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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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