Tools of Paleomicrobiology

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Studying ancient microbes requires creativity. Contamination and  preservation are the primary problems, dealing with limited and degraded tissues. We don’t find corpses in permafrost every day! Most of the time tissue is confined to bones and mummies kept in a wide variety of environments. This post will review some of the major tools I have found for investigating ancient microbes. I do not have experience working with ancient materials, so this is based completely on what I have read.

With all of these methods, not detecting the microbe does not mean that it’s not there. It may be simply too degraded for the method to have worked. These methods also can’t determine when the microbe entered the material.

Tools for hunting ancient microbes:

  1. Sampling and cultures: Culturing ancient microbes is limited to specimens that have been kept frozen. It might be possible that some bacterial spores could endure long enough to culture (this being both a risk and boon).
  2. Light Microscopy depends on cellular and tissue preservation. Since most bacterial stains rely on an intact cell wall, I am not very confident about the effectiveness of the standard stains. The gram stain becomes unreliable on old living bacterial cultures much less material that is centuries old. Light microscopy should be able to give at least preliminary identification of parasites like lice, ticks, fleas, worms, etc.
  3. Immunology:
    Direct fluorescent antibody (DFA) of Yersinia pestis (CDC, PHIL #1918)

    Antibody stains of tissue sections for specific bacterial components like the Yersinia pestis fluorescent antibody stain to the right may be more effective than more traditional stains if preservation is good enough. It has been done for tissue stored in old paraffin blocks (the way  tissue that has been used for histological purposes is stored). However, loss of antigenic properties in decayed tissue makes immunohistological methods difficult on archaeological remains (Lepidi, 2008). Plague F1 antigen has also been detected from pulverized tissue with an antibody-based dipstick. Antigen detection has been showed to be significantly more sensitive than DNA  for Yersinia pestis (Carsten et al, 2004). This makes a lot sense when you consider the relative amounts of antigen vs. the specific region of DNA to be amplified. If this works for Y. pestis, it should also work for other species specific proteins. The presence of microbes in a population can also be  detected by a specific host response against it, essentially by detecting antibodies that can still bind antigen.

  4. Electron Microscopy: Both transmission electron microscopy (TEM) and scanning electron microscopy (SEM) are useful for paleomicrobiology. The three-dimensional image produced by SEM can better identify the dried remains of parasites like the human lice identified among Napoleon’s soldiers (Raoult et al, 2006). TEM is particularly useful for finding viruses in fresh tissue; smallpox has been identified in a 16th century Italian mummy and old formalin fixed tissues (Lepidi, 2008). Syphilis was also identified in another Italian mummy by TEM (Lepidi, 2008). However, TEM is particularly sensitive to preservation conditions and the quality of the tissue. Molecular methods are more likely to securely identify bacteria and viruses.
  5. Molecular methods: There are two basic types of molecular detection: detection of unique biomolecules specific to a species like the F1 antigen of Yersinia pestis, or analysis of microbial DNA. Microbes produce an array of biomolecules that are genus if not species specific such as tetracyclines produced by Streptomyces species. However, the primary molecular method is the amplification of specific microbial DNA. It can be done by looking for specific microbes, usually by the suicide PCR technique, or by attempting to amplify all bacterial species in a specimen by amplifying well characterized ribosomal genes, identifying species by their specific sequences. Both DNA-based methods have their pros and cons. I will come back to the DNA based methods in posts on the method and with specific examples in the near future.
  6. Osteology – I don’t know much about osteology and paleomicrobiology sources don’t write about osteology as a tool of paleomicrobiology but it seems to me that there are indications in the bone of a few pathogens. To be fair, the bones reveal a minority of major pathogens. Tuberculosis, leprosy, and syphillis-like pathogens are the only agents I know of off-hand that leave distinctive bone lesions. (I have to chuckle every time I read that because there is no sign of these three diseases that the population seems healthy, or some similar gross exaggeration.) Otherwise, the bones can give non-specific signs of infection that can be followed up on by other methods.

So these are the basic tools I have come across in my reading. I’ll be back with discussions of some of the major considerations and things to look out for in paleomicrobiology projects soon.

References:

Drancourt, M., & Raoult, D. (2005). Palaeomicrobiology: current issues and perspectives Nature Reviews Microbiology, 3 (1), 23-35 DOI: 10.1038/nrmicro1063

Lepidi, H. “Histologic detection of past pathogens”, pp. 69-72 in Paleomicrobiology: Past Human Infections. D. Raoult & M. Drancourt, Eds. Springer, 2008.

Raoult D, Dutour O, Houhamdi L, Jankauskas R, Fournier PE, Ardagna Y, Drancourt M, Signoli M, La VD, Macia Y, & Aboudharam G (2006). Evidence for louse-transmitted diseases in soldiers of Napoleon’s Grand Army in Vilnius. The Journal of infectious diseases, 193 (1), 112-20 PMID: 16323139

Carsten M Pusch, Lila Rahalison, Nikolaus Blin, Graeme J Nicholson, and Alfred Czarnetzki. (2004) Yersinial F1 antigen and the cause of the Black Death. The Lancet Infectious Disease, 4, 284-284.

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