Abstract

Yersinia pestis has been identified as the causative agent of the Black Death pandemic in the 14th century. However, retrospective diagnostics in human skeletons after more than 600 years are critical. We describe a strategy following a modern diagnostic algorithm and working under strict ancient DNA regime for the identification of medieval human plague victims. An initial screening and DNA quantification assay detected the Y. pestis specific pla gene of the high copy number plasmid pPCP1. Results were confirmed by conventional PCR and sequence analysis targeting both Y. pestis specific virulence plasmids pPCP1 and pMT1. All assays were meticulously validated according to human clinical diagnostics requirements (ISO 15189) regarding efficiency, sensitivity, specificity, and limit of detection (LOD). Assay specificity was 100% tested on 41 clinically relevant bacteria and 29 Y. pseudotuberculosis strains as well as for DNA of 22 Y. pestis strains and 30 previously confirmed clinical human plague samples. The optimized LOD was down to 4 gene copies. 29 individuals from three different multiple inhumations were initially assessed as possible victims of the Black Death pandemic. 7 samples (24%) were positive in the pPCP1 specific screening assay. Confirmation through second target pMT1 specific PCR was successful for 4 of the positive individuals (14%). A maximum of 700 and 560 copies per µl aDNA were quantified in two of the samples. Those were positive in all assays including all repetitions, and are candidates for future continuative investigations such as whole genome sequencing. We discuss that all precautions taken here for the work with aDNA are sufficient to prevent external sample contamination and fulfill the criteria of authenticity. With regard to retrospective diagnostics of a human pathogen and the uniqueness of ancient material we strongly recommend using a careful strategy and validated assays as presented in our study.

Highlights

  • Yersinia (Y.) pestis evolved from Y. pseudotuberculosis in Central Asia about 1,500–20,000 years ago [1,2]

  • Study collectives To trace the plague agent Y. pestis we investigated ancient DNA (aDNA)

  • One major goal of the study focused on the testing of medieval human samples using a similar diagnostic approach as recommended for the detection of highly pathogenic bacteria such as Y. pestis in clinical human samples of recent plague victims [39]

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Summary

Introduction

Yersinia (Y.) pestis evolved from Y. pseudotuberculosis in Central Asia about 1,500–20,000 years ago [1,2]. The beginning of the second pandemic became known as the Black Death originating from the Latin expression atra mors, whereas “atra” can be translated with “black” or “terrible” [5]. It was part of the so-called second pandemic, which started in 1345/6 and lasted for several centuries [1,5]. Since 1894 the most recently evolved biovar Orientalis spread to various countries Named modern plague, it is still endemic in Asia, Africa, and America causing 1,000 to 3,000 noted human cases each year including up to 230 deaths [3]

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