Toward a Molecular History of Yersinia pestis (AHA)

This post a resource for the presentation I gave at the AHA meeting in New Orleans on January 5, 2013. A color handout of the slides can be downloaded here.

This map will be continually updated as new finds are published. Some of the balloons mark sites with multiple studies. Click on the balloons for citations.

View Larger Map

References:

Achtman, M. (2012). Insights from genomic comparisons of genetically monomorphic bacterial pathogens. Philosophical Transactions of the Royal Society B: Biological Sciences, 367(1590), 860–867. doi:10.1098/rstb.2011.0303

Bos, K. I., Schuenemann, V. J., Golding, G. B., Burbano, H. A., Waglechner, N., Coombes, B. K., et al. (2011). A draft genome of Yersinia pestis from victims of the Black Death. Nature, 1–5. doi:10.1038/nature10549

Bos, K. I., Stevens, P., Nieselt, K., Hendrik N Poinar, DeWitte, S. N., & Krause, J. (2012). Yersinia pestis: New Evidence for an Old Infection. PLoS ONE, 7(11), e49803.

Drancourt, M., & Raoult, D. (2005). Palaeomicrobiology: current issues and perspectives. Nature Reviews Microbiology, 3(1), 23–35. doi:10.1038/nrmicro1063

Drancourt, M., Houhamdi, L., & Raoult, D. (2006). Yersinia pestis as a telluric, human ectoparasite-borne organism. The Lancet Infectious Diseases, 6(4), 234–241. doi:10.1016/S1473-3099(06)70438-8

Haensch, S., Bianucci, R., Signoli, M., Rajerison, M., Schultz, M., Kacki, S., et al. (2010). Distinct Clones of Yersinia pestis Caused the Black Death. (N. J. Besansky, Ed.)PLoS Pathogens, 6(10), e1001134. doi:10.1371/journal.ppat.1001134.t001

Houhamdi, L., Lepidi, H., Drancourt, M., & Raoult, D. (2006). Experimental model to evaluate the human body louse as a vector of plague. The Journal of Infectious Diseases, 194(11), 1589–1596. doi:10.1086/508995

Little, L. K. (2011). Plague Historians in Lab Coats*. Past & Present, 213(1), 267–290. doi:10.1093/pastj/gtr014

Malou, N., Tran, T.-N.-N., Nappez, C., Signoli, M., Le Forestier, C., Castex, D., et al. (2012). Immuno-PCR – A New Tool for Paleomicrobiology: The Plague Paradigm. (S. Bereswill, Ed.)PLoS ONE, 7(2), e31744. doi:10.1371/journal.pone.0031744.g006

Morelli, G., Song, Y., Mazzoni, C. J., Eppinger, M., Roumagnac, P., Wagner, D. M., et al. (2010). Yersinia pestis genome sequencing identifies patterns of global phylogenetic diversity. Nature Genetics. doi:10.1038/ng.705

Nguyen-Hieu, T., Aboudharam, G., Signoli, M., Rigeade, C., Drancourt, M., & Raoult, D. (2010). Evidence of a Louse-Borne Outbreak Involving Typhus in Douai, 1710-1712 during the War of Spanish Succession. PLoS ONE, 5(10), e15405. doi:10.1371/journal.pone.0015405

Parkhill, J., Wren, B. W., Thomson, N. R., Titball, R. W., Holden, M. T., Prentice, M. B., et al. (2001). Genome sequence of Yersinia pestis, the causative agent of plague. Nature, 413(6855), 523–527. doi:10.1038/35097083

Pusch, C. M., Rahalison, L., Blin, N., Nicholson, G. J., & Czarnetzki, A. (2004). Yersinial F1 antigen and the cause of Black Death. The Lancet Infectious Diseases, 4(8), 484–485. doi:10.1016/S1473-3099(04)01099-0

Raoult, D., Dutour, O., Houhamdi, L., Jankauskas, R., Fournier, P.-E., Ardagna, Y., et al. (2006). Evidence for louse-transmitted diseases in soldiers of Napoleon’s Grand Army in Vilnius. The Journal of Infectious Diseases, 193(1), 112–120. doi:10.1086/498534

Schuenemann, V. J., Bos, K., Dewitte, S., Schmedes, S., Jamieson, J., Mittnik, A., et al. (2011). PNAS Plus: Targeted enrichment of ancient pathogens yielding the pPCP1 plasmid of Yersinia pestis from victims of the Black Death. Proceedings of the National Academy of Sciences, 1–22. doi:10.1073/pnas.1105107108

Tran, T., Forestier, C., & Drancourt, M. (n.d.). Brief communication: Co‐detection of Bartonella quintana and Yersinia pestis in an 11th–15th burial site in Bondy, France. American Journal of ….

Tran, T.-N.-N., Signoli, M., Fozzati, L., Aboudharam, G., Raoult, D., & Drancourt, M. (2011). High throughput, multiplexed pathogen detection authenticates plague waves in medieval venice, Italy. PLoS ONE, 6(3), e16735. doi:10.1371/journal.pone.0016735

Wiechmann, I., & Grupe, G. (2004). Detection ofYersinia pestis DNA in two early medieval skeletal finds from Aschheim (Upper Bavaria, 6th century A.D.). American Journal of Physical Anthropology, 126(1), 48–55. doi:10.1002/ajpa.10276

Wiechmann, I., Harbeck, M., & Grupe, G. (2010). Yersinia pestis DNA Sequences in Late Medieval Skeletal Finds, Bavaria. Emerging Infectious Diseases, 16(11), 1806–1807.

Remodeling the Plague Phylogenetic Tree

Understanding the molecular history of any organism requires fitting together ancient DNA with the phylogenetic tree constructed with living exemplars. Constructing a bacterial phylogenetic tree is a snapshot of a moving target because its impossible to sample all of the strains.  A recent study by the East Smithfield group ( Bos et al, 2012 [2]) seeks to fit the recent near complete genomic sequence of Yersinia pestis from the Black Death cemetery at East Smithfield into the current phylogenetic tree.

They pooled their SNP database with those used by Morelli et al [3] for a total of 311 strains, plus the parental species Yersinia pseudotuberculosis as its foundation.  The East Smithfield group expect that the SNP comparison “could provide a qualitative indication of phylogenetic signals that were lost via our original, more conservative analytical approach based strictly on complete genomes.” [2]

New phylogeny of Yersinia pestis (Bos et al, 2012)

Their analysis confirmed that the Black Death strain settles into the base of split between branch 1 & 2. This matches what Haensch et al [4] found in 14th century sites at Hereford and Saint-Laurent-de-Cabrerisse. This indicates that the split occurred after the Black Death, probably due to microevolution in geographically distinct regions. Branch 2 is localized primarily along the Silk Road route in Central Asia, while branch 1 is far more widely distributed  and produced the third pandemic strain [3].  Bos et al further identified two living strains, designated 3.ANT, with SNP profiles that match their East Smithfield Black Death SNP profile [2]. These strains have not been completely sequenced and the plasmid profiles of these strains and the Black Death strain have not been characterized, so we can not yet say that these strains are genetically identical in sequence or genomic architecture to the Black Death strain [2]. Note that genomic architecture (placement of genes in chromosome) will mostly likely effect gene expression and therefore function of the microbe.

The East Smithfield group  observed that a small group of three strains diverged from the main descent line immediately before the Black Death, designated here as 0.ANT3, were all isolated from China [2]. They suggest that these strains may have been produced during a diversifying event that produced the main Black Death strain, possibly in Asia before it reached Europe.

They also observed 11 strains of Yersinia pestis clumped at the 0.ANT1 branch point [2]. By their calculations this split would have occurred between the 8th and 10th century (732-980 AD) overlapping with the documented period of the Plague of Justinian. They suggest that these strains represent genetic radiation that occurred during the Justinian expansion. This is a change from their observations based solely on comparisons of complete genomes [1].

The East Smithfield genomic group still have not incorporated ancient DNA data from any other group in their analysis.

References:

[1] Bos KI, Schuenemann VS, Golding GB, Burbano HA, Waglechner N, et al. (2011) A draft genome of Yersinia pestis from victims of the Black Death. Nature 478: 506.510.

[2] Bos KI, Stevens P, Nieselt K, Poinar HN, DeWitte SN, et al. (2012) Yersinia pestis: New Evidence for an Old Infection. PLoS ONE 7(11): e49803. doi:10.1371/journal.pone.0049803

[3] Morelli G, Song Y, Mazzoni CJ, Eppinger M, Roumagnac P, et al. (2010) Yersinia pestis genome sequencing identifies patterns of global phylogenetic diversity. Nat Genet 42: 1140.1143.

[4] Haensch S, Bianucci R, Signoli M, Rajerison M, Schultz M, et al. (2010) Distinct Clones of Yersinia pestis Caused the Black Death. PLoS Pathog 6(10): e1001134. doi:10.1371/journal.ppat.1001134

AHA 2013: The Power of Cartography: Remapping the Black Death in the Age of Genomics and GIS

This coming year’s American Historical Association meeting will be held in New Orleans, Jan 2 – Jan 6, 2013. The schedule went online within about the last day. I’ll be at the AHA meeting for the first time this year at this session. The links below are to the session and individual speaker abstracts. I think it will be a great session and hopefully something more permanent will come of it.

The Power of Cartography: Remapping the Black Death in the Age of Genomics and GIS

AHA Session 143
Medieval Academy of America 4

Saturday, January 5, 2013: 9:00 AM-11:00 AM

Bayside Ballroom A (Sheraton New Orleans)

Chair: Nukhet Varlik, Rutgers University–Newark

Papers:

Remapping the Black Death
David Mengel, Xavier University

Toward a Molecular History of Yersinia pestis
Michelle Ziegler, Saint Louis University

In Search of the Black Death in Central Eurasia
Uli Schamiloglu, University of Wisconsin–Madison

Plague Patterns in Fifteenth Century Milan
Ann Carmichael, Indiana University Bloomington

The Public Practice of History in and for a Digital Age (session theme tag)

Plague at the Siege of Caffa, 1346

The first stage of the Black Death among Europeans was said to begin with the whoosh of a Mongol trebuchet. Gabriele De’ Mussi, a lawyer from near Genoa writing in about 1348, is believed to have recorded the account of the earliest use of plague as  weapon of war at Caffa in 1346.

Mongol siege with trebuchet. Edinburgh University Library via Wikipedia Commons.

“The dying Tartars, stunned and stupefied by the immensity of the disaster brought about by the disease, and realizing that they had no hope of escape, lost interest in the siege. But they ordered corpses to be placed in catapults and lobbed into the city in the hope that the intolerable stench would kill everyone inside. What seemed like mountains of dead were thrown into the city, and the Christians could not hide or flee or escape from them, although they dumped as many bodies as they could into the sea. As soon as the rotting corpses tainted the air and poisoned the water supply, and the stench was so overwhelming that hardly one in several thousand was in a position to flee the remains of the Tartar army. Moreover one infected man could carry the poison to others, and infect people and places with the disease by look alone. No one knew, or could discover, a means of defense.” (Horrox, p. 17).

As Mark Wheelis observes in his 2002 article, infected corpses flung into the fortress by trebuchet are a plausible means of infecting the inhabitants. It is not unlikely that corpses would be flung into fortresses, as a terror tactic, with or without disease. Imagine being besieged for months and then having rotting corpses flung at you. By choosing the strongest smelling corpses, it is likely that when they were hurled they came down hard and very sticky. Body fluids can certainly spread Yersinia pestis to people with abrasions and the mess these would have caused upon landing would draw rats from within the fortress. Rats will feed on dead corpses. Direct contact may account for more medieval cases of plague that it is generally credited; in the United States, direct contact is the likely form of transmission for 20% of cases between 1970 and 1995 (Wheelis, 2002).  Wheelis suggests that this had an additional bonus,  helping the Mongols (Tartars)  deal with the mortuary problem that plague always causes. While it is possible that rat transmission brought the plague into the fortress, there is no reason to doubt Gabriele De’ Mussi’s association between the flung corpses and the beginning of the outbreak.

Gabriele De’ Mussi is also revealing his own thoughts about contagion and transmission. Following the miasma theory of contagion, infection is passed through the contamination of the air and water/food. According to Gabriele De’ Mussi, “intolerable stench would kill everyone inside”; the contagion is in the foul-smelling air that can also permeate and therefore contaminate food or water. Although he recognized that it could be transmitted person to person, it was done “by look alone”. Without understanding germ theory, they could not understand what was actually being passed but did recognize respiratory and oral routes of transmission.

Using corpses to foul land or water is a method of biological warfare that is probably as old as warfare itself. If you read all of Gabriele De’ Mussi’s account, he is not really condemning the Tartars (Mongols) as doing anything out-of-bounds or evil. To Gabriele, God was striking down both the Tartars and the Christians. It is unclear if the Tartars were using the corpses as a tactic of biological warfare  or as an act of terrorism and desperation, a ‘share the pain’ or revenge tactic.   Gabriele De’ Mussi says “realizing that they had no hope of escape, lost interest in the siege” and then began to fling the corpses. At this point the Tartars had nothing more to lose. Unlike virtually every other biological warfare strategy, the Tartars did not have to be concerned with the greatest risk of biological warfare, blow-back, that is, the infection of your own army. Although many Italians fled the fortress, it remained under Italian control and the Tartars did abandon their siege (Wheelis, 2002).

Far from blaming the Tartars, Gabriele De’ Mussi places the responsibility for bringing the plague to the Mediterranean squarely on his own people, the Genoese and Venetians.

“among those who escaped from Caffa by boat were a few sailors who had been infected with the poisonous disease. Some boats were bound for Genoa, others for Venice, and to other Christian areas. When sailors reached these places and mixed with people there, it was as if they had brought evil spirits with them: every city, every settlement, and their inhabitants, both men and women, died suddenly. … We Genoese and Venetians bear responsibility for revealing the judgements of God. Alas, once our ships had brought us to port we went to our homes. And because we had long been delayed by tragic events, and because among us there were scarcely ten survivors from a thousand sailors, relations, kinsmen and neighbors flocked to us from all sides. But, to our anguish, we were carrying darts of death. While they hugged and kissed us we were spreading poison from our lips even as we spoke.” (Horrox, p. 18-19)

Wheelis argues that Gabriele De’ Mussi must be mistaken that a single or few ships fleeing from Caffa brought the plague to Europe.  He notes that the spread into the Mediterranean shown in the figure below took too long for a direct voyage from Caffa to Italy. Wheelis points out that this is probably the work of multiple streams of infected ships. He also suggests that caravan routes over land did spread plague to the south of Caffa, though as his maps shows, not nearly as effectively as by sea. Wheelis concludes that “the siege of Caffa, for all its dramatic appeal, probably had no more than anecdotal importance in the spread of the plague, a macabre incident in terrifying times.” Perhaps, but Gabriele De’ Mussi’s rhetoric may be read in other ways.

Transmission from Caffa. (Wheelis, 2002)

Gabriele De’ Mussi deploys a dizzying array of rhetorical devices to express the trauma and tragedy of the plague. He is inconsistent in his point of view, sometimes writing as through he were at Caffa or on the fleeing ship, other times writing from  a distance. As Wheelis notes, Gabriele De’ Mussi did not leave Italy during this years. The entire treatise is wailing to God over the tragedy, and at times talking directly or even conversing with God. When Gabriele De’ Mussi writes that “we Genoese and Venetians bear responsibility for revealing the judgements of God”, he is not referring to a few refugees. This seems to be an indictment of an entire people and could reflect transmission of the plague through the Italian trading network. It should also be considered that ships carrying refugees from the East had a powerful symbolic importance to Italians. Mythologically, the Romans claimed descent from Aeneas who led refugees from Troy to Italy where they won a home. Just as refugees from the East founded the Roman people, Italians fleeing from the East have now brought destruction to Italy and all of Europe. Gabriele De’ Mussi does not mention Aeneas, Troy or the founding of Rome but simplifying his story of plague transmission to a few refugees fleeing from the East makes his story more powerful.

Although refugees from Caffa may have only been one stream of disease transmission, ships from Caffa could have been important for two reasons. First, ships from Caffa could have been a primary disease stream even if they didn’t directly reach Genoa or Venice. Following what we now know about super-spreading phenomena a few ships stopping at multiple ports could radically effect transmission dynamics as plague entered Europe. Additionally, at least in Genoa and possibly Venice, the events at Caffa and its refugees formed a foundation narrative for a tragedy of biblical proportions. A few survivors from Caffa who eventually made their way home, possibly changing ships multiple times, may have arrived about the same time as the plague with horror stories of the plague in ships and ports along their route.

References:

Wheelis M (2002). Biological warfare at the 1346 siege of Caffa. Emerging infectious diseases, 8 (9), 971-5 PMID: 12194776

Horrox, R. ed & trans. (1994) The Black Death. Manchester Medieval Sources series. Manchester and New York: Manchester University Press.

sciseekclaimtoken-4ffce35ac9df7

A Flock of Plague Doctors

 

See the you-tube page for full credits.

Gothic Epidemiology? or Gothic Historiography?

I was reading David Mengel’s recent article on plague in Bohemia and he kept referring to this apparently well-known concept, gothic epidemiology. Being the early medieval geek that I am, my first thought was Ostrogoth or Visigoth, and what do they have to do with epidemiology, especially in Bohemia? Feeling that I was clearing missing out on an important concept in plague studies, I looked up the original paper by Faye Marie Getz in 1991.

It turns out that Getz was referring to the genre of Gothic literature that began in the 18th century when Gothic came to mean anything that “offended Enlightenment sensibilities”, anything anti-modern to the new men of the age of reason. ‘Gothic’ architecture gave way to neo-classical architecture, and Roman and Greek revival artistic motifs were everywhere. Yet, Gothic literature typified by Mary Shelly’s Frankenstein, and the works of Lord Byron and Edgar Allen Poe also developed during this time.  Getz characterized the essential elements of Gothic literary sensibility as

“an interest in distant and exotic places and times, especially in the Middle Ages and the Orient; the celebration of the power of nature and the ineffability of nature’s essence; the unity of disparate elements – of good and evil, the hideous and the beautiful, the dead and the living; the seduction of the primitive and wild in nature, of the bizarre; the insignificance of human beings against nature; the existence of geniuses; the importance of individual experience; and finally the emphasis on suffering, death, and redemption.” (p. 279)

Getz and others have found that from the mid-eighteenth century historians wrote of the plague as typifying and glorifying this Gothic sensibility. It was these early historians who made the plague a symbol of all that is dark, deadly and medieval.

The most influential gothic epidemiologist was Justin Hecker whose influential book Der schwarze Tod im vierzehten Jahrhunder  (The Black Death in the Fourteenth Century) in 1832 began modern plague studies. Getz characterizes Hecker’s book as seeing the plague as an inescapable force of nature that swept away the old, bringing historical change and most importantly progress.

“The miasma of plague was in Hecker both literally and figuratively atmospheric. It rolled like a fog out of the mysterious East, a crawling miasma exhaled by earthquakes and volcanoes, by the rotting dead in graveyards and battlefields, by decaying matter in marshes and swamps. Plague leveled all those who stood before it and spared no person. It seeped into churches, castles, and cottages. There was no escape. Nature herself spoke of the coming disaster. Comets, earthquakes, and volcanoes shattered man’s complacency. A pillar of fire appeared over the papal palace at Avignon. Plants and animals behaved in a bizarre manner. The very heavens rained disaster.” (Getz 1991: 276-277)

The plague itself was as heroic as disastrous.   Nature becomes God’s avenging army that sweeps away a whole civilization that has gone astray. Getz believes that Hecker set the foundation for most 19th and 20th century plague scholarship on four basic pillars: 1) the Black Death marked the end of an era and the start of a reinvigorated, more industrious society; 2) the plague was a natural phenomenon that was beyond human understanding,  awesome in its power, and unlike anything before or since;  3) the Black Death was a double-edged sword that was both terrible  in its destruction but also culturally transformative, begetting the Renaissance, and 4) a focus on the bizarre and morbid facts and behaviors. Getz notes that Hecker was the first person to focus on Flagellants or the massacre of Jews during the Black Death, two aspects of the pandemic that still draw an extraordinary amount of attention.  These four elements compromise what Getz calls Gothic epidemiology.

Many plague historians followed in Hecker’s footsteps (and occasionally still do). The morbid and bizarre feature more in plague history than in any other area of the history of medicine. The plague doctor has become an icon of  the Middle Ages.  Claims that the plague is the root cause behind vampires, werewolves and revenants/zombies are still made. The awesomeness of the plague as a natural phenomenon is still with us and can almost border on nature worship. Nearly 150 years after Hecker’s paradigm emerged, Robert Gottfried’s The Black Death: Natural and Human Disaster in Medieval Europe (1983) opens with a Chaucer  quotation, “Nature, the vicaire of the almyghty lorde” (The Parliament of Fowls, c. 1380). So two of Hecker’s four pillars are still with us in varying degrees. Given how popular Gothic culture still is, these elements are likely to remain.

The dilemma for plague historians and scientists comes in attracting interdisciplinary and public attention for their work without distorting the history and/or science. The easiest way to attract attention is by focusing on either the bizarre and morbid, or on nature’s power and plague’s uniqueness. This is the same compromise documentary films must make.  To avoid this fate some will be happy writing only for other academics of their ilk, but for all except the most specialized topics this is short-sighted. First, as one of the most interdisciplinary topics available today it is necessary to write so that other disciplines can understand and follow the argument, and to highlight aspects that are interesting to other disciplines. It not fair to complain about the misuse of our work if we don’t write at an interdisciplinary level. Given the wide range of disciplines involved in plague studies this is essentially a college-level general public audience. Second, the plague’s attraction for those who like elements of  Gothic culture means that there are educational opportunities for history and science. The CDC received a fair amount of attention for its Zombie apocalypse  preparedness educational program and the Zombie Research Society has a real epidemiologist on their advisory board. Of course, zombies being entirely fictional gives them a lot of latitude. However, if done carefully the plague can still be used to teach history and science without over focusing on only the most bizarre aspects or distorting its history.

The other two of Hecker’s pillars are the creation of academics rather than appealing to the public. Did the plague end an era and create a positive cultural transformation? Most plague historians can dispatch the idea that the Black Death was a great boundary in time after which everything changed. This is not to say that the plague didn’t have a cultural impact but it did not beget the Renaissance or the Reformation. It is just as likely that in periods of cultural transformation and upheaval, humans are more vulnerable to the plague and its transmission patterns. Meaning the plague gathered momentum because of cultural changes rather than being the cause of cultural change.

This also makes me think of the questions that surround the Plague of Justinian that began in 541. Did it end antiquity? Did it prevent the Western Roman Empire from recovering and reasserting itself? No. How odd that we place a plague pandemic at the beginning and end of the Medieval period.  Who determined when ‘classical antiquity‘ was as a period? Those same 17-18th century elites who developed Gothic literature. As modern historians show more continuity across time periods, there is a tendency to chip away at the length of the medieval period. The new designation of Late Antiquity is chipping the period of c. 500-750 away from early medieval, taking the Plague of Justianian with it. Fitting for a plague named for a Roman emperor?

In the Gothic epidemiology paradigm, the plague of Justianian must have been minimized to the point that it was forgotten for a long time. Afterall, it didn’t create a reinvigorated society; it marked the beginning of the medieval period, the ‘dark ages‘. The term ‘dark ages’ being contrasted with the supposed light of Rome. Perhaps we should refer to Gothic historiography rather than epidemiology since the paradigm clearly goes beyond just the Black Death.

References

Getz FM (1991). Black death and the silver lining: meaning, continuity, and revolutionary change in histories of medieval plague. Journal of the history of biology, 24 (2), 265-89 PMID: 11612554

Mengel DC (2011). A plague on Bohemia? Mapping the Black Death. Past & present, 211 (1), 3-34 PMID: 21961188

Editor’s Selection Icon

Plague Detection by Immuno-PCR

Once again the Marseille research group is pushing the bounds of plague detection. This time their target is looking for a more sensitive method of detecting non-nucleic acid biomolecules from Yersinia pestis, ‘the plague’. We have now moved into an era where PCR is being used in the mechanics of testing, rather than amplifying the ultimate target of the test.

Immuno-PCR

The immuno-PCR (iPCR) method is outlined in the figure. The selective component of the assay is the mouse polyclonal anti-Yersinia pestis antibody. Polyclonal antibodies are the products of several different B lymphocytes reacting to the same antigen, protein in this case. This means the antibodies in preparation will bind to different parts (epitopes) of the same protein. This should be an advantage in working with badly degraded material.

In iPCR the selective reagent is the non-human antibody generated against the microbial target. The second antibody is against the non-human antibody (mouse, rabbit, etc) and carries biotin as a marker. These biotinylated antibodies are a very common and widely available immunology reagent. The third antibody is against the biotin and carries a reporter sequence of DNA. Quantitative real-time PCR is then done on the reporter DNA sequence attached to the antibody. This amplified reporter DNA can easily measure very tiny amounts of DNA. The iPCR system utilizes advances in PCR technology to measure specific protein levels.

Malou et al (2012) took  known positive and negative teeth, and subjected them to iPCR, PCR and the standard ELISA antibody assay.   They first determined the detection limit for ELISA and iPCR, and set a threshold for a positive iPCR result with 10 known negative teeth (5 ancient and 5 modern). They then coded and randomized  34 historically known positive teeth, the 10 negative teeth (5 ancient and 5 modern) and two blanks for testing with ELISA, PCR, and iPCR. The results and how they compare are shown in the diagram below. The ELISA only picked up four teeth with one being a known negative resulting in a sensitivity of 9% and specificity of 90%. Of the three ELISA true positives, two were from a 17th century site at Lariey and one from a 6th century site at Sens, both in France. For the iPCR, 14 of 34 exceeded the threshold and were considered positive for a 41% sensitivity with a 100% specificity (all positives were historically positive, no false positives). These teeth came from Lariey, Bourges, Sens, Bondy, and Venice with a date range from the 6th to 17th century. Traditional PCR identified 10 out of 34 historically positive teeth fora  sensitivity of 29% with 8 of 10 also being identified by iPCR.

Venn diagram of positive teeth detected by ELISA, PCR, and iPCR.

Immuno-PCR compares well with the efficiency and specificity of standard PCR for Yersinia pestis DNA and is more sensitive than the standard ELISA antibody assay. A standard ELISA produced the worst results. Although not quantitative, it would have been interesting if they had also done the Rapid Diagnostic Dipstick Test (RDT), another antibody based protein detection method, that has been used in several studies.  They are suggesting that iPCR be added to traditional PCR as a method of further confirmation of Yersinia pestis at a site. Immuno-PCR can be done on the same teeth as traditional PCR and should be easily doable with the expertise and equipment in labs that conduct traditional aDNA PCR.  By identifying both aDNA and protein, the confirmation of Yersinia pestis in the ancient remains should become quite strong.

Reference:

Malou, N., Tran, T., Nappez, C., Signoli, M., Le Forestier, C., Castex, D., Drancourt, M., & Raoult, D. (2012). Immuno-PCR – A New Tool for Paleomicrobiology: The Plague Paradigm PLoS ONE, 7 (2) DOI: 10.1371/journal.pone.0031744