Ancient bacterial DNA (aDNA) in dental calculus from archaeological human remains

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/232393245

Ancient bacterial DNA (aDNA) in dental calculus from archaeological human remains Article in Journal of Archaeological Science · August 2011 DOI: 10.1016/j.jas.2011.03.020

CITATIONS

READS

24

448

4 authors, including: Hans Preus

Pia Bennike

University of Oslo

University of Copenhagen

79 PUBLICATIONS 1,411 CITATIONS

44 PUBLICATIONS 452 CITATIONS

SEE PROFILE

SEE PROFILE

All content following this page was uploaded by Hans Preus on 06 January 2014. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original document and are linked to publications on ResearchGate, letting you access and read them immediately.

This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright

Author's personal copy

Journal of Archaeological Science 38 (2011) 1827e1831

Contents lists available at ScienceDirect

Journal of Archaeological Science journal homepage: http://www.elsevier.com/locate/jas

Ancient bacterial DNA (aDNA) in dental calculus from archaeological human remains Hans R. Preus a, *, Ole J. Marvik b, Knut A. Selvig c, Pia Bennike d a

Department of Periodontology, Institute of Clinical Odontology, Dental Faculty, University of Oslo, P.O. Box 1109 Blindern, N-0317 Oslo, Norway Affitech AS, Oslo Research Park, Oslo, Norway c Dept of Anatomy, Faculty of Dentistry, University of Bergen, Norway d Saxo Institute, University of Copenhagen, Denmark b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 October 2010 Received in revised form 8 March 2011 Accepted 17 March 2011

The purpose of this study was to identify reactive bacterial aDNA in archaeological human dental calculus. Dental calculus was collected from a middle/late Neolithic human skull from Hulbjerg passage grave, Langeland, Denmark and prepared for transmission electron microscopy (TEM) or gold-labeled antibody TEM. TEM showed calcified, as well as non-calcified bacteria. Immunogold labeling occurred over the cytoplasmic portions of the sectioned bacteria. The result demonstrated that it is possible to identify aDNA sequences from bacteria in archaeological material of considerable age by this technique. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Bacteria Dental calculus Transmission electron microscopy Bioarchaeology Immunolabeling

1. Introduction There is a growing interest for ancient biomolecules in several fields of science. Researchers are striving to develop more precise techniques to trace human migration and behavior in the past (Campana, 2008; Ricaut et al., 2004). Analyzing and comparing ancient DNA (aDNA) may also facilitate distinction between sites within an archeologically interesting area (Bollingono and Vigne, 2008; Fortea et al., 2008). Forensic scientists search for evidence of historic crimes through studies of (aDNA) (Schlablitsky et al., 2006; O’Rourke et al., 2000), and physicians use aDNA to study hereditary and infectious diseases in the past (Witas and Zawicki, 2006; Herrmann, 1998). It has also been suggested that studies of DNA from prehistoric bacteria or viruses may lead to the discovery of new approaches to fight modern versions of these infectious agents. A high-profiled example is the excavation and study of victims of the Spanish flu on the Island of Spitsbergen, Norway, in 1998 (Duncan, 2003; Annan et al., 2000). In this particular endeavor they searched for the Spanish flu virus DNA/RNA in order to study and produce antibodies against a future viral pandemic of similar origin. However, recent reports on current diseases, like

* Corresponding author. Tel.: þ47 22 85 21 63; fax: þ47 22 85 23 96. E-mail address: [email protected] (H.R. Preus). 0305-4403/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jas.2011.03.020

tuberculosis and syphilis, have focused on historical information about the original phenotype and genetic development of the responsible bacteria (Hunniusvon et al., 2007; Hummel et al., 1995), also emphasizing the limitations of these techniques. The interest in the historic and original infectious agents is boosted by the growing concern that modern versions have evolved to escape the treatment of available antibiotic drugs (Høiby et al., 2010), and the desire to develop new antimicrobials based on the original DNA of the microorganisms. A major problem in this field of research is the lack of specimens suitable for systematic analysis. Teeth and calcified material are identified in most, if not all, archaeological digs where human remains are found. Dental calculus is therefore a common find, and may serve as a systematic source of information in a field where only occasional findings of preserved aDNA have been the order of the day. The DNA from bacteria, fungi or viruses, and more seldom human cells, are obviously more preserved against contamination and deterioration embedded in amorphous calcifications than on the inner or outer surfaces of other human tissues. Studies of archeological calculus material have been reported (Gobetz and Bozarth, 2001; Herschkovitz et al., 1997; Middleton and Rovner, 1994), but to our knowledge, reporting it as a source of aDNA, has not been done. The potential problem of historic and modern contamination of aDNA samples has been strongly emphasized by many researchers

Author's personal copy

1828

H.R. Preus et al. / Journal of Archaeological Science 38 (2011) 1827e1831

(Bouwman et al., 2006; Gilbert et al., 2005; Yang and Watt, 2005; Pruvost and Geigl, 2004; Yang et al., 2003; Preus, 1998; Burger et al., 1997; Dutour, 1997; Schmidt et al., 1995). Thus the need for highly preserved aDNA sources, with less contamination potential, is of interest to the archaeological community. In this communication we propose a way to overcome several of these obstacles, and suggest dental calculus as a systematic source of aDNA from archaeological material. Dental calculus contains bacteria and intercellular substance forming a matrix for deposition of amorphous and crystalline calcium phosphates and silicate (Lang et al., 2008). In this highly calcified substance the encapsulated bacteria are to a great extent protected against post mortem degradation, unless embedded in an acidic environment. Since nucleic acids are normally stabilized by divalent cat-ions, DNA is well preserved in calculus with its abundance of Ca2þ containing amorphous crystals. Moreover, as skulls and teeth are often the best preserved specimens in archaeological excavation sites, archeological dental calculus might be an ideal material for systematic studies of aDNA. Thus, the aim of the present investigation was to demonstrate the presence of reactive sequences of DNA from oral bacteria embedded in dental calculus from human remains of considerable age. 2. Materials and methods 2.1. Sample Calculus was collected from the roots of the lower central incisors of a skull from the Stone Age Hulbjerg passage grave built in the Middle Neolithic on the island of Langeland, Denmark. The skull was found together with skeletal remains of 52 individuals including men, women and children. The archaeological artifacts and 14C-analyses showed that the passage grave had been used periodically between 3200 and 1800 BC (Bennike and Bohr, 1990), indicating that the age of the calculus sample is between 4000 and 5000 years. In passage graves, the bones of individual skeletons are usually scattered in anatomical disorder and are found in variable state of preservation. Some of the bones, like the skull from which the actual calculus samples were taken, were intact and well preserved, and the femora have been used in a study of bone mineral content in various periods (Skaarup, 1985). After the passage grave was closed, it has apparently remained undisturbed until its excavation in 1960. The skeletal material from the site was stored at the Laboratory of Biological Anthropology in Copenhagen. More recently an anthropological study has revealed evidence of both dental (a drilled hole) and surgical intervention (trephination) (Bennike, 1985). Dental calculus from the drilled tooth has previously been examined by scanning electron microscopy, revealing bacteria-like structures (Bennike and Fredebo, 1986). These specimens were different from those presented in the present article. 2.2. Methods The dry calculus specimens from the root of the lower, central inscisor (2.1 Sample) were dehydrated in an aqua solution of graded alcohol, treated with osmium tetroxide fixative to enhance contrast, and embedded in epoxy resin following routine procedures for transmission electron microscopy (TEM). Sixteen specimens of either untreated sections, or sections treated with ultraviolet light and gold-labeled monoclonal antibodies specific for thymine dimers (Marvik et al., 1997), were processed for TEM. Thin sections were collected on electron microscopic grids and exposed to ultraviolet light irradiation (UV). Non-irradiated controls were also included. Moderate doses of UV at 310 nm will,

in the presence of acetophenone, preferentially induce the formation of the 50 60 cyclobutane type of thymine dimers, for which the selected H3 antibodies are specific (Roza et al., 1988). The H3 antibodies were produced by a mouse hybridoma cell line (Roza et al., 1988) that has previously been characterized for the use in photo-immunodetection (Marvik et al., 1997). They show a sensitivity in membrane assays of about 0.5 pg with a specificity of >10E6 higher affinity for irradiated versus non-irradiated samples. Thus for practical purposes they are unreactive toward DNA without 50 -60 cyclobutane type of thymine dimers. Importantly, the reactivity of the H3 antibodies toward the 50 -60 cyclobutane dimer decreases dramatically (>100 fold) when a thymine dimer is positioned closer than about 15 residues from the end of the chain (Marvik unpublished results; Roza et al., 1988). This makes the antibody a useful probe for irradiated DNA fragments larger than at least 20e30 residues. In short, series of 50 ml droplets of reactants and washes were prepared on sheets of parafilm. All grids were treated with each reactant by placing them inverted on the relevant series of droplets for the times indicated below. The grids were pretreated for 15 min with 2 mM acetophenone diluted 1:5000 in 1-x PBS. Subsequently, half of both the test and the control grids were irradiated with 80 kJ/m2 in a Vilber Lourmat UV-crosslinker using tubes with peak emission at 310 nm. All grids were blocked with 10% inactivated Fetal Calf Serum (FCS) in 1-x PBS for 20 min before exposure to the anti-thymine dimer antibodies (either undiluted or diluted 1:10 in FCS/PBS) for another 20 min. Following washing on droplets of PBS for 5 times 5 min, the grids were treated with 10 nm gold-labeled secondary anti-mouse antibodies in 5% FCS/PBS for 20 min. Washing was repeated 4 times with PBS and 6 times with water and contrasted with 1% uranyl acetate with lead citrate before microscopy (Reynolds, 1963). Irradiated and nonirradiated sections of fresh mammalian connective tissue (dog gingiva), served as positive control. The experimental design is shown in Fig. 1. 3. Results 3.1. Transmission electron microscopyduntreated sections Transmission electron microscopy of untreated calculus revealed a highly calcified substance with scattered voids corresponding to non-calcified bacteria (Fig. 2). Circular and elongated profiles within the calcified substance could be identified as calcified bacterial cells. Electron diffraction analysis indicated that the mineral crystals were predominantly hydroxyapatite with occasional, presumably post mortem, deposition of whitlockite in the voids. 3.2. Transmission electron microscopydtreated sections The gold-labeled thymine dimer treated sections showed metal particles bound over the cytoplasmic portions of several noncalcified bacteria (Fig. 3). Eight clusters of 6e8 bacteria each were investigated by TEM, showing varying binding of particles from section to section. Gold particles were not observed in the intercellular matrix. The bacterial cell wall was distinctly outlined; however, the cytoplasm appeared shrunken and few or no ultra structural details were visible. Due to the high electron density of the mineral crystals, the presence or absence of labeling on calcified cells could not be determined. 3.3. Transmission electron microscopydcontrols The specificity of the reaction was verified by the presence of gold-labeled thymine dimer antibodies only over the nuclear

Author's personal copy

H.R. Preus et al. / Journal of Archaeological Science 38 (2011) 1827e1831

1829

Dental Calculus (test specimen) Dog gingiva (pos. control specimen)

No UV- irradiation

Direct Transmission Electron Microscopy

UV- irradiation

Immuno-Gold-labeling (Anti-TT dimer abs)

Immuno-Gold-labeling (Anti-TT dimer abs)

Transmission Electron Microscopy

Transmission Electron Microscopy

Neg. control technique

Pos. control technique

Fig. 1. Experimental design.

region in the positive controls (Fig. 4aec), and only over the intracellular area of the calculus bacterial cells (Fig. 3). All negative, non-irradiated controls remained antibody negative (not shown).

Paleoepidemiology is a new approach in the studies of diseases of the past. This approach requires well-documented, and systematic series of samples in order to minimize biases caused by contamination and partial degeneration of the aDNA (Dutour, 1997). Only few skeletal samples are compatible with this set of conditions. However, aDNA in dental, arterial or renal calculus is well preserved due to its historic inclusion in the amorphous calcium. The scope of this study was to identify possible ancient DNA in calculus of considerable age, and did not engage in identifying aDNA from specific bacteria. However, having shown that one can identify fairly long sequences of aDNA in calculus by our technique, techniques like in situ PCR may be used in the future to identify specific microorganisms in archaeological dental, renal or arterial calcifications. In dental plaque there are more than 500 different bacterial types, of which many have not been identified by name or family (Lang et al., 2008). Dental calculus reflects this abundance of different bacteria in addition to an occasional, although rare,

immunological cell migrated through the pocket epithelium (Lang et al., 2008). To identify these, specific probes against the bacteria or cells of interest are needed. The present technique only describes the presence of reactive bacterial aDNA in dental calculus, “reactive” meaning that the DNA present in the sample has a sufficient length to a) produce thymine dimers upon UV irradiation and b) serve as a reactive antigen for the H3 antibodies. In humans, the oral cavity has a fairly stable microflora (Lang et al., 2008). However it is the most common entrance for intruding pathogens, and transient establishment of pathogenic microorganisms in the oral flora has been documented (Scannapieco et al., 1998; Eguchi et al., 2003; Sumi et al., 2007; Eskandari et al., 2010). However, as a person attracts a fatal infection, and lives his last weeks or days, the microorganisms causing his death will be found in saliva, and therefore his post mortem dental calculus. Moreover, different people, in different locations on the globe, may have different bacteria specific to their oral floras (Cutress et al., 1982; Preus et al., 1995). If such different, specific bacteria are possible to find in different populations in our globalized society, it would undoubtedly be true for ancient cultures with no, or far less exchange of people, and thereby microorganisms. Studies of ancient DNA carries a heavy duty to substantiate that the DNA claimed to be ancient, is actually located within the ancient cell compartment, and is not the result of artifacts, or

Fig. 2. Dental calculus harvested from a human skull at an archaeological site dated to the period 3200e1800 BC. The transmission electron micrograph shows a highly calcified substance containing voids corresponding to non-calcified bacteria, as well as circular profiles of completely calcified cells (arrows). Magnification bar: 1.0 mm.

Fig. 3. A UV-treated section from the ancient dental calculus showing gold labeling of the cytoplasmic portion of the non-calcified bacteria indicating presence of thymine dimers in aDNA (arrows). There is no labeling of the cell wall or intercellular matrix. In the electron dense calcified areas individual crystals of hydroxyapatite are barely identifiable. Magnification bar: 1.0 mm.

4. Discussion

Author's personal copy

1830

H.R. Preus et al. / Journal of Archaeological Science 38 (2011) 1827e1831

study was prepared with several levels of positive and negative controls. Moreover, with no gold particles observed outside the perimeter of the bacterial cells investigated, we dear draw the conclusion that our observations were of bacterial aDNA located within the calcified bacteria of the calculus. One may argue that the darker, intercellular substance of the calculus will mask any gold particles bound in this area. This would be a pertinent assumption, but if so, the gold particles would also be expected to be seen over the lighter areas of the section and outside the perimeter of the calcified cells. In Fig. 3 approximately 50% of the TEM image has a background on which gold particles would contrast, and still the particles are only seen within the perimeter of the ancient bacterial cell. Moreover, if this had been the result of artifacts during handling or preparation, or contamination from any modern and present DNA source, we would expect to find DNA also on the amorphous phases of the ancient calculusdand more evenly distributed on the slide section. Previous studies have shown that background binding to nonirradiated DNA is at least six orders of magnitude lower than binding to the thymine dimer epitope. Although the method does not allow determination of fragment sizes, the antibody is known to react very poorly with TT dimers being part of below 20 residues (Marvik et al., 1997; Roza et al., 1988). The fact that the positive controls comprising irradiated sections of fresh dog gingiva showed similar label density clearly indicates that the DNA in the samples is reasonably intact, and of some length. Our study suggests that it is possible to identify reactive aDNA sequences in archaeological material of considerable age by this technique.

Acknowledgments The expertise of Ms. Rita Greiner-Simonsen in processing the material for TEM is gratefully acknowledged.

References

Fig. 4. a) Gingival fibroblast, nucleus with gold labeling. b) Detail. Gold labeling in nucleus and on nuclear membrane. c) Gingival cell with gold labeling in nucleus. Original magnification 10,000.

modern DNA contamination, during handling or preparation (Yang and Watt, 2005; Yang et al., 2003; Preus, 1998). The chance for contamination will vary with the methods used, and increase with increasing sensitivity of the method employed (Preus, 1998). Our

Annan, A.P., Berdal, B.P., Smith, C.R., Heginbottom, J.A., Skehel, J.J., Davis, J.L., Oxford, J.S., Duncan, K.E., Roberts, N., Lewin, P.K., Daniels, R.S., Bergan, T., 2000. Ground penetrating radar surveys to locate 1918 Spanish flu victims in permafrost. J. Forensic. Sci. 45, 68e76. Bennike, P., 1985. Stenalderbefolkningen på øerne syd for Fyn. (The Neolithic population on the islands South of Funen). In: Skaarup, J. (Ed.), Yngre Stenalder På Øerne Syd for Fyn. Langelands Museum, Rudkøbing, pp. 467e491. Bennike, P., Bohr, H., 1990. Bone mineral content in past and present. In: Christiansen, C., Overgaard, K. (Eds.), Osteoporosis. Proceedings from the Third International Symposium on Osteoporosis, Munksgaard, Copenhagen, pp. 89e91. Bennike, P., Fredebo, L., 1986. Dental treatment in the stone age. Bull. Hist. Dent. 34, 81e87. Bollingono, R., Vigne, J.-D., 2008. Temperature monitoring in archaeological animal bone samples in the near east arid area, before, during and after excavation. J. Archaeol. Sci. 35, 873e881. Bouwman, A.S., Chilvers, E.R., Brown, K.A., Brown, T.A., 2006. Identification of the authentic ancient DNA sequence in a human bone contaminated with modern DNA. Am. J. Phys. Anthropol. 131, 428e431. Burger, J., Hummel, S., Herrmann, B., 1997. Detection of DNA single-copy sequences of prehistoric teeth. Site milieu as a factor for preservation of DNA. Anthropol. Anz. 55, 193e198. Campana, M.G., 2008. The use of ancient microsatellites to detect past migrations. Arch. Rev. Cambridge 23, 147e160. Cutress, T.W., Powell, R.N., Ball, M.E., 1982. Differing profiles of periodontal disease in two similar South Pacific island populations. Community Dent. Oral Epidemiol. 10, 193e203. Duncan, K.E., 2003. Hunting the 1918 Flu. One Scientist’s Search for the Killer Virus. University of Toronto press, Toronto, Canada. Dutour, O., 1997. Paleoepidemiology of plague: its application to the study of tuberculosis in the past. The First Symposium of the Center for Emerging Diseases; Program and Abstracts. Jerusalem, Israel. Eguchi, J., Ishihara, K., Watanabe, A., Fukumoto, Y., Okuda, K., 2003. PCR method is essential for detecting Mycobacterium tuberculosis in oral cavity samples. Oral Microbiol. Immunol. 18, 156e159.

Author's personal copy

H.R. Preus et al. / Journal of Archaeological Science 38 (2011) 1827e1831 Eskandari, A., Mahmoudpour, A., Abolfazli, A., Lafzi, A., 2010. Detection of Helicobacter pylori using PCR in dental plaque of patients with and without Gastritis. Med. Oral Patol. Oral Cir. Bucal. 15, 28e31. Fortea, J., de la Rasilla, M., Garcia-Tabernero, A., Gigli, E., Rosas, A., Lalueza-Fox, C., 2008. Excavation protocol of bone remains for neanderthal DNA analysis in El Sidron cave (Asturias, Spain). J. Hum. Evol. 55, 353e357. Gilbert, M.T., Rudbeck, L., Willerslev, E., Hansen, A.J., Smith, C., Penkman, K.E.H., 2005. Biochemical and physical correlates of DNA contamination in archaeological human bones and teeth excavated in Matera, Italy. J. Archaeol. Sci. 32, 785e793. Gobetz, K.E., Bozarth, S.R., 2001. Implications for late pleistocene mastodont diet from opal phytoliths in tooth calculus. Quat. Res. 55, 115e122. Høiby, N., Bjarnsholt, T., Givskov, M., Molin, S., Ciofu, O., 2010. Antibiotic resistance of bacterial biofilms. Int. J. Antimicrob. Agents 35, 322e332. Herrmann, B., 1998. Paleoepidemiology of infectious diseases using a DNA analysis: pros and cons. Bull. Mem. Soc. D’antropol 10, 195e200. Herschkovitz, I., Kelly, J., Latimer, B., Rotschild, B.M., Simpson, S., Polak, J., Rosenberg, M., 1997. Oral bacteria in Miocene Sivapithecus. J. Hum. Evol. 33, 507e512. Hummel, S., Nordsiek, G., Rameckers, J., Lassen, C., Zierdt, H., Baron, H., Herrmann, B., 1995. aDNA: ein neuer zugang zu alten fragen. Z. Morphol. Antropol. 81, 41e65. Hunniusvon, T.E., Yang, D.Y., Eng, B., Waye, J.S., Saunders, S.R., 2007. Digging deeper into the limits of ancient DNA research on syphilis. J. Archaeol. Sci. 34, 2091e2100. Lang, N.P., Mombelli, A., Attström, R., 2008. Oral biofilms and calculus. In: Lang, N.P., Karring, T. (Eds.), Clinical Periodontology and Implant Dentistry, fifth ed. Blackwell Munksgaard, Oxford, pp. 183e206. Marvik, O.J., Isaksen, M.L., Roza, L., Lindquist, H., 1997. Photoimmunodetection: a nonradioactive labelling and detection method for DNA. Biotechnology 23, 892e896. Middleton, W.D., Rovner, I., 1994. Extraction of Opal Phytolitis from herbivore dental calculus. J. Archaeol. Sci. 21, 469e473. O’Rourke, D.H., Hayes, M.G., Carlyle, S.W., 2000. Ancient DNA studies in physical anthropology. Ann. Rev. Anthropol. 29, 217e242. Preus, H.R., 1998. Ancient mitochondral DNA, is it reproducibly detectable? dA comment to current methodology. In: Wallin, P. (Ed.), No Barrier Seminar

View publication stats

1831

Papers, vol 1. The KonTiki Museum and Institute for Pacific Archaeology and Cultural History, Oslo, Norway, pp. 8e10. Preus, H.R., Ånerud, Å, Boysen, H., Dunford, R.G., Zambon, J.J., Löe, H., 1995. The natural history of periodontal disease. The correlation of selected microbiological parameters with disease severity in Sri Lankan tea workers. J. Clin. Periodontol. 22, 674e678. Pruvost, M., Geigl, E.-M., 2004. Real-time PCR to assess the authenticity of ancient DNA amplification. J. Archaeol. Sci. 31, 1191e1197. Reynolds, E.S., 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell. Biol. 17, 208e212. Ricaut, F.-X., Keyser-Tracqui, C., Burgois, J., Crubezy, E., Ludes, B., 2004. Genetic analysis of a Scytho-Siberian skeleton and its implications for ancient central Asian Migration. Hum. Biol. 76, 109e126. Roza, L., van der Wulp, K.J.M., MacFarlane, S.J., Lohman, P.H.M., Baan, R.A., 1988. Detection of cyclobutane thymine dimers in DNA of human cells with monoclonal antibodies raised against a thymine dimer-containing tetranucleotide. Photochem. Photobiol. 48, 627e633. Scannapieco, F.A., Papandonatos, G.D., Dunford, R.G., 1998. Associations between oral conditions and respiratory disease in a national sample survey population. Ann. Periodontol. 3, 251e256. Schlablitsky, J., Dixon, K.J., Leney, M., 2006. Forensic technology and the historical archaeologist. Hist. Archaeol. 40, 1e7. Schmidt, T., Hummel, S., Herrmann, B., 1995. Evidence of contamination in laboratory disposables. Naturwissenschaften 82, 423e431. Skaarup, J. (Ed.), 1985. Neolithic on the Islands South of Funen. Langelands Museum, Rudkøbing, Denmark. Sumi, Y., Miura, H., Michiwaki, Y., Nagaosa, S., Nagaya, M., 2007. Colonization of dental plaque by respiratory pathogens in dependent elderly. Arch. Gerontol. Geriatr. 44, 119e124. Witas, H.W., Zawicki, P., 2006. Allele protecting against HIV (CCR5-32) identifies in early medieval specimens from central Poland. Preliminary results. Prz. Antropol. 69, 49e53. Yang, D.Y., Watt, K., 2005. Contamination controls when preparing archaeological remains for ancient DNA analysis. J. Archaeol. Sci. 32, 331e336. Yang, D.Y., Eng, B., Saunders, S.R., 2003. Hypersensitive PCR, ancient human mtDNA, and contamination. Hum. Biol. 75, 355e364.