Sharon N. DeWitte

A draft genome of Yersinia pestis from victims of the Black Death

Technological advances in DNA recovery and sequencing have drastically expanded the scope of genetic analyses of ancient specimens to the extent that full genomic investigations are now feasible and are quickly becoming standard. This trend has important implications for infectious disease research because genomic data from ancient microbes may help to elucidate mechanisms of pathogen evolution and adaptation for emerging and re-emerging infections. Here we report a reconstructed ancient genome of Yersinia pestis at 30-fold average coverage from Black Death victims securely dated to episodes of pestilence-associated mortality in London, England, 1348–1350. Genetic architecture and phylogenetic analysis indicate that the ancient organism is ancestral to most extant strains and sits very close to the ancestral node of all Y. pestis commonly associated with human infection. Temporal estimates suggest that the Black Death of 1347–1351 was the main historical event responsible for the introduction and widespread dissemination of the ancestor to all currently circulating Y. pestis strains pathogenic to humans, and further indicates that contemporary Y. pestis epidemics have their origins in the medieval era. Comparisons against modern genomes reveal no unique derived positions in the medieval organism, indicating that the perceived increased virulence of the disease during the Black Death may not have been due to bacterial phenotype. These findings support the notion that factors other than microbial genetics, such as environment, vector dynamics and host susceptibility, should be at the forefront of epidemiological discussions regarding emerging Y. pestis infections.

Yersinia pestis and the Plague of Justinian 541–543 AD: a genomic analysis

BACKGROUND Yersinia pestis has caused at least three human plague pandemics. The second (Black Death, 14-17th centuries) and third (19-20th centuries) have been genetically characterised, but there is only a limited understanding of the first pandemic, the Plague of Justinian (6-8th centuries). To address this gap, we sequenced and analysed draft genomes of Y pestis obtained from two individuals who died in the first pandemic. METHODS Teeth were removed from two individuals (known as A120 and A76) from the early medieval Aschheim-Bajuwarenring cemetery (Aschheim, Bavaria, Germany). We isolated DNA from the teeth using a modified phenol-chloroform method. We screened DNA extracts for the presence of the Y pestis-specific pla gene on the pPCP1 plasmid using primers and standards from an established assay, enriched the DNA, and then sequenced it. We reconstructed draft genomes of the infectious Y pestis strains, compared them with a database of genomes from 131 Y pestis strains from the second and third pandemics, and constructed a maximum likelihood phylogenetic tree. FINDINGS Radiocarbon dating of both individuals (A120 to 533 AD [plus or minus 98 years]; A76 to 504 AD [plus or minus 61 years]) places them in the timeframe of the first pandemic. Our phylogeny contains a novel branch (100% bootstrap at all relevant nodes) leading to the two Justinian samples. This branch has no known contemporary representatives, and thus is either extinct or unsampled in wild rodent reservoirs. The Justinian branch is interleaved between two extant groups, 0.ANT1 and 0.ANT2, and is distant from strains associated with the second and third pandemics. INTERPRETATION We conclude that the Y pestis lineages that caused the Plague of Justinian and the Black Death 800 years later were independent emergences from rodents into human beings. These results show that rodent species worldwide represent important reservoirs for the repeated emergence of diverse lineages of Y pestis into human populations. FUNDING McMaster University, Northern Arizona University, Social Sciences and Humanities Research Council of Canada, Canada Research Chairs Program, US Department of Homeland Security, US National Institutes of Health, Australian National Health and Medical Research Council.

Selectivity of Black Death mortality with respect to preexisting health

Was the mortality associated with the deadliest known epidemic in human history, the Black Death of 1347–1351, selective with respect to preexisting health conditions (“frailty”)? Many researchers have assumed that the Black Death was so virulent, and the European population so immunologically naïve, that the epidemic killed indiscriminately, irrespective of age, sex, or frailty. If this were true, Black Death cemeteries would provide unbiased cross-sections of demographic and epidemiological conditions in 14th-century Europe. Using skeletal remains from medieval England and Denmark, new methods of paleodemographic age estimation, and a recent multistate model of selective mortality, we test the assumption that the mid-14th-century Black Death killed indiscriminately. Skeletons from the East Smithfield Black Death cemetery in London are compared with normal, nonepidemic cemetery samples from two medieval Danish towns (Viborg and Odense). The results suggest that the Black Death did not kill indiscriminately—that it was, in fact, selective with respect to frailty, although probably not as strongly selective as normal mortality.

Stature and frailty during the Black Death: the effect of stature on risks of epidemic mortality in London, A.D. 1348–1350

Recent research has shown that preexisting health condition affected an individuals risk of dying during the 14th-century Black Death. However, a previous study of the effect of adult stature on risk of mortality during the epidemic failed to find a relationship between the two; this result is perhaps surprising given the well-documented inverse association between stature and mortality in human populations. We suggest that the previous study used an analytical approach that was more complex than was necessary for an assessment of the effect of adult stature on risk of mortality. This study presents a reanalysis of data on adult stature and age-at-death during the Black Death in London, 1348-1350 AD. The results indicate that short stature increased risks of mortality during the medieval epidemic, consistent with previous work that revealed a negative effect of poor health on risk of mortality during the Black Death. However, the results from a normal, non-epidemic mortality comparison sample do not show an association between stature and risks of mortality among adults under conditions of normal mortality. Fishers exact tests, used to determine whether individuals who were growing during the Great Famine of 1315-1322 were more likely to be of short stature than those who did not endure the famine, revealed no differences between the two groups, suggesting that the famine was not a source of variation in stature among those who died during the Black Death.

What Do We Know about the Agricultural Demographic Transition

The Agricultural Revolution accompanied, either as a cause or as an effect, important changes in human demographic systems. The consensus model is that fertility and mortality increased and health declined with the adoption of agriculture, compared to those for hunter‐gatherers. Analysis of the agricultural transition relies primarily on archaeological and paleodemographic data and is thus subject to the errors associated with such data. The assumptions needed to use these data can profoundly affect the inferences that are drawn. While it is clear that, in general, population growth accompanied the agricultural transition, it is not as clear exactly how fertility and mortality changed or whether the transition caused a decline in health. Although the model of the agricultural demographic transition as outlined here may be correct, researchers should remain aware of the underlying assumptions and be open to future empirical evidence.

Ancient pathogen DNA in archaeological samples detected with a Microbial Detection Array.

Ancient human remains of paleopathological interest typically contain highly degraded DNA in which pathogenic taxa are often minority components, making sequence-based metagenomic characterization costly. Microarrays may hold a potential solution to these challenges, offering a rapid, affordable, and highly informative snapshot of microbial diversity in complex samples without the lengthy analysis and/or high cost associated with high-throughput sequencing. Their versatility is well established for modern clinical specimens, but they have yet to be applied to ancient remains. Here we report bacterial profiles of archaeological and historical human remains using the Lawrence Livermore Microbial Detection Array (LLMDA). The array successfully identified previously-verified bacterial human pathogens, including Vibrio cholerae (cholera) in a 19th century intestinal specimen and Yersinia pestis (“Black Death” plague) in a medieval tooth, which represented only minute fractions (0.03% and 0.08% alignable high-throughput shotgun sequencing reads) of their respective DNA content. This demonstrates that the LLMDA can identify primary and/or co-infecting bacterial pathogens in ancient samples, thereby serving as a rapid and inexpensive paleopathological screening tool to study health across both space and time.

Mortality risk and survival in the aftermath of the medieval Black Death.

The medieval Black Death (c. 1347-1351) was one of the most devastating epidemics in human history. It killed tens of millions of Europeans, and recent analyses have shown that the disease targeted elderly adults and individuals who had been previously exposed to physiological stressors. Following the epidemic, there were improvements in standards of living, particularly in dietary quality for all socioeconomic strata. This study investigates whether the combination of the selective mortality of the Black Death and post-epidemic improvements in standards of living had detectable effects on survival and mortality in London. Samples are drawn from several pre- and post-Black Death London cemeteries. The pre-Black Death sample comes from the Guildhall Yard (n = 75) and St. Nicholas Shambles (n = 246) cemeteries, which date to the 11th–12th centuries, and from two phases within the St. Mary Spital cemetery, which date to between 1120-1300 (n = 143). The St. Mary Graces cemetery (n = 133) was in use from 1350–1538 and thus represents post-epidemic demographic conditions. By applying Kaplan-Meier analysis and the Gompertz hazard model to transition analysis age estimates, and controlling for changes in birth rates, this study examines differences in survivorship and mortality risk between the pre- and post-Black Death populations of London. The results indicate that there are significant differences in survival and mortality risk, but not birth rates, between the two time periods, which suggest improvements in health following the Black Death, despite repeated outbreaks of plague in the centuries after the Black Death.

Yersinia pestis: new evidence for an old infection

The successful reconstruction of an ancient bacterial genome from archaeological material presents an important methodological advancement for infectious disease research. The reliability of evolutionary histories inferred by the incorporation of ancient data, however, are highly contingent upon the level of genetic diversity represented in modern genomic sequences that are publicly accessible, and the paucity of available complete genomes restricts the level of phylogenetic resolution that can be obtained. Here we add to our original analysis of the Yersinia pestis strain implicated in the Black Death by consolidating our dataset for 18 modern genomes with single nucleotide polymorphism (SNP) data for an additional 289 strains at over 600 positions. The inclusion of this additional data reveals a cluster of Y. pestis strains that diverge at a time significantly in advance of the Black Death, with divergence dates roughly coincident with the Plague of Justinian (6th to 8th century AD). In addition, the analysis reveals further clues regarding potential radiation events that occurred immediately preceding the Black Death, and the legacy it may have left in modern Y. pestis populations. This work reiterates the need for more publicly available complete genomes, both modern and ancient, to achieve an accurate understanding of the history of this bacterium.

Differential survival among individuals with active and healed periosteal new bone formation

Periosteal new bone formation is frequently used in paleopathological and paleoepidemiological studies to diagnose particular diseases or to assess non-specific stress in past populations. Many researchers distinguish between active (woven or unremodeled) and healed (sclerotic or remodeled) periosteal lesions during data collection, but few published studies maintain a distinction between these two activity categories in analysis or interpretation. Though it has been suggested that healed periosteal lesions might indicate relatively good health and enhanced survivorship, no study has explicitly examined this possible relationship in a large skeletal sample that includes both children and adults. This study examines the relationship between periosteal lesion activity (active vs. healed) and survival using a sample of 538 individuals from several medieval London cemeteries, which in combination span the period 1120-1538. The results of Kaplan-Meier survival analysis indicate that healed periosteal lesions are associated with survival advantages compared to both those with active lesions and those without any lesions at all. These results suggest that active periosteal lesions might most closely reflect high frailty and bioarchaeological studies should focus on the distinction between the presence or absence of healing rather than merely on the presence of periosteal lesions irrespective of their activity.

Sex differences in periodontal disease in catastrophic and attritional assemblages from medieval london

Periodontal disease is one of the most common chronic diseases in living populations, and most studies that have examined sex differences in periodontal disease have found higher frequencies in men compared to women. This study examines sex differences in periodontal disease in two cemeteries from medieval London: the East Smithfield cemetery (c. 1349-1350), an exclusively Black Death cemetery that represents catastrophic mortality (n = 161), and the St. Mary Graces cemetery (c. 1350-1538), a post-Black Death attritional assemblage that represents normal medieval mortality (n = 100). The results reveal a significantly higher frequency of periodontal disease, independent of age, among males compared with females in St. Mary Graces, but no significant difference between the sexes in East Smithfield. The sex differences in the attritional assemblage might reflect heightened susceptibility to periodontal disease in the living population or sex differences in frailty. The differences in the sex patterns of periodontal disease between the two cemeteries might be the result of disproportionately negative effects of the Great Bovine Pestilence and consequent decreases in dairy availability on female oral health among victims of the Black Death.