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J APPL POULT RES 2007. 16:563-573. doi:10.3382/japr.2006-00105
© 2007 Poultry Science Association
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Research Reports

Determination of Ileum Microbial Diversity of Broilers Fed Triticale- or Corn-Based Diets and Colonized by Salmonella1

F. B. O. Santos*, B. W. Sheldon*,2, A. A. Santos, Jr.*, P. R. Ferket*, M. D. Lee{dagger}, A. Petroso{dagger} and D. Smith{dagger}

* Department of Poultry Science, College of Agriculture and Life Sciences, North Carolina State University, Raleigh 27695; and {dagger} Department of Avian Medicine, College of Veterinary Medicine, University of Georgia, Athens 30602

Correspondence: 2 Corresponding author: brian_sheldon{at}ncsu.edu


   SUMMARY
TOP
 SUMMARY
DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
Diversity of the bacterial communities in the ileum of broilers was characterized using denaturing gradient gel electrophoresis. Denaturing gradient gel electrophoresis separation of polymerase chain reaction amplicons of the V2–V3 variable regions of the 16S rDNA is a common method to profile community diversity and has been used to assess the effects of diet and antibiotics on the ileal bacterial community of chickens. Broilers raised either on litter floor or in cage batteries were fed either a finely ground corn- (control), a finely ground triticale-, or a whole triticale-based diet from 0 to 42 d. Microbial DNA was extracted from the ileum content of 42-d-old broilers, and the 16S rDNA gene was amplified by polymerase chain reaction and the amplicons separated by denaturing gradient gel electrophoresis. Diversity indexes including richness, evenness, diversity, and pairwise similarity coefficients were calculated. Diversity indexes were related to the dietary treatments, housing designs, and to changes in Salmonella colonization of broiler ceca as characterized by the most probable number method. Higher microbial diversity indexes were observed among birds fed whole triticale-based diets and reared on litter floors. In contrast, finely ground grain treatments had lower diversity and higher Salmonella prevalence than the whole triticale treatment. The data indicated that combination of high dietary fiber content and increased coarseness of the diet by feeding whole triticale stimulated microbial community diversity and discouraged Salmonella colonization, perhaps through a competitive exclusion-type mechanism.

Key Words: Salmonella • broiler • microbial diversity • triticale • whole grain • fiber


   DESCRIPTION OF PROBLEM
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
The intestinal flora generally affects the health of the host by influencing digestion and nutrient absorption, intestinal morphology, and defense of the host against infection [1, 2]. Through different inhibitory effects, which include competition for nutrients [3], production of antimicrobial substances such as bacteriocins [4], and physical binding to the surface of the intestinal epithelium, the resident microflora can prevent opportunistic pathogens from obtaining an attachment site along the intestinal mucosa [5]. The composition and diversity of the microbial community of the avian intestinal tract can be influenced by many factors including bird age [6, 7], intestinal infections [8], and diet [6, 9].

Promoting the development of the beneficial bacteria within the avian intestinal tract could help to reduce foodborne pathogen colonization, which would reduce human exposure to these pathogenic organisms and related illness and deaths [10]. Several alternative products intended to control Salmonella in poultry have been developed in response to the anticipated reduction in use of antibiotics as growth promoters. Nutritional strategies that have been used in the poultry industry to promote intestinal health and to increase the resistance to pathogen colonization include the use of probiotics [9], prebiotics [11], and enzyme supplementation [12]. Netherwood et al. [9] monitored the response of the avian intestinal bacterial microflora to probiotic administration and detected a shift in the composition of the microflora after probiotic use. Prebiotics, which are carbohydrates or other organic compounds that are not digestible by the host animal but digestible by specific microbial populations of the intestinal tract [10], have also been used to promote intestinal health of poultry [11].

Cereals such as wheat and triticale are rich in nonstarch polysaccharides (NSP; dietary fiber) that comprise a major part of dietary CF. Non-starch polysaccharides are a heterogeneous group of polysaccharides having varying degrees of water solubility, size, and structure and are not digested by the avian digestive tract [13]. Nonstarch polysaccharides have been used for many years in poultry diets as dietary fiber, and, more recently, they have been evaluated as potential prebiotics [12].

An alternative strategy for pathogen control and growth promotion is the rearing of broilers on nonlitter systems. The Farmer Automatic Broilermatic cage system is an example of a nonlitter system. The system is a cage facility that allows broilers to be raised on a surface nearly free of feces, yet avoiding many of the problems associated with standard cage systems such as breast blisters, folliculitis, and wing and leg breakage-downgrade problems [14].

The study reported herein was designed to evaluate the effects of grain, particle size, and type of housing system on the intestinal microbial diversity and Salmonella colonization in broilers. Changes in ileal bacterial populations of broilers fed triticale- or corn-based diets were investigated by the separation of 16S rDNA polymerase chain reaction (PCR) amplicons by denaturing gradient gel electrophoresis (DGGE). In addition, the changes in the intestinal microbial community diversity characterized by PCR-DGGE were related to changes in Salmonella fecal and cecal populations of turkeys.


   MATERIALS AND METHODS
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
Bird Husbandry
One thousand nine hundred twenty 1-d-old Ross 508 [15] broiler chickens were weighed, neck-tagged, and orally gavaged with 1 mL (8 x 105 colony forming units; cfu) of a cocktail of 4 serovars of Salmonella enterica subspecies enterica before being randomly assigned to 1 of 2 experimental housing designs, a conventional litter-floored house, or the Broilermatic system [16], a nonlitter cage-based design [14]. The bedding material used for the litter-floored house was composed of fresh pine shavings. In each house, broilers were assigned to 1 of 3 dietary treatments consisting of either finely ground corn for treatment 1 (C, control) or finely ground or whole triticale for treatments 2 (T) and 3 (WT), respectively. Birds were kept on a 24 h/d light schedule in both houses. Feed and water were provided ad libitum. Broilers were inspected daily and birds with visual health problems were removed and humanely killed.

Experimental Design and Diets
The experimental design consisted of 3 dietary treatments (C, T, WT), each with 8 replicate pens or cages of 40 broiler chickens per housing design. All broilers were fed either a corn- or a triticale-soybean meal-based diet over the entire experimental period (1 to 42 d). Feed was offered in crumble form from 1 to 14 d of age (starter diet) and in pellet form from 15 to 42 d (grower and finisher diets; Table 1Go). The experimental diets were formulated using least-cost linear programming to meet or exceed the NRC [17] nutrient requirements. The corn was supplied and ground by Southern States Feed Mill (Siler City, NC), resulting in a final average particle size of 560 µm. The triticale [18] was supplied by Resource Seeds (Gilroy, CA) and ground at the North Carolina State University feed mill (Raleigh) in a hammermill [19] equipped with a 3-mm screen to produce a final average particle size of 560 µm. The third experimental diet was prepared using the whole triticale grain. Average particle size of the pelleted diets was 3.39, 4.25, and 2.99 mm in diameter for C, T, and WT, respectively. As recommended by the supplier, triticale-based diets were supplemented with Avizyme 1502 [20], which provided 600 endo-1,4-ß-xylanase units (EXU; EC 3.2.1.8) per kilogram of feed [21]. The enzyme preparation also contained standardized activities of at least 8,000 units of subtilisin (EC 3.2.1.8) and 800 units of (– amylase (EC 3.4.21.6 [EC] 2) per gram of product. The feed did not contain any antimicrobials or coccidiostats.


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  		Table 1. Composition and nutrient content of the experimental diets containing different particle sizes of corn and triticale fed to broilers from 1 to 42 d of age
 
Inoculum Preparation and Salmonella Enumeration
A cocktail of Salmonella enterica subspecies enterica serotypes Typhimurium (ATCC 700408), Newport (ATCC 6962), Heidelberg (ATCC 8326), and Kentucky (field serovar previously isolated from turkey feces and serotyped by the National Veterinary Service Laboratories [22]) was used as the inoculum. For the purpose of this document, serovars of Salmonella enterica subspecies enterica will be called Salmonella accompanied by the serovar name (e.g., Salmonella Typhimurium or Salmonella Typhimurium). For preparation of the inoculum, the 4 serovars were grown separately overnight at 37° C in brain-heart infusion broth [23]. The cultures were then mixed together and serially diluted in buffered peptone water [23] to an estimated final concentration of 106 cfu/mL. The cell count was determined by direct plating on brain-heart infusion agar plates. Plates were incubated overnight at 37° C. Inoculum concentration was confirmed to be 8 x 105 cfu/mL after plate count. Negative controls were used for all plating procedures to ensure that the media had been properly sterilized.

At 42 d of age, 8 birds per dietary treatment per house were euthanized by cervical dislocation, and the ceca were aseptically removed, weighed, and stored on ice for circa 1 h before being quantitatively cultivated for Salmonella isolation. Cultivation of Salmonella was performed immediately after sampling using the most probable number (MPN) procedure as previously described [24]. Populations of Salmonella for each sample were determined using Thomas’ approximation [25].

Bacterial DNA Isolation and PCR-DGGE Analysis
Fifteen centimeters of the distal section of the ileum (above the ileocecal-colonic junction) were aseptically removed from the same birds used for Salmonella enumeration and frozen at – 20° C until further analysis.

To isolate bacterial DNA, ileum samples were first defrosted and their contents removed and pooled. Each pool was composed of the ileal contents of 4 birds per treatment per house. Bacteria were subsequently collected from the pooled sample using differential centrifugation [6]. Community DNA from the bacterial suspensions was extracted using a ballistic lysis method [6]. Briefly, lysed cells were treated with sodium dodecyl sulfate (final concentration, 0.5%) and proteinase K (final concentration, 0.1 mg/mL) and incubated at 37° C for 30 min. The sample was extracted twice with an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) and once with chloroform-isoamyl alcohol (24:1). Deoxyribonucleic acid was concentrated with a 0.6 volume of isopropanol and resuspended in 50 µ L of sterile water. Deoxyribonucleic acid concentrations were measured with a Beckman DU640 spectrophotometer [26]. For PCR amplification, each DNA sample was amplified using primers HDA1-GC and HDA2 as described by Walter et al. [27]. Diversity of the communities was characterized by separation of PCR amplicons using DGGE analysis as described by Knarreborg et al. [28] with some modifications. Denaturing gradient gel electrophoresis was performed using a 16 cm x 16 cm x 1 mm 6% polyacrylamide gel (ratio of acrylamide to bisacrylamide, 37.5:1) containing a 15 to 55% gradient of urea and formamide. Amplicons were separated by electrophoresis at 200 V for 3 h, and gels were stained using Sybr Green [29]. To compare gels run in different trials, samples were repeated on subsequent gels allowing alignment of the gels using band patterns from the repeated samples.

Examination of DGGE Gels
Examination of DGGE gels was based on methods previously described by McCracken et al. [30], Konstantinov et al. [31], and Høj et al. [32]. Gels were compared using the BioNumerics software [33] as follows. The number of bands per lane was assessed using a 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 constituting 1% or more of the total area of all bands were considered as dominant DGGE bands and included in the analysis. Subsequently, band migration distance and intensity of the bands within each lane of the gel were measured [34]. This information was then used to calculate several measures of microbial ecology including number of bands (microbial community richness; S), Shannon’s equitability (microbial community evenness; EH [35]), and Shannon’s index (microbial community diversity, H' [36]) [37, 38, 39]. In the description of the indexes that follows, species refers to individual bands on the DGGE gels. However, because the bands on the DGGE gels correspond to the percentage of G + C content within the melting domains for the V2 and V3 PCR amplicons, bacterial species with similar G + C content in the amplified V2 and V3 region may form assemblages and appear as a single band [40].

Band surface area corresponds to the optical density measurements of each band. The optical densities were measured based on the plotted band intensity and migration distance. Each band formed a peak relative to its intensity and migration distance, and the area underneath the peaks was measured using the BioNumerics software. Sorenson’s similarity index or coefficient of similarity index (CS) [41] was used to compare average percentage similarities of DGGE banding patterns within each treatment group (intragroup comparison) and between treatment groups (intergroup comparison).

The similarity between the DGGE profiles was determined by calculating the band similarity coefficient (SD) [42]. Cophenetic correlation coefficients were calculated and are represented on the root of each cluster in the dendrogram (relatedness tree). This parameter is used to express the consistency of a cluster and represents the correlation between distance values calculated during tree building and the observed distance. Clusters (groups) were determined by sequential comparison of band patterns, and the results (relative similarities) are represented in the dendrogram.

Statistical Analysis
All data were analyzed using the GLM procedure for ANOVA of SAS [43, 44]. Before statistical analysis, all MPN data were transformed to the base-10 logarithm, and replicate pens or cages of 40 birds each served as experimental units. For microbial diversity indexes, pooled samples served as the experimental units for statistical analysis. The DGGE patterns were compared using BioNumerics software. Cluster analysis was conducted using the dice coefficient [42] for band matching with 1% position tolerance and the unweighted pair group method with arithmetic means to generate the dendrogram, which describes the relationship between bacterial communities among dietary treatments and house designs.

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 and euthanasia practices were performed with full consideration of animal welfare.


   RESULTS AND DISCUSSION
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
Because corn and triticale differ considerably in their NSP content [12], it was possible to evaluate the effect of varying levels of dietary NSP on intestinal microbial ecology. On average, the difference between these 2 finisher diets was 43% in total dietary fiber, 16% of cellulose-lignin content, and 90% of hemicellulose content. Several researchers have demonstrated that an increase in dietary hemicellulose (also known as NSP) from 2 to 40 g/kg can change animal performance, intestinal viscosity, nutrient digestibility, and microbial community structure [45, 46, 47].

As hypothesized, distinct clusters (Figure 1Go) were clearly distinguished based on diet, specifically with respect to type of grain. Microbial composition within the triticalefed broilers was similar. Similarly, a cluster of about 60% relatedness was detected by the microbial populations derived from the corn-fed birds. Cophenetic correlation values were estimated by Bio-Numerics software, and the values are shown in the dendrogram (Figure 1Go). The cophenetic value for the whole dendrogram (89%) indicates that the dendrogram did not distort the original structure in the input data [48].


Figure 1
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  		Figure 1. Cluster analysis of the denaturing gradient gel electrophoresis profiles of microbial populations present in the ileum contents of 42-d broilers fed finely ground corn (C) and finely ground or whole triticale (T and WT, respectively)-based diets and reared in a litter floor house ({star}) or in the Broilermatic nonlitter cage system (*). The dendrogram was band-based and was constructed using the unweighted pair group method with arithmatic means using the BioNumerics software [33]. Cophenetic correlation coefficients are represented on the root of each cluster in the dendrogram (relatedness tree). Similarity between samples is indicated by the percentage coefficient bar located above the dendrogram. Numbers accompanying the dietary treatments (C, T, and WT) represent the replicate samples (4 replicates/treatment). Each sample represents a pool of ileum contents of 4 birds. Distances are measured in arbitrary units.

 
The cluster analysis results were supported by the Sorenson’s CS analysis. The comparison was based on the average number of bands in common within each treatment group (Figure 2aGo) and between treatment groups (Figure 2bGo). The CS among samples within each treatment group and between treatment groups were significantly different (P < 0.05, Figure 2Go). Statistical differences in similarities within dietary treatments showed that the similarities between samples within each dietary treatment were higher for treatments C and WT (60 and 63%, respectively) than T (31%). The CS between treatment groups revealed that the highest value was observed among the triticale-based diets (T-WT, CS = 33%, P = 0.019). The lowest Cs value was observed in the comparison between the corn-control diet and whole triticale (C-WT, CS = 13.2%, P = 0.019). Therefore, grain type and coarseness altered the intestinal community structure probably by increasing the levels of microbial competition in the intestinal tract for the birds fed whole triticale.


Figure 2
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  		Figure 2. Percentage of similarities of denaturing gradient gel electrophoresis banding patterns from bacterial DNA of the ileum contents of 42-d broilers fed finely ground corn (C) and finely ground or whole triticale (T and WT, respectively)-based diets. Sorenson’s similarity index (% similarities, y-axis) is based on the average number of bands in common within each dietary treatment (a) and between dietary treatments (b) across housing designs. Values represent means from each group of comparison (dietary treatments, x-axis). Values sharing different letters within the charts are statistically different (P < 0.05).

 
Depending on housing design, the microbial community richness and diversity (Table 2Go) indexes were affected by the dietary treatments (diet x housing interaction, P < 0.05). Birds raised on litter were influenced by dietary treatments, whereas no differences were observed between dietary treatments for broilers raised in cages (P > 0.05). Additionally, evenness, total band surface area, and average band surface area were not affected by diet or housing design (Table 3Go, P > 0.05). Generally, the most significant contrasts observed in this study were between birds fed the whole triticale-based diets and those fed the finely ground grain (regardless of grain type) diets in the litter-reared broilers. Feeding whole triticale significantly increased richness by 71% as compared with feeding the finely ground grain diets (9.0 vs. 5.3 bands, P < 0.05, Table 2Go) when broilers were reared on litter. However, species evenness was not affected by the dietary treatments (Table 3Go). Therefore, as a result of increased microbial community richness, diversity increased by 32% (P = 0.04) when broilers were reared on litter and fed the whole triticale-based diet (2.05 vs. 1.55, Table 2Go).


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  		Table 2. Species richness and diversity of microbial populations present in the ileum contents of broilers fed corn-or triticale-soybean meal diets and raised in a conventional litter floored house or in the Broilermatic system at 42 d of age
 

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  		Table 3. Species evenness of microbial populations, average and total band surface area plots of denaturing gradient gel electrophoresis bands from bacterial DNA present in the ileum contents of broilers fed corn- or triticale-soybean meal diets and raised in a conventional litter-floored house or in the Broilermatic system at 42 d of age
 
The cecal Salmonella population data (Table 4Go) support the intestinal microbial diversity findings. At d 42, contrast analysis showed that broilers fed whole triticale had significantly lower Salmonella populations than birds fed finely ground grain (3.5 vs. 4.4 log MPN/g, P = 0.0379). The data suggest that whole grain feeding stimulated microbial community diversity, resulting in a higher level of microbial competition in the intestinal tract, which perhaps discouraged Salmonella colonization. Similar results have been reported by Engberg and coworkers [49], who demonstrated that whole wheat feeding reduced the number of enterococci and lactose-negative enterobacteria in the small intestine of broilers as compared with feeding pellets made with finely ground grain.


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  		Table 4. Cecal Salmonella population of broilers fed corn- or triticale-soybean meal diets and raised in a conventional litter-floored house or in the Broilermatic system at 42 d of age
 
The absence of any beneficial effect from feeding the finely ground triticale-based diet on microbial diversity indexes may be associated with low exogenous enzyme activity in the diet. Recently, Santos et al. [50] reported that supplementation of endoxylanase to wheat-based diets at 5,500 EXU/kg of feed yielded a significantly better turkey performance than did the supplementation at 2,250 EXU/kg of feed. Some investigators have attributed the lack of response to the use of inappropriate enzymes for the type of grain [51]. Thus, the data suggest that the concentration of added enzyme to the triticale-based diets was insufficient to completely degrade the insoluble NSP molecules present.

Furthermore, the microbial community diversity of birds fed whole triticale-based diets may have been influenced by the presence of higher concentrations of endogenous glycanases. The incorporation of whole grains into pelleted broiler diets has been shown to increase the level of grain endogenous enzymes helping in the digestive processes [52]. Therefore, it is feasible that the presence of endogenous enzymes in the whole triticale-based diet complemented the exogenous-supplemented endoxylanase, resulting in the growth of different bacterial species and therefore greater microbial diversity.

The PCR-DGGE technique has been previously used to evaluate dietary effects on changes in the microbial profile of chickens [6, 53]. However, the method does have some limitations. Although DGGE separation of 16S variable sequences provides a convenient method to evaluate entire microbial ecosystems in many samples [30], multiple bands of the same bacterial species can be present in the analysis, especially when determining shifts in predominant microbial populations [54]. Buchan and coworkers [55] demonstrated that DGGE banding patterns of environmental Escherichia coli isolates obtained from different sources, including poultry, had multiple banding patterns indicating a high degree of diversity. It is possible that multiple bands representing the Salmonella species were present in the samples having elevated populations of this pathogen, which would have resulted in a measure of increased richness of the samples.

The difference in Salmonella populations between the 2 housing systems was 0.53 log or about 11,000 more cells/g in the intestinal samples taken from birds reared in the Broilermatic system. Furthermore, the richness of the flora of cage-reared broilers (regardless of dietary treatment) was equivalent to that of litter-reared broilers fed whole triticale (8.5 vs. 9.0 bands). Therefore, the increase in richness of cage-reared broilers may be partially attributed to the higher Salmonella populations observed in these birds. Similarly, the observed microbial richness and diversity detected in corn-fed birds raised on litter may have been caused by higher Salmonella populations.

In conclusion, different intestinal microbial population profiles were observed among the dietary treatments, especially among broilers reared on litter. Rearing broilers on litter and feeding whole triticale encouraged the growth of a greater variety of bacterial species and consequently a more diverse microbial community in the intestinal tract. Conversely, the diversity of intestinal microflora of broilers raised in cages was not influenced by the dietary treatments. Litter-reared broilers fed finely ground grain diets, regardless of grain type, had lower microbial community diversity but higher Salmonella populations than did those fed whole triticale. Thus, microbial community diversity seems to be influenced by the coarseness of the grain, which may be an important factor affecting Salmonella colonization of the broiler intestine. Therefore, feeding whole high-NSP content cereals, such as triticale, may be a useful approach to control enteric pathogens in poultry with the added benefit of improved intestinal health and food safety.


   CONCLUSIONS AND APPLICATIONS
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
REFERENCES AND NOTES
 

  1. Broilers reared in a conventional litter house and fed whole triticale had a more diverse intestinal microflora than those fed the finely ground grain, regardless of grain type.
  2. Rearing broilers in a litter house and feeding whole triticale increased microbial community diversity and discouraged Salmonella colonization, resulting in reduced cecal Salmonella populations. Therefore, microbial community diversity may be an important condition affecting Salmonella colonization of the broiler intestine.
  3. Feeding whole high-NSP content cereals may be a useful approach to control enteric pathogens by improving intestinal health of poultry and benefiting food safety.


   ACKNOWLEDGMENTS
 
This study was supported by the USDA Initiative for Future Agriculture and Food Systems grant. We wish to thank Annette Israel, Jamie Warner, Jean de Oliveira, Ondulla Foye, Renee Plunske, Mike Mann, Robert Neely, and the North Carolina State University Poultry Educational Unit farm employees, Raleigh, for their technical assistance during this study. Appreciation is also extended to Southern States Feed Mill (Farmville, NC) for providing and grinding the corn used in the experimental diets, to Resource Seeds Inc. (Golroy, CA) for providing the triticale used in the experimental diets, and to Sophia Kathariou and Robin Siletzky from the Food Science Department, North Carolina State University, Raleigh, for technical assistance and equipment support with the BioNumerics software.


   FOOTNOTES
 
1 Use of trade names in this publication does not imply endorsement by the North Carolina Agriculture Research Service or the North Carolina Cooperative Extension Service of the products mentioned nor criticism of similar products not mentioned. Back


   REFERENCES AND NOTES
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 

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  35. EH index. Community evenness was calculated using the Shannon’s equitability index based on the formula: EH = H' /lnS, where H' = the Shannon’s index and lnS = the natural log of the total number of species in the community (total number of bands in the DGGE gel, richness) [56].
  36. H' index. The Shannon index was calculated using the following function: H' = – {sum}PilogPi, where Pi = the proportion of individuals in the population belonging to the ith species, thus Pi corresponds to the proportional abundance of band i. Therefore, H' values were calculated based on the intensity of each band as measured by the peak height of the band in the densitometric curves. The importance probability, Pi, was calculated as: Pi = ni/T, where ni = the peak height and T = the sum of all peak heights in the densitometric curve for a specific lane.
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  41. CS index. Calculations for the Sorenson’s similarity index are based on the formula: CS = [2j/(a + b)] x 100, where a = the number of bands in lane 1, b = the number of bands in lane 2, and j = the number of common bands between lanes 1 and 2. The CS values of 100% indicate that DGGE profiles are identical, whereas CS values of 0% indicate that the DGGE profiles are different [56].
  42. Band similarity coefficient. Dice: SD = 2nAB/(nA + nB), where nA = the number of bands in line 1, nB = the number of bands in lane 2, and nAB = the number of common bands.
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  44. Statistical analysis. All data were analyzed using the general linear models procedure for ANOVA of SAS [43] according to the following model: Yijk = µ + {delta} i + {tau}j + ({delta} {tau})ij + Eijk, where Yijk = the observed dependent variable (microbial diversity indexes or Salmonella population), µ = overall mean, {delta} i = house design effect, {tau}j = dietary treatment effect, ({delta} {tau})ij = interaction between house and dietary treatment effect, and Eijk = random error. Variables having a significant F-test were compared using the least squares means (LSMEANS) function of SAS [43] and were considered to be significant at P < 0.05 unless otherwise stated.
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  58. IKA Werke Labortechnik, Staufen, Germany.
  59. Labconco Corporation, Kansas City, MO.
  60. Sybron Corporation, Dubuque, IA.




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