Category Archives: aDNA

Keeping Bronze Age Yersinia pestis in Perspective

Graphic abstract from ____
Graphic abstract from Rasmussen et al, 2015.

The latest plague news to splash across headlines is the discovery of Yersinia pestis aDNA in seven Bronze Age remains from Eurasia.  The most important findings in this new study are not anthropological; they are evolutionary. This paper allows us to drop a couple more evolutionary mile markers. Finding  7% of the tested remains  (7 out of 101) positive for plague is surprising, but I’m not yet ready to believe that it was endemic over such a huge area scattered over 2000 years. Not yet anyway.

The new phylogenetic tree places Y. pestis in humans since the Bronze Age and the origin of the species as far back 50,000 years ago.  It also opens up questions on the original reservoir species and the location of the birth of the species, although central Asia is still the most likely location.

Stretching out the Yersinia pestis tree. Blue arrows are gains and red arrows are losses. (Rasmussen et al, 2015)


So let’s look at the genetic results in three areas highlighted by Rasmussen et al: flea transmission, Pla activity, and suppression of the immune response stimulating flagellin production. These traits are critical to producing bubonic plague as we know today.

Late phase flea transmission of modern Yersinia pestis is dependent on the ability to survive in and colonize the flea. The Bronze Age strains have all of the plasmids and virulence genes of modern strains except one, the ymt gene that encodes the murine toxin. The basic tool set of modern strains also have deactivated or knocked-out the protein products of three ancestral genes that hinder Yersinia pestis biofilm formation. Remnants of these genes persist as pseudogenes in modern strains. (A pseudogene is the corpse of a former gene.) This genetic combination allows Y. pestis to survive in the mid-gut of the flea, persist longer and form a biofilm; a necessity for late phase flea transmission. However, as Monica Green reminded me,  ymt is not required for early phase flea transmission, dirty-needle style (Johnson et al, 2014). In fact, since Y. pestis does not need to persist long or multiply at all, there are no known genes needed to be present or absent for early phase transmission.  As I recently reviewed, early phase transmission is very common and effective (see Eisen, Dennis & Gage, 2015). Based on the dates of their samples, they estimate that ymt was gained in about 1000 BC. In the RISE509 strain from Afanasievo Gora in southern Siberia, the pde3 is inactive but the other two, pde2 and rcsA, are still functional. Taken together this genetic combination should allow early phase flea transmission but not late phase flea transmission that requires biofilm formation. They are still mid-way in developing late phase flea transmission. This makes sense for a microbe being transmitted dirty-needle style, providing the opportunity for natural selection to develop late phase transmission bit by bit. While early phase transmission can support regional epizootics and epidemics,  late phase flea transmission is probably important for long distance transmission by fleas in grain or textiles, or by sea.

The recent discovery of the Pla gene in Citrobacter koseri and Escherichia coli, other enteric opportunistic flora, but not found in Yersinia pseudotuberculosis, suggests that lateral gene transfer  brought the plasmid to the young Y. pestis while still in the enteric environment (Hänsch et al, 2015) . This is consistent with Y. pestis Pla and Salmonella enterica PgeE both evolving from the same ancestral omptin ancestor in an enteric environment (Haiko et al, 2009).  This suggests that Y. pestis may have remained an enteric organism for some time after it split from Y. pseudotuberculosis.

Six of seven Bronze Age Y. pestis strains contain the Pla gene required for deep tissue invasion and bubo formation. Rasmussen et al (2015) suggest that the strain lacking the Pla gene has lost it and that this gene has been lost more than once in the phylogenetic tree. In other words, Pla was present in the common ancestral strain. However, to support development of bubonic plague Pla needed to gain a mutation at position 259 that these strains lack (Haiko et al, 2009). So the Pla gene without the mutation at position 259 can support pneumonic plague but not bubonic in the Pestoides F strain (0.PE2) of Y. pestis (Zimbler et al, 2015).  On the other hand, Sebbane et al (2006) showed that strains completely lacking Pla can still develop primary septicemic plague following flea transmission. They can envision an “evolutionary scenario in which plague emerged as a flea-borne septicemic disease of limited transmissibility”(Sebbane et al, 2006).  Without the polymorphism at position 259, bubo formation should be retarded, if not suppressed.

A third genetic difference of possible significance is the apparent ability to produce flagellin, a major activator of the human innate immune system. Modern Y. pestis strains have deactivated the production of flagellin by a frameshift mutation in the regulatory gene flhD. The Bronze Age strains lack this frameshift and so presumably had normal flagellin production. However, Y. pseudotuberculosis and  Y. enterocolitica down regulate production at mammalian body temperatures. If the ancestral Y. pestis did also then its possible that it wasn’t a factor in human infections.  Experimentally recreating the regulatory environment from  Y. pseudotuberculosis would be much more difficult than simply inserting an intact copy of the gene in a modern strain of Y. pestis.

Predicting the impact of these ancestral genes is highly conjectural. This combination of genes has never been studied together. Since these strains were isolated from human remains we can assume that there is a path for transmission and pathogenesis. The reliance on early phase flea transmission, the less virulent pla allele and the possible production of flagellin suggest that Bronze Age local (dermal) infections from flea bites would be less virulent (more survivable). Interestingly, these milder local infections may have been immunogenic.

As Y. pestis moved away from an enteric lifestyle, producing a septicemia was necessary for either flea transmission or development of a secondary pneumonia with aerosol transmission. I find it hard to believe that Bronze Age Siberia or Estonia had a large enough population for sustained pneumonic transmission. Since Pulex irritans can transmit Y pestis without development of a biofilm, there is no reason to see humans as a dead-end to flea transmission even as early as the Bronze Age.

Humans could have also contracted septicemic plague by ingesting infected meat. Although natural ingestion infections are very rare today, this mode remains effective. A village size outbreak could easily occur from sharing a large infected animal as happened in Afghanistan in 2007. In that outbreak a single infected camel shared among two villages produced 83 probable cases of plague with 17 deaths, a case fatality rate of 20.5%. (Leslie et al, 2011).  Last but certainly not least, the further back we go in Yersinia pestis‘ evolution the more likely ingestion is to be a mode of transmission like its ancestor Yersinia pseudotuberculosis.

Its takes more than good transmission to cause a demographic changing epidemic over large areas like the Eurasian continent. It also requires a fairly high human density and good trade or communication routes. Humans play the the most important role in transmitting plague of pandemic size. I can’t say if the cultural factors that make such large epidemics possible were in place in Bronze Age Eurasia.

Let’s keep things in perspective before we conjure up the specter of virgin soil epidemics of plague in the Bronze Age. Yersinia pestis is the kind of over achiever that may have been a player in Bronze Age demographics but it would be nice to have a lot more evidence before jumping to that conclusion.


Rasmussen, S., Allentoft, M. E., Nielsen, K., Orlando, L., Sikora, M., Sjögren, K.-G., et al. (2015). Early Divergent Strains of Yersinia pestis in Eurasia 5,000 Years Ago. Cell, 163(3), 571–582.

Eisen, R. J., Dennis, D. T., & Gage, K. L. (2015). The Role of Early-Phase Transmission in the Spread of Yersinia pestis. Journal of Medical Entomology, tjv128–10.

Johnson, T. L., Hinnebusch, B. J., Boegler, K. A., Graham, C. B., MacMillan, K., Montenieri, J. A., et al. (2014). Yersinia murine toxin is not required for early-phase transmission of Yersinia pestis by Oropsylla montana (Siphonaptera: Ceratophyllidae) or Xenopsylla cheopis (Siphonaptera: Pulicidae). Microbiology, 160(Pt_11), 2517–2525.

LESLIE, T., WHITEHOUSE, C. A., YINGST, S., BALDWIN, C., KAKAR, F., MOFLEH, J., et al. (2011). Outbreak of gastroenteritis caused by Yersinia pestis in Afghanistan. Epidemiology and Infection, 139(5), 728–735.

Sebbane, F., Jarrett, C. O., Gardner, D., Long, D., & Hinnebusch, B. J. (2006). Role of the Yersinia pestis plasminogen activator in the incidence of distinct septicemic and bubonic forms of flea-borne plague. Proceedings of the National Academy of Sciences of the United States of America, 103(14), 5526–5530.

Zimbler, D. L., Schroeder, J. A., Eddy, J. L., & Lathem, W. W. (2015). Early emergence of Yersinia pestis as a severe respiratory pathogen. Nature Communications, 6, 1–10.

Hänsch, S., Cilli, E., Catalano, G., Gruppioni, G., Bianucci, R., Stenseth, N. C., et al. (2015). The pla gene, encoding plasminogen activator, is not specific to Yersinia pestis. BMC Research Notes, 1–3.

Haiko, J., Kukkonen, M., Ravantti, J. J., Westerlund-Wikstrom, B., & Korhonen, T. K. (2009). The Single Substitution I259T, Conserved in the Plasminogen Activator Pla of Pandemic Yersinia pestis Branches, Enhances Fibrinolytic Activity. Journal of Bacteriology, 191(15), 4758–4766.

A Migration Age Anglo-Saxon Leper

Paleomicrobiology and isotopic analysis has the ability to completely change what we know of past infectious diseases. A study published this month on a fifth century Anglo-Saxon skeleton is one of the most complete I have read.

Lesions on skeletons found at Great Chesterfield in Essex, England, suggested possible leprosy. To confirm this diagnosis, they chose one skeleton that is nearly complete and in good shape for further analysis.

Grave GC86 from Great Chesterford, excavated in a rescue archaeology operation in 1953-4.
Grave GC86 from Great Chesterford, excavated in a rescue archaeology operation in 1953-4. (Inskip et al, 2015)

The skeleton (GC96) shown to the right is of a 25 to 35-year-old male buried in modestly furnished grave in an area of the cemetery with other visibly disabled people. Radiocarbon dating places these remains at AD 415-545, and thus Migration Age for the Anglo-Saxons. The Great Chesterford cemetery is located roughly in an approximate border area between the kingdom of the East Saxons and East Angles at the site of a ford of the River Cam (or Granta) downriver from Cambridge. He was buried with a slender knife secured by a belt with an oval buckle. Over his left shoulder, a spear and a conical ferrule were found.  Lesions consistent with lepromatous leprosy were found on the lower legs with extensive remodeling of the right foot. A bronze shoelace tag found near the right foot suggests the diseased foot covered with a shoe.  Given the lesions found on the foot and lower legs, the ferrule may have capped a walking staff. His facial bones were missing losing a common, distinctive site of leprosy lesions. The disorganized and rough appearance of new bone growth suggest that the lesion was active at the time of death.

Profile of the mycolic acids extracted from the indicated bones.
Profile of the mycolic acids extracted from the indicated bones. (Inskip et al, 2015)

Selections of bone were taken and powdered to extract aDNA and for lipid analysis. Mycobacterium species that cause leprosy and tuberculosis have distinctive lipid profiles that have been successfully extracted and identified by archaeological remains in the past. Their analysis of lipids from the bones confirmed the presence of Mycobacterium leprae and excluded the presence of Mycobacterium tuberculosis.  The aDNA analysis confirmed identified the presence of Mycobacterium leprae strain 3I-1, that has been previously found in later medieval England, Denmark and Sweden. Inskip et al (2015) suggest a possible Scandinavian origin for the strain.  The VNTR analysis used to produce ‘genetic fingerprints’ shows that this strain of M. leprae is unique among other ancient isolates and should be useful in the comparative analysis of other early remains. Other remains in the same cemetery have similar lesions and will be investigated in the future.

Isotopic analysis of his tooth enamel provide an indication of childhood location and adult nutrition. Carbon analysis showed a diet of primarily C3 plants, consistent with southern Britain. Analysis of oxygen and strontium isotopes suggest he did not spend his childhood in the area of Great Chesterford.

The combination of the two isotopes gives his best probable origin to be between north-central France and the north-central Germany, in other words, the region of the Anglo-Saxon homeland. A continental origin coupled with the dating range between 415 and 545 suggests that he was part of the migration of the peoples who later called themselves Anglo-Saxons. He was likely no more Scandinavian than any of the other migration era ‘English’. This is further supported by a relatively high level of leprosy (by osteological analysis) in medieval city of Schleswig, the very area where the Angles are most specifically located. Further analysis of migration era remains should refine the origins of this strain of leprosy and determine its frequency.


Inskip, S. A., Taylor, G. M., Zakrzewski, S. R., Mays, S. A., Pike, A. W. G., Llewellyn, G., et al. (2015). Osteological, Biomolecular and Geochemical Examination of an Early Anglo-Saxon Case of Lepromatous Leprosy. PLoS ONE, 10(5), e0124282. doi:10.1371/journal.pone.0124282.s001

Kristina Killgrove, 14 May 2015 “Earliest Case of Leprosy in Britain reveals Scandinavian Origins of the Disease”,

SIMON MAYS, SONIA R. ZAKRZEWSKI, SARAH A. INSKIP, STEPHANIE WRIGHT and JOANNA R. SOFAER. (2015) Anglo-Saxon concepts of dis/ability: placing disease at Great Chesterford in its wider context. Poster at The 84th Annual Meeting of the American Association of Physical Anthropologists.

Contours of the Black Death Cemetery at Charterhouse Square, London

Excavations for the Crossrail Extension project discovered the second major Black Death cemetery in London in 2013. This week the first peer-reviewed publication of findings from the site appeared (in press).  As a rescue excavation in the midst of a construction project, the site had to be quickly surveyed for the extent of the cemetery and this is what is contained in this publication.

This site is part of 13 acres leased by Sir Walter de Mauny from St Bartholomew’s Priory for an emergency cemetery for plague victims in 1349 AD.  The site has been used for a variety of purposes over the centuries and currently is a four acre green space called Charterhouse square. The site is graphically displayed below with the locations of later structures.

Crossrails site, London
Crossrails site in Charterhouse Square, London (Dick et al., 2015)

The initial discovery came in a shaft just to the southwest of the Charterhouse Square. There they found three layers of graves with a total of 25 bodies lacking signs of trauma and with pottery shards from 1270-1350 AD. Subsequent radiocarbon dating and aDNA analysis confirmed that they were victims of the Black Death.

The surveys conducted over just two days were able to outline the broad contours of features at the site. These included a 15th century building, a priory kitchen, a probable World War II submerged emergency water tank, and a possible ditch and bank along the cemetery that is mentioned in descriptions. They believe that a disturbed area in the southwest corner represents about 200 individual graves, although only excavation can confirm these graves. They concluded that their ability to detect medieval objects in such an intensely used urban area suggests these methods are a good option for similar future situations.

The scans also revealed some surprises. There are not as many graves as descriptions suggest should have been there, though bodies may be more dense that suggested by the scans. They also did not find any large pits of  stacked bodies. This indicates that even during the height of the Black Death, many people were still buried in individual graves. Graves were found in three phases with layers of clay-rich earth in between perhaps in an attempt to seal the graves. These scans should allow them to target future excavations to areas with a high probability of dense graves.


Dick, H. C., Pringle, J. K., Sloane, B., Carver, J., Haffenden, A., Stephen Porter, H. A., et al. (2015). Detection and characterisation of Black Death burials by multi-proxy geophysical methods. Journal of Archaeological Science, 1–50. doi:10.1016/j.jas.2015.04.010 [In press, accepted manuscript]