title: 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.
authors.affiliation: Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves, 9500, Prédio 43421, C.P. 15005, CEP 91501-970, Porto Alegre, RS, Brazil.
authors.forename: Mateus Matiuzzi
authors.initials: MM
authors.lastname: da Costa
authors.forename: Catia Silene
authors.initials: CS
authors.lastname: Klein
authors.forename: Raquel
authors.initials: R
authors.lastname: Balestrin
authors.forename: Augusto
authors.initials: A
authors.lastname: Schrank
authors.forename: Itamar Antonio
authors.initials: IA
authors.lastname: Piffer
authors.forename: Sérgio Ceroni
authors.initials: SC
authors.lastname: da Silva
authors.forename: Irene Silveira
authors.initials: IS
authors.lastname: Schrank
mesh_terms: D000188:Actinobacillus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D000189:Actinobacillus Infections / Q000382:microbiology / Q000662:veterinary; D016977:Actinobacillus pleuropneumoniae / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D015373:Bacterial Typing Techniques; D004269:DNA, Bacterial / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D005798:Genes, Bacterial; D020459:Genes, rRNA / Q000235:genetics; D006460:Hemolysin Proteins; D006461:Hemolysis; D010325:Pasteurella / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D012703:Serotyping; D013552:Swine / Q000382:microbiology; D014510:Urease / Q000032:analysis
chemical_list: C085361:ApxII toxin, bacteria; C079721:ApxIII toxin protein, Actinobacillus; C122177:ApxIVA protein, Actinobacillus pleuropneumoniae; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D006460:Hemolysin Proteins; D012336:RNA, Ribosomal, 16S; C087744:ApxI toxin, Bacteria; D014510:Urease