A microbial muramidase improves growth performance and reduces inflammatory cell infiltration in the intestine of broilers chickens under Eimeria and Clostridium perfringens challenge.

The objective of the present studies was to evaluate muramidase (MUR) supplementation in broilers under Eimeria and/or Clostridium perfringens challenge. For this, 2 experiments were conducted. Experiment 1. A total of 256 one-day old male Cobb 500 chicks were placed in battery cages in a completely randomized design, with 5 treatment groups, 7 replicate cages per treatment and 8 birds per cage. The treatments were: nonchallenged control (NC), challenged control (CC), CC + MUR at 25,000 or 35,000 LSU(F)/kg, and CC + Enramycin at 10 ppm (positive control-PC). Challenge consisted of 15× the recommended dose of coccidiosis vaccine at placement, and Clostridium perfringens (108 CFU/bird) inoculation at 10, 11, and 12 d. Macro and microscopic evaluation, immunohistochemistry, and gene expression were evaluated at 7, 14, 21, and 28 d of age. Experiment 2. A total of 1,120 one-day old male Cobb 500 chicks were placed in floor pens with fresh litter in a completely randomized design, with 4 treatment groups, 8 replicate pens per treatment, and 35 birds per pen. The treatments were: Control, supplementation of MUR at 25,000 or 45,000 LSU(F)/kg, and a positive control (basal diet plus Enramycin). At 10, 11, and 12 d of the experiment all the birds were inoculated by oral gavage with a fresh broth culture of a field isolate Clostridium perfringens (0.5 mL containing 106 CFU/bird). It was observed that in Experiment 1 MUR supplementation reduced the infiltration of macrophages and CD8+ lymphocytes in the liver and ileum of infected birds, downregulated IL-8 and upregulated IL-10 expression. In Experiment 2, MUR linearly improved the growth performance of the birds, increased breast meat yield, and improved absorption capacity. MUR supplementation elicited an anti-inflammatory response in birds undergoing a NE challenge model that may explain the improved growth performance of supplemented birds.

Host-mediated beneficial effects of phytochemicals for prevention of avian coccidiosis.

Both in vitro and in vivo studies were conducted to evaluate the beneficial effects of green tea extract (GT), cinnamon oil (CO), and pomegranate extract (PO) on avian coccidiosis. In experiment (EXP) 1, an in vitro culture system was used to investigate the individual effects of GT, CO, and PO on the proinflammatory cytokine response and integrity of tight junction (TJ) in chicken intestinal epithelial cells (IEC), on the differentiation of quail muscle cells and primary chicken embryonic muscle cells, and anticoccidial and antibacterial activities against Eimeria tenella sporozoites and Clostridium perfringens bacteria, respectively. In EXP 2 and 3, in vivo trials were carried out to study the dose-dependent effect of blended phytochemicals (GT, CO, PO) on coccidiosis in broiler chickens infected with E. maxima. For EXP 2, one hundred male broiler chickens (0-day-old) were allocated into the following five treatment groups: Control group for non-infected chickens (NC), Basal diet group for E. maxima-infected chickens (PC), PC group supplemented with phytochemicals at 50 (Phy 50), 100 (Phy 100), and 200 (Phy 200) mg/kg feed diets for E. maxima-infected chickens. For EXP 3, one hundred twenty male broiler chickens (0-day-old) were allocated into the following six treatment groups: NC, PC, PC supplemented with phytochemicals at 10 (Phy 10), 20 (Phy 20), 30 (Phy 30), and 100 (Phy 100) mg/kg feed for E. maxima-infected chickens. Body weights (BW) were measured on days 0, 7, 14, 20, and 22, and jejunum samples were used to measure cytokine, TJ protein, and antioxidant enzyme responses at 8 days post-infection (dpi). Fecal samples for oocyst enumeration were collected from 6 to 8 dpi. In vitro, CO and PO reduced LPS-induced IL-1ß and IL-8 in IEC, respectively, and GT enhanced the gene expression of occludin in IEC. PO at 1.0 and 5.0 mg/mL exerted antimicrobial effect against E. tenella sporozoites and C. perfringens bacteria, respectively. In vivo, chickens fed a diet supplemented with phytochemicals showed enhanced BW, reduced oocyst shedding, and decreased proinflammatory cytokines following E. maxima challenge. In conclusion, the combination of GT, CO, and PO in the diet of broiler chickens infected with E. maxima induced enhanced host disease resistance including innate immunity and gut health, which contributed to improved growth and reduced disease responses. These findings provide scientific support for the development of a novel phytogenic feed additive formula that enhances the growth and intestinal health of broiler chickens infected with coccidiosis.

Spray-dried porcine plasma enhances feed efficiency, intestinal integrity, and immune response of broilers challenged with necrotic enteritis.

Re-emergence of enteric diseases in the postantibiotic era has imposed severe loss to the poultry industry leading to the urgent need for appropriate additives to maintain gut health. Recently, more attention has been paid to animal plasma due to its high concentrations of active components such as albumins and globulins. The objective of this study was to evaluate the effects of spray-dried porcine plasma (SDP) supplementation during the starter phase (d 0-10) on growth performance, intestine health, and immune response of broilers under necrotic enteritis (NE) challenge. A total of 720 day-old male broiler parental line chicks (Ross 308) were randomly assigned to a 2 (NE challenge: no, yes) × 2 (SDP: 0, 2%) factorial arrangement with 12 replications of 15 chicks each. To induce NE, birds were inoculated with live Eimeria vaccine on d 9 and Clostridium perfringens on d 14. The body weight of birds and feed consumption were measured per pen on d 8, 10, 24, and 29 to calculate performance parameters. On d 16, three birds per pen were sampled to analyse the intestinal lesion score, gut permeability, villi morphology, relative weight of organs, and immune response. Results showed that SDP improved (P < 0.001) FCR in the pre-challenge phase (d 0-8). The results indicated that supplementing SDP lowered (P < 0.01) FCR at the end of the experiment (d 29). Dietary SDP decreased (P < 0.05) the concentration of FITC-d in serum samples of challenged broilers, although it did not affect the intestinal morphology and lesion score. Birds fed with SDP had a higher (P < 0.05) relative weight of bursa (g/kg live body weight) compared to non-supplemented birds. Supplementing SDP reduced the concentration of interleukin-6 (P < 0.05) and α-1 acid glycoprotein (P = 0.051) in serum samples of broilers. In conclusion, supplementation of SDP in the starter phase enhanced feed efficiency and gut integrity in NE challenged broilers, possibly through manipulating the immune response, while further studies targeting intestinal microflora and key genes are required to explore the mode of action.

Phytochemical composition and In-vitro activity of ethanolic extract of Eucalyptus globulus leaves against multidrug resistant poultry pathogens.

Aim of the present study was to determine the In-vitro antibacterial activity of ethanolic extract of E. globulus leaves against common multidrug resistant poultry pathogens. Phytochemical analysis through HPLC revealed that kaempeferol (7.315min) followed by querecetin (6.655min) and myrecetin (3.655min). Percent area of kaempeferol (6826.88%) was highest, followed by myrecetin (5516.22%) and querecetin (163.748%). Phytochemical investigation of ethanolic extract of E. globulus leaves through GCMS showed highest retention time (min) α-pinene (20.43) and α-terpineol (20.15) accompanied by spathulenol (11.97), piperitone (11.04). The ethanolic extracts of E. globulus leaves showed a highest zone of inhibition against S. pullorum SP6; 20.64± 2.08, E. coli SE 12; 19.75± 2.83, C. perfringens type A (CPM38-01); 19.46± 2.02. The highest level of MIC of E. globulus noted were against S. gallinarum S22; 133.37±53.294, S. gallinarum S1; 130.20±45.10, S. gallinarum S4; 129.47±24.182, S. gallinarum S3; 126.83±72.392. In conclusion, the study confirmed that the ethanolic extract of E. globulus is composed of active ingredients having antibacterial activity and can be referred as an alternate to antibiotics.

Activity of ethanolic extract of Eucalyptus globulus leaves against multi drug resistant poultry pathogens in broiler chicks.

The present study was designed to evaluate the antimicrobial activity of E. globulus leaves in broiler chicks. Total (n=255) day-old chicks were segregated into five groups i.e. Pathogenic E. coli, S. pullorum, S. gallinarum and C. perfringens type A and control negative group. Each bacterial challenged (1x 107 CFU) group was divided into control positive, antibiotic, probiotic and E. globulus group. Experimental birds were exposed to E. coli, S. pullorum, S. gallinarum and C. perfringens type A at different ages. At 35th day of experiment the log reduction for each group was determined. The highest log reduction in E. coli and C. perfringens Type A colonies count were found in E. globulus (3.26) (2.33) treated group followed by antibiotic (2.85) (1.59) and probiotic (2.84) (1.50) respectively. The log reduction in S. pullorum colonies count was highest in E. globulus (2.50) followed by probiotic (2.24) and antibiotic (2.16). The S. gallinarum colonies count log reduction was found highest for antibiotic (2.84) followed by probiotic (2.48) and E. globulus group. The results of in-vivo experiment revealed that ethanolic extract of E. globulus has antibacterial activity and it can be used as a replacement to low level of antibiotics added in poultry feed.

Efficacy of Trace Mineral-Amino Acid Complexes in a Necrotic Enteritis Challenge Model.

Necrotic enteritis (NE) is a common and costly disease of poultry caused by virulent toxigenic strains of Clostridium perfringens. Although the importance of trace minerals for intestinal integrity and health is well documented, there is little information on their role in ameliorating the effects of NE. The two studies reported here examined the effects of replacing a portion of the dietary zinc (Zn), copper (Cu), and manganese (Mn) supplied as sulfates in the control diets with metal-amino acid-complexed minerals in a NE-challenge model consisting of coccidiosis and Clostridium perfringens. In a 28-day battery study, the treatments were the following: (1) no additional Zn or Mn, unchallenged (negative control); (2) no added Zn or Mn, challenged (positive control); (3) added ZnSO4 and MnSO4 at 100 ppm each, challenged; (4) additional ZnSO4 at 60 ppm, Availa-Zn at 40 ppm (Low), and MnSO4 at 100 ppm, challenged; (5) added ZnSO4 at 60 ppm, Availa-Zn at 60 ppm (high), and MnSO4 at 100 ppm, challenged; and (6) added ZnSO4 at 60 ppm, Availa-Zn at 40 ppm, MnSO4 at 60 ppm, and Availa-Mn at 40 ppm, challenged. None of the treatments ameliorated gross lesion scores, but all reduced NE-associated mortality compared with the positive control. At 28 days, the group supplemented with Availa-Zn at 40 ppm (low) had a lower body weight than challenged groups supplemented with Zn and the negative control. In a floor pen study, the five treatment groups were the following: (1) Zn, Mn, and Cu from sulfate sources at 100, 100, and 20 ppm respectively; (2) Zn, Mn, and Cu from sulfate sources at 40, 100, and 20 ppm, respectively, plus Zn from Availa-Zn at 60 ppm; (3) Zn and Mn from sulfate sources at 40 and 100 ppm, respectively, plus Zn from Availa-Zn at 60 ppm and Cu from Availa-Cu at 10 ppm; (4) Zn, Mn, and Cu from sulfate sources at 60, 60, and 20 ppm, respectively, plus Zn and Mn from Availa-Zn/Mn at 40 and 40 ppm, respectively; and (5) bacitracin methylene disalicylate at 55 g/metric ton with Zn, Mn, and Cu from sulfate sources at 100, 100, and 20 ppm, respectively (Zoetis, Inc., Kalamazoo, MI). None of the treatments reduced lesion scores. The Availa-Zn and Availa-Zn/Mn had lower mortality than the sulfate-supplemented feed, whereas Availa-Zn/Cu and bacitracin methylene disalicylate were intermediate and did not differ from the other groups. Considering both trials together, and by using NE mortality as the discriminating factor, we found that adding Zn and Mn exceeding National Research Council requirements reduced NE-associated mortality, and in the floor pen study, complexed Zn and complexed Zn plus Mn appeared to be superior to sulfates.

Effects of Replacing In-feed Antibiotics with Synergistic Organic Acids on Growth Performance, Health, Carcass, and Immune and Oxidative Statuses of Broiler Chickens Under Clostridium perfringens Type A Challenge.

This study was conducted to investigate the effects of replacing in-feed antibiotics with synergistic organic acids on growth performance, health, carcass, and immune and oxidative statuses of broiler chickens under Clostridium perfringens (CP) type A challenge. Two organic acid products were tested: organic acid 1 (OA1), consisting of butyrate, medium-chain fatty acids, organic acids, and phenolics; and organic acid 2 (OA2), consisting of buffered short-chain fatty acids. Six hundred 1-day-old male Arbor Acres broiler chicks were randomly assigned to one of five treatments: Control 1, basal diet, nonchallenged birds; Control 2, basal diet, with CP challenge; antimicrobial growth promoters (AGP), basal diet supplemented with Aureomycin (chlortetracycline), with CP challenge; OA1, basal diet supplemented with OA1, with CP challenge; and OA1OA2, basal diet supplemented with OA1 and OA2, with CP challenge. Each treatment had eight replicate pens of 15 birds. The experiments lasted for 29 days. The disease challenge was performed on days 15-17, with an oral gavage of 0.5 mL of CP culture (2.0 × 108 colony-forming units [CFU]/mL) for each bird. Body weights (BWs), intestinal lesion scores, immune organ indices, and serum malondialdehyde (MDA) concentrations were measured on days 19, 22, and 29, respectively, in three birds per pen. Carcass characteristics were determined on day 29. No treatment-related differences in mortality were noted before (P = 0.28) or after (P = 0.64) challenge or over the whole study period (days 0-28; P = 0.66). On day 19, the BW of Control 2 was lower than other treatments (P < 0.0001). On day 22, AGP, OA1, and OA1OA2 had higher BW than Control 2 (P = 0.001). The breast muscle yield of OA1 and OA1OA2 was higher than AGP (P < 0.05). The abdominal fat yield of OA1OA2 was lower than AGP and Control 2 (P < 0.05). On day 22, the birds fed OA1OA2 showed lower intestinal lesion scores than OA1 (P < 0.05). No treatment-related differences in immune organ (spleen, thymus, and bursa) indices were noted (P > 0.05). On day 29, the MDA concentration of OA1 and OA1OA2 was lower than those of Control 1 and AGP (P < 0.05). In conclusion, the addition of organic acids may protect broiler chickens from severe intestinal lesions and oxidative stress and may help reduce abdominal fat mass deposition. There is potential for organic acid-based products as alternatives for AGP in preventing necrotic enteritis in broilers.

Effect of Bacillus subtilis DSM 32315 under Different Necrotic Enteritis Models in Broiler Chickens: A Meta-Analysis of Five Independent Research Trials.

Challenge models are needed to understand the pathogenesis of necrotic enteritis (NE) and provide the basis of evaluating nonantibiotic feed-additive interventions. In the category of nonantibiotic feed additives, the application of probiotics to improve intestinal health and growth performance of broiler chickens in the face of an NE challenge has been well described. However, it is crucial to evaluate the consistency of specific probiotics for mitigating the disease challenge and improving performance. Therefore, a meta-analysis of five independent research trials was conducted with the objective of evaluating the effect of Bacillus subtilis DSM 32315 (probiotic) on body weight gain (BWG), feed conversion ratio (FCR), NE mortality, and lesion score (LS) of broiler chickens challenged with NE. These independent studies were conducted in three countries (the United States, Thailand, and Finland). The statistical analysis used fixed and random effects to estimate the mean effect size (MES) of the difference between NE-challenged birds (control) and NE-challenged probiotic-fed birds and the 95% confidence interval of MES. A meta-regression was performed to evaluate heterogeneity (MES variance) among studies. The statistical analysis was performed using a robust variance estimation strategy with a SAS macro. Probiotic-supplemented birds had a significantly higher BWG (MES = 1.04, P = 0.009) and a significantly lower FCR (MES = -1.39, P = 0.020), NE mortality (MES = -1.15, P = 0.012), and LS (MES = -1.29, P = 0.045). Response variables of BWG (Q = 2.81, P = 0.560) and NE mortality (Q = 5.60, P = 0.354) did not present heterogeneity. Heterogeneity was found for FCR (Q = 10.34, P = 0.035) and LS (Q = 16.13, P = 0.001). Overall, dietary supplementation of B. subtilis DSM 32315 significantly improved BWG and reduced FCR, mortality, and LS in a repeatable large-scale manner.

Cannabis-derived cannabidiol and nanoselenium improve gut barrier function and affect bacterial enzyme activity in chickens subjected to C. perfringens challenge.

Revealing the multifocal mechanisms affecting cross-talk between Clostridium perfringens pathogenesis and the host response is an urgent need in the poultry industry. Herein, the activity of Cannabis sativa-derived cannabidiol (CBD) and selenium nanoparticles (Nano-Se) in modulating the host response to Clostridium perfringens challenge was investigated in broiler chickens subjected to a mild infection model. The infected chickens exhibited no clinical manifestations, confirming the potential hazard of pathogen transmission to the food chain in the commercial sector. However, both CBD and Nano-Se affected the responses of chickens to C. perfringens challenge. The beneficial actions of both agents were manifested in the upregulated expression of genes determining gut barrier function. Both CBD and Nano-Se promoted shifts in gut bacterial enzyme activity to increased energy uptake in challenged chickens and upregulated potential collagenase activity. There was no opposite effect of CBD and Nano-Se in mediating the host response to challenge, whereas an additive effect was evidenced on the upregulation of gene determining gut integrity. Collectively, these findings indicate that understanding the action mechanisms of CBD and Nano-Se is of great interest for developing a preventive strategy for C. perfringens infection in broilers.

Interactive effect of dietary calcium and phytase on broilers challenged with subclinical necrotic enteritis: 3. Serum calcium and phosphorus, and bone mineralization.

Calcium is chelated by phytic acid and forms phytate-mineral complexes reducing Ca availability and the ability of phytase to hydrolyze phytate. An increased Ca concentration in the gut favors the activity of Clostridium perfringens (C. perfringens). Therefore, it was hypothesized that high dietary calcium with high dietary phytase would decrease serum Ca and P and bone mineralization during necrotic enteritis occurrence. A total of 768 one-day-old Ross 308 male chicks were randomly allocated to 8 treatments with 6 replicate pens, each housing 16 birds. A 2 × 2 × 2 factorial arrangement of treatments was applied: dietary Ca (0.6 or 1.0%), phytase (500 or 1,500 FTU/kg), and challenge (no or yes). Half of the birds (384) were challenged with Eimeria spp. on day 9 and C. perfringens strain EHE-NE18 on day 14 and 15. Blood was collected from 2 birds per pen to determine Ca, P, and parathyroid hormone in the serum. The middle toe, tibia, and femur were excised from 2 birds per pen on day 16 and 29 for determination of ash, breaking strength (BS), and mineral concentration. The challenge decreased (P < 0.05) serum Ca+ in birds regardless of dietary Ca level (day 16). There was a challenge × Ca interaction (P < 0.05) for tibial BS (day 16), with challenge being more severe in birds fed high Ca than low Ca diets. A challenge × phytase interaction (P < 0.05) was present for femur ash (day 16), with high phytase only increasing ash in challenged birds. The challenge decreased (P < 0.05) the BS of femur and tibia at each time point. Birds fed high dietary Ca had lower tibial Mg (P < 0.001), Fe (P < 0.001), Na (P < 0.001), and Zn (P < 0.05) concentrations (day 29). Altogether, high dietary Ca and phytase improved bone mineralization showing that attention to Ca and P nutrition and phytase matrix values is warranted when high levels of phytase are used.

Research Note: Effect of synbiotic supplementation on caecal Clostridium perfringens load in broiler chickens with different necrotic enteritis challenge models.

Studies were conducted to determine the efficacy of synbiotic applications to combat the negative effects of necrotic enteritis (NE). An in vitro study was conducted to test the effect of probiotics species supernatants to decrease Clostridium perfringens (CP) proliferation. Lactobacillus reuteri, Enterococcus faecium, Bifidobacterium animalis, and Pediococcus acidilactici culture supernatants decreased the proliferation of CP at 1:1 supernatant-to-pathogen dilution in vitro. Two in vivo studies were conducted to determine the in vivo response of synbiotic supplementation containing the aforementioned probiotic strains on broiler production performance and caecal CP load in broilers induced with NE infection. In experiment 1, 75 broiler chicks were randomly allotted to 3 treatment groups, control (basal diet), ionophore (Salinomycin), and synbiotic (PoultryStar me), from day of hatch, and NE was induced in all birds. There were no significant treatment effects on BW, feed consumption, and feed gain ratio. However, at 35 D, ionophore or synbiotic supplementation increased (P < 0.05) villi height and decreased interleukin (IL)-1 mRNA abundance, while synbiotic supplementation increased (P < 0.05) IL-10 mRNA abundance compared with the control group, respectively. In experiment 2, 360 broiler chicks were randomly allotted to 3 treatments, an unchallenged negative control (control; basal diet), challenged positive control (NE; basal diet), or NE + synbiotic group (synbiotic). At both 21 and 42 D of age, NE birds had decreased (P < 0.05) BW, feed conversion, and jejunal villi height compared with control, while NE + synbiotic birds were not different from control groups. At 42 D of age, NE birds had 2.2 log/g increased CP in the ceca contents compared with control, while synbiotic birds had CP load that was not different than that of the control group. NE + synbiotic birds had significantly greater amounts of bile anti-CP IgA than the control and NE groups. It can be concluded that synbiotic supplementation decreased CP proliferation in vitro and caecal CP load in vivo while improving production parameters during an NE infection in broilers.

The effect of different dietary ratios of lysine and arginine in diets with high or low methionine levels on oxidative and epigenetic DNA damage, the gene expression of tight junction proteins and selected metabolic parameters in Clostridium perfringens-challenged turkeys.

Two experiments were performed to investigate the effect of different ratios of arginine (Arg) to lysine (Lys) in diets with low (30% Lys; Experiment 1) and high (45% Lys; Experiment 2) methionine (Met) levels on selected metabolic parameters, oxidative and epigenetic DNA damage, and the mechanisms underlying intestinal barrier integrity in turkeys challenged with Clostridium perfringens. In each experiment, 108 one-day-old Hybrid Converter female turkeys were placed in 6 pens (18 birds per pen) and reared for 42 days. At 34, 36 and 37 days of age, half of the birds were subjected to C. perfringens challenge. A 3 × 2 factorial design with three levels of Arg relative to Lys (90, 100 and 110%; Arg90, Arg100 and Arg110, respectively) and C. perfringens infection (-, +) was employed. Challenging birds with C. perfringens increased lipid oxidation and the oxidation and methylation of DNA of intestinal mucosa, and down-regulated the activities of DNA-repairing enzymes. Neither the dietary treatment nor the challenge affected the markers of liver function or metabolism. Arg110 diets with the high Met level induced DNA oxidation and methylation whereas these processes were downregulated in birds fed Arg90 diets. The results indicate that Arg90 diets with high Met levels have a beneficial influence on the indicators of intestinal barrier integrity in turkeys with necrotic enteritis (NE). Despite the analyzed amino acid ratios interacted with the systems responsible for the maintenance of gut integrity in the host organism, this dietary intervention probably enabled birds to cope with NE.

Influence of meat and bone meal, phytase, and antibiotics on broiler chickens challenged with subclinical necrotic enteritis: 1. growth performance, intestinal pH, apparent ileal digestibility, cecal microbiota, and tibial mineralization.

This study investigated the influence of meat and bone meal (MBM), phytase, and antibiotics (AB) on the performance, intestinal pH, ileal digestibility, cecal microbiota, and tibial mineralization in Ross 308 broilers challenged with necrotic enteritis (NE). A total of 672-day-old male Ross 308 chicks were allocated to 8 treatments with 6 replicate pens, with 14 birds each. The study employed a 2 × 2 × 2 factorial arrangement of treatments: MBM (no or yes), AB (no or yes, zinc bacitracin + salinomycin), and phytase level (500 or 1,500 FTU/kg; both using 500 matrix recommendations). Diets were based on wheat-soybean meal-canola meal. All birds were challenged with Eimeria spp on day 9 and Clostridium perfringens (C. perfringens) strain EHE-NE18 on day 14 and day 15. On day 21 (postchallenge), birds fed MBM had reduced weight gain (WG; P < 0.05) relative to without MBM. A 2-way phytase × AB interaction for WG on day 14 (P < 0.001) and day 21 (P < 0.001) and feed conversion ratio on day 21 (P < 0.001) and day 42 (P < 0.01) indicated positive effects of high phytase on bird performance in the presence of AB. On day 42, a 3-way MBM × phytase × AB interaction (P < 0.01) was observed for WG, showing high phytase increased WG with AB, relative to the birds without AB in the presence of MBM. A 2-way MBM × phytase interaction (P < 0.01) was observed for apparent ileal digestibility of Ca and P on day 16, whereby there was a notable reduction in Ca and P digestibility in birds fed MBM-free diets and a low phytase level, but with the high phytase level, Ca and P digestibility was not influenced by MBM. In conclusion, in NE challenged birds, high phytase has a beneficial effect on leg health and mineral utilization to the extent that it can replace MBM and has beneficial effects on bird performance in the presence of AB.

Effect of Bacillus subtilis DSM 32315 on the intestinal structural integrity and growth performance of broiler chickens under necrotic enteritis challenge.

The effect of dietary inclusion of Bacillus subtilis DSM 32315 on the intestinal health and growth performance of Cobb 500 male broilers subjected to a Clostridium perfringens-induced necrotic enteritis (NE) challenge was determined in 2 experiments. In experiment 1, chicks were randomly assigned to 4 treatments of 10 replicate/treatment. In experiment 2, chicks were randomly assigned to 4 treatments of 12 replicates/treatment. The experimental treatments were non-infected, non-supplemented control, infected, non-supplemented control (IC), infected + Bacillus subtilis DSM 32315 (B. subtilis DSM 32315), infected + bacitracin methylene disalicylate (BMD). In both experiments, NE was induced by oral inoculation of toxin producing C. perfringens on 3 consecutive days between 17 and 20 D of age, following exposure of birds to pre-disposing conditions. At day 28 (experiment 1), broilers fed diets with B. subtilis DSM 32315 exhibited a significantly higher body weight, lower mortality, and intestinal NE lesion score, compared to the IC treatment. At day 42 (experiment 2), B. subtilis DSM 32315 supplementation significantly improved BW, feed conversion ratio, production efficiency factor, NE lesion score, and mortality, compared to IC treatment. The effect of B. subtilis DSM 32315 on intestinal integrity of NE challenged chickens was evaluated with histomorphometry. A significantly shallower crypt depth and higher villus height to crypt depth ratio were observed in the mid-intestine of birds belonging to the B. subtilis DSM 32315 group, compared to the IC group. Furthermore, B. subtilis DSM 32315 supplementation significantly reduced the enteritis index associated with NE. In both experiments, the effect of B. subtilis DSM 32315 on the phenotypic measurements of NE and performance was comparable to the effect observed with BMD supplementation. In conclusion, supplementation of the direct fed microbial strain B. subtilis DSM 32315 can ameliorate the pathology and performance detriments associated with NE.

Can dietary zinc diminish the impact of necrotic enteritis on growth performance of broiler chickens by modulating the intestinal immune-system and microbiota?

The objective of this study was to compare the effects of inorganic and proteinate Zn in chickens challenged with coccidia and Clostridium perfringens. A 3 × 2 factorial design was used, with 3 dietary formulations (0 or 90 mg/kg supplemental Zn from ZnSO4 or Zn proteinate, with or without challenge). On day 14, challenged birds were orally gavaged with approx. 5,000 Eimeria maxima sporulated oocysts, and on day 19 to 21 with C. perfringens (108 CFU/D). Productive performance was assessed at day 21 and 28. At 21 D, necrotic enteritis (NE) lesion severity, intestinal permeability, gene expression, and ileal and cecal microbiota were evaluated. An interaction of Zn source by challenge was observed for lesion score and mortality, wherein Zn supplementation decreased the degree of NE lesions (P = 0.02) and mortality due to NE (P = 0.008). In the jejunum, an interaction of Zn source by challenge was observed for the expression of IL-8 (P = 0.001) and INF-γ (P = 0.03), wherein the NE challenge upregulated their expression, but not in the Zn proteinate supplemented birds. Zn proteinate supplementation downregulated iNOS vs. ZnSO4 supplemented birds (P = 0.0003), and supplemental Zn downregulated TLR-2 (P = 0.05) and ZnT5 (P = 0.04), regardless of the source. In the ileal microbiota, Zn proteinate supplementation decreased the frequency of Lactobacillus (P = 0.01), and the challenge increased Enterobacteriaceae (P = 0.01). Dietary Zn decreased NE lesion severity and mortality due to NE; Zn proteinate led to lower expression of IL-8 and INF-γ in challenged birds which may be an indicative of a lessened inflammatory response.

Equilibrium Isotherm Approach to Measure the Capability of Yeast Cell Wall to Adsorb Clostridium perfringens.

Yeast cell wall (YCW) products are currently used as substitutes to antibiotic growth promoters, to improve animal performances, and to reduce the incidence of infectious diseases in livestock. They are claimed to bind enteropathogens, thus interfering with their colonization in the intestinal mucosa. Although the anti-infectious activity of YCW products on Gram-positive pathogens like Clostridium perfringens has been reported in vivo, in vitro evidences on the adsorption of C. perfringens by YCW fractions are not yet available. Preliminary results showed that purified YCW products exert antimicrobial activity toward C. perfringens. Using the adsorption isotherm approach, we measured the ability of YCW products in adsorbing C. perfringens, thus reducing its viability. Dosages of YCW products >1 mg/mL adsorbed 4 Log colony-forming unit (CFU)/mL of C. perfringens in buffered solution. The maximum adsorption of the bacterium was reached in 3 h, whereas only one product of four YCW products retained the adsorption up to 6 h. The analysis of equilibrium isotherms and adsorption kinetics revealed that all products adsorb C. perfringens in a dose- and time-dependent manner, with high affinity and capacity, sequestering up to 4 Log CFU/mg of product. The determination of adsorption parameters allows to differentiate among adsorbents and select the most efficient product. This approach discriminated among YCW products more efficiently than the antimicrobial assay. In conclusion, this study suggests that the ability of YCW products in reducing C. perfringens viability can be the result of an adsorption mechanism.

Zinc source modulates intestinal inflammation and intestinal integrity of broiler chickens challenged with coccidia and Clostridium perfringens.

Two dietary sources of zinc (ZnSO4 or organic Zn) were tested in chickens challenged with coccidiosis (Co) or coccidiosis plus Clostridium perfringens (CoCPF). On day 14, the chickens were orally gavaged with ∼5,000 Eimeria maxima sporulated oocysts. On day 19, 20, and 21 chickens challenged with C. perfringens were given a broth culture containing 108 cfu of this bacterium. Productive performance parameters were determined at d 14, 21, and 28. On day 21, necrotic enteritis (NE) lesions were scored, and intestinal permeability was evaluated. Jejunum and cecal tonsils were collected for morphology and gene expression analysis. On day 21, organic Zn improved BW gain by 18.6% (P = 0.07), and FCR by 12% (P = 0.09) in CoCPF challenged chickens vs. birds fed ZnSO4. From 1 to 28, organic Zn increased BW gain (P = 0.02), and improved FCR (P = 0.03) vs. birds fed ZnSO4. At 21 d, NE lesions were only observed in CoCPF birds (P < 0.001), and mortality due to NE was only observed when CoCPF birds were fed ZnSO4 (P = 0.001). Organic Zn fed birds had increased villus height in the jejunum (P = 0.005) and decreased intestinal permeability (P = 0.01) vs. ZnSO4. In the jejunum, organic Zn fed birds showed a downregulation of expression of IL-8 (P = 0.02), and upregulation of IL-10 (P = 0.05) in CoCPF birds vs. ZnSO4- CoCPF birds. As main effect, birds supplemented with organic Zn had higher mRNA expression of TLR-2 (P = 0.02) and IgA (P = 0.01). In the cecal tonsils, organic Zn fed birds showed upregulation of iNOS (P = 0.008) in CoCPF birds vs. ZnSO4-CoCPF birds. Organic Zn supplementation reduced intestinal permeability and attenuated intestinal inflammation of broilers co-challenged with coccidia and C. perfringens.

Zinc source influences the gene expression of zinc transporters in jejunum and cecal tonsils during broiler challenge with Eimeria maxima and Clostridium perfringens.

The objective of this experiment was to study the effects of dietary zinc (Zn) source on gene expression of Zn transporters (metallothionein [MT], ZIP 3, 5, 8, 9, 10, 11, 13, and 14, and ZnT 1, 4, 5, 6, 7, 9, and 10) in the jejunum and cecal tonsils of broilers challenged with coccidia or coccidia plus Clostridium perfringens. A 2 × 2 factorial design was used with 2 Zn sources (90 mg Zn/kg from either ZnSO4 or an organic Zn, Bioplex® Zn) and challenged with approximately 5,000 oocysts of Eimeria maxima at 14 d of age with or without C. perfringens (108 CFU/bird) at 18, 19, and 20 d of age (8 pens per treatment and 8 birds per pen) after which 1 bird/pen was sampled at 21 d of age. In the jejunum, co-infection resulted in higher ZnT 5 and 6 gene expression, while organic Zn fed birds had lower ZIP 5 and 11, and higher ZnT1. Additionally, an interaction of challenge by Zn source was noted wherein ZnT10 was unaffected by the C. perfringens in the organic Zn treatment but was 2.7-fold lower in the co-infected ZnSO4 fed birds. S100A9 gene expression, a biomarker of inflammatory response in necrotic enteritis, increased 2 and 2.8-fold in the cecal tonsils and jejunum with the co-infection, respectively. Supplementation with organic Zn lowered S100A9 by 1.9 and 4.4-fold in the cecal tonsils and jejunum, respectively, when birds were supplemented with ZnSO4. Notably, MT, ZIP 3, 8, 9, 10, 13, or 14, and ZnT 4, 7, and 9 were unaffected by Zn source and/or method of challenge. An interaction of challenge by Zn source was also noted for serum Zn concentration, which was reduced when birds were challenged with C. perfringens and fed ZnSO4 but no difference between challenge method when birds were fed organic Zn. Based on the expression of ZnT and ZIP genes, more Zn trafficking due to treatment occured in the jejunum than cecal tonsils, but further studies are needed to ascertain how Zn source regulates intracellular free Zn concentrations and whole-body Zn status during an enteric challenge.

Growth of Clostridium perfringens in sous vide cooked ground beef with added grape seed extract.

The growth of Clostridium perfringens from spore inocula was studied in sous vide cooked ground beef with added 0 to 3% grape seed extract (GSE). C. perfringens did not grow at 4 °C with or without GSE present. Lag time (LT) was 95 h in control samples at 15 °C, whereas 1-3% GSE addition significantly (p < .05) extended LT to 244 h or longer. Generation time (GT) in 3% GSE added beef was similar to that of control (19 h, 3% GSE versus 18 h, control) at 15 °C. At 20 °C, GT was 1.5 h in samples without GSE; however, 1-3% GSE addition extended GT about 2-3 folds (p < .05). Lag time at 20 °C was 23 h in control samples, while LT was 40-59 h in samples containing GSE. Interestingly, GSE did not affect LT at 25 °C; however, significantly (p < .05) longer GT was observed in 3% GSE added samples than the other sample groups. Additionally, GSE from 1 to 3% in beef extended the period needed to reach 6 log cfu/g at 15 or 20 °C, while 3% GSE was required at 25 °C. The findings suggest that GSE exhibits concentration and temperature dependent inhibitory effect on growth of C. perfringens in sous vide cooked ground beef. Grape seed extract can be used to extend the shelf-life and ensure the microbiological safety of sous vide cooked meat products.

Histological parameters to evaluate intestinal health on broilers challenged with Eimeria and Clostridium perfringens with or without enramycin as growth promoter.

The maintenance of integrity of the gastrointestinal tract is an important aspect for animal productivity, since it is able to absorb nutrients more efficiently and serves as a barrier against microorganisms. To control agents detrimental to intestinal integrity, growth-promoting antibiotics (AGP) are used, which reduce the number of toxin-producing microorganisms in the intestinal lumen, acting as anti-inflammatory agents. There is a demand for restriction of use of AGP in animal feed, but there are few studies showing what parameters we should observe to search for alternative additives. The aim of this study was to establish histological parameters that explain the effect of enramycin as growth promoter on intestinal health in broilers challenged with Eimeria and Clostridium perfringens. The zootechnical performance and the histology by I See Inside (ISI) methodology were evaluated on liver and ileum samples. Chickens challenged without AGP have the worst BWG, FCR, and histological ISI score (ISI score 9) in the ileum compared to non-challenged (ISI score 5). The use of enramycin on challenged group significantly minimized the ISI score in the ileum at 21 and 28 d (ISI score 7.4 and 8.0, respectively) compared with the challenged group not fed with enramycin (ISI score 9.2 and 9.9, respectively), associated with reduced lamina propria thickness and inflammatory cell infiltration. We suggest these 2 histological parameters as a standard to compare products for gut health.