Bartonella quintana caused trench fever in the framework of two World Wars and is now recognized as an agent of re-emerging infection. Many reports indicated the popularity of B. quintana exposure since the 90s. For evaluating its prevalence in ancient populations, we used real-time PCR to detect B. quintana DNA in 400 teeth collected from 145 individuals dating from the 1st to 19th centuries in nine archeological sites with the presence of negative controls. Fisher’s exact test was used to compare the prevalence of B. quintana detection in civil and military populations. B. quintana DNA was confirmed in a total of 28/145 (19.3%) individuals, comprising 78 citizens and 67 soldiers, 20.1% and 17.9% of which were positive for B. quintana bacteremia, respectively. This study collected previous studies on these ancient samples and showed that the presence of B. quintana infection followed the course of time in human history; a total of 14/15 sites from five European countries had a positive prevalence. The positive rate in soldiers was higher than those of citizens, with 20% and 18.8%, respectively, in the 18th - 19th centuries, but the difference in frequency was not significant. These results confirmed the role of dental pulp in diagnosing B. quintana bacteremia in ancient populations and showed the incidence of B. quintana in both citizens and soldiers. Many recent findings contributed to understanding the coevolution of the relationship between B. quintana and humans.

22Bartonella quintana. 23 24 2 ABSTRACT 25Photogrammetry and cascading microscopy investigations of dental pulp specimens 26 collected from 2,000-year-old individuals buried in a Roman necropolis in Besançon, 27 France, revealed unprecedented preserved tissular and cellular morphology. 28 Photogrammetry yielded 3-D images of the smallest archaeological human remain 29 ever recovered. Optical microscopy examinations after standard hematoxylin-30 phloxine-saffron staining and anti-glycophorin A immunohistochemistry exposed 31 dental pulp cells, in addition erythrocytes were visualized by electron microscopy, 32 which indicated that the ancient dental pulp trapped a blood drop. Fluorescence in 33 situ hybridization applied on red blood cells revealed the louse-borne pathogen 34 Bartonella quintana, a finding confirmed by polymerase chain reaction assays. 35 Through paleohistology and paleocytology, we demonstrate that ancient dental pulp 36 preserved intact blood cells at the time of the individual's death, offering an 37 unprecedented opportunity to engage in direct and indirect tests to diagnose 38 pathogens in ancient buried individuals. 39 40 41 42 3