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    Further development of a cell culture based approach to model the diet-derived impacts on the faecal microbiome and potential host health in the domestic dog : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (Ph.D.) in Animal Science at Massey University, Palmerston North, New Zealand
    (Massey University, 2023) Phimister, Francis
    Globally, diets that promote and optimise health and a ‘healthy microbiome’ are becoming increasingly popular for pets. However, the impacts of novel diet ingredients and formulations on the health of the host and their microbiome require testing to ensure there are no unforseen detrimental effects. However, there are currently a very limited number of canine-specific intestinal cell lines and the capacity to utilise these cells to model host-food interactions is limited. Thus, this doctoral project aimed to develop an in vitro model of the canine intestine, using a previously established canine intestinal epithelial cell (cIEC) line. This could then be used to characterise the canine response to dietary challenges to the gut microbiota. As there is limited research that has assessed the interactions of the gut microbiota with the canine intestine, the initial step of this project was to evaluate the current knowledge of the intestinal inflammatory response to bacterial ligands and diet-derived metabolites (Chapter Two). This literature review indicated that prior to investigating the interactions between bacteria and the canine intestine an evaluation of the canine intestinal response to these challenge compounds was required. Building on the knowledge base established in Chapter Two, key microbes associated with health in the dog required identification. Thus, this thesis provided the first meta-analysis of the available literature on the relationship of dietary nutrients and their impact on the gut microbiota in the dog (Chapter Three). The hypothesis of this meta-analysis was that dietary protein and dietary fats would have singificant impacts on the faecal microbiota of the dog and additionally, that this analysis would reveal bacterial genera associated with these dietary macronutrients.In the meta-analysis the novel discovery was made that despite its low relative abundance,Sharpea was the genera most associated with causing the shifts in microbial profiles in response to changes in both crude protein, and crude fats, thus confirming the hypotheses. Early results indicated that the methods required to further refine the existing cIEC line as an in vitro model of the canine intestine were sub-optimal and required further development. These method developments are detailed in Chapter Four. Experiments in this chapter assessed methods to promote cell growth and differentiation in a cellZscope system, which automatically performed barrier integrity assessments, whilst inside a temperature-controlled incubator. The inclusion of 150 nM hydrocortisonein cell culture media during the initial 48 hours of cellular differentiation, and subsequent removal of hydrocortisone after this period successfully enabled the cIEC to differentiate and form confluent monolayers in the cellZscope system. These methods were intended to be used going forwards in an apically anaerobic system that would more closely resemble conditions seen in vivo and would have allowed the simultaneous culture of the oxygen-requiring cIEC and anaerobic bacteria. Work utilising this model was stopped due to complications that arose from the Covid-19 pandemic, but work conducted and experiments that were planned are explored in Chapter Six. Utilising the refined methods from Chapter Four, the inflammatory response of the cIEC to butyrate and bacterial lipopolysaccharides (LPS) were characterised (Chapter Five). This was performed to address gaps in the literature highlighted in Chapter Two.It was hypothesised that the stimulation with bacterial LPS would cause a pro-inflammatory response, whilst the stimulation with butyrate would cause an anti-inflammatory response. Furthermore, it was also hypothesised that the stimulation of the cIEC with both LPS and butyrate would cause the butyrate to reduce the pro-inflammatory response, and the LPS to reduce the anti-inflammatory response. It was observed that LPS induced a pro-inflammatory response in the cIEC, which butyrate was able to mitigate in most instances. Overall, the methods developed and refined in this project will be able to be utilised in future experiments utilising these cells, such as evaluating new pet food ingredients for beneficial effects and exploring how changes in the gut microbiome impact gut health in the dog. It can take the knowledge established in Chapter Three to further investigate the impacts of the bacterial genera on the health of the dog. Futhermore, it can utilise the immune responses observed in Chapter Five to better understand the relationship between inflammation and diet in the dog. Future work can build on the knowledge discovered and presented in this thesis to fully understand the impact of diet changes on the health of the dog, and further define the microbial profile of the ‘healthy microbiome’ for the dog
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    Methodology of culture maintenance and inoculum development for production of solvents by Clostridium acetobutylicum : a thesis presented in partial fulfilment of the requirements for the degree of Master of Technology in Biotechnology at Massey University
    (Massey University, 1985) Gutierrez, Noemi A
    Various methods of culture maintenance and inoculum develop­ment were evaluated for their effectiveness in conserving and improving the property of 2 strains of Clostridium acetobutyicum, namely NCIB 2951 and NRRL B-594, to produce solvents by fermentation of whey permeate. The majority of the methods were effective in maintaining the viability and solventogenic property of the organism. However, since in some cases the viability was maintained but the solventogenic property was not, it is clear that the latter should be used as the index in determining the storage life and time of reprocessing of the stock culture. The methods of culture maintenance investigated included refrigeration at 4°c in distilled water, in phosphate buffer and in Cooked Meat Medium containing glucose ( CMMG) ; by freezing at -20°c in distilled water and in phosphate buffer; by drying in soil and by lyophilization ( freeze drying); and by periodic transfer in CMMG and in whey permeate containing yeast extract. Maintenance of the stock cultures at -20° C in distilled water was found to be the most efficient for the storage stability of both strains of organism. The viability and the potential to produce high solvent concentrations, primarily butanol were maintained without any significant loss after 9 months and 12 months, for strain NCIB 2951 and strain NRRL B-594, respectively. The criteria important for a commercial fermentation, i.e., sugar utilization, yield and butanol production rate, remained stable during storage by this method. It was observed that periodic transfer was a poor method as the culture lost their solventogenic property despite remaining viable. The other preservation methods were not as satisfactory as freezing in distilled water at -20°c since the fermentation ability degenerated to some extent after 9 months of storage. Therefore, after such a period reprocessing of the stock cultures kept by these methods is necessary to revive the cultures and minimize degeneration. The repeated use of the stock cultures was found to be deleterious and should be avoided. The inoculum development procedure investigated to maximize fermentation efficiency included the conventional heat shocking of the stock culture; variation in the number of culture stages; use of gassing as an index of transfer time; and the use of different levels of inoculum size. The strain differences which exist between NRRL B-594 and NCIB 2951 influenced how the inocula from these strains should be propagated prior to fermentation. Strain NRRL B-594 responded to heat shocking while strain NCIB 2951 did not. Neither ethanol nor butanol treatment of the stock cultures of the latter were advantageous. Using a 3-stage inoculum development procedure, the ferment­ation efficiency of strain NRRL B-594 was improved by employing heat shocking at 80C for 15 min in the revival stage of the stock culture. The germination factors for the spores of NCIB 2951 await identification. However, by using the presence of highly motile cells as an index in transferring from the revival stage, the inoculum develop­ment procedure resulted in a significantly higher butanol concentration value and production rate. Thus, the revival stage was the most critical.
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    A statistical approach to medium optimization for growth and toxin production by the bacterium Bacillus thuringiensis : a thesis presented in partial fulfilment of the requirements for the degree of Master of Technology in Biotechnology at Massey University, Palmerston North, New Zealand
    (Massey University, 1976) Tran, Quan Dat
    Bacillus thuringiensis is a Gram positive spore-forming bacterium. Knowledge of its pathogenicity against the larval stages of certain lepidopterous insects has been known for over 70 years. Ishiwata (1902) was the first to isolate it from dying silkworm larvae. Later Berliner (1915) isolated it from sick larvae of Anagasta kuhniella. He noted the existence of a parasporal body or "Rostkorper" in sporulated cells. These observations were confirmed by Mattes (1927), but it was not until 1953 that Hannay (1953) characterized the parasporal body as a crystal. The crystal has since been known to play a key role in the pathogenicity towards the most susceptible lepidopterous larvae. In recent years a considerable degree of interest has been aroused on the use of biological methods for insect control as against the use of chemical insecticides. Among many biological products considered as insect control agents, the crystal produced in the bacterium B. thuringiensis is one of the most hopeful. Industrial organisations in several countries are presently engaged in fundamental research and commercial scale production of insecticidal preparations based on this bacterium (Falcon, 1971; Pendleton, 1969). Apart from the crystal, which might be considered as an enterotoxin, a number of exotoxins are also known or postulated to be produced by B. thuringiensis. Heimpel (1967a) suggested the following nomenclature: δ -endotoxin, or the proteinaceous crystal; α-exotoxin, a lecithinase C or phospholipase C; β -exotoxin, a thermostable exotoxin or "fly factor"; γ -exotoxin (an enzyme that clears egg yolk agar, not yet identified). The toxicity for insects of the β -exotoxin and the δ -endotoxin has been substantiated, but the efficiency of the so-called γ -exotoxin has not been proved. Quite recently Krieg (1971) suggested that the α -exotoxin is not identical with lecithinase C. He concluded that the α -exotoxin is a thermosensitive exotoxin of proteinaceous character which is produced during growth phase by strains of B. thuringiensis and of B. cereus. Krieg (1970) also isolated a relative heat stable bacteriocin produced by B. thuringiensis which he called Thuricin. He suggested that this is a polypeptide and can cause inhibition of growth of Gram positive bacteria and antagonism between several strains of B. thuringiensis.[FROM INTRO]