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Veterinary Immunology and Immunopathology 118 (2007) 1–11
Bacillus cereus var. toyoi enhanced systemic
immune response in piglets
Peter Schierack a,*, Lothar H. Wieler a, David Taras b, Volker Herwig c,Babila Tachu d, Andreas Hlinak e, Michael F.G. Schmidt f, Lydia Scharek f
a Institut fur Mikrobiologie und Tierseuchen, FU Berlin, Philippstr. 13, 10115 Berlin, Germanyb Institut fur Tierernahrung, FU Berlin, Brummerstr. 34, 14195 Berlin, Germany
c Impfstoffwerk Dessau-Tornau GmbH, Streetzer Weg 15a, 06862 Rodleben, Germanyd Institut fur Molekulare Parasitologie, HU Berlin, Philippstr. 13, 10115 Berlin, Germany
e Landeslabor Brandenburg, Ringstr. 1030, 15236 Frankfurt (Oder), Germanyf Institut fur Immunologie und Molekularbiologie, FU Berlin, Philippstr. 13, 10115 Berlin, Germany
Received 24 August 2006; received in revised form 7 March 2007; accepted 21 March 2007
Abstract
Probiotic bacteria have been suggested to stimulate the host immune system. In this study we evaluated the immunomodulatory
effects of probiotic Bacillus cereus var. toyoi on the systemic immunity of piglets. A pool of 70 piglets was divided into a probiotic
or control group. We determined the ratios of peripheral blood mononuclear cell (PBMC) subsets and measured proliferative
responses and cytokine production of PBMCs and effects on vaccination responses. Blood samples of probiotic-treated piglets
showed a significantly lower frequency of CD8high/CD3+ T cells and CD8low/CD3+ T cells and a significant higher CD4+/CD8+
ratio. IL-4 and IFN-g production of polyclonally stimulated PBMCs was on average higher in the probiotic group. Specific
proliferative responses of PBMCs to Influenza vaccination antigens were significantly higher and antibody titers against H3N2
Influenza and Mycoplasma vaccination antigens were on average higher in the probiotic group. In conclusion, B. cereus var. toyoi
therefore alters the immune status of piglets as indicated by changes in the ratios as well as functionalities of systemic immune cell
populations.
# 2007 Elsevier B.V. All rights reserved.
Keywords: Probiotic; Bacillus cereus var. toyoi; Proliferation; PBMCs; Pig; Vaccination
1. Introduction
The indigenous bacterial flora plays a key role in the
development of the gut, including the gut-associated
immune system, and thus plays an important role in
Abbreviations: CCU, colour changing unit; PC5, R-phycoerythrin
covalently linked to cyanin 5.1; TCID, tissue culture infectious dose
* Corresponding author at: Postfach 040225, D-10116 Berlin, Ger-
many. Tel.: +49 30 2093 6704; fax: +49 30 2093 6067.
E-mail address: schierack.peter@vetmed.fu-berlin.de
(P. Schierack).
0165-2427/$ – see front matter # 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetimm.2007.03.006
defense against enteropathogenic bacteria. Prior studies
have shown that bacterial colonization of the gut
contributes to the development and maintenance of oral
tolerance, and therefore not only local immune
reactions but also systemic immunity is modulated
by the intestinal flora (McGhee et al., 1992; Tanaka and
Ishikawa, 2004). Non-pathogenic bacteria appear to
possess only moderate immune stimulatory effects on
the mature, gut-associated immune system and, under
normal physiological conditions, indigenous bacteria
should be tolerated by the local immune system of the
host (Scharek et al., 2005; Westendorf et al., 2005).
P. Schierack et al. / Veterinary Immunology and Immunopathology 118 (2007) 1–112
However, some probiotics have been shown to affect
gut-associated immunity in animals and humans (Mack
and Lebel, 2004), and certain probiotic bacteria may
even affect systemic immunity (Schultz et al., 2003). It
has previously been shown that the probiotic Lactoba-
cillus rhamnosus enhanced the activity of circulating
natural killer (NK) cells in healthy elderly (Gill et al.,
2001), elevated IFN-g production of PBMCs in infants
with cow’s milk allergy (Pohjavuori et al., 2004), and
induced T cell hyporesponsiveness, including impaired
ex vivo T helper subsets 1 and 2 responses without up-
regulation of immune regulatory cytokines (Braat et al.,
2004).
Bacillus cereus var. toyoi is used as a probiotic feed
supplement in livestock farming, where it has been
reported to contribute to higher weight gain, improved
feed conversion ratios, a reduction in the incidence of
liquid feces and post-weaning diarrhea and lower
mortality rates of piglets (Alexopoulos et al., 2001;
Baum et al., 2002; Gedek et al., 1993; Kirchgessner
et al., 1993; Taras et al., 2005). However, B. cereus
strains can be internalized by epithelial cells (Minnaard
et al., 2004), and B. cereus var. toyoi, like almost all
species of the Bacillus group, produces a hemolysin
with lytic activity for epithelial cells (Pruss et al., 1999).
In other studies, other Bacillus species were shown to be
immunogenic in a murine model marked by enhanced
expression of the cytokines TNF-a and IFN-g (Braat
et al., 2004).
Since this bacterium is not a member of the
indigenous bacterial flora of swine, we hypothesized
an immunomodulatory potential for B. cereus var. toyoi
in pigs. In this study, we show that feed supplementation
of piglets with B. cereus var. toyoi affects several
aspects of the immune system, modulates the T cell
status, and appears to possess adjuvant effects in
vaccination studies.
2. Materials and methods
Studies on the systemic immune response of piglets
were part of the Deutsche Forschungsgemeinschaft
(DFG) Research Unit ‘‘An Integrative Analysis of
Mechanisms of Probiotic Action in Pigs’’. During
studies with the probiotic B. cereus var. toyoi, 186
piglets were randomly assigned to two groups: a B.
cereus var. toyoi fed group and an untreated control
group. Seventy piglets were included in this study.
Twenty animals from each group from different
litters were sacrificed at ages between days 7 and 56
post partum for studies on the effects on intestinal
anatomy, physiology and immunology. Data concerning
performance and other parameters of these animals
have previously been published (Reiter et al., 2006;
Taras et al., 2005).
2.1. Animal study
Lactating sows were housed individually in farrow-
ing facilities on straw bedding together with their litters
until weaning. Piglets were weaned after 28 days and
reared together with littermates as pairs or triplets in
pens of flat-deck batteries. Sows and piglets of both
treatment groups were kept in separate housing
facilities of identical construction and environmental
conditions, which were spatially discrete from each
other.
Pigs were fed according to the recommendations of
the National Research Council (National Research
Council, 1998). Piglets had ad libitum access to pre-
starter feed from day 15 to day 28 and to starter feed
from day 29 to day 56 of age. Dietary supplementation
of B. cereus var. toyoi for gestating sows in the
probiotic group started 24 days after mating, while all
other probiotic diets for the different feeding phases
were supplemented right from the start of the
respective phase. The mean concentration (�S.D.)
of the supplemented B. cereus var. toyoi in the food
for piglets were measured as 1.35 (�0.45) � 106 cfu/g
dry weight food, respectively. During this study,
piglets were sacrificed for other purposes. B. cereus
var. toyoi cells (vegetative cells and spores) were
present in all sacrificed piglets in each intestinal
section of the probiotic group with at least 1.8
(�0.9) � 104 cfu/g dry mass, whilst in the control
group B. cereus var. toyoi cells were not detectable
(for further details see Taras et al., 2005). The
application of prophylactic and therapeutic antibiotics
to gestating and lactating sows as well as piglets was
prohibited during and at least 3 months prior to the
trial.
In this study, piglets of a pool of 70 animals
(crossbred Landrasse and Duroc) were randomly
assigned to two groups: a B. cereus var. toyoi fed
group (probiotic group, n = 34) and an untreated control
group (n = 36). Twenty animals of the probiotic group
and 19 animals of the control group were randomly
assigned to the Influenza vaccination group, whereas 14
animals of the probiotic group and 17 animals of the
control group were randomly assigned to the Myco-
plasma vaccination group. Piglets from the Influenza
vaccination group were used to obtain data from flow
cytometry, cytokine production, polyclonally stimu-
lated and antigen-driven proliferation, and antibody
P. Schierack et al. / Veterinary Immunology and Immunopathology 118 (2007) 1–11 3
Fig. 1. Outline of the experiment. A total of 70 piglets was randomly assigned to the Influenza (A, 39 animals) or the Mycoplasma (B, 31 animals)
vaccination group. Shown are the assignment of the tests to the vaccination groups and the age of the animals.
titers of vaccinated animals. Piglets of the Mycoplasma
vaccination group were used to obtain data from
antibody titers. Fig. 1 describes the outline of the
experiment.
2.2. Flow cytometry
Both vaccinated and non-vaccinated animals of the
Influenza vaccination group were included in this study.
Mononuclear cell fractions of peripheral blood from 19
control (11 vaccinated and 8 non-vaccinated) and 20
probiotic (10 vaccinated and 10 non-vaccinated) piglets
(age 96 days) were isolated from citrated venous blood
by density gradient sedimentation using Ficoll-Hypa-
que (Pharmacia Biotech Products, Germany). Primary
antibodies against CD4 and CD8 surface antigens
(mouse anti-porcine CD4a, clone 74-12-4, conjugated
to FITC and mouse anti-porcine CD8a, clone 76-2-11,
conjugated to R-phycoerythrin) were obtained from
Southern Biotechnology Associates (Birmingham,
USA).
The primary CD4 antibody recognizes both CD4+/
CD8� as well as CD4+/CD8low T cells, which are
predominantly memory helper T cells (Pescovitz et al.,
1984; Zuckermann, 1999). The CD8a antibody reacts
with the CD8a chain of the CD8 co-receptor, which can
be expressed as either CD8aa homodimer or a CD8ab
heterodimer. The antibody stains both CD8high T cells
as well as CD8low, comprising memory helper and NK
cells (Krog et al., 2003). In a one-step incubation, for
each reaction, 5 � 105 cells were labeled with the
respective antibody in a volume of 100 ml of PBS and
0.2% BSA for 20 min at 4 8C. Cells were washed with
3 ml PBS–BSA (300 � g, 10 min) and resuspended in
1 ml of PBS–BSA. The primary CD8a antibody was
also used in combination with a biotinylated antibody
against CD3 (mouse anti-porcine CD3e, Biotin con-
jugate, Clone BB238E6, Southern Biotechnology
P. Schierack et al. / Veterinary Immunology and Immunopathology 118 (2007) 1–114
Associates, Birmingham, USA) followed by a wash,
and incubation with streptavidin conjugated to PC5
(streptavidin–PC5, Beckman Coulter, Krefeld, Ger-
many). CD14, CD21, and TcR1 (gd-T cell receptor)
were detected using unlabelled primary antibodies
(mouse anti-porcine CD21, Clone BB6-11C9.6; South-
ern Biotechnology Associates, Birmingham, USA;
mouse anti-porcine CD14, Clone MIL-2; Serotec,
Dusseldorf, Germany; mouse anti TcR1, Clone
PGBL22-A, Acris, Bad Nauheim, Germany), followed
by a wash, and incubation with a FITC-labeled
secondary antibody (goat anti-mouse IgG, Beckman
Coulter, Krefeld, Germany), carried out under the same
conditions. After a second washing process, cells were
resuspended in 1 ml of PBS–BSA and used for flow
cytometry determinations using an EPICS XL flow
cytometer equipped with a 488 nm argon laser (Beck-
man Coulter, Krefeld, Germany). To rule out non-
specific labeling, secondary antibodies alone were also
incubated with the porcine immune cells and showed no
significant binding.
2.3. Quantification of cytokines in cell culture
supernatant
IFN-g as well as IL-4 production of PBMCs was
quantified on day 96 for 7 non-vaccinated control and 6
non-vaccinated probiotic piglets of the Influenza
vaccination group. 3.5 � 105 PBMCs/well were cul-
tured in 96-well flat bottom plates (Costar, Corning,
NY) at 37 8C and stimulated with 2 mg/ml Concana-
valin A (Amersham Pharmacia Biotech, Germany).
Culture supernatants were collected after 24 h of
incubation. Cytokine assays were performed in dupli-
cate and quantified by ELISA according to the
manufacturer’s instructions (Swine Immunoassay Kits,
Biosource Europe, Belgium).
2.4. Antigens for proliferation studies
The Influenza and Mycoplasma antigens used in
proliferation studies were the same used in the
formulated vaccines. Influenza H1N1 respectively
H3N2 antigens were provided by R. Durrwald (IDT,
Germany). Influenza virus was grown on MDBK cells
(Madin Darby bovine kidney) and inactivated with
ethylenimine. Tissue culture infecting dose 50
(TCID50) before inactivation was 106.66/ml (H1N1)
and 105.66/ml (H3N2) respectively. HA titers were 256/
512 before inactivation and 512/1024 after inactivation
(H1N1) and 256/512 before inactivation and 512 after
inactivation (H3N2). Inactivated Mycoplasma hyop-
neumoniae antigen was provided by S. Springer (IDT,
Germany) at a concentration of 9 � 107 CCU/ml.
Mycoplasma was grown in Friis medium, inactivated
using ethylenimine and neutralized using sodium
thiosulfate.
2.5. Polyclonally stimulated and antigen-driven
proliferation of PBMCs
The peripheral mononuclear cell fraction (PBMCs) of
piglets was isolated from citrated venous blood by
density gradient sedimentation using Ficoll-Hypaque
(Pharmacia Biotech Products, Germany). Cells were
resuspended in RPMI 1640 (Biochrom KG, Germany),
supplemented with 100 U/ml penicillin, 100 mg/ml
streptomycin, 2 mM glutamine and 5% FCS (Biochrom
KG, Germany). Viability of the cells was controlled by
trypan blue exclusion and 3.5 � 105 PBMCs/well were
cultured in 96-well flat bottom plates (Costar, Corning,
NY) at 37 8C. For polyclonally stimulated proliferation
we used 0.5 mg/ml Concanavalin A (Amersham Phar-
macia Biotech, Germany) for 72 h. Nineteen control (11
vaccinated and 8 non-vaccinated) and 20 probiotic (10
vaccinated and 10 non-vaccinated) animals of the
Influenza vaccination group were included in this
polyclonally stimulated proliferation study.
For antigen-driven proliferation we used Influenza
antigen (1 ml/well with TCID50 for H1N1 105.66/ml or
1 ml/well with TCID50 for H3N2 104.66/ml) or
Mycoplasma antigen (1 ml/well with 9 � 104 CCU/
ml) for 96 h. Since Influenza virus hemagglutinin
glycoprotein (HA) as well as whole HA-expressing
viral particles are considered strong polyclonal B cell
activators (‘‘mitogens’’) of mice and humans (Rott
et al., 1995), we decided to use low concentrations
(1 ml/well) of H1N1 or H3N2 inactivated viral
vaccination particles for the antigen-specific prolifera-
tion studies to avoid polyclonal stimulation. Prolifera-
tion was quantified by [3H]-thymidine incorporation
during the last 20 h of incubation. Counts determined
for cells grown in medium alone (=spontaneous
proliferation) were subtracted from the stimulated
cultures to compare only the counts driven by the
specific stimulus. All assays were performed in
triplicate. The values of vaccinated animals were
compared with those of non-vaccinated animals to
exclude natural infections of the animals with antigens
used in the study. Polyclonal proliferation assays with
PBMCs of 20 piglets of each group was determined on
days 56 and 96. Influenza antigen-specific proliferation
was determined for 19 animals of the control group (11
animals vaccinated and 8 animals non-vaccinated) and
P. Schierack et al. / Veterinary Immunology and Immunopathology 118 (2007) 1–11 5
for 20 animals of the probiotic group (10 animals
vaccinated and 10 animals non-vaccinated). Myco-
plasma antigen-specific proliferation was determined
for 17 animals of the control group (9 animals
vaccinated and 8 animals non-vaccinated) and for 14
animals of the probiotic group (10 animals vaccinated
and 4 animals non-vaccinated).
2.6. Vaccination of piglets
Vaccinations with Respiporc FLU1 (IDT, Germany)
with inactivated Influenza virus H1N1 and H3N2
antigen and Respiporc M.hyo one shot1 (IDT,
Germany) with inactivated bacterial M. hyopneumoniae
antigen were chosen for our vaccination studies.
For Influenza immunization, 11 control and 10
probiotic piglets were vaccinated on day 63 and 84
intramuscularly according to the manufacturer’s recom-
mendations. Eight control and 10 probiotic piglets
remained non-vaccinated. Both the proliferative
response and antibody titers were determined in the
same animals on day 96. For M. hyopneumoniae
immunization, 9 control and 10 probiotic piglets were
vaccinated on day 28 intramuscularly according to the
manufacturer’s recommendations. Eight control and 4
probiotic piglets remained non-vaccinated. Proliferative
responses were determined on day 39 and antibody
titers on day 68. Antibody titers against Influenza
antigens were detected using hemagglutination inhibi-
tion test (HAI) as previously described (Lang et al.,
2004) and antibody titers against Mycoplasma antigens
were detected using the ELISA Hyoptest-II (Bommeli
Diagnostics, Switzerland). The vaccinated animals
were compared with matching numbers of non-
vaccinated animals to exclude natural infections of
the animals with Influenza virus or M. hyopneumoniae.
2.7. Statistical analysis
The statistical analysis was performed using the
software SPSS 12.0 (SPSS Inc., Chicago, IL). Group
means were compared using the parameter-free Mann–
Whitney U-test. p-Values were calculated for two-sided
comparison of group means and were considered
significant at an alpha of p < 0.05.
3. Results
3.1. FACS analysis
The relative numbers of lymphocyte subsets and
monocytes of 19 control and 20 probiotic animals were
determined by flow cytometry. There were no differ-
ences in the immune cell populations between
vaccinated and non-vaccinated animals. The data
summarized from all probiotic and all control animals
(Fig. 2A) showed that the percentage of CD8+ T cells
was significantly lower ( p < 0.05) in the blood of
piglets from the probiotic group. The percentages of
helper T cells (CD4+), gd T cells (TcR1+), mature B
cells (CD21+) and monocytes (CD14+) were similar in
both groups (Fig. 2A). As shown in Fig. 2B, the lower
percentage of CD8+ T cells in the blood of piglets from
the probiotic group was due to the lower percentage of
bright CD8+ (CD8high/CD3+; p < 0.05) and dim CD8+
T cells (CD8low/CD3+; p < 0.05) but not assumed
Natural Killer cells (CD8+/CD3�) (Fig. 2B and C).
The CD4+:CD8+ ratio might be indicative for the
effectiveness of a secondary immune response (Apple-
yard et al., 2002). Therefore we compared this ratio
between the probiotic and the control group. Probiotic
piglets (n = 18) showed a higher ratio of CD4+:CD8+ T
cells with a ratio of 1.06 � 0.35 than control piglets
(n = 19) with a ration of 0.83 � 0.24 ( p < 0.05).
3.2. Cytokine production of PBMCs
To determine whether feeding probiotic B. cereus
var. toyoi affected cytokine production of peripheral
immune cells, PBMCs from seven non-vaccinated
control and six non-vaccinated probiotic piglets were
stimulated polyclonally with 2 mg/ml Concanavalin A
for 24 h on day 96 h. Supernatants were tested for IL-4
and IFN-g production. PBMCs of the probiotic group
(53.0 � 17.2 pg/ml) produced on average more IL-4
than those of the control group (38.1 � 10.5 pg/ml). In
addition, PBMCs of the probiotic group
(324.7 � 161.0 pg/ml) produced more IFN-g than
PBMCs of the control group (207.5 � 70.2 pg/ml).
PBMCs cultured without Concanavalin stimulation in
RPMI medium alone showed no detectable cytokine
levels.
3.3. Polyclonally stimulated proliferation of
PBMCs
To determine the proliferative response of peripheral
immune cells after B. cereus var. toyoi food supple-
mentation, PBMC preparations of 19 control and 20
probiotic animals were stimulated polyclonally with
0.5 mg/ml Concanavalin A (ConA) for 72 h. The
proliferative response of both the probiotic and the
control groups were similar on days 56 and 96 (Fig. 3).
In addition, there were no differences between
P. Schierack et al. / Veterinary Immunology and Immunopathology 118 (2007) 1–116
Fig. 2. Comparison of lymphocyte subsets and monocytes from piglets of the probiotic and the control group. On day 96, PBMCs of piglets from
both groups were isolated by density gradient sedimentation and surface antigens were stained with the indicated antibodies. Lymphocyte subsets
and monocytes were determined using flow cytometry. (A) Lymphocyte subsets and monocytes, (B) CD8+ T lymphocytes, separated by their
different subtypes, (C) one exemplary flow cytometric visualization of one sample of each group. Data shown are means � standard deviation.
Numbers of samples measured are given on top of the error bars. *p < 0.05.
vaccinated (11 animals of the control and 10 animals of
the probiotic group) and non-vaccinated animals (8
animals of the control and 10 animals of the probiotic
group). However, both RPMI (spontaneous prolifera-
tion, p < 0.001) and ConA (p < 0.005) stimulated
proliferative response was higher on day 96 compared
to day 56.
3.4. Vaccination
We were also interested to know to what extent the
probiotic B. cereus var. toyoi supplementation would
affect an immune reaction in piglets. To determine the
immune responses of piglets to vaccination, both the
antigen-specific proliferation and the antigen-specific
antibody production against viral (Influenza) and
bacterial (Mycoplasma) antigens were measured.
Eleven control and 10 probiotic piglets were
vaccinated with a commercially available combined
inactivated Influenza virus vaccine, containing the
strains H1N1 and H3N2. Control, non-vaccinated
groups consisted of 8 untreated and 10 probiotic-
treated piglets. On day 96 (33 days after first
immunization and 11 days after boosting) we deter-
mined the antigen-specific proliferative response of
PBMCs and serum antibody titers. PBMCs were
stimulated with H1N1 or H3N2 Influenza antigens.
As shown in Fig. 4A/4B, both the spontaneous
proliferation ( p < 0.05) as well as the proliferative
responses to both H1N1 ( p < 0.001) and H3N2
( p < 0.005) Influenza antigens increased in the vacci-
nated and non-vaccinated groups from day 56 (before
vaccination) and day 96 (after vaccination), indepen-
dent of a probiotic feed supplementation. Additionally,
there was no difference between the proliferative
responses of PBMCs of the vaccinated and non-
vaccinated control animals. However, there was a
significant increase of the proliferative response of
P. Schierack et al. / Veterinary Immunology and Immunopathology 118 (2007) 1–11 7
Fig. 3. Comparison of polyclonal stimulation of T cells from piglets
on days 56 and 96. PBMCs were stimulated with 0.5 mg/ml Con-
canavalin A for 72 h. Proliferation was quantified by [3H]-thymidine
incorporation during the last 24 h. Data shown are means � standard
deviation. Numbers of samples measured are given on top of the error
bars. (A) Day 56; (B) day 96.
PBMCs of vaccinated probiotic animals compared to
vaccinated control animals and non-vaccinated probio-
tic animals ( p < 0.05; Fig. 4A/4B). Since the mock
control values (PBMCs stimulated with cell culture
supernatant of non-infected cultures of the same cells
used to propagate Influenza vaccination virus) were
identical to values of spontaneous proliferation in
preliminary experiments (total of at least 20 animals),
we only included spontaneous proliferation as standard
in our experiment.
Fig. 4. Comparison of vaccination antigen-specific stimulation of T cells fr
PBMCs were stimulated with H1N1 (A) or H3N2 (B) vaccination antigens f
during the last 24 h. Data shown are means � standard deviation. Numbers o
day 96. *p < 0.05.
Average serum antibody titers against H1N1 anti-
gens were similar in both the probiotic and in the control
group (Fig. 5A). However, nine piglets out of ten (90%)
of the probiotic group had antibody titers against H1N1
antigen higher than 160 compared to six out of eleven
(55%) of the probiotic group. Average serum antibody
titers against H3N2 antigens were only slightly higher
in the probiotic compared to the control group (Fig. 5B).
We found no correlation between antibody titers and
antigen-specific proliferation rates in single animals.
In addition, 10 control and 9 probiotic piglets were
also vaccinated with a commercially available M.
hyopneumoniae vaccine, with 8 and 4 non-vaccinated
piglets serving as controls for each group, respectively.
Proliferative T cell response to the Mycoplasma
vaccination antigen was only detectable in very few
PBMC samples and showed no correlation to vacci-
nated or non-vaccinated animals. Antibody titers on day
40 after vaccination were on average higher for the
probiotic group compared to the control group (Fig. 6).
4. Discussion
It is generally acknowledged that the commensal
intestinal flora has an impact on the immune functions
of the host. As autochthonous bacteria contribute to the
development and maintenance of oral tolerance, the
immune stimulatory effects of indigenous bacteria have
generally not been well studied (McGhee et al., 1992;
Talham et al., 1999; Tanaka and Ishikawa, 2004). Since
many of the bacterial species used for probiotic food
supplementation are also members of the indigenous
om piglets on day 56 (before vaccination) and 96 (after vaccination).
or 96 h. Proliferation was quantified by [3H]-thymidine incorporation
f samples measured are given on top of the error bars. (A) Day 56; (B)
P. Schierack et al. / Veterinary Immunology and Immunopathology 118 (2007) 1–118
Fig. 5. Comparison of serum antibody titers after Influenza vaccina-
tion. Piglets were vaccinated intramuscularly at the age of 63 days
(first vaccination) and 84 days (boostering) simultaneously with
inactivated H1N1 (A) and H3N2 (B) antigens. Antibody titers were
measured by hemagglutination inhibition test (HAI) before vaccina-
tion and 12 days after boostering. Data shown are means � standard
deviation. Numbers of samples measured are given below the dot
plots.
Fig. 6. Comparison of serum antibody titers after Mycoplasma vac-
cination. Piglets were vaccinated intramuscularly at the age of 28 days
with inactivated M. hyopneumoniae antigen. Antibody titers were
measured by ELISA 40 days after vaccination. Data shown are
means � standard deviation. Numbers of samples measured are given
below the dot plots. (A) Day 56; (B) day 96.
intestinal flora (de Champs et al., 2003; Jin et al., 2000),
such bacterial species would not be expected to
contribute to a systemic immune response (Scharek
et al., 2005; Westendorf et al., 2005). B. cereus var.
toyoi, a Gram-positive spore-forming bacterium, is not a
member of the indigenous intestinal flora of pigs and
produces a potent hemolysin, this species might
therefore be more immunogenic than other probiotics.
Indeed, the immunogenic properties of B. cereus spores
have been shown in mice, where the expression of
inflammatory cytokines in the gut with modest spore-
specific IgG titers was observed, and the in vitro-
germination of spores in macrophages resulted in the
induction of IFN-g and the pro-inflammatory cytokine
TNF-a (Duc le et al., 2004).
In this study we show that feed supplementation with
B. cereus var. toyoi modulated both the composition as
well as the activities of blood immune cells in piglets.
Our vaccination studies further indicated that B. cereus
var. toyoi improved the vaccination outcome, mimick-
ing an ‘‘adjuvant effect’’.
Blood samples of probiotic-treated piglets showed
significant differences in the relative immune cell
populations compared to the untreated, control group.
Although we did not carry out double-stainings for CD4
and CD8, and therefore cannot draw conclusions
concerning the development of CD4/CD8 double
positive T cells (predominantly memory helper T
cells), the results suggest that the relative amount of
memory helper cells was diminished in the probiotic
group. Piglets of the probiotic group showed signifi-
cantly reduced numbers of weakly staining CD8+ T
cells (CD8low/CD3+), which should be memory helper
T cells (Zuckermann, 1999). Furthermore, the popula-
tion of bright CD8+ T cells (CD8high/CD3+), generally
considered cytotoxic T cells, was also lower.
Intraepithelial CD8+ T cells have also generally been
assigned to the cytotoxic lymphocyte (CTL) population,
responsible for killing of infected or stressed target cells
(Cheroutre, 2005; Wilson et al., 1986). However, it was
recently shown that the vast majority (up to 90%) of
perforin-positive PBMCs in pigs are CD3 negative and
therefore resemble NK cells (Denyer et al., 2006).
Perforin was absent from most ab T cells and also
absent from gd T cells in porcine blood. This finding is
consistent with the observation that an MHC-restricted
P. Schierack et al. / Veterinary Immunology and Immunopathology 118 (2007) 1–11 9
antigen-specific cytotoxic activity in pigs is difficult to
demonstrate (Pauly et al., 1996; Saalmuller et al., 1994).
Surprisingly a subset of CD4+ CD8low T cells was
perforin-positive and showed cytotoxic activity (Denyer
et al., 2006). Moreover not all porcine CD4+/CD8+
double positive T cells are memory helper cells.
Apparently, the phenotypes and functions of porcine
CD8+ T cells differ from the CD8+ populations found
in mice and humans. The functions of the different
subsets of porcine CD8+ T cells therefore, remain to be
verified. The consequences of the reduced CD8+ T cell
populations in blood observed in our study for probiotic
piglets will require further clarification.
The results also indicated that that probiotic-treated
piglets showed a significant higher ratio of CD4+ to
CD8+ T cells. A recent study with vaccinated and
challenged pigs noted a change in the CD4+:CD8+ ratio
during a secondary immune response of piglets infected
with low doses of bacterial antigens applied as aerosols
(Appleyard et al., 2002). An increase in the
CD4+:CD8+ ratio correlated with a reduction in
clinical signs during the following high dose challenge
with the same bacterium. In our study, the elevated
CD4+:CD8+ ratio in the Bacillus group might result
from continuous feeding of B. cereus and the
subsequent prolonged presence of Bacillus antigens
which might be equivalent to a secondary immune
response of an infection.
Differences in the reactivities of PBMCs were also
observed in the vaccination trials. Vaccinations were
chosen to evaluate immune parameters during a
challenge of the immune system. The immune
stimulation by intramuscular vaccination was expected
to be better defined and the immune reaction was
expected to be more moderate compared to infection
studies with pathogens. Additionally, bacteria coloniz-
ing the host have been found to affect adhesion and
invasion of pathogens to mucus and epithelial cells in
the absence of immune cell responses (Kleta et al.,
2006), which might also affect validations of infection
studies with pathogens. In the vaccination trials we
determined both the antigen-specific proliferations of
PBMCs in vitro as well as antigen-specific serum
antibody concentrations. The Influenza antigens showed
enhanced proliferation only with PBMC preparations
from vaccinated piglets, suggesting an expansion of the
memory cell/antigen presentating populations. The
probiotic group reacted significantly better to an
application with Influenza vaccination antigens. The
enhanced proliferation rates did not correlate with
higher numbers of blood monocytes. Changes in the
numbers of helper T cells are also not expected to be
responsible for the higher proliferation counts (Mos-
mann and Coffman, 1989). Since IL-4 supports the
antigen-presenting functions of monocytes and macro-
phages by up-regulating expression of MHCII mole-
cules, and IL-4 activated monocytes/macrophages
enhance T cell proliferation (Lutz et al., 1996; Ruppert
et al., 1991), the average elevated IL-4 levels in the
probiotic group would provide at least a partial
explanation for the improved proliferation after antigen
challenge. For reasons which remain unclear, Myco-
plasma antigens did not result in a proliferative response
of PBMCs for either the control or probiotic group
samples.
Interestingly, in all proliferation assays, spontaneous
proliferation, proliferative responses to Concanavalin A
and Influenza antigens (H1N1 and H3N2) were higher
on day 96 compared to day 56 possibly indicating
maturation of the immune system of the piglets.
In vitro studies in our laboratory showed strong dose-
dependent cytotoxic/cytolytic effects of high infection
rates with B. cereus var. toyoi on cells of a porcine
intestinal epithelial cell line, IPEC-J2 (unpublished
data). These cytotoxic/cytolytic effects were likely due
to B. cereus var. toyoi hemolysin, since it was shown
that other B. cereus strains lead to necrosis after
infection of enterocyte-like cells due to their hemolysin
(HBL) which is produced by most species of the B.
cereus group (Minnaard et al., 2004; Pruss et al., 1999).
Apoptotic enterocytes and remnants can be carried by
dendritic cells (DCs) to T and B cell areas and stimulate
an immune response (Huang et al., 2002). Additionally,
DCs can be induced by luminal bacteria to extend cell
processes between intestinal epithelial cells and capture
and translocate bacteria into the Peyers’ patches
(Rescigno et al., 2001). Previous studies have also
shown that the activity of DCs can be affected by the
composition of the gut microflora (Christensen et al.,
2002; Karlsson et al., 2004; Mohamadzadeh et al.,
2005), and Bacillus subtilis flagellin induced MIP-3a
release by epithelial cells, a chemokine responsible for
the recruitment of immature DCs (Rimoldi et al., 2004).
The stimulated DCs can, in turn, activate T and B cells
(Moulin et al., 2000) and induce TH1 or TH2 responses
(Iwasaki and Kelsall, 1999; Maldonado-Lopez et al.,
1999). Bacillus species (B. cereus, B. subtilis) can also
be internalized by epithelial cells, and B. subtilis spores
have been shown to germinate efficiently in macro-
phages and to activate gene expression (Duc le et al.,
2004; Minnaard et al., 2004). These observations
suggest that B. cereus var. toyoi may irritate the
intestinal mucosa, resulting in activation of the mucosal
immune system.
P. Schierack et al. / Veterinary Immunology and Immunopathology 118 (2007) 1–1110
This suggestion is supported by other data from our
group. In parallel studies, Scharek et al. investigated gut-
associated immune cell populations of B. cereus var.
toyoi-treated piglets. Probiotic piglets showed signifi-
cantly more CD8+ T cells in the upper jejunal epithelium
shortly after weaning (28 days), and concluded that this
was a result of a stimulation of the mucosal immune
system by B. cereus var. toyoi (unpublished observa-
tions). However, histological changes were not observed
which would have indicated severe inflammation in the
intestine of piglets of our studies (Weyrauch et al.,
personal communication). Similar observations of an
increase of mucosal CD8+ cells without visible signs of
inflammation were reported using the probiotic E. coli
Nissle1917 in swine (Duncker et al., 2006), also
consistent with the idea that probiotics are capable of
stimulating the gut-associated immune system without
overt signs of inflammation.
In conclusion, we show that feed supplementation of
piglets with the probiotic B. cereus var. toyoi affects the
systemic immune response of piglets, with an improved
post-vaccination response to viral antigens and changes
in the circulating, blood immune cell populations.
While further studies will be required to determine the
long-term impact of the apparent irritation of the
mucosa and the subsequent modulation of the systemic
immune response, other studies on this same group of
animals found that B. cereus var. toyoi also affected the
growth performance and health status of piglets.
Weaned probiotic piglets had a higher gain to feed
ratio than control piglets and probiotic supplementation
led to a reduction in the incidence of liquid feces and
post-weaning diarrhea (Taras et al., 2005). These
observations suggest that at least during the first few
months of life, any apparent intestinal irritation or
changes in immune cell populations has no averse
affects on the overall health of piglets, and may simply
reflect additional effects of the same immunological
processes contributing to the observed improved
general health status of the animals.
Acknowledgements
This work was supported by grant FOR 438/1-1 from
the Deutsche Forschungsgemeinschaft. We thank K.
Tedin (Institut fur Mikrobiologie und Tierseuchen,
Berlin, Germany) for helpful suggestions. Additionally,
we thank B. Seeger (Neurowissenschaftliches For-
schungszentrum, Berlin, Germany) for assistance with
the proliferation assays, C. Menge (Institut fur Hygiene
und Infektionskrankheiten der Tiere, Giessen, Ger-
many) for helpful suggestions on the manuscript and M.
Filter, P. Wrede (Institut fur Molekularbiologie und
Biochemie, Charite, Humboldt-Universitat Berlin,
Germany) and F. Antonelli (Instiut fur Tierernahrung,
Berlin, Germany) for statistical analysis.
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