Samples included breasts, tenderloins (comprised of the muscle

pectoralis minor) and thighs. All samples were tray packs of approximately 1–2 lbs. More samples were processed during the months of summer than in winter. Sample preparation and enrichment procedures From each tray pack, 25 g of product were weighed and placed in sterile Whirl-Pak® bags (Nasco, Fort Atkinson, WI). Meat samples DNA Synthesis inhibitor were enriched at a 1:4 ratio (w:v) in modified Bolton broth supplemented only with cefoperazone (33 mg per l), amphotericin B (4 mg per l) and 5% lysed horse blood. Samples were enriched for 48 h at 42°C under microaerobic atmosphere (10% CO2, 5% O2, and 85% N2, AirGas South, Inc., Montgomery, AL), which was added to anaerobic jars with an evacuation replacement system (MACSmics Jar Gassing System, Microbiology International, Frederick, MD). Isolation of Campylobacter spp Enriched samples (broth) were plated (~0.1 ml) on modified charcoal cefoperazone AZ 628 manufacturer deoxycholate agar (mCCDA) for isolation and identification of Campylobacter spp. In 2009, 2010 and 2011, a slight modification was made to the protocol. For each sample, 0.1 ml of the enrichment

broth was transferred to an mCCDA plate using a filter membrane as described elsewhere [12]. All agar plates were incubated at 42°C under microaerobiosis for 48 h. selleck chemical Suspected Campylobacter colonies were observed under phase contrast microscopy (Optiphot-2, Nikon Instruments Inc., Melville, NY, or BX51, Olympus America Inc., Center Valley, PA) for their spiral morphology and darting motility. A small amount of growth from each plate was transferred to modified Campy-Cefex (mCC) agar plates supplemented with cefoperazone (33 mg), amphotericin B (4 mg) and 5% lysed horse blood. Plates were incubated at 42°C for 24 h under microaerobic conditions, and from these plates DNA was extracted using the Wizard® Genomic DNA Purification Kit as described by the manufacturer Calpain (Promega, Madison, WI) but without

the RNA digestion step, and plugs were made for PFGE analysis. Isolates were stored at −80°C in tryptic soy broth (TSB, Difco, Detroit, MI) supplemented with 30% glycerol (vol/vol) and 5% horse blood. Identification of isolates using mPCR assays Isolates were identified as C. jejuni or C. coli using two multiplex PCR (mPCR) assays: one based on primers targeting the ask gene of C. coli[13] and the hipO gene of C. jejuni[14], and the other targeting the ask gene of C. coli (different primers from the previous mPCR) and the glyA gene of C. jejuni[15]. PCR assays were performed in 25 μl aliquots using pre-made mixes of GoTaq® (Promega) or EconoTaq® PLUS (Lucigen, Middleton, WI). The assays were performed in a DNA Engine® Thermal Cycler (Bio-Rad Laboratories, Hercules, CA) as previously described [10, 15]. Amplified products were detected by gel electrophoresis stained with ethidium bromide and visualized using the VersaDoc™ Imaging System (Bio-Rad Laboratories).