Identification of Actinobacillus pleuropneumoniae biovars 1 and 2 in pigs using a PCR assay

Abstract

Actinobacillus pleuropneumoniae causes swine pleuropneumonia worldwide. Previously, we described a gene sequence of approximately 800 bp in A. pleuropneumoniae serotype 1 that encodes a metalloprotease of 24 kDa, (Genbank accession no. AY217757). We selected primers carrying the forward and reverse 5′-terminal sequences of this region of the gene for the development of a species-specific PCR assay. The primers amplified an 800 bp sequence from isolated DNA and lysed bacteria of the 13 A. pleuropneumoniae biovar 1 serotypes, with the exception of subtype 1b. The primers also amplified the sequence in nasal secretion cultures from pigs with chronic and acute experimental pleuropneumonia. No PCR products were detected when A. pleuropneumoniae serotypes of biovar 2 were used. Internal primers from this gene sequence detected biovar 2 and subtype 1b, leading to the production of a 350 bp PCR product. The primers did not amplify DNA from other related species from the Pasteurellaceae family. The 800 bp PCR assay was sensitive in vitro, with a detection limit of 5.5 pg of extracted DNA, and an average of 120 CFU. The specificity and sensitivity of this PCR assay make it a useful method for the rapid identification and diagnosis of A. pleuropneumoniae.