Host-mediated beneficial effects of phytochemicals for prevention of avian coccidiosis

Abstract

Both in vitro and in vivo studies were conducted to evaluate the beneficial effects of green tea extract (GT), cinnamon oil (CO), and pomegranate extract (PO) on avian coccidiosis. In experiment (EXP) 1, an in vitro culture system was used to investigate the individual effects of GT, CO, and PO on the proinflammatory cytokine response and integrity of tight junction (TJ) in chicken intestinal epithelial cells (IEC), on the differentiation of quail muscle cells and primary chicken embryonic muscle cells, and anticoccidial and antibacterial activities against Eimeria tenella sporozoites and Clostridium perfringens bacteria, respectively. In EXP 2 and 3, in vivo trials were carried out to study the dose-dependent effect of blended phytochemicals (GT, CO, PO) on coccidiosis in broiler chickens infected with E. maxima. For EXP 2, one hundred male broiler chickens (0-day-old) were allocated into the following five treatment groups: Control group for non-infected chickens (NC), Basal diet group for E. maxima-infected chickens (PC), PC group supplemented with phytochemicals at 50 (Phy 50), 100 (Phy 100), and 200 (Phy 200) mg/kg feed diets for E. maxima-infected chickens. For EXP 3, one hundred twenty male broiler chickens (0-day-old) were allocated into the following six treatment groups: NC, PC, PC supplemented with phytochemicals at 10 (Phy 10), 20 (Phy 20), 30 (Phy 30), and 100 (Phy 100) mg/kg feed for E. maxima-infected chickens. Body weights (BW) were measured on days 0, 7, 14, 20, and 22, and jejunum samples were used to measure cytokine, TJ protein, and antioxidant enzyme responses at 8 days post-infection (dpi). Fecal samples for oocyst enumeration were collected from 6 to 8 dpi. In vitro, CO and PO reduced LPS-induced IL-1β and IL-8 in IEC, respectively, and GT enhanced the gene expression of occludin in IEC. PO at 1.0 and 5.0 mg/mL exerted antimicrobial effect against E. tenella sporozoites and C. perfringens bacteria, respectively. In vivo, chickens fed a diet supplemented with phytochemicals showed enhanced BW, reduced oocyst shedding, and decreased proinflammatory cytokines following E. maxima challenge. In conclusion, the combination of GT, CO, and PO in the diet of broiler chickens infected with E. maxima induced enhanced host disease resistance including innate immunity and gut health, which contributed to improved growth and reduced disease responses. These findings provide scientific support for the development of a novel phytogenic feed additive formula that enhances the growth and intestinal health of broiler chickens infected with coccidiosis.

Keywords: Eimeria maxima; alternatives to antibiotics; broiler chicken; coccidiosis; growth; gut health; intestinal immunity; phytochemicals.

Figure 1

Gene expression of IL-1β in phytochemical-induced chicken intestinal epithelial cells (IECs). The IECs were treated with three different concentrations (0.5, 1.0, and 10.0 mg/mL) of each phytochemical [cinnamon oil (CO, A), green tea extract (GT, B), and pomegranate (PO, C)] for 18 h with LPS (1μg/mL). The data represent the average of six independent experiments. P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), and P < 0.0001 (****) were considered statistically significant compared to the control (0.0 mg/mL phytochemicals). The fold changes in each transcript were normalized to glyceraldehyde-3-phosphate dehydrogenase and are relative to the transcript expression in unstimulated control group (normalized to 1) using the comparative ΔΔ Ct method.

Figure 2

Gene expression of IL-8 in phytochemical-induced chicken intestinal epithelial cells. Cells were treated with three different concentrations (0.5, 1.0, and 10.0 mg/mL) of each phytochemical [cinnamon oil (CO, A), green tea extract (GT, B), and pomegranate (PO, C)] for 18 h with LPS (1μg/mL). The data represent the average of six independent experiments. P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), and P < 0.0001 (****) were considered statistically significant compared to the control (0.0 mg/mL phytochemicals). The fold changes in each transcript were normalized to glyceraldehyde-3-phosphate dehydrogenase and are relative to the transcript expression in unstimulated control group (normalized to 1) using the comparative ΔΔ Ct method.

Figure 3

Viability of E. tenella sporozoites against phytochemical administration. Antiparasitic activities against E. tenella sporozoites were determined by counting viable sporozoites after 3 h incubation with cinnamon oil (A), green tea extract (B), and pomegranate extract (C). The data represent the average of six independent experiments. P < 0.0001 (****) was considered statistically significant compared to the control (0.0 mg/mL phytochemicals). 100% value of the control is 2.23 × 10⁷ ± 2.0 × 10⁶ of E. tenella sporozoites. Pomegranate administration reduced the viability of sporozoites by less than 20% in the experimental range.

Figure 4

Viability of C.m perfringens against phytochemical administration. Antibacterial activities against C. perfringens were determined by counting colonies after 18 h incubation with cinnamon oil (A), green tea extract (B), and pomegranate extract (C). The data represent the average of six independent experiments. P < 0.05 (*) and P < 0.001 (***) were considered statistically significant compared to the control (0.0 mg/mL phytochemicals). 100% value of the control is 1 × 10⁹ ± 1.1 × 10⁸ CFU of C. perfringens. Pomegranate and tea extract at 5.0 mg/mL reduced the viability of C. perfringens by less than 40%.

Figure 5

Transcripts of proinflammatory cytokines (A; IL-1β, B: IL-8, C: TNFSF-15 and D: IFN-γ) in jejunum of chickens fed diet supplemented with phytochemical mixture at 100 mg/kg feed during infection with E. maxima in experiment 2. NC, basal diet; PC, basal diet for infected chickens; Phy 100, phytochemical at 100 mg/kg feed; IL, interleukin; TNFSF, tumor necrosis factor superfamily; IFN, interferon. All chickens, except for NC, were infected by oral gavage on day 14 with 1.0 × 10⁴ oocysts/chicken of E. maxima. P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***) were considered statistically significant. The data were collected from jejunal tissues of 5 chickens per treatment on d 22 (8 days post-infection). Transcript levels of the cytokines were measured using quantitative RT-PCR and normalized to GAPDH transcript levels.

Figure 6

Transcripts of tight junction proteins (A; Claudin, B: JAM-2, C: Occludin and D: ZO-1) in jejunum of chickens fed diet supplemented with phytochemical mixture at 100 mg/kg feed during infection with E. maxima in experiment 2. NC, basal diet; PC, basal diet for infected chickens; Phy 100, phytochemical at 100 mg/kg feed; JAM, junctional adhesion molecule; ZO, zonula occludins. All chickens, except for NC, were infected by oral gavage on day 14 with 1.0 × 10⁴ oocysts/chicken of E. maxima. The data were collected from jejunal tissues of 5 chickens per treatment on d 22 (8 days post-infection). Transcript levels of the cytokines were measured using quantitative RT-PCR and normalized to GAPDH transcript levels.

Figure 7

Fecal oocyst shedding (A, B) and three-dimensional response plot (C) on final body weight (BW) of chicken fed diet supplemented with phytochemical mixture. The oocyst shedding was measured from fecal samples which were collected on a tray installed under the cage from 6 to 8 days post-infection. NC group, basal diet without infection, was excluded in oocyst results because there was no oocyst. In the three-dimensional plot, the average values of PC on final BW and oocyst number were considered as 100%. The surface was drawn with each value of BWs, oocyst numbers, and Phy concentrations without statistic calculation.

Figure 8

Transcripts of proinflammatory cytokines (A; IL-1β, B: IL-8, C: TNFSF-15 and D: IFN-γ) in jejunum of chickens fed diet supplemented with phytochemical during infection with E. maxima in experiment 3. NC, basal diet; PC, basal diet for infected chickens; Phy 20, phytochemical mixture at 20 mg/kg feed; IL, interleukin; TNFSF, tumor necrosis factor superfamily; IFN, interferon. All chickens, except for NC, were infected by oral gavage on day 14 with 1.0 × 10⁴ oocysts/chicken of E. maxima. P < 0.05 (*) and P < 0.01 (**) were considered statistically significant. The data were collected from jejunal tissues of 5 chickens per treatment on d 22 (8 days post-infection). Transcript levels of the cytokines were measured using quantitative RT-PCR and normalized to GAPDH transcript levels.

Figure 9

Transcripts of tight junction proteins (A; Claudin, B: JAM-2, C: Occludin and D: ZO-1) in jejunum of chickens fed diet supplemented with phytochemicals during infection with E. maxima in experiment 3. NC, basal diet; PC, basal diet for infected chickens; Phy 20, phytochemical mixture at 20 mg/kg feed; ZAM, junctional adhesion molecule; ZO, zonula occludins. All chickens, except for NC, were infected by oral gavage on day 14 with 1.0 × 10⁴ oocysts/chicken of E. maxima. P < 0.05 (*) was considered statistically significant. The data were collected from jejunal tissues of 5 chickens per treatment on d 22 (8 days post-infection). Transcript levels of the cytokines were measured using quantitative RT-PCR and normalized to GAPDH transcript levels.