Identification of immunogenic candidate antigens, proteins expressed in vivo, and development of attenuated strains of Flavobacterium psychrophilum for vaccine development /by Benjamin Ryan LaFrentz.

Flavobacterium psychrophilum, the etiological agent of bacterial coldwater disease (CWD) and rainbow trout fry syndrome (RTFS), has emerged as one of the most important bacterial pathogens in salmonid aquaculture worldwide. A number of experiments were conducted to identify immunogenic candidate antigens as well as unique proteins expressed in vivo and to develop attenuated strains for vaccine development.;Electrophoretic and Western blot analyses identified the presence of two distinct carbohydrate-handing patterns, corresponding to the lipopolysaccharide (LPS) and the glycocalyx F. psychrophilum. Carbohydrate components previously speculated to be LPS with O-polysaccharide and a possible vaccine candidate antigen were determined to actually be the glycocalyx capsular layer. This research suggests that the glycocalyx of F. psychrophilum may he an important antigen to consider for vaccine development.;The ability of a crude LPS extract to provide enhanced protection against F. psychrophilum infections was evaluated in rainbow trout (Oncorhynchus mykiss Walbaum). The results suggested that antibodies specific for low molecular mass proteins present in the crude LPS preparation of F. psychrophilum may play a greater role in protection against CWD than the LPS.;An immunoproteomic analysis of F. psychrophilum was performed using serum from rainbow trout immunized with high and mid-molecular mass fractions of the bacterium. A total of 15 immunogenic proteins were positively identified following sequence comparison to the F. psychrophilum genome and at least five proteins appear promising as vaccine candidates based on known function. These include the DNA chaperone DnaK (heat shock protein 70), outer membrane protein OmpA, trigger factor TF, elongation factor EF-TU, and gliding motility protein GldN.;A proteomic approach was used to identify proteins of F. psychrophilum that are expressed during in vivo and iron-limited growth conditions. A number of proteins exhibited increased expression, some of which included chaperone and heat shock proteins, the gliding motility protein GldN, outer membrane protein OmpH, two probable outer membrane proteins (OmpA family), and several proteins with unknown function. The results suggest that some of these proteins may be involved in the pathogenesis of F. psychrophilum and may serve as vaccine candidate antigens.;Finally, an attenuated strain of F. psychrophilum, 259-93B.17, was produced by selecting for resistance to rifampicin. Immunization of rainbow trout with this strain by intraperitoneal injection resulted in significant protection against challenge with a virulent strain. The results demonstrate that the attenuated 259-93B.17 strain may serve as an effective live vaccine for the prevention of F. psychrophilum infections.Thesis (Ph. D., Natural Resources)--University of Idaho, December 2007