Anterior cruciate ligament rupture (ACL) is a major source of morbidity in the dog, leading to severe osteoarthritis of the knee joint and marked lameness. Following rupture, the ACL will not heal and in the dog, ACL rupture is thought to be the end stage of degenerative ligament disease (non-contact ACL injury). The extracellular matrix (ECM) of CLs has been extensively studied but little is known of the role of elastic fibres in the physiology of the ECM, the mechanics of CL function and in CL degeneration. Elastic fibres include polymers of fibrillins (microfibrils), bundles of microfibrils (oxytalan fibres) and elastin fibres (bundles of microfibrils with an elastin core). The hypothesis of this thesis is that elastin has a mechanical and a biological role in the canine cruciate ligament complex. It is further hypothesised that the distribution and function of elastic fibres will vary between three breeds of dog with differing risk of ACL rupture are: the greyhound with a low risk, the beagle with a low-to-moderate risk and the Labrador retriever with a high risk. The distribution of elastic fibres, fibrillins and cells was investigated throughout the CL complex using a combination of histochemical staining and immunofluorescence. CL microanatomy was studied using Nomarski differential interference microscopy. Elastin was measured biochemically and compared to histologic assessment of tissue architecture, elastic fibre staining and other biochemical parameters. The biological effect of elastin degradation products (EDPs) was assessed in an in vitro ACL cell culture model. A low risk dog breed to ACL rupture (greyhound) was used in all investigations and comparisons were made with other breeds with regard to cellular and elastic fibre anatomy. Differences in cell morphology between breeds with differing risk of ACL rupture may reflect fundamental differences in CL physiology possibly through altered cell-to-cell communication. Cellular and matrix changes, considered degenerative, were seen throughout the CL complex and may reflect adaptation rather than degeneration in certain dog breeds such as the greyhound. Elastin content ranged from 5.9 to 19.4% of ligament dry weight. This was a far greater proportion of canine CLs than previously. Elastin fibres may have a mechanical role in bundle reorganization following ligament deformation. The distribution of fibrillins 1 and 2 was different from the pattern previously reported in tendon and may represent a fundamental difference between ligament and tendon. In the greyhound CL there was a significant proportional increase in oxytalan fibre staining with advancing CL degeneration. This response was seen also in the Labrador retriever and the beagle but the increase in oxytalan fibre staining was less marked with advancing degeneration. Therefore production of oxytalan fibres may reflect a healing response in damaged CL tissue in breeds at a low risk of ligament rupture. Fragments of elastin containing the VGVAPG motif affect canine ACL cells in vitro resulting in increased transcription of fibrillin 2 mRNA. Additionally, there was synergism with TGF-β1 resulting in upregulation of the elastin laminin receptor 1, through which EDPs are transduced. EDPs may thus have a role in response to injury in the CL.

The racing Greyhound presents us with an opportunity to study the characteristics of a successful athlete and the costs and risks such specialisation entails. This thesis investigates the nature of the injuries suffered by racing Greyhounds and how adaptation of the musculoskeletal system to the unique pattern of stresses encountered during racing and training might impact upon the risk of injury. Racing Greyhounds sustain a number of musculoskeletal injuries. Several of these, notably fatigue fractures of distal limb bones, are very similar to those seen in human athletes and military recruits (Armstrong et al. 2004; Beck et al. 2000; Brukner et al. 1996; Kowal 1980; Matheson et al. 1987). The most common, often leading to the dog being euthanatised, is fracture of the right tarsus. Evaluation of tarsal fractures via radiography alone frequently resulted in an underestimation of the severity of the injuries, whereas the use of computed tomography provided a more detailed, accurate assessment. Evidence of asymmetric bone remodelling was found in the distal limb bones of racing Greyhounds. Rail‐side bones had significantly higher bone density and increased levels of bone resorption and formation markers compared to contralateral bones. Greyhound bones also have regional differences in trabecular architecture. In contrast, Staffordshire Bull Terrier (SBT) bones did not show these differences. Additionally, Greyhound distal limb tendons appear well adapted to withstand the high stresses of racing; they are stronger, stiffer, and in the pelvic limbs, return more elastic strain energy than the corresponding SBT tendons. Greyhounds had left‐to‐right asymmetries in the tensile properties of their pelvic limb tendons, which SBTs did not. SBTs are not bred for racing and are unlikely to encounter asymmetric stresses. Therefore, the adaptive changes observed in the Greyhound bones and tendons appear to result from the asymmetric stresses encountered by the Greyhounds during racing around ovoid tracks.

The aim of the present study was to investigate the hormone, sexual behaviour and neuropeptide profiles of follicular phase ewes and examine alterations after the application of acute stressors. In study 1, follicular phases of intact ewes were synchronised with progesterone vaginal pessaries. Ewes then received saline vehicle, insulin (4 iu/kg) or endotoxin (LPS; 100ng/kg) at 28h after progesterone withdrawal (PW; time zero). In study 2, this protocol was repeated, but animals were killed at 0h, 16h, 31h and 40h after PW and brain tissue retrieved. In study 1, there was a delay of 17.6h and 7.2h (P<0.05), respectively, in half the insulin-treated animals (‘insulin-delayed’) but not in the other half; and a delay of 22.5h and 20.7h (P<0.001), respectively, in all LPS-treated animals. Plasma oestradiol concentrations decreased after both stressors (P<0.001) and cortisol increased in all groups (P<0.05); whereas progesterone increased in the insulin-delayed and LPS groups only (P<0.05). In study 2, immunohistochemistry was used to examine transcriptional activation (co-expression with c-Fos) of various neuropeptides in the hypothalamus and preoptic area. In control ewes, the maximum percentage of dynorphin cells co-localising c-Fos (i.e., activated) was observed at 31h after PW (52%; P<0.05), whereas maximum activated kisspeptin and neurokinin B cells occurred at 40h after PW (49 and 42%, respectively; P<0.05). The percentage of activated dopamine cells decreased before the onset of sexual behaviour (from 70 to 26%; P<0.05) whereas β-endorphin activation was lower during the LH surge (from 41 to 10%; P<0.05). In contrast, neuropeptide Y and somatostatin activation was higher during the surge (from 21 to 36%; P<0.08; and from 14 and 9% to 47 and 73%, respectively; P<0.05). However, LPS decreased the percentage of activated dynorphin cells (to 11%; P<0.05) and kisspeptin cells (to 22%; P<0.05). On the contrary, insulin decreased the percentage of activated dynorphin cells (to 27%) in two of the insulin-treated animals (insulin-responders) but not in the other two; whereas the percentage of activated kisspeptin cells increased in all insulin-treated animals (52%; P<0.05). Neurokinin B was not altered by either treatment. Furthermore, insulin increased the percentage of activated β-endorphin, neuropeptide Y and somatostatin cells in the ARC (to 71, 72 and 63%, respectively, P<0.05) but LPS did not have the same effect. In the VMN, activation of somatostatin cells was greater in all LPS treated animals (from 8 to 27%; P<0.05) but only in two of the insulin-treated animals (to 55 and 76%; insulin-responders) but not in the other two (to 5 and 6%; insulin-non-responders). These results indicate that there is a specific hormonal, behavioural and neuropeptide pattern during the follicular phase of intact ewes and this is disturbed by acute LPS or insulin administration in the late follicular phase, leading to the disruption of the LH surge.

The work in this thesis falls into three parts. The first part relates to the time spent with the industry as part of this CASE Studentship, whilst the second and third parts relate to stochastic transmission models and the analysis of interventions imposed upon these models. The second and third parts are linked by a common aim, which is to develop models to understand the dynamics of Salmonella transmission on a pig farm and thus identify key drivers of Salmonella. The thesis begins with an assessment and analysis of a Farm Tool Questionnaire that was developed by the industry. A total of 28 farms were visited, had pooled faecal samples taken and completed the Farm Tool Questionnaire. The main aim of this study was to pilot the developed tool and identify any areas that could be modified in order to enhance its usability. Furthermore, the results from the study were used in an attempt to highlight any possible areas of farm management that differ between Platinum farms and non-Platinum farms. It was shown that Platinum farms were likely to adopt a subset of biosecurity practices, which should consequently encourage farms to adopt a range of biosecurity practices rather than focusing on one aspect of biosecurity. The thesis then turns to the development of mathematical models in order to try and understand how the components of the system interact by using both numerical simulation and mathematical analysis. As farming methods differ considerably between farms, two key forms of unit structure were analysed: a fully slatted unit and a solid floored unit. The models were developed using a semi-stochastic transmission model similar to Xiao et al. [2006] (Y. Xiao, D. Clancy, N. P. French & R. G. Bowers. A semi-stochastic model for Salmonella infection in a multi-group herd. Mathematical Biosciences, 200(2):214-233, 2006). These were then used to assess any differences in dynamics as a result of farm structure. Finally, both sets of models were analysed in order to identify any possible interventions that could have some form of control on Salmonella prevalence at slaughter. The models showed that the key drivers of Salmonella transmission were the amount of bacteria shed and the probability of infection after exposure. As such, interventions focusing on these aspects should be implemented in order to see the most beneficial results. The rate at which infection was able to spread when shedding was high was found to be of great importance within the various models; indicating that solid flooring is a potential risk factor. Furthermore, as infection was able to spread quickly within the solid-floored unit, the time interval at which cleaning and disinfection were carried out could be of importance. However, this would require further investigation.

Relatively little is known about Salmonella Virchow as a pathogen. Its prevalence varies from country to country, however it is constantly associated with invasive disease, particularly in children and the immuno-compromised. The main sources of S. Virchow are humans and poultry. The aims of this study were to determine the genetic relatedness of S. Virchow isolates from difference sources in England, to characterise its infection biology using in vitro and in vivo based models, to establish the immune response produced by poultry in response to infection with the serovar and to begin to determine the level of protection and cross-protection that could be achieved against the serovar and S. Typhimurium. The genetic relatedness of the S. Virchow isolates was determined using molecular typing techniques including MLST and PFGE. The isolates were screened for the presence of 12 virulence genes that have been associated with adhesion, invasion and persistence. Human and avian cell lines and in vivo poultry infection experiments were used to characterise S. Virchow’s invasiveness, persistence and ability to elicit an immune response, compared to a well characterised S. Typhimurium isolate. Immune responses were evaluated by immunohistochemistry, RT-PCR and ELISA, to establish aspects of the innate, cellular and humoral response, as well as cytokine and chemokine expression. An in vivo poultry infection experiment was performed to gain an indication of the level of protection and cross-protection offered by primary infection with S. Virchow against secondary infection. Bacteriology, ELISA and western blot methods were used analyse this. Overall, S. Virchow appears to be a relatively clonal serovar, regardless of the source and the results indicate this is widespread and not solely in the UK. All of the isolates possessed the 12 virulence genes, which could contribute to its virulence in some hosts. S. Virchow was particularly persistent and inflammatory in the human Caco2 cells, which is consistent with the increased virulence previously reported in humans. The in vitro HD11 assay and the in vivo poultry infection experiments were consistent in showing S. Virchow colonises the chicken intestine to high levels, causes transient systemic infection and stimulates a moderate inflammatory response, very similar to S. Typhimurium infection. S. Virchow infection stimulated all aspects of the chicken immune system, characteristic of a broad-range serovar. Initial results from the in vivo protection experiment showed primary infection with S. Virchow does offer some protection against systemic invasion, although adequate protection against caecal colonisation was not found. However, 2 proteins were identified that strongly reacted and cross-reacted with sera from infected chickens, providing optimism that a vaccine to protect against S. Virchow colonisation could be developed with further research.

Canine infection from pathogenic Leptospira serovars remains an issue within the UK, despite the availability of a canine vaccine. Canine leptospirosis cases are non-reportable and data regarding current levels for both suspected and confirmed cases is limited. A questionnaire based survey was undertaken to determine the number of canine leptospirosis cases within UK practices over a 12 month period. Average canine vaccination coverage across responding practices was determined as 60%, with 1669 vaccines administered per practice on average within the responding practices. No significant difference was witnessed between doses administered in either mixed or dedicated small animal practices (1692.40 and 1653.38 respectively), demonstrating that vaccination habits vary between individual clinicians rather than practice type. Diagnosing leptospirosis remains an issue, particularly relating to vague clinical signs during early infection. Survey results emphasised the priority that clinicians base on initially vague signs, with leptospirosis being typically considered once icteric signs present, where mortality rates are greater and treatment is less effective. Despite well documented associations linking leptospirosis and rodents, a current uncertainty remains regarding serovars maintained within the UK. In an attempt to clarify the situation, 283 wild rodents were sampled from rural (n=7) and urban sites (n=8). Infection was identified within 23 (8.13%) samples belonging to wood mice (n=16/152), bank voles (n=5/47) and field voles (n=2/10). Initial Leptospira identification using direct sequencing of PCR amplicons showed a single infecting pathogenic species (Leptospira interrogans). Serology data was obtained for 71 rodents using the microscopic agglutination test (MAT). Positive samples from pooled antigen testing (n=7/71; 9.86%) were further tested using four individual antigens. Data further confirmed a single infecting species (L. interrogans) and serogroup (Australis). Interestingly, we did not detect Leptospira within the portion of 67 rat kidney samples investigated. Stained kidney sections (n=11) showed limited association between inflammation and leptospire presence, indicating that rodents may shed the bacteria asymptomatically. Multi-locus sequence typing (MLST) schemes have been successfully applied to identify the sequence types (STs) of pathogenic Leptospira strains. The PCR positive rodent samples were tested using MLST (n=23). All samples with a full profile (n=11) were shown to belong within ST-24, with five partial profiles also likely to belong to the same ST. To date, three serovars are within ST-24 (Jalna, Bratislava and Muenchen), that belong to the Australis serogroup. This was the first study to utilise DNA extracted directly from kidney tissue to perform Leptospira MLST analysis. MLST data further emphasises a single infecting species and presents evidence for a single infecting serogroup. Further work involved full genome sequencing of ten strains not previously investigated, covering pathogenic, intermediate and saprophytic species. Sequence data for each strain was obtained using the MiSeq platform. The ‘core’ genome was identified across 17 strains (n=1,095; 28.76%), with pathogenic strains being more conserved with a greater shared core genome (n=2,859; 69.30%). Single nucleotide polymorphism (SNP) data was generated for strains (n=6), with an average of 35,346 SNPs per strain (range=686 to 55,303). L. interrogans serovar Icterohaemorrhagiae had the lowest SNP count (686) and was the only strain with a greater number of non-synonymous SNPs compared to synonymous (1.8:1); indicating close relatedness between serovars Icterohaemorrhagiae and Copenhageni. Coding sequences were identified within genome regions that may relate to antigenic differences (high SNP variation) or relate to key cellular processes (low SNP variation). Due to poor gene characterisation, hypothetical proteins make up a high proportion of coding sequences within such regions. Further work to characterise identified coding sequences may identify future therapeutic or diagnostic targets. This project aimed to investigate the current situation concerning canine and rodent Leptospira research within the UK. Results presented within this thesis demonstrate several wild rodent species within England are capable of maintaining and potentially shedding pathogenic strains known to infect both humans and dogs. Serogroup Australis (found infecting rodents) is now protected within a tetravalent canine vaccine; however annual booster vaccinations are required for optimal immunity. Extended urban sampling would be of great benefit considering the absence of positive urban samples. Suspected and confirmed canine cases are still witnessed within UK practices despite the reported majority having current vaccinations. Continued monitoring of serogroups would benefit vaccination strategies, and an emphasis on early detection within infected dogs would allow for the greatest chance of survival.

Bluetongue (BT) is a seasonal vector-borne, viral, disease that causes significant economic and welfare problems in ruminants. It is transmitted by species of Culicoides midges (Diptera: Ceratopogonidae), and as such, the distribution of the disease is restricted to regions where the vectors are present. Once restricted to tropical and subtropical regions of the world, serotypes of BT have been causing outbreaks in southern Europe, following its introduction in 1998, and in 2006, BT serotype-8 emerged in northern Europe, causing devastating economic, welfare and production consequences. The northwards expansion of BT has been attributed to a shift in the geographic limit of the Culicoides imicola Meigen vector, and the involvement of the newly implicated Palaearctic vectors, the Obsoletus and Pulicaris Groups. Little is known about the ecological characteristics of the newly implicated vectors, or indeed those believed to be non-vectors, including their distribution and abundance, making disease risk assessment and management difficult. Within this thesis, a series of field experiments were initiated on a group of farms to gain insight into the distribution and abundance of Culicoides species. The results highlighted that a very high level of variation is seen when trapping Culicoides at the local-scale, yet it is possible to build a strong model explaining this variation using a mixture of host and environmental variables, with satellite-derived ecological correlates. This high level of variation in midge catches present between farms undermines attempts to record their nationwide distribution in larger scale models. The results uniquely model Obsoletus Group abundance, and highlight a difference in host involvement between vector and non-vector models. Further field studies which showed a lack of significant variation both between years and at the within-farm level highlight the robustness of this model in predicting the distribution of the BT vectors species, such that it could prove useful for exploring targeted surveillance and control methods. Culicoides distributions do not remain static, therefore an understanding of their flight behaviour is critical to determining the distance over which an insect may transmit a disease agent and the size of the area over which control should be applied. Laboratory studies were undertaken to validate the use of commercial fluorescent dusts as a quick and effective method of marking Culicoides for both field and laboratory studies, and a ‘self-marking’ technique was conceived. Dispersal studies, using the dusts, determined the distances that Obsoletus Group females and males, as well as C. pulicaris females, are able to disperse over a set period of time. This knowledge of flight speed and distance is of utmost value as a critical component in the modelling of BT disease and other Culicoides-borne diseases. The Obsoletus Group contains four members (C. obsoletus, C. scoticus, C. chiopterus and C. dewulfi¬) which are difficult to differentiate down a microscope. Using morphometric analyses, female C. obsoletus and C. scoticus individuals could be separated under a stereomicroscope based on abdominal measurements. Studies such as those contained in this thesis, therefore, are of utmost value in providing information on critical components in the modelling of BT disease and other Culicoides-borne diseases.

Culicoides biting midges (Diptera: Ceratopogonidae) are biological vectors of economically important arboviruses of livestock. Two such arboviruses, bluetongue virus (BTV) and Schmallenberg virus (SBV) have recently emerged in northern Europe inflicting unprecedented outbreaks of disease in this region. The aim of the current investigation was to explore both host seeking behaviour and surveillance methods for livestock-associated Culicoides species in the UK. To achieve this aim, a series of field-based, manipulative experiments were conducted using three farm sites in southern England. These studies demonstrated that host preference had a significant impact upon several parameters important in determining arbovirus transmission. Culicoides were found to be differentially attracted to different breeds of sheep (p<0.05) and blood feeding efficiency was shown to be determined in part by whether the sheep had been sheared (p<0.05). In addition the presence of an alternative host (a cow and its calf) was demonstrated to lead to an increased Culicoides biting rate on sheep held in close proximity (p<0.05), increasing the risk of arbovirus transmission. Preliminary studies of volatile chemicals produced by hosts illustrated that while these attracted livestock-associated Culicoides at rates higher than those recorded in un-baited traps (p<0.05), collections only represented a small proportion of those collected on hosts themselves. These studies, however, provided a platform for future investigations of this area. Finally, the use of light-emitting diode (LED) baited suction traps was trialled as a means of improving detection sensitivity in surveillance of Culicoides populations. This study found that certain Culicoides species demonstrated increased sensitivity to specific wavelengths (p<0.05) and integration of these commercially available traps could improve our understanding of the abundance, geographic distribution and behaviour of these species.

This thesis presents work to examine the genetic basis of CCL rupture in dogs. It describes research to identify causative mutations that will help to develop a genetic screening test to identify dogs that have a high risk of developing CCL rupture. Cranial Cruciate Ligament (CCL) rupture is the most common cause of hind limb lameness in dogs and is especially common in large and giant breeds such as Newfoundlands, Rottweilers and Staffordshire Bull Terriers. CCL rupture cases and from two continents (Europe n = 48 and North America n = 48) were examined using genome wide association studies (GWAS). A candidate gene study was performed using Sequenom iPlex genotyping, on cases and controls from Newfoundlands (99 cases, 172 controls) and three other susceptible breeds: Labradors (124 cases, 165 controls), Rottweilers (57 cases, 81 controls) and Staffordshire Bull Terriers (13 cases, 38 controls). One hundred and eighty-six SNPs across 26 candidate genes were investigated for association with CCL rupture. To investigate downstream events, gene expression was compared between healthy and CCL rupture tissue. An in-vitro laboratory model of CCL rupture was investigated by examining ligamentocytes induced with and without TNFα. To investigate whether there was an auto-immune component to CCL rupture, the two main loci of the dog leucocyte antigen (DLA) system were assessed for disease association. Principle component analysis of the GWAS data revealed population stratification within the Newfoundland breed, indicative of the continent of origin (Europe or North America). GWAS identified three main regions associated with CCL rupture (on chromosomes 1, 3 and 33). Significantly associated genes SORCS2 and SEMA5B function in neurological pathways; this may indicate that mechanotransduction, neurological and neuromuscular pathways play an important role in the pathogenesis and susceptibility to CCL rupture. Candidate gene analysis identified associations with two collagen genes (collagen type-V and collagen type-I) and three extracellular matrix proteins; Aggrecan (ACAN), Opticin (OPTC) and Latent transforming growth factor beta 2 (LTBP2). Gene expression analysis revealed significant differential expressions in COL1A1 and COL1A2. These results indicate that the strength and stability of the ligament is probably important in susceptibility to CCL rupture. Gene expression results also revealed that genes involved in degradation (TRAP and DIRC2) are upregulated, indicating that the cells are trying to repair themselves whilst a simultaneous degradative process is still on going. The TNFα model may be used as an in-vitro model to study CCL rupture, but may be more useful as a model for examining changes that occur after CCL rupture rather than the early stages of the disease. We showed no association with the DLA region and CCL rupture. The identified associated regions should be further investigated and refined using next generation, targeted re-sequencing and transcriptomic approaches. This could identify the specific causative mutations involved in CCL rupture susceptibility. This study confirms that there are complex multigenetic and environmental factors involved in CCL rupture susceptibility. The work has contributed to the understanding of causative factors involved in CCL rupture susceptibility and may be an important step in the development of a screening test(s) to reduce the incidence of CCL rupture in dogs and thus improve their health and welfare.

African horse sickness (AHS) is a devastating disease of many equids and in the absence of effective treatment, vaccination plays an important role. Concerns of the commercially available live attenuated vaccine have stimulated the development of novel recombinant vaccines. The viral protein VP7 of AHSV induces high levels of antibody in infected horses. Both viral replication, and expressed VP7 in insect cells, results in the generation of hexagonal crystalline structures composed entirely of VP7 (~5pm in diameter). The aim of this thesis was to explore the immunogenicity of VP7 crystals and their potential as a novel vaccine delivery platform. Firstly, successful expression of AHSV-4 VP7 was characterised in vitro using recombinant baculovirus and live vaccinia vectored systems. These provided the necessary tools to explore VP7 crystal immunogenicity. Pilot VP7 vaccination studies were conducted in the equine and mouse animal model. Secondly, insertions of AHSV-4 VP2a peptide into VP7 at amino acid position 200 did not compromise the ability of the modified VP7 constructs to form crystals and allowed investigations of the immunogenicity of VP7/VP2a200 in the mouse animal model. This project demonstrated that VP7 crystals were poor immunogens in the absence of adjuvant and multiple vaccinations. However, immunogenicity was Improved when crystals were partially solubilised with urea, indicating that soluble VP7 was more immunogenic than VP7 crystals. Hence, crystal generation may provide a mechanism for immune evasion in AHSV infection. Nevertheless, both VP7/VP2a200 untreated crystals and urea treated VP7/VP2a200 crystals induced insert-specific antibodies. Importantly, VP2a peptide antibodies raised against urea treated crystals induced low levels of AHSV-4 neutralisation in vitro. Future work should include exploring the immunological and protective potential of soluble VP7 and further Investigating the use of VP7 as a vaccine platform.