Carney Matheson

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.

Hominin dispersal into the Nefud Desert and Middle palaeolithic settlement along the Jubbah Palaeolake, Northern Arabia.

The Arabian Peninsula is a key region for understanding hominin dispersals and the effect of climate change on prehistoric demography, although little information on these topics is presently available owing to the poor preservation of archaeological sites in this desert environment. Here, we describe the discovery of three stratified and buried archaeological sites in the Nefud Desert, which includes the oldest dated occupation for the region. The stone tool assemblages are identified as a Middle Palaeolithic industry that includes Levallois manufacturing methods and the production of tools on flakes. Hominin occupations correspond with humid periods, particularly Marine Isotope Stages 7 and 5 of the Late Pleistocene. The Middle Palaeolithic occupations were situated along the Jubbah palaeolake-shores, in a grassland setting with some trees. Populations procured different raw materials across the lake region to manufacture stone tools, using the implements to process plants and animals. To reach the Jubbah palaeolake, Middle Palaeolithic populations travelled into the ameliorated Nefud Desert interior, possibly gaining access from multiple directions, either using routes from the north and west (the Levant and the Sinai), the north (the Mesopotamian plains and the Euphrates basin), or the east (the Persian Gulf). The Jubbah stone tool assemblages have their own suite of technological characters, but have types reminiscent of both African Middle Stone Age and Levantine Middle Palaeolithic industries. Comparative inter-regional analysis of core technology indicates morphological similarities with the Levantine Tabun C assemblage, associated with human fossils controversially identified as either Neanderthals or Homo sapiens.

Assessing PCR Inhibition from Humic Substances

Inhibition remains the greatest methodological challenge in molecular analysis of buried biological remains. Inhibitory compounds associated with soil environments comprise primarily of humic acids and fulvic acids, collectively referred to as humic substances. We examined the sensitivity of 13 DNA polymerases to both humic acids (11ng-110� g) and fulvic acids (9.4ng-94� g) and the concentration at which successful amplification can be achieved. This research identified that all 13 DNA polymerases tested exhibited inhibition with varying concentrations of humic acids and that 5 out of the 13 DNA polymerase tested exhibited inhibition with varying concentrations of fulvic acid. The most tolerant DNA polymerase to inhibition due to the presence of humic and fulvic acids is pfu DNA polymerase followed by KlenTaq ® LA DNA polymerase and RealTaq DNA polymerase that were both only inhibited by 11� g and 110� g of humic acids. In addition, we present the use of size exclusion chromatography to remove small molecular weight humic substance, dramatically increasing the success of molecular analysis on material associated with burial. This research has implications to the fields of environmental microbiology, soil science, forensic science and archaeological science.

Molecular Exploration of the First-Century Tomb of the Shroud in Akeldama, Jerusalem

The Tomb of the Shroud is a first-century C.E. tomb discovered in Akeldama, Jerusalem, Israel that had been illegally entered and looted. The investigation of this tomb by an interdisciplinary team of researchers began in 2000. More than twenty stone ossuaries for collecting human bones were found, along with textiles from a burial shroud, hair and skeletal remains. The research presented here focuses on genetic analysis of the bioarchaeological remains from the tomb using mitochondrial DNA to examine familial relationships of the individuals within the tomb and molecular screening for the presence of disease. There are three mitochondrial haplotypes shared between a number of the remains analyzed suggesting a possible family tomb. There were two pathogens genetically detected within the collection of osteological samples, these were Mycobacterium tuberculosis and Mycobacterium leprae. The Tomb of the Shroud is one of very few examples of a preserved shrouded human burial and the only example of a plaster sealed loculus with remains genetically confirmed to have belonged to a shrouded male individual that suffered from tuberculosis and leprosy dating to the first-century C.E. This is the earliest case of leprosy with a confirmed date in which M. leprae DNA was detected.

Ancient Leishmaniasis in a Highland Desert of Northern Chile

Background Leishmaniasis is an infectious disease endemic today in many areas of South America. Methodology We discovered morphologic and molecular evidence of ancient infections in 4 female skulls in the archaeological cemetery of Coyo Oriente, in the desert of San Pedro de Atacama, Northern Chile. The boney facial lesions visible in the skulls could have been caused by a number of chronic infections including chronic Leishmaniasis. This diagnosis was confirmed using PCR-sequenced analyses of bone fragments from the skulls of the affected individuals.Leishmaniasis is not normally found in the high-altitude desert of Northern Chile; where the harsh climate does not allow the parasite to complete its life cycle. The presence of Leishmaniasis in ancient skulls from the region implies infection by the protozoan in an endemic area–likely, in our subjects, to have been the lowlands of North-Eastern Argentina or in Southern Bolivia. Conclusions We propose that the presence of the disease in ancient times in the high altitude desert of San Pedro de Atacama is the result of an exogamic system of patrilocal marriages, where women from different cultures followed their husbands to their ancestral homes, allowing immigrant women, infected early in life, to be incorporated in the Atacama desert society before they became disfigured by the disease. The present globalization of goods and services and the extraordinary facile movement of people across borders and continents have lead to a resurgence of infectious diseases and re-emergence of infections such as Leishmaniasis. We show here that such factors were already present millennia ago, shaping demographic trends and the epidemiology of infections just as they do today.

Technical note: removal of metal ion inhibition encountered during DNA extraction and amplification of copper-preserved archaeological bone using size exclusion chromatography.

A novel technique for the removal of metal ions inhibiting DNA extraction and PCR of archaeological bone extracts is presented using size exclusion chromatography. Two case studies, involving copper inhibition, demonstrate the effective removal of metal ion inhibition. Light microscopy, SEM, elemental analysis, and genetic analysis were used to demonstrate the effective removal of metal ions from samples that previously exhibited molecular inhibition. This research identifies that copper can cause inhibition of DNA polymerase during DNA amplification. The use of size exclusion chromatography as an additional purification step before DNA amplification from degraded bone samples successfully removes metal ions and other inhibitors, for the analysis of archaeological bone. The biochemistry of inhibition is explored through chemical and enzymatic extraction methodology on archaeological material. We demonstrate a simple purification technique that provides a high yield of purified DNA (>95%) that can be used to address most types of inhibition commonly associated with the analysis of degraded archaeological and forensic samples. We present a new opportunity for the molecular analysis of archaeological samples preserved in the presence of metal ions, such as copper, which have previously yielded no DNA results.

Titanic's unknown child: The critical role of the mitochondrial DNA coding region in a re-identification effort

This report describes a re-examination of the remains of a young male child recovered in the Northwest Atlantic following the loss of the Royal Mail Ship Titanic in 1912 and buried as an unknown in Halifax, Nova Scotia shortly thereafter. Following exhumation of the grave in 2001, mitochondrial DNA (mtDNA) hypervariable region 1 sequencing and odontological examination of the extremely limited skeletal remains resulted in the identification of the child as Eino Viljami Panula, a 13-month-old Finnish boy. This paper details recent and more extensive mitochondrial genome analyses that indicate the remains are instead most likely those of an English child, Sidney Leslie Goodwin. The case demonstrates the benefit of targeted mtDNA coding region typing in difficult forensic cases, and highlights the need for entire mtDNA sequence databases appropriate for forensic use.

Evolutionary changes in the genome of Mycobacterium tuberculosis and the human genome from 9000 years BP until modern times

The demonstration of Mycobacterium tuberculosis DNA in ancient skeletons gives researchers an insight into its evolution. Findings of the last two decades sketched the biological relationships between the various species of tubercle bacilli, the time scale involved, their possible origin and dispersal. This paper includes the available evidence and on-going research. In the submerged Eastern Mediterranean Neolithic village of Atlit Yam (9000 BP), a human lineage of M. tuberculosis, defined by the TbD1 deletion in its genome, was demonstrated. An infected infant at the site provides an example of active tuberculosis in a human with a naïve immune system. Over 4000 years later tuberculosis was found in Jericho. Urbanization increases population density encouraging M. tuberculosis/human co-evolution. As susceptible humans die of tuberculosis, survivors develop genetic resistance to disease. Thus in 18th century Hungarian mummies from Vác, 65% were positive for tuberculosis yet a 95-year-old woman had clearly survived a childhood Ghon lesion. Whole genome studies are in progress, to detect changes over the millennia both in bacterial virulence and also host susceptibility/resistance genes that determine the NRAMP protein and Killer Cell Immunoglobulin-like Receptors (KIRs). This paper surveys present evidence and includes initial findings.

Archaeobotany in Australia and New Guinea: practice, potential and prospects

Abstract Archaeobotany is the study of plant remains from archaeological contexts. Despite Australasian research being at the forefront of several methodological innovations over the last three decades, archaeobotany is now a relatively peripheral concern to most archaeological projects in Australia and New Guinea. In this paper, many practicing archaeobotanists working in these regions argue for a more central role for archaeobotany in standard archaeological practice. An overview of archaeobotanical techniques and applications is presented, the potential for archaeobotany to address key historical research questions is indicated, and initiatives designed to promote archaeobotany and improve current practices are outlined.