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METABOLISM AND NUTRITION |
* Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Canada; School of Biochemistry and Molecular Biology, Faculty of Science and ANU Medical School, Australian National University, Canberra; and Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand
1 Corresponding author: doug.korver{at}ualberta.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: broiler • antimicrobial • lactobacilli • bile salt • ileum
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Antimicrobial drugs could influence the numbers and types of bacteria resident in the small bowel of broilers, which is a major region of nutrient absorption (Renner, 1965). The metabolic activity of specific bacteria in the small bowel might be important in broiler nutrition, especially the deconjugation of conjugated bile salts by bacterial bile salt hydrolase activity, an activity particularly associated with lactobacilli (Moser and Savage, 2001). Reduced concentrations of conjugated bile salts might impair lipid absorption and lead to reduced weight gain, analogous to the pathological condition of humans known as the "contaminated small bowel syndrome" (Gracey, 1983; Van Eldere, 1999). Overgrowth of the small bowel of humans by bacteria that produce bile salt hydrolases results in steatorrhea and weight loss due to impaired digestion and absorption of dietary lipids. Antibiotic therapy results in the reduction of symptoms (Gracey, 1983). In support of this hypothesis, Feighner and Dashkevicz (1987) reported that bacterial bile salt hydrolase activity was reduced in the ileum of chickens administered various antimicrobial drugs in the feed. This reduction in bacterial bile salt hydrolase activity correlated with improved growth performance (Feighner and Dashkevicz, 1987).
We measured the weight gain, efficiency of gain, feed conversion ratio, fat digestion, concentrations of conjugated bile salts, and bacterial populations in ileal contents of broilers fed antimicrobial drugs that are commonly used in broiler production (bacitracin, monensin) alone or in combination. In the present study, we focused our studies on the ileum, because it is known to provide a habitat for bacteria (Gong et al., 2002; Zhu et al., 2002; Guan et al., 2003; Lu et al., 2003) and because lipid absorption occurs maximally in the distal jejunum and proximal ileum (Renner, 1965). Due to the "enterogastric reflex," the small bowel contents are refluxed by antiperistaltic movements at 15- to 20-min intervals (Duke, 1986). Bacterial activities in the ileum could therefore affect the physiology of the whole small bowel as a result of this retrograde movement. We report here the association of increased weight gain and improved feed conversion ratio in broilers caused by dietary antimicrobial drugs, with a reduction in ileal Lactobacillus salivarius populations and reduced deconjugation of bile salts.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Trial 1.
Birds (straight-run) were raised either in floor pens with fresh straw as the litter material or in Specht (Specht Canada Inc., Stony Plain, Alberta, Canada) wire-floored pullet cages without litter. In each of 8 floor pens per treatment, 125 chicks were placed at a stocking density of 609 cm2/bird. Pullet cages (n = 8 cages per treatment) housed 20 birds per cage at a stocking density of 318 cm2/bird. Birds in both environments had free access to feed and water for the duration of the experiment. Experimental wheat-based diets were formulated to meet or exceed NRC (1994) recommended levels of all nutrients (Table 1
). Bacitracin (bacitracin methylene disalicylate, BMD 110 G Medicated Premix, Alpharma Canada Corp., Mississauga, Ontario, Canada) and monensin (monensin sodium, Coban Premix, Elanco Animal Health, Guelph, Ontario, Canada) were each administered in the feed at 0.5 g/kg of diet. Body weight gain and feed intake of birds (on a pen basis) were measured from d 0 to 10, and feed conversion ratio (g of feed/g of gain) was calculated.
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Trials 3 and 4.
At day of hatch, birds (straight-run) were placed in floor pens (n = 2 pens per treatment, 51 birds per pen) to mimic the stocking density in the floor pens in trials 1 and 2. The experimental treatments were the same as those used in trial 2, and performance data were gathered as described above. In trial 4 only, 1% of an inert digestibility marker [acid-insoluble ash; celite (Celite Corp., Lompar, CA)] was included in the diets at the expense of wheat. At 10 d of age, 8 birds per pen were killed by cervical dislocation, and the ileal contents were collected by removing the ileum (Meckel’s diverticulum to the ileocecal junction) and gently rolling a test tube along the length of the intestinal section to force the digesta into a sample bag. The samples were stored at – 20 ° C until assay for fat and acid-insoluble ash content. Acid-insoluble ash content was measured as described by Ravindran et al. (2000), fat was extracted from the feed and ileal samples using the biphasic solvent method of Folch et al. (1957), and the digestibility of dietary fat was calculated as described by Ravindran et al. (2000).
Conjugated Bile Salts in Ileal Contents
Within each trial, chicks (10, 5, 8, and 8 chicks per treatment of trials 1, 2, 3, and 4, respectively) were randomly selected from floor pens or cages and killed by cervical dislocation at the end of the starter period. The abdominal cavity was opened, the ileum was removed, and the ileal contents were collected and stored at – 80 ° C until assayed. Bile salts for use as standards were purchased from Sigma (Sigma Chemical Co., St. Louis, MO). The concentrations of the predominant conjugated bile salts present in broiler ileal contents [taurocholate (TCA) and taurochenodeoxycholate (TCDCA)] were analyzed using an HPLC method modified from that of Torchia et al. (2001). Briefly, 500 mg of ileal contents were diluted with 1 mL of HPLC-grade methanol containing hyodeoxycholic acid (HDCA; 1 mg/mL) as an internal reference for peak identification. The tubes were then vortexed for 30 s and incubated at room temperature for 5 min, followed by centrifugation (5,000 x g, 10 min, 4 ° C). The supernatants were filtered (0.22 µm) into sterile microfuge tubes. A 400-µL volume was transferred and concentrated 4-fold under vacuum. The concentrated samples were then transferred to crimp cap vials and analyzed by a HPLC evaporative light-scattering detector using a C18 guard column (Phenomex, Torrence, CA) on a Varian 9010 HPLC (Varian Inc., Walnut Creek, CA) equipped with a Hewlett Packard Series 1050 auto sampler (Agilent Technologies, Wilmington, DE) and an All-tech 500 evaporative light scattering detector (Alltech Associates Inc., Deerfield, IL). The concentrated samples (10 µL) were run isocratically using a methanol:acetonitrile:water (53:23:24, vol/vol) mixture containing 30 mM ammonium hydroxide and adjusted to pH 5.6 with glacial acetic acid. A bile acid standard solution was made by combining 1 mg/mL each of taurodeoxycholic acid, TCDCA, HDCA, cholic acid, and chenodeoxycholic acid in methanol. High-performance liquid chromatography was performed with a solvent flow rate of 0.7 mL/min, and an evaporative light-scattering detector was used with a gas flow of 3.0 L/min and a drift tube temperature of 100 ° C. Bile acid peaks were identified based on their retention times compared with bile acid standards within a run time of approximately 45 min. Peak integration was performed using a Shimadzu CLASS-VP Chromatography Laboratory Automated Software System (Shimadzu Scientific Instruments Inc., Columbia, MD).
Enumeration of Bacteria in Ileal Contents
Two methods were used to determine bacterial numbers in the ileum of birds. First, homogenates of ileal contents (10% wt/vol) in PBS (pH 7.4; Sigma Chemical Co.) were prepared, and the colony-forming units per gram were determined by plating serial dilutions of the homogenates on Lactobacilli deMan, Rogosa, and Sharpe (MRS) agar plates (Difco Laboratories, Detroit, MI) and counting colonies, differentially according to colony morphology, after 48 h of incubation at 37 ° C under anaerobic conditions. Second, total bacterial counts from ileal contents were determined by flow cytometry of fluorescent bacteria, following the directions provided with the Bacteria Counting Kit (Molecular Probes Inc., Eugene, OR). This method was used to determine the total numbers of bacteria, because many bacterial types inhabiting the gut have not yet been cultivated under laboratory conditions (Zhu et al., 2002; Lu et al., 2003). Briefly, a 10% (wt/vol) homogenate of ileal contents prepared in filtered PBS was vortexed for 5 min with 5 to 10 glass beads (3-mm diameter). Debris was removed by centrifugation (700 x g for 1 min), and the remaining supernatant was centrifuged at 8,000 x g for 3 min. The pellet was washed once in filtered PBS and suspended in 1 mL of filtered TE (10 mM Tris hydrochloride, 1 mM EDTA, pH 8.0) containing 1 mg of lysozyme/mL and incubated at room temperature for 10 min. Lysozyme was added to permeabolize the cell and allow the probe access to the cells (Snart et al., 2006). Serial dilutions were prepared in 5% (vol/vol) Tris-buffered saline (pH 8.0) in 0.15 M NaCl before incubation (37 ° C, 15 min) with 1 µL of SYTO BC stain (Molecular Probes Inc.). Immediately before analysis, 10 µL of standard microspheres (Molecular Probes Inc) was added. Flow cytometric analysis was performed using a FACSCalibur flow cytometer (Becton, Dickenson and Co., San Jose, CA) with an Ar ion laser. The fluorescence of the standard microspheres and SYTO BC stain were collected in the FL1 (515 to 545 nm, fluorescein) channel. System threshold was set on forward scatter signals, and all bacterial analyses were performed at the low rate settings (12 µL/min). The stained bacteria and the standard microspheres were distinguished on a plot of forward scatter vs. fluorescence (FL1). The number of bacteria was determined by comparing the fluorescence intensity with a fixed number of microspheres, as described by the kit manufacturers.
Identification of Bacterial Isolate A6
Bacterial DNA was extracted from an overnight culture (Lactobacilli MRS broth) as described previously (Guan et al., 2003). Polymerase chain reaction primers SacI-POmod and HDA-2 (Rodtong and Tannock, 1993; Walter et al., 2000) were used to amplify a partial sequence of the 16S ribosomal RNA (rRNA) gene (528 bp; nucleotides 11 to 539). Polymerase chain reactions were performed with a GeneAmp PCR System 9700 thermocycler (Applied Bio-systems, Foster City, CA) using the Qiagen Taq Core Kit (Qiagen Inc., Mississauga, Ontario, Canada) in a total volume of 50 µL, according to the manufacturer’s instructions. The PCR program included preheating at 95 ° C for 4 min, 25 cycles of denaturation (1 min at 95 ° C), annealing (1 min at 58 ° C), and extension (2 min at 72 ° C), with a final extension at 72 ° C for 7 min. The 16S rRNA gene amplicons were ligated in vector pCR2.1-TOPO (Invitrogen Canada Inc., Burlington, Ontario), and One Shot Top10 (Invitrogen Canada Inc.) competent Escherichia coli cells were chemically transformed, as described by the manufacturer. Recombinant cells were grown on Luria-Bertani agar plates containing 100 µg of ampicillin/mL and 40 µg of X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galacto-pyranoside)/mL. Cloned 16S rRNA gene sequences were amplified using plasmid-targeted primers (M13 forward: 5'-GTAAAACGACGGCCAG-3' ; M13 reverse: 5'-CAGGAAACAGCTATGAC-3' ) and the following PCR program: preheating at 94 ° C for 4 min, 25 cycles of denaturation (1 min at 94 ° C), annealing (1 min at 56 ° C), and extension (2 min at 72 ° C), with a final extension at 72 ° C for 7 min. The 16S rRNA partial gene sequence was determined by the Molecular Biology Services Unit at the University of Alberta, and the retrieved sequence was compared with those in the National Center for Biotechnology Information database using the BLASTN algorithm (Altschul et al., 1990).
Bile Salt Deconjugation in Vitro by L. salivarius Strain A6
Bile salt hydrolase activity in culture was assessed using a method modified from that of Knarreborg et al. (2002). Lactobacilli were cultured in MRS broth (Difco Laboratories) overnight and used to inoculate (0.1% vol/vol) MRS broth containing 2mM TCDCA or TCA. The cultures were incubated anaerobically for 24 h at 37 ° C. Then, 1-mL volumes were centrifuged, and 100 µL of supernatant was diluted 10-fold in methanol containing 0.1 mg of HDCA/mL for HPLC analysis of bile salt deconjugation. The preparation was incubated at room temperature for 5 min before centrifugation (5,000 x g, 10 min, 4 ° C), filtered (0.22 µm), and concentrated 10-fold by evaporation and dissolving in one-tenth the volume of methanol. These samples and preparations made from uninoculated culture media were then analyzed by HPLC, as described above.
Bile Salt Deconjugation In Vivo by L. salivarius Strain A6
Chickens were hatched and raised under "clean-room" conditions to exclude colonization of the birds by lactobacilli, as described previously (Hagen et al., 2005). Clean-room bird experimentation was approved by the University of Alberta Health Sciences Animal Policy and Welfare Committee. Briefly, the incubator and bird cage were maintained under positive pressure and chemically disinfected before and after experiments. Eggs were obtained from a local supplier, chemically disinfected, and incubated at 38 ° C and 65% humidity for 21 d. On day of hatch, 5 to 10 birds were transferred to a cage in which they were given sterile water and 
-irradiated feed ad libitum for 14 d. The temperature in the cage was maintained from 35 to 40 ° C, and the birds were supplied with fresh sterile bedding (shavings) daily.
At d 2 of age, each chick was inoculated by gavage with 0.5 mL of a bacterial suspension (~109 bacteria/mL) in PBS prepared from an overnight MRS broth culture of L. salivarus strain A6. At d 10 of age, the birds were killed by cervical dislocation, and the ileal contents were collected as described above. Rogosa SL agar (Difco Laboratories) plate counts were used to determine total Lactobacillus numbers in the ileal contents. Bile salt concentrations in the ileal contents of A6-colonized birds and of uninoculated birds (raised Lactobacillus-free under identical conditions) were measured as described above.
Statistical Analysis
For statistical analysis of chicken performance data, the PROC GLM procedure of SAS (SAS Institute, 1999) was used with a 2-way ANOVA with bacitracin and monensin as main effects. The pen was the experimental unit for BW gain, feed intake, and feed conversion ratio data. The individual bird was the experimental unit for d 10 or 11 BW and digestibility data. Significant differences between means were detected using the pdiff procedure of SAS. Statistical analysis of other measurements was by the Mann-Whitney nonparametric test or Kruskal-Wallis nonparametric ANOVA.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; P < 0.05). Birds in floor pens had lower concentrations of TCA in ileal contents than those housed in cages (Figure 1; P < 0.05).
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). Although there was variation in the results obtained between trials 2 and 3, birds administered antimicrobial drugs had more conjugated bile salts in ileal contents than did chicks that were untreated (Table 3). In trial 2, addition of bacitracin to the diet reduced feed conversion ratios from 12 to 35 (Table 4) and from 0 to 42 d of age (data not shown; P < 0.02) relative to diets containing no bacitracin. Likewise, in trial 3, broiler BW and BW gain were greater (P < 0.05), and feed intake and the feed conversion ratio were improved (P = 0.0925 and 0.0800, respectively) when bacitracin was added to the feed as compared with diets containing no bacitracin (Table 5). Broiler production characteristics were not affected by dietary treatment in trial 4 (Table 5).
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). Differential colony counts on Lactobacilli MRS agar plates showed that a large white colony was common on plates inoculated with dilutions from untreated birds but in low numbers in cultures of ileal homogenate from chicks fed antimicrobial drugs (Table 6). The partial 16S rRNA gene sequence obtained from a representative of the large white colony-type (strain A6) identified the isolate by BLASTN search of the National Center for Biotechnology Information database as L. salivarius (99% identity over total sequence length of 528 bp).
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). The mean population (log10) of L. salivarius A6 was 5.8/g of ileal contents (SEM = 0.2; n = 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The crop of chickens is colonized by lactobacilli that are consequently present in the digesta throughout the remainder of the digestive tract (Gong et al., 2002; Guan et al., 2003; Lu et al., 2003). These bacteria are noted for the production of bile salt hydrolases, which are active in the small bowel (Tannock et al., 1994; Moser and Savage, 2001). The absorption of lipids by chicks is influenced by the chemical structure of fats and is age-dependent, improving with age (Katongole and March, 1980; Ortiz et al., 1998). Because of this, lipids of animal origin are poorly absorbed in chickens < 2 wk of age (Hakansson, 1974). Therefore, suboptimal concentrations of conjugated bile salts resulting from bacterial hydrolytic activity due to lactobacilli could compound the lesser ability of young birds to absorb lipids.
We observed in trial 1 that broiler chicks held in floor pens had lower concentrations of TCA in the ileal contents than did caged birds and that the floor pen birds gained less weight. Therefore, all of our subsequent observations were made on birds maintained in floor pens, because we postulated that growth-promoting effects would be observed optimally under these conditions. The differential effect of housing method might have been due to different microbial exposures resulting from living on litter compared with living in wire-floored cages. For example, the isolation rate of Campylobacter from a naturally exposed population of broilers was lower when the birds were subsequently housed in cages than in floor pens (Willis et al., 2002). This may be because airborne microorganism concentrations are higher when broilers are raised in floor pens with litter than in cages with raised net floors (Madelin and Wathes, 1989).
Trials 2 and 3 enabled the effect of antimicrobial drug administration in the feed on levels of conjugated bile salts to be measured. Perhaps due to the difficulty in maintaining identical environmental conditions among large-scale production trials, we noted variation in results obtained from trials 2 and 3. In trial 2, the administration of monensin alone or in combination with bacitracin resulted in increased levels of conjugated bile salts (TCA and TCDCA) in the ileum, whereas bacitracin and monensin, alone or in combination, increased the concentrations of TCDCA in trial 3. The general outcome from the 2 trials, however, was that at least 1 of the predominant conjugated bile salts in the ileum was "protected" from deconjugation by antimicrobial drug administration. Increased BW and improved feed conversion ratio at the end of the starter period in trial 3 was associated with the administration of antimicrobial drugs, in particular, bacitracin, and an increased concentration of TCDCA. These higher concentrations of conjugated bile salts should have improved lipid absorption in the digestive tract. Indeed, measurement of lipid digestion in trial 4 revealed that monensin, which had resulted in higher levels of conjugated bile salts in both trials 2 and 3, improved lipid digestion in the birds. The inconsistent effect of dietary antimicrobials on performance reported in this study may be due to the relatively small number of replications per treatment, particularly in trials 3 and 4. In spite of the positive effect of monensin on lipid digestibility in trial 4, this drug did not improve performance relative to the control treatment. Other researchers have reported a lack of a growth-promoting effect of monensin in the absence of a coccidial challenge (Izat et al., 1991; Riddell and Classen, 1992). The effect of either monensin or bacitracin on the presence of conjugated bile salts in the ileal digesta and subsequent effects on broiler performance warrants further investigation.
To determine the antimicrobial target of the drugs in the ileum, bacterial numbers were estimated by fluorescence-activated cell sorting and by bacteriological culture. The latter method revealed that L. salivarius was present in much lower numbers (usually undetectable) in birds administered antimicrobial drugs. A representative strain of L. salivarius isolated from ileal contents deconjugated TCDCA and, to a lesser extent, TCA in vitro. Both TCA and TCDCA were deconjugated by this representative strain in the digestive tract of ex-Lactobacillus-free birds. We were not able to compare production data from Lactobacillus-free and A6-colonized birds, because the number of chicks that can be maintained under clean-room conditions is very limited. The results of our study did, however, identify L. salivarius as a target in future research, for elimination from the small bowel of broilers by methods other than the administration of antimicrobial drugs.
Lactobacillus salivarius is a common inhabitant of the digestive tract of broilers (Knarreborg et al., 2002; Guan et al., 2003). In previous studies, L. salivarius has been detected in older birds rather than during the starter period but was not detected when birds were fed a diet containing animal fat and avilamycin and salinomycin (Knarreborg et al., 2002). We postulate that the microbial ecology of L. salivarius varies among broiler production units as a result of as yet undetermined factors. Further, it can be proposed that although L. salivarius has the potential to inhabit the gut of young birds, antimicrobial drug administration suppresses these bacteria and their associated bile salt hydrolase activity, which, in turn, increases conjugated bile salt levels and improves lipid digestion and absorption by the chickens.
Future studies could investigate the potential of strains of lactobacilli that do not produce bile salt hydrolases to competitively exclude L. salivarius from the ileum of broilers. Because bile salt hydrolase activity is common among lactobacilli, this might require the use of strains in which the genetic determinants of bile salt hydrolases have been deleted. The effect of gene deletion on ecological performance of the strain would, however, need to be examined. Alternatively or additional to this probiotic approach, dietary additives (prebiotics) that would encourage the proliferation of bacterial types that do not produce bile salt hydrolases and suppress L. salivarius populations might be investigated.
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ACKNOWLEDGMENTS |
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Received for publication March 23, 2006. Accepted for publication July 18, 2006.
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