The limits of biomolecular palaeopathology: ancient DNA cannot be used to study venereal syphilis
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.
Section snippets
Bones
Bones are listed in Table 1. All were taken from non-disarticulated skeletons. Those displaying treponemal pathology were obtained from six cemetery sites: Rivenhall, Kingston-upon-Hull, Magdalen Street, Farringdon Street, Newcastle Infirmary and Poultry One. Five tuberculous bones were also studied, from Farringdon Street, Poultry One and Royal Mint.
Rivenhall cemetery in Essex was in use during the 9th–19th centuries. The one recovered skeleton to show signs of treponemal disease (bone 204)
Presence of human mtDNA in the bones
There are approximately 8000 copies of the mitochondrial genome per human cell [8]. This is substantially higher than the anticipated relative copy number for treponemal genomes, so PCRs directed at a mitochondrial locus must be successful if there is to be any chance that treponemal aDNA will be detectable in a bone.
Each of the 46 bones listed in Table 1 was tested for the presence of human mtDNA by PCRs directed at the hypervariable segment 1 (HVS1). Evidence for the presence of mtDNA was
Conclusions
The aim of the work described in this paper was to determine if ancient DNA can be used to study the palaeopathology of venereal syphilis. We assembled a set of relatively recent bones, many with osteological indications of venereal syphilis, and tested these for the presence of human mtDNA, M. tuberculosis DNA and treponemal DNA. The results are summarised in Table 6. We carried out nine different treponemal PCRs on each of 46 bones but none of these gave an authentic positive result. There
Acknowledgements
We thank Sheila Lukehart for supplying the modern treponemal control DNA, and Andrew Chamberlain, Brian Connell, Simon Mays, Charlotte Roberts, Ann Stirland, Mike Taylor and Bill White for supplying bone samples. We also thank Mike Taylor for allowing us to report his unpublished results for bones from Kingston-upon-Hull, Rivenhall and Ipswich Blackfriars, and Keri Brown for helpful discussions during the course of this work. A.S.B. was supported by a Natural Environment Research Council
References (47)
- et al.
Mycobacterium tuberculosis complex DNA in ancient human bones
J. Archaeol. Sci.
(1996) - et al.
The potential significance of dietary offsets for the interpretation of radiocarbon dates: an archaeologically significant example from medieval Norwich
J. Archaeol. Sci.
(2004) - et al.
DNA from Mycobacterium tuberculosis complex identified in North American pre-Columbian human skeletal remains
J. Archaeol. Sci.
(1998) - et al.
DNA sequences of Mycobacterium leprae recovered from ancient bones
FEMS Microbiol. Lett.
(2003) - et al.
A mediaeval case of lepromatous leprosy from 13–14th century Orkney, Scotland
J. Archaeol. Sci.
(2000) - et al.
The origin and antiquity of syphilis
Curr. Anthropol.
(1998) - et al.
Comparison between silica-based methods for the extraction of DNA from human bones from 18th–mid-19th century London
Ancient Biomol.
(2002) - A. Boylston, M. Holst, C.A. Roberts, The human skeletal remains from Hull Magistrates Court, skeletal report submitted...
Directions for future research with ancient DNA in forensic archaeology
Ancient Biomol.
(2001)Genomes II
(2002)
The flanking region sequences of the 15-kDA lipoprotein gene differentiate pathogenic treponemes
J. Infect. Dis.
Treponema pallidum major sheath protein homologue TprK is a target of opsonic antibodies and the protective immune response
J. Exp. Med.
The tprK gene is heterogeneous among Treponema pallidum strains and has multiple alleles
Infect. Immun.
Assessment of Human Bone from Farringdon Street
Ancient DNA: do it right or not at all
Science
Mycobacterium tuberculosis complex DNA in calcified pleura from remains 1400 years old
Lett. Appl. Microbiol.
Detection of a 400-year old Yersinia pestis DNA in human dental pulp: an approach to the diagnosis of ancient septicemia
Proc. Natl. Acad. Sci. USA
Prevalence of human tuberculosis in a medieval population of Lithuania studied by ancient DNA analysis
Ancient Biomol.
Widespread occurrence of Mycobacterium tuberculosis DNA from 18th–19th century Hungarians
Am. J. Phys. Anthropol.
Absence of Yersinia pestis-specific DNA in human teeth from five European excavations of putative plague victims
Microbiology
Molecular evidence for different stages of tuberculosis in ancient bone samples from Hungary
Am. J. Phys. Anthropol.
Detection of leprosy in ancient human skeletal remains by molecular identification of Mycobacterium leprae
Am. J. Clin. Pathol.
On the origin of the human treponematoses
Bull. WHO
Cited by (82)
Examining pathogen DNA recovery across the remains of a 14th century Italian friar (Blessed Sante) infected with Brucella melitensis
2022, International Journal of PaleopathologyAncient Bacterial Genomes Reveal a High Diversity of Treponema pallidum Strains in Early Modern Europe
2020, Current BiologyCitation Excerpt :The treponemal spirochetes survive poorly outside their host organism and are present in extremely low quantities during late-stage infections, often evading detection even in living patients [48]. The final, tertiary-stage treponematoses produce the most frequently detected skeletal alterations, but in these cases, the disease has often already reached the latent stage, thwarting attempts to retrieve its DNA [49, 50]. Most notably, the bones most likely containing large amounts of treponemal agents belong to congenitally infected neonates.
Treponematosis in a pre-Columbian hunter-gatherer male from Antofagasta (1830 ± 20 BP, Northern Coast of Chile)
2020, International Journal of PaleopathologyGenetic affiliations within a 19th century burial ground at Darwen, Lancashire, UK
2019, Journal of Archaeological Science: ReportsAncient DNA in the study of ancient disease
2019, Ortner's Identification of Pathological Conditions in Human Skeletal Remains