Development and application of an ERIC-PCR method for genotyping and differentiating Actinobacillus pleuropneumoniae isolates

ABSTRACT

Background: The pleuropneumonia caused by Actinobacillus pleuropneumoniae is one the most important swine respiratory diseases. Biochemical and serological tests are widely applied for the diagnosis and characterization of this bacterium. However, in some isolates, confl icting results are found. There are at least 15 serotypes with signifi cant differencesin virulence that have been identifi ed until now. Moreover, cross reactions between serotypes are not uncommon. Theserotype determination from isolates occurring in outbreaks is an important procedure in prophylaxis and control of thedisease. The present work focuses on the application of an ERIC-PCR technique for genotyping and differentiation ofA. pleuropneumoniae isolates.Materials, Methods & Results: Fifteen reference strains for the recognized A. pleuropneumoniae serotypes were analyzedin this work, alongside with 27 fi eld isolates that had been previously characterized regarding biochemical, serological andmolecular features. Total DNA from each sample was purifi ed and subjected to PCR amplifi cation using ERIC-specifi cprimers (ERIC1R and ERIC2). The resulting amplicons were analyzed by agarose gel electrophoresis and their sizes wereestimated from the gel images. Bands with similar sizes were identifi ed and used to construct a binary matrix that tookinto account the presence or absence of individual bands in all lanes. Pair-wise similarity coeffi cients were computed fromthe binary matrix and the similarity matrices obtained were utilized to construct an UPGMA-based dendrogram. The amplicons obtained from the A. pleuropneumoniae reference strains generated a very distinctive pattern for each one of thetested strains. Moreover, all samples presented a large enough number of amplicons (bands) as to enable an unequivocaldifferentiation of each sample. Reproducibility of the developed ERIC-PCR method was assessed by means of duplicate...(AU)