Bacillus subtilis PB6 based probiotic supplementation plays a role in the recovery after the necrotic enteritis challenge.
ABSTRACT: In poultry production, birds are raised under intensive conditions, which can enable rapid spread of infections, with Clostridium perfringens-caused necrotic enteritis (NE) being one of the most devastating for the industry. The current investigation was conducted to evaluate the effectiveness of Bacillus subtilis PB6 probiotic supplementation on bird's post NE recovery, based on chicken performance, cecal microbiota composition, ileum histomorphometric measurements, and short-chain fatty acid production in the cecum of the birds that were challenged with NE mid-production. Birds were split into four groups, including a negative control, positive control challenged with C. perfringens, group supplemented with B. subtilis probiotic, and NE challenged birds supplemented with B. subtilis probiotic. Following NE challenge birds were allowed to reach the end of production time at 40 days, and samples were collected to estimate if probiotic supplementation resulted in better post-NE recovery. Intestinal lesion score across the duodenum, jejunum, and ileum indicated that at the end of production timeline NE challenged birds supplemented with B. subtilis probiotic had lower intestinal lesion scores compared to NE challenged birds without probiotic supplementation implying improved recovery. Probiotic supplementation improved performance of NE challenged birds only in the post-NE recovery stage. NE challenged birds had a significant increase in cecal propionic acid, which was not observed in NE challenged birds supplemented with B.subtilus. Both B. subtilis supplemented groups (challenged and unchanged) were characterized by a significant rise in cecal acetic and butyric acid. Our results demonstrate that B. subtilis supplementation can assist the birds in dealing with NE outbreak and long term recovery.
Project description:Necrotic enteritis (NE) continues to present major challenges to the poultry industry, and the etiologic agent Clostridium perfringens is the fourth leading cause of bacterially-induced food- borne illnesses in the US. This study was designed to evaluate the effects of a probiotic during naturally occurring NE. On day of hatch, 1080 Cobb 500 male broilers were randomly allocated to three groups (12 replicate pens/treatment, 30 birds/pen) including 1) negative control (NC): corn-soybean meal diet; 2) positive control (PC): NC + 20 mg virginiamycin/kg diet (0.450 kg Stafac®20/ton); and 3) NC + PrimaLac (1.36 and 0.91 kg/ton from 1-21 and 22-42 days, respectively). One day (d) post placement, all birds were challenged by a commercial live oocyst coccidia vaccine as a predisposing factor to NE. Body weight and feed intake were measured at the onset of NE (d 8) and end of each feeding phase. On d 8, small intestines of two birds/pen were examined for NE lesions, and jejunum samples from one bird were collected for mRNA gene expression analysis of tight junction proteins, cytokines, and nutrient transporters. Data were analyzed using the Jump (JMP) software and significance between treatments identified by LSD (P < 0.05). Compared to NC, supplementation of probiotic reduced d 1-42 mortality; however, PC was the only group with significantly lower mortality. Despite significantly improved feed conversion ratio (FCR) in PC and probiotic groups during d 1-42, average daily gain was only higher in PC (77.69 g/bird) compared with NC (74.99 g/bird). Furthermore, probiotic and PC groups had significantly reduced lesion scores in the duodenum and jejunum compared to NC. Expression of claudin-3 was higher, while expression of zonula occluden-2 tended (P = 0.06) to be higher in probiotic-supplemented birds compared to NC. Moreover, birds fed the probiotic diet had significantly higher expression of IL-10, IL-17, AMPK-?1, and SGLT1 mRNA compared to NC birds. The expression of PepT1 was higher for the probiotic-supplemented group compared to PC. IFN-? expression was lower in PC compared to NC, while there was no difference between probiotic and NC. There were no differences in gene expression of sIgA, TNF-?, IL-1?, and IL-22 among treatments. Collectively, these data indicate that in a naturally occurring NE model, supplementation of a probiotic helps to improve FCR and reduce lesions, potentially due to the improvements in mRNA expression of tight junctions, cytokines, and nutrient transporters.
Project description:Background:The poultry industry is in need of effective antibiotic alternatives to control outbreaks of necrotic enteritis (NE) due to Clostridium perfringens. In the present study, we investigated the effects of dietary supplementation with a blend of encapsulated essential oils and organic acids (BLJ) on growth performance and gut health using a coinfection model of NE in broiler chickens. Methods:Two hundred and eighty-eight one-day-old male Arbor Acres broiler chicks were randomly assigned using a 2?×?2 factorial design into two groups fed either 0 or 500?mg/kg dietary BLJ and co-challenged (or not challenged for the control) with Eimeria spp./C. perfringens. Results:Infected birds fed the BLJ-supplemented diet exhibited an improved feed conversion ratio throughout the trial (P <?0.01), a higher villus height and villus height/crypt depth ratio, and reduced intestinal C. perfringens counts, liver C. perfringens carriage, gut lesion scores and serum fluorescein isothiocyanate dextran (FITC-D) concentrations at 7?d post-infection compared with those of birds without BLJ supplementation (P <?0.05). NE-infected birds fed BLJ exhibited significantly upregulated claudin-1 and IGF-2 mRNA levels (P <?0.05), increased A20 mRNA expression and significantly downregulated TRAF-6, TNFSF15 and TOLLIP mRNA levels in the jejunum at 7?d post-infection compared with those in birds without BLJ supplementation (P <?0.05). Compared with the uninfected and untreated birds, the uninfected birds fed BLJ displayed increased relative abundances of Lactobacillus and Coprococcus but reduced Rikenellaceae levels. Compared with the unsupplemented NE-challenged birds, infected birds fed BLJ showed an increased relative abundance of Unclassified_Lachnospiraceae and a significantly decreased relative abundance of Erysipelotrichaceae. Conclusion:BLJ supplementation improved growth performance and gut health in NE-infected broiler chickens by strengthening the intestinal barrier function, positively modulating the gut microbiota community and differentially regulating intestinal immune responses. Our results also suggested that adding BLJ effectively controlled NE infections after experimental Eimeria and Clostridium perfringens coinfection.
Project description:Salmonella Enteritidis (SE) has been the most common Salmonella serotype associated with foodborne infections in the last several years. Dietary applications of yeast-based preparations in feed have shown to reduce Salmonella colonization in chickens augmenting SE control strategies. This study was conducted to evaluate the effects of a mannan-rich yeast cell wall-derived preparation (Actigen®) administered in feed at a rate of 400 g/ton on SE colonization in the cecum and internal organs of commercial layer chickens. Sixteen week-old layer pullets were orally challenged with a selected nalidixic acid resistant SE strain at a dose of 1.7×10^9 colony forming units (CFU) per bird. SE colonization was assessed by evaluating isolation rates from ovary and pooled liver/spleen samples as well as enumeration of SE in cecal pouches one week post-challenge. Recovery rates of SE from the ovaries of directly challenged birds receiving Actigen® were significantly lower (P <0.02) than those in directly challenged birds fed an unsupplemented control diet. Recovery rates of SE from pooled liver/spleen samples were not significantly different between Actigen®-treated pullets and controls (P = 0.22). Using direct plate count methods, cecal SE concentrations were 1 log10 lower (P <0.001) in challenged pullets in the Actigen®-supplemented group than in the challenged controls. The SE concentration distributions in the ceca were similar in groups testing positive and groups testing negative for SE in the ovaries and liver/spleens tissues. As a result, SE concentrations in the ceca could not be directly related to the occurrence or prevalence of SE in these tissues. In conclusion, Actigen® supplementation appears to decrease the prevalence of SE in ovarian tissue and concentrations of SE in cecal contents and may be useful as a tool for reducing the risk of eggshell contamination and transovarian transmission of SE in eggs.
Project description:Necrotic enteritis (NE) is an infection of the gastrointestinal tract and is estimated to cost the global poultry industry billions of dollars annually. A study was conducted to examine whether reducing the crude protein might offset the severity of NE in broilers experimentally challenged with Eimeria spp. on day 9 and Clostridium perfringens on days 14 and 15. Furthermore, increasing the dietary amino acid (AA) density of the diet was also examined owing to identified benefits of improving performance compromised from low protein (LP) diets or NE. A 2 × 2 × 3 factorial arrangement of treatments at 6 replicates per treatment was used with 972 Ross 308 cockerels fed wheat-sorghum-soy-based diets to 35 D. Factors were NE challenge: no or yes; protein: standard (SP) or LP; and AA density: 100% AA, 115% with only essential AA (115% EAA) increased, and 115% AA with both essential and nonessential AA (115% AA) increased. The performance was measured in grower (days 7-21), finisher (days 21-35), and overall (day 7-35) periods. In addition, on day 16, intestinal lesion score and cecal short-chain fatty acids were measured. Only in nonchallenged birds fed LP diets, 115% AA increased grower feed intake (P < 0.01) and body weight gain (P < 0.05) compared to 115% EAA treatments. Challenge increased jejunal lesions (P < 0.001) with no difference between dietary treatments. Finisher body weight gain was greater in nonchallenged birds fed the 115% AA diets than in challenged birds (P < 0.05). Feeding diets with higher nonessential AA encouraged faster recovery from NE challenge. When fed the SP diets, NE challenge increased cecal butyric acid (P < 0.01) and total short-chain fatty acids (P < 0.05). The nutrient matrix used in LP diets does not favor beneficial butyric acid-producing bacteria. Using LP diets to mitigate NE severity does not offset the predisposing effect of E. spp. when attacking the gastrointestinal tract, and NE recovery is favored when feeding SP diets or additional AA.
Project description:Subclinical necrotic enteritis (NE) was induced in broiler chicks using a high dose of Eimeria spp. vaccine in the drinking water on day 9, and Clostridium perfringens (Cp) culture mixed in the feed on days 14 and 15. The aim was to evaluate the effects of probiotic Bacillus amyloliquefaciens strain H57 (H57) in preventing NE in chicks. Day-old Ross 308, male broilers were weighed and randomly assigned to 6 treatment groups (6 replicate cages/treatment and 8 birds/cage). Birds in group 1 (control) were fed the basal wheat-soybean diet without H57 or NE infection; in group 2 (Eimeria) were treated with Eimeria alone; in group 3 (Cp) were treated with Cp alone; in group 4 (NE) received both Eimeria and Cp; in group 5 (NE-H57) received NE infection and H57; and group 6 (H57) received H57. The basal diet of chicks in groups 5 and 6 was supplemented with H57 at a density of 2 × 108 spores/g feed from 1 D of age. On day 21, there were no significant treatment effects on BW and feed intake between control and H57 birds. However, on day 21, the feed conversion ratio of NE-H57 birds was significantly improved when compared with NE birds (1.28 vs. 1.36; P < 0.001). Birds challenged with NE had a higher occurrence of pasty vent than birds infected with either Eimeria, Cp, or NE-H57 (41 vs. 27 vs. 29 vs. 19%, respectively; P < 0.001). Intestinal lesion scores of NE birds were also higher than those of Eimeria, Cp, and NE-H57 birds (5.67 vs. 2.56 vs. 2.78 vs. 2.10, respectively; P < 0.001) and correlated with pasty vent (Pearson's r = 0.56; P < 0.001). Microscopic evaluation showed mucosal damage and necrosis in NE birds. In contrast, villi from NE-H57 birds were normal, with no damage or infiltration with Eimeria or Cp. H57 appears to be effective in challenged birds, as it maintained epithelial barrier integrity and improved feed efficiency.
Project description:OBJECTIVE:This study assessed the effects of probiotics on cecal microbiota, gene expression of intestinal tight junction proteins, and immune response in the cecal tonsil of broiler chickens challenged with Salmonella enterica subsp. enterica. METHODS:One-day-old broiler chickens (n = 240) were randomly allocated to four treatments: negative control (Cont), multi-strain probiotic-treated group (Pro), Salmonella-infected group (Sal), and multi-strain probiotic-treated and Salmonella-infected group (ProSal). All chickens except those in the Cont and Pro groups were gavaged with 1×108 cfu/mL of S. enterica subsp. enterica 4 days after hatching. RESULTS:Our results indicated that body weight, weight gain, and feed conversion ratio of birds were significantly reduced (p<0.05) by Salmonella challenge. Chickens challenged with Salmonella decreased cecal microbial diversity. Chickens in the Sal group exhibited abundant Proteobacteria than those in the Cont, Pro, and ProSal groups. Salmonella infection downregulated gene expression of Occludin, zonula occludens-1 (ZO1), and Mucin 2 in the jejunum and Occludin and Claudin in the ileum. Moreover, the Sal group increased gene expression of interferon-? (IFN-?), interleukin-6 (IL-6), IL-1?, and lipopolysaccharide-induced tumor necrosis factor-alpha factor (LITAF) and reduced levels of transforming growth factor-?4 and IL-10 compared with the other groups (p<0.05). However, chickens receiving probiotic diets increased Lactobacillaceae abundance and reduced Enterobacteriaceae abundance in the ceca. Moreover, supplementation with probiotics increased the mRNA expression of Occludin, ZO1, and Mucin 2 in the ileum (p<0.05). In addition, probiotic supplementation downregulated the mRNA levels of IFN-? (p<0.05) and LITAF (p = 0.075) and upregulated IL-10 (p = 0.084) expression in the cecal tonsil. CONCLUSION:The administration of multi-strain probiotics modulated intestinal microbiota, gene expression of tight junction proteins, and immunomodulatory activity in broiler chickens.
Project description:Background:The ban of in-feed antimicrobial additives has negatively affected the poultry industry by causing necrotic enteritis (NE) to emerge in the flocks. Alternatives such as Bacillus probiotics have shown to be effective on eliminating the negative effects of this disease. Two experiments were conducted to investigate the effect of Bacillus amyloliquefaciens CECT 5940 (BA) in broiler chickens under NE challenge and/or fed diets with different protein levels. Methods:In both experiments, 480?day-old mix-sexed Ross-308 broilers were arranged in a 2 × 2 factorial arrangement of treatments. In experiment 1, the factors were NE challenge (yes or no) and probiotic (yes or no). In experiment 2, the factors were dietary crude protein levels (standard or reduced) and probiotic (yes or no) and were used under NE challenge condition. Oral administration of Eimeria oocysts (day 9) followed by inoculation with Clostridium perfringens (day 14 and 15) was used to induce NE challenge. On day 16, two birds from each treatment were gavaged with fluorescein isothiocyanate-dextran (FITC-d) and blood samples were collected for gut integrity evaluation, and jejunal samples were collected for gene expression assay. Results:In experiment 1, BA supplementation decreased caspase-3 (CASP3) (P < 0.001) and caspase-8 (CASP8) (P?<?0.05) and increased occludin (OCLD) (P?<?0.05) expression regardless of the challenge. Additionally, BA supplementation downregulated interfron-? (IFN-?) expression (P < 0.01) and upregulated immunoglobulin-G (IgG) (P?<?0.01) and immunoglobulin-M (IgM) (P?<?0.05) only in challenged birds. In experiment 2, the expression of genes encoding mucin-2 (MUC2) (P?<?0.001), tight junction protein-1 (TJP1) (P?<?0.05) and OCLD (P?<?0.05) were upregulated by the addition of BA in the diet, regardless of the crude protein level. Further, BA supplementation downregulated INF-? (P?<?0.01) and upregulated immunoglobulin-A (IgA) (P?<?0.05), IgM (P?<?0.05) and IgG (P?<?0.01) regardless of the crude protein level. Conclusion:These findings suggest that supplementation of BA in broiler diets can improve gut health by modulation of genes related to the mucosal barrier, tight junction, and immunity in broilers challenged by unfavourable conditions such as NE challenge.
Project description:Clostridium perfringens can induce necrotic enteritis of chickens, which causes large economic losses every year. Bacillus licheniformis, a probiotic, can inhibit the growth of pathogenic bacteria such as Clostridium perfringens, thereby improving the health status of chickens. However, from a microbial ecology perspective, the mechanisms by which alterations to the gut microbiota improve health remain unknown. In this study, we used Illumina MiSeq sequencing to investigate the cecal microbiota of a negative control group (NC), a C. perfringens and Eimeria challenge group with fishmeal supplementation (PC), a group supplemented with fishmeal and infected with coccidia (FC), and group PC with B. licheniformis supplementation (BL). We found that the health status of C. perfringens-challenged chickens was compromised, and that B. licheniformis improved the growth of the chickens challenged with pathogens. Microbial diversity analysis and taxonomic profiling of groups NC, PC, and FC revealed a disturbed cecal microflora of the birds with C. perfringens. We also characterized the microbiota of the chickens in the BL group using several methods. Principal coordinate analysis demonstrated that, compared with group PC, the bacterial community structure of group BL was more similar to that of group NC. Linear discriminant analysis with effect size revealed less differentially represented bacterial taxa between groups BL and NC than between groups PC and NC. In addition, groups BL and NC appeared to have similar overrepresented microbial taxa (such as Bacteroides, Helicobacter, Megamonas, and Akkermansia) compared with group PC. Finally, a phylogenetic investigation of communities by reconstruction of unobserved states analysis indicated that large differences existed between group PC and groups NC and BL. In conclusion, pre-treatment with B. licheniformis reduced the disturbance of the cecal microbiome induced by challenge with C. perfringens and other factors in broiler chickens.
Project description:Probiotics have become increasingly popular in the poultry industry as a promising nutritional intervention to promote the modulation of intestinal microbial communities and their metabolic activities as a means of improving health and performance. This study aimed to determine the influence of different probiotic formulations on the taxonomic and metabolic profiling of cecal microbial communities, as well as to define associations between cecal microbiota and growth parameters in 21 and 42-day-old broilers. Probiotics investigated included a synbiotic (SYNBIO), a yeast-based probiotic (YEAST), and three single-strain formulations of spore-forming Bacillus amyloliquefaciens (SINGLE1), B. subtilis (SINGLE2) and B. licheniformis (SINGLE3). Dietary inclusion of SYNBIO, YEAST, SINGLE2, and SINGLE3 into the diets supported a significant stimulation of BW and BWG by 7 days of age. Besides, SYNBIO reduced the overall mortality rate by 42d (p<0.05). No significant variation was observed among different probiotic-based formulations for cecal microbiota composition. However, there was a treatment-specific effect on the metabolic profiles, with a particular beneficial metabolic adaptation by the microbiota when supplemented by SYNBIO and SINGLE2. Furthermore, the population of Lactobacillales was identified to be strongly associated with lower Enterobacteriales colonization, higher BW means, and lower mortality rate of growing broilers. Overall, the results emphasize that probiotic supplementation may enhance the microbial energy metabolism in the ceca of young broilers.
Project description:Three hundred and sixty 1-day-old male broiler chicks were randomly allocated to 4 treatments of 6 replicates to evaluate the effects of cLFchimera, a recombinant antimicrobial peptide (AMP), on gut health attributes of broiler chickens under necrotic enteritis (NE) challenge. Treatments were as follows: (T1) unchallenged group fed with corn-soybean meal (CSM) without NE challenge and additives (NC); (T2) group fed with CSM and challenged with NE without any additives (PC); (T3) PC group supplemented with 20 mg cLFchimera/kg diet (AMP); (T4) PC group supplemented with 45 mg antibiotic (bacitracin methylene disalicylate)/kg diet (antibiotic). Birds were sampled for villi morphology, ileal microbiota, and jejunal gene expression of cytokines, tight junctions proteins, and mucin. Results showed that AMP ameliorated NE-related intestinal lesions, reduced mortality, and rehabilitated jejunal villi morphology in NE challenged birds. While the antibiotic non-selectively reduced the count of bacteria, AMP restored microflora balance in the ileum of challenged birds. cLFchimera regulated the expression of cytokines, junctional proteins, and mucin transcripts in the jejunum of NE challenged birds. In conclusion, cLFchimera can be a reliable candidate to substitute growth promoter antibiotics, while more research is required to unveil the exact mode of action of this synthetic peptide.