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.     

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.     

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.     

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.     

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.     

New data on relevant ancient Egyptian wooden artifacts: Identification of wooden species and study of the state of conservation with multidisciplinary analyses

Wood species identification and characterization of its weathering processes are crucial steps in the scientific approach of conservation of wooden cultural heritage. Many precious wooden objects of ancient Egypt are largely present in museums, nevertheless relatively little information is available concerning the nature of timber used and on their status of conservation. To address this gap, the wooden species of three relevant archaeological wood objects (statue, box, and coffin) arising from different Egyptian archaeological sites dated from the Old Kingdom (2,686–2,181 BC) to New Kingdom (1,550–1,069 BC) were deeply studied. Five hardwood and softwood species were identified belonging to Tamarix mannifera, T. gennessarensis, Ficus sycomorus, Vachellia nilotica, and Cedrus sp. Such data confirmed the recurrence of Vachellia and Tamarix among the most common timbers found in ancient Egypt. Scanning electron microscope, Fourier transform spectroscopy, and synchrotron x-ray radiation diffraction were conducted to evaluate the archaeological wood deterioration. The formation of microcracks, biological degradation patterns (fungal colonization), or chemical characterization (accumulation of salts on and in-between wooden cells) were detected. SEM micrographs showed the presence of fungal hyphae and conidial spores on the wooden cells. Significant changes in the chemical wood composition and decrease in the crystallinity index were detected.     

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.     

Late third‐millennium elite burials in Bahrain

Studies of old aerial photographs of the Bahrain burial mound fields have revealed that a small number of both Early Type (c. 2200–2050 BC) and Late Type (c. 2050–1750 BC) mounds are encircled by an outer ring wall, apparently marking out the mound as belonging to an elite. Four of these mounds have been excavated, and the results are presented. The geological differences between the Early Type and the Late Type mound landscapes are discussed.     

Migration in the Nile Valley during the New Kingdom period: a preliminary strontium isotope study

The value of strontium isotope analysis in identifying immigrants at numerous archaeological sites and regional areas has been demonstrated by several researchers, usually by comparing ⁸⁷Sr/⁸⁶Sr values of human tooth enamel and/or bone with the local strontium isotope signature determined by faunal and environmental samples. This paper examines the feasibility of using ⁸⁷Sr/⁸⁶Sr ratios to investigate residential mobility in the Nile Valley region, specifically at the New Kingdom period (∼1050–1400 BC) archaeological site of Tombos (ancient Nubia). Archaeological and textual information regarding this period indicates that immigrant Egyptians and local native Nubians were likely interacting at this site during a period of Egyptian colonial occupation. The results of this study suggest that non-local individuals may be distinguished from locals using ⁸⁷Sr/⁸⁶Sr values and that colonial agents in the Tombos population were probably both local native Nubians and immigrants.     

First evidence of white wine in ancient Egypt from Tutankhamun's tomb

Wine has been considered to be mainly red in ancient Egypt linked with the blood of Osiris, the God of resurrection. No text that refers to white wines from the Dynastic Period (3150–332 BC) exists. The first white wine from ancient Egypt was made near Alexandria during the third century AD. To investigate the presence of white wine in ancient Egypt, dry residue samples from King Tutankhamun's amphorae are studied in this article using the LC/MS/MS method for wine markers. This investigation into the existence of white wines in Tutankhamun's tomb allows us to shed new light on the symbolism of white wine in ancient Egypt.     

Characterization of ancient building lime mortars of Anhui province,China: A multi-analytical approach

The characterization of lime mortars has become of primary importance in order to obtain information about the raw ingredients and building technology of ancient masonry structures. Five different samples from Anhui province, China, representing two types of lime mortar-based materials: joint mortars between the bricks of the city wall and lime mortar sealing a tomb coffin, were collected for analysis. Archaeological information about the samples was reported and studies into the microtextural features and mineralogical compositions of those mortars were performed via a multi-analytical approach. The joint lime mortars between the bricks of city walls, including Mingzhongdu city wall, Mingzhongdu Xihua gate foundation, Zhengyangguan city wall and She county city wall, were found to be aerial lime mortars. The lime mortar from Nanling Tieguai Song dynasty tomb was analysed and found to be a pozzolanic mortar consisting of lime, clay, sand and cocciopesto-like materials. The analytical results serve as a critical reference for the maintenance and restoration of ancient city walls in Anhui province, and as a starting point for searching for ancient Chinese pozzolanic mortars and technology for making those mortars.     

First ancient bovine DNA evidence from India: difficult but not impossible

Ancient DNA isolation from the tropical countries has been shown to be very difficult in the past. Here for the first time we have been successful in isolating ancient DNA from Indian cattle samples. We were able to obtain DNA and sequence the partial mitochondrial D-loop in 3 of the 15 bovine fossil samples ranging in age from 2000 BC to 1000 AD, and were able to further identify the most recent sample as being of Bos indicus origin. Our results on ancient DNA extraction from India will encourage other researchers in this field to carry out further studies of ancient DNA from Indian bovine samples. Our results represent the first successful extraction and amplification of bovine ancient DNA from India, and thus may pave the road for a better understanding of demographic and historical processes of cattle domestication that has taken place in this region.     

The production technology of Egyptian blue and green frits from second millennium BC Egypt and Mesopotamia

The microstructures and chemical compositions of some 55 Egyptian blue and green frit samples from New Kingdom Egypt and 15th century BC Mesopotamia are determined using analytical scanning electron microscopy, the main focus being on frit cakes, powder residues, and frit vessel and bead fragments from one of the “factory areas” at Amarna in Middle Egypt. Replicate Egyptian blue frits produced in the laboratory are similarly investigated. Comparison of the microstructures of the ancient and replicate samples suggest that frit cakes are the primary product, and that these were ground to produce the powder, and then moulded to shape and refired to produce the vessels and beads. Egyptian blue and green frits are shown to be distinct pigments, the pigment produced depending on the relative proportions of copper oxide and lime in the mixtures. The bulk and glass phase compositions are used to try to infer the different sources of the quartz, lime, copper and alkali flux used in the production of frits from Egypt and Mesopotamia. An estimate is made of the scale of production of Egyptian blue frit in New Kingdom Egypt.     

韩国坟丘墓社会的性质

在古代韩国,马韩盛行过坟丘墓,其主要特征是在地上的坟丘中,由多个埋葬主体通过追加葬形式形成。坟丘墓最晚到公元前1世纪开始出现,其存在从方形木棺向圆形石室的变迁过程。韩国坟丘墓社会中只有百济国发展成为国家百济,因其与先进地区临近,容易接收先进文化,其它小国在酋长社会状态下被吞并,因其农业共同体性质过强,并且与周边国家对等交流困难。     

Essentials in the use of mycolic acid biomarkers for tuberculosis detection: response to “High-throughput mass spectrometric analysis of 1400-year-old mycolic acids as biomarkers for ancient tuberculosis infection” by

High molecular weight long-chain mycolic acids are key structural components of the cell envelope of Mycobacterium tuberculosis and they are established as biomarkers for the identification of both ancient and modern tuberculosis. Mycolic acids from M. tuberculosis have a characteristic profile, reflecting contributions from five major distinct homologous series of mycolate structural types. Diagnosis of tuberculosis in archaeological material, using mycolic acid biomarkers, depends on objective recognition of the key characteristic mycolic acid components. A recent article in this journal claimed that tuberculosis could be confirmed in ancient bones by high throughput mass spectrometric analysis of mycolic acids. Scrutiny of the data presented reveals no convincing evidence for the presence of mycolic acids, characteristic of the M. tuberculosis complex, in the skeletal remains examined. This communication reviews the essential criteria necessary for positive tuberculosis diagnosis, using mycolic acids.     

Paleopathological and Molecular Study on Two Cases of Ancient Childhood Leprosy from the Roman and Byzantine Empires

This study is based on the paleaopathology of leprosy on human skeletal remains and the detection of ancient Mycobacterium leprae DNA. Two cases of childhood leprosy were recognized. The first case was in a Roman necropolis at Martellona (Rome, Central Italy), dated to the 2nd to 3rd centuries ce . The skeleton of a child aged 4–5 years, from tomb 162, is the youngest individual in Italy from this time period, with the clear rhino‐maxillary syndrome and other bony changes indicative of leprosy. The second case from a burial at Kovuklukaya, in the Sinop region of Northern Turkey, was from the 8th to the 10th centuries, during the Byzantine era. The endocranium of a 4–5‐month‐old infant with new bone formation—an indication of chronic inflammation—was positive for M. leprae DNA. Infant and childhood leprosy is uncommon today, and there is a scarcity of information in the osteoarchaeological literature of leprosy in the past, especially in children. The significance of these cases is that it adds to an understanding of the history of the disease in the former Roman Empire. It is hoped that over time sufficient data can be obtained to understand the epidemiological dynamics and clinical evolution of leprosy from the ancient period until today. Copyright © 2012 John Wiley & Sons, Ltd.     

Changes in shell gathering in an early agricultural society at the head of Ise Bay,Japan

We examined changes in shell gathering in an early agricultural society at the head of Ise Bay, Japan based on the analysis of almost 20,000 individual shell remains from four coastal settlements. We analyzed historical changes in the composition and size range of the shell species Meretrix lusoria. The most common species went from Crassostrea gigas in the early Middle Yayoi period (about 400–350BC) to M. lusoria in the mid-Middle Yayoi period (about 320–200BC). It is possible that the seacoast tideland, an environment suitable for C. gigas, was buried in sediment from the rivers and thereby transformed into muddy sand bottoms, an environment more suitable for M. lusoria. We also analyzed historical changes in the size range of M. lusoria. The mean size of M. lusoria increased significantly from the early Final Jomon (about 1250–1000BC) to the late Early Yayoi (about 500–400BC), from the late Early Yayoi to the early Middle Yayoi, and from the early to the mid-Middle Yayoi periods. Evidence suggests that during the Yayoi period, the demand for shells decreased and therefore, the pressure to collect M. lusoria also decreased. We concluded that the larger specimens of M. lusoria were collected selectively.     

郭店一号楚墓年代析论

郭店一号楚墓出土的各类器物,具有战国中期偏晚和战国晚期偏早的若干特征,其中部分因素与包山一、二号墓接近,部分因素又与包山四、五号墓,马山一号墓及九店乙组墓四期六段的若干特征一致。包山二号墓下葬于公元前316年或稍晚;包山四、五号墓,马山一号墓和九店乙组四期六段的墓葬,是战国晚期早段较有代表性的楚墓,按照考古学分期断代以最晚的器物断定其时代下限的一般规律,郭店一号楚墓的时代应属于战国晚期的早段,其下限不晚于白起拔郢,具体年代应在公元前300年稍后不久。     

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.