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Cowdria ruminantium (Cowdria) is an obligate intracellular pathogen that causes heartwater in ruminants. Cellular immunity and the type I cytokine IFN-γ have been implicated in protective immunity to heartwater. The aim of this thesis was to identify proteins of the Welgevonden isolate of Cowdria that induce lymphocyte proliferation and IFN-γ production. Differential centrifugation was found to be the simplest and most efficient method of Cowdria purification. Cowdria organisms were fractionated into their constituent proteins of between 11 and 168 kDa by continuous flow electrophoresis. The resulting fractions were tested for their ability to stimulate lymphocyte proliferation in vitro. In an attempt to simulate the natural infective process, peripheral blood mononuclear cells (PBMC) were obtained from two cattle rendered immune by infection and treatment and assayed in proliferation assays with the proteins fractions. In a parallel study, four cattle were immunised with inactivated Cowdria to determine whether their lymphocytes responded similarly. Cowdria-specific proliferation was detected for only a brief period after immunisation by infection with live organisms. This response was only detected again two to three years later. In contrast, PBMC from animals immunised with inactivated organisms were continuously responsive for at least three years. Only Cowdria proteins with molecular masses of 11, 12, 14 to 17 and 19 to 23 kDa induced proliferative responses in PBMC obtained from all six animals. Cell surface phenotypic analysis of Cowdria specific T-cell lines indicated that CD4⁺ lymphocytes were enriched over time with a concomitant increase in antigen-specific proliferation and IFN-γ production. Proteins of molecular masses 13 to 18 kDa induced CD4⁺ lymphocyte proliferation and IFN-γ production by T-cell lines from all the animals tested. Antibodies raised in a chicken and in rabbits to these low molecular weight proteins had low titres and specificity. Two-dimensional electrophoresis indicated that proteins within a single molecular weight range comprised several components with different pIs, revealing the complexity of the Cowdria proteome. This complicates the search for potentially protective antigens. Nevertheless, since they cause proliferation and IFN-γ production by lymphocytes from immunised cattle, these low molecular weight proteins merit further investigation as potential vaccine antigens. / Author: Mirinda van Kleef neé Rossouw

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the degree of Master of Pharmacy, Johannesburg, 2017 / Oral diseases in South Africa remain a huge public health problem due to the high cost, prevalence, severity and the influence on the patients well-being. Treatment for oral diseases requires the need for specialist dental health care workers and come at a high cost causing a great burden on the health system. The three most important oral diseases are dental caries, caused by Streptoccocus mutans, Streptococcus sanguis, Lactobacillus acidophilus and Lactobacillus casei, periodontal diseases caused by Porphrymonas gingivalis and Fusobacterium nucleatum and oral candidiasis caused by Candida albicans, Candida glabrata and Candida krusei. An ethnobotanical review has revealed that over a 120 South African medicinal plants are used for the treatment of oral diseases. This coupled with the lack of research on the subject, allowed for the investigation of the antimicrobial efficacy of some South African plants against oral pathogens. A total of 140 aqueous and organic extracts and six essential oils were prepared from 31 different plant species. These plant samples were screened for antimicrobial efficacy against nine oral pathogens using the micro-titre plate dilution assay. Plant extracts that were found to have noteworthy antimicrobial activity against Streptococcus mutans were further evaluated on the effect on S. mutans biofilm formation using the glass slide method. The toxicity profiles of plant samples that were found to have noteworthy antimicrobial activity were evaluated using the brine shrimp lethality assay. The plants did not exhibit antimicrobial efficacy against all nine pathogens in this study, instead, most were very specific to disease conditions. Some plants did show good antimicrobial activity against four of the nine pathogens tested (A. afra leaves, C. torulosa stems, C, brachiata leaves and H. natalensis leaves).The organic extract of Cissampelos torulosa stems displayed the lowest MIC value of 0.05 mg/ml against both Lactobacillus spp. This antimicrobial activity was also observed with the organic extract of Spirostachys africana leaves against Candida albicans. In some instances, a direct relationship was found between the traditional use of the plant and the antimicrobial activity observed. For example, noteworthy activity (MIC < 1.00 m/ml) was observed against all three Candida spp. for Clematis brachiata (leaves), a plant traditionally used to treat oral thrush. Englerophytum vi magalismonatanum (stems) displayed notable activity against both Streptococcus spp. (MIC 0.83 mg/ml against S. mutans and MIC 0.67 mg/ml against S. sanguis). Spirostachys africana leaves displayed the greatest anti-adherent properties against S. mutans biofilm formation at both 24 and 48 h, reducing the biofilm by 97.56% and 86.58% respectively. The majority of plant samples tested in the brine shrimp lethality assay (BSLA) were considered safe, however, 13 plant samples were considered toxic, at a concentration of 1 mg/ml, and their LC50 values were determined. The findings from the results favour the potential use of these plants in treating oral diseases such as dental caries, periodontal diseases and oral thrush, and scientifically validates the traditional use of some of these plants. / XL2017

Enteric pathogens have a substantial impact on human health as they can cause outbreaks and severe disease outcomes. These pathogens employ many virulence strategies to evade host defenses and cause disease. While some virulence strategies have been carefully studied, other mechanisms remain largely uncharacterized. In addition, there are a number of putative virulence factors that have yet to be phenotypically or biochemically characterized. In order to facilitate the development of novel and effective treatment strategies for enteric pathogens, an understanding of how these putative virulence-associated proteins contribute to pathogenesis is required. In this work, the characterizations of two proteins implicated in the processes of motility and type III secretion are presented. The Escherichia coli O157:H7 protein Z0021 is found in an O-island unique to the most virulent serotypes of Shiga-toxin producing E. coli. Z0021 was found to encode for a repressor of motility that exerted its regulatory effect prior to the activation of class II promoters in the flagellar cascade. This work provided the first identification and characterization of a fimbrial operon-encoded motility repressor in E. coli O157:H7. The second protein, SsaN, from Salmonella enterica is a putative type III secretion system ATPase. This work examined the role of SsaN in virulence and effector secretion, and moreover provided insight into the mechanism by which SsaN binds to a chaperone to facilitate effector secretion. Together, these findings contribute to the understanding of the virulence strategies of two enteric pathogens that have had, and continue to have, significant impacts on human health worldwide. / Thesis / Doctor of Philosophy (PhD)

Fusarium head blight is one of the devastating diseases of cereal crops caused by Fusarium graminearum. This fungal pathogen produces mycotoxins like deoxynivalenol (DON), depositing it in wheat kernels and making them unfit for consumption. In addition, it causes decreases in the nutritional content of the wheat. Since wheat contaminated with DON above permissible levels must be discarded, it also leads to huge economic losses to the farmers. Fungi have a complex network of hyphae that lets them sense their surroundings. These are advantageous for nutrition and pathogenesis needs. Fungi have evolved nuanced mechanisms to orient hyphae towards external cues. Through our studies described in Chapter 2, we have elucidated the role of Ste3 GPCR in mediating fungal chemotropism towards peroxidases (previouly shown for Ste2). Both of these receptors were shown to activate the CWI-MAPK pathway in response to peroxidases. In addition, pathology assays on germinating wheat coleoptiles and detached Arabidopsis leaves showed that a Ste3 knockout strain was significantly compromised in its' ability to cause lesion development. In Chapter 3, we investigated the heterodimerization between FgSte3 and FgSte2 in response to peroxidase and a potential HRP-derived ligand of fungal origin. BRET and pull-down experiments confirmed the interaction. Chapter 4 consists of ongoing projects that go beyond the scope of the timeline for this dissertation. This includes establishing an Sf21 insect cell expression platform for the expression and purification of full length FgSte3 with a goal to elucidate the structure of the protein. Together these studies enhance our understanding of the mechanistic aspects of fungal pathogenesis and represent a step forward toward the development of novel anti-fungal compounds.

Due to the rapid nature of microwave heating, the microbiological safety of foods prepared in the microwave has been in question for several years. Because foods are heated from the inside out and are strictly governed by their own internal properties such as ionic content, moisture level and specific heat, work must be done to further master control of such properties so microwave cooking can be more predictable, controlled and ensure control pathogens. This study concentrated on the effect of fat content, evaporative cooling and food type on the rate of food borne pathogen survival rates in microwave heated foods. Foods investigated in this study included fresh, raw broccoli spears; a regular, whole muscle breaded chicken patty and a fat free, breaded, formed chicken patty; and raw ground beef patties at three differing fat percentages. All foods were tested in triplicate. A Sharp® 1000W Light-Duty Commercial Microwave Oven was used to treat inoculated samples according to their recommended cooking times. Two sets of samples were treated, one wrapped with Saran™ Wrap and the other without wrap. F-values were determined for each product. Raw ground beef patties at fat contents of 30%, 15% and 7%, heated for the same time had F-values ranging from 0.03 to 126.20. The lower the fat content, the lower the lethality. Regular and fat free chicken tenders had similar patterns. F-values for fresh broccoli indicted that vegetative pathogens survived the recommended microwave process. Covering in Saran™ Wrap had some preventive effect on evaporative cooling depending on the food tested and significantly (p < 0.05) increased most F-values. Inoculated pack studies were performed in triplicate on each food with Listeria monocytogenes, Salmonella and Escherichia coli O157:H7. Survival was determined by presence or absence of growth of each pathogen after enrichment. Listeria monocytogenes survived in all samples except for the 30% fat ground beef patties. The Salmonella species had a lower survival rate; however, it was still present in uncovered 15% fat ground beef, covered 7% fat ground beef, uncovered chicken patties (both types) and in all broccoli samples tested. E. coli O157:H7 survived in all samples except the 30% fat ground beef samples. Results indicate that higher fat contents seem to ensure lower rates of pathogen survival. This was especially true for the raw ground beef, which had received no prior processing other than the grinding of the whole muscle. There were fewer survival differences in the preprocessed, frozen chicken patties. Both were shown to support no pathogen survival in covered samples, except the fat free chicken patties. Listeria monocytogenes was shown to consistently survive the suggested cooking time in these samples. This is consistent with expectations that fat free food samples would display more survival than regular fat samples. Overall, covering samples with Saran™ had little effect on pathogen survival rates. There were survival differences in some covered and uncovered samples consistent with expectations that covered samples would show less survival than uncovered, but further work including more samples would be necessary to ensure that the covered or uncovered variable made the true difference in pathogen survival. Finally, broccoli demonstrated consistent pathogen survival in all categories of testing. This indicates microwave oven prepared vegetables could be a prime source of pathogen transmission to consumers. Further work needs to concentrate on determining the correct processing times and parameters that need to be met to ensure safe food. / Master of Science

The goal of this study was to assess the effect of residence time on the chemical and microbial quality of reclaimed water in two distribution systems located in southern Arizona. Utility A produced Class A water and utilized chlorine as a means of disinfection whereas Utility B produced Class A+ water and utilized UV radiation as a means of disinfection. Water-based pathogens were consistently detected in both distribution systems beyond the point of compliance, while microbial indicators like Escherichia coli was only detected in Utility B suggesting that treatment eliminated waterborne pathogens. Heterotrophic plate concentrations in samples from both utilities initially increased rapidly with increased distance from the point-of-compliance and were as high as 10⁹ CFU/100ml. Regardless of the initial level of treatment, the microbial quality deteriorated with increased residence time in the distribution systems. The second study was designed to evaluate the effect of reclaimed water storage on microbial and chemical quality of two classes of reclaimed water (Class A and Class A+). In Class A water, nitrification was observed during both field scale trials resulting in concentrations greater than 10mg/L while nitrification was not observed in Class A+. Chlorine residuals rapidly decreased within 48hours of storage. HPC concentration were as high as 10⁷ - 10⁸ /100ml. In both field scale trials, there was no observed growth of HPC during storage and waterborne indicator bacteria were rarely detected, and if detected, only at low concentrations. Based on this data, deterioration of microbial water quality during storage is minimal.

Potato (Solanum tuberosum) is one of the most popular food crops grown as a substitute staple in Zimbabwe. Its production is constrained by pest and diseases which reduce the yield drastically. The blackleg / soft rot disease complex caused by Pectobacterium and Dickeya species (formerly known as Erwinia) has been identified as a potential threat to potato production. The soft rot pathogens can cause systemic and vascular infections in potatoes, which result in the development of various symptoms on the stem and tubers. The symptoms that develop on the plant are not species specific but depend on climatic conditions prevailing at the infection stage. The pathogens affect the crop at all stages of production, which include in the field, storage and in transit. The primary objectives of this study were to identify the pathogens which cause blackleg and soft rot on potatoes in Zimbabwe; document the grower’s knowledge of blackleg and soft rot diseases in Zimbabwe; evaluate cultivars grown in Zimbabwe and South Africa for tolerance to Pectobacterium and Dickeya species; determine the role of calcium in blackleg and soft rot development; and determine the genetic diversity of Pectobacterium carotovorum subsp. brasiliensis isolates from South Africa, Zimbabwe and mini-tubers imported from China. The first chapter introduces the research highlighting the importance of potatoes globally and in Zimbabwe. It discusses production constraints caused by soft rot pathogens, as well as the pathogenesis of Pectobacterium and Dickeya spp. It also summarises the research objectives and thesis outline. Chapter 2 provides an overview of published literature on morphology, pathogenicity and identification of blackleg and soft rot pathogens, epidemiology, host range, defense mechanisms in plants, host nutrition and disease management strategies. Taxonomic revisions have led to the reclassification of pectolytic Erwinia into several genera. Strains formerly described as Erwinia carotovora have been incorporated into the genus Pectobacterium, and strains classified as Erwinia chrysanthemi are now assigned to the genus Dickeya. Several species and subspecies of Pectobacterium and Dickeya have isolated from infected potatoes. In Zimbabwe only Pectobacterium atrosepticum and Pectobacterium carotovorum subsp. carotovorum have until now been identified as the causal agents of blackleg and soft rot diseases, respectively. Although other species and subspecies of Pectobacterium and Dickeya have been isolated from infected potato plants in Zimbabwe, their potential to cause diseases on potatoes should not be underestimated. In chapter 3 bacterial isolates collected from infected plants showing typical blackleg / soft rot disease symptoms were identified using biochemical and physiological methods, as well as rep-PCR, Amplified Fragment Length Polymorphism (AFLP) and single gene sequencing using two genes, viz. gyrB and recA. Amplified Fragment Length Polymorphisms (AFLPs) were used to study the genetic diversity among potato isolates from Zimbabwe, South Africa and mini-tubers imported from China. Pectobacterium and Dickeya species enter the plant through wounds and natural openings such as lenticels. When they invade the plant they colonise the vascular tissue and spaces between the thin walled parenchyma cells. They remain inside these cells as latent infections and disease symptoms develop when host resistance is impaired or when environmental conditions become conducive for disease development. Chapter 4 investigates the role of defense related enzymes, polyphenol oxidase, phenylalanine ammonia lyase, peroxidases, chlorogenic acid and total soluble phenols in host resistance to potato soft rot. The potato varieties were assayed for activity of polyphenol oxidase and phenylalanine ammonia lyase, peroxidases and concentration of chlorogenic acid and total soluble phenols in tuber tissue. The role of these four components in imparting resistance against the soft rot pathogens was also investigated. Blackleg and soft rot diseases are seed-borne and difficult to control using chemical, physical and cultural methods. Several approaches aimed at controlling blackleg and tuber rot have been studied but the degree of success has been variable. Plant nutrition is an important component of natural disease resistance. The effect of calcium soil amendments in enhancing resistance to potato soft rot pathogens was investigated in Chapter 5. A management strategy based on calcium-induced defense responses of potato to Pectobacterium and Dickeya spp. was investigated. Calcium promotes production of phenolics and strengthens the cell wall, making the plants resistant to maceration by pectolytic enzymes. In Chapter 6 a survey was conducted in nine potato growing regions of Zimbabwe using an informal structured questionnaire. The findings highlighted the distribution and impact of blackleg and soft rot diseases on the Zimbabwean potato industry. A broad spectrum of information on blackleg / soft rot disease complex was gathered and this information can help growers to make informed decisions about control strategies to apply. Each chapter in this thesis has been treated as an independent entity. Thus redundancy between chapters could not be avoided. It is my hope that the results of these studies on soft rot pathogens in Zimbabwe will contribute to a better understanding of the blackleg / soft rot disease complex that they cause. I also hope that these studies will form the basis of detailed and future investigations in epidemiology and disease control strategies. / Thesis (PhD)--University of Pretoria, 2012. / Microbiology and Plant Pathology / unrestricted

Yes / Peptidyl-prolyl isomerase (PPIase) lipoproteins have been shown to influence the virulence of a number of Gram-positive bacterial human and animal pathogens, most likely through facilitating the folding of cell envelope and secreted virulence factors. Here, we used a proteomic approach to demonstrate that the Streptococcus equi PPIase SEQ0694 alters the production of multiple secreted proteins, including at least two putative virulence factors (FNE and IdeE2). We demonstrate also that, despite some unusual sequence features, recombinant SEQ0694 and its central parvulin domain are functional PPIases. These data add to our knowledge of the mechanisms by which lipoprotein PPIases contribute to the virulence of streptococcal pathogens.

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