Published January 23, 2015
In vitro test on the ability of a yeast cell wall based product to inhibit the
Escherichia coli F4ac adhesion on the brush border of porcine intestinal villi1
P. Trevisi,*2 D. Priori,* G. Gandolfi,* M. Colombo,* F. Coloretti,† T. Goossens,‡ and P. Bosi*
*University of Bologna, Department of Agri-food Protection and Improvement, 40127-Bologna, Italy; †University of
Bologna, Department of Food Science, 40127- Bologna, Italy; and ‡Nutriad, 9200 - Dendermonde, Belgium
ABSTRACT: The ability of a yeast cell wall (YCW)based product (SENTIGUARD C; Nutriad) to inhibit
the enterotoxigenic Escherichia coli F4ac (ETEC)
adhesion on the brush border of porcine intestinal villi
was tested. The ETEC suspensions were preincubated
with 2 batches of the product (A and B) at different
concentrations (10, 5, and 0.5%, wt/vol) or with their
filtrates (AF and BF) and then with intestinal villi
susceptible to ETEC adhesion. In all the trials, ETEC
suspensions were also preincubated with egg yolk
(E) immunized against ETEC to assess the maximum
inhibition of the adhesiveness or directly with the
villi [control group (Con)] to verify the maximum
adhesiveness of the pathogen. For each treatment, 20
different villi were observed, brush border measured,
and the adherent pathogens counted. A scanning
electron microscope analysis was used to confirm the
ability of ETEC to adhere on the YCW. The E treatment
significantly reduced the pathogen adhesion on the villi
compared with the C group in all the trials (P < 0.001).
Both batches of SENTIGUARD C significantly reduced
the pathogen adhesion on the villi compared with the
C group at the concentration of 10 and 5% (P < 0.001)
but not at the concentration of 0.5%. The BF did not
significantly reduce the ETEC adhesion whereas the AF
significantly increased bacterial adhesion (P = 0.015).
The microscopy results confirm the ability of ETEC to
adhere on YCW. Taken together, our results indicate the
ability of the SENTIGUARD C to contain the intestinal
infection from ETEC in young pigs with the affinity of
ETEC to YCW.
Key words: adhesion, brush border, Escherichia coli F4ac, pig, yeast cell wall
© 2012 American Society of Animal Science. All rights reserved.
INTRODUCTION
Colibacillosis is associated with pathogenic
Escherichia coli colonization of the digestive tract,
which is mediated by binding of bacterial fimbriae to
ligands on gut epithelial cells. This is also the case for
enterotoxigenic E. coli F4ac (ETEC), the pathogen
most frequently associated with diarrhea in piglets.
Over the last years, the use of nonantibiotic dietary
strategies to prevent intestinal diseases has gained
considerable interest. The inclusion of yeast cell wall
(YCW) in animal feed might be a promising strategy to
reduce colibacillosis in pigs. Indeed, several serotypes
of E. coli and Salmonella spp. have been shown to
This work was supported in part by Nutriad INTERNATIONAL
nv; authors are grateful to Marzia Benevelli for the technical
support and Istituto Zooprofilattico Sperimentale Bruno Umbertini
for providing Escherichia coli F4ac.
2
Corresponding author: [email protected]
1
J. Anim. Sci. 2012.90:275–277
doi:10.2527/jas53771
adhere to the surface of dried brewer’s yeast products
in agglutination tests (Spring et al., 2000; White et
al., 2002). These results warrant further study of the
applicability of YCW to prevent ETEC adhesion to
porcine intestinal epithelium. We therefore set up in
vitro studies to test if (i) YCW can prevent the adhesion
of ETEC to intestinal villi, (ii) the action of YCW is
dose dependent, and (iii) there are soluble factors that
can inhibit the ETEC adhesion.
MATERIALS AND METHODS
In Vitro Villus Adhesion Assay
The ability of 2 different batches (batch A and B)
of the YCW-based product SENTIGUARD C, provided
by Nutriad, Turnhout, Belgium, and of their filtrates to
inhibit in vitro ETEC adhesion was assessed using the
protocol described by Bosi et al. (2004) adapted for this
275
276
Trevisi et al.
Table 1. Effect of 2 batches of a yeast cell wall-based
product to inhibit the adhesion of enterotoxigenic
Escherichia coli F4ac (ETEC) on the pig intestinal
brush border
Treatment1
C
E
A10
B10
SEM
ETEC (n/250 μm)
P-value2
68.39
14.98
17.04
26.56
5.54
<0.001
<0.001
<0.001
1
C = control group (incubation of villi + ETEC); E = ETEC preabsorbed
with of egg yolk immunized against ETEC; A10 = ETEC preabsorbed with
batch A at 10%; B10 = ETEC preabsorbed with batch B at 10%.
2
The groups were compared with the control, considered as a reference.
purpose. The modification consisted of preincubation of
ETEC with the tested product prior to incubation with
the intestinal villi. The in vitro assay consisted of 3 trials.
In each of them, a subset of villi was incubated with
ETEC suspension preincubated with 10% of egg yolk
immunized against this bacterial strain, able to maximize
the inhibition of the adhesiveness of ETEC through its
anti-F4ac immunoglobulins content (E treatment), and a
subset of villi was incubated directly with ETEC without
preincubation [control group (Con) treatment], to obtain
the maximum potential adhesiveness of the ETEC.
In trial 1, intestinal villi collected from 1 piglet were
divided in 4 subsets and incubated with the following
treatments: ETEC preincubated with (i) batch A at the
concentration of 10% (wt/vol; A10), (ii) batch B at 10%
(wt/vol; B10), (iii) E treatment, and (iv) C treatment. In
trial 2, the intestinal villi collected from 1 piglet were
divided in 6 subsets and incubated with the following
treatments: ETEC preincubated with (i) batch A at 0.5%
(wt/vol; A0.5), (ii) batch B at 0.5% (wt/vol; B0.5), (iii)
batch A at 5% (wt/vol; A5), (iv) batch B at 5% (wt/vol;
Table 2. Effect of 2 doses of a yeast cell wall-based
products to inhibit the adhesion of enterotoxigenic
Escherichia coli F4ac (ETEC) on the pig intestinal
brush border
Treatment1
C
E
A0.5
A5
B0.5
B5
SEM
ETEC (n/250 μm)
P-value2
70.66
20.64
57.37
39.70
55.65
34.33
4.49
<0.001
0.144
<0.001
0.073
<0.001
1
C = control group (incubation of villi + ETEC); E = ETEC preabsorbed
with of egg yolk immunized against ETEC; A0.5 or A5 = ETEC preabsorbed
with batch A at 0.5 or 5%; B0.5 or B5 = ETEC preabsorbed with batch B at
0.5 or 5%.
2
The groups were compared with the control, considered as a reference.
B5), (v) E treatment, and (vi) C treatment. For trial 3,
batches A and B were incubated in PBS at a concentration
of 10% (wt/vol), and after 1 h the suspensions were
filtered and the resulting solutions were used for the in
vitro test. Intestinal villi were collected from 2 piglets
and each was divided in 6 subsets and incubated with
the following treatments: ETEC preincubated with
(i) batch A at 10% (wt/vol; A10), (ii) batch B at 10%
(wt/vol; B10), (iii) filtrate solution from batch A at the
concentration of 10% (vol/vol; AF), (iv) filtrate solution
from batch B at 10% (vol/vol; BF), (v) E treatment, and
(vi) C treatment. After incubation, intestinal villi were
examined by phase-contrast microscopy coupled with a
digital camera at 100× magnification. For each group,
pictures from 20 different villi were acquired, brush
border length was measured, and adherent pathogens
counted. The values were reported as number of bacteria
adhered along a 250-μm length of villous brush border.
Figure 1. Ability of Escherichia coli F4ac to adhere on the yeast cell wall (A) 10,000× magnification and (B) 20,000× magnification.
Yeast cell wall inhibits E. coli F4 adhesion
Table 3. Effect of a yeast cell wall-based products and
its filtrate to inhibit the adhesion of enterotoxigenic
Escherichia coli F4ac (ETEC) on the pig intestinal
brush border
Treatment1
ETEC (n/250 μm)
P-value2
70.65
41.46
85.64
71.26
55.33
52.17
3.74
<0.001
0.015
1.000
0.001
0.001
C
E
AF
BF
A10
B10
SEM
1
C = control group (incubation of villi + ETEC); E = ETEC preabsorbed
with of egg yolk immunized against ETEC; AF = ETEC preabsorbed with
filtrate from batch A; BF = ETEC preabsorbed with filtrate from batch B; A10
= ETEC F4 preabsorbed with batch A at 10%; B10 = ETEC preabsorbed with
batch B at 10%.
2
The groups were compared with the control, considered as a reference.
Scanning Electron Microscope Analyses
After incubation with ETEC as described above,
samples were prepared following the protocol reported
by Bottazzi and Bianchi (1980). The pictures were
acquired by scanning electron microscope coupled with
a digital camera.
Statistical Analysis
Data were analyzed by ANOVA testing the effect of
different incubation treatments. In trial 3 the effect of
repetition was also included. The groups were compared
with the control, considered as a reference, by the
Dunnett test.
RESULTS AND DISCUSSION
Three concentrations were tested for 2 batches
of the product and the results shown in Tables 1 and 2
indicated that at the concentration of 10 and 5%, the
product reduced (P < 0.001) the ETEC adhesion on the
intestinal brush border membrane. On the other hand,
when the concentration dropped to 0.5%, the effect
disappeared. The agglutination test conducted by White
et al. (2002) suggested the ability of dried yeast to capture
277
ETEC reducing its pathogenicity. The in vitro model
demonstrated that a preincubation of the ETEC with a
YCW-based product reduced the ability of this pathogen
to adhere to its specific intestinal receptors.
To exclude the potential presence of soluble factors
able to inhibit ETEC adhesion on the brush border
membrane we set up a specific in vitro test. The results
reported in Table 3 demonstrate for both batches the
absence of soluble factors able to reduce ETEC adhesion
on the villi. Also in this case, the highest products dose
interfered with the ETEC adhesion (P = 0.001). The
reliability of our test was supported by the observation
that in the group preabsorbed with the immunized egg
yolk, the adhesion of ETEC on the brush border was
lower (P < 0.001) than in the C in all the three trials,
in agreement with Jin et al. (1998). Finally, by scanning
electron microscopy, we observed the ability of the YCW
to capture the E. coli F4 (Figure 1). On the whole, these
results confirm the ability of the yeast wall composing
SENTIGUARD C to capture the ETEC, reducing the
amount of this pathogen free to adhere on the intestinal
wall.
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