uberis based on colonial appearance, Gram stain reaction and catalase test (National Mastitis Council, 2004) and by conventional identification (Odierno et al., 2006). The selected colonies were maintained frozen at −20 °C in Todd–Hewitt broth (Sigma-Aldrich

Co.) containing 20% glycerol for further characterization. Isolates were identified as representing S. uberis by restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene according to Jayarao find more et al. (1992). All the isolates were additionally confirmed by RFLP analysis of the 16S rRNA gene, using the restriction enzymes RsaI and AvaII (Khan et al., 2003). Streptococcus uberis ATCC 27958 and Streptococcus parauberis ATCC 13386 were used as reference strains. The target genes, the oligonucleotide primers used and the sizes of the amplicons are summarized in Table 1. Synergistic CAMP-like haemolytic

activities were determined together with a β-toxin-producing Staphylococcus aureus on sheep blood agar plates (Odierno et al., 2006). Genomic DNA was isolated as described by Jayarao et al. (1992), purified by ethanol precipitation and dissolved in a buffer containing 10 mM Tris/HCl (pH 7.6) and 0.1 mM EDTA. Specific oligonucleotide primers for the detection of the cfu, lbp and sua genes of S. uberis were designed for this study with primer3 software (http://frodo.wi.mit.edu/primer3/). buy SP600125 DNA amplification for the hasA, hasB, hasC, oppF, pauA/B and skc genes was performed using oligonucleotide primers derived from published sequences. All the oligonucleotides were synthesized by Promega

Corporation. The PCR was standardized for the detection of each virulence-associated gene following the methodologies described with suitable modifications to optimize the different conditions that affect the sensitivity and specificity of the reaction. Flavopiridol (Alvocidib) Details of the primer sequences are shown in Table 1. To amplify the genes, 50 μL of reaction mixture was made containing 20 ng template DNA, 1 μM oligonucleotide primers, 0.4 μM of each of the four dNTPs, 1.50 U Taq polymerase and 1.5 mM MgCl2. The annealing temperature was varied from 48 to 58 °C depending on the gene being amplified. The reactions were carried out in a thermal cycler and genes of each isolate were tested at least twice. A positive and a negative control were included in each run. PCR products were resolved on 1.2% agarose gel at 70 V for 1.5 h. Gels were stained with ethidium bromide solution (0.5 mg mL−1) and photographed under UV light with MiniBisPRO gel documentation. RFLP analysis of the 16S rRNA gene successfully identified 78 isolates as S. uberis at the molecular level based on comparisons with reference strain S. uberis ATCC 27958 (Fig. 1). A synergistic haemolytic CAMP-like reaction on sheep blood agar within the zone of staphylococcal β-toxin could be observed for 18 of the 78 (23%) S. uberis strains. The standardized PCR allowed the amplification of putative and known virulence-associated genes of S.