Wisconsin Public Television University Place Wednesday Nite at the Lab  David Blehert, "The Discovery of Bat White Nose Syndrome"  March 9, 2011 UP562     cc >> Tom Zinnen: Welcome, everybody, to Wednesday Nite at the Lab. I'm Tom Zinnen. I work here at the Wisconsin Biotechnology Center and for UW Extension Cooperative Extension and on behalf of our partners, Wisconsin Public Television and the Wisconsin Alumni Association, thanks for coming to Wednesday Nite at the Lab. We do this every Wednesday night, 50 times a year. Tonight, I'm delighted to introduce to you David Blehert. He earned a bachelor of science disagree in biology from the University of Minnesota in 1993 and doctorate here in bacteriology in 1999. He then completed a postdoc at the National Institutes of Health in Bethesda, Maryland, before joining the United States Geological Survey, National Wildlife Health Center here in Madison, Wisconsin. He is the head of Diagnostic Microbiology. He started there 2003. His current research projects include characterization of the emergence and pathogenesis of bat white nose syndrome, which will be the subject of his talk tonight. 

The use of molecular markers to understand the epidemiology of avian cholera among wild waterfowl and the identification of environmental factors that precipitate outbreaks of avian botulism. His lab's collaborative efforts to identify the fungus that causes the skin infection hallmark of white nose syndrome in bats were published in "Science Magazine" 2009. It doesn't get any better than being published in Science. He has since co-authored six additional peer reviewed papers relevant to white nose syndrome, mycology, pathology, epidemiology, and diagnostics. This particular splendid topic I think because it's always amazing when something new pops up and may change the look and feel of our landscape here in Wisconsin. It's great to have a representative of the National Wildlife Health Center Lab here. I think it's one of the great resources that Madison is home to. Please join me in welcoming David Blehert to Wednesday Nite at the Lab. (applause) >> David Blehert: Thank you, Tom, for the invitation to speak and for the nice introduction. Can people hear me okay? As Tom said, I come from the National Wildlife Health Center. 

We are a federal government facility under the United States Department of Interior. We are a wildlife diagnostic and research lab. We conduct wildlife diagnostics for the entire country. We function similar to the CDC for human disease, but for wildlife. Today I'm going to present a summary of the white nose syndrome wildlife disease investigation and research that's been conducted at our center for about the past two and a half to three years now in collaboration with numerous university, federal and state partners. So, I wanted to start, hopefully this works, with a short clip. I have always been very interested in the things I have done as a scientist, graduate student or postdoc. I don't know that other people necessarily shared that enthusiasm when I studied bacteria that degraded compounds in soil or bacteria that colonized human tooth surfaces, but there is something about this disease that seems to have captivated public attention like nothing I have ever worked on before. It was very interesting to come in to work one morning and have people say, did you see this on television last night? >> The hair I recovered from the truck? It's a bat hair. That's the look on my face when I figured it out. 

There was a white powdery fungus on the hair called Geomyces. Apparently, bats in New York and all over the northeast are dying from something called white-nose syndrome. >> So they must have tracked that into the cab, maybe that can help pinpoint where they're holding Connor. >> Exactly. I'm compiling a known list of where the fungus has been found. (sighs heavily) (laughter) >> That's what I thought. I still want that list. That was actually a pretty accurate representation. They didn't even call us. I think that tells us that we are at least doing part of our job in getting the word out to the public. 

Before I begin, I want to get this out of the way before I forget. There are a lot of people I need to thank for the work that I'll present to you. A lot of this was done by one of my graduate students, Jeff Lorch here with the UW-Madison Environmental Toxicology program. I have another USGS colleague Paul Cryan, out in Fort Collins, Colorado, who's a bat ecologist that's prepared most of the maps that I'll show you today. Our national wildlife health center, wildlife pathologists, especially Dr. Carol Meteyer have been instrumental in the discovery of this disease. Also, folks in my laboratory including one of my UW postdocs, Michelle Verant, as well as the lead technician in my laboratory, Brenda Berlowski-Zier. We have another person I'll talk about later, Melissa Behr, who used to be at the State Health Department of New York, who now we're fortunate to have at the Wisconsin Veterinary Diagnostic Lab, just on that end of campus, as well as numerous other people and funding from sources like Bat Conservation International, National Speleological Society, Indiana Fund, United States Fish and Wildlife Service and the United States Geological Survey. So before I get into the meat of this presentation, here's a few quick facts relevant to bats of the United States, or bats of the world, for that matter. Bats are the second most species diverse group of mammals on the planet only behind rodents, so of about 5,000 mammalian species, over 1100 of those species are bats. There's 45 bat species in the United States, most of which are insectivorous, or eat insects as their food source. 

About half of those bat species use hibernation as their obligate or required strategy to survive the winter when it gets cold, obviously, and when their insect prey disappears. The other half are considered migratory, and they move to warmer climates. The bats that are affected by white-nose syndrome to date are all obligate hibernators and they are specifically infected by this disease while they hibernate, which is highly unusual. As far as I know, this is the first disease that specifically targets hibernating animals. So to provide some brief background on the disease itself, white-nose syndrome is a disease that has caused mass mortalities of insectivorous hibernating bats at a growing number of hibernation sites in the northeastern and mid-Atlantic United States. It's moving westward and has been documented up into Canada and in Tennessee, with total mortality estimates in excess of 1,000,000 bats. The mortality event on the order of white-nose syndrome and caused by infectious disease is unprecedented, not only on the bat in the United States, those 45 species I mentioned, but among all of the 1100+ bat species of the world. To further make that point, a population modeling paper that came out in Science last summer estimated a 99% chance for regional elimination of little brown bats, once the most numerous species in the eastern United States, within the next 16 years if declines continue at today's rates. To look at the history of the disease, based on a photograph taken by a recreational caver in February 2006, let's see if I can get this pointing right here. You can see the white nose on the bat. 

The photograph was taken in February 2006, but wasn't made public or brought to our attention until 2008. But this photograph indicates Howes Cave in New York as the likely index or initial site for this disease. Interestingly, Howes Cave is connected to Howes Cavern, which is a commercial tourist cave, that entertains up to a quarter-million visitors per year. Although this still is just a hypothesis, and we don't have conclusive evidence, it's possible that this disease emerged at this location in New York as a result of an accidental human introduction because of the high-human traffic at this site. To date, extensive global and travel trade networks have effectively broken down all those natural barriers that used to prevent spread of pathogens around the world, such as oceans and mountain ranges, and global travel and trade are one of the largest drivers in the emergence and subsequent spread of infectious diseases among humans, plants and animals around the world. So the next year the disease-- or during the course of normal bat hibernation population surveys being conducted by the New York State Department of Environmental Conservation, the disease was in fact discovered by biologists. When they found these unusual clinical signs, in some cases, including dead bats on cave floors, in other cases, bats exhibiting unusual behaviors like instead of hibernating deep in caves, they were hanging within the zones of light penetration near cave entrances. Also, bats flying around during the daytime over the snowy winter landscape, when they're supposed to otherwise be hibernating. Large numbers of bats on the landscape, on the snow, which also manifested as increased submissions to the New York State Department of Health Rabies Laboratory, and of course these white noses. New York state realized something was unusual. 

Out of all of the caves they surveyed that spring in 2007, they found either dead bats or bats with these unusual signs in five sites within a 15-kilometer radius circle, now encompassing two counties. It was the next winter that we were contacted, as well as other laboratories, and became significantly involved in the disease investigation. By the end of the season, we and our group of investigators had identified white-nose syndrome, now on 33 sites, extending 210-kilometers from that original detection site in Albany County New York, now placing the disease into four states, New York, Massachusetts, Vermont and Connecticut. This takes us to the winter of 2008-2009. Things got much worse, as you can see from the distribution map. I guess I never said this initially, but counties where disease has been documented are shown in yellow. By now, by the end of winter 2008-2009, we document the disease out to a distance of 900-kilometers from the epicenter. At this point, there is no precedence for studying or responding to wildlife disease in somewhat cryptic hibernating mammals like bats, so really, white-nose syndrome represents a new wildlife management paradigm or even a new disease management paradigm. Last winter we further documented continued expansion, confirming detections in Canada, in Quebec and Ontario, as well as detecting disease into the state of Tennessee. I'll tell you about some of the additional techniques we brought on, but these sites through Tennessee, we are actually able to isolate fungus and DNA, but we hadn't at this point yet documented disease on bats. 

Yes? >> Do you know how it was spread? Was it spread by people, or did you realize that bats had spread that far? >> I think the answer was both. I have slides to address that later, but we do know that bats, even the non-migratory hibernating bats, they move distances of up to 200-miles between their summer feeding grounds and winter hibernation sites. They are very social, gregarious animals, so there are many opportunities for bat to bat spread, and we are still investigating some of these further detections of what I'll call nucleic acid only detection, that extend all the way out to Oklahoma. We have not yet seen disease manifest out that far. So the current situation, we have now confirmed clinical disease in bats all the way out to western Tennessee. We have new detections of disease in southern Indiana, as well as detections in North Carolina. To date we have confirmed clinical disease in these six bat species, the Northern Long-eared Bat-- I'll say that they belong to three different genera. There are three myota species here, the Northern Long-eared Bat, the Little Brown Bat, and the Indiana bat, as well as the Big Brown Bat, which belongs to the genus Eptesicus, then the tri colored bat, otherwise known as the Eastern Pipistrelle, and the genus Para Myotis. As I've mentioned, the one thing that all of these bats species have in common, is that they all obligately hibernate to survive the winters. So let's briefly look at the ranges of these bat species. 

Here again we have the counties where either white-nose syndrome or other signs of the pathogen have been detected by various techniques in yellow, overlaid with the red polygons that represent the species distributions, first of the eastern small footed bat. Then we add the northern bat on top. Add in the tri colored bat, or the eastern Pipistrelle. I recognize it's only half the country, but when we add in the little brown bat, we're talking almost the entire distribution all the way to the west coast, and similarly with the big brown bat. This nucleic acid only detection in the state of Oklahoma was in a different species called the cave bat. This is interesting because this species, you can see, only exists west of the great plains. In addition to the little brown bat and big brown bat, it could further serve as a bridge into some of these other twelve hibernating bat species that, again, only exist west of the great plains. Now, this last series of maps shows the distributions of some of the endangered bat species that would potentially be susceptible. In the case of the Indiana bat, I showed you a picture previously, this bat has been documented to be affected by white-nose syndrome. We also have the gray bat, one of the nucleic acid only detections in the state of Missouri was in skin samples that we received from an endangered gray bat. 

We didn't have enough material to do further follow up on those samples, so those don't have the full level of confidence of our disease identification. The problem with gray bats is that there are about 1.5 million of these animals left, and they all hibernate primarily in about eight caves during the winter, so if there is bat to bat transmission, that could make these hibernating populations very susceptible if they prove to be sensitive to disease and it gets into their populations. We also have the Virginia big-eared bat. You can see there's three purplish polygons in the known range of white-nose syndrome. Interestingly, we have documented white-nose syndrome in little brown bats in caves shared with Virginia big eared bats, and we have not yet seen clinical disease in a Virginia big eared bat. These bats prefer cooler and dryer areas to hibernate which may be less conducive to the development of this disease. We don't yet know the answer to that. It could be the case with it being such a small population, and it being protected, it's more difficult to get specimens. Specimens are harder to find in addition to protections, so it could be as a result of less opportunity to find them, but that's also something that we would like to further investigate with regard to species susceptibility among the hibernators. Last, we have the Ozark big-eared bat, approximately only 2,000 individuals left. 

We have not yet documented disease in this species. Now let me transition to a brief discussion of disease ecology. In order to understand the emergence and subsequent spread of a disease, we have to understand how susceptible hosts, in this case, bats, pathogens and environments come together. This is applicable across the board, be it a stomach virus outbreak among passengers on a cruise ship or to the way that a cold virus can spread among children at a daycare center or for that matter, a white-nose syndrome outbreak among hibernating bats in high densities and close proximity to each other within an environment such as a cave, which is conducive to the maintenance of this pathogen. So hopefully, I have now convinced you that the hosts for white-nose syndrome are bats. As we just reviewed, clinical white-nose syndrome infections have been documented in six different species, three different genera, all obligate hibernators. Now I would like to move on to this environmental component of this disease. If you remember from the series of maps that I have shown you, that pattern of spread, what's special about that? The red dots on this map show the locations of caves in the eastern United States. These caves are what serve as prime bat hibernation sites. If we overlay the two, the positive counties for disease with the red cave locations, you can see in the Midwest, these concentrations of caves, as well as through Tennessee and Kentucky, and up the Appalachian Ridge and into New York in this area. 

So this definitely implicates caves as an environment conducive to the development of white-nose syndrome. Caves provide very stable and uniformly cool temperatures with near saturating humidity conditions that our data to date indicates are likely to be ideal for promoting growth and maintenance of the white-nose syndrome pathogen. Now to complete the discussion of this white-nose syndrome disease triad, I'll transition to pathogen aspects of the disease. So at the National Wildlife Health Center, I'm a member of the diagnostic branch, or the disease investigation branch which consists of laboratories-- Let me back up. Basically, what we do is we receive animals that are found dead in the field or parts of those animals and they go through our diagnostic process. A diagnostic pathologist is assigned to the case. They examine the specimens, and they submit tissues to our various laboratories. So this particular investigation got under way after we at the National Wildlife Health Center were contacted by a state biologist in the state of New York, Allen Hicks, in 2008. So bats were submitted to our center. What we do, as I mentioned, is receive the sick animals from biologists in the field, and look for some common thread among them such as a pathogenic microorganism that's present among sick animals, but absent from healthy animals. 

So specimens arrive in our lab. They go through our suite of diagnostic laboratories, and with regard to chemical analysis, no toxins were found, and a group from New York state has recently published similar results. No disease causing parasites were found. Of course, we did find parasites, but nothing in unusual burdens or burdens that would be unusual for a wildlife species. We employed both classical virology techniques to isolate viral pathogens and didn't find any known pathogens. We also collaborated with one of the virus hunter labs that uses all molecular techniques to sequence all DNA present in sample and look for potential pathogens, and nothing was found. Additionally, there were no consistent findings on bacteriology in my laboratory. When it came to our fungal work, however, the situation was more complicated. So we are getting pictures of bats with white noses. We are all suspecting it's a fungus, so of course, we're trying to collect cultures from the bat wings where you can see the white fungal material on the wings, on the ears, on the muzzles. 

What was interesting is that this material was very fragile. By the time the bats are packaged in a bio secure manner and shipped to our center for investigation, the white stuff was gone, and so how do you culture something, or how do you target samples when you can't see it? In the meantime, fungi are all over in the air. If I take a Q-tip and swab my skin, I'm going to grow fungi, unfortunately, probably from everybody in the audience as well. So we were growing fungi. We were growing hundreds of different fungi, and how do you make any sense of that? So the pathologist I mentioned in the initial slide, Melissa Behr, who now works at the Wisconsin Vet Lab, she was in Albany, New York, at the time, which was literally ten miles from some of these infected mines. She made that same observation. Even if bats were collected from a mine and driven ten miles to her lab, the white stuff was gone, so she made an arrangement to go into one of these mines and collect samples for microscopy analysis from bats right there in the mines when she could see the white stuff. She then took these two pictures and shared them widely, so she presented these pictures of what looks like a pure growth of fungus with a very unusual spore morphology. You can see this hook shape. In the meantime, based on discussions that we had with numerous people, when you conduct classical microbiology analyses, you can only grow what your laboratory conditions are permissive to grow. 

When bats hibernate, they actually drop their body temperatures to about the equivalent of the temperature inside your refrigerator, which is about 7-degrees centigrade, maybe a little warmer than your fridge. We recognize that and initially, rather than our typical work on warm-blooded animals is done at 98.6 Fahrenheit or 37 centigrade, we initially made the decision to cultivate the bat tissues at room temperature, a compromise between getting things to grow at an appreciable rate but not expecting something growing on a hibernating cold animal to grow at 98.6. From those analyses, we were growing, I'll call it a fungal weed after fungal weed, likely just representative of the many, many fungi present in these cool dark caves where bats live. So at some point we also made the decision to take some of these cultures and incubate them in the laboratory refrigerator, and very, very slowly, they were growing. It takes months to grow these cultures. So about the time that Melissa shared these photographs, we had some cultures coming up in our refrigerator. So when we saw these photographs, sometimes in today's world when we rely more heavily on molecular biology, I remembered that the prefix to microbiology is the same as that for microscope, so we looked at our cultures under the microscope and lo and behold, they were the same thing, so now we finally knew that we had found something. So what is this fungus? It's known as a cycrophile. It requires cold for growth. It can't grow at room temperature. 

It's common on sick bats, absent from healthy bats. All of the fungal isolates within the genetic markers that we have looked at have been identical which can suggest spread of a pathogen from a single point source introduction, based on early ribosomal RNA sequencing, we determined that the fungus was a member of an actually relatively common group of soil fungi, called Geomyces. Based on its growth properties and its unique morphology, and what it does to bats, we named it Geomyces destructans. So now it leads to the question of, where might this fungus have come from? As word got out about this around the world through the popular press and through scientific contacts, it came to our attention that people in Europe had reported similar manifestations of white fungus on the muzzles of bats with historical records dating back several decades, but there were never any reports of unusual mortality associated with this and the fungus had never been identified. So three groups, one which we collaborated with, plus two others have now identified genetically identical Geomyces destructans on bats in all of these countries shown here. The work we were involved in is shown by the solid symbols. The Czech Republic and Slovakia symbols, I've just summarized, but in the Czech Republic and Slovakia, they've actually identified the fungus in upwards of 50 different sites. So this fungus appears to be quite common in Europe, yet their bats aren't dying. There is no bat species common between Europe and North America. Bat population demographics are also quite different in Europe compared to North America, whereas we in the east coast and other places in the country -- in the east coast we used to and in other places we still do, have very large hibernating aggregations of bats, thousands to hundreds of thousands in single caves or mines. 

In Europe, there's maybe only one or two large aggregations on average. Their bat hibernating populations are like 30-100 bats. It's possible their bat species are resistant. It's possible that they previously underwent an unreported decline, who knows a hundred years ago, a thousand years ago, even longer, and that they effectively co-exist with this fungus which may be behaving as an introduced invasive exotic species in North America. Yes? >> In Asia, do you have any data from there? >> We don't have any data yet. I would be interested to know, in Russia, for example, they have large populations with myota species, or in the temperate regions of Asia, but we don't know that yet. >> Are there bats in tropical climates? >> Yes, I believe even more species, but those would tend to be the non-hibernating species. It's hard to transition from when we say we believe to we know. We have good data to indicate that a bat that doesn't drop its body temperature below 14-degrees centigrade or below 50 some degrees Fahrenheit would actually be resistant to this disease. >> That's good because there are a lot of cave areas in the Caribbean. 

>> Right. So bats worldwide, and in North America and temperate regions, they play a very important role in insect control, but in tropical areas of the world, they also play important roles in seed dispersal and in pollination. In the United States, bats are, I believe, the sole pollinator of the agave plant, which is used to make tequila, so we don't want to lose that. But thankfully, that is a non-hibernating bat. Presumably, that won't be affected. In terms of what we are doing in the laboratory now, we have three techniques for diagnosing this disease. We have fungal culture so we can incubate tissues from bats in the cold and grow the fungus. We have histopathology in which we can actually see the interactions of the fungus with the tissues of the bats. This is what we use to actually define the clinical state of the disease. I'll touch on that later. 

We also have a molecular technique, PCR, where we can more rapidly screen for the presence of DNA from the fungus on bat tissues. In terms of the overall summary of the diagnostic work we've been doing since 2007, here I show in red the states where clinical disease has been documented. The numbers in each state shows the numbers of bats that we received from that state. Our sample is opportunistic, so we don't have an active surveillance program where we're saying give us 100 bats from every state. Rather, biologists are doing surveys on the sites and if they find anything unusual, they submit bats and we perform diagnostics. Although we don't have perfect representation, we have decent representation across the United States. The asterisks show states from which we have only received bats in the summertime, and we do have information that the bats, or that the fungus effectively goes away if the bats are able to survive hibernation, so those aren't necessarily the best samples for ruling out disease in states like Washington, Oregon, Idaho, for example. But we're also seeing this temporal or wave like pattern spread, so we don't have any real reason to believe that the disease is that far west. Just briefly in terms of the genetic work that we've done, in microorganisms and higher organisms, one of the markers used to determine species is a conserved region known as the ribosomal RNA, part of what makes our protein synthesis machinery or protein synthesis machinery of a fungus or a bat or bacterium. In the case of Geomyces destructans, this insertion sequence is present at the end of the small ribosomal RNA subunit, so initially using amplification primers that could bind about here at this arrow and here at this arrow, we can amplify and sequence through this entire region, which is about 800-900 nucleotides long and demonstrate genetic identity among the isolates. 

We also did complete analyses of this ribosomal RNA small subunit. Now we have developed specific primers that target both this insertion sequence or enteron as well as the junction between the 5.8-S ribosomal RNA subunit and what's called the internal transcribed spacer region two. And I won't get too much more into the details. That's the basis for our PCR. In terms of histopathology, if I run through this slide quickly, the dark purple, I think that is, that I'm pointing at, this is fungus, using the particular stain that we use called PAS. Fungus stains dark purple. This material here represents the cross section of a bat wing. So these are pigmented cells on the epidermis at the top of the wing. Here we have pigmented cells on the bottom of the wing. The wing is a very delicate skin tissue, essentially two cell layers thick with a thin layer of connective tissue here in the middle. 

Here you can see, first of all, the fungus with the characteristic curved spores colonizing the surface of the bat wing, but not only colonizing the surface, forming this large aggregation of filaments and then actually eroding through the epidermis and invading into the connective tissue, so this isn't a superficial skin infection like athlete's foot. Athlete's foot fungus is called a dermatophyte. By definition, dermatophytes colonize the dead skin layer on the surface of our skin, around the surface of an animal's skin. Geomyces destructans and white-nose syndrome is actually invasive and actively invades and destroys living tissue. Here in this slide is a lower magnification image of cross section of bat wing that I'm tracing through. You can see these very proliferative and invasive huge pockets of fungal infection literally destroying the bat's wings. This is a section of muzzle, a piece from a bat's face, and this here represents either a sweat or herbaceous gland where the fungus has entered and breeched through the basement membrane and you can see the purple throughout the tissue, so it's invaded the entire muzzle here. This structure here represents a cross section of a hair shaft, and the fungus has invaded around that hair shaft and again, breeched the basement membrane and is invading the regional tissues. As I have said, this is not by any means a superficial infection. It's very invasive. 

Bat wings, in addition to being critical for flight, and if a bat can't fly, it can't feed itself, are also critical for heat dissipation during flight. During hibernation, bats aren't drinking. Bats aren't eating. Their wing skin surface actually represents eight times more surface area than all of the skin on the rest of their body, so destruction of the membrane of the wings is basically destroying the water control integrity function of the wing skin. One of our hypothesis is that bats are actually dehydrating because they are losing water through their wing membrane. There is also documented passive gas exchange, co2 removal through the dense capillary networks in their wings. Yes? >> There are many kinds of fungi, so has only one kind been detected? >> I won't say that we never detect other fungi, but this pattern of invasion is always associated with Geomyces destructans. We see this pattern on histopathology, and then we back this result by growing the fungus out of the bat wing, and/or demonstrating that nucleic acid from this fungus is present. >> So there hasn't been a discovery of another kind of fungus that is responsible? >> Correct. >> Another question, are there any bats that are immune, somehow? >> Right. 

That's something we are actively investigating. There is the Virginia big-eared bat, which is an endangered species, that we have not yet documented to be infected, even though it's inhabiting caves with large numbers of infected little brown bats, for example. We don't know if that's an immunology based resistance, or perhaps it's behavioral, that those bats are inhabiting regions of the caves not conducive to proliferation of the fungus. Then there's also the European bat species in which we have recently documented the fungus and there are anecdotal records suggesting that the fungus has been there for decades but has never been documented to decline or die as a result of it, so again, we don't know if it's the result of immunological resistance or if it's behavioral or situational based on different population densities, different population interactions. Hmm hmm? >> Do you think there is a possibility that a human factor might be involved? I was thinking maybe somebody that went caving in Europe came over here to do some spelunking? >> That is one of our hypotheses as to how it got here. We are doing a large scale sequencing project of isolates from Europe and North America to see if we can infer that type of relationship based on rates of genetic change. I would certainly say for any microorganism, humans and the things we carry around can certainly serve to move microbes from one environment to another. That's why hand sanitation is so critical among physicians as they move between patients in the hospital. So certainly, and I'll talk about our work in environmental detection, but to cut to the chase, yes. I think if you walk into a cave that has this on the cave floor and then immediately walk into another cave with those same shoes on, the chance of an inadvertent introduction is certainly there. 

Back to the wing skin, to make an analogy, I don't know if people are familiar, there's a fungal disease of amphibians called Chytridiomycosis which has been implicated in global amphibian declines. It's probably the closest analogy we have to white-nose syndrome in bats. Interestingly, Chytridiomycosis in frogs, salamanders, toads, is very superficial compared to this fungal infection, but yet, just as deadly. There was a paper published in Science, I think about a year ago now, in October 2009, I think, so two years ago, where they showed-- I told you about the functions of bat wing skin, reasons bat wing skin is critical other than for flight. Frogs, which live in water, also rely on their skin for more than connective tissue purposes. It's also very important in regulating water balance and electrolyte balance, so in this recent publication, they showed that when frogs become infected with the Chytrid fungus, it causes their electrolytes to go out of balance, and they ultimately die of cardiac arrest. So a frog gets a superficial skin infection, and it has a heart attack, so we are still working to better understand this mechanism in bats, but here we are not talking a superficial infection. We are talking about something an organ, its skin, on a region of its body, its wings, especially that makes up eight times more surface area than the whole rest of its body. So one of the things we have been working on is a transmission study to conclusively demonstrate that Geomyces destructans is the white-nose syndrome pathogen. This falls under the guise of fulfilling Koch's postulates for infectious disease, whereby you demonstrate that infected animals are colonized by a particular pathogen and unaffected animals don't, and we have seen that. 

You then have to isolate that pathogen in pure culture, put it on a healthy animal, demonstrate that that healthy animal develops clinical disease and then re-isolate the same organism from the experimentally infected animal. So to conduct this study, we used little brown bats collected here in Wisconsin that were demonstrated to be uninfected. We also brought in some positive control infected bats from New York state. Our facility has a bio secure/biosafety level three animal isolation facility, so this work was done using hibernating little brown bats that we housed in refrigerators, and we set up three different groups. In one, we co-housed our infected New York bats with healthy Wisconsin bats to see if the pathogen could transmit bat to bat, so we had two cages like that on either side. Then we had another cage with healthy Wisconsin bats in the middle to determine if the disease could transfer by aerosol, as I'm showing here. We had an inoculated group where we had healthy Wisconsin bats in the outer cages, infected by putting a small drop of fungus suspension on their wing skin with healthy bats in a central cage to see if the agent would transmit by aerosol. Then we had our control animals that just had a sham inoculum. The results of that study, first and importantly, none of our Wisconsin bats had any signs of infection. All of the positive control New York bats, 100% that we brought in had clinical white-nose syndrome as determined by histopathology culture and PCR. 

And then as determined by histopathology, both the Wisconsin bats that were co-housed with New York bats, 95% of the Wisconsin bats co-housed with the New York bats as well as 95% of the bats that we inoculated with fungus, developed clinical white-nose syndrome. Interestingly, none of the bats in the aerosol exposure cage developed white-nose syndrome, so it suggests that bat to bat contact or direct application as we did, is the most likely means by which the disease is transmitted. In terms of histopathology, I've already shown you this image, but essentially, the lesions that occurred in the experimentally infected animals or the bat to bat contact animals were identical to those we see in wild bats. Then, because the environment is likely a critical component in white-nose syndrome, we worked with a large network of volunteers to collect soil samples from numerous sites from states bordering on and east of the Mississippi River with the goal of determining the distribution of this fungus. The states shown on this map in yellow show the states where white-nose syndrome was known to exist at the time that the samples were collected. In blue, I have some of the states shown, ones that were analyzed for this portion of the study, where white-nose syndrome wasn't yet diagnosed, but where we screened samples. From this initial analysis, we did find the fungus in soil both viable and its DNA signature at three sites in the northeast. We didn't find it at any of these other sites, interestingly. One of the things that has confounded this analysis is-- maybe it shouldn't have been to our surprise, but we were using our PCR technique that we had validated or qualified on bat wing skin. The normal environment for Geomyces fungi is in the soil. 

So perhaps what we should have anticipated is that the diversity of these fungi would be greater in soil than in the background that we had previously tested our molecular technique for detecting the fungus, bat wing skin. So when we first started on this project, effectively every sample we screened seemed positive. As we went deeper into the samples and did DNA sequencing analyses, by only analyzing 24 samples, we believe we've discovered possibly up to 12 new species of this fungus that were previously unknown, that have presumably always coexisted with bats in their hibernation sites and that are not pathogenic. So in terms of another way forward and better understanding this pathogen is to learn more about these Geomyces soil fungi. We have now gone back and done the culture analyses so we have representatives of these -- in the laboratory and we can begin to look at how the non-pathogenic soil Geomyces differ from the Geomyces destructans, which is the bat pathogen. Now I have a series of slides where I'll sum up. A lot of what is on the slides was developed by a colleague of mine, Dr. Tom Diliberto, who works for the United States Department of Agriculture Wildlife Services. So here is some of the thoughts from these slides. First of all, white-nose syndrome is not an ordinary wildlife disease. Few wildlife diseases have affected as many species over such a large geographic region in such a short period of time with this much impact on populations. 

If we compare it to other wildlife diseases like West Nile virus, rabies, H5N1, high path influenza and take these criteria in mind, unfortunately, white-nose syndrome seems to have it all when it comes to multiple species affected, high morbidity, high mortality, seemingly near optimal transmission and rapid spread, affecting animals over a large geographic area with the potential for long term population impacts, and even though bats are little tiny animals that people might liken to flying rodents, they're actually more like top level predators like polar bears or moose. They live a very long time, 12 to 25 years for an animal that can weigh six grams and its body is literally that big. They only have, in terms of little brown bats, for example, one offspring per year, so the potential for population recovery is slow at best. Some of the confounding factors that have likely contributed to the severity of white-nose syndrome among hibernating bats is the fact that they form high density clusters of  highly susceptible individuals during hibernation. There appears to be a high infection rate, high mortality rate. There likely is environmental transmission. We have found fungus in the environment, which suggest environmental persistence. During hibernation, bats don't just go into a cave, hibernate, and wake up in the spring, or arouse, technically, when they're hibernating, they're not sleeping, but they go into bouts for nine to 20 days, and then they have inter-arousals for a couple hours, restore their metabolic functions, warm up their bodies for a couple hours. They might move around within their clusters, among different clusters, and then they go back into hibernation. There is movement in bat hibernation caves. 

Even among the hibernating non-migratory species, they do effectively migrate between where they hibernate in the winter, and where they have their young or otherwise hang out in the summer, especially the females. Then, as I mentioned, low natural reproductive rates. I also previously mentioned this comparison to Chytridiomycosis, an amphibian fungal disease. Both are cutaneous diseases. Chytridiomycosis has caused and continues to cause global declines and extinctions of amphibians. Both diseases likely involve complex combinations of biotic and abiotic factors including an environmental component. Even if you could treat an individual animal, for example, if it goes back into a hibernation site that's contaminated, it will likely be reinfected. These caves are not like a poultry barn where you can remove the sick animals, decontaminate the barn and then have it be repopulated. These caves are very complex ecosystems with very little influence from the outside. Going into a cave and spraying a fungicide could have very adverse implications. 

Therefore effective prevention and management options for white-nose syndrome or for Chytridiomycosis among free ranging amphibians, which we have known for longer are either unavailable, impractical or have been ineffective. White-nose syndrome likely will have ecological and economic implications. Bats are primary predators of insects and population declines could influence ecosystem functions in ways that we can't predict yet such as forest health. Bats do consume a lot of moths, which can be forest pests. Bats are also keystone species in cave environments and the guano that bats drop in caves provide nutrients for the other organisms that obligately inhabit those caves. Although there is not likely to be direct human health implications, because I think that we're too warm to be infected by this fungus, there could be indirect implications such as perhaps increased vector-borne disease to the degree that bats control mosquito populations or other problems that result as ecosystem integrity degenerates. Bat population declines likely will have economic implications for agriculture. In one study done in Texas with Brazilian fruit-tailed bats and cotton farming, just over an eight county region, bats were documented to provide about a million dollars in pest control services a year to these farmers. It's also been calculated that, these are rough numbers, of course, but if a million bats have died of this disease, that over the course of a spring, summer and fall, that a million bats consume about 700-tons of insects. That's a lot of insects. 

The disease has been primarily in the northeastern United States where agriculture is not necessarily a major part of the economy, but as it moves into the Midwest, that could become a concern with regard to those ecosystem services provided by bats that will be lost. In terms of future directions and what we are working on now, we worked with the Broad Institute out at MIT and Harvard to sequence the Geomyces destructans genome. We are now mining that data and using that to do resequencing studies to look at rates of genetic change of the fungus as it moves across the United States and in isolates from Europe to infer origins. Our studies into the mechanisms of white-nose syndrome pathogenesis are ongoing. We are also conducting detailed comparative studies of cave associated Geomyces species with Geomyces destructans and working to sequence additional genetic markers so we have techniques to better go into environments and rapidly screen samples for the fungus and then also working on environmental persistent studies so we can better understand why disease manifests in one location versus another. If we can understand that, perhaps we can look at how we can manipulate environments in non-destructive ways to maybe tilt things slightly out of favor for the fungus and maybe a little more in favor of the bats without completely perturbing the cave ecosystem. To end with some final thoughts, managing disease in free ranging wildlife is very different than managing disease in domestic animals or in humans, and requires different strategies. In particular, disease management in epidemiology have not traditionally been part of the core curriculum for becoming a wildlife ecologist or a speleologist or cave explorer, so therefore there is a learning curve among our relevant stakeholders in this problem regarding infection control measures that are routine in human or veterinary medicine, but that are not routine among wildlife biologists or cavers. One thing we can control, or prevent, is that we ourselves don't inadvertently move this fungus site to site. I think that we have a responsibility to do that. 

The environmental reservoir for Geomyces destructans, as well as sensitive cave ecology both present unique challenges toward managing this disease and I think, in terms of developing solutions or management options, it's going to come from thinking outside the box to identify practical solutions that we can actually apply over large populations with reasonable budgets that are available to us and wildlife management that first and foremost do no harm to the ecosystem. To expand on that point briefly, the state of Wisconsin has been very progressive through the Department of Natural Resources Endangered Resources Division, and they recently approved-- it was initially an emergency order and has now been made permanent, but even ahead of detecting the disease in Wisconsin, the four cave bat species that exist in Wisconsin that are known to be susceptible to this disease have been listed at the state level as endangered, which affords them protections. Also, Wisconsin is unique as a state in having a very expansive, exotic species law, and Geomyces destructans the fungus implicated in the disease has been designated as an exotic invasive species, so both the endangered status for the bat and invasive status for the fungus provide the wildlife resource managers with authorities to enact management regulations with regard to gear decontamination, cave entrance restrictions and things of that nature. With that, I'll pause. I'm happy to take questions. (applause)