Recipient Organization
MISSISSIPPI STATE UNIV
(N/A)
MISSISSIPPI STATE,MS 39762
Performing Department
College Of Veterinary Medicine
Non Technical Summary
Channel catfish farming is the largest aquaculture industry in the U.S., and Edwardsiella ictaluri, causing enteric septicemia of catfish (ESC), is recognized as the most important pathogen. Efficacious live attenuated Edwardciella ictaluri vaccine is expected to deliver the antigen to antigen presenting cells (APCs) that can elicit potent protective immune responses resulting in generation of memory cells able to respond upon infection by either killing of the infected targets or induce killing mechanisms in the infected cells. E. ictaluri has acquired multiple strategies to overcome immune barriers, using monocytes and macrophages as residences and source for dissemination, thus impairing their participation in generation of protective antibacterial immunity. We hypothesize that efficacious E. ictaluri live attenuated vaccine (LAV) by activating monocytes and macrophages will be able to induce early activation of immune system. Our long-term goal is to identify immunological mechanisms that underscore E. ictaluri pathogenesis to enable development of effective control strategies for the enteric septicemia of catfish (ESC). The overall objective of this project is to evaluate APC-dependent innate and adaptive immune effector mechanisms between an E. ictaluri LAV candidate we developed (EiΔevpB) and a commercial LAV (Aquavac-ESC).The rationale of the proposed research is that B cell-mediated immunity is not central to the intracellular pathogen E. ictaluri control. T cell-inducing vaccines must aim to deliver the antigen to APCs that it can be presented on MHC molecules on the cell surface thus bridging innate and adaptive immunity. This study is significant because of the economic impact ESC has on the commercial channel catfish industry, which is larger than all other aquaculture industries in the U.S. combined. However, the channel catfish industry is currently facing severe economic stress from increased feed prices and pressure from foreign competition. E. ictaluri is a primary pathogen that affects all size classes of fish, and it often predisposes secondary infections with other pathogens.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Our long-term goal is to identify immunological mechanisms that underscore E. ictaluri pathogenesis to enable development of effective control strategies for the enteric septicemia of catfish (ESC). The overall objective of this project is to evaluate APC-dependent innate and adaptive immune effector mechanisms between an E. ictaluri LAV candidate we developed (EiΔevpB) and a commercial LAV (Aquavac-ESC). We will achieve our objective through the following specific aim: Determine vaccine induced productive innate immune responses through catfish APC. Our hypothesis for this aim is based on precedence that vaccination against E. ictaluri, an intracellular pathogen, must aim to deliver the antigens to APCs that they can be presented on MHC molecules on the cell surface to generate a pool of memory T cells. This proposal is innovative because we will conduct morphological and functional assessment of professional APCs, including monocyte/macrophages, B cells and dendritic cells (DCs) in catfish.
Project Methods
Macrophage isolation: Macrophages will be isolated from non-vaccinated and vaccinated catfish by peritoneal lavage as described previously (Jenkins and Klesius, 1998). Briefly, 360 specific pathogen free juvenile catfish will stocked into 12 40-L tanks (30 fish/tank) and four tanks will be assigned to EiΔevpB, Aquavac-ESC, and Wild-type groups. Five catfish from each tank will be injected intraperitonelly at time 0 (before vaccination), and 10, 20, 30, 45, and 60 days (after vaccination) with squalene and cells will be harvested 4-5 days after squalene injection, washed in PBS, resuspended in channel catfish macrophage medium (CCMM) prepared as described (Booth et al., 2006), counted and assessed for their viability.Isolation of cells from blood, head kidney and spleen: Peripheral blood, head kidney, and spleen cell separation from vaccinated and non-vaccinated catfish will be performed as described previously (Zhao et al., 2008). Tissues will be collected at the time points after macrophage collection. Briefly, peripheral blood will be collected from the caudal vein followed by centrifugation at 500g and resuspended in RPMI 1640. Head kidney and spleen will be dissected from the fish and placed in a sterile culture dish containing tissue culture medium (TCM). To obtain single-cell suspension, tissue will be minced with sterile forceps, repeatedly aspirated using a 1 ml syringe and passed through nylon filter. The resulting cell suspension will be washed and resuspended in TCM. Cell suspensions will be layered over Histopaque 1077 and centrifuged at 500g for 40 min. Cells then will be collected from the interface, washed 3 times in TCM, counted and assessed for viability by trypan blue exclusion.Isolation of B cells: Cells derived from head kidney, spleen and peripheral blood leukocytes (PBL) of vaccinated and non-vaccinated catfish will be stained with catfish IgM-specific and matching isotype control mAbs (kindly provided by Drs. Bengten and Wilson, University of Medical Center, Jackson, MS) followed by the addition of isotype-specific phycoerythrin conjugates. After the staining procedure cells will be washed and assessed for their ability to phagocytose live attenuated E. ictaluri. Phagocytosis in vitro: To evaluate E. ictaluri phagocytosis in fish macrophages and B cells, live and opsonized E. ictaluri 93-146 carrying pAKgfp1 will be added for 60 min to the catfish APCs at 1:20 and 1:100 ratio and incubated at 28o C (Karsi and Lawrence, 2007). The macrophages will be treated with fluorescent E. ictaluri according to the following regimens: (1) bacteria not opsonized, (2) bacteria opsonized with serum from non-vaccinated catfish; (3) bacteria opsonized with serum from vaccinated catfish. To determine background levels ofphagocytosis (negative controls) cells will be incubated in the presence of E. ictaluri at 4o C. Cells will washed three times by centrifugation in cold PBS and analyzed by one and dual color Flow Cytometry using a FACSCalibur as described (Boyd et al., 2004).Bacteria killing assay: Bacterial killing assay will be done as described and modified previously by Booth et al. and Russo et al. respectively (Booth et al., 2006; Russo et al., 2009). Briefly, macrophages from vaccinated and non-vaccinated fish will be incubated for 48 hrs at 28oC with untreated or opsonized with immune serum from vaccinated catfish green-fluorescent E. ictaluri in a ratio 1:1 in a 96-well plate in the presence of CCMM without antibiotics. Extracellular bacteria will be killed by incubation with CCMM supplemented with high doses (100 μg/ml) of gentamicin, streptomycin and penicillin for 1 hr. Macrophages then will be washed and cultured in CCMM supplemented with low doses (10 μg/ml) of the antibiotics followed by lysis with 1% Triton at 12 hrs and thereafter at intervals of 24 hrs up to a total of 48 hrs. The lysed cells will be centrifuged at 5000g, washed and plated on SOB agar with ampicillin, and incubated overnight at 27oC.Assessment of DC-like Langerin/CD207+ cells in the haemopoietic tissues of catfish: A DC-specific Langerin-like protein was found in fish and this protein shows conservation between humans and fish based on cross-reactivity between the species (Lovy et al., 2009). The intracellular staining procedure will be performed as described elsewhere (Fellman et al., 2011). Briefly, cells derived from head kidney, spleen and peripheral blood leukocytes (PBL) of vaccinated and non-vaccinated catfish will be fixed and permeabilized using the BD Cytofix/Cytoperm Plus Kit according to the manufacturer's instructions, followed by incubation with human Langerin-specific mAbs that are commercially available (R&D Systems) and isotype control antibodies. Cells will be washed and analyzed by one color flow cytometry using a FACSCalibur.Statistical Analysis: Analysis of variance (ANOVA), followed by Fisher's LSD multiple comparison post hoc test will be used to evaluate differences in mean fluorescence intensity (MFIs) and will be presented as means + SD. The level of significance for all tests will be set as P<0.05.