Figure 2. Amplification of the arp gene fragment from the extracts obtained at the UAC¸ . M, molecular weight marker, 100 bp DNA ladder (Sigma); 1, ELS 551; 4,7, ELS 558; 2,5 unaffected ancient samples; 3,6,8 extraction blanks (KE); 9, PCR blank (K2). doi:10.1371/journal.pone.0036371.g002 sequences were identical to the sequence of T. pallidum subsp. pallidum (Figure S3). Discussion In this study we amplified ancient Treponemal DNA from four specimens belonging to at least 2 different individuals. This represents the highest rate of success in amplifying ancient Treponemal DNA. Previously, just one ancient individual yielded positive result [10], despite several attempts in dozens of individuals analyzed [15–17]. The main difference between our study and previous reports lies in the fact that we analyzed neonate remains, with clear signs of being affected by congenital syphilis The Molecular Study of Ancient Syphilis (CS). This lead us to hypothesize that in remains from CS-affected neonates the probability of preservation of Treponemal DNA is highly increased, due to a rapid dissemination in the skeleton of a high number of spirochetes, which after the death would leave their DNA that would be preserved by its association to hydroxyapatite in bones. Recently a newborn infant affected by CS showed long-bone lesions upon X-ray examination [23], and Armangil et al. [24] reported a newborn infant with unusual clinical findings of CS such as a non-fluctuant mass surrounding the left calf, without any additional system involvement such as hepatic or skin involvement or lymph nodes. Furthermore, a study analyzing 121 infants below 3 months of age, affected by CS, also found bone damage in 54.3% of the cases [25]. This indicates that newborns are sensitive to bone damage at early stages of the disease, supporting the findings of Schneider (cited by [26]) who reported that, in congenitally syphilitic infants, resting spirochetes may be found in the bone cell lacunae, and demonstrate the rapid spread of T. pallidum throughout the whole skeleton in CS. In 1931 Bauer demonstrated the presence of T. pallidum in the pulp, the dentinoid, the dentinal tubules, the tooth sac, the ameloblast layer and even in the stellate reticulum, by microscopic studies in four congenitally syphilitic fetuses and infants [27]. Further studies on the jaws of six congenitally syphilitic but nonmacerated subjects (four fetuses of 6, 8, and 9 months, and two infants of 2 1/2 weeks and 1 1/2 months of age) demonstrated the presence of T. pallidum in the bone and tooth tissues, although it was noted that the older the infant the fewer were the spirochetes observed [27]. Some years later Je et al. [28] found T. pallidum spirochetes in the ameloblast layer, stellate reticulum of enamel epithelium, odon- toblast layer, and predentin layer, analyzing two cases of CS. These data support the hypothesis that in neonates the number of spirochetes in the skeleton is good enough to warrant their DNA preservation, and that the younger the individual affected by CS, the bigger the probability of amplifiable DNA preservation. This is in contrast to the case of adults affected by venereal syphilis, in which the number of spirochetes in bones is reduced as the disease advance to later stages. Furthermore, von Hunnius et al. [17] infected rabbits with T. pallidum pallidum in order to assess the feasibility of isolating treponemal DNA from bones. Their results Figure 3. Amplification of the 119-bp fragment of the 59UTR of the 15-kDa lipoprotein gene of T. pallidum, from DNA extracts obtained at the UA C¸. M, molecular weight marker, 100 bp DNA ladder (Sigma); 3, ELS 551; 10,14, ELS 558; 1,2,4,5,7,9,11,13 unaffected ancient samples; 6,8,12,15 extraction blanks (KE); 16, PCR blank. doi:10.1371/journal.pone.0036371.g003 PLoS ONE | www.plosone.org 3 May 2012 | Volume 7 | Issue 5 | e36371