Thesis (M.S. in engineering)--Washington State University, December 2009. / Title from PDF title page (viewed on Feb. 4, 2010). "College of Engineering and Architecture." Includes bibliographical references (p. 50-55).

Thesis (Ph.D.) -- McMaster University, 1997. / Includes bibliographical references. Also available via World Wide Web.

Three studies were conducted to evaluate the relationship between insulin and nutritional parameters in rainbow trout. In the first study effects on growth parameters and tissue composition of rainbow trout were investigated following injection of bovine insulin at two dose levels every 48 hr for 56 days. In addition, [¹⁴C]-leucine incorporation into plasma, liver, and skeletal muscle was studied for the two insulin treatments and a group of the saline-injected controls given a single shock-dose of insulin (5.0 IU/kg). Hypoglycemic responses were observed with all insulin treatments. In comparison to controls, high insulin treatment gave a significant body weight increase. At both levels, insulin increased the content of protein, lipid, and also the incorporation of [¹⁴C]-leucine activity in skeletal muscle. Simultaneous decreases in specific activity of plasma and liver tissue indicated a net movement of [¹⁴C]-leucine toward the peripheral musculature. No effect of the hormone was seen on the glycogen content of liver or muscle tissue over the 56-day period. In a second study, the effect of bovine insulin on tissue incorporation of [¹⁴C]-glucose and [³H]-leucine was investigated in fed and fasted rainbow trout reared on a control and high-protein diet. Insulin produced marked hypoglycemia and mobilization of liver glycogen in all treatments. Although insulin gave no evidence of glycogenic stimulation it did appear to promote oxidative clearance of [¹⁴C]-glucose. Compared to [¹⁴C]-glucose much greater tissue incorporation of [³H]-leucine was observed in fasted fish; insulin stimulated the incorporation of [³H]-leucine into skeletal muscle protein. In plasma, liver, and skeletal muscle of all treatments, the summed specific activities of [³H]-leucine was considerably greater than that of the summed values of [¹⁴C]-glucose following insulin administration. Four weeks of fasting apparently lowered basal metabolism but no changes were observed in plasma glucose and glycogen stores. There was some evidence of gluconeogenic activity in the high protein-fasted fish and the data indicated in all fasted treatments a stimulation of [¹⁴C]-glucose and [³H]-leucine metabolism following insulin administration. As a third investigation, [¹²⁵I]-iodoinsulin binding studies in the presence of a concentration range of bovine insulin were conducted to establish specific insulin binding levels in erythrocytes, skeletal muscle plasma membranes and isolated hepatocytes of rainbow trout reared on control-, high-protein and high-carbohydrate diets. Negative cooperativity was observed and receptor concentrations and apparent dissociation constants established for each preparation. No differences of specific binding attributed to diet were detected in either erythrocytes or skeletal muscle plasma membrane preparations, However, the receptor concentration of isolated hepatocytes from high-carbohydrate reared trout was increased. This contrasted to comparable mammalian studies. In view of an apparent depression of receptor concentration in skeletal muscle plasma membranes and isolated hepatocytes of high-protein reared trout, these data were interpreted according to the reciprocal relationship observed between endogenous insulin and insulin receptor concentrations established for mammals. Unlike mammals where glucose was shown to be the primary insulin secretagogue, endogenous insulin levels in rainbow trout have been closely correlated to circulatory amino acids and are thought to be primarily controlled by these protein metabolites. / Graduation date: 1982

Several compounds have been shown to induce the mixed function oxidase enzyme system of animals, and also to inhibit tumor formation. Included in this category are several vegetables and isolated vegetable components from the Cruciferae family. In this study rainbow trout were fed blanched freeze-dried cauliflower, broccoli, or Brussel sprouts at the 20% supplemental level for two months, or 500 ppm benzyl or phenethyl isothiocyanate. At this time the livers were removed and hepatic microsomal enzymes were studied. This treatment did not cause any increases in the metabolism of p-nitroanisole or benzo(a)pyrene and cytochrome P-450 levels were not elevated. On the contrary, benzo(a)pyrene monooxygenase, 7-ethoxycoumarin 0-dealkylase, resorufin 0-dealkylase, p-nitroanisole 0-demethylase, and cytochrome P-450 levels were increased in trout treated orally and intraperitoneally with β-napthoflavone. For all parameters studied, a greater increase in activity was seen in the trout treated intraperitoneally. The vegetable and isothiocyanate supplemented diets also did not inhibit tumor formation in trout challenged with 20 ppb aflatoxin B₁ for one month. A study with rats showed that both blanched freeze-dried cauliflower and air-dried cauliflower cause significant increases in p-nitroanisole 0-demethylase activity and benzo(a)pyrene monooxygenase activity and increased cytochrome P-450 levels in rat hepatic microsomes when fed at the 20% supplemental level for 25 days. It was concluded that the null effects seen with the trout fed the vegetables and isothiocyanates were not due to the heat treatment of the vegetables since rats fed blanched freeze-dried cauliflower had significant increases in enzymatic activity and increased cytochrome P-450 levels over controls. The results of the β-napthoflavone experiment verified the validity and reliability of the experimental assays and conditions and showed that trout hepatic microsomal enzymes can be induced by orally administered compounds. / Graduation date: 1981

A dietary level of Aroclor 1254 (100 ppm) was fed to rainbow trout (Salmo gairdneri) for 15 weeks to determine the effects on hepatic microsomal enzyme induction. Fish were also fed combined polychlorinated biphenyl (PCB) (100 ppm) and cyclopropene fatty acids (CPFA) (50 ppm) to determine the effects on mixed function oxidase (MFO) induction. Dietary PCBs markedly induced the microsomal activities of 7-ethoxyresorufin O-deethylase, 7-ethoxy-coumarin O-deethylase, and benzo(a)-pyrene monooxygenase. Ethoxyresorufin O-deethylase activity continued to increase to a level 77-fold higher than control at week 15. Ethoxycoumarin O-deethylase and benzo(a)-pyrene monooxygenase activities increased to 7.1-fold and 48-fold over control at week 9 and then slightly decreased to 6.8-fold and 45-fold over control at week 15, respectively. Cytochrome P450 values remained approximately 2-fold above controls from week 5 through week 15. At weeks 1 and 3, cytochrome P450 levels were not significantly different from control. Ethoxyresorufin O-deethylase, ethoxycoumarin O-deethylase, and benzo(a)pyrene monooxygenase activities in the combined PCB and CPFA-fed trout were significantly higher than in controls and CPFA-fed fish, and significantly lower than in PCB-fed fish. There was no significant difference in cytochrome P450 levels after week 5. This is the first time dietary PCBs have been shown to induce the MFC system in PCB-fed rainbow trout. / Graduation date: 1980

No description available.

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.

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.

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