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A PCR-based procedure for detection and serotype identification of Actinobacillus pleuropneumoniae strains was developed and evaluated. The A. pleuropneumoniae tbpA and tbpB genes were used as targets for amplification of DNA fragments, with a pair of specific primers for each gene. Amplification with tbpA primers rendered a 2.8-kb PCR product from all 12 A. pleuropneumoniae reference strains as well as from Actinobacillus suis strain CCM 5586, while amplification of a 1.9-kb PCR product was observed when testing ten Haemophilus parasuis strains of different serovars. Amplification of the tbpB gene from A. pleuropneumoniae serotypes 1, 6, 8 and 12, and A. suis CCM 5586 rendered an identical 1.8-kb fragment, while from A. pleuropneumoniae serotypes 2, 3, 4, 7, 9, 10 and 11, and H. parasuis strains it produced a 1.7-kb fragment. No PCR amplification product was observed when examining strains of 19 other swine pathogens or closely related species. The minimal detection limit for whole-cell A. pleuropneumoniae templates was between 5-50 and 3 x 10(2)-3 x 10(3) CFU when tbpA and tbpB specific primers, respectively, were used. Restriction fragment length polymorphism (RFLP) analysis of the PCR-generated products rendered different patterns, easily allowing us to discriminate between A. pleuropneumoniae, H. parasuis and A. suis and, more importantly, to distinguish ten RFLP A. pleuropneumoniae groups (the highest discrimination reported so far for a PCR assay with A. pleuropneumoniae), in such a way that the only serotypes with profiles identical to each other were 4 to 11 and 7 to 9. Moreover, the PCR-RFLP analysis was assayed in 36 A. pleuropneumoniae field isolates and in porcine samples (lungs and nasal swabs from experimentally infected animals). In both cases the system proved to be very efficient in A. pleuropneumoniae identification and serotype discrimination.

A total of 229 Spanish Actinobacillus pleuropneumoniae isolates recovered from diseased pigs with pleuropneumonia from 1997 to 2004 was tested for their susceptibility to 11 antimicrobials in a broth microdilution method. All the isolates were susceptible to florfenicol and most of them to cephalothin; however, a high rate of resistance was observed to tetracycline. A bimodal or multimodal distribution of isolates over the MIC range were observed for penicillins, tetracycline, trimethoprim, sulfisoxazole and nalidixic acid, suggesting the development of acquired resistance. Eight resistance patterns were established, and 21.1% of the isolates were resistant to at least two antimicrobials. In addition, a considerable increase in the resistance to tetracyclines was observed during the last decade in Spain, when compared with other A. pleuropneumoniae strains isolated during 1987–1988 (Gutierrez, C.B., Piriz, S., Vadillo, S., Rodriguez Ferri, E.F., 1993. In vitro susceptibility of Actinobacillus pleuropneumoniae strains to 42 antimicrobial agents. Am. J. Vet. Res. 54, 546–550); this finding was also observed for gentamicin in minor percentage.