Effects Of Probiotics,bidens Pilosa And Piper Sarmentosum Extract On Eimeria Tenella Infection In Chinese Native Yellow Chickens

Avian coccidiosis is a protozoan parasitic disease caused by several species of Eimeria belonging to the phylum Apicomplexa that mainly affects the intestinal tract of birds.Seven species of Eimeria have been recognized for coccidiosis in chicken.Among them,Eimeria tenella(E.tenella)has been found one of the most divesting specie,which resides in the cecum of chicken and causes destruction of mucosa through villi epithelial cells,resulting in severe epithelial damage,bloody feces,reduced weight gain,decreased feed efficiency and ultimate death.Various control measures,including drugs and vaccines,have been implemented to overcome the Eimeria infection.However,with the passage of time,the Eimeria species have emerged resistant due to repeated and prolonged use of the drugs and the poor use of vaccines may cause disease reversion.In order to overcome these problems,there is an urgent need of novel approaches.Therefore,some probiotics and herbs [Bidens pilosa(B.pilosa)and Piper sarmentosum(P.sarmentosum)extract,(individually and/or in combination)] was studied as prophylactic and therapeutic approaches for control of coccidiosis,where some clinical parameters(body weight,oocyst shedding in feces,bloody diarrhea score and cecal lesion score)were evaluated to examine the potential therapeutic mechanisms of probiotics,B.pilosa and P.sarmentosum extract(PSE).To further confirm the deep mechanisms of probiotics,B.pilosa and P.sarmentosum extract,RNA sequencing,Mi Seq sequencing and quantifying the m RNA expression patterns related with pro-inflammatory and inflammatory cytokines,tight junction proteins,antioxidant enzymes and apoptosis factors were carried out.1.Transcriptome analysis revealed ameliorative effects of Bacillus based probiotic on immunity,gut barrier system and metabolism of chicken under an experimentally induced Eimeria tenella infection in chickenIn this experiment,we performed transcriptome analysis in the chicken cecum tissues in order to examine the underlying potential therapeutic mechanisms of Bacillus based probiotic feeding under an experimental E.tenella infection.For that purpose,sixty three one-day-old Chinese native yellow chickens were randomly divided into one of three experimental groups with 21 birds in each group.The three groups consisted of:(1)negative control untreated non-challenged(NC),(2)positive control untreated challenged(PC)and(3)B.subtilis fed challenged chickens(BS + ET).The results for clinical parameters showed that birds in BS + ET group decreased the bloody diarrhea scores,oocyst shedding and lesion scores compared to PC group.RNA-sequencing(RNA-seq)analysis revealed that in total,2509 up-regulated and 2465 down-regulated differentially expressed genes(DEGs)were detected in the PC group versus NC group comparison.In the comparison of PC group versus BS + ET group,a total of 784 up-regulated and 493 down-regulated DEGs were found.Among them,several DEGs encoding proteins involved in immunity,gut barrier integrity,homeostasis and metabolism were up-regulated by the treatment of probiotic.Functional analysis of DEGs also revealed that some gene ontology(GO)terms related with immunity,metabolism and cellular development were significantly affected by the exposure of probiotic.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis showed that the DEGs in the cecum of BS + ET group mainly participated in the pathways related with immunity and gut barrier integrity,including mitogen-activated protein kinase(MAPK)signaling pathway,toll-like receptor(TLR)signaling pathway,extracellular matrix(ECM)-receptor interaction,tight junction and so on.These results suggested that Bacillus based probiotic modulate immunity,maintain gut homeostasis as well as barrier system and improve metabolism during E.tenella infection.2.Effect of Bacillus subtilis probiotics on the intestinal microbiota of chickens infected with Eimeria tenellaThis trial was then aimed to investigate the chicken gut whole microbiota responses to B.subtilis probiotic feeding in the presence and absence of Eimeria infection.For that purpose,84 newly hatched chicks were assigned into four groups,including(1)non-treated non-challenged control group(CG-ET),(2)non-treated challenged control group(CG + ET),(3)B.subtilis-fed non-challenged group(BS-ET)and(4)B.subtilis-fed challenged group(BS+ ET).CG + ET and BS + ET groups were challenged with E.tenella on 21 day of housing.The results for Alpha diversity revealed that CG + ET and BS+ ET had the lowest indexes of Ace,Chao 1 and Shannon,while the highest indexes of Simpson were found in comparison to CG-ET and BS-ET.Firmicutes was the most affected phylum in all experimental groups following Proteobacteria and Bacteroidota,which showed increased abundance in CG-ET and BS-ET groups,whereas Proteobacteria and Bacteroidota affected in CG + ET and BS + ET groups.The linear discriminant analysis effect size method(l Ef Se)analysis revealed that compared to the CG + ET group,the BS+ ET group increased the abundances of some commensal genera,including Clostridium sensu stricto 1,Corynebacterium,Enterococcus,Romboutsia,Subdoligranulum,Bacillus,Turicibacter and Weissella,with roles in butyrate production,anti-inflammation,metabolic reactions and the modulation of protective pathways against pathogens.These findings evidenced that supplementation of B.subtilis probiotic was positively influenced with commensal genera,thereby alleviating the Eimeria-induced intestinal disruption.3.Role of probiotic and Bidens pilosa on the prevention and control of chicken Eimeria tenellaIn this study,individual and combined anticoccidial mechanisms of probiotic and B.pilosa were evaluated.For that purpose,a total of one hundred and fifty one-day-old chicks were equally distributed into 5 treatment groups with three biological replicates: two groups were allocated as control groups[control group untreated unchallenged(CG)and control positive untreated challenged(CPG)] and three groups were fed diets with probiotic(PG),B.pilosa(BPG)and probiotic + B.pilosa(PG + BPG)and challenged with E.tenella.Birds of all groups were assessed for pre and post-infection body weights,oocyst shedding,cecal lesion scores and m RNA expression levels of apoptosis related proteins(Bcl-2,Bax and caspase-3),antioxidant enzymes(CAT and SOD),pro-inflammatory cytokines(IL-6 and IL-8)and tight junction proteins(CLDN 1 and ZO 1).The results revealed that during infection(day21-29),CPG chickens significantly decreased the body weight compared with CG chickens;however,there was no significant effect on the body weight of chickens fed with probiotic,B.pilosa and probiotic + B.pilosa was observed.CPG birds increased(P < 0.05)oocyst shedding,destructive ratio of ceca and mortality as compared to PG,BPG and PG + BPG birds.CPG group up-regulated the m RNA expression levels of Bcl-2 and down-regulated the m RNA patterns of Bax relative to PG,BPG and PG + BPG groups.Moreover,chickens in groups PG,BPG and PG + BPG enhanced the activities of antioxidant enzymes,pro-inflammatory cytokines and tight junction proteins with the comparison of chickens in group CPG.These results elaborated that the supplementation of probiotic and B.pilosa(individually and/or in combination)appeared to be effective in inhibiting the occurrence of disease and decreasing the severity of Eimeria infection in chickens.4.Effects of feed supplemented Piper sarmentosum extract on Eimeria tenella in chickensThe final experiment was carried out to investigate the potential protective effects of dietary PSE supplementation on body weight,cecal lesion scores,oocyst shedding and m RNA levels of pro-inflammatory and anti-Inflammatory cytokines,tight junction(TJ)related proteins,antioxidant enzymes and apoptosis related mechanism of chickens during induced E.tenella infection.A total of 96 one-day-old Chinese native yellow chickens were randomly allocated to 1 of 3 treatment groups,including [1] control negative untreated uninfected group(CN),[2] control positive untreated infected group(CP)and [3] P.sarmentosum extract-treated infected group(PSE).The results demonstrated that CP group showed a reduction(P < 0.05)in post-infection(PI)body weight compared to control negative group.However,PSE had no significant effects on body weight and cecal lesions compared with CP group.PSE group decreased(P < 0.05)the bloody diarrhea scores and oocyst shedding(during the d 4 to 7 post-infection)than that of CP chickens.CP chickens up-regulated(P < 0.05)the m RNA expression of IL-8and Bcl-2 compared to PSE chickens,while IFN-γ compared to CN chickens.On the other hand,PSE tended to increase(P < 0.05)the transcription patterns of IL-4,IL-10,CLDN 1,SOD and Bax with the comparison of CP;however,there were no significant effects on IL-8,ZO 1 and CAT expression between PSE and CP groups.These findings elaborated that dietary P.sarmentosum extract exhibits potential anticoccidial effects in controlling the coccidia infection.In summary,the present thesis project demonstrated that supplementation of probiotics,B.pilosa and P.sarmentosum extract are effective in increasing the survival of chickens against coccidia by improving overall performance and alleviating negative influences on gut health.The collected results provide sufficient knowledge for probiotics,B.pilosa and P.armentosum extract to be used as alternatives of anticoccidials in feed.