A series of investigations were conducted in order to characterise the GIT microbiota of salmonids and to determine the effect of microbial modulating feed additives on the intestinal microbiota, immunity and growth of salmonids. The first experiment, Chapter three, used PCR-DGGE and 16S rRNA gene sequence analysis of cultivable bacteria were used to investigate the GIT microbiota of brown trout. 16S rRNA gene sequence analysis demonstrated that Citrobacter freundii and Carnobacterium maltaromaticum were the predominant culturable viable bacteria and lactic acid bacteria, respectively in all regions of the GIT. DGGE revealed complex communities with a diverse range of microbes from the Firmicutes and Proteobacteria phyla. The latter chapters focused not only identifying the gut microbiota of salmonids, but also on the ability of probiotics and prebiotics to modulate these communities. In Chapter four, rainbow trout were fed a commercial diet supplemented with P. acidilactici for four weeks. P. acidilactici was detected in the GIT of the probiotic group by multiple methods and P. acidilactici was able to persist for at least 24h at the cessation of probiotic feeding. Histological appraisal on the intestine revealed significantly higher microvilli density in the posterior mucosa and a higher density of goblet cells in the anterior mucosa of the probiotic fed fish. RT-PCR results demonstrated that IL-1β, IL-8 and IgT gene expression were up-regulated in the P. acidilactici fed fish at the end of the study. Whilst mRNA of PCNA, HSP70 and casp-3 were down-regulated in the probiotic group at both sampling points. In Chapter five, the efficacy of dietary administration of P. acidilactici and short chain fructooligosaccharide (scFOS) on Atlantic salmon (Salmo salar L.) was evaluated at 63 and 132 days. Compared to the control group, total bacterial cell counts in all regions of the intestine with exception of the anterior digesta were significantly lower in the synbiotic group at the mid sampling point. PCR-DGGE revealed that species richness, diversity and the number of OTUs were significantly higher in the synbiotic group in the anterior digesta at the mid sampling point. Intestinal microvilli and villi length were increased in the anterior intestine of the synbiotic fed group at the end sampling point. IEL levels were increased in the synbiotic group in the posterior intestine at both sampling points. The expression of immunological genes were significantly up-regulated in the synbiotic fed salmon. In Chapter six, rainbow trout were fed three diets fishmeal (FM), soybean meal (SBM) and PlantMix diets supplemented with or without P. acidilactici for 12 weeks. At both sampling points, with exception of fish fed FM, LAB levels were significantly higher in all probiotic groups compared to the control groups. Serum lysozyme activity was significantly higher in fish fed FM and SBM diets containing P. acidilactici than that of fish fed the control diets. This body of research demonstrates that P. acidilactici can modulate immune response via up-regulation of immune genes as well as modulate IEL and goblet cell levels. Despite these benefits, P. acidilactici had no detrimental effects on growth performance.

Only a few studies have looked at microbial biogeography in soils and whether microorganisms are endemic to an area is still debatable. Tramway Ridge, a geothermal area on Mount Erebus, Antarctica, provides a unique opportunity due to its isolation and extreme conditions to explore the possibilities of microbial endemism and to identify novel Bacteria and Archaea. This site was chosen for a culture-independent study with a preliminary culturing survey for bacterial communities along three temperature gradients (65 C - 2.5'C). In addition, a physico-chemical analysis was undertaken to identify which environmental factors were driving the different diversity along the transects. An automated rRNA intergenic spacer analysis (ARISA) was used to assess the diversity across the transects using Bacteria and Cyanobacteria-specific primers and results showed that temperature and pH were the main drivers for these communities. Due to its unique physico-chemical and ARISA profile, a hot temperature site (T-3A, 65'C) was chosen for further investigation by bacterial and archaeal 16S rDNA clone libraries. Unique rDNA types among the 78 bacterial and 83 archaeal clones were identified by restriction fragment length polymophisms and 18 bacterial and 5 archaeal operational taxonomic units (gt97% identity) were observed. All of the bacterial sequences were deeply branching and loosely affiliated with other recognised bacterial divisions, with 40% of the sequences not affiliated to any genus. The archaeal clones were found to be deep-branching and sequences clustered together within the Crenarcaheota. In addition, two strains of Bacilli were isolated. The novel microorganisms show that the Tramway Ridge communities are unique from organisms found in other environments and show that quotEverything is (not) everywherequot.

At the commencement of this study, some sensitive overseas markets were rejecting chilled vacuum-packed New Zealand lamb due to higher than expected total viable counts, and counts of Enterobacteriaceae, a family of bacteria used to indicate sanitary condition. Of the many factors that influence the bacterial composition of chilled lamb in the overseas marketplace, the meat producer can only exert significant control over: Hygiene, ensuring the bacterial viable count on the meat prior to packaging is as low as possible, and comprised of as few species as possible that are capable of anaerobic growth at chilled meat temperatures. Maintaining the pH of the meat within acceptable limits, by careful animal selection and minimal pre-slaughter stress. Refrigeration temperatures, through rigorous maintenance of the cold-chain. The type of preservative packaging used, which is often limited by regulation in the marketplace. Initial work established that the bacterial microbiota present on the meat contact surfaces in the butchering facilities at some premises, in particular conveyor belting, was excessive and comprised of species that contributed to the high counts on the meat reported above. As a means of improving the hygiene of this process, this study investigated the hypothesis that some species of bacteria were able to form biofilms on the conveyor belt contact surfaces, becoming reservoirs for cross-contamination. This hypothesis was not been proven by this work; the results showing that biofilms were not present and that adequate hygiene of these surfaces instead depends on the ability to remove all meat-based residues from them at the completion of each day's processing. For premises operating interlocking belts from one manufacturer (Intraloxreg), a clean-in-place system is now available that is able to achieve this. Premises operating conventional disinfectant and water sanitisation of either continuous or interlocking belts must ensure that meat residue is completely removed before disinfection. The majority of New Zealand meat industry premises can now demonstrate that their hygienic processes in this area are under control. The microbiota of conveyor belting in this study was found to consist of bacteria from five taxonomic groups; the Flavobacteriaceae, the Actinomycetales, the Bacillus/Clostridium group, and the alpha and gamma branches of the Proteobacteria. The genera present on belts from premises whose hygiene was found to be in control did not contain species known to cause food-borne disease or spoilage of vacuum packaged meats. The bacterial viable count remains the most effective method available at this time for monitoring conveyor belt hygiene. Attempts to develop a monitoring system based on microscopy of an in-situ sampling device were unsuccessful due to an inability to penetrate the meat residue matrix. Denaturing Gradient Gel Electrophoresis (DGGE) may offer an alternative for rapid investigation of diversity, but further work is required before this can be validated for routine use.

Thesis (Ph. D.)(Science of Religion and Missiology))--University of Pretoria, 2000. / Includes bibliographical references.

>Magister Scientiae - MSc / The extreme conditions of Antarctic desert soils render this environment selective towards a diverse range of psychrotrophic microbial communities. Cracks and fissures in translucent quartz rocks permit an adequate amount of penetrating light, sufficient water and nutrients to support cryptic microbial development. Hypolithons colonizing the ventral surface of these quartz rocks have been classified into three types: cyanobacterial dominated (Type I),moss dominated (Type II) and lichenized (Type III) communities. Eukaryotic microbial communities were reported to represent only a minor fraction of Antarctic communities. In this study, culture independent techniques (DGGE, T-RFLP and clone library construction) were employed to determine the profile of the dominant eukaryotes, fungi and microalgae present in the three different hypolithic communities. The 18S rRNA gene (Euk for eukaryotes), internal transcribed spacer (ITS for fungi) and microalgal specific regions of the 18S rRNA gene, were the phylogenetic markers targeted for PCR amplification from hypolith metagenomic DNA. Results suggest that the three hypolith types are characterized by different eukaryotic, fungal and microalgal communities, as implied by nMDS analysis of the DGGE and T-RFLP profiles. Sequence analysis indicates close affiliation to members of Amoebozoa, Alveolata, Rhizaria (general eukaryote), Ascomycota (fungal) and Streptophyta (microalgal). Many of these clones may represent novel species. This study demonstrates that Dry Valley hypolithons harbour higher eukaryote diversity than previously recognised.Each hypolithon is colonized by specialized microbial communities with possible keystone species. The ecological role of the detected microorganisms in the hypolith environment is also theorized, and a trophic hierarchy postulated.

The nature and extent of microbial biodiversity remain controversial with persistent debates over patterns of distributions (i.e. cosmopolitanism vs. endemism) and the processes that structure these patterns (neutrality vs. selection). We used culture-independent approaches to address these issues focusing on two groups of ciliates, the Oligotrichia (Spirotrichea) and Choreotrichea (Spirotrichea). To assess the diversity of these ciliates, we designed primers specific to SSU rDNA of ciliates within these clades, and investigated (1) geographic and temporal distributions along three coastal sites in the Northwest Atlantic; (2) the relationship between ciliate communities in the benthos and the plankton along the New England coast; and (3) diversity in ciliate communities across an environmental gradient at six stations in Long Island Sound spanning the frontal region that separates the fresher Connecticut River outflow plume from the open Sound. Each collection had its own distinct assemblage of rare and abundant ciliate haplotypes, and genealogical analyses of our samples combined with published sequences from identified morphospecies reveal that haplotype diversity at these sites is greatest within the genus Strombidium, in the Oligotrichia. Clustering of phylogenetic types indicates that benthic assemblages of oligotrichs and choreotrichs appear to be more like those from spatially distinct benthic communities than the ciliate communities sampled in the water above them. Neither ciliate diversity nor species composition showed any clear relationship to measured environmental parameters (temperature, salinity, accessory pigment composition, and chorophyll), although we observed that diversity decreased moving from nearshore to offshore. We find no strong fit of our communities to log series, geometric, or log normal distributions, though one of the 3 clusters is most consistent with a log series distribution. These analyses suggest that Oligotrich and Choreotrich communities in coastal environments may be distributed in a neutral manner. We investigated the effectiveness of molecular approaches in characterizing ciliate diversity in our samples. Estimates of diversity based on molecular markers are similar to estimates from morphological observations for Choreotrich ciliates, but much greater for Oligotrich ciliates. Sediment and plankton subsamples differed in their robustness to repeated subsampling. Sediment gave variable estimates of diversity while plankton subsamples produced consistent results.

No description available.

Thesis (MSc (Microbiology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The relationship between various land management practices, soil properties and the soil microbial communities are complex and little is known about the effect of these interactions on plant productivity in agricultural systems. Although it would be advantageous to have a single organism or property that can be used as a measure of soil health, it may not be possible. Soil organisms which include both the microorganisms as well as soil fauna are subjected to the effect of their immediate environment. This microenvironment in turn is determined by the soil properties as well as above ground flora and their interactions. Most soil indicators interact with each other, and these interactions can modify or influence the soil properties. The complexities of the interactions between critical soil indicator values often preclude its practical use by land managers and policy makers. However, soil microbial communities (e.g. diversity and structural stability) may serve as a relative indicator of soil quality. These communities are sensitive to land management practices and changes in the microenvironment. The objective of this study was to gain an understanding of the complex relationships by investigating the effect of conventional, integrated and organic apple production systems on the physical, chemical and biological (particularly soil microbial diversity) properties of the soil. Automated Ribosomal Intergenic spacer analysis (ARISA) was used to characterise fungal (F-ARISA) and bacterial (B-ARISA) communities from soil samples obtained from an experimental apple orchard in Elgin, Grabouw. The intergenic spacer (ITS) region from the fungal rRNA operon was amplified using ITS4 and fluorescently FAM (6- carboxylfluorescein) labelled ITS5 primers. Similarly, the 16S-23S intergenic spacer region from the bacterial rRNA operon was amplified using ITSR and FAM-labelled ITSF primers. The sensitivity of the technique allowed us to discriminate between the soil microbial communities of the different treatments. From our results we observed significant increase (p < 0.05) in the fungal community diversity between the February and April samples, while the bacterial community diversity was consistent (p > 0.05). Also, treatments with mulch showed a significantly higher microbial diversity than the other treatments at a 5 % significance level. Fungal communities showed significant correlation with the potassium concentration in the soil, while bacterial communities depicted a significant correlation with the soil phosphorous concentration. Based on the results we concluded that different management practices have a significant effect on the soil microbial communities and that these communities are particularly sensitive to small changes in the environment. However, there is still a need to determine what the composition of the soil microbial communities are to be able to correlate our observations with soil health. / AFRIKAANSE OPSOMMING: Die verhouding tussen verskillende landboubestuurspraktyke, grondeienskappe en die mikrobiese gemeenskappe in grond is kompleks en weinig is bekend oor die uitwerking van hierdie interaksies op die produktiwiteit van landboustelsels. Alhoewel dit voordelig sou wees om ‘n enkele organisme of eienskap te kan hê wat die gesondheid van grond kan meet, sal dit dalk nie moontlik wees nie. Grondorganismes wat die mikroörganismes sowel as die grondfauna insluit, is onderworpe aan die invloed van hulle onmiddelike omgewings. Hierdie mikro-omgewings op hulle beurt word weer beïnvloed deur die grondeienskappe sowel as die die oppervlak flora en hulle wisselwerkinge. Meeste van die grondaanwysers toon ook wisselwerkinge met mekaar, en hierdie wisselwerkinge kan die grondeienskappe beïnvloed or selfs verander. Die kompleksiteit van die wisselwerkinge tussen kritiese grond aanwysers is meestal die rede waarom dit nie deur grondbestuurders en beleidsmakers gebruik word nie. Dit is ongeag die feit dat grond mikrobiese gemeenskappe (bv. diversiteit en stukturele stabiliteit) mag dien as ‘n relatiewe aanwyser van grondkwaliteit. Hierdie gemeenskappe is sensitief vir bestuurspraktyke en veranderinge in die mikro-omgewing. Die doel van die studie was om die ingewikkelde verhoudings in die grondgemeenskappe te bestudeer en die uitwerking van konventionele, geïntegreerde en organiese appel produksie sisteme op die fisiese, chemiese en biologiese eienskappe (veral die grond mikrobiologiese diversiteit) te bepaal. Geoutomatiseerde Ribosomale Intergeniese Spasie Analise (ARISA) is gebruik om die fungus (F-ARISA) en bakteriese (B-ARISA) gemeenskappe van grondmonsters wat vanaf ‘n proef appelboord in Elgin (Grabouw) verkry is, te bepaal. Die intergeenspasie (ITS) area van die fungus rDNA operon is vermeerder deur die ITS4 en fluoresserende FAM (6-karboxylfluorescein) gemerkte ITS5 inleiers te gebruik. Soortgelyk is die 16S-23S intergeenspasie area van die bakteriese rDNA operon vermeerder deur ITSR en FAM-gemerkte ITSF inleiers te gebruik. Die sensitiwiteit van die tegniek laat ons toe om te onderskei tussen die grond mikrobiese gemeenskappe vanaf verskillende grondbehandelings. Vanuit die resultate kon ons aflei dat daar ‘n toename (p < 0.05) in die fungus gemeenskap diversiteit vanaf Februarie to April was terwyl die bakteriese gemeenskap ‘n konstante diversteit getoon het (p > 0.05). Behandelings met grondbedekking het ook ‘n beduidend hoër mikrobiese diversiteit getoon as ander behandelings. Fungus gemeenskappe het beduidende korrelasies getoon met kalium konsentrasies in die grond, terwyl bakteriese gemeenskappe ‘n beduidende korrelasie getoon het met grond fosfor konsentrasies. Gebaseer op die resultate kon ons aflei dat verskillende bestuurspraktyke ‘n uitwerking kan hê op die grond mikrobiese gemeenskappe en dat hierdie gemeenskappe sensitief is vir klein veranderinge in die omgewing. Dit sal egter nog nodig wees om die spesifieke samestelling van die grond mikrobiese gemeenskappe te bepaal voor ons hierdie waarnemings kan korreleer met grondgesondheid.

M. Tech. (Department of Biotechnology, Faculty of Applied and Computer Science), Vaal University of Technology| / Pigeon droppings, found in abundance in most cities and towns where pigeons are found, are a source of potential yeast and molds into the environment. Invasive fungal infections are a cause of morbidity and often mortality in immunocompromised individuals. The objective of this study was to the identification of bacterial and mold agents from pigeon droppings. Pigeon droppings samples were collected from three locations during the winter and summer months and studied for the occurrence of bacteria, yeast and molds by utilising culture-independent techniques. Amplification of the 16S rDNA gene and the internal transcribed spacer (ITS) region, cloning and ARDRA and DGGE were used for the characterisation of the microbial populations followed by sequencing. Several mold and yeasts, as well as bacteria were found to be present in pigeon droppings, which can spread into the environment and be transmitted to immunocompromised individuals and children. DGGE analysis of the bacterial communities revealed banding patterns that clustered all but one winter samples and all summer samples, showing a high similarity among the microbial members in both seasons and sample locations. Fungal DGGE analysis revealed clusters that grouped summer and winter samples from Johannesburg and Pretoria while VUT samples were clustered on their own. From the identification of fungal and bacterial DNA, Cryptococcus species was the majority of fungi isolated from the dropping samples. Geotrichum, Kazachstania and Fusarium species were isolated from phylotypes obtained from ITS amplicons analysed by ARDRA. Lactobacillus and Enteroccoccus species, organisms usually found in the gastrointestinal tract were the common bacterial members identified. The results showed no difference in microbial communities across all sample locations, while seasonal changes also had no impact in microbial community patterns.

O objetivo deste trabalho foi avaliar o efeito da substituição total ou parcial do leite (M-x1) por extrato de soja (S-x2) e/ou WPI combinado com inulina (PI-x3) em formulações de sorvete simbiótico de maçã sobre a viabilidade e sobrevivência de L. acidophilus La-5 e B. animalis Bb-12 através de condições gastrointestinais simuladas in vitro e monitorar mudanças morfológicas das células durante períodos de estresse gastrointestinal em uma das formulações, bem como investigar características tecnológicas (overrun, taxa de derretimento e dureza instrumental) e aceitação sensorial. Para este fim, um desenho experimental para misturas simples (simplex-centroid) foi utilizado, incluindo um ponto axial, utilizando diferentes proporções de x1, x2, x3. Foram estudadas oito formulações de sorvete simbiótico de maçã durante o armazenamento congelado por até 84 dias. Os sorvetes mostraram-se satisfatórios como veículos para La-5 e Bb-12 (cerca de 7,5 log ufc/g), mesmo ao final do período de 84 dias de armazenamento a -18 °C. Em todas as formulações, a análise por qPCR combinada com o uso de propidium monoazide (PMA-qPCR) evidenciou que os probióticos resistiram ao ensaio in vitro, com níveis significativos de sobrevivência, atingindo taxas de sobrevivência superiores a 50%. Além disso, imagens de microscopia eletrônica de varredura evidenciaram células com diferenças morfológicas, sugerindo alterações fisiológicas em resposta ao estresse induzido durante o ensaio in vitro. Um modelo linear e um especial cúbico foram obtidos para os parâmetros de overrun e de dureza, respectivamente, mostrando que os fatores leite, extrato de soja e isolado proteico de soro + inulina influenciaram fortemente esses parâmetros tecnológicos. No entanto, a taxa de derretimento foi pouco afetada por tais fatores. O modelo especial cúbico mostrou que a interação binária (S-PI) e ternária (M-S-PI) resultou em maiores valores de dureza. Para este parâmetro específico, a combinação recomendada dos ingredientes é de 3,7% de leite em pó, 1,3% de extrato de soja e 5,0% PI. Os escores médios de aceitabilidade sensorial variaram de 5,6 a 8,0. A aceitabilidade geral do sorvete produzido apenas com o PI foi menor quando comparada a ao sorvete de leite (M), embora uma grande frequência de escores tenha sido observada na área da escala compreendendo as opiniões \"gostei ligeiramente\" (escore 6) e \"gostei extremamente\" (escore 9) para todas as formulações de sorvete avaliadas. Apesar de todas as formulações terem conferido resistência às cepas sob estresse, a variação encontrada na sobrevivência de La-5 e Bb-12 sugere que a tolerância é, de fato, afetada pela escolha da matriz alimentar. Os resultados também mostraram que, além de ser um alimento nutritivo, as formulações de sorvete que tiveram a substituição parcial do leite em pó poderiam assegurar aos consumidores um produto funcional com baixo teor de lactose, enquanto sorvetes sem leite em pó oferecem um alimento funcional sem lactose. / The aim of this study was to assess the effect of the total or partial replacement of milk (M-x1) by soy extract (S-x2), and/or WPI combined with inulin (PI-x3) in synbiotic apple ice cream formulations on the viability and survival of L. acidophilus La-5 and B. animalis Bb-12 through in vitro simulated gastrointestinal (GI) conditions and to monitor cells morphological changes during gastrointestinal stress in one of the formulations, as well as investigating technological features (overrun, meltdown rate, instrumental hardness) and sensory acceptability. For this purpose, an experimental design for simple mixtures (simplex-centroid) was used including an axial point, using different proportions of x1, x2, x3. Eight formulations of synbiotic apple ice cream were studied during frozen storage for up to 84 days. The ice creams showed to be satisfactory vehicles for La-5 and Bb-12 (populations around 7.5 log cfu/g), even after the whole storage period (84 days/-18°C). In all formulations, the propidium monoazide qPCR (PMA-qPCR) analysis evidenced that probiotics could resist to the in vitro GI assay, with significant survival levels, achieving survival rates exceeding 50%. Additionally, scanning electron microscopy images evidenced cells with morphological differences, suggesting physiological changes in response to the induced stress during the in vitro assay. A linear and a special cubic models were obtained for the overrun and hardness parameters showing that factors milk, soy extract, and whey protein isolate + inulin strongly influenced these technological parameters. However meltdown rate was slightly affected by the factors studied. The special cubic model showed that the binary (S-PI) and tertiary (M-S-PI) interactions resulted in higher hardness values. For this specific parameter, combination of the factors is recommended to be 3.7% milk, 1.3% soy extract, and 5.0% PI. The sensory acceptability mean scores ranged from 5.6 to 8.0. The overall acceptability of the ice cream produced only with PIn was lower when compared to milk based ice cream (M), even though a high frequency of scores was observed in the scale area comprising the opinions \"liked slightly\" (score 6) and \"liked extremely\" (score 9) for all ice cream formulations evaluated. Although all formulations provided resistance to the probiotic strains under GI stress, the variation found in La-5 and Bb-12 survival suggests that GI tolerance is indeed affected by the choice of the food matrix. The results also showed that, besides being a nutritious food, the ice cream formulations that had the partial replacement of milk powder could assure consumers a functional product with low content of lactose, whereas ice creams with no milk powder offer a lactose-free functional food.