• Ancient DNA;
  • Mycobacterium leprae;
  • Twelfth century;
  • Paleobacteriology

The use of DNA techniques to detect pathogenic agents in ancient remains has exponentially increased recently. Reports include detection of Mycobacterium tuberculosis, Treponema pallidum, Yersinia pestis, Bacillus anthracis, and Mycobacterium leprae[1], among others. However, many of these studies have been criticised and doubts have been cast on the authenticity of their results [2,3]. One of the main problems indicated is the use of positive controls to test the primers and conditions used for polymerase chain reaction (PCR). This practice could produce millions of potentially contaminant fragments of target DNA. We have amplified, cloned and sequenced DNA from M. leprae by following strict procedures to avoid contamination [4,5] and without the use of positive controls. In this study we present a sequence from M leprae retrieved from mediaeval bones.

The samples analysed are from the necropolis of the Almohade fortress site of the ‘Capilla y Castillo de San Jorge’ (12th century AD, Seville, Spain).

We studied four adult individuals (A-326, A-120, A-43, A-343), each characterised by bone destruction, especially of foot bones. The lesions found were diagnosed by osteological criteria as leprosy [6]. These are the oldest Spanish cases of Hansen foot.

DNA was extracted from the metacarpal bones of each individual [4,5,7]. Primers were designed to amplify a portion of the central domain pertaining to a family of dispersed repetitive sequences (RLEP), specific to M. leprae, which contain the +2678 ClaI restriction site [8]. The use of this repetitive element increases chances of preservation, and therefore of detection. To further increase the sensitivity, the primers were designed to perform nested PCR, amplifying short fragments: 149 bp [F-2575 (5′-CCATTTCTGCCGCTGGTATCG-3′) and R-2723 (5′GCGCTAGAAGGTTGCCGTATG-3′)] and 97 bp [F-2621 (5′-TCAGCCAGCAAGCAGGCATG-3′) and R-2717 (5′-GAAGGTTGCCGTATGTGCC-3′)].

Blank controls were used that underwent all the extraction, purification, amplification, and nested re-amplification procedures.

Only one extract, from A-120, provided positive amplification for the 149-bp target; however, positive results were obtained from A-43 and A-120 for the 97-bp target through nested PCR.

The PCR products were subjected to restriction analysis with ClaI. All of these showed the expected pattern: two fragments of 105 and 44 bp for the 149-bp fragment and two fragments of 59 and 38 bp for the 97-bp fragment.

In addition, the 97-bp fragment from individual A-120 was cloned and sequenced. The sequence was identical to the central domain (from position 2621 to 2717) of the RLEP family [9]. Furthermore, a homology search in the NCBI gene bank produced no significant alignment with any other organism except for M. leprae.

As we did not use positive controls, and as Mycobacterial DNA had never been used in our laboratory, we can conclude that the only possible sources of the amplified specific fragments of M. leprae are the samples themselves. Some isolated cases have been processed [1,10], but in this paper, we present the first analysis from different individuals from a possible leprosy community.

These results show that it is possible to retrieve authentic DNA sequence information from ancient pathogens, supporting the idea that a new discipline could be established, namely palaeobacteriology [11].

In particular, the study of the RLEP in ancient M. leprae specimens could shed light on the origin of this family of dispersed repeats, which has few features in common with classical bacterial insertion sequences [8].


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  2. References
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