Studies thus far, have concentrated

on the recovery of O157 from the rumen, the in vitro O157 growth dynamics in modified rumen fluid or media with additives to mimic the rumen environment, expression of select O157 genes under controlled pH and VFA conditions, dietary effects on bacterial survival, and effects of select flora/metabolite on the growth/survival of O157 in the rumen or rumen fluid [6–11]. Despite this, however, a comprehensive study of the mechanisms used by O157 to survive the rumen environment is yet to be undertaken. Hence, as an initial step, we determined the repertoire of O157 proteins Wnt inhibitor (proteome) as expressed in vitro in harvested, rumen fluid (RF). We included RF of varying www.selleckchem.com/products/tpx-0005.html compositions (with and without normal flora, or depleted of nutrients essential for bacterial growth), with no additives, and used diverse culture conditions, to identify bacterial factors that may enable O157 adaptation to the rumen. Methods Bacterial strain, inoculum preparation and animals Wild-type O157 strain 86–24 (Shiga toxin (Stx) 1-negative, Stx 2-positive; motile; clinical isolate) was used in this study [12]. Overnight culture of O157 in Luria-Bertani (LB) broth, grown at 39°C

with aeration was used to prepare log-phase sub-cultures of the same in 50 ml LB broth, under the same growth conditions. tuclazepam Bacteria harvested from the log-phase cultures at an OD600 0.5-0.6, washed and re-suspended in sterile 0.9% saline, were used to inoculate various rumen fluid (RF) or LB aliquots as described under ‘Culture conditions and processing for proteomics’. All O157 cultures were confirmed serologically using latex agglutination kits (Remel Inc., Lenexa, KS). Two rumen-fistulated SIS3 cost Holstein cows, routinely used as rumen fluid ‘donors’ at the National Animal Disease Center (NADC, Ames, IA)

with approval from the NADC-Animal Care and Use Committee, were used in this study. Both animals, approximately 1 year of age, were fed the NADC Maintenance Diet (corn silage, grass hay, 520 pellets, protein supplements) at 25% fiber and 10% protein, with ad-lib access to water through out. Unfiltered (uRF), Filtered (fRF), and Depleted RF (dRF) Rumen fluid samples collected from the two animals (Samples A and B; Tables 1 and 2), on separate days, were used to prepare the RF-preparations for each experiment set (Experiment I and II). Two liters of RF was collected 2–3 hr post-feeding to allow for rumination to occur, at each sampling time [10, 13]. RF was strained through cheesecloth to remove large feed particles, and poured into collection flasks; pH was recorded on site and an aliquot frozen at –80°C for volatile fatty acid (VFA) analysis. Approximately 500 ml of the strained RF was stored as the unfiltered RF (uRF) at 4°C.