This thesis tests the hypothesis that innate immunity may be enhanced immediately following a stressful event. The experiments characterize the acute effects of the fight or flight response on some immunological and endocrine parameters in rainbow trout (Oncorhynchus mykiss). Plasma cortisol and catecholamines were elevated within seconds of the initiation of an acute handling stressor consisting of 30 seconds in the air and five, 10 or 20 minutes in a shallow bucket of water. Plasma lysozyme activity increased after stress, however, the increases were not statistically significant unless variation was reduced by serial bleeding of the same individual trout before and after stress. A more "resting" fish was achieved by use of the anesthetic 2-phenoxy-ethanol which was surreptitiously introduced into the tanks before the initial bleed. Individual fish were then revived in freshwater and stressed as before. Enhancement of lysozyme activity was evident although levels of plasma stress hormones in fish that were anesthetized, revived and stressed were less than when fish were similarly stressed without anesthetic. Levels of cortisol and catecholamines increased within seconds of capture and aerial exposure, returned to near pre-stress levels after the fish had been placed in a shallow bucket of water for 30 seconds, then increased again. Evaluation of the influence of acute stress on survival following challenge with the pathogen Vibrio anguillarum yielded equivocal data. Results presented here suggest that enhancement of innate defenses as part of the fight or flight response merits further evaluation. / Graduation date: 1997

Graduation date: 1992

Rainbow trout were exposed to aluminum at pH 7.25 and 8.25 and four hardnesses (10, 30, 80, and 120 ppm CaCO₃) for 96 hours in a continuous-flow system and mortality and aluminum accumulation in the gills were determined. Temperature, pH, and dissolved oxygen were measured daily for each treatment. Dissolved and total aluminum concentrations and hardness were determined following exposure periods of 48 and 96 hours. Aluminum was most toxic at pH 8.25, and was more toxic at lower than at higher hardnesses. Water hardness provided a significant protective effect against aluminum-induced mortality (p < 0.05), and there were no significant effects for water hardness on gill accumulation at either of pH. At pH 7.25 no mortalities occurred under any conditions. At pH 8.25, the accumulation of aluminum in gill tissues was higher than for pH 7.25 following exposure for 96 hours. In addition, aluminum concentration and exposure time had a significantly cumulative effect on fish mortality (p < 0.05). Possible mechanisms for aluminum toxicity and the accumulation of aluminum in the gills of rainbow trout were attributed to the forms and solubilities of aluminum species at different pH values. Competition between Ca²⁺ and aluminum for binding sites on the gills likely influenced aluminum toxic action. / Graduation date: 1992

The peripheral-type benzodiazepine receptor (PTBR) of trout brain was pharmacologically characterized and pyrethroid interaction with this site investigated. High-affinity binding sites for [³H]PK 11195 were detected in brain membranes of rainbow trout; these shared some of the characteristics of the PTBR of rodent brain (i.e., high affinity for PK 11195 and an endogenous ligand protoporphyrin IX) but were unique in the low affinity for Ro5-4864. Permethrin displaced [³H]PK 11195 binding with micromolar affinity while deltamethrin had less than 50% efficacy at displacement. Thus the PTBR appeared not to be relevant to pyrethroid toxicity in rainbow trout. Pyrethroid interaction with the GABA, receptor was investigated using [³⁵S]TBPS as a radioligand probe and by measurement of GABA-stimulated ³⁶c1- influx in vesicle preparations. At micromolar concentrations, deltamethrin, cypermethrin isomers and other pyrethroids inhibited [³⁵S]TBPS binding by 55- 95% with limited stereoselectivity. Pyrethroids were found to effect a GABAdependent inhibition of [³⁵S]TBPS binding. Ro5-4864, which showed micromolar affinity for the trout PTBR, produced a GABA-modulated interaction with [³⁵S]TBPS binding. These results delineate the reciprocal allosteric interactions between a pyrethroid binding site, a Ro5-4864 binding site, the GABA recognition moiety and the TBPS binding site in trout brain. However, pyrethroids exhibited a modest affinity for this binding site on the GABAA receptor. Pyrethroids indirectly inhibited the GABA-dependent influx of ³⁶Cl⁻into trout brain synaptoneurosomes by increasing the basal uptake of chloride, thereby compromising the ability of the vesicles to respond to applications of GABA. This pyrethroid effect was of nanomolar potency, stereospecific, tetrodotoxinsensitive and mimicked by veratridine. These results suggest that the primary effect of pyrethroids in trout brain, as measured by this assay, was due to an interaction with voltage-dependent sodium channels, increasing sodium conductance and thereby increasing the basal uptake of ³⁶Cl⁻ through a voltagesensitive channel. The convulsant activity of deltamethrin was tested in rainbow trout. The EC₅₀ for convulsant severity was 32 μg /kg body weight. By comparison, pyrethroids at these concentrations in rodents produce no overt toxicity but act as potent proconvulsants. / Graduation date: 1990

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.