Molecular Identification of Bovine Pathogens

PCR with species-specific primers was used to identify the four bacterial species from isolates obtained from beef cattle, and to confirm the biochemical (aerobic) and morphologic (Mycoplasma) identification of the isolates from the dairy cattle. Primers were designed in Primer3 (Koressaar and Remm 2007 (link); Untergrasser et al. 2012 (link)) to targeted genes specific to each species (Table 3). PCR protocols for M. haemolytica (Alexander et al. 2008 (link)) and P. multocida (Ullah et al. 2009 ) were performed as described previously. Protocols for H. somni and M. bovis were developed during this study. PCR was performed on an Eppendorf Mastercycler (Eppendorf, Hamburg, Germany) with 1× GoTAQ Green Master Mix (Promega Biosciences LLC, San Luis Obispo, CA), 10 μM of each primer and 10 ng of DNA for 5 min at 95°, 35 cycles of 30 sec at 95°, 30 sec at 56°, and 45 sec at 72°, followed by 10 min at 72°. Products were visualized on a 1.5% agarose gel using a ChemiDoc-It^TS2 Imager (UVP, LLC, Upland, CA), purified using the QIAquick PCR Purification Kit (Qiagen), Sanger sequenced (www.davissequencing.com) and aligned to their respective reference genomes using Bowtie2–default v2.2.8 (Langmead and Salzberg 2012 (link)) to verify that the appropriate target regions were amplified.