Effects Of Bacillus Licheniformis On Ileum Microbiota In Broiler Chickens Infected With Necrotic Enteritis

Necrotic enteritis(NE)is a severe intestinal disease,which can change gut microbiota.NE outbreaks in broiler chickens have a severe economic impact.However,little is known regarding how the gut microbiota of NE chicken ileum are changed by Bacillus licheniformis.The objective of this study was to investigate how the ileum microbiota structure was changed by Bacillus licheniformis in broiler chickens challenged with Clostridium perfringens fishmeal and coccidium-induced necrotic enteritis.Furthermore,the data of this study may provide a new insight into the prevention and treatment of NE in broilers.Simply stated,240 chickens were born on the same day with similar body masses and were purchased from a local commercial hatchery.All the samples were randomly assigned to four groups(NC,FC,PC and BL),five replicates per treatment,and twelve chickens per replicate in a pen.During this experiment,all the chickens were fed with free access to water and food in a temperature controlled room,and light was provided24 hours a day.The process is distributed into five stages:(1)The birds were fed with a basal diet from day 1 to day 13.(2)From day 14 onward,the diets of all the birds were changed to the basal diets supplemented with 30%fishmeal(w/w),except for those of the NC group.(3)On day 15,all the birds,with the exception of those in the NC group,were inoculated with a 10-fold coccidiosis vaccine by oral gavage.In contrast,the birds in the NC group received sterile phosphate buffered saline.(4)On days 18,19,and 20,the birds in the PC and BL groups were individually infected with 1 mL of C.perfringens through a plastic tube containing approximately 2.2×10~8 CFU/mL.(5)On days 21 to 28,The birds were fed with a basal diet.The feed of the BL group was dosed with 1.0×10~6 CFU/g B.licheniformis throughout the experiment.The results showed that:1.During the experiment,only 4 broilers died in the PC group,and the death rate was 6.7%.The growth performance of the chickens showed that the body weight gain and feed conversion ratio of group BL were significantly different from other groups(P<0.05),and others three groups were not significant between each others(P>0.05)in14 days of age.In 22 days of age,the body weight gain and feed conversion ratio of group NC were significantly different from those of group PC(P<0.05),and other groups were not significant between each others(P>0.05).In 28 days of age,the body weight gain and feed conversion ratio of group NC and BL were significantly different from those of group PC(P<0.05),and others groups were not significant between each other(P>0.05).2.A total of 60 chicken ileum samples(15 NC,15 PC,15 FC and 15 BL)were collected and sent for sequencing in 22 days of ages.After selecting the OTU and checking for chimaera,a total of 1,532,759 reads were assigned to 4,883 non-singleton OTUs from 60 samples.Each sample had 25,546±9,653 reads and 428±172 OTUs on average.PC was significantly higher than NC(P<0.05)in community richness.However,no significant difference in Alpha diversity analysis was observed between the samples of NC and BL(P>0.05).Compared to the PC,beta diversity were more similar in BL and NC.At the phylum level,35 phylum taxa were detected.And the dominant phylum taxa were classified into 7 phyla taxa,and 6 were identified as a phylum taxa.Firmicutes,Proteobacteria and Bacteroidetes were significantly different(P<0.05)in NC and PC.At the class level,81 class taxa were detected.And the dominant class were classified into 6 class taxa,and 5 were identified as a class taxa.Bacilli,Gammaproteobacteria and Bacteroidia were significantly different(P<0.05)in NC and PC.Bacilli,was significantly different(P<0.05)in NC and BL.Bacteroidia was significantly different(P<0.05)in BL and PC.At the order level,147 order taxa were detected.And the dominant order taxa were classified into 8 order taxa,and 7were identified as a order.Lactobacillales,Bacillales and Enterobacteriales were significantly different(P<0.05)in NC and PC.Lactobacillales and Bacillales were significantly different(P<0.05)in NC and BL.Bacteroidales and Bacillales were significantly different(P<0.05)in NC.At the family level,248 family taxa were detected.And the dominant family were classified into 13 family taxa,and 11 were identified as a family.Lactobacillaceae,Enterobacteriaceae and Lachnospiraceae were significantly different(P<0.05)in NC and PC.Lactobacillaceae was significantly different(P<0.05)in NC and BL.At the genera level,422 genera taxa were detected.And the dominant genera were classified into 14 genera taxa,and 9 were identified as a genera.21 bacterial taxa were significant difference compared NC with PC,and only 2bacterial taxa were significant difference compared NC and BL by Lefse.The PICRUSt analysis revealed that the enrichment of"Glycan Biosynthesis and Metabolism"pathways in the PC group is remarkable,which was correlated with Ruminococcus.And a tumour suppressor gene,p53,which was negatively correlated with Helicobacter,was enriched in the BL group.3.Real-time PCR demonstrated that four groups were no significant difference(P>0.05)with each other in 14 days of age.In 22 days of age,compared with NC,the level of total bacteria were significant difference(P<0.05)with PC and FC,and Firmicutes was significant difference(P<0.05)with FC.In 28 days of age,the level of total bacteria in the PC group increased significantly(P<0.05)than NC.Compared with the PC group,Bacteroidetes in the NC and FC significantly decreased(P<0.05).Firmicutes in the PC group increased significantly(P<0.05)than NC.Compared with the FC group,Enterobacteriaceae in the NC and FC significantly increased(P<0.05).NC was significant difference(P<0.05)with FC and PC in Lactobacillus.And BL was also significant difference(P<0.05)with PC in Lactobacillus.4.We analyze flora structure by PCR-DGGE.In 14 days of age,NC and BL group were clustered into two cluster respectively.In 22 days of age,the samples from NC and BL were more similar than PC,and NC and BL were lowly difference within intra-group.In 28 days of age,ileal flora of broilers in each group were clustered into two clusters,BL group and the control group(NC)clustered into a cluster,the other two groups gather in another cluster.In summary,the results of our study showed that dietary supplementation of B.licheniformis in necrotic enteritis chickens were not only promote growth,but also against disease,normalize the ileum microbiota and restore host to health.