No description available.

No description available.

No description available.

No description available.

Gram-positive pathogens are responsible for a wide range of global diseases, including nosocomial infections. The increasing incidence of antibiotic-resistant strains warrants the development of novel therapeutic strategies to combat these organisms.

No description available.

Twelve isolates of Ichthyophthirius multifiliis were successfully established and maintained by serial passage through naïve carp, for a maximum of 39 laboratory cycles. The management system employed was such that large numbers of the parasite were available for all investigations. The ability to induce exit of immature trophonts through media incubation was used to confirm events in the initial stages of host colonisation. The normal course of primary infection was also established providing useful criteria for assessing success of the in vitro systems tested. Survival of both theronts and tomonts within selected monophasic media was investigated. Theronts in Eagles Minimum Essential medium (EMEM), survived and were viable for 120 hours, 72 hours longer than water controls. No further development of the theronts was observed. Tomonts also demonstrated an increased survival time in comparison to the controls with tomites surviving within the cyst for 22 days within EMEM-S media diluted 50:50 with sterile distilled water. Division of tomonts was identified as being precystic, post divisional cystic or cystic, and the frequency of such divisions was dependent upon dilution of media. Sterile viable theronts were recovered at 168h from tomonts that had been incubated within EMEM diluted 30:70 with distilled water. Delayed encystment was achieved by incubation in concentrated media, theront production being delayed for 96h, 72h later than seen in the aquatic environment. Cultured cell monolayers were used as associates within culture systems. Behaviour of theronts on introduction into the culture systems indicated recognition of the cultured tissue as potential host material, sustained contact of up to l20hours was observed between the introduced parasite and cells. However, no developmental markers were identified within the cultured parasite and no significant growth was achieved. Attempts to simulate the situation in vivo by use of multilayered systems and crude cell explants were also unsuccessful. Transmission electron microscopy of the parasite within a cell aggregate system was undertaken at daily intervals up to 120h providing evidence that the parasite was attempting to gain nutrients by phagocytosis. However, increased vacuolation of the parasite during the period of culture was clearly evident leading eventually to parasite death. The significance of the results is discussed in relation to the normal course of infection and the future promise of a long term culture method for this important pathogen.

Little is known about the effect of different modes of expression of an antigen in rBCG on immune response. An appropriate wing of the immune system, with different degrees, is activated upon encounter with a foreign antigen. Knowledge of these responses is vital to the development of future recombinant vaccine. Various E. coli-mycobacterial species shuttle vector constructs were made using a combination of mycobacterial promoters and signal sequences. Thus enabling foreign antigens to be expressed cytoplasmically or secreted outside rBCG as native proteins or membrane-associated lipoproteins. A pivotal study using an E. coli beta-lactamase as a reporter gene is described for the evaluation of the strength of promoter and signal sequence constructs both in vitro and most importantly in vivo using the mouse macrophage cell line J-774. Expression of the diphtheria toxin fragment B as a foreign antigen was detected in vitro with all constructed plasmid vectors in rBCG using a western blot as a means of detection. It was observed that all hsp60 promoter-based constructs exhibited a high frequency with variable degree of plasmid DNA deletions Using three different rBCG substrains, the BCG Tokyo was found to be more stable (P < 0.01) and exhibited less degree of deletion (P < 0.001) compared to either BCG Moreau or BCG Pasteur. Sequence analysis of deleted plasmid DNA revealed a specific region common with nearly all plasmid deletions. Such a region of the DNA was found to correspond to the first transcriptional starting site of the hsp60 promoter. Furthermore no differences were observed in the level of expression among the three-rBCG substrains, retaining plasmid DNA, when detected by immunoblotting.

No description available.

Burkholderia pseudomallei is the aetiological agent of melioidosis and a potential bioterror threat. Infections are difficult to treat due to extensive antibiotic resistance and there is no prophylactic vaccine available. Studies have shown that the capsular polysaccharide (CPS) of B. pseudomallei is a virulence factor, immunogen and candidate antigen for a glycoconjugate vaccine. However, polysaccharides are complex to synthesise. One approach is to genetically engineer Escherichia coli to express the CPS; however, previous attempts at cloning the CPS coding locus from B. pseudomallei into E. coli were unsuccessful. This project proposes to clone only the essential genes from B. pseudomallei and to use native E. coli mechanisms to complete CPS synthesis. This would contribute to development of a new platform for the expression of any bespoke polysaccharide in E. coli. Six biosynthetic genes for the nucleotide sugar precursor were successfully expressed in E. coli. The structure of the precursor was verified by mass spectrometry. Precursor synthesis was also performed in an in vitro microfluidics system. This minimised the quantity of substrates and enzymes required, in preparation for the characterisation of glycosyltransferases required for CPS assembly. A novel assay for characterising glycosyltransferase activity was also developed, as current available options are prohibitively expensive and require significant quantities of glycosyltransferase which are difficult to purify. Finally, plasmids for the expression of additional glycosyltransferases to link the nascent B. pseudomallei CPS to truncated polysaccharides in E. coli were constructed. The aim of this project was to contribute to the development of a platform for the expression of bespoke polysaccharides in E. coli. The CPS of B. pseudomallei was chosen as the model polysaccharide as it has a simple structure and its manufacture is desirable for use in a vaccine against melioidosis.