Monoclonal antibodies (mAbs) have been essential tools in the elucidation of the immune system of mammals, and their application to identify surface molecules on leukocytes have allowed important functions of these cell to be identified (such as receptors that bind antigens, ligands involved in cell to cell signaling or in initiating immune response activity). Not only have mAbs been used to discriminate cells during different stages of cell development, but have also assisted in understanding the dynamics of molecules expressed during functional processes. Such molecules detected on human leukocytes are called human leukocyte differentiation antigens or HLDA. In order to group the antibodies that detect similar molecules and have similar patterns of reaction, immunologists have organised the mAbs that bind to these antigens into Clusters of Differentiation (CD). So far, there are about 350 leukocyte surface molecules detected by mAbs with a CD nomenclature for human leukocytes (www.hcdm.org). In fish immunology there is a great need to produce mAbs that are able to differentiate the various components of the fish immune system to assist in the elucidation of the fish immune system. The present study was an endeavour to develop and characterise mAbs that could be accredited to such scheme. A better understanding of the fish immune system is urgently required so that effective strategies of control can be developed for significant diseases during fish farming. Monoclonal antibodies were prepared by immunizing mice with thymic leukocytes from rainbow trout. The leukocytes were activated with the lectin Concanavalin A to promote the activation and proliferation of the target T-cell population. The selection of clones producing antibodies during screening was performed on the basis of the response of the supernatant from hybridomas using three consecutive assays. First, selection was determined by the positive staining of cells from the thymus in a Dot blot assay. Secondary screening was performed by means of flow cytometry (FCM) and the criterion for selection was the preferential detection of leukocytes gated in the lymphocyte region. Finally, the positive supernatants from hybridomes were evaluated to determine their effectiveness in the detection of modifications in the labelled cells during a multiple way activation by detection of foreign histocompatibility complex enhanced with mitogens. Monoclonal antibody TcOm15 was selected from 564 hybridomas produced and then used to stain cells from various Rainbow Trout tissues. It was clear from FCM, microscopy and Western blot analysis that mAb TcOm15 not only reacted with thymic cells but also with cells from other tissues. Differential staining of cells with mAb TcOm15 was observed with 27.1 ±1.4 % of leukocytes from peripheral blood leukocytes (PBL) stained in comparison to 2.0 ±0.2 % from the thymus, 13.8 ±0.4 % from the spleen, and 5.6 ±0.6 % cells stained from head kidney. The labeled cells showed characteristics of lymphocytes and monocytes, presenting a distinctive staining in immunohistochemistry and confocal microscopy. Western blot analysis, using electrophoresed proteins under denaturing conditions with leukocytes from several different tissues, showed that mAb TcOm15 did not detect a single protein. At least three proteins appeared to be identified by the mAb at 105, 160 and 200 kDa. The proteins were identified as α Actinin-4, non-erythroid Spectrin αII chain or Ig-like protein and non-muscle Myosin (MYH10) by MALDI-TOF analysis. Three of these identities are for compositional molecules for the cytoskeleton of different types of cells, and one it is associated to immunoglobulin superfamily. The identification of these proteins by mAb TcOm15 suggests an ability of this mAb to detect a specific function, possibly related with the synchronicity of expression or interaction of cytoskeleton-membrane proteins forming a multiprotein complex. Another possibility is as a carrier role for a protein during interactions. Colocalization of the mAb with F actin from the cytoskeleton was also observed suggesting the possibility that mAb TcOm15 detects a specific site in a multi-protein complex from the cytoskeleton. The molecule detected showed down-regulation in a dose dependant way with Concanavalin A and the expression was almost lost following stimulation of cells with phorbol 12-myristate 13-acetate stimulation. Leukocytes from the PBL and thymus up-regulated the expression of the TcOm15 molecule under mitogenic conditions in vitro, and results from in vivo experiments suggested the possibility of up-regulation on thymic cells. In conclusion, the results obtained in the present study provide information on a potentially useful marker (mAb TcOm15) for a cytoskeleton-membrane antigen that is modulated during stimulation of teleost lymphocytes. Additionally, this may enable insights into the relationship between cytoskeletal proteins and membrane associated immunoglobulin. Future research is necessary in order to explain this relationship and to determine the functional participation of the TcOm15 molecule during the activation of rainbow trout cells.

From Introduction: Rainbow trout (Salmo gairdneri) were introduced into Europe from North America in the latter half of the last century. They can tolerate higher water temperatures and lower oxygen concentrations than the native brown trout (Salmo trutta). Rainbows grow faster than brown trout under similar conditions and are thus particularly attractive for artificial rearing methods. In Denmark there is a thriving rainbow trout farming industry producing about 9,000 metric tons annually which is largely exported for table use (Mills 1971). In Britain production of rainbow trout for food is not on such a large scale but they form the basis of a considerable sport fishery. In Scotland and Northern England although rainbow trout reach sexual maturity they do not generally breed so the population is entirely dependent on restocking with hatchery reared fish, thus although living more or less wild in many British waters , this species is essentially an artificially managed resource upon which man can impose genetic selection (Donaldson and Olson 1957) as well as normal fishery controls. A detailed understanding of the biology and physiology of this species is hence of particular importance.

Through the implementation of in vivo feeding trials, the efficacy of three wheat gluten (WG) products, vital (Amytex®), hydrolysed (Merripro®) and soluble hydrolysed (Solpro®) wheat gluten as replacement of soy protein concentrate, and scFOS prebiotic (Profeed®) supplementation were analysed to assess their impacts on intestinal health and production of juvenile rainbow trout. Microbial community analysis in experiment one revealed a degree of diet based modulation with 7.5% and 15% inclusions of wheat gluten (WG) products. Bacterial species diversity was significantly reduced with 15% hydrolysed wheat gluten (HWG) inclusion compared to the plant protein control and 15% vital wheat gluten (VWG) treatments, with sequenced OTUs dominated by the phylum Firmicutes and possible promotion of probiotic species. No detrimental effects were observed on intestinal morphology. These findings led onto a longer duration feed trial with a more holistic, higher resolution approach. Experiment two revealed modulation of the allochthonous intestinal microbiota, with increased proportions of Enterococcus and Weissella in the 10% and 20% VWG treatments. Bacillus and Leuconostoc relative abundances were significantly increased with 10% HWG and soluble hydrolysed (Sol) wheat gluten inclusions. HSP 70 transcripts were significantly down-regulated in all WG treatments compared to the basal soy protein concentrate treatment (SPC) and increased intraepithelial leukocyte counts were observed with 10% VWG inclusion. Growth performance was unaffected by 10% dietary inclusions of WG, however, FCR’s were significantly improved in the 20% VWG treatment compared to the 10% HWG and Soluble treatments. This led to the investigation of increased inclusion levels of WG products in experiment three. All WG treatments in experiment three yielded significantly improved growth performance. Somatic indices were significantly increased with 30% blended WG inclusion compared to the SPC treatment. Modulation of allochthonous intestinal microbiota was observed to a lower degree than the previous experiments, with a dose response observed with increasing blended WG inclusion. In the final experiment two basal diets (SPC and 20% Blended) and two scFOS supplemented diets (SPC + FOS and 20% Blended + FOS) were investigated for the effect on growth performance, gut health and allochthonous microbial population. Growth performance was unaffected, however, modulation of the allochthonous microbial population was observed with an apparent synergistic effect of scFOS supplementation in WG diets. This synergistic trend was also observed in the transcription level expression of immune relevant genes. 20% WG inclusion with additional scFOS supplementation observed significant down regulation of the pro-inflammatory cytokine TNF-α, as well as HSP 70, CASP 3 and Glute ST compared to the 20% Blend treatment. The present research demonstrates dietary inclusions of WG products, solely or blended, at the expense of soy protein concentrate to modulate the allochthonous microbial population, potentially promoting probiotic species, whilst reducing the levels of intestinal stress in juvenile rainbow trout. Supplementation of the prebiotic scFOS modulated the microbial populations, enhancing the proportion of potential probiotic species, and combined with WG inclusions, reduce intestinal and oxidative stress and inflammation biomarkers, with no observed deleterious effects.

The critical swimming speed (Ucrit) of rainbow trout parr (Oncorhynchus mykiss) and three life stages of Galaxias maculatus, larval (whitebait), postlarval inanga and adult inanga, were tested at temperatures from 5oC to 25oC. All fish were swum at their acclimation temperature under normoxic conditions to determine the optimal aerobic exercise temperature. To determine whether acclimation affected swimming ability, trout parr acclimated to either 10oC or 20oC were swum at 20oC and 10oC, respectively. The potential effect of mild hypoxia (75% saturation) on trout parr and whitebait was also examined at 10oC, 15oC and 20oC, and also tested separately and in combination were the effects of mild hypoxia and severe anaemia on the prolonged swimming ability of trout smolts at temperatures from 10oC to 20oC. For all trout experiments, blood samples were taken from non-exercised and exercised fish by acute caudal venepuncture to determine haematological responses to both acclimation and exercise. Under normoxic conditions, Ucrit max for trout parr (7.0 0.5 cm fork length) was calculated to be 5.8 body lengths per second (BL s-1) at 15.1oC, but declined at lower and higher temperatures. This result implies that swimming performance was limited by temperature below 15oC, whereas performance at higher temperatures was limited by oxygen availability. In support of this hypothesis, mild hypoxia (75% saturation) had no effect at 10oC or 15oC but caused a significant reduction in Ucrit at 20oC. However, fish acclimated at 20oC showed an adaptive elevation in oxygen carrying capacity due to an increase in mean erythrocyte volume and haemoglobin content. Furthermore, acclimation to 20oC improved warm water swimming performance. Trout parr acclimated to 10oC performed significantly worse than fish acclimated to 20oC when swum at 20oC. However, trout parr acclimated to 20oC performed as well as fish acclimated to 10oC when swum at 10oC. Following exercise, haematocrit was elevated under both normoxic and hypoxic conditions. However, the primary cause of this apparent increase in oxygen carrying capacity was splenic release of erythrocytes under normoxic conditions, whereas stress-induced erythrocytic swelling contributed to the observed increase in hypoxia. This contrasting response was most pronounced at 10oC. Larval whitebait (4.7 - 5.0 cm total length (TL)) also showed a temperature dependence of prolonged swimming ability with Ucrit max calculated to be 5.1 BL s-1 at 17.7oC. Hypoxia significantly reduced Ucrit at 15oC and 20oC, lowering the optimal aerobic temperature to 13.9oC and reducing Ucrit to 4.2 BL s-1. Mild hypoxia therefore had a more pronounced impact on inanga whitebait than trout. Postlarval inanga (3.9 - 4.0 cm TL) performed poorly at higher temperatures with Ucrit max of 5.6 BL s-1 at 9.4oC indicating an ontogenetic change in swimming ability, possibly resulting from a developmental shift in red muscle kinetics or a greater dependence on anaerobic muscle. Adult inanga (5.5 - 6.8 cm TL) prolonged swimming ability showed similar temperature dependence to that of inanga whitebait but lower relative swimming speeds due to their larger size. The dramatic decline in performance exhibited by juveniles at warmer temperatures was not apparent in adults. Ucrit max for adults was 4.0 BL s-1 at 18.3oC. The critical swimming speed of trout smolts, subjected to mild hypoxia (6.8 mg

The characteristics of an inhibitor of infectious pancreatic necrosis virus (IPNV) found in normal rainbow trout serum (RTS) were studied. The serum inhibitor had a molecular weight of approximately 150 kDa and was dependent on divalent cations, either Ca����� or Mg�����. It was stable at temperatures up to 50��C and at a pH range between 4-10. The inhibitor directly inactivated the virus and the inhibition level was dependent on cell densities and on the time at which virus was exposed to RTS. The level of virus inhibition by RTS was altered by the cell line in which virus was produced. IPNV was more efficiently inhibited by RTS in salmonid cell lines than in non-salmonid cell lines. Most of the salmonid sera tested showed inhibition, while non-salmonid sera did not inhibit IPNV replication. Rainbow trout continuously showed a significant level of inhibition in their serum after 23 weeks post hatch. Three isolates of IPNV were passaged five times in RTG-2 cells with either MEM-10 or MEM-10 with 1% rainbow trout serum and virus from each passage were tested for RTS sensitivity in vitro and virulence in vivo. The mortality level in brook trout fry was highly variable during viral passages, ranging between 30-89%. The RTS sensitivity and virulence were changed during viral passages, and these changes were dependent on cell culture conditions and IPNV isolate used. It was found that an IPNV crayfish isolate passaged in RTG-2 cells with MEM-10 showed significantly increased RTS sensitivity. This was, however, not correlated with decreased virulence. All three isolates showed identical antigenicity patterns with a panel of 11 monoclonal antibodies, irrespective of viral passage conditions. Clones prepared from an IPNV-Jasper (Ja) population which had been twice passed through brook trout were heterogeneous with respect to RTS sensitivity, serotype, and cDNA sequences. Eight percent of clones (4/50) were very sensitive to RTS (Ja-S), as was the parent strain, and eighty four percent of clones (42/50) showed a mid-range of RTS sensitivity. The final eight percent of clones (4/50) were RTS resistant (Ja-R). Enzyme immunodot assay revealed that Ja-S clones and Ja-R clones differed by several epitopes. Ja-S and Ja-R had significant differences in their cDNA sequences for the capsid protein VP2. These two strains shared 80.7% and 86% identity in nucleic acid and in amino acid sequences, respectively. / Graduation date: 2001

Previous reports indicate the presence of multiple CYP1A sequences in rainbow trout, but their functional differences are unknown. This report describes the cloning and partial characterization of four trout CYP1A cDNAs, which are given the tentative designations CYP1A1v2, v3, v4, and v5. Comparison among these four and three previously reported trout CYP1A sequences reveals that all of the nucleotide and translated amino acid sequences all are closely related (96.9-99.4% cDNA identity; 95.2-99.4% amino acid sequence identity) but none are identical. Six of these sequences encode proteins of 522 amino acids, and one encodes a protein of 536 amino acids. Expression vectors containing the cDNAs for CYP1A1v2, v3, and v4 were transformed into yeast, yielding microsomal hemoprotein CYP contents (63, 156, 96 pmol/mg) comparable to those reported for human CYP1A1 (68-156 pmol/mg) expressed in this system (Eugster et al., 1990, Biochem. Biophys. Res. Commun. 737-744). Kinetic analysis of CYP1A1v2 and v3 proteins indicated similar but not identical Michaelis constants (20��3 vs 13��2 ��M) and molar activities (508��47 vs 218��19 pmol/min/nmol P450) for oxidation of aflatoxin B��� (AFB���) to aflatoxin M a reaction characteristic of human CYP1A2. Trout CYP1A1v2 and v3 exhibited lower activity for production of AFB,-8,9-exo-epoxide, also a human CYP1A2 activity. Kinetic data for ethoxyresorufin 0deethylation, a prototypical mammalian CYP1A1 activity, also revealed modest but distinct differences in which CYP1A1v3 was more active for this substrate (Km=0.07 �� 0.01 ��M, Vm=1398 �� 95 pmol/min/nmol P450) than was CYP1A1v2 (Km=0.15 �� 0.03 ��M, Vm=684 �� 83 pmol/min/nmol p450). Interestingly, CYP1A1v4 showed no catalytic activity towards AFB ethoxyresorufin, or 7,12-dimethylbenzanthracene despite formation of a hemoprotein. These results together with previous studies demonstrate the presence in various rainbow trout populations of at least seven CYPIA cDNAs representing gene duplication or allelic variation. Present results show that one of three such cDNA sequences encodes a CYP1A hemoprotein with no apparent catalytic activity, that two of the encoded proteins possess certain catalytic properties common to both human CYP1A1 and CYP1A2, and that the sequence differences, though small, are reflected in enzymic properties that can be distinguished. / Graduation date: 1998

Graduation date: 1993

Thesis (PhD)--Montana State University--Bozeman, 2008. / Typescript. Chairperson, Graduate Committee: Thomas E. McMahon. Includes bibliographical references.

Thesis (Ph. D. biochemistry)--Washington State University, May 2008. / Includes bibliographical references.

Thesis (Ph. D.)--University of Wyoming, 2006. / Title from PDF title page (viewed on June 30, 2008). Includes bibliographical references (p. 96-98).