Thesis (M. Sc.)--University of Canterbury, 2007. / Typescript (photocopy). Includes bibliographical references (leaves 65-75). Also available via the World Wide Web.

Thesis (Ph. D.)--Montana State University--Bozeman, 2004. / Title from PDF t.p. (viewed on June 12, 2006). Chairperson, Graduate Committee: Thomas McMahon. Includes bibliographical references.

Thesis (M.S.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains xi, 155 : ill., maps (some col.). Includes abstract. Includes bibliographical references (p. 80-91).

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

Thesis (M.S.)--Oregon State University, 2007. / Printout. Includes bibliographical references (leaves 50-58). Also available on the World Wide Web.

The aim of the work will be based on the diet composition monitoring of the introduced rainbow trout and wild brown trout and grayling in defined time intervals, and assessing the degree of the food competition among the monitored species. The experiment was held in the second half of September 2010 in the river Blanice in the protected fish areas close to the village Těšovice, where several dozens of rainbow trout were stocked. In a predefined several - day interval of were caught 30 pieces of the rainbow trout (SL = 266 ? 14 mm, m = 347 ? 54 g), 33 brown trout (SL = 244 ? 19 mm, m = 219 ? 51 g), 14 graylings (SL = 256 ? 27 mm, m = 240 ? 91 g) and 12 resident rainbow trout (SL = 255 ? 13 mm, m = 280 ? 47 g) originated from the last plant. Within these groups a fish stomach content and consequently food selectivity was examined by using of the Ivlev's selectivity index (1961), a food competition was examined by using of the Schoener's index food overlap (1970) and the importance of various food components was examined by using of the index of preponderance according to Natarajan (1961). Rainbow trout began to ingest food immediately after the stocking into the stream and its occurrence was seen in all fish crop caught during the period. The insect (Insecta) was given as a preferential food of the rainbow trout and according to the index of preponderance (IP), it formed 54%, Trichoptera (IP) 24% and Ephemeroptera (IP) 19% orders belonged to the most preferable kinds of the food. According to the index of preponderance, gastropod (Gastropoda) formed (IP) 25% of a secondary food. The rainbow trout was accepting the same dietary components as well as the original sorts of salmonoid fish within the river. According to the index of a food overlap (S), the food competition between rainbow trout and brown trout was equal to the value of (S = 0.566) during the whole monitored period and the grayling was equal to (S = 0.520). Even though both of these values are slightly below the significant food overlap (S = 0.6), we can still say that the food competition is relatively high among these kinds.

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

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