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

Abstract

A method has been developed which allows the determination of the activator, the structural and the secretion genes of the three toxins ApxI, ApxII and ApxIII in Actinobacillus pleuropneumoniae in only two PCR reactions. The oligonucleotide primers were designed to amplify a significant part of the activator and structural genes apxICA, apxIICA and apxIIICA together in a single PCR reaction giving amplification products which differ in length, in order to be clearly separated by agarose gel electrophoresis. Variations in the apxIA and apxIIIA genes which were found in different serotypes were taken into account in the design of the primers to give a uniform amplification product for both variants of the apxIA and the apxIIIA genes. The secretion genes apxIBD and apxIIIBD are also detected in a single PCR reaction containing two pairs of oligonucleotide primers which yield two differently sized fragments to differentiate between apxIBD and apxIIIBD genes. The reference strains of A. pleuropneumoniae serotypes 1-12 and 104 field strains representing all serotypes obtained from various laboratories worldwide were analysed for their content of apx genes. The two PCR reactions give toxin gene patterns which are characteristic for different groups of serotypes in A. pleuropneumoniae and allow the rapid differentiation of five toxin type groups, group 1 including serotypes 1, 5a, 5b, 9 and 11, group 2 including serotypes 2, 4, 6, 8, group 3 with serotype 3, group 4 with serotype 7 and 12 and group 5 with serotype 10. The method enhances and facilitates differentiation of A. pleuropneumoniae strains for diagnostics and epidemiology and allows the detection of serotypes with atypical toxin patterns.