Nasopharyngeal Swab (NPS) Collection and Culture

Double guarded swabs (#022964 MWI, Nampa, ID, USA) were used to sample the nasopharynx of cattle as previously described (7). One swab was collected from each nostril then the two swabs were placed together into transport media (Modified Amies Clear gel, SP130X, Starplex Scientific Inc. Etobicoke, Ontario, Canada) and transported back to the laboratory on ice for culture within 6 h of collection. Both swabs were streaked together on the first quadrant of 5 sequential plates containing tryptic soy agar + 5% sheep's blood (blood agar) plates. Five sequential plates were streaked in order to account for the possibility that overgrowth of contaminant bacteria might prevent identification of M. haemolytica on the first plate. For each of the 5 plates a new sterile loop was used to streak from the first quadrant to the remaining 3 quadrants. Plates were streaked and evaluated in a biosafety cabinet to prevent contamination. After streaking, plates were incubated at 37°C, 5% CO₂ for 18–24 h, then colonies phenotypically consistent with M. haemolytica (round white/gray with a glossy edge and beta hemolysis) were collected, with one colony tested by the oxidase (slow +), indole (–), catalase (+), and KoH (+) tests to confirm identity. If there were fewer than 3 colonies on a plate the biochemical tests were not performed until the isolates on the primary plate were subcultured. Isolated colonies consistent with M. haemolytica were subcultured to a new plate; a maximum of 20 colonies were collected from each of the 5 plates, for up to 100 colonies per NPS. The subcultures were incubated at 37°C in 5% CO₂ for 18–24 h, then for each subculture plate all bacteria were swabbed off and transferred to 1 ml of 50% glycerol in 1X phosphate buffered saline, and stored at −80°C.