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A Bacillus subtilis (QST 713) spore-based probiotic for necrotic enteritis control in broiler chickens
©2013 Poultry Science Association, Inc.
A Bacillus subtilis (QST 713) spore-based probiotic for necrotic enteritis control in broiler chickens G. B. Tactacan, J. K. Schmidt, M. J. Miille, and D. R. Jimenez1 Bayer CropScience, Biologics, 1540 Drew Avenue, Davis, CA 95618
SUMMARY Necrotic enteritis (NE) is a common poultry disease that significantly affects the profitability of commercial broiler production. Traditionally, antibiotic feed supplements are administered to poultry to combat the negative effects of NE. However, due to the ban on growth-promoting antibiotics in the European Union, increasing concern for antibiotic resistance, and demand for drug residue free poultry products, alternative approaches are necessary to maximize broiler performance against NE. A study was conducted to determine the efficacy of feeding Bacillus subtilis (QST 713) spores in broilers using an NE-induced challenge condition. High and low concentrations of B. subtilis (QST 713) spores (1 × 106 and 1 × 105 cfu/g of feed) in broiler feeds were used to identify the appropriate dose to overcome the adverse effects of NE. A treatment group supplemented with bacitracin methyl disalicylate (BMD) at a therapeutic level (50 g/t) was included to compare the efficacy of B. subtilis to a conventionally used antibiotic. Based on these results, NE was successfully induced in the nonmedicated broiler chickens orally inoculated with coccidial oocyst and Clostridium perfringens. Relative to the nonchallenged group, birds infected with NE with no medication showed a significantly (P < 0.05) decreased BW gain, increased FCR, increased NE lesion score, and increased NE mortality. Supplementation of B. subtilis at 1 × 106 cfu/g of feed and BMD mitigated the negative effects of NE in all production parameters and measures of NE infection. No significant differences (P > 0.05) were observed between NE-challenged birds fed with high dose of B. subtilis (QST 713) and those birds supplemented with BMD. Low dose supplementation (1 × 105 cfu/g of feed) of B. subtilis (QST 713) increased NE mortality (P < 0.05) relative to high dose supplementation. Overall, based on these data, adequate supplementation of B. subtilis (QST 713) could serve as an alternative approach in controlling NE infection in broilers without continuously depending on antibiotics during their growing period. Key words: Bacillus subtilis, bacitracin methyl disalicylate, Clostridium perfringens, necrotic enteritis, broiler chicken 2013 J. Appl. Poult. Res. 22:825–831 http://dx.doi.org/10.3382/japr.2013-00730
DESCRIPTION OF PROBLEM Necrotic enteritis (NE) is a common poultry disease which affects the growth and feed 1
Corresponding author: [email protected]
conversion of broiler chickens in subclinical cases and causes high mortality in clinical cases [1, 2]. Caused by Clostridium perfringens, the colonization of the avian gastrointestinal tract
Downloaded from http://japr.oxfordjournals.org/ at Purdue University Libraries ADMN on August 28, 2014
Primary Audience: Nutritionists, Veterinarians, Researchers, Broiler Producers
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ing broiler chickens dietary supplemented with B. subtilis (QST 713).
MATERIALS AND METHODS General The research facility at the Southern Poultry Research Inc. in Athens, Georgia, was used to conduct this study. A medicated treatment group supplemented with bacitracin methyl disalicylate (BMD) at therapeutic level (50 g/t) was included to compare the efficacy of B. subtilis spores (QST 713) to a conventionally used antibiotic in poultry feeds. The isolate of C. perfringens used to induce NE infection in the challenge study had shown susceptibility to BMD, as described previously [2]. All animal procedures were conducted according to the Guidelines for Good Target Animal Study Practice [7] and were consistent with applicable laws and regulations governing the humane care of animals. Experimental Design A total of 400 one-day-old Cobb × Cobb male broiler chicks were purchased from CobbVantress Hatchery in Cleveland, Georgia. Upon arrival to the research facility the chicks were placed in Petersime battery cages and were randomly assigned to receive 1 of 5 dietary treatments: (1) nonmedicated, non-NE challenged; (2) nonmedicated, NE challenged; (3) BMD 50 g/t, NE challenged; (4) B. subtilis spores (QST 713) at a dose of 1 × 106 CFU/g of feed, NE challenged; and (5) B. subtilis spores (QST 713) at a dose of 1 × 105 CFU/g of feed, NE challenged. The study began when the birds were placed (d 0), at which time they were allocated to the experimental cages and fed their corresponding dietary treatments. Feed and water were made available ad libitum throughout the study. A randomized complete block design with 10 blocks and 5 treatments per block was used in this study. Eight birds per cage were placed in each battery with a floor space of 0.63 ft2/bird (588 cm2/bird). The feeder space per bird was 68 cm2. Lighting was provided 24 h/d, and a thermostatically controlled gas furnace and air conditioner maintained uniform temperature throughout the study.
Downloaded from http://japr.oxfordjournals.org/ at Purdue University Libraries ADMN on August 28, 2014
by this pathogen leads to NE infection, resulting in severe economic losses in commercial broiler operation. Traditionally, antibiotic feed supplements have been administered to poultry to combat the negative effects of NE. However, due to the increasing concern in antibiotic resistance, the ban on growth-promoting antibiotics to broilers in the European Union, and the increasing demand for naturally produced poultry products, there is a growing interest in finding alternatives to antibiotics for the control and prevention of NE in broiler chickens. Dietary supplementation with probiotics is one of several approaches that have the potential to overcome NE infection. The term probiotic or direct-fed microbial has been defined as a “live microbial feed supplement which beneficially affects the host animal by improving its intestinal balance” [3]. Hofacre et al. [2] challenged broiler chickens experimentally with Clostridium perfringens and observed that probiotics reduced NE-associated mortality and its subclinical effects on FE. Similarly, Craven [4] reported a reduction in C. perfringens colonization and decreased incidence of NE in chickens treated with probiotics. In a field study, Kaldhusdal and Lovland [1] found that posthatch use of microflora prepared from adult birds was associated with delayed intestinal proliferation of C. perfringens, delayed appearance of NE gross lesions, and better production performance at slaughter. Therefore, taken together, the use of probiotics in broiler chickens may show promise in controlling and preventing NE. Among the probiotic preparations currently available for poultry are several products composed of bacterial spores, principally of the genus Bacillus. Spore-forming Bacillus species are ideally suited as commercial probiotic products due to the ability of their spores to survive harsh environmental conditions and long-term storage [5, 6]. Bacterial spores are extremely robust during distribution and administration in animal feeds (e.g., pelleting) and are able to pass through the acidic gastric environment of the target host species and enter the small intestine in a viable state. At present, a limited number of published studies have demonstrated the efficacy of Bacillus subtilis spores as prophylactic agents against NE in broiler chickens. Therefore, an NE challenge study was conducted us-
Tactacan et al.: PROBIOTIC FOR NECROTIC ENTERITIS
Statistical Analysis The statistical analysis was conducted using the SAS software [8]. Performance and lesion score data were analyzed as a randomized complete block design using PROC MIXED, whereas mortality data were analyzed using PROC FREQ. Data are presented as least squares means with differences between means determined using Tukey’s honestly significance difference. All statements of significance were based on P < 0.05.
RESULTS AND DISCUSSION The results from the colony-forming units analysis of the feed samples confirmed that the B. subtilis (QST 713) spores were supplemented at the appropriate high (9.12 × 105 cfu/g of feed) and low (1.13 × 105 cfu/g of feed) doses in the broiler chickens. The NE challenge suppressed the BW gain and increased the FCR of the nonmedicated broiler chickens orally inoculated with coccidial oocysts and C. perfringens. Compared with the non-NE-challenged birds, the BW gain and feed conversion performance of the nonmedicated NE-challenged birds decreased and increased, respectively (P < 0.05), from d 0 to 28 of the study (Figures 1 and 2). The supplementation of B. subtilis (QST 713) spores at 1 × 106 cfu/g of feed (high dose) in broiler chickens mitigated the negative effects of NE. When supplemented at a high concentration, no differences (P > 0.05) in BW gain and FCR were observed between the B. subtilis-treated and the non-NE-challenged birds. However, when B. subtilis (QST 713) spores were supplemented at 1 × 105 cfu/g of feed (low dose), BW gain and FCR decreased and increased, respectively (P < 0.05), relative to the non-NE-challenged group. No significant differences were observed between NE-challenged birds supplemented with B. subtilis (QST 713) spores and BMD. Necrotic enteritis challenge resulted in increased NE lesions in the intestine of the nonmedicated birds on d 22. Compared with the nonchallenged group, NE lesion scores increased significantly in NE-challenged birds that did not receive medication (Figure 3). The supplementation of B. subtilis (QST 713) spores and BMD ameliorated the intestinal lesions caused by NE. The NE lesion scores of birds supplemented with B. subtilis (QST 713) spores and BMD decreased significantly relative to the nonmedicated NE-challenged birds. Consequently, the percentage of NE mortality in the nonmedicated broiler chickens was significantly increased by NE challenge. Mortality due to NE rose to 25%, demonstrating the high virulence of the E. maxima and C. perfringens isolates used in the study. No significant differences were observed in the NE mortality of birds supplemented with a high dose of B. subtilis (QST
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A nonmedicated, corn-soybean meal-based commercial type broiler starter feed compounded with feedstuffs commonly used in the United States was formulated for this study. From this basal starter feed, the experimental treatment feeds were prepared. All diets were in mash form. Three feed samples were collected, one each from the beginning, middle, and end of the batch of treatment diet and mixed to form a composite sample. One composite sample was taken from the composite for each treatment and was used for colony-forming units determination. On d 14, all birds were orally inoculated with a coccidial inoculum containing approximately 5,000 oocysts of Eimeria maxima per bird. Starting on d 19, all birds except treatment 1 were given 1 mL of a broth culture of C. perfringens at approximately 1 × 108 cfu/bird. The birds were administered a fresh broth culture once daily for 3 d (on d 19, 20, and 21). All birds were weighed by cage on 0, 14, 22, and 28 d of age. Feed was weighed on d 0 and remaining feed was weighed at 14, 22, and 28 d of age. On d 22, 3 birds from each cage were selected, euthanized, weighed, and examined for the presence of NE lesions. The scoring was done blindly and based on a score of 0 to 3, with 0 being normal and 3 being the most severe [2]. Means for cage weight gain, feed consumption, feed conversion [adjusted for mortality: feed consumed/(final live weight + mortality weight)], NE lesion scores, and NE mortality were calculated. The mortality was assessed by gross lesions on necropsy-enlarged, dark-colored livers and the pseudomembrane appearance of classical NE in the small intestine.
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Figure 2. The FCR of broiler chickens with or without necrotic enteritis challenged and fed with or without Bacillus subtilis (QST 713) or bacitracin methyl disalicylate (BMD) from 1 to 28 d old. Bars with different letters (a–c) are significantly different (P < 0.05). Data represent means ± SEM (n = 10). med. = medicated.
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Figure 1. The BW gain of broiler chickens with or without necrotic enteritis challenge and fed with or without Bacillus subtilis (QST 713) or bacitracin methyl disalicylate (BMD) from 1 to 28 d old. Bars with different letters (a–c) are significantly different (P < 0.05). Data represent means ± SEM (n = 10). med. = medicated.
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713) spores, BMD, and the non-NE-challenged group. Low dose supplementation of B. subtilis (QST 713) spores decreased NE mortality compared with the nonmedicated NE-challenged birds (Figure 4); however, it increased NE mortality compared with the high dose B. subtilis (QST 713) supplemented, BMD supplemented, and the non-NE-challenged birds. The non-NEchallenged birds had no mortality and did not develop NE. The purpose of this study was to determine the efficacy of B. subtilis (QST 713) spores in overcoming the adverse effects of NE in the production performance of broiler chickens. To identify the appropriate dietary concentration against NE, high (1 × 106 cfu/g of feed) and low (1 × 105 cfu/g of feed) doses of B. subtilis (QST 713) spores were supplemented in the broiler feeds. A treatment group supplemented with BMD was added in the study to gain insights on the potential of B. subtilis (QST 713) spores to replace an antibiotic growth promoter that is used conventionally in controlling and preventing NE. The dietary supplementation of B. subtilis at 1 × 106 cfu/g of feed and BMD was equally ef-
fective in controlling the subclinical and clinical effects of NE in broiler chickens. However, a partial loss in efficacy in terms of reducing NE-associated mortality was observed when B. subtilis (QST 713) spores were incorporated at the lower concentration of 1 × 105 cfu/g of feed. Knap et al. [9] reported a similar decrease in efficacy of Bacillus licheniformis spores when supplemented at a low dose in NE-challenged broiler chickens. In their study, the supplementation of B. licheniformis spores at 8 × 105 cfu/g of feed resulted in decreased BW gain and increased FCR compared with birds fed with B. licheniformis spores at higher concentrations (8 × 106 and 8 × 107 cfu/g of feed). At these concentrations, B. licheniformis-fed birds showed no differences in BW gain, FCR, NE lesion score, and NE mortality in birds treated with virginiamycin (15 g/t). In reports, B. subtilis supplementation in poultry without NE challenge resulted in an increase in BW gain and improvement in FE when fed to turkeys [10] and broiler chickens [11]. Therefore, beneficial effects of B. subtilis may be present in the production performance of poultry even in the absence of clinical NE. Future studies on B. subtilis (QST 713) will be
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Figure 3. The necrotic enteritis (NE) lesion score of broiler chickens with or without NE challenge and fed with or without Bacillus subtilis (QST 713) or bacitracin methyl disalicylate (BMD) at d 22. Bars with different letters (a–c) are significantly different (P < 0.05). Data represent means ± SEM (n = 30). Necrotic lesion scoring was based on Hofacre et al. [2] with a lesion score of 0 = none, 1 = mild, 2 = moderate, 3 = severe. med. = medicated.
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focused on its effect in the production performance of broiler chickens without NE challenge. At present, the mechanism of action of B. subtilis as a probiotic for the control and prevention of NE is not totally clear. Previously, when 1- and 20-d-old chickens were challenged with 1 × 109 spores of B. subtilis strain and infected 24 h later with 1 × 105 cfu of C. perfringens, colonization and persistence of C. perfringens was suppressed [12]. Bacillus spp. are grampositive, spore-forming microorganisms capable of germinating in the gastrointestinal tract of the chickens [13, 14]. The germination of the spores gives rise to metabolically active vegetative cells; therefore, it can be assumed that Bacillus-based probiotics may exert their effects through mechanisms involved in the metabolism of the vegetative cells. Indeed, Teo and Tan [15] reported the production of an antimicrobial factor typical of gram-positive bacteriocin by B. subtilis (PB6) which was found to be active against various strains of Clostridium spp. The production of antimicrobials by probiotics is considered one of the principal mechanisms that inhibit pathogenic microorganisms in the gastrointestinal tract. Bacillus spp. are known to produce a large number of antimicrobials [16];
these include bacteriocins and bacteriocin-like inhibitory substances (e.g., Subtilin, Coagulin), as well as antimicrobials based on peptides and polyketides (e.g., Surfactin, Bacilysin, Difficidin, and Macrolactin). Bacillus subtilis (QST 713) are known to produce some of these antimicrobial compounds (internal data). Stimulation of the bird’s immune system or immunomodulation is another important mechanism that may explain the role of B. subtilis spores against NE. In several studies, it was shown that germinating Bacillus spores can elicit potent immune responses in the gastrointestinal tract, and that this immune stimulation may be the underlying reason why spores exert a probiotic effect [17, 18]. Rhee et al. [19] examined the effects of orally administered bacteria on the development of the gut-associated lymphoid tissue in infant rabbits and found that B. subtilis was of greater importance than other commensal bacteria in gut-associated lymphoid tissue development. Accordingly, Molnar et al. [20] reported increased immunological tissue development in the ileal mucosa of broiler chickens, which paralleled the increasing B. subtilis concentrations in the feed. Gut-associated lymphoid tissue development reflects its role in the defense of
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Figure 4. The necrotic enteritis (NE) mortality of broiler chickens with or without NE challenge and fed with or without Bacillus subtilis (QST 713) or bacitracin methyl disalicylate (BMD) from 1 to 28 d old. Bars with different letters (a–c) are significantly different (P < 0.05). Data represent means ± SEM (n = 80). med. = medicated.
Tactacan et al.: PROBIOTIC FOR NECROTIC ENTERITIS mucosal surfaces against enteric pathogens such as C. perfringens.
CONCLUSIONS AND APPLICATIONS
REFERENCES AND NOTES 1. Kaldhusdal, M., and A. Lovland. 2000. The economic impact of Clostridium perfringens is greater than anticipated. World’s Poult. Sci. J. 16:50–51. 2. Hofacre, C. L., T. Beacorn, S. Collett, and G. Mathis. 2003. Using competitive exclusion, mannan-oligosaccharide and other intestinal products to control necrotic enteritis. J. Appl. Poult. Res. 12:60–64. 3. Fuller, R. 1999. Probiotics for farm animals. Pages 15–22 in Probiotics: A Critical Review. G. W. Tannock, ed. Horizon Scientific Press, Wymondham, UK. 4. Craven, S. E. 2000. Colonization of the intestinal tract by Clostridium perfringens and fecal shedding in diet-stressed and unstressed broiler chickens. Poult. Sci. 79:843–849. 5. Fuller, R. 1989. Probiotics in man and animals. J. Appl. Bacteriol. 66:365–378. 6. Hong, H. A., L. H. Duc, and S. M. Cutting. 2005. The use of bacterial spore formers as probiotics. FEMS Microbiol. Rev. 29:813–835.
7. Food and Drug Administration Center for Veterinary Medicine. 1997. Good target animal study practices: Clinical investigators and monitors. Guidance for industry #58. FDA. Rockville, MD. 8. SAS Institute. 1998. SAS/STAT User’s Guide, Release 6.03 Edition. SAS Inst. Inc., Cary, NC. 9. Knap, I., B. Lund, A. B. Kehler, C. L. Hofacre, and G. Mathis. 2010. Bacillus licheniformis prevent necrotic enteritis in broiler chickens. Avian Dis. 54:931–935. 10. Jiraphocakul, S., T. W. Sullivan, and K. M. Shahani. 1990. Influence of a dried Bacillus subtilis culture and antibiotics on performance and intestinal and microflora in turkeys. Poult. Sci. 69:1966–1973. 11. Santoso, U., K. Tanaka, and S. Ohtani. 1995. Effect of dried Bacillus subtilis culture on growth, body composition and hepatic lipogenic enzyme activity in female broiler chicks. Br. J. Nutr. 74:523–529. 12. La Ragione, R. M., and M. J. Woodward. 2003. Competitive exclusion by Bacillus subtilis spores of Salmonella enterica serotype Enteritidis and Clostridium perfringens in young chickens. Vet. Microbiol. 94:245–256. 13. Jadamus, A., W. Vahjen, and O. Simon. 2001. Growth behavior of a spore forming probiotic strain in the gastrointestinal tract of broiler chickens and piglets. Arch. Tierernahr. 54:1–17. 14. Cartman, S. T., R. M. La Ragione, and M. J. Woodward. 2008. Bacillus subtilis spores germinate in the chicken gastrointestinal tract. Appl. Environ. Microbiol. 74:5254– 5258. 15. Teo, A. Y., and H. Tan. 2005. Inhibition of Clostridium perfringens by a novel strain of Bacillus subtilis isolated from the gastrointestinal tracts of healthy chickens. Appl. Environ. Microbiol. 71:4185–4190. 16. Urdaci, M. C., and I. Pinchuk. 2004. Antimicrobial activity of Bacillus probiotics. Pages 171–182 in Bacterial Spore Formers: Probiotics and Emerging Applications. E. Ricca, A. O. Henriques, and S. M. Cutting, ed. Horizon Bioscience, Oxford, UK. 17. Duc, H., H. A. Hong, N. Fairweather, E. Ricca, and S. M. Cutting. 2003. Bacterial spores as vaccine vehicles. Infect. Immun. 71:2810–2818. 18. Duc, H., H. A. Hong, N. Q. Uyen, and S. M. Cutting. 2004. Intracellular fate and immunogenicity of B. subtilis spores. Vaccine 22:1873–1885. 19. Rhee, K. J., P. Sethupathi, A. Driks, D. K. Lanning, and K. L. Knight. 2004. Role of commensal bacteria in development of gut-associated lymphoid tissues and preimmune antibody repertoire. J. Immunol. 172:1118–1124. 20. Molnár, A. K., B. Podmaniczky, P. Kurti, I. Tenk, R. Glavits, G. Y. Virag, and Z. S. Szabo. 2011. Effect of different concentrations of Bacillus subtilis on growth performance, carcase quality, gut microflora, and immune response of broiler chickens. Br. Poult. Sci. 52:658–665.
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1. Necrotic enteritis was successfully induced in nonmedicated broiler chickens orally inoculated with coccidial oocyst and C. perfringens in this study. The adverse effects of the NE infection in broiler chickens resulted in decreased BW gain, increased FCR, increased intestinal lesions, and increased mortality. 2. Adequate level of dietary B. subtilis (QST 713) spores supplementation was equally effective as BMD in mitigating the subclinical and clinical effects of NE in broiler chickens. 3. Under the conditions of this study, the supplementation of B. subtilis (QST 713) spores could serve as an alternative approach in controlling NE infection in broiler chickens without continuously using antibiotic growth promoter in the feed throughout their growing period.
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A Bacillus subtilis (QST 713) spore-based probiotic for necrotic enteritis control in broiler chickens G. B. Tactacan, J. K. Schmidt, M. J. Miille, and D. R. Jimenez1 Bayer CropScience, Biologics, 1540 Drew Avenue, Davis, CA 95618
SUMMARY Necrotic enteritis (NE) is a common poultry disease that significantly affects the profitability of commercial broiler production. Traditionally, antibiotic feed supplements are administered to poultry to combat the negative effects of NE. However, due to the ban on growth-promoting antibiotics in the European Union, increasing concern for antibiotic resistance, and demand for drug residue free poultry products, alternative approaches are necessary to maximize broiler performance against NE. A study was conducted to determine the efficacy of feeding Bacillus subtilis (QST 713) spores in broilers using an NE-induced challenge condition. High and low concentrations of B. subtilis (QST 713) spores (1 × 106 and 1 × 105 cfu/g of feed) in broiler feeds were used to identify the appropriate dose to overcome the adverse effects of NE. A treatment group supplemented with bacitracin methyl disalicylate (BMD) at a therapeutic level (50 g/t) was included to compare the efficacy of B. subtilis to a conventionally used antibiotic. Based on these results, NE was successfully induced in the nonmedicated broiler chickens orally inoculated with coccidial oocyst and Clostridium perfringens. Relative to the nonchallenged group, birds infected with NE with no medication showed a significantly (P < 0.05) decreased BW gain, increased FCR, increased NE lesion score, and increased NE mortality. Supplementation of B. subtilis at 1 × 106 cfu/g of feed and BMD mitigated the negative effects of NE in all production parameters and measures of NE infection. No significant differences (P > 0.05) were observed between NE-challenged birds fed with high dose of B. subtilis (QST 713) and those birds supplemented with BMD. Low dose supplementation (1 × 105 cfu/g of feed) of B. subtilis (QST 713) increased NE mortality (P < 0.05) relative to high dose supplementation. Overall, based on these data, adequate supplementation of B. subtilis (QST 713) could serve as an alternative approach in controlling NE infection in broilers without continuously depending on antibiotics during their growing period. Key words: Bacillus subtilis, bacitracin methyl disalicylate, Clostridium perfringens, necrotic enteritis, broiler chicken 2013 J. Appl. Poult. Res. 22:825–831 http://dx.doi.org/10.3382/japr.2013-00730
DESCRIPTION OF PROBLEM Necrotic enteritis (NE) is a common poultry disease which affects the growth and feed 1
Corresponding author: [email protected]
conversion of broiler chickens in subclinical cases and causes high mortality in clinical cases [1, 2]. Caused by Clostridium perfringens, the colonization of the avian gastrointestinal tract
Downloaded from http://japr.oxfordjournals.org/ at Purdue University Libraries ADMN on August 28, 2014
Primary Audience: Nutritionists, Veterinarians, Researchers, Broiler Producers
JAPR: Research Report
826
ing broiler chickens dietary supplemented with B. subtilis (QST 713).
MATERIALS AND METHODS General The research facility at the Southern Poultry Research Inc. in Athens, Georgia, was used to conduct this study. A medicated treatment group supplemented with bacitracin methyl disalicylate (BMD) at therapeutic level (50 g/t) was included to compare the efficacy of B. subtilis spores (QST 713) to a conventionally used antibiotic in poultry feeds. The isolate of C. perfringens used to induce NE infection in the challenge study had shown susceptibility to BMD, as described previously [2]. All animal procedures were conducted according to the Guidelines for Good Target Animal Study Practice [7] and were consistent with applicable laws and regulations governing the humane care of animals. Experimental Design A total of 400 one-day-old Cobb × Cobb male broiler chicks were purchased from CobbVantress Hatchery in Cleveland, Georgia. Upon arrival to the research facility the chicks were placed in Petersime battery cages and were randomly assigned to receive 1 of 5 dietary treatments: (1) nonmedicated, non-NE challenged; (2) nonmedicated, NE challenged; (3) BMD 50 g/t, NE challenged; (4) B. subtilis spores (QST 713) at a dose of 1 × 106 CFU/g of feed, NE challenged; and (5) B. subtilis spores (QST 713) at a dose of 1 × 105 CFU/g of feed, NE challenged. The study began when the birds were placed (d 0), at which time they were allocated to the experimental cages and fed their corresponding dietary treatments. Feed and water were made available ad libitum throughout the study. A randomized complete block design with 10 blocks and 5 treatments per block was used in this study. Eight birds per cage were placed in each battery with a floor space of 0.63 ft2/bird (588 cm2/bird). The feeder space per bird was 68 cm2. Lighting was provided 24 h/d, and a thermostatically controlled gas furnace and air conditioner maintained uniform temperature throughout the study.
Downloaded from http://japr.oxfordjournals.org/ at Purdue University Libraries ADMN on August 28, 2014
by this pathogen leads to NE infection, resulting in severe economic losses in commercial broiler operation. Traditionally, antibiotic feed supplements have been administered to poultry to combat the negative effects of NE. However, due to the increasing concern in antibiotic resistance, the ban on growth-promoting antibiotics to broilers in the European Union, and the increasing demand for naturally produced poultry products, there is a growing interest in finding alternatives to antibiotics for the control and prevention of NE in broiler chickens. Dietary supplementation with probiotics is one of several approaches that have the potential to overcome NE infection. The term probiotic or direct-fed microbial has been defined as a “live microbial feed supplement which beneficially affects the host animal by improving its intestinal balance” [3]. Hofacre et al. [2] challenged broiler chickens experimentally with Clostridium perfringens and observed that probiotics reduced NE-associated mortality and its subclinical effects on FE. Similarly, Craven [4] reported a reduction in C. perfringens colonization and decreased incidence of NE in chickens treated with probiotics. In a field study, Kaldhusdal and Lovland [1] found that posthatch use of microflora prepared from adult birds was associated with delayed intestinal proliferation of C. perfringens, delayed appearance of NE gross lesions, and better production performance at slaughter. Therefore, taken together, the use of probiotics in broiler chickens may show promise in controlling and preventing NE. Among the probiotic preparations currently available for poultry are several products composed of bacterial spores, principally of the genus Bacillus. Spore-forming Bacillus species are ideally suited as commercial probiotic products due to the ability of their spores to survive harsh environmental conditions and long-term storage [5, 6]. Bacterial spores are extremely robust during distribution and administration in animal feeds (e.g., pelleting) and are able to pass through the acidic gastric environment of the target host species and enter the small intestine in a viable state. At present, a limited number of published studies have demonstrated the efficacy of Bacillus subtilis spores as prophylactic agents against NE in broiler chickens. Therefore, an NE challenge study was conducted us-
Tactacan et al.: PROBIOTIC FOR NECROTIC ENTERITIS
Statistical Analysis The statistical analysis was conducted using the SAS software [8]. Performance and lesion score data were analyzed as a randomized complete block design using PROC MIXED, whereas mortality data were analyzed using PROC FREQ. Data are presented as least squares means with differences between means determined using Tukey’s honestly significance difference. All statements of significance were based on P < 0.05.
RESULTS AND DISCUSSION The results from the colony-forming units analysis of the feed samples confirmed that the B. subtilis (QST 713) spores were supplemented at the appropriate high (9.12 × 105 cfu/g of feed) and low (1.13 × 105 cfu/g of feed) doses in the broiler chickens. The NE challenge suppressed the BW gain and increased the FCR of the nonmedicated broiler chickens orally inoculated with coccidial oocysts and C. perfringens. Compared with the non-NE-challenged birds, the BW gain and feed conversion performance of the nonmedicated NE-challenged birds decreased and increased, respectively (P < 0.05), from d 0 to 28 of the study (Figures 1 and 2). The supplementation of B. subtilis (QST 713) spores at 1 × 106 cfu/g of feed (high dose) in broiler chickens mitigated the negative effects of NE. When supplemented at a high concentration, no differences (P > 0.05) in BW gain and FCR were observed between the B. subtilis-treated and the non-NE-challenged birds. However, when B. subtilis (QST 713) spores were supplemented at 1 × 105 cfu/g of feed (low dose), BW gain and FCR decreased and increased, respectively (P < 0.05), relative to the non-NE-challenged group. No significant differences were observed between NE-challenged birds supplemented with B. subtilis (QST 713) spores and BMD. Necrotic enteritis challenge resulted in increased NE lesions in the intestine of the nonmedicated birds on d 22. Compared with the nonchallenged group, NE lesion scores increased significantly in NE-challenged birds that did not receive medication (Figure 3). The supplementation of B. subtilis (QST 713) spores and BMD ameliorated the intestinal lesions caused by NE. The NE lesion scores of birds supplemented with B. subtilis (QST 713) spores and BMD decreased significantly relative to the nonmedicated NE-challenged birds. Consequently, the percentage of NE mortality in the nonmedicated broiler chickens was significantly increased by NE challenge. Mortality due to NE rose to 25%, demonstrating the high virulence of the E. maxima and C. perfringens isolates used in the study. No significant differences were observed in the NE mortality of birds supplemented with a high dose of B. subtilis (QST
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A nonmedicated, corn-soybean meal-based commercial type broiler starter feed compounded with feedstuffs commonly used in the United States was formulated for this study. From this basal starter feed, the experimental treatment feeds were prepared. All diets were in mash form. Three feed samples were collected, one each from the beginning, middle, and end of the batch of treatment diet and mixed to form a composite sample. One composite sample was taken from the composite for each treatment and was used for colony-forming units determination. On d 14, all birds were orally inoculated with a coccidial inoculum containing approximately 5,000 oocysts of Eimeria maxima per bird. Starting on d 19, all birds except treatment 1 were given 1 mL of a broth culture of C. perfringens at approximately 1 × 108 cfu/bird. The birds were administered a fresh broth culture once daily for 3 d (on d 19, 20, and 21). All birds were weighed by cage on 0, 14, 22, and 28 d of age. Feed was weighed on d 0 and remaining feed was weighed at 14, 22, and 28 d of age. On d 22, 3 birds from each cage were selected, euthanized, weighed, and examined for the presence of NE lesions. The scoring was done blindly and based on a score of 0 to 3, with 0 being normal and 3 being the most severe [2]. Means for cage weight gain, feed consumption, feed conversion [adjusted for mortality: feed consumed/(final live weight + mortality weight)], NE lesion scores, and NE mortality were calculated. The mortality was assessed by gross lesions on necropsy-enlarged, dark-colored livers and the pseudomembrane appearance of classical NE in the small intestine.
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Figure 2. The FCR of broiler chickens with or without necrotic enteritis challenged and fed with or without Bacillus subtilis (QST 713) or bacitracin methyl disalicylate (BMD) from 1 to 28 d old. Bars with different letters (a–c) are significantly different (P < 0.05). Data represent means ± SEM (n = 10). med. = medicated.
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Figure 1. The BW gain of broiler chickens with or without necrotic enteritis challenge and fed with or without Bacillus subtilis (QST 713) or bacitracin methyl disalicylate (BMD) from 1 to 28 d old. Bars with different letters (a–c) are significantly different (P < 0.05). Data represent means ± SEM (n = 10). med. = medicated.
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713) spores, BMD, and the non-NE-challenged group. Low dose supplementation of B. subtilis (QST 713) spores decreased NE mortality compared with the nonmedicated NE-challenged birds (Figure 4); however, it increased NE mortality compared with the high dose B. subtilis (QST 713) supplemented, BMD supplemented, and the non-NE-challenged birds. The non-NEchallenged birds had no mortality and did not develop NE. The purpose of this study was to determine the efficacy of B. subtilis (QST 713) spores in overcoming the adverse effects of NE in the production performance of broiler chickens. To identify the appropriate dietary concentration against NE, high (1 × 106 cfu/g of feed) and low (1 × 105 cfu/g of feed) doses of B. subtilis (QST 713) spores were supplemented in the broiler feeds. A treatment group supplemented with BMD was added in the study to gain insights on the potential of B. subtilis (QST 713) spores to replace an antibiotic growth promoter that is used conventionally in controlling and preventing NE. The dietary supplementation of B. subtilis at 1 × 106 cfu/g of feed and BMD was equally ef-
fective in controlling the subclinical and clinical effects of NE in broiler chickens. However, a partial loss in efficacy in terms of reducing NE-associated mortality was observed when B. subtilis (QST 713) spores were incorporated at the lower concentration of 1 × 105 cfu/g of feed. Knap et al. [9] reported a similar decrease in efficacy of Bacillus licheniformis spores when supplemented at a low dose in NE-challenged broiler chickens. In their study, the supplementation of B. licheniformis spores at 8 × 105 cfu/g of feed resulted in decreased BW gain and increased FCR compared with birds fed with B. licheniformis spores at higher concentrations (8 × 106 and 8 × 107 cfu/g of feed). At these concentrations, B. licheniformis-fed birds showed no differences in BW gain, FCR, NE lesion score, and NE mortality in birds treated with virginiamycin (15 g/t). In reports, B. subtilis supplementation in poultry without NE challenge resulted in an increase in BW gain and improvement in FE when fed to turkeys [10] and broiler chickens [11]. Therefore, beneficial effects of B. subtilis may be present in the production performance of poultry even in the absence of clinical NE. Future studies on B. subtilis (QST 713) will be
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Figure 3. The necrotic enteritis (NE) lesion score of broiler chickens with or without NE challenge and fed with or without Bacillus subtilis (QST 713) or bacitracin methyl disalicylate (BMD) at d 22. Bars with different letters (a–c) are significantly different (P < 0.05). Data represent means ± SEM (n = 30). Necrotic lesion scoring was based on Hofacre et al. [2] with a lesion score of 0 = none, 1 = mild, 2 = moderate, 3 = severe. med. = medicated.
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focused on its effect in the production performance of broiler chickens without NE challenge. At present, the mechanism of action of B. subtilis as a probiotic for the control and prevention of NE is not totally clear. Previously, when 1- and 20-d-old chickens were challenged with 1 × 109 spores of B. subtilis strain and infected 24 h later with 1 × 105 cfu of C. perfringens, colonization and persistence of C. perfringens was suppressed [12]. Bacillus spp. are grampositive, spore-forming microorganisms capable of germinating in the gastrointestinal tract of the chickens [13, 14]. The germination of the spores gives rise to metabolically active vegetative cells; therefore, it can be assumed that Bacillus-based probiotics may exert their effects through mechanisms involved in the metabolism of the vegetative cells. Indeed, Teo and Tan [15] reported the production of an antimicrobial factor typical of gram-positive bacteriocin by B. subtilis (PB6) which was found to be active against various strains of Clostridium spp. The production of antimicrobials by probiotics is considered one of the principal mechanisms that inhibit pathogenic microorganisms in the gastrointestinal tract. Bacillus spp. are known to produce a large number of antimicrobials [16];
these include bacteriocins and bacteriocin-like inhibitory substances (e.g., Subtilin, Coagulin), as well as antimicrobials based on peptides and polyketides (e.g., Surfactin, Bacilysin, Difficidin, and Macrolactin). Bacillus subtilis (QST 713) are known to produce some of these antimicrobial compounds (internal data). Stimulation of the bird’s immune system or immunomodulation is another important mechanism that may explain the role of B. subtilis spores against NE. In several studies, it was shown that germinating Bacillus spores can elicit potent immune responses in the gastrointestinal tract, and that this immune stimulation may be the underlying reason why spores exert a probiotic effect [17, 18]. Rhee et al. [19] examined the effects of orally administered bacteria on the development of the gut-associated lymphoid tissue in infant rabbits and found that B. subtilis was of greater importance than other commensal bacteria in gut-associated lymphoid tissue development. Accordingly, Molnar et al. [20] reported increased immunological tissue development in the ileal mucosa of broiler chickens, which paralleled the increasing B. subtilis concentrations in the feed. Gut-associated lymphoid tissue development reflects its role in the defense of
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Figure 4. The necrotic enteritis (NE) mortality of broiler chickens with or without NE challenge and fed with or without Bacillus subtilis (QST 713) or bacitracin methyl disalicylate (BMD) from 1 to 28 d old. Bars with different letters (a–c) are significantly different (P < 0.05). Data represent means ± SEM (n = 80). med. = medicated.
Tactacan et al.: PROBIOTIC FOR NECROTIC ENTERITIS mucosal surfaces against enteric pathogens such as C. perfringens.
CONCLUSIONS AND APPLICATIONS
REFERENCES AND NOTES 1. Kaldhusdal, M., and A. Lovland. 2000. The economic impact of Clostridium perfringens is greater than anticipated. World’s Poult. Sci. J. 16:50–51. 2. Hofacre, C. L., T. Beacorn, S. Collett, and G. Mathis. 2003. Using competitive exclusion, mannan-oligosaccharide and other intestinal products to control necrotic enteritis. J. Appl. Poult. Res. 12:60–64. 3. Fuller, R. 1999. Probiotics for farm animals. Pages 15–22 in Probiotics: A Critical Review. G. W. Tannock, ed. Horizon Scientific Press, Wymondham, UK. 4. Craven, S. E. 2000. Colonization of the intestinal tract by Clostridium perfringens and fecal shedding in diet-stressed and unstressed broiler chickens. Poult. Sci. 79:843–849. 5. Fuller, R. 1989. Probiotics in man and animals. J. Appl. Bacteriol. 66:365–378. 6. Hong, H. A., L. H. Duc, and S. M. Cutting. 2005. The use of bacterial spore formers as probiotics. FEMS Microbiol. Rev. 29:813–835.
7. Food and Drug Administration Center for Veterinary Medicine. 1997. Good target animal study practices: Clinical investigators and monitors. Guidance for industry #58. FDA. Rockville, MD. 8. SAS Institute. 1998. SAS/STAT User’s Guide, Release 6.03 Edition. SAS Inst. Inc., Cary, NC. 9. Knap, I., B. Lund, A. B. Kehler, C. L. Hofacre, and G. Mathis. 2010. Bacillus licheniformis prevent necrotic enteritis in broiler chickens. Avian Dis. 54:931–935. 10. Jiraphocakul, S., T. W. Sullivan, and K. M. Shahani. 1990. Influence of a dried Bacillus subtilis culture and antibiotics on performance and intestinal and microflora in turkeys. Poult. Sci. 69:1966–1973. 11. Santoso, U., K. Tanaka, and S. Ohtani. 1995. Effect of dried Bacillus subtilis culture on growth, body composition and hepatic lipogenic enzyme activity in female broiler chicks. Br. J. Nutr. 74:523–529. 12. La Ragione, R. M., and M. J. Woodward. 2003. Competitive exclusion by Bacillus subtilis spores of Salmonella enterica serotype Enteritidis and Clostridium perfringens in young chickens. Vet. Microbiol. 94:245–256. 13. Jadamus, A., W. Vahjen, and O. Simon. 2001. Growth behavior of a spore forming probiotic strain in the gastrointestinal tract of broiler chickens and piglets. Arch. Tierernahr. 54:1–17. 14. Cartman, S. T., R. M. La Ragione, and M. J. Woodward. 2008. Bacillus subtilis spores germinate in the chicken gastrointestinal tract. Appl. Environ. Microbiol. 74:5254– 5258. 15. Teo, A. Y., and H. Tan. 2005. Inhibition of Clostridium perfringens by a novel strain of Bacillus subtilis isolated from the gastrointestinal tracts of healthy chickens. Appl. Environ. Microbiol. 71:4185–4190. 16. Urdaci, M. C., and I. Pinchuk. 2004. Antimicrobial activity of Bacillus probiotics. Pages 171–182 in Bacterial Spore Formers: Probiotics and Emerging Applications. E. Ricca, A. O. Henriques, and S. M. Cutting, ed. Horizon Bioscience, Oxford, UK. 17. Duc, H., H. A. Hong, N. Fairweather, E. Ricca, and S. M. Cutting. 2003. Bacterial spores as vaccine vehicles. Infect. Immun. 71:2810–2818. 18. Duc, H., H. A. Hong, N. Q. Uyen, and S. M. Cutting. 2004. Intracellular fate and immunogenicity of B. subtilis spores. Vaccine 22:1873–1885. 19. Rhee, K. J., P. Sethupathi, A. Driks, D. K. Lanning, and K. L. Knight. 2004. Role of commensal bacteria in development of gut-associated lymphoid tissues and preimmune antibody repertoire. J. Immunol. 172:1118–1124. 20. Molnár, A. K., B. Podmaniczky, P. Kurti, I. Tenk, R. Glavits, G. Y. Virag, and Z. S. Szabo. 2011. Effect of different concentrations of Bacillus subtilis on growth performance, carcase quality, gut microflora, and immune response of broiler chickens. Br. Poult. Sci. 52:658–665.
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1. Necrotic enteritis was successfully induced in nonmedicated broiler chickens orally inoculated with coccidial oocyst and C. perfringens in this study. The adverse effects of the NE infection in broiler chickens resulted in decreased BW gain, increased FCR, increased intestinal lesions, and increased mortality. 2. Adequate level of dietary B. subtilis (QST 713) spores supplementation was equally effective as BMD in mitigating the subclinical and clinical effects of NE in broiler chickens. 3. Under the conditions of this study, the supplementation of B. subtilis (QST 713) spores could serve as an alternative approach in controlling NE infection in broiler chickens without continuously using antibiotic growth promoter in the feed throughout their growing period.
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