Development of an efficient PCR method for toxin typing of Actinobacillus pleuropneumoniae strains

Frey, Joachim; Beck, Marco; Bosch, J. F. van den; Segers, R. P. A. M.; Nicolet, Jacques

doi:10.1016/s0890-8508(95)90158-2

Cited by 63 publications

(59 citation statements)

References 17 publications

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“…Specific Aqx PCR, based on the aqxA gene, was done as described by Berthoud et al [2], while specific ApxI, ApxII and ApxIII PCR, amplifying apxIA, apxIIA, apxIIIA plus a small part of the corresponding C genes, was carried out as described by Frey et al [8]. Specific ApxIV PCR was finally done as reported by Schaller et al [17].…”

Section: Dna Isolation and Pcr Analysismentioning

confidence: 99%

Phylogenetic relationship of equine Actinobacillus species and distribution of RTX toxin genes among clusters

et al. 2003

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-Equine Actinobacillus species were analysed phylogenetically by 16S rRNA gene (rrs) sequencing focusing on the species Actinobacillus equuli, which has recently been subdivided into the non-haemolytic A. equuli subsp. equuli and the haemolytic A. equuli subsp. haemolyticus. In parallel we determined the profile for RTX toxin genes of the sample of strains by PCR testing for the presence of the A. equuli haemolysin gene aqx, and the toxin genes apxI, apxII, apxIII and apxIV, which are known in porcine pathogens such as Actinobacillus pleuropneumoniae and Actinobacillus suis. The rrs-based phylogenetic analysis revealed two distinct subclusters containing both A. equuli subsp. equuli and A. equuli subsp. haemolyticus distributed through both subclusters with no correlation to taxonomic classification. Within one of the rrs-based subclusters containing the A. equuli subsp. equuli type strain, clustered as well the porcine Actinobacillus suis strains. This latter is known to be also phenotypically closely related to A. equuli. The toxin gene analysis revealed that all A. equuli subsp. haemolyticus strains from both rrs subclusters specifically contained the aqx gene while the A. suis strains harboured the genes apxI and apxII. The aqx gene was found to be specific for A. equuli subsp. haemolyticus, since A. equuli subsp. equuli contained no aqx nor any of the other RTX genes tested. The specificity of aqx for the haemolytic equine A. equuli and ApxI and ApxII for the porcine A. suis indicates a role of these RTX toxins in host species predilection of the two closely related species of bacterial pathogens and allows PCR based diagnostic differentiation of the two.Actinobacillus / horse / RTX toxin / phylogeny / diagnostic

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Section: Dna Isolation and Pcr Analysismentioning

confidence: 99%

Phylogenetic relationship of equine Actinobacillus species and distribution of RTX toxin genes among clusters

et al. 2003

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“…a,12,21 Actinobacillus pleuropneumoniae biovars I and II were identified by the Apx toxin gene (apx) detection. 10 For the identification of A. pleuropneumoniae serotype 2 biovars I and II, a cps2-based polymerase chain reaction (PCR) was used.…”

Section: Genetic Typing and Identificationmentioning

confidence: 99%

Routine Phenotypic Identification of Bacterial Species of the Family Pasteurellaceae Isolated from Animals

et al. 2008

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Abstract. Pasteurellaceae are bacteria with an important role as primary or opportunistic, mainly respiratory, pathogens in domestic and wild animals. Some species of Pasteurellaceae cause severe diseases with high economic losses in commercial animal husbandry and are of great diagnostic concern. Because of new data on the phylogeny of Pasteurellaceae, their taxonomy has recently been revised profoundly, thus requiring an improved phenotypic differentiation procedure to identify the individual species of this family. A new and simplified procedure to identify species of Actinobacillus, Avibacterium, Gallibacterium, Haemophilus, Mannheimia, Nicoletella, and Pasteurella, which are most commonly isolated from clinical samples of diseased animals in veterinary diagnostic laboratories, is presented in the current study. The identification procedure was evaluated with 40 type and reference strains and with 267 strains from routine diagnostic analysis of various animal species, including 28 different bacterial species. Type, reference, and field strains were analyzed by 16S ribosomal RNA (rrs) and rpoB gene sequencing for unambiguous species determination as a basis to evaluate the phenotypic differentiation schema. Primary phenotypic differentiation is based on b-nicotinamide adenine dinucleotide (b-NAD) dependence and hemolysis, which are readily determined on the isolation medium. The procedure divides the 28 species into 4 groups for which particular biochemical reactions were chosen to identify the bacterial species. The phenotypic identification procedure allowed researchers to determine the species of 240 out of 267 field strains. The procedure is an easy and cost-effective system for the rapid identification of species of the Pasteurellaceae family isolated from clinical specimens of animals.

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“…ApxI is produced by serotypes 1, 5, 9, 10, 11 and 14, and ApxII is produced by all serotypes except serotype 10 and 14. ApxIII is produced by serotypes 2, 3, 4, 6, 8, and 15 [2,8,21], and the ApxIV toxin is produced by all 15 serotypes [2,21,22]. The apxIV gene is species-specific rather than serotype-specific [5,22], and because of this, the apxIV gene may serve as a sensitive and specific probe for the diagnosis of A. pleuropneumoniae infection.…”

Section: Introductionmentioning

confidence: 99%

In situ hybridization for the detection of the apxIV gene in the lungs of pigs experimentally infected with twelve Actinobacillus pleuropneumoniae serotypes

2002

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-The detection of the apxIV gene in lung tissues from pigs experimentally infected with the 12 major A. pleuropneumoniae serotype (1 to 12) reference strains was studied by in situ hybridization using a non-radioactive digoxigenin-labeled DNA probe. In situ hybridization produced a distinct positive signal in all pigs inoculated with the 12 A. pleuropneumoniae serotypes. Positive hybridization typically exhibited a dark-brown to black reaction product in intracellular and extracellular locations, without background staining. A strong hybridization signal was seen in degenerated alveolar leukocytes ("oat cells") adjacent to the foci of coagulative necrosis and in the alveolar spaces. The in situ hybridization methodology developed for the detection of the apxIV gene is a valuable tool for the diagnosis of porcine pleuropneumonia caused by A. pleuropneumoniae when only formalin-fixed tissues are submitted for diagnosis.Actinobacillus pleuropneumoniae / Apx toxin / in situ hybridization / lung / pig Résumé -Détection par hybridation in situ du gène apxIV dans les poumons de porcs infectés expérimentalement par douze sérotypes de Actinobacillus pleuropneumoniae. La détection du gène apxIV dans les tissus pulmonaires de porcs infectés expérimentalement par les douze sérotypes majeurs des souches de référence (1 à 12) de Actinobacillus pleuropneumoniae a été effectuée par hybridation in situ utilisant une sonde à ADN non radioactive marquée à la digoxigénine. L'hybridation in situ a produit un signal positif distinct chez tous les porcs inoculés par les douze sérotypes de A. pleuropneumoniae. Un résultat positif d'hybridation entraînait typiquement un produit de réaction marron foncé à noir localisé en intra-et en extra-cellulaire, sans bruit de fond. Un signal fort d'hybridation a été observé dans les leucocytes alvéolaires dégénérés adjacents aux foyers de nécrose et dans les espaces alvéolaires. La méthode d'hybridation in situ développée pour la détection du gène apxIV est un outil précieux pour le diagnostic de la pleuropneumonie porcine causée par A. pleuropneumoniae lorsque seuls les tissus fixés dans le formol sont soumis pour le diagnostic.Actinobacillus pleuropneumoniae / Apx toxine / hybridation in situ / poumon / porc 653 Vet.

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Development of an efficient PCR method for toxin typing of Actinobacillus pleuropneumoniae strains

Cited by 63 publications

References 17 publications

Phylogenetic relationship of equine Actinobacillus species and distribution of RTX toxin genes among clusters

Phylogenetic relationship of equine Actinobacillus species and distribution of RTX toxin genes among clusters

Routine Phenotypic Identification of Bacterial Species of the Family Pasteurellaceae Isolated from Animals

In situ hybridization for the detection of the apxIV gene in the lungs of pigs experimentally infected with twelve Actinobacillus pleuropneumoniae serotypes

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