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The search for alternative therapies to antimicrobial growth promoters (AGP) in poultry production has gained momentum in the past years because of consumer preference and government restrictions on the use of AGP in animal production. Flavonoids are plant-derived metabolites that have been studied for their health-promoting properties that could potentially be used as an alternative to AGP in poultry. In a previous study, we showed that the inclusion of a flavonoid-rich corn cultivar (PennHFD1) in the diet improved the health of broilers undergoing necrotic enteritis. However, the mechanisms of action by which the PennHFD1-based diet ameliorated necrotic enteritis are unknown. This study describes the microbial diversity and composition of the jejunum and ileum of chickens co-infected with Eimeria maxima and Clostridium perfringens and treated with a high-flavonoid corn-based diet. Luminal content and mucosal samples from the jejunum and ileum were collected for DNA extraction, 16S rRNA amplicon sequencing and data analyses. The infection model and the dietary treatments significantly changed the alfa diversity indices (Mucosal samples: ASVs, P = 0.04; Luminal content samples: ASVs, P = 0.03), and beta diversities (Mucosal samples: P < 0.01, Luminal content: P < 0.01) of the ileal samples but not those of the jejunal samples. The microbial composition revealed that birds fed the high-flavonoid corn diet had a lower relative abundance of C. perfringens compared to birds fed the commercial corn diet. The treatments also changed the relative abundance of other bacteria that are related to gut health, such as Lactobacillus. We concluded that both the infection model and the dietary high-flavonoid corn changed the broilers' gut microbial diversity and composition. In addition, the decrease in the relative abundance of C. perfringens corroborates with a decrease in mortality and intestinal lesions due to necrotic enteritis. Collecting different segments and sample types provided a broader understanding of the changes in the gut microbiota among treatments. [ABSTRACT FROM AUTHOR]

No study has demonstrated the use of dietary Cannabis-derived cannabidiol (CBD) to alter the stress response in chickens or examined its effects on meat volatile compounds (VOCs). Here, we subjected chickens to dysbiosis via C. perfringens infection or Escherichia coli lipopolysaccharide (LPS) treatment and investigated the potential link between meat VOCs and cecal bacterial activity and the ameliorative effect of CBD. The cecal bacterial production of short-chain fatty acids (SCFAs) was closely correlated with meat VOCs. CBD supplementation reduced the formation of breast meat spoilage VOCs, including alcohols, trimethylamine and pentanoic acid, in the challenged birds, partly by decreasing cecal putrefactive SCFA production. Meat VOC/cecal SCFA relationships differed according to the challenge, and CBD attenuated the effects of C. perfringens infection better than the effects of LPS challenge on meat VOCs. These findings provide new insights into the interactions among bioactive agent supplementation, gut microbiota activity and meat properties in birds. [ABSTRACT FROM AUTHOR]

Background: Two experiments were conducted to establish an optimal NE challenge model and evaluate the efficacy of stimbiotic (STB) supplementation in necrotic enteritis (NE) challenged broilers. In Exp. 1, a total of 120 Arbor Acres (AA) broilers (45.0 ± 0.21 g) were randomly assigned to 6 treatments in a 3 × 2 factorial arrangement. Vaccine treatments included non-challenge (0), × 10 the recommended dose (× 10) or × 20 the recommended dose (× 20) by the manufacturer. Clostridium perfringens (CP) treatments were non-challenge (No) or 3 mL of 2.2 × 107 CFU CP challenge (Yes). In Exp. 2, a total of 72 AA broilers (40.17 ± 0.27 g) were randomly assigned to 6 treatments in a 3 × 2 factorial arrangement. Dietary treatments included non-additive (CON), 100 mg/kg STB (STB) and 100 mg/kg STB on top of a typical commercial blend including an essential oil, probiotics, and enzyme (CB). Challenge treatments included non-NE challenge (No) and NE challenge (Yes) as established in Exp. 1. Results: In Exp. 1, CP and vaccine challenge decreased (P < 0.05) body weight (BW), body weight gain (BWG) and feed intake (FI), and increased (P < 0.05) the number of broilers with diarrhea and intestinal lesions. The oral administration of × 20 recommended dose of vaccines coupled with 3 mL of 2.2 × 107 CFU CP resulted in (P < 0.01) a significantly increased incidence of wet litter and intestinal lesions. Thus, this treatment was chosen as the challenge model for the successful inducement of NE in Exp. 2. In Exp. 2, the NE challenge negatively affected (P < 0.01) growth performance, ileal morphology, immunoglobulin contents in blood, caecal microbiota in the caecum, footpad dermatitis, intestinal lesion scores, tumour necrosis factor (TNF-α) and endotoxin in the serum compared with the non-NE challenged birds. The supplementation of STB and CB in diets enhanced (P < 0.05) growth performance, intestinal microbiota, and blood profiles by stimulating ileal morphology (VH and VH:CD) and propionate production in the cecum, and there were no differences in measured variables between STB and CB supplemented birds. Conclusion: Overall, these results indicate that STB supplementation was able to reduce the inflammatory response and improve the performance of NE challenged birds, and the supplementation of STB alone was as effective as a typical commercial blend containing a number of other additives. [ABSTRACT FROM AUTHOR]

The objective of this study was isolating and characterising Clostridium perfringens from chickens in Vietnam and identifying virulence factors involved with enteritis. Five hundred thirty-one faecal and sixty-eight intestinal samples were collected from healthy and diseased chickens for the C. perfringens isolation. The presence of virulence factors was determined by multiplex PCR. The netB gene of the selected isolates was sequenced and checked for its expression by SDS-PAGE. Two hundred seventy-two C. perfringens isolates were collected. All of them were shown to be positive for the cpa gene. The netB gene was detected in 26.56% of the C. perfringens isolates from the healthy chickens, while 43.45% of the isolates from the faeces and 45% of the isolates from the intestinal samples were positive for this gene in the diseased birds. All eight isolates positive to netB from the diseased chickens showed 100% identity in the netB sequence and produced the NetB toxin in vitro, whereas only two out of eight healthy chicken-derived isolates produced this toxin. Nine out of ten chickens experimentally infected with the C. perfringens netB-positive isolate showed typical signs of enteritis. The cpa gene was the most prevalent virulence factor identified in the bacteria C. perfringens, but the netB gene could be a major player responsible for necrotic enteritis progression in chickens. [ABSTRACT FROM AUTHOR]

The use of probiotics in poultry production has increased rapidly, and this movement has been promoted by global events, such as the prohibition or decline in the use of antibiotic growth promotants in poultry feeds. There has been a persistent search for alternative feed additives, and probiotics have shown that they can restore the composition of the gut microbiota, and produce health benefits to the host, including improvements in performance. Probiotics have shown potential to increase productivity in poultry, especially in flocks challenged by stressors. However, the outcomes of probiotic use have not always been consistent. There is an increasing demand for well defined products that can be applied strategically, and currently, probiotic research is focusing on delineating their mechanisms of action in the gut that contribute to an improved efficacy. In particular, mechanisms involved in the maintenance and protection of intestinal barrier integrity and the role of the gut microbiota are being extensively investigated. It has been shown that probiotics modulate intestinal immune pathways both directly and through interactions with the gut microbiota. These interactions are key to maintaining gut homeostasis and function, and improving feed efficiency. Research has demonstrated that probiotics execute their effects through multiple mechanisms. The present review describes recent advances in probiotic use in poultry. It focuses on the current understanding of gut homeostasis and gut health in chickens, and how it can be assessed and improved through supplementation of poultry diets with probiotics in poultry diets. In particular, cellular and molecular mechanisms involved in the maintenance and protection of gut barrier structure and function are described. It also highlights important factors that influence probiotic efficacy and bird performance. Following the banning or decline in the use of antibiotic growth promotors (AGPs), the use of probiotics in poultry production has increased rapidly. The present review describes recent advances in probiotic use in poultry, and focuses on the current understanding of gut health, how to assess it in chickens, and how to improve it via probiotic supplementation in poultry feeds. In particular, it explains the mechanisms how probiotics produce health benefits to the host, including improvements in bird performance. [ABSTRACT FROM AUTHOR]

Matrix metalloproteinases (MMPs) play an important role in intestinal extracellular matrix homeostasis. An overexpression of MMPs results in tissue destruction and local inflammation and has been associated with multiple inflammatory diseases. These host proteases might also be important in tissue damage caused by infectious agents, such as in intestinal damage in Clostridium perfringens-induced avian necrotic enteritis (NE). The aim of the present study was to elucidate the effect of a C. perfringens infection on the MMP activity in the small intestine of birds with a pre-disposing coccidial infection to obtain a more thorough understanding of the pathogenesis of NE. For this purpose, the gelatinolytic activity present in jejunal tissue of Eimeria infected birds which were challenged with either a pathogenic C. perfringens type G strain or a commensal C. perfringens type A strain was analyzed using substrate zymography. The results show that infection of broilers with Eimeria and different C. perfringens strains, independent of their pathogenicity, decreases the expression of a 40–45 kDa host collagenase in the jejunum, as compared to the expression in Eimeria-infected control birds. It was also shown that the expression of 2 MMPs with molecular weights of approximately 50–60 and 60–70 kDa was significantly lower in necrotic tissue as compared to the activity in macroscopically healthy tissue adjacent to the lesion. These results indicate that host collagenases are not elicited by the C. perfringens infection for permeabilizing the host mucosa to allow penetration of the NetB toxin in Eimeria infected broilers. [ABSTRACT FROM AUTHOR]

Necrotic enteritis is rarely reported because it is often misdiagnosed as coccidiosis due to similarity in clinical and pathological features. A field outbreak of necrotic enteritis in a flock of 13 weeks old 4,500 commercial pullets was investigated, the onset of the disease, morbidity and mortality rates were recorded. Post mortem examinations were conducted and gross lesions were documented. Tissues were collected and fixed in 10 % neutral buffered formalin and processed for histopathological examinations. Clinical signs observed were ruffled feathers, weakness, somnolence, loss of weight and diarrhoea; while the gross lesions observed were emaciated carcasses, lean abdominal fat, enlarged, pale and haemorrhagic liver; enlarged, mottled and congested spleen; mucus and diphtheritic membrane on the jejunal mucosa and enlarged kidneys. The histopathological findings of the intestine were diffused necrotic epithelial cells with marked mononuclear cells infiltration in the mucosa with severe oedema fluid. The necrotic enteritis was diagnosed based on clinical signs, pathology as well as isolation and identification of Clostridium perfringes. Triplesulfa® (sulfadimidine sodium, sulfadiazine sodium and sulfamerazine sodium); Tridox® L.A (20% Oxytetracycline long acting) and Enterocillin® (Amoxycillintrihydrate and Colistin sulphate) were ineffective, while copper sulphate at 1g/5L of drinking water was found to be effective for the treatment of the disease. The haematological values indicated lymphocytosis due to damage of the tissue caused by C. perfringes and the toxins produced. [ABSTRACT FROM AUTHOR]

Global poultry production is facing many challenges and is currently under pressure due to the presence of several diseases like Necrotic Enteritis (NE). It is estimated that NE-caused global economic losses has increased from 2 billion to 6 billion US$ in 2015 because it is not easy to diagnose and control disease at the earlier stage of occurrence. Additionally, ban on the in-feed antibiotics and some other genetic and non-genetic predisposing factors affect the occurrence of the disease. Though the incidence of the disease can be reduced by minimizing the predisposing factors and through immunization of birds but there is no single remedy to control the disease. Therefore, we suggest that there is need to find out the genetic variants that could help to select the birds resistant to NE. The current review details the pertinent features about the genetic and genomics of susceptibility and immune response of birds to Necrotic Enteritis. We report here the list of candidate gene reported for their involvement with the susceptibility and/or resistance to the disease. However, most of these genes are involved in immune-related functions. For better understanding of the role of Clostridium perfringens and its toxins in the pathogenesis of disease there is need to unveil the association between any specific genetic variation and clinical status of NE. However, the presence of substantial genetic variations among different breeds/strains of chicken shows that it is possible to develop broiler strain with genetic resistant against NE. It would help in the cost-effective and sustainable production of safe broiler meat. [ABSTRACT FROM AUTHOR]

The present research work was designed to know the effect of carbohydrate rich diet on the experimental pathology of necrotic enteritis in broilers. For experimental purpose, 15 birds of 14 days of age were grouped into 3 (A, B and C). Birds of group A were fed with 400gm maize/kg (40% increase) and group B were fed with 200gm maize/kg (20% increase) of normal feed from day 14 to day 27 and challenged from day 21 to 25 days with 1x108 CFU/3ml of inoculums of Cl. perfringens consecutive 5 days. Group C was kept in control without additional maize. Birds of all groups were observed up to 27 days of age. The clinical signs diarrhea, ruffled feather were common in both group A and B, and less feed intake in group A than group B. There was no mortality in any groups. Birds of all groups were sacrificed at the day 28. Necrosis and hemorrhage in intestine, enlarged liver, and hemorrhage in the base of heart were found in group A (+++). In impression smear of intestines, the average 10-12 bacteria were found in group A and 8-10 bacteria were found in group B in higher magnification (100X). In histopathology, severe infiltration of inflammatory cells including heterophils and lymphocytes and sloughing off mucosal epithelium that changed by pink color cytoplasm with pyknotic nuclei were observed in group A (+++) while group B showed infiltration of inflammatory cells and sloughing off mucosal epithelium (++). The birds of group C were normal in all respects. Anaerobic bacteria counted from intestinal content of group A was 2.8x107 CFU/ml, group B was 2.2x107 CFU/ml and group C was 1.0x107CFU/ml. From the findings, it may be said that carbohydrate rich diet is a predisposing factor for necrotic enteritis in broilers. [ABSTRACT FROM AUTHOR]

This study was designed to know the effect of protein rich diet (50% fish meal) on the experimental pathology of necrotic enteritis in broilers. The Clostridium (Cl.) perfringens was obtained from the Department of Pathology, Bangladesh Agricultural University. Reconfirmation and recharacterization of Cl. perfringens were performed by culture, microscopic examination, staining and biochemical tests. The experimental pathologic studies were performed with supplementation of protein rich diet and challenged with Cl. perfringens in broilers. The dose of the inoculum for experimental infection with Cl. perfringens was 1x108 CFU/2.5ml. Fifteen birds of 21 days old were divided into 3 (A, B and C) groups each containing 5 birds. Birds of group A were fed with 50% fish meal at a rate of 500gm /kg of feed from day 21 to day 34 and challenged from day 28 to day 32 with 1x108 CFU/2.5ml. Birds of group B were fed with normal feed and challenged on day 28 for consecutive five days. Group C was kept as control with commercial normal pellet without Cl. perfringens. Birds of all groups were observed up to 34 days of age for clinical signs. Eighty percent (4/5) of the birds of group A developed moderate clinical signs like diarrhoea, ruffled feather and less feed intake whereas 40% (2/5) birds of group B developed same clinical signs like group A but in mild form. There was no mortality in any groups. All the birds were sacrificed at Day 35. Severe necrosis and hemorrhage in intestine, enlarged liver and hemorrhage in the base of heart were noted in the birds of group A. On an average 2-5 bacteria were found in impression smear of intestines in higher magnification (100x), and anaerobic bacteria counted from intestinal content was 1.51x107CFU/ml. In histopathology, necrosis and reactive cells were found in liver, heart, lung and sloughing off intestinal epithelium was also found in intestines. On the other hand similar lesions like group A were observed in the birds of group B but in moderate form and no bacteria was found in impression smears of intestines. Anaerobic bacteria counted from intestinal content of this group was 1.1x107CFU/ml. In histopathology necrosis, reactive cells were found but less than group A. The birds of group C were normal in all parameters. However, anaerobic bacteria count from the intestinal content was 0.8x107CFU/ml. From this study, it may be concluded that protein rich diet is a predisposing factor for necrotic enteritis in broilers. [ABSTRACT FROM AUTHOR]

Type A Clostridium perfringens causes poultry necrotic enteritis (NE), an enteric disease of considerable economic importance, yet can also exist as a member of the normal intestinal microbiota. A recently discovered pore-forming toxin, NetB, is associated with pathogenesis in most, but not all, NE isolates. This finding suggested that NE-causing strains may possess other virulence gene(s) not present in commensal type A isolates. We used high-throughput sequencing (HTS) technologies to generate draft genome sequences of seven unrelated C. perfringens poultry NE isolates and one isolate from a healthy bird, and identified additional novel NE-associated genes by comparison with nine publicly available reference genomes. Thirty-one open reading frames (ORFs) were unique to all NE strains and formed the basis for three highly conserved NE-associated loci that we designated NELoc-1 (42 kb), NELoc-2 (11.2 kb) and NELoc-3 (5.6 kb). The largest locus, NELoc-1, consisted of netB and 36 additional genes, including those predicted to encode two leukocidins, an internalin-like protein and a ricin-domain protein. Pulsed-field gel electrophoresis (PFGE) and Southern blotting revealed that the NE strains each carried 2 to 5 large plasmids, and that NELoc-1 and -3 were localized on distinct plasmids of sizes ∼85 and ∼70 kb, respectively. Sequencing of the regions flanking these loci revealed similarity to previously characterized conjugative plasmids of C. perfringens. These results provide significant insight into the pathogenetic basis of poultry NE and are the first to demonstrate that netB resides in a large, plasmid-encoded locus. Our findings strongly suggest that poultry NE is caused by several novel virulence factors, whose genes are clustered on discrete pathogenicity loci, some of which are plasmid-borne. [ABSTRACT FROM AUTHOR]

Background: Clostridium perfringens (Cp) is a Gram-positive anaerobic bacterium that causes necrotic enteritis (NE) in poultry when it overgrows in the small intestine. NE disease has previously been controlled through the use of growth-promoting antibiotics. This practice was recently banned in European countries, leading to significantly increased incidence of NE threatening the poultry industry. Control strategies and technology as substitutes to dietary antibiotics are therefore urgently required. To develop the substitutes, it is important to understand host immune responses to Cp infection. However, the knowledge is still lacking. We therefore investigated gene expression profiles within immunologically-relevant tissue, the spleen, in order to identify factors that are involved in immunity to NE and have potential as therapeutic targets. Results: Use of a 44 K Agilent chicken genome microarray revealed significant up-regulation of many immune-associated genes in Cp-challenged chickens, including galectin 3, IFNAR1, IgY-receptor, TCRã, granzyme A, and mannose-6-P-R, which were subsequently validated by quantitative PCR assays. Functional annotation of differentially expressed genes was conducted using the High Throughput Gene Ontology Functional Annotation database. Medicated and Non-medicated chickens had similar annotation profiles with cell activities and regulation being the most dominant biological processes following Cp infection. Conclusion: Broiler chickens demonstrated an intricate and holistic magnitude of host response to Cp challenge and the development of NE. Although the influence of dietary antibiotics appeared to be less significant than the disease process, both had a considerable impact on the host response. Markers previously identified in intestinal inflammatory diseases of other species, including humans, and indicators of enhanced antibody responses, appeared to be involved in the chicken response to Cp challenge. The significance in host immune responses of immune mediators identified from the present study warrants further studies to verify their functions during NE development and to determine their potential application to control NE disease. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Necrotic enteritis (NE) caused by Clostridium perfringens infection has reemerged as a prevalent poultry disease worldwide due to reduced usage of prophylactic antibiotics under consumer preferences and regulatory pressures. The lack of alternative antimicrobial strategies to control this disease is mainly due to limited insight into the relationship between NE pathogenesis, microbiome, and host responses. Here we showed that the microbial metabolic byproduct of secondary bile acid deoxycholic acid (DCA), at as low as 50 µM, inhibited 82.8% of C. perfringens growth in Tryptic Soy Broth (P < 0.05). Sequential Eimeria maxima and C. perfringens challenges significantly induced NE, severe intestinal inflammation, and body weight (BW) loss in broiler chickens. These negative effects were diminished (P < 0.05) by 1.5 g/kg DCA diet. At the cellular level, DCA alleviated NE-associated ileal epithelial death and significantly reduced lamina propria cell apoptosis. Interestingly, DCA reduced C. perfringens invasion into ileum (P < 0.05) without altering the bacterial ileal luminal colonization. Molecular analysis showed that DCA significantly reduced inflammatory mediators of Infγ, Litaf, Il1β, and Mmp9 mRNA accumulation in ileal tissue. Mechanism studies revealed that C. perfringens induced (P < 0.05) elevated expression of inflammatory mediators of Infγ, Litaf, and Ptgs2 (Cyclooxygenases-2 (COX-2) gene) in chicken splenocytes. Inhibiting the COX signaling by aspirin significantly attenuated INFγ-induced inflammatory response in the splenocytes. Consistent with the in vitro assay, chickens fed 0.12 g/kg aspirin diet protected the birds against NE-induced BW loss, ileal inflammation, and intestinal cell apoptosis. In conclusion, microbial metabolic product DCA prevents NE-induced BW loss and ileal inflammation through attenuating inflammatory response. These novel findings of microbiome protecting birds against NE provide new options on developing next generation antimicrobial alternatives against NE. [ABSTRACT FROM AUTHOR]