Veterinary Immunology and Immunopathology 128 (2009) 60–66

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Veterinary Immunology and Immunopathology

journal homepage: www.elsevier.com/locate/vetimm

Research paper

Effect of b-glucans on an ETEC infection in piglets

E. Stuyven a,*, E. Cox a, S. Vancaeneghem a, S. Arnouts a, P. Deprez c, B.M. Goddeeris a,b
a
Laboratory of Immunology, Faculty of Veterinary Medicine, UGent, Salisburylaan 133, B-9820 Merelbeke, Belgium
b
Faculty of Bioscience Engineering, Department of Biosystems, K.U.Leuven, Kasteelpark Arenberg 30, B-3001 Heverlee, Belgium
c
Department of Internal Medicine and Clinical Biology of Large Animals, Faculty of Veterinary Medicine, University Ghent, Merelbeke, Belgium

A R T I C L E I N F O A B S T R A C T

Keywords: The effect of orally administered b-glucans in protecting pigs against an ETEC infection
b-Glucans after weaning was analysed in this study. Three b-glucans that differed in origin
Immunostimulation (Saccharomyces cerevisiae (MCG (Macrogard) and G2) or Sclerotium rolfsii (G3)) and/or
In vivo extraction procedure were tested. Pigs fed for 2 weeks after weaning with these glucans
Pigs
were less susceptible to an F4+ ETEC infection in comparison with the control group. This
was evidenced by a reduction in the faecal excretion of F4+ Escherichia coli as well as a
reduced F4-specific serum antibody response. This decrease in faecal excretion was
statistically significant for pigs fed with the MCG glucan in a first experiment and with the
G3 glucan in a second experiment; diarrhoea was milder in the glucan-supplemented
groups and was significantly reduced in the MCG-supplemented group. Furthermore, a
lower amount of F4-specific IgM antibody-secreting cells (ASC) was found in the lymphoid
tissues of pigs fed with G2 or G3 glucans in comparison with the control pig, as well as
lower F4-specific IgA ASC in G3-fed pigs in comparison with the control pig. This study
showed that b-glucans can protect against an ETEC infection. Both MCG from S. cerevisiae
and G3 from S. rolfsii, resulted in significant effects. To our knowledge, this is the first in
vivo study, in which the use of b-glucans as feed ingredient for just-weaned piglets was
tested for their protective effects against ETEC infection.
ß 2008 Elsevier B.V. All rights reserved.

1. Introduction against a challenge infection that occurs 24 days later

Enterotoxigenic Escherichia coli (ETEC) are an important
cause of diarrhoea in neonatal, suckling and newly weaned
piglets. During the neonatal and suckling period, the
piglets can be passively protected by antibodies preset in
milk (Rutters and Jones, 1973; Deprez et al., 1986; Osek
et al., 1995). This protection disappears at weaning,
making weaned piglets susceptible again to an ETEC
infection (Hampson, 1994). Weaned piglets need an active
immunity to be protected against ETEC. We have shown
that an oral immunisation of weaned piglets with purified
F4 fimbriae results in an immune response (Van den
Broeck et al., 1999a,b) that completely protects piglets
* Corresponding author. Tel.: +32 92647384; fax: +32 92647496.
E-mail address: Edith.Stuyven@UGent.be (E. Stuyven).
(Verdonck et al., 2004). This response, however, comes too
late to protect piglets against ETEC-induced weaning
diarrhoea, since ETEC infections most often occur the first
week after weaning. Attempts to immunize piglets during
the suckling period did not result in a complete protection
(Snoeck et al., 2003). Therefore a vaccine inducing
protective immunity at weaning that can be given during
the suckling period is not available yet. The prophylactic
use of antibiotics could also protect piglets, but is
prohibited in Europe since January 2006. Therefore,
alternatives are searched for in order to prevent infection
on problem farms.
b-Glucans, polymers of D-glucose extracted from the
cell wall of bacteria, fungi or yeast, are known as
immunostimulators. b-Glucans have the capacity to
activate the innate immune system, thereby enhancing
the defence barriers and thus providing protection against

0165-2427/$ – see front matter ß 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetimm.2008.10.311
 E. Stuyven et al. / Veterinary Immunology and Immunopathology 128 (2009) 60–66
an otherwise severe or lethal infection (Raa, 1996;
Vetvicka et al., 2002). Experimental studies have demon-
strated the protective efficacy of different glucans against a
variety of experimentally induced infections in mouse, rat,
guinea pigs and fish (Williams et al., 1978; Cook et al.,
1980; Robertsen et al., 1990; Chang et al., 2003;
Drandarska et al., 2005). The b-glucans activate mono-
cytes, macrophages, neutrophils and NK cells and indir-
ectly T- and B-lymphocytes via cytokines (Sherwood et al.,
1987; Williams et al., 1988; Suzuki et al., 1990; Adachi
et al., 1994; Ohno et al., 1995; Lowe et al., 2002). As a
consequence b-glucans can be used as adjuvants in
immunisations (Mohagheghpour et al., 1995; Markevich
et al., 1996; Hetland et al., 2000). They can be given by
parenteral or oral route. Careful selection of the appro-
priate administration route is advised, because some b-
glucans, like lentinan and schizophyllan, are not effective
by oral administration, while SSG from the fungus
Sclerotinia sclerotiorum possess immunomodulating and
antitumor activities in mice after oral administration in
high doses (over 80 mg/kg) (Suzuki et al., 1989). The orally
administered SSG can activate Peyer’s patch cells, thus
enhancing antigen-specific and non-specific production of
IgA at several mucosal sites (Hashimoto et al., 1991).
Most studies on the effect of b-glucans have been
performed in mice, rats and fish. Little is known about the
effects of b-glucans in pigs. Dritz et al. (1995) studied the
effect of dietary b-glucans on growth performance,
neutrophil and macrophage functions, haptoglobin pro-
duction and resistance to Streptococcus suis challenge in
weaning pigs. They concluded that pigs fed with a 0.025%
b-glucan diet had the best growth performance.
The aim of the present study was to determine whether
oral administration of b-glucans to piglets could increase
their resistance against an infection with ETEC.
2. Materials and methods
2.1. Animals
Twenty-eight piglets (Belgian Landrace  Piétrain),
which were seronegative for F4-specific antibodies, were
weaned at the age of 4–5 weeks, transported to the
experimental facilities at the faculty and subsequently
housed in isolation units where they obtained water and
food ad libitum. All piglets were treated orally with
colistine (150,000 U/kg of body weight/day, Promycine
pulvis, VMD, Berendonk, Belgium) from 2 days before until
3 days after weaning to prevent colonization with ETEC
due to transport and handling.
The animals were euthanized during or at the end of
each experiment (see Section 2.5) to determine the
number of F4-specific antibody-secreting cells (ASC), the
production of specific cytokines and the presence of the
small intestinal F4 receptor (F4R). In the absence of this
receptor, pigs are not susceptible to an F4 ETEC infection.
Euthanasia was performed by intravenous injection of
pentobarbital (24 mg/kg; Nembutal, Sanofi Santé Animale,
Brussels, Belgium) followed by exsanguination.
All experimental and animal management procedures
have been approved by the animal care and ethics
61
committee of the Faculty of Veterinary Medicine, Ghent
University.
2.2. Bacterial inoculum
The hemolytic E. coli strain GIS 26, serotype O149:F4ac+,
LT STa+, STb+ was cultured during 18 h (37 8C, 85 rpm) in
+
Tryptone Soya Broth (Oxoid, Basingstoke, Hampshire,
England) and bacteria were collected by centrifugation
(2808  g, 45 min, 4 8C). Subsequently, the bacteria were
washed three times in PBS (150 mM, pH 7.4) and
resuspended in PBS at a concentration of 109 bacteria/ml.
2.3. b-Glucans
In the present study three different b-glucan prepara-
tions, Macrogard and two other from INVE Technologies
NV (Dendermonde, Belgium) were tested. These b-glucans
differed in origin and/or extraction procedure (Table 1).
The b-glucans were mixed in the food and were given for 2
weeks each at their advised dose.
2.4. In vitro villous adhesion assay for presence of F4
receptors (F4R)
The in vitro villous adhesion assay was performed as
described by Van den Broeck et al. (1999c).
2.5. Experimental procedures
2.5.1. Effect of Macrogard
Ten piglets were weaned at the age of 5 weeks and
divided into two groups. Six piglets were given Macrogard in
the feed (500 g/ton feed) for 2 weeks (MCG group). The
remaining four piglets served as untreated controls (control
group). Seventeen days postweaning (3 days after the last
administration of glucan), all animals were orally inoculated
with the F4+ ETEC strain as described previously (Van den
Broeck et al., 1999a) with minor modifications. Briefly, pigs
were pretreated orally for 2 days with florfenicol (300 mg/kg
BW SID). On the third day the animals were deprived of food
and water for 3 h. Subsequently, 60 ml NaHCO3 (1.4% [w/v]
in distilled water) was administered intragastrically to
neutralise the gastric pH. 15–30 min later the pigs were
given 1010 F4+ ETEC intragastrically and 2 h after the
infection, the piglets were given food and water ad libitum.
F4-specific serum IgM, IgA and IgG were measured at 0,
5, 7 and 10 days post-infection (dpi), whereas the faecal F4+
ETEC excretion was determined daily from 2 to 7 dpi.
Table 1
Characteristics of the experimental groups of the second experiment
regarding the type of b-glucan administered.
Group n Dose Origin of b-glucan Extraction procedure
(g/ton)
MCG 5 500 Saccharomyces Not known
cerevisiae
G2 5 750 Saccharomyces Lysis + drying
cerevisiae
G3 5 500 Sclerotium rolfsii Fermentation + drying
Control 4 – – –
62 E. Stuyven et al. / Veterinary Immunology and Immunopathology 128 (2009) 60–66
2.5.2. Comparison of Macrogard with two other b-glucan
preparations
In a second experiment, Macrogard and two other b-
glucan preparations (G2 and G3) were tested for their
protective effect (Table 1). Hereto 19 piglets were weaned at
the age of 4 weeks and divided into four groups. Each group
contained five piglets, except for the control group, which
consisted of four piglets. Administration of glucan, infection
with F4+ ETEC and the analyses of faecal excretions were
performed as described in the first experiment. Serum IgM,
IgA and IgG antibodies specific for F4 were measured at 0, 5,
8, 11 and 16 dpi. To have an indication if there were effects
on intestinal or systemic production of IgA and/or IgM,
numbers of F4-specific IgM and IgA ASC numbers were
measured in mesenteric lymph nodes (MLN), Peyer’s
patches, lamina propria and in spleen of one piglet of each
group at 14 dpi. To determine the effect of MCG and G3 on
production of IL-6, IL-10, TGFb and IFNg, mRNA for these
cytokines was quantified in cells of lymphoid tissues
(mesenteric lymph nodes, ileal and jejunal Peyer’s patches
(JPP)) at 2 dpi in one pig of the control and the G3 group and
at 14 dpi in one pig of the control, MCG and G3 group.
2.6. Samples
2.6.1. Serum
Blood was taken from the vena jugularis. Serum was
collected and inactivated at 56 8C during 30 min. Subse-
quently the serum was treated with kaolin (Sigma–Aldrich,
Bornem, Belgium) to decrease the background reading in
ELISA (Van den Broeck et al., 1999a) and frozen at 20 8C.
2.6.2. Spleen and lymph node monomorphonuclear cells
After euthanasia, the spleen and some mesenteric
lymph nodes were aseptically dissected. After removing
surrounding fat, the monomorphonuclear cells (MC) were
isolated by teasing the tissues apart, followed by lysis of
erythrocytes with ammonium chloride (0.8%, w/v). After
centrifugation, the pelleted cells were washed and
resuspended at 107 cells/ml in leukocyte medium (RPMI-
1640 (Gibco BRL, Life Technologies, Merelbeke, Belgium)
containing FCS (10%, v/v), non-essential amino acids, Na-
pyruvate (100 mg/ml), L-glutamine (200 mg/ml), penicillin
(100 IU/ml) and kanamycin (100 mg/ml)).
2.6.3. Lamina propria and Peyer’s patches MC
Segments of the small intestine were dissected from the
piglets and cut into pieces of 5 cm2 and washed three times
with PBS, twice with PBS without Ca2+ or Mg2+ (CMF
buffer) and once with CMF buffer supplemented with EDTA
(3.7%, w/v). After washing, the pieces were incubated in
CMF–EDTA at 37 8C for 15 min. The Peyer’s patches MC
were scraped from the mucosa. The lamina propria MC
were collected after 30 min incubation (37 8C) with RPMI
supplemented with FCS (5%, w/v), collagenase (0.015%, w/
v) (Sigma–Aldrich, Brussels, Belgium) and DNAse I (0.010%,
w/v) (Boehringer Mannheim, Brussels, Belgium). The
isolated cells were washed with RPMI supplemented with
FCS (2%, v/v), penicillin (100 IU/ml) and streptomycin
(100 mg/ml) and resuspended in leukocyte medium at
107 cells/ml.
2.7. ELISA for F4-specific IgM, IgA and IgG
F4-specific IgM, IgA and IgG were detected by ELISA as
described by Van der Stede et al. (2002).
2.8. ELIspot assay for F4-specific IgM- and IgA-secreting cells
F4-specific IgM and IgA ASC were determined by the
protocol described by Van den Broeck et al. (1999a).
2.9. Faecal excretion of F4+ ETEC
To determine the faecal F4+ ETEC excretion after the
infection, faeces was collected daily for 7 days. In the first
experiment faeces was stored at 20 8C before analyses,
whereas analyses of F4+ ETEC in the sample was done on
the fresh sample in the second experiment. The number of
F4+ ETEC in the faeces was quantified by dot-blotting
haemolytic colonies after inoculation of 10-fold dilutions
of faeces on blood agar plates and immunodetection of F4
fimbriae production using the F4-specific monoclonal
antibody IMM01 as described by Van den Broeck et al.
(1999b).
2.10. Diarrhoea score
After challenge infection, the severity of the diarrhoea
was evaluated by scoring the consistency of the faeces:
0 = normal; 1 = pasty; 2 = semi-liquid; 3 = watery. Scoring
was performed twice a day by two persons, independently
of each other (Van der Stede et al., 2003).
2.11. Statistical analysis
Differences in log10 F4+ ETEC per gram faeces between
the groups and differences in log2 antibody serum titres
(IgM, IgA and IgG) were tested for statistical significance
using the General Linear Model (Multivariate). The Mann–
Whitney U test was used to demonstrate statistical
differences in duration of diarrhoea and diarrhoea scores
between groups. P < 0.05 was considered as statistically
significant.
3. Results
3.1. F4R characterization of the experimental animals
All experimental animals expressed the F4R as evi-
denced by >5 bacteria/250 mm villous length in the villous
adhesion assay (Rasschaert et al., 2007).
3.2. Faecal excretion of F4+ ETEC upon infection with ETEC
In the first experiment, F4+ E. coli were isolated for 5
consecutive days from faecal samples of two pigs (>106 F4+
ETEC per gram faeces) and for 2 days of the 5 days of the
study from the other two pigs (between 2  106 and
2  107 F4+ ETEC per gram faeces) of the control group,
whereas no F4+ E. coli could be isolated from the faeces of
any of the pigs fed with Macrogard (Fig. 1A). Excretion of
F4+ ETEC is significantly different between experimental
 E. Stuyven et al. / Veterinary Immunology and Immunopathology 128 (2009) 60–66 63

Fig. 2. Evolution of F4-specific IgM, IgA and IgG antibody titres in serum
(+SEM) in the control and MCG group after infection with F4+ ETEC (dpi:
days post-infection). *MCG group presents statistically significant
differences from the control group with P < 0.05.

Fig. 1. Mean log10 (number of hemolytic F4+ ETEC per gram faeces) (+SEM)
per group at different days post-infection (dpi) in the first experiment (A)
and second experiment (B). *P < 0.05 in comparison with the control
group.

and control groups on day 2 post-infection; the variation

seen among excretion in the pigs of the control group and
the limited number of pigs in each group may explain why
this difference did not extend to all other days of the study.
In the second experiment, where Macrogard and two
other glucan preparations were tested, overall higher
amounts of F4+ ETEC per gram faeces were isolated from
the freshly examined faecal samples of all groups than in
the first experiment. Furthermore mean excretion in the
control group was higher than in the glucan groups on
every day post-infection except on day 5. However only in
the G3 group, the excretion was significantly lower in
comparison with the control group on day 4 post-infection
(Fig. 1B).

3.3. Diarrhoea

In the first experiment, diarrhoea in the control group

was seen for a mean period of 4.3 days (range 2–6 days),
whereas this was slightly shorter in the MCG group (mean
duration of 3.4 days (range 3–4 days)). The severity of the
diarrhoea as expressed by the diarrhoea score was similar
for both groups the first day after the challenge infection,
but was lower in the glucan group from day 2 onwards
with a significant difference on day 3 (P = 0.032; median
score (range) for the MCG group 1 (0–1) and for the control
group 2.5 (1–3)).
In the second experiment, the same effect was seen for
the MCG group in comparison with the control group as in
the first experiment. Pigs of the MCG group presented with
a significantly shorter period of diarrhoea than the control
pigs (P = 0.046; mean duration (range) of 1.6 days (0–3) for
the MCG group and 3.7 (3–4) for the control group) and
also the severity of the diarrhoea, reflected by the
Fig. 3. Evolution of the F4-specific IgM, IgA and IgG antibody titres in
diarrhoea score, was clearly lower for the MCG group serum (+SEM) in the control and glucan-treated groups after infection
from day 2 onwards with significant differences on days 4 with F4+ ETEC infection (dpi: days post-infection). *Number of group:
(P = 0.042) and 5 (P = 0.042). For both other glucan groups, P < 0.05 between these groups and the control group.
64 E. Stuyven et al. / Veterinary Immunology and Immunopathology 128 (2009) 60–66
effects on duration and severity of diarrhoea were not
significant, although mean duration and median scores
were lower than for the control group.
3.4. F4-specific immune responses in serum
In the first experiment, infection with F4+ ETEC induced
a rapid increase in the mean F4-specific IgM, IgA and IgG
titres in the control group with maximum IgM and IgA
7 dpi and IgG 11 dpi. F4-specific IgM immediately declined
again whereas IgA remained stable during 14 days before it
slowly declined. F4-specific IgG titres, however, did not
decline during the experiment. This F4-specific antibody
response was similar to the response seen in previous
studies (Verdonck et al., 2002). In the MCG group, however,
the IgM, IgA and IgG titres did not exceed background
levels suggesting absence of or low ETEC infection in this
group (Fig. 2).
In the second experiment (Fig. 3), similar F4-specific
antibody responses were seen in the control group upon
ETEC infection as in the first experiment. And also here, the
F4-specific IgM, IgA and IgG responses were clearly lower
in the glucan-treated groups than in the control group.
Differences were most pronounced for the G3 group, which
showed significantly lower responses for all three isotypes.
3.5. The F4-specific B cells in the spleen and intestinal mucosa
In order to confirm the lower F4-specific serum
antibody responses following glucan supplementation
Fig. 4. F4-specific IgM and IgA ASC per 107 mononuclear cells (MC) in
spleen (Sp), mesenteric lymph node (MLN), jejunal (JPP) and ileal Peyer’s
patches (IPP) and lamina propria (LP), 14 days post-infection (dpi) in the
control and glucan-treated groups (n = 1).
and to see if glucans could have a similar effect on the
intestinal mucosal immune response, the number of F4-
specific IgM and IgA ASC were determined in the
mesenteric lymph nodes, the Peyer’s patches, the lamina
propria and in the spleen for one pig of each group at 14 dpi
(Fig. 4).
The control pig had a clear IgM response with highest
numbers of IgM ASC in the spleen, reflecting the strong
systemic IgM response in the control group and less
numbers of ASC in the mesenteric lymph nodes, the jejunal
and the ileal Peyer’s patches (IPP) and the lamina propria,
similar to what was seen in a previous study in the gut-
associated lymphoid tissues 11–15 dpi (Verdonck et al.,
2002). The pigs of the G2 and G3 group had little or no IgM
ASC in their tissues, reflecting the low systemic IgM
responses in both groups. The MCG pig, however, had
higher numbers of ASC in the mesenteric lymph nodes
versus 2.5-times less ASC in spleen and jejunal Peyer’s
patches, which was not in accordance with the low IgM
responses in pigs of this group.
Overall the numbers of IgA ASC were lower than the
numbers of IgM ASC. For the control pig, the highest
numbers of IgA ASC were found in the mesenteric lymph
nodes and decreasing numbers in the jejunal Peyer’s
patches, in the spleen and the ileal Peyer’s patches,
which agrees with results in previous experiments
(Verdonck et al., 2002). As for IgM, the G2 and G3 pigs
showed almost no IgA ASC and the MCG pig a high
number of IgA ASC, especially in the mesenteric lymph
nodes.
4. Discussion
Studies in which b-glucans are tested to protect piglets
against an infection are few. A study of Dritz et al. (1995)
shows that administration of glucans originating from
Saccharomyces cerevisiae in the food resulted in an
increased growth performance of the piglets but also in
an increased susceptibility to S. suis infection. These results
are in contradiction with most other studies performed in
mice, humans and fish, where b-glucans are shown to
induce protection against several bacterial (Kokoshis et al.,
1978; Robertsen et al., 1990), fungal (Williams and
DiLuzio, 1980), parasitic (Cook et al., 1980) and viral
(Williams et al., 1978) infections.
The present study was undertaken to screen three
different b-glucans for their protective effects against an
F4+ ETEC infection in newly weaned piglets. Results show
that pigs fed b-glucans were less susceptible to coloniza-
tion with F4+ E. coli and/or diarrhoea. Indeed, in a first
experiment, oral supplementation with Macrogard for 14
days reduced the faecal excretion of F4+ E. coli and the
accompanying diarrhoea; even though the experimental
groups were small, these effects were significant at some of
the days evaluated post-infection. The reduced bacterial
excretion and diarrhoea suggested a correspondingly
reduced colonization of the small intestine with F4+ ETEC.
The observed reduction could also explain the significantly
lower F4-specific serum IgM, IgA and IgM response in the
MCG group in comparison with the control group (Newby
et al., 1981).
 E. Stuyven et al. / Veterinary Immunology and Immunopathology 128 (2009) 60–66
In the second experiment, a less pronounced protective
effect of the MCG glucan was observed relative to the first
experiment, but there was still a reduction of approximately
2 log10 F4+ E. coli in the faeces on days 2–4 post-infection.
This less pronounced protection could be due to a more
severe ETEC infection in this experiment than in the first one,
a fact which is reflected by the higher excretion of E. coli and
the higher IgM and IgA responses detected in the control
group in the second relative to the first experiment.
However, the effects of Macrogard on duration and severity
of diarrhoea were nevertheless significant.
Both of the other tested b-glucans also seem to have
some beneficial effects, although these effects were clearly
less pronounced with only a significant reduction in F4+ E.
coli excretion for the G3 group on day 4 post-infection.
Several factors could be responsible for the different effect
of the glucans. A first factor may be the dose of the
oligosaccharide. Raa (1996) pointed out that the effective
dose is different for different b-glucans preparations. In
the present study, the oligosaccharides were tested at the
dose suggested by the company. Secondly, the immunos-
timulatory capacity of b-glucans is influenced by their
ultrastructure (Ohno et al., 1995; Okazaki et al., 1995,
1996). The ultrastructure of G3 must differ from that of
Macrogard and G2, since the G3 glucan is a gel forming b-
glucan whereas both other glucans are particulates. Gel
forming b-glucans have a triple or a single helix
conformation, whereas particle b-glucans have a non-
uniform structure because of irregular branches and
backbone units (Nagi et al., 1993; Okazaki et al., 1996).
In fact, b-1,3-glucans with a high degree of polymerization
(DP > 100) are completely insoluble in water (Zeković and
Kwiatkowski, 2005). Solubility increases as the degree of
polymerization of b-1,3-glucans is lowered. Their solubi-
lity appears to depend also on length of side substituted
branches and the frequency of side branches (Fleet and
Manners, 1976). Data suggest that higher ordered struc-
tures like the triple helices are responsible for the
immunomodulating activity of b-1,3-glucans (Hamuro
et al., 1971; Maeda et al., 1988). Furthermore, b-glucans
with a degree of branching of 0.2–0.33 seem to be the most
active ones (Bohn and BeMiller, 1995).
For the most part, a decrease in faecal excretion of F4+
ETEC was accompanied by a decrease in the serum
antibody response. This seems to indicate that prevention
of adhesion and/or of colonization of the small intestine
could be one of the effects of the glucans. A direct effect on
adhesion would suggest that b-glucans act as an analogue
for a receptor or ligand. In this experiment the piglets were
infected 3 days after administration of b-glucan, so it is
unlikely that b-glucans were still present in the small
intestine at that moment. Furthermore, for one pig of the
G3 group euthanatized 2 days after the challenge infection,
the villi allowed in vitro the adhesion of F4+ E. coli.
Furthermore, attempts to block in vitro the adhesion with
all three glucans were unsuccessful (unpublished find-
ings). Therefore, a direct effect on adhesion seems unlikely.
Indirect effects on colonization could occur via activation
of enterocytes which subsequently can secrete antimicro-
bial peptides. However, Dectin-1, the main receptor of b-
glucans, has not been found on enterocytes (Rice et al.,
65
2005). Nevertheless, Rice et al. (2005) reported inter-
nalisation of soluble glucans in enterocytes through a
Dectin-1 independent mechanism. This could initiate
activation of enterocytes. Subsequently they observed
the glucans in cells of the GALT. Dectin-1 has been found on
macrophages, monocytes, dendritic cells, neutrophils,
some subsets of T cells and in humans also on B cells,
making these cell populations in the gut targets for
activation by b-glucans (Pyz et al., 2006). It should be
mentioned, however, that it is unclear until now if
insoluble particulate glucans are absorbed (Rice et al.,
2005).
In contrast to the G2 and G3 pigs and seemingly in
contrast to the serum antibody response, important
numbers of F4-specific ASC were found in the different
lymphoid tissues of the MCG pig in the second experiment.
This could either suggest a strong activation of the GALT
with rapid clearance of the F4+ ETEC infection or the pig did
not respond appropriately on the glucan administration.
Therefore, this finding needs further confirmation.
5. Conclusion
Piglets receiving food supplemented with b-glucans for
2 weeks after weaning show a decreased susceptibility to
ETEC, suggesting that b-glucans could be an alternative for
antibiotics for prevention of postweaning infections with
E. coli.
Acknowledgements
The work was financially supported by INVE Technol-
ogies (Dendermonde, Belgium), by the FOD Health of the
Federal government, by Ghent University and the IWT-
Flanders. We thank G. De Smet, D. Slos and R. Cooman for
technical assistance.
Conflict of interest
None.
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