An investigation of 5186 hock maps, from 3691 cows with hair loss on the hocks from 76 UK dairy farms, identified six risk factors associated with area of hair loss: higher locomotion score; poor cleanliness; higher mean milk yield; low body condition score, prolonged winter housing and certain combinations of stall base and bedding materials. Another finding was significantly poor correlation between a categorical scale and the continuous measure of hair loss. Hair loss on the hocks of 70 heifers (three herds) was observed monthly from Sep 2008-Mar 2010. The results imply that lameness precedes hair loss on the hock and not vice versa, i.e. lame animals develop hair loss rather than hock hair loss leads to future lameness. Poor cleanliness score and higher milk yield were associated positively with the risk of having hair loss on the hock. Based on the total 6889 observations from 731 cows in four herds, cows with a greater decrease in BCS (compared to BCS at calving) had higher probability of becoming lame and lower probability of recovering. Also, increase in BCS from calving was associated with lower probability of cows moving from non-lame to lame state and higher probability from lame to non-lame. Days of lactation, months of calving and parity impacted upon both non-lame to lame and lame to non-lame transitions. Analysis of 678 cows from four herds found that cows with chronic lameness had a lower probability of conception and a longer calving to conception period compared with healthy cows. Further, cows with lower average BCS had a lower probability of conception and were more likely to be lame. However, these factors didn’t influence the likelihood of an individual AI leading to pregnancy.

Bovine tuberculosis (bTB) is one of the principal concerns currently facing the livestock industry in England. The disease has spread dramatically in recent years and is costing the country millions of pounds each year. Tens of thousands of cattle are being slaughtered annually; a huge financial and emotional burden to affected farmers. While various measures to control the disease have been taken, none have been successful in bringing it under control. Instead bTB continues to spread unabated. The essence of the bTB problem is that it necessitates industry buy-in in order to implement disease control measures. It is therefore not simply an issue of regulation. Current government bTB control policy emphasises communication and cooperative working across the government and the farming industry, coupled with cost and responsibility sharing. However, previous studies have shown that relationships between farmers and the government are already strained, engendered by a sense of distrust and a lack of confidence. Although some social science work has been conducted within the field of disease control and particularly bTB, the majority focuses on farmers‘ attitudes towards government policy and disease control. However, in order to implement successful disease control measures it is necessary to explore the ways in which farmers currently respond to bTB, and how their responses may be recognised by, and incorporated into, successful policy. While previous research has identified the important role of the wider social context in influencing farmers‘ attitudes and behaviour, no studies have yet provided an in-depth analysis of farmers‘ social networks in relation to bTB. In response, this study uses the lens of social capital to explore the various social ties which enhance or constrain farmers‘ capacity to respond to bTB. An iterative, mixed methods approach is adopted across two phases of research. The first incorporates twenty in-depth qualitative farmer interviews, exploring various aspects of bTB risk and response strategies as well as the core features of social capital. This informs a second, quantitative phase, in which data are iii gathered through a self-completion postal survey of 374 farmers in the South West of England. A farmer segmentation model is developed using factor and cluster analysis and two farmer groups are identified. The first group represents vulnerable farmers who are concerned about the negative impacts of bTB, and who are internally focused with respect to their networks. Characteristically, they exhibit strong relationships with others from within the farming community. In comparison, the second group are more resilient and less concerned about the impacts of bTB on their farm business. These farmers are externally focused, mainly seeking information from the government, the National Farmers‘ Union and their vet. The role of various forms of social capital is explored and an important distinction between the two farmer groups is found. Vulnerable farmers tend to be members of close networks of other farmers (bonding social capital), while resilient farmers are more likely to enjoy positive relationships with those from outside the farming community including vets (bridging social capital) and the government (linking social capital). However, while the research findings suggest that bridging and linking social capital can positively influence farmers‘ attitudes towards bTB, they do not necessarily lead to positive disease control behaviour. Statistical analysis of the data reveals no significant differences between the farmer groups in terms of their uptake of biosecurity measures, which represents an important disease avoidance strategy. A disjuncture between farmers‘ attitudes and their behaviour is therefore identified. The research concludes that investment in social capital between the government and farmers should form a core area of policy through providing opportunities for consistent and regular contact, allowing for the development of trusting and productive relationships. The current situation, characterised by low levels of trust and limited uptake of recommended disease control measures by farmers, indicates incoherence with contemporary policy discourses. A better understanding of the role of social capital in influencing farmer attitudes and behaviour will enable policy makers to increase the ability of farmers to respond to bTB risk, either through disease avoidance or through more effective management and coping mechanisms.

Clinical reasoning is the skill used when veterinary surgeons make a decision regarding the diagnosis, treatment plan or prognosis of a patient. Despite its necessity and ubiquity within clinical practice, very little is known about the development of clinical reasoning during undergraduate training. Even less is understood about how veterinary schools should be helping students improve this skill. The aim of the research presented within this thesis was to, firstly, examine the development of clinical reasoning ability within veterinary students and, secondly, to investigate possible methods to aid this process. The University of Nottingham School of Veterinary Medicine and Science (SVMS) was used as a case study for this research. In study one, focus groups and interviews were conducted with SVMS staff, students and graduates to investigate the development of clinical reasoning. A curriculum document content analysis was also performed. The findings suggested that clinical reasoning development is not optimal, with alumni facing a steep learning curve when entering practice. These results were used to design study two, in which a simulated consultation exercise utilizing standardised clients was created and implemented for final year students. The success of the simulation was measured using both quantitative and qualitative methods – all of which supported the use of the session for clinical reasoning development. The final study, also building on the findings of study one, aimed to improve the accessibility of veterinary surgeons’ decision-making processes during student clinical extramural studies placements (CEMS). A reflective Decision Diary was created and trialled with third and fourth year SVMS students. Diary content analysis showed the study aim was met, triangulated by survey and focus group findings. During the research, wider issues relating to clinical reasoning integration into veterinary curricula were unearthed. These included low student awareness of the subject and the misalignment between the skill learnt during training and the skill required when in practice. Several recommendations have been made to improve the design of the undergraduate curriculum in relation to clinical reasoning.

Articular cartilage can be damaged directly through injury or osteoarthritis (OA). This tissue is very poor at regenerating itself due to its avascular nature and the immobility of chondrocytes within the tissue. There are a range of surgical techniques to repair cartilage lesions. Cellular therapies such Autologous Chondrocyte Implantation (ACI) and later modifications have been used to repair cartilage lesions for the past two decades. However, there is currently no completely successful treatment of cartilage lesions, with the newly generated cartilage often possessing very poor mechanical properties. Also, cell-based therapies require large numbers of chondrocytes which have to be expanded in monolayer. A consequence of this expansion is a loss of the chondrocyte phenotype (dedifferentiation). The overall aim of this thesis was to develop a greater understanding of chondrocyte dedifferentiation and redifferentiation in vitro using canine chondrocytes. Dogs can also suffer with OA and have been used extensively as a model for OA. Firstly, canine chondrocytes were expanded in monolayer up to P5 to confirm dedifferentiation. These cells were shown to have lost their typical chondrocytic phenotype through decreased expression of collagen type II and increased expression of collagen type I and CD44. A considerable part of this element of the thesis also involved identifying antibodies that would cross-react with the target canine antigens. The next aim of the thesis was to redifferentiate dedifferentiated chondrocytes through three-dimensional (3D) culture. Initial problems with high density pellet culture led to the selection of a supporting material. Alginate was chosen as it is a naturally occurring polymer which has previously been used to culture chondrocytes in 3D. After making several adjustments to the set-up and downstream analysis of the beads, chondrocytes from different passages were seeded into them. Alginate beads seeded with P2 chondrocytes appeared to contain cells with a more chondrocyte-like phenotype compared to P3- and P4-seeded beads. However, expression of collagen type I was still relatively high in P2-seeded beads, indicating 3D culture alone is not enough to induce complete redifferentiation. Therefore, the final aim of this thesis was to enhance the redifferentiation of dedifferentiated canine chondrocytes. Two initial conditions were selected; addition of 25μg/ml ascorbate to the culture medium and incubating the beads under reduced oxygen conditions (2.4%). Culturing the beads under reduced oxygen conditions (2.4%) appeared to enhance redifferentiation. However addition of ascorbate to the culture medium had mixed results. This culture system can now be further adapted and modified to better enhance chondrocyte redifferentiation. This work could include combining the two conditions already tested as well as adding growth factors to the culture medium. More successful maintenance of the chondrocyte phenotype in vitro, could potentially lead to better articular cartilage regeneration both in vitro and in vivo.

The aim of this Ph.D. was to develop advanced methods for quantitative proteomics and use these methods to investigate the presence of protein biomarkers of sperm performance, differential expression of sperm membrane proteins and differential expression of E.coli proteins. Quantitative analysis of E.coli generated analytical samples that were analysed with multiple mass spectrometers and with multiple software packages. Through these samples an optimal label free quantitative proteomic workflow was generated and software was thoroughly tested to determine the optimal software to be used for data analysis on varying biological questions. Identification of protein(s) that correlate with increased or decreased fertility would be economically beneficial. Currently semen samples are subject to quality control where general movement and morphological defects are studied, but this does not always correlate with the ejaculate passing a post cryopreservation quality control check or that specific bull generating offspring. Identification of a protein or set of proteins with abundance variation in bulls of known high or low fertility would allow lower fertility bulls to be removed from the breeding programme at an early age, reducing rearing costs, and would allow longitudinal health monitoring of individual bulls. Discovery of differentially expressed proteins in the membrane of sperm with the X or Y chromosome would allow the generation of a method to separate the two sperm populations. This will be beneficial as most livestock farmers would prefer offspring of a specific sex, either to sell or replenish animal stock. Quantitative analysis of proteins present in bovine seminal plasma led to the identification and quantitative comparison of the seminal plasma proteins present in two breeds of bull, Holstein and Belgian Blue and a quantitative comparison of the seminal plasma from two domestic farm animal species, bovine and porcine. Intra species comparisons determined no quantitative variation between the two breeds, while the inter species comparison determined variation between the proteins present in both species seminal plasma and the corresponding amounts of proteins present in both species. A quantitative comparison was performed to determine the expression of proteins from two strains of E.coli, a wild type strain (MG1655) and a genome depleted strain (MDS66), this led to the confirmation of gene deletions in the genome depleted strain due to their lack of protein products in mass spectrometric analysis, and the identification of proteins that were differentially expressed due to pleiotropic effects of these genome deletions. To investigate the proteins expressed in the sperm membrane a mass spectrometer compatible enrichment method was generated and membrane proteins were identified, quantified and compared between sperm expressing X and Y chromosomes. This study did not lead to the determination of any proteins with differential expression in the X or Y bearing sperm.

The Apicomplexan parasite Toxoplasma gondii is an obligate intracellular parasite. Infection by T .gondii causes the disease toxoplasmosis, which is one of the most prevalent parasitic diseases of animals and humans. It has been 100 years since the first discovery of the parasite in 1908; research on T. gondii has been carried out in many scientific disciplines consistently expanding the understanding of this parasite. In the last ten years, the developments of EST, microarray, genome sequencing and continuing efforts towards genome annotation has centralized the focus of T. gondii research on the understanding of gene expression and gene functions on the genome scale. Equipped with the technical advances in mass spectrometry and bioinformatics, proteomics has become established as an integral component in the post-genomics era by providing first-hand data on the functional products of gene expression. In this study, three complementary proteomic strategies, 1-DE, 2-DE and MudPIT, have been used to characterise the proteome of T. gondii tachyzoites. Protein identifications have been acquired for more than two thousand (2252) unique release 4 genes, representing almost one third (29%) of the predicted proteome of all life cycle stages. Functional predictions for each protein were carried out, which provided valuable insights into the composition of the expressed proteome and their potential biological roles. The T. gondii proteomic data has been integrated into the publically accessible ToxoDB, where 2477 intron-spanning peptides provided supporting evidence for correct splice site annotation of the release 4 genome annotation. The incompleteness of the release 4 genome annotation has been highlighted using peptide evidence, confirming 421 splice sites that are only predicted by alternative gene models. Analysis has also been carried out on the proteomic data in the light of other genome wide expression data. The comparison of the proteome and transcriptome of Toxoplasma and other Apicomplexa parasites has revealed important discrepancies between protein and mRNA expression where interesting candidates have been highlighted for further investigation. A preliminary DIGE study has been developed to characterize protein expression changes in T. gondii grown in the presence or absence of glucose. In conclusion, this study has demonstrated the importance of proteomic applications in understanding gene expression profiles and regulation in T. gondii and highlighted the importance and potential of proteogenomic approaches in genome annotation process. The importance of temporal and quantitative proteomics as well as the future of systems biology has been discussed.

Canine atopic dermatitis (cAD) is a common and severe pruritic, inflammatory skin disease that can be considered a naturally-occurring, spontaneous model of human Atopic Dermatitis (hAD). The genetics of cAD are poorly understood and therefore the aim of this project was to investigate the genetic factors involved in the pathogenesis of cAD and to identify specific gene associations with cAD within and between dog breeds. It was hoped that this study would further strengthen the evidence that dogs are a suitable model hAD. Using dogs as a model to study the genetic basis of AD is advantageous because dog breeds form genetically isolated populations exhibiting strong linkage disequilibrium (LD). In contrast to humans where LD across the genome is weak (10-100 kb), domestic dog breeds have strong LD which extends over long distances (0.8 -5 Mb). This is highly advantageous in genetic studies because fewer genetic markers and smaller sample sizes are needed to find disease associations in dogs. To study the genetic basis of cAD a dual approach of candidate gene association study and genome wide association study (GWAS) was used. This gave not only a novel unbiased approach but also used information from previous studies on which gene selection was based. Therefore increasing the likelihood that the causative genes involved in cAD pathogenesis could be identified. This thesis demonstrated altered mRNA expression in 54 genes out of 22,000 transcripts by mRNA microarray in cAD. Further to this qPCR was used to confirm the microarray results and quantify gene expression in potential cAD candidate genes. This approach identified 11 genes with altered expression in cAD. The qPCR results were further correlated 2 with the clinical outcomes: Canine Atopic Dermatitis and Severity Index (CADESI-03) and number of responses on intra-dermal allergen tests. Eleven novel SNPs and 1 novel microsatellite were identified by transgenomic WAVE analysis. The microsatellite was further typed in 659 dogs and but no association with cAD was found. A GWAS with 22,362 SNPs was performed. The significant results were validated by Sequenom along with the SNPs from the candidate gene study (literature selected genes) in 659 dogs across 8 breeds. In total, 232 SNPs across 54 genes and 41 intergenic regions were genotyped on Sequenom. From this 45 putative associations were found in various breeds. A large number of these associations had relevant functions to AD and/or previous association with hAD.

Campylobacter spp. are the most common causes of bacterial gastroenteritis in humans worldwide, and although poultry and cattle are considered major sources of Campylobacter spp., infection has also been associated with dogs. In order to investigate the potential zoonotic risk to humans, dog faeces were examined for the presence of Campylobacter spp. from several different dog populations including; vet-visiting, boarding, rescue and hunt dogs. The Campylobacter spp. prevalence, and species distribution was determined for all studies, and some studies were analysed for possible risk factors for Campylobacter spp. carriage in dogs. Longitudinal studies were carried out on kennelled dogs to investigate shedding patterns, and possible transmission. All C. jejuni, and 41 C. upsaliensis isolates from these studies underwent multilocus sequence typing (MLST), along with nine C. upsaliensis isolates originating from human clinical cases, in order to identify possible sources of infection, and assess the potential zoonotic risk to humans. Additionally a pilot study was performed to annotate a plasmid as part of a C. upsaliensis genome project. The findings of this thesis found that the overall prevalence of Campylobacter spp. ranged from 0-73%, although the majority of studies had a prevalence greater than 30%. The prevalence and species distribution differed depending upon the dog population. Kennelled dogs generally demonstrated the highest overall Campylobacter spp. prevalence, whilst the greatest species diversity was found in hunt dogs. C. upsaliensis dominated in most of the populations sampled, except for two hunt kennels where C. lari and C. jejuni dominated. The prevalence of C. jejuni was relatively high in some of the rescue and hunt kennels, reaching 20% and 26% respectively, whereas in vet-visiting and boarding dogs it was relatively low, 1.2-9%. Longitudinal studies indicated that the majority of dogs entered the kennels already carrying Campylobacter spp. but when possible transmission events occurred they often involved C. jejuni. Rescue dogs appeared to be exposed to sources of C. jejuni before and after entry to the kennel, but boarding dogs were only exposed after entry. The shedding of C. jejuni in dogs appeared to be over short durations, whereas dogs that carried C. upsaliensis shed the bacterium in nearly every sample. Data suggested that dogs carried the same C. upsaliensis strain throughout the study, providing further evidence that the species may act as a commensal in dogs. Further to this no associations could be made between Campylobacter spp. carriage, specifically C. upsaliensis, and disease in dogs in any of the studies. Younger dogs were significantly more likely to carry C. upsaliensis than older dogs in the vet-visiting study (OR for every additional month 0.99) and living with another dog carrying Campylobacter spp., was significantly associated with Campylobacter spp. carriage in dogs. A considerable amount of genetic diversity was observed within the C. jejuni and C. upsaliensis isolates originating from dogs, and MLST results suggested that strains of both species were the same, or highly similar to strains found in humans. This suggests that there may be common sources of infection for both humans and dogs and that dogs remain a potential zoonotic risk to humans. Although only a small number of household dogs carry C. jejuni, infected dogs should still be considered a potential zoonotic risk to humans, particularly if the dogs originate from kennelled or hunt kennel populations where the prevalence may be higher. Dogs remain a significant reservoir of C. upsaliensis, but the relationship between the presence of C. upsaliensis and gastroenteritis in both dogs and humans is still unclear.

The aim of this thesis was to investigate the mechanisms that mediate the effects of beta-adrenergic agonists (BA) and Growth Hormone (GH) in sheep, by examining the changes in skeletal muscle transcriptome and blood metabolome in order to identify the predominant metabolic mechanisms by which muscle hypertrophy was mediated. Male lambs (120 days old) were all fed a high protein/energy feed ad-libitum, with the GH group (n=10) receiving a single subcutaneous injection of bovine GH (3.75mg/kg body weight, POSILAC, Monsanto) on day 1; the BA group (n=10) receiving BA (cimaterol) at 10mg/kg feed, whereas the control group (CO, n=11) only had the ad-libitum feed. After 6 days sheep were slaughtered, plasma and samples of the Longissimus dorsi (LD), Supraspinatus (SS) muscles and liver were collected. The effect of treatments on the LD transcriptome was assessed on a subset of samples (n=3 from each treatment) via a cross-species approach using the Affymetrix Human U133+2 GeneChip array (47K human microarray). Verification of identified differentially expressed genes and proteins was by quantitative RT-PCR or western blotting, respectively, on all animals. Metabolomics analysis of plasma samples was carried out by Metabolon Inc. (USA) using GC/MS and LC/MS/MS platforms. BA, but not GH, significantly (P<0.05) increased muscle weights and this was associated transition to large fast-glycolytic muscle fibre types. In GH, but not BA treated animals, there was an increase in liver weights (P<0.001). This was associated with an increase in the whole liver content of glycogen (P<0.001), protein (P<0.01), and lipid (P<0.05) content. Analysis of the LD transcriptome of the treated sheep identified 477 and 316 transcripts were significantly altered (P<0.05 and 1.5 fold change) by BA and GH respectively, relative to controls. This muscle was selected as it is a commercial valuable muscle and is commonly used for muscle biochemical studies therefore this would allow us to make comparisons to other studies, including our own. In addition it is a fast glycolytic muscle fibre type there could be compared against SS muscle (oxidative muscle fibre type). BA decreased the expression of genes involved with oxidative phosphorylation and upregulated those serine biosynthetic pathways. Subsequent qRT-PCR analysis showed a BA induced increase in expression of phosphoglycerate dehydrogenase (PHGDH) (P<0.05) and phosphoserine-aminotransferase (PSAT) (P<0.05) mRNA in both LD and SS but not liver. In LD there was also an increase (P<0.001) in PHGDH protein in muscle from BA treated sheep relative to GH treated sheep. Up-regulation of this pathway has been previously reported in cancer cells which has a tendency to be associated with an increase in gene expression of a specific isoform of the glycolysis enzyme pyruvate kinase (PKM2) which has reduced activity. Total PKM and PKM1 and PKM2 isoforms were increased in the SS and LD of BA treated sheep (P<0.05). Previous studies in cancer cells have suggested that increases in serine synthesis are mediated by changes in PKM2 expression and associated enzyme activity. The lack of a differential increase in PKM2 suggested that the regulation of muscle PK in BA treated animals was not critical to the potential increase in serine synthesis capacity. No clear change in PKM gene expression suggested this was not the mechanism by which the serine synthesis pathway was stimulated. There was an increase (P<0.05) in the expression the mitochondrial form of phosphoenolpyruvate carboxykinase (PCK2) in the LD of BA treated sheep, which might be expected to increase gluconeogenic potential thereby increasing intermediates that could be used for serine synthesis. There was no effect of this gene on sheep treated with GH. An increase in the gene expression of asparagine synthetase (ASNS) was also seen in the muscles of BA but not GH treated sheep (P<0.001) and there was no effect on their livers, which further suggested that BA was influencing the production of nonessential amino acids. Metabolomics analysis showed that products of triacylglycerol breakdown, glycerol and free fatty acids, were all elevated in the plasma of both BA and GH treatments, indicating lipolytic effects but the increase in the free fatty acid profile were more pronounced with GH treatment (P<0.05). Likewise GH rather than BA had a greater impact on elevating plasma glucose and associated metabolites such as pyruvate (P<0.05). There was no effect of either treatment on plasma serine or asparagine concentrations. However there was a decrease in glycine (P<0.05) and glutamine (P<0.05) in GH relative to control, with BA decreasing histidine (P<0.05) and methionine (P<0.01) relative to control. Cell culture experiments were carried out in the myogenic C2C12 cell line to determine if the genes associated with the GH and BA response in sheep were affected during myogenesis and whether there was an effect of des (1-3) IGF-I and dibutyryl cyclic adenosine monophosphate (dbcAMP) that stimulates GH and BA signalling pathways respectively. During differentiation, without treatment, gene expression of PHGDH and PSAT enzymes declined (P<0.05), which might be expected as cells move from a proliferative to a terminally differentiate state. There was no clear effect of treatment on genes associated with the serine synthesis pathway suggesting that the effects of BA, in particular, are on muscle fibres rather than differentiating cells. Of the two growth promoters examined in this thesis BA appears to be the most potent in skeletal muscle. A clear effect of this agent was an increase in the gene expression of the serine biosynthetic pathway, which has been shown to be upregulated in various cancers and, in this pathology, is thought to be a novel mechanism for hyperplastic growth. The associated changes in the expression of genes such as ASNS and PCK2 indicate that their co-ordinated upregulation could be mediated via endoplasmic reticulum stress response mechanisms. Unlike GH, BA does not appear to have a major effect upon the systemic mobilisation of nutrients, but instead seems to targets muscle fibres, activating muscle biosynthetic pathways that potentially provide the substrates required for growth.

This thesis develops the scant existing literature which explores the ethical justifications for living animal donation in the veterinary setting and contributes to the considerable research on social and ethical aspects of human living donation. The work argues that whilst some justifications for human living donation are not-transferable, others may be adapted and applied to the veterinary setting. The unique social context of veterinary donation is analysed, using novel empirical analysis to refine and contextualize the ethical arguments made. The research methods entail an innovative comparative ethical analysis and a qualitative empirical study which are integrated using an empirical bioethics approach. The main justificatory arguments for human living donation are identified as informed consent and donor best interest. These arguments stem from a human’s acknowledged rights to bodily integrity and the human medical professional’s duty not to harm their patients. The reduced capacity of animal donors means that donor consent arguments are not directly transferable to the veterinary setting and an animal owner’s informed consent is shown to have reduced moral authority. Whilst animal donors may lack comparable bodily rights, veterinary living donation practices can conflict with a veterinary surgeon’s professional obligations. A comparable justification for living animal donation is argued to exist only when the procedure is in the donor animal’s best interest. An empirical analysis of canine blood donation to a UK animal blood bank develops understanding of the social context of living animal donation. The analysis indicates that animal owners may not always be motivated by the best interest of donor animals; furthermore, consistent themes such as trust, optimism and human-animal comparisons have implications for the quality of a donor owner’s consent. These ethical and empirical findings are used to construct an ethical framework for living animal donation with detailed provisions for owner consent, donor best interest, donor harm and benefit, recipient benefit, fairness and transparency. The ethical framework is used to argue for the development of regulatory approaches to companion animal blood banking and feline renal transplantation in the UK. The work also has wider implications for veterinary ethics, veterinary policy and the social and ethical understanding of human living donation.