Category Archives: Lyme disease

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

General Principles of Zoonotic Landscape Epidemiology

Zoonoses, pathogens with animal reservoirs, exist as part of a complex system of interactions between animal reservoirs, vectors, ecological factors and human interaction. Landscape epidemiology has existed as a field of study since Russian epidemiologist E.N. Pavlovsky coined the term and laid the groundwork in the 1960s. Landscape epidemiology is in essence the study of environmental foci of zoonotic disease, what Pavlovsky called a nidas. Many of the variables have been identified and studied in individual pathogen systems.

Each system seems so complex and unique that it can be easy to think that they each exist as separate entities with little to do with each other. It is necessary to develop some general principles to both see the bigger picture, and guide research and response to less studied and newly discovered pathogens. Lambin et al. set out to do just that by doing a meta-analysis of eight regional case studies of zoonotic diseases in Europe and East Africa: West Nile Virus in Senegal, Tick-borne Encephalitis in Latvia, Sandfly abundance (leishmaniasis vector) in the French Pyrenees, Rift Valley Fever in Senegal, West Nile Virus hosts in Camargue, Rodent-borne Puumala hantavirus in Belgium, human cases of Lyme borreliosis in Belgium, and risk of malaria re-emergence in Camargue. Obviously, as indicated, not all of these studies look at all factors involved in landscape epidemiology so validation is not solely based on the number of case studies that support each principle.

The ten proposed principles by Lambin et al are shown graphically below where they fit into the system of variables.

Graphical representation of the landscape determinants of disease transmission. The numbers refer to the ten propositions formulated in this paper. Lambin et al. International Journal of Health Geographics 2010 9:54   doi:10.1186/1476-072X-9-54
Graphical representation of the landscape determinants of disease transmission. The numbers refer to the ten propositions formulated in this paper.
Lambin et al. International Journal of Health Geographics 2010 9:54 doi:10.1186/1476-072X-9-54

Proposed general principles (Lambin et al, 2010):

  1. Landscape attributes may influence the level of transmission of an infection” This proposal is found in all case species. Features of the landscape influence vector and host distribution across the region of study. Distribution and type of water (fresh, brackish, or salt water) is a common landscape feature that influences density of insect vectors.
  2. Spatial variations in disease risk depend not only on the presence and area of critical habitats but also on their spatial configuration“.   The sheer size of the critical area is not the only or necessarily the most important characteristic to determine risk in an area. Some vectors like ticks thrive along border zones between ecosystems, like edges between woodland and grasslands.
  3. Disease risk depends on the connectivity of habitats for vectors and hosts” Creating contact zones or contiguous zones that create linked areas are also important. The spatial configuration can create corridors for disease persistence in harsh landscapes. Type and connectivity of  vegetation is as important as terrain for vector habitats. Connectivity between suitable habitat for rodents and insects allows the disease to spread from one patch to the next amplifying the pathogen to a level that increases risks of human transmission. Connections between patches of critical habitats allows for recolonization after local extinction.
  4. The landscape is a proxy for specific associations of reservoir hosts and vectors linked with the emergence of multi-host disease.” Their principle could be better fleshed out; their primary evidence coming from West Nile Virus (WNV). Like other multi-host pathogens, WNV has some hosts that are much more important than others for transmission across wide regions. In WNV migratory birds are a key to understanding its spread and epidemic dynamics. WNV is also an example of a disease with different proxies and amplification hosts in different regions of the world.
  5. To understand ecological factors influencing spatial variations of disease risk, one needs to take into account the pathways of pathogen transmission between vectors, hosts, and the physical environment.” Vector-borne diseases require direct contact between humans and the vector. For other zoonoses like hantavirus contact between humans and animal hosts can be via aerosols of material with rodent feces or dust containing rodent remains. For example, people have contracted hantavirus by vacuuming up rodent remains in homes. When estimating risk of transmission to humans, abiotic (non-living) environmental conditions that can preserve or transmit to humans have to be considered. Climate and moisture content of the soil are common abiotic factors to be concerned about. Additional support for this principle comes from the role of the rodent burrow system on plague (Yersinia pestis) hosts and vectors.
  6. The emergence and distribution of infection through time and space is controlled by different factors acting at multiple scales” In their discussion of this principle, they focus on human interaction with the environment and particularly urbanization altering disease risk. They note that climate change and natural environmental change do not account for all emerging and re-emerging disease but the activities of humans including urbanization and ecological change like deforestation. Ben-Ari et al‘s study on plague and climate change also looks at the many factors at all levels from micro to macro scales effect the abundance and likelihood of transmission of the plague.

    Plague cycle including hosts and vectors with abiotic influences
    Plague cycle including hosts and vectors with abiotic influences (Ben-Ari et al, 2011).
  7. Landscape and meteorological factors control not just the emergence but also the spatial concentration and spatial diffusion of infection risk” This principle just adjusts the previous principles to take account of primarily rainfall by looking at temporary ponds or wetlands. This particularly affects mosquito abundance, but as the graphic above demonstrates also effects soil moisture.
  8. Spatial variation in disease risk depends not only on land cover but also on land use, via the probability of contact between, on one hand, human hosts and, on the other hand, infectious vectors, animal hosts or their infected habitats” Land use has been long known to affect mosquito abundance and disease transmission. Clearing land for settlements or agriculture always increases standing water in ditches, tire ruts, railroad ditches, animal troughs, incomplete building projects, and due to loss of water absorbing vegetation. A century of malaria research and management has focused on land use and the elimination of standing water.  Mature water management programs for cultivation or flood control can also alter vector abundance and human contact rates. For example flooding fields to grow rice not only provides habitat for mosquito production but also brings people into the fields to cultivate increasing contact rates. Irrigation canals would have a similar effect.
  9. The relationship between land use and the probability of contact between vectors and animal hosts and human hosts is influenced by land ownership” In Lambin et al, they looked at the contact rates between public (state) land and private ownership. In these studies state ownership increased access to forestland over private ownership.By the same token, state ownership could also prevent deforestation and urbanization by preserving the wilderness or reserving the land for other uses. Forest age and maturity also varies significantly between state forests and private land.
  10. Human behaviour is a crucial controlling factor of vector-human contacts, and of infection.”  Humans bring themselves into contact with vectors by risky behavior and can control exposure vectors and infections. Obviously, vaccination is one of the controlling factors of infection, although many zoonotic infections have either no or poor vaccines. Occupational and recreational exposure to vectors often explains gender difference in infection rates.

In conclusion these principles begin to mark out the three sides of a zoonotic triangle: biology of pathogen, vector and host; ecological system where they exist; and human behavior and ecological interaction. Human behavior including land use and constructed environments is as important as the other two sides of the triangle. Humans are not passive victims or collateral damage.

Reference:

Lambin, E. F., Tran, A., Vanwambeke, S. O., Linard, C., & Soti, V. (2010). Pathogenic landscapes: Interactions between land, people, disease vectors, and their animal hosts. International Journal of Health Geographics, 9(1), 54. doi:10.1186/1476-072X-9-54 [open access]

Ben-Ari T, Neerinckx S, Gage KL, Kreppel K, Laudisoit A, et al. (2011) Plague and Climate: Scales Matter. PLoS Pathog 7(9): e1002160. doi:10.1371/journal.ppat.1002160

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:

ResearchBlogging.org

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.