2012 Annual Report
1a.Objectives (from AD-416):
1. Identify the frequency of mature B cells, plasmablasts, transitional plasma cells, and mature plasma cells, in freshly isolated trout immune tissues from ARS-Fp-R-, C-, and S-line fish. 2. Analyze the in vitro LPS activation response in the ARS-Fp-R-, C-, and S-line fish and analyze the relative capabilities of B cells to differentiate from naïve B cells, to plasmablast and finally to plasma cells. 3. Determine the frequency of gated macrophages and activated, proliferating T helper cells in each of the three trout lines after in vitro or in vivo induction. 4. Measure levels of the cytokines TNF-alpha and IL-1 beta in cell subpopulations.
1b.Approach (from AD-416):
The relative frequency of resting B, plasmablasts, plasma cells, proliferating T cells, and activated macrophages will be determined in the three trout lines using flow cytometry. Fluorescently labeled antibodies specific to trout IgM, IgD, Pax-5, the proliferation marker Edu, CD3, TNF-alpha, and IL1-beta will be used. Tissue from four immune sites, peripheral blood, spleen, anterior kidney, and posterior kidney will be cultured in vitro with one of two mitogens, either E.coli lipopolyssacharide (LPS) or Flavobacterium LPS, and cells analyzed at selected time points after 0-7 days of induction. Undergraduate students from William and Mary, Department of Biology will work on this project during the academic year and during summer internships.
Research carried out this year at William and Mary addressed objective 2 of the research proposal. Specifically, we addressed whether there are differences in the type and/or the percentage of antibody secreting cells between resistant and susceptible lines of rainbow trout. Flow cytometry was used to explore the possibility that resistant fish have either more antibody secreting cells to begin with or, are able to produce such cells more quickly in response to an infection. Also, we tested whether resistant fish have a greater capacity to expand their population of effective, Flavobacterium-specific immune cells. We have recently optimized our approaches to obtain the best combination of immune markers (fluorescently tagged antibodies) for flow cytometry. Using flow cytometry, we investigated how the resistant and susceptible rainbow trout lines differ in their immune response to a specific, artificial “test vaccine”, TNP. This year, a total of three William and Mary undergraduate students worked on this project.