The morphology of the fungus Candida albicans was characterised by measurement of cell dimensions with the aid of computerised image analysis. The dimensions were used to calculate a mathematical ratio, the morphology index, which fell in the range 1 to 4.5. Spherical yeast cells gave low Mi values, whilst true hyphal cells gave Mi values greater than 3.2. This study shows that Mi can be used reliably in the place of subjective descriptions of C. albicans morphology. The mean Mi of a cell population varied according to the growth environment and the strain of C. albicans. Mi was found to reveal a continuum of morphologies in C. albicans cells both in vitro and in vivo, with no clear relationship observed between cellular morphology and the pathogenic status of C. albicans. Chemical and cellular analyses were performed on a variety of morphological forms of C. albicans, as determined by Mi. The chitin content of C. albicans cells increased linearly with Mi, although no significant correlation was found between the activity of the polysaccharide degradation enzymes, chitinase and glucanase, and morphology. Autoradiography and analysis of cell wall expansion suggested that the apex of the cell was the main region of expansion, regardless of morphology. General wall expansion was found to be repressed in cells with Mi greater than 2. The rate of overall cell wall expansion increased linearly with Mi. Investigations with "hypha-specific" monoclonal antibodies indicated a correlation between antibody reactivity and Mi, epitope expression appeared to be induced in cells with Mi greater than 3. The linear relationship observed between some properties (polysaccharide composition and overall wall expansion) and Mi suggests that quantitative regulation mechanisms may determine cell morphology.

In vitro infection is a fundamental part of HIV research. A successful detectable infection forms the basis for most experimentation on drug design or vaccine development. Deviation from this infection gives insight into whether a particular treatment regimen may be effective or not. For example, neutralization assay used in vaccine studies to evaluate induction of neutralizing antibodies requires in vitro infection of cells and measuring the ability of serum antibodies to reduce or prevent an infection. Drug screening also uses the ability to limit infection as the proof of a successful interference with virus production. Infecting peripheral blood mononuclear cells (PBMCs) is a very important tool for generating viral stocks (cell free or PBMC-associated) which can be used to infect other cells. Generating these stocks is costly and limiting the use of large volumes of cellfree viral stocks as well as increasing virus yield makes research more cost effective. Thus, studying factors that increase in vitro infection can help us limit virus stock use and provide information on how one can in future gain higher yields from co-culture. This work focused on subtype C since it is the strain that infects most people worldwide and is most abundant in South Africa. Objectives: We wanted to evaluate methods used for enhancement of in vitro infection and possibly develop an in vitro infection protocol for consistent and persistent infection. DNA PCR is not valued as a means of detecting in vitro HIV-1 infections and measuring the secretion of p24 by specialisedELISA is preferred. We therefore wanted to evaluate whether the enhancement of in vitro infection would lead to a better detection of infection in vitro by standard DNA PCR or Real-time(RT)-PCR. Since NF-êâ (a host transcription factor) was identified as playing an essential role in the production of virus the next goal was to evaluate the effect known enhancers of this transcription factor would have on detection of in vitro infection because of a potential increase in virus production. Hypothesis: Spinoculation and NF-êâ enhancement by inducting stimulus gives a higher thus more consistent infection in vitro that can be detected using standard molecular techniques. Methods: Spinoculation and polybrene addition was applied to PM1, CEM.NKR-CCR5 or PBMC cultures to boost infection. Further increase in virus production was attempted using three NF-êâ enhancers. DNA PCR, RT-PCR and p24 ELISA was applied to detect enhancement of infection using the viral strain Du-151. Results: Spinoculation (1200 x g for 3 hours) was superior to polybrene as an enhancer of in vitro infection and this was demonstrated with p24-ELISA, DNA PCR and Real-time PCR. NF-êâ enhancement through UV-C irradiation enhanced viral production in the macrophage/T-cell tropic cell line PM1 (p<0.05) and was superior to H2O2 and LiCl. LiCl had a more pronounced effect in the case of PBMCs. (p<0.05) A viral concentration of 500 TCID50 was sufficiently DNA PCR detectable following 7 day incubation provided that spinoculation and UV-C irradiation was also applied. PM1 was persistently infected in vitro and is in our opinion better suited for experimentation due to the fact that it does not show the degree of cytotoxicity of CEM.NKR-CCR5. This cell line also is known to produce infectious virus that sustain the infection. Conclusion: Detectable PCR results were obtained with 500 TCID50 and the use of enhancing factors. One of these enhancing factors is spinoculation. Spinoculation is a better technique to use to enhance cell virus contact and lacks the toxicity of polybrene. NF-êâ enhancement by UV-C has the best effect on virus production in PM1 where LiCl was found to be better suited for PBMC. DNA PCR can be used to successfully detect infection when enhancing techniques are applied. / Dr. Debra Meyer

M.Tech.

M.Tech.

Pseudomonas aeruginosa is an opportunistic pathogen capable of infecting the human cornea. Such infections are difficult to treat, and are often fulminative, in that the infected eye is lost, or severely scarred. The use of alternative therapeutic agents has been necessitated by the frequent failure of conventional antibiotic therapy. Equine hyperimmune antilipopolysaccharide plasma (Anti-LPS) was obtained by the plasmapheresis of suitably immunized horses. The plasma contained 1,O- 1 ,5g/ml of LPS-precipitible IgG antibodies. Topical administration of Anti-LPS as a lavage was shown to be effective against Pseudomonas keratitis in rabbits and guinea pigs. Subsequent use of topical corticosteroids was found to further reduce corneal pathology. The improvement noted in these experimental infections involved all three parameters measured, area of keratitis, depth of lesion, and degree of vascularization. In vitro , Anti-LPS was shown to be rapidly bactericidal for Gram negative bacteria. The plasma can therefore be said to have a dual mechanism of action: antitoxic, and antibacterial. Ocular administration of Anti-LPS, by both the topical and subconjunctival routes, was well tolerated by both rabbits and baboons. In conclusion, Anti-LPS is a potentially useful immunotherapeutic agent with many applications in both veteriary and human medicine, particularly in the treatment of surface infections involving antibiotic-resistant Gram negative bacteria / KMBT_363 / Adobe Acrobat 9.53 Paper Capture Plug-in

The distribution and abundance of terrestrial snails which inhabit the Rocky Mountain bighorn sheep (Ovis canadensis Canadensis Shaw) ranges in the East Kootenay region of British Columbia were related to edaphic and climatic factors. Emphasis was placed upon those snail species which have been implicated as intermediate hosts of the sheep lungworm, Protestrongylus stilesi Dikmans. Retinella electrina (Gould), Euconulus fulvus (Muller), and Vitrina alaskana Dall were found to be widely distributed on all ranges and were present in all but the driest plant communities. On low elevation ranges, Euconulus is the most abundant but Retinellai.is the most widely distributed. In alpine regions, however, Vitrina is the dominant species. These hydrophilic species were found mainly on organic soils under leaf litter or logs in aspen and coniferous forest communities where moisture conditions were suitable. The relatively xerophilic species, Vallonia cyclophorella (Sterki), Gastrocopta holzingeri Sterki, and Pupilla muscorum (L.) are restricted to the dry, sandy soils of the bunchgrass communities, where they live under rocks. Of these species, Vallonia is the most abundant, but on the Columbia Lake range, Pupilla, which is restricted to this range, is almost as numerous. The clay and silt soils of the bitterbrush communities appear to be unsuitable for the survival of any snails. Wide temperature and moisture fluctuations, resulting in part from soil texture, appear to be the main factors limiting the occurrence of snails in these sites. With the possible exception of Vitrina and Pupilla, the distribution and abundance of snails on the East Kootenay sheep ranges can not be explained by variations in soil calcium, even though calcium was shown to affect reproductive and growth rates. Vallonia and Pupilla appear to be the most suitable intermediate hosts for sheep lungworm. However, no infected snails were found on any of the ranges and it was established that snails live in a habitat which is inaccessible to sheep. This indicates that terrestrial snails may not play a role in the life cycle of sheep lungworm in the East Kootenay region of B.C. / Science, Faculty of / Zoology, Department of / Graduate

A semi-quantitative indirect immunofluorescence assay was developed which distinguishes two types of patients from whom yersiniae are recovered: those who produce a strong yersiniae specific coproantibody secretory IgA (SIgA) response and those who do not. This SIgA response appeared to be yersiniae specific as faecal supernatant controls from patients whose stools where shown to yield negative or positive cultures for Salmonella, Campylobacter, or Clostridia were SIgA negative. Organisms isolated from patients with high SIgA titers had a higher incidence of virulence associated characteristics although SIgA response was not associated with most other commonly recognized assays of virulence. A strong association was shown to exist between SIgA titre and mouse virulence, the gold standard of bacterial virulence. Clinical examination of patients culture positive with yersiniae documented a strong association between acute enteric illness and high SIgA titre. This association was not dependant on the cultured yersiniae species. No single in vitro virulence associated assay was found to be a reliable predictor of animal virulence. The virulence of nine Y.frederiksenii and one Y.kristensenii, previously thought to be non-pathogenic in man, was also documented. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Intact bovine Longissimus dovsi muscle was subjected to a mild washing procedure in order to reduce the concentration of the sarcoplasmic fluid. Intact and washed muscle samples were inoculated with Pseudomonas fragi to evaluate the effect of a sarcoplasmic reduction on bacterial growth and subsequent spoilage during storage at 4°C for 12 days. Aseptic controls were stored under similar conditions. Alterations in the water-soluble, salt-soluble, urea-soluble and urea-insoluble protein fractions, as well as the total carbohydrate, pH and bacterial numbers, were monitored in both intact and washed inoculated muscle samples. Scanning and transmission electron microscopy were employed to monitor ultrastructural changes on the muscle surface as a consequence of the growth of P. fragi. Analysis of water-soluble components (non-protein nitrogen, water-soluble proteins and carbohydrates) indicated that the washing procedure effectively removed the majority of these components. Increases in the extractability of the water-soluble and salt-soluble protein fractions were observed in the intact inoculated muscle sample. Alterations in the SDS-gel electrophoretic pattern of the water-soluble, salt-soluble, urea-soluble and urea-insoluble proteins were evident. Total carbohydrate decreased as a result of growth of P. fragi. An increase in pH of the intact muscle occurred as bacterial numbers increased. Significantly (P < 0.01) higher growth rates were observed on the intact muscle tissue than the washed muscle tissue. Relatively little change in the non-protein nitrogen, water-soluble and salt-soluble protein content was observed in the washed inoculated muscle tissue. A slight decrease in total carbohydrate was seen. Minor changes in the SDS-gel electrophoretograms of the salt-soluble proteins were apparent. Little change in pH of the washed inoculated sample occurred due to the growth of P. fragi. Scanning electron micrographs indicated that surface degradation of both intact and washed inoculated muscle samples were apparent only in areas of localized colonization. Glycocalyx appeared to mediate not only cell to cell attachment, but also cell to muscle surface adhesion. Bacteria were observed growing between muscle fibers. Transmission electron micrographs of intact inoculated muscle tissue confirmed the mediation of glycocalyx in bacterial adhesion. Cellular evaginations were present on the surface of the bacteria. Autolysis was minimal in both intact and washed aseptic muscle controls. / Land and Food Systems, Faculty of / Graduate

No description available.

In this thesis I will review the current knowledge of community-acquired pneumonia, including its classification, pathogenesis, pathology, aetiology, diagnosis, and antibiotic therapy and report my experience with severe community-acquired pneumonia in patients requiring admission to our respiratory intensive care unit. The original research in this thesis comprises a prospective, descriptive analysis of 196 cases of severe community-acquired pneumonia requiring admission to the Respiratory Intensive Unit at Groote Schuur Hospital from January 1987 until December 1992, with emphasis on the influence of aetiology on the severity of pneumonia, the· aetiological diagnosis of pneumonia in severely ill patients, measures of severity of pneumonia, and an audit of ICU therapy and outcome. In addition, different aspects of novel therapies and specific aetiological varieties of pneumonia which have been investigated over the past ten years will be presented.