Bacillus Coagulans Enhance the Immune Function of the Intestinal Mucosa of Yellow Broilers

Abstract

This experiment was conducted to investigate the effects of Bacillus coagulans on the growth performance and immune functions of the intestinal mucosa of yellow broilers. Three hundred and sixty one-day-old yellow chicks were randomly allocated to four treatments groups with six replicates of 15 chicks each. The broilers were randomly subjected to one of the following treatments for 28 days: control group (group1, fed a basal diet) and three treatments (group 2, 3, 4) fed the basal diet supplemented with 100, 200, or 300 mg/kg Bacillus coagulans , respectively). The results showed that for 28 days, compared with the control diet, the dietary addition of 200 mg/kg Bacillus coagulans significantly decreased the feed/gain ratio (F/G) (p<0.05), improved the thymus index, spleen index and bursa index (p<0.05), increased the villus height to crypt depth ratio (V/C) in the duodenum (p<0.05), increased the number of secretory immunoglobulin (sIgA) positive cells ( p<0.05). The dietary addition of 200 mg/kg Bacillus coagulans promoted a significant increase in Lactobacillus spp. populations and suppressed Escherichia coli replication in cecum, compared with the control (p<0.05). Moreover, the dietary addition of 200 mg/kg Bacillus coagulans also significantly enhanced the levels of interferon alpha (IFNα), toll-like receptor (TLR3), and melanoma differentiation-associated protein 5(MDA5) in the duodenum (p<0.05). In conclusion, the dietary addition of Bacillus coagulans significantly improved broiler performance, and enhanced the intestinal mucosal barrier and immune function. The optimal dosage of Bacillus coagulans for yellow broilers was determined as 2×108 cfu/kg.

Full text

115
Brazilian Journal of Poultry Science
Revista Brasileira de Ciência Avícola
ISSN 1516-635X Jan - Mar 2017 / v.19 / n.1 / 115-122
http://dx.doi.org/10.1590/1806-9061-2015-0180
Bacillus Coagulans Enhance the Immune Function of
the Intestinal Mucosa of Yellow Broilers
Author(s)
Xu LI
Fan QI
Zhuang YI,II
Wang QI,II
Gao YI
Wang CI
I College of Animal Sciences – Fujian Agri-
culture and Forestry University, Fuzhou
350002, China.
II Fujian Key Laboratory of Traditional Chine-
se Veterinary Medicine and Animal Health
(Fujian Agriculture and Forestry University)
Mail Address
Corresponding author e-mail address
Quanxi Wang
College of Animal Sciences – Fujian
Agriculture and Forestry University, Fuzhou
350002, China.
Tel: (+086)059183758852
E-mail: wqx608@126.com
Keywords
Bacillus coagulans, yellow broilers, growth
performance, intestinal mucosa immune
function.
Submitted: August/2016
Approved: November/2016
ABSTRACT
This experiment was conducted to investigate the effects of Bacillus
coagulans on the growth performance and immune functions of the
intestinal mucosa of yellow broilers. Three hundred and sixty one-day-old
yellow chicks were randomly allocated to four treatments groups with
six replicates of 15 chicks each. The broilers were randomly subjected
to one of the following treatments for 28 days: control group (group1,
fed a basal diet) and three treatments (group 2, 3, 4) fed the basal
diet supplemented with 100, 200, or 300 mg/kg Bacillus coagulans,
respectively). The results showed that for 28 days, compared with
the control diet, the dietary addition of 200 mg/kg Bacillus coagulans
signicantly decreased the feed/gain ratio (F/G) (p<0.05), improved
the thymus index, spleen index and bursa index (p<0.05), increased
the villus height to crypt depth ratio (V/C) in the duodenum (p<0.05),
increased the number of secretory immunoglobulin (sIgA) positive
cells (p<0.05). The dietary addition of 200 mg/kg Bacillus coagulans
promoted a signicant increase in Lactobacillus spp. populations
and suppressed Escherichia coli replication in cecum, compared with
the control (p<0.05). Moreover, the dietary addition of 200 mg/kg
Bacillus coagulans also signicantly enhanced the levels of interferon
alpha (IFNα), toll-like receptor (TLR3), and melanoma differentiation-
associated protein 5(MDA5) in the duodenum (p<0.05). In conclusion,
the dietary addition of Bacillus coagulans signicantly improved broiler
performance, and enhanced the intestinal mucosal barrier and immune
function. The optimal dosage of Bacillus coagulans for yellow broilers
was determined as 2×108 cfu/kg.
INTRODUCTION
Moore rstly reported that the animal weight was signicantly
enhanced by the addition of antibiotics in the feed in 1946 (Moore et
al., 1946). In the 1950s, the Food and Drug Administration (FDA) of the
United States rstly approved antibiotics as feed additives, after which
they were widely applied in the poultry industry for the treatment and
prevention of bacterial diseases and as growth promoter. However, the
negative effects of the extensive use – and even abuse – of antibiotics
gradually emerged, such as antibiotic residues in meat (Smither et
al., 1980), bacterial resistance (Krushna Chandra Sahoo et al., 2010),
intestinal ora imbalance, and environmental pollution (Pan et al.,
2011), leading several countries to introduce legislation to limit the
application of antibiotics in animal feeds.
The ban on the use of antibiotics as feed additives is an inevitable
trend in China, and therefore, the research on alternatives for in-
feed antibiotics is urgent. At present, there are many studies on such
alternatives, such as benecial bacteria, prebiotics, enzymes, acidulants,
and plant extracts (Vahjen et al., 2007; Natsir et al., 2010). Probiotics

116
Xu L, Fan Q, Zhuang Y,
Wang Q, Gao Y, Wang C Bacillus Coagulans Enhance the Immune Function of
the Intestinal Mucosa of Yellow Broilers
maintain intestinal ora balance, enhance the intestinal
barrier function (Anderson et al., 2010; Dai et al., 2012;
Furrie E., 2005), and the immune function. Probiotics
also can effectively improve the activity of interferon,
which stimulates the immune cells to produce specic
antibodies, such as sIgA, improve the discrimination of
immune system, and induce cytokine expression in T
and B lymphocytes and macrophages (Russell et al.,
2013). Rajput reported that the dietary inclusion of a
yeast (Saccharomyces boulardii) and Bacillus subtilis
signicantly increased the weights of the bursa and
the thymus, and increased the mRNA expression levels
of the occluding, cloudin 2 and cloudin 3 nucleotides,
the number of IgA-positive cells in the jejunum, as well
as the intestinal levels of interleukin (IL)-6, IL-10, tumor
necrosis factor (TNF) alpha, transforming growth factor
beta (TGFβ), and sIgA (Rajput et al.,2013).
Intestinal-related immune system can identify gut
microbes by pattern recognition receptors (PRRs), such
as toll-like receptors (TLRs) (Gómez-Llorente et al.,
2010). Probiotics can activate the mitogen-activated
protein kinase (MAPK) and nuclear factor kappa(NF-
kB) to activate TLRs, thereby regulating the immune
function (Lebeer et al., 2010; Kawai et al., 2010; Wells
et al., 2011). Probiotics can also regulate the immune
function by regulating the inammatory reaction
(Castillo et al., 2011).
In this experiment, the effect of Bacillus coagulans
on the growth performance and the immune function
of the intestinal mucosa of yellow-feathered broilers
was investigated.
MATERIALS AND METHODS
Birds and management
A total of 360 one-day-old healthy Lingnan yellow-
feathered chickens were provided by Guangdong
Wens Food Group Co., Ltd.
The experiment was performed at the poultry
laboratory of Fujian Agriculture and Forestry University
in Fuzhou, Fujian province, China. Fifteen chickens
were reared per cage equipped with a drinker and a
feeder. The water changed and feed was added to the
feeders once daily. Birds were submitted to 23 hours of
light. Vaccination was carried out according to Table 1.
Bacillus coagulans powder, containing that
contained 1×109cfu/g living bacteria, was provided by
Luodong Bio-Technology CO. LTD.
Experiment design
Chickens were randomly divided into four
treatments with six replicates of 15 birds each. The
chickens in the control group (group 1) were fed with
a basal diet, and those in other groups (groups 2, 3, 4)
were fed with the basal diet supplemented with 100,
200, or 300 mg/kg Bacillus coagulans, respectively. The
experiment lasted for 28 days.
The basal diet was formulated according to China’s
poultry industry standards for Chinese color-feathered
chicken between 1 to 28 days old. The diet was formu-
lated for the entire period and supplied as mash. The
Bacillus coagulans powder was added in the premix.
The composition of the basal diet is shown in Table 2.
Table 2 – Composition and nutrient level of basal diet (air
dry basis) %
Items Content(0-28d)%
Corn 58
Soybean meal 27
Expanded soybean 10
Limestone 1.0
Premix14.0
Total 100.0
Nutrient levels
Metabolizable Energy (ME), MJ·kg-1 11.87
Crude Protein (CP), % 21.20
Calcium (Ca), % 1.02
Total Phosphorus (TP), % 0.55
Available Phosphorus (AP), % 0.31
Lysine (Lys), % 1.12
Methionine (Met) + Cystine (Cys), % 0.83
1) The nutrient values in the table are calculated values.
2) The premix supplied per kg diet: Cu (as copper sulfate) 10mg, Fe (as ferrous sulfa-
te) 72mg, Zn (as zinc sulfate) 60.2mg, Mn (manganese) 78mg, I (iodine) 0.4mg, Se
(selenium) 0.24mg, choline 600mg, Vit. A (Vitamin A) 10000IU, Vit. D3 (Vitamin D3)
2600IU, Vit. E (Vitamin E) 26mg, Vit. K3 (Vitamin K3) 2.6mg, Vit. B1 (Vitamin B1) 2.6mg,
Vit. B2 (Vitamin B2) 6.5 mg, Vit. B6 (Vitamin B6) 2.60mg, Vit. B12 (Vitamin B12) 19.5μg,
nicotinic acid 26mg, D-pantothenic acid 13mg, Folic acid 1.3mg, biotin 104μg,
methionine 2364IU.
Table 1 – The immune program
Age Vaccine Dose Immune method
1 day old Marek’s disease vaccine
Combined vaccination of Newcastle disease and infectious bronchitis
One feather
One feather
Intramuscular injection
Eye droppings
5 day old Avianinuenzavaccine
Fowlpox vaccine
One feather
One feather
Intramuscular injection
Hypodermic injection
10 day old Newcastle disease vaccine One feather Intramuscular injection
20 day old
infectious bursal disease vaccine
Newcastle disease vaccine
Avianinuenzavaccine
One feather
One feather
One feather
eye droppings
Intramuscular injection
Intramuscular injection

117
Xu L, Fan Q, Zhuang Y,
Wang Q, Gao Y, Wang C Bacillus Coagulans Enhance the Immune Function of
the Intestinal Mucosa of Yellow Broilers
Growth performance parameters
Chicks were individually weighed on day 1 of the
trial, and no signicant differences were detected. On
day 28, chicks were fasted for 12h and individually
weighed. Feed was offered daily at 5:00 pm, after feed
residues were measured. Average daily feed intake,
average daily weight gain, and feed to gain ratio were
calculated per experimental group. Livability (%) was
calculated weekly.
Immune organ index
On day 28, three chicks in per replicate were selected
and sacriced by decapitation. The thymus, spleen and
bursa were collected and weighed. Thymus, spleen,
and bursa indexes were calculated as = [organ weight
(g)/ body weight (g)] ×100.
Bacterial flora detection in the cecum by
SYBR-PCR
Standard curve preparation
Plate count: Escherichia coli strain k88 was used as
reference strain. It was cultured in lysogeny broth (LB)
at 37 oC for 7 h, and then was serially diluted to 10-9.
Dilutions were then cultured in Macconkey medium at
37oC for 24 h. Finally, bacterial colonies were counted.
Standard curve: The DNA of Escherichia coli strain
k88 was serially diluted to 10-6., and was detected by
Quantitative Real-time Polymerase Chain Reaction
(SYBR-PCR), and the linear equation was calculated.
Detection of Escherichia coli and
Lactobacillus spp.
The universal primers of bacterial genera and
the specic PCR primers of Escherichia coli and
Lactobacillus genus were designed (Table 3). On day
28, the cecal content (200 mg) from the three chicks
per replicate sacriced for organ index were collected,
and total DNA was extracted using a genomic DNA
extraction kit (Taingen, Beijing, China). Escherichia
coli and Lactobacillus spp. in the cecal content were
detected by SYBR-PCR.
Reaction volumes of 25 μL consisted of 12.5 μL
SYBR® Premix Ex TaqTM (2×) (Promega, Wisconsin,
U.S.A), ROX reference dye ( 50× ) 0.5 μL, 1 μLDNA,
1 μL forward primer (10 μM), 1 μL reverse primer (10
μM), and 10 μL RNase free ddH2O. PCR conditions were
initial denaturation at 95oC for 0.5 min followed by 40
cycles at 95oC for 5 s, 60oC for 34 s, and 95oC for 15
s, 60oC for 1 min, 95oC for 15 s. Data was calculated
by delta delta CT(rrCT), and were analyzed by
SPSS19.0.
Villus height / crypt depth ratio
On day 28, three birds per treatment were
sacriced for organ index determination, and the
intestinal contents were rinsed with physiological
saline (PBS, 0.01mol/L, pH=7.1). Duodenal sections
(1.5cm×1.5cm×0.5cm) were collected, xed in 4%
paraformaldehyde xing solution for 24 hours, and
were submitted to routine histology procedures,
including dehydration, clearing, and embedding
in parafn. Samples were cut into 4 to 6μm semi-
serial cross sections and stained with hematoxylin
and eosin (HE). Intestinal villus height and crypt
depth were measured under an optical microscope
at 40x magnication using the software HPIAS-5100
(Qianping, Shanghai, China).
Detection of sIgA by immunohistoche-
mistry
The sections of duodenum from the chicks sacriced
for organ index were prepared and inhibited by
preincubating the tissues in 3% H2O2, incubated in 5%
normal goat serum for 0.5h followed by
an overnight incubation at 4oC with 1:40
dilution of rabbit anti-chicken IgA serum.
Sections were then incubated with goat
anti-rabbit IgG (1:300, Vektor ABC kit,
PK-6101) at room temperature for 1h,
followed by incubation with an avidin-
biotin-peroxidase conjugate solution at
room temperature for 1h. The sections
were then rinsed three times with
phosphate salt buffer (PBS) and were
incubated with 3,3’-diaminobenzidine
tetrahydrochloride (Sigma, Calif, U.S.A)
solution dissolved in 0.05 M Tris-HCl
buffer (pH 7.4) at room temperature.
Table 3 – Primer sequences
Items Primer sequences Amplied
fragments(bp)
Bacterium universal primer (16s) F: 5’-CCTACGGGAGGCAGCAG-3’
R: 5’-ATTACCGCGGCTGCTGG-3’194
Escherichia coli(16s) F: 5’-GTTAATACCTTTGCTCATTGA-3’
R: 5’-ACCAGGGTATCTTAATCCTGTT-3’340
Lactobacillus genus(16s) F: 5’- AGCAGTAGGGAATCTTCCA-3’
R: 5’ -CACCGCTACACATGGAG -3’341
IFN-αF: 5’ -GGACATGGCTCCCACACTAC-3’
R: 5’ -ATCCGGTTGAGGAGGCTTT-3’204
TLR3 F: 5’ -CCATTTGATTGCACCTGTGA-3’
R: 5’ -GCAACACCAGAGTACCGTGA-3’133
MDA5 F: 5’ -GAAGAAGGTGTCCGCTTATCA-3’
R: 5’ -GAATCTGAGGCTGTGGAATCA-3’169
β-actin F: 5’ -CCAAAGCCAACAGAGAGAAGAT-3’
R: 5’ -CATCACCAGAGTCCATCACAAT-3’138

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Xu L, Fan Q, Zhuang Y,
Wang Q, Gao Y, Wang C Bacillus Coagulans Enhance the Immune Function of
the Intestinal Mucosa of Yellow Broilers
Ten minutes later the enzyme-substrate reaction were
stopped with 0.05M Tris-HCl buffer (pH 7.4). Sections
were then rinsed in PBS and counterstained with
hematoxylin. Finally, sections were cleared and sealed
with a glass coverslip. In the duodenum, IgA-positive
lymphocytes were identied by their characteristic
morphology: round, with a nucleus surrounded by a
yellow-brown stained ring.
Expression of INF, TLR-3 and MDA-5 mRNA
Quantitative real-time PCR was performed with
the primers shown in Table 3 to analyze the mRNA
expression levels of interferon (IFN) and pattern
recognition receptors (TLR-3, MDA-5) to verify if the
Bacillus coagulans product stimulated
interferon production to enhance
intestinal mucosal immune function.
Data was calculated and analyzed by
rrCT.
RESULTS
Growth performance
Table 4 shows the performance results
obtained during the 28 days of the
experiment. Average daily feed intake
was not different among treatments
(p>0.05). Average daily weight gain
was 0.40% (p>0.05), 3.75% (p<0.05),
3.23% (p>0.05) higher in groups 2,
3, and 4, respectively, compared with
group 1. Average body weight gain of
group 3 was 3.73% higher than in group
1 (p<0.05). The feed conversion ratio of
group 3 was 2.55% lower than that of
group 1 (p<0.05). Compared with group
1, groups 2, 3 and 4 presented 1.15%,
1.15%, and 2.30% survival rates,
but the difference was not signicant
(p>0.05). These results showed that the
growth performance of chickens fed
with 200 mg/kg Bacillus coagulans was
signicantly enhanced.
Immune organ index
Immune organ index indicates status of the avian
immune function. As shown in Table 5, the thymus
index of groups 2, 3, and 4 was 7.18% (p<0.05),
7.27% (p<0.05), and 0.56% (p>0.05) higher compared
with group 1. The spleen index of groups 2, 3, and 4
was 2.75% (p<0.05), 14.66% (p<0.05), and 10.91%
(p>0.05), respectively, higher than that of group 1.
The bursa index of groups 2, 3, and 4 was 14.24%,
14.28%, 14.24% (p<0.05) than that of group 1. These
results conrm that the dietary inclusion of 100 and
200 mg/kg Bacillus coagulans can signicantly increase
the immune organ index of chickens, particularly at
200 mg/kg.
Table 4 – Effects of Bacillus coagulans on the growth performance in yellow broilers
Treatments Average daily feed intake Average daily weight gain Feed conversion ratio Survival rate
1 29.75±0.81 15.18±0.46b1.96±0.04a 96.67±3.65
2 29.70±0.68 15.24±0.30ab 1.95±0.05ab 97.78±3.44
3 30.14±0.82 15.75±0.44a1.91±0.02b97.78±3.44
4 30.52±0.85 15.67±0.48ab 1.95±0.02 ab 98.89±2.72
Means followed by different superscripts in the same column are statistically different (p<0.05).
Figure 1 – Bacillus coagulans optimized the bacterial ora in the yellow
feather broiler chickens
A: Primer specicity identication showed that the designed primers can be used for real-time uorescence
quantitative PCR reaction. (1: Escherichia; 2: Negative control of Escherichia; 3: Lactobacillus; 4: Negative control
of Lactobacillus; M: 2000 DNAmarker)
B: Standard curve of standard samples (Escherichia Coli. strain k88 DNA)
C: Melting curve of standard sample
D: Amplication curve of standard sample
E: Melting curve of E. coli
F: Melting curve of Lactobacillus

119
Xu L, Fan Q, Zhuang Y,
Wang Q, Gao Y, Wang C Bacillus Coagulans Enhance the Immune Function of
the Intestinal Mucosa of Yellow Broilers
Table 5 – Effects of Bacillus coagulans on the immune
organ index (%) of yellow broilers
Treatments Thymus index Spleen index Bursal index
1 0.4292±0.0435a0.1623±0.0216a0.2844±0.0466a
2 0.4600±0.0246b0.1830±0.0236b0.3249±0.0345b
3 0.4604±0.0277b0.1861±0.0264b0.3250±0.0517b
4 0.4316±0.0393ab 0.1800±0.0194ab 0.3249±0.0432b
Means followed by different superscripts in the same column are statistically different
(p<0.05).
Cecal bacterial flora
Plate count results showed that the number of
Escherichia coli strain k88 in the cecum was 1.6×1010
cfu/mL. The regression coefcient (R2) of the curve
was 0.9994, indicating that the counts were linearly
correlated with the dietary inclusion levels of the
product. The equation of the standard curve was y=-
3.006x+43.031 (y: Ct value’x: log value of standard
DNA samples).
Total bacterial counts in three groups fed with three
different doses Bacilllus coagulans were not different
(p>0.05) compared with the control group (Table 6).
Escherichia coli counts in the cecum of broilers were
not signicantly different among treatments (p>0.05).
However, Lactobacillus spp. counts were signicantly
increased in the cecum of broilers fed with 200 mg/kg
Bacillus coagulans in basal diet (p<0.05).
Table 6 – Effects of Bacillus coagulans on the microbial
ora ( lgcfu·g-1 ) of yellow broilers.
Treatments Total bacterial count Escherichia coli Lactobacillus spp.
1 11.85±0.21 10.67±0.38 9.34±1.14a
2 11.87±0.13 10.36±0.32 9.73±1.16ab
3 11.88±0.22 10.42±0.41 10.18±0.76b
4 11.84±0.19 10.37±0.41 9.40±0.78ab
Means followed by different superscripts in the same column are statistically different
(p<0.05)
Duodenal villus to crypt ratio
On day 28, intestinal villus height in three groups
fed with three doses of Bacilllus coagulans was not
different change compared with the control group
(p>0.05), but the duodenal crypts of group 3 were
signicantly deeper compared with the control group
(p<0.05). Therefore, the villus height to crypt depth
ratio (V/C) value was higher in the duodenum of
broilers fed with 200 mg/kg Bacillus coagulans in the
basal diet (Table 7).
Table 7 – Effects of Bacillus coagulans on the villus height
to crypt depth ratio (V/C value) of duodenum in yellow
broilers.
Treatments Intestinal villi
height (μm) Crypt depth (μm) V/C value
1 1120.91±41.24 114.48±8.54a9.83±0.78a
2 1157.63±35.33 108.93±7.75ab 10.68±0.96ab
3 1160.20±45.00 103.39±9.19b11.30±1.11b
4 1134.50±28.22 105.79±7.54ab 10.76±0.72ab
Means followed by different superscripts in the same column are statistically different
(p<0.05)
Levels of sIgA-positive cells lever in
duodenum
In the duodenum, sIgA-positive lymphocytes were
identified by immunohistochemistry. These cells were
present in the lamina propria of duodenal villi (Fig. 2
A-D). Fig. 2 E shows that there were more sIgA-positive
cells in the duodenum of group 3 (p< 0.05) compared
with group 1. No differences were observed between
the other groups (p>0.05).
mRNA expression of IFN-α, MDA-5 and
TLR-3
A-D: SIgA-positive lymphocytes were identified by immunohistochemistry method with
characteristic antibody. These cells were present in the lamina propria of villi in the
duodenum. A: group I, B: group II, C: group III, D: group IV. E: Statistical analysis on the
number of SIgA-positive cells. *: p< 0.05.1-4: group I-IV.
Figure 2 – Bacilllus coagulans increase SIgA-positive cells in the duodenum
IFN-α is an important immune regulator, and may
stimulate the innate immune and antiviral responses.

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Wang Q, Gao Y, Wang C Bacillus Coagulans Enhance the Immune Function of
the Intestinal Mucosa of Yellow Broilers
The objective of this measurement was to investigate if
the Bacilllus coagulans was able to regulate the innate
immune function of the intestinal tract. The results
showed that, on day 28, the mRNA expressions of IFN-α,
MDA-5 and TLR3 were signicantly higher in group 3
compared with group 1 (p<0.05 or p<0.01) (Fig. 3),
indicating that Bacilllus coagulans may stimulate the
innate immune function of the intestinal tract.
A: IFN- α, B: MDA5, C: TLR3, 1: group I, 2: group II, 3: group III, 4 group IV
Figure 3 – Bacilllus coagulans promoted the mRNA expression of IFN-α, MDA5 and
TLR3
DISCUSSION
The results of this experiment showed that the
growth performance of chickens fed 200 mg/kg
Bacilllus coagulans was effectively improved, which
is consistent with the other reports. Lei reported
that the dietary inclusion of spores of a lactobacillus
improved the conversion ratio and reduced mortality
and culling rate of broilers (Lei et al., 2015). Lactobacilli
improve calcium and phosphorus utilization, and lactic
acid promotes the transformation of pepsinogen
into protease, stimulating the peristalsis of the small
intestine to enhance nutrient digestion (Ma et al.,
2014; Giang et al., 2010).
Thymus, spleen, and bursa of Fabricius are important
immune organs of poultry, and their index reects the
immune function of the poultry. Our results are in
agreement with the ndings of Jin Er-hui (2013), who
showed that the 21-day old and 42-day old AA chickens
fed] a bacillus-based probiotic contained 1x1011cfu/g
provided by Jiaxing Kori Biological Technology Co., LTD
at 200 mg/kg presented better development of the
immune organs and immune function than 400 mg/
kg (Jin et al., 2013). Other studies have shown that
Bacillus spp. regulate the intestinal bacterial ora and
improve the immune function of the intestinal mucosa
(Isolauri et al., 2001).
The ceca are the main site of intestinal microbial
replication and activity. Lactobacillus spp. and
bidobacteria are the predominant genera (Apajalahti
et al., 2004). These are commensal bacteria and can
suppress the harmful bacteria and inammation
(Sheil et al., 2007). Escherichia coli is a conditionally
pathogenic bacterium, and may become harmful and
cause diarrhea when the intestinal microbial ora is
imbalanced (Zhang et al., 2010).
Bacillus coagulans is able to produce bacteriocins,
such as lactosporin, which have signicant antibacterial
activity (Riazi et al., 2009). In addition, it produces
lactic acid and other organic acids, reducing the
colonization of harmful bacteria in the intestinal tract
(Cui et al., 2005). Bacillus coagulans also breaks down
polysaccharides into oligosaccharides, promoting
the growth of lactobacilli, bidobacteria and other
benecial bacteria, maintaining intestinal ora balance
(Zheng et al.,2011). In recent years, a new compound,
dysprosium, produced from Bacillus coagulans was
shown to have broad antibacterial spectrum and to
be highly efcient (Honda et al., 2011). Studies found
that morphological changes in the small intestine were
closely related to a toxin produced by enterotoxigenic
Escherichia coli (ETEC) in the gut Jindal et al.,2006.
The small intestine is the main organ for the digestion
and absorption of nutrients. Villus height, crypt depth
and V/C ratio are indication of nutrient absorption
capacity by the intestine (Caspary et al., 1992). In the
present experiment, there was a positive and signicant
correlation between the intestinal villus height and the
number of epithelial cells. Increases in the number of
intestinal epithelial cells and in villus height indicates
better nutrient absorption capacity ability of the body
enhanced. Crypt depth reects the secretory function
of small intestine. Bacillus spp. can stimulate the
differentiation and proliferation of intestinal epithelial
cells and improve nutrient utilization (Artis et al., 2008;
Wells et al., 2011; Duerr et al., 2012). Lei reported
that broilers receiving a direct-fed microbial based
on Bacillus amyloliquefaciens signicantly increased
crypt depth and villus height to crypt depth ratio in
the duodenum, jejunum, and ileum (Lei et al., 2015).
Similarly, in broilers fed diets with 200 mg/kg Bacillus
coagulans, an increase in V/C ratio was also observed
(Lin et al., 2014).
The function of the intestinal barrier is to maintain
epithelial integrity and to protect the body from the
environment. The intestinal barrier functions include
the mucous layer, secretory IgA and epithelial junction
adhesion complex (Miriam et al., 2012). If this barrier
is disrupted, antigens reach the submucosa and induce

121
Xu L, Fan Q, Zhuang Y,
Wang Q, Gao Y, Wang C Bacillus Coagulans Enhance the Immune Function of
the Intestinal Mucosa of Yellow Broilers
inammatory response (Wang et al., 2012). The
integrated intestinal mucosa barrier is important for
the defense against pathogenic bacteria (Blikslager et
al., 2007).
Secretory IgA is produced and secreted by IgA-
positive plasma cells in the lamina propria of the
intestinal mucosa. It is released in the intestinal
lumen and mixed with the normal ora. It allows the
establishment of the normal ora and inhibit pathogen
colonization. Medici fed BALB mice with probiotic
fresh cheese (PFC) and found the PFC enabled
Bifidobacterium bifidum, Lactobacillus acidophilus and
L. paracasei to exert important immunomodulating
effects in the gut (Medici et al.,2004). In our study,
the number of IgA-positive cells was signicantly
increased when broilers were fed 200 mg/kg Bacillus
coagulans, suggesting that this probiotic may enhance
the immune function of the intestinal mucosa.
The intestinal mucosa is rich in lymphoid tissues,
called the gut-associated lymphoid tissue (GALT). GALT
can secret multiple cellular factors, such as interferon,
colony stimulating factors (CSFs), interleukins, etc.,
which are able to kill pathogens and regulate the
immune function of the mucosa. Interferons, in
particularly, mediate the innate immune. Interferons are
produced in response to pathogen infection. However,
the infecting pathogen need to be recognized by
pattern recognition receptors (PRRs), such as toll-like
receptors (TLRs), melanoma differentiation-associated
protein 5 (MDA-5)-like receptors, increasing the
expression of IFN type I in the infected cells. MDA5 is
one of the most important PRRs (Durbin et al.,2013).
MDA-5 plays the critical role in the recognition of
pathogens in the cytoplasm and transmits a signal
to induce expression of interferon and cytokines. The
newly-synthesized IFN-I is then secreted and binds to
the IFN-I receptor (IFNAR), inducing the expression of
hundreds of IFN stimulating genes (ISGs) that promote
immune regulation.
Kailova reported that mice suffering from
necrotizing enterocolitis (NEC) were orally received
Bidobacterium OLB 6378, which stimulated the
mRNA expression of TLR2, cyclo-oxygenase2 (COX-2),
and increased the synthesis intestinal of prostaglandin
estradiol 2 (Kailova et al.,2010). Liu also veried that
Roy’s lactobacillus DSM 17938 rat NEC has a positive
effect on the prevention on rat NEC, which can
signicantly reduce the mRNA expression of TNF alpha
and TLR 4 and TNF alpha, TLR 4 protein levels (Liu et al.,
2012). Rajput veried that boundens yeast and bacillus
B10 stimulated chicken bone marrow dendritic cells,
increasing TLR 1, TLR 2, TLR 4, and TLR 15 expression
(Rajput et al., 2014).
Bacillus coagulans not only has the characteristics
of lactobacilli and bidobacteria, but is also resistant to
acid environments and high temperatures (Hyronimus
et al., 2000; Ripamonti et al., 2009). Previous
experimental studies showed that Bacillus coagulans
was able to regulate cell factors, enhanced the
phagocytosis of phagocytic cells, enhanced the activity
of NK, T and B cells, and increased the expression of
IgA, IgG and IgM (Kodali et al., 2008). Consistently, the
results of the present experiment showed that Bacillus
coagulans can enhance the immune function of the
gut of yellow-feathered broilers.
ACKNOWLEDGMENTS
This work was supported by a program from
China Fujian laying hen industry system program (NO.
K83139297 (2013-2017)).
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