The limits of biomolecular palaeopathology: ancient DNA cannot be used to study venereal syphilis

Abstract

To determine whether ancient DNA (aDNA) can be used to study the palaeopathology of venereal syphilis, we carried out a comprehensive analysis of the preservation of human and pathogen DNA in a set of 46 bones of various ages, most of which displayed osteological indications of the disease. Bones came from seven English cemetery sites that were in use during the 9th–19th centuries. Twelve of the 46 bones consistently yielded mitochondrial DNA (mtDNA) sequences after replicate polymerase chain reactions (PCRs), and a further 13 bones yielded mtDNA sequences with less reproducibility. The sequence data enabled tentative mitochondrial haplogroups to be assigned to nine of the bones, and the identities and frequencies of these haplogroups were compatible with the geographical origins of the bones. Twenty-one bones consistently gave negative results with all mtDNA PCRs, indicating that at least these bones were not contaminated with modern human DNA, and those bones that gave positive results only yielded one sequence each, again suggesting that widespread modern contamination had not occurred. Mycobacterium tuberculosis sequences were obtained from seven bones, including three of five bones with tuberculous lesions. The cloned and direct sequences obtained from both the mtDNA and M. tuberculosis PCR products showed features typical of degraded aDNA. All of these results suggest that at least some of the 46 bones that we studied were suitable for aDNA analysis. All 46 bones were tested with nine different treponemal PCRs, each optimised to give a detection limit of ≤5 genomes. Although various bones gave PCR products of the expected size with one or more of these PCRs, sequencing showed that none of these products were authentic treponemal amplicons. Our failure to detect treponemal DNA in bones that were suitable for aDNA analysis, using highly sensitive PCRs, suggests that treponemal DNA is not preserved in human bone and that it is therefore not possible to use aDNA analysis to study venereal syphilis. Any past or future paper claiming detection of treponemal aDNA should therefore be accompanied by a detailed justification of the results.

Introduction

Ancient DNA is looked on as having enormous potential in the study of palaeodisease [7]. Theoretically, any pathogen that invades the blood system and/or hard tissues could leave an ancient DNA signature in the skeletal remains after the death of the host. Detection of this ancient DNA could be used to identify the pathogen and hence assign a possible cause of death, and analysis of variable regions of the pathogen genome could, potentially, be used to study the evolution of disease-causing organisms.

To date, most of the attention in biomolecular palaeopathology has been focused on tuberculosis, with a growing body of literature describing use of the polymerase chain reaction (PCR) to amplify Mycobacterium tuberculosis complex DNA from human bones of various ages [2], [6], [14], [16], [17], [19], [25], [37], [38], [42], [44], but there have also been reports of detection of ancient DNA (aDNA) for leprosy [20], [28], [30], [45], malaria [36], [43], plague [15], [31] and syphilis [22]. Because the aDNA being sought is pathogen rather than human, studies of palaeodisease are less susceptible to artefactual results due to contamination with modern DNA during handling of bones. However, such contamination is still possible, and the veracity of some published accounts of ancient pathogen DNA is difficult to assess as the comprehensive procedures required to authenticate the ancient origin of a DNA sequence [13] are not always described.

One of the most interesting palaeopathological questions that could be answered through use of aDNA is the origin of venereal syphilis in Europe. The first historical records for this disease are thought to relate to the epidemic of 1496, which affected large parts of Europe. As no equivalent disease of such virulence can be recognised earlier than 1496 it has been suggested that this epidemic represents the emergence of a new pathogen, Treponema pallidum subsp. pallidum, not previously present in Europe. The close proximity between the date of the epidemic and the return of the Columbus expedition in 1493 led to the popular view that venereal syphilis was introduced from the Americas by infected crewmen [1], but there is little solid support for this theory in the archaeological and historical records, and it is contradicted by those Old World skeletons, dated to pre-1493, that appear to show osteological signs of venereal syphilis [26], [33]. Alternative theories are, therefore, that venereal syphilis was present in Europe before 1493 and that the epidemic occurred due to low population immunity or to a mutation that increased the virulence of T. pallidum subsp. pallidum [21], [24], or that another treponemal disease which was present in Europe adapted to different climatic conditions and/or cultural practices to become venereal syphilis around the end of the 15th century [23]. These questions are potentially testable by aDNA analysis. The underlying hypothesis of the Columbian Theory — that T. pallidum subsp. pallidum was absent in the Old World prior to 1493 — can be tested by examining the pre-Columbian syphilitic skeletons for aDNA of this organism. If T. pallidum subsp. pallidum is present in the Old World pre-1493 then analysis of the aDNA sequences of those genes thought to underlie the virulence of the organism might reveal genetic changes responsible for the increase in venereal syphilis in the 15th and 16th centuries.

For aDNA analysis of palaeodisease to be successful, pathogens must be incorporated into bone material, either through remodelling during the latter stages of the disease (as probably occurs with tuberculosis and leprosy) or by transfer of a blood-borne pathogen to the bone matrix after death (a possible explanation of the reports of Plasmodium DNA in some skeletal remains). In the tertiary stages of treponemal diseases, when bone remodelling occurs, the number of organisms within the host is reduced and there is hence little opportunity for pathogen DNA to enter the bone [32]. Pathogen load is higher during the secondary stages of venereal syphilis but during this period there is very little remodelling and the only opportunity for treponemal DNA to enter bones would be via normal turnover. To determine whether these considerations place limitations on biomolecular studies of venereal syphilis, we carried out a comprehensive analysis of the preservation of human and pathogen DNA in 46 bones of various ages, most of which displayed osteological indications of venereal syphilis.