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Sheep scab is a highly contagious ectoparasitic disease caused by the mite, Psoroptes ovis, which causes intensely pruritic lesions with severe dermatitis and is a major welfare and production issue in the UK national flock. In an attempt to improve disease control, sheep scab was recently made notifiable in Scotland indicating that early diagnostic tests will be crucial to the success of this legislation. A sensitive and specific antibody based assay has been developed which can diagnose early infestation, but which does not indicate current disease status post-treatment due to residual circulating antibody levels. However, studies into host biology and response to disease have illustrated the potential use of biomarkers (BMs) in diagnostics as indicators of disease progression and the effectiveness of treatment regimes, including a recent microarray analysis which identified over 600 host genes differentially expressed in circulating leukocytes following P. ovis infestation. As many of these genes encoded proteins known to be involved in inflammatory responses, this data was used in the search for potential BMs. Initially the genes were filtered and ranked, using bioinformatic analysis, to identify the most promising BM candidates and then evaluated using Western blot analysis against a range of sera from P. ovis infested and naïve sheep. Promising results were obtained for a complement binding protein, C4BPB, showing it was rapidly up-regulated following infestation and correlated with disease progression as determined by lesion size development. The ovine C4BPB gene was successfully sequenced for the first time and a recombinant form of this protein expressed in E. coli. Antibodies, raised in rabbits against ovine rC4BPB, were used to develop a sandwich ELISA, results from which suggested the potential of C4BPB as a BM for sheep scab as it indicated current disease status postinfestation and post-treatment. The major ruminant acute phase proteins (APPs) serum amyloid A (SAA) and haptoglobin (Hp) were then investigated using commercially available assays, as previous studies indicated they were effective markers of inflammatory disease in ruminants. Results from these analyses indicated that both APPs responded positively to infestation with P. ovis but this was not statistically significant until 4 weeks post-infestation. After treatment, the APPs declined rapidly, as described by their short half life of less than 3 days following successful treatment, compared with 56 days for the estimated half life of the host antibody against the mite antigen Pso o 2. Further statistical analysis of the APP response suggested that SAA was the more discriminatory marker, with lower pre-infestation levels and higher sensitivity at the estimated optimum cut-off values. The possibility of using a signature of BMs, as an alternative to a single BM, was discussed as a method of increasing the sensitivity and specificity of the improved test, along with the potential of combining the BM diagnostic with the existing antibody assay. It was concluded that this would provide a highly sensitive and specific test for sheep scab which would diagnose early infestation as well as indicating current disease status post-treatment, providing a highly beneficial tool to the sheep industry to aid the control of this disease.

This thesis reports on an unknown/orphan gene from the sheep tick <i>Ixodes ricinus, </i>so named <i>RicOrph17, </i>that was identified by mass spectrometry while attempting to isolate a hydrolase protein by a chromatographic approach.  <i>Ric</i>Orph17 was “deorphanised” employing bioinformatics, gene-knockdown and recombinant protein production to determine its function. Primers were designed to screen an <i>I.</i><i> ricinus </i>cDNA library to obtain the full length sequence of the gene.  The <i>RicOrph17</i> open reading frame encodes a 145 amino acid polypeptide, including a putative 26-amino acids signal peptide and a 26-coding region with a putative GPI-anchoring site.  The deduced <i>Ric</i>Orph17 protein shared some structural features of the three finger protein family, including a cysteine skeleton responsible for the formation of disulfide bonds.  <i>Ric</i>Orph17 has close homologues in many different insects species. RT-PCR analysis showed that <i>RicOrph17</i> was expressed in various tissues and at different life stages. <i>RicOrph17</i> was not up regulated upon bacterial challenge.  Injection of <i>RicOrph17-</i>dsRNA into unfed adult ticks silenced the target gene expression in the whole tick.  Such dsRNA-injected ticks challenged with <i>E. coli </i>showed higher morality compared to the control injected ticks.  Recombinant <i>Ric</i>Orph17 lacking the signal peptide and the GPI-anchor peptide was produced in <i>E. coli </i>Origami cells to assist the determination of its function.  Rec<i>Ric</i>Orph17 possessed potent antibacterial activity against Gram +ve, -ve bacteria and yeast. FITC-labelled rec<i>Ric</i>Orph17 accumulated inside bacteria and did not remain embedded in the bacterial membrane.  Further, rec<i>Ric</i>Orph17 was demonstrated to bind DNA in gel retardation studies.  Taken together, these results suggest that <i>Ric</i>Orph17 is a novel GPI-linked antimicrobial peptide with an intracellular target involved in tick immunity.

Fostering is a method used by shepherds that allows the successful rearing of abandoned lambs onto other ewes, or the ability to provide surplus lambs a new mother in the case of triplets. Past research has focused on the success rates of the varying methods available, however, more research is needed to increase the knowledge about commonly selected fostering methods and the behaviour, welfare and production implications of the different methods used. Questionnaires were distributed at national farming events targeting registered sheep farms around the UK to establish which foster methods were currently in use and to collate the farmers opinions of their usage in modern day sheep farming. An experimental study was carried out to assess the welfare and production implications of the use of these methods. 84 ewes were allocated to one of the three experimental foster methods or the control group (twin lambing). They were also classified according to their lambing experience (multiparous or primiparous). Behavioural observations were conducted post-foster on the ewes and their lambs. The ewes’ salivary cortisol concentration and heart rate frequency were also monitored at 0, 30, 60, 90 and 180 minutes post-foster. To assess the production implications of the different foster methods, lamb weights and body measurements were taken at 0, 7, 30, 90 and 180 days of age to asses growth rates. Lambs were weaned at 3 months of age and remained at pasture until slaughter, at approximately 6 months of age. The lambs’ carcass quality was assessed by means of weights, zoometric measurements and conformation scores and some meat quality parameters (ultimate pH, water holding capacity and colour) were also investigated. 93% of farmers used fostering, preferring to foster rather than artificially rear lambs. Almost two-thirds favoured birth fluids (64%) and 19% of farmers used restraint crates, The most popular combination of foster methods was cervical stimulation plus birth fluids (CSBF). Exploratory factor analysis showed two main components helping farmers to decide which foster method to use, the ewes’ health and welfare and the farmers previous knowledge and success of a foster method. Birth fluids, restraint and CSBF were methods selected for behavioural data, showing that negative behaviours were significantly higher for restrained ewes compared to other treatments. Restrained ewes also showed significantly higher heart rates and salivary cortisol concentrations. Production data showed that ewe reared lambs gained significantly more weight than artificially reared lambs up to 90 days of age. However, there was no difference from that time until slaughter. Conformation and chest roundness scores were significantly better for ewe reared lambs compared to artificially reared ones. Foster methods did not have any significant effect on the growth rates, carcass or meat quality measurements for the lambs studied. The majority of farmers selected to use birth fluids, seen as a welfare friendly and less-invasive method of fostering. However, some farmers selected the restraint method based on the urgency of the lambs to receive milk and to avoid artificial rearing. The restraint crates caused significant changes to the ewes’ behaviour and increased their heart rates and cortisol concentrations, indicating a higher level of distress that could be compromising the ewes welfare. Lamb growth rates, carcass and meat quality were not affected by the different foster methods, proved that successfully established foster methods of any kind have no differential implications for the farms productivity (under UK commercial practices). Artificially reared lambs did show worse carcass conformation, suggesting that ewe rearing, and therefore fostering, offers advantages for the productivity of sheep farmers.

In seven experiments carried out under natural-daylength conditions at 57oN, Scottish Blackface and Border Leicester x Scottish Blackface ewes of varying physiological states and nutritional regimes received daily at 1500 h an oral dose of either melatonin dissolved in water and alcohol or the vehicle alone. Throughout the experiments blood samples were collected thrice weekly for progesterone, prolactin and follicle stimulating hormone concentrations (FSH) and at 10 to 15 minute intervals for periods of up to 24 h for luteinizing hormone (LH). Ovulation rates were determined by laparoscopy. When given from the beginning of June and mid-March respectively melatonin advanced and reversed the breeding season, the reversal being as effective in ewes with an extended lactation as in those weaned early or those that had not bred in the previous year. Short-term treatment (30 days) with melatonin during mid-anoestrus not only failed to advance the breeding season but delayed it. Longer term treatment (60 to 90 days) produced transient oestrous cyclicity with ewes returning to anoestrus within one month after the end of treatment. For ewes on a low plane of nutrition melatonin increased ovulation rate at first behavioural oestrus and appeared to reduce embryo mortality. In all experiments melatonin suppressed plasma prolactin 10 to 15 days after the start of treatment with levels remaining low throughout the period of treatment except during late pregnancy. There was no evidence that melatonin promoted a progressive increase in the frequency of the pulsatile release of LH or had any stimulatory effect on FSH secretion in the first 6 weeks of treatment. It did however increase plasma progesterone concentrations in the post-oestrus period.