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The Relationship Between Streptococcus Suis and Haemophilus Parasuis, Two Important Residents of the Tonsils of the Soft Palate in Swine

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The Relationship Between Streptococcus Suis and Haemophilus Parasuis, Two Important Residents of the Tonsils of the Soft Palate in Swine The relationship between Streptococcus suis and Haemophilus parasuis, two important residents of the tonsils of the soft palate in swine by Allison M. E. Barre A Thesis presented to The University of Guelph In partial fulfillment of requirements for the degree of Master of Science In Pathiobiology Guelph, Ontario, Canada © Allison Barre, August, 2015 ABSTRACT The relationship between Streptococcus suis and Haemophilus parasuis, two important residents of the tonsils of the soft palate in swine Allison Barre Advisor: University of Guelph, 2015 Dr. J.I. MacInnes Haemophilus parasuis and Streptococcus suis are residents of tonsils of the soft palate in healthy pigs but can also cause severe disease. Planktonic growth curves indicated that S. suis growth was enhanced in the presence of H. parasuis, while H. parasuis was at a disadvantage. It was also found that H. parasuis and S. suis biofilm biomass was decreased in co-culture. These effects were demonstrated to be partly attributable to secreted factors. Finally, H. parasuis protected S. suis from antibiotics in co- culture biofilms, and S. suis protected H. parasuis against swine complement proteins. The results here suggest H. parasuis and S. suis may increase each others’ virulence by preventing entrance into a quiescent biofilm form, as well as providing synergistic protection against antimicrobials and complement. A further understanding of the interplay between S. suis and H. parasuis could lead to the development of new approaches to reduce swine respiratory disease. Acknowledgements The journey to completion of this Master of Science program has been full of challenging experiences and fulfilling successes. There are many people who contributed during the last two years, and I would like to thank them all for their support. First, I would like to thank my advisor, Dr. Janet MacInnes, who gave my research a strong direction, while helping me decide which questions to ask. She also gave me the chance to think and grow independently and always encouraged me to use my voice. Next, thanks to the members of my committee, Dr. Carlton Gyles and Dr. Terri O’Sullivan, both of whom were full of suggestions and valuable feedback. As well, thank you to Dr. John Prescott who has graciously agreed to be a part of my examination committee. In the lab, I need to give special thanks to Elizabeth Kunkel, an undergraduate summer student who helped me finish up last minute experiments throughout this final semester. Also, thank you to the rest of my lab members, Glenn Soltes, Adina Bujold, Shivani Ojha, and Vivian Nicholson, all of whom gave me critical feedback and helped to get my experiments started. I also would like to thank both NSERC and the OVC for providing me scholarships to support my work throughout this program. Thank you especially to Derrick Yeung, who has been the major source of encouragement for me throughout the last two years. Without his immense patience and understanding (even amidst the efforts of his own thesis), I would not have managed to stay nearly as sane. You have been amazing. Last, but not least, I want to thank both my parents for their love, their support, their genes, and their microbiomes, all of which were integral to me reaching this point. iii Declaration of Work Done I declare that this thesis ‘The relationship between Streptococcus suis and Haemophilus parasuis, two important residents of the tonsils of the soft palate tonsil in swine’ is entirely my own work and that where any material could be construed as the work of others, it is fully cited and referenced, and/or with appropriate acknowledgement given. All experiments were designed and completed solely by me with the exception of some work found in Chapter 6, which was completed with the assistance of Elizabeth Kunkel. Signature: Name of student: Allison Barre Name of supervisor: Dr. Janet MacInnes iv Table of Contents Acknowledgements ...................................................................................................................... iii Declaration of Work Done .......................................................................................................... iv List of Tables .............................................................................................................................. viii List of Figures ............................................................................................................................... ix Chapter 1 – Review of the literature ............................................................................................1 1.1 The swine tonsil environment ..........................................................................................1 1.1.1 Swine tonsillar microbiota .................................................................................................... 2 1.2 Polymicrobial disease .......................................................................................................4 1.2.1 The ecology of microbial communities ................................................................................ 6 1.2.1.1 Antagonistic relationships ......................................................................................... 7 1.2.1.2 Synergistic relationships ............................................................................................ 9 1.2.2 Biofilms .............................................................................................................................. 13 1.2.3 New approaches to the treatment of polymicrobial infections ........................................... 14 1.3 Polymicrobial interactions in the respiratory tract .....................................................16 1.4 Swine pathogens of interest ............................................................................................19 1.4.1 Haemophilus parasuis ........................................................................................................ 19 1.4.2 Streptococcus suis .............................................................................................................. 23 1.4.3 Epidemiological relationship between H. parasuis and S. suis .......................................... 28 1.5 References ........................................................................................................................29 Chapter 2 – Purpose and objectives ...........................................................................................45 Chapter 3 – Comparison of mono- and co-culture growth of H. parasuis and S. suis ..........46 3.1 Introduction .....................................................................................................................46 3.2 Methods ...........................................................................................................................47 3.2.1 Bacterial strains and growth conditions. ............................................................................. 47 3.2.2 Optimization of H. parasuis growth conditions ................................................................. 48 3.2.3 Growth interactions on a solid agar surface ....................................................................... 48 3.2.4 Bactericidal assay ............................................................................................................... 49 3.2.5 Evaluation of hemolysis on blood agar .............................................................................. 49 3.2.6 Mono- and co-culture growth curves .................................................................................. 50 3.2.7 Bioinformatic analysis of the biochemical profiles of H. parasuis and S. suis .................. 50 3.2.8 Statistical analysis .............................................................................................................. 51 3.3 Results ..............................................................................................................................51 3.3.1 Growth interactions on a solid agar surface ....................................................................... 51 3.3.2 Evidence of bactericidal effects on solid agar surface ....................................................... 52 3.3.3 Changes in blood agar hemolysis as a result of co-culture ................................................. 52 3.3.4 Optimization of H. parasuis growth conditions ................................................................. 52 3.3.5 Comparison of mono- and co-culture growth curves ......................................................... 54 3.3.6 Bioinformatic analysis of the biochemical profiles of H. parasuis and S. suis .................. 54 3.4 Discussion ........................................................................................................................55 3.5 References ........................................................................................................................60 v Chapter 4 – Characteristics of co-culture biofilms of H. parasuis and S. suis .......................71 4.1 Introduction .....................................................................................................................71 4.2 Methods ...........................................................................................................................73 4.2.1 Cultivation of mono- and co-culture biofilms .................................................................... 73 4.2.2 Crystal violet assay ............................................................................................................
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