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Brazilian Journal of Poultry Science

Print version ISSN 1516-635XOn-line version ISSN 1806-9061

Rev. Bras. Cienc. Avic. vol.19 no.1 Campinas Jan./Mar. 2017

http://dx.doi.org/10.1590/1806-9061-2015-0180 

Articles

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

L XuI 

Q FanI 

Y ZhuangI  II 

Q WangI  II 

Y GaoI 

C WangI 

ICollege of Animal Sciences - Fujian Agriculture and Forestry University, Fuzhou 350002, China.

IIFujian Key Laboratory of Traditional Chinese Veterinary Medicine and Animal Health (Fujian Agriculture and Forestry University)

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.

Keywords: Bacillus coagulans; yellow broilers; growth performance; intestinal mucosa immune function.

INTRODUCTION

Moore firstly reported that the animal weight was significantly 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 firstly 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 flora 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 beneficial bacteria, prebiotics, enzymes, acidulants, and plant extracts (Vahjen et al., 2007; Natsir et al., 2010). Probiotics maintain intestinal flora 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 specific 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 significantly 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 (TGF), 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 inflammatory 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.

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 influenza 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 influenza vaccine One feather One feather One feather eye droppings Intramuscular injection Intramuscular injection

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 formulated 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
Premix1 4.0
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 sulfate) 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.

Growth performance parameters

Chicks were individually weighed on day 1 of the trial, and no significant 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 sacrificed 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 specific 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 sacrificed 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.

Table 3 Primer sequences 

Items Primer sequences Amplified 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-α F: 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
-actin F: 5' -CCAAAGCCAACAGAGAGAAGAT-3' R: 5' -CATCACCAGAGTCCATCACAAT-3' 138

Reaction volumes of 25 μL consisted of 12.5 μL SYBR(r) 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(CT), and were analyzed by SPSS19.0.

Villus height / crypt depth ratio

On day 28, three birds per treatment were sacrificed 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, fixed in 4% paraformaldehyde fixing solution for 24 hours, and were submitted to routine histology procedures, including dehydration, clearing, and embedding in paraffin. 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 magnification using the software HPIAS-5100 (Qianping, Shanghai, China).

Detection of sIgA by immunohistoche-mistry

The sections of duodenum from the chicks sacrificed 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. 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 identified 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 CT.

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 significant (p>0.05). These results showed that the growth performance of chickens fed with 200 mg/kg Bacillus coagulans was significantly enhanced.

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±0.46b 1.96±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±0.44a 1.91±0.02b 97.78±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).

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 confirm that the dietary inclusion of 100 and 200 mg/kg Bacillus coagulans can significantly increase the immune organ index of chickens, particularly at 200 mg/kg.

Table 5 Effects of Bacillus coagulans on the immune organ index (%) of yellow broilers 

Treatments Thymus index Spleen index Bursal index
1 0.4292±0.0435a 0.1623±0.0216a 0.2844±0.0466a
2 0.4600±0.0246b 0.1830±0.0236b 0.3249±0.0345b
3 0.4604±0.0277b 0.1861±0.0264b 0.3250±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 coefficient (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 significantly different among treatments (p>0.05). However, Lactobacillus spp. counts were significantly 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 flora ( 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 significantly 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±8.54a 9.83±0.78a
2 1157.63±35.33 108.93±7.75ab 10.68±0.96ab
3 1160.20±45.00 103.39±9.19b 11.30±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).

A: Primer specificity identification showed that the designed primers can be used for real-time fluorescence 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: Amplification curve of standard sample E: Melting curve of E. coli F: Melting curve of Lactobacillus

Figure 1 Bacillus coagulans optimized the bacterial flora in the yellow feather broiler chickens 

mRNA expression of IFN-a, 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-a is an important immune regulator, and may stimulate the innate immune and antiviral responses. 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 significantly 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 reflects the immune function of the poultry. Our results are in agreement with the findings 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 flora 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 bifidobacteria are the predominant genera (Apajalahti et al., 2004). These are commensal bacteria and can suppress the harmful bacteria and inflammation (Sheil et al., 2007). Escherichia coli is a conditionally pathogenic bacterium, and may become harmful and cause diarrhea when the intestinal microbial flora is imbalanced (Zhang et al., 2010).

Bacillus coagulans is able to produce bacteriocins, such as lactosporin, which have significant 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, bifidobacteria and other beneficial bacteria, maintaining intestinal flora 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 efficient (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 significant 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 reflects 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 significantly 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 inflammatory 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 flora. It allows the establishment of the normal flora 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 significantly 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 Bifidobacterium 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 verified that Roy's lactobacillus DSM 17938 rat NEC has a positive effect on the prevention on rat NEC, which can significantly reduce the mRNA expression of TNF alpha and TLR 4 and TNF alpha, TLR 4 protein levels (Liu et al. , 2012). Rajput verified 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 bifidobacteria, 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)).

REFERENCES

Anderson RC, Cookson AL, McNabb WC, Park Z, Mccann MJ. Lactobacillus plantarum MB452 enhances the function of the intestinal barrier by increasing the expression levels of genes involved in tight junction formation. BMC Microbiology 2010; 10: 316. [ Links ]

Apajalahti J, Kettunen A, Graham H. Characteristics of the gastrointestinal microbial communities, with special reference to the chicken. World Poultry Science Journal 2004;60 (2): 223-232. [ Links ]

Jindal A, Kocherginskaya S, Mehboob A, Robert M,Mackie RI,Zillles LR. Antimicrobial use and resistance in swine waste treatment systems. Applied and Environmental Microbiology 2006;72 (12): 7813-7820. [ Links ]

Artis D. Epithelial-cell recognition of commensal bacteria and maintenance of immune homeostasis in the gut. Nature Reviews Immunology 2008;8 (6): 411-420. [ Links ]

Blikslager AT, Moeser AJ, Gookin JL, Jones SL, Odle J. Restoration of barrier function in injured intestinal mucosa. Physiological Reviews 2007;87(2): 545-564. [ Links ]

Caspary WF. Physiology and pathophysiology of intestinal absorption. American Journal of Clinical Nutrition 1992; (55): 299-308. [ Links ]

Castillo NA, Perdigón G, Leblanc A de M. Oral administration of a probiotic Lactobacillus modulates cytokine production and TLR expression improving the immune response against Salmonella enterica serovar typhimurium infection in mice. BMC Microbiology 2011; 11: 177-189. [ Links ]

Cui YL, Run SC, Wan FC. Bacteriostasis of Bacillus coagulans TBC 169 to enteropathogenic bacteria. Chinese Journal of Microecology 2005; 17 (5): 333-338. [ Links ]

Dai C, Zhao D H, Jiang M. VSL3 probiotics regulate the intestinal epithelial barrier in vivo and in vitro via the p38 and ERK signaling pathways. International Journal of Molecular Medicine 2012; 29 (2): 202-208. [ Links ]

Duerr CU, Hornef MW. The mammalian intestinal epithelium as integral player in the establishment and maintenance of host-microbial homeostasis. Seminars Immunology 2012; 24 (1): 25-35. [ Links ]

Furrie E, Macfarlane S, Kennedy A, Cummings JH, Walsh SV, O'Neil DA. Synbiotic therapy ( Bifidobacterium longum/Synergy 1) initiates resolution of inflammation in patients with active ulcerative colitis: a randomised controlled pilot trial. Gut 2005; 54(2): 242-249. [ Links ]

Gómez-Llorente C, Muñoz S, Gil A. Role of Toll-like receptors in the development of immunotolerance mediated by probiotics. Proceeding of Nutrition Society 2010; 69(3): 381-389. [ Links ]

Giang HH, Viet T Q, Linberg J E, Linberg JE. Groeth performance, digestibility, gut environment and health status in weaned piglets fed a diet supplemented with potentially probiotic complexes of lactic acid bacteria. Livestock Science 2010;129(1-3): 95-103. [ Links ]

Honda H, Gibson GR, Farmer S, Keller D, McCartney AL. Use of a continuous culture fermentation system to investigate the effect of GanedenBC30 (Bacillus coagulans GBI-30, 6086) supplementation on pathogen survival in the human gut microbiota. Anaerobe 2011; 17 (1): 36-42. [ Links ]

Hyronimus B, Le Marrec C, Sassi AH, Deschamps A. Acid and bile tolerance of spore-forming lactic acid bacteria.International Journal of Food Microbiology 2000;61(2-3):193-197. [ Links ]

Isolauri E, Sutas Y, Kankaanpaa P,Arvilommi H,Salminen S. Probiotics: effects on immunity. American Journal of Clinical Nutrition 2001; 72 (2): 444-450. [ Links ]

Jin Er-hui, Chen Yao-xing,Wang Qun,Qiao En-mei,Wu Guo-zhong,Li Sheng-he. Effect of the bacillus probiotics on blood cells anf structure of immune organs in broilers. Acta veterinaria et zootechnica sinica2013;44(5):778-787. [ Links ]

Kailova L, Mount Patrick SK, Arganbright KM, Halpem MD, Kinouchi T,Dvorak B. Bifidobacterium bifidum reduces apoptosis in the intestinal epithelium in necrotizing enterocolitis. American Journal of Physiology Gastrointestinal Liver Physiology 2010; 299(5):1118-1127. [ Links ]

Kawai T, Akira S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nature Immunology 2010;11: 373-384. [ Links ]

Kodali VP, Sen R. Antioxidant and free radical scavenging activities of an exopolysaccharide from a probiotic bacterium. Journal of Biotechnology 2008;3(2): 245-251. [ Links ]

Sahoo KC, Tamhankar AJ, Johansson E, Lundborg CS. Antibiotic use, resistance development and environmental factors: a qualitative study among healthcare professionals in Orissa, India. BMC Public Health 2010; 10(1):1-10. [ Links ]

Lebeer S, Vanderleyden J, De Keersmaecker CJ. Host interactions of probiotic bacterial surface molecules: comparison with commensals and pathogens. Nature Review Microbiology 2010; 8(3): 171-184. [ Links ]

Lei XJ, Piao X , Ru Y, Zhang H, Peron A, Zhang H. Effect of bacillus amyloliquefaciens-based direct-fed microbial on performance, nutrient utilization, intestinal morphology and cacal microflora in broiler chickens. Asian-Australasian Journal of Animal Science 2015;28(2):239-246. [ Links ]

Lin LH, Ke FR, Zhan TT, Xu LH, Wang QX, Wang CK, Huang SW. Effects of Bacillus coagulans on Performance, Serum Biochemical Indices and Antioxidant Function of Yellow Broilers. Chinese Journal of Animal Nutrition. 2014, 26(12): 2806-2813. [ Links ]

Liu Y, Fatheree NY, Mangalat N, Rhoads JM. Lactobacillus reuteri strains reduce incidence and severity of experimental necrotizing enterocolitis via modulation of TLR4 and NF- (B signaling in the intestine. American Journal of Physiology Gastrointestinal Liver Physiology 2012; 302(6): 608-617. [ Links ]

Ma K, Maeda T, You H, Shirai Y. Open fermentative production of L-lactic acid with high optical purity by thermophilic Bacillus coagulans using excess sludge as nutrient. Bioresource Technology 2014;151: 28-35. [ Links ]

Medici M, Vinderola C, Perdigon G. Gut mucosal immunomodulation by probiotic fresh chess. International Dairy Journal 2004,14(7):611-618. [ Links ]

Miriam BB, Julio PD, Sergio MQ, Carolina G L, Angel G. Probiotic Mechanisms of Action. Annal of Nutrition Metabolism 2012;61:160-174. [ Links ]

Moore PR, Evension A, Luckey TD, Mccoy E, Elvehjem CA, Hart EB. Use of sulfasuxidine, streptothricin and streptomycin in nutritional studies with the chick.The Journal of Biological Chemistry 1946;165: 437-441. [ Links ]

Natsir MH, Sjofjan O, Umam K, Manab A, Widodo E. Effects of liquid and encapsulated lactic acid in broiler diets on performances, intestinal characteristics and intestinal microflora.The Journal of Poultry Science 2010; 47: 240-243. [ Links ]

Pan X, Qiang Z, Ben W, Chen M. Residual veterinary antibiotics in swine manure from concentrated animal feeding operations in Shandong Province, China. Chemosphere 2011; 84 (5): 695-700. [ Links ]

Rajput IR, Li LY, Xin X, Wu BB, Juan ZL, Cui DY, Li WF. Effect of Saccharomyces boulardii and Bacillus subtills B10 on intestinal ultrastructure modulation and mucosal immunity development mechanism in broiler chickens. Poultry Science 2013;92 (4): 956-965. [ Links ]

Rajput IR, Hussain A, Li YL, Zhang X, Xu X, Long MY, You DY, Li WF. Saccharomyces boulardii and Bacillus subtilis B10 modulate TLRs mediated signaling to induce immunity by chicken BMDCs. Journal of Cell Biochemistry 2014; 115 (1): 189-198. [ Links ]

Riazi S, Wirawan RE, Badmaev V, Chikindas ML. Characterization of lactosporin, a novel antimicrobial protein produced by Bacillus coagulans ATCC 7050. Journal of Applied Microbiology 2009; 106(4): 1370-1377. [ Links ]

Ripamonti B, Agazzi A, Baldi A, Balzaretti C, Bersani C, Pirani S, Rebucci R, Savoini G, Stilla S, Stenico A, Domeneghini C. Administration of Bacillus coagulans in calves:recovery from faecal samples and evaluation of functional aspects of spores. Veterninary Research Communication 2009; 33(8):991-1001. [ Links ]

Durbin RK, Kotenko SV, Durbin JE.Interferon induction and function at the mucosal surface. Immunological reviews 2013;255(1): 25-39. [ Links ]

Sheil B, Shanahan F, Omahony L. Probiotic effects on inflammatory bowel disease. The Journal of Nutrition 2007; 137 (3): 819S-824S. [ Links ]

Smither R, Lott AF, Dalziel RW, Ostler DC. Antibiotic residues in meat in the United Kingdom; an assessment of specific tests to detect and identify antibiotic residues. Journal of Hygiene, Camb 1980; 85(3):359- 359. [ Links ]

Vahjen W, Osswald T, Schäfer K, Simon O. Comparison of a xylanase and a complex of non starch polysaccharide-degrading enzymes with regard to performance and bacterial metabolism in weaned piglets. Archives of Animal Nutrition 2007; 61(2):90-102. [ Links ]

Wang N, Wang G, Hao JX, Ma JJ, Wang Y, Jiang XY, Jiang HQ.Curcumin ameliorates hydrogen peroxide-induced epithelial barrier disruption by upregulating heme oxygenase-1 expression in human intestinal epithelial cells.Digestive Diseases and Science 2012;57(7):1792-1801. [ Links ]

Wells JM. Immunomodulatory mechanisms of lactabacilli. Microbial Cell Factories 2011; 10 (suppl 1): S17. [ Links ]

Wells JM, Rossi O, Meijerink M, Baarlen P. Epithelial crosstalk at the microbiota-mucosal interface. Proceedings of the National Academy Sciences 2011;108 (1): 4607-4614. [ Links ]

Zhang L, Xu Y Q, Liu H Y, Lai T,Ma JJ, Wang JF,Zhu YH.Evaluation of Lactobacillus rhamnosus GG using an Escherichia coli K88 model of piglet diarrhoea: Effects on diarrhoea incidence, faecal microflora and immune responses. Vet Microbiol 2010; 141 (1-2): 141-148. [ Links ]

Zheng Z, Ma C, Gao C,Li F,Qin J, Zhang H,Wang K,Xu P. Efficient conversion of phenylpyruvic acid to phenyllactic acid by using whole cells of Bacillus coagulans SDM. PLoS One 2011;6 (4): e19030. [ Links ]

Received: August 2016; Accepted: November 2016

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

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