7th

Annual White-Nose Syndrome Workshop

Oral Presentation and Poster Abstracts

September 8-12, 2014

St. Louis, Missouri

Abstracts and the agenda are available online at:

https://www.whitenosesyndrome.org/wns-symposia-workshops

Oral Presentation Abstracts (Alphabetical by first author)

In vivo evaluation of fungistatic volatile compounds from native soil bacteria Rhodococcus

rhodochrous DAP96253 for inhibition of Psuedogymnoascus destructans in bats. Sybill K. Amelon

1, Christopher T. Cornelison

2, Sarah Hooper

3, and Dan Lindner

1

1USDA Forest Service,

2Georgia State University,

3Department of Pathology Microbiology and

Immunology University of Missouri

Initial ex vivo anti-infectivity assays using bat wing explants were conducted at Georgia State University

in shared airspace with induced Rhodococcus rhodochrous DAP96253 (RRDAP) at 7°C. In all samples

(n=9) induced RRDAP completely inhibited fungal colonization of bat wing explants as compared to

controls incubated in the absence of induced RRDAP. Our objectives in this study were to assess the

short-term impact of RRDAP to non-diseased little brown bats (Myotis lucifugus) in in vivo exposure

trials, and conduct in vivo clinical treatment trials with White-nose syndrome (WNS) infected little brown

bats to determine treatment effects on disease progression and bat survival. We hypothesized RRDAP

VOCs would have no short term negative effect on condition, activity or mortality rate in otherwise

normal hibernating little brown bats, would reduce or eliminate the viability of Pseudogymnoascus

destructans (Pd) spores, and would reduce mortality and levels of arousal activity of WNS infected bats.

In January 2014, exposure trials were conducted on “healthy” little brown bats in vivo in hibernation

chambers according to IACUC Protocol at the University of Missouri, Columbia, MO. Outcomes for

exposed bats indicated no short-term adverse effects on bats tested. Additionally, results of treatment

trials will be reviewed.

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* indicates presenting author

Summer surveillance for Pseudogymnoascus destructans at contaminated hibernacula - Part 2 Anne Ballmann

1, Miranda Torkelson

2, Elizabeth A. Bohuski

1, David Blehert

1, Carol Meteyer

3, and Robin

Russell1

1 U.S. Geological Survey-National Wildlife Health Center,

2 University of Wisconsin-Madison,

3 U.S.

Geological Survey National Center

Last year we reported preliminary findings from a cross-sectional survey conducted in late summer 2012

to detect Pseudogymnoascus destructans (Pd) on bat wings and from research equipment and caving gear

used at 8 hibernacula within the WNS endemic area of the Ohio River Valley. Pd was detected from a

small number (4/617) of wing swabs of bats captured from inside 3 of 8 study sites, including 1 of 2

negative control sites that remained visibly free of signs of WNS as of the end of Spring 2012. Although

the overall apparent prevalence of Pd exposure among bats using these hibernacula during late summer

was low (0.5%), viable fungus was isolated from at least one bat in August. Additionally, Pd was

detected on trapping equipment and items carried into contaminated hibernacula at another two study

sites. This year we report on the results of real-time PCR (IGS region) performed on opportunistically

collected feces from individual bats (223) and compare Pd prevalence of bats and the levels of

environmental Pd contamination among study sites with stable post-WNS winter bat populations versus

those sites with reduced post-WNS winter bat populations. Feces yielded a higher apparent prevalence of

Pd exposure (18%) among bats sampled during the summer within the WNS endemic region than wing

swabs alone. For environmental sampling paired by location inside the hibernaculum, cave sediments

demonstrated greater detection probabilities and higher concentrations of Pd than wall/ceiling swabs

taken of the equivalent surface area. Low recapture rates of marked bats at all locations on subsequent

trap nights suggested a high degree of movement among bats using these sites in late summer and thus,

summer-exposed bats may be contributing to pathogen dissemination on the landscape more than

previously thought.

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Surveillance for the presence of Pseudogymnoascus destructans on captured bats in the

Southeastern United States Riley Bernard

1*, Jeffrey Foster

2, Emma Willcox

1, and Gary McCracken

1

1University of Tennessee,

2University of New Hampshire

Pseudogymnoascus destructans (Pd), the causal agent of White-nose Syndrome (WNS), is responsible for

the mortality of hibernating bats across eastern North America. To determine the effect of WNS on active

bats in the Southeastern United States, we collected fungal swabs from bats captured outside of several

large hibernacula and on the landscape in Tennessee. Samples were collected from 960 captured bats,

representing 11 species, during winters 2011−12, 2012−13, and 2013−14 and summer 2013.

Approximately 42% of the bats captured were found positive for Pd, with fungal load and prevalence

varying among species, between sites and across seasons. Fungal swabs samples taken from two

Rafinesque’s big-eared bats (Corynorhinus rafinesquii), two eastern red bats (Lasiurus borealis) and one

silver-hair bat (Lasionycteris noctivagans) were found Pd positive. This is the first confirmation of P.

destructans fungus on Rafinesque’s big-eared bats and eastern red bats and second record of the WNS

fungus on a silver-haired bat.

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* indicates presenting author

Hibernating ecology of gray bats (Myotis grisescens) at white-nose syndrome infected hibernacula Eric Britzke

1*, Michael Whitby

2, Rick Toomey

3, Steve Thomas

4, and Jessica Hawkins

5

1US Army Engineer Research and Development Center, Vicksburg, MS;

2University of Nebraska-Lincoln,

Lincoln, NE, 3NPS Mammoth Cave National Park, Mammoth Cave, KY,

4NPS, Cumberland Piedmont

Network, Mammoth Cave, KY, 5U.S. Forest Service Ozark-St. Francis National Forest

The status of the federally endangered gray bat (Myotis grisescens) was being re-evaluated prior to the

emergence of White Nose Syndrome (WNS), but this effort was placed on hold until the impacts of WNS

on gray bats was known. Initial work with tissue explants showed comparable growth rates of the fungus

that causes WNS on gray bat tissue to growth rates on explants of severely impacted species; suggesting

that gray bats are susceptible to this disease. However, although WNS has been documented in the range

of the gray bat for over 3 years, there have been no documented declines in the species. One possible

explanation for this lack of impacts is that gray bats naturally arouse more commonly, thereby minimizing

the impact of WNS. Thus, we investigated the hibernation ecology of gray bats to examine if the

hibernation ecology of this species may be responsible for the lack of population declines. In the winter

of 2013-2014 we attached 15 transmitters to the back of gray bats during hibernation at 3 sites (Long and

Coach Caves, Kentucky and Blanchard Springs Caverns, Arkansas). We used data logging receivers in

each cave to monitoring body temperature of the gray bats for the life of the transmitter. Body

temperatures during torpor ranged from ~8-10° C. Initial analyses show that gray bats do not arouse more

frequently than other vespertilionid bats. Additionally, comparisons will be made between this effort and

data collected during the winter of 2012-2013 at Long Cave and Blanchard Springs Caverns. This data

suggest something other than the hibernation ecology of this species is responsible for the lack of

observed population declines due to WNS.

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Impacts of probiotic treatment by the bacteria, Pseudomonas fluorescens, on Pseudogymnoascus

destructans infection in vivo Tina L. Cheng

1, Joseph Hoyt

1, Kate Langwig

1, Craig Willis

2, Winifred F. Frick*

1, and A. Marm

Kilpatrick1.

1University of California, Santa Cruz,

2University of Winnipeg, Canada

We conducted an in vivo laboratory experiment to test the efficacy of probiotic treatment by a naturally-

occurring skin bacteria in bats, Pseudomonas fluorescens (Ps. fluorescens), on infection by

Pseudogymnoascus destructans (Pd) in Myotis lucifugus. This work follows previous in vitro challenge

experiments demonstrating promising antifungal properties by Ps. fluorescens against Pd growth. Our

experiment asked two questions: (1) What are the impacts of Ps. fluorescens treatment on fungal infection

and disease severity in M. lucifugus? (2) Can treating bats with Ps. fluorescens prior to Pd infection

provide protection against a later-acquired Pd infection? To investigate these questions, we conducted

two probiotic treatment groups – one group simultaneously treated with probiotic and inoculated with Pd,

the second group treated with probiotic 3 weeks prior to Pd inoculation. In addition, we conducted three

control groups – probiotic control, Pd control, sham control. We found relative increases in survival for

probiotic-treated groups compared to our sham control group. We also found evidence for decreased

fungal infection and severity in probiotic-treated groups. Our results suggest that probiotic treatment can

reduce incidence of White-nose Syndrome in M. lucifugus although timing of treatment is an important

factor.

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* indicates presenting author

Evaluation of induced Rhodococcus rhodochrousstrain DAP96253 as a potential biological control

agent of Pseudogymnoascus destructans Christopher T. Cornelison

1*, Sybill K. Amelon

2, Kyle T. Gabriel

1, and Sidney A. Crow Jr

1

1Georgia State University – Applied and Environmental Microbiology,

2U.S. Forest Service – Northern

Research branch

The in vitro activity of induced Rhodococcus rhodochrous strain DAP96253 against Pseudogymnoascus

destructans has been well established. In an effort to effectively forecast the in situ efficacy of this

potential biocontrol at reducing the mortality of bats with WNS, induced R. rhodochrous was investigated

for potential toxicological effects on healthy bats, as well as its ability to influence disease outcome in

naturally infected bats in vitro. Congruently, a diverse array of North American, hibernacula-associated,

fungi were assayed for their potential susceptibility to induced R. rhodochrous as a measure of the

potential collateral impact on the mycoflora of hibernacula treated with induced R. rhodochrous. In all

cases favorable experimental outcomes were observed and further analysis established a statistical

significance to the survival of naturally-infected bats exposed to induced R. rhodochrous. Cumulatively,

these results represent a significant step towards in situ management of this disease and warrant large-

scale in situ trials.

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Observed WNS resiliency in little brown bats at Fort Drum Military Installation, NY? Christopher Dobony

*

1Department of Army, Fort Drum Military Installation, New York

We monitored a maternity colony of little brown bats (Myotis lucifugus) on Fort Drum Military

Installation in northern New York between 2006 and 2014. Declines in colony numbers presumed to be

caused by white-nose syndrome were initially discovered in the spring of 2009. Although numbers

declined drastically, the colony size “stabilized” in 2010 and has fluctuated between approximately 95-

105 adult females. While it has remained at that approximate size for the last five years, the apparent

reproductive rate has steadily increased from approximately 42% since the initial decline to

approximately 95% in 2014, leading to an overall colony size of approximately 185 individuals. We

determined that a minimum of 58 individual banded female little brown bats survived over multiple years

despite suspected continued exposure to white-nose syndrome. We determined that a minimum of 26

female bats that were originally captured and banded as juveniles have returned in subsequent years,

many returning over multiple years. Our results provide evidence that female little brown myotis are

likely infected each year by white-nose syndrome, however, are able to survive, heal from damage and

infection associated with the disease, and raise young.

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Responding to threats of white-nose syndrome in Canada Charles M. Francis

1

1Environment Canada, Canadian Wildlife Service

Environment Canada is proposing to list 3 species of bats Myotis lucifugus, Myotis septentrionalis, and

Perimyotis subflavus as Endangered under the Species at Risk Act (SARA) in Canada. Listing would

provide a number of protections on public lands and enhance the profile of bat conservation needs in

Canada. To complement the listing, Environment Canada is proposing to support a number of actions to

* indicates presenting author

address threats from the disease, to the extent possible, and to reduce negative population level impacts on

listed bats from other sources. These include developing and promoting guidelines (including

decontamination protocols) to minimize the risk of human-assisted spread of white-nose syndrome

(WNS); continuing to support research and monitoring activities related to WNS as outlined in the

National Plan to Manage White Nose Syndrome in Bats in Canada; and developing guidelines to

minimize the risk of mortality and loss of habitat to bats associated with wind turbines, exclusion of bats

from human dwellings, mining operations, and forestry practices. Environment Canada plans to draw on

information in existing guidelines in other jurisdictions, as well as the expertise of bat biologists in

Canada and elsewhere to develop guidelines that will help bats, while being sufficiently practical that they

may actually be adopted by industry and other stakeholders.

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Transmission, detection, and progression of white-nose syndrome across North America Winifred F. Frick

1*, Tina Cheng

1, Kate Langwig

1, Amanda Janicki

2, Katy Parise

3, Kevin Drees

3,

Jeff Foster2, and A. Marm Kilpatrick

1

1University of California, Santa Cruz,

2University of Tennessee, Knoxville,

3Northern Arizona University,

Progression and impacts of white-nose syndrome vary considerably among species and across regions in

North America. Understanding the progression of disease over time is useful for determining effective

surveillance and management strategies, particularly at the disease invasion front. We investigate how

and why prevalence of infection, infection intensity and disease impacts vary among species and across

sites across North America. We examine the influence of geographic factors, such as latitude and

elevation, as well as climatic drivers, such as winter season length and temperatures, on transmission and

impacts. We also compare the sensitivity of qPCR diagnostics from swab surveillance surveys to visual

and histopathological confirmation of disease presence in detecting P. destructans on the invasion front.

We show that qPCR diagnostics are far more sensitive to detecting infection on individual bats, but

transmission is so high that visual surveys often detect P. destructans at the site level in the same year it

is detected by qPCR.

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Exploring the recovery phase of white-nose syndrome Nathan W. Fuller

1*, Liam P. McGuire

2, Evan L. Pannkuk

3, Heather W. Mayberry

4, Thomas S. Risch

5, and

Craig K. R. Willis6

1Boston University,

2Texas Tech University,

3Georgetown University,

4University of Toronto,

Mississauga, 5Arkansas State University,

6University of Winnipeg

Wing damage resulting from P. destructans infection is a lasting injury posing significant challenges to

WNS survivors. Wings are vital structures to bats in that they facilitate flight, but wings also play

important physiological roles. Wings are the site where cutaneous water loss, thermoregulation, and some

gas exchange occur during both torpor and euthermy. Despite the importance of wings in bat physiology,

effects of wing damage resulting from P. destructans infection is an understudied topic. Moreover,

healing following wing damage, the physical and physiological changes to wing tissue that accompany

healing, and the fate of surviving bats have received little focused study from the WNS community. Our

studies over the last three years have touched on some aspects of WNS recovery. We show that 1) bat

wing tissue has an exceptional ability to recover from WNS damage; 2) skin surface lipid profiles vary

with the extent of wing damage and time after emergence from hibernation; and 3) Pd load drops

* indicates presenting author

precipitously after 2 weeks post-emergence, but some bats retain a small load into the latter part of the

active season. We have also conducted detailed histological analysis of WNS lesions and how these

wounds change over the course WNS recovery. Surviving bats represent a faint glimmer of hope that

WNS will not totally eliminate affected populations. Thus, it is imperative that more research efforts

focus on remnant bat populations so that effective treatment and recovery strategies can be developed and

deployed.

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Heterogeneity in contact rates leads to explosive amplification of white-nose syndrome in bats Joseph R. Hoyt

1, Kate E. Langwig

1, J. Paul White

2, Heather Kaarakka

2, Jennifer Redell

2, Winifred F.

Frick1, and A. Marm Kilpatrick

1

1University of California, Santa Cruz,

2Wisconsin Department of Natural Resources

Heterogeneity in host social behavior can have profound impacts on pathogen transmission and thus

disease outcome. White-nose syndrome (WNS), caused by the fungal pathogen, Pseudogymnoascus

destructans, has caused precipitous declines in bat populations across eastern North America. However,

impacts from WNS vary considerably among species and populations, and social behavior during

hibernation, such as clustering, may contribute to these differences. To investigate the influence of social

behavior of bats on transmission, we marked two species of bats (8 bats marked per site) in four sites with

a fluorescent tracer dust at the beginning of the hibernation season. We found that contact rates among

hibernating individuals increased with colony size in the more social species, Myotis lucifugus, but not for

the solitary species, Perimyotis subflavus. We also found substantial heterogeneity in contact rates among

individuals and sites, suggesting that a few individuals can infect a large fraction of the bats in a

hibernaculum with P. destructans. This provides evidence that high contact rates of M. lucifugus may be

responsible for driving the explosive transmission of P. destructans within hibernacula.

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Estimating little brown bat (Myotis lucifugus) survival rates post-white-nose syndrome at Aeolus

Cave in Dorset, VT using PIT tag technology Morgan K. Ingalls

1, Alyssa B. Bennett

2*, Scott R. Darling

2, Joel E. Flewelling

2, Carl J. Herzog

3,

and Al C. Hicks4

1Antioch University New England,

2Vermont Fish and Wildlife Department,

3New York Department of

Environmental Conservation, 4Vesper Environmental

An important factor in determining the status of bat species affected by White-nose Syndrome (WNS) is

an accurate estimate of current post-WNS survivorship and the potential for some levels of resistance to

the disease. Winter 2012 and 2013 observations at the entrance to Aeolus Cave revealed that bats were

still demonstrating WNS-associated behavior such as flying out early and dying. The objective of this

research was to estimate the current over-winter survival rate of little brown bats (by gender and age) at

Aeolus Cave using PIT (Passive Integrated Transponder) tag technology.

During the 2013 fall swarm, 442 little brown bats were captured in harp traps at the cave entrance and a

PIT-tag was glued to the back of each. Two custom-made PIT tag antennas and solar-powered readers

were placed near the entrance to Aeolus Cave and were powered on after bats entered for hibernation. A

total of 192 bats were recorded between 28 October and 2 June. Of these, seven bats passed through the

readers pre-emergence (3 April 3) and 185 between 3 April 3 and 2 June, when temperature and weather

* indicates presenting author

conditions would suggest bats could survive outside the cave. We conclude a minimum overwinter

survival rate of 42% for little brown bats at Aeolus Cave, a significant increase from the estimated 10%

survival rate when WNS first devastated this species in 2009.

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Immune responses to Pseudogymnoascus destructans Joseph S. Johnson

1, Thomas M. Lilley

1, DeeAnn M. Reeder

1, Gábor Á. Czirják, Christian Voigt, and Ken

A. Field1

1Bucknell University

White-nose syndrome (WNS) is a fungal disease caused by Pseudogymnoascus destructans (Pd) that

affects bats during hibernation. Although millions of bats have died from WNS in North America,

mortality does not occur equally among all North American species or among species of European bats.

These differences in susceptibility to WNS may be due to differences in the strength or type of immune

response to Pd. To test this hypothesis, we quantified antibodies reactive to Pd among free-ranging bats in

North America and Europe, and captive little brown myotis (Myotis lucifugus) immunized against, or

surviving infection with, Pd. Our results will help determine whether antibody responses are sufficient to

provide protection to WNS, or whether they contribute to WNS pathology.

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Mechanisms allowing persistence of remnant populations and actions to restore them

A. Marm Kilpatrick1, Kate E. Langwig

1, Tina L. Cheng

1, Joseph R. Hoyt

1, Jeffrey T. Foster

2, and

Winifred F. Frick1

1University of California, Santa Cruz,

2University of New Hampshire

White-nose syndrome has devastated bat populations across large regions of northeast North America and

threatens multiple species with extinction. However, counts at multiple winter hibernacula of little brown

myotis have stabilized in New York, while they have declined to zero or near zero in many other

locations. The mechanisms enabling these populations to persist in places where WNS has been for more

than five years are unknown but understanding them may suggest strategies for managing bat populations,

and restoring populations that have been extirpated. We review the currently available strategies for

managing WNS, including successes and challenges. We use field data and models to examine the long

term outcomes of bat populations under the different mechanisms that could be allowing persistence of

populations with WNS. Under even the most optimistic outcome – that remnant populations have

developed resistance to or tolerance of WNS – bat populations will take several decades to recover.

Under several other mechanisms allowing persistence populations never recover to their former

abundance. Results suggest that in areas where substantial populations still exist and are under threat

from WNS (i.e. the invasion front), the most promising management action at present is manipulating

hibernacula microclimates. Treatment, such as the use of biocontrols, also offers potential, if effective

agents can be developed. We outline remaining research questions that need to be answered to manage

and restore bat populations affected by WNS.

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* indicates presenting author

Inconsistent messages in state agency and media communication about bats facing precipitous

decline from white-nose syndrome Heidi E. Kretser

1,2*, Graham Dixon

3,4, Judy Zwillenberg

3, T. Bruce Lauber

2, and Katherine McComas

3

1Wildlife Conservation Society ,

2Cornell University – Department of Natural Resources,

3Cornell

University - Department of Communication, 4Washington State University

Most conservation recovery efforts require communication to influence human behavior that might

exacerbate conservation problems. However, communicating conservation recovery messages related to

bats may be challenging because bats are a risk‐laden species, as potential carriers of rabies, a disease

fatal to humans. To determine the consistency of messages potentially received by the public, we

conducted: a) a content analysis of 108 press releases from state wildlife and public health agency

communication about bats in eight states from 2006 to 2013; and b) a media analysis of major newspaper

articles about bats within the same states. We coded for mention of 1) negative impacts that bats pose to

humans or the environment, e.g., rabies; 2) negative impacts to bats posed by humans or environment,

e.g., WNS; 3) positive impacts of bats on humans or environment, e.g., pest control, and 4) positive

actions by humans to benefit bats. We found that public health agencies focused more on human health

risks, such as rabies, while rarely mentioning WNS. Wildlife agencies focused more on WNS with less

mention of rabies. Newspaper articles also tended to focus on rabies or WNS, but not both. Our results

reveal competing priorities of public health and wildlife agencies and underscore challenges of

communication for conserving a risk-laden species. Research should examine how the public reacts to

potentially conflicting messages about bats. We suggest a need to develop and align communication

messages across agencies and in the media to promote recovery and conservation for bat species.

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Transmission of Pseudogymnoascus destructans: Insights from the invasion front Kate E. Langwig

1*, Joseph R. Hoyt

1, Katy L. Parise

2, Jeffrey T. Foster

2, Winifred F. Frick

1, and

A. Marm Kilpatrick1

1University of California, Santa Cruz,

2 Northern Arizona University

Hibernating bat species affected by white-nose syndrome (WNS) vary in a number of important ways

which may influence infection prevalence and intensity of the pathogen Pseudogymnoascus destructans.

Furthermore, the timing of pathogen arrival may affect transmission and impacts to bat populations. We

investigated pathogen prevalence and intensity on five bat species at sites in Illinois where P. destructans

was currently invading, and in the year following pathogen arrival. Swabs from exposed wing and muzzle

tissue were collected and analyzed using real-time quantitative PCR. We found that in the first year of P.

destructans invasion, infection prevalence was only 0-5% during early hibernation for all five species. For

little brown myotis and Northern long-eared myotis, infection prevalence increased to 100% by the end of

hibernation in the first year of P. destructans invasion. However, for three other species, infection

prevalence was more variable at the end of hibernation (10-50%). Colony counts of most bat species were

stable in the first year, and environmental contamination with P. destructans was mainly restricted to

areas underneath roosting bats. In the year following P. destructans invasion, infection prevalence was

much higher in early hibernation, with all but big brown bats having 75%-100% infection prevalence, and

infection intensity was 10,000 times higher than in the invasion year. In contrast to the previous year,

populations decreased 40-98% and environmental contamination was higher and more widespread.

Management actions taken the year of WNS arrival are likely to be most successful in preventing the

collapse of bat populations.

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* indicates presenting author

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Evaluation of B-23, a fungally derived volatile organic compound, for the control of WNS Lisa Last

1, and Katrina Morris

2

1 Southeastern Cooperative Wildlife Disease Study,

2 Georgia Department of Natural Resources

Chemical control of White-Nose Syndrome (WNS) has proven challenging due to the unique physiology

of bats and the physical difficulties encountered in their chosen hibernacula. Volatile organic compounds

(VOCs) hold promise as contact independent chemical control solutions to inhibit the growth of

Pseudogymnoascus destructans (Pd) and limit the spread of WNS. B-23 is a long-acting, synthetically

produced compound derived from the active ingredients of the endophyte Muscodor crispans with broad

spectrum antimicrobial activity. It has been successfully utilized in a variety of settings, including

healthcare, agriculture, and food storage. Commercial products containing B-23 have been safely used

with multiple mammalian species and all components are classified as GRAS. This study examined the

efficacy of B-23 to inhibit the growth of Pd in a laboratory setting. Significant inhibitory zones were

produced at concentrations of 4, 8, and 16 ul of B-23. Inhibition was maintained for over 9 months, with

the greatest effects seen with the highest concentration. Free-ranging Pd-free bats were then exposed to

sachets of B-23 for a period of one month. Bats were apparently healthy at initial visit and no deleterious

physical or behavioral changes were noted following exposure to B-23. Additional laboratory and field

studies are needed to better characterize the optimal application methods, efficacy, and safety of B-23 for

the control of WNS.

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Understanding and potentially manipulating the genetics of Pseudogymnoascus destructans:

Implications for WNS management Daniel L. Lindner

1*, Jonathan Palmer

1

1 U.S. Forest Service – Northern Research Station, Center for Forest Mycology Research

Genetics, and more recently genomics, are powerful tools that have been used to understand the

pathogenicity, life cycle, and ecology of many disease-causing organisms. Fungi have traditionally been

used as model systems in genetics, but extremely little is known about the genetics of the fungus

Pseudogymnoascus destructans (Pd), the casual agent of white-nose syndrome in bats. We used genetic

techniques to elucidate the sexual capacity of Pd and unexpectedly found that, unlike many close

relatives, Pd appears to not be self-fertile (homothallic), but rather requires two different mating types to

initiate sexual reproduction. To date, all isolates of Pd from North America that have been tested are of

the same mating type, while both mating types can be found in close proximity in central Europe. This

suggests the presence of mating populations of Pd in Europe and underscores the need for continued

vigilance regarding additional introductions of Pd into North America, given that sexual recombination

could allow the fungus to adapt more quickly and overcome host resistance mechanisms. Additionally,

we continue to use comparative genomics to identify potential virulence factors in Pd, which could lead

to targeted management techniques, such as gene silencing or drug development.

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* indicates presenting author

Overview of ongoing white-nose syndrome diagnostic and research projects at the USGS – National

Wildlife Health Center Jeffrey Lorch

1*, Katie Schmidt

2, Sébastien Puechmaille

3, and David Blehert

2

1University of Wisconsin – Madison,

2U.S. Geological Survey – National Wildlife Health Center,

3University College Dublin

The U.S. Geological Survey – National Wildlife Health Center (NWHC) continues efforts to advance

white-nose syndrome (WNS) diagnostics and research. Here we provide updates of two ongoing projects.

The first project involves the development of an in situ hybridization technique to better discriminate

between infections caused by Pseudogymnoascus destructans and those caused by other fungi.

Histopathology is considered the current “gold standard” for diagnosing WNS. However, in some P.

destructans-positive bats (particularly in certain bat species), atypical lesions and/or noncharacteristic

fungal invasion patterns have been observed. Since other fungi are known to invade bat skin, determining

whether P. destructans is the fungus causing an infection in such cases is challenging. We designed, and

are in the process of qualifying, multiple P. destructans-specific DNA probes to assist with discriminating

infections caused by P. destructans and those caused by other fungi. The resulting assays will be useful

to better diagnose these potentially atypical presentations of WNS. The second project targeted the

isolation of P. destructans sister taxa. Recent phylogenetic analyses of North American cave-dwelling

Pseudogymnoascus spp. demonstrated that these species are distant relatives of P. destructans and may

have limited utility in helping to understand the basic biology, evolutionary history, and virulence factors

of P. destructans. Since P. destructans is thought to be native to Europe, we hypothesized that the fungus

may have sister taxa in that region. While a culture-based survey revealed a high diversity of

Pseudogymnoascus spp. in European soils, no close relatives to P. destructans were found.

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Ecological and phylogenetic modeling shows that the white-nose syndrome fungus is a generalist

pathogen of hibernating bats Natália Martínková

1*, Hana Bandouchova

2, Tomáš Bartonička

3, Jan Zukal

1, Jiri Pikula

2 1Institute of

1Vertebrate Biology, Academy of Sciences of the Czech Republic, Brno, Czech Republic,

2University of

Veterinary and Pharmaceutical Sciences, Brno, Czech Republic, 3Masaryk University, Brno, Czech

Republic

Multiple species of bats emerge from European hibernacula where Pseudogymnoascus [Geomyces]

destructans infection was identified previously in numbers relatively stable between years. Using UV

fluorescence, we examined 276 bats of 15 species from hibernacula in the Czech Republic over 2012 and

2013 to biopsy their wings for histopathological investigation. Prior to the UV examination, dorsal side of

the left wing of each individual was swabbed for P. destructans spore load quantification using qPCR.

We found that 11 bat species were positive for the white-nose syndrome (WNS). These included

representatives from genera Myotis, Eptesicus, Plecotus, Barbastella and Rhinolophus, and in two species

we found WNS lesions that spanned the full thickness of the wing membrane. The prevalence of WNS in

hibernation survivors, evidenced by histopathology, ranged from 4% in Rhinolophus hipposideros to 55%

in Myotis myotis. It is important to note that WNS affects species from both Chiroptera suborders. Our

results challenge views that European bats are resistant to the infection and we therefore tested whether

relatedness, ecological or behavioral traits of bats in North America and Europe explain the pattern of

infection. Using phylogenetic generalized least square modeling and phylogenetic representation, we

found that the infected species are statistically phylogenetically related, although our new data begin to

lower the significance of the test, and WNS affects species with diverse ecology. We conclude that P.

destructans is a generalist pathogen that can infect any bat species hibernating within its distribution

* indicates presenting author

range. The different outcome of WNS between the continents and the reasons for the discrepancy are the

key focus of our further research.

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Implications of ameliorating survival of little brown bats (Myotis lucifugus) infected with white-

nose syndrome of the direction of future conservation efforts

Brooke Maslo1*

, Mick Valent2, John F. Gumbs

3, and Winifred F. Frick

4

1The State University of New Jersey- Department of Ecology, Evolution and Natural Resources, Rutgers,

2New Jersey Division of Fish and Wildlife,

3BATS Research Center,

4University of California-

Department of Ecology and Evolutionary Biology

Long-term management of bat populations impacted by White-nose Syndrome (WNS) is challenged by a

lack of post-disturbance demographic data. Early demographic analyses can inform rapid management

and be modified iteratively as additional empirical data are generated. Using four years of post-WNS

mark-capture data on little brown bats (Myotis lucifugus), we estimate the first robust apparent annual

survival rates for bats at a WNS infected site. We found strong support for a time-varying amelioration

effect in survival, which increases from 0.69 (95% CI = 0.46, 0.85) to 0.74 (95% CI = 0.52, 0.88) for

males and 0.66 (95% CI = 0.46, 0.81) to 0.71 (95% CI = 0.41, 0.89) for females. We incorporate these

estimates into a stochastic matrix population model and calculate a long-term negative population growth

rate of = 0.95 (despite ameliorating survival rates), supporting a call for immediate management

intervention of remnant bat populations. Our subsequent vital rate sensitivity analysis (VRSA) suggests

that management intervention targeting survival of all age classes may maximize short-term (15 years)

persistence of infected little brown bat populations. While mitigative measures to alleviate the intensity of

infection (e.g., antifungal treatments, supplemental winter resources) may be infeasible over a long

timeframe, short-term implementation of these strategies may allow declining populations to persist long

enough to be rescued by conservation actions informed by more complete analyses of vital rates.

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Metabolic rate and evaporative water loss in hibernating little brown bats inoculated with

Pseudogymnoascus destructans Liam McGuire

1*, Heather Mayberry

1, and Craig Willis

1

1University of Winnipeg, Winnipeg, MB

Bats with WNS experience increased arousal frequencies leading to the exhaustion of energy stores and

death by starvation. However the cause of the increased arousal frequency is not known. In healthy bats,

dehydration has been proposed as a cue for periodic arousals. Torpid bats lose water at a greater rate than

can be replenished by metabolic processes, and therefore bats must arouse to drink. The ‘dehydration

hypothesis’ proposes that wing damage caused by WNS leads to increased evaporative water loss (EWL),

and therefore more frequent arousals. Furthermore, WNS may lead to an increase in torpid metabolic rate

which would also contribute to premature exhaustion of fat stores. We used respirometry to test the

predictions that bats inoculated with Pd would 1) have elevated rates of EWL, and 2) have elevated torpid

metabolic rates compared to healthy control bats. We collected wild bats from a WNS-negative cave in

central Manitoba, inoculated them with Pd (or sham inoculated controls), and held them in cages in a

temperature and humidity controlled environmental chamber for hibernation. After > 114 days of

hibernation, we transferred bats to respirometry chambers to measure torpid metabolic rate and EWL in

dry and humid air. Consistent with the predictions arising from the dehydration hypothesis, bats

* indicates presenting author

inoculated with Pd had higher rates of EWL. Inoculated bats also had higher torpid metabolic rates.

Therefore bats with WNS use more energy during torpor bouts, and suffer increased EWL which may be

the trigger for increased arousal frequency, further depleting energy stores.

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Testing of actinobacteria isolated from twelve western bat species against Pseudogymnoascus

destructans: Clues to potential bat natural defenses Diana E. Northup

1*, Nicole A. Caimi

1, Andrea Porras-Alfaro

2, Ara S. Kooser

1, Debbie C. Buecher

3, Jesse

M. Young1, Jason C. Kimble

1, Kaitlyn J. Hughes

1, and Ernie W. Valdez

4

1University of New Mexico,

2Western Illinois University,

3Buecher Biological Consulting,

4U.S.

Geological Society

We characterized the bacterial and fungal microbiota that reside on fur and membranes of bats captured in

caves and near drinking sources in New Mexico and Arizona. Our study revealed the presence of many

Actinobacteria, a bacterial phylum known for the production of important secondary metabolites.

Different bat species varied in the abundance of Actinobacteria leading us to hypothesize that some bat

species may possess natural defenses against pathogens. To test this hypothesis, in relation to

Pseudogymnoascus destructans, we swabbed bats at El Malpais National Monument (ELMA) southwest

of Grants, NM; Fort Stanton Cave near Capitan, NM; BLM Caves 45 and 55 in NM, and in Parashant

National Monument in northwest Arizona. Swabs were immediately used to inoculate a suite of media

that target Actinobacteria, including humic acid vitamin agar, Actinomycete Isolation Agar, and gellum

gum medium. Parent cultures were subcultured and two hundred pure isolates were obtained. DNA was

extracted and sequenced. Sequences were identified with NCBI BLAST, and grouped by similarity. The

majority of the cultures were Streptomyces spp. The ability of forty representative isolates to inhibit P.

destructans was tested using an agar diffusion technique in which the actinobacterial isolate is first grown

on R2A, followed by an overlay of fungal-appropriate medium on which a lawn of P. destructans was

plated. Plates were monitored for the development of zones of inhibition, which were observed in four

isolates. Our preliminary results suggest that microbiota present on bats may have the potential to

produce anti-fungals.

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Canada’s response to white-nose syndrome: Plans, infrastructure, actions Allysia Park

1* (on behalf of the Inter-agency WNS Committee), and Craig Stephen

1

1Canadian Wildlife Health Cooperative

White-Nose Syndrome (WNS) is an unprecedented North America challenge for wildlife protection.

Since its first detection in Canada in 2010 in Quebec and Ontario, the disease has expanded into the three

Atlantic provinces. In 2013-14, WNS was detected in several new counties in eastern Canada along with

substantial declines in previously-affected populations. No westward extension of the range of WNS or

the pathogen has yet to be detected. Little Brown and Northern Myotis and Tri-colored bats have been

given provincial endangered status in Ontario, Nova Scotia and New Brunswick. National status as

endangered species has been recommended by COSEWIC, but, the response to this recommendation is

pending.

As the disease expands across the continent, it is important to have strategies that are consistent across

jurisdictions. Environment Canada supports the Inter-agency WNS Committee, which works with the

* indicates presenting author

approval of the Canadian Wildlife Directors Committee, to ensure a pan-Canadian approach to detection,

prevention and mitigation of WNS across provinces and territories. The Canadian Wildlife Health

Cooperative serves as secretariat and coordinator for this committee and its working groups. Many action

items have been completed (ex. creation of a national management plan, developing necropsy and

culturing Pseudogymnoascus destructans (P.d.) protocols) and a number of activities are ongoing (ex.

WNS and P.d. surveillance updates, WNS communications). Canada-US collaborations have sped the

development of standard approaches to WNS, including Best Management Plans, which could be adapted

to different jurisdictions. They have also lead to international projects such as product testing

disinfectants for decontamination against P.d. spores and supporting participation in NABat. Future

priorities include developing nation-wide conservation strategies for bat populations and a citizen science

program for monitoring.

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Evaluation of Epicoccum nigrum link and Polyethylene Glycol (PEG 8000) in the control of

Pseudogymnascus destructans (Blehert & Gargas) Minnis & D.L. Lindner Jake Perryman

1, Gregory Turner

2 and Barrie Overton

1*

1Lock Haven University,

2Pennsylvania Game Commission.

In an effort to control Pseudogymnoascus destructans (Pd) infection in bats, Epicoccum nigrum, a known

biological control agent used in the fruit industry to control fungi and bacteria was evaluated in dual-

culture against Pd. Polyethelene Glycol (PEG 8000) is an inert water soluble polymer used in the

pharmaceutical industry and for medical procedures in humans to control hospital-acquired infections.

PEG 8000 was evaluated in-vitro as a potential chemical control of Pd in liquid broth and applied to cave

sediment. Epicoccum nigrum showed characteristic hyphal coiling against Pd when grown in dual-

culture and visualized with fluorescent and DIC microscopy. Confirmation of secondary metabolite

diffusion was observed from E. nigrum as an orange pigment. This secondary metabolite stained Pd

conidia and hyphae causing them to fluoresce at different wavelengths. This is the first report of this

secondary metabolite (epicocconone) staining a filamentous fungus, as it has been previously reported

and patented, as a stain for yeasts. Additionally, secondary metabolites from E. nigrum were evaluated

using a modified Kirby-Bauer disk diffusion assay and preliminary results show that metabolites from E.

nigrum slightly inhibit germination of Pd conidia. PEG 8000 reduced mycelia growth and conidial

germination in liquid broth and completely inhibited conidial germination and mycelial growth of Pd on

sterilized cave sediment compared to the controls. Epicoccum nigrum has a greater tolerance to PEG

8000 than Pd suggesting a combination therapy is possible. Studies in a cave environment or with live

bats have not been completed.

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Distribution and ecology of Geomyces destructans in Eurasia and its relationship with bats Sebastien J. Puechmaille.

Institute of Zoology, Univ of Greifswald, Greifswald, Germany, School of Biological and Environmental Sciences,

Univ. College Dublin, Dublin, Ireland, Groupe Chiroptères de Midi-Pyrénées (CREN-GCMP), Toulouse, France

Geomyces destructans (Gd), the fungus associated with White-Nose Syndrome and mass mortalities in

North America, has been found in many European countries and infecting many species, yet no mass

mortality comparable to North America has ever been reported during contemporary times. Despite being

infected by the fungus, it is not yet known to what extent European species are affected from such

infection. It is nevertheless clear that understanding factors influencing the fungus presence and

* indicates presenting author

prevalence on bats is of prime importance. From an ecological perspective, our work addresses the

following questions: (1) Does Gd presence explain mortalities observed at hibernacula, (2) does Gd

prevalence (estimated with the naked eye) on bat vary over the course of the winter and over space, (2) is

Gd present in the bat environment and to what extent, (3) is the environment a reservoir for Gd, (4) which

species are most infected by Gd, (5) which biotic or abiotic factors influence the presence of Gd in the

environment and on bats. To answer these questions and with the help of a large network of bat

researchers/biologists and conservationist, we sampled sediments and walls at more than 250 hibernation

sites across the Western Palearctic and to a lesser extent beyond. These sites encompass the range of most

common underground bat hibernacula types known, e.g. caves, mines, cellars, tunnels, bunkers, quarries,

castles. To be able to compare the results across the study area with as little biases as possible, the same

sampling protocol, specifically developed for the current project, was applied at every site. Analyses for

the presence of Gd in the collected samples were carried out using a combination of state of the art

genetic and culture techniques. The main results show that Gd presence does not explain the occasional

mortalities observed at hibernacula. The prevalence of Gd on bats drastically varies over the course of the

winter and over space. Although not easily detectable, Gd is present in the environment around bats

(walls, sediments) and such environment is hypothesized to act as a reservoir for Gd.

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Species differences in susceptibility to WNS and the cost of infection DeeAnn M Reeder

1*, Melissa Meierhofer

1, Shayne Lumadue

1, Marianne S. Moore

1,

Joseph S. Johnson1, Melissa J. Behr

2, and Kenneth A Field

1

1Bucknell University,

2School of Veterinary Medicine, University of Wisconsin-Madison

White-nose syndrome is caused by the emerging fungal pathogen Pseudogymnoascus destructans (Pd).

Interestingly it does not affect all species equally and surviving infection with Pd is presumably

influenced by a number of factors, including within- and between-species variation in thermoregulatory

traits, life history and behavioral traits, and immunological responsiveness. We conducted captive

experimental Pd infections of little brown myotis (Myotis lucifugus) and big brown bats (Eptesicus

fuscus). Housed in identical conditions, and thus controlling for differences in thermoregulatory

preferences between the species, big brown bats developed much lower levels of infection than little

brown myotis and displayed adaptive changes in torpor patterns, rather than the too frequent arousal bouts

known to occur in little brown myotis. In a separate study aimed at understanding the consequences of

surviving WNS, we found no difference in the reproductive rates of surviving big brown bats (field

sampled) and little brown myotis (captive Pd inoculation study) relative to uninfected animals. For Pd-

infected female little brown myotis, significant wing tissue degradation was evident by 19 days after

emergence from hibernation and these bats exhibited significantly higher mass specific metabolic rate

(MSMR) at this time. While these females were able to reproduce with ad lib food and no flight

requirements, further research investigating the long-term consequences for both mothers and pups in the

field is necessary.

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Demographic and physiological responses by populations of surviving little brown bats Myotis

lucifugus to white-nose syndrome Brent J. Sewall

1*, Gregory G. Turner

2, Cynthia Hauser

2, and James Sinclair

2

1Temple University,

2Pennsylvania Game Commission

* indicates presenting author

White-nose syndrome (WNS) has devastated colonies of little brown bats Myotis lucifugus across the

eastern United States, leaving few surviving bats in areas where they were formerly abundant. A major

unanswered question, however, is whether these remaining bats are simply the last individuals to succumb

to an ongoing regional extirpation, or, alternatively, whether these bats could continue to survive in a

remnant population capable of persisting over time. To evaluate this question, we examined multiple

lines of evidence to determine responses of bat populations to WNS. Data included bat counts in

hibernacula, measurements of pre-hibernation body condition, and evaluation of infection load during

hibernation. Data were collected repeatedly to evaluate changes over time since the onset of WNS.

Alteration in the rate of decline at hibernacula, increases in pre-hibernation body-condition, and decreases

in infection load over time were considered evidence of population-level response that may facilitate M.

lucifugus persistence over time in areas affected by WNS. Our results indicate that M. lucifugus bat

colonies declined at a less rapid rate, pre-hibernation body condition improved, and infection load in

hibernating bats decreased since local onset of white-nose syndrome. Together, these results suggest that

M. lucifugus populations in the eastern United States have responded to the severe threat posed by WNS

in ways that may support persistence of a remnant population over time.

Notes:_______________________________________________________________________________

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Examination of survivors: Responses of exposed colonies of little brown bats (Myotis lucifugus)

across time. Greg Turner

1*, Carl J. Herzog

2, Natália Martínková

3, Brent Sewell

4, Jim Sinclair

1, and Cynthia Hauser

1

1Pennsylvania Game Commission,

2New York Department of Environmental Conservation,

3Academy of

Sciences of the Czech Republic, 4Temple University

WNS-contaminated sites were first confirmed in Pennsylvania during the 2008-2009 winter. Mass

mortality typically proceeded for 2 years post-contamination and an examination of 34 sites with

consistent survey effort both before and following contamination has exceeded 99% for the little brown

bat (Myotis lucifugus) in Pennsylvania. Following infection a significant increase in arousal patterns in

little brown bats occurred, explaining the emaciated condition noted among the perished. Despite the

severity of reduction, a surviving population of little brown bats has persisted at two Pennsylvania

hibernacula with prior monitoring. A comparison of body mass prior to contamination and following the

2 years of mass mortality demonstrates a significant increase in body mass for adults. Additionally, a new

technique which makes sub-dermal points of infection fluorescence, documents significant annual

reductions in the quantity of fluorescence since contamination.

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Playing the odds on bat white-nose syndrome: Information from the field to inform surveillance

strategies Michelle L. Verant

1*, Elizabeth A. Bohuski

2, Kevin J. Olival

3, Jonathan H. Epstein

3, and

David S. Blehert2

1University of Wisconsin-Madison,

2U.S. Geological Survey – National Wildlife Health Center,

3EcoHealth Alliance

Recent advances in diagnostic tools have allowed for early detection of Pseudogymnoascus destructans

(Pd) and white-nose syndrome (WNS) in hibernating bat populations. Viable Pd has also been isolated

from environmental substrates within WNS-affected hibernacula, demonstrating that environmental

reservoirs can serve as alternative targets for pathogen detection. However, dynamics of Pd transmission

* indicates presenting author

between bats and environmental substrates within a hibernaculum are poorly defined. We used qPCR to

temporally characterize prevalence and loads (intensity) of Pd on bats (N=558) and in environmental

substrates (N=1050) within five bat hibernacula. To define relationships between Pd infections in bats

and environmental loads of the fungus from initial introduction of WNS to an endemic state, hibernacula

were categorized based upon the length of time since WNS was first confirmed at each site (WNS years).

Prevalence and intensities of Pd on bats were low during WNS year-1 and increased significantly to near

100% prevalence and peak intensities by WNS year-3. Detections of Pd in environmental substrates

lagged one year behind detections on bats. Once detected, however, environmental fungal loads initially

increased across subsequent WNS-years, then exhibited evidence of decline. These results indicate that

bats are more profitable targets than environmental substrates for early detection surveillance strategies

and are likely primary drivers in the establishment of environmental reservoirs within a hibernaculum.

Increased understanding of pathogen spread and persistence within bat populations and environmental

reservoirs will help to inform surveillance strategies and identify critical control points for disease

management.

Notes:_______________________________________________________________________________

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Population genomics of little brown myotis (Myotis lucifugus) reveal adaptive genetic diversity and

varied connectivity of populations across North America Aryn P. Wilder

1*, Thomas H. Kunz

1, Michael D. Sorenson

1

1Boston University

The little brown myotis (Myotis lucifugus) is a wide-ranging species that has suffered massive declines

from white-nose syndrome (WNS) in the eastern part of its distribution, leading to predictions of eventual

extirpation from this region. We analyzed population genetic structure across the range of little brown

myotis to: 1) infer patterns of gene flow across the continent and across putative subspecies boundaries;

2) infer male and female dispersal patterns; 3) scan for regionally adaptive genetic diversity that may be

threatened by WNS; and 4) assess the potential for WNS to continue its spread across the continent in

light of population genetic patterns. We used restriction-site associated DNA sequencing to generate ~440

kb of sequence data per sample for more than 300 individuals. Genome-wide data suggest two moderately

differentiated populations (FST = 0.121) divided roughly by the Rocky Mountains, with minimal

geographic structure within the larger eastern population, and greater structure within the smaller western

population. Differentiation in the West indicates that seasonal dispersal is far more limited than in the

East. Maternally-inherited markers had relatively restricted geographic distributions, whereas gene flow

of biparentally-inherited and paternally-inherited markers was high across these distributions, suggesting

male-biased gene flow. Outlier analysis indicates divergent natural selection between the populations, and

the possibility that unique, adaptive genetic variation may be threatened by declines. Range-wide

population genetics of this species suggests that the spread of WNS, at least as mediated by this species,

will slow as it moves westward, where populations are smaller and connectivity is lower.

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* indicates presenting author

Development of an Agrobacterium-mediated transformation system for the cold-adapted fungi

Pseudogymnoascus destructans and Geomyces pannorum sensu lato Tao Zhang

1, Vishnu Chaturvedi

1, 2, and Sudha Chaturvedi

1, 2*

1Mycology Laboratory, Wadsworth Center, New York State Department of Health,

2Department of

Biomedical Sciences, School of Public Health, University at Albany, NY

The mechanisms of cold adaptation by fungi remain unknown. This topic is of high interest due to the

recent emergence of white-nose syndrome (WNS), a skin infection of hibernating bats caused by

Pseudogymnoascus destructans (Pd). We developed an Agrobacterium tumefaciens-mediated

transformation (ATMT) system for Pd and related human pathogen Geomyces pannorum sensu lato (Gp).

URE1 gene encoding the enzyme urease was used as an easy to screen marker to facilitate molecular

genetic analyses. A user-specific excision reagent (USER) friendly pRF-HU2 vector containing Pd or Gp

ure1::hygromycin (HYG) disruption cassette was introduced into A. tumefaciens AGL-1 cells by

electroporation and the resulting strains were co-cultivated with Pd or Gp conidia for various durations

and temperatures to optimize the ATMT system. The transformation efficiency of Pd was 0.006% and of

Gp was 0.018% and these were strongly correlated with the length of the incubation period (96 h for Pd;

72h for Gp) and with temperature (15-18°C for Pd; 25°C for Gp). The homologous recombination

efficiency for Pd was 3.1%, and for Gp was 16.7%. The lower transformation and homologous

recombination efficiency for Pd could be attributed to the lower co-cultivation temperature and slower

growth rate in comparison with Gp. The availability of a transformation system for cold-adapted fungi

would facilitate future molecular genetic analyses of Pd and Gp.

Notes:_______________________________________________________________________________

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Poster Presentation Abstracts (Alphabetical by first author)

Expanded surveillance for the detection of Pseudogymnoascus destructans distribution and spread

in the continental United States

Anne Ballmann1, David Blehert

1, Elizabeth A. Bohuski

1, and Robin Russell

1

1U.S. Geological Survey-National Wildlife Health Center, Madison, WI

To date, national surveillance efforts for white-nose syndrome (WNS) have focused on identifying

clinically-affected bats for diagnostic testing to confirm histopathological lesions caused by the fungus,

Pseudogymnoascus destructans (Pd), during winter when disease is most prevalent. Pd detection on bats

(and less commonly on environmental substrates) has been demonstrated in advance of recognizable

clinical disease in hibernating bat populations. As sites are typically entered once per winter season to

minimize disturbance, early infections and/or low disease prevalence can easily go undetected in bat

populations by visual inspection alone. Furthermore, site selection has often been based on untested

prioritization criteria presumed to increase the risk for Pd contamination and diagnostic submissions have

often been limited to the county-level so as not to overburden laboratory capacity. Improvements to the

sensitivity and specificity of the Pd PCR and high thru-put technology allows for expanded non-lethal

sampling to identify Pd range expansion in advance of clinical disease. The main objectives for this 3-

year project are to: 1) expand the existing national surveillance program by incorporating swab sampling

* indicates presenting author

methods to assist with early detection of Pd into new areas or on new species; 2) to clarify knowledge

gaps related to patterns and rates of Pd movement on the landscape; 3) to provide diagnostic support for

priority field research within the WNS-affected area; and 4) to address the concomitant expansion of

diagnostic capacity and standardization needed to successfully implement active Pd

surveillance. Findings obtained from the expanded national Pd surveillance strategy will provide a better

understanding of the geographic distribution of the fungus and assist management agencies with the

timely implementation of biosecurity restrictions which may help slow pathogen spread. It also allows

for clearer understanding of patterns of pathogen dispersal and disease progression among hibernacula to

critically evaluate site characteristics thought to be associated with a greater risk of Pd contamination and

more accurately model pathogen movement. Finally, on-going surveillance within the WNS affected

region can provide insight into behavioral and/or biological resistance among remnant bat populations or

critical assessments of field efficacy for various Pd control strategies. Results from Year 1 of this project

will be presented. Participation in Years 2 and 3 is being sought.

Notes:_______________________________________________________________________________

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Winter roosts of little Brown Bats in Juneau, Alaska: An Ace in the Hole?

Karen Blejwas1, Michael Kohan

1, Laura Beard

1, Dylan Rhea-Fournier

1, and Grey Pendleton

1

1Alaska Department of Fish and Game

Little brown bats in eastern North America typically hibernate in caves and mines, often in large numbers,

however few large hibernacula have been identified in the western part of their range and none have been

found west of the Rocky Mountains. We used a combination of radiotelemetry and acoustic monitoring to

investigate the overwintering strategy of little brown bats in Juneau, Alaska. We radiotracked 4 little

brown bats to their presumed winter roosts; 1 was in a hole beneath the root wad of an old stump and 3

were in holes in old rock slide areas on steep, forested hillsides. Bats entered their roosts between Oct 1 –

17; first spring detections of little brown bat echolocation calls at the roost sites ranged from April 13 –

25. No little brown bat calls were detected during the winter months. We compared temperatures and

relative humidity inside (~0.3 – 0.5 m from the opening) and outside the roosts. Relative humidity

dropped as low as 40-60% outside of roosts, but remained near 100% within the roosts throughout the

winter. Average temperature at midnight ranged from -2.33 to -0.63 outside the roosts and -1.04 to 2.03

inside; corresponding standard deviations ranged from 3.69 to 4.10 outside and 1.05 to 1.85 inside the

roosts, indicating conditions inside the holes were more stable than outside. If roosting solitarily in holes

in the ground is a common overwintering strategy of little brown bats in the west, western populations

may be much less vulnerable to white-nose syndrome than their eastern counterparts.

Notes:_______________________________________________________________________________

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Compensatory Pulmonary Gas Exchange is an Unlikely Mechanism of WNS-related Dehydration

Charleve S. Carey1, and Justin G. Boyles

1

1Cooperative Wildlife Research Laboratory, Southern Illinois University

The fungus, Pseudogymnoascus destructans (Pd), is the causative agent of WNS, but to date, we have

limited understanding of how an infection with Pd can lead to mortality in hibernating bats. Evidence

suggests dehydration is an important part of the pathogenesis of WNS. Cryan et al. (2010) proposed four

possible mechanisms by which infection could lead to dehydration. In this study, we tested one of these

* indicates presenting author

hypotheses - Pd infection could cause disruption to passive gas-exchange pathways across the wing

membranes, thereby causing a compensatory increase in water-intensive pulmonary respiration. We

hypothesized total evaporative water loss would be greater when passive gas-exchange was inhibited,

especially at low ambient temperatures. Contrary to expectations, we found that bats did not lose more

water when passive gas-exchange was retarded (at least within the resolution of our equipment). This

study provides evidence against the proposed proximal mechanism that disruption to passive gas-

exchange causes dehydration and ultimately death to WNS-infected bats.

Notes:_______________________________________________________________________________

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Resistance to white-nose syndrome in bat populations of the Northern Forest: Exploring the critical

disease-genotype-microbiome link

Deahn Donner 1, Leah Berkman

2, Paula Marquardt

1, Jacqueline Frair

2, Daniel Lindner

1, Jonathan Palmer

1,

and B. Prom1

1U.S. Forest Service-Northern Research Station,

2State University of New York – College of

Environmental Science and Forestry

The WNS epidemic in North America has exposed the lack of information on the immune system of

bats. Although much research on immune response and broad genomic patterns has appeared recently,

resistance conferred by Major Histocompatibility Complex (MHC) variants has received little

attention. We are examining the MHC pre- and post-WNS exposure to identify where disease resistant

individuals may exist, and to determine whether genotype and resistance to WNS may be related to

naturally occurring skin microbiota of bats. Skin microbes can play an important role in moderating

immune responses, and fighting the fungus, so finding associations between host genotypes and the

microbiome will illuminate relationships among the natural defenses against Pd. Living bats in the

Northeast States that have been continuously exposed to WNS in hibernacula may harbor resistant

genotypes and protective microbial communities. Comparison with unaffected regions may indicate a

selective advantage in the presence of WNS. Bat tissue and wing swabs (n=90) were collected from little

brown bats at hibernacula across post-(NY) and pre- (IL, WI) affected areas. We are evaluating the

potential for next generation sequencing to accurately and thoroughly genotype MHC-DRB loci (n=16)

using locus-specific primers designed from the little brown bat genome and Sanger

sequencing. Metagenomic analysis using the internal transcribed spacer region will be used to

characterize the fungal community. Conservation of disease resistant populations and their habitat will be

important to recovery efforts so individuals can disperse and reproduce, thereby facilitating the natural

expansion of genetically resistant bats.

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National Speleological Society WNS Support Summary

Jennifer Foote1

1National Speleological Society

The National Speleological Society (NSS) is the nation’s oldest and largest organization dedicated to the

study, exploration, and conservation of caves and their natural resources. The NSS has been involved at

the forefront of the investigation into white-nose syndrome since its discovery in New York in 2007. Our

* indicates presenting author

individual members have volunteered for numerous bat field surveys, both underground during

hibernation season, and above ground conducting emergence surveys and summer acoustical monitoring.

Since 1969 members of the Huntsville Grotto have supported bat research at Fern Cave, the largest Gray

Bat hibernaculum in the country. The NSS has supported active education and outreach goals as well. In

2009 the NSS was asked to take the lead in raising and granting money for critical WNS research, as state

and federal funding streams could not react quickly enough through their lengthy budget processes. Our

members stepped up to the plate, and hundreds of them have made individual contributions, totaling over

$120,000, supporting 21 Rapid Response Fund grants for critical and timely white-nose syndrome

research.

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Use of anti-Pseudogymnoascus destructans VOCs and formulations with a novel automated

dispersal device for the treatment and prevention of white-nose syndrome

Kyle T. Gabriel1*

, Christopher T. Cornelison1, Tom Tomasi

2, M. Kevin Keel

3, and Sidney A. Crow Jr.

1

1Georgia State University, Applied and Environmental Microbiology,

2Missouri State University,

Department of Biology, 3University of California, Davis, School of Veterinary Medicine

Bacterially-produced volatile organic compounds (VOCs) have demonstrated significant anti-P.

destructans activity at low concentrations (< 1 ppm) (Cornelison et al., 2013, Mycopathologia).

Additionally, these VOCs demonstrated their effect through a contact-independent antagonism which may

present many advantages in the control of WNS. As bats have been known to hibernate in areas that make

direct access difficult or impossible, it is believed that contact-independent antagonism may be a viable

option for treating bats in vivo and in situ. The development of a novel device to utilize these VOCs has

enabled the treatment of infected hibernacula in karst environments as well as treatment chambers. The

device calculates accurate application intervals and autonomously dispenses specific volumes of VOCs as

microscopic vapor droplets (0.5-5 µm diameter), facilitating rapid evaporation in order to attain a desired

gaseous concentration. Furthermore, the design allows for a wide range of essential oils, VOCs, and VOC

formulations to be used, and with the addition of a rugged enclosure and battery-power, prolonged use in

harsh environments is possible. Toxicity testing was conducted on torpid bats (n=37) with the device and

a highly-effective VOC formulation (10x higher than the effective dose). Thorough examination of the

histopathology demonstrated no detrimental effects from acute or chronic exposure.

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Communicating about white-nose syndrome with a unified voice

Catherine J. Hibbard1 White-nose Syndrome Communications and Outreach Working Group

1U.S. Fish and Wildlife Service, Northeast Regional Office

The White-Nose Syndrome Communications and Outreach Working Group is one of seven working

groups developed under the National White-nose Syndrome Plan to address research and management

priorities. Comprised of about 20 members from federal and state agencies and non-governmental groups,

the working group’s mission is to develop and carry out a plan for communicating information about

white-nose syndrome to partners involved in the white-nose syndrome response, and to the public.

* indicates presenting author

As part of this effort, the Communications and Outreach Working Group developed messages to use

when speaking to audiences not familiar with white-nose syndrome. This poster will introduce working

team members, highlight our messages and provide workshop attendees with other working group

updates.

Working group members at the workshop will be available during the poster session to interact with the

white-nose syndrome community and out outreach materials.

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The status of Pseudogymnoascus destructans in temperate Asia

Joseph R. Hoyt1, Guanjun Lu

2, Kate E. Langwig

1, A. Marm Kilpatrick

1, Katy L. Parise

4, Winifred F.

Frick1, Jeffery T. Foster

3, Keping Sun

2, and Jiang Feng

2

1University of California, Santa Cruz,

2Northeast Normal University,

3University of New Hampshire,

4Northern Arizona University

The current known distribution of Pseudogymnoascus destructans includes parts of North America and a

large portion of the European continent, but the origins of this fungal pathogen remain unknown. A

missing gap in the sampling distribution is temperate Asia, where the conditions are suitable for pathogen

growth, and large numbers of hibernating bats exist in cave habitats. Determining the global distribution

of P. destructans is important for understanding the origins and global threat of white-nose syndrome. We

sampled for P. destructans across northeastern China, including four provinces and one municipality

(Heilongjiang, Jilin, Liaoning, Shandong, and Beijing). We collected samples from bats, soil, and

hibernacula surfaces in 19 caves and mines in June-July, 2014 where bats are suspected to hibernate

during winter. We are testing the samples using a qPCR assay targeting the IGS region of the P.

destructans genome. This study will help determine the risk of human-mediated movement of P.

destructans to and from Asia.

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Hibernating bats and abandoned mines of the Upper Peninsula of Michigan

Allen Kurta1 and Steven M. Smith

1

1Eastern Michigan University

Prior to arrival of white-nose syndrome, we found bats hibernating in 82 of 119 abandoned mines in

northern Michigan. Unoccupied sites typically were short (19 ± 17 m SD) and/or experienced chimney-

effect airflow, which led to temperatures near or below freezing (-0.8 ± 2.9 ºC). Overall, occupied sites

were more structurally complex, longer (307 ± 865 m), and warmer (5.7 ± 3.0 ºC) than unoccupied mines.

Number of bats varied from 1 to > 55,000, although the median was 115. Eastern Pipistrelles (Perimyotis

subflavus) and Big Brown Bats (Eptesicus fuscus) accounted for only 0.5% of the total of 244,341 bats

that were observed. Ninety percent of hibernating animals were Little Brown Bats (Myotis lucifugus), and

almost 10% were Northern Bats (M. septentrionalis). Relative to Little Brown Bats, Northern Bats were

more common in the mines of the Upper Peninsula than in hibernacula in the East and Ohio River Valley.

Maximum ambient temperature, presence of standing water, and water vapor pressure deficit were

potential predictors of the number of Myotis that was present. Seventy-five percent of Northern Bats and

* indicates presenting author

22% of Little Brown Bats roosted alone, rather than cluster with other bats. Little Brown Bats in

Michigan were solitary much more often than in the East.

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Hibernating bats can offset arousal costs by not warming up to normal body temperatures:

implications for energy budgeting in WNS

Heather Mayberry1, Liam McGuire

1, and Craig Willis

1

1University of Winnipeg, Winnipeg, MB

Periodic arousals account for the majority of the energy cost of hibernation. With WNS, increased arousal

frequency depletes energy stores leading bats to die of starvation. We hypothesized that bats could use

physiological mechanisms to reduce arousal energy expenditures. Bats sometimes use heterothermy

(lowering body temperature) during arousals, therefore we predicted bats could save energy by arousing

without increasing body temperature. Furthermore, we predicted non-warming arousals would be

associated with extreme energy distress at the end of hibernation. To test these predictions, we collected

hibernating bats from a WNS-negative cave in Manitoba. Bats were inoculated with either Pd or a sham

control, and placed in temperature and humidity controlled environmental chambers for hibernation. We

recorded activity with motion-sensitive IR video cameras, and skin temperature (Tsk) with iButtons

affixed to the bats. We observed some bats experiencing slow, shaky and uncoordinated movements

during arousals which were strikingly different from typical arousal activities. These abnormal arousals

corresponded with Tsk <15°C, whereas normal arousals occurred at Tsk >25°C. These “cold arousals”

were observed in both inoculated and control animals, suggesting it is not a physiological response to

disease. As we predicted, “cold arousals” typically occurred in the final days of hibernation, when energy

stores were nearing exhaustion. However, we also observed “cold arousals” associated with disturbance

in mid-hibernation. Therefore we suggest that “cold arousals” are a facultative strategy that may be used

to manage energy stores, rather than being related to disease and extreme energy distress.

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Uncovering skin immune proteins as predictors of resistance against WNS Marianne Moore

1, Liliana Dávalos

1, and Amy Russell

1Stony Brook University, New York,

2Grand Valley State University

We are launching a new study to investigate the composition of bat skin immune proteins as predictors of

resistance to white-nose syndrome (WNS), and to discover the mechanisms underlying the survival of

remnant populations in the WNS-affected area. The project uses proteomics to characterize and compare

the diversity and relative abundance of skin immune proteins of five bat species that vary in observed

rates of WNS-associated mortality (Myotis lucifugus, Eptesicus fuscus, M. austroriparius, M. grisescens,

and Corynorhinus townsendii virginianus). We will compare protein profiles among species to test the

prediction that certain proteins related to anti-fungal responses are more prevalent in species that appear

to suffer less from the effects of WNS, such as M. grisescens and C. townsendii virginianus. To test the

prediction that proteins prevalent in survivors of more highly susceptible species are similar to those

found in resistant species, M. lucifugus and E. fuscus are being more extensively sampled both within and

outside of the WNS-affected area and their protein profiles will be compared between sites. Microsatellite

genotyping will be used to quantify levels of relatedness among sampled individuals, which will allow for

* indicates presenting author

functional and adaptive similarity in immunological proteins to be differentiated from similarity due to

common ancestry. We focus on antimicrobial peptides (AMPs), a set of proteins that is known to kill or

inhibit the growth of invading microorganisms such as fungi. Finally, we will investigate these peptides

as a control for WNS.

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Florida caves: Suitable for the growth of Pseudogymnoascus destructans and used by wintering

tricolored bats

Kevin J. Oxenrider1*, Jeff Gore

2, L. Smith

3, Terry Doonan

1, and M. Tucker

1

Florida Fish and Wildlife Conservation Commission, 1Lake City, FL,

2Panama City, FL,

3Gainesville, FL

While Pseudogymnoascus destructans has not been detected in any Florida cave as of February 2014, it

has now been reported in Alabama, Georgia, and Mississippi. Since 2011, Florida Fish and Wildlife

Conservation Commission (FWC) biologists have monitored ambient cave temperatures and found

Florida’s caves to be within the range for P. destructans growth, causing concern that the fungus could

impact cave-hibernating species such as the tricolored bat (Perimyotis subflavus). The tricolored bat is

the most common bat species known to enter extended torpor in Florida and likely the most susceptible to

P. destructans and WNS. However, little is known about current hibernacula and winter abundance for

this species in Florida. In February 2014, FWC biologists surveyed 32 caves to locate tricolored bat

hibernacula. A total of 377 tricolored bats were recorded in 20 caves. In caves containing >10 tricolored

bats, 10–20 individuals were swabbed to test for the presence of P. destructans. Wall temperatures near

torpid tricolored bats were similar in northwest and north-central Florida caves (t = 1.66, p = 0.311) and

were appropriate for the growth of P. destructans (6.9 ± 2.8°C). However, all swab samples were

negative for the presence of P. destructans. FWC will continue to survey caves in winter to document

hibernacula and abundance of tricolored bats, and to monitor for the presence of P. destructans. FWC

biologists will also work with land managers and the public to increase WNS education, and implement

decontamination procedures at public caves where appropriate.

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