Twenty-gram packets were made with NP-51, individual packets were used once daily and any remaining material was

discarded. Viable cultures of NP-51 were mixed into sterile, powdered mouse chow (7012 Teklad LM-485 Mouse/Rat Sterilizable Diet; Selleck BLZ945 Harlan Teklad Diets, Madison WI) using a KitchenAid® 5-Quart Tilt-Head Artisan Series Stand Mixer (Bed Bath & Beyond; Lubbock, TX) at setting 2 or 3, for 15–20 minutes in a BSL-2 safety cabinet (this insured even distribution of NP-51 in the powdered chow). Non-viable NP-51 was prepared by heating samples at 180°C in a dry oven for 20 min (Fisher Scientific Convection Gravity Oven; Fisher Sci, Houston, TX). Non-viable cultures selleckchem were mixed with an identical mixer system, separately, www.selleckchem.com/products/pf-03084014-pf-3084014.html using sterile bowls and utensils. Each chow was replaced daily with new feed according to experimental conditions. Animal cages and feed containers were handled under a BSL-2 safety cabinet. Feed containers were cleaned and sterilized weekly by autoclaving (121°C for 15 min), new feed containers were replaced along with sterilized cages and bedding every 3rd or 7th day. Utensils for preparing chow including bowls, mixing utensils, and glassware were cleaned daily and sterilized with baking at 180°C in a convection gravity oven at a minimum of 4 hours or overnight, before use. MAP infection

and sampling schedule On day 46, through intraperitoneal (IP) injection experimental Reverse Transcriptase inhibitor groups were injected with 100 μl of sterile PBS containing 1×107 CFU/ml viable or non-viable MAP. Controls were injected with 100 μl PBS only. Animals were observed closely for 48 h for negative physiological reactions to IP injections. Every

45 days post infection – Days 90, 135, and 180- necropsies were performed. Serum/ tissue collection & cytokine analysis At each necropsy, blood was collected into serum separation tubes, and serum was pooled from each experimental group (n =5) (13×100 mm, SST™ Serum Separation Tubes; Beckton- Dickinson; San Jose, CA). Blood samples were refrigerated for 24-48 h after collection, followed by centrifugation at 5,000 × g for 5 min (Marathon 2100R, Thermo-Fisher Scientific; Houston, TX). Serum was transferred, using disposable, sterile serological pipettes to sterile, 2 ml cryogenic tubes and stored at −20°C (Fisher Scientific; Houston, TX). Two-hundred microliters of serum from each experimental condition and for all collection time points were shipped to TTUHSC, at El Paso and analyzed using a Mouse Cytokine 20-Plex Panel for the Luminex® platform – according to manufacturer protocol (Invitrogen/Life Technologies; Carlsbad, CA). Serum was analyzed in triplicate wells and compared to standards. Tissue RNA/DNA extractions, cDNA synthesis, & cDNA analysis Colon tissues were ground with mortar and pestle in liquid nitrogen to preserve RNA/DNA and prevent nuclease activity in tissues.