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 )