Accounting for much of the landscape of southern England, chalk stream environments hold significant cultural, economic and ecological value. However, attempts to retain this value are often hindered by the remnants of historic management practices that have occurred across several millennia, as well as contemporary demands upon chalk stream amenity, including water abstraction, recreational use and fisheries management. One land-use that is believed to have a detrimental effect upon chalk streams, but which has been inadequately researched, is cattle grazing. Within this thesis the effects of cattle grazing in English chalk streams are assessed using a range of techniques. Terrestrial laser-scanning is employed to show that cattle can cause small, local changes in river bank topography. Direct and remote observations are used to link cattle behaviour to landscape utilisation, and a staticially significant correlation between air temperature and in-stream cattle activity is identified. Laboratory faecal analysis is conducted to establish the nutrient loading due to cattle, with results showing that cattle faeces contain signfiicant concentrations of phosphate. In-stream water turbidity monitoring is combined with remotely sensed cattle behaviour data to demonstrate that in-stream cattle activity has a minimal effect upon suspended sediment concentrations in an English chalk stream. A study using the diffuse fine sediment risk model, SCIMAP, highlights the hydrologically disconnected nature of English chalk streams, with model outputs concluding that topography, rather than land-use (cattle grazing), is the key control on diffuse fine sediment risk in English chalk streams. Combined, these individual findings provide a detailed, inter-disciplinary assessment that concludes the effects of cattle grazing in English chalk streams are different to those recorded in research from other environments, with physio-chemical effects (i.e. nutrient loading) being of greater significance than geomorphological agency (i.e. river bank destabilisation). This overarching conclusion has implications for the management of cattle grazing in English chalk streams, and these are discussed.

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As nutrition is of central importance to cattle fertility, this study sought to assess how veterinarians and nutritional advisers manage trace element imbalance in the UK; diagnosis and treatment. The study also sought to develop a robust system for oocyte recovery (ovum pick-up (OPU)) and in vitro embryo production (IVP) for commercial use, and to identify key factors influencing success, including oestrus synchrony and ovarian stimulation prior to OPU. The intention originally was to use OPU/IVP to investigate the impact of mineral imbalances on bovine oocyte quality, early embryo development and pregnancy establishment following embryo transfer (ET). In the first survey of its kind in the UK, the understanding and approach of advisers to mineral nutrition on farms was investigated. Of the 173 respondents, 78% were vets in practice. The overall importance of minerals was recorded by vets as low 33%, medium 37%, and high 30%, while non-vets scored importance as low 17%, medium 48%, and high 35%. There was little consensus amongst the advisers, or within the vet and non-vet subgroups about mechanisms and interactions associated with deficiency, and particularly of copper responsive conditions. The most frequently identified deficiencies were selenium, copper and iodine, while the most commonly identified toxicity was molybdenum. For copper responsive conditions, all of the listed treatments were used at least "occasionally"; the most frequently being glass boluses, in-feed supplementation, matrix boluses, and then copper injections. While there was a diverse choice of treatments, altering the ration was relatively rarely selected. This thesis also provides the first large-scale retrospective analysis of factors influencing the establishment of a commercially robust ovum pick up (OPU) and in vitro embryo production (IVP) platform in the UK. Over a 5-year period, a system was developed and validated for use in the UK with 2,138 cycles of OPU. These cycles were analysed as four sets of data and included two IVP laboratories and 6 OPU teams. Factors in these analyses included OPU team, IVP laboratory, ovarian stimulation protocol and semen type (unsorted vs sex-sorted). The mean number of follicles aspirated by the OPU teams ranged from 6.5 to 14.9 (P < 0.001), while the number of oocytes collected was between 4.0 and 12.4 (P < 0.001). There was an indication (P=0.055) that the blastocyst per oocyte rate varied between teams. The proportion of blastocysts from oocytes that cleaved was higher (P=0.01) for unsorted than sexed semen. Two commercial products containing different ratios of follicle stimulating hormone (FSH) to luteinising hormone (LH) (Folltropin® and Pluset®) were compared in ovarian stimulation programs. The addition of 'coasting' (short-term (typically 48h) hormonal withdrawal after FSH stimulation), prior to OPU was also investigated. Pluset® resulted in a greater (P < 0.001) mean number of follicles aspirated, more (P=0.003) blastocysts per oocyte matured and more (P < 0.001) embryos per cycle (2.45), compared with Folltropin® (1.17) or with no stimulation (1.24). Throughout the study there was a steady improvement in blastocyst production per OPU cycle. In a separate analysis, Grade 1 cumulus oocyte complexes (COCs) as a proportion of COCs recovered, oocytes that cleaved as a proportion of total COCs, and blastocysts as a proportion of total COCs, were all greater (P < 0.05) for stimulated than non-stimulated cycles, irrespective of FSH/LH product. A composite score of oocyte quality and quantity was proposed (sCOC); Log Total Mean sCOC was correlated (P < 0.001) with both the proportion of blastocysts per oocyte collected, and the total number of embryos produced per cycle. Finally, twelve peri-pubertal heifers (approximately 10 months old) participated in a crossover trial which compared PRID® (Delta®) vs CIDR® progesterone releasing intravaginal devices for use in OPU/IVP cycles. Vaginoscopic examination found higher vaginal inflammation grades for PRID® than CIDR® (P < 0.001). There was evidence of vaginal inflammation continuing for at least 2 weeks after device withdrawal. The proportion cleaved of oocytes inseminated was higher for PRID® than CIDR® (P < 0.05). Numerically but not significantly there was a higher proportion of blastocysts per cycle and a higher Log Total Mean sCOC score per cycle with PRID® than CIDR® treatments, but blastocyst yield was low throughout, suggesting a need to repeat the trial. Data collection and analyses are ongoing, to identify other key performance indicators within the OPU/IVP embryo transfer (ET) system, with a view to refining the sCOC composite score model. A robust OPU/IVP/ET system has been developed and this could be used to investigate further how mineral imbalances impact oocyte competence and blastocyst yield.

To date there are no effective treatments for prion diseases, and these diseases are always fatal in both humans and animals. Additionally, the gold standard for diagnosis of these disease remains to be the analysis of biopsied brain tissue obtained post mortem. Consequently, there is a continued demand for therapeutics and ante-mortem diagnostics for prion diseases. This project addresses these demands by investigating candidate therapeutic and diagnostic ligands for prion diseases. This study investigated recombinant prion proteins (rPrPs) as inhibitors in scrapie and bovine spongiform encephalopathy (BSE) in vitro amplification by protein misfolding cyclic amplification (PMCA). Three ovine rPrPs with the polymorphisms VRQ, ARQ and ARR and hamster rPrP were tested against scrapie PMCA in dilution series to calculate IC50 values. The two most potent inhibitors, VRQ and ARQ, were then similarly tested against bovine spongiform encephalopathy (BSE) amplification. The most potent inhibitor of both disease types, the ovine rPrP VRQ, was then observed to inhibit a range of different scrapie and BSE strains at a fixed concentration. It is recommended that further investigation into rPrP inhibitors is performed. Strain characterisation of scrapie was investigated using rPrP inhibitors, following observations that the rPrP inhibitors generate a pattern of inhibition at a set concentration. Although this pattern of inhibition was repeatable in scrapie amplification by PMCA, this was limited to a single round of PMCA. Ultimately, this limited the application of this method to only amplification efficient prion strains and isolates. It is recommended that this method be investigated further in combination with the amplification of different isolates in substrates of different genotypes over multiple rounds of PMCA, as well as the analysis of glycoform ratios by western blotting. Here it was also identified that the imidazole used in the elution buffer for immobilised metal affinity chromatography (IMAC) can inhibit prion amplification in a strain dependent manner. This inhibition could be used in combination with the proposed method as a multi-faceted assay of prion strain characterisation. The use of next generation phage display (NGPD) to map the epitopes of autoantibodies in the sera of scrapie infected sheep was also investigated. This was performed to identify peptides that were immunoreactive to autoantibodies specific to the disease state. The identification of diagnostic peptides would then enable the development of an ante-mortem serological diagnostic test for scrapie. NGPD successfully selected immunoreactive peptides, of which 39 were selected for validation by peptide enzyme-linked immunosorbent assays (ELISAs). Although none of the peptides demonstrated diagnostic specificity by peptide ELISA, an optimised ELISA methodology was developed for future use in the validation of NGPD selected peptides. Further variations in the NGPD method, as well as validation by immunoassay, can be investigated to identify diagnostic peptides immunoreactive to scrapie specific autoantibodies.

Foot-and-mouth disease virus (FMDV) causes the most contagious transboundary disease of animals, affecting both wild and domestic cloven-hoofed animals. Similarly to other RNA viruses, FMDV is highly variable as a result of the inherent low fidelity of the viral RNA-dependent RNA polymerase. The accumulation of this variability and relatedness between FMDV sequences was used to provide evidence for modes of transmission (fomite) as well as a constant clock rate across two FMDV topotypes (~8.70 x 10-3 substitutions/site/year), during the 1967 UK FMD epidemic, using full genome consensus sequencing. However, during an epidemic, virus replicates within multiple animals, where it is also replicating and evolving within different tissues and cells. Each scale of evolution, from a single cell to multiple animals across the globe, involves evolutionary processes that shape the viral diversity generated below the level of the consensus. During this PhD project, next-generation sequencing (NGS) was used to dissect the fine scale viral population diversity of FMDV. Collaboration with the Institute of Biodiversity, Animal Health and Comparative Medicine at the University of Glasgow provided the specialist bioinformatic and statistical capabilities required for the analysis of NGS datasets. As part of this collaboration, a new systematic approach was developed to process NGS data and distinguish genuine mutations from artefacts. Additionally, evolutionary models were applied to this data to estimate parameters such as the genome-wide mutation rate of FMDV (upper limit of 7.8 x 10-4 per nt). Analysis of the mutation spectra generated from a clonal control study established a mutation frequency threshold of 0.5% above which there can be confidence that 95% of mutations are real in the sense that they are present in the sampled virus population. This threshold, together with an optimized protocol, was used for the more extensive investigation of within and between host viral population dynamics during transmission. Analysis of mutation spectra and site-specific mutations revealed that intra-host bottlenecks are typically more pronounced than inter-host bottlenecks. NGS analysis has distinguished between the population structure of multiple samples taken from a single host, which may provide the means to reconstruct both intra- and inter-host transmission routes in the future. A more sophisticated understanding of viral diversity at its finest scales could hold the key to the better understanding of viral pathogenesis and, therefore development of effective and sustainable disease treatment and control strategies.

The major reservoirs for most influenza A virus subtypes are wild aquatic birds, especially ducks. However, they are typically resistant to the effects of the infection and usually do not develop clinical disease. In contrast, some influenza viruses cause severe illness or even death in susceptible hosts like chickens and turkeys. Paradoxically, infection of primary duck cells results in rapid cell death, whereas in chicken cells, death occurs less rapidly. Duck cells produce fewer infectious virions in comparison with the longer surviving chicken cells. In order to understand this variation in infectious virus production, chicken and duck embryo fibroblast cells (CEF and DEF) were infected with low pathogenic avian H2N3, and the viruses produced from the two hosts ware characterised. Infectious virus production from chicken cells was significantly greater than that observed from duck cells, from 8–48 hr after infection. Influenza matrix gene and protein expression, analysed by quantitative real time PCR and western blotting of culture supernatants, showed comparable levels between species at 8 and 24 hr post infection. These findings led to investigation of virus budding and morphology following infection of duck and chicken cells with the virus. Differences in morphology of released virions were observed. Budding viruses from duck cells were elongated, while chicken cells produced almost spherical virions. There was a similar clear difference in virus morphology in the duck and chicken culture supernatants. Spherical viruses were observed in chicken supernatants while duck cell supernatants contained pleomorphic virions. No differences between any genes of chicken– and duck–derived viruses were found, suggesting that host cell determinants might be responsible for such variations in virus morphology. DEF cells showed extensive production of filamentous or short filament virions following infection with filamentous (equine H3N8) and non–filamentous (avian H2N3) virus strain, respectively. This was observed even after actin disruption with cytochalasin D (Cyt.D). CEF cells infected with equine H3N8 virus produced extensive filamentous virus, which decreased markedly after disruption of actin with Cyt.D, whereas, following infection with H2N3, spherical virions were observed in the presence or absence of the actin inhibitor. Cells were also transfected with green fluorescent protein – microtubule-associated protein 1A/1B-light chain 3 (GFP–LC3) expression vector and then infected or mock infected with avian H2N3. Short filaments were observed from untransfected and transfected duck cells, while spherical and short filaments were observed from untransfected and transfected chicken cells, respectively. Filamentous virus formation could be enhanced as a result of autophagy which is more marked in duck cells than chicken cells. Further studies such as studying the structure of chicken and duck fibroblast cell membranes, the use of other drugs that inhibit actin in a mechanistically different way, and the role of other cellular proteins in modulating virus morphology should be considered.

Latest epidemiological data suggests 1.5 billion adults worldwide are either overweight or obese. With increasing weight and obesity, adipocytes increase in size. The enlargement of adipocytes has been associated with low grade chronic inflammation via elevated adipokine secretion. Previous epidemiological studies in humans and experimental studies in animals have shown that during different periods of pregnancy (gestation) the offspring that are born to maternal nutritional manipulation are more susceptible to developing metabolic diseases in later adult life. Therefore, the aim of this thesis was to investigate the role of maternal nutritional manipulation on adipose tissue depots and in particular the consequences the effect on markers of adipokine secretion. Studies were conducted on both large and small animals (i.e. sheep and rats). Sheep studies focused on mid to late and late gestation periods of maternal nutritional restriction. Rat studies concentrated on long term fructose feeding during pregnancy and its effect on both the mother and offspring. Gene expression analysis identified an up-regulation in inflammatory related genes in pericardial and subcutaneous adipose tissue in the sheep studies. This was also seen in the rat studies with protein and gene expression displaying an up-regulation of inflammatory and metabolic related genes and proteins. The main conclusion of my thesis is that after following maternal nutrient restriction, females appear to be much more sensitive to inflammatory and metabolic adaptations compared to males, possibly due to sex hormones playing a role. Whilst fructose feeding during pregnancy concluded the possibility of homeorhesis playing a protective role against potentially detrimental inflammatory pathways being activated in the mothers, the offspring however displayed signs of low level chronic inflammation in the retroperitoneal depot from early infancy to later adult life.

This thesis examines effects of companion animals on human cardiovascular reactivity. An examination of previous research investigating effects of companion animals on human cardiovascular reactivity suggested that previous mixed and mainly non-significant results in this area might be due to failures in methodology. This led to the development of recommendations for future studies. Three studies are presented which examined the effect of presence of an unfamiliar dog on participants' cardiovascular levels during a standardised reactivity study. The consistent finding from these studies was that the presence of an unfamiliar dog had no discernible effects on cardiovascular levels throughout the experiment (baseline and task levels combined) or on reactivity to stressors (difference between task and baseline levels). The fourth study investigated the effect of presence of the participants own pet on cardiovascular levels during a reactivity study. The study also included a condition of human companion presence. The results of the study indicate significant moderation of reactivity from the presence of both a pet dog and a human friend. The design of the study allows elimination of certain explanations such as differential vocal styles, distraction, threat of setting and perception of the experimenter. Whether social support is the mechanism which accounts for stress moderation in either companion condition is debatable. However in the case of pet dogs, it is argued that presence of ones pet during an everyday setting where one encounters stressful events would occur too infrequently to provide regular moderation of the stress response in the manner which has been proposed to lead to health benefits.

The interaction between host genetics and epidemiological processes in ovine footrot was investigated using a combination of data analysis and simulation modelling. The study’s aims were to determine the potential for genetic selection to be used to reduce the prevalence of footrot in the UK and to assess different strategies for use of conventional epidemiological interventions. A stochastic simulation model was developed, incorporating host genetics for traits controlling footrot resistance, bacterial population dynamics, sheep population dynamics and epidemiological processes. Sensitivity analysis of the model showed survival time of Dichelobacter nodosus in the environment and infection rate were the key determinants of disease outcomes. Antibiotics were predicted to be the most effective conventional control method, reducing prevalence of footrot to 1-2% when administered promptly. Pasture rotation, selective culling and vaccination were all predicted to reduce prevalence but to a lower extent. Analysis of field data confirmed the likely role for some degree of host genetic control of footrot resistance, i.e. resistance appears to be lowly to moderately heritable. Using the simulation model it was then shown that genetic selection could be effective at reducing footrot prevalence. In combination with antibiotic treatment or pasture rotation elimination of footrot from an individual flock could be achieved. Genetic selection was predicted to be effective at reducing prevalence and improving resistance but the choice of selection criteria impacts the results seen. It is likely that progress would be slower in field situations because footrot traits would be diluted by simultaneous selection for other traits affecting profitability. Field studies are required to determine optimal combinations of interventions and genetic selection and to validate modelling outcomes. Combined data from longitudinal disease observations, genetic information and bacterial samples are necessary to address current knowledge gaps and to further advance understanding of host and disease processes in ovine footrot.

A seroepidemiological study was undertaken in a pedigree dairy herd that had a history of abortions due to neosporosis. The infection in this closed herd was thought to have arisen from a point-source infection, after which sporadic abortions have occurred. All cattle were bled twice, once in the winter and again the following summer and antibodies to N. caninum measured using an ELISA. The overall seroprevalence of Neospora was found to be 18 %. Three data sets; age-prevalence data, dam-daughter pair analysis and family tree data showed vertical transmission to be an important route of transmission of neosporosis in this herd. Analysis of anti- Neospora antibody titres with respect to the stage in the breeding cycle of cows appeared to show no association on a herd level. Data was collected on the number of Artificial Insemination (AI) services per successful pregnancy which showed a significantly greater number of Al services in Neospora-seropositive cattle compared with Neospora-seronegative cattle. This is the first study to assess the effect of neosporosis on cattle fertility in a quantitative manner and suggests that a wider study is justified. N. caninum shares many similarities with T gondii and has widely been assumed also to have a world-wide distribution. Two regions of Africa, Ghana in West Africa and Tanzania in East Africa, were studied in a cross-sectional survey of neosporosis in cattle indigenous to these areas. A prevalence of 8.1 % and 2% was found in two different areas in cattle native to Tanzania. Despite sampling a significant number of cattle in all three ecological zones of Ghana and of several different breeds, no Neospora-seropositive cattle were found. Possible reasons for the apparent absence of N. caninum in West Africa are discussed. To determine the overall genetic diversity in laboratory isolates of N. caninum, RAPD and AFLP methods were used. Genetic diversity was found to be low amongst Neospora laboratory isolates, relative to T. gondii, but demonstrated that genetic heterogeneity does exist within the species. Both RAPD and AFLP data were subjected to pair-wise similarity and cluster analysis and showed that there was no clustering with respect to host or geographical origin. The genetic similarity between cattle and dog isolates suggests that these hosts are epidemiologically related. In order to exploit the genetic heterogeneity in N. caninum to analyse a wider range of clinical field samples, several methods were attempted to devise PCR-based sequence-specific typing approaches that could be used on infected bovine tissue. Microsatellite markers were identified in N. caninum DNA sequences, however none of the microsatellite regions gave rise to detectable size differences, although they remain to be tested on a wider range of field samples. Laboratory isolates of N. caninum were also analysed for polymorphisms with two conserved minisatellite probes, 33.6 and 33.15, but although hybridisation occurred to digested parasite DNA, identical fingerprints were obtained for each isolate. In a final attempt to identify sequence-specific polymorphic markers, intron regions from two genes, actin and tubulin, were amplified and sequenced in both laboratory and field isolates. This approach revealed a number of single nucleotide polymorphisms (SNPs) that were able to differentiate between some isolates of N. caninum and might serve as useful molecular markers. SNPs were found more frequently in the clinical field samples, suggesting that the diversity of N. caninum is greater than that represented by current laboratory isolates. Further genotyping of field samples will enable the genetic population structure of N. caninum to be determined to facilitate molecular epidemiological studies.