2012 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 first year of this project, we examined the effect of the genetically acquired resistance to F. psychrophilum on the generation of specific immunity to two types of antigen. For these studies, resistant (ARS-Fp-R) and susceptible (ARS-Fp-S) stocks were immunized with either haptenated lipopolysaccharide (TNP-LPS; T-independent) or haptenated protein (TNP-KLH; T-dependent). LPS is a highly immunogenic / protective antigen found in Gram-negative bacterial pathogens such as F. psychrophilum. KLH a highly immunogenic test protein antigen commonly used in immunology. Prior to initiating this study, we examined post-splenectomy recovery and the general effects of this procedure on the trout. We also set up two experiments to more closely define the general responsiveness of these stocks to our antigens. After obtaining this preliminary data, we splenectomized or mock-splenectomized 160 R and 160 S fish, rested them for 90 days, then immunized them with either TNP-LPS or TNP-KLH. In the end, we produced 8 groups of 40 fish (R vs. S; splenectomizd vs. mock-splenectomized; vs. TNP-LPS vs. TNP-KLH). We sacrifice 10 fish of each group at weeks 0, 5, 10 and 15 post immunization and are examining the serum anti-TNP titers, affinity; anti-TNP plasma cells / plasmablasts, leukocytic cellularity in the four major immune tissues (blood, anterior kidney, posterior kidney, and spleen (in mock-splenectomized fish)); hematocrits, PBL differential staining, and weights. This particular study should be completed by September 28, 2012. We believe that these studies will reveal potential impacts of this genetic resistance on inducible immunity and may afford us the possibility of determine its potential for enhanced immunity upon vaccination, and to determine the relative importance of innate vs. adaptive immunity in these resistant fish.