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J. Anim. Sci. 2006. 84:567-578
© 2006 American Society of Animal Science

ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION

Ontogeny of T lymphocytes and intestinal morphological characteristics in neonatal pigs at different ages in the postnatal period1

D. C. Brown*,2, C. V. Maxwell*, G. F. Erf{dagger}, M. E. Davis*,3, S. Singh*,4 and Z. B. Johnson*

* Department of Animal Science and and {dagger} Center for Excellence in Poultry Science, University of Arkansas, Division of Agriculture, Fayetteville 72701


   Abstract
Top
 Abstract
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 IMPLICATIONS
 LITERATURE CITED
 
To evaluate morphological characteristics and development of the immune system at different ages in neonatal pigs, 4 piglets were euthanized at 7, 14, and 18 d of age for collection of blood, bile, and intestinal tissue for morphological measurements. Blood was collected for differential cell counts, lymphocyte blastogenesis, immunoglobulin (Ig) concentrations, cytokine concentrations, and flow cytometric analysis. Bile was collected for quantification of Ig-A and Ig-M. Villus width and crypt depth from duodenum sections, as well as ileum crypt depth, were reduced (P ≤ 0.08) in 18-d-old pigs compared with 7-d-old pigs. No age-related differences (P ≥ 0.11) were observed in the number of goblet cells with neutral and acidic mucins, serum or enteric Ig concentrations, IL-2, IL-4, spontaneous lymphocyte proliferation, or leukocyte concentrations. When measured as counts per minute (cpm) and as a stimulation index (SI), lymphocyte proliferation responses to phytohaemagglutinin increased (P = 0.05) between 7 and 14 d of age; no changes (P = 0.10) occurred at 18 d of age. No age-related changes (P = 0.39) were observed in response to pokeweed mitogen (PWM) when measured as cpm; however, the SI from PWM-induced lymphocytes decreased (P = 0.04) 4-fold between 7 and 18 d of age. The CD4+:CD8+ and populations of lymphocytes expressing CD2+CD4+CD8– (T helper cells) and CD25+CD4+CD8– (activated T helper cells) were greater (P ≥ 0.04) at 7 d of age than at 14 and 18 d. Populations of T lymphocytes, cytotoxic T cells (CD2+CD4–CD8+), activated lymphocytes (CD25+), and activated cytotoxic T cells (CD25+CD4–CD8+) were greater (P ≥ 0.02) in 18-d-old pigs compared with 7-d-old pigs, whereas CD2+CD4–CD8– [double negative cells] were lower (P = 0.08) in 18-d-old pigs compared with 14-d-old pigs. The percentage of CD2+ T cells was 8.4% at 7 d of age, and by the time the pigs reached 18 d of age, the percentage of CD2+ T cells was 33.8%. Moreover, the percentage of {gamma}{delta} T cells was greater (P = 0.02) in 18-d-old pigs than in 7-d-old pigs (74.8 vs. 46.1%, respectively). Results indicate that the porcine immune system and gut are continuously changing as the young pig matures. Changes occurred in lymphocyte phenotypic expression and functional capabilities, as well as morphology and mucin production, and their role may be to further protect the neonate from antigenic challenge as protection from passive immunity declines.

Key Words: development • goblet cell • immune system • morphology • preweaning • swine


   INTRODUCTION
Top
 Abstract
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 IMPLICATIONS
 LITERATURE CITED
 
Survival of the neonatal pig against enteric pathogens is dependent on gut development. At this time, the mucosal epithelium of the small intestine is anatomically and functionally immature (Tang et al., 1999Go). Postnatal gut development is important not only to protect the neonate from enteric pathogens but for digestion and absorption of nutrients for growth. Because the piglet immune system is immature at this stage, the neonate is dependent on specific immunity, either innate or passively acquired from the dam (Stokes and Bourne, 1989Go) to further protect it from enteric pathogens. However, development of the immune system of the neonatal pig is not well defined. Age-related changes have been observed in immunoglobulins (Ig) during the first 3 wk of life in pigs (McCauley and Hartmann, 1984Go). Studies have also reported limited responses to mitogens (substances that simulate cellular division; Valpotic et al., 1989Go; Becker and Misfeldt, 1993Go), whereas Hoskinson et al. (1990)Go demonstrated lymphocyte proliferation greatest at 0.5 wk of age. Percentages of cells expressing CD2+, CD4+, and CD8+ molecules in the blood change as the piglet ages (Becker and Misfeldt, 1993Go; Yang and Parkhouse, 1996Go), as do the number of leukocytes and lymphocytes (Becker and Misfeldt, 1993Go). Research identifying the populations of activated T cells and T cells with the {gamma}{delta}T cell receptor in pigs is limited. Therefore, the objectives of the experiment were to further evaluate gut morphology and the development of the porcine immune system based on Ig concentrations, cytokine concentrations, as well as determining cell numbers and function and phenotypic expression of surface antigens of lymphocytes isolated from the blood at different ages in the postnatal period.


   MATERIALS AND METHODS
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 IMPLICATIONS
 LITERATURE CITED
 
Animals
This study was conducted in accordance with the Animal Care Protocol No. 04014 for swine experiments issued by the University of Arkansas Animal Care Committee. Twelve crossbred sex pigs (Yorkshire, Landrace, and Duroc sows mated to DeKalb EB sires) were used for the experiment. Ten sows from one farrowing group were selected and maintained under conventional management conditions and practices at the University of Arkansas Research Station. Immediately after birth, teeth were clipped, tails were docked, iron injections were administered, and ears were notched for identification. Male piglets were surgically castrated 3 d after farrowing, and piglets had access to creep feed (a common 1.57% lysine Phase 1 corn-soybean diet containing 3.75% spray-dried plasma, 7% soy protein concentrate, and 17% lactose) 7 d after farrowing and thereafter. Piglets were allowed to suckle until weaning (d 19 of age) or until euthanized for the experiment (d 7, 14, or 18 of age). During the study, piglets in each farrowing crate were observed at least twice daily (early in the morning and again in the afternoon) to monitor health status. Six piglets appearing ill were removed and not utilized for the experiment.

At 7, 14, and 18 d of age, groups of 4 healthy piglets (2 barrows and 2 gilts) were selected from the sows, BW were recorded, and blood samples (20 mL) were collected into Vacutainer tubes containing EDTA (Becton Dickinson, Franklin Lakes, NJ) via vena cava puncture to determine differential leukocyte counts and to obtain blood for flow cytometric analysis. For each of the collection days, one piglet that represented the average BW was selected from 4 randomly selected sows so that all litters were represented at least once and only 2 litters were represented twice. Piglets were also selected so that piglets from the same litter were not euthanized on the same collection day. After blood was taken, piglets were rendered unconscious and insensitive to pain by captive-bolt stunning and, subsequently, were exsanguinated, which was carried out in accordance with the Animal Care Protocol No. 04014 for swine experiments issued by the University of Arkansas Animal Care Committee. Furthermore, bile was collected from each piglet after exsanguination and stored at –80°C until analyzed for IgA and IgM by ELISA.

Ig Analysis
After exsanguination, bile and serum were collected for quantification of IgA and IgM. Bile and serum concentrations of IgA and IgM were determined using pig IgA and IgM ELISA kits in accordance to manufacturer (Bethyl Laboratories, Montgomery, TX) instructions. The range of the IgA and IgM assays was 7.81 to 1,000 ng/mL; sensitivity was 2.0 ng/mL. The intraassay CV for IgA and IgM in bile was 1.9 and 4.9%, respectively, whereas the intraassay CV for serum IgA and IgM was 2.9 and 3.1%, respectively.

Differential Blood Leukocyte Concentrations
Whole blood samples from pigs at 7, 14, and 18 d of age were analyzed for differential leukocyte proportions and concentrations on a multiparameter, automated hematology analyzer (CELL-DYN 3500SL System, Abbott, Abbot Park, IL) calibrated for porcine blood. Proportions of lymphocytes, neutrophils, monocytes, and eosinophils were calculated as a percentage of the leukocyte concentration.

Lymphocyte Blastogenesis
In vitro cellular immune activity was measured using a lymphocyte blastogenesis assay adapted from Blecha et al. (1983)Go and previously described in detail (Davis et al., 2002Go). Phytohemagglutinin (PHA; Sigma Chemical Co., St. Louis, MO, which stimulates mainly T cells and some B cells) and pokeweed mitogen (PWM; Sigma Chemical Co., which stimulates mainly B cells) were administered at a concentration of 50 and 25 µg/mL, respectively, to stimulate lymphocyte proliferation. Incubation, labeling with [3H]thymidine, and cell harvesting procedures followed those outlined by van Heugten and Spears (1997)Go. Cells were harvested on glass fiber mats and were placed in scintillation tubes containing scintillation fluid; radioactivity was measured as counts per minute (cpm) on a liquid scintillation analyzer (TRI-CARB 2200CA, Packard Instrument Co., Downers Grove, IL). To standardize the data for comparison, a stimulation index (SI) was calculated by subtracting the cpm of the unstimulated cultures from cpm of the the stimulated cultures and dividing by cpm of the the unstimulated cultures.

Cytokine Analysis
For IL-2 and IL-4 production, peripheral blood mononuclear cells were resuspended in media at 2 x 106 cells/mL and plated in duplicate in 24-well plates in 1,000-µL aliquots with or without 10 µg of Concanavalin-A [ConA (stimulates T cells); Sigma Chemical Co.]/mL to stimulate cytokine production. Cultures were incubated for 48 h at 39.2°C and 5% CO2, and media were collected from wells and stored at –20°C until analyzed for IL-2 and IL-4 concentrations. Production of IL-2 and IL-4 by cultures was determined using pig IL-2 and IL-4 ELISA kits following manufacturer instructions (Biosource, Camarillo, CA). The dynamic ranges for the IL-2 and IL-4 assays were 2.78 to 2,850 pg/mL and 4.88 to 2,500 pg/mL, respectively, with a sensitivity of 2.0 pg/mL. The intraassay CV for IL-2 and IL-4 assays were 6.3 and 11.5%, respectively.

Tissue Collection and Histology
Following exsanguination, samples of duodenum (15 cm proximal to the pyloric junction), jejunum (55 cm proximal to the pyloric junction), and ileum (15 cm distal to the ileocaecal junction) were obtained from each piglet and placed on ice. Duodenal, jejunal, and ileal samples were cut longitudinally at the antimesenteric attachment and immediately fixed in 10% neutral-buffered formalin as described by Jaegar et al. (1990) and Nunez et al. (1996)Go. After fixation, samples were embedded in paraffin.

Sections, 4 to 6 µm, were sliced on a microtome (Lipshaw, Pittsburgh, PA), mounted on slides, and stained with hematoxylin and eosin. Villus height, area, and crypt depth were evaluated using the Image-Pro Plus image analysis program (version 5.0, Media Cybernetics, Houston, TX) and a bright-field microscope at 4x magnification. Villus height, area, and crypt depth were measured according to the method of Jaeger et al. (1990)Go and Nunez et al. (1996)Go, and values for each tissue were based on the average measurements of 10 villi or 10 crypts.

Additionally, 6-µm sections were sliced on a microtome (Lipshaw) subsequently mounted on glass slides, and stained with alcian blue (pH 2.5) and periodic acid-Schiff’s for determination of acidic (sialomucins) and neutral carbohydrates, respectively, or with high iron diamine and alcian blue (pH 1.0) for determination of sulfate (sulphomucins)-containing goblet cells. Positively stained goblet cells were counted within 10 randomly selected villi from each tissue from each piglet and the average of the 10 villi were used for analysis.

Monoclonal Antibodies
Monoclonal antibodies used in this study specific for swine leukocytes and dilutions are reported in Table 1Go. A panel of 7 commercially available mouse monoclonal antibodies were used to identify pig T lymphocytes [CD2, T cell subset (70% of T cells); VMRD, Inc., Pullman, WA], T lymphocytes (CD3, all T cells; Southern Biotechnology Assoc., Inc., Birmingham, AL), T helper lymphocytes (CD4; Southern Biotechnology Assoc., Inc.), cytotoxic T lymphocytes (CD8; Southern Biotechnology Assoc., Inc.), {gamma}/{delta}T cell receptor ({gamma}/{delta}TCR; VMRD, Inc.), activated T and B lymphocytes [CD25 (IL-2 receptor); VMRD, Inc.], and major histocompatibility class II (MHC-II; VMRD, Inc).


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  		Table 1. Swine-specific monoclonal antibodies and secondary antibodies used to define cell surface molecule expression and differential populations of leukocytes derived from peripheral blood
 
Monoclonal antibodies specific for CD3 and CD4 were directly labeled with R-phycoerythrin (PE) and fluorescein isothiocyanate (FITC), respectively, whereas, biotinylated primary antibodies (CD8) were detected by Quantum Red (QR)-labeled streptavidin (Sigma Chemical Co.). Primary antibodies specific for CD2, {gamma}/{delta} TCR, CD25, and MHC-II were identified with PE-conjugated goat polyclonal antibodies specific for mouse subclasses (Sigma Chemical Co.). Isotype control monoclonal antibodies with irrelevant specificity were included to assess nonspecific labeling.

Staining of Lymphocytes
All immunoreagents were tested for appropriate binding of monoclonal antibodies to leukocytes of interest. Unlabeled cells were used as a negative control for innate fluorescence detectable in cell suspensions. Labeled isotype controls were used to assess nonspecific binding of the directly conjugated monoclonal antibodies, whereas incubating cells with an unlabeled isotype control in the place of the primary antibody assessed nonspecific binding by labeled secondary antibodies. To conduct compensation [e.g., subtract detection of fluorescent laser (FL)-1 (FITC) from the FL-2 detector (PE), subtract detection of FL-2 from the FL-1, and subtract detection of FL-3 detector (QR) from the FL-1 and FL-2 detectors], single color-labeled cell suspensions were used.

Peripheral blood mononuclear cells were prepared for flow cytometric analysis using the procedures of Mishell and Shiigi (1980)Go. Peripheral blood mononuclear cells were resuspended to a minimum concentration of 105 cells/mL in RPMI medium devoid of phenol red (Sigma Chemical Co.). Cell suspensions were then administered in 50-µL aliquots to wells of 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 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 washing plates twice with 180 µL of PBS+ and centrifuging at 180 x g for 4 min at 4°C. After washing, if the primary antibody was unconjugated or biotinylated, 50 µL of conjugated goat polyclonal antibodies for mouse Ig subclasses (with an appropriate fluorochrome or QR-labeled streptavidin; 1:50 dilution) were administered for 20 min at room temperature for double indirect staining. Excess antibody was removed by washing plates twice with 180 µL of PBS+ and centrifuging at 180 x g for 4 min at 4°C. Contents of wells were removed and placed into Falcon tubes (Sigma Chemical Co.) to evaluate cell populations. Dual fluorescence staining was performed to identify CD4+CD8–, CD4–CD8+, CD4+CD8+, and CD4–CD8– lymphocyte proportions.

For triple indirect staining, double-stained cells (CD4+CD8+) were incubated for 30 min at 4°C with 50 µL of unconjugated monoclonal antibodies specific for CD2, CD25 (IL-2), or {gamma}/{delta} TCR. Following this incubation, excess antibody was removed by washing plates twice with 180 µL of PBS+ and centrifuging at 180 x g for 4 min at 4°C. After washing, 50 µL of PE-conjugated goat polyclonal antibody (1:50 dilution) was administered for 20 min at room temperature. Excess antibody was removed by washing plates twice with 180 µL of PBS+ and centrifuging at 180 x g for 4 min at 4°C. Contents of wells were removed and placed into 5-mL Falcon tubes to evaluate cell populations.

Flow Cytometry
A FACSort flow cytometer and CellQuest software (Becton-Dickinson Immunocytometry Systems, San Jose, CA) were used to conduct one-, 2- and 3-color cell population analyses. Unlabeled cells were used to display the cell populations based on their light scattering properties in the forward and 90-degree direction (to indicate size and internal complexity of cells, respectively). A gate was drawn around the live cell population to exclude dead cells and debris that might have remained in the cell suspension. Gated unlabeled cells were also used as a negative control such that any autofluorescence detected from these cells was held into account by positioning the unlabeled cells in the lower left quadrant (negative fluorescence) in a dot plot fluorescence display of PE vs. FITC, PE vs. QR, and FITC vs. QR. Similarly, in the absence of nonspecific binding, the cells in the cell suspensions stained to determine nonspecific binding of the detection antibodies (nonspecific staining controls described previously) were displayed in the lower left quadrant on the appropriate dot plot fluorescence display. Hence, using these negative staining controls, the quadrants delineating fluorescence positive and fluorescence negative cell populations could be placed with confidence.

Statistical Analysis
All experimental data are presented as least squares means ± SEM. The data were analyzed as a completely randomized design with treatments arranged in a 2 x 3 factorial. The model included terms for day, sex, and the day x sex interaction; individual piglets were the experimental units. Data were subjected to an ANOVA using the GLM procedure of SAS (SAS Inst., Inc., Cary, NC). When a significant interaction was observed, interaction treatment means were separated using the PDIFF option of the LSMEANS statement in PROC GLM. Main effect means were evaluated when the interaction was not significant (P > 0.10). There were no sex or day x sex effects for any of the immunological or morphology data except for the proportions of CD25+ lymphocytes, so this table is the only data that incorporates the day x sex interaction.

For evaluating the different goblet cell types over time, the model included terms for day, sex, goblet cell type, sex x goblet cell type interaction, day x goblet cell type interaction, and the day x sex x goblet cell type interaction. When a significant interaction was observed, interaction treatment means were separated using the PDIFF option of the LSMEANS statement in PROC GLM. Main effect means were evaluated when the interaction was not significant (P > 0.10).


   RESULTS AND DISCUSSION
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
IMPLICATIONS
 LITERATURE CITED
 
Differential Blood Leukocyte Concentrations
Numbers and percentages of neutrophils, lymphocytes, monocytes, and eosinophils did not (P = 0.14) change with age (Table 2Go) and were similar to those reported by others in porcine blood (Duncan and Prasse, 1978Go). However, Becker and Misfeldt (1993)Go observed that the numbers of leukocytes and lymphocytes obtained from pigs at d 1, 18 to 19, and 27 to 30 increased with age, whereas the number of neutrophils did not change from d 1 to 30. Other researchers have reported that the percentage of neutrophils exceeds the percentage of lymphocytes at birth, but their ratio is reversed by about 10 d of age (Gardiner et al., 1953Go).


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  		Table 2. Differential leukocyte counts (total cells and percentage of white blood cells; WBC) of pigs sampled at 7, 14, and 18 d of age
 
In the current study, the percentage of neutrophils exceeded the percentage of lymphocytes at 14 d of age. Differences in the percentages of neutrophils and lymphocytes observed between studies could be due to different environments in which the pigs were reared. A study by McTaggart and Rowntree (1969)Go found that pigs reared under conventional conditions had greater total leukocyte number because of increased numbers of neutrophils than pigs reared under minimal disease conditions, suggesting that the environment may have a role in the presence of this defensive, inflammatory cell.

Flow Cytometry
Several studies have characterized T lymphocyte subpopulations in the mature pig (Jonjic et al., 1987Go; Lunney and Pescovitz, 1987Go; Saalmuller et al., 1989Go) and in the neonatal pig at 1, 16, and 28 d of age (Becker and Misfeldt, 1993Go). Although there has been research on the characterization of the T lymphocyte subpopulations at earlier stages in the life of the pig, there has been limited research to further identify the populations of activated T cells and T cells with the {gamma}{delta} TCR in neonatal pigs. There are 4 {alpha}ß TCR T-cell subsets (CD2+CD4+CD8–, CD2+CD4–CD8+, CD2+CD4+CD8+, and CD2+CD4–CD8–) and 3 T cell subsets with the {gamma}{delta} TCR such as, CD2+CD4–CD8+, CD2+CD4–CD8–, and CD2–CD4–CD8– (Yang and Parkhouse, 1996Go; Sinkora et al., 1998Go), that have been identified in porcine peripheral blood.

The current study also detected these T cell subpopulations in the peripheral blood of neonatal pigs (Tables 3Go, 4Go, 5Go, 6Go, and 7Go). The current study, as well as other research (Becker and Misfeldt, 1993Go; Yang and Parkhouse, 1996Go), has detected age-related differences in T cell subpopulations in the neonatal pig. The proportion of cytotoxic T cells expressing CD2 (Table 6Go), activated cytotoxic T cells (Table 7Go), and activated T and/or B cells (Table 7Go) increased (P ≤ 0.02) as the young pig aged, regardless of pig sex. As the pig ages and maternal Ig decrease in the milk, the pig must rely on its own immune system for protection from antigenic stimuli. Therefore, the increase in the proportion of activated T cells as the pig becomes older may be due to antigenic stimuli in the environment of the pig initiating a cellular immune response and may also indicate that the piglet is more dependent upon its own immune system to eliminate an antigenic challenge rather than passive immunity from the sow.


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  		Table 3. Blood T lymphocytes and the proportions of CD4+, CD8+, {gamma}{delta} T cell receptor (TCR), and CD25+ blood lymphocytes in pigs at 7, 14, and 18 d of age
 

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  		Table 4. Blood CD4- and CD8-defined T cell subsets (%) in pigs at 7, 14, and 18 d of age
 

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  		Table 5. The proportions of {gamma}{delta} T cell receptor (TCR) lymphocytes and their CD4- and CD8-defined subsets in pigs at 7, 14, and 18 d of age
 

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  		Table 6. The proportions of CD2 blood lymphocytes and their CD4- and/or CD8-defined subsets in pigs at 7, 14, and 18 d of age
 

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  		Table 7. The proportions of CD25 (IL-2 receptor) blood lymphocytes and their CD4- and CD8-defined subsets in male and female pigs at 7, 14, and 18 d of age1
 
In the current study, the CD4+:CD8+ ratio in 7-d-old pigs was 1.16, which was greater (P = 0.04) than those of 14- and 18-d-old pigs (Table 3Go). This high CD4+:CD8+ ratio was due to elevated (P = 0.001) populations of T helper cells in the peripheral blood on d 7 (Table 6Go). The CD4+:CD8+ ratios of 14- and 18-d-old pigs in the current study were 0.40 and 0.27, respectively, which were similar to those reported by Becker and Misfeldt (1993)Go where the CD4+:CD8+ ratio in peripheral blood of 18- to 19-d-old pigs and 27- to 30-d-old pigs was 0.19 and 0.36, respectively. The population of activated T helper cells was greater (P = 0.02) in 7-d-old pigs compared with 14- and 18-d-old pigs; female piglets had a lower (P = 0.02) population of activated T helper cells compared with male piglets at d 7 (day x sex interaction; Table 7Go). Additionally, the population of activated memory cells was greater in 7-d-old mixed sex pigs compared with 14-d-old mixed sex pigs and 18-d-old male pigs but was similar (P ≥ 0.53) to 18-d-old female pigs (day x sex interaction; Table 7Go).

Studies have shown that infections in chicks (Lillehoj, 1994Go) and children (Hashimoto et al., 1994Go) can cause greater CD4+:CD8+ ratios. A rise in the number of helper T cells indicates that antigen-presenting cells, such as macrophages, are presenting a greater amount of foreign antigens (Matis, 1990Go), which may result in more cytokine production and subsequent cellular and humoral immune activation (Tonegawa, 1985Go). Therefore, the changes in the ratio of CD4+:CD8+ T cells may indicate that the 7-d-old pigs are undergoing an antigenic challenge that requires cellular and humoral immune activation.

In the current study, age-related differences (P = 0.02) were detected in the percentage of CD2+ T cells (Table 6Go). The percentage of CD2+ T cells was 8.4% at 7 d of age, and by the time the pigs reached 18 d of age, the percentage of CD2+ T cells was 33.8%. Research has shown that young pigs have a low frequency of the CD2+ T cells in their peripheral blood (Yang and Parkhouse, 1996Go). Yang and Parkhouse (1996)Go reported that the percentage of CD2+ T cells (with the {alpha}ß TCR) found in the peripheral blood of 4-wk-old pigs was only about 12% of the T cell population; however, the population of this T cell subset was greater in 8-, 12- and 16-mo-old pigs (30, 48.8, and 34.2%, respectively). Other studies have shown that the majority of T cells are {gamma}{delta} T cells (~20.0%) in blood of neonatal pigs (Yang and Parkhouse, 1996Go; Solano-Aguilar et al., 2001Go). This was similar to the findings of the current study, where there were high percentages of {gamma}{delta} T cells found in circulation of 7-, 14-, and 18-d old pigs (46.1, 51.6, and 74.8%, respectively; Table 3Go). Additionally, the percentage of {gamma}{delta} T cells was greater (P = 0.02) in 18-d-old pigs compared with 7-d-old pigs [74.8 vs. 46.1%, respectively (Table 5Go)]. In the current study, younger pigs (7 d) appear to have a low percentage of CD2+ (all {alpha}ß TCR and a small subset of {gamma}{delta} TCR) cells and a high percentage of {gamma}{delta} T cells, and as the piglet ages, the percentages of CD2+ (all {alpha}ß TCR and a small subset of {gamma}{delta} TCR) cells and {gamma}{delta} T cells also increase. Results from the current study indicate that during the first few weeks of life, the young pig may rely on {gamma}{delta} T-cell activity more than its {alpha}ß T-cell repertoire. As the pig gets closer to the weaning age (19 to 21 d of age) and experiences antigens, it relies on the further development of {gamma}{delta} T cell repertoire, as well as the development of the {alpha}ß T-cell repertoire.

The T cell subset with the phenotype CD4-CD8– (CD2+ and CD2– subsets) expressing the {gamma}{delta} TCR are a unique T cell population observed in the peripheral blood of swine (Binns et al., 1992Go), cattle, and sheep, but not in human or rodent circulation (Haas et al., 1990Go; Hein and Mackay, 1991Go). Studies have shown that most of the population of peripheral CD4–CD8– [double negative (DN)] cells are negative for CD2 and express {gamma}{delta} TCR chains (Saalmuller et al., 1989Go). In the current study, however, we observed that the populations of DN cells expressing CD2 or {gamma}/{delta} TCR chains were similar in number (Tables 5Go and 6Go, respectively). There were no (P ≥ 0.18) age-related changes in the populations of DN cells expressing the {gamma}/{delta} TCR, and the populations of DN expressing CD2 were greater (P = 0.08) at d 14 of age than at d 18 of age. The exact function of this subpopulation of T cells is still unclear because there is no information concerning the antigens recognized or surface molecules involved in their functional capabilities. However, there is evidence that {gamma}/{delta} TCR T cells can recognize proteins directly without antigen processing in association with MHC molecules, such as nonpolymorphic MHC-like molecules (Abbas et al., 1997Go). Furthermore, the populations of DN cells expressing CD25 were greater (P = 0.08) at d 14 of age than at d 18 of age (Table 7Go).

Another population of T cells detected in peripheral blood of piglets in the current study that is unique to the porcine immune system are CD4+CD8+ (double positive; DP) cells with {alpha}ß TCR chains. Although there were no (P ≥ 0.31) age-related changes in the proportion of DP cells (Table 4Go) or DP cells expressing CD2 (Table 6Go), the population of activated DP cells (Table 7Go) decreased (P = 0.08) with age. Circulating DP T cells are found in high proportion in swine and have a morphological phenotype similar to mature resting T lymphocytes (Saalmuller, 1998Go). Saalmuller (1998)Go observed that these extrathymic DP T lymphocytes are different from DP thymocytes in both size and surface marker expression (they show no expression of the thymocyte-specific CD1 antigen). Similarly to CD4+CD8– (T helper cells), extrathymic DP T lymphocytes respond to mitogen and to alloantigen in mixed leukocyte cultures (Saalmuller, 1998Go). Furthermore, both subpopulations (CD4+CD8+ and CD4+CD8–) can induce an MHC-II restricted proliferative immune response and synthesis of cytokines (IL-2 and 4; Summerfield et al., 1996Go), as well as show T helper cell function for the generation of alloantigen-specific cytolytic T cells and T-cell-dependent in vitro synthesis of Ig (Saalmuller, 1998Go). Although both T helper lymphocytes and DP lymphocytes are able to react during a primary response, only DP lymphocytes show a significant antigen-specific secondary immune response that is MHC-II restricted, and the additional expression of CD8 molecule seems to maintain no CD8-specific functional activity (Summerfield et al., 1996Go).

Lymphocyte Proliferation
Proliferative responses for unstimulated and mitogen-stimulated lymphocytes isolated from the peripheral blood on d 7, 14, and 18 are presented in Table 8Go. Spontaneous proliferation by unstimulated lymphocytes isolated from peripheral blood was not (P ≥ 0.16) affected by age. These results are similar to those of Becker and Misfeldt (1993)Go, who reported that spontaneous lymphocyte proliferation was not affected in lymphocytes isolated from peripheral blood in 1-, 16-, and 28-d-old pigs; however, spontaneous proliferation in lymphocytes from the spleen and thymus increased with age between d 16 and 28. In contrast, when using lymphocytes isolated from peripheral blood from pigs at 0.5, 1, 3, and 6 wk of age, Hoskinson et al. (1990)Go reported that the rate of spontaneous proliferation by unstimulated lymphocytes varied with age. Spontaneous lymphocyte proliferation was greatest at 0.5 wk, decreased 75% by 1 wk of age, and then decreased more gradually through 6 wk of age.


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  		Table 8. Lymphocyte proliferation, cytokine concentrations, and peripheral and enteric immunoglobulin (Ig) concentrations pigs sampled at 7, 14, and 18 d of age
 
Differences between studies could be due to variation in age in which each study isolated lymphocytes from the piglet. Another possible reason for the variation could be due to the different levels of antigenic exposure within the surrounding environment of the piglet and its effects on the maturation or activation of lymphocytes. If the young pig is at a greater level of antigentic exposure, there may be a greater proportion of immune cells in an activated state as well as a greater proportion of immature immune cells being produced to mature into functional T helper or cytotoxic T cells that can facilitate antigen elimination. These immune cell populations (activated and immature T cells) seem to have an inherently greater spontaneous proliferation rate, which could lead to differences observed between the studies.

In the current study, responses to PHA were greater (P = 0.04) at 14 and 18 d of age in the peripheral blood than at 7 d of age (Table 8Go). This increase in proliferation of lymphocytes (mainly T cells) isolated from the blood in response to PHA has also been observed in pigs between 16 and 18 d of age in peripheral blood and thymus; however, the proliferation of cells isolated from the spleen were not affected by age (Becker and Misfeldt, 1993Go). The observed increase in response to PHA may also be a result of age-related differences in circulating cortisol concentrations in the piglet. Pigs are born with elevated concentrations of cortisol (Dvorak, 1972Go), which may suppress immune function in the neonate. Physiologic concentrations of cortisol have been shown to reduce the capability of porcine lymphoid cells to proliferate in vitro (Kelley et al., 1982Go). Restraining 8-wk old piglets increased concentrations of cortisol and decreased responses to intradermal PHA (Westly and Kelley, 1984Go). Furthermore, lymphocytes isolated from the peripheral blood have a lower proliferative response to mitogen in pigs undergoing social and environmental stressors (Hicks et al., 1998Go). Therefore, as the age of the pig increases, peripheral lymphocytes have a greater functional capacity, which may be due to decreased concentrations of cortisol.

Lymphocyte proliferation induced by PWM was not (P = 0.39) affected by age (Table 8Go) of neonatal pigs in the current study. These results are consistent with those of Hoskinson et al. (1990)Go, who reported that proliferative responses to PWM from blood lymphocytes (mainly B cells) did not change between 0.5 and 3 wk of age. However, in the current study, the SI from PWM-induced lymphocytes decreased (P = 0.04) 4-fold between 7 and 18 d of age. These results contradict studies indicating that SI from PWM-induced lymphocytes increased about 5-fold between birth and 6 wk of age in pigs (Hoskinson et al., 1990Go) and from 1 to 10 d of age in calves (Manak, 1986Go).

Cytokine Profiles and Ig
There were no (P ≥ 0.59) age-related differences observed in the production of IL-2 or IL-4 from ConA-stimulated peripheral mononuclear cells (Table 8Go). Additionally, no age-related differences (P ≥ 0.21) were observed in serum or bile Ig concentrations (Table 8Go). To our knowledge, this study is the first to establish Ig concentrations from the bile in the developing piglet. Bile Ig concentrations may provide a better determination of the secretion of enteric IgA and IgM in the individual piglet, given that there may be contamination of maternal Ig in the gastrointestinal tract because of injection of milk from the sow. Greater concentrations of IgA were observed in the bile at 7, 14, and 18 d of age compared with concentrations of IgM. Several studies have shown that IgA derived from the mucosal lamina propria eludes the epithelial secretory component and leaves the site of the plasma cell secretion via circulation to the liver, which is then transported through the biliary tract back to the upper intestine (Jackson et al., 1978Go; Manning et al., 1984Go). The function of retrieval of IgA for the gut is performed by hepatocytes that synthesize secretory component, which appears at the sinusoidal plasma membrane border of the hepatic cell. Dimeric IgA binds to the hepatocyte and is transported to the bile cannaliculis similar to the process of transport in the gut (Schreiber and Walker, 1988Go).

The exact role of hepatic plasma cells and biliary secretory IgA production, in terms of mucosal defense, is not yet fully appreciated. A study by Altorfer et al. (1987)Go has demonstrated that after a primary mucosal immunization with cholera toxin, specific IgA-secreting plasma cells appeared in the liver before these cells were found in the lamina propria, suggesting that the liver may have a central role in protecting against newly acquired intestinal antigens. Therefore, in young pigs that have a compromised immune system, this hepatic-derived secretory IgA may be essential to protect the neonate from an enteric pathogenic challenge by neutralizing viruses, inhibiting bacterial attachment, and by opsonizing or lysing bacteria at the mucosal level (Porter, 1986Go).

Villus and Crypt Architecture
Morphometric measurements of villus height and width, crypt depth, and the villus height:crypt depth ratio of the duodenum, jejunum, and ileum from 7-, 14-, and 18-d-old pigs are presented in Table 9Go. There were no (P ≥ 0.15) age-related changes observed in duodenum, jejunum, and ileum villus height or the villus height:crypt depth ratio. These results are similar to those of 15-d-old pigs, where villus height was not markedly different from those of newborn pigs (Smith, 1984Go). However, in the current study, villus width and crypt depth from duodenal sections, as well as ileal crypt depth, were reduced (P ≤ 0.08) in 18-d-old pigs compared with 7-d-old pigs. This reduction (P ≤ 0.07) in villus width and crypt was also apparent in jejunal sections in 18-d-old pigs compared with 14-d-old pigs.


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  		Table 9. Morphological measurements of villus height and width, crypt depth, and villus height:crypt depth ratio (V:C) from duodenum, jejunum, and ileum intestinal sections taken from pigs at 7, 14, and 18 d of age
 
During normal neonatal development, changes in villus height are normally accompanied by secondary changes in the 3-D structure of villi (Paterson and Smith, 1983Go). Furthermore, similar changes in intestinal villus shape can also be detected in neonatal pigs. Smith (1984)Go observed that the intestinal villus of 15-d-old pigs was cylindrical and changed to a conical shape over the next 6 d. Alterations in intestinal villi may account for the changes observed in villus width in the present experiment between 7- and 18-d-old pigs.

Before the weaning period, piglets can consume small quantities of sow feed or creep feed causing local immunological responses to the soy protein. Bourne (1984)Go has explained that feeding soy protein before weaning can compromise intestinal morphology by increasing crypt cell division, and the appearance of immature enterocytes on the villus, causing the piglet to become more susceptibile to pathogenic challenges. However, Hampson (1986)Go observed that introducing creep feed at 10 d of age did not influence small intestine structure in unweaned pigs up to 32 d of age. They concluded that changes observed in intestinal morphology may be due to an interaction between introduced creep feed and intestinal microflora. The succession of microbes colonizing the gastrointestinal tract of the piglet occurs during early development and can shift in response to diet (Mackie et al., 1999Go). Therefore, morphological alterations in the gastrointestinal tract of piglets before weaning may be associated with changes in gut microbial populations in response to creep feed as well as sow feed.

Gastrointestinal Mucin Production
Mucins are considered to be an important determinant of gut health and disease; however, results with pigs are scarce and inconsistent. Interspersed among the absorptive cells of the intestinal epithelia, goblet cells function in the synthesis of water-soluble mucins and trefoil peptides to form a continuous gel on the mucosal surface (Kindon et al., 1995Go; Matsuo et al., 1997Go). The mucus layer constitutes a physical barrier between the lumen and epithelium as well as an important framework for host-bacteria and bacteria-bacteria interactions (Bourlioux et al., 2003Go). Mucin released from goblet cells protects the epithelial cells from digestive enzymes produced by intestinal flora and provides an extensive barrier to prevent penetration of potential pathogens into epithelium and bacterial overgrowth (Neutra and Forstner, 1987Go). Bacterial infection (Cohen et al., 1983Go), resident intestinal flora (Mantle et al., 1989Go), toxins (Roomi et al., 1984Go), and parasitic infestation (Miller et al., 1981Go) are all factors that can stimulate the release of mucins from goblet cells.

In the current study, there were no (P ≥ 0.36) age-related changes in the number of goblet cells with neutral or acidic mucins within the duodenum, jejunum, or ileum of the gastrointestinal tract of piglets (Table 10Go). However, there was a greater (P = 0.001) proportion of sulfomucins found in the duodenum at d 7, 14, and 18 of age and in the jejunum at d 7 and 14 of age than neutral and acidic mucins (Table 10Go). Furthermore, this greater proportion of sulfomucins at 7, 14, and 18 d of age than neutral and acidic mucins was also detected in the ileum as well as a greater proportion of sulfomucins on d 18 than on d 14 (day x goblet cell, P = 0.03; Table 10Go). This greater proportion of sulfomucins may protect the neonate from enteric infections.


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  		Table 10. The production of 3 different mucins (neutral, acidic, and sulfuric) within the duodenum, jejunum, and ileum of the gastrointestinal tract taken from pigs at 7, 14, and 18 d of age
 
For example, the occurrence of severe colitis is correlated with depletion of sulfate mucins in humans (Probert et al., 1995Go). A study by Strous and Dekker (1992)Go found that the presence of abundant sulfate and sialic acid on the mucins of goblet cells are important for gel formation, and, thus, for maintenance of the protective layer on the mucosal surface of the small intestine to defend the animal against enteric pathogens. Brunsgaard (1997)Go showed that during the first 3 to 4 mo of life, pigs had increased production of mucin secretion (especially the sulfomucins) in their small intestine, which may contribute to decreased susceptibility to enteric infections as the pig matures.

Conclusions
In conclusion, results of the current study indicate that the immune system is continuously changing as the young pig matures. There are changes in the phenotypic expression of peripheral blood lymphocytes, and these cells appear to have a greater functional capacity, as determined by mitogen-induced proliferation. During the first few weeks of life, the young pig may rely on {gamma}{delta} T cell activity, and later, once the piglet experiences antigen, it relies on further development of the {gamma}{delta} T cell repertoire, as well as the development of the {alpha}ß T cell repertoire and the population of cytotoxic T cells for protection from antigenic challenges. Furthermore, as the young pig ages, there are also alterations in villus-to-crypt architecture and mucin production from goblet cells in the intestinal tract. These changes in lymphocyte phenotypic expression and functional capabilities, as well as mucin production, may be to further protect the neonate from antigenic challenge as protection from passive immunity declines.


   IMPLICATIONS
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 IMPLICATIONS
LITERATURE CITED
 
Changes are occurring in lymphocyte phenotypic expression and functional capabilities, as well as morphology and mucin production, as the young pig matures. These changes may be important for survival against an antigenic challenge as protection from maternal passive immunity declines. It becomes important to understand and define the development of the immune system of the piglet and gut morphology throughout production. This knowledge can provide valuable information on how to augment this complex system to allow the neonatal pig to respond more efficiently to immunological challenges improving survival rates and growth response.


   Footnotes
 
1 Authors acknowledge Agtech Products, Inc. for their financial support, Jason K. Apple and Elizabeth Kegley for their critical review of the manuscript, Jerry D. Stephenson for his assistance in collection of pig tissues, and Ashley Hayes, Casey Whiteside, Misty Smith, and Butch Watson for animal husbandry. Back

3 Present address: Agtech Products, Inc., Waukesha, WI 53186. Back

4 Present address: Harrison Memorial Animal Hospital, Denver, CO 80223. Back

2 Corresponding author: dlance{at}uark.edu

Received for publication June 7, 2005. Accepted for publication October 12, 2005.


   LITERATURE CITED
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 IMPLICATIONS
 LITERATURE CITED
 


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