Two-color hybridization assay for simultaneous detection of Bordetella bronchiseptica and toxigenic Pasteurella multocida from swine

Abstract

Bordetella bronchiseptica and toxigenic Pasteurella multocida are the etiologic agents of swine atrophic rhinitis. Methods currently used for their identification are time-consuming and suffer from a lack of sensitivity. We describe a colony lift-hybridization assay for detection of B. bronchiseptica and toxigenic P. multocida that can be performed with a single colony lift derived from a primary isolation plate without the need for pure subcultures of suspect bacteria. Membranes are hybridized simultaneously to probes derived from the B. bronchiseptica alcA gene and the P. multocida toxA gene. A multicolor development procedure permits sequential detection of bound probes. The assay was tested with 84 primary isolation plates generated from nasal swabs from swine with clinical signs of atrophic rhinitis. Comparison of the results from the colony lift-hybridization assay with those from conventional testing, based on a combination of colony morphology, biochemical reactions, mouse lethality, and enzyme-linked immunosorbent assay, indicated that the colony lift assay has superior sensitivity and comparable specificity. This technique has wide application for diagnostic and experimental studies.

FIG. 1

Colony lift from a plate streaked with toxigenic P. multocida (A), nontoxigenic P. multocida (B), and B. bronchiseptica (C), hybridized with 30 ng each of fluorescein-ToxA and digoxigenin-AlcA per ml, and developed by the multicolor detection procedure.

FIG. 2

Colony lift from a plate streaked with swine isolates of E. coli (locations 1 to 6) or S. typhimurium (location 7), S. choleraesuis (location 8), S. anatum (location 9), S. heidelberg (location 10), S. infantis (location 11), S. montevideo (location 12), and S. derby (location 13); hybridized with digoxigenin-ToxA; and developed with a chemiluminescent substrate. The location of the toxigenic P. multocida strain used as a positive control is indicated with an asterisk.

FIG. 3

Colony lifts from a primary isolation plate reported to be negative for toxigenic P. multocida by conventional methods. Membranes were hybridized with 30 ng each of fluorescein-ToxA and digoxigenin-AlcA per ml and developed by the multicolor detection procedure (A) or subjected to a colony blot assay with a monoclonal antibody specific for the P. multocida dermonecrotic toxin (B).