Leishmaniasis is a parasitic protozoal disease affecting humans and animals with phlebotomine sand flies as intermediate vectors. The parasite infects phagocytic cells of the mammalian host where they transform from the flagellated promastigote to non-flagellated amastigote phase. There is no effective vaccine in use against this parasite and production relies on finding potent immunogenic antigens with a Th1 bias and long lasting memory response. In this study the immunogenicity of L. mexicana Soluble Leishmania Antigens (SLA) prepared by two different methods (SLA1&2) was investigated by immunisation of Balb/c mice and challenge with live L. mexicana and an in vitro immunological analysis. Immunisation of Balb/c mice with SLA mixed with IFA adjuvant significantly protected against challenge with live L. mexicana parasites. The SLA2 was also further fractionated into six sub fractions by fast protein liquid chromatography (FPLC) using Mono Q columns and the immunogenicity of each fraction was analysed either by ability to stimulate CTL activity against dendritic cells (DCs) target cells loaded with SLA2 and SLA2 fractions or by tritiated thymidine uptake proliferation assay. Immunisation of Balb/c mice with whole SLA as well as the SLA2 fractions induced a significant CTL activity, but responses were higher for the whole SLA. Splenocytes stimulated in vitro for 7 and 14 days with SLA2 and SLA2 fractions induced significant proliferation responses which was increased when splenocytes were stimulated with DCs loaded with these antigens. Leishmania parasites require a number of immune-evasion mechanisms to resist phagolysosome fusion and prevent activation of more-potent acquired immune responses. Down regulation of MHC class I and II expression on infected phagocytic cells may be one of the immune evasion strategies used by the Leishmania parasite. In this study the effect of L. mexicana infection on the expression of surface molecules was investigated in DCs. Unlike treatment with autoclaved parasite, infection of DCs with live L. mexicana parasite down regulated the expression of MHC class I, class II, CD11c, CD80 and CD40. Also, in vitro treatment of DCs with fungizone as early as 1 hour after the initiation of infection with L. mexicana restored their MHC class I expression, as determined by antibody staining and flow cytometry analysis. Interestingly treatment of L. mexicana infected DCs with fungizone also restored their susceptibility to CTL activity. As part of searching for new Leishmania antigens of a potential vaccine application, the immunogenicity of L. donovani centrin-3 (Ldcen-3) was investigated in a Balb/c model. The immunogenicity of Ldcen-3 has not previously been investigated. Ldcen- 3 is a calcium binding protein that has been shown to be involved in duplication and segregation of the centrosome in higher and lower eukaryotes. The Ldcen-3 gene was cloned in various vectors and coated on gold particles for gene gun immunisation. Significant protection was induced by immunisation with 1μg DNA of pcDNA3.1- Ldcen-3 or pCRT7/CT-TOPO-Ldcen-3 constructs. Protection against challenge with live parasite was vector dependent where better protection was induced by pCR T7/CT-TOPO-Ldcen-3. Splenocytes from Balb/c mice immunised with pcDNA3.1- Ldcen-3 or pCRT7/CT-TOPO-Ldcen-3 has a potent CTL response against DC targets loaded with SLA or tumour cells transfected with Ldcen-3 plasmid construct. Collectively, results presented in this study suggest that the whole SLA was more immunogenic than any SLA fractions produced by fast protein liquid chromatography. Results also suggest that L. mexicana could use down regulation of MHC I as a possible mechanism to evade killing by CTL and susceptibility to CTL could be restored by treatment of infected cells with fungizone. These findings also suggest the potential benefit of combination therapy in controlling Leishmania infection. This study has also investigated for the first time the immunogenicity of Ldcen-3 gene which was shown to be highly immunogenic via protection against challenge with live parasite and induction of CTL in immunised mice.

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Many human populations spend approximately 90 % of their time indoors, yet relatively little is known about the microbial communities associated with indoor environments. This is despite knowledge that these microorganisms can contribute to adverse health effects, including the acquisition of healthcare-associated infections, which cause significant morbidity and mortality. The concept of the ‘indoor microbiome’ is relatively new and to date, few studies have been field-based, systematic and long-term. Hospitals in particular, are unique environments which have been shown to drive microbial evolutionary processes as they contain a different sub-set of the human population. The study of the hospital microbiome could have important implications for healthcare and infection control. This thesis explores a range of methods for investigating microorganisms in different indoor environments, including a classroom and outpatient’s waiting areas and wards in a hospital. Results show that the classroom is much more heavily contaminated in terms of total viable counts (TVCs) of bacteria recovered than the hospital environment. This was thought to be attributed to the absence of a strict cleaning regime in the classroom. High-touch items were less contaminated than other objects, likely due to them being obvious cleaning targets. Potential pathogens, including a number of Enterobacteriaceae were cultured from the classroom, outpatient’s waiting area and ward. Virus nucleic acid was recovered from an outpatient’s area, including norovirus and rotavirus RNA. Adenovirus DNA was frequently isolated throughout a 3 month screening protocol and there appeared to be evidence to suggest that a viral marker may be more appropriate than TVCs for identifying viral contamination. Human-associated bacteria were found to be dominant on a hospital ward over a 12 month longitudinal screening study and the presence of numerous bacterial taxa, which may be of concern in the context of paediatrics and immunodeficient patients, was also demonstrated.

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Ovine enzootic abortion (OEA) is an economically important disease of ewes caused by a type of <i>Chlamydia psittaci</i>. Vaccines have been available to control this disease for more than 30 years. In the past decade, vaccine efficacy has been poor despite efforts to improve the inactivated whole organism vaccine currently in use. The initial aim of this project was to characterise the antigenic structure of OEA <i>C.psittaci</i> and to identify potentially immunoprotective antigens. Retrospective analyses of sera taken from ewes of different immune status were carried out using immunoblotting. A single 39 to 40 kDa antigen, thought to be the major outer membrane protein (MOMP) of OEA <i>C.psittaci</i>, was implicated as an important immunogen. A series of biochemical analyses including gel electrophoresis, immunoblotting, peptide mapping, surface radio-iodination, detergent fractionation, <i>in vitro</i> oligomerisation and protein microsequencing, as well as electron microscopy, showed conclusively that this antigen was indeed the MOMP of OEA <i>C.psittaci</i>, the analogue of well-characterised MOMPs of other chlamydial strains. OEA MOMP was found to be a major surface-exposed component of the outer membrane fraction of the chlamydial elementary body (EB), and appeared as fine ultrastructural particles (3 to 4 nm in diameter) densely packed on the outermost surface of chlamydial EBs. It possessed epitopes cross-reactive with other chlamydial MOMPs and is a site of heterogeneity between ovine <i>C.psittaci</i> types. Its solubility was enhanced in the presence of reducing agent and MOMP monomers formed disulphide cross-linked oligomers under non-reducing conditions <i>in vitro</i>. It was found to possess an N-terminus identical to <i>C.trachomatis</i> MOMP indicating that cleavage of the signal sequence occurs at the same site to produce a mature protein of 39.5kDa. A method was adapted to isolate MOMP by detergent extraction. An outer membrane fraction, highly enriched in MOMP, was prepared in sufficient quantities for a vaccination-challenge experiment. The results showed that both purified whole elementary bodies and the outer membrane preparation, given in a single dose, could protect ewes from infection and abortion. To test the hypothesis that MOMP was a protective component in these vaccine preparations, sufficient quantities of purified MOMP were needed. A recombinant DNA approach was taken. Firstly, the MOMP gene from a vaccine strain, S26/3, was completely sequenced and extensively analysed in order to develop good strategies of expressing recombinant MOMP (rMOMP). This monocistronic gene contained putative tandem promoter and rho-independent terminator sequences flanking a 1,167 base-pair open reading frame. Comparison with other MOMP gene sequences revealed four highly variable domains interdigitating five constant domains. Using the polymerase chain reaction and specially designed primers, a specific sequence corresponding to the mature MOMP was amplified, cloned into M13 mptac18 viral expression vector and subcloned into three plasmid vectors, pUC8, pRIT5 and pRIT2T, for expression. Several rMOMPs representing the complete mature MOMP and a truncated MOMP were successfully expressed in <i>Escherichia coli</i>. Finally, further analyses showed that these rMOMPs retained antigenic properties of MOMP and that large quantities could be obtained in a highly purified form. Such rMOMPs can now be tested for the presence of epitopes which can protect pregnant ewes from ovine enzootic abortion.

Chronic respiratory colonisation by the adaptable opportunistic pathogen <i>Pseudomonas aeruginosa</i> is a major debilitating feature of the inherited disease cystic fibrosis (CF). This thesis considers various aspects of the pathogenesis of <i>P.aeruginosa</i> in CF, including the serological response to bacterial colonisation, and possible factors involved in early colonisation. Anti-<i>P.aeruginosa</i> lipopolysaccharide (LPS) antibodies in sera, saliva and sputa from patients with CF were measured by enzyme-linked immunosorbent assay (ELISA) incorporating either a polyvalent pseudomonas smooth LPS extract vaccine, or <i>P.aeruginosa</i> core, rough LPS. Elevated levels of anti-LPS IgG antibodies in serum, and IgA antibodies in saliva and sputum were demonstrated in patients chronically colonised by <i>P.aeruginosa</i>. Low levels of serum anti-LPS IgG antibodies were detected in some patients intermittently colonised by <i>P.aeruginosa</i>, but not in non-<i>P.aeruginosa</i> colonised patients. Anti-LPS IgA antibodies were detected in some of both intermittently and non-colonised patients. Immunoblot analysis of serum IgG and sputum IgA antibodies to <i>P.aeruginosa</i> LPS revealed a response directed towards O-antigenic LPS in the initial stages of pulmonary colonisation with non-mucoid <i>P.aeruginosa</i> and a response towards common core LPS during subsequent chronic infection with mucoid <i>P.aeruginosa</i>. Flagellar preparations from <i>P.aeruginosa</i> strains were characterised and used in ELISA and immunoblot studies to detect anti-<i>P.aeruginosa</i> flagellar antibodies in sera, saliva and sputum. Serum anti-flagellar IgG antibodies were detected, particularly in those CF patients intermittently or chronically colonised by <i>P.aeruginosa</i>. Antibodies to both type -a and -b flagella were detected; in some patients a pronounced antibody response to only one of the flagellar types was evident. Anti-<i>P.aeruginosa</i> LPS monoclonal antibodies (MAbs) were produced for use in a sandwich ELISA for the detection of <i>P.aeruginosa</i> in respiratory secretions of patients with CF. LPS defective mutants expressing only common core LPS were used to immunise mice for preparation of MAbs. Antibodies were screened in ELISA and the antigenic component(s) of LPS recognised by the most cross-reactive MAbs was checked by immunoblotting. Five IgG MAbs were characterised and found to recognise the core component of <i>P.aeruginosa</i> LPS. Two of the MAbs were particularly reactive against core LPS from all O-antigenic serotypes of <i>P.aeruginosa</i> and were included in the sandwich ELISA for detection of <i>P.aeruginosa</i> LPS. A biotin-streptavidin amplification system was used to increase assay sensitivity. The sensitivity of the assay was 0.1 ng/ml <i>P.aeruginosa</i> LPS; the assay was able to detect <i>P.aeruginosa</i> LPS in the respiratory secretions from patients with CF.

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