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 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.

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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

 

Detecting pathogens in medieval Venice

Medieval Venice was a trading empire, one of the busiest ports of the late medieval world. As a hub of commerce waves of plague visited and revisited Venice in 1348, 1462, 1485, 1506, 1575-1577, and 1630-1632 with the last two producing mortality rates around 30% of the population (Tran et al, 2011).

Three medieval plague graves in Venice. Grave B (upper right) produced one of the positive Yersinia pestis results. (Tran et al, 2011; doi:10.1371/journal.pone.0016735.g001)

As we all know, Venice has a land problem, or rather a lack of land problem. Thriving economies draw large populations and burial space becomes difficult to come by. Adding the plague on top and we have the perfect conditions for the discovery of mass plague burials.

On the island of Lazzaretto Vecchio in Venice 92 mass burials of 5-184 people have been uncovered and were used by the Marseille team for mass pathogen aDNA screening (Tran et al, 2011). Burials for the 14th, 15th, 16th, and 17th centuries were uncovered. Teeth in good condition (closed apex and no caries or trauma) were collected by the archaeologist who bagged them individually to prevent contamination. The team chose 173 specimens divided equally over the 14th-17th centuries. aDNA extracted from these teeth were screened by multiplex PCR for seven epidemic-causing pathogens: Yersinia pestis (plague), Bacillus anthracis (anthrax), Salmonella enterica Typhi (typhoid fever), smallpox, Barontella quintana (trench fever), Borrelia recurrentis (louse-borne relapsing fever), and Rickettsia prowazekii (epidemic typhus).

Of the seven pathogens screened for, only two were found in aDNA isolated from these teeth. Bartonella quintana, the causative agent of trench fever, was found in five teeth (2.9%) from the 15th and 16th centuries (Tran et al, 2011). B. quintana is a human louse transmitted disease that can explode in epidemic fashion, getting it’s name from epidemics in the trenches of World War I. However it does not always cause death so it’s presence does not necessarily indicate cause of death (Fournier et al, 2011; Foucault, Brouqui, & Raoult, 2006). Yersinia pestis, the causative agent of the plague, was found in three (1.7%) of the multiplex PCR screened specimens from the 14th and 16th centuries. One of these three was confirmed by suicide PCR and sequencing of the glpD gene, and a further positive specimen was found by PCR that the multiplex real-time PCR screening had missed (Tran et al, 2011). If I am reading their report correctly that would be overall four positive specimens by the two tests combined.

If these mass graves are primarily plague victims as Tran et al (2011) assert then efficiency of detection leaves much to be desired. It is possible that the aDNA is in bad shape. The graves pictured above are densely packed and I wonder about decomposition conditions. Does aDNA survive as well in these mass graves as it does in single graves?  A control  for aDNA integrity really needs to be established. They also remind us that aDNA detection is less sensitive than immunological methods, but even if they have detected only a small fraction of those that immunological methods would have found, that would still be a minority of skeletons sampled. The advantage of this study is that they were able to screen for seven pathogens at once.

The Marseille group is working under a hypothesis that plague was transmitted at least in part by the human louse. They have aired this hypothesis primarily in earlier publications and I may post on those papers someday. Their hypothesis explains the inclusion of two louse transmitted pathogens in their screening panel. If plague was transmitted by human lice this would change the transmission dynamics of the plague significantly and may explain at least some of the epidemiological conflicts with modern flea-transmitted plague dynamics.

Tran TN, 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) PMID: 21423736

Fournier PE, Ndihokubwayo JB, Guidran J, Kelly PJ, & Raoult D (2002). Human pathogens in body and head lice. Emerging infectious diseases, 8 (12), 1515-8 PMID: 12498677

Foucault C, Brouqui P, & Raoult D (2006). Bartonella quintana characteristics and clinical management. Emerging infectious diseases, 12 (2), 217-23 PMID: 16494745

The Vampire in the Plague Pit

Amid the chaos of a mass grave of plague victims, the 2006-2007 summer project team from the Archeoclub of Venice got a surprise. Among the dead they found evidence of belief in the undead, fear of the vampire.

Photo from the University of Florence. Image credit: "Reuters/handout". Also given in Nuzzulese and Borrini, 2010.

Photo of the skeleton in the grave from the University of Florence. Image credit: "Reuters/handout".

So how do you stop the undead from feasting on the corpses in the mass grave?  The sexton’s solution was to insert a brick in the mouth and get on with the rest of the burials. The fear was real. These sextons risked contagion to open the mouth of a decaying corpse in a plague pit to insert the brick (carefully enough not to break any teeth).

On 8 July 1468 the authorities of Venice passed a quarantine decree that sent plague victims to the island of Lazzaretto Nuovo for care and where the bodies were buried along a wall in mass graves. The excavation found two levels of skeletons: an older level with “postmortem fragmentation” and an upper level that was undisturbed. Devotional medals including the 1600 Jubilee medal dated the undisturbed bodies to the 17th century and the older level to the plague of 1576.

The body in question is that of the upper half of an adult female skeleton with a brick wedged in her mouth. Arrangement of the upper skeleton suggests burial in a shroud. The lower skeleton was probably destroyed by later burials. Analysis of the fill of the grave ruled out the brick working its way into the skull as the ground settled. There were no similar bricks or rocks in the grave trench. Forensic, odontogical, and chemical analysis of the skull and teeth indicates that this was an elderly woman of about 61 years old +/- 5 years of European descent (by DNA analysis) with old healed injuries who ate a primarily vegetarian diet indicating a lower social status. If the plague pit is representative of a cross- section of Venetian society, this woman would have been considered quite old. The bones and teeth revealed no signs of childhood disease or malnutrition.

Borrini and Nuzzolese (2010) hypothesize that the sextons revealed the shrouded remains of the old woman when digging a grave for a recent death. They believe that the body was still then shrouded except for the face which seemed to them to be eating the shroud. The sextons concluded that she was (or was becoming) a vampire or revenant (undead flesh eater) and employed the easiest method for preventing her from feasting on the dead by jamming an inedible object into her mouth.

So why did the sextons believe that this old woman was a vampire or more accurately a revenant? My understanding is that anthropologists currently credit the normal processes of decomposition for the traits of the vampire or revenant. Bloated corpses appeared to have feasted after death. Seeping red liquid from the mouth and nose contributed to the belief that they drank blood, though this is actually decomposition fluid. Fluid seeping from the mouth and nose of a decomposing corpse will cause the shroud above the face to decompose exposing the face. The exposed face through the partially decomposed shroud could look like it was trying to gnaw its way out of the shroud. Dehydration of the flesh also made it look like the hair and nails were continuing to grow. Ever-young, handsome, even sexy vampires are a completely modern creation that flow mostly from Bram Stoker’s Dracula published at the turn of the 20th century. Late medieval to early modern vampires/revenants were more like independent-minded, flesh-eating zombies.

Vampire mother from 'Vampires of Venice' episode of Doctor Who, broadcast May 2010.

In an even further departure from 17th century beliefs, could the discovery and press over this old woman have inspired the 16th century female vampires of Venice in last season’s Doctor Who? The “Vampires of Venice” episode was set during a plague in 1580 Venice. The mother vampire, the secular authority in Venice, uses the quarantine law  and fear of the plague to keep people in Venice so that they can be prayed upon by her children.

References:

Nuzzolese E, & Borrini M (2010). Forensic Approach to an Archaeological Casework of “Vampire” Skeletal Remains in Venice: Odontological and Anthropological Prospectus* Journal of forensic sciences PMID: 20707834

Interviews with Matteo Borrini on National Geographic Explorer: “Vampire Forensics” originally aired 2/23/2010.

Christine Dell’Amore, “Vampire of Venice” Unmasked: Plague Victim or Witch? National Geographic Daily News, February 26, 2010.

Daniel Flynn. March 12, 2009. “Vampire” unearthed in Venice plague grave. Reuters.