Within the last few years a considerable number of molecular studies have provided evidence for the presence of Mycobacterium tuberculosis complex DNA in ancient skeletal and mummified material (1,7,9,18,27,28,34,38,41,47). Besides the mere evidence of M. tuberculosis complex DNA, initial information suggested a high frequency of tuberculosis in ancient populations (13), and we have recently provided evidence that this also holds true for pharaonic Egypt (47 (20), which is based on the variation of the direct-repeat (DR) region in M. tuberculosis complex members. Using this technique, differentiation up to a subspecies level is possible. Spoligotyping is widely used and accepted in medical microbiology for the initial genotyping of the M. tuberculosis complex at the population level. In addition, spoligotyping seems to be the most suitable method for analyzing ancient material, since usually only minute amounts of a significantly fragmented mycobacterial ancient DNA (aDNA) remains in the samples under investigation. Likewise, other recent methods, such as IS6110 restriction fragment length polymorphism (43), ligation-mediated PCR (31), and variable number of tandem repeat typing (24), require cell culture conditions or at least high-molecular-weight bacterial DNA and are therefore not applicable to ancient tissue material.In addition, spoligotyping seems to be suitable for investigating evolutionary aspects of human tuberculosis (36) and may clarify the origin and transmission of the disease in humans of various historical periods and populations (35). When its results are combined with other data, they can be used to construct phylogenetic trees reaching back to the beginning of the pathogenesis and spread of the disease in humans and animals (37).In this regard, there is still an open debate about the origins of tuberculosis in human and animal species. One previous hypothesis (5) suggests that M. bovis is the probable ancestor which was transmitted from cattle to humans during domestication. Other theories assume that an M. tuberculosis complex precursor evolved from M. africanum and that the present-day M. tuberculosis and M. bovis developed in parallel (39). This theory is supported by nucleotide sequence analyses of current M. tuberculosis isolates, revealing an absence of allelic variation. The evolutionary origin of M. tuberculosis was therefore suggested to be 15,000 to 20,000 years ago (39).In a recent paper, a new evolutionary scenario was presented based on the occurrence of several deletions in the genomes of the tubercle bacilli (4). These findings allow a differentiation of M. tuberculosis strains into modern and ancestral strains depending on the presence or absence of an M. tuberculosis-* Corresponding author. Mailing address:

We describe the molecular identification of the M. tuberculosis complex DNA in bone tissue samples from recent and historic populations. In a first set, archival paraffin material from vertebral bodies of 12 recent cases with clinically/microbiologically proven tuberculosis was compared to 12 further cases without tuberculosis. While eight TB cases revealed a specific mycobacterial amplification product, none of the controls was positive. Interestingly, one case with tuberculous sepsis (Landouzy sepsis), five cases with tuberculous spread beyond the primarily affected organ (i.e., lymph node or miliar involvement), and also two of six cases with restricted pulmonary tuberculosis reacted positively in the vertebral specimens. This indicates that a molecular analysis can detect mycobacteria even in unremarkable bone tissue, proving that organ tuberculosis is present. In addition, the extent of spread is of high significance for the frequency of positive reactions. In addition, we investigated a series of vertebral samples coming from an Egyptian population of the necropolis of Thebes-West dating to approximately 1450-500 BC. In this group of 36 cases, three of five cases with typical macromorphological signs for tuberculous spondylitis, 2 of 12 cases with nonspecific alterations, and 2 of 19 cases without macroscopic pathology revealed a specific amplicon of the M. tuberculosis complex. This suggests a significant frequency of infected people in that ancient population. Finally, a fourth group of 51 long bone samples with pathological alterations coming form a southern German ossuary (between AD 1400-1800) was investigated, and 10 cases were positive for the M. tuberculosis complex. These studies of historic material clearly support the notion that tuberculous infections can be unequivocally identified by molecular techniques. The relatively high frequency of ancient bacterial DNA amplifications in unremarkable bone is well-explained by our analysis of the recent material. Our data form an important basis for the investigation of tuberculosis frequency and spread in historic periods.

We describe the molecular identification of human tuberculosis (TB) from vertebral bone tissue samples from three different populations of ancient Egypt. The specimens were obtained from the predynastic to early dynastic necropolis of Abydos (7 individuals, c. 3500-2650 B.C.), from a Middle Kingdom to Second Intermediate Period tomb of the necropolis of Thebes-West (37. c. 2100-1550 B.C.) and from five further Theban tombs used in the New Kingdom and the Late Period (39, c. 1450-500 B.C.). A total of 18 cases tested positive for the presence of ancient DNA (aDNA) of the M. tuberculosis complex. Out of the 9 cases with typical macromorphological signs of tuberculous spondylitis, 6 were positive for mycobacterial aDNA (66.7%). Of 24 cases with non-specific pathological alterations, 5 provided a positive result (20.8%). In 50 cases of normally appearing vertebral bones 7 tested positive (14.0%). There were only minor differences in the frequencies between the three populations. These data strongly support the notion that tuberculosis was present and prevalent in ancient Egypt since very early periods of this civilization. The unexpectedly high rate of mycobacterial aDNA in normal bone samples is presumably due to a pre- to perimortal systemic spread of the bacteria and indicates a generalized infection by M. tuberculosis.

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.

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