Veterinary Immunology and Immunopathology 111 (2006) 239250

www.elsevier.com/locate/vetimm

Effect of orally administered probiotic E. coli strain Nissle 1917

on intestinal mucosal immune cells of healthy young pigs
Swantje C. Duncker a,1, Axel Lorentz b, Bernd Schroeder a,
Gerhard Breves a, Stephan C. Bischoff b,*
a
Department of Physiology, School of Veterinary Medicine, Bischofsholer Damm 15, 30173 Hannover, Germany
b
Department of Nutritional Medicine and Prevention, University of Hohenheim, Fruwirthstr. 12, 70593 Stuttgart, Germany
Received 8 August 2005; received in revised form 8 December 2005; accepted 16 January 2006

Abstract

Several beneficial effects of probiotics have been described in studies using rodent disease models and in human patients;
however, the underlying mechanisms remained mostly unclear. Only a few studies focused on the effects of probiotics on the
intestinal mucosal immune system. Here, we studied the effect of the probiotic strain E. coli Nissle 1917 (EcN) administered
orally to young pigs at two concentrations (109 and 1011 CFU/d for 21 days) on the gut-associated lymphatic tissue. This
probiotic strain was shown recently to reduce recurrence of inflammation in ulcerative colitis patients. We quantified the number
and distribution of intestinal immune cells (granulocytes, mast cells, CD4+, CD8+, CD25+, IgA+ lymphocytes) and the mucosal
mRNA expression of cytokines (IFN-gamma, TNF-alpha, TGF-beta, IL-10) and antimicrobial peptides (PR-39, NK-lysin,
prepro-defensin-beta 1, protegrins). The number and distribution of cells were highly different between small intestinal and
colon segments in all groups, but were not influenced by EcN, except high dose EcN fed pigs (1011 CFU/d) showing an increase
in mucosal CD8+ cells in the ascending colon. The mRNA analysis revealed no changes associated with EcN feeding. In
conclusion, according to our analyses EcN has only minor effects on the distribution of mucosal immune cells in the gut of
healthy individuals. The well-established preventive effects of EcN might therefore be relate to other mechanisms than simple
modulation of immune cell distribution.
# 2006 Elsevier B.V. All rights reserved.

Keywords: Probiotics; Intestine; Porcine; GALT; Lymphocytes; E. coli Nissle 1917; CD8

Abbreviations: AMP, anti-microbial peptide; CD, cluster of differentiation; EcN, Escherichia coli Nissle 1917; GALT, gut associated
lymphoid tissue; IEL, intraepithelial lymphocytes; LP, lamina propria; UC, ulcerative colitis
* Corresponding author. Tel.: +49 711 459 4101; fax: +49 711 459 4343.
E-mail addresses: duncker@mcmaster.ca (S.C. Duncker), bischoff.stephan@uni-hohenheim.de (S.C. Bischoff).
1
Present address: Brain-Body Institute (BBI), 50 Charlton Ave. E, Hamilton, Ont., Canada L8N 4A6. Tel.: +1 905 522 1155x2277/5284.

0165-2427/$ see front matter # 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetimm.2006.01.017
240 S.C. Duncker et al. / Veterinary Immunology and Immunopathology 111 (2006) 239250
1. Introduction
Particular probiotic strains have been successfully
used for prophylaxis of intestinal infection in live-
stock animals (Vanbelle et al., 1990; Alexopoulos
et al., 2004). In humans, they are known to prevent
atopic dermatitis (Lodinova-Zadnikova et al., 2003;
Kalliomaki et al., 2003), gastrointestinal infectious
and antibiotic associated diarrhoea (McFarland et al.,
1995; DSouza et al., 2002), as well as inflammatory
bowel disease such as ulcerative colitis (Kruis et al.,
1997, 2004; Rembacken et al., 1999). Furthermore,
some probiotics such as Lactococcus lactis have been
used for delivery of cytokines towards the intestinal
mucosa (Steidler et al., 2003). Even though selective
probiotics such as Lactobacillus GG (Kalliomaki
et al., 2001, 2003), Saccharomyces boulardii (McFar-
land et al., 1995) and E. coli Nissle 1917 (Kruis et al.,
1997, 2004; Rembacken et al., 1999) have been
proven to be clinically effective, the modes of action
by which they achieve their beneficial effects
remained unclear. In vitro experiments suggested
that both modulation of the gut flora and of the
intestinal cytokine production subsequently leading
to modulation of the adaptive immune response might
be of relevance (Christensen et al., 2002; Vaarala,
2003). So far, only a few studies have investigated the
influence of probiotics on the number and distribution
of intestinal immune cells in vivo (Perdigon et al.,
1995; Pestka et al., 2001).
The probiotic E. coli strain Nissle 1917 (EcN)
used in this study is of the serotype O6:K5:H1 and
was isolated in 1916 by the German physician Alfred
Nissle (Loew, 2000). Since then this bacterial strain
has been used as a probiotic drug and is considered
to be safe (Blum et al., 1995; Grozdanov et al., 2002,
2004; Westendorf et al., 2005). Oral administration
of up to 7.2
109 CFU of EcN per animal did not
cause colitis or urogenital infection in neonatal
gnotobiotic piglets (Gunzer et al., 2002). EcN
contains different so-called fitness factors such
as siderophores and microcins (Patzer et al., 2003;
Grozdanov et al., 2004). Moreover, the bacterium
shows a distinct LPS-structure supposed to exert
particular immune modulating properties (Grozda-
nov et al., 2002). EcN is the active component of
Mutaflor1, a drug showing equivalent effectiveness
as 50 -amino-salicylates in maintaining remission in
ulcerative colitis (UC) in humans (Kruis et al., 1997,
2004; Rembacken et al., 1999). The mechanisms
underlying the beneficial effects of EcN are unclear
as yet, immunomodulating effects have been
discussed (Hockertz, 1997; Schultz et al., 2004;
Sturm et al., 2005). Here, we aimed to further
elucidate the mechanisms of action in an animal
system thought to have a rather similar intestinal
anatomy and structure of the mucosal immune
system compared to man. This seems to be of
relevance, since beneficial clinical effects of EcN
were only shown in humans and calves (von Buenau
et al., 2005) so far. Therefore, we chose pigs for our
ex vivo studies and examined the effects of EcN on
immune cell distribution and cytokine expression as
well as AMP (antimicrobial peptides) expression in
the intestinal mucosa.
2. Material and methods
2.1. Animals
Fifteen young adult pigs with a mean weight of
16  2.7 kg were used for experiments. The pigs
originated from different litters of a conventional
crossbred program of German Landrace and
Pietrain carried out by the university research
farm. All animals were on a regular deworming
schedule with 0.3 mg/kg ivermectin two times a year.
Upon their arrival the pigs were housed on straw.
They were maintained on a regular mixed diet for
growing pigs. Following a 2 weeks adaptation period
the animals were divided into three groups of five pigs
each. The first group served as control. The second
group received 109 CFU EcN/d and the third group
1011 CFU EcN/d. EcN was given as a suspension in
dilution medium (2.5 g NaCl, 2.5 g KCl, 0.2 g
MgSO4, 0.2 g CaCl2, 0.2 g MgCl26H2O, 20 ml
NaOH (32%) add distilled H2O to a total of
1000 ml) orally for 21 days. The control group
received the dilution medium without EcN. The
animals were killed by exsanguination following
mechanical stunning. The abdominal contents were
removed immediately after death. The protocol of
animal treatment was approved and the procedure
was supervised by an animal protection officer of the
University.
 S.C. Duncker et al. / Veterinary Immunology and Immunopathology 111 (2006) 239250 241

2.2. Fecal samples 2.4. Histology

To ensure proper administration of EcN, fecal Paraformaldehyde-fixed paraffin slides were

samples were taken from every animal at days 7
and 14 of the experiment and after slaughter. They
were analyzed for EcN content by multiplex PCR
using strain-specific DNA primer sequences as
described elsewhere (Blum-Oehler et al., 2003).
All EcN-treated animals were tested positive for
EcN at days 7 and 14 whereas control animals
always yielded negative EcN RT-PCR results (data
not shown).
2.3. Tissue samples
Tissue samples were taken immediately after
slaughter from the duodenum (10 cm aboral from
the pylorus), jejunum (4.50 m aboral from the
pylorus), ileum (5 cm oral from the ileocolic
orifice), ascending (10 cm aboral from the caeco
colic junction) and descending (60 cm aboral from
the end of the ascending colon) colon. Mucosa
preparations and small tissue specimen (0.5 cm)
were immediately snap frozen in liquid nitrogen for
RNA isolation and cryostat sectioning, respectively.
An additional tissue specimen was collected at each
location (0.5 cm) and fixed either in 4% parafor-
maldehyde at pH 7.2 for 1824 h or in Carnoys
solution (60% ethanol absolute, 30% chloroform,
10% glacial acetic acid) for 2 h. The samples were
embedded in paraffin wax and sectioned at 2 mm,
placed on SuperfrostTM plus slides (Menzel,
Braunschweig, Germany) and dried overnight at
37 8C.
deparaffinized, hydrated and stained using Meyers
hemalum solution (Merck, Darmstadt, Germany) and
aqueous eosin Y Solution (SigmaAldrich, Stein-
heim, Germany) according to standard protocols
(Romeis, 1989). Carnoys solution fixed paraffin
sections were stained with toluidine blue O (Sigma
Aldrich) for an hour and counterstained with Meyers
haemalum. Following dehydration with increasing
concentrations of alcohol and finally xylene, the
slides were mounted in Entellan1 (Merck).
2.5. Monoclonal antibodies
The antibodies used in this study are listed in
Table 1. Biotinylated secondary Ab (sAb) were
obtained from Southern Biotechnology, Birmingham,
USA.
2.6. Immunohistochemistry
Different immune cell populations were identified
by immunohistochemistry using the enzyme-linked
immunoperoxidase technique. Incubation steps were
carried out in a humid chamber at room temperature
if not indicated otherwise. After each step slides were
washed for 5 min in TBS (pH 7.4) supplemented with
0.035% Tween120 (SigmaAldrich). CD4+ and
CD8+ subpopulation analysis was performed using
cryostat sections of 5 mm thickness placed on
Superfrost1 plus glass slides (Menzel-Glaeser,
Braunschweig, Germany). Sections were air-dried

Table 1
Antibody dilutions
Primary Ab Clone Company Dilution Secondary Ab
a
CD4 74-12-4 VMRD, USA 1/50 Goatanti-mouse IgG2b
CD8 MIL-12 Serotec, UK 1/100 b Goatanti-mouse IgG2a
CD25 231.3B2 Serotec, UK 1/20a Goatanti-mouse IgG1
IgA K61 1B4 Serotec, UK 1/10a Goatanti-mouse IgG1
Mouse IgG1 Dianova, Germany 1/20a Goatanti- mouse IgG1
Mouse IgG2a Dianova, Germany 1/100 c Goatanti-mouse IgG2a
Mouse IgG2b Dianova, Germany 1/50b Goatanti-mouse IgG2b
a
Dilutions were prepared with Monet Blue (Biocare, Germany).
b
Dilutions were prepared with VanGogh Yellow (Biocare, Germany).
242 S.C. Duncker et al. / Veterinary Immunology and Immunopathology 111 (2006) 239250
for 30 min and fixed in a mix of equal amounts of
acetone and methanol at 4 8C. Tissues were blocked
with 5% normal swine serum diluted in Tris-buffered
saline (TBS) for 30 min to reduce unspecific Ab-
binding, and then incubated overnight with the
primary Ab at 4 8C. The following day slides were
blocked again with 5% normal swine serum diluted in
TBS, and then incubated with a biotinylated
secondary Ab (1/50 dilution) for 30 min. Streptavi-
dinhorse radish peroxidase (Biocare, Hamburg,
Germany) and 3-amino-9-ethyl carbazol (AEC) were
used to visualize specific binding of the Ab (Zymed,
San Francisco, CA). Slides were counterstained with
Meyers hemalum solution for improved cell differ-
entiation and finally mounted in Faramount1 Aqu-
eous Mounting Medium (Dako, Hamburg, Germany).
Each staining series was accompanied by one slide
treated with an isotype control replacing the primary
Ab. Because of the lack of endogenous peroxidase
activity in the cryostat sections, blocking with
hydrogen peroxide was not necessary using this
staining protocol.
For the detection of CD25 and IgA paraffin
embedded specimen were used. Slides were depar-
affinized and hydrated. To ensure antigen de-masking
the samples were treated with Dako1 Target Retrival
Solution (pH 6) (Dako) in a water bath at 95 8C. The
subsequent immunohistochemistry was performed as
described above; however, an additional incubation in
methanol containing 1.2% hydrogen peroxide was
added following the incubation with the sAb to ensure
quenching of endogenous peroxidase.
2.7. Cell counts
Cells counts within the intestinal mucosa were
performed at 1000-fold magnification using a light
microscope Labophot-2 (Nikon, Tokyo, Japan). Cells
were counted separately in the epithelium and the
lamina propria (LP). Data were expressed in percent of
positive cells per total number of epithelial cells or
total number of cells in the LP, respectively. Cells were
assigned to the intraepithelial compartment only if
they were located with the epithelial cell band or at
least half of their plasma membrane was in contact
with the basement membrane of the epithelium. For
CD8+ cells, we differentiated between luminal and
basal section of the mucosa. Therefore, the luminal
compartment of the mucosa was considered to be the
villi in the small intestine and the most luminal
microscopic field of view (1000 times magnification)
in the large intestine. The basal compartment of the
mucosa contained the crypts in the small intestine and
the most basal microscopic field of view in the large
intestine. The assignment made in the large intestine
ensured that cells were not accidentally counted for
both compartments. CD4+ cells could be evaluated on
a semi-quantitative basis only by assignment to four
groups defined as follows: (1) <25% of LP cells
CD4+; (2) 2549% of LP cells CD4+; (3) 5075% of
LP cells CD4+; (4) >75% of LP cells CD4+.
2.8. RNA isolation and RT-PCR
Total RNA was prepared from washed and stripped
mucosa samples (0.90.8 g) using the RNeasy Mini
Kit (Qiagen, Hilden, Germany) following the manu-
facturers manual. The amount of total RNA was
quantified by spectrometry. For RT-PCR 200 ng of
total RNA was used and processed as described
previously (Gebhardt et al., 2002). Briefly, RNA was
treated with 10 U RNase-free DNAse (Promega,
Madison, WI) to remove genomic DNA. After
denaturation at 70 8C cDNA was synthesized at
49 8C adding SuperscriptTM plus reverse transcriptase
(Invitrogen, Karlsruhe, Germany) and oligo-dT
primers (Pharmacia, Uppsala, Sweden). 1/10 of the
total cDNA was used for one PCR reaction. PCR was
performed with 30 s at 94 8C, 45 s at 5558 8C, 35 s at
72 8C. Numbers of cycles and annealing temperatures
differed for each pair of primers: IFN-gamma (34
cycles, annealing 58 8C), TNF-alpha (35 cycles,
annealing 58 8C), TGF-beta (32 cycles, annealing
58 8C), IL-10 (40 cycles, annealing 58 8C) prepro-
defensin-beta 1 (39 cycles, annealing 58 8C), prote-
grins and PR-39 (42 cycles, annealing 55 8C), NK-
lysin (36 cycles, annealing 55 8C) and GAPDH (28
cycles, annealing 56 8C). To ensure that a saturation of
primers were not misinterpreted as an increase of
mRNA, as many cycles of PCR were used as were
necessary to ensure the amplification reaction was still
in its exponential phase. The number of cycles for
every primer pair was determined by preliminary
experiments with samples from at least three different
untreated pigs and for every intestinal location tested.
The cycle number was than used in treated and
 S.C. Duncker et al. / Veterinary Immunology and Immunopathology 111 (2006) 239250 243

Table 2
PCR-primer
Sense-primer Antisense-primer
IFN-gamma 50 -GCAAGTACCTCAGATGTACC-30 50 -TGGCCTTGGAACATAGTCTG-30
TNF-alpha 50 -ACTGCACTTCGAGGTTATCG-30 50 -AGAGGACCTGGGAGTAGATG-30
TGF-beta 50 -CAGAGAGGCTATAGAGGGTT-30 50 -TGTCTAGGCTCCAGATGTAG-30
IL-10 50 -TTGCCAAGCCTTGTCAGAGA-30 50 -TCACCCATGGCTTTGTAGAC-30
PR-39 50 -ACCCATCCATTCACTCAC-30 50 -AGCCACAACAATAAGATCC-30
NK-lysin 50 -GAGCAGTTCTGCCAGAACCT-30 50 -GCAGGAGTTAGGTGAGAGAA-30
Protegrines 50 -TGGATCAGATCAAGGACC-30 50 -ACACAGACGCAGAACCTAC-30
Prepro-defensine-beta 50 -CCTCCTTGTATTCCTCCTCA-30 50 -TTGCAGCATTTGACTTGGGG-30
GAPDH 50 -ACCACAGTCCATGCCATCAC-30 50 -TCCACCACCCTGTTGCTGTA-30

untreated animals to allow comparative semi-quanti- significantly different when comparing small and
tative detection of mRNA expression. PCR was large intestinal sites, their density within the LP
performed with 2.5 U Taq DNA polymerase (Invitro- ranging from 0.4% in the jejunum to 3.7% in the
gen) and 20 pmol of specific sense and antisense duodenum and colon (Fig. 1B). Mast cells were
primers shown in Table 2. The PCR products equally distributed throughout the intestinal LP,
contained cDNA fragments of 359 bp (IFN-gamma), and accounted for about 3% of LP cells at all
266 bp (TNF-alpha), 371 bp (TGF-beta), 243 bp
(IL-10), 168 bp (prepro-defensin-beta 1), 262 bp
(PR-39), 100 bp (protegrins), 479 bp (NK-lysin)
and 452 bp (GAPDH), respectively. Eleven micro-
liters of the PCR products were visualized on 1%
agarose gel containing ethidium bromide (500 ng/ml)
and photographed.

2.9. Statistics

Data were expressed as mean (S.D.) if not

indicated otherwise. Differences in cell counts were
accessed using the Students t-test and differences
between experimental groups by one-way ANOVA.

3. Results

3.1. Number and distribution of granulocytes and

mast cells

In untreated animals, granulocytes and mast cells

were detected in the lamina propria of the mucosa, but
not in the epithelium. Eosinophils were concentrated
within the basal part of the LP. They were much more
frequent in the small compared to the large intestine,
with highest numbers in the mucosa of the ileum
(11  2.2% of total LP cells). In the colon almost no
eosinophils were visible (0.6  0.3% of total LP
cells) (Fig. 1A). Neutrophil numbers were not
Fig. 1. Eosinophil (A), neutrophil (B) and mast cell (C) density in
the intestinal lamina propria mucosae (LP) shown as per cent of all
lamina propria cells. Animals were either untreated (white bars), or
treated with 109 (hatched bars) or 1011 CFU/d (black bars) of E. coli
Nissle 1917 (EcN). Bars show the mean of five pigs per group with
error bars indicating the standard deviation.
244 S.C. Duncker et al. / Veterinary Immunology and Immunopathology 111 (2006) 239250
investigated sites of the gut (Fig. 1C). No basophils
could be detected in the intestinal mucosa of the pigs.
The numbers of the three cell types examined here
did not change following treatment of the animals
with either low or high dose of EcN, as shown in
Fig. 1.
3.2. Number and distribution of T lymphocyte
subsets and plasma cells
IgA producing plasma cells, CD4+ cells, CD25+
cells and CD8+ cells were analyzed by immunohis-
tochemistry. IgA plasma cells were predominantly
found in the LP of the crypts. They were virtually
absent in the epithelium and in submucosal layers.
Number of IgA plasma cells were highest in the
duodenum with a density of 21.1% (3.1% of LP
cells). Plasma cell counts were significantly lower at
Fig. 2. Density of IgA+ plasma cell (A), CD4+ cells (B) and CD25+
cells (C) in the intestinal lamina propria mucosae (LP) shown as per
cent of all lamina propria cells. Animals were either untreated (white
bars), or treated with 109 (hatched bars) or 1011 CFU/d (black bars)
of E. coli Nissle 1917 (EcN). Bars show the mean of five pigs per
group with error bars indicating the standard deviation.
the more caudal sites of the GI tract (jejunum, ileum,
colon), where only 3% of LP cells were IgA plasma
cells. The density of IgA plasma cells was not
changed by treatment of the animals with EcN
(Fig. 2A).
Cells expressing the CD4 co-receptor were
frequently found in the LP but were absent in the
epithelium. Within the LP 5075% of cells were CD4
positive. Therefore, we only performed a semi-
quantitative analysis. CD4+ cells were evenly
distributed throughout cross-sections of the mucosa
in the ileum and colon; however, the mucosa of the
duodenum and jejunum contained higher numbers of
CD4+ cells in the LP of the villi compared to the
crypts (Fig. 2B). CD25+ cells were localized in small
amounts (2.43.7% of total LP cells) in the intestinal
mucosa. They were evenly distributed in the LP
(Fig. 2C) and only sporadically detected in the
epithelium. Again, neither CD4+ cell density nor
CD25+ cell density changed following EcN treatment
(Fig. 2B and C).
CD8+ cells were the only ones found to be
consistently present within the epithelium and the LP
of the intestinal mucosa. The density of CD8+ cell
was higher in the luminal compared to the basal
Fig. 3. Density of CD8+ cells in the luminal compartment of the
mucosa (A) and the basal compartment of the mucosa (B), shown in
percent of all epithelial cells, LP cells, respectively. Animals were
either untreated (white bars), or treated with 109 (hatched bars) or
1011 CFU/d (black bars) of E. coli Nissle 1917 (EcN). First bar of
every colour pattern indicating cells in the epithelium and the second
bar cells in the LP. Bars show the mean of five pigs per group with
error bars indicating the standard deviation. *p < 0.05 compared to
same intestinal segment in the control group.
 S.C. Duncker et al. / Veterinary Immunology and Immunopathology 111 (2006) 239250 245

compartment of the mucosa. Highest counts were
found in the luminal epithelium of the duodenum.
Along the intestinal mucosa the CD8+ cell counts
in the LP were reverse to those in the epithelium
of the same compartment. For example in the
luminal compartment of the mucosa the epithelium
contained much higher numbers of CD8+ cells than
the corresponding LP (Fig. 3A). In the basal
compartment of the mucosa the opposite was true
(Fig. 3B). Animals receiving high amounts of EcN
(1011 CFU/d) showed significantly higher CD8+
cells counts in the luminal LP of the Ileum, in the
epithelium and the lamina propria of the ascending
colon compared to placebo-fed pigs (Figs. 3 and 4).
In low dose fed animals CD8+ cells where increased
in the basal epithelium of the jejunum and in the
luminal LP of the ascending colon (Fig. 3). No
significant changes in CD8+ cell counts were obse-
rved in the mucosa of other intestinal segments on
EcN treatment.

Fig. 4. Pictures showing cells bearing the CD8 antigen (red) in the mucosa of the ascending colon. The animal treated with 1011 CFU/d (B) of
EcN shows an increase in the number of CD8 positive cells compared to the untreated animal (A). Slides were stained by immunohistochemistry
using the MIL-12 Ab (Serotec, UK) or a mouse IgG2a (Dianova, Germany) isotype as negative control (C). Pictures from one representative
animal per group are shown. Metering bar = 25 mm.

Fig. 5. mRNA expression of cytokines (IFN-gamma, TNF-alpha, TGF-beta, IL-10) and antimicrobial peptides (prepro-defensin-beta 1, NK-
lysin) in the ileum and the ascending colon compared to housekeeping gene (GAPDH). Pictures show two representative animals of the control
group and the group treated with 1011 CFU/d of E. coli Nissle 1917 (EcN). Numbers indicate the PCR cycles accomplished.
246 S.C. Duncker et al. / Veterinary Immunology and Immunopathology 111 (2006) 239250
3.3. mRNA expression of different cytokines
and AMP
Total RNA from intestinal mucosa of duodenum,
ileum and ascending colon in control animals and
animals fed 1011 CFU EcN/d was investigated.
mRNA expression of different cytokines and anti-
microbial peptides was evaluated semi-quantitatively
by comparison to GAPDH as housekeeping gene to
check for possible changes in immune cell sub-
populations. mRNA expression of IFN-gamma, TNF-
alpha, TGF-beta and IL-10 as well as the mRNA
expression of the AMP: prepro-defensine-beta 1 and
NK-lysin was detected in the duodenum (data not
shown), ileum and ascending colon (Fig. 5). In all
samples PR-39 was only partly seen in the intestinal
mucosa and mRNA for protegrins was absent in all
animals and intestinal segments (data not shown).
Transmural duodenal tissue samples of 3-week-old
piglets were included as positive controls. Neither in
the investigated cytokines nor in the AMP changes
were detected after feeding high amounts of EcN
(1011 CFU/d).
4. Discussion
During the last decades pigs health became a huge
economic factor in livestock farming because of
intensive animal husbandry and increasing produc-
tion rates. Simultaneously, the opportunities of
prophylactic treatment against the major porcine
pathogens were limited due to governmental restric-
tion (prohibition of antibiotics in animal feed, etc.).
Therefore, alternatives are needed. Probiotics showed
the potential to fulfil such requirements, even though
the mechanisms of their beneficial effects are mostly
unknown. This study was performed to investigate the
effects of EcN, a probiotic E coli strain successfully
used in human and veterinary medicine, on the
intestinal immune system of healthy young pigs. The
aim was to further understand the modes of action of
this probiotic strain, which obviously has beneficial
properties both in man (Kruis et al., 2004) and in
calves (von Buenau et al., 2005). We selected the pig
as a model not only because this species might as well
benefit of probiotic treatment (Alexopoulos et al.,
2004), but also because the pig intestinal immune
system is considered to be much closer to the human
one than the rodent immune system (Miller and
Ullrey, 1987; Boes and Helwigh, 2000). Studies have
been performed in pigs on the distribution of
particular immune cells in the blood and in different
tissues (Bianchi et al., 1992; Yang and Parkhouse,
1996; Boeker et al., 1999; Saalmuller et al., 2002;
Sinkora et al., 2005) including studies on T cells in the
intestine (Vega-Lopez et al., 1993; Pabst and
Rothkotter, 1999). Nevertheless, it is surprising to
note that no published data are available on the
distribution of cells of the innate immune system
within the intestine of the pig. Therefore, we were
interested not only in studying the influence of EcN on
intestinal immune cells, but also in quantifying the
number and distribution of granulocytes, mast cells
and lymphocyte subsets in the gut mucosa.
Eosinophils were mainly found in the LP of the
crypts in the small intestine and the basal part of the
crypts in the large intestine, as described previously
(Bianchi et al., 1992). Eosinophils could be found at
all sites of the small and large intestine, the highest
numbers being seen in the ileum. Interestingly, we
found for the first time that the density of eosinophils
in the colon LP was lower than 1% and thus much
lower than that described in humans (Bischoff et al.,
1996). Because none of the animals showed signs of
parasite infection and all were dewormed on a routine
schedule, an increase of eosinophils in the small
intestine due to parasitosis can be largely ruled out.
There is evidence from the literature that gastro-
intestinal eosinophils not only act as inflammatory
effector cells of allergy and parasitic infection, but are
also capable of functioning as immunomodulating
cells (Kato et al., 1998). Kato et al. showed a
spontaneous minimal degranulation of intestinal
eosinophils in the healthy gut that does not occur in
other normal tissues. These data suggest that
eosinophils may also act as immune-modulating cells
in the small intestine of pigs.
Compared to eosinophils the amount of neutro-
phils in the intestinal mucosa of healthy pigs was
much lower. Neutrophil counts were highest in the
duodenum and descending colon. The amount of
neutrophils was higher than the numbers regarded
to be normal in humans (0.4%) (Ogra et al., 1999). In
the duodenum the reason might be that pigs are
more prone to contact with microorganisms because
 S.C. Duncker et al. / Veterinary Immunology and Immunopathology 111 (2006) 239250
of their usual environment. Therefore, more neutro-
phils may ensure a fast reaction to potential
pathogens. The relatively high amount of neutrophils
in the colon, compared to humans, is likely to be a
result of higher physiologic amounts of bacteria in
this part of the pig intestine compared to the human
colon. In contrast to neutrophils, the counts of mast
cells were in the expected ranges, since our data were
similar to those reported by Xu et al. (1993) who did
similar studies in pigs. Mast cells, unlike granulo-
cytes, were evenly distributed throughout the intest-
inal segments.
Treatment with EcN had no effect on the number
and distribution of granulocytes and mast cells in
healthy pigs. Moreover, the absence of major changes
in tested cytokine and AMP mRNA expression makes
an influence of EcN on the cellular metabolism
unlikely too. Even though, a definite conclusion on
cytokine-mRNA concentration would require a quan-
titative method for mRNA detection like real time
PCR, because the semi-quantitative method used can
only detect major changes. However, the unaltered
number of cells in healthy EcN-treated animals does
not rule out a positive effect on disease-induced cell
recruitment (Sturm et al., 2005; Kamada et al., 2005).
From experiments with other probiotics like lactoba-
cilli it is known, that they are able to decrease
infection-induced, transmucosal migration of neutro-
phils (Michail and Abernathy, 2002). Keeping in mind
EcN being a probiotic, the fact that it does not recruit
neutrophils in healthy animals rather underlines its
apathogenicity than its ineffectiveness. Even though in
the present study no changes were detected with
respect to the number and distribution of intestinal
eosinophils, it still needs to be clarified if the
administration of EcN leads to alterations in mediator
composition and release in this cells.
In accordance with findings of other authors
(Rothkotter et al., 1991; Bianchi et al., 1992) IgA-
producing plasma cells were mainly found in the basal
part of the mucosa with highest amounts in the crypts
of the duodenum. No changes were detected following
EcN treatment. An increase in the number of intestinal
IgA producing plasma cells, does not seem to be a
mechanism of action of EcN in conventionally reared,
healthy pigs. Nevertheless, unchanged IgA plasma
cell counts do not rule out an influence of EcN on the
luminal secretory IgA concentration as reported for
247
other probiotics like Saccharomyces boulardii (Buts
et al., 1990; Rodrigues et al., 2000).
Heterogeneous results were obtained with regard to
lymphocyte subsets. We found no changes in CD4+
and CD25+ cells, but an increase of CD8+ cells in the
ascending colon induced by EcN. In contrast to the
results of others (Vega-Lopez et al., 1993), we found in
our experiments polarization of mucosal CD4+ cells
towards the LP of the villi only in the duodenum and
jejunum. In the ileum and colon, the CD4+
lymphocytes were equally distributed throughout
the mucosa. An explanation of this difference might
be the lower age of the animals used in the study by
Vega-Lopez et al. Even though no alterations in the
CD4+ cell counts were evident between treatment and
control groups, an influence EcN on the balance of
CD4+ subpopulations cannot be ruled out. Different
from CD4+ cells significantly more CD8+ cells were
counted in the luminal epithelium than in the lamina
propria of the intestine, as reported earlier by other
groups (Vega-Lopez et al., 1993, 2001; Pabst and
Rothkotter, 1999). This holds true for all intestinal
segments investigated. We extended these findings by
distinguishing between the epithelium and the LP in
the luminal and the basal part of the mucosa,
respectively. The highest number of CD8+ cells
(one-third of total cell count) was found in the luminal
epithelium of the duodenum. Different from the
findings of Vega-Lopez et al. we could not confirm that
the CD8+ cells of the LP were localized near the
basement membrane of the epithelium, possibly
because of different methodological approaches. We
report here for the first time that EcN treatment
increased the amount of CD8+ cells in the ascending
colon in healthy young pigs. Feeding 1011 CFU EcN/d
for 21 days resulted in an increase of CD8+ cells in the
ascending colon, both in the luminal as well as in the
basal compartment of the mucosa. In the absence of
mucosal inflammation (no increase in neutrophils, no
differences of proinflammatory cytokine-mRNA
expression in comparison between treated and
untreated groups, no activation of lymphocytes) the
high numbers of CD8+ cells cannot be interpreted as
an inflammatory response. From our experiments it
cannot be concluded whether the increased number of
CD8+ cells detected in the epithelium of the ascending
colon is due to proliferation of CD8+ cells or to
recruitment of cells from other sites like the lamina
248 S.C. Duncker et al. / Veterinary Immunology and Immunopathology 111 (2006) 239250
propria or the mesenteric lymph nodes. However, it
has been shown in mice that intestinal IEL are able to
proliferate in the epithelium (Lin et al., 1994). The
accumulation of lymphocytes originating from the
peripheral blood as an explanation of the increase of
CD8+ cells due to EcN treatment is unlikely, since
Sturm et al. (2005) showed a down-regulation of
proliferation in peripheral blood T cells by EcN in
vitro, whereas the proliferation of LP T cells was
unchanged. The cell population expressing CD8 co-
receptor in the intestine consists of different cell types
including cytotoxic T cells, gamma/delta T cells and
NK cells. In this study we did not differentiate
between subtypes of CD8+ cells as CD8high and
CD8low. Therefore, no conclusion can be drawn on
changes within a special subtype of CD8+ cells;
however, the beneficial effects of probiotics such as
EcN could be related to known effects of CD8+
subpopulations, such as defence against pathogens by
cytotoxic CD8+ cells, or maintenance and enhance-
ment of the epithelial integrity by gamma/delta CD8+
cells (Chen et al., 2002). Other than in humans and
mice intraepithelial CD8+ gamma/delta T cells have
not yet been identified in the intestine of pigs, but that
does not rule out the possibility that equivalent
subtypes of CD8+ cells exist also in pigs within the
epithelium. Especially, because there is increasing
evidence that pigs have a distinct repertoire of
intestinal gamma/delta TCR (Holtmeier et al., 2002)
and T cells expressing the delta chain of the TCR have
been shown in the colonic epithelium (Hontecillas
et al., 2005). Future experiments are needed to further
characterize the phenotype of CD8+ cells in the pig,
and to establish their biological relevance in disease
and disease prevention.
Even though the porcine intestinal immune
system might be an excellent model in terms of
transfer of results to the situation in humans, there are
of course some limitations. The lack of EcN effects
on the density of innate immune cells such as
granulocytes or mast cells shown in this study, as
well as on the density of CD4+ cells (mostly T helper
lymphocytes), CD25+ (activated T cells) and IgA
cells might be the result of the limited number of
animals examined here. Unfortunately, the repertoire
of antibodies directed against porcine cell surface
antigens is limited compared to that available for
rodents. A future improvement in availability of
directly fluorescent-labeled antibodies to perform
fluorescent immunohistochemistry and an improve-
ment in isolation techniques for intestinal immune
cells will be important to defining specialized subsets
of lymphocytes and innate immune cells in situ. This
will help to further investigate an influence of EcN on
intestinal immune cells in the pig. Furthermore,
using EcN in an infection model might be useful to
reveal changes in the reaction and migration of
intestinal immune cell population that cannot be
detected in healthy animals.
5. Conclusions
We showed in this study as described earlier in
humans (Kruis et al., 2004) and mice (Waidmann
et al., 2003; Schultz et al., 2004), that EcN did not
induce any signs of inflammation and did not exhibit
any signs of pathogenicity, even when fed to young
pigs at high doses. The EcN-induced enhancement of
the number of CD8+ cells in the ascending colon
might be linked to the beneficial role of maintaining
porcine intestinal health and preventing disease.
Intestinal epithelial cells have been shown in vitro to
produce keratinocyte growth factor (KGF) (Chen
et al., 2002) and might thereby increase the epithelial
integrity. Mice lacking gamma/delta TCR IEL have
impaired epithelial development in the intestine
(Boismenu and Havran, 1994). It has also been
shown recently that IEL express junctional molecules
like epithelial cells, whereas lymphocytes from other
body sites (spleen, mesenteric lymph nodes, thymus,
Payers patches) do not (Inagaki-Ohara et al., 2005).
This opens up new possibilities for the involvement of
IEL in the epithelial barrier and for the communica-
tion with epithelial cells. To further evaluate the
beneficial effects of EcN in pigs and the underlying
mechanisms, experiments with pig infection models
are of special interest.
Acknowledgements
We thank Gisela Weier and Gerhild Becker for
excellent technical assistance and Yvonne Armbrecht
and Michael Rohde for help with the animals as well
as Anne Luschert for advice in immunohistochem-
 S.C. Duncker et al. / Veterinary Immunology and Immunopathology 111 (2006) 239250
istry. This work was supported by Ardeypharm
GmbH, Herdecke, Germany and the Deutsche For-
schungsgemeinschaft (SFB 280).
References
Alexopoulos, C., Georgoulakis, I.E., Tzivara, A., Kyriakis, C.S.,
Govaris, A., Kyriakis, S.C., 2004. Field evaluation of the effect
of a probiotic-containing Bacillus licheniformis and Bacillus
subtilis spores on the health status, performance, and carcass
quality of grower and finisher pigs. J. Vet. Med. A Physiol.
Pathol. Clin. Med. 51, 306312.
Bianchi, A.T., Zwart, R.J., Jeurissen, S.H., Moonen-Leusen, H.W.,
1992. Development of the B- and T-cell compartments in
porcine lymphoid organs from birth to adult life: an immuno-
histological approach. Vet. Immunol. Immunopathol. 33, 201
221.
Bischoff, S.C., Wedemeyer, J., Herrmann, A., Meier, P.N., Traut-
wein, C., Cetin, Y., Maschek, H., Stolte, M., Gebel, M., Manns,
M.P., 1996. Quantitative assessment of intestinal eosinophils and
mast cells in inflammatory bowel disease. Histopathology 28, 1
13.
Blum, G., Marre, R., Hacker, J., 1995. Properties of Escherichia coli
strains of serotype O6. Infection 23, 234236.
Blum-Oehler, G., Oswald, S., Eiteljorge, K., Sonnenborn, U.,
Schulze, J., Kruis, W., Hacker, J., 2003. Development of
strain-specific PCR reactions for the detection of the probiotic
Escherichia coli strain Nissle 1917 in fecal samples. Res.
Microbiol. 154, 5966.
Boeker, M., Pabst, R., Rothkotter, H.J., 1999. Quantification of B, T
and null lymphocyte subpopulations in the blood and lymphoid
organs of the pig. Immunobiology 201, 7487.
Boes, J., Helwigh, A.B., 2000. Animal models of intestinal nema-
tode infections of humans. Parasitology 121 (Suppl.), S97
S111.
Boismenu, R., Havran, W.L., 1994. Modulation of epithelial cell
growth by intraepithelial gamma delta T cells. Science 266,
12531255.
Buts, J.P., Bernasconi, P., Vaerman, J.P., Dive, C., 1990. Stimulation
of secretory IgA and secretory component of immunoglobulins
in small intestine of rats treated with Saccharomyces boulardii.
Dig. Dis. Sci. 35, 251256.
Chen, Y., Chou, K., Fuchs, E., Havran, W.L., Boismenu, R., 2002.
Protection of the intestinal mucosa by intraepithelial gamma
delta T cells. Proc. Natl. Acad. Sci. U.S.A. 99, 1433814343.
Christensen, H.R., Frokiaer, H., Pestka, J.J., 2002. Lactobacilli
differentially modulate expression of cytokines and maturation
surface markers in murine dendritic cells. J. Immunol. 168, 171
178.
DSouza, A.L., Rajkumar, C., Cooke, J., Bulpitt, C.J., 2002. Pro-
biotics in prevention of antibiotic associated diarrhoea: meta-
analysis. BMJ 324, 1361.
Gebhardt, T., Sellge, G., Lorentz, A., Raab, R., Manns, M.P.,
Bischoff, S.C., 2002. Cultured human intestinal mast cells
express functional IL-3 receptors and respond to IL-3 by enhan-
249
cing growth and IgE receptor-dependent mediator release. Eur. J.
Immunol. 32, 23082316.
Grozdanov, L., Raasch, C., Schulze, J., Sonnenborn, U., Gottschalk,
G., Hacker, J., Dobrindt, U., 2004. Analysis of the genome
structure of the nonpathogenic probiotic Escherichia coli strain
Nissle 1917. J. Bacteriol. 186, 54325441.
Grozdanov, L., Zahringer, U., Blum-Oehler, G., Brade, L., Henne,
A., Knirel, Y.A., Schombel, U., Schulze, J., Sonnenborn, U.,
Gottschalk, G., Hacker, J., Rietschel, E.T., Dobrindt, U., 2002. A
single nucleotide exchange in the wzy gene is responsible for the
semirough O6 lipopolysaccharide phenotype and serum sensi-
tivity of Escherichia coli strain Nissle 1917. J. Bacteriol. 184,
59125925.
Gunzer, F., Hennig-Pauka, I., Waldmann, K.H., Sandhoff, R., Grone,
H.J., Kreipe, H.H., Matussek, A., Mengel, M., 2002. Gnotobiotic
piglets develop thrombotic microangiopathy after oral infection
with enterohemorrhagic Escherichia coli. Am. J. Clin. Pathol.
118, 364375.
Hockertz, S., 1997. Augmentation of host defence against bacterial
and fungal infections of mice pretreated with the non-pathogenic
Escherichia coli strain Nissle 1917. Arzneimittelforschung 47,
793796.
Holtmeier, W., Kaller, J., Geisel, W., Pabst, R., Caspary, W.F.,
Rothkotter, H.J., 2002. Development and compartmentalization
of the porcine TCR delta repertoire at mucosal and extraintest-
inal sites: the pig as a model for analyzing the effects of age and
microbial factors. J. Immunol. 169, 19932002.
Hontecillas, R., Bassaganya-Riera, J., Wilson, J., Hutto, D.L.,
Wannemuehler, M.J., 2005. CD4+ T-cell responses and distribu-
tion at the colonic mucosa during Brachyspira hyodysenteriae-
induced colitis in pigs. Immunology 115, 127135.
Inagaki-Ohara, K., Sawaguchi, A., Suganuma, T., Matsuzaki, G.,
Nawa, Y., 2005. Intraepithelial lymphocytes express junctional
molecules in murine small intestine. Biochem. Biophys. Res.
Commun. 331, 977983.
Kalliomaki, M., Salminen, S., Arvilommi, H., Kero, P., Koskinen, P.,
Isolauri, E., 2001. Probiotics in primary prevention of atopic
disease: a randomised placebo-controlled trial. Lancet 357,
10761079.
Kalliomaki, M., Salminen, S., Poussa, T., Arvilommi, H., Isolauri,
E., 2003. Probiotics and prevention of atopic disease: 4-year
follow-up of a randomised placebo-controlled trial. Lancet 361,
18691871.
Kamada, N., Inoue, N., Hisamatsu, T., Okamoto, T., Matsuoka, K.,
sato, T., Chinen, H., Su Hong, K., Yamada, T., Suzuki, Y.,
Suzuki, T., Watanabe, N., Tsuchimoto, K., Hibi, T., 2005.
Nonpathogenic Escherichia coli Nissle1917 prevents murine
acute and chronic colitis. Inflamm. Bowel. Dis. 11, 455463.
Kato, M., Kephart, G.M., Talley, N.J., Wagner, J.M., Sarr, M.G.,
Bonno, M., McGovern, T.W., Gleich, G.J., 1998. Eosinophil
infiltration and degranulation in normal human tissue. Anat. Rec.
252, 418425.
Kruis, W., Fric, P., Pokrotnieks, J., Lukas, M., Fixa, B., Kascak, M.,
Kamm, M.A., Weismueller, J., Beglinger, C., Stolte, M., Wolff,
C., Schulze, J., 2004. Maintaining remission of ulcerative colitis
with the probiotic Escherichia coli Nissle 1917 is as effective as
with standard mesalazine. Gut 53, 16171623.
250 S.C. Duncker et al. / Veterinary Immunology and Immunopathology 111 (2006) 239250
Kruis, W., Schutz, E., Fric, P., Fixa, B., Judmaier, G., Stolte, M.,
1997. Double-blind comparison of an oral Escherichia coli
preparation and mesalazine in maintaining remission of ulcera-
tive colitis. Aliment Pharmacol. Ther. 11, 853858.
Lin, T., Matzusaki, G., Yoshida, H., Kobayashi, K., Kenai, H.,
Omoto, K., Nomoto, K., 1994. CD3-CD8+ intestinal intrae-
pithelial lymphocytes (IEL) and the extrathymic development of
IEL. Eur. J. Immunol. 24, 10801087.
Lodinova-Zadnikova, R., Cukrowska, B., Tlaskalova-Hogenova, H.,
2003. Oral administration of probiotic Escherichia coli after
birth reduces frequency of allergies and repeated infections later
in life (after 10 and 20 years). Int. Arch. Allergy Immunol. 131,
209211.
Loew, D., 2000. Leben und Werken von Alfred Nissle. 1119.
McFarland, L.V., Surawicz, C.M., Greenberg, R.N., Fekety, R.,
Elmer, G.W., Moyer, K.A., Melcher, S.A., Bowen, K.E., Cox,
J.L., Noorani, Z., 1995. Prevention of beta-lactam-associated
diarrhea by Saccharomyces boulardii compared with placebo.
Am. J. Gastroenterol. 90, 439448.
Michail, S., Abernathy, F., 2002. Lactobacillus plantarum reduces
the in vitro secretory response of intestinal epithelial cells to
enteropathogenic Escherichia coli infection. J. Pediatr. Gastro-
enterol. Nutr. 35, 350355.
Miller, E.R., Ullrey, D.E., 1987. The pig as a model for human
nutrition. Annu. Rev. Nutr. 7, 361382.
Ogra, P.L., Lamm, M.E., Bienenstock, J., Mestecky, J., Strober, W.,
McGhee, J.R., 1999. Mukosal Immunology. Academic Press,
San Diego.
Pabst, R., Rothkotter, H.J., 1999. Postnatal development of lym-
phocyte subsets in different compartments of the small intestine
of piglets. Vet. Immunol. Immunopathol. 72, 167173.
Patzer, S.I., Baquero, M.R., Bravo, D., Moreno, F., Hantke, K., 2003.
The colicin G, H and X determinants encode microcins M and
H47, which might utilize the catecholate siderophore receptors
FepA, Cir, Fiu and IroN. Microbiology 149, 25572570.
Perdigon, G., Alvarez, S., Rachid, M., Aguero, G., Gobbato, N.,
1995. Immune system stimulation by probiotics. J. Dairy Sci. 78,
15971606.
Pestka, J.J., Ha, C.L., Warner, R.W., Lee, J.H., Ustunol, Z., 2001.
Effects of ingestion of yogurts containing Bifidobacterium and
Lactobacillus acidophilus on spleen and Peyers patch lympho-
cyte populations in the mouse. J. Food Prot. 64, 392395.
Rembacken, B.J., Snelling, A.M., Hawkey, P.M., Chalmers, D.M.,
Axon, A.T., 1999. Non-pathogenic Escherichia coli versus
mesalazine for the treatment of ulcerative colitis: a randomised
trial. Lancet 354, 635639.
Rodrigues, A.C.P., Cara, D.C., Fretez, S.H.G.G., Cunha, F.Q.,
Vieira, E.C., Nicoli, J.R., Vieira, L.Q., 2000. Saccharomyces
boulardii stimulates sIgA production and the phagocytic system
of gnotobiotic mice. J. Appl. Microbiol. 89, 404414.
Romeis, B., 1989. Mikroskopische Techniken. Urban & Schwarzen-
berg, Munchen.
Rothkotter, H.J., Ulbrich, H., Pabst, R., 1991. The postnatal devel-
opment of gut lamina propria lymphocytes: number, prolifera-
tion, and T and B cell subsets in conventional and germ-free pigs.
Pediatr. Res. 29, 237242.
Saalmuller, A., Werner, T., Fachinger, V., 2002. T-helper cells
from naive to committed. Vet. Immunol. Immunopathol. 87,
137145.
Schultz, M., Strauch, U.G., Linde, H.J., Watzl, S., Obermeier, F.,
Gottl, C., Dunger, N., Grunwald, N., Scholmerich, J., Rath, H.C.,
2004. Preventive effects of Escherichia coli strain Nissle 1917 on
acute and chronic intestinal inflammation in two different
murine models of colitis. Clin. Diagn. Lab. Immunol. 11,
372378.
Sinkora, M., Butler, J.E., Holtmeier, W., Sinkorova, J., 2005.
Lymphocyte development in fetal piglets: facts and surprises.
Vet. Immunol. Immunopathol. 108, 177184.
Steidler, L., Neirynck, S., Huyghebaert, N., Snoeck, V., Vermeire,
A., Goddeeris, B., Cox, E., Remon, J.P., Remaut, E., 2003.
Biological containment of genetically modified Lactococcus
lactis for intestinal delivery of human interleukin 10. Nat.
Biotechnol. 21, 785789.
Sturm, A., Rilling, K., Baumgart, D.C., Gargas, K., Abou-Ghazale,
T., raupach, B., Eckert, J., Schumann, R.R., Enders, C., Sonnen-
born, U., Wiedenmann, B., Dignass, A.U., 2005. Escherichia
coli Nissle 1917 distinctively modulates T-cell cycling and
expansion via toll-like receptor 2 signaling. Infect. Immun.
73, 14521465.
Vaarala, O., 2003. Immunological effects of probiotics with special
reference to lactobacilli. Clin. Exp. Allergy 33, 16341640.
Vanbelle, M., Teller, E., Focant, M., 1990. Probiotics in animal
nutrition: a review. Arch. Tierernahr. 40, 543567.
Vega-Lopez, M.A., Arenas-Contreras, G., Bailey, M., Gonzalez-
Pozos, S., Stokes, C.R., Ortega, M.G., Mondragon-Flores, R.,
2001. Development of intraepithelial cells in the porcine small
intestine. Dev. Immunol. 8, 147158.
Vega-Lopez, M.A., Telemo, E., Bailey, M., Stevens, K., Stokes,
C.R., 1993. Immune cell distribution in the small intestine of the
pig: immunohistological evidence for an organized compart-
mentalization in the lamina propria. Vet. Immunol. Immuno-
pathol. 37, 4960.
von Buenau, R., Jaekel, L., Schubotz, E., Schwarz, S., Stroff, T.,
Krueger, M., 2005. Escherichia coli strain Nissle 1917: signifi-
cant reduction of neonatal calf diarrhea. J. Dairy Sci. 88, 317
323.
Waidmann, M., Bechtold, O., Frick, J.-S., Lehr, H.-A., Schubert, S.,
Dobrindt, U., Loffler, J., Bohn, E., Autenrieth, I.B., 2003.
Bacteroides vulgatus protects against escherichia coli-induced
colitis in gnotobiotic interleukin-2-deficient mice. Gastroenter-
ology 125, 162177.
Westendorf, A.M., Gunzer, F., Deppenmeier, S., Tapadar, D., Hun-
ger, J.K., Schmidt, M.A., Buer, J., Bruder, D., 2005. Intestinal
immunity of Escherichia coli NISSLE 1917: a safe carrier for
therapeutic molecules. FEMS Immunol. Med. Microbiol. 43,
373384.
Xu, L.R., Carr, M.M., Bland, A.P., Hall, G.A., 1993. Histochemistry
and morphology of porcine mast cells. Histochem. J. 25, 516
522.
Yang, H., Parkhouse, R.M., 1996. Phenotypic classification of
porcine lymphocyte subpopulations in blood and lymphoid
tissues. Immunology 89, 7683.