Molecular Confirmation of Yersinia pestis in 6th century Bavaria

Erasing any lingering doubts about the agent of the Plague of Justinian, a group of German biological anthropologists have shown conclusively that Yersinia pestis caused an epidemic in a 6th century Bavarian cemetery at Aschheim. Harbeck et al (2013) provide a convincing refutation of previous theories about the etiologic agent of the Plague of Justinian.   Returning to the same cemetery where plague was previously reported, two independent labs using the most modern standards to prevent contamination confirmed Yersinia pestis from multiple burials within the cemetery making this the best characterized Early Medieval plague cemetery.

The cemetery, called Aschheim, is in Bavaria outside of Munich. It contains the remains of 438 people with an unusually high number of multiple graves but no disordered mass graves. The 19 multiple burials contained two to five individuals arranged in lines. The cemetery was dated archaeologically to 500-700 AD with remains being carbon dated ranging from 530 to 680, all consistent with the 541 pandemic and its aftermath. Harbeck et al (2013) tested 19 individuals from 12 multiple graves. From these, there were eight positive samples, but only one produced enough aDNA to do some SNP genotyping. Added to the previous paper, this makes 11 positive individuals from this cemetery. Given the tenuous survival of aDNA, 11 positive individuals out of 21 tested in the two combined papers is a very good success rate. This is a cemetery that the F1 antigen test would be interesting since it could be used on the entire cemetery without great cost or labor. More sensitive than aDNA, the antigen test could tell us the percentage of plague deaths in the cemetery.

Individual A120 was screened with several SNPs that mapped it to an early region of the phylogenetic tree in the 0.ANT section. This makes the Plague of Justinian isolate ancestral to the Black Death isolates (yellow boxes below) from East Smithfield. This section whose only point of diversity is 0.ANT1 at node 4. Date predictions for the nodes of diversity in the tree fits with the Plague of Justinian falling in this region.  Modern isolates that  form this region of the phylogenetic tree all come from central Asia (around Tibet), suggesting that like the Black Death, the Plague of Justinian also originated in Asia. Overall, everything fits in well with expectations for the first pandemic.

(Harbeck et al, 2013. Fig. 1)

Reference:

Harbeck M, Seifert L, Hänsch S, Wagner DM, Birdsell D, et al. (2013) Yersinia pestis DNA from Skeletal Remains from the 6th Century AD Reveals Insights into Justinianic Plague. PLoS Pathog 9(5): e1003349. doi:10.1371/journal.ppat.1003349

Wiechmann I, & Grupe G (2005). Detection of Yersinia 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 PMID: 15386257

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

Siberian Mummy Yields 300-year-old Smallpox DNA

Five mummies in one grave. Benigini et al. NEJM, 2012. DOI: 10.1056/NEJMc1208124

It was the mass grave that got their attention. Four bodies crammed into one casket, with one child outside but with the casket. Multiple graves are not common in Yakutia, Siberia. Examination of the late 17th to early 18th century mummies indicates that burial came quickly after death. The casket contains one adult male over age 30, an adult female, an adult female under age 23,  a male child about 5 years old, and outside the casket a child about 4 years old.

The French and Russian team led by Philippe Biagini undertook pathological and genetic analysis of all five mummies. They were able to confirm that the older woman is the mother of  the young adult woman and the adult male. They took lung and tooth specimens from each mummy at the site (in situ). Finding iron inclusions in the lungs of the young female (mummy 2), suggested to the team that she suffered a pulmonary hemorrhage shortly before death. They don’t say how they jumped from there to screening for smallpox or what other pathogens were considered.  Oddly, they make no mention of any smallpox lesions on the mummy. (Without other bioarchaeological data, is it possible that this team only received the tooth and lung specimens, but not the remainder of the mummy?)

The DNA was divided among three labs. Three short sections of Variola  (smallpox) genome were amplified by at least two labs each. They failed to amplify long stretches of the virus, suggesting that there are no intact virons left in the mummy. Their phylogenetic analysis grouped this virus, PoxSib, with Variola but distinct from both clade 1 and clade 2. They suggest that PoxSib could be an ancestral strain to both clade 1 and clade 2 or a strain that has not been previously sampled. Biagini et al. suggest this virus may have come to Siberia with the Russian conquest in early 18th century, possibly connected with a documented outbreak in 1714. This grave comes from the same culture as previously analyzed graves that isolated the first ancient whooping cough.

Reference:

Biagini, P., Thèves, C., Balaresque, P., Géraut, A., Cannet, C., Keyser, C., Nikolaeva, D., Gérard, P., Duchesne, S., Orlando, L., Willerslev, E., Alekseev, A., de Micco, P., Ludes, B., & Crubézy, E. (2012). Variola Virus in a 300-Year-Old Siberian Mummy New England Journal of Medicine, 367 (21), 2057-2059 DOI: 10.1056/NEJMc1208124 See supplemental appendix for most of the detail.

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)

Insights from Plague Genomics, Part 1: The Chromosome

Most of the news lately has been about the plague phylogenetic tree produced by looking at single nucleotide polymorphisms (SNPs). The plague tree is remarkably simple and can lead to the mistaken impression that the rest of plague genomics are/will be simple. Michel Drancourt has recently compiled an array of genomic information that shows that SNPs are only part of the story.

A more broad view of plague genomics illustrates why the four biovars will continue to be used in scientific and clinical work. The four biovars are easily distinguished by their phenotype (traits that you can see or measure), the most common and easiest way for plague to be typed in clinical settings. It is important to note that the biovars/phenotypes tell clinicians most of what they need to know to treat the patient(s), their only real goal. Naturally, the biovars reflect genomic clusters beyond the metabolic skills measured in the standard phenotype.

Table and figure from Drancourt, 2012.

Chromosomal rearrangements have been the primary evolutionary mechanism of Yersinia pestis. The figure above shows clones representing the four biovars illustrating rearrangements (follow the lines) and inversions (shown underneath the center line representing each clone’s chromosome). These rearrangements are important for two reasons. First, this is a primary mechanism for DNA loss. Recombination errors can cause  sections of the chromosome to be lost. If the section does not contain vital genes, it will make the clone a leaner specialist. This makes sense of the 13% of parental Yersinia pseudotuberculosis genome lost by Y. pestis, while only gaining two coding sequences among eight new loci. Therefore, other Y. pestis specific genes are all contained on plasmids or other mobile elements.   Second, gene rearrangements can change control of gene expression. Although bacteria do not control their genes individually like eukaryotes, they are controlled in sets called operons. Presumably, genomic rearrangements, that do not respect gene or operon  structure, could change the gene compliment of an operon or destroy the operon control regions deregulating its genes. It can also destroy gene function resulting in pseudogenes (relics or wrecks of former genes). Natural selection will eliminate any damaging rearrangements and favor rearrangements that enhance efficient control. Natural selection works so well on operons that they often contain only genes related to specific metabolic pathways and functions.

Genomic rearrangements continue today. The North American strains provide a datable short-range evolutionary history. In only about 100 years the North American clones have gained one genome rearrangement, six inversions, and several SNPs. Despite all the rearrangements shown in the figure above and the loss of Y. pseudotuberculosis sequences, sequenced clones from all the biovars represented above have similar sized genomes. With the current set of hosts, this suggests that the genome is pared down to near its optimal size. For all the little extras that make Y. pestis an effective pathogen, the plasmids take center stage, and I’ll cover those in part 2.

Drancourt, M (2012). Plague in the genomic era Clinical Microbiology and Infection, 18, 224-230

Metagenomics, Lyme Disease, and the Tyrolean Iceman’s Tattoos

When the genetic analysis of the 5,300 year old Tyrolean Iceman, better known as Ötzi, was published in February, most of the attention was naturally focused on his genomic DNA. His genomic DNA produced some interesting results: he had brown eyes, blood type O+, was probably lactose intolerant and from a southern European gene pool. He also had a collection of alleles that associate with atherosclerosis that correlate with calcifications found by CT scan in Ötzi’s arteries.

To round out a complete analysis of the single 100 mg specimen they took from Ötzi’s ileum, the largest bone of the pelvis, they did a metagenomic analysis to identify all of the non-human DNA sequences amplified. Pelvis is not really an ideal bone to take a specimen from given its proximity to the intestinal organisms that play a role in decomposing the body. Surprisingly, bacterial DNA was a very small 0.84% of the identified sequences. They oddly make no reference to the 18% of DNA reads identified as “other eukaryote”.  Of the bacterial species, 72% of the sequences were from the genus Clostridia, who are primarily spore-forming anaerobes found in the soil. The one pathogen of significance discovered was Borrelia burgdorferi, the agent of Lyme disease.

Iceman metagenome (Keller et al, 2012)

Dark field image of Borrelia burgdorferi. Photo Credit: CDC

The break down of the Iceman’s microbial phylum yielded an impressive array of bacterial diversity.  The Firmicutes include the anaerobic Clorstridium species that are found in the soil. The Proteobacteria include the enteric bacteria like Escherichia coli, many of which are facultative anaerobes. Both of these phylum would be included in decomposition of the body and as anaerobes could grow in the corpse. Borrelia burgdorferi, the agent of Lyme disease, belongs to the phylum Spirochaetes. They were able to sequence approximately 60% of the Borrelia burgdorferi genome. To find B. burgdorferi in the pelvis suggests that the infection was in a systemic phase.

There are two pieces of correlating data to support a Borrelia burgdorferi infection. The international team that did this work linked the infection with Ötzi’s atherosclerosis, an association previously shown between Lyme disease and several other systemic infections.

Tattoos on the Iceman cover or align with major joints and muscles. (South Tyrol Museum of Archaeology site)

Yet, a common symptom of systemic Lyme disease is joint and muscle pain. One of the earliest observations of Ötzi’s mummy is that he has a lot of tattoos specifically placed over joints and muscle groups in places where strain would be expected. These tattoos do not appear to be decorative or signs of inclusion in a community. Consensus appears to have formed early on that these tattoos were medicinal, probably for pain relief. Scans of the mummy do suggest some arthritis. With his lifestyle, an approximately 45-year-old man is expected to have some arthritis and pain.  Both atherosclerosis, and evidence of joint pain and some arthritis can be explained by other means, but when taken together with the B. burgdorferi DNA make a compelling case that Lyme disease contributed to his overall state of health.

Reference:

Keller, A., Graefen, A., Ball, M., Matzas, M., Boisguerin, V., Maixner, F., Leidinger, P., Backes, C., Khairat, R., Forster, M., Stade, B., Franke, A., Mayer, J., Spangler, J., McLaughlin, S., Shah, M., Lee, C., Harkins, T., Sartori, A., Moreno-Estrada, A., Henn, B., Sikora, M., Semino, O., Chiaroni, J., Rootsi, S., Myres, N., Cabrera, V., Underhill, P., Bustamante, C., Vigl, E., Samadelli, M., Cipollini, G., Haas, J., Katus, H., O’Connor, B., Carlson, M., Meder, B., Blin, N., Meese, E., Pusch, C., & Zink, A. (2012). New insights into the Tyrolean Iceman’s origin and phenotype as inferred by whole-genome sequencing Nature Communications, 3 DOI: 10.1038/ncomms1701

South Tyrol Museum of Archaeology permanently houses and studies the mummy.