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,† M. E. Davis,*3, S. Singh,*4 and Z. B. Johnson*
*Department of Animal Science and †Center for Excellence in Poultry Science, University of Arkansas,
Division of Agriculture, Fayetteville 72701
ABSTRACT: 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 PWMinduced lymphocytes decreased (P = 0.04) 4-fold be-
tween 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 γδ 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
2006 American Society of Animal Science. All rights reserved.
J. Anim. Sci. 2006. 84:567–578
the mucosal epithelium of the small intestine is anatomically and functionally immature (Tang et al., 1999).
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, 1989) 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, 1984). Studies have also reported limited
responses to mitogens (substances that simulate cellular division; Valpotic et al., 1989; Becker and Misfeldt,
INTRODUCTION
Survival of the neonatal pig against enteric pathogens is dependent on gut development. At this time,
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.
2
Corresponding author: dlance@uark.edu
3
Present address: Agtech Products, Inc., Waukesha, WI 53186.
4
Present address: Harrison Memorial Animal Hospital, Denver,
CO 80223.
Received June 7, 2005.
Accepted October 12, 2005.
567
568
Brown et al.
1993), whereas Hoskinson et al. (1990) 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, 1993; Yang and Parkhouse, 1996), as do
the number of leukocytes and lymphocytes (Becker and
Misfeldt, 1993). Research identifying the populations
of activated T cells and T cells with the γδ 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
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 accor-
dance 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) and previously described in detail (Davis et
al., 2002). 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 ␮g/mL,
respectively, to stimulate lymphocyte proliferation. Incubation, labeling with [3H]thymidine, and cell harvesting procedures followed those outlined by van Heugten
and Spears (1997). 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 × 106 cells/
mL and plated in duplicate in 24-well plates in 1,000-
Neonatal pig intestinal morphology and T cell ontogeny
␮L aliquots with or without 10 ␮g 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). After fixation, samples were embedded in paraffin.
Sections, 4 to 6 ␮m, 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 4×
magnification. Villus height, area, and crypt depth were
measured according to the method of Jaeger et al. (1990)
and Nunez et al. (1996), and values for each tissue
were based on the average measurements of 10 villi or
10 crypts.
Additionally, 6-␮m sections were sliced on a microtome (Lipshaw) subsequently mounted on glass slides,
and stained with alcian blue (pH 2.5) and periodic acidSchiff’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 1.
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 Biotech-
569
nology Assoc., Inc.), γ/δ T cell receptor (γ/δ TCR; VMRD,
Inc.), activated T and B lymphocytes [CD25 (IL-2 receptor); VMRD, Inc.], and major histocompatibility class
II (MHC-II; VMRD, Inc).
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, γ/
δ TCR, CD25, and MHC-II were identified with PEconjugated 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 FL2 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). 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-␮L 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-␮L 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 ␮L of PBS+
and centrifuging at 180 × g for 4 min at 4°C. After
washing, if the primary antibody was unconjugated or
biotinylated, 50 ␮L 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 ␮L of PBS+ and centrifuging at 180 × 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 fluo-
570
Brown et al.
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
(mAb)1
Clone
Isotype
Specificity
Dilution
Cell type(s)
expressing molecule
CD2
CD3
CD4
CD8
MSA42
PPT34
74-12-44
76-2-114
IgG2a3
IgG1κ
IgG2bκ
IgG2aκ
CD2
CE3ε
CD4a
CD8 α chain
1:100
1:50
1:100
1:100
γ/δ T cell receptor (TCR)
PGBL22A2
IgG1
Po-TcR1-N4
(γδ)
1:100
CD25 [IL-2 receptor (IL-2R)]
PGBL25A2
IgG1
CD25 (IL-2R)
1:100
Major histocompatibility
class-II (MHC-II)
Secondary antibodies
R-phycoerythrin (PE)5
MSA32
IgG2a
MHC-II
molecule
1:50
Not listed
IgG mAb
1:50
Anti-mouse Ig conjugates
Fluorescein isothiocyanate
(FITC)5
Not listed
IgG mAb
1:50
Anti-mouse Ig conjugates
Quantum Red (QR)-labeled
streptavidin5
Not listed
IgG mAb
1:50
Anti-mouse Ig conjugates
Unlabeled isotype control5
TS
Mouse IgG
whole
molecule
Mouse IgG
whole
molecule
Mouse IgG
whole
molecule
IgG1
Nonspecific
reagent
1:50
PE isotype control5
TS
IgG1
Nonspecific
reagent
1:50
FITC isotype control5
TS
IgG1
Nonspecific
reagent
1:50
QR-labeled streptavidin
isotype control5
TS
IgG1
Nonspecific
reagent
1:50
Testing nonspecific reagent
binding to the target
cell population
Testing nonspecific reagent
binding to the target
cell population
Testing nonspecific reagent
binding to the target
cell population
Testing nonspecific reagent
binding to the target
cell population
70% T lymphocytes
T lymphocytes
T helper lymphocytes
Cytotoxic T lymphocytes and
natural killer cells
Helper and cytotoxic
T lymphocytes
with the γδ TCR
Activated helper and cytotoxic
T lymphocytes
Monocytes/macrophages, B
and T lymphocytes, etc.
1
Monoclonal antibodies are mouse anti-pig.
Purchased from Veterinary Medical Research and Development, Inc., Pullman, WA.
3
Ig = immunoglobulin.
4
Purchased from Southern Biotechnology Associates, Inc., Birmingham, AL.
5
Purchased from Sigma Chemical Co., St. Louis, MO.
2
rescence 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
␮L of unconjugated monoclonal antibodies specific for
CD2, CD25 (IL-2), or γ/δ TCR. Following this incubation, excess antibody was removed by washing plates
twice with 180 ␮L of PBS+ and centrifuging at 180 ×
g for 4 min at 4°C. After washing, 50 ␮L 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
␮L of PBS+ and centrifuging at 180 × 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 fluores-
Neonatal pig intestinal morphology and T cell ontogeny
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
Day of age1
Item
7
14
18
SEM
Leukocyte count
(total cells, thousands/␮L)
WBC
Neutrophil
Lymphocytes
Monocytes
Eosinophils
7.4
2.5
4.6
0.04
0.01
9.0
5.1
3.7
0.04
0.12
Neutrophil
Lymphocytes
Monocytes
Eosinophils
43.5
53.4
0.42
0.23
56.0
41.9
0.45
1.61
6.7
3.5
3.1
0.02
0.07
2.7
1.6
2.6
0.02
0.08
Leukocyte (%)
50.2
48.3
0.31
1.10
17.7
14.9
0.18
1.28
1
Values represent the mean of 4 pigs sampled each day of age.
cence 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 ×
3 factorial. The model included terms for day, sex, and
the day × 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 × 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 × sex interaction.
For evaluating the different goblet cell types over
time, the model included terms for day, sex, goblet cell
type, sex × goblet cell type interaction, day × goblet cell
type interaction, and the day × sex × 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
Differential Blood Leukocyte Concentrations
Numbers and percentages of neutrophils, lymphocytes, monocytes, and eosinophils did not (P = 0.14)
change with age (Table 2) and were similar to those
reported by others in porcine blood (Duncan and Prasse,
571
1978). However, Becker and Misfeldt (1993) 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., 1953).
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) 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., 1987; Lunney
and Pescovitz, 1987; Saalmuller et al., 1989) and in the
neonatal pig at 1, 16, and 28 d of age (Becker and Misfeldt, 1993). 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 γδ TCR in neonatal pigs.
There are 4 αβTCR T-cell subsets (CD2+CD4+CD8−,
CD2+CD4−CD8+,
CD2+CD4+CD8+,
and
CD2+CD4−CD8−) and 3 T cell subsets with the γδ TCR
such as, CD2+CD4−CD8+, CD2+CD4−CD8−, and
CD2−CD4−CD8− (Yang and Parkhouse, 1996; Sinkora
et al., 1998), 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
3, 4, 5, 6, and 7). The current study, as well as other
research (Becker and Misfeldt, 1993; Yang and Parkhouse, 1996), has detected age-related differences in T
cell subpopulations in the neonatal pig. The proportion
of cytotoxic T cells expressing CD2 (Table 6), activated
cytotoxic T cells (Table 7), and activated T and/or B
cells (Table 7) 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.
572
Brown et al.
Table 3. Blood T lymphocytes and the proportions of
CD4+, CD8+, γδ T cell receptor (TCR), and CD25+ blood
lymphocytes in pigs at 7, 14, and 18 d of age
Table 5. The proportions of γδ T cell receptor (TCR) lymphocytes and their CD4- and CD8-defined subsets in pigs
at 7, 14, and 18 d of age
Day of age1
Item
7
T lymphocytes (% of peripheral
blood mononuclear cells)
T cell subsets with αβ and γδ
T cell receptor (% of
T lymphocytes)
CD4+
CD8+
CD25+
γ/δ TCR
CD4+:CD8+
13.6
Day of age1
14
b
25.1
38.6a
41.6
51.5
46.1d
1.16a
b
18.9b
43.3
52.2
51.6cd
0.40b
18
57.9
a
7.8b
57.6
34.6
74.8d
0.27b
SEM
13.1
8.7
27.7
18.8
16.3
0.42
a,b
Within a row, least squares means lacking a common superscript
letter differ (P < 0.01).
c,d
Within a row, least squares means lacking a common superscript
letter differ (P < 0.08).
1
Values are means of 4 pigs representing each day sampled. Double
staining procedures were used to immunofluorescently label peripheral blood mononuclear cell suspensions from pigs at 7, 14, and 18
d of age. Mouse anti-pig mAb specific for CD3 (mouse anti-pig CD3;
phycoerythrin), CD4 (mouse anti-pig CD4; fluorescein isothiocyanate), CD8 (biotinylated mouse anti-pig CD8), γδ T cell receptor
(mouse anti-pig γδ TCR; phycoerythrin), CD25 (mouse anti-pig CD25;
phycoerythrin), and major histocompatability class-II (MHC-II)
(mouse anti-pig MHC-II; phycoerythrin) were used to identify CD3-,
CD4-, CD8-, γδ TCR-, CD25-, and MHC-II-defined populations. Flow
cytometry was utilized to examine blood lymphocyte populations.
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 3). 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 6).
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) 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
Item
γ/δ TCR
CD4- and CD8-defined T cell
subsets within γδ TCR-defined
populations
CD4+CD8−3
CD4−CD8+3
CD4+CD8+3
CD4−CD8−3
Day of age1
Item
CD4- and CD8-defined
T cell subsets (%)
CD8+CD4−
CD8+CD4+
CD8−CD4−
CD8−CD4+
7
29.9
6.7
43.1
20.3
14
44.8
11.9
27.6
15.8
18
30.7
11.2
37.0
21.0
SEM
14.4
6.0
17.3
12.1
1
Values are means of 4 pigs representing each day sampled. Double
staining procedures were used to immunofluorescently label peripheral blood mononuclear cell suspensions from pigs at 7, 14, and 18
d of age. Mouse anti-pig monoclonal antibody specific for CD4 (mouse
anti-pig CD4; fluorescein isothiocyanate), and CD8 (biotinylated
mouse anti-pig CD8) were used to identify CD4- and CD8-defined
lymphocyte populations. Flow cytometry was utilized to examine
blood lymphocyte populations.
5.6
14
b
2.6
78.5
11.8
7.2
12.9
18
ab
11.8
62.6
15.9
9.7
SEM
b
6.2
0.10
87.8
9.5
2.6
13.2
22.0
12.4
6.0
20.7
a,b
Within a row, least squares means lacking a common superscript
letter differ (P < 0.03).
1
Values are means of 4 pigs representing each day sampled. Triple
staining procedures were used to immunofluorescently label peripheral blood mononuclear cell (PBMC) suspensions from pigs at 7, 14,
and 18 d of age. Mouse anti-pig mAb specific for γδ TCR (mouse antipig γδ TCR; phycoerythrin), CD4 (mouse anti-pig CD4; fluorescein
isothiocyanate), and CD8 (biotinylated mouse anti-pig CD8) were
used to identify TCR-, CD4-, and CD8-defined lymphocyte populations. Flow cytometry was utilized to examine blood lymphocyte populations.
2
Percentage of PBMC.
3
Percentage of γδ TCR+ lymphocytes.
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 × sex interaction;
Table 7). Additionally, the population of activated memory cells was greater in 7-d-old mixed sex pigs compared
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
Day of age1
Item
2
Table 4. Blood CD4- and CD8-defined T cell subsets (%)
in pigs at 7, 14, and 18 d of age
7
2
CD2
CD4- and CD8-defined T cell
subsets within CD2-defined
populations
CD4+CD8−3
CD4−CD8+3
CD4+CD8+3
CD4−CD8−3
7
8.4
14
b
3.8a
65.5b
26.6
4.1cd
20.1
ab
0.30b
70.5ab
19.8
9.4c
18
33.8
a
0.57b
82.2b
14.5
2.6d
SEM
10.0
1.0
6.4
8.2
3.8
a,b
Within a row, least squares means lacking a common superscript
letter differ (P < 0.02).
c,d
Within a row, least squares means lacking a common superscript
letter differ (P < 0.10).
1
Values are means of 4 pigs representing each day sampled. Triple
staining procedures were used to immunofluorescently label peripheral blood mononuclear cell (PBMC) suspensions from pigs at 7, 14,
and 18 d of age. Mouse anti-pig monoclonal antibodies specific for
CD2 (mouse anti-pig CD2; phycoerythrin), CD4 (mouse anti-pig CD4;
fluorescein isothiocyanate), and CD8 (biotinylated mouse anti-pig
CD8) were used to identify CD2-, CD4-, and CD8-defined lymphocyte
populations. Flow cytometry was utilized to examine blood lymphocyte populations.
2
Percentage of PBMC.
3
Percentage of CD2+ lymphocytes.
573
Neonatal pig intestinal morphology and T cell ontogeny
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
7 d of age
Item
Male
2
CD25
CD4- and CD8-defined T cell subsets
within CD25-defined populations
CD4+CD8−3
CD4−CD8+3
CD4+CD8+3
CD4−CD8−3
3.74
14 d of age
Female
c
7.1a
59.3c
30.7a
2.9
4.8
c
3.6b
57.6c
29.0a
9.8
Male
23.2
a
0.82c
70.8b
15.4b
13.0
18 d of age
Female
10.6
b
0.81c
67.4bc
17.4b
14.4
Male
25.7
a
0.11c
91.9a
3.9c
4.1
Female
13.8
b
0.56c
70.3b
27.1a
2.1
SEM
2.5
0.66
4.8
4.5
6.6
Within a row, least square means lacking a common superscript letter differ (P ≤ 0.05).
Values are means of 4 pigs representing each day sampled. Triple staining procedures were used to
immunofluorescently label peripheral blood mononuclear cell (PBMC) suspensions from pigs at 7, 14, and
18 d of age. Mouse anti-pig monoclonal antibody specific for CD25 (mouse anti-pig CD25; phycoerythrin),
CD4 (mouse anti-pig CD4; fluorescein isothiocyanate), and CD8 (biotinylated mouse anti-pig CD8) were
used to identify CD25-, CD4-, and CD8-defined lymphocyte populations. Flow cytometry was utilized to
examine blood lymphocyte populations.
2
Percentage of PBMC.
3
Percentage of CD25+ lymphocytes.
a,b,c
1
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
× sex interaction; Table 7).
Studies have shown that infections in chicks (Lillehoj,
1994) and children (Hashimoto et al., 1994) 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, 1990), which may result in
more cytokine production and subsequent cellular and
humoral immune activation (Tonegawa, 1985). 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 6). 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, 1996). Yang and Parkhouse (1996) reported
that the percentage of CD2+ T cells (with the αβ 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 16mo-old pigs (30, 48.8, and 34.2%, respectively). Other
studies have shown that the majority of T cells are γδ
T cells (∼20.0%) in blood of neonatal pigs (Yang and
Parkhouse, 1996; Solano-Aguilar et al., 2001). This was
similar to the findings of the current study, where there
were high percentages of γδ T cells found in circulation
of 7-, 14-, and 18-d old pigs (46.1, 51.6, and 74.8%,
respectively; Table 3). Additionally, the percentage of
γδ 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 5)]. In the current study, younger pigs (7 d) ap-
pear to have a low percentage of CD2+ (all αβ TCR and
a small subset of γδ TCR) cells and a high percentage
of γδ T cells, and as the piglet ages, the percentages of
CD2+ (all αβ TCR and a small subset of γδ TCR) cells
and γδ T cells also increase. Results from the current
study indicate that during the first few weeks of life,
the young pig may rely on γδ T-cell activity more than
its αβ 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 γδ T cell
repertoire, as well as the development of the αβ Tcell repertoire.
The T cell subset with the phenotype CD4-CD8−
(CD2+ and CD2− subsets) expressing the γδ TCR are
a unique T cell population observed in the peripheral
blood of swine (Binns et al., 1992), cattle, and sheep,
but not in human or rodent circulation (Haas et al.,
1990; Hein and Mackay, 1991). Studies have shown
that most of the population of peripheral CD4−CD8−
[double negative (DN)] cells are negative for CD2 and
express γδ TCR chains (Saalmuller et al., 1989). In the
current study, however, we observed that the populations of DN cells expressing CD2 or γδ TCR chains were
similar in number (Tables 5 and 6, respectively). There
were no (P ≥ 0.18) age-related changes in the populations of DN cells expressing the γδ 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 γδ TCR T
cells can recognize proteins directly without antigen
processing in association with MHC molecules, such
as nonpolymorphic MHC-like molecules (Abbas et al.,
1997). 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 7).
574
Brown et al.
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 αβ TCR chains. Although there
were no (P ≥ 0.31) age-related changes in the proportion
of DP cells (Table 4) or DP cells expressing CD2 (Table
6), the population of activated DP cells (Table 7) 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, 1998). Saalmuller (1998) 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 thymocytespecific CD1 antigen). Similarly to CD4+CD8− (T helper
cells), extrathymic DP T lymphocytes respond to mitogen and to alloantigen in mixed leukocyte cultures
(Saalmuller, 1998). 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., 1996), 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, 1998). 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., 1996).
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 8.
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), 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)
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.
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 lympho-
cytes. 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 8). 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, 1993). 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,
1972), 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., 1982). Restraining 8-wk old piglets increased concentrations of
cortisol and decreased responses to intradermal PHA
(Westly and Kelley, 1984). Furthermore, lymphocytes
isolated from the peripheral blood have a lower proliferative response to mitogen in pigs undergoing social and
environmental stressors (Hicks et al., 1998). 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 8) of neonatal pigs in
the current study. These results are consistent with
those of Hoskinson et al. (1990), 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 PWMinduced 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., 1990) and from 1 to 10 d of age
in calves (Manak, 1986).
Cytokine Profiles and Ig
There were no (P ≥ 0.59) age-related differences observed in the production of IL-2 or IL-4 from ConAstimulated peripheral mononuclear cells (Table 8). Additionally, no age-related differences (P ≥ 0.21) were
observed in serum or bile Ig concentrations (Table 8).
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 determina-
575
Neonatal pig intestinal morphology and T cell ontogeny
Table 8. Lymphocyte proliferation, cytokine concentrations, and peripheral and enteric
immunoglobulin (Ig) concentrations pigs sampled at 7, 14, and 18 d of age
Day of age1
Item
7
14
18
SEM
340
38,997a
31,261
270
28,491a
12,382
127
17,955
25,480
124a
100ab
87a
36b
54
53
2
Lymphocyte proliferation, cpm
Unstimulated
Phytohemagglutinin
Pokeweed mitogen
461
1,316b
55,153
Lymphocyte proliferation,3 stimulation index
Phytohemagglutinin
Pokeweed mitogen
2.3b
149a
Cytokine concentrations, pg/mL
IL-2
IL-4
Ins4
Ins4
686
1,025
464
411
482
913
53,307
1,242
21,360
960
36,362
1,331
22,022
278
996
1,121
1,093
966
853
1,154
132
93
Bile Ig, ng/mL
IgA
IgM
Serum Ig, ng/mL
IgA
IgM
Within a row, least squares means lacking a common superscript letter differ (P < 0.05).
Values represent the mean of 4 pigs sampled each day of age.
2
Proliferation of peripheral blood mononuclear cells in counts per minute (cpm).
3
The stimulation index was calculated by subtracting the cpm of the unstimulated cultures from cpm of
the stimulated cultures and dividing by cpm of the unstimulated cultures.
4
Ins = insufficient numbers of cells were harvested to conduct analyses.
a,b
1
tion 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., 1978; Manning et al., 1984). 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, 1988).
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) 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 hepaticderived 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, 1986).
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 9. 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, 1984).
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.
During normal neonatal development, changes in villus height are normally accompanied by secondary
changes in the 3-D structure of villi (Paterson and
Smith, 1983). Furthermore, similar changes in intestinal villus shape can also be detected in neonatal pigs.
Smith (1984) observed that the intestinal villus of 15d-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 immu-
576
Brown et al.
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
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
Day of age1
Item
Day of age1
7
14
18
SEM
Duodenum
Villus height, ␮m
Villus width, ␮m
Crypt depth, ␮m
V:C, ␮m/␮m
764
181a
345a
2.20
727
188a
201b
3.68
716
154b
192b
4.25
249
16
55
1.39
Jejunum2
Villus height, ␮m
Villus width, ␮m
Crypt depth, ␮m
V:C, ␮m/␮m
—
—
—
—
825
158c
186c
4.62
629
139d
113d
6.23
224
12
48
2.63
Ileum2
Villus height, ␮m
Villus width, ␮m
Crypt depth, ␮m
V:C, ␮m/␮m
782
204
286c
2.96
619
196
201cd
3.15
633
167
160d
4.24
144
31
70
1.10
2
a,b
Within a row, least squares means lacking a common superscript
letter differ (P < 0.05).
c,d
Within a row, least squares means lacking a common superscript
letter differ (P < 0.10).
1
Values are means of 4 pigs representing each day sampled.
2
Tissues samples for light microscopy were immediately fixed in
10% neutral buffered formalin and embedded in paraffin. Hisotlogical
sections were stained with hematoxylin and eosin, and the image
was captured at a magnification of 4× and analyzed using Image-Pro
Plus Software (Meyers Instruments, Houston, TX) to evaluate villus
height and width, crypt depth, and the V:C from the small intestine
of neonatal pigs.
nological responses to the soy protein. Bourne (1984)
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) 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., 1999). 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
Goblet cells2
7
14
18
12.23 ± 2.8c
8.88 ± 2.8c
20.94 ± 2.8b
12.55 ± 2.8c
11.13 ± 2.8c
24.42 ± 2.8b
12.15 ± 2.8c
11.50 ± 2.8c
31.70 ± 2.8a
Jejunum
Acidic
Neutral
Sulfuric
—
—
—
6.38 ± 1.6
6.98 ± 1.6
15.79 ± 1.6
6.73 ± 1.6
6.47 ± 1.6
16.18 ± 1.6
Ileum
Acidic
Neutral
Sulfuric
10.08 ± 2.5f
10.85 ± 2.5f
29.51 ± 2.5de
10.85 ± 2.5f
11.08 ± 2.5f
22.93 ± 2.5e
10.10 ± 2.5f
10.73 ± 2.5f
35.46 ± 2.5d
Duodenum
Acidic
Neutral
Sulfuric
a–c
Within intestinal section, day × goblet cell type interaction (P =
0.17).
d–f
Within intestinal section, day × goblet cell type interaction (P <
0.05).
1
Values are means of 4 pigs representing each day sampled.
2
Tissue samples for light microscopy were immediately fixed in
10% neutral buffered formalin and embedded in paraffin. Histological
sections were stained with hematoxylin and eosin, alcian blue and
periodic acid-Schiff’s, or high iron dye [high iron diamine and alcian
blue (pH 1.0)], and analyzed using a light microscope using 40× magnification to evaluate the different mucins produced from enteric goblet
cells in the small intestine of neonatal pigs. Values represent the
least squares means (%) ± SEM.
and trefoil peptides to form a continuous gel on the
mucosal surface (Kindon et al., 1995; Matsuo et al.,
1997). 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., 2003). 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, 1987). Bacterial infection
(Cohen et al., 1983), resident intestinal flora (Mantle
et al., 1989), toxins (Roomi et al., 1984), and parasitic
infestation (Miller et al., 1981) are all factors that can
stimulate the release of mucins from goblet cells.
In the current study, there were no (P ≥ 0.36) agerelated 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
10). 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 10). 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 × goblet cell, P = 0.03;
Table 10). This greater proportion of sulfomucins may
protect the neonate from enteric infections.
Neonatal pig intestinal morphology and T cell ontogeny
For example, the occurrence of severe colitis is correlated with depletion of sulfate mucins in humans (Probert et al., 1995). A study by Strous and Dekker (1992)
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) 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
γδ T cell activity, and later, once the piglet experiences
antigen, it relies on further development of the γδ T
cell repertoire, as well as the development of the αβ 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
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.
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Altorfer, J., S. J. Hardesty, J. H. Scott, and A. L. Jones. 1987. Specific
antibody synthesis and biliary secretion by the rat liver after
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