Analysis of intestinal microbial diversity
OTU clustering and Species annotation
After high-throughput sequencing, a total of 319,140 valid sequences were obtained from the 12 samples (Table 1). Under the 97% similarity level, the blank group sequence clustering analysis for 403 OTUs, cluster analysis for 405 OTUs group Ⅰ, cluster analysis for 422 OTUs group Ⅱ, cluster analysis for 381 OTUs group Ⅲ.
Table 4 | Basic information of intestinal microbial sequencing of chickens
The group
|
Sample number
|
Number of OTUs
|
Effective sequence number
|
Con
|
A1
|
267
|
17247
|
A2
|
284
|
27310
|
A3
|
299
|
28044
|
Total
|
403
|
72601
|
Ⅰ
|
B1
|
273
|
27511
|
B2
|
276
|
27125
|
B3
|
324
|
22475
|
Total
|
405
|
77111
|
Ⅱ
|
C1
|
246
|
25580
|
C2
|
277
|
28907
|
C3
|
320
|
25698
|
Total
|
422
|
80185
|
Ⅲ
|
D1
|
254
|
31401
|
D2
|
288
|
30673
|
D3
|
272
|
27169
|
Total
|
381
|
89243
|
Species community analysis
Common and unique OTU statistical analysis
The Venn diagram was made by analyzing the total number of OTUs. As shown in FIG. 3-1 A, there were 286 OTUs in the four groups and 303 OTUs in the test group. In addition, each sample has 91 OTUs (Figure 1 B). Blank group special OTUs and Ⅰ respectively 4 OTUs, unique characteristic OTUs Ⅲ group number is three, but no specific OTUs Ⅱ group.
Community composition analysis
The relative abundance of each taxonomic group was calculated. At the same time, the taxonomic group whose relative abundance was above 1% (default value) was selected, and the first 15 (default value) were classified, and the relative abundance distribution diagram and the relationship diagram between samples and species were drawn. As shown in figure 3-2 A, the first advantage of 15 species belong to Barnesiella respectively (genus name, OTU - 2), Bacteroides (Bacteroides, OTU - 3, OTU - 4, OTU - 5, OTU - 9), the other branches of bacteria genera (Alistipes, OTU - 6 and OTU - 7), fecal coli (Faecalibacterium, OTU - 8), Campylobacter, Campylobacter, OTU - 10), Muribaculaceae (OTU - 11), Plesiomonas (OTU-12), Negativibacillus (OTU-13), Bacteroides Gallinaceum (OTU-17), Bacteroides Caecicola (OTU-18), and Ruminococcus (OTU-20).
In the blank group chicks caecum of species relative abundance within the top ten respectively OTU - 11 (18.25%) > OTU - 2 (7.86%) > OTU - 8 (6.24%) > OTU - 4 (5.30%) > OUT 18 (5.04%) > OTU - 13 (4.28%) > OTU - 7 (3.89%) > OTU - 6 (3.35%) > OTU - 20 (2.45%) > OTU - 9 (1.29%).GroupⅠ chicks in the cecum relative abundance of top ten species respectively OTU - 12 (23.33%) > OTU - 5 (13.79%) > OTU - 4 (8.60%) > OTU - 8 (4.64%) > OTU - 20 (4.37%) = OTU - 18 (4.37%) > OTU - 10 (2.07%) > OTU - 6 (1.95%) > OTU - 13 (1.91%) > OTU - 9 (1.02%). Strains of chicken caecum group Ⅱrelative abundance the top 10 were OUT - 2 (25.13%) > OTU - 17 (8.93%) > OTU - 7 (3.71%) > OTU - 6 (3.65%) > OTU - 20 (3.36%) > OTU - 8 (3.26%) > OTU - 10 (2.61%) > OTU - 13 (1.33%) > OTU - 5 (1.04%) > OTU - 9 (0.61%). Group Ⅲ chicks, the relative abundance of cecum strains within the first ten OTU - 3 (18.79%) > OTU - 4 (13.79%) > OUT - 2 (10.66%) > OTU - 20 (7.37%) > OTU - 9 (5.51%) > OTU - 8 (3.62%) > OTU - 10 (2.17%) > OTU - 7 (1.81%) > OTU - 13 (0.58%) > OUT - 6 (0.04%).
Therefore, the main dominant species in the intestinal tract of broiler chickens in the four groups were Muribaculaceae (Otu-11), Plesiomonas (Otu-12), Barnesiella (Otu-2) and Bacteroides (Otu-3). Muribaculaceae (Otu-11) mainly existed in the cecum of the chicks in the blank group, and the relative abundance of the other three groups was about 0.0065%, 0.14% and 0.0034%. Barnesiella (OTU - 2) exists in the in the cecum of broiler chickens of the blank group, group Ⅱ and group Ⅲ, but in group Ⅰ chicks the the cecum content was very low (0.084%). OTU - 3 mainly exist in the cecum chicks of the chick of group Ⅲ, the relative abundance of the other three groups in the cecum of chicks was low (0.045%, 0.031% and 0.062%). OTU - 12 mainly exists in the in the cecum of group Ⅰ broiler chicks, the relative abundance of the other three group in the cecum of chicks was only about 0.0069%, 0.0037% and 0.0090% (FIG.1).
According to the OTU table, the relative abundance of Lactobacillus was counted.
According to the results (FIG. 3-3), there was no significant difference (p = 0.572 > 0.05) among the number of intestinal lactobacilli in experimental group and the blank group chick’s , but in group and Ⅲ the lactobacillus abundance was increased by about 7.33 times, 66% and 6.33 times respectively.
Species abundance cluster analysis
By the classification information drawn through heat map (OTU) (figure 3-4), displayed blank group and groupⅡ for a class, group I and Ⅲ together for a class.
Phylogenetic analysis
The top 20 species with relative abundance above 0.01% were selected to construct the species evolutionary tree by grouping. As shown, blank group and groupⅠ chicks caecum microorganism were similar, however groupⅡ and Ⅲchicken caecum microorganism were similar (figure 3-5).
At different classification levels, the cluster tree was established by clustering analysis of samples under classification, and the horizontal bar chart was formed by the classification of samples at different levels. As shown in Figure 3-7, the distribution of OTU-11 was different from that of other species, and its main distribution was in the cecum of the chickens in the blank group. OTU - 14 and OTU - 17 was a category, mainly distributed in the cecum of broiler chickens of groupⅡ. OTU - 5 and OTU - 12 were, mainly distributed in the chicken’s cecum of groupⅠ. OTU - 8 and OTU - 20 were distributed with all the four groups, and total abundance, in the group oⅠ and Ⅱ to prevent the abundance in the cecum is very close.
LEfSe analysis
Species with significant difference between groups were identified by linear discriminant analysis Efect Size (LEfSe). As shown in Figure 7, it was found that the abundance of two strains, both belonging to Ruminiclostridium 9, significantly increased in the cecum of broiler chickens in the control group from the level of "gate" to the level of OTU. Three kinds of bacteria were found to be abundant in the cecum of groupⅠ broiler chickens, Melaina bacteria, Gastranaerophilales, Cyanobacteria (Cyanobacteria) respectively. Five kinds of bacterial abundance increased significantly in the cecum of group Ⅱ chickens Negativicutes, Selenomonadales, Phascolarctobacterium sp. 377 (377) the koala coli, Acidaminococcaceae aureus (amino acid), Phascolarcto bacterium Respectively. Abundance in only one flora blind field significantly in group Ⅲ, it belongs to the Phylum Firmicutes.
Difference test between groups
Kruskal-wallis rank sum test was used to compare the distribution of species in control group and experimental group. As shown in figure 8, although the distribution of OTUs in the four groups was different, the results showed that the distribution of OTUs in the cecum of chicks in the experimental group and the control group was not significant (p>0.05).
Network and predictive analysis
Through the analysis of collinear network, the similarity and difference between samples are highlighted. As shown in Figure 9A, the difference between the samples in the test group and the control group was not significant. Spearman correlation coefficient (threshold value is 0.8) was calculated based on the relative abundance between species samples based on OTU table to make the interaction network diagram. As shown in 14 B, the correlation between OTU-42 and OTU-85 is relatively high, while the correlation between OTU-102 and OTU-24, OTU-194, OTU-360 and OTU-47 is relatively high. The correlation between OTU-360 and OTU-47, OTU-31 and OTU-43 is relatively high. However, OTU-46 only has a high correlation with OTU-47. Otu-13 has only a high correlation with OTU-24.
Relative expression of TLRs mRNA
As shown in (Figure 9 A) relative expression of TLR1A gene in jejunum and cecum of broiler chickens in the test group was significantly increased compared with the control group (P =0.004<0.05 and P =0.036<0.05). Group Ⅲ jejunum TLR1A transcription level increased 30.4%; Group Ⅱ and Ⅲ cecum TLR1A transcription level raised 29.2% (p < 0.001) and 62.0% (p < 0.001) respectively. Relative expression of the ileum TLR1A genes significantly lower (p = 0.003 < 0.01), group Ⅰ and Ⅱ fell 20.8%, respectively (p = 0.002 < 0.01) and 11.1% (p = 0.036 < 0.05).
Compared with the control group, experimental group jejunum TLR1B genetic relative expression quantity of no significant change (p = 0.052 > 0.05), but group Ⅱ jejunum TLR1B transcription level increased by 23%. Experimental group in the ileum TLR1B relative expression increased significantly (p = 0.001 < 0.01), group Ⅰ, Ⅱ and Ⅲ ileum TLR1B transcription level by 30.4% (p = 0.001 < 0.01), 37.33% (p < 0.001), 15.34% (p = 0.023 < 0.05) respectively. Within the cecum TLR1B gene expression quantity significantly reduced relatively, although groupⅠ and Ⅱ set of changes was not significant, but group Ⅲ decreased by 54.1% (p < 0.001) (Figure 9 B)
Compared with the control group, the relative expression of TLR2A in the jejunum of experimental group significantly increased (p = 0.003 < 0.01), group Ⅰ and Ⅱ raised 27.9% (p = 0.011 < 0.05) and 46.9% (p = 0.001 < 0.01) respectively. TLR2A relative expression in the in the ileum of experimental group decreased significantly (p = 0.002 < 0.01), for group of Ⅱ and Ⅲ were reduced by 24.1% (p = 0.003 < 0.01) and 27.2% (p = 0.001 < 0.01). Within the cecum TLR2A significantly lower (p < 0.01), group Ⅱ and Ⅲ respectively by 12.8% (p = 0.019 < 0.05) and 39.8% (p < 0.001). Relative expression in the liver of TLR2A significantly increased (p < 0.01), group Ⅰ and Ⅲ raised about 16.7% respectively (p = 0.011 < 0.05) and 45.9% (p < 0.01). Spleens TLR2A relative expression significantly increased (p < 0.01), a group of Ⅰ TLR2A relative expression reduced, but not significant (p = 0.192 > 0.05). However, group Ⅱ and Ⅲ respectively, the relative expression of about 8 times (p < 0.01) and 2.5 (p < 0.01). Within the kidney TLR2A gene expression quantity increased (p < 0.01), group of kidney Ⅰ TLR2A gene expression quantity is reduced, but not dramatically, but group Ⅱ increases by about 69.2% (p < 0.01) (Figure 9 C)
Compared with the control group, TLR2B gene expression in jejunum of the experimental group was significantly decreased (P <0.01). Group Ⅱ and Ⅲ reduced by 23.8% (p < 0.01) and 54.4% respectively (p < 0.01). Group Ⅰ ileum TLR2B genes relative expression was significantly increased by 24.1% (p = 0.007 < 0.01), but the group Ⅱ and Ⅲ were reduced by 34.4% (p = 0.001 < 0.01) and 15.6% (p = 0.047 < 0.05). The relative expression of TLR2B gene in cecum was significantly decreased (P <0.01), and decreased by 25.2%, 44.4% and 60.4%, respectively, in the three groups (Figure 9 D)
Compared with the control group, TLR3 gene expression quantity in th ofe jejunum experimental group increased significantly (p = 0.008 < 0.01), in group Ⅰ and Ⅱ increased by 69.48% (p = 0.002 < 0.01) and 52.29% (p = 0.009 < 0.01) respectively. TLR3 gene expression was significantly increased in the ileum (P <0.01). These three groups were significantly increased by 47.s70%, 57.58% and 97.60%, respectively. TLR3 gene expression in the cecum of the all three experimental group was increased significantly, which was about 1.56, 6.56 and 3.4 times, respectively. Relative expression in the liver TLR3 gene significantly increased, and only set Ⅱ significantly by 93.2% (p < 0.01). TLR3 gene expression in spleen volume increased significantly (p < 0.01), group Ⅱ and Ⅲ raised 13.79 times and 1.92 times respectively (p = 0.014 < 0.05). There was no significant change in TLR3 gene expression in the kidney (Figure 9 E)
Compared with the blank group, jejunum TLR4 gene expression quantity in experimental groups increased significantly (p = 0.002 < 0.01), group Ⅱ and Ⅲ raised 64.3% respectively (p = 0.001 < 0.01) and 59.4% (p = 0.002 < 0.01). Ileum amount of TLR4 gene expression in the experimental group significantly raised (p = 0.001 < 0.01), in group Ⅰ and Ⅱ by 64.8% (p < 0.01) and 35.7% (p = 0.007 < 0.01) respectively. However, there was no significant change in TLR4 gene expression in the cecum of the experimental group (Figure 9 F)
Compared with the blank group, TLR5 gene expression in the jejunum of the experimental group was significantly increased (P <0.01), which was about 1.9-fold (P <0.01), 1.94-fold (P <0.01) and 1.14-fold (P <0.01), respectively, in the three groups. The amount of living gene expression in the illium of experimental groups was significantly influenced, such as, group Ⅰ and Ⅱ living gene expression fell by 31.8% (p < 0.01) and 31.5% (p < 0.01), while Ⅲ group increased by about 9.2% (p = 0.015 < 0.05). TLR5 gene expression in cecum of experimental group was significantly increased (P <0.01). The increase in the three groups was about 1.25-fold (P <0.01), 0.8 fold (P =0.002<0.05) and 1.47 fold (P <0.01), respectively (Figure 9 G)
Compared with the blank group, TLR7 gene expression in the jejunum was significantly increased (p<0.01), by 144% (p <0.01), 92.7% (p <0.01) and 88.3% (p <0.01), respectively. TLR7 gene expression in the illium of experimental groups was significantly decreased (p = 0.001 < 0.01), but group Ⅱ and Ⅲ by 29.0% (p < 0.01) and 21.7% (p = 0.002 < 0.01) respectively. TLR7 gene expression quantity within the cecum of group Ⅰ and Ⅲincreased significantly (p < 0.01), about 1.5 times and 46.9%, respectively (Figure 9 H).
Compared with the blank group, TLR15 gene expression in the jejunum of the experimental group was significantly decreased, by 44.8% (p <0.01), 64.8% (p <0.01) and 63.2% (p <0.01), respectively. There was no significant change in TLR15 gene expression in the ileum (p=0.120>0.05). But groupⅢ increased significantly (p = 0.029 < 0.05). TLR15 gene expression quantity in the cecum of, group Ⅰ and Ⅱ significantly increased by 39.3% (p = 0.005 < 0.01) and 35.5% (p = 0.008 < 0.01), but group Ⅲ significantly reduced by 30.6% (p = 0.016 < 0.01). TLR15 gene expression volume in the liver increased significantly (p = 0.005 < 0.01), group Ⅱ and Ⅲ increased by 17.8% (p = 0.024 < 0.05) and 28.8% (p = 0.002 < 0.01) respectively. TLR15 gene expression quantity in the spleen of experimental group Ⅱ and Ⅲ increased significantly about 12.62 times (p < 0.01) and 4.19 (p < 0.01). The relative expression of TLR15 in the kidney of the experimental group was significantly changed. However, in group Ⅰ it was reduced by 29.8% (p = 0.034 < 0.05), in group Ⅱ and Ⅲ it was increased about 56.5% (p = 0.001 < 0.05) and 2.89 (p < 0.01) (Figure 9 I)
Compared with the blank group, TLR21 gene expression in jejunum, ileum and cecum in the experimental group was decreased (p <0.01). Within the group Ⅱ and Ⅲ jejunum TLR21 decreased about 30.1% amount of gene expression (p = 0.012 < 0.05) and 51.2% (p = 0.001 < 0.05). Within the ilium of group Ⅰ, Ⅱ and Ⅲ it was reduced by 45.3% (p = 0.001 < 0.01), 67.9% (p < 0.01) and 46.5% (p = 0.001 < 0.01). In the cecum the expression of TLR21 was decreased by 32.4% (p <0.01), 26.3% (p =0.001<0.01), and 53.7% (p <0.01), respectively (Figure 9 J)
Compared with the control group, MyD88 gene expression in the jejunum of the experimental group was significantly increased (p <0.01), and it was up-regulated by 5.3, 5.7 and 3.4 times in the three groups, respectively. Experimental group MyD88 gene expression in the ileum volume decreased significantly, the expression in the group Ⅰ although improved, but not statistically significant. However, groups Ⅱ and Ⅲ MyD88 gene expression reduced about 29.55% respectively (p = 0.002 < 0.01) and 39.2% (p < 0.01). MyD88 gene expression was significantly down-regulated in the cecum of the experimental group. The three groups significantly decreased by 32.70%, 47.56% and 45.4%, respectively. MyD88 gene expression in the liver volume of experimental groups increased significantly, especially group Ⅱ significantly increased by 93.31% (p < 0.01). MyD88 gene expression in the spleen of the experimental groups was significantly increased, 72.55%, 1.64 times and 1.61 times, in the three groups, respectively. MyD88 gene expression in kidney volume of experimental group Ⅰ was significantly reduced (p = 0.024 < 0.05), but in the group Ⅱ it was significantly improved (p = 0.004 < 0.01) (Figure 9 K).
There was no significant change in Il-1 gene expression in the liver compared with the blank group (p=0.131>0.05). Gene expression quantity of IL - 1β in the spleen of experimental group Ⅲ was significantly increased (p = 0.041 < 0.05), by 28.26% (p = 0.007 < 0.05). The expression of IL-1 gene in the kidney was significantly increased in the three groups by 32.37%, 68.73% and 1.18 times (p <0.01), respectively (Figure 9 L).