Dietary Eugenol Nanoemulsion Potentiated Performance of Broiler Chickens: Orchestration of Digestive Enzymes, Intestinal Barrier Functions and Cytokines Related Gene Expression With a Consequence of Attenuating the Severity of E. coli O78 Infection

Abstract

Recently, the use of essential oils (EOs) or their bioactive compounds encapsulated by nanoparticles as alternative supplements for in-feed antimicrobials is gaining attention, especially in organic poultry production. Focusing on eugenol, its incorporation into the nanoformulation is a novel strategy to improve its stability and bioavailability and thus augment its growth-boosting and antimicrobial activities. Therefore, we explored eugenol nanoemulsion activities in modulating growth, digestive and gut barrier functions, immunity, cecal microbiota, and broilers response to avian pathogenic E. coli challenge (APEC) O78. A total of 1,000 one-day-old broiler chicks were allocated into five groups; negative control (NC, fed basal diet), positive control (PC), and 100, 250, and 400 mg/kg eugenol nanoemulsion supplemented groups. All groups except NC were challenged with APEC O78 at 14 days of age. The results showed that birds fed eugenol nanoemulsion displayed higher BWG, FI, and survivability and most improved FCR over the whole rearing period. Birds fed 400 mg/kg of eugenol nanoemulsion sustained a higher growth rate (24% vs. PC) after infection. Likely, the expression of digestive enzymes' genes (AMY2A, CCK, CELA1, and PNLIP) was more prominently upregulated and unaffected by APEC O78 challenge in the group fed eugenol nanoemulsion at the level of 400 mg/kg. Enhanced gut barrier integrity was sustained post-challenge in the group supplemented with higher levels of eugenol nanoemulsion as evidenced by the overexpression of cathelicidins-2, β-defensin-1, MUC-2, JAM-2, occludin, CLDN-1, and FABP-2 genes. A distinct modulatory effect of dietary eugenol nanoemulsion was observed on cytokine genes (IL-1β, TNF-α, IL-6, IL-8, and IL-10) expression with a prominent reduction in the excessive inflammatory reactions post-challenge. Supplementing eugenol nanoemulsion increased the relative cecal abundance of Lactobacillus species and reduced Enterobacteriaceae and Bacteriods counts. Notably, a prominent reduction in APEC O78 loads with downregulation of papC, iroN, iutA, and iss virulence genes and detrimental modifications in E. coli morphological features were noticed in the 400 mg/kg eugenol nanoemulsion group at the 3rd-week post-challenge. Collectively, we recommend the use of eugenol nanoemulsion as a prospective targeted delivery approach for achieving maximum broilers growth and protection against APEC O78 infection.

Keywords: APEC O78; barrier function; broiler chickens; eugenol nanoemulsion; immunity; performance; virulence gene expression.

Figure 1

Fourier transform infrared (FTIR) spectroscopy (a) and transmission electron microscopy (TEM, b) of eugenol nanoemulsion.

Figure 2

Heat map demonstrating the expression levels of genes related to digestive enzymes; AMY2A (alpha 2A amylase), PNLIP (pancreatic lipase, CCK (cholecystokinin), and CELA1 (chymotrypsin-like elastase family, member 1) in the pancreas of broiler chickens received different levels of eugenol nanoemulsion pre- (at day 14) and post- (at 3rd week) E. coli O78 challenge detected by RT-qPCR technique. Values are means with their SE. The intensity of orange color denotes the degree of upregulation of the investigated genes. NC (negative control): birds fed basal diet without eugenol nanoemulsion and were not challenged, PC (positive control): birds fed a control diet without eugenol nanoemulsion and were challenged, eugenol nanoemulsion 100, 250, and 400: birds fed basal diet supplemented with 100, 250, and 400 mg/kg diet eugenol nanoemulsion. All groups except NC were challenged with E. coli O78 at 14 days of age. Different letters within the same row indicate a statistical significance (P < 0.05).

Figure 3

Heat map illustrating the gene expression profiles associated with barrier functions; cathelicidins-2, β-defensin-1, MUC-2 (mucin-2), JAM-2 (junctional adhesion molecule-2), occludin, CLDN-1 (claudins-1), and FABP-2 (fatty acid binding protein-2) in the jejunal tissues of broiler chickens supplemented with different levels of eugenol nanoemulsion pre- (at day 14) and post- (at 3rd week) E. coli O78 challenge determined by RT-qPCR assay. Values are means with their SE. The intensity of orange color denotes the degree of upregulation of the investigated genes. NC (negative control): birds fed basal diet without eugenol nanoemulsion and were not challenged, PC (positive control): birds fed a control diet without eugenol nanoemulsion and were challenged, eugenol nanoemulsion 100, 250, and 400: birds fed basal diet supplemented with 100, 250, and 400 mg/kg diet eugenol nanoemulsion. All groups except NC were challenged with E. coli O78 at 14 days of age. Different letters within the same row indicate a statistical significance (P < 0.05).

Figure 4

Heat map depicting RT-qPCR analysis of the relative expression levels of mRNAs encoding cytokines; IL-6 (interleukin-6), IL-8, IL-1β, IL-10, and TNF-α (tumor necrosis factor alpha) in the jejunal segments of broiler chickens fed different levels of eugenol nanoemulsion pre- (at day 14) and post- (at 3rd week) E. coli O78 challenge. Values are means with their SE. The intensity of orange and blue colors denotes the degree of upregulation and downregulation of the investigated genes, respectively. NC (negative control): birds fed basal diet without eugenol nanoemulsion and were not challenged, PC (positive control): birds fed a control diet without eugenol nanoemulsion and were challenged, eugenol nanoemulsion 100, 250, and 400: birds fed basal diet supplemented with 100, 250, and 400 mg/kg diet eugenol nanoemulsion. All groups except NC were challenged with E. coli O78 at 14 days of age. Different letters within the same row indicate a statistical significance (P < 0.05).

Figure 5

The impact of dietary inclusion of various levels of eugenol nanoemulsion on the populations (log₁₀ CFU) of total bacteria, Lactobacillus, Enterobacteriaceae, and Bacteriods as was estimated by qPCR assay in the cecal digesta of broiler chickens pre- (at day 14) and post- (at 3rd week) E. coli O78 challenge. Values are means with their SE. NC (negative control): birds fed basal diet without eugenol nanoemulsion and were not challenged, PC (positive control): birds fed a control diet without eugenol nanoemulsion and were challenged, eugenol nanoemulsion 100, 250, and 400: birds fed basal diet supplemented with 100, 250, and 400 mg/kg diet eugenol nanoemulsion. All groups except NC were challenged with E. coli O78 at 14 days of age.

Figure 6

Quantification of cecal APEC O78 loads (log₁₀ CFU) in response to eugenol nanoemulsion supplementation at the 1st and 3rd weeks post-challenge as was measured by qPCR assay. Values are means with their SE. NC (negative control): birds fed basal diet without eugenol nanoemulsion and were not challenged, PC (positive control): birds fed a control diet without eugenol nanoemulsion and were challenged, eugenol nanoemulsion 100, 250, and 400: birds fed basal diet supplemented with 100, 250, and 400 mg/kg diet eugenol nanoemulsion. All groups except NC were challenged with E. coli O78 at 14 days of age.

Figure 7

RT-qPCR investigation of the relative mRNA expression levels of E. coli papC, iutA, iroN, and iss virulence genes in the cecal contents of broiler chickens fed different levels of eugenol nanoemulsion at the 1st- and 3rd-week post-challenges. Values are means with their SE. NC (negative control): birds fed basal diet without eugenol nanoemulsion and were not challenged, PC (positive control): birds fed a control diet without eugenol nanoemulsion and were challenged, eugenol nanoemulsion 100, 250, and 400: birds fed basal diet supplemented with 100, 250, and 400 mg/kg diet eugenol nanoemulsion. All groups except NC were challenged with E. coli O78 at 14 days of age.