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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 18(th) 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.

Chemical decomposition and fragmentation may limit the detection of ancient host and microbial DNA while some proteins can be detected for extended periods of time. We applied paleoproteomics on 300-year-old dental pulp specimens recovered from 16 individuals in two archeological funeral sites in France, comprising one documented plague site and one documented plague-negative site. The dental pulp paleoproteome of the 16 teeth comprised 439 peptides representative of 30 proteins of human origin and 211 peptides representative of 27 proteins of non-human origin. Human proteins consisted of conjunctive tissue and blood proteins including IgA immunoglobulins. Four peptides were indicative of three presumable Yersinia pestis proteins detected in 3/8 dental pulp specimens from the plague-positive site but not in the eight dental pulp specimens collected in the plague-negative site. Paleoproteomics applied to the dental pulp is a new and innovative approach to screen ancient individuals for the detection of blood-borne pathogens and host inflammatory response.

While negative controls remained negative, 1/29 paleoserum sample collected from soldier 521 showed reactivity against the Influenza viruses A and B contained in the 2020 vaccine (FluarixTetra, GSK vaccines, Brentford, UK) and 5/29 (24%) collected from soldiers 500, 508, 511, 512, 528 showed anti-Coronavirus reactivity; exhibiting area under curve > 500 units (determined on the basis of the negative and positive control area under curve values) against SARS-CoV-2 in one sample, 229E in two samples and OC43 in four samples; with the individual 528 exhibiting a significant reactivity against the three Coronaviruses (Fig. 1). Individual Age class (years) a 500 30–59 508 30–59 511 20–39 512 30–59 521 20–49 528 20–49 533 30–59 aAge was estimated via the evaluation of pubic symphysis morphology (Schmitt, 2008) and auricular surface of the pelvic bone (Schmitt, 2005), in agreement with the French Army archives indicating that these soldiers were between 20 and 50 years (Verna et al., 2021). [...]due to extensive cross-reactivity between Coronaviruses, mainly supported by the relatively conserved nucleocapsid protein antigenicity, it was not possible to derive from our data, the exact Coronavirus species implied in the ‘Russian flu’. [...]it is necessary to consolidate these indirect diagnostic data by incorporation of recombinant Coronavirus antigens into the mini-line blot assay; and in case of suspected ancient viremia to attempt direct diagnosis based on the detection of specific Coronavirus peptide sequences using paleoproteomics methods (Barbieri et al., 2017), or even nucleotide sequences by metagenomics, in order to support the Coronavirus hypothesis in the controversial aetiology of ‘Russian flu’.

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

Methanogens are acknowledged archaeal members of modern dental calculus microbiota and dental pathogen complexes. Their repertoire in ancient dental calculus is poorly known. We therefore investigated archaea in one hundred dental calculus specimens collected from individuals recovered from six archaeological sites in France dated from the 14 th to 19 th centuries AD. Dental calculus was demonstrated by macroscopic and cone-beam observations. In 56 calculus specimens free of PCR inhibition, PCR sequencing identified Candidatus Methanobrevibacter sp. N13 in 44.6%, Methanobrevibacter oralis in 19.6%, a new Methanomassiliicoccus luminyensis- like methanogen in 12.5%, a Candidatus Nitrososphaera evergladensis-like in one and Methanoculleus bourgensis in one specimen, respectively. One Candidatus Methanobrevibacter sp. N13 dental calculus was further documented by fluorescent in situ hybridization. The prevalence of dental calculus M. oralis was significantly lower in past populations than in modern populations (P = 0.03, Chi-square test). This investigation revealed a previously unknown repertoire of archaea found in the oral cavity of past French populations as reflected in preserved dental calculus.

Historical records suggest that multiple burial sites from the 14th-16th centuries in Venice, Italy, were used during the Black Death and subsequent plague epidemics. High throughput, multiplexed real-time PCR detected DNA of seven highly transmissible pathogens in 173 dental pulp specimens collected from 46 graves. Bartonella quintana DNA was identified in five (2.9%) samples, including three from the 16th century and two from the 15th century, and Yersinia pestis DNA was detected in three (1.7%) samples, including two from the 14th century and one from the 16th century. Partial glpD gene sequencing indicated that the detected Y. pestis was the Orientalis biotype. These data document for the first time successive plague epidemics in the medieval European city where quarantine was first instituted in the 14th century.

The lack of well-preserved material upon which to base the paleo-microbiological detection of parasites has prevented extensive documentation of past outbreaks of malaria in Europe. By trapping intact erythrocytes at the time of death, dental pulp has been shown to be a suitable tissue for documenting ancient intraerythrocytic pathogens such as parasites. Total DNA and proteins extracted from 23 dental pulp specimens collected from individuals exhumed from the 9th to 13th century archaeological site in Mariana, Corsica, were analyzed using open-mind paleo-auto-immunohistochemistry and direct metagenomics, -targeting immunochromatography assays. All experiments incorporated appropriate negative controls. Paleo-auto-immunohistochemistry revealed the presence of parasites spp. in the dental pulp of nine teeth. A further immunochromatography assay identified the presence of at least one antigen in nine individuals. The nine teeth, for which the PfHRP-2 antigen specific of was detected, were also positive using paleo-autoimmunohistochemistry and metagenomics. Dental pulp erythrocytes proved to be suitable for the direct paleomicrobiology documentation of malaria in nine individuals buried in medieval Corsica, in agreement with historical data. This provides additional information on the millennial dynamics of spp. in the Mediterranean basin.

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 β -globin gene demonstrated the absence of inhibitors in these preparations. The incorporation of primers specific for Y. pestis rpoB (the RNA polymerase β -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.

The cause of past plague pandemics was controversial but several research teams used PCR techniques and dental pulp as the primary material to reveal that they were caused by Yersinia pestis. However, the degradation of DNA limits the ability to detect ancient infections. We used for the first time immuno-PCR to detect Yersinia pestis antigens; it can detect protein concentrations 70 times lower than the standard ELISA. After determining the cut-off value, we tested 34 teeth that were obtained from mass graves of plague, and compared previous PCR results with ELISA and immuno-PCR results. The immuno-PCR technique was the most sensitive (14 out of 34) followed by the PCR technique (10 out of 34) and ELISA (3 out of 34). The combination of these three methods identified 18 out of 34 (53%) teeth as presumably being from people with the plague. Immuno-PCR is specific (no false-positive samples were found) and more sensitive than the currently used method to detect antigens of ancient infections in dental pulp. The combination of three methods, ELISA, PCR and immuno-PCR, increased the capacity to identify ancient pathogens in dental pulp.

Ancient dental pulps are highly precious samples because they conserve DNA from humans and blood-borne pathogens for ages. However, little is known about the microbial communities present in dental pulps. Here, we analyzed ancient and modern dental pulp samples from different time periods and geographic regions and found that they are colonized by distinct microbial communities, which can be differentiated from other oral cavity samples. We found that despite the presence of environmental bacteria, ancient dental pulps conserve a clear and well-conserved record of oral microbes. We were able to detect several different oral pathogens in ancient and modern dental pulps, which are commonly associated with periodontal diseases. We thus showed that ancient dental pulps are not only valuable sources of DNA from humans and systemic infections, but also an open window for the study of ancient oral microbiomes.