Necrotic enteritis (NE) is an important enteric disease in poultry production that has re-emerged as a major problem following an EU wide ban on the use of in-feed antimicrobials. Although the primary aetiological agent of disease is Clostridium perfringens type A, a commensal in the gastrointestinal tract (GIT) of chickens, numerous additional influential factors have been reported that can predispose chickens to NE. These precipitating factors mainly include diet, co-infection with other pathogens particularly coccidia, as well as environmental and management factors. Despite being first described almost more than 50 years ago, a reliable, consistently reproducible experimental model for NE induction is still lacking. Here, a series of experiments were conducted to investigate the importance of the various potential predisposing factors, in isolation and in combination, that are believed to play a role in sub-clinical NE development: feed withdrawal, dietary protein sources, co- infection with coccidia, C. perfringens dose and contact with reused litter. In addition, chicken breed sensitivity to NE was assessed, and last, but not least, blood and gut tissue samples were used to identify novel biochemical markers for sub-clinical NE. Chapter 3 reports an experiment showing that feed withdrawal up to 24 hrs in experimentally challenged birds did not result in NE specific lesions. Chapter 4 shows that replacing dietary soyabean meal with potato protein concentrate or canola meal or adding synthetic trypsin inhibitor to the soyabean meal control diet did not induce sub-clinical NE in birds housed on reused litter, a natural source of C. perfringens challenge. Chapter 5 describes that in vitro growth of C. perfringens on in vitro digested grower diets was prolonged following the addition of fishmeal, suggesting that the role of fish meal as a predisposing factor for in vivo sub-clinical NE cannot be excluded. All subsequent diets therefore contained high levels of fish meal. When this was used in combination with high dose of coccidial vaccine, a repeated in-feed challenge for three days at 102 colony forming units (cfu) C. perfringens per g feed did not result in sub-clinical NE, though at 109 cfu/g resulted in 10% of challenged birds (3 out of 30) showing NE-specific lesions (Chapter 6). Further study is needed to determine if the two Ross birds with gross NE lesions compared to the one Hubbard bird (out of 15 birds each) was due to a lower level of NE resistance. 3 The failure to significantly induce sub-clinical NE in the previous experiments suggests that challenging the birds with C. perfringens in the isolated presence of suspected predisposing factors may not provide a suitable experimental model. Indeed, when birds were dosed twice daily with 108 cfu C. perfringens for three days in the presence of high levels of fishmeal, canola meal as main protein source, coccidial and IBD vaccinations, and feed withdrawal prior to challenge, 40.6% of the challenged birds developed lesions of sub-clinical NE without inducing mortality (Chapter 7). This concurred with reduced growth performance relative to the sham-infected control birds, and thus is a successful model for induction of sub-clinical NE. Finally this work has, for the first time provided novel information on potential biomarkers (Chapter 8). Whilst challenge did not impact on the expression of genes previously shown to be differentially expressed upon C. perfringens toxin exposure, the serum ceruloplasmin concentration increased, suggesting that monitoring this acute phase protein may indicate the presence of C. perfringens infection in poultry. However, as such markers generally lack specificity, further research confirming its role in response to sub-clinical NE is needed to provide a fully effective diagnostic and prognostic marker for flock health and welfare, as well as ultimately helping to gain better understanding of the pathophysiology of sub-clinical NE. Improved knowledge of the effect of different dietary components on the growth of C. perfringens may help in the formulation of broiler diets to assist in further reducing the incidence of NE particularly in the absence of antimicrobial growth promoters. It is hoped that host responses in terms of acute phase proteins, and possibley gene expression, will also provide greater insight into the pathogenesis of NE. Provided that the developed experimental sub-clinical NE model is reproducible, this will benefit the understanding of this billion dollar disease and enable further investigation of various chemical and non-chemical interventions to reduce its severity and impact on poultry production.

Four studies consisting of 6 experiments were conducted to investigate the likely mechanisms of actions of feed additives used in the place of antibiotics to improve performance and enteric health in broilers. In the first study, the relationship between digesta pH, body weight and nutrient utilisation in broilers of the same breed but with different propensity for weight gain at different ages were investigated. It was noted that birds in group H (heavy) consumed more feed (P < 0.001) than those in group L (light) during the starter (day 10 to 14) and grower (day 15 to 28) phases. Birds in group H had lower (P < 0.05) caecal pH in the starter and grower phases and lower (P < 0.05) proventriculus pH in grower and finisher (day 29 to 42) phases. In the grower phase, caecal pH was correlated (r = 0.553) with total tract retention of DM and energy utilisation at both the ileal and total tract levels, whereas during the finisher phase, crop pH was correlated with ileal nutrient utilisation, and jejunum pH was correlated with total tract energy utilisation. The data showed that differences in body weight are also reflected in differences in gut pH which is likely indicative of differences in intestinal condition between birds with heavier or lighter body weight. The differences in the gut pH explained about half of the variations in total tract nutrient and energy utilisation. Lower gut pH is advantageous for beneficial bacteria colonization but disadvantageous for pathogenic ones colonization and hence it is likely that birds with the same genetic potential may have differences in growth performance based on the type of bacteria colonizing their gut. In the second study, the response of broiler chickens to the supplementation of benzoic acid (BA) was investigated using growth performance, nutrient and energy utilization, intestinal acidity and histomorphology of the intestine as response criteria, using 945 Ross 308 male broilers in 3 treatments with 7 replicates each for 42 days. In the grower (day 11 to 21) phase, BA supplementation at 0.53 g/kg (BA1) increased (P < 0.001) body weight gain and reduced (P < 0.01) FCR, whereas supplementation at 3.20 g/kg (BA2) reduced (P < 0.005) the feed intake without affecting the body weight gain, resulting in a better FCR. Compared with the control, BA supplementations increased (P < 0.001) the protein and energy efficiency ratios in starter and grower phases and tended (P < 0.10) to increase the energy efficiency ratio in finisher phase (day 22 to 42) or the overall experimental period (day 0 – 42). BA supplementations at both rates reduced the caecal pH. Supplementation of 3.20 g/kg BA stimulated the proliferation of the absorptive cells in the jejunum, as shown in the improvement of the villus and crypt dimensions. The data from this study indicated that dietary supplementation of BA beneficially modified intestinal milieu and improved the growth performance of broiler chicks at 42 d of age. In third study, two experiments were conducted to investigate the benefit of using BA and turmeric meal (TM) individually or in combination using growth performance, nutrient utilization, and intestinal health as response criteria. A total of 300 male one-day old broilers (Ross 308) were assigned in 5 treatments in randomized complete block design with 2x2 + 1 factorial arrangement, with 6 replicate pens and 10 birds each pen. Combination of 1 g/kg BA and 5 g/kg TM improved (P < 0.05) body weight gain relative to the control. Supplementation of 2 g/kg BA reduced the pH in the crop (P < 0.001) and jejunum (P < 0.01), whereas combination of BA and TM at 2 and 10 g/kg respectively reduced digesta pH in the crop (P < 0.001), jejunum (P < 0.01), and caeca (P < 0.05). All of the dietary treatments increased (P < 0.005) villus height, crypt depth and width relative to the control. All dietary treatments increased (P < 0.001) AME and AMEn relative to the control diet, whilst supplementation of 10 g/kg TM only increased energy digestibility (P < 0.05) and ileal digestible energy (P < 0.01). Orthogonal contrasts showed that BA and TM were additive in their effects on the growth performance, digesta pH in the proventriculus, jejunum, and ileum, and energy utilization, but associative on the energy digestibility, as well as the crop and caecal pH. None of the treatment altered the relative weight and length of the digestive tract of 21 days old broiler. These studies pointed out that BA and TM can be used in the diet individually or in combination to improve the enteric health, nutrient and energy utilization, and growth performance of broiler chickens. In the fourth study, two experiments were designed to investigate the efficacy of TM and garlic meal (GM) using growth performance, intestinal pH, and energy and nutrient utilisation as response criteria. Three hundreds male one-day old broilers (Ross 308) were assigned in 5 treatments in randomized complete block design with 2x2 + 1 factorial arrangement, with 6 replicate pens and 10 birds each pen. Results showed that combination of GM and TM at 10 g/kg each increased (P < 0.05) the body weight gain, final body weight, and gain to feed ratio relative to the control and the diet with GM supplementation alone. The crop and caecal pH were reduced (P < 0.05) when the diets were supplemented with TM alone at 10 g/kg. The proventriculus pH also dropped (P < 0.05) following GM and TM mixture supplementation at 10 g/kg each relative to the control. Supplementation of 10 g/kg TM alone or in combination with GM at 5 g/kg each increased (P < 0.05) the apparent ileal energy digestibility and ileal digestible energy. All of the dietary treatments increased (P < 0.001) both AME and AMEn compared with the control. Orthogonal contrasts showed that GM and TM were additive for feed intake, nutrient and energy utilization at both the ileal and total tract levels, but associative in their effects on body weight gain and gain to feed ratio. These studies indicated that GM and TM can be used alone or in combination to support intestinal health, improve energy and nutrient utilization, and stimulate growth performance of broiler chickens. Combination of GM and TM at the rate of 5 g/kg each was optimum for enhancing nutrient and energy utilization and promoting growth performance of broiler chickens. Taken together, these studies showed that benzoic acid and herbal products (garlic and turmeric meal) can be supplemented in the diet alone or in combination to improve the enteric health, nutrient and energy utilization, and growth performance of broiler chickens. Improvements on the growth performance might be attributed to the reduction of the entero-pathogens in the gut, enhancement of intestinal health, alteration of the absorptive cells in the intestinal wall, and improvement in the nutrient and energy utilization.

K99 was chosen as a model target for this study, to explore the application of recombinant antibody technology to livestock infection. Our aims were to isolate and characterise single-chain Fv antibodies against K99 using phage display. Escherichia coli B41, a clinical isolate that expresses K99 fimbriae, was grown and fimbriae were extracted by heat-shock treatment and then precipitated by ammonium sulphate. The major K99 subunit (Fan C) was purified from crude fimbriae extract by an ion-exchange chromatography method using SP-XL columns. The semi-synthetic Tomlinson I and J libraries (Center of Protein Engineering, Cambridge, UK) were used to select phage antibodies against K99 using immunotubes coated with the major fimbrial subunit. After three rounds of selection, phage were transfected into E. coli HB2151 for the expression of soluble scFvs. Fifteen scFv clones with high activity against K99 fimbriae were identified by ELISA and sequenced. Of these, six scFvs carried sequences that were reasonably diverse. These proteins were purified for further characterization. The recombinant antibodies were shown to react with fimbriae present at the surface of E. coli B41 using immunofluorescence microscopy and immunogold electron microscopy. Some of the purified scFv antibodies were also able to inhibit the agglutination of sheep erythrocytes by E. coli B41 grown at 37°C. To pursue this observation, attempts were made to use the scFvs in an in vitro model of bacterial colonisation in which bacteria were tested for attachment to isolated bovine intestinal villi. Although bacteria could be observed adhering to the brush border, the scFvs appeared unable to prevent this attachment. Further experiments with this in vitro model or a mouse model of ETEC infection, allied with epitope mapping studies should determine if anti-colonisation activity is attributable to binding of scFvs to the receptor-recognition site on the major subunit of the adhesin.

Hyperlipidaemia describes increased plasma concentrations of cholesterol and/or triglyceride and may arise from primary, often inherited defects in lipoprotein metabolism or appear secondary to systemic disease. The aim of this study was to characterise the abnormalities of lipoprotein metabolism responsible for hyperlipidaemia in the dog, to establish their effect upon the health of the individual, and to evaluate approaches to therapy. A combined ultracentrifugation/precipitation technique for the measurement of very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL) was adapted and validated for use with canine plasma. Alternative methods of analysis were evaluated, but none of the commercially available precipitation reagents for the measurement of human lipoproteins were found to be suitable for use in the dog. Lipoprotein electrophoresis was useful in the investigation of hyperlipidaemic patients by allowing the identification of other lipoprotein classes, namely chylomicrons, HDL1 and B-VLDL. The effects of age, breed, sex and health status on lipoprotein concentrations in the dog were established. The HDL concentration was greater in entire females than males and certain disease processes were found to displaycharacteristic lipoprotein abnormalities. Dogs with diabetes mellitus and hypothyroidism presented with defects in the metabolism of LDL and HDL and triglyceride-rich lipoproteins, while hyperadrenocorticism, obstructive jaundice and protein-losing nephropathies were predominantly associated with abnormalities of cholesterol metabolism. A decreased lipoprotein lipase activity was partially responsible for the hyperlipidaemia in hypothyroid dogs. Overt hyperlipidaemia was not identified in obese dogs, but aberrations of triglyceride metabolism were identified by kinetic analysis and found to be a result of decreased lipoprotein lipase activity. Impaired triglyceride clearance in the postprandial period may therefore predispose obese dogs to the development of acute pancreatitis.

The overall objective of this thesis was to study the effects of regular and high (super-) doses of phytase in the gut of broilers, with the aim of documenting the mechanism of their action leading to improvements in animal health. Phytase is often supplemented to commercial broiler diets to facilitate the hydrolysis of plant phytate and release of phosphorus for utilisation. Although not the original intention of its addition, phytase supplementation leads to improvements in growth performance parameters and enhanced nutrient utilisation. Further benefits have also been observed following the addition of super-doses of phytase which are not explained by an increase in phosphorus release, and thus have been termed ‘extra-phosphoric effects’. Using diets formulated to be adequate or marginally deficient in available phosphorus (aP) forming the negative control, NC), phytase was supplemented at 1,500 and 3,000 FTU/kg phytase in the first study (both super-doses) and the partitioning of nutrients within the body was investigated. It appeared that there were some metabolic changes between 1,500 and 3,000 FTU/kg, switching between protein and fat accretion, potentially as a consequence of nutrient availability, although these changes were not reflected by changes in growth performance parameters. However, the loss of the NC treatment without phytase on day 12 limits the comparison of the phytase within the NC treatment, but does allow for comparison of each dose at adequate or low dietary aP levels. As expected, a greater degree of phytate hydrolysis was achieved with 3,000 than with 1,500 FTU/kg phytase, but changes in carcass accretion characteristics were greater with 1,500 than 3,000 FTU/kg. Using these findings and the observation that there were no further changes in the parameters measured by increasing phytase from 1,500 to 3,000 FTU/kg (aside from phytate hydrolysis), 1,500 FTU/kg phytase was selected as the super-dose to be used in subsequent studies. The next study considered the influence of regular (500 FTU/kg) and super doses (1,500 FTU/kg) of phytase from within the gut. Overall, it was observed that changes were occurring to the gut environment, which ultimately would influence the absorptive capacity and conditions for further phytate hydrolysis. Dietary treatment influenced gut conditions such as pH, intestinal morphology and bacterial populations which can subsequently influence nutrient utilisation and potential for growth. The subsequent study was designed to investigate the effects within the gut in more detail. The release of nutrients from phytate hydrolysis and their bioavailability within the digesta can influence conditions within intestine, facilitating enhanced absorption. One of the parameters investigated was the expression of genes involved in the transport of nutrients in the intestine. Overall, there were few significant dietary treatment influences on gene expression in the intestine, however there was a dose-dependent response of phytase on the expression of the jejunual divalent mineral transporter. This indicates a change in divalent mineral bioavailability in the intestine, with correlations with inositol phosphate esters (IPs) being identified. This is likely explained by the IPs produced by phytase hydrolysis and accumulating in the digesta, differing between regular and high doses of phytase. It became apparent that interactions between the products of phytate hydrolysis (IP3, IP4) and minerals in the digesta had the potential to influence the gut environment and subsequent nutrient bioavailability and overall phytase action. The final study was designed to increase the content of the IPs, and investigate the influence of phytase under these conditions. As the complete hydrolysis of phytate to myo-inositol has been reported to be beneficial due to its proposed insulin mimetic effects, myo-inositol was also supplemented to one of the diets to see if any further benefits would be observed when supplemented alongside super-doses of phytase. Neither increased concentrations of the higher IP esters (IP6, IP5 and IP4) nor myo-inositol (myo-) had any effect on broiler growth performance, however there were still apparent beneficial influences of phytase supplementation. The results suggest considerable and important interactions between minerals and IP esters within the digesta, which ultimately have the potential to influence gut conditions and thus nutrient utilisation and growth performance. Reduced concentrations of blood glucose in the high IP ester diet with additional phytase supplementation suggest some insulin-like effects of myo- production. Additionally, the lack of effect of myo- supplementation on blood glucose and insulin concentrations suggests a difference between the structure of phytase-produced myo- and supplemented myo-. Although there were no improvements in growth performance by increasing phytase from 500 to 1,500 FTU/kg, there were changes occurring at the level of the gut and expression of genes in the intestine, influencing nutrient utilisation and the partitioning of nutrients within the body. There are many factors to be considered when supplementing phytase, with dietary nutrient content and nutrient release and IP production during phytate hydrolysis having an influence on phytase action, nutrient absorption and conditions within the gut. Super-doses of phytase may be beneficial for maintaining optimal gut conditions, clearing IP esters from the digesta, reducing their potential to form complexes with minerals and other nutrients, ultimately influencing the efficiency of production.

This project set out to determine the importance of tick-borne protozoan and bacterial pathogens in Scottish livestock. The study comprised several aspects including a national survey of large animal veterinary surgeons, an appraisal of cases of tick-borne disease observed by Scottish Disease Surveillance Centres, the development of a novel assay to detect piroplasms and a targeted cross- sectional study of livestock and deer in the north of Scotland. A survey of the experiences of veterinary surgeons treating livestock in Scotland revealed cases of babesiosis (Babesia divergens) in cattle and tick-borne fever (Anaplasma phagocytophilum) in sheep. Examination of the records of the Scottish Agricultural College Consulting Disease Surveillance Centres for the years 2000 - 2013 revealed cases of babesiosis in cattle (n = 55) along with tick- borne fever and related disease in sheep (n = 116) and cattle (n = 6). Taking a combined passive and active surveillance approach, clinical material was obtained from a large number of healthy sheep, cattle and wild red deer (Cervus elaphus) together with a number of cattle and sheep suspected or confirmed as having a tick-borne disease. All samples were examined using a genus-specific nested PCR targeting the v4 region of the Babesia/Theileria 18S rRNA gene, which was developed and validated in the course of this study. This gene segment was confirmed as being capable of differentiating a diverse range of Babesia and Theileria spp. based on direct sequencing of PCR amplicons. A nested PCR assay targeting the 16S rRNA gene of A. phagocytophilum was also applied to each clinical sample and, if positive, the msp4 locus was also amplified and sequenced. Babesia ventatorum, a parasite typically associated with the roe deer (Capreolus capreolus) in Europe, was detected in 9 % of healthy sheep. Significantly, this is the first description of this parasite in sheep or in a vertebrate host in the United Kingdom. Babesia divergens was found in 11 % of wild red deer, confirming the presence of this parasite in this host species by molecular means for the first time in Scotland. Additionally a Babesia odocoilei-like parasite was found in 15 % of wild red deer, again for the first time in Scotland and only the second time in Europe. In cattle, B. divergens was confirmed as the cause of three clinical cases of babesiosis and was also found in the blood of 6 % of healthy cattle in December. Anaplasma phagocytophilum was found at a high prevalence in healthy sheep (73 %) and red deer (40 %) and at lower levels in healthy cattle (2.8 %). Comparison of msp4 gene sequences confirmed identical or highly similar msp4 genotypes in sheep and deer. Red deer were infected with larger numbers of msp4 genotypes than sheep and infection with multiple genotypes increased over the course of a grazing season on tick-infested hill land. Anaplasma phagocytophilum is zoonotic and can also have negative welfare and economic impacts in both sheep and, to a lesser extent, cattle and so these findings are highly significant. An incidental finding was Sarcocystis sp. similar to S. tenella in 3 % of healthy sheep. These results of this work clearly show what can be achieved by an active surveillance approach, using a ‘catch all’ molecular assay. In summary, this study discovered a novel, zoonotic pathogen in Scottish livestock and demonstrated that an endemic and arguably largely overlooked bacterium, A. phagocytophilum, is highly prevalent in the sheep population in tick-risk areas. Moreover, genotyping of this pathogen and B. divergens in both livestock and deer has provided new insights into potential reservoirs of infection for these organisms.

Influenza A viruses (IAVs) are common infections of certain avian reservoir species, and they periodically transfer to mammalian hosts. These cross-species jumps are usually associated with sporadic outbreaks, and on rare occasions lead to the establishment of a lineage in the new host species. The immune pressure exerted by the new host on the emergent virus forces it to evolve and adopt strategies to evade immunity in order to survive in nature. Understanding the biological mechanisms that allow successful inter-species transmission and adaptation to mammals is crucial to develop the theoretical tools required to predict and/or control emergence of new viruses in humans and animals. H3N8 equine influenza virus (EIV) represents an interesting model to study the dynamic of within-host variation of an avian-origin IAV. Indeed, this virus has emerged from birds in 1963 and has circulated in horse populations for more than fifty years despite the availability of vaccines. Evidence of evolution of EIV virulence factor non-structural protein 1 (NS1) also exists. NS1 is the main viral antagonist of the host interferon (IFN) response, and it relies on different strategies for overcoming these responses, which varies depending on the viral strain. While some NS1 proteins effectively block the induction of IFN and IFN stimulated genes (ISGs), others block general gene expression at a post-transcriptional level, and therefore reduce the synthesis of IFN and ISGs indirectly. Importantly, little is known about the contribution of these NS1 functions to EIV infection phenotype and adaptation to horses. In this work, we characterised NS1 proteins spanning the entire EIV lineage and showed that NS1s from different time periods after EIV emergence counteract the IFN response using different and mutually exclusive mechanisms. While EIVs circulating in the early 1960s blocked general gene expression by a NS1-mediated blockade of the cleavage and polyadenylation specificity factor 30 (CPSF30), NS1s from contemporary EIVs specifically inhibit the induction of ISGs by interfering with the JAK/STAT pathway. These contrasting anti-IFN strategies are associated with two mutations that appeared sequentially during EIV evolution, E186K substitution and C-terminal truncation. These changes in NS1 allowed contemporary EIVs to replicate in the presence of high levels of IFN. The results shown here with EIV indicate that the interplay between virus evolution and immune evasion plays a key role in IAV mammalian adaptation.

Equine fetlock region disease is responsible for significant morbidity and mortality. Diagnosis of sesamoidean ligament, cartilage and subchondral bone injury has been obtained by clinical MRI. Low-field MRI provides images helpful in the investigation of MCPJ/MTPJ region pathology in horses in the clinical setting but the greater resolution of high and ultra-field MR images has the potential to aid interpretation through a better understanding of MRI anatomy. Quantitative MRI could provide a non-invasive technique to determine tissue biochemical properties associated with the early onset of articular cartilage degenerative conditions such as osteoarthritis. So far, ultra-high field MRI has not been used in equine research and practice. However, recently 3T MRI has been introduced in equine hospitals in Europe and the US. The general objectives of this project, which utilised cadaver limbs, was to improve understanding of the MRI anatomy of the equine MCPJ/MTPJ region and to evaluate the use of MRI for the non-invasive, quantitative assessment of articular cartilage from the same region. The first specific objective was to describe the appearance of the normal anatomy of the equine MCPJ/MTPJ region, especially the SDFT & DDFT and DSLs, using high field (1.5T) and ultra high field (7T) MRI and to compare the images obtained with the two systems. The second objective was to determine the accuracy and precision of articular cartilage thickness measurements using 1.5T and 7T MRI and comparing the measurements with those made from histological sections of the MCPJ/MTPJ. The third objective was to measure T1 & T2 MRI sequence relaxation times for normal horse articular cartilage pre and post gadolinium contrast (dGEMRIC) administration and to determine their correlation with GAG concentration, including a description of topographical variation. The fourth objective was to compare sodium concentration in normal equine MCPJ/MTPJ articular cartilage measured using 7T MR imaging with a dual tuned quadrature 23Na/1H coil with the biochemical properties (sodium concentration determined by flame photometry and GAG concentration). The final objective was to evaluate MR sodium imaging for the assessment of enzymatically degraded equine cartilage. The findings demonstrated that 7T MRI produces high resolution images, which enable better evaluation of the hard and soft tissues of the equine MCPJ/MTPJ region than images from lower field MR systems and which permit accurate and precise articular cartilage thickness measurements to be made. Moreover, it was found that the dGEMRIC technique appears to provide a feasible quantitative tool for evaluating the articular cartilage properties. However, the quantitative parameters determined by the dGEMRIC method cannot fully characterise the biochemical properties of the cartilage. Moreover, delayed gadolinium-enhanced (dGEMRIC) techniques are time consuming, requiring relatively long incubation and scanning times. The measurement of T2 time is a very complex method. The work described in the last chapters demonstrated that sodium MRI was significantly correlated with the biochemical properties of the equine articular cartilage. Therefore the sodium MRI technique showed promise in imaging articular cartilage and providing useful information on the biochemical properties of the cartilage.

Although diarrhoea caused by Cryptosporidium is prevalent in livestock species throughout the world relatively little is known about the species and subtypes of Cryptosporidium found in cattle on Scottish farms. In particular, little is known about the shedding profiles (age when calves become infected and duration of shedding) of the different species found in cattle and how calves become infected. There are several theories about how neonatal calves first become infected with the parasite but the role which adult cattle play in the transmission of the parasite has not been fully addressed. It was previously thought that adult cattle did not become infected with the same species of Cryptosporidium which causes disease in the young calves. Some studies have shown that this may not be true and with the advance of new techniques to discriminate species this is an area which should be revisited. In addition, it is known that it is possible for humans to become infected with Cryptosporidium and show clinical disease early in life and then again later in adulthood. In livestock however, diarrhoea caused by the parasite is generally only seen in neonatal livestock while older animals tend to be asymptomatic. It is not known if this resistance to clinical disease at an older age is due to changes in the host with an increase in age or if prior infection “immunises” the animal and provides protection against re-infection. It is also not known if infection with one isolate of C. parvum will provide protection against infection with another or if the protection formed is species/isolate specific. The main aims of this thesis were to: determine the species and subtypes of Cryptosporidium found in calves on a study farm over a one year period from birth; assess the role which adult cattle play in the transmission of the parasite to newborn calves; develop new typing tools to enable the rapid and easy differentiation of Cryptosporidium species found in cattle and to examine the host-pathogen interactions in animals given serial experimental challenges with distinct Cryptosporidium parvum isolates to determine if the resistance seen in older animals on farms is due to an increase in age or as a result of prior infection. iii A variety of different approaches were taken to achieve these aims. Longitudinal experiments carried out on a study farm revealed that in calves < 9 weeks of age the most common species of Cryptosporidium is C. parvum and that all calves in the group became infected with Cryptosporidium within the first two weeks of life. Sample collection from the same animals later in life (at 6 months of age) showed that contrary to most previous studies the most common species detected at in this age group was also C. parvum although, interestingly, the subtype which the calves were shedding was not the same subtype that they were shedding previously. The longitudinal study which investigated the role of adult cattle in the transmission of Cryptosporidium also yielded some interesting results. It was found that most of the adult cattle on this farm were shedding Cryptosporidium albeit intermittently. Speciation of the positive samples revealed that, on this farm, the most predominant species of Cryptosporidium in adult cattle was also C. parvum. This is very unusual as most previous studies have not found this level of infection in older cattle and C. parvum is not usually found in this age group. A number of different subtypes were found in adult cattle and some animals shed more than one subtype over the course of the study. This contradicts prior findings which demonstrated that only one subtype is found on a single farm. The experimental infection trial involving infection of young (<1 week old) and older (6 week old) lambs with distinct C. parvum isolates demonstrated that an increase in age at primary infection reduces the effect of clinical disease. Animals which were infected at <1 week of age were re-challenged at 6 weeks of age with either a homologous or heterologous infection. Results revealed that previous exposure does not protect against re-infection with the same or a different isolate of C. parvum. This study also demonstrated that an increase in infective dose leads to a shorter pre-patent period and that there are variations in the clinical manifestations of different isolates of the same Cryptosporidium species.

Acute phase proteins (APPs) are proteins synthesised predominantly in the liver, whose plasma concentrations increase (positive APP) or decrease (negative APP) as a result of infection, inflammation, trauma and tissue injury. They also change as a result of the introduction of immunogens such as bacterial lipopolysaccharide (LPS), turpentine and vaccination. While publications on APPs in chickens are numerous, the limited availability of anti-sera and commercial ELISAs has resulted in a lot of information on only a few APPs. Disease is a threat to the poultry industry, as pathogens have the potential to evolve, spread and cause rapid onset of disease that is detrimental to the welfare of birds. Low level, sub-acute disease with non-specific, often undiagnosed causes can greatly affect bird health and growth and impact greatly on productivity and profitability. Developing and validating methods to measure and characterise APPs in chickens will allow these proteins to be used diagnostically for monitoring flock health. Using immune parameters such as APPs that correlate with disease resistance or improvements in production and welfare will allow the use of APPs as selection parameters for breeding to be evaluated. For APPs to be useful parameters on which to evaluate chicken health, information on normal APP concentrations is required. Ceruloplasmin (Cp) and PIT54 concentrations were found to be much lower in healthy birds form commercial production farms than the reported normal values obtained from the literature. These APPs were found to be significantly higher in culled birds from a commercial farm and Cp, PIT54 and ovotransferrin (Ovt) were significantly higher in birds classified as having obvious gait defects. Using quantitative shotgun proteomics to identify the differentially abundant proteins between three pools: highly acute phase (HAP), acute phase (AP) and non-acute phase (NAP), generated data from which a selection of proteins, based on the fold difference between the three pools was made. These proteins were targeted on a individual samples alongside proteins known to be APPs in chickens or other species: serum amyloid A (SAA), C-reactive protein (CRP), Ovt, apolipoprotein A-I (apo-AI), transthyretin (Ttn), haemopexin (Hpx) and PIT54. Together with immunoassay data for SAA, Ovt, alpha-1-acid glycoprotein (AGP) and Cp the results of this research reveal that SAA is the only major APP in chickens. Ovotransferrin and AGP behave as moderate APPs while PIT54 and Cp are minor APPs. Haemopexin was not significantly different between the three acute phase groups. Apolipoprotein AI and Ttn were significantly lower in the HAP and AP groups and as such can be classed as negative APPs. In an effort to identify CRP, multiple anti-sera cross reacting with CRP from other species were used and a phosphorylcholine column known to affinity purify CRP were used. Enriched fractions containing low molecular weight proteins, elutions from the affinity column together with HAP, AP and NAP pooled samples were applied to a Q-Exactive Hybrid Quadrupole–Orbitrap mass spectrometer (Thermo Scientific) for Shotgun analysis and CRP was not identified. It would appear that CRP is not present as a plasma protein constitutively or during an APR in chickens and as such is not an APP in this species. Of the proteins targeted as possible novel biomarkers of the APR in chickens mannan binding lectin associated serine protease-2, α-2-HS-glycoprotein (fetuin) and major facilitator superfamily domain-containing protein 10 were reduced in abundance in the HAP group, behaving as negative biomarkers. Myeloid protein and putative ISG(12)2 were positively associated with the acute phase being significantly higher in the HAP and AP groups. The protein cathepsin D was significantly higher in both HAP and AP compared to the NAP indicating that of all the proteins targeted, this appears to have the most potential as a biomarker of the acute phase, as it was significantly increased in the AP as well as the HAP group. To evaluate APPs and investigate biomarkers of intestinal health, a study using re-used poultry litter was undertaken. The introduction of litter at 12 days of age did not significantly increase any APPs measured using immunoassays and quantitative proteomics at 3, 6 and 10 days post introduction. While no APP was found to be significantly different between the challenged and control groups at anytime point, the APPs AGP, SAA and Hpx did increase over time in all birds. The protein apolipoprotein AIV (apo-AIV) was targeted as a possible APP and because of its reported role in controlling satiety. An ELISA was developed, successfully validated and used to measure apo-AIV in this study. While no significant differences in apo-AIV plasma concentrations between challenged and control groups were identified apo-AIV plasma concentrations did change significantly between certain time points in challenged and control groups. Apoliporotein AIV does not appear to behave as an APP in chickens, as it was not significantly different between acute phase groups. The actin associated proteins villin and gelsolin were investigated as possible biomarkers of intestinal health. Villin was found not to be present in the plasma of chickens and as such not a biomarker target. Gelsolin was found not to be differentially expressed during the acute phase or as a result of intestinal challenge. Finally a proteomic approach was undertaken to investigate gastrocnemius tendon (GT) rupture in broiler chickens with a view of elucidating to and identify proteins associated with risk of rupture. A number of proteins were found to be differentially expressed between tendon pools and further work would enable further detailing of these findings. In conclusion this work has made a number of novel findings and addressed a number of data poor areas. The area of chicken APPs research has stagnated over the last 15 years with publications becoming repetitive and reliant on a small number of immunoassays. This work has sought to characterise the classic APPs in chickens, and use a quantitative proteomic approach to measure and categorise them. This method was also used to take a fresh approach to biomarker identification for both the APR and intestinal health. The development and validation of assays for Ovt and apo-AIV and the shotgun data mean that these proteins can be further characterised in chickens with a view of applying their measurement to diagnostics and selective breeding programs.