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

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

SUBMITTER: Whelan RA

PROVIDER: S-EPMC6698186 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Necrotic enteritis (NE) is an important enteric disease in poultry and has become a major concern in poultry production in the post-antibiotic era. The infection with NE can damage the intestinal mucosa of the birds leading to impaired health and, thus, productivity. To gain a better understanding of how NE impacts the gut function of infected broilers, global mRNA sequencing (RNA-seq) was performed in the jejunum tissue of NE challenged and non-challenged broilers to identify the pathways and genes affected by this disease. Briefly, to induce NE, birds in the challenge group were inoculated with 1 mL of Eimeria species on day 9 followed by 1 mL of approximately 108 CFU/mL of a NetB producing Clostridium perfringens on days 14 and 15. On day 16, 2 birds in each treatment were randomly selected and euthanized and the whole intestinal tract was evaluated for lesion scores. Duodenum tissue samples from one of the euthanized birds of each replicate (n = 4) was used for histology, and the jejunum tissue for RNA extraction. RNA-seq analysis was performed with an Illumina RNA HiSeq 2000 sequencer. The differentially expressed genes (DEG) were identified and functional analysis was performed in DAVID to find protein-protein interactions (PPI). At a false discovery rate threshold <0.05, a total of 377 DEG (207 upregulated and 170 downregulated) DEG were identified. Pathway enrichment analysis revealed that DEG were considerably enriched in peroxisome proliferator-activated receptors (PPAR) signaling (P < 0.01) and β-oxidation pathways (P < 0.05). The DEG were mostly related to fatty acid metabolism and degradation (cluster of differentiation 36 [CD36], acyl-CoA synthetase bubblegum family member-1 [ACSBG1], fatty acid-binding protein-1 and -2 [FABP1] and [FABP2]; and acyl-coenzyme A synthetase-1 [ACSL1]), bile acid production and transportation (acyl-CoA oxidase-2 [ACOX2], apical sodium-bile acid transporter [ASBT]) and essential genes in the immune system (interferon-, [IFN-γ], LCK proto-oncogene, Src family tyrosine kinase [LCK], zeta chain of T cell receptor associated protein kinase 70 kDa [ZAP70], and aconitate decarboxylase 1 [ACOD1]). Our data revealed that pathways related to fatty acid digestion were significantly compromised which thereby could have affected metabolic and immune responses in NE infected birds.

Project description:Clostridium perfringens-induced necrotic enteritis (NE) is an economically important disease of broiler chickens. The present study evaluated the effect of C. perfringens on the intestinal histomorphometry, enteric microbial colonization, and host immune responses using 3 experimental NE reproduction methods. The experimental groups consisted of 1) unchallenged Control diet (corn-soybean meal), 2) Control diet + Eimera inoculation at d 11 followed by C. perfringens challenge at d 15 (ECp), 3) Wheat-based diet + C. perfringens challenge (WCp), and 4) Wheat-based diet + Eimeria inoculation followed by C. perfringens challenge (WECp). The results showed that chickens receiving ECp and WECp had reduced (P < 0.05) bird performance coupled with enteric gross lesions and epithelial damage at d 17 and 24 of age compared to unchallenged control birds. These ECp and WECp administered birds also had increased (P < 0.05) ileal colonization by clostridia and E. coli at d 17 and 24, while the resident Lactobacillus counts were reduced (P < 0.05) at d 24 of age. Furthermore, at d 24, jejunal transcription of IL-6, IL-10, annexin-A1 and IL-2 genes was upregulated (P < 0.05) in the ECp group, whereas the transcription of TNF receptor associated factor (TRAF)-3 gene was increased (P < 0.05) in WECp treated birds when compared to unchallenged control group. Additionally, stimulation of chicken splenocytes and cecal tonsilocytes with virulent C. perfringens bacilli or their secretory proteins resulted in a higher (P < 0.05) frequency of T cells and their upregulation of MHC-II molecule, as determined by flow cytometry. These findings suggest that C. perfringens, while inducing epithelial damage and changes in microbiota, can also trigger host immune responses. Furthermore, NE reproduction methods using coccidia with or without the wheat-based dietary predisposition seem to facilitate an optimal NE reproduction in broiler chickens and thus, may provide better avenues for future C. perfringens research.

Project description:Necrotic enteritis (NE) is one of the most common and costly diseases in the modern broiler industry, having an estimated economic impact of $6 billion dollars annually. Increasing incidents of NE have resulted from restrictions on the use of antibiotic feed additives throughout the broiler industry. As such, finding effective antibiotic alternatives has become a priority. In this study, an experimental model of NE was used, comprising a commercial infectious bursal disease virus vaccine and Clostridium perfringens (C. perfringens) inoculation. Yeast cells wall (YCW) components, β-glucan (BG), and mannoproteins (MPTs) were evaluated for their effects on disease development. Chicken-specific immunometabolic kinome peptide arrays were used to measure differential phosphorylation between control (uninfected), challenged (infected), and challenged and treated birds in duodenal, jejunal, and ileal tissues. Treatment groups included crude YCW preparation, BG, MPT, or BG+MPT as feed additives. Data analysis revealed kinome profiles cluster predominantly by tissue, with duodenum showing the greatest relative signaling and jejunum showing the greatest response to treatment. BG, MPT, and BG+MPT cluster together, separate from controls and challenge birds in each tissue. Changes in signaling resulting from the treatments were observed in cell growth and survival responses as well as immune responses. None of the treatments of disease challenge returned the profiles to control-like. This is attributable to immune modulation and metabolic effects of the treatments generating distinct profiles from control. Importantly, all the treatments are distinct from the challenge group despite being challenged themselves. Only BG+MPT treatment had a significant effect on bird weight gain compared with the NE challenge group, and this treatment had the greatest impact on gut tissue signaling in all segments. The signaling changes elicited by BG+MPT during an NE challenge were increased cell growth and survival signaling, reducing cell death, apoptosis and innate inflammatory responses, and generating compensatory signaling to reduce disease severity.

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

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