DNA and/or RNA extracted from freshly collected tissue fragments from the calves and the aborted fetus (Boom et al. 1990 (link)), associated with proteinase K (Ambion, Grand Island, NY, USA), and a combination of the phenol/chloroform/isoamyl alcohol and silica/guanidine isothiocyanate method (Alfieri et al. 2006 (link)), was used in PCR assays designed to amplify specific amplicons of principal infectious disease agents of cattle. These PCR protocols targeted the ovine herpesvirus type 2 (OvHV-2) tegument protein gene (Baxter et al. 1993 (link)), the glycoprotein C gene of bovine herpesvirus-1 (BoHV-1) and −5 (Claus et al. 2005 (link)), the listeriolysin (hlyA) gene of Listeria monocytogenes (Wesley et al. 2002 (link)), the 16S rRNA gene of H. somni (Angen et al. 1998 (link)), and the 5′-UTR region of pestivirus (Vilček et al. 1994 (link)). Additionally, the feces from the calf with clinical diagnosis of enteritis were evaluated for the presence of the nucleoprotein gene of bovine coronavirus BCoV (Takiuchi et al. 2006 (link)) and G (VP7) and P (VP4) genes of bovine group A rotavirus, BoRV-A (Gouvea et al. 1990 (link); Gentsch et al. 1992 (link)).
Positive controls consisted of DNA/RNA from previous cases: OvHV-2 (Headley et al. 2012b ), L. monocytogenes (Headley et al. 2012a ), cell culture-adapted Los Angeles and AA Par strains of BoHV-1 and −5, respectively (Claus et al. 2005 (link)), BVDV (NADL strain)-infected Madin Darby bovine kidney cells, and BCoV (Takiuchi et al. 2006 (link)). No positive control was included in the H. somni PCR assays. Nuclease-free water (Invitrogen Corp. Carlsbad, CA, USA) was used as negative controls in all PCR assays. All PCR products were separated by electrophoresis in 2 % agarose gels, stained with ethidium bromide, and examined under ultraviolet light.
The amplified PCR products were then purified (illustra GFX PCR DNA and Gel Band Purification Kit, GE Healthcare, Little Chalfont, Buckinghamshire, UK) and submitted for direct sequencing using the forward and reverse primers. The partial nucleotide sequences were initially compared by the BLAST (http://www.ncbi.nlm.nih.gov/BLAST ) program with similar selected sequences deposited in GenBank. Phylogenetic tree and sequence alignments based on the 16S rRNA gene of the Pasteurellaceae family were then created by using MEGA 5.10 (Tamura et al. 2011 (link)), constructed by the neighbor-joining method, based on 1,000 bootstrapped data sets; distance values were calculated by using the Kimura 2 parameter model. Escherichia coli was used as the out-group to provide stability to the generated tree.
Positive controls consisted of DNA/RNA from previous cases: OvHV-2 (Headley et al. 2012b ), L. monocytogenes (Headley et al. 2012a ), cell culture-adapted Los Angeles and AA Par strains of BoHV-1 and −5, respectively (Claus et al. 2005 (link)), BVDV (NADL strain)-infected Madin Darby bovine kidney cells, and BCoV (Takiuchi et al. 2006 (link)). No positive control was included in the H. somni PCR assays. Nuclease-free water (Invitrogen Corp. Carlsbad, CA, USA) was used as negative controls in all PCR assays. All PCR products were separated by electrophoresis in 2 % agarose gels, stained with ethidium bromide, and examined under ultraviolet light.
The amplified PCR products were then purified (illustra GFX PCR DNA and Gel Band Purification Kit, GE Healthcare, Little Chalfont, Buckinghamshire, UK) and submitted for direct sequencing using the forward and reverse primers. The partial nucleotide sequences were initially compared by the BLAST (
