The Isolation Of Actinobacillus Pleuropneumoniae By Immonumagnetic Bead Techniques

Porcine contagious pleuropneumonia (PCP) is a common, severe, contagious pulmonary disease of porcine. The clinical signs vary from high mortality to chronic, retarded growth. Actinobacillus pleuropneumoniae (APP) is its etiological agent. Isolation of the pathogens is an important precondition for diagnosis and prevention of disease and for epidemiological investigation. At present, bacterial isolation depends on the direct inoculation of the suspected samples to the normal TSA. The selective medium was used to improve the isolation efficiency. However, the vulnerability of actinobacillus pleuropneumoniae, which reduces the number of live pathogen in the samples, limits the traditional methods. Therefore, the effort to develop a novel method to improve bacterial isolation will contribute to the research on this disease and set up an exemplary to the identical study on other diseases Immonumagnetic bead techniques is a new popular immunological technology in recent years, which was developed in the middle of 1970s'. It possessed a lot of advantages of solid reagen, such as good specificity, rapidness, highly efficacy, and repeatability.it is also cost-effective method due to no requirement of expensive instruments during detection. These merits contribute to it wider application in the fields of immunodetection, immunoadsorption, cell detachment and cultivation.In the experiment, outer membrane lipoprotein of Actinobacillus pleuropneumoniae is expressed and purificied, used as antigen to immunize the rabbit for antiserum production. The purified IgG was used to coat the immonumagnetic beads. The optimal concentration of IgG and time length for coating immonumagnetic beads were determined and the procession for detection of actinobacillus pleuropneumoniae was developed.. All of the 14 reference strains could be detected by the new method. The analytical sensitivity of the assay was 10.6CFU/ml. Twenty suspected strains were subjected to analysis, from which ten strains were identified as Actinobacillus pleuropneumoniae, further confirmed by gel diffusion test (GDT) using reference serotyping sera and ApxIV-based PCR. The pathogens could be recovered from infected animals. Finally, forty clinical samples were tested by this method and five actinobacillus pleuropneumoniae were separated. These results indicated the establishment of new immunomagnetic bead-based method to isolate actinobacillus pleuropneumoniae, providing a useful tool for future work.