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J APPL POULT RES 2006. 15:229-235
© 2006 Poultry Science Association
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Research Reports

Effect of Bacillus subtilis PB6 (CloSTAT) on Broilers Infected with a Pathogenic Strain of Escherichia coli1

A. Y.-L. Teo and H.-M. Tan2

Kemin Industries (Asia) Pte. Ltd., Research and Development Department, 12 Senoko Drive, Singapore 758200, Republic of Singapore

Correspondence: 2 Corresponding author: haimeng.tan{at}kemin.com


   SUMMARY
TOP
 SUMMARY
DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSIONS
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
In an attempt to demonstrate the efficacy of Bacillus subtilis PB6, isolated from the gut of a healthy chicken, on broiler performance, an experimental trial was conducted in which broilers, infected or uninfected with a pathogenic strain of Escherichia coli, were supplemented with B. subtilis PB6 (CloSTAT) or zinc bacitracin-colistin sulfate and compared against negative controls given no antibiotic supplementation. We observed 10- and 8-point improvements in feed conversion ratio (FCR) in 42-d-old uninfected broilers treated with B. subtilis PB6 when compared with the negative and antibiotic controls, respectively. However, infected birds supplemented with B. subtilis PB6 registered a significant 15-point FCR improvement over those in the negative control group (P < 0.05). Compared with the negative control, the increase in body weights of uninfected and infected 42-d-old broilers receiving B. subtilis PB6 were 97 and 152 g, respectively (P < 0.05). The growth-promoting and protective results from this study indicated that B. subtilis PB6 not only helped in the maintenance of beneficial bacteria but also could act as a replacement for antimicrobial growth promoters in broilers. The percentages of mortality of infected birds within antibiotic-treated groups and B. subtilis PB6-treated groups were reduced from 14% to 6 and 8%, respectively. Numerically, uninfected birds supplemented with B. subtilis PB6 had elevated levels of lactobacilli (1.4 to 4.5 x 107 cfu/g) in their intestinal tracts (32- and 42-d-old broilers) compared with the controls. Even after being challenged with a pathogenic strain of E. coli, the lactobacilli counts in the B. subtilis PB6-treated birds tended to remain the same as those receiving antibiotic and were considerably higher than those of the untreated birds.

Key Words: Bacillus subtilis • Escherichia coli • Lactobacillus spp. • antibiotic growth promoter replacement • feed conversion ratio


   DESCRIPTION OF PROBLEM
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
MATERIALS AND METHODS
 RESULTS AND DISCUSSIONS
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
A number of species from the Bacillus genus have been considered safe for application in the food and agricultural industries [1, 2]. Compared with other bacteria that are used for probiotic purposes, Bacillus spp. can be administered orally as cells or spores. The fact that Bacillus spp. can exist as vegetative cells or spores can be advantageous because they are heat resistant and tolerant of bile salts [3]. These characteristics are especially important for those bacteria intended for use in agricultural feed, in which they would have to survive high temperature during the pelleting process and, upon ingestion, the lytic action of bile salts in the intestinal tract of the animal. Previous studies have demonstrated that many strains of lactic acid bacteria are not suitable for use as probiotics in the agricultural industry because they are not resistant to heat and the effect of bile salts [4]. Our studies have shown that cells of Bacillus subtilis can withstand heating of up to 100°C for several minutes [5]. In addition, results from our laboratory have also shown that spores of Bacillus spp. could survive in 0.5% bile salts and continue to germinate into vegetative cells. In terms of beneficial effects, not much is known about the metabolites produced by Bacillus spp. in the intestinal tracts of animals. However, studies in our laboratory have demonstrated the production of antimicrobial factor(s) by certain strains of Bacillus spp. that are capable of inhibiting Clostridium spp., Campylobacter spp., and Streptococcus spp. [6, 7], the first 2 being of particular health importance in the poultry industry. Other studies have suggested that there is immunostimulating activity associated with the use of B. subtilis as probiotic [8, 9].

Therefore, in the present study, our primary objective was to evaluate the effect of B. subtilis PB6 on the performance of broilers. In addition, the protective effect of B. subtilis PB6 was evaluated on broilers challenged with a strain of pathogenic Escherichia coli.


   MATERIALS AND METHODS
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
RESULTS AND DISCUSSIONS
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
Experimental Design
Six hundred 1-d-old broilers were randomly divided into 3 groups. Each group was then divided into 2 subgroups: A and B. The broilers in subgroup B were infected with a pathogenic strain of E. coli (108 cfu/mL) isolated from the intestinal tract of an infected broiler. The broilers in subgroup A were not infected with the microorganism. Each subgroup had 5 replicates with 20 birds in each replicate. There were even numbers of female and male birds in each replicate. The design of trial and feed formulation are shown in Tables 1Go and 2Go, respectively. No antibiotic growth promoter was supplemented in the diets of the negative control group except for maduramicin-ammonium (Cygro) [10]. The antibiotic control feed contained 16.7 mg of zinc bacitracin/kg and 3.3 mg of colistin sulfate/kg. The treatment group contained cells of B. subtilis PB6 (CloSTAT) [11] added at an inclusion rate of 109 cfu/t of feed. All diets, upon the addition of coccidiostat, antibiotic or B. subtilis PB6, were subjected to 80 to 85°C steam pelleting. All broilers were maintained in cages with free access to feed and water except for the period before infection with E. coli. Faint lighting was provided during the night to assure continuous feeding and water intake.


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  		Table 1. Design of experiment
 

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  		Table 2. Composition of diets and nutrient contents
 
E. coli Challenge Method
Broilers of 18, 28, and 38 d of age in the B subgroups were infected with a pathogenic strain of E. coli. The strain was subcultured in a nutrient broth medium for 24 h and then diluted in sterile water. Birds in subgroups B from each treatment group were not provided water for 2 h before being treated with water containing E. coli. The doses of E. coli fed to the 18-, 28-, and 38-d-old broilers were 8 x 107, 2 x 108, and 4 x 108 cfu/chick, respectively, to ensure all challenged birds received the same concentration of the target pathogen. As controls, birds from each subgroup A were treated with sterile water.

Enumeration and Identification of Bacterial Cells
On d 32 and 42, 8 broilers (4 in each subgroup, 2 males and 2 females) from each group were euthanized, and their intestinal tracts were removed immediately. Segments between the duodenum and jejunum portions were tied with thread to prevent further agitation of the intestinal contents. A 0.5-g sample of chyme from the jejunum portions was collected, mixed, and resuspended in sterile physiological saline solution. Cells of Lactobacillus spp. were plated onto deMan, Rogosa, and Sharpe (MRS) agar media acidified with lactic acid to pH 5.3. The MRS agar plates were then incubated at 37°C under 5% CO2 for 48 h. Similarly, 0.5-g of chyme from the jejunum portions was collected from E. coli-challenged birds, mixed, and resuspended in sterile physiological saline solution before plating onto eosin methylene blue (EMB) agar. After 48 or 24 h of incubation at 37°C, cells of Lactobacillus spp. and E. coli, respectively, were enumerated and recorded. Representative colonies from the MRS (pH 5.3) or EMB agar plates were Gram stained and microscopically examined to confirm their morphological features. A biochemical test kit, API 50 CHB/L [12] was used to identify Lactobacillus pp. from the rest of the bacteria. The carbohydrate fermentation profiles were then computed using the APILAB Plus software [12] against a database.

Statistics and Analysis of Data
The measurements of feed consumption and mortality of broilers in each replicate of subgroups were recorded daily. The body weights of all birds were measured on d 21 and 42 posthatching. Experimental data were then analyzed using Microsoft Excel 2000 [13] other statistical analysis was conducted using ANOVA in the SPSS statistical package [13] and Duncan’s multiple range test [14] to analyze for statistical significance between the subgroups.


   RESULTS AND DISCUSSIONS
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSIONS
CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
Bird Performance
The performance of broilers in each group and subgroup is summarized in Table 3Go. Poor performance in terms of both body weight and FCR was associated with broilers infected with a pathogenic strain of E. coli within the negative control group. Infected and uninfected broilers had similar FCR and weight gains after supplementation with B. subtilis PB6. Throughout the entire 42-d posthatching, we observed an increasing trend in weight gain and improved FCR of uninfected and infected broilers supplemented with B. subtilis PB6 compared with those in the antibiotic and negative control groups.


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  		Table 3. The effects of antibiotic, Bacillus subtilis PB6, and Escherichia coli challenge on broiler performance
 
During the first 21 d posthatching, uninfected broilers treated with B. subtilis PB6 resulted in increased weight gain of 2.1 and 3.9% compared with the antibiotic and negative controls, respectively (Table 3Go). For the period from 21 to 42 d, 10- and 11-point FCR improvements were observed in uninfected broilers treated with B. subtilis PB6 compared with the negative and antibiotic control groups, respectively. Overall, the body weights of 42-d-old uninfected broilers treated with B. subtilis PB6 increased signifi-cantly at 4.4 and 4.9% when compared with the negative and antibiotic controls, respectively (P < 0.05). A 10-point improvement in FCR was observed in uninfected broilers treated with B. subtilis PB6 when compared with the negative control and an 8-point enhancement in FCR when compared with the antibiotic control.

The positive effect of B. subtilis PB6 supplementation on FCR was observed for 21-, 21-to-42-, and 42-d-old infected broilers when compared with the negative control (Table 3Go). Statistical significance was observed in the FCR of 21-d-old infected broilers supplemented with B. subtilis PB6 (1.64) compared with the FCR of the negative control (1.77; P < 0.05). Overall, there was statistical significance in the improvement of FCR for infected broilers treated with B. subtilis PB6 (1.81) compared with the FCR of the negative infected control (1.96) for the entire period of 42 d (P < 0.05). When compared with the antibiotic control, the effect of B. subti-lis PB6 supplementation on FCR of 21-, 21-to-42-, and 42-d-old infected broilers improved by 5, 11, and 7 points, respectively.

A statistically significant increase in weight gain (5.9%) was observed in 21-d-old infected broilers treated with B. subtilis PB6 compared with those in the negative control group (P < 0.05; Table 3Go). A 7.6% increase in weight gain was observed in 21-to-42-d-old infected birds treated with B. subtilis PB6 compared with the negative control birds. Compared with the negative control, an overall increase of 7% in body weight of 42-d-old infected broilers treated with B. subtilis PB6 was observed (P < 0.05). Throughout the entire duration of 42 d, an increase of 3.8% in weight gains of infected birds treated with B. subtilis PB6 was observed in comparison to the antibiotic treatment.

The results of mortality are shown in Table 4Go. The mortality rate appeared to be greater among infected broilers than among uninfected broilers. Although there was a lack of significance, mortalities of infected birds within antibiotic-treated and B. subtilis PB6-treated groups were 6 and 8%, respectively, compared with 14% in the negative control group (Table 4Go).


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  		Table 4. The effects of antibiotic, Bacillus subtilis PB6, and Escherichia coli on mortality1
 
In contrast to some studies that reported no inherent benefits with the use of probiotic micro-organisms, others have shown that these micro-organisms enhance digestion and absorption of nutrients [15, 16, 17, 18, 19] in animals. In this regard, our present study showed a trend in broilers treated with B. subtilis PB6, which improved the FCR of broilers up to 42 d of age, particularly in those that were challenged with E. coli (1.81; P < 0.05). Furthermore, there was a significant increase in the body weight of infected 42-d-old broilers treated with B. subtilis PB6 when compared with the birds from the negative control group (P < 0.05).

Comparison of Lactobacillus Cell Counts Among Treatments
Among the unchallenged 32-d-old broilers, a numerical increase in cells of Lactobacillus was observed in B. subtilis PB6-treated broilers (~7.6-log) compared with the negative (~6.5-log) and antibiotic (~6.5-log) controls (Table 5Go). Although no statistical significance was observed, the challenged 32-d-old broilers that were treated with B. subtilis PB6 seemed to maintain higher numbers of Lactobacillus (~6.9-log) compared with ~6.3 and ~6.5-log, respectively, for the negative and antibiotic controls (Table 5Go).


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  		Table 5. Comparison of the Lactobacillus cell counts between challenged and unchallenged broilers (32- and 42-d-old broilers)1,2
 
A similar trend was also observed in the unchallenged 42-d-old birds from various treatment groups (Table 5Go). Although no statistical significance was observed, the cell counts of Lactobacillus in uninfected 42-d-old broilers treated with B. subtilis PB6 were higher than the negative control. After challenge with E. coli, the population of Lactobacillus in 42-d-old broilers treated with B. subtilis PB6 and antibiotic were similar in counts (~6.5 to 7.0 log) but greater than those of the negative control group (~5.8 log) (Table 5Go). Counts for lactoba-cilli were highest in birds fed with B. subtilis PB6 when unchallenged with E. coli. Counts of lactobacilli were similar to antibiotic-treated birds after the E. coli challenge for the 32- and 42-d-old birds.

There are many beneficial effects associated with the use of microbial probiotics in animal feed [20, 21, 22]. These include the competitive exclusion of pathogenic E. coli [23], Campylo-bacter jejuni [24], and Salmonella enteritidis [25] to enhance growth and viability of benefi-cial gut microflora, such as lactic acid bacteria [26]. Similar to findings by Hosoi et al. [26], the current study showed that the level of viable Lactobacillus cells in the intestinal tracts of unchallenged 42-d-old broilers treated with B. sub-tilisPB6 was at least 1 log10 greater than those from the negative and antibiotic control groups. Even after being challenged with a pathogenic strain of E. coli, the levels of lactobacilli in birds supplemented with B. subtilis PB6 were similar to those of the antibiotic-treated birds.

Other criteria used for defining probiotic bacteria include bile and acid stability [27, 28], production of antimicrobial substances [29], and meeting safety or generally recognized as safe (GRAS) status [30, 31]. Previous studies in our laboratory have indicated that B. subtilis PB6 is stable in bile or acid solutions [5] and produces an antimicrobial factor(s) of a proteinaceous nature that is stable under high heat and solvents [6]. Cytotoxicity studies have also confirmed that B. subtilis PB6 is nonpathogenic or toxi-genic for use in the feed industry [7]. These results taken together indicate that B. subtilis PB6 can act as a replacement for the antimicrobial growth promoters in broilers.


   CONCLUSIONS AND APPLICATIONS
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSIONS
 CONCLUSIONS AND APPLICATIONS
REFERENCES AND NOTES
 

  1. Poor body weight and feed efficiency were associated with broilers infected with a pathogenic strain of E. coli compared with the uninfected ones within the negative control group.
  2. The body weight and FCR of 42-d-old infected broilers treated with CloSTAT, a product containing B. subtilis PB6, improved significantly at 6.9% and 15 points, respectively, when compared with those infected with E. coli in the negative control group.
  3. CloSTAT increased the concentrations of Lactobacillus spp. within the intestinal tracts of 32- and 42-d-old uninfected broilers when compared with birds fed the negative and antibiotic controls.
  4. The cell counts of lactobacilli were similar in infected and uninfected birds fed diets containing CloSTAT and antibiotic.
  5. Although no statistical significance was observed, a decreasing trend in the percentage of mortality of infected birds within antibiotic-treated and CloSTAT-treated groups was observed relative to the negative control group.


   ACKNOWLEDGMENTS
 
We thank Zhou Yan Min (Nanjing Agricultural University, PR China) for help with the trial and microbiological analyses.


   FOOTNOTES
 
1 The use of trade names in this publication does not imply endorsement of the products mentioned or criticism of similar products not mentioned. Back


   REFERENCES AND NOTES
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSIONS
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 

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