2013 Annual Report
1a.Objectives (from AD-416):
1) Determine if differences in the phagocytic response to F. psychrophilum exists in macrophages and neutrophils isolated from ARS-Fp-R, -C, and –S lines..
2)Determine if antibody-producing (B) lymphocytes from the major tissues of these trout respond differentially to F. psychrophilum vs. control E.coli LPS..
3)Determine if differential cellular and cytokine mechanisms are involved in the observed differences in the antibody responses to T-independent and T-dependent antigens in ARS-Fp-R, -C, and –S lines.
1b.Approach (from AD-416):
Generally, via in vitro or in vivo analyses of ARS-Fp-R, -C, and-S lines:.
1)The ability of phagocytic cells to uptake and destroy live F. psychrophilum will be quantitatively determined using fluorescence microscopy and luminescence assays..
2)In vitro cultures of leukocytes from various immune tissues will be stimulated with LPS from F. psychrophilum and their relative abilities to develop antibody-producing B cells will be assessed. These studies will be coordinated with scientist who will be conducting cytometric characterization of these cells..
3)Trout of all three strains will be immunized with T-dependent and T-independent antigens and ex vivo and in vivo analyses of antibody production and antibody-producing cell development ascertained the effects of splenectomy on the relative ability to generate a productive antibody response will be compared between strains to determine the role splenomegaly may play in resistance.
In this reporting period, splenectomy or mock surgery were performed on 160 disease resistant and disease susceptible trout. These fish were then apportioned into 8 groups of 40 fish, which were then either immunized with either a protein or carbohydrate antigen. Ten fish from each of these 8 groups were assessed on weeks 0, 5, 10, and 15 for antibody titers, affinity, and total numbers of B cells in various tissues. These analyses, together with other physical parameters, provided over 10,000 data points which are currently being statistically analyzed. In preparation for the next 2013 study, splenectomies have been completed and fish will be immunized with Fp carbohydrate or a similar carbohydrate from a control bacterium. Groups will be analyzed on weeks 0, 10, 20, and 30 to provide long-term analyses of the antibody response.