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PROCESSING, PRODUCTS, AND FOOD SAFETY |
* Department of Health and Biomedical Sciences, Florida Hospital College of Health Sciences, Orlando 32803; Department of Poultry Science, North Carolina State University, Raleigh 27695-7608; and Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana 61801
1 Corresponding author: anael.santos{at}fhchs.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-amylase (Danisco, , Wiltshire, UK). The dietary activity levels of XY1 and XY2 were 2,500 and 650 endo-1,4-β-xylanase units/kg of feed, respectively. Microbial DNA was extracted from the ileal content of 16-wk-old turkeys, and the 16S rDNA gene was amplified by PCR and analyzed by denaturing gradient gel electrophoresis. Diversity indexes, including richness (number of species, S), evenness (relative distribution of species, EH), diversity (using Shannon’s index, H'), and Sorenson’s pairwise similarities coefficient (measures the species in common between different habitats, Cs) were calculated. Additionally, diversity indexes were associated with Salmonella prevalence determined from fresh fecal droppings collected from each pen. On the basis of contrast analysis, the wheat-based diets resulted in higher microbial diversity indexes than the corn-based diets (S = 10 vs. 12; EH = 0.9 vs. 0.8; H' = 2.2 vs. 1.9, P < 0.05). Likewise, enzyme supplementation stimulated growth of the microbiota and increased the diversity indexes in comparison with unsupplemented treatments (S = 13 vs. 10; EH = 0.9 vs. 0.8; H' = 2.2 vs. 1.9, P < 0.05). Salmonella prevalence was higher (P < 0.05) at 15 wk in turkeys fed the corn-based diet (Salmonella prevalence = 50%) than in turkeys fed the corn-enzyme (Salmonella prevalence = 13%) and wheat-based (Salmonella prevalence = 0%) dietary treatments. Therefore, contrast analysis showed that birds fed the corn control diet had lower microbiota diversity but higher Salmonella prevalence than birds fed the enzyme-supplemented and wheat-based diets. In contrast, birds fed the wheat-based diets had higher diversity but lower Salmonella prevalence than birds fed the corn-based diets. High dietary nonstarch polysaccharides from wheat and dietary exogenous enzyme supplementation promoted microbial community diversity and apparently discouraged Salmonella colonization through competitive exclusion. Nonstarch polysaccharides and dietary exogenous enzyme supplementation may be practical tools to control enteric pathogens and benefit the intestinal health and food safety of the birds.
Key Words: nonstarch polysaccharide • enzyme • polymerase chain reaction-denaturing gradient gel electrophoresis • microbial ecology • turkey
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The intestinal microbiota constitute a diverse collection of cultivable and uncultivable microbial species. Most of the knowledge concerning intestinal bacterial species has been determined by culture methods (i.e., cultivation of samples in selective media, generation of pure cultures, and subsequent taxonomic identification of the unknown bacterium). Although cultivation-based techniques have been useful for analysis of specific groups of bacteria, they have several limitations for surveying the intestinal ecosystem (McCracken et al., 2001). In addition to being time-consuming and labor-intensive, the use of selective media specific for different types of bacteria dictates the types of bacteria that can be enumerated (McCracken et al., 2001). Furthermore, estimation of culturability of bacteria in the GIT varies from 20 to 50% (Zoetendal et al., 1998). Thus, up to 80% of intestinal bacterial species may not be detected by using cultivation-based techniques (Suau et al., 1999; Vaughan et al., 2000).
The use of molecular biology methods has greatly enhanced the knowledge of GIT bacterial communities. One major advantage is the rapidity and sensitivity of the determination as compared with culture methods. Ribosomal DNA (rDNA) and ribosomal RNA (rRNA) have been shown to be excellent markers for grouping bacteria according to their phylogenetic origin (Lane et al., 1985). Comparison of bacterial rDNA sequences has demonstrated similarities that can be categorized into cluster and subcluster groups (Simon et al., 2004). These phylogenetic trees correlate well with existing taxonomic systems while also emphasizing relationships, which has led to the generation of new taxa (Simon et al., 2004).
Currently, ribosomal RNA or DNA analysis is the most commonly used measure of environmental diversity (Liu and Stahl, 2002). The most important advance has been the use of 16S rRNA or rDNA as a molecular fingerprint to identify and classify organisms, which has allowed for the development of cultivation-independent techniques for analyzing community diversity (Amann et al., 1995; Raskin et al., 1997). Denaturing gradient gel electrophoresis (DGGE) of 16S rDNA amplicons is a quick, economical, and reliable technique for the analysis of microbial community fingerprints (Muyzer et al., 1993). In addition, this cultivation-independent technique is less labor-intensive than traditional microbiological approaches, and it can be applied to evaluate dietary-, drug-, or disease-associated alterations of the intestinal microbial population (McCracken et al., 2001). This method has commonly been used for community profiling and analysis of shifts in the GIT microbiota of humans (Liu and Stahl, 2002), pigs (Collier et al., 2003a; Konstantinov et al., 2003), mice (McCracken et al., 2001), broiler chickens (Collier et al., 2003b; Hume et al., 2003), and turkeys (Waters et al., 2005); in composting processes (Ishii and Takii, 2003); and in soil (Torsvik et al., 1998; Kirk et al., 2004). However, little research has investigated the mode of action of dietary NSP and enzyme supplementation to monitor the composition of the microbial community and the exclusion of pathogens through an analysis of PCR-DGGE amplicons of the ileal digesta of turkeys.
The study reported herein tested the hypothesis that diets high in NSP content increase the diversity of the microbial community and discourage Salmonella colonization in the posterior gut, especially when the diet is supplemented with a NSP-hydrolyzing enzyme that increases substrate availability. To test this hypothesis, we investigated the change in ileal bacterial populations of turkeys fed wheat- and corn-based diets with or without supplementation of exogenous endoxylanase, as determined by PCR-DGGE of 16S rDNA amplicons. In addition, the changes in diversity of the microbial community were associated with changes in Salmonella spp. colonization of turkeys.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-amylase (EC 3.4.21.6
[EC]
2) per gram of product. The genetically modified T. longibrachiatum produces an endoxylanase stable to heat processing of up to approximately 85°C, which allows the enzyme preparation to be added to the feed in the mixer before pelleting. The dietary XY1 and XY2 activity levels were 2,500 and 650 EXU/kg of feed, respectively.
Diets
The experimental diets are presented in Table 1
. Four feed phases were used during the course of the experiment: feed 1 (1 to 28 d), feed 2 (29 to 56 d), feed 3 (57 to 84 d), feed 4 (85 to 113 d). All diets were formulated by using least-cost linear programming software, such that the diets met or exceed NRC (1994) recommendations for amino acids and energy. Diets of treatments 1 and 2 consisted of the same wheat and soybean meal (SBM) basal diet without and with XY1 supplementation, respectively. Diets of treatments 3 and 4 consisted of the same corn and SBM basal diet without and with XY2 supplementation, respectively. All feed was pellet-processed and fed in crumble form up to 4 wk of age, and subsequently in whole 8-mm pellet form. Composite feed samples from each diet were taken immediately after manufacture and analyzed for CP, fat, ash, calcium, and phosphorus. The chemical analyses were performed as shown on the footnote of Table 1. Additionally, all feed samples were analyzed for total dietary fiber, soluble dietary fiber (SDF), and insoluble dietary fiber (IDF) by using the Megazyme Total Dietary Fiber Assay kit (Total Dietary Fiber Assay Kit, Megazyme International Ireland Ltd., Co. Wicklow, Ireland). Pooled samples from the corn-based diets were sampled independently from the wheat-based diets before fiber analysis. The pooled samples consisted of 500 grams of feed sampled from each feed phase that were blended together.
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Bird Husbandry
The facility, located at the North Carolina State University Turkey Education Unit (Raleigh, NC), was a curtain-sided house containing forty-eight 9.3-m2 pens. The floor-ing of each pen was top-dressed with 4 cm of soft pine shavings at the start of the experiment. Ventilation was provided by natural air movement through appropriately adjusted curtain sides and air-mixing fans located on the ceiling throughout the house. High and low ambient temperatures within the house were recorded at 4 places twice daily throughout the duration of the trial. The house temperature was kept at 29 to 31°C during the first week, and then gradually stepped down to the ambient outside temperature, which ranged from 2 to 28°C. The house was illuminated with incandescent lights for 23 h per day on the first week and thereafter by natural daylight. Heat lamp units with 125-W bulbs provided supplemental heat for each pen. Feed and water were provided ad libitum throughout the duration of the study. A visual inspection of the health of all birds was performed daily and the weights of culled birds and reasons they were removed were recorded. Crippled or dead birds were removed and recorded.
A total of 960 one-day-old male Nicholas turkeys (Aviagen, Huntsville, AL) were randomly assigned to 32 pens in the experimental house. The house contained 4 sections with 8 pens each. Each treatment combination was replicated twice in each section of the house (8 pens/treatment). The 4 dietary treatments were randomly assigned to pens by using the Proc-Plan procedure (SAS Institute, 1996). Each pen of 30 turkeys was subjected to 1 of 4 dietary treatments from 1 to 113 d (0 to 16 wk) of age: wheat control (WC), wheat with Avizyme 1302 at 2,500 EXU/kg (WE), corn control (CC), and corn with Avizyme 1500 at 650 EXU/kg (CE).
Salmonella spp. Prevalence
In the present study, poults were naturally infected with Salmonella spp. from an unknown source. The poults were not tested for Salmonella colonization when they were received from the hatchery, so it could not be confirmed that the Salmonella came from the hatchery. In addition, the turkey experimental house was not tested before receiving the birds, so it could not be confirmed that the birds were colonized by Salmonella present in the house environment. However, all feed was tested for the presence of Salmonella, and it was always negative. Nonetheless, Salmonella colonization was detected when the flock was first tested at 3 wk of age, with nearly all pens being Salmonella positive, and Salmonella colonization was not significantly influenced by grain type (corn or wheat) or enzyme supplementation.
At 3, 9, and 15 wk of age, a composite sample of fresh fecal droppings from each pen was collected throughout the entire pen. Approximately 50- and 150-g samples of fresh fecal droppings were collected from each pen housing younger or older birds, respectively. An attempt was made to collect all available fresh fecal samples scattered in each pen. At 18 wk of age, 2 birds per pen were killed with carbon dioxide, and cecal samples were collected by cutting the ceca vertically to expose the content and mucosa. Samples collected at 3, 9, 15, and 18 wk of age were used to determine the prevalence of Salmonella spp. among the treatments according to the method described by Santos et al. (2005).
Ileal DNA Isolation and PCR-DGGE Analysis.
At 16 wk, 8 birds per treatment were killed by cervical dislocation and ileal contents were immediately placed into microcentrifuge tubes and snap-frozen in liquid nitrogen and stored at –80°C until DNA isolation. Deoxyribonucleic acid was isolated from the samples following a modification of extraction methods described previously (Tsai and Olsen, 1992; Wilson and Blitchington, 1996). Specifically, ileal samples were vortexed in 20 mL of sterile PBS solution for 10 min and then centrifuged for 2 min at 30 x g. The supernatant, which contained the bacteria, was removed and centrifuged for an additional 5 min at 12,000 x g. The supernatant from this step was discarded and the pellet was subjected to lysozyme treatment for 30 min at 37°C, at which time a stop solution (0.1 mol/L of NaCl, 0.48 mol/L of Tris, pH 8.0, 10% SDS) was added for 30 min at 37°C. These samples were subjected to 3 freeze-thaw cycles (–80°C and room temperature, respectively), proteinase K treatment (30 min at 37°C), and extraction by phenol, phenol:chloroform:isoamyl alcohol (25:24:1), and chloroform, followed by isopropanol precipitation in ammonium acetate (2.5 mol/L final concentration).
For PCR-DGGE analyses, each DNA sample was amplified by using primers specific for conserved sequences flanking the variable V3 region of the 16S rDNA, as described previously (Muyzer et al., 1998). After visual confirmation of the approximately 200-bp PCR product by agarose gel electrophoresis, mung bean nuclease (Stratagene, La Jolla, CA) was added to remove single-stranded DNA (Simpson et al., 1999). For each sample, 3 µL of 10x mung bean buffer and 0.75 U of mung bean nuclease were added to 15 µL of the PCR product. After 10 min of incubation at 37°C, the mung bean nuclease reactions were stopped by adding 10 µL of DGGE gel loading buffer (0.05% bromophenol blue, 0.05% xylene cyanol, and 70% glycerol in sterile nanopure H2O). Reactions were stored at –20°C until PCR-DGGE analysis, which was performed within 5 d of PCR.
Denaturing gradient gel electrophoresis was performed with the Bio-Rad D-Code System (Bio-Rad, Hercules, CA) as described previously (Simpson et al., 1999). To separate PCR fragments, 35 to 60% linear DNA-denaturing gradients (100% denaturant was equivalent to 7 mol/L of urea and 40% deionized formamide) were formed in 8% polyacrylamide gels by using a Bio-Rad Gradient Former. Gels were polymerized on GelBond PAG gel support films (FMC, Rockland, ME). Polymerase chain reaction products were loaded in each lane and electrophoresis was performed at 150 V for 2 h at 60°C, then for 1 h at 200 V. Additionally, bacterial reference ladders representing known bacterial strains were loaded to allow standardization of band migration and gel curvature among different gels (Simpson et al., 2000). After electrophoresis, gels were silver-stained (Muyzer et al., 1998) and scanned by using a GS-710 Calibrated Imagining Densitometer (Bio-Rad). Some samples did not yield high-quality PCR-DGGE gel lanes; therefore, only 7 samples from 7 individual turkeys from each treatment group were used for examination of the PCR-DGGE gels.
Examination of the DGGE Gels.
Examination of the DGGE gels was based on methods described previously by McCracken et al. (2001), Konstantinov et al. (2003), and Hoj et al. (2005). First, the gels were examined by using BioNumerics software version 3.5 (Applied Maths BVBA, Austin, TX). Several bands per lane were assessed by using the band-searching algorithm within the program. A manual check was done and the DGGE fragments constituting less than 1% of the total area of all bands were omitted. Bands greater than 1% of the total area of all bands were considered dominant DGGE bands and were included in further analyses. Subsequently, the PCR-DGGE banding patterns were measured by determining the migration distance and intensity of the bands within each lane of the gel (Simpson et al., 2000). This information was then used to analyze the banding patterns via several measures of community diversity, including band number (S), the Shannon index of general diversity (H'), and Shannon’s equitability index (EH; Shannon and Weaver, 1949; Sneath and Sokal, 1973; Magurran, 1988). These indices measure ecological diversity by using various parameters, including species richness (the number of different species) and evenness (the distribution of individuals within each species in the ecosystem; Magurran, 1988). In the description of the indices that follows, "species" refers to individual bands on the PCR-DGGE gels. However, because the bands on the PCR-DGGE gels correspond to the percentage of guanine and cytosine (G + C) content within the melting domains for the V3 PCR amplicon, bacterial species with a similar G + C content in the amplified V3 region may form assemblages and appear as a single band, resulting in fewer bands (Muyzer and Smalla, 1998).
Band number corresponds to the number of individual bands in a single lane (Table 2). Band frequency was calculated by measuring the percentage of all samples containing each individual band (Figure 1). Sorenson’s similarity index (Cs) was used to compare average percentage similarities of PCR-DGGE banding patterns (based on the average number of bands in common) within each treatment group (intragroup comparison, Figure 2A) and between treatment groups (intergroup comparison, Figure 2B). Calculations for Sorenson’s similarity index are based on the formula Cs = [2j/(a + b)] x 100, where a is the number of PCR-DGGE bands in lane 1, b is the number of PCR-DGGE bands in lane 2, and j is the number of common PCR-DGGE bands within bands 1 and 2. As a parameter for the structural diversity of the microbial community, Shannon’s index of general diversity (H') were calculated using the following function: H' = –
Pi logPI, where Pi is the proportion of individuals in the population belonging to the ith species; for analysis of DGGE patterns, Pi corresponds to the proportional abundance of band i (Table 2). Therefore, H' values were calculated on the basis of the bands on the gel tracks that were applied for the generation of dendrograms by using the intensities of the bands, as judged by peak height in the densitometric curves. The importance probability, Pi, was calculated as Pi = ni/T, where ni is the height of a peak and T is the sum of all peak heights in the densitometric curve. Community evenness was calculated by using Shannon’s equitability index (EH index) based on the formula EH = H'/lnS, where H' is the H' index calculated as previously mentioned and S is the total number of species in the community (total number of PCR-DGGE bands; Foucher et al., 2004; Table 2).
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i + Eij, where Yij is the observed dependent variable (microbial diversity indexes), µ is the overall mean, i is the difference between means for treatment i and µ (treatment effect), and Eij is the random error. The individual bird served as the experimental unit for statistical analysis unless otherwise stated. Variables having a significant F-test were compared by using the least squares means function (SAS Institute, 1996), and they were considered to be significant at P < 0.05. Dietary treatments were also compared by using the planned comparisons of least square means (contrast analysis) function (SAS Institute, 1996; Furr, 2003). The contrast analyses were used to contrast the effects of the different grains (wheat vs. corn) and enzyme (enzyme vs. no enzyme) treatments. All percentage data were transformed to arcsine of the square root before analysis. Because no statistical differences were observed between the transformed and original data (not transformed), the statistics presented in this paper are from the untransformed data. The microbiology data were examined by using the following procedure. Pen means served as the experimental unit for statistical analysis. The results of Salmonella prevalence were examined by using the frequency analysis procedure (chi-square test; SAS Institute, 1996). The results were considered to be significant at P < 0.05.
Animal Ethics
The experiments reported herein were conducted according to the guidelines of the Institutional Animal Care and Use Committee at North Carolina State University. All husbandry practices and euthanasia were done with full consideration of animal welfare.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. At 15 wk, Salmonella spp. were not recovered from the fecal samples from turkeys fed the wheat-based diets, which were statistically lower (P < 0.05) than samples from pens receiving the corn-based diets: 32% of the pens fed the corn-based diets were positive for Salmonella presence. Supplementation of the corn-based diets with enzyme significantly reduced Salmonella spp. presence in the fecal content of turkeys at 15 wk of age such that it was statistically equivalent to those fed the wheat-based diets. At 18 wk, Salmonella spp. prevalence in the cecal content of turkeys was not statistically different among treatment groups. However, Salmonella spp. from cecal contents were not recovered from any toms fed the wheat-based diets or those fed the CE diets, whereas approximately 13% of the toms receiving the CC diet were positive for Salmonella spp.
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. The number of bands in any individual lane ranged from 5 to 21. Enzyme supplementation significantly increased (P < 0.05) the number of bands by approximately 45 and 16% from birds fed the WE and CE diets, respectively, as compared with the corresponding unsupplemented control treatments (WC and CC, respectively). The average number of bands from birds fed wheat and corn without enzyme were the same (S = 9.6).
The percentage of samples containing a specific frequency of bands was calculated to characterize the distribution frequency of bands among the different samples (Figure 1). The maximum number of distinct bands for each treatment was 23 for the WC and CC diets, 26 for the CE diet, and 32 for the WE diet. Although the total number of distinct bands combining all samples was 41 and the mean indicated 26 distinct bands observed across treatments, the majority of bands were expressed in a low percentage of the samples (Figure 1B). Only 5 common bands were present in 
40% of the samples across treatments, and there were no common bands present in > 80% of the samples.
Sorenson’s similarity coefficients (Cs) are shown in Figure 2A (average number of bands in common within each treatment group) and Figure 2B (average number of bands in common between treatment groups). A Cs of 100% indicates that the DGGE profiles were identical, whereas a Cs of 0% indicates that the DGGE profiles were completely different (Waters et al., 2005). The Cs among samples within each treatment group were not significantly different (P < 0.05; Figure 2A). The Cs between treatment groups revealed several diet- and enzyme-dependent differences in the bands of the ileal microbial population (Figure 2B). The similarity value for the intergroup comparison of the WC with the CC diets had the highest Cs value (34.3 vs. 23.8%, WC-CC vs. others, P < 0.001) as compared with other intergroup treatment comparisons. The lowest observed Cs was the comparison between CC and WE treatments (19 vs. 27%, CC-WE vs. others, P < 0.001).
Polymerase chain reaction-DGGE banding patterns of the ileal microbiota were analyzed by using Shannon’s index (microbial community diversity, H') and Shannon’s equitability index (microbial community evenness, EH; Table 2). In the current study, enzyme supplementation significantly increased (P < 0.05) community diversity in comparison with the unsupplemented groups (2.2 vs. 1.9). Enzyme supplementation of the corn-based diet increased (P < 0.05) community diversity such that it was statistically equivalent to the wheat-based diets. In addition, the microbial community in birds fed the wheat-based diets was more evenly distributed (P < 0.05) than that in birds fed the corn-based diets (0.9 vs. 0.8; Figure 3B). Enzyme supplementation of the corn-based diets increased (P < 0.05) community evenness such that it was statistically equivalent to the wheat-based diets.
The similarities between the DGGE profiles were determined and a dendrogram was constructed (Figure 3). The effects of diet or enzyme supplementation on microbial composition were more clearly distinguished by the cluster analysis based on the unweighted pair group method with arithmetic averages. In the dendrogram, distinct clusters by diet were observed such that clusters were formed based on the type of grain and the presence of enzyme. The microbial populations of the enzyme-supplemented treatments resembled each other. Similarly, a cluster of approximately 50% similarity was formed from the microbial population of the unsupplemented treatments. Cophenetic correlation values were estimated by BioNumerics software and the values are shown in the dendrogram (Figure 3). The cophenetic value for the whole dendrogram (72) indicates that the dendogram did not distort the original structure in the input data of the current study.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Salmonella in the experimental turkey flock was not significantly influenced by grain type (corn or wheat) or enzyme supplementation at 3 and 9 wk. At 15 wk, however, Salmonella spp. were not recovered from any pens receiving the wheat-based diets, whereas 38% of the pens receiving the corn-based diets were positive. Salmonella prevalence in the fecal content of corn-fed turkeys at 15 wk of age was not statistically different from that of turkeys fed either wheat- or wheat-enzyme-based diets. Additionally, the fact that Salmonella spp. was not recovered from cecal samples collected from 18-wk-old toms fed the CE diets or the wheat-based diets indicates that the enteric Salmonella population of turkeys fed wheat-based diets and the CE diet was reduced between 16 and 18 wk of age compared with those fed the CC treatment. Presumably, dietary NSP or fermentable oligosaccharides generated by dietary enzyme supplementation encouraged a microbial population that competitively excluded Salmonella.
Corn and wheat were chosen to be the major components of the experimental diets because of their common use in poultry feed throughout the world and their great difference in NSP content. Therefore, the use of corn and wheat to formulate the experimental diets allowed us to test the hypothesis that the dietary NSP level would alter the gut microbial ecosystem by changing the availability of fermentable carbohydrates. The corn-SBM experimental diet had an average of 1.6% SDF and 14.5% IDF on a DM basis (Table 1), whereas the wheat-based diets contained 2.0% DM-SDF and 17.9% DM-IDF. Thus, the difference in total NSP between these 2 diets averaged 37.2 g/kg. Several researchers have demonstrated that a dietary NSP content from 2 to 40 g/kg can change animal performance, intestinal viscosity, nutrient digestibility, and microbial community structure (Choct and Annison, 1992; Annison, 1993; Langhout et al., 2000; Svihus, 2001; Lan, 2004). In agreement with these studies, distinct PCR-DGGE banding pattern clusters by diet were observed in the current experiment such that clusters were easily associated based on the type of grain and dietary enzyme supplementation. The microbial populations of the enzyme-supplemented treatments resembled each other, and a cluster was formed with approximately 50% similarity among the bacterial communities. In addition, a cluster of approximately 52% similarity was formed from the microbial population sampled from turkeys fed the diets that were not supplemented with enzyme. The Cs between treatment groups supported the cluster analysis. The highest Cs values were observed among the WC and CC diets and the enzyme-supplemented diets. The lowest Cs value was observed for the comparison between the CC diet and the WE diet. Therefore, NSP and enzyme supplementation presumably altered the intestinal community structure.
Dietary fiber is composed of NSP and lignin, which are resistant to hydrolysis by the endogenous digestive enzymes of man (Trowell et al., 1976). Nonstarch polysaccharides are the primary substrate for Lactobacillus and Bifidobacterium, which are important commensal bacteria of the GIT. Lan (2004) theorized that an increase in substrate availability increases overall microbial fermentation. However, the intestinal environment, which influences the proliferation of microbial communities, will be influenced by the source and level of dietary fiber (Hogberg, 2003).
A corn-SBM diet contains a low amount of NSP, which is largely insoluble and provides a limited amount of slowly fermentable substrate for microbial growth in the hindgut of poultry (Bach Knudsen, 1997, 2001; Hogberg, 2003). Low-NSP diets result in decreased passage rate through the proximal GIT and increased cecal contents, associated with a low cecal turnover rate (Fraga et al., 1991). Thus, the growth of organisms that are permanent members of the normal microbiota, such as Lactobacillus and Bifidobacterium, are promoted on this stagnated environment. However, transient bacteria such as Salmonella spp., which are present only under unusual environmental conditions, may also grow (Hogberg, 2003). The increase in transient microbes may be supported by the higher prevalence of Salmonella present in the toms fed corn-based diets compared with toms fed wheat-based diets. Furthermore, as indicated by the decrease in H' values of birds fed corn-SBM diets, the diversity of the resident flora decreased because of the limited amount of substrate.
A diet with a high NSP content and a large amount of insoluble NSP, as in a wheat-based diet, provides ample substrate to support increased microbial growth (Bach Knudsen et al., 1991). In the present research, all the diversity indexes were higher for wheat-based diets than for corn-based diets, which indicates an increased number of microbes in the overall community. Other researchers have also reported that fiber-containing diets increased the populations of bacteria in the GIT of different animals (Wise et al., 1986; Gestel et al., 1994; Le Blay et al., 1999) and increased the diversity of intestinal microbiota in mice (McCracken et al., 2001) and pigs (Hogberg, 2003).
The WC and CC diets resulted in a lower richness index compared with the enzyme-supplemented treatments, indicating that the 2 control diets supported a lower number of bacterial species. The richness of the WC treatment was low, probably because the majority of the NSP substrate available in this diet was insoluble fiber, characterized by low digestibility, a slow degradation rate (Bach Knudsen and Jorgensen, 2001), and an increased GIT passage rate (Kass et al., 1980; Bach Knudsen, 2001). Therefore, only a more specialized microbiota capable of fermenting the kind of substrate present in the wheat-based diets was probable able to grow (Hogberg, 2003). In contrast, the richness of the CC diets was possibly low because of strong competition for substrate (Hogberg, 2003). In this highly competitive ecosystem, only dominant microbial niches were able to grow, which also resulted in decreased microbial community evenness (EH value). Therefore, the diversity of the enteric microbial community in turkeys fed corn-SBM diets decreased because of decreases in both richness (number of species) and evenness (the distribution of individuals within each species in the community).
Dietary exogenous enzyme supplementation apparently increased the availability and variety of substrates that supported the growth of more diverse microbiota. Both enzyme preparations used in this study contained endoxylanase. Oligosaccharides of intermediate length, such as xylobiose, xylotriose, and xylopentaose, are products of the hydrolysis by endoxylanase (Uchino and Nakane, 1981; Akiba and Horikoshi, 1988; Bataillon et al., 2000), as used in this experiment. These xylooligosaccharides have been shown to be readily fermented by the GIT microbiota (Van Laere and et al., 1997), especially for Lactobacillus and Bifidobacterium (Van Laere et al., 1997; Hogberg and Lindberg, 2004). In agreement, in the current experiment enzyme supplementation increased the number of PCR-DGGE bands by approximately 27% as compared with the unsupplemented treatments. Similarly, Hogberg and Lindberg (2004) studied the influence of cereal NSP and enzyme supplementation on digestion and the gut environment and showed that the substrate for the growth of lactic acid bacteria (i.e., Lactobacillus) was released in the diet when enzyme was added. Apparently, dietary supplementation of NSP-hydrolyzing enzymes promoted growth of the microbiota through increased substrate availability in the hindgut.
The higher diversity index observed among the enzyme-supplemented treatment groups compared with the unsupplemented treatments supports the assumption that xylooligosaccharides released by endoxylanase stimulated the growth of a greater variety of bacterial species that efficiently hydrolyzed these substrates. Thus, the release of substrate by the enzyme not only increased some bacterial niches, but also allowed the overall microbiota to grow evenly and probably maintained a more stable enteric community. Similarly, Bartelt et al. (2002) measured the prececal digestibility of arabinoxylans in piglets and reported considerable digestibility of insoluble arabinoxylans, but not the entire soluble fiber fraction. They demonstrated that the digestibility of soluble and insoluble NSP increased with dietary xylanase supplementation. The authors suggested that this improved NSP digestibility might be a direct effect of the exogenous enzyme, but also of a stimulated degradation by supporting a specialized group of NSP-hydrolyzing bacteria.
Microbial community diversity seems to be an important condition to control Salmonella colonization of the turkey intestine. Turkeys fed the CC diet had a lower community diversity but a higher Salmonella prevalence than the birds fed the CE and wheat-based diets. In contrast, turkeys fed the wheat-based diets had a higher microbial community diversity but a lower Salmonella prevalence than those fed the corn-based diets. In addition, dietary enzyme supplementation increased the microbial diversity in turkeys fed either the corn- or wheat-based diets, and it decreased the presence of Salmonella in turkey toms as compared with those birds fed diets not supplemented with the enzyme. Therefore, these results support the hypothesis that diets with a high NSP content increase microbial community diversity and discourage Salmonella colonization, especially when the diet is supplemented with NSP-degrading enzymes.
As claimed by Ferket (1991), the stability of the microbiota population and its ability to cope with minor changes in the gut environment increase as the number of microbial species increases. An increase in community diversity increases the symbiotic relationship within the community, which helps maintain a dynamic intestinal ecosystem (Hentschel et al., 2000) that competitively excludes Salmonella. These symbiotic groups of microorganisms probably competitively excluded Salmonella by competing for available nutrients and maintaining their habitat by consuming and metabolizing substrate resources of the intestine (Roberfroid et al., 1995; Falk et al., 1998). In addition, the increased microbiota community diversity and associated dynamics of the intestinal ecosystem increased competitive exclusion against Salmonella as follows: 1) by competing for gut lining attachment (Roberfroid et al., 1995; Falk et al., 1998); 2) by producing bacteriocins and antimicrobial peptides (Hancock and Rozek, 2002; Joerger, 2003); 3) by stimulating the intestinal associated immune system through cell wall components (Nousiainen and Setala, 1998); and 4) by increasing the production of short-chain fatty acids, which have bacteriostatic and bactericidal properties (Fuller, 1977), and by stimulating intraepithelial lymphocytes and natural killer cells that enhance the host’s immunological defense mechanisms (Ishizuka and Tanaka, 2002; Ishizuka et al., 2004; Lan, 2004).
In theory, the high amount of readily fermentable substrate from NSP-rich diets, especially when supplemental enzymes are used to facilitate NSP hydrolysis, would encourage transient microbes such as Salmonella because the microbial competition for substrate is diminished. However, birds fed wheat-based diets had a lower Salmonella prevalence as compared with those fed the corn-based diets, especially when supplemented with enzyme. The mechanism by which NSP acts as a prebiotic compound (selectively metabolized by beneficial members of the intestinal microbiota) is not completely understood at the present time. It is presumed that symbiotic bacteria are able to produce prebiotic-hydrolyzing enzymes, whereas transient microbes express very low enzyme activity (Rastall et al., 2005). Although, this hypothesis has not been tested in this study, the current experimental results suggest that Salmonella colonization is inhibited as commensal microbiota proliferation is promoted by dietary NSP and endoxylanase supplementation.
The PCR-DGGE technique has been used to evaluate dietary effects on changes in the microbiota profile of chickens (Hume et al., 2003) and turkeys (Waters et al., 2005), but it has some limitations. Polymerase chain reaction-DGGE provides a convenient method to evaluate entire microbial ecosystems and it allows the analysis of a large number of samples, but this technique is most useful for determining shifts in predominant microbial populations (McCracken et al., 2001). Microbial populations constituting less than 9% of the total intestinal microbial ecosystem are not detected (Zoetendal et al., 1998). Moreover, the apparent community diversity determined by PCR-DGGE may be lower than expected because different bacterial species, possessing similar G + C content of the 16S rDNA gene, may be represented in the same PCR-DGGE band (Palys et al., 1997; Muyzer, 1999). Consequently, the number of PCR-DGGE bands is generally lower than the number of bacterial species detectable by cultivation-based methods and direct cloning strategies (Zoetendal et al., 1998; Muyzer, 1999; Simpson et al., 1999; Lesser et al., 2000). These limitations may account in part for the decreased band number in the present study. Analysis of the turkey ileal bacterial community in the current study identified 41 distinct bands when combining distinct PCR-DGGE bands of all samples. There was a maximum of 21 bands within a single sample. These results are consistent with those of other authors who observed 38 distinct bands from fecal samples of humans (Zoetendal et al., 1998), 35 bands from fecal samples of pigs (Simpson et al., 2000), 32 bands from fecal samples of mice (McCracken et al., 2001), and 41 bands from cecal samples of turkeys (Waters et al., 2005).
This study demonstrated the utility of PCR-DGGE analysis for monitoring diet- and enzyme-induced alterations of the complex intestinal microbial ecosystem and associating these changes with Salmonella infection in turkeys. This cultivation-independent technique is less time-consuming and less labor-intensive than traditional microbiological techniques. It can also be used to evaluate the effect of other dietary treatments, drug treatments, or disease conditions on intestinal microbial populations. The results of the current study confirm that PCR-DGGE is a useful tool to study shifts in the gastrointestinal microbiota of birds (Hume et al., 2003; Waters et al., 2005).
In conclusion, the total microbial population as well as the diversity of the bacterial community was influenced by dietary NSP and enzyme supplementation. The NSP content of cereal grain-based diets (i.e., wheat, triticale, and rye) and enzyme dietary supplementation increased microbial community diversity and discouraged Salmonella colonization in the turkey intestine. Thus, NSP and dietary exogenous enzyme supplementation may be a practical tool to control enteric pathogens and benefit intestinal health and food safety.
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ACKNOWLEDGMENTS |
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Received for publication December 26, 2006. Accepted for publication March 6, 2008.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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