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Title: ANALYTICAL VERIFICATION OF A MULTIPLEX PCR FOR IDENTIFICATION OF BORDETELLA BRONCHISEPTICA AND PASTEURELLA MULTOCIDA FROM SWINE

Author
item Register, Karen
item DEJONG, KEITH - SELF-EMPLOYED

Submitted to: Veterinary Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/5/2006
Publication Date: 10/31/2006
Citation: Register, K.B., Dejong, K.D. 2006. Analytical verification of a multiplex PCR for identification of Bordetella bronchiseptica and Pasteurella multocida from swine. Veterinary Microbiology. 117(2-4):201-210.

Interpretive Summary: The bacteria Bordetella bronchiseptica and Pasteurella multocida cause swine atrophic rhinitis, a respiratory disease associated with considerable morbidity and economic loss to producers. Only dermonecrotic toxin-producing strains of P. multocida play a role in this disease while both toxigenic and nontoxigenic strains have been associated with pneumonia. The methods currently available for identification of these bacteria are slow and costly, depend on subjective observations, and have low sensitivity. This report describes the development, optimization, and performance characteristics of a novel multiplex PCR assay for simultaneous identification of both bacteria which also distinguishes toxigenic from nontoxigenic P. multocida. Based on analysis of a large number of isolates the assay is highly sensitive and specific and can detect the presence of only a few hundred bacteria. The data presented establish this multiplex PCR as a reliable method for identification of B. bronchiseptica and both toxigenic and nontoxigenic P. multocida that may greatly simplify investigations of swine atrophic rhinitis and bronchopneumonia.

Technical Abstract: Bordetella bronchiseptica and Pasteurella multocida are etiologic agents of progressive atrophic rhinitis (PAR) and bronchopneumonia in swine. Only dermonecrotic toxin-producing strains of P. multocida play a role in atrophic rhinitis while both toxigenic and nontoxigenic strains have been associated with pneumonia. Monitoring and investigation of outbreaks involving these bacteria require sensitive and accurate identification and reliable determination of the toxigenic status of P. multocida isolates. In the present study we report the development, optimization, and performance characteristics of a multiplex PCR assay for simultaneous amplification of up to 3 different targets, one common to all P. multocida strains, one found only in toxigenic P. multocida strains, and one common to B. bronchiseptica strains. Based on analysis of 94 P. multocida isolates (31 toxigenic) and 90 B. bronchiseptica isolates assay sensitivity is 100% for all amplicons. Evaluation of 22 isolates of other bacterial genera and species commonly found in the swine respiratory tract demonstrated a specificity of 100% for all gene targets. However, considering data previously reported by others for the P. multocida gene target a specificity of 98.2% is more accurate. The limit of detection for simultaneous amplification of all targets is 1-10pg of DNA per target, corresponding to a few hundred genomes or less. Amplicon mobility in agarose gels and sequence analysis indicate the amplicons are highly stable. The data presented establish this multiplex PCR as a reliable method for identification of B. bronchiseptica and both toxigenic and nontoxigenic P. multocida that may greatly simplify investigations of swine PAR and bronchopneumonia.