J ANIM SCI-2007-Emmanuel-233-9

D. G. V. Emmanuel, A. Jafari, K. A. Beauchemin, J. A. Z. Leedle and B. N. Ametajinflammatory response in feedlot steersinduces anSaccharomyces cerevisiae and Enterococcus faecium Feeding live cultures of doi: 10.2527/jas.2006-2162007, 85:233-239.J ANIM SCI /content/85/1/233the World Wide Web at:The online version of this article, along with updated information and services, is located onFeeding live cultures of Enterococcus faecium and Saccharomyces cerevisiaeinduces an inflammatory response in feedlot steersD.G.V.Emmanuel,*A.Jafari,*1K.A.Beauchemin,†J.A.Z.Leedle,‡2and B.N.Ametaj*3*Department of Agricultural,Food and Nutritional Science,University of Alberta,Edmonton,Canada T6G 2P5;†Research Center,Agriculture and Agri-Food Canada,Lethbridge,Canada T1J 4B1;and ‡Chr.Hansen Inc.,Milwaukee,WI 53214ABSTRACT:Two experiments were conducted to in-vestigate the effects of oral supplementation of the lac-tic-acid-producing bacterium Enterococcus faecium EF212alone or in combination with Saccharomyces cer-evisiae (yeast)on mediators of the acute phase response in feedlot steers.Eight fistulated steers were used to study the effects of E.faecium alone or with yeast in a crossover design with 2Latin squares,4steers within each square,and 2periods.The length of each period was 3wk,with a 10-d adaptation and an 11-d measure-ment period.The experimental diet contained 87%steam-rolled barley,8%whole-crop barley silage,and 5%supplement (DM basis).In Exp.1,treatments were control vs.the lactic-acid-producing bacterium E.fae-cium (6×1010cfu/d).In Exp.2,treatments were control vs.E.faecium (6×1010cfu/d)and S.cerevisiae (6×1010cfu/d).The bacteria and yeast supplements were blended with calcium carbonate to supply 6×1010cfu/d when top-dressed into the diet once daily at the time Key words:acute phase protein,direct-fed microbial,feedlot steer,probiotic,yeast©2007American Society of Animal Science.All rights reserved.J.Anim.Sci.2007.85:233–239doi:10.2527/jas.2006-216INTRODUCTIONThere is growing interest in feeding direct-fed micro-bials (DFM )to cattle to improve digestion and enhance BW gain,as well as to prevent acidosis and outbreaks of foodborne pathogens.For example,Enterococcus fae-cium ,a common DFM strain was reported to reduce the risk of acidosis when fed to dairy cows (Nocek et al.,2002).In addition,E.faecium and other lactic acid-producing bacteria reduce fecal shedding of important enteropathogens like Escherichia coli O157:H7,Salmo-1Present address:Isfahan University of Technology,Isfahan,Iran 84156.2Present address:JL Microbiology Inc.,Hartland,WI.3Corresponding author:burim.ametaj@ualberta.ca Received April 5,2006.Accepted July 26,2006.233of feeding (10g/d).Steers fed the control diet received only carrier (10g/d).Blood samples were collected from the jugular vein on d 17and 21of each period,and serum amyloid A (SAA),lipopolysaccharide binding protein (LBP),haptoglobin,and alpha 1-acid glycopro-tein (α1-AGP)were measured.Supplementation of feed with E.faecium had no effect on concentrations of SAA,LBP,haptoglobin,or α1-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 α1-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.nella ,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 (Iso-lauri et al.,2001).For example,oral administration of E.faecium stimulated the mucosal and systemic im-Emmanuel et al. 234mune responses in young dogs with increased produc-tion of immunoglobulin A(Benyacoub et al.,2003).Sim-ilarly,a short-term oral administration of S.cerevisiae resulted in enhanced resistance of mice toward infec-tions with Klebsiella pneumoniae,Streptococcus pneu-moniae,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 glycopro-tein(α1-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 feed-ing E.faecium alone or in combination with S.cerevisiae on selected mediators of acute phase response in beef cattle fed high proportions of grain.MATERIALS AND METHODS Animals and TreatmentsAs previously reported by Beauchemin et al.(2003), 8cannulated steers(Exp.1BW=507±45kg;Exp.2 BW=538±46kg)were used in2experiments.Steers were kept in individual stalls bedded with rubber mats and cared for according to the guidelines of the Cana-dian Council on Animal Care(1993).The experimental design was a2×2Latin square with2squares,4 steers within each square,2periods,and2diets in each experiment.The squares within each experiment were conducted concurrently,and experiments were run con-secutively.The length of each period was21d,which was divided into a10-d adaptation and an11-d mea-surement.To minimize carry over effects from period to period, on the last day of periods1and2,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 correspond-ing 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 EF212with S.cerevisiae(yeast).The bacteria and yeast were blended with calcium carbonate(car-rier)to supply6×109cfu 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(10g/d).Both E.faecium EF212and S.cerevisiae were supplied by Chr.Hansen Inc.(Milwaukee,WI).The viability of the Table1.Ingredients and chemical composition of the total mixed diet(DM basis)Item% Ingredient1Barley silage2,37.94 Barley,steam-rolled487.13 Barley,ground0.97 Canola meal 1.52 Calcium carbonate 1.85 Trace mineral/vitamin mix50.05 Salt0.54 Chemical compositionME allowed gain,6kg/d 2.01 NE g,6Mcal/kg 1.17 NE m,6Mcal/kg 1.80 OM,%94.20 CP,%12.50 MP allowed gain,6kg/d 1.53 NDF,%22.00 Effective NDF,%8.09 ADF,%10.20 1All ingredients pelleted,excluding steam-rolled barley and silage. 2Physical effectiveness was66%,measured as the sum of the pro-portion of sample retained on the top(4.2%)and bottom(61.9%) sieves of the Pennsylvania State University particle separator.3Composition was37.1%DM,12.3%CP,45.8%NDF,28.3%ADF, and4.8%lignin based on4samples composited by period.4Processing index,calculated as the volume weight(DM basis)of the barley after processing,expressed as a percentage of its volume weight(DM basis)before processing,was79%.5Supplied per kilogram of DM of diet:15mg of Cu;63mg of Zn; 27mg of Mn;0.65mg of Io;0.2mg of Co;0.3mg of Se;4,200IU of vitamin A;415IU of vitamin D;and13IU of vitamin E.6Estimated from the NRC(1996).Animal BW used in the model was450kg.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,effectivefiber,mineral,and vitamin needs for cattle weighing450kg and gaining 1.5kg/d(Table1).A feed mixer was used for preparing the diet each day.The diet was fed once a day at0900. Feed and water were available ad libitum,and orts were approximately10%of the diet.Blood Sampling and Laboratory AnalysesBlood samples were obtained from each steer on d17 and21of each period.The reason for choosing these sampling days was the time needed for the host to re-spond to the feeding of DFM.At5h after feeding,blood samples were collected from the jugular vein into10-mL vacuum tubes containing Na-heparin(Vacutainer, Becton Dickinson,Franklin Lakes,NJ).Samples were centrifuged(5,000×g,20min,4°C)within20min, and plasma was collected,immediately placed on ice, transported to the laboratory,and frozen at−20°C un-til analysis.Concentrations of SAA in the plasma were deter-mined by commercially available bovine ELISA kits (Tridelta Development Ltd.,Greystones Co.,Wicklow, Ireland)according to the manufacturer’s instructionsDirect-fed microbials and acute phase response235and as described by McDonald et al.(1991).All samplesincluding standards were tested in duplicate.Sampleswere initially diluted1:500.Optical density values wereread on a microplate spectrophotometer(model SpectraMax190,Molecular Devices Corporation,Sunnyvale,CA)at450nm.The intra-and interassay CV were below10%.According to the manufacturer,the detection limitof the assay was0.30␮g/mL.Concentrations of haptoglobin in plasma were deter-mined by bovine ELISA kits(Tridelta DevelopmentLtd.),as described by Godson et al.(1996),using a poolof bovine serum as the standard.All samples includingstandards were tested in duplicate.Optical density val-ues were read on the Spectra Max190microplate spec-trophotometer at630nm.The intra-and interassay CVwere below10%,and the detection limit of the assaywas at0.05␮g/mL.Concentrations ofα1-AGP in plasma were measuredwith bovine radial immunodiffusion(RID)assay kits(Tridelta Development Ltd.).Single RID assays wereprepared to measure plasma concentrations ofα1-AGP.Calibrators and samples were applied to wells in5.0-␮L volumes.Plates were placed in humidified chambers at37°C and allowed to incubate for24h before readingthe test results.For the calibrators,a plot of the diame-ter squared on the y-axis and the concentration of theantigen on the x-axis,gave a linear function,as de-scribed previously by Mancini et al.(1965).On the basisof this linear function,sample concentrations were cal-culated.The intra-and interassay CV were below4%,and the detection limit of the assay was at50␮g/mL.Concentrations of LBP in the plasma were deter-mined with a commercially available multispeciesELISA kit that crossreacts with bovine LBP(Cell Sci-ences Inc.,Norwood,MA).Plasma samples were ini-tially diluted1:1,500,and samples with optical densityvalues lower than the range of the standard curve werediluted1:1,200and reassayed according to the manu-facturer’s instructions.The optical density at450nmwas measured on the Spectra Max190microplate spec-trophotometer.The intra-and interassay CV were be-low10%,and the detection limits of the assay were1.6to100ng/mL.The concentration of LBP was calculatedby extrapolating from a standard curve of knownamounts of human LBP.Statistical AnalysesData were analyzed using the MIXED procedure(SAS Inst.Inc.,Cary,NC)with thefirst autoregressivecovariance structure.For variables measured overtime,the model included treatment,day,and the2-way interaction asfixed effects.The random effectswere square,steer within square,and period.Periodwithin square was not considered in the model becauseboth squares were conducted simultaneously,and thusthe effect of period was considered to be the same forboth squares.The REML method was used to estimatethe variance components,and the Bayesian information criterion was used to determine the bestfitting model,whereas the Kenward-Roger method was used to ap-proximate the denominator degree of freedom.Data forsampling time were analyzed as repeated measures.Significance was declared at P<0.05.RESULTS AND DISCUSSION Previously,we showed the metabolic and productiveaspects of feeding E.faecium alone or combined withyeast in feedlot steers(Beauchemin et al.,2003).Otherresearchers also have investigated the productive as-pects of supplementation of E.faecium and yeast indairy cattle(Krehbiel et al.,2003;Nocek et al.,2003).To our knowledge,however,this is thefirst study toevaluate immunomodulatory effects of DFM in cattle.Results of Exp.1showed no significant overall treat-ment effects of feeding feedlot steers E.faecium onplasma concentrations of SAA,LBP,haptoglobin,andα1-AGP(Table2).No significant differences were ob-served in the concentrations of SAA,LBP,haptoglobin,andα1-AGP in blood collected on d17vs.21betweencontrols and those supplemented with E.faecium.Al-though we did notfind treatment effects in Exp.1forthe concentrations of SAA in plasma,values were con-sistent with the value of29␮g/mL that was reportedrecently for healthy steers(Tourlomoussis et al.,2004).The SAA values for our control steers were about40␮g/mL and about35␮g/mL in steers fed E.faecium (Table2).In contrast,results of Exp.2,in which steerswere supplemented with E.faecium and yeast,showedelevated concentrations of SAA in plasma comparedwith control steers(P=0.02;Figure1).No significantday effect or treatment×day interaction was obtainedfor concentrations of SAA in plasma in Exp.2(Figure1).Serum amyloid A is a protein produced by the liverand is associated with high-density lipoproteins in theplasma.Although the precise physiological role of SAAin the host defense mechanism is not well understood,SAA is involved in binding,neutralization,and rapidremoval of endotoxin from circulation(Baumberger etal.,1991).Production and release of SAA from liverhepatocytes is stimulated by cytokines IL-1,IL-6,andTNF-αsecreted by activated liver macrophages afterremoval of endotoxin from circulation(Watanabe et al.,2000;Elam et al.,2003).The mechanism by which addi-tion of yeast to E.faecium enhanced production of SAAby the liver is not well understood;however,some of thecontributing factors might include cytokines producedlocally by gastrointestinal immune cells or the translo-cation of yeast antigenic compounds such as glucan ormannan into the bloodstream and subsequent activa-tion of liver macrophages.Recent research indicatesthat glucan and mannan derived from S.cerevisiae in-duce production of TNF-αby monocytes(Tada et al.,2002;Majtan et al.,2005).These data are thefirst reported on concentrationsof LBP in feedlot steers.In clinically healthy Holsteindairy cows in midlactation,plasma LBP was reportedEmmanuel et al.236Table 2.Acute phase proteins in the plasma of feedlot steers with or without Enterococcus faecium (n =8;Exp.1)Day 2Treatment 11721P -valueItemCont. E.faec.Cont. E.faec.Cont. E.faec.SEM Treatment Day T ×D 3SAA,␮g/mL 41.035.139.036.542.933.720.90.220.900.47LBP,␮g/mL 21.315.712.420.530.310.87.20.570.660.30Hp,␮g/mL2732242702462752012170.360.710.63α1-AGP,␮g/mL6646826766966516682590.210.900.461Eight cannulated steers were used in a 2×2Latin square design.Steers were fed a diet that was top-dressed with either the control treatment (Cont.)or E.faecium EF212(E.faec.).The bacteria were blended with calcium carbonate (carrier)to supply 6×109cfu of bacteria per gram of carrier.The diet of each steer was top-dressed with blend or carrier,once daily at the time of feeding (10g/d).Enterococcus faecium EF212was supplied by Chr.Hansen Inc.(Milwaukee,WI).2Blood samples were obtained from each steer on d 17and 21of each period and analyzed for plasma serum amyloid A (SAA),lipopolysaccharide binding protein (LBP),haptoglobin (Hp),and alpha 1-acid glyco-protein (α1-AGP).3T ×D =Treatment ×day.to be approximately 37␮g/mL (Bannerman et al.,2003).The same authors reported that within 8h of adminis-tering lipopolysaccharide into the blood of dairy cows,plasma LBP increased more than 3.5-fold,reaching av-erage values of 137␮g/mL and remaining high during the entire 72h of the experimental period (Bannerman et al.,2003).Feedlot steers in our experiment had LBP values greater than 20␮g/mL 21d 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 inExp.2,a treatment ×day interactionFigure 1.Least squares means ±SEM (Exp.2)of plasma serum amyloid A (SAA)in control steers supplemented with 10g/d of calcium carbonate (solid bars)and steers supplemented with Enterococcus faecium EF212and Sa-charomyces cerevisiae (open bars;n =8/group)at 6×109cfu/d for 11d in a 2×2Latin square experiment (10-d adaptation and 11-d measurements).a,b Means with differ-ent superscripts differ,P =0.02.for plasma concentrations of LBP (P =0.02;Figure 2)was detected.An effect also was observed for concentra-tions of LBP between controls and steers treated with E.faecium and yeast on d 21of the experiment (P <0.05).The LBP is a liver-derived acute phase protein that is implicated in modulating host responses to endo-toxin from gram-negative bacteria.The protein inter-acts with circulatory endotoxin to form complexes that bind to CD14,which facilitates binding and activation of TLR4/MD-2complex on cells of the monocytic lineage and neutrophils,resulting in their activation (Fitzger-ald et al.,2004).This triggers release of cytokines,which areresponsible for initiating the acute phaseFigure 2.Least squares means ±SEM (Exp.2)of plasma lipopolysaccharide binding protein (LBP)in control steers supplemented with carrier (solid bars)and steers supple-mented with Enterococcus faecium EF212and Sacharomyces cerevisiae (open bars;n =8/group)at 6×109cfu/d for 11d in a 2×2Latin square experiment (10-d adaptation and 11-d measurements).a,b Means with different super-scripts differ,P =0.02.Direct-fed microbials and acute phase response237Figure3.Least squares means±SEM(Exp.2)of plasma haptoglobin in control steers supplemented with carrier (solid bars)and steers supplemented with Enterococcus faecium EF212and Sacharomyces cerevisiae(open bars;n= 8/group)at6×109cfu/d for11d in a2×2Latin square experiment(10-d adaptation and11-d measurements). a,b Means with different superscripts differ,P=0.01. response(Moshage,1997).Lipopolysaccharide binding protein also facilitates transferring of endotoxin to lipo-proteins and its rapid removal from circulation by the liver(Kitchens and Thompson,2003).Increased plasma concentrations of LBP in our steers support the hypoth-esis that feeding yeast may increase translocation of endotoxin,or yeast-derived antigenic compounds like glucans and mannans,or both.In support of this hy-pothesis are results showing that enhanced production of TNF-αby monocytes stimulated with the S.cerevisiae membrane-product mannan required presence of LBP (Tada et al.,2002).Tourlomoussis et al.(2004)reported haptoglobin con-centration of110␮g/mL in plasma of healthy beef cat-tle;however,cattle under different pathological condi-tions had average plasma haptoglobin values of approx-imately270␮g/mL.Results of Exp.1showed haptoglobin concentrations of about270␮g/mL in con-trol steers and about225␮g/mL in steers supplemented with E.faecium(Table2).Elevated plasma haptoglobin values suggest translocation of bacteria into the blood-stream of feedlot steers fed high proportions of grain.In our study,E.faecium supplementation reduced plasma haptoglobin values.Further,Gozho et al.(2005)re-ported increased concentrations of endotoxin in the ru-men of male Jersey cattle fed high-grain diets and that this was associated with elevated concentrations of hap-toglobin 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 bac-teria and bacterial products(Deitch,1990). Treatment affected plasma concentration of hapto-globin in steers fed E.faecium and yeast(P<0.01; Figure3);however,no effect of day or treatment×dayFigure4.Least squares means±SEM(Exp.2)of alpha1-acid glycoprotein(α1-AGP)in control steers supple-mented with carrier(solid bars)and steers supplemented with Enterococcus faecium EF212and Sacharomyces cerevis-iae(open bars;n=8/group)at6×109cfu/d for11d in a2×2Latin square experiment(10-d adaptation and11-d measurements).interaction was observed(Figure3).Typically,concen-trations of haptoglobin in plasma are low but increase when there is an inflammatory response and transloca-tion of bacteria into the bloodstream(Deignan et al., 2000).By binding to hemoglobin,haptoglobin prevents utilization of iron in the hemoglobin by bacteria translo-cated into the bloodstream(Wassell,2000).Thus,the greater plasma haptoglobin concentration in steers fed yeast might be due to increased translocation of bacte-ria into the bloodstream.The mechanism by which yeast increases translocation of bacteria is not well un-derstood and remains to be elucidated.This is thefirst report on concentrations ofα1-AGP in plasma of feedlot steers.In healthy dairy cows,Ta-mura et al.(1989)obtained plasma values ofα1-AGP of approximately283␮g/mL.In addition,cows suffering from traumatic pericarditis,arthritis,mastitis,pneu-monia,and mesenteric liponecrosis hadα1-AGP values at or greater than450␮g/mL(Tamura et al.,1989). When steers were supplemented with E.faecium alone (Exp.1),results showed values of plasmaα1-AGP greater than600␮g/mL(Table2).Elevated concentra-tions ofα1-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 proteinα1-AGP also were elevated(greater than600␮g/mL)in plasma of all steers in Exp.2,and again,no differences were found between controls and steers fed E.faecium and yeasts(Figure4).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, 6of the8steers in period1and5of the8steers inEmmanuel et al. 238period2experienced subclinical ruminal acidosis.In Exp.2,5steers experienced subclinical ruminal acido-sis in period1and4in period2(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).In a previous publication involving metabolic and production aspects of the same experiments,we re-ported that steers supplemented with E.faecium had almost4-fold greater total number of coliform bacteria in the feces than those of controls(16×106vs.3.8×106cfu/g)(Beauchemin et al.,2003).This effect was negated,however,when yeast was provided.Interest-ingly,data reported in this paper showed that supple-menting 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 bac-teria(Nes and Holo,2000).Dead gram-negative bacte-ria release endotoxin,and the latter may transfer into the bloodstream and stimulate production of cytokines and increase gut permeability to gastrointestinalflora (Deitch,1990).Enhanced production of proinflamma-tory cytokines like IL-1,TNF-α,and IL-6has been re-ported following intake of probiotics(Wold,2001).The proinflammatory cytokines stimulate hepatocytes to se-crete acute phase proteins(Gruys et al.,2005).In conclusion,results reported in this study show for thefirst time that feeding live probiotic bacteria such as E.faecium to feedlot steers under high-grain diet for a period of11d had no effects on acute phase proteins measured(i.e.,SAA,LBP,haptoglobin,andα1-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. 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