Foot-and-mouth disease (FMD), a disease of cloven hooved animals caused by FMD virus (FMDV), is one of the most economically devastating diseases of livestock worldwide. The global burden of disease is borne largely by livestock-keepers in areas of Africa and Asia where the disease is endemic and where many people rely on livestock for their livelihoods and food-security. Yet, there are many gaps in our knowledge of the drivers of FMDV circulation in these settings. In East Africa, FMD epidemiology is complicated by the circulation of multiple FMDV serotypes (distinct antigenic variants) and by the presence of large populations of susceptible wildlife and domestic livestock. The African buffalo (Syncerus caffer) is the only wildlife species with consistent evidence of high levels of FMDV infection, and East Africa contains the largest population of this species globally. To inform FMD control in this region, key questions relate to heterogeneities in FMD prevalence and impacts in different livestock management systems and to the role of wildlife as a potential source of FMDV for livestock. To develop FMD control strategies and make best use of vaccine control options, serotype-specific patterns of circulation need to be characterised. In this study, the impacts and epidemiology of FMD were investigated across a range of traditional livestock-keeping systems in northern Tanzania, including pastoralist, agro-pastoralist and rural smallholder systems. Data were generated through field studies and laboratory analyses between 2010 and 2015. The study involved analysis of existing household survey data and generated serological data from cross-sectional livestock and buffalo samples and longitudinal cattle samples. Serological analyses included non-structural protein ELISAs, serotype-specific solid-phase competitive ELISAs, with optimisation to detect East African FMDV variants, and virus neutralisation testing. Risk factors for FMDV infection and outbreaks were investigated through analysis of cross-sectional serological data in conjunction with a case-control outbreak analysis. A novel Bayesian modeling approach was developed to infer serotype-specific infection history from serological data, and combined with virus isolation data from FMD outbreaks to characterise temporal and spatial patterns of serotype-specific infection. A high seroprevalence of FMD was detected in both northern Tanzanian livestock (69%, [66.5 - 71.4%] in cattle and 48.5%, [45.7-51.3%] in small ruminants) and in buffalo (80.9%, [74.7-86.1%]). Four different serotypes of FMDV (A, O, SAT1 and SAT2) were isolated from livestock. Up to three outbreaks per year were reported by households and active surveillance highlighted up to four serial outbreaks in the same herds within three years. Agro-pastoral and pastoral livestock keepers reported more frequent FMD outbreaks compared to smallholders. Households in all three management systems reported that FMD outbreaks caused significant impacts on milk production and sales, and on animals’ draught power, hence on crop production, with implications for food security and livelihoods. Risk factor analyses showed that older livestock were more likely to be seropositive for FMD (Odds Ratio [OR] 1.4 [1.4-1.5] per extra year) and that cattle (OR 3.3 [2.7-4.0]) were more likely than sheep and goats to be seropositive. Livestock managed by agro-pastoralists (OR 8.1 [2.8-23.6]) or pastoralists (OR 7.1 [2.9-17.6]) were more likely to be seropositive compared to those managed by smallholders. Larger herds (OR: 1.02 [1.01-1.03] per extra bovine) and those that recently acquired new livestock (OR: 5.57 [1.01 – 30.91]) had increased odds of suffering an FMD outbreak. Measures of potential contact with buffalo or with other FMD susceptible wildlife did not increase the likelihood of FMD in livestock in either the cross-sectional serological analysis or case-control outbreak analysis. The Bayesian model was validated to correctly infer from ELISA data the most recent serotype to infect cattle. Consistent with the lack of risk factors related to wildlife contact, temporal and spatial patterns of exposure to specific FMDV serotypes were not tightly linked in cattle and buffalo. In cattle, four serial waves of different FMDV serotypes that swept through southern Kenyan and northern Tanzanian livestock populations over a four-year period dominated infection patterns. In contrast, only two serotypes (SAT1 and SAT2) dominated in buffalo populations. Key conclusions are that FMD has a substantial impact in traditional livestock systems in East Africa. Wildlife does not currently appear to act as an important source of FMDV for East African livestock, and control efforts in the region should initially focus on livestock management and vaccination strategies. A novel modeling approach greatly facilitated the interpretation of serological data and may be a potent epidemiological tool in the African setting. There was a clear temporal pattern of FMDV antigenic dominance across northern Tanzania and southern Kenya. Longer-term research to investigate whether serotype-specific FMDV sweeps are truly predictable, and to shed light on FMD post-infection immunity in animals exposed to serial FMD infections is warranted.

The PARABAN project has been a Scotland-wide initiative to develop and deliver farm-specific ‘best practice’ for the control of Mycobacterium avium ssp. paratuberculosis (MAP) in cattle using ‘Knowledge Exchange’. A range of partners have been involved, including nine ‘Champion Farms’. With input from the farmer, his/her vet and PARABAN advisors, a tailored monitoring and control programme was devised for each ‘Champion Farm’, taking into account the history of the disease on the farm, the physical facilities available and farmer objectives. Culling decisions based on live animal test results were incorporated into each farm-specific programme to complement the management programme already in place to maintain each herd. Results were analysed and discussed with all the partners throughout the project and then offered for wider scrutiny at farm open days. Feedback and questions from these open days have been used to complete the ‘Knowledge Exchange’ cycle. As a major component of the PARABAN project the author collected samples from all adult animals culled from ‘Champion Farms’ at slaughter or as fallen stock, irrespective of in-life MAP test status. These were then subjected to histopathological examination by experienced veterinary pathologists and the results compared with the results from in-life MAP testing. This was intended to evaluate the contribution slaughterhouse sampling could make towards decision making for disease control on farm and formed the main aim of this thesis. In total, samples of terminal ileum and draining lymph node were collected from three-hundred and fifty-two animals. A positive result on histopathology was defined as the presence of lesions typical of MAP and also the presence of acid-fast bacteria within the sections. There was found to be fair agreement between the overall results from histopathology and serum ELISA (Kappa = 0.33), though there appeared to be some variation in agreement between the tests on the individual ‘Champion Farms’. The presence of MAP was confirmed in seven of the eight farms which contributed animals to this study, despite sometimes prolonged efforts at controlling the disease. A separate study was undertaken to make use of the archives of the Scottish Centre for Production Animal Health and Food Safety at the Veterinary School, University of Glasgow. The archive contained records of cases from across southern Scotland and northern England. Analysis of the data generated from examination of these records suggested that MAP is widespread within the Scottish cattle herd and may well be increasing.

Periodontal disease is a common and painful condition in the horse. Although awareness of the condition is growing amongst the veterinary profession and horse owners, the presence of the disease is often overlooked and treatment can be difficult. Despite this, there have been few recent studies of the aetiopathogenesis of the condition. Certain species of bacteria may act as periodontal pathogens, stimulating a destructive inflammatory response in periodontal tissues and this has been well recognised as being important to the aetiopathogenesis of the disease in man. However few equine studies on this aspect of the disease have been carried out. The main aims of this study were: - 1) to identify the bacteria associated with a healthy oral cavity and periodontitis in horses using culture dependent and independent methods; 2) to assess the differences in bacterial populations between the healthy and periodontitis groups and identify putative pathogens; 3) to quantify the expression patterns of TLRs 2, 4 and 9, the pro-inflammatory cytokines IL-1β and TNFα, anti-inflammatory cytokine IL-10 and Th1/Th2/Th17 cytokines IL-4, IL-6/ IL-12, IFNɣ/ IL-17, within gingival tissue from each sample group; 4) to use matched data to establish if associations exist between the presence and quantity of bacterial species present and TLR expression and 5) to determine activation of TLRs 2, 4 and 9 by putative pathogens using specific in- vitro TLR assays. Swabs were taken from the gingival sulcus of 42 orally healthy horses and plaque samples were taken from the periodontal pockets of 61 horses with periodontal disease. The location and grade of the lesion was noted and an equine dental chart completed for each case. Bacteria were identified using high throughput 16S rRNA gene sequencing, QPCR, whole genome sequencing and conventional culture followed by 16S gene sequencing. Gingival biopsies were taken from 13 orally healthy horses and 20 horses with periodontitis and gene expression of TLR 2, TLR 4, TLR 9, IL-1β, IL-4, IL-6, IL-10, IL-12, IL-17, TNFα and IFNɣ was measured. THP-1X Blue, MyD88 THP-1X Blue, HEK hTLR 2 Blue and HEK hTLR 4 Blue human cell lines were co-cultured with putative periodontal pathogens and their response measured via level of secreted embryonic alkaline phosphatase. Clinical, microbiological and immunological data underwent cross-matching analysis. Microbial populations showed 89% dissimilarly between oral health and periodontitis with a less diverse population present in diseased equine periodontal pockets. The most discriminative bacteria between health and disease identified at genus level were Fusobacteria and Acinetobacter in health and Pseudomonas and Prevotella in periodontitis. The most abundant genera were Gemella (36.5%), Pseudomonas (14%) and Acinetobacter (8%) in orally healthy samples and Pseudomonas (25%), Prevotella (14%) and Acinetobacter (9.4%) in periodontitis samples. Whole genome sequencing revealed the presence of 75 species of Prevotella in the equine oral cavity and a significantly higher number of reads corresponding to Prevotella bivia, Prevotella dentalis, Prevotella denticola, Prevotella intermedia, Prevotella melaninogenica, Prevotella nigrescens were noted in diseased samples. Significant increases in expression of TLR 4 mRNA, TLR 9 mRNA and, in particular TLR 2, mRNA were noted in diseased equine gingival tissue in addition to increased pro-inflammatory and anti-inflammatory cytokine mRNA expression. Presence of P. intermedia was significantly positively correlated with expression of TLR 2 in equine periodontitis. In addition, the presence of Aggregatibacter actinomycetemcomitans was positively associated with disease severity and expression of TLR 4 mRNA in the horse. Co-culture of periodontal pathogens with human cell lines revealed that the innate immune response to the presence of these bacteria is mainly mediated through TLR 2 activation. The use of both culture dependent and culture independent methods to investigate the equine oral microbiome has provided significant breadth and depth of information on the microbiology of equine periodontal disease. Microbial populations are significantly different as expected and bacteria belonging to the Prevotella genus have been strongly implicated in the aetiopathogenesis of the condition. The innate immune response produced in periodontally diseased equine gingival tissue has been characterised for the first time in the horse.

Bovine mastitis, inflammation of the mammary gland, is one of the most costly and prevalent diseases in the dairy industry. It is commonly caused by bacteria, and Streptococcus uberis is one of the most prevalent causative agents. With advancements in omics technologies, the analysis of system-wide changes in the expression of proteins and metabolites in milk has become possible, and such analyses have broadened the knowledge of molecular changes in bovine mastitis. The work presented in this thesis aims to understand the dynamics of molecular changes in bovine mastitis caused by Streptococcus uberis through system-wide profiling and integrated analysis of milk proteins and metabolites. To this end, archived milk samples collected at specific intervals during the course of an experimentally induced model of Streptococcus uberis mastitis were used. Label-free quantitative proteomics and untargeted metabolomics data were generated from the archived milk samples obtained from six cows at six time-points (0, 36, 42, 57, 81 & 312 hours post-challenge). A total of 570 bovine proteins and 690 putative metabolites were quantified. Hierarchical cluster analysis and principal component analysis showed clustering of samples by the stage of infection, with similarities between pre-infection and resolution stages (0 and 312 hours post-challenge), early infection stages (36 and 42 hours post-challenge) and late infection stages (57 and 81 hours post-challenge). The proteomics and metabolomics data were analysed at both individual omics-layer level and combined inter-layer-level. At individual omics layer-level, the temporal changes identified include changes in the expression of proteins in acute-phase response signalling, FXR/RXR activation, complement system, IL-6 and IL-10 pathways, and changes in the expression of metabolites related to amino acid, carbohydrate, lipid and nucleotide metabolisms. The combined inter-layer-level analyses revealed functional relevance of proteins and metabolites enriched in the co-expression modules. For example, possible immunomodulatory role of bile acids via the FXR/RXR activation pathways could be inferred. Similarly, the actin-binding proteins could be linked to endocytic trafficking of signalling receptors. Overall, the work presented in this thesis provides deeper understanding of molecular changes in mastitis. On a secondary note, it also serves as a case study in the use of integrative polyomics analysis methods in the investigation of host-pathogen interactions.

No description available.

Lymphoma is the most common haematopoietic malignancy of dogs, but little is known about its aetiology and pathogenesis. Canine lymphoma is composed of a clinically heterogeneous group of tumours, as in human non-Hodgkin lymphoma (hNHL), which shares many similarities with the canine disease. In humans, lymphoma is classified into many different subtypes, each with its own clinicopathological characteristics, prognosis, and in many cases, treatment regime. In dogs, diagnosis is often rudimentary, and many cases are not routinely or robustly subtyped. This has hampered efforts to develop accurate prognostic indicators and novel therapies, as heterogeneous groups of cases are analysed. More accurate classification of canine lymphomas is required before real progress can be made. In the work described in this thesis, molecular techniques were used to develop diagnostic tests to aid diagnosis of canine lymphoma, with a view to improving disease classification, and gaining a greater understanding of disease pathogenesis. The diagnosis of canine lymphoma can be assisted by PCR for antigen receptor rearrangements (PARR), which can determine clonality and tumour cell lineage. Diagnostic samples from suspected lymphoma patients were screened with multiple published PARR primers to establish a panel of assays for routine use. Primer coverage was assessed, and modifications to both primer sequences and primer combinations made where necessary. The optimised PARR assay was robust, highly sensitive and specific, comparable with published studies and provided useful diagnostic information unavailable from other tests. In humans, the Epstein-Barr virus (EBV) is associated with several lymphoma subtypes. Recently, it has been suggested that EBV or an EBV-like virus is circulating in dogs. Serological and molecular techniques were used to investigate whether EBV, or a novel herpesvirus, is associated with canine lymphoma. In an assay designed to detect antibodies to EBV viral capsid antigens, 41% of dogs were positive. Dogs with cancers, including lymphoma, were more frequently positive than controls, but no particular association with B-cell lymphoma was noted. EBV-specific RNA and DNA sequences were not detected in lymphoma tissue by in situ hybridisation or PCR and herpesvirus genomes were not detected using multiple degenerate PCR assays with the ability to detect novel herpesviruses. No evidence that herpesviruses are directly involved in common types of canine lymphoma was found, although the presence of an EBV-like virus in the canine population cannot be excluded. Gene expression profiling has been used to subtype human diffuse large B-cell lymphoma, and to provide insight into pathogenesis. Next-generation sequencing was used to generate transcriptome data from a panel of canine B- and T-cell lymphomas. Gene expression analysis showed clear clustering of B-cell and T-cell types, and suggested that differentiation within the B-cell group was possible. Pathway analysis demonstrated up-regulation of genes in pathways appropriate to the tumour type. Sequence data were also explored to identify variants in important genes and pathways which may have functional consequences. Many affected genes were identified, and comparison with hNHL suggested that common driver mutations are present. By identifying novel targets, such studies may pave the way for development of new therapies to benefit both man and dog.

Human papillomavirus type 16 (HPVI6) is a causative agent of cervical cancer. The HPV16 viral transcription/replication factor E2 is essential for the viral life cycle. The function of E2 is dependenton the interaction with cellular partner proteins. The amino- terminal domain of E2 is an acidic protein-protein interaction domain essential for all of the functions of E2. A yeast-2-hybrid screen using the amino-terminal domain of HPV16 E2 as bait identified multiple possible partner proteins for E2 function (Boner & Morgan 2002) including the DNA replication/repair protein TopBPl. E2 molecules from HPVI6 and HPV18 interact with multiple proteins involved in the cellular response to DNA damage (e.g. p53, BRCAl, PARP and TopBPl). The role of TopBPl in E2 function and the functional response of E2 to DNA damage stimuli were investigated. Overexpression of TopBPl enhances the transcription and replication activation functions of E2. Overexpression of an amino-terminal truncation mutant of TopBPl has no effect on the transcription/replication functions of E2. During this study a novel method for the detection of E1/E2 DNA replication function by real-time PCR was developed. The UVB irradiation of cells resulted in the significant reduction of both E2 transactivation function and E2 protein amount. These results demonstrated that E2 function is altered by cellular DNA damage response proteins and signaling pathways. In many HPV induced cancers the HPV genome is either present integrated into the cellular chromosomes or is maintained episomally with large DNA deletions/rearrangements. Therefore HPV genome stability is a significant risk factor for the development of HPV induced cancer. Thus the fidelity of DNA replication activated by the HPV 16 E1/E2 replication factors was investigated on undamaged and UVC damaged templates in a variety of genetic backgrounds. On undamaged DNA templates there were a significant amount of mutations due to DNA deletions/rearrangements and the frequency of mutation increased when the template was damaged. This increase on damaged templates was marked in cells with defects in key DNA replication or repair proteins. These results highlight the instability of HPV16 genome replication. The interaction of E2 with DNA damage response proteins and the reduction of E2 function in response to DNA damage may be an evolutionary response by the virus to ensure genetic integrity and host cell viability.

The historical development, uses, pharmacology, chemistry and metabolism of phanothiazine and the phenothiazine tranquillisers are reviewed along with the analytical techniques which have been used for their detection and determination. The metabolism and excretion by the horse of promazine chlorpromazine, acepramazine and propionylpromazine, tranquillisers commonly used in equine practice, have been examined qualitatively and quantitatively. Methods of collection, storage and analysis of horse urine have been developed, and the use of mass spectroscopy in combination with gas liquid chromatography for the detection of phenothiazine derivatives in biological fluids has been examined. Qualitative results have shown that many biotransformations of such derivatives take place in the horse. 30 metabolites of promazine were detected in addition to the parent drug, 20 of chlorpromazine and 11 and 6 respectively from acepromazine and propion ylpromazine. Hydroxylation followed by conjugation, prademinantly with glucoronic acid and to a smaller extent with sulphuric acid, is the major route of metabolism of promazine and chlorpromazine. Acapromazine and propionylpromezine, on the other hand, are not conjugated to a great extent. Instead they lose the side chain ketone grouping attached to the nucleus at the 2-position and are excreted in the sulphoxide form. Quantitative results have shown that excretion is irregular and prolonged, in some cases lasting for a week after administration. The percentage of dose detected as urinary metabolites res low, being approximately 19% for chlorpromazine, 10% for promazine and 3% for acepromazine. Conjugated metabolites were excreted predominantly as sulphide derivatives, whereas the unconjugated fraction were predominantly in the sulphoxide form. The applicability of the techniques used for the detection of phenothiazine derivatives, after administration in small doses just sufficient to produce an effect, has also been examined.

A variety of inflammatory markers, coupled with changes in a number of haematological and biochemical parameters have classically been used to diagnose, monitor, and prognosticate disease in horses. Unfortunately these traditional markers respond fairly slowly to the presence of disease and inflammation and have wide normal ranges (Allen and Cold 1988; Pepys et al., 1989). Serum amyloid A (SAA), and haptoglobin are acute phase proteins common to humans, cattle, sheep, mice, and several other species, including the horse. In several of these species, plasma concentration of SAA has been shown to increase 1000-fold following tissue injury, cellular necrosis, inflammation, and infection and decline rapidly in the recovery phase. In the studies presented here the concentration of serum amyloid A (SAA), haptoglobin and fibrinogen and other indices of health were measured in a number of different groups of horses, including; normal horses, those subjected to operative surgery, horses with surgical colic, racehorses in training, some of which had evidence of gastric ulceration, and foals with respiratory disease. Acute phase protein concentration was modeled with the outcome and with other commonly measured indices of health relevant to the disease states of interest. The study indicates that there is an association between acute and chronic inflammation and between the present of disease both overt and latent and suggests that the concentration of a number of acute phase proteins could be used to aid decision making when planning diagnostic or treatment interventions in horses.

No description available.