Beadblaster 24 homogenizer

The regulatory regions of ipf and klf were cloned into pCS26 upstream to a promoterless luxCDABE operon (pCS26::Pipf and pCS26::Pklf, respectively) and transformed into S. Infantis 119944. The reporter strains were grown for 16 h in LB supplemented with kanamycin at 37°C and ~8×10⁸ CFU were used to infect by oral gavage female C57BL/6 mice (Envigo, Israel) that were pretreated with streptomycin. Similarly, one day old SPF White Leghorns chicks (Charles River) were infected orally (inter crop) with ~1×10⁷ CFU of the reporter strains in 0.2 ml saline. At 24h p.i., mice and chicks were sacrificed and their intact gastrointestinal tracts were removed and imaged using a photon-counting in-vivo imaging system (Photon-Imager, Biospace Lab or IVIS Lumina LT, PerkinElmer). To determine bacterial loads, the organs were homogenized in 0.7 ml saline using a BeadBlaster 24 homogenizer (Benchmark Scientific), serially diluted and plated on XLD agar plates supplemented with kanamycin.

Swabs, as well as tissue samples, were re-suspended in 1 mL of phosphate-buffered saline (pH 7.4 +/− 0.2, supplemented with 10,000 IU/mL penicillin, 10 mg/mL streptomycin, 0.25 mg/mL gentamycin, and 5000 IU/mL nystatin), thoroughly homogenized by using Bead Blaster 24 Homogenizer (Benchmark Scientific, 2600, Sayreville, NJ, USA). After centrifugation at 1500× g for 10 min at 4 °C, total RNA was purified with the NucleoSpin^® RNA Virus Kit (Macherey-Nagel, Düren, Germany) following the manufacturer’s instructions. RNA was then eluted in 50 µL RNase-free water. All samples were tested by RT PCR for the influenza A matrix gene as described by Spackman, E. et al. (2002) [44 (link)]. The positive samples were then tested for the H9 gene detection by RT-PCR according to Monne, I. et al. (2008) [45 (link)] by using the AgPath-IDTM One-Step RT-PCR Kit (Ambion, Applied Biosystems, Grand Island, NY, USA). The rRT-PCR was performed with Aria Mx (Agilent Technologies, Santa Clara, CA, USA) and ABI 7500 Fast (Applied Biosystems, Foster City, CA, USA) 96-well plate real-time PCR devices.