SE Colonization in the Intestinal Tract
At 7 dpi, SE colonization in the digestive tract showed that the number of SE in cecum of the treated group was significantly higher than that in other sections of the intestine (6.96×106 CFU/g ), followed by ileum (1.41×106 CFU/g respectively) (P < 0.05) (Table 1). At 14 dpi, the number of SE in cecum of the treated group was the highest (1.59×103 CFU/g ) across all sections of the intestine, and was significantly lower than that in the treated group at 7 dpi (P < 0.05) (Table 1). The colonization of SE in the control group was negative.
Table 1
The logarithm of SE colonization for each tissue at different time points
|
7 d
|
|
14 d
|
|
CFU/g = mean number of colonies/100*1000/quality of contents(A)
|
LOG(A + 1)
|
|
CFU/g = mean number of colonies/100*1000/quality of contents(A)
|
LOG(A + 1)
|
esophagus
|
0.00
|
0.00
|
|
0.00
|
0.00
|
crop
|
227.27
|
2.36
|
|
1201.31
|
3.08
|
glandular stomach
|
3333.33
|
3.52
|
|
99.50
|
2.00
|
muscular stomach
|
144.93
|
2.16
|
|
0.00
|
0.00
|
duodenum
|
0.00
|
0.00
|
|
0.00
|
0.00
|
jejunum
|
0.00
|
0.00
|
|
1275.42*
|
3.11
|
ileum
|
140571.43
|
5.15
|
|
661.79*
|
2.82
|
cecum
|
6965174.13
|
6.84
|
|
1594.20*
|
3.20*
|
feces
|
741490.83
|
5.87
|
|
1504.30*
|
3.18
|
Note: “*” indicated P < 0.05. |
Immune Organ Index (Dup: Abstract ?)
At 7dpi, the thymus and spleen index of the treated group were 2.49 and 1.66 respectively, which were higher than that of the control group (2.24 and 1.13, respectively), and the difference in spleen index was significant between the two groups (P < 0.05). At 14 dpi, the thymus and spleen index of the treated groups were reduced compared with the control group (Table 2).
Table 2
Comparison of thymus and spleen index between the two groups (n = 3)
DPI
|
7 d
|
14 d
|
|
thymus
|
spleen
|
thymus
|
spleen
|
CK
|
2.24 ± 0.17
|
1.13 ± 0.21
|
1.78 ± 0.16
|
1.08 ± 0.31
|
T
|
2.49 ± 0.27
|
1.66 ± 0.19*
|
1.67 ± 0.18
|
0.99 ± 0.16
|
Note: “*” indicated P < 0.05. |
Serum Cytokine Responding To Se Challenge
Compared with the control group, the contents of IL-2 and IL-18 in serum of the treated group significantly increased by 131.56 pg·mL− 1 and 98.07 pg·mL− 1 (P < 0.05) at 7 dpi, respectively. The contents of TNF-α, IL-2 and IL-18 significantly increased by 29.65 pg·mL− 1, 96.06 pg·mL− 1 and 126.18 pg·mL− 1 at 14 dpi (P < 0.05), respectively (Fig. 1).
Histopathological Results
The pathological sections of the liver, jejunum, ovary, spleen, oviduct, and pancreas were prepared and observed (Fig. 2).
The liver cells of the control group were neatly arranged, and there was no vacuole in the cytoplasm. In contrast, the liver cell structure in the treated group was destroyed and the arrangement was disordered, and there was slight bleeding in the liver, and the cavity appeared in the cytoplasm of cells (Fig. 2 1a-1d).
The jejunal villi in the control group were closely arranged, and the mucosal structure was uniform. In the treated group, the jejunal villi became shorter, broken, swollen, sparse, and irregular in shape, the serous layer of the intestinal wall was partially shed, and the mucosal layer became chaos and thickening (Fig. 2 2a-2d).
The ovarian follicles in the control group were evenly distributed, and there was no damage to the membrane structure. The ovarian hemorrhage, destruction of follicle membrane structure, follicle and follicle membrane hemorrhage, and plasma cell infiltration were observed in the treated group. (Fig. 2 3a-3d).
The oviducal staining showed the control group had a regular shape of the oviduct, and the cells were arranged tightly and neatly. After the infection, the oviducal gland was congested and flushed, the mucosa thickened, and the mononuclear cells infiltrated (Fig. 2 4a-4d).
The staining of pancreas slices showed the islet cells of the control group were arranged tightly and evenly, the pancreas acinar of the treated group had reduced, the islet cells atrophied, and some islets were irregularly shaped (Fig. 2 5a-5d).
The lymphocytes in the spleen of the treated group decreased, bleeding appeared in the white pulp, and there was a homogeneous red-stained substance (Fig. 2 6a-6d).
Quantitative Real-time Pcr (Dup: Abstract ?)
The expression of TLR1a, TLR1b, TLR2, TLR4, TLR5, TLR7, and TLR15 genes in the cecum were significantly increased in the treated group at 7 dpi (P < 0.05), of which TLR15 expression was 3.97 times that in the control group. At 14 dpi, TLR4 and TLR15 gene expression in the treated group were significantly increased (P < 0.05), which were 1.42 and 2.23 times that in the control group, respectively. The expression levels of TLRs decreased at 14 dpi compared with 7 dpi. In the oviduct, the expression of TLR1a, TLR5, and TLR7 increased significantly at 7 dpi (P < 0.05), which were 3.45, 3.52, and 3.13 times that in the control group, respectively. At 14 dpi, TLR1b and TLR3 expressions were 0.3 and 0.22 times that in the control group, respectively (P < 0.01). The expression levels of TLR4 and TLR5 of the ovary at 7 dpi were higher than that in the control group (P < 0.05), which were 1.62 and 2.04 times, respectively. The expression level of TLR15 at 14 dpi was significantly higher than that of the control group (P < 0.01) (Fig. 3).
Microbiota Composition Of Cecal And Oviducal Content
Microbial Composition in Cecum
Heatmap results indicated that SE infection caused dramatic changes in the cecal microbial composition (Fig. 4). The average relative abundance of Firmicutes was 76.19%, which was the dominant bacteria in the cecum contents. In addition, Bacteroidetes and Actinobacteria accounted for an average of 15.01% and 6.13%, respectively, followed by Proteobacteria (1.69%). The remaining categories combined with the unknown group accounted for 0.98%. After the infection, the proportion of Firmicutes in the cecum content was significantly increased, while the proportion of Bacteroides was reduced significantly (Fig. 4a). At the genus level, Anaerotruncus, Butyricicoccus, Parabacteroides, and Lactobacillus accounted for a large proportion of the control group. The predominant genera were Blautia, SMB53, Faecalibacterium and Turicibacter in the treated group (Fig. 4b).
Microbial Composition In Oviduct
The heatmap was used to identify differentially abundant taxonomic features of oviduct microbial composition at the phylum and genus levels (Fig. 5). At the phylum level, among the three groups, Proteobacteria accounted for 44.56% on average, followed by Firmicutes and Bacteroidetes (23.02% and 19.50% on average), and Actinobacteria accounted for an average of 5.03%. The remaining categories were below 1.00%, and the total proportion of mergers with unknown categories was 7.88% (Fig. 5a). Therefore, Proteobacteria was the dominant strain in the contents of the oviduct. SE infection altered the microbial community in the oviduct, of which the Proteobacteria was significantly reduced while the Firmicutes was significantly increased (P < 0.05). At the genus level, the predominant genera in the control group were Mycoplana, Phyllobacterium, Methylobaterium, and Erythrobacter. In the treated group, the predominant genera were Sutterella, Pseudomonas, and Ruegeria (Fig. 5b).
Microbial Diversity Affected By Se Infection In Cecum
Venn diagrams were constructed to visualize differences and overlaps of OTUs between the control group and the treated group (Fig. 6). The three groups shared 1,444 OTUs. The number of unique OTUs was 4,614 in CK, 2,761 in T1 and 3,222 in T2, indicating that SE infection lead to the number of unique OTUs in the treated group decreased, especially at 7dpi. In pairwise sharing, the treated group on the 7dpi and the 14dpi shared the most OTUs.
Principal Component Analysis
We performed PCA analysis on the composition of bacterial genera community structure in different groups. PCA analysis of cecal content extracted two principal components, PC1 (80.7%) and PC2 (17.9%), and showed that the cecal bacterial genera community in CK was separated from that in T1 and T2, the similarity between T1 and T2 in the genus level was relatively high. Among the three groups, the differences between Faecalibacterium, Bacteroides, Bifidobacterium, Lactobacillus, and Rumenococcus were greater than that of other genera (Fig. 7a).
The PCA results of oviduct microbial composition showed that Ochrobactrum, Bacteroides, Sediminibacterium, Acinetobacter, Agrobacterium, Cupriavidus, Blautia, and Lactobacillus were different compared to other bacteria among the three groups. There was no overlap among the three groups, the control group and the treated groups were separated (Fig. 7b).
Effects Of Se Infection On Probiotic And Harmful Bacteria In Cecum Contents
At 7 dpi, the relative abundance of Bifidobacterium and Lactobacillus in the treated group was 0.031% and 1.030%, and in the control group were 0.056% and 2.770%, respectively. Compared with the control group, the relative abundance of Bifidobacterium in the treated group was reduced and the relative abundance of Lactobacillus was reduced significantly (P < 0.05). At 14 dpi, the relative abundance of Bifidobacterium and Lactobacillus increased significantly compared with that at 7dpi (P < 0.05), which was 0.036% and 1.046%, respectively (Fig. 8a).
At 7 dpi, the relative abundance of Enterobacteriaceae and Enterococcus in the treated group were 1.199% and 0.072%, respectively, which was significantly higher than that of the control group of 0.056% and 0.017% (P < 0.05). At 14 dpi, the relative abundance of Enterobacteriaceae and Enterococcus decreased compared with the 7dpi, which was 0.010% and 0%, respectively (Fig. 8b).
Relationship Between Tlrs Expression And Microbial Abundance Of The Cecum
Pearson correlation analysis was performed between the expression of TLRs and the abundance of microbes in the cecum. The abundance of Bifidobacterium and Lactobacillus were negatively correlated with the expression of TLRs except for TLR3. While the abundance of Enterobacteriaceae and Enterococcus were positively correlated with the expression of TLRs except for TLR3. Enterobacteriaceae were significantly correlated with the expression of TLR1a and TLR5 (P < 0.05), and Enterococci were significantly correlated with TLR1b and TLR2 (P < 0.05). TLRs expression was not correlated with the total number of OTUs (Table 3).
Table 3
Pearson correlation coefficient between TLRS gene expression and cecal microbial community abundance
|
Pearson correlation coefficient
|
|
Bifidobacterium
|
Lactobacillus
|
Enterobacteriaceae
|
Enterococcus
|
OTU
|
TLR1A
|
-0.514
|
-0.35
|
0.999*
|
0.99
|
0.245
|
TLR1B
|
-0.626
|
-0.474
|
0.982
|
1**
|
0.111
|
TLR2
|
-0.644
|
-0.495
|
0.978
|
1*
|
0.087
|
TLR3
|
0.606
|
0.452
|
-0.987
|
-1
|
-0.136
|
TLR4
|
-0.795
|
-0.672
|
0.909
|
0.972
|
-0.131
|
TLR5
|
-0.423
|
-0.252
|
0.999*
|
0.971
|
0.344
|
TLR7
|
-0.556
|
-0.396
|
0.995
|
0.996
|
0.197
|
TLR15
|
-0.908
|
-0.817
|
0.796
|
0.897
|
-0.344
|
TLR21
|
-0.751
|
-0.619
|
0.935
|
0.986
|
-0.062
|
Note: The correlation is positively correlated with the absolute value of the correlation coefficient. “*” indicated P < 0.05, “**” indicated P < 0.01. |