Beak and feather disease (BFD), caused by Beak and feather disease virus (BFDV) is a dermatological condition afflicting parrot species. It is becoming increasingly difficult to ignore not only the significant negative economic impact that the virus has on the parrot breeding industry but also the detrimental effect it has on the survival of the endemic Cape parrot (Poicephalus robustus). The virus, a member of the Circoviridae, is known to possess a non-enveloped, circular, single-stranded DNA genome. Two major open reading frames (ORFs) encode the replication associated protein (Rep) and the coat protein (CP). The study was set out to evaluate and improve the current diagnostic strategy for BFDV, with both molecular and serological techniques. The following objectives were attempted: 1. To evaluate polymerase chain reaction (PCR) and quantitative real-time polymerase chain reaction (qPCR) as diagnostic tools for BFDV. Detection of BFDV with conventional PCR is not always sensitive, especially in birds without clinical symptoms. Furthermore, genetic variance was suggested to have a detrimental effect on primer hybridisation. A real-time assay was designed to address these problems. It amplified a 115 bp fragment of ORF V1 and was able to quantify viral load. 2. To recombinantly express BFDV coat protein. A sustainable source of the main immunogen, coat protein, was needed for use in serological test development. Bacterial expression of BFDV CP was unsuccessful; however, BFDV CP from an alternative expression study was used as a serological diagnostic antigen. 3. To develop serological diagnostic tests for BFDV. A novel slide agglutination test was developed and will serve as an initial screening tool in serological diagnosis. Steps were made in the development of a competitive Enzyme Linked Immunosorbent Assay (ELISA) for a quantitative indication of immune response to BFDV. A significant proportion of asymptomatic BFDV infections exist. Using a combination approach of both molecular and serological tests increases the capacity to detect infections or exposure to virus. New techniques described should be used in conjunction with existing tests and should not completely replace conventional techniques for diagnosis of BFDV infection or detection of exposure to the virus.

In order to study the decrease of yogurt fermentation time, the effects of a wide range of supplements on yogurt fermentation time were evaluated. YE was identified as the only supplement which showed potential. Unfortunately it resulted in a product with an unacceptable flavour. It was therefore important to identify and isolate the specific component responsible for the decrease in yogurt fermentation time. YE was fractionated with size exclusion chromatography and it was subsequently determined that the accelerating fraction had a low molecular weight. It was important to establish whether the accelerating component was of mineral, vitamin or amino acid origin. Three cocktails containing the most common minerals, vitamins and amino acids were prepared and their respective effects on yogurt fermentation were determined. Results indicated that when compared to the respective controls, no decrease in fermentation time was observed with the mineral and vitamin cocktails. A decrease in fermentation time was observed with the amino acid cocktail, indicating that the accelerating component present in YE was of amino acid origin. It was however not clear whether it was a single amino acid or a peptide. The accelerating fraction was further analysed by SDS-PAGE and due to no visible bands in the respective region (<1kDa), it could not be analysed with mass spectrometry. The fraction obtained directly after size exclusion chromatography was however analysed by using mass spectrometry in order to determine the total amino acid content of the accelerating fraction after which it was evident that the fraction containing the accelerant contained an abundance of peptides. The individual effects of the 17 identified amino acids were determined in respective yogurt fermentations. Results indicated that no single amino acid was responsible for the decrease in yogurt fermentation time. Although a combination of the 17 amino acids in one fermentation run resulted in a decrease in fermentation time in comparison to unsupplemented yogurt, the decrease was not as considerable as that of the accelerating fraction obtained after size exclusion chromatography. Due to the fact that it was not practical to evaluate the effect of all possible combinations of the 17 amino acids, the combinations that were evaluated were based on literature reports on stimulation of Streptococcus thermophilus growth. Focus was placed on the growth of Streptococcus thermophilus due to this organism being the growth limiting organism between the two starter organisms used for yogurt fermentation. None of the evaluated amino acid combinations decreased yogurt fermentation time, and it was therefore concluded that the accelerating component was a peptide and not a free amino acid. In order to establish the mechanism of acceleration of the isolated YE fraction, it was important to determine whether the addition of YE to milk increased the rate of starter bacteria growth or whether it completed the growth requirements of starter bacteria. The latter could either result in earlier initiation of lactic acid production or in increased levels of lactic acid production. Results indicated that YE had no effect on the total growth rate of the starter bacteria. However, when examining the effect on the respective bacteria individually, it became clear that YE increased Streptococcus thermophilus cell numbers in comparison to the unsupplemented control. Using resazurin, it seems YE increased the metabolic rate of the starter bacteria. Although supplementation of yogurt with YE did not influence combined starter bacteria growth, it influenced lactic acid production. The addition of YE to yogurt resulted in increased lactic acid levels as well as an increase in lactic acid production rate. Lactic acid was also initiated earlier in the fermentation process, resulting in higher lactic acid levels in comparison to unsupplemented yogurt. It was therefore evident from this study that YE did not provide nutrients that are not already present in milk, but rather provided nutrients in a readily available form at the beginning of the yogurt fermentation process resulting in the reduction of the lag phase of lactic acid production.

Milk is a complex and complete food for the specific nutritional requirements of the neonate. For the dairy industry, milk is a suitable raw material for the production of other high value products. Although extensive research has been carried out on milk of economically exploited dairy animals such as cow, goat, sheep, buffalo and camel, there are properties which are not explicit in the milk of these, that are not completely understood. Research of the milk from non-dairy animals, in which these properties are explicit, may provide answers. One of the unique properties is the content of oligosaccharides, which is low in the dairy animals, but high in some species. This property points to a specialized saccharide synthesis in the latter, where the whey protein α-lactalbumin may play a role. Another unique property is the structure of casein micelles, which in the dairy animals is stabilized by the presence of a specific ratio of four casein types, as well as their specific structural properties. The most important is the κ-casein with its amphipathic nature. In some non-dairy species, stable casein micelles are formed in spite of the absence, or low content, of some of the casein types, specifically the κ- casein. The milk of the African elephant (Loxodonta africana) displays several unique properties. In this research the proteome of its milk was investigated, with a focus on α-lactalbumin and the caseins, in order to shed light on the mentioned unique properties. The proteomics approach was used, which includes gel electrophoresis and mass spectrometry. Computer modeling was also employed. The major proteins αs1-, αs2-, β-casein, α-lactalbumin, β-lactoglobulin and serum albumin of African elephant milk were identified with one-dimensional electrophoresis and mass spectrometry. Better results were obtained with two-dimensional electrophoresis and orbitrap mass spectrometry, with which α-lactalbumin, lactoferrin, β- and κ-casein were identified. The multiple sequence alignment of α-lactalbumins showed that there are six amino acid positions that are unique to that of African elephant. Most of the amino acid substitutions in this protein were found to be conserved, and the structure model of African elephant α-lactalbumin was found to be homologous to the X-ray crystallography structures of several species. Consequently the structure models of β- 1,4-galactosyltransferase 1 and the lactose synthase complex were built, again showing homology to crystallographic data from other species. It may therefore be concluded that structures of α-lactalbumin and β-1,4-galactosyltransferase 1 are highly conserved amongst species. The saccharide synthesis in the African elephant milk would probably not differ from that of other mammals, and may therefore not be the reason for high levels of oligosaccharides in its milk. The comparison of amino acid sequences and hydropathy plots of African elephant β- casein with that of other species showed that it would self-aggregate and interact via hydrophobic interactions with other caseins to form casein micelles, similar to the model proposed for cowâs milk micelles. However, the African elephant β-casein displays several more hydrophilic regions, compared to the cowâs protein. The amino acid sequence and hydropathy plots predicted that African elephant κ- casein would function in the same way as the equivalent of other species. The ratio of African elephant milk κ-casein to β-casein was calculated to be approximately 1:8.5, which is in the same order as camel and rat milk, compared to the 3:8 of cowâs milk. This means that there would be very little κ-casein on the surface to effect repulsion of the micelles. There should therefore be other protein regions protruding from the micelle surface to aid in this function. It is suggested that in African elephant milk the hydrophilic regions of β-casein carry out this role.

Previous unpublished research by Kock and co-workers indicated that the antimalarial drug chloroquine (CQ) stimulates yeast sexual stages (biosensors). Consequently several indicator yeasts (Eremothecium ashbyi, Dipodascopsis uninucleata var. uninucelata, Lipomyces yamadae and Scheffersomyces stipitis) were exposed to concentration gradients of CQ in the Anti-mitochondrial Antifungal Assay (3A) system and their ascospore release mechanics were subjected to Auger architectomics. Auger architectomics is the study of the structure and atomic composition of cells by making use of Nano Scanning Auger Microscopy (NanoSAM) as well as other techniques (http://en.wikipedia.org/wiki/Auger_architectomics). Investigation of the ascospore release mechanics revealed that L. yamadae and S. stipitis were sequestrate (making use of passive ascospore release) while E. ashbyi and D. uninucleata made use of active ascospore release. The sensors of L. yamadae have smooth, spherical ascospores that are released by destruction of the sensor wall. The spherical sensors of S. stipitis each contain two brimmed (âhatâ-shaped) ascospores that are released when the sensor wall breaks apart. The sensors of E. ashbyi are mostly intercalary in long chains with up to sixteen sickle-shaped ascospores in each ellipsoidal sensor. The V-shaped fins at the base of each ascospore of E. ashbyi are coated with 3-hydroxy (3-OH) oxylipins, making them hydrophobic. This facilitates the movement of ascospores by water flow. The tapered tips of the ascospores pierce through the sensor wall to allow release (http://vimeo.com/61521401). In D. Uninucleata, the inside surface of the sensors are lined with flexible sheaths, surrounding the ascospores inside the central channel. Inflation of the sheaths due to water uptake, generates turgor pressure that forces the ascospores out of the sensor. This is in sharp contrast to the morphologically similar yeast Dipodascus geniculatus, where the inflation of sheaths surrounding each individual ascospore is responsible for ascospore release. In all cases the sensors with ascospores were observed to have increased mitochondrial activity compared to surrounding cells. It was found with the 3A system that CQ is indeed a potent yeast fertility drug, having pro-fertility effects on all the yeast sensors used, including yeasts with decreased ability to sporulate due to preservation by sub-cultivation. Chloroquine caused increased formation of mature sensors of L. yamadae and S. stipitis, and increased the rate of ascospore release from the sensors of E. ashbyi and D. uninucleata. This data becomes even more compelling when considered that the S. stipitis strain used had lost the ability to sporulate prior to this study. An investigation of the relevant literature showed that the pro-fertility effects of CQ are highly conserved in the Eukarya, having similar effects on mammals (including humans) as well as the malaria causing parasite Plasmodium falciparum. This highlights the need to re-evaluate future and current CQ based treatment regimes.

The nutritional composition of agave flowers was determined and the following nutrients were analysed: moisture (86.62%); energy (226 kJ/100 g); proteins (1.71 g/100 g); fat (0.46 g/100 g); dietary fibre (2.12 g/100 g); K (207.77 mg/100 g); Mg (53.06 mg/100 g); Ca (48.33 mg/100 g); P (32.12 mg/100 g); Na (1.27 mg/100 g); Fe (1.03 mg/100 g); Zn (0.66 mg/100 g); Cu (0.04 mg/100 g); and Mn (0.15 mg/100 g). In contrast to many vegetables, the flower samples contained sugars in the form of sucrose (0.52 g/100 g), glucose (0.77 g/100 g), fructose (1.06 g/100 g) and maltose (0.69 g/100 g). The vitamin C content was 1.03 mg /100 g, but no vitamin A was detected. When compared to other flower vegetables, the agave flower had the lowest contents for protein, P, K, Mn, Na, Cu and vitamin C, but the highest value for fat. The agave flower and artichoke had similar values for energy, moisture and Mg, while the cauliflower and agave compared well in regard to Ca and Zn contents. Broccoli had similar contents for protein, fibre, P and Cu. The agave had higher values for energy, and fat than cauliflower, and higher fat and Ca, Fe, Mg, P and Zn values than broccoli. Artichokes had lower contents for moisture, fat, Ca and Zn than the agave flowers. Broccoli and cauliflower were moister than the agaves. Descriptive sensory analysis was used to investigate how various treatments would influence the sensory properties of Agave americana flowers. Blanched and unblanched flowers, subjected to three treatment methods (steaming, stir frying and pickling), were analyzed by ten trained panelists, in three replications. The data was analyzed using principle component analysis. A lexicon of 20 attributes was generated, including 11 for the steamed treatment, an additional six for the stir fried treatment and another four for the pickled treatment. Of these, three descriptors were for the attribute aroma, six for mouthfeel, five for appearance, four for taste and one for aftertaste. The unblanched pickled agave flowers were characterized by crunchy, fibrous and chewy mouthfeel, bitter taste, green pepper colour and cactus appearance, and a cucumber odour. For the blanched pickled agave flowers, taste descriptors were prominent, namely sweet, sour and sweet-sour, followed by a sweet aftertaste, combined with a moist appearance. For all the unblanched flowers, regardless of treatment, some of the descriptors had negative connections, like fibrous, bitter, cactus and rancid. Descriptors for the blanched flowers, again regardless of treatment, were more favourable and included sweet, sour and sweet-sour taste, and green bean and nutty odour. Three panels of 50 members each participated in the consumer acceptance tests, one each for the steamed breads and chocolate cakes, and one for the stew and deep fried flowers. Apart from acceptability, aroma, taste and texture were also evaluated for the baked products. The breads, cakes and stews were defrosted at 4 ºC overnight. The breads and cakes were left at room temperature (22ºC) before serving, while the stews were served heated. The breads, fritters and stews were prepared with blanched flowers, while the cake was made with unblanched flour. All the products were acceptable, but in different degrees. The scores were lower for aroma and final acceptability of the bread, due to unfamiliar texture experienced by the consumer panel. The agave cake scored lower for aroma and taste, but higher than the bread on texture. The stews and battered agave fritters were liked by the consumer panels and scored between 6.92 and 7.26 on the hedonic scale.

It has previously been implicated in literature that intracellular gas bubbles cannot form in yeast cells even under high gas supersaturation conditions. Furthermore, not even protein-coated gas vesicles found in Cyanobacteria are expected in yeasts. The lack of intracellular gas bubbles has been attributed to the increased structuring of water and lack of water with nucleation properties. This, however, is considered a missing link since yeasts, the workhorses of the baking and brewing industry, are known to produce and vigorously release carbon dioxide (CO2) gas during fermentation. Here we resolve the missing link between CO2 production by glycolysis and the eventual release of CO2 from the cells, and show that yeasts are capable of producing intracellular gas bubbles which were found to occupy a significant part of the cell. These gas bubbles do not contain a membrane that surrounds them. Furthermore, addition of zinc to the growth medium resulted in the âgalvanizationâ of the bubbles suggesting that the gas bubbles may possibly contain CO2. These findings should pave way for future research on CO2 behaviour under pressurized conditions that may have an impact on fermentation biotechnology. Furthermore we show that these intracellular gas bubbles deform cell organelles such as the nucleus. The skin surrounding the gas bubbles is able to withstand tension as they do not disintegrate when they come in contact with organelle membranes. Further research should now be performed on the mechanical effects of the gas bubbles on metabolic and coding functions of yeasts as gas bubbles deform and contort cell organelles. From these findings careful consideration is required during optimization of fermentation parameters to prevent CO2 toxicity effects on fermentation performance and flavor formation in practical brewing.

The aim of this project was to sequence the whole genome of Thermus sp. NMX2 A.1 strain and compare it to the whole genome sequence of T. scotoductus SA-01.Therafter attempt to use experimental data to confirm functionality within the genomes and lastly isolate a new Thermus sp. from the fissure water samples routinely collected from the deep gold mines of South Africa and the Northam Platinum mine. The genus Thermus has been extensively studied since the discovery of T. aquaticus in 1969 by Brock and Freeze and hundreds of species had been isolated ever since. However, up to date only eight validly described species are comprised in the genus Thermus. Moreover, amongst this handful amount of species from the genus Thermus; great discoveries have been made and highlighted parts drew further attention in studying this genus even more. Their metabolism is one of the aspects looked into especially the denitrification respiration. With the application of a nitrate operon it is known that a few species within this genus are able to grow under such conditions, meanwhile; others are modified by genetically manipulating them to do so, for instance T. thermophilus HB27. Subsequently, T. scotoductus SA-01 is reported to naturally possess the nitrate operon allowing it to grow in oxygen restricted conditions without any genetic manipulation applied. Recently, another strain was discovered to have the same functionalities as T. scotoductus SA-01 when grown under denitrification respiration with the supplement of potassium nitrate. In addition to that, many phylogenetic similarities and identical remarks were also observed between T. scotoductus SA-01 and the newly sequence Thermus sp. NMX2 A.1 strain such as the ones carried out using the BOX-PCR fingerprinting. Comparison was, therefore, carried out on genome level to verify the phylogenetic similarities; of which was seen that the two strains shared up to (81.6%) similarities. The remaining percentage represented a set of genes that where uniquely found in the newly sequenced strain such entailed the Calvin cycle. It is however, understandable as to why there might be differences between the two strains since the T. scotoductus SA-01 was isolated in the deep gold mines of South Africa and Thermus sp. NMX2 A.1 strain from the thermal hot springs of New Mexico in the USA. As a result; T. scotoductus SA-01 also indicated islands within its genome in which were not found in the newly sequenced strain. A detailed experimental procedure was carried out to further support the theoretical similarities of the two strains with the presence of their genes that participate within the nitrate operon. Their functionalities were therefore analyzed using the profile of the nitrite detection as initial step of nitrate reduction, of which they showed an identical profile from the beginning of the incubation period until the end. Lastly, with the advantages of a variety of fissure water samples collected from the different deep gold mines of South Africa and the Northam platinum mine a search for possible Thermus isolate was in addition performed. A range of different applications were implemented to obtain pure Gram negatives cells. This however, didnât successfully yield the expected results; even though there were numerous indications that the Gram negative cells where present and one could even hypothesize that those Gram negative cells were a genus Thermus since the ârotund bodiesâ characteristics were seen amongst these fissure water-samples. Unfortunately this directive was not achieved but the search continues to extend the known Thermus sp. isolations and exploration into their metabolic versatility.

Uranium (U) and chromium (Cr) in groundwater are a serious public health concern due to their chemical toxicity. Even so, microorganisms have developed mechanisms which permit them to thrive under previously perceived uninhabitable conditions. A number of bacteria have been isolated from areas impacted with the soluble heavy metals, and can be exploited as bioremediation agents since they are well adapted to these metals. To date, the use of microbial mechanisms for bioremediation processes is a growing industry since it provides green and sustainable technologies. In this study, the upflow bioreactors were used as a low cost, low maintenance effective bioremediation strategy in comparison to the available methods of remediation. Two metals known to be toxic in their soluble state were treated. The first was Cr(VI) from an impacted site in Limpopo and the second was U(VI) from the Wonderfonteinspruit catchment, North West Province. The system was efficient for the removal of soluble Cr(VI) and U(VI) from the impacted water through biostimulation of indigenous bacterial communities. This system can be up scaled and employed for the remediation of impacted sites, and it will be useful especially at low levels of U(VI). Indigenous bacterial community from impacted sites have the capability to reduce Cr(VI) and U(VI) effectively over a sustainable period. The shortage of electron donor and continuous oxygen exposure in the case of U(VI) act as a limiting factor. However, in this study successful Cr(VI) and U(VI) reduction rates were increased by the addition of an electron donor to stimulate the indigenous bacterial community. Furthermore, a third upflow bioreactor showed that it is even possible with gradual increases of U(VI) concentration that U(VI) bioreduction is possible at very high levels. The influent water was spiked step wise with uranyl acetate, allowed to reach maximal U(VI) reduction/removal and then the diversity was assessed. Despite the 10 mg/l U(VI) fed to the bioreactor, the established microbial community was able to tolerate, adapt and thereby remove the U(VI) from the spiked water. Even though biofilm could not sturdily adhere to the matrix from the bioreactor, high levels of U(VI) removal could be achieved and the planktonic community maintained. No biofilm could be observed from SEM analysis from the TEM it was observed that the planktonic microbial community have an interaction with uranium. Since no U(VI) could be detected from the effluent samples, it is thus postulated the uranium in contact with the microbial cells is in another form, probably U(IV) as previously shown in this laboratory. This study allows for the understanding of the metal microbe interactions in impacted environments, the use of this biome to remediate the water in an effective, low cost and maintenance bioreactor.

Prof. S. Kruger

Thesis (Ph. D., Microbiology, Molecular Biology and Biochemistry)--University of Idaho, September 2006. / Major professor: Gregory Bohach. Includes bibliographical references. Also available online (PDF file) by subscription or by purchasing the individual file.