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Understanding the Relationship Between the Tonsil Microbiota and Clinical Streptococcus Suis Infection in Nursery Pigs

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Understanding the Relationship Between the Tonsil Microbiota and Clinical Streptococcus Suis Infection in Nursery Pigs Understanding the Relationship Between the Tonsil Microbiota and Clinical Streptococcus suis Infection in Nursery Pigs by Sarah Victoria Hill A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Pathobiology Guelph, Ontario, Canada © Sarah Hill, September 2019 ABSTRACT UNDERSTANDING THE RELATIONSHIP BETWEEN THE TONSIL MICROBIOTA AND CLINICAL STREPTOCOCCUS SUIS INFECTION IN NURSERY PIGS Sarah Hill Advisor(s): University of Guelph, 2019 Dr. Nicole Ricker Dr. Vahab Farzan The objective of this study was to determine if the tonsillar microbiota is different in nursery pigs with Streptococcus suis disease compared with that of healthy pigs. Fifty- six pigs from 9 farms were classified as confirmed cases, probable cases and healthy controls. Illumina MiSeq-sequencing of the 16S V3-V4 hypervariable region was done to assess the microbiota composition and the mothur pipeline was used for clustering and taxonomical assignment. Overall, 453, 600 and 334 different taxa were identified in confirmed cases, probable cases and controls, respectively. The beta diversity differed significantly between the farms (p=0.035). Alpha diversity differed between probable cases and control (p<0.001) and tended to be different between confirmed and probable cases (p=0.088). The inverse Simpson tended to be different between confirmed cases and controls (p=0.083). Phylogenetic analysis demonstrated no differences between the microbiota of nursery pigs with clinical S. suis infection and that of heathy animals. iii ACKNOWLEDGEMENTS First and foremost, I’d like to thank my entire advisory committee: Dr. Vahab Farzan, Dr. Nicole Ricker, Dr. Janet MacInnes and Dr. Emma Allen-Vercoe for all their guidance and support throughout my master’s. I would like to thank Cole Siegel and his entire family for their unconditional support. Cole, all the late nights spent editing my papers and the chores you did around the house were so helpful and I cannot thank you enough. Mum, thank you for all your love and unconditional support; the daily phone calls kept me sane. Pregent Family, thank you for always hosting family gatherings, its always nice to see you all and it’s a nice break from school! The Hill family, thank you for everything you have done for me. Uncle Dale and Aunt Sonja, I would not be here today without your support. Laura and Michelle, you were the sisters I never had; hanging out with you both, whether it was in Ottawa or Toronto would always cheer me up. Oma, thank you for always treating me like a grand- daughter. I would also like to thank Oma for my first car, it was a saving grace for commuting back and forth during my master’s. Dad and Amanda, thank you for all the effort you’ve put in for me to have a relationship with my baby brothers Logan and Atlas. I hope one day when they are older, they too follow their passions and enjoy school. Aunt Andi and Uncle Dave, thank you for all your Facebook message checkups. It was really nice knowing I had family who care so much about my well-being. I would like to thank the entire pig group for being so welcoming, the coffee and the Friday afternoon visits. Dr. Bob Friendship, thank you for always having your door open; from the assistance in pig knowledge to the casual chats, I really appreciate it all. Linda Kraemer, thank you for giving me a desk in the pig palace- it was super convenient being close to my friends and the pig family. Outbreaks softball team, thank you having me on your team- the weekly Tuesday games were so much fun and a good break from school work! I would also like to thank the Canadian First Research Excellence Fund for financial support and the Ontario pork producers who very generously granted us access to their facilities and for their patient assistance during the sampling processes. Finally, King George and Eli - thank you for always being the best fur friends one could have. iii iv DECLARATION OF WORK All the work described was performed by me with the exception of: 1. Leann Denich, Jeremy Wong, Chris McLaren-Almond, Maria Amezcua, Kaushalya Kuruppu, Eric Perrin, Karen De Bruyn, assisted in farm visits 2. Leann Denich and Emily Arndt performed the culture-dependent study and identified S. suis isolates 3. Jordan Buchan and Alison Jeffery assisted in tonsil preparation for DNA extraction 4. Jeff Gross from Advanced Analytical Centre at the University of Guelph performed Qubits for most of the samples and performed Illumina MiSeq on all samples iv v TABLE OF CONTENTS Abstract ............................................................................................................................ ii Acknowledgements ......................................................................................................... iii Declaration of Work ......................................................................................................... iv Table of Contents ............................................................................................................. v List of Tables .................................................................................................................. viii List of Figures .................................................................................................................. ix List of Abbreviations ......................................................................................................... x List of Appendices ........................................................................................................... xi 1 Literature Review ...................................................................................................... 1 1.1 Introduction ........................................................................................................ 1 1.1.1 Streptococcus suis ...................................................................................... 1 1.1.2 Clinical diagnosis ........................................................................................ 1 1.1.3 Epidemiology .............................................................................................. 2 1.1.4 Virulence ..................................................................................................... 3 1.1.5 Treatment and management ...................................................................... 4 1.2 Tonsils, a natural habitat of S. suis .................................................................... 5 1.2.1 Anatomy and the role of tonsils of the soft palate ....................................... 5 1.3 Tonsil microbiome .............................................................................................. 6 1.3.1 Bacterial communities in the tonsils of the soft palate ................................ 6 1.3.2 Presence of bacteria and viruses associated with swine diseases ............ 9 1.4 Roles within bacterial communities .................................................................. 10 1.4.1 Microbial interactions ................................................................................ 10 v vi 1.4.2 Polymicrobial infections ............................................................................ 13 1.5 Methods for identifying microbial communities ................................................ 14 1.5.1 DNA extraction kits ................................................................................... 14 1.5.2 Quantification of DNA ............................................................................... 15 1.5.3 16S rRNA gene sequences ...................................................................... 15 1.5.4 DNA sequencing ....................................................................................... 17 1.5.5 Analysis of microbiota data ....................................................................... 17 1.6 Conclusion ....................................................................................................... 18 1.7 Figures ............................................................................................................. 19 1.8 Tables .............................................................................................................. 20 2 A descriptive analysis of the microbiota of the tonsil of the soft palate in nursery pigs in Ontario farms ...................................................................................................... 21 2.1 Introduction ...................................................................................................... 21 2.2 Methods and materials ..................................................................................... 23 2.2.1 Farm and Pig Selection ............................................................................ 23 2.2.2 Sample collection ...................................................................................... 23 2.2.3 S. suis isolation and identification ............................................................. 24 2.2.4 DNA extraction from tonsil tissues ............................................................ 25 2.2.5 16rRNA sequencing .................................................................................. 25 2.2.6 Data analysis ............................................................................................ 26 2.2.7 Statistical analysis ..................................................................................... 27 2.3 Results and discussion ...................................................................................
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