The Preparation And Evaluation Of Immune Efficacy Of Actinobacillus Pleuropneumoniae Ghosts

Porcine contagious pleuropneumonia (PCP) is a highly contagious disease which is caused by Actinobacillus pleuropneumoniae (APP) and characterized by severe fibrinous necrotizing hemorrhagic pleuropneumonia in pigs. Since this disease was reported by Patttison in 1957, it spreaded worldwidely and caused important economic losses in industrialized pig production. Inactivated vaccines were long used vaccines in many countries to prevent PCP, however, inactivated vaccine can not provide surfficient protection to other serotypes of APP. Development novel vaccine is the main focus of the researchers who worked on APP.In the present study, lysis gene E from phage plasmid PhiX174 was amplified by PCR and inserted into pBV220 vector which include theλPL/PR-cI857 regulatory system. Lysis gene E was connected in series with theλPL/PR-cI857 regulatory system to become the temperature-sensitive lysis case that was in constructed bacteriolysis plasmid pBV-E. The lysis case including gene E was inserted into the pGZRS-18 vector in order to produce a lysis plasmid pGZRS-Eλ. The plasmid pGZRS-Eλwas transformed to APP1 by calcium chloride method. APP1 harboring the bacteriolysis plasmid was grown in a waterbath shaker at 28°C under aerobic conditions until mid log-phase. Then chloromycin(final concentration 1μg/ml) was added into the culture that was transferred to a 42°C water-bath shaker. Finnally APP1 ghosts were produced, which were in intact forms under transmission electron microscope, and their contents were released to extracellular region. The efficacy of bacteriolysis which caused by pBV-E and pGZRS-Eλin each host bacterium was 99.86% and 99.99%. This experiment laid a foundation of study on bacterial ghost as a novel vaccine and adjuvant.The pigs were immunized on day 0 and 14 in following groups: GroupⅠ(intramuscular, 6 pigs), GroupⅡ(aerosol, 6 pigs)and Control group (distilled water, 4 pigs), and challenged with APP1 (5×109cfu) and APP2 (5×109cfu) on day 28. The serum antibody titers were determined using indirect enzyme-linked immunosorbent assay (iELISA) coated with APP1 whole-cells. The results of indirect ELISA showed the antibody level in intramuscular immunization group were significantly higher than those of immunization and the control group (p < 0.01). Two weeks after first immunization, the level of antibodies was lower than one week after first immunization. Two weeks after second immunization, the antibody level in intramuscular immunization group was significantly higher than those of immunization and the control group (p < 0.01). Two weeks after the challenge the immune protection efficacy of Actinobacillus pleuropneumoniae ghosts vaccine was evaluated based on survival rates, lung pathological changes and indirect immune fluorescence (IIF). Results showed that intramuscular immunization with Actinobacillus pleuropneumoniae ghosts can induce immune response which surpassed that indued by immunization. This experiment is a preliminary study of the protective efficacy of Actinobacillus pleuropneumoniae ghosts.