Investigating distinct immunization routes in zebrafish on mucosal immunity
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AbstractModel organisms such as Danio rerio are used to explore and model physiological reactions to stress, illness, and disease. Immune response is measured by quantifying lymphocyte presence and the production of mRNA encoding different immunoglobulin proteins. Because the method through which D. rerio is exposed to an antigen affects its immune response and the degree to which immunity is developed, we will expand on experiments comparing the immune response after visceral or mucosal introduction of DNP-KLH in D. rerio. In our visceral treatment, we will combine DNP-KLH with Freunds incomplete adjuvant and inject the mixture into the fish (100 uL at 1 mg/mL). For our mucosal exposure, we will combine DNP-KLH with cholera toxin as an adjuvant and introduce the mixture to their water (35 mg/L). Two control groups will account for intravenous immune response by injections solely with sterile saline solution as well as a control for the anesthetic used, MS-222. Viscera tissue samples will be collected from the immunized fish and two control groups of fish at weekly points throughout the experiment and will be analyzed for lymphocyte versus granulocyte ratios with flow cytometry and expression of IgM, IgZ-1, and IgZ-2 levels via qPCR. We expect to see an increase in lymphocyte percentages for both immunized groups compared to the control groups, with a higher lymphocyte ratio in the mucosal group than the visceral group. Furthermore, we expect more mucosal IgZ isotypes in the mucosal group and higher levels of the IgM, IgZ-1, and IgZ-2 antibodies in the visceral group than in the mucosal group. For future experiments, we would like to determine specific tissues of interest that provide data for analysis of mucosal and visceral immunological responses.
