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

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.

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

 

Mapping Malaria in Anglo-Saxon England

Guthlac at Croyland in the marshes of the Wash.

England once looked very different. Much of southern Britain was marshland for most of the island’s occupied history. These bogs, fens, and marshes ensured that areas of virtual wilderness persisted  from before Roman Britain through the Norman period and beyond. Despite the difficulties of using fenlands, these areas were not only occupied throughout the Anglo-Saxon period, but important centers like Croyland, Bardney, and Ely eventually developed in the marsh.

The largest fenland region was known as ‘the Wash’.  This low-lying region drained four rivers into  a square bay of the North Sea that forms the corner between Lincolnshire and Norfolk. In Anglo-Saxon times, this tidal marsh and bog was a vast border region between the region of Lindsey and East Anglia.  Places like Croyland and Ely were islands in the wetlands.  The eighth century Life of Guthlac describes the environment of Croyland when Guthlac arrived:

There is in the Midland district of Britain a most dismal fen of immense size, which begins at the banks of the river Granta not far from the camp which is called Gronte (Cambridge) and stretches from the south as far north as the sea. It a very long tract, now consisting of marshes, now of bogs, sometimes with black waters overhung by fog, sometimes studded with woodland islands and traversed by the windings of tortuous streams. (Hill, 1981:11 cited in Gowland & Western, 2011).

These marshes are ideal for malaria, but evidence of malaria in Anglo-Saxon England has been lacking. It is supposed that malaria would have been brought to Britain with the Romans (1). Unfortunately, there is no evidence that I know of that malaria became endemic in Roman Britain much less lasted into the early medieval (Anglo-Saxon) period. It has also been speculated that ‘spring fever’ (lecten adl) found in Anglo-Saxon leechbooks is the spring manifestation of tertian malaria (1) caused by Plasmodium vivax. This would fit the pattern of malaria in cool or cold climates like that found in Finland discussed in a recent post. Indoor transmission in Anglo-Saxon earthen floored, open structured wooden homes with thatched roofs would be an ideal way to concentrate malaria in a thinly populated marsh.  (Without chimneys homes had to open enough to allow smoke to escape from a central hearth.)

Incidence of Malaria in England, 1840-1910 (2)

It has long been known that Britain can environmentally support endemic malaria. Malaria was fairly wide-spread in 19th century Britain when it was first mapped (figure to left) (2). The upper black area on the map includes much of ‘the Wash’. However, proof of malaria is more tenuous for the medieval period.  Together with the unhealthy reputation of the brackish marshlands there is at least enough evidence to suggest that endemic malaria reached back into the late medieval period.

Malaria went by a variety of local names before the early modern period. Malaria-like fevers are mentioned in literature from Geoffrey Chaucer to William Shakespeare (2, 3). Terminology for malaria was not settled upon the Italian ‘malaria’ until the early modern period. Before then, it went by a variety of terms the most universal being ‘ague’, meaning the shakes, and sometimes  ‘fever and ague’ referring to the cyclic breaking of a fever.

Gowland and Western (2011) took a new approach to finding evidence of malaria in Anglo-Saxon England (400-1100 AD) (4). Malaria caused by Plasmodium vivax causes chronic hemolytic anemia that may result in cribra orbitalia due to expansion of the bone marrow in the cranium. Gowland and Western correlated the presence of cribra orbitalia in Anglo-Saxon skeletal remains with the presence of the malarial vector Anopheles atroparavus and reports of ‘ague’ in 19th century England.

The Anglo-Saxon cemeteries used in their study are mapped in the figure below on the left. Note that not many cemeteries are located near the modern coastline of ‘the Wash’. This area would have likely been too wet for settlement.

Anglo-Saxon cemeteries (4)

Map of A. atroparvus with 19th century "ague" records. (4)

Gowland and Western  determined areas capable of sustaining malaria by mapping the presence of A. atroparvus from a 1900 AD British Museum survey (shown above on the right) (4).  The darker the shading the more reports of mosquitoes. This survey was reported to not have been systematic, so they augmented it with 19th century ‘ague’ reports (triangles).  There are some notable areas with high levels of mosquitoes that lack ague reports. This map was use to determine malarial regions for correlation with either cribra orbitalia or the poor nutrition control enamel hypoplasia. It also roughly correlates with the 1840-1910 malaria incidence in the color map above by Kuhn et al (2).

An inverse distance map showing A. atroparus incidence vs. hot and cold spots for cribra orbitalia. (4)

In this last map, malarial areas are plotted with hot and cold spots for cribra orbitalia.  Purple and blue areas on the map indicate the highest areas of A. atroparvus in 1900, while red and orange circles indicate the cribra orbitalia ‘hot’ spots. Areas of cribra orbitalia correlate very well with malarial areas around the Wash.  Cribra orbitalia ‘cold’ spots (blue circles) correlate with areas of low A. atroparvus. They found no correlation between enamel hypoplasia with either ‘malarial’ or ‘non-malarial’ areas (4).

If this cribra orbitalia is due to malaria, it is likely an underestimate of the amount of malaria in the English wetlands. Cribra orbitalia forms in children so it will not indicate adults who contract malaria. Communities like Ely, Croyland and Peterborough were large monasteries who probably drew many into the marsh as adults.

Confirmation of malaria in Anglo-Saxon England will have to wait for molecular evidence, but this skeletal evidence strengthens the hypothesis that it was endemic in early medieval Britain. It also should be informative for the areas to concentrate efforts to find molecular evidence.

References:

(1) Cameron, M.L. (1993, repr. 2006) Anglo-Saxon Medicine. Cambridge University Press.

(2) Kuhn, K., Campbell-Lendrum, D., Armstrong, B., & Davies, C. (2003). Malaria in Britain: Past, present, and future Proceedings of the National Academy of Sciences, 100 (17), 9997-10001 DOI: 10.1073/pnas.1233687100

(3) Reiter P (2000). From Shakespeare to Defoe: malaria in England in the Little Ice Age. Emerging infectious diseases, 6 (1), 1-11 PMID: 10653562

(4) Gowland RL, & Western AG (2011). Morbidity in the marshes: Using spatial epidemiology to investigate skeletal evidence for malaria in Anglo-Saxon England (AD 410-1050). American journal of physical anthropology PMID: 22183814