Evaluation of PCR Based on Gene apxIVA Associated with 16S rDNA Sequencing for the Identification of Actinobacillus pleuropneumoniae and Related Species

Abstract

The pleuropneumonia caused by Actinobacillus pleuropneumoniae (App) is one the most important swine respiratory diseases. Biochemical and serological tests are widely applied for App diagnosis and characterization. However, in some isolates, conflicting results are found. The present work focus on the characterization of 29 isolates biochemically classified as A. pleuropneumoniae, collected from swine in herds with or without a clinical history of pleuropneumonia. Sixteen isolates were from healthy swine, initially classified as nonserotypable A. pleuropneumoniae; they displayed differences in the molecular characterization patterns of App (genes cpx and apxI, II, and III). Those bacteria that could not be serotyped were submitted to rDNA 16S sequencing. All 29 isolates were analyzed by PCR for the presence of the apxIVA gene. Thirteen isolates (45%) were confirmed to be A. pleuropneumoniae by PCR, nine being from diseased animals (31%) and four from healthy animals (14%) with conclusive serotyping. The rDNA 16S sequencing was used to classify the other 16 isolates in related species other than A. pleuropneumoniae, resulting in eleven A. minor, three A. porcinus, and two Pasteurella sp. Because of conflicting results between biochemical tests and rDNA 16S sequencing, the biochemical characterization was repeated, and the new results were in agreement with the rDNA 16S sequencing data. Biochemical characterization proved to be efficient for the majority of the A. pleuropneumoniae isolates. Nevertheless, conventional tests can render conflicting results, and other methodologies, such as amplification of A. pleuropneumoniae specific apxIVA gene and rDNA 16S sequencing, are very useful for improved classification. We also observed a great variety in rDNA 16S sequences from different A. minor isolates.

Development and Use of a Multiplex Polymerase Chain Reaction Assay Based on Apx Toxin Genes for Genotyping of Actinobacillus Pleuropneumoniae Isolates

“…Molecular methods such as amplification of the apxIV genes and 16S ribosomal DNA sequencing have been reported as alternative and reliable methods to classify A. pleuropneumoniae. 3,8,19 This study uses the apxIV gene and the other apx genes for identification and genotyping of A. pleuropneumoniae.…”

A BOX-SCAR fragment for the identification ofActinobacillus pleuropneumoniae

“…When using the primer pair APXIV‐Up and APXIV‐Do and the amplification conditions suggested by Costa et al . (), only 84% of our clinical isolates, which had been previously identified by biochemical tests and characterised by multiplex PCR (Rossi et al ., ), were positive for the apxIVA DNA fragment. Similarly, 94.5% of isolates that had previously been identified as A. pleuropneumoniae using biochemical tests were positive for the apxIVA gene in a study by Urbaniak & Markowska‐Daniel ().…”

Identificação diferencial de Rhodococcus equi e Dietzia maris em bubalinos

“…O emprego de métodos fenotípicos e moleculares para identificação de bactérias é amplamente descrito na literatura (Soedermanto et al, 1998;Costa et al, 2004). Bactérias corineformes pertencem ao grupo de microrganismos cujos métodos de diagnóstico convencionais, baseados exclusivamente nas características fenotípicas, são inconclusivos (Bizet et al, 1997).…”