Donoghue, HD

Spigelman, M

English

PubMed

Udgivelsesdato: 22 March 2006We agree with Donoghue &amp; Spigelman (2005) that, although pathogen studies hold great potential, any discussion requires a critical assessment of the results to date. However, we did note, as did Pääbo et al. (2004), that the field of ancient pathogen DNA still lacks a series of well-controlled and rigorous studies that address technical issues and reliability criteria. This is unfortunate, as the rapid evolutionary rate of many pathogens offers a unique means to establish the authenticity of ancient pathogen sequences-since they should clearly be ancestral to modern genetic diversity (e.g. Reid et al. 1999). Such characterisation of pathogen evolutionary rates would also be of major importance to studies of the origin and spread of diseases, and so the relative lack of such investigation is particularly regrettable. Very few ancient pathogen studies have followed established aDNA protocols such as independent replication and cloning. Only 3 out of 47 studies have performed independent replication at all (Drancourt &amp; Raoult 2005), and none of the studies (Spigelman et al. 2002; Fletcher et al. 2003a,b; Donoghue et al. 2005) mentioned by Donoghue &amp; Spigelman (2005) as following aDNA criteria included cloning. Yet these studies have used mobile insertion elements (e.g. IS 6110 in tuberculosis) or conserved loci (e.g. 16S) to detect the presence of pathogens, and very similar or identical sequences have been reported from environmental bacteria (Gilbert et al. 2004). For example, Rollo &amp; Marota (1999) report that direct sequencing of ancient microbial DNA produced a sequence resembling (for example) Treponerma pallidum (the causative agent of venereal syphilis) even in the absence of real T. pallidum, simply due to the presence of diverse bacterial DNA in the experiment. In addition, the limited knowledge of current microbial diversity (only 1-5% of extant microbial diversity is believed to be known) makes it very difficult to use short aDNA sequences to establish unequivocally the presence of a given pathogen, as even supposed ‘obligate' species of humans or plants may possess currently undetected close relatives in the surrounding environment (Hebsgaard et al. 2005). Lastly, where specific studies have set out to replicate reports of ancient pathogenic bacterial DNA it has not been possible (Gilbert et al. 2004). </p

Willerslev, Eske

Cooper, Alan

Copenhagen University Research Information System

Pathogenic microbial ancient DNA: a problem or an opportunity?

E. Willerslev

A. Cooper

Crossref

Donoghue H.D

UCL Discovery

Comment Pathogenic microbial ancient DNA - a problem or an opportunity? The recent review on ancient DNA in this journal by Willerslev and Cooper (2005) is timely. It follows more than a decade of intensive research in this area, and others have also seen the need to review progress and reflect on the future (Pääbo et al. 2004; Cipollaro et al. 2005; Nicholls 2005). Since the first papers in this field, the majority of studies have dealt with population genetics, diet, domestication, and evolution of mammals and birds. The study of ancient microbial DNA developed shortly after, and has led, for example, to studies of bacteria in amber (Bada et al. 1999; Greenblatt et al. 2004) and microbes retrieved from the depths of the permafrost (Christner et al. 2003).   A distinctive branch of ancient DNA research is the study of pathogenic microbial DNA within the bodies of infected host species, primarily humans. There is increasing interest in this field, but in order to critically assess this body of research, readers should be aware of the differences caused by the characteristics of the microbial pathogens, and their distribution within the host. Microorganisms differ in the susceptibility of their DNA to the initial decay processes and the physico-chemical changes over time. Mycobacteria, including the causative agents of tuberculosis and leprosy, have resistant hydrophobic cell walls that have been shown to physically persist for at least 250 years (Donoghue et al.2004). In addition, they are members of a family with DNA rich in guanine and  cytosine which confers greater stability (Pääbo et al.2004). Therefore, it is not surprising that M. tuberculosis and M. leprae are the subjects of the majority of ancient microbial pathogen papers (Donoghue et al.2004; Drancourt & Raoult 2005). In contrast, Treponema pallidum, the causative agent of syphilis, is an exquisitely environmentally sensitive spirochaete, and its DNA very labile. Gram-negative bacteria are a little more robust and the DNA may persist in especially protected sites such as dental pulp.   Pathogenic microorganisms and their DNA will always be a minor component within a host, so it is necessary to know the sites where the particular pathogen will be localised. Initially, attention was directed to bony lesions in suspected tuberculosis cases, yet even in the pre-antibiotic era these were rare, occurring in only 3-6% of cases.  The majority of patients died from disseminated disease before lesions had time to form. Lack of understanding this point led to classical palaeopathologists querying reports that M.tuberculosis DNA could be detected in bones without lesions (reviewed by Donoghue et al.2004; Zink et al. 2005). We have detected M.tuberculosis DNA in over 50% of ribs from an 18th century Hungarian population yet <5% showed any pathological changes (updated data from Fletcher et al. 2003a). Here, the visceral surface closest to the lung was examined, not the inner tissue, and usually the upper ribs where a primary focus of infection is most likely.    Other diseases are highly localised. M. leprae DNA is primarily found in the cavum nasale and maxilla; diarrhoeal diseases are restricted to the intestinal tract, although they may spread to the liver, e.g. Schistosoma mansoni (Rutherford 1999). In meningitis, pathogens are restricted to the CSF and meninges, so only vertebrae and skull samples may be positive. Septicaemic diseases, such as bubonic plague and disseminated tuberculosis, will result in pathogen DNA being distributed around the body via the blood. In these cases bone marrow and dental pulp should be examined (Wiechmann & Grupe 2004; Drancourt & Raoult 2005). These sites should also be examined in suspected cases of blood-borne parasites, for example, of malaria (Sallares & Gomzi 2001).  Syphilis was an early focus of interest because of the clear palaeopathological markers of the disease. However, this is a disease where knowledge of its natural history is crucial. Although there is a septicaemic stage of the disease with high numbers of organisms, this is rarely the cause of death. It is tertiary syphilis that kills, yet any medical textbook will point out that the characteristic lesions of tertiary syphilis contain few, if any spirochaetes. This, together with the fragility of the pathogen, explains the negative findings (Bouwman & Brown 2005). Examination of human tissues for microbial pathogens requires modification of procedures used for extraction of host DNA. Pathogenic species are not normally present in healthy individuals, and several, such as Mycobacterium  tuberculosis and  Mycobacterium leprae have no known environmental reservoir. Therefore, their detection indicates infection and the site may demonstrate whether this was localized or disseminated. Hemi-nested and nested PCR are commonly used, due to the initial low number of target molecules. More rigorous DNA extraction procedures may be necessary for microbes with persistent cell walls. Contamination from investigators is less of a problem, compared with the need to exclude modern human DNA from ancient DNA studies. Even so, strict precautions against cross-contamination are taken and it is common practice for independent verification to be carried out, often in more than one other laboratory (Spigelman et al. 2002; Fletcher et al 2003a,b; Donoghue et al 2005). The use of species-specific loci for PCR is widespread, and for those used in clinical diagnostic microbiology, there are extensive data on their stability and specificity. This has led to the possibility of molecular epidemiological studies (see Donoghue et al. 2004, Drancourt & Raoult 2005) that throw light on the evolution and spread of microbial pathogens in the past. In conclusion, rather than ancient microbial DNA being a problem (Willerslev & Cooper 2005), we agree with Pääbo et al. (2004) that this field, in particular that of pathogenic microbial ancient DNA, is potentially very exciting, and is already reaping results. Helen D. Donoghue* & Mark Spigelman Centre for Infectious Diseases and International Health, Department of Infection, University College London, 46, Cleveland Street, London W1T 4JF, UK *h.donoghue@ucl.ac.uk REFERENCES Bada, J.L., Wang, X.S. & Hamilton, H. 1999 Preservation of key biomolecules in the fossil record: current knowledge and future challenges. Phil. Trans. R. Soc. B 354, 77–86. (doi:10.1098/rstb.1999.0361) Bouwman, A.S. & Brown, T.A. 2005 The limits of biomolecular palaeopathology: ancient DNA cannot be used to study venereal syphilis. J. Archaeol. Sci. 32, 703–713. (doi:10.1016/j.jas.2004.11.014) Christner, B.C., Mosley-Thompson, E., Thompson, L.G. & Reeve, J.N. 2003 Bacterial recovery from ancient glacial ice. Environ. Microbiol. 5, 33–36. (doi:10.1046/j.1462-   2920.2003.00422.x) Cipollaro, M., Galderisi, U. & Di Bernardo, G. 2005 Ancient DNA as a multidisciplinary experience. J. Cell. Physiol. 202, 315–322. (doi:10.1002/jcp.20116) Donoghue, H.D., Spigelman, M., Greenblatt, C.L., Lev-Maor, G., Bar-Gal, G.K., Matheson, C., Vernon, K., Nerlich, A.G. & Zink, A.R. 2004 Tuberculosis: from prehistory to Robert Koch, as revealed by ancient DNA. Lancet Infect. Dis. 4, 584–592. (doi:10.1016/S1473-   3099(04)01133-8) Donoghue, H.D., Marcsik, A., Matheson, C., Vernon, K., Nuorala, E., Molto, J.E., Greenblatt,  C.L. & Spigelman, M. 2005 Co-infection of Mycobacterium tuberculosis and Mycobacterium leprae in human archaeological samples: a possible explanation for the historical decline of leprosy. Proc. R. Soc. B 272, 389–394. (doi:10.1098/rspb.2004.   2966) Drancourt, M. & Raoult, D. 2005 Palaeomicrobiology: current issues and perspectives. Nat. Rev. Microbiol. 3, 23–35. (doi:10.1038/nrmicro1063) Fletcher, H.A., Donoghue, H.D., Holton, J., Pap, I. & Spigelman, M. 2003a Widespread occurrence of Mycobacterium tuberculosis DNA from 18th–19th century Hungarians. Am. J. Phys. Anthropol.  120, 144–512. (doi:10.1002/ajpa.10114) Fletcher, H.A., Donoghue, H.D., Taylor, G.M., van der Zanden, A.G. & Spigelman, M. 2003b Molecular analysis of Mycobacterium tuberculosis DNA from a family of 18th century Hungarians. Microbiology 149, 143–151. (doi:10.1099/mic.0.25961-0) Greenblatt, C.L., Baum, J., Klein, B.Y., Nachshon, S., Koltunov, V. & Cano, R.J. 2004 Micrococcus luteus -- survival in amber. Microb. Ecol. 48, 120–127. (doi:10.   1007/s00248-003-2016-5) Nicholls, H. 2005 Ancient DNA comes of age. PLoS Biol. 3, 0192-0196. (doi:10.1371/journal.pbio.0030056) Pääbo, S., Poinar, H., Serre, D., Jaenicke-Despres, V., Hebler, J., Rohland, N., Kuch, M., Krause, J., Vigilant, L. & Hofreiter, M. 2004 Genetic analyses from ancient DNA. Annu. Rev. Genet. 38, 645–679. (doi:10.1146/annurev.genet.37.110801.143214) Rutherford, P. 1999 Immunocytochemistry and the diagnosis of schistosomiasis: ancient and modern. Parasitol. Today 15, 390–391. (doi:10.1016/S0169-4758(99)01503-3) Sallares, R. & Gomzi, S. 2001 Biomolecular archaeology of malaria. Ancient Biomolecules 3, 195–213. Spigelman, M., Matheson, C., Lev, G., Greenblatt. C. & Donoghue, H.D. 2002 Confirmation of the presence of Mycobacterium tuberculosis complex-specific DNA in three archaeological specimens. Int. J. Osteoarchaeol. 12, 393–401. (doi:10.1002/oa.638) Wiechmann, I. & Grupe, G.  2005 Detection of Yersinia pestis DNA in two early medieval skeletal finds from Aschheim (Upper Bavaria, 6th century AD). Am. J. Phys. Anthropol. 126, 48–55. (Published online 30 June 2004.) (doi:10.1002/ajpa.10276) Willerslev, E. & Cooper, A. 2005 Review Paper. Ancient DNA. Proc. R. Soc. B 272, 3–16. (doi:10.1098/rspb.2004.2813) Zink, A.R., Grabner, W. & Nerlich, A.G. 2005 Molecular identification of human tuberculosis in recent and historic bone tissue samples: the role of molecular techniques for the study of historic tuberculosis. Am. J. Phys. Anthropol. 126, 32–47. (doi:10.1002/ajpa.10409) 

Confirmation of the presence of Mycobacterium tuberculosis complex-specific DNA in three archaeological specimens.

Immunocytochemistry and the diagnosis of schistosomiasis: ancient and modern.

Molecular analysis of Mycobacterium tuberculosis DNA from a family of 18th century Hungarians.

Preservation of key biomolecules in the fossil record: current knowledge and future

Pathogenic microbial ancient DNA: a problem or an opportunity?

Abstract

Similar works

Full text

Available Versions

Copenhagen University Research Information System

Crossref

Crossref

UCL Discovery

Crossref

Adelaide Research & Scholarship