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Insufficient phylogenetic analysis may not exclude candidacy of typhoid fever as a probable cause of the Plague of Athens (reply to Shapiro et al.)

      The cause of the Plague of Athens (430–426 BC) has been debated among scientists, who have relied exclusively on Thucydides’ historical narrations

      Thucydides. History of the Peloponnesian War. Books I & II, §2.47–55. London, UK: Harvard University Press; 1919. p. 341–59.

      to introduce several possible diagnoses.
      • Durack D.T.
      • Littman R.J.
      • Benitez R.M.
      • Mackowiak P.A.
      Hellenic holocaust: a historical clinico-pathologic conference.
      • Cunha B.A.
      The cause of the plague of Athens: plague, typhoid, typhus, smallpox or measles?.
      The application of DNA analysis on skeletal remains taken from the Kerameikos mass grave
      • Baziotopoulou-Valavani E.A.
      Mass burial from the cemetery of Kerameikos.
      has been acknowledged as an ideal material that might provide clues for a definite evidence-based diagnosis of the epidemic.
      • Holden C.
      (Random samples) Athenian plague probe.
      Following a research methodology of proven accuracy and validity (‘suicide’ PCR),
      • Raoult D.
      • Aboudharam G.
      • Crubezy E.
      • Larroy Ludes B.
      • Drancourt M.
      Molecular identification by suicide PCR of Yersinia pestis as the agent of medieval black death.
      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.
      • 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.
      Despite this evidence-documented approach, Shapiro et al.

      Shapiro B, Rambault A, Gilbert TP. No proof that typhoid caused the Plague of Athens. Int J Infect Dis 2006; doi:10.1016/j.ijid.2006.02.006.

      have argued against the validity of these results. Through the application of a simple phylogenetic analysis of the published sequence of one gene (narG), Shapiro et al. concluded that, although the sequenced ancient DNA quite possibly corresponds to a Salmonella strain, this might not be typhoid. Instead, they argue that this sequence is more closely matched to other Salmonella species such as S. bongori, S. arizonae, and S. diarizonae rather than S. enterica Typhi. The authors base this assumption on an inferred evolutionary timescale of Salmonella and other related bacterial species. Shapiro et al. eventually make the assumption that the identified sequence most probably represents a modern and currently unknown free-living soil bacterium instead of an ancient one.
      Against these arguments we must once again state the extreme preventative measures that were taken in our study
      • 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.
      to exclude any possibility of environmental contamination. These included the absence of the pathogens themselves or a previously attempted extraction or PCR amplification of the target DNA sequences in the implemented laboratories, and also the ‘suicide’ PCR methodology that was followed, which excluded positive controls from this study. In addition, since possible environmental contamination is a major problem with ancient DNA studies as Shapiro et al. suggest, soil wash was actually used as a negative control in addition to DNA extracts from modern teeth. As is clearly stated in the original publication of our results, no product was yielded following the application of the same primers under the same laboratory conditions on the negative controls as well as on the soil sample washed off the ancient teeth,
      • 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.
      thus excluding the possibility of any contamination of the investigated ancient material. Besides, Salmonella species do not survive for long in soil, which is typically regarded as a transitional environment for this pathogen prior to its infecting a host.
      • Winfield M.D.
      • Groisman E.A.
      Role of nonhost environments in the lifestyles of Salmonella and Escherichia coli.
      Even if the soil of the mass grave was indeed contaminated by a modern Salmonella strain, such as a close relative of S. bongori, S. arizonae, and S. diarizonae (which are naturally found in reptiles), as suggested by Shapiro et al.,

      Shapiro B, Rambault A, Gilbert TP. No proof that typhoid caused the Plague of Athens. Int J Infect Dis 2006; doi:10.1016/j.ijid.2006.02.006.

      it would not be possible for any of these pathogens to survive during the long storage of the skeletal material and not be identified in the subsequently conducted investigation of the soil wash.
      On the other hand, the application of phylogenetic models, as suggested by Shapiro et al.,

      Shapiro B, Rambault A, Gilbert TP. No proof that typhoid caused the Plague of Athens. Int J Infect Dis 2006; doi:10.1016/j.ijid.2006.02.006.

      undoubtedly constitutes a powerful tool for the introduction of theoretical assumptions and hypotheses where clear-cut evidence is not available. Nevertheless, it has been supported that there is no ‘golden rule’ to fit all cases of phylogenetic analysis, whereas a minimum of 6–10 comparable gene sequences are needed to permit a complete phylogenetic study.
      • Cummings M.P.
      • Meyer A.
      Magic bullets and golden rules: data sampling in molecular phylogenetics.
      In contrast to these guidelines, the use of only one gene sequence for the application of phylogenetic analysis in bacteria, in which lateral gene transfer occurs widely between species,
      • Hoshino T.
      • Fujiwara T.
      • Kilian M.
      Use of phylogenetic and phenotypic analyses to identify nonhemolytic streptococci isolated from bacteremic patients.
      might yield misleading results. Such seems to be the case for Shapiro et al.'s approach

      Shapiro B, Rambault A, Gilbert TP. No proof that typhoid caused the Plague of Athens. Int J Infect Dis 2006; doi:10.1016/j.ijid.2006.02.006.

      that applied their proposed model on only one gene sequence. Besides, while there have been some reports of molecular data, which are useful for construction of accepted phylogenetic trees of Salmonella species,
      • Kanluk N.A.
      • Monteiro M.A.
      • Parker C.T.
      • Whitfield C.
      Molecular diversity of the genetic loci responsible for lipopolysaccharide core oligosaccharide assembly within the genus Salmonella.
      • Chan K.
      • Baker S.
      • Kim C.C.
      • Detweiler C.S.
      • Dougan G.
      • Falkow S.
      Genomic comparison of Salmonella enterica serovars and Salmonella bongori by use of an S. enterica serovar Typhimurium DNA microarray.
      other reports yield conflicting results. For example, the evolutionary relationships of Salmonella species based on synonymous site variation in the fimA, fimI, and fimZ genes, which belong in the same operon, result in diverse phylogenetic trees, possibly because of variable horizontal transmission of all or part of the same gene cluster.
      • Boyd E.F.
      • Hartl D.L.
      Analysis of the type 1 pilin gene cluster fim in Salmonella: its distinct evolutionary histories in the 5′ and 3′ regions.
      Another phylogenetic study of three genes placed most studied Salmonella species in monophyletic lineages, but one serotype was placed into two statistically well supported separate lineages which contained either bovine only or avian only isolates.
      • Sukhnanand S.
      • Alcaine S.
      • Warnick L.D.
      • Su W.L.
      • Hof J.
      • Craver M.P.J.
      • et al.
      DNA sequence-based subtyping and evolutionary analysis of selected Salmonella enterica serotypes.
      In brief we doubt Shapiro et al.'s assumptions since:
      • Environmental contamination of the investigated ancient material or its surrounding soil was specifically excluded by the precise methodology that was followed in our research study.
      • A phylogenetic analysis of only one gene might be insufficient and/or misleading.
      • The speculative results of the conducted phylogenetic analysis (“a modern and currently unknown free-living soil Salmonella species”) cannot be more valid than the known clear evidence – Salmonella species do not survive for long in soil; dental pulp DNA sequences for two other genes have been detected, which not only are closest to S. enterica serovar Typhi, but one of these genes is lacking in its closest known relative, 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.
      We agree with Shapiro et al.

      Shapiro B, Rambault A, Gilbert TP. No proof that typhoid caused the Plague of Athens. Int J Infect Dis 2006; doi:10.1016/j.ijid.2006.02.006.

      that there is not yet definite proof that the cause of the Plague of Athens was typhoid fever. The concurrent presence of a plurality of infectious diseases in besieged Athens of 430–426 BC was not excluded in the first place
      • 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.
      allowing for the variable clinical manifestation of the epidemic as reported by Thucydides.
      • Cunha B.A.
      The cause of the plague of Athens: plague, typhoid, typhus, smallpox or measles?.
      Conflict of interest: No conflict of interest to declare.

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      Linked Article

      • No proof that typhoid caused the Plague of Athens (a reply to Papagrigorakis et al.)
        International Journal of Infectious DiseasesVol. 10Issue 4
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          The etiological agent of the Plague of Athens (430–426 BC) has been a hotly debated topic.1 Recently Papagrigorakis et al.2 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.
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