Immunogenicity and safety of RplB vaccine in adult horse.

Six yearlings will be used for the study. The RplB vaccine will be administrated intramuscularly to all animals in two doses, two weeks apart. Blood samples will be collected from horses before the first vaccination, before the second vaccination and again two weeks after the second vaccination. Bronchoalveolar fluid will be taken previous vaccination and 2 weeks after the second vaccination. To determine the safety of the antigenic preparation, animals will be observed daily and clinically assessed based on once a week physical examinations, including heart rate, respiratory rate, and rectal temperature recording. Local effects at the injection site will be recorded by visual inspection and palpation, for any sign of pain and/or swelling. Horses will be screen for anti-Vap antibodies using ELISA prior to vaccination. Detection of specific anti-RplB and anti-GST tag antibodies will be carried out by ELISA and western immnoblotting. Determination of relative concentrations of RplB-specific total IgG and specific immunoglobulin (Ig) subclasses will be carried out using specific antisera to horse Ig isotypes and IgG subisotypes (IgA, IgE, IgM, IgGa, IgGb, IgGc, IgGT) in an ELISA test. Pili-specific cytokine response (Th1, TH2, CTL, etc.) will be measured by RNA isolation and RT-PCR after in vitro stimulation of lymphocytes.