Murid herpesvirus 4 (MHV-68) has been widely studied as a model of gammaherpesvirus infection. Infection of Apodemus sylvaticus, a natural host for MHV-68, revealed that a virally-encoded chemokine binding protein (M3) influences the composition of the perivascular and peribronchiolar inflammatory infiltration and the formation of BALT in the lung during lytic infection. In addition, host genes were identified which were expressed at higher levels in the presence of MHV-68 M3 at 14 days post infection (dpi), including Clara cell secretory protein (CCSP), Short palate lung and nasal epithelium clone 1 (SPLUNC1) and Anterior gradient 3 (AGR3). The aim of this work was to further investigate the expression of these genes and their corresponding proteins in relation to respiratory viral infection. CCSP and SPLUNC1 have previously been shown to have antiinflammatory properties in models of virus, bacteria and allergen induced inflammation. AGR3 is thought to be homologous to AGR2, which is associated with the transition of Clara cells to mucous cells in the lung. Following MHV-68 infection in A. sylvaticus, levels of both CCSP and SPLUNC1 were reduced in the bronchioles at 7 dpi and increased at 14 dpi, compared to mock-infected controls. In the absence of M3, the level of CCSP was reduced compared to wild type MHV-68 infected animals at both timepoints, whereas no significant difference in the expression of SPLUNC1 in the bronchioles was present. The regulation of both of these genes has previously been associated with interferon γ (IFNγ); infection of 129 wild type and IFNγR-/- mice revealed that CCSP expression was increased and SPLUNC1 expression decreased in the presence of IFNγ. However, this effect was smaller than that due to MHV-68 infection. Expression of AGR3 in the respiratory tract was increased in response to MHV-68 infection, whereas AGR2 was decreased. To investigate whether these effects were specific to MHV-68, infection with other respiratory viruses, with different cellular tropisms in the respiratory tract were examined in BALB/c mice. Infection with Human respiratory syncytial virus, Sendai virus and several strains of Influenzavirus A led to a decrease in both CCSP and SPLUNC1 expression during acute infection, when this was associated with a significant inflammatory response in the lung. The findings of this work showed that CCSP and SPLUNC1 are constitutively expressed in the non-ciliated cells of the respiratory epithelium and support the hypothesis that they have an antiinflammatory role in the lung. Expression of both proteins is reduced in the event of acute viral infection resulting in significant inflammation. In MHV-68 infection of A. sylvaticus, increased expression of CCSP and SPLUNC1 at later timepoints suggests that these proteins are implicated in the resolution of the inflammatory response.

Mastitis remains one of the most common diseases of dairy cows and represents a large economic loss to the industry as well as a considerable welfare issue to the cows affected. Decisions are routinely made about the treatment and control of mastitis despite evidence being sparse regarding the likely consequences in terms of clinical efficacy and return on investment. The aim of this thesis was to enhance decision making around the treatment and prevention of mastitis using probabilistic methods. In Chapter 2 and Chapter 3, decision making around the treatment of clinical mastitis was explored using probabilistic sensitivity analysis. The results from Chapter 2 identified transmission to be the most influential parameter affecting the cost of clinical mastitis at cow level and, therefore, highlighted how important the prevention of transmission was in order to minimise losses associated with clinical mastitis. The cost-effectiveness of an on-farm culture (OFC) approach to the treatment of clinical mastitis was explored in Chapter 3, and compared with the cost-effectiveness of a ‘standard’ approach commonly used in the UK. The results of this study identified that the OFC approach could be cost-effective in some circumstances but this was highly dependent on the proportion of Gram-negative infections and the reduction in bacteriological cure rate that may occur as a result of the delay before treatment. Therefore, in the UK, this approach is unlikely to be cost beneficial in the majority of dairy herds. In Chapters 4, 5 and 6, decision making around the control of mastitis was explored utilising data from UK dairy herds that had participated in a nationwide mastitis control plan. In Chapter 4, mastitis control interventions were identified that were not currently practised by a large proportion of herds, and the frequency at which they were made a priority by the plan deliverers was also reported. In Chapter 5 and Chapter 6, the cost-effectiveness of specific mastitis control interventions was explored within an integrated Bayesian cost-effectiveness framework from herds with a predominance of environmental intramammary infections. Results from the Bayesian microsimulations identified that a variety of interventions would be cost effective in different farm circumstances. The cost-effectiveness of different interventions has been incorporated in a decision support tool to assist optimal decision making by veterinary practitioners in the field.

The research in this thesis deals with the influence of charge on the adsorption of milk proteins to surfaces. A variety of charged surfaces were used including negatively charged and zwitterionic liposomes prepared from phosphatidylglycerol and phosphatidylcholine respectively and positively and negatively charged polystyrene latices. Adsorption was determined by measuring the increase in the hydrodynamic radius of the particles by photon correlation spectroscopy and also by solution depletion techniques. In some instances, electrophoretic mobility measurements were also used in order to determine changes in the surface charge of the particle as a result of protein adsorption. The ionic strength and pH of the buffer were found to be important in the adsorption of protein to liposomes. In the absence of NaCl, adsorption did not occur. At low pH values, addition of both k-casein and B-Iactoglobulin to negatively charged liposomes caused very large increases in size presumably as a result of aggregation. At pH6.2, protein layer thicknesses on the negatively charged liposomes were significantly greater than on the zwitterionic ones due to charge repulsion between the negatively charged surface and the negatively charged regions of the proteins. Removal of the negatively charged phosphate groups which form a cluster in the hydrophilic region of B-casein resulted in a reduction in the thickness of the adsorbed protein layer on the negatively charged liposome but did not have any effect on the thickness on the zwitterionic surface. The thickness of adsorbed layers of as1-, k-, and B-casein and B-lactoglobulin on the phosphatidylcholine liposomes were all very similar at around 6nm. Addition of as1-casein to the negatively charged liposomes appeared to cause aggregation as a result of protein molecules bridging between liposomes. Attempts to determine the fraction of added protein which bound to the surface of the liposomes were unsuccessful and therefore, the binding of native, dephosphorylated and methyl-esterified l3-casein to small, monodisperse, positively and negatively charged polystyrene latices was studied. As with the liposomes, the thickness of the adsorbed B-casein layer was greater on the negatively charged surface. Removal of the phosphate groups from the protein decreased the layer thickness by about 4nm on the negatively charged surface but had relatively little effect on the thickness on the positively charged surface, once again showing the effect of charge interactions. As with dephosphorylation, methylation also reduces the net negative charge of the protein, but by a different mechanism. This also resulted in a reduction in the thickness of the adsorbed protein layers but only after a significant proportion of the free carboxyl groups had been esterified. Thus methylation of 35 % of these groups had relatively little effect on the thickness of the layer on the positively charged latex and no effect on the negatively charged, but esterification of a further 9% (equivalent to two residues) caused a substantial decrease in thickness on both surfaces. These changes are believed to result from alterations in both the charge and hydrophilicity of particular regions of the B-casein molecule. Bridging was found to occur when low levels of native or modified B-casein were added to the positively charged latex. Protein loading was found to range from 2.5 to 5.5mg m-2 depending on the nature of the protein and the charge on the surface. The thickness of adsorbed native and dephosphorylated B-casein layers on the negatively charged latex was found to be influenced by the presence of calcium and increasing ionic strength. Increasing levels of either calcium ions or NaCI in the medium resulted in a very pronounced decrease in the thickness of pre-adsorbed phosphorylated B-casein layers. The changes in dephosphorylated protein layers were less pronounced. The results are discussed in terms of the proposed loop-and-train configuration of the B-casein at the surface of the latex. The influence of protein phenotype and the extent of glycosylation on the adsorption of k-casein was also determined. The more highly glycosylated protein molecules, which also had a higher net negative charge, formed thicker layers on the negatively charged surface. Again, layer thicknesses were less on the positively charged surface, but for each x-cn phenotype glycosylation increased the thickness, presumably as a result of the increased hydrophilicity of the protein. k-Casein A, which has one more negative charge than the B phenotype, was found to give a slightly thicker layer on the negatively charged latex. Under certain conditions, adsorbed k-casein could be cleaved by the enzyme chymosin as shown by the reduction in the size of the coated latex.

The aims of the first study were to estimate the incidence rate of clinical mastitis (IRCM) for suckler ewes (ewes rearing lambs for meat production) in England and to identify potential risk factors. A postal questionnaire was sent to 999 randomly selected English sheep farmers in 2010 to gather data on the IRCM and flock management practices for the calendar year 2009. The mean IRCM per flock was 1.2 / 100 ewes / flock / year (0.0-19.1). The IRCM was 2.0, 0.9 and 1.3 / 100 ewes / year for flocks that were always housed, always outdoors and a combination of both respectively. Six mixed effects over-dispersed Poisson regression models offset by flock size were developed to identify management practices associated with IRCM. Of particular interest were environmental factors, as indicated by the affect flooring and indoor versus outdoor rearing had on the IRCM, and host susceptibility as indicated by the association of IRCM with udder conformation (a potentially heritable trait). Based on these findings, the role of the environment was first considered by investigating the potential sources of mastitis-causing pathogens. Milk samples and udder skin swabs were taken from 27 pedigree Texel ewes with clinical mastitis and 3 ewes showing no clinical signs in order to identify whether udder skin could act as a bacterial reservoir for intramammary infections. Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS) was used to identify isolates to species level and compared to pulsed-field gel electrophoresis (PFGE) to assess the capabilities of MALDI-ToF-MS as a strain differentiation tool. MALDI-ToF-MS had good correspondence with PFGE for all species tested in this study including Staphylococcus warneri, Staphylococcus equorum, Rhodococcus corprophilus, Bacillus pumilus and Micrococcus luteus. The same strain type was found in mastitic milk and on ewe udder skin for Staphylococcus warneri and Rhodococcus corprophilus indicating a possible reservoir of bacteria that might enter the mammary gland. The third study built upon the findings from the second study by identifying the role of lamb mouths and ewe udder skin as potential transmission pathways of mastitis causing bacteria by sampling these environments and ewe milk over time in a longitudinal study of 21 ewes over 10 weeks. Twenty-six bacterial species were found in more than one location (lamb mouth, ewe udder skin and/or ewe milk), many of which have previously been associated with mastitis. Potential transmission events and persistence of the same bacterial strains between and within lamb mouths, ewe udder skin and/or ewe milk over time were identified for the first time in suckler ewes using MALDI-ToF-MS for a variety of bacterial species. Having identified udder conformation as the most significant factor associated with IRCM in the first study, the final study aimed to investigate whether udder and teat conformation are heritable traits that affect mastitis. A total of 968 records were collected from 10 Texel flocks over a period of 3 years (2012-2014) from England, Wales and Scotland to assess whether udder and teat conformation were heritable traits associated with higher levels of chronic mastitis. Univariate quantitative genetic parameters were estimated using individual animal and sire models. The heritabilities for teat length and teat placement were greatest (0.42 and 0.35, respectively). The remaining traits (traits that generally describe the volume of the udder) were of moderate to low heritability. Univariable logistic regression was used to identify the phenotypic association between udder traits and chronic mastitis. The work in this thesis has addressed several gaps in the knowledge in mastitis epidemiology by providing the first estimate for the IRCM in suckler flocks in England, and generating hypotheses for factors that may affect the IRCM. The role of the ewe’s environment and heritable traits that could potentially affect a ewe’s likelihood of getting mastitis were subsequently investigated. The results have contributed to our understanding of the factors associated with the development of mastitis.

Ovine footrot is an infectious disease of sheep which causes serious economic losses to countries worldwide and costs the UK £84 M per annum. For effective control of this disease which is a major animal welfare issue, it is essential to study mechanisms of transmission. One of the key aims of the project was to improve our understanding of the potential for Dichelobacter nodosus, the causal agent, transmission via the environment within a flock of ewes and lambs. Newborn lambs were free from D. nodosus presence on their feet, but rapidly became contaminated with D. nodosus within 5-13 h after birth as detected using the specific molecular markers rpoD and 16S rRNA gene. The likely source of contamination of lambs was the straw bedding from the communal pens. A diverse population of D. nodosus was observed on the feet of ewes and lambs as determined by the presence of multiple strains with variable numbers of pgrA tandem repeats in the R1 region. This was further supported by Multi Locus Variable Number Tandem Repeats (MLVA) typing of the isolates, which also indicated high variation in the alleles present on the ewes and lambs. The primary aim was to determine if multiple strains present on the ewes were vertically transmitted to the lambs. This work has clearly demonstrated that no vertical transmission occurred between ewes and lambs and some strains, but not all were shared between ewe and its lamb suggesting transmission from their mother or other ewes sharing the same lambing pen. D. nodosus was detected in a range of environmental samples such as bedding, faecal balls compacted within the interdigital space and soil suggesting that shedding into the environment is the main route of D. nodosus transmission. Survival studies provided evidence that the pathogen persisted in soil microcosms for at least 40 days with viable cells persisting for a minimum of 30 days in four soil types. A lower temperature of 5 oC and clay soil was associated with longer duration of survival. Single Nucleotide Polymorphism (SNP) analysis of isolates from the ewes and lambs indicated that two main clonal populations existed that represented two clusters α and β within clade I, a virulent clade from the recent genome study including 103 global D. nodosus strains. No UK strains grouped with the benign clade II. Previous diversity studies on the isolates indicated diversity in MLVA types, pgr alleles and pgrA tandem repeats, but these did not correlate with the clustering; clusters α and β contained a mix of pgrA and pgrB but both were in clade I. This conflicts with the role of pgrB as a non-virulent allele although pgrA expression was induced by hoof horn in vitro, whereas pgrB was not. The work reported in this thesis has improved our understanding of the environmental transmission of D. nodosus between sheep, longevity of the pathogen in soil and diversity of strains in the UK.

This PhD thesis aimed to identify the motivators and barriers of farmers and veterinary surgeons to the implementation of vaccination strategies on British dairy farms. There is no universal cattle vaccination schedule in Britain, therefore vaccination decisions are made on a farm by farm basis, however there is a paucity of research investigating the decision-making behind dairy cattle vaccination. Twenty-four semi-structured interviews were carried out with dairy farmers and fourteen with veterinary surgeons. The transcripts were subject to thematic analysis which generated five key themes from each of the interview studies. Farmers and vets perceive vaccines to be an effective and useful tool to control and prevent disease on British dairy farms and are motivated to vaccinate cattle if there is evidence of disease on-farm, or a perceived risk of disease entering a farm. Challenges to cattle vaccination chiefly arise from differences in how risk is perceived by vets and farmers, and farmers’ potential lack of awareness of their herd’s disease status. Enhancing the relationship between farmers and vets is therefore crucial to optimising vaccination decision-making. In order to optimise implementation of vaccination strategies further research including farmer vaccination compliance, vaccine efficacy, methods of communication and risk perception is needed. This will support creation of a practical vaccination strategy, and could provide a basis for national collaborative disease control strategy.

No description available.

No description available.

Skeletal muscle comprises about half of the body weight in mammals and its diverse roles in metabolism and innate immune functions makes cultured muscle as a useful tool in biomedical research. In this thesis, some of the major technical obstacles in porcine primary muscle cell cultures such as inadequate differentiation of myoblasts into myotubes and the premature detachment of the formed myotubes have been overcome with the achievement of high differentiation and myotube fusion rate of over 85% along with prolonged maintenance of myotubes in excess of 70 days. Myosin heavy chain (MyHC) expression profile of differentiated myotubes recapitulated adult muscle fibres and displayed phenotypic plasticity of gene expression in response to different media. Growth factor ractopamine (Ract) treatment (1h and 6h) of myotubes followed with subsequent bioinformatics analysis of a stable isotope labelling by amino acids in cell culture (SILAC) based proteomics study suggested differential expression of proteins associated with anti-viral innate immune response and increased protein accretion. Porcine muscle cells were infected with influenza A viruses to evaluate their immune function. Porcine muscle cells expressed influenza virus sialic acid α-2,3 and α-2,6 receptors and were fully permissive to influenza virus infection. Myoblasts produced more virus particles than myotubes. Muscle cells expressed the pro-inflammatory genes tumor necrosis factor alpha (TNF-α) and antiviral gene Mx-1 and the infected cells had elevated caspase 3/7 activity to indicate apoptosis. However, myotubes pre-treated with Ract appeared to confer no reduction in influenza virus output. Evidence presented herein suggests that the functional myotubes developed by this work can be used as a tool to study the molecular mechanisms of growth and innate immune pathways in muscle.

Abdominal pain (colic) in the horse is one of the most common emergency problems in the horse, but there are major gaps in the current evidence. Five original studies were undertaken to build evidence on the primary assessment of abdominal pain in the horse. The first study was a systematic review of risk factors for abdominal pain. This identified 83 publications on risk factors for colic; 47 met inclusion criteria and were reviewed and categorised. Twenty four studies were evaluated using the JBI-Mastari critical appraisal tool. Evidence from the ten publications with least susceptibility to bias was collated. There was consensus of evidence for increasing age, recent change in diet and previous history of abdominal pain are risk factors for colic. This evidence should inform veterinary practitioner’s clinical decision making and approach to disease prevention. The second study was a systematic review of diagnostic tests for abdominal pain. This identified 190 publications; 46 publications met inclusion criteria and were reviewed and categorised. Thirty studies were evaluated using the QUADAS critical appraisal tool. None of the studies used a suitable methodology to assess the value of a diagnostic test to differentiate critical cases. The outcomes of this systematic review were a description of the current evidence and recommendations for future research using appropriate study designs. Both systematic reviews highlighted a lack of evidence from primary practice. The third study was a prospective survey of the primary assessment of horses with abdominal pain by veterinary practitioners. Data was collected from 1016 cases, including clinical history, presenting signs, diagnostic and treatment approaches and case outcome. Cases were categorised as critical or non-critical. Multivariate logistic regression was used to identify clinical features associated with critical cases. Five variables remained in the final model: pain, heart rate, capillary refill time, pulse character and gastrointestinal borborygmi. These should be considered essential components of the initial assessment and triage of horses presenting with colic. The fourth study was an online survey of veterinary practitioner’s opinions of diagnostic tests for colic. Responses from 228 participants were analysed. The most frequently used tests were ‘response to analgesia’, ‘rectal examination’ and ‘nasogastric intubation’, but there was a wide variation in practitioners’ approaches. The main reasons for not using tests were that practitioners considered they were not required, or had concerns around personal safety, lack of facilities or financial constraints. The final phase was two multi-disciplinary workshops to review current evidence and generate recommendations. Fifty participants attended the first workshop and generated 84 statements on the recognition of colic. Forty one participants attended the second workshop and generated 160 statements on the assessment of horses with colic. These statements form the first phase of a multi-disciplinary Delphi process to develop best practice guidelines on the recognition and primary assessment of horses with abdominal pain. This research makes significant contributions to the current evidence on abdominal pain in the horse. It has consolidated existing information, made recommendations for future research, and worked with practitioners to address evidence gaps, and involved different stakeholders in deciding how this evidence should be applied.