Food-borne salmonellosis is a major cause of gastrointestinal disease in humans and domestic animals worldwide. In this study, the population structure and genetic relationship of various Salmonella serovars were investigated using Multilocus Sequence Typing (MLST). The MLST scheme, using seven housekeeping genes, was used to characterize and differentiate 153 Salmonella isolates of 34 serovars into 51 sequence types (STs) with fourteen novel STs identified among 13 serovars in the collection. The scheme provided good sub-type discrimination among various Salmonella serovars. Comparison of 51 allelic profiles against the database using START and eBURST software indicated a strong association of STs with serovars. The phylogenetic structure of each serovar was represented in one lineage of a cluster, with the exceptions that isolates of serovars Newport and Java were clustered into three and two distinct lineages, respectively. In addition, 17 STs of seven serovars were differentiated into nine clonal complexes. These results allowed the development of a molecular serotyping scheme based on detection of serovar specific single nucleotide polymorphism~SNPs) seen in the MLST data. The SNaPshot system is a multiplex primer extension assay (MPEA) that enables multiplex SNP analysis. Here, the method has been developed for the identification of five Salmonella serotypes commonly detected in the UK, based on the above serotype specific SNPs. The SNPs, in genes hemD, thrA, purE and sucA, acted as surrogate markers for serovars Typhimurium, Enteritidis, Virchow, Infantis and Bracnderup. The MPEA was performed using two separate panels of MPEA reactions and evaluated using 152 S. enterica isolates that had been characterized by MLST. The MPEA was shown to be 100% specific and sensitive for this collection of isolates. Furthermore, the MPEA was applied to identify the serovar or ST of DNA recovered from clinical specimens (faecal samples from human Salmonella infection) (n=15) and food samples (n= 10). The isolation of Salmonella from these specimens/samples and their subsequent serotyping indicated that use of the MPEA had allowed accurate identification of the serovar of 96% of isolates present in the samples. Interestingly, the assay also allowed identification of the serovar of two Salmonella isolates is from the above samples that were not able to be fully typed using serological methods. The method could be applied in less than six hours and has potential for improved patient care, public health investigation of Salmonella outbreaks and source tracing. Recently, the DiversiLab rep-Pf'R system has been developed using microfluidic chips to provide standardized semi-automated fingerprinting for pathogens including S. enterica. In the current study, 71 isolates of S. enterica, representing 21 different serovars, were analysed using MLST and the DiversiLab rep-Pf'R system. MLST was able to identify 31 STs, while the DiversiLab system revealed 38 Diversil.ab types (OTs). The DiversiLab rep-P(R approach distinguished isolates of different serovars and showed a greater discriminatory power (0.95) than MLST (0.89). The OiversiLab system exhibited 92% concordance with MLST and 90% with serotyping, while the concordance level of MLST with serotyping was 96%, representing a strong association. MLST and the DiversiLab reppeR system may provide useful additional informative techniques for the molecular identification of S. enterica isolates. In addition, the OiversiLab rep-PCR system may provide a rapid (less than 4 hours) and standardized method for the identification of S. enterica isolates.

High-intensity exercise has been associated with an increased susceptibility to illness and infections, yet the underlying metabolic mechanism underpinning the onset of a suppressed immune system has yet to be determined. In view of the importance of preventing overtraining and the associated impact on health and sporting performance, the aim of the current thesis was to determine the role of 5'AMP-activated Protein Kinase (AMPK) in the maintenance of cellular energy within immune cells, and thus its influence on the functional abilities of immune cells. Two experimental approaches were employed: in vitro studies involving cultured monocytic monomac6 cells to confirm the importance of AMPK in maintaining correct cellular energy status, and in vivo studies to determine the effects of acute bouts of highintensity exercise of ~70% VO2max in various exercise modes (cycling, running and rowing), on AMPK activation within human mononuclear cells, and several markers of global immune function. Also, an 8-week training programme was employed to investigate the chronic effects of high-intensity exercise on mononuclear cell AMPK activation, and markers of global immune function. In vitro data confirmed AMPKα1 isoform expression in monocytes, which is activated by hypoxia-induced decreases in cellular ATP levels (mimicked by in vitro oligomycin treatment) to phosphorylate inducible phosphofructokinase-2, and activate anaerobic glycolysis to replenish cellular energy stores. In contrast, bouts of high intensity exercise (~70% VO2max for 45 minutes) brought about transient dephosphorylation of AMPK for ~1 hour post-exercise, which correlated to transient decreases in immune function (detected as decreased salivary IgA and suppressed cytokine release). Dephosphorylation of AMPK occurred via an AMP:ATP-independent mechanism, and coincided with a decrease in intracellular levels of reactive oxygen species. Importantly, AMPK inactivation lessened in extent in mononuclear cells following an 8-week training programme. In conclusion, these studies detail the potential involvement of AMPK inactivation, and the consequent disruptions in cellular energy homeostasis in mononuclear cells following an acute bout of exercise. Longitudinal data suggests improvements by chronic training adaptations, but in general, propose a role that AMPK inactivation may repress immune cell function.

Parasitic diseases remain a major public health problem affecting hundreds of millions of people, particularly in tropical developing countries. Pharmaceutical research in natural products represents a major strategy for discovering and developing new drugs. The use of medicinal plants for the treatment of parasitic diseases is well known and documented since ancient times. Saponins are a structurally diverse class of compounds occurring in many plant species, and possessing a number of desirable biological and pharmacological activities. In this work methodology was developed to extract, isolate and purify desirable triterpenoid saponins based on the unusual aglycone backbone hederagenin from the fruits and leaves of common ivy (Hedera helix) and by chemical modification of the glycosides and aglycone, further derivatives were prepared. These novel compounds which were used to conduct trials of their biological activities to identify the best means of optimising these activities. The goal was to identify effective antileishmanial, antiacanthamoebal and antiplant parasitic agents from plant sources using a systematic approach. The compounds were evaluated against Leishmania major, Leishmania mexicana and Leishmania donovani. This study concluded that modified triterpenoid saponins from Hedera helix show promising in vitro and in vivo activity against promastigote and amastigote stages and can be considered as new lead structures in the search for novel antileishmanial drugs. Some compounds showed a remarkable amoebicidal effect on Acanthamoeba castellanii. These compounds had been tested against plants parasitic and were found to be moderately effective in reducing egg hatching and mortality of the second juveniles. Four derivatives of eugenol were studied for their antileishmanial activity against L. major and L. mexicana. These results showed moderate activity. The project also determined the effect ofbioactive plant extract against in controlling microbial growth for three kinds of bacteria, Pseudomonas aeruginosa, Eschericha coli, and Staphylococcus aureus. One type of fungus, Candida albicans was also examined. The results had activity against bacterial and fungal infections and could be used to control these organisms.

This thesis aims to explore whether cells of the CNS express TLRs and whether they are capable of responding to different stimuli. To explore gene expression a novel custom microarray was designed, developed and validated to assay the expression of selected gene transcripts involved in innate immune responses. Gene transcript levels were first studied in glial cells at rest. Cells were stimulated with bacterial lipopolysaccharide, or by infection with the neuro-invasive Semliki Forest virus (SFV). Both microglia and astrocytes in culture expressed a multitude of TLRs that were differentially modulated in a specific manner depending on the nature of the stimulus. The expression of TLR suggests glial cells are capable of recognising a vast array of microbial-associated molecules. Such a strategy may be an essential requirement for an organ mostly devoid of recognisable immune processes. <i>In vivo</i>, the resting CNS exhibited extensive TLR expression with TLR 3 expressed at exceptionally high levels, comparable to that of lymphoid tissue, but varying with mouse strain. The data reported here show for the first time that TLRs in the brain are up-regulated during viral encephalitis. Furthermore, this response was appropriate to the pathogen, with selective up-regulation of TLRs that sense infection. Intracerebral inoculation with either SFV or rabies virus initiated substantial upregulation of TLR 2, 3 and 9. Type-I IFN independent mechanisms mediated the up-regulation of TLR 2 following SFV infection, whilst for the two TLRs that mediate recognition of viral nucleic acids, TLR3 and TLR 9, up-regulation of gene expression was dependent upon and proportional to the type-I IFN response. It is likely that by up-regulating TLR 3 and 9, type-I IFN acts to increase the sensitivity of cells in the vicinity of virally infected cells. Transmissible spongiform encephalopathies are a group of diseases characterised by chronic neurodegeneration and glial cell activation. This thesis demonstrates that the CNS significantly up-regulated several TLRs, and in the case of TLR 2, by 10-fold towards terminal disease. This response further describes the apparent non-productive innate immune activation of these cells during these diseases. In summary, the finding that the brain has the ability via TLR expression to detect infection and discern its type provides an important contribution to understanding pathological processes in this organ.

Clostridium botulinum and some strains of C. baratii and C. butyricum produce one of the most potent toxins known to man, botulinum neurotoxin, and are responsible for the disease botulism. This severe neuroparalytic disease IS the result of botulinum neurotoxin negotiating a complex path to the cholinergic nerve endings. There, it interferes with the release of excitatory neurotransmitters resulting in flaccid paralysis and if untreated, death. The neurotoxin, itself a multi-faceted protein, does not act alone but is produced as part of a large, hetero-multimeric complex with the associated non-toxic proteins. This complex, known to protect the toxin from acids and proteases in the gut, has recently been suggested to play a more active role in toxicity. Here, the proteins from the lesser-studied toxin' complex type (the OrfX type) are shown for the first time to share sequence similarity and syriteny with clusters of proteins that are co-localised with various putative toxin genes in diverse other species. The extracellular supernatant proteome of C. botulinum is characterised and mined for potential novel virulence factors, with metabolic cost of extracellular protein being highlighted as a potential marker of virulence associated extracellular proteins. The supernatant of 22 clinical strains of C. botulinum were investigated for the presence of the toxin complex; all toxin complex components were jdentified in the majority of strains indicating the importance of these proteins in the causation of botulism. A relationship between ' OrfX-encoding strains and infant botulism was also uncovered in clinical strains from the UK. Hypotheses to explain this association are explored. Finally, the transcriptome of C. botulinum was investigated using RNA-sequencing. This uncovered a complex and diverse picture of transcription in C. botulinum and raised questions as to the role of the alternative sigma factor BotR in the regulation of bont.

A 75 kb gene cluster in Pseudomonasfl uorescens NCIMB 10586 encodesa modular polyketide synthase responsible for biosynthesis of the antibiotic mupirocin. All seven PKS modules lack intrinsic acyl transferase (AT) domains. Two AT domains within the multifunctional polypeptide MmpC are thought to operate in trans. An unusually large tailoring region encodes 26 discrete proteins that modify the polyketide/fatty acid backbone, in addition to providing resistance and regulatory functions. This study investigated the functions of four tailoring region genes. MupJ, MupK and mAcpC were revealed to be involved in insertion of a β-methyl group, while MupN was shown to have phosphopantetheinyl transferase activity. Five tailoring acyl carrier proteins (mAcps) were found to be specific and non-interchangeable, and the influence of C-termini on mAcp-specificity was investigated. Attempts to overexpress mAcps in P. fluorescens revealed that a broad-host-range IncP-1 vector is unstablein this host,p romptingt he constructiono f a new IncP-9v ector for use in P. fluorescens. The importance of the two ATs and the third domain of MmpC were demonstrated by mutagenesis and complementation. Overexpression of AT domains did not increase mupirocin production but specific heterologous trans-AT domains could malonylate the mupirocin PKS.

No description available.

The zoonotic bacterium Campylobacterjejlllli is the major cause of gastrointestinal disease in developed countries. The source ofhuman infections is believed to be animal food products, but the relative contributions ofdifferent sources to the overall burden of disease remain unclear. By improving our knowledge ofthe variable genome of Campylobacterjejlllli it may be possible to uncover links between variable gene distribution and strain phenotypes such as host preference or virulence. Our current knowledge ofvariable genes may be unrepresentative of strains from potential disease reservoirs such as ruminants and wildlife species. The technique of suppression subtraction hybridization can be used to identify gene fragments present in the genome of a strain of interest (tester strain) but absent from the genome ofa reference strain (driver strain). We carried out subtractions using strains from novel hosts, and various MLST clonal complexes. The distribution ofeight subtracted sequences was analysed using PCR and dot blot hybridization. These sequences included four with putative roles in the use of alternative electron acceptors, which have been hypothesized to contribute to selective advantages in specific ecological niches. For these eight subtracted sequences there was no evidence for a link between sequence distribution and host species. However, there was a clear correlation between subtracted sequence distribution and clonal complex, suggesting minimal transfer ofthese genes between clonal complexes. To further characterise genomic variation in C. jejzl11i populations, comparative genomic hybridization was carried out on a panel of C. jejlllli isolates representing diverse host species and MLSTs. DNA from these isolates was interrogated using a newly developed oligonucleotide microarray based on the genomes of C. jejllni strains NCTC11168, RM1221 and 81-176. The microarray also included strain variable genes from other strains and genetic loci in the database, and a selection of genes from our subtractions. The strain panel included several isolates from wildlife and water sources with unusual MLST genotypes not currently found in clinical isolates from humans (WW isolates). . Clustering analysis ofM-CGH data confirmed that genomic content was assocfated with the MLST rather than the host species ofthe isolates. Strikingly the WW isolates had a number of genes deleted or significantly divergent from the arrayed genes, which were present in all or most other isolates. Many ofthese genes had functions which related to colonisation, invasion and virulence. This suggests that the WW strains may reach humans but may be unable to colonise or cause disease in humans. The genome sequence of a WW isolate revealed several novel genomic insertions not present in the genome of C. jejzl11i NCTC11168. These novel insertions may contain genes which contribute to the colonisation of water and wildlife sources, or the unusual epidemiology of the WW strains. Further studies will include the genome sequencing of several more WW isolates and characterisation oftheir colonisation phenotypes using a chick colonisation model, expression M-CGH, and defined gene mutants.

One of the most important areas of research regarding adaptation of malaria vectors that has received very little attention is the effect of selection on the population structure of An. arabiellsis and the ecological significance of melanisation on the ability ofAn. gambiae to surVive in different habitats. Most microsatellite-based studies of population structure have selected loci that are located within inversions many of which may be subject to selection. The structure of the population will therefore depend on the nature of the selection on the selected loci and the effect of other nearby loci. Locus 33Cl within chromosome 3Ra inversion of An. gambiae showed abberation in the genetic differentiation of An. arabiellsis population sampl~d from the north-south transect of East Africa (Donnelly and Townson 2000).The aim of the first part of this thesis which constituted Chapter 3 is to determine if there is any signature of positive selection on some candidate loci located within the 3Ra inversion and investigate its effect on nucleotide variation ofAn. arabiellsis populations. In line with this objective, we obtained 20 sequences each of 4 Epsilon class Glutathione S-transjerases (GSTs) i.e. (GSTEl, 441 bp (n=20), GSTE2, 800bp (n=20), GSTE6, 735bp (n=20) and GSTE8, 796bp (n=20) from samples of An. arabiellsis captured from Sudan, Ethiopia, Malawi and Tanzania. Glutathione s-transferases (GSTs) are detoxication enzymes that are involved in the metabolism, detoxication and excretion of a large number of endogenous and exogenous compounds including DDT from the cells of organisms. Using evolutionary models, we showed that 3 out of the 4 loci deviated from neutral expectations. This is indicative of selective constraints on these loci. Pairwise estimates of population differentiation, Fsr values were far in excess of those observed from microsatellite-based studies of the same samples with specimen from Ethiopia much more genetically differentiated from the rest.

This thesis investigated the interaction of humoral mechanisms of immunity in the sheep with antigens of <i>Pasteurella haemolytica</i>. An assay was developed to detect toxic activity in crude preparations of <i>P. haemolytica</i> cytotoxin (CC). A cytotoxin neutralisation (CN) assay showed that a serotype A2 CC vaccine could raise CN antibodies in a rabbit and that immune sera and lung washings from lambs 'convalescent' from serotype A2 infection had CN titres. Thus CN titres correlated with protection in lambs, indicating A2 CC had potential as a vaccine component. Analysis of the CC showed it to contain outer membrane proteins, lipopolysaccharide (LPS), serotype specific capsular antigens and proteases as well as cytotoxin antigens. Microtitre plate assays were developed to detect antibodies involved in bactericidal activity and opsonophagocytosis. Convalescent lamb sera and lung washings, in combination with complement, were bactericidal for <i>P. haemolytica</i> A2 organisms, and adsorption studies indicated LPS to be a target for antibodies involved in bactericidal activity. In contrast, <i>P. haemolytica</i> serotype T10 organisms were resistant to bactericidal activity, this resistance being associated with the 'smooth' type LPS found in all T serotype organisms. All A serotype organisms had 'rough' type LPS, except for serotypes A5 and all which were intermediate in LPS profile. Both complement and antibodies in convalescent serum opsonised serotype A2 organisms causing their increased uptake of phagocytes. Adsorption studies were not conclusive, but indicated that LPS was not a target for opsonic antibodies, whereas opsonins were removed by adsorption with a sodium salicylate extract (SSE) of serotype A2 cells which contained capsular polysaccharide antigens and outer membrane proteins as well as LPS. Serotype A2 organisms grown <i>in vivo</i> were susceptible to bactericidal and opsonophagocytic mechanisms of immunity. Also, passive protection studies in lambs indicated that sera, which variously stimulated CN, bactericidal and opsonophagocytic mechanisms, were highly protective. These combined findings suggested that humoral mechanisms of immunity were active against organisms grown both <i>in vivo</i> and <i>in vitro</i>, and that humoral immunity alone was highly protective against serotype A2 infection. As a vaccine, the serotype A2 CC gave 86% protection against experimental homologous challenge. Protection correlated (P < 0.001) with serum CN titres and bactericidal capacity, but opsonophagocytosis was not stimulated. CN and bactericidal activity appeared to be stimulated by cytotoxin and LPS antigens in the CC. This study, therefore, identified <i>in vitro</i> correlates of immunity to <i>P. haemolytica</i> infection in lambs, and demonstrated the ability of a cytotoxin based vaccine to stimulate some of these mechanisms of immunity and engender a high degree of protection against infection.