Evaluation of a PCR for detection of Actinobacillus pleuropneumoniae in mixed bacterial cultures from tonsils

Abstract

A PCR for the detection of Actinobacillus pleuropneumoniae was evaluated. All of 102 field, isolates of A, pleuropneumoniae reacted in the PCR by amplification of a 985 bp product. No PCR amplification product was observed when examining strains of A. ureae, A. capsulatus, A. hominis, A. equuli, A, rossii, A. suis, Escherichia coli, Bordetella bronchiseptica. Streptococcus suis, Pasteurella haemolytica, Pasteurella multocida, Haemophilus parasuis, Haemophilus taxon Minor group, Haemophilus taxon D/E and haemophilus taxon F. Amplification of a 985 bp product was, however, observed when testing strains of A. lignieresii. The lower detection limit of the PCR test was 10(3) A. pleuropneumoniae CFU/PCR test tube and was not affected by addition of 10(6) E. coli CFU/PCR test tube. Mixed bacterial cultures from tonsils of 101 pigs from 9 different herds were tested by culture and by PCR using four different bacteriological media. While 65% reacted positive in the PCR only 23% were positive by culture, thereby suggesting a superior sensitivity of the PCR test to that of culture. The use of selective media, large inoculum and incubation for 48 h gave the highest number of positive PCR reactions from mixed bacterial cultures. Tonsil cultures from 50 pigs from an A. pleuropneumoniae-negative herd did not react in the PCR. The results show that PCR on mixed bacterial cultures from tonsils may be a highly sensitive method for the detection of A. pleuropneumoniae in pig herds.