Disease has been identified as a major problem in the aquaculture industry for the welfare of the fish stocked as well as for its economic impact. The number of diseases affecting cultured fish has increased significantly during recent years with the emergence of several conditions that have added to the overall impact of disease on the industry. Frequently, a lack of scientific knowledge about these diseases is compounded by an absence of effective treatment and control strategies. This has been the case with rainbow trout gastroenteritis (RTGE), an emerging disease of rainbow trout (Oncorhynchus mykiss Walbaum). This study investigated several aspects related to its aetiology and control. A retrospective survey of UK rainbow trout farmers was undertaken to ascertain the extent and severity of RTGE in the UK as well as to identify RTGE risk factors at the site level. Participants in this study accounted for over 85% of UK rainbow trout production in 2004. It was found that the total number of RTGE-affected sites had risen from 2 in the year 2000 to 7 in 2005. The disease was only reported from sites producing more than 200 tonnes of trout/year for the table market. Analysis of risk factors associated with RTGE at the site level showed that this syndrome was associated with large tonnage and rapid production of rainbow trout for the table market. The data collected during this study enabled the identification of those sites that were most likely to present with RTGE the following year and this information was used to study the epidemiology of RTGE at the unit level. A prospective longitudinal study was undertaken in 12 RTGE-affected UK sites. It described in detail the impact, presentation, current control strategies and spread pattern of RTGE within affected UK sites. The risk factors associated with RTGE presence and severity were also investigated. Data were collected for each productive unit (i.e. cage, pond, raceway or tank) on the mortalities, fish origin, site management and environmental factors. RTGE was identified using a case definition based on gross pathological lesions. Analysis of these data revealed that RTGE behaved in an infectious manner. This conclusion was supported by the presence of a pattern typical of a propagating epidemic within affected units. Also, the risk of an unaffected unit becoming RTGE positive was increased if it had received fish from or was contiguous to a RTGE-affected unit. The presentation also suggested an incubation period of 20-25 days. Risk factor analysis identified management and environmental risk factors for RTGE, including high feed input and stressful events, which could be used to generate a list of control strategies. A study of the histopathological and ultrastructural presentation of RTGE was conducted. The location of segmented filamentous bacteria (SFB) and pathological changes found in affected fish were examined. Pyloric caeca were the digestive organ where SFB were found more frequently and in higher numbers, suggesting that this was the best location to detect SFB in RTGE-affected trout. Scanning and transmission electron microscopy revealed a previously undescribed interaction of SFB with the mucosa of distal intestine and pyloric caeca and this included the presence of attachment sites and SFB engulfment by enterocytes, as previously described in other host species. The SFB were not always adjacent to the pathological changes observed in the digestive tract of RTGE-affected trout. Such changes included cytoskeletal damage and osmotic imbalance of enterocytes, with frequent detachment. These observations suggested that if SFB are indeed the cause of RTGE their pathogenesis must involve the production of extracellular products. Analysis of the gross presentation and blood biochemistry in RTGE-affected fish was used to examine the patho-physiologic mechanisms of RTGE. To enable identification of positive RTGE cases for this study, a case definition was created from the information available on RTGE gross presentation in the literature. This case definition was assessed in a sample including 152 fish cases and 152 fish controls from 11 RTGE-affected UK sites, matched by unit of origin. The analysis of these fish using bacteriology, packed cell volume (PCV) and histopathology revealed that RTGE occurred simultaneously with other parasitic and bacterial diseases in a percentage of fish identified with this case definition. With the information gained after analysing the gross presentation, RTGE-affected fish without concurrent disease were selected for the study of the pathogenesis, which included blood biochemical analyses. These analyses revealed a severe osmotic imbalance, and a reduced albumin/globulin ratio suggesting selective loss of albumin, typical for a protein losing enteropathy. The role of the SFB “Candidatus arthromitus” in the aetiology of RTGE was assessed using a newly developed “C. arthromitus”-specific polymerase chain reaction assay (PCR) in conjunction with histological detection. This technique was applied to eight different groups of trout, including an RTGE-affected group and seven negative control groups. This analysis was conducted on DNA extracted from paraffin wax-embedded tissues as well as fresh intestinal contents. The results revealed the presence of “C. arthromitus” DNA in apparently healthy fish from sites where RTGE had never been reported. Additionally, SFB were observed histologically in two trout from an RTGE-free hatchery. These findings do not permit the exclusion of “C. arthromitus” as the aetiological agent for RTGE, although they suggest that the presence of these organisms in the digestive system of healthy trout is not sufficient to cause clinical disease, and therefore other factors are necessary. In conclusion, this study has used a multidisciplinary approach to the study of RTGE which has generated scientific information related to the epidemiology, pathogenesis and aetiology of this syndrome. The results of this project have suggested priority areas where further work is required, including experimental transmission of RTGE, field assessment of the control strategies proposed and further investigation into the aetiology of RTGE.

This dissertation describes two different projects. The first is the development of an oral DNA vaccine delivery system for fish. A novel oral DNA vaccine delivery system was developed for Rainbow Trout by combining non-viral vectors (polycationic liposomes or polycationic polymer) to facilitate the DNA vaccine's uptake by cell membranes along with enteric-coated protection of the DNA embedded in microparticles to prevent DNA degradation in the gastrointestinal tract. Spray drying and spray coating bead techniques were employed in the preparation of the DNA vaccine microparticles. The spray drying technique allowed production of spherical shape enteric-coated microparticles with a particle size range of 0.18 to 20 ��m. Larger particle sizes of 40-50 mesh were obtained from the spray-coated bead technique. The resultant DNA vaccine microparticles were granulated with regular fish feed and given to fish to investigate the efficacy of the delivery system in providing protection against IHNV, and to demonstrate the ease of administration in fish. An in vivo fish trial experiment showed improvement in fish survival rate when fish were immunized with larger particle size DNA vaccine microparticles. Further research to find effective vector carriers for the DNA vaccine delivery system and to seek modifications of the delivery system that will still prevent the denaturation of plasmid DNA that will also facilitate membrane uptake of the DNA vaccine is needed in order to develop a safe, effective, and commercially viable vaccine to control the outbreak of IHNV. The second project of the dissertation is prediction of in vitro drug release profiles from a novel matrix tablet spray-coated with a barrier membrane using mathematical and statistical models. Tablets were prepared by direct compression followed by spray coating. The relationship of the amount of hydrophilic materials in the core tablets and barrier thickness on drug release mechanism was investigated using factorial design and regression analysis. Drug release characteristics were influenced and can be controlled by modifying the amount of hydrophilic materials in the core tablet and the barrier thickness. Mathematical equation generated from regression analysis of n-value, lag time, and percent drug release as a function of the amount of hydrophilic material and the amount of coating material applied can now be used as a tool for predicting and optimizing in vitro drug release from matrix tablets spray-coated with a barrier membrane. / Graduation date: 2003

Interest in the subject of fish welfare is continuing to grow, with increasing public awareness and new legislation in the UK. Water quality has long been recognised as being of prime importance for welfare: water provides the fish with oxygen and removes and dilutes potentially toxic waste metabolites. This thesis investigates the interactions between water quality and the welfare of farmed rainbow trout (Oncorhynchus mykiss Walbaum). A literature review was undertaken to identify current recommended water quality limits for the health and welfare of farmed rainbow trout. Contradictions in the literature regarding suggested ‘safe’ water quality limits were also identified, as were deficiencies in some of the methods used to arrive at conclusions for recommended limits. The literature relating to the effects of poor water quality on welfare were also reviewed. The review ends with a discussion about water quality monitoring in the context of on-farm welfare assessment and how the information might be used in such a scheme. A telephone survey of UK rainbow trout farmers was undertaken to ascertain the level of water quality monitoring currently conducted. Participants in this study accounted for over 80% of 2005 UK rainbow trout production. It was established that 54% of farmers monitored dissolved oxygen to some extent and 69% monitored temperature, the most commonly measured water quality parameters and among the most important for health, welfare and growth. Subsequent visits were made to a sample of the participants in the telephone survey to obtain more detailed information of the farming operations, such as frequency of water quality monitoring, retention of production data and slaughter methods. Monitoring water quality will be an integral part of any on-farm welfare assessment scheme, and while measuring some water quality parameters requires specialist equipment, farmers should be able to monitor the essential parameters, dissolved oxygen and temperature. Any on-farm welfare assessment scheme for rainbow trout should incorparate fish-based measures in addition to resource-based parameters in order to provide as complete an overview of trout welfare as possible. An epidemiological study was undertaken to investigate the current status of welfare on UK rainbow trout farms and to identify risk factors for welfare. Forty-four trout farms from throughout the British Isles were visited between July 2005 and April 2007, sampling a total of 3700 fish from 189 different systems. Farms were visited twice, once in winter and once in summer, to account for any seasonal differences in fish physiology and environmental conditions. Data were collected on a range of fish parameters, together with background information on the batch from which the fish originated. Particular emphasis was placed on water quality due to the potential effects this can have on welfare. The water in each system sampled was monitored for 24 hours, with measurements of dissolved oxygen, temperature, pH, specific conductivity and ammonia taken every 15 minutes. A welfare score was developed for each fish using a multifactorial method, combining data on the condition of the fins, the condition of the gills, the stress hormone cortisol, the splenosomatic index and the mortality levels for the population of fish in the system. Using this welfare score and the individual components of the score as response variables, multi-level models were developed using the water quality, system and husbandry data collected. The primary risk factor that was associated with deteriorating welfare was disease. The purpose for which the fish was being farmed was also important, as fish farmed for the table market had on average worse welfare than those farmed for restocking fisheries. Seasonal effects, linked to higher water temperatures in summer, were associated with poorer welfare scores. Aside from seasonal effects, there is not much evidence that poor water quality is a major problem for the welfare of farmed rainbow trout in the UK. While deteriorating water quality certainly has the potential to affect the welfare of farmed rainbow trout, water quality measurements were within recommended ranges for the majority of farms visited. The results of this epidemiological study suggest that factors other than water quality may have a greater impact on trout welfare, such as exposure to diseases and production differences between farming for the table and restocking markets.

La truite arc-en-ciel (Oncorhynchus mykiss), espèce d'élevage majeure en Europe et notamment en France, est l'une des espèces de poisson les mieux connues dans un grand nombre de domaines, y compris l'immunologie. Les virus qui l'infectent ont aussi été bien caractérisés, en particulier deux Novirhabdovirus, le virus de la septicémie hémorragique virale (VSHV) et le virus de la nécrose hématopoïétique infectieuse (VNHI), tous deux connus pour provoquer des pertes importantes dans les élevages aquacoles. Quelques travaux, conduits notamment à l'INRA, ont mis en évidence l'existence d'une variabilité génétique de la résistance à ces infections chez la truite (Quillet et al., 2007). Une approche combinant analyse génétique et étude des réponses cellulaires a été développée pour tenter de mieux caractériser la réponse de la truite contre le VSHV. L'objectif est de développer des outils d'amélioration de la santé dans les élevages piscicoles et de mieux comprendre les mécanismes de résistance antivirale chez les vertébrés. Tout d'abord, une démarche de cartographie de QTL (quantitative trait locus) a permis de détecter un QTL majeur de résistance au VSHV dans la région télomérique du groupe de liaison 31 de la truite arc-en-ciel. Ce QTL contrôle la survie des poissons et la croissance in vitro du virus sur explants de nageoire (VREFT), ce qui suggère fortement l'implication de mécanismes innés dans la résistance. Le QTL est retrouvé dans des croisements impliquant des reproducteurs de résistance variée, et peut expliquer jusqu'à 65% (survie) et 49% (VREFT) de la variance phénotypique observée. Enfin, l'effet du QTL est conservé quel que soit le mode d'infection employé (balnéation ou injection intrapéritonéale), suggérant que la résistance n'est pas liée à des particularités des tissus superficiels (peau, mucus), premiers sites de contact entre le virus et son hôte. En parallèle, des lignées cellulaires ont été dérivées à partir d'ovaires de truites appartenant à des lignées isogéniques présentant des niveaux de résistance variable à l'infection par le VSHV. Une corrélation remarquable est observée entre la résistance à l'infection des lignées cellulaires et la survie des poissons dont elles sont issues, confirmant définitivement le rôle déterminant de mécanismes innés dans la résistance. Ce modèle cellulaire a également permis de montrer que le contrôle précoce de la prolifération virale était une étape clé de la résistance. Le parallélisme entre résistance in vitro et in vivo semble conservé lors de l'infection par un second rhabdovirus, le VNHI, bien qu'aucune corrélation dans la résistance à ces deux infections n'ait été observée dans cette étude. Par ailleurs, le QTL à effet fort identifié pour la résistance au VSHV ne joue pas un rôle majeur dans la variabilité de résistance au VNHI. Ceci suggère que, même si ils concourent à l'activation de voies de signalisation communes, les facteurs clés de la résistance aux deux virus sont différents, et leur expression contrôlée par des zones génomiques distinctes. Les résultats obtenus dans cette étude ont permis de démontrer sans équivoque le rôle clé des mécanismes innés dans la résistance de la truite à l'un de ses principaux virus, et l'existence d'une forte variabilité génétique sous-tendant l'expression des facteurs impliqués. En proposant des bases nouvelles pour aborder l'analyse des interactions hôte-virus chez la truite, ils ouvrent la voie à la découverte de mécanismes potentiellement nouveaux dans la réponse des poissons à ces infections et à une meilleure compréhension de ces mécanismes chez les vertébrés. / The rainbow trout (Oncorhynchus mykiss) is one of most significant fish model in many scientific fields, including immunology. Due to its importance in aquaculture, viruses that can infect this species have been well characterized. Two well-known Novirhabdoviruses, the viral haemorrhagic septicemia virus (VHSV) and the infectious hematopoietic necrosis virus (IHNV) cause serious damage in fish farms and represent a significant threat for aquaculture in a number of countries. Our laboratories have previously reported a wide range of susceptibility to these infections in rainbow trout depending on the host genetic background (Quillet et al., 2007). In this work, we undertook a dual approach to better characterize the antiviral response in fish. A without a priori approach led to the detection of a major QTL (quantitative trait locus) for resistance to VHSV in the telomeric region of the rainbow trout linkage group 31. This QTL controls both fish survival and viral replication in excised fin tissue (VREFT), suggesting the involvement of innate mechanisms in the resistance, and can explain up to 65% (survival) and 49% (VREFT) of the observed phenotypic variation. Additionally, this major locus was retrieved in a number of genetic backgrounds, and regardless of the infection route (waterborne infection or injection), suggesting that the virus entry in fish is not the main factor of resistance. In parallel, cell lines were derived from ovaries of several rainbow trout isogenic lines with various levels of susceptibility to infection with VHSV. Resistance of cell lines to infection by the virus was remarkably correlated with the survival of fish from which they were derived, confirming the importance of innate factors for the resistance. This model also showed that the early stage response is critical for the cellular fate after infection. The parallelism between resistance in vitro and in vivo has finally been observed after infection by a second rhabdovirus, IHNV, although no correlation in resistance to these two viruses could be detected. Moreover, no major QTL for IHNV resistance was found in the region of the VHSV QTL. This observation suggests that the key factors of resistance are different, even if they contribute to the activation of common signaling pathways. The expression of these factors is in any case controlled by distinct regions of the genome. Our work demonstrates a strong genetic determinism of resistance to a major virus in rainbow trout, based on innate mechanisms. We believe that these results pave the way for the discovery of new host response mechanisms against viruses, leading to a better understanding of antiviral immunity in vertebrates.

Life history within a single species can vary significantly. Many of these differences are associated with varying environmental conditions. Understanding what environmental conditions cue alternate life histories within a single species has been researched extensively. In salmonid fishes, more than almost any other group, varying environmental conditions give rise to individuals within species that take markedly different life history trajectories. Oncorhynchus mykissis a species of salmonid native to the Pacific Northwest region of North America. This species has two life history forms, anadromous and resident. The anadromous form spends a portion of its life in ocean while the resident life history form completes its entire life history in freshwater. Until the decision to migrate and morphological changes associated with smoltification occur, the two life history variants of this species are indistinguishable from each other. This ambiguity in juvenile O. mykiss morphology presents challenges for conservation managers charged with protecting and increasing threatened O. mykiss populations around the Pacific Northwest because conservation efforts cannot be evaluated until juvenile fish make the decision to migrate. Microarray gene expression analysis was used to profile gene expression in juvenile populations of wild and hatchery O. mykiss to identify gene expression variation associated with alternate life history variants. This analysis identified 8 DNA sequences present in both brain and gill tissues that differ in expression in rainbow trout and steelhead hatchery stocks. Differential expression as quantified by microarrays was validated with quantitative real-time PCR. Lastly, the expression of these putative life history markers was preliminarily evaluated in a wild population of O. mykiss at sample locations in the South Fork John Day River Basin, Oregon with known ratios of juvenile anadromous and resident fish.

Investigations of effects of starvation and time at stocking on the survival of catchable rainbow trout in two areas of Mammoth Creek in Dixie National Forest, Utah, were conducted from May 24 to December 6, 1969. Fish were starved for 6 days and 1 day and stocked in the morning (5:40-7:00 a.m.) and in the afternoon (1:45-5:30 p.m.). Out of 7,000 tagged fish stocked, 4,751 tags were returned by fishermen. The 6-day starved fish, stocked in the morning returned to the creel in highest numbers (1,240); followed by 1-day starved, afternoon-stocked fish (1,194); 6-day starved, afternoon-stocked fish (1,163); and 1-day starved, morning-stocked fish (1,154). For the entire stream, the main effects of starvation and time at stocking were not significant at the 10 percent level, but their interaction was significant at the 1 percent level. In the upper area, however, both 6-day starved lots returned to the creel in greater numbers (692) than the 1-day starved lots (643), and the main effect of starvation was significant at the 1 percent level in addition to the interaction being significant at the 5 percent level. About 81 percent of the tags returned were from fish caught in the sections stocked. One percent of the tags were from fish that moved upstream and 18 percent from fish that moved approximately 3 miles or less downstream. No consistent downstream movement patterns were related to either starvation or time at stocking. One week after the fishing season, 68 percent of all (19) tagged fish captured by electrofishing were 6-day starved fish, but this difference was not significant at the 10 percent level.

In the past few years the Utah Fish and Game Department, as well as the fish and game departments of other states, has been spending fisheries money for fish eradication on both lakes and streams. The Utah Fish and Game Department recently suggested to the University that research be initiated on the subject of toxaphene as a fish poison. Consequently, a better insight into conditions that exist for a given water type could be developed. To date there is a very small amount of material written on toxaphene. This is due primarily to the fact that toxaphene was not developed until the 19hO's. Toxaphene is used mainly as an insecticide. Such crops as tomatoes, beans, alfalfa, clover and cotton are protected by the use of toxaphene (Hudd and Genelly, 1956).

Users of the Instream Flow Incremental Methodology CTFIM) commonly assume that there is a positive linear relationship between available habitat (WUA) and stream fish biomass. However, several studies have shown a lack cf correlation between WUA and biomass. Such poor relationships may occur if other factors limit fish abundance. Food availability is a potentially important factor limiting abundance of stream fishes and can affect blomass by influencing fish condition (individual weight). I collected rainbow trout (Oncorhynchus mykiss) exposed to different levels of invertebrate-drift density to determine if condition was greater where food was more abundant. I also examined whether size-selective predation and gut fullness varied with drift density. Size-selective predation was not related to drift density. However, condition and gut fullness were both significantly correlated with drift density. Furthermore, changes in drift density appeared to have a stronger influence on small-trout condition than large-trout condition. The results of this study and others support the contention that the reliability of the IFIM might be improved if food availability were incorporated into models.

Chlorophyllin (CHL), a water-soluble derivative of the green plant pigment, chlorophyll, is an effective antimutagen and anticarcinogen in various model systems when used as a modulator against a class of carcinogens that, in general, have a structure consisting of at least three fused rings. Dibenzo[a,l]pyrene (DBP), an extremely potent environmental carcinogen, has been isolated from urban air samples, tobacco smoke, and coal smoke condensate. A study was conducted to evaluate the complex interrelationships among dietary DBP doses with co-exposure to a range of CHL doses. In order to achieve adequate statistical power in the generation of multiple dose-response curves, this dose-dose matrix experiment utilized over 12,000 rainbow trout. The resulting DNA adducts were assessed and evaluated as biomarkers of exposure to discern their relationship with the final tumor outcome. CHL was highly effective in reducing DBP-initiated DNA adduct formation in the liver and stomach and strongly inhibited tumor formation in the liver (56-79% inhibition), stomach (30-68%), and swim bladder (over 80% at the highest DBP dose). Molecular dosimetry revealed adduct formation to be predictive of final tumor response in both organs regardless of CHL dose. Other parameters evaluated were consistent with CHL-mediated protection. A clinical CHL preparation, evaluated in a human population subsequent to the seminal demonstration of CHL chemopreventive properties against AFB��� in trout (1), revealed CHL to be just as effective in reducing biomarkers of alfatoxin exposure to humans (2). Dietary administration of this clinical preparation along with DBP in the rainbow trout demonstrated CHL protective capacity against DBP-initiated multi-organ DNA adduct formation and final tumor incidence. Sucrose was evaluated, deemed unlikely to be sequestered in a complex with CHL, and was used as a control in a pharmacokinetic study evaluating the biodistribution of DBP with and without CHL. The results provide evidence against a non-specific masking mechanism for CHL-mediated blocking of DBP (or aflatoxin)-initiated tumorigenesis. CHL at multiple doses provided significant protection against multi-dose DBP-initiated DNA adduction and tumor formation in multiple organs. CHL-mediated protection, primarily by reduced carcinogen biouptake and consistent with a complexation mechanism, is supported by these results. / Graduation date: 2003

During the past 30 years, numerous studies have focused on the toxicities of polycyclic aromatic hydrocarbons (PAH). Laboratory and field studies have helped elucidate the detrimental effects of these chemicals on growth, reproduction and immune response. Polycyclic aromatic hydrocarbons are in the priority list of chemicals to be studied by different governmental agencies and universities and understanding their mechanisms of action is the focus of the current research. The manuscripts presented in this dissertation are focused on the effects and mechanism of action of PAH on disease susceptibility. After a dietary exposure to PAH for up to 50 days (chapter II) and samplings after 3, 7, 14, 28 and 50 days, a number of biomarkers of PAH exposure were measured: Fluorescent aromatic compounds (FACs) in bile, ethoxyresorufin-o-deethylase (EROD) in liver microsomes, cytochrome P450 1A immunohistochemistry in liver and kidney and adduct formation in liver. Additionally markers of oxidative stress were measured: comet assay in blood, protein nitration in kidney and F2-isoprostanes in kidney. Oxidative stress was a probable factor in PAH induced responses in fish adapted to long-term PAH exposures and aryl hydrocarbon activation was not necessarily involved in this process. Disease challenge with Aeromonas salmonicida (chapter III) resulted in differences in mortalities that demonstrated that fish exposed to PAH were more susceptible to disease than fish not exposed to PAH. Determination of gene expression in head kidney of fish exposed and not exposed to PAH challenged with A. salmonicida using microarray and RT-PCR technologies 2, 4, 10 and 20 days after challenge (chapter IV), suggested that PAH exposure was associated with down regulation of interleukin 8, transport associated protein 1, NF-kB modulator, recombination activating gene and major histocompatibility complex II two days after challenge in fish exposed to PAH. The transcript levels were closer to control levels 20 days after challenge, this indicated a recovery from the effect of PAH exposure. / Graduation date: 2006