Tris edta buffer

Nasopharyngeal samples were collected from each animal at entry (day 0) and then 60 days after feedlot placement, according to Alexander et al.4 (link) Double-guarded swabs were used for sampling and were transported to the lab on ice for processing, within 1 hour of collection. The end of each swab was removed and vortexed in 0.7 ml brain heart infusion (BHI) broth containing 20% glycerol and 0.1 ml of serial 10-fold dilutions were spread onto BHI, MRS, or 5% sheep blood agar. All agar plates were incubated at 37 °C for 24–48 h in a 5% CO₂-enriched environment. Up to seven randomly selected bacterial isolates were chosen from each plate and subcultured onto fresh agar plates. The same individuals were responsible for picking all the colonies at both time points. Subcultured isolates were transferred to 50 μl of Tris-EDTA (TE) buffer (Sigma Aldrich, St. Louis, MO, USA) and kept at −80 °C until use. The remaining swab-BHI mixtures were also stored at −80 °C.

Total RNA was extracted from skin samples of 10 fish per diet from both Trial#1 and #2 using TRI reagent (Sigma), as instructed by the manufacturer. RNA was also extracted in the same manner for the Trial#1 gut samples (n = 6). The concentration and quality of the RNA was assessed by both the ND-1000 Nanodrop Spectrophotometer (Labtech Inc., East Sussex, UK) and the Agilent 2100 Bioanalyzer (Agilent Technologies, Cheshire, UK). RNA (2 μg) was used for cDNA synthesis using Bioscript reverse transcriptase kit (Bioline, London, UK) and 0.2 μg of Random Hexamer Primer (Thermo Scientific, Northumberland, UK). The mixture was incubated at 70°C for 5 minutes and then left to rest on ice for 2 minutes. The master mix for 25 μL reactions was prepared as described in the kit protocol. The tubes were incubated at 25°C for 10 minutes, 45°C for 60 minutes and 72°C for 10 minutes. The cDNA was diluted five-fold using 1x Tris-EDTA (TE) buffer (Sigma).

Fifteen Holstein-Friesian cows, of mixed parity, within the same university dairy herd were sampled 7 and 21 days postpartum (DPP) in the morning after milking. A clinical examination and sample collection were conducted by a veterinarian and involved recording the rectal temperature, body condition score, heart rate and vaginal discharge. At each time-point, an endometrial biopsy was taken from the same post-gravid horn as previously described [67 (link)]. Uterine culture swabs were placed (in duplicate per cow) in 1 ml of Tris-EDTA buffer (Sigma Aldrich® Vale Road, Arklow, Wicklow, Ireland). Immediately after collection, the biopsy was dissected in two - one half was snap frozen in liquid nitrogen for transcriptomic analysis, and the other half was fixed in 10 % neutral-buffered formalin solution (Sigma Aldrich® Vale Road, Arklow, Wicklow, Ireland) for histopathological assessment. Whole blood was collected in spray-coated K₂EDTA Vacutainer®, for haematology and plasma protein analysis, followed by 4 ml of blood in one sodium fluoride/Na₂ EDTA Vacutainer®, for blood metabolite analysis at pre- and postpartum time points. All experimental procedures were carried out under license from the Irish Department of Health and Children in accordance with the European Community Directive 86-609-EC and were approved by the Animal Research Ethics Committee, University College Dublin.

Samples for DNA extraction (1 ml) were collected from above experiments in RNase/DNase-free tubes. Cell pellets were obtained by removing 900 μl of the supernatant, and the pellets were immediately flash frozen in liquid nitrogen and stored at −70 °C. Prior to extraction, 100 μl of lysozyme (1 mg/ml, Sigma-Aldrich) in Tris-EDTA buffer (Sigma-Aldrich) were added to the pellet and left for 20 min at room temperature. For DNA extraction, the AllPrep DNA/RNA mini kit (Qiagen, USA) was used according to the manufacturer’s protocol. The final DNA extractions were stored at −70 °C until further processing.

Polymorphisms in the genes for proteins used in the ACVs (PtxA, Prn, Fim2 and Fim3) were analysed as described previously [5 (link), 15 (link), 44 (link), 45 (link)]. The pertussis toxin promoter, ptxP, was also included, as previous studies have shown that the ptxP3 allele is an important characteristic of successful isolates [5 (link), 20 (link)–22 (link), 25 (link), 27 (link)–30 (link)]. For DNA isolation, bacterial cells were lysed in Tris-EDTA buffer (Sigma-Aldrich, Zwijndrecht, the Netherlands, 1.0 M Tris–HCl, containing 0.1 M EDTA, 100× concentrated) at 95 °C for 5 min, centrifuged briefly and used in a polymerase chain reaction (PCR) assay.