Vaccination and the use of immune stimulants are two important ways to mitigate the costs of disease in fish aquaculture. A vaccine to Enteric redmouth disease (ERM) was the first licensed fish vaccine in the world. Although effective in protecting fish from the motile bacterial (Yersinia ruckeri) infection, ERM can occur in ERM vaccinated fish due to the rise of non-motile Y. ruckeri that does not express flagellin. This highlighted the need for continual improvement of vaccine efficacy and the importance of flagellin in fish immune responses. In this thesis the immune response to the ERM vaccine was studied first to give insights for vaccine development. A recombinant flagellin from Y. ruckeri (YRF) was then produced and its bioactivities were investigated in vitro and in vivo. The immune response to ERM vaccination was studied in rainbow trout in two major and relevant immune organs, the spleen and gills. Intraperitoneal injection of the ERM vaccine induces an early balanced expression of pro- and anti-inflammatory cytokines and adaptive cytokines in the spleen, with a heightened expression of acute phase proteins (APPs) and anti-microbial peptides (AMPs) in both spleen and gills. The analysis suggests that ERM vaccination activates host innate immunity and the expression of specific IL-12 and IL-23 isoforms leading to a Th1 and Th17 biased immune responses. This study has increased our understanding of the host immune response to ERM vaccination and the adaptive pathways involved. The early responses of a set of genes established in this study may prove useful as biomarkers in future vaccine development in aquaculture. YRF was next produced in a bacterial system, and purified. Its bioactivity was investigated first in the trout macrophage cell line RTS-11 and head kidney primary cell cultures. YRF is a potent activator of pro-inflammatory cytokines, APPs, AMPs and subunits of the IL-12 cytokine family in vitro. This property was further confirm in vivo in multiple tissues after intraperitoneal injection of YRF. These results suggest that flagellins are important pathogen-associated molecular patterns (PAMPs) that can activate an inflammatory response in fish not only in vitro but also in vivo. Furthermore, YRF was shown to be the most potent PAMP in vitro, in terms of activation of an inflammatory response, compared to pure LPS and peptidoglycan. In addition, YRF mixed with complete Freund's adjuvant can induce YRF-specific IgM antibodies in rainbow trout. These antibodies are able to neutralize YRF bioactivity, and react against the middle domain of YRF, as assessed in Western blot analysis. When YRF was fused with a protein antigen, it increased the antigen-specific IgM antibody response. This analysis reveals that YRF is a potent activator of host immune responses and can be used as an immune stimulant and adjuvant to improve vaccine efficacy.

Graduation date: 2001

Graduation date: 2000

Recent advances in DNA vaccine technology has brought about a promising strategy for the control of viruses that contain surface membrane glycoproteins. This type of vaccine involves the intramuscular injection of a bacterial plasmid containing a gene encoding a viral protein. The strategy uses eukaryotic processing of the protein as would naturally occur during a viral infection. In this study, plasmid DNA encoding the glycoproteins of infectious hematopoietic necrosis virus (pcDNA3-IHNV-g), snakehead rhabdovirus (pcDNA3-SHRV-g), or spring viremia of carp virus (pcDNA3-SVCV-g) was injected into the skeletal muscle of rainbow trout fry. At 30 days post-vaccination, fish were challenged with IHNV. Protection against IHNV was observed among all DNA vaccinated groups. Fish injected with plasmid pcDNA3-IHNV-g, pcDNA3-SHRV-g, or pcDNA3-SVCV-g had relative survival rates of 93.2%, 98.3% and 94.9%, respectively. The mechanisms for the viral mediated resistance induced by these glycoprotein based DNA vaccines is unknown. A parallel study conducted by Dr. Carol Kim on the production of Mx proteins in these fish indicates that the observed protection might be a consequence of the stimulation of interferon. / Graduation date: 1999

Rainbow trout cystatin C cDNA has been isolated from trout liver. The full-length cystatin cDNA (674 bp) included the 5'untranslated region and the polyadenylation signal sequence AATAAA in the 3' region. Translation of the cDNA defines 132 amino acid residues. Comparison of the amino acid sequence with those of family 2 cystatins indicates that the 21 amino acids at the N-terminal end is a signal peptide necessary for cystatin secretion, and the remaining 111 amino acids represent mature cystatin. Four cysteine residues in the cystatin may form two disulfide bonds producing a molecule with the properties of a family 2 cystatin. Trout cystatin C gene expression was analyzed by Northern blot. This gene is expressed at various levels in all tissues examined. This difference may reflect differences in degree of regulation of cysteine proteinase activities. A high level of trout cystatin C expressed in trout hepatic tissue or cell cultures suggested that cystatin C expression might be related to tumorigenesis. Southern blot of trout genomic DNA showed that the copy number of the trout cystatin gene is probably one per haploid genome. Trout cystatin C was expressed in E. coli at a yield of 3-5 mg/L culture, but no inhibitory activity was detected for the untreated recombinant protein. However, after refolding, recombinant cystatin C displayed inhibitory activity against papain. The dissociation constant of recombinant cystatin C against papain is 1.2 x 10������ nM, similar to that of human cystatin C. Trout cystatin C was also expressed in yeast cells, but no inhibitory activity was detected either. No cystatin C was secreted in the yeast expression system using either the trout cystatin C secretion signal, or the yeast invertase secretion signal. The expression levels of trout cystatin C in our expression systems are still low for industrial requirements. Therefore, further investigation will be needed to construct more efficient expression systems and vectors for trout cystatin C heterologous production. / Graduation date: 1999

Graduation date: 1996

The immune systems of various teleost fish have been studied in some detail for the past several decades. One aspect of fish immunity, that of endogenously produced modulating factors, has recently received a great deal of attention. Understanding the functions and roles of endogenous factors that regulate fish immunity is paramount to expanding the fields of fish immunology and vaccinology. It is know that several lymphoid cell derived factors are detectable in in vitro cell culture systems and exhibit immune modulating effects similar to well studied proteins in mammals. However in comparison, few genes or gene products involved in the modulation of the trout immune responses have been isolated, cloned and characterized. The studies described herein were designed to isolate specific genes from rainbow trout (0ncorhynchus mykiss) and characterize their involvement in the modulation and regulation of the trout immune system. Two distinct genes were isolated cloned and sequenced. The first, non-specific cytotoxic cell enhancement factor (NCEF) gene is closely related to a human gene termed "natural killer enhancement factor" (NKEF) which is important in the modulation of human natural killer cell activity. The second gene is closely related to a group of recently characterized mammalian genes involved in the signal transduction of cytokines termed "STATs". The role of these genes and their respective protein products will be examined and discussed. The antigenic structures of the fish proteins (NCEF and STAT5) were examined by western blot and immunohistochemistry. Monoclonal antibodies derived against the respective human proteins were found to cross react with the corresponding trout proteins, demonstrating antigenic relatedness. The monoclonal regents were also used to analyze the expression of these proteins in fish cells of lymphoid and non lymphoid origin. In vitro cell culture analysis was used to determine the effects and roles of NCEF and STAT5 gene products in the trout immune system. The cytolytic and apoptotic killing activities of spleen, head kidney and peripheral blood leukocytes were found to be enhanced by NCEF. Mitogenic stimulation of peripheral blood lymphoid cells resulted in the trout STAT5 protein binding to a know sequences contained with in the promoters of genes transcriptionally activated in response to cytokine exposure. / Graduation date: 1996

Graduation date: 1996

Graduation date: 1995

Graduation date: 1995