A programme of research was undertaken to assess the feasibility of the application of gene cloning techniques to Renibacteriun salmoninarum, an important, but as yet poorly characterized pathogen of salmonid fish. Difficulties encountered during in vitro culture, have restricted our knowledge of the bacterium, especially with respect to ita mechanisms and molecules of pathogenicity. A collection of R.salmoninarum isolates was made, and an initial study conducted to determine target molecules for cloning. All strains tested were found to have uniform protein profiles when analysed with SOS-PAGE, and uniform antigenic profiles when subject to immunoblotting experiments utilizing rabbit antisera raised against both whole cell and ECP preparations of the bacterium. The presence of haemolytic and proteolytic activity, putative pathogenicity determinants of R.salmoninarum, could not be established. Genomic DNA extracted from isolates of R.salmoninarum was used to construct gene libraries in a variety of E. coli K/2 host vector systems. Gene libraries were screened with antisera for the presence of R.salmoninarum antigens, and with erythrocytes and a range of natural and synthetic substrates for the presence of haemolytic and proteolytic components. As a result of the screening process, a stable haemolytic E. coli "clone was detected and isolated from a pHC79/R.salmoninarum/Sau3A gene library. This clone contained the recombinant cosmid molecule pRHLY1 bearing a gene for RHLY, a putative R.salmoninarum haemolysin on a DNA insert of approximately 32 kbp. The RHLY encoding gene, designated rsh, was subcloned on a 1.65 kbp DNA fragment to form the recombinant plasmid pRHLYll, for which a preliminary restriction endonuclease cleavage map was constructed. Southern blot hybridization was performed, and established that the fragment containing the rsh gene was a true representative of a R.salmoninarun genomic DNA sequence, present in all isolates studied. Mini cell analysis of the cloned rsh gene and immunoblot of the pRHLY -containing E. coli clone with antiserum to R.salmoninarum whole cells, revealed that haemolytic activity is mediated by a protein of an approximate size of 48-50 kDa. Haemolytic activity could not be detected in cell-free assay and RHLY was not purified, but experiments conducted with RHLY -containing E. coli indicated that the protein is active against a wide spectrum of animal erythrocytes, across a wide temperature range. The specific mode of cytolytic activity was not established. Immunoblot analysis utilizing an antiserum raised to a β-galactosidase/RHLY fusion protein, revealed that a similar protein is found in whole cell preparations of R.salmoninarum. The nucleotide sequence of the rsh gene was determined, and was shown to contain an ORF of 1248 bp. Putative transcriptional/translational control regions were identified for the rsh gene, and the primary amino acid sequence of the RHLY protein determined. A computer-aided search of the PIR protein sequence database, failed to detect any protein with significant homology to RHLY, therefore, conclusive evidence for structure/function of the molecule was not obtained. However, the RHLY protein was found to have a 30-35 amino acid motif, conserved and repeated twice, that shares significant homology with a repeated motif in the sequence of dniR protein, the product of a gene in E. coli K12 that regulates the expression of the enzyme nitrite reductase. Future studies of the RHL Y protein, the rsh gene, and other components of R.salmoninarum are discussed.

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This research investigates the links between nutrient enrichment, bryozoan abundance and the development of myxozoan parasites in bryozoans. It was found that bryozoan abundances are elevated in nutrient-rich rivers, demonstrating the potential bio-indicator value of bryozoans and predicting increased bryozoan abundances with increased planktonic productivity when space is not limiting. A comprehensive set of laboratory microcosm studies showed also that experimental nutrient enrichment increased growth rates and statoblast production in Fredericella sultana, corroborating the results from field surveys.

L'impact de la nutrition sur l'immunité intestinale de la truite arc-en-ciel est encore mal connu. C'est pourquoi cette thèse avait pour objectifs de mieux caractériser son système immunitaire intestinal et d'évaluer les possibilités de modulation de la réponse immune intestinale par l'ajout de nucléotides libres dans son alimentation. Nos résultats indiquent que les phagocytes intestinaux présentent une activité de phagocytose plus faible que ceux du rein antérieur. La cytotoxicité naturelle mesurée au niveau intestinal est deux fois plus élevée que celle du rein antérieur et cette observation est corrélée à une augmentation du transcrit codant NKEF (Natural Killer Enhancement Factor). Nous avons également montré que les lymphocytes intestinaux ne répondent pas à une stimulation mitogénique in vitro et que ceci n'est pas due à l'apoptose des cellules. Une forte expression des transcrits codant CD8a et CD3 a été détectée dans les leucocytes intestinaux, ce qui suggère une importante proportion de lymphocytes T exprimant la forme homodimérique aa de CD8 dans ce tissu. Enfin, nous avons montré que l'ajout de nucléotides libres à l'alimentation de truites saines stimule la prolifération spontanée ainsi que la phagocytose des leucocytes intestinaux in vitro. Par contre, aucune modulation de la cytotoxicité naturelle ou de l'expression des transcrits codant les marqueurs spécifiques des lymphocytes T et B et les cytokines inflammatoires n'a été observée. Il serait à présent intéressant de renouveler ces essais en utilisant des poissons infectés afin de pouvoir observer l'effet des nucléotides sur la réponse inflammatoire et sur la réponse spécifique / The impact of nutrition on rainbow trout intestinal immunity, a farmed fish with high economic value, remains unclear. Consequently, the objectives of this thesis were to better characterize the intestinal immune system of that fish and to determine if it is possible to modulate its intestinal immune response by dietary free nucleotides. Our results show that intestinal phagocytes are less activated by yeast cells but when they are activated they can ingest as many yeast cells as their head kidney (HK) counterparts. We noted that the natural cytotoxic activity of intestinal leukocytes is twice higher than the one of HK leukocytes. This natural cytotoxic activity is correlated with an increase of transcripts encoding the natural killer enhancement factor (NKEF). Intestinal leukocytes did not respond to an in vitro mitogenic stimulation. This lack of response is not due to apoptosis. We also observed a high expression of CD8a and CD3 transcripts in gut leukocytes, suggesting that the intestine could contain a high proportion of T cells expressing the aa homodimeric form of CD8. Finally, we observed that dietary free nucleotides stimulate the spontaneous proliferation and the phagocytic activity of intestinal leucocytes in vitro. However, they did not modulate natural cytotoxicity activity nor did they affect the amounts of transcripts encoding specific markers of T and B lymphocytes and inflammatory cytokines. In the future, it will be interesting to repeat these experiments using infected fish in order to study the effect of nucleotides on the inflammatory and specific immune responses