INTRODUCTION Pathogenic Escherichia coli causes a variety of dis-

eases known as colibacillosis, which causes enteric dis-ease and increases mortality rate in chickens (He et al., 2011; oh et al., 2012), and results in economic losses in the poultry industry (Kumar et al., 2004; Alonso et al., 2011). Escherichia coli K88 causes damage in the intestine of animals and produces lipopolysaccharide. Immunological stress emanating from lipopolysaccha-ride affects the physiological and pathological processes of poultry and interferes with their normal functioning (Huff et al., 2008; Shini et al., 2008; Yang et al., 2008;

Munyaka et al., 2012). Antibiotics are usually used to treat or control colibacillosis; however, the emergence of antibiotic-resistant bacteria has reduced the effec-tiveness of antibiotics and may also cause problems to human health (Asai et al., 2011; Belanger et al., 2011; Dheilly et al., 2011; Kobayashi et al., 2011; Dheilly et al., 2012). The development of antibiotic resistance has aroused a worldwide concern about limiting the usage of antibiotics in animal agriculture.

Clostridium butyricum is a butyric acid-producing, spore-forming, gram-positive anaerobe, which is found in soil and intestines of healthy animals and humans (Pan et al., 2008a; Yang et al., 2010). It is able to survive in media of low pH and relatively high bile con-centrations, and it produces endospores. These prop-erties of C. butyricum make it suitable as a probiotic supplement in animal feed (Sato and Tanaka, 1996; He et al., 2004; Kong et al., 2011). Dietary supplementa-

Effects of Clostridium butyricum on growth performance, immune function, and cecal microflora in broiler chickens challenged with Escherichia coli K88

L. Zhang ,*† G. T. Cao ,† X. F. Zeng ,‡ L. Zhou ,† P. R. Ferket ,§ Y. P. Xiao ,† A. G. Chen ,† and C. M. Yang *†1

* College of Animal Science and Technology, Zhejiang A & F University, Lin’an 311300, China; † College of Animal Sciences, Zhejiang University, Hangzhou 310058, China;

‡ Zhejiang Huijia Biological Technology Ltd., Anji 313307, China; and § Department of Poultry Science, North Carolina State University, Raleigh 27695

ABSTRACT This study was conducted to investigate the effects of Clostridium butyricum on growth perfor-mance, immune function, and cecal microflora in broil-er chickens challenged with Escherichia coli K88. Three hundred sixty 1-d-old broiler chickens were randomly divided into 4 treatments: negative control (NC) birds were fed a basal diet and not challenged with E. coliK88; positive control (PC) birds were fed a basal diet and challenged with E. coli K88; C. butyricum treat-ment (CB) birds were fed a diet containing 2 × 107

cfu C. butyricum/kg of diet and challenged with E. coliK88; and colistin sulfate treatment (CS) birds were fed a diet containing 20 mg of colistin sulfate/kg of diet and challenged with E. coli K88. Birds fed CB had greater (P < 0.05) BW than the PC birds from 3 to 21 d postchallenge. Birds fed CB had greater (P < 0.05) serum IgA and IgY at 14 d postchallenge, greater (P< 0.05) serum IgM at 21 d postchallenge, and greater

(P < 0.05) mucosal secreted IgA at 3 and 7 d postchal-lenge than the PC birds. Birds fed CB had greater con-centrations of serum complement component 3 at 14 d postchallenge, and greater (P < 0.05) concentrations of serum complement component 4 at 3, 7, and 14 d postchallenge than the PC birds. Birds in the CS or CB treatments had less cecal E. coli population at 3, 7, and 21 d postchallenge, and less cecal Clostridium perfrin-gens counts at 21 d postchallenge compared with the PC birds. The CB treatment increased (P < 0.05) the population of cecal Lactobacillus at 3 d postchallenge and the number of cecal Bifidobacterium at 3, 14, and 21 d postchallenge in comparison with the PC treat-ment. The results indicate that dietary supplementa-tion of CB promotes growth performance, improves immune function, and benefits the cecal microflora in Escherichia coli K88-challenged chickens.

Key words: Clostridium butyricum , growth performance , immune function , cecal microflora , Escherichia coli K88

2014 Poultry Science 93 :46–53http://dx.doi.org/ 10.3382/ps.2013-03412

Received June 13, 2013. Accepted September 23, 2013. 1 Corresponding author: yangcaimei2012@163.com

© 2014 Poultry Science Association Inc.

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tion of C. butyricum has been demonstrated by several researchers to promote growth performance (Han et al., 1984; Song et al., 2006; Mountzouris et al., 2010), im-prove immune function (Chen et al., 1993; Murayama et al., 1995; Wang et al., 1996; Song et al., 2006; Zhang et al., 2009; Cao et al., 2012; Yang et al., 2012), and benefit the balance of the intestinal flora (Seki et al., 2003; Takahashi et al., 2004; Imase et al., 2008; Li et al., 2009; Zhang et al., 2009; Kong et al., 2011). To our knowledge, however, the effects of C. butyricum on infected animals have not been reported. The present experiment was conducted to investigate the effects of C. butyricum on growth performance, immune function, and cecal microflora in broiler chickens challenged with Escherichia coli K88.

MATERIALS AND METHODSAll the procedures were approved by the Institutional

Animal Care and use Committee of Zhejiang univer-sity.

Birds, Diets, and Experimental DesignThree hundred sixty 1-d-old male Cobb broiler chick-

ens were obtained from a local commercial hatchery (Charoen Pokphand Group, Haining, China). on d 1, all birds were randomly assigned to 4 treatments, with 15 birds per cage and 6 replication cages per treatment. All birds were raised in wired cages. The treatments were as follows: negative control (NC) birds fed a basal diet and not challenged with E. coli K88; positive con-trol (PC) birds fed a basal diet and orally challenged with 0.5 mL of E. coli K88 (2 × 108 cfu/mL) on d 7; C. butyricum treatment (CB) birds fed a diet supplement-ed with 2 × 107 cfu C. butyricum/kg of feed and orally challenged with 0.5 mL of E. coli K88 (2 × 108 cfu/mL) on d 7; and colistin sulfate treatment (CS) birds fed a diet supplemented with 20 mg of colistin sulfate/kg of feed and orally challenged with 0.5 mL of E. coli K88 (2 × 108 cfu/mL) on d 7. The birds in NC treatment were placed in one room, and the birds in other 3 treatments were placed in another room to prevent cross-contami-nation. The 2 rooms used in this study were of identical configuration, and previous growth studies revealed no significant room effects.

All birds were offered the same antibiotic-free basal diets. The nutrient levels of the diets met or exceeded the NRC (1994) recommendations. Experimental diets and water were available ad libitum. The temperature were adjusted to 32°C in the first week and gradually lowered to 25°C.

The C. butyricum strain (HJCB998) was provided by Zhejiang Huijia Biological Technology Ltd., Anji, China. It was grown anaerobically in a liquid fermen-tation tank at 37°C for 48 h. After centrifugation, the cells were harvested and dried by spray drying technol-ogy. Then the powder of C. butyricum was added to the

basal diet. Colistin sulfate was obtained from Zhejiang Qianjiang Biochemical Ltd., Haining, China.

Oral ChallengeThe E. coli K88 strain was originally obtained from

College of Animal Sciences, Zhejiang university (Hang-zhou, China) and was grown at 37°C. The birds were initially fed different experimental diet for the first 6 d. on d 7, birds in PC, CB, and CS treatment groups were orally challenged into the crop with 0.5 mL (2 × 108 cfu/mL) of the freshly grown E. coli K88 inoculants by using a polyethylene tube attached to a syringe. The NC treatment was administrated similarly with the same amount of saline solution.

Growth PerformanceBirds were weighed individually at 3, 7, 14, and 21

d postchallenge to evaluate growth performance. Feed consumption and feed-to-gain ratio could not be de-termined because of an indeterminate amount of feed wastage.

Sample CollectionAt 3, 7, 14, and 21 d postchallenge, 6 birds per treat-

ment were randomly selected, and blood samples were taken from the wing vein. The blood samples were al-lowed to clot for 20 min at 4°C. After centrifugation (3,000 × g, 10 min) at 4°C, the serum was harvested and stored at −20°C. The birds were then killed by Co2 inhalation and jejunum and cecum samples were collected. The jejunum section was gently flushed with PBS, and the mucosa was scraped from the jejunum with a sterile blade and stored in a 1.5-mL sterile mi-crocentrifuge tube at −20°C. The posterior end of both ceca were ligated with surgical thread and then cut to aseptically remove the ceca and put in a sterilized bag-gie and stored on ice for subsequent enumeration of the microbial population.

Complement Components and Immunoglobulins

The concentrations of serum lysozyme, complement component 3 (C3), and complement component 4 (C4) were measured using the chicken-specific ELISA kits (Jiancheng Biological Engineering Institute, Nanjing, China). The concentrations of IgA, IgM, IgY in serum and secreted IgA (sIgA) in jejunal mucosa were de-termined through microtiter plates and chicken-specif-ic IgA, IgM, IgY, and sIgA ELISA quantitation kits (uscn Life Science Inc., Wuhan, China). The ELISA procedures were done as described by the manufactur-er’s protocol. All ELISA kits have high sensitivity and specificity for chickens. No significant cross-reactivity was observed.

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Cecal MicrobiotaThe cecal contents (0.5 g) were diluted with 4.5 mL of

PBS in a flask containing glass beads and then diluted 10-fold from 10−2 to 10−9. Cecal contents were plated on MacConkey’s agar at 37°C for 24 h to enumerate E. coli, and plated on tryptose-sulfite-d-cycoserine agar in an anaerobic incubator at 37°C for 24 h to enumerate Clostridium perfringens. Lactobacillus and Bifidobacte-rium colonies were identified by plating cecal contents on Lactobacillus select agar, and Beerens agar, respec-tively, incubating in an anaerobic incubator at 37°C for 48 h. Results were reported as log10 cfu per gram of ce-cal digesta. All agars were obtained from Land Bridge Technology, Beijing, China.

Statistical AnalysisAll data was analyzed using SPSS16.0 (SPSS Inc.,

Chicago, IL) for one-way ANoVA analysis. Differences among means of treatments were compared using least significant difference. An α value of 0.05 was used as the criterion for statistical significance.

RESULTS

Growth PerformanceBirds in the PC treatment group had less (P < 0.05)

overall BW than the NC birds (Table 1). Birds fed CB diet had greater (P < 0.05) overall BW than those fed the PC diet. In comparison with the PC, dietary supplementation of CS increased (P < 0.05) BW from 7 to 21 d postchallenge. No significant difference in BW was observed between the birds fed CB and CS diet.

Serum ImmunoglobulinBirds challenged with E. coli K88 had greater (P

< 0.05) serum IgA concentrations than unchallenged birds at 3 d postchallenge (Table 2). Birds fed CB had greater (P < 0.05) IgA than either NC or PC birds at 14 d postchallenge. There were no significant differenc-es in IgA among the 4 treatments at 7 and 21 d postch-allenge. Birds fed CB or CS had greater (P < 0.05) IgY

than NC birds at 3 d postchallenge. In comparison with the NC or PC treatments, dietary supplementation of CB increased (P < 0.05) serum IgY at 14 d postchal-lenge. No significant differences were observed in serum IgY among the 4 treatments at 7 and 21 d postchal-lenge. In comparison with NC, dietary supplementation of CB increased (P < 0.05) serum IgM at 3, 14, and 21 d postchallenge, and CS increased (P < 0.05) serum IgM at 7 and 21 d postchallenge. Broilers fed CB had greater (P < 0.05) jejunum mucosal sIgA concentra-tions than the other treatment groups at 3 d postchal-lenge, and CB birds had greater (P < 0.05) mucosal sIgA than the PC birds at 3 and 7 d postchallenge.

Complement Components and LysozymeThe concentrations of serum C3 in the birds fed C.

butyricum were greater (P < 0.05) than those in the unchallenged birds from 3 to 14 d postchallenge (Table 3). Broilers fed CB had greater (P < 0.05) serum C3 than the PC broilers at 14 d postchallenge. In compari-son with the NC or PC birds, these CB birds also had greater (P < 0.05) serum C4 at 3, 7, and 14 d postchal-lenge. Birds challenged with E. coli K88 had greater (P < 0.05) lysozyme than unchallenged birds at 7 and 14 d postchallenge. Dietary treatments did not affect serum lysozyme at 3, 7, and 14 d postchallenge.

Cecal Microflora PopulationBirds in PC treatment group had a greater (P < 0.05)

number of E. coli in cecal contents than the NC birds from 3 to 21 d postchallenge (Figure 1). Birds fed CB or CS had a lower number of cecal E. coli than the PC birds at 3, 7, and 21 d postchallenge. Broilers in the CS treatment group had a lower number of C. perfringens than those in the other treatment groups at 3 d postch-allenge (Figure 2). Birds in CS or CB treatment groups had lower C. perfringens counts than the PC birds at 21 d postchallenge. Birds fed CB had greater (P < 0.05) Lactobacillus population in cecal contents at 3 d post-challenge than the PC birds (Figure 3). There were no differences in the number of Lactobacillus among the 4 treatment groups at 7 and 21 d postchallenge. Birds in the PC treatment group had a lower (P < 0.05) number

Table 1. Effects of Clostridium butyricum on growth performance in broiler1

ItemDays

postchallenge2

Experimental treatment

SEM P-valueNC PC CB CS

BW (g) 3 342.7a 309.0c 332.0ab 319.9bc 3.6 0.0047 398.5a 346.7c 369.9b 382.6ab 4.6 <0.001

14 724.5a 621.0c 689.0ab 711.9ab 9.3 <0.00121 1,297.2a 1,086.9b 1,202.9a 1,274.8a 20.9 <0.001

a–cMeans in the same row with different superscript letters differ significantly (P < 0.05).1Each mean represents 6 birds. NC = birds fed a basal diet and not challenged with E. coli K88. PC = birds fed a basal diet and challenged with

E. coli K88. CB = birds fed 2 × 107 cfu C. butyricum/kg of diet and challenged with E. coli K88. CS = birds fed 20 mg of colistin sulfate/kg of diet and challenged with E. coli K88.

2The days after challenge with E. coli K88.

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of Bifidobacterium in cecal contents at 3, 7, and 14 d postchallenge than the unchallenged birds (Figure 4). In comparison with PC, the CB treatment increased (P < 0.05) the cecal Bifidobacterium counts at 3, 14, and 21 d postchallenge.

DISCUSSIONProbiotics have been used as a substitute for anti-

biotics in animal feed for many years. They have been shown to promote growth performance and improve nutrient utilization efficiency in chickens (Han et al., 1984; Grimes et al., 2008; Mountzouris et al., 2010; Nakanishi and Tanaka, 2010), although some studies

reported probiotics have little to no effect on growth performance (Biernasiak and Slizewska, 2009; Lee et al., 2010; Rahimi et al., 2011). In contrast, several stud-ies demonstrated that dietary supplementation of the probiotic, C. butyricum, improves growth performance in broiler chickens (Han et al., 1984; Mountzouris et al., 2010; Cao et al., 2012; Yang et al., 2012). Moreover, results of the study reported herein demonstrated that dietary supplementation of C. butyricum was effective in alleviating the growth suppression caused by E. coli challenge in broilers. Clostridium butyricum and colistin sulfate had equally effective prophylactic effects among the E. coli-challenged broilers, as there were no sig-nificant differences in BW between these 2 treatment

Table 2. Effects of Clostridium butyricum on inmunoglobulins in broilers1

ItemDays

postchallenge2

Experimental treatment

SEM P-valueNC PC CB CS

IgA3 (μg/mL) 3 98.59b 133.75a 144.18a 132.14a 10.62 0.027 191.24 201.37 222.06 226.09 17.63 0.4

14 245.09c 265.04bc 331.88a 308.88ab 16.93 0.0121 281.43 304.77 319.36 284.38 14.29 0.49

IgY3 (μg/mL) 3 183.39b 222.25ab 224.18a 236.26a 13.55 0.057 301.37 291.24 305.4 326.09 14.62 0.54

14 345.09b 365.04b 431.01a 398.88ab 16.9 0.0221 381.43 394.77 419.36 384.38 15.58 0.35

IgM3 (μg/mL) 3 71.70b 77.76ab 87.85a 84.98ab 4.23 0.017 89.08b 97.20ab 103.82ab 108.26a 4.99 0.02

14 134.86b 136.56ab 161.89a 145.58ab 8.26 <0.0121 96.06c 116.89b 143.64a 116.95b 6.85 <0.01

sIgA4 (ng/mL) 3 1.75b 1.69b 3.30a 2.27b 0.25 <0.017 1.89ab 1.75b 2.55a 1.89ab 0.2 0.03

14 2.76a 1.88b 2.65ab 2.61ab 0.26 0.0521 2.3 2.12 2.53 2.63 0.37 0.74

a–cMeans in the same row with different superscript letters differ significantly (P < 0.05).1Each mean represents 6 birds. NC = birds fed a basal diet and not challenged with E. coli K88. PC = birds fed a basal diet and challenged with

E. coli K88. CB = birds fed 2 × 107 cfu C. butyricum/kg of diet and challenged with E. coli K88. CS = birds fed 20 mg of colistin sulfate/kg of diet and challenged with E. coli K88.

2The days after challenge with E. coli K88.3The concentration in serum.4The concentration in jejunal mucosa.

Table 3. Effects of Clostridium butyricum on serum complement components and lysozyme in broilers1

ItemDays

postchallenge2

Experimental treatment

SEM P-valueNC PC CB CS

C3 (mg/mL) 3 0.067b 0.079ab 0.092a 0.075b 0.004 0.047 0.082b 0.091ab 0.105a 0.109a 0.006 0.01

14 0.243b 0.219b 0.298a 0.245b 0.016 0.0221 0.213 0.208 0.258 0.233 0.014 0.09

C4 (mg/mL) 3 0.033b 0.034b 0.045a 0.037b 0.003 0.017 0.048b 0.049b 0.055a 0.052ab 0.002 0.02

14 0.065c 0.085b 0.104a 0.080b 0.007 <0.0121 0.117 0.129 0.134 0.118 0.009 0.07

Lysozyme (μg/mL) 3 1.292b 1.514ab 1.819a 1.658ab 0.138 0.027 1.263b 1.952a 2.035a 1.895a 0.071 <0.01

14 1.469b 1.882a 1.947a 1.920a 0.119 <0.0121 2.462b 2.026b 2.478b 3.234a 0.148 <0.01

a–cMeans in the same row with different superscript letters differ significantly (P < 0.05).1Each mean represents 6 birds. NC = birds fed a basal diet and not challenged with E. coli K88. PC = birds fed a basal diet and challenged with

E. coli K88. CB = birds fed 2 × 107 cfu C. butyricum/kg of diet and challenged with E. coli K88. CS = birds fed 20 mg of colistin sulfate/kg of diet and challenged with E. coli K88. C3 = complement 3; C4 = complement 4.

2The days after challenge with E. coli K88.

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Figure 1. Effects of Clostridium butyricum on cecal Escherichia coli population in broilers on the days after challenge with E. coli K88. Bars represent means for the 6 birds per treatment ± SEM. Within the same day, bars with different letters (a–c) differ significantly (P < 0.05). Bac-terial number is expressed as log10 cfu per gram of wet digesta. Each mean represents 6 birds. NC = birds fed a basal diet and not challenged with E. coli K88. PC = birds fed a basal diet and challenged with E. coli K88. CB = birds fed 2 × 107 cfu Clostridium butyricum/kg of diet and challenged with E. coli K88. CS = birds fed 20 mg of colistin sulfate/kg of diet and challenged with E. coli K88.

Figure 2. Effects of Clostridium butyricum on cecal Clostridium perfringens population in broilers on the days after challenge with Escherichia coli K88. Bars represent means for the 6 birds per treatment ± SEM. Within the same day, bars with different letters (a,b) differ significantly (P < 0.05). Bacterial number is expressed as log10 cfu per gram of wet digesta. Each mean represents 6 birds. NC = birds fed a basal diet and not challenged with E. coli K88. PC = birds fed a basal diet and challenged with E. coli K88. CB = birds fed 2 × 107 cfu C. butyricum/kg of diet and challenged with E. coli K88. CS = birds fed 20 mg of colistin sulfate/kg of diet and challenged with E. coli K88.

Figure 3. Effects of Clostridium butyricum on cecal Lactobacillus population in broilers on the days after challenge with Escherichia coli K88. Bars represent means for the 6 birds per treatment ± SEM. Within the same day, bars with different letters (a,b) differ significantly (P < 0.05). Bacterial number is expressed as log10 cfu per gram of wet digesta. Each mean represents 6 birds. NC = birds fed a basal diet and not challenged with E. coli K88. PC = birds fed a basal diet and challenged with E. coli K88. CB = birds fed 2 × 107 cfu C. butyricum/kg of diet and challenged with E. coli K88. CS = birds fed 20 mg of colistin sulfate/kg of diet and challenged with E. coli K88.

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groups. Apparently, C. butyricum improved BW of the E. coli K88-challenged birds by decreasing the cecal E. coli population via competitive exclusion. other re-searchers have shown that dietary supplementation of probiotics decreased the number of pathogenic E. coli (Bhandari et al., 2010; Giang et al., 2011; Roodposhti and Dabiri, 2012). our previous research also demon-strated that C. butyricum decreased the number of cecal E. coli, which contributed to the growth performance in unchallenged broiler chickens (Yang et al., 2012).

Complement components and immunoglobulin are usually used to evaluate the immune status of animals due to their important roles in immune function. Di-etary supplementation of probiotics may modulate mu-cosal and systemic immune activity and epithelial func-tion, including mucosal T cells, B cells, epithelial cells, dendritic cells, macrophage, nature killer cells, antibod-ies, and cytokines (Fedorak and Madsen, 2004). Dietary supplementation of Saccharomyces cerevisiae fermenta-tion product significantly increased sIgA content in the cecal tonsil (Gao et al., 2009). Dietary supplementation of C. butyricum enhanced IgM antibody formation and the activity of macrophage and NK cells in mice (Wang et al., 1996). Clostridium butyricum supplementation also increased the serum lysozyme activity and IgM in Miichthys miiuy (Song et al., 2006). Phagocytic activi-ties of the head kidney macrophages, the lysozyme ac-tivities of serum and gut mucosa, and immunoglobulin in Chinese drum, Miichthys miiuy, were increased after oral administration of live or dead cells of C. butyrium (Pan et al., 2008a). In the experiment reported herein, the birds fed C. butyrium had greater serum IgA and IgY at 14 d postchallenge, greater IgM at 21 d postch-allenge, and greater mucosal sIgA at 3 and 7 d post-challenge than the E. coli K88-challenged birds fed the basal diet. These results were similar to those reported by Murayama et al. (1995), who demonstrated C. bu-tyricum MIYAIRI588 increased the production of IgA, IgM, and IgY in mouse Peyer’s patch cell culture. our

previous results also showed that C. butyrium improved serum IgA, IgY, and IgM in broiler chickens (Yang et al., 2012).

Probiotics have been shown to benefit immune func-tion in a variety of animal models. Dietary supplement of the fermentation product of Saccharomyces cerevisiae has been shown to increase serum lysozyme in broiler chickens challenged with Eimeria tenella (Gao et al., 2009). During the whole feeding period of grouper (Epi-nephelus coioides), supplementation of Psychrobacter sp. increased serum C3 and C4 (Sun et al., 2011). Yang et al. (2012) showed that dietary supplementation of C. butyricum increased serum C3 in broiler chickens. However, not until the current study has the effect of C. butyricum on the immune function of E. coli-challenged birds been reported. In the current study, birds fed C. butyrium had greater C3 at 14 d postchallenge, and greater C4 from 3 to 14 d postchallenge than the chal-lenged birds fed the basal diet. Apparently, C. butyrium may promote the immune function of enteric disease-challenged birds, which may contribute to improved growth performance.

The intestine harbors a complex microbial commu-nity that plays a key role in nutrition and health (Kong et al., 2011). It is well known that probiotics and their metabolites are able to promote a symbiotic balance of microorganisms in the gut of the host (Seki et al., 2003; Imase et al., 2008; Zhang et al., 2009; Mountzouris et al., 2010; Kong et al., 2011; Rahimi et al., 2011). In-hibition of pathogen replication has been shown to be mediated by low-molecular-weight substances, primar-ily short-chain fatty acids (SCFA; oelschlaeger, 2010). These SCFA exert therapeutic effects on some human and experimental animal diseases. Zhang et al. (2011) showed that dietary supplementation of C. butyricum increased the concentrations of acetic acid, n-butyric acid, n-valeric acid, and total SCFA in cecal digesta of broiler chickens. Clostridium butyricum produces high levels of SCFA in the gut lumen and competes with the

Figure 4. Effects of Clostridium butyricum on cecal Bifidobacterium population in broilers on the days after challenge with E. coli K88. Bars represent means for the 6 birds per treatment ± SEM. Within the same day, bars with different letters (a,b) differ significantly (P < 0.05). Bacte-rial number is expressed as log10 cfu per gram of wet digesta. Each mean represents 6 birds. NC = birds fed a basal diet and not challenged with E. coli K88. PC = birds fed a basal diet and challenged with E. coli K88. CB = birds fed 2 × 107 cfu C. butyricum/kg of diet and challenged with E. coli K88. CS = birds fed 20 mg of colistin sulfate/kg of diet and challenged with E. coli K88.

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pathogens for limiting resources (Araki et al., 2002). For example, the growth of enterohemorrhagic E. coli o157:H7 and the production of Shiga-like toxins in broth cultures are inhibited by co-incubation with C. butyricum (Takahashi et al., 2004). Imase et al. (2008) reported that C. butyricum reduced the production of C. difficile toxin A, likely by inhibiting the growth of this pathogen. Pan et al. (2008b) showed that C. bu-tyricum had strong adhesion property and a higher an-tagonistic activity to Aeromonas hydrophila and Vibrio anguillarum, both on agar plate and cell model. Howev-er, little has been published on the effects of probiotics on cecal microflora of enteric disease-challenged birds. Grimes et al. (2008) reported that direct-fed microbial reduced the Salmonella population in Salmonella-chal-lenged birds. Li et al. (2009) demonstrated that probi-otics increased the numbers of Lactobacilli and Bacillus cereus and reduced the numbers of E. coli in chicks. In the present experiment, the C. butyricum increased the population of Bifidobacterium and Lactobacillus, and reduced the population of E. coli in E. coli-challenged birds. These results indicate that C. butyrium may pro-mote a more symbiotic intestinal microflora favoring the host when challenged with an enteric pathogen such as E. coli.

In conclusion, the results of the present experiment confirm that enteric disease challenge with E. coli K88 decreases growth performance of broiler chickens, but dietary supplementation of C. butyrium can serve as an effective prophylactic treatment to alleviate this growth suppression. Dietary supplementation of C. bu-tyrium improves growth performance, promotes immu-nity function, and benefits the cecal microflora in E. coli K88-challenged birds.

ACKNOWLEDGMENTSThis work was partially supported by the National

Development and Reform Commission of China (proj-ect no. 2011-1158).

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