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The influence of copper concentration and source on ileal microbiota
The influence of copper concentration and source on ileal microbiota Y. Pang, J. A. Patterson, and T. J. Applegate1 Department of Animal Sciences, Purdue University, West Lafayette, IN 47907-2054 number of lactobacilli or number of E. coli. The effects of Cu and Cu source on ileal microbiota and growth performance in broiler chickens were determined in the second study. Bird performance was not affected by Cu source or concentration. The bacterial culture enumeration results revealed that supplementation with 187.5 mg/kg of Cu from Cu sulfate pentahydrate and TBCC had no effect on number of ileal lactobacilli of birds. The denaturing gradient gel electrophoresis analyses of ileal microbial communities revealed that neither Cu supplementation nor source had effects on the number of bacterial species predominant in the ileal digesta or associated with the ileal mucosa. Supplementation with TBCC supplementation significantly increased the similarity coefficients of microbiota in the ileal mucosa compared with cross-products of all individuals. This suggests that TBCC may alter the intestinal microbiota, yet this shift had no effect on bird performance.
ABSTRACT Copper is normally supplemented in poultry diets as a growth promotant and antimicrobial. However, there are conflicting reports about the growth benefits and little information about how Cu affects the microbiota in the intestinal tract of poultry. Therefore, in vitro and in vivo experiments were conducted with broilers to determine the effects of Cu source and supplementation on ileal microbiota. The influence of Cu on growth of lactobacilli and Escherichia coli in media inoculated with ileal contents was determined in the first study. When Cu sulfate pentahydrate was supplemented to the cultures, quadratic increases in lactobacilli to graded concentrations of Cu up to 125 mg/kg and quadratic decreases in E. coli up to 250 mg/kg of Cu were observed after 24 h of incubation at 37°C. However, when tribasic Cu chloride (TBCC) was supplemented, neither linear nor quadratic responses to graded concentrations of dietary Cu were observed on
Key words: chicken, copper, Escherichia coli, lactobacilli, intestinal microbiota 2009 Poultry Science 88:586–592 doi:10.3382/ps.2008-00243 that supplementation of 250 mg/kg of Cu had no significant effect on broiler BW gain and feed conversion efficiency. Banks et al. (2004a) noted that Cu supplementation with 250 mg/kg from Cu Sul decreased BW gain, feed consumption, and feed conversion efficiency. Although conflicting results about growth benefits of high dietary Cu on broilers have been reported, the poultry industry still adds high concentrations of Cu to diets as a growth promoter and for its purported bactericidal and bacteriostatic activity. Little research has been conducted, however, to study mechanisms by which high dietary Cu affects the growth of birds. One of the possible mechanisms by which Cu may benefit birds is by shifting the gastrointestinal microbiota, thereby reducing susceptibility of birds to disease, reducing intestinal lymphocyte recruitment and infiltration (Arias and Koutsos, 2006) and thus increasing nutrient absorption (Hawbaker et al., 1961; Bunch et al., 1965). Copper has been used for centuries to disinfect both liquids and solids. Currently, Cu is used as a water purifier, algicide, fungicide, nematocide, molluscicide, and antifouling and antibacterial agent (Borkow and Gabbay, 2004).
INTRODUCTION Copper is an essential micromineral for proper collagen growth and development, proper iron metabolism, and hematopoiesis, as well as other important physiological activities (Pond et al., 1995). The Cu requirement of broilers is 8 mg/kg (NRC, 1994); however, upwards of 125 to 250 mg/kg of Cu from Cu sulfate pentahydrate (Cu Sul) is routinely added into broiler and turkey diets to protect the health of birds and increase growth (Pesti and Bakalli, 1996). An increase in growth and feed intake was observed when birds were fed 250 mg/kg of Cu (Jenkins et al., 1970). Supplementation of either 125 or 250 mg/kg of Cu resulted in improved growth and feed conversion efficiency; inclusion of 375 mg/kg provided no further benefit (Pesti and Bakalli, 1996). However, Robbins and Baker (1980) and Banks et al. (2004b) reported ©2009 Poultry Science Association Inc. Received June 17, 2008. Accepted November 12, 2008. 1 Corresponding author: [email protected]
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Copper is toxic to some species of bacteria such as Klebsiella aerogenes (Silver and Phung, 1996), whereas others have developed complex resistance mechanisms. Some experiments have been conducted to determine how high dietary Cu affects microorganisms in swine and poultry. Supplementation of Cu Sul to pig diets reduces the number of ureolytic bacteria as a group, of which streptococci make up approximately 75% (Varel et al., 1987), inhibits the coliforms in the cecum and the colon (Hojberg et al., 2005), and reduces the number of streptococci in fecal samples (Fuller et al., 1960). In chickens, Xia et al. (2004) reported that supplementation of 36.75 mg/kg of Cu from Cu sulfate to broiler chicken diets had no influence on intestinal microbiota; however, supplementation with 36.75 mg/ kg of Cu from Cu-bearing montmorillonite reduced the total viable counts of Escherichia coli and Clostridia in the small intestine and cecum. However, no research has been conducted to determine how high dietary Cu affects ileal microbiota of broiler chickens fed industry standard diets and raised in floor pens and whether being an antimicrobial is one of the mechanisms for the growth benefits of Cu. Moreover, the bactericidal action of Cu is dependent on concentration of free ionic Cu (Zevenhuizen et al., 1979; Menkissoglu and Lindow, 1991). Because different sources of Cu have different solubility, they may affect intestinal microbiota differently. Copper sulfate pentahydrate is the most common Cu source for poultry and is very soluble in both water and acidic solvents (Guo et al., 2001; Pang and Applegate, 2006). Tribasic Cu chloride (TBCC) is a relatively new and concentrated source of Cu (58%). It is nearly insoluble in water and yet very soluble in acidic solvents (Cromwell et al., 1998; Pang and Applegate, 2006). When fed to poultry, TBCC can have positive effects on performance (Arias and Koutsos, 2006). Therefore, these 2 sources of Cu were chosen for this study. The in vitro experiment was designed to study the influence of Cu concentration (125 or 250 mg/kg) and Cu source (Cu Sul or TBCC) on growth of lactobacilli and E. coli in media inoculated with ileal contents from broilers in vitro. The in vivo experiment determined the effects of 187.5 mg/kg of Cu from Cu Sul or TBCC on ileal microbiota by bacterial enumeration by culture and PCR combined with denaturing gradient gel electrophoresis (DGGE) and growth performance in broilers fed standard industry diets and raised in floor pens with recycled litter. Based on its reported growth-promoting property, we hypothesized that high dietary Cu may selectively inhibit some bacteria, such as E. coli, and stimulate beneficial bacteria, such as lactobacilli.
MATERIALS AND METHODS All experiments described herein were approved by the Purdue University Animal Care and Use Committee.
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In Vitro Experiment A 49-d-old broiler chicken raised in the floor pen from a control treatment in a separate experiment was killed by CO2 asphyxiation. The ileal contents were placed into a sterile 15-mL centrifuge tube. Eight grams of the ileal contents was weighed and transferred under a flow of CO2 gas into sterile 125-mL serum bottles containing 72 mL of anaerobic buffered peptone water (g/L: peptone water, 15, Difco, Detroit, MI; KH2PO4, 1.5; Na2HPO4, 3.5; cysteine·HCl, 0.5; Na2CO3, 4.0 and 0.1 mL 0.1% resazurin, boiled and autoclaved under a CO2 atmosphere). Serial 10-fold dilutions of the inoculum were performed anaerobically by the technique of Miller and Wolin (1974) and plated, as described below, to determine initial numbers of lactobacilli and E. coli in ileal contents. The in vitro experiment was repeated once. One milliliter of 10-fold diluted ileal contents was inoculated using a 1-cc tuberculin syringe (Sherwood Medical, St. Louis, MO) into a sealed 20-mL serum tube containing 0.5 g of ground feed, formulated to contain a basal concentration of 32.5 mg/kg of Cu (from CuSO4·5H2O) and 4.0 mL of sterile, anerobic PBS (g/L: NaCl, 8.48; K2HPO4, 8.72; KH2PO4, 6.8; cysteine·HCl, 0.5; Na2CO3, 4.0 and 0.1 mL of 0.1% resazurin, Bryant and Robinson, 1961). Five treatment groups were examined in this experiment: 2 Cu sources (Cu Sul or TBCC) at 2 Cu concentrations (125 or 250 mg/kg) and 1 control treatment (no additional supplemental Cu). Copper was added to growth media and sterilized in a capped serum tube. Each treatment was done in triplicate. Cultures were incubated anaerobically at 37°C for 24 h and then were serially diluted, by syringe, in anaerobic buffered peptone water and plated on Rogosa agar plates (Merck KGaA, Darmstadt, Germany) for lactobacilli and Levine eosin methylene blue agar plates (VM219142 411, Merck KGaA) for E. coli, respectively. The E. coli plates were incubated aerobically at 37°C for 24 h; lactobacilli plates were incubated anaerobically at 37°C for 48 h in 2-L jars flushed with CO2 and were made anaerobic using GasPak (VWR, West Chester, PA) plus H and CO2 and anaerobic indicator strips (VWR). The colonies on each plate were counted and the results are expressed as base-10 logarithm colonyforming units per milliliter.
Broiler Chicken Trial Nine hundred thirty six male Ross 308 broiler chicks were individually weighed at hatch and randomly assigned to 24 pens, such that BW differences were minimized between pens. The chicks were started on the experiment diets on d 0. There were 39 birds per pen (953 cm2/bird) and 8 replicate pens per diet. Recycled litter was used from a previous study. Birds had ad libitum access to water and feed. The chicks were on a lighting schedule of 24 h of light from hatch to d 4, 16L:8D from
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d 5 to 18, and 22L:2D per day from d 19 to 34. Mortality was recorded by pen. The 3 diets were made from a large basal diet to minimize any mixing error (Table 1). The experimental diets were made by adding Cu to the basal diet and mixing well, ignoring the weight of the Cu supplementation added. Birds were fed 1 of 3 experimental diets: a basal diet formulated to contain 8 mg/kg of Cu (from Cu Sul), a diet containing 187.5 mg/kg of Cu from Cu Sul, and a diet containing 187.5 mg/kg of Cu from TBCC (Micronutrients Inc., Indianapolis, IN). Celite (diatomaceous earth, Celite Corporation, Lompac, CA) was added as an indigestible marker to measure apparent ileal P digestibility. Each experimental diet was analyzed for DM, total P, acid insoluble ash, CP, Ca, and Cu. Birds and feed were weighed on d 18, 28, and 34 by pen. On d 28, five chicks per pen were killed by CO2 asphyxiation. Ileal contents were collected for determining apparent ileal P digestibility. Ileal contents and mucosa of 1 bird per pen were collected and mixed for enumerating lactobacilli and E. coli using the same methods as in the in vitro experiment. Ileal contents were collected by gently squeezing contents from the Table 1. Basal diet formulation (%, as-fed basis) and nutrient composition for broiler chickens Item Ingredient, % Corn Soybean meal, 48% CP Soy oil NaCl dl-Methionine l-Lysine-HCl Limestone Monocalcium phosphate Vitamin and mineral premix3 Celite Calculated analysis MEn, kcal/kg CP, % Lysine, % Ca, % Total P, % Nonphytate P, % Determined analysis4 CP, % Ca, % Total P, % 1
Starter basal diet1 (d 1 to 17)
Grower basal diet2 (d 18 to 34)
57.94 34.87 3.42 0.42 0.19 0.01 1.44 1.36 0.35 —
60.20 30.25 4.71 0.42 0.12 0.06 1.37 1.02 0.35 1.5
3,100 22.00 1.20 0.90 0.71 0.45
3,175 20.00 1.10 0.80 0.61 0.36
22.31 1.00 0.71
20.77 0.90 0.59
Experimental diets from the starter phase containing a formulated 8.0, 187.5, and 187.5 mg/kg of Cu from Cu sulfate pentahydrate, Cu sulfate pentahydrate, or tribasic Cu chloride were analyzed (triplicate) to contain 22.3, 179.2, and 175.3 mg/kg of Cu, respectively. 2 Experimental diets from the grower phase containing a formulated 8.0, 187.5, and 187.5 mg/kg of Cu from Cu sulfate pentahydrate, Cu sulfate pentahydrate, or tribasic Cu chloride were analyzed (triplicate) to contain 16.2, 162.2, and 172.1 mg/kg of Cu, respectively. 3 Supplied per kilogram of diet: vitamin A, 13,233 IU; vitamin D3, 6,636 IU; vitamin E, 44.1 IU; vitamin K, 4.5 mg; thiamine, 2.21 mg; riboflavin, 6.6 mg; pantothenic acid, 24.3 mg; niacin, 88.2 mg; pyridoxine, 3.31 mg; folic acid, 1.10 mg; biotin, 0.33 mg; vitamin B12, 24.8 µg; choline, 669.8 mg; Fe from ferrous sulfate, 50.1 mg; Cu from Cu sulfate, 7.7 mg; Mn from Mn oxide, 125.1 mg; Zn from Zn oxide, 125.1 mg; I from ethylene diamine dihydroidide, 2.10 mg; Se from Na selenite, 0.30 mg. 4 All analyses were conducted in duplicate.
ileum. The ileum was then rinsed with sterile saline, cut and opened lengthwise, and ileal mucosa samples were collected by scraping the mucosal layer with a glass slide. Samples were diluted by 104, 105, and 106 for lactobacilli enumeration and by 102, 103, and 104 for E. coli enumeration in anaerobic buffered peptone water. Birds were fed the experimental diets until 34 d when ileal contents and mucosa were collected as described previously for further analyses of microbial community structure. Changes in microbial community structure in ileal contents and mucosa samples were also determined using DGGE. Samples for DGGE analysis were frozen and stored at −20°C for future analysis.
DNA Isolation Genomic DNA was isolated from intestinal digesta and tissue samples using the Ultraclean Fecal DNA kit (MoBio, Solana Beach, CA) as described by Lu et al. (2008) and Thompson et al. (2008). Samples were diluted 1:1 with sterile distilled water, and 0.25 g of the diluted sample was added to a bead-beating tube containing beads, bead solution, and lysis solution. Cells were lysed by a combination of detergent and mechanical action using a MoBio Vortex Adapter on a standard vortex. From the lysed cells, the released DNA was bound to a silica spin filter. The filter was washed and DNA was eluted using DNase-free Tris buffer. The DGGE was performed according to previously described methods with modification (Muyzer et al., 1993; Muyzer and Smalla, 1998), using bacteria-specific PCR primers to conserved regions flanking the variable V3 region of 16S rDNA. Each PCR reaction mixture contained 0.02 nmol of reverse primer (534r): 5′-ATT ACC GCG GCT GCT GG-3′ and 0.02 nmol of forward primer with a GC clamp (341FGC): 5′CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG GCC TAC GGG AGG CAG CAG-3′ (Integrated DNA Technologies, Skokie, IL), 3.75 units of Promega Taq DNA polymerase (Promega, Madison, WI), 5 to 10 ng of template DNA, 10× DNA polymerase buffer (containing 10 mM Tris-HCl, 50 mM KCl, and 0.1% Triton X-100), and 25 mM MgCl2. Amplifications were performed using a Gene Amp PCR System 9700 (PE Applied Biosystems, Foster City, CA) using the following program: 1) denaturation at 94°C for 3 min; 2) subsequent denaturation at 94°C for 1 min; 3) annealing at 65°C for 1 min, −0.5°C per cycle; 4) extension at 72°C for 1 min; 5) steps 2 to 4 repeated for 30 cycles; 6) denaturation at 94°C for 1 min; 7) annealing at 55°C for 1 min; 8) extension at 72°C for 1 min; steps 6 to 8 repeated for 7 cycles; 9) extension at 72°C for 7 min; and 10) 4.0°C final holding temperature. Polyacrylamide gels (8% acrylamide-bisacrylamide ratio 37:5:1) were cast with a 40 to 60% urea-deionized formamide gradient. The 100% denaturing acrylamide was 7 M urea and 40% deionized formamide. Amplified samples were mixed with a 20% volume of 5× loading buffer [0.025% (wt/vol) bromophenol blue, 0.025%
COPPER EFFECTS ON INTESTINAL MICROBIOTA
(wt/vol) xylene cyanol, 47% (vol/vol) 0.1 M EDTA, and 47% (vol/vol) glycerol] and 20 µL was loaded into each sample well (20-well comb). Gels were placed in a DCode Universal Mutation Detection System (Bio-Rad, Hercules, CA) and electrophoresed in 0.5× Tris-acetate-EDTA buffer at 60°C for 10 min at 200 V, followed by 16 h at 70 V. Gels were silver stained as described by Sambrook et al. (1989), using the following procedure: fixation solution for 3 min (80% ethanol, 4% glacial acetic acid, 16% distilled water), silver stain for 10 min (0.2% AgNO3, 99.8% fixation solution), developed for 15 to 20 min (0.001% NaBH4, 25% 1.5 M NaOH, 75% formaldehyde), and preservation solution for 7 min (27% ethanol, 12% glycerol, 61% distilled water).
Feed Sample Analysis Feed samples were ground using a centrifugal grinder (ZM100, Retsch GmbH & Co., Haan, Germany) through a 0.5-mm screen and dried at 105°C overnight for DM determination. Crude protein for the feed samples was measured with the use of a Leco model FP 2000 N combustion analyzer (Leco Corp., St. Joseph, MI; method 990.03, AOAC International, 2000). Determination of acid insoluble ash was done according to the procedures described by Scott and Boldaji (1997). Total P was determined according to the procedures of Sands et al. (2001). Calcium and Cu were analyzed by atomic absorption spectrophotometry (Varian FS240 AA, Varian Inc., Palo Alto, CA; method 968.08, AOAC International, 2000).
Statistical Analysis The in vitro microbial data from 2 experiments were analyzed using initial values as covariates. For each Cu source, linear and quadratic contrasts were conducted. All DGGE data were analyzed using the MIXED model of SAS (SAS Institute Inc., Cary, NC). Similarity indices of microbial communities in ileal samples were analyzed using the MIXED model of SAS and treatment differences were determined by the Tukey-Kramer means separation. Fragment pattern relatedness was determined using Bionumerics Software (version 2.5, Applied Maths, Austin, TX), which determined number of bands per sample and similarity coefficients for banding patterns between pairs of samples. A distance matrix was calculated using the DICE function and dendrograms were constructed from this matrix using the unweighted pair group means average function. The degree of similarity of banding patterns between pairs of samples was represented as a similarity coefficient. Similarity coefficients between pairs of samples (individual birds within a treatment) were segregated by treatment and similarity coefficients across treatments (all birds across all treatments; hereafter referred to as cross-products) were used as an estimate of similarity assuming no treatment effect. Comparisons of crossproducts represent all pairwise comparisons across
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treatments. Therefore, treatment similarity coefficients that are greater than the cross-product similarity coefficients indicate that the homogeneity of bacterial communities within treatment is greater than that between treatments. Tukey-Kramer means separation was used to identify treatment differences. Significance was determined using P-value <0.05. Other data were analyzed by ANOVA, using the GLM procedures of SAS. Differences between means were determined by Tukey’s means comparison. The significance level (α) was 0.05. Microbial populations were expressed and analyzed as base-10 logarithm (cfu/ mL).
RESULTS The In Vitro Experiment When Cu Sul was supplemented to the cultures, lactobacilli increased quadratically to 125 mg/kg of Cu and E. coli decreased quadratically up to 250 mg/kg of Cu (P < 0.05; Table 2). Changes in bacterial populations of 0.3 to 0.4 log10 cfu/mL may not be biologically relevant in vivo; however, with the large inoculum and background number of lactobacilli and E. coli, dramatic changes would be unexpected. However, when TBCC was supplemented, neither linear nor quadratic responses to Cu were observed in the number of lactobacilli or number of E. coli.
The Broiler Chicken Experiment Supplementation with 187.5 mg/kg of Cu from Cu Sul or TBCC had no effects on ileal lactobacilli populations of birds at 28 d of age and averaged 8.90, 8.35, and 8.07 log10 cfu/mL (SEM; ±0.24) for control, CuSul, and TBCC, respectively (data not shown). Ileal E. coli populations were below the dilutions tested. Neither Cu supplementation at 187.5 mg/kg nor Cu source affected the number of predominant bacterial species (DGGE band number) in ileal contents (mean = 9.6) and ileal mucosa (mean = 8.6; data not shown). Neither Cu supplementation at 187.5 mg/kg nor Cu source affected the similarity coefficients of the microbial community structure in ileal digesta (Table 3). Supplementation with TBCC at 187.5 mg/kg increased the similarity coefficients of ileal mucosal bacteria; however, compared with the cross-products, Cu Sul and control were not different. Therefore, individual birds fed TBCC had more similar microbiota associated with the ileal mucosa than the rest of the birds in the population. Additionally, the microbiota associated with the ileal mucosa from birds fed CuSul or the control was as unaffected by these treatments due to no statistical differences with the pairwise comparisons with individual birds in the entire population. Supplementation with 187.5 mg/kg of Cu from Cu Sul or TBCC had no effect on apparent ileal P digestibility of birds at 28 d (mean = 53.6%; data not shown), BW
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(mean ± SEM; 0.453 ± 0.068, 1.188 ± 0.012, and 1.744 ± 0.018 kg at 17, 28, and 34 d of age, respectively), feed intake, and G:F from 0 to 34 d of age.
Table 2. Effects of Cu on lactobacilli and Escherichia coli populations in media inoculated with chicken ileal contents1 Lactobacilli, log cfu/mL
Treatments2
DISCUSSION The poultry industry routinely adds high concentrations of Cu to broiler and turkey diets as a growth promotant and antimicrobial (Pesti and Bakalli, 1996). Copper easily interacts with radicals, especially with molecular oxygen, and produces hydroperoxide radicals, making Cu very toxic (Rodriguez-Montelongo et al., 1993; Suwalsky et al., 1998). In addition, ionic Cu can bind to the plasma membrane by electrostatic attraction and then penetrate into the cell membrane through opening or closing of the membrane channel (Suwalsky et al., 1998). This alters the permeability of cellular membranes and results in leakage of intracellular ions and low molecular weight metabolites (Suwalsky et al., 1998). Concurrently, Cu2+ enters the cell, catalyzes lipid oxidation, and strongly combines with the intracellular S-containing amino acids, which leads to denaturation of protein and bacteria death (Solioz et al., 1994; Suwalsky et al., 1998; Guo et al., 2005). When Cu Sul was supplemented to the cultures, quadratic responses to Cu supplementation were observed on number of lactobacilli, increasing when 125 mg/kg of Cu was fed but decreasing when up to 250 mg/kg of Cu was fed. This suggested that concentrations of Cu Sul greater than 125 mg/kg may inhibit lactobacilli growth. Supplementation with TBCC at either 125 or 250 mg/ kg had no effects on number of lactobacilli, which may result from its low in vitro solubility at pH 6.5 (10.63 and 9.11% at 125 and 250 mg/kg, respectively; Pang and Applegate, 2006). In the in vivo experiment, supplementation with 187.5 mg/kg from either Cu Sul or TBCC had no effect on number of ileal lactobacilli. Xia et al. (2004) reported that supplementation with 36.75 mg/kg of Cu from Cu Sul did not affect the growth of lactobacilli in the small intestine and the cecum in broiler chickens. However, dietary concentration of 175 mg/kg of Cu from Cu Sul reduced lactic acid bacteria and lactobacilli counts of stomach contents in weanling piglets (Hojberg et al., 2005). When Cu Sul was supplemented to the cultures, linear and quadratic responses to graded concentration of Cu were observed on number of E. coli. Copper supplementation at 250 mg/kg decreased the growth of E. coli but not with 125 mg/kg of Cu. The solubilities of Cu Sul at 125 and 250 mg/kg at pH 6.5 are 86.57 and 75.51%, respectively (Pang and Applegate, 2006). Due to the bactericidal action of Cu, depending on concentration of free ionic Cu in solution (Zevenhuizen et al., 1979; Menkissoglu and Lindow, 1991), the cultures treated with 125 mg/kg of Cu from Cu Sul may not have enough soluble Cu to inhibit the E. coli. The cultures treated with 250 mg/kg of Cu had much more soluble Cu than the ones treated with 125 mg/kg of Cu, which may inhibit E. coli. Supplementation with TBCC
Initial3 Control CuS125 CuS250 TBCC125 TBCC250 SEM
7.26 8.20 8.31 8.15 8.20 8.13 0.03 Probability <0.0001
Source of variation Probability of treatment effect Cu Sul Linear Quadratic TBCC Linear Quadratic
E. coli, log cfu/mL 7.00 7.90 7.91 7.49 7.88 7.83 0.04 <0.0001
0.29 0.004
<0.0001 0.0002
0.06 0.25
0.11 0.59
1
Means represent 6 replicates (n = 6). Cultures were incubated anaerobically at 37°C for 24 h. 2 Control = no Cu supplemented; CuS125 = 125 mg/kg of Cu from CuSO4·5H2O; CuS250 = 250 mg/kg of Cu from CuSO4·5H2O; TBCC125 = 125 mg/kg of Cu from tribasic copper chloride (TBCC); TBCC250 = 250 mg/kg of Cu from TBCC. 3 Initial data were not included in statistical analysis and were before incubation.
at either 125 or 250 mg/kg had no effects on number of E. coli, which may result from the low solubilities of TBCC at 125 and 250 mg/kg at pH 6.5 (10.63 and 9.11%, respectively; Pang and Applegate, 2006). In the in vivo experiment, the ileal E. coli population in the birds was much lower than what was expected and was diluted beyond detection. Xia et al. (2004) reported Table 3. Influence of Cu supplementation similarity indices of microbial communities in the ileal contents and ileal mucosa of broiler chicken at 34 d of age1 2
Cu source Control Cu Sul4 TBCC5 Cross-products6 SEM Probability of diet effect a,b
Dietary Cu, mg/kg 8.0 187.5 187.5
Similarity coefficient,1 % Ileal contents Ileal mucosa 64.49ab,3 50.98b 67.16a 57.65ab 3.61 0.012
69.62ab 67.61ab 71.39a 63.07b 3.78 0.018
Means within a column with no common superscript differ significantly (P ≤ 0.05) as a result of a Tukey-Kramer adjustment means comparison. 1 Indicates similarity value for the comparison of denaturing gradient gel electrophoresis banding patterns across all individuals analyzed across all treatments. 2 The degree of similarity of banding patterns between pairs of samples is represented as a similarity coefficient. Treatment similarity coefficients that are greater than the cross-product similarity coefficients indicate that the homogeneity of bacterial communities within treatment is greater than that between treatments. 3 Means represent 8 replicate pens of 1 bird per pen (n = 8). 4 Cu Sul = Cu sulfate pentahydrate. 5 TBCC = tribasic Cu chloride. 6 Indicates similarity value for the comparison of denaturing gradient gel electrophoresis banding patterns across all individuals analyzed across ages.
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that supplementation with 36.75 mg/kg of Cu from Cu Sul to broiler chicken diets had no influence on intestinal E. coli. However, supplementation with 36.75 mg/ kg of Cu from Cu-bearing montmorillonite significantly decreased the growth of intestinal E. coli (Xia et al., 2004). It was found that Cu(II)-exchanged montmorillonite inhibited the growth of E. coli K88 through bacterial cell wall injury, bacterial enzyme leakage, and an inhibition on bacterial respiration metabolism (Guo et al., 2005). Due to the tedious and time-consuming work of traditional culture techniques, only lactobacilli and E. coli were enumerated in this study. In addition, the molecular technique of DGGE was used to determine the ileal microbiota change of high dietary Cu addition and Cu source in broiler chickens. With DGGE, differences in gene primary sequence can be detected at the level of 1-bp substitution (Fischer and Lerman, 1983). The DGGE results showed that neither Cu addition concentration nor Cu source affected the number of predominant bacterial species in ileal contents or ileal mucosa. Neither Cu addition nor Cu source affected the similarity coefficients of the microbial community structure in ileal contents. This suggests that Cu addition or Cu source did not change the bacterial diversity in the ileal contents. However, in ileal mucosa, birds fed diets supplemented with TBCC had greater similarity coefficients than all individuals analyzed (i.e., crossproducts), which suggests that only TBCC addition significantly changed the microbial community associated with the ileal mucosa. High dietary Cu is added into the poultry diets to prevent disease and improve growth performance, which may replace antibiotics due to the concerns about antibiotic resistance bacteria. Some researchers did observe that high dietary Cu affected intestinal microbiota profile and reduced the growth of pathogenic bacteria in birds (Xia et al., 2004; Guo et al., 2005; Hojberg et al., 2005). However, high dietary Cu may cause some problems such as the development of Cu-resistant bacteria. Some Cu resistance genes have been detected in many bacteria. For example, CuiD is crucial for survival at high-Cu environment in Salmonella enterica serovar Typhimurium (Lim et al., 2002). Another, CueO, is a multi-Cu oxidase that confers Cu tolerance in E. coli. (Grass and Rensing, 2001). A plasmid-borne Cu resistance operon (lco) was identified from Lactococcus lactis ssp. lactis LL58-1 by Liu et al. (2002). In addition, the transferable gene tcrB encoding resistance to Cu Sul was identified in enterococci by Hasman and Aarestrup (2002), which was found in all Cu-resistant enterococci isolates from the pigs in Denmark, Spain, and Sweden (Aarestrup et al., 2002). These bacteria developed complex resistance mechanisms such as Cu transport ATPases and metallothionein cation-binding proteins (Silver and Phung, 1996), secretion of extracellular polysaccharides to sequester ionic Cu (Bitton and Freihofer, 1978), and Cu efflux systems to rid cyto-
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plasm of toxic concentrations of Cu (Silver and Phung, 1996). High amounts of Cu in diets may reduce the capacity of phytin P to be hydrolyzed by phytase because of its high affinity for phytate. In this experiment, supplementation of 187.5 mg/kg of Cu from either Cu Sul or TBCC did not affect apparent ileal P digestibility. This does not agree with the reports of Banks et al. (2004a), who reported that up to 250 mg/kg of supplementation of Cu Sul did not affect the efficacy of phytase in broilers but lowered apparent P retention regardless of phytase supplementation by 11.0 to 15.0%. Banks et al. (2004b) also observed that supplementation with 250 mg/kg of Cu from Cu citrate or Cu Sul decreased apparent P retention; however, supplementation with 250 mg/kg of Cu Lys or Cu chloride did not affect apparent P retention. In conclusion, when Cu Sul was supplemented to the cultures, lactobacilli showed quadratic increases up to 125 mg/kg of Cu, and E. coli showed quadratic decreases up to 250 mg/kg of Cu (P < 0.05) after 24 h of incubation at 37°C. However, neither linear nor quadratic responses to graded concentrations of TBCC were observed on number of lactobacilli or number of E. coli. Supplementation with 187.5 mg/kg of Cu from either Cu Sul or TBCC had no effects on number of ileal lactobacilli in broiler chickens. No growth benefits or inhibition on apparent ileal P digestibility were noted when 187.5 mg/kg of Cu from Cu Sul or TBCC was added to broiler diets. More work needs be done to clarify the growth benefits, mechanisms, and effects on intestinal microbiota when high dietary Cu is fed to birds.
REFERENCES Aarestrup, F. M., H. Hasman, L. B. Jensen, M. Moreno, I. A. Herrero, L. Dominguez, M. Finn, and A. Franklin. 2002. Antimicrobial resistance among enterococci from pigs in three European countries. Appl. Environ. Microbiol. 68:4127–4129. AOAC International. 2000. Official Methods of Analysis of AOAC International. 17th ed. AOAC International, Gaithersburg, MD. Arias, V. J., and E. A. Koutsos. 2006. Effects of copper source and level in intestinal physiology and growth of broiler chickens. Poult. Sci. 85:999–1007. Banks, K. M., K. L. Thompson, P. Jaynes, and T. J. Applegate. 2004a. The effects of copper on the efficacy of phytase, growth, and phosphorus retention in broiler chicks. Poult. Sci. 83:1335– 1341. Banks, K. M., K. L. Thompson, J. K. Rush, and T. J. Applegate. 2004b. Effects of copper source on phosphorus retention in broiler chicks and laying hens. Poult. Sci. 83:990–996. Bitton, G., and V. Freihofer. 1978. Influence of extracellular polysaccharides on the toxicity of copper and cadmium toward Klebsiella aerogenes. Microb. Ecol. 4:119–125. Borkow, G., and J. Gabbay. 2004. Putting copper into action: Copper-impregnated products with potent biocidal activities. FASEB J. 18:1728–1730. Bryant, M. P., and I. M. Robinson. 1961. Studies on the nitrogen requirements of some ruminal cellulolytic bacteria. Appl. Microbiol. 9:96–103. Bunch, R. J., J. T. McCall, V. C. Speer, and V. W. Hays. 1965. Copper supplementation for weanling pigs. J. Anim. Sci. 24:995– 1000.
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Cromwell, G. L., M. D. Lindemann, H. J. Monegue, D. D. Hall, and D. E. Orr Jr. 1998. Tribasic copper chloride and copper sulfate as copper sources for weanling pigs. J. Anim. Sci. 76:118–123. Fischer, S. G., and L. S. Lerman. 1983. DNA fragments differing by single base-pair substitutions are separated in denaturing gradient gels: Correspondence with melting theory. Proc. Natl. Acad. Sci. USA 80:1579–1583. Fuller, R., L. G. M. Newland, C. A. E. Briggs, R. Braude, and K. G. Mitchell. 1960. The normal intestinal flora of the pig. IV. The effect of dietary supplements of penicillin, chlortetracycline or copper sulphate on the faecal flora. J. Appl. Bacteriol. 23:195– 205. Grass, G., and C. Rensing. 2001. CueO is a multi-copper oxidase that confers copper tolerance in Escherichia coli. Biochem. Biophys. Res. Commun. 286:902–908. Guo, R., P. R. Henry, R. A. Holwerda, J. Cao, R. C. Littell, R. D. Miles, and C. B. Ammerman. 2001. Chemical characteristics and relative bioavailability of supplemental organic copper sources for poultry. J. Anim. Sci. 79:1132–1141. Hasman, H., and F. M. Aarestrup. 2002. tcrB, a gene conferring transferable copper resistance in Enterococcus faecium: Occurrence, transferability, and linkage to macrolide and glycopeptide resistance. Antimicrob. Agents Chemother. 46:1410–1416. Hawbaker, J. A., V. C. Speer, V. W. Hayes, and D. V. Catron. 1961. Effects of copper sulfate and other chemoprophylactics in growing swine rations. J. Anim. Sci. 20:163–167. Hojberg, O, N. Canibe, H. D. Poulsen, M. S. Hedemann, and B. B. Jensen. 2005. Influence of dietary zinc oxide and copper sulfate on the gastrointestinal ecosystem in newly weaned piglets. Appl. Environ. Microbiol. 71:2267–2277. Jenkins, N. K., T. R. Morris, and D. Valamotis. 1970. The effect of diet and copper supplementation on chick growth. Br. Poult. Sci. 11:241–248. Lim, S. Y., M. H. Joe, S. S. Song, M. H. Lee, J. W. Foster, Y. K. Park, S. Y. Choi, and I. S. Lee. 2002. CuiD is a crucial gene for survival at high copper environment in Salmonella enterica serovar Typhimurium. Mol. Cells 14:177–184. Liu, C. Q., P. Charoechai, N. Khunajakr, Y. M. Deng, Widodo, and N. W. Dunn. 2002. Genetic and transcriptional analysis of a novel plasmid-encoded copper resistance operon from Lactococcus lactis. Gene 297:241–247. Lu, J., J. W. Santo Domingo, R. Lamendella, T. Edge, and S. Hill. 2008. Phylogenetic diversity and molecular detection of bacteria in gull feces. Appl. Environ. Microbiol. 74:3969–3976. Menkissoglu, O., and S. E. Lindow. 1991. Relationship of free ionic copper and toxicity to bacteria in solutions of organic compounds. Phytopathology 81:1258–1263. Miller, T. L., and M. J. Wolin. 1974. A serum bottle modification of the Hungate technique for cultivating obligate anaerobes. Appl. Microbiol. 27:985–987. Muyzer, G., E. C. de Waal, and A. G. Uitterlinden. 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16s rRNA. Appl. Environ. Microbiol. 59:695–700. Muyzer, G., and K. Smalla. 1998. Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie van Leeuwenhoek 73:127–141.
NRC. 1994. Nutritional Requirements of Poultry. 9th rev. ed. National Academy Press, Washington, DC. Pang, Y., and T. J. Applegate. 2006. Effects of copper source and concentration on in vitro phytate phosphorus hydrolysis by phytase. J. Agric. Food Chem. 54:1792–1796. Pesti, G. M., and R. I. Bakalli. 1996. Studies on the feeding of cupric sulfate pentahydrate and cupric citrate to broiler chickens. Poult. Sci. 75:1086–1091. Pond, W. G., D. C. Church, and K. R. Pond. 1995. Pages 199–203 in Basic Animal Nutrition and Feeding. 4th ed. John Wiley & Sons Inc., New York, NY. Robbins, K. R., and D. H. Baker. 1980. Effect of sulfur amino acid level and source on the performance of chicks fed high levels of copper. Poult. Sci. 59:1246–1253. Rodriguez-Montelongo, L., L. C. de la Cruz-Rodriguez, R. N. Farias, and E. M. Massa. 1993. Membrane-associated redox cycling of copper mediates hydroperoxide toxicity in Escherichia coli. Biochim. Biophys. Acta 1144:77–84. Sambrook, J., H. Schiehle, and T. Bosch. 1989. Page 6.44 in Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Sands, J. S., D. Ragland, C. Baxter, B. C. Joern, T. E. Sauber, and O. Adeola. 2001. Phosphorus bioavailability, growth performance, and nutrient balance in pigs fed high available phosphorus corn and phytase. J. Anim. Sci. 79:2134–2142. Scott, T. A., and F. Boldaji. 1997. Comparison of inert markers [chromic oxide or insoluble ash (Celite)] for determining apparent metabolizable energy of wheat- or barley-based broiler diets with or without enzymes. Poult. Sci. 76:594–598. Silver, S., and L. T. Phung. 1996. Bacterial heavy metal resistance: New surprises. Annu. Rev. Microbiol. 50:753–789. Solioz, M., A. Odermatt, and R. Krapf. 1994. Copper pumping ATPases: Common concepts in bacteria and man. FEBS Lett. 346:44–47. Suwalsky, M., B. Ungerer, L. Quevedo, F. Aguilar, and C. P. Sotomayor. 1998. Cu2+ ions interact with cell membranes. J. Inorg. Biochem. 70:233–238. Thompson, K. L., K. M. Burkholder, J. A. Patterson, and T. J. Applegate. 2008. Microbial ecology shifts in the ileum of broilers during feed withdrawal and dietary manipulations. Poult. Sci. 87:1624–1632. Tong, G., M. Yulong, G. Peng, and X. Zirong. 2005. Antibacterial effects of the Cu(II)-exchanged montmorillonite on Escherishia coli K88 and Salmonella choleraesuis. Vet. Microbiol. 105:113–122. Varel, V. H., I. M. Robinson, and W. G. Pond. 1987. Effect of dietary copper sulfate, aureo SP250, or clinoptilolite on ureolytic bacteria found in the pig large intestine. Appl. Environ. Microbiol. 53:2009–2012. Xia, M. S., C. H. Hu, and Z. R. Xu. 2004. Effects of copper-bearing montmorillonite on growth performance, digestive enzyme activities, and intestinal microflora and morphology of male broilers. Poult. Sci. 83:1868–1875. Zevenhuizen, L. P. T. M., J. Dofing, E. J. Eshuis, and I. J. ScholtenKoerselman. 1979. Inhibitory effects of copper on bacteria related to the free ion concentration. Microb. Ecol. 5:139–146.
ABSTRACT Copper is normally supplemented in poultry diets as a growth promotant and antimicrobial. However, there are conflicting reports about the growth benefits and little information about how Cu affects the microbiota in the intestinal tract of poultry. Therefore, in vitro and in vivo experiments were conducted with broilers to determine the effects of Cu source and supplementation on ileal microbiota. The influence of Cu on growth of lactobacilli and Escherichia coli in media inoculated with ileal contents was determined in the first study. When Cu sulfate pentahydrate was supplemented to the cultures, quadratic increases in lactobacilli to graded concentrations of Cu up to 125 mg/kg and quadratic decreases in E. coli up to 250 mg/kg of Cu were observed after 24 h of incubation at 37°C. However, when tribasic Cu chloride (TBCC) was supplemented, neither linear nor quadratic responses to graded concentrations of dietary Cu were observed on
Key words: chicken, copper, Escherichia coli, lactobacilli, intestinal microbiota 2009 Poultry Science 88:586–592 doi:10.3382/ps.2008-00243 that supplementation of 250 mg/kg of Cu had no significant effect on broiler BW gain and feed conversion efficiency. Banks et al. (2004a) noted that Cu supplementation with 250 mg/kg from Cu Sul decreased BW gain, feed consumption, and feed conversion efficiency. Although conflicting results about growth benefits of high dietary Cu on broilers have been reported, the poultry industry still adds high concentrations of Cu to diets as a growth promoter and for its purported bactericidal and bacteriostatic activity. Little research has been conducted, however, to study mechanisms by which high dietary Cu affects the growth of birds. One of the possible mechanisms by which Cu may benefit birds is by shifting the gastrointestinal microbiota, thereby reducing susceptibility of birds to disease, reducing intestinal lymphocyte recruitment and infiltration (Arias and Koutsos, 2006) and thus increasing nutrient absorption (Hawbaker et al., 1961; Bunch et al., 1965). Copper has been used for centuries to disinfect both liquids and solids. Currently, Cu is used as a water purifier, algicide, fungicide, nematocide, molluscicide, and antifouling and antibacterial agent (Borkow and Gabbay, 2004).
INTRODUCTION Copper is an essential micromineral for proper collagen growth and development, proper iron metabolism, and hematopoiesis, as well as other important physiological activities (Pond et al., 1995). The Cu requirement of broilers is 8 mg/kg (NRC, 1994); however, upwards of 125 to 250 mg/kg of Cu from Cu sulfate pentahydrate (Cu Sul) is routinely added into broiler and turkey diets to protect the health of birds and increase growth (Pesti and Bakalli, 1996). An increase in growth and feed intake was observed when birds were fed 250 mg/kg of Cu (Jenkins et al., 1970). Supplementation of either 125 or 250 mg/kg of Cu resulted in improved growth and feed conversion efficiency; inclusion of 375 mg/kg provided no further benefit (Pesti and Bakalli, 1996). However, Robbins and Baker (1980) and Banks et al. (2004b) reported ©2009 Poultry Science Association Inc. Received June 17, 2008. Accepted November 12, 2008. 1 Corresponding author: [email protected]
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Copper is toxic to some species of bacteria such as Klebsiella aerogenes (Silver and Phung, 1996), whereas others have developed complex resistance mechanisms. Some experiments have been conducted to determine how high dietary Cu affects microorganisms in swine and poultry. Supplementation of Cu Sul to pig diets reduces the number of ureolytic bacteria as a group, of which streptococci make up approximately 75% (Varel et al., 1987), inhibits the coliforms in the cecum and the colon (Hojberg et al., 2005), and reduces the number of streptococci in fecal samples (Fuller et al., 1960). In chickens, Xia et al. (2004) reported that supplementation of 36.75 mg/kg of Cu from Cu sulfate to broiler chicken diets had no influence on intestinal microbiota; however, supplementation with 36.75 mg/ kg of Cu from Cu-bearing montmorillonite reduced the total viable counts of Escherichia coli and Clostridia in the small intestine and cecum. However, no research has been conducted to determine how high dietary Cu affects ileal microbiota of broiler chickens fed industry standard diets and raised in floor pens and whether being an antimicrobial is one of the mechanisms for the growth benefits of Cu. Moreover, the bactericidal action of Cu is dependent on concentration of free ionic Cu (Zevenhuizen et al., 1979; Menkissoglu and Lindow, 1991). Because different sources of Cu have different solubility, they may affect intestinal microbiota differently. Copper sulfate pentahydrate is the most common Cu source for poultry and is very soluble in both water and acidic solvents (Guo et al., 2001; Pang and Applegate, 2006). Tribasic Cu chloride (TBCC) is a relatively new and concentrated source of Cu (58%). It is nearly insoluble in water and yet very soluble in acidic solvents (Cromwell et al., 1998; Pang and Applegate, 2006). When fed to poultry, TBCC can have positive effects on performance (Arias and Koutsos, 2006). Therefore, these 2 sources of Cu were chosen for this study. The in vitro experiment was designed to study the influence of Cu concentration (125 or 250 mg/kg) and Cu source (Cu Sul or TBCC) on growth of lactobacilli and E. coli in media inoculated with ileal contents from broilers in vitro. The in vivo experiment determined the effects of 187.5 mg/kg of Cu from Cu Sul or TBCC on ileal microbiota by bacterial enumeration by culture and PCR combined with denaturing gradient gel electrophoresis (DGGE) and growth performance in broilers fed standard industry diets and raised in floor pens with recycled litter. Based on its reported growth-promoting property, we hypothesized that high dietary Cu may selectively inhibit some bacteria, such as E. coli, and stimulate beneficial bacteria, such as lactobacilli.
MATERIALS AND METHODS All experiments described herein were approved by the Purdue University Animal Care and Use Committee.
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In Vitro Experiment A 49-d-old broiler chicken raised in the floor pen from a control treatment in a separate experiment was killed by CO2 asphyxiation. The ileal contents were placed into a sterile 15-mL centrifuge tube. Eight grams of the ileal contents was weighed and transferred under a flow of CO2 gas into sterile 125-mL serum bottles containing 72 mL of anaerobic buffered peptone water (g/L: peptone water, 15, Difco, Detroit, MI; KH2PO4, 1.5; Na2HPO4, 3.5; cysteine·HCl, 0.5; Na2CO3, 4.0 and 0.1 mL 0.1% resazurin, boiled and autoclaved under a CO2 atmosphere). Serial 10-fold dilutions of the inoculum were performed anaerobically by the technique of Miller and Wolin (1974) and plated, as described below, to determine initial numbers of lactobacilli and E. coli in ileal contents. The in vitro experiment was repeated once. One milliliter of 10-fold diluted ileal contents was inoculated using a 1-cc tuberculin syringe (Sherwood Medical, St. Louis, MO) into a sealed 20-mL serum tube containing 0.5 g of ground feed, formulated to contain a basal concentration of 32.5 mg/kg of Cu (from CuSO4·5H2O) and 4.0 mL of sterile, anerobic PBS (g/L: NaCl, 8.48; K2HPO4, 8.72; KH2PO4, 6.8; cysteine·HCl, 0.5; Na2CO3, 4.0 and 0.1 mL of 0.1% resazurin, Bryant and Robinson, 1961). Five treatment groups were examined in this experiment: 2 Cu sources (Cu Sul or TBCC) at 2 Cu concentrations (125 or 250 mg/kg) and 1 control treatment (no additional supplemental Cu). Copper was added to growth media and sterilized in a capped serum tube. Each treatment was done in triplicate. Cultures were incubated anaerobically at 37°C for 24 h and then were serially diluted, by syringe, in anaerobic buffered peptone water and plated on Rogosa agar plates (Merck KGaA, Darmstadt, Germany) for lactobacilli and Levine eosin methylene blue agar plates (VM219142 411, Merck KGaA) for E. coli, respectively. The E. coli plates were incubated aerobically at 37°C for 24 h; lactobacilli plates were incubated anaerobically at 37°C for 48 h in 2-L jars flushed with CO2 and were made anaerobic using GasPak (VWR, West Chester, PA) plus H and CO2 and anaerobic indicator strips (VWR). The colonies on each plate were counted and the results are expressed as base-10 logarithm colonyforming units per milliliter.
Broiler Chicken Trial Nine hundred thirty six male Ross 308 broiler chicks were individually weighed at hatch and randomly assigned to 24 pens, such that BW differences were minimized between pens. The chicks were started on the experiment diets on d 0. There were 39 birds per pen (953 cm2/bird) and 8 replicate pens per diet. Recycled litter was used from a previous study. Birds had ad libitum access to water and feed. The chicks were on a lighting schedule of 24 h of light from hatch to d 4, 16L:8D from
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d 5 to 18, and 22L:2D per day from d 19 to 34. Mortality was recorded by pen. The 3 diets were made from a large basal diet to minimize any mixing error (Table 1). The experimental diets were made by adding Cu to the basal diet and mixing well, ignoring the weight of the Cu supplementation added. Birds were fed 1 of 3 experimental diets: a basal diet formulated to contain 8 mg/kg of Cu (from Cu Sul), a diet containing 187.5 mg/kg of Cu from Cu Sul, and a diet containing 187.5 mg/kg of Cu from TBCC (Micronutrients Inc., Indianapolis, IN). Celite (diatomaceous earth, Celite Corporation, Lompac, CA) was added as an indigestible marker to measure apparent ileal P digestibility. Each experimental diet was analyzed for DM, total P, acid insoluble ash, CP, Ca, and Cu. Birds and feed were weighed on d 18, 28, and 34 by pen. On d 28, five chicks per pen were killed by CO2 asphyxiation. Ileal contents were collected for determining apparent ileal P digestibility. Ileal contents and mucosa of 1 bird per pen were collected and mixed for enumerating lactobacilli and E. coli using the same methods as in the in vitro experiment. Ileal contents were collected by gently squeezing contents from the Table 1. Basal diet formulation (%, as-fed basis) and nutrient composition for broiler chickens Item Ingredient, % Corn Soybean meal, 48% CP Soy oil NaCl dl-Methionine l-Lysine-HCl Limestone Monocalcium phosphate Vitamin and mineral premix3 Celite Calculated analysis MEn, kcal/kg CP, % Lysine, % Ca, % Total P, % Nonphytate P, % Determined analysis4 CP, % Ca, % Total P, % 1
Starter basal diet1 (d 1 to 17)
Grower basal diet2 (d 18 to 34)
57.94 34.87 3.42 0.42 0.19 0.01 1.44 1.36 0.35 —
60.20 30.25 4.71 0.42 0.12 0.06 1.37 1.02 0.35 1.5
3,100 22.00 1.20 0.90 0.71 0.45
3,175 20.00 1.10 0.80 0.61 0.36
22.31 1.00 0.71
20.77 0.90 0.59
Experimental diets from the starter phase containing a formulated 8.0, 187.5, and 187.5 mg/kg of Cu from Cu sulfate pentahydrate, Cu sulfate pentahydrate, or tribasic Cu chloride were analyzed (triplicate) to contain 22.3, 179.2, and 175.3 mg/kg of Cu, respectively. 2 Experimental diets from the grower phase containing a formulated 8.0, 187.5, and 187.5 mg/kg of Cu from Cu sulfate pentahydrate, Cu sulfate pentahydrate, or tribasic Cu chloride were analyzed (triplicate) to contain 16.2, 162.2, and 172.1 mg/kg of Cu, respectively. 3 Supplied per kilogram of diet: vitamin A, 13,233 IU; vitamin D3, 6,636 IU; vitamin E, 44.1 IU; vitamin K, 4.5 mg; thiamine, 2.21 mg; riboflavin, 6.6 mg; pantothenic acid, 24.3 mg; niacin, 88.2 mg; pyridoxine, 3.31 mg; folic acid, 1.10 mg; biotin, 0.33 mg; vitamin B12, 24.8 µg; choline, 669.8 mg; Fe from ferrous sulfate, 50.1 mg; Cu from Cu sulfate, 7.7 mg; Mn from Mn oxide, 125.1 mg; Zn from Zn oxide, 125.1 mg; I from ethylene diamine dihydroidide, 2.10 mg; Se from Na selenite, 0.30 mg. 4 All analyses were conducted in duplicate.
ileum. The ileum was then rinsed with sterile saline, cut and opened lengthwise, and ileal mucosa samples were collected by scraping the mucosal layer with a glass slide. Samples were diluted by 104, 105, and 106 for lactobacilli enumeration and by 102, 103, and 104 for E. coli enumeration in anaerobic buffered peptone water. Birds were fed the experimental diets until 34 d when ileal contents and mucosa were collected as described previously for further analyses of microbial community structure. Changes in microbial community structure in ileal contents and mucosa samples were also determined using DGGE. Samples for DGGE analysis were frozen and stored at −20°C for future analysis.
DNA Isolation Genomic DNA was isolated from intestinal digesta and tissue samples using the Ultraclean Fecal DNA kit (MoBio, Solana Beach, CA) as described by Lu et al. (2008) and Thompson et al. (2008). Samples were diluted 1:1 with sterile distilled water, and 0.25 g of the diluted sample was added to a bead-beating tube containing beads, bead solution, and lysis solution. Cells were lysed by a combination of detergent and mechanical action using a MoBio Vortex Adapter on a standard vortex. From the lysed cells, the released DNA was bound to a silica spin filter. The filter was washed and DNA was eluted using DNase-free Tris buffer. The DGGE was performed according to previously described methods with modification (Muyzer et al., 1993; Muyzer and Smalla, 1998), using bacteria-specific PCR primers to conserved regions flanking the variable V3 region of 16S rDNA. Each PCR reaction mixture contained 0.02 nmol of reverse primer (534r): 5′-ATT ACC GCG GCT GCT GG-3′ and 0.02 nmol of forward primer with a GC clamp (341FGC): 5′CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG GCC TAC GGG AGG CAG CAG-3′ (Integrated DNA Technologies, Skokie, IL), 3.75 units of Promega Taq DNA polymerase (Promega, Madison, WI), 5 to 10 ng of template DNA, 10× DNA polymerase buffer (containing 10 mM Tris-HCl, 50 mM KCl, and 0.1% Triton X-100), and 25 mM MgCl2. Amplifications were performed using a Gene Amp PCR System 9700 (PE Applied Biosystems, Foster City, CA) using the following program: 1) denaturation at 94°C for 3 min; 2) subsequent denaturation at 94°C for 1 min; 3) annealing at 65°C for 1 min, −0.5°C per cycle; 4) extension at 72°C for 1 min; 5) steps 2 to 4 repeated for 30 cycles; 6) denaturation at 94°C for 1 min; 7) annealing at 55°C for 1 min; 8) extension at 72°C for 1 min; steps 6 to 8 repeated for 7 cycles; 9) extension at 72°C for 7 min; and 10) 4.0°C final holding temperature. Polyacrylamide gels (8% acrylamide-bisacrylamide ratio 37:5:1) were cast with a 40 to 60% urea-deionized formamide gradient. The 100% denaturing acrylamide was 7 M urea and 40% deionized formamide. Amplified samples were mixed with a 20% volume of 5× loading buffer [0.025% (wt/vol) bromophenol blue, 0.025%
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(wt/vol) xylene cyanol, 47% (vol/vol) 0.1 M EDTA, and 47% (vol/vol) glycerol] and 20 µL was loaded into each sample well (20-well comb). Gels were placed in a DCode Universal Mutation Detection System (Bio-Rad, Hercules, CA) and electrophoresed in 0.5× Tris-acetate-EDTA buffer at 60°C for 10 min at 200 V, followed by 16 h at 70 V. Gels were silver stained as described by Sambrook et al. (1989), using the following procedure: fixation solution for 3 min (80% ethanol, 4% glacial acetic acid, 16% distilled water), silver stain for 10 min (0.2% AgNO3, 99.8% fixation solution), developed for 15 to 20 min (0.001% NaBH4, 25% 1.5 M NaOH, 75% formaldehyde), and preservation solution for 7 min (27% ethanol, 12% glycerol, 61% distilled water).
Feed Sample Analysis Feed samples were ground using a centrifugal grinder (ZM100, Retsch GmbH & Co., Haan, Germany) through a 0.5-mm screen and dried at 105°C overnight for DM determination. Crude protein for the feed samples was measured with the use of a Leco model FP 2000 N combustion analyzer (Leco Corp., St. Joseph, MI; method 990.03, AOAC International, 2000). Determination of acid insoluble ash was done according to the procedures described by Scott and Boldaji (1997). Total P was determined according to the procedures of Sands et al. (2001). Calcium and Cu were analyzed by atomic absorption spectrophotometry (Varian FS240 AA, Varian Inc., Palo Alto, CA; method 968.08, AOAC International, 2000).
Statistical Analysis The in vitro microbial data from 2 experiments were analyzed using initial values as covariates. For each Cu source, linear and quadratic contrasts were conducted. All DGGE data were analyzed using the MIXED model of SAS (SAS Institute Inc., Cary, NC). Similarity indices of microbial communities in ileal samples were analyzed using the MIXED model of SAS and treatment differences were determined by the Tukey-Kramer means separation. Fragment pattern relatedness was determined using Bionumerics Software (version 2.5, Applied Maths, Austin, TX), which determined number of bands per sample and similarity coefficients for banding patterns between pairs of samples. A distance matrix was calculated using the DICE function and dendrograms were constructed from this matrix using the unweighted pair group means average function. The degree of similarity of banding patterns between pairs of samples was represented as a similarity coefficient. Similarity coefficients between pairs of samples (individual birds within a treatment) were segregated by treatment and similarity coefficients across treatments (all birds across all treatments; hereafter referred to as cross-products) were used as an estimate of similarity assuming no treatment effect. Comparisons of crossproducts represent all pairwise comparisons across
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treatments. Therefore, treatment similarity coefficients that are greater than the cross-product similarity coefficients indicate that the homogeneity of bacterial communities within treatment is greater than that between treatments. Tukey-Kramer means separation was used to identify treatment differences. Significance was determined using P-value <0.05. Other data were analyzed by ANOVA, using the GLM procedures of SAS. Differences between means were determined by Tukey’s means comparison. The significance level (α) was 0.05. Microbial populations were expressed and analyzed as base-10 logarithm (cfu/ mL).
RESULTS The In Vitro Experiment When Cu Sul was supplemented to the cultures, lactobacilli increased quadratically to 125 mg/kg of Cu and E. coli decreased quadratically up to 250 mg/kg of Cu (P < 0.05; Table 2). Changes in bacterial populations of 0.3 to 0.4 log10 cfu/mL may not be biologically relevant in vivo; however, with the large inoculum and background number of lactobacilli and E. coli, dramatic changes would be unexpected. However, when TBCC was supplemented, neither linear nor quadratic responses to Cu were observed in the number of lactobacilli or number of E. coli.
The Broiler Chicken Experiment Supplementation with 187.5 mg/kg of Cu from Cu Sul or TBCC had no effects on ileal lactobacilli populations of birds at 28 d of age and averaged 8.90, 8.35, and 8.07 log10 cfu/mL (SEM; ±0.24) for control, CuSul, and TBCC, respectively (data not shown). Ileal E. coli populations were below the dilutions tested. Neither Cu supplementation at 187.5 mg/kg nor Cu source affected the number of predominant bacterial species (DGGE band number) in ileal contents (mean = 9.6) and ileal mucosa (mean = 8.6; data not shown). Neither Cu supplementation at 187.5 mg/kg nor Cu source affected the similarity coefficients of the microbial community structure in ileal digesta (Table 3). Supplementation with TBCC at 187.5 mg/kg increased the similarity coefficients of ileal mucosal bacteria; however, compared with the cross-products, Cu Sul and control were not different. Therefore, individual birds fed TBCC had more similar microbiota associated with the ileal mucosa than the rest of the birds in the population. Additionally, the microbiota associated with the ileal mucosa from birds fed CuSul or the control was as unaffected by these treatments due to no statistical differences with the pairwise comparisons with individual birds in the entire population. Supplementation with 187.5 mg/kg of Cu from Cu Sul or TBCC had no effect on apparent ileal P digestibility of birds at 28 d (mean = 53.6%; data not shown), BW
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(mean ± SEM; 0.453 ± 0.068, 1.188 ± 0.012, and 1.744 ± 0.018 kg at 17, 28, and 34 d of age, respectively), feed intake, and G:F from 0 to 34 d of age.
Table 2. Effects of Cu on lactobacilli and Escherichia coli populations in media inoculated with chicken ileal contents1 Lactobacilli, log cfu/mL
Treatments2
DISCUSSION The poultry industry routinely adds high concentrations of Cu to broiler and turkey diets as a growth promotant and antimicrobial (Pesti and Bakalli, 1996). Copper easily interacts with radicals, especially with molecular oxygen, and produces hydroperoxide radicals, making Cu very toxic (Rodriguez-Montelongo et al., 1993; Suwalsky et al., 1998). In addition, ionic Cu can bind to the plasma membrane by electrostatic attraction and then penetrate into the cell membrane through opening or closing of the membrane channel (Suwalsky et al., 1998). This alters the permeability of cellular membranes and results in leakage of intracellular ions and low molecular weight metabolites (Suwalsky et al., 1998). Concurrently, Cu2+ enters the cell, catalyzes lipid oxidation, and strongly combines with the intracellular S-containing amino acids, which leads to denaturation of protein and bacteria death (Solioz et al., 1994; Suwalsky et al., 1998; Guo et al., 2005). When Cu Sul was supplemented to the cultures, quadratic responses to Cu supplementation were observed on number of lactobacilli, increasing when 125 mg/kg of Cu was fed but decreasing when up to 250 mg/kg of Cu was fed. This suggested that concentrations of Cu Sul greater than 125 mg/kg may inhibit lactobacilli growth. Supplementation with TBCC at either 125 or 250 mg/ kg had no effects on number of lactobacilli, which may result from its low in vitro solubility at pH 6.5 (10.63 and 9.11% at 125 and 250 mg/kg, respectively; Pang and Applegate, 2006). In the in vivo experiment, supplementation with 187.5 mg/kg from either Cu Sul or TBCC had no effect on number of ileal lactobacilli. Xia et al. (2004) reported that supplementation with 36.75 mg/kg of Cu from Cu Sul did not affect the growth of lactobacilli in the small intestine and the cecum in broiler chickens. However, dietary concentration of 175 mg/kg of Cu from Cu Sul reduced lactic acid bacteria and lactobacilli counts of stomach contents in weanling piglets (Hojberg et al., 2005). When Cu Sul was supplemented to the cultures, linear and quadratic responses to graded concentration of Cu were observed on number of E. coli. Copper supplementation at 250 mg/kg decreased the growth of E. coli but not with 125 mg/kg of Cu. The solubilities of Cu Sul at 125 and 250 mg/kg at pH 6.5 are 86.57 and 75.51%, respectively (Pang and Applegate, 2006). Due to the bactericidal action of Cu, depending on concentration of free ionic Cu in solution (Zevenhuizen et al., 1979; Menkissoglu and Lindow, 1991), the cultures treated with 125 mg/kg of Cu from Cu Sul may not have enough soluble Cu to inhibit the E. coli. The cultures treated with 250 mg/kg of Cu had much more soluble Cu than the ones treated with 125 mg/kg of Cu, which may inhibit E. coli. Supplementation with TBCC
Initial3 Control CuS125 CuS250 TBCC125 TBCC250 SEM
7.26 8.20 8.31 8.15 8.20 8.13 0.03 Probability <0.0001
Source of variation Probability of treatment effect Cu Sul Linear Quadratic TBCC Linear Quadratic
E. coli, log cfu/mL 7.00 7.90 7.91 7.49 7.88 7.83 0.04 <0.0001
0.29 0.004
<0.0001 0.0002
0.06 0.25
0.11 0.59
1
Means represent 6 replicates (n = 6). Cultures were incubated anaerobically at 37°C for 24 h. 2 Control = no Cu supplemented; CuS125 = 125 mg/kg of Cu from CuSO4·5H2O; CuS250 = 250 mg/kg of Cu from CuSO4·5H2O; TBCC125 = 125 mg/kg of Cu from tribasic copper chloride (TBCC); TBCC250 = 250 mg/kg of Cu from TBCC. 3 Initial data were not included in statistical analysis and were before incubation.
at either 125 or 250 mg/kg had no effects on number of E. coli, which may result from the low solubilities of TBCC at 125 and 250 mg/kg at pH 6.5 (10.63 and 9.11%, respectively; Pang and Applegate, 2006). In the in vivo experiment, the ileal E. coli population in the birds was much lower than what was expected and was diluted beyond detection. Xia et al. (2004) reported Table 3. Influence of Cu supplementation similarity indices of microbial communities in the ileal contents and ileal mucosa of broiler chicken at 34 d of age1 2
Cu source Control Cu Sul4 TBCC5 Cross-products6 SEM Probability of diet effect a,b
Dietary Cu, mg/kg 8.0 187.5 187.5
Similarity coefficient,1 % Ileal contents Ileal mucosa 64.49ab,3 50.98b 67.16a 57.65ab 3.61 0.012
69.62ab 67.61ab 71.39a 63.07b 3.78 0.018
Means within a column with no common superscript differ significantly (P ≤ 0.05) as a result of a Tukey-Kramer adjustment means comparison. 1 Indicates similarity value for the comparison of denaturing gradient gel electrophoresis banding patterns across all individuals analyzed across all treatments. 2 The degree of similarity of banding patterns between pairs of samples is represented as a similarity coefficient. Treatment similarity coefficients that are greater than the cross-product similarity coefficients indicate that the homogeneity of bacterial communities within treatment is greater than that between treatments. 3 Means represent 8 replicate pens of 1 bird per pen (n = 8). 4 Cu Sul = Cu sulfate pentahydrate. 5 TBCC = tribasic Cu chloride. 6 Indicates similarity value for the comparison of denaturing gradient gel electrophoresis banding patterns across all individuals analyzed across ages.
COPPER EFFECTS ON INTESTINAL MICROBIOTA
that supplementation with 36.75 mg/kg of Cu from Cu Sul to broiler chicken diets had no influence on intestinal E. coli. However, supplementation with 36.75 mg/ kg of Cu from Cu-bearing montmorillonite significantly decreased the growth of intestinal E. coli (Xia et al., 2004). It was found that Cu(II)-exchanged montmorillonite inhibited the growth of E. coli K88 through bacterial cell wall injury, bacterial enzyme leakage, and an inhibition on bacterial respiration metabolism (Guo et al., 2005). Due to the tedious and time-consuming work of traditional culture techniques, only lactobacilli and E. coli were enumerated in this study. In addition, the molecular technique of DGGE was used to determine the ileal microbiota change of high dietary Cu addition and Cu source in broiler chickens. With DGGE, differences in gene primary sequence can be detected at the level of 1-bp substitution (Fischer and Lerman, 1983). The DGGE results showed that neither Cu addition concentration nor Cu source affected the number of predominant bacterial species in ileal contents or ileal mucosa. Neither Cu addition nor Cu source affected the similarity coefficients of the microbial community structure in ileal contents. This suggests that Cu addition or Cu source did not change the bacterial diversity in the ileal contents. However, in ileal mucosa, birds fed diets supplemented with TBCC had greater similarity coefficients than all individuals analyzed (i.e., crossproducts), which suggests that only TBCC addition significantly changed the microbial community associated with the ileal mucosa. High dietary Cu is added into the poultry diets to prevent disease and improve growth performance, which may replace antibiotics due to the concerns about antibiotic resistance bacteria. Some researchers did observe that high dietary Cu affected intestinal microbiota profile and reduced the growth of pathogenic bacteria in birds (Xia et al., 2004; Guo et al., 2005; Hojberg et al., 2005). However, high dietary Cu may cause some problems such as the development of Cu-resistant bacteria. Some Cu resistance genes have been detected in many bacteria. For example, CuiD is crucial for survival at high-Cu environment in Salmonella enterica serovar Typhimurium (Lim et al., 2002). Another, CueO, is a multi-Cu oxidase that confers Cu tolerance in E. coli. (Grass and Rensing, 2001). A plasmid-borne Cu resistance operon (lco) was identified from Lactococcus lactis ssp. lactis LL58-1 by Liu et al. (2002). In addition, the transferable gene tcrB encoding resistance to Cu Sul was identified in enterococci by Hasman and Aarestrup (2002), which was found in all Cu-resistant enterococci isolates from the pigs in Denmark, Spain, and Sweden (Aarestrup et al., 2002). These bacteria developed complex resistance mechanisms such as Cu transport ATPases and metallothionein cation-binding proteins (Silver and Phung, 1996), secretion of extracellular polysaccharides to sequester ionic Cu (Bitton and Freihofer, 1978), and Cu efflux systems to rid cyto-
591
plasm of toxic concentrations of Cu (Silver and Phung, 1996). High amounts of Cu in diets may reduce the capacity of phytin P to be hydrolyzed by phytase because of its high affinity for phytate. In this experiment, supplementation of 187.5 mg/kg of Cu from either Cu Sul or TBCC did not affect apparent ileal P digestibility. This does not agree with the reports of Banks et al. (2004a), who reported that up to 250 mg/kg of supplementation of Cu Sul did not affect the efficacy of phytase in broilers but lowered apparent P retention regardless of phytase supplementation by 11.0 to 15.0%. Banks et al. (2004b) also observed that supplementation with 250 mg/kg of Cu from Cu citrate or Cu Sul decreased apparent P retention; however, supplementation with 250 mg/kg of Cu Lys or Cu chloride did not affect apparent P retention. In conclusion, when Cu Sul was supplemented to the cultures, lactobacilli showed quadratic increases up to 125 mg/kg of Cu, and E. coli showed quadratic decreases up to 250 mg/kg of Cu (P < 0.05) after 24 h of incubation at 37°C. However, neither linear nor quadratic responses to graded concentrations of TBCC were observed on number of lactobacilli or number of E. coli. Supplementation with 187.5 mg/kg of Cu from either Cu Sul or TBCC had no effects on number of ileal lactobacilli in broiler chickens. No growth benefits or inhibition on apparent ileal P digestibility were noted when 187.5 mg/kg of Cu from Cu Sul or TBCC was added to broiler diets. More work needs be done to clarify the growth benefits, mechanisms, and effects on intestinal microbiota when high dietary Cu is fed to birds.
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