Progress 07/01/22 to 07/31/23
Outputs
Target Audience: We aim to reach four primary audiences: aquaculture farmers, animal feed producers, academic institutions housing fish health researchers, and federal agencies like USDA NIFA. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two scientists led the studies and mentored post-docs and undergraduates in the project. One technical staff at PhycoVax assisted in diatom transformation, quality control, large-scale cultivation and downstream processing. One post-doctoral researcher at UC Davis was involved in the planning and execution of the challenges. She also assisted in developing and carrying out vaccine protocols and training of undergraduates. Two undergraduate students at UC Davis were also trained in fish husbandry, sample collection and molecular analysis, including gene expression and quantification of bacterial loads via qPCR. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
APPROACH: A particle bombardment method was employed to transform the antigen IglC into the diatom, which resulted in transgene integration into the nuclear genome at random sites. The IglC was tagged with eGFP to sort diatom clones with a high recombinant protein expression level by a fluorescence cell sorter. The best-expressing lines were subjected to large-scale cultivation for whole-cell oral vaccination of tilapia. Tilapia were acclimated for four weeks before the experiment and fed at 2% body weight daily. Tilapia were immunized by providing an experimental diet composed of 50% regular feed plus 50% diatom biomass expressing eGFP-IglC, eGFP-adjuvant, and eGFP-adjuvant-IglC for two consecutive days a week for 1, 2, or 4 weeks after 24 hr fasting to ensure that fish consume all the diet provided. Two different sets of controls were used. The first control was fed commercially available feed alone, and the second control treatment was fed feed mixed with wild-type diatom cells without transformation. Five treatments were immunized as follows: A. Commercial feed alone (control 1), B. Commercial feed mixed with wild-type diatom cells without transformation, C. Commercial feed mixed with eGFP-IglC, D. Commercial feed mixed with eGFP-adjuvant, and E. Commercial feed mixed with eGFP-adjuvant-IglC vaccines orally administered for 1, 2, or 4 weeks. Four replicate tanks with 20 fish per tank (n=80 fish per treatment) were used for each condition. Five weeks post-initial vaccination, fish were challenged by immersion for 5 hrs at 25±1°C using 1x105 CFU/mL of wild type F. orientalis. Another set of treatments were maintained in similar conditions but weren't challenged. Twenty-four hours post-challenge, eight fish per treatment were collected and euthanized, and internal organs and gills were collected to quantify transcript abundance of interferon-gamma and IL-12 (markers of cell immunity) and of interleukin 10 and transforming growth factor beta (regulatory molecules) using reverse transcriptase qPCR. Twenty-one days post-challenge, relative percent survival was calculated for each treatment. The internal organs of ten surviving fish per treatment were collected and used to evaluate bacterial load by quantifying F. orientalis DNA using qPCR. We observed a noteworthy reduction in TGFβ levels in both the gills and internal organs, coupled with an elevation in IFNγ levels within the gills. These findings underscore the potential of our innovative delivery method, prompting additional investigations to develop a potent oral vaccine to combat fish francisellosis disease.
Publications
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