci</surname>
<given-names>N</given-names>
</name>
, <name><surname>Schultz</surname>
<given-names>M</given-names>
</name>
, <name><surname>Özkul</surname>
<given-names>M</given-names>
</name>
, <etal>et al</etal>
(<year>2008</year>
) <article-title>First <italic>Homo erectus</italic>
from Turkey and implications for migrations into temperate Eurasia</article-title>
. <source>Am J Phys Anthropol</source>
<volume>135</volume>
: <fpage>110</fpage>
–<lpage>116</lpage>
<pub-id pub-id-type="pmid">18067194</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Roberts1"><label>47</label>
<mixed-citation publication-type="journal"><name><surname>Roberts</surname>
<given-names>CA</given-names>
</name>
, <name><surname>Pfister</surname>
<given-names>L-A</given-names>
</name>
, <name><surname>Mays</surname>
<given-names>S</given-names>
</name>
(<year>2009</year>
) <article-title>Was tuberculosis present in <italic>Homo erectus</italic>
in Turkey</article-title>
? <source>Am J Phys Anthropol</source>
<volume>139</volume>
: <fpage>442</fpage>
–<lpage>444</lpage>
<pub-id pub-id-type="pmid">19358292</pub-id>
</mixed-citation>
</ref>
<ref id="pone.0041923-Donoghue1"><label>48</label>
<mixed-citation publication-type="journal"><name><surname>Donoghue</surname>
<given-names>HD</given-names>
</name>
, <name><surname>Lee</surname>
<given-names>OY-C</given-names>
</name>
, <name><surname>Minnikin</surname>
<given-names>DE</given-names>
</name>
, <name><surname>Besra</surname>
<given-names>GS</given-names>
</name>
, <name><surname>Taylor</surname>
<given-names>JH</given-names>
</name>
, <etal>et al</etal>
(<year>2010</year>
) <article-title>Tuberculosis in Dr. Granville’s mummy: a molecular re-examination of the earliest known Egyptian mummy to be scientifically examined and given a medical diagnosis</article-title>
. <source>Proc Roy Soc B</source>
<volume>277</volume>
: <fpage>51</fpage>
–<lpage>56</lpage>
</mixed-citation>
</ref>
<ref id="pone.0041923-Taylor1"><label>49</label>
<mixed-citation publication-type="journal"><name><surname>Taylor</surname>
<given-names>GM</given-names>
</name>
, <name><surname>Blau</surname>
<given-names>S</given-names>
</name>
, <name><surname>Mays</surname>
<given-names>S</given-names>
</name>
, <name><surname>Monot</surname>
<given-names>M</given-names>
</name>
, <name><surname>Lee</surname>
<given-names>OY-C</given-names>
</name>
, <etal>et al</etal>
(<year>2009</year>
) <article-title><italic>Mycobacterium leprae</italic>
genotype amplified from an archaeological case of lepromatous leprosy in Central Asia</article-title>
. <source>J Archaeol Sci</source>
<volume>36</volume>
: <fpage>2408</fpage>
–<lpage>2414</lpage>
</mixed-citation>
</ref>
</ref-list>
</back>
</pmc>
</record>
Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Wicri/Archeologie/explor/PaleopathV1/Data/Pmc/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 000361 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd -nk 000361 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien |wiki= Wicri/Archeologie |area= PaleopathV1 |flux= Pmc |étape= Corpus |type= RBID |clé= PMC:3408397 |texte= Mycobacterium tuberculosis Complex Lipid Virulence Factors Preserved in the 17,000-Year-Old Skeleton of an Extinct Bison, Bison antiquus }}
Pour générer des pages wiki
HfdIndexSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/RBID.i -Sk "pubmed:22860031" \ | HfdSelect -Kh $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd \ | NlmPubMed2Wicri -a PaleopathV1
![]() | This area was generated with Dilib version V0.6.27. | ![]() |