Enterococcus faecium NCIMB 10415 shows no beneficiary effects in ...

AEM Accepts, published online ahead of print on 27 April 2012
Appl. Environ. Microbiol. doi:10.1128/AEM.00395-12
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
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Enterococcus faecium NCIMB 10415 shows no beneficiary effects in weaned
pigs infected with Salmonella enterica serovar Typhimurium DT 104.
Susanne Kreuzer 1 *, Pawel Janczyk 2 * § , Jens Aßmus 1 , Michael F.G. Schmidt 3 , Gudrun A.
Brockmann 1 , Karsten Nöckler 2
1 Humboldt-Universität zu Berlin, Breeding Biology and Molecular Genetics, Invalidenstr. 42,
D-10115 Berlin, Germany
2 Federal Institute for Risk Assessment, Department of Biological Safety, Unit Molecular
Diagnostics and Genetics, Diedersdorfer Weg 1, 12277 Berlin, Germany
3 Freie Universität Berlin, Institut für Immunologie und Molekularbiologie, Philippstr. 13,
Berlin, Germany
*These authors contributed equally to this work
§ corresponding author: pawel_janczyk@t-online.de; pawel.janczyk@bfr.bund.de
Running title: Effects of E. faecium supplementation in an infection model in piglets
SK: Susanne.Kreuzer.1@agrar.hu-berlin.de
PJ: pawel_janczyk@t-online.de; pawel.janczyk@bfr.bund.de
JA: Jens.Assmus@staff.hu-berlin.de
MFGS: michael.schmidt3@fu-berlin.de
GAB: gudrun.brockmann@agrar.hu-berlin.de
KN: karsten.noeckler@bfr.bund.de
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Abstract
Salmonella enterica serovar Typhimurium DT 104 (S. Typhimurium) is the major pathogen
for salmonellosis outbreaks in Europe. We tested if the probiotic bacterium Enterococcus
faecium NCIMB 10415 (E. faecium) can prevent or alleviate samonellosis. Therefore, piglets
of the German Landrace breed that were treated with E. faecium (n=16) as a feed additive and
untreated controls (n=16) were challenged with S. Typhimurium 10 days after weaning. The
presence of salmonellae in feces and selected organs as well as the immune response were
investigated. Piglets treated with E. faecium gained less weight than control piglets (p=0.05).
Feeding of E. faecium had no effect on fecal shedding of salmonellae and resulted in higher
abundance of the pathogen in tonsils of all challenged animals. The specific (anti-Salmonella
IgG) and unspecific (haptoglobin) humoral immune responses as well as the cellular immune
response (T helper cells, cytotoxic T cells, regulatory T cells, γδ T cells and B cells) in the
lymph nodes, Peyer’s patches of different segments of the intestine (jejunal, ileo-cecal), the
ileal papilla and in the blood were affected in the course of time after infection (p

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to the combined feed were widely used to prevent infection outbreaks in the herds. This led to
development of bacterial resistance against many antibiotics, and finally the EU banned their
use in pig feed in 2006. Since then, much interest has been raised on the potential use of
alternatives such as probiotics, i.e. live microorganisms, which when administered in
adequate amounts confer a health benefit to the host (5). Probiotics are believed to possess
beneficial effects on the gastrointestinal tract (GIT) of farm animals, especially the lactic acid-
producing bacteria (LAB), and thus result in increased performance of piglets. Among the
different mode of actions of the LAB, modulation of the mucosal and systemic immune
systems are considered to play a major role e.g. in prophylaxis against infections (15). The
gut-associated lymphoid tissue (GALT) is the largest collection of lymphoid tissues in the
body and is therefore an important first barrier against intestinal pathogens e.g. Salmonella sp.
or Escherichia coli (7). It consists of organized tissues such as jejunal and ileo-cecal
mesenterial lymph nodes, Peyer’s patches and diffusely scattered intraepithelial lymphocytes
(7), which are expected to respond to pathogen infection.
LAB, such as several strains of lactobacilli and some enterococci, are in the focus of research
as they are traditionally used in a range of industrial food fermentations with a long history of
safe use (16). Evidence was provided (14) that Enterococcus faecium NCIMB 10415 (E.
faecium) reduces the Chlamydia sp. load of healthy piglets. Furthermore, experiments in
piglets fed with E. faecium have shown a reduction of diarrhea (22, 24). Those data suggested
a potential beneficial effect of using E. faecium as probiotic to reduce infectious diseases in
pigs. In contrast, (21) reported that E. faecium NCIMB 10415 tended to increase the shedding
of S. Typhimurium in feces and the numbers of salmonellae in internal organs after gastric
administration of S. Typhimurium DT104 to weaning piglets (crossbred German Landrace
and Duroc).
The aim of the present study was to test the probiotic effect of E. faecium on the immune
response after infection with salmonellae. To investigate whether the effects published
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previously (21) were incidental or possibly race-specific, the present study was performed
under a similar approach using the same S. Typhimurium and E. faecium strains in piglets of
the pure German Landrace. In order to assess the activation of the adaptive systemic and
mucosal immune systems, we determined anti-Salmonella IgG as well as the levels of B and
T lymphocyte subpopulations in the different tissues of the GALT. Additionally, the effect of
the probiotic on the performance and feed intake was investigated.
Materials and Methods
Animals
We used German Landrace piglets (N=32) of sows that were feed either a diet supplemented
with a probiotic (P group) or a diet without supplementation (C group). The probiotic
supplemented group contained Enterococcus faecium NCIMB 10415 (E. faecium) as a
probiotic additive, the control group was fed the same basic feed without E. faecium. Sows of
the P group received E. faecium already for three weeks before parturition, and the piglets
were offered the respective combined feed with or without E. faecium from the age of 12 days
on. Piglets of both feeding groups (n=16 in both feeding groups) were weaned at 28 days (d)
of age and were then pair wise allocated to pens. The leftovers were collected daily and feed
intake was recorded on dry matter basis. Water was provided via nipple drinkers ad libitum.
The pens’ floors and the feeding troughs were cleaned thoroughly twice daily. After 10 days
of adaptation (at the age of 38 days), all piglets were challenged with Salmonella enterica
serovar Typhimurium DT104 (S. Typhimurium) by intragastric application as described
elsewhere (21). Six piglets from each group were sacrificed on d 2 post infection (pi) and ten
piglets on d28 pi.
The experiment has been approved by the local authority (Landesamt für Gesundheit und
Soziales, Berlin) under the ID: G0348/09.
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Sampling
After infection with S. Typhimurium, the following clinical and zootechnical parameters were
recorded for all pigs: (i) general condition and fecal score (from 1 to 5, where 1=liquid and
5=firm); (ii) rectal temperature and (iii) shedding of salmonellae in feces were determined
daily for 5 days pi and then once a week; (iv) prevalence of anti-Salmonella IgG in blood
samples, which were obtained before infection and then at weekly intervals, (v) daily feed
intake, and (vi) weekly body weight for calculation of the feed conversion ratio (kg feed / kg
weight gain).
During necropsy, liver, spleen, palatine tonsils, segments of mid-jejunum, ileum, cecum and
ascendent colon (3 rd centripetal gyrus), jejunal and colonic mesenterial lymph nodes, and
muscle samples from the fore (Musculus triceps brachii) and hind limbs (Musculus gluteus
superficialis) were collected for cultivation of S. Typhimurium.
For the characterization of the immune cells, mesenterial jejunal and ileo-cecal lymph nodes
as well as Peyer’s patches and ileal papilla were collected from six animals per group on d2
and d28 pi.
Bacterial strains
The E. faecium strain NCIMB 10415 is a commercial probiotic feed additive (Cylactin ® LBC
ME10, DSM nutritional Products Ltd, Switzerland). It was provided in a microencapsulated
form and mixed to the diets of sows, suckling and weaned piglets at concentrations of 1-5x10 9
colony forming units (CFU) / kg feed.
For the infection with S. Typhimurium, the strain DT104 was chosen as it was obtained from
a swine with sepsis (21) and was characterized by multiple resistance against antibiotics
(chloramphenicol, ciprofloxacin, tetracycline, florfenicol, ampicillin, sulfamethoxazole,
colistin, streptomycin, nalidixic acid). The resistance against nalidixic acid was used for later
selective cultivation of the strain from the samples. The S. Typhimurium was cultured in
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Luria-Bertani broth at 37°C for 20-22 hours to reach an optical density of 0.69 to 0.72,
corresponding with 2-3x10 9 CFU/mL, which was subsequently confirmed by plating. Each
piglet was infected with 7 ml of such culture broth (1.4-2.1x10 10 CFU in total).
Quantitation of salmonellae
The quantitative detection of salmonellae in feces was performed using a spiral plater
(Whitley, Meintrup DWS, Germany) with a detection limit of 4x10 2 CFU/g. For each sample,
50 µl from 5 dilutions were streaked onto 3 xylose-lysine-deoxycholate agar plates
supplemented with 50 µg/ml of NAL (XLD-NAL) and incubated at 37°C for 20-24 h. For
Quantitation of salmonellae in internal organs tissue samples were immersed in 95% ethanol,
flamed, minced aseptically, and homogenized with BPW (1:10) in filter bags using a
stomacher for 2 min at high speed. All samples were quantified for S. Typhimurium by
plating of 100 µL of filtrates using a spiral plater. The detection limit was 2x10 2 CFU/g. In
addition, 1 ml of the 10-fold dilution was streaked directly onto 3 XLD-NAL agar plates to
increase the detection limit to 10 CFU/g. Five hundred µl of blood collected in tubes
containing K-EDTA was streaked onto 3 XLD-NAL agar plates in order to test if there was
bacteraemia after the challenge.
The obtained colony numbers were transformed into log CFU/g to obtain nearly normal
distribution of trait values. Mean values and standard deviations are provided in the text.
Isolation of immune cells
Immune cells were characterized for six animals in the E. faecium supplemented group and
the control group at d2 and d28 pi (Σ n=24). The dissected lymph nodes were transferred into
15 ml tubes filled with 5 ml phosphate buffered saline containing 0.2% bovine serum albumin
(PBS/BSA 0.2 %). The Peyer’s Patches and the ileal papilla were put into Hank's Buffered
Salt Solution (HBSS). The individual tissue samples were pressed through a 70 μm Cell
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Strainer (Becton Dickinson GmbH, Heidelberg). The remaining cell suspensions containing
lymphocytes and peripheral blood mononuclear cells (PBMC) were further purified by
centrifugation for 30 min at 700g at 20°C in a ficoll gradient (Histopaque®-1077, Sigma-
Aldrich Corporation). After final lysis of erythrocytes in Erylyse-Puffer pH 7.2–7.4
(Morphisto GmbH) for 5 min on ice, the immune cells were washed with 10 ml of PBS/BSA
0.2 % and centrifuged for 15 min at 390 g at 4°C.
Flow cytometry
Flow cytometry was performed with purified immune cells. The cells were stained with
primary anti-CD4 conjugates in a one step-incubation. For each reaction, 1x10 6 cells were
exposed to saturating concentrations of the antibodies in a volume of 30 µl of PBS for 30 min
on ice in the darkness. Cells were washed with 1 ml of PBS, spun at 2500 g for 5 min, and
resuspended in 300 µl PBS. Immune cell antigens CD2, CD25, CD8β, TcR1 and IgM (Table
1) were detected using unlabelled primary antibodies followed by washing and incubation
with a fluorescence-labeled secondary antibody (Table 1). Table 1 displays the source of each
antibody. Different lymphocyte subpopulations were designated by their phenotypes
according to (18) (γδ T cells, B cells) and (8) (αβ T cells). Per sample, 40,000 lymphocytes
were assayed by flow cytometry (FCM) using an BD FACSCaliburTM flow cytometer within
the lymphocyte gate corresponding to their forward light and sideward light scatter signals
(Figure 1A). From these cells only living ones, which were negative for the propidium iodide
(PI) staining (0.5 µg/ml) were further analyzed (Figure 1B). T helper cells (Th), cytotoxic T
cells (Tc) and regulatory T cells (Treg) were determined by detection of the surface markers
CD4, CD8β (Figure 1C) and CD25 (Figure 1D), respectively. For phenotypic analysis of γδ-
TCR cells, a marker for the δ chain and the surface marker CD2 were chosen in the same
scheme as shown for CD4, CD8. Immature and mature naive B cells were identified by
staining with the surface marker IgM.
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Serology
Blood samples of all animals were collected from the cranial vena cava at d2 and d28 pi.
Blood was incubated at 37°C for 2 hours, centrifuged for 15 min at 1400 g, and serum was
collected and stored at -20°C until analysis.
The presence of anti-Salmonella antibodies was tested with the Salmotype ® PIGSCREEN
enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions
(Labordiagnostik Leipzig, Leipzig, Germany). The IgG levels were calculated using a
reference standard method and presented as percents of the optical density (OD%).
Haptoglobin concentrations in the sera were analysed by use of the Pig Haptoglobin ELISA
Test Kit (Life Diagnostics, West Chester, USA) following the manufacturer’s instructions.
The haptoglobin concentrations in sera were determined by applying standard curves and
calculated in mg/ml.
Statistical analysis
Statistical analysis was performed using R 2.11.1 and SPSS 12.0.2. (SPSS, Inc., Chicago, IL).
For the phenotypes of immune cells, we analyzed 6 samples per group (d pi x diet).The
experiment had a statistical power level of 0.8 for an anticipated correlation (r²) between diet
and phenoytype of r² ≥ 0.35 and a p-value of 0.1. Alternatively, it had a power of 0.7 to detect
effects with a p-value of 0.05. All data values higher than 2-times the interquartiles range
(IQR) below the 1st quartile and above the 3rd quartile were identified as outliers. Outliers
had no significant effects on test statistics due to less than one percent of outliers in our data.
The Shapiro-Wilk test was used to check raw data for normal distribution. Since raw data
were not normally distributed numbers of salmonellae were log-transformed.
To test the effects of supplementation with E. faecium on the phenotypes, we applied an
analysis of variance (ANOVA) with E. faecium supplementation in the diet, days post
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infection and sex as fixed effects. The following linear model was used to assess the variation
(V) for every trait for each tissue:
Vtrait = Vdiet group + Vdays post infection + Vsex + residual error
The amounts of S. Typhimurium in feces and organs were compared using the non-
parameteric Kruskal-Wallis test because salmonellae could not be continuously detected in all
samples. Differences were considered significant at p

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case, constipation occurred and the faecal score was 5 (hard firm stool) until d4 pi, indicating
high water demand.
Increased rectal body temperatures (≥40°C) were measured in three and two piglets on d1 pi
and in seven and eight piglets on d2 pi in the C and P groups, respectively. On average,
piglets in the C and P groups had elevated rectal temperatures for 1.8±1.0 and 0.9±1.4 days
within the first 7 days pi (p=0.13), and thereafter all piglets had a physiological temperature.
Diarrhea was recorded at score 1 (watery) and 2 (mushy). Before infection, diarrhea (score 1)
was observed in one animal from the P group and in two control animals (score 2). At the day
of infection, no diarrhea was noticed. One day pi, one control piglet had faeces of score 2. On
d2 pi, watery or mushy diarrhea was observed in three and one C piglet; and in two and three
piglets from the P group, respectively. Three days pi, additional two control and one P piglets
developed diarrhea. The diarrhea lasted for one to two days without statistical differences
between the treatment groups (p=0.442).
Detection of Salmonella Typhimurium in feces and organs
None of the animals shed salmonellae before challenge. One day pi, all animals shed S.
Typhimurium, except one and two piglets from the C and P groups, respectively, which
started to shed salmonellae on day two pi. Shedding of salmonellae lasted from 1 to 28 days,
with levels of 4-8 log CFU per g feces. At the end of the trial, on d28 pi, S. Typhimurium was
detected in feces from 1 pig from C (2.8 log CFU/g) and 3 pigs from P (0.9-3.0 log CFU/g)
without significant differences between groups (Figure 2A).
S. Typhimurium was detected in the wall of GIT (2.4-6.4 log CFU/g) and in mesenterial LN
(2.5-3.1 log CFU/g) of all pigs on d2 pi. S. Typhimurium was also present on d2 pi in tonsils
from one pig from C (2.8 log CFU/g) and 2 from P (2.1-3.7 log CFU/g); and on d28 pi in one
C (5.7 log CFU/g) and in four pigs from P (1.2–5.9 log CFU/g). Sporadically S. Typhimurium
could be also detected in liver and spleen on d2 pi. On d28 pi, S. Typhimurium was detected
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in the P group in the wall from ileum of 2 and in the wall from jejunum of 5 piglets. At this
time, 3 piglets from C and 3 piglets from P had S. Typhimurium in the wall of cecum; 2
piglets from C and one piglet from P in colon. No effect of the probiotic on the CFU numbers
could be observed (Figure 2B, 2C). Furthermore, no salmonellae could be isolated from the
blood showing that no bacteraemia had occurred.
Humoral immunity - anti-Salmonella IgG
For all samples measured, the anti-Salmonella IgG titers were below the 20%-cut-off value
(Figure 2D). Although low throughout, the levels of specific antibodies were significantly
higher in E. faecium treated than in untreated piglets (p0.5). However,
the high standard deviations of the mean IgG titers in both groups implicate high individual
variability in the immunological response to the S. Typhimurium challenge, which did not
correlate with the numbers of salmonellae shed in faeces.
Acute phase protein haptoglobin as part of innate immunity
At weaning, haptoglobin concentrations in serum were 2.18±1.1 and 1.98±0.44 mg/ml in C
and P, respectively. They remained at the elevated level in C, and increased in P (p=0.048)
one week later (Figure 2E). After the adaptation period, at the day of challenge the
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haptoglobin was slow and reached a significantly lower level only on d28 pi (p

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In an ANOVA analysis over the whole test period, there were no significant effects in the
tested immune cell populations between the probiotic treated group and the control group, and
no effects between sexes.
Discussion
E. faecium NCIMB 10415 has been widely approved as a probiotic feed supplement for use in
animals in the European Union. To obtain the approval for commercial use, a feed additive
must possess at least one of the characteristics stated in Article 5 (3) of the Regulation No
1831/2003 (2). When applying for approval of a bacterial strain as a probiotic feed additive, it
is therefore sufficient to show that a given strain will “favourably affect animal production,
performance or welfare, particularly by affecting the gastro-intestinal flora or digestibility of
feeding stuffs” (2). Studies to test the beneficial effect of E. faecium mostly focused on
zootechnical parameters such as feed intake, daily weight gain, and feed conversion ratio
under commercial farm conditions, without focus on targeted infection (11, 24).
In the present study, we examined the effect of E. faecium administration with respect to the
response to S. Typhimurium infection in weaned piglets of the German Landrace breed. In our
bacterial challenge trial, E. faecium fed piglets gained even less weight than the untreated
control animals. This shows that there are no positive effects of this probiotic on the pig’s
bodily development in face of an infection with S. Typhimurium. Similarly, the same
probiotic (Cylactin ® ) reduced daily weight gain in uninfected piglets fed the probiotic in the
same way, without further effects on feed conversion ratio (12, 22). In contrast, (24) reported
improvement of daily weight gain (17g/day) of unweaned piglets kept under farm conditions,
fed the same probiotic strain suspended in liquid or gel form. Therefore, it remains unclear if
different form of application (liquid, microencapsulated; once or for several days) or other
factors such as farm conditions, genetic background of the animals or gestation number could
influence the effects of the E. faecium on the body weight gain.
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In the present study, we investigated the effect of E. faecium as a probiotic on shedding of
pathogen bacteria in a model challenging experiment with Salmonella enterica serovar
Typhimurium DT104 infection. The virulence of the S. Typhimurium strain used for infection
could be confirmed in one infected piglet. All others piglets developed mild clinical signs
only. Nevertheless, colonization of the gastrointestinal tract with S. Typhimurium occurred in
all pigs since all of them shed salmonellae for at least two days after infection. It is well
known that in the course of pathogenesis after salmonellae infection, the wall of the
gastrointestinal tract is actively invaded by the pathogen using different mechanisms (9)
before it multiplies and spreads to other organs through macrophages thereby reaching other
lymphatic organs such as the mesenterial lymph nodes of the small and large intestine, the
spleen and tonsils (6). In the present study we could not observe any protective effects of E.
faecium, neither on clinical symptoms of salmonellosis, nor on S. Typhimurium shedding or
distribution into internal organs. This is in line with the results obtained from a similar
experimental setting (21). In contrast, (1) reported a reduced number of shed S. Typhimurium
without effects on the duration of the shedding in Large White x Landrace weaned piglets fed
with a cocktail of five different probiotic strains of LAB. It is intriguing that reports on the
effects of probiotics on pathogen infection often provide controversial results. We suspect that
the reasons can be manifold. The choice of the pathogen, the way of infection (oral once or
for several days, intragastric, “troyan model”), the infection pressure and other controlled or
uncontrollable experimental conditions as well as genetic predisposition could play a role in
yielding variable effect of probiotics. We would like to emphasize for the present study, that
the pens and feeding troughs were cleaned thoroughly twice daily and that this rigorous
hygienic regime in our experiment was obviously sufficient to reduce the number of
pathogens in the environment and therefore to eliminate or at least reduce the source of re-
infection. This could have an impact on the lack of differences between the two experimental
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groups. On the other hand, the control pigs coped very well and recovered from the infection
without any additional help which underlines the importance of proper management.
The present study confirms that feeding E. faecium to piglets resulted in higher numbers of S.
Typhimurium in tonsils, as reported previously (21). The higher abundance of salmonellae in
the tonsils lasting for at least 28 days after infection could become a source for re-infection of
the pigs and thus be responsible for subclinical and chronic infections of a pig herd.
Furthermore, infected tonsils could also be a potential source of contamination of meat at
slaughter. From this perspective, our data would provide evidence against the use of E.
faecium NCIMB 10415 as a probiotic feed supplement in pigs to prevent infection of meat
and thereby potentially humans via the slaughter house.
Probiotics are considered to modulate the host’s immune system resulting in an improvement
of protection against infections (20). In the previous study (21) higher levels of serum IgM
and IgA against S. Typhimurium could be observed in the group fed E. faecium without any
effects on serum anti-S. Typhimurium IgG. Also in the present study, E. faecium had no
increasing effect on serum IgG after infection. However, we found that the increase of
monomeric cell surface bound IgM was more prominent in the E. faecium treated group
(p

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magnitude than those in viral infection studies in pigs (17). The level remained elevated for a
longer time in probiotic-fed pigs than in controls, indicating either longer lasting synthesis
and release of the acute phase protein, or its slower degradation when E. faecium was in the
diet. Thus, these data suggest that the non-specific reaction of the immune system was
activated for a longer time under the probiotic treatment. As we failed to find differences in
the relative cell count of B cells with cell surface bound monomeric IgM between the E.
faecium supplemented and the control groups in GALT, we hypothesize that there was no
stimulation of the immune response against S. Typhimurium by E. faecium. This is in
agreement with previous results obtained in Landrace x Duroc piglets (21). The same strain of
E. faecium that we fed to sows and piglets could reduce the rate of naturally occurring
infection with Chlamydia sp. in pigs (14). Considering the effects of E. faecium on infection
with salmonellae or chlamydiae in the previous studies (14, 21), the authors observed a
reduction of the cytotoxic CD8 + subpopulation of intraepithelial and circulating lymphocytes
and therefore discussed as possible mechanism that E. faecium activates the production and
secretion of IgM and IgA, and the reduction of the CD8 + lymphocytes could be a result of
increased binding to the intestinal pathogen bound by the antibodes. In our study, the
frequency of cytotoxic CD8 + T cells in tissues responsible for the removal of cells infected by
S. Typhimurium was lower in E. faecium treated pigs than in control animals. This is in line
with the finding, that the frequency of salmonellae was also higher in tissues of the probiotic
feed group. Otherwise, the proportion of circulating effector CD8 + cytotoxic T lymphocytes
was enhanced in the peripher blood mononuclear cells in the E. faecium treated group two
days post infection. Therefore, one could assume an alternative mechanism caused by E.
faecium. Maybe E. faecium potentially inhibits efficient homing of the effector cytotoxic T
cells. This would explain the lower clearance of salmonellae in the E. faecium treated group.
That goes with the assumption that E. faecium would enhance the IgM and IgA production
and secretion, and therefore the stimulation of more naive T cells to differentiate into
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circulating effector T cells. Although the assumptions of the possible mechanisms of the
effects of an E. faecium supplementation on a S. Typhimurium infection made here are in line
with previous studies, one has to consider, that we did not measure antigen-specific T or B
cell response. Therefore the mechanism has to be elucidated in further research. However, the
same pattern of low shedding of salmonellae in both groups suggests that this mechanism
does not result in an improved elimination of the pathogen from the gastrointestinal tract of
the piglets. According to previous published evidence (19, 23), we detected a high percentage
of CD2 - γδ T cells in the peripheral blood with only few such cells accumulated in the
lymphoid tissues of the intestine. CD2 + γδ T cells were present in all examined tissues and in
blood with a frequency of 1-2%. Neither the CD2 - γδ T cells nor the CD2 + γδ T cells showed
a difference between the E. faecium and control group. Although the CD2 + γδ T cells, which
are additionally expressing CD8αα, are believed to be potentially cytotoxic (18) and hence are
able to clear salmonellae infected cells, γδ T cells seemed to be no target of activation for E.
faecium derived signaling in our salmonellae challenge experiment. The number of B cells in
ileal Peyer’s patches, and ileal papilla significantly (p

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Acknowledgements
The authors thank Dr. Robert Pieper, Freie Universität Berlin, for providing the piglets and
Enno Luge and the team of Dr. Stefanie Banneke from the Research Institute for Risk
Assessment, Berlin for the excellent animal care and technical support during the experiment.
We further thank Prof. Klaus Osterrieder for permission to use his flow cytometer.
The study was funded by the German Research Foundation (Deutsche
Forschungsgemeinschaft, DFG) within the Collaborative Research Group (SFB,
Sonderforschungsbereich) 852/1 “Nutrition and intestinal microbiota - host interactions in the
pig”. The authors are solely responsible for the data and do not represent any opinion of
neither the DFG nor other public or commercial entity.
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FIGURE 1 Gating and selection strategies of the flow cytometry data for the analyses of
lymphocyte subpopulations
The x and y axes show the intensity of the light scatter or fluorescent signal. Framed cells
populations in A were further analysed in B etc. A) Lymphocytes gated according to their
forward sideward light scatter signals. Circulated lymphocytes represent 40,000 cells. B) only
living PI negative cells were taken for analyses. C) Relative cell counts of living lymphocyte
subpopulations (CD8pos = cytotoxic T cells; CD4pos,CD8pos = pig specific double positive
cells, CD4pos = T helper cells) detected with antibodies CD8β and CD4 were calculated. D)
the relative number of CD25high cells of the CD4pos cells were calculated.
FIGURE 2 Colonization of piglets with salmonellae and the reaction of their immune
systems on the infection
Shedding of Salmonella Typhimurium in feces (A) and in organs either two (B) or 28 days
(C), as well as serum anti-salmonella IgG titres (D) and haptoglobin concentration (E) after
intra-gastric S. Typhimurium infection in groups of piglets that were either untreated controls
(open squares - C) or treated with E. faecium (gray squares - P). Colony forming units (CFU)
per gram feces were log transformed to obtain normal distribution. Box-Whisker plots
indicate the median (horizontal lines), the lower and upper quartiles (lower and upper borders
of the boxes); minimum and maximum as well as the outliers (mild - open circles; extreme -
asterix) are presented.
FIGURE 3 Relative cell counts to the living lymphocyte population of αβ T cells
A) CD8β pos (cytotoxic T cells), B) CD8β and CD4 pos cells and D) CD4 pos (T helper cells)
in peripher blood mononuclear cells (PBMCs), ileal lymph nodes (IL LN), ileal Peyers Patch
(IL PP) and papilla (PAP IL) of the E. faecium supplemented group (P) and the non E.
faecium supplemented control group (C) for the time points 2 dpi and 28 dpi. 3C) FACS
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scatter plots of one female piglet derived from P at d2 pi as an example for the appearance of
T helper cells, cytotoxic T cells and CD4 + CD8 + T cells in the PBMCs, IL LN, ILPP and PAP
IL. All piglets were infected with S. Typhimurium.
FIGURE 4 Relative cell counts to the living lymphocyte population of γδ T cells
A) CD2 and γδ TCR positive cells B) γδ TCR positive (CD2 negative) cells in the in peripher
blood mononuclear cells (PBMCs), ileal lymph nodes (IL LN), ileal Peyer’s patch (IL PP) and
papilla (PAP IL) of piglets from E. faecium supplemented group (P) and the control group (C)
2d and 28 d after challenge with S. Typhimurium.
FIGURE 5 Relative cell counts to the living lymphocyte population of B cells
A) IgM positive cells (B cells) of one piglet representative for E.faecium supplemented group
(P), 2 dpi in the ileal lymph node (IL LN) and one piglet representative for control (C), 2 dpi
in IL LN. B), C), D) IgM positive cells (B cells) of 12 piglets from P and of 12 piglets from C
at the time points 2 dpi (n=6) and 28 dpi (n=6) in B) mesenteric lymph nodes (LN) of jejunum
(Je LN) and ileum (IL LN), in C) peripher blood mononuclear cells (PBMCs) and in D) the
ileal Peyer’s patch (IL PP) and papilla (PAP_IL). All piglets were infected with S.
Typhimurium.
23

TABLE 1 – primary and secondary antibodies (AB) used for flow cytometry staining*
Primary AB Clone Isotype Labelling Company
CD2 MSA-4 IgG2a none VMRD
CD4 74-12-4 IgG2b FITC Southern Biotech
CD8β PG164A IgG2a none VMRD
TcR1-N4 (δ) PGBL22A IgG1 none VMRD
CD25 K231.3B2 IgG1 none Biozol
IgM PIG45A IgG2b none VMRD
Secondary AB Clone Isotype Labelling Company
Goat Anti-Mouse IgG1 pooled IgG1 APC Southern Biotech
Goat Anti-Mouse IgG2b pooled IgG2b FITC Southern Biotech
Goat Anti-Mouse IgG2a pooled IgG2a PE Southern Biotech
* The references for each antibody are provided by the manufacturers in the datasheets.