Clostridium perfringens α toxin damages the immune function, antioxidant capacity and intestinal health and induces PLCγ1/AMPK/mTOR pathway-mediated autophagy in broiler chickens

Abstract

Clostridium perfringens α toxin is generated by all types of C. perfringens and is closely related to necrotic enteritis in poultry. This study was conducted to investigate the effects of α toxin on immune function, antioxidant capacity, intestinal health and the underlying mechanisms in broiler chickens. A total of 144 twenty-day-old broiler chickens were randomly assigned to four treatments. On d 21, the birds were intraperitoneally injected with PBS (control group) or α toxin at 0.025, 0.1 or 0.4 U/kg of body weight. Samples were collected at 3 h and 24 h post injection (p.i.). Results showed that α toxin challenge linearly decreased the average daily gain during the 3 days after infection and decreased plasma IgA and IgM levels 3 h p.i. Plasma diamine oxidase and d-lactate levels were linearly elevated by α toxin challenge at 3 h p.i. and 24 h p.i. Alpha toxin challenge linearly decreased plasma and jejunal mucosal catalase, glutathione peroxidase and total superoxide dismutase activities at 3 h p.i. and linearly decreased glutathione peroxidase and total superoxide dismutase activities at 24 h p.i. The ileal villus height to crypt depth ratio decreased linearly with increasing α toxin levels at 3 h p.i. and 24 h p.i. Alpha toxin challenge linearly elevated jejunal IL-1β, IL-6, IL-8 and tumor necrosis factor α mRNA expression at 3 h p.i. Additionally, α toxin challenge linearly reduced the jejunal claudin-1, claudin-3 and zonula occludens 1 mRNA expression at 3 h p.i. and the claudin-3, occludin and zonula occludens 1 mRNA expression at 24 h p.i. What's more, α toxin linearly increased the jejunal PLCγ1, AMPKα1 and ATG5 mRNA expression and linearly decreased the mTOR mRNA expression. In conclusion, C. perfringens α toxin challenge decreased body weight gain, impaired immune function, antioxidant capacity and intestinal health, and induced PLCγ1/AMPK/mTOR pathway-mediated autophagy. The recommended intraperitoneal injection dose for moderate injury was 0.1 U/kg of body weight and the recommended sampling time was 3 h p.i. in broiler chickens.

Keywords: Antioxidant capacity; Autophagy; Broiler chickens; Clostridium perfringens α toxin; Immune function; Intestinal health; Signaling pathway.

Fig. 1

The average daily gain of broiler chickens during 3 days after α toxin injection (d 21 to 24). The four groups were injected with either PBS or α toxin at doses of 0.025, 0.1, and 0.4 U/kg of body weight, respectively. Values are means with their SEM represented by vertical bars. Mean values with different letters were significantly different (P < 0.05).

Fig. 2

Effects of α toxin on the jejunal relative mRNA expression of inflammatory genes of broiler chickens at 3 h. The four groups were injected with either PBS or α toxin at doses of 0.025, 0.1, and 0.4 U/kg of body weight, respectively. TNF-α, tumor necrosis factor alpha. Values are means with their SEM represented by vertical bars. Mean values with different letters were significantly different (P < 0.05).

Fig. 3

Effects of α toxin on the jejunal relative mRNA expression of inflammatory genes of broiler chickens at 24 h. The four groups were injected with either PBS or α toxin at doses of 0.025, 0.1, and 0.4 U/kg of body weight, respectively. TNF-α, tumor necrosis factor alpha. Values are means with their SEM represented by vertical bars. Mean values with different letters were significantly different (P < 0.05).

Fig. 4

Effects of α toxin on the jejunal relative mRNA expression of tight junction proteins of broiler chickens at 3 h. The four groups were injected with either PBS or α toxin at doses of 0.025, 0.1, and 0.4 U/kg of body weight, respectively. ZO-1, zonula occludens 1. Values are means with their SEM represented by vertical bars. Mean values with different letters were significantly different (P < 0.05).

Fig. 5

Effects of α toxin on the jejunal relative mRNA expression of tight junction proteins of broiler chickens at 24 h. The four groups were injected with either PBS or α toxin at doses of 0.025, 0.1, and 0.4 U/kg of body weight, respectively. ZO-1, zonula occludens 1. Values are means with their SEM represented by vertical bars. Mean values with different letters were significantly different (P < 0.05).

Fig. 6

Effects of α toxin on the jejunal relative mRNA expression of autophagy-related signaling pathway of broiler chickens at 3 h. The four groups were injected with either PBS or α toxin at doses of 0.025, 0.1, and 0.4 U/kg of body weight, respectively. PLCγ1, Phospholipases Cγ1; AMPKα1, AMP-activated protein kinase α1; mTOR, mammalian target of rapamycin; Beclin1, programmed cell death 1; ATG5, autophagy-related gene 5. Values are means with their SEM represented by vertical bars. Mean values with different letters were significantly different (P < 0.05).

Fig. 7

Effects of α toxin on the jejunal relative mRNA expression of autophagy-related signaling pathway of broiler chickens at 24 h. The four groups were injected with either PBS or α toxin at doses of 0.025, 0.1, and 0.4 U/kg of body weight, respectively. PLCγ1, Phospholipases Cγ1; AMPKα1, AMP-activated protein kinase α1; mTOR, mammalian target of rapamycin; Beclin1, programmed cell death 1; ATG5, autophagy-related gene 5. Values are means with their SEM represented by vertical bars. Mean values with different letters were significantly different (P < 0.05).