Evaluation of the Performance and Intestinal Gut Microflora of Broilers Fed on Corn-Soy Diets Supplemented With Bacillus subtilis PB6 (CloSTAT) 1

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1 2007 Poultry Science Association, Inc. Evaluation of the Performance and Intestinal Gut Microflora of Broilers Fed on Corn-Soy Diets Supplemented With Bacillus subtilis PB6 (CloSTAT) 1 A. Y. Teo and H.-M. Tan 2 Research and Development Department, Kemin Industries (Asia) Pte Ltd., Singapore , Singapore Primary Audience: Veterinarians, Nutritionists, Researchers, Production Managers SUMMARY In this study, broilers provided feed containing 10 9 cfu/t Bacillus subtilis PB6 in the finisher phase had a FCR similar to those on Zn bacitracin and significantly better than that of broilers provided nonmedicated feed with no added B. subtilis PB6 (P < 0.05). Over a 42-d period, broilers provided feed with B. subtilis PB6 had comparable feed intake and FCR as the antibiotic control. The counts of Lactobacillus species and Bifidobacterium species of broilers provided feed supplemented with B. subtilis PB6 were not significantly different from the number of these bacteria recovered from broilers provided feed supplemented with antibiotics. Numerically, up to 1- to 2- log 10 reduction in the number of Clostridium species recovered was observed in broilers provided feed supplemented with B. subtilis PB6 when compared with both negative and antibiotic controls. In terms of immunological response, birds provided feed supplemented with B. subtilis PB6 had significantly heavier bursas, heterophils with higher in vitro phagoctyosis for Escherichia coli, and lower ileal E. coli populations, indicating a potentiating role of B. subtilis PB6 as a probiotic on the chicken innate immune system. Key words: Bacillus subtilis, Lactobacillus species, Clostridium species, Escherichia coli, direct-fed microbial, immune response, feed conversion ratio 2007 J. Appl. Poult. Res. 16: DESCRIPTION OF PROBLEM Bacterial microflora can have both favorable and unfavorable effects on the intestinal health of the host and its susceptibility to disease [1]. Beneficial bacteria, such as Lactobacillus species and Bifidobacterium species, in the intestine have been recognized for their ability to improve the health of host animals [2]. Substantial progress has been made in the development of probiotics, prebiotics, and synbiotics, which are effective in increasing and maintaining the population of lactic acid bacteria in the intestine [3]. Previous studies have suggested that Lactobacillus species, Bifidobacterium species, as well as Bacillus subtilis can be used to increase and maintain beneficial bacteria in the intestine [4]. The latter, B. subtilis, 1 The use of trade names in this publication does not imply endorsement of the products mentioned or criticism of similar products not mentioned. 2 Corresponding author: haimeng.tan@kemin.com

2 TEO AND TAN: BACILLUS SUBTILIS 297 is considered generally recognized as safe and has found application in the feed industry [5]. In addition to improving intestinal microflora [6], beneficial effects such as recovery from diarrhea [7], enhanced BW gain, and improved feed efficiency [8] in the hosts have been observed. Previous in vivo trials have shown that the inclusion of B. subtilis PB6 as a feed additive improved the weight gain and feed efficiency of broilers [9]. Microbiological evaluation of the intestinal bacterial microflora of the tested broilers has demonstrated that the counts of Clostridium perfringens decreased with increasing dosage of B. subtilis PB6 in the feed [10]. These results confirmed our findings from the in vitro studies, which have demonstrated the inhibitory effect of B. subtilis PB6 on C. perfringens [11]. A similar study revealed that beneficial intestinal bacteria, such as Lactobacillus species, increased when birds were provided feed supplemented with B. subtilis PB6 [9]. Therefore, the purpose of the present study was to evaluate the animal performance, desirable and undesirable intestinal bacterial flora, and the immunological response of broilers fed on corn-soy diets supplemented with B. subtilis PB6. MATERIALS AND METHODS Bacterial Strain and Culture Conditions Cells of B. subtilis PB6 or CloSTAT [12] were grown in tryptic soy broth [13] supplemented with 0.6% yeast extract [14] and incubated at 37 C under agitation (100 rpm) in a shaker incubator. The culture was transferred weekly to fresh tryptic soy broth supplemented with 0.6% yeast extract and then stored at 4 C before use as seed cultures. Freshly grown B. subtilis PB6 cultures were resuspended in 40% glycerol and kept at 80 C. Scale-Up Fermentation and Spray-Drying of Bacterial Culture A 2-L B. Braun Biostat B-DCU stirred tank fermenter [15] was used to produce 10 L of a culture containing approximately 10 9 B. subtilis PB6/mL. The medium was sterilized in the fermenter by autoclaving at 121 C for 20 min, cooled to room temperature, and inoculated with a 1% overnight seed culture of B. subtilis Table 1. The composition and calculated analysis of the basal diets 1 Ingredient, % Starter Finisher Corn Soybean meal Vegetable oil Dicalcium phosphate Limestone Lys HCl DL-Met Salt Mineral premix Vitamin premix Calculated analysis AME, kcal/kg 2,960 3,090 CP, % Lys, % Met + Cys, % Ca, % Total P, % Phytate P, % Nonphytate P, % Probiotics or Zn bacitracin were added to the basal diet to form the 4 dietary treatments. PB6. The fermentation temperature was maintained at 37 C. The culture was allowed to grow in the fermenter for 24 h. The 24-h culture was then spray-dried using a pilot-scale Büchi minispray dryer [16] with inlet and outlet temperatures set at 115 and 65 C, respectively. The final spray-dried products were adjusted to contain 10 8 and 10 9 cfu/kg of B. subtilis PB6 with silica. Feed Characteristics Diets were formulated to meet or exceed the requirements of all nutrients [17] during the starter and finisher phases of the broilers throughout the 1 to 42-d feeding duration (Table 1). Two sets of basal diets were used: 1) a broiler starter (1 to 21 d) and 2) a broiler finisher (22 to 42 d). Four treatment diets were used in the present study. The negative control cornsoybean meal diet contained no antibiotic supplements, whereas the positive control basal diet was supplemented with 100 mg/kg of Albac Zn bacitracin [18]. Feed that was not supplemented with antibiotics was also used to prepare basal diets supplemented with 10 8 or 10 9 cfu/t of B. subtilis PB6. No coccidiostat was

3 298 added to any of the treatments used in the present study. Feed Mixing Procedures During the diet mixing, the CloSTAT was first mixed into the premix and then into the diet according to standard operating procedures for feed additive mixing at Massey University [19]. All mineral and vitamin supplements (including limestone, inorganic phosphate source, trace mineral premix, and vitamin premix), known as the filler, were accurately weighed and then hand-mixed. CloSTAT was then handmixed with the filler at a predetermined ratio using the quartering technique. In this technique, the filler was divided into 4 quarters. The CloSTAT product was first mixed with 1 portion of feed, then mixed with the next filler portion. The resulting mix, CloSTAT filler, was blended in a small Hobart mixer for 10 min. Eventually, the CloSTAT filler mix was added to the basal diet containing major ingredients and mixed in a horizontal mixer for another 5 min. All diets used in this study were coldpelleted (65 to 70 C) using a 3-mm die and stored in airtight containers until use. Pretreatment of Study Birds One-day-old male broiler (Ross) chicks of average 45-g weights were obtained from a commercial hatchery [20]. As per normal commercial practice in New Zealand, broilers were not vaccinated, because the country is free from infectious diseases, including infectious bronchitis. Bird Management Chicks were randomly assigned to 40 pens (10 birds per pen) in 3-tier electrically heated battery brooders (60 60 cm). The brooders were housed in an environmentally controlled room with 24 h of fluorescent lighting. The 4 dietary treatments were then randomly assigned to 10 pens of 10 chicks each. The birds were transferred to colony cages in an environmentally controlled room on d 14. The pens allowed 450 cm 2 of floor space per bird (i.e., 22 birds/ m 2 ) from d 1 to 42. Room temperature was maintained at 32 ± 1 C during the first week and gradually decreased to 24 C by the end of JAPR: Research Report the third week. Ventilation was controlled using mechanical fans mounted onto the walls. Procedures of the Broiler Trial Body weights and feed intake for each pen were recorded at weekly intervals. Feed and water were provided ad libitum throughout the 42-d trial period. Mortality and morbidity were observed and recorded on a daily basis. Any bird that died was weighed, and the FCR was adjusted accordingly. Feed conversion ratios were calculated by dividing total feed intake by weight of live plus dead birds. Necropsies were performed on birds that died during the current study. On d 21, two birds with approximate mean BW from replicate pens were selected for weighing and then euthanized by i.v. injection of Na pentobarbitone. The ileal section of the small intestine, from the vitelline diverticulum to a point about 40 mm proximal to the ileocecal junction, was ligated with nylon string. The ileal digesta were collected and immediately transported to the laboratory for evaluation and analysis of gut microflora [21]. Lymphoid organs including spleen and bursa were removed and weighed. Blood samples were collected for immunological testing. Bacteriological Examinations Ileal digesta collected from each bird were placed on ice and then analyzed for bacterial concentrations of Salmonella species, Bifidobacterium species, Lactobacillus species, Clostridium species, and coliform bacteria within 1 h after sample collection. Lactobacilli were enumerated on deman, Rogosa, and Sharpe broth [13]; bifidobacteria were enumerated on a Bifidobacterium selective Beeren s agar [22]; clostridia were enumerated on tryptose-sulfitecycloserine agar containing egg yolk emulsion [14]; coliform bacteria and Salmonella were enumerated on MacConkey and bismuth sulphite agar media [14], respectively. Selective agar used to enumerate bifidobacteria, lactobacilli, and clostridia species were incubated anaerobically for 48 h at 37 C, whereas selective agar used to enumerate coliform bacteria and Salmonella species were incubated aerobically for 24 h at 37 C.

4 TEO AND TAN: BACILLUS SUBTILIS 299 Table 2. Influence of Bacillus subtilis PB6 on the weight gain, feed intake, and FCR of male broilers (1 to 21 d posthatching) 1 Weight gain Feed intake FCR 2 Mortality Treatment (g/bird) (g/bird) (g/g) (no. died/total) Negative control 777 a 1,091 a a 4/100 Positive control (100 mg/kg of Zn bacitracin) 773 a 1,086 a a 3/100 B. subtilis PB6 (10 8 cfu/t) 780 a 1,099 a a 2/100 B. subtilis PB6 (10 9 cfu/t) 725 b 1,037 b a 2/100 Pooled SEM Significance, P = LSD, P < a,b Values with different superscripts in the same column are significantly different (P < 0.05). 1 Each mean value represents an average of 10 replicate pens of 10 birds each. 2 Corrected for mortality. Immunological Assay Blood heterophils were obtained from 2 birds per cage, pooled, and heterophil cell numbers were adjusted to /ml for use in the phagocytosis assay [23, 24]. Fluorescein isothiocyanate labeled E. coli were opsonized with mixed chicken serum before the addition of the heterophil suspension and incubation for 30 min at 41 C. The heterophils were then stained with trypan blue and analyzed for phagocytic activity by flow cytometry. Statistics and Analysis of Data The data were analyzed using the GLM procedure of SAS [25] with pen means as the experimental unit. Where F-values are significant (P < 0.05), the means are then separated using the LSD method. RESULTS AND DISCUSSION Performance Data There was no significant difference in FCR among treatments during the starter phase (Table 2), although the weight gain and feed intake of birds provided feed supplemented with 10 9 cfu/t of B. subtilis PB6 were less than the other treatments. During the finisher phase, feed intakes of birds fed the negative control diet (3,297 g/ bird) and the diet containing 10 8 cfu/t of B. subtilis PB6 (3,292 g/bird) were similar (P > 0.05); however, the weight gain of the latter treatment was no different from birds receiving Zn bacitracin (Table 3). It was unclear why feed intake was lower (P < 0.05) in the birds treated with either 10 9 cfu/t of B. subtilis PB6 or Zn bacitracin. In contrast, when compared Table 3. Influence of Bacillus subtilis PB6 on the weight gain, feed intake, and FCR of male broilers (22 to 42 d posthatching) 1 Weight gain Feed intake FCR 2 Mortality Treatment (g/bird) (g/bird) (g/g) (no. died/total) Negative control 1,965 a 3,297 a a 5/80 Positive control (100 mg/kg of Zn bacitracin) 1,971 a 3,144 b b 5/80 B. subtilis PB6 (10 8 cfu/t) 1,996 a 3,292 a a 2/80 B. subtilis PB6 (10 9 cfu/t) 1,924 a 3,101 b b 2/80 Pooled SEM Significance, P = NS LSD, P < a,b Values with different superscripts in the same column are significantly different (P < 0.05). 1 Each mean value represents an average of 10 replicate pens of 8 birds each. 2 Corrected for mortality.

5 300 JAPR: Research Report Table 4. Influence of Bacillus subtilis PB6 on the weight gain, feed intake, and FCR of male broilers (1 to 42 d posthatching) 1 Weight gain Feed intake FCR 2 Mortality Treatment (g/bird) (g/bird) (g/g) (no. died/total) Negative control 2,742 a 4,388 a a 9/100 Positive control (100 mg/kg of Zn bacitracin) 2,744 a 4,229 ab b 8/100 B. subtilis PB6 (10 8 cfu/t) 2,776 a 4,391 a ab 4/100 B. subtilis PB6 (10 9 cfu/t) 2,649 b 4,138 b ab 3/100 Pooled SEM Significance, P = LSD, P < a,b Values with different superscripts in the same column are significantly different (P < 0.05). 1 Each mean value represents an average of 10 replicate pens of 10 birds each during 1 to 21 d posthatching and 10 replicate pens of 8 birds each during 22 to 42 d posthatching. 2 Corrected for mortality. with the negative control diet, the feed efficiencies improved by 4.4 and 4.8 points with the addition of 10 9 cfu/t of B. subtilis PB6 and Zn bacitracin, respectively (P < 0.05). Over the 42-d trial period, the addition of Zn bacitracin had no significant effect (P > 0.05) on weight gains and feed intake but improved feed efficiency by 3.7 points compared with the negative control (P < 0.05; Table 4). Weight gain of birds fed the diet containing 10 8 cfu/t of B. subtilis PB6 was higher than the 10 9 cfu/t treatment. Feed efficiency was not different in birds treated with the CloSTAT product or 100 mg/kg of Zn bacitracin, although the Zn bacitracin was better than the negative control. One of the objectives of this study was to evaluate the effects of B. subtilis PB6 on the performance and intestinal gut flora of broilers fed with corn-soy diets. Studies by Jiraphocakul et al. [8] have demonstrated that when cells of B. subtilis were fed to turkeys, there was an enhancement in terms of BW gain and feed efficiency. In this regard, the results of this trial showed that broilers treated with B. subtilis PB6 had comparable feed intake and FCR as the antibiotic control. Greater improvements in weight gain and feed efficiency have also been reported in the recent broiler trials with B. subtilis PB6 [9]. In that study, a 10-point and 8- point FCR improvement was observed in 42- d-old broilers treated with 10 9 cfu/t of B. subtilis PB6 when compared with the negative and antibiotic controls, respectively. Statistical analysis of mortality data, after transformation of percentage values to n + 1, was conducted to determine the treatment effects. Mortality in the broiler groups treated Table 5. Influence of probiotic products on the relative weights of spleen and bursa in male broilers fed corn-soy diets at 21 and 42 d of age 1 Spleen weight 2 Bursa weight 3 Treatment 21 d 42 d 21 d 42 d Negative control 0.93 a 1.16 a 2.11 b 1.58 b Positive control (100 mg/kg of Zn bacitracin) 1.04 a 1.12 a 2.16 b 1.47 b Bacillus subtilis PB6 (10 8 cfu/t) 0.97 a 1.16 a 2.47 a 1.88 a B. subtilis PB6 (10 9 cfu/t) 0.98 a 1.24 a 2.50 a 1.83 a Pooled SEM Significance, P = NS NS LSD, P < a,b Values with different superscripts in the same column are significantly different (P < 0.05). 1 Each mean value represents an average of 20 observations at 21 d of age and an average of 10 observations at 42 d of age. 2 Spleen weight in grams; BW in kilograms. 3 Bursa weight in grams; BW in kilograms.

6 TEO AND TAN: BACILLUS SUBTILIS 301 Table 6. Influence of Bacillus subtilis PB6 on ileal populations (cfu/g) of Lactobacillus, Bifidobacterium, Clostridium, Escherichia, and Salmonella cultures 1 Lactobacillus Bifidobacterium Clostridium Escherichia Treatment species species species coli (cfu/g) Negative control Positive control (100 mg/kg of Zn bacitracin) B. subtilis PB6 (10 8 cfu/t) B. subtilis PB6 (10 9 cfu/t) Pooled SEM Significance, P = LSD, P < Each mean value represents an average of 10 birds. Ileal digesta samples were taken on d 21. All ileal digesta samples were negative for Salmonella. Values are not significantly different (P < 0.05). with diets containing 10 8 or 10 9 cfu/t of the CloSTAT products was 4 and 3%, respectively, compared with 8 and 9%, respectively, for the antibiotic and negative control treatments (Table 4). Most of the deaths occurred during the finisher period. However, postmortem examination of the dead birds revealed no abnormalities in the gross pathology of major organs. Weights of Lymphoid Organs An increase in the weight of spleen or bursa correlates with the ability of the body to produce lymphoid cells during an immune response. In the current study, none of the treatments used had any effect on the relative weights of spleen (Table 5). However, the relative weights of bursa were higher (P < 0.01) in 21- and 42-d-old birds that were fed diets containing 10 8 or 10 9 cfu/t of B. subtilis PB6. The bursa weights of these birds were not influenced by the antibiotic treatment. Gut Flora Composition The influence of treatments on the ileal microbial population is presented in Table 6. Treatments used in the present study had no effect on the populations of lactobacilli species and Bifidobacterium species. Compared with the negative control, the number of Clostridium species decreased to 3.69, 2.68, and 3.17 log 10 in the ileum of broilers fed with diets containing Zn bacitracin and 10 8 and 10 9 cfu/t of B. subtilis PB6, respectively (P < 0.10). The population of E. coli was not influenced by the addition of Zn bacitracin but decreased to 5.1 and 4.2 log 10 with the inclusion of 10 8 and 10 9 cfu/t of the CloSTAT products, respectively (P < 0.06). In the present study, all samples were found to be negative for Salmonella species. Reports have indicated that when cells of B. subtilis were fed to the experimental animals, beneficial microorganisms such as lactobacilli began to recolonize within the intestinal tract [26]. Other studies have also demonstrated that the ingestion of Bacillus species helped to restore the normal microbial flora following extensive antibiotic usage or illness [7]. In our study, broilers provided feed supplemented with B. subtilis PB6 experienced no reduction in the counts of beneficial intestinal bacteria, Lactobacillus species and Bifidobacterium species. examined. On the contrary, a 1- to 2-log 10 reduction in the cell counts of Clostridium species and E. coli was observed in broilers provided feed supplemented with B. subtilis PB6 (Table 6). This in vivo observation is in Table 7. Influence of Bacillus subtilis PB6 on immune response in male broilers 1 Treatment Phagocytosis Negative control 6.59 Positive control (100 mg/kg of Zn bacitracin) 6.23 B. subtilis PB6 (10 8 cfu/t) B. subtilis PB6 (10 9 cfu/t) 8.85 Pooled SEM 2.81 Significance, P = 0.09 LSD, P < Each mean value represents an average of 10 birds. Ileal blood samples were taken on d 21, and heterophils were stained with trypan blue and analyzed by flow cytometry for phagocytic activity toward Escherichia coli.

7 302 agreement with previous findings from our laboratory that demonstrated the killing of Clostridium species by B. subtilis PB6 [11]. Immune Response Measurements Phagocytosis is a key mechanism by which pathogens are engulfed and eradicated from the body. In the current study, broilers provided feed supplemented with B. subtilis PB6 tended to have a higher degree of phagocytosis compared with the antibiotic and negative control diets (P = 0.09; Table 7). The increase in in vitro phagocytosis toward E. coli also correlated with a decrease in ileal E. coli counts (Table 6) and earlier observations [9]. Such a potentiating effect on the innate immune system has also been recently observed [27]. Furthermore, this is JAPR: Research Report 1. Halzapfel, W. H., P. Haberer, J. Snel, U. Schillinger, and J. H. J. Huis in t Veld Overview of gut flora and probiotics. Int. J. Microbiol. 41: Collins, M. D., and G. R. Gibson Probiotics, prebiotics and synbiotics: Approaches for modulating the microbial ecology of the gut. Am. J. Clin. Nutr. 69:1052S 1057S. 3. Klein, G., A. Pack, C. Bonaparte, and G. Reuter Taxonomy and physiology of probiotic lactic acid bacteria. Int. J. Food Microbiol. 41: Goldin, B. R Health benefits of probiotics. Br. J. Nutr. 80:S203 S Sharp, R. J., M. D. Scawen, and T. Atkinson Fermentation and downstream processing of Bacillus. Pages in Bacillus. C. R. Harwood, ed. Plenum Press, New York, NY. 6. Hosoi, T., A. Ametani, K. Kiuchi, and S. Kaminogawa Changes in fecal microflora induced by intubation of mice with Bacillus subtilis (natto) spores are dependent upon dietary components. Can. J. Microbiol. 45: Maruta, K., H. Miyazaki, S. Masuda, M. Takahashi, T. Marubashi, Y. Tadano, and H. Takahashi Exclusion of intesconsistent with the heavier relative bursa weights in birds treated with B. subtilis PB6, suggesting a greater degree of immune stimulation (Table 5). In healthy animals, the upper part of the small intestine contains relatively few bacteria, but the concentration of the bacteria increases in jejunum, ileum, and the colonic portions of the gastrointestinal tract, and the flora consists of mainly nonpathogenic bacteria [23]. Nevertheless, opportunistic bacteria such as Clostridium species, E. coli, Klebsiella species, Staphylococcus species, or Enterococcus species can proliferate to high numbers and cause disease under certain conditions [24]. Beneficial probiotic bacteria, on the other hand, have been reported to stimulate healthy gut structure and systemic immune system [28]. CONCLUSIONS AND APPLICATIONS 1. Broilers fed on a diet containing 10 9 cfu/t of B. subtilis PB6 in the finisher phase had a similar FCR as those on Zn bacitracin that was significantly better than that of broilers provided nonmedicated feed with no added B. subtilis PB6. 2. Birds provided feed supplemented with B. subtilis PB6 had significantly heavier bursa weights compared with the antibiotic and negative control groups. 3. Heterophils isolated from broilers provided feed supplemented with B. subtilis PB6 had higher phagocytic activity against E. coli in contrast to broilers provided medicated or nonmedicated feed without any B. subtilis PB6 (P < 0.1). 4. Compared with negative control, broilers provided feed supplemented with B. subtilis PB6 tended to have lower ileal populations of Clostridium species and E. coli (P < 0.1). REFERENCES AND NOTES tinal pathogens by continuous feeding with Bacillus subtilis C-3102 and its influence on the intestinal microflora in broilers. Anim. Sci. Technol. 67: Jiraphocakul, S., T. W. Sullivan, and K. M. Shahani Influence of a dried Bacillus subtilis culture and antibiotics on performance and intestinal microflora in turkeys. Poult. Sci. 69: Teo, A. Y., and H.-M. Tan Effect of Bacillus subtilis PB6 (CloSTAT) on broilers infected with a pathogenic strain of Escherichia coli. J. Appl. Poult. Res. 15: Teo, A. Y., and H.-M. Tan The effect of CloSTAT on pathogenic and beneficial bacteria in broilers. Page 174 in Abstr. XXII World Poult. Congr., Istanbul, Turkey. World s Poult. Sci. Assoc., Beekburgen, the Netherlands. 11. Teo, A. Y., and H.-M. Tan Inhibition of Clostridium perfringens byanovelstrainofbacillus subtilis isolated from the gastrointestinal tracts of healthy chickens. Appl. Environ. Microbiol. 71: CloSTAT, Kemin Industries Inc., Des Moines, IA.

8 TEO AND TAN: BACILLUS SUBTILIS Becton, Dickinson and Co., Cockeysville, MD. 14. Oxoid Ltd., Hampshire, UK. 15. B. Braun Biotech International GmbH, Melsungen, Germany. 16. Büchi, Flawil, Switzerland. 17. NRC Nutrient Requirements of Poultry. 9th ed. Natl. Acad. Press, Washington, DC. 18. Albac, Alpharma AS, Oslo, Norway. 19. Poultry Research Unit Standard Operating Procedures for Mixing Feed Additives into Diets. Monogastric Res. Cent., Massey Univ., Palmerson North, New Zealand. 20. Tegel Foods Ltd, New Plymouth, New Zealand. 21. Sutter, V. L., D. M. Citron, M. A. C. Rdelstein, and S. M. Finegold Wadsworth Anaerobic Bacteriology Manual. 4th ed. Star Publ. Co., Belmont, CA. 22. Beerens, H An elective and selective culture medium for Bifidobacterium. Lett. Appl. Microbiol. 11: Moreau, M. C., and V. Gaboriau-Routhiau Influence of resident intestinal microflora on the development and functions of the intestinal-associated lymphoid tissue. Pages in Probiotics 3. Immunomodulation by the Gut Microflora and Probiotics. R. Fuller and G. Perdigon, ed. Kluwer, London, UK. 24. Cebra, J. J Influences of microbiota on intestinal immune system development. Am. J. Clin. Nutr. 69:1046S 1051S. 25. SAS Institute SAS User s Guide. Version 8 ed. SAS Inst. Inc., Cary, NC. 26. Maruta, K., H. Miyazaki, Y. Tadano, S. Masuda, A. Suzuki, H. Takahashi, and M. Takahashi Effects of Bacillus subtilis C-3102 intake on fecal flora of sows and on diarrhea and mortality rate of their piglets. Anim. Sci. Technol. 67: Farnell, M. B., A. M. Donoghue, F. Solios de los Santos, P. J. Blore, B. M. Hargis, G. Tellez, and D. J. Donoghue Upregulation of oxidative burst and degranulation in chicken heterophils stimulated with probiotic bacteria. Poult. Sci. 85: Maassen, C. B. M., J. D. Laman, W. J. A. Boersma, and E. Claassen Modulation of cytokine expression by lactobacilli and its possible therapeutic use. Pages in Probiotics 3. Immunomodulation by the Gut Microflora and Probiotics. R. Fuller and G. Perdigon, ed. Kluwer, London. UK. Acknowledgments We thank V. Ravindran, D. Thomas, and B. Camden from the Monogastric Research Centre, Institute of Food, Nutrition and Human Health, Massey University, Palmerston North, New Zealand, for assistance with the trial. Special thanks also go to M. R. Alley from the Institute of Veterinary, Animal and Biomedical Sciences, Palmerston North, New Zealand, for conducting the pathological and veterinary studies on the broilers.