Development of an in vitro fluorometric assay to study adherence of Pasteurella haemolytica to bovine cells

OBJECTIVE: To develop an in vitro fluorometric assay to assess Pasteurella haemolytica adherence to bovine respiratory and epithelial cells and compare adherence of single strains of P. haemolytica serovars A1 and A2 (PhA1 and PhA2, respectively). SAMPLE POPULATION: Monolayers of bovine turbinate and Madin Darby bovine kidney (MDBK) cells. PROCEDURE: To determine optimal inoculum concentration and incubation time, various concentrations of P. haemolytica were labeled with fluorescein isothiocyanate and incubated with monolayers of bovine cells for various times. Bovine cells were washed to remove nonadherent bacteria, and percentage of bacteria adhered (percentage of adherence) was estimated fluorometrically. Percentage of adherence of PhA1 was compared with that of PhA2. RESULTS: The optimal inoculum concentration that resulted in measurable fluorescence of adherent bacteria was 1 x 10(8) colony-forming units/ml, and the optimal incubation time was 45 minutes. Percentage of adherence of PhA1 to MDBK and turbinate cells was significantly greater than that determined for PhA2. CONCLUSIONS: The in vitro fluorometric assay is a time-efficient, inexpensive, and labor-saving method for evaluation of P. haemolytica adherence to bovine cells. The concentration of bacteria used to inoculate bovine cells in this assay is similar to that typically used in other types of in vitro adherence assays. The predominance of PhA1 over PhA2 during the early stages of bovine respiratory disease may be attributable to the ability of PhA1 to adhere more avidly to nasopharyngeal tissue.