The substantial increase in production by the aquaculture industry in the last few decades has been accompanied by a greater demand for prophylactic measures to control fish health, since fish diseases are a major impediment for the profitability of fish farms. To date vaccination has played a major role in the success of fish farming, offering resistance to a limited number of infectious diseases. In order to expand the availability of effective vaccines, a better knowledge of the piscine immune system is crucial. Although a tremendous effort has been put into studies of the fish immune system over recent years, our understanding is still far behind when compared to that of mammals, with many gaps in knowledge still requiring to be addressed. To gain a better understanding of fish immune responses, this thesis has focused on characterising a number of immune molecules considered crucial for disease resistance against extracellular pathogens, in a commercially important species, the rainbow trout (Oncorhynchus mykiss). Adaptive immunity is regulated by a group of specialised lymphocytes, the T Helper (T H) cells. They exert their helper functions through the release of a range of cytokines, which can be used to further categorise them into subsets. One of the latest subsets to be characterised was the T H 17 lineage, which secretes various cytokines, including interleukin (IL )-17 A and IL-22, pivotal in the eradication of extracellular pathogens. To gain a better insight into TH17-type cytokines and their responses in fish, Chapters II and III focused on the characterisation of such molecules, through expression studies, and by analysing their bioactivity as recombinant proteins for the rust time in piscine species. Homology studies confirmed that both molecules are likely to belong to the IL- 22 and IL-17 cytokine families, noting that the trout IL-17 AlP protein shared a close relationship with other piscine IL-17 AlP2 molecules. Interestingly, analysis of their biological activities in splenocyte primary cultures indicated that the two proteins had an effect on the expression of antimicrobial peptides, with IL-22 displaying a potentially more important role than IL-17 AlF. Cytokines are only able to conduct their effects through binding to specific cell surface receptors, acting as ligands. The presence of these particular receptors dictates which cells can be targeted by such cytokines and highlights the importance of the ligand-receptor interaction. To further understand the already identified cytokines, or their family members, a range of receptors was characterised in Chapter IV, with two of them (CRFB4 and IL-17RA) being potentially involved in the signalling of IL-22 and IL-17 AlF ligands. Although homology studies confirmed they belong to the IL-17 and class II cytokine receptor families, orthology of all nine receptors to mammalian homologues could not be inferred. Therefore, further work is required to achieve a better understanding of the homology of these molecules with the mammalian receptors, and their potential involvement with IL-22 and IL-17 AlF intracellular signalling. TH17 cells are characterised not only by the unique range of effector cytokines they secrete, but also through the presence of a master transcription factor, the retinoid-related orphan receptor (ROR)-yt. In Chapter V, two members of the ROR family were characterised, sharing 90% identity between each other. Trout ROR-y1 and -y2 were found to have a close relationship with known ROR-y family members, sharing a particularly high homology with the mammalian splice variant ROR-yt. This suggests that the piscine molecules are likely to be homologues of ROR-yt. However, it still remains to be elucidated whether they can drive piscine TH17 cell differentiation and indeed trigger a TH17 response in fish. T Helper (TH) cells are vital in promoting immune responses. Thus, Chapter VI aimed at developing a potentially effective antibody which could recognise the CD4 marker, typically expressed by this cell subset. The polyclonal antibody, produced using a recombinant protein approach, revealed encouraging results. It was found by Western blot analysis that this antibody could recognise proteins with a molecular weight approximating to that expected for the trout CD4 molecule and its potential splice variant. However, to confirm these observations, further work is still required t? better characterise this antibody and the cells it targets. The results of this thesis have revealed that despite the similarities between the mammalian and piscine immune systems, clear differences exist and also that fish have developed unique molecules and mechanisms not found in mammals. It is therefore of great interest to understand in more detail the role of these novel components so that future progress can be made towards gaining a better comprehension of fish immunity and help advance new vaccination strategies.

Fish diseases endanger the aquaculture industry worldwide. Approaches to combat fish diseases are hampered by the relative poor knowledge of immune relevant genes in fish. This thesis describes the cloning and expression of several immune relevant genes in rainbow trout (Oncorhynchus mykiss). The full-length cDNA and genomic DNA of the inducible nitric oxide synthase (iNOS) gene have been cloned and sequenced. The gene structure was the first to be determined outwith the mammals. The expression of the iNOS gene is induced by virus (VHSV) infection, but its detection by RT-PCR might be impaired by the secondary structure formed in the iNOS mRNA. Screening of a genomic library resulted in the isolation of a second allele of IL-1b1 gene with a large intron III. This allele appears to have resulted from a recent retroposition of a HpaI SINE into intron III. Two other retroposition events have also been recognised in the same intron. The survival mechanism of this SINE is discussed and a model of trout IL-1b gene formation proposed. Both alleles of the IL-1b1 gene are constitutively expressed and induced by LPS and recombinant trout IL-1b1 in heterozygous RTS-11 cells. The promoter of the IL-1b1 allele S gene was isolated and the transcription start site was determined. A series of promoter-reportor gene constructs were transfected into RTG cells to study their activities under different conditions. The first intron is an important part of the promoter of IL-1b1 allele S and NFkB is a transcription factor required for IL-1b1 expression. The cytokine receptor common gamma chain (gC) was also isolated from rainbow trout, the first outwith mammals. Its expression can be detected in blood, gill, kidney, brain and liver, and in macrophage cultures. The expression of gC in macrophage cell cultures is upregulated by recombinant IL-1b1 and LPS stimulation. The expression of gC is also detectable in RTG-2 cells with an increased transcript level after stimulation with recombinant IL-1b1.

No description available.

No description available.

No description available.

Diplostomiasis, or eyefluke is caused by the metacercarial stage of the digenean parasite <i>Diplostomum spathaceum</i> which selectively invades the lenses of freshwater fish and can cause partial and total blindness. This study investigates the host-parasite relationship of <i>D. spathaceum</i> infections in rainbow trout with particular reference to the diplostomule or post-penetration stage of the parasite. The point of cercarial penetration influenced the speed with which diplostomules reached the lens and the length of time they were exposed to the fish defence mechanisms. The further away the point of cercarial penetration from the eye, the longer diplostomules take to reach the lens and the fewer establish as metacercariae. Total body immersion using cercariae resulted in significantly more metacercariae establishing in the lens compared with entry via intramuscular, intraperitoneal or intracardial injection of either cercariae or diplostomules. Cercariae may be drawn in by the respiratory current and enter the fish via the gills and buccal cavity. The gills provide an immediate source of entry into the circulatory and lymphatic system of the fish. There did not appear to be any preferred route of migration. The optimum conditions for the <i>in vitro</i> collection of diplostomules were developed. The optimum conditions for <i>in vitro</i> transformation of <i>D. spathaceum</i> cercariae were established. The optimum conditions for the short-term <i>in vitro</i> maintenance of diplostomules were investigated. L-15 medium supplemented with 5% heat-inactivated foetal calf serum maintained approximately 80% survival for the first 24-48 h of culture. Rainbow trout were immunized with cercariae and diplostomules of <i>D. spathaceum</i>. Circulating antibody was detected by ELISA to both cercariae and diplostomules by 6-7 weeks post-immunization. Cross-reactivity occurred between anti-cercarial serum and diplostomule and metacercarial antigens, and anti-diplostomule serum cross-reacted with cercarial and metacercarial antigens in ELISA. Immunofluoresence studies indicated that the tail region of the cercriae was strongly antigenic; a differential fluorescence was observed for both anti-cercarial and anti-diplostomule sera with the cercarial tails fluorescing more strongly than the bodies. No cross-reactivity was observed in fluorescence between anti-cercarial or anti-diplostomule sera and metacercarial antigen although anti-cercariae serum cross-reacted with diplostomule antigen and <i>vice versa</i>. Normal trout serum and anti-diplostomule serum were cytotoxic to diplostomules of <i>D. spathaceum in vitro</i>. Immunized fish exhibited a significant degree of protection against further infection by <i>D. spathaceum</i>. However, there was no correlation between protection and the level of serum antibody. Isolated normal trout macrophages kill significant numbers of diplostomules <i>in vitro</i> at effector: parasite ratios of 150:1 or greater. <i>In vitro</i> killing was not increased using diplostomules opsonized with anti-serum or <i>in vivo</i>-activated macrophages individually, but when combined, increased killing occurred. Diplostomules were capable of eliciting respiratory burst activity from macrophages <i>in vitro</i>, suggesting that reactive oxygen species may have a role to play in the killing mechanism. Diplostomule antigen was capable of activating macrophages <i>in vivo</i>.

Diphyllobothrium dendriticum and D. ditremum have a wide distribution in the trout of Scottish lochs, although no specific trends in their overall distribution have been observed. The seasonal recruitment trends and development of infections with Diphyllobothrium spp. pleroceroids in wild rainbow trout, in one particular Scottish loch, were monitored regularly for the period 1986 to 1989. Infection varied between these years, but in 1987 intensities from March to November reached their highest levels in November when sampling ended. These two species reached infection intensities, in individual fish, higher than any previous reports and trout stocks appeared to be affected. Plerocercoids, in host-derived cysts, were located on the peritoneal stomach surface, the gross pathology of which is described. The cellular components of the host-derived cyst were examined in detail by light and electron microscopy. A number of leucocyte types, found within a collagenous matrix with associated fibroblasts, were observed. Leucocyte types included neutrophils, eosinophilic granular leucocytes, macrophages and occasional plasma cells. Antibody production, in response to natural infection, was examined by indirect immunofluorescence using serum on cryostat sections of plerocercoids obtained from wild-caught rainbow trout. The tegument of these larvae showed a positive fluorescence, indicating the presence of serum antibody in these trout. Semi-quantitative estimates of antibody titres were estimated by an optimised enzyme-linked immunosorbent assay (ELISA). To facilitate further examination of the events associated with the establishment of plerocercoids and associated host-derived cyst, under controlled conditions, a routinely maintained laboratory life cycle of D. dendriticum was established. This laboratory life cycle utilised Herring gulls (Larus agentatus) and the copepod, Cyclops abyssorum, as the definitive and first intermediate hosts respectively, and provided plerocercoid infections in trout which were examined at various times post infection. Tegumental extracts from the plerocercoids of D. dendriticum, obtained by freezing and thawing specimens, and conditioned medium obtained after in vitro maintenance of live plerocercoids, were prepared. These extracts were tested in respiratory burst assays and were found to stimulate the production of both hydrogen peroxide and superoxide anion by rainbow trout macrophages. The migratory responses of trout macrophages and neutrophils to these agents were also investigated, by an optimised Boyden technique using a 48 Well Micro Chemotaxis Chamber. Leucocytes were found to have an increased chemokinetic motility following stimulation/contact with these antigen preparations. Finally, to investigate if antigens on live parasites were attractive/stimulatory, an in vitro adherence assay was carried out. Procercoid stages, which share common antigens with the plerocercoid stage, were maintained in vitro and incubated with rainbow trout leucocyte suspensions, in the absence and presence of normal or immune serum from infected fish. Leucocytes adherence was considerably increased by the presence of immune serum, indicating the possible interaction of the non-specific and specific immune response in the host inflammatory reaction.

The reproductive characteristics of a wide range of freshwater-resident and anadromous Scottish forms of Salmo trutta were examined. A total of 653 females were sampled from 27 wild and two long-term domestic populations covering a fork length range of 116-725mm. Total fecundities of the wild fish were 51-5952 eggs. Egg diameters ranged from 3.6 to 6.4mm. Gonadosomatic indices ranged from 5-26%. The youngest females were 2+ years. Trout from fast-growing populations matured earlier and were shorter-lived than slow-growing trout. Regression equations for the relationship between body size and fecundity and egg diameter are provided for the national data set and for individual and grouped populations. Significant differences in fecundity and egg diameter were found between populations. After standardisation to a common body length, fecundity and GSI were found to be positively and egg diameter negatively correlated with trophic status of the water bodies and with early growth rate. The domestic populations provided the overall highest relative fecundities and smallest egg diameters. Fecundity of wild trout was negatively and egg diameter positively correlated with latitude. There was no relationship with numbers of other fish species present. Above-falls trout were short-lived, yet had high relative fecundity and small egg size. In these populations, age was more important than length in determining egg diameter. The rate at which maturity is reached, varying between populations, may be more important than absolute growth rate in determining fecundity and egg size. Lifetime eggs per female and instantaneous production by 1000 females were estimated for individual populations. The significance of the estimates for the River Ewe System in north west Scotland, an area of recent sea trout population decline, is discussed.

Thesis (M.S. (zoology))--Washington State University, December 2007. / Includes bibliographical references.

Graduation date: 1969