Human epidemiological studies have indentified obesity as an independent risk factor for renal disease. In addition, maternal nutrient restriction (MNR) during gestation results in a series of adaptations that may predispose those offspring to obesity and hypertension. Recent reports demonstrated that obese sheep exposed to MNR during early to mid gestation have a predisposition to ectopic lipid deposition in the heart and a rise in necrotic adipocytes, which are markers of severe metabolic dysfunction. Surprisingly, in this model of MNR, the renal tissue of those offspring showed an apparent reduction in cell apoptosis. However, renal diseases associated with obesity have a slow progression and their mechanisms are not completely understood. In the light of these results, the main hypothesis of my thesis is that the renal amelioration observed in those nutrient restricted (NR) obese offspring is a product of post-injury responses, inducing scarring and others adaptations to obesity. Therefore, some of the main regulatory factors in renal and perirenal adipose tissue (PAT) development were analysed in seven-day-old and one-year-old obese sheep offspring exposed to MNR (3.5 KJ/days) from 30 to 80 gestational days (term ≈ 145 days). At one week of age, the renal composition and gene expression showed small changes between NR offspring and those born to control mothers. However, in PAT of NR offspring, an increased in expression of the methyltransferase DNMT-1 and a decrease in mRNA abundance of IGF-2 were observed. At six months of age, obesity onset was accompanied by raised plasma cortisol and leptin concentration in NR offspring compared to control. By one year of age, whilst plasma leptin concentration was similar between the obese groups, in the PAT of the NR offspring there was an increase in gene expression of pro-inflammatory factors and DNMT-1, suggesting advanced adipose tissue remodelling. In kidney, regardless of in utero diet, obesity induced similar amounts of oxidative stress, activation of cellular proliferative factors and collagen deposition. Although, both obese groups had equal activation of pro-apoptotic factors (e.g p-53 and Bax), renal iron and mRNA abundance of the death receptor, Fas only increased in obese offspring born to control fed mothers. A major finding in the NR kidney was increased ectopic triglyceride deposition, indicating early onset post-injury in response to sympathetic activation and lipotoxity. The main conclusion of my thesis is that functional changes observed in the adipose tissue lead the kidney to an initial cycle of cell proliferation, apoptosis and arrest, followed by tissue remodelling, characterised by the presence of collagen. However, this adaptation to obesity is accompanied by an increase in lipid deposition in the kidney of the NR group that may be a sign of an advanced state of metabolic dysfunction.

BACKGROUND Chronic respiratory inflammation is a major cause of recurrent airway obstruction (RAO) in mature horses. RAO has aetiological and clinical similarities to human asthma. Remodelling of airway tissue after bronchial inflammation is evident in RAO and human asthma. Severe asthma in humans is associated with defective lipoxin A4 (LXA4) synthesis and abnormal expression of specific lipoxin receptor (ALX). Arachidonic acid metabolite LXA4 modulates acute inflammation in a number of species and models of acute inflammation. Dysfunctional LXA4 synthesis and/or expression of the ALX receptor may contribute to the chronic inflammatory response seen in asthma. Abnormal LXA4 and/or ALX expression may also be present in horses with RAO thereby promoting airway remodelling. HYPOTHESIS Equine inflammation and its resolution can be modelled in vitro using respiratory epithelial and smooth muscle models. AIMS To develop an in vitro equine respiratory model of respiratory epithelium and airway smooth muscle. Characterise the response of this model to exogenous lipolysaccharide (LPS) and lipoxin A4 on selected molecules associated with inflammation and inflammatory resolution. METHODS Primary equine tracheal epithelial (ETE) cells were obtained from trypsindissociation of tracheal epithelial tissue derived from healthy horses with no sign of inflammation at post-mortem. Primary airway smooth muscle (ASM) cells were cultured from explants of equine trachealis muscle. Near confluent (70-75%) ETE and ASM cells were stimulated with 0.1, 10 and 100µg/ml LPS at 4 and 24 hrs (ETE cells) or 12, 24 and 72hrs (ASM cells). Expression of COX-2 mANA in these cells was used to determine a suitable time point and LPS concentration to induce inflammation. Inflammatory resolution was then examined by comparing the selected time points and LPS concentrations with the response of ETE and ASM cells to 15 minutes incubation with 100µM LXA4 and LXA4/LPS treatment. Finally, the inflammatory relationship between the epithelium and smooth muscle layer was examined by a co-culture model of ETE and ASM cells. Conditioned media from ETE cells treated with 0.1µg/ml LPS, 100µM LXA4 and LXA4/LPS treatment for 24hrs was used to culture ASM cells for a further 24hrs. To examine inflammation and its resolution, selected genes, namely ALX, toll-like receptor 4 (TLA-4), tumour-necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were amplified and quantified by real time PCR. ALX and COX-2 proteins were monitored by Western blot and nitric oxide (NO) levels measured by fluorometric analysis. Values for statistical analysis of ETE and ASM cells were obtained with a two and one-way ANOVA and Tukey's multiple comparisons test. RESULTS Primary ETE and ASM cells were identified by positive immunocytochemical staining with pan cytokeratin-26 and alpha-smooth muscle actin respectively. Treatment of ETE cells with 0.1µg/ml LPS for 24hrs increased iNOS and COX-2 mRNA levels, and significantly increased mRNA expression for ALX, TLR-4 TNF-α and IL-1 j β mRNA (p values <0.05). ASM cells incubated over 72hrs incubation with 0.1119/ml LPS showed increased expression of selected genes but only significant increases in COX-2 mRNA were observed (p values <0.05). Incubation of ETE and ASM cells with LXA4 did not significantly increase or inhibit inflammation as measured by real time PCR, Western blot and fluorometric anlaysis. Western blotting showed some inhibition of COX-2 protein in ASM cells but not ETE cells at 72hrs after LXA4 treatment. Fluorometric analysis of NO levels in ETE and ASM cells showed no significant difference after treatment in either cell type. No noticeable evidence of inflammation or inhibition of inflammation was observed in the co-culture model of ETE and ASM cells. CONCLUSION It was concluded that an in vitro equine model of respiratory epithelium and smooth muscle was successfully established. It was possible to induce partial inflammation in ETE and ASM cells but inflammatory resolution could not be definitively shown in either cell type.

This thesis presents research on the dynamics and control of porcine reproductive and respiratory syndrome (PRRS) caused by the PRRS virus (PRRSV) in the pig population of Great Britain (GB). The roles of the metapopulation of pig herds (metaherd) and individual herd characteristics are examined, and different control and intervention strategies assessed. A novel stochastic model of a metaherd was created, incorporating the births, deaths, slaughter, culls and movement of pigs within and between herds. The metaherd was structured to have characteristics representative of the GB metaherd: the distribution of herd sizes, ‘source’ herds per herd, and numbers of pigs moved per movement. The metaherd was arranged into a typical pyramidal structure. A stochastic infectious process of PRRSV was included. Herd size was found to be key to within herd persistence of PRRSV, with infection failing to persist in smaller (~250 sow) herds. Fadeout did not occur in larger herds once infection established in the rearing herd. PRRSV reduces productivity of herds and the metaherd. There was variability in productivity both between herds and within herds over time. The number of source herds did not influence the dynamics, persistence or prevalence of infection within a herd. Breeding herd production was further decreased by PRRSV when the herd also had a rearing herd (breeder finisher). The model was extended to test the effects of control and intervention strategies. Vaccination effect increased with herd size, and reduced variability in production. Vaccination in small herds was ineffective in increasing production due to PRRSV failing to persist regardless of vaccination. Vaccination of the breeding herd produced higher gains per vaccine dose than vaccination of the rearing herd only. Vaccination of the rearing herd only resulted in higher total herd and metaherd gains, with less variability. Partial de-population combined with vaccination increased the probability of increasing herd performance unless the herd was small (<100 sows) or very large (>1000 sows). Results highlighted the value of modelling to support the decisions of individual farmers to vaccinate and partially depopulate, showing that the optimal decision is influenced by the herd size. Results also demonstrated that the decision to introduce interventions is different for individual farmers.

Constructive alignment (CA) describes an approach to education where teaching, learning and assessment are aligned to allow the learner to achieve the intended learning outcomes (ILOs) of the course. Assessment has a strong influence on learning and therefore the potential to have either a positive or negative impact on CA. The aim of the research in this thesis is to explore the effects of assessment on CA. The context for the research is the final year of study within the School of Veterinary Medicine and Science (SVMS), at the University of Nottingham. Five mixed methods studies were conducted utilising questionnaires, semi-structured interviews and focus groups. In study one the ILOs of the course were defined and subsequently used in study two as a framework for an alumni survey to determine how well prepared graduates feel for clinical practice. Studies three and four investigated the impact of assessments on learning behaviour, namely multiple-choice questions (MCQ), directly observed procedural skills (DOPS) and the script concordance test (SCT). Finally in study five the influence of the transition to practice on learning behaviour during final year was explored. Graduates felt well prepared by the SVMS curriculum for a career in practice with respect to all ILOs. However, assessments were not rated so highly when considered alone. DOPS and to some extent the SCT are considered to be authentic assessment formats and encourage a deep approach to learning. The MCQ in this context results in surface learning strategies being adopted. The imminent transition to practice has a positive effect on learning behaviour, however this conflicts with the preparation required for final year examinations. Elements of the assessment strategy that have a positive and negative impact on CA are identified and discussed. Changes to the curriculum are proposed to enhance CA and ease the transition to practice.

Routinely collected herd management data in a variety of formats were collated from 468 dairy herds, and novel objective measures of data recording quality were developed and applied. This revealed that there was a substantial amount of variation in data quality between herds, and the vast majority of herds failed to meet the threshold level for at least one of the data quality measures used. Analysis of trends in reproductive performance across the herds with good quality fertility event recording suggested that their fertility was generally declining through the first half of the 2000s, but there was some evidence that improvements in submission rate were beginning to reverse this decline in the later years studied (up to 2007). Associations between reproduction and two endemic diseases common in dairy cattle (mastitis and lameness) were explored using multilevel discrete time survival modelling, and probabilistic sensitivity analysis (PSA) used to contextualise and illustrate the results. In both cases, statistical modelling revealed significant and sizeable associations between disease events and reproductive outcomes at lactation level. However, simulation and application of PSA showed that a herd’s incidence rate of either disease was highly unlikely to influence its overall reproductive performance to a clinically relevant degree when other inputs to herd fertility were also considered. Factors associated with the proportion of serves leading to a pregnancy (pregnancy rate) were explored using multilevel logistic regression modelling. This revealed that relatively little of the variation in herd pregnancy rate is explainable by routinely recorded milk recording data (including constituent concentration in early lactation as well as daily and lactation yields). A large amount of the unexplained variation was revealed to be at herd level and very little at cow level, suggesting that investigation of herd management practices associated with pregnancy rate would be rewarding.

The small East African shorthorn zebu (EASZ) is an indigenous stable zebu x taurine admixed cattle population highly adapted to the local African environment, i.e. disease resistance, thermotolerance, sustain themselves on poor feed quality, in comparison to the exotic productive cattle. Understanding the genetic control of their adaptations will help sustainable breeding improvement programs, both within populations and through crossbreeding. In this thesis, genome-wide SNP data, from two commercially available SNP arrays; the Illumina BovineSNP50 BeadChip v.1 and the Illumina BovineHD BeadChip, and full genome sequence of 10 pooled EASZ samples were used to define candidate regions with signatures of positive selection in the genome of EASZ. In chapter 2, the low-density SNP chip has defined 24 candidate regions (21 in the autosomes and three in the sex chromosome X (BTA X)) by two Extended Haplotype Homozygosity (EHH)-based (intra-population iHS and inter-population Rsb) analyses and an inter-population FST approach. Characterising the EASZ genome with high-density genome-wide SNP analyses (meta-SS, Rsb, iHS and ΔAF) and pooled heterozygosity (Hp) sequence analysis, in chapters 3 and 4, identified a total of 266 autosomal and 20 BTA X candidate regions. Analysing genome-wide SNP data of zebu cattle populations from Uganda and Nigeria classified some of these regions as East African zebu-sharing and East and West African zebu-sharing candidate regions. Genes and quantitative trait loci (QTL) associated with adaptive traits (e.g., reproduction, immunity and heat stress) were found within the identified candidate regions. Moreover, Nine non-synonymous variants were found in five candidate genes and considered as possible causative mutations under selection. Finally, the mitochondrial DNA (mtDNA) of 13 EASZ were investigated and affiliated to T1a, T1b and T1b1 sub-haplogroups. No selective advantage was found to be associated with taurine mtDNA over the zebu type. Although purifying selection is the main selective factor influencing African cattle mtDNA, in Cox-2 gene of the T1b/T1b1 sub-haplogroups a non-synonymous mutation may confer a selective advantage. The results of this thesis are just the first milestone to define the valuable genetic material in EASZ, which should be conserved in future composite cattle upon informed selective crossbreeding and/or the recently introduced gene modification techniques (TALEN and CRISPER/Cas9).

Obesity is a growing problem for humans and their horses and ponies, yet emaciated animals still form an important part of the work of equine welfare charities. Non-invasive yet reliable methods of assessing equine body fat are required in order to promote management procedures to improve animal welfare. The overall objective of this work was to investigate the application of a horse-specific body condition scoring system in ponies in order to either validate or revise it, or even replace it with a novel system if necessary. Seasonal differences in appetite, body mass (BM) gain, body condition score (BCS) change and direct (ultrasound) and indirect (morphometry and D2O dilution) measures of body fat were explored in two relatively homogeneous groups of mature Welsh mountain pony mares, studied over summer (June –September 2007) and winter (January-April 2008). The ponies in each group were paired so that, at study outset, two ponies were ‘thin’ (BCS, 1-3/9); two were ‘moderate’ (BCS, 4-6/9); and two were ‘obese’ (BCS, 7-9/9). The greatest appetites (peak 4.6% BM as DMI), increases in body mass (~60kg) and in BCS (~3 points) were recorded for ponies of non-obese outset condition in summer (non-ObS, n=4). For ponies of non-obese outset condition in winter (non-ObW, n=3), appetites peaked at 3.5% BM as DMI, BM increased by a mean of 50kg and BCS increased by ~2 points over the 3 month study period. Appetites for all obese (Ob, n=4) ponies remained almost constant (~2% BM as DMI; peak 2.3% BM as DMI) and minimal changes in BM (n=3) and BCS (n=4) were recorded, regardless of season. All measures of body fat increased for non-Ob ponies (non-ObS>non-ObW). An exponential relationship was determined between body fat content and BCS and for values > 6, BCS was not a useful predictor of actual body fat content. The endogenous circannual mechanisms to encourage winter weight loss were insufficient to prevent the development of obesity in ad libitum fed ponies. The effects of dietary restriction to 1% BM as DMI were studied in a group of 5 overweight or obese mature pony mares (BCS 5.6-8/9). Those measures outlined above were likewise recorded. All ponies remained healthy throughout the 12 week trial. Overall, BM reduced by 1% of outset BM per week. Approximately half the lost BM comprised fat, but fatter animals lost relatively more fat. Despite an average loss of ~30 kg BM, BCS did not change appreciably suggesting that BCS was a relatively poor indicator of early weight/fat loss in obese ponies. The relationships between BCS, direct (ultrasonic) and indirect (morphometric and D2O dilution derived) measures of body fat and actual body fat content determined by both physical dissection and chemical cadaver analysis were explored using 7 donated mature Welsh pony mares (BCS 1.25 to 7/9). Body ‘fat’ content (dissected white adipose tissue or chemically-extracted lipid fractions) was the most variable constituent of the cadavers (up to 1/3rd body mass), and was non-linearly related to BCS. From these studies, it was also possible to validate the D2O dilution technique for the measurement of total body water and fat in ponies. Contemporaneously gathered data for BCS and body fat (D2O dilution) from 48 separate observations were explored statistically. A non-linear association between body fat content and BCS was confirmed, with a cut off value of BCS 7/9, above which BCS was less useful for determining body fat content. A novel BCS system was created and is undergoing field trials.

Equine laminitis manifests as a painful condition of the foot and is characterised by disruption of the dermo-epidermal lamellar bond within the equine hoof. Chronic pain in laminitis leads to debilitation and if unchecked euthanasia of the horse and is therefore a significant welfare issue for horses worlwide. P2X receptors are shown to have a pivotal role in pain pathways as well as other tissue functions making them attractive candidates for research in naturally occurring diseases such as equine laminitis. In this study, the presence and distribution of P2X receptors in tissues of the equine digit and its associated vasculature and innervation was investigated. The investigation was extended to tissues from horses suffering from naturally occurring chronic active lamintis in an attempt to identify changes in distribution or expression of the receptors in these tissues. Finally, the functionality of the P2X receptors expressed in equine nervous tissue was investigated. Gene expression (mRNA) for all seven receptors was found in the studied tissues and protein expression for P2X1-3 and 7 receptor subtype proteins in these tissues was confirmed with Western blot analysis. Immunohistochemical analysis of P2X1-3 and 7 receptor subtypes distribution showed differential cellular distribution within normal equine tissue and differential distribution and expression between normal and diseased tissues too. In particular, in a horse with shorter duration of chronic laminitis (1 month), P2X3 receptor subtype immunostaining in DRG neurons presented a marked increase in large neurons which coincided with a decrease in the proportion of these neurons in the DRG. Also, horses suffering from chronic laminitis displayed expression of P2X3 receptor subtype in smooth muscle cells of the palmar digital artery which was absent in normal horses. In chronic laminitic hooves, P2X7 receptor subtype showed particularly strong staining in partially keratinised cells. Finally, intracellular calcium measurements provided evidence for the presence of functional purinergic and P2X receptors in equine spinal cord and DRG cells, respectively. The expression of P2X receptor subtypes in key equine tissues for laminitis and chronic pain in laminitis and their differential expression in laminitic tissues highlights a potential role in chronic laminitis molecular mechanisms. Further research in this area may provide insights into putative targets for disease modulation and pain management in chronic laminitis and other conditions of the horse associated with chronic pain.

Canine infections with antimicrobial resistant (AMR), particularly multi-drug resistant (MDR) bacteria are increasing, severely limiting therapeutic options, and representing an animal health issue. In addition, with potential transfer of AMR bacteria between dogs, their environment, humans and other animals, there may also be a public health risk. Commensal isolates can be a source of clinical infections and studies reporting the prevalence of AMR and risk factors for such isolates are important. Furthermore, one of the most significant impacts upon commensal bacterial populations is antimicrobial therapy that may select for pre-existing AMR organisms or transmission of resistance determinants. The aim of this work was to investigate AMR amongst canine commensal bacterial populations and the effects of five different antimicrobials, authorised to treat dogs in the UK, on these populations both during and after therapy. Three groups of dogs were enrolled: healthy non-antimicrobial treated, non-vet visiting dogs (n = 28), to investigate longitudinal carriage of faecal E. coli; healthy non-antimicrobial treated, non-vet visiting, dogs (n = 73) and antimicrobial treated, non-hospitalised dogs (n = 127) to investigate longitudinal carriage of mucosal staphylococci and faecal E. coli. Staphylococci and E. coli isolated from swabs (nose/perineum) and faecal samples respectively, were tested for phenotypic AMR and carriage of resistance genes by PCR assay. Staphylococci were assigned to species by PCR assay (nuc gene), MALDI-TOF-MS and sequencing (tuf gene). Healthy dog E. coli underwent phylo-typing, and a selection of longitudinal healthy dog E. coli isolates were genotyped. Questionnaire data were used to formulate independent variables. Statistical analysis included Pearson’s Chi-square, survival analysis and multivariable logistic regression; multilevel for clustered data. The prevalence of meticillin-resistant (MR; 42%) and MDR staphylococci (resistant to ≥ 3 antimicrobial classes; 34%) was high amongst healthy dogs, however MR-coagulase positive staphylococci were not detected. The most common species detected was S. epidermidis (52% of dogs), followed by S. pseudintermedius (44%). S. aureus was only detected in a small number of dogs (8%). Faecal E. coli with AMR to at least one tested drug (63%), MDR (30%) and AmpC-production (16%) were prevalent in healthy dogs, however ESBL-producers (1%) were rare. Healthy dogs carried a predominance of phylogenetic group B1; group B2 E. coli isolates were less likely to have AMR while group D isolates were more likely. Carriage of E. coli with AMR to at least one tested drug was common and persistent, whereas MDR, AmpC- and ESBL-types were intermittent or transient. Genotyping revealed high intra-dog diversity with frequent new genotypes and resistance phenotypes detected over time. AMR staphylococci and E. coli were detected in more dogs following antimicrobial treatment than baseline, but generally returned to pre-treatment levels within three months. Eating raw meat/animal stools, living with other dogs/in-contact humans or pets that had been hospitalised/in-contact humans working with farm animals were associated with the detection of AMR canine commensal bacteria. In particular, following treatment with beta-lactams or fluoroquinolones there was a significant increase in the detection of MDR and AmpC-producing E. coli or MR- and MDR staphylococci, respectively. However significant differences were not detected at one month after the end of treatment. Antimicrobial therapy is a risk factor for antimicrobial resistant commensal bacteria in dogs and recovery may take up to three months after the end of treatment. This highlights the importance of prudent antimicrobial use and prescribing guidelines. However other factors, such as diet, in-contacts, co-selection and bacterial fitness may be involved in the carriage of resistant bacteria and should be considered.

Feline calicivirus (FCV) is an important veterinary pathogen of domestic cats. It is highly variable and rapidly evolving such that many different strains of varying pathogenicity exist from avirulent, to mildly virulent and hypervirulent. FCV is one of only a small number of viruses within the Caliciviridae to propagate readily in vitro. This property has made FCV a model for the study of the viruses of the calicivirus family. However, relatively little remains known about the way in which these viruses affect host cellular mechanisms. Despite the increasing use of post-genomic technologies in other areas of science, what is known about the effects of calicivirus replication upon host cells has generally relied on relatively conventional technologies to target known defined pathways. In order to investigate FCV modulation of the host cell proteome a 2D-DIGE experiment was performed upon a single viral strain (FCV-F9) with four time points analysed. The results of this analysis showed that 30 host proteins were differentially expressed in the cells infected with FCV-F9 and of these 30 proteins, 14 were successfully identified using mass spectrometry and 11 of these had cytoskeletal proteins within their identifications. To further evaluate these host proteome changes and to explore whether variations in strain pathogenicity were associated with varying affects in the host proteome, a second 2D-DIGE experiment was performed upon four strains of FCV isolated from cats exhibiting a range of clinical signs. As in the first experiment, host cytoskeletal proteins appeared to be targeted for modulation during infection. Overall, this experiment uncovered 131 modulated host proteins with 61 proteins successfully identified by mass spectrometry and 16 of these had cytoskeletal proteins within their identifications. Also found within this experiment was evidence of the putative virulent FCV strain UKOS-A having a different affect upon 14 of the differentially expressed proteins to the other three FCV strains tested. UKOS-A was found to down-regulate these proteins whereas the other strains had an up-regulatory effect, these proteins could be a potential marker of hypervirulence but would need further study to confirm their validity. To follow the modulation of the identified host cytoskeletal proteins, immunofluorescent staining coupled with confocal microscopy was used to track FCV capsid protein and two cytoskeletal proteins vimentin and tubulin. Results showed that during infection with the four FCV strains tested both vimentin and tubulin were both significantly altered during infection with the intensity of fluorescent staining increasing in all cases. To complement the proteomic analyses, an experiment designed to discover differentially expressed gene transcripts was conducted using RNA-seq technology. Overall, there were 354 genes found to be differentially transcribed within the infected cells at both four and seven hrs post infection (p.i.) with the majority of modulated genes experiencing down-regulation at four hrs p.i. and up-regulation at seven hrs p.i.. Once again, genes within the cytoskeletal regulation pathway were up-regulated alongside genes from the apoptosis regulatory pathway and immune system response pathways. To further characterise the putative virulent strain used in earlier studies, attempts were made to sequence the rest of this virus using polymerase chain reaction and conventional Sanger sequencing technologies. Although almost 50% of the UKOS-A strain was sequenced, the sequence obtained was no more similar or different to other virulent systemic disease (VSD) causing strains. Overall, the most marked effects of FCV infection upon the host were found amongst the cytoskeletal genes and proteins which have been demonstrated to undergo extensive modification. This is a completely novel finding in this family of viruses. Further studies will be needed to identify the consequences of the identified cytoskeletal changes to both the host and the virus.