Preliminary Studies On The Roles Of Clostridium Butyricum For Ameliorating Gut Mucosal Barrier Of Broiler Chickens With Necrotic Enteritis

Necrotic enteritis(NE)in chicken caused by Clostridium perfringens(C.perfringens)is one of the main deseases that dramatically harm chicken farming.In the context of prohibiting or restricting the use of antibiotic feed additices,it is an urgent need to develop alternatives for antibiotic feed additives and explore their mechanism of action.Clostridium butyricum(C.butyricum)is a probiotic preparation that has been widely used in animal production,but whether it can promote the repair of the function integrity of the intestinal epithetlial barrier in chickens with NE and its impacting mechanism is nearly unknown.Therefore,this study developed a model of NE disease in chickens and added C.butyricum in feed to analyze the changes in weight gain,intestinal mucosal cell tight junction protein,short circuit current,etc.in sick chickens,and explored its mechanism of action through transcriptiomics and metabolomics and in vitro organoids model,to lay a theoretical and experimental foundation for the probitic prevention and treatment of NE in chickens.The main methods and results of this study are as follows:1)Firstly,we investigated the effects of C.butyricum supplementations on weight gains,gut findings,gut barrier modes,and the alterative expressions of immune-related cytokines to NE with bioilers.Chickens were divided into five groups: control group(NC),supplement C.butyricum only group(CB),NE group(PC),supplement C.butyricum from Day 14 to Day 22 with the NE group(NECB1),and supplement C.butyricum from Day 1 to Day 22 with the NE group(NECB2).The results showed a significantly decreased average daily weight gain and an increased feed conversion rate in the infected group(PC)compared with the C.butyricum-supplemented groups(NECB1 and NECB2).Histopathological observation on the Hematoxylin–Eosin stain small intestine sections revealed that supplementation of C.butyricum(NECB1 and NECB2)could increase the intestinal villus height/crypt depth and lessen the intestinal damage under NE.ELISA and Limulus tests showed that broilers infected with NE(PC)had higher serum Ig A and lipopolysaccharide content,however,after C.butyricum supplementation(NECB1 and NECB2),they returned to a normal level.Furthermore,real-time PCR and Western blot results indicated that compared with PC,supplementing C.butyricum(NECB1 and NECB2)could initialize the expression of genes related to the intestinal barrier-associated proteins(such as CLDN-1,CLDN-3,OCLN,MUC-2,ZO-1,and CLDN5),cytokines(such as IL-10,IL-6,and TGFB1),and C.perfringens plc gene expression.Moreover,the results detected by the Ussing chamber suggested that C.butyricum(NECB1 and NECB2)could amend the decrease in tissue conductance value and short-circuit current value caused by NE.In addition,NECB2 significantly reduced the upregulation of fluorescein isothiocyanate-dextran flux caused by the NE disease.In conclusion,these findings suggested that dietary supplementation of C.butyricum in broilers with NE improved chicken growth performance,intestinal integrity and barrier function,and immunological status.2)Secondly,we investigated the potential mechanisms of C.butyricum in restoring the intestinal health of broiler chickens with NE by high-throughput sequencing.Chickens were divided into three groups: the control group(Control),NE group(PC),and NE group supplemented with C.butyricum from Day 1 to Day 22(NECB).The contents of the small intestine as well as the tissue of the small intestine were collected.Transcriptomics sequencing of intestine tissue samples and metabolomics analyses of intestine contents were performed.Transcriptome results showed that compared to NECB,456 differentially expressed genes were upregulated and 338 were downregulated in the NE group.KEGG analysis revealed alterations in pathways including MAPK signaling,Toll-like receptor signaling,NOD-like receptor signaling,and arginine synthesis,and so on.Also,the metabolomic analysis identified 199 differentinally produced metabolites between the two groups both in the positive and negative ion models.Several metabolic pathways were also altered,including the calcium signaling pathway,arachidonic acid metabolism,galactose metabolism,carbon metabolism,and iron death,and some others.Furthermore,integrative transcriptomics and metabolomics analysis demonstrated that both of them targeted the MAPK pathway,and the expression levels of c-JUN and JUND in the MAPK pathway in NE broilers dropped following supplementation with C.butyricum.And JNK is a positive regulator of c-JUN,and we postulated,as a result,that C.butyricum might have a function in JNK regulation.3)Finally,to further investigate the repairring mechanism of C.butyricum to the intestinal structural barrier in NE broilers,we used organoid culture systems to investigate the physiological mechanisms of bacterial-epithelial interaction.Data from IFA showed that JNK localized to the nucleus after C.perfringens infected chicken 2D small intestine enteroids,while JNK was inhibited activation after the addition of C.butyricum.In addition,real-time PCR,western blot and immunofluorescence results showed the tight junctions(CLDN-1 and OCLN)were decreased by C.perfringens infection.However,the addition of C.butyricum amended the decrease in CLDN-1 and OCLN expression caused by C.perfringens,which was similar to the addition of the JNK pathway inhibitor SP600125.These results showed that C.butyricum can repair intestinal structure barrier damage through the JNK pathway caused by C.perfringens.In conclusion,our studies have proved that C.butyricum can significantly promote the repair(/recovery)of the gut mucosal barrier function of NE in chickens,improve the mucosal immune responses,and ameliorate the daily weight gain and growth performance of chickens.Furthermore,we also suggested the potential roles of the JNK signaling pathway in the regulation of cell tight junction protein expression by the transcriptomic,metabolomic and 2D enteroid models.These results will give theoretical and/or experimental foundations for the wide application of C.butyricum to the prevention and treatment of NE with chicken.