IMPROVED DIAGNOSTIC PCR ASSAY FOR ACTINOBACILLUS-PLEUROPNEUMONIAE BASED ON THE NUCLEOTIDE-SEQUENCE OF AN OUTER-MEMBRANE LIPOPROTEIN

The gene (omlA) coding for an outer membrane protein of Actinobacillus pleuropneumoniae serotypes 1 and 5 has been described earlier and has formed the basis for development of a specific PCR assay, The corresp onding regions of all 12 A. pleuropneumoniae reference strains of biov ar 1 were sequenced, Alignment of the sequences revealed conserved ter minal 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 of A. pleuropneumoniae obtained from lun gs. Their identity was verified by sequencing approximately 500 bp of the amplification product from 50 of the A. pleuropneumoniae isolates, which all showed the expected DNA sequence characteristic of the sero type, To test the specificity of the reaction, 23 other bacterial spec ies related to A. pleuropneumoniae or isolated from pigs were assayed. They were all found negative in the PCR, as were tonsil cultures from 50 pigs of an A. pleuropneumoniae-negative herd. The sensitivity asse ssed by agarose gel analysis of the PCR product was 10(2) CFU/PCR test tube. The specificity and sensitivity of this PCR compared to those o f culture suggest the use of this PCR for routine identification of A. pleuropneumoniae.