Epidemiological methods for use with Actinobacillus pleuropneumoniae: New developments in serotyping and future prospects.

摘要

Actinobacillus pleuropneumoniae, the cause of porcine pleuropneumonia, is responsible for significant economic losses throughout the world. Serotyping, currently the "gold standard" method for strain identification, has been the mainstay technique for epidemiological monitoring and also can inform on decisions as to appropriate therapy, disease prevention or eradication. There are 16 serotypes (1-4, 5a, 5b, 6-15) with different ones predominating in particular geographical regions. Serotyping is typically determined by the immunological reactivity of rabbit serum to different surface polysaccharides. Conventional serotyping methods include complement fixation, indirect haemagglutination, ELISA,agglutination and coagulation, latex agglutination, indirect fluorescent antibody labeling, immunodiffusion and ring precipitation. These methods are not without their limitations: individual batches of test sera show variation and there is cross reaction between different serotypes e.g. between serotypes 3, 6 and 8. To circumvent problems associated with immunological methods, investigators have used gene-based methods e.g. multiplex PCR reactions that can identify strains of serotype 2, 5, 6 and 8. We now describe a serotype 3 specific PCR that can be used in a multiplex format with the A. pleuropneumoniae apxⅣ specific gene. Other methods that have been used in epidemiological/identification studies include ribotyping, sequence analysis of ribosomal intergenic regions, pulsed field gel electrophoresis,restriction endonuclease fingerprinting, aroA gene PCR-restriction fragment length polymorphism, 16S RNA sequencing, rapid amplified polymorphic DNA analysis and pulsed field gel electrophoresis. Typing methods based on the genes involved in the expression of ApxⅠ-Ⅲ toxin and the outer membrane protein OmlA have been used to inform on the serotype. However, based on the three published multilocus enzyme electrophoresis studies (MLEE) with A.pleuropneumoniae we have hypothesised that multilocus sequence typing (MLST) would be a valuable tool in epidemiological and evolutionary studies with this important pathogen. MLST involves the DNA sequencing of 7housekeeping genes that are distributed around the genome and are not proximal to others that may be under immune selection. MLST has the advantage over conventional laboratory based techniques that the data obtained are unambiguous and can be rapidly shared with other laboratories throughout the world via the intemet. Preliminary data,using isolates from Denmark, China and the UK, indicate that MLST is more discriminatory than serotyping and will be of considerable value in epidemiological studies both at the micro (within herd) and macro (global) levels and in understanding the evolution of A. pleuropneumoniae.