Graduate Thesis Or Dissertation
 

Development of novel oral enteric-coated aquaculture vibrio vaccines

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  • An oral Vibrio vaccine for salmonids was developed. The vaccine was produced by spray coating lyophilized formalin-killed whole cells of Vibrio anguillarum (VA LS 1- 74) onto non-pareil sugar beads. Then methacrylic acrylic acid copolymer (Eudragit L-30D) was applied as an enteric protective coating. Using x-ray radiographic techniques, it was found that large particles (> 1.1 mm) remain in the fish stomach for more than 2 hours before they would enter the pyloric caeca. The pyloric sphincter which has an opening of 0.94 mm, acts as barrier to prevent the passage of large food particles in the stomach to the pyloric caeca. Based on this information non-pareil sugar beads of 18-20 mesh or smaller should be used as the vaccine carriers. A 15% (w/w) Eudragit L-30D coating is needed to provide enteric protection of the vaccine loaded sugar beads of 18-20 mesh size. Lower levels of coating resulted in the bead breaking down in the stomach and releasing contents prior to entering the pyloric caeca. Since the lymphoid tissues are diffuse throughout the whole GI tract, it may not be necessary to target a vaccine to deliver antigens to a specific area of the intestinal tract, but only protect the antigens from gastric fluids. In vitro dissolution studies indicate that 10% VA LS 1- 74 loading was sufficient for rapid vaccine release (42% released in 30 minutes) and a 15% Eudragit L-30D coating was suitable for providing protection against stomach acid. The vaccine product used in vivo studies contained 10% VA LS 1- 74 and 15% Eudragit L-30D on non-pareil sugar seeds of 18-20 mesh size. Coho salmon were given the vaccine orally, and 30 days afterward a live challenge test was performed. There was no significant difference in the survival rates in a live bacteria challenge test with the positive control (83.3%) and test (80.3%) groups. Both had higher survival rates than the no vaccine fed control group. The serum and mucosal antibody levels to Vibrio were significantly higher (p<0.01) in the test group (19700 units/ml) than the other two groups (2530 units/ml in the positive control group and 617 units/ml in the negative control group). The antibody titer appears to be a better indicator for vaccine efficacy than survival rate of live bacteria challenge tests. The oral Vibrio vaccine developed is effective, and the technique to protect the antigen can be applied to other antigens or proteins for oral delivery producing an economical pathway for mass vaccination of fish.
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