Nonradioactive colony lift-hybridization assay for detection of Bordetella bronchiseptica infection in swine

Abstract

Current methods for the isolation and identification of Bordetella bronchiseptica from clinical samples are time-consuming and are based, in part, on subjective observations. We describe the use of a Bordetella-specific DNA probe in a nonradioactive colony lift-hybridization assay for the identification of B. bronchiseptica. Eleven of 82 clinical specimens were found to contain B. bronchiseptica by this method, while only 5 of these were reported to contain the organism when the specimens were analyzed by traditional methods. The chromosomal fragment containing a sequence complementary to the probe appeared to be conserved in B. bronchiseptica isolates from swine from a variety of sources. The assay is more rapid than culture and biochemical testing since it can be performed directly on primary culture plates, even when they are heavily contaminated with other bacterial species. Only minimal training is required to accomplish the assay successfully, and the results are easy to interpret.

Two-Color Hybridization Assay for Simultaneous Detection of Bordetella bronchiseptica and Toxigenic Pasteurella multocida from Swine

“…We conclude that the colony lift-hybridization assay described here is more sensitive than standard methods since it permits the detection of even a few colonies whose presence and morphology are masked by overgrowth of other bacteria in the sample. Similar findings were reported in a previous study in which 6 of 77 clinical specimens reported to be negative by conventional identification methods were subsequently shown to contain colonies of B. bronchiseptica in an area of confluent growth (19).…”

“…A previous report from this laboratory described the use of a Bordetella-specific probe in a highly sensitive colony lifthybridization assay for identification of B. bronchiseptica infection in swine (19). This procedure is faster, more objective, and better suited to the analysis of large numbers of samples than standard identification methods based on colony morphology and biochemical testing.…”

“…The sensitivity and specificity of the AlcA probe for identification of B. bronchiseptica have previously been demonstrated with colony lifts derived from diagnostic samples (19). However, the membranes used in that study were developed with a chemiluminescent substrate, which is approximately 10 times more sensitive than multicolor detection (2).…”

“…The Bordetella-specific probe pDLA5, which contains a 4.7-kb fragment that includes the B. bronchiseptica alcA gene, has been described (6). A previous study demonstrated the specificity and sensitivity of this probe for identification of B. bronchiseptica (19). Commercially obtained plasmid purification columns were used to prepare plasmids pPMT and pDLA5, as instructed by the manufacturer (Qiagen, Chatsworth, Calif.).…”

“…Previously, we reported on a simple, nonradioactive colony hybridization assay for detection of B. bronchiseptica from primary culture plates (19). However, an assay that could detect both toxigenic P. multocida and B. bronchiseptica would be of great benefit to diagnostic laboratories and would facilitate experimental studies.…”