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      Evidence of a Louse-Borne Outbreak Involving Typhus in Douai, 1710-1712 during the War of Spanish Succession

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          Abstract

          Background

          The new field of paleomicrobiology allows past outbreaks to be identified by testing dental pulp of human remains with PCR.

          Methods

          We identified a mass grave in Douai, France dating from the early XVIII th century. This city was besieged during the European war of Spanish succession. We tested dental pulp from 1192 teeth (including 40 from Douai) by quantitative PCR (qPCR) for R. prowazekii and B. quintana. We also used ultra-sensitive suicide PCR to detect R. prowazekii and genotyped positive samples.

          Results and Discussion

          In the Douai remains, we identified one case of B. quintana infection (by qPCR) and R. prowazekii (by suicide PCR) in 6/21 individuals (29%). The R. prowazekii was genotype B, a genotype previously found in a Spanish isolate obtained in the first part of the XX th century.

          Conclusion

          Louse-borne outbreaks were raging during the XVIII th century; our results support the hypothesis that typhus was imported into Europe by Spanish soldiers from America.

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          Most cited references 22

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          Molecular identification by "suicide PCR" of Yersinia pestis as the agent of medieval black death.

          Medieval Black Death is believed to have killed up to one-third of the Western European population during the 14th century. It was identified as plague at this time, but recently the causative organism was debated because no definitive evidence has been obtained to confirm the role of Yersinia pestis as the agent of plague. We obtained the teeth of a child and two adults from a 14th century grave in France, disrupted them to obtain the pulp, and applied the new "suicide PCR" protocol in which the primers are used only once. There were no positive controls: Neither Yersinia nor Yersinia DNA were introduced in the laboratory. A negative result is followed by a new test using other primers; a positive result is followed by sequencing. The second and third primer pair used, coding for a part of the pla gene, generated amplicons whose sequence confirmed that it was Y. pestis in 1 tooth from the child and 19/19 teeth from the adults. Negative controls were negative. Attempts to detect the putative alternative etiologic agents Bacillus anthracis and Rickettsia prowazekii failed. Suicide PCR avoids any risk of contamination as it uses a single-shot primer-its specificity is absolute. We believe that we can end the controversy: Medieval Black Death was plague.
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            Detection of 400-year-old Yersinia pestis DNA in human dental pulp: an approach to the diagnosis of ancient septicemia.

            Ancient septicemic plague epidemics were reported to have killed millions of people for 2 millenniums. However, confident diagnosis of ancient septicemia solely on the basis of historical clinical observations is not possible. The lack of suitable infected material has prevented direct demonstration of ancient septicemia; thus, the history of most infections such as plague remains hypothetical. The durability of dental pulp, together with its natural sterility, makes it a suitable material on which to base such research. We hypothesized that it would be a lasting refuge for Yersinia pestis, the plague agent. DNA extracts were made from the dental pulp of 12 unerupted teeth extracted from skeletons excavated from 16th and 18th century French graves of persons thought to have died of plague ("plague teeth") and from 7 ancient negative control teeth. PCRs incorporating ancient DNA extracts and primers specific for the human beta-globin gene demonstrated the absence of inhibitors in these preparations. The incorporation of primers specific for Y. pestis rpoB (the RNA polymerase beta-subunit-encoding gene) and the recognized virulence-associated pla (the plasminogen activator-encoding gene) repeatedly yielded products that had a nucleotide sequence indistinguishable from that of modern day isolates of the bacterium. The specific pla sequence was obtained from 6 of 12 plague skeleton teeth but 0 of 7 negative controls (P < 0.034, Fisher exact test). A nucleic acid-based confirmation of ancient plague was achieved for historically identified victims, and we have confirmed the presence of the disease at the end of 16th century in France. Dental pulp is an attractive target in the quest to determine the etiology of septicemic illnesses detected in ancient corpses. Molecular techniques could be applied to this material to resolve historical outbreaks.
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              The body louse as a vector of reemerging human diseases.

              The body louse, Pediculus humanus humanus, is a strict human parasite, living and multiplying in clothing. Louse infestation is associated with cold weather and a lack of hygiene. Three pathogenic bacteria are transmitted by the body louse. Borrelia recurrentis is a spirochete, the agent of relapsing fever, recently cultured on axenic medium. Historically, massive outbreaks have occurred in Eurasia and Africa, but currently the disease is found only in Ethiopia and neighboring countries. Bartonella quintana is now recognized as an agent of bacillary angiomatosis bacteremia, trench fever, endocarditis, and chronic lymphadenopathy among the homeless. Rickettsia prowazekii is the agent of epidemic typhus. The most recent outbreak (and the largest since World War II) was observed in Burundi. A small outbreak was also reported in Russia in 1997. Louse infestation appears to become more prevalent worldwide, associated with a decline in social and hygienic conditions provoked by civil unrest and economic instability.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2010
                27 October 2010
                : 5
                : 10
                Affiliations
                [1 ]Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, UMR CNRS 6236, IRD198, IFR48, Faculté de Médecine, Université de la Méditerranée, Marseille, France
                [2 ]Unité d'anthropologie, UMR CNRS 6578, Faculté de médecine, Université de la Méditerranée, Marseille, France
                Charité-Universitätsmedizin Berlin, Germany
                Author notes

                Conceived and designed the experiments: DR. Performed the experiments: TNH MS CR. Analyzed the data: GA DR MD. Contributed reagents/materials/analysis tools: MS GA DR CR. Wrote the paper: TNH GA DR.

                Article
                PONE-D-10-01268
                10.1371/journal.pone.0015405
                2965176
                21060879
                Nguyen-Hieu, 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.
                Counts
                Pages: 8
                Categories
                Research Article
                Biology
                Biochemistry
                Nucleic Acids
                DNA
                DNA amplification
                Evolutionary Biology
                Paleontology
                Microbiology
                Bacteriology
                Emerging Infectious Diseases
                Medical Microbiology
                Medicine
                Infectious Diseases

                Uncategorized

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