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* Department of Diagnostic Medicine and Pathobiology,
and
Department of Animal Sciences and Industry, Kansas State University, Manhattan 66506
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: Escherichia coli O157 • fecal shedding • finishing cattle • grain processing • preharvest intervention
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Many preharvest intervention strategies aimed at reducing prevalence of E. coli O157 have been tested with varying levels of success (Callaway et al., 2003). Fecal shedding of E. coli O157 in cattle is reflective of the ability of the organism to persist in or colonize the gastrointestinal tract. Evidence suggests that the site of persistence or colonization is in the hindgut and not the rumen (Rasmussen et al., 1993; Brown et al., 1997; Grauke et al., 2002; Van Baale et al., 2004). Although the reasons are not known, it is likely that the ecosystem of the hindgut is relatively more hospitable than the rumen.
Therefore, we hypothesize that dietary factors that promote the supply of substrates (starch, fiber, protein, or lipids) to the hindgut will have a significant effect on the ability of E. coli O157 to survive and colonize and thereby influence shedding in feces. Our objective is to use processed grains to effect changes in hindgut fermentation that will have detrimental effects on the survival, growth, and colonization of E. coli O157. Grains that are less extensively digested within the rumen present more starch to the hindgut, thereby increasing fermentative activity and acid production in the hindgut. Steam-flaking of grains has been shown to enhance ruminal starch digestion compared with dry-rolling, effectively reducing the amount of starch reaching the hindgut (Huntington, 1997).
The objective of this study was to evaluate the effects of grain type (sorghum or wheat) and grain processing (dry-rolled or steam-flaked) in finishing diets on prevalence of E. coli O157 in cattle.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Heifers positive for fecal shedding of E. coli O157 were retested within a week, and 40 heifers (initial BW = 287 ± 5 kg) were selected for use in the study. Heifers were then grouped based on prescreen E. coli O157-positive samples and within group were randomly assigned to 1 of 4 treatments. Treatments consisted of a 2 x 2 factorial arrangement with factor 1 being grain type (sorghum- or wheat-based diets) and factor 2 being the method of grain processing (steam-flaking or dry-rolling). Within treatment, animals were randomly assigned to 1 of 2 barns and 1 of 20 individual pens within each barn.
A series of transition diets were used to adapt animals to high-concentrate finishing diets consisting of 81.4% (DM basis) dry-rolled or steam-flaked sorghum, or 52.0% (DM basis) dry-rolled or steam-flaked wheat (Table 1). Steam-flaked corn was added to wheat diets to achieve a similar forage:concentrate ratio among all diets. Each transition diet was fed for 4 d to achieve the final diet on d 16 of the study.
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). Bunks were evaluated every morning at approximately 0700 h, and feed was delivered once daily at 0900 h. Heifers were fed amounts sufficient to result in only traces of feed remaining on the following day, and any feed remaining from the previous day was weighed and DM was measured to determine feed intake of each animal.
Fecal and RAMS Samples Collection and E. coli O157 Culture
Fecal and RAMS samples were collected from each heifer 3 times a week for 30 d (d 0, 2, 5, 7, 9, 12, 14, 16, 19, 21, 23, 26, 28, and 30). Fecal samples were deposited into Whirl-pack bags (Nasco, Ft. Atkinson, WI) and placed on ice. Rectoanal mucosal swab samples were placed directly into test tubes containing 3 mL of gram-negative broth (Becton Dickinson Co., Franklin Lakes, NJ) supplemented with cefixime, cefsulodin, and vancomycin (GNccv; Greenquist et al., 2005).
Whirl-pack bags and RAMS test tubes were then transported on ice to the laboratory. Approximately 1 g of feces from each heifer was placed into a test tube containing 9 mL of GNccv. Fecal and RAMS tubes were vortexed for 1 min and incubated at 37°C for 6 h. Following enrichment, tubes were vortexed for 1 min and 1 mL from each tube was subjected to immunomagnetic separation (Dynal Inc., New Hyde Park, NY). After the immunomagnetic separation procedure, samples were plated onto sorbitol MacConkey agar (Becton Dickinson Co.) containing cefixime (50 ng/mL) and potassium tellurite (2.5 µg/mL; CT-SMAC). Plates were incubated overnight (16 to 18 h) and up to 6 sorbitol-negative colonies were grown on blood agar (Remel, Lenexa, KS) for 12 to 18 h at 37°C. Cultures from blood agar were tested for indole production and latex agglutination for the O157 antigen (Oxoid Limited, Basingstoke, Hampshire, UK). The species was confirmed by API (Rapid 20E, Biomerieux Inc., Hazelwood, MO) on prescreening samples and samples collected on d 0, 2, and 5.
Fecal pH
Fecal pH was measured in samples collected on d 9, 16, 23, and 30 of the study. Approximately 5 g of feces from each animal were suspended in 25 mL of distilled water inside a 50-mL polypropylene tube (Becton Dickinson and Co.). The tube was vortexed to make a fecal suspension before pH was measured with an Accumet model AR 10 pH meter (Fisher Scientific International, Pittsburgh, PA).
Statistical Analyses
Mean DM values for each dietary ingredient were calculated and used to determine daily DM intake for each animal. Intake expressed as average daily DM intake from d 9 to the end of the study was analyzed using the MIXED procedure (SAS Inst. Inc., Cary, NC) utilizing grain type, grain processing, and the interaction as fixed effects and barn as a random effect.
The prevalence of E. coli O157, expressed as a binomial response (positive or negative) on each sampling day, was analyzed with animal as the experimental unit. Cattle were considered positive if E. coli O157 was detected by fecal or RAMS culture. Samples collected on d 0, 2, 5, and 7 were not included in the analysis because cattle were being fed transition diets consisting of less than 60% sorghum or less than 40% wheat. Data were analyzed using the GLIMMIX procedure of SAS in a split-split-plot design, with the whole-plot factor being prescreen, positive sample group in a randomized complete block design with barn as block. Grain-type, grain processing method and the 2-way interaction made up the subplot, and the sub-sub-plot was repeated measures over sampling days utilizing first order autoregression.
Fecal pH data were analyzed with the MIXED procedure of SAS in a split-plot design with the whole plot factors being grain type, grain processing method, and the 2-way interaction, with repeated measures as the subplot. Barn was included as a random effect in this analysis.
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RESULTS |
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Dry-Matter Intake
Mean daily DM intakes from d 9 to the end of the study for heifers fed steam-flaked sorghum, dry-rolled sorghum, steam-flaked wheat, and dry-rolled wheat diets were 6.12 ± 0.38, 6.79 ± 0.36, 6.14 ± 0.38, and 7.21 ± 0.36 kg, respectively. Grain processing affected (P = 0.026) DMI, but grain type and grain type x grain processing interaction did not (P > 0.10). Dry-matter intake of heifers fed diets with dry-rolled grains (7.00 ± 0.26 kg) was greater (P = 0.026) compared with heifers fed diets containing steam-flaked grains (6.13 ± 0.27 kg).
Prevalence of E. coli O157
Mean prevalence of E. coli O157 in all heifers across all sampling days was 50.0%. Analysis of prevalence data began on d 9 when animals were on the third transition diet. Mean prevalences of E. coli O157 from d 9 in heifers fed the steam-flaked sorghum, dry-rolled sorghum, steam-flaked wheat, and dry-rolled wheat diets were 73, 30, 58, and 29%, respectively. Grain-type did not affect (P = 0.29) prevalence of E. coli O157, but the grain type x sampling day interaction was significant (P = 0.04). It should be noted that the grains (sorghum and wheat) were included in diets at different levels to avoid potential ruminal digestive disorders associated with wheat-based diets. On d 12 and 19 of the study, prevalence of E. coli O157 was greater (P < 0.05) in heifers fed sorghum-based compared with heifers fed wheat-based diets, but the difference was not significant on other sampling days (Figure 1).
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Van Baale et al., 2004; Bach et al., 2005), experimental trials utilizing large numbers of animals to compare prevalence of natural infection (Brashears et al., 2003; Berg et al., 2004), or observational studies (Garber et al., 1995; Dargatz et al., 1997; Herriott et al., 1998). Potential limitations to these types of studies include the need to euthanize experimentally inoculated animals, the cost incurred with sampling large numbers of animals, and external factors that are difficult to evaluate effectively in observational studies. For this study, we prescreened a population of cattle and selected a subpopulation that was positive for fecal E. coli O157 to impose treatments. This model proved useful in that prevalence was great enough to detect statistical differences among treatment groups with only 10 animals per treatment. We included RAMS method because RAMS has been shown to be more sensitive than fecal testing in detecting animals that are positive for E. coli O157 (Greenquist et al., 2005).
Fecal shedding of E. coli O157 is reflective of the ability of the organism to persist or colonize the gastrointestinal tract. Published data suggest that the hindgut may be the major site of E. coli O157 persistence. Naylor et al. (2003) suggested that the primary site of E. coli O157 colonization was the rectum, specifically the region approximately 2 to 5 cm proximal to the rectoanal junction. In studies with experimentally inoculated cattle (Grauke et al., 2002, 2003; Van Baale et al., 2004) or prevalence data in gut contents collected from necropsied or slaughtered cattle (Laven et al., 2003; Van Baale et al., 2004), E. coli O157 was detected more frequently in the cecum and colon than in the rumen. Possibly, the conditions in the cecum and colon (greater pH, lower VFA concentrations, absence of ciliated protozoa, slower rate of passage of digesta, etc., compared with the rumen) are more hospitable to the survival and growth of E. coli O157.
Sorghum and wheat grains were chosen for this study because their ruminal digestibilities are substantially different, thus differing in the amount of starch reaching the hindgut (Huntington, 1997). Wheat diets (steam-flaked or dry-rolled) in our study contained only 52.0% wheat, and steam-flaked corn was added to achieve similar forage:concentrate ratio among all diets. Because wheat has one of the fastest rates of ruminal starch digestion (Stock and Britton, 1993) with increased propensity to induce metabolic disorders, such as subacute acidosis, feeding high levels of wheat in cattle diets may increase the likelihood of digestive disorders (Axe et al., 1987; Stock and Britton, 1993).
In the current study, DMI was affected by grain processing method. Intakes were lower in cattle fed diets with steam-flaked grains. This observation is consistent with those presented in a review by Owens et al. (1997) in which steam-rolling sorghum, wheat, or corn decreased (P < 0.05) daily DMI of feedlot cattle compared with dry-rolling the grains. Dry matter intakes of wheat-based diets have been reported to be lower than DMI of sorghum-based diets (Owens et al., 1997). In our study, we did not observe differences in DMI among heifers fed sorghum or wheat diets. Differences in DMI of diets containing different grain types or grain processing methods are likely a combination of differences in metabolizable energy and ruminal degradation of starch yielding differences in ruminal acid concentrations (Owens et al., 1997).
Previous studies have shown that cattle diets containing grains with lower ruminal starch degradation are associated with lower prevalence of E. coli O157 (Buchko et al., 2000; Berg et al., 2004). This may be due to a greater amount of starch being fermented in the hindgut, ultimately yielding greater organic acid concentrations and reduced pH in the region of the gastrointestinal tract that has been identified as the primary colonization site of E. coli O157 (Grauke et al., 2002; Van Baale et al., 2004). Although starch fermentation was not measured in this study, we believe that increasing hindgut fermentation of starch and consequent acid production will create inhospitable conditions for E. coli O157. Short-chain fatty acids (acetic, propionic, butyric) have been shown to suppress and inhibit growth of E. coli O157 at pH values of 6.0 and 5.5, respectively (Shin et al., 2002). Van Kessel et al. (2002) evaluated differences in pH and microbial populations in cannulated steers with starch hydrolysate infused into the rumen or abomasum. They reported that infusing starch into the abomasum decreased pH and increased total anaerobic bacteria, total aerobic bacteria, and generic E. coli in the hindgut compared with infusing starch into the rumen. Increased availability of substrate in the hindgut increases the accumulation of organic acids (VFA) and reduces pH (Van Kessel et al., 2002). In our study, differences in fecal pH were not consistently detected among dietary treatments. It is possible that fecal pH may not truly reflect the pH of cecum or colon. In addition to grain processing, greater intake of grains allows more starch to reach the hindgut (Owens et al., 1997). The greater intake of DR grains compared with SF grains observed in our study would further enhance the effects of grain processing on E. coli O157 in the hindgut.
It is documented that dry-rolled grains have decreased ruminal degradation of starch compared with steam-flaked grains, thus presenting more starch to the hindgut and possibly increasing fecal starch content (Zinn et al., 1995; Huntington, 1997). Previous studies have shown that fecal pH of cattle fed steam-flaked grains was greater (P < 0.05) than that of cattle fed dry-rolled grains (Oliveira et al., 1995; Barajas and Zinn, 1998). In these studies, cattle fed steam-flaked grains also had reduced fecal starch compared with cattle fed dry-rolled grains. Berg et al. (2004) observed decreased (P < 0.01) fecal pH and lower (P < 0.05) prevalence of E. coli O157 and in cattle fed corn compared with cattle fed barley. Because barley is more digestible than corn in the rumen, corn diets would present more starch to the hindgut and increase organic acid production, thus reducing pH and potentially reducing survivability of E. coli O157.
In conclusion, prescreening and selecting cattle positive for shedding of Escherichia coli O157 is a useful model to evaluate potential preharvest intervention strategies. Because grain processing influences site and extent of starch digestion in ruminants, it offers a simple and effective method of targeting specific regions of the gastrointestinal tract to affect changes in fermentation (acid production, pH, microflora, etc.). Utilizing grains processed by methods that are known to increase the amount of starch reaching the hindgut and enhance fermentation may be useful in reducing E. coli O157 in cattle if fed prior to slaughter.
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Footnotes |
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2 Corresponding author: tnagaraj{at}vet.k-state.edu
Received for publication February 13, 2006. Accepted for publication January 2, 2007.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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