| This study aims to use Illumina MiSeq high-throughput sequencing technology,quantitative PCR and PCR-DGGE technology to research on caecum microorganism composition of normal chickens and regulation effects of bacillus licheniformis on caecum flora of broilers infected with necrotic enteritis.240 day-old broilers with similar weight were randomly divided into four groups.The design of the four groups was: control group feeding with basal diet;Infection group feeding with high fishmeal,clostridium perfringens and coccidia;Predisposing group feeding with high fishmeal and coccidia;Prevention group feeding with B.licheniformis,and the remaining treatment was same as that of the infection group.On day 22,15 chickens were randomly picked and cecal samples were collected to conduct high-throughput sequencing,qPCR and PCR-DGGE.On day 14 and 28,5 chickens were randomly picked and cecal samples were collected to conduct qPCR and PCR-DGGE.Using three methods as above,the cecum samples of four groups were analyzed.The results were as follows:The high-throughput sequencing analysis of the cecum samples of healthy broilers showed that the dilution curve had entered the platform stage,and sequencing depth met the requirements of analysis;Only a few samples were different from other samples revealed by Beta diversity;Species composition showed that caecum of healthy chickens was mainly composed of Bacteroidetes,Firmicutes,Proteobacteria and Tenericutes,which consist of 98.8% of the whole microbiota;KEGG database comparative analysis found that the top three gene functions were: transporter,purine metabolism,DNA repair and recombinant protein.The high-throughput analysis of how necrotic enteritis effected cecal microbiota of chickens showed that: Alpha diversity analysis indicated Shannon index of control,predisposing and infection group increase in order.As for Chao1 and observed species,there were significant differences between predisposing and control group,and there is no significant difference between infection and control group;The PCoA analysis showed that the separation degree of sample points between predisposing and infection group washigher.Sample points of infection and control group were nearly separated completely,indicating that microflora structure of them was significantly different.Via Venn graph,it was found that of 162 taxa,the number of specific taxa in infection group was the highest(37,22.8% of total).These results showed that the microbiota of infection group was more specific,which proved that C.perfringens could disturb the cecal microbiota.Regulation effects of B.licheniformis by high-throughput analysis showed: Alpha diversity indicated there is no significant difference in Shannon index between prevention group and other 2 groups.There was no significant difference among three groups in the Chao1 and observed species index neither;For PCoA analysis,in the group,sample points of prevention group were closer and the aggregation degree was higher,indicating that the differences in the group were small.Between the groups,the sample points of prevention group were closer to those of control group,indicating that the two groups were more similar in the group structure.For the proportion of the top 15 species,according to the analysis three groups shared 12 species together,accounts for two-thirds of the total,which showed that they had higher consistency in species composition.However,the relative abundance of each bacterium was different in the groups.Using LDA Effect Size analysis to explore the differences between species,results showed that while control and prevention group had less significantly different species,control and infection group had more significantly different species(21).At the same time,compared with infection group,the relative abundance of four species(Bacteroides,Helicobacter,Megamonas and Akkermansia)increased both in prevention group and control group.Compared with infection group,the cecal bacterial composition of prevention group was closer to that of control group,indicating that B.licheniformis could normalize the cecal microbiota;PICRUSt analysis indicated that there are more significantly different gene functions of chicken caecum flora between infection group and other 2 groups.And the differences between control group and prevention group were smaller,which illustrated the similarity of gene function in chicken caecum between control group and prevention group.Quantitative PCR and PCR-DGGE analysis on different ages of chickens showed that on day 14,there was no significant difference of copy number of chicken caecum targeting five detection of bacteria in control group and prevention group.PCR-DGGE showed thatclustering was divided into two clusters from the beginning,indicating the difference in the microstructure of cecum between control group and prevention group.On day 22,the copy number of total bacteria,firmicutes and enterobacteriaceae in control group were significantly higher than that in infection group.PCR-DGGE showed that there was a disorder in samples of infection group,not only among the samples within the group,but also different from other three groups.On day 28,the microbiota of cecum gradually stabilized,and there was no significant difference in the number of copies of the total bacteria,firmicutes and bacteroidetes among four groups.PCR-DGGE results found the similarity between two samples of prevention group and three samples of control group was above 0.74,indicating that there was still a high similarity between control and prevention group.The experimental results showed that,after B.licheniformis treatment,the microbiota structure and function of chicken caecum tend to be similar with that of healthy chickens,which proved that B.licheniformis could regulate cecal microflora,and improve the health of chickens. |
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