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The aims of this thesis were to use a serum-free culture system to investigate the role of the IGF system during early bovine follicular development. The study focused on investigating the actions and regulation of IGF in three key stages of follicle growth. These key stages were: primordial follicle initiation, the transition from preantral to antral stages, and early antral follicle growth. No effects of IGF-I on primordial follicle initiation or activated follicle growth were found, but IGF-1 that was not regulated by IGFBPs was found to have a negative effect on the oocyte health of growing follicles after 6 days of culture. Hence, during the early stages of follicle development the regulation of the bioavailability of IGF is crucial to maintain the health of the oocyte. A 6 day culture of bovine early antral follicles in the presence of IGF-1 was found to stimulate both follicle proliferation in the early stages of development, and differentiation at all developmental stages, as exhibited by oestradiol production. Furthermore, these effects were found to occur in a dose and stage dependent manner. Additionally, oocyte health was improved by the addition of recombinant IGF-I in the more mature antral follicles. These results highlight the importance of follicle developmental stage when deciding the best in vitro culture conditions. The biological actions of IGF depend in part on the ability of specific proteases to break down the IGF/IGFBP complex. The secretion of proteases capable of degrading IGFBP-2 by different bovine follicular compartments was identified, and the effect of IGF-1 and/or follicle stimulating hormone (FSH) on modulating IGFBP-2 proteolytic degradation was also investigated. Proteolysis of IGFBP-2 caused by the incubation of IGFBP-2 with oocytes was not detected. However, incubation of IGFBP-2 with granulosa cells did cause a small level of IGFBP-2 degradation. The ability of IGF-1 and FSH to enhance or inhibit the degradation of IGFBP-2 was also studied. The use of a serum-free culture system in this study has improved our understanding of how the complex IGF system is regulated throughout bovine follicular development. It has highlighted that the regulation of IGF bioavailability to a growing follicle is governed by different levels of regulation, such as the level of expression and degree of proteolysis of IGFBPs, which are follicle stage dependent. A fuller understanding of the mechanisms that control the access and regulation of local growth factors will allow us to move closer to developing an in vitro culture system capable of producing large numbers of viable oocytes for use in reproductive technologies.

The mechanisms governing development of mammalian oocytes are not well understood. Isolation and in vitro growth of immature cattle follicles will enable determination of the factors affecting bovine follicular development, have potential applications in assisted reproduction and provide a suitable model for studying human infertility. Intercommunication of the oocyte and somatic cells is necessary for normal oocyte and follicle development. Studies using systems where oocyte-somatic cell communication is preserved allows an accurate assessment of the factors affecting follicular development. The aims of this project were to examine early follicle and oocyte development in cattle and determine whether the bovine oocyte plays a role in follicular development. A non-enzymatic isolation procedure was developed which allowed intact bovine follicles to be isolated. On the basis of follicle size, these could be divided into 3 distinct stages: large preantral, large preantral/early antral and antral follicles. A culture technique was devised which supported in vitro follicle and oocyte development, the key elements of which were: volume of medium (0.25 ml/follicle), serum and insulin minimal number of medium changes and a substrate of collagen. The effect of FSH on preantral to early antral follicles in culture was examined. Initial experiments on large preantral/early antral follicle growth found that all FSH doses stimulated an increase in follicle diameter. The dose of FSH was important as low levels did not stimulate proliferation or affect oocyte size whilst high levels reduced proliferation, inhibited oocyte growth and reduced oocyte quality. Oocyte localised granulosa cell proliferation was observed in some follicles only when a healthy oocyte was present, demonstrating the importance of oocyte-somatic cell communication in granulosa cell proliferation and differentiation. The intensity of oocyte localised proliferation was reduced at high FSH doses, confirming its dose dependent inhibitory effect on follicular development. FSH stimulated the growth of large preantral/early antral and antral follicles but not oocyte growth in any of the stages. The increase in size was due to an increase in intercellular spacing and, as antral cavities were neither maintained or formed during culture, this may be analogous to antrum development. FSH maintained granulosa cell proliferation in all follicle size classes. No detectable effect of FSH on preantral follicles were found, therefore the effect of FSH depends on the stage of follicle examined.

Salmonella is an important zoonotic pathogen and 10,000 cases of human salmonellosis are reported annually in the UK. The most commonly implicated serovars are S. Enteritidis and S. Typhimurium. Since a quarter of British pigs carry Salmonella in their gut at slaughter, there is an urgent requirement for improved control strategies that could benefit human health. A literature review showed that hygiene, biosecurity and feed exposures were important risk factors for Salmonella infection in pigs, which originates from environmental contamination or introducing infected pigs into the herd. The aim of this research was to design and test an intervention to control Salmonella in pigs. The following objectives were achieved: 1. An evaluation of tests for Salmonella in pigs: isolation by culture and the meat juice (MJ) ELISA, to inform test selection for the intervention study. 2. A national farm-level survey to estimate the variation in Salmonella prevalence between farms and to investigate risk factors associated with infection. 3. An analysis of a merged MJ ELISA dataset with a quality assurance dataset to provide additional information on risk factors. 4. A randomised controlled trial of an enhanced hygiene and biosecurity protocol intended to control Salmonella infection in finisher pigs. The intervention was tested on 48 farms. The primary outcome was the pen incidence rate of Salmonella infection, measured by culture of pooled pen floor faecal samples. No important change in incidence between intervention and comparison groups was seen. Analysis by reported behaviour showed that improved attention to between-batch cleaning and disinfection was beneficial. The prevalence of infected pens shortly after re-stocking had an overwhelming effect on incidence whilst improved hygiene during production had relatively little effect. Therefore, enhanced hygiene and biosecurity may yield benefits in Salmonella control, but these may be overwhelmed by the introduction of infection at re-stocking or through residual environmental contamination.

Epizootic Hemorrhagic Disease Virus (EHDV) is an insect-transmitted pathogen of ruminants, causing periodic and significant losses in wild and captive deer populations and less frequently, a bluetongue-like disease in cattle. The serogroup of EHDV within the Orbivirus genus of the Reoviridae family consists of seven serotypes, in which emerging serotypes pose an increasing risk either regionally or globally, due to the insect vectors. To date, no vaccine against EHDV is commercially available, apart from the live-attenuated vaccine for EHDV-2 (IBAV). In this study, Virus-Like Particles (VLPs) of EHDV-1 and heterologous VLPs of EHDV-2 were generated using baculovirus multigene expression system for the synthesis of the two outer and two inner capsid proteins, essential for the formation of VLPs. The assembly of EHDV-1 recombinant structural proteins into Core-Like particles (CLPs, two proteins) and (VLPs, four proteins) was confirmed by EM analysis. The biological activity of the raised antisera to neutralise EHDV-1 was efficiently confirmed by neutralisation assay at 1:64 dilution. Cross neutralising activities were also detected against EHDV-2 and EHDV-6 serotypes at 1:8 dilution. Results presented in this study validate the potential efficacy of the VLP as a neutralising vaccine and strongly suggest its use as vaccine candidate. Additionally, an alternative approach was also initiated in this research to develop a rational vaccine against EHDV-2 using the reverse genetics system (RG). Towards this, it was first established that in vitro synthesised transcripts from purified EHDV-2 cores could generate infectious virus upon cell transfection. Note that both the generation of core transcripts and recovery of infectious virus of EHDV were not demonstrated previously. Subsequently a complete set of 10 T7 transcripts was synthesised, however, it was not possible to recover any infectious virus, likely to be some unwarranted mutations. Nevertheless, these transcripts will be further investigated for future RG studies.

Culicoides midges are important haematophagous insects. Their principal significance is as vectors of> 75 diseases worldwide. Surveillance over a four-year period using Onderstepoort UV-light traps, found that Culicoides Obsolelus and Pulicaris groups dominated the species assemblage in Northern Ireland, at 74.8% and 20.7% of the total catch, respectively. Should a Culicoides vectored disease be introduced into Northern Ireland during the vector active period (early April to mid December), there are ample potential vectors to enable disease transmission. Principal Component Analysis of habitats surrunding trap locations confirmed that Culicoides abundance was positively correlated to characteristics of a livestock-rearing farm, whilst negatively correlated to arable and non-vegetated land. There was no significant difference between numbers pf Obsoletus Culicoides trapped outdoors or within cattle housing. However, fewer Pulicaris group Culicoides were captured in indoor compared to outdoor traps. Obsoletus group Culicoides breed mostly in cattle dung, whilst most Pulicaris group emerged from sheep dung. Cattle slurry and on-farm middens were also identified as suitable breeding sites for Obsoletus group Culicoides, although emergence from these substrates was sporadic. Culicoides alighting on farm livestock were mostly Obsoletus and Pulicaris groups, representing 92.0% and 7.7% of all Culicoides captured, respectively. Pulicaris group Culicoides had an intrinsic attraction to sheep when compared with cattle on a per liveweight basis. However, there was no evidence to suggest that Obsoletus group Culicoides fed preferentially on cattle over sheep, suggesting that this group is prone to opportunistic host seeking behaviour. Visual cues played a significant role in the host seeking behaviour of Obsoletus group Culicoides, with white and clear traps more attractive than yellow or blue.

Parasites of livestock have major negative economic impacts worldwide; as such, enhanced control of key parasitic diseases would augment farm profitability. However, control has been complicated in recent years through the advent of resistance to commonly used anthelmintic drugs. With the emergence of helminth populations resistant to four of the five major classes of anthelmintic drugs comes the need to develop sustainable control strategies for animal welfare. The first steps in developing future control strategies are; the quantification of the prevalence of resistance within a geographical area, and, investigations on the ways in which such levels of resistance have arisen. The aims of this Thesis were - 1. To investigate the prevalence of Anthelmintic Resistance (AR) in sheep flocks in NI; 2. To examine potential management factors associated with the development of AR; and 3. To attempt to identify non-operational factors which may exacerbate the development of AR. As the levels of AR are most pronounced in small ruminants, the majority of work was undertaken in sheep. Many of the management practices that select for AR in ovine nematodes are conserved between bovine, porcine and avian parasites; as such, data collated in this Thesis will have relevance to all animal enterprises within the Agri-Food industry of NI. Included within this Thesis are - 1. The the results of two Questionnaire surveys which detail the methods used to control gastrointestinal nematode, trematode and cestode species parasitising sheep flocks; 2. The results of three coprological surveys investigating anthelmintic treatment efficacy in controlling nematodiasis, fasciolosis and trichostrongylosis/teladorsagiosis; and, 3. The publications which came about through the completion of the above. All research was carried out in the Parasitology laboratory, Veterinary Sciences Divison, Agri-Food and Biosciences Institute, Stormont, during the years of 2010 - 2013.

Canine Transmissible Venereal Tumour (CTVT) is a neoplastic disease occurring naturally in dogs. CTVT is usually sexually transmitted, but transmission may occur by licking, biting, or scratching tumour-affected areas. Cytogenetic studies directed to verify the cell transmissibility of the CTVT showed that the tumour cells are characterized by a rearranged karyotype, which is similar in tumours from in different parts of the world. Thus it was postulated that that the transmissible agent causing the CTVT is the tumour cell itself and that all worldwide CTVT have a clonal origin. Given the lack of a definitive proof of the cellular transmission and clonal origin of CTVT, in this PhD project, I tested this hypothesis by analysing the genetic distance between host and tumour and between tumours, using molecular genetic markers including major histocompatibility (MHC) genes, microsatellite loci, and mitochondrial (mt) DNA in matched tumour and normal tissues from naturally occurring tumours collected worldwide. Here I demonstrate that tumours are genetically distinct from their hosts and that the tumours obtained from 5 different continents are derived from a single neoplastic clone. During its evolution CTVT has diverged into two distinct phylogenetic subclades. Although naturally occurring CTVTs are observed only in dogs, CTVT has been reported to be experimentally transmitted by inoculation of tumour cells in other species of the Canidae family such as foxes, coyotes and jackals, thus raising questions about its capacity to grow as an allograft or xenograft and its phylogenetic origin of CTVT. I therefore examined MHC gene transcription in a progressive growing tumour and observed MHC class I and II downregulation. In this study phylogenetic analyses indicate that CTVT most likely originated from a wolf or an East Asian breed of dog. CTVT has been described for the first time in 1876 by Novinsky, thus arguing for an age of at least 130 years old. Here phylogenetic analysis based on microsatellites suggests that CTVT originated between 250 and 2500 years ago.

African trypanosomes (<i>Trypanosoma brucei spp.</i>) are the causative agents of Human African Trypanosomiasis and Nagana in cattle and livestock. The complex biphasic lifecycle of <i>T. brucei</i> involves differentiation from the mammalian host to the tsetse fly vector, a process that involves highly regulated changes in gene expression, morphology and surface antigen expression. Three members of the <i>T. brucei </i>Zinc Finger Protein (TbZFP1, 2 and 3) family have been characterised, which contain the unusual C_x8C_x5C_x3H RNA binding zinc finger motif, as well as a PY or WW protein-protein interacting motif. The TbZFP family are important regulators of differentiation and all three molecules have been shown <i>in vivo</i> to be essential for effective differentiation, although no proteins or RNA transcripts involved in these mechanisms have as yet been identified. Here, data obtained from screens for proteins and RNA transcripts that interact with the TbZFP family is presented. Using the yeast two-hybrid system, TbZFP1 was shown to interact directly with TbZFP2 and 3, this interaction being dependent on the WW domain. Furthermore, a yeast two-hybrid screen to identify novel protein interactions using TbZFP2 and 3 was undertaken, though no genuine interactions were identified. In addition, a systematic evolution of ligands by an exponential enrichment (SELEX) approach was used to identify the sequence TCAGT/C as a putative RNA binding motif for TbZFP3 and the ability of TbZFP3 to bind RNA was verified using an <i>in vitro</i> EMSA. Finally, a screen was undertaken for RNA transcripts whose abundance was affected by the ablation of TbZFP2.

An <i>in vitro</i> method in which to investigate transgene expression in the chicken oviduct prior to the generation of transgenics is desirable. The egg white protein genes are expressed in the tubular gland cells (TGCs) of the oviduct of laying hens. TGCs were isolated from the magnum region of the oviduct of adult hens and were found to be sufficiently viable in culture for use to investigate transgene expression. Egg white mRNAs and proteins were detected in TGCs maintained <i>in vitro</i> for 72 hours but tests of transfection using several commercially available reagents, electroporation and lentiviral transduction indicated that the frequency of gene transfer was too low to be useful. As an alternative, the use of oviduct tissue explants was investigated. Explants were isolated from the magnum region of the oviduct of sexually mature laying hens and the cells in the explants were found to be sufficiently viable in culture for use to investigate transgene expression. Egg white mRNAs and proteins were detected in the explants maintained <i>in vitro</i> for 4-5 days. Transfection using reagents was not successful but gene transfer into the explants was successfully achieved through electroporation. Expression of a therapeutic protein was detected in the explants after electroporation with a transgene constructs carrying a ubiquitous promoter or the ovalbumin promoter. Gene transfer into the explants was also achieved through transduction with EIAV and HIV vectors pseudotyped with a variety of envelope proteins. The highest levels of transduction were achieved using vectors pseudotyped with the vesicular stomatitis virus G envelope protein. Expression of a therapeutic protein was detected in the explants after transduction with viral vectors carrying a ubiquitous promoter or the ovalbumin promoter. Chicken oviduct explants were identified as suitable for use <i>in vitro</i> to investigate transgene expression in the chicken oviduct.