Inoculation of bats with European Geomyces
destructans supports the novel pathogen hypothesis
for the origin of white-nose syndrome
Lisa Warneckea,1, James M. Turnera,1, Trent K. Bollingerb, Jeffrey M. Lorchc,d, Vikram Misrae, Paul M. Cryanf,
Gudrun Wibbeltg, David S. Blehertd, and Craig K. R. Willisa,2
aDepartment of Biology and Centre for Forest Interdisciplinary Research, University of Winnipeg, Winnipeg, MB, Canada R3B 2E9;bDepartment of
Veterinary Pathology, Canadian Cooperative Wildlife Health Centre, andeDepartment of Veterinary Microbiology, Western College of Veterinary Medicine,
University of Saskatchewan, Saskatoon, SK, Canada S7N 5B4;cMolecular and Environmental Toxicology Center, University of Wisconsin, Madison, WI 53706;
dUS Geological Survey, National Wildlife Health Center, Madison, WI 53711;fUS Geological Survey, Fort Collins Science Center, Fort Collins, CO 80526; and
gLeibniz Institute for Zoo and Wildlife Research, 10315 Berlin, Germany
Edited by Jitender P. Dubey, US Department of Agriculture, Beltsville, MD, and approved March 9, 2012 (received for review January 9, 2012)
White-nose syndrome (WNS) is an emerging disease of hibernat-
ing bats associated with cutaneous infection by the fungus Geo-
myces destructans (Gd), and responsible for devastating declines
of bat populations in eastern North America. Affected bats appear
emaciated and one hypothesis is that they spend too much time
out of torpor during hibernation, depleting vital fat reserves re-
quired to survive the winter. The fungus has also been found at
low levels on bats throughout Europe but without mass mortality.
This finding suggests that Gd is either native to both continents
but has been rendered more pathogenic in North America by mu-
tation or environmental change, or that it recently arrived in North
America as an invader from Europe. Thus, a causal link between
Gd and mortality has not been established and the reason for its
high pathogenicity in North America is unknown. Here we show
that experimental inoculation with either North American or Eu-
ropean isolates of Gd causes WNS and mortality in the North
American bat, Myotis lucifugus. In contrast to control bats, indi-
viduals inoculated with either isolate of Gd developed cutaneous
infections diagnostic of WNS, exhibited a progressive increase in
the frequency of arousals from torpor during hibernation, and
were emaciated after 3–4 mo. Our results demonstrate that al-
tered torpor-arousal cycles underlie mortality from WNS and pro-
vide direct evidence that Gd is a novel pathogen to North America
fungal pathogen|infectious disease|invasive species|Chiroptera|
destructans (Gd) (1). WNS has killed millions of bats across 16
US states and four Canadian provinces since its emergence in
New York State in 2006 (2). So far, nine bat species from three
genera, all of which hibernate in caves or mines, have been found
to carry Gd, and mortality of infected bats has been observed in
six of these species (2). The population dynamics of most af-
fected species are not well understood but the effects of current
declines are likely to be drastic; for example, the little brown bat
(Myotis lucifugus) was the most widespread and common bat
species in North America before WNS, but is now predicted to
face local extinction in WNS-affected areas within two decades
(3). During hibernation, the skin of WNS-affected bats is co-
lonized by Gd, which invades cutaneous tissues of the muzzle,
ears, and wings (4, 5). Major inflammation is usually not ob-
served in infected tissues (6), possibly because immune respon-
ses in hibernating animals are suppressed (7). Mortality occurs
in the second half of the hibernation season and affected bats
are typically emaciated. Recently Lorch et al. (1) showed that
experimental inoculation of M. lucifugus with Gd caused the
characteristic wing lesions associated with WNS, and confirmed
hite-nose syndrome (WNS) is a rapidly spreading wildlife
disease caused by the cold-tolerant fungus Geomyces
that Gd can be spread by direct contact between bats. However,
no study has established a causal mechanism linking Gd with
One possible explanation for mortality from WNS is that Gd
causes a disruption of energy balance during hibernation. Hi-
bernating mammals spend the majority of their time in torpor,
a state of controlled reduction in body temperature (Tb) and
metabolic rate, which is interrupted by brief periodic arousals to
normothermic Tb(8). Although these arousals last less than 24 h
in most species, the high metabolic cost of thermoregulation
during normothermia at a low ambient temperature (Ta) means
they account for the vast majority of over-winter energy expen-
diture (8, 9). Food is unavailable for most temperate-zone bats
during winter, so they must survive on stored fat (9). Therefore,
one hypothesis to explain WNS-related mortality is that Gd
causes bats to increase the duration and/or frequency of peri-
odic arousals, resulting in premature depletion of fat and con-
sequently starvation (10). Preliminary support for this hypothesis
was found based on an energetic model (11) but, to date, there is
no experimental evidence that bats infected with Gd spend more
time out of torpor than uninfected controls.
In addition to the mechanism underlying mortality, the origin
of WNS is still unknown. There are two competing explanations
for the origin of any emerging infectious disease (12). Such a
disease may result from a pathogen that has been present his-
torically but is rendered more pathogenic by a genetic mutation
or environmental change (i.e., the endemic pathogen hypothe-
sis). Alternatively, a pathogen may arrive in a new geographic
area and encounter a naive host population (the “novel” or in-
vasive pathogen hypothesis) (12). It is now established that Gd
occurs at low levelsonbats throughout Europe, whereit has been
isolated from eight Myotis spp., but with no evidence of mass
mortality (13, 14). Given that Gd went undiscovered in Europe
until WNS was observed in North America, one possibility is that
Gd has occurred historically at low levels on bats from both
continents but went unnoticed until mass mortality of bats in
North America led to intensive sampling for a potential patho-
gen. This theory is cause for concern because European bats
could be at risk from the accidental introduction of North
Author contributions: P.M.C., D.S.B., and C.K.R.W. designed research; L.W., J.M.T., T.K.B.,
V.M., and C.K.R.W. performed research; J.M.L., G.W., and D.S.B. contributed new re-
agents/analytic tools; L.W., J.M.T., and C.K.R.W. analyzed data; and L.W., J.M.T., and
C.K.R.W. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1L.W. and J.M.T. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
www.pnas.org/cgi/doi/10.1073/pnas.1200374109 PNAS Early Edition
| 1 of 5
American Gd to bat hibernacula in Europe. Alternatively, Gd
may have arrived in North America as a recent invader from
Europe, perhaps introduced by tourists visiting caves. Wibbelt
et al. (14) hypothesized that under this novel pathogen scenario
Europeanbats may have coevolved with Gd over many years, and
differences in its apparent pathogenicity for North American
versus European bats could reflect differences in the physiology
or behavior of the bats or differences in their environments,
rather than intercontinental differences in Gd. Confirming one
or the other of these hypotheses is essential because different
disease-management strategies are warranted for invasive versus
endemic pathogens (12).
We conducted an inoculation experiment with M. lucifugus to
evaluate three hypotheses important for our understanding of
WNS. First, we tested a key prediction of the novel pathogen
hypothesis, which predicts that Gd isolated from Europe should
cause the same clinical signs in a North American bat species as
Gd isolated from North America. Therefore, we inoculated in-
dividual M. lucifugus with either a North American isolate of Gd
(NAGd) or a European isolate (EUGd) and assessed clinical
signs following several months of infection. Second, we tested
whether inoculation with Gd, alone, is sufficient to cause mor-
tality, a fundamental question about WNS that has still not been
addressed. Third, by monitoring skin temperatures of bats
following inoculation, we assessed the hypothesis that infection
with Gd causes bats to increase the frequency and/or duration of
periodic arousals during hibernation, leading to premature fat
depletion (10, 11). Importantly, we kept animals in environmental
conditions closely matched to those of M. lucifugus hibernacula
(9), particularly in terms of high relative humidity (RH).
All bats entered multiday torpor bouts (i.e., began hibernating)
within the first week of the study (Fig. 1). Average torpor bout
duration over the entire study period was 9.0 ± 1.0 d for NAGd
bats (individual range 1.2–32.4 d), 6.1 ± 0.6 d for EUGd bats
(1.0–21.8 d), and 16.0 ± 0.9 d for sham-infected control group
(CO) bats (2.0–33.4 d). Both NAGd and EUGd caused a pro-
gressive increase in the frequency of periodic arousals over the
course of the experiment. There was no significant difference
among groups during Interval 1, but treatment groups aroused
significantly more often throughout the rest of hibernation
(Table 1). In fact, during Interval 3, arousal frequency of NAGd
bats was three times—and EUGd bats four times—that of CO
bats (Fig. 2 A and B). The effect of time on arousal frequency
was significant for each group (Table 2) with a significant in-
crease over time for both NAGd and EUGd bats, and a decrease
for CO bats (Fig. 2A). In contrast, the duration of periodic
arousals was not affected by inoculation (Fig. 2C and Table 1),
nor by time for any group (Table 2).
Both isolates of Gd caused all known clinical signs of WNS
(5, 6), including loss of elasticity, irregular pigmentation and
stickiness of wing tissue, and white surface growth. Histopa-
thology confirmed infection in all NAGd and EUGd bats as
fungal hyphae penetrated the epidermis and damaged underlying
tissues, consistent with previous studies (5, 6) (Fig. 3 A and B).
Gd was also cultured from sections of wing tissue for bats from
both treatment groups but no CO bats showed any evidence of
infection by Gd (Fig. 3C).
There was a highly significant effect of inoculation on survival
for both NAGd (x2= 17.1, P < 0.001) and EUGd bats (x2= 26.4,
P < 0.001) compared with controls, and NAGd bats survived
significantly longer than EUGd bats (Fig. 4) (x2= 20.3, P <
0.001). Mortality was first observed for EUGd bats on day 71,
and surviving bats from the group were euthanized on day 91
after 16 bats reached moribund status. Mortality first occurred
for NAGd bats on day 88 and surviving bats from this group were
terminated on day 114 after 12 bats reached moribund status.
Two EUGd and four NAGd bats were unable to arouse from
torpor when removed from the chamber at the end of the ex-
periment and were therefore also considered moribund. Based
on necropsies, bats from both treatment groups had virtually no
fat reserves remaining. On day 119, when we terminated the CO
two each from the following groups: (A) inoculated with NAGd; (B) in-
oculated with EUGd; (C) sham-inoculated control. The x axis shows the day
of study, where day 1 is November 27, 2010; the bars at the bottom indicate
the division of the study period into 26-d intervals, and months.
Representative traces of skin temperature (Tskin) for six M. lucifugus,
and arousal duration
Sample sizes and ANOVA results for arousal frequency
Interval NAGd (n) EUGd (n) CO (n) df1df2
36 20.79 <0.001
40 52.82 <0.001
Sample sizes and ANOVA results for arousal frequency (arousal bat−1·d−1)
and arousal duration (length of time above skin temperature threshold) for
the four time intervals for M. lucifugus inoculated with NAGd and EUGd com-
comparisons (SNK) are indicated in Fig. 2. Significant results are in bold.
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| www.pnas.org/cgi/doi/10.1073/pnas.1200374109 Warnecke et al.
group, all bats were alive, capable of endogenous arousal from
torpor, and still had subcutaneous fat reserves.
The susceptibility of a North American bat species to both EUGd
and NAGd strongly supports the novel pathogen hypothesis that
accidental introduction of Gd from Europe is responsible for the
WNS-related mass mortality of bats in North America. Our data
suggest that the absence of mortality observed among European
bats infected with Gd reflects different physiological and be-
havioral responses of European versus North American bats
rather than a heightened pathogenicity of NAGd (14). This
finding also supports the hypothesis of Wibbelt et al. (14) that
Gd may have impacted European bat populations in the past and
that bats in Europe have coevolved resistance to (e.g., via im-
mune system responses), or tolerance of (e.g., via behavioral
adaptations), infection with Gd. These findings have significant
implications for management and future research. Endemic
pathogens are best addressed via management of factors that
enhance virulence of the pathogen (e.g., environmental or biotic
cofactors), and novel pathogens are best dealt with by managing
the agents that spread the disease (12). Managing agents of
spread for WNS will be impractical, if not impossible, because
the putative agents (i.e., the bats) are highly cryptic, widely dis-
persed for much of the year, and wide-ranging. However, our
results support the high priority of research aimed at under-
standing temporal and spatial aspects of Gd transmission in the
wild, as this work could aid in the development of management
strategies focused on critical locations or times of year when Gd
is likely to be transmitted. Encouragingly, our findings suggest
that European bats face little risk from the possible reintro-
duction of Gd from North America to Europe, although it would
be useful to repeat our experiment with a European bat species.
Interestingly, we found that EUGd affected M. lucifugus more
quickly than NAGd (Figs. 1 and 4). Rapid evolution of the host–
pathogen interaction between Gd and bats could help explain
this pattern (12, 15). For example, if European bats exhibit re-
sistance to infection, Gd in Europe may face intense selection
pressure for increased production of potential virulence factors
and more rapid growth to facilitate its propagation and trans-
mission. However, if the production of virulence factors and
rapid growth are costly for Gd, selective trade-offs could quickly
favor a less pathogenic, slower growing variant of the fungus as it
infected a naive host population in North America. Moreover,
dramatic population declines of North American bats in the
early years of the epizootic could have reduced the potential for
transmission among bats, enhancing selection for reduced patho-
genicity in North America. Despite this potentially encouraging
finding, clearly the version of Gd now present in North America is
highly pathogenic to a number of bat species. Thus, more labo-
ratory and field experiments are necessary to better understand
interactions between bats and Gd, particularly studies aimed at
better understanding transmission of the fungus in the wild.
Our study also confirms that Gd causes mortality of hiber-
nating bats and provides direct evidence for the hypothesis that
an increase in arousal frequency during hibernation is the
mechanism underlying mortality. The three- to fourfold increase
in arousal frequency we observed for infected bats is similar
to the pattern predicted by Boyles and Willis (11) based on an
energetic model. The additional arousals would prematurely
deplete the stored energy of a small hibernator like M. lucifugus
which, in its northern distribution, must survive >190 d exclu-
sively on fat reserves (9, 16). Periodic arousals account for only
1.2% of the hibernation time budget, yet the thermoregulatory
cost of each arousal amounts to about 5% of the winter energy
budget (9). Hence, each additional arousal shortens the time
a bat is able to hibernate by about 9 d. WNS-affected bats are
often observed flying outside hibernacula during the daytime
in winter (4), possibly searching for food and, like the Gd-in-
oculated bats in our study, WNS-affected carcasses collected
from hibernacula after mass mortality events were emaciated (4).
Hence, we conclude that infection with Gd causes an increase in
arousal frequency, leading to emaciation because fat reserves are
with NAGd, EUGd, or CO. Frequency of arousals based on skin temperature
(A), total count of arousals based on video observations (B), and mean
arousal duration (C). Within intervals, different letters above bars indicate
significant differences between groups (SNK post hoc tests following sig-
nificant ANOVA in Table 1).
Changes in torpor patterns in M. lucifugus following inoculation
effects of time on arousal frequency and arousal duration
Repeated-measures ANOVA results for within-group
Repeated-measures ANOVA results for within-group effects of time on
arousal frequency (arousal bat−1·d−1) and arousal duration (length of time
above skin temperature threshold) for M. lucifugus inoculated with NAGd
and EUGd compared with sham inoculated controls (CO). Significant results
are in bold.
Warnecke et al. PNAS Early Edition
| 3 of 5
One explanation for infected bats spending more time out of
torpor during hibernation is that, after rewarming, bats intensify
grooming because of skin irritation (17). Another possibility is
that infected bats elevate Tbto mount an immune response (18).
Both of these hypotheses predict that infected bats should pro-
long the duration of each periodic arousal, for which we found
no evidence. A third hypothesis is that infection influences
physiological processes that trigger arousal from torpor. One of
the leading explanations for periodic arousals is that evaporative
water loss during hibernation leads to dehydration over time,
even at high RH, which eventually triggers rewarming (19).
Cryan et al. (20) suggested that wing damage caused by Gd
infection could elevate cutaneous water loss, reducing the time
bats are able to spend in torpor before dehydration triggers
arousal, and this hypothesis has circumstantial support (21). We
found a progressive increase in arousal frequency, presumably as
fungal proliferation increased, with no change in arousal dura-
tion. This pattern is counter to the immune response or skin
irritation hypotheses but consistent with dehydration. The in-
creased arousal frequency we observed, combined with past work
demonstrating the importance of high RH for successful hiber-
nation in M. lucifugus (9), implicates susceptibility to dehydration
as an explanation for the high rates of mortality from WNS in
this species. Dehydration, prompting arousal from torpor to
search for water, could also explain the winter flights of affected
bats outside hibernacula (20). Interestingly, a previous in-
oculation experiment performed under much drier conditions
(∼82% RH at 6.5 °C) than those experienced by little brown bats
in the wild, or the bats in our study, observed 20% mortality of
control bats after 3 mo with no significant difference in survival
between inoculated bats and controls (1). This difference be-
tween studies could reflect the influence of humidity and de-
hydration on survival of uninfected bats, or a reduction in the
proliferative abilities of Gd under drier conditions. Future de-
tailed studies examining hygric aspects of bat hibernation, as well
as the effects of humidity on the growth and pathogenicity of Gd,
may help clarify the influence of environmental conditions and
water loss on the progression of WNS.
Our study confirms that Gd is the cause of mortality from
WNS and strongly implicates premature fat depletion because of
increased arousal frequency as the ultimate cause of death. The
study also lends strong support to the novel pathogen hypothesis
that Gd is an invasive species from Europe. Our findings have
implications for future studies on the ecophysiology and sus-
ceptibility of WNS-affected bats, as well as the pathogenicity and
transmission of Gd.
Materials and Methods
Bats. Fifty-four male M. lucifugus (8.6 ± 0.1 g) were collected from a WNS-
negative cave in Manitoba, Canada, in November 2010 and transported to
the University of Saskatchewan. Bats were randomly divided into three
groups of 18 individuals each: (i) inoculated with NAGd; (ii) inoculated with
EUGd; and (iii) a sham-inoculated control group, CO. Each group was housed
in a mesh enclosure contained within a separate environmental chamber at
Ta= 7.0 and >97% RH. Bats were not fed but were provided with ad libitum
water. In March 2011, surviving bats were removed from the environmental
chambers, anesthetized, and humanely euthanized. Methods were ap-
proved by the University Committee on Animal Care and Supply of the
University of Saskatchewan (Protocol #20100120) under Manitoba Wildlife
Scientific Permit WB11145.
Inoculation. For NAGd and EUGd bats, 20 μL of inoculum containing
∼500,000 Gd conidia suspended in PBS-Tween-20 solution was pipetted
onto the dorsal surface of each wing. NAGd was designated type isolate
representative M. lucifugus (A) 88 d after inoculation with NAGd (arrow-
head shows cup-shaped accumulation of fungal hyphae within the epider-
mis, growing into the underlying subcutis; arrow shows hyphae deep within
the subcutis; brown granular pigment is melanin within the epidermis); (B)
77 d after inoculation with EUGd (arrowhead shows thick mat of fungal
hyphae in the epidermis growing into the underlying dermis and subcutis;
arrow shows fungal hyphae deep within the wing membrane); or (C) sham-
inoculated controls after 119 d.
Light micrographs of wing membrane in transverse section for three
group NAGd (dashed line), EUGd (dotted line), and CO (solid line). The closed
circle at the end of each line indicates the day when the group was termi-
nated, day 1 is November 27, 2010.
Survival of individual M. lucifugus over the course of the study for
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| www.pnas.org/cgi/doi/10.1073/pnas.1200374109 Warnecke et al.
20631–21 (American Type Culture Collection, ATCC MYA-4855) (5) isolated Download full-text
from a M. lucifugus collected in New York on February 2, 2008. The EUGd
isolate (MmyotGER2) was obtained from a greater mouse-eared bat
(Myotis myotis) collected in Thuringa, Germany, on March 7, 2009 (14). CO
bats were sham-inoculated with 20 μL of PBS-Tween-20 solution lacking
Skin Temperature. All bats were equipped with one of two types of device to
record skin temperature (Tskin): either temperature-sensitive radio trans-
mitters (LB-2NT; Holohil Systems) or data loggers (DS1922L-F5 Thermochron
iButton, Maxim; and iBBat, Alpha Mach). Tskinwas recorded every 15 min.
Behavior. Infrared cameras inside each environment chamber allowed us to
monitor behavior and count the total number of arousals from torpor for
each group within each interval (Fig. 2B). These data were clearly consistent
with Tskin(compare Fig. 2 A and B).
Histopathology. We examined multiple sections from the left wing, as well as
nose and ear, following Meteyer et al. (6). Tissues were fixed in formalin
immediately after bats were euthanized and later stained for histopatho-
logical examination using the periodic-acid Schiff method. All NAGd and
EUGd bats exhibited the epidermal lesions typical of WNS (5, 6).
Analyses. The study period was divided into four intervals of 26.3 d each. We
tabulated the number of arousals from torpor for each individual to generate
differences in torpor bout duration and arousal duration among groups within
on torpor patterns (i.e., arousal frequency and arousal duration) we used re-
peated-measures ANOVA testing for differences among the first three intervals
within each group. A Breslow–Gehan survival analysis was used to test for dif-
ferences in the time to mortality/moribund status for the three groups with a
Bonferroni correction to account for multiple comparisons between each pair of
groups. All analyses were conducted using statistiXL v7.0 and Systat v11.0.
ACKNOWLEDGMENTS. We thank M. Burmester, P. Mason, and M. Weiss for
animal care support; C. Rainbow, C. Wilson, and M. Zimmer for pathology
assistance; P. Withers for assistance with statistical analyses; M. Kilpatrick and
W. Frick for helpful discussions; and M. Brigham, B. Fenton, and an anonymous
reviewer for excellent comments on drafts of the manuscript. Funding was
provided by a US Fish and Wildlife Service grant (to C.K.R.W., D.S.B., and
P.M.C.); grants from the Natural Sciences and Engineering Research Council,
the Canada Foundation for Innovation and Manitoba Research, and Innova-
tion Fund (to C.K.R.W.); and a Government of Canada Post-Doctoral Research
Fellowship and a fellowship within the Postdoc Programme of the DAAD,
German Academic Exchange Service (to L.W.).
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