Thesis (MSc (Microbiology))--University of Stellenbosch, 2005. / Intraspecific diversity among yeasts, including basidiomycetous yeasts has mostly been studied from a taxonomic point of view. The heterobasidiomycetous genus Cryptococcus is no exception and it was found to contain species that display heterogeneity both on a genetic and physiological level, i.e. diversity among strains originating from different geographical areas. It was stated that this diversity within yeast species is possibly caused by intrinsic attributes of the different habitats the strains of a particular species originate from. However, little is known about the diversity of a species within a specific habitat. Thus, in this study intraspecific diversity among selected cryptoccoci isolated from a single soil sample originating from pristine Fynbos vegetation , was investigated.

Thesis (MSc (Microbiology))--Stellenbosch University, 2008. / The genus Fusarium contains members that are phytopathogens of a number of agricultural commodities causing severe diseases such as wilts and rots. Fusarium species also secrete mycotoxins that have devastating effects on humans and animals. The ability of Fusarium species to change their genetic makeup in response to their immediate environment allows these fungi to exist in diverse habitats. Due to the ubiquitous nature of Fusarium, it forms part of the fungal communities in both agricultural and native soils. Fynbos is the major vegetation type of the Cape Floristic Region (CFR), which is a region that is renowned for its high plant species diversity and endemism. In this study, the occurrence and distribution of Fusarium species in indigenous fynbos soils and associated plant debris is investigated. In addition, the phylogenetic relationships between Fusarium species occurring in this particular habitat are evaluated. Fusarium isolates were recovered from soils and associated plant debris, and identified based on morphological characteristics. The morphological identification of isolates was confirmed using Polymerase Chain Reaction (PCR) based restriction fragment length polymorphism (RFLP) analyses of the translation elongation factor 1 alpha (TEF-1α) and internal transcribed spacer (ITS) regions. Furthermore, phylogenetic relationships between Fusarium species were based on the TEF-1α, ITS and β-tubulin gene regions. One-hundred-and-twenty-two (122) Fusarium strains were isolated from the fynbos soils in the Cape Peninsula area (Western Cape). Based on both morphological and molecular identification, the most prevalent Fusarium species in the fynbos soils were F. oxysporum Schlecht. emend. Snyd. and Hans., F. solani (Martius) Appel and Wollenw. emend. Snyd. and Hans., F. equiseti (Corda) Sacc. and an undescribed Fusarium species. Fusarium oxysporum was the dominant species in fynbos soils and strains of this species displayed significant genetic variability. Some strains of both F. oxysporum and F. solani showed close phylogenetic affinities to formae speciales (strains pathogenic to specific plant hosts) in the phylogenetic analyses. However, no diseased plants were observed in and within the vicinity of our sampling sites. In the third chapter, the undescribed Fusarium strains are described as Fusarium peninsulae prov. nom. Morphologically these strains are characterized by falcate macroconidia produced from brown sporodochia. The macroconidia are pedicellate, falcate to curved with hooked apical cells. Also, this fungus produces apedicellate mesoconidia on polyphialides in the aerial mycelium and forms microconidia sparsely. Chlamydospores are formed abundantly on aerial mycelium and submerged hyphae. All these morphological characteristics closely relate this fungus to F. camptoceras species complex in Fusarium section Arthrosporiella. However, phylogenetic analysis based on the ITS sequences differentiate these strains from F. camptoceras and other related species in section Arthrosporiella. Considering the fact that both as phytopathogens and saprophytic fungi, Fusarium species secrete a variety of cell wall degrading enzymes such as cellulases and xylanases. These enzymes allow the fungi to degrade the plant cell wall components to obtain nutrients. In Fusarium, notably endoxylanases play a role in phytopathogenesis of these fungi. Endoxylanase enzymes from F. oxysporum f. sp. lycopersici, F. verticillioides and F. graminearum have been characterized. In this final chapter, the use of the endoxylanase encoding gene, as a molecular marker in phylogenetic analysis was evaluated using F. graminearum (Fg) clade species as model. Degenerated primers were designed and the endoxylanase region amplified by PCR, cloned and sequenced. PAUPgenerated neighbour-joining analysis of the endoxylanase (XYL) region enabled all species to be distinguished and was as informative as the analysis generated with UTPammonia ligase (URA), phosphate permase (PHO), reductase (RED) and trichothecene 3- О-acetyltransferase (TRI101). Furthermore, the results of the phylogenetic analysis of XYL showed better species resolution in comparison to the analysis of the structural genes (TEF-1α and histone H3). Overall, the results demonstrated that phylogenetic analysis of XYL combined with other functional genes (URA, PHO, RED and TRI101) clearly distinguished between the Fg clade species far better than the analysis of structural genes (TEF-1α and histone H3).

No description available.

No description available.

No description available.

No description available.

Achieving transplant tolerance in the autoimmune environment will require targeting multiple immunologic dysregulations in T-B lymphocyte collaboration that drive the aggressive anti-graft response. At a biologic level, my findings reveal the necessity of overcoming B lymphocyte mediated restriction of CD4 Treg function, failed HSC mobilization, and enhanced T cell metabolism in achieving transplant tolerance in murine models of Type 1 Diabetes (T1D) and Systemic Lupus Erythematosus (SLE). At a cellular level, my dissertation demonstrates that specific failures in CD4 and CD8 Treg mediated suppression of the effector cell response in autoimmune T1D and SLE contributes to a generalized resistance to transplant tolerance observed in these strains. Overall, identification of and surmounting the key dysregulations in T-B cell collaboration that permit loss of tolerance in autoimmunity will advance the clinical potential of transplantation as a cure for autoimmune disease.

Seasonal influenza remains a worldwide health concern and recently, the novel avian influenza virus, H5N1, has infected and caused disease in humans, though the virus is not currently capable of human-to-human transmission. Since 2003, 850 human cases of the novel influenza virus H5N1 have been reported with a 50% mortality rate. Recently two labs have shown, very few mutations may be necessary for efficient transmission between humans. In order to examine the immune response to H5N1, a panel of antibodies from subjects in a phase I clinical trial of an experimental H5N1 vaccine were isolated and characterized. We choose a potent and specific anti-H5N1 antibody, H5.3, for further studies in order to determine the molecular mechanism of neutralization used by H5.3 and how the antibody developed. The structure of the H5.3 Fab in complex with the H5 head domain showed H5.3 interacts with the highly conserved receptor binding site and polymorphic residues on the edges of the interface, indicating breadth and potency of the antibody conflict due to variability outside the receptor-binding site. As evidenced by the structures of the H5.3 Fab in complex with H5 respiratory droplet transmissible variants, the receptor preference of the virus may not be critically important for recognition by a receptor binding site directed antibody. The H5N1 vaccine elicited a primarily naïve antibody response, as the H5-specific antibodies had a lower number of somatic mutations than the broadly neutralizing influenza antibodies. The H5.3 somatic mutations do not stabilize the protein conformation, as it remains flexibility after affinity maturation, and do not have a large effect on increasing the affinity of H5.3 for H5. Overall, this research will contribute to influenza vaccine design.

The role of bacteria in the decay of building stone from ancient monuments was examined using the framework of Koch's postulates. This involved a stepwise approach to investigate the occurrence, nature and decay potential of bacteria on stone. Prior to investigating the occurrence of bacteria on stonework it was necessary to develop a standardised procedure of high precision for the recovery and enumeration of these bacteria. A number of different methods to remove bacteria from stone were studied including physical agitation, chemical desorption and surfactant treatment. Finally a method was adopted in which stone samples were powdered, homogenised in a dilute solution of surfactant (Tween 80) and counted on an automatic plating system. A range of growth media were used to examine three different bacterial types, namely, sulphur-oxidising, nitrifying and heterotrophic. To investigate the occurrence and distri but10n of bacteria on both sound and decayed stone extensive bacteriological surveys were conducted on stonework at two monuments, Portchester Castle and Tintern Abbey. All types of bacteria were widely distributed on both sandstone and limestone at the monuments. At each monument, significantly more sulphur-oxidising and heterotrophic bacteria were associated with severely decayed stone than undecayed stone. Electron microscopy confirmed that large populations of bacteria could be found predominantly 5-10mm below the surface of decayed stone. Approximately 200 bacteria were isolated into pure culture during the field surveys of the two monuments. All isolates were screened for decay potential using a liquid culture system involving static growth of bacteria in the presence of 1cm stone discs. From the 200 isolates, about 30 were capable of causing substantial weight loss in sandstone discs under heterotrophic conditions. Five isolates were able to cause a large weight loss using only mineral nutrients. Some isolates caused a significant weight gain in the stone discs under these conditions. Statistical analysis of the data from this decay screen indicated that weight loss of stone could be directly correlated to a decrease in pH of the medium and a release of calcium and silicate from the stone. Futher decay studies carried out on selected isolates suggested that under heterotrophic conditions the bacteria secreted quantities of organic acids in to the medium which could attack the stone. However, in the presence of an inorganic nutrient source, the generation of mineral acids may be involved. Under both conditions different stones had varying resistance to bacterial decay and this appeared to be dependent upon the level of calcite in the stone. Specific antibody techniques such as BLISA and FAT were examined and proved very useful in demonstrating the presence of certain principal decay species on samples of decayed stone.

The objective of this project was to evaluate various culturing and isolation techniques of Campylobacter jejuni and to develop methods to detect the organism in foods. The morphological, cultural and biochemical characteristics of C. jejuni were studied using developed microbiological methods. A variety of media, broths, microaerophilic atmospheres and diluents, now available, were tested for their applicability to detect low numbers of the organism in food samples. Direct plating, filtration, double incubation enrichment, milk separation enrichment and swabbing methods were used to recover C. jejuni from seeded milk and fowl samples. As few as 16 organisms per ml of milk could be recovered using the double incubation enrichment. Raw milk samples from retail supermarkets and the Oregon State University Dairy Herd were tested for the presence of C. jejuni with the double incubation enrichment. No positive confirmation of the organism was made, although suspect microorganisms were observed microscopically. The survival of C. jejuni in foods and effect of sanitizers was studied. Raw and underprocessed foods pose the greatest risks as vehicles of Campylobacter infections. If contaminated foods are held at refrigeration temperatures C. jejuni could survive. Properly sanitized dairy equipment poses no apparent health problem and water should have a residual chlorine level of greater than 5 ppm to be safe. / Graduation date: 1982