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
Project Number: 1930-32000-005-05
General Cooperative Agreement
Start Date: Jan 15, 2012
End Date: Jan 14, 2016
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.
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.