Indicator strains included E. coli FUA1036, E. coli FUA1063, E. coli FUA1064, www.selleckchem.com/products/azd5363.html Listeria innocua ATCC33090, and Enterococcus facaelis FUA3141. The deferred inhibition assay was repeated with the addition of 20 g L-1 proteinase K in 100 mmol L-1 Tris-Cl, pH 8.5, which was spotted adjacent to test strain colonies and plates were incubated for four hours at 55°C to maximize proteinase activity before overlayering was conducted. Identification of library clones via sequencing PCR-DGGE analysis was initially carried out characterise bovine vaginal microbiota by a culture-independent approach. The DNA concentration of samples from healthy cows, however, was below the detection limit of PCR-DGGE

analysis and DGGE patterns could be obtained only for two samples from animals #2373 #2409 (data not shown). Total bacterial DNA was isolated from

these two vaginal swab samples via both phenol chloroform extraction and Wizard MagneSil® Tfx™ System (click here Promega). Nested PCR was conducted to maximize DNA amplification by amplifying with 616V and Vistusertib cost 630R primers prior to amplification with HDA primers (Table 2). PCR products that were amplified with HDA primers were cloned into a pCR 2.1-TOPO vector using the TOPO TA Cloning® Kit (Invitrogen) according to manufacturer’s instructions. The Promega’s Wizard® Plus SV A clone library was constructed using PCR products that were amplified with HDA primers, which were then cloned into a pCR 2.1-TOPO vector, using the TOPO TA Cloning® Kit (Invitrogen) according to manufacturer’s instructions. The Promega’s Wizard® Plus SV Minipreps DNA Purification System was used for plasmid isolation. To confirm the cloning of the inserts, sequencing of the amplified insert was performed according to the Invitrogen TOPO TA Cloning® Kit manual. Quantitative PCR Quantitative PCR was conducted with vaginal mucus samples collected from ten cows, using syringes fitted with an approximately 30 cm long collection tube. Samples from 10 animals that developed metritis Doxacurium chloride after calving were randomly selected from samples of a larger cohort of animals. Total

bacterial DNA was extracted using the Wizard MagneSil® Tfx™ System (Promega) and DNA concentrations were measured using the NanoDrop spectrophotometer system ND-1000, software version 3.3.0 (Thermo Fisher Scientific Inc., Wilmington, USA). All dagger-marked primer pairs that are listed in Table 2 were used in the preparation of standards and qPCR analyses. Standards were prepared using purified PCR products, which were serially diluted ten-fold. Diluted standards (10-3 to 10-8) were used to generate standard curves. TaqMan probes were used for the pedA gene and the total bacteria qPCR experiments. In both cases, each probe was labelled with 5’-FAM and 3’-TAMRA as fluorescent reporter dye and quencher respectively. The total reaction volume was set to 25 μL, which contained 12.5 μL TaqMan Universal PCR Master Mix (Applied Biosystems), 2.