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

Graduation date: 1993

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.

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.

M.Sc. / Please refer to full text to view abstract

A series of experiments with Aeromonas salmonicida and infectious hematopoietic necrosis virus (IHNV) were carried out to determine dynamics of the spread of infection in chinook salmon (1.2-1.98g) and rainbow trout (1.2-3.1g). It was found in experiments with A. salmonicida that fish infected by bath immersion became infectious at 4 days postexposure (dpe), one day prior to dying from furunculosis. In cohabitation experiments with a single infected fish donor, an average of 75% disease specific mortality was obtained. There was suggestive evidence that there is a positive relationship between holding volumes and furunculosis prevalence in cohabitation experiments with single donor fish. Median day to infection was inversely correlated with density. The threshold density at density of 1.97 fish/L was approximately 30 times less than the density of 0.08 fish/L, 13.33 and 320 fish respectively. Reproductive ratio (R₀) and transmission coefficient (β) in the furunculosis epizootic were 3.23 and 0.021 (individuals*day)⁻¹ respectively. The mortality rate (α) of infected animals was 28.7% per day. The models constructed successfully mirrored the results of laboratory experiments. Data produced by simulation of the models were significantly associated with the data obtained from laboratory experiments for susceptible (S) class and also for infected class. In similar experiments carried out with IHNV, it was found that donor fish became infectious 3 dpe. The virus levels in donor fish and prevalence levels were also highly associated. Smaller volumes of that led to higher prevalence levels than observed in bigger volumes with 60 or 30 fish in each. A single donor fish was able to transfer virus to recipient fish. However, unlike the A. salmonicida experiment, transmission was insufficient to initiate a full-scale infectious hematopoietic (IHN) epizootic. Estimated parameters for dynamics of infection were approximately half of the values for A. salmonicida (R₀=2.57,β=0.008 (individuals*day)⁻¹ and α=0.15). The models constructed for IHNV spread were used to simulate the results of density experiment. However, it was not possible to test the association between susceptible and infected classes due to inadequate number of infected fish. / Graduation date: 2001

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

One of the greatest challenges in finfish aquaculture is combating losses caused by infectious bacterial diseases, and a better understanding of the interactions between the host immune system and pathogens is essential for developing new methods to manage infections and outbreaks. Extracellular traps (ETs) are decondensed nuclear chromatin released by neutrophils into the extracellular matrix that can ensnare and kill microbes. Since the discovery of ETs in humans, these innate immune effectors have been characterised across the animal kingdom, including in some fish species, though their existence the salmonids has yet to be confirmed. Therefore, the aim of this thesis was to confirm and characterise the release of ETs in the rainbow trout (Oncorhynchus mykiss) and investigate the interaction of these structures with fish pathogenic bacteria. To do this, a triple-layer Percoll gradient technique was employed to give highly enriched cell suspensions of polymorphonuclear cells (PMNs) derived from head-kidney tissue preparations. Treatment of PMN-enriched cell suspensions with the nucleic-acid-specific stain, SYTOX Green, revealed the presence of ET-like structures that had been released without stimulation. These ET-like structures were confirmed by immunostaining techniques to contain the diagnostic proteinaceous markers of ETs: neutrophil elastase, myeloperoxidase and the H2A histone. Previously characterised inhibitors and inducers of ET release from phagocytic immune cells in other animals confirmed that calcium ionophore (CaI), flagellin, and cytochalasin D shared similar activities for ET-release by rainbow trout PMNs. However, interestingly, as the common ET-inducer phorbol-myristate acetate (PMA) and ET-inhibitor diphenyleneiodonium (DPI) did not exert their expected potency in ET release assays with the PMNs, perhaps indicating that these fish cells are less dependent on NADPH oxidase signalling for ET release compared to mammals and most invertebrate species. The PMN-derived ETs were demonstrated to bind to and trap the extracellular nuclease-deficient bacterial fish pathogen, Vibrio anguillarum (Vib 87) when co-cultured. Finally, extracellular nuclease activity produced by a V. anguillarum isolate (Vib 6) during culture was able to degrade ETs released by rainbow trout PMNs in a dose-dependent manner. Moreover, viable colony counts, fluorescent and phase contrast microscopy demonstrated that V. anguillarum Vib 6 eluded trapping by ETs, while an extracellular nuclease-deficient isolate did not. These observations are consistent with the suggestion that nucleases are a microbial virulence factor during host infection. Confirming the existence and antimicrobial potential of extracellular traps released by rainbow trout PMNs may provide a platform towards the development of novel therapeutics to reduce mortalities in finfish aquaculture caused by infectious microbial pathogens.