also benefit from molecular techniques for identification of ancient strains specific to syphilis and then generate information useful in the phylogenetic reconstruction of modern strains. This could also help to shed light on the historical development of the disease, regarding continental origin, geographic distribution and epide- miology. Here, we present results from an analysis of newborn specimens sharing similar osteoporotic pathology. They were recovered from the crypt of ‘‘La Ermita de la Soledad’’, located in the province of Huelva in the southwest of Spain, the province from where the first Columbus expedition departed to America in 1492. This Hermitage was built between the XVI and XVII centuries [22]. Although the exact date of its construction is unknown, the human remains buried in the crypt cannot be dated before the discovery of the Americas. Nevertheless, this is the most ancient case, reported to date, from which detection of Treponemal DNA has been successful. We put forward a hypothesis on why we succeeded taking into account the course of the disease in neonate individuals. Results Individual’s Genetic Characterization Four newborn specimens with similar osteoporotic pathology were analyzed: one left femur (ELS 646), two left humeri (ELS 551 and ELS 558) and one right hemi-frontal (ELS 944) (Fig. 1). We obtained partial sequences of the mitochondrial Hypervariable Segment I (HVSI) for each of the four specimens analyzed (Table 1). HVSI is the most informative segment of the control region of mitochondrial DNA, and the most used in human population studies. For all but one of the specimens (ELS646) independent replication was accomplished in at least two laboratories. From the pattern of substitutions observed among the sequences, we can identify at least two different sequences. Specimens ELS 551, ELS646, and ELS 994 share the 16270T, 16311C distinctive motif, indicating they belong either to the mitochondrial haplogroup U5 or to haplogroup K2; while ELS 558 is differentiated by the absence of this motif having a Figure 1. The presence of two left humeri (ELS 551 and ELS 558) indicates that at least two individuals are represented in these remains. Molecular data confirmed the presence of at least two individuals, one that might be represented by ELS 551, ELS646, and ELS 944, and one represented by ELS558 (see results). doi:10.1371/journal.pone.0036371.g001 The Molecular Study of Ancient Syphilis Table 1. Partial mitochondrial DNA sequences (16210–16319) obtained from the analyzed specimens in the different laboratories involved in the study. 16270T, 16311C 16270T, 16311C LANGEBIO ND 16270T, 16311C Lab Sample UAB ELS551 ELS558 ELS646 ELS944 ND CRS 16270T, 16311C 16270T, 16311C UAC¸ CRS Only differences to the CRS are shown. ND, not determined because amplification was not possible in the independent extract obtained at the specified laboratory; NA, amplification was not attempted (sample was not sent) at the specified laboratory; CRS, identical to the Cambridge Reference Sequence. doi:10.1371/journal.pone.0036371.t001 haplotype identical to the Cambridge Reference Sequence (CRS) for this fragment. In summary, along with morphological information, HVSI sequences indicate these samples come from at least two different individuals, one that might be represented by ELS 551, ELS646, and ELS 944, and another represented by ELS 558. Treponema pallidum DNA amplification At the UAB laboratory, the four samples (ELS 551, ELS 558, ELS 646 and ELS 944) amplified for an arp gene fragment (see Material and Methods). For all samples, the 106 bp fragment was digested with NaeI, after which, the expected fragment of 84 bp was observed, while the 22 bp fragment was not, due to the presence of primer-dimer products (data submitted but not shown). As most of the amplification product was used in the digestion assay, the sequencing of this fragment was not attempted, considering also that the identity of the fragment was confirmed by the presence of the NaeI restriction site at the expected position. At the UAC¸ , DNA from specimens ELS 551 and ELS 558 was independently extracted and analyzed. A weak amplification of the arp gene fragment was obtained only for ELS 551 (Fig. 2). The band was cloned and 3 positive clones were sequenced in both directions. The sequence from the three inserts was identical among them, and to the sequence of T. pallidum spp. (Figure S1). The restriction site for NaeI was identified at the expected position. We have not a definite explanation regarding why ELS 558 was not amplified for this fragment at the UAC¸ , considering it worked at the UAB. There are many reasons that could explain this result, including differences in PCR equipments or primers qualities, and/or differences in the content of inhibitors in the extracts used, since the attempts were made from independent extracts at each laboratory. From the amplification of the 59UTR fragment of the 15-kDa lipoprotein gene, clear bands were obtained from both specimens ELS 551 and ELS 558, and cloned (Fig. 3). From ELS551 three clones were sequenced in both directions. From ELS558 three clones were also sequenced in both strands. The 119 bp sequences from all six clones were identical to the sequence of T. pallidum subsp. pallidum (Figure S2). Therefore, the Eco47III restriction site characteristic of this subspecies was present in all the sequences. At Langebio, amplification of the 59UTR fragment of the 15- kDa lipoprotein gene was obtained from the three analyzed specimens (ELS 551, ELS 558 and ELS 944). From ELS 551 we obtained sequences from 14 clones, from ELS 558 six clones were sequenced, and from ELS 944 only one clone was sequenced. All PLoS ONE | www.plosone.org 2 May 2012 | Volume 7 | Issue 5 | e36371