High-throughput mass spectrometric analysis of 1400-year-old mycolic acids as biomarkers for ancient tuberculosis infection

The cell wall of mycobacteria includes an unusual outer membrane of extremely low permeability. This cell envelope consists of a characteristic cell wall skeleton, a mycoloyl arabinogalactan peptidoglycan complex, and related hydrophobic components that contribute to the cell surface properties. In this study 1400-year-old mycolic acids as unique tuberculosis biomarkers have been extracted and identified for the first time by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) and Fourier transform infrared spectroscopy (FT-IR). The data suggest that the MALDI TOF MS has potential as a rapid and reproducible technique for the detection and identification of ancient mycobacterial infections.     

Analysis of ancient mycolic acids by using MALDI TOF MS: response to “Essentials in the use of mycolic acid biomarkers for tuberculosis detection” by Minnikin et al., 2010

The mycobacterial cell wall is consists of several long chain lipid and glycolipid components. Mycolic acids are major and unique fraction of the cell envelope of mycobacteria that include the ancient causative agents of tuberculosis and leprosy. The analytical investigation of these lipid biomarker residues is one of the most promising perspectives in the field of molecular paleopathology. Recently published in this journal, Minnikin et al. present a systematic critique of the MALDI TOF MS analysis for determination of ancient mycolic acids, focusing on our previous paper. In this study, our mass spectrometric investigations by commonly used 2,5 DHB matrix were presented with our comments to the critique authors. On the base of our previous and recent mass spectrometric results we have to realize that the clinical protocols and standards cannot directly be used for the biomolecular paleopathological investigations. The applicability of the recent mycobacterial and clinical results is very limited in the biomolecular archaeology, thus the recent scientific results and protocols should be adapted carefully to bioarchaeological sciences.     

Molecular history of tuberculosis from ancient mummies and skeletons

The origin and evolution of the infectious disease tuberculosis (TB) and its pathogens is still not fully understood. An important effort for a better understanding of the underlying mechanisms of TB evolution lies within the investigation of skeletal and mummified material dating back several thousand of years. In this work, molecular data from mummified and skeletal material from different time periods of the Old World are compared, and the current status of ancient mycobacterial DNA analysis in ancient human remains is discussed, with particular reference to the genetic evolution of human TB. The molecular analysis of material from southern Germany (1400–1800 AD), Hungary (600–1700 AD) and Egypt (3500–500 BC) revealed high frequencies of TB in all time periods. In several individuals from ancient Egypt the mycobacterial DNA could be further characterised by spoligotyping. Thereby, evidence for ancestral M. tuberculosis strains was found in the pre‐ to early dynastic material from Abydos (3500–2650 BC), while typical M. africanum signatures were detected in the Middle Kingdom tomb in Thebes‐West (2050–1650 BC). Samples from the New Kingdom to Late Period tombs (1500–500 BC) were characterised as modern M. tuberculosis strains. In concordance with other studies on ancient skeletal and mummified samples, no evidence for the presence of M. bovis was found. These results contradict the theory that M. tuberculosis evolved from M. bovis during domestication, but supports the new scenario that M. tuberculosis probably derived from an ancestral progenitor strain. Copyright © 2007 John Wiley & Sons, Ltd.     

Tuberculosis among Iron Age individuals from Tyva,South Siberia: palaeopathological and biomolecular findings

The study focuses on the evidence for tuberculosis apparent in an Iron Age population recovered from the cemetery of Aymyrlyg, Tyva (Tuva), South Siberia. A recent wholly molecular study of five of the cases confirmed the presence of Mycobacterium tuberculosis (MTB) complex DNA in four of the individuals. In all cases the disease was caused by strains of Mycobacterium bovis rather than Mycobacterium tuberculosis and represents the first positive identification of the bovine form of the disease in archaeological human remains. Details of the palaeopathological characteristics of the cases are provided in the current paper, while the molecular observations are extended to include a quantitative evaluation of the surviving mycobacterial DNA using real-time PCR. The observation that bovine tuberculosis was the pathogen responsible is discussed in terms of current understanding of the evolution of the MTB complex as well as the implications for future ancient DNA studies in this area.     

Molecular identification and characterization of Mycobacterium tuberculosis complex in ancient Egyptian mummies

A considerable number of molecular studies have provided evidence for the presence of Mycobacterium tuberculosis complex (MTB) DNA in ancient skeletal and mummified material. Moreover first studies on the differentiation of sub‐types of the MTB (M. tuberculosis, M. bovis, M. africanum, M. microti, M. canettii) have successfully been performed on ancient tissue samples. In our present study we extend previous analyses and investigate bone and soft tissue samples from 118 ancient Egyptian mummies and skeletons from the Pre‐ to Early Dynastic site of Abydos and different tomb complexes in Thebes West, which were built and used between the Middle and New Kingdom until the Late Period (c. 2050–500 BC). The samples were tested for the presence of MTB DNA and further identified by spoligotyping. Twenty‐six samples provided molecular evidence for the presence of ancient mycobacterial DNA by amplification of a 123 base pair fragment of the repetitive element IS6110. Out of the 26 positive samples, 12 provided a complete spoligotyping signature, which was compared to an international database. Ten further cases showed an incomplete, patchy hybridization pattern, while in four cases no spoligotyping signature could be obtained. Interestingly, they all show either a M. tuberculosis or M. africanum pattern, but none revealed a M. bovis specific pattern. In the material from a Middle Kingdom tomb (used exclusively between c. 2050–1650 BC) several samples revealed a M. africanum type specific spoligotyping signature, while samples from later periods provided patterns typical for M. tuberculosis. This study clearly shows that spoligotyping can be used for the characterization of members of the MTB in historic tissue samples. In addition, our results do not support the theory that M. tuberculosis originated from the M. bovis type. Copyright © 2004 John Wiley & Sons, Ltd.     

Earliest evidence of spinal tuberculosis from the Aneolithic Yayoi period in Japan

Tuberculosis has existed from early prehistoric days to modern times. The main causative agents of tuberculosis worldwide are Mycobacterium tuberculosis (M. tuberculosis) and M. bovis, along with M. africanum, M. cenettii and M. microti; these species make up the ‘M. tuberculosis complex’. This worldwide infection has been of special interest to palaeopathologists due to its characteristic bone lesions as well as its great antiquity. Historically, tuberculosis has been recognised in Japan for more than a thousand years. However, the origin and early prevalence of tuberculosis remain unknown. In the present study, we present the earliest evidence of skeletal tuberculosis found in the Aneolithic Yayoi period in Japan (ca. 300 BC to AD 300). The skeletal remains showing typical pathological changes of spinal tuberculosis were dated to between 454 BC and AD 124 by dendrochronological methods using coburied arrow-shield board and house columns made of Japanese cedar. We discuss the early prevalence of this infectious disease and its influence on the population history of the Japanese from prehistoric to Aneolithic times. Copyright © 2006 John Wiley & Sons, Ltd.     

Osteological and Biomolecular Study of Two Possible Cases of Hypertrophic Osteoarthropathy from Mediaeval England

Two skeletons from Mediaeval Wharram Percy, England, show osteological lesions consistent with hypertrophic osteoarthropathy. The primary cause of hypertrophic osteoarthropathy is generally chronic pulmonary disease, usually either cancer or infection; in the pre-antibiotic era it was predominantly the latter. Biomolecular analyses indicate the presence of Mycobacterium tuberculosis DNA in one of the specimens, strongly indicating that pulmonary tuberculosis was the eliciting factor in this case. This is the first time that a primary cause for hypertrophic osteoarthropathy has been firmly identified in an ancient skeleton and illustrates the potential of a dual approach using both osteological and biomolecular techniques for enhancing our understanding of early disease. The other specimen proved negative for M. tuberculosis complex DNA, however the presence of infectious rib lesions allowed us to suggest that some non-tuberculous pulmonary infection was likely the primary cause of hypertrophic osteoarthropathy in this case.     

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

This journal published the first reported identification of Mycobacterium tuberculosis complex (MTB) DNA in ancient human remains but concerns were raised about the article two years after publication. These were based on methodology which, in the field of ancient DNA, was still developing. Here we present a re‐examination of the 1993 research conducted on three specimens which exhibited palaeopathologies indicative of tuberculosis. The specimens were: an ulna from pre‐European‐contact Borneo, a spine from Byzantine Turkey, and a lumbar‐sacral spine from 17th century Scotland. There was insufficient material to permit re‐examination of all of the original samples. The earlier results were confirmed in two independent laboratories using different methodologies. MTB DNA complex‐specific DNA amplicons were obtained, and sequenced in both laboratories, in a re‐analysis of samples which supported the earlier findings. Copyright © 2002 John Wiley & Sons, Ltd.     

Deficiencies and challenges in the study of ancient tuberculosis DNA

We explore the standards of research and reporting needed to justify the destructive analysis of archaeological human bone for biomolecular studies of ancient tuberculosis (TB). Acceptable standards in osteological interpretation have been met in some biomolecular papers, but there are also cases where insufficient care has been taken in distinguishing between pathognomonic lesions and those that are ‘consistent with’ a diagnosis of TB. Some biomolecular studies have failed to recognize that archaeological bones might be contaminated with environmental mycobacteria whose DNA could give rise to false positives in polymerase chain reactions directed at members of the Mycobacterium tuberculosis complex. The difficulties of applying spoligotyping to ancient DNA have also been underestimated and conclusions drawn from such analyses are often weakly supported. Assumptions that mycobacterial DNA preserves better than human DNA, and that contamination with modern DNA is less of a problem, has led in some cases to a laxity in research standards with insufficient attention paid to the need to authenticate ancient DNA results. We illustrate our concerns by reference to a recent paper reporting biomolecular detection of ancient TB DNA in skeletons from the eastern Mediterranean Neolithic settlement of Atlit-Yam. We are unconvinced that the skeletal evidence presented in this paper gives sufficient indication of TB to warrant destructive analysis, and we are concerned that during the biomolecular part of the project inadequate attention was paid to the possibility that results might be due to laboratory cross-contamination or to amplification of environmental mycobacterial DNA present in the bones.     

A Mediaeval Case of Lepromatous Leprosy from 13–14th Century Orkney, Scotland

Erosion in the 1960s resulted in exposure of human skeletal remains from a Norse Christian cemetery at Newark Bay, Orkney, Scotland. One set of remains showed osteological evidence of advanced lepromatous leprosy, but the absence of bones from the lower limbs precluded definitive diagnosis. The aim of the present study was to determine whether Mycobacterium leprae could be detected in bone extracts, as a means of confirming the diagnosis of leprosy. Bone samples were examined from the suspected leprosy case and from a second contemporary burial thought to be free of disease. DNA was amplified by polymerase chain reaction (PCR) using primers specific for a repetitive element (RLEP) characteristic of M. leprae. Additional PCR tests specific for Mycobacterium tuberculosis and for amelogenin (a human gene suitable for sex determination) were also applied to the samples. M. leprae DNA was detected only in the skull sample from the suspected leprosy case. The DNA sequence was identical to that found in present day isolates of M. leprae. Positive results were obtained only using a PCR reaction designed to amplify relatively short stretches of DNA (<175 bp), suggesting the microbial DNA had undergone extensive fragmentation. There was no evidence of M. tuberculosis DNA in bones from the leprosy suspect or control individual. The ability to recover ancient samples of DNA provides an opportunity to study long-term evolutionary changes that may affect the epidemiology of microbial pathogens.     

Tuberculosis on the north coast of Peru: skeletal and molecular paleopathology of late pre-Hispanic and postcontact mycobacterial disease

This paper examines skeletal and ancient DNA evidence in the study of suspected tuberculosis infection in the late pre-Hispanic and Colonial-era Lambayeque Valley Complex, north coast Peru (A.D. 900–1750). We integrate information on macroscopic lesion characteristics and distribution, radiographic and CT scan imagery, and analysis of Mycobacterium tuberculosis complex rpoB and IS6110 ancient DNA (aDNA) sequences. Destructive lesions were observed in the vertebral bodies of three precontact indigenous adult males, one colonial adolescent female, and in the cranium of a Colonial-period subadult. Assessment of lesion morphology and distribution led us to consider multiple diseases, but tuberculosis represents the most likely diagnostic option in all individuals. DNA was poorly preserved in all samples, but an IS6110 sequence was amplified in one precontact individual consistent with macroscopic diagnosis. These findings expand the geographic and temporal extent of tuberculosis to the late pre-Hispanic and Colonial north coast of Peru to highlight potential synergisms between diet, settlement patterns, and the evolution of Andean tuberculosis before and after European conquest. Moreover, this study helps focus several key questions in Andean tuberculosis research, including possible reassessment of the presence of the IS6110 sequence in the pre-Columbian Americas. Methodological considerations include differential diagnosis – especially with incomplete skeletons – and limitations of aDNA studies underscoring an approach integrating macroscopic, radiographic, and molecular lines of evidence in the paleopathological investigation of one of humankind’s most devastating and destabilizing diseases.     

Biomolecular archaeology of ancient tuberculosis: response to “Deficiencies and challenges in the study of ancient tuberculosis DNA” by Wilbur et al. (2009)

It is sixteen years since the first detection of Mycobacterium tuberculosis DNA in archaeological specimens, yet the validity of findings continues to be questioned. Rigorous scientific scrutiny and debate is valuable and has led to a coalescence of procedures and precautions amongst those actively engaged in this work. It is disappointing that these good practices are not recognised by certain scientists whose primary expertise is in the related fields of archaeology, palaeopathology, and eukaryote ancient DNA. There is a danger that by constant repetition, disputable and inadequately justified concerns will assume the status of self-perpetuating myths and misunderstandings. We discuss these issues with reference to a recent article in this journal, in which clear peer-reviewed scientific data were specifically targeted as part of a general critique of the field of the palaeomicrobiology of tuberculosis. We believe we have given sufficient evidence and cogent argument to persuade the unbiased reader that the views in the critique by Wilbur et al. are unjustified.     

The use of the polymerase chain reaction (PCR) to detect Mycobacterium tuberculosis in ancient skeletons

The polymerase chain reaction has been used to extract ancient DNA from a wide range of different types of material. We have considered the possibility of finding residual bacterial DNA in bone that may have been infected with Mycobacterium tuberculosis using this technique. We propose a method of collecting samples and testing for the presence of degraded genetic material in ancient bone. The steps of the polymerase chain reaction are detailed and discussed, as are those for the preparation of the bone sample. Results obtained would suggest that this technique could have wide application in osteoarchaeological research by giving us new information on diseases of antiquity. Future avenues for the investigation of bacterial DNA in ancient bone are suggested.     

Investigating archaeological site formation processes on the battleship USS Arizona using finite element analysis

Scientists from the National Institute of Standards and Technology (NIST) worked in a collaborative partnership with archaeologists from the National Park Service's (NPS) Submerged Resources Center (SRC) to develop a finite element model (FEM) of the battleship USS Arizona. An FEM is a computer-based engineering model that calculates theoretical stresses, propagation of force, and shape changes to a structure under loads using thousands or even millions of individual elements whose individual responses are well understood. NIST researchers created an FEM of an 80 ft. (25 m) midships section of the Arizona site to analyze archaeological site formation processes on the sunken battleship, in particular to determine the current condition of the wreck and predict its future strength and structural integrity as it continues to corrode. The NIST's FEM study is one aspect of a larger project under the direction of the NPS, the USS Arizona Preservation Project, whose goal is to determine the nature and rate of corrosion affecting USS Arizona, and to model its long-term structural deterioration. The FEM incorporates findings from other key components of the USS Arizona Preservation Project, such as steel hull corrosion rates, structural surveys of the vessel, sediment compaction studies, and analysis of the concretion that covers the ship's hull, into a single tool that is being used to predict how the wreck will degrade in the future.     

Chemical characterisation of anthro-technosols from Bronze to Middle Age in Bologna (Italy)

The aim of this research was the understanding of a peculiarly expanded soil sequence in Bologna (i.e., Roman Bononia, Etruscan Felsina), one of the most important archaeological poles for Etruscan colonisation and settlement in northern Italy. The uppermost part of the chronological sequence dating from the Bronze Age up to medieval and modern times was analysed for the first time from geochemical and pedological viewpoints. The integration of archaeological, stratigraphical and pedological data supported by soil chemical analysis, a fundamental key for reconstructing a correct evolutionary sequence, made it possible to recognise seventeen buried Soil Units (ancient Technosols, Anthrosols and Cambisols) characterised by different kinds and degrees of intensity of anthropogenic influence. In some cases, the results show a relatively severe element concentration (Cu, Sn) mainly characterising the Iron Age (Etruscan and Villanovan) stratigraphic units, representing an ancient industrial pollution linked to local dispersal of melting activity dump products generated in near-site surrounding areas. Therefore, a comparison was possible between natural blank and anthropogenically polluted geochemical values for some local soils.     

Tikal timbers and temples: ancient Maya agroforestry and the end of time

Tikal, a major lowland Maya civic-ceremonial center in the heart of the Petén region of Central America, relied heavily on the adjacent lowland rainforest as a resource base for fuel and construction materials. In this study, we analyzed 135 wood samples from timbers used in the construction of all six of the city's major temples as well as two major palaces to determine which tree species were being exploited and to better understand ancient Maya agroforestry practices during the Late Classic period. We found evidence for a change in preference from the large-growing, upland forest species, Manilkara zapota, to a seasonal wetland species Haematoxylon campechianum in A.D. 741 as well as a decrease in lintel beam widths over time. Though M. zapota later returned as the wood species of choice in A.D. 810, beam widths were found to be significantly smaller. These findings concur with models that hypothesize widespread deforestation during the Late Classic period and indicate a declining forest resource base by the 9th century A.D. Because of the many large timbers available for temple construction in the 8th century, some system of forest conservation is indicated for the ancient Maya prior to the Late Classic period.     

Molecular markers for the discrimination of Triticum turgidum L. subsp. dicoccum (Schrank ex Schübl.) Thell. and Triticum timopheevii (Zhuk.) Zhuk. subsp. timopheevii

The persistent uncertainty on the classification of the “new” glume wheat found in Neolithic and Bronze Age sites from Greece and other European settlements might be resolved only through analysis of its ancient DNA. Tools able to discriminate among different Triticum species on the basis of scarce, very damaged DNA, are therefore essential. While current attempts concentrate on DNA fragments sequencing and comparison, in some instances PCR-based selective amplification techniques might offer a cheaper and quicker alternative. The purpose of this research was therefore the identification of species-specific primers, able to distinguish caryopses of Triticum timopheevii subsp. timopheevii from those of Triticum turgidum subsp. dicoccum. Primers and their working conditions were defined and optimized using DNA from modern accessions. The ribosomal primers ITS1 tim and ITS2 tim, and the nuclear primer acetyl-coenzyme A tim clearly discriminated the sequences of Triticum timopheevii from other species. Finally, Neolithic charred wheat grains found in the sites of Sammardenchia (Pozzuolo del Friuli, Udine) and La Marmotta (Lago di Bracciano, Roma), belonging to the “new” wheat type or to emmer, were tested with the three selected primers. However, the results were not conclusive, because the samples analysed were apparently too degraded to yield useful DNA.     

The ‘walking’ megalithic statues (moai) of Easter Island

Explaining how the monumental statues (moai) of Easter Island were transported has remained open to debate and speculation, including their resource expenditures and role in deforestation. Archaeological evidence including analysis of moai variability, particularly those abandoned along ancient roads, indicates transport was achieved in a vertical position. To test this proposition we constructed a precise three-dimensional 4.35 metric ton replica of an actual statue and demonstrate how positioning the center of mass allowed it to fall forward and rock from side to side causing it to ‘walk.’ Our experiments reveal how the statue form was engineered for efficient transport by a small number of individuals.     

Ectopic paragonimiasis from 400-year-old female mummy of Korea

Of the parasite eggs discovered in ancient samples from Korean archaeological sites, Paragonimus spp. are of particular importance in that they are regarded as one of the most insidious trematode infection sources. Although their infection prevalence decreased rapidly in the early 20th century, archaeoparasitological studies on the species are still required, as historical knowledge of paragonimiasis remains far from comprehensive. Fortunately, we recently were given a chance to examine a medieval Korean mummy in a good state of preservation. Using morphological techniques, we discovered evidence of ectopic paragonimiasis in the sample from the 17th-century female. When the ITS 2 gene was extracted, amplified and sequenced from Paragonimus eggs, it showed 100% homology to the sequences of modern Paragonimus westermani reported from Korea and Japan, forming a cluster distinct from South Asian P. westermani. Our report is the first-ever analysis of ancient Paragonimus DNA from any archaeological field in the world. The ectopic paragonimiasis diagnosis made in this study, especially involving the liver sample, also is the first of its kind in archaeoparasitology.     

应用热辅助水解甲基化裂解气相色谱质谱技术对古代漆器漆膜的分析研究

为了改良漆器漆膜的性能,古代漆工在制作过程中通常向生漆中加入干性油等添加剂。为了确定生漆及添加剂的种类,本工作采用四甲基氢氧化铵(TMAH)作为甲基化试剂,利用热辅助水解甲基化裂解气相色谱质谱(THM-Py-GC/MS)技术对所选的参考样品进行分析,然后将成熟的方法和实验条件应用到古代漆器的分析研究中。在古代样品中检测出了3-十五烷基邻苯二酚(C15)、3-十五烯基邻苯二酚(C15:1)等来源于中国、日本地区种植的Rhus vernicifera树种的生漆的特征热裂解化合物,确定了所用生漆的种类。另外,本实验在古代漆器中检测到了棕榈酸、硬脂酸、庚酸、辛酸和壬二酸等脂肪酸,推断该漆膜中添加了干性油。利用三维视频显微镜、扫描电镜、X射线衍射分析方法发现古代漆器采用了在胎体上依次髹漆灰、底漆和色漆的髹漆工艺,其中漆灰主要由漆液和钠长石、石英调制而成,黑色漆膜的呈色颜料为含铁化合物,揭示了古代漆器的髹漆工艺。该研究结果不仅增强人们对古代漆器制作材料和工艺的科学认知,而且为保护漆器这一独特的文化遗产提供科学依据。