Genetic diversity and population structure of the intestinal spirochaete Brachyspira pilosicoli Neo Zhi Yu Eugene Bachelor of Science This thesis is presented for the degree of Doctor of Philosophy of Murdoch University 2012 School of Veterinary and Biomedical Science, Murdoch University, Murdoch, Western Australia, 6150 Table of Contents Table of Contents Abstract ....................................................................................................................................................... iv Acknowledgements ............................................................................................................................ vii Declaration ............................................................................................................................................. viii CHAPTER 1: LITERATURE REVIEW ................................................................. 1 1.1 GENERAL INTRODUCTION .......................................................................................................... 1 1.1.1 Preface ......................................................................................................................................... 1 1.2 GENERAL DESCRIPTION OF INTESTINAL SPIROCHAETES ......... 2 1.2.1 Introduction to the spirochaetes ................................................................................... 2 1.2.2 Current taxonomy of the family Spirochaetaceae ................................................ 4 1.2.2.1 Borrelia .............................................................................................................................. 4 1.2.2.2 Brevinema......................................................................................................................... 4 1.2.2.3 Cristispira ......................................................................................................................... 5 1.2.2.4 Spirochaeta ...................................................................................................................... 5 1.2.2.5 Spironema ......................................................................................................................... 6 1.2.2.6 Treponema ....................................................................................................................... 6 1.2.3 Family Leptospiraceae ......................................................................................................... 6 1.2.3.1 Leptonema ........................................................................................................................ 7 1.2.3.2 Leptospira ......................................................................................................................... 7 1.2.3.3 Turneriella ....................................................................................................................... 7 1.2.4 Family Brachyspiraceae ...................................................................................................... 8 1.2.4.1 Brachyspira ..................................................................................................................... 8 1.2.4.2 Brachyspira hyodysenteriae ................................................................................... 8 1.2.4.3 Brachyspira intermedia ............................................................................................ 9 1.2.4.4 Brachyspira innocens ................................................................................................. 9 1.2.4.5 Brachyspira alvinipulli ............................................................................................10 1.2.4.6 Brachyspira murdochii ............................................................................................10 1.2.4.7 Brachyspira aalborgi ................................................................................................12 1.2.4.8 Other unofficial species ..........................................................................................12 1.2.4.9 Brachyspira pilosicoli ...............................................................................................12 1.2.5 B. pilosicoli infection and pathogenesis- in humans and animals ............. 14 1.2.5.1 Introduction ..................................................................................................................14 1.2.5.2 Intestinal spirochaetosis in animals ...............................................................15 1.2.5.3 Human intestinal spirochaetosis (HIS) ..........................................................16 i Table of Contents 1.2.6 Genome sequences of Brachyspira species ............................................................1 7 1.2.7 Genetic variation in B. pilosicoli ..................................................................................17 1.3 METHODS USED FOR STRAIN TYPING AND ASSESSING POPULATION STRUCTURES OF BRACHYSPIRA SPECIES ........... 20 1.3.1 Introduction ............................................................................................................................20 1.3.2 DNA restriction enzyme analysis (REA) ..................................................................21 1.3.3 Restriction Fragment Length Polymorphism (RFLP) Analysis ...................22 1.3.4 Randomly amplified polymorphic DNA assay (RAPD) ....................................24 1.3.5 Pulsed-Field Gel Electrophoresis (PFGE) ................................................................25 1.3.6 Multilocus Enzyme Electrophoresis (MLEE) .........................................................27 1.3.7 Multiple-Locus Variable number tandem repeat Analysis (MLVA) ......... 28 1.3.8 Multilocus Sequence Typing (MLST) .........................................................................29 1.3.8.1 Use of MLST for analyzing Brachyspira species ........................................32 1.3.8.2 Use of MLST for Brachyspira pilosicoli ...........................................................33 1.4 BETA-LACTAMASES ................................................................................ 33 1.5 AIMS AND OBJECTIVES OF THIS STUDY .......................................... 37 CHAPTER 2: MULTILOCUS SEQUENCE TYPING (MLST) OF BRACHYSPIRA PILOSICOLI ................................................................................ 38 2.1 INTRODUCTION ..............................................................................................................................38 2.2 MATERIALS AND METHODS ....................................................................................................38 2.2.1 Brachyspira pilosicoli strains ..................................................................... 38 2.2.2 Spirochaete culture .................................................................................... 39 2.2.3 DNA extraction ........................................................................................... 40 2.2.4 Multilous sequence typing primers ......................................................... 40 2.2.5 Polymerase Chain Reaction (PCR) .............................................................. 41 2.2.6 Electrophoresis ........................................................................................... 42 2.2.7 Purification of PCR products ...................................................................... 43 2.2.8 Sequencing of PCR products ...................................................................... 43 2.2.9 Analysis ....................................................................................................... 44 2.3 RESULTS .............................................................................................................................................49 2.4 DISCUSSION ......................................................................................................................................59 2.5 CONCLUSION ....................................................................................................................................65 CHAPTER 3: MULTIPLE LOCUS VARIABLE NUMBER TANDEM REPEAT ANALYSIS (MLVA) OF BRACHYSPIRA PILOSICOLI ................... 66 ii Table of Contents 3.1 INTRODUCTION ..............................................................................................................................66 3.2 MATERIALS AND METHODS ....................................................................................................67 3.2.1 Detection and selection of VNTR loci ........................................................ 67 3.2.2 Selection of primers for MLVA ................................................................... 67 3.2.3 Detection of VNTRs in B. pilosicoli ............................................................. 69 3.3 RESULTS .............................................................................................................................................71 3.4 DISCUSSION ......................................................................................................................................75 3.5 CONCLUSION ....................................................................................................................................78 CHAPTER 4: DISTRIBUTION AND PHYLOGENETIC ANALYSIS OF BLAOXA-63, A GENE ENCODING A GROUP IV CLASS D BETA- LACTAMASE IN BRACHYSPIRA PILOSICOLI STRAINS ............................. 79 4.1 INTRODUCTION ..............................................................................................................................79