Potential to reduce Escherichia coli shedding in cattle feces by using sainfoin (Onobrychis viciifolia) forage, tested in vitro and in vivo

Abstract

There is a growing concern about the presence of pathogens in cattle manure and its implications on human and environmental health. The phytochemical-rich forage sainfoin (Onobrychis viciifolia) and purified phenolics (trans-cinnamic acid, p-coumaric acid, and ferulic acid) were evaluated for their ability to reduce the viability of pathogenic Escherichia coli strains, including E. coli O157:H7. MICs were determined using purified phenolics and acetone extracts of sainfoin and alfalfa (Medicago sativa), a non-tannin-containing legume. Ground sainfoin or pure phenolics were mixed with fresh cattle feces and inoculated with a ciprofloxacin-resistant strain of E. coli, O157:H7, to assess its viability at -20 degrees C, 5 degrees C, or 37 degrees C over 14 days. Forty steers were fed either a sainfoin (hay or silage) or alfalfa (hay or silage) diet over a 9-week period. In the in vitro study, the MICs for coumaric (1.2 mg/ml) and cinnamic (1.4 mg/ml) acids were 10- to 20-fold lower than the MICs for sainfoin and alfalfa extracts. In the inoculated feces, the -20 degrees C treatment had death rates which were at least twice as high as those of the 5 degrees C treatment, irrespective of the additive used. Sainfoin was less effective than coumaric acid in reducing E. coli O157:H7 Cip(r) in the inoculated feces. During the animal trial, fecal E. coli numbers declined marginally in the presence of sainfoin (silage and hay) and alfalfa silage but not in the presence of hay, indicating the presence of other phenolics in alfalfa. In conclusion, phenolic-containing forages can be used as a means of minimally reducing E. coli shedding in cattle without affecting animal production.

FIG. 1.

Death rates of E. coli O157:H7 Cip^r cells inoculated in cattle feces, with the addition of either 0.5% (wt/wt) ground sainfoin silage or 0.5% (wt/wt) p-coumaric acid and incubated at different temperatures (−20, 37, or 5°C). Death rates (log₁₀ CFU/g dry feces/day) were calculated as the slope from the average linear regression of each additive and temperature treatment (data not shown). Results shown are death rate averages, with respective standard errors of the means. Statistical significance (P < 0.05) between means is denoted by different letters.

FIG. 2.

Effects of outdoor temperature (A) and diet (B) on the viability of fecal E. coli spp. over time. Outdoor temperature data (°C) during the trial (A) are presented to show the apparent relationship between outdoor temperature and fecal E. coli numbers (log₁₀ CFU/g dry feces) (B). Temperature data were from Environment Canada (http://www.climate.weatheroffice.ec.gc.ca/climateData/canada_e.html) and are presented as mean weekly temperatures. Fecal samples were collected from animals fed one of the following four diets: sainfoin silage (•), sainfoin hay (▪), alfalfa silage (○), and alfalfa hay (□). Week 2 is representative of the control period when all animals were fed an alfalfa (hay or silage) diet prior to the start of the experimental diets. Fecal E. coli sp. numbers are a pooled average for each treatment. Statistical significance (P < 0.05) between means is denoted by different letters. Bracketed points have the same letter(s).

FIG. 3.

Average fecal E. coli sp. numbers measured from the feces of cattle fed either a sainfoin (silage or hay) or alfalfa (silage or hay) diet. Average E. coli sp. numbers (log₁₀ CFU/g dry feces) were measured in fecal samples pooled by treatment (diet). Fecal E. coli sp. numbers were subsequently averaged per treatment during the experimental period (weeks 4 to 11). Data presented are averages, with their respective standard errors of the means shown as vertical lines. Statistical significance (P < 0.05) between means is denoted by different letters.