Ancient Remnants: Biomolecules in Paleomicrobiology

Ancient DNA is not the only method of detecting and identifying ancient pathogens. Survival challenges for ancient DNA place very real limitations on its usefulness and sensitivity as a detection method. The main advantage of aDNA is that it can be genotyped to compare with modern species. For archaeological purposes, other biomolecules may be detected better and cheaper than aDNA.

Sources of Ancient Biomolecules

Proteins and lipids can be extracted from a variety of materials. Bone tissue and especially teeth are still the primary source for non-nucleic acid biomolecules. Hair and mummified skin, muscle, and internal organs have all been successfully used to extract usable proteins. Both proteins and DNA can be extracted old paraffin embedded human tissues previously used for past histological examination. Frozen tissue can yield protein, lipid and DNA.

Materials and methods for nonnucleic acid biomolecule detection in ancient material (Fig 2, Tran, Aboudharam, Raoult, and Drancourt, 2011)

Methods of Detection

• Immunohistochemistry: Immunohistochemistry combines antibody detection of microbial components with cellular morphology. These methods are similar, if not identical to, standard immunohistochemical assays done on modern patient tissue. Although protein and tissue degradation can be a problem, immunohistochemistry has been done successfully on mummified tissue, detecting 16th century smallpox and syphilis.
• ELISA and Immunochromography: Extracted protein can be assayed by immunochromatography (potentially as a dipstick) or ELISA (enzyme linked immunosorbent assay). Both immunochromatography and ELISA has been used to detect Yersinia pestis‘ F1 antigen in late medieval and early modern specimens. Pusch et al (2004) does a good comparison of the sensitivity of Y. pestis F1 antigen immunochromography versus ancient DNA amplification on the same specimens.  Immunodetection methods have been used to detect Plasmodium falciparum  (malaria) in natural mummies from ancient Nubia and Egypt, as well as in skeletal tissue from the 16th century Medici family of Italy.
• High Performance Liquid Chromatography (HPLC) can be used to differentiate ancient lipids extracted from specimens. It has been used primarily for the detection of mycolic acids and mycocerosic acids found in the cell wall of Mycobacterium tuberculosis. Mycolic acids have been used to confirm tuberculosis in remains as old as 9000 years before present.
• Mass Spectrometry: Mass spectrometry has been successfully used on ancient proteins and peptides. Studies on collagen proteins have been able to distinguish ancient animal species and tuberculosis. Mass spectrometry can also identify microbial products like tetracycline identified and analyzed in Late Antique Nubian bones (Nelson et al, 2010).  Their analysis was able to prove that the tetracycline was deposited in the living bone tissue rather than infiltration from soil bacteria.
• Late Serology: Active immunoglobulins (IgG) have been extracted from ancient bones. When exposed to “pathogen-specific antigen” the antibody still reacts. Antibodies against syphilis reacted from extracts of ancient bones that also showed osteological lesions of syphilis and PCR confirmation of Treponema pallidum (Tran et al, 2011). I haven’t read any studies using this technique yet.
• ImmunoPCR: ImmunoPCR is a somewhat odd technique that detects protein, not DNA. The DNA is essentially an amplified reporter of the antigen-antibody reaction monitored by real-time PCR. I haven’t read any studies using immunoPCR yet either.

Any of these methods can be used in conjunction with aDNA analysis to confirm a diagnosis on an ancient specimen. A developing consensus for diagnosing ancient infectious disease calls for the identification of species specific aDNA and some species specific non-nucleic acid biomolecule. Like ancient DNA studies, consensus standards for non-nucleic acid biomolecules have yet to be agreed upon. Determining what the appropriate positive and negative controls are is vital.The entire field is still rapidly evolving and it is likely to take years to develop. The one thing we know for sure is that these new molecular techniques will revolutionize archaeology over the next generation.

Tran TN, Aboudharam G, Raoult D, & Drancourt M (2011). Beyond ancient microbial DNA: nonnucleotidic biomolecules for paleomicrobiology. BioTechniques, 50 (6), 370-80 PMID: 21781037.
[Review article] This review article is available free online.

Pusch CM, Rahalison L, Blin N, Nicholson GJ, & Czarnetzki A (2004). Yersinial F1 antigen and the cause of Black Death. The Lancet infectious diseases, 4 (8), 484-5 PMID: 15288817

Nelson ML, Dinardo A, Hochberg J, & Armelagos GJ (2010). Brief communication: Mass spectroscopic characterization of tetracycline in the skeletal remains of an ancient population from Sudanese Nubia 350-550 CE. American journal of physical anthropology, 143 (1), 151-4 PMID: 20564518