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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Two cases of leprosy from Žatec (Bohemia), dated to the turn of the 12th century and confirmed by DNA analysis for Mycobacterium leprae

The leprosy known today primarily from tropical areas was a relatively common disease in European Middle Ages. This article describes two skeletons bearing palaeopathological indications of leprosy from a cemetery at ?atec in North-West Bohemia (Czech Republic). The archaeological context clearly shows that these individuals were buried prior to the second decade of the 12th century, and most probably in the second half of 11th century. This rules out the possibility that these individuals might have contracted the disease in connection with the Crusades, in which a Bohemian contingent under Prince Vladislav II participated from 1141 to 1142. Molecular genetic methods were applied to detect specific DNA fragments of the causative agent of leprosy, Mycobacterium leprae. The nasal concha of one individual yielded DNA that could be directly sequenced after isolation and amplification. The vertebral body of the second individual, on the other hand, did not provide DNA of sufficient quality for direct sequencing and only weak amplification was detectable. The morphological and genetic analyses both indicate that leprosy existed prior to the Crusades in medieval Bohemia, albeit that its prevalence was probably not as great as in northern or western Europe.     

The limits of biomolecular palaeopathology: ancient DNA cannot be used to study venereal syphilis

To determine whether ancient DNA (aDNA) can be used to study the palaeopathology of venereal syphilis, we carried out a comprehensive analysis of the preservation of human and pathogen DNA in a set of 46 bones of various ages, most of which displayed osteological indications of the disease. Bones came from seven English cemetery sites that were in use during the 9th–19th centuries. Twelve of the 46 bones consistently yielded mitochondrial DNA (mtDNA) sequences after replicate polymerase chain reactions (PCRs), and a further 13 bones yielded mtDNA sequences with less reproducibility. The sequence data enabled tentative mitochondrial haplogroups to be assigned to nine of the bones, and the identities and frequencies of these haplogroups were compatible with the geographical origins of the bones. Twenty-one bones consistently gave negative results with all mtDNA PCRs, indicating that at least these bones were not contaminated with modern human DNA, and those bones that gave positive results only yielded one sequence each, again suggesting that widespread modern contamination had not occurred. Mycobacterium tuberculosis sequences were obtained from seven bones, including three of five bones with tuberculous lesions. The cloned and direct sequences obtained from both the mtDNA and M. tuberculosis PCR products showed features typical of degraded aDNA. All of these results suggest that at least some of the 46 bones that we studied were suitable for aDNA analysis. All 46 bones were tested with nine different treponemal PCRs, each optimised to give a detection limit of ≤5 genomes. Although various bones gave PCR products of the expected size with one or more of these PCRs, sequencing showed that none of these products were authentic treponemal amplicons. Our failure to detect treponemal DNA in bones that were suitable for aDNA analysis, using highly sensitive PCRs, suggests that treponemal DNA is not preserved in human bone and that it is therefore not possible to use aDNA analysis to study venereal syphilis. Any past or future paper claiming detection of treponemal aDNA should therefore be accompanied by a detailed justification of the results.     

Pre‐Columbian Tuberculosis in Northwest Argentina: Skeletal Evidence from Rincón Chico 21 Cemetery

Systematic excavation of collective burial sites makes possible the recovery of skeletal series which may show bony evidence of infectious pathological conditions. This paper presents the first evidence of the existence of tuberculosis in prehistoric populations of NW Argentina with a subsistence economy based on agriculture and pastoralism. The study was carried out on individuals from Rincón Chico 21 cemetery, a burial site located in the Santa María Valley, Catamarca, used between the Late Ceramic Period and the onset of the Inca empire expansion (AD 1000–1400). Six individuals out of the 70 so far excavated showed destructive lesions in the vertebral bodies and periosteal reactions in other bones. The morphology and distribution of bone lesions led us to rule out several diseases from a broad spectrum of possible diseases that could have affected the skeletal system. Thus, the lesions were interpreted as caused by mycobacterial infections (Mycobacterium tuberculosis Complex). Considering previous studies on the dynamics of biocultural interactions which take into account information related from contextual associations and chronology, we can conclude that a tuberculosis‐like disease was present in prehistoric populations from NW Argentina. Copyright © 2011 John Wiley & Sons, Ltd.     

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.     

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.     

Clarifying prehistoric parasitism from a complementary morphological and molecular approach

This paper reports an approach to the identification of prehistoric parasitic infection, which integrates traditional morphological methods with molecular methods. The approach includes the strengths of each method while mitigating the limitations. Demonstrating the efficacy of this approach, we provide a case study from a 1400 year old desiccated fecal sample from La Cueva de los Muertos Chiquitos, archaeological site, near Rio Zape, Durango, Mexico. Traditionally prepared microscope slides were processed via microscopy and tentative ascarids were identified. Information regarding the parasites' developmental stage was recorded. DNA was then extracted directly from the slide material. From this DNA extract, a small segment of the 18S ribosomal RNA gene variant that is specific to Ascaris, and its phylogenetically close relatives, was targeted for PCR amplification and sequencing. Phylogenetic analysis of the DNA sequence best matched a member of physalopterids, rather than ascarids, with a single exception of a match to Contracaecum spiculigerum. Subsequent extractions, amplifications and sequencing of the original rehydrated coprolite material confirmed these results. The C. spiculigerum sequence represented a phylogenetic anomaly and subsequent analysis determined the sequence was an error in the BLAST database, likely attributable to misidentification of juvenile specimens prior to sequencing and submission. Physaloptera are a difficult genus to identify morphologically and can carry major health burdens. They may be underreported in humans, in part, because of morphological similarities to the more common human parasites belonging to ascarids. We conclude that integrating traditional morphological methods with molecular methods can help resolve this issue, in both contemporary and prehistoric populations.     

Variable nucleotide tandem repeat (VNTR) typing of two palaeopathological cases of lepromatous leprosy from Mediaeval England

Using ancient DNA methods, we have examined in detail two archaeological cases of leprosy from Mediaeval England. The first was a child skeleton with rhino-maxillary changes typical of lepromatous leprosy (LL). The second case was the skeleton of a male adult who showed both typical rhino-maxillary changes and osteitis/periostitis on the leg and foot bones. Bone powder was sampled from both cases and DNA extracts were prepared. These were subjected to a series of polymerase chain reactions (PCRs) specific for regions on the Mycobacterium leprae genome. The repetitive element RLEP was used for confirmation of M. leprae DNA and then three polymorphic regions were successfully amplified and sequenced to determine the number of variable nucleotide tandem repeats (vntr) at these loci. These were the microsatellite regions ML2344 and ML2172 and the minisatellite region ML0058. Genotyping data from the strains preserved within the skeletal remains were compared with those obtained for a reference strain of M. leprae. Variation at these three loci was found between both burials and the reference strain, indicating that vntr typing of LL cases from the archaeological record is a useful way of confirming disease and an additional means of authenticating aDNA data. This demonstrates the feasibility of targeting multiple loci for phylogenetic studies of leprosy strains from archival sources.     

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.     

Digging deeper into the limits of ancient DNA research on syphilis

The search for the origins of syphilis has a long history in the medical and anthropological literatures. If we know more about the emergence of the pathogen that causes the disease in humans we will understand its evolution through time and space as well as shed light on its current state in living populations. Ancient DNA techniques used to isolate Treponema pallidum subsp. pallidum DNA from archaeological human specimens provide direct evidence of its existence in the past. However to date, only Kolman et al. (1999) have been successful in this endeavour, while other attempts have failed (e.g., Barnes and Thomas, 2006; Bouwman and Brown, 2005). Why has there been little success? This paper serves to compliment and add relevant information to Bouwman and Brown's and Barnes and Thomas' discussion concerning our inability to apply ancient DNA techniques to study venereal syphilis in past human populations.Our approach utilized 15 different human specimens from different geographies and different temporal periods: eight samples come from medically diagnosed individuals archived during the American Civil War period; six originate from the United Kingdom and predate 1492 with four of these samples having been previously analyzed by Bouwman and Brown and one sample comes from historic Canada. Human mitochondrial and amelogenin DNA, as well as several genes from the Treponema organism were analyzed revealing the relatively good preservation of human multi-copy and single copy DNA but not treponemal DNA. This study also incorporates a unique molecular experiment using rabbits infected with venereal syphilis to help illustrate that treponemal DNA disseminates to bone early during the first stages of infection but is not present in later stages of the disease using the techniques presented in this study.     

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.