Prion diseases or transmissible spongiform encephalopathies (TSE) are characterised by the accumulation of a misfolded conformer (PrPSc) of a host encoded protein (PrPC). The misfolding event that leads to the formation PrPSc can be replicated in the in vitro amplification technique, protein misfolding cyclic amplification (PMCA). This thesis focuses on the application PMCA to study multiple aspects of prion misfolding in relation to ruminant prion diseases, specifically developing techniques to detect and characterise PrPSc in scrapie and BSE infections. Utilising recombinant hamster PrP (rPrP) as substrate in PMCA, multiple genotypes of scrapie were successfully amplified in an attempt to describe a quantifiable technique applicable to a wide range of scrapie isolates. Observations of non-specific protease resistant rPrP formation was investigated with modifications to the PMCA methodology, which ultimately proved unsuccessful in reducing non-specific protease resistant rPrP. Using brain PrPC as substrate, the quantitative PMCA technique was piloted with BSE to correlate in vitro replication efficiency with infectious titre in mouse bioassay, but no correlation was identified. Atypical forms of BSE occur primarily in older cattle, are asymptomatic and thought to be spontaneous diseases. None the less, infection models in rodents and primates have identified the zoonotic potential of H-type and L-type BSE. Therefore PMCA methods were developed which were able to successfully amplify both atypical forms of BSE. In particular, sensitive detection and discrimination from classical BSE was demonstrated for H-type BSE, which has not previously been amplified in PMCA. H-type BSE could be detected in 1x10¬-12 g brain material and was discriminated from classical BSE by increased protease sensitivity, relatively high molecular weight and antibody reactivity. Evidence exists for co-infection of TSE strains, yet scrapie and BSE co-infection in an ovine host remains unaddressed. To study the disease progression and tissue dissemination of co-infections a PMCA assay capable of specifically amplifying BSE PrPSc in the presence of excess scrapie was applied to artificially mixed brain homogenates containing BSE and scrapie, and compared to current statutory strain typing methods. The PMCA was found to have sensitivity and specificity of 100% in mixes containing 0.1% BSE and 99.9% scrapie brain material, which was more effective than conventional strain typing methods. The assay was then applied to the brain, spleen and lymph of scrapie and BSE experimental co-infections in two genotypes of sheep, and to animals which belonged to a flock with endemic natural scrapie and that also received experimental BSE infections. The PMCA data demonstrated that sheep with PRNP genotype ARQ/ARQ (at amino acid positions 134, 154 and 171) were resistant to BSE in a co-infection scenario. In sheep with PRNP genotype of VRQ/ARQ, mixed infections could occur, and animals with scrapie PrPSc only in the brain could harbour BSE PrPSc in peripheral tissues. Co-infection was also possible in sheep with natural scrapie infections. The assay was compared to conventional testing methods of western blotting, PrPd profiling and immunohistochemistry and displayed superior sensitivity in BSE detection. PMCA amplification of bovine BSE isolates in ovine substrates identified several instances in which the molecular characteristics of the PrPSc was scrapie-like in terms of molecular weight, antibody reactivity and glycoform profile, and in some cases PrPSc characteristic of BSE could no longer be recovered. This occurred in a genotype specific manner, ‘molecular switching’ was only apparent in ovine substrate VRQ/VRQ in accordance with previous findings. These results raise the possibility of such an event occurring in in vivo ovine BSE infections and the zoonotic potential of these scrapie like conformers are yet to be fully addressed.

Understanding both the mechanistic basis of virulence and the evolutionary processes under which it can arise, is fundamental if we are to increase our knowledge of disease causing bacteria in an era of ever increasing antibiotic resistance. To date, there has been a substantial effort to understand virulence evolution both theoretically and experimentally. However, comparatively little experimental work has focussed on the evolution of virulence in opportunistic pathogens, and how virulence varies across multiple host organisms. In this thesis, the opportunist Pseudomonas aeruginosa was used to study virulence and its evolution in hosts. The virulence levels of different strains of P. aeruginosa, both laboratory and clinical, were tested in the nematode Caenorhabditis elegans. It was found that virulence of P. aeruginosa was lower in clinical strains isolated from chronic infections, and laboratory strains initially isolated from chronic infections, while a strain isolated from an acute infection was substantially more virulent. Using the C. elegans host, a selection experiment was carried out to test the effect of host density on virulence evolution. This resulted in a varied array of P. aeruginosa virulence phenotypes, but the worm evolved bacteria did not exhibit drastically different phenotypes from the worm negative control experiments. However, the degree of evolution observed across all treatment groups, compared with the wild type, highlighted the changes a bacterial population can undergo in vitro, even from a relatively short period of sub culture. Lastly, P. aeruginosa virulence was tested in alternative host organisms, Galleria mellonella (waxmoth larvae) and Vigna radiate (mung bean seedlings). Virulence varied according to the host, with virulence in G. mellonella being universally high, while no virulence was observed in V. radiate. This work highlights that high or low virulence cannot automatically be assumed in different hosts for opportunistic bacteria such as P. aeruginosa. Furthermore, the results highlight the unpredictable nature of virulence evolution, and how dependent the emergence of virulence is on the interaction between the pathogen and the host. A more complete understanding of the principles underlying bacterial evolution and virulence could allow for the construction of more specific and accurate studies, with experimental conditions carefully engineered to best replicate the clinical conditions of interest.

The aim of the work described here was to examine the antiprotozoal activity of the pericarp from Sapindus rarak DC (Sapindaceae) for its potential to defaunate the rumen, either partially or completely, without having a detrimental effect on the bacterial population and on rumen fermentation, and hence enhancing rumen productivity. An approach to improving microbial efficiency in the rumen is to eliminate protozoa (defaunation). Elimination of protozoa by chemical means is potentially the most convenient method. However, it is likely that some chemicals are not toxic specifically to the protozoa and probably kill other microorganisms and host cells in the rumen, therefore novel natural antiprotozoal agents are being sought. A possible role for saponins has been of interest to many researchers as saponins induce marked reductions in rumen protozoa numbers, particularly when the animal is fed on high-concentrate diets. The susceptibility of rumen protozoa and lack of susceptibility of rumen bacteria to saponins is explained by the reaction of saponins with membrane sterols, which are present only in eukaryotic membranes and not in prokaryotic bacterial cells. Recent in vitro results suggest that S. rarak exhibited higher immobilising activity and produce higher activity to lyse the cells compared to other saponin-containing plants. A preliminary study on the antiprotozoal activity of the active agent present in the pericarp of fruits of S. rarak, well known for the high saponin content of its fruits, was carried out. An antiprotozoal assay was developed. The results were consistent within experiments, but not between experiments, possibly due to the number of protozoa fluctuating widely with time. A study on the effect of Sapindus rarak on rumen bacteria, using a quantitative plating method, showed that there was no effect on rumen bacteria. Raw material and extract of pericarp of S. rarak were screened for their effects on rumen protozoa and bacteria (with a view to predicting its safety as a feed supplement and for its potential to defaunate the rumen, either partially or completely). The pericarp of S. rarak was extracted using selective extraction of saponins with CIS support. The fractions were collected and monitored by Thin Layer Chromatography (TLC). The active compounds were tested in vitro for toxicity to rumen protozoa by visual assessment of protozoal viability (Nottingham studies) and by measuring the degradation of labelled bacterial protein by rumen protozoa (Aberdeen studies). The influence of methanol extract of S. rarak on growth of pure cultures of rumen bacteria was also examined (Aberdeen studies). The results showed that the saponin fractions of S. rarak exhibit antiprotozoal activity as confirmed by visual assessment as well as by measurement of the breakdown of 14C-Ieucine-Iabelled Prevotella bryantti in rumen fluid incubated in vitro. No evidence was found of protozoal resistance to S. rarak. Inclusion of methanol extract of S. rarak in the growth medium of pure cultures of rumen bacteria had no effect, except that cellulolytic bacteria showed susceptibility. Extracts appeared in this study to prolong the lag phase following inoculation of Streptococcus bovis or inhibit the growth of Butyrifibrio fibrisolvens, Ruminococcos albus, and Ruminococcos flavefaciens. It seems that antibacterial properties were more pronounced against gram-positive bacteria, similar to the action ofionophores (McGuffey et al., 2001). A study on the effects of S. rarak on ruminal digestion, fermentation and ammonia concentration, using ruminally and duodenally cannulated dairy cows, showed that direct administration of S. rarak into the rumen did not decrease protozoal numbers in vivo. However, there were some indicators of selective activity against holotrichs. Changes in propionate concentration in the rumen and lack of change in microbial flow parameters suggest no adverse effects on other fermentation measures. Increased production of propionate is beneficial to the animal by affecting the capture of fermentation energy in the rumen. S. rarak caused a decrease in ammonia concentration. The observed ammonia levels represent a balance between the processes of degradation of feed protein and uptake of ammonia for synthesis of microbial protein. The lower ammonia levels could be due to higher incorporation of ammonia, peptide, or amino acids into microbial protein. In conclusion, S. rarak tested in this study has a great potential for suppressing rumen ciliate populations, but exerted negligible general effects on ruminal fermentation. Therefore, controlling rumen ciliate protozoa would be expected to lessen the dependence on protein supplementation under high-production conditions and would also be beneficial under conditions where the quantity of protein absorbed from the post-ruminal gut limits animal productivity, which occurs frequently in animals receiving low-quality tropical forages.

Bovine mastitis is recognised worldwide as the most important and costly disease affecting dairy cattle. The reduction of herd mastitis rates is crucially needed to improve animal welfare and profitability, and lessen the reliance on antibiotics. Single nucleotide polymorphisms (SNPs) within genes that have a critical role in the innate immune response, such as Toll-like receptors (TLRs) and the chemokine receptors CXCR1 and CXCR2, could impact on establishment and progression of intramammary infection, and therefore influence an animal’s susceptibility to disease. The genetic selection of animals with favourable TLR and CXCR1/2 mutations, with no impact on production traits, could be incorporated into dairy breeding programmes. In order to investigate any associations with clinical mastitis (CM) incidence and milk quality and quantity, this study identified and analysed SNPs alongside actual CM and production data from a Holstein-Friesian herd. This revealed 46 SNPs, 9 of which are novel, within boTLR1/4/5, boCD14, boCXCR1 and boCXCR2; selected SNPs were then tested for association with CM. This is the first report of boTLR1 SNPs and a non-coding boCXCR1 SNP that associate significantly with susceptibility to CM. Favourable linkage of reduced CM with increased milk fat and protein was observed, indicating selection for these markers would not be detrimental to milk quality. Furthermore, this study provides evidence that some of these SNPs underpin functional variation in bovine TLR1 and CXCR1, and possibly underlie an immunological mechanism for disease susceptibility. SNPs in boTLR1 and boCXCR1 were significantly associated with impaired transcript levels in milk somatic cells. In addition boTLR1 SNPs associated with impaired cytokine responses from cell populations when exposed to ligand or heat-killed mastitis-causing bacteria. The potential impact of boTLR1 variation on the immune response to Staphylococcus aureus is demonstrated, and this has implications for boTLR1-mediated immune responses to other pathogens.

In order to develop novel antibacterial therapies that combine anti-adhesion, anti-quorum sensing and the delivery of conventional antibiotics, the effects of polymers on bacterial aggregation and quorum sensing (QS) were studied. QS is a term used to describe method by which bacteria use chemical signal molecules to modulate pre-infection behaviour such as surface attachment. Polymers that can interfere with bacterial adhesion or the signal molecules used for QS are therefore a potential means to control bacterial population responses. In this thesis, the ability of the cationic polymers poly (N-[3-(dimethylamino) propyl] methacrylamide) (p(DMAPMAm), P1) and poly (N-dopamine methacrylamide-co-N-[3-(dimethylamino) propyl] methacrylamide) (p(DMAm-co-DMAPMAm), P2) to cluster a range of bacteria, such as Staphylococcus aureus(Gram-positive), Vibrio harveyi, Escherichia coli and Pseudomonas aeruginosa(Gram-negative) under conditions of varying pH and polymer concentration was investigated. It was identified that clustering ability was strongly dependent on the balance between charge and hydrophobicity. The results also suggested that catechol moieties might have a positive effect on adhesive properties. Moreover, the potency of polymers against QS of Vibrio harveyi was assayed via testing bioluminescence. P1 which was able to bind to the surface of bacteria through electrostatic interactions enhanced the expression of QS and P2 which could bind to both the bacteria and QS signals showed the ability to both enhance and reduce light production. Furthermore, polymeric vesicles made of copolymers containing poly (3,4-dihydroxy-L-phenylalanine methacrylamide) (p(L-DMAm)) which displayed similar dual affinity compared toP2 were prepared and their ability to modulate QS responses in Vibrio harveyi was demonstrated. All the vesicles showed higher potency in quenching bioluminescence than their linear polymer analogues. To explore the feasibility of using self-assembled polymers for anti-microbial drug delivery, silver loaded DOPG lipid vesicles were made and were found to interfere with QSwhile reducing bacterial viability when the concentration of Ag+ was above the MIC (0.1 μg/mL). The results overall suggested that combined antimicrobial therapies might be possible using polymers and both QS and cytostatic or cytotoxic agents.

Bacteria communicate with each other by releasing chemicals called quorum sensing molecules. A common gram-negative bacteria, Pseudomonas aeruginosa, releases a number of such molecules including, N-3-(oxododecanoyl)-L-homoserine lactone (3OC12-HSL) and Pseudomonas quinolone signal (2-heptyl-3,4-dihydroxyquinoline, PQS). The quorum sensing molecules have immunomodulatory effects on mammalian cells in addition to their role in bacteria. There has been very little study of the vascular effects of quorum sensing molecules on the host. The aim of this thesis was; therefore, to investigate the effect of Pseudomonas aeruginosa quorum sensing molecules, 3OC12-HSL and PQS, on vascular function and endothelial cell permeability and to study the potential mechanisms involved. Both 3OC12-HSL and PQS caused slowly developing vasorelaxation in arterial and venous preparations isolated from pigs. PQS was slightly more potent (pIC50) as a vasorelaxant than 3OC12-HSL. The endothelium was not a prerequisite for 3OC12-HSL-induced vasorelaxation since removal of endothelium and the nitric oxide synthase inhibitor (L-NAME) did not attenuate responses. Indeed, the opposite is true since 3OC12-HSL vasorelaxation responses were larger in the absence of the endothelium and nitric oxide. The mechanism of 3OC12-HSL does not involve either prostanoids, cyclic GMP, mitochondrial targets, calcium channels, chloride channels, PPAR-γ receptors, pannexin receptors, gap junctions or cystic fibrosis transmembrane conductance regulator. However, ouabain and depolarisation induced by potassium chloride decreased 3OC12-HSL responses suggest an impact on membrane potential. Overnight exposure of the porcine coronary artery to wild type Pseudomonas aeruginosa (PAO-L), produced a selective reduction in the magnitude of contractions to potassium chloride. However, neither 3OC12-HSL nor PQS produced an inflammatory response with prolonged exposure since they did not modify tumor necrosis factor alpha or vascular tone under these conditions. Both 3OC12-HSL and PQS increased endothelial cell permeability. Both caused a fall in endothelial resistance and an increase in FITC-dextran transport across human brain microvascular endothelial cells (HBMECs). The immunoblotting data showed that the levels of adherens junction and tight junction protein expression were decreased in HBMECs exposed to 3OC12-HSL, but not PQS. Rather PQS, but not 3OC12-HSL, caused a concentration-dependent phosphorylation of p38 MAPK, suggesting these molecules may modify vascular permeability by different mechanisms. In summary, these data show that 3OC12-HSL and PQS affect mammalian vascular function by decreasing vascular tone and increasing endothelial permeability. The potential advantage of these effects of quorum sensing molecules on host cells allowing bacteria to promote increase local circulation or produce oedema or allow access to promote systemic infection.

The detection and control of microorganisms such as bacteria is important in a wide range of industries and clinical settings. Detection, binding and removal of such pathogenic contaminants can be achieved through judicious consideration of the targets which are available at or in the bacterial cell. Polymers have the ability to present a number of binding ligands for cell targeting on one macromolecule and so avidity of interaction can be greatly increased. The goal of the project was to test whether polymers generated with bacteria in situ would have their composition significantly altered to determine if a templating process was occurring. It was also anticipated that the templated polymers would have better re-binding properties than those produced in the absence of bacteria. A series of chemical functionalities were analysed for their binding properties to bacteria. The functionalities were chosen with consideration to the cell surface characteristics. Further to identification of the most binding and least binding functionalities the polymers were tested for their cytotoxicity against bacteria and human epithelial cells. Concentration ranges were determined which could facilitate bacterial binding and templating yet minimise the lethality of the processes. Templated polymers of the bacteria were generated using a novel method of atom transfer radical polymerisation (ATRP) which we have termed bacterial activated atom transfer radical polymerisation (b-ATRP). This polymerisation method has maximised the potential for templating processes to occur during the polymerisation. Templated polymers differed in both their composition and their binding behaviour to non-templated polymers. The bacterial organic reduction process has also been demonstrated to have greater scope for use within the organic chemistry field as demonstrated by the use of this system to enable in "click-chemistry" via the reduction of copper.