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Cell surface mannan oligosaccharides have been implicated in die pathogenesis of C. albicans through roles such as adhesion to host cells and modulation of the immune system (Calderone, 1993, Chaffin, 1998). Understanding the molecular biology of glycosylation is therefore critical to elucidating host-fungus interactions. The CaMNTI gene, responsible for adding the second mannose on 0-linked mannan has previously been shown to be important for adhesion and virulence in C. albicans (Buurman et ai, 1998). In this project, the CaMNT1 gene family was studied as a means to understand more about the roles of oligosaccharide synthesis in Candida-host interactions. The Camntlp enzyme was expressed heterologously in P. pastoris and its cofactor and acceptor specificities were determined. The key residues for the retaining mechanism of CaMntlp catalysis and for cofactor binding were determined by site-directed mutagenesis as Asp350, Glu-318, His-377 and His377. This knowledge may aid in the rational design of antifungals against the MNT1 family of Candida mannosyltransferases. Two further members of the gene fainily; CaMNT3 and CaMNT5 were disrupted using the ura-blaster technique. Deletion of CaMNT3 and to a lesser extent CaMNT5 led to strains unable to form hyphae on solid Spider medium. This phenotype is distinct from that reported for CaMNT1 and suggests a role for these genes upstream or downstream of the Cphlp MAP Itinase cascade that regulates yeast-hypha morphogenesis. The Camnt3 and CamntS nuU mutants were attenuated in virulence in a systemic mouse model of candidosis. The heterozygous mutants were more attenuated in virulence than the nuU mutants indicating that a compensation mechanism may upregulate expression of other MNT genes when both copies of CaMNTS or CaMNT3 are disrupted. This study has shown that glycan synthesis affects morphogenesis in C. albicans and indicates many cell wall proteins require to be glycosylated to perform their function.

The main aim of the studies was to evaluate the modes of actions of Bio-Mos and the effect that it has on intestinal health as well as performance in broiler chickens. For the purpose of this study there were 2 main objectives. The first was to determine the effect of Bio-Mos as well as soluble mannan on salmonella colonization and to do this it was necessary to develop an in vivo pathogen challenge model, specifically designed for salmonella, using the chicken as animal model. The aim with this salmonella assay was to design a model that could accurately determine the efficacy of different components of the yeast cell wall at reducing or eliminating salmonella colonisation in chickens. The second objective was to evaluate the effect of Bio-Mos with or without the addition of a soluble mannan, fed at different inclusion levels, on chicken health. Specific parameters measured included feed conversion ratios (FCR), volatile fatty acid (VFA) analysis, antibiotic resistance amongst coliform populations, immunoglobulin quantification and gut morphology. Gut morphology measurements included villi height and width, crypt depth, muscularis thickness, goblet cell size and goblet cell density. The salmonella assay trial was not able to yield positive results for either the cell wall preparations or the positive control, indicating that there are some external factors that have to be addressed before this assay can be used to draw any accurate conclusions from. The second section of this study did show FCR differences between some of the treatments, but did not show numerically large differences for VFA production or antibiotic resistance, however the histological evaluation did yield interesting results. Measurements based on the villi height and width, crypt depth and muscularis thickness showed no significant differences between treatments but there was a treatment effect on the goblet cells. The goblet cells of chickens receiving cell wall preparations were statistically significantly larger and present at a higher density than those of the control treatment birds. In an attempt to develop the salmonella assay several aspects of the existing assay model were altered or eliminated. It is possible that the assay can work with some more adjustments, but due to time constrictions it was not possible to further explore alternative approaches. Little research has been done on the effect of nutrition on the goblet cells in chicken intestines. The results noted in this report warrant a more in-depth investigation into the exact modes of action resulting in the differences in goblet cells observed. The use of cell wall preparations on a commercial level holds many advantages, as cell wall preparations appear to affect animal health in a positive way. / Dissertation (MSc (Agric) : Animal Nutrition)--University of Pretoria, 2008. / Animal and Wildlife Sciences / MSc (Agric) / unrestricted

The Candida albicans cell wall provides an architecture that allows for the organism to survive environmental stress as well as interaction with host tissues. Previous work has focused on growing C. albicans on media such as Sabouraud or YPD at 30 °C. Because C. albicans normally colonizes a host, we hypothesized that cultivation on blood or serum at 37 °C would result in structural changes in cell wall mannan. C. albicans SC5314 was inoculated onto YPD, 5% blood, or 5% serum agar media three successive times at 30 °C and 37 °C, then cultivated overnight at 30 °C in YPD. The mannan was extracted and characterized using 1D and 2D 1H NMR techniques. At 30 °C cells grown in blood and serum contain less acid-stable terminal β-(1→2)-linked d-mannose and α-(1→2)-linked d-mannose-containing side chains, while the acid-labile side chains of mannan grown in blood and serum contain fewer β-Man-(1→2)-α-Man- (1→ side chains. The decrement in acid-stable mannan side chains is greater at 37 °C than at 30 °C. Cells grown on blood at 37 °C show fewer →6)-α-Man-(1→ structural motifs in the acid-stable polymer backbone. The data indicate that C. albicans, grown on media containing host-derived components, produces less complex mannan. This is accentuated when the cells are cultured at 37 °C. This study demonstrates that the C. albicans cell wall is a dynamic and adaptive organelle, which alters its structural phenotype in response to growth in host-derived media at physiological temperature.

Diese Arbeit untersucht das Auftreten falschpositiver Mannan- und Galactomannanergebnisse bei hämatologischen Patienten nach allogener Stammzelltransplantation, die parenteral ernährt werden müssen oder eine antibiotische Therapie mit Piperacillin-Tazobactam erhalten. Die Kenntnis von Quellen, die die Spezifität des Platelia™ Candida Ag plus und den Platelia™ Aspergillus EIA beeinflussen, ist ein wichtiger Beitrag zur Diagnostik systemischer Pilzinfektionen. / This study investigates the occurrence of false-positive mannan and galactomannan results in hematological patients undergoing allogeneic hematopoietic cell transplantation with current parenteral nutrition or antibiotic treatment with piperacillin-tazobactam. The knowledge of sources influencing specificity of Platelia™ Candida Ag plus and Platelia™ Aspergillus EIA contributes to the diagnosis of invasive fungal infections.

Orientador: Aulus Cavalieri Carciofi / Resumo: Derivados da parede celular de levedura têm sido estudados por seu efeito prebiótico. Recentemente, preparações purificadas e mais solúveis foram desenvolvidas, com vista a aumentar seu efeito fisiológico. Este estudo teve por objetivo avaliar os efeitos da inclusão de dois diferentes extratos da parede celular de leveduras, um convencional (PCL) e outro com maior porcentagem de mananoligossacarídeos solúveis (PCLs) sobre a digestibilidade, produtos de fermentação nas fezes e alguns parâmetros imunológicos de cães adultos hígidos. Foi empregada uma única formulação, desdobrada em três tratamentos: CON – controle, sem adição de parede celular de levedura; PCL – adição de 0,3% de parede celular de levedura convencional; PCLs - adição de 0,3% de parede celular de levedura com elevada solubilidade de mananoligossacarídeos (MOS). Foram utilizados 24 cães beagle adultos, com oito repetições por ração, em delineamento em blocos casualizados. Cada bloco teve duração 30 dias, sendo avaliados no início e no final do período as concentrações séricas de fator de necrose tumoral alfa e interleucinas 6 e 10, produção ex vivo de intermediários reativos do oxigênio e nitrogênio por células mononucleares e polimorfonucleares de sangue periférico, capacidade fagocítica de monócitos e neutrófilos e concentração de IgA nas fezes. Adicionalmente, foi avaliada a digestiblidade aparente dos nutrientes, produção e qualidade das fezes, ácidos graxos de cadeia curta e ramificada, lactato, amônia, pH e... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Derivates of yeast cell wall have been studied by its prebiotic effect. Recently, more purified and soluble preparations were developed, attempting to increase their biological actions. This study evaluated the inclusion of two yeast cell wall preparations, one conventional (YCW), and another with higher solubility of the mannan oligosaccharide fraction (YCWs), on their effects on nutrient digestibility, fermentation products on feces, and some immunological parameters of dogs. A single food formulation was used, unfolded on the following treatments: CON – control, without yeast cell wall addition; YCW – addition of 0.3% of a conventional yeas cell wall extract; YCWs – addition of 0.3% of a yeast cell wall extraction with elevated mannan oligosaccharides solubility. Twenty-four adult beagle dogs were used, eight dogs per food, distributed on a randomized block design. Blocks lasted 30 days, and tumor necrosis factor alpha and interleukins 6 and 10, ex vivo production of hydrogen peroxide and nitric oxide by peripheral neutrophils and monocytes, phagocytic index, and fecal IgA were evaluated at the beginning and ending of each period. Additionally, nutrient digestibility, feces production and quality, and fermentation products, pH, and biogenic amines were quantified on feces. Results were evaluated by variance analysis and compared by Tukey test (P<0.05). For the immunological parameters, the basal values were used as a covariate. The inclusion of YCWs reduced fat digestibili... (Complete abstract click electronic access below) / Mestre

C. albicans is a polymorphic fungal pathogen which has the ability to shift from yeast to hyphae. C. albicans cell wall is composed of glucan, chitin, mannoprotein and mannan. It is not possible, using standard extraction methods, to isolate mannan from C. albicans hyphae. To isolate hyphal mannan, we developed a simplified alkali extraction method. Using this method it was determined that hyphal mannan has a much lower molecular weight, a smaller polymer distribution and altered conformation structure when compared to yeast mannan. The hyphal mannan was found to contain little to no acid-labile portion with only α-Man-PO4 groups and no long chains of β-1, 2-linked mannosyl repeat units, when compared to the yeast mannan. It was concluded that the C. albicans hyphal mannan is substantially different from the mannan found in the yeast form. This is an entirely new observation that extends the existing knowledge about the structural biology of C. albicans hyphae and may provide insights into the role of hyphae in pathogenesis.

Three experiments were conducted to evaluate the impacts of removing antibiotic growth promoters (AGP) on broiler performance and yield and to evaluate alternative products as potential replacements. In experiment one, approximately 552,000 broilers were reared in four solid-wall, tunnel ventilated houses that were divided into two paired-house facilities, each assigned one of two dietary treatments. The treated group received basal diets containing salinomycin (SAL), roxarsone (ROX) and AGP, while the control group received the same diets without ROX and AGP. Removal of ROX and AGP had no affect on average body weight and feed efficiency, while livability was significantly affected negatively by the removal of ROX and AGP. Tender, wing, drum and percentage of total white meat showed significant improvements in yield during the study, while all other parts were not affected by removal of ROX and AGP. In experiment two, an investigation was conducted to evaluate the effects on performance from feeding Bacillus subtilis spores (Gallipro®, Chr Hansen A/S, Denmark), as a direct-fed microbial additive, to commercial broiler chickens. Birds were divided among two paired-house facilities. The treatment group received basal diets supplemented with B. subtilis spores, while the control group was fed the same basal diets containing an AGP. Feed conversion ratio was significantly lower for the treatment group, while average body weight, coccidiosis lesion scores, and footpad scores were not affected by the treatments. In experiment three, 6,000 broiler chickens were equally divided among four treatment groups and reared to 49 d to determine the effectiveness mannan oligosaccharides (MOS, Bio-Mos®, Alltech, Nicholasville, Kentucky, USA) as an alternative for an AGP program and MOS plus Natustat™ (NAT, Alltech, Nicholasville, Kentucky, USA) as an alternative to an enteric health program (AGP+anticoccidial drug). Average body weight for the control (CON) and antibiotic (ANT) groups was significantly different from the MOS+NAT group, but not the MOS group. Carcass front half, carcass hind half, frame and skin yields were improved for all treatments when compared to the MOS+NAT group. Conversely, percent total white meat yield was improved with the inclusion of MOS when compared to the ANT group. The findings of this research suggest that the removal of AGP from the diets of commercial broiler chickens does not affect the performance and yield of the birds over a one year production period. Furthermore, B. subtilis spores and mannan oligosaccharides provide acceptable alternatives to an AGP program.

The objective of these studies was to evaluate the effect of supplying mannan oligosaccharides (MOS) to pig diets on both weanling pig and sow performances. Nursery pigs challenged with LPS to stimulate an immune challenge had increased body temperature and respiratory rate as well as elevated serum cortisol and TNF-α concentration, MOS-supplemented pigs had a lower rectal temperature and respiratory rate which implied that MOS improves some aspect of the immune function of piglets. MOS supplementation in sow diets during late gestation and lactation had no effect on litter size, but the piglets from MOS-fed sows were heavier at birth (P = 0.04), at weaning (P = 0.03), and during the entire nursery period (P < 0.01). Moreover, milk fat and protein levels as well as the Ig concentrations in milk from MOS-fed sows were numerically higher (2 – 12%; P > 0.10) than control sows. Overall, MOS supplementation in the nursery diet may have limited effects on the growth performance, but may have some beneficial influence on pigs under immune challenge. Furthermore, including MOS to the sow diet during late gestation and lactation can potentially improve piglet body weight as well as growth during the suckling and nursery periods.

The effect of intestinal microbiota and dietary supplementation of mannan oligosaccharides (MOS) on mucosal architecture and digestive physiology in broiler chicks was examined. In experiment 1, pre-sterilized eggs (Ross x Ross 308) were placed in three HEPA (high efficiency particulate air)-filtered isolator units at day 19 of incubation. Germ-free chicks in one isolator were conventionalized by exposure to cecal contents from a laying hen. Bacterial contamination occurred in one germ-free isolator such that these birds were monoassociated by a bacterium within the Acinetobacter spp. resulting in 3 categories of microbial status including germ-free (GF, n=10), conventionalized (CV, n=19) and monoassociated (Mono, n=13) birds. Dietary treatments assigned to each isolator consisted of a negative control (NC, 0 g/kg of MOS in the basal diet) and MOS (2 g/kg of MOS in the diet) resulting in a 2X3 factorial treatment arrangement. At 7 d of age, body weight was recorded and birds were killed to permit collection of visceral organs, intestinal tissues and cecal contents. Body weight, relative length of small intestinal segments and relative bursa weight were significantly increased in CV birds. These birds also had increased crypt depth and lamina propria area. Dietary MOS increased villus height and villus surface area in CV birds compared with GF and Mono birds. Transcripts for all housekeeping genes tested in ileal tissue were increased by MOS such that transcripts were normalized to unit mass of total RNA. In comparison to birds fed the NC diet, MOS significantly increased the abundance of proliferative cell nuclear antigen (PCNA), toll-like receptor (TLR)-4, avian β-defensin (GAL)-6, interleukin (IL)-8, peptide transporter 1 (PepT1) and sodium-dependent glucose transporter (SGLT)-1 transcripts in ileum per unit total RNA. However, the effect of microbial status on selected gene expression profiles was surprisingly limited. A second experiment was conducted to confirm the conventionalization protocol produced a complex microbiota similar to conventionally reared birds. Twenty day-old broiler chicks (Ross x Ross 308) were assigned to one of two wire-floored battery cages provided the NC and MOS diets ad libitum and killed at 7 d of age. Terminal restriction fragment length polymorphism (TRFLP) analysis demonstrated that microbial diversity indices (Richness, Evenness, Shannon, and Simpson) were greater in conventionalized gnotobiotic birds compared to the conventionally reared birds confirming a successful conventionalization strategy in the gnotobiotic trial. These studies demonstrate that under good hygienic conditions, CV chicks thrive similar to GF animals. Based on responses to MOS observed in GF birds, evidence indicates that MOS, independent of changes in microbial composition, directly modifies host response parameters including innate immune activation, digestive and absorptive function.