Monday, October 31, 2011

Bring out your dead! (so their plague genome can be analyzed)

Many of us may have had our first introduction to medieval history thought this great Monty Python movie. And as is their nature, this bit from “The Holy Grail” is pretty accurate. The Black Death, the epidemic of plague that ripped through medieval England and Europe, killed thousands of people and led to creation of the middle class (people were finally in demand once 30-60% of them died). But I digress. The reason I’m writing about the Black Death is because a paper came out last week in the journal Nature describing a new genomic analysis of bacterial DNA samples collected from infected people buried in a cemetery outside of London. There was a great summary paper published in the same issue that described what the current paper found and some of the controversies that exist between scientists in this field. I highly recommend reading this, I think it is very widely accessible. Below I discuss some of the things I learned from the papers and why I think it’s interesting. (Note: unless you have access through a university you may not be able to see the primary source paper. Hopefully everyone can get to the summary paper, though)

First, a little background. Bubonic plague is caused by a flea-vectored bacteria, Yersinia pestis. Human cases of the plague are rare but still occur (are easily treatable with antibiotics) but plague epidemics are a big problem in some wildlife populations, such as prairie dogs in the American west. The Black Death describes the plague epidemic that occurred in Europe between 1347-1351. Since the sanitation conditions in a big city like London was probably more abysmal that we can imagine today, you can expect there to be fleas everywhere. So, the main questions people studying these old strains of the bacteria are trying to answer are, Why did the Black Death kill so many people?, and Why are cases today much less severe than symptoms described during this ancient period?

Two hypotheses that tried to explain these observed differences between the 14th century plague and cases today are that the Black Death wasn’t actually caused by Yersinia and that a more virulent strain of the bacteria was the culprit for the deaths during medieval times. The first of these hypotheses was rejected when Y. pestis bacteria was recovered from the teeth of two individuals known to have died of the plague during the 14th century. However, some researchers weren’t convinced of this conclusion because it was very difficult to repeat these methods, i.e. it is really had to get usable DNA from human remains that are 600 + years old and make sure the results aren’t contaminated. Subsequently, if it is hard to recover samples that are in good enough condition to be analyzed, it is almost impossible, with the tools available in the early 2000s, to construct a genome with a good amount of confidence, so scientists were not able to test that second hypothesis.

Enter this new paper and new fancy technology! Researchers from Canada, Germany, and the United States began collaborating to unearth new information from bodies buried in the East Smithfield cemetery (or “plague pit” as some called it) that was used during the Black Death. Samples that had been collected and stored over many years were re-analyzed using what is called next-generation pyroseqencing. This is a really amazing method that amplifies DNA from a sample thousands of times with extreme precision. This means that even if there is a very small amount of bacterial DNA in a tissue sample, it will be amplified to the point of detection. Older methods, like PCR (polymerase chain reaction), which is still the workhorse of a lot of microbiology, were not sensitive enough to extract detailed DNA sequence information from these medieval tissue samples. So, this new analysis was able to compile this new sequence data into a genome of Yersinia pestis and compare it to modern samples of this bacteria and see how they are related.

What they found was that the ancient and modern strains of the bacteria are very similar, showing not a whole lot of evolutionary changes between the 14th century and now. It also showed that the Black Death strain of Yersinia is probably the ancestor to modern strains, meaning that the Black Death strain of the bacteria is the source of most modern cases (see the paper for a picture of the phylogeny if you’re interested). This means that the “strain with increased virulence” hypothesis that was proposed is not supported by this data. What this suggested to researchers was that there was some kind of environmental interaction with the Yersinia infection that caused people to be much more susceptible or have more severe symptoms. This could be due to wars that were happing around this time, wearing down the men that came back from fighting. Also, there are other flea hosts besides the rats living in the city, which may change the probably transmission of the bacteria to humans if the fleas were infected from this alternate reservoir.

These findings are interesting for a lot of reasons, but some are very close to my research. First, I am planning on using the sequencing method used by the authors to analyze the bacterial communities in my ticks and rodent blood samples. Analyzing “meta-communities” is one of the main applications of next-generation sequencing because it gives you so much data on the composition, in terms of number and quantity, of microbial communities that wouldn’t be possible with previously used methods. Second, the finding that the ancient and modern strains of the bacteria are very closely related highlights how complex the pathogen-host interaction is. How a host will respond to an infection depends on a whole suite of characteristics, of both host and pathogen. Scaling up from the individual, how a population will respond to an epidemic will depend greatly on population composition, taking into account age structure, host quality or health, contact rates, and immuneocompetence. Some of these aspects may be very difficult to measure when your population lived over 600 years ago, but I bet the researchers in this field have methods they can use to figure them out. It is quite amazing what we are able to do now to investigate questions that have been unanswered for so long. And since this is a hot topic, I am sure there will be competing genomes and other analyses published in the near future.

I haven’t watched “Monty Pyton and the Holy Grail” for a long time, but I have the feeling I’ll be bringing this paper up next time. Sorry in advance to any family or friends that will have to bear with my extreme nerdyness!

Wednesday, October 5, 2011

A parasite more disgusting than ticks

** Warning: This post is going to maybe too gross for some people, so if you really are not too cool with parasites you may just want to skip this one (hehe).

The little white-footed mice that are the focal host of most of my research aren’t just hosts for ticks. They are the habitat for a multitude of macro- and microparasites. For instance, in our research we have collected three kinds of ectoparasites, ticks, fleas and mites. Many other researchers have studied intestinal worms, both acquired trophically (through eating infected food, like crickets), and by simple contact with worm eggs in the habitat. They can carry many vector-borne pathogens, bacteria and other microbes carried by ticks and fleas, such as Borrelia burgdorferi, the causative agent of Lyme disease, Bartonella, a flea-borne bacteria, and Babesia, a tick-borne protozoa similar to the causative agent of Malaria. And one of the more disgusting parasites that uses these mice as hosts is the bot fly.

You may have heard of bot flies in association with people coming back from the tropics with weird bumps, to then have a squishy larva pop out of them a few weeks later (see NBC story here). I had heard of them infecting primates and other tropical vertebrates, but didn’t know they were around in temperate areas as well. But, in our 2009 field season we were finding mice with what looked like tumors or extra testes (they were usually in their lower torso area). The size of the growth in relation to the mouse’s body size is pretty significant, like half the size of its torso in some cases (see below). We really had no idea what these were, so we took some pictures and sent them to John Whitaker, a mammal expert at Indiana State University. He identified the growths as bot flies right away. This year I saw a few more mice with similar infections, and one of the other researchers in my lab did too. And being nerdy scientists, we did some online searching to find out more about this gross parasite that is infecting our animals.

One of the mice we captured in 2009 with a botfly infection. Notice how big it is compared to the size of its body!

The most useful source we found was a Catts 1982 paper in the Annual Review of Entomology. It starts out with a great line:

“Maggots of cuterebrid bot flies are conspicuous, repulsive, and frequently encountered cutaneous parasites of mammals in the New World.”

I don’t know if I’ve seen the word “repulsive” in an academic paper before.* This went through a detailed overview of the life history of this parasite and what is known about different aspects of this larval and adult biology in the Americas. A drawing of a typical bot fly life-cycle is below, and is simply the adult fly lays eggs (1000-3000 per female, depending on species) usually close to host burrows, those eggs hatch into microscopic larvae when they sense and increase in temperature (a potential host), the larva get in to the host through eyes, nose, mouth or open cuts on the skin, they then migrate through the host’s body over a few days and then settle in the abdomen of the host to develop into the final larval stage. They create a “warble pore” through the host’s skin to breathe out of while developing. The larva then emerged out of the pore, develops into a pupae under leaf litter and can overwinter in this stage, and then emerges as an adult. Adults do not eat at all, they only mate and lay eggs.

Illustration of bot fly lifecycle from Catts 1982. The host is on the right side, and two alternate pathways are shown for infection (direct contact with larvae or females laying eggs on host, which is less common).

Yes, this is pretty gross and you may have visions of “Aliens” in your head right now. But surprisingly, it seems like hosts don’t suffer too many long-term effects of the infection. The Catts paper summarizes studies that found hosts have lower than usual protein levels during infection but seem to make up for the loss by eating more. The warble pore usually heals completely in a couple days with little infection, possibly due to compounds in the mucus the larva surrounds itself in.

Last week I did my last field sampling of the year and found a mouse with a really huge bot fly infection. I took a picture to show Angie, the other researcher who had found these infections while doing some mammal trapping. I said it would be really interesting if I caught the same guy again to see if the larva emerged, since it was so big already. 

Animal 595 with a big bot fly infection. The dark circle is the warble pore made by the developing larva. Hopefully you can see how huge this infection is at the time.
And, low and behold, I caught that same mouse the next day and the larvae had emerged. 
Same mouse, no bot fly. The pore is bloody from the larva emerging and his torso looks much more normal-shaped. But the mouse doesn't look too happy!
The little guy did not look like he was in good shape, go figure since a parasite the size of his torso just squirted out of him. When I released him after doing all the handling of the other animals, he just kind of sat on the ground, instead of running away quickly like they usually do. He was all hunched up and kind of wobbled as he walked away. I was even able to reach down and grab him off the ground, which I would never be able to do with a healthy animal. I am thinking he must have been in some significant pain after that lava emerged. I am hoping he is ok, after reading that the wounds heal pretty quickly. Poor little guy! But I guess since I do study parasites, I should be glad that I am studying a host that is so desirable for all these different things, and emphasizes that the host really is a community and that ecology matters at so many different levels.

* If you want to take a look at the paper, there is a lot of other somewhat non-academic language used. It’s kind of funny to see what you could get away with in older publications.