| Porcine contagious pleuropneumonia is a severe respiratory contagious disease caused by Actinobacillus pleuropneumoniae. The disease is widely endemic all over the world and often results in considerable economic losses. At present, the inactivated vaccine is still the main measures to prevent and control the disease, due to some limitations in practice, the genetic engineering attenuated vaccine is becoming one of the important research directions in future for its advantages such as stability, efficiency and good cross immunity. This study based on domestic epidemic situation of APP and research foundation in our laboratory, we carried out experiments as follows:Apx Ⅰ-ELISA specific antibody detection methods, the trial research of Apx Ⅰ C gene-deleted vaccine, and cross-immunity, security and immune efficacy of the vaccine.1. Research of Apx Ⅰ-ELISA Antibody Detection MethodApx Ⅰ secreted by APP serotype10was extracted with steps of salting out, dialysis and concentration, then the crude protein was purified by gel filtration chromatography, the molecular weight of the purified protein is identified by SDS-PAGE electrophoresis. The purified Apx I was used as the antigen of indirect ELISA, we established antibody-specific diagnosis method through series of optimization of reaction conditions. The optimal coating antigen concentration of Apx Ⅰ was9.17μg/mL, the optimal blocking time was90min by5%skim milk, the optimal serum dilution was1:320and optimal reaction time was60min, the optimal working concentration of rabbit anti-pig IgG-HRP wasl:6000and best working time was60min, the optimal TMB substrate reaction time was15min. the cut-off value of ELISA was0.34. The Apx Ⅰ-ELISA method had high sensitivity, strong specificity, and good reproducibility. The compliance rate with commercial IHA kits reached95%.2. Trial Research of Apx I C Gene-deleted Vaccine(1) Screening Test of Lyoprotectant for SWI△I C. The growth curve of Apx I C gene-deleted strains(SWl△I C) was measured by Plate counting method and spectrophotometric method. According to the growth curve, we knew that when the OD600of culture reached about3.0, the number of viable bacteria reached its maximum, approximately8.50×109CFU/mL.10%Skim milk was chose as the basic ingredient of the lyoprotectant. sucrose, glucose, trehalose, glycerol, sorbitol, sodium glutamate, gelatin were used in single-factor test for determination of different components on SWI△I C lyophilized protective effect, on the basis of single-factor test results we further designed orthogonal experiment and measured the protection effect of the various components with the order as follows:skim milk> sucrose> glycerol> sodium glutamate, while determing the protection formulations as skim milk12%, sucrose5%, glycerol3%and sodium glutamate3%, and the freeze-dried survival rate of SWI△I C strain reached about17%. Survival rates of SWI△I C strain had been greatly improved compared with the previous5%sucrose skim milk protestant.(2) Preparation and Quality Inspection of Vaccine. With the optimized lyoprotectant we prepared three batches of vaccine, and followed by serial inspections such as physical properties, purity, moisture content and the survival rate, the results of quality inspection showed that all prepared vaccine were met with relevant requirements of the veterinary biological products procedure.3. Cross-immunity, Security and Immune Potency Test of Apx I C Gene-deletion Vaccine(1) Cross-immunity Test. The Apx I C gene-deleted vaccine prepared previously were used for immunizing mice at the dose of2X107CFU (0.ILD50) every two weeks and for three times, and after the third immunization all mice were challenged with APP strain serotypes1,5,7at the dose of10LD50separately. The results showed that mice were not only resistant to the virulent strain of parental serotype but also resistant to heterologous strong strains such as serotypes1and7(75%,87.5%respectively). (2) Security Test of Piglets. When inoculated with the Apx I C gene-deleted vaccine at the dose of5×109CFU, piglets only appeared transient rise in body temperature, but no respiratory symptoms, lethargy and appetite abnormality, two weeks later piglets were killed for the necropsy we found that there were no pleuropneumonia in lung or other pathological changes, it indicated that in the range of5×109CFU immunizing dose the piglets are all secure.(3) Immune Efficacy Test of Piglets. Piglets were inoculated with Apx Ⅰ C gene-deleted vaccine at the dose of5×108CFU intratracheally and intramuscularly every two weeks for three times. Serum samples was collected before inoculation, IHA kits as well as Apx I-ELISA established in this study were all used to detect the antibody titre. After the third immunization piglets were challenged by APP serotype5(standard virulent strain) at the dose of1×109CFU. The results showed that two immunization pathways could all induce the piglets generating higher Apx Ⅰ antibody titre and can be completely resistant at the challenge of parental virulent strain. |
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