Psittacine beak and feather disease (PBFD) is a readily recognisable dermatologic condition in wild and captive psittacines worldwide. It is caused by Beak and feather disease virus (BFDV) which is classified in the family Circoviridae and the genus Circovirus. BFDV has a circular ss-DNA genome consisting of seven open reading frames (ORFs), three being conserved in all BFDV isolates, ORF 1 which encodes the Rep protein, ORF 2 which encodes the coat or capsid protein (CP) and ORF 5 which encodes a protein whose function is as yet unknown. General symptoms of the disease include the symmetrical loss of feathers, feather abnormalities, beak and claw deformities, weight loss, anorexia and immunosuppression. The inability to grow BFDV in tissue culture or in embryonated eggs has hindered the routine diagnosis of PBFD affected birds and the development of reliable diagnostic tests and an effective vaccination program. PBFD is widespread in South Africa, leading to a loss of at least 10% of psittacine breeding stocks annually. The disease is also a major threat to the already endangered Cape Parrot (Poicephalus robustus) and the black-cheeked lovebird (Agapornis nigrigenis) and it is only a matter of time before we may see the extinction of these and other parrot species due to the lack of a preventative vaccine. The economical and natural implications of the attack by PBFD led to the aims of the present study which were to develop a potential DNA vaccine candidate, develop an expression system for production of recombinant CP as antigenic protein and establish an enzyme linked immunosorbent assay for the detection of BFDV-specific antibodies in parrots. The entire CP gene which has been suggested to encode for the epitopic protein of the virus was amplified by polymerase chain reaction (PCR) and ligated into a bacterial vector, pBAD/His B or a yeast vector, pKOV136 for expression of recombinant CP in Escherichia coli or Yarrowia lipolytica, respectively. Alternatively, CP gene PCR products were ligated into the mammalian expression vector pcDNAâ¢3.1D/V5-His-TOPO® which was the vector of choice for DNA vaccine design and used to transiently transfect Chinese hamster ovary cells. Subsequently, the candidate DNA vaccine was used in a basic vaccine trial where budgerigars (Melopsittacus undulatus) were vaccinated either with the DNA vaccine candidate or a sub-unit vaccine consisting of purified recombinant CP. Expression of recombinant CP was monitored using polyacrylamide gel electrophoresis (PAGE), chemiluminescent and colorimetric detection on Western blots and ELISAs. While expression of the recombinant CP was unsuccessful in the yeast system using pKOV136, expression of recombinant CP was achieved in E. coli cells using the pBAD vector. Recombinant CP was partially purified and applied in both indirect and indirect competitive ELISAs as coating antigen for the detection of BFDV specific antibodies. Using the established ELISAs, BFDV specific antibodies could be detected in naturally infected parrots as well as in budgerigars vaccinated with the DNA vaccine and sub-unit vaccine. Comparable results were obtained when nonpurified recombinant CP was applied in the ELISAs in lieu of partially purified recombinant CP. Vaccinated budgerigars formed BFDV specific antibodies in response to the DNA vaccine and sub-unit vaccine that were detected using the indirect competitive ELISA established in the study. The antibody responses to the sub-unit vaccine were higher than those in response to vaccination with the DNA vaccine candidate. Although the indirect competitive ELISA could not provide an indication of whether these antibody responses are protective, the results obtained during the trial are a preliminary indication that both the DNA vaccine and sub-unit vaccine may be functional in parrots and safe to use as no adverse reactions were observed.

Mannan polysaccharides occur in hemicellulose fraction of the plant cell walls. The hydrolysis of these polymers involves the action of enzymes such as β-mannanase, β-mannosidase and α- galactosidase which are produced by both fungi and bacteria. The current study reports on the production of β-mannanase, β-mannosidase and α-galactosidase by newly isolated Scopulariopsis candida strains LMK004 and LMK008. The effect of medium composition and carbon source on growth and enzyme production was evaluated in a liquid culture. A combination of Vogelâs medium and locust bean gum was found to stimulate growth and increase β-mannanase production. Optimal β-mannanase production of 7800 nkat/g biomass for LMK004 and 13300 nkat/g biomass for LMK008 was achieved in media containing 10% NaCl, 1X Vogelâs medium, 1% yeast extract and 1% locust bean gum. Both strains secreted trace amounts (less than 1 nkat/ml) of β-mannosidase and α-galactosidase indicating that these enzymes may be retained intracellularly. Native-PAGE and SDS-PAGE were used together with the zymogram to assess purity and to estimate the molecular weight of the proteins. The molecular weight of LMK004 β-mannanase was estimated to be â41 kDa whereas that of LMK008 β-mannanase could not be determined due to excessive loss of protein material during dialysis. The β-mannanase from LMK004 was most active at pH 5 and 50 °C, and retained ⥠80% of its activity at pH 5 â 6.5 after 24 hrs of incubation at 4 °C. In contrast, the LMK008 β- mannanase retained ⥠60% activity between pH 6 â 7. Both enzymes remained stable for 3 hrs at temperature between 30 °C and 40 °C, and showed loss of activity at higher temperatures. The two enzymes displayed different degrees of halotolerance. The LMK008 β-mannanase tolerated high NaCl concentrations with 60% activity remaining after incubation for 2 hrs at 20% NaCl, whereas the LMK004 β-mannanase was only active between 0% - 10% NaCl. It is clear from the current study that the two strains of S. candida produce distinct β-mannanases which may be useful candidates in low water activity reactions.

The genus Dipodascopsis was extensively studied with regards to reproductive cycles as well as the presence, distribution and function of 3-OH oxylipins. Most of this research was carried out on D. uninucleata var. uninucleata (Canadian strain) as well as D. tóthii. However, little is known concerning D. uninucleata var. uninucleata isolated from South African soil, as well as D. uninucleata var. wickerhamii. Consequently, using gas chromatography-mass spectrometry, electron microscopy and confocal laser scanning microscopy, the two varieties were compared regarding their morphologies, oxylipin production, mitochondrial activity as well as life cycles and ascospore release. According to literature, the two varieties differ only in their ability to assimilate certain carbon sources. During this study, differences in ascospore size as well as differences in ascospore clustering, after release, was observed. Furthermore, differences in the type of 3-OH oxylipin produced by the two varieties, also existed. 3-OH oxylipin production was found to be associated mainly with the sexual stage and concentrated in the ascus surrounding the ascospores, in both varieties. Furthermore, increased mitochondrial activity was also observed during the sexual stage and found to be concentrated in close vicinity of the ascospores. Since mitochondria produce 3-OH oxylipins, it is suggested that the increased activity during sexual development would be to aid in the production and release of the ascospores, as well as the accumulation of these 3-OH oxylipins. In addition, acetylsalicylic acid was found to inhibit the production of 3-OH oxylipins by probably decreasing mitochondrial activity resulting in the inhibition of ascospore release.

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).