Bioarchaeological investigation of individuals with suspected multibacillary leprosy from the mediaeval leprosarium of St Mary Magdalen, Winchester, Hampshire, UK.

Introduction. We have examined four burials from the St Mary Magdalen mediaeval leprosarium cemetery in Winchester, Hampshire, UK. One (Sk.8) was a male child, two (Sk.45 and Sk.52) were adolescent females and the fourth (Sk.512) was an adult male. The cemetery was in use between the 10th and 12th centuries. All showed skeletal lesions of leprosy. Additionally, one of the two females (Sk.45) had lesions suggestive of multi-cystic tuberculosis and the second (Sk.52) of leprogenic odontodysplasia (LO), a rare malformation of the roots of the permanent maxillary incisors.Gap statement. Relatively little is known of the manifestations of lepromatous leprosy (LL) in younger individuals from the archaeological record.Aims and Methodology. To address this, we have used ancient DNA testing and osteological examination of the individuals, supplemented with X-ray and microcomputed tomography (micro-CT) scan as necessary to assess the disease status.Results and Conclusions. The presence of Mycobacterium leprae DNA was confirmed in both females, and genotyping showed SNP type 3I-1 strains but with a clear genotypic variation. We could not confirm Mycobacterium tuberculosis complex DNA in the female individual SK.45. High levels of M. leprae DNA were found within the pulp cavities of four maxillary teeth from the male child (Sk.8) with LO, consistent with the theory that the replication of M. leprae in alveolar bone may interfere with root formation at key stages of development. We report our biomolecular findings in these individuals and review the evidence this site has contributed to our knowledge of mediaeval leprosy.

Leprosy at the edge of Europe-Biomolecular, isotopic and osteoarchaeological findings from medieval Ireland.

Relatively little is known of leprosy in Medieval Ireland; as an island located at the far west of Europe it has the potential to provide interesting insights in relation to the historical epidemiology of the disease. To this end the study focuses on five cases of probable leprosy identified in human skeletal remains excavated from inhumation burials. Three of the individuals derived from the cemetery of St Michael Le Pole, Golden Lane, Dublin, while single examples were also identified from Ardreigh, Co. Kildare, and St Patrick's Church, Armoy, Co. Antrim. The individuals were radiocarbon dated and examined biomolecularly for evidence of either of the causative pathogens, M. leprae or M. lepromatosis. Oxygen and strontium isotopes were measured in tooth enamel and rib samples to determine where the individuals had spent their formative years and to ascertain if they had undertaken any recent migrations. We detected M. leprae DNA in the three Golden Lane cases but not in the probable cases from either Ardreigh Co. Kildare or Armoy, Co. Antrim. M. lepromatosis was not detected in any of the burals. DNA preservation was sufficiently robust to allow genotyping of M. leprae strains in two of the Golden Lane burials, SkCXCV (12-13th century) and SkCCXXX (11-13th century). These strains were found to belong on different lineages of the M. leprae phylogenetic tree, namely branches 3 and 2 respectively. Whole genome sequencing was also attempted on these two isolates with a view to gaining further information but poor genome coverage precluded phylogenetic analysis. Data from the biomolecular study was combined with osteological, isotopic and radiocarbon dating to provide a comprehensive and multidisciplinary study of the Irish cases. Strontium and oxygen isotopic analysis indicate that two of the individuals from Golden Lane (SkCXLVIII (10-11th century) and SkCXCV) were of Scandinavian origin, while SkCCXXX may have spent his childhood in the north of Ireland or central Britain. We propose that the Vikings were responsible for introducing leprosy to Ireland. This work adds to our knowledge of the likely origins of leprosy in Medieval Ireland and will hopefully stimulate further research into the history and spread of this ancient disease across the world.

Ancient genomes reveal a high diversity of Mycobacterium leprae in medieval Europe.

Studying ancient DNA allows us to retrace the evolutionary history of human pathogens, such as Mycobacterium leprae, the main causative agent of leprosy. Leprosy is one of the oldest recorded and most stigmatizing diseases in human history. The disease was prevalent in Europe until the 16th century and is still endemic in many countries with over 200,000 new cases reported annually. Previous worldwide studies on modern and European medieval M. leprae genomes revealed that they cluster into several distinct branches of which two were present in medieval Northwestern Europe. In this study, we analyzed 10 new medieval M. leprae genomes including the so far oldest M. leprae genome from one of the earliest known cases of leprosy in the United Kingdom-a skeleton from the Great Chesterford cemetery with a calibrated age of 415-545 C.E. This dataset provides a genetic time transect of M. leprae diversity in Europe over the past 1500 years. We find M. leprae strains from four distinct branches to be present in the Early Medieval Period, and strains from three different branches were detected within a single cemetery from the High Medieval Period. Altogether these findings suggest a higher genetic diversity of M. leprae strains in medieval Europe at various time points than previously assumed. The resulting more complex picture of the past phylogeography of leprosy in Europe impacts current phylogeographical models of M. leprae dissemination. It suggests alternative models for the past spread of leprosy such as a wide spread prevalence of strains from different branches in Eurasia already in Antiquity or maybe even an origin in Western Eurasia. Furthermore, these results highlight how studying ancient M. leprae strains improves understanding the history of leprosy worldwide.

Leprosy in pre-Norman Suffolk, UK: biomolecular and geochemical analysis of the woman from Hoxne.

PURPOSE: A woman's skull, exhibiting features of lepromatous leprosy (LL), was recovered from a garden in Hoxne, Suffolk. The absence of post crania and lack of formal excavation meant that diagnosis and dating was uncertain. The aim of this research was to confirm the diagnosis using biomolecular means and second, to place it in context with other British leprosy cases using SNP genotyping and radiocarbon dating. METHODOLOGY: Bone from the skull was analysed by ancient DNA (aDNA) methods and subjected to radiocarbon dating. As a result, stable carbon and nitrogen isotope values were produced, both useful for assessing aspects of the woman's diet.Results/Key findings. aDNA confirmed the presence of mycobacterium leprae and genotyping demonstrated an ancestral variant of subtype 3I, the same lineage recently identified in living squirrels in the south of England. Radiocarbon dating revealed the woman lived approximately between 885-1015 AD, providing evidence for endurance of this subtype in East Anglia, having been previously identified as early as the fifth-sixth century (Great Chesterford) and as late as the thirteenth century (Ipswich). CONCLUSIONS: The confirmation of a new pre-Norman leprosy case in East Anglia is of interest as this is where a high proportion of cases are located. Possible factors for this may include preservation and excavation biases, population density, but also connection and trade, possibly of fur, with the continent. Future research on other British LL cases should focus on exploring these aspects to advance understanding of the disease's history, here and on the continent.

Investigation of a Medieval Pilgrim Burial Excavated from the Leprosarium of St Mary Magdalen Winchester, UK.

We have examined the remains of a Pilgrim burial from St Mary Magdalen, Winchester. The individual was a young adult male, aged around 18-25 years at the time of death. Radiocarbon dating showed the remains dated to the late 11th-early 12th centuries, a time when pilgrimages were at their height in Europe. Several lines of evidence in connection with the burial suggested this was an individual of some means and prestige. Although buried within the leprosarium cemetery, the skeleton showed only minimal skeletal evidence for leprosy, which was confined to the bones of the feet and legs. Nonetheless, molecular testing of several skeletal elements, including uninvolved bones all showed robust evidence of DNA from Mycobacterium leprae, consistent with the lepromatous or multibacillary form of the disease. We infer that in life, this individual almost certainly suffered with multiple soft tissue lesions. Genotyping of the M.leprae strain showed this belonged to the 2F lineage, today associated with cases from South-Central and Western Asia. During osteological examination it was noted that the cranium and facial features displayed atypical morphology for northern European populations. Subsequently, geochemical isotopic analyses carried out on tooth enamel indicated that this individual was indeed not local to the Winchester region, although it was not possible to be more specific about their geographic origin.

Osteological, biomolecular and geochemical examination of an early anglo-saxon case of lepromatous leprosy.

We have examined a 5th to 6th century inhumation from Great Chesterford, Essex, UK. The incomplete remains are those of a young male, aged around 21-35 years at death. The remains show osteological evidence of lepromatous leprosy (LL) and this was confirmed by lipid biomarker analysis and ancient DNA (aDNA) analysis, which provided evidence for both multi-copy and single copy loci from the Mycobacterium leprae genome. Genotyping showed the strain belonged to the 3I lineage, but the Great Chesterford isolate appeared to be ancestral to 3I strains found in later medieval cases in southern Britain and also continental Europe. While a number of contemporaneous cases exist, at present, this case of leprosy is the earliest radiocarbon dated case in Britain confirmed by both aDNA and lipid biomarkers. Importantly, Strontium and Oxygen isotope analysis suggest that the individual is likely to have originated from outside Britain. This potentially sheds light on the origins of the strain in Britain and its subsequent spread to other parts of the world, including the Americas where the 3I lineage of M. leprae is still found in some southern states of America.

Mycobacterium leprae genomes from a British medieval leprosy hospital: towards understanding an ancient epidemic.

BACKGROUND: Leprosy has afflicted humankind throughout history leaving evidence in both early texts and the archaeological record. In Britain, leprosy was widespread throughout the Middle Ages until its gradual and unexplained decline between the 14th and 16th centuries. The nature of this ancient endemic leprosy and its relationship to modern strains is only partly understood. Modern leprosy strains are currently divided into 5 phylogenetic groups, types 0 to 4, each with strong geographical links. Until recently, European strains, both ancient and modern, were thought to be exclusively type 3 strains. However, evidence for type 2 strains, a group normally associated with Central Asia and the Middle East, has recently been found in archaeological samples in Scandinavia and from two skeletons from the medieval leprosy hospital (or leprosarium) of St Mary Magdalen, near Winchester, England. RESULTS: Here we report the genotypic analysis and whole genome sequencing of two further ancient M. leprae genomes extracted from the remains of two individuals, Sk14 and Sk27, that were excavated from 10th-12th century burials at the leprosarium of St Mary Magdalen. DNA was extracted from the surfaces of bones showing osteological signs of leprosy. Known M. leprae polymorphisms were PCR amplified and Sanger sequenced, while draft genomes were generated by enriching for M. leprae DNA, and Illumina sequencing. SNP-typing and phylogenetic analysis of the draft genomes placed both of these ancient strains in the conserved type 2 group, with very few novel SNPs compared to other ancient or modern strains. CONCLUSIONS: The genomes of the two newly sequenced M. leprae strains group firmly with other type 2F strains. Moreover, the M. leprae strain most closely related to one of the strains, Sk14, in the worldwide phylogeny is a contemporaneous ancient St Magdalen skeleton, vividly illustrating the epidemic and clonal nature of leprosy at this site. The prevalence of these type 2 strains indicates that type 2F strains, in contrast to later European and associated North American type 3 isolates, may have been the co-dominant or even the predominant genotype at this location during the 11th century.

Genome-wide comparison of medieval and modern Mycobacterium leprae.

Leprosy was endemic in Europe until the Middle Ages. Using DNA array capture, we have obtained genome sequences of Mycobacterium leprae from skeletons of five medieval leprosy cases from the United Kingdom, Sweden, and Denmark. In one case, the DNA was so well preserved that full de novo assembly of the ancient bacterial genome could be achieved through shotgun sequencing alone. The ancient M. leprae sequences were compared with those of 11 modern strains, representing diverse genotypes and geographic origins. The comparisons revealed remarkable genomic conservation during the past 1000 years, a European origin for leprosy in the Americas, and the presence of an M. leprae genotype in medieval Europe now commonly associated with the Middle East. The exceptional preservation of M. leprae biomarkers, both DNA and mycolic acids, in ancient skeletons has major implications for palaeomicrobiology and human pathogen evolution.

Detection and strain typing of ancient Mycobacterium leprae from a medieval leprosy hospital.

Nine burials excavated from the Magdalen Hill Archaeological Research Project (MHARP) in Winchester, UK, showing skeletal signs of lepromatous leprosy (LL) have been studied using a multidisciplinary approach including osteological, geochemical and biomolecular techniques. DNA from Mycobacterium leprae was amplified from all nine skeletons but not from control skeletons devoid of indicative pathology. In several specimens we corroborated the identification of M. leprae with detection of mycolic acids specific to the cell wall of M. leprae and persistent in the skeletal samples. In five cases, the preservation of the material allowed detailed genotyping using single-nucleotide polymorphism (SNP) and multiple locus variable number tandem repeat analysis (MLVA). Three of the five cases proved to be infected with SNP type 3I-1, ancestral to contemporary M. leprae isolates found in southern states of America and likely carried by European migrants. From the remaining two burials we identified, for the first time in the British Isles, the occurrence of SNP type 2F. Stable isotope analysis conducted on tooth enamel taken from two of the type 3I-1 and one of the type 2F remains revealed that all three individuals had probably spent their formative years in the Winchester area. Previously, type 2F has been implicated as the precursor strain that migrated from the Middle East to India and South-East Asia, subsequently evolving to type 1 strains. Thus we show that type 2F had also spread westwards to Britain by the early medieval period.

Multiple loci variable number tandem repeat (VNTR) analysis (MLVA) of Mycobacterium leprae isolates amplified from European archaeological human remains with lepromatous leprosy.

Molecular typing methods based on polymorphisms in single nucleotides and short tandem repeat motifs have been developed as epidemiological typing tools for Mycobacterium leprae. We have used a variable number tandem repeat method based on three variable loci to identify strain variation in archaeological cases of lepromatous leprosy. The panel of polymorphic loci used revealed unique profiles in five cases of leprosy, including those with identical SNP type and subtype. These were also different from profiles of three previously studied lepromatous skeletons. Whilst examination with SNP typing provides evidence for disease origins, dissemination and phylogeny, tandem repeat typing may be useful for studying cases from within a defined area or community where SNP types may be identical due to geographical constraints. We envisage the technique may be useful in studying contemporaneous burials such as those associated with leprosaria and will prove invaluable in authentication of ancient DNA analyses.

Comparative genomic and phylogeographic analysis of Mycobacterium leprae.

Reductive evolution and massive pseudogene formation have shaped the 3.31-Mb genome of Mycobacterium leprae, an unculturable obligate pathogen that causes leprosy in humans. The complete genome sequence of M. leprae strain Br4923 from Brazil was obtained by conventional methods (6x coverage), and Illumina resequencing technology was used to obtain the sequences of strains Thai53 (38x coverage) and NHDP63 (46x coverage) from Thailand and the United States, respectively. Whole-genome comparisons with the previously sequenced TN strain from India revealed that the four strains share 99.995% sequence identity and differ only in 215 polymorphic sites, mainly SNPs, and by 5 pseudogenes. Sixteen interrelated SNP subtypes were defined by genotyping both extant and extinct strains of M. leprae from around the world. The 16 SNP subtypes showed a strong geographical association that reflects the migration patterns of early humans and trade routes, with the Silk Road linking Europe to China having contributed to the spread of leprosy.

Rapid detection of Mycobacterium bovis DNA in cattle lymph nodes with visible lesions using PCR.

BACKGROUND: We have evaluated a sensitive screening assay for Mycobacterium tuberculosis (MTB) complex organisms and a specific assay for detecting Mycobacterium bovis DNA in lymph nodes taken from cattle with evidence of bovine tuberculosis. Underlying these series of experiments was the need for a versatile DNA extraction protocol which could handle tissue samples and with the potential for automation.The target for the screening assay was the multi-copy insertion element IS1081, present in 6 copies in the MTB complex. For confirmation of M. bovis we used primers flanking a specific deletion in the genome of M. bovis known as region of difference 4 (RD4). The sensitivity and specificity of these PCRs has been tested on genomic DNA from MTB complex reference strains, mycobacteria other than tuberculosis (MOTT), spiked samples and on clinical material. RESULTS: The minimum detection limits of the IS1081 method was < I genome copy and for the RD4 PCR was 5 genome copies. Both methods can be readily adapted for quantitative PCR with the use of SYBR Green intercalating dye on the RotorGene 3000 platform (Corbett Research). Initial testing of field samples of bovine lymph nodes with visible lesions (VL, n = 109) highlighted two shortfalls of the molecular approach. Firstly, comparison of IS1081 PCR with the "gold standard" of culture showed a sensitivity of approximately 70%. The sensitivity of the RD4 PCR method was 50%. Secondly, the success rate of spoligotyping applied directly to clinical material was 51% compared with cultures. A series of further experiments indicated that the discrepancy between sensitivity of detection found with purified mycobacterial DNA and direct testing of field samples was due to limited mycobacterial DNA recovery from tissue homogenates rather than PCR inhibition. The resilient mycobacterial cell wall, the presence of tissue debris and the paucibacillary nature of some cattle VL tissue may all contribute to this observation. Any of these factors may restrict application of other more discriminant typing methods.A simple means of increasing the efficiency of mycobacterial DNA recovery was assessed using a further pool of 95 cattle VL. Following modification of the extraction protocol, detection rate with the IS1081 and RD4 methods increased to 91% and 59% respectively. CONCLUSION: The IS1081 PCR is a realistic screening method for rapid identification of positive cases but the sensitivity of single copy methods, like RD4 and also of spoligotyping will need to be improved to make these applicable for direct testing of tissue extracts.

First report of Mycobacterium bovis DNA in human remains from the Iron Age.

Tuberculosis has plagued humankind since prehistoric times, as is evident from characteristic lesions on human skeletons dating back to the Neolithic period. The disease in man is due predominantly to infection with either Mycobacterium tuberculosis or Mycobacterium bovis, both members of the M. tuberculosis (MTB) complex. A number of studies have shown that when conditions permit, surviving mycobacterial DNA may be amplified from bone by PCR. Such ancient DNA (aDNA) analyses are subject to stringent tests of authenticity and, when feasible, are invariably limited by DNA fragmentation. Using PCRs based on single-nucleotide polymorphic loci and regions of difference (RDs) in the MTB complex, a study was made of five Iron Age individuals with spinal lesions recovered from the cemetery of Aymyrlyg, South Siberia. A sensitive screening PCR for MTB complex mycobacteria was positive in four out of the five cases. Genotyping evidence indicated that all four cases were due to infection with M. bovis rather than M. tuberculosis and the data were consistent with the proposed phylogenetic model of the MTB complex. This is believed to be the first report of M. bovis causing Pott's disease in archaeological human remains. The study shows that genotyping of ancestral strains of MTB complex mycobacteria from contexts of known date provides information which allows the phylogeny of the model to be tested. Moreover, it shows that loss of DNA from RD4, which defines classic M. bovis, had already occurred from the genome over 2000 years before the present.

Bovine tuberculosis (Mycobacterium bovis) in British farmland wildlife: the importance to agriculture.

Bovine tuberculosis (bTB) is an important disease of cattle and an emerging infectious disease of humans. Cow- and badger-based control strategies have failed to eradicate bTB from the British cattle herd, and the incidence is rising by about 18%per year. The annual cost to taxpayers in Britain is currently 74 million UK pounds. Research has focused on the badger as a potential bTB reservoir, with little attention being paid to other mammals common on farmland. We have conducted a systematic survey of wild mammals (n=4393 individuals) present on dairy farms to explore the role of species other than badgers in the epidemiology of bTB. Cultures were prepared from 10397 samples (primarily faeces, urine and tracheal aspirates). One of the 1307 bank voles (Clethrionomys glareolus) live-sampled, and three of the 43 badgers (Meles meles), yielded positive isolates of Mycobacterium bovis. This is the first time the bacterium has been isolated from the bank vole. The strain type was the same as that found in cattle and badgers on the same farm. However, our work indicates that the mean prevalence of infectious individuals among common farmland wildlife is extremely low (the upper 95% confidence interval is < or =2.0 for all of the abundant species). Mathematical models illustrate that it is highly unlikely the disease could be maintained at such low levels. Our results suggest that these animals are relatively unimportant as reservoirs of bTB, having insufficient within-species (or within-group) transmission to sustain the infection, though occasional spill-overs from cattle or badgers may occur.

Genotypic analysis of the earliest known prehistoric case of tuberculosis in Britain.

The earliest known case of human tuberculosis in Britain dates to the middle period of the Iron Age, approximately 2,200 years before present. Bone lesions on the spine of a male skeleton excavated at Tarrant Hinton in Dorset, United Kingdom, show evidence of Pott's disease and are supported by molecular evidence of Mycobacterium tuberculosis complex DNA amplified by IS6110 PCR (19). In the present study, we used a further series of sensitive PCR methods to confirm the diagnosis of tuberculosis and to determine the genotype of the infecting strain. These tests demonstrated that this individual was infected with a strain of M. tuberculosis rather than Mycobacterium bovis. The strain had undergone the tuberculosis D1 deletion affecting the mmpS6 and mmpL6 genes and can therefore be identified as a member of the family of "modern" M. tuberculosis isolates. All evidence obtained was consistent with surviving mycobacterial DNA being highly fragmented in this case.

Microsatellite mapping of Mycobacterium leprae populations in infected humans.

To investigate genetic diversity in a bacterial population, we measured the copy numbers of simple sequence repeats, or microsatellites, in Mycobacterium leprae from patients living in and around Hyderabad, India. Three microsatellite loci containing trinucleotide or dinucleotide repeats were amplified from infected tissues, and the copy numbers were established by sequence analysis. Extensive diversity was observed in a cross-sectional survey of 33 patients, but closely related profiles were found for members of a multicase family likely to share a common transmission source. Sampling of multiple tissues from single individuals demonstrated identical microsatellite profiles in the skin, nasal cavity, and bloodstream but revealed differences at one or more loci for M. leprae present in nerves. Microsatellite mapping of M. leprae represents a useful tool for tracking short transmission chains. Comparison of skin and nerve lesions suggests that the evolution of disease within an individual involves the expansion of multiple distinct subpopulations of M. leprae.

Microsatellite mapping of Mycobacterium leprae populations in infected humans

To investigate genetic diversity in a bacterial population, we measured the copy numbers of simple sequence repeats, or microsatellites, in Mycobacterium leprae from patients living in and around Hyderabad, India. Three microsatellite loci containing trinucleotide or dinucleotide repeats were amplified from infected tissues, and the copy numbers were established by sequence analysis. Extensive diversity was observed in a cross-sectional survey of 33 patients, but closely related profiles were found for members of a multicase family likely to share a common transmission source. Sampling of multiple tissues from single individuals demonstrated identical microsatellite profiles in the skin, nasal cavity, and bloodstream but revealed differences at one or more loci for M. leprae present in nerves. Microsatellite mapping of M. leprae represents a useful tool for tracking short transmission chains. Comparison of skin and nerve lesions suggests that the evolution of disease within an individual involves the expansion of multiple distinct subpopulations of M. leprae.

Molecular analysis of Mycobacterium tuberculosis DNA from a family of 18th century Hungarians.

The naturally mummified remains of a mother and two daughters found in an 18th century Hungarian crypt were analysed, using multiple molecular genetic techniques to examine the epidemiology and evolution of tuberculosis. DNA was amplified from a number of targets on the Mycobacterium tuberculosis genome, including DNA from IS6110, gyrA, katG codon 463, oxyR, dnaA-dnaN, mtp40, plcD and the direct repeat (DR) region. The strains present in the mummified remains were identified as M. tuberculosis and not Mycobacterium bovis, from katG and gyrA genotyping, PCR from the oxyR and mtp40 loci, and spoligotyping. Spoligotyping divided the samples into two strain types, and screening for a deletion in the MT1801-plcD region initially divided the strains into three types. Further investigation showed, however, that an apparent deletion was due to poor DNA preservation. By comparing the effect of PCR target size on the yield of amplicon, a clear difference was shown between 18th century and modern M. tuberculosis DNA. A two-centre system was used to confirm the findings of this study, which clearly demonstrate the value of using molecular genetic techniques to study historical cases of tuberculosis and the care required in drawing conclusions. The genotyping and spoligotyping results are consistent with the most recent theory of the evolution and spread of the modern tuberculosis epidemic.

Spoligotyping of Mycobacterium tuberculosis isolates from multiple-drug-resistant tuberculosis patients from Bombay, India.

Spoligotyping was undertaken in 65 multiple-drug-resistant Mycobacterium tuberculosis isolates from Bombay, India. The spoligotype patterns showed seven closely related clusters, a cluster with 2 Beijing-like isolates, and unique spoligotypes (43%). Of the clusters, one with 29% of all the isolates suggested transmission of a dominant resistant clone.