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.

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 (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.