Mark Spigelman

Detection and Molecular Characterization of 9000-Year-Old Mycobacterium tuberculosis from a Neolithic Settlement in the Eastern Mediterranean

Background Mycobacterium tuberculosis is the principal etiologic agent of human tuberculosis. It has no environmental reservoir and is believed to have co-evolved with its host over millennia. This is supported by skeletal evidence of the disease in early humans, and inferred from M. tuberculosis genomic analysis. Direct examination of ancient human remains for M. tuberculosis biomarkers should aid our understanding of the nature of prehistoric tuberculosis and the host/pathogen relationship. Methodology/Principal Findings We used conventional PCR to examine bone samples with typical tuberculosis lesions from a woman and infant, who were buried together in the now submerged site of Atlit-Yam in the Eastern Mediterranean, dating from 9250-8160 years ago. Rigorous precautions were taken to prevent contamination, and independent centers were used to confirm authenticity of findings. DNA from five M tuberculosis genetic loci was detected and had characteristics consistent with extant genetic lineages. High performance liquid chromatography was used as an independent method of verification and it directly detected mycolic acid lipid biomarkers, specific for the M. tuberculosis complex. Conclusions/Significance Human tuberculosis was confirmed by morphological and molecular methods in a population living in one of the first villages with evidence of agriculture and animal domestication. The widespread use of animals was not a source of infection but may have supported a denser human population that facilitated transmission of the tubercle bacillus. The similarity of the M. tuberculosis genetic signature with those of today gives support to the theory of a long-term co-existence of host and pathogen.

Mycobacterium tuberculosis Complex DNA from an Extinct Bison Dated 17,000 Years before the Present

In order to assess the presence of tuberculosis in Pleistocene bison and the origin of tuberculosis in North America, 2 separate DNA extractions were performed by 2 separate laboratories on samples from the metacarpal of an extinct long-horned bison that was radiocarbon dated at 17,870+/-230 years before present and that had pathological changes suggestive of tuberculosis. Polymerase chain reaction amplification isolated fragments of tuberculosis DNA, which were sequenced, and on which spoligotyping was also performed to help determine its relationship to the various members of the Mycobacterium tuberculosis complex. Extensive precautions against contamination with modern M. tuberculosis complex DNA were employed, including analysis of paleontologic and modern specimens in 2 geographically separate laboratories.

Tuberculosis: from prehistory to Robert Koch, as revealed by ancient DNA

During the past 10 years palaeomicrobiology, a new scientific discipline, has developed. The study of ancient pathogens by direct detection of their DNA has answered several historical questions and shown changes to pathogens over time. However, ancient DNA (aDNA) continues to be controversial and great care is needed to provide valid data. Here we review the most successful application of the technology, which is the study of tuberculosis. This has provided direct support for the current theory of Mycobacterium tuberculosis evolution, and suggests areas of investigation for the interaction of M tuberculosis with its host.

Co–infection of Mycobacterium tuberculosis and Mycobacterium leprae in human archaeological samples: a possible explanation for the historical decline of leprosy

Both leprosy and tuberculosis were prevalent in Europe during the first millennium but thereafter leprosy declined. It is not known why this occurred, but one suggestion is that cross–immunity protected tuberculosis patients from leprosy. To investigate any relationship between the two diseases, selected archaeological samples, dating from the Roman period to the thirteenth century, were examined for both Mycobacterium leprae and Mycobacterium tuberculosis DNA, using PCR. The work was carried out and verified in geographically separate and independent laboratories. Several specimens with palaeopathological signs of leprosy were found to contain DNA from both pathogens, indicating that these diseases coexisted in the past. We suggest that the immunological changes found in multi–bacillary leprosy, in association with the socio–economic impact on those suffering from the disease, led to increased mortality from tuberculosis and therefore to the historical decline in leprosy.

PCR primers that can detect low levels of Mycobacterium leprae DNA

There are several specific PCR-based methods to detect Mycobacterium leprae DNA, but the amplicons are quite large. For example, primers that target the 36-kDa antigen gene and are in common diagnostic use yield a 530-bp product. This may be a disadvantage when examining samples in which the DNA is likely to be damaged and fragmented. Therefore, two sets of M. leprae-specific nested primers were designed, based on existing primer pairs which have been shown to be specific for M. leprae. Primers that targeted the 18-kDa antigen gene gave an outer product of 136 bp and inner product of 110 bp. The primers based on the RLEP repetitive sequence yielded a 129-bp outer product and 99-bp nested product. With dilutions of a standard M. leprae killed whole-cell preparation as the source of DNA, both single-stage and nested PCR were performed after optimisation of the experimental conditions. Compared with the 36-kDa antigen gene primers, the 18-kDa antigen gene outer primers were 100-fold more sensitive and the RLEP outer primers were 1000-fold more sensitive. As an illustration of two possible applications of these new primers, positive results were obtained from three skin slit samples from treated lepromatous leprosy patients and three archaeological samples from human remains showing typical leprosy palaeopathology. It was concluded that these new primers are a useful means of detecting M. leprae DNA which is damaged or present at a very low level.

Mycobacterium tuberculosis complex DNA in calcified pleura from remains 1400 years old

Mycobacterium tuberculosis complex DNA was isolated and identified in calcified pleura from remains 1400 years old, with the polymerase chain reaction. This is the first demonstration of tuberculosis in non‐mummified archaeological tissue other than bone; the presence of mycobacterial mycolic acids in the sample supports this conclusion. The study of ancient DNA from microbial pathogens is of interest as it enables verification of traditional diagnoses, may answer long‐standing questions in the history of disease, and provides ancient DNA sequences that can be compared with those of modern isolates.

Metagenomic Analysis of Tuberculosis in a Mummy

Metagenomic analysis, which involves the sequencing of unenriched DNA from uncultured samples, is probably best known for its use in the analysis of microbes. Here, the method is applied to obtain the sequence of two strains of tuberculosis from a mummified human body.

Tuberculosis in Dr Granville's mummy: a molecular re-examination of the earliest known Egyptian mummy to be scientifically examined and given a medical diagnosis

‘Dr Granvilles mummy’ was described to the Royal Society of London in 1825 and was the first ancient Egyptian mummy to be subjected to a scientific autopsy. The remains are those of a woman, Irtyersenu, aged about 50, from the necropolis of Thebes and dated to about 600 BC. Augustus Bozzi Granville (1783–1872), an eminent physician and obstetrician, described many organs still in situ and attributed the cause of death to a tumour of the ovary. However, subsequent histological investigations indicate that the tumour is a benign cystadenoma. Histology of the lungs demonstrated a potentially fatal pulmonary exudate and earlier studies attempted to associate this with particular disease conditions. Palaeopathology and ancient DNA analyses show that tuberculosis was widespread in ancient Egypt, so a systematic search for tuberculosis was made, using specific DNA and lipid biomarker analyses. Clear evidence for Mycobacterium tuberculosis complex DNA was obtained in lung tissue and gall bladder samples, based on nested PCR of the IS6110 locus. Lung and femurs were positive for specific M. tuberculosis complex cell-wall mycolic acids, demonstrated by high-performance liquid chromatography of pyrenebutyric acid–pentafluorobenzyl mycolates. Therefore, tuberculosis is likely to have been the major cause of death of Irtyersenu.

Leishmaniasis in ancient Egypt and upper Nubia

To the Editor: Leishmaniasis is a disease caused by parasites of the genus Leishmania. The infection is transmitted to humans through the bites of female sandflies and manifests mainly in 3 forms: visceral, cutaneous, and mucocutaneous. Visceral leishmaniasis or kala-azar, the often fatal form of the disease, is caused by species of the Leishmania donovani complex. These parasites were responsible for severe recent outbreaks in Sudan and other countries and are thought to originate in East Africa (1–4). In this report, we describe the successful amplification of L. donovani DNA in ancient Egyptian and Christian Nubian mummies dating back 4,000 years. Besides the first proof for visceral leishmaniasis in paleopathology, we provide evidence that leishmaniasis was present in Nubia in the early Christian period and that the organism also infected ancient Egyptians, probably because of close trading contacts to Nubia, during the Middle Kingdom. We analyzed 91 bone tissue samples from ancient Egyptian mummies and skeletons and 70 bone marrow samples from naturally mummified human remains from Upper Nubia. The Egyptian material derived from the Pre- to Early Dynastic site of Abydos (n = 7; 3500–2800 BC), a Middle Kingdom tomb in Thebes West (42; 2050–1650 BC), and different tomb complexes in Thebes West, which were built and used between the Middle and New Kingdom until the Late Period (42; c. 2050–500 BC). The Nubian samples were taken before the flooding caused by the Aswan Dam from 2 early Christian burial sites at Kulubnarti, between the second and third cataracts of the Nile River in northern Sudan. One site was on an island in the Nile and dated from 550 to 750 AD. The other was on the western bank of the Nile and was in use from c.750 to 1500 AD. All samples were tested for Leishmania spp. DNA and further characterized by direct sequencing. In 4 of the 91 Egyptian and 9 of the 70 Nubian samples, a 120-bp fragment of a conserved region of the minicircle molecule of kinetoplastid mitochondrial DNA of the parasite (5,6) could be successfully amplified and, with the first primer pair, unambiguously related to L. donovani species after sequencing (Figure). The positive samples from ancient Egypt exclusively originated from the Middle Kingdom tomb, while no molecular evidence for ancient Leishmania DNA was found in the Pre- to Early Dynastic and the New Kingdom to Late Period specimens. Figure PCR amplification of a 120-bp fragment of kinetoplastid mitochondrial DNA of Leishmania spp. in Egyptian and Nubian mummies. Lane 1, 50-bp ladder lanes 2–8, mummy samples; lanes 9,10, extraction controls; lane 11, PCR controls. Lane 6 provides ... In the Middle Kingdom, the Egyptians extended trade relationships and military expeditions to Nubia, the modern Sudan, with particular interest in the gold resources of the country and in obtaining slaves to serve as servants or soldiers in the pharaoh’s army. Today, the Sudan is one of the highly endemic countries for visceral leishmaniasis or kala-azar, which is thought to have originated in East Africa and later spread to the Indian subcontinent and the New World (4). Therefore, the high incidence of Leishmania DNA in the Middle Kingdom samples (4 [9.5%] of 42) and the lack of findings in earlier or later time periods, may indicate that leishmaniasis was introduced into Egypt at this time. Leishmaniasis did not likely become endemic in the Egyptian Nile Valley because the disease is closely linked to its vector, the phlebotomine sandfly, and the distribution of Acacia-Balanites woodland (7). That ancient Egyptians became infected because of close trade contacts and associated travel with Nubia during the Middle Kingdom seems more plausible. The high frequency of Leishmania DNA–positive samples in the Nubian mummies (12.9%) suggests that leishmaniasis was endemic in Nubia during the Early Christian period and, in light of the data on the ancient Egyptian mummies, probably already several thousand years before. Taken together, our results support the theory that Sudan could have been indeed the original focus of visceral leishmaniasis (4). Our study shows a completely new aspect of molecular paleopathology. The detection of ancient pathogen DNA is not only used to identify a certain disease and gain information on its frequency and evolutionary origin but also to trace back cultural contacts and their role in the transmission and spread of infectious diseases.

Gymnophalloides seoi Eggs from the Stool of a 17th Century Female Mummy Found in Hadong, Republic of Korea

It was previously reported that paleoparasitological clues for parasites infecting humans could be found in the feces of mummies of the Joseon Dynasty (1392–1910) in the Republic of Korea. Here, we report the presence of trematode eggs, including Clonorchis sinensis, Metagonimus yokogawai, and Gymnophalloides seoi (a human parasite known in Korea since 1993) in the feces of a recently excavated female mummy in Hadong, Republic of Korea. This is the first report of the discovery of a G. seoi infection in a human mummy. Since Hadong is currently not an endemic area for G. seoi, we speculate that the parasite might have occurred frequently along coastal areas of the Korean peninsula several hundred years ago and that the endemic areas contracted to, more or less, restricted regions since that time.