Yersinia pestis DNA Sequences in Late Medieval Skeletal Finds, Bavaria

To the Editor: Yersinia pestis, the causative agent of plague, is held responsible for 3 human pandemics: the Justinian plague (5th–7th century), the Black Death (13th–15th century), and modern plague (1870s to present). In 1894, Alexandre Yersin identifi ed Y. pestis during an epidemic of plague in Hong Kong (1). However, whether Y. pestis was indeed responsible for the medieval epidemic is still controversial. Y. pestis specifi c DNA has been detected in medieval skeletal fi nds (2,3), although some investigators have failed to do so (4), leading to the suggestion that a viral hemorrhagic fever was the agent of these medieval pandemics (5). Against this background, we investigated a mass burial site that was discovered under the sacristy of the St. Leonhard Catholic church in Manching-Pichl, near Ingolstadt in Bavaria, Germany. In 1984, during the renovation of this church, 75 human skeletons and several scattered skeletal elements were discovered under the sacristy. The skeletons lay densely packed in 4 layers. Apparently, no grave pit had been dug at all, but the bodies were obviously deposited side by side and covered with dirt layer by layer. An approximation of the time of burial was possible only by means of accompanying building structures, which were dated to the Gothic period (1250–1500 CE). In the course of research preceding this study, our research group found Y. pestis DNA in 10 of 33 examined individual skeletal remains from the mass grave beneath the sacristy (6) by using the primer pair YP12D/YP11R (3). In the current study, the remains of 6 persons from the mass burial site that had positive Y. pestis DNA results before were further investigated. DNA from additional tooth samples of these persons was extracted, and more markers mapped on the Y. pestis high copy number plasmid pPCP1 were included. In addition to the primer systems YP12D/YP11R and YP11D/YP10R (3), the following primer systems were used. The primer pair YP14F (5′TCCGGGTCAGGTAATATGGA-3′)/ YP13R (5′-ACCAGCCTTTCACAT TGAGG-3′) amplifi es another sequence section (positions 6953–7082, reference: Y. pestis strain CO92 plasmid pPCP1 sequence AL109969.1) on the Y. pestis pla gene (encoding plasminogen activator). The plasminogen activator belongs to the virulence factors of Y. pestis. The primer pair pst-F (5′-GGTAA ATCGCTGAACCGAAG-3′)/pst-R (5 ′-AACAGCACCTCTGACGCT TT-3′) amplifi es a 129-bp sequence section (positions 5026–5154, GenBank accession no. AL109969.1) on the pst gene (encoding pesticin activity protein). Pesticin is a bacteriocin that is active against only a few closely related microorganisms. The primer pair PCP-F (5′CATCCACATGCTCAACCCTA-3′)/ PCP-R (5′-CTGAACGCATTTCAG TGGTG-3′) can amplify a 128–130bp sequence section (positions 8428– 8555, GenBank accession no. AF053945.1; positions 8428–8557, GenBank accession no. AL109969.1) on plasmid pPCP1. These 3 new primer sets, designed with the aid of the software component Primer3 (7), were applied by using a suicide PCR method (3,8); i.e., new primer pairs targeting sequences not previously amplifi ed in the laboratory were used. At no time during all examinations was any modern Y. pestis DNA included. The sample preparation, DNA extraction, PCR setup, electrophoretic separation, and sequencing of amplicons are described elsewhere (6,9); however, we used 0.20 μmol/L of each primer, an annealing temperature