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ANIMAL PRODUCTION |
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* Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Canada T6G 2P5;
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Research Center, Agriculture and Agri-Food Canada, Lethbridge, Canada T1J 4B1; and
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Chr. Hansen Inc., Milwaukee, WI 53214
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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1-AGP) were measured. Supplementation of feed with E. faecium had no effect on concentrations of SAA, LBP, haptoglobin, or 1-AGP in plasma compared with those of controls. However, feeding E. faecium and yeast increased (P = 0.02) plasma concentrations of SAA, LBP, and haptoglobin but had no effect on plasma 1-AGP. In conclusion, oral supplementation of E. faecium alone had no effect on the mediators of the acute phase response that were measured, whereas feeding of E. faecium and yeast induced an inflammatory response in feedlot steers fed high-grain diets. Further research is warranted to determine the mechanism(s) by which E. faecium and yeast stimulated production of acute phase proteins in feedlot steers.
Key Words: acute phase protein • direct-fed microbial • feedlot steer • probiotic • yeast
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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). In addition, E. faecium and other lactic acid-producing bacteria reduce fecal shedding of important enteropathogens like Escherichia coli O157:H7, Salmonella, shigella, and clostridia (Lewenstein, et al., 1979; Zhao et al., 1998; Ohya et al., 2000). Similarly, the yeast Saccharomyces cerevisiae stimulates cellulolytic and lactate-utilizing bacteria and improves weight gain in beef cattle (Yoon and Stern, 1996). Therefore, live cultures of E. faecium and S. cerevisiae might be useful to improve animal health.
Although the influence of DFM and other probiotic bacteria on blood chemistry, ruminal acidosis, ruminal microflora, BW gain, digestion, and feed intake has been studied in feedlot steers (Ghorbani et al., 2002; Beauchemin et al., 2003), little information is available concerning their effect on the immune system. Several studies performed in other animal models show that live DFM are capable of modulating the innate and acquired immunity at the local and systemic level (Isolauri et al., 2001). For example, oral administration of E. faecium stimulated the mucosal and systemic immune responses in young dogs with increased production of immunoglobulin A (Benyacoub et al., 2003). Similarly, a short-term oral administration of S. cerevisiae resulted in enhanced resistance of mice toward infections with Klebsiella pneumoniae, Streptococcus pneumoniae, and Streptococcus pyogenes (Bizzini and Fattal-German, 1990).
Activation of the immune system in conditions like inflammation, tissue injury, and infection is associated with release of acute phase proteins by the liver, known as the acute phase response (Suffredini et al., 1999). The acute phase proteins commonly studied in cattle are serum amyloid A (SAA), lipopolysaccharide binding protein (LBP), haptoglobin, and alpha1-acid glycoprotein (1-AGP; Ametaj et al., 2005; Gozho et al., 2005).
Although the favorable effects of DFM in modulating the different aspects of metabolism and production have been studied in feedlot cattle, little attention has been paid to their immunomodulatory effects. Therefore, the objective of this study was to investigate effects of feeding E. faecium alone or in combination with S. cerevisiae on selected mediators of acute phase response in beef cattle fed high proportions of grain.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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As previously reported by Beauchemin et al. (2003), 8 cannulated steers (Exp. 1 BW = 507 ± 45 kg; Exp. 2 BW = 538 ± 46 kg) were used in 2 experiments. Steers were kept in individual stalls bedded with rubber mats and cared for according to the guidelines of the Canadian Council on Animal Care (1993). The experimental design was a 2 x 2 Latin square with 2 squares, 4 steers within each square, 2 periods, and 2 diets in each experiment. The squares within each experiment were conducted concurrently, and experiments were run consecutively. The length of each period was 21 d, which was divided into a 10-d adaptation and an 11-d measurement.
To minimize carry over effects from period to period, on the last day of periods 1 and 2, the rumen of each steer was emptied manually, and the contents were placed into the rumen of the next steer within the square that was to receive that treatment. Thus, each steer began the period with rumen contents corresponding to the same treatment it was fed.
In Exp. 1, steers were fed a diet that was top-dressed with the control treatment (carrier) or E. faecium EF212; and in Exp. 2, steers were fed a diet that was top-dressed with the control treatment (carrier) or E. faecium EF212 with S. cerevisiae (yeast). The bacteria and yeast were blended with calcium carbonate (carrier) to supply 6 x 109 cfu of bacteria or yeast/g of carrier. The diet of each steer was top-dressed with blend or carrier once daily at the time of feeding (10 g/d). Both E. faecium EF212 and S. cerevisiae were supplied by Chr. Hansen Inc. (Milwaukee, WI). The viability of the preparations was tested by Chr. Hansen Inc. before beginning the experiments. Experimental diets were formulated based on the NRC requirements (1996) to meet or exceed the CP, effective fiber, mineral, and vitamin needs for cattle weighing 450 kg and gaining 1.5 kg/d (Table 1). A feed mixer was used for preparing the diet each day. The diet was fed once a day at 0900. Feed and water were available ad libitum, and orts were approximately 10% of the diet.
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Blood samples were obtained from each steer on d 17 and 21 of each period. The reason for choosing these sampling days was the time needed for the host to respond to the feeding of DFM. At 5 h after feeding, blood samples were collected from the jugular vein into 10-mL vacuum tubes containing Naheparin (Vacutainer, Becton Dickinson, Franklin Lakes, NJ). Samples were centrifuged (5,000 x g, 20 min, 4°C) within 20 min, and plasma was collected, immediately placed on ice, transported to the laboratory, and frozen at –20°C until analysis.
Concentrations of SAA in the plasma were determined by commercially available bovine ELISA kits (Tridelta Development Ltd., Greystones Co., Wicklow, Ireland) according to the manufacturer’s instructions and as described by McDonald et al. (1991). All samples including standards were tested in duplicate. Samples were initially diluted 1:500. Optical density values were read on a microplate spectrophotometer (model Spectra Max 190, Molecular Devices Corporation, Sunnyvale, CA) at 450 nm. The intra- and interassay CV were below 10%. According to the manufacturer, the detection limit of the assay was 0.30 µg/mL.
Concentrations of haptoglobin in plasma were determined by bovine ELISA kits (Tridelta Development Ltd.), as described by Godson et al. (1996), using a pool of bovine serum as the standard. All samples including standards were tested in duplicate. Optical density values were read on the Spectra Max 190 microplate spectrophotometer at 630 nm. The intra- and interassay CV were below 10%, and the detection limit of the assay was at 0.05 µg/mL.
Concentrations of 1-AGP in plasma were measured with bovine radial immunodiffusion (RID) assay kits (Tridelta Development Ltd.). Single RID assays were prepared to measure plasma concentrations of 1-AGP. Calibrators and samples were applied to wells in 5.0-µL volumes. Plates were placed in humidified chambers at 37°C and allowed to incubate for 24 h before reading the test results. For the calibrators, a plot of the diameter squared on the y-axis and the concentration of the antigen on the x-axis, gave a linear function, as described previously by Mancini et al. (1965). On the basis of this linear function, sample concentrations were calculated. The intra- and interassay CV were below 4%, and the detection limit of the assay was at 50 µg/mL.
Concentrations of LBP in the plasma were determined with a commercially available multispecies ELISA kit that crossreacts with bovine LBP (Cell Sciences Inc., Norwood, MA). Plasma samples were initially diluted 1:1,500, and samples with optical density values lower than the range of the standard curve were diluted 1:1,200 and reassayed according to the manufacturer’s instructions. The optical density at 450 nm was measured on the Spectra Max 190 microplate spectrophotometer. The intra- and interassay CV were below 10%, and the detection limits of the assay were 1.6 to 100 ng/mL. The concentration of LBP was calculated by extrapolating from a standard curve of known amounts of human LBP.
Statistical Analyses
Data were analyzed using the MIXED procedure (SAS Inst. Inc., Cary, NC) with the first autoregressive covariance structure. For variables measured over time, the model included treatment, day, and the 2-way interaction as fixed effects. The random effects were square, steer within square, and period. Period within square was not considered in the model because both squares were conducted simultaneously, and thus the effect of period was considered to be the same for both squares. The REML method was used to estimate the variance components, and the Bayesian information criterion was used to determine the best fitting model, whereas the Kenward-Roger method was used to approximate the denominator degree of freedom. Data for sampling time were analyzed as repeated measures. Significance was declared at P < 0.05.
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RESULTS AND DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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). Other researchers also have investigated the productive aspects of supplementation of E. faecium and yeast in dairy cattle (Krehbiel et al., 2003; Nocek et al., 2003). To our knowledge, however, this is the first study to evaluate immunomodulatory effects of DFM in cattle.
Results of Exp. 1 showed no significant overall treatment effects of feeding feedlot steers E. faecium on plasma concentrations of SAA, LBP, haptoglobin, and 1-AGP (Table 2). No significant differences were observed in the concentrations of SAA, LBP, haptoglobin, and 1-AGP in blood collected on d 17 vs. 21 between controls and those supplemented with E. faecium. Although we did not find treatment effects in Exp. 1 for the concentrations of SAA in plasma, values were consistent with the value of 29 µg/mL that was reported recently for healthy steers (Tourlomoussis et al., 2004). The SAA values for our control steers were about 40 µg/mL and about 35 µg/mL in steers fed E. faecium (Table 2). In contrast, results of Exp. 2, in which steers were supplemented with E. faecium and yeast, showed elevated concentrations of SAA in plasma compared with control steers (P = 0.02; Figure 1). No significant day effect or treatment x day interaction was obtained for concentrations of SAA in plasma in Exp. 2 (Figure 1). Serum amyloid A is a protein produced by the liver and is associated with high-density lipoproteins in the plasma. Although the precise physiological role of SAA in the host defense mechanism is not well understood, SAA is involved in binding, neutralization, and rapid removal of endotoxin from circulation (Baumberger et al., 1991). Production and release of SAA from liver hepatocytes is stimulated by cytokines IL-1, IL-6, and TNF- secreted by activated liver macrophages after removal of endotoxin from circulation (Watanabe et al., 2000; Elam et al., 2003). The mechanism by which addition of yeast to E. faecium enhanced production of SAA by the liver is not well understood; however, some of the contributing factors might include cytokines produced locally by gastrointestinal immune cells or the translocation of yeast antigenic compounds such as glucan or mannan into the bloodstream and subsequent activation of liver macrophages. Recent research indicates that glucan and mannan derived from S. cerevisiae induce production of TNF- by monocytes (Tada et al., 2002; Majtan et al., 2005).
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). The same authors reported that within 8 h of administering lipopolysaccharide into the blood of dairy cows, plasma LBP increased more than 3.5-fold, reaching average values of 137 µg/mL and remaining high during the entire 72 h of the experimental period (Bannerman et al., 2003). Feedlot steers in our experiment had LBP values greater than 20 µg/mL 21 d after feeding a diet with a high proportion of grain. Addition of E. faecium in the diet had no effect on LBP concentrations in plasma.
In contrast to results of Exp. 1, when E. faecium and yeast were fed in Exp. 2, a treatment x day interaction for plasma concentrations of LBP (P = 0.02; Figure 2) was detected. An effect also was observed for concentrations of LBP between controls and steers treated with E. faecium and yeast on d 21 of the experiment (P < 0.05). The LBP is a liver-derived acute phase protein that is implicated in modulating host responses to endotoxin from gram-negative bacteria. The protein interacts with circulatory endotoxin to form complexes that bind to CD14, which facilitates binding and activation of TLR4/MD-2 complex on cells of the monocytic lineage and neutrophils, resulting in their activation (Fitzgerald et al., 2004). This triggers release of cytokines, which are responsible for initiating the acute phase response (Moshage, 1997). Lipopolysaccharide binding protein also facilitates transferring of endotoxin to lipoproteins and its rapid removal from circulation by the liver (Kitchens and Thompson, 2003). Increased plasma concentrations of LBP in our steers support the hypothesis that feeding yeast may increase translocation of endotoxin, or yeast-derived antigenic compounds like glucans and mannans, or both. In support of this hypothesis are results showing that enhanced production of TNF- by monocytes stimulated with the S. cerevisiae membrane-product mannan required presence of LBP (Tada et al., 2002).
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reported haptoglobin concentration of 110 µg/mL in plasma of healthy beef cattle; however, cattle under different pathological conditions had average plasma haptoglobin values of approximately 270 µg/mL. Results of Exp. 1 showed haptoglobin concentrations of about 270 µg/mL in control steers and about 225 µg/mL in steers supplemented with E. faecium (Table 2
). Elevated plasma haptoglobin values suggest translocation of bacteria into the bloodstream of feedlot steers fed high proportions of grain. In our study, E. faecium supplementation reduced plasma haptoglobin values. Further, Gozho et al. (2005) reported increased concentrations of endotoxin in the rumen of male Jersey cattle fed high-grain diets and that this was associated with elevated concentrations of haptoglobin in plasma. Endotoxin is a cell-wall component of gram-negative bacteria and, when released in great amounts, has been documented to affect gut mucosal barrier functions and subsequent translocation of bacteria and bacterial products (Deitch, 1990).
Treatment affected plasma concentration of haptoglobin in steers fed E. faecium and yeast (P < 0.01; Figure 3); however, no effect of day or treatment x day interaction was observed (Figure 3). Typically, concentrations of haptoglobin in plasma are low but increase when there is an inflammatory response and translocation of bacteria into the bloodstream (Deignan et al., 2000). By binding to hemoglobin, haptoglobin prevents utilization of iron in the hemoglobin by bacteria translocated into the bloodstream (Wassell, 2000). Thus, the greater plasma haptoglobin concentration in steers fed yeast might be due to increased translocation of bacteria into the bloodstream. The mechanism by which yeast increases translocation of bacteria is not well understood and remains to be elucidated.
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1-AGP in plasma of feedlot steers. In healthy dairy cows, Tamura et al. (1989) obtained plasma values of 1-AGP of approximately 283 µg/mL. In addition, cows suffering from traumatic pericarditis, arthritis, mastitis, pneumonia, and mesenteric liponecrosis had 1-AGP values at or greater than 450 µg/mL (Tamura et al., 1989). When steers were supplemented with E. faecium alone (Exp. 1), results showed values of plasma 1-AGP greater than 600 µg/mL (Table 2). Elevated concentrations of 1-AGP in control and experimental animals in our experiment suggest that feeding high proportions of grain solicits an inflammatory condition in feedlot steers.
Concentrations of the acute phase protein 1-AGP also were elevated (greater than 600 µg/mL) in plasma of all steers in Exp. 2, and again, no differences were found between controls and steers fed E. faecium and yeasts (Figure 4). Elevated concentrations of this acute phase protein are again indicative of an inflammatory response in feedlot steers fed E. faecium and yeast. As previously stated (Beauchemin et al., 2003), in Exp. 1, 6 of the 8 steers in period 1 and 5 of the 8 steers in period 2 experienced subclinical ruminal acidosis. In Exp. 2, 5 steers experienced subclinical ruminal acidosis in period 1 and 4 in period 2 (Beauchemin et al., 2003). Prolonged exposure of the ruminal epithelium to high acid concentrations (i.e., acidosis) can result in inflammation of the rumen wall (i.e., rumenitis) and then to hyperkeratosis and parakeratosis (Fell and Weekes, 1975). Alpha1-acid glycoprotein is produced by the liver to control inappropriate or extended activation of the immune system (Fournier et al., 2000).
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). This effect was negated, however, when yeast was provided. Interestingly, data reported in this paper showed that supplementing diets of the same feedlot steers with E. faecium alone did not elicit an acute phase response; however, supplementing E. faecium and yeast was associated with increased concentrations of acute phase proteins in the plasma. The reason for elevated production of acute phase proteins when E. faecium and yeast were fed to steers is not well understood at present and may be due to lysis of coliform bacteria when yeast is added. Yeast is known to support gram-positive bacteria, which in turn might produce bacteriocins, antibiotic-like substances that can kill certain gram-negative bacteria (Nes and Holo, 2000). Dead gram-negative bacteria release endotoxin, and the latter may transfer into the bloodstream and stimulate production of cytokines and increase gut permeability to gastrointestinal flora (Deitch, 1990). Enhanced production of proinflammatory cytokines like IL-1, TNF-, and IL-6 has been reported following intake of probiotics (Wold, 2001). The proinflammatory cytokines stimulate hepatocytes to secrete acute phase proteins (Gruys et al., 2005).
In conclusion, results reported in this study show for the first time that feeding live probiotic bacteria such as E. faecium to feedlot steers under high-grain diet for a period of 11 d had no effects on acute phase proteins measured (i.e., SAA, LBP, haptoglobin, and 1-AGP). On the other hand, feeding a combination of E. faecium and S. cerevisiae increased concentrations of SAA, LBP, and haptoglobin in the plasma of experimental animals. Our finding that combined feeding of E. faecium and yeast stimulated an inflammatory response in feedlot cattle fed high proportions of grain suggests that further research is needed to understand whether the effect is due to the yeast alone or due to a combination effect with E. faecium. Further, it would be important to understand the mechanism by which the DFM stimulate production of these proteins and whether elevated plasma concentrations of SAA, LBP, and haptoglobin are beneficial or detrimental to the host.
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Footnotes |
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2 Present address: JL Microbiology Inc., Hartland, WI.
3 Corresponding author: burim.ametaj{at}ualberta.ca
Received for publication April 5, 2006. Accepted for publication July 26, 2006.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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release by fungal cell wall polysaccharides. Z. Naturforsch. [C] 60:921–926.This article has been cited by other articles:
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  I. Szabo, L. H. Wieler, K. Tedin, L. Scharek-Tedin, D. Taras, A. Hensel, B. Appel, and K. Nockler Influence of a Probiotic Strain of Enterococcus faecium on Salmonella enterica Serovar Typhimurium DT104 Infection in a Porcine Animal Infection Model Appl. Envir. Microbiol., May 1, 2009; 75(9): 2621 - 2628. [Abstract] [Full Text] [PDF]
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 B. N. Ametaj, K. M. Koenig, S. M. Dunn, W. Z. Yang, Q. Zebeli, and K. A. Beauchemin Backgrounding and finishing diets are associated with inflammatory responses in feedlot steers J Anim Sci, April 1, 2009; 87(4): 1314 - 1320. [Abstract] [Full Text] [PDF]
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