Isolation Of Actinobacillus Pleuropneumoniae And Development Of ApxII-and ApxIV-ELISA Kits

Actinobacillus pleuropneumoniae (APP) is the infectious agent of porcine contagious pleuropneumonia (PCP). This infection causes great economic loss in the pig industry worldwide. Until now, 15 serotypes have been described on the basis of the antigenic diversity of the capsular polysaccharides and lipopolysaccharides. The main virulence factors are CPS, LPS, OMP, TBP, adhesion factors, Apx, Urease and so on. A. pleuropneumoniae produces proteinaceous cytotoxins that is the main virulence factors, ApxⅠ, ApxⅡ, ApxⅢand ApxⅣ. ApxⅡis expressed by all serotypes of A. pleuropneumoniae except serotypes 10 and 14. ApxⅣis secreted by all serotypes only after infection of pigs, but not under in vitro conditions. This study contains the following 3 parts of works:1. Isolation and identification of A. pleuropneumoniaeSamples were inoculated on the media from pneumonic lungs and arthrosis of pigs, then the microscopic morphology of bacteria is observed as gram-negative by microscope. The bacterium was characterized by routine bacteriology and apxⅣ-PCR with purified bacteria, which included 4 strains serotype 4, 1 strain serotype 3 and 1 strain no serotyping by GDT.2. Development and application of ApxⅡ-ELISA diagnostic kit for A. pleuropneumoniaeAn ApxⅡ-ELISA was established in our previous studies based on the characterizations of ApxⅡ. In this study, we developed this method a diagnostic kit, whose specificity, sensitivity, repeatability and stability were evaluated and compared with the indirect haemagglutination assay (IHA). A total of 1453 clinical sera from China, USA, Canada, Denmark, Australia and UK were screened using our ApxⅡ-ELISA kit.The results showed that the ApxⅡ-ELISA kit has very high specificity,sensitivity and repeatability [the inter- and intrabatch imprecision (CV%)＜15%] and stability (very stable at 4-8 C for 9 months). Compared with the indirect haemagglutination assay (IHA), ApxⅡ-ELISA was more sensitive than IHA. Kinetics of ApxⅡantibodies showed that the ApxⅡantibodies could be detected after 3 or 4 weeks post inoculation with the trivalent vaccine, the peak value of ApxⅡtiter is 1:1280. The positive ratio of samples from China, USA, Canada, Denmark, Australia and UK were 49.56%, 85.51%, 1.92%, 73.33%, 1.85% and 100%, respectively. Our data confirmed that the ApxⅡ-ELISA kit can be used to detect the antibodies of the toxin ApxⅡand evaluated the effect of App vaccine. 3. Development and application of ApxⅣ-ELISA diagnostic kit for A. pleuropneumoniaeAn ApxⅣ-ELISA was established in our previous studies based on the characterizations of ApxⅣ. In this study, we developed this method a diagnostic kit, whose specificity, sensitivity, repeatability and stability were evaluated and compared with an analogous ELISA kit (CHEKIT-APP-APXⅣ) from the company IDEXX. A total of 1453 clinical sera from China, USA, Canada, Denmark, Australia and UK were screened using our ApxⅡ-ELISA kit.The results showed that the ApxⅣ-ELISA kit has very high specificity,sensitivity and repeatability [the inter- and intrabatch imprecision (CV%)＜15%] and stability (very stable at 4-8 C for 9 months). Compared with CHEKIT-APP-APXIV, the overall agreement rate was up to 91.37%. Kinetics of ApxⅣantibodies showed that the ApxⅣantibodies could be detected after 3 or 4 weeks post infection with APP strain, the peak value of ApxⅡtiter was 1:640. Sera were negative that from pigs immunized (IM) with the trivalent vaccine and control pig (NC). The positive ratio of samples from China, USA, Canada, Denmark, Australia and UK were 22.30%, 39.72.%, 0.64%, 42.22%, 1.85% and 93.94%, respectively. Our data confirmed that the ApxIV-ELISA kit can be used to distinguish the vaccinated pigs from APP-infected ones.A. pleuropneumoniae is currently difficult to isolate in the pig farms, the reason should be investigated. Six strains of APP have been isolated from different Chinese pig farm, which provided important materials for etiological and epidemiological study of porcine contagious pleuropneumonia. At the same time, ApxⅡ- and ApxIV-ELISA kits were developed in this study. The latter one reached the level of clinical application. The ELISA kits provided important tools for the diagnosis and control of porcine contagious pleuropneumonia.