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Muzafar, Mohd
Languages: English
Types: Doctoral thesis
Subjects: SF
Ovine footrot is an infectious disease of sheep which causes serious economic losses to countries worldwide and costs the UK £84 M per annum. For effective control of this disease which is a major animal welfare issue, it is essential to study mechanisms of transmission. One of the key aims of the project was to improve our understanding of the potential for Dichelobacter nodosus, the causal agent, transmission via the environment within a flock of ewes and lambs. Newborn lambs were free from D. nodosus presence on their feet, but rapidly became contaminated with D. nodosus within 5-13 h after birth as detected using the specific molecular markers rpoD and 16S rRNA gene. The likely source of contamination of lambs was the straw bedding from the communal pens. A diverse population of D. nodosus was observed on the feet of ewes and lambs as determined by the presence of multiple strains with variable numbers of pgrA tandem repeats in the R1 region. This was further supported by Multi Locus Variable Number Tandem Repeats (MLVA) typing of the isolates, which also indicated high variation in the alleles present on the ewes and lambs. The primary aim was to determine if multiple strains present on the ewes were vertically transmitted to the lambs. This work has clearly demonstrated that no vertical transmission occurred between ewes and lambs and some strains, but not all were shared between ewe and its lamb suggesting transmission from their mother or other ewes sharing the same lambing pen. D. nodosus was detected in a range of environmental samples such as bedding, faecal balls compacted within the interdigital space and soil suggesting that shedding into the environment is the main route of D. nodosus transmission. Survival studies provided evidence that the pathogen persisted in soil microcosms for at least 40 days with viable cells persisting for a minimum of 30 days in four soil types. A lower temperature of 5 oC and clay soil was associated with longer duration of survival. Single Nucleotide Polymorphism (SNP) analysis of isolates from the ewes and lambs indicated that two main clonal populations existed that represented two clusters α and β within clade I, a virulent clade from the recent genome study including 103 global D. nodosus strains. No UK strains grouped with the benign clade II. Previous diversity studies on the isolates indicated diversity in MLVA types, pgr alleles and pgrA tandem repeats, but these did not correlate with the clustering; clusters α and β contained a mix of pgrA and pgrB but both were in clade I. This conflicts with the role of pgrB as a non-virulent allele although pgrA expression was induced by hoof horn in vitro, whereas pgrB was not. The work reported in this thesis has improved our understanding of the environmental transmission of D. nodosus between sheep, longevity of the pathogen in soil and diversity of strains in the UK.
  • The results below are discovered through our pilot algorithms. Let us know how we are doing!

    • 4.3.4. Genome Sequencing of Isolates ...................................................................... 91 4.3.5. pgr sequencing and phylogenetic analysis...................................................... 92 4.3.6. Comparison of pgrA and pgrB upstream regions............................................ 93 4.3.7. Prediction of PgrA structure............................................................................ 96 4.3.8. Determination of the expression of the Pgr locus of D. nodosus .................... 96 4.3.9. Extraction of bacterial DNA from cultured cells ............................................. 96 4.3.10. Extraction of bacterial RNA from cultured cells ........................................... 97 4.3.11. Normalization using rpoD, D. nodosus 16S rRNA, pgrA.............................. 99 Table 2.1. Ewes and lambs included in the study, with regards to,........................... 34 date of birth (D.O.B.) and time of sampling after birth.
    • Table 2.2. List of primers and probes used in the study........................................... 38 Recipient of Warwick University Award, Chancellor's Scholarship/School of Life Sciences - 1150257, 2011 - 2015.
    • Muzafar, M., Calvo-Bado, L.A., Green, L.E., Smith, E.M., Russell, C.L., GrogonoThomas, R., Wellington, E.M.H., 2015a. The role of the environment in transmission of Dichelobacter nodosus between ewes and their lambs. Veterinary microbiology 179, 53-59.
    • Muzafar, M., Green, L.E., Calvo-Bado, L.A., Tichauer, E., King H., James P., Wellington, E.M.H., 2015b. Survival of ovine footrot pathogen Dichelobacter nodosus in different soils. Anaerobe 38, 81-87.
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