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Examination of several Oklahoma bat hibernacula cave soils for
Pseudogymnoascus destructans, the causative agent of White-Nose Syndrome
Author(s): James P. Creecy, William Caire, and Kylie A. Gilcrest
Source: The Southwestern Naturalist, 60(2-3):213-217.
Published By: Southwestern Association of Naturalists
DOI: http://dx.doi.org/10.1894/JKF-53.1
URL: http://www.bioone.org/doi/full/10.1894/JKF-53.1
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2. THE SOUTHWESTERN NATURALIST 60(2-3): 213–217 JUNE-SEPTEMBER 2015
EXAMINATION OF SEVERAL OKLAHOMA BAT HIBERNACULA CAVE
SOILS FOR PSEUDOGYMNOASCUS DESTRUCTANS, THE CAUSATIVE
AGENT OF WHITE-NOSE SYNDROME
JAMES P. CREECY, WILLIAM CAIRE,* AND KYLIE A. GILCREST
Biology Department, University of Central Oklahoma, 100 North University Drive, Edmond, OK 73034
*Correspondent: wcaire@uco.edu
ABSTRACT—White-nose syndrome is an often lethal fungal infection of bats that is caused by the fungus
Pseudogymnoascus destructans, formerly Geomyces destructans. The fungal spores can persist for extended periods
of time in the soil and on surfaces in caves where it might be found even after the bats depart. In 2010, a single
bat, Myotis velifer, from a western Oklahoma gypsum cave, was initially diagnosed by the U.S. Geological Survey
National Wildlife Health Center, as ‘‘suspect white-nose syndrome.’’ Based on this, we decided to examine soil
samples from various bat caves across Oklahoma for the presence of P. destructans. We used Real-time
Polymerase Chain Reaction to analyze 83 soil samples from 17 caves in Oklahoma. None of the soil samples
were found to contain genetic material from P. destructans. We postulate that P. destructans has not yet reached
Oklahoma because of the negative Real-time Polymerase Chain Reaction results, because a National Wildlife
Health Center reexamination of the original bat suggested the bat was negative for P. destructans, and
subsequent analyses of bat nose and wing swabs, bat tissues, and physical examination of hibernating bats have
all been negative for white-nose syndrome.
RESUME—El s´ındrome de nariz blanca es una infecci´on mic´otica a menudo letal en murci´elagos que es
causada por el hongo Pseudogymnoascus destructans, anteriormente Geomyces destructans. Las esporas de los
hongos pueden persistir por largos per´ıodos de tiempo en el suelo y en superficies en las cuevas donde se
pueden encontrar incluso despu´es de que los murci´elagos se van. En 2010, un s´olo murci´elago Myotis velifer, de
una cueva de yeso al oeste de Oklahoma fue diagnosticado inicialmente por el USGS Nacional Wildlife Health
Center (NWHC), como ‘‘sospechoso del s´ındrome de nariz blanca’’. Bas´andonos en esto, decidimos examinar
las muestras de suelo de varias cuevas de murci´elagos a trav´es de Oklahoma por la presencia de P. destructans.
Se utiliz´o PCR en tiempo real para analizar 83 muestras de suelo de 17 cuevas en Oklahoma. Ningunas de las
muestras de suelo conten´ıa el material gen´etico del P. destructans. Postulamos que P. destructans todav´ıa no ha
llegado a Oklahoma debido a los resultados negativos de PCR en tiempo real, debido a que una
reexaminaci´on del NWHC del murci´elago original sugiri´o que el murci´elago result´o negativo para el P.
destructans y debido a que unos an´alisis posteriores de hisopos de nariz y ala de murci´elagos, tejidos de
murci´elagos, y el examen f´ısico de murci´elagos en hibernaci´on han sido negativos para el s´ındrome de nariz
blanca.
White-nose syndrome (WNS) is an often lethal
parasitic infection of bats that is caused by Pseudogymnoas-
cus destructans, formerly Geomyces destructans (Minnis and
Linder, 2013), a psychrophilic and keratinophylic asco-
mycete fungus (Blehert et al., 2009, 2011; Cryan et al.,
2010). WNS has been described as the most devastating
disease ever reported for hibernating bats (Moore et al.,
2011) and it has caused widespread mortality to millions
of bats in the United States (Verant et al., 2012). The
disease has progressively, since the initial discovery in New
York in 2006 (Blehert et al., 2009), spread to the south
and west. In 2014, bats with WNS were confirmed in
Missouri and Arkansas near the border with Oklahoma
(White-Nose Syndrom.org; http://whitenosesyndrome.
org/resources/map).
A cave myotis, Myotis velifer, found with white crusty
material on the skin, was submitted from Woodward
County in western Oklahoma to the United States
Geological Service National Wildlife Health Center
(NWHC) to determine whether the white material was
attributed to P. destructans. In May 2010, the NWHC
reported that the submitted bat was ‘‘suspect WNS’’ for P.
destructans (NWHC Diagnostic Final Report 23042, May
2010). For four years following the initial report from the
NWHC, hibernating M. velifer in the same cave where the
suspect WNS bat was found, as well as several other major
hibernation sites in western Oklahoma, were examined

3. during late hibernation (January and February) for visible
signs of WNS. To date, no bats have been seen that appear
to have WNS (W. Caire, pers. observ.).
The absence of signs of WNS on the bats was an
encouraging observation for the health of the bat
hibernacula, but it is known that P. destructans produces
spores that can persist for extended periods in the soil
and on surfaces in caves where bats hibernate even after
the bats depart (Lorch et al., 2013). Because of the report
of a WNS suspect bat from western Oklahoma mentioned
earlier, we analyzed soils from selected bat hibernacula in
Oklahoma in 2011 and 2012 to determine whether
genetic material indicative of P. destructans might be
present.
METHODS AND MATERIALS—Sample Collection and DNA
Extraction—During the 2011 and 2012 bat hibernation
season, we collected 83 soil samples from 17 Oklahoma
cave systems (Table 1) known to be inhabited by bat
colonies. We followed universal precautions to prevent
the spread of P. destructans (U.S. Fish and Wildlife Service,
http://www.fws.gov/WhiteNoseSyndrome/) during cave
visits and the processing of soil samples. Procedures for
sampling in the bat caves were approved by the
Institutional Animal Care and Use Committee of the
University of Central Oklahoma. We also followed
guidelines of the American Society of Mammalogists for
the Use of Wild Mammals in Research (Sikes et al., 2011).
The soil collections taken in the caves and near the bats
were under the auspices of a Scientific Collecting Permit
issued by the Oklahoma Department of Wildlife Conser-
vation or the United States Fish and Wildlife Service.
During each soil sample collection, we took care to
prevent cross-contamination (sterilized sample contain-
ers, a new pair of latex gloves, use of sterilized soil-
collecting spatulas, placing each sample in a separate
ziplock bag, etc.) among soil samples and to minimize the
impact to the hibernacula. We collected 1–4 soil samples;
usually 1 from the entrance to the cave, in the twilight
region, and usually 2 from separate interior locations in
TABLE 1—Caves in Oklahoma from which 83 soil samples were screened for Pseudogymnoascus destructans DNA, the causative agent
of white-nose syndrome. Included are locations in the caves where soil samples were analyzed: E, entrance; TW, twilight; UB, under
bats; BC, back of cave in total darkness. AD-6, AD-7, AD-14, DL-39 and DL-91 are cave codes used by the U.S. Fish and Wildlife Service
to protect the location of caves with endangered species of bats. Touchdown PCR and Real-time PCR analysis was conducted to detect
Geomyces or a closely related fungal species and P. destructans DNA, respectively. All samples that were analyzed using Real-time PCR
(represented as RT) were negative for P. destructans DNA. Three of the sixteen soil samples analyzed by touchdown PCR (represented
as PCR) were positive for Geomyces or a closely related fungal species DNA and are identified by an *. The remaining samples analyzed
by touchdown PCR were negative. No data is indicated by the em dash (—).
Year Cave, Provenience
Soil sample sites and method of PCR analysis
E TW UB BC
2011 AD-6, Adair Co. RT RT RT RT
2011 AD-7, Adair Co. RT RT RT RT
2011 AD-14, Adair Co. (Back Door) RT RT RT RT
2011 AD-14, Adair Co. (Main Entrance) RT RT RT RT
2011 AD-14, Adair Co. (Third Cave) RT RT RT RT
2011 AD-14, Adair Co. (Side Door) RT RT RT RT
2011 Alabaster Caverns, Woodward Co. — — PCR/RT PCR/RT
2011 Broken Horn, Woodward Co. RT — — —
2011 Cattle Cave, Woodward Co. — — RT RT
2011 DL-39, Delaware Co. — RT RT —
2011 DL-91, Delaware Co. RT RT — —
2011 Faulkner Cave, Woods Co. — — PCR/RT PCR/RT
2011 Jester Cave, Greer Co. RT RT RT RT
2011 Merihew Cave, Woods Co. RT RT PCR/RT PCR/RT
2011 Nescatunga Cave, Major Co. — — PCR/RT PCR/RT
2011 Selman Bat Cave, Woodward Co. RT RT RT RT
2011 Selman Cave System-Skylight, Woodward Co. RT RT PCR*/RT PCR/RT
2011 Selman Cave System-Skunkeater, Woodward Co. RT RT RT RT
2011 Washita Cave Bat Cave, Washita Co. — — PCR/RT PCR*/RT
2012 Gnarled Knuckle, Murray Co. RT RT RT —
2012 Jester Cave, Greer Co. RT RT RT RT
2012 Nescatunga Cave, Major Co. RT RT PCR/RT PCR*/RT
2012 Selman Bat Cave, Woodward Co. RT RT RT —
2012 Selman Cave System-Skunkeater, Woodward Co. RT RT RT RT
2012 Washita Cave Bat Cave, Washita Co. RT RT PCR/RT PCR/RT
2012 Vickery Bat Cave, Major Co. RT RT — —
214 vol. 60, no. 2-3The Southwestern Naturalist

4. total darkness. If bats were present, we took a soil sample
from under or near where the bats were roosting. Soil
samples were kept cool and transported to the University
of Central Oklahoma (Edmond, Oklahoma) where they
were stored at -808C until DNA was extracted. Although
the cave environments were not a focus of this study, we
included the air temperatures and relative humidities
recorded (HOBO data loggers; Onset Computer Corpo-
ration, Bourne, Maine) on 28 January 2011 from near the
soil collection sites in the Selman Cave System. This is the
cave where the NWHC, (Diagnostic Services Case report
23042, May 2010) first indicated that P. destructans was
present in western Oklahoma.
We isolated total environmental DNA from each soil
sample using the PowerSoil DNA Isolation Kit (MoBio
Laboratories Inc., Carlsbad, California) as recommended
by the manufacturer. We performed proper experimental
procedures and controls throughout the DNA extraction
process to evaluate the potential for contamination. All
soil samples were opened individually and never shared
time or space with another soil sample. Between each
sample, we decontaminated the laboratory bench and
applied new bench coat. We included extraction blank
controls, containing all reagents but lacking a soil sample,
with each set of extracted samples. All extraction blank
controls performed as expected, and supported the
conclusion that the samples were not contaminated
during DNA extraction. We quantified total environmen-
tal DNA from extracted samples using a Nanodrop 2000
(Thermo Scientific, Wilmington, Delaware) and stored it
at -208C prior to subsequent Polymerase Chain Reaction
(PCR) and Real-time PCR analyses.
Touchdown PCR—DNA quality and quantity obtained
from soil samples was variable; therefore, we employed
touchdown PCR as a sensitive and highly specific method
for the amplification of DNA from Geomyces and close
relatives (Don et al., 1991; Lindner and Banik, 2009).
Each touchdown PCR reaction contained a final volume
of 25 lL. Within each reaction was 3.5 lL Failsafe Buffer
I, 1.2 lL of each 10 lM PCR primer, 5U of AmpliTaq
Gold, and 5 lL template DNA (4–178 ng total DNA;
Epicentre Technologies, Madison, Wisconsin). We uti-
lized the previously published primer set nu-SSI(1506)-
184-50
-Gd and nu-5.8S-144-30
-Gd (Lorch et al., 2010)
under the following thermocycling conditions: 988C for 2
min; 40 cycles of denaturation at 958C for 30 s, 3-min
annealing period, and 728C extension for 1 min; and a
final extension at 728C for 7 min. The annealing
temperature for the first 20 cycles consisted of 0.58C
temperature step-downs every cycle (from 608C to 508C);
the annealing temperature for the final 20 cycles was
508C. Positive and negative controls were run concurrent-
ly during PCR amplification. Positive controls contained
of 10 ng of P. destructans genomic DNA purchased from
American Type Culture Collection (MYA-4855). We
evaluated PCR inhibition in an independent set of
reactions by adding 50 ng of P. destructans genomic
DNA (American Type Culture Collection MYA-4855) in
place of 1 lL of water to the reaction while keeping all
other conditions consistent.
Real-time PCR Survey for P. destructans—We subjected
DNA extracted from each soil sample to Real-time PCR
analysis using the validated Real-time IGS PCR test
established at the United States Geological Service-
National Wildlife Health Center (Muller et al., 2013).
The primers and probe published by Muller et al. (2013)
were previously characterized as specific for the IGS
region of P. destructans. Both primers and the probe were
synthesized by Biosearch Technologies Inc. (Novato,
California), and Real-time PCR of the IGS region of P.
destructans was conducted using an ABI 7500 Real-Time
PCR system (Life Technologies, Grand Island, New York)
and the QuantiFaste Probe PCR + ROX Vial Kit master
mix, (QIAGEN Inc., Valencia, California) according to
the manufacturer’s instructions. We avoided cross-con-
tamination by following standard laboratory practices and
monitored cross-contamination by analysis of reagent
blank samples. Each Real-time PCR reaction had a total
volume of 25 lL. Contained within each reaction was 12.5
lL 2· master mix, 0.5 lL 50· ROX dye solution, 0.5 lL
of each 20 lM PCR primer, 0.25 lL of 20 lM probe and 5
lL template DNA (Muller et al., 2013). We used known P.
destructans genomic DNA as a positive control for all runs.
PCR cycling conditions included a 958C for 3 min initial
activation step followed by 40 cycles of 958C for 3 s and
608C for 30 s. We classified a sample as positive if
maximum fluorescence exceeded 10% of the maximum
fluorescence of the positive control samples (King and
Guidry, 2004), and we classified failure to exceed this level
of fluorescence as a negative result.
RESULTS—Environmental DNA Extraction, Amplification
and Real-time PCR analysis—We extracted 83 total soil
samples using the Powersoil Isolation Kit, and DNA
concentration from each sample ranged from 0.8 to 35.6
ng/lL. PCR amplification of DNA obtained from soil
samples has been known to present a number of
challenges, including PCR inhibition and uncertainty of
fungal DNA abundance in the extract. As stated above, we
evaluated PCR inhibition by addition of known P.
destructans DNA to an otherwise standard PCR reaction.
For all soil samples evaluated, addition of 5 lL of DNA
extract to a traditional PCR reaction did not result in the
observation of PCR inhibition by gel electrophoresis. We
analyzed 16 soil samples from 8 cave systems by
touchdown PCR, and 3 samples indicated the presence
of DNA from Geomyces or a species closely related to
Geomyces (see Table 1). In order to more accurately
evaluate the possible presence of P. destructans DNA, we
conducted a Real-time PCR test of the species-specific IGS
region. Following Real-time PCR analysis of all 83 soil
samples collected over the 2-year period, all evidence
June 2015 Creecy et al.—Examination of bat hibernacula soils for Pseudogymnoascus destructans 215

5. indicates that there is an absence of P. destructans DNA
within the Oklahoma hibernacula tested.
DISCUSSION—This study is the first attempt to determine
whether P. destructans is present in caves soils on the
western edge of the area where WNS has been reported in
the United States. It appears that at the time of the
analysis of the soil samples, there was no P. destructans
DNA within the Oklahoma hibernacula tested. This
provides baseline chronological data should P. destructans
enter Oklahoma in the future.
Several possibilities exist as to explain why P. destructans
has not been found in any of the Oklahoma cave soils
examined. The first possibility is that the fungus has not
yet been dispersed into Oklahoma by wildlife, humans, or
other means. The single M. velifer from western Oklaho-
ma that was originally reported in 2010 as P. destructans
suspect by the NWHC was recently reexamined by
histopathology, fungal culture, and PCR, using a more
specific procedure (Muller et al., 2013); and upon review,
it was reclassified as negative for WNS (NWHC Supple-
mental Report for Diagnostic Final Report 23042, April
2014). During the hibernating season of 2010–2011, five
M. velifer, from the same cave in northwestern Oklahoma
from which the first suspect WNS bat was reported, were
submitted to the NWHC for testing. Those bats also
yielded negative results (NWHC, Diagnostic Final Report
23571, June 2011). A tri-colored bat (Perimyotis subflavus),
collected in Adair County in eastern Oklahoma in 2011,
also tested negative for WNS (NWHC, Diagnostic Final
Report 23570, July 2011). In addition, 81 combination
wing/muzzle swabs from hibernating M. velifer in Wood-
ward, Washita, and Greer counties in Oklahoma were
submitted to the NWHC during the winter of 2014 for
testing, and all were negative for P. destructans (NWHC
Diagnostic Services Final Reports Case Reports 24764,
24765, 24766, 24767, April 2014). Thus it appears likely
that P. destructans has not yet spread to western Oklahoma
bats and thus would not be in the soils.
A second possibility is that P. destructans reached
Oklahoma, but either did not become established or
persists at such low abundance that it was not easily
detected. Boyles and Willis (2010) suggested that local-
ized warm areas inside cold hibernacula might reduce
mortality of hibernating bats affected by WNS. Verant et
al. (2012) described temperature-dependent growth
performances of P. destructans isolates and noted that
optimal growth temperatures were between 12.58 and
15.88C, with an upper critical temperature for growth
between 19.08 and 19.88C. Above 128C, all P. destructans
isolates displayed atypical morphology. Their study
demonstrated that variations in hibernacula temperatures
could affect the growth and physiology of P. destructans,
which might impact the persistence and abundance of P.
destructans in western bat gypsum-cave hibernacula. Myotis
velifer and other bat species usually hibernate in western
Oklahoma gypsum caves from October to March.
Temperatures, relative humidity, and number of bats
counted and checked for WNS in various cave sections of
the largest known M. velifer gypsum-cave hibernaculum in
western Oklahoma are presented in Table 2 for 28
January 2011. This is the cave from which the original
WNS suspect positive bat (now negative) was tested by the
NWHC. Although the stability and microhabitats of
western Oklahoma gypsum and eastern limestone bat
caves have not yet been compared, we suspect that the
limestone caves in eastern Oklahoma are probably more
similar in geology and environmental conditions to the
TABLE 2—Temperature, relative humidity, soil sample locations, and the number of bats counted (total bats = 46,988) and surveyed
for white-nose syndrome in various sections of the Selman Cave System, the largest known Myotis velifer gypsum-cave hibernaculum in
western Oklahoma (Woodward Co.), on 28 January 2011.
Skunkeater entrance total of 23,253 bats
Cave section Temperature Relative humidity No. of bats
Entrance (soil sample) 16.08C 23% 0
Twilight (soil sample) 11.78C 34% 0
Base of Chimney 8.78C 50% 18
Stream passage (soil sample) 7.28C 58% 15,277
Past First Breakdown (soil sample) 8.68C 63% 4,652
Big Dome 13.48C 58% 16
Past Big Dome 8.78C 65% 3,290
Skylight entrance total of 23,735 bats
Cave section Temperature Relative humidity No. of bats
Entrance (soil sample) 18.18C 23% 0
Twilight (soil sample) 18.18C 31% 10,075
Pass Twilight (soil sample) 8.78C 52% 13,609
Under Breakdown (soil sample) 9.38C 53% 49
Rear Entrance 9.98C 54% 2
216 vol. 60, no. 2-3The Southwestern Naturalist

6. caves farther east in the United States where WNS is
prevalent. Thus, we suspect that WNS will soon be seen in
eastern Oklahoma caves where several endangered bat
species (Myotis grisescens, Myotis sodalis, and Corynorhinus
townsendii ingens) reside. The possibility that microcli-
mates in western bat gypsum-cave hibernacula might not
be conducive for survival of P. destructans should be
examined over several seasons if no WNS is found in
western Oklahoma in the next few years. We encourage
biologists to continue to monitor Oklahoma cave soils for
the possible existence of P. destructans. This would provide
baseline information related to the ecology and physiol-
ogy of P. destructans and help improve our understanding
of the spread of P. destructans and WNS.
We appreciate the cooperative efforts of the following groups
and individuals who assisted in the field and lab work during this
project: Central Oklahoma Grotto, students and other faculty at
the University of Central Oklahoma, including: E. York, T.
Payne, L. Loucks, T. Cloud, S. Frasse, and J. Bowen. We thank
the following individuals on staff with the National Wildlife
Health Center for their help with the testing of the bats for
white-nose syndrome and providing the final diagnostic reports:
D. E. Green, V. Shearn-Boschler, D. Blehert, A. Ballmann, and C.
L. White. We appreciate the financial support from U.S. Fish
and Wildlife Service and their providing soil samples from bat
caves in eastern Oklahoma. We appreciate the help and
cooperation of the Oklahoma Department of Tourism, Alabaster
Caverns State Park, and the Oklahoma Department of Wildlife
Conservation. We are also grateful to the Oklahoma landowners
who allowed us access to the caves on their properties.
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Acceptance recommended by Associate Editor, Jennifer K. Frey, 7 October
2014.
June 2015 Creecy et al.—Examination of bat hibernacula soils for Pseudogymnoascus destructans 217