The effect of butyric acid and nucleotides supplementation on broiler (Gallus gallus domesticus) growth performance, immune status, intestinal histology, and serum parameters.

Background: Butyric acid and its derivatives support the immune system, lessen inflammation, and lessen oxidative stress in broilers in addition to preserving gut homeostasis and epithelial integrity. Broiler performance has also been demonstrated to rise with the addition of nucleotides to the diet. Aim: The purpose of the study was to ascertain the effects of butyric acid and nucleotides added to feed on the overall performance, immunity, oxidant/antioxidant enzyme levels, intestinal histology, and hepatic functions of broilers. Methods: Four experimental groups of thirty chickens, each were used in the present study. The groups were assigned as a control group that received normal diet without additives, butyrate (B) group received the diet supplemented with butyric acid (250 g/ton feed), nucleotides (N) group received the diet supplemented with nucleotides (200 g/ton feed), and the fourth group received the diet supplemented with a combination of butyrate and nucleotide (BN) (250 g/ton B feed, and 200 g/ton N feed, respectively). Necrotic enteritis was produced in ten birds from each group to assess the immune-modulatory effect of these supplements, antioxidant status, intestinal histology, and liver functions were measured in all experimental groups. Results: The addition of butyric acid and nucleotides to feed enhanced body weight, growth performance, hepatic functions, and antioxidant capabilities. Histological sections of the gut from challenged or unchallenged (with necrotic enteritis) groups in the BN group showed considerable improvement, as shown by strong proliferation in intestinal crypts and villus enterocytes. Conclusion: Nucleotides and butyric acid can be added to broiler feeding regimens to enhance growth and health.

Bromelain can reduce the negative effects of a subclinical necrotic enteritis in broiler chickens.

This study was conducted to examine the efficacy of a bromelain-based supplementation coded ANR-pf on growth performance and intestinal lesion of broiler chickens under necrotic enteritis (NE) challenge. A total of 540 Ross 308 day-old male chicks were randomly allocated into 6 treatments of 6 replicates. The bromelain formulation was delivered to chickens through gavaging or in drinking water method twice, on d 8 and 13. Nonchallenged groups included 1) without or 2) with the specific bromelain formulation gavaged at 0.8 mL/kg. NE-challenged groups included 3) without the specific bromelain formulation; 4) gavaged with 0.4 mL/kg; 5) gavaged with 0.8 mL/kg and 6) supplemented with 0.8 mL/kg via drinking water. Birds were challenged with Eimeria spp. on d 9 and Clostridium perfringens (NE-18 strain) on d 14 and 15. On d 14 and 19, fresh faecal contents were collected for the determination of oocyst counts. Intestinal lesion scores were determined on d16. Performance and mortality were recorded throughout the entire experiment. Among challenged groups, birds received additive via drinking water had higher weight gain (WG) compared to the remaining groups (P < 0.001) in the grower phase and had lower FCR compared to 0.4 mL/kg inoculated group in the grower and finisher phases (P < 0.001). Bromelain supplementation via drinking water improved the WG of challenged birds, similar to that of the nonchallenged birds (P < 0.001), and lowered FCR compared to other challenged groups (P < 0.001). Nonchallenged birds and birds that received bromelain formulation in drinking water did not have lesions throughout the small intestine whereas challenged birds, either un-supplemented or supplemented with bromelain via inoculation route recorded similar lesion score levels in the jejunum. At d 19, birds received bromelain in drinking water had lower fecal oocyst numbers compared to challenged birds without additive (P < 0.001). In conclusion, bromelain administration via drinking water could ameliorate the negative impacts of NE-infection in broilers by improving performance, lowering the oocyst numbers and lesion scores.

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.

Enterococcus faecium supplementation prevents enteritis caused by Escherichia coli in goats.

The probiotic Enterococcus faecium is a gut microbe with immunomodulatory effects, which has been widely used to prevent diarrhoea in pigs and birds. Escherichia coli is a common pathogen that causes inflammatory bowel disease in animals. The aim of this study was to investigate the protective effects of E. faecium on enteritis in goats. Forty goats were randomly divided into 4 treatment groups: control, E. faecium, E. coli, and E. faecium + E. coli. The changes of physiological indicators and diarrhoea scoring were evaluated on days -4, -2, 0, 2, 4, 6, and 8. The pathological examination, inflammatory cytokines mRNA expression and bacterial counts in jejunum and caecum were detected on day 4 and 8. The results showed that body temperature, respiratory rate, heart rate and leukocyte counts all increased from the 2nd to the 6th day after feeding with E. coli, and the diarrhoea score was significantly increased. However, E. faecium-pretreated goats had lower body temperatures and fewer leukocytes than E. coli-treated goats on day 2, as well as decreased diarrhoea scores. E. coli treatment caused histopathological damage and morphological changes in the jejunum and caecum, while pretreatment with E. faecium significantly alleviated these injuries. E. faecium pretreatment can reduce the load of E. coli and increase the prevalence of Lactobacillus, thereby balancing the microbiota in the intestine. Furthermore, E. coli-infected goats pretreated with E. faecium showed obvious inhibition of Toll-like receptor 4, interleukin (IL)-1ß, IL-6, IL-8 and tumour necrosis factor-α mRNA expression in the jejunum and caecum compared to that in the E. coli treatment group. In conclusion, the addition of E. faecium to goat feed is beneficial for improving clinical symptoms, maintaining intestinal mucosa integrity, balancing the microbiota and decreasing inflammatory responses in E. coli-induced intestinal injury.

Alleviating effects of essential oil from Artemisia vulgaris on enteritis in zebrafish via modulating oxidative stress and inflammatory response.

Artemisia vulgaris (A. vulgaris) is a traditional Chinese medicine widely distributed in China and contains many bioactive compounds with pharmacological effects. However, the anti-inflammatory effects and mechanism of essential oil from A. vulgaris on enteritis in fish are still unclear. In this study, in order to elucidate the underlying mechanism of essential oil from A. vulgaris on zebrafish enteritis, zebrafish were used for establishing animal models to observe the histopathological changes of intestines, determine the activities of immune-related enzymes and oxidative stress indicators, and the mRNA expression of genes in MyD88/TRAF6/NF-KB signaling pathways. The results showed that different doses of A. vulgaris essential oil could effectively alleviate zebrafish enteritis in a dose- and time-dependent manner by improving the intestinal histopathological damage, decreasing the intestinal oxidative stress, repairing the intestinal immune ability, changing the expression levels of IL-1ß, IL-10 and genes in MyD88/TRAF6/NF-κB pathway. In addition, co-treatment with oxazolone and MyD88 inhibitor could alleviate the morphological damage, the induction of oxidative stress, and the levels of immune-related enzymes and the mRNA expression of genes in MyD88/TRAF6/NF-κB signaling pathway. Moreover, essential oil from A. vulgaris had more significantly therapeutic effects on enteritis of male zebrafish than that of female zebrafish. This result will clarify the therapeutic effect and anti-inflammatory mechanism of essential oil from A. vulgaris on zebrafish enteritis, and provide a theoretical basis for further research on the rationality of A. vulgaris to replace feed antibiotics.

A mixture of organic acids and thymol protects primary chicken intestinal epithelial cells from Clostridium perfringens infection in vitro.

Necrotic enteritis causes economic losses estimated to be up to 6 billion US dollars per year. Clinical and subclinical infections in poultry are also both correlated with decreased growth and feed efficiency. Moreover, in a context of increased antibiotic resistance, feed additives with enhanced antimicrobial properties are a useful and increasingly needed strategy. In this study, the protective effects of a blend of thymol and organic acids against the effects of Clostridium perfringens type A (CP) on chicken intestinal epithelial cells were investigated and compared to bacitracin, a widely used antibiotic in poultry production. Primary chicken intestinal epithelial cells were challenged with CP for a total time of 3 h to assess the beneficial effect of 2 doses of citric acid, dodecanoic acid, and thymol-containing blend, and compare them with bacitracin. During the challenge, different parameters were recorded, such as transepithelial electrical resistance, cell viability, mRNA expression, and reactive oxygen species production. CP induced inflammation with cytokine production and loss of epithelial barrier integrity. It was also able to induce reactive oxygen species production and increase the caspase expression leading to cellular death. The high dose of the blend acted similarly to bacitracin, preventing the disruptive effects of CP and inducing also an increase in zonula occludens-1 mRNA expression. The low dose only partially prevented the disruptive effects of CP but successfully reduced the associated inflammation. This study shows that the usage of thymol combined with 2 organic acids can protect primary chicken intestinal epithelial cells from CP-induced damages creating a valid candidate to substitute or adjuvate the antibiotic treatment against necrotic enteritis.

Effects of therapeutic levels of dietary antibiotics on the cecal microbiome composition of broiler chickens.

Dietary antibiotics, including antibiotic growth promoters (AGPs), have been commonly used to improve health and growth of poultry. The present study investigated the effects of therapeutic doses of dietary antibiotics, including bacitracin methylene disalicylate (BMD), penicillin G potassium (PP) and an ionophore (salinomycin, SA), on the cecal microbiome of chickens. BMD and SA treatments were given as dietary supplements from d 1 to 35 of age. The SAPP (salinomycin+ penicillin G potassium) group was given SA as a dietary supplement from d 1 to 35 of age and PP was added to drinking water from d 19 to 24 of age to simulate common practices for control of necrotic enteritis in broilers. The cecal contents were collected from all treatment groups on d 10, 24, and 35 of age and DNA was extracted for metagenomic analysis of the cecal microbiome. The results revealed that dietary or water supplementation of therapeutic levels of antibiotics and ionophores to chickens significantly altered the cecal microbial homeostasis during different stages of the chicken life. The alpha diversity analysis showed that BMD, SA, and SAPP treatments decreased diversity and evenness of the cecal microbiome of treated chickens on d 10 of age. Species richness was also reduced on d 35 following treatment with BMD. Beta diversity analyses revealed that SAPP and BMD induced significant changes in the relative abundance of Gram-positive and -negative bacteria on d 10, while no significant differences were observed on d 24. On d 35, the non-treated control group had higher relative abundance of unclassified Gram-positive and -negative bacteria compared to SA, SAPP, and BMD treatment groups. Overall, despite their beneficial role in controlling necrotic enteritis outbreaks, the findings of this study highlight the potential negative effects of dietary supplementation of therapeutic levels of antibiotics on the gut microbiome and suggest that adjusting gut bacteria may be required to restore microbial richness and diversity of the gut microbiome following treatment with these antibiotics.

Effects of Dietary Astragalus Polysaccharide Supplementation on the Th17/Treg Balance and the Gut Microbiota of Broiler Chickens Challenged With Necrotic Enteritis.

This study aimed to investigate the effects of dietary astragalus polysaccharide (APS) supplementation on the immune function, gut microbiota and metabolism of broiler chickens challenged with necrotic enteritis (NE). Two hundred forty Arbor Acres broiler chicks (one day old) were randomly assigned using a 2 × 2 factorial arrangement into two groups fed different levels of dietary APS (0 or 200 ppm of diet) and two disease challenge groups (control or NE challenged). The results showed that NE infection significantly increased FCR, mortality rate, Th17/Treg (Th17 cells% in blood and ileum, Th17/Treg, IL-17 and IL-17/IL-10 in blood), NO, lysozyme activity and IL-1ß in blood, intestinal immune cell proportion and activity (Tc%, Treg% and monocyte phagocytic activity in ileum), intestinal inflammatory cytokines (TLR2, NF-κB, TNF-α and IL- 6) gene expression levels, and the number of Clostridium perfringens in cecum. NE infection significantly reduced body weight gain, thymus index, lymphocyte proliferation activity in blood and ileum, villus height and V/C in jejunum, Th cells% and Mucin2 gene expression in ileum. Dietary APS supplementation significantly increased body weight, feed intake, proportion of immune cells (T cells in blood and Tc, Treg in ileum), lymphocyte proliferation activity, V/C in jejunum, and ZO-1 gene expression in ileum. Dietary APS supplementation significantly reduced FCR and mortality rate, Th17/Treg, Th17%, intestinal pathology scores, intestinal inflammatory cytokine gene expression levels, and the number of Clostridium perfringens in cecum. In addition, broilers challenged with NE significantly increased Staphylococcus and Turicibacter and reduced α diversity of microbiota in ileum. Dietary APS supplementation significantly increased α diversity, Romboutsia, Halomonas, propionic acid, butyric acid, formononetin, taurine, cholic acid and equol and downregulated uric acid, L-arginine and serotonin in ileum. Spearman's correlation analysis revealed that Romboutsia, Turicibacter, Staphylocpccus, Halomonas, Streptococcus, Escherichia-Shigella, Prevotella, uric acid, L-arginine, jerivne, sodium cholate and cholic acid were related to inflammation and Th17/Treg balance. In conclusion, APS alleviated intestinal inflammation in broilers challenged with NE probably by regulating intestinal immune, Th17/Treg balance, as well as intestinal microbiota and metabolites.

Mesoporous silica nanoparticles-based formulations of a chimeric proteinous vaccine candidate against necrotic enteritis disease.

To develop a nanoparticle-based vaccine against necrotic enteritis, a chimeric antigen (rNA) consisting of the main antigens of Clostridium perfringens, NetB, and Alpha toxin, was prepared. Then, the rNA molecules were loaded onto the functionalized mesoporous silica nanoparticles (MSNPs) using physical adsorption or covalent conjugation methods. The characterization of synthesized nanoparticles was performed by scanning electron microscopy, dynamic light scattering, zeta potential measurement, Fourier transform infrared spectroscopy, and thermogravimetry techniques. The results revealed that the spherical nanoparticles with an average diameter of 90 ±â€¯12 nm and suitable surface chemistries are prepared. MSNPs-based formulations did not show any significant toxicity on the chicken embryo fibroblast cells. The results of the challenge experiments using subcutaneous or oral administration of the as-prepared formulations in the animal model showed that the as-prepared nanosystems, similar to those formulated with a commercial adjuvant (Montanide), present stronger humoral immune responses as compared to that of the free proteins. It was also indicated that the best protection is obtained in groups vaccinated with MSNPs-based nanovaccine, especially those who orally received covalently conjugated nanovaccine candidates. These results recommend that the MSNPs-based formulated chimeric proteinous vaccine candidates can be considered as an effective immunizing system for the oral vaccination of poultry against gastrointestinal infectious diseases.

Buffered formic acid and a monoglyceride blend coordinately alleviate subclinical necrotic enteritis impact in broiler chickens.

The objective of this study was to evaluate the effect of 2 different doses of a partially buffered formic acid product (Amasil NA; 61% formic acid, 20.5% sodium formate), and a monoglyceride blend of short- and medium-chain fatty acids (BalanGut LS P) on necrotic enteritis (NE) infected broilers in terms of performance, intestinal microbial population and short-chain fatty acids concentrations in the gastrointestinal tract. A total of 528-day-old as hatched Ross 308 broilers were allocated to 48 pens with 11 birds in each pen. Six dietary treatments applied in the study were: T1) nonsupplemented diet (Control); T2) antibiotic supplemented diets; T3) and T4) high (Starter: 0.5%; Grower and Finisher: 0.5%) and low (Starter: 0.3%; Grower and Finisher: 0.2%) dose of Amasil NA; and groups T5) and T6) high (Starter: 0.3%; Grower and Finisher: 0.2%) and low dose (Starter: 0.3%; Grower: 0.15%; Finisher: 0.075%) of (BalanGut LS P). All birds in this study were fed starter (d 0-10), grower (d 11-24) and finisher (d 25-35) diets and challenged with NE. To induce subclinical NE, oral administrations of Eimeria oocysts (d 9) followed by inoculation of Clostridium perfringens strains (d 14 and 15) were applied. Results showed that birds fed the high dose of Amasil NA, had a higher feed conversion ratio (FCR,P < 0.05) compared to the nonsupplemented group during the starter period. Antibiotic supplementation reduced FCR during the grower (P < 0.001), finisher (P < 0.05) and overall (P < 0.001) periods of the experiment. Both levels of BalanGut LS P and low levels of Amasil NA enhanced overall FCR (P < 0.05) compared to the birds in the nonsupplemented group. Compared to the nonsupplemented group, high levels of Amasil NA and low levels of BalanGut LS P improved FCR in the finisher stage (P < 0.05). On d 16, cecum digesta of birds fed with antibiotic supplemented diets showed a significantly lower number of C. perfringens (P < 0.001) compared to the nonsupplemented and high level of BalanGut LS P group. Bacillus (P < 0.01) and Ruminococcus numbers were significantly lower in the birds fed with high level of Amasil NA (P < 0.05) compared to the antibiotic supplemented diets. High doses of Amasil NA, showed the highest propionate concentration in the cecum (P < 0.001). The study suggests that supplementation of BalanGut LS P and Amasil NA at different feeding phases may achieve optimal performance improvement in broilers under NE challenge.

Effects of feed supplementation with 3 different probiotic Bacillus strains and their combination on the performance of broiler chickens challenged with Clostridium perfringens.

The application of probiotics in broiler feed, to alleviate performance deficiencies due to mild infections by coccidia and Clostridium perfringens, is of increasing interest for the poultry industry. Therefore, our objective was to evaluate the capacity of 3 Bacillus strains and their combination as probiotics in vitro and in vivo. Thus, protein and carbohydrate degradation and C. perfringens growth inhibition capabilities were assessed by colometry measurement and an agar diffusion bioassay, respectively. A total of 2,250 1-day-old male broiler chicks were assigned to 5 dietary treatments: 1) non-probiotic-supplemented control (control); 2) control + DSM 32324 at 0.8 × 106 cfu/g of feed; 3) control + DSM 32325 at 0.5 × 106 cfu/g of feed; 4) control + DSM 25840 at 0.3 × 106 cfu/g of feed; and 5) control + DSM 32324 + DSM 32325 + DSM 25840 at 1.6 × 106 cfu/g of feed. A pathogenic field strain of C. perfringens was used to induce the necrotic enteritis challenge on day 19, 20, and 21. All birds and remaining feed were weighed on pen basis on day 0, 21, 35, and 42, to calculate BW gain and mortality-adjusted feed conversion. Mortality and mortality due to necrotic enteritis were recorded daily. On day 21, 45 birds per treatment were evaluated for macroscopic intestinal necrotic enteritis lesions. Performance data were statistically analyzed using an ANOVA and subjected to a least significant difference comparison. Necrotic enteritis lesion scores were statistically analyzed using nonparametric Kruskal-Wallis test. Dunn's test was used for treatment comparison. The tested strains showed different abilities of degrading protein and carbohydrates and inhibiting C. perfringens growth in vitro. The birds fed the multi-train combination presented significantly better performance and lower necrotic enteritis lesion score than those in the control group. Dietary supplementation with probiotics resulted in significantly lower necrotic enteritis mortality. The results demonstrate the suitability of the evaluated Bacillus multistrain combination as an effective probiotic in C. perfringens-challenged chickens.

Direct evidence of the preventive effect of milk replacer-based probiotic feeding in calves against severe diarrhea.

Diarrhea is a major cause of death in calves and this is linked directly to economic loss in the cattle industry. Fermented milk replacer (FMR) has been used widely in clinical settings for calf feeding to improve its health and growth. However, the protective efficacy of FMR on calf diarrhea remains unclear. In this study, we verified the preventive effects of FMR feeding on calf diarrhea using an experimental infection model of bovine rotavirus (BRV) in newborn calves and a field study in dairy farms with calf diarrhea. In addition, we evaluated the protective efficacy of lactic acid bacteria-supplemented milk replacer (LAB-MR) in an experimental infection model. In the experimental infection, calves fed FMR or high-concentrated LAB-MR had diarrhea, but the water content of feces was lower and more stable than that of calves fed normal milk replacer. The amount of milk intake also decreased temporarily, but recovered immediately in the FMR- and LAB-MR-fed calves. As compared with the control calves, FMR- or LAB-MR-fed calves showed less severe or reduced histopathological lesions of enteritis in the intestinal mucosa. In a field study using dairy calves, FMR feeding significantly reduced the incidence of enteritis, mortality from enteritis, duration of a series of treatment for enteritis, number of consultations, and cost of medical care for the disease. These results suggest that feeding milk replacer-based probiotics to calves reduces the severity of diarrhea and tissue damage to the intestinal tract caused by BRV infection and provides significant clinical benefits to the prevention and treatment of calf diarrhea.

Analysis of miRNA Expression in the Ileum of Broiler Chickens During Bacillus licheniformis H2 Supplementation Against Subclinical Necrotic Enteritis.

Subclinical necrotic enteritis (SNE) is one of the serious threats to the poultry industry. Probiotics have been proven to exert beneficial effects in controlling SNE. However, their exact mechanisms have not been fully elucidated. Moreover, few studies have focused on their impact on microRNAs (miRNAs). Therefore, the present study aimed to explore the miRNA expression profiles in the ileum of broiler chickens during probiotic supplementation for controlling SNE. A total of 180 newly hatched male broilers were randomly allocated into three groups, including a negative control group, an SNE infection group, and a Bacillus licheniformis H2 pretreatment group. Illumina high-throughput sequencing was conducted to identify the miRNA expression of the three groups. Results showed that 628 miRNAs, including 582 known miRNAs and 46 novel miRNAs, were detected in the miRNA libraries. The target genes of 57 significantly differentially expressed miRNAs were predicted and annotated. Moreover, they were found to be partly enriched in pathways related to immunity and inflammation such as tumor necrosis factor receptor binding, immune response-regulating signaling pathway, Toll-like receptor 2 signaling pathway, interleukin-15 production, activation of NF-κB-inducing kinase activity, and MAP kinase tyrosine/serine/threonine phosphatase activity. Some of the target genes of 57 miRNAs were related to the MAPK signaling pathway. Furthermore, the expression of several miRNAs, which may be involved in the MAPK signaling pathway, was significantly affected by SNE induction and showed no significant difference in the presence of H2. All these findings provide comprehensive miRNA expression profiles of three different treatment groups. They further suggest that H2 could exert beneficial effects in controlling SNE through immune and inflammatory response associated with altered miRNA expression, such as the MAPK signaling pathway.

Varying starch to fat ratios in pelleted diets: II. Effects on intestinal histomorphometry, Clostridium perfringens and short-chain fatty acids in Eimeria-challenged broiler chickens.

1. The hypothesis behind the study was that a high dietary starch level (HS) would lead to impaired gut health compared to a low-starch diet (LS) in Eimeria-challenged broilers. The effects of two diets with different starch to fat ratios on intestinal histomorphometry, Clostridium perfringens counts and toxin profile, necrotic enteritis prevalence and abundance of short-chain fatty acids (SCFAs) were examined. 2. A total of 1,920 one-day-old Ross 308 broiler chickens were fed one of two isocaloric diets formulated either with high (32:1) or low (2:1) starch to fat ratios from d 10 to 29 of age. Each treatment group had 12 pen replicates containing 80 broilers each. On d 17, the chickens were challenged with Eimeria vaccine strains. Samples were collected on d 16, 21-23 and 29. 3. Whereas villus length increased gradually throughout the study in the HS group, a peak level was reached on d 21-23 in the LS group. On d 29, the HS group had significantly longer villi than the LS group. 4. Caecal SCFA concentrations were higher in the HS group compared to the LS group on d 16. In both groups, the SCFA level peaked on d 21-23, with the most pronounced increase seen in the LS group. 5. The C. perfringens netB:cpa ratio increased from d 16 to 29 in the HS group. C. perfringens counts and necrotic enteritis prevalence were similar between the two groups. 6. Diet affected the dynamics of small intestinal villus length and caecal SCFA abundance. These findings suggest that structural remodelling of the small intestine is an adaptation to different dietary starch levels, and that caecal SCFA abundance is associated with the availability of substrate for the microbiota in the posterior intestinal segments. Chickens adapted to higher levels of dietary starch might be more robust against Eimeria infections due to increased mucosal surface area. Studies with other dietary starch sources are required to clarify the impact of dietary starch levels on intestinal health in Eimeria-challenged broilers.

Insect (black soldier fly, Hermetia illucens) meal supplementation prevents the soybean meal-induced intestinal enteritis in rainbow trout and health benefits of using insect oil.

Black solider fly larvae (BSFL) and their oils (BSFLO) are receiving increasing attention as sustainable ingredients in fish feeds, but mostly as replacements to marine sources. There were two aims to this study; in exp. 1, soybean meal (SBM)-based diets were formulated to contain BSFL as supplements at 0 (SBM), 8 (SBM + BSFLlow) or 16% (SBM + BSFLhigh) with a control diet being fishmeal-based (FM). In exp. 2, diets included only fish oil (FO), soybean oil (SBO), BSFLO or BSFLO + bile acid (BA), and all lipid sources were added at 16%. Both experiments were run at the same time and fed to rainbow trout (32 g) with each treatment being triplicated. After 10 weeks the fish were sampled for liver and distal intestine histology, expression of genes responsible for inflammation in the intestine and kidneys, and serum peroxidase and lysozyme activities. In exp. 1, supplementations of BSFL effectively prevented SBM-induced intestinal enteritis, down-regulated intestinal prostaglandin and interferon regulatory factor 1 (IRF-1), while the SBM + BSFLhigh diet significantly increased serum lysozyme activity. In exp. 2, BSFLO caused no histomorphological change to the liver or intestine, but kidney interluekin-8, tumor necrosis factor and IRF-1 were significantly upregulated along with significantly higher serum peroxidase activity. The inclusion of BA in the BSFLO diets significantly upregulated intestinal prostaglandin gene expression. Overall, BSFL supplementations of 8 or 16% prevented SBM-induced intestinal enteritis based on histological observations, which was supported by a down-regulation in pro-inflammatory genes and enhanced innate immunity. Meanwhile, the use of BSFLO showed some immunological benefits. Therefore, these sustainable resources are recommended in the diets of rainbow trout, especially when using elevated levels of plant-based proteins.

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.

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.

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.

Interactive effect of 2 dietary calcium and phytase levels on broilers challenged with subclinical necrotic enteritis: part 1-broiler performance, gut lesions and pH, bacterial counts, and apparent ileal digestibility.

This study investigated the hypothesis that high dietary calcium (Ca) would stimulate necrotic enteritis (NE) and reduce performance, gut health, and nutrient digestibility, and if increased, phytase would reduce NE. Ross 308 male broilers (n = 768) were randomly distributed to 8 treatments in a factorial arrangement. Factors were NE challenge (no or yes), phytase level (500 or 1,500 FTU/kg using 500 FTU/kg matrix values), and Ca level (0.6 or 1.0% starter, 0.5 or 0.9% grower, 0.4 or 0.8% finisher) with the same level of available P (0.40 S, 0.35 G, and 0.35 F). There were 48 pens, 16 birds per pen and 6 replications. Half of the birds were challenged with Eimeria spp on day 9 and 108 CFU per mL of Clostridium perfringens strain EHE-NE18 on day 14 and 15. Gain was higher in birds fed high phytase on day 14 (P < 0.01), day 21 (P < 0.01), day 28 (P < 0.01), and day 35 (P < 0.01). Birds fed high phytase had greater livability on day 21 (P < 0.01). Ca was more digestible in high-Ca diets on day 16, and an NE × Ca interaction (P < 0.05) showed this effect to be more pronounced in unchallenged than in challenged birds. A challenge × Ca interaction for apparent ileal digestibility (AID) of crude protein (CP) (P < 0.05) indicated lower AID of CP in challenged birds fed high Ca. The challenge decreased AID of Ca (P < 0.01). Ca level had no impact on C. perfringens count, but it decreased Lactobacillus (P < 0.05) and Bifidobacteria (P < 0.05) populations in the ceca. High dietary Ca decreased feed conversion ratio. Overall (42 D), the highest WG was observed in unchallenged birds fed high Ca and high phytase with the lowest WG observed in NE-challenged birds fed low Ca and low phytase. The results suggest that full matrix values for high doses of phytase may be appropriate during NE challenge.

Interactive effect of dietary calcium and phytase on broilers challenged with subclinical necrotic enteritis: part 2. Gut permeability, phytate ester concentrations, jejunal gene expression, and intestinal morphology.

Calcium has the capacity to interact with phytate-P to form Ca-phytate complexes and decrease the ability of exogenous phytase to degrade phytic acid. This study investigated the hypothesis that high dietary Ca would impair gut permeability, phytate esters (inositol x-phosphate, IPx: IP3, IP4, IP5, and IP6) degradation, jejunal gene expression, and intestinal morphology. Ross 308 day-old male broilers (n = 768) were distributed into 48-floor pens each housing 16 birds in a factorial arrangement. Factors were NE challenge-no or yes; phytase level of 500 or 1,500 FTU/kg, and Ca level 0.6 or 1.0% starter, 0.5 or 0.9% grower, 0.4 or 0.8% finisher with available P in each phase. Challenged birds were gavaged with 3 field strains of Eimeria on day 9 and 108 CFU per mL of Clostridium perfringens Strain EHE-NE18 on day 14 and day 15. A phytase × Ca interaction was observed in the ileum for IP3 (P < 0.01), IP4 (P < 0.05), and IP6 (P < 0.01). The IP3 and IP4 concentrations were similar for both doses of phytase in the presence of low Ca, but with high Ca, both increased significantly but to a greater extent when the high dose of phytase was used. While IP6 concentrations were low and similar between both doses of phytase at low Ca levels, increasing dietary Ca levels increased IP6 concentrations regardless of phytase dose, but the effect was greater in the low phytase diet. A phytase × Ca interaction was detected for vitamin D receptor (VDR) (P < 0.05) expression where bird fed low phytase and low Ca recorded the highest expression of VDR, all other treatments being equivalent. The challenge decreased crypt depth to villus height ratio (P < 0.001). Challenge birds had higher fluorescein isothiocyanate dextran (P < 0.05) in blood compared with unchallenged birds. Thus, high Ca and high phytase, while not the best for IP6 destruction, did not lead to huge reductions in indicators of gut health.