 |
MB 451 Microbial Diversity |
Scientific paper summary
 Audio recording of this lecture |
Detection of 400-year old Yersinia pestis DNA in human dental pulp
Question : Was Yersinia pestis really the cause of the Black Death?
ABSTRACT Ancient septicemic plague epidemics were reported to have killed millions of people for 2 millenniums. However, confident diagnosis of ancient septicemia solely on the basis of historical clinical observations is not possible. The lack of suitable infected material has prevented direct demonstration of ancient septicemia; thus, the history of most infections such as plague remains hypothetical. The durability of dental pulp, together with its natural sterility, makes it a suitable material on which to base such research. We hypothesized that it would be a lasting refuge for Yersinia pestis, the plague agent. DNA extracts were made from the dental pulp of 12 unerupted teeth extracted from skeletons excavated from 16th and 18th century French graves of persons thought to have died of plague (‘‘plague teeth’’) and from 7 ancient negative control teeth. PCRs incorporating ancient DNA extracts and primers specific for the human -globin gene demonstrated the absence of inhibitors in these preparations. The incorporation of primers specific for Y. pestis rpoB (the RNA polymerase -subunit-encoding gene) and the recognized virulence-associated pla (the plasminogen activator- encoding gene) repeatedly yielded products that had a nucleotide sequence indistinguishable from that of modern day isolates of the bacterium. The specific pla sequence was obtained from 6 of 12 plague skeleton teeth but 0 of 7 negative controls (P < 0.034, Fisher exact test). A nucleic acid-based confirmation of ancient plague was achieved for historically identified victims, and we have confirmed the presence of the disease at the end of 16th century in France. Dental pulp is an attractive target in the quest to determine the etiology of septicemic illnesses detected in ancient corpses. Molecular techniques could be applied to this material to resolve historical outbreaks.
This paper is an example of "molecular microbial archaeology". The authors describe their attempt to demonstrate septicemic Yersinia pestis infections in the remains of children that died of Black Death some 400 years ago. It's amazing how contentious this issue is - was Black Death really bubonic plague, as most most historians believe, or was it a viral haemorrhagic fever like Ebola, anthrax, typhus, or something else? Or different things at different times or places?
The authors claim to show that the Black Death was really caused by Yersinia pestis, but as we'll see, this paper doesn't completely put this to rest. In any case, they show the utility of examining DNA extracted from the unerupted teeth of old skeletal remains for the detection of septicemic infections.
Unerupted teeth were thought to be the best source of DNA for several reasons: first and foremost, of course, is that this is probably the part of a skeletal remain containing organisms in an infected person that is most isolated from the decay process. The authors collected unerupted teeth from the skeletal remains of children from plague hospital mass grave sites, and from other skeletons of about the same age that did not apparently die of plague. The skeletons were available because of excavations required for urban development. The teeth were cleaned, opened, and DNA was extracted from the remains of their pulp. This DNA was used as template in a series of PCR reactions using primers specific for beta-globin (a positive control for human DNA), and the Y. pestis rpoB (RNA polymerase beta subunit) and pla (plasminogen activating protein, a virulence protein) genes:
13 teeth from 5 "plague" children were tested, and 7 teeeth from 7 control childern were tested. All of the control teeth were negative for pla PCR product, but positive for the control beta-hemoglobin, showing that they were able to extract DNA from the teeth and that there were no PCR inhibitors in the samples. pla PCR product was detected in at least 1 tooth from 4 of the 5 children were thought to have died of plague, although not all teeth of apparently infected children gave a positive result. Teeth from these same children were also positive for the rpoB PCR product, although in order to see the PCR product, they had to go through 2 sequential rounds of 40-cycle PCR. The sequence of the rpoB PCR products confirmed that they were from Y. pestis (although it would not be possible to distinguish different strains of this specie). Sequences of the pla PCR product showed that the children were infected with what is now known as the KIM5 strain of Y. pestis.
In the end, they believe that they've identified the cause of the plague to be Yersinia pestis. They later reported reproducing these results (and expanding on them), using an apparently contamination-proof method they call "suicide PCR". All this means is that they never PCR the same fragment twice (DNA generated in previous reactions being the prime source of potential contamination), and never amplify aanything within a prior PCR fragment. Every PCR is a new one, with at least one primer outside of the region PCRed in any prior experiment. And of course they never use positive controls, or work in a lab that has ever had Y. pestis or its DNA in it.
It turns out, however, that others were not convinced. In a 2004 paper, some folks have tried unsuccessfully to repeat these kinds of experiments from plague skeletal remains, using perhaps more stringent means to keep the dental pulp free of contamination (they imbedded them in plastic, clipped off the root, extracted the pulp out of the hole).
But the authors of the original paper dispute these findings:
Drancourt & Raoult 2004 Microbiol. 150:263 Molecular detection of Yersina pestis in dental pulp.
And so these authors believe they've shown that Y. pestis caused the Black Death, but others were not convinced. So who's right? Do you believe the unrepeatable (at least by others) result or the negative result? My inclination would be provisional acceptance the positive results, given the historical evidence in support of bubonic plague, the sequence variant they identified, the fact that their negative controls were clean, and the apparent lengths they went to to prevent contamination.
In the end, more specificity is needed; what kind of Y. pestis was it? Did the same strain cause all three major pandemics of the Black Death? How did this/these old strains differ from modern strains?
In 2004, Drancourt & Rault published another paper designed to identify which of the major types of Y. pestis (biovars Orientalis, Medievalis, and Antiqua) caused these three major pandemics, and how they compared genotypically to modern strains of this pathogen.
Hypothesis: The 3 plague pandemics were caused by Y. pestis serovars Antiqua, Medevalis, and Orientalis, respectively.
Three pandemics have been attributed to plague in the last 1,500 years. Yersinia pestis caused the third, and its DNA was found in human remains from the second. The Antiqua biovar of Y. pestis may have caused the first pandemic; the other two biovars, Medievalis and Orientalis, may have caused the second and third pandemics, respectively. To test this hypothesis, we designed an original genotyping system based on intergenic spacer sequencing called multiple spacer typing (MST). We found that MST differentiated every biovar in a collection of 36 Y. pestis isolates representative of the three biovars. When MST was applied to dental pulp collected from remains of eight persons who likely died in the first and second pandemics, this system identified original sequences that matched those of Y. pestis Orientalis. These data indicate that Y. pestis caused cases of Justinian plague. The two historical plague pandemics were likely caused by Orientalis-like strains.
With complete genome sequences from representatives of all three serovars of Y. pestis in hand, the authors design a series of PCR primer targeting varible intergenic regions they hope will distinguish in both size and sequence DNA from any Y. pestis strain. In the first part of the paper, the authors test 35 modern Y. pestis clinical isolates (about a dozen of each serovar) with these primers, and show that they can readily identify the correct serovar based on the sequences of these intergenic spacers.
Phylogenetic analysis of the spacer sequences generates trees that have three main branches, one of each of the serovars - and one leftover, a odd strain that seems to represent a forth type.
The real experiment, of course, was to test teeth from remains from each of the 2 ancient plagues (the last, "modern" plague is not in doubt, it was strain Orientalis). They were able to get PCR products from some primer sets and remains (all negative controls were clean):
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.
To the authors surprize, all PCR products, from both ancient pandemics, were from serovar Orientalis! One of their positives is a sequence variant not yet known in modern cultures, suggesting that it couldn't be contaimination. And so it seems that they've laid this issue to rest.