Leprosy is still one of the most important diseases in the world today: it afflicts 15 million people and is the most important cause of deformity in man. The causative organism, Mycobacterium leprae (M. leprae) has an affinity for skin and nerve and is remarkably nontoxic; therefore, most of the clinical manifestations are due to the host response. The nerve destruction with consequent deformity, which is the most important complication of the disease, usually occurs during borderline leprosy reactions. Surprisingly there has been little research into this type of reaction, though it seems likely that they result from an increase in cell-mediated immunological reactivity. This thesis describes a prospective study of 83 patients who attended the Addis Ababa Leprosy Hospital with borderline leprosy, the purpose of which was to clarify the mechanisms involved in borderline leprosy reactions. Seventeen of the 83 patients developed reactions during the period of follow up (between one and two years) and the clinical, histological and immunological findings were compared in this group of patients with those patients who did not develop reactions. It was observed that in the reaction patients there was a significant rise in lymphocyte transformation (LT) responses to both 'whole'and 'sonicated' preparations of M. leprae, confirming that these reactions are due to an increase in cell-mediated immunity to mycobacterial antigens. However it was found that in those patients who presented with reactions involving the skin alone, there was a very marked rise in the responses to whole M. leprae (and a smaller rise in response to sonicated M. leprae) whereas those who had reactions involving nerve alone had a rise in LT responses only to sonicated M. leprae. Those with involvement of skin and nerve in the reaction had a marked rise in responses to both antigens. The reason for this remains obscure, but it seems likely that bacillary membrane antigens are associated with 'skin' reactions, and cytoplasmic antigens with 'nerve' reactions; it is possible that this results from different handling of the bacilli by macrophages in skin and Schwann cells in nerve. There were two other findings of considerable importance related to the mechanisms which might be involved in reactions. Firstly, bacilli were often found in dermal and peripheral nerves without surrounding chronic inflammatory infiltrate, though there was marked granulomatous response in skin. It seems likely that this is of importance as sudden 'immune recognition' of bacilli in nerve tissue might account for the increased cell-mediated reactivity that occurs in this type of reaction. Secondly, autologous plasma, which is normally suppressive to LT responses to phytohaemagglutinin in leprosy, developed an augmenting effect during reaction. it is possible that plasma factors normally act as a form of 'brake' mechanism preventing delayed hypersensitivity reactions but during reaction this effect is lost. Probably the most important observation of the study in the short term was the effect of dapsone on reactions. It has always been taught that dapsone should be given initially at low dosage in an attempt to prevent reactions: however, there have been no published reports to support this hypothesis. In a study of 68 patients it was found that of those receiving dapsone 5 mg daily, 11 patients developed reactions whereas only 3 of those receiving 50 mg daily did so. This would suggest that dapsone in higher dosage does not predispose to borderline leprosy reactions, and indeed, may prevent them. When those patients who did not develop reactions were studied it was found that those with borderline tuberculoid (BT) leprosy had significantly higher LT responses to whole and sonicated M. leprae than those with borderline lepromatous (BL) leprosy. However, there was considerable variation in both groups. Those patients with inflamed skin lesions had higher responses in both BT and BL patients than those with non-inflamed lesions, and indeed, those with BL leprosy and inflamed skin lesions had higher responses than those with BT leprosy with non-inflamed lesions. This would suggest that the LT test in leprosy reflects the degree of delayed hypersensitivity rather than that of protective immunity, and that as in tuberculosis, the two phenomena are not necessarily related. Thus some of the mechanisms involved in borderline leprosy reactions and the immune response in leprosy have been clarified, and it is hoped that as a result of this study reactions in some patients will be prevented. It is also hoped that the encouraging results of the study will stimulate further research into these reactions to minimise the morbidity of leprosy in the future.

The literature on the epidemiology, pathogenesis, and laboratory identification procedures for group B, beta-haemolytic streptococci (Streptococcus agalactiae) is reviewed. Sampling methods for optimum isolation of beta-haemolytic streptococci, particularly group B, were investigated and the role of transport media in maintaining the survival of these bacteria on swabs was assessed. The results indicated that, in general, none of the recognised transport media preparations offered any advantage over the use of plain cotton-wool swabs in preserving beta-haemolytic streptococci during storage for periods up to 48h. Storage temperature of swabs did however have a profound effect on survival of organisms. The prevalence and significance of group B streptococci (GBS) in the upper respiratory tract of a group of Edinburgh schoolchildren was investigated. During the period of study a number of sampling and laboratory techniques for the isolation and identification of GBS were compared. The overall carriage rate of beta-haemolytic streptococci in the throats of the children sampled and the role of this area in the isolation of GBS is discussed. A new commercially-available kit for the identification of beta-haemolytic streptococci was compared with the standard 'Lancefield' technique for grouping streptococci. In addition, a system of streptococcal grouping based on a modified enzyme-linked immunosorbent assay (ELISA) is presented. The value of this technique was assessed against the two previously mentioned systems. A degree of difficulty in obtaining the required specificity in the ELISA studies prompted further investigations into the immuno-chemical character of the cell wall of strains of GBS. Unlike many previous studies, cell walls were collected and purified by sodium dodecyl sulphate (SDS) treatment and the secondary wall polymers were separated from the peptidoglycan component by avariety of procedures. Extracted antigens were visualised by reacting with specific antisera in a crossed inmunoelectrophoresis system. Further purification of antigen complexes were achieved by chromatographic methods and chemical analysis was performed using paper and gas-liquid chromatography. The relevance of the specific antigen complexes isolated from GBS cells in relation to serological grouping and typing methods is discussed. Comment is made on the possibilities of further applications of these studies.

Campylobacter jejuni is a common cause of human gastrointestinal infections. Invasion of host epithelial cells is believed to be an important virulence Mechanism of this bacterium. C. jejuni strains vary in their ability to invade the human epithelial cells and some of the strains are hyperinvasive. The aim of this work was to find the molecular basis of this hyperinvasive phenotype. The previously studied hyperinvasive phenotype of C. jejuni 01/51 in INT-407 cells was verified using Caco-2 cell based invasion assay and the assay was set up at Nottingham Trent University by selecting blood agar for 48 hours as the pre-assay bacterial growth conditions and 100 multiplicity of infection as the starting inoculum. Seven hundred and sixty eight mutants generated in this strain by random transposon insertional mutagenesis were screened for their ability to invade human intestinal epithelial cells in an in vitro model and 174 mutants were selected for further studies. The motility of selected mutants was determined and 40 mutants that showed more than 75% motility compared to wildtype strain, with a very low level of invasion were selected for reconfirmation of this reduced invasion phenotype using standard INT-407 and Caco-2 cells based invasion assays. Localisation of the transposon insertion site by plasmid rescue was attempted in 15 mutants that showed very low levels of invasion in both eukaryotic cell lines. Preliminary DNA sequencing data from these mutants has identified, amongst others: cipA; an anion-uptake ABC-transport system permease; a glycosyltransferase; a membrane protein; a capsule polysaccharide biosynthesis protein, a putative histidine triad (HIT) family protein; a putative restriction modification enzyme; a putA; a putative cytochrome C and 2 hypothetical proteins. DNA sequence from one mutant had no database match. Targeted mutagenesis was done in 6 genes to verify the reduced invasion phenotype observed in the transposon mutants and Caco-2 cell-based adhesion and invasion assays were performed. All the six targeted mutants showed reduced invasion of Caco-2 cells compared to the wildtype strain but interestingly adhesion was variable. Complementation of 4 mutated genes partially restored the reduced invasion phenotype in the mutants. The reduced invasion of the mutants into human intestinal epithelial cells was not due to a reduction in their growth, motility, atmospheric air survival, culture media or intracellular survival of the mutants. The Cj1136 mutant showed significantly reduced ability to colonise the chick gut and the gene was also found to be crucial for lipooligosaccharides biosynthesis in C. jejuni.

No description available.

No description available.

A survey of clinical samples revealed that LBs are a universal feature of tubercle bacilli in sputum. A number of conditions including hypoxia, Nitric Oxide (NO) exposure, pH, heat and cold shock were shown to promote LB formation in M. tuberculosis in vitro. The formation of LBs in NO exposed M. tuberculosis was shown to correlate with the level of antibiotic tolerance displayed by the population. Antibiotic tolerance was thought to be a result of transitory growth arrest; however attempts to assess the growth status of LB positive M. tuberculosis cells were unsuccessful. The morphology of LBs in mycobacteria varied according to the growth condition of the cell and may be due to a change in lipid composition. The mechanism by which LBs are formed in mycobacteria remains unknown; however, there was some evidence to suggest that it follows a scheme similar to that which has been previously demonstrated in Rhodococcus opacus. It was concluded that LB formation in mycobacteria may depend on a number of environmental factors, including conditions that promote growth arrest. The formation of LBs in M. tuberculosis may anticipate antibiotic tolerance. The presence of LBs in sputum tubercle bacilli may be used to assess treatment response in patients with tuberculosis; however, it remains to be shown that LB positive M. tuberculosis cells in vitro represent the physiological LB positive sputum bacilli.

Estuary is a unique area of important economic, cultural and environ- mental value. It is also a region with very complicated hydrodynamic mechanisms, partly due to the interaction of freshwater and seawater. Much research e®ort has been invested in improving the application of estuary and preventing estuarine environment from any undue damage. With the rapid development of computational resources, mathematical model has become a popular approach used for the investigation of es- tuarine hydrodynamics. The aim of this research study is to set up the numerical models which can be used for investigating the saline-wedge purging process, astronomical tidal circulation and typhoon-induced storm surge in the estuarine regions. Two mathematical models have been developed for this purpose. One objective of this project is to set up a two-dimensional model for exploring the °ushing process of trapped saltwater subject to upstream freshwater turbulent °ow. Most numerical simulations currently ap- plied are based on single-phase models, which are not suitable for the two-phase °ow before the mixture of saltwater and freshwater. The multiphase Eulerian model, a part of commercial code FLUENT6.2, has been applied for the ¯rst time to study this complex mixing inter- action in estuary. The distinguishing characteristic of this model is to treat saltwater and freshwater as two single miscible phases instead of a mixture phase with density variation, and the advantage of using a multiphase approach over a single-phase model is that it can e±ciently and accurately treat both the free water surface and relatively high density excess between two °uids simultaneously. The other objective of this project is to develop a three-dimensional model based on the FVCOM open source code, with the aim to better understand the estuarine hydrodynamics with or without the presence of typhoon. It is found that the original FVCOM code can not re- produce an accurate tidal hydrodynamics in estuary, mainly due to the inaccurate calculation of bottom friction in shallow water. To overcome this di±culty, an improved simulation of the bed friction has been in- corporated into the existing code for estuarine tide. This model has also been developed by including air-pressure gradient term to study the hydrodynamic response to cyclonic typhoon. To include the e®ect of typhoon (wind stress and pressure de¯cit), a symmetrical cyclone model is adopted. However, the typhoon-induced wind ¯eld has been predicted poorly when the typhoon enters the near-shore region. This is because the typhoon quickly loses its symmetrical property in the near-shore region. To overcome this di±culty, an asymmetrical cyclone model is derived on the basis of characteristic isobar. The accuracy of open sea boundary for storm surge model has also been improved by using large scale model. The numerical models have been compared with laboratory experiments or ¯eld observations. The comparison results show a good agreement numerical simulations and physical measurements. It is anticipated that the models developed in this research can make signi¯cant contribution in estuarine application and protection.

This is the first study on human and animal infections with Cryptosporidillm spp. in I Gizan and Maddina regions of Saudi Arabia. Between March 2005 and September 2006, stool Isamples from 1641 people (predominantly children) were screened for Cryptosporidillm spp. , These included 454 patients with diarrhoea and 62 controls in Gizan and 1125 patients from IMaddina. In addition, 279 animal samples were examined from Gizan, including cows, goats and i sheep. j The human samples from Gizan were initially screened by miGroscopy using modified j Ziehl-Neelsen (ZN) and Safranin Methylene Blue staining techniques, while the animal samples ; were only screened using a modified ZN staining technique. The control samples were all i negative by both methods. ZN identified Cryptosporidium oocysts in 21 (4.6%) samples and i SM-B identified oocysts in 26 (5.7%) out of454 human samples from Gizan. ZN only identified i Cryptosporidium oocysts in one sheep sample. Stools from Maddina were not routinely examined j by microscopy unless positive by other means. . All 1920 faecal samples (human or animal) were evaluated using an Enzyme Immunoassay (EIA), which detected Cryptosporidium Specific Antigen in 104 (5.4 %) ofthe samples (103 human and one animal). Comparison ofthe detection rates between the diagnostic methods, Le. microscopy and EIA, using a subgroup of454 samples from Gizan, showed 100% agreement in specificity between the two methods, but EIA was more sensitive (5.7% vs 9.9%). In both Gizan and Maddina regions, cryptosporidiosis was more common in children less than two years ofage and the maximum prevalences were observed during the cooler months. All 104 samples (46 from Gizan, 58 from Maddina) containing Cryptosporidium oocysts were initially amplified by I8S rRNA-based PCR. The 101 (97.1%) samples positive for Cryptosporidium were further genotyped by an 18S rRNA-based PCR-Restriction Fragment Length Polymorphism (RFLP) technique. The 104 samples were also amplified using PCR for GP60 and HSP70 genes, for multilocus sub-genotyping. Among the 101 samples which were positive by 18S rRNA PCR, 79 (78.2 %) isolates were subsequently classified by RFLP as C. parvum, 13 (12.9 %) as C. hominis and one (1 %) was C.fe/is (from a sheep in the Gizan area). 8 human samples from the Gizan area contained mixed infections with C. parvum and C. hominis, as determined using RFLP of I8S rRNA gene. 95 (91.3%) and 88 (84.6%) ofthe 104 samples containing Cryptosporidium oocysts were successfully amplified for sub-genotype studies, using PCR for the GP60 and HSP70 genes respectively. Sequence analysis of study isolates ofthese two loci in general confirmed the species identification obtained by thel8S rRNA gene PCR- RFLP analysis. In total, allele groups IIa, IIc and IId of C. parvum and allele groups Ib and Ie of C. hominis were obtained by GP60 sequencing. HSP70 sequence analysis was less successful and did not add much to discrimination. Cryptosporidiosis was common in children, and screening of stools by EIA was twice as sensitive as light microscopy after special staining. The predominant species in humans was zoonotic C. parvum, with some C. parvum subtypes associated with anthroponotic spread also identified by multilocus sequencing. PCR amplification of both GP60 and HSP70 genes was successful, but GP60 was the most useful additional tool, showing extensive genetic heterogeneity in Cryptosporidium spp. at the subtype level. Although this may correlate with origin of the infection, studies in local animals were unsuccessful in confirming possible animal sources.

No description available.

Clostridium difficile is a Gram-positive, anaerobic bacterium that is the most frequent cause of antibiotic-associated colitis and healthcare-acquired diarrhoea worldwide. In many Gram-positive bacteria, a membrane bound sortase enzyme covalently anchors surface proteins to the cell wall, a process that is essential for virulence. Sortase protein anchoring is mediated by a conserved cell wall sorting signal on the anchored protein, containing the “LPXTG-like” motif. Sequence analysis confirmed that C. difficile strain 630 encodes a single sortase, CD2718, but little is known about its function. In this study, we identify seven predicted cell wall proteins with the (S/P)PXTG sorting motif, four of which are conserved across all five C. difficile lineages and include potential adhesins and cell wall hydrolases. A FRET-based assay was developed to confirm that recombinant CD2718 catalyses the cleavage of fluorescently labelled peptides containing (S/P)PXTG motifs in vitro. Mass spectrometry reveals the cleavage site to be between the threonine and glycine residues of the (S/P)PXTG peptide. Replacement of the predicted catalytic cysteine residue at position 209 with alanine abolishes CD2718 activity, as does addition of the cysteine protease inhibitor MTSET to the reaction. The activity of CD2718 can also be inhibited by several small-molecule inhibitors identified through an in silico screen. CD2718-mediated cleavage of a recombinant fusion protein containing the full length predicted sortase substrate CD0183 was also observed. These results demonstrate for the first time that C. difficile encodes a single sortase enzyme that recognises (S/P)PXTG sequences. The activity of CD2718 can be inhibited by rationally designed small-molecule inhibitors, and may be an appropriate target for downstream anti-infective therapies against C. difficile infection.