The equine sarcoid is the most commonly detected skin tumour in equidae worldwide and has been reported in horses, donkeys, mules and zebra. Sarcoids can be defined as locally aggressive, fibroblastic, benign tumours of the equine skin and although they do not metastasise, they exhibit variable clinical presentations ranging from small alopecic areas to rapidly growing, ulcerated fleshy masses. Clinical behaviour may also vary, from aggressive infiltrative growth to spontaneous regression. Diverse treatment modalities have been reported, and these have been used separately, or have been combined, with variable efficacy. No single therapeutic approach has been found to be universally successful. Although rarely life-threatening, equine sarcoids can have important welfare and economic implications. There is a large body of evidence now supporting the hypothesis that bovine papilloma virus (BPV) is the aetiological agent of equine sarcoids and our understanding of the general pathophysiology of the disease continues to progress; however, several fundamental aspects of the disease remain unclear. In the first chapter, the clinical aspects of the disease are discussed and evidence to support a viral aetiology is presented, along with background information regarding papilloma virus infections in the natural host. An epidemiological overview of the disease that considers the most current theories and understanding of equine sarcoids is also given. Chapter II describes the materials and methods used in the course of carrying out the investigations detailed in Chapters III, IV and V. The aim of Chapter III was to investigate the potential role of flies as vectors in transmission of equine sarcoids between infected and susceptible animals. We found that BPV-1 DNA can be detected in flies trapped from areas where animals with BPV-1 infected sarcoids are housed. This study provides evidence to support the hypothesis that flies may therefore be significant in disease transmission. Such evidence will be of benefit in formulating management control strategies for fly control measures that will reduce the incidence and spread of equine sarcoids. The work described in Chapter IV was carried out to investigate the hypothesis that the development of the sarcoid tumour arises primarily through inhibition of apoptotic pathways by viral gene products. Assessment of DNA-damage-induced apoptosis in BPV transformed equine fibroblasts revealed that sarcoid derived cells and BPV-1 transfected fibroblasts are less resistant to apoptosis than normal, BPV-negative control equine fibroblasts, but are more likely to recover from DNA damage and continue to grow and divide. Further research was directed towards identifying the viral genes responsible for conferring resistance to apoptosis through siRNA knock-down and monitoring expression of endogenous cellular proteins known to be involved in apoptotic pathways. Using an siRNA targeted to a particular viral protein, we were able to reduce the ability of the BPV transformed cells to survive after DNA damage. Potentially, this information can now be used to develop novel therapeutic strategies. The final chapter describes the results of a study in to the expression of the p53 protein in sarcoids in vivo and in BPV transformed cells in vitro in tissue culture. In vivo, analysis of 51 equine sarcoid lesions showed that 48% of tumours are associated with nuclear p53 protein expression and that high levels of p53 were associated with clinically aggressive tumour types (fibroblastic). Interestingly, we observed high levels of cytoplasmic p53 staining with no nuclear staining in one tumour in vivo. In vitro, p53 mRNA levels were assessed in BPV transformed and BPV negative fibroblasts. The half-life of the p53 protein, the cellular location and functionality were also investigated in vitro. The results showed that BPV transformed equine fibroblast cells also exhibit increased nuclear p53 protein expression and one cell line (EqS04b) showed a cytoplasmic staining pattern similar to that observed in vitro. However, the abnormal level and location of the p53 does not appear to affect the transactivational functionality of p53 since p21 expression was induced by exposing the cells to UVB irradiation.

The principal aim of this study was to investigate the epidemiology of recurrent airway obstruction (RAO) in horses in Great Britain using risk-screening questionnaires (RSQ). Three processes were used to aid construction of the RSQ for RAO, namely: a review of the scientific literature, a survey of equine practitioners in the UK and a modified Delphi consultation with experts in the field of RAO. The demographic information, generated by the questionnaire, enabled investigation of risk factors associated with the disease using multilevel, multivariable logistic regression. Two models were constructed. The first related to host and environmental risk factors and the second explored the effect of early life factors. The host and environmental model identified an increased risk of RAO in association with increasing age and the horse residing in an urban or semi-urban environment. There were also some associations that were contrary to what would be expected from knowledge of the aetiology of RAO. In particular, horses fed soaked (wet) hay had increased odds of having RAO, whilst horses fed dry hay had decreased odds. The early life model identified an increased risk of a horse having RAO if its owner had acquired them after the age of two years or that in early life it had been fed hay or had a respiratory infection. The final stage of the study was to develop and assess an educational package for horse owners regarding the disease. RAO appears to be worryingly prevalent in the horse population of Great Britain; a real concern in terms of welfare. Much can be done to alleviate this chronic disease by controlling a horse’s environment to reduce respiratory challenge. Greater emphasis could be placed on assisting horse owners in making this transition by providing detailed guidance.

The gene dense major histocompatibility complex (MHC) region, present in all jawed vertebrates, encodes molecules involved in self-non-self discrimination and the binding and presentation of antigenic peptides to T cells during the adaptive immune response. Variation in MHC genes is thought to be driven largely by pathogen-mediated selection, with diversity at MHC loci believed to benefit populations by allowing responses to rapidly evolving disease pathogens. However, in economically important dairy cattle, there are concerns that intensive selection for production and fitness traits may override natural selection. It had been hypothesised that these focussed dairy breeding practices may lead to a reduction in MHC diversity and leave cattle populations susceptible to new disease pathogens. The purpose of this study was to estimate current levels of MHC class I diversity in the UK Holstein-Friesian dairy cattle population, primarily through the assessment of diversity in bull populations with genetic input into the UK herd. In a sample of Canadian Holstein bulls, levels of class I allelic diversity were low given the size of the population sampled, but no significant loss of diversity over a twenty year period of selection was detected. Simulations of gene flow implicated trait selection as an influential force shaping diversity in the Canadian Holstein bull population. Haplotypes detected at high frequency were often negatively associated with selection traits indicating the action of heterozygote advantage. A SNP-based assay has been designed to facilitate rapid detection of common haplotypes and thus enable breeders to make more efficient selective breeding decisions whilst also maintaining MHC diversity in cattle populations. Investigations of class I diversity were expanded to incorporate the British Friesian bull population which were shown to have a markedly different pattern of class I diversity to that observed in the Canadian Holstein sample. A number of novel allele sequences and haplotypes were detected in the British Friesian bulls, the characterisation of which has contributed to our knowledge of the mechanisms driving diversity in the cattle class I region. MHC class I typing data from two bull populations and statistical analysis of trait associations with MHC haplotypes provides a comprehensive picture of MHC class I diversity in the wider UK herd and the selective forces integral to shaping diversity.

M.Tech.

Trypanosomiasis is one of the most important animal diseases in Ethiopia causing many thousands of deaths each year and the disease is not limited to Ethiopia alone. Approximately quarter of the total land surface of the African continent is estimated to b infested by tsetse flies and virtually allot this infested area 1s south of the Sahara. The geographical distribution of the various species of trypanosomas and their vector host, the tsetse flles. has been studied by a number of investigators. While all the species of the cyclically transmitted trypanosomes are distributed over Africa south of the Sahara, the predominant species vary between east, central, and west Africa.

The aim of this research work was to investigate and quantify the risk of fatal and fracture injury for Thoroughbreds participating in flat racing in the US and Canada so that horses at particular risk can be identified and the risk of fatal injury reduced. Risk factors associated with fatalities and fractures were identified and predictive models for both fatalities and fractures were developed and their performance was evaluated. Our analysis was based on 188,269 Thoroughbreds that raced on 89 racecourses reporting injuries to the Equine Injury Database (EID) in the US and Canada from 1st January 2009 to 31st December 2015. This included 2,493,957 race starts and 4,592,162 exercise starts. The race starts reported to the EID represented the starts for 90.0% of all official Thoroughbred racing events in the United States and Canada during the 7-year observation period. The annual average risk of fatal and fracture equine injuries for the period 2009 - 2015 was estimated and a description of the different injury types that resulted in fatalities and fractures was given, based on the cases recorded in the EID. Possible risk factors were pre-screened using univariable logistic regression models; risk factors with an association indicated by p < 0.20 were then included in a stepwise logistic regression selection process. A forward bidirectional elimination approach using Akaike's Information Criterion was utilised for the stepwise selection. We identified more than 20 risk factors that were found to be significantly associated with fatal injury (p < 0.05) and more than 20 risk factors associated with fracture injury, across the final multi-variable models. The risk factors identified are related to the horse’s previous racing history, the trainer, the race, the horse's expected performance and the horse's racing history. Five different algorithms were used to develop predictive models based on the data available from the period 2009 - 2014 for both fatal and fracture injuries. Firstly, we used Multivariable Logistic Regression, commonly used in risk factor analysis. Secondly, Improved Balanced Random Forests were developed, a machine learning algorithm based on a modification of the random forests algorithm. Because fatal injuries are extremely rare events, less than 2 instances per 1000 starts on average, balanced samples were used to develop the Random Forest model to deal with the class-imbalance problem. Furthermore, we trained an Artificial Neural Network with a single layer and two networks with deep architecture, a Deep Belief Network and a Stacked Denoising Autoencoder. As artificial neural networks and deep learning models have been successfully used to solve complex problems in a diverse field of domains we wanted to explore the possibility of using them to successfully predict equine injuries. The performance of each classifier was evaluated by calculating the Area Under the Receiver Operating Characteristic Curve (AUC), using the data available from 2015 for validation. AUC results ranged from 0.62 to 0.64 for the best performing algorithm and similar predictive results were obtained from the wide array of different models created. This is the first study to make use of the extensive information contained in the EID to identify risk factors associated with equine fatal and fracture injuries in the US and Canada for this period. To our knowledge, this is the largest retrospective observational study investigating the risk of equine fatal and fracture injuries during flat racing in the literature. This is also the first study to train logistic regression and machine learning models to predict equine injuries using such an extensive amount of data and a full year of horse racing events for prediction and evaluation. We believe the results could help identify horses at high risk of (fatal) injury on entering a race and inform the design and implementation of preventive measures aimed at minimising the number of Thoroughbreds sustaining fatal injuries during racing in North America.

Porcine congenital splayleg (PCS) is the most important congenital condition of piglets, associated with lameness and immobility, of unknown aetiology and pathogenesis. The aim of this study is to investigate the cellular and molecular changes in skeletal muscles of PCS, thereby gaining new molecular insights into this clinical condition. Based on immunohistochemistry and histological image analyses on 4 sets of 2-day- old splayleg piglets, each with a corresponding normal litter mate, a consistent discovery has been that PCS muscles [semitendinosus (ST), longissimus dorsi (LD) and gastrocnemius (G)] showed extensive fibre atrophy without apparent tissue damage. At present, it is not certain if PCS-associated fibre atrophy is accompanied by fibre hypoplasia. Both normal and PCS muscle fibres showed similar widespread distribution of lipid- and oxidative-positive fibres. Although there was no significant difference in fibre type composition, several structural myosin heavy chain (MyHC) genes were significantly down-regulated in PCS affected muscles. Interestingly, MAFbx, a major atrophy marker, was highly up-regulated in almost all PCS muscles, when compared with controls from normal litter mates. In contrast, P311, a novel 8- kDa protein, was relatively down-regulated in all PCS muscles examined. To further investigate the functional role of P311 in skeletal muscle, its full-length cDNA was sequenced (Accession. N0. EF416570) and over-expressed in murine C2C12 muscle cells. P311 over-expression enhanced cell proliferation and reduced myotube formation in C2C12 cells. The over-expression of calcineurin, a key intracellular calcium-dependent signalling factor of muscle differentiation, down- regulated P311 expression. Reduced P311 expression in PCS piglets might contribute to atrophy through reduced myotube contribution. To investigate the functional role of SPARCL-1, a matricellular secreted glycoprotein that belongs to SPARC family, its full-length cDNA was sequenced (Accession. N0. EF416571) and over-expressed in murine C2C12 muscle cells. SPARCL-1 overexpression led to reduced cell proliferation and down-regulation of MyHC genes during late differentiation. SPARCL-1 might be associated as a negative regulator of skeletal muscle cell proliferation and cell differentiation. However, endogenous SPARCL-1 expression was similar between PCS and normal muscles. Hence, although SPARCL-1 could play a role in muscle development, it is unlikely to be a main factor in the development of PCS. In summary, PCS is shown to be a condition characterised by extensive fibre atrophy and raised fibre density, and it is proposed that the combined differential expression of MAFbx and P311 is of potential value in the diagnosis of sub-clinical PCS.

Theileria annulata is a protozoan parasite of cattle, that causes the disease tropical theileriosis throughout sub-tropical regions of the Old World. Theileria parasites have the ability to immortalise the host leukocyte they infect causing clonal expansion and dissemination of infected leukocytes throughout the host. This property has allowed the development of an in vitro system for the culture of bovine cells infected by the macroschizont stage of the parasite. In addition, differentiation of the parasite towards the next life cycle stage, the merozoite, can be induced in culture. The signals that cause the macroschizont to differentiate into merozoites are not fully understood, although it is known that this event is associated with a major elevation in merozoite gene expression (Shiels et al., 1994). Recently a small family of parasite genes that are negatively regulated early during differentiation to the merozoite were identified. One member, known as TashAT2 contained predicted AT hook DNA binding motifs and was shown to be localised to the host cell nucleus. It has been postulated that the TashAT2 polypeptide may play a role in the regulation of macroschizont or modulation of host cell gene expression (Swan et al., 1999). The focus of this project was to characterise TashAT1, a second member of the TashAT gene family. To this end, the TashAT1 gene was sub-cloned and sequenced and mapped to a region of the genome containing TashAT2 and a third Task AT gene, TashAT3. The 1.4kb open reading frame of TashAT1 was virtually identical to the five prime end of TashAT3, indicating that TashAT1 or TashAT3 (TashAT1/3) were derived from a recent duplication event. The predicted amino acid sequence of TashAT1/3 contained four AT hook motifs, a nuclear localisation signal and a signal sequence. Northern blot analysis revealed that TashAT1, TashAT2 and TashAT3 mRNA were down regulated early, during differentiation to the merozoite in vitro. However, no down regulation was observed for any of the TashAT transcripts in a cell line that was severely attenuated with respect to parasite differentiation. Sequence analysis of the upstream regions of TashAT1/3 identified a motif element (TashUM) located 43bp upstream of the putative transcription start site of TashAT1/3 that was highly related to a sequence upstream of TashAT1 and another, unrelated macroschizont gene, Tash1. Preliminary electromobility band shift analysis of TashUM revealed that it bound to a factor found in host and parasite enriched nuclear extract, which appeared to decrease in abundance as the parasite differentiated towards merogony. Antisera generated against a region of TashAT1 failed to recognise a TashAT1 polypeptide by Western blot analysis. However, a 180kDa polypeptide that was down regulated with respect to merogony and co-localised to the host nucleus was specifically recognised. The detected polypeptide was identified as TashAT3 on the basis of size, sequence identity and predicted expression profile. Immunofluorescence analysis showed that the anti-TashAT1 antisera reacted against both the host nucleus and parasite. This reactivity was lost as the parasite differentiated to the merozoite. The host reactivity was probably due to recognition of TashAT3, while it could not be concluded that the parasite reactivity was directed against TashAT1. Taken together, the results indicated that TashAT3 and possibly TashAT1 are additional candidates for parasite encoded factors that are translocated to the host nucleus, bind to DNA and alter host cell gene expression. This modulation of gene expression could directly or indirectly alter the phenotype of the host cell and be involved in parasite dependent regulation of leukocyte cell division.

The hypothesis was that the cellular architecture of the lymphoid tissue was determined by antigenic stimulation. In order to test that hypothesis chickens were produced and maintained in an environment as free from antigens as possible. The chickens were then challenged with an antigen and the cellular changes in the lymphoid tissue, especially the spleen, examined. The thesis thus falls into three sections. In Section 1, after the concept of germ-free life was introduced, the practical problems of producing and maintaining germ-free chickens were discussed in relation to the needs of the present work. A detailed description then followed of the method by which germ-free chickens were produced and maintained in this study. The use of the germicide "Portex D.C.R." was described for sterllizing the surface fertile a eggs in order to obtain germ-free chickens. In Section II the lymphoid tissue of the conventional chickens was described. In order to form bose-line or norm for the study of the effect of antigens in germ-free chickens the lymphoid tissue of the four weeks old unstimulated germ-free chicken was stufied. Serum proteins from these birds were immuncelectrophoresed. It was shown that in the unstimulated four week old germ-free chicken no germinal centres were present in the spleen and that there were fewer cells of the plasmacellular series when compared with four week old conventional chickens. The level of immune-globulins was lower in germ-free chickens than in conventional chickens. Having established this norm it was then possible (Section III) to challenge the germ-free chickens with an antigen in an attempt to induce germinal centre formation and study the way in which germinal centres were formed. The tissues from the germ-free chickens stimulated with a known antigen were examined using conventional histology and immunofluorescence; serum antibodies were estimated and serum proteins were immunoelectrophoresed. In experiment I an attempt was made to induce germinal centre formation in conventional birds using Shigella glexneri as antigen. In the next two experiments a soluble protein antigen (Human serum albumin) was administered to seven day old germ-free chickens (expt. 2) and to seven day old conventional chickens (expt. 3) to induce germinal centre formation. In the remaining three experiments a staphylococcus isolated from a chickens was used as antigen. Germ-free chickens were given the staphylococcus as a primary injection at seven days old (expt. 4) and at twenty-one days old (expt. 5) and as a secondary injection at twenty-one days old (expt. 6) following a primary at seven days old. From there experiments it was concluded that the cellular architecture of the same age was not the same. In germ-free chickens both the soluble protein antigens (HSA) and the particulate antigen (staphylococcus) induced proliferation of cells of the plasmacellular series but germinal centres were found in the spleen following stirculation with staphylococcus. Further, the response to the same dose of the same antigen (staphylococcus) varied with the age of the chicken during the neonatal period. A greater number of germinal centres and a greater proliferation of cells of the plasmacellular series was induced following injection of staphylococcus into 21 day old germ-free chickens than into 7 day old germ-free chickens. A secondary challenge with staphylococcus at 21 days old in germ-free chickens following a primary injection at 7 days old induced a greater number of germinal centres than a primary injection at 21 days old. The germinal centre appeared to be formed in the spleen not by rapid multiplication of a small focus of cells but by aggregation of haemocytoblasts in the periarteriolar lymphocyte sheath. Lymphocyres did not appear to be incorporated in germinal centre formation but seemed to be formed from haemocytoblasts within the germinal centre. No cellular changes were induced in the thymus or bursa of Fabricius following antigenic stimulation of germ-free chickens. It was clear from the experimental work that the cellular architecture of the spleen of the chicken was dependent upon immunogenic stimulus.

This thesis is principally concerned with the construction and development of a sustained release bolus system supplying a range of trace elements to ruminant livestock. The system is a patented invention of the University of Glasgow. Section 1 describes the technology of construction. A compressed mixture of common inorganic salts in cylindrical form (25 mm diameter, 40-100 mm length) is coated by dipping in a polyester resin leaving one flat end uncoated. Release of material into the reticulo-rumen is partly by dissolution and partly by mutual erosion of two boluses administered together. The initial prototype contained copper oxide, manganese sulphate, zinc oxide, zinc sulphate, sodium selenite, cobalt sulphate and potassium iodide with Vitamins A, D3 and E. Section 2 presents results of the examination of a range of factors which might affect the rate of release of nutrients from the bolus. These included the number of coats of resin and the initial length of the bolus. The compositional specification of zinc oxide (a major ingredient) was found to be of great importance and strict control was necessary to produce the required overall release rate. Changes in the inclusion of zinc sulphate could also markedly affect the dissolution/erosion characteristics. An increased number (1-3) of boluses simultaneously administered and the presence of metallic residues (end weights, tubes, cylinders) from other bolus systems was demonstrated to greatly increase the rate of release of material from the bolus. Section 3 describes a series of trials at different sites with grazing cattle judged by the local veterinary surgeons to require supplementation with one or more of the trace elements. The adequacy of supplementation was assessed by the measurement of blood parameters in comparison with untreated animals and/or animals given alternative supplementation as injections or alternative boluses. It was concluded that the bolus system provided adequate copper and selenium to cattle (130-500 kg liveweight) as judged by the responses in plasma copper and glutathione peroxidase activity and that these responses were as favourable as those found by injection of copper and/or selenate containing products. No sound conclusions regarding the effectiveness of the bolus system in supplying cobalt could be made due to the possible limitations of the assay method (and its interpretation) for Vitamin B12. There were no indications of cobalt inadequacy in the cattle. Section 4 of the thesis examined the possibility that the bolus construction with a modified matrix might be capable of providing a constant release of a variety of medicaments. Limited exploratory trials were conducted to examine the possible inclusion of materials such as levamisole hydrochloride, ivermectin, oxfendazole, laidlomycin propionate and Vitamin E. Distinct possibilities were found but much further work would be required to establish formulations giving appropriate and constant release of materials in the normally accepted therapeutic range. Section 5 examined the possibilities for the development of a comparable, but smaller (19 mm diameter) bolus appropriate for use in sheep. Regurgitation was a major problem but was effectively eliminated by increasing the overall density to 3.0 g/ccm. The cost of manufacture would be such as to allow the use of only a single bolus. The absence of loss by mutual erosion between two boluses was found to lead to little further weight loss in prototypes after about 60 days. Nevertheless, analyses of faeces and of livers recovered at slaughter demonstrated the effectiveness of the copper contained in the bolus. These were such as to give concern about the possibility of potential copper toxicity. In Section 6 an assessment was made of the effectiveness of a high density carbo-wax matrix as a carrier for avoparcin in an alternative bolus system. Comparisons were made by evaluation of the avoparcin concentrations in faeces with that resulting from a constant daily in-feed addition of avoparcin. Assessment of faecal output indirectly by estimation of the chromium content of faeces resulting from constant addition of the inert marker to the constant diet given to all cattle showed the experimental bolus construction to be irregular and erratic in relation to the direct inclusion in feed.