Abstract From AD 1347 to AD 1353, the Black Death killed tens of millions of people in Europe, leaving misery and devastation in its wake, with successive epidemics ravaging the continent until the 18th century. The etiology of this disease has remained highly controversial, ranging from claims based on genetics and the historical descriptions of symptoms that it was caused by Yersinia pestis to conclusions that it must have been caused by other pathogens. It has also been disputed whether plague had the same etiology in northern and southern Europe. Here we identified DNA and protein signatures specific for Y. pestis in human skeletons from mass graves in northern, central and southern Europe that were associated archaeologically with the Black Death and subsequent resurgences. We confirm that Y. pestis caused the Black Death and later epidemics on the entire European continent over the course of four centuries. Furthermore, on the basis of 17 single nucleotide polymorphisms plus the absence of a deletion in glpD gene, our aDNA results identified two previously unknown but related clades of Y. pestis associated with distinct medieval mass graves. These findings suggest that plague was imported to Europe on two or more occasions, each following a distinct route. These two clades are ancestral to modern isolates of Y. pestis biovars Orientalis and Medievalis. Our results clarify the etiology of the Black Death and provide a paradigm for a detailed historical reconstruction of the infection routes followed by this disease.

Author Summary Several historical epidemic waves of plague have been attributed to Yersinia pestis, the etiologic agent of modern plague. The most famous of these was the second pandemic which was active in Europe from AD 1347 until 1750, and began with the ‘Black Death’. The most informative method to establish the etiological nature of these ancient infections should be the analysis of ancient DNA, but the results of this method have been controversial. Here, by combining ancient DNA analyses and protein-specific detection, we demonstrate unambiguously that Y. pestis caused the Black Death. Furthermore, we show that at least two variants of Y. pestis spread over Europe during the second pandemic. The analysis of up to 20 diagnostic markers reveals that the two variants evolved near the time that phylogenetic branches 1 and 2 separated and may no longer exist. Our results thus resolve a long-standing debate about the etiology of the Black Death and provide key information about the evolution of the plague bacillus and the spread of the disease during the Middle Ages.

Citation: Haensch S, Bianucci R, Signoli M, Rajerison M, Schultz M, Kacki S, et al. (2010) Distinct Clones of Yersinia pestis Caused the Black Death. PLoS Pathog 6(10): e1001134. https://doi.org/10.1371/journal.ppat.1001134 Editor: Nora J. Besansky, University of Notre Dame, United States of America Received: May 28, 2010; Accepted: September 7, 2010; Published: October 7, 2010 Copyright: © 2010 Haensch et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by grants from the Deutsche Forschungsgemeinschaft (DFG Br 2965/1-1 and Br 2965/1-2), the University of Mainz (FP1-2007) and the Science Foundation of Ireland (05/FE1/B882). The RDT analysis was supported by Compagnia di San Paolo (2007.0171). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Of the numerous epidemics in human history, three pandemics are generally accepted as having been caused by plague. Justinian's plague (AD 541–542) spread from Egypt to areas surrounding the Mediterranean [1]. In 1347, an epidemic known as the Black Death spread from the Caspian Sea to almost all European countries, causing the death of one third of the European population over the next few years [2]. This second pandemic persisted in Europe until 1750, causing successive and progressively declining epidemic waves. A third plague pandemic began in the Yunnan region of China in the mid-19th century, and spread globally via shipping from Hong Kong in 1894. During this last pandemic, the etiological cause of plague was identified as Yersinia pestis, a Gram-negative bacterium [3], [4]. Most microbiologists and epidemiologists believe that Y. pestis was also the etiological agent of the first two pandemics. This belief is supported by ancient DNA (aDNA) analyses which identified sequences specific for Y. pestis in the teeth of central European plague victims from the first and second pandemics [5]–[7]. Moreover, the Y. pestis F1 protein capsule antigen has been detected in ancient plague skeletons from Germany and France by immunochromatography [8], [9]. Based on studies on modern strains, microbiologists have subdivided Y. pestis into three biovars: Antiqua, Medievalis, and Orientalis. These biovars can be distinguished depending on their abilities to ferment glycerol and reduce nitrate [10]. The Medievalis biovar is unable to reduce nitrates due to a G to T mutation that results in a stop codon in the napA gene [11], while the Orientalis biovar cannot ferment glycerol because of a 93 bp deletion in the glpD gene [11], [12]. Conversely, the Antiqua biovar is capable of performing both reactions [10]. An apparent historical association of the routes of the three pandemics with the modern geographical sources of the three biovars led Devignat to propose that each plague pandemic was caused by a different biovar [10]. There is no doubt that the ongoing third pandemic was caused by biovar Orientalis, but an attribution of the first and second pandemics to Antiqua and Medievalis, respectively, is questionable. Unlike Devignat's hypothesis, recent aDNA analyses of samples from the 7th–9th and 18th centuries yielded Orientalis-specific microsatellites [13] and the characteristic 93 bp glpD deletion [14], thus suggesting that the Orientalis biovar also caused Justinian's plague and the second pandemic. Despite these results, a debate continues regarding whether Y. pestis really was the causative agent of the Black Death, as summarized by Byrne [15]. Some epidemiologists and historians have denied this conclusion due to inconsistencies between the clinical and epidemiological characteristics of plague in historical records and those observed in India in the early 20th century [16]–[19]. Alternative putative etiologies of the Black Death include a viral hemorrhagic fever [16] or a currently unknown pathogen [19]. In part, these alternative etiologies reflect apparent discrepancies between historical observations of extremely rapid spread of mortality during the Black Death with the dogma based on Indian epidemiology that plague is associated with transmission from infected rats via blocked fleas, which can first transmit Y. pestis approximately 30-days after a blood meal. However, recent data show that transmission by fleas can occur continuously after a blood meal and does not depend on blockage [20]. Even the aDNA studies have not been considered to be conclusive because other teams were initially unable to find ancient Y. pestis DNA in human teeth from plague pits in central and north Europe [21], thus supporting the interpretation that the massive human deaths that occurred in these areas at the time of the second pandemic were not caused by Y. pestis. Here, we combined analyses of aDNA with detection of the Y. pestis F1 antigen on skeletons from mass graves throughout Europe that date to the time of the Black Death and later epidemic waves. Our results show that historical plague was caused by Y. pestis throughout Europe. We also genotyped the causative agent for these mass fatalities by informative single nucleotide polymorphisms (SNPs) [11], and show that the strains causing mass deaths were unrelated to either Medievalis or Orientalis biovars.

Discussion From historical accounts we know that the Black Death was imported into southern Europe from Asia, and reached Marseilles (southeast France) by November 1347. Plague then spread to western France by land and sea, reaching Narbonne and Carcassonne at the beginning of 1348 [17], [25]. This extension to the west was probably responsible for a plague epidemic in Saint-Laurent-de-la-Cabrerisse, which lies between Narbonne and Carcassonne. Although written records are not available to confirm that supposition, radiocarbon measurements on skeletons from the three multiple graves in Saint-Laurent-de-la-Cabrerisse date those skeletons to the outbreak of AD 1348 or 1374. Plague continued to spread to northern France in early 1348, and is thought to have been exported from France to England via shipping and trading in the summer of that year [26]. Hereford, a busy English market town near the Welsh border, and a centre for religious pilgrimage, recorded its first plague deaths in the spring of 1349, followed by a second outbreak in 1361, and a third in 1369 [27]. The Hereford plague pits have been AMS radiocarbon dated to calAD 1335±54 and our specimens can therefore be attributed to one of these three epidemic waves. Our finding of identical genotypes (based on 20 markers) in Saint-Laurent-de-la-Cabrerisse and Hereford thus lends support to historical evidence [2], [25] which suggest that plague spread from France to England (Fig. 1) in the second half of the 14th century. Bergen op Zoom was a thriving port city in the south of the Low Countries. There are no written reports describing plague in Bergen op Zoom for the years 1348–1351 or in subsequent decades because administrative records were destroyed by fire in 1397. Radiocarbon dating has not been performed on the skeletal remains of the circa 800 individuals buried in the mass graves from Bergen op Zoom, but soil stratigraphy, artifacts and coins allow dating to the mid-14th century. Historical records indicate that the Black Death reached the southern Low Countries from France or England in 1349 and that the northern Low Countries were infected from Friesland in 1350 [25]. However, the Y. pestis genotype identified in our skeletons from Bergen op Zoom differed from those found in Hereford and Saint-Laurent-de-la-Cabrerisse, implying that Bergen op Zoom (and possibly other parts of the southern Netherlands) was not directly infected from England or France in AD 1349. Instead, our results are more consistent with the idea that the genotype in Bergen op Zoom represents a different route of plague spread, possibly from the northern to the southern Low Countries in AD 1350 (Fig. 1). Bergen op Zoom was in intensive commercial contact with the north of the Netherlands in the 14th century, exporting pottery to Amsterdam [28]. Friesland, in turn, was infected from Norway, which during the Middle Ages traded extensively with both England and the Hanseatic cities along the North Sea coast of Germany. Only one SNP, s12, was identified that distinguished the genotypes in Bergen op Zoom and Hereford, indicating that they are closely related. That single SNP is highly informative because it is on branch 1, showing that the genotype found in Bergen op Zoom evolved on the phylogenetic path to 1.ORI, the causative agent of the third pandemic that erupted in Hong Kong in 1894. If SNP s12 had evolved anywhere within Europe, then Y. pestis must have subsequently spread from Europe to Central and East Asia, the source of the third pandemic associated with 1.ORI [11]. Alternatively, and probably more likely, multiple genotypes were imported to Europe via the usual routes of trade from Central Asia during the 14th century and afterwards. In that event, the lineage from Bergen op Zoom could represent a distinct wave of infection relative to those from Hereford and Saint-Laurent-de-la-Cabrerisse that reached the Low Countries from Norway, the Hanseatic cities, or another site that is not intuitively obvious from the historical records. Together with prior analyses from the south of France [5], [6] and Germany [7], our data from widely distributed mass plague pits ends the debate about the etiology of the Black Death, and unambiguously demonstrates that Y. pestis was the causative agent of the epidemic plague that devastated Europe during the Middle Ages. However, there is an apparent discrepancy between the genotypes indentified in this study and those reported elsewhere on aDNA from Justinian's plague and the second pandemic [13], [14]. In the first of these analyses, aDNA from Justinian's plague and the second pandemic was concluded to correspond to biovar Orientalis (now designated 1.ORI) on the basis of microsatellites. That assignment is not definitive because only isolates of four modern populations (1.ORI, 2.MED, 1.ANT and 2.ANT) were used for comparative purposes, and it is not clear whether microsatellites could distinguish 1.ORI from other historical populations on branch 1 [29]. The second analysis demonstrated the presence of the 96 bp glpD deletion that is characteristic of 1.ORI in samples from the 7th to 9th century (Justinian's plague) and about 1720 (the end of the second pandemic). In our study we did not find the glpD deletion in Y. pestis aDNA from the Black Death period. We did not test aDNA from the same sources and dates, and the possibility that 1.ORI bacteria were imported from East Asia both before and after the Black Death cannot be excluded. However, new data suggesting that 1.ORI evolved in the last 212 years [30] argue against this possibility. The ancient genotype from Bergen op Zoom described here differs from all known modern populations from three continents [30], and might now be extinct. The strains from France or England may still exist because the SNPs that have currently been tested match the genotype of isolates from China that have recently been assigned to a new branch, designated branch 3 [30]. Additional SNPs that are specific for branch 3 will first need to be identified and tested to determine whether the ancient populations from France and England matches existing isolates. These tests should involve further genotyping of large, global collections of extant lineages in combination with extensive palaeogenetic analyses of other mass graves from the historical routes travelled by the Black Death and Justinian's plague. Such analyses are very promising because they could potentially fully reconstruct the history of ancient plague pandemics within the modern phylogeographical context of Y. pestis.

Materials and Methods Samples For aDNA analyses, one or more teeth or bone samples were taken from 76 presumed plague victims from various locations in Europe (Table 1). Archaeological information on the single plague sites can be found in more detailed form in Text S1. Teeth were preferred as sample material due to better aDNA preservation, and where possible, two different teeth per individual were submitted for genetic investigation. If no teeth were available, femoral bone samples were used. Negative controls were obtained from individuals who either died before or after the Black Death, in a context that did not involve a major epidemic. Sample preparation and DNA extraction aDNA analyses were carried out in the laboratories of the Institute of Anthropology in Mainz, Germany. The pre-PCR laboratories are situated in a humanities building physically separated from the post-PCR area, have dedicated equipment and are subjected to overnight UV-light exposure. Other anti-contamination measures used are extensively reported in previous publications [31], [32]. After arrival in the laboratory, samples were submitted to decontamination procedures consisting of 45 minutes of UV irradiation on each side, mechanical removal of the outer surface by sandblasting (Harnisch und Rieth, Winterbach, Germany) and a second UV irradiation. The samples were then fine powdered using a mixer mill (Retsch, Haan, Germany) and stored at 4°C or −20°C until use. Aliquots of 0.38–0.5 g powder were incubated overnight on a rotary mixer at 37°C in a decalcification and digestion solution consisting of 0.5 M EDTA (pH 8; Roth, Karlsruhe, Germany), 0.4% N-Laurylsarcosine and 0.46 mg/ml Proteinase K (Roche, Germany). Post-digestion, DNA was extracted using phenol/chloroform/isoamyl alcohol (25∶24∶1 Roth, Karlsruhe, Germany) and trichlormethan/chloroform (ROTIPURAN ≥99%, p.a.; Roth, Germany); then desalted and concentrated using micro-concentrators (Centricons 100, 50 or Amicon Ultra-15 Centrifugal Filter Units, Millipore, Schwalbach/Ts., Germany). When possible, several independent extracts were obtained from two or more different samples from each individual (see Table S3). In addition to blank extraction controls, a negative control consisting of hydroxyl apatite was generally co-processed by milling, and further co-extracted and co-amplified throughout the analyses (milling blank). Sterile aliquot reagents were changed frequently. To monitor the bacterial content of the soil, 500 mg of earth associated with the sample Man30 (Augsburg) was analyzed. The sample was incubated in 1 ml buffer consisting of 100 mM NaCl, 10 mM Tris/HCl (pH 8), 50 mM EDTA (pH 8) and 0.5% SDS and submitted to 20 minutes sonication (EMMI 30HC, EMAG Technologies, Germany) at 50°C. Incubation on a rotary mixer overnight at 37°C followed after adding 0.1 mg/ml Proteinase K. DNA extraction was carried out with phenol/chloroform/isoamyl alcohol as described above. For desalting and concentration, both Centricons 50 and Amicon Ultra-15 Centrifugal Filter Units were used. Amplification of Y. pestis specific genes The amplifications of the Y. pestis specific genes were performed using the primers described in Table S2. The amplification reaction was set up with 2–10 µl of extracts in a final volume of 50 µL, 1–1,2× PCR Gold Buffer (Applied Biosystems, Darmstadt, Germany), 2.5 U Ampli-TaqGold (Applied Biosystems, Darmstadt, Germany), 2.5–3 mM MgCl 2 (Applied Biosystems), 0.2 mM dNTP mix (Qiagen GmbH, Hilden, Germany), 0.2 µM each Primer (Biospring, Frankfurt am Main, Germany), 8 µg/ml BSA (Roche) and UV irradiated HPLC water (Arcos Organics/Fisher Scientific, Germany). The amplifications were carried out in a Mastercycler Gradient (Eppendorf, Hamburg, Germany). The cycle program consisted of an initial denaturation at 94°C for 6 minutes, followed by 50 cycles of 40 seconds at 94°C, 40 seconds at 52–62°C and 40 seconds at 72°C. In the different PCR sets, samples were co-processed together with milling, extraction and amplification controls. Amplification of the soil sample was performed with two different concentrations of the target (1∶1 and 1∶50, each time 0.1–5 µL target) using the primer pairs 16S F3/R3 and rpoB F1/R1 as described above. Sequencing Amplification products were purified using the Invisorb Rapid PCR Purification Kit (Invitek, Berlin-Buch, Germany) according to the manufacturer's instructions. Alternatively, the purification was carried out by digestion with ExoI (20 U/µL; Fermentas, St. Leon-Rot, Germany)/SAP (1 U/µL; Fermentas, St. Leon-Rot, Germany) enzymes by incubating the reaction mix for 45–60 minutes at 37°C. The successive enzyme inactivation at 80°C for 15 minutes concluded the procedure. Sequencing of successfully amplified fragments was carried out with the DNA Sequencing Kit (BigDye Terminator v3.1 Cycle Sequencing Kit; Applied Biosystems) using 25 cycles at 92°C for 30 seconds, 15 seconds at 52–62°C and 2.5–3 minutes at 60°C. Cycle sequencing products were purified by using Sephadex-G50 Fine (GE Healthcare, Uppsala, Sweden) and analyzed by capillary electrophoresis on ABI PRISM 3130 Genetic Analyzers (ABI PRISM Applied Biosystems). Sequence analyses and alignments The sequences were further analyzed using the program Seqman II and MegAlign from the DNA Star software package (version 7.0.0). The sequence alignments are listed in Text S2 (Fig.s S1, S2, S3, S4, S5 and S6). The reference sequences used to align the sequenced products are listed below (paragraph ‘Accession numbers’). Strain CO92 belongs to branch 1.ORI and strain KIM belongs to branch 2.MED according to the proposed classification [11]. The position of the single SNPs was previously published [11]. Rapid diagnostic test (RDT) Nineteen samples from Parma, seven samples from Augsburg, five samples from Bergen op Zoom, seven samples from Hereford and nine from Saint-Laurent-de-la-Cabrerisse were tested along with soil samples and 28 negative controls with double blind procedures. The analyses were carried out at the Laboratory of Parasitology and Parasitic Diseases, Department of Animal, Production, Epidemiology and Ecology at the University of Turin, Italy. Spongy bone from femora was chosen as a preferential source of material [22]. The bone samples were cleaned with dry brushes and decontaminated by UV light. The external bone surface was removed with a drill (Kavo Intramatic Lux 2) while mounted on a micromotor turning at 9,000 rpm. Next, the spongy bone samples (1–2 g) were powdered by hand in sterile conditions using a mortar and pestle. Powder from each sample was stored in 15 ml sterile vials until use. Tooth samples were prepared as described for aDNA analyses. To detect Yersinia pestis F1 antigen, we used the plague dipstick assay developed and tested on extant sufferers by the Institute Pasteur of Madagascar and Paris (rapid diagnostic test for plague- RDT). This immunochromatographic-based assay detects the F1 envelope glycoprotein specific to Y. pestis [33], [34]. The principles of the dipstick assay and methods for semi-quantitation of the AgF1 concentrations in ancient specimens have been described elsewhere [9]. For each sample, extracts were prepared from 50 mg of bone material, tooth or soil samples reconstituted in 200 µl of sterile saline solution and subjected to the following protocol: three freeze/thaw cycles, sonication for 15 minutes, and a fourth freeze/thaw cycle. The suspensions were incubated for 24 hours at 4°C to solubilize the remaining antigens. The crude extracts were then centrifuged at 5,000 rpm at room temperature and the supernatant submitted to dipstick assay. The tests were repeated five times on the same specimen and the results were read after 15 minutes. The detection threshold of the test (0.5 ng/ml) is diagnostic for Y. pestis infection in ancient skeletal remains [9], [22], [23]. The results of the investigation are detailed in Table S1. Accession numbers The GenBank (http://www.ncbi.nlm.nih.gov) accession numbers for DNA sequences longer than 50 bp determined in this paper are HM752036-HM752097. All alignments are published in Text S2 (Fig.s S1, S2, S3 S4, S5 and S6). To align sequenced products, the reference sequences from Y. pestis CO92 (NC_003134; AL590842), Y. pestis KIM (AE009952.1) and Y. pseudotuberculosis IP32953 (BX936398) were used for genomic markers and SNPs, whereas caf1- and pla-sequences were aligned with NC_003132 (AL109969) and NC_003134 (AL117211).

Acknowledgments The authors are grateful to Maria Giovanna Belcastro (Dept. of Experimental Evolutionary Biology, University of Bologna, Italy), Michaela Hermann (Kunstsammlungen und Museen, Abt. Römisches Museum/Stadtarchäologie, Augsburg, Germany), Anthea Boylston and Alan Ogden (Div. of Archaeological Sciences, University of Bradford, UK) for the samples from Parma, Augsburg, and Hereford, respectively. We are indebted to Lila Rahalison (Pasteur Institute of Madagascar) for providing the dipstick assay, and Ezio Ferroglio (Dept. of Animal Production, Epidemiology and Ecology, Faculty of Veterinary Medicine, University of Turin, Italy) for technical support for the RDT analysis. Thanks to Mauro Gasparini (Department of Mathematics, Polytechnic of Turin, Italy) for statistical assistance, to Joachim Burger (Institute for Anthropology, University of Mainz, Germany) for his comments and to Otto Appenzeller (New Mexico Health Enhancement and Marathon Clinics Research Foundation, Albuquerque, New Mexico, USA) for important editing suggestions.

Author Contributions Conceived the experiment: R. Bianucci, M. Schultz, B. Bramanti. Carried out aDNA research: S. Haensch, B. Bramanti. Performed the RDT analysis: R. Bianucci, M. Rajerison. Carried out the data analysis together with E. Carniel and M. Achtman: S. Haensch, B. Bramanti. Contributed with archaeological and historical data: M. Signoli, S. Kacki, M. Vermunt, D. Weston, D. Hurst. Wrote the paper: S. Haensch, M. Achtman, E. Carniel, B. Bramanti.