Direct PCR analysis for toxigenic Pasteurella multocida.

Abstract

A more rapid, accurate method to detect toxigenic Pasteurella multocida is needed for improved clinical diagnosis, farm biosecurity, and epidemiological studies. Toxigenic and nontoxigenic P. multocida isolates cannot be differentiated by morphology or standard biochemical reactions. The feasibility of using PCR for accurate, rapid detection of toxigenic P. multocida from swabs was investigated. A PCR protocol which results in amplification of an 846-nucleotide segment of the toxA gene was developed. The PCR amplification protocol is specific for toxigenic P. multocida and can detect fewer than 100 bacteria. There was concordance of PCR results with (i) detection of toxA gene with colony blot hybridization, (ii) detection of ToxA protein with colony immunoblot analysis, and (iii) lethal toxicity of sonicate in mice in a test set of 40 swine diagnostic isolates. Results of an enzyme-linked immunosorbent assay for ToxA agreed with the other assays except for a negative reaction in one of the 19 isolates that the other assays identified as toxigenic. In addition to accuracy, as required for a rapid direct specimen assay, toxigenic P. multocida was recovered efficiently from inoculated swabs without inhibition of the PCR. The results show that PCR detection of toxigenic P. multocida directly from clinical swab specimens should be feasible.