The nasopharyngeal microbiota of beef cattle before and after transport to a feedlot

Holman, Devin B.; Timsit, Edouard; Amat, Samat; Abbott, D. Wade; Buret, Andre G.; Alexander, Trevor W.

Nasopharyngeal swabs were suspended in 1.2 mL of brain heart infusion (BHI) broth with 20% glycerol and vortexed. For isolation of M. haemolytica and P. multocida, a 100 μl aliquot of the swab suspension was plated onto tryptic soy agar (TSA) plates containing 5% sheep blood, supplemented with 15 μg bacitracin ml −1 (Dalynn Biologicals, Inc., Calgary, AB, Canada), and incubated overnight at 37°C. For culturing of H. somni, a 100 μl aliquot of the swab suspension was plated onto TSA plates containing 5% sheep blood without the bacitracin supplement and incubated for 48 h in a 10% CO 2enriched environment at 37°C. Colonies displaying morphology of M. haemolytica (white-grey, round, medium-sized, non-mucoid, exhibiting β-haemolysis), P. multocida (translucent, greyish in colour, and mucoid in consistency) and H. somni (yellowish hue, haemolytic) were confirmed through polymerase chain reaction (PCR) analysis using HotStarTaq Plus Master Mix (Qiagen Canada Inc., Toronto, ON) according to manufacturer's specifications with primers and annealing conditions described in Table S1 (Additional file 2). Colonies were lysed in Tris-ethylenediaminetetraacetic acid (EDTA) buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, pH 8.0) at 95°C for 5 min and used as deoxyribonucleic acid (DNA) template (2 μl) in PCR. The swabs were placed in the remaining swab suspension and stored at −80°C until DNA extraction.