Mycobacterium leprae genotype amplified from an archaeological case of lepromatous leprosy in Central Asia

We have amplified Mycobacterium leprae DNA from the skeleton of an adult human female exhibiting signs of lepromatous leprosy (LL). The remains were excavated from the site of Devkesken 6 on the Ustyurt plateau of Uzbekistan and date to between the 1st and 4th centuries AD. Recovered DNA was fragmented but of sufficient quality and quantity to allow a series of biomolecular genotyping methods to be applied. These methods included variable nucleotide tandem repeat (VNTR) typing of two microsatellite and one minisatellite regions and also single-nucleotide polymorphism (SNP) typing for nine informative loci.Genotyping showed that the causative strain of M. leprae exhibited a SNP-type 3 profile, characteristic of cases associated geographically with Europe and North Africa. Further SNP sub-typing was performed and the data obtained from the Uzbek leper was compared with the same loci amplified from a case of LL recovered from Blackfriars, Ipswich, UK dating to between the 13th and 16th centuries AD. Unique group 3 subtypes were found in both the Uzbek case and Ipswich 1914. These appear to be ancestral to recent type 3 strains. Mycolic acid analysis confirmed the presence of M. leprae in the Uzbek samples. Phylogenetically informative SNPs and other polymorphic loci will contribute to the study of human migrations, as well as the origin and spread of leprosy.     

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.     

Paleopathological and Molecular Study on Two Cases of Ancient Childhood Leprosy from the Roman and Byzantine Empires

This study is based on the paleaopathology of leprosy on human skeletal remains and the detection of ancient Mycobacterium leprae DNA. Two cases of childhood leprosy were recognized. The first case was in a Roman necropolis at Martellona (Rome, Central Italy), dated to the 2nd to 3rd centuries ce . The skeleton of a child aged 4–5 years, from tomb 162, is the youngest individual in Italy from this time period, with the clear rhino‐maxillary syndrome and other bony changes indicative of leprosy. The second case from a burial at Kovuklukaya, in the Sinop region of Northern Turkey, was from the 8th to the 10th centuries, during the Byzantine era. The endocranium of a 4–5‐month‐old infant with new bone formation—an indication of chronic inflammation—was positive for M. leprae DNA. Infant and childhood leprosy is uncommon today, and there is a scarcity of information in the osteoarchaeological literature of leprosy in the past, especially in children. The significance of these cases is that it adds to an understanding of the history of the disease in the former Roman Empire. It is hoped that over time sufficient data can be obtained to understand the epidemiological dynamics and clinical evolution of leprosy from the ancient period until today. Copyright © 2012 John Wiley & Sons, Ltd.     

Ancient-DNA reveals an Asian type of Mycobacterium leprae in medieval Scandinavia

Leprosy is a chronic infection of the skin and peripheral nerves caused by the pathogen Mycobacterium leprae. Its impact on human populations and societies of the past as well as its phylogeographic patterns around the world – at least in modern times – has been well documented. This slow growing bacterium has been shown to exist in distinct ‘SNP types’ that occur in relatively defined parts of the globe. The routes that the disease followed in the past are, however, still uncertain. This study of ancient-DNA typing of archaeological human remains from Sweden dated to early Medieval times provides genetic evidence that a transmission of M. leprae ‘SNP subtype’ 2G – found mainly in Asia – took or had already taken place at that time from the Middle East to Scandinavia. This finding is unique in the history of leprosy in Europe. All human specimens from this continent – both modern and ancient – that have been tested to date showed that the one responsible for the infection strains of M. leprae belong to ‘SNP type’ 3, whereas our results show that there were some European populations that were hosts to bacteria representing ‘SNP type’ 2 of the species as well.     

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.     

Metastatic carcinoma in a leper skeleton from a Medieval cemetery in Chichester England

Both leprosy and metastatic cancer are well documented in the literature on human skeletal palaeopathology. The manifestation of both pathological conditions in a single archaeological skeleton has not been reported. A case from a Medieval site in Chichester, England exhibits bone lesions and patterns of skeletal involvement indicative of both these diseases. Evidence of leprosy is largely restricted to the lower tibiae, fibulae and the bones of the feet. Fine destructive foci and reactive fibre bone associated with metastatic cancer are distributed in many of the remaining areas of the skeleton. There is minimal overlap in the areas of the skeleton involved in the two pathological processes.     

Lepromatous leprosy in an early mediaeval cemetery in Central Italy (Morrione, Campochiaro, Molise, 6th–8th century AD)

The aim of this study is to investigate and discuss the cases of leprosy diagnosed on two skeletal individuals from an early mediaeval necropolis at Morrione (Campochiaro, Molise, Central Italy), dated to the 6th–8th century AD. At present only two inhumation areas have been found in Campochiaro: one at Morrione and the other at Vicenne. Since no signs of a stable settlement or built-up area have been found, it seems the cemeteries can be attributed to a semi-nomadic Lombard-Avar group. This site may have represented a military outpost to control the area against Byzantine invasions. The skeletons of a female (40–46 years) and a male (50–55 years), from tombs 68 and 108, revealed several bony changes indicative of leprosy with clear rhino-maxillary syndrome in both specimens. There is a scarcity of information in the osteoarchaeological literature of leprosy in ancient Italy. The significance of these cases is that they add to an understanding of the history of the disease in Italy and to the data set necessary to understand the epidemiological dynamics in the Mediterranean basin during the ancient period.     

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.     

Wild or domesticated? An ancient DNA approach to canid species identification in South Africa's Western Cape Province

The fragmentary remains typical of archaeological fauna can prove impossible to identify when confronted with closely related sympatric species. In southern Africa domesticated dogs (Canis familiaris) and black-backed jackals (Canis mesomelas) pose just such a problem, rendering difficult an accurate assessment of the timing of arrival of domesticated dogs in the region. Contextual evidence has suggested that canid remains from four Later Stone Age archaeological sites were likely to be domesticated dogs rather than jackals, and were subjected to ancient DNA analyses to determine species. Every tested specimen proved to derive from black-backed jackals. These data provide not only an unexpected window on southern African prehistory, but also highlight the value in applying ancient DNA techniques to archaeological species identification.     

Skeletal manifestations of vitamin D deficiency osteomalacia in documented historical collections

The frequency with which changes related to vitamin D deficiency are recorded in juvenile bone from archaeological contexts makes it clear that conditions conducive to such deficiency were fairly widespread at a number of points in the past. Although changes will take longer to be manifest in the adult skeleton than in juveniles, and may not be as obvious, the scarcity of reported cases suggests that it is likely that cases of osteomalacia are being overlooked in archaeological human bone. Vitamin D is probably better described as a hormone, rather than a vitamin, and the production of vitamin D within the body following exposure to sunlight allows adequate mineralisation of bone to take place. Lack of exposure to sunlight, which can be caused by a range of factors, is probably one of the main causes of vitamin D deficiency. The result of such a deficiency is a general weakening of the skeleton. The range of skeletal changes recorded across different bones of the skeleton in two documented historical pathology collections (the Galler collection, Basel, and the collection of the Federal Museum for Pathological Anatomy, Vienna), are discussed for scapulae, vertebrae, ribs, sterna, pelves and femora. The likelihood of each feature being preserved in archaeological skeletal material is considered. Although the changes associated with osteomalacia may lead to fragmentation of the skeleton, the presence of characteristic changes on bones from across the skeleton should make the condition identifiable using macroscopic examination, even where the skeleton is not well preserved. The identification of cases of osteomalacia in archaeological skeletal material is potentially significant because of the socio‐cultural information that can be implied from diagnosis of the condition. Copyright © 2005 John Wiley & Sons, Ltd.     

Leprosy in a skeleton from the 7th century necropolis of Vicenne‐Campochiaro (Molise,Italy)

Analysis of the skeleton from tomb 144 of the early medieval necropolis of Vicenne‐Campochiaro in Central Italy revealed several features indicative of leprosy. The skeleton belongs to a male estimated to be between 20 and 25 years of age at death. The distal halves of the 1st and 2nd left metatarsals present acro‐osteolysis and both legs show severe subperiosteal bone reaction. The facial skeleton shows changes compatible with a chronic inflammatory process, possibly due to an infectious disease. The anatomical distribution of the lesions and their association with other skeletal lesions seems to be compatible with a near‐lepromatous form of leprosy. A differential diagnosis is made, and the skeletal traits pathognomonic of leprosy are discussed. Copyright © 2005 John Wiley & Sons, Ltd.     

DNA analysis of archaeological sheep remains from China

Recent research has thrown considerable light on the history of the domestic sheep, but has not extended to ancient sheep specimens. In the present study, ancient DNA analysis was carried out on eight archaeological sheep remains recovered from Erlitou archaeological site in Henan Province (ca. 2100–1800 B.C.) to explore the genetic structure of ancient sheep and the phylogenetic relationship between ancient and modern sheep. We analyzed the control region sequences and coding regions of mitochondrial DNA from the remains by direct sequencing and restriction fragment length polymorphism analysis, respectively. Our results reveal that all ancient sheep belong to lineage A defined by modern sheep sequences. Phylogenetic analysis shows that neither argali (Ovis ammon) nor urial (Ovis vignei) mtDNA is closely related to Erlitou ancient sheep. In addition, our results suggest that ancient DNA analysis can serve as a powerful tool in tracing prehistoric population movement.     

Molecular and morphological analyses of avian eggshell excavated from a late thirteenth century earth oven

Using ancient DNA (aDNA) extracted from eggshell of the extinct moa (Aves: Dinornithiformes) we determined the species composition and number of eggs found in a late thirteenth century earth oven feature at Wairau Bar (South Island, New Zealand) – one of New Zealand’s most significant archaeological sites. Mitochondrial and nuclear DNA signatures confirmed this oven feature contained fragments of at least 31 moa eggs, representing three moa genera: Emeus; Euryapteryx; Dinornis. We demonstrate through the genetic identification of 127 moa eggshell fragments that thickness is an unreliable character for species assignment. We also present a protocol for assessing the preservation likelihood of DNA in burnt eggshell. This is useful because eggshell fragments found in archaeological contexts have often been thermally modified, and heat significantly increases DNA fragmentation. Eggshell is widely used in radiocarbon dating and stable isotope research, this study showcases how aDNA can also add to our knowledge of eggshell in both archaeological and palaeoecological contexts.     

Possible cases of leprosy and tuberculosis in medieval Sigtuna,Sweden

In Sigtuna, Sweden, a medieval cemetery, including 227 skeletons, was analysed in 2006. On the outskirts of the churchyard, six skeletons with bone changes indicating systemic inflammatory disease were observed. Two out of three individuals with well‐preserved facial bone regions displayed signs of rhinomaxillary remodelling. Four of the afflicted exhibit severe bilateral alterations of the lower legs and phalanges of the feet and concentric atrophy of the metatarsals. In addition, one of the individuals exhibited a kyphosis in the lumbar vertebrae. In a discussion about alternative diagnoses, lepromatous leprosy and tuberculosis were identified as the causes of the destructive lesions in two individuals. Though the skeletal changes of the lower legs and feet in four cases demonstrate a close resemblance to secondary lesions of leprosy, the disease could not be confirmed. The skeletal changes of the last individual were unspecific and the possible causes several, rendering diagnosis difficult. The burial locations imply that the afflicted persons belonged to a lower social stratum. Due to the significantly higher frequency of pathological changes in the cemetery compared to other cemeteries in the town, the individuals could be regarded as fellow sufferers among others with various medical conditions. The bioarchaeological identification of systemic infectious diseases of a group of individuals of this size is unique to north of Scania in Sweden, where only a few cases of leprosy and tuberculosis have previously been diagnosed. The significance of the present study is emphasised by the interconnection between the afflicted, the archaeological context and the knowledge of the medieval society in Sigtuna. Copyright © 2010 John Wiley & Sons, Ltd.     

Septic bone changes in leprosy: A clinical,radiological and palaeopathological review

Infective lesions of bone in leprosy are of two types. In the rhinomaxillary syndrome, superficial infective changes in the bones of the maxilla are due to the presence of Mycobacterium leprae. Lytic foci in the medulla or the outer cortical surface may be due to Mycobacterium leprae at the site as granulomatous lesions. The most frequent infective bone changes in leprosy are, however, sequelae of regional anaesthesia and soft tissue ulceration. The bone lesions are due to pyogenic bacteria and the nature of these infective lesions is similar to that in non-leprous conditions. The pyogenic lesions in leprosy are restricted to the bones of the hands, feet and lower legs.     

Ancient Iberian horses: a method to recover DNA from archaeological samples buried under sub-optimal conditions for preservation

Existing methods to extract, amplify, and sequence ancient DNA (aDNA) from horse bone and teeth were optimized to recover DNA from a depositional environment of highly permeable acidic soil. DNA was successfully retrieved using 0.10g of bone powder from horse (Equus sp.) remains dating to 25 K years utilizing the methods optimized for this archaeological material. The genetic analyses were performed in a facility that is dedicated to ancient DNA research (Paleo-DNA Laboratory, Thunder Bay, ON, Canada) and has not been previously used to analyse modern or ancient horse DNA. Research was replicated to obtain reliable sequencing results for six samples from the Iberian Peninsula that were consistent with published sequences of Equus caballus. The archaeological sequence data obtained support hypotheses that promote the significance that the Iberian Peninsula has had to the multi-focal centres of origin for horse domestication and distribution of modern horse breeds. The data presented may provide evidence of the existence of an Iberian refugium for Equus during the last glacial period, 10 K years BP. Further molecular data analyses will enhance the ideas presented by this data and our understanding of horse domestication and phylogeny. The optimization of molecular techniques to successfully obtain DNA using minimally destructive, cosmetically sensitive techniques from archaeological remains endeavours to foster further cooperation between museums and researchers.     

Childhood scurvy in a medieval burial from Mačvanska Mitrovica,Serbia

The type and distribution of abnormalities in the skeleton of a child 2 years of age indicates that the child likely suffered from scurvy at the time of death. The burial is from the archaeological site of Zidine, Mačvanska Mitrovica, Serbia dated to the Medieval Period. Abnormal porosity and reactive woven bone formation are present affecting both the axial and appendicular skeleton. This case of scurvy is important because archaeological evidence of scurvy in this geographical location and at this time period is unknown. It is also important because the relative completeness of the skeleton and the widespread evidence of skeletal abnormalities provide additional insight regarding the skeletal manifestations of scurvy in young children. Copyright © 2009 John Wiley & Sons, Ltd.     

SEX IDENTIFICATION OF SLAVE SACRIFICE VICTIMS FROM QIN STATE TOMBS IN THE SPRING AND AUTUMN PERIOD OF CHINA USING ANCIENT DNA

Ancient DNA (aDNA) analysis was employed to determine the sex of slave sacrifice victims from Qin State tombs during the Spring and Autumn Period of China. It is difficult to obtain sex information from fragmentary skeleton samples with the aid of skeletal morphology methods. aDNA was extracted from the dentine in selected tooth samples of sacrificial slaves using a modified traditional method, which combines together phenol/chloroform extraction, silicon dioxide adsorption and ultrafiltration concentration. Based on the sequence differences between the amelogenin homologous gene in the X and that in the Y chromosome, a pair of specific primers was designed to identify the sex of the selected samples. In the selected eight typical samples, the aDNA analytical results revealed that three were males and two were females. These findings indicate that molecular sex identification might provide more valuable information for archaeological research on the institution of slave sacrifice in the Spring and Autumn Period of China.