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For more information, please contact the WRAP Team at: [email protected] warwick.ac.uk/lib-publications Persistence of Dichelobacter nodosus, the causal agent of ovine footrot By Katharina Giebel A THESIS SUBMITTED TO THE UNIVERSITY OF WARWICK FOR THE DEGREE OF DOCTOR OF PHILOSOPHY School of Life Sciences University of Warwick June 2017 Table of contents Acknowledgements i Declaration ii Summary iii List of figures iv List of tables viii Abbreviations xii CHAPTER 1 General Introduction 1 1.1 Ovine footrot and its impact on economy and health and welfare of sheep 1 1.2 Global footrot prevalence and prevalence of lameness in the United Kingdom 1 1.3 Disease expression and epidemiology 3 1.4 Treatment of footrot 4 1.5 Characterization of Dichelobacter nodosus 5 1.5.1 Main virulence factors of Dichelobacter nodosus 6 1.5.2 Benign and virulent strains of Dichelobacter nodosus in sheep populations 8 1.5.3 Antigenic diversity of Dichelobacter nodosus 8 1.6 The role of climate and environment in footrot initiation, severity and elimination 10 1.6.1 The environment in disease initiation and transmission of footrot 10 1.6.2 The role of the environment in control and elimination of footrot 11 1.7 Persistence of Dichelobacter nodosus on sheep and in the farm environment 12 1.7.1 Detection of Dichelobacter nodosus on feet, in the oral cavity and in faecal samples 13 1.7.2 Distribution of Dichelobacter nodosus in the farm environment 14 1.8 Methods for the detection, quantification and characterization of Dichelobacter nodosus 15 1.8.1 Molecular epidemiology 15 1.8.2 Culture dependent methods 15 1.8.3 Culture independent methods 16 1.8.4 Dichelobacter nodosus 16s rRNA gene sequence analysis 16 1.8.5 Real-time quantitative PCR (qPCR) 17 1.8.6 Multiple Loci Variable Number Tandem Repeat (VNTR) Analysis (MLVA) 17 1.11.6.1 Assay technology 18 1.11.6.2 Previous uses of MLVA for the typing of bacteria and in farm animal disease research 19 1.11.6.3 The Dichelobacter nodosus MLVA assay 21 1.9 Aims, objectives and hypotheses 23 1.10 Thesis structure 24 CHAPTER 2 Materials, methods and laboratory tool development 25 2.1 Bacterial strains and control DNA samples used throughout the project 26 2.2 Culture media and bacterial growth conditions 27 2.2.1 Isolation of Dichelobacter nodosus from field samples (swabs) on Hoof-Horn Agar (HA) 28 2.2.2 Culturing of Dichelobacter nodosus isolates on Eugon Agar 29 2.2.3 Identification of pure isolates by examination of colony morphology and colony lysis PCR 29 2.3 DNA extractions 29 2.3.1 DNA extractions from swabs, soil, grass, and faecal samples 29 2.3.2 DNA extractions from pure cultured isolates (DNeasy®Blood & Tissue Kit) 30 2.3.3 DNA Extractions from plasmid DNA (QIAprep®Miniprep plasmid extraction kit) 31 2.4 Purification of PCR products 31 2.4.1 QIAquick® PCR purification kit 31 2.4.2 QIAquick® Nucleotide removal kit 31 2.5 Sanger sequencing of PCR amplicons 31 2.6 Quantification of DNA 32 2.7 Gel electrophoresis for visualization of PCR amplicons 32 2.8 Cloning 32 2.9 A PCR for quantifying Dichelobacter nodosus: Amplification of the Dichelobacter nodosus rpoD gene 33 2.9.1 TaqMan® Probe chemistry 33 2.9.2 Dichelobacter nodosus quantitative PCR primer and probes 33 2.9.3 Dichelobacter nodosus quantitative PCR cycling parameters 34 2.9.4 Dichelobacter nodosus quantitative PCR plasmid standard curves and detection limit 34 2.9.5 Spiking of swabs, soil and faeces 35 2.9.6 Cloning of the Dichelobacter nodosus rpoD amplicon for sequencing 37 2.10 Generic bacterial 16S rRNA gene PCR for the detection of Dichelobacter nodosus 37 2.10.1 Non-specificity of Dichelobacter nodosus 16S rRNA gene detection primers 37 2.10.2 Development of Dichelobacter nodosus specific 16S rRNA gene primers 40 2.10.3 Specificity of the developed 16S rRNA gene primers 41 2.10.4 Dichelobacter nodosus specific final 16S rRNA gene PCR protocol 42 2.10.5 Nested PCR: Modification of the Universal 16S rRNA gene primers 43 2.10.6 Testing of the developed Dichelobacter nodosus specific 16S rRNA gene primers 43 2.11 Multiple Loci Variable Number Tandem Repeat (VNTR) Analysis (MLVA) 44 2.11.1 Assay optimizations and cycling conditions 44 2.11.2 Fragments analysis and data analysis 45 2.12 Presence of Dichelobacter nodosus in areas where sheep are historically absent 46 CHAPTER 3 Detection and quantification of Dichelobacter nodosus on sheep and in environmental samples: Evidence from two field studies 47 3.1 Introduction 47 3.2 Ethical approval 47 3.3 Materials and methods 48 3.3.1 Farms and animals 48 3.3.2 Animal and environmental sampling procedures (Studies 1 and 2) 49 3.3.2.1 Procedures for collection of samples from sheep 50 3.3.2.2 Sampling procedures unique to study 1 53 3.3.2.3 Sampling procedures unique to study 2 53 3.3.2.4 Sampling procedures for collection of soil and grass samples (Studies 1 and 2) 54 3.3.2.5 Collection of climate data (Studies 1 and 2) 55 3.3.3 Analysis of samples in the laboratory (Studies 1 and 2) 56 3.3.3.1 DNA extractions 56 3.3.3.2 Quantification of Dichlelobacter nodosus using real-time PCR (Studies 1 and 2) 56 3.3.3.3 Determination of soil moisture content (Study 2) 57 3.3.4 Data analysis 57 3.3.4.1 Differences in Dichelobacter nodosus load and detection frequencies over time 57 3.3.4.2 Kaplan Meier survival curve (Study 1) 57 3.3.4.3 Correlations and associations between variables (Studies 1 and 2) 58 3.3.4.4 Binomial mixed effects regression model (Studies 1 and 2) 58 3.4 Study 1: Results 60 3.4.1 Disease status of the study group 60 3.4.2 Climate during study 1 63 3.4.3 Detection and quantification of Dichelobacter nodosus in all sample types 64 3.4.4 Kaplan-Meier survival curve 69 3.4.5 Binomial mixed effects logistic regression model 71 3.4.6 Correlations and associations of predictor variables 76 3.5 Study 2: Results 79 3.5.1 Disease status of the study group and animal selection for analysis 79 3.5.2 Climate during study 2 82 3.5.3 Detection and quantification of Dichelobacter nodosus 84 3.5.3.1 Dichelobacter nodosus detection and quantification on feet, in the gingival cavity and in faeces 84 3.5.3.2 Detection and quantification of Dichelobacter nodosus in soil and grass samples 85 3.5.4 Dichelobacter nodosus detection on lesion-free feet 86 3.5.5 Dichelobacter nodosus loads on feet from week 1-3 88 3.5.6 The effect of climate on disease scores and Dichelobacter nodosus detection 88 3.5.7 Binomial mixed effects logistic regression model 90 3.5.8 Correlations and associations of predictor variables 96 3.6 Discussion 98 CHAPTER 4 Optimization and validation of a multiple locus variable number tandem repeat analysis for differentiation of Dichelobacter nodosus strains from mixed DNA samples 102 4.1 Introduction 102 4.2 Materials and methods 105 4.2.1 Assay optimizations and cycling conditions 105 4.2.2 Amplification of Dichelobacter nodosus from swabs, faecal and environmental samples 105 4.2.3 Fragment analysis 106 4.2.4 Determination of repeat sizes 106 4.2.5 MLVA specificity for Dichelobacter nodosus 107 4.2.6 MLVA sensitivity to Dichelobacter nodosus load 107 4.2.7 MLVA profiles of Dichelobacter nodosus isolates and determination of the fragment analysis threshold level 108 4.2.8 Testing recovery of Dichelobacter nodosus communities through the creation of model communities 108 4.2.9 Assessment of the MLVA assay on mixed DNA samples in two contrasting field studies (studies 1 and 2) 109 4.2.9.1 Farms, animals and sample collection 109 4.2.9.2 Laboratory analysis 109 4.2.9.3 Data analysis 110 4.3 Results 111 4.3.1 Dichelobacter nodosus MLVA PCR specificity 111 4.3.2 Determination of repeat sizes for the targeted MLVA loci 114 4.3.3 Sensitivity of the MLVA assay 115 4.3.4 Determination of minimum peak size in fragment analysis using the Dichelobacter nodosus MLVA assay 116 4.3.5 Dichelobacter nodosus model communities to test the recovery of all VNTR amplicons after fragment analysis 118 4.3.6 Detection of the Dichelobacter nodosus VNTR loci in mixed DNA swabs and environmental samples 120 4.3.6.1 Study 1 120 4.3.6.2 Study 2 120 4.3.7. MLVA profile of isolates and mixed DNA samples 121 4.3.7.1 Study 1 121 4.3.7.2 Study 2 126 4.4 Discussion 129 CHAPTER 5 Persistence of Dichelobacter nodosus during periods of non-transmission in Southern Spain 133 5.1 Introduction 135 5.2 Materials and Methods 135 5.2.1 Research collaboration and ethical approval 135 5.2.2 Farms and Animals 135 5.2.3 Sampling procedure: Sheep 135 5.2.4 Sampling procedure: Environment 136 5.2.4 Sample storage and shipping 137 5.2.6 Collection of climate data 137 5.2.7 Laboratory analysis 137 5.3 Results 138 5.3.1 Climate in Córdoba from November 2015 to April 2016 138 5.3.2 Climate in Córdoba from May 2015 to July 2015 138 5.3.3 Farm 1 138 5.3.3.1 Disease status of the flock on Farm 1 in April 138 5.3.3.2 Disease status of the flock on Farm 1 in July 139 5.3.3.3 Dichelobacter nodosus bacterial loads and communities on sheep and in the farm environment in April and July 139 5.3.4 Farm 2 141 5.3.4.1 Disease status of farm 2 in April 141 5.3.4.2 Dichelobacter nodosus bacterial loads and communities on sheep and in the farm environment 142 5.4 Discussion 143 CHAPTER 6 General discussion, conclusions and future research 146 6.1 Key findings 146 6.2 Discussion of key findings 146 6.3 Limitations 151 6.4 Conclusions 151 6.5 Future work 152 References 153 Appendices 172 Acknowledgements There are many people to thank for their help, support and guidance during the last three and a half years.