Feeding a combination of lactate-utilizing and lactateproducing bacteria modulates acute phase response in feedlot steers D. G. V. Emmanuel1, A. Jafari2, K. A. Beauchemin3, J. A. Z. Leedle4, 5, and B. N. Ametaj1, 6 1Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton Canada T6G 2P5 (e-mail
[email protected]); 2Isfahan University of Technology, Isfahan, Iran 84156, 3Research Center, Agriculture and Agri-Food Canada, Lethbridge, Canada T1J 4B1, 4Chr. Hansen, Inc., Milwaukee, WI 53214, USA. Received 1 August 2006, accepted 18 January 2007.
Emmanuel, D. G. V., Jafari, A., Beauchemin, K. A., Leedle, J. A. Z. and Ametaj, B. N. 2007. Feeding a combination of lactateutilizing and lactate-producing bacteria modulates acute phase response in feedlot steers. Can. J. Anim. Sci. 87: 251–257. Six ruminally cannulated steers were used in a replicated 3 × 3 Latin square with 2 wk for adaptation and 1 wk for measurements to study the effects of Propionibacterium P15 (P15) alone or in combination with Enterococcus faecium EF212 (PE) on acute phase response. Treatments were: (i) carrier (control), (ii) P15, and (iii) PE. The bacterial treatments [109 colony forming units (CFU) d–1] mixed in whey powder (carrier), or whey powder alone for control animals, were top-dressed once daily at the time of feeding (10 g steer–1 d–1). Blood samples were obtained from jugular veins on the last day of each period at 0, 6, and 12 h after feeding of bacteria and serum amyloid A (SAA), haptoglobin, lipopolysaccharide de-binding protein (LBP) were measured by ELISA and alpha1-acid glycoprotein (α1-AGP) by radial immunodiffusion. Results indicate that feeding P15 alone increased SAA and lowered plasma haptoglobin, whereas, feeding PE elevated concentrations of SAA, but had no effect on plasma haptoglobin. No significant differences were obtained for plasma α1-AGP among the experimental groups. In conclusion, feeding direct-fed microbials induced an inflammatory response in feedlot steers. Key words: Acute phase response, feedlot steers, direct-fed microbials Emmanuel, D. G. V., Jafari, A., Beauchemin, K. A., Leedle, J. A. Z. et Ametaj, B. N. 2007. L’alimentation d’une combinaison des bactéries de allaiter-utilisation et allaiter-productrices module la réponse de phase aiguë dans des boeufs de fourrage. Can. J. Anim. Sci. 87: 251–257. Six cannulated ruminally des boeufs ont été employés dans 3 une place latine repliée du × 3 avec 2 sem. d’adaptation et mesures de 1 sem. seul pour étudier les effets de la propionobactérie P15 (P15) ou en combination avec le faecium EF212 (PE) d’enterocoque sur la réponse de phase aiguë. Les traitements étaient comme suit : (i) porteur (commande), (ii) P15, et (iii) PE. Seuls les traitements bactériens (109 CFU d–1) mélangés dans la poudre de lait (porteur), ou la poudre de lait pour des animaux témoins, dessus-ont été habillés une fois quotidiennement à l’heure de l’alimentation (10 g steer–1 d–1). Des échantillons de sang ont été obtenus à partir des veines jugulaires le dernier jour de chaque période à 0, 6, et 12 h après alimentation des bactéries et de l’amyloïde A (SAA), haptoglobin, la protéine obligatoire de lipopolysaccharide (LBP) de sérum ont été mesurés par ELISA et glycoprotéine d’alpha1-acid (α1-AGP) par l’immunodiffusion radiale. Les résultats indiquent cela seul P15 de alimentation SAA accru et haptoglobin abaissé de plasma ; considérant que, les concentrations élevées par PE de alimentation de SAA mais n’ont eu aucun effet sur le haptoglobin de plasma. Aucune différence significative n’a été obtenue pour le plasma α1-AGP parmi les groupes expérimentaux. En conclusion, l’alimentation diriger-a alimenté des microbials induit une réponse inflammatoire dans des boeufs de fourrage. Mots clés: La réponse de phase aiguë, boeufs de fourrage, dirige-a alimente des microbials
al feeding trials to improve feed efficiency and reduce acidosis by stimulating lactate-utilizers in dairy and beef cattle (Nocek et al. 2002, 2003; Beauchemin et al. 2003). Both Propionibacterium and Enterococcus strains have been suggested to help rumen microflora adapt to the presence of excess lactic acid within the rumen (Nocek et al. 2002, 2003; Beauchemin et al. 2003).
There is a growing interest in using live microbial cultures, known also as direct-fed microbials (DFM), to replace or reduce the use of antibiotics in beef cattle. Several of these bacteria, such as Propionibacterium strains, have been shown to be beneficial because they convert lactate into propionate and prevent rumen acidosis (Baldwin et al. 1962: Beauchemin et al. 2003). In addition, lactate-producing bacteria such as Enterococcus strains have been tested in sever-
Abbreviations: α1-AGP, alpha1-acid glycoprotein; CFU, colony forming units; DFM, direct-fed microbials; IL, interleukin; LBP, lipopolysaccharide-binding protein; RID, radial immunodiffusion; SAA, serum amyloid A; TNF, tumor necrosis factor
5Present address: J.L. Microbiology, Inc., Hartland, WI, USA. 6To whom correspondence should be addressed.
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In addition to their beneficial effects in the gastrointestinal tract, DFM have been shown to modulate the host’s immune responses. For example, recent evidence indicates that DFM used in yogurt manufacturing, like Streptococcus thermophilus, Lactobacillus bulgaricus, Bifidobacterium adolescentis, and Bifidobacterium bifidum, stimulate production of proinflammatory cytokines like interleukin (IL)1, IL-6, and tumor necrosis factor (TNF)-alpha by macrophages and peripheral blood mononuclear cells (Marin et al. 1998). Moreover, proinflammatory cytokines stimulate the release of a variety of proteins from the human hepatoma cells including serum amyloid A (SAA), lipopolysaccharide-binding protein (LBP), haptoglobin, and alpha1-acid glycoprotein (α1-AGP), which are part of a general non-specific immune response known as acute phase response (Conti et al. 1995; Wan et al. 1995). Although the favorable effects of Propionibacterium and E. faecium strains in modulating the microbial population in the digestive tract as well as in improving weight gain and feed efficiency of cattle have been evidenced, there are no reports on their immunomodulatory effects. Therefore, the objective of this study was to investigate the effects of feeding live cultures of Propionibacterium P15 alone or in combination with E. faecium EF212 on mediators of acute phase response in feedlot cattle fed high-grain diets. MATERIALS AND METHODS Animals and Treatments Six cannulated steers were used in these experiments. Steers were kept in individual stalls bedded with rubber mats, in accordance with the guidelines of Canadian Council on Animal Care (1993). A more detailed description of the animals and the experiments has been reported previously by Ghorbani et al. (2002), Briefly, the experimental design was a replicated 3 × 3 Latin square design, balanced for residual effects, with two squares, three steers within each square, three periods and three diets. The length of each period was 21 d, divided into 14 d of adaptation and 7 d of experimentation. To minimize the carryover effects from period to period, the rumen of each steer was emptied manually on the last day of periods 1 and 2, and the contents were placed into the rumen of the next steer within the square that was to receive that treatment. Thus, each steer started the period with rumen contents corresponding to the same treatment it was fed. Steers were fed the same diet that was top-dressed with one of the following treatments: (i) whey powder (ii) Propionibacterium strain P15 (P15), and (iii) P15 plus E. faecium strain EF212 (PE). Both Propionibacterium and E. faecium were supplied by Chr. Hansen, Inc. (Milwaukee, WI). The viability of the preparation was tested by Chr. Hansen, Inc., before starting the experiments. The experimental groups were fed DFM blended with whey powder (carrier) to supply each 1 × 109 colony forming units (CFU) of bacteria per day. The diet of each control steer was topdressed with carrier alone, once daily at the time of feeding (10 g steer–1 d–1). The selection of DFM dose was based on
previous research conducted by Chrs. Hansen, Inc with doses ranging between 1 × 106 and 1 × 109 CFU per day and indicating beneficial effects of the highest dose. The experimental diet contained 87% steam-rolled barley, 9% whole crop barley silage, and 4% supplement (DM basis), and is shown in Table 1. The criteria used to formulate the diet were based on the National Research Council (1996) recommendations to meet or exceed the metabolizable energy, CP, effective fibre, mineral, and vitamin requirements of cattle weighing 670 kg. A feed mixer was used for preparing the diet that was fed once daily at 0900. Feed and water were available ad libitum, and orts were at least 10% of feed provided. Blood Sampling and Laboratory Analysis Blood samples were obtained from each steer on the 21st d of the experiment of each period at 0, 6, and 12 h after feeding of the DFM. Samples were drawn from the jugular vein into 10-mL vacuum tubes containing Na-heparin (Becton Dickinson, Franklin Lakes, NJ). Then, samples were centrifuged (5000 × g, 20 min at 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 plasma were determined by commercially available ELISA kits (Tridelta Development Ltd., Greystones Co., Wicklow, Ireland) according to the manufacturer’s instructions. The monoclonal antibodies and the ELISA were originally described by McDonald et al. (1991). All samples including the standards were assayed in duplicate. Samples were initially diluted 1:500. Optical density values were read on a microplate spectrophotometer (model Spectra Max 190, Molecular Devices Corporation, CA) at 450 nm. According to the manufacturer, the detection limit of the assay was 0.3 µg mL–1. Concentrations of LBP in plasma were determined with a commercially available LBP ELISA kit that cross-reacts with bovine LBP (Cell Sciences, Inc., Norwood, MA). Plasma samples were initially diluted 1:1500, and samples with optical density values lower than the range of the standard curve were diluted 1:1200 and re-assayed according to the manufacturer’s instructions. The optical density at 450 nm was measured on a microplate spectrophotometer. The concentration of LBP was calculated based on a standard curve of known amounts of human LBP. Concentrations of haptoglobin in plasma were determined by ELISA kits (Tridelta Development Ltd., Greystones Co., Wicklow, Ireland) as described by Godson et al. (1996) using a pool of bovine serum as standard. All samples including the standards were assayed in duplicate. Optical density values were read on microplate at 630 nm. Concentrations of α1-AGP in plasma were measured with radial immunodiffusion (RID) assay plates (Tridelta Development Ltd., Greystones Co., Wicklow, Ireland). Single RID assays were prepared to measure plasma concentrations of α1-AGP. Standards 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 standards, a plot of the diameter squared on the y axis and the concentration of the anti-
EMMANUEL ET AL. — DIRECT-FED MICROBIALS FOR FEEDLOT STEERS Table 1. Ingredient and chemical composition (mean and SD) of the diet (DM basis) Item Ingredientz Barley silagey Steam-rolled barley Ground barley Canola meal Calcium carbonate Trace mineral mixx Dry molasses Canola oil Flavor Vitamin A, D, Ew Chemical composition, % DMv DM Organic matter Crude protein NDF ADF Ca P Mg K
%
SD
9.00 87.00 0.858 0.966 1.029 0.831 0.199 0.084 0.002 0.031 82.8 96.0 14.6 19.31 8.09 0.39 0.34 0.16 0.57
1.75 0.62 0.52 0.36 0.18 0.13 0.01 0.02 0.01
zAll ingredients pelleted excluding rolled barley and silage. yComposition was 40.6% DM, 92.6% OM, 11.3% CP, 42.3%
NDF, 27.5% ADF, based on three samples composited by period. xComposition: NaCl, 73.47%; Dynamate (Pitman Moore Inc., Mundelein, IL; 18% K, 11% Mg, 22% S and 1,000 mg/kg of Fe) 20%; ZnSO4•H2O, 2.5%; CoSO4•6H2O, 0.01%; MnSO4•4H2O, 3%; Na2SeO3, 0.01%; ethylenediamine dihydroiodide, 0.02%; CuSO4•5H2O, 1%. wComposition: 680 000 IU of vitamin A kg–1, 160 000 IU of vitamin D kg–1, and 2000 IU of vitamin E kg–1. vWeekly fresh samples of barley silage, concentrate, and diet were composited by period and analyzed for chemical components (means ± SD).
gen 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. Statistical Analysis Data were analyzed using the MIXED model procedure of SAS (SAS Institute, Inc. 2002). The model included treatment and time as fixed effects. The random effects were square, steer within square, and period within square. The restricted maximum likelihood method was used to estimate the variance components and the Kenward-Rogers method was used to approximate the degrees of freedom. The data for sampling time within day were analyzed as a repeated measure. The differences among means were determined using single degree of freedom contrasts. Significance was declared at P < 0.05, whereas the tendency of significance was declared at P < 0.10. RESULTS Overall means and statistics for the three samplings at 0, 6, and 12 h after feeding of the DFM during the last day of the period (i.e., day 21) are presented at Table 2. A treatment effect was obtained for plasma SAA (P < 0.001; Table 2, Fig. 1); however no time effect or time × treatment interaction was observed for plasma SAA among the experimental groups (P > 0.10). Steers supplemented
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with P15 had nearly 25% higher concentrations of SAA in the plasma compared with those of the control group (P < 0.001). Furthermore, the group of steers supplemented with the PE had almost threefold higher plasma SAA compared with steers in the control group (P < 0.001). The group of steer fed PE also had greater concentrations of SAA than the group of steers fed P15 (P < 0.001). Overall concentrations of LBP in plasma are presented in Table 2, whereas time-related changes in the concentrations of LBP in plasma are shown in Fig. 2. Although steers fed PE had nearly 50% greater concentrations of plasma LBP compared with control steers, and 70% greater concentrations compared with steers fed P15 alone, the differences did not reach significant levels (P = 0.12) . No time effect or treatment by time interaction occurred for plasma LBP values. Plasma LBP concentrations were similar for the three groups of steers at 0, 6, and 12 h after feeding (P = 0.5). Concentrations of haptoglobin in the plasma are presented in Table 2, whereas time-related changes of plasma haptoglobin are presented in Fig. 3. There were treatment effects, as well as time effects, for plasma haptoglobin (P < 0.001 and P < 0.05, respectively). Steers receiving PE had the highest concentrations of haptoglobin in plasma, whereas steers fed P15 alone had the lowest concentrations (P < 0.05), while concentrations in control steers were intermediate. Concentrations of haptoglobin decreased with time after feeding (P < 0.05). Concentrations of plasma α1-AGP were not affected by treatment, time or treatment by time (P > 0.1; Table 2, Fig. 4). DISCUSSION Previously, we showed metabolic and productive aspects of feeding Propionibacterium, alone or in combination with E. faecium, in feedlot steers fed a high-grain diet (Ghorbani et al. 2002). In the present study, we report concentrations of several acute phase proteins such as SAA, LBP, haptoglobin and α1-AGP in the plasma of the same feedlot steers. Tourlomoussis et al. (2004) reported plasma SAA values of 26 ± 21 µg mL–1 in healthy steers. Data from our study indicate similar values with those reported by Tourlomoussis et al. (2004) in control steers (19 µg mL–1), those fed Propionibacterium alone (25 µg mL–1), and those fed Propionibacterium and Enterococcus (59 µg mL–1). Results of this study, also, show that feeding lactate-utilizing Propionibacterium for 7 consecutive days increases concentrations of SAA in plasma of steers by 25% compared with those in the control group. However, feeding Enterococcus with Propionibacterium tripled concentration of SAA in plasma compared with those of the controls. This effect is not due to feeding of Enterococcus alone because in a previous investigation we showed no effect of feeding steers E. faecium EF212 (at 1010 CFU d–1) on plasma SAA, LBP, haptoglobin, and α1-AGP (Emmanuel et al. 2007). The reason for this synergetic effect of Propionibacterium and Enterococcus on plasma SAA is not clear at present. Serum amyloid A is an acute phase protein produced by the liver in response to inflammatory conditions associated with enhanced concentrations of plasma cytokines such as TNF-
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Table 2. Acute phase proteins in the plasma of feedlot steers fed Propionibacterium strain P15 with or without E. faecium strain EF212 (n = 6) Treatmentz
P value
Item
Control
P15
PE
SEMy
SAAx (µg mL–1) LBPw (µg mL–1) Hpv (µg mL–1) α1-AGPu (µg mL–1) zSteers were fed a diet
19.92a 18.83 551a 398
25.01b 16.70 439b 473
59.56c 28.34 645a 442
6.74 5.86 43 45
Treatment
