Looking for the syphilis spirochete in ancient bones

PCR is a powerful tool that has been used to detect microbial DNA in human remains unearthed by archaeologists.  This approach has helped reveal when and where infectious diseases such as tuberculosis and the plague have afflicted human populations in the past.  With the controversy raging over the question of whether the syphilis spirochete was present in Europe before Columbus sailed to America, one would think that scientists would have tried PCR to detect Treponema pallidum DNA in skeletal remains.  Well, they have, but in almost every case they failed to detect T. pallidum DNA, even in bones bearing the lesions of syphilis.  The problem is that adults who die in the later stages of syphilis do not have many T. pallidum spirochetes in their bones.

On the other hand, spirochetes are relatively abundant in the bones of infants afflicted with congenital syphilis.  Therefore the skeletal remains of the very young may be a better source for detection of T. pallidum DNA by PCR.  As reported in their recent PLoS One article, Montiel and colleagues looked for T. pallidum DNA in skeletal remains gathered from a 16th-17th century crypt in Spain.  The investigators found four infant bones with lesions that were consistent with congenital syphilis.  Since there were two left humeri (specimens ELS551 and ELS558 in the image below), the bones must have belonged to at least two newborns.

Figure 1 from Montiel et al., 2012.  Source.

The PCR reactions were conducted on specimens from both newborns in each of three different laboratories.  Two segments along the T. pallidum chromosome were targeted.  One lab targeted the arp gene, the second lab targeted the 5' UTR of the 15 kDa lipoprotein gene, and the third targeted both sequences.  All attempts but one led to the generation of PCR products.  To confirm that they derived from T. pallidum sequences, the PCR products were either analyzed by restriction digestion or cloned and sequenced.  The 5' UTR of the lipoprotein gene was critical to this effort because its sequence can be used to distinguish the syphilis spirochete from the other disease-causing treponemes.  The PCR products from both newborns turned out to have the Eco47III restriction site that is unique to the syphilis spirochete among modern treponemes.  The molecular analysis therefore supported the diagnosis of congenital syphilis in the two long-deceased infants.

The investigators took special precautions to minimize the risk of contamination, which is always a concern of paleomicrobiologists running PCR reactions.  For example, the experiments were done in laboratories in which Treponema-containing samples had never been handled.  The investigators even excluded positive controls from their PCR reactions.

Scientists are still not certain whether congenital syphilis can be correctly diagnosed by examining bone pathology alone (see pp. 102-103 of this paper for a nice discussion of this issue).  The PCR method will therefore aid scientists wishing to identify skeletal remains afflicted with congenital syphilis.  It also gives paleomicrobiologists hope that PCR methods will help answer the centuries-old question about the origin of syphilis.

References

Montiel R, Solórzano E, Díaz N, Álvarez-Sandoval BA, González-Ruiz M, Cañadas MP, Simões N, Isidro A, & Malgosa A (2012). Neonate human remains: a window of opportunity to the molecular study of ancient syphilis. PloS one, 7 (5) PMID: 22567153

Bouwman, AS, & Brown, TA (2005). The limits of biomolecular palaeopahology: ancient DNA cannot be used to study venereal syphilis Journal of Archaeological Science, 32, 703-713 DOI: 10.1016/j.jas.2004.11.014

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