Identifying infection reservoirs of digital dermatitis in dairy cattle Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy By Jennifer Bell September 2017 1 Table of Contents Abstract .................................................................................................................................... 7 Acknowledgements .................................................................................................................. 9 List of knowledge exchange contributions relating to this thesis ......................................... 10 Abbreviations ......................................................................................................................... 11 Chapter 1: Introduction ......................................................................................................... 14 1.1 Bovine digital dermatitis – overview ........................................................................... 14 1.2.1 Anatomical location of DD lesions ........................................................................ 14 1.2.2 Clinical appearance and histopathology of DD lesions ......................................... 15 1.2.3 DD lesion classification.......................................................................................... 17 1.3 Epidemiology of DD...................................................................................................... 18 1.3.1 Geographic spread of DD ...................................................................................... 18 1.3.2 Prevalence ............................................................................................................. 18 1.3.3 Seasonality ............................................................................................................ 19 1.4 The implications of DD ................................................................................................. 19 1.4.1 Welfare of affected cattle ..................................................................................... 19 1.4.2 Economic impact ................................................................................................... 20 1.5 The aetiology of DD ...................................................................................................... 21 1.5.1 A polymicrobial disease ........................................................................................ 21 1.5.2 Treponemes associated with DD .......................................................................... 23 1.5.3 Experimental models of DD .................................................................................. 24 1.6 The Treponema genus .................................................................................................. 25 1.6.1 Treponema biology ............................................................................................... 25 1.6.2 Pathogenic treponemes important in medical and veterinary health ................. 27 1.6.3 Non-pathogenic commensal treponemes ............................................................ 28 1.6.4 Detection methods for treponemes ..................................................................... 28 1.7 The promiscuous nature of DD treponemes ............................................................... 31 1.7.1 Manifestations of DD in animals other than dairy cattle ...................................... 31 1.7.2 Detection of DD treponemes in other types of dairy cattle infections................. 31 1.8 Diagnosis, treatment and control of DD ...................................................................... 32 2 1.8.1 Diagnosis of DD on farm ....................................................................................... 32 1.8.2 Treatment and control of DD ................................................................................ 33 1.9 Risk factors for DD........................................................................................................ 35 1.9.1 Animal level risk factors ........................................................................................ 35 1.9.2 Farm level risk factors ........................................................................................... 37 1.10 Infection reservoirs of DD-associated treponemes ................................................... 41 1.10.1 Infection reservoirs within dairy cattle tissues. .................................................. 41 1.10.2 Infection reservoirs within the dairy farm environment .................................... 43 1.10.3 Other species and infection types in DD spread ................................................. 44 1.11 Aims of the project..................................................................................................... 45 Chapter 2: General materials and methods ........................................................................... 47 2.1 Media and supplements .............................................................................................. 47 2.2 Buffers and reagents .................................................................................................... 48 2.3 Farm information ......................................................................................................... 50 2.4 Bovine faecal samples .................................................................................................. 50 2.5 Treponeme cultivation ................................................................................................. 51 2.5.1 Cultivation of isolated digital dermatitis treponemes in liquid media ................. 51 2.5.2 Cultivation of digital dermatitis treponemes on solid media ............................... 51 2.5.3 Cultivation of isolated commensal GI tract treponemes in liquid media ............. 53 2.5.4 Cultivation of commensal GI tract treponemes on solid media ........................... 54 2.5.5 Treponeme culture storage .................................................................................. 54 2.6 DNA extraction ............................................................................................................. 54 2.6.1 Treponeme culture DNA extraction ...................................................................... 54 2.7 Polymerase chain reaction (PCR) assays ...................................................................... 54 2.7.1 Universal bacterial 16S rRNA gene PCR assay ...................................................... 54 2.7.2 Digital dermatitis treponeme phylogroup specific 16S rRNA gene nested PCR assay ............................................................................................................................... 55 2.7.3 Treponema genus specific 16S rRNA gene PCR assay ........................................... 56 2.7.4. Agarose gel electrophoresis ................................................................................. 57 2.8 16S rRNA gene sequencing .......................................................................................... 57 2.8.1 PCR purification ..................................................................................................... 57 2.8.2 16S rRNA gene sequencing ................................................................................... 57 2.8.3 Phylogenetic tree analysis of 16S rRNA gene sequencing .................................... 58 2.9 Ethical approval ............................................................................................................ 58 3 Chapter 3: Development of optimised techniques for the detection of DD treponeme DNA from bovine faeces ................................................................................................................ 59 3.1 Introduction ................................................................................................................. 59 3.2 Materials and methods ................................................................................................ 62 3.2.1 Optimisation of digital dermatitis treponeme phylogroup specific polymerase chain reaction assays ..................................................................................................... 62 3.2.2 Treponema pedis phylogroup polymerase chain reaction primer analysis and optimisation ................................................................................................................... 63 3.2.3 Digital dermatitis treponeme detection faeces inhibition assays ........................ 63 3.2.4 Faeces spiking for DNA extraction optimisation ................................................... 64 3.2.5 DNA extraction optimisation ................................................................................ 64 3.2.6 Evaluation of DNA extraction methods for digital dermatitis treponeme culture and faeces spiked with digital dermatitis treponemes .................................................. 66 3.2.7 Faeces spiking for alternative DNA extraction protocols ...................................... 66 3.2.8 Alternative DNA extraction protocols optimisation ............................................. 66 3.3 Results .......................................................................................................................... 69 3.3.1 Optimisation of single step PCR assays specific for each of the three digital dermatitis associated treponeme phylogroups ............................................................