Ancient Mycobacterium leprae genome reveals medieval English red squirrels as animal leprosy host.

Leprosy, one of the oldest recorded diseases in human history, remains prevalent in Asia, Africa, and South America, with over 200,000 cases every year.1,2 Although ancient DNA (aDNA) approaches on the major causative agent, Mycobacterium leprae, have elucidated the disease's evolutionary history,3,4,5 the role of animal hosts and interspecies transmission in the past remains unexplored. Research has uncovered relationships between medieval strains isolated from archaeological human remains and modern animal hosts such as the red squirrel in England.6,7 However, the time frame, distribution, and direction of transmissions remains unknown. Here, we studied 25 human and 12 squirrel samples from two archaeological sites in Winchester, a medieval English city well known for its leprosarium and connections to the fur trade. We reconstructed four medieval M. leprae genomes, including one from a red squirrel, at a 2.2-fold average coverage. Our analysis revealed a phylogenetic placement of all strains on branch 3 as well as a close relationship between the squirrel strain and one newly reconstructed medieval human strain. In particular, the medieval squirrel strain is more closely related to some medieval human strains from Winchester than to modern red squirrel strains from England, indicating a yet-undetected circulation of M. leprae in non-human hosts in the Middle Ages. Our study represents the first One Health approach for M. leprae in archaeology, which is centered around a medieval animal host strain, and highlights the future capability of such approaches to understand the disease's zoonotic past and current potential.

Genetic history of Cambridgeshire before and after the Black Death.

The extent of the devastation of the Black Death pandemic (1346-1353) on European populations is known from documentary sources and its bacterial source illuminated by studies of ancient pathogen DNA. What has remained less understood is the effect of the pandemic on human mobility and genetic diversity at the local scale. Here, we report 275 ancient genomes, including 109 with coverage >0.1×, from later medieval and postmedieval Cambridgeshire of individuals buried before and after the Black Death. Consistent with the function of the institutions, we found a lack of close relatives among the friars and the inmates of the hospital in contrast to their abundance in general urban and rural parish communities. While we detect long-term shifts in local genetic ancestry in Cambridgeshire, we find no evidence of major changes in genetic ancestry nor higher differentiation of immune loci between cohorts living before and after the Black Death.

Investigating the association between intestinal parasite infection and cribra orbitalia in the medieval population of Cambridge, UK.

OBJECTIVE: Cribra orbitalia is believed to be a skeletal indicator of chronic anaemia, scurvy, rickets or related metabolic diseases. It has been suggested that it may be used as a proxy indicator for intestinal parasite infection, as parasites often cause anaemia today. Our aim is to investigate this association in the medieval population of Cambridge, UK. MATERIALS: Individuals excavated from the cemeteries of the Augustinian friary and All Saints by the Castle parish church, and aged from 7 to adulthood. METHODS: We undertook parasite analysis of the pelvic sediment and control samples of 46 burials with intact orbital roofs. RESULTS: Human roundworm (Ascaris lumbricoides) and/or whipworm (Trichuris trichiura) were identified in the pelvic sediment of 22 individuals, and cribra orbitalia noted in 11 individuals. Barnards test showed no association between parasite infection and cribra orbitalia (p = .882). CONCLUSION: We found no association between infection and cribra orbitalia infection in this medieval adult population, calling into question this hypothesis, at least for adults. SIGNIFICANCE: High or low cribra orbitalia prevalence in adults should not be used to infer rates of intestinal parasite infection. LIMITATIONS: The individuals in the study were over the age of 7, with no younger children. It is possible that only parasites which cause marked anaemia (such as hookworm, schistosomiasis or malaria) may cause cribra orbitalia, while less marked anaemia from roundworm and whipworm may not do so. SUGGESTIONS FOR FURTHER RESEARCH: Repeating this study in younger children, when most cribra orbitalia appears to form.

Tuberculosis before and after the Black Death (1346-1353 CE) in the Hospital of St John the Evangelist in Cambridge, England.

This research explores how the prevalence of tuberculosis (TB) in a medieval hospital was affected by the demographic and social changes that following the Black Death (1346-1353 CE), the initial years of the Second Plague Pandemic. To do this, skeletal remains of individuals buried at the Hospital of St John the Evangelist in Cambridge, England, that could be dated to living before (n = 77) or after (n = 55) the Black Death were assessed for evidence of TB (indicated by destructive lesions of the spine, ribs, large joints, and other recognised criteria). Overall, the odds of females having skeletal lesions caused by TB were over four times higher than males. No significant difference was detected in the prevalence rates in those who lived before and after the Black Death (7.8%, 6/77 before and 11.0%, 6/55 after). However, the odds of females having skeletal evidence of TB were over five times greater after the Black Death than they were before. These findings indicate that women may have been 1) more susceptible to TB, 2) surviving longer post-infection than men, and/or 3) that women with TB were more likely to be admitted to the Hospital especially following the Black Death. It is also possible that impairment due to TB infection may have been a contributing factor for entry into the Hospital for women but not men.

Metabolomics in archaeological science: A review of their advances and present requirements.

Metabolomics, the study of metabolites (small molecules of <1500 daltons), has been posited as a potential tool to explore the past in a comparable manner to other omics, e.g., genomics or proteomics. Archaeologists have used metabolomic approaches for a decade or so, mainly applied to organic residues adhering to archaeological materials. Because of advances in sensitivity, resolution, and the increased availability of different analytical platforms, combined with the low mass/volume required for analysis, metabolomics is now becoming a more feasible choice in the archaeological sector. Additional approaches, as presented by our group, show the versatility of metabolomics as a source of knowledge about the human past when using human osteoarchaeological remains. There is tremendous potential for metabolomics within archaeology, but further efforts are required to position it as a routine technique.

Human Archaeological Dentin as Source of Polar and Less Polar Metabolites for Untargeted Metabolomic Research: The Case of Yersinia pestis.

Metabolomic approaches, such as in clinical applications of living individuals, have shown potential use for solving questions regarding the past when applied to archaeological material. Here, we study for the first time the potential of this Omic approach as applied to metabolites extracted from archaeological human dentin. Dentin obtained from micro sampling the dental pulp of teeth of victims and non-victims of Yersinia pestis (plague) from a 6th century Cambridgeshire site are used to evaluate the potential use of such unique material for untargeted metabolomic studies on disease state through liquid chromatography hyphenated to high-resolution mass spectrometry (LC-HRMS). Results show that small molecules of both likely endogenous and exogenous sources are preserved for a range of polar and less polar/apolar metabolites in archaeological dentin; however, untargeted metabolomic profiles show no clear differentiation between healthy and infected individuals in the small sample analysed (n = 20). This study discusses the potential of dentin as a source of small molecules for metabolomic assays and highlights: (1) the need for follow up research to optimise sampling protocols, (2) the requirements of studies with larger sample numbers and (3) the necessity of more databases to amplify the positive results achievable with this Omic technique in the archaeological sciences.

Examination of human osteoarchaeological remains as a feasible source of polar and apolar metabolites to study past conditions.

Metabolomics is a modern tool that aids in our understanding of the molecular changes in organisms. Archaeological science is a branch of archaeology that explores different archaeological materials using modern analytical tools. Human osteoarchaeological material are a frequent finding in archaeological contexts and have the potential to offer information about previous human populations, which can be illuminating about our current condition. Using a set of samples comprising different skeletal elements and bone structures, here we explore for the first time the possibility of extracting metabolites from osteoarchaeological material. Here, a protocol for extraction and measurement of extracted polar and less-polar/apolar metabolites by ultra-high performance liquid chromatography hyphenated to high resolution mass spectrometry is presented to measure the molecules separated after a reversed phase and hydrophilic interaction liquid chromatography column. Molecular information was obtained, showing that osteoarchaeological material is a viable source of molecular information for metabolomic studies.

Caring for the injured: Exploring the immediate and long-term consequences of injury in medieval Cambridge, England.

OBJECTIVE: To combine paleopathological and biomechanical analysis to reconstruct the impact that a severe skeletal injury had on an individual's ability to function and participate in medieval society. MATERIALS: Three medieval individuals from Cambridge, England with ante-mortem fractures to the lower limb were analyzed. METHODS: Plain X-rays were used to determine the degree of malunion, rotation and overlap of each fracture. Cortical bone architecture of the injured individuals and 28 uninjured controls were analyzed using micro-computed tomography (µCT). Clinical and functional consequences were examined using the Bioarcheology of Care framework. RESULTS: The mechanism of injury, the secondary complications, and the extent of the care received was reconstructed for each individual. Bilateral asymmetry in the cortical bone architecture revealed the long-term alterations to each individual's gait. CONCLUSION: Each of these individuals survived a severe injury resulting in chronic physical impairment, though not all would have been considered 'disabled'. SIGNIFICANCE: This research contributes to the discussion about medieval care provision and social constructions of disability by illustrating how an interdisciplinary approach provides insight into the experiences of those with physical impairments. The integration of µCT imaging within the Bioarcheology of Care model is a novel approach with great potential for application across the field. LIMITATIONS: Biomechanical analysis was restricted to cortical geometry. SUGGESTIONS FOR FUTURE RESEARCH: Further study of bilateral asymmetry in trabecular architecture could complement our understanding of altered loading modalities in past societies.

Intestinal parasite infection in the Augustinian friars and general population of medieval Cambridge, UK.

OBJECTIVE: To investigate how lifestyle may have impacted the risk of contracting intestinal parasites in medieval England . Regular clergy (such as those living in monasteries) and the lay population form interesting groups for comparison as diet and lifestyle varied significantly. Monasteries were built with latrine blocks and hand washing facilities, unlike houses of the poor. MATERIALS: Sediment samples from the pelvis, along with control samples from feet and skull, of 19 burials of Augustinian Friars (13th-16th century), and 25 burials from All Saints by the Castle parish cemetery (10th-14th century), Cambridge. METHODS: We analysed the sediment using micro-sieving and digital light microscopy to identify the eggs of intestinal parasites. RESULTS: Parasite prevalence (roundworm and whipworm) in the Augustinian friars was 58%, and in the All Saints by the Castle parishioners just 32% (Barnards Test score statistic 1.7176, p-value 0.092). CONCLUSIONS: It is interesting that the friars had nearly double the infection rate of parasites spread by poor hygiene, compared with the general population. We consider options that might explain this difference, and discuss descriptions and treatment of intestinal worms in medical texts circulating in Cambridge during the medieval period. SIGNIFICANCE: This is the first study to compare prevalence of parasite infection between groups with different socioeconomic status from the same location. LIMITATIONS: Quality of egg preservation was suboptimal, so our data may under-represent the true prevalence. SUGGESTIONS FOR FURTHER RESEARCH: Larger studies with greater statistical power, covering different time periods and regions.

Ancient herpes simplex 1 genomes reveal recent viral structure in Eurasia.

Human herpes simplex virus 1 (HSV-1), a life-long infection spread by oral contact, infects a majority of adults globally. Phylogeographic clustering of sampled diversity into European, pan-Eurasian, and African groups has suggested the virus codiverged with human migrations out of Africa, although a much younger origin has also been proposed. We present three full ancient European HSV-1 genomes and one partial genome, dating from the 3rd to 17th century CE, sequenced to up to 9.5× with paired human genomes up to 10.16×. Considering a dataset of modern and ancient genomes, we apply phylogenetic methods to estimate the age of sampled modern Eurasian HSV-1 diversity to 4.68 (3.87 to 5.65) ka. Extrapolation of estimated rates to a global dataset points to the age of extant sampled HSV-1 as 5.29 (4.60 to 6.12) ka, suggesting HSV-1 lineage replacement coinciding with the late Neolithic period and following Bronze Age migrations.

An invasive Haemophilus influenzae serotype b infection in an Anglo-Saxon plague victim.

BACKGROUND: The human pathogen Haemophilus influenzae was the main cause of bacterial meningitis in children and a major cause of worldwide infant mortality before the introduction of a vaccine in the 1980s. Although the occurrence of serotype b (Hib), the most virulent type of H. influenzae, has since decreased, reports of infections with other serotypes and non-typeable strains are on the rise. While non-typeable strains have been studied in-depth, very little is known of the pathogen's evolutionary history, and no genomes dating prior to 1940 were available. RESULTS: We describe a Hib genome isolated from a 6-year-old Anglo-Saxon plague victim, from approximately 540 to 550 CE, Edix Hill, England, showing signs of invasive infection on its skeleton. We find that the genome clusters in phylogenetic division II with Hib strain NCTC8468, which also caused invasive disease. While the virulence profile of our genome was distinct, its genomic similarity to NCTC8468 points to mostly clonal evolution of the clade since the 6th century. We also reconstruct a partial Yersinia pestis genome, which is likely identical to a published first plague pandemic genome of Edix Hill. CONCLUSIONS: Our study presents the earliest genomic evidence for H. influenzae, points to the potential presence of larger genomic diversity in the phylogenetic division II serotype b clade in the past, and allows the first insights into the evolutionary history of this major human pathogen. The identification of both plague and Hib opens questions on the effect of plague in immunocompromised individuals already affected by infectious diseases.

Assessing the relative benefits of imaging with plain radiographs and microCT scanning to diagnose cancer in past populations.

OBJECTIVE: To determine the degree to which plain radiographs (x-rays) and microCT scans can improve accuracy in the diagnosis of cancer in human remains from past populations. MATERIALS: The skeletal remains of 143 individuals from medieval Cambridgeshire, dating from 6th-16th century CE. METHODS: Visual inspection of the skeletons for lesions compatible with malignancy, coupled with plain radiographs and microCT scans of the pelvis, femora and vertebra. RESULTS: Three individuals had visually apparent metastases on their skeletons. Plain radiographs did not identify further individuals with cancer, but did show further lesions in bones with normal external appearance. MicroCT scans identified cancer in two further individuals with normal visual appearance and normal plain radiographs. CONCLUSIONS: Imaging human skeletal remains increases the detection rate of cancer in human skeletal remains. We found microCT scanning to be a much more sensitive imaging modality than plain radiography. It improved our diagnostic accuracy and enabled us to more reliably distinguish between malignant lesions and taphonomic change. SIGNIFICANCE: Future studies investigating the prevalence and nature of malignancy in past populations would benefit from systematic microCT scanning of pelvis, femora and vertebrae of skeletons to optimise their diagnostic accuracy. LIMITATIONS: MicroCT scanning is more expensive than plain radiographs, and may not be easily accessible to biological anthropologists. SUGGESTIONS FOR FURTHER RESEARCH: To apply this approach to skeletal series from different time periods and geographical regions, where the types of cancer existing in the local population may differ from those we studied in medieval Britain.

Ancient dental calculus preserves signatures of biofilm succession and interindividual variation independent of dental pathology.

Dental calculus preserves oral microbes, enabling comparative studies of the oral microbiome and health through time. However, small sample sizes and limited dental health metadata have hindered health-focused investigations to date. Here, we investigate the relationship between tobacco pipe smoking and dental calculus microbiomes. Dental calculus from 75 individuals from the 19th century Middenbeemster skeletal collection (Netherlands) were analyzed by metagenomics. Demographic and dental health parameters were systematically recorded, including the presence/number of pipe notches. Comparative data sets from European populations before and after the introduction of tobacco were also analyzed. Calculus species profiles were compared with oral pathology to examine associations between microbiome community, smoking behavior, and oral health status. The Middenbeemster individuals exhibited relatively poor oral health, with a high prevalence of periodontal disease, caries, heavy calculus deposits, and antemortem tooth loss. No associations between pipe notches and dental pathologies, or microbial species composition, were found. Calculus samples before and after the introduction of tobacco showed highly similar species profiles. Observed interindividual microbiome differences were consistent with previously described variation in human populations from the Upper Paleolithic to the present. Dental calculus may not preserve microbial indicators of health and disease status as distinctly as dental plaque.

One Health Approaches to Trace Mycobacterium leprae's Zoonotic Potential Through Time.

Hansen's disease (leprosy), mainly caused by infection with Mycobacterium leprae, has accompanied humanity for thousands of years. Although currently rare in Europe, there are over 200,000 new infections annually in South East Asia, Africa, and South America. Over the years many disciplines - palaeopathology, ancient DNA and other ancient biomolecules, and history - have contributed to a better understanding of leprosy's past, in particular its history in medieval Europe. We discuss their contributions and potential, especially in relation to the role of inter-species transmission, an unexplored phenomenon in the disease's history. Here, we explore the potential of interdisciplinary approaches that understand disease as a biosocial phenomenon, which is a product of both infection with M. leprae and social behaviours that facilitate transmission and spread. Genetic evidence of M. leprae isolated from archaeological remains combined with systematic zooarchaeological and historical analysis would not only identify when and in what direction transmission occurred, but also key social behaviours and motivations that brought species together. In our opinion, this combination is crucial to understand the disease's zoonotic past and current potential.

Mycobacterium leprae diversity and population dynamics in medieval Europe from novel ancient genomes.

BACKGROUND: Hansen's disease (leprosy), widespread in medieval Europe, is today mainly prevalent in tropical and subtropical regions with around 200,000 new cases reported annually. Despite its long history and appearance in historical records, its origins and past dissemination patterns are still widely unknown. Applying ancient DNA approaches to its major causative agent, Mycobacterium leprae, can significantly improve our understanding of the disease's complex history. Previous studies have identified a high genetic continuity of the pathogen over the last 1500 years and the existence of at least four M. leprae lineages in some parts of Europe since the Early Medieval period. RESULTS: Here, we reconstructed 19 ancient M. leprae genomes to further investigate M. leprae's genetic variation in Europe, with a dedicated focus on bacterial genomes from previously unstudied regions (Belarus, Iberia, Russia, Scotland), from multiple sites in a single region (Cambridgeshire, England), and from two Iberian leprosaria. Overall, our data confirm the existence of similar phylogeographic patterns across Europe, including high diversity in leprosaria. Further, we identified a new genotype in Belarus. By doubling the number of complete ancient M. leprae genomes, our results improve our knowledge of the past phylogeography of M. leprae and reveal a particularly high M. leprae diversity in European medieval leprosaria. CONCLUSIONS: Our findings allow us to detect similar patterns of strain diversity across Europe with branch 3 as the most common branch and the leprosaria as centers for high diversity. The higher resolution of our phylogeny tree also refined our understanding of the interspecies transfer between red squirrels and humans pointing to a late antique/early medieval transmission. Furthermore, with our new estimates on the past population diversity of M. leprae, we gained first insights into the disease's global history in relation to major historic events such as the Roman expansion or the beginning of the regular transatlantic long distance trade. In summary, our findings highlight how studying ancient M. leprae genomes worldwide improves our understanding of leprosy's global history and can contribute to current models of M. leprae's worldwide dissemination, including interspecies transmissions.

The greatest health problem of the Middle Ages? Estimating the burden of disease in medieval England.

OBJECTIVE: To identify the major health problems of the Middle Ages. Bubonic plague is often considered the greatest health disaster in medieval history, but this has never been systematically investigated. MATERIALS: We triangulate upon the problem using (i) modern WHO data on disease in the modern developing world, (ii) historical evidence for England such as post-medieval Bills of Mortality, and (iii) prevalences derived from original and published palaeopathological studies. METHODS: Systematic analysis of the consequences of these health conditions using Disability Adjusted Life Years (DALYs) according to the Global Burden of Disease methodology. RESULTS: Infant and child death due to varied causes had the greatest impact upon population and health, followed by a range of chronic/infectious diseases, with tuberculosis probably being the next most significant one. CONCLUSIONS: Among medieval health problems, we estimate that plague was probably 7th-10th in overall importance. Although lethal and disruptive, it struck only periodically and had less cumulative long-term human consequences than chronically endemic conditions (e.g. bacterial and viral infections causing infant and child death, tuberculosis, and other pathogens). SIGNIFICANCE: In contrast to modern health regimes, medieval health was above all an ecological struggle against a diverse host of infectious pathogens; social inequality was probably also an important contributing factor. LIMITATIONS: Methodological assumptions and use of proxy data mean that only approximate modelling of prevalences is possible. SUGGESTIONS FOR FURTHER RESEARCH: Progress in understanding medieval health really depends upon understanding ancient infectious disease through further development of biomolecular methods.

Fancy shoes and painful feet: Hallux valgus and fracture risk in medieval Cambridge, England.

OBJECTIVE: Hallux valgus, the lateral deviation of the great toe, can result in poor balance, impaired mobility and is an independent risk factor for falls. This research aims to compare the prevalence of hallux valgus in subpopulations of medieval Cambridge, England, and to examine the relationship between hallux valgus and fractures to examine the impact of impaired mobility and poor balance caused by this condition. MATERIALS: 177 adult individuals from four cemeteries located in Cambridge, England. METHODS: Human remains were macroscopically and radiographically assessed. RESULTS: Hallux valgus was identified in 18 % of individuals and was significantly more common during the 14th-15th centuries than the 11th-13th centuries. The highest prevalence was observed in the friary (43 %), followed by the Hospital (23 %), the rurban parish cemetery (10 %), and the rural parish cemetery (3%). Fractures from falls were significantly more common in those with hallux valgus than those without. CONCLUSION: The increased prevalence of hallux valgus identified in individuals from the 14th to 15th centuries coincided with the adoption of new footwear with pointed toes. Those that adopted this fashion trend appear to have been more likely to develop balance and mobility problems that resulted in an increased risk of falls. SIGNIFICANCE: This is the first study to explore the relationship between foot problems and functional ability by studying hallux valgus in archaeological assemblages. LIMITATIONS: Falls are complex and determining the mechanism of injury in human skeletal remains is not always possible. FURTHER RESEARCH: Fracture prevalence rates may have been affected by biological factors and underlying pathological conditions.

Gout and 'Podagra' in medieval Cambridge, England.

OBJECTIVE: To estimate the prevalence rate of gout and to explore the social factors that contributed to its development in the various sub-populations in medieval Cambridge. MATERIALS: 177 adult individuals from four medieval cemeteries located in and around Cambridge, UK. METHODS: Lesions were assessed macroscopically and radiographically. Elements with lytic lesions were described and imaged using micro-computed tomography (µCT) to determine their morphology. RESULTS: Gout was identified in 3 % of the population. Individuals buried in the friary had highest prevalence (14 %), with low prevalence rates in the Hospital (3 %) and town parish cemetery (2 %), with no cases in the rural parish cemetery. Gout was more prevalent during the 14th-15th centuries than the 10th-13th centuries. CONCLUSION: The high prevalence rate of gout in the friary is at least partly explained by the consumption of alcohol and purine-rich diets by the friars and the wealthy townsfolk. Medieval medical texts from Cambridge show that gout (known as podagra) was sometimes treated with medications made from the root of the autumn crocus. This root contains colchicine, which is a medicine that is still used to treat gout today. SIGNIFICANCE: This is one of the first studies to assess the epidemiology of gout in medieval England and suggests that gout varied with social status. LIMITATIONS: Our sample size precludes statistical analysis. SUGGESTIONS FOR FURTHER RESEARCH: Additional studies that assess the epidemiology of gout in medieval Europe is needed in order to be able to fully contextualize these findings.