Recipient Organization
MISSISSIPPI STATE UNIV
(N/A)
MISSISSIPPI STATE,MS 39762
Performing Department
College Of Veterinary Medicine
Non Technical Summary
Edwardsiella ictaluri, a Gram-negative, intracellular, facultative bacterium, causes enteric septicemia of catfish (ESC), which is one of the most devastating diseases in the U.S. catfish industry. Dendritic cells (DCs) are the most powerful professional antigen-presenting cells (APCs), bridging and initiating innate and adaptive immune responses in vertebrates by presenting the antigens to T cells, which results in the generation of memory cells that can prevent disease or at least reduce the severity of disease during re-infection. Multiple studies documented the morphological and functional characterization of DCs in teleost fish, such as zebrafish, rainbow trout, and barramundi. In channel catfish, Langerhans cells (LCs), which are a subset of DCs in mammals, have been identified in the immunocompetent tissue by our research group. Also, our group developed two protective E. ictaluri live attenuated vaccine (LAV) strains (EiΔevpB and ESC-NDKL1). Recently, we documented that the number of LCs significantly increased in the lymphoid organs of catfish exposed to two LAV strains. However, the effects of two LAVs on antigen uptake in DCs and the T cell proliferation ability of DCs in channel catfish are still unexplored. Thus, it is critical to describe the antigen-presenting function of DCs in channel catfish. Without this knowledge we cannot evaluate the role of DCs in the generation of protective T cell-mediated immunity against ESC.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Identification of the morphological and functional properties of dendritic cells in the lymphoid tissues of channel catfishAssessment of dendritic cell-dependent T cell proliferation in peripheral blood of channel catfish
Project Methods
-EffortsIdentification of the morphological and functional properties of dendritic cells in the lymphoid tissues of channel catfishIsolation of cells from blood, head kidney, and spleen: We will set up cell cultures and enrich DC-like cells from channel catfish peripheral blood (PB), anterior kidney (AK), and spleen. Briefly, PB will be collected from the caudal vein and diluted with RPMI 1640 with L-glutamine (Gibco) at room temperature (RT, 22-25 °C). AK and spleen will be dissected from catfish, macerated by using sterile forceps, and passed through cell dissociation sieves (Sigma, St. Louis, MO) to obtain a single-cell suspension.Hematopoietic cell culture: The single-cell suspension from PB, AK, and spleen will be counted and assessed for viability by using a hemocytometer and trypan blue exclusion. Then, cell number will be adjusted to a final concentration of approximately 1x10^6-1x10^7 cells/ml in supplemented L-15 media as described (Bassity et al., 2012). Cells will be plated in culture flasks with phenolic style caps (Corning, Corning, NY) and cultured at room temperature for 7-14 days before harvest. Non-adherent cells will be harvested several times in this period and removed media will be replaced by fresh supplemented media (Bassity et al., 2012).Harvest and enrichment of catfish DC-like cells: Cell culture flasks will be gently agitated to suspend non-adherent cells, and media containing the suspended non-adherent cells will be collected. Then, the cell suspension will be layered over Nycoprep/One-step monocytes and centrifuged according to the manufacturer's instructions. The buffy coat containing enriched DC-like cells will be collected and washed in fresh media before further use (Bassity et al., 2012).Morphological characterization of DC-like cells in channel catfish: Harvested non-adherent mononuclear cells will be used for cytospins, which will be prepared at 500 rpm for 1 min by using a Cyto-Tek centrifuge machine, then samples will be analyzed by light microscopy as described (see preliminary data) to determine the morphology of DC-like cells in catfish.Phagocytosis in vitro: To evaluate the phagocytosis ability of DC-like cells in catfish, E. ictaluri 93-146 carrying pAKgfp1 will be used (Karsi et al., 2007). Catfish DC-like cells will be transferred to a 6-well plate (Fisher Scientific, Pittsburgh, PA). Then, GFP-labeled E. ictaluri WT and two LAV (EiΔevpB and ESC-NDKL1) strains will be added to catfish DC-like cells at 1:20, 1:50, and 1:100 ratio and incubated 1 hour at 28oC. Catfish DC-like cells will be exposed to GFP-labeled E. ictaluri according to the following conditions: 1) bacteria not opsonized, 2) bacteria opsonized with serum from non-vaccinated catfish, 3) bacteria opsonized with sera from vaccinated fish. Also, cells will be treated with E. ictaluri at 4o C to determine the background levels of phagocytosis (negative controls). Following the incubation, cells will be collected and washed three times by centrifugation in cold PBS and analyzed by one and dual-color Flow Cytometry using a NovoCyte Flow Cytometry (ACEA Biosciences, Inc.) (Boyd et al., 2004).Assessment of dendritic cell-dependent T cell proliferation in peripheral blood of channel catfishMixed leukocyte reaction (MLR): Catfish DC-like cells (stimulators) will be treated with mitomycin C (100 µg/ml) to prevent the proliferation of DC-like cells as described previously (Pinchuk et al., 1994). Then, cells will be re-suspended at 2x10^6 cells/ml in L-15 medium supplemented with 5% catfish serum (MLR medium). Stimulators will be plated in 100 ml MLR medium at 2x10^5 cells per well in 96-well round-bottom plates (Corning). Peripheral blood will be collected, and peripheral blood mononuclear cells (PBMCs) will be isolated by using Histopaque 1077 (Sigma) gradient. T cells will be responders in PBMCs. A total of 2x10^5 PBMCs will be added to all wells, thus resulting in a stimulator to responder ratio of 1:1. Also, ratios of 1:2, 1:4, and 1:8 will be performed. PBMCs alone will be used as a negative control (Bassity et al., 2012). MLR plates will be incubated for 6 days, and cells will be collected, resuspended in PBS, and analyzed by a NovoCyte Flow Cytometry.