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ANIMAL PRODUCTION |
* Department of Animal Science and
and
Center for Excellence in Poultry Science, University of Arkansas, Fayetteville 72701 and
and
Department of Agricultural and Human Sciences, Morehead State University, Morehead, KY 40351
Abstract
Phosphorylated mannans derived from the yeast cell wall of Saccharomyces cerevisiae may beneficially modulate immune function in the weanling pig, possibly providing an alternative to the use of dietary growth-promoting antibiotics. Therefore, in this study, 32 pigs averaging 19 d of age and 5.7 ± 0.2 kg initial BW were randomly assigned to 16 pens in an environmentally controlled nursery to determine the effects of dietary supplementation with phosphorylated mannans on growth and immune function. Average daily gain and G:F ratio increased (P < 0.05) when pigs were fed diets supplemented with mannans from d 0 to 14 after weaning and in the overall experiment. Percentage of neutrophils was lower (P < 0.08) and percentage of lymphocytes was higher (P < 0.05) in blood from pigs fed mannans than when pigs were fed the basal diet. Lamina propria macrophages isolated from pigs fed diets containing mannans phagocytosed a greater (P < 0.05) number of sheep red blood cells (2.63 ± 0.11) than did lamina propria macrophages isolated from pigs fed the basal diet (2.31 ± 0.11). On d 19 after weaning, pigs fed diets supplemented with mannans tended to have a greater (P < 0.10) percentage of CD14+ lamina propria leukocytes than did pigs fed the basal diet. On d 21 following weaning, the percentage of CD14+MHCII+ leukocytes isolated from lamina propria tissue tended (P < 0.10) to be lower when pigs were fed mannans than when pigs were fed the basal diet. Pigs fed diets containing mannans had a lower (P < 0.05) ratio of CD3+CD4+:CD3+CD8+ T lymphocytes isolated from jejunal lamina propria tissue only on d 21 after weaning compared with pigs fed the basal diet. Supplementation of mannans in the diets of weanling pigs improved gain and efficiency, and intermittently affected selected components of the young pigs’ immune function both systemically and enterically.
Key Words: Acquired Immunity • Intestinal Immunity • Mannan • Phagocytosis • Swine
Introduction
Although prohibited in many European countries, the addition of antibiotic growth promoters to swine diets is a common practice in the United States, particularly to the diets of newly weaned pigs. However, there has been increasing pressure on the livestock industry to decrease or discontinue these additions because of the potential development of antibiotic resistance. The need for alternative methods to improve growth and efficiency of livestock production and to modulate an animal’s natural ability to fight disease has prompted the scientific investigation of several feed additives and their ability to positively alter immune function (Berg, 1998
; Turner et al., 2001).
The supplementation of swine diets with phosphorylated mannans derived from the yeast cell wall of Saccharomyces cerevisiae has the potential to provide an alternative to the dietary addition of growth-promoting antibiotics. In a manner similar to the action of antibiotics added to the diet, mannans have the ability to alter the microbial population in the intestinal tract. This modification seems to be accomplished by the ability of mannans to attach to mannose-binding proteins on the cell surface of some strains of bacteria, thereby preventing these bacteria from colonizing the intestinal tract by interfering with the binding of carbohydrate residues on epithelial cell surfaces (Spring et al., 2000). Mannans have also been reported to alter immune function in swine (Kim et al., 2000), and this may be an additional mechanism by which mannans improve growth performance. However, the mechanism by which mannans modulate health is not well defined. Consequently, a better understanding of its mechanism of action is needed before the benefits of phosphorylated mannans can be successfully used in swine production systems. Therefore, the specific objectives of this study were to evaluate growth performance and the immunomodulatory effects of dietary phosphorylated mannans fed to weanling pigs.
Materials and Methods
Animals, Diets, and Tissue Collection
All experimental procedures performed in this study were approved by the University of Arkansas Animal Care and Use Committee. Thirty-two barrows and gilts (Yorkshire x Landrace females mated to DeKalb EB sires) were weaned at an average of 19 d of age and 5.7 ± 0.2 kg initial BW to an on-site, environmentally controlled nursery facility. Temperature was maintained at 28.3°C and decreased by 1.2°C until the end of the experiment. Pigs were distributed randomly within 16 pens (1.22 m x 1.52 m) such that two pigs were housed in each pen, and gender and genetics were distributed equally among treatments. Dietary treatments (Table 1) consisted of a typical starter pig diet with and without the addition of phosphorylated mannans at 0.3% (as-fed basis) of the diet. Both experimental diets contained a growth-promoting antibiotic supplement. Diets were formulated to meet or exceed the pigs’ dietary nutrient requirements as determined by the NRC (1998) and were assigned to pens in a completely randomized design. Phosphorylated mannans were substituted at the expense of corn in the basal diet, and dietary treatments were administered throughout the entire experimentation period.
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1-acid glycoprotein (AGP) analysis. On d 14, pigs were bled again to obtain a serum sample for the determination of AGP concentration and a whole blood sample in tubes containing EDTA for the isolation of peripheral blood mononuclear cells to determine lymphocyte proliferation responses and the phagocytic ability of monocytes/macrophages. Pigs were killed by lethal injection of sodium pentobarbital (Delmarva Laboratories, Inc., Midlothian, VA) on d 19, 21, 24, and 26 after weaning, such that four randomly selected pens (two pens representing each dietary treatment) were sampled on each day. Before euthanasia, blood was collected via vena cava puncture into tubes containing EDTA for determination of differential leukocyte counts and to obtain blood for flow cytometric phenotyping. Then, two jejunal samples were obtained from each pig and placed on ice. To obtain the first jejunal sample, 15 cm of the jejunum was dissected after measuring 55 cm from the pyloric junction. This tissue was used to isolate intraepithelial lymphocytes for flow cytometric analysis. An additional 40-cm sample of the jejunum was then dissected, and lamina propria leukocytes were isolated for flow cytometric phenotypic analysis and macrophage phagocytosis. Fecal matter was removed from the lumen of each sample by flushing with cold Roswell Park Memorial Institute medium (RPMI, Sigma Chemical Co., St. Louis, MO) using a 10-mL syringe.
Differential Blood Leukocyte and 1-Acid Glycoprotein Determination
Differential leukocyte proportions and concentrations were analyzed on a multiparameter, automated hematology analyzer (Abbott, Abbot Park, IL) calibrated for porcine blood. A single radial immunodiffusion assay was performed to determine AGP concentrations at weaning and 14 d postweaning using a commercial test kit (Cardiotech Services, Inc., Louisville, KY). Briefly, 5 µL of serum was administered to each well in an agar gel plate containing antiserum to porcine AGP. Plates were incubated at 37°C for 24 h. The diameter of the precipitin ring was measured and compared with a standard curve derived from standards ranging from 50 to 1,500 µL provided by the manufacturer of the commercial test kit.
Isolation of Intraepithelial Lymphocytes
The isolation procedure for intraepithelial lymphocytes was adapted from the methods of Kearsey and Stadnyk (1996), Poussier and Julius, (1997), Todd et al. (1999), and Solano-Aguilar et al. (2000). Briefly, the 15-cm jejunal sample for the isolation of intraepithelial lymphocytes was incubated on ice for approximately 90 min. Following the cold incubation, samples were flushed with 10 mL of HEPES (Sigma)-buffered, Hank’s balanced salt solution containing 1 mM dithioerythritol (Sigma), and 10% fetal bovine serum (Atlanta Biologicals, Atlanta, GA). Weakly adherent epithelial cells and cells within the mucus coating were obtained by gently squeezing the tissue by manual compression. The flushing procedure was repeated five times, and the lumenal eluate containing the intraepithelial lymphocytes was collected in a polystyrene container. Cells within the luminal eluate were washed by centrifuging at 350 x g for 5 min, and the resulting pellet was subjected to density gradient centrifugation for further purification by resuspension in 25 mL of a 30% Percoll (Sigma) solution. Cells resuspended in 30% Percoll were centrifuged at 350 x g for 15 min. The supernatant was discarded and the pellet was resuspended in 15 mL of 45% Percoll, underlayed with 15 mL of 75% Percoll, and centrifuged at 350 x g for 30 min. Intraepithelial lymphocytes were collected at the interface between the 45 and 75% Percoll layers and washed by centrifuging in RPMI at 130 x g for 10 min. Evaluation of isolated cells using a hemocytometer and Trypan Blue (Sigma) exclusion determined leukocyte yield to be approximately 4.9 x 106 leukocytes isolated from 15 cm of jejunal tissue with >90% viability.
Isolation of Lamina Propria Leukocytes
The procedure for the isolation of lamina propria leukocytes was adapted from the method of Bailey et al. (1994), Poussier and Julius (1997), and Solano-Aguilar et al. (2000). Briefly, the 40-cm jejunal sample for the isolation of lamina propria leukocytes was incubated on ice for approximately 30 min. The jejunal sample was then examined for the presence of Peyers patches, which were dissected and discarded. The jejunal sample was cut longitudinally, and then cut into small pieces of approximately 2 cm and placed in an Erlenmeyer flask containing Hank’s balanced salt solution and a magnetic stir bar. Samples were stirred vigorously for 20 min at room temperature. Following the 20-min wash, the supernatant was discarded and the wash was repeated to remove the epithelium from the intestinal mucosa. After discarding the supernatant from the second wash, 25 mL of medium warmed to 39.2°C and containing 180 U of collagenase (Sigma) was added to the flask containing the jejunal tissue and stirred vigorously for 20 min. Following the 20-min digestion, the supernatant was collected and the procedure was repeated two more times. A fourth digestion was performed after adding 25 mL of warmed medium containing 260 U of collagenase for 30 min and the supernatant was collected. The collected supernatants from the four digestions were combined into one 50-mL polystyrene tube and washed three times by centrifugation at 200 x g for 10 min. Evaluation of isolated cells using a hemocytometer and Trypan Blue (Sigma) exclusion determined leukocyte yield to be approximately 16.8 x 106 leukocytes isolated from 40 cm of jejunal tissue with >90% viability.
Lymphocyte Blastogenesis.
In vitro cellular proliferative response was measured using a lymphocyte blastogenesis assay adapted from the methods of Blecha et al. (1983). Briefly, peripheral blood mononuclear cells were isolated by gradient centrifugation using Ficoll gradient (Histopaque 1077, density = 1.077 g/mL; Sigma). Remaining erythrocytes were lysed by adding 1 mL of sterile water to the isolated cell pellet for 20 s, and isotonicity was restored by the addition of RPMI. Cells were resuspended in medium at 2 x 106 cells/mL and plated in triplicate in 96-well, round-bottom plates in 100-µL aliquots. Phytohemagglutinin (PHA, Sigma), pokeweed mitogen (PWM, Sigma), and concanavalin A (ConA, Sigma) were administered in 50-µL aliquots to each well at a concentration of 40, 15, and 25 µg/mL, respectively, to stimulate lymphocyte proliferation, whereas wells containing cells unstimulated with mitogen were administered 50 µL of medium. Concentrations chosen for each respective mitogen elicited the maximum proliferative response during pretesting. Cells were incubated with the mitogens for 48 h at 39.2°C and 5% CO2. Following the 48-h incubation, 1 µCi of tritiated-thymidine in 50 µL of RPMI was added to each well, and the cells were incubated for an additional 18 h. Cells were harvested on glass fiber mats and the radioactivity was measured on a liquid scintillation analyzer (TRI-CARB 2200CA, Packard Instrument Co., Downers Grove, IL). Incubation, labeling with tritiated thymidine, and cell harvesting followed procedures outlined by van Heugten and Spears (1994).
Macrophage Phagocytosis
The method used to measure monocyte/macrophage phagocytic ability of peripheral blood and lamina propria macrophages was adapted from the method of Nibbering et al. (1987), Heggen et al. (1998), and Monteleone et al. (1999). Peripheral blood mononuclear cells from blood and leukocytes from jejunal lamina propria tissue were isolated to collect monocyte/macrophages via adherence to glass surfaces, and measure their ability to phagocytose sheep red blood cells (SRBC). The incubation temperature for monocyte/macrophage assays and the lymphocyte blastogenesis assay was set at 39.2°C rather than 37°C to facilitate monocyte/macrophage activation under conditions simulating pig body temperature (Natale and McCullough, 1998). Briefly, cell suspensions isolated from blood and intestinal tissue were diluted to approximately 2 x 106 cells/mL in Leibovitz’s L-15/McCoy’s Hahn media (LM Hahn, Atlanta Biologicals) medium. A glass coverslip was added to each well of a six-well plate and 2 mL of cell suspension containing monocytes/macrophages was added to each well in duplicate for each sample. Each coverslip was completely covered by cell suspension. Cells were incubated for 5 h at 39.2°C and 5% CO2. Following the 5-h incubation period, medium from each well was removed and replaced by 2 mL of fresh LM Hahn medium warmed to 39.2°C. Plates containing cell suspensions isolated from blood were incubated for an additional 11 h, and those containing cell suspensions isolated from intestinal lamina propria tissue were incubated for an additional 21 h. Following the respective incubations, plates were removed from the incubator, excess medium was removed from each well, and 2 mL of a 5% SRBC suspension was added to each well. Plates were incubated with SRBC for 2 h, after which cover slips were removed and non-adherent cells and excess SRBC were washed from the cover slip by rinsing with warmed LM Hahn medium. Cover slips were then stained in Heme 3 (Fisher Scientific, Pittsburgh, PA) and the percentage of phagocytic monocytes/macrophages and the average number of SRBC consumed by each phagocytic monocyte/macrophage were determined.
Monoclonal Antibodies
Monoclonal antibodies used in this study were specific for swine leukocytes (Table 2). A panel of five commercially available mouse monoclonal antibodies were used to identify porcine T lymphocytes (CD3, Southern Biotechnology Associates, Inc., Birmingham, AL), T helper lymphocytes (CD4, Southern Biotechnology Associates, Inc.), cytotoxic T lymphocytes (CD8, Southern Biotechnology Associates, Inc.), monocytes/macrophages (CD14, VMRD, Inc., Pullman, WA), and major histocompatability complex class 2 (MHCII, VMRD, Inc.). Monoclonal antibodies specific for CD3 and CD4 were directly labeled with phycoerythrin (PE) and fluorescein 5(6)-isothiocyanate (FITC), respectively. The monoclonal antibody used to identify CD8 was biotinylated, and strepavidin, labeled with a tandem fluorescent dye consisting of PE coupled with Cy5 reactive dye, was used to identify the biotinylated primary antibody. Primary antibodies specific for CD14 (mouse anti-pig IgG1) and MHC-II (mouse anti-pig IgG2a) were identified with PE (goat anti-mouse IgG2a, Southern Biotechnology Associates, Inc.) and FITC (goat anti-mouse IgG1, Southern Biotechnology Associates, Inc.), respectively. Labeled and unlabeled isotype control monoclonal antibodies with irrelevant binding specificity were included to account for nonspecific labeling.
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Erythrocytes were lysed in whole blood samples that were obtained for flow cytometric analysis using published procedures (Mishell and Shiigi, 1980). Leukocytes from erythrocyte-lysed whole blood and jejunal intraepithelial and lamina propria tissue were resuspended to a minimum concentration of 105 cells/mL in RPMI 1640 medium devoid of phenol red (Sigma). Fifty-microliter aliquots of the cell suspensions were added to a 96-well microtiter plate. Mouse monoclonal antibodies specific for swine cell surface markers were diluted in PBS containing 1% BSA and 0.1% sodium azide (PBS+), and then administered in 50 µL aliquots to appropriate wells at optimal dilutions determined for each antibody during pretest trials. Plates were then incubated for 30 min at 4°C. Following the cold incubation, excess antibody was removed by two washings of plates with 200 µL of PBS+ and centrifuging at 180 x g for 4 min at 4°C. If the primary antibody was unconjugated, 50 µL of secondary antibody conjugated with an appropriate fluorochrome was administered following the PBS+ washes. Dual fluorescence staining was performed to identify CD3+CD4–, CD3–CD4+, CD3+CD4+, CD3+CD8–, CD3–CD8+, CD3+CD8+, CD4+CD8–, CD4–CD8+, CD4+CD8+, CD14+MHCII–, CD14–MHCII+, and CD14+MHCII+ leukocyte proportions.
FACSort flow cytometer and CellQuest software (Becton-Dickinson Immunocytometry Systems, San Jose, CA) were used to conduct one- and two-color analysis of stained cell populations. The proportions among CD4+- and CD8+-defined T subsets were expressed as a ratio of CD4+ to CD8+ T (CD3+) lymphocytes.
Statistical Analysis
Data were analyzed as a completely randomized design with pen as the experimental unit. The model included the effects of dietary treatment when analyzing ADG, ADFI, G:F, lymphocyte blastogenesis, and monocyte/macrophage phagocytosis. Initial BW was used as a covariate when analyzing ADG, ADFI, and G:F. The model included the effects of dietary treatment when analyzing the flow cytometric data, and dietary treatment, sampling day, and the treatment x day interaction when analyzing differential blood leukocytes and AGP concentrations. Data were analyzed using the GLM procedure of SAS (SAS Inst., Inc., Cary, NC).
Results and Discussion
In a previous experiment conducted in our laboratory, phosphorylated mannans altered the proliferation of lymphocytes isolated from peripheral blood (Davis et al., 2004). However, the means by which the dietary inclusion of phosphorylated mannans may result in an alteration of the proliferative responses of peripheral blood lymphocytes was unknown. We speculated that because phosphorylated mannans have been reported to alter the gastrointestinal microbial population (Spring et al., 2000), the alterations in systemic immune function were possibly an indirect response to changes that were occurring in gastrointestinal immunity. The immune assays measured in this experiment were selected to give an indication of the effects of phosphorylated mannans on systemic and intestinal immune function, as well as to confirm previous peripheral blood lymphocyte responses and improvements in gain and efficiency.
Growth Performance
Average daily gain, ADFI, G:F, and pig BW are reported in Table 3. Pigs fed diets supplemented with mannans had greater (P < 0.05) ADG and G:F than pigs fed the basal diet from d 0 to 14 postweaning. Although ADG, ADFI, and G:F were unaltered as a result of dietary treatment from d 14 to 21 postweaning, the improvement in ADG and G:F was maintained in the overall experiment (P < 0.05). This was reflected by the greater (P < 0.05) BW of pigs fed mannans on d 14 and 21 of the experiment compared with pigs fed the basal diet.
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). In this experiment, the benefits to supplementing weanling pig diets with mannans seem to manifest during the first 2 wk after weaning. In contrast, other work has reported no benefit when adding mannans to weanling pig diets when compared with a positive-control diet with antibiotics or a negative control diet devoid of antibiotics (Ko et al., 1998). These inconsistent responses to mannan supplementation in weanling pig diets may be because of varying environmental conditions and health status within herds. The improvement in growth performance from mannan addition may be greater when it is supplemented to slower-growing pigs, where slow growth is speculated to be indicative of herd health challenges (Pettigrew, 2000).
Differential Blood Leukocyte Proportions, 1-Acid Glycoprotein, and Lymphocyte Proliferation
The percentage of lymphocytes increased (P < 0.05) and the percentage of neutrophils tended to decrease (P < 0.08) in blood obtained from pigs fed mannans compared with those fed the basal diet (Table 4). Because there is often an increase in blood neutrophils as the first line of defense associated with subclinical and clinical infection (Roth, 1999), the observed tendency for neutrophil proportions to decrease in the blood of mannan-fed pigs suggests less of an inflammatory challenge. Proinflammatory cytokines (interleukin-1, interleukin-6, and tumor necrosis factor-) produced during an immune response to infection alter protein and lipid metabolism and, as a result, influence growth and efficiency of gain (Johnson, 1997; Spurlock, 1997). The indication that inflammation is decreased in mannan-fed pigs is supported by the improvements in gain and efficiency when pigs were fed phosphorylated mannans. Although not significantly different, the increase in the concentration of lymphocytes and the decrease in the concentration of neutrophils resulted in a numerically lower (P = 0.14) neutrophil:lymphocyte ratio when pigs were fed diets containing mannans compared with pigs fed the basal diet. An increase in the neutrophil:lymphocyte ratio is associated with stress in poultry (Gross and Siegel, 1983) and in the weaned pig (Stull et al., 1999). Pigs exposed to social stressors, such as the stress of mixing at weaning, have an elevated proportion of neutrophils in the blood (Morrow-Tesch et al., 1994). The greater proportion of blood lymphocytes and decrease in blood neutrophils observed when pigs were fed mannans may be an indication that mannan supplementation alleviates the alterations in immune function that result in an inflammatory response during the stress of weaning.
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), the proliferation of unstimulated lymphocytes, or the proliferation of lymphocytes stimulated with PHA, PWM, and ConA in vitro (Table 5). Alpha1-acid glycoprotein is one of many acute-phase proteins that have been reported to increase in association with an inflammatory challenge (Klasing and Johnstone, 1991). An increase in acute-phase proteins has usually been associated with an acute immune stimulus, such as the administration of LPS or a direct pathogenic challenge (Dritz et al., 1996; Williams et al., 1997). Because there was no direct immune challenge administered in this experiment, any alteration in AGP concentrations that may have occurred in response to dietary treatments in the presence of a disease challenge was not manifested, or may have been masked by the inclusion of antibiotic growth promoters. Although the lymphocyte proliferation response to mitogens administered in vitro was not impacted by dietary treatments in this experiment, there is some evidence that phosphorylated mannans may have an inhibitory effect on lymphocyte function (Muchmore et al., 1990; Podzorski et al., 1990).
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).
Gelderman et al. (1998) reported that macrophage function could be enhanced by the binding of mannose to receptors on the macrophage cell surface. In the Gelderman study, exposure of macrophages to myeloperoxidase, a high-mannose-containing enzyme released into the extracellular environment by phagocytic neutrophils, enhanced macrophage function by binding to the macrophage mannose receptor. Specifically, it induced an increase in the respiratory burst, phagocytosis, and intracellular killing of Candida albicans. The increase in the ability of jejunal lamina propria macrophages isolated from pigs fed mannans to phagocytose SRBC in this experiment may be a result of the macrophages’ exposure to mannose in the enteric environment
Flow Cytometry
On d 19 postweaning, pigs fed diets supplemented with mannans tended to have a greater (P < 0.10) proportion of CD14+ lamina propria leukocytes than did pigs fed the basal diet, whereas on d 21 after weaning, pigs fed mannans tended to have a lower (P < 0.10) percentage of CD14+MHCII+ leukocytes isolated from lamina propria tissue compared with pigs fed the basal diet (Table 6). On d 21 postweaning, the proportion of CD14+ blood leukocytes was lower (P < 0.05) when pigs were fed mannans compared with those fed the basal diet. Neither the proportion of CD14+ nor CD14+MHCII+ leukocytes differed in response to dietary treatment on d 24 and 26 after weaning.
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). Basta et al. (1999) assessed the differences in phenotypic markers and functions of porcine monocytes as they became activated and differentiated into macrophages. They reported that CD14 expression on monocytes was downregulated as they differentiated into macrophages and as their phagocytic and bacteriocidal activities increased. In addition, as the monocyte advanced in its state of activation, its ability to act as an antigen-presenting cell via MHCII expression declined. So, as the monocyte differentiates into an activated macrophage, expression of CD14 and MHCII on its cell surface decreases, and its phagocytic activity becomes its predominant function. The greater proportion of CD14+ leukocytes on d 19 after weaning indicates a greater proportion of macrophages present in the lamina propria tissue of pigs fed mannans compared with pigs fed the basal diet. This may indicate a greater infiltration of monocytes to the enteric lamina propria in mannan-fed pigs. Although only transiently observed on d 21 after weaning, the decrease in the proportions of CD14+MHCII+ lamina propria leukocytes observed in this study when pigs were fed mannans may indicate a maturation of tissue macrophage function, and supports the observed increase in phagocytic ability of jejunal lamina propria macrophages to phagocytose SRBC.
Although there were no differences in the ratios of T cells isolated from blood or intraepithelial tissue, pigs fed diets containing mannans had a lower (P < 0.05) ratio of CD3+ CD4+:CD3+CD8+ T lymphocytes isolated from jejunal lamina propria tissue on d 21 postweaning compared with pigs fed the basal diet (Table 7), indicating a greater proportion of CD8+ T cells in the lamina propria of mannan supplemented pigs. The establishment of the T cell population within the gastrointestinal tract of the young pig progresses from almost no T cells present in the intestine of the newborn pig, to the emergence of CD4+ T cells into the intestinal mucosa at approximately 2 to 4 wk of age, to the infiltration of CD8+ T cells within the intestinal lamina propria of the villi from 4 wk of age onward (Bailey et al., 2001). Additionally, McCracken et al. (1999) reported that the number of CD8+ T lymphocytes per 100 µm of villus isolated from the intestinal jejunum was increased postweaning. The low ratio of CD3+CD4+:CD3+CD8+ lymphocytes observed in this experiment is in agreement with the observations of McCracken et al. (1999) and the developmental progression of the CD8+ T cell populations reviewed by Bailey et al. (2001). The lower ratio of CD3+CD4+ :CD3+CD8+ lymphocytes observed when pigs were fed mannans suggests that there is a greater infiltration of CD3+CD8+ lymphocytes within the lamina propria of mannan-fed pigs, and could be an indication that mannan supplementation enhances the establishment of a mature T cell repertoire within the gastrointestinal tract of the 3- to 4-wk-old weaned pig. The isolated observation of this alteration in T lymphocyte ratios only on d 21 postweaning may be indicative of the time needed for the dietary supplementation of phosphorylated mannans to elicit an immune response.
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). Alternatively, because phosphorylated mannans have been documented to alter bacterial populations within the intestinal tract (Spring et al., 2000), the alterations in immune function observed in this study may have resulted from changes in the enteric microbial population by the presence of mannans in the luminal environment of the intestinal tract. The microflora present in the gastrointestinal tract are known to be a factor in the development of the young pig’s immune system, both enterically and systemically (Gaskins, 1997). It should be noted that alterations in immune responses as a result of phosphorylated mannan supplementation may have been masked by the dietary inclusion of growth-promoting antibiotics, and this may explain the inconsistent immune responses observed in this study. The immune function alteration observed in response to mannan supplementation may provide health benefits for combating disease challenges in addition to an improvement in growth performance of the weanling pig. Implications
Phosphorylated mannans may serve as a growth-enhancing additive in the diets of newly weaned pigs. The addition of phosphorylated mannans to weaned pig diets resulted in intermittent alterations of some of the immune system aspects measured in this study. Further investigation is warranted to better discern the mechanisms by which mannans periodically alter systemic and enteric immune function when supplemented to swine diets.
Footnotes
1 The authors express thanks to Alltech (Nicholasville, KY) for the kind donation of the phosphorylated mannan product (Bio-Mos) used in this study, T. Bersi for her technical assistance with the flow cytometric analysis, the University of Arkansas Cell Isolation and Characterization Facility for use of the flow cytometer and workstation, and A. Hays, C. Whiteside, M. Smith, and S. Arthur for animal husbandry and laboratory assistance. 
2 Correspondence: 1120 Maple St. AFLS B-107A (phone: 479-575-5036; fax: 479-575-7294; e-mail: medavis{at}uark.edu).
Received for publication August 25, 2003. Accepted for publication December 23, 2003.
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