Subclinical Doses of Combined Fumonisins and Deoxynivalenol Predispose Clostridium perfringens- Inoculated Broilers to Necrotic Enteritis

Abstract

Fumonisins (FB) and deoxynivalenol (DON) are mycotoxins which may predispose broiler chickens to necrotic enteritis (NE). The objective of this study was to identify the effects of subclinical doses of combined FB and DON on NE. A total of 480 day-old male broiler chicks were divided into four treatment groups; 1) control group (basal diet + Clostridium perfringens); 2) necrotic enteritis group (basal diet + Eimeria maxima + C. perfringens); 3) FB + DON group (basal diet + 3 mg/kg FB + 4 mg/kg DON + C. perfringens); and 4) FB + DON + NE group (basal diet + 3 mg/kg FB + 4 mg/kg DON + E. maxima + C. perfringens). Birds in NE and FB + DON + NE groups received 2.5 × 10³ E. maxima on day 14. All birds were inoculated with C. perfringens on days 19, 20, and 21. On day 35, birds in the NE, FB + DON, and FB + DON + NE groups had 242, 84, and 339 g lower BWG and a 19-, 2-, and 22-point increase in FCR respectively, than in the control group. Subclinical doses of FB + DON increased (p < 0.05) the NE lesion scores compared to the control group on day 21. On day 21, birds in the NE, FB + DON, and FB + DON + NE groups had increased (p < 0.05) serum FITC-D, lower (p < 0.05) jejunal tight junction protein mRNA, and increased (p < 0.05) cecal tonsil IL-1 mRNA compared to control group. On day 21, birds in the NE group had decreased (p < 0.05) villi height to crypt depth ratio compared to the control group and the presence of FB + DON in NE-induced birds further decreased the villi height to crypt depth ratio. Birds in the NE, FB + DON, and FB + DON + NE groups had increased (p < 0.05) C. perfringens, lower (p < 0.05) Lactobacillus loads in the cecal content, and a lower (p < 0.05) CD8⁺: CD4⁺ cell ratio in the cecal tonsils compared to the control group. It can be concluded that subclinical doses of combined FB and DON predispose C. perfringens-inoculated birds to NE, and the presence of FB + DON in NE-induced birds exacerbated the severity of NE.

Keywords: broiler chicken; deoxynivalenol; fumonisins; immune response; necrotic enteritis; tight junction proteins.

FIGURE 1

Effect of subclinical dose of FB + DON and E. maxima/C. perfringens challenge on production performances. Day-old chicks were distributed into four treatment groups: control, necrotic enteritis (NE), fumonisin + deoxynivalenol (FB + DON), and FB + DON + NE groups. Birds in the NE and FB + DON + NE groups received 2.5 × 10³ Eimeria maxima oocyst per bird on day 14. All birds received 1 × 10⁸ CFU/bird of Clostridium perfringens on days 19, 20, and 21. Body weight and feed consumption was measured on days 0, 7, 14, 21, 28, and 35 of age to calculate body weight gain (Panel 1A) and feed consumption ratio (Panel 1B). Mortality-corrected body weight gain and feed conversion ratio are presented. Bars (+SEM) without a common superscript differ significantly (p < 0.05). n = 8 pens of 15 birds/pen.

FIGURE 2

Effect of subclinical dose of FB + DON and E. maxima/C. perfringens challenge on gut permeability to FITC-dextran. Day-old chicks were distributed into four treatment groups: control, necrotic enteritis (NE), fumonisin + deoxynivalenol (FB + DON), and FB + DON + NE groups. Birds in the NE and FB + DON + NE groups received 2.5 × 10³ Eimeria maxima oocyst per bird on day 14. All birds received 1 × 10⁸ CFU/bird of Clostridium perfringens on days 19, 20, and 21. One bird/pen was orally gavaged with 2.2 mg/bird of 4,000 MW fluorescein isothiocyanate dextran (FITC-D), and blood was collected 2 h later. Serum FITC-D concentration was determined in a microplate reader. Bars (+SEM) without a common superscript differ significantly (p < 0.05). n = 8 (8 pens of 15 birds/pen).

FIGURE 3

Effect of subclinical dose of FB + DON and E. maxima/C. perfringens challenge on jejunal tight junction protein mRNA expression. Day-old chicks were distributed into four treatment groups: control, necrotic enteritis (NE), fumonisin + deoxynivalenol (FB + DON), and FB + DON + NE groups. Birds in the NE and FB + DON + NE groups received 2.5 × 10³ Eimeria maxima oocyst per bird on day 14. All birds received 1 × 10⁸ CFU/bird of Clostridium perfringens on days 19, 20, and 21. Tight junction protein mRNA content was analyzed after correcting for the housekeeping gene RPS13 mRNA content and normalizing to the mRNA content of the control group at D21 (A), D28 (B) and D35 (C), so all bars represent fold change compared to the control group. Bars (+SEM) without a common superscript differ significantly (p < 0.05). n = 8 (8 pens of 15 birds/pen).

FIGURE 4

Effect of subclinical dose of FB + DON and E. maxima/C. perfringens challenge on cecal tonsil cytokine mRNA expression. Day-old chicks were distributed into four treatment groups: control, necrotic enteritis (NE), fumonisin + deoxynivalenol (FB + DON), and FB + DON + NE groups. Birds in the NE and FB + DON + NE groups received 2.5 × 10³ Eimeria maxima oocyst per bird on day 14. All birds received 1 × 10⁸ CFU/bird of Clostridium perfringens on days 19, 20, and 21. IL-1β, IL-10, LITAF, and IFN-γ mRNA content was analyzed after correcting for the housekeeping gene RPS13 mRNA content and normalizing to the mRNA content of the control group, so all bars represent fold change compared to the control group. Bars (+SEM) without a common superscript differ significantly (p < 0.05). n = 8 (8 pens of 15 birds/pen).

FIGURE 5

Effect of subclinical dose of FB + DON and E. maxima/C. perfringens challenge on the spleen and cecal tonsil CD8⁺: CD4⁺ ratio. Day-old chicks were distributed into four treatment groups: control, necrotic enteritis (NE), fumonisin + deoxynivalenol (FB + DON), and FB + DON + NE groups. Birds in the NE and FB + DON + NE groups received 2.5 × 10³ Eimeria maxima oocyst per bird on day 14. All birds received 1 × 10⁸ CFU/bird of Clostridium perfringens on days 19, 20, and 21. CD4⁺ and CD8⁺ cells were identified using fluorescent-linked anti-chicken CD4 and CD8 in a flow cytometer. Bars (+SEM) without a common superscript differ significantly (p < 0.05). n = 8 (8 pens of 15 birds/pen).

FIGURE 6

Effect of subclinical dose of FB + DON and E. maxima/C. perfringens challenge on jejunal and ileal histomorphology. Day-old chicks were distributed into four treatment groups: control, necrotic enteritis (NE), fumonisin + deoxynivalenol (FB + DON), and FB + DON + NE groups. Birds in the NE and FB + DON + NE groups received 2.5 × 10³ Eimeria maxima oocyst per bird on day 14. All birds received 1 × 10⁸ CFU/bird of Clostridium perfringens on days 19, 20, and 21. Jejunal and ileal sections were stained with hematoxylin and eosin. Villi height and crypt depth were measured using cellSens Imaging software, and villi height:crypt depth ratio was calculated. Bars (+SEM) without a common superscript differ significantly (p < 0.05). n = 8 (8 pens of 15 birds/pen).

FIGURE 7

Effect of subclinical dose of FB + DON and E. maxima/C. perfringens challenge on C. perfringens, total Lactobacillus, and total Bifidobacteria loads in the cecal content. Day-old chicks were distributed into four treatment groups: control, necrotic enteritis (NE), fumonisin + deoxynivalenol (FB + DON), and FB + DON + NE groups. Birds in the NE and FB + DON + NE groups received 2.5 × 10³ Eimeria maxima oocyst per bird on day 14. All birds received 1 × 10⁸ CFU/bird of Clostridium perfringens on days 19, 20, and 21. Cecal content was analyzed for C. perfringens, (A) total Lactobacillus, (B) and total Bifidobacteria (C) through PCR. Bars (+SEM) without a common superscript differ significantly (p < 0.05). n = 8 (8 pens of 15 birds/pen).

FIGURE 8

Heat map representing Pearson’s r correlation coefficient matrix between cytokine amounts and body weight gain. Heat map showing the transcriptional fold change of LITAF, IL-1β, IL-10, and IFN-γ in the cecal tonsils of birds fed mycotoxin contaminated diet and induced with necrotic enteritis. The color scale, −1 (blue) to +1 (red), and the negative value of the coefficient indicate that increased IL-1β and IL-10 mRNA expression levels are inversely affected the body weight.