The bacterial suspension was boiled for 5 min and the cell debris was pelleted by centrifugation at 13,000 g, and the supernatant was used as template DNA for PCR analysis. The template DNA was used at 10% of the final PCR volume in the presence of 10 ρmoles of forward and reverse primer (Table 2), 10 μM dNTPs, 1x polymerase reaction buffer, 1 unit of thermal stable DNA polymerase and 3.5 mM MgCl2. The PCR reaction was performed as follows; 95°C for 5 mins for 1 repeat, 95°C for 30 seconds, 50°C for 1 minute and 72°C for 1 minute for 45 repeat cycles followed by a final extension of 72°C for 5 minutes. Presence of PCR

product amplification was determined by agarose gel electrophoresis.

Stattic mw Table 2 Primers used in this study Primer name 5`-3` primer sequence Tlp1p F TTG TTA TCG TTT ACG CTG ATG Tlp1p R TGG AAG ATC TTT ATT ATA ATT TTT TAA GGG TTT AA Tlp2p F CAT ATG CAA GCA ATT TTT CAT GAA GTT GTG A Tlp2p R CTC GAG TTA TTT ATA AAC TGG AGC TTC TAT TTG TT Tlp3p F CAT ATG ACC TCA CTA TAT GAA AGC ACT CTT Tlp3p R CTC GAG TTA TGC AGC TTT ATA AAT AGG TTT ATT TAT A Tlp4p F CTC Vactosertib purchase GAG GAT TCG AGA AAC AAT ACA TAT GAA TT Tlp4p R CTC GAG TTA TTG TTT CAT TAA AAT AGA ATT AAC AGC Tlp7p F CAT AGT TTT AAA AAT ACT GCC AAT AAA ATG AG Tlp7p R CTC GAG TTA AGA TTG ACT GGT TTT GCT TAT ATC Tlp7i F CTG CGA TCT CAT CCA TCA TTT GAG TTG C Tlp7i R CAT GCT AAA GAA TTA GCT CAA GGA AGT GGC Tlp10p F CAT ATG AAC TAT TCT TCA TCT AAA GAT AAT AA Tlp10p R CTC GAG TTA TTT AAA TAA ATT AGA TTG TTC TAT AGT Selleckchem MDV3100 Tlp11mid F CTC TGA TGG CAA AAG TGT AAC Tlp11mid R CTC TTC AGA TTG AGC GAT AAC Therm 1 (23SRNA) TTA TCC AAT ACC AAC ATT AGT Therm 2.1 (23SRNA) GAA GAT ACG GTG CTA TTT TG Preparation of C. jejuni inoculum C. jejuni cells were harvested from Columbia agar plates in 1 mL of PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4, pH 7.5) and the concentration was adjusted to 1 x 108 cfu/mL using spectrophotometry followed by a viable count. Inoculation of chickens with C. jejuni Ross breed chickens (Barters, Rochdale, Qld), with maximum age difference of 2 hours

and at one day after hatching, were placed into groups Idelalisib research buy of five, colour marked and pre-inoculation faecal samples were taken from the cloaca and cultured. Chickens were housed in clean barrier cages at 28°C and allowed access to sterilised food and water. All experiments were approved by the Griffith University Animal Ethics Committee (approval number: MSC/04/08/AEC). Following a pre-inoculation cloacal swab, one day old chickens were orally inoculated with 30 μL PBS containing 1 x 108 cfu bacterial cells as previously described [22]. On day 6, euthanasia was performed by cervical dislocation.