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Two types of estrogen binding sites were found in the rainbow trout liver cytosol, using the equilibrium binding assay. The higher affinity, lower capacity type I site had a Kd range of 0.53-5.9 nM and a concentration range of 14-95 pmoles/g protein. The lower affinity, higher capacity type II sites had a Kd range of 65-265 nM and a concentration range of 20-180 pmoles/g protein. These estrogen binding sites are both bound by ^3H-moxestrol. The estrogen binding sites in the serum were also examined and two components were found with the higher affinity component having a Kd of 2.63 nM and a concentration of 25 pmoles/g protein while the lower affinity component had a Kd of 79 nM and a concentration of 200 pmoles/g protein. However, these estrogen binding components are unable to bind the synthetic estrogen, DES, and therefore cannot account for either of the binding components found in the liver cytosol which bind DES as readily as 17β-estradiol. The cytosol estrogen binding sites bind non-estrogens in addition to estrogens. Sucrose gradient centrifugation of the cytosol yielded two estrogen binding peaks, one at 4.4S, the other at 3.7S. The heavier peak contained binding sites able to bind progesterone as well. Gel filtration of the cytosol also resulted in two peaks, one at 43,000 daltons, the other at 33,000 daltons. Again the heavier peak could be partially competed out by progesterone. The half life of these binding components was 60 minutes at 37°C, while no decrease in binding was observed after 4 hours at either 0 or 12 0 c. After 17β-estradiol treatment in vivo type I sites (relative to type II sites) were 74% depleted after 8.5 hours and 40% depleted after 24 hours. Finally, o,p'-DDT and p,p'-DDT, components of technical grade preparations of the insecticide DDT were both able to compete for estrogen binding sites in the rainbow trout liver cytosol and therefore may be able to affect the expression of estrogen inducible genes. / Thesis / Master of Science (MSc)

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.

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

Graduation date: 1993

Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (M.S.)--Montana State University--Bozeman, 2005. / Title from PDF t.p. (viewed on June 10, 2006). Chairperson, Graduate Committee: Thomas McMahon. Includes bibliographical references (p. 52-62).

Understanding that disease is a limiting factor to the aquaculture industry together with the knowledge that drugs and chemotherapeutics can cause newly resistant bacterial strains, has driven attention to finding new prophylactic measures to control diseases that include vaccination and the use of “functional feeds” to modulate the fish immune system. The supplementation of immunostimulants into fish diets is already widely used in aquaculture. However, searching for new and effective substances is one of the targets of many fish feed suppliers, including EWOS Ltd. who have co-funded the work presented in this study. There are a number of immunostimulant molecules used at present. Some bacterial components such as LPS are used to enrich fish diets and have been described to improve the natural immune defences. In contrast, peptidoglycan (PG), another ubiquitous component of the bacterial cell wall, has so far received less attention and is therefore investigated in this present study. Its ability to stimulate innate immunity is assessed using antimicrobial peptides (AMPs) as molecular markers, which are known to be involved in the early response against a broad range of pathogens. To date, AMPs in fish are not well characterised and in most cases the mechanisms of pathogen killing as well as the pathways inducing their expression still remain to be elucidated. Initially the cloning and characterisation of three novel trout β-defensin genes (omDB-2, omDB-3, omBD-4) was performed, and the molecules compared to the previously reported omDB-1. Each β-defensin gene was fully cloned and preliminary expression work in vivo and in vitro revealed the ability of these genes to be induced by bacteria and viruses. Analysis of the gene organization found that all three new genes contained three exons divided by two introns. Constitutive expression of these genes was detected by real time PCR ofmucosal and systemic tissues from healthy fish, with omDB-3 and omDB-4 showing the highest expression levels. Following bacterial challenge in vivo, the defensin genes were induced at the three mucosal sites examined (skin, gill, gut), with levels of omDB-2 and omDB-3 increased some 16-fold in gut and gill respectively. Using polyinosinic polycytosinic RNA (polyI:C) as a viral mimic, all of the four trout -defensin genes were induced in head kidney primary leucocyte cultures at 4h post-stimulation, with omDB-1 and omDB-3 showing particularly high expression. To determine the -defensin spectrum of activity against 10 strains of Gram negative and Gram positive bacteria, transfected RTG-2 cell lines over expressing GFP and the target genes omDB-1, omDB-3 and omDB-4 -defensins were produced and their supernatants used. Results showed highest bioactivity against Gram negative bacteria, in particular the supernatant from omDB-1 transfected cells showed the widest range of activity towards the majority of selected bacteria. In addition immune relevant genes (Toll-like receptors, genes involved in the anti-inflammatory response and in the apoptosis process) were screened in normal cell lines stimulated with the supernatant of omDB-1, as well as in the RTG-2 cells transfected with the three different defensins. Results showed for all the cell lines, a clear link with the viral recognition receptors TLR 3 and TLR 9, which supported the poly I:C data reported in Chapter 2 and by the induction in omDB-1 and omDB-3 transfected cell lines of the IFN- gene known to be involved in the antiviral response. Trout β-defensins also up-regulated MHC II and the CCR6 receptor. To determine the effects of fish diets enriched with different concentrations of PG, three in vivo feeding trial experiments in rainbow trout were carried out. Effectiveness of the diets was assessed using gene expression of selected AMPs, including β-defensins, cathelicidins and liver expressed antimicrobial peptide molecules. Fish fed with diets containing either 10 mg/Kg or 50 mg/Kg of PG respectively, showed the highest up-regulation of AMPs at 14 days of feeding. Data showed omDB-2 in the gut as the most inducible gene in agreement with the results obtained in the first experiment and omDB-3 was the fastest to respond in skin and gill. In addition, after ceasation of feeding the enriched diet, modulation of AMP expression was still detectable 28 days later, although a lower degree of induction was found in such fish relative to those maintained on the enriched diet. A final PG feeding trial was combined with a Yersinia ruckeri bacterial challenge which used two PG supplemented diets containing 10 mg/Kg and 50 mg/Kg of immunostimulant, and a commercial β-glucan supplemented diet (as a positive control), and fed to trout for 7 and 14 days before intraperitoneal injection challenge of the fish. Only a delay in the mortality rate was found in fish fed for 14 days with the 10 mg/Kg diet, with no clear protection from any of the functional feeds assessed. Finally, at least 500 bp of the regulatory 5’ end flanking region of two defensin (omDB-1 and omDB-2) and two liver expressed (hepcidin and LEAP-2A) genes were cloned and sequenced. In addition, the promoter sequence already known for the cathelicidin-1 gene was used in this study. Bioinformatic tools were used to search for putative transcription factor binding sites, and revealed the presence in all promoters of regulatory elements which could enhance or inhibit the expression of these genes, in response to different stimuli.