Effect Of Condensed Tannins From Purple Prairie Clover On Fecal Shedding Of Escherichia Coli By Beef Cattle And On Rumen Fermentation And Rumen Bacteria

The native legume Purple prairie clover (PPC, Petalostemon purpureum) is well adapted to the prairie region and is considered an important palatable component of prairie hay. PPC contained high concentration of condensed tannin (CT) that possesses strong antimicrobial activity against Escherichia coli O157:H7. Therefore, PPC could be a valuable diet component for sustainable beef production and food safety. However, there is no information available on the nutritive value of PPC and the effects of PPC tannin on nutrient metabolism of ruminant. The overall objectives of this research included (1) evaluating the in vivo effect of incorporating PPC into mixed forages on the reduction of fecal shedding of E. coli and possible antibiotic mechanism. (2)Determine the effect of condensed tannin on PPC two growth stages rumen nitrogen digestibility and on rumen microbial protein synthetic efficiency by mixed cool season grass. Meanwhile,δ¹⁵N alfalfa mixed PPC were used for assess of condensed tannin capacity in reducing nitrogen from alfalfa to ammonia, and (3) rumen bacteria reaction by condensed tannin and adaptation were evaluated by pure culture technique.For further understanding the effect of CT on E. coli, In vitro study was conducted to assess the inhibition of Condensed tannins isolated from PPC on E. coli or E. coli O157:H7 and possible mechanism. Tannin-mediated alterations in E. coli cell walls were detected by transmission electron microscopy (TEM), and scanning electron microscopy (SEM) revealed large amounts of extra cell material present on E. coli. The permeability of bacteria membrane was reduced when CT were added at levels of 200μg/ml. Grazing studies were conducted at Swift Current, Saskatchewan during two seasons (summer and fall) in two consecutive years (2009 and 2010) to assess the effect of including PPC in cool season pastures on fecal shedding of E.coli in beef cattle. Twenty five steers were allocated into five paddocks distributed in three treatments. One paddock containing pure brome grass (Check; C), two paddocks (Simple) and two paddocks (Complex). Purple prairie clover was mainly in vegetative/early flowering stage during summer and in later flowering/early seeding stage during fall. Fecal samples were collected from rectum of the cattle by hand grabbing. Fecal samples collected in 2010 were mixed for each animal into a single sample for summer and fall grazing periods and analyzed for organic matter (OM), total N, ammonia-N (NH3-N) and volatile fatty acids (VFA). Check and PPC mixed plants collected from all paddocks were harvested during summer and fall periods for CT analysis. Concentration of CT in PPC mixed pasture was higher (P < 0.01) in fall than in summer. Compared to the Check, counts of E. coli in feces of cattle grazing pasture containing PPC was lower (P < 0.05) in fall for both years, and in summer during 2010. There was no difference in fecal E. coli counts between cattle grazing the simple and complex PPC-containing pastures in 2009 and 2010. Fecal pH, total N, NH3-N, VFA and acetate: propionate ratio (A: P) did not change during summer grazing in 2010. . However, cattle grazing C in fall had higher (P < 0.05) pH, N, NH3-N, VFA, and lower (P < 0.05) A:P than cattle grazing simple and complex pastures containing PPC. Determination of microbial population revealed that total 16S rDNA gene copies were higher in C, but not significant. Amount of 16S rDNA gene copies of Fibrobacter succinogenes, Ruminococcus albus, and Ruminobacter amylophilus in fecal samples from animals grazing C or PPC were low but not different. Other main rumen bacteria species (Ruminococcus flavefaciens, Prevotella bryantii, Streptococcus bovis and Selenomonas ruminantium) were not detectable. These results suggest that inhibitory effects of CT on E. coli are related to shifts in the cell membrane. Incorporation of PPC into forage has potential to reduce the prevalence of fecal E. coli.For understanding the chemical changes in PPC at different growth stage. The chemical composition of the whole plant and condensed tannin content from leaf, stem and flower were determined in two stages. Whole plants of PPC were harvested from artificial pastures at Swift Current, SK at vegetative (VEG) and full-flowering/early seeding (FL) stages. Proportions of leaf, stem and flower were determined after freeze drying. Whole plants were analyzed for OM, total N, neutral detergent fibre (NDF) and acid detergent fibre (ADF). Leaf, stem, flower and whole plant were also analyzed for total phenolics, total tannins and condensed tannin (CT). Condensed tannins were detected in all tissues (i.e. leaf, stem and flower) of the plant, with flower containing the highest (198-213 g/kg DM) and stem the lowest (17-18 g/kg DM) contents. Concentrations of total phenolics, total extractable tannins and total CT (extractable and protein- and fibre- bound) in leaf were higher (P<0.01) in VEG than FL stage, but similar in stem and flowers at both growth stages.The following three in vitro were conducted to evaluate the effect of condensed tannin on rumen fermentation and nitrogen digestion by mixed cool season grass andδ¹⁵N lablled alfalfa. The first In vitro experiment was conducted to assess the effects of condensed tannins (CT) on the ruminal degradability of PPC by using three DAISYII fermentor units. Whole PPC plants were harvested at vegetative (VEG) and full-flowering/early seeding (FL) stages from pastures located at three different sites.δ¹⁵N labelled ammonium sulfate was included in the inoculum to assess microbial protein synthesis and feed colonization. Half of the jars in each unit were supplemented with polyethylene glycol (PEG), yielding a 2 x 2 factorial arrangement of treatments in each unit. Plants harvested at VEG stage had higher (P<0.001) TDMD, total nitrogen degradability (TND) and potential degradable fraction (b) of DM and N than those harvested at FL stage. Inclusion of PEG increased (P<0.01) TND and the potentially degradable N fraction of PPC harvested at FL, but not at VEG stage.Effect of PPC tannins on ruminal fermentation and ture dry matter disappearance of mixed forages from grazing trial were determined in vitro (48-h batch culture) by incubating mixtures of PPC and cool season grasses containing 7, 14, 29 and 42 g CT/kg DM with mixed rumen microbes.δ¹⁵N labelled ammonium sulfate was added to quantify microbial protein (MP) synthesis. Polyethylene glycol (PEG) was included in half of the vials for each mixture yielding a 4 x 2 factorial arrangement of treatments. Substrate, PEG and substrate x PEG interaction had no effect on potential gas production (A) or Lag time. However, rate of gas production was increased (P<0.05) by the inclusion of PEG. As the proportion of PPC increased in the forage mixtures, true dry matter disappearance (TDMD) increased (P < 0.01) at both 12 and 48-h incubation, whereas efficiency of MP synthesis (mg/g truly digested DM) decreased (P<0.01) at 12 but not at 48 of the incubation. Inclusion of PEG increased (P<0.01) TDMD at 12-h but reduced (P<0.01) the efficiency of MP synthesis at 12 and 48h incubation. As the concentration of CT increased, efficacy of PEG treatment in increasing ammonia accumulation and decreasing MP synthesis was increased. The results indicated that incorporation of PPC into cool season grasses improved ruminal digestibility. Condensed tannins in PPC/grasses mixture at concentrations up to 42 g/kg DM had no negative effects on the extent of DM digestibility, but increased MP synthesis.Effect of PPC on retard of alfalfa protein transferred into ammonia, methane production and rumen bacteria were determined in vitro (48-h batch culture) by incubating mixtures of PPC andδ¹⁵N labeled alfalfa at different ratios (100:0, 75: 0, 50:50, 25: 75, and 0:100). Half of the vials were supplemented with polyethylene glycol (PEG), yielding a 5 x 2 factorial arrangement of treatments in each batch culture. The experiment was conducted at three time and incubations were carried out at 6, 12, 24 and 48h. Determination of alfalfa protein conversion to ammonia and changes in main ruminal bacteria were detected usingδ¹⁵N diffusion and real-time polymerase chain reaction techniques, respectively. Inclusion of PEG did not affect methane produced per unit of digested DM (DDM). As the tannin concentration increased, addition of PEG increased (P<0.01) ammonia and NH3-15N accumulation, and the amount of 15N from alfalfa converted into ammonia. Bacterial gene copies of cellulolytic (Fibrobacter succinogenes, Ruminococcus flavefaciens, Ruminococcus albus), non-cellulolytic (Streptococcus bovis and Ruminobacter amylophilus) bacteria and Archae were reduced as PPC ratio increased, but these changes were statistically similar. Overall, the alfalfa protein can be protected by CT from PPC to convert into ammonia, but the CT also has a negative effect on ruminal bacteria.Our previous study showed PPC contained high concentration of tannins. However, the ecological role of PPC in the mixed forage pasture and the effects of PPC tannins on the nutritive value of mixed forages have not been assessed. Thus, two of in vitro experiments were conducted to assess the effect of CT from PPC on ruminal fermentation, degradability and bacteria population.A study using pure cultures of main ruminal bacteria was conducted to assess if ruminal bacteria were able to overcome the negative effects of isolated CT from PPC. Three cellulolytic bacteria (Fibrobacter succinogenes, Ruminococcus flavefaciens, Ruminococcus albus) and four non-cellulolytic bacteria (Prevotella bryantii, Ruminobacter amylophilus, Streptococcus bovis, and Selenomonas ruminantium) were used. Ruminal bacteria was initially cultured in a media containing 0 or 25μg CT/ml (cellulolytic) during 21-d and 0 or 50μg CT/ml (non-cellulolytic) during 15-d doing transferences every three days. At these levels of CT, cellulolytic and non-cellulolytic bacteria were able to overcome the potential negative effects of tannins. To observe the persistence of this effect, cellulolytic bacteria were then cultured for 4-d on Whatman N°1 filter paper in medium containing 0, 75, 150, 300 or 450μg CT/ml, whereas non-cellulolutic bacteria were cultured for 24-h in ruminal fluid medium containing 0, 100, 200, 400 or 600μg CT/ml. The growth curve showed that among non-cellulolytic bacteria, P. bryantii, R. amylophilus, and S. bovis were more sensitive to CT than S. ruminantium, which grew well up to 600μg CT/ml. The fiber digestion of F. succinogenes, R. flavefaciens, and R. albus was markedly reduced by CT at 75μg CT/ml and little bacteria grow was observed at CT concentrations up to 150μg/ml (P <0.01). Scanning electron micrographs indicated that the attachment of cellulolytic bacteria was depressed by CT and no adaptation was found in this group.Overall, Purple prairie clover could be characterized as good quality forage with higher N content and uique bioactive compounds and also has the potential to alter membrane of E coli and reduce fecal shedding of E. coli in cattle. Condensed tannins in PPC/grasses mixture at concentrations up to 42 g/kg DM had no negative effects on the extent of DM digestibility, but increased MP synthesis. The CT can protect alfalfa nitrogen from ruminal degradation into ammonia but, the attachment of cellulolytic bacteria can be affected by CT even at very low concentration. The cellulotic bacteria were very sensitive to CT from PPC and their attachment can be affected.