Improved Diagnostic PCR Assay forActinobacillus pleuropneumoniaeBased on the Nucleotide Sequence of an Outer Membrane Lipoprotein

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Abstract
The gene (omlA) coding for an outer membrane protein ofActinobacillus pleuropneumoniaeserotypes 1 and 5 has been described earlier and has formed the basis for development of a specific PCR assay. The corresponding regions of all 12A. pleuropneumoniaereference strains of biovar 1 were sequenced. Alignment of the sequences revealed conserved terminal and variable middle regions, which divided the reference strains into four distinct groups. Primers were selected from the conserved 5′ and 3′ termini of the gene. A 950-bp amplicon was obtained from each of 102 tested field isolates ofA. pleuropneumoniaeobtained from lungs. Their identity was verified by sequencing approximately 500 bp of the amplification product from 50 of theA. pleuropneumoniaeisolates, which all showed the expected DNA sequence characteristic of the serotype. To test the specificity of the reaction, 23 other bacterial species related toA. pleuropneumoniaeor isolated from pigs were assayed. They were all found negative in the PCR, as were tonsil cultures from 50 pigs of anA. pleuropneumoniae-negative herd. The sensitivity assessed by agarose gel analysis of the PCR product was 102CFU/PCR test tube. The specificity and sensitivity of this PCR compared to those of culture suggest the use of this PCR for routine identification ofA. pleuropneumoniae.

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