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Identifying the Infection Reservoirs of Digital Dermatitis in Beef Cattle and Sheep

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Identifying the Infection Reservoirs of Digital Dermatitis in Beef Cattle and Sheep Student Final Report No. 7792 October 2015 Identifying the infection reservoirs of digital dermatitis in beef cattle and sheep October 2015 Student Final Report No. 7792 Identifying the infection reservoirs of digital dermatitis in beef cattle and sheep L E Sullivan1, 1 Department of Infection Biology, School of Veterinary Science, Institute of infection and Global Health, Liverpool, Merseyside, UK, L3 5RF. Supervisor: Nick Evans and Stuart Carter. This is the final report of a PhD project (AHDB Beef and Lamb Ref: 7792) that ran from October 2012 to September 2015. The work was funded by AHDB Beef and Lamb, HCC and QMS. While the Agriculture and Horticulture Development Board, operating through its AHDB Beef and Lamb division, seeks to ensure that the information contained within this document is accurate at the time of printing, no warranty is given in respect thereof and, to the maximum extent permitted by law, the Agriculture and Horticulture Development Board accepts no liability for loss, damage or injury howsoever caused (including that caused by negligence) or suffered directly or indirectly in relation to information and opinions contained in or omitted from this document. Reference herein to trade names and proprietary products without stating that they are protected does not imply that they may be regarded as unprotected and thus free for general use. No endorsement of named products is intended, nor is any criticism implied of other alternative, but unnamed, products. AHDB Beef and Lamb is the beef and lamb division of the Agriculture and Horticulture Development Board for levy payers in England. Hybu Cig Cymru - Meat Promotion Wales (HCC) is the Welsh red meat organisation for levy payers in Wales. Quality Meat Scotland (QMS) is the Scottish red meat organisation for levy payers in Scotland. CONTENTS 1. ABSTRACT ................................................................................................ 8 2. INTRODUCTION ........................................................................................ 9 2.1. Digital Dermatitis ........................................................................... 9 2.1.1. General Introduction ................................................................. 9 2.2. Lameness in ruminants - definition, causes and magnitude of the problem ............................................................................................. 10 2.2.1. Definition and observations .................................................... 10 2.2.2. Dairy cattle lameness in the UK .............................................. 11 2.2.3. Beef cattle lameness in the UK ............................................... 12 2.2.4. Sheep lameness in the UK ..................................................... 13 2.2.5. Common causes of cattle lameness ....................................... 13 2.2.6. Common causes of sheep lameness ...................................... 16 2.3. Importance of bovine digital dermatitis (BDD) and contagious ovine digital dermatitis (CODD) ............................................................. 17 2.3.1. Economic impact of BDD and CODD ..................................... 17 2.3.2. Welfare implications ............................................................... 18 2.4. Epidemiology ............................................................................... 19 2.4.1. Introduction and spread of digital dermatitis ........................... 19 2.4.2. Prevalence ............................................................................. 19 2.4.3. Seasonality ............................................................................. 20 2.4.4. Risk factors associated with the prevalence of BDD and CODD..................................................................................... 20 2.5. Clinical signs and management ................................................. 24 2.5.1. Clinical manifestations of BDD in Dairy cattle ......................... 24 2.5.2. Clinical manifestations of CODD in sheep .............................. 30 2.5.3. Diagnosis and treatment of BDD ............................................ 32 2.5.4. Diagnosis and treatment of CODD ......................................... 33 2.6. Pathogenesis of digital dermatitis ............................................. 33 2.7. Introduction to the Spirochaetes ............................................... 36 2.7.1. Spirochaete phylogeny ........................................................... 36 2.7.2. Spirochaete structure and biology .......................................... 39 2.7.3. Spirochaete motility ................................................................ 40 2.7.4. Metabolic requirements of spirochaetes ................................. 40 2.7.5. Antibiotic resistance of spirochaetes ....................................... 41 2.7.6. Free living and host-associated non-pathogenic spirochaetes 41 2.7.7. Host-associated pathogenic spirochaetes .............................. 42 2.8. The Genus Treponema................................................................ 42 2.8.1. Introduction ............................................................................ 42 2.8.2. Non-pathogenic Treponema ................................................... 43 2.8.3. Pathogenic Treponema .......................................................... 44 2.9. Treponemes and Digital Dermatitis............................................ 45 2.9.1. Aetiology of BDD .................................................................... 45 2.9.2. Aetiology of CODD ................................................................. 46 2.9.3. Isolation and detection of BDD and CODD associated treponemes ............................................................................ 47 2.9.4. Transmission and carriage sites of DD treponemes ................ 48 2.10. Aims of the project ...................................................................... 49 3. MATERIALS AND METHODS ................................................................. 51 3.1. Reagents and buffers .................................................................. 51 3.2. Collection and transportation of clinical samples .................... 55 3.2.1. Collection of, BDD and CODD lesion samples and ruminant healthy foot tissues ................................................................. 55 3.2.2. Rectal tissue ........................................................................... 55 3.2.3. Gingival tissue ........................................................................ 56 3.2.4. Faecal samples ...................................................................... 56 3.2.5. Swab samples ........................................................................ 56 3.3. Bacterial culture .......................................................................... 57 3.3.1. Inoculation into liquid and solid media .................................... 57 3.3.2. Storage of treponeme cultures ............................................... 59 3.4. DNA extraction ............................................................................ 59 3.4.1. Treponeme cultures ................................................................ 59 3.4.2. Tissues and swabs ................................................................. 59 3.4.3. Faeces ................................................................................... 59 3.5. Polymerase Chain Reaction (PCR) assays ................................ 59 3.5.1. Primers ................................................................................... 59 3.5.2. Universal bacterial 16S rRNA gene PCR ................................ 61 3.5.3. Treponeme phylogroup specific 16S rRNA gene PCR assays 62 3.5.4. Treponeme genus specific 16S rRNA gene PCR assay ......... 62 3.5.5. Dichelobacter nodosus specific 16S rRNA gene PCR assay .. 62 3.5.6. Fusobacterium necrophorum specific lktA PCR assay ............ 63 3.5.7. Agarose Gel Electrophoresis .................................................. 64 3.6. Gene sequencing and assembly ................................................ 64 3.6.1. Purification of PCR products ................................................... 64 3.6.2. Gene sequencing and assembly ............................................. 64 3.7. Phylogenetic analysis of bacterial 16S rRNA gene sequences 65 3.8. Statistical analyses ..................................................................... 65 3.8.1. Chi-square test for significance .............................................. 65 3.8.2. Fisher’s exact test for significance .......................................... 66 3.9. Serological methods ................................................................... 66 3.9.1. Blood collection ...................................................................... 66 3.9.2. Antigen preparation ................................................................ 67 3.9.3. Quantification of protein concentration in antigen preparations ........................................................................... 68 3.9.4. Trichloroacetic acid (TCA) protein precipitation ...................... 68 3.9.5. Enzyme-linked immunosorbent assays (ELISAs) ................... 68 3.9.6. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D SDS-PAGE) ............................................ 70 3.9.7. Western Blotting ..................................................................... 73 4. RESULTS ................................................................................................
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