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.     

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.     

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.     

Multiple bone tuberculosis in a child from predynastic Upper Egypt (3200 BC)

Tuberculosis, one of the most ancient human diseases, was present in ancient Egypt and has been observed since predynastic times. Excavations in the predynastic to early dynastic necropolis of Adaima, Upper Egypt (3500–2700 BC) led to the discovery of a number of remarkably well‐preserved skeletons of children. The skeletal remains of a 4.5–5‐year‐old child dated from Nagada III A2 (3200–3100 BC) displayed various lesions on the post‐cranial skeleton: spondylitis on the thoracic (T12) and lumbar (L1) vertebrae, partial lytic destruction of the right radio‐ulnar joint, lytic lesions on the scapula and a clavicle, dactylitis on the short bones of hands and feet, enlargement (spina ventosa) and periosteal new bone formation on the long bones. Radiographs show well‐defined radiolucent (cyst‐like) lesions in the metaphysis and the diaphysis of long tubular bones (ulna, radius, femur, tibia, fibula). The lesions recorded during macroscopic and radiological analysis strongly suggest a case of multiple bone tuberculosis. The occurrence of this case of tuberculosis in a child provides a picture of a period where tuberculosis must have been endemic throughout the population living during the origins of urban settlement in Upper Egypt during the predynastic period. Copyright © 2009 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.     

Dental morphological evidence for biological continuity between the A-group and C-group periods in lower Nubia

Data are presented on non-metric dental morphological traits in skeletal samples from the Lower Nubian A-Group (ca. 3100–2500 BC) and C-Group (ca. 2000–1500 BC) cultural periods. In order to examine possible biological differentiation between these two periods, their trait frequencies were analysed using the mean measure of divergence statistic. Results indicate biological continuity, consistent with in situ evolution, although the problem of small samples requires that these results be accepted with caution. Although the diffusion of ideas or material culture into the area through military and trade contacts is likely, any archaeologically observable cultural differences between these two phases are more consistent with local cultural evolution than with the importation of a new cultural system through the migration of a foreign population into the area. The timing of a population replacement in ancient Nubia that has been identified by other scholars should now be sought either before the A-Group period or after the C-Group.     

The 4.2 ka BP Climate Event in Egypt: Integration of Archaeological,Geoarchaeological, and Bioarchaeological Evidence

The 4.2 ka BP climate event was a phase of severe global drought. Its evidence has been attested in many regions of the world and was reported to have played an important role in the collapse of major ancient civilizations. This article provides a comprehensive literature review using several proxies on the 4.2 ka BP climate event in Ancient Egypt. It applies a multidisciplinary approach that integrates archaeological, geoarchaeological, and bioarchaeological evidence. The conclusion is that the event had critical environmental, economic, and political impacts. It led to aridification, a decline in the Nile level and Nile-fed lakes, encroachment of aeolian desert sand into the Nile Valley, lower crop yields, and famine across ancient Egypt. Therefore, as a compound event, it played an important role in the collapse of the Old Kingdom (ca. 2181 BC), and its effect continued in the First Intermediate Period (ca. 2181–2055 BC) when Egypt witnessed political fragmentation.

     

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.     

The hyaena in dynastic Egypt: Fancy food or fantasy food?

The extensive city of Tell el‐Amarna was constructed by the Pharaoh Akhenaten, and occupied for only 15 years until his death in 1332 BCE. Among the sites excavated, there is the ‘Workmen's Village’, a small detached village with closely grouped houses, yards and animal pens. Among the identified animal bones excavated from there are those of the striped hyaena, Hyaena hyaena. Tomb and temple scenes, mainly from the Old Kingdom, show hyaenas as ritual offerings or associated with hunting events, even under close husbandry, restraint and special feeding. The meaning of these scenes is considered in relation to the definite evidence for butchery and muscle stripping seen on these bones. Hyaena meat may well have posed a threat to the consumer as a probable carrier of trichinosis, a disease now known from mummified human tissues from ancient Egypt. Copyright © 2010 John Wiley & Sons, Ltd.     

Cats of the Pharaohs: Genetic Comparison of Egyptian Cat Mummies to their Feline Contemporaries

The ancient Egyptians mummified an abundance of cats during the Late Period (664 - 332 BC). The overlapping morphology and sizes of developing wildcats and domestic cats confounds the identity of mummified cat species. Genetic analyses should support mummy identification and was conducted on two long bones and a mandible of three cats that were mummified by the ancient Egyptians. The mummy DNA was extracted in a dedicated ancient DNA laboratory at the University of California - Davis, then directly sequencing between 246 and 402 bp of the mtDNA control region from each bone. When compared to a dataset of wildcats (Felis silvestris silvestris, F. s. tristrami, and F. chaus) as well as a previously published worldwide dataset of modern domestic cat samples, including Egypt, the DNA evidence suggests the three mummies represent common contemporary domestic cat mitotypes prevalent in modern Egypt and the Middle East. Divergence estimates date the origin of the mummies' mitotypes to between two and 7.5 thousand years prior to their mummification, likely prior to or during Egyptian Predyanstic and Early Dynastic Periods. These data are the first genetic evidence supporting that the ancient Egyptians used domesticated cats, F. s. catus, for votive mummies, and likely implies cats were domesticated prior to extensive mummification of cats.     

Loose‐footed Sails of the Egyptian New Kingdom Ships

The relief from the temple of Ramesses III (1184–1153 BC) in Medinet Habu is often considered to be the most ancient representation of a brailed sail. According to many researchers, the loose‐footed sail and the system of brails were invented in the ancient countries of the Syro‐Canaanite coast and were later adopted by the Ancient Egyptians. However, iconographic material from Ancient Egypt testifies to the indigenous origin of a loose‐footed sail. The article presents an unknown relief from the Amarna period (1352–1336 BC) that further supports this hypothesis.     

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.     

First identification of an ancient Egyptian mummified human placenta

In the course of excavations at Thebes‐West, Upper Egypt, a human organ was recovered from the poorly preserved torso of a female mummy, which was archaeologically dated to the New Kingdom. In the field, the organ was tentatively identified as a liver, but without much certainty. After rehydration and fixation, histological observations led to a rejection of this diagnosis and resulted in the hypothesis that this organ could be a placenta. Comparative histology, performed on an experimentally mummified modern human placenta, revealed a close similarity of microstructural features, which strongly supports the diagnosis of the organ as a placenta. In this paper, we can therefore present the first report of an ancient Egyptian mummified human placenta and provide new insight into Egyptian funeral practices in general, and the fate of the excavated female in particular. Copyright © 2004 John Wiley & Sons, Ltd.     

Did Ancient Egyptian Ships have Keels? The evidence of Thonis‐Heracleion Ship 17

This paper presents the debate over the presence or absence of keels in the construction of Egyptian ships of the dynastic period. It contains an analysis of ship representations and models dating to the 18th–20th dynasties of the New Kingdom (c.1539–1077 BC) that provide evidence for the introduction of keels in Egyptian shipbuilding, and new archaeological material from excavations at Thonis‐Heracleion that sheds more light on the question.     

On the probable genotype of domestic cats in ancient Egypt

Genetic and archaeological evidence suggests that the majority of mummified ritual cats (Felis libyca subsp.) in ancient Egypt carried the t⁺ allele and exhibited the striped tabby phenotype. A few of these ritual cats, however, appear to be F. chaus subsp., most likely carried the T^a allele and were characterized by the Abyssinian phenotype. Several types of evidence suggest that the T^a allele first became fixed in a Felis sp. population in either southeast Asia or northeast Africa. A number of Felis subspecies appear to be plausible candidates for the population of T^a origin.     

Biological continuity between the A- and C-groups in lower Nubia: Evidence from cranial non-metric traits

Data are presented on 45 cranial non-metric traits in skeletal samples of the Lower Nubian A-Group (ca. 3100–2500 BC) and C-Group (ca. 2000–1500 BC) cultural periods. Results, based on the mean measure of divergence statistic, reveal biological continuity, consistent with interpretations of in situ evolution as opposed to models of discontinuity resulting from gene flow. Any differences between the A-and C-Group cultural phases that may be evident in the archaeological record are therefore more likely the result of local cultural evolution, rather than the introduction of new ideas, customs, and materials through the migration of a new population into this area. The problem of small samples necessitates that our results be accepted cautiously.