Thesis (M.Sc.)--Memorial University of Newfoundland, 2002. / Includes bibliographical references.

Red mark syndrome (RMS) is a non-lethal skin condition, of unknown aetiology, affecting rainbow trout (Oncorhynchus mykiss) in the United Kingdom since 2003. It has now spread to 50% of the rainbow trout farms, resulting in great economic losses due to the downgrading of the product. There are also similar skin conditions in rainbow trout, for instance strawberry disease (SD) in the USA. As with RMS, the aetiological agent of this disease is also unknown. Several potential aetiological agents have been proposed, including a Rickettsia-like organism (RLO) in SD in the USA and Flavobacterium psychrophilum in RMS in the UK. The aim of the research presented here was to investigate the causative agent of RMS and to establish if there is a relationship between RMS and SD. An RLO was found to be associated with both RMS and SD-affected fish using immunohistochemistry (IHC) and polymerase chain reaction (PCR). The results of the IHC, together with the similarities in the pathology between the two conditions, suggest that RMS and SD are most likely the same disease (Chapter 2). In an attempt to isolate the RLO, F. psychrophilum or other suspected causative agents of RMS, several artificial bacteriological media, cell culture methods and novel techniques such as MagnaBind™ IgG beads (magnetic beads) were utilized. Although initial results appeared promising, no specific bacterial or viral agent was isolated using these methods. Transmission electron microscopy was used to analyse samples in an attempt to visualise any viruses and/or the RLO suspected of causing RMS (Chapter 3), but none were seen. Investigation into the involvement of both the RLO and F. psychrophilum in RMS using primary culture and IHC, together with the more advanced techniques of MALDI-TOF–MS and 16s rRNA gene sequencing, showed no association between F. psychrophilum and RMS. A quantitative PCR (qPCR), together with IHC, showed a positive correlation between the RLO and RMS-affected tissue, but this did not v distinguish between primary or secondary involvement of the organism. Results following analysis of samples using other assays, including ELISA and IHC, both using serum from naturally infected individuals, 16s rRNA gene PCR and bacterial isolation, were inconclusive, with methods requiring further optimisation for future use. The qPCR used in the study also needs to be fully optimised, as the results of a ring trial between three laboratories were considerably different (Chapter 4). Cohabitation challenges were conducted in the USA to investigate the involvement of the RLO in the early stages of SD. Clinical signs of SD were clearly evident in a small percentage of the cohabitated naïve fish. In most of these cases the DNA of the RLO could be detected, but again primary or secondary involvement could not be determined due to the small sample size (Chapter 5). In conclusion, the results from the analysis of samples by PCR, IHC with anti-F. psychrophilum PAbs, MALDI-TOF-MS and 16s rRNA gene sequencing indicate that F. psychrophilum is unlikely to be the causative aetiological agent of RMS. Although Koch’s postulates were not fulfilled, a strong correlation was obtained between the RLO and RMS-affected fish in the IHC, PCR and qPCR using RLO specific primers. It is unclear however, if the involvement of the RLO is as a primary or secondary pathogen. The RLO associated with RMS appears to have antigens in common with Piscirickettsia salmonis (from the results of the IHC), the causative agent of Salmon Rickettsial Syndrome, for which commercial vaccines are available, and should therefore be investigated as a form of mitigation for RMS, since the RLO has not yet been isolated and a traditional inactivated whole cell vaccine is not possible at this time. Efforts to isolate the RLO should continue and the involvement of other pathogens in RMS should be investigated further with new cutting edge techniques such as next generation sequencing or random multiplex (RT)-PCR to rule out viral involvement in the disease.

Thesis (M.S.)--Montana State University, 2004. / Title from PDF title page (viewed Feb. 11, 2005). Includes bibliographical references (p. 54-60).

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 110-124).

Thesis (M.S.)--Oregon State University, 2011. / Printout. Includes bibliographical references. Also available on the World Wide Web.

Rainbow trout represents the most prominent species in freshwater farming in UK aquaculture. One of the common diseases constraining rainbow trout production and increasingly causing problems in Atlantic salmon (Salmo salar L.) hatcheries worldwide is rainbow trout fry syndrome (RTFS) or bacterial cold water disease (BCWD). During the last 20 years, the development of a commercial vaccine against RTFS has been hindered by the prevalence of a wide range of the fish pathogen F. psychrophilum, thus the current treatment of choice is the use of antibiotics. Studies involved in understanding the innate and adaptive immune response of vaccinated rainbow trout fry using inactivated whole cell are still lacking. Therefore, the aim of this thesis is to characterise the strain diversity and antibiotic susceptibility of UK F. psychrophilum isolates, evaluate the efficacy of a whole-cell formalin-killed polyvalent vaccine, which was developed based on the characterisation results of this study, and investigate the immune response in trout fry following the immersion vaccination via the changes in expression of relevant immune genes. A total of 315 F. psychrophilum isolates, 293 of which were collected within the UK, were characterised using four genotyping methods and a serotyping scheme. A high strain diversity was identified among the isolates with 54 pulsotypes, ten (GTG)5-PCR types, two 16S rRNA allele lineages, seven plasmid profiles and three serotypes. The predominant profile observed within the F. psychrophilum isolates examined was PFGE cluster II – (GTG)5-PCR type r1 – 16S rRNA lineage II – serotype Th (n= 70/156, 45%). The characterisation results not only revealed the wide distribution within the UK and the persistence within a site of predominant pulsotypes, but also the presence of unique genotypes in certain sites or countries. Co-existence of genetically and serologically heterogeneous isolates within each farm was detected, highlighting the reasons this disease is so difficult to control, especially by vaccination. The occurrence over time of F. psychrophilum pulsotypes within a site could provide important epidemiological data for farm management and the development of site-specific vaccines. The antimicrobial susceptibilities of 140 F. psychrophilum strains, 125 of which were from the UK, were evaluated by the broth microdilution (MIC) and disc diffusion methods. There was evidence of reduced susceptibilities to three of the main antimicrobials used in UK aquaculture. Broth microdilution testing showed that only 12% of 118 UK isolates tested were WT to oxolinic acid (MIC COWT 0.25 mg L-1), 42% were WT for oxytetracycline (MIC COWT 0.25 mg L-1), and 66% were WT for amoxicillin. In contrast, all the isolates tested were WT (MIC COWT 2 mg L-1) for florfenicol, the antimicrobial of choice for RTFS control in the UK. Despite the imprecision of disc diffusion-based COWT values due to high standard deviations, there was a high categorical agreement between the classification of the strains (into WT or NWT) by MIC and disc diffusion methods for florfenicol (100%), oxolinic acid (99%), amoxicillin (97%) and oxytetracycline (94%). In general, this study showed that the UK F. psychrophilum isolates examined remain susceptible to florfenicol and also stresses the importance of performing susceptibility testing using standardised methods and COWT values. Several statistically significant associations between genotypes and the reduced susceptibilities of F. psychrophilum strains were revealed. A whole-cell formalin killed polyvalent vaccine against RTFS/BCWD was developed by combining three genetically and serologically divergent strains, recently collected from UK farms. The efficacy of this polyvalent vaccine was evaluated after immersion vaccination in 5 g trout and bath challenge using hydrogen peroxide as a pre-stressor with a virulent heterologous isolate of F. psychrophilum strain. Significant protection was achieved with an RPS of 84%. The combination of exposure to hydrogen peroxide prior to bath challenge may be an alternative to an injection challenge with 12 g trout, although further standardisation and optimisation of the challenge model is required. Changes in the innate immune response of trout fry following the initial vaccination included the up-regulation of the interleukin 1 β (IL-1β) gene in head kidney at 4 h and the up-regulation of toll-like receptor-2 (TLR-2) in skin at day 2. While the expression levels of C3 was unchanged, the down regulation of CD8-α in head kidney and spleen and CD4-1 in spleen were documented. IgM and IgT transcripts were found to be up-regulated in hind-gut two days post-vaccination. Understanding the strain diversity and the antibiotic susceptibility of UK F. psychrophilum isolates could help improve the control strategies, such as preventing the spreading of pathogenic F. psychrophilum clones between fish farms, reducing the use of antibiotics in RTFS/BCWD treatment and monitoring the development of acquired antibiotic resistance mechanisms. Moreover, strain characterisation data of UK F. psychrophilum species has assisted in selecting suitable candidates for developing an effective RTFS vaccine.

The study of the gut microbiota of fish began in the 1930’s and since that time a considerable amount of information has been collated on its composition and diversity. These studies have revealed that the microbial communities of the fish gastrointestinal tract are generally difficult to culture on bacteriological media and mainly consist of bacteria, archaea, viruses, yeasts and protists. The bacteria appear to be the most abundant of these microbial groups and their activity may have major implications for host health, development, immunity and nutrition. Therefore, much of the most recent published research has focused on developing improved methods of identifying the extent of the bacterial diversity within the fish gut and unravelling the potential influence of these microorganisms on the health of farmed fish species. However, whilst such studies have improved our knowledge of the dominant bacterial groups present in the rainbow trout gastrointestinal tract, the limited resolution capacity of many of the methods used has meant that our understanding of their baseline composition in healthy fish remains poorly understood. In this study, the bacterial communities that inhabit the intestine, now commonly referred to as the ‘microbiome’, of farmed Rainbow trout (Oncorhynchus mykiss) were characterized using a culture independent high-throughput molecular sequencing method. The microbiome of the intestinal lumen and mucosa was investigated to ascertain the true extent of the bacterial diversity present in this fish species prior to further experiments. It was found that the diversity of the intestinal microbiome was greater than previous studies had reported with a total of 90 and 159 bacterial genera being identified in both the lumen and mucosal regions respectively. The dominant bacterial phyla identified in both of the regions investigated were Proteobacteria, Firmicutes, Fusobacteria, Bacteroidetes and Actinobacteria. Furthermore, the data collected suggested that the intestinal microbiome may be similar in structure between individual fish, and illustrate the utility of next generation molecular methods in the investigation of the fish gut microbiome. A study was conducted to examine the effect of diet on the composition of the intestinal microbiome of rainbow trout. Two diets, one control and one treatment, were prepared which were identical apart from that the treatment diet contained a microalgal component at 5% of the total formulation. These diets were fed to rainbow trout for a total of 15 weeks. At the end of the trial period a total of 12 fish, three from each of four tanks, were sacrificed from each of the control and treatment groups and their intestinal tissue was sampled in order to compare the composition of the microbiome of both groups. The results revealed that both groups of fish shared similar microbiome compositions, with the Tenericutes being by far the most dominant phylum observed. The structure of the intestinal microbiome was not significantly different between both populations of trout tested. An increased level of bacterial diversity was noted in the treatment fish, however, this was not found to be statistically significant. A limited number of bacterial taxa were discriminatory between diets and were significantly elevated in the treatment group. These taxa were predominantly lactic acid bacteria of the genera Streptococcus, Leuconstoc, Lactobacillus, Lactococcus and Weissella. The results of this study suggested that the minor difference in the diets fed resulted in a correspondingly minor alteration in the intestinal microbiome of the tested rainbow trout. This may indicate that diet composition can modify the composition of the intestinal microbiome of these fish. A further study was conducted to investigate the structure of the intestinal microbiome from groups of fish reared in both freshwater cages and aquarium systems, in order to assess whether or not fish raised in different environments share similar microbiomes. This study also employed a novel computational tool, PICRUSt, to analyse the predicted functional capacity of the microbial communities of individual fish sampled from both environments. The data collected suggested that the structure of the intestinal microbiome was similar regardless of where the fish were raised, with the Tenericutes, Firmicutes, Proteobacteria, Spirochaetae and Bacteroidetes representing the dominant bacterial phyla recorded in the rainbow trout intestine. This suggests that the host may regulate the formation of the intestinal microbiome. A significant difference was however noted in community membership between the fish populations tested, which may point to an environmental influence on the intestinal microbiome. These data suggest that both deterministic host factors and stochastic environmental influences play important roles in shaping the composition of the bacterial communities in the intestine of these fish. The PICRUSt analysis revealed that gene pathways relating to metabolism, transport and cellular processes were enhanced in all of the fish studied, which may signal an involvement of these communities in the digestive processes of rainbow trout. In conclusion, this study used high-throughput sequencing methods in order to improve our understanding of the intestinal microbiome of farmed rainbow trout, and the effect of dietary and environmental factors on its composition. This research has generated scientific information relating to baseline bacterial community compositions in healthy fish, which may be used in future experiments including screening these baselines against the effects of novel aquafeed formulations, environmental perturbations or pathogenic challenges.

The survival and physiology of rainbow trout in alkaline waters was the focus of this thesis. It is known that salmonids have problems with ammonia excretion in alkaline water (Cameron and Heisler, 1983; Wright and Wood, 1985). The first set of studies attempted to increase the survival rates of rainbow trout planted into alkaline lakes by acclimating them to the alkaline conditions before their release. The first field study acclimated rainbow trout to alkaline waters by acidifying the lake water with C0₂ in order to reduce the magnitude of the pH change experienced by the fish. The second field study acclimated rainbow trout to alkaline waters by increasing the alkalinity of the hatchery water in which the fish were held, over a six day period. In both studies the acclimated fish experienced higher survival rates relative to non-acclimated fish. Plasma sodium concentrations ([Na⁺]p1) of the fish were shown to increase, while plasma chloride concentrations decreased. These changes were attributed to an increase in the exchange of external Na⁺ with endogenous H⁺, and the decrease in the exchange of endogenous HC0₃⁻ with external Cl⁻, respectively. The increased [Na⁺]pl may have also been the result of the exchange of plasma ammonium (NH₄⁺) with external Na⁺. The second set of studies investigated the physiological response of rainbow trout to alkaline waters. The first study, the chronic exposure of rainbow trout to alkaline water, showed that trout in hard alkaline water experienced higher survival rates and regulated plasma ammonia and ion concentrations more competently than trout in soft alkaline water. This increased ability to regulate plasma ammonia and ion concentrations was attributed to the possible "reactivation" of the Na⁺/NH₄⁺ exchange. The purpose of the second study, the acute exposure of rainbow trout to alkaline water, was to further investigate the mechanisms that enable fish in hard alkaline water to survive better than fish in soft alkaline water. The possible activity of the Na⁺/NH₄⁺ exchange was again observed in the hard alkaline water. The addition of amiloride to the alkaline hard treatment water increased plasma total ammonia and stabilized [Na⁺]pl′, which supported the "reactivation" of the Na⁺/NH₄⁺ exchange in hard alkaline water. As a result of these studies, the acclimation of rainbow trout to hard alkaline water before being planted into any alkaline body of water was recommended. / Land and Food Systems, Faculty of / Graduate

This study examines three possible methods for improving the digestibility and bioavailability of zinc to rainbow trout (Oncorhynchus mikiss). The first method was to examine the availability of the zinc utilisation from commonly used protein sources; the second was to assess the efficacy of the upstream use of an enzyme treatment of the raw materials; the third was to assess the use of organically complexed mineral supplements as opposed to the inorganic salts widely used at present. The first section indicated that the zinc from the soyabean meal was the most available (49.4%). The zinc digestibilities of the animal based protein used in this current study were 15.1% for LT94 fish meal, 26.6% for the Provimi 66 white fishmeal and 15.8% for poultry meat meal. The zinc in the maize/corn gluten meal was 31.9% digestible and from the NuPro 26.1%. Gram for gram maize gluten meal supplied the least amount of zinc to the fish (3.66 mg per kg). Based on these results the diets for the subsequent supplementation trials were formulated. The liver, eye and caudal fin were identified biomarkers of a severe zinc deficiency. The second part of the study revealed a soybean product, treated by exogenous enzymes, had a higher phosphorus digestibility, (49.0%, vs. 36.6%) and zinc digestibility (30.7% vs. 7.9%) The treatment did not improve the protein digestibility (85%). The third part of the study showed the organic source proved more digestible than the inorganic, 37.4% and 26.9% respectively. The fish fed the organic source maintained a higher level of zinc in both the eye and caudal fin. The liver zinc levels were unaffected by both dietary level and zinc source. Analysis of the liver for a zinc dependant protein showed that under stress conditions only the organic supplemented fish were able to synthesis this protein. The analysis of the mRNA levels coding for this protein indicate the fish on both zinc forms up regulated the production of the mRNA to the same extent when stressed. Finally this study also examined the viability of using a stable isotope to identify different ‘preferences’ for one form of supplementation over the other in different tissues. This method illustrated a tissue dependant difference to how the fish attempted physiologically to compensate for zinc deficiency. The rate of turnover was fastest in the liver, then the caudal fin and then the eye, and also showed that when the diet was more deficient there was an increased ability for the tissues to take up the organic form.

Research report presented in partial fulfilment of the requirements for the degree of Master of Business Administration at the University of Stellenbosch / Thesis (MBA (Graduate School of Business))--Stellenbosch University, 2011. / ENGLISH ABSTRACT: The purpose of the study is to investigate the feasibility and sustainability of producing biodiesel from Jatropha, which contains oil in its seeds by using the Rainbow Biotech economic intervention or also known as the Distributed Food and Fuel Plantation (DFFP) model. The DFFP model is an effective mechanism for producing food and fuel in conjunction with each other, without the biodiesel production influencing the production of food in an adverse way. This is very important, as food security is a high priority for Africa, in order to address the problem of hunger and poverty. Biodiesel produced from Jatropha is an eco-friendly and sustainable alternative to fossil fuel diesel, as it is a carbon neutral fuel. Jatropha cultivation will also create much needed employment in the rural areas of Africa, which has the highest need for socio-economic development on the continent. Jatropha also has the ability to grow on marginal soils and wastelands. Africa has vast open spaces on which Jatropha, as a biodiesel source, can be cultivated without infringing on food production areas. The energy return of the whole production cycle of Jatropha biodiesel is nevertheless a source of much debate. Most stakeholders felt that the energy return is negative. No agreement has yet been reached about whether the energy content of the by-products from biodiesel must be included in the integrated energy balance equation. In order to make biodiesel from Jatropha a sustainable alternative for small farmers in the Southern African Development Community (SADC), government and regional policies will have to support it by promoting it actively. This will generate interest from global biodiesel investors who will then be willing to invest in projects based on the DFFP model of economic development. The DFFP model offers investors economic returns on investment of between 29 and 33 per cent and payback periods of less than four years. It is a very attractive economic development instrument, as it will ensure equitable and sustainable economic and rural expansion in SADC. Ultimately, this model has the potential to create a better life for all the inhabitants on the African continent. Rainbow Biotech (the DFFP model), as an economic development mechanism, will therefore indeed be the next big thing for the SADC in the future.