showed that although treponemal DNA could be isolated from the site of injection in the ‘‘chronic rabbit’’, it could only be isolated from the bone during the acute stage (not in advanced stages of the disease) [17]. It is worth noting that a number of studies have shown the presence of bone lesions in early syphilis [29–31], and this opens up the possibility that affected skeletons of young adults, who died during the early stages of syphilis, might also contain amplifiable T. pallidum DNA. However, the doubt remains on how to identify those cases before attempting destructive analysis. Our study demonstrates that neonates affected by CS are in fact a good source of Treponemal ancient DNA. Material and Methods Samples The remains of at least eighty individuals of both sexes were recovered during archaeological excavation works prior to restoring ‘‘La Ermita de la Soledad’’ crypt. Seventeen of these individuals were neonates ranging in age from birth to six months at the time of death [32]. Historical data indicates that the crypt was built between the XVI and XVII centuries [22]. The analysis of this material was originally requested by Diputacio´ n de Huelva (Autonomous Government of Huelva Province, Spain), therefore no specific permits were required for the studies described below. In this study, 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). Age at death was diagnosed from bone length [33–35] along with the thickness and dimensions of cranial vault bones [36,37]. The specimens were studied both morphologically and radiolog- ically. A high degree of osteoporoses, detachment of the outer layer of the cortical bone, osteolytic juxtametaphysial areas and distal epiphysis destruction were observed. Integrating this data, differential diagnosis was conducted taking into account the subject’s age at death and the probability of suffering the illness considering the site’s geographical location and chronology; taken together, these observations supported the hypothesis of an infectious etiology such as congenital treponematosis or haema- togenous osteomyelitis [38]. Initial molecular analysis was conducted at the Universitat Auto` noma de Barcelona (UAB). Subsequently, bone fragments from two left humeri (ELS 551 and ELS 558) were sent to the Universidade dos Ac¸ores (UAC¸ , Portugal), for independent replication. Samples from the same left humeri and a fragment from the right hemi-frontal (ELS944) were also sent to the Laboratorio Nacional de Geno´mica para la Biodiversidad (Langebio, CINVESTAV-IPN, Mexico). DNA Extraction and Purification In the three laboratories involved in the present study, DNA was extracted according to the methodology developed at the aDNA laboratory of the Biological Anthropology Unit of the UAB, with the conditions of sterility and the suitable precaution- ary measures previously described [39]. The extraction process started with the elimination of the outer bone surface, followed by bone powdering by using a micro motor with a hand piece and diamond drill for dentist use; washings with EDTA; incubation at 37uC with extraction buffer and Proteinase K; DNA extraction with phenol/chloroform; and centrifugation dialysis with sterile water, and concentration with a Centricon 30 filter MilliporeH. Finally, the extracts were stored at 4uC for four days to overcome PCR inhibitors [40,41]. In order to monitor contamination, The Molecular Study of Ancient Syphilis extraction blanks (KE), containing only reagents, were subjected to analysis. PCR negative controls (K2) were also included. At the UAB and Langebio, sample preparation, DNA extraction and PCR set-up were performed in laboratories dedicated specifically to ancient DNA, positively pressurized and physically isolated from the PCR thermal cycler and the laboratories for post-PCR or any other molecular biology research; additionally, full-body suits, breathing masks and protective lenses were used. At the UAC¸ , DNA extraction was conducted in a biological security hood, where no previous DNA extraction of any organisms had been performed before. PCR reactions were prepared in a different cabinet (horizontal filtered laminar flux), located in a different laboratory and dedicated solely to this purpose. In all three laboratories, no work with modern Treponema spp. had been previously conducted. Individual’s Genetic Characterization According to anatomical information, the four bone fragments analyzed belonged to at least two individuals (ELS 551 and ELS 558 are two left humeri). To clarify if the other remains represented different individuals, the hypervariable segment I (HVSI) of the mitochondrial control region was amplified. At the UAB the primers L15997-H16401, or two sets of overlapping primers (L15997-H16254 and L16209-H16401) were used. Amplified fragments were purified and directly sequenced in both strands. At the UAC¸ and at Langebio, primers L16190-H16209 and L16320-H16339 were used. Amplified fragments were cloned and sequenced in both strands. Only the region comprising 16210–16319 was obtained in at least two independent laborato- ries and this is the region presented here. Numbers refers to nucleotide positions in the Cambridge Reference Sequence (CRS) [42]. Treponema pallidum DNA amplification At the UAB, T. pallidum DNA was amplified with specific primers designed for this study (F284 59-GTGATCCTCTGT- CATCCCCG-39 and R370 59-TCCACCTCACGAGA- CAAAGG-39), that amplify a fragment of 106 bp, between positions 284 and 370, of the acidic repeat protein gene (arp) [43]. A Blast search [44] performed at the NCBI GenBank showed that this fragment presents 100% of similarity only with T. pallidum subspecies, with an E-value of 2610247 for all of the subspecies of T. pallidum that were matched. The primers were tested independently at General lab facilities (Barcelona) by using DNA from T. pallidum subsp. pallidum Nichols strain as a positive control. Although both Blast and experimental results gave us confidence on the specificity of this fragment to T. pallidum subspecies, it is not useful to discriminate among subspecies, and therefore a new set of primers was also used in subsequent experiments (see below). Amplification of the arp fragment was performed in a 50 ml volume. Thermal cycler conditions consisted of an initial denaturation at 94uC for 5 min, followed by 39 cycles at 94uC for 50 seconds, 55uC for 1 min and 72uC for 1 min and final extension at 72uC for 5 min. Blank controls (K2) were included in all PCR assays. The target fragment presents a NaeI restriction site that produces two fragments of 84 and 22 bp after digestion. Digestions with NaeI were performed according to the supplier instructions (New England Biolabs). At the UAC¸ , besides the fragment of the arp gene, amplification of a 119-bp fragment of the 59 untranslated region (UTR) of the 15-kDa lipoprotein gene that contains an Eco47III restriction site specific to T. pallidum subsp. pallidum [20] was also attempted; using the primers and amplification conditions described by Kolman et PLoS ONE | www.plosone.org 4 May 2012 | Volume 7 | Issue 5 | e36371