PROBIOTIC APPLICATION OF SYMBIOTIC BACTERIA ISOLATED FROM WESTERN
BAT SPECIES ONTO CAPTIVE AND FIELD BATS TO PREVENT WHITE-NOSE
SYNDROME

by

NICOLAS FONTAINE
Bachelor of Science, Major in Animal Biology, Thompson Rivers University, 2017

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR
THE DEGREE OF MASTER OF SCIENCE IN ENVIRONMENTAL SCIENCE
In the Department of Biological Sciences
Thesis examining committee:
Naowarat Cheeptham (PhD), Professor and Thesis Supervisor, Department of
Biological Sciences
Cori Lausen (PhD), Adjunct Faculty of Biological Sciences and Co-Supervisor, Wildlife
Conservation Society of Canada
Mark Paetkau (PhD), Associate Teaching Professor and Committee Member,
Department of Physical Sciences
Dave Sedgman (DVM), Committee Member, Retired Veterinarian
Dan Durall (PhD), External Examiner, Associate Professor, UBC Department of Biology

April 2021
Thompson Rivers University

Nicolas Fontaine, 2021
Thesis Supervisor: Professor Naowarat Cheeptham
Thesis Co-Supervisor: Wildlife Conservation Scientist Cori Lausen
i

ABSTRACT
Wildlife diseases can have drastic consequences for species at an unprecedented rate.
Without proper intervention, diseases can threaten population viability and result in
species extinction. The delicate balance of an ecosystem is associated with the niche of
an individual species and can quickly become unstable upon the introduction of an
invasive disease. Managing these diseases is often quite challenging and typically
requires immediate action to prevent further ecological loss. The psychrophilic fungus,
Pseudogymnoascus destructans, Pd, has devastated populations of several North
American bat species. White-nose syndrome (WNS) is a deadly disease that indirectly
causes hibernating bat mortality by growing on the cutaneous surfaces of the bat and
causing more frequent arousal periods. In addition to physiological imbalances caused
by the degradation of wing tissue, the decreased torpor length and associated
increased metabolic rate will burn precious fat stores, cause rapid dehydration, and lead
to bat mortality. Reducing the severity of the Pd infection could result in lower mortality
rates. Experiments on captive bats and free-flying wild bats were conducted to alter
native wing microflora found on Myotis yumanensis, using a prophylactic topical
probiotic cocktail. The probiotic contains Pseudomonas bacterial species that were
isolated from wings of healthy British Columbia bats and were found to inhibit Pd. The
four main objectives of this study were: (1) to develop a technique of applying the antiPd microbes to Myotis bats roosting in buildings and/or bat boxes; (2) to test whether an
microbiome enhanced with anti-Pd microbes can be sustained on bat wings; (3) to
replicate captive trial tests and procedures in a field trial setting; and (4) to quantify the
ability of the probiotic to inhibit Pd germination/growth on live bat skin. Two captive bat
trials at the B.C. Wildlife Park and one lab hibernation trial were conducted. I developed
an effective two-step application process of misting a roosting substrate with water,
followed by powdered clay containing freeze-dried probiotic cells. Quantitative PCR
analyses confirmed successful long term adherence of the probiotic cells to bat boxes,
and successful transfer of these microbes to bats’ wings. The probiotic was field-trialed
at summer maternity roosts in the Greater Vancouver area, successfully applying
probiotic at building and bat box roosts, and through swab sampling of captures of freeflying bats, determined the probiotic was successfully transferred. In a laboratory
ii

experiment, I placed probiotic bacteria and Pd onto separated bat patagia, with the goal
of adapting and refining tissue explant chamber technology to keep bat skin cells
partially alive on which to test the interaction of Pd and probiotic cells. This probiotic tool
provides one potential prevention measure for reducing the spread, or at minimum the
severity, of WNS in the Pacific Northwest of North America.
Key-words: bats, Pseudogymnoascus destructans, probiotic, Pseudomonas, whitenose syndrome (WNS), disease management, captive trial, field trial, prophylaxis, bats,
Myotis yumanensis

Acknowledgements
I would like to thank my supervisors, Dr. Naowarat Cheeptham and Dr. Cori
Lausen for providing me the opportunity and support to work on this project. As well, I
would like to acknowledge the support, expertise and contributions of Dr. Adrian
Forsythe, Dr. Jianping Xu and Heather Yoell from McMaster University. Thank you to
my advisory committee members, Dr. Mark Paetkau and Dr. Dave Sedgman, for
assisting me throughout the project, as well as my external advisor Dr. Dan Durall.
For advice, support, and development of study design and methodology, I would
like to thank: Dr. Glenna McGregor, Dr. Helen Schwantje, Dr. Purnima Govindarajulu,
Orville Dyer, Dr. Craig K.R. Willis, Dr. Yvonne Dzal, Mandy Kellner, Dr. Leigh Anne
Isaac, Dr. Karen Hodges, and Dr. Nancy Flood.
For assistance with laboratory work and captive trials I would like to thank Aaron
Wong, Karen Vanderwolf, Gabrielle Kam, Brandon Hayashi, Julianna Bissonnette,
Chadabhorn Insuk, Monique Nijjer, Paul Grand, Brian Burciaca, Ethan Herndier, Susan
Dulc, Maria Collins, Taylor Kutzley, Soumya Ghosh, Robyn McArthur, Rory Mckerchar,
Sara Lawrence, Linda Qjan, Keegan Koning, Skylah Mcleod Van Wagoner, Cailin
McNeely, Leanne Mills, Timothy Crowe.
For help with field work I would like to thank Leah Rensel, Aimee Mitchell, Chris
Currie, Patrick Burke, Laura Kaupas, Suhani Srihari, Nick Hindley, Laura Tessaro, AJ
iii

Fedoruk, Robyn Worchester, Kristen Mancuso, Kirk Miles, Lorraine Ens, Milena Wilson,
Greg Michalowski, John Saremba, Christina Saremba, Isabelle Groc, Cam Hiebert,
Joanna Burgar, Deanna MacTavish, Jenn Barrett, Julie Fournier, Matt Jones, Melodie
Lioret, Sarah McLaughlin, Stephanie Lee, Thea Warren, Timothy Silva. For permissions
I would to thank BC Parks, Lower Shuswap Indian Band, Metro Vancouver Regional
Parks, Creston Valley Wildlife Management Area, BC Hydro, and individual landowners
provided logistic support, and access to study sites.
For support of the captive trials, I would like to thank the BC Wildlife Park and in
particular Glenn Grant, Ardice Fleck, Paige McKnight, Jorrit Koedooder, Tracy
Reynolds, and others. For volunteer assistance to design the bat enclosures I thank
Tom Haag and I thank the numerous volunteers who supported the building of these
including Frank Ritcey and Doug Burles. For assistance with necropsies and in-kind
support, I thank Dr. Glenna McGregor of BC Animal Health Lab and Dr. Helen
Schwantje BC Ministry of Forest Lands and Natural Resource Operations. For guidance
on explant use, I thank Dr. Kevin Keel from the University of California. For logistical
support I thank Ashley Doyle and Marilyn Carpenter, Kwantlen First Nation.
This project is funded in part by the following: Environment and Climate Change
Strategy, US Fish and Wildlife Service, National Fish and Wildlife Federation (Bats for
the Future Fund); TRU Undergraduate Research Enhancement Fund (UREAP); TRU
Graduate Student Research Mentor Fellowship Award (awarded to Nicolas Fontaine);
TRU Internal Research Fund; TRU Tri-University Grant (Lausen, Hodges, and
Cheeptham); BC graduate scholarship (awarded to Nicolas Fontaine); Kamloops
Naturalist Club; Habitat Conservation Trust Foundation and Forest Enhancement
Society of B.C.; fightWNS, MicroGrants for MicroBats; Fish and Wildlife Compensation
Program Coastal; BC Ministry of Environment and Climate Change Strategy;
Neskonolith Indian Band (graduate award support to Nicolas Fontaine); Suncor Energy
Foundation (graduate scholarship to Nicolas Fontaine); TD Bank Group bursary with
matching funds from the Government of Canada (Awarded to Nicolas Fontaine); Global
Jeff Metcalf Fellowship (to Linda Qian, University of Chicago for lab assistance at TRU);
Mitacs Indigenous Business Internship.
iv

Table of Contents
ABSTRACT ......................................................................................................................ii
Acknowledgements ......................................................................................................... iii
Table of Contents ............................................................................................................ v
Chapter 1: INTRODUCTION ........................................................................................... 1
Chapter 2: Testing the Efficacy of a Topical Probiotic on Captive Bats to Prevent WhiteNose Syndrome ............................................................................................................ 19
Chapter 3: Field Application of Pseudomonas fluorescens Species Complex Bacteria
onto Bat Boxes in the Greater Vancouver Area ............................................................ 61
Chapter 4: Inoculation and Quantification of Pseudomonas fluorescens Species
Complex Bacteria and Pseudogymnoascus destructans on Myotis Patagia ................. 86
Chapter 5. CONCLUSION........................................................................................... 115
APPENDIX .................................................................................................................. 123

List of Figures
Figure 2.1 Enclosure design…………………………………………………………………..24
Figure 2.2 Captive trial timeline…………………………………………………………….…27
Figure 2.3 Four-chamber bat box and probiotic application……………………………..…28
Figure 2.4 Timeline of four-chamber bat box experiment……………………………….....29
Figure 2.5 Modified beverage cooler used for hibernation experiment……….………..…32
Figure 2.6 qPCR results from Fall captive trial…………………………………………..…..34
Figure 2.7 Mean cell concentrations of 2019 captive bats before probiotic inoculation….35
Figure 2.8 Mean cell concentrations of the 2019 control bat box……………………….....35
Figure 2.9 Mean cell concentrations of the Treatment 1 bat swabs…………...................37
Figure 2.10 Mean cell concentrations of the Treatment 1 bat box swabs…………….....37
Figure 2.11 Mean cell concentrations of the Treatment 2 bat swabs ………………….....39
Figure 2.12 Mean cell concentrations of the Treatment 2 bat box……………………..….39
v

Figure 2.13 Mean cell concentrations of the four-chamber bat box…………………….....41
Figure 2.14 Average daily temperature of the four-chamber bat box……………………...42
Figure 2.15 Average daily maximum temperature of the four-chamber bat box………….44
Figure 2.16 Mean starting quantities of the hibernation bat swabs………………………..48
Figure 3.1 Map of the Greater Vancouver region inoculation sites……………………..…66
Figure 3.2 Number of probiotic cells found within Stave Lake bat box 2 ………………….71
Figure 3.3 Number of probiotic cells found within Hayward bat box……………………….72
Figure 3.4 Number of probiotic cells found on boiler room entrance in Stave Lake……..73
Figure 3.5 Number of probiotic cells found on bat swabs from Stave Lake……………….74
Figure 4.1 Explant chambers with separated bat tissue…………………………………....94
Figure 4.2 Probiotic bacteria CFU versus OD at 600nm……………………………………97
Figure 4.3 Separated bat wing inoculated with Pd……………………………………..….. 98
Figure 4.4 Pd mycelia qPCR results, summer trial……………………………………….....99
Figure 4.5 Pd mycelia qPCR results, hibernation trial……………………………………..100

List of Tables
Table 2.1 One way ANOVA and Tukey test analysis of the four-chamber bat box average
daily temperatures………………………………………………………………..……………43
Table 2.2 One way ANOVA and Tukey test analysis of the Four-chamber bat box average
daily maximum temperatures…………………………………………………………………45
Table 2.3 General Linear Model (GLM) of frozen water swab samples from the fourchamber bat box…………………………………………………………………………….….46
Table 2.4 Ratio of Synxantha A to other probiotic bacteria…………………………….…..47
Table 2.5 Concentrations of probiotic at the start and end of the hibernation trial……….49
Table 3.1 Characteristics of bats caught at Stave Lake..................................................75
Table 4.1 Number of samples in each category of the explant experiment……………….95
Table 4.2 Cq results of Pd inoculation from the explant experiment……………………..102
Table 4.3 Cq results of the 3.5 day explant group………………………………….………104
Table 4.4 Cq results of the 7.5 day explant group…………………………………..……..105

vi

Appendix
Figure A.1 Synergistics inhibition testing of anti-Pd isolates……………………………...123
Table C.1 Captive bat swabbing area from the captive trial….…………………………...126
Table C.2. Swab schedule of Treatment 1 and 2 bats…………………………………….127
Figure C.1 Swab locations of Treatment 1 and 2 bats……………………………………127
Table C.3. Swab schedule of bats in the hibernation trial…………………………………127
Figure C.2 Swab locations of Hibernation bats…………………………………………….128
Figure D.1. Temperature and Relative Humidity within the hibernation chamber……...129
Figure E.1. Broth mean cell concentrations of the Treatment 1 bat swabs……………..130
Figure E.2. Broth mean cell concentrations of the Treatment 1 bat box swabs…………131
Figure E.3. Broth mean cell concentrations of the Treatment 2 bat swabs……………..131
Figure E.4. Broth mean cell concentrations of the Treatment 2 bat box swabs…………132
Figure E.5. Broth mean cell concentrations of the hibernation trial bat swabs………….132
Figure E.6. Broth mean cell concentrations of the four-chamber bat box…………….....133
Table F.1 Histology score results from captive bats, preliminary trial……………………134
Table F.2 Histology score results of myotis wing tissue, summer trial…………………..137
Table F.3 Histology score results of myotis tail tissue, summer trial………………….....138
Table F.4 Histology score results of myotis ear tissue, summer trial…………………….139
Table F.5 Histology score results of myotis nose tissue, summer trial…………………..140
Table F.6 Histology score results of myotis lungs and spleens, summer trial…………..141
Table G.1 Treatment 1 group sample and replicate numbers…………………………….142
Table G.2 Treatment 2 group sample and replicate numbers…………………………….143
Table G.3 Hibernation group sample and replicate numbers……………………………143

Glossary of terms
ANOVA Analysis of Variance
BCWP BC Wildlife Park
BSC

Biosafety Cabinet

CFU

Colony Forming Units

DAPG

Diacetylphloroglucinol

DNA

Deoxyribonucleic Acid
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EPFU

Big brown bat (Eptesicus fuscus)

EWL

Evaporative Water Loss

GLM

General Linear Model

LB

Lysogeny Broth

MYLU

Little brown bat (Myotis lucifugus)

MYYU Yuma myotis (Myotis yumanensis)
NA

Nutrient Agar

OD

Optical Density

PBS

Phosphate Buffer Saline

PCR

Polymerase Chain Reaction

Pd

Pseudogymnoascus destructans

PIT

Passive Integrated Transponder

qPCR Quantitative Polymerase Chain Reaction
RNA

Ribonucleic Acid

SDA

Sabouraud Dextrose Agar

TMR

Torpor Metabolic Rate

TRU

Thompson Rivers University

WCS

Wildlife Conservation Society (Of Canada)

WNS

White-Nose Syndrome

viii

Chapter 1: INTRODUCTION
Chiroptera is the second largest mammalian order with over 1,400 species found
worldwide excluding the arctic poles (Simmons and Cirranello 2021). One hundred and
nine of these species are considered vulnerable, eighty-three are currently endangered,
and twenty-one are critically endangered (BCI 2021). New bat species are continuously
being discovered and 242 bat species are data deficient with an unknown conservation
status (BCI 2021). British Columbia is home to 17 of the 19 bat species found in
Canada (Naughton 2012).
All bat species in Canada are insectivores that feed on arthropods (Harvey, et al.
2011; Nagorsen and Brigham 1995). As such, when insects are not present, starting in
fall, bats must either migrate or hibernate until insect prey are again abundant. Most bat
species in Canada are hibernating species, with only 3 that are considered migrating
species: Lasiurus borealis, L. cinereus, and Lasionycteris novtivagans (Harvey, et al.
2011; Nagorsen and Brigham 1995). Hibernating species generally roost within mines,
caves, rock crevices, and in some areas some species will hibernate in trees and
buildings (Naughton 2012). Hibernation is thought to occur shortly after mating swarms
in September-October or later (i.e. Myotis. lucifugus, Eptesicus fuscus, M. yumanensis,
M. californicus, Euderma maculatum, M. septentrionalis, M. evotis, Antrozous pallidus,
M. thysanodes, M. volans, M. ciliolabrum, M. leibii, L. noctivagans, Parastrellus
hesperus, Corynorhinus townsendii). Bats may undergo torpor to conserve energy and
reduce water loss when their ambient temperature is less than their thermoneutral zone
(Herreid and Schmidt-Nielsen 1966). Torpor decreases their body temperature to match
their ambient temperature and lower their metabolic rate (Geiser 2004). Basic bodily
functions such as breathing and heart rate are slowed immensely and their immune
system is suppressed throughout torpor, making them vulnerable to infection (Bouma et
al., 2010; Geiser 2004; Moore et al., 2011). Durations of torpor are intermittent and
often delayed by brief arousals that vary among species, in which body temperature and
metabolic rate reach normothermic levels briefly before bats re-enter torpor. These
arousals are the main contributor of stored energy usage when in hibernation (Thomas
et al. 1990). Feeding, drinking, mating, grooming, and replenishing electrolytes have all
1

been documented in between bouts of torpor (Boyles et al. 2006; Wilcox et al. 2014).
After emergence from hibernation, which typically occurs between March-May the
following spring, females will typically return to previously used maternity roosts whether
it be a bat box, building, or tree roost. Males will instead form smaller colonies or roost
by themselves (Naughton 2012).
Temperate bats choose where to roost based on many factors such as
thermoregulation, reproduction status, risk, coloniality, and roost quality (Boyles 2007;
kerth et al. 2008; Lausen and Barclay 2006; Racey et al. 1973). Bats often choose roost
based on their thermoneutral zone, in which they do not have to regulate their body
temperature to match their roost (Boyles 2007). This will eliminate unneeded energy
expenditure in colder temperatures and dehydration from warmer areas (Genoud et al.
1990). Furthermore, bats undergoing gestation or lactation will choose roosts that do
not require torpor, to avoid negative effects such as parturition and reduced milk supply
(Racey 1973; Studier and O’Farrell, 1972). Once maternity colonies are formed in the
summer, individual bats must decide how to balance risks such as parasitism and
disease risk in larger maternity colonies, with the benefits of coloniality such as social
thermoregulation, predator avoidance, and maintenance of social relationships
(Altrignham 2011; Bartonička and Gaisler 2007; Kerth 2008; Reckardt and Kerth 2007;
Russo et al. 2017; Webber et al. 2018; Willis and Brigham 2007).
Roost switching is common to balance these factors and is different between
artificial and natural roosts (Whitaker 1998). Man made structures such as bridges, and
buildings often mimic natural roosts quite well, and provide better thermal stability,
warmer microclimates, and greater protection from predators than natural roosts
(Lausen and Barclay 2006; Rueegger 2016; Russo and Ancillotto 2015). These
conditions promote reproductive success, better body condition, faster juvenile
development, and inevitably less roost switching (Allen et al. 2010; Godhino et al. 2015;
Lausen and Barclay 2006; Webber et al. 2016; Zahn 1999).
Species such as, but not limited to, C. townsendii, E. fuscus, and more
commonly found, M. lucifugus, and M. yumanensis are all found in buildings throughout
2

the summer season (Nagorsen 1995). In BC, most bats raise a single young each
summer, although some migratory species can give birth to up to 4 pups. Most species
give birth in June each year and pups will depend on their mother’s milk for roughly 6
weeks until they learn to fly and hunt on their own. Many maternity colonies break up in
late summer, and it is thought that most bats will arrive at hibernation areas by October,
and bats mate somewhere either en-route to hibernacula, or at the hibernacula. It is
now known that some species of bats in B.C. will continue to mate during winter and in
spring (C. Lausen, pers. comm.)
Because bats consume thousands of insect prey nightly in the summer, they are
important environmentally and economically; the value of insectivorous bats in the
United States is estimated to be roughly 22.9 billion USD a year (range $3.7 - $53
billion, Boyles et al. 2011). Humans have a significant impact on bat populations worldwide, through roost disturbance, habitat loss, direct mortality such as for food
consumption and persecution, and indirect mortality including exposure to chemicals
like pesticides (Berthinussen et al. 2014). In North America, deforestation, and
developments such as roads, buildings, and windfarms contribute to habitat
fragmentation. Such expansions can result in roost destruction, loss of traditional land
used for migration and insect feeding and forced movements into uninhabited areas.
Roads are particularly bad because they restrict foraging access for bats and directly
contribute to fragmentation and in some cases, casualties (Kerth and Melber 2009;
Lesinski et al. 2011). Bat casualties are often seen from wind turbines, in which
hundreds of thousands of bat deaths each year from barotrauma (Baerwald et al. 2008;
Smallwood 2013), due to migratory movements and unnatural attraction towards the
turbines (Cryan and Barclay 2009). North American bat species face many cumulative
conservation threats, however the biggest threat in the last decade, which can likely
lead to the extinction of several bat species, is White-nose syndrome (WNS).
White-nose syndrome is a fatal bat disease that is a direct result of infection from
the fungal pathogen Pseudogymnoascus destructans. The fungus was first discovered
in a cave near Albany, New York in 2006 (Blehert et al. 2009; Frick et al. 2010). Since
3

then it has caused an unknown number of bat mortalities, having been estimated at
more than 6 million bat deaths in 2012 (USFW 2012). Mortality rates as high as 90100% have been reported (Drees et al. 2017; Frick et al. 2010; Frick et al. 2015;
Leopardi et al. 2015). Pd is currently detected in 39 states and seven Canadian
provinces (USFW 2020) and has not been officially documented in British Columbia yet.
However, bat hibernacula in British Columbia (and the Pacific Northwest in its entirety)
are largely unknown and un-surveyed (Weller et al. 2018). Therefore it is reasonable to
think WNS has already reached British Columbia and we simply have not detected it
yet, especially considering the cases found in Washington State came from Kentucky
(Thapa at al. 2021).
Differential rate of mortality caused by WNS is due to many factors such as
species, hibernacula microclimates, and individual bat interactions associated with
cluster size (Lorch et al. 2011; Wilder et al. 2011). Certain species are more affected
than others, particularly M. lucifugus), Perimyotis subflavus, M. sodalis, and more
recently, M. septentrionalis (Frick et al. 2015). The origin of Pd is speculated to be from
Europe (Drees et al. 2017; Leopardi et al. 2015). Eurasian bats have not suffered the
same population declines or mass mortalities as seen in North American bats. This is
likely due to WNS being an invasive novel fungus that North American bat species have
never encountered or adapted to, unlike bats from Eurasia thought to have co-evolved
with this fungus (Leopardi et al. 2015). Furthermore, there is genomic evidence that
Eurasian bats may have co-evolved resistance or tolerance to WNS infection in the past
(Hoyt et al. 2016; Leopardi et al. 2015).
Bats have suffered high mortality rates because of Pd’s persistence, growth
conditions, mechanism of infection, and difficulty to remove from hibernacula. Pd is a
psychrophilic saprotroph that grows optimally between 12.5-15.8oC in high relative
humidity conditions of 81.5% (Marroquin et al. 2017; Verant et al. 2012). It may persist
in caves for centuries and may reinfect bats that use the same area for hibernation
(Reynolds et al. 2015). Bats who survive until emergence will groom off WNS hyphae
and resume their normal life cycle until the next hibernation period with a normal body
4

mass and some wing tissue injuries (Fuller et al. 2020; Reichard and Kunz 2009).
However, survival of bats after hibernation within infected caves has been largely
dependant on body weight and greater fat reserves (Cheng et al. 2019; Jonasson and
Willis 2011) with physiological disturbance and starvation being the main mechanism of
mortality (Storm and Boyles 2011; Cryan et al. 2013; Warneke et al. 2013; Verant et al.
2014). E. fuscus can have similar hibernation conditions to vulnerable species, however
it has exhibited resistance presumably due to its skin microbiota (Frank et al. 2014;
Lemieux-Labonté et al. 2020). Bat species exposed to re-occurring infection from the
same hibernacula either reach 100% infection or have a lesser mortality rate, possibly
due to behavioural or skin microbiota adaptions that previous bats did not have (Frick et
al. 2017; Hoyt et al. 2015; Langwig et al. 2017).
An augmented skin microbiota that provides resistance to Pd infection has been
a promising method of controlling WNS spread. Many ex-vivo laboratory trials have
demonstrated antagonistic interactions from certain bacteria due to the release of 2,4Diacetylphloroglucinol antibiotic metabolite (Delany et al. 2000; Bangera and
Thomashow 1999). Pseudomonas isolates are commonly used in research studies
because of its natural abundance within environments, its successful history as an antifungal agent in agriculture, and detected presence within some wild bat wing microbiota
(Cheng et al. 2016; Lemieux-Labonté et al. 2017; Hoyt et al. 2015).
Several studies have used bacteria from the P. fluorescens species complex for
anti-Pd experiments, both in the lab and field (Cheng et al. 2016; Lemieux-Labonté et
al., 2017; Hoyt et al. 2015; Hoyt et al. 2019). Bacteria within the Pseudomonas genus
are characterized by their rod shape (0.5-1.0 µm diameter, 1.5-5.0 µm in length) and
gram negative morphology. They are aerobic with an optimum growth temperature
between 4-42oC and are commonly found within soils (Bossis et al. 2000; Chakravarty
and Gregory 2015). P. fluorescens is a species complex that was previously noted as
an individual species but is instead composed of a wide range of ~52 diverse species
that form their own phylogenetic group and comprise the same core proteome (GarridoSanz et al. 2017; Mulet et al. 2010; Nikolaidis et al. 2020). This classification was due to
5

the advantages of 16s rRNA genotyping in the late 20th century that has resulted in
many bacteria to be re-classified from the Pseudomonas genus (Anzai et al. 2000). In
this thesis, we use a probiotic cocktail of synergistic P. fluorescens bacteria species that
display anti-Pd properties. All four species belong with the P. fluorescens species
complex, P. azotoformans and P. synxantha strains A and B, and P. antarctica
(Nikolaidis et al. 2020). P. azotoformans exhibits antifungal properties and have
previously been used as a biocontrol agent against cucumber Colletotrichum orbiculare
(Sang et al. 2014). It was isolated from an adult C. townsendii caught from a maternity
roost in Deroche, British Columbia. P. synxantha is a bacteria species commonly found
within the rhizosphere of plants and exhibits nematocidal and antifungal properties
(Wechter et al. 2002)(Janakiev et al 2019). Strain A was isolated from a M. yumanensis
at a mine hibernaculum near Salmo, British Columbia. Strain B was isolated from an
adult female E. fuscus caught from a mine near Salmo, British Columbia. P. antarctica
was first isolated by Gundlapalli et al. in 2004 in Wright Valley, Antarctica. It is a
psychrophilic aerobic species that grows optimally between 4-30oC. It is the most exotic
species in our cocktail and was isolated from a female juvenile M. evotis at a mine
hibernaculum near Nelway, British Columbia.
Very few studies have conducted field trials that implement anti-Pd bacteria into
wild bat populations due to the inherit difficulty of treatment, especially in western bat
species, and risks of upsetting the natural balance of skin microbiota (Cheng et al.
2016; Hoyt et al. 2019). Researchers have considered creative solutions such as raising
bat hibernacula temperatures to reduce torpor and introducing UV lights to prevent Pd
growth however these are largely dependent on knowledge of hibernacula (Boyles and
Willis 2010; Palmer et al. 2018;). Studies have also considered and/or tested vaccines,
antifungal drugs, chitosan, other fungi, propolis, orange oil and other volatile organic
compounds to fight WNS (Boire et al. 2016; Chaturvedi et al. 2011; Cornelison et al.
2014a; Cornelison et al. 2014b; Ghosh et al. 2017; Hoyt et al. 2015; Kulhanek 2016;
Lemieux-Labonté et al., 2017; Micalizzi and Smith 2020; Robbins et al. 2011; Rocke et
al. 2019; Zhang et al. 2015).

6

There are currently no methods available to prevent the spread of WNS,
especially in western bat species. There has been no official documentation of WNS in
British Columbia (WNS 2019), therefore a proactive approach to preventing infection is
still possible. Once infected, it would be difficult to treat bats in British Columbia due to
hibernacula being largely unknown, likely remote, and all known hibernacula contain
few bats and few species (Fletcher et al. 2020; Weller et al. 2018). Fletcher et al. (2020)
recommends that treatment measures should be considered that do not require
knowledge of bat hibernacula in western North America. This study proposes the first
bioactive treatment of WNS for western bat species.
This project’s goal was to test a newly developed anti-Pd prophylaxis (Forsythe
et al. 2021) to prevent western bat mortality due to WNS. The first objective was to test
the probiotic in a captive setting to confirm that a sustained changed in wing microbiota
can be achieved. I hypothesized that if I introduce the probiotic bacteria onto bat wings,
then probiotic bacterial species will be detected on their wings for some period of time
following application. I wanted to determine how long the probiotic can be detected on
captive and wild bat wings following inoculation. The second objective is to develop an
application method that is safe, replicable, easy to do, and inexpensive for widespread
use in the Pacific Northwest. If a universal application method was developed, then it
could be transitioned into field testing and widespread use. Since probiotic cells cannot
be inoculated directly, a carrier agent needs to be utilized, specifically one that does not
harm bats and replicates a substance found in the wild. The third objective is to
successfully apply probiotic onto wild bat roosts and monitor levels of detected probiotic
on their wings. If I were to replicate the methodology from the captive trial into a field
trial on wild bats, then there should be a detectable concentration of probiotic on their
wings. This is especially important because widespread use is dependant on successful
field trial results. Finally, the fourth objective involves describing and quantifying
probiotic and Pd interactions on live bat skin. If I inoculate Pd onto wing tissue from the
captive trials, then I should see some antagonistic interactions from the probiotic
bacteria concentrations on their wings. This can be further examined using explant

7

chambers, in which wing tissue is inoculated with Pd and the probiotic bacteria at the
same time and monitored for abundance using qPCR.
Here I present, in chronological order, the research that was conducted during
the testing and field pilot implementation of the probiotic. The first chapter describes the
captive trials, with the preliminary findings which facilitated field implementation; in the
second chapter I present the field trial application of the prophylaxis; and in the third
chapter I describe a pilot ex-vivo Pd-challenge experiment on severed bat wings that
were inoculated with the probiotic.

8

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Chapter 2: Testing the Efficacy of a Topical Probiotic on Captive Bats to Prevent
White-Nose Syndrome
INTRODUCTION
White-nose syndrome (WNS) is one of the deadliest wildlife diseases in recorded
history and is causing devastating consequences for bat populations in North America
(Frick et al. 2010). White-nose syndrome is caused by Pseudogymnoascus destructans
(Pd) and has resulted in greater than 90% mortality rates in some bat roosts (Frick et al.
2015; Lorch et al. 2011). It was introduced to the east coast of North America in 2006
and has been spreading ever since (Frick et al. 2010, Lorch et al. 2016), including a
giant leap of the fungus into Washington state (USFWS 2019), the only western state
where WNS has been found to date.
Pseudogymnoascus destructans is a psychrophilic fungus that grows optimally
between 12.5-15.8°C and 70-81.5% relative humidity and causes mortality of
hibernating bats (Johnson et al. 2014; Marroquin et al. 2017; Verant et al. 2012;
Warnecke et al. 2012). Hibernating bats have arousals periods for several physiological
reasons, however, once infected with Pd, a bat will arouse much more than usual,
experience higher rates of evaporative water loss (EWL), and blood electrolyte
disruption (Jonasson and Willis 2012, McGuire et al. 2017, Reeder et al. 2012, Verant et
al. 2014, Warnecke et al. 2013). These physiological imbalances will eventually kill the
bat because of greater energy expenditure, thus burning fat stores built up from the fall
season. Bats will arouse from hibernation due to WNS disturbance and attempts to
groom the fungus off of their bodies (Reeder et al. 2012). Mechanically, the fungal
hyphae will penetrate the connective tissue of the cutaneous sections of the bat and
cause “cup-like” epidermal erosions on the wings, among small tears and patches of
rough skin. The hyphae will noticeably invade hair follicles, sebaceous and apocrine
glands and regional connective tissues; while producing a noticeable opaque white
texture among the cutaneous sections of the muzzle, wings and tail regions (Meteyer et
al. 2009).

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If the bat has survived until spring, it will move to summer roosts where continued
grooming, and a warm body temperature above that which Pd can survive, will reduce
Pd spore loads to nearly zero for the duration of the reproductive period if they do not
return to the winter roosts. The Pd fungus has been known to persist in caves because
of the continuous lower temperature and high humidity (Hoyt et al. 2015a).
For the most vulnerable bat species, such as Myotis septentrionalis and M.
lucifugus, once a winter colony has been exposed to Pd there is reduced chance of
survival; however, some populations, especially of less vulnerable species, have shown
less infection intensity after surviving WNS (Langwig et al. 2017). Hypotheses for why
some bats survive WNS included gene selection pertaining to immune response and
wound healing, skin microbiota, and behavioural adaptions (Frick et al. 2017; Hazim et
al. 2018; Langwig et al. 2017; Lemieux-Labonté et al. (2017). However, cumulative
mortality risk models suggest that even populations of less susceptible bat species may
follow a stabilized negative growth rate after a major mortality event (Maslo et al. 2017),
leading to a false sense of security in the fate of some bat species.
WNS along with other fungal diseases such as chytridiomycosis
(Batrachochytrium dendrobatidis) and ophidiomycosis (Ophidiomyces ophiodiicola),
which has caused rapid decline in amphibian and snake species, respectively, are
threatening wildlife in unprecedented ways, and urgency surrounds the management of
these diseases (Becker et al. 2015, Lorch et al. 2015). Reducing the mortality of bats
from WNS could have important benefits for the ecosystem and economy. It is
especially important to consider the economic benefits bats provide. They control some
insect populations and reduce the use of pesticides in North American agriculture and
forestry industries; without the ecosystem services provided by bats, it is estimated that
the US could see an economic loss of 3.7-53 billion dollars a year (Boyles et al. 2011).
A vaccine showing promising results has recently been conducted in vivo and is
awaiting field testing (Rocke et al. 2019). However, administering a vaccine can be
difficult on free ranging or wild animals. Administering requires consistent human
intervention as it would require vaccination of all individuals recruited into a population
20

each year, and depending on its efficacy, could require repeated vaccinations of the
same individual. It can be expensive and could cause repeated disturbance to bats
(Langwig et al. 2015).
The microflora present on an animal’s skin plays a vital role in the health of the
individual. Healthy bacteria have been proven to provide increased protection from
pathogen colonization by increased processing of skin proteins and overall inhibition of
invasive transient microorganisms (Clay 2014; Grice and Segre 2011). Probiotics offer
advantages over chemical antifungal treatments, being safer and longer lasting
(Thomas and Willis 1998, TUFTS University 2011). Traditionally, fungicides have been
used to combat diseases in agriculture (e.g., Knight et al. 1997). Broad use of
fungicides can be dangerous to native microbiomes and expensive (Thomas and Willis
1998, TUFTS University 2011). However, bacteria belonging to the P. fluorescens
species complex have been increasingly considered for biocontrol of pests in agriculture
because of its production of bioactive metabolites and rapid growth within the
rhizosphere (Prasad et al. 2018). Other examples of probiotics assisting with fungal
diseases include clearing chytridiomycosis infection through bacterial control (Becker et
al. 2015); defending crustacean embryos with symbiotic marine bacteria (Gil-Turnes et
al. 1989); and protecting wasp larvae from fungal infection (Kaltenpoth et al. 2004).
Anti-Pd bacteria have shown to be a promising choice for protecting bats from
WNS mortality (Hamm et al. 2017, Hoyt et al. 2015b, Hoyt et al. 2019). Utilizing bacteria
found on native bat species can prevent complications and assist with anti-Pd
colonization into bat wing microbiomes (Bletz et al. 2013). Further, there is potential for
probiotics to coevolve with the pathogen and provide long lasting solutions not seen in
chemical fungicides (Thomas et al 1998).
Studies using microbials to inhibit Pd include Trichoderma polysporum fungi
(Zhang et al. 2015), and Rhodococcus rhodochrous to inhibit Pd growth when it is
induced with urea (Cornelison et al. 2014). Three other studies have tested the
Pseudomonas fluorescens species complex for its anti-Pd activity (Cheng et al. 2017;
Hoyt et al. 2015b; Hoyt et al. 2019). Utilizing naturally occurring Pseudomonas bacteria
21

to inhibit Pd as a biocontrol agent was first proposed by Hoyt et al. (2015b). Cheng et al.
(2016) produced mixed results, in which bacteria from the P. fluorescens species
complex were applied in large doses to bats which were hibernated in a laboratory
setting. Hoyt et al. (2019), applied the same probiotic in a controlled field trial at a mine
hibernaculum, reporting obvious survival benefits from application of the probiotic -- fivefold survival of the probiotic-treated group compared to the control group.
We have developed a prophylaxis composed of Pseudomonas bacteria naturally
found on bats’ wings in British Columbia, Canada. Candidate isolates were identified
based on Pd inhibition, and combinations of isolates were previously tested for
synergistic activity ex-vivo (Appendix A; unpublished data). Four isolates were selected
for the final probiotic cocktail based on their mix of partial and full inhibition, and additive
inhibition against Pd: P. azotoformans, P. synxantha strains A and B, and P. antarctica.
The premise of our WNS prophylaxis approach is that through summer
application of probiotic on wings, bats arrive at winter hibernacula with enhanced wing
microbiomes containing anti-Pd probiotic bacteria. This may be able to delay or prevent
Pd growth for a critical portion of the winter.
Here, we test this probiotic cocktail in varying dosages, on captive free-flying
bats. Our goals were to develop a passive method of application that could also be used
in field applications; confirm that a sustained change in wing microbiome could be
achieved, in which probiotic bacteria would become incorporated into the wing
microbiome; compare temperature and probiotic cell counts on an empty bat box; and
test the health of captive bats in ‘treatment’ and ‘control’ groups to ensure the probiotic
caused no ill effects.
I describe two captive trials on captive M. yumanensis bats free-flying in large
open air enclosures during summer months – one pilot trial in 2018 and a
comprehensive trial in 2019. The latter trial expanded into fall and early winter to include
a laboratory hibernation experiment of captive bats. The hibernation experiment

22

examined the response of our probiotic bacteria on hibernating bat in a controlled
environmental chamber.
METHODS
Captive Bat Enclosures
Two large bat enclosures were built at the BC Wildlife Park (BCWP) in
Kamloops, located away from the public and enclosed in an area only accessible by
employees. Each of the two enclosures were covered by a large carport to provide
shelter from sun and weather; however, bats were still exposed to environmental
fluctuations including changes in temperature, humidity, and wind because the ends of
the carport was left open and the enclosures’ outer walls were constructed of wire
mesh. A solid middle wall divided each enclosure in half, forming two flight chambers of
equal area and design (each chamber 3.05m long x 1.37m wide x 3.05m high). The two
enclosures were approximately 30 metres from each other. Some of the mesh walls
were strategically covered with corrugated plastic board to reduce wind turbulence
which could result in air exchange between sides, and to ensure that bat feeding and
water dishes were not upset by strong gusts of wind. All human entry was through a
vestibule on one end of each flight chamber, and these vestibules were on opposite
ends of the chambers to further reduce the chance of contamination. Vestibules had a
door on each side – one for entry/exit from the vestibule to the outdoors, and the other
door which entered into the flight chamber. Vestibules’ outside doors were hinged solid
wood with spring hinges for automatic closure, and the inside doors were zippered vinyl
to ensure bats could not escape from the chamber. A Tyvek suit, boot covers, shower
caps, and disposable gloves were donned while standing in the vestibule so that all
entry in and out of the chamber reduced chance of cross-contamination. Each day, the
‘control’ bats were tended to first and then the ‘treatment’ bats to reduce chance of
accidental introducing probiotic to the control side. There was a large Rubbermaid
storage bin outside each vestibule which held items specific to each enclosure group.
Items that did move between sides were sterilized with 70% ethanol before reuse.

23

Each flight chamber had a single chamber bat house (with hinged front door to
allow access to bats) mounted on the middle dividing wall for bats to roost in. On each
end (opposite ends of each flight chamber), there was a watering dish, and a food
station. For details on bat care see Appendix B.
Only one enclosure had been built at the time of the 2018 pilot captive trial, and
one flight chamber was used for the Treatment bats and the other for the Control bats.
In 2019, we used two enclosures, one enclosure housed two groups of Treatment bats
(Treatment 1, Treatment 2), each in their own flight chamber (Figure 2.1). And the other
enclosure housed the Control bats, all on one side in one flight chamber. The second
flight chamber of this enclosure was not used. A HOBO microclimate data logger (Onset
Corporation, SN 20340415 for treatment 1 and SN 20340414 for treatment 2) was
installed into each treatment chamber to measure temperature and relative humidity.
Each logger had two probes: one was placed ¾ of the way into the left side of the box
and the other probe was attached to the wall of the same enclosure within a cut-out
yogurt container to shield it.
A.

B.

Figure 2.1. A. Floor plan of control and treatment enclosures. The control enclosure was
approximately 30m away from the treatment enclosure. B. Outside view of treatment
enclosure.
24

Animal Collection and Handling
M. yumanensis bats were captured from Vernon, Chase, Greater Vancouver, or
Creston, BC, under the permit issued to C. Lausen, WCS Canada, 287882. Each bat
used in the trial had a colored or numbered forearm band on its wing to differentiate
them from one another. They were taught to self-feed on mealworms and self-drink out
of a water dishes. In 2018, 11 adult M. yumanensis were taken captive (10 males, 1
female), and in 2019, 20 adult M. yumanensis were taken captive (19 males, 1
females).
Probiotic Composition
In 2018, two synergistic strains, which were Pseudomonas strains that exhibited
full inhibition of Pd growth in vitro were used in our pilot probiotic. Both isolates were
sourced from a Eptesicus fuscus female adult bat from a mine hibernaculum near
Salmo, BC. One of these strains were identified as P. synxantha strain B and was used
in our 2019 experiment probiotic. Full inhibition isolates are characterized by their ability
to fully inhibit Pd, which is contrast to a partial inhibitor, that only suppresses hyphal
growth.
In 2019, four synergistic Pseudomonas strains were included into a final probiotic:
Pseudomonas synxantha strains A (full inhibitor) and B (full inhibitor), P. azotoformans
(full inhibitor), and P. antarctica (partial inhibitor). Each isolate was previously sourced
from bats in BC. Pseudomonas synxantha strain A was isolated from a M. yumanensis
at a mine hibernaculum near Salmo, British Columbia; strain B was isolated from an
adult female E. fuscus captured free-flying at a mine near Salmo, BC (as told above); P.
azotoformans was isolated from an adult Corynorhinus townsendii caught from a
maternity roost in Deroche, British Columbia; and P. antarctica was sourced from a
female juvenile M. evotis at from a mine hibernaculum near Nelway, BC.
Probiotic strains were made in large quantities and pelleted and lyophilized using
a benchtop freeze-dryer (Labconco). All water was extracted from cell pellets after 3

25

hours at -50°C. Viability and inhibition of probiotic cocktail was confirmed after the
freeze-drying process. Dry pellets were ground into a fine powder using a sterile pestle.
In 2018, probiotic was applied directly to bats’ wings in sterile water using a
pipette and in 2019, probiotic was instead applied by freeze drying bacteria and
combining it with sterile potter’s clay. Two hundred and fifty million Colony Forming
Units (CFU) of each bacterium were used as a cumulative dose of about 1 billion cells
for each dosage. Each dose was applied with sterile water onto each bat house to
reactivate the freeze-dried bacteria.
Development of Probiotic Application
An application method for the four probiotic bacteria underwent different revisions
throughout the 2019 trial. The first method incorporated a combination of our bacteria
directly with water before application. 1 billion lyophilized cells were combined with
275mL of sterile water and sprayed onto the Treatment 1 bat house on April 26th. Bats
were removed from their bat house and placed into sterile cotton bags before removing
their bat house from the treatment chamber. The bat house was put flat onto the ground
and the door was opened to expose the roosting chamber. A hand pump sprayer was
used to apply the probiotic-water solution evenly onto the bat house chamber.
Afterwards, the bat house sat overnight until it was dry and re-established within their
treatment enclosure. A different sterile bat house was used as a substitute.
Water and clay were added separately for the second application method and
were used in a two-step process. Bats were removed from their bat box and placed into
cloth bags during the inoculation. The bat box was removed and sprayed with a minimal
amount of sterile water evenly over the entire surface. The probiotic was prepared in
sterile potter’s clay and spread evenly over the bat box within the enclosure. After ten
minutes the bat box was placed into the same location previously and bats were placed
back into the box. Bacteria amounts were consistent for repeated doses, however
different amounts of clay and water were utilized for our Treatment 1 bat house in order
to establish an ideal consistency for repeated applications: 125mL water and 10g of clay
26

on May 12th and 24th, 50mL water and 30g clay on June 24th, and 29th. The
Treatment 2 bat house received the same later dosage on June 15. Finally, two
dosages of 1 billion cells were applied to our treatment 2 captive bat boxes on
September 30th and October 7th before acclimation began on November 3rd for the
hibernation experiment. The method of applying water and then powered clay became
the final application method used, although there was further work done on how to
create a sprayer to put clay powder up into bat box chambers in the field where
chambers are vertical and cannot be accessed via a door as they were in the single
chamber bat boxes used in the enclosures (see Chapter 3 methods).

April 26th

June 15th

Treatment 1

Treatment 2

group
probiotic
application

group
probiotic
application

July 8th

August 9th

September 30th
and October 7th

November 3rd

Treatment 2

Probiotic

Acclimation

Treatment 1

group ends.

applications

to

group

Bats wait

of bat box

hibernation

euthanasia

now until

used by

chamber

hibernation

bats

began

December 21st

Euthanasia
of bats in
hibernation
trial

Figure 2.2. Timeline of probiotic application and euthanasia of Treatment 1, 2 and
Hibernation groups. *Bats remained in outdoor enclosure throughout September and
October.
Four-chambered Bat Box
In 2019, a four-chambered bat box (Chamber 1 directly above landing platform;
Chamber 4 was furthest from the landing platform) was treated with probiotic-laden clay,
just as were the single-chamber bat boxes within each enclosure’s flight chamber.
However, this box was mounted on a pole in direct sunlight, as would a typical field27

based bat box for wild bats. The bat box was hung 3m off the ground and faced the
sunset (west) starting May 17th, 2019. A HOBO microclimate datalogger (Onset
Corporation, SN 20577875 for chamber 1, SN 20577881 for chamber 2, SN 20577876
for chamber 3, and SN 20569683 for chamber 4 ) was placed into each chamber of the
bat house to measure temperature and relative humidity (chamber 4’s sensor recorded
temperature only). This box’s entrance was screened off to prevent it from being used
by wild bats in the area; probiotic concentrations and internal microclimate were
measured throughout the summer.
The loggers were strategically placed for different experiments: initially at a depth
of 15.25cm from May 17th-July 14th for the first experiment, and 30.5cm into the box
from July 14th until the end of the trial for the second experiment. Each chamber was
inoculated with a ‘dose’ of probiotic clay powder and a clay paste on May 23rd, both of
which were 15.25cm deep into the bat box (Figure 2.3). On July 20th a second powder
treatment was inoculated deeper within each bat box chamber at 30.5cm. Each
treatment was separated and swabbed individually by chamber each week until the end
of August.
A

B

Figure 2.3. A. Four-chamber bat box hanging off the ground and away from our captive
bat enclosures. B. Clay paste application (left), powder application (right) within the fourchamber bat box.
28

May 17th

May 23rd

July 20th

August 24th

Four-

Clay paste

Second

Last swab

chamber

and

clay

of four-

bat box

powder

powder

chamber

hung up

application

application

experiment

Figure 2.4. Timeline of four-chamber bat box experiment.

Sampling of Microbes from Wings and Roost Substrates
Treatment 1 group of captive bats were swabbed with sterile polyester tips from
April 28th until July 8th. The second treatment group was swabbed from June 15th until
August 24th. Finally, the hibernation trial bats were swabbed from November 16th to
December 21st. Swabs were performed once a week to prevent bat stress and
interference with treated bats microflora. Each bat was individually swabbed by rolling
the polyester tip evenly onto the bat’s wings.
Swabbing protocol evolved as the experiment proceeded and this meant that
Treatment 1 bats were swabbed differently throughout the trial. Wing area was mapped
by measuring left and right arm length (see Appendix C). On May 26th and June 1st,
the ventral and dorsal side were swabbed together. One swab constituted both wings,
wing tip to armpit along the wing membrane, on one side of the bat (ventral or dorsal).
One swab for both wings, wingtip to armpit on the dorsal side, were used permanently
from June 8th until the end of the trial.
Bats from the hibernation trial were removed from the cooler every 7-10 days to
offer water and food, if needed, and to perform swab sampling. Bats were warmed with
29

heat pads for approximately 5 minutes before food and water were offered. A polyester
swab was rolled along the top portion of one wing from tip to armpit. Left and right wings
were alternated when swabbing to prevent accidental rubbing off of probiotic from
previous swabbing. We swabbed along the myotis finger bones. Each week a different
fingerbone was used. polyester swab tips were broken off into Eppendorf tubes
containing 1mL of sterile water and were immediately transported to the lab.
Processing of Swab Samples
Using sterile polyester-tipped swabs, bats and roost substrates were periodically
sampled to quantify presence of each probiotic bacterial strain using qPCR. Custom
probes were designed specific to each bacterial strain. Upon swabbing of bat wings or
roosting substrates, swabs were immediately placed into Eppendorf tubes, refrigerated,
and shipped for qPCR analysis at McMaster University. In 2018, the wing area for each
bat was measured using photography and subsequent calculation of area
(SketchAndCalc 2021). Subsequent analyses showed no significant difference among
wing areas, and thus for 2019, mean swabbing areas were used to calibrate all cell
concentration for wings depending on swab location (/cm 2; see Appendix C for
measurements and dates used).
An additional test of viability on our samples was introduced on June 8th until the
end of the trial. This test required the swab sample to be split: each Eppendorf vial
containing a swab was vortexed on medium-high for 20 seconds and the tips were
removed from the water inside the tube. The remaining 1mL was divided into a separate
Eppendorf containing 1mL of LB broth, and the remaining water was frozen. The LB
broth containing half our sampled cells were placed onto a shaker at 25 oC on medium
for 12 hours. Afterwards the broth was frozen and transported to McMaster University
alongside our original frozen water samples for qPCR analysis. The surface area was
measured of each wing section to account for differences in inoculated area.
Collaborative researchers at McMaster University used the following protocol for
qPCR analysis. Species-specific gyrB primers (modified from Yamamoto et al. 2000)
30

were used to quantify cell concentrations in each swab compared to background levels.
Swabs tips were washed with PBS in sterile tubes and vortexed. This suspension was
then sonicated for 30 seconds in a sterile glass vial. Standards were created through
serial dilution of cells (105, 104, 103, and 102) which were prepared identically to wing
swab samples. All reactions were carried out in triplicate containing: 10μl of 2X NEB
Luna qPCR (SYBR green dye), 0.5μL of each primer at 10μM, and 2μL of sonicated
cells. Reactions were run on a BioRad CFX96 machine (Mississauga, Ontario). Initial
denaturation was performed at 95°C for 2 minutes, followed by 40 cycles of additional
denaturation at 95°C for 30 seconds and annealing/extension for 30 seconds at 60°C.
Using CFX Manager software (V3.0) a standard curve was created using the Cycle
Threshold (Cq) values from samples of known concentration of cells. Fitting the Cq
values from samples of unknown concentration allow for us to extrapolate the number of
cells present in each swab. These swabs were corrected for the average wing
area. Standard error was calculated for all means. See appendix G for sample sizes
and details on mean and error calculations. Unfortunately, due to unreliability with the P.
antarctica probe, only some of the samples managed to be salvaged and presented
appropriately.
Hibernation Trial
Three M. yumanensis bats from our previous captive trial were acclimated into
the hibernation chamber gradually over 5 days, followed by another decrease of 1 oC 14
days later (see Appendix D for environmental chamber conditions). A modified glassdoor wine fridge of approximate dimensions was used as a hibernation chamber: width
of 59.4cm, height of 85.6cm and depth of 62.5cm (Figure 2.5). The fridge was
maintained at a temperature of 4.5-8oC throughout the trial, using a digital thermostat
controller which was accessible on the outside of the fridge. Internal humidity was
measured and controlled to range between 90-100% humidity by using a terrarium
humidifier (Zoo Med Reptile Fogger) ; humidity control was automated using a humidity
controller (Inkbird) to replicate M. yumanensis hibernating environmental conditions. A
reptile humidifier was used to achieve high humidity and an electronic humidity sensor
31

powered the humidifier when humidity dropped below 90%. Temperature and humidity
were monitored using a HOBO microclimate monitor (SN 20577876) and conditions
were modified upon observation.
Mesh screen lined the fridge to allow bats to move around in the chamber. A
cloth roosting pouch, a tin food dish, and a small water dish (with marbles and sponge
to prevent drowning) were placed into the hibernation chamber. A wide-angle infrared
camera (Model number: GXLFPW7WE58LHJS2111A) was placed into the chamber
which enabled monitoring of the bats without disturbance. Video footage was automated
and could be accessed via a smartphone app (iCookyCam). The glass door of the fridge
was covered with a thick blanket to keep out light, and the room housing the fridge was
kept dark. An oxygen tank was connected by an external hose which reached into the
chamber; oxygen was monitored using an oxygen sensor and air levels were manually
maintained between 19-24% oxygen using an external air tank. An oxygen monitor was
installed inside the chamber. Once every ~10 days the door of the fridge was opened,
bats were weighed, warmed, swab-sampled and then offered food (mealworms) and
water.

Figure 2.5. A modified beverage cooler was converted into an environmental chamber
for the bat hibernation experiment. The temperature and humidity were controlled by a
thermostat and reptile humidifier, respectively. Humidity was monitored with a sensor to

32

retain >90% relative humidity. A wireless camera was installed to view the bats and
record activity. The roost pouch and food were removed shortly after acclimation.
Necropsies and Histology
All captive bats were ultimately euthanized (see figure 2.2 for dates). Necropsies
and wing tissue histology (n = 9 bats in 2018; n = 13 bats in 2019) were conducted by
Dr. Glenna McGregor (BC Animal Health Lab, Abbotsford, BC examined skin of wings,
ears, nose and tail looked for the following potential pathologies: acanthosis, bacterial
epidermal, bacterial invasion, dermal inflammation, epidermal inflammation,
hemorrhage, hyperkeratosis, hyphae, mites, monocytes, overall inflammation, rabies,
segs (mature neutrophiles), ulceration and yeast).

RESULTS
Swab Sample Results - 2018 Pilot
From wing samples taken at the start of the 2018 pilot captive trial, we detected
large quantities of the probiotic strains on bats from the treatment side of the enclosure
(Figure 2.6B). The probiotic abundance on samples taken from the control side of the
enclosure was low at first, but increased over time, increasing to levels similar to that of
treated bats (Figure 2.6A), providing conclusive evidence of cross-contamination, likely
through probiotic travelling in the air between sides of the enclosure during spraying of
probiotic on treatment bats. As such, the wing concentrations from the 2018 captive bat
trial were not used in further analyses, and are presented here (Figure 2.6) only to
provide context for the necropsies and histology examination. Refinement of methods
from the 2018 trial informed the 2019 captive trial, including the building of a separate
enclosure 30 metres away for the Control bats to eliminate the problem of crosscontamination.

33

A)

B)
Date

Date

Figure 2.6. A) The number of probiotic cells detected on treated bats was determined
using qPCR of wing swabs, relative to pre-exposure concentrations, corrected by wing
area. B) The number of probiotic cells detected on control bats using qPCR of wing
swabs, corrected by wing area. Each colour is one individual captive bat. Figures
prepared by Adrian Forsythe.
2019 Captive Summer Trial
Baseline and control swabs
Concentrations of each probiotic bacteria were quantified on every bat when they
were first introduced into captivity on April 21st (Figure 2.7). Background levels of all
three probiotic bacteria were detectable from their wings and averaged to find a mean.
There were 4.99 ± 1.21 cells/cm2 (31 replicates) of P. synxantha B, 88.4 ± 19.9
cells/cm2 (n=30 replicates) of P. synxantha A, and 1.62 ± 0.37 cells/cm2 (n=21
replicates) of P. azotoformans. Probiotic bacteria was found on the control bat box
throughout the captive trial however it was very low relative to the treatment bat boxes
(Figure 2.8, 2.10 and 2.12).

34

Bacteria Concentration/mL/cm2

1000

100

10

1
P. synxantha strain B

P. synxantha strain A

P. azotoformans

Bacteria species

Figure 2.7. Number of probiotic cells present on captive bats on April 21st, before
probiotic application began. Note the logarithmic scale.

Concentration of cells/mL/cm2

1000
100
10
1
0.1
0.01
2019-04-28

2019-05-05

2019-05-19

2019-06-15

2019-06-29

2019-07-08

Date
P. synxantha strain B

P. synxantha strain A

P. azotoformans

Figure 2.8. Number of probiotic cells present on the control bat box throughout the trial.
Note the logarithmic scale.

35

Broth Results
The broth results were dropped from our analysis because of too many
confounding variables and randomness of the data. Factors that lowered the reliability
of our results included: competing microbes growing in the broth, only incubating the
samples for 12 hours, and inability to reliably split a suspension 50/50 in terms of cell
count. Results for the broth test are found in Appendix E.
Treatment 1
Concentrations were measured on bat wings from May 26th until June 29th.
Concentrations of each bacteria followed a similar trend and P. synxantha A was
significantly higher throughout the trial than P. azotoformans and P. synxantha B
(Figure 2.9) per cm2 of bat wings. P. synxantha A ranged between 5.95 ± 3.77 x102 to ±
4.88 ± 2.57 x105 cells/cm2 throughout the trial and was consistently at least ~15 times
higher in concentration if we do not consider the similar concentrations of P. synxantha
A and P. synxantha B on June 1st. Concentrations of the other 3 bacteria ranged from
19.1 ± 12.4 cells/cm2 (P. azotoformans strain B on June 1st) to 1.74 ± .904x104 cells/cm2
(P. synxantha B on May 26th).
Bat box concentrations (figure 2.10) show a similar pattern seen in the bat
swabs, in which P. synxantha A was much higher than the other two probiotic bacteria
in quantity. P. synxantha A cell concentration ranged between 1.95 ± ,144 x104
cells/cm2 to 3.95 ± .197 x104 cells/cm2 throughout the trial period. The other three
bacteria ranged from 563 ± 32.5 cells/cm2 (P. azotoformans on June 1st) to 2.08 ± .12
x103 cells/cm2 (P. synxantha B on June 8th). The concentration of P. synxantha A was
consistently at least ~18.5 times higher than the other two bacteria concentrations on
each swabbing date.

36

Concentration of cells/ml/cm2

1000000
100000
10000
1000
100
10
1
2019-05-26

2019-06-01

2019-06-08

2019-06-15

Date
P. synxantha strain B

P. synxantha strain A

P. azotoformans

Figure 2.9. Starting quantities of probiotic cells detected on treated bats in the
Treatment 1 group using qPCR for each probiotic bacteria species. Concentrations are
corrected by wing area to represent cm2. Note the logarithmic scale.

Concentration of cells/mL/cm2

100000

10000

1000

100
2019-06-01

2019-06-08

2019-06-15

Date
P. synxantha strain B

P. synxantha strain A

P. azotoformans

Figure 2.10. Concentrations of probiotic cells detected within the bat box in the
Treatment 1 group using qPCR for each probiotic bacteria species. Concentrations are
corrected by wing area to represent cm2. Note the logarithmic scale.

37

Enclosure and bat box temperatures were compared between Treatment 1 and
Treatment 2 groups for any significant differences. The temperature of each enclosure
and bat box within treatment group 1 and 2 were measured from May 12th until August
7th (n=41,601 for each location and treatment group). There were no significant
differences between enclosure temperatures (19.1 ± 0.03 oC treatment 1; 19.00 ± 0.03
oC treatment 2; P>0.05), however, bat box temperatures were significantly different

(19.6 ± 0.03 oC treatment 1; 19.4 ± 0.03 oC treatment 2; P<0.05). The significant
difference is very small with a mean difference (T1-T2) of 0.190 ± .001 oC.
Treatment 2
Concentrations were calculated based on area of the skin in cm2 and a mean
was found between all bat wing swabs. Bat swabs taken from the Treatment 2 group
have a detectable concentration of each species throughout the trial from June 29th
until August 14th (Figures 2.11). P. synxantha A was overall the most abundant among
each probiotic species, however relative concentrations between the probiotic species
are mixed. P. synxantha A had a range of 13.6 ± 8.63 cells/cm2 to 667 ± 0 cells/cm2 and
was only significantly higher than the other 3 bacteria on July 13th and August 9th (at
least ~19 times higher). The other 3 bacteria ranged between 12.7 ± 0.86 cells/cm2 (P.
antarctica on August 9th) and 1.36 ± 1.35 x103 cells/cm2 (P. azotoformans on August
14th)
Treatment 2 bat box swabs showed an abundant probiotic concentration
throughout the trial since application on June 13th (Figure 2.12). P. synxantha A ranged
between 1.68 ± .81 x104 cells/cm2 to 3.99 ± 2.07 x104 cells/cm2 and was consistently at
least ~18 times higher than the other bacteria species. Each of the other three probiotic
species ranged between 91.2 ± 42.5 cells/cm2 (P. antarctica on August 18th) and 2.17 ±
1.14 x103 (P. synxantha B on July 27th) throughout the swabbing period.

38

Concentration of cells/ml/cm2

10000
1000
100
10
1
0.1
2019-06-29

2019-07-13

2019-08-09

2019-08-14

P. azotoformans

P. antarctica

Date
P. synxantha strain B

P. synxantha strain A

Figure 2.11. Starting quantities of probiotic cells detected on treated bats within the
Treatment 2 group using qPCR for each probiotic bacteria species that was frozen in
H2O. Note the logarithmic scales.

Concentration fo cells/mL/cm2

100000

10000

1000

100

10
2019-07-13

2019-07-27

2019-08-09

Date
P. synxantha strain B

P. synxantha strain A

P. azotoformans

P. antarctica

Figure 2.12. Starting quantities of probiotic cells detected on the bat box within the
Treatment 2 group using qPCR for each probiotic bacteria species that was frozen in H2O.
Note the logarithmic scales.

39

Four-chamber Bat box
The probiotic bacteria were still detectable using qPCR when exposed to
summer heat within the four-chamber bat box three months after original application,
from July 20th until August 24th (Figure 2.13). The results from the paste application
were excluded due to small amounts of detectable bacteria found immediately after
application. All chambers were combined to form a mean of detectable bacteria found in
the four-chamber bat box across all chambers for each date swabbed. This allowed us
to look for general trends across all samples of our data.
P. synxantha A was significantly more abundant than P. synxantha B, P.
antarctica, and P. azotorformans, which all had similar quantities throughout the trial.
Results are similar to the bat box results for treatment 1 and treatment 2. In which P.
synxantha A is approximately 18.5 times higher than other individual probiotic bacteria
species across all sampling dates except for June 9th, in which it is only 4.4 times
higher. P. synxantha A concentration ranged from 9.73 ± 1.88 x103 to 1.58 ± .860 x105
cells/cm2 throughout the trial, whereas the other 3 bacteria ranged from 23.9 ± 7.26
cells/cm2 (P. antarctica on August 24th) to 1.49 ± 1.14 x104 (P. antarctica on August 9th).

40

Concentration of cells/mL/cm2

1000000
100000
10000
1000
100
10
1
2019-07-20

2019-07-27

2019-08-09

2019-08-17

2019-08-24

Date
P. synxantha strain B

P. synxantha strain A

P. azotoformans

P. antarctica

Figure 2.13. Concentration of probiotic cells detected in combined chambers of the fourchamber bat box using qPCR for each probiotic bacteria species frozen in H 2O. Note
the logarithmic scales.
Daily temperatures within each chamber averaged between 15.78oC-33.75oC
across the entire monitoring period (Figure 2.14). Chamber 4 (front chamber) exhibited
the highest average daily temperature from the end of July and into August, however
none of the chambers average values were significantly different throughout the trial
period (P=0.456; Table 2.1 ). Chamber 1 had a statistically significantly higher maximum
daily temperature when compared to chambers 2, 3 and 4 (P<0.001) (Figure 2.15)
throughout the trial period. The maximum temperatures seen in chambers 1-4 were
57.4oC, 49.7oC, 49.5oC, and 51.8oC, and the minimum daily temperatures were 5.5oC,
6.3oC, 6.5oC, and 6.4oC, respectively. Chamber 1 had a significantly higher maximum
temperature than other chambers (P<0.05; Table 2.2). We saw no significant
differences in average daily relative humidity or minimum temperatures among
chambers (P>0.5) (results not presented).

Individual chambers differed significantly in probiotic concentrations (table 2.3).
Chamber 1 was the highest, followed by chamber 2, 3 and then 4 (P<0.05). When
41

bacteria concentrations are compared, P. synxantha A was the only significant probiotic
bacteria (P<0.05), whereas P. azotoformans, P. synxantha B, P. antarctica are not
significantly different from each other (P<0.05).
35

Temperature oC

32.5
30
27.5
25
22.5
20
17.5
15

Date
Chamber1Mean

Chamber2Mean

Chamber3Mean

Chamber4Mean

Figure 2.14. Average daily temperature for each chamber of the four-chamber bat box
between 2019-05-17 and 2019-08-15.

42

Table 2.1. One Way Analysis Of Variance and Post Hoc test between each chamber of
the four-chamber bat box comparing average daily temperature. Means that do not
share a letter are signficantly different.
Analysis of Variance for Average Daily Temperature
Source

DF

Adj SS

Adj MS

F-Value

P-Value

Factor

3

46.41

15.47

0.87

0.456

Error

268 4756.41

Total

271 4802.81

Factor

N

Mean

StDev

95% CI

Chamber1Mean

68

23.638

4.01

(22.633, 24.644)

Chamber2Mean

68

23.345

4.138

(22.339, 24.351)

Chamber3Mean

68

23.525

4.185

(22.519, 24.531)

Chamber4Mean

68

24.426

4.503

(23.420, 24.431)

17.75

Grouping Information Using the Tukey Method and 95% Confidence
Factor

N

Mean

Grouping

Chamber4Mean

68

24.426

A

Chamber1Mean

68

23.638

A

Chamber3Mean

68

23.525

A

Chamber2Mean

68

23.345

A

43

Temperature oC

60
55
50
45
40
35
30
25
20
15

Date
Chamber1Max

Chamber2Max

Chamber3Max

Chamber4Max

Figure 2.15. Average daily maximum temperature for each chamber of the fourchamber bat box between 2019-05-17 and 2019-08-15.

44

Table 2.2. One Way Analysis Of Variance and Post Hoc test between each chamber of
the four-chamber bat box comparing daily maximum temperatures. Means that do not
share a letter indicate statistically signifcant differences.
Analysis of Variance for Maximum Daily Temperature
Source

DF

Adj SS

Adj MS

F-Value

P-Value

Factor

3

1349

449.71

8.28

0.000

Error

268 14557

54.32

Total

271 15906

Factor

N

Mean

StDev

95% CI

Chamber1Mean

68

23.638

4.01

(22.633, 24.644)

Chamber2Mean

68

23.345

4.138

(22.339, 24.351)

Chamber3Mean

68

23.525

4.185

(22.519, 24.531)

Chamber4Mean

68

24.426

4.503

(23.420, 25.431)

Grouping Information Using the Tukey Method and 95% Confidence
Factor

N

Mean

Grouping

Chamber1Mean

68

24.426

A

Chamber4Mean

68

23.638

B

Chamber2Mean

68

23.525

B

Chamber3Mean

68

23.345

B

45

Table 2.3. General Linear Model for details of frozen water samples on chamber,
bacteria species, and interactions between the two. Means that do not share a letter
indicate statistically signifcant differences.
Analysis of Variance for Frozen Water Samples
Source

DF

Adj SS

Adj MS

F-Value

P-Value

Chamber

3

28.723

9.5743

15.06

0.000

Bacteria Species

3

79.773

26.591

41.84

0.000

Chamber*Bacteria Species

9

1.142

0.1269

0.2

0.994

Error

171 108.683

Total

186 218.205

0.6356

Tukey Method and 95% Confidence
Chamber

N

Mean

Grouping

1

47

4.23486

A

2

46

3.64363

B

3

47

3.40292

4

47

3.18837

Bacteria Species

N

Mean

P. synxantha A

48

4.72339 A

P. synxantha B

48

3.44183

B

P. azotoformans

48

3.18439

B

P. antarctica

43

3.12017

B

B, C
C

Grouping

Bat Box Probiotic Comparison
P. synxantha A has a higher ratio of cell abundance compared to other probiotic
bacteria found within each bat box (Table 2.4). When the ratio of P. synxantha to the
average value of other probiotic bacteria is tested, an increase in concentration of P.
synxantha is seen over time. Treatment 1 bat box P. synxantha A concentration
increased by 4% from June 6th to June 15th, Treatment 2 bat box increased by 9.1%
46

from July 13th to August 9th, and the four-chamber bat box increased by 8.3% from July
20th to August 24th.
Table 2.4. Ratio of P. synxantha strain A to all other probiotic bacteria found within the
bat box, from first swabbing date to last. A higher ratio signifies greater abundance of P.
synxantha A to other probiotic bacteria.
Treatment 1 Bat Box
2019-06-01
2019-06-15

(P. synxantha strain A)/((P. synxantha B + P. azotoformans/2))
24.3
25.3

Treatment 2 Bat Box
2019-07-13
2019-08-09

(P. synxantha strain A)/((P. synxantha B + P. azotoformans+P. antacrtica/3))
32.0
34.9

Four-chamber Bat Box
2019-07-20
2019-08-24

(P. synxantha strain A)/((P. synxantha B + P. azotoformans+P. antacrtica/3))
32.6
35.3

Hibernation Captive Trial
Primers specific to each of the probiotic species were used to quantify the
abundance of probiotic present on wing tissue throughout the hibernation trial. Bats
were not exposed to probiotic bacteria since the application dates of September 30 th
and October 7th. Results suggest that each of the probiotic species stabilized in
concentration throughout the testing period from November 16th until December 21st
(Figure 2.16). However, P. synxantha largely dominated with a higher concentration and
was ~18 times higher than the other individual bacteria concentrations except for the
last swab on December 21st. Detectable concentrations of each bacteria significantly
dipped in concentration on December 18th but returned to previous levels on December
21st, in which P. antarctica reached 6.95 ± 3.9 x109 cells/cm2 and trumped P. synxantha
A by 141,919-fold. Each bacteria species significantly increased in concentration from
their original starting quantities from November 16th, however P. antarctica increased
47

4.66x106 fold higher in comparison to the other bacteria which increased ~14 fold from
their starting quantities, although this could be an outlier in the data (Table 2.5). This
increase suggests that the probiotic bacteria were proliferating on the captive bat wings
during hibernation when exposed to the high humidity and low temperature conditions.
Captive myotis body weights slowly decreased throughout the trial and can be attributed
due to their disturbance from weekly swabbing and artificial hibernating conditions. Bats
were largely torpor throughout the hibernating conditions but were removed once they
reached a critical body mass of ~4.0g.

Concentration of cells/ml/cm2

1.E+11
1.E+10
1.E+09
1.E+08
1.E+07
1.E+06
1.E+05
1.E+04
1.E+03
1.E+02
2019-11-16

2019-11-28

2019-12-08

2019-12-18

2019-12-21

Date
P. synxantha strain B

P. synxantha strain A

P. azotoformans

P. antarctica

Figure 2.16. Concentration of probiotic cells detected on treated bats using qPCR for
each probiotic bacteria species after being placed in sterile water and
immediately frozen. Cell concentrations are corrected by wing area to represent cm2.
Note the logarithmic scale.

48

Table 2.5. Concentrations of probiotic detected on bats in hibernation chamber, at the
start and end of the 35 day experiment. Concentrations (adjusted by wing area, cm2)
were determined using qPCR. N=3 bats. Note: the final value of P. antarctica may be an
outlier.
P. synxantha B

P. synxantha A

P. azotoformans

P. antarctica

Start

1.56 ± .186 x103

2.79 ± ,337 x104

870 ± 103

1.49 ± .096x103

concentration

(.691 - 2.32 x103)

(1.24 - 4.1 x104)

(.395 - 1.3 x103)

(1.21 - 1.81 x103)

End

2.24 ± .148 x104

4.11 ± .275x105

1.08 ± .081 x104

6.95 ± 3.9 x109

concentration

(1.88 - 2.82 x104)

(3.27 - 4.96 x105)

(8.72 - 1.37 x104)

(8.35x101 -2.02x1010)

14.4

14.7

12.4

4.66x106

(/cm2)

(/cm2)
Fold increase

Necropsies and Histology
Fall Trial
Necropsies were performed by Dr. Glenna McGregor, BC Animal Health Lab. A
summary of microscopic test scores from the fall captive trial (Appendix F) reveals that
most of the bats that remained in captivity until the end of the trial period showed overall
high scores (negative health), ranging from 9 - 94 (mean = 46; n = 6). Bats that died
prior to the end of the trial and were thus only in captivity for less than 1 month, showed
overall lower scores (better health), ranging from 1 – 13 (mean = 5.3; n = 3), and
showing overall less inflammation. In general, G. McGregor commented that the
inflammation was higher in all bats that were examined in contrast to what she would
typically see in wild bats; this included more acanthosis (thickening of the skin),
potentially due to repeated swabbing. Bats that were euthanized at the end of the trial
were examined for neutrophils in the lung and spleen and it was found that there was a
large number of these indicative of a systemic inflammatory response (G. McGregor,
pers. comm.), but also commonly associated with a response of hibernators when
entering deep bouts of torpor (Bouma et al. 2010).

49

Summer Captive Trial
Necropsies performed on the control group were compared to the Treatment 1
group. Wings, ears, tails, noses, lungs, spleens were examined for a lengthy number of
histology criteria (Appendix F) such as inflammation, neutrophil and monocyte count,
acanthosis and hyperkeratosis. Results were similar to the fall trial histology
examination; however, the control bats more closely resembled the treatment bats
based on criteria and no significant differences were found between the two groups
(P>0.05; not shown here) when a General Linear Model is used to compare histology
criteria (Appendix F). The only significant outlier was dermal inflammation in the tail
(P=0.045), in which the control had more inflammation than our treatment group, which
is a negligible finding and is likely due to handling.
DISCUSSION
We saw no detrimental effects from the prophylaxis application in two separate
captive trials and the probiotic can easily be transported and implemented using the
clay substrate with freeze dried cells application method that we developed. Probiotic
bacteria were persistent and presumably viable on captive bats and bat boxes months
after application, particularly P. synxantha A, including when exposed to high summer
heat in the 40-50oC range, and substantially increased in viability and growth when
exposed to environmental conditions in the hibernation trial. P. synxantha A abundance
was significantly higher in treatments 1 and 2 and the four-chamber bat box, whereas P.
antarctica was significantly higher at the end of the hibernation experiment.
Solutions are urgently needed to prevent WNS infection from inevitably causing
bat mortalities in Western Canada. Pd infection has not been officially documented in
British Columbia (WNS 2019), however, bat mortalities due to WNS are already evident
in Washington state (WDFW 2021). M. septentrionalis is threatened and other bat
populations could collapse without human intervention (Frick et al. 2015). British
Columbia has the largest number of bat species in Canada, and thus stands to lose
significant biodiversity if WNS affects many western species. Few studies have tested
50

WNS treatment methods on live bats, and fewer have investigated use of bacteria
antagonistic against Pd (Cheng et al. 2016; Hoyt et al. 2019). This study is the first one
to test an anti-Pd probiotic cocktail on captive bats in summer on free-flying bats.
I describe the cellular concentration of bacteria on wings in relation to length of
time swabbing – in summer (captive enclosures roosting in bat boxes) and winter
(hibernation chamber) conditions. There is a clear pattern in probiotic bacteria numbers
that I describe. I also describe the relationship between roost microclimate temperatures
in the four-chamber bat box in relation to changes in bacterial concentrations on
roosting substrates. We determined that the final probiotic cocktail is safe to use, having
found no negative effects on the health of bats associated with its application.
Treatment 1 probiotic bat swab concentration decreased by more than half from
the May 26th to June 15th samples. By June 15th, bacteria concentrations were 6.62 ±
6.5 x103 cells/cm2 for P. synxantha B (62% lower), 1.0 ± .98 x105 cells/cm2 for P.
synxantha A (79.5% lower), and 3.66 ± 3.59 x103 cells/cm2 for P. azotoformans (55.6%
lower). The treatment 2 probiotic bat swab concentrations changed in concentration
from June 29th to August 14th with final concentrations of 77.0 ± 75.2 cells/cm2 for P.
synxantha B (31% lower), 58.4 ± 24.1 cells/cm2 for P. synxantha A (4.29x higher), and
1.36 ± 1.35 x103 cells/cm2 for P. azotoformans (23.2x higher). The standard error is
quite large for some values which is because of the large variability seen in the qPCR
triplicate values of each swab. The larger values in treatment 1 bats are likely because
of the multiple treatments and revisions of probiotic application they underwent on April
26th, May 12th, and May 24th. Therefore, it is likely that treatment 1 bats had a
background concentration of probiotic from their previous treatments leading into May
26th, when their probiotic concentrations were highest. Treatment 2 bats were only
exposed to one probiotic application on June 15th and have a relatively lower
concentration. These results encourage the use of multiple treatments to increase
probiotic bacteria concentration on bat wings. Overview on experimental design and
justification can be found in Appendix G.

51

Concentration of probiotic species are comparable between the treatment 1 and
2’s bat box and the four chamber bat box, and each species appears to follow a similar
trend of relative concentrations. P. synxantha A consistently hovers between 1x105 to
1x106 cells/cm2 in the treatment 1 and 2 and four-chamber bat box. P. synxantha A has
nearly always remained approximately 18 times higher in concentration than the other
species (~18.5 fold for treatment 1, ~18 fold for treatment 2, and ~18.5 fold for the four
chamber bat box). This contrasts with P. antarctica, which appears to have the lowest
abundance in bat boxes and on bat wings relative to the other probiotic bacteria except
for the hibernation trial. Arguably this pattern suggests that P. synxantha A is either
proliferating within the bat boxes and on the bat wings, or is dying off at a slower rate
than the other three bacteria. A clear pattern is evident from the data that P. synxantha
A is outnumbering each of the other three probiotic bacteria, which all show similar
values. This idea is further promoted by table 2.4 when we see an increase of P.
synxantha A’s ratio to other bacteria from start to finish. It is difficult to determine the
specific reason or variables for this difference, but it may be due to differences in
generation time and/or resilience of environmental factors such as temperature,
moisture, and pH.
There are many factors that can influence the growth and proliferation of
bacterial strains on bat skin, however, they are largely unknown, particularly for this
study. Weather, nutrients, humidity, where bats were sourced, grooming behaviour, and
other competing microbes are all covariates that could influence the results. The
microbiota likely varied significantly between our treatment bats and interacted in
unknown ways with our probiotic treatment. Bats were captured from different locations
and previous research has shown that microbiota can vary between populations
significantly (Avena et al. 2016; Lemieux-Labonté et al. 2016; Winter et al. 2017).
Further study is warranted to examine what constitutes ideal growing conditions of the
probiotic bacteria on bat wings. For example, what conditions promote anti-Pd
antifungal compounds to be secreted when Pd is present within the environment? Clay
has been shown to promote biofilm in Pseudomonas bacteria (Alimova, et al. 2009). As
such, the use of clay to adhere the microbes on the roosting surfaces of the bat boxes
52

might additionally act as nutrient supplementation. This might in fact be essential to the
production of secondary metabolites and thus the efficacy of this treatment (Duffy and
Défago 1999). Testing the proliferation and persistence of the probiotic on bat box
substrates in absence of clay, would be needed to determine this.
The four-chamber bat box had a larger abundance of P. synxantha A than the
other probiotic bacteria. When the chamber data is pooled (figure 2.13), there is an
obvious trend of P. synxantha A being significantly larger than the other 3 probiotic
bacteria as seen in treatment 1 and 2 bat boxes. This result is replicated when each
chamber is looked at individually and P. synxantha A is still the highest (Table 2.3).
However bacteria concentrations significantly (P<0.001) differed between individual
chambers of the four-chamber bat box and each bacteria species (Table 2.3).
Concentrations of all probiotic bacteria remained stable from July 20th until the end of
the trial on August 24th. Each chamber significantly differed from at least one other
chamber, with the largest difference being between the deepest one, chamber 1, and
the outside one, chamber 4. Variables such as humidity, average daily temperature, and
minimum daily temperatures were all similar (P>0.05) however chamber 1 had
significantly hotter maximum daily temperatures when compared to chamber 2, 3 and 4
throughout the entire trial (P<0.001). Interestingly, chamber 1 had a significantly higher
concentration of probiotic bacteria than chambers 2,3 and 4 (P<0.001). Based on the
humidity and temperature variables we tested, bacteria proliferation and viability
increased when maximum temperatures increased within the bat box. Furthermore, the
lowest maximum temperatures seen were in chamber 4, although not a significantly
different temperature in comparison between chambers; this chamber had a
significantly smaller number of bacteria. The only correlation that is evident from the
data, based on the variables we tested, is that the higher maximum temperature may
have encouraged probiotic bacteria growth based on the significantly higher abundance
of probiotic bacteria, whereas a lower maximum temperature decreased abundance of
probiotic bacteria. In the future, a controlled laboratory experiment that tested different
temperatures may give clearer results when correlating temperature to bacteria growth.

53

There are many variables that may have interfered with these results such as weather,
UV light, and overall sun exposure to each of the chambers.
Histology scores between Treatment 1 and the control group were not
significantly different. I can therefore conclude that there are no negative effects from
the probiotic application that hindered the treatment bats when compared to the control.
Although both groups had what was thought to be more inflammation than wild bats,
captivity is likely to explain this (G. McGregor, pers. comm.). Additionally, bats from the
pilot trial had increased numbers of neutrophils in the lungs and spleen, but these bats
were euthanized shortly after cold fall temperatures set in (November, when bats were
using long bouts of torpor and likely preparing for hibernation). Leukocytes are less
commonly found in the blood when bats are torpid; instead, immune system cells will
move into organs to support torpor once bats transition into hibernation (Bouma et al.
2010).
Because the higher wing cell concentrations occur shortly after bat box
inoculation (Figure 2.9), in field use with wild bats, we would recommend applying the
probiotic late in the summer season, close to the time when bats leave maternity roosts
for their hibernacula. However, there are other considerations that just be made such as
frequency and timing of bat visits to inoculated roosts (roost switching) and thus it might
actually be better to inoculate well before the end of summer (see next chapter), with
optional reinoculation of roosts as opportunity allows. Even if wing concentrations at the
end of summer are low when bats leave their maternity roosts, results of our hibernation
experiment are encouraging, showing at least a 12.4-14.7 fold increase (Table 2.5) in
probiotic cells within ~ one month of hibernation for P. azotoformans, P. synxantha A
and B, and as such, even small concentrations of probiotic on bats entering into
hibernation may be enough to protect bats from Pd growth. Unsurprisingly, P. antarctica
grew to extremely significant concentrations by the end of the trial and was 4.66x10 6
times higher with a final concentration of 6.95 ± 3.9 x109 within a single month. However
this large increase may be because of the final value being an outlier. Concentration of
P. antarctica outnumbers the other 3 probiotic bacteria significantly and future studies
54

may consider utilizing only P. antarctica for probiotic applications that occur in late fall
before bats leave for hibernation because of its psychrophilic characteristics. We did not
perform a histological examination of the hibernation bats however, they did not differ in
behaviour throughout the experiment.
In conclusion, the probiotic we have developed is safe to use on bats, can be
successfully transferred to bats’ via their roosting substrates, and presents promise of
preventing WNS disease in hibernating bats. P. synxantha A and P. antarctica are
promising probiotic bacteria because of their distinguishably high concentrations in
summer and hibernation experiments. Future researchers should consider testing only
P. synxantha A and P. antarctica together in higher concentrations, specifically in a
large comprehensive field trial.
.

55

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Chapter 3: Field Application of Pseudomonas fluorescens Species Complex
Bacteria onto Bat Boxes in the Greater Vancouver Area
INTRODUCTION
White-nose syndrome (WNS) is a fungal disease that can kill hibernating bats
(Lorch et al. 2011; Frick et al. 2010). Mortality rates of up to 90-100% result from a
cascade of physiological events and wing damage stemming from dehydration,
electrolyte depletion, and starvation (Cryan et al. 2012; Turner at al. 2011; Verant et al.
2014; Frick et al. 2015; Warnecke et al. 2013). White-nose syndrome is caused by the
fungal pathogen Pseudogymnoascus destructans (Pd) and is touted as one of the most
catastrophic wildlife diseases in recorded history (Drees et al. 2017; Frick et al. 2010;
Frick et al. 2015; Leopardi et al. 2015). Three species are highly vulnerable to WNS,
including Myotis lucifugus, Perimyotis subflavus, and M. septentrionalis, all listed as
endangered in Canada (Committee on the Status of Endangered Wildlife in Canada,
2013). The first infection occurred near New York Albany in 2006, presumably
transferred from Eurasia (Blehert et al. 2009; Frick et al. 2010; Puechmaille et al. 2011a;
Puechmaille et al. 2011b). Pd has been documented in seven Canadian provinces and
39 American states and west of the Rockies Mountains, is found only in one state so
far, Washington state (WNS 2019), although Pd has been detected in California. Pd is
predicted to persist in cave hibernacula for up to a century after it is introduced due to
its ability to act as a saprotroph and expand once introduced (Frick et al. 2017; Lindner
et al. 2011; Lorch et al. 2013a; Lorch et al. 2013b; Reynolds and Barton 2014; Reynolds
et al. 2015). Spores, when airborne, spread easily and are found on surfaces of infected
bat hibernacula each hibernation cycle (Langwig et al. 2015a; Kokurewicz et al. 2016;
Hoyt et al. 2019; Zhelyazkova et al. 2020). Bats returning to hibernation are rapidly reinfected each year until the infection rate reaches near 100% after 2-3 years (Frick et
al., 2017). However, this infection rate can be reduced if spore loads in the environment
are kept low (Hoyt et al. 2020). Strategies to reduce growth of Pd on hibernating bats
can reduce spore loads on individual bats, reducing likelihood of mortality, and reduce

61

overall environmental loads and thus reduce population-level infection rate in a
hibernaculum.
Locations of bat hibernacula in the Pacific Northwest and Western North America
are mostly unknown (Weller et al. 2018). This challenges most mitigation methods
relying on knowledge of bat hibernacula (Fletcher et al. 2020; Weller et al. 2018).
However, bat boxes used by maternity colonies present an alternative treatment site to
roost hibernacula. Used annually by large numbers of adult females typically showing
high fidelity to roosts (AK Department of Fish and Game 2021; www.batwatch.ca;
www.bcbats.ca), maternity roosts are increasingly being identified by government
outreach initiatives (e.g., AK Department of Fish and Game 2021) and community bat
programs (e.g., www.batwatch.ca; www.bcbats.ca).Therefore a WNS mitigation method
that targets maternity colonies could be highly impactful at a local population level to
reducing mortality caused by WNS.
With the goal of preventing Pd infection through application of a summer
prophylaxis, rather than treating WNS in winter, we conducted a field trial on maternity
colonies in the greater Vancouver area of Western Canada in August of 2019. Roosting
substrates of maternity colonies were sprayed with a probiotic of anti-Pd bacteria (see
Chapter 1) that were naturally sourced from wild bats wings. We selected four study
sites, each with a large mixed colony of Myotis yumanensis and M. lucifugus. The
roosts of two of these colonies were treated with probiotic, while there was no
application of probiotic at the other two sites, leaving them as controls for comparison.
Bats were studied at all four sites to establish baseline ecological and behavioural data
and identify main roosts used during the pup-rearing season (June – August; Rensel
2021).
Bats and roosting substrates were inoculated with probiotic and then
opportunistically swabbed for analysis of probiotic cell concentrations using quantitative
PCR (qPCR). In order to track individual bats over time, including post-hibernation
return (survival) rates, bats were tracked using Passive Integrated Transponder (PIT)tags and arm bands in collaboration with the University of British Columbia Okanagan
62

(Rensel 2021). Although we performed the pilot probiotic application late in the season,
we still managed to compare treatment bats to controls in their separate locations prior
to bats leaving for hibernation.
The probiotic cocktail we developed and field test here is comprised of 4 anti-Pd
bacteria species belonging to the Pseudomonas fluorescens species complex: P.
azotoformans, P. antarctica, and two strains of P. synxantha. These bacteria
demonstrated inhibition of Pd growth in vitro and were isolated from Eptesicus fuscus,
M. yumanensis, Corynorhinus townsendii, and M. evotis. Many species of bacteria
within the P. fluorescens species complex are known for secreting antimicrobial
secondary metabolites, and numerous studies have now shown that several strains of
bacteria from the P. fluorescens species complex inhibit Pd growth both in vitro and in
vivo in lab settings (Cheng et al. 2016; Lemieux-Labonté et al., 2017; Hoyt et al. 2015)
and recently in a controlled hibernation field trial (Hoyt et al. 2019). Hoyt et al. (2019)
demonstrated that certain bacteria from the P. fluorescens species complex reduce the
mortality rate of WNS. Here I describe the first study of bacteria from the Pseudomonas
fluorescens species complex to be used as a prophylaxis and applied at maternity
roosts, in advance of hibernation. We used autoclaved clay powder mixed with freezedried probiotic to dilute the probiotic and distribute it within bat boxes. Clay is inert and
resembles similar substrates found in natural crevice roosts used by bats such as rock
crevices. The premise of our approach is that bats coat their wings and bodies with
probiotic clay as they roost against the inoculated surfaces of their bat box or building
roosts. My objectives were to inoculate maternity roost substrates with the four-strain
probiotic cocktail, targeting human-built structures in summer, and subsequently monitor
the presence, concentration and viability of probiotic bacteria on treated roosting
substrates and wings of bats that have roosted on these substrates. Additionally, I
aimed to examine longevity of persistence on roosting surfaces and wings between
seasons, and monitor return rates (survival) of bats post-hibernation following exposure
to the probiotic cocktail. To date, I have been able to test roosting surfaces, but due to
COVID-19 field restrictions, no swab-sampling of bat wings has occurred. Additionally,
because Pd has not yet been detected in the study area and PIT tag surveillance is not
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possible at all four field sites, comparison of return/survival rates among sites is not yet
possible.

METHODS
Applicator design and probiotic preparation
Four different species of bacteria were included in the probiotic mixture:
Pseudomonas azotoformans isolated from a C. townsendii, P. synxantha A isolated
from a M. yumanensis, P. antarctica isolated from a M. evotis, and P. synxantha B
isolated from a E. fuscus. These bacterial species inhibit the growth of Pd and are a mix
of partial and full inhibitors (see Chapter 2). Bacteria were individually grown in 100mLof
lysogeny broth (LB) and incubated at 30oC, shaking at ~150rpm over night. Culture’s
were then centrifuged at 5000 rpm for 10 min and rinsed with Phosphate Buffer Saline
(PBS) three times. The cells were mixed with 1g of sterile clay powder and freeze-dried
in a cryo-lyophilizer for a minimum of 2 hours. Viability and bacteria counts were tested
by diluting and spread-plating and serial diluting 0.01g of freeze-dried suspension onto
LB agar , incubating at 25oC for 24hrs and counting the Colony Forming Units (CFU) the
next day. The synxantha A species final yield was 2.54x109 cells per gram of freezedried suspension, the P. synxantha B species final yield was 1.02x1012 cells per gram of
freeze-dried suspension, the P. antarctica species was 9.82x1010 cells per gram of
freeze-dried suspension, and the P. azotoformans species final yield was 6.64x1011
cells per gram of freeze-dried suspension. Each dosage was composed of 250 million
cells of each species that was mixed with 30g of clay for treatment of an individual bat
box chamber. Clay was added and vortexed on medium speed for 20 seconds in small
increments of 5g to promote homogeneity in each dosage. The final product was 40
doses of probiotic containing 1 billion freeze-dried probiotic per dose per bat box
chamber.
The probiotic-containing clay powder then needed to be propelled up into the bat
box chambers. I devised a propellant technique using a can of emzone compressed air
attached to a 50ml centrifuge tube containing the probiotic-clay mixture. I drilled a small
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hole into each end of the centrifuge tube; using a silicone seal, I attached the thin
1.5mm diameter tube that is supplied with the compressed air can to one end of the
centrifuge tube, and a large 10mm diameter rubber straw to the other end which was
the centrifuge tube’s lid. The rubber straw was 30cm long and flexible for directing the
spray up into each chamber of the bat box. I loaded the probiotic-clay powder into the
centrifuge tube by opening and closing the screw cap lid that was now attached to the
rubber straw. During deployment, powdered clay placed in the tube was propelled out of
the straw into each chamber of the bat box by depressing the nozzle of the spray can. A
light mist of water (~50ml of sterile de-ionized water per bat box chamber) was sprayed
into the box using a manual pump garden wand sprayer (with a misting wand with a
nozzle small enough in diameter to reach into each chamber) beforehand to enable the
propelled powder to adhere to the wood and assisted with activating the freeze-dried
bacteria. In subsequent field deployments, we substituted a manual action tire pump in
place of the aerosol spray can. This reduces cost and is quicker, because the aerosol
air cans can become very cold, requiring a resting period before continuing to spray,
and they are non-refillable.
Field Application
In 2018 and 2019, 464 bats were individually marked with either PIT-tags (Biomark, ID,
USA; 8 mm) or metal arm bands (Porzana, UK; 2.4 or 2.7 mm diameter, split-ring
lipped). Bats were captured and marked at three study sites in the lower mainland of
British Columbia (Figure 3.1): 220 bats were banded at Colony Farm Regional Park,
213 bats were banded and 493 were PIT-tagged at Stave Lake BC Hydro (of which 15
received both PIT-tags and bands), and 235 bats were banded and 337 were PITtagged at Alice Lake Provincial Park (of which16 bats received both PIT-tags and
bands. Each band and/or PIT tag enabled individual identification of bats in subsequent
monitoring. PIT tagged bats, when entering/exiting a roost equipped with a PIT tag
reader of probiotic levels and diversification of controls vs treatment.
Probiotic was applied to roosting surfaces at two study locations: Colony Farm
Regional Park and Stave Lake BC Hydro. On August 7th, 2019, I inoculated the four
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main bat boxes used by bats at Colony Farm park (numbered 1-4 from left to right of the
park). I used a standard dose one billion cells (250 million cells of each of the four
probiotic species) per chamber: each bat box had 7 chambers, for a total of 28 doses of
probiotic-clay deployed at this site. Three bat boxes were sprayed at night when bats
left to forage and one bat box was sprayed during daylight hours when no bats were
present.
At Stave Lake BC Hydro, probiotic-clay powder was sprayed into each chamber
of three four–chambered bat boxes, and on the outside of the roost entrance to a
building (Stave Lodge) on August 8th, 2019. Bats were not present in the bat boxes
during the day when probiotic was applied, but were present inside the lodge roost.

Figure 3.1. Map of field study sites in the Greater Vancouver region in southwestern
British Columbia. Treatment sites were Colony Farm Regional Park and Stave Lake BC
Hydro. Control location was Alice Lake. Each location has multiple roosts used by a mix
of M. yumanensis and M. lucifugus.
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Roost Substrate and Wing Swab Samples
To track the presence of each probiotic bacteria, bats’ wings were swabbed with
a sterile polyester swab is lightly dragged along the edge of both bat’s arm bones on the
dorsal surface (Appendix H). Each polyester swab tip was stored in a sterile Eppendorf
tube, and kept frozen prior to processing. Swabs were washed with 0.5ml PBS solution
and vortexed to dislodge cells picked up in the swab. The PBS suspension was then
sonicated in a sonicating water bath for 30 seconds in a sterile glass vial. Similarly, for
swab sampling of roost substrates, a polyester swab with a long handle was dragged
across a roost surface along a predetermined line or within a predetermined area.
Swabbing occurred at the entrances of each inoculated bat box chamber and the boiler
room entrance. Swabbing areas were kept consistent between bats and between roost
surfaces; therefore the measured starting quantities are only comparable relative to
their own groups. Bat swabs were divided based on mean wing area seen in the captive
trial of 19.7cm due to measurement of field trial bats not taken at the time. The boiler
room entrance and bat box swabs were not measured for swabbing area.
Baseline and control swabs of bats were taken before the bat box probiotic
inoculations. Adult females from each site were swab-sampled for baseline
concentrations of naturally-occurring bacteria matching the probiotic strains constituting
the probiotic cocktail: one M. yumanensis/lucifugus bat from Colony Farm on July 11th;
one M. yumanensis/lucifugus bat from Stave Lake on July 29th. An individual M.
yumanensis/lucifugus bat was swabbed from Alice Lake on July 24th, July 31st, and
August 14th as a control. This latter sample from the Control site was one week after
application of the probiotic at the treatment sites. All samples were qPCR-analyzed with
the custom probes for 3 of the 4 bacteria making up the probiotic. These baseline levels
formed the zero baseline when measuring the starting quantity of the treatment bat
swabs.
Treatment of bat boxes occurred on August 7th for Colony Farms and August 8th
for Stave Lake. There were three bat boxes in total at Stave Lake, two of which
attached to a lodge and designated as “#1” and “#2”, and a third bat box known as
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“Hayward Bat Box”. Four bat boxes were inoculated at Colony Farms. Bat boxes were
monitored manually and through PIT tag readers for bat activity in the following weeks
after probiotic inoculation. Bats were not seen roosting in any bat boxes at Colony
Farms when inspected on August 13th and a lack of a PIT tag reader at this site
precluded knowing if bats were present on other days. An adult female M. lucifugus and
a juvenile female M. yumanensis/lucifugus species were captured in the Colony Farm
site using mist nets and swabbed for qPCR analysis of the probiotic species. It could not
be confirmed that they had been using the probiotic-inoculated bat box roosts. There
were no bats detected in roost boxes from Colony Farms throughout the rest of the trial,
and this was unsurprising given that this colony leaves these roosts by mid-August each
year (J. Saremba, Burke Mountain Naturalists, pers. comm.).
Each chamber of the three inoculated bat boxes and the boiler room roost
entrance were swabbed on August 22nd, 14 days following application. Swabs were
attached to a wood dowel and rubbed against a pre-determined and consistent bat box
surface area vertically in each chamber. Relative quantity of each of the probiotic
bacteria in each swab was measured using qPCR. On August 27th, 19 days after
probiotic application at roosts, 33 bats were swabbed at the Stave Lake study site. Bats
were captured using a harp trap from the Hayward Lake bat box.
On March 3rd, 2020, overwinter survivability of the probiotic species was tested
by swab-sampling previously inoculated roosting surfaces. We sampled bat boxes 1-4
from Colony Farms three times (n=12 swabs), 2 swabs were frozen in water and the
third swab was placed in broth and allowed to grow in an incubator for 12 hours before
being frozen. Box 1 from Stave Lake was sampled three times (n=3), 2 swabs were
frozen in water and the third swab was placed in broth and allowed to grow in an
incubator for 12 hours before being frozen.
qPCR Analysis
All swab samples were processed using a relative-quantity multiplex probebased qPCR assay. Species-specific gyrB primers (modified from Yamamoto et al.
68

2000) were used to quantify cell concentrations in swabs for each probiotic isolate. We
designed strain-specific qPCR probes, with a 5’ fluorophore tag and 3’ quencher, for
use in multiplex reaction, measuring the relative combination of 3 of the 4 probiotic
species in each reaction. Species measured with qPCR were P. azotoformans, P.
synxantha A and B. Standards were created through serial dilution of cells suspension
(105, 104, 103, and 102) from each probiotic strain and all reactions were carried out in
triplicate. We then created a standard curve using the Cycle Threshold (Cq) values from
cell standards of known concentration. Fitting the Cq values from samples of unknown
concentration enable extrapolation of the number of cells present in each swab.
Standard error is presented for all means.
RESULTS
Summer Swab Samples – Bats and Roosts
Three strains of the probiotic bacteria were detected on both bats from Colony
Farms, with P. synxantha strain A 18.7-36.2 times higher in concentration than the other
two bacteria when both swab concentrations are combined. P. synxantha strain A
concentration was 2.36 ± .602 x103 cells/cm2 (1.28 - 3.36 x103, n= 3 replicates) on a
juvenile and 3.87 ± 3.18x104 cells/cm2 (5.47 - 102 x103, n= 3 replicates) and on an adult
female; P. synxantha strain B was 136 ± 36.0 cells/cm2 (70.6-195, n= 3 replicates) and
2.06 ± 1.67 x103 cells/cm2 (295-5429, n= 3 replicates); P. azotoformans was 67.8 ± 18
cells/cm2 (37.1-99.3, n=3 replicates) and 1.06 ± .865 x103 cells/cm2 (.153-2.79 x103,
n=3 explicates), respectively. Unfortunately due to problems with the 4th PCR probe,
presence of P. antarctica could not be tested. High variance between replicates likely
reflects variability occurring from sampling during qPCR.
Three bat boxes (Lodge Bat Box #1, Lodge Bat Box #2, and Hayward Lake Box)
and the boiler room roost entrance Stave Lodge were swabbed at Stave Lake BC Hydro
on August 22nd for detection of probiotic 22 days after the roosts were first inoculated.
All but one of the Lodge boxes had detectable amounts of probiotic bacteria within each
chamber; Lodge Bat Box 1 inexplicably had no detectable amount of probiotic in any of
the chambers. At the other roosts, P. synxantha strain A was the most abundant
69

bacteria (10-12.5 times more; Figures 3.2 and 3.3) within each chamber of both bat
boxes and at the boiler room roost entrance (Figure 3.4). Lodge Bat Box 2 and 3 each
varied in the amount of probiotic among chambers; the former had the greatest
abundance of probiotic bacteria within its third chamber, followed by the first, fourth, and
second and each probiotic bacteria species was detected in measurable quantities.
Hayward Bat Box had a greater number of probiotic bacteria present within each
chamber compared to Lodge Bat Box 2. These results might reflect the randomness of
the application process given that not all clay powder sticks when sprayed into the bat
box chambers, and the spraying consistency across the substrate is largely uncontrolled
which means that depending on where the swab sample is taken, probiotic
concentrations can vary widely. Interestingly, bacteria in Hayward Bat Box was detected
highest in the outer chamber 4, and progressively less bacteria found in each chamber,
with chamber 1 having the least (Figure 3.3). Bats typically roost in chamber 1 as that
chamber is the first one they enter from the landing platform. Bats can move
progressively from one chamber to the next moving outward, and therefore all bats will
pass through chamber 1, each picking up probiotic clay, and depending on the number
of bats in the box, they may not proceed past chamber 1.
At the entrance of the boiler room roost entrance, two swabs were taken (Figure
3.4) and each had a detectable number of bacteria: P. synxantha A had a marginally
greater number of cells compared to P. synxantha B and P. azotoformans.
At the control site, Alice Lake Provincial Park, 3 bats were captured (one per
night on 24 July, 31 July and 14 August, 2019). No detectable probiotic bacteria were
found on their wings through qPCR analysis. The two baseline swabs from each bat at
Colony Farms and Stave Lake on July 11th and July 29th had no detectable probiotic.

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Concentration of cells/swab

100000
10000
1000
100
10
1
1

2

3

4

Chamber
P. synxantha strain B

P. synxantha strain A

P. azotoformans

Figure 3.2. qPCR probiotic data comparison of P. azotoformans, and strains A and B of
P. synxantha from 4 swabbed chambers of Bat Box 2 from Stave Lake on August 22nd.
Note the logarithmic scale.

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A.

Concentration of Cells

30000
25000
20000
15000
10000
5000
0
1

2

3

4

Chamber
P. synxantha strain B

P. synxantha strain A

P. azotoformans

B.

Concentration of Cells

100000
10000
1000
100
10
1
1

2

3

4

Chamber
P. synxantha strain B

P. synxantha strain A

P. azotoformans

Figure 3.3. qPCR probiotic data comparison of P. azotoformans, and P. synxantha A
and B from each of four swab-sampled chambers of Hayward Bat Box from Stave Lake
on August 22nd. A. shows linear scale and the notable magnitude of difference in cell
concentrations, with >10x greater P. synxantha A strain. B. Same data plotted on
logarithmic scale to visualize concentrations of the other two probiotic isolates.

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Number of Cells

10000

1000

100

10

1
1

2

Boiler Room Entrance Swab
P. synxantha strain B

P. synxantha strain A

P. azotoformans

Figure 3.4. qPCR probiotic data of P. azotoformans, and strains A and B of P.
synxantha from 2 swabs of the boiler room entrance at Stave Lake on August 22nd,
2019. Note the logarithmic scale.
PIT tag readers at probiotic-treated Stave Lake roost sites confirmed that bats
used all 3 bat boxes as a night roost for at least 5 days after inoculation with probiotic.
The boiler room roost was consistently occupied. At Hayward Bat Box, 33 bats were
captured with harp traps and swab-sampled on August 27th, almost three weeks since
the roosting chambers were inoculated on August 8th. The majority of swabbed bats
were adult female M. lucifugus, except for one juvenile female and one adult M.
yumanensis (Table 3.1). Ten of the 33 samples (30%) had detectable amounts of
probiotic on their wings.
Of the 10 bats with a quantifiable number of probiotic bacteria on their wings,
bacterial strains and concentrations varied widely. P. synxantha strain B was the most
widespread (8 of 10 swabs) and its starting quantities were higher than P. azotoformans
in samples where both bacteria were found; however, the most abundant probiotic
bacteria was clearly P. synxantha strain A (5 of 10 swabs; Figure 3.5). P. azotoformans
was detected on 3 of the 10 swabs (Swab 1, 4, and 9 in figure 3.5).

73

Concentration of cells/ml/cm2

10000
1000
100
10
1
0.1
1

2

3

4

5

6

7

8

9

10

Bat number
P. synxantha strain B

P. synxantha strain A

P. azotoformans

Figure 3.5. Quantitative PCR analysis of wings of 10 captured bats (for which probiotic
was detected) on August 27th, 2019 at Stave Lake study site. Presence of three
probiotic bacteria were tested: P. azotoformans, and strains A and B of P. synxantha.
Note the logarithmic scale.

74

Table 3.1. Characteristics of bats caught at Stave Lake on August 27th, 2019.
Bat
Swab
#
1
1
1
2
2
2
3
3
3
4
4
4
5
5
5
6
6
6
7
7
7
8
8
8
9
9
9
10
10
10
11
11
11

Probiotic
Species
P. synxantha B
P. synxantha A
P. azotoformans
P. synxantha B
P. synxantha A
P. azotoformans
P. synxantha B
P. synxantha A
P. azotoformans
P. synxantha B
P. synxantha A
P. azotoformans
P. synxantha B
P. synxantha A
P. azotoformans
P. synxantha B
P. synxantha A
P. azotoformans
P. synxantha B
P. synxantha A
P. azotoformans
P. synxantha B
P. synxantha A
P. azotoformans
P. synxantha B
P. synxantha A
P. azotoformans
P. synxantha B
P. synxantha A
P. azotoformans
P. synxantha B
P. synxantha A
P. azotoformans

# of
qPCR
replicates Age Sex Species
2
A
F
MYLU
1
A
F
MYLU
1
A
F
MYLU
1
J
F
MYLU
J
F
MYLU
J
F
MYLU
1
A
F
MYLU
A
F
MYLU
A
F
MYLU
1
A
F
MYLU
1
A
F
MYLU
1
A
F
MYLU
1
A
F
YULU
A
F
YULU
A
F
YULU
A
F
MYLU
3
A
F
MYLU
A
F
MYLU
1
A
F
MYLU
A
F
MYLU
A
F
MYLU
A
F
MYLU
1
A
F
MYLU
A
F
MYLU
1
A
F
MYLU
1
A
F
MYLU
1
A
F
MYLU
1
A
F
MYLU
A
F
MYLU
A
F
MYLU
J
M
MYLU
1
J
M
MYLU
J
M
MYLU

* Concentration deemed to be an outlier and thus this sample was removed from
analysis.
75

Spring Swab Samples of Roosts
P. azotoformans, P. synxantha A, and P. synxantha B were not present in any of
the swabs of roost substrates in March 2020. Broth was inoculated with swab
suspension as a way to try to grow any trace probiotic cells, however and only one
swab from a bat box at Colony Farm (Bat Box 2) grew one of the strains -- P. synxantha
A, however it was not present in the water sample.
DISCUSSION
We determined that probiotic could be transferred to wild free-flying bats by
applying it to their roost substrates. We found evidence at both of our treatment sites,
that bats had probiotic bacteria on their wings several weeks after roost inoculation. At
Colony Farm, only 2 bats were captured, and they had both detectable amounts of
probiotic on their wings one week after bat box inoculation. Inoculated bat boxes from
Colony Farm were empty after inoculation throughout the rest of the trial, which is
consistent with their annual pattern of leaving the bat box array by mid-August each
year (John Saremba, Burke Mountain Naturalists, unpublished data). These bats may
leave the area for mating and hibernation, and this colony may leave earlier than others
if they have a long ways to travel to hibernacula (Norquay et al. 2013), or they have
more suitable roosts for late summer/early fall). Bats were still present at Stave Lake at
least three weeks after inoculation of the three bat boxes and the boiler room roost
entrance. Three of the 4 probiotic species were detected at two of the three bat box
roosts and at the Lodge roost entrance at Stave Lake, several weeks following roost
inoculation. It was not possible to test for the presence of the fourth bacteria species, P.
antarctica because the PCR probe did not anneal properly during qPCR DNA
amplification. The other 3 probiotic species were present in abundant numbers with P.
synxantha A being greater than the others by 17.1-43.9 fold in Bat Box 2, 15.9-44.7 fold
in Hayward Bat Box, and 15.9-47.8 fold at the boiler room roost entrance. Although the
protocol used to create the probiotic-clay and the application of the clay into each
roosting chamber was designed to apply each probiotic bacteria in approximately equal
concentrations, one of the bacteria (P. synxantha A) amplified over time on roost and
76

wing substrates. The reason is unknown, but may due to its ability to proliferate in the
warm bat roost conditions. This same uneven proliferation was not seen in hibernation
conditions (see Chapter 2). What has not been confirmed is whether the bacterial cells
of all probiotic cells swabbed from bats and roosts are viable or whether the DNA of
nonviable cells remains and is being amplified. For example, if P. synxantha B and P.
azotoformans are not viable and only DNA remains to be amplified, but P. azotoformans
is viable, then it would appear that this latter strain has drastically outnumbered the
other strains when in fact its cell division is just indicative of viability. This strain,
however, might truly be more prolific in the warm summer roosts conditions and on
warm-bodied bats (adult females raising young maintain warm bodies, e.g., Lausen and
Barclay 2003). Further experimentation and sampling will need to be done to shed light
on viability in relation to relative concentrations of the four probiotic bacteria. We had
planned for our broth inoculations of each sample to test for viability, but this proved to
be unreliable; this may stem from an underestimate of the time needed for cell division,
or from competition of other microbes in a rich nutrient environment.
We know from our captive bat trials that probiotic remains on bat box substrates
for several months after application in summer months (see Chapter 2). However, this
was the first test of whether the probiotic would still be detectable on bat box roosting
substrates the following spring. We detected none of the probiotic species in the
overwinter subsampling of bat boxes at Stave Lake in March. Only one sample of one
chamber in one box at Colony Farm showed a trace quantity of P. synxantha A from a
broth sample. This failure to find probiotic cells on the roosting surfaces suggests
significant die off of the probiotic in the bat boxes overwinter. However the presence of
P. synxantha A in one broth sample may suggest a small quantity of cells were able to
grow when exposed to broth, but were too small to be quantified in water. Application
protocol, frequency, and/or cell concentrations should be adjusted in future field
activities to verify conclusions and determine a potential cause for the overwintering dieoff; for example, freezing temperatures might be responsible for no detection of cells in
the broth, but the fact that the water samples also proved to have no bacterial DNA to
amplify suggests that the DNA of the bacteria was also gone and perhaps there was
77

enzymatic breakdown of the probiotic cells by other microbes on the roosting surface.
Application and sampling of a roost substrate in a heated building will help to determine
if temperature is a factor, or whether there other potential causes like microbial
competition. In this study we did not swab at any other time points between winter and
spring, therefore we do not know the exact time frame in which the probiotic species
were present. Multiple sampling periods and environmental condition analysis, such as
temperature and humidity, throughout winter could provide better insight to each
bacteria species survivability.
Bat boxes were not swabbed at Colony Farm due to no bats being observed
within the bat boxes after the first and second week after inoculation. Unlike at Stave
and Alice Lake sites, bats at this site were not PIT-tagged, and so presence of bats
could not be known unless visually observed. This together with the fact that this colony
is known to leave these bat boxes annually in early to mid August, resulted in few bats
being sampled.
Bat hibernacula in western United States is largely unknown, with 95% of counts
containing less than or equal to 10 myotis species in each roost (Weller et al., 2018). At
this time it is impossible to apply probiotic to hibernating sites, and even then, they
would likely be heavily disturbed. Future methods should consider a quicker, and more
efficient method of application that would only be conducted with 1 or 2 people.
Our results found that 30% of swabbed bats from Stave Lake had a detectable
amount of applied probiotic on their wings. All bats found with our probiotic on their
wings were female MYLU species. The period between bat box inoculation and bat
swabbing was 19 days from August 8th until August 27th. This would presume that
swabbed bats likely came into direct contact with the inoculated probiotic within the bat
boxes. To further re-iterate our findings, we saw none of our probiotic species in the
baseline wild bat swabs from the Stave Lake bat swab, the Colony farms bat swab, and
the 2 bat swabs from Alice Lake from July and early August. Like in the bat box
chambers, P. synxantha A was much more abundant on each bat swab compared to
the other probiotic species. These results give us an idea of how much probiotic was
78

present on their wings with the dosage we used. Although an estimated 250 million cells
of each anti-Pd bacteria species were inoculated onto each bat box, our highest result
was 2.37 ± .237 x104 (.190 - 2.64 x104) cells of synxantha A as seen in chamber 4 of
the Hayward Bat Box. The highest result seen on bats was 1.11x103 ± 0 cells/cm2 of P.
synxantha A that used the Hayward Bat Box. Interestingly, the 2 most highly used
chambers of the Hayward box (chambers 1 and 2 near the landing platform entrance)
were noticeably lower in quantity of all microbes compared to the least used chambers
(3 and 4;Figure 3.3). This may reflect the overall use of the bat box by bats and the
adhering of cells to bat skin and fur that is expected to occur. The bats caught at Colony
Farms had a much higher result of 3.87 ± 3.18 x104 (8.38 - 102 x103) P. synxantha
cells/cm2, however, we do not have bat box swabs to compare to. A larger abundance
of bacteria on bats and within bat boxes may stimulate biofilm growth. Ideally, we would
like to see biofilm formation within the bat box and eventually hibernacula from bats
transferring it. Biofilm could form and survive in bat hibernacula because of its above
freezing, humid and stable refrigerator-like temperatures (Fenton and Robert 1980;
Gennari and Dragotto 1992). Therefore spore loads within the environment and on
individual bats would likely be lower from the anti-Pd metabolites released from the
biofilm.
For further studies we would recommend using repeated, and possibly higher,
dosages of anti-Pd bacteria from the P. fluorescens species complex and further
monitoring of the bat box chambers throughout winter and into spring. Consideration of
repeated dosing of bat boxes is warranted because of the high concentrations of
probiotic bacteria seen in the captive trial hibernation experiment (chapter 2). The
captive bats from the hibernation trial in chapter 2 used a bat box that was doused twice
with probiotic on September 30th and October 7th before moving into the hibernation trial
on November 7th. Probiotic concentrations greatly increased throughout the hibernation
trial into the hundreds of thousands per cm2. Wild bats may have a greater
concentration of detectable probiotic on their wings if the bat boxes were inoculated
multiple times, similar to our captive trial. Clay has previously been documented as a
great mineral source for Pseudomonas bacteria (Alimova, et al. 2009). Furthermore,
79

repeated dosages of anti-Pd bacteria to maintain bacteria proliferation and growth has
been recommended by other researchers when using anti-Pd bacteria to reduce WNS
impacts (Hoyt et al. 2019). Utilizing P. synxantha A, P. azotoformans and P. synxantha
B colonization could be a useful management practice when hibernacula are not known.
These results give an insight into new types of management practices that rely on
natural anti-Pd probiotics found on a smaller percentage of wild bats and spreading it
throughout bat species populations.
In conclusion, we inoculated bat boxes and a building roost with our anti-Pd
probiotic naturally found on wild bats, and later detect the same probiotic species on
bats that were using the bat boxes. Inoculating the roost substrate directly would have
likely been a better choice than the roost entrance of the building, as not all bats likely
encountered the probiotic. Probiotic species were not detectable in the same bat boxes
following exposure to winter conditions and seem to have died off by spring. Further
research is needed to examine different dosage concentrations and treatment
frequency needed to sustain probiotic cell numbers both on roost surfaces, and bat
wings throughout the entire hibernation period into spring. Monitoring cell
concentrations on bat species while they hibernate in the wild would be ideal, however,
this may be impossible due to bat hibernacula being mostly unknown. Instead, sampling
efforts should be bolstered to increase sampling size and frequency throughout the fall
season to create a timeline of probiotic abundance on bats and bat boxes. Methods
should also be implemented to determine viability and metabolite abundance of
probiotic bacteria on the bat box and bats throughout fall and into winter if possible. This
study is one step towards alleviating the selective pressure that western WNS impacted
species are facing during hibernation. In the future, affected bats may survive
hibernation and Pd exposure if they were to maintain anti-Pd bacteria numbers and
indirectly defend themselves from mass mortality.

80

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Chapter 4: Inoculation and Quantification of Pseudomonas fluorescens Species
Complex Bacteria and Pseudogymnoascus destructans on Myotis Patagia
INTRODUCTION
White-nose syndrome (WNS) is a deadly infectious disease that results in bat
mortality during hibernation (Frick et al. 2010). This disease is caused by the fungal
pathogen Pseudogymnoascus destructans (Pd) which has caused millions of bat deaths
across eastern North America (Frick et al. 2010; USFWS 2012). Surviving bats will
groom off the fungus and persist, though survival rates for some populations have been
less than 10%; , Pd fungus can persist in caves and infect bats in each subsequent
hibernation period (Frick et al. 2015; Lorch et al. 2011; Hoyt et al. 2015). Pd is a
psychrophilic fungus and grows optimally between 12.5-15.8oC in the presence of high
relative humidity (80.5%; Verant et al. 2012; Marroquin et al. 2017). These temperature
and humidity conditions are found in caves in which many bats choose to hibernate
(Davis 1970; Fenton 1970; Anderson and Robert 1971; Raesly and Gates 1987; Cryan
et al. 2010; Vanderwolf et al. 2012; Kurta 2014). White-nose syndrome was introduced
into North America in 2006 and bat mortalities were first noted in 2007 near Albany,
New York (Blehert et al. 2009; Frick et al. 2010). Today it now resides in 39 states and
seven Canadian provinces (USFWS 2020). Bats are thought to fill crucial roles in
ecosystems, agriculture, and forests (Boyles et al. 2011; Kunz et al. 2011). They are
voracious predators of insect pests and provide a natural method of insect control
without the use of pesticides. Vector-borne diseases that utilize insects as a method of
transmission may in part managed be by insectivorous bats. Natural pest control
services of bats there is estimated to be worth billions of dollars to the US agricultural
industry (Boyles et al. 2011; Kunz et al. 2011).Therefore it is especially important to
understand how to control WNS infection in bats to prevent or reduce further mortalities.
This is especially important as the disease spreads in western North America where the
species diversity of bats far exceeds that of the eastern part of the continent. In British
Columbia (B.C.) in particular, bats represent nearly 20% of small mammal diversity
(CDC 2021). White-nose syndrome affects hibernating bats. Fourteen of the 17 bat
86

species in B.C. are known to hibernate (Naughton 2012), suggesting that WNS could
substantially decrease mammal diversity in the province (and in Canada). With a
growing understanding of the importance of biodiversity to the function and health of
ecosystems (e.g., Battista et al. 2016), control over this invasive bat fungus takes on
even greater urgency as it infects new western bat species for which susceptibility is as
of yet unknown.
White-nose syndrome fungal infection is subtle to detect in bat wings and deadly
during hibernation (Meteyer et al. 2009). Signs of infection are inconsistent but typically
include rough patches on the face, ears, forearms, wing membranes, and feet. Skin
tissues may show irregular pigmentation or small tears in wing membranes from Pd
infection. The wing membrane epidermis is composed of 2 single cell layers that are
separated by a thin layer of blood and lymphatic vessels, nerves, muscles and
specialized connective tissues (Makanya et al. 2007). Pd has shown to highly express
94 genes during the cutaneous infection of M. lucifugus, and 34 genes that directly
contribute to its virulence (Reeder at al. 2017). These genes include expression of
enzymes lipase 1 and squalene monooxygenase which degrade surface lipids and
sebum found on the bat wing epidermis. Other genes significantly improved Pd’s heat
shock response, cell wall remodelling and micronutrient acquisition to likely evade host
pattern recognition receptors and antibody responses. Pseudogymnoascus destructans
will commonly produce cup like erosions that are filled with fungal hyphae on bat wing
membranes and ulcerate the underlying connective tissue as infection continues.
Pseudogymnoascus destructans hyphae are septate and will branch in either uniform,
parallel walls, or irregular bulging walls that are larger in diameter (Makanya et al 2007;
Meteyer et al. 2009). Hyphae will destroy apocrine glands, hair follicles and sebaceous
glands, and replace host connective tissue, blood and lymphatic vessels, glandular
structures, elastin and muscle fibers with Pd digests.
Ulceration and invasion of Pd into host tissue can cause many physiological
complications for its host. Damage to the epidermis has been shown to cause eventual
electrolyte depletion, hypotonic dehydration and respiratory acidosis during torpor and
87

hibernation (Verant et al. 2014; Warneke et al. 2013). Pseudogymnoascus destructans
hyphae will damage underlying connective tissue and increase vascular permeability,
which further accelerates fluid loss and can cause hypovolaemia which would trigger
bats to awake from torpor and drink water (Warnecke et al. 2013). Furthermore,
destruction of epidermis can prevent normal blood flow and proper CO2 expiration,
increasing ventilation rates and possibly hyperventilation, which further contributes to
arousal frequency and evaporative water loss (EWL; Verant et al. 2014; Warnecke et al.
2013). Sodium and chloride levels will drastically decrease due to hibernacula not
containing sufficient electrolytes (eg. insects) to replenish losses caused by the disease
(Cryan et al. 2013). Imbalance of electrolyte levels can interfere with homeostatic
function and lead to possible impaired neural and/or heart function as seen in other
mammals. It has been proven that bats exhibit heightened energy expenditure and EWL
after infection during torpor and this directly contributes to WNS pathophysiology
(McGuire et al. 2017). Bats will initially express a heightened torpor metabolic rate
(TMR) and this is possibly due to a small immune response when bats initially try to
fight off the infection (Field et al. 2015, Lilley et al. 2017). However, torpor will typically
suppress a bat’s immune system making them more vulnerable to infection as
hibernation continues (Bouma et al., 2010; Geiser 2004; Moore et al., 2011). Put simply,
bat mortality is the result of premature arousal from torpor because of physiological
imbalances and results in depletion of energy reserves for winter survival (Storm and
Boyles 2010; Cryan et al. 2013; Warneke et al. 2013; Verant et al. 2014).
Methods of controlling Pd infection in wild bats are being researched, but more
than a decade after the disease’s discovery in North America, field-ready preventative
or treatment tools do not yet exist. Anti-fungal agents such as fungicidal drugs that are
commonly used in agriculture, vaccines, and UV light are being studied (Palmer et al.
2018; Rocke et al. 2019.). Fungicidal drugs were among the first treatment methods
tested and proven to be unsuccessful. Not only can anti-fungal drugs be dangerous to a
bat’s natural microbiome and expensive, but they can be toxic and have effects on nontarget taxa when deployed in bat roosts. Vaccines are expensive, require extensive
testing, and are difficult to implement into wild animals. Finally, UV light is non-specific
88

and does not reach under bats arms when they hibernate, leaving reservoirs for the
fungus to persist. Utilizing anti-Pd bacteria has been a promising method of controlling
Pd infection and an increasingly interesting form of controlling WNS infection. In vitro
tests utilizing antifungal metabolites from bacteria can limit growth of Pd mycelia and
spores (Cheng et al. 2016; Chaturvedi et al. 2011; Cornelison et al. 2014a; Cornelison
et al. 2014b; Lemieux-Labonté et al., 2017; Micalizzi and Smith 2020; Hoyt et al. 2015).
Researchers from University of California have developed an in vivo screening method
using explant chambers to examine Pd infected bat tissue in the presence of R.
rhodochrous (strain DAP 96253 induced with urea; Cornelison et al. 2014b). In vivo
tests prevent other fast growing fungal microbes and mold from interfering with Pd and
anti-Pd bacterial inoculation.
Here, I assess whether severed Myotis yumanensis bat wings treated with a
cocktail of anti-Pd Pseudomonas bacteria can prevent or slow Pd spore germination
and/or hyphae growth. The anti-Pd cocktail contains four synergistic bacterial strains
from Western Canada that were originally found on wild bat wings and inhibit the growth
of Pd (see Chapter 2). Here I investigate whether the probiotic reduces Pd growth or
germination. I used two different approaches to testing this: one using wings of bats
who had been indirectly inoculated via application of a probiotic-clay powder (see
Chapter 2) on their roosting surfaces; the other using direct inoculation of live wing skin
excised from a freshly euthanized bat. Here I present results of Pd-challenge
experiments. where I exposed captive bats to the probiotic at their bat box roosts,
humanely euthanized the bats, severed their wings, and challenged them with Pd to
quantify and describe subsequent Pd spore germination or Pd hyphae growth time. I
also performed an experiment using wings of wild bats that were not exposed to
probiotic, whereby the skin tissue is kept partially alive using tissue media in custom
explant chambers. Probiotic cells and Pd spores are applied to the explants and the
subsequent germination is examined and compared between explants with and without
probiotic cells.

89

METHODS
Probiotic Cocktail
The first Pd challenge experiment used two synergistic bacterial strains of
Pseudomonas. Both were isolated from an adult female Eptesicus fuscus caught in a
mine outside of Salmo in the Kootenay area of British Columbia. Only one of these were
carried through to the next probiotic cocktail and it is now identified as P. synxantha B.
The present anti-Pd cocktail contains four synergistic bacterial strains from
western Canada: Pseudomonas synxantha strains A and B, P. azotoformans, and P.
antarctica. Each isolate was previously sourced from bats in BC. Pseudomonas
synxantha strain A was isolated from a M. yumanensis at a mine hibernaculum near
Salmo, BC; strain B was isolated from an adult female E. fuscus captured free-flying at
a mine near Salmo, BC (as told above); P. azotoformans was isolated from a
Corynorhinus townsendii adult bat caught at a maternity roost in Deroche, BC; and P.
antarctica was sourced from a female juvenile M. evotis at from a mine hibernaculum
near Nelway, BC.

Pd Challenge - Ex vivo wing culture
After application of the probiotic cocktail to captive bats indirectly via treatment of
their roosting surfaces (see Chapter 2), wings of euthanized bats were challenged with
Pd to investigate growth of Pd on these previously inoculated wings versus wings of
bats that had not roosted on an inoculated roosting surface.
In all experiments, bats were humanely euthanized and separated into control
(no probiotic) and treatment (probiotic) groups for fungal Pd inoculation. Each wing was
inoculated with fungal Pd spores as described below. Reduced Pd growth on treatment
group wings compared to control group wings would suggest previously applied
probiotic was inhibiting Pd growth.
In Experiment A, after removal from the carcasses, the wings were immediately
spread out onto a sterile cork board and pinned using sterile toothpicks within a sealed
90

bioassay plate (n=12 wings). Two petri dishes were filled with water to generate high
humidity and the bioassay plates were placed into a 15oC incubator to simulate wild Pd
growth conditions. Six wings were used from our trial for testing Pd inoculation. Three
‘types’ of wings were used: wings of control bats, and wings from two types of treatment
groups -- one that stopped receiving probiotic several weeks before the end of the trial,
and one that received the higher dose of probiotic near the end of our trial. Two sets of
these three types of wings were used.
One set of 3 wings were inoculated with 5x105 conidia/mL water suspension on
two equal portions of the wing. One portion was scratched with an inoculation loop to
promote germination and this was compared to unscratched areas of the wings. Pd
hyphae are known to penetrate bat wing tissue (Meteyer et al. 2009) and scratching
could assist Pd growth. The other set of three wings were grown in the same conditions;
however, they were placed directly onto SDA agar instead of cork board. Wings were left
for one month and examined daily for fungal growth. Observed fungal growth was
swabbed with sterile polyester tips and transferred onto SDA plates. It should be noted
that the probiotic cocktail present on these captive bats were a pilot blend of two
synergistic bacterial strains of Pseudomonas.
In Experiment B, I Pd-challenged wings of another set of captive bats
(euthanized in July 2019; control vs treatment; refer to Chapter 2 for details) that had
been inoculated with the final probiotic cocktail blend of four bacteria (see Probiotic
Cocktail section above). There were 6 treatment bat wings (n = 6 bats) that were
previously exposed to the probiotic that inhibits WNS, and 5 control wings (n = 5 bats)
that had not been exposed to the probiotic bacteria. The bat wings were used
immediately after necropsy to reduce wing decay as a confounding factor. The other
wing that was not used, was for the histological examination (see chapter 2).
Two different methods of Pd application were used. The first method used a
polyester swab that was rubbed onto a mature Pd plate that was established onto
Sabouraud Dextrose Agar (SDA) and had uniform mycelia growth. A swab was pushed
into the fungus and rubbed uniformly over the plate to collect spores with the swab. The
91

swab was then rubbed onto a wing. An individual swab was rubbed onto two treatment
group bat wings and two control bat wings, for a total of four swabs. With this method,
there was an unknown amount of Pd added, but hyphae would be added not just spores
to ensure sufficient inoculation of Pd to the wings.
The second method used a different approach to applying Pd, which required
application of spores only, not mycelia. Seven different doses of 150,000 spores were
added together in Phosphate Buffer Solution (PBS) and vortexed in two intervals at
5000rpm for 10 minutes. Each pellet was diluted with 1ml of yeast extract broth for 5
hours on a 200rpm shaker at 14oC to assist with germination. Spores were vortexed at
7500 rpm for ten minutes after the 5 hours and resuspended with sterile water, twice.
Before the spores were applied to the wings, they were vortexed at 7500 rpm for ten
minutes and the sterile water was decanted. A sterile swab was pushed into the
centrifuge tube and rubbed against the pellet in the tube before being rubbed onto the
wings. There were four doses for treatment bat wings and 3 doses were for control bat
wings.
Wings were placed into petri dishes and stretched into place with water agar
anchor points. The water agar prevented the wing from closing and raised humidity
within the plate. Petri dishes containing the wings were sealed profusely with a
generous amount of parafilm and placed into a 14oC cooler for one month. After one
month the wings were removed and placed into 50ml centrifuge tubes containing PBS
solution. The wings were gently vortexed at medium-high for 30 seconds before being
sent to McMaster University for qPCR analysis.
In Experiment C, three bats (n=6 wings) from the captive hibernation trial were
euthanized and immediately the skin tissue was exposed to Pd. These bats had shown
substantial growth of probiotic bacteria on their wings during a 35 day hibernation period
(see Chapter 2 Results). A new in-house method was used to grow Pd without
interference from competing mold or fungi. Methodology was changed because of other
distinguishable fungi growing on the bat wings previously, presumably due to the slow

92

growth rate of Pd. We developed a new method that attempted to prevent fast growing
fungi from outcompeting Pd.
Five SDA plates growing a uniform Pd mycelium were scraped and blended in
saline solution for 30 seconds. The suspension was then centrifuged into concentrated
micro doses of 0.2 mL PBS solution containing Pd mycelia – defined as a ‘dose’ for this
experiment. A single ’dose‘ was inoculated onto each treatment wing (n = 6) and a
previously frozen severed wing (n = 1) from an untreated bat of the same species. For
comparison, as a ‘standard positive’ for Pd, one ‘dose’ was immediately frozen and sent
as a comparison to treated wings. Treated wings were placed into a 14oC incubator for
1 week before being placed into PBS solutions and frozen to stop Pd growth. Wings
were analyzed by qPCR for growth over the time period.
To analyze Pd growth/quantities in control versus treatment wings post
incubation, in all experiments, we visually inspected the wings and fungus for similarities
to Pd using a dissecting microscope before sending for qPCR analysis. Fungi was reisolated onto SDA and examined for growth rate and morphology. Examination of
isolates was compared to Pd cultures previously grown on SDA plates.
Pd Challenge - Ex vivo explants
The purpose of the explant experiment was to test the Pd inhibition threshold of
the anti-Pd bacteria -- Pseudomonas synxantha, strains A and B; P. azotoformans; and
P. antarctica -- on live bat tissue. Severed tissue explants can be kept partially alive
within specialized chambers in an ex vivo experiment, thus preventing the associated
interference of decomposition (Figure 4.1). Wild captured M. yumanensis bats (n=3)
were euthanized by overdosing via isoflurane at Thompson Rivers University in
Kamloops, BC. Each bat’s patagium was separated into 1 cm diameter samples using a
biopsy punch to collect full-thickness samples of skin (n=24). Biopsy punches were
placed into the individual explant chambers quick enough to ensure constant source of
nutrients to maintain cellular activity in skin cells. One side of the explant was exposed
to Eagle’s modified minimal essential medium supplemented with or without antibiotics
(gentamycin, 10 µg/ml).
93

Explants were separated into two groups, 3 days and 7 days, and two
subgroups, with and without gentamycin (Table 4.1). Two lengths of time were used
because, due to this being a novel experiment, we were unsure how long the Pd would
take to grow and/or colonize the skin tissue within the explant chambers. Ultimately, the
time periods ended up being 3.5 and 7.5 days to test different timeframes. Seven and a
half days was chosen to give the Pd enough time to grow but hopefully not be
outcompeted by other fungi. Three and a half days was chosen to see if Pd numbers
grew in quantity and to avoid any competing fungi possibly growing on the explant if left
longer.
Samples that were exposed to antibiotic media (n = 13) were compared to
samples that did not have any exposure to antibiotic media using qPCR (n=11). Postexperiment, a subsample of the explants (n=5) were examined using Scanning Electron
Microscope (SEM) analysis to visualize Pd colonization and microscopic interactions
between the probiotic bacteria, Pd and gentamycin, at the end of the 3 and 7 day trials.
SEM samples were also swabbed for qPCR and were used in both the qPCR and SEM
groups.
A.

B.

Figure 4.1. Explant chambers that contain separated myotis patagium A) Myotis
patagium with antibiotic, Pd and probiotic; and B) Myotis patagium with only Pd and no
probiotic or antibiotics, with white fungal growth evident.
94

Table 4.1. Number of samples in each category that were exposed to media with
antibiotics, media without antibiotics, and sent for SEM analysis. Group 1 consists of
samples that had endpoints of 3.5 days, and group are samples with an endpoint of 7.5
days.

Add Probiotic (3.5
days)
No Probiotic (3.5
days)
Probiotic + Pd
(3.5 days)
Pd only (3.5
days)
Add Probiotic (7.5
days)
No Probiotic (7.5
days)
Probiotic + Pd
(7.5 days)
Pd only (7.5
days)

Media with
antibiotic (qPCR)

Media without
antibiotic (qPCR)

Sent for SEMMedia without
antibiotic

Sent for
SEM-Media
with antibiotic

3

2

0

0

2

2

0

0

2

2

0

0

0

0

1

0

2

1

0

1

2

1

0

1

2

2

0

1

0

1

1

0

Probiotic bacteria were grown in individual flasks of LB broth in a 25oC incubator
4 hours before bat euthanasia. Cell concentrations in each probiotic dosage were as
follows: 500 cells of P. synxantha strain B, 8500 cells of P. synxantha strain A, 200
cells of P. azotoformans, and 200 cells of P. antarctica. Cell concentrations were
estimated using Colony Forming Units (CFU) vs Optical Density (600nm) curves (Figure
4.2). Six hundred nanometer wavelength was used due to the yellow/golden tint of LB
broth that cells were grown in. Each inoculated broth was portioned based on CFU
curve estimations and centrifuged at 4000 rpm for 5 minutes in a tabletop centrifuge.
This process was repeated three times and rinsed with Phosphate Buffer Solution
(PBS) to remove any broth from the final dosage. The final probiotic dosage contained
all four bacteria species in 20µl of PBS which was pipetted onto the opposite surface of
explants either exposed or not exposed to Eagle’s modified minimal essential medium.

95

Pd spores were separated into individual dosages from a larger stock
concentration and centrifuged at 10,000 rpm for 5 minutes. The Pd spores stock
concentration was constituted of PBS with 0.02% Tween 20 to achieve even distribution
of spores within inoculum once pipetted onto the explant. Eighteen hundred Pd spores
were inoculated onto explants to monitor interactions with probiotic bacteria and
antibiotics.
Explant chambers were sealed with parafilm and placed into a 14 oC incubator.
After 0.5 days, half of the surface area of each explant (except the Pd control) were
swabbed with a sterile polyester-tipped swab before being placed into the incubator.
This swab was placed into sterile water and frozen until qPCR analysis. The other side
of the explants were swabbed at the end of the experimental periods (3.5 and 7.5 days,
for Groups 1 and 2, respectively) to quantify probiotic and Pd spore quantity. Swabs
were placed into sterile water and frozen until qPCR analysis.
Five explants were sent for SEM analysis: two with Pd spores inoculum only with
no exposure to antibiotic media over 3.5 and 7.5 days, respectively; one with probiotic
inoculum only and exposed to antibiotic media over 7.5 days; one with no inoculum and
was exposed to antibiotic media for 7.5 days; and one with probiotic and Pd inoculum
and was exposed to antibiotic media for 7.5 days. The quantitative PCR was conducted
using the previous methodology in this thesis (See chapter 2 and 3 methods).

96

Figure 4.2. Plotted CFU versus OD at 600nm of Pseudomonas synxantha, Strains A
and B; P. azotoformans; and P. antarctica when grown in LB broth at 25oC.
97

RESULTS
Ex vivo Wing Cultures
Neither treatment nor control group wings exhibited any significant Pd growth
in experiments A, B and C. This was determined through all methods of evaluation:
visual examination, examination under a microscope, and through qPCR analysis of
mycelia DNA quantity. Other species of fungi grew on the wings that were different
visually and morphologically different from Pd. Fungi isolates exhibited rapid,
irregular growth once they were plated onto SDA agar and visualized for growth rate
and morphology. I concluded that bat wings plated onto SDA agar exhibited an
increased number of bacteria and fungi growth that were not associated with our
experiment. As this was first noted in Experiment A, a revised protocol was used in
Experiment B, as described in Methods, to minimize the likelihood of other microbes
dominating the bat tissue, however, this too was unsuccessful – Pd growth on both
groups of wings was minimal and other forms of unidentified fungi and mold were
present (Figure 4.3).

Figure 4.3. Separated bat wing from a control bat one week after Pd inoculation
using the first application method, in which Pd was swabbed onto the bat tissue.
Obvious fungal growth is evident that does not represent Pd.
98

The germination of Pd spores was minimal despite having grown Pd spores in
yeast broth before inoculation. One bat in Experiment B exhibited increased number
of Pd mycelia (L Metal R Grey individual; Figure 4.4), however, this was the only
sample. In Experiment C (Figure 4.5), qPCR results suggests that Pd either
degraded on the wing tissue or, more likely, sampling and qPCR procedures
interfered with the analysis. The frozen ‘dose’ was approximately the same number
of Pd that was placed onto each wing in the frozen, control and treatment groups.
Therefore, measuring the number of Pd in the mycelial sample in PBS may have
worked much better in comparison to sending the entire wing submerged in PBS in
for analysis. This major difference in quantity between the mycelial sample and wing

LBlack

samples is likely attributed to the sampling procedure.

Figure 4.4. Number of Pd mycelia detected on control and treatment wings from the
summer trial. The x axis is each individually identified captive bat. There were two
bats designated as “nobands”, one in the control group and one in the treatment
group. Figure prepared by Adrian Forsythe.
99

A

B

Figure 4.5. Average Pd mycelia concentrations on the treated bat wings and
controls. A wing from a previously frozen bat of the same species was used as a
control and dosed with the same concentration as the treated wings (frozen). The
sample ‘mycelia’ is not a wing sample, but the Pd stock solution (standard) used for
comparison. Suspensions were concentrated 10x in an attempt to quantify lower
values. Picture B is a zoomed in version of A. Prepared by Adrian Forsythe.
100

Ex vivo Explant Results
Pd Quantities
Quantitative PCR Cq results were compared between the 3.5 and 7.5 day groups
and the subgroups that did or did not use antibiotics when inoculated with either Pd
alone, probiotic alone, Pd and probiotic, or nothing (Table 4.2). All samples exposed to
antibiotic medium had inconsistent results and/or showed little to no Pd or probiotic in
the qPCR analysis. This suggests that gentamycin antibiotic destroyed Pd spores and
probiotic cells on the explants, ultimately resulting in DNA degradation of these cells
and inconclusive qPCR results. Inoculated samples in both Groups 1 and 2 provided
evidence of Pd spores upon first swabbing. However, at the termination of each
experiment, 3.5 and 7.5 days later, respectively, qPCR of swab samples revealed no Pd
was evident (Tables 4.2).
Swabs of wing tissues with probiotic that were not exposed to antibiotic were
more consistent. Pd concentrations in Group 1 (3.5 days) decreased when exposed to
bacteria inoculation on the same surface. The Pd Cq numbers changed from 37.75 to
38.44, and from 38.16 to 38.19, signifying decreases in the amount of Pd DNA present
on these two explants (Table 4.2). One of the explants in Group 2 (7.5 days) produced
similar Pd reduction (Cq from 36.5 to 37.0; Table 4.2). The other explant changed in Cq
values from 36.72 to 39.54, signifying a massive increase in Pd concentration. The
inconsistencies observed may be largely due to swab sampling error, confounded by
small sample sizes. Some contamination of Pd was evident among some of the
explants and could largely be due to the explants sharing the same incubator.
The Pd control was not exposed to antibiotic and was swabbed after 3.5 days
and 7.5 days, demonstrating a higher detection count (lower Cq) after 7.5 days than at
3.5 days (Table 4.2). Notably, although only a single sample, the Cq concentration of Pd
is highest in this control sample compared to all other explant samples.

101

Table 4.2. Baseline and endpoint swab sample Cq results of Pd from the Group 1 and 2
explants. Inconsistent or unexpected results are highlighted in yellow.
With antibiotic
Without antibiotic
Cq after 0.5 Cq after 3.5
Cq after 0.5 Cq after 3.5
Group 1
days
days
days
days
With probiotic (A)
30.44
0 With probiotic (A)
0
0
With probiotic (B)
0
0 With probiotic (B)
0
0
With probiotic (C)
0
0
With no probiotic (A)
With no probiotic (B)
Probiotic + Pd (A)
Probiotic + Pd (B)
Group 2
With probiotic (A)
With probiotic (B)

0
0

35.7 With no probiotic (A)
0 With no probiotic (B)

38.5
39.49

Probiotic + Pd (A)
Probiotic + Pd (B)
Pd Only

Cq after 0.5 Cq after 7.5
days
days
0
0 With probiotic (A)
0
0

With no probiotic (A)
With no probiotic (B)

0
32.79

0 With no probiotic (A)
0

Probiotic + Pd (A)
Probiotic + Pd (B)

0
40.4

0
0

Probiotic + Pd (A)
Probiotic + Pd (B)
Pd Only

0
0

37.74
0

37.75
38.16

38.44
38.19
37.36
Cq after 0.5 Cq after 7.5
days
days
0
39.02

0

0

36.5
39.54

36.99
36.72
37.26

Probiotic Quantities
Each of the three probiotic bacteria, P. azotoformans, P. synxantha strain A, and
P. synxantha strain B were analyzed for each of the two experimental groups: 0.5 day
baseline, 3.5 day endpoint, and 7.5 day endpoint, with and without antibiotics. Results
for the fourth bacteria, P. antarctica, are largely inconsistent due to difficulties with
custom gyrB probe for this species not annealing and thus resulting in poor DNA
amplification. A change in probiotic Cq values could be calculated for 8 explants only
due to failure of some samples to amplify DNA. Six of these 8 samples showed an
overall decrease in probiotic cells from the start of the experiment to the end of the
102

experiment (ie. Cq values increased; Tables 4.3 and 4.4). Two explants, one with (3.5
day group) and one without (7.5 day group) Pd inoculation, showed an overall increase
in probiotic cells during the course of the experiment.
In conclusion, I saw many inconsistencies in the explants, most notably those
exposed to the gentamycin antibiotic, similar to the results of the Pd qPCR results
above (Tables 4.3 and Table 4.4). However, other inconsistencies point towards
additional methodological concerns such as small sample sizes, sources of crosscontamination of Pd spores, sampling biases (possibly due to adherence of cells on
swabs and small surfaces being swabbed).

103

Table 4.3. qPCR results of P. azotoformans, P. synxantha strain A, P. synxantha strain
B, and P. antarctica from the 3.5 day group 1. Inconsistent or unexpected results are
highlighted in yellow.
Group 1 Cq values from qPCR
Starting Point (qPCR 0.5 days)
with antibiotic
P.
P. synxantha
synxantha
P.
P.
P. synxantha
B
A
azotoformans antarctica
B
With
probiotic
(A)
With
probiotic
(B)
With
probiotic
(C)
With no
probiotic
(A)
With no
probiotic
(B)
Probiotic +
Pd (A)
Probiotic +
Pd (B)

With no
probiotic
(A)
With no
probiotic
(B)
Probiotic +
Pd (A)
Probiotic +
Pd (B)

P.
antarctica

0

0

0

0

0

0

0

0

33.61

35.81

33.81

37.37

38.96

38.98

38.34

0

38.09

39.64

38.97

40.58*

40.08

42.34

40.01

0

44.95

0

38.37

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

38.62

25.27

27.71

25.53

27.5

37.2
38.79
37.93
Starting Point (qPCR 0.5 days)

39.82

35.29

37.27
35.5
Endpoint (qPCR 3.5 days)

36.55

without antibiotic
With
probiotic
(A)
With
probiotic
(B)

Endpoint (qPCR 3.5 days)
with antibiotic
P.
synxantha
P.
A
azotoformans

without antibiotic

38.78

39.88

39.07

0

0

0

0

0

35.21

36.98

35.46

39.56

38.86

39.01

38.99

38.26

0

0

0

0

0

0

0

0

0

0

0

0

43.94

0

40.28

0

36.83

38.5

37.15

0

0

0

0

0

36.52

38.08

36.92

38.62

36.87

38.42

37.34

41.11

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Table 4.4. qPCR results of P. azotoformans, P. synxantha strain A, P. synxantha strain
B, and P. antarctica from the 7.5 day group 2. Inconsistent or unexpected results are
highlighted in yellow.
Group 2 Cq values from qPCR
Starting Point (qPCR 0.5 days)

P. synxantha
B
With
probiotic

With no
probiotic
(A)
With no
probiotic
(C)
Probiotic +
Pd (A)
Probiotic +
Pd (B)

with antibiotic
P.
synxantha
P.
A
azotoformans

P.
antarctica

Endpoint (qPCR 7.5 days)

P. synxantha
B

with antibiotic
P.
synxantha
P.
A
azotoformans

P.
antarctica

42.11

43.9

42.64

0

0

0

0

0

37.95

39.55

38.72

0

41.49

0

42.04

42.12

0

0

0

0

40.92

42.24

42.02

38.73

0

0

0

0

41.29

42.79

42.1

0

0

0

0

0

36.24

38.53

36.88

0

37.74
39.36
38.24
Starting Point (qPCR 0.5 days)

37.88

0

0
0
Endpoint (qPCR 7.5 days)

0

without antibiotic

without antibiotic

With
probiotic
(A)

38.97

40.82

38.82

42.05

23.71

25.75

24.11

31.66

With no
probiotic
(A)

0

0

0

0

0

0

0

0

37.2

38.53

37.77

45.04

37.93

39.32

38.52

0

36.9

37.86

36.88

0

40.68

42.35

41.24

0

Probiotic +
Pd (A)
Probiotic +
Pd (B)

105

DISCUSSION
Here we tested methods of inoculating Pd onto both dead and live bat tissue in
multiple experiments, as well as testing interactions between the anti-Pd probiotic
bacteria and Pd. We found that there were inherent difficulties with utilizing dead tissue
as a replicate for Pd infection and other variables may have impeded the results. Using
live wing tissue explants allowed us to hurdle some of the dead tissue complications
and Pd was more easily detectable by qPCR and did not have other competing molds to
interact with. However, low cycle numbers were evident and likely due to inadequate
sampling procedures. Each experiment provided insight into what Pd inoculation studies
should consider before an experiment begins.
Wings that were inoculated ex vivo produced few spores or mycelial growth when
analyzed by qPCR. Pd was difficult to visualize based on the fast growth of competing
decay molds. Natural microbes on the wing grew faster on the decaying tissue and out
competed Pd even while growing in optimum Pd growth conditions of 14oC and high
humidity. A growing fungi was easily distinguishable as not being Pd when it was
isolated into subculture, based on its morphology and fast growth within 1-2 days
compared to Pd which naturally takes at least 3-5 days to see any growth on SDA.
Spores were grown in yeast extract to promote mycelia growth before inoculation still
could not out compete decaying molds on the bat wings. Utilizing blended Pd mycelia
was successful on frozen bat wings and SDA agar in previous trials (Not shown here),
however it was unsuccessful on the final set of bat wings from the hibernation trial.
There was some slow growing white fungal growth seen on the bat wings before
submerging it into PBS for analysis. Pd mycelia were detected in the PBS solution
control in the correct concentration however it was not detectable on the severed bat
wings when the submerged PBS was tested for Pd. This is likely due to interference
from skin tissue preventing qPCR probes from detecting Pd, or difficulties with sampling
and extracting the spores from the large suspension in which the wing was submerged
in. Future efforts would need to consider utilizing a swab or smaller wing sample for
qPCR, histological cross examination, or SEM methods to better quantify growth and
106

colonization of wing tissues. Furthermore, although Pd mycelia can grow quickly on
frozen wings, this dosage does not reflect natural Pd infection in wild bats regarding
concentration and mechanism (Makanya et al 2007; Meteyer et al. 2009). Finally, dead
wing tissue is considerably different than live tissue in terms of growth conditions.
Sebum and skin composition, which are crucial factors for skin microbes, are
particularly different in dead tissue than live tissue and do not replicate typical growth
medium for Pd (Wei et al. 2020).
Non-antibiotic results from the explant experiment suggest that there was some
interaction between Pd spores and anti-Pd bacteria. Both “Probiotic+Pd” explant
samples from group 1 that were not exposed to antibiotic decreased in Pd concentration
in relation to the Pd control, which instead increased. This could signify that probiotic
bacteria were antagonistic towards Pd growth. Pd concentration in one sample
increased in group 2 by nearly 3 fold (“Probiotic+Pd (B), without antibiotics”), however
bacteria concentration was quite low in comparison to other explants after 7 days. We
failed to detect any probiotic after 3.5 days on samples “With probiotic (A)” and
“Probiotic+Pd (A). P. antarctica was largely the most inconsistent result of all the
probiotic bacteria and was not detected when other bacteria were. This can be seen in
nearly all samples of probiotic+Pd in group 2. Low Cq numbers are likely stemmed from
swab sampling and inadequate pick-up of spores and/or probiotic cells. Due to the
design of the explant chamber and low number of cells and spores involved, swabbing
was likely an inferior method of extraction compared to utilizing the entire explant
submerged in PBS for qPCR.
Antagonism is still highly suggestive from every other sample exposed to
probiotic bacteria decreased in Pd concentration. It is difficult to draw conclusions from
qPCR bacteria results, however nearly all samples decreased in probiotic concentration
when compared to their baselines. One sample from group 2 increased nearly 14 fold
(“Probiotic (A)”), however this is likely due to sampling or inoculation error. Some
samples had probiotic and Pd contamination which may have led to further ambiguous
results. Future experiments should consider separate incubators to prevent this.
107

A similar experiment by Cornelison et al. in 2014b saw inhibition of Pd growth on
explants that shared the same airspace as anti-Pd bacteria. In our experiment, each
explant chamber was sealed with parafilm and inoculated on the same surface instead
of different surfaces. The anti-Pd bacteria in our study has been shown to inhibit Pd on
cultured agar, presumably through naturally released antibiotic metabolites such as 2,4Diacetylphloroglucinol (Delany et al. 2000; Bangera and Thomashow 1999). Bacteria
were inoculated onto the surface of the explant before their growth phase and
exponential growth to encourage its anti-Pd properties. Furthermore, the only explant in
which we saw any fungal growth was on the control 3.5 and 7.5 day explants, in which a
slow growing white fungus was forming without any probiotic interaction. This is
encouraging to state that the inoculated probiotics may have impeded Pd growth
however without a larger sample size it is difficult to make any assumptions from our
results.
Our results also suggested that utilizing antibiotic’s to prevent unwanted bacteria
infection and mold growth were not necessary from our observations. No mold or visible
morphological change of the explants occurred and explants that were not exposed to
antibiotics were indistinguishable from ones that did. Furthermore, many qPCR results
were ambiguous from explants that were exposed to antibiotic media for both the 3.5
and 7.5 day groups. Although we were very diligent in attempting to prevent antibiotic
exposure to the inoculum surface and did not visibly see leakage, there is a possibility
that gentamycin antibiotic perfused through the epithelial matrix and interacted with both
the probiotic and/or Pd inoculum. Future experiments should consider avoiding
antibiotics unless necessary to prevent this interfering variable.
We did not test for skin viability once the experiment concluded for both groups
of explants. Although skin had the same texture and no signs of decay were present, we
did not conclude this with a cross section and/or histological examination. No decay
mold and similar feel to a live bat patagium may hint at live skin tissue. Future
experiments should also consider utilizing a larger sample size for more robust results.
Here, nearly half our explant samples were unusable due to antibiotic interference. We
108

would recommend using a larger sample size to monitor Pd and probiotic interactions
on the live explant tissue.
In conclusion, there are many obstacles and variables to consider when
inoculating Pd onto bat tissue for ex vivo experiments. Refining ex vivo experiments are
important considerations when live bats are not available, or researchers do not want to
replicate the rigorous methods of hibernating wild animals. Here we found that live
tissue explants can be an encouraging possibility for future Pd inoculation experiments.
Researchers need to consider the difficulty of using explant chambers and the
forewarning of small sample sizes as seen here. Experiments should seriously consider
not using antibiotics to prevent unwanted variables. We did not see any visual
difference between explants that were or were not exposed to antibiotics, however
qPCR results were mixed and ambiguous. The explant experiment was novel; however
it has tremendous potential for future WNS research given enough refinement to its
methods.

109

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Chapter 5. CONCLUSION
This thesis covered three aspects of implementing an anti-Pd probiotic to fight
WNS: Captive trial testing, field trial testing, and Pd challenge lab experiments.
Following two separate captive trials, the probiotic has been shown to have no
detrimental effects to bats, and can easily be transferred to bats indirectly using a
mixture of clay powder and freeze-dried bacteria, a method of application that I
developed. Probiotic bacteria were persistent on captive bats and bat boxes months
after application, including when exposed to high summer heat in the 40-50oC range.
Although viability of cells following these extreme temperatures has yet to be tested, P.
synxantha A showed signs of proliferating in the bat boxes. Probiotic cells grew
substantially, confirming viability when exposed to hibernation conditions.
The field trial was on-the-ground evidence that probiotic can be successfully
transferred from inoculated human-made structures onto wild bat wings. Wings of wild
Myotis lucifugus and M. yumanensis bats roosting in bat box or building roosts at our
study sites had detectable amounts of the probiotic species used in our probiotic
cocktail, following inoculation of their roosting substrates.
While the probiotic cocktail has performed well in vitro against Pd, it has yet to be
properly tested to quantify its efficacy in vivo in the lab and in the field when bats
hibernate in Pd infected sites. Through multiple experiments, I determined that Pd does
not grow on dead bat tissue, presumably due to competition from fast growing molds.
Explant experiments were an improvement because they utilized live tissue; although
challenging and requiring euthanizing of live bats, explant chambers present a
promising tool for answering the question of in vivo efficacy of probiotic against Pd
germination. However, low sample sizes of the pilot that I performed made it difficult to
make conclusions. Refined methodology including using separate Bio-safety Cabinets
(BSC) will help reduce cross-contamination; more euthanized bats will enable larger
sample sizes; direct DNA extraction of tissue explants instead of swabs may reduce
high variability; histology examination will enable viability testing; and inoculating
probiotic bacteria one day before Pd could encourage bacteria inhibition of Pd.
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In the field, realized reduction of WNS-caused mortality will not be possible until
the bats in our study area are exposed to Pd in winter hibernation. Each spring Pd
surveillance efforts are ongoing, and automated PIT tag readers track return rates of
bats after hibernation allowing for these to be compared between Control and
Treatment sites.
There were many challenges associated with this project. It is near impossible to
replicate a roost in a captive setting without an extensively large enclosure and minimal
disturbance. Stress and capture myopathy are unpredictable variables that may prevent
desired results due to unprecedented events such as weight loss, un-natural behaviour,
and death (Jung et al. 2002). These factors were evident when conducting the captive
trial experiments, in which some bats stopped eating and/or were not able to participate
in the trial further. Most experiments had an acceptable sample size however the
hibernation experiment, which is arguably one of the most important experiments in this
thesis, falls short in sample size because of our bats falling victim to environmental
conditions and stress from captivity. Further experiments should heavily consider these
variables and start with a larger sample size to offset any unpredicted losses.
In particular, the captive trial in 2019 (Chapter 2) had limitations such as number
of assistants, space to construct enclosures, number of bats, having to develop an
application method, problems with the P. antarctica primer, and using a novel study
design. This led to smaller sample sizes over a shorter period than what we would have
preferred. These problems resulted in an irregular study design that was not well suited
to comparing treatment 1 to treatment 2 groups over different time periods, although
comparing these two groups was not an objective. What would have been useful, in
hindsight, was to have had a 3rd treatment group which could have received one dose of
probiotic with exposure for a few weeks only, and then swapping of the bat boxes to see
how long the bats retained probiotic on their wings. While this was done at the end of
the captive trial, it was cut short after a few weeks to apply probiotic to the bats that
would then enter into the hibernation experiment. If a second hibernation fridge and
experiment could have been conducted, it would have been interesting to see how the
116

probiotic increased from small concentrations present, more closely simulating what
might be the case if a bat in the wild receives probiotic application from its late summer
roost, and then leaves to transition roosts for a few weeks or months prior to
hibernation.
The study design could have been improved by utilizing separate treatment
enclosures in different locations with their own control enclosures also in different
locations (to separate treatments into their own groups better), to ensure absolutely no
cross-contamination. Treatments could have occurred at similar times and tested
different concentrations or more frequent dosages. A larger sample size of bats would
have been beneficial for more rigorous comparisons, but was limited by permitting.
Similarly, testing the probiotic on additional species of bats was not possible due to
permitting, but may have been interesting to compare results.
Finally, we did not look at the metagenomics of the bat skin. In the future,
researchers should strongly consider looking at the entire microflora of the inoculated
bat species, to examine the shifts in their microbiome from introducing the probiotic
species bacteria. While we attempted to do this, our samples contained small samples
of DNA that were not easily analyzed. Some results from the metagenomic sampling
may be forthcoming, but were not included in this thesis.
Researchers have explored many routes to prevent WNS infection in hibernating
bats. Chitosan, polyethelene glycol, P. fluorescens species complex bacteria,
Rhodococcus rhodocrous, Trichoderma sp, terbinafine, vaccines, valencia orange oil,
and propolis have all been considered for preventing Pd infection, as summarized in a
paper by Hoyt et al. (2019). Similarities are evident between every manuscript because
they overlap with objectives of developing an effective method of preventing Pd
germination. This can otherwise be said in the general hypothesis, “If I apply X to this
media (or bat) then Pd will not grow”. Two field trials using bacteria from the P.
fluorescens species complex have already been successful (Cheng et al. 2016; Hoyt et
al. 2019). The study by Hoyt et al. (2019) provides strong evidence that bats can be
augmented to survive overwinter despite exposure to Pd. They found that bats
117

inoculated with anti-Pd bacteria from the P. fluorescens species complex emerged from
hibernation later with a five fold increase in survivability. The study by Cheng et al.
suggested that bats must be inoculated upon exposure to Pd but still provided evidence
of certain bacteria within the P. fluorescens species complex lowering disease severity
when Pd is present. Current knowledge and proposals of fighting Pd infection in wild
bats are consistently changing however anti-Pd probiotics have been considered the
most promising method, particularly in British Columbia where hibernacula are not
known and preventative measures are needed instead (Fletcher et al. 2020; Weller et
al. 2018). Probiotics are beneficial because anti-Pd bacteria can be isolated from bat
wings and reintroduced into other local populations with little risk to or the bat’s health or
natural microbiota (Thomas and Willis 1998). E. fuscus and some persisting populations
of bats that survive WNS are known to have an abundance of anti-Pd microbiota that
can prevent Pd infection during hibernation (Langwig et al. 2017; Lemieux-Labonté et
al. 2020). I am hopeful that the anti-Pd bacterial cocktail described in this thesis can be
applied as a prophylaxis to bats in BC to reduce overwinter WNS mortality rates bats
from this province come in contact with Pd.
Bats are an important contributor of downwards pressure on arthropods,
specifically nocturnal flying insects such as mosquitos, moths, and beetles, and are
considered one of the most important predators of nocturnal flying insects (Nagorsen
1995). Adult little brown females eat thousand of insects a night (Anthony and Kunz
1977; Kurta et al. 1989; Nagorsen 1995). Studies have shown that exclusion of bats
dramatically increase the abundance of insects prevalent on plants (Kalka et al. 2008;
Williams-Guillen et al. 2008), sometimes 2 fold. Boyles et al. (2011) estimated the value
of insectivorous bats to be 3.7-53 billion USD a year in North America. Cost savings are
due to decreased use of pesticides and applications needed to fight resistance in
agricultural pests. This is worrying, because White-nose syndrome, among other
conservation threats such as wind turbines, roads, habitat fragmentation and loss, and
deforestation have incredibly devastated bat populations (Berthinussen et al. 2014).
White-nose syndrome is responsible for over 6 million bat deaths and recently emerged
in Washington state (Frick et al. 2010; Frick et al. 2015; WNS 2019). Extinction risk for
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many species is real and often the result of emerging infectious diseases (Daszak 2000;
De Castro and Bolker 2004).
The overarching conservation goal of this project was to prevent, or lessen, the
number of deaths caused by WNS once it arrives in British Columbia. This project is
unique in all of North America as there are no other studies of using probiotic as a
prophylaxis applied at maternity roosts. Much of the methodology employed in this
project, and use of bio-augmentation via probiotics is novel (Thomas and Willis 1998).
This work has established a foundation for other researchers to replicate and build
upon.
Future research directions are limited in preventing and/or stopping WNS from
causing mass mortalities in western hibernating bat species. Studies should consider
two goals, 1. Build on the preventative measures that we suggest in this thesis, and 2.
Urgently increase bat population monitoring and assess hibernacula for signs of WNS.
Bats mostly hibernate in small clusters in the western North America (Weller et al. 2018)
making it difficult to locate bat hibernacula. Here we propose a solution using bat boxes
and man made structures to transfer anti-Pd probiotic onto roosting bats. However, this
may limit the application potential of rural areas where bat boxes may be impossible to
maintain or dose properly. Ideally, we would want to apply our probiotic to known areas
of hibernacula and monitor for Pd and probiotic levels. Because of the short time frame,
incredible difficulty and sparse groupings, it is likely impossible to directly apply the
probiotic to bat hibernacula. Refining a preventative measure is the only plausible option
currently available. Bat monitoring should continue with the thought of direct hibernacula
inoculation, but not relied on. Future field trials would benefit from a repeated, and
higher dosage of anti-Pd bacteria on bat boxes in October with minimal disturbance to
the roosting colony. It would be beneficial to investigate treatment frequency and
dosages needed to maintain a viable concentration of bacteria that release the anti-Pd
metabolites needed to prevent WNS infection. Further, if a nutrient supplement that
stimulated pseudomonas metabolite production was proven safe in another captive trial,
perhaps it could be added to the final prophylactic cocktail (Duffy and Défago 1999).
119

There are many different research directions to take in preventing WNS in western bat
species and time is running out. Researchers will likely have to compromise on the best
possible approach with whichever prophylaxis they finalize on and where to apply it.
WNS will inevitably be found in British Columbia and it is up to researchers to decide on
what is the best possible approach to prevent mass bat mortality.
Studies performed in this thesis were in collaboration with the BC Ministry of
Agriculture, McMaster University, UBC Okanagan, and Wildlife Conservation Society
Canada. Capture information came from Leah Rensel of UBCO, and the qPCR results
which I present in many tables and figures were generated by Adrian Forsythe,
McMaster University.

120

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Cheng T, Mayberry H, McGuire L, Hoyt J, Langwig K, Nguyen H, Parise K, Foster J,
Willis C, Kilpatrick A et al. 2016. Efficacy of a probiotic bacterium to treat bats
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54(3):701-708.
Daszak P. 2000. Emerging Infectious Diseases of Wildlife-- Threats to Biodiversity and
Human Health. Science 287(5452):443-449.
De Castro F, Bolker B. 2004. Mechanisms of disease-induced extinction. Ecology
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Duffy, B. K., & Défago, G. 1999. Environmental factors modulating antibiotic and
siderophore biosynthesis by Pseudomonas fluorescens biocontrol strains. Appl.
Environ. Microbiol 65(6):2429-2438.
Jung T, Thompson I, Hickey M, Titman R. 2002. Apparent capture myopathy in hoary
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Kurta A, Bell G, Nagy K, Kunz T. 1989. Energetics of Pregnancy and Lactation in
Freeranging Little Brown Bats (Myotis lucifugus). Physiological Zoology
62(3):804-818.
Fletcher Q, Webber Q, Willis C. 2020. Modelling the potential efficacy of treatments for
white‐nose syndrome in bats. Journal of Applied Ecology 57(7):1283-1291
Frick W, Pollock J, Hicks A, Langwig K, Reynolds D, Turner G, Butchkoski C, Kunz T.
2010. An Emerging Disease Causes Regional Population Collapse of a Common
North American Bat Species. Science 329(5992):679-682.
Frick W, Puechmaille S, Hoyt J, Nickel B, Langwig K, Foster J, Barlow K, Bartonička T,
Feller D, Haarsma A et al. 2015. Disease alters macroecological patterns of
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Hoyt J, Langwig K, White J, Kaarakka H, Redell J, Parise K, Frick W, Foster J, Kilpatrick
A. 2019. Field trial of a probiotic bacteria to protect bats from white-nose
syndrome. Scientific Reports 9(1):9158.
Langwig K, Hoyt J, Parise K, Frick W, Foster J, Kilpatrick A. 2017. Resistance in
persisting bat populations after white-nose syndrome invasion. Philosophical
Transactions of the Royal Society B: Biological Sciences 372(1712):20160044.
Lemieux-Labonté V, Dorville N, Willis C, Lapointe F. 2020. Corrigendum: Antifungal
Potential of the Skin Microbiota of Hibernating Big Brown Bats (Eptesicus fuscus)
Infected with the Causal Agent of White-Nose Syndrome. Frontiers in
Microbiology. 11:588889.
Nagorsen D, Brigham R. 1995. Bats of British Columbia. Vancouver: UBC Press.
Thomas M, Willis A. 1998. Biocontrol—risky but necessary? Trends in Ecology &
Evolution 13(8):325–329.
Weller T, Rodhouse T, Neubaum D, Ormsbee P, Dixon R, Popp D, Williams J, Osborn
S, Rogers B, Beard L et al. 2018. A review of bat hibernacula across the western
United States: Implications for white-nose syndrome surveillance and
management. PLoS One 13(10):e0205647.
Williams-Guillen K, Perfecto I, Vandermeer J. 2008. Bats Limit Insects in a Neotropical
Agroforestry System. Science 320(5872):70-70.
WNS, Whitenosesyndrome.org. 2019. Spread Maps. [accessed 2021 Apr 4]. Available:
https://www.whitenosesyndrome.org/static-spread-map.

122

APPENDIX A. Synergistic Testing of Anti-Pd Isolates
A)

B)

C)

D)

Figure A.1. Inhibition of Pd in pairwise combinations A) measuring full zone of clearing;
B) partial zone of clearing; C) Fractional Inhibitory Concentration Index for full inhibition;
and D) partial inhibition. Figures prepared by Adrian Forsythe.

123

APPENDIX B. Animal care
Summer 2019 Trial
Twenty M. yumanensis were used in our captive trial, and were taken from
Creston, Kuskanook, Chase, and Lardeau areas in British Columbia. Bats were brought
into captivity in two batches. The first batch of 15 bats were captured within two days
apart and were progressively placed into small, medium and large cages. Each bat was
banded with one or two coloured bands on their forearms to differentiate them from
each other. They were taught the principle of self feeding (mealworms) and self-drinking
out of dishes throughout the acclimation process. This was performed by having
mealworms and a water dish present within the mesh chamber for several days.
Similarly to our fall trial, bats were manually fed mealworms and water for the first few
days, with close observation on subsequent nights to ensure they are each drinking and
eating without human intervention (observing colored bands). Bats were released from
the large mesh enclosure and placed into our control enclosure for further acclimation.
They were monitored daily for weights until evidence of self feeding and watering were
shown. The amount of food and water accepted by each bat was recorded daily. Food
stations consist of half aluminum foil cupcake tins filled with mealworms held in place by
wooden boards to prevent shifting if the bats climbed into the food dishes. A water
station was a dish kept topped up with an upside down bottle of water, and each dish
was filled with marbles and had sponges on top which were regularly changed; these
measures were taken to ensure sufficient water and prevent accidental entrapment or
submersion of bats. Additionally, a mesh overlay on one end allowed bats to easily land
above the water dish and climb down to approach its surface or the soaked sponges.
Once the bats learned how to feed and water on their own, hand feeding and watering
were not required. Bats were separated into Control and Treatment 1 groups when they
were comfortable living within captivity, sustained their weight, and independently drank
water. Mealworms were fed the same rich Omega 3 fatty acid diet used in the fall
captive trial. This diet is a better approximation of one consisting of wild caught insects.
The second batch of 5 bats were introduced into the Treatment 2 group at a later date
with the same protocol followed as above.
124

Bats were monitored daily within their enclosure and were visited on a consistent
time schedule each day. Little time was spent within the enclosure and noise was kept
to a minimum to prevent disturbance and stress to the bats. They were removed from
their bat house once a week for a health inspection by a registered Veterinarian
Technician at the Kamloops BC Wildlife Park. Bats were placed into cloth bags and
individually weighed before looking for details such as behaviour, body condition, injury,
dehydration, and the condition of the eyes.
Hibernation Trial
The bats were introduced to the beverage cooler and it was set at 12-13oC,
mimicking outside temperatures. Humidity was immediately introduced at >90% with
mealworms, water and a roost pouch readily available. Each subsequent day, the
temperature was dropped by 1oC until it reached 7-9oC. Bats were taught the principle
of self-feeding and self-drinking out of a water dish during this time. Bats were manually
fed mealworms and water for the first few nights, and closely observed on each
subsequent night to ensure they are eating and drinking without human intervention.
Each bat’s weight was monitored for weight loss or gain to presume self feeding. Once
the optimum temperature was reached and bats had a steady weight of >6.0g to survive
hibernation, we removed the mealworms and the roost pouch to entice hibernation. Two
weeks later the temperature was dropped by another 1oC to approximately 6-8oC to
further entice the bats to hibernate. Bats were meticulously monitored throughout the
acclimation process and were checked daily with a Bluetooth infrared camera placed
inside the fridge. Past and current bat activity was examined with the camera and
identified with a coloured forearm band previously attached onto their wing. A red light
was also used to watch the bats through the glass door and the checks were always
completed within a dark room to prevent further disturbance. Notes were taken daily
such as, roosting position of each bat, individual behaviour of each bat, temperature
and humidity levels, water and food levels, and level of water in terrarium humidifier.
Particularly, notes such as torpor time and environmental conditions were of greatest
importance.

125

APPENDIX C. Swabbing Area and Sampling Dates
Table C.1. Swabbing area of the left arm, right arm, and both arms combined of bats
from the 2019 captive trial. Excluded width of body.
Right arm
Bat Code

Left arm length

length

Left+right arm length combined

LMRP

9.91

10.16

20.07

LMRG

10.80

10.29

21.08

LM

9.33

9.21

18.54

LMRB

9.72

10.41

20.13

LMRLB

9.46

9.72

19.18

LG

9.21

9.33

18.54

LMRG

9.72

9.84

19.56

NB

9.97

9.78

19.75

LB

10.73

10.60

21.34

LMRB

9.65

9.59

19.24

LR

9.53

9.65

19.18

LBRP

9.97

10.10

20.07

LB

9.46

9.33

18.80

LBRLB

9.53

9.65

19.18

LG

10.22

10.35

20.57

LLB

9.53

9.59

19.11

RP

10.16

10.48

20.64

LDB

9.72

9.84

19.56

Mean

9.81

9.88

19.70

SE

0.10

0.10

0.19

126

Table C.2. Swab schedule of treatment 1 and 2 bats.
Treatment group

Date

Swab Location

Length

Baseline Bat Swabs

April 28th

Right wing

9.88

Treatment 1

Entire Sampling period

Both wings

19.70

Treatment 2

June 15th

Right wing

9.88

Treatment 2

June 29th

Left wing

9.81

Treatment 2

July 27th

Left wing

9.81

Treatment 2

Aug 9th

Right wing

9.88

Treatment 2

Aug 14th

Left wing

9.81

Treatment 2

Aug 24th

Left wing

9.81

Left wing

Right wing

Both wings

Figure C.1 Representation of bat swab areas for Treatment group 1 and 2. Note: scale
of photo is not accurate.

Table C.3. Swab schedule of bats in the hibernation trial.
Sample Dates
Wing section

oct 30 and dec 21

07-Nov Nov 18 and 28

Shoulder to

First and Second

elbow (1)

finger bone (2)

dec 8 and 18
Fourth finger

Third finger bone (3)

bone (4)

L Silver

4.3815

5.334

5.08

2.921

L Silver R Green

4.445

6.35

4.6355

4.191

No Bands

4.0005

5.334

4.8895

4.445

Mean

4.27482

5.67182

4.86664

3.85064

0.054645321

0.133333333

0.050689688

0.185592145

SE

127

2.
3.
4.
1.

Figure C.2 Representation of bat swab areas for the Hibernation group. Note: scale of
photo is not accurate.

128

APPENDIX D. Hibernation Fridge Temperature and Relative Humidity.

Figure D.1. Temperature and relative humidity percentage within the hibernation
chamber throughout the trial period.

129

APPENDIX E. Swab Broth Suspension Results.

Concentration of cells/ml/cm2

10000

1000

100

10

1
2019-06-08

2019-06-15

2019-06-22

Date
P. synxantha strain B

P. synxantha strain A

P. azotoformans

Figure E.1. Concentration of probiotic cells detected on treated bats in the Treatment 1
group using qPCR for each probiotic bacteria species. Swab suspension was grown in
broth for 12 hours and incubated at 25oC. Concentrations are corrected by wing area to
represent cm2. Note the logarithmic scale.

130

Concentration of cells/ml/cm2

100000
10000
1000
100
10
1
2019-06-08

2019-06-15

2019-06-22

2019-06-29

2019-07-08

Date
P. synxantha strain B

P. azotoformans

P. synxantha strain A

Figure E.2. Concentration of probiotic cells detected on the bat box in the Treatment 1
group using qPCR for each probiotic bacteria species. Swab suspension was grown in
broth for 12 hours and incubated at 25oC. Note the logarithmic scale.

Concentration of cells/ml/cm2

10000

1000

100

10

1
2019-07-13

2019-08-09

2019-08-14

Date
P. synxantha strain B

P. synxantha strain A

P. azotoformans

P. antarctica

Figure E.3. Concentration of probiotic cells detected on treated bats in the Treatment 2
group using qPCR for each probiotic bacteria species. Swab suspension was grown in
broth for 12 hours and incubated at 25oC. Concentrations are corrected by wing area to
represent cm2. Note the logarithmic scale.

131

Concentration of cells/mL/cm2

100000
10000
1000
100
10
1
2019-07-13

2019-07-27

2019-08-09

Date
P. synxantha strain B

P. synxantha strain A

P. azotoformans

P. antarctica

Figure E.4. Concentration of probiotic cells detected on the bat box in the Treatment 2
group using qPCR for each probiotic bacteria species. Swab suspension was grown in
broth for 12 hours and incubated at 25oC. Note the logarithmic scale.

Concentration of cells/ml/cm2

100000000
10000000
1000000
100000
10000
1000
100
10
1
2019-11-16

2019-11-28

2019-12-08

2019-12-18

2019-12-21

Date
P. synxantha strain B

P. synxantha strain A

P. azotoformans

P. antarctica

Figure E.5. Concentration of probiotic cells detected from bat swabs in the hibernation
trial using qPCR for each probiotic bacteria species. Swab suspension was grown in
broth for 12 hours and incubated at 25oC. Concentrations are corrected by wing area to
represent cm2. Note the logarithmic scale.

132

Concentration of Cells

100000
10000
1000
100
10
1
2019-07-20

2019-07-27

2019-08-09

2019-08-17

2019-08-24

P. azotoformans

P. antarctica

Date
P. synxantha strain B

P. synxantha strain A

Figure E.6. Concentration of probiotic cells detected within the four-chamber bat box
using qPCR. Swab suspension was grown in broth for 12 hours and incubated at 25 oC.
Results from all chambers are pooled into one figure. Note the logarithmic scale.

APPENDIX F. HISTOLOGY SCORE RESULTS
133

Table F.1. Histology score results from the 2018 captive bat trial.

Bat

Band

Identification# Colour

19-1004-A

19-1004-C

18-5528-C

18-5528-A

Purple
C
Blue
C
Light
blue

Dark
blue

18-5528-B

Grey

19-1004-F

Black

Start

End

Treatment/control mass

mass

(g)

(g)

Control

5.3

7.1

4-Nov-18

Good

Control

3.6

6.2

4-Nov-18

Good

4.2

4.2

11-Sep-18

3.9

3.9

11-Sep-18

4.6

4.7

9-Oct-18

4

6.3

4-Nov-18

Date
deceased

Control,
deceased before
treatment began
Treatment,
deceased before
treatment began
Control*
1st Treatment
Period only

Post

Necropy

mortem

Comments (G.

preservation

McGregor)

mod,
freeze-thaw

mod,
freeze-thaw
mod,
freeze-thaw
Good
A LOT of large
foamy
macrophages in
the lungs in

19-1004-B

Purple

1st Treatment

P

Period only

4.6

7

4-Nov-18

Good

septa and
alveoli with grey
material
inside.** Mild
renal
coccidiosis.

19-1004-D

Blue P

1st & 2nd
treatments

Moderate
4.8

6.8

4-Nov-18

Good

numbers of
large foamy

134

macrophages in
lung. **
Mod number of
19-1004-E

Green

1st & 2nd
treatments

5.2

6.2

4-Nov-18

Good

large foamy
macrophages in
lung. **

*bat died of fall while torpid
* This was attributed to bats preparing for
hibernation (Bouma et al. 2010; see
Chapter 2 Results).

135

B)
BAT
No Probiotic Applied
BODY

Purple

Light

Dark

PART

C

blue*

blue*

WING

30

23

0

TAIL

24

15

EAR

0

0

NOSE

33

Blue C

Probiotic Applied
Grey*

Black

2

1

1

13

0

0

0

0

0

Purple

Blue P

Green

24

2

16

2

20

19

17

0

1

0

0

12

0

0

0

0

3

3

3

4

2

3

4

3

3

5

4

1

94

56

9

54

27

37

P

LUNG neutrophil
count
SPLEEN neutrophil
count

OVERALL
COUNT

13

2

1

AVERAGE
SCORE

33.2

31.75

these 3 bats died and were sent for necropsy; the others were all euthanized at the
*

end of the trial period in early Nov.
Segs and Monos - these tests are on a scale of 0 - 100 but are shown here instead as
1/100 (e.g. 25 shown as 2.5) to enable a crude weighting for summing and average

Note: scoring.
Dark bats that were Controls or died before treatments began; italics are bats that were not
shade in captivity as long as the other bats (died prematurely)
bats that received probiotic treatment; italics are bats that received a large dose of
Light probiotic in last week or trial vs non-italics are Treatment bats that did not receive
shade treatment for the last 2 weeks of trial

136

Table F.2. Histology scores of wing tissue from bats within the 2019 summer captive trial. These
consisted of Control and Treatment 1 groups.
Wing
PM preservation

Tx group

acanthosis

hyperkeratosis

overall
inflammation

epidermal
inflammation

dermal
inflammation

Segs

Monos

Good

control

1

0

2

0

2

0

100

Good

control

2

1

2

2

1

0

100

Good

control

3

2

3

0

3

0

100

Good

control

1

0

2

1

1

0

100

Good

control

2

1

3

1

2

25

75

Good

control

1

0

3

0

3

0

100

Good

T1

2

1

3

1

3

25

75

Good

T1

3

1

3

0

3

0

100

Good

T1

2

0

1

0

1

0

100

Good

T1

2

0

3

0

3

25

75

Good

T1

1

0

1

0

1

0

100

Good

T1

2

2

2

0

2

25

75

Good

T1

2

1

3

3

3

50

50

Control mean

1.67

0.667

2.5

0.667

2

Treatment mean

2

0.714

2.29

0.571

2.286

Hyphae

Bacterial
epidermal

Bacterial
invasion

Ulceration

Hemorrhage

Mites

Yeast

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

137

Table F.3. Histology scores of tail tissue from bats within the 2019 summer captive trial. These
consisted of Control and Treatment 1 groups.
Tail
PM
preservation

Tx group

acanthosis

hyperkeratosis

overall
inflammation

epidermal
inflammation

dermal
inflammation

Segs

Monos

Good

control

0

0

2

0

3

25

75

Good

control

1

1

0

0

0

0

0

Good

control

2

0

4

1

3

25

75

Good

control

2

1

3

0

3

25

75

Good

control

1

0

1

0

1

25

75

Good

control

1

0

2

0

2

0

100

Good

T1

1

1

1

0

1

0

100

Good

T1

1

0

2

0

2

0

100

Good

T1

0

0

0

0

0

0

0

Good

T1

1

0

1

0

1

25

75

Good

T1

0

0

0

0

0

0

0

Good

T1

0

0

2

2

0

100

0

Good

T1

1

0

1

0

1

0

100

1.17

0.333

2

0.167

2

0.571

0.143

1

0.286

0.714

Control
mean
Treatment
mean

Hyphae

Bacterial
epidermal

Bacterial
invasion

Ulceration

Hemorrhage

Mites

Yeast

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

138

Table F.4. Histology scores of ear tissue from bats within the 2019 summer captive trial. These
consisted of Control and Treatment 1 groups.
Ear
PM
preservation

Tx group

acanthosis

hyperkeratosis

overall
inflammation

epidermal
inflammation

dermal
inflammation

Good

control

0

0

2

0

2

Good

control

0

0

2

0

2

Good

control

0

0

2

0

2

Good

control

0

0

0

0

0

Good

control

0

0

0

0

0

Good

control

0

0

0

0

0

Good

T1

0

0

4

0

4

Good

T1

0

0

1

0

1

Good

T1

0

0

2

0

2

Good

T1

0

0

3

0

3

Good

T1

0

0

0

0

0

Good

T1

0

0

0

0

0

Good

T1

0

0

1

0

1

0

0

1

0

1

0

0

1.571

0

1.571

Control
mean
Treatment
mean

Hyphae

Bacterial
epidermal

Bacterial
invasion

Ulceration

Hemorrhage

Mites

Yeast

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

139

Table F.5. Histology scores of nose tissue from bats within the 2019 summer captive trial.
These consisted of Control and Treatment 1 groups
Nose
PM
preservation

Tx group

overall
inflammation

epidermal
inflammation

dermal
inflammation

Segs

Monos

Good

control

0

0

0

0

0

Good

control

0

0

0

0

0

Good

control

5

5

5

75

25

Good

control

1

0

1

0

100

Good

control

0

0

0

0

0

Good

control

0

0

0

0

0

Good

T1

0

0

0

0

0

Good

T1

0

0

0

0

0

Good

T1

0

0

0

0

0

Good

T1

0

0

0

0

0

Good

T1

0

0

0

0

0

Good

T1

1

0

1

0

100

Good

T1

0

0

0

0

0

1

0.833

1

0.143

0

0.143

Control
mean
Treatment
mean

Hyphae

Bacterial
epidermal

Bacterial
invasion

Ulceration

Hemorrhage

Mites

0

0

0

0

0

0

0

0

0

0

0

0

0

3

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0.5

0

0

0

0

0

0

0

0

0

0

140

Table F.6. Histology scores of lungs and spleens from bats within the 2019 summer captive trial.
These consisted of Control and Treatment 1 groups.
Lung

Spleen

Tx group

Lung neuts

Lung histocytic
cells

control

2

3

1

control

1

2

1

control

2

1

1

control

2

2

2

control

2

3

2

control

1

3

1

T1

1

1

n/a

T1

2

3

1

T1

1

1

1

T1

1

2

n/a

T1

1

2

1

T1

1

3

0

T1

2

3

n/a

1.67

2.33

1.33

1.28

2.14

0.75

Control
mean
Treatment
mean

spleen
neuts

141

APPENDIX G. Captive Trial Experimental Design and Justification
The experimental design involves 3 groups of bats which belong to the
Treatment 1 group, Treatment 2 group, and the Hibernation group. Each group was
independent of one another because of how they differed in their experimental design.
The Treatment 1 group was inoculated with two probiotic dosages which were on May
12th and May 24th, followed by weekly swabbing (swab areas can be found in Appendix
C) of their wings. Treatment 2 group was only inoculated with one dosage of probiotic
bacteria on June 15th. The bat box in the Treatment 1 group was changed out upon
inoculating another dosage, whereas the Treatment 2 group bats used the same bat
box that was initially inoculated. Therefore we are comparing two groups of bats that
were exposed to differing concentrations of probiotic, one dosage versus two dosages
of probiotic bacteria. The Hibernation group was independent of the other treatment
groups because it was placed into entirely different environmental conditions within a
hibernacula chamber (Appendix D).
All replicates from each species of bacteria and on each swab date formulate a
mean abundance of cells in each category (Table G.1, G.2, G.3). The standard error is
calculated among all replicates due to the mean of probiotic bacteria being derived from
these individual replicates. Of which, the mean of means of each bacteria from each bat
would derive the same result as the mean of all replicates they originated from.
Table G.1. Number of swabbed bats in the Treatment 1 group and qPCR data replicates
of each bacteria species found across all bats.
Date of bat
swab
2019-05-26
2019-06-01
2019-06-08
2019-06-15

Number of
bats swabbed
7
7
7
7

P. synxantha strain B
replicates
14
9
2
7

P. synxantha
strain A replicates
15
8
2
9

P. azotoformans
replicates
15
7
2
7

142

Table G.2. Number of swabbed bats in the Treatment 2 group and qPCR data replicates
of each bacteria species found across all bats.

Date of bat
swab
2019-06-29
2019-07-13
2019-08-09
2019-08-14

Number of
bats
swabbed
3
3
3
3

P. synxantha
strain B
replicates
4
1
1
2

P. synxantha
strain A
replicates
4
1
1
2

P.
azotoformans
replicates
4
1
1
2

P. antarctica
replicates
0
0
2
0

Table G.3. Number of swabbed bats in the Hibernation group and qPCR data replicates
of each bacteria species found across all bats.

2019-11-16
2019-11-28
2019-12-08
2019-12-18
2019-12-21

Number of
bats
swabbed
3
3
3
3
3

P. synxantha
strain B replicates
9
9
9
3
6

P. synxantha
strain A
replicates
9
9
9
3
6

P.
azotoformans
replicates
9
9
9
3
6

P. antarctica
replicates
6
9
9
0
6

143

APPENDIX H. Swab Protocol
Step-by-step protocol
INTRODUCTION:
The objective of this sampling is to collect metagenomics data on the bat species
and populations for conservation efforts. Metagenomic sampling of bats will provide
total genomic data of a bat’s wing microbiome.
Materials needed for each bat / environmental sample:
Included in kit:
1 vial of sterile NaCl (0.15M) buffer solution (for moistening swab and storing
swab after sampling)
1 ziploc bag (to place vial in when sampling is completed)
1 sticker OR 1 piece of rite on rain paper (label with sample information)
1 sterile swab with a predefined breakpoint on the stick
Included in kit:
One large Ziploc bag is included for each sampling session
Also needed per sample:
1 small Ziploc bag for individual samples
1 pair of latex/nitrile gloves
1 permanent marker
Decontamination chemical (for WNS spread prevention) for any surface that
happens to touch the bat

Methods: swabbing
i.

Put on latex/nitrile gloves.

ii. Fill in information on sticker/rite on rain paper. Include the following 7 information
fields:
NAME of Principal Investigator:
DATE (MMM DD/YY)
SITE
BATID#
SPECIES: (4 letter code)
SEX: M/F
AGE: A/Juv
144

If stickers are provided in your kit, this information should be filled out on the label with
permanent marker and adhered to the inside of the small Ziploc bag. But if there is
only rite on the rain paper, please write this information on the paper and place it
inside the Ziploc bag.

iii. Open the Whatman Omni swab package so that the swab handle is presented to the
handler. Keep the tip of the swab inside the package to keep it sterile. Avoid contact
with the tip of the swab.
iv. Soak the tip of the swab in 0.15M NaCl buffer tube. Remove excess liquid from
the swab by pressing the swab tip against the inside of the tube wall.
v. Part 1 (to be completed for all bats). Swab the bat:
a) Please read the precautions at the bottom of this protocol.
b) Carefully swab the inside of the right wing (forearm and wing) in linear
strokes. Cover the entire wing surface, slowly rolling or lightly brushing the
swab over the skin. This should take at least 12 seconds.
c) Immediately place the swab into the vial -- Release the tip of the swab into
the collection tube by pressing on the swab handle.
d) Place collection tube into small Ziploc bag. Be sure there is a label on the
inside of this bag (either a sticker or piece of paper).

vi. Part 2 (To be completed only once at each capture site (Environmental Reference
Swab -- to be completed only if there are known bat roosts at the sampling area).
a) Using another Whatman omni swab, vial and fresh gloves, create a label with the
following information fields:
NAME of Principal Investigator:
DATE (MMM DD/YY)
SITE:
ENVIRONMENTAL SUBSTRATE SWABBED: (e.g., inside rock crevice,
mine wall, rafter of attic)
KNOWN USE BY ROOSTING BATS? y/n
145

b) Swab the wall/surface closest to where the bats are roosting or would typically
roost for 10 seconds in a circle of approximately 5cm.
•

For example, if bats are captured outside of a rocky bluff area, and bats
are thought to be using the rock crevices in the area, swab the inside of a
randomly selected crevice. If an actual crevice roost is known, sample this
instead.

c) Release the tip of the swab into the collection tube by pressing on the swab
handle.
d) Store swab sample immediately after swabbing by releasing tip into collection
tube

146

APPENDIX I. Explant Protocol
Myotis Explant SOP
Introduction:
The purpose of this SOP is to test the Pd inhibition threshold of the anti-Pd bacteria
Pseudomonas synxantha, Strains A and B; P. azotoformans; and P. antarctica on live
bat tissue. Severed tissue explants can be kept partially alive within specialized
chambers, thus preventing the associated interference of decomposition.
Materials needed:
Autoclave and/or sterilize the following:
→ Isoflurane
→ Cotton balls
→ Small mason jars with lids
→ Large sterile scissors
→ Surgical scalpel
→ Clean cutting board
→ Biohazard waste bag
→ Sterile forceps
→ Biosafety cabinet (BSC)
→ Fume hood
→ Calipers
→ Gloves
→ P20 & P200 pipettes and tips
→ Explant chamber
→ Explant hole-punch
→ Eagles minimal essential medium
→ Gentamycin
→ Probiotic dosage
→ Gas mask
→ Prepared probiotic bacteria
→ Prepared Pd spores
147

→ Scanning Electron Microscope (SEM)
→ Phosphate Buffer Solution (PBS)
→ Centrifuge
→ Tinfoil

Procedure
1. Gather necessary material in both the fume hood and BSC. Make sure to wear a
mask that protects against volatile gases (i.e. Isoflurane). Add media with and
without gentamycin antibiotics into the explant chambers and seal off with tinfoil.
Leave the chambers in the BSC until the explants are extracted from the bats.

2. To prepare for euthanasia, soak a cotton ball with a generous amount of
isoflurane and place it into the bottom of a mason jar. Be sure to have positive
airflow in the fume hood to avoid leaking of the anesthetic.

3. Take the bat and place it into the jar containing the cotton ball soaked in
isoflurane. After 1-2 minutes, the bat should be knocked out and unconscious
from the anesthetic. The bat should overdose quite quickly.

4. After the bat is no longer moving and at least 5 minutes have passed, remove the
bat from the jar and move it to the BSC. Quickly decapitate the bat using a sharp
pair of scissors to confirm euthanasia. Do not stop halfway through.

5. Turn off laminar airflow going into the BSC. The anesthetic is contained within
the separate fume hood and a sterile environment is not needed for sampling the
explants.

6. Using the explant hole-punch, sample skin tissue from the myotis wing patagium
one at a time. Work quickly and diligently because samples can dry up and
become unusable.
148

7. Place the sampled tissue into the explant chamber and screw both pieces of the
apparatus together to seal the explant into place. Place tinfoil over top of the
explant chambers. Repeat steps 6-7 until sufficient explants have been sampled
or the tissue is starting to dry out.

8. Turn on the laminar air flow and prepare probiotic dosages for application.

a. Bacteria will be grown before application in LB broth. Utilizing growth
curves, OD readings, and dilution calculations, a proper dosage can be
achieved for all explants. Remove designated amount of each of the 4
probiotics and mix them together. Prepare the same number of dosages
as explants.

b. Centrifuge at 4000rpm for ten minutes. Remove supernatant and flush
with 1ml of PBS. Vortex on medium high.

c. Repeat step b and centrifuge once more to remove any LB broth from the
bacteria. Remove supernatant and add 250ul of PBS. Vortex on medium
high to displace the bacteria pellet.

9. Inoculate each of the explants with the probiotic.

10. Inoculate the designated number of explants with Pd spores from previously
prepared spore isolations. Refer to the spore isolation SOP for more info.

11. Fully seal the explant chambers and place the bat corpses into the -80 freezer.
Monitor the explants daily.

149

12. After 3-4 days, half of the explants will be removed and monitored through SEM
for spore germination and hyphae growth. Control will be compared to probiotic
treatments at different spore loads, with and without antibiotics.

13. After 1 week, monitor the other half of the explants for spore growth and
germination. Remove explants and place them into PBS solutions. Freeze the
samples. Pd and probiotic numbers can then be monitored through qPCR
analysis.

150