Multiple Cases with Probable Treponemal Infection from 16th to 19th Centuries Romania

In this paper, we describe pathological lesions identified in seven skeletons discovered in the Saint Sava necropolis in Bucharest, Romania, dating to the Late Medieval/Early Modern period. The pathological changes observed in the skeletons were analysed using macroscopic examination. Additionally, computed tomography scanning was performed on two individuals displaying advanced lesions on the cranial surface. For the differential diagnosis, we took into consideration treponemal infection, tuberculosis, osteomyelitis, leprosy, fluorosis, melorheostosis, hypertrophic pulmonary osteoarthropathy, Paget's disease and mycotic infection, along with the possibility of multiple afflictions occurring simultaneously. The morphology and distribution of the lesions are suggestive of treponematosis, which, to our knowledge, makes this the first case of this disease on the Romanian territory in archaeological populations. Copyright © 2015 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.     

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.     

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.     

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.     

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.     

Living and dying as subjects of the Inca Empire: Adult diet and health at Puruchuco-Huaquerones,Peru

Imperial strategies varied as the Inca expanded their empire from the capital in Cusco. Some communities were conquered violently while others were ruled indirectly with little evidence of Inca presence. According to ethnohistorical evidence, the central coast was peacefully annexed by the Incas ca. A.D. 1470, but little is known about how Inca imperialism may have affected the quality of life of subjects of the Inca Empire. We integrate multiple lines of evidence to assess diet, health and disease for a sample of human remains from the Late Horizon cemetery of Puruchuco-Huaquerones, Peru. Specifically, we examine and analyze osteological, dental and stable isotope data (n = 162, 90 and 46 respectively) in order to investigate whether the Inca period population at Puruchuco-Huaquerones experienced nutritionally insufficient diets and poor health under imperial policies. Diet at Puruchuco-Huaquerones incorporated a variety of foods, both plant and animal. Osteological lesions and stature indicate periods of stress, with males experiencing more illness relative to females. Stature sexual dimorphism, the presence of healed lesions and isotope data indicate a sufficiently nutritious diet and support the conclusion that, although disease was present, individuals were healthy enough to survive and recover. Geographical and temporal comparisons suggest that health changed little with the Inca annexation of this region, but future work is needed.     

Human‐Aided Movement of Viral Disease and the Archaeology of Avian Osteopetrosis

The term avian osteopetrosis is used to describe alterations to the skeletal elements of several species of domestic bird, most typically the chicken, Gallus gallus domesticus (L. 1758). Such lesions are routinely identified in animal bones from archaeological sites due to their distinctive appearance, which is characterised by proliferative diaphyseal thickening. These lesions are relatively uncomplicated for specialists to differentially diagnose and are caused by a range of avian leucosis viruses in a series of subgroups. Only some avian leucosis viruses cause the development of such characteristic lesions in osteological tissue. Viraemia is necessary for the formation of skeletal pathology, and avian osteopetrosis lesions affect skeletal elements at different rates. Lesion expression differs by the age and sex of the infected individual, and environmental conditions have an impact on the prevalence of avian leucosis viruses in poultry flocks. These factors have implications for the ways in which diagnosed instances of avian osteopetrosis in archaeological assemblages are interpreted. By integrating veterinary research with archaeological evidence for the presence of avian leucosis viruses across Western Europe, this paper discusses the nature of these pathogens, outlines criteria for differential diagnosis, and offers a fresh perspective on the human‐aided movement of animal disease in the past through investigation of the incidence and geographic distribution of avian osteopetrosis lesions from the first century BC to the post‐medieval period. © 2017 The Authors International Journal of Osteoarchaeology Published by 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.     

Bone reactions on a pliocene cetacean rib indicate short‐term survival of predation event

There is little osteological evidence of non‐lethal predation events in the archaeological or vertebrate paleontological record. A small section of Pliocene cetacean rib collected from the Yorktown Formation within the PCS Phosphate Mine (formerly Lee Creek Mine), Aurora, North Carolina, U.S.A., shows evidence of this kind of trophic interaction. In this case study, we offer a diagnosis of bone traumatic pathology in which three bone‐forming lesions on the partial rib are interpreted as the reaction to a bite inflicted by a macro‐predator, the first such report from a marine environment. In addition to the three well‐defined lesions, a thin layer of woven bone covers much of the remaining cortical bone. The combination of the three bone‐forming lesions and the thin layer of woven bone suggest the presence of an inflammatory process almost certainly caused by infection secondary to the trauma. Survival following the traumatic event was probably less than 6 weeks. The Neogene chondrichthyan fauna from this locality preserves several large predators, including Carcharocles megalodon, Carcharodon carcharias, Isurus xiphidon and Parotodus benedeni, which were all capable, at least in terms of their size, of having bitten the cetacean. Although most of the odontocetes known from the Yorktown Formation were too small to have inflicted this wound, some of the physeterids may have been large enough to have caused the lesions on the partial rib. This evidence of predation in Pliocene whale bone raises the possibility of similar lesions being found in whale bone recovered from archaeological sites. Copyright © 2010 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.     

A DNA test for sex identification in cattle confirms osteometric results

It is of vital importance to be able to sex identify cattle remains to understand the strategies and importance of cattle husbandry in an ancient society. This is usually done from osteoarchaeological assemblages and often relies on measurements of metapodials. The breadth measurement of the distal trochlea is considered an easy way to identify the sex. Bones from males appears to be easily distinguishable from female counterparts, although it has been complicated to find an external control for the morphological results. Here we investigate the reliability of these particular morphometrics for sex identifying cattle bones with molecular genetics. We use a sex discriminating single nucleotide polymorphism in the ZFXY gene and we apply it to DNA from the bones. To keep the fragment size short and suitable for ancient DNA we base the test on a SNP. The test confirms the osteological sex identification in all cases were DNA could be retrieved. This molecular method can also be used when no fragments suitable for osteological sex identification can be found or when the measurements are non-conclusive.     

The first archaeological example of Freiberg's infraction

Freiberg described a condition in which collapse of the juvenile second metatarsal head gave rise to localized pain and swelling.¹ This eponymous disease is traditionally classed as one of the osteochondroses, a group of disparate lesions that typically manifest as destruction of an immature epiphysis. A medieval second metatarsal is presented, which by osteological and radiological examination is diagnostic of Freiberg's infraction. This is the first reported archaeological example of the condition.     

Distinguishing between archaeological sheep and goat bones using a single collagen peptide

We describe a method of isolating and analyzing a single collagen peptide able to distinguish between sheep and goat bone collagen. The 33 amino acid peptide from both sheep and goat collagen was sequenced and shown to differ between the two species at two positions. Analysis of a range of caprines indicated that the sequence changes occurred between the divergence of the Himalayan tahr (Hemitragus jemlahicus) and the ibex (Capra ibex) and that the proposed goat marker is diagnostic of all Capra species and breeds. The survival of these markers in archaeological bones was tested using a set of 26 ovicaprid specimens from Domuztepe, a Neolithic site in south central Turkey. These markers were used to test the osteological determination of 24 of the Domuztepe bones, and determine the species for two immature specimens. The collagen-peptide method has advantages over other non-morphological methods of sheep/goat distinction because of the long-term survival of collagen over other biomolecules such as ancient DNA. The results also highlighted the problems in relying upon one morphological criterion, in this case on the distal radius, to distinguish between sheep and goat bones.     

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.     

New light on the personal identification of a skeleton of a member of Sir John Franklin’s last expedition to the Arctic, 1845

In 1845, an expedition, commanded by Sir John Franklin, set out to try and discover the north-west passage. All 129 men on this ill-fated voyage perished. Over the years, skeletal remains associated with the final throes of the expedition have been located on and near King William Island, Nunavut, in the Canadian arctic. In general, even a tentative personal identification for these remains has proved impossible. An exception is some skeletal remains that were recovered in 1869 and brought back to England and interred beneath the memorial to the Franklin expedition in Greenwich. In the 19th century, these were tentatively identified as of one of HMS Erebus’s lieutenants, Henry Le Vesconte, a conclusion that has been widely accepted in studies of the Franklin voyage. Renovations to the monument in 2009 provided an opportunity for scientific examination of the remains, and to re-evaluate the personal identification made nearly 140 years before. The current work, which is the first modern scientific analysis of a fairly complete skeleton associated with the Franklin voyage, describes the remains and the artefacts interred with them, discusses the pathological conditions present, and evaluates the personal identification using osteological techniques and isotope geochemistry. Results indicate that the remains are of an adult male of European ancestry. Although some writers have suggested that scurvy or tuberculosis may have been important causes of morbidity and mortality on the Franklin expedition, osteological analysis and, in the case of tuberculosis, DNA analysis, provided no evidence for their presence in these remains. Isotopic studies indicate that the personal identification as Le Vesconte is unlikely to be correct. From the isotopic results and forensic facial reconstruction, HDS Goodsir, an assistant surgeon on the expedition, appears a more likely identification, but the results do not allow a firm conclusion.     

Trace elements in palaeopathology: Quantitative analysis of a case of hypertrophic osteoarthropathy by instrumental neutron activation analysis

Instrumental neutron activation analysis (INAA) of the bones of a 6th century case (AD ) of hypertrophic osteoarthropathy (HOA) showed high levels of Zn, Mn and K in the periosteal new bone. The concentration of Zn in the periosteal new bone appeared to be biogenetic in origin and connected with active mineralization processes in the abnormal bone tissue. Manganese and potassium concentrations were, at least partly, related to diagenetic processes.     

In sickness or in health? Assessment of Early Iron Age human skeletons from Toutswe sites,east central Botswana

The general health of Early Iron Age (AD 700 to 1300) mixed farmers in east central Botswana has not been studied before. In this study the bones and teeth of 84 individuals from ten Toutswe sites were analysed for osteological manifestations of disease, with the aim of assessing the general health of the Toutswe communities. These individuals were aged between newborn and 75 years old, and include both sexes. Results indicate the presence of diseases commonly found in archaeological populations such as osteoarthritis, spina bifida occulta, cribra orbitalia, porotic hyperostosis and enamel hypoplasias. One possible case of DISH was found. The frequency of lesions was comparable with or even lower than at other similar sites, such as K2 and Mapungubwe and other parts of southern Africa. The samples represent communities with relatively low levels of stress and infections. This seems to support the results of the palaeodemographic analysis, which indicated that the Toutswe people were better off than those of neighbouring K2 and Mapungubwe. Copyright © 2008 John Wiley & Sons, Ltd.     

Scurvy as a factor in the loss of the 1845 Franklin expedition to the Arctic: a reconsideration

In 1845, an expedition, commanded by Sir John Franklin, set out to discover the Northwest Passage. The ships entered the Canadian Arctic, and from September 1846 were beset in ice off King William Island. A note left by the expedition in May 1847 reported all was well, but by April 1848, 24 of the 129 men had died. The ice‐locked ships were deserted in April 1848, but the 105 survivors were so weakened that all perished before they could reach safety. The causes of the morbidity and mortality aboard the ships have long been debated, and many commentators have argued that scurvy was an important factor. This study evaluates the historical evidence for the likely effectiveness of anti‐scorbutic precautions taken on polar voyages at that time, and investigates whether the skeletal remains associated with the expedition provide evidence for scurvy. Skeletal remains available for study were carefully examined for pathological changes, and lesions potentially consistent with scurvy were subject to histological analysis. Where remains were no longer accessible, use was made of published osteological work. It is argued that the anti‐scorbutic measures customarily taken on mid 19^th century British naval polar voyages were such that there is no a priori reason to suppose that scurvy should have been a problem prior to the desertion of the ships. The analysis of the skeletal evidence provided little in the way of bony lesions consistent with the disease, and cannot therefore be used to support the presence of scurvy. Factors other than scurvy may been the main causes of morbidity and mortality in the 11 months prior to the desertion of the ships. Copyright © 2012 John Wiley & Sons, Ltd.