Evaluation of a multiplex PCR test for simultaneous identification and serotyping of Actinobacillus pleuropneumoniae serotypes 2, 5, and 6

Abstract

Serotype-specific DNA regions involved in the biosynthesis of capsular polysaccharides (cps region) were used to develop a multiplex PCR test for the simultaneous species identification and serotyping of Actinobacillus pleuropneumoniae serotypes 2, 5, and 6. Primers specific for serotypes 2, 5, and 6 were combined with the already existing species-specific primers used in a PCR test based on the omlA gene. The PCR test was evaluated with serotype reference strains of A. pleuropneumoniae as well as 182 Danish field isolates previously serotyped by latex agglutination or immunodiffusion. For all serologically typeable strains, a complete correspondence was found between the results obtained by the multiplex PCR test and the results obtained by the traditional serotyping methods. Six of eight serologically nontypeable strains could be allocated to a serotype on the basis of the multiplex PCR results. The species specificity of the assay was evaluated with a collection of 93 strains representing 29 different species within the family Pasteurellaceae, as well as species normally found in the respiratory tracts of swine. All of these strains were negative by the multiplex PCR test, including 50 field isolates of the phylogenetically closely related species Actinobacillus lignieresii. When the multiplex PCR test was used to test Danish field strains, it was able to identify the serotypes of approximately 94% of all strains isolated from swine with clinical disease. More than 90% of the isolates that cross-reacted by the latex agglutination test were of serotype 2, 5, or 6. Determination of the serotype by PCR represents a convenient and specific method for the serotyping of A. pleuropneumoniae in diagnostic laboratories.

FIG. 1.

Agarose gel electrophoresis of DNA fragments generated by multiplex PCR with the reference A. pleuropneumoniae strains of serotypes 1 to 15 (as indicated above the lanes) (Table 1). All strains gave rise to a species-specific band of approximately 950 bp, in addition to serotype-specific bands: approximately 500 bp (serotype 2), approximately 1,100 bp (serotypes 5A and 5B), and approximately 720 bp (serotype 6). Lane N, nontemplate control; lanes M, DNA molecular weight marker VI (Boehringer Mannheim).