The inulin neoseries, trisaccharide, neokestose was produced by the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma Y4-3) during growth on sucrose. To produce neokestose, whole cells harvested from the late exponential growth phase were incubated for 36 to 40 h at 25 oC in 0.2 M citratephosphate buffer (pH 7) containing 220 g.l-1 sucrose. Neokestose made up about 50 % of this mixture, which was purified equally well by both a carbon:celite chromatography as well as a batch filtration process, when eluting with similar amounts of water followed by a 50 % ethanol elution step. A final product was combined from various purification runs which consisted of 82.6 % neokestose, 8.7 % sucrose, 7.6 % GF3, 1.2 % glucose and 0.1 % fructose. Lactobacillus and Bifidobacterium genera are considered part of the beneficial group in in the intestine of animal and man. Bifidobacterium levels were higher than Lactobacillus levels in the caeca of New Hampshire layers, whereas in this study only Lactobacillus species were found in broilers. The reason for the absence of the Bifidobacterium species in the caecum of broilers was not determined. The prebiotic effect was evaluated on 5 week old broiler caecal material in vitro over 24 hours based on the viable levels of the total anaerobic bacteria, Lactobacillus and coliforms. The prebiotic effect was also evaluated on viable levels of added Salmonella Typhi, Escherichia coli and Campylobacter jejuni. Volatile fatty acids and pH were measured. The effect of neokestose on these groups was compared to that of inulin, a known prebiotic, and glucose. The total anaerobe and Lactobacillus levels increased over 24 hours for neokestose, inulin and glucose. Although there was no significant difference between the treatments higher levels were found for neokestose and glucose than for inulin. A decrease in the viable levels of E. coli, S. Typhi and C. jejuni were seen over 24 hours. The production of acetic acid, butyric acid and propionic acid was not significantly different for the treatments and the control. The pH decrease over 24 hours for the treatments was significantly different from the control, which indicated that lactate (not measured) production was probably higher in the neokestose, inulin and glucose treatments. In vivo tests are, however, required to fully evaluate the prebiotic and âbifidogenicâ effect of neokestose for broilers.

From the extensive literature review given in Chapter 1, it is evident that milk is an important part of the diet and can also serve as a good medium for growth of microorganisms. These microorganisms can be either pathogenic or being undesired causing spoilage. The pathogenicity and incidence of undesired microorganisms were reviewed and as a result one of the aims of this study was to assess the hygienic quality of milk sold in and around Bloemfontein. The results obtained during this study proved interesting as it showed an alarming high percentage of these milk samples were of a poor microbial quality as they did not confirm to the National Legislation regarding milk sold to consumers. The importance of yeasts in the dairy industry has been highlighted on a number of occasions by various authors. Despite indications of yeasts associated with dairy products, especially in yoghurts and cheeses, and milk being the raw material of these products, surprisingly few studies have been conducted on the specific occurrence of yeasts in either raw or pasteurized milk. The results obtained showed an ability of these yeasts to survive and proliferate in both raw and pasteurized milk. However, the number of yeast cells was low and insignificant to cause major problems. A wide diversity, including 14 different species was isolated and characterized. The alarming effect remains that predominant species like Candida albicans was found, a severe human pathogen. Due to concerns that some potentially dangerous and high numbers of undesired microorganisms may derive from the dairy farm, the ability to efficiently control these populations at the farm level seemed desirable. Consequently, the effect of ultraviolet irradiation on the microbial loads and chemical composition of raw milk was investigated. The results showed a 90% reduction on the microbial populations, except yeast numbers showing more resistance being reduced by 73%. Chemical analysis compared from results performed before and after UV radiation showed no significant alterations in the milk composition. Based on the results obtained, it was suggested that the usage of UV radiation on the milk resulted in an enhanced shelf-life and better microbial quality.

In this study, the construction of a forecasting model, using intracellular fatty acid composition as indicator, was attempted to assist in the search for yeasts capable of producing 3-hydroxy oxylipins. In order to achieve this, it was first attempted to establish a database mapping the distribution of fatty acids (FAs) associated with the neutral-, glyco- and phospholipid fractions of the 10 species representing the genus Saccharomycopsis. It was possible to identify nine of the 10 species i.e. Saccharomycopsis capsularis, S. crataegensis, S. fibuligera, S. javanensis, S. malanga, S. schoenii, S. selenospora, S. synnaedendra and S vini with the exception of S. fermentans. Saccharomycopsis crataegensis was unique since it produced by far the highest percentage neutral lipids (52.4% w/w) while S. schoenii produced the highest percentage phospholipids (35.9% w/w). All strains produced palmitic- (16:0), stearic- (18:0), oleic- (18:1) and linoleic acid (18:2) in all lipid fractions analysed. The major FAs produced were 18:1 and 18:2, while palmitoleic- (16:1) and a-linolenic acid [18:3 (w-3)] varied between species. Saccharomycopsis capsularis produced the highest percentage 18:2 in the neutral lipid fraction while S. crataegensis, S. malanga and S. selenospora produced the highest percentages of 18:1, 18:0 and, 18:3 (w-3) respectively, in the neutral lipids. Saccharomycopsis vini produced the lowest percentage 16:0 in this fraction. Saccharomycopsis fibuligera and S. schoenii produced the highest percentages of 16:0 and 18:2 respectively in the glycolipid fraction. Saccharomycopsis javanensis and S. synnaedendra produced the highest percentages of 18:1 and 16:1 respectively in the phospholipid fraction. Although it was possible to differentiate between most species using this phenotypic character, these FAs could not be used to predict what kind of 3-OH oxylipins these species are capable of producing. Saccharomycopsis fermentans (novel unidentified 3-OH oxylipin), S. malanga (3-OH 16:0), S. synnaedendra (3-OH 16:0, 3-OH 17:0, 3-OH 18:0, 3-OH 18:1, 3-OH 19:0, 3-OH 19:1, 3-OH 20:0, 3-OH 22:0) and S. vini (3-OH 9:1, 3-OH 10:1) could be separated using this character. Although, S. capsularis and S. javanensis both produced 3-OH 9:1, fatty acids with uneven carbon atoms which may serve as precursors could not be detected in the neutral-, glyco- or phospho-lipid fractions.

Not available

3-OH oxylipins are saturated and unsaturated oxidized fatty acids which are produced in mitochondria via incomplete b-oxidation or fatty acid synthesis type II. These compounds possibly play an important role in the sexual cycle of yeasts by assisting with ascospore liberation. Literature suggests that 3-OH oxylipins act as a lubricant during ascospore liberation, thereby ensuring efficient release of ascospores. Since the discovery of oxylipins i.e. 3-hydroxy (OH) oxylipins in the early 1990âs, these compounds were found to be distributed in various species of the fungal domain. Studies performed thus far, however, focused mainly on non-fermenting yeast species such as Ascoidea, Dipodascopsis and Eremothecium. According to various studies performed so far, 3-OH oxylipins were found to accumulate specifically in the sexual structures (asci and surrounding ascospores) of various nonfermenting yeasts. These studies also revealed that the sexual stages of nonfermenting yeasts are most susceptible to acetylsalicylic acid (ASA), a known mitochondrial (respiration and 3-OH oxylipin production) inhibitor. No information regarding oxylipin accumulation in asci or ASA-sensitivity of fermentative yeasts, however, has so far been reported. Using confocal laser scanning microscopy in combination with an oxylipin probe for 3-OH oxylipins and coupled to a fluorescing secondary antibody, the accumulation of these oxylipins was discovered in the asci of the following fermentative yeasts i.e. Pichia anomala, Pichia farinosa and Schizosaccharomyces octosporus. Interestingly, no 3-OH oxylipin accumulation was observed in the asci of the fermenting yeast Zygosaccharomyces bailii. This could be ascribed to the fact that this yeast depends more on a fermentative pathway for growth and sexual reproduction, than on mitochondrial respiration. Since 3-OH oxylipins are produced in the mitochondria, it is expected that there should be an increase in mitochondrial activity associated with these sexual structures. Using confocal laser scanning microscopy and a mitochondrial fluorescing probe (Rhodamine 123), an increase in mitochondrial activity was also observed in the asci of the fermenting yeasts tested, again with the exception of Z. bailii. Furthermore, during this study links between yeast sexual reproduction, 3-OH oxylipin accumulation/production, mitochondrial activity and oxygen requirement were established. This study revealed that fermenting yeasts are more resistant to ASA than non-fermenting yeasts when grown in liquid media. This is probably due to the fact that these yeasts can use either aerobic respiration or a fermentative pathway for growth and reproduction. This research prompted the development of a bio-assay that may find application in screening for effective antimitochondrial antifungals.

Mycoplasma gallisepticum is the most pathogenic avian Mycoplasma species and leads to great economic losses worldwide (Kleven, 2003). Prevention and control of this organism has been achieved by vaccination and antibiotic administration. Ferraz and Danelli (2003) reported on the most widely used live vaccines which are MG-F, Ts-11 and 6/85. The MG-F strain is currently not registered for use in South Africa. M. imitans is the most closely related organism to M. gallisepticum and was tentatively identified as M. gallisepticum until Bradbury and co-workers (1993) defined it as a different species. However, this species is still misidentified as M. gallisepticum due to serological cross-reactivity and M. gallisepticum specific primers also detecting M. imitans (Markham et al., 1999). While M. gallisepticum has been characterized in some countries, very little information is documented on the South African isolates. It therefore became the aim of this study to investigate the presence and diversity of this organism in Southern Africa, investigate the presence of M. imitans as well as to establish the relatedness of the Southern African strains to those isolated outside Africa. Samples were collected from serologically positive birds from different farms in South Africa and Zimbabwe. Two PCR (Polymerase Chain Reaction) assays were optimized, one to detect M. gallisepticum of a specific gene Mgc2, the other to detect both M. gallisepticum and M. imitans. A total of five samples were detected with the Mgc2-PCR, while almost all samples could be detected with the 16S rRNA gene-PCR. This is a possible indication of a different M. gallisepticum isolate that can be detected on the 16S rRNA gene level but not at the Mgc2 level, or the isolate could indeed be M. imitans. In a study by Woese and co-workers (1985), the Mgc2 gene was implicated as one of the rapidly mutating genes due to adaptation, a possible reason for the non- detection of Mgc2 but the detection of the 16S rRNA gene. Of the sequenced 16S rRNA gene-PCR amplicons, M. gallisepticum and M. imitans had the highest homology. However, there are only two complete sequences of the 16S rRNA genes that belong to two M. gallisepticum strains deposited in Genbank, thus limiting the information on the isolates detected. Restriction Fragment Length Polymorphisms (RFLPs) were performed and well optimized. Ts-11 gave the expected profile while tested samples could not be placed in any of the reference groups. It was observed, however, that the RFLP profile of one of the amplicons was similar to the 6/85 vaccine strain and subsequent results correlated with this finding. Two of the amplicons could be sequenced and further analyzed. A phylogenetic tree showed one of the amplicons clustering away from the other Mycoplasma species though its sequence was found to be that of M. gallisepticum or M. imitans. In another tree, one of the amplicons showed more homology to the pathogenic strains while the other showed more homology to the vaccine strain 6/85. The DGGE analyses showed that the amplicons consist of a mixed template which was the reason why some samples could not be properly sequenced. This might be an indication of mixed infection within the flocks. However, it was expected of the 16S rRNA to give these results. Furthermore, the DGGE results indicated that the vaccine strain Ts-11 is used to vaccinate some of the flocks, while other flocks are infected with wild-type Mycoplasma. The results also suggest the possibility of the presence of two copies of the amplified region of the 16S rRNA gene in this vaccine strain. The study has confirmed the presence of M. gallisepticum and the possible presence of M. imitans. Different yet closely related Mycoplasma isolates are also present in South Africa. These could represent novel strains or species and warrant further investigation.

The effect of ASA [(Aldrich, Steinheim, Germany); 80 g L-1 stock solution in absolute ethanol (Merck, Darmstadt, Germany)] on yeast growth was assessed in test tubes containing 5 mL of YNB (6.7 g L-1; Difco Laboratories, Detroit, Michigan, USA) broth supplemented with 2 % glucose (Saarchem, Wadeville, South Africa) (Petrou & Shanson, 2000). The tubes were incubated aerobically with 2 day old cultures on YM agar while agitating on a Rollordrum (30 oC for 4 days) according to the assimilation tests in liquid medium protocol (Yarrow, 1998). All yeasts were subjected to the following ASA concentration gradient: 0 mM, 1 mM, 2 mM, 3 mM, 4 mM and 5 mM ASA. Ethanol (ETOH) control (equivalent to the ethanol volume used to reconstitute 5 mM ASA) as well as a negative control (i.e. no ASA, ethanol or inoculum) were included. Growth was determined visually using a white card containing black lines as described by Yarrow (1998). Here, +++ indicates good growth (no black lines visible), ++ indicates growth (black lines just visible) and + indicates weak or no growth (black lines similar to that of inoculated tubes at the start of growth).

The focus of this survey study was to investigate the perceptions of the people regarding the seshoeshoe dress as part of the cultural identity for Basotho women in Lesotho. The population for the study was derived from the Lesotho College of Education staff and students, representing consumers, and the seshoeshoe dressmakers within the Central Business District area of Maseru as producers of the seshoeshoe dress. A quantitative research design with both a questionnaire and a structured interview was used to obtain information from the staff and students; an interview schedule was used to gather information from the dressmakers. The study was carried out within the theoretical framework of the cultural, contextual and semiotic perspectives, in which the contextual perspective facilitated an understanding of the interface between the individualsâ appearance and cultural processes. The cultural perspective provided a shared symbolic order within which people interpreted and developed meanings, while the semiotic analysis allowed the investigator to document the functions of dress in terms of its use within communities as well as within the Basotho society. Findings emerging from the study indicate that seshoeshoe dress is a symbol of national identity for Basotho women. Seshoeshoe dress is expensive. Appropriate new styles are acceptable but there is a concern regarding over- modification of the traditional style. Despite the emergence of the modernised seshoeshoe dress styles, and while a certain amount of change is allowed, the traditional style should be maintained and safeguarded as a cultural heritage. Current styles were found to be attractive to both the youth and the elderly, although mostly to the youth. Current styles, in addition to being attractive, demand less fabric and labour. The modified seshoeshoe dress styles are more popular than the traditional styles. Dressmakers are more familiar with the names of motifs than are their customers. A way for classifying these motifs was developed during the study. Preferred colours of consumers are lebete (spleen), blue, brown, golden blue and golden brown. Style, taste, acceptance and fashionability all play an important role in the popularity of seshoeshoe dress. Improved economy and technology have resulted in the emergence of a variety of new styles offering a wider choice to consumers. Seshoeshoe fabric should be used for purposes that will continue to dignify it as part of the cultural identity of the Basotho people. The study recommends that in order for the contributions of the Morija Arts and Cultural Festival and, the Cultural Days held at schools to be sustainable in terms of the significance of seshoeshoe as a cultural identity for Basotho women, the government through the Ministry of Tourism, Culture and Environment should formalise these activities at national level.

Arxula adeninivorans strain LS3, a dimorphic yeast with potential for biotechnological applications, has in recent years has been studied for the production of heterologous proteins. The growth kinetics of this species and the effect of environmental conditions on its morphology under controlled cultivation conditions have not been well documented. Because genetic transformation might affect the yeast physiology, a comparative investigation of the growth characteristics of strains LS3, G1211 (LEU2+) (the auxotroph derived from strain LS3) and LS3/pXynA, derived from strain LS3 by transformation with the Thermomyces lanuginosus xylanase gene under the control of the TEF1 promoter, was conducted, focusing on the effect of temperature and dissolved oxygen tension (DOT) on the morphology and growth parameters of the strains. This xylanase gene (XynA) was integrated in the 25S rDNA locus and expressed under control of the strong constitutive Arxula-derived TEF1 promoter. The plasmid copy number integrated into the rDNA locus was unknown. Little to no activity was found with the A. adeninivorans LS3 transformants, namely only 5.86 nkat ml-1-1 (0.35 U ml) compared to the 4 418 nkat ml-1-1 (265 U ml) obtained with the T. lanuginosus strain SSBP positive control. The protein itself might have been defective and since it accumulated intracellularly, this could also have resulted in the diminished activity observed. Cultivation in a temperature gradient incubator over a wide temperature range revealed significant differences in the maximum specific growth rates of the strains LS3 and G1211(LEU2+), namely 0.48 and 0.52 h-1, respectively. The minimum and maximum temperatures were 18 and 46°C, respectively, with the optimum temperature in the range of 37 to 39°C. In accordance with the literature, in shake flask cultures the morphology of LS3 changed with an increase in temperature from predominantly budding cells at 30°C to pseudohyphae at 42°C and resembled a mycelial culture at 45°C. By contrast, the morphology of strain G1211 (LEU2+) remained pseudohyphal at 45°C. During batch cultivations no true hyphae were observed, but a change in morphology, from budding cells to pseudohyphae, was observed during cultivation at different dissolved oxygen concentrations at different temperatures for LS3 and LS3/pXynA. G1211 (LEU2+) remained a pseudohyphal culture. The critical dissolved oxygen concentration (Ccrit) value, determined from the intersection of tangent lines of a plot of specific rate of oxygen uptake as function of dissolved oxygen concentration, of strains LS3 and G1211 (LEU2+) was 0.016 mmol l-1-1 (9% of saturation) and 0.013 mmol l (7% of saturation), respectively. This revealed that A. adeninivorans strain G1211 (LEU2+) had a slight but significantly lower Ccrit than strain LS3. This study showed that insertion of plasmid pAL-ALEU2m has a significant effect on the specific growth rate and on the morphology of Arxula adeninivorans LS3. Insertion of plasmid pXynA had only a slight effect on the morphology but no effect on the specific growth rate.

Incidences of an Adenoviral infection have been recently reported in South African psittacine birds. These birds have been diagnosed by histopathology post-mortem. This Psittacine Adenovirus (PsAdV) has been reported as the second most deadly disease-causing virus in psittacine species with Psittacine Beak and Feather Disease Virus (PBFD) being the first. High mortalities with no symptoms have been observed amongst the birds. There is limited information on the distribution and antigenic characterisation of PsAdV in South Africa. This prompted the investigation into the isolation and characterisation of this PsAdV by virtue of molecular and conventional techniques. Two birds suspected of being exposed to PsAdV were donated to the laboratory. Histopathological tests were already performed on these birds by a consulting veterinarian who found evidence supporting the Adenoviral infection. The livers of the parrots received were homogenised and DNA was then extracted from the suspension. These birds were from the same parrot breeding farm in the Free State region and their deaths occurred around September a year apart, inferring a seasonal occurrence of the infected birds. A polymerase chain reaction (PCR) was established as a rapid test to confirm the Adenoviral infection amongst the birds received. A primer pair amplifying the hexon gene loop1 variable region (L1) located in two conserved pedestral regions was modified using an existing primer pair as this gene codes for the hexon protein where the group, subgroup and type specific antigenic determinants are located. PCR resulted in the expected amplicon size of ~587bp for all the samples tested with the use of a Fowl Adenovirus (FAdV) received from the Faculty of Veterinary Science, Onderstepoort (Pretoria) as a positive control. The positive DNA products were then subjected to restriction fragment length polymorphism (RFLP) in order to investigate differences in the restriction profiles. The suspected PsAdV samples provided a similar profile compared to the different one elicited by the fowl sample where only two bands were similar to the other profile with two extra unique bands observed. These samples were then ligated into the pGem-Teasy vector and the positive clones were selected and sent to Inqaba Biotech for sequencing. The results where blasted on the NCBI GenBank Database and a high degree of similarity was found with the PsAdV sequences already on the database. Both the nucleotide and amino acid sequence alignments performed using DNAssist v2.2 showed a high homology with all the sequences. There were some differences observed and with the protein alignment the different amino acids observed were of the same group of amino acids. This suggested that the sequences were of the same genogroup. A neighbour-joining phylogenetic tree was constructed and this showed 4 major clusters where one of them represented the PsAdV sequences. The PsAdV sequences were separated from the other clusters by a 100% bootstrap value. The two minor branches within this cluster separating the UFS sequences from the reference PsAdV sequences suggested that the UFS samples could be different isolates. Attempts to cultivate the virus in primary chicken embryonated liver cells and SPF embryonated eggs were successful and a cytopathic effect (CPE) typical of Adenoviruses was seen in the cells. With the SPF eggs definite differences were observed between the infected embryos and the negative control embryos. These were also typical of Adenoviruses, particularly of group I Aviadenoviruses (AAVs). We propose that further studies be concentrated on the development of antibodies against this PsAdV in order to establish serological techniques so to be able to differentiate between different serogroups and isolates. We also propose the development of a vaccine against this psittacine Adenovirus so to put biosecurity measures in place.