Yeast culture supplement during nursing and transport affects immunity and intestinal microbial ecology of weanling pigs

S. M. Weedman, M. H. Rostagno, J. A. Patterson, I. Yoon, G. Fitzner and S. D. Eicher J ANIM SCI 2011, 89:1908-1921. doi: 10.2527/jas.2009-2539

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Yeast culture supplement during nursing and transport affects immunity and intestinal microbial ecology of weanling pigs1,2
S. M. Weedman,* M. H. Rostagno, J. A. Patterson,* I. Yoon, G. Fitzner, and S. D. Eicher3
*Purdue University, West Lafayette, IN 47907; Livestock Behavior Research Unit, USDA-ARS, West Lafayette, IN 47907; and Diamond V Mills Inc., Cedar Rapids, IA 52407-4570

ABSTRACT: The objectives of this study were to determine the influence of a Saccharomyces cerevisiae fermentation product on innate immunity and intestinal microbial ecology after weaning and transport stress. In a randomized complete block design, before weaning and in a split-plot analysis of a 2 2 factorial arrangement of yeast culture (YY) and transport (TT) after weaning, 3-d-old pigs (n = 108) were randomly assigned within litter (block) to either a control (NY, milk only) or yeast culture diet (YY; delivered in milk to provide 0.1 g of yeast culture product/kg of BW) from d 4 to 21. At weaning (d 21), randomly, one-half of the NY and YY pigs were assigned to a 6-h transport (NY-TT and YY-TT) before being moved to nursery housing, and the other one-half were moved directly to nursery housing (NY-NT and YY-NT, where NT is no transport). The yeast treatment was a 0.2% S. cerevisiae fermentation product and the control treatment was a 0.2% grain blank in feed for 2 wk. On d 1 before transport and on d 1, 4, 7, and 14 after transport, blood was collected for leukocyte assays, and mesenteric lymph node, jejunal, and ileal tissue, and jejunal, ileal, and cecal contents were collected for Toll-like receptor expression (TLR); enumeration of Escherichia coli, total coliforms, and lactobacilli; detection of Salmonella; and microbial

analysis. After weaning, a yeast transport interaction for ADG was seen (P = 0.05). Transport affected (P = 0.09) ADFI after weaning. Yeast treatment decreased hematocrit (P = 0.04). A yeast transport interaction was found for counts of white blood cells (P = 0.01) and neutrophils (P = 0.02) and for the neutrophil-tolymphocyte ratio (P = 0.02). Monocyte counts revealed a transport (P = 0.01) effect. Interactions of yeast transport (P = 0.001) and yeast transport day (P = 0.09) for TLR2 and yeast transport (P = 0.08) for TLR4 expression in the mesenteric lymph node were detected. Day affected lactobacilli, total coliform, and E. coli counts. More pigs were positive for Salmonella on d 7 and 14 than on d 4, and more YY-TT pigs were positive (P = 0.07) on d 4. The number of bands for microbial amplicons in the ileum was greater for pigs in the control treatment than in the yeast treatment on d 0, and this number tended to decrease (P = 0.066) between d 1 and 14 for all pigs. Similarity coefficients for jejunal contents were greater (P = 0.03) for pigs fed NY than for those fed YY, but pigs fed YY had greater similarity coefficients for ileal (P = 0.001) and cecal (P = 0.058) contents. The number of yeast transport day interactions demonstrates the complexity of the stress and dietary relationship.

Key words: immune response, intestinal microbiota, pig, transport, weaning, yeast culture 2011 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2011. 89:19081921 doi:10.2527/jas.2009-2539

INTRODUCTION
After weaning, piglet immunity is compromised by exposure to multiple stressors. These stressors lead

Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. 2 Funded in part by Diamond V Mills Inc. (Cedar Rapids, IA). 3 Corresponding author: Susan.Eicher@ars.usda.gov Received September 30, 2009. Accepted January 11, 2011.

to reduced feed intake and alter the development and community structure of the commensal bacterial population in the intestine. Consequently, weaning and transport stress enhances the vulnerability to colonization by pathogenic bacteria. Thus, developing methods to stimulate the innate immune system of the pig may decrease susceptibility of the host. Several attempts have been made to alleviate weaning stress by feeding dietary modulators, including spraydried plasma (Pierce et al., 2005), vitamin E (Fragou et al., 2006), altered CP and fermentable carbohydrates (Bikker et al., 2006), nondigestible oligosaccharides and nonstarch polysaccharides (Van Nevel et al., 2005),

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and yeasts (Li et al., 2006) and yeast products (Li et al., 2005; Eicher et al., 2006; Hahn et al., 2006). Yeast supplements are promising because they are capable of enhancing peripheral as well as intestinal immunity. Yeast culture is a dried fermented product composed of residual dead yeast (Saccharomyces cerevisiae) and the media on which it was grown. Portions of the yeast cell wall, including -glucans and mannans, have immunomodulating properties. The -glucan component can modulate both the innate and adaptive arms of the immune system (Xiao et al., 2004). Studies of the effects of yeast culture or yeast extracts on immunity in mice are abundant, but few publications have addressed the effects of yeast on immune modulation in swine. Even fewer studies have incorporated the effects of yeast extracts or yeast cultures on commensal bacteria. The objectives of this study were to determine the influence of a S. cerevisiae fermentation product on innate immunity and intestinal microbial ecology after weaning and transport stressors in pigs.

MATERIALS AND METHODS

Animal use was approved by the Purdue Animal Care and Use Committee.

Animals, Experimental Design, and Housing

The experiment was conducted at the Purdue University Swine Unit in 2 replications, 1 in the fall and 1 in the spring. One hundred eight piglets (54 per replication) from 24 multiparous sows (12 per replication) were individually weighed at 72 h after farrowing. Within each litter, 4 piglets were chosen based on similar BW, and BW and sex were balanced among litters and piglets were assigned to 1 of 4 treatments arranged in a factorial array. Factors were yeast supplementation [yeast (YY) or no yeast (NY)] and transport [transported (TT) and no transport (NT)]. The experiment was analyzed as 2 separate experiments, preweaning and postweaning. The preweaning data was treated as a randomized complete block design, with yeast treatment and the control diet as treatments and litter as the blocking factor. The postweaning data were analyzed as a split plot in time with a 2 2 factorial arrangement of the treatments yeast culture (YY) and transport (TT). At experimental d 0, the yeast supplement (XPC, Diamond V Yeast Culture, Diamond V Mills Inc., Cedar Rapids, IA) was delivered in milk to provide 0.1 g of yeast culture product/kg of BW from d 4 to 21. At weaning (d 21), one-half of the NY and YY pigs (randomly assigned within litter) were transported for 6 h (NY-TT and YY-TT) before being moved to nursery housing, and the other one-half were moved directly to nursery housing (NY-NT and YY-NT). Pigs in the YY-NT and YY-TT treatments were supplemented with 0.2% yeast supplement and those in the

NY-NT and NY-TT treatments received a 0.2% grain blank in feed for 2 wk. The yeast culture was fed in warmed (37C) whole bovine milk at 0.1 g of yeast culture/kg of BW. Yeast culture [(0.1 g of yeast culture/kg of mean BW of the pigs) 54] was mixed into 54 mL of milk (1 mL/piglet). Individual volumes of milk containing yeast culture were administered orally to each piglet to achieve 0.1 g of yeast culture/kg of BW. Pigs in the control treatments received only milk adjusted for BW. Individual BW of piglets were recorded twice weekly, and yeast culture in the milk aliquots was adjusted until weaning at approximately 21 d of age. One additional piglet from each of 12 litters at each replication was randomly assigned to the nontransported treatments (NY-NT or YY-NT) as the baseline controls before transport. Piglets were color coded with paint markers for treatment identification. At weaning, 48 piglets (24 per replication), 1 NY and 1 YY randomly assigned from each litter, were transported (TT) for 6 h by trailer (bedded with shavings with ample space allowance, 0.18 m2/piglet; Sutherland et al., 2009) over a designated route to include highway and interstate roads (2 transports). Piglets were penned within the transport vehicle with littermates because it is well established that isolation is an extreme stressor (Herskin and Jensen, 2000; Kanitz et al., 2004). Piglets rested and did not fight at any point in the transport, presumably because they were with littermates and dominance was already established, and because they were not in competition for food or water. The remaining piglets (NT) were moved into the nursery environment at the same time the transported piglets were loaded onto the trailer. Piglets were housed individually after weaning. Pens were arranged by day of euthanasia within the room, with treatments randomly placed within the day of euthanasia pens. This placement was tested as a random variable, and placement within the room was found not to be significant (P > 0.10). Each treatment was represented in an end pen at least once. Experimental diets were composed of phase 1 and phase 2 diets (Table 1) with a grain blank (grain mixture that was used to make the yeast culture) to replace the yeast culture. For the yeast treatments, yeast culture was included at 0.2% of the nursery diet and the diet was adjusted for the 15% CP supplied by the yeast product. Weanling pigs were fed free choice for up to 2 wk after weaning. On the day of weaning before transport, 6 baseline piglets from each of the dietary treatments (NY or YY) were humanely killed with 0.22 mL of BeuthanasiaD (Schering-Plough Animal Health Corporation, Summit, NJ) per kilogram of BW, followed by exsanguination. These served as end points (d 21) for the preweaning pigs and as the baseline values for postweaning pigs. Immediately after the piglets returned from transport and at 4, 7, and 14 d after transport, 6 piglets from

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each treatment group (NY-NT, YY-NT, NY-TT, and YY-NT) were killed for each replication.

Table 1. Ingredient composition of experimental diets (as-fed basis) fed to pigs after weaning (d 21 to 35) that included the yeast supplement or a grain blank
Ingredient, % Finely ground corn Spray-dried whey Soybean meal, 47.5% Animal plasma White swine grease Fishmeal, menhaden Lactose Blood meal, swine Dicalcium phosphate Calcium carbonate Zinc oxide, 72% Salt plain (Kleer Mixing1) Supplement2 dl-Methionine l-Lysine hydrochloride Vitamin premix3 Nonsulfur trace mineral premix4 Selenium, 0.05%
1 2

Sample Collection and Measurements Performance. After birth, individual BW of piglets were recorded twice weekly until weaning. Piglets were weighed at weaning and on d 4, 7, or 14 after transport. Individual feed consumption was recorded at each weighing interval after weaning and feed conversion was determined. Hematology. Before euthanasia, piglets were restrained using a V-trough, and jugular blood was collected into 2 vacuum tubes: one 5-mL EDTA tube (12 mg in a 7-mL draw tube; Becton Dickinson, Franklin Lakes, NJ) and one 10-mL tube containing acid citrate dextrose [ACD; 1.5 mL of solution A (22.0 g of trisodium citrate; 8 g of citric acid; and 24.5 g of dextrose/L) in an 8.5-mL draw tube; Becton Dickinson]. Blood was stored on ice until assayed. Blood from the EDTA tubes was used for a 5-part differential hematology analysis using a Hemavet 950 Hematology Analyzer (Drew Scientific, Dallas, TX). Blood cell measures consisted of hematocrit percentage; counts of total white blood cells (WBC), neutrophils, monocytes, and lymphocytes; and percentages of neutrophils, monocytes, and lymphocytes. Blood collected into vacuum tubes containing ACD was used for peripheral leukocyte flow cytometric analysis. Leukocyte Function. Cluster of differentiation (CD) 14 is part of the lipopolysaccharide receptor that is primarily on monocytes, macrophages, and dendritic cells involved in antigen processing and presentation. The expression of CD18, as part of an adhesion molecule, is upregulated on activated cells, particularly on neutrophils. Cell surface expression of CD14 and CD18 was analyzed using 3 aliquots of 500 L each of whole blood from the ACD tubes. Blood was incubated in a 37C water bath for 30 min before addition of antibodies. To the first tube, 10 L of phycoerythrin-conjugated, monoclonal mouse anti-human CD14, clone TUK4 (DakoCytomation, Carpinteria, CA) and 20 L of monoclonal mouse anti-human leukocyte functional antigen-1, -chain/fluorescein isothiocyanate CD18, clone 6.7 antibodies (BDPharmingen, San Jose, CA; 100 mg/L) were added to detect cell surface marker expression. FluoSpheres, carboxylate-modified microspheres (12.5 L; 1.0 m; Molecular Probes, Eugene, OR), were added to the second tube for microbead phagocytosis analysis. Another tube remained without antibody to serve as unstained control cells and allowed gating of autofluorescence. Samples were incubated in a 37C water bath for 30 min. Cells were lysed by hypotonic lysis, in which 900 L of cold sterile water was added to each sample tube for 45 s. After red blood cell lysis, 0.1 mL of 10 Hanks Balanced Salt Solution (HBSS) was added to restore isotonicity. Samples were centrifuged for 3 min at 1,800 g at 4C and cell

Phase 1 34.8 25.0 12.5 6.7 6.0 6.0 5.0 1.7 0.5 0.4 0.375 0.25 0.20 0.15 0.15 0.15 0.125 0.05

Phase 2 39.3 25.0 20.0 5.0 2.5 2.5 2.5 1.1 0.6 0.375 0.30 0.20 0.15 0.15 0.15 0.125 0.05

Morton Kleer Mixing Salt (Morton Salt, Chicago, IL). Grain blank or Saccharomyces cerevisiae yeast supplement (XPC, Diamond V Mills Inc., Cedar Rapids, IA). The yeast supplement provides not less than 15% CP, 1.50% fat, and 22.0% fiber. 3 Provided the following per kilogram of diet (as-fed basis): vitamin A, 6,600 IU; vitamin D3, 660 IU; vitamin E, 44.0 IU; menadione, 2.2 mg; vitamin B12, 38.5 g; riboflavin, 8.8 mg; d-pantothenic acid, 22.0 mg; niacin, 33.0 mg. 4 Provided the following per kilogram of diet (as-fed basis): Fe, 84.88 mg; Zn, 84.88 mg; Mn, 10.5 mg; Cu, 7.88 mg; I, 0.29 mg; Se, 0.3 mg.

lysis was repeated. Samples were washed with 1 mL of 1 HBSS and centrifuged at 1,800 g for 3 min at 4C. To preserve samples for flow cytometric analysis, cells were suspended in 500 L of 2% paraformaldehyde in 1 HBSS. Flow cytometry was conducted using a Coulter Epics XL-MCL Flow Cytometer and System II software (Beckman Coulter Inc., Miami, FL). The flow cytometer used a 488-nm air-cooled argon laser for excitation, a 525-band pass filter for detection of fluorescein isothiocyanate emissions, and a 575-band pass filter for detection of phycoerythrin emission. For each sample, a total population of 10,000 cells was analyzed. RNA Extraction and PCR. Mesenteric lymph nodes (MLN) located proximally to the ileocecal junction, and one 3-cm section of the ileum and the jejunum were harvested aseptically from each piglet after exsanguination. Sections were stripped of the serosal layer and cut along the mesentery to expose the inner area of the tissue. Tissue was rinsed with sterile 1 HBSS and placed in 3.6-mL cryovial tubes with 3 mL of RNAlater (Ambion Inc., Austin, TX) for RNA extraction and stored at 80C. Samples were homogenized and RNA was extracted by using a tissue grinder and RNeasy Mini Kit (Qiagen, Valencia, CA). Extracted RNA was quantified using the GeneQuant pro RNA/DNA Calculator (Amersham Biosciences Corp., Piscataway, NJ). Complementary DNA was amplified with master mix, composed of reagents included in the TaqMan

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Reverse Transcription Reagent Pack (Applied Biosystems, Foster City, CA) and prepared in a sterile disposable reservoir (Vistalab Technologies, Mt. Kisco, NY), for a 100-L final volume. It contained 50 units/L of Multi-Scribe reverse transcriptase, 25 mM MgCl2, 2.5 M random hexamers, 0.4 units/L of ribonuclease inhibitor, 50 M deoxynucleotide 5-triphosphate, and TaqMan reverse-transcription buffer (TaqMan reversetranscription reagents, Applied Biosystems). Sample RNA and ribonuclease-free water (Ambion Inc.) were added to the master mix at appropriate quantities. Complementary DNA was amplified (Hybaid PCR Express Thermo Cycler, Midwest Scientific, St. Louis, MO) using the following cycling conditions: 50C for 2 min of activation, followed by 40 cycles of 95C for 10 min, 95C for 15 s, 60C for 1 min, and a final stage of 60C for 5 min, with a holding temperature of 4C. The resulting cDNA was stored at 80C until PCR was performed. After reverse transcription, the relative abundance of Toll-like receptor (TLR) 2 and TLR4 were determined by quantitative reverse-transcription PCR using primers and probes (Table 2) developed using Primer Express and synthesized by Applied Biosystems. The abundance of genes of interest relative to the quantity of 18S rRNA in total RNA isolated from homogenized MLN, jejunal, and ileal tissue was quantified using an ABI Prism 7000 Sequence Detection System (Applied Biosystems). Reactions were carried out using the appropriated TaqMan probe (200 nM), forward primer and reverse primer, PCR master mix (Applied Biosystems), and 5 L of the cDNA sample and a program consisting of an initial step at 50C for 2 min, followed by 50 cycles of 10 min at 95C, 20 s at 95C, and 1 min at 60C. Commercially available eukaryotic 18S rRNA primers and probes (Applied Biosystems) were used as an endogenous control. Microbial Analysis. Intestinal contents (2 mL) were collected aseptically after slaughter from the jejunum, ileum, cecum, and MLN for microbial analysis. Individual contents of the jejunum, ileum, and cecum were divided into 2 aliquots, one for microbial profiling using denaturing gradient-gel electrophoresis (DGGE) and one for enumeration of Escherichia coli, total co-

liforms, and Lactobacillus. Part of the second aliquot and MLN were selectively enriched for isolation of Salmonella. To detect E. coli, total coliforms, and Lactobacillus, intestinal contents were serially diluted in buffered peptone water (Neogen, Lansing, MI) and incubated overnight at 37C. Samples (1 mL) were plated on Petrifilm (3M, St. Paul, MN) for enumeration of E. coli and total coliforms and on Rogosa agar (Difco, Lawrence, KS) for enumeration of lactobacilli and were incubated for 24 h at 37C. To detect Salmonella, cecal contents and MLN were diluted 10-fold in tetrathionate broth (Neogen) and incubated overnight at 37C. Samples (1 mL) were transferred to 10 mL of Rappaport-Vassiliadis broth (Neogen) and incubated for 24 h at 42C. Loops of Rappaport-Vassiliadis enrichment were streaked onto XLT4 plates (Neogen) and incubated for 24 h at 37C. Black-colored colonies were identified as Salmonella positive by using Rambach agar (DRG International Inc., Mountainside, NJ). For microbial profiling, jejunal, ileal, and cecal contents were collected into 1.5-mL microfuge tubes and frozen at 80C until assayed. Tissues were homogenized (UltraTurrax T 25, IKA Works Inc., Wilmington, NC), and genomic DNA was extracted from both tissues and contents by using a Fast DNA Spin Kit for Soil (MP Biomedicals LLC, Solon, OH). Extracted DNA was quantified using the GeneQuant pro RNA/ DNA Calculator (Amersham Biosciences Corp.) and then diluted for PCR analysis. Polymerase chain reaction procedures were as described by Williams et al. (2008). Briefly, the V3 region of the 16S rDNA was amplified and bands were separated on 30 to 60% denaturing gradient polyacrylamide gels (8%) using a DCode Universal Mutation Detection System (Bio-Rad, Hercules, CA). Samples were electrophoresed at 60C for 5.5 h. Fragment pattern relatedness was determined using Bionumerics Software (version 2.5, Applied Maths, Austin TX). Similarity coefficients were derived from the pairwise comparison of banding patterns between 2 samples and were calculated using the DICE algorithm for bands within sample, as described by Burkholder et al.(2008).

Table 2. Primers and probes used for quantitative reverse-transcription PCR for Toll-like receptor (TLR) 2 and TLR4 gene expression
Gene TLR2 TLR4 Primer or probe Forward primer (sense) Reverse primer (antisense) Probe Forward primer (sense) Reverse primer (antisense) Probe Sequence TGT GGC CAT CGC TGC TAA C GGG ACA CCA CGA CAA TAA CCT T VIC1 TCA TCC AGG AAG GTT TCC ACA AAA GTC G MGBNFQ2 TGT GGC CAT CGC TGC TAA C GGG ACA CCA CGA CAA TAA CCT T VIC TCA TCC AGG AAG GTT TCC ACA AAA GTC G MGBNFQ Accession number AY392087 AB232527

1 2

VIC is a proprietary designation (Applied Biosystems, Foster City, CA). MGBNFQ = minor groove binder nonfluorescent quencher.

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Statistical Analysis
Data were tested for normality using the Univariate procedure (SAS Inst. Inc., Cary, NC) and transformed as necessary: WBC were square root transformed, all microbial analysis were natural log-transformed, and ileal TLR2 and TLR4 and jejunal TLR2 were log-transformed. Data are presented as least squares means SE. The treatments were analyzed as a factorial arrangement of yeast and transport. Performance (BW, ADG, and feed conversion) was analyzed as a preweaning segment and as a postweaning segment. Body weight, ADG, ADFI, and G:F were analyzed as repeated measures using mixed model procedures (SAS Inst. Inc.), with pig as the experimental unit. For the first 21-d analysis, the fixed effects, consisting of the 2 dietary treatments (yeast and control), day, and their interactions, were tested as a repeated measure. For the first 21 d, d 0 BW was used as a covariate. The first covariate was used because BW differences between treatments were detected at experimental d 0. For postweaning data, the mixed models procedures of SAS were used to analyze the split-plot design, with the factorial arrangement of yeast and transport as the whole plots and day as the subplot. The fixed effects tested were day and treatments (yeast, transport, and their interaction). For the last 2 wk when the yeast was delivered in dry feed, BW at d 21 was used as a covariate. The second covariate was used because of anticipated differences caused by the treatments during the nursery phase of the experiment. Season (fall and spring) and room were tested as random variables and removed because they were not significant. Error terms were determined by the mixed models procedures, which eliminates the need for error term construction by automatically incorporating the correct error term into test statistics. Replication and sex were tested as random variables for each and were included in the model only when they were significant. The same statistical analyses were used for blood analysis and leukocyte functions (hematocrit percentages; WBC counts; neutrophil, lymphocyte, and monocyte counts and percentages; neutrophil-to-lymphocyte ratio; CD14 and CD18 cell surface expression; and phagocytic cells), microbial analysis (E. coli, lactobacilli, and total coliforms), and RNA expression of pathogen recognition molecules (TLR2 and TLR4). Treatment differences were determined using Fishers protected LSD and Tukey-Kramer means separation. Significance is reported at = 0.10. However, exact probability values are provided for the discretion of the reader. Salmonella data were analyzed by 2, and the effects of replication, treatment, day, and sex were tested. Chi-squared analysis was used to separate day effects within treatments and treatment effects within days for Salmonella counts. The total number of bands, within a sample, and similarity coefficients, within treatments and across treatments (cross-products),of DGGE data

were statistically analyzed as ANOVA using the PROC MIXED command of SAS as described by Burkholder et al.(2008), with treatment and day as the variables. Cross-products were used as an estimate of no treatment effect for instances in which similarity values were numerically similar but banding patterns were different (Burkholder et al., 2008).

RESULTS Performance
Initial BW (Table 3) was greater for piglets in the YY than in the NY treatment (P = 0.02). Therefore, d 0 BW was used as a BW covariate for the first 21 d. Yeast was not significant (P = 0.11), but a day effect (P = 0.001) was detected in the first 21 d. Body weight at d 21 was used as a covariate for the remaining 2 wk of growth analysis. A yeast transport interaction (P = 0.05) and a day effect (P = 0.001) were detected in the analysis of ADG for d 21 to 35 of the study. Average daily feed intake (Table 3) was affected (P = 0.09) by transport during wk 3 (the first week after weaning). Feed efficiency was not affected by treatments (Table 3). Pigs supplemented with yeast had lesser hematocrit percentages than nonsupplemented pigs (P = 0.04), and after d 1, hematocrit percentages increased for all pigs (P = 0.001) through the end of the study.

Hematology
White blood cell counts (Table 4) were affected by a yeast transport interaction (P = 0.01) and a yeast transport day interaction (P = 0.04). On d 1, WBC counts were less for the YY-TT treatment than for the NY-NT treatment. By d 4, piglets in the NY-TT and YY-NT groups had greater WBC counts than piglets in the NY-NT or YY-TT group. A yeast transport interaction (P = 0.02) and a day effect (P = 0.01) were detected for neutrophil counts (Table 4), such that pigs in the YY-TT group had smaller neutrophil counts than did pigs in the NY-TT or YY-NT group, and neutrophil numbers increased throughout the study. Lymphocyte counts had a day effect (P = 0.01) and a yeast transport day interaction (P = 0.02) such that on d 4, pigs in the YY-TT group had the least lymphocytes compared with pigs in the NY-TT and YY-NT group, but on d 14, piglets in the YY-NT group had the least lymphocyte numbers compared with piglets in the NY-NT group. Monocyte counts revealed a transport effect (P = 0.01) and a day effect (P = 0.01). Monocyte counts of transported pigs were greater than those of nontransported pigs, and counts increased throughout the study following transport. Analysis of neutrophil-to-lymphocyte ratios (Table 4) revealed a yeast transport day interaction (P = 0.04), a yeast transport interaction (P = 0.04), a day effect (P = 0.04), and a transport effect (P = 0.06). On d 1, pig-

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Table 3. Performance of piglets from birth to 35 d of age1 for pigs fed a yeast culture or the carrier only from d 3 to 35 and transported for 6 h or not transported at weaning (d 21) in a 2 2 factorial arrangement of treatments for d 21 to 35
Treatment mean2 NY-NT 1.99 6.22 2.74 (0.20) 4.75 (0.29) 5.88 (0.23) 6.02 0.16 (0.02) 0.16 0.26 (0.02) 2.05 0.06 0.02 0.11 0.001 0.001 NY-TT YY-NT YY-TT SEM Yeast Day Trn3 P-value Yeast day 0.45 0.05 0.24 0.22 0.68 0.45 0.84 0.18 0.83 0.37 0.84 0.77 0.09 0.89 0.16 0.71 0.21 Yeast trn Yeast trn day

Item 2.72 (0.23) 4.61 (0.30) 6.43 (0.26) 7.48 (0.17) 8.69 (0.25) 0.23 0.58 0.45 0.64 7.05 (0.12) 9.4 (0.29) 0.22 0.64 0.46 0.71 7.14 (0.18) 9.58 (0.29) 0.23 0.63 0.47 0.65 7.82 (0.26) 9.10 (0.24) 0.19 0.58 0.43 0.69 0.26 (0.02) 0.26 (0.04) 0.02 0.05 0.05 0.08

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Initial BW,4 d 3, kg Wk 1,4 d 3 to 7 BW at d 7, kg (ADG, kg/d) Wk 2,4 d 7 to 14 BW at d 14, kg (ADG, kg/d) Wk 3,4 d 14 to 21 BW at d 21, kg (ADG, kg/d) Postweaning wk 4 and 5,5 d 21 to 35 BW at d 28, kg (ADG, kg/d) BW at d 35, kg (ADG, kg/d) Wk 3 ADFI, kg/d, d 21 to 28 ADFI, kg/d, d 28 to 35 G:F wk 4, d 21 to 28 G:F wk 5, d 28 to 35

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Least squares means; none were transformed. NY-NT = control diet and not transported; NY-TT = control diet and transported; YY-NT = yeast culture diet (XPC, Diamond V Mills Inc., Cedar Rapids, IA) and not transported; YY-TT = yeast culture diet and transported. 3 Trn = transport. 4 NY-NT (n = 29), NY-TT (n = 24), YY-NT (n = 30), and YY-TT (n = 23). 5 NY-NT, NY-TT, YY-NT, and YY-TT (n = 6).

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Table 4. Effects of yeast culture and transport by day on hematology1 for pigs fed a yeast culture or the carrier only from d 3 to 35 and transported for 6 h or not transported at weaning (d 21) in a 2 2 factorial arrangement of treatments2
Item Day and treatment d0 NY YY d 1 NY-NT NY-TT YY-NT YY-TT d 4 NY-NT NY-TT YY-NT YY-TT d 7 NY-NT NY-TT YY-NT YY-TT d 14 NY-NT NY-TT YY-NT YY-TT Effect, P-value Yeast Transport Day Yeast transport Yeast transport day
a,b 1

HCT, % 33.6 2.4 32.2 2.4 25.8 1.85 28.4 1.85 30.3 1.85 26.1 1.85 27.9 1.77 33.2 1.77 14.5 1.77 26.1 1.77 29.5 2.26 31.2 2.26 28.2 2.26 30.2 2.26 30.4 2.40 35.6 2.40 31.3 2.40 35.5 2.40 0.04 0.40 0.001 0.36 0.87

WBC,3 106/mL 10.1 2.4 11.2 2.4 11.1 0.80a 10.4 1.80ab 9.3 0.80ab 7.7 0.80b 11.4 0.98b 13.5 0.98a 16.9 0.98a 7.7 0.98b 12.2 0.98 14.2 0.98 10.9 0.98 15.0 0.98 15.3 1.04 18.4 1.04 15.4 1.04 12.7 1.04 0.17 0.66 0.01 0.01 0.04

Neutrophils, 106/mL 6.4 1.8 6.0 1.8 6.0 0.46 4.8 0.46 5.4 0.46 3.9 0.46 4.9 0.56 5.7 0.56 7.1 0.56 3.9 0.56 6.4 0.56 8.0 0.56 5.0 0.56 7.0 0.56 6.5 0.60 9.1 0.60 8.9 0.60 4.9 0.60 0.08 0.30 0.01 0.02 0.10

Lymphocytes, 106/mL 2.3 1.8 4.0 4.3 4.4 0.37 5.0 0.37 3.4 0.37 3.4 0.37 4.9 0.45ab 5.9 0.45a 7.4 0.45a 3.4 0.45b 5.1 0.45 5.0 0.45 5.5 0.45 7.0 0.45 7.4 0.48a 6.7 0.48ab 4.2 0.48b 5.3 0.48ab 0.15 0.81 0.01 0.49 0.02

Monocytes, 106/mL 0.34 0.4 0.25 0.2 0.36 0.04 0.25 0.04 0.20 0.04 0.27 0.04 0.24 0.05 0.39 0.05 0.38 0.05 0.27 0.05 0.41 0.05 0.44 0.05 0.28 0.05 0.58 0.05 0.69 0.05 1.16 0.05 1.05 0.05 1.20 0.05 0.23 0.01 0.01 0.84 0.18

Neutrophil:lymphocyte ratio

4.42 3.4 3.53 3.2 1.46 0.10ab 1.04 0.10b 1.81 0.10a 1.16 0.10b 1.03 0.11 1.00 0.11 1.19 0.11 1.16 0.11 1.39 0.12 1.77 0.12 1.08 0.12 1.11 0.12 0.86 0.13b 1.54 1.13ab 2.23 1.13a 0.90 0.13b 0.87 0.06 0.04 0.02 0.04

Means with different superscripts within a day for each variable differ (P < 0.05). Nontransformed means SE. 2 HCT = hematocrit; WBC = white blood cells; NY = control diet; YY = yeast culture diet (XPC, Diamond V Mills Inc., Cedar Rapids, IA); NY-NT = control diet and not transported; NY-TT = control diet and transported; YY-NT = yeast culture diet and not transported; YY-TT = yeast culture diet and transported. 3 Values were square root transformed for statistical analysis.

lets in the YY-NT group had a greater ratio than did piglets in the NY-TT or YY-TT group, and on d 14, piglets in the YY-NT group had a greater ratio than piglets in the NY-NT or YY-TT group.

Leukocyte Function and RNA Expression of TLR

Percentage of cells positive for fluorescence resulting from bead phagocytosis did not differ (P > 0.10) among the treatments or by day (data not shown). Fluorescence from CD14 expression was affected by day (P = 0.04) but not by treatments. Fewer cells expressed CD14 on d 1 and 7 than on d 4 and 14. The percentage of cells positive for CD18 fluorescence was affected by day (P = 0.001) but not treatment. More cells expressed CD18 on d 14 than on d 1, 4, or 7 after transport. In jejunal tissues, a day effect was detected (P = 0.07) for TLR4 (Figure 1). No main effects or interac-

tions were observed for expression of TLR2 or TLR4 in ileal tissues (P > 0.10). Day effects were detected for RNA expression of TLR2 (P = 0.01) and TLR4 (P = 0.01) in MLN. Yeast transport interactions were detected in MLN for TLR2 (P = 0.001) and TLR4 (P = 0.08) expression. Additionally, a yeast transport day interaction (P = 0.09) for TLR2 in the MLN was observed. For both TLR2 and TLR4 expression, the YY-NT treatment had the greatest peak expression on d 1 after weaning, but NY-TT and YY-TT treatments also had greater expression of TLR2 compared with the NY-NT treatment.

Microbial Analysis
Lactobacilli, total coliforms, and E. coli were enumerated from jejunal, ileal, and cecal contents (Table 5). Lactobacillus counts were affected by day in the jejunum, ileum, and cecum such that counts were greater (P < 0.001) on d 4 and 7 compared with d 1 and 14. Total

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Figure 1. Relative abundance of Toll-like receptor (TLR) 2 (panels a, c, and e) and TLR4 (panels b, d, and f) compared with the 18S internal standard from total RNA extracted from jejunal tissues (panels a and b), ileal tissues (panels c and d), and mesenteric lymph nodes (panels e and f) at weaning (d 0) and d 1, 4, 7, and 14 after transport. Treatments were a factorial arrangement of diet and transport. Pigs were fed yeast culture (YY; XPC, Diamond V Mills Inc., Cedar Rapids, IA) or control (NY) diets from d 1 through 35, and one-half of the pigs on each of those dietary treatments were transported for 6 h (YY-TT and NY-TT) or not transported (YY-NT and NY-NT) at weaning (d 21). Data for ileal TLR2 and TLR4 and for jejunal TLR2 were log-transformed for statistical analysis. Yst = yeast; Trn = transport.

coliform counts were affected by day in the jejunum (P = 0.04) and ileum (P = 0.03) such that d 1 had smaller counts than d 7. Total coliform counts in the cecum (P = 0.059) had a day effect such that counts were less on

d 1 than on d 7. Escherichia coli counts in the jejunum tended (P = 0.06) to differ by day such that counts were greater on d 1 compared with d 14. On d 1, E. coli counts in the ileum were greater (P = 0.007) compared

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Table 5. Effects of yeast culture and transport by day on intestinal microbial ecology1 for pigs fed a yeast culture or the carrier only from d 3 to 35 and transported for 6 h or not transported at weaning (d 21) in a 2 2 factorial arrangement of treatments2
Lactobacilli Ileum Cecum Jejunum Ileum Cecum Jejunum Ileum Coliforms Escherichia coli Cecum

Item

Jejunum

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Day and treatment d 0 YY NY d 1 NY-NT NY-TT YY-NT YY-TT d 4 NY-NT NY-TT YY-NT YY-TT d 7 NY-NT NY-TT YY-NT YY-TT d 14 NY-NT NY-TT YY-NT YY-TT Effect,3 P-value Yeast Transport Day Yeast transport Yeast transport day 9.3 0.29 8.9 0.14 8.3 0.33 8.1 0.37 7.7 0.37 8.1 0.33 8.7 0.30 8.9 0.30 9.0 0.30 8.8 0.30 9.2 0.30 8.7 0.30 9.4 0.30 9.2 0.30 8.3 0.30 7.4 0.30 7.6 0.30 7.6 0.30 0.86 0.39 0.03 0.89 0.32 0.99 0.48 0.01 0.70 0.52 0.17 0.44 0.04 0.84 0.80 0.52 0.39 0.03 0.89 0.32 0.99 0.90 0.06 0.81 0.47 0.55 0.59 0.06 0.24 0.52 8.9 0.17 8.7 0.23 8.6 0.32 8.1 0.32 8.3 0.32 8.2 0.35 8.5 0.32 9.3 0.32 9.1 0.32 9.0 0.32 9.0 0.32 8.7 0.32 9.4 0.32 9.1 0.32 8.5 0.32 8.3 0.32 8.1 0.32 8.0 0.32 7.0 0.40 7.4 0.43 8.3 0.32 9.5 0.41 7.8 0.51 8.5 0.32 9.2 0.32 8.8 0.32 9.2 0.29 8.7 0.32 9.1 0.29 9.3 0.29 9.3 0.29 9.2 0.32 9.3 0.32 9.0 0.32 9.0 0.32 9.1 0.29 7.8 0.32 7.8 0.35 8.6 0.38 9.0 0.38 8.5 0.38 8.3 0.38 9.3 0.38 8.3 0.38 8.7 0.38 9.0 0.38 9.4 0.38 9.4 0.38 9.5 0.38 9.4 0.38 9.1 0.38 8.9 0.42 9.1 0.47 8.6 0.42 8.0 0.23 8.3 0.03 7.7 0.43 8.7 0.43 8.4 0.43 8.8 0.43 9.0 0.43 8.2 0.43 8.3 0.43 8.5 0.43 9.2 0.43 9.4 0.43 9.4 0.43 8.8 0.43 8.9 0.43 8.9 0.43 8.8 0.43 8.6 0.47 4.0 0.29 3.1 0.32 3.9 0.79 6.0 0.79 3.3 0.88 4.5 0.79 5.4 0.88 4.3 0.79 4.9 0.72 2.9 0.72 3.7 0.72 3.4 0.88 4.0 0.88 4.2 0.88 3.6 0.88 3.2 1.01 3.6 1.24 3.0 0.88

7.7 0.23 7.9 0.17 8.1 0.42 7.2 0.54 6.9 0.66 7.8 0.46 8.2 0.38 8.8 0.38 8.9 0.42 9.1 0.42 9.2 0.38 8.8 0.38 9.3 0.38 8.7 0.38 7.9 0.38 7.2 0.41 7.5 0.41 7.6 0.38

5.5 0.69 5.2 0.55 6.6 0.96 6.8 0.96 6.1 0.96 6.5 0.86 5.5 0.79 5.3 0.79 4.8 0.86 4.8 0.86 4.8 0.79 4.7 0.79 4.7 0.79 4.8 0.79 5.3 0.86 4.0 0.79 3.7 0.86 4.1 0.86 0.31 0.88 0.01 0.49 0.91

6.3 0.32 6.4 0.55 6.8 0.54 7.8 0.54 6.0 0.54 7.4 0.54 6.8 0.54 5.4 0.54 5.9 0.54 5.8 0.54 5.6 0.54 5.0 0.54 6.7 0.54 4.8 0.54 5.6 0.54 6.6 0.54 5.5 0.54 5.6 0.54 0.41 0.85 0.01 0.70 0.29

0.82 0.62 0.01 0.22 0.28

Nontransformed means SE. YY = yeast culture diet (XPC, Diamond V Mills Inc., Cedar Rapids, IA); NY = control diet; NY-NT = control diet and not transported; NY-TT = control diet and transported; YY-NT = yeast culture diet and not transported; YY-TT = yeast culture diet and transported. 3 All values were log-transformed (natural logarithm; ln) before statistical analysis.

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with those on d 7 and 14. Escherichia coli counts in the cecum were greatest (P = 0.002) on d 1. Isolation of Salmonella from MLN and cecal contents (P > 0.05) did not differ among treatments. However, across all treatments, cecal Salmonella differed (P < 0.0001) by day such that more pigs were positive for Salmonella on d 7 and 14 than on d 1 and 4 (Table 6). On d 4, more pigs in the YY-TT group tended (P = 0.07) to be positive for cecal Salmonella, whereas all other treatments had more pigs negative for cecal Salmonella (Table 6). Cecal Salmonella analysis showed a replication effect on d 0 (P = 0.0005) and d 1 (P < 0.0001) such that all pigs from the fall replication were negative for Salmonella, whereas all pigs from the spring replication were positive (data not shown). On d 4, there also tended to be a replication effect (P = 0.08) for cecal Salmonella in which all but 2 pigs from the fall replication were negative for Salmonella and one-half were negative from the spring replication (data not shown). Similar to cecal Salmonella, a day effect was detected in MLN, but this occurred earlier, with most pigs being positive by d 4. Additionally, there was a replication effect (P = 0.003) for MLN Salmonella such that on d 0, all pigs from the fall replication were negative for Salmonella, whereas all but 1 pig from the spring replication were positive (data not shown). Microbial amplicon number and pattern relatedness were determined for jejunal, ileal, and cecal contents. Because d 0 samples were collected from pigs in both the TT and NT pens, they were pooled to increase replicates and analyzed separately. Control pigs had more bands (P = 0.037) in the ileum on d 0 than did the pigs fed yeast (15.5 and 9.0, respectively), but there were no differences between treatments in number of bands in the jejunum and cecum on d 0. Dietary treatment and transportation did not affect (P > 0.10) the number of bands in the jejunal, ileal, or cecal contents after transportation (data not shown).

A day effect (P = 0.066) in number of bands in cecal contents was detected such that band numbers on d 1 were greater than those on d 14 (22.9, 19.0, 19.5, and 17.6 bands for d 1, 4, 7, and 14, respectively). Numbers of bands over all posttransport days were similar for jejunal and ileal contents and were less (P = 0.001) than in cecal contents (11.6, 11.1, and 19.8 bands, respectively). Similarity coefficients of the DGGE data were more complex, with significant differences (P < 0.05) between dietary treatments, transportation, and days after transportation, as well as interactions (Table 7). Similarity coefficients on d 0 were least (P < 0.05) in the jejunum and increased from 55.1 to 71.9 and 70.7 in the ileum and cecum, respectively. Similarity coefficients were numerically greater than in our earlier work (Williams et al., 2008), but in both studies, similarity coefficients increased along the digestive tract (P < 0.05). Means of similarity coefficients for day, yeast supplementation, and transport values within the jejunum and ileum were not different (P > 0.10) and were less (P < 0.05) than values for the cecum (52.8, 51.6, and 67.8, respectively). Day effects (P < 0.05) were such that similarity coefficients decreased between d 0 and 1 irrespective of diet or transport status, and then increased over time toward the initial d 0 values, although there was considerable variation in response (Table 7). Over all days, similarity coefficients for the control treatments (NY-NT and NY-TT) were greater (P = 0.029) in the jejunum than for the pigs fed yeast (YY-NT and YY-TT), whereas similarity coefficients were greater in the ileum (P < 0.001) and cecum (P = 0.058) for pigs fed yeast.

DISCUSSION
Piglets experience several stressors that accompany weaning: a new environment, mixing with unfamiliar

Table 6. Effects of yeast culture and transport by day on the pig mesenteric lymph node (MLN) and cecal Salmonella occurrences1 (n = 6 pigs/treatment per day)
d0 Tissue and treatment2 Cecum NY-NT NY-TT YY-NT YY-TT MLN3 NY-NT NY-TT YY-NT YY-TT
a,b 1

d1 Positive 3 3 3 4 3 3 3 2 6 5 5 2 3 3 3 3 0 1 1 3 Negative Positive 1a 2a 1a 4b 5 5 6 6

d4 Negative 5 4 5 2 1 1 0 0 6 6 6 6 5 4 6 5 Positive

d7 Negative 0 0 0 0 1 2 0 1 5 6 6 6 6 5 6 5

d 14 Positive 1 0 0 0 0 1 0 1 Negative

Positive 3 3 3 2

Negative

Means of cecal tissue positive for Salmonella with differing superscripts within a day differ (P = 0.07). Positive = positive for detection of Salmonella; negative = negative for detection of Salmonella. 2 NY-NT = control diet and not transported; NY-TT = control diet and transported; YY-NT = yeast culture diet (XPC, Diamond V Mills Inc., Cedar Rapids, IA) and not transported; YY-TT = yeast culture diet and transported. 3 Within the MLN, a day effect was detected for all treatments (P < 0.05).
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Table 7. Denaturing gradient gel electrophoresis similarity coefficients of jejunal, ileal, and cecal bacterial species in piglets1
Tissue and treatment Jejunum NY-NT NY-TT YY-NT YY-TT Cross-product SEM Ileum NY-NT NY-TT YY-NT YY-TT Cross-product SEM Cecum NY-NT NY-TT YY-NT YY-TT Cross-product SEM
ac xz

d0 56.23 53.93 61.63 6.25 65.83b 77.92a 64.46b 2.61 69.89 71.96 75.47 3.07

d1 40.64c,z 60.61a,xy 35.65c,z 44.74bc 48.72b 5.89 44.81b,yz 54.66a,x 57.11a 47.85ab,y 52.92ab 4.26 62.20bc 57.86c,y 70.55a,xy 66.67ab,y 65.26b 6.10

d4 62.75a,x 43.94c,z 48.21bc,y 56.24ab 50.09bc 4.17 38.53c,z 55.19ab,x 54.30b 64.18a,x 52.35b 3.30 68.32b 78.52a,x 66.67b,xy 63.44b,y 68.17b 6.10

d7 56.80a,y 66.05a,x 56.96a,x 46.46b 56.51a 5.89 37.67c,z 43.87bc,y 56.50a 46.32b,y 46.56b 3.30 67.06c 69.03bc,x 77.76ab,x 83.08a,x 73.78b,c 3.52

d 14 62.00x 56.01xy 55.44xy 51.11 56.00 10.2 59.94x 49.21xy 56.82 58.74x 58.02 6.02 66.59b 60.00b,y 74.93a,x 66.07b,y 67.47b 6.10

Cross-product2 50.72y 53.65y 48.22y 48.64 44.90y 49.39xy 55.07 46.51y 65.32 61.39y 66.55y 65.86y

SEM 4.17 10.2 5.89 5.89 4.26 6.02 4.26 3.30 3.52 6.10 6.10 6.10

Means within columns of a tissue with different superscript letters are significantly different (P < 0.05). Means within rows of a tissue with different superscript letters are significantly different (P < 0.05). The NY-TT d-4 value of 43.94 was significantly different from all the other values, and thus the z designation. The xy designation for the NY-TT d-14 value of 56.01 indicates that it is similar to the d-1 value of 60.61 and is also similar to and between the d-7 value of 66.05 and the cross-product value of 53.65, which are statistically different. In the cecum for d 4, the NY-TT value of 78.52 is significantly different from all other d-4 values, except for the YY-NT value of 66.67 (because of greater variation within these values), even though the YY-NT value is less than the NY-NT value of 68.32. 1 Least squares means. NY-NT = control diet and not transported; NY-TT = control diet and transported; YY-NT = yeast culture diet (XPC, Diamond V Mills Inc., Cedar Rapids, IA) and not transported; YY-TT = yeast culture diet and transported. 2 Cross-products are comparisons of individual samples within and between treatments and day. ,Because of large SE within one of the treatment sets, means within rows or within columns of a tissue, respectively, were not different (P > 0.05).

pigs, maternal separation, a new source of nourishment, and sometimes transportation. Exposing the piglet to a nutrient either through the sow diet (K. Scott, Purdue University, West Lafayette, IN, unpublished data) or through direct supplementation (Bruininx et al., 2004; Mei and Xu, 2005; Kuller et al., 2007) is a method that has shown some promise in alleviating part of the weaning stress. Using a S. cerevisiae fermentation product to aid in the dietary transition of weaning and stressors that often accompany weaning was the focus of this study. We observed only limited effects of yeast on BW before weaning, which is consistent with the growth response to yeast in other studies with pigs for the first 2 wk after weaning (van der Peet-Schwering et al., 2007) and with dairy calves (Magalhes et al., 2008). Transport effects were the only effect on BW after weaning in this study. Additionally, ADFI was affected only by transport. However, neither van der Peet-Schwering et al. (2007) nor Magalhes et al. (2008) provided yeast culture during suckling, as was done in the present study. Although pigs fed yeast had greater BW gain in the first 3 wk, those differences were abrogated by the end of the first week postweaning. By the end of the study, feed intake was more similar, implying only brief

effects of transport and differing effects of yeast in dry feed than in liquid feed. We found that only transport decreased ADFI in the first week after transport and weaning, but it did not change G:F. However, Carlson et al. (2005) found greater ADG and ADFI when nursery pigs were fed yeast extract, but this did not translate into improved G:F. Similarly, Shen et al. (2009) found improved ADG and ADFI when pigs were fed yeast culture. Part of that improvement was attributed to changes in gut morphology and immune modulations reflected by plasma IFN- concentrations that were less than in controls at d 7 postweaning but that were greater than in controls at d 21 after weaning. In this study, changes in immune cell populations over time were related to yeast culture treatment and transport. Transport, together with yeast supplementation, suppressed neutrophil and WBC counts that would typically increase after a stressor. This study had weaning stress followed immediately by transport stress, which created a compound stress situation typically seen in production. Transport treatments also decreased the neutrophil-to-lymphocyte ratio early, and with yeast treatment it remained at a smaller ratio at d 14. Age-related increases in CD14 and CD18 expression were evident, but no treatment differences were

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detected. Cluster of differentiation 14 is part of the lipopolysaccharide receptor that functions in interactions with and identification of gram-negative bacteria, such as E. coli and Salmonella. The distribution of monocytes, macrophages, and dendritic cells along with the frequencies of CD14 expression on those cells in intestinal and systemic lymphoid tissues respond to intestinal colonization by lactic acid bacteria differently than does an enteric pathogen such as the human rotavirus (Zhang et al., 2008). Lactobacilli counts as well as the CD14 expression on immune cells were not different among treatments. Lipopolysaccharide binding can be accomplished through other substances found in porcine milk (Shahriar et al., 2006), such as lactoferrin, soluble CD14, and serum amyloid A. However, only lactoferrin could withstand digestion (Lee et al., 1998). Serum amyloid A concentrations were found to increase significantly in older pigs after a long transport (Pieiro et al., 2007). Therefore, in this study, if lipopolysaccharide was being controlled by molecules other than cell surface-expressed CD14, expression of cell surface CD14 may not have been stimulated. The lack of phagocytic cell response is curious. In addition to neutrophils, a subpopulation of leukocytes, natural killer (NK) cells, are the predominant innate immune cells for pigs (Pintari and Saalmller, 2008). These cells and T cells typically express the CD8 cell surface protein (Denyer et al., 2006). Feeding yeast culture to nursery pigs decreased the CD4:CD8 ratio (Shen et al., 2009), implying that yeast culture enhances the NK cell or T cell populations. In vitro work (Jensen et al., 2008) showed that yeast culture enhanced expression of CD69 and CD25. Increased lymphocyte numbers, in addition to neutrophil populations, may be reflective of NK and cytotoxic cell activity in this study. That occurrence may have resulted in less neutrophil stimulation and reduced CD18 cell surface expression and phagocytosis by the neutrophils. Intestinal microbial ecology differed for E. coli in the jejunum in the current study. Previously, E. coli concentrations in the cecum, but not in the colon or rectum, were reduced by feeding a yeast culture to weaning pigs (Shen et al., 2009). When agglutination of pathogenic bacteria by a yeast extract was tested, 64% of E. coli strains that were tested and 67% of Salmonella spp. strains that were tested were found to agglutinate to the yeast product (Kogan and Kocher, 2007). Salmonella enterica serovar Typhimurium strains (70%) and S. enterica serovar Enteritidis (86%) were even greater. The authors cited 3 mechanisms for the effect of the yeast product on pig health: 1) inhibition of pathogen adhesion to gastrointestinal epithelial tissue, 2) stimulation of immune cells in Peyers patches, or 3) adsorption of mycotoxins and inhibiting their actions, or all 3. Diet had no effect on the number of predominant bacteria (bands) in the various sections of the intestinal tract. This is not surprising in that the yeast product was not fed at the substrate level. Even though there

was no diet effect on the predominant bands, the data did not rule out an effect on less dominant bacterial populations that affect pig health. Davis et al. (2007) saw an increase in the number of bands 20 d after weaning in pigs fed an antibiotic compared with control pigs, but no time course was addressed. There was also no effect of transportation, which is somewhat surprising considering that stress is thought to disturb the gastrointestinal tract and allow for fluctuation in bacterial populations in the intestinal tract. However, one may have fluctuation in bacterial species and still have the same number of predominant bacteria. There was a day effect in cecal contents, in which the number of bands was greatest at d 1 and declined thereafter. Konstantinov et al. (2003) found a difference in the number of bands after weaning, as in the present study. The increase in the number of predominant bands in cecal contents is consistent with the well-known increase in bacterial numbers and species in the cecum compared with the jejunum and ileum. Even though the number of predominant species increased from the jejunum to the cecum, suggesting more complex microbiota, the similarity coefficients were greater in the cecum than in the jejunum, suggesting that the digesta flux in the proximal small intestine maintains a greater diversity of bacteria in the jejunum compared with the cecum. Numerically, the d 0 similarity coefficients were greater in all sections of the intestinal tract than at any day postweaning. Postweaning, similarity coefficients were least at d 1 and generally increased over time, suggesting that weaning may cause a disruption of the intestinal microbiota. The greater similarity coefficients in the ileum and cecum, but not the jejunum, for pigs fed yeast may suggest that the yeast has less effect in the jejunum. Either the increased flux rate or the greater microbial diversity (smaller similarity coefficient) in the jejunum causes the reduced response to the yeast product. This is in contrast to the data of van der Peet-Schwering et al. (2007) in which they observed a decrease in diversity in pigs fed yeast. The data demonstrate that weaning does cause fluctuation in the predominant bacterial populations in the intestinal tract. An interesting question is whether there is a greater effect on nonpredominant bacterial populations, such as coliforms, that may have significant effects on animal health. However, our data showed only day effects on total coliforms. Overall, many complex interactions were detected. However, a time effect persisted across most variables. We first saw increased TLR2 and TLR4 in MLN on d 1. That response was greatest in pigs in the YY-NT group, suggesting that the yeast culture had primed immune cells for the changes that occurred with weaning. By d 4, Salmonella spp. had increased in the MLN, and by d 7, cecum Salmonella spp. had increased. The immune response of increased monocytes was observed by d 7 and 14. There were many transport and yeast interactions with day. However, the most striking ef-

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fect of the combination was reduced WBC, specifically reduced neutrophils. The yeast treatment had a production response similar to yeast culture treatments in other studies. Although much variability in responses depends on size, -glucan origin, and extraction processes (Sonck et al., 2010), it appears that providing a smaller fragment of a specific portion of the yeast, as in a yeast extract, provides a different response than when the whole yeast culture is fed (Kogan and Kocher, 2007). Additionally, the quality of the housing environment and the stressors that were experienced could explain some of the differences that were noted between this and other studies. Piglets in our study did not have to compete for feed or dominance after weaning because of the individual housing. Although they had visual, auditory, and some tactile access to other piglets, the relative isolation may have been a negative factor. Overall, the results suggest that yeast culture has immunomodulating properties that may alter interactions with intestinal microbial ecology. However, more complex studies are required to elucidate this relationship. It appears that early delivery of a yeast supplement to piglets has some benefits.

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