Temporal Effects of High Fishmeal Diet on Gut Microbiota and Immune Response in Clostridium perfringens-Challenged Chickens.
ABSTRACT: Necrotic enteritis (NE) caused by Clostridium perfringens is responsible for huge financial losses in the poultry industry annually. A diet highly supplemented with fishmeal is one factor predisposing chickens to the development of clinical NE. However, the effects of fishmeal-rich diets on the gut microbiota and immune response in chickens with C. perfringens challenge over the long-term are not well-understood. Here, a chicken NE model was established in which chickens were fed high fishmeal diet and subsequently infected with C. perfringens (FM/CP). Two control groups of chickens, one that was not infected and had a high fishmeal feeding (FM) and another group only infected with C. perfringens with basic diets (CP), were used as comparators. We analyzed the gut microbiota and immune response of the three groups at the age of 20, 24 [1 day post-infection (dpi)] and 30 days (7 dpi) using 16S rDNA sequencing and real-time PCR, respectively. We found that the composition of the gut microbiota had significant shifted in both the CP and FM/CP groups, although the CP group did not have intestinal lesions. The structure of the gut microbiota in C. perfringens-challenged chickens, independent of a high fishmeal diet, had the tendency to return to their non-infection state if the chickens no longer received C. perfringens challenge. Gut microbiota variation with time in challenged chickens with high fishmeal diet feeding was superimposed upon that of non-infected chickens with high fishmeal feeding. For the immune response, the relative expression of IL-8 in the ileum was significantly higher in infected chickens independent of high fishmeal feeding than in non-infected chickens. However, the expression of alpha 1-acid glycoprotein (AGP) and serum amyloid A (SAA) genes in chicken liver were significantly increased in FM/CP compared to the other groups. In conclusion, high fishmeal feeding induced significant changes to the structure of chicken gut microbiota over time and such changes provided an opening for C. perfringens infection to progress to NE. The relative expression of AGP and SAA in liver tissue may be used as diagnostic biomarkers for poultry NE but such an indication requires further investigation.
Project description:Necrotic enteritis (NE) causes huge economic losses to the poultry industry. Probiotics are used as potential alternatives to antibiotics to prevent NE. It is known that Clostridium butyricum can act as a probiotic that can prevent infection. However, whether or not it exerts a beneficial effect on NE in chickens remains elusive. Therefore, we investigated the impact of C. butyricum on immune response and intestinal microbiota during the development of NE in chickens, including experimental stages with basal diets, high-fishmeal-supplementation diets, and Clostridium perfringens challenge. Chickens were divided into two groups from day 1 to day 20: one group had its diet supplemented with C. butyricum supplementation and one did not. At day 20, the chickens were divided into four groups: C. perfringens challenged and unchallenged chickens with and without C. butyricum supplementation. All groups were fed a basal diet for 13 days and thereafter a basal diet with 50% fishmeal from day 14 to 24. Chickens were infected with C. perfringens from day 21 to 23. At days 13, 20 and 24, samples were collected for analysis of the relative expression of immune response and intestinal mucosa barrier-related genes and intestinal microbes. The results show that C. butyricum can inhibit the increase in IL-17A gene expression and the reduction in Claudin-1 gene induced-expression caused by C. perfringens challenge. Moreover, C. butyricum was found to increase the expression of anti-inflammatory IL-10 in infected chickens. Although C. butyricum was found to have a significant beneficial effect on the structure of intestinal bacteria in the basal diet groups and decrease the abundance of C. perfringens in the gut, it did not significantly affect the occurrence of intestinal lesions and did not significantly correct the shift in gut bacterial composition post C. perfringens infection. In conclusion, although C. butyricum promotes the expression of anti-inflammatory and tight junction protein genes and inhibits pro-inflammatory genes in C. perfringens-challenged chickens, it is not adequate to improve the structure of intestinal microbiota in NE chickens. Therefore, more effective schemes of C. butyricum supplementation to prevent and treat NE in chickens need to be identified.
Project description:Necrotic enteritis (NE) is a severe intestinal disease, which can change gut microbiota and result in a high cost for the poultry industry worldwide. However, little is known regarding how the gut microbiota of NE chicken ileum are changed by Bacillus licheniformis. This study was conducted to investigate how ileum microbiota structure was changed by B. licheniformis in broiler chickens challenged with Clostridium perfringens-induced NE through Illumina MiSeq sequencing. The broilers were randomly separated into four groups: the negative control group (NC), the positive control group (PC), the fishmeal and coccidia group (FC), and the PC group supplied with feed containing B. licheniformis (BL). Compared to the PC and FC, alpha diversity, beta diversity, and the bacterial taxa of the ileum microbiota were more similar in BL and NC. Some genera, which were related to the NE control, became insignificant in BL with NC, such as Lactobacillus, Lactococcus, Bacteroides, Ruminococcus and Helicobacter. The PICRUSt analysis revealed that a tumour suppressor gene, p53, which was negatively correlated with Helicobacter, was enriched in the BL group. Our findings showed that the ileum microbiota disorder caused by NE in chickens was normalized by dietary B. licheniformis supplementation.
Project description:Clostridium perfringens can induce necrotic enteritis of chickens, which causes large economic losses every year. Bacillus licheniformis, a probiotic, can inhibit the growth of pathogenic bacteria such as Clostridium perfringens, thereby improving the health status of chickens. However, from a microbial ecology perspective, the mechanisms by which alterations to the gut microbiota improve health remain unknown. In this study, we used Illumina MiSeq sequencing to investigate the cecal microbiota of a negative control group (NC), a C. perfringens and Eimeria challenge group with fishmeal supplementation (PC), a group supplemented with fishmeal and infected with coccidia (FC), and group PC with B. licheniformis supplementation (BL). We found that the health status of C. perfringens-challenged chickens was compromised, and that B. licheniformis improved the growth of the chickens challenged with pathogens. Microbial diversity analysis and taxonomic profiling of groups NC, PC, and FC revealed a disturbed cecal microflora of the birds with C. perfringens. We also characterized the microbiota of the chickens in the BL group using several methods. Principal coordinate analysis demonstrated that, compared with group PC, the bacterial community structure of group BL was more similar to that of group NC. Linear discriminant analysis with effect size revealed less differentially represented bacterial taxa between groups BL and NC than between groups PC and NC. In addition, groups BL and NC appeared to have similar overrepresented microbial taxa (such as Bacteroides, Helicobacter, Megamonas, and Akkermansia) compared with group PC. Finally, a phylogenetic investigation of communities by reconstruction of unobserved states analysis indicated that large differences existed between group PC and groups NC and BL. In conclusion, pre-treatment with B. licheniformis reduced the disturbance of the cecal microbiome induced by challenge with C. perfringens and other factors in broiler chickens.
Project description:Gut microbiota has been demonstrated to be involved in intestinal nutrition, defense, and immunity, as well as participating in disease progression. This study was to investigate gut microbiota changes in chickens challenged with netB-positive Clostridium perfringens strain (CP1) and/or the predisposing Eimeria species (Eimeria) and fed diets with fishmeal supplementation. In addition, the effects of lauric acid, a medium-chain fatty acid (MCFA), on necrotic enteritis (NE) reduction and modulation of microbiota were evaluated. The results demonstrated that microbial communities in the jejunum were distinct from those in the cecum, and the microbial community change was more significant in jejunum. Challenge of CP1 in conjunction with Eimeria significantly reduced species diversity in jejunal microbiota, but cecal microbiota remained stable. In the jejunum, CP1 challenge increased the abundance of the genera of Clostridium sensu stricto 1, Escherichia Shigella, and Weissella, but significantly decreased the population of Lactobacillus. Eimeria infection on its own was unable to promote NE, demonstrating decrements of Clostridium sensu stricto 1 and Lactobacillus. Co-infection with CP1 and Eimeria reproduced the majority of NE lesions with significant increment of Clostridium sensu stricto 1 and reduction in Lactobacillus. The advance of changes on these two taxa increased the severity of NE lesions. Further analyses of metagenomeSeq, STAMP, and LEfSe consistently showed significant overgrowth of Clostridium sensu stricto 1 was associated with NE. The supplementation of lauric acid did not reduce NE incidence and severity but decreased the relative abundance of Escherichia Shigella. In conclusion, significant overgrowth of C. perfringens as well as other Clostridium species in Clostridium sensu stricto 1 with the decrement of Lactobacillus in the jejunum is the featured microbiota correlated with NE. Controlling proliferation of Clostridium sensu stricto 1 and manipulation of Lactobacillus in the jejunum should be the strategy to prevent NE.
Project description:Background:The poultry industry is in need of effective antibiotic alternatives to control outbreaks of necrotic enteritis (NE) due to Clostridium perfringens. In the present study, we investigated the effects of dietary supplementation with a blend of encapsulated essential oils and organic acids (BLJ) on growth performance and gut health using a coinfection model of NE in broiler chickens. Methods:Two hundred and eighty-eight one-day-old male Arbor Acres broiler chicks were randomly assigned using a 2?×?2 factorial design into two groups fed either 0 or 500?mg/kg dietary BLJ and co-challenged (or not challenged for the control) with Eimeria spp./C. perfringens. Results:Infected birds fed the BLJ-supplemented diet exhibited an improved feed conversion ratio throughout the trial (P <?0.01), a higher villus height and villus height/crypt depth ratio, and reduced intestinal C. perfringens counts, liver C. perfringens carriage, gut lesion scores and serum fluorescein isothiocyanate dextran (FITC-D) concentrations at 7?d post-infection compared with those of birds without BLJ supplementation (P <?0.05). NE-infected birds fed BLJ exhibited significantly upregulated claudin-1 and IGF-2 mRNA levels (P <?0.05), increased A20 mRNA expression and significantly downregulated TRAF-6, TNFSF15 and TOLLIP mRNA levels in the jejunum at 7?d post-infection compared with those in birds without BLJ supplementation (P <?0.05). Compared with the uninfected and untreated birds, the uninfected birds fed BLJ displayed increased relative abundances of Lactobacillus and Coprococcus but reduced Rikenellaceae levels. Compared with the unsupplemented NE-challenged birds, infected birds fed BLJ showed an increased relative abundance of Unclassified_Lachnospiraceae and a significantly decreased relative abundance of Erysipelotrichaceae. Conclusion:BLJ supplementation improved growth performance and gut health in NE-infected broiler chickens by strengthening the intestinal barrier function, positively modulating the gut microbiota community and differentially regulating intestinal immune responses. Our results also suggested that adding BLJ effectively controlled NE infections after experimental Eimeria and Clostridium perfringens coinfection.
Project description:Our previous reports suggested that Dietary l-arginine supplementation attenuated gut injury of broiler chickens infected with Clostridium perfringens by enhancing intestinal immune responses, absorption and barrier function, but its effect on the gut microbiome of broiler chickens remains unclear. This experiment aimed at evaluating the effects of Dietary l-arginine supplementation on the gut bacterial community composition and function of broiler chickens challenged with C. perfringens. In total, 105 1-day-old male Arbor Acres broiler chickens were assigned to three groups: Control (CTL), C. perfringens-challenged (CP), and C. perfringens-challenged and fed diet supplemented with 0.3% l-arginine (ARGCP) groups. The challenge led to macroscopic and histomorphological gut lesions, decreased villus height and increased the number of Observed species, Shannon, Chao1 and ACE indices of ileal microbiota, whereas l-arginine addition reversed these changes. Moreover, the three treatments harbored distinct microbial communities (ANOSIM, P < 0.05). At the genus level, 24 taxa (e.g., Nitrosomonas spp., Coxiella spp., Ruegeria spp., and Thauera spp.) were significantly more abundant in CP group than in CTL group (P < 0.05), whereas the levels of 23 genera of them were significantly decreased by l-arginine supplementation (P < 0.05). The abundances of only 3 genera were different between CTL and ARGCP groups (P < 0.05). At the species level, the challenge promoted the relative abundance of Nitrospira sp. enrichment culture clone M1-9, Bradyrhizobium elkanii, Nitrospira bacterium SG8-3, and Pseudomonas veronii, which was reversed by l-arginine supplementation (P < 0.05). Furthermore, the challenge decreased the levels of Lactobacillus gasseri (P < 0.05). Predictive functional profiling of microbial communities by PICRUSt showed that compared with CP group, ARGCP group had enriched pathways relating to membrane transport, replication and repair, translation and nucleotide metabolism and suppressed functions corresponding to amino acid and lipid metabolisms (P < 0.05). The relative abundances of KEGG pathways in l-arginine-fed broilers were almost equal to those of the controls. In conclusion, l-arginine alleviated the gut injury and normalized the ileal microbiota of C. perfringens-challenged chickens to resemble that of unchallenged controls in terms of microbial composition and functionality.
Project description:Necrotic enteritis (NE) caused by Clostridium perfringens is one of the most detrimental infectious diseases in poultry. This study examined the effect of blends of essential oils (BEOs) (25% thymol and 25% carvacrol) on NE and bacterial dynamics and functions in chicks challenged with C. perfringens. Chicks were assigned to a Control diet and BEOs diet (Control diet +?120?mg/kg BEOs), were challenged with C. perfringens from days 14 to 20 and were killed on day 21 for assessment. Supplementation with BEOs decreased the mortality, alleviated gut lesions, and decreased the virulence factors of pathogenic bacteria (VF 0073-ClpE, VF0124-LPS, and VF0350-BSH). Lack of supplementation also changed the nutrient and immunological dynamics of host microbiota in responding to C. perfringens infection. Adding BEOs changed the host ileum microbial population by increasing the numbers of Lactobacillus crispatus and Lactobacillus agilis, and decreasing Lactobacillus salivarius and Lactobacillus johnsonii. The functional roles of these changing host bacterial populations coupled with the putative reduced pathogenicity of C. perfringens by BEOs contributed to the reduction in gut lesions and mortality in infected chickens. It suggests that dietary supplementation with BEOs could significantly reduce the impact of NE caused by C. perfringens on broilers.
Project description:The poultry industry is in need of effective antibiotic alternatives to control outbreaks of necrotic enteritis (NE) due to Clostridium perfringens.This study was conducted to investigate the effects of feeding Bacillus coagulans on the growth performance and gut health of broiler chickens with C. perfringens-induced NE. Two hundred and forty 1-day-old broiler chicks were randomly assigned to a 2 × 2 factorial arrangement with two dietary B. coagulans levels (0 or 4 × 109 CFU/kg of diet) and two disease challenge statuses (control or NE challenged).NE-induced reduction in body weight gain was relieved by the addition of B. coagulans into broiler diets compared with the NE-infected birds. NE infection damaged intestinal morphological structure, promoted intestinal C. perfringens growth and liver invasion, and enhanced anti-C. perfringens specific sIgA concentrations in the gut and specific IgG levels in serum compared with the uninfected birds. NE infection significantly (P < 0.05) decreased mucin-2 (at 14 d post-infection (DPI), toll -like receptor 2 (TLR2, at 7 and 14 DPI), TLR4 (at 7 and 14 DPI), tumor necrosis factor super family 15 (TNFSF15, at 7 and 14 DPI), lysozyme (LYZ, at 14 DPI) and fowlicidin-2 (at 7 and 14 DPI) mRNA levels, whereas it dramatically (P = 0.001) increased IFN-γ mRNA levels at 7 DPI. However, challenged birds fed diets supplemented with B. coagulans showed a significant (P < 0.01) decrease in gut lesion scores, decreased C. perfringens numbers in the cecum and liver, and an increase in fowlicidin-2 mRNA levels in compared with the uninfected birds. In addition, compared with the non-supplemented group, dietary inclusion of B. coagulans improved intestinal barrier structure, further increased specific sIgA levels and alkaline phosphatase (IAP) activity in the jejunum, enhanced the expression of jejunum lysozyme mRNA, and inhibited the growth, colonization, and invasion of C. perfringens; in contrast, it reduced serum-specific IgG concentrations and jejunum IFN-γ mRNA levels.These results indicated that dietary B. coagulans supplementation appeared to be effective in preventing the occurrence and reducing the severity of C. perfringens-induced NE in broiler chickens.
Project description:Necrotic enteritis (NE), caused by Clostridium perfringens, has cost the poultry industry $2 billion in losses. This study aimed to investigate the effect of Bacillus licheniformis as dietary supplement on the growth, serum antioxidant status, and expression of lipid-metabolism genes of broiler chickens with C. perfringens-induced NE.A total of 240 one-day-old broilers were randomly grouped into four: a negative control, an NE experimental model (PC), chickens fed a diet supplemented with 30 % of fishmeal from day 14 onwards and challenged with coccidiosis vaccine (FC), and NE group supplied with feed containing 1.0 × 10(6) CFU/g B. licheniformis (BL).Body weight gain, feed conversion ratio, serum antioxidant status, and lipid-metabolism-gene expression were analyzed. In the PC group, FCR increased significantly whereas serum catalase and glutathione peroxidase activity decreased compared with NC group. Dietary B. licheniformis supplementation improved FCR and oxidative stress in experimental avian NE. Using Bacillus licheniformis as a direct-fed microbial (DFM) could also significantly upregulate catabolism-related genes, namely, peroxisome proliferator-activated receptor-? and carnitine palmitoyltransferase-1, in livers and changed the expression of lipid-anabolism genes.These results suggested that dietary B. licheniformis supplementation can enhance growth and antioxidant ability, as well as change the expression of genes related to fatty-acid synthesis and oxidation in the livers of NE-infected broilers.
Project description:Necrotic enteritis (NE) is a poultry disease caused by Clostridium perfringens and characterized by severe intestinal necrosis. The incidence of avian NE has been progressively increasing following the removal of antibiotics from poultry feed. We evaluated the effect of diets supplemented with the thermally-processed clays, calcium montmorillonite (CaMM) on clinical signs, immunopathology, and cytokine responses in broiler chickens using an experimental model of NE consisting of co-infection with Eimeria maxima and C. perfringens. In Trial 1, Ross/Ross chickens were fed from hatch with a normal basal diet or a CaMM-supplemented diet with or without a fermentable fiber, an organic acid, and/or a plant extract, and co-infected with E. maxima and C. perfringens under conditions simulating clinical infection in the field. Chickens fed a diet supplemented with CaMM plus a fermentable fiber and an organic acid had increased body weight gain, reduced gut lesions, and increased serum antibody levels to C. perfringens ?-toxin and NetB toxin compared with chickens fed the basal diet alone. Levels of transcripts for interleukin-1? (IL-1?), IL-6, inducible nitric oxide synthase, and tumor necrosis factor-? superfamily-15 were significantly altered in the intestine and spleen of CaMM-supplemented chickens compared with unsupplemented controls (p<0.05). In Trial 2, Cobb/Cobb chickens were fed an unsupplemented diet or a diet supplemented with CaMM or Varium®, each with a fermentable fiber and an organic acid, and co-infected with E. maxima and C. perfringens under subclinical infection conditions. Compared with unsupplemented controls, broilers fed with CaMM plus a fermentable fiber and an organic acid had increased body weight gain, and reduced feed conversion ratio, mortality, and intestinal lesions, compared with chickens fed an unsupplemented diet (p<0.05). Dietary supplementation of broiler chickens with CaMM plus a fermentable fiber and an organic acid might be useful to control avian NE in the field.