A secondary bile acid from microbiota metabolism attenuates ileitis and bile acid reduction in subclinical necrotic enteritis in chickens

Background:Clostridium perfringens-induced chicken necrotic enteritis(NE)is responsible for substantial economic losses worldwide annually.Recently,as a result of antibiotic growth promoter prohibition,the prevalence of NE in chickens has reemerged.This study was aimed to reduce NE through titrating dietary deoxycholic acid(DCA)as an effective antimicrobial alternative.Materials and methods:Day-old broiler chicks were assigned to six groups and fed diets supplemented with 0(basal diet),0.8,1.0 and 1.5 g/kg(on top of basal diet)DCA.The birds were challenged with Eimeria maxima(20,000 oocysts/bird)at d 18 and C.perfringens(109 CFU/bird per day)at d 23,24,and 25 to induce NE.The birds were sacrificed at d 26 when ileal tissue and digesta were collected for analyzing histopathology,mRNA accumulation and C.perfringens colonization by real-time PCR,targeted metabolomics of bile acids,fluorescence in situ hybridization(FISH),or terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)assay.Results:At the cellular level,birds infected with E.maxima and C.perfringens developed subclinical NE and showed shortening villi,crypt hyperplasia and immune cell infiltration in ileum.Dietary DCA alleviated the NE-induced ileal inflammation in a dose-dependent manner compared to NE control birds.Consistent with the increased histopathological scores,subclinical NE birds suffered body weight gain reduction compared to the uninfected birds,an effect attenuated with increased doses of dietary DCA.At the molecular level,the highest dose of DCA at 1.5 g/kg reduced C.perfringens luminal colonization compared to NE birds using PCR and FISH.Furthermore,the dietary DCA reduced subclinical NE-induced intestinal inflammatory gene expression and cell apoptosis using PCR and TUNEL assays.Upon further examining ileal bile acid pool through targeted metabolomics,subclinical NE reduced the total bile acid level in ileal digesta compared to uninfected birds.Notably,dietary DCA increased total bile acid and DCA levels in a dose-dependent manner compared to NE birds.Conclusion:These results indicate that DCA attenuates NE-induced intestinal inflammation and bile acid reduction and could be an effective antimicrobial alternative against the intestinal disease.