Bacillus amyloliquefaciens CECT 5940 improves performance and gut function in broilers fed different levels of protein and/or under necrotic enteritis challenge.

Two studies were conducted to investigate the effect of Bacillus amyloliquefaciens CECT 5940 (BA) as a probiotic on growth performance, amino acid digestibility and bacteria population in broiler chickens under a subclinical necrotic enteritis (NE) challenge and/or fed diets with different levels of crude protein (CP). Both studies consisted of a 2 × 2 factorial arrangement of treatments with 480 Ross 308 mix-sexed broiler chickens. In study 1, treatments included 1) NE challenge (+/-), and 2) BA (1.0 × 106 CFU/g of feed) supplementation (+/-). In study 2, all birds were under NE challenge, and treatments were 1) CP level (Standard/Reduced [2% less than standard]) and 2) BA (1.0 × 106 CFU/g of feed) supplementation (+/-). After inducing NE infection, blood samples were taken on d 16 for uric acid evaluation, and cecal samples were collected for bacterial enumeration. In both studies, ileal digesta was collected on d 35 for nutrient digestibility evaluation. In study 1, the NE challenge reduced body weight gain (BWG), supressed feed conversion ratio (FCR) and serum uric acid levels (P < 0.001). Supplementation of BA increased BWG (P < 0.001) and reduced FCR (P = 0.043) across dietary treatments, regardless of challenge. Bacillus (P = 0.030) and Ruminococcus (P = 0.029) genomic DNA copy numbers and concentration of butyrate (P = 0.017) were higher in birds fed the diets supplemented with BA. In study 2, reduced protein (RCP) diets decreased BWG (P = 0.010) and uric acid levels in serum (P < 0.001). Supplementation of BA improved BWG (P = 0.001) and FCR (P = 0.005) and increased Ruminococcus numbers (P = 0.018) and butyrate concentration (P = 0.033) in the ceca, regardless of dietary CP level. Further, addition of BA reduced Clostridium perfringens numbers only in birds fed with RCP diets (P = 0.039). At d 35, BA supplemented diets showed higher apparent ileal digestibility of cystine (P = 0.013), valine (P = 0.020), and lysine (P = 0.014). In conclusion, this study suggests positive effects of BA supplementation in broiler diets via modulating gut microflora and improving nutrient uptake.

Modulations of genes related to gut integrity, apoptosis, and immunity underlie the beneficial effects of Bacillus amyloliquefaciens CECT 5940 in broilers fed diets with different protein levels in a necrotic enteritis challenge model.

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-γ (IFN-γ) 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-γ (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.

Bacillus amyloliquefaciens CECT 5940 alone or in combination with antibiotic growth promoters improves performance in broilers under enteric pathogen challenge.

A study was conducted to investigate the effects of Bacillus amyloliquefaciens CECT 5940 as a direct-fed microbial (DFM) alone or in association with bacitracin methylene disalicylate (BMD) in broilers under enteric pathogen challenge. A total of 1,530-day-old male Cobb500 chicks were randomly assigned to 5 treatments, with 9 replicate pens with 34 birds each. Treatments included positive control (PC, basal diet without additives or challenge); negative control (NC, basal diet without additive and challenged birds); NC + 0.05 g/kg BMD; NC + 1 g/kg DFM (106 CFU B. amyloliquefaciens CECT 5940/g of feed); and NC + 0.05 g/kg BMD + 1 g/kg DFM. The challenge consisted of oral gavage with Eimeria maxima and Clostridium perfringens inoculum. Body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR) were evaluated on days 21, 35, and 42. Ileal and cecal content were collected on days 21 and 28 for C. perfringens enumeration by real-time PCR assay and the intestinal health was evaluated by scores. Uniformity (UN), carcass (CY), and breast meat yields (BMY) were evaluated on day 42. After 14 and 21 d post-inoculation, birds in the challenged groups had significant lower FI and BWG compared to the PC group (P < 0.05). However, the groups receiving DFM, BMD, or its combination presented better FCR, CY, BMY, UN, and lower incidence of footpad lesion and litter quality visual scores, compared to the NC group without feed additives (P < 0.05). Mortality was not affected by treatments (P > 0.05). Broilers fed DFM, BMD, or its combination presented lower C. perfringens in ileal content at 21 and 28 d compared to NC group without additives (P < 0.05) and also maintained gut health by keeping the frequency of ballooning, abnormal content, and swollen mucosa comparable to the PC group (P > 0.05). The study indicates that Bacillus amyloliquefaciens CECT 5940 is effective as BMD to provide similar performance and gut health in challenged broilers.

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

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.

The impact of Bacillus subtilis DSM 32315 on the pathology, performance, and intestinal microbiome of broiler chickens in a necrotic enteritis challenge.

It was hypothesized that dietary inclusion of Bacillus subtilis DSM 32315 could inhibit Clostridium perfringens induced necrotic enteritis (NE), thereby improving broiler performance. Male, d 0 chicks were randomly assigned 14 birds/pen, 11 pens/treatment in 3 treatments: a basal diet (control), a coccidiostat fed control (Narasin), and a direct fed microbial (DFM) B. subtilis DSM 32315 treatment. Necrotic enteritis was induced in all birds by oral inoculation of Eimeria maxima oocysts on d 12 and a virulent C. perfringens on d 16. Mortality was reduced (P < 0.001) in DFM and Narasin compared to control. DFM reduced (P < 0.001) feed conversion ratio (FCR) compared to control. Furthermore, DFM and Narasin reduced (P < 0.001) footpad lesions. The DFM was shown to increase (P < 0.05) Bacillus spp. and decrease (P < 0.05) C. perfringens in the ileum and cecum at several time points. To investigate microbiome changes in the cecum, digesta samples were analyzed with % guanine and cytosine (%G+C) microbial profiling which fractionates bacterial chromosomes based on the %G+C in DNA. The method revealed treatment profile peaks in low (27.0 to 34.5%), mid (40.5 to 54.0%), and high (59.0 to 68.0%) G+C fractions. 16S rRNA gene amplification and high throughput sequencing was conducted on each of these fractions in order to elucidate specific bacterial population differences. In the low and mid %G+C fractions, DFM had greater abundance of Lactobacillaceae family members (P = 0.03 and P = 0.01, respectively) and Lactobacillus salivarius (P = 0.04 and P = 0.01, respectively) than control or Narasin. Lactobacillus johnsonii was also greater in the low %G+C fraction compared to control and Narasin (P = 0.01). Lachnospiraceae (P = 0.04) and Ruminococcaceae (P < 0.01) in the mid %G+C fraction were reduced in the DFM compared to control. Positive alterations to the microbial populations in the gut of broilers may at least be a partial mechanism by which B. subtilis DSM 32315 reduced pathology and improved performance of broilers in the NE challenge.

Feeding oscillating dietary crude protein concentrations increases nitrogen utilization in growing lambs and this response is partly attributable to increased urea transfer to the rumen.

Our objective was to determine the effects of feeding oscillating compared with static dietary crude protein (CP) concentrations on nitrogen (N) retention and urea flux across ruminal epithelia. Twenty-seven Suffolk wether lambs (n = 9) were assigned to a medium-CP diet [MEDIUM; 127 g CP⋅kg dry matter (DM)(-1)] or to diets with oscillating CP content (OSC) fed in 2 different sequences, i.e. 2 d of low CP (103 g CP⋅kg DM(-1)) followed by 2 d of high CP (161 g CP⋅kg DM(-1); OSC-HIGH) or vice versa (OSC-LOW). Diet adaptation was for 24 d, followed by 8 d of total urine and feces collection. On d 33, lambs were slaughtered 4 h after the morning feeding, such that those receiving OSC-LOW and OSC-HIGH diets were slaughtered on d 3 of receiving the low- or high-CP diets, respectively. Ruminal epithelia were collected and mounted in Ussing chambers and the serosal-to-mucosal urea flux (J(sm-urea)) was measured using (14)C-urea. Ruminal NH(3)-N concentration was lower (P = 0.001) in lambs fed OSC-LOW compared with those fed OSC-HIGH. Although N intake was similar, retained N (P = 0.001) and microbial N supply (P = 0.001) were greater in lambs fed OSC compared with those fed MEDIUM. The total J(sm-urea) was higher (P = 0.001) in lambs fed OSC-LOW compared with those fed OSC-HIGH. Across diets, the addition of phloretin [a known specific inhibitor of facilitative urea transporter (UT)-B] reduced J(sm-urea) by 19.5-22.3% (P = 0.001); however, phloretin-insensitive J(sm-urea) was the predominant route for transepithelial urea transfer. Taken together, these data indicate that feeding oscillating dietary CP concentrations improves N retention partly by increasing urea recycling to the rumen when animals are fed low-CP diets, but the greater rates of urea transfer cannot be attributable to upregulation of UT-B.

Feeding sunflower oil to partially defaunate the rumen increases nitrogen retention, urea-nitrogen recycling to the gastrointestinal tract and the anabolic use of recycled urea-nitrogen in growing lambs.

The objective of the present study was to delineate how interactions between feeding sunflower oil (SFO) to partially defaunate the rumen and altering dietary ruminally fermentable carbohydrate may alter urea-N kinetics and N metabolism in lambs. In a 4 × 4 Latin square with a 2 × 2 factorial arrangement of treatments, four Suffolk ram lambs (61·5 (se 4·0) kg) were used. Treatments were 0 ( - SFO) v. 6 % (+SFO) SFO and dry-rolled barley (DRB) v. pelleted barley (PB). N balance was measured over 4 d, with concurrent measurement of urea-N kinetics using continuous intra-jugular infusions of [(15)N(15)N]urea. Feeding SFO decreased (P = 0·001) ruminal protozoa and NH3-N concentrations. Urinary N excretion was lower (P = 0·003), and retained N was higher (P = 0·002) in +SFO lambs compared with - SFO lambs. Endogenous production of urea-N (urea-N entry rate; UER) was similar across treatments. Urea-N transfer to the gastrointestinal tract (GIT) (GIT entry rate; GER), expressed as absolute amounts (16·4 v. 13·1 g/d) or as a proportion of the UER (0·693 v. 0·570), its anabolic use (9·0 v. 6·0 g/d) and microbial N supply (14·6 v. 10·9 g/d) were higher (P ≤ 0·001) in +SFO lambs compared with -SFO lambs. As a proportion of the UER, GER was higher, whereas urinary urea-N loss was lower, in lambs fed PB compared with those fed DRB (P = 0·01). In summary, feeding SFO increased urea-N recycling to the GIT and microbial non-NH(3)-N supply, thus providing new evidence that the improved efficiency of N utilization in partially defaunated ruminants could be partly mediated by an increase in urea-N recycling.