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Characterization of the Bacterial Communities of the Tonsil of the Soft Palate of Swine

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Characterization of the Bacterial Communities of the Tonsil of the Soft Palate of Swine by Shaun Kernaghan 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 © Shaun Kernaghan, December, 2013 ABSTRACT CHARACTERIZATION OF THE BACTERIAL COMMUNITIES OF THE TONSIL OF THE SOFT PALATE OF SWINE Shaun Kernaghan Advisor: University of Guelph, 2013 Professor Janet I. MacInnes Terminal restriction fragment length polymorphism (T-RFLP) analysis and pyrosequencing were used to characterize the microbiota of the tonsil of the soft palate of 126 unfit and 18 healthy pigs. The T-RFLP analysis method was first optimized for the study of the pig tonsil microbiota and the data compared with culture-based identification of common pig pathogens. Putative identifications of the members of the microbiota revealed that the phyla Firmicutes, Proteobacteria and Bacteroidetes were the most prevalent. A comparison of the T-RFLP analysis results grouped into clusters to clinical conditions revealed paleness, abscess, PRRS virus, and Mycoplasma hyopneumoniae to be significantly associated with cluster membership. T-RFLP analysis was also used to select representative tonsil samples for pyrosequencing. These studies confirmed Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria, and Proteobacteria to be the core phyla of the microbiota of the tonsil of the soft palate of pigs. Acknowledgements I would like to thank my advisor Janet MacInnes for her support and endless patience during this project. I would like to thank my committee, Patrick Boerlin and Emma Allen-Vercoe, for their insights and support, as well as Zvoninir Poljak for his help through this project. I would also like to thank the members of the MacInnes lab, Allen-Vercoe lab, AHL diagnostic lab, and Laboratory Services for their help. Finally, a big thank you to my family for their patience and support throughout my studies. iii Declaration of Work Done All the work described in this thesis was performed by me with the exception of: 1. Julie McDonald aided in DNA extraction comparison by providing two commercial kits and explaining the use of the bead-beater. 2. Dr. Zvonimir Poljak wrote the STATA code for statistical comparisons. 3. Charlotte Swanson performed DNA extractions of several tonsil cultures. 4. Pyrosequencing was performed by Marcio Costa in the laboratory of Dr. Scott Weese. iv Table of Contents Acknowledgements .............................................................................................................. iii Declaration of Work Done .................................................................................................. iv Table of Contents .................................................................................................................. v List of Tables ..................................................................................................................... viii List of Figures .................................................................................................................... viii List of Abbreviations ........................................................................................................... xi Chapter 1 Review of the Literature ....................................................................................................... 1 1.1. Methods for Profiling Bacterial Communities .............................................................. 1 1.1.1. Study of Microbial Communities using 16S rRNA Genes ........................................... 1 1.1.2. Clone Library Analysis .............................................................................................. 2 1.1.3. Biases Associated with Clone and PCR-Based Studies ............................................... 3 1.1.4. Denaturing and Temperature Gradient Gel Electrophoresis ...................................... 6 1.1.5. Terminal Restriction Fragment Length Polymorphism Analysis ................................. 8 1.1.6. Diversity Microarrays ............................................................................................. 11 1.1.7. 16S rRNA Gene Pyrosequencing.............................................................................. 13 1.2. Studying Bacterial Communities................................................................................. 16 1.2.1. Studying Bacterial Communities in Macro-Organisms............................................. 16 1.2.2. Tonsil of the Soft Palate of Pigs ............................................................................... 18 1.2.3. Bacteria associated with Pig Tonsils ....................................................................... 19 1.3. References .................................................................................................................... 22 Chapter 2 Objectives ............................................................................................................................ 39 Chapter 3 Optimization of the terminal restriction fragment length polymorphism analysis (T- RFLP) method for the characterization of bacterial communities at the tonsil of the soft palate of swine ..................................................................................................................... 41 v 3.1. Introduction ................................................................................................................. 41 3.2. Materials & methods ................................................................................................... 43 3.2.1. Sample collection and processing ............................................................................ 43 3.2.2. Comparison of DNA extraction kits ......................................................................... 44 3.2.3. Checking lysis of ‘difficult-to-lyse’ bacteria ............................................................. 45 3.2.4. Tonsil tissue homogenization ................................................................................... 45 3.2.5. T-RFLP analysis PCR amplification and digestion .................................................. 46 3.2.6. Capillary electrophoresis ........................................................................................ 47 3.2.7. Comparison of tissue and culture samples ............................................................... 48 3.2.8. Mock community analysis ........................................................................................ 48 3.2.9. Filtering and binning of results................................................................................ 49 3.2.10. Creation of database for bacterial pathogen identification .................................... 49 3.2.11. Diagnostic Comparison ......................................................................................... 50 3.3. Results .......................................................................................................................... 50 3.3.1. DNA extraction kit comparison ................................................................................ 50 3.3.2. Evaluation of the Qiagen-bb protocol ...................................................................... 51 3.3.3. Tissue homogenization protocol comparison ........................................................... 51 3.3.4. PCR optimization .................................................................................................... 51 3.3.5. Mock community analysis ........................................................................................ 52 3.3.6. Comparison of T-RFLP analysis and culture identifications .................................... 52 3.4. Discussion ..................................................................................................................... 53 3.5. References .................................................................................................................... 62 Chapter 4 T-RFLP analysis of the bacterial communities associated with pig tonsils ...................... 76 4.1. Introduction ................................................................................................................. 76 4.2. Material & methods ..................................................................................................... 77 4.2.1. Sample collection and T-RFLP analysis................................................................... 77 4.2.2. Non-metric multidimensional scaling (NMS) analysis and beta diversity ................. 78 4.2.3. Putative identification of T-RFLP analysis fragments .............................................. 79 4.2.4. Cluster analysis and comparison with clinical information ...................................... 80 vi 4.3. Results & Discussion .................................................................................................... 80 4.3.1. Comparison of OTUs observed in both healthy and unfit pigs .................................. 80 4.3.2. The ability of T-RFLP analysis to match fragments to putative identifications ......... 83 4.3.3. Putative identifications of the bacterial communities from healthy and unfit pigs ..... 84 4.3.4. Comparison of bacterial community data to clinical information collected from unfit pigs ................................................................................................................................... 88 4.4. Conclusion ...................................................................................................................
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