Poult. Sci.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Poult Sci 2007. 86:1633-1639
© 2007 Poultry Science Association
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dunkley, K. D.
Right arrow Articles by Ricke, S. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dunkley, K. D.
Right arrow Articles by Ricke, S. C.

ENVIRONMENT, WELL-BEING, AND BEHAVIOR

Molting in Salmonella Enteritidis-Challenged Laying Hens Fed Alfalfa Crumbles. I. Salmonella Enteritidis Colonization and Virulence Gene hilA Response

K. D. Dunkley*, J. L. McReynolds*,{dagger},1, M. E. Hume{dagger}, C. S. Dunkley*,2, T. R. Callaway{dagger}, L. F. Kubena{dagger}, D. J. Nisbet{dagger} and S. C. Ricke*,3

* Department of Poultry Science, Texas A&M University, College Station 77843; and {dagger} Southern Plains Agricultural Research Center, Food and Feed Safety Research Unit, Agricultural Research Service, USDA, College Station, TX 77845

1 Corresponding author: mcreynolds{at}ffsru.tamu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The objectives of this study were to enumerate Salmonella enterica serovar Enteritidis colonization in fecal, cecal, and internal organs, and to compare the level of virulence gene expression (hilA) of experimentally challenged laying hens fed different dietary molt-induction regimens. Twelve Salmonella-free Single Comb Leghorn hens (>50 wk old) hens were randomly assigned to each of 6 treatment groups designated based on diet in 2 trials: 1) feed withdrawal Salmonella Enteritidis-positive (FW+), 2) fully fed Salmonella Enteritidis-positive (FF+), 3) 100% alfalfa crumble Salmonella Enteritidis-positive (ALC+), 4) feed withdrawal Salmonella Enteritidis-negative, 5) fully fed Salmonella Enteritidis-negative, and 6) 100% alfalfa crumble Salmonella Enteritidis-negative. A forced molt was induced by a 12-d alfalfa diet and a feed-withdrawal regimen. On d 4 of the molt, all hens in groups 1, 2, and 3 were challenged by crop gavage with 1 mL of inocula containing approximately 106 cfu of nalidixic acid- and novobiocin-resistant Salmonella Enteritidis (phage type 13A). At the conclusion of both trials, all hens were euthanized and Salmonella Enteritidis colonization was enumerated in the cecal contents, liver, spleen, and ovaries. In addition, fecal (d 4 and 8) and cecal samples (necropsy at d 12) were collected postchallenge from treatment groups 1, 2, and 3 (Salmonella Enteritidis-positive) to quantify hilA expression by PCR. In both trials, all nonchallenged birds were Salmonella Enteritidis-negative; therefore, no further analysis was done. In trial 1, a 2-fold reduction in Salmonella Enteritidis colonization was observed in the ALC+ hens (log10 Salmonella Enteritidis of 1.99) compared with the FW+ hens (log10 Salmonella Enteritidis of 3.89). In trial 2, a 4-fold reduction in Salmonella Enteritidis colonization was observed in the ALC+ hens (log10 Salmonella Enteritidis of 1.27) compared with the FW+ hens (log10 Salmonella Enteritidis of 5.12). In trial 2, Salmonella Enteritidis colonization in spleens was higher (P ≤ 0.05) in FW+ hens compared with ALC+ and FF+ hens. Relative expression of hilA was higher (P ≤ 0.05) in FW+ compared with FF+ hens, whereas the FF+ and ALC+ groups were not different (P > 0.05). In trial 2, hilA expression in FW+ hens was higher (P ≤ 0.05) for d 6, 11, and 12, respectively, when compared with ALC+ and FF+ hens. The results of these studies support the concept that changes in the gastrointestinal tract microenvironment, such as those created during feed deprivation, encourage Salmonella Enteritidis virulence and susceptibility in molted hens.

Key Words: Salmonella • hilA • alfalfa • molting • virulence


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
To induce molting, feed withdrawal has been the primary method of choice to achieve a new egg-laying cycle rapidly and economically (Brake, 1993; Holt, 1995). However, feed withdrawal compromises the birds’ immune system, making them susceptible to infection by a number of microorganisms, including Salmonella enterica serovar Enteritidis (Holt, 2003). Feed withdrawal-associated stress causes increased susceptibility to Salmonella Enteritidis infection in the gastrointestinal (GI) tract (Holt, 1993, 2003; Corrier et al., 1997; Durant et al., 1999; Ricke, 2003), usually marked by increased intestinal shedding and colonization in the internal organs, such as the liver, spleen, and ovaries (Holt, 1993; Thiagarajan et al., 1994; Holt et al., 1995). The genes required for Salmonella pathogenesis and subsequent infection are located on Salmonella pathogenicity island 1, at centisome 63 on the chromosome (Galán and Sansonetti, 1996). The transcriptional activator HilA, encoded by Salmonella pathogenicity island 1, coordinately regulates the expression of invasion genes in Salmonella in response to environmental conditions, including stress and low nutrient concentrations (Bajaj et al., 1996). In vitro experiments have indicated a positive relationship between increased hilA expression and potential for increased Salmonella Enteritidis infection in hens during feed withdrawal (Durant et al., 1999, 2000). Therefore, evaluation of hilA expression could be an important consideration for estimating the potential susceptibility of hens to Salmonella Enteritidis colonization and infection.

Alternative molting methods are known to reduce Salmonella Enteritidis invasion in the GI tract of experimental hens (Seo et al., 2001; Holt, 2003; Ricke, 2003; Moore et al., 2004; Park et al., 2004a,b; Ricke et al. 2004; McReynolds et al., 2005, 2006; Woodward et al., 2005). Fermentable high-fiber diets such as alfalfa meal have been examined as potential molting approaches that retain normal microbial flora and reduce proliferation of Salmonella Enteritidis (Woodward et al., 2005). Alfalfa meal and pellets, alone or in combination with a layer ration, have been shown to be capable of causing ovarian regression during molting and restoration of optimal postmolt egg production comparable to feed withdrawal-induced molt (Donalson et al., 2005; Landers et al., 2005a,b). The use of alfalfa meal as a single dietary source appears to support microbial fermentation in the chicken ceca sufficient to restrict Salmonella Enteritidis colonization, but inconsistent intake can be a problem (Woodward et al., 2005). The use of crumble and pelleted physical forms of feeds has been examined as a means to increase the feed-conversion ratios of birds (Kilburn and Edwards, 2001). Furthermore, Nir et al. (1994) noted that crumble feed is more suitable in the development of the chicken digestive tract compared with mash, which is uniform in particle size. The objectives of this study were to enumerate Salmonella Enteritidis colonization in fecal, cecal, and internal organs and compare the level of virulence gene expression (hilA) of experimentally challenged laying hens fed an alfalfa crumble diet, with hens either undergoing feed withdrawal or fed a layer ration.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Experimental Design
Experiments 1 and 2 were conducted with Single Comb White Leghorn hens, >50 wk of age, obtained from a local commercial laying flock. The hens were placed in wire layer cages and given free access to water and an unmedicated corn-soybean meal-based mashed layer ration that met NRC (1994) recommendations for nutrients. The alfalfa crumble diet used in the study was considered high in crude fiber (24 to 25%), moderate in CP (17 to 18%), and low in ME (1,200 kcal/kg; NRC, 1994). Feed samples and fecal samples (1 g) were collected and examined for Salmonellae. Samples were cultured in tetrathionate broth (Becton, Dickinson and Company, Sparks, MD) and on brilliant green agar (BGA) plates (Becton, Dickinson and Company) as previously described by Andrews et al. (1995). All hens and feed used in both trials tested negative for Salmonella.

Trials 1 and 2
The hens were allowed to acclimate to the cages for 2 wk, then were exposed to an 8L:16D photoperiod for 1 wk before changing the diets or, for hens in the feed-withdrawal group (FW), removing feed. This light schedule continued for a 12-d period, after which the experiment was terminated. Twelve hens were randomly assigned to 6 treatment groups, designated as follows: 1) feed withdrawal Salmonella Enteritidis-positive (FW+), 2) fully fed Salmonella Enteritidis-positive (FF+), 3) 100% alfalfa crumble Salmonella Enteritidis-positive (ALC+), 4) feed withdrawal Salmonella Enteritidis-negative (FW–), 5) fully fed Salmonella Enteritidis-negative (FF–), and 6) 100% alfalfa crumble Salmonella Enteritidis-negative (ALC–). Treatment diets were applied to each treatment on d 1 of the molt at the same time feed was removed from hens in the FW group. Treatment diets were administered for 12 d to coincide with the time period that hens in the FW group were deprived of feed. Hens in all the treatment groups were provided water ad libitum. On d 4 of the molt, all hens in groups 1, 2, and 3 were challenged by crop gavage with 1 mL of inocula containing approximately 106 cfu of nalidixic acid (NA)- and novobiocin (NO)-resistant Salmonella Enteritidis (Sigma Aldrich Co., St. Louis, MO). Groups, 4, 5, and 6 were not challenged with Salmonella Enteritidis. The Salmonella Enteritidis-positive and Salmonella Enteritidis-negative hens were placed in separate rooms. Footbaths were located at the doors of all the rooms in the facilities and noninfected hens were cared for before the infected hens each day. At the conclusion of both trials, all hens were euthanized and Salmonella Enteritidis colonization was enumerated in the cecal contents, liver, spleen, and ovaries. In addition, fecal (d 4 and 8) and cecal samples (necropsy at d 12) were collected from 5 postchallenged hens per treatment group 1, 2, and 3 (Salmonella Enteritidis-positive) for determination of hilA expression by PCR.

Bacterial Strain
A primary poultry isolate of Salmonella Enteritidis (phage type 13A) from the National Veterinary Services Laboratory (Ames, IA), selected for resistance to NO and NA in the USDA-ARS facility (College Station, TX), was used. The media used to culture the resistant isolate contained 25 µg of NO and 20 µg of NA/mL (Sigma Aldrich Co.). The culture was prepared from an overnight culture previously transferred 3 times in trypticase soy broth (Becton, Dickinson and Company). The challenge inoculum was prepared by serially diluting the culture in sterile PBS to a concentration of approximately 106 cfu/mL. The colony-forming units of the challenge inoculum were confirmed by plating on BGA plates (Becton, Dickinson and Company).

Necropsy
At the conclusion of both studies, hens were euthanized and the ceca, liver, spleen, and ovaries were excised aseptically. Serial dilutions were performed using 0.25 g of the cecal contents. One hundred microliters from each dilution tube was subsequently placed onto a BGA plate (Becton, Dickinson and Company) containing NA and NO and spread-plated using a bacterial cell spreader. Plates were incubated for 24 h at 37°C, and colony-forming units enumerated and expressed as log10 Salmonella Enteritidis/g of cecal contents. Samples of the ceca, liver, spleen, and ovaries of each hen were cultured for Salmonella Enteritidis. The organ samples were incubated for 24 h at 41°C in Rappaport-Vassiliadis R10 broth (Difco Laboratories, Detroit, MI). After incubation, the broth was streaked onto NA/NO BGA plates (Becton, Dickinson and Company) and incubated for an additional 24 h at 37°C. On the following day, plates were examined for Salmonella Enteritidis colonies and were recorded as either negative or positive for Salmonella Enteritidis.

Immunomagnetic Separation of Salmonella Enteritidis with Dynabeads
Anti-Salmonella Dynabeads (Dynal Biotech ASA, Oslo, Norway) were used in an immunomagnetic separation technique to remove Salmonella Enteritidis from feces (Olsvik et al., 1994) for hilA detection by real-time PCR. Fecal and cecal samples were suspended in a 1:2 (wt/vol) ratio of RNAlater (Sigma Aldrich Co.) in Whirl-Pak filter bags (Nasco, Fort Atkinson, WI). Triplicate 1-mL portions were placed into 1.5-mL sterile microcentrifuge tubes with 20 µL of anti-Salmonella Dynabeads. Microcentrifuge tubes were transferred to a Dynal MPC-M rack (Dynal Biotech ASA) and samples were processed according to the manufacturer’s instructions. Dynabead-bacterial complexes were resuspended in 100 µL of RNAlater (Sigma Aldrich Co.) and frozen until RNA was extracted.

RNA Extraction and Primer Design
Ribonucleic acid was extracted from the Dynabead-Salmonella Enteritidis complexes according to the manufacturer’s instructions (RNeasy Mini Kit, Qiagen, Valencia, CA). Salmonella RNA was subjected to reverse transcription (RT)-PCR to obtain cDNA. Primers were designed for hilA and 16S rRNA genes using sequence data obtained from the GenBank Web site and optimized using Primer Express 1.0 software (Perkin-Elmer Applied Bio-systems, Foster City, CA). Optimized sequences were processed on the National Center for Biotechnology Information Web site to determine their cross-reactivity with other species of bacteria. The primers used in this study are listed in Table 1Go (McClelland et al., 2001).


View this table:
[in this window]
[in a new window]

  		Table 1. Primer sequences used in real-time PCR reactions1
 
RT Reaction
To obtain Salmonella cDNA for real time-PCR, RT reactions were performed using the reagents from the Taq-Man OneStep RT-PCR Kit (Perkin-Elmer Applied Biosystems). Each reaction contained 10x RT-PCR buffer, 500 µM dNTPs Mix (Perkin-Elmer Applied Biosystems), 20 U/reaction of RNase inhibitor (Perkin-Elmer Applied Biosystems), 25 mM MgCl2, 200 ng of RNA, 2.0 µL of each primer (2.5 µM), 1.25 U/µL of MultiScribe RT (Perkin-Elmer Applied Biosystems), and RNase-free water to a final volume of 20 µL. A positive RT reaction was run to ensure the proper procedure. The positive reaction contained all the same components, except that in place of the RNA sample template, a DNA sample supplied with the kit was used as the template. To determine whether RNA samples were contaminated with DNA, 2 negative RT reactions were run on each RNA sample. One negative RT reaction contained the same components as the positive RT reactions, except that it lacked the RNA sample template and contained more water to ensure that the final concentrations of the remaining components were the same. The other negative RT reaction did not contain the RT enzyme to ensure that there was no DNA contamination. All 1-step RT reactions were performed on a Gene Amp PCR System (Perkin-Elmer GeneAmp PCR Systems, Wellesley, MA) under the following conditions: incubation for 10 min at 25°C, RT for 30 min at 45°C, RT inactivation for 5 min at 95°C, and 3-step cycling for 1 min at 94°C, 1 min at 53°C, and 1 min at 72°C for 40 cycles. The samples were held at 4°C until they could be removed.

Real-Time PCR Reaction
Real-time PCR reactions were performed on an ABI Prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystems). Each 20 µL of SYBR Green PCR reaction contained 1 µL of cDNA, 0.2 µL of each primer, 10 µL of 1x SYBR Green PCR Master Mix (Perkin-Elmer Applied Biosystems), and PCR water. Thermal cycling conditions were as follows: 48°C for 30 min, 95°C for 10 min, and 40 repeats of 95°C for 15 s and 60°C for 1 min. The same positive and negative RT reactions were run for the real-time PCR as for the RT reactions.

Gene Analysis and Expression
Samples for real-time PCR reactions (Orlando et al., 1998; Bustin, 2000; Livak and Schmittgen, 2001; Pierson et al., 2003) were run in triplicate to determine Salmonella Enteritidis expression. Data were analyzed using the relative quantification method (2–{Delta}{Delta}CT; Livak and Schmittgen, 2001), which describes the change in expression of the target gene (hilA) relative to the 16S rRNA reference gene (rsmC) from an untreated Salmonella Enteritidis control sample (Tscherne et al., 1999; Kundinger et al., 2007). Data were analyzed by averaging the CT values (cycle at which each sample amplification curve crosses a specific threshold) for triplicate samples. The {Delta}CT value of the target gene (hilA) was determined by normalizing to the endogenous control gene 16S rRNA. These samples were subsequently subtracted from the 16S rRNA gene from the untreated Salmonella Enteritidis control sample, which was prepared according to Fey et al. (2004). The {Delta}{Delta}CT was used to calculate relative expression using the formula 2–{Delta}{Delta}CT (Giulietti et al., 2001; Livak and Schmittgen, 2001; Lehman and Kreipe, 2001).

Statistical Analysis
A chi-squared analysis was used to determine significant differences among treatment groups for incidences of Salmonella Enteritidis colonization of the ceca, liver, spleen, and ovaries (Luginbuke and Schlotzhauer, 1987). Data from gene expression levels were analyzed by 1-way ANOVA subjected to linear regression using PC-SAS software (SAS Institute, 2001). Differences between means were determined using least squares means and Tukey’s honest significant test. Statistical variation was also estimated by the standard error of the mean. All statistical analyses were considered significant at P ≤ 0.05.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Salmonella Enteritidis Colonization in the Organs
The results of Salmonella Enteritidis colonization in the liver, spleen, and ovaries are shown in Table 2Go. In both trials, all nonchallenged birds (FF–, FW–, and ALC– treatments) were Salmonella Enteritidis-negative (data not shown). In trial 1 of this study, the ALC+ treatment group showed a trend of numerically lower Salmonella Enteritidis colonization in the ovaries 8% (1/12), spleen 17% (2/12), and liver 25% (3/12), compared with FW+ hens [ovaries 33% (4/12), spleen 33% (4/12), and liver 50% (6/ 12)], but these differences were not statistically significant (P > 0.05). In the same trial, FF+ hens were Salmonella Enteritidis-negative for the liver, spleen, and ovaries; however, only Salmonella Enteritidis colonization in the liver was less (P ≤ 0.05) compared with FW+ and ALC+ hens. In general, trial 2 demonstrated significantly (P ≤ 0.001) higher Salmonella Enteritidis infection compared with trial 1. In trial 2, the FW+ hens exhibited 42% Salmonella Enteritidis infection in the liver, and 63% of the hens were positive in the spleen. Salmonella Enteritidis colonization in the spleen (trial 2) was not statistically different for both FF+ (5%) and ALC+ (10%) but was significantly (P ≤ 0.001) less than in FW+ hens. This indicated that the alfalfa crumble diet effectively limited the incidence of colonization in these organs. The ALC+ treatment group exhibited a 25% infection of Salmonella Enteritidis in the ovaries, compared with 47% in the ovaries in FW+ hens; only FF+ ovaries were less (P < 0.05). Woodward et al. (2005) reported that Salmonella Enteritidis colonization generally increased in the liver, spleen, and ovaries in feed-withdrawal hens, compared with alfalfa meal molt-induced hens. In a molt induction study of hens fed alfalfa meal and alfalfa combined with a standard commercial layer diet, McReynolds et al. (2006) observed substantial reductions in Salmonella Enteritidis colonization in the liver of hens fed the 100% alfalfa meal diet and more than a 3-fold reduction in alfalfa meal-layer ration combinations when compared with feed-withdrawal hens.


View this table:
[in this window]
[in a new window]

  		Table 2. Colonization of Salmonella Enteritidis in the organs of molted laying hens (trials 1 and 2)
 
Salmonella Enteritidis Colonization of the Cecal Contents
In both trials, based on a chi-squared analysis, there were no differences between the ALC+ diet and the FF+ control in cecal Salmonella Enteritidis enrichment positives; however, hens in the FW+ treatment yielded significantly (P ≤ 0.05) higher Salmonella Enteritidis infectivity colony-forming units per gram of cecal contents (Figure 1Go). In trial 1, ALC+ hens exhibited a 2-fold (log10 1.99) Salmonella Enteritidis reduction in colonization when compared with FW+ (log10 3.89). In trial 2, a 4-fold (log10 1.29) Salmonella Enteritidis reduction in colonization occurred for ALC+ hens compared with FW+ hens (log10 5.12). Woodward et al. (2005) saw a 2- to 7-fold reduction in Salmonella Enteritidis colonization in the ceca of alfalfa meal-fed hens compared with feed-withdrawal hens. McReynolds et al. (2006) reported similar reductions of Salmonella Enteritidis ceca colonization in birds fed a 100% alfalfa meal diet and a 70% alfalfa meal:30% standard commercial layer diet, respectively, compared with molted feed-withdrawal hens.


Figure 1
View larger version (9K):
[in this window]
[in a new window]

  		Figure 1. Salmonella enteritidis colonization in the ceca of molted laying hens (trials 1 and 2). a,bMean values within trial 1 without a common letter differ (P ≤ 0.05). A,BMean values within trial 2 without a common letter differ (P ≤ 0.05). FW+ = feed withdrawal, challenged with Salmonella Enteritidis; FF+ = fully fed, challenged with Salmonella Enteritidis; ALC+ = alfalfa crumble, challenged with Salmonella Enteritidis.

 
HilA Response
The relative expression of Salmonella hilA genes was examined from fecal (d 6 and 11) and cecal contents (d 12 necropsy) of post-Salmonella Enteritidis-challenged molted hens (Figure 2Go). Relative expression of hilA was higher (P ≤ 0.05) on all days examined in trial 2 compared with trial 1. There were no significant (P > 0.05) differences in hilA expression between fecal and cecal contents. In trial 1, fecal and cecal Salmonella hilA expression in FW+ hens was higher (P ≤ 0.05) than in FF+ hens (Figure 2Go, panels A, C, and E). However, there were no significant differences (P > 0.05) between hilA expression in the ALC+ group compared with the FF+ group. In trial 2, hilA expression was higher (P ≤ 0.05) in FW+ hens for d 6, 11, and 12, respectively (Figure 2Go, panels B, D, and F), when compared with ALC+ and FF+ hens. This corresponds to previous in vitro studies by Durant et al. (1999), in which sterile crop contents were used to incubate Salmonella Enteritidis; they observed that hilA expression was nearly doubled in crop contents from hens undergoing feed withdrawal compared with crops from layer ration-fed hens, corresponding to a 5- to 6-fold increase in Salmonella Enteritidis colonization in the spleen and liver on molted hens compared with those of layer ration-fed hens. In addition, they reported a 3.5- to 5.5-fold increase of Salmonella colonization in the ceca of feed-withdrawal hens compared with nonmolted hens.


Figure 2
View larger version (16K):
[in this window]
[in a new window]

  		Figure 2. Relative expression of hilA in Salmonella Enteritidis-challenged fully fed (nonmolted) and molted hens (A) d 6 fecal samples, trial 1; (B) d 6 fecal samples, trial 2; (C) d 11 fecal samples, trial 1; (D) d 11 fecal samples, trial 2; (E) d 12 cecal content, trial 1; (F) d 12 cecal contents, trial 2. Error bars indicate SEM. a,bMeans within trials 1 and 2 without a common letter differ (P < 0.05). FF+ = fully fed (nonmolted) challenged with Salmonella Enteritidis; ALC+ = alfalfa challenged with Salmonella Enteritidis; FW+ = feed withdrawal, challenged with Salmonella Enteritidis.

 
It has previously been demonstrated that molted hens shed significantly more Salmonella Enteritidis in their feces (Holt and Porter, 1992; Holt, 1993; Holt et al., 1995) and generally exhibit much higher levels of Salmonella Enteritidis survival and invasion in their internal organs (Holt et al., 1995). The quantification of hilA expression has been proposed as an accurate indicator of the level of Salmonella virulence in the GI tract and potential susceptibility of hens (Durant et al., 1999, 2000; Ricke, 2003). In the current study, the lower hilA expression in the ALC+ and FF+ diets generally paralleled organ infection levels and suggests that both diets provided available substrates that minimized Salmonella Enteritidis virulence. In addition to limiting Salmonella Enteritidis infection, the effectiveness of FF+ and ALC+ diets in reducing cecal Salmonella colonization may rely on their ability to support normal microflora fermentation in the intestines (Nurmi and Rantala, 1973; Barnes et al., 1980; Nisbet et al., 1994; Corrier et al., 1995; Van der Wielen et al., 2000, 2001, 2002; Ricke, 2003; Woodward et al., 2005). The results of the current study suggest that alfalfa would be capable of retaining protective microflora and providing the desired fermentative capacity needed during molt to establish resistance to enteropathogens such as Salmonella Enteritidis. However, determining this will require in vitro and in vivo profiling of both the molecular ecology and fermentation capacity of the cecal microorganisms in hens fed these diets.


   ACKNOWLEDGMENTS
 
This research is supported by Hatch grant H8311 administered by the Texas Agricultural Experimental Station, USDA-NRI grant number 2002-02614, and US Poultry and Egg Association grant 485. Thanks are given to Denise Caldwell for technical assistance and Cassie Woodward for helpful discussion in preparing the manuscript.


   FOOTNOTES
 
2 Current address: Dept. of Poultry Science, University of Georgia, PO Box 748, Tifton, GA 31793-0478. Back

3 Current address: Center for Food Safety and Microbiology, IFSE, University of Arkansas, 2650 N. Young Ave., Fayetteville, AR 72704. Back

Received for publication November 15, 2006. Accepted for publication April 15, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Andrews, W. H., G. A. June, P. S. Sherrod, T. S. Hammack, and R. M. Amaguana. 1995. Salmonella. Pages 5.10–5.20 in Bacteriological Analytical Manual. 8th ed. Assoc. Off. Anal. Chem., Arlington, VA.

Bajaj, V., R. L. Lucas, C. Hwang, and C. A. Lee. 1996. Co-ordinate regulation of Salmonella typhimurium invasion genes by environmental and regulatory factors is mediated by control of hilA expression. Mol. Microbiol. 22:703–714.[Web of Science][Medline]

Barnes, E. M., C. S. Impey, and D. M. Cooper. 1980. Manipulation of the crop and intestinal flora of the newly hatched chick. Am. J. Clin. Nutr. 33:2426–2433.[Abstract/Free Full Text]

Brake, J. 1993. Recent advances in induced molting. Poult. Sci. 72:929–931.[Web of Science]

Bustin, S. 2000. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol. 25:169–193.[Abstract]

Corrier, D. E., D. J. Nisbet, B. M. Hargis, P. S. Holt, and J. R. DeLoach. 1997. Provision of lactose to molting hens enhances resistance to Salmonella enteritidis colonization. J. Food Prot. 60:10–15.[Web of Science][Medline]

Corrier, D. E., D. J. Nisbet, C. M. Scanlan, A. G. Hollister, and J. R. DeLoach. 1995. Control of Salmonella typhimurium colonization in broiler chicks with a continuous-flow characterized mixed culture of cecal bacteria. Poult. Sci. 74:916–924.[Web of Science][Medline]

Donalson, L. M., W. K. Kim, C. L. Woodward, P. Herrera, L. F. Kubena, D. J. Nisbet, and S. C. Ricke. 2005. Utilizing different ration of alfalfa and layer ratios for molt induction and performance in commercial laying hens. Poult. Sci. 84:362–369.[Abstract/Free Full Text]

Durant, J. A., D. E. Corrier, J. A. Byrd, L. H. Stanker, and S. C. Ricke. 1999. Feed deprivation affects crop environment and modulates Salmonella enteritidis colonization and invasion of Leghorn hens. Appl. Environ. Microbiol. 65:1919–1923.[Abstract/Free Full Text]

Durant, J. A., D. E. Corrier, L. H. Stanker, and S. C. Ricke. 2000. Expression of the hilA Salmonella typhimurium gene in a poultry Salm. enteritidis isolate in response to lactate and nutrients. J. Appl. Microbiol. 89:63–69.[Medline]

Fey, A., S. Eichler, S. Flavier, R. Christen, M. G. Höfle, and C. A. Guzmán. 2004. Establishment of a real-time PCR-based approached for accurate quantification of bacterial RNA targets in water, using Salmonella as a model organism. Appl. Environ. Microbiol. 70:3618–3623.[Abstract/Free Full Text]

Galán, J. E., and P. J. Sansonetti. 1996. Molecular and cellular bases of Salmonella and Shigella interaction with host cells. Pages 2757–2773 in Escherichia coli and Salmonella: Cellular and Molecular Biology. Vol. 2. F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger, ed. Am. Soc. Microbiol. Press, Washington, DC.

Giulietti, A., L. Overbergh, D. Vakckx, B. Decallonne, R. Bouillon, and C. Mathieu. 2001. An overview of real-time quantitative PCR: Applications to quantify cytokine gene expression. Methods 25:386–401.[Web of Science][Medline]

Holt, P. S. 1993. Effect of induced molting on the susceptibility of White Leghorn hens to a Salmonella enteritidis infection. Avian Dis. 37:412–417.[Web of Science][Medline]

Holt, P. S. 1995. Horizontal transmission of Salmonella enteritidis in molted and unmolted laying chickens. Avian Dis. 39:239–249.[Web of Science][Medline]

Holt, P. S. 2003. Molting and Salmonella enterica serovar Enteritidis infection: The problem and some solutions. Poult. Sci. 82:1008–1010.[Abstract/Free Full Text]

Holt, P. S., N. P. Macri, and R. E. Porter, Jr. 1995. Microbiological analysis of the early Salmonella enteritidis infection in molted and unmolted hens. Avian Dis. 39:55–63.[Web of Science][Medline]

Holt, P. S., and R. E. Porter, Jr. 1992. Use of pilocarpine-induced alimentary secretions to measure intestinal shedding of Salmonella enteritidis in chickens. Avian Dis. 36:54–58.[Web of Science][Medline]

Kilburn, J., and H. M. Edwards, Jr. 2001. The response of broilers to the feeding of mash or pelleted diets containing maize of varying particle sizes. Br. Poult. Sci. 42:484–491.[Web of Science][Medline]

Kundinger, M. M., I. B. Zabala-Diaz, V. I. Chalova, W. K. Kim, R. W. Moore, and S. C. Ricke. 2007. Characterization of rsmC as a potential reference gene for Salmonella Typhimurium gene expression during growth in spent media. Sens. Instr. Food Qual. Saf. doi:10.1007/S11694-007-9011-3

Landers, K. L., Z. R. Howard, C. L. Woodward, S. G. Birkhold, and S. C. Ricke. 2005b. Potential of alfalfa as an alternative molt induction diet for laying hens: Egg quality and consumer acceptability. Bioresour. Technol. 96:907–911.[Web of Science][Medline]

Landers, K. L., C. Woodward, X. Li, L. F. Kubena, D. J. Nisbet, and S. C. Ricke. 2005a. Alfalfa as a single dietary source for molt induction in laying hens. Bioresour. Technol. 96:565–570.[Web of Science][Medline]

Lehman, U., and H. Kreipe. 2001. Real-time PCR analysis of DNA and RNA extracted from formalin-fixed and paraffin-embedded biopsies. Methods 25:409–418.[Web of Science][Medline]

Livak, K. J., and T. D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-{Delta}{Delta}CT method. Methods 25:402–408.[Web of Science][Medline]

Luginbuke, R., and S. D. Schlotzhauer. 1987. SAS/SAT Guide for Personal Computers. 6th ed. SAS Inst. Inc. Cary, NC.

McClelland, M., K. E. Sanderson, J. Speith, S. W. Clifton, P. Latreille, L. Courtney, S. Porwollik, J. Ali, M. Dante, F. Du, S. Hou, D. Layman, S. Leonard, C. Nguyen, K. Scott, A. Holmes, N. Grewal, E. Mulvaney, E. Ryan, H. Sun, L. Florea, W. Miller, T. Stoneking, M. Nhan, R. Waterston, and R. K. Wilson. 2001. Complete genome sequence of Salmonella enterica serovar Typhimurium LT2. Nature 413:852–856.[Medline]

McReynolds, J., L. Kubena, J. Byrd, R. Anderson, S. Ricke, and D. Nisbet. 2005. Evaluation of Salmonella enteritidis in molting hens after administration of an experimental chlorate product (for nine days) in the drinking water and feeding an alfalfa molt diet. Poult. Sci. 84:1186–1190.[Abstract/Free Full Text]

McReynolds, J. L., R. W. Moore, L. F. Kubena, J. A. Byrd, C. L. Woodward, D. J. Nisbet, and S. C. Ricke. 2006. Effects of various combinations of alfalfa and standard layer diet on susceptibility of laying hens to Salmonella Enteritidis during forced molt. Poult. Sci. 85:1123–1128.[Abstract/Free Full Text]

Moore, R. W., S. Y. Park, L. F. Kubena, J. A. Byrd, J. L. McReynolds, M. R. Burnham, M. E. Hume, S. G. Birkhold, D. J. Nisbet, and S. C. Ricke. 2004. Comparison of zinc acetate and propionate addition on gastrointestinal tract fermentation and susceptibility of laying hens to Salmonella enteritidis during forced molt. Poult. Sci. 83:1276–1286.[Abstract/Free Full Text]

Nir, L., R. Hillel, G. Shefet, and Z. Nitsan. 1994. Effects of grain particle size on performance. 2. Grain texture interactions. Poult. Sci. 73:781–791.[Web of Science][Medline]

Nisbet, D. J., S. C. Ricke, C. M. Scanlan, D. E. Corrier, A. G. Hollister, and J. R. DeLoach. 1994. Inoculation of broiler chicks with a continuous-flow derived bacterial culture facilities early cecal bacterial colonization and increases resistance to Salmonella typhimurium. J. Food Prot. 57:12–15.[Web of Science]

NRC (National Research Council). 1994. Nutrient Requirements of Poultry. 9th rev. ed. Natl. Acad. Press, Washington, DC.

Nurmi, E., and M. Rantala. 1973. New aspects of Salmonella infection in broiler production. Nature 241:210–211.[Medline]

Olsvik, Ø., T. Popovic, E. Skjerve, K. S. Cudjoe, E. Hornes, J. Ugelstad, and M. Uhlén. 1994. Magnetic separation techniques in diagnostic microbiology. Clin. Microbiol. Rev. 7:43–45.[Abstract/Free Full Text]

Orlando, C., P. Pinzani, and M. Pazzagli. 1998. Development in quantitative PCR. Clin. Chem. Lab. Med. 36:255–269.[Web of Science][Medline]

Park, S. Y., S. G. Birkhold, L. F. Kubena, D. J. Nisbet, and S. C. Ricke. 2004b. Review on the role of dietary zinc in poultry nutrition, immunity, and reproduction. Biol. Trace Elem. Res. 101:147–163.[Web of Science][Medline]

Park, S. Y., W. K. Kim, S. G. Birkhold, L. F. Kubena, D. J. Nisbet, and S. C. Ricke. 2004a. Induced moulting issues and alternative dietary strategies for the egg industry in the United States. World’s Poult. Sci. J. 60:196–209.[Web of Science]

Pierson, S. N., J. N. Butler, and R. G. Foster. 2003. Experimental validation of novel and conventional approaches to quantitative real-time PCR data analysis. Nucleic Acids Res. 31(E73):1–7.[Abstract/Free Full Text]

Ricke, S. C. 2003. The gastrointestinal tract ecology of Salmonella Enteritidis colonization in molting hens. Poult. Sci. 82:1003–1007.[Abstract/Free Full Text]

Ricke, S. C., S. Y. Park, R. W. Moore, Y. M. Kwon, C. L. Woodward, J. A. Byrd, D. J. Nisbet, and L. F. Kubena. 2004. Feeding low calcium and zinc molt diets sustains gastrointestinal fermentation and limits Salmonella enterica serovar Enteriditis colonization in laying hens. J. Food Safety 24:291–308.

SAS Inst. 2001. SAS/STAT User’s Guide: Statistics. Release 8.2. SAS Institute Inc. Cary, NC.

Seo, K.-H., P. S. Holt, and R. K. Gast. 2001. Comparison of Salmonella Enteritidis infection in hens molted via long-term withdrawal versus full-fed wheat middling. J. Food Prot. 64:1917–1921.[Web of Science][Medline]

Thiagarajan, D., A. M. Saeed, and E. K. Asem. 1994. Mechanism of transovarian transmission of Salmonella enteritidis in laying hens. Poult. Sci. 73:89–98.[Web of Science][Medline]

Tscherne, J. S., K. Nurse, P. Popienick, and J. Ofengand. 1999. Purification, cloning, and characterization of the 16 S RNA m2G1207 methyltransferase from Escherichia coli. J. Biol. Chem. 274:924–929.[Abstract/Free Full Text]

Van der Wielen, P. W. J. J., S. Biesterveld, L. J. A. Lipman, and F. van Knapen. 2001. Inhibition of a glucose-limited sequencing fed-batch culture of Salmonella enterica serovar Enteritidis by volatile fatty acids representative of the ceca of broiler chickens. Appl. Environ. Microbiol. 67:1979–1982.[Abstract/Free Full Text]

Van der Wielen, P. W. J. J., S. Biesterveld, S. Notermans, H. Hofstra, B. A. P. Urlings, and F. Van Knapen. 2000. Role of volatile fatty acids in development of the cecal microflora in broiler chickens during growth. Appl. Environ. Microbiol. 66:2536–2540.[Abstract/Free Full Text]

Van der Wielen, P. W. J. J., L. J. A. Lipman, F. van Knapen, and S. Biesterveld. 2002. Competitive exclusion of Salmonella enterica serovar Enteritidis by Lactobacillus crispatus and Clostridium lactatifermentans in a sequencing fed-batch culture. Appl. Environ. Microbiol. 68:555–559.[Abstract/Free Full Text]

Woodward, C. L., Y. M. Kwon, L. F. Kubena, J. A. Byrd, R. W. Moore, D. J. Nisbet, and S. C. Ricke. 2005. Reduction of Salmonella enterica serovar Enteritidis colonization and invasion by an alfalfa diet during molt in Leghorn hens. Poult. Sci. 84:185–193.[Abstract/Free Full Text]




This article has been cited by other articles:


Home page
 Poult. Sci.Home page
L. M. Donalson, J. L. McReynolds, W. K. Kim, V. I. Chalova, C. L. Woodward, L. F. Kubena, D. J. Nisbet, and S. C. Ricke
The Influence of a Fructooligosaccharide Prebiotic Combined with Alfalfa Molt Diets on the Gastrointestinal Tract Fermentation, Salmonella Enteritidis Infection, and Intestinal Shedding in Laying Hens
Poult. Sci., July 1, 2008; 87(7): 1253 - 1262.
[Abstract] [Full Text] [PDF]

Home page
 Poult. Sci.Home page
C. S. Dunkley, T. H. Friend, J. L. McReynolds, C. L. Woodward, W. K. Kim, K. D. Dunkley, L. F. Kubena, D. J. Nisbet, and S. C. Ricke
Behavioral Responses of Laying Hens to Different Alfalfa-Layer Ration Combinations Fed During Molting
Poult. Sci., June 1, 2008; 87(6): 1005 - 1011.
[Abstract] [Full Text] [PDF]

Home page
 Poult. Sci.Home page
C. S. Dunkley, T. H. Friend, J. L. McReynolds, W. K. Kim, K. D. Dunkley, L. F. Kubena, D. J. Nisbet, and S. C Ricke
Behavior of Laying Hens on Alfalfa Crumble Molt Diets
Poult. Sci., May 1, 2008; 87(5): 815 - 822.
[Abstract] [Full Text] [PDF]

Home page
 Poult. Sci.Home page
C. S. Dunkley, J. L. McReynolds, K. D. Dunkley, L. F. Kubena, D. J. Nisbet, and S. C. Ricke
Molting in Salmonella Enteritidis-Challenged Laying Hens Fed Alfalfa Crumbles. III. Blood Plasma Metabolite Response
Poult. Sci., December 1, 2007; 86(12): 2492 - 2501.
[Abstract] [Full Text] [PDF]

Home page
 Poult. Sci.Home page
C. S. Dunkley, J. L. McReynolds, K. D. Dunkley, L. N. Njongmeta, L. R. Berghman, L. F. Kubena, D. J. Nisbet, and S. C. Ricke
Molting in Salmonella Enteritidis-Challenged Laying Hens Fed Alfalfa Crumbles. IV. Immune and Stress Protein Response
Poult. Sci., December 1, 2007; 86(12): 2502 - 2508.
[Abstract] [Full Text] [PDF]

Home page
 Poult. Sci.Home page
K. D. Dunkley, J. L. McReynolds, M. E. Hume, C. S. Dunkley, T. R. Callaway, L. F. Kubena, D. J. Nisbet, and S. C. Ricke
Molting in Salmonella Enteritidis-Challenged Laying Hens Fed Alfalfa Crumbles. II. Fermentation and Microbial Ecology Response
Poult. Sci., October 1, 2007; 86(10): 2101 - 2109.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dunkley, K. D.
Right arrow Articles by Ricke, S. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dunkley, K. D.
Right arrow Articles by Ricke, S. C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS