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No proof that typhoid caused the Plague of Athens (a reply to Papagrigorakis et al.)

      The etiological agent of the Plague of Athens (430–426 BC) has been a hotly debated topic.
      • Cunha B.A.
      The cause of the plague of Athens: plague, typhoid, typhus, smallpox or measles?.
      Recently Papagrigorakis et al.
      • Papagrigorakis M.J.
      • Yapijakis C.
      • Synodinos P.N.
      • Baziotopoulou-Valavani E.
      DNA examination of ancient dental pulp incriminates typhoid fever as a probable cause of the Plague of Athens.
      claimed to have isolated typhoid fever DNA (caused by Salmonella enterica serovar Typhi) from three 2500-year-old teeth from putative plague victims. DNA amplifications from these teeth, which come from the ancient Kerameikos mass burial site in Athens, resulted in two fragments of DNA, both showing some similarity to previously published Salmonella species.
      The authors’ diagnosis is based on a similarity score, resulting from BLAST comparisons between their amplified fragments and published sequences. Specifically, the authors report a 7% divergence between their sequences and S. enterica serovar Typhi, and 8% divergence between their sequence and the next most closely-related Salmonella strain, S. typhimurium. Based on the closer match to S. enterica, the authors conclude that “[if] another, yet unknown pathogen… was the actual cause of the Plague of Athens, it would have to be closely related to S. enterica and definitely closer than S. typhimurium.”
      • Papagrigorakis M.J.
      • Yapijakis C.
      • Synodinos P.N.
      • Baziotopoulou-Valavani E.
      DNA examination of ancient dental pulp incriminates typhoid fever as a probable cause of the Plague of Athens.
      This statement, however, is simply not true. Although the Athens sequence is indeed slightly more similar to S. enterica, the two cited Salmonella species are actually much more closely related to each other, with less than 1% divergence for the sequenced gene. In fact, if a simple phylogenetic analysis is performed, the ancient sequence is shown to fall outside both S. enterica and S. typhimurium, as well as several other Salmonella species (Figure 1). While this analysis confirms that the Athens sequence is possibly Salmonella, it demonstrates clearly that it is not typhoid (97% bootstrap value; Figure 1). Based on the evolutionary timescale inferred for Salmonella and E. coli,
      • Ochman H.
      • Elwyn S.
      • Moran N.A.
      Calibrating bacterial evolution.
      the Athens sequence and typhoid would have shared a common ancestor in the order of millions of years ago.
      Figure thumbnail gr1
      Figure 1Neighbor-joining (NJ) tree constructed by PAUP* 4.0b10
      • Swofford D.L.
      PAUP* Phylogenetic methods using parsimony (and other methods).
      using the HKY-Γ model of substitution (ts/tv = 2.4, Γ shape = 0.29; estimated using maximum likelihood (ML)) for 360-bp of the nitrate reductase gene. Bootstrap values are given for pertinent branches (10 000 replicate NJ trees using ML distances as above). Bacterial sequences other than the Athens sequence were obtained from GenBank, Washington University, St. Louis, USA (http://genomeold.wustl.edu/projects/bacterial/) and the Sanger Institute (http://www.sanger.ac.uk/Projects/Salmonella/).
      While we cannot exclude the possibility that the Athens sequence is a previously unidentified infectious agent which caused the Plague of Athens, it is quite reasonable to assume that the sequence is actually that of a modern, free-living soil bacterium, a possibility that could have been explored by extracting DNA from surrounding soil samples as additional negative controls. What is certain is that the sequences obtained do not implicate typhoid as the cause of the plague.
      This study highlights one of the most significant and recurring problems in ancient DNA research: that of authentication of results. While many labs are now careful to use strict experimental controls (as outlined in Cooper and Poinar
      • Cooper A.
      • Poinar H.N.
      Ancient DNA: do it right or not at all.
      ), the recommendation of phylogenetic verification of results continues to be ignored, despite the relative simplicity and lack of expense of such tests, particularly in comparison to experimental procedures. It should be noted that many of the most embarrassing mistakes in the ancient DNA literature (including the incorrect report of dinosaur DNA in the early 1990s
      • Woodward S.R.
      • Weyland N.J.
      • Bunnell M.
      DNA sequence from Cretaceous Period bone fragments.
      ) could have been avoided with a simple phylogenetic test.
      Conflict of interest: No conflict of interest to declare.

      References

        • Cunha B.A.
        The cause of the plague of Athens: plague, typhoid, typhus, smallpox or measles?.
        Infect Dis Clin N Am. 2004; 18: 29-43
        • Papagrigorakis M.J.
        • Yapijakis C.
        • Synodinos P.N.
        • Baziotopoulou-Valavani E.
        DNA examination of ancient dental pulp incriminates typhoid fever as a probable cause of the Plague of Athens.
        Int J Infect Dis. 2006; 10: 206-214
        • Ochman H.
        • Elwyn S.
        • Moran N.A.
        Calibrating bacterial evolution.
        Proc Natl Acad Sci USA. 1999; 96: 12638-12643
        • Cooper A.
        • Poinar H.N.
        Ancient DNA: do it right or not at all.
        Science. 2000; 289: 1139
        • Woodward S.R.
        • Weyland N.J.
        • Bunnell M.
        DNA sequence from Cretaceous Period bone fragments.
        Science. 2004; 266: 1229-1232
        • Swofford D.L.
        PAUP* Phylogenetic methods using parsimony (and other methods).
        Sinauer, Sunderland, MA, USA1999

      Linked Article

      • Insufficient phylogenetic analysis may not exclude candidacy of typhoid fever as a probable cause of the Plague of Athens (reply to Shapiro et al.)
        International Journal of Infectious DiseasesVol. 10Issue 4
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          The cause of the Plague of Athens (430–426 BC) has been debated among scientists, who have relied exclusively on Thucydides’ historical narrations1 to introduce several possible diagnoses.2,3 The application of DNA analysis on skeletal remains taken from the Kerameikos mass grave4 has been acknowledged as an ideal material that might provide clues for a definite evidence-based diagnosis of the epidemic.5 Following a research methodology of proven accuracy and validity (‘suicide’ PCR),6 it was shown by analysis of three genes (osmC, clyA, narG) that an ancient strain of Salmonella enterica serovar Typhi was present in the investigated dental pulp material of three putative victims of the plague, thus incriminating typhoid fever as a probable cause.
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