Optimization of surfactin production from Bacillus subtilis
 in fermentation and its effects on Clostridium perfringens
-induced necrotic enteritis and growth performance in broilers

Clostridium perfringens (CP) is the principal pathogenic bacterium of chicken necrotic enteritis (NE), which causes substantial economic losses in poultry worldwide. Although probiotics are known to provide multiple benefits, little is known about the potential effects of Bacillus subtilis (B. subtilis) application in preventing CP-induced necrotic enteritis. In this study, 450 male Arbor Acres broilers were divided into 5 experimental treatments: A: basal diet (control group); B: basal diet and CP challenge (model group); C: CP challenge+10 mg/kg enramycin (positive control group); D: CP challenge+ 4 × 10 7 CFU / kg of feed B. subtilis PB6 (PB6 low-dosage group); and E: CP challenge+ 6 × 10 7 CFU / kg of feed B. subtilis PB6 (PB6 high-dosage group). There were 6 replicate pens per treatment with 15 broilers per pen. The present research examined the effect of Bacillus subtilis PB6 (B. subtilis PB6) on growth performance, mRNA expression of intestinal cytokines and tight junctions, and gut flora composition in broilers challenged with CP. The entire experiment was divided into two phases: the non-CP challenge phase (d0–18) and the CP challenge phase (d18–26). PB6 did not increase the growth performance during the first stage, but the PB6 high-dosage group was found to have larger body weight gain and ADFI during the CP challenge stage. Feed supplementation with PB6 reduced the lesion score of challenged chicks, with increased tight junction-related gene expression (occludin and ZO-1) and decreased TNF-α expression compared with CP-infected birds. A decrease in the abundance of Clostridium XI, Streptococcus, and Staphylococcus was observed after CP infection ( P < 0.05 ), while supplementation with PB6 restored the ileal microbial composition. In conclusion, administration of B. subtilis PB6 improved growth performance, enhanced intestinal barrier function, and mitigated intestinal inflammation/lesions, which might be due to its restoring effects on the ileal microbial composition in CP-challenged broilers.

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Supplementing Garlic Nanohydrogel Optimized Growth, Gastrointestinal Integrity and Economics and Ameliorated Necrotic Enteritis in Broiler Chickens Using a Clostridium perfringens Challenge Model

Animals ◽

10.3390/ani11072027 ◽

2021 ◽

Vol 11 (7) ◽

pp. 2027

Author(s):

Doaa Ibrahim ◽

Tamer Ahmed Ismail ◽

Eman Khalifa ◽

Shaimaa A. Abd El-Kader ◽

Dalia Ibrahim Mohamed ◽

...

Keyword(s):

Growth Performance ◽

Clostridium Perfringens ◽

Broiler Chickens ◽

Body Weight Gain ◽

Economic Benefits ◽

Production Costs ◽

Broiler Chicks ◽

Necrotic Enteritis ◽

Feed Conversion ◽

Intestinal Lesion

Necrotic enteritis (NE) caused by Clostridium perfringens (C. perfringens) results in impaired bird growth performance and increased production costs. Nanotechnology application in the poultry industry to control NE outbreaks is still not completely clarified. Therefore, the efficacy of dietary garlic nano-hydrogel (G-NHG) on broilers growth performance, intestinal integrity, economic returns and its potency to alleviate C. perfringens levels using NE challenge model were addressed. A total of 1200 male broiler chicks (Ross 308) were assigned into six groups; four supplemented with 100, 200, 300 or 400 mg of G-NHG/kg diet and co-challenged with C. perfringens at 21, 22 and 23 d of age and two control groups fed basal diet with or without C. perfringens challenge. Over the total growing period, the 400 mg/kg G-NHG group had the most improved body weight gain and feed conversion efficiency regardless of challenge. Parallel with these results, the mRNA expression of genes encoding digestive enzymes (alpha 2A amylase (AMY2A), pancreatic lipase (PNLIP) and cholecystokinin (CCK)) and intestinal barriers (junctional adhesion molecule-2 (JAM-2), occludin and mucin-2 (Muc-2)) were increased in groups fed G-NHG at higher levels to be nearly similar to those in the unchallenged group. At 14 d post challenge, real-time PCR results revealed that inclusion of G-NHG led to a dose-dependently decrease in the C. perfringens population, thereby decreasing the birds’ intestinal lesion score and mortality rates. Using 400 mg/kg of G-NHG remarkably ameliorated the adverse effects of NE caused by C. perfringens challenge, which contributed to better growth performance of challenged birds with rational economic benefits.

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Bacillus subtilis DSM 32315 Supplementation Attenuates the Effects of Clostridium perfringens Challenge on the Growth Performance and Intestinal Microbiota of Broiler Chickens

Microorganisms ◽

10.3390/microorganisms7030071 ◽

2019 ◽

Vol 7 (3) ◽

pp. 71 ◽

Cited By ~ 9

Author(s):

Cristiano Bortoluzzi ◽

Bruno Serpa Vieira ◽

Juliano Cesar de Paula Dorigam ◽

Anita Menconi ◽

Adebayo Sokale ◽

...

Keyword(s):

Bacillus Subtilis ◽

Clostridium Perfringens ◽

Intestinal Microbiota ◽

Broiler Chickens ◽

Body Weight Gain ◽

Dietary Supplementation ◽

Necrotic Enteritis ◽

Feed Conversion ◽

Negative Effects ◽

Infected Control

The objective of this study was to evaluate the effects of the dietary supplementation of Bacillus subtilis DSM 32315 (probiotic) on the performance and intestinal microbiota of broiler chickens infected with Clostridium perfringens (CP). One-day-old broiler chickens were assigned to 3 treatments with 8 replicate pens (50 birds/pen). The treatments were: non-infected control; infected control; and infected supplemented with probiotic (1 × 106 CFU/g of feed). On day of hatch, all birds were sprayed with a coccidia vaccine based on the manufacturer recommended dosage. On d 18–20 the infected birds were inoculated with CP via feed. Necrotic enteritis (NE) lesion score was performed on d 21. Digestive tract of 2 birds/pen was collected on d 21 to analyze the ileal and cecal microbiota by 16S rRNA sequencing. Performance was evaluated on d 28 and 42. On d 21, probiotic supplementation reduced (p < 0.001) the severity of NE related lesion versus infected control birds. On d 28, feed efficiency was improved (p < 0.001) in birds supplemented with probiotic versus infected control birds. On d 42, body weight gain (BW gain) and feed conversion ratio (FCR) were improved (p < 0.001) in probiotic supplemented birds versus infected control birds. The diversity, composition and predictive function of the intestinal microbial digesta changed with the infection but the supplementation of probiotic reduced these variations. Therefore, dietary supplementation of Bacillus subtilis DSM 32315 was beneficial in attenuating the negative effects of CP challenge on the performance and intestinal microbiota of broilers chickens.

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Inhibition of Clostridium perfringens by a Novel Strain of Bacillus subtilis Isolated from the Gastrointestinal Tracts of Healthy Chickens

Applied and Environmental Microbiology ◽

10.1128/aem.71.8.4185-4190.2005 ◽

2005 ◽

Vol 71 (8) ◽

pp. 4185-4190 ◽

Cited By ~ 82

Author(s):

Alex Yeow-Lim Teo ◽

Hai-Meng Tan

Keyword(s):

Bacillus Subtilis ◽

Campylobacter Jejuni ◽

Clostridium Perfringens ◽

Mass Spectroscopy ◽

Campylobacter Coli ◽

Necrotic Enteritis ◽

Spectroscopy Technique ◽

Antimicrobial Spectrum ◽

Optimum Growth Temperature ◽

Optimum Ph

ABSTRACT The objectives of this study were to isolate beneficial strains of microorganisms from the gastrointestinal tracts of healthy chickens and to screen them against Clostridium perfringens, a causative agent of necrotic enteritis in poultry. One of the bacteria isolated, a strain of Bacillus subtilis, was found to possess an anticlostridial factor that could inhibit the C. perfringens ATCC 13124 used in this study. The anticlostridial factor produced by B. subtilis PB6 was found to be fully or partially inactivated in the presence of pronase, trypsin, and pepsin. In contrast, the antimicrobial activity of the anticlostridial factor was not affected by treatment at 100 or 121°C or by treatment with any of the organic solvents used in the study. The optimum growth temperature and optimum pH for production of the anticlostridial factor were 37°C and 6.20, respectively. Using the mass spectroscopy-mass spectroscopy technique, the apparent molecular mass of the anticlostridial factor was estimated to be in the range from 960 to 983 Da. In terms of the antimicrobial spectrum, the anticlostridial factor was inhibitory toward various strains of C. perfringens implicated in necrotic enteritis in poultry, Clostridium difficile, Streptococcus pneumoniae, Campylobacter jejuni, and Campylobacter coli.

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Bacillus subtilis DSM29784 Alleviates Negative Effects on Growth Performance in Broilers by Improving the Intestinal Health Under Necrotic Enteritis Challenge

Frontiers in Microbiology ◽

10.3389/fmicb.2021.723187 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yuanyuan Wang ◽

Yibin Xu ◽

Shengliang Xu ◽

Jinyong Yang ◽

Kaiying Wang ◽

...

Keyword(s):

Bacillus Subtilis ◽

Growth Performance ◽

Economic Losses ◽

Broiler Chicks ◽

Necrotic Enteritis ◽

Negative Effects ◽

Treatment Groups ◽

Beneficial Effects ◽

Intestinal Lesions ◽

Colony Forming Units

Along with banning antibiotics, necrotic enteritis (NE), especially subclinical enteritis (SNE), poses a significant threat to the chicken industry; however, probiotics are a potentially promising intervention. We aimed to investigate the beneficial effects of Bacillus subtilis DSM29784 (BS) on the treatment of Clostridium perfringens (CP)-induced SNE in broilers. A total of 360 1-day-old broiler chicks were divided into three treatment groups, namely control (Ctr), SNE, and BS treatment (BST) groups, all of which were fed with a basal died for 21days, and then from day 22 onward, only the BST group had a BS supplemented diet (1×109 colony-forming units BS/kg). On day 15, all chicks, except the Ctr group, were challenged with a 20-fold dose coccidiosis vaccine and 1ml CP (2×108) on days 18–21 for SNE induction. Beneficial effects were observed on growth performance in BST compared to SNE broilers. BST treatment alleviated intestinal lesions and increased the villus height/crypt depth ratio. Further, BST broilers showed increased maltase activity in the duodenum compared with SNE chicks, and a significantly decreased caspase-3 protein expression in the jejunum mucosa. Moreover, an increased abundance of Ruminococcaceae and Bifidobacterium beneficial gut bacteria and an altered gut metabolome were observed. Taken together, we demonstrate that the manipulation of microbial gut composition using probiotics may be a promising prevention strategy for SNE by improving the composition and metabolism of the intestinal microbiota, intestinal structure, and reducing inflammation and apoptosis. Hence, BS potentially has active ingredients that may be used as antibiotic substitutes and effectively reduces the economic losses caused by SNE. The findings of this study provide a scientific foundation for BS application in broiler feed in the future.

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Recombinant Bacillus subtilis Expressing the Clostridium perfringens Alpha Toxoid Is a Candidate Orally Delivered Vaccine against Necrotic Enteritis

Infection and Immunity ◽

10.1128/iai.00686-08 ◽

2008 ◽

Vol 76 (11) ◽

pp. 5257-5265 ◽

Cited By ~ 54

Author(s):

Tran H. Hoang ◽

Huynh A. Hong ◽

Graeme C. Clark ◽

Richard W. Titball ◽

Simon M. Cutting

Keyword(s):

Bacillus Subtilis ◽

Clostridium Perfringens ◽

Vegetative Cell ◽

Active Agent ◽

Secretory Iga ◽

Toxin Gene ◽

Spore Coat ◽

Necrotic Enteritis ◽

Alpha Toxin

ABSTRACT Recombinant Bacillus subtilis endospores have been used to vaccinate against tetanus and anthrax. In this work, we have developed spores that could be used to vaccinate against Clostridium perfringens alpha toxin and that could be used to protect against gas gangrene in humans and necrotic enteritis in poultry. The primary active agent in both cases is alpha toxin. A carboxy-terminal segment of the alpha toxin gene (cpa) fused to the glutathione-S-transferase (GST) gene was cloned in B. subtilis such that the encoded GST-Cpa247-370 polypeptide had been expressed in the following three different ways: expression in the vegetative cell, expression on the surface of the spore coat (fused to the CotB spore coat protein), and a combined approach of spore coat expression coupled with expression in the vegetative cell. Mice immunized orally or nasally with three doses of recombinant spores that carried GST-Cpa247-370 on the spore surface showed the most striking responses. This included seroconversion with anti-Cpa247-370-specific immunoglobulin G (IgG) responses in their sera, a Th2 bias, and secretory IgA responses in saliva, feces, and lung samples. Neutralizing IgG antibodies to alpha toxin were detected using in vitro and in vivo assays, and a toxin challenge established protection. Mice immunized nasally or orally with recombinant spores were protected against a challenge with 12 median lethal doses of alpha toxin. Existing use of spores as competitive exclusion agents in animal feeds supports their use as a potentially economical and heat-stable vaccine for the poultry industry.

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