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* Department of Animal Sciences and Industry and
and
Department of Statistics, Kansas State University, Manhattan 66506-1600 and
and
Minnesota Corn Processors, Marshall 56258
2 Correspondence:
Call Hall, Room 133 (phone: 785-532-1204; fax: 785-532-5681; E-mail:
jdrouill{at}oznet.ksu.edu).
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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Key Words: Cattle • Escherichia coli • Finishing • Flaking • Maize • Maize Gluten
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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Previously, researchers have indicated that the replacement of grain with hay in cattle diets 5 d prior to slaughter can impact fermentation patterns by lowering VFA concentrations, thus reducing acid-resistant E. coli populations in the rumen and feces of cattle (Diez-Gonzalez et al., 1998). Abruptly changing cattle diets to lower ruminal and colonic acid-resistant E. coli populations preceding slaughter will likely compromise cattle performance by: 1) reducing the energy density of the diet and decreasing the likelihood that cattle will reach choice quality grade; 2) increasing gut fill, thereby reducing dressing percentage and complicating strategies to avoid exposing gut contents during evisceration; and 3) exacerbating the potential for dark cutting beef due to abrupt changes in the diet prior to slaughter. We hypothesized that the fibrous, low-starch characteristics of WCGF may complement steam-flaked corn (SFC) finishing diets and could be utilized to manipulate ruminal and fecal organic acid concentrations and potentially prevent development of acid resistance in E. coli and coliform bacteria residing in the gastrointestinal tracts of cattle.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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). Diets were formulated to be isonitrogenous and provided 33 mg/kg of monensin and 11 mg/kg of tylosin (Elanco Animal Health, Indianapolis, IN). Steers were offered ad libitum access to their respective diets fed once daily at 0800 for 152 d.
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Dietary NEg values were calculated according to procedures outlined by Löest et al. (2001) using NRC (1984) equations for implanted, medium-framed steers. Steer performance and carcass characteristics were analyzed as a randomized complete-block design using the GLM procedure of SAS (SAS Inst. Inc., Cary, NC). A pen of steers comprised the experimental unit. Block (growing treatment) and finishing treatments were included in the model statement and linear and quadratic effects of dietary WCGF were tested using orthogonal contrasts.
Microbiological Analysis
Ruminal fluid and feces were collected from 180 steers (three animals per pen daily) from d 114 through 118. Sampling commenced at 0600 on each sampling day and approximately 15 min per animal was required to obtain samples. Fecal samples were obtained from the rectum and ruminal fluid was collected via rumenocentesis, as described by Nordlund and Garrett (1994). Approximately 5 to 10 mL of fluid was aspirated from the ventral sac of the rumen using a 10-mL disposable syringe equipped with a 12.7-cm, 16-gauge, sterile, stainless steel needle. Following collection of the ruminal fluid and feces, portions of each sample were subdivided immediately on site to provide a portion for pH measurement. Fecal samples (10 g) were mixed with 20 mL of deionized water and analyzed with a portable pH electrode. The pH of the corresponding ruminal sample was measured concurrently. Aliquots (4 mL) of these liquefied fecal and ruminal samples were pipetted into vials containing 1 mL of 25% (wt/vol) meta-phosphoric acid and frozen for future determination of VFA concentrations. The remainder of each original fecal and ruminal sample was placed on ice and immediately transported to the Kansas State University food safety laboratory.
In the laboratory, sterile deionized water (20 mL) was added to feces (10 g) to liquefy the samples. Ruminal and fecal samples were homogenized for 2 min in a Stomacher Lab Blender 400 (Tekmar, Cincinnati, OH). Following homogenization, samples were tested for acid resistance in coliform bacteria by incubating 1-mL aliquots at 32°C for 1 h in filter-sterilized, citric acid/sodium phosphate, McIlvaine buffers (9 mL) adjusted to pH 2, 4, or 7 with 1 M sodium phosphate or 85% phosphoric acid. These three levels were chosen to provide a neutral (pH 7), an intermediate (pH 4), and a strong (pH 2) acid shock. The intermediate level was chosen to emulate the acidity of many common organic carcass washes, whereas the strong level was chosen because the human gastric stomach is frequently pH 2. After 1 h, 1 M NaOH was added to neutralize samples exposed to pH 2 and pH 4. Serial dilutions (three for pH 2 and six for pH 4 and 7) were prepared in 0.1% sterile peptone diluent. Appropriate dilutions were plated in duplicate on E. coli Count Petrifilm (3M, St. Paul, MN). All plates were incubated at 35°C for 24 ± 2 h and typical colonies were enumerated as E. coli and total coliforms. Microbial populations in ruminal fluid samples were reported as colony-forming units (CFU)/mL. Fecal samples were dried at 105°C in a forced-air oven and the population counts recorded from the Petrifilm were expressed as CFU/g of dry feces. Concentrations of VFA were measured using gas chromatography (Hewlett-Packard 5890A gas chromatograph, 183 x 0.635 cm column; Supelco SP 1200 packing, N2 carrier at 80 mL/min; flame-ionization detector at 225°C, Palo Alto, CA). Ruminal samples were analyzed for total lactate as described by Barker and Summerson (1941).
Ruminal and fecal VFA, pH, and ruminal lactate were analyzed as a randomized complete-block design using the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC), with a pen of steers comprising the experimental unit. The model statement included effects of block (previous treatment) and finishing treatment, and the random statement included effects of sampling day, sampling day x finishing treatment, and block x finishing treatment. Linear and quadratic effects of dietary WCGF were tested using orthogonal contrasts. Ruminal and fecal E. coli and total coliforms were analyzed as a split-plot design using the MIXED procedure of SAS. The model statement included effects of block, finishing treatment, buffer treatment (pH), and finishing treatment x pH; the random statement included effects of sampling day, sampling day x finishing treatment, and sampling day x block x finishing treatment. Linear and quadratic effects of dietary WCGF, pH, and their interactions were tested using orthogonal contrasts.
For statistical analysis, a value of one-half of the detection limit was entered for samples in which a target population was not detected. Because ruminal and fecal samples were diluted differently and the respective buffer treatments required different amounts of acid or base, the detection limit varied with sample type and buffer treatment.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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). Ham et al. (1995) and Hussein and Berger (1995) observed quadratic increases in DMI as WCGF was included in finishing diets. They found that feeding 25 and 40% WCGF produced maximal intakes. Firkins et al. (1985) also noticed an increase in DMI when WCGF replaced corn in finishing diets. However, Krehbiel et al. (1995) reported that cattle fed 86.5 and 94.5% WCGF had lower intakes than cattle fed 0 or 35% WCGF in ground corn-based diets.
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Dietary NEg values (based on steer performance) changed as WCGF was added to the diet (linear, P = 0.02; quadratic P = 0.07). Ham et al. (1995) found that in finishing diets, WCGF contained 90 to 100% the NEg of corn. However, Richards et al. (1998) reported that finishing diets containing up to 50% WCGF supplied more NEg than finishing diets based on dry-rolled corn. By combining a source of highly digestible fiber with SFC, we likely diluted the dietary starch supply in the rumen and reduced the acidic insult that often accompanies rapid ruminal degradation of starch. This positive association seems to be most evident when moderate levels of WCGF are used to replace SFC. Feeding higher levels of WCGF resulted in an appreciable reduction in dietary energy, which would override the benefits provided by the added fiber.
Carcass traits for steers fed WCGF were similar to steers fed SFC. Although not significant, slight reductions in carcass weight, dressing percentage, longissimus muscle area, marbling score, and the percentage of carcasses grading USDA Choice were observed when steers were fed 60% WCGF. Krehbiel et al. (1995) observed reductions in final weight, hot carcass weight, fat thickness, yield grade, and quality grade with high levels of WCGF. In our study, the occurrences of dark cutters and liver abscesses were low, but increased linearly (dark cutters, P = 0.08; liver abscesses, P = 0.03) as the level of WCGF increased. Firkins et al. (1985) reported an increase in the percentage of livers condemned when cattle were fed WCGF compared to corn. They fed WCGF in corn-based diets with or without corn silage as the only source of roughage. Cattle fed WCGF and corn silage had a lower percentage of condemned livers than cattle fed WCGF and no roughage. They concluded that using WCGF as the only source of roughage might have contributed to the higher percentage of liver abscesses. This is in disagreement with Hussein and Berger et al. (1995), who found a linear reduction in the percentage of liver abscesses with increasing amounts of WCGF.
Microbiological Analysis
Increasing dietary WCGF decreased (linear, P < 0.05) total ruminal VFA, propionate, and valerate, and increased (linear, P < 0.05) isovalerate and acetate:propionate (Table 3) ratio. Consequently, ruminal pH increased (P < 0.05) linearly with increasing levels of WCGF (Figure 1). Ruminal lactate was similar among diets.
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Diez-Gonzalez et al. (1998) decreased ruminal VFA and increased pH by switching cattle from grain- to hay-based diets. Cattle fed 90% grain had approximately 89 mM total VFA, whereas switching to 100% hay resulted in approximately 69 mM total VFA, and ruminal pH increased from 6.3 to 6.8. Tkalcic et al. (2000) also reported a similar reduction in total ruminal VFA from 102.6 mM to 92.3 mM when calves were fed high-concentrate and high-roughage diets, respectively.
In our study, exposure of ruminal samples to pH 2 and 4 during the incubation period reduced (P < 0.001) the populations of E. coli and coliforms (Table 4). Exposing ruminal samples to pH 2 for 60 min decreased E. coli and total coliforms by 2.9 and 3.1 log10 CFU/mL, respectively, when compared to exposure at pH 7. Similarly, reductions of 1.1 and 0.8 log10 CFU/mL for ruminal E. coli and total coliforms, respectively, were observed by comparing the difference between populations at pH 7 and pH 4.
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Recovery of ruminal E. coli and total coliforms was similar across dietary treatments at pH 2 and pH 7. However, increasing dietary WCGF resulted in a quadratic response (P < 0.05) at pH 4. Steers fed 30% WCGF had higher (0.7 log10 CFU/mL) populations of ruminal E. coli and total coliforms than steers fed 0 or 60% WCGF. Because of the absence of other quadratic effects in our study, it is difficult to speculate as to why this occurred. Perhaps the optimal ruminal pH or VFA concentration for coliform bacteria was maintained by steers receiving 30% WCGF, and this may have conditioned these bacteria to withstand a mild pH 4 shock. Others have reported that bacteria grown in the presence of weak acids develop tolerance and can withstand subsequent exposure to extreme acid conditions (Lin et al., 1996; Garren et al., 1997).
Ruminal VFA are considered to be toxic to coliform bacteria (Brownlie and Grau, 1967; Wolin, 1969; Wallace et al., 1989), but some strains of E. coli can develop tolerance if they are grown in the presence of undissociated VFA, including acetate, propionate, and butyrate (Diez-Gonzalez and Russell, 1999). Cattle fed 30% WCGF had slightly higher ruminal populations of E. coli and total coliforms at pH 7, and at pH 4 this difference became larger. Perhaps the proportion of undissociated:dissociated VFA supplied by 30% WCGF enabled these bacteria to withstand a mild acid shock. The reason why this response is not demonstrated at pH 2 is unknown, but pH 2 was highly lethal (average = 1.6 log10 CFU/mL) to all coliform bacteria. Steers fed 0% WCGF exhibited the highest concentration of ruminal VFA and had the lowest ruminal pH. The undissociated:dissociated ratio of VFA was higher in these cattle and should have been conducive to bacterial acid adaptation and presumably development of acid resistance by E. coli. Despite the numerically higher E. coli populations observed at pH 2 for steers fed no WCGF, this trend was not reproduced at pH 4.
Increasing dietary WCGF linearly reduced total fecal VFA (P = 0.06), propionate (P < 0.01), butyrate (P < 0.01), and isovalerate (P < 0.05), and linearly increased (P < 0.05) isobutyrate and acetate:propionate ratio (Table 2
). Consequently, fecal pH increased (linear, P < 0.01) with each addition of WCGF to the diet (Figure 1). Similarly, Diez-Gonzalez et al. (1998) reported a decrease in fecal VFA and a subsequent increase in colonic pH when hay replaced grain. In their study, cattle fed grain had colonic VFA concentrations of approximately 85 mM, whereas cattle fed hay had approximately 25 mM VFA. They reported that colonic pH increased from 5.4 to 7.4 by switching cattle from grain to hay. Scott et al. (2000) reported colonic pH increased from 6.52 to 7.94 when cattle were switched from grain diets to hay diets.
The rate of WCGF disappearance in the rumen is rapid, largely due to the soluble steep liquor fraction (Firkins et al., 1985; Krehbiel et al., 1995; McCoy et al., 1997). Because diets containing WCGF have less starch and are rapidly degraded, the majority of fermentation of diets containing WCGF may have occurred in the rumen, and this would have decreased the substrate available to fermentation in the large intestine. Stock (2000) reported that the fiber fraction of WCGF may be digested more postruminally, resulting in less total acid production in the rumen. However, it appears that ruminal escape of starch from grain in diets containing more SFC and less WCGF resulted in higher large intestinal VFA concentrations, regardless of large intestinal fiber digestion that may take place when dietary WCGF is increased.
Acid-resistant fecal E. coli and total coliforms were not affected by dietary treatments (Table 4). The acid challenge effectively reduced (P < 0.001) fecal E. coli and total coliforms. Fecal E. coli and total coliforms were reduced from pH 7 to pH 2 by 5.8 log10 CFU/g, whereas exposure to pH 4 only reduced the population by 0.5 log10 CFU/g. Similar to the populations in ruminal samples, fecal coliforms were numerically higher for steers fed 30% WCGF, and this was most evident again at pH 4. Fecal E. coli populations at pH 7 averaged 7.3 log10 CFU/g of dry feces across all dietary treatments. Diez-Gonzalez et al. (1998) reported populations of E. coli isolated from the feces of cattle fed grain diets to be 6.9 log10 CFU/g, whereas cattle fed hay diets had 4.3 log10 CFU/g. Jordan and McEwen (1998) reported fecal E. coli to be 6.4 and 6.7 log10 CFU/g for cattle fed high-roughage and high-energy feedlot diets, respectively. Scott et al. (2000) found total E. coli numbers isolated from the feces of feedlot cattle to be 8.25 to 8.46 log10 CFU/g.
The results of our study disagree with those of Diez-Gonzalez et al. (1998) and Scott et al. (2000), who found that increasing colonic pH by feeding hay instead of grain resulted in fewer acid-resistant E. coli and total coliforms. However, it should be noted that by feeding hay instead of grain, Diez-Gonzalez et al. (1998) lowered the colonic VFA concentration by 60 mM, whereas the fecal VFA concentration was lowered by 10 mM in this study. Similar to our findings, Scott et al. (2000) reported that diets containing WCGF increased colonic pH but did not reduce acid-resistant E. coli shedding. They found that diets containing WCGF significantly increased acid-resistant E. coli shedding in cattle compared with diets containing dry-rolled corn or high-moisture corn.
Conceivably, by consuming a 90% cracked corn diet, the cattle in the study by Diez-Gonzalez et al. (1998) passed a large portion of undigested grain starch to the large intestine and fermentation of this starch created an acidic environment. Unprocessed or less extensively processed grains may contribute to larger populations of acid-resistant E. coli as a result of increased fermentative activity in the large intestine. Highly processed grains, such as SFC and high-moisture corn, are digested to a greater extent in the rumen than cracked or rolled corn (Huntington, 1997; Owens et al., 1997). Because we used SFC-based diets, perhaps a potential acidic environment was avoided in the large intestine. However, Scott et al. (2000) observed no differences in acid-resistant E. coli shedding from cattle fed dry-rolled corn or high-moisture corn diets.
Tkalcic et al. (2000) observed a positive correlation between ruminal VFA concentrations and fecal shedding of acid-resistant E. coli O157:H7. Although we did not measure E. coli O157:H7 specifically in our study, and, therefore, cannot determine if we influenced shedding of this organism, our procedure challenged E. coli and total coliforms recovered from the animal, which is a practical approach to determine whether E. coli isolates will be able to withstand acidic extremes and potentially cause illness in humans. The uniqueness of E. coli to develop acid resistance and its relationship to commensal and pathogenic strains has been debated (Miller and Kaspar, 1994; Garren et al., 1998; Duffy et al., 2000). Some research has tried to demonstrate that EHEC is more acid resistant than generic E. coli (Miller and Kaspar, 1994; Garren et al., 1998). However, Duffy et al. (2000) used strains of generic E. coli and EHEC in an acid resistance challenge study and determined that generic E. coli could be used as a model for studies designed to test compliance or standards for controlling EHEC. Additionally, McClure and Hall (2000) stated "there are no definitive studies to support the commonly held belief that E. coli O157:H7 is any more acid tolerant than other E. coli present as part of the normal enteric flora." We feel that examining the growth of acid-resistant generic E. coli and total coliforms provides inclusive information that encompasses several pathogenic strains of bacteria and is an appropriate model to determine reduction strategies for controlling acid-resistant, pathogenic, enteric bacteria. Studies that inoculate cattle with acid-resistant strains of E. coli assume that acid-resistance is maintained after recovery from the animal. Testing for acid-resistance postrecovery will enable researchers to understand if these organisms have the potential to survive the human gastric barrier and result in human pathogenesis.
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Implications |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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Footnotes |
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Received for publication January 24, 2002. Accepted for publication July 10, 2002.
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Literature Cited |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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