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.

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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.

One of the greatest proximate threats facing biodiversity is habitat change as a result of the combined effects of agricultural development and livestock grazing. Extensive livestock systems are increasingly competing with wildlife for access to land and natural resources in African rangelands. Ethiopia has the highest numbers of livestock in Africa with most livestock production taking place in highland systems. The Bale Mountains contain the largest extent of afroalpine habitat in Africa and are the most important conservation area in Ethiopia as one of thirty-four Conservation International Biodiversity Hotspots. The Bale Mountains National Park was established forty years ago to protect the endemic, and rodent-specialist Ethiopian wolf (Canis simensis) and the afroalpine habitats upon which they depend. I use a 21 year time-series of livestock counts in the park to detect changes in the Oromo traditional livestock production system or godantu. I show that a seasonal transhumant livestock production regime, akin to godantu, only appears to persist in the Web valley today and I also find some evidence that the type of livestock is changing with smaller stock, such as sheep and goats, being more frequently kept. Despite reportedly growing numbers of households in the Web valley, I do not detect an overall increase in livestock numbers, suggesting that the area may have reached its carrying capacity in terms of livestock. In contrast, the number of livestock on the Sanetti Plateau increased over the monitoring period, including the remote and inhospitable western section of the plateau. Furthermore, I detect an increase in the risk of contact and disease transmission between Ethiopian wolves and free-roaming dogs in wolf optimal habitats. I subsequently estimate, through the use of transects, the densities of livestock in the Web valley, Morebawa and Sanetti and calculate the ratio of observed to maximum sustainable livestock density (based on rainfall and vegetation productivity). The conservative results suggest that the Web valley and Morebawa are overstocked given the rangeland predicted productivity for those areas, and that the problem of overstocking is at its worst during the dry season. I then establish critical relationships between vegetation conditions, livestock grazing pressure and rodent populations (Tachyoryctes macrocephalus, Lophuromys melanonyx and Arvicanthis blicki) in the afroalpine, under both natural and controlled experimental conditions. Rodent biomass declines as the livestock index increases along a natural grazing gradient, but rodents‟ body condition, use of the habitat or breeding ecology and most vegetation variables measured do not significantly vary across the livestock grazing gradient. Rodent populations do respond to the experimental removal of livestock inside exclosures, although this response is site and species-specific, and I find some evidence that these responses are concurrent with changes in the vegetation. Grazed plots have higher plant species richness and diversity compared to the exclosure sites which harbour reduced bareground cover and taller vegetation. Differences among rodent species in their responses to grazing may be mediated by interaction between the direct effects of grazing on habitat quality and species-specific habitat requirements, although the specific mechanism of this interaction could not be tested. Livestock may negatively affect rodents by increasing their predation risk (through removing vegetation cover), by reducing the soil suitability for maintaining burrow systems (through trampling) or, less likely, by competing for food resources. A series of simple dynamic food chain models are developed to explore the interactions between Ethiopian wolves, rodents and vegetation and how they may be affected by increasing levels of livestock grazing. I also explore how predictions made about these trophic dynamics are affected by the type of functional response linking the different levels. The models suggest that the pastures of the Web valley and Morebawa are likely to be incapable of maintaining wolves at current livestock densities, while the Sanetti plateau may be able to sustain only slight increases in livestock density before tipping into a trophic configuration unable to sustain wolves. This model is a first step in assessing the seriousness of conflict between pastoralists and wildlife in BMNP. Resource selection functions are developed and validated for cattle and sheep/goats grazing in the Bale afroalpine in an attempt to understand some of the drivers behind the heterogeneous use of the landscape by livestock. Habitat use by livestock is focused on lower-lying pastures in the vicinity of water sources (rivers or mineral springs). Only cattle strongly select for/against particular vegetation types. The probability of habitat use is also linked to the distance from the nearest villages. The models developed highlight a strong association between livestock use and rodent biomass in Morebawa and Eastern Sanetti, suggesting that livestock grazing poses a threat to Ethiopian wolves‟ persistence in marginal habitats in which rodent availability is already limited. Furthermore, the concentration of livestock around water sources has serious implications for the degradation of the park‟s hydrological system and the livelihoods and food security of the millions of people living in the dependent lowlands. Integrating research and practice is a fundamental challenge for conservation. I discuss how the methodological tools developed and the insights gained into the dynamics of the afroalpine system can contribute to the management of livestock grazing pressure in Bale Mountains National Park and highlight gaps in the knowledge of the afroalpine ecosystem where more research is needed.

Footrot (FR) is a highly infectious and debilitating disease of sheep, which has a significant economic impact on the sheep farming industry, in the UK and worldwide and causes significant suffering of sheep. Despite some recent advances, FR remains a scientifically challenging disease to understand. To help improve our understanding of disease pathogenesis, two culture-independent techniques were developed to examine the microbial succession events between the causative agent, Dichelobacter nodosus and an accessory agent, Fusobacterium necrophorum, the latter also postulated to be involved in disease initiation. The two populations were monitored in relation to disease initiation and progression during a longitudinal study and disease presentation in tissue biopsies (in situ). Finally, the distribution of these two species of bacteria in the environment was examined to highlight possible sources of infection. The work in this thesis has demonstrated that FR is a disease where expression is related to D. nodosus load present in the ovine interdigital space. D. nodosus (rpoD) load increased from that on a healthy foot to one presenting with interdigital dermatitis (ID) and feet with a higher D. nodosus (rpoD) load were more likely to go on to develop FR one week later. FISH analysis of the D. nodosus population present within the epidermis also revealed similar findings; D. nodosus cell counts increased during stages of ID, but the organism was less frequently detected in biopsies from feet with FR. Suggesting that ID might be the most infectious stage of the disease process. A fact that needs to be highlighted to farmers to encourage treatment at this stage of disease. In contrast, F. necrophorum (rpoB) load did not correlate with ID presentation or prior to the development of FR, but increased the week of FR onset. FISH analysis also revealed that F. necrophorum cell counts were higher in feet with FR than those with ID. It is possible therefore that F. necrophorum may thrive in the altered environment of a foot presenting with FR, possibly contributing to disease persistence and severity. Finally, both pathogens were detected in a range of environmental samples from a farm with endemic FR, highlighting possible sources of infection and material, which once contaminated with D. nodosus and F. necrophorum may contribute to the spread of FR. This study has provided an improved understanding of the microbial population dynamics involved in the development of ID and FR in sheep, which may have implications for control and treatment practices not only in the UK, but world-wide.

Four visits were made to 50 dairy farms in England and Wales between February 2003 and February 2004 and a further three visits to 42 of these farms between February 2005 and February 2006. At each visit the locomotion of all cows was scored (15,597 cows, 34,643 measures). Multivariable regression analyses were preformed to identify risk factors associated with increased lameness (poor locomotion). Factors associated with mean herd poor locomotion were dry cows kept in straw yards compared with cubicle houses, pregnant heifers kept with milking cows compared with dry cows in winter, passage way widths <3m compared with 2:,3m, a kerb height of ~15cm compared with >15cm, routine trimming of claws ofall cows by a claw trimmer or by- the farmer compared with no routine claw trimming, feeding maize silage to milking cows compared with other forage types, and the use of automatic scrapers compared with tractor scrapers in the cubicle house. Farmers recorded the lesions they observed while treating lame cows. Sole ulcer, white line disease and digital dermatitis were the three most frequently recorded lesions. The occurrence of sole ulcer, white line disease or digital dermatitis compared with having no lesion was used as the outcome variable in three multilevel binomial logistic regression models with month from calving nested within cow nested within farm. Risk factors associated with increased risk of sole ulcer were being housed on sparse bedding for four months or more, having grooved concrete floors on ~e farm, large herd sizes and parity numbers of four and above. Risk factors associated with increased white line disease were grooved concrete floors increasing parity number. Risk factors associated with increased digital dermatitis were grooved concrete floors and large herd sizes. A large clinical trial was used to assess the effect of intervening on known risk factors associated with lameness. Farmers in the treatment group received farm specific recommendations grouped under five targets areas which reflected the hypothesised aetiologies of sole ulcer, white line disease and digital dermatitis. There were small reductions in locomotion score and sole ulcer rate on treatment farms compared with control. These suggested that the hypotheses that reduced standing time and encouraged increased lying time through improved cow comfort may reduce sole ulcer were, at least in part, correct. There was no clear effect of treatment on the rate white line disease and digital dermatitis. It is likely that more than one of the following factors explain the lack of significant results from these initial investigations; poor recognition oflameness by farmers, insufficient uptake of recommendations, predisposition to lameness from previous lameness event and insufficiently specific lesion definitions.