The fitness of white nose syndrome-

infected Myotis lucifugus when treated

with Pantoea ananatis.
A RESEARCH PROPOSAL

Allison Smith
DRURY UNIVERSITY | 900 N BENTON AVENUE, SPRINGFIELD, MO. 65802
TOTAL FUNDS: $599,203
SUBMITION DATE: APRIL 11, 2018
 Summary

As mass die-offs of bats in North America have occurred since 2006 because of the
presence of the disease White Nose Syndrome, current research is attempting to find feasible
options to stop the spread of this disease and to treat bats from it. The following proposal focuses
on identifying one such feasible bat treatment option. This experiment will analyze the effect
Pantoea ananatis, a known inhibitor of Pseudogymnoascus destructans, has on the survival rate
of Myotis lucifugus infected with White Nose Syndrome. This will be done by collecting young
bats that have not been exposed to P.s destructans from the wild and introducing various
treatments to the bats: no treatment, P. ananatis only, P. destructans only, P. destructans + P.
ananatis, and P. ananatis + P. destructans. The bats will be kept in environmental chambers
over a period of five hibernation periods (five years), where survival rates will be recorded each
year, to allow for the analysis of treatment survival rate over time. First, it will be determined
whether the mean survival rates between treatments is different or not. If it is, further
determination of which treatments had different survival rates will ensue. This study has the
potential to identify a viable treatment option for White Nose Syndrome in the wild.

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 Table of Contents
Summary .........................................................................................................................................1

Introduction ....................................................................................................................................3

Methodology ...................................................................................................................................5

Relevant Institutional Resources ..................................................................................................9

Budget ...........................................................................................................................................10

Literature Cited ...........................................................................................................................12

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Introduction

The loss of insectivorous bats due to white-nose syndrome (WNS) would lead to larger
agricultural, economic, and ecological costs because of increased insect populations, increased
crop damage, and decreased pollination (Boyles et al., 2011). It was estimated in 2011 that the
loss of bats in North America would cause more than $3.7 billion/year in only agricultural
damages (Boyles et al., 2011). As bats also pollinate a variety of flowers, including agricultural
plants such as peaches and bananas (National Park Service, 2015), if fewer bats were present,
less pollination could occur, causing for decreasing fruit availabity for consumption.

White-nose syndrome, a disease caused by the fungus Pseudogymnoascus destructans,

has led to mass die-offs of hibernating bat species across North America for over a decade
(Warnecke et al., 2012). The fungus was first discovered in North America in 2006, and it is
believed that the fungus was introduced from Europe and then introduced in North America
(U.S. Fish and Wildlife Service, 2016; Zukal et al., 2016). Since P. destructans introduction into
New York, it has spread to 33 states in the U.S. and 5 provinces in Canada (U.S. Fish and
Wildlife Service, 2017). The disease is expected to continue spreading (O’Regan et al., 2015)
and could threaten over half of all bat populations in North America (U.S. Fish and Wildlife
Service, 2016), with estimated bat deaths between 5.7 million to 6.7 million individuals (U.S.
Fish and Wildlife Service, 2012). The fungus has caused extirpations of bat populations and its
expected spread may cause it to endanger even more.

WNS is caused by P. destructans compromising the skin of hibernating bats; the disease
develops only on hibernating bats because the fungus develops on exposed skin at cool
temperatures when bats lower their body temperature during hibernation to near the temperature
of the hibernacula (U.S. Geological Survey, 2017 and Warnecke et al. 2012). The fungus can
survive in bat hibernacula-like conditions because of the high humidity and cool temperatures
found in the hibernacula, typically caves and mines. During hibernation, P. destructans is lethal
to bats as is it causes the following effects: weakened immune system, increased energy demand,
deterioration of blood vessels and muscle tissues, electrolyte imbalances, depletion of fat
reserves, dehydration, and emaciation (U.S. Geological Survey, 2017; Verant et al., 2014). The
combined above effects contribute to the ultimate death of the bat (U.S. Geological Survey,
2017; Cryan et al., 2010). While bats surviving the winter can rid themselves of P. destructans

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(Langwig et al., 2015) and repair damaged skin (Fuller et al., 2011); the fungus remains in the
hibernacula as a saprotroph, possibly infecting healthy bats which enter the contaminated space
(Langwig et al., 2015; Reynolds et al., 2015). Much about the saprotrophic growth of P.
destructans is still uncertain, but Micalizzi et al.suggested that reducing the fungus’s growth in
the hibernacula may prevent or lessen the severity of bat population collapses from WNS (2017).
Therefore, finding a way to increase the survival rate among hibernating bats exposed to P.
destructans by inhibiting the growth of P. destructans in hibernaculas could be important to
decreasing bat population die-offs across North America and controlling the spread of WNS.

To date, nearly 150 inhibitors of P. destructans have been identified from research
studies utilizing petri dish cultures (Micalizzi et al., 2017; Cornelison et al. 2014; Zhang and
Chaturvedi, 2015). One recent study identified 145 inhibitors, where 53 had inhibition rates
above 85% within the petri dish cultures (Micalizzi et al. 2017). From this study, the identified
inhibitor Pantoea ananatis will be used for this research. P. ananatis has been chosen for the
following reasons: 1) it is known to inhibit the growth of P. destructans within a petri dish by
99%, 2) has been found to inhibit the growth of P. destructans via shared airspace, 3) has had the
volatile compounds it produces identified, 4) naturally occurs in Canada, 5) can develop at cave
hibernacula conditions (dark, high humidity, and moist), 6) produced the only volatile substances
out of the studied antagonists which had an inhibition rate of 100% after 2 weeks, and 7) stopped
P. destructans from redeveloping after the antagonist’s initial introduction to the protagonist
(Micalizzi et al., 2017). Thus, P. ananatis is a viable feasible option as an inhibitor of P.
destructans on live bats because the antagonist due to the above reasons allows for ease of
introduction to the bats, reduces the risk of introducing foreign invasive species, offers the
possibility of preventative treatments. While other antagonist could have been chosen for this
study, P. ananatis has shown the above combined characteristics, where other inhibitors have
only shown some of these characteristics or similar characteristics without being as effective as
P. ananatis is as an inhibitor of P. destructans.

Little Brown Bats, Myotis lucifugus, will be used as a host to perform this study. M.
lucifugus have been chosen because their life span is typically 6-7 years in the wild (Fenton and
Barclay, 1980), but can be up to 30 years in captivity (Nowak, 1994); this allows for the long-
term study of the bat species without high concern of them dying from old age or captivity.

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Because most bats infected with WNS die within three years of infection (Bernard and
McCracken, 2017) and M. lucifugus live longer than three years both in the wild and in captivity,
the effects of P. destructans and P. destructans with treatment can be studied past the critical
three-year time frame of WNS. M. lucifugus is well-known to hibernate in the cave hibernacula
setting (Fenton and Barclay, 1980; Nowak, 1994; Wilson and Ruff, 1999) in which P.
destructans thrives, and they are known to be affected by WNS (Meteyer et al., 2011; Fuller et
al., 2011; Blehert et al., 2009). Thus, M. lucifugus is a great candidate for this study as they are
affected by the protagonist of interest, P. ananatis and are naturally found in the same
environmental conditions. M. lucifugus is known to have large roost sizes, allowing for the
collection of a large number of the bats with relatively the same age and exposed to similar
environmental conditions, minimizing variables in the study (Fenton and Barclay, 1980; Nowak,
1994). Lastly, M. lucifugus are present in the geographic area where the study is to take place,
adding a convenience factor for the collection and transport of this host species, and occur over
the same range the chosen antagonist, P. ananatis (Fenton and Barclay, 1980; Nowak, 1994;
Micalizzi, 2017).

This study investigates survival rates of WNS-infected Myotis lucifugus with and without
P. ananatis, a known P. destructans antagonist, and when antagonist introduction is pre-/post-P.
destructans exposure. It is hypothesized that the mean survival rate for all treatment groups are
different. The importance of this study lies in determining whether P. ananatis will affect the
survival fitness of P. destructans-infected bats; if P. ananatis increases the survival rate of P.
destructans-infected bats, P. ananatis may be a viable treatment option for WNS in the wild.

Methodology

Myotis lucifugus Collection

To account for the four weeks required for young to become independent after birth
(Fenton and Barclay, 1980) and as M. lucifugus birth young during June and July (Fenton and
Barclay, 1980; Wai-Ping and Fenton, 1988), collection of M. lucifugus will occur during mid-
late August before the first hibernation period. Juvenile bats less than 1 year in age will be
collected by measuring the length of forearm (where size is an indicator of chronological age) as
reference standards are known and this method provides relatively accurate age estimates up to
95% with intervals of ±1-2 days (Anthony, 1988; Brunet-Rossinni and Wilkinson, 2009). The

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bats will be collected from the wild, preferably from one maternity colony if possible. Collection
will take place during the day, while bats are less likely to be active. The bats will be collected
from a cave which is found to not contain P. destructans. Determining the presence or absence of
P. destructans at the collection site will follow techniques from the Western College of
Veterinary Medicine (2015), where swabs will be taken randomly from the cave’s surfaces and
tested for the presence of P. destructans in fungal cultures (samples and tests will take place
three months prior to bat collection). The bats will be collected after receiving a Permit to Import
or Transport Live Bats from the Centers for Disease Control and Prevention. Bats will be
collected by using gloved hands from a roost and will be stored and transported as described by
the USGS Standard Operating Procedure for the Study of Bats in the Field (U.S. Geological
Survey, 2013). Briefly, each bat will be individually placed in a cloth bag with a draw string,
placed in a cool and dark area in a solid ventilated container, and transported by motor vehicle to
the environmental containment site (U.S. Geological Survey 2013). Following capture, all
equipment will be decontaminated with Lysol or Clorox to reduce the risk of transmitting
diseases. During transport, the bags will be kept away from objects capable of crushing the bats
and monitored for audible distress sounds. If necessary, bats in the bags will be assessed for
distress at least every 2-4 hours, but travel time will attempt to be as little as possible. Bats will
be taken straight to the containment site. Bats will be randomly assigned to environmental
containment units and their corresponding treatment conditions.

Environmental Chambers

A total of 25 environmental chambers (see chamber model DT72LGD made by Powers

Scientific Inc. Pipersville, Pennsylvania in Grieneisen et al., 2015) will be used to house the
collected bats, with 20 individuals housed in each container. The environmental chambers will
be controlled for temperature and humidity M. lucifugus undergoes in their natural habitat. Food
(see Diet & Nutrition) will be provided during non-hibernacula conditions. The following
environmental chamber conditions will be used for four periods throughout a year for five years:

Time Period (months) Nov.-Feb. March-May June-Aug. Sep.-Oct.

Temperature (˚C; Day/Night) 10/10 13/14 13/25 13/17
Humidity (%; Day/Night) 95/95 95/7 95/60 95/15

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 The hibernacula setting will be replicated in each chamber from November to the end of
February, allowing the overall hibernation period lasting between 4 months as it would in the
wild (Fenton and Barclay, 1980; Nowak, 1994) and as other studies have found this time period
to work for captively held Myotis (Grieneisen et al., 2015; Warnecke et al., 2012). During this
hibernation period, the temperature will be gradually reduced to about 10˚C. No food will be
provided, but water will still be provided for the bats, as done in other research (Grieneisen et al.,
2015). A high level of humidity, 95%, will be maintained in the hibernacula to replicate natural
conditions (Pierce et al., 2017). For non-hibernacula, day temperatures are near the average cave
temperature in Missouri (Missouri Department of Conservation, 2000), as bats tend to remain in
caves during the day, and night temperatures are based on the average months’ night
temperatures while bats would be out (U.S. Climate Data, 2018). For non-hibernacula months,
humidity for the day is the same as the average humidity for a cave (Pierce et al., 2017), while
night changes to the average months’ humidity (Weather Spark, 2016).

Diet & Nutrition

Food and water dishes will also be washed daily. Prior research has noted that Myotis
take about one week to learn how to find food on their own captivity; thus, hand-feeding will
occur if necessary (Grieneisen et al., 2015; Orr, 1958). To ensure dietary deficiency does not
occur, bats will be fed mealworms, Tenebrio molitor, and a vitamin supplement mixture (see Bat
World Sanctuary, 2010).

Exposed Treatment Conditions

Five different treatment conditions will be applied during this study: no treatment, P.
ananatis only, P. destructans only, P. destructans + P. ananatis, and P. ananatis + P.
destructans. The no treatment condition will act as a control, demonstrating how M. lucifugus
would survive in the previously described conditions. P. ananatis only will demonstrate the
effect of the antagonist on M. lucifugus. P. destructans only will show the effect of WNS on M.
lucifugus. P. destructans + P. ananatis will illustrate the effect of introducting the WNS-causing
agent to M. lucifugus followed by introducing the bats to the antagonist. P. ananatis + P.
destructans will exhibit the effect of introducing the WNS antagonist followed by the fungus to
M. lucigugus. The post/prior exposure of the treatment will be tested as a previous study found
an antagonist of P. destructans to reduce disease exposure when the antagonist was applied post
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P. destructans exposure, while found that probiotic treatment worsened the severity of the
disease (Cheng et al., 2017).

Each of the 5 treatment conditions will have 5 replicates with 20 bats in each replicate.
Treatments will be applied randomly using a random sequence generator. The treatment
conditions will be applied during the first hibernation period following the collection of the bats.
As done with other treatments by Pierce et al. (2017), the treatments will be applied by the 14th
day of the hibernation period.

Pseudogymnoascus destructans and Pantoea ananatis Inoculation

Inoculation procedures will follow Warnecke et al. (2012). Briefly, 20µL of inoculum
containing about 500,000 conidia suspended in PBS-Tween-20 solution will be pipetted onto the
dorsal surface of each wing of bats being infected with P. destructans. The type isolate used for
P. destructans is 20631-21 (American Type Culture Collection, ATCC MYA-4855) (Gargas et
al., 2009), which was isolated from M. lucifugus being studied at New York in February 2008
(Wibbelt et al., 2010). Using the time frame by Micalizzi et al. (2017), The bats being treated
with the antagonist post P. destructans exposure will be introduced to P. ananatis (type isolate
RFA4P2) 14 days after P. destructans incolulation. Following methodology of Pierce et al.
(2017) when treating M. lucifugus with Rhodococcus rhodochrous, induced P. ananatis cells in
petri dishes will be put in the necessary exposure chambers at ~1gft-3 of the total air volume; the
petri dishes will be removed after 72 hours. The bats being pre-treated with the antagonist will
follow the same method, but 14 days prior to being exposed to P. destructans.

Determining Fitness

Survival fitness will be determined both per year and overall after the five-hibernation
period study. Survival fitness will be determined as a percent of the initial bats (ones present at
the first hibernation period) to the end of each hibernation period and at the end of the study; this
will be achieved by taking exact accounts of the bats present in each containment unit.

Euthanasia

All bats used in this experiment will be euthanized post-experimentation for the safety of
life forms outside of the experiment and in accordance with Missouri’s Wildlife Collector’s

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Permit. Euthanasia will follow instructions set by the Missouri Department of Conservation and
work to be as humane as possible. Thus, bats will be euthanized via cervical dislocation as this is
a recommended method by the American Society of Mammologists and the American Veterinary
Medical Association for small mammals (U.S. Geological Survey, 2013; American Veterinary
Medical Association, 2013). After this, each bat’s status will be checked to ensure it has been
euthanized. If it has not been, a secondary method will be used to euthanize it, such as
decapitation.

Data Analysis

A one-way analysis of variance (ANOVA) test will be performed to determine if there is

a difference between the mean survival rate of all treatment groups. The experiment-wise alpha
will be set to 0.05 for this test. If a difference is found, multiple comparison tests will be used to
determine which mean survival rate(s) is(are) different to one another; the alpha for these tests
will be set at 0.05.

Approach

For the experiment to stay on track during the proposed time period, the following outline
of desired accomplishments is tabulated by year:

Year 1 Year 2 Year 3 Year 4 Year 5

-introduce M. -determine -determine -determine -determine
lugifugus to survival rates of survival rates of survival rates of survival rates of
treatments 2nd hibernation 3rd hibernation 4th hibernation 5th hibernation
-determine period period period period
survival rates of -process and
1st hibernation analyze data
period -present findings

Relevant Institutional Resources

The presented institution should be chosen to perform this research as prior bat research
regarding live bats and WNS has been performed as the institution; thus, relevant personnel and
knowledge is already present. Internet access, computers, data analysis programs, rental vehicles,
glassware, and some lab space is available for use at this institution. Furthermore, this institution
is within hours of multiple possible sites of M. lucifugus, allowing for simpler collection and
transport of live bats.

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Budget

Budget summary by year:

Items Total 1st Year 2nd Year 3rd Year 4th Year 5th Year
Personnel $70,000 $14,000 $14,000 $14,000 $14,000 $14,000
Consultants $1,400 $14,000 $0 $0 $0 $0
Equipment $390,000 $378,500 $3,000 $3,000 $3,000 $3,500
Materials& Supplies $27,840 $7,040 $5,200 $5,200 $5,200 $5,200
Travel $1,000 $1,000 $0 $0 $0 $0
Indirect Costs $108,742 $54,371.00 $27,185.50 $13,592.75 $6,796.38 $6,796.38

Budget summary by items:

Expense Type Sponsor Institution Total

Personnel
Research Assistant $60,000 $0 $60,000
Project Associate $0 $10,000 $0
Subtotal $60,000 $10,000 $60,000
Consultants
Bat Biologist, $200/day, 7 days $1,400 $0 $1,400
Equipment
Decontamination Equipment $1,000 $0 $1,000
Environmental Chambers (x25) $375,000 $0 $375,000
Lab Rental Space $15,000 $0 $15,000
Subtotal $391,000 $0 $391,000
Materials & Supplies
Glassware $200 $0 $200
Permits $1,000 $0 $1,000
Transport Containers $750 $0 $750
Cloth Bags $350 $0 $350
P. destructans strain $350 $0 $350
P. ananatis strain $100 $0 $100
PBS-Tween-20 Solution $90 $0 $90
Animal Care $25,000 $0 $25,000
Subtotal $27,840 $0 $27,840
Travel
Transportation $1,000 $0 $1,000
Cost Totals
Total Direct Costs $481,240 $10,000 $491,240
Indirect Costs $106,263 $2,200 $108,463
(22% modified total direct costs)
Grand Total $587,003 $12,200 $599,203

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Budget Justification

Personnel

Research Assistant (effort = 12 calendar months). This individual will assist with the study’s
coordination, ordering supplies and materials, collection of data, daily management of the study,
data entry, and overseeing bat health.

Project Associate (effort = 24 calendar months). This individual will assist with data collection
and analysis over the five-year period (for a total of 24 calendar months).

Consultants

Bat Biologist (effort = .25 calendar months). This individual will assist in determining the bat
collection site, bat collection, and preparing the bats for transport.

Equipment

Funds are requested to purchase twenty-five environmental chambers ($15,000 each);

decontamination equipment for post bat collection, transport, and study; and lab rental space
($3,000/year). The environmental chambers will control humidity, photoperiod, and temperature.
These chambers will be used throughout the five-year study period. Decontamination equipment
will be used to ensure any spores from WNS or other harmful substances are not transmitted
from one location to another or from animal to animal. Lab rental space will be needed
throughout the study in order to keep the bats; thus, lab space will be used throughout the five-
year study.

Materials and Supplies

$25,000 is requested for animal care expenses. This covers the necessary expenses for dietary
needs of all bats over the entirety of the five-year study. $750 is requested for the transport
containers needed to keep the bats calm during transport; an additional $350 is requested for the
cloth bags needed to put each individual bat in during transport to ensure its safety. $1,000 is
requested to obtain the necessary permits to transport, collect, and test on animals; this request
includes license renewal expenses for the study period. $350 is to purchase the P. destructans
strain, while $100 is for the purchase the P. ananatis strain. $200 is requested for the purchase of
all necessary glassware used for the study, also accounting for pipette expenses.

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Travel

Transportation will be required to transport the bats to the study site and rent a motor vehicle.
$500 is requested to cover this transportation expense.

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