Epidemiology of Salmonella and Antimicrobial Resistant Enteric Bacteria in Raccoons and the ...

222 Pages·2016·2.31 MB·English

by  Kristin Bondo

Epidemiology of Salmonella and Antimicrobial Resistant Enteric Bacteria in
Raccoons and the Environment on Swine Farms and Conservation Areas in
Southern Ontario

by
Kristin J. Bondo

A Thesis
Presented to The
University of Guelph

In partial fulfilment of requirements
for the degree of
Doctor of Philosophy
in
Pathobiology

Guelph, Ontario, Canada

© Kristin J. Bondo, December 2015
ABSTRACT

EPIDEMIOLOGY OF SALMONELLA AND ANTIMICROBIAL RESISTANT ENTERIC
BACTERIA IN RACCOONS AND THE ENVIRONMENT ON SWINE FARMS AND
CONSERVATION AREAS IN SOUTHERN ONTARIO

Kristin Bondo Advisor
University of Guelph, 2015 Dr. C. Jardine

Relatively little is known about the epidemiology of Salmonella and antimicrobial
resistant enteric bacteria in wildlife living in different habitat types. My objective was to
investigate demographic, temporal, and climatic factors associated with carriage of Salmonella
and resistant Escherichia coli isolates in raccoons (Procyon lotor) and their environment on
swine farms and conservation areas. Using a repeated cross-sectional study design, we collected
fecal samples from raccoons and environmental samples (soil, manure pits, dumpsters) on 5
swine farms and 5 conservation areas in Ontario, Canada once every five weeks from May
through November, 2011–2013. Salmonella was detected in 26% of raccoon fecal samples, 27%
of raccoon paw samples, 6% of soil samples, 30% of manure pit samples, and 23% of dumpster
samples. Of samples testing positive for Salmonella, antimicrobial resistance was detected in 5%
of raccoon fecal, 0.2% of paw, 8% of soil, 10% of manure pit, and no dumpster samples. Of
samples testing positive for E. coli, antimicrobial resistance was detected in 7% of raccoon fecal,
6% of paw, 10% of soil, 57% of manure pit, and 14% of dumpster samples. Using multi-level
logistic regression analyses, I found location type (swine farm or conservation area) was not a
significant explanatory variable for Salmonella occurrence in raccoon feces or soil. However,
swine farm origin was significantly associated with the presence of resistant E. coli in soil, but
not raccoon feces. I found similar Salmonella serotypes, and resistance patterns and resistance
genes in raccoon fecal E. coli and environmental samples from the same sites, suggesting that
raccoons may act as sentinels of both Salmonella and antimicrobial resistant bacteria in the
environment. A variety of Salmonella serovars that infect humans and livestock in the feces of
raccoons were detected indicating that raccoons living near humans may play a role in the
epidemiology of salmonellosis in livestock and humans in southwestern Ontario. The role of
raccoons in maintaining and disseminating antimicrobial resistant bacteria is less clear because
the prevalence of resistant isolates found in raccoon fecal and paw samples was much lower than
what has been reported in livestock and poultry in Ontario.
iv

ACKNOWLEDGEMENTS
I would like to express my sincerest gratitude to my thesis advisor, Dr. Claire Jardine, for
providing me with the opportunity to grow as a researcher within her lab and for being a
supportive mentor. I would also like to thank my committee member, Dr. David Pearl, for
providing statistical and epidemiological advice on the project. Without the vision, guidance, and
understanding of Drs. Jardine and Pearl, this thesis would not have been possible. I would also
like to thank committee members, Drs. Patrick Boerlin, Jane Parmley, and Richard-Reid Smith,
for their guidance over the last several years.
This project also would not have been possible without the hard work and dedication of
the many lab technicians, Erin Scharf-Harkness, Samantha Allen, Tami Harvey, and Barbara
Jefferson, and summer students, Samantha Kagan and Mary Thompson, who all helped with
field work and/or submitting lab samples. I would also like to thank Nicol Janecko and Gabhan
Chalmers for their expertise and attention to detail when performing lab work, to Bryan
Bloomfield for maintaining relations with landowners, and to Bill Morton and Mario Paroutis for
re-foaming radio collars. I would also like to thank Andrea Desruisseau and Chad Gill of the
Canadian Integrated Program for Antimicrobial Resistance Surveillance AMR lab, and the
technicians of the OIE Reference Laboratory for Salmonellosis, National Microbiology
Laboratory at Guelph (formerly the Laboratory for Foodborne Zoonoses), Public Health Agency
of Canada for antimicrobial susceptibility testing and Salmonella serotyoing and phagetyping,
respectively. In addition, I would like to thank the McEwen lab group at the Canadian Research
Institute for Food Safety for isolating Salmonella and E. coli.
Many thanks goes to Lenny Shirose and David Christo who provided friendship and
humor when our lab shared space with CWHC and my fellow graduate students in the Jardine
v

lab: Katie Clow, Christine James, Ariel Porty, Jamie Rothenburger, and Diana Sinclair, who
have all been supportive lab members as well. I am also thankful to the hospitality of the Grand
River Conservation Authority employees and swine farm owners who allowed us to conduct
research on their property.
My deepest appreciation goes to my family, including my parents Gail and Paul Bondo,
and my brothers, Eric and Mark Bondo, who provided love and support throughout the journey
and to my uncle, Quentin Papach, who provided invaluable assistance in helping to design and
create a database in Access. I would also like to thank Michelle Da’Costa for being an awesome
roommate throughout the years and Michael Frase for his friendship and permission to use his
wildlife artwork in my presentations. Last but not least, I would like to thank the Canadian band,
Strange Advance, for musically accompanying me on my medical journey and for helping to
bring me back to life with “We Run” and then “This Island Earth”. Drew Arnott, in particular,
not only has been my best friend for the last three years but also provided much friendship and
moral support that literally and figuratively was “World’s Away”. As Novalis said and which
was also quoted by Oliver Sacks’ in Musicophiliia, “Every disease is a musical problem; every
cure is a musical solution”.
Funding for this project was provided by the National Science and Engineering Research
Council (NSERC) and the United States Department of Agriculture (USDA). Some lab supplies
as well were provided in-kind by the Public Health Agency of Canada. K.J.B. also received
support through an Ontario Graduate Scholarship (OGS)
vi

TABLE OF CONTENTS
ACKNOWELDGEMENTS……………………………………………………………………. iv
TABLE OF CONTENTS………………………………………………………………………..vi
LIST OF TABLES……………………………………………………………………………….ix
LIST OF FIGURES…………………………………………………………………………… xiv
LIST OF ABBREVIATIONS…………………………………………………………………. xv
DECLARATION OF WORK………………………………………………………………… xvi
1. INTRODUCTION, LITERATURE REVIEW AND STUDY RATIONALE
1.0 INTRODUCTION………………………………………………………………………………………… 1
1.1 MECHANISMS OF ANTIMICROBIAL RESISTANCE……………………………………………….. 3
1.2 NON-TYPHOIDAL SALMONELLA AND COMMENSAL E. COLI……………………………………….. 7
1.3 ANTIMICROBIAL RESISTANT BACTERIA FOUND IN WILDLIFE………………………………. 15
1.4 DETECTION OF ANTIMCROBIAL RESISTANCE IN BACTERIA FROM WILDLIFE…………… 20
1.5 ROLE OF WILDLIFE IN THE EPIDEMIOLOGY OF ANTIMICROBIAL RESISTANCE………..... 35
1.6 STUDY RATIONALE………………………………………………………………………………….. 38
1.7 OBJECTIVES………………………………………………………………………………………….... 39
1.8 REFERENCES………………………………………………………………………………………....... 40
2. EPIDEMIOLOGY OF SALMONELLA ON THE PAWS AND IN THE FECES OF
FREE-RANGING RACCOONS (PROCYON LOTOR) IN SOUTHERN ONTARIO,
CANADA.
1. INTRODUCTION………………………………………………………………………………………… 66
2. MATERIALS AND METHODS…………………………………………………………………………. 68
3. RESULTS…………………………………………………………………………………………………. 71
4. DISCUSSION……………………………………………………………………………………………... 73
5. REFERENCES……………………………………………………………………………………………. 82
vii

3. IMPACT OF SEASON, DEMOGRAPHIC AND ENVIRONMENTAL FACTORS ON
SALMONELLA OCCURRENCE IN RACCOONS (PROCYON LOTOR) FROM
SWINE FARMS AND CONSERVATION AREAS IN SOUTHERN ONTARIO
1. INTRODUCTION………………………………………………………………………………………… 87
2. MATERIALS AND METHODS…………………………………………………………………………. 89
3. RESULTS………………………………………………………………………………………………… 96
4. DISCUSSION…………………………………………………………………………………………….. 99
5. REFERENCES…………………………………………………………………………………………... 118
4. EPIDEMIOLOGY OF ANTIMICROBIAL RESISTANCE IN ESCHERICHIA COLI
ISOLATED FROM RACCOONS (PROCYON LOTOR) AND THEIR ENVIRONMENT
ON SWINE FARMS AND CONSERVATION AREAS
1. INTRODUCTION……………………………………………………………………………………….. 125
2. MATERIALS AND METHODS………………………………………………………………………... 128
3. RESULTS………………………………………………………………………………………………... 134
4. DISCUSSION……………………………………………………………………………………………. 139
5. REFERENCES………………………………………………………………………………………….....166
5. GENERAL DISCUSSION AND CONCLUSIONS
1. SUMMARY…………………………………………………………………………………………….. 172
2. INFLUENCE OF LOCATION TYPE…………………………………………………………………... 172
3. EPIDEMIOLOGY………………………………………………………………………………………. 174
4. ZOONOTIC RISK………………………………………………………………………………………. 177
5. ADVANTAGES AND LIMITATIONS OF STUDY DESIGN………………………………………… 178
6. POTENTIAL IMPLICATIONS AND OUTCOMES OF RESEARCH………………………………… 180
7. FUTURE WORK…….………………………………………………………………………………….. 180

8. REFERENCES…………………………………………………………………………………………... 183

APPENDIX A
Attributes of swine farms and conservation areas used in study………………………………………… 187

APPENDIX B
Antimicrobial susceptibility breakpoints used for Salmonella and Escherichia coli: Panel type
CMV2AGNF……………………………………………………………………….................................. 188
viii

APPENDIX C
Distribution of minimum inhibitory concentrations among Salmonella isolates from all sample
types……………………………………………………………………………………………………… 190

APPENDIX D
Distribution of minimum inhibitory concentrations among E. coli isolates from all sample
types……………………………………………………………………………………………………… 194

APPENDIX E
Antimicrobial resistance patterns and genes detected in Salmonella positive raccoon fecal, soil,
and manure pit samples on five conservation areas and five swine farms in southwestern Ontario
from May to November 2011–2013……………………………………………………………………... 198

APPENDIX F
Antimicrobial resistance phenotypic patterns for Salmonella detected overall and by location type
for raccoon fecal, paw, soil, manure pit, and dumpster samples……………………………………........ 200

APPENDIX G
Number of the most common antimicrobial resistance phenotypic patterns detected for E. coli
overall and by location type for raccoon fecal, paw, soil, manure pit, and dumpster samples.…………. 201

APPENDIX H
Number of times and years individual raccoons were trapped over a three year period………………….. 203
APPENDIX I
Number of individual raccoons that were trapped two or more times in consecutive sessions over a
three year period…………………………………………………………………………………………. 204

APPENDIX J
Number of individual raccoons testing positive for Salmonella according to how many times and
number of years they were trapped ……………………………………………………………………... 205

APPENDIX K
History of individual raccoons trapped in consecutive sessions (Appendix I) that tested positive for
Salmonella two or more times in a consecutive trapping session……………………………………….. 206
ix

LIST OF TABLES
Table 2.1 ……………………………………………………………………………………….. 77
Proportion of raccoons (Procyon lotor) with Salmonella in feces and on paws
by age, sex, location type, and season categories in Ontario from May–October 2012
based on 416 paired observations
Table 2.2 ……………………………………………………………………………………….. 78
Salmonella serovars detected in paw and fecal samples of raccoons testing positive for
Salmonella in southwestern Ontario from May–October 2012
Table 2.3 ………………………………………………………………………………………... 79
Univariable multi-level logistic regression models showing associations between the
occurrence of Salmonella in raccoons with respect to sample type, raccoon age, sex,
location type, and season and multi-level logistic regression model showing significant
associations between the presence of Salmonella on raccoon paws and feces with respect
to raccoon sex, sample type, season, and interaction effects
Table 2.4 ……………………………………………………………………………………….. 81
Contrasts derived from the logistic regression model (Table 2.3) for the presence of
Salmonella in raccoons to determine the differences among sex, season, and sample type
Table 3.1 ………………………………………………………………………………………. 106
Percentage (95% CI) of raccoon fecal, soil, manure pit, and dumpster samples testing
positive for Salmonella overall and by age, sex, location type, season, and year in Ontario
from May–November 2011–2013
x

Table 3.2 ………………………………………………………………………………………. 108
Results from univariable multi-level and exact logistic regression models showing
associations between the occurrence of Salmonella in raccoon fecal, soil, and manure
pit samples with respect to raccoon age (adult or juvenile) and sex (male or female)
where applicable, location type (swine farm or conservation area), year (2011–2013)
and season (May–July or August–November) in Ontario, Canada
Table 3.3 ………………………………………………………………………………………. 110
Results from multi-level logistic regression models showing associations between
the occurrence of Salmonella in raccoon fecal and soil samples with respect to raccoon
sex (male/female) where applicable, year (2011–2013), season (May–July or August–
November), rainfall and interaction effects in Ontario, Canada
Table 3.4 ………………………………………………………………………………………. 111
Contrasts derived from the multi-level logistic regression model for the presence of
Salmonella in raccoon feces (Table 3.3) from Ontario, Canada to interpret interaction
effects between raccoon sex (male or female) and season (May–July or August–
November)
Table 3.5 ………………………………………………………………………………………. 112
Percentage (95% CI) of raccoon fecal, soil, manure pit, and dumpster samples testing
positive for Salmonella that were detected and serotyped for each serovar overall and
according to location type (conservation area or swine farm) in southwestern Ontario
from May–November 2011–2013