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

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 differentiationassociated 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.


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 infeed 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

Bacillus Coagulans Enhance the Immune Function of the Intestinal Mucosa of Yellow Broilers
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 (TGFβ), and sIgA (Rajput et al.,2013).
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.

Birds and management
A total of 360 one-day-old healthy Lingnan yellowfeathered 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.

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.

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.

Standard curve preparation
Plate count: Escherichia coli strain k88 was used as reference strain.It was cultured in lysogeny broth (LB) at 37 o C for 7 h, and then was serially diluted to 10 -9 .Dilutions were then cultured in Macconkey medium at 37 o C 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.

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 semiserial 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 immunohistochemistry
The sections of duodenum from the chicks sacrificed for organ index were prepared and inhibited by preincubating the tissues in 3% H 2 O 2 , incubated in 5% normal goat serum for 0.5h followed by an overnight incubation at 4 o C with 1:40 dilution of rabbit anti-chicken IgA serum.

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 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 rrCT.

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.

Cecal bacterial flora
Plate count results showed that the number of Escherichia coli strain k88 in the cecum was 1.6×10 10 cfu/mL.The regression coefficient (R 2 ) 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).

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).

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).

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 significantly higher in group 3 compared with group 1 (p<0.05or p<0.01) (Fig. 3), indicating that Bacilllus coagulans may stimulate the innate immune function of the intestinal tract.

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 1x10 11 cfu/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

Bacillus Coagulans Enhance the Immune Function of the Intestinal Mucosa of Yellow Broilers
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 IgApositive 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.
(10 μM), and 10 μL RNase free ddH 2 O. PCR conditions were initial denaturation at 95 o C for 0.5 min followed by 40 cycles at 95 o C for 5 s, 60 o C for 34 s, and 95 o C for 15 s, 60 o C for 1 min, 95 o C for 15 s.Data was calculated by delta delta CT(rrCT), and were analyzed by SPSS19.0.

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

Table 2 -
Composition and nutrient level of basal diet (air dry basis) %

Table 1 -
The immune program

Table 4 -
Effects of Bacillus coagulans on the growth performance in yellow broilers Means followed by different superscripts in the same column are statistically different (p<0.05).

Table 5 -
Effects of Bacillus coagulans on the immune organ index (%) of yellow broilers
ab Means followed by different superscripts in the same column are statistically different (p<0.05)

Table 7 -
Effects of Bacillus coagulans on the villus height to crypt depth ratio (V/C value) of duodenum in yellow broilers.