The Biological Characteristics And Immunogenicity Of Three Mutant Strains Of Actinobacillus Pleuropneumoniae Of Deleting Apx Gene

Porcine contagious pleuropneumonia (PCP) is a highly contagious disease that is caused by Actinobacillus pleuropneumoniae (APP) and has caused severe economic losses worldwide in the swine industry. Compared with inactivated vaccines and subunit vaccines, the attenuated vaccine could present antigens and stimulate immune responses. It is an important research direction for APP attenuated vaccine. In this experiment, we evaluated biological characteristics and immunogenicity of three mutant strains in mice. The main contents and results are as follows: 1. Biological characteristics of the mutant stainsThe mutant strains apxⅡC-/kan+, apxⅡC-/IN+ of APP-7 and SW1△IC of APP-5 are as research subjects, we evaluated biological characteristics of three mutant strains.The haemolytic activity of both the parental and mutant strains was examined. Briefly, the bacteria were inoculated onto a Rabbit blood agar plate and E. coli DHsa used as negative control. The results showed that APP-5 had strong hemolytic activity, APP-7 had weak hemolytic activity and the mutant strains apxIIC-/kan+, apxIIC-/IN+ and SW1△IC produced no hemolysis.After a single bacterial colony was inoculated into TSB+NAD, the OD600 was determined every hour. The results showed that no obvious difference was observed in the growth curves of the APP-7 parent strain and the apxⅡC-/kan+ and apxⅡC-/IN+ mutant strains on in vitro growth, implying that the deletion of the apxIIC gene had no significant influence on the growth of A. pleuropneumoniae, and the APP-5 parent strain and the SW1△IC mutant strain was the same result, implying that the deletion of the apxIC gene had no also significant influence on the growth of A. pleuropneumoniae.Mice were inoculated with different doses of the mutant and parent strains. The number of surviving mice of each group was recorded 3 days post-challenge. The LD50 data shown that the SW1△IC mutant strain was attenuated by fifteen-fold, which was compared with the APP-5 parent mutant; the apxⅡC-/kan+ and apxⅡC-/IN+ mutant strains were attenuated by five-fold, and seven-fold respectively, which were compared with the APP-7 parent mutant. 2. The immune and protective efficiency of the mutant strains in miceOne hundred sixty-eight 6-week-old female mice that were shown sero-negative for A. pleuropneumoniae by ApxⅠ-based and ApxⅡ-based ELISA were randomly divided into A, B, C and D groups. Group A of 48 mice were vaccinated three time subcutaneously with 4×107CFU of apxⅡC/kan+mutant in 200μL TSB. Group B of 48 mice were vaccinated with 4×107CFU of apxⅡC/ⅠN+ mutant. Group C of 48 mice were vaccinated with 4×107CFU of SW1△ⅠC mutant. Group D of 24 were inoculated with TSB as control. Two weeks after the immunization, mice challenged with difference dose of serovar 1, serovar 5 and serovar 7.In apxⅡC/kan+, apxⅡC/ⅠN+ and SW1△ⅠC vaccinated group, all mice which were challenged with 10LD50 were gained protection efficiency of 100% against homologous and heterologous serovars. In the TSB control groups, there was no mouse survived after post-challenge. With the 20LD50 dose challenge, the apxⅡC-/kan+ could offer 25% protection against serovar 1 and serovar 5, and 62.5% protection against serovar 7; the apxⅡC-/ⅠN+ could offer 50% protection against serovar 1, serovar 5 and serovar 7; the SW1△ⅠC could offer 37.5% protection against serovar 1, and offer 100% protection against serovar 5, and offer 50% protection against serovar 7.Sera were isolated from blood samples collected from mice by tail venipuncture preimmunization at weeks 0,2,4 and 6 after the primary immunization. ApxⅠand ApxⅡ-specific antibody responses were determined by ELISA. There was significant differences in antibody titers between apxⅡC-/kan+ vaccinated group and control group (P<0.01). There were the same result in the axpⅡC-/ⅠN+ vaccinated group, and the SW1△ⅠC vaccinated group. The results showed that the mutant strains apxⅡC-/kan+, axpⅡC-/ⅠN+ and SW1△ⅠC could generate high toxin antibodies.