Location: Foreign Animal Disease Research
Project Number: 1940-32000-057-41
Start Date: Jun 01, 2012
End Date: May 31, 2015
1. Bovine immune cells will be cultured with live or inactivated viruses. FMDV-specific antibody production will be quantified and characterized from the culture supernatants and FMDV-specific antibody secreting cells will also be determined. Cell cultures will be sorted for specific surface cell markers to determine FMDV-specific antibodies. 2. The levels of interferon will be measured to determine antibody concentrations. Specific antibody detection will be performed to quantify the amount of stimulated cells and measure the magnitue of response multiple bond connection strenght. Activation markers will be detected through flow cytometery and to determine activation methodologies. 3. A recombinant antigen comprising 40 tandem copies (a multi-peptide construct) of the FMDV viral protein (VP)1 site A will be assessed as thymus (T)-cell independent antibody inducer to study on the structural features of FMDV T-cell independent antigens. Specifically, we will produce the recombinant antigen and will study the cellular requirement to induce the antibody response against it. If positive results are obtained, the immunogenicity of the recombinant antigen will be assessed in experimental model (mice) and natural host (cattle). Amendment 1: To determine the role that FMDV vaccination plays on the induction of memory cells and antigenic production, naive cattle will be vaccinated with inactivated FMDV O1/Campos strain vaccine at INTA. Sera will be collected and mononuclear cell cultures prepared and tested for memory B-cell presence. FMDV-specific antibody secreting-cells (ASC) will be detected by ASC ELISpot assay developed by INTA. Vaccinated cattle will be euthanized and necropsied to obtain lymph nodes and tissue from the inoculation site. Mononuclear cell suspensions will be prepared to perform the detection of FMDV ASC by ELISpot. Serum samples from vaccinated cattle will be taken before each immunization and 15 days after the last booster and analyzed by the presence of FMDV-specific antibodies in an indirect sandwich ELISA. The ELISAs will be developed to assess anti-FMDV humoral responses and determine the presence of FMDV antigens in the processed cattle lymph nodes.