Identification and Interpretation of Historical Cemeteries Linked to Epidemics

Medieval Black Death is believed to have killed up to one-third of the Western European population during the 14th century. It was identified as plague at this time, but recently the causative organism was debated because no definitive evidence has been obtained to confirm the role of Yersinia pestis as the agent of plague. We obtained the teeth of a child and two adults from a 14th century grave in France, disrupted them to obtain the pulp, and applied the new "suicide PCR" protocol in which the primers are used only once. There were no positive controls: Neither Yersinia nor Yersinia DNA were introduced in the laboratory. A negative result is followed by a new test using other primers; a positive result is followed by sequencing. The second and third primer pair used, coding for a part of the pla gene, generated amplicons whose sequence confirmed that it was Y. pestis in 1 tooth from the child and 19/19 teeth from the adults. Negative controls were negative. Attempts to detect the putative alternative etiologic agents Bacillus anthracis and Rickettsia prowazekii failed. Suicide PCR avoids any risk of contamination as it uses a single-shot primer-its specificity is absolute. We believe that we can end the controversy: Medieval Black Death was plague.

Yersinia pestis, a group A bioterrorism agent ( 1 ), causes plague, a reemerging zoonotic disease transmitted to humans through flea bites and typically characterized by the appearance of a tender and swollen lymph node, the bubo ( 2 ). This organism has been subdivided into three biovars on the basis of their abilities to ferment glycerol and to reduce nitrate. Based on their current geographic niche and on historical records that indicate the geographic origin of the pandemics, researchers have postulated that each biovar caused a specific pandemic ( 2 , 3 ). Biovar Antiqua, from East Africa, may have descended from bacteria that caused the first pandemic, whereas Medievalis, from central Asia, may have descended from the bacteria that caused the second pandemic. Bacteria linked to the third pandemic are all of the Orientalis biovar ( 3 ). In this study, we tested this hypothesis for the first time by detecting biovars in ancient human remains. No molecular biology–based method proved reliable and convenient for Y. pestis genotyping. Genome sequences of Y. pestis strain CO92, a Orientalis biovar, and Y. pestis strain KIM, a Medievalis biovar, are now available ( 4 , 5 ), which provides an opportunity to examine them for differences associated with the biovar and for genotyping. Genome analysis of the closely related Rickettsia prowazekii ( 6 ) and R. conorii ( 7 ) showed that intergenic spacers, which have been submitted to less evolutionary pressure than coding sequences, may be variable enough to differentiate closely related microorganisms. We, therefore, hypothesized that sequencing of several intergenic spacers would allow determination of a biovar-specific spacer pattern in Y. pestis. We named this method multiple spacer typing (MST). We first demonstrated that MST allowed biovar genotyping of a large collection of Y. pestis isolates and further applied it to the dental pulp collected from persons whose deaths are attributed to the first and second pandemics. Methods Bacterial Strains Thirty-five strains representative of the three Y. pestis biovars (11 Antiqua isolates, 12 Medievalis isolates, and 12 Orientalis isolates) isolated from 1947 to 1996 from various host species in 13 countries are presented in Table 1. Nineteen of these isolates have been previously characterized by Achtman et al. ( 8 ). Nucleic acid was extracted as previously described ( 9 ), and species identification was confirmed for all the strains by partial sequencing of the rpob gene ( 10 ). Table 1 Alleles of eight spacers in three Yersinia pestis biovars Biovar YP no. strains Country YP1 YP3 YP4 YP5 YP7 YP8 YP9 YP10 Isolate type Antiqua 611/Japan Japan 1 4 1 3 1 1 1 1 1 552/Margaret Kenya 1 3 1 1 4 1 1 1 2 548/343 Belgium 1 3 1 1 5 1 1 1 3 544 Congo 1 3 1 1 7 1 1 1 4 549 Kenya 1 3 1 1 8 1 1 1 5 542 Belgium 1 3 1 1 6 1 1 1 6 550 Congo 1 3 1 1 9 1 1 1 7 553 Kenya 1 3 1 1 7 1 1 1 4 566 Kenya 1 3 1 1 6 1 1 1 6 677 Kenya 1 3 1 1 9 1 1 1 7 545 Kenya 1 3 1 1 7 1 1 1 4 Medievalis 519/PKH-4 Kurdistan 2 2 2 2 1 1 1 1 10 616/PAR-13 Iran 2 2 2 2 1 1 1 1 8 557/PKR292 Kurdistan 2 2 2 2 4 1 1 1 9 564 Kurdistan 2 2 2 2 6 1 1 1 10 565 Turkey 2 2 2 2 5 1 1 1 8 557 Kurdistan 2 2 2 2 4 1 1 1 9 518 Kurdistan 2 2 2 2 5 1 1 1 8 520 Kurdistan 2 2 2 2 5 1 1 1 8 560 Kurdistan 2 2 2 2 5 1 1 1 8 561 Kurdistan 2 2 2 2 5 1 1 1 8 617 Iran 2 2 2 2 5 1 1 1 8 670 Kurdistan 2 2 2 2 5 1 1 1 8 1594 Kurdistan 2 2 2 2 9 1 1 1 11 Orientalis 304/6-69 Madagascar 1 5 1 1 1 2 2 1 12 685 Germany 1 5 1 1 2 2 2 1 13 Hamburg10 USA 1 5 1 1 2 2 2 2 14 CO92 USA 1 5 1 1 2 2 2 1 13 507 Vietnam 1 1 1 1 6 2 2 1 15 1513 Madagascar 1 5 1 1 2 2 2 1 16 571 Brazil 1 5 1 1 4 2 2 1 17 613 Myanmar 1 5 1 1 3 2 2 1 18 643 Madagascar 1 5 1 1 3 2 2 1 18 695 Germany 1 1 1 1 4 2 2 1 17 772 Vietnam 1 5 1 1 4 2 2 1 17 989 Vietnam 1 5 1 1 1 2 2 1 19 Spacer Sequence Database and Phylogenetic Analyses We analyzed the complete genome sequences of Y. pestis strain CO92, biovar Orientalis (GenBank accession no. NC-003143) ( 4 ) and Y. pestis strain KIM, biovar Medievalis (GenBank accession no. NC-004088) ( 5 ), which were obtained from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database ( 11 ). We used the Primer3 program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) to determine the primer sequences specific for the genomic segments of interest ( 12 ). The primers flanked intergenic sequences of Y. pestis CO92 that exhibited large sequence differences with the homologous Y. pestis KIM strain sequences. We generated a list of Y. pestis CO92 intergenic sequences of 50 to 300 bp and carried out BLASTN searches to identify the homologous intergenic sequences in Y. pestis KIM strain by using the Y. pestis CO92 genes flanking the intergenic sequences as queries ( 13 ). When both genes flanking the intergenic sequence exhibited best-matches with the BLAST score >120 bits, we estimated the length of the corresponding intergenic sequence in the Y. pestis KIM strain. We then aligned the homologous intergenic sequences ( 8 x 106 combinations (223 molecular events). The 387-bp spacer YP1 exhibited a 183-bp deletion specific for the Medievalis isolates. For spacer YP3, we observed five alleles: Orientalis isolates 1513 and 695 exhibited a complete, 340-bp sequence, and the other Orientalis isolates exhibited a 16-bp deletion; Medievalis isolates featured a 48-bp deletion and a mutation G → A at position 135 of the spacer; Antiqua isolates exhibited a 32-bp deletion except for isolate 611, which had a 16-bp deletion and mutations C164 → T, C166 → T and A191 → G. Spacer YP3 thus differentiated every biovar. As for spacer YP4, Medievalis isolates were characterized by a 36-bp deletion, whereas Antiqua and Orientalis isolates exhibited a complete spacer. For spacer YP5, Medievalis isolates exhibited a full-length 292-bp spacer, whereas Antiqua and Orientalis isolates exhibited a 32-bp deletion at position 101 of the spacer. For spacer YP7, a total of nine alleles were found; Antiqua isolates were classified within seven alleles, Medievalis isolates within four alleles, and Orientalis isolates within five alleles. Alleles two and three were found only among Orientalis isolates. A full-length sequence of 330 bp was found for strain 549; the other isolates exhibited deletions. At position 126 in the spacer, strains 643 and 695 exhibited a 56-bp deletion; at position 133, strains 304, 1092, 507 and 571 exhibited a 49-bp deletion; at position 140, strains 611, 685, and 537 exhibited a 42-bp deletion; at position 142, strains 616, 548, 565, 518, 520, 560, 561, and 670 exhibited a 35-bp deletion; at position 149, strains 519, 542, 566, 564, and 1513 exhibited a 28-bp deletion; at position 155, strains 552, 613, 772, and 989 exhibited a 21-bp deletion; at position 162, strains 550, 677, and 1594 exhibited a 14-bp deletion; and at position 169 in the spacer, strains 544, 553, and 545 exhibited a 7-bp deletion. As for spacer YP8, Antiqua and Medievalis isolates exhibited a full-length 236-bp spacer, whereas Orientalis isolates had an 18-bp deletion at position 36 of the spacer. As for spacer YP9, Antiqua and Medievalis isolates exhibited a full-length 292-bp sequence; Orientalis isolates had an 18-bp deletion located at position 123 of the spacer. As for YP10 spacer, Y. pestis CO92 strain had a unique sequence of 369-bp; all the other isolates exhibited an 18-bp deletion located at position 177 of the spacer. Sequences herein determined were deposited in GenBank (Table A1). Phylogenetic Analysis Among the 35 studied Y. pestis strains, we identified three main phylogenetic clusters, each of which included only strains from a single biovar, i.e., Orientalis, Medievalis, or Antiqua, as observed in the unrooted dendrogram (Figure 1). The same topology was obtained when data were analyzed by parsimony, neighbor-joining, and maximum likelihood analyses (Figure A3). Within the Medievalis cluster, strains 519 and 616, and strains 557 and 564 were grouped by pairs; no other subgroup was identified. Within the Orientalis cluster, three groups were identified; one group included strains 989, 613, 772, and 1513; a second group was made up of three pairs of strains, i.e., 304 and CO92, 507 and 571, and 685 and 1537; the third group diverged before the differentiation of the other two groups and contained strains 695 and 643. Within the Antiqua cluster, two groups were identified; one group included strains 553, 544, 545, 550, and 677, with the last two clustered; the second group comprised strains 552, 542, and 566; strains 548 and 549 did not group with either of these two groups but diverged before the differentiation of the other Antiqua strains. The Antiqua strain 611 exhibited a unique phylogenetic position. This strain differed from all other studied Y. pestis strains and appeared to have diverged before the separation of the three main clusters. Figure 1 Unrooted tree showing the phylogenetic relationships among the 35 studied Yersinia pestis isolates inferred from sequence analysis of the combination of the eight intergenic spacers using the unweighted pair group method with arithmetic mean method. O, Y. pestis Orientalis biovar; M, Y. pestis Medievalis biovar; A, Y. pestis Antiqua biovar. Numbers refer to the isolate number as in Table 1. MST of Ancient Dental Pulp Specimens In the 46 PCR experiments we performed on ancient tooth samples, we obtained 10 Y. pestis sequences (Figure 2); no sequences were found in the 51 PCR experiments with control teeth (p < 10–4). The teeth from 7 of the 8 persons' remains yielded 10 specific sequences, 3 persons were positive for two molecular targets, but none of the teeth of 17 persons used as negative controls yielded specific sequences (p < 10–4). YP1 PCR yielded an amplicon in one of six tested persons, YP8 PCR yielded an amplicon with identical sequence in six of six tested persons, and YP3 PCR yielded an amplicon in three of seven tested persons. When compared with GenBank database (Table A2), theYP1 sequence obtained in skeleton 5 yielded complete similarity with the homologous region in Y. pestis C092 strain over 390 positions and 99% sequence similarity with the homologous region in Y. pestis KIM strain over 174 positions; the YP8 sequence obtained in the six persons yielded 99% sequence similarity with homologous region in Y. pestis C092 strain over 178 positions and complete sequence identity with homologous region in Y. pestis KIM strain over 116 positions; the YP3 sequence obtained in skeletons 4 and 8 yielded complete sequence identity with that of homologous region in Y. pestis strain CO92 over 364 positions and 98% sequence similarity with homologous region in Y. pestis KIM strain over 206 positions; the YP3 sequence obtained in human remain 2 yielded a 98% similarity with homologous region in Y. pestis CO92 strain over 283 positions and a 95% similarity with homologous region in Y. pestis KIM strain over 162 positions. For each one of these 10 amplicons, further matches dropped to <90% sequence similarity over short sequences of 10 nt to 50 nt. When blasted to our local Y. pestis spacer sequence database, the YP1 spacer sequence obtained in human skeleton 5 was identical to that of the Orientalis and Antiqua reference sequences (Table A3), whereas smaller BLAST scores were obtained for the Medievalis reference sequences. Regarding the six YP8 spacer sequences obtained in skeletons 1–6, the 12 first best scores were obtained with Orientalis reference sequences. For the YP3 spacer sequence obtained in skeletons 4 and 8, the 10 first best scores were obtained with Orientalis reference sequences. For the YP3 spacer sequence obtained in skeleton 2, the 10 first best scores were obtained with Orientalis reference sequences, and this amplicon exhibited two specific nucleotide substitutions (Figure 2; Table A2). These mutations were consistently obtained in six clones. Figure 2 Molecular detection of Yersinia pestis was achieved in the dental pulp of remains of humans excavated from one Justinian and two Black Death mass graves in France by spacer amplification and sequencing (+, positive polymerase chain reaction [PCR] amplification and sequencing; –, absence of PCR amplification; ND, not done). Sequence analyses showed strains were of Orientalis genotype in all sets of remains; one of them exhibited two mutations numbered according to Y. pestis CO92 strain genome sequence (GenBank accession no. NC-003143). Negative control teeth remained negative. Discussion Our data show that MST differentiates the three biovars of Y. pestis in a collection of 35 isolates representative of the three biovars and originating from various sources and 13 countries. This finding suggests that MST data can be extrapolated to the entire Y. pestis species. Pulsed-field gel electrophoresis (PFGE) has been the only other technique that allows for biovar and strain differentiation, but it requires large amounts of cultured microorganisms, and the stability of PFGE profiles in subculture has been questioned ( 9 ). Ribotyping did not classify isolates into their respective biovars ( 9 , 21 , 22 ). Specific insertion sequences (IS), including IS100 ( 23 ), IS285 ( 24 , 25 ), and IS1541 ( 26 , 27 ), were used as markers in restriction fragment length polymorphism (RFLP) analyses ( 8 , 28 ) and in PCR-based technique ( 29 ). The last approach produced identical patterns of IS100 distribution in Antiqua and Medievalis isolates ( 29 ). A variable-number tandem repeat technique ( 30 , 31 ) had a greater discrimination capacity than did ribotyping, but isolates from different areas were found to harbor identical types. Sequencing of fragments of five housekeeping genes in 36 Y. pestis isolates from various locations and from 12 to 13 isolates from Y. pseudotuberculosis and Y. enterocolitica did not show diversity in any Y. pestis housekeeping gene ( 8 ). Indeed, 19 of these 36 Y. pestis isolates were also included in the present work and featured 12 different MST profiles, thus demonstrating the validity of our hypothesis and the usefulness of MST for Y. pestis genotyping. Morever, its format is applicable to microbial analyses of ancient samples since it requires small amounts of DNA and targets small genomic fragments. Few studies have aimed to disclose intraspecifc phylogenetic relationships of Y. pestis isolates. RFLP, probed with the IS100, indicated that, among 36 Y. pestis isolates, the three biovars formed distinct branches of the phylogenetic tree and that Y. pestis was a clone that evolved from Y. pseudotuberculosis 1,500–20,000 years ago ( 8 ). Isolates of biovars Antiqua and Medievalis clustered altogether apart from those belonging to biovar Orientalis. Likewise, a dendrogram constructed by the UPGMA clustering method on a PCR-based IS100 fingerprint database clearly discriminated Y. pestis isolates of the Orientalis biovar that formed a homogeneous group, whereas isolates of the Antiqua and Medievalis biovars mixed together ( 29 ). Isolates of biovar Antiqua showed a variety of fingerprinting profiles, whereas Medievalis isolates clustered with the Antiqua isolates originating from Southeast Asia, which suggests their close phylogenetic relationships. In this study, one isolate (strain Nicholisk 51) displayed a genotyping pattern typical of biovar Orientalis isolates, although this isolate was biovar Antiqua and lacked a 93-bp deletion within the glycerol-3-phosphate dehydrogenase glpD gene characteristic for the glycerol-negative Orientalis biovar. Our data support the view that most Y. pestis isolates cluster according to their biovar, but isolates of biovar Antiqua are more distantly related to other isolates than biovars Orientalis and Medievalis are. MST-based phylogenetic reconstructions unexpectedly found that one Y. pestis Antiqua isolate, number 611, formed a fourth branch, which suggests that Y. pestis may comprise four different lineages instead of the three that have been recognized so far. This unique isolate had not been included in Achtman and collaborators' study ( 8 ). MST was applied to ancient human specimens to test the hypothesis that three biovars were responsible for the three historical pandemics. Contamination of ancient samples by modern Y. pestis DNA and cross-contamination were prevented in our experiments. Indeed, we carried out two independent experiments, each with new reagents, in a laboratory where Y. pestis had never been introduced or studied, without positive controls ( 17 ). Both experiments produced consistent results, and negative controls were always negative. That we obtained a unique YP3 sequence further excludes the possibility of contamination, since this sequence differs from all the currently known sequences. This unique sequence was consistently found in six of six clones and thus did not result from the false incorporation of nucleotides by the DNA polymerase. In our previous work on the Black Death, we also reported a unique sequence ( 17 ). In the present study, the accurate identification of Y. pestis was confirmed by using two successive sequence analyses. We first blasted the sequences derived from ancient specimens against the GenBank database and observed best matches for Y. pestis homologous sequences, thus ensuring accurate species identification of the amplicons. We then blasted these sequences against our local Y. pestis spacer sequence database and found best matches for homologous sequence in Orientalis reference isolates for the three tested spacers. As our local database has 35 different reference sequences representative of the three Y. pestis biovars, there is no doubt regarding the identification nor the fact that Orientalis-like Y. pestis alone was implicated in the personal remains that we investigated. DNA of Y. pestis was recovered from remains of persons in one mass grave established to be of the Justinian pandemic era on the basis of radiocarbon dating (Figure A4). We found that the genotype Orientalis, which now occurs worldwide, was involved in all three pandemics. Also, we detected Y. pestis in additonal human remains from Black Death sites, which adds more evidence for its role in the second pandemic in southern France (four sites tested positive) ( 18 , 19 ). Indeed, historical descriptions were suggestive of bubonic plague in medieval southern Europe; in northern Europe, historical data indicated that the Black Death had a different epidemiologic pattern. This finding may indicate that latter outbreaks in the north were not caused by transmission of Y. pestis by blocked rat fleas but rather by mechanical transmission of plague bacteria by another ectoparasite that used humans as their primary hosts. Alternatively, another pathogen may have caused these outbreaks, and a search for Y. pestis in the dental pulps of suspected plague victims in Copenhagen (two persons' remains) and Verdun (five persons' remains) dating from the 18th century failed to show Y. pestis DNA ( 32 ). Further studies may elucidate the respective role of Y. pestis and other pathogens that may have contributed to deaths in these times.