Identifying the cause of past epidemics replies on the specific detection of pathogens in buried individuals, so-called paleomicrobiology, an emerging field of research which gained from technological advances in microbiology. For almost 15 years, the detection, identification and characterization of microbes in ancient environmental and human specimens emerged on the basis of ancient DNA analyses. Limitations in ancient DNA amplification and contamination led to explore alternative methods of detection and characterisation of non-DNA biomolecules including mycolic acids for detecting ancient mycobacteria and proteins. Also, immunochemistry, immuno-chromatography, enzyme linked immunosorbent assay and immuno-PCR have been developed for the specific detection of microbes in ancient human and environmental specimens. As for ancient DNA, strict protocols have to be enforced in order to ensure the authenticity of data. We herein review that the plural analysis of non-DNA biomolecules from ancient microbes, opens new opportunities for the identification of ancient microbes and new venues to help resolving the controversies about the cause of some historical pandemics and to study the co-evolution of microbes with hosts.
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Paleomicrobiology, the quest for microbes in ancient environmental and human remains, is an emerging field of research which gained from technological advances in microbiology. The most abundant corpus of research used molecular biology techniques, aimed to detect and to analyse ancient microbial DNA (Drancourt and Raoult, 2005). This DNA-based approach is prone to contamination of the ancient material by environmental DNA, including the aDNA previously PCR-amplified in the laboratory (Green et al., 2009) which may lead to false positive results. Also, the natural decaying of DNA resulting from chemical modifications and from its fragmentation, along with the presence of poorly characterised PCR inhibitors in ancient specimens, may limit the molecular detection of microbial DNA in some ancient specimens (Paabo, 1989). A theoretical limit of the D- and L-enantiomers (D/L) of aspartic acid ratio > 0.08 has been calculated, beyond which ancient DNA could be non-detectable (Poinar et al., 1996). For example, it was shown that ancient treponema DNA was not preserved in human bone specimens dating dating back to the 9th -19th centuries (Bouwman and Brown, 2005). For these reasons, interpreting the results of some initial studies has been questioned (Spencer and Howe, 2004) and strict rules of experimental processes and interpretation of data have been enforced in order to ensure the authenticity of data (Drancourt and Raoult, 2005).
Bibliography indicates a shift in the methods used in paleomicrobiology from aDNA analysis forwards non-DNA biomolecule analyses (Figure XX). [NY, DECRIRE LE RESULTAT DE VOTRE RECHERCHE BIBLIOMETRIQUE]. In fact, methods based on the immunodetection of microbial antigens have been early developed in paleomicrobiology (Fornaciari and Marchetti, 1986;Fornaciari et al., 1989;Dumler, 1991;Dumler et al., 2003;Bruschi et al., 2006;Cerutti et al., 2007;Bianucci et al., 2008b;Bianucci et al., 2009;Fornaciari et al., 2010a) and these approaches are now in the framework of extensive demonstration that ancient proteins at large, including plant and animal proteins could persist across geological times (Schweitzer et al., 2007;Asara et al., 2007a;Asara et al., 2007b;Organ et al., 2008;Schweitzer et al., 2009; Travis2010). Several studies further indicated that proteins may better resist than aDNA to taphonomic decaying (Nielsen-Marsh et al., 2005;Organ et al., 2008;Heaton et al., 2009).
These observations opened new perspectives for the detection and characterization of microbial non-DNA biomolecules in ancient remains. Two lines of methods have been developed, i.e. the immunodetection aiming to the detection of pathogen specific antigens and the analytical techniques aiming to analyze a set of pathogen specific biomolecules. Methods and first results on such non-DNA microbial biomolecules detection in ancient remains are herein reviewed. Analysed papers have been retrieved through Pubmed and Medline databases using the following combined keywords: ancient, past, virus, parasite, bacteria, infection, pathogen, proteome, proteomics, DNA contamination, paleomicrobiology, quantification protein, immunodetection, immunological assay, late serology, mass spectrometry, MALDI-TOF, mycolic acid, dental pulp. References from a previous review (Drancourt and Raoult, 2005) as well as references from selected papers were also reviewed. We searched within the database of Journal of Archaeological Science (Journal homepage: http://www.elsevier.com/locate/jas) and the database of American Journal of Physical Anthropology (Journal homepage: http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1096-8644). The selected papers were evaluated by the criteria for strength and quality of evidence based on previously publication (Drancourt and Raoult, 2005) (Box 1) (Table 1).
Ancient proteins for paleomicrobiology.
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The techniques. After a seminal study suggesting that ancient proteins retained some integrity and antigenic specificity (Smith and Wilson, 1990), the demonstration that proteins could be detected in some insects preserved in amber (Poinar et al., 1996) paved the way towards the analysis of non-DNA, ancient biomolecules (Heaton et al., 2009). Paleopathologists have used a variety of methods to demonstrate the presence of microbial antigens and immunoglobulins in archeological tissues (Willcox, 2002). A first group of methods aims to the detection and quantification of antigenic epitopes of selected microbial proteins extracted from ancient specimens. Indeed, microbial antigens can be extracted from ancient specimens and their presence can be detected by several techniques including immunochromatographic detection, enzyme-linked immunosorbent assay (ELISA), and XXXXXXX [NY, LA IL FAUT DETAILLER LES METHODES D'EXTRACTION ET LES TECHNIQUES COMME VOUS L'AVEZ FAIT POUR BIANUCCI]. As for immunochromatographic detection, a protocol indicated that 50 mg of bone material or 7.5 mg of ancient dental pulp were reconstituted in 200 ml of sterile saline solution and subjected to three freeze/thaw cycles (1 min each cycle); sonication for 15 min, a fourth freeze/thaw cycle; 24 - hours incubation at 4°C in order to solubilise the remaining antigens; centrifugation at 5,000 rpm et room temperature and then they could use the supernatant with the dipstick assay (Bianucci et al., 2009). In this study, the yield of protein extraction was not reported and how to stock the protein for the next using ????. The capacity of immunodetection to trace minute quantities of microbial materials could be further increased by the application of the immuno-PCR, a technique combining the advantages of immunodetection with the logarithmic, PCR-based amplification of the signal (Sano et al., 1992). Immuno-PCR was first presented as a technique in which a specific antibody-DNA conjugate is used to detect antigen (Sano et al., 1992). Accordingly, immuno-PCR has a sensitivity grater than any existing antigen detection system because of the tremandous amplification capability and specificity of PCR (Sano et al., 1992). For example, we are developing quantitative immuno-PCR for the detection of the plague agent Yersinia pestis in the dental pulp of past plague's victims (D. Raoult, unpublished data).
A derived method aims to the detection of immunoglobulins in ancient specimens. Indeed, it was demonstrated that immunoglobulins could be extracted from ancient bone tissues (Torres et al., 2002) [NY, DETAILLER LA TECHNIQUE; EST-CE UN DES PROTOCOLES PRESENTES PLUS HAUT ? SI OUI? LE COPIER/COLLER]. It was further demonstrated that in these immunoglobulins could retain their antibody property of binding to a specific antigen, thus founding so-called late serology (Kolman et al., 1999). In this approach, immunoglobulins extracted from ancient bone specimens are made reacting with a pathogen-specific antigen (Kolman et al., 1999).
A third group of methods uses immunohistochemistry for detecting microbial antigens and even entire microbial cells without extraction directly in ancient tissues [NY, DETAILLER UN PEU LES TECHNIQUES: IMMUNOPEROXYDASE ET MICROSCOPIE OPTIQUE; IMMUNOGOLD ET MICROSCOPIE ELECTRONIQUE].
Immunodetection techniques may however yield false negative results since antigenic proteins may be extracted from ancient tissues in concentration below that detectable by techniques. This major challenge may explain the few publications in paleomicrobiology concerning this method (Lepidi, 2008). Also, false positive result may result from cross-reactivity between primary antibody and non-specific antigen in ancient samples. For example, the initial claim of detection of haemoglobin in 4,500-year-old human bones (Ascenzi et al., 1985) was further shown to be a false result due to cross-reactivity of the primary antibody with non-haemoglobin bone proteins (Lendaro et al., 1991); former study lack appropriate negative controls, such as identical tissue in non-related, individuals (Fornaciari et al., 2010b). In the experiments of immunodetection, assessing the specificity of the primary antibody is crucial for the accurate interpretation of the data. As for ancient pathogens, the primary antibody has to be specific for the targeted pathogen and thus not cross-react with other microorganisms and not cross-react with non-pathogen components of the tissue under analysis. This can be demonstrated by testing a collection of relevant organisms prior to the test and by the incorporation of the appropriate negative controls into the test. At last, the secondary antibody has to have no cross-reactivity with the tissue under analysis, as demonstrated by another negative control consisting in the incubation with non-infected, ancient tissue. In contrast, immunological detection methods are easy to perform as they require a few specific equipments, for example ELISA technique or immunohistochemistry technique (Cerutti et al., 2007). Also, it is possible to localize microbial cells in ancient, mummified and fixed tissues (Dumler et al., 2003; Fornaciari and Marchetti, 1986). A third advantage of the immunological detection is the capacity to accurately detect a pathogen from even closely related organisms, depending on the specificity of the primary antibody. This fact has been illustrated by the late diagnosis of fatal Rocky Mountain spotted fever in the remains of patient died in 1901 with an erroneous diagnosis of typhus due to Rickettsia prowazekii (Dumler, 1991). A last advantage is its capacity to detect minute quantities of antigenic material. Immunodetection could thus complement that of aDNA, by providing an independent demonstration with a low risk of cross-contamination; and by providing additional data in the case of false-negative aDNA-based detection.
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Although several studies reported the use of the matrix-assisted XXXX (MALDI-TOF MS) analysis of ancient animal proteins or peptides, these techniques have been used only once for the detection of ancient microbial proteins (Hollemeyer et al., 2008). The application of mass spectrometry in the field of archaeology and evolutionary biology is the work of Hollemeyer et al. 2008 which identified species from 5300-year-old Tyrolean mummy, also called iceman or Oetzi (Hollemeyer et al., 2008). With the successful obtain of mass data from individual hairs in their study, these authors suggested that mass spectrometry constitutes a powerful analytical method for the rapid and high-throughput characterization of ancient samples (Hollemeyer et al., 2008). It is however noteworthy that the accurate identification of ancient animals is contributive to the study of potential reservoirs of ancient pathogens as illustrated by the rapid identification of mammals which could have been ancient reservoirs for ancient Bartonella organisms [PAPIER DE DANG SUR LES CHATS; PAPIER MD + HENRI DE LUMLEY SUR B. QUINTANA]. [NY, IL FAUT QUE NOUS REPARLIONS DE CETTE PARTIE]. We indeed developed a simple and efficient protocol to extract total ancient proteins from ancient dental pulp specimens for the MALDI-TOF MS analysis. After incubating the dental pulp with 1 ml of 500 mM EDTA, pH 8.0 with agitation at room temperature for 24 hours, the specimen is then sonicated five times for 1 min each and centrifuged at 17,900 x g for 40 min at room temperature. The specimen is then dialysied overnight in 2 L of a solution containing 50 mM Tris-Hcl, pH 8.0, and 150 mM NaCl. After proper extraction, protein concentration can be determined using the Bradford protein quantification protocol (Bradford, 1976). With dental pulp of eight individuals from four burial sites dating between 700 and 8,500 years ago, we obtained the protein concentrations of 0.28 ± 0.12 g/l yielded by Bradford protein quantification protocol (Figure 1). We stocked the extract protein at -80°C for the future using. In our experiments, the stock of the extract protein from ancient specimens at -80°C yielded for one year reproducible MALDI-TOF MS profiles and we have obtained an identifying MALDI-TOF peptidic profile from a two 8,500-year-old human dental pulp specimens, including two deciduous teeth collected in two different Neolithic children in Syria (6500 BC)(T-N-N. Tran and M. Drancourt, unpublished data). However, when applying the same ways to ancient microbial proteins, there are some difficulties because of low concentration of microbial proteins in total extract or ancient proteins. For example, MALDI-TOF MS comparison of dental pulp peptides profiling between a negative control group and ancient teeth collected from plague victims failed to find the different between two groups. A recent publication analysed ancient mycobacterial proteins in ancient remain was achieved by using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI TOF/TOF MS) (Boros-Major et al., 2010). Just as for microbial protein analyses, an advantage of analytical techniques of protein is less subject to false positives or contamination by comparing with PCR techniques (Schweitzer, 2004).
Protein-based detection of ancient pathogens. Immunodetection has been mostly applied to fastidious organisms as illustrated by the immunohistochemical detection of the syphilis bacterium Treponema pallidum in the mummy of Maria d'Aragona (1503-68) recovered from an abbey in Naples, Italy (Fornaciari et al., 1989). Further demonstration was achieved in a 200-year-old skeleton [NY, IL FAUT EN DIRE UN PEU PLUS](Kolman et al., 1999). [NY, QU'EST-CE QUE CES TRAVAUX NOUS APPRENNENT ?]. The same approach has been used to detect the emerging bacterium Tropheryma whipplei responsible for so-called Whipple's disease, in the intestinal tissues preserved from the necropsy index case described by Dr. Whipple in 1907; numerous intracellular bacteria were observed and a majority of the foamy macrophages identified in the widened lamina propria stained intensely (Dumler et al., 2003). This work confirmed that the disease such as described by Dr Whipple was indeed due to T. whipplei (La Scola 2001). Likewise, an immunoperoxydase test detected Rickettsia rickettsii, the Rocky Mountain spotted fever bacterium but not Rickettsia prowazekii, the typhus bacterium, in different tissues collected from a patient who died in 1901 with the diagnosis of typhus (Dumler, 1991). This work illustrated the capacity of these techniques to provide an accurate, retrospective diagnosis of an deadly infection in forensic cases. As for ancient viruses, the immunostaining of skin incubated with anti-vaccinia-virus antiserum followed by protein-A/gold and electron microscopy has been successfully employed in the study of the detection of intact smallpox virus particles in an Italian mummy dated from the sixteenth century (Fornaciari and Marchetti, 1986). This result demonstrated that the antigenic structure of the viral particles is well preserved in mummies tissues (Fornaciari and Marchetti, 1986).
Detection of the plague bacterium Yersinia pestis-specific F1 antigen has been helpful to demonstrate that several burial sites in Europe were plague sites including the Black Death sites. (Pusch et al., 2004;Bianucci et al., 2008b;Bianucci et al., 2009). Using a rapid diagnostic test for plague (RDT), plague was demonstrated in four Benedictine nuns and two priests in two burial sites in France, Poitiers and La Chaize-le-Vicomte, between 1578 and 1632 (Bianucci et al., 2008c). ELISA and immunohistochemical analysis was also used to identify Y. pestis in ancient plague victims in Venice (San Leonardo in Fossa Mala, XIV century) and Genoa (Bastione dell'Acquasola, XIV century), Italy in addition with a subject suspected Y. pestis infection from Briançon (France, XVII century) (Cerutti et al., 2007). They used four specimens from the same period deriving from graves not related to epidemics for negative controls (Cerutti et al., 2007). ELISA has been also used for the detection of circulating schistosome antigen in Egyptian mummies (Deelder et al., 1990). Cheek and colon from a mummy (1198 - 1150 BC) in which Schistosoma haematobium eggs had been found and shin tissue from a late predynastic (3200 BC) were used; negative control samples were gut tissue from mummies from the Atacama (2000 BC) (Deelder et al., 1990). Likewise, the detection of the histidine-rich protein-2 antigen (PfHRP-2) derived from Plasmodium falciparum for the identification of malaria in samples from naturally desiccated 1,450 -5200-year-old mummies from Egypt and Nubia, stands as a particularly successful use of a dual antibody immunoassay in paleopathology (Miller et al., 1994). These authors suggested that the stability over several thousand years of PfHRP-2 indicated the potential value of paleo-immunological diagnosis in investigating the distribution of such an important disease as malaria during prehistoric and even Palaeolithic times (Miller et al., 1994). These authors used gut tissue from unembalmed, desiccated mummies >3,000 years old from the site of Camarones in the Atacama desert near Arica, northern Chile as negative control (Miller et al., 1994). A recent example of detection past pathogens via immunological technique is the use of two different qualitative-double antibody immunoassays for examine the presence of Plasmodium falciparum ancient proteins in the skeletal remains of four members of the Medici family, Italia 16th century (Fornaciari et al., 2010a;Fornaciari et al., 2010b). Because of pig is the unique reservoir for T. solium; the finding of this parasite suggested that swine farming was current in Hellenistic Egypt, as supported by other archeological evidences confirmed an ancient case of cysticercosis in an Egyptian mummy of 20 year-old woman about who lived in the late Ptolemaic period (second to first centuries B.C.) (Bruschi et al., 2006) [NY, IL FAUT REPRENDRE CET EXEMPLE, IL N'EST PAS COMPLET].
Other non-DNA biomolecules for paleomicrobiology.
The techniques. The detection of mycolic acids and mycocerosic acids, two components of the tuberculosis bacterium Mycobacterium tuberculosis cell wall, has been used as a surrogate to detect this pathogen in ancient human skeletons [REF; Kremer et al., 2000). Because of these lipids are particularly robust, they appeared as ideal biomarkers in complementing the detection of M. tuberculosis DNA for the diagnosis of ancient tuberculosis (Redman et al., 2009). With mycolic avec biomarkers, there are some advantages for the detection of M. tuberculosis. First, it can avoid the problem of contamination with DNA/PCR technique. Second, this non-DNA biomolecule is robust and the technique is sensitive for detection M. tuberculosis in ancient remains (Donoghue et al., 2010) Indeed, mycolic acids have been detected in ancient human remains by using high performance liquid chromatography (HPLC) and mass spectrometry (Gernaey et al., 2001;Donoghue, 2008;Mark et al., 2010). These approaches are sensitive enough to detect the molecules directly without amplification (Donoghue, 2008). In a limited number of studies, these biomarkers have been used in complement to the detection of M. tuberculosis DNA (Donoghue et al., 2009).[NY, IL FAUT DETAILLER UN PEU].
Mycolic acid-based detection of ancient tuberculosis. Human tuberculosis caused by Mycobacterium tuberculosis, is thought to have emerged in Africa at the end of the Neolithic Period with further evolution towards M. tuberculosis complex organisms with specialized ecological niches (Gagneux and Small, 2007). For example, the findings of M. tuberculosis DNA the Karkur calcified pleura (AD 600) by PCR were confirmed by parallel HPLC-detection of mycolic acids (Donoghue et al., 1998), establishing the first detection of M. tuberculosis in a non-mummified tissue other than bone (Donoghue et al., 1998). After testing two biomarkers for detection M. tuberculosis in the ribs of skeletons 1000-year-old derived from Addingham, West Yorkshire, from an early medieval (Anglo-Saxon) cemetery, Gernaey et al. 2001 concluded that mycolic acids seemed more reliable to diagnose ancient tuberculosis than IS6110 and this biomarker could be of value in tracing paleomicrobiology of tuberculosis back into antiquity (Gernaey et al., 2001). Another study detected M. tuberculosis DNA and used HPLC as an independent method of confirmation by directly detecting mycolic acids in the ribs, arm bones (adult) and long bones (infant) from human skeletons at the site of Atlit-Yam, 10 km south of Haifa, calibrated radiocarbon dates range from 9250-8160 years BP, indicating a date during the last phase of the Pre-Pottery Neolithic C period (Hershkovitz et al., 2008). Mycocerosic acid has been detected to trace M. tuberculosis in the ribs from 49 individuals of the 19th and 20th century Coimbra Identified skeletal collection, half with documentary data indicating tuberculosis as a cause of death (Redman et al., 2009). They have found mycocerosic acids in archaeological remains for the first time and they showed that mycocerosic acid are valuable biomarkers for the diagnosis of ancient tuberculosis (Redman et al., 2009). Also, ancient mycolic acids from archaeological bone samples (1,400-year-old) have been detected by MALDI-TOF-MS (Mark et al., 2010). Their data suggested that the MALDI-TOF-MS has potential as a rapid and reproducible technique for the detection and identification of ancient mycobacterial infections (Mark et al., 2010).
The first publication of the application MALDI-TOF technique in the field of paleomicrobiology is the work of Mark et al. 2010 (Mark et al., 2010). In fact, these authors analyzed five anthropological bone samples from different archaeological periods (from 600 AD to 1600 AD) in which four samples were investigated for DNA of Mycobacterium tuberculosis complex and the test was positive in all cases (Mark et al., 2010). After mycolic acid extraction from these bones, they used MALDI-TOF mass spectrometry to obtain the spectra and they compared with the spectra obtained from negative control which was non-infected, healthy bone samples from the processed cemeteries (Mark et al., 2010).[NY, IL FAUT REFERENCER ET COMMENTER LA RECENTE CRITIQUE DE CE TRAVAIL ++].
Provisional recommendations for using non-DNA biomolecules in paleomicrobiology.
As for the analysis of ancient microbial DNA, the common types of sample preserved in archaeological records and that may contain ancient proteins, are bone tissues (Brandt et al., 2002;Schweitzer et al., 2007;Asara et al., 2007a;Asara et al., 2007b;Organ et al., 2008;Schweitzer et al., 2009). Another source of ancient protein might come from mummified tissues. In the case of mummified tissues, an autopsy revealed a bundle of linen bandages, enveloping a hollow muscular organ, identified as the stomach was used to diagnosis the cysticercosis in an Egyptian mummy dating back to the late Ptolemaic period (second to first centuries B.C.) (Bruschi et al., 2006). Other sources for ancient protein might be materials such as hair (Hollemeyer et al., 2008), teeth (Bianucci et al., 2009), skin tissues (Fornaciari and Marchetti, 1986) and other mummified tissues: muscle (Bianucci et al., 2008a), brain, lung (Miller et al., 1994). However, the degradation of ancient proteins is important and it is the challenge for this domain (Willcox, 2002). In our lab, we use dental pulp as materials of choice for the study in paleomicrobiology (Figure 2).
4.2. Principles for obtaining reliable data in immunodetection
Principles for obtaining reliable data in analytical techniques
Due to the difficulty of obtaining ancient microbial DNA and the critical question of contamination by using PCR, it is not always possible to utilise the PCR/sequencing technique to determine the cause of past infections. For example, in the study investigated the cause of Black Death, the authors used PCR method and the dipstick assay (F1 antigen) for the 17th century skeletal remains, their results suggested that in 83% of samples the PCR reactions started from less template molecules than is needed for positive identification (Pusch et al., 2004). Even if detection and characterisation of DNA is the most widely used approach for the study of ancient pathogens (Drancourt and Raoult, 2008), for the infectious diseases that kill rapidly leave no traces in teeth, bone, or coprolites, which is problematic (Cunha and Cunha, 2008) in DNA technique. The studies of past infection via the way of macromolecules can be a complement method to resolve the problems in the past. As for immunodetection technique and late serology, these techniques can only answer one question at a time. However, a versatile immunodetection technique named auto-immunochemistry has been developed for the diagnosis of modern infectious diseases (Lepidi, 2008). The other difficulty in paleoproteomic is the reservation of ancient protein; it is the same difficulty when we investigate about the ancient samples. Immunodetection of infectious agent in ancient specimens have proven that ancient proteins can retain their integrity and antigenic speficity and it may open the new door beyond of DNA for paleomicrobiology. But, until now there are fewer studies concerning the detection of past pathogens by proteomic methods than studies by DNA methods in the same field. Lepidi gave one explanation about this limitation that may be due to the antigenic determinants in ancient tissues are often impaired or destroyed (Lepidi, 2008). Since it has been shown that antibodies could survive for centuries, this method could potentially be applied to ancient specimens, in order to demonstrate the presence of microorganisms. This technique relies on the immunodetection of a pathogen by using the patient's serum. This approach could be sometimes applied to ancient diseases specimens.