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

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1 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 , China; College of Animal Sciences, Zhejiang University, Hangzhou , China; Zhejiang Huijia Biological Technology Ltd., Anji , China; and Department of Poultry Science, North Carolina State University, Raleigh ABSTRACT This study was conducted to investigate the effects of Clostridium butyricum on growth performance, immune function, and cecal microflora in broiler 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. coli K88; positive control (PC) birds were fed a basal diet and challenged with E. coli K88; C. butyricum treatment (CB) birds were fed a diet containing cfu C. butyricum/kg of diet and challenged with E. coli K88; 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 INTRODUCTION Pathogenic Escherichia coli causes a variety of diseases known as colibacillosis, which causes enteric disease 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 lipopolysaccharide 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; (P < 0.05) mucosal secreted IgA at 3 and 7 d postchallenge than the PC birds. Birds fed CB had greater concentrations 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 perfringens 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 treatment. The results indicate that dietary supplementation 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 K Poultry Science 93 : /ps Poultry Science Association Inc. Received June 13, Accepted September 23, Corresponding author: yangcaimei2012@163.com Munyaka et al., 2012). Antibiotics are usually used to treat or control colibacillosis; however, the emergence of antibiotic-resistant bacteria has reduced the effectiveness 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 concentrations, and it produces endospores. These properties 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- 46

2 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), improve 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. CLOSTRIDIUM BUTYRICUM IN CHALLENGED BROILERS basal diet. Colistin sulfate was obtained from Zhejiang Qianjiang Biochemical Ltd., Haining, China. Oral Challenge The E. coli K88 strain was originally obtained from College of Animal Sciences, Zhejiang University (Hangzhou, 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 ( 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. 47 MATERIALS AND METHODS All the procedures were approved by the Institutional Animal Care and Use Committee of Zhejiang University. Birds, Diets, and Experimental Design Three hundred sixty 1-d-old male Cobb broiler chickens 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 control (PC) birds fed a basal diet and orally challenged with 0.5 ml of E. coli K88 ( cfu/ml) on d 7; C. butyricum treatment (CB) birds fed a diet supplemented with cfu C. butyricum/kg of feed and orally challenged with 0.5 ml of E. coli K88 ( 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 ( 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-contamination. 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 fermentation tank at 37 C for 48 h. After centrifugation, the cells were harvested and dried by spray drying technology. Then the powder of C. butyricum was added to the Growth Performance Birds 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 determined because of an indeterminate amount of feed wastage. Sample Collection At 3, 7, 14, and 21 d postchallenge, 6 birds per treatment were randomly selected, and blood samples were taken from the wing vein. The blood samples were allowed 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 CO 2 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 microcentrifuge 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 baggie 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 determined through microtiter plates and chicken-specific IgA, IgM, IgY, and siga ELISA quantitation kits (Uscn Life Science Inc., Wuhan, China). The ELISA procedures were done as described by the manufacturer s protocol. All ELISA kits have high sensitivity and specificity for chickens. No significant cross-reactivity was observed.

3 48 Zhang et al. Cecal Microbiota The 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 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 Bifidobacterium colonies were identified by plating cecal contents on Lactobacillus select agar, and Beerens agar, respectively, incubating in an anaerobic incubator at 37 C for 48 h. Results were reported as log 10 cfu per gram of cecal digesta. All agars were obtained from Land Bridge Technology, Beijing, China. Statistical Analysis All 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. Growth Performance RESULTS Birds 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 Immunoglobulin Birds 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 differences in IgA among the 4 treatments at 7 and 21 d postchallenge. 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 postchallenge. No significant differences were observed in serum IgY among the 4 treatments at 7 and 21 d postchallenge. 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 concentrations than the other treatment groups at 3 d postchallenge, and CB birds had greater (P < 0.05) mucosal siga than the PC birds at 3 and 7 d postchallenge. Complement Components and Lysozyme The 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 comparison with the NC or PC birds, these CB birds also had greater (P < 0.05) serum C4 at 3, 7, and 14 d postchallenge. 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 Population Birds 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 postchallenge (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 postchallenge 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 broiler 1 Item Days postchallenge 2 Experimental treatment NC PC CB CS SEM P-value BW (g) a c ab bc a c b ab 4.6 < a c ab ab 9.3 < ,297.2 a 1,086.9 b 1,202.9 a 1,274.8 a 20.9 <0.001 a c Means in the same row with different superscript letters differ significantly (P < 0.05). 1 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 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. 2 The days after challenge with E. coli K88.

4 CLOSTRIDIUM BUTYRICUM IN CHALLENGED BROILERS Table 2. Effects of Clostridium butyricum on inmunoglobulins in broilers 1 49 Item Days postchallenge 2 Experimental treatment NC PC CB CS SEM P-value IgA 3 (μg/ml) b a a a c bc a ab IgY 3 (μg/ml) b ab a a b b a ab IgM 3 (μg/ml) b ab a ab b ab ab a b ab a ab 8.26 < c b a b 6.85 <0.01 siga 4 (ng/ml) b 1.69 b 3.30 a 2.27 b 0.25 < ab 1.75 b 2.55 a 1.89 ab a 1.88 b 2.65 ab 2.61 ab a c Means in the same row with different superscript letters differ significantly (P < 0.05). 1 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 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. 2 The days after challenge with E. coli K88. 3 The concentration in serum. 4 The concentration in jejunal mucosa. 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. DISCUSSION Probiotics have been used as a substitute for antibiotics 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 studies 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 significant differences in BW between these 2 treatment Table 3. Effects of Clostridium butyricum on serum complement components and lysozyme in broilers 1 Item Days postchallenge 2 Experimental treatment NC PC CB CS SEM P-value C3 (mg/ml) b ab a b b ab a a b b a b C4 (mg/ml) b b a b b b a ab c b a b < Lysozyme (µg/ml) b ab a ab b a a a < b a a a < b b b a <0.01 a c Means in the same row with different superscript letters differ significantly (P < 0.05). 1 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 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. 2 The days after challenge with E. coli K88.

5 50 Zhang et al. 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). Bacterial number is expressed as log 10 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 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 log 10 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 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 log 10 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 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.

6 CLOSTRIDIUM BUTYRICUM IN CHALLENGED BROILERS 51 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). Bacterial number is expressed as log 10 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 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. groups. Apparently, C. butyricum improved BW of the E. coli K88-challenged birds by decreasing the cecal E. coli population via competitive exclusion. Other researchers 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 demonstrated 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. Dietary supplementation of probiotics may modulate mucosal and systemic immune activity and epithelial function, including mucosal T cells, B cells, epithelial cells, dendritic cells, macrophage, nature killer cells, antibodies, and cytokines (Fedorak and Madsen, 2004). Dietary supplementation of Saccharomyces cerevisiae fermentation 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 activities of the head kidney macrophages, the lysozyme activities 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 postchallenge, and greater mucosal siga at 3 and 7 d postchallenge 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. butyricum 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 function 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 (Epinephelus 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 challenged birds fed the basal diet. Apparently, C. butyrium may promote the immune function of enteric diseasechallenged birds, which may contribute to improved growth performance. The intestine harbors a complex microbial community 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). Inhibition of pathogen replication has been shown to be mediated by low-molecular-weight substances, primarily 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

7 52 Zhang et al. 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. butyricum had strong adhesion property and a higher antagonistic activity to Aeromonas hydrophila and Vibrio anguillarum, both on agar plate and cell model. However, 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-challenged birds. Li et al. (2009) demonstrated that probiotics 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 promote 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. butyrium improves growth performance, promotes immunity function, and benefits the cecal microflora in E. coli K88-challenged birds. ACKNOWLEDGMENTS This work was partially supported by the National Development and Reform Commission of China (project no ). REFERENCES Alonso, M. Z., N. L. Padola, A. E. Parma, and P. M. A. Lucchesi Enteropathogenic Escherichia coli contamination at different stages of the chicken slaughtering process. Poult. Sci. 90: Araki, Y., A. Andoh, Y. Fujiyama, J. Takizawa, W. Takizawa, and T. Bamba Oral administration of a product derived from Clostridium butyricum in rats. Int. J. Mol. Med. 9: Asai, T., K. Masani, C. Sato, M. Hiki, M. Usui, K. Baba, M. Ozawa, K. Harada, H. Aoki, and T. Sawada Phylogenetic groups and cephalosporin resistance genes of Escherichia coli from diseased food-producing animals in Japan. Acta Vet. Scand. 53: Belanger, L., A. Garenaux, J. Harel, M. Boulianne, E. Nadeau, and C. M. Dozois Escherichia coli from animal reservoirs as a potential source of human extra intestinal pathogenic E. coli. FEMS Immunol. Med. Microbiol. 62:1 10. Bhandari, S. K., F. O. Opapeju, D. O. Krause, and C. M. Nyachoti Dietary protein level and probiotic supplementation effects on piglet response to Escherichia coli K88 challenge: Performance and gut microbial population. Livest. Sci. 133: Biernasiak, J., and K. Slizewska The effect of a new probiotic preparation on the performance and faecal microflora of broiler chickens. Vet. Med. (Praha) 54: Cao, G. T., Y. P. Xiao, C. M. Yang, A. G. Chen, T. T. Liu, L. Zhou, L. Zhang, and P. R. Ferket Effects of Clostridium butyricum on growth performance, nitrogen metabolism, intestinal morphology and cecal microflora in broiler chicken. J. Anim. Vet. Adv. 11: Chen, H. Y., S. L. Wu, M. Y. Yeh, C. F. Chen, Y. Mikami, and J. S. Wu Antimetastatic activity induced by Clostridium butyricum and characterization of effector cells. Anticancer Res. 13: Dheilly, A., A. Bouder, L. Le Devendec, G. Hellard, and I. Kempf Clinical and microbial efficacy of antimicrobial treatments of experimental avian colibacillosis. Vet. Microbiol. 149: Dheilly, A., L. Le Devendec, G. Mourand, A. Bouder, E. Jouy, and I. Kempf Resistance gene transfer during treatments for experimental avian colibacillosis. Antimicrob. Agents Chemother. 56: Fedorak, R. N., and K. L. Madsen Probiotics and prebiotics in gastrointestinal disorders. Curr. Opin. Gastroenterol. 20: Gao, J., H. J. Zhang, S. G. Wu, S. H. Yu, I. Yoon, D. Moore, Y. P. Gao, H. J. Yan, and G. H. Qi Effect of Saccharomyces cerevisiae fermentation product on immune functions of broilers challenged with Eimeria tenella. Poult. Sci. 88: Giang, H. H., T. Q. Vieti, B. Ogle, and J. E. Lindberg Effects of supplementation of probiotics on the performance, nutrient digestibility and faecal microflora in growing-finishing pigs. Asianaustralas. J. Anim. Sci. 24: Grimes, J. L., S. Rahimi, E. Oviedo, B. W. Sheldon, and F. B. O. Santos Effects of a direct-fed microbial (primalac) on turkey poult performance and susceptibility to oral Salmonella challenge. Poult. Sci. 87: Han, I. K., S. C. Lee, and J. D. Kim Studies on the growth promoting effects of probiotics, 2: The effects of Clostridium butyricum ID on the performance and the changes in the microbial flora of the feces and intestinal contents of the broiler chicks. Hanguk. Chuksan. Hakhoe. Chi. 26: He, C. L., B. D. Fu, H. Q. Shen, X. L. Jiang, C. S. Zhang, S. C. Wu, W. Zhu, and X. B. Wei Xiang-Qi-Tang increases avian pathogenic Escherichia coli-induced survival rate and regulates serum levels of tumor necrosis factor alpha, interleukin-1 and soluble endothelial protein C receptor in chicken. Biol. Pharm. Bull. 34: He, G. Q., Q. Kong, and L. X. Ding Response surface methodology for optimizing the fermentation medium of Clostridium butyricum. Lett. Appl. Microbiol. 39: Huff, G. R., W. E. Huff, N. C. Rath, N. B. Anthony, and K. E. Nestor Effects of Escherichia coli challenge and transport stress on hematology and serum chemistry values of three genetic lines of turkeys. Poult. Sci. 87: Imase, K., M. Takahashi, A. Tanaka, K. Tokunaga, H. Sugano, M. Tanaka, H. Ishida, S. Kamiya, and S. Takahashi Efficacy of Clostridium butyricum preparation concomitantly with Helicobacter pylori eradication therapy in relation to changes in the intestinal microbiota. Microbiol. Immunol. 52: Kobayashi, R. K. T., I. Aquino, A. L. D. Ferreira, and M. C. Vidotto EcoR phylogenetic analysis and virulence genotyping of avian pathogenic Escherichia coli strains and Escherichia coli isolates from commercial chicken carcasses in Southern Brazil. Foodborne Pathog. Dis. 8: Kong, Q., G. Q. He, J. L. Jia, Q. L. Zhu, and H. Ruan Oral administration of Clostridium butyricum for modulating gastrointestinal microflora in mice. Curr. Microbiol. 62: Kumar, A., N. Jindal, C. L. Shukla, R. K. Asrani, D. R. Ledoux, and G. E. Rottinghaus Pathological changes in broiler chickens fed ochratoxin A and inoculated with Escherichia coli. Avian Pathol. 33: Lee, K. W., S. H. Lee, H. S. Lillehoj, G. X. Li, S. I. Jang, U. S. Babu, M. S. Park, D. K. Kim, E. P. Lillehoj, A. P. Neumann, T. G. Rehberger, and G. R. Siragusa Effects of direct-fed microbials on growth performance, gut morphometry, and immune characteristics in broiler chickens. Poult. Sci. 89:

8 CLOSTRIDIUM BUTYRICUM IN CHALLENGED BROILERS 53 Li, S. P., X. J. Zhao, and J. Y. Wang Synergy of Astragalus polysaccharides and probiotics (Lactobacillus and Bacillus cereus) on immunity and intestinal microbiota in chicks. Poult. Sci. 88: Mountzouris, K. C., P. Tsitrsikos, I. Palamidi, A. Arvaniti, M. Mohnl, G. Schatzmayr, and K. Fegeros Effects of probiotic inclusion levels in broiler nutrition on growth performance, nutrient digestibility, plasma immunoglobulins, and cecal microflora composition. Poult. Sci. 89: Munyaka, P. M., G. Tactacan, M. Jing, K. O, J. D. House, and J. C. Rodriguez-Lecompte Immunomodulation in young laying hens by dietary folic acid and acute immune responses after challenge with Escherichia coli lipopolysaccharide. Poult. Sci. 91: Murayama, T., N. Mita, M. Tanaka, T. Kitajo, T. Asano, K. Mizuochi, and K. Kaneko Effects of orally administered Clostridium butyricum MIYAIRI 588 on mucosal immunity in mice. Vet. Immunol. Immunopathol. 48: Nakanishi, S., and M. Tanaka Sequence analysis of a bacteriocinogenic plasmid of Clostridium butyricum and expression of the bacteriocin gene in Escherichia coli. Anaerobe 16: NRC Nutrient Requirements for Poultry. 9th rev. ed. National Academy Press, Washington, DC. Oelschlaeger, T. A Mechanisms of probiotic action A review. Int. J. Med. Microbiol. 300: Oh, J. Y., M. S. Kang, H. Yoon, H. W. Choi, B. K. An, E. G. Shin, Y. J. Kim, M. J. Kim, and J. H. Kwon The embryo lethality of Escherichia coli isolates and its relationship to the presence of virulence- associated genes. Poult. Sci. 91: Pan, X., T. Wu, Z. Song, H. Tang, and Z. Zhao. 2008a. Immune responses and enhanced disease resistance in Chinese drum, Miichthys miiuy (Basilewsky), after oral administration of live or dead cells of Clostridium butyrium CB2. J. Fish Dis. 31: Pan, X., T. Wu, L. Zhang, Z. Song, H. Tang, and Z. Zhao. 2008b. In vitro evaluation on adherence and antimicrobial properties of a candidate probiotic Clostridium butyricum CB2 for farmed fish. J. Appl. Microbiol. 105: Rahimi, S., S. Kathariou, J. L. Grimes, and R. M. Siletzky Effect of direct-fed microbials on performance and Clostridium perfringens colonization of turkey poults. Poult. Sci. 90: Roodposhti, P. M., and N. Dabiri Effects of probiotic and prebiotic on average daily gain, fecal shedding of Escherichia coli, and immune system status in newborn female calves. Asianaustralas. J. Anim. Sci. 25: Sato, R., and M. Tanaka Multiplication of orally administered Clostridium butyricum in rats. Microb. Ecol. Health Dis. 9: Seki, H., M. Shiohara, T. Matsumura, N. Miyagawa, M. Tanaka, A. Komiyama, and S. Kurata Prevention of antibiotic-associated diarrhea in children by Clostridium butyricum MIYAIRI. Pediatr Int. 45: Shini, S., P. Kaiser, A. Shini, and W. L. Bryden Differential alterations in ultrastructural morphology of chicken heterophils and lymphocytes induced by corticosterone and lipopolysaccharide. Vet. Immunol. Immunopathol. 122: Song, Z. F., T. X. Wu, L. S. Cai, L. J. Zhang, and X. D. Zheng Effects of dietary supplementation with Clostridium butyricum on the growth performance and humoral immune response in Miichthys miiuy. J. Zhejiang Univ. Sci. B 7: Sun, Y. Z., H. L. Yang, R. L. Ma, C. X. Zhang, and W. Y. Lin Effect of dietary administration of Psychrobacter sp. on the growth, feed utilization, digestive enzymes and immune responses of grouper Epinephelus coioides. Aquacult. Nutr. 17:e733 e740. Takahashi, M., H. Taguchi, H. Yamaguchi, T. Osaki, A. Komatsu, and S. Kamiya The effect of probiotic treatment with Clostridium butyricum on enterohemorrhagic Escherichia coli O157:H7 infection in mice. FEMS Immunol. Med. Microbiol. 41: Wang, G. R., H. Y. Chen, C. H. Chen, M. Y. Yeh, and Y. Mikami Immunopotentiating activity of Clostridium butyricum in mice. Proc. Natl. Sci. Counc. Repub. China B 20: Yang, C. M., G. T. Cao, P. R. Ferket, T. T. Liu, L. Zhou, L. Zhang, Y. P. Xiao, and A. G. Chen Effects of probiotic, Clostridium butyricum, on growth performance, immune function, and cecal microflora in broiler chickens. Poult. Sci. 91: Yang, X., Y. Guo, X. He, J. Yuan, Y. Yang, and Z. Wang Growth performance and immune responses in chickens after challenge with lipopolysaccharide and modulation by dietary different oils. Animal 2: Yang, X., B. Zhang, Y. Guo, P. Jiao, and F. Long Effects of dietary lipids and Clostridium butyricum on fat deposition and meat quality of broiler chickens. Poult. Sci. 89: Zhang, B. K., X. Yang, Y. M. Guo, and F. Y. Long Effects of dietary lipids and Clostridium butyricum on the performance and the digestive tract of broiler chickens. Arch. Anim. Nutr. 65: Zhang, H. Q., T. T. Ding, J. S. Zhao, X. Yang, H. X. Zhang, J. J. Zhang, and Y. L. Cui Therapeutic effects of Clostridium butyricum on experimental colitis induced by oxazolone in rats. World J. Gastroenterol. 15: