Category Archives: paleomicrobiology

Ötzi’s Lyme Disease in Context

One of the ancient DNA finds that continues to intrigue me is the discovery of Borrelia burgdorferi, the agent of Lyme disease, in Ötzi the 5300-year-old ice mummy from the Italian Alps. As far as I know, this is the only finding of B. burgdorferi in ancient remains of any date.  I discussed the initial report of these findings back in the summer of 2012. 

 

The more we learn about Ötzi’s environment and lifestyle, the less mysterious it seems. There are no signs of human habitation or land management in these high Alpine regions. Indicators of deforestation, farming, and pasture maintenance are lacking from lake sediment and pollen studies. Festi, Putzer and Oeggl (2013) found the first signs of human land management in the Ötztal Alps to began about 1000 years after Ötzi’s time. During the Copper Age, subsistence occupation of the valley floor was sufficient for the population of Ötzi’s time. They did minimal farming, and breeding of caprines (sheep, goats, and ibex). Festi, Putzer and Oeggl (2013) note that Ötzi’s mummy is the only piece of evidence for humans that high in the Otztal Alps before the Bronze Age.

Before Ötzi’s time, landscape management in the Mesolithic was to support red deer herds that were “in a state of semi-domestication by means of active hunting” (Rollo et al, 2002). (Native Americans managed deer populations in similar ways by promoting a landscape where deer thrive near their hunting grounds.) The importance of deer to Ötzi is underscored by everything about him from the red deer meat in his stomach to the roe deerskin that made up his quiver and antler in some of his tools (Rollo et al, 2012). Two different species of deer have been confirmed by genetic analysis.  Most of his clothing was made of sheep and goat skins (O’Sullivan et al, 2016).

The agent of Lyme disease, B. burgdorferi, is transmitted primarily by the tick Ixodes ricinus, common on deer, sheep, cattle, humans and dogs as adults and feed on rodents and small mammals as nymphs. Ticks often thrive at the forest edge where there are grasses for them to climb up to catch passing deer. It seems likely that they would also thrive in along upland forest edges as well. Ixodes ricinus is found throughout the Alps.  It is feasible that Lyme disease was a greater problem for humans when we relied on deer as a staple food.

Ötzi’s B. burgdorferi has yet to be confirmed by a second group. Interestingly, a recent study of B. burgdorferi’s phylogeny suggests that it originated in Europe and later spread to ‘post-Columbian’ North America (Margos et al, 2008). Although Lyme disease was only recognized in the 20th century, it is apparently an ancient disease caused by multiple Borrelia species. And Ötzi’s sequence has not been added to any phylogeny I’ve found, odd. Overlooked, or a problematic sequence?


References

Festi, D., Putzer, A., & Oeggl, K. (2013). Mid and late Holocene land-use changes in the Otztal Alps, territory of the Neolithic Iceman “Otzi”. Quaternary International, 353, 1–18. http://doi.org/10.1016/j.quaint.2013.07.052

Margos, G., Gatewood, A. G., Aanensen, D. M., Hanincová, K., Terekhova, D., Vollmer, S. A., et al. (2008). MLST of housekeeping genes captures geographic population structure and suggests a European origin of Borrelia burgdorferi. Proceedings of the National Academy of Sciences, 105(25), 8730–8735. http://doi.org/10.1073/pnas.0800323105

O’Sullivan, N. J., Teasdale, M. D., Mattiangeli, V., Maixner, F., Pinhasi, R., Bradley, D. G., & Zink, A. (2016). A whole mitochondria analysis of the Tyrolean Iceman’s leather provides insights into the animal sources of Copper Age clothing. Scientific Reports, 6, 1–9. http://doi.org/10.1038/srep31279

Rollo, F., Ubaldi, M., Ermini, L., & Marota, I. (2002). Otzi’s last meals: DNA analysis of the intestinal content of the Neolithic glacier mummy from the Alps. Proceedings of the National Academy of Sciences of the United States of America, 99(20), 12594–12599. http://doi.org/10.1073/pnas.192184599

Plague in 6th century Aschheim and Altenerding, Bavaria

Since I last wrote about Bavaria, the aDNA centers have been busy. With the accepted manuscript of the second new paper available this past week, its time for an update. The fourth paper on Aschheim not only confirmed the first three, but it also produced the first full genome of Yersinia pestis for the Plague of Justinian (Wagner et al, 2014). This paper also confirmed the Bavarian strain’s placement in the phylogeny of Y. pestis. The availability of the first full genome will primarily be important for comparison to newly discovered samples from elsewhere. Using newer technology, the newest paper refined some of the Aschheim sequence and produced a full genome of Y. pestis from a woman buried at Altenerding, about 20 km from Aschheim (Feldman et al, 2016). Radiocarbon dating from both sites places the epidemic in the mid-sixth century; it can not differentiate which specific epidemic ‘wave’.  The Altenerding epidemic was from the same Y. pestis lineage as Aschheim proving that this was a regional epidemic, possibly the same epidemic event. The phylogeny for the first pandemic is still based on a single epidemic from one geographic region, so the time is not yet ripe to use the phylogeny to tell inform us on the transmission or route of the pandemic.

6th cent Bavaria
Map of Roman Bavaria showing the Roman roads with Aschheim and Altenerding marked. The half circle/mound mark designates Roman villas. (modified from the Pelagios project)

It is, however,  time to start thinking a little more about the environment of these sites. They are both located on the Munich gravel plain, foreland (foothills) north of the Alps. Aschheim is located closer to the Alps at an elevation of 500 meters with Altenerding 20 km further north at a lower elevation in small valley formed by a tributary of the River Isar. The Roman road running horizontally across the map runs west to Augsburg, the capital of the Roman province of Raetia Secunda and east to the city of Batavia, a colony in the province of Noricum. The road running by Altenerding would take traffic eventually north toward Regensburg (Casta Regina).

Large water feature is Speichersee lake with a man-made 20th century reservoir used to power hydroelectric plants and serve some of the water needs of the Munich region. As far as I can tell, none of this would have been present in the Late Antique period. The River Isar is the green line to the west of both sites. Munich will later be founded where the road crosses the river from monastic land in about 1158. There was nothing special at the river crossing in the sixth century. Although the road crosses the river, there is no indication of a Roman bridge on the map.

Both Aschheim and Altenerding are located in what would have been the province of Raetia II. While they are along Roman roads, this would have been a rural area. Both Aschheim and Altenerding were sites of Roman villas and Dornach near Aschheim was a small settlement. How much of this would have been occupied and further developed (or not) after the Roman army left is unclear. The cemetery at Altenerding is triple the size of Aschheim. Yet, there is reason to think that Aschheim was hit harder by the plague and based on the carbon dates of graves with some molecular plague signal, probably more than once. Michael McCormick (2015:83) suggests that the Aschheim cemetery gathered graves from a dispersed settlement that probably had fewer than 70 people at any one time.

A living history museum in Munich area at Kirchheim has reconstructed typical buildings from the early medieval Merovingian period. Although this area was nominally under Merovingian Frankish hegemony there is little specifically Frankish about the archaeology. They were all wooden construction. Below is a picture of a sunken pit building, an ‘out building’ and a long house.

1280px-Bajuwarenhof_Kirchheim_Übersicht_2012-08-05
Reconstruction of 6th-7th century Bavarian buildings at Kirchheim in the Munich district close to Aschheim. (Photo by Leporollo, Wikipedia CC3.0)

Continue to think of the Plague of Justinian in Constantinople and Pelusium, it was surely there. Just remember that most of its geographic spread may have looked more like this picture.

References:

Feldman, M., Harbeck, M., Keller, M., Spyrou, M. A., Rott, A., Trautmann, B., et al. (2016). A high-coverage Yersinia pestis Genome from a 6th-century Justinianic Plague Victim. Molecular Biology and Evolution, 1–31. [Accepted manuscript]

McCormick, M. (2015). Tracking mass death during the fall of Rome’s empire (I). Journal of Roman Archaeology, 28, 325–357.

Wagner, D. M., Klunk, J., Harbeck, M., Devault, A., Waglechner, N., Sahl, J. W., et al. (2014). Yersinia pestis and the Plague of Justinian 541–543 AD: a genomic analysis. The Lancet Infectious Diseases, 14(4), 1–8. http://doi.org/10.1016/S1473-3099(13)70323-2

The Schistosoma in the Reliquary

The 800th anniversary of the birth of Saint Louis, King of France, in 2014 provided an opportunity to obtain a sample of his relics for “scientific identification”. With all relics the chain of custody and its backstory are critical for evaluation. Most of Louis’ relics held in the Basilica of Saint-Denis  were destroyed during the religious violence of  sixteenth century Paris. Fortunately the process of preserving and transferring Louis home to France from the site of death on crusade in Tunis, North Africa left bits of him in several locations.  Part of the preservation process used at the time removed his intestines and other internal organs to be embalmed separately while the remainder of the body was boiled to clean the bones to return to Paris. The bones were enshrined in Paris, while the heart and some viscera were enshrined at the abbey of Monreale near Palermo by his brother Charles, King of Sicily, who oversaw the preparation of the body and its transport back to France.

San_Domenico47
13th century Reliquary of Saint Louis, Basicilla of St. Dominic, Bologna Italy. (Source: Photo of Georges Jansoone, public domain on Wikipedia)

During a stop over at Bologna en route to Lyon and then Paris, some of his viscera were removed and interned in the Basilica of Saint Dominic. In 1297 Louis, who had died on 25 August 1270, was officially canonized as Saint Louis of France.  A portion of these visceral relics were given for the consecration of the cathedral of Turin in 1895, and these were transferred to the cathedral of Versailles in 1985. It is from this visceral relic that the 2 g specimen was obtained for scientific evaluation.

Microscopic Examination

The plan is to do a full “medical and forensic anthropological analysis” of the remains. The first result released by Phillipe Charlier’s team is the discovery of a semicircular parasite viewed by Scanning Electron Microscope (SEM) analysis, identified as a male Schistosoma based on its size and morphology.  Schistomsoma are a sexually dimorphic flat worm, also known as a blood fluke, that inhabit the capillaries of the abdomen (mesentery or bladder plexus depending on species) and release their eggs into either feces or urine. The eggs hatch in fresh water and must pass through a fresh water snail before emerging as larvae that can inhabit a mammalian host. Only about 50% of the eggs produced actually exit the body.  The adult worms and eggs that do not reach the feces or urine can cause extensive inflammation resulting in granulomas and fibrosis (scar tissue) to the abdominal organs (liver, spleen, intestines, bladder) and the blood vessels of the abdomen causing an accumulation of fluid in the peritoneal cavity.  Eggs that do breach into the lumen of the intestine cause chronic blood loss into the lumen producing chronic bloody stools. In the worst cases the blood loss can cause anemia.

Saint Louis
Source: Charlier, Bouchet, Weil & Bonnet, 2015.

Compare to a SEM preparation of a modern (non-mummified) male Schistosoma:

800px-Schistosoma_20041-300
A C-shaped male schistosoma; the smaller female resides in the canal. (Source: David Williams, Illinois State University made public domain, Wikipedia)

King Louis had not been in Tunis long enough for him to contracted schistosomiasis upon his arrival only a month before his death. When and where he contracted the flat worm infestation is open to more speculation.  Schistosoma have been observed in archaeological remains of one individual from 9th century France, but have not yet been commonly observed. Charlier et al. (2015) suggest that Louis’ previous crusade in North Africa between 1250 and 1254, spending some time imprisoned in Damietta, Egypt, is the most likely period for start of the infection. If this is true, then Louis would have had a chronic infection for about 20 years at the time of his death. Charlier’s team do not believe that Schistosomiasis contributed to his death.

So far they have not observed any other parasites in the sample. This is not necessarily surprising considering that they have not yet identified the anatomic source of the specimen. It is not possible to even guess at the anatomic source from the crumbling, blackened specimen pictured in their study (fig. 1). Their analysis is continuing.

Debating Saint Louis’ Cause of Death

As soon as the schistosoma report was published, the debate on the cause of Louis’ death began in the letters of Forensic Science, Medicine and Pathology (where the report was published). So lets begin with the best account of Louis’ death, and go from there.

Beyond skirmishing and entrenching the camp nothing was done, as King Louis was awaiting the arrival of his brother Charles of Anjou (now King of Sicily). Whilst they were waiting encamped, John Tristan fell sick, and died on board one of the ships on August 3rd. A few days later the Legate also died and many other persons, some of fever, some of dysentery. Philip, the King’s eldest son, fell sick with fever; and the King was taken with dysentery (the complaint to which he nearly succumbed in his first Crusade) and died on August 25th. (Guillaume de Nangis account in the Memoirs of Lord Joinville, Book 4, Ch. 4)

Strangely, the plague has traditionally been claimed as Louis’ cause of death. This is completely unfounded since the Black Death will not bring epidemic Yersinia pestis back to the Mediterranean for another 77 years! There is nothing in the account above to suggest plague. This has rightly been dismissed as Louis’ cause of death.

Eric Faure wrote a letter arguing that malaria was a possible cause based on reports of Louis’ history of tertian fevers dating back to the 1242. Faure suggests that Louis went on his first crusade in thanksgiving for surviving “cerebral malaria with a coma” after a relapse in 1244. Cerebral malaria is usually caused by Plasmodium falciparum, which is not a chronic (relapse causing) infection. If Louis suffered relapses of malaria contracted in France then it was most likely Plasmodium vivax, which rarely causes cerebral malaria. Whether or not Louis had cerebral malaria in the 1240s, this doesn’t really inform of his his health in 1270.  Faure also notes that some of the men on Louis’ last crusade had intermittent fevers suggestive of malaria. Faure reaches too far suggesting that the dysentery was a symptom of malaria. Gastrointestinal symptoms are possible but rare in malaria and usually then in children. Philippe Charlier responded with a letter dismissing Faure’s suggestion to look for Plasmodium in the remains, because they would not have caused Louis’ death. Following the report in Lord Joinville’s memoir (quoted above), Charlier reports in his letter that his team is now looking for evidence of bacteria, viruses or amoeba in the embalmed “intestines” that are more likely to be the cause of the “dysenteric syndrome” reported in “Louis and his court”.

I will be watching for the final report, but the idea of a single enteric pathogen being the cause of death may not really represent reality. Based on Joinville’s memoir is appears that the “court” was suffering from a variety of camp diseases found in most medieval armies on prolonged campaigns. In such a situation, co-infection is highly likely particularly with chronic parasites. Indeed, Louis was probably not the only one in camp with schistosomiasis lingering from previous travels.  Although I doubt malaria caused Louis’ dysentery, it is quite possible he was suffering from chronic malaria and that it contributed to weakening his health, making him more susceptible to other pathogens. Indeed co-infection with Schistosoma and Plasmosdium could have made him quite anemic.  It would still be worth knowing if Louis had an active malaria infection, even if Shigella or another enteric pathogen was the primary cause of death.

References:

Charlier, P., Bouchet, F., Weil, R., & Bonnet, B. (Oct. 2015). Schistosomiasis in the mummified viscera of Saint-Louis (1270 AD). Forensic Science, Medicine, and Pathology, 1–2. http://doi.org/10.1007/s12024-015-9722-4

Faure, E. (Dec. 2015). The infections of Saint-Louis: possible involvement of malaria.[Letter]  Forensic Science, Medicine, and Pathology, 1–1. http://doi.org/10.1007/s12024-015-9732-2

Charlier, P. (2016). Neither plague nor malaria, but dysentery as a cause of death for St. Louis. [Letter]  Forensic Science, Medicine, and Pathology, 1–1. http://doi.org/10.1007/s12024-015-9738-9

The Memoirs of Lord Joinvilletranslated by Ethel Wedgwood, E-text. University of Virginia library

Louis IV of France, Wikipedia.

WHO fact sheet: Schistosomiasis

Schistosoma, Wikipedia

Michael Walsh, Schistosomiasis on the Infection Landscapes blog. See this page for the best description of the medical effects of schistosomiasis.