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United States Department of Agriculture

Agricultural Research Service

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Research Project: Cytometric Comparison Between Flavobacterium-Resistant and -Susceptible Lines of Rainbow Trout During the in Vitro B-Cell Response.

Location: Cool and Cold Water Aquaculture Research

2013 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.


3.Progress Report:

Research carried out this year at William and Mary addressed objective 2 of the research proposal. Five undergraduate students were trained and then worked on this project during the spring and fall semesters (5-10 hours/week/student for 15 weeks). Furthermore, 13 undergraduates worked on this project as part of an Immunology Laboratory course (W&M), where they explored changes in immune cell number and response following immunization. One student completed a 10 week summer internship at the NCCCWA.

Two projects have been ongoing: one involved analysis of immune tissues from immunized resistant and susceptible line fish, which also tested possible differential effects of splenectomy on humoral immune responsiveness. The percentage of antigen-specific B lymphocytes were determined, data analyzed independently by two students, and entered in spreadsheets for statistical analysis. Second, antibodies to two pro-inflammatory markers were used to stain cells and differential protein expression patterns in immune tissues are currently being explored. Immune cell cultures from various tissues were analyzed after 2, 4, or 7 day exposure to antigen. This project is still in its early stages, but all protocols are streamlined and students are analyzing data.


Last Modified: 12/21/2014
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