3.1. HQD reduced the mortality of colibacillosis and increased the average daily gain in chicks
Compared with the CG, chicks challenged with E. coli O78 at 27 days of age showed ruffled feathers, closed eyes, reluctance to move, defecated white or green stools and soiled vent. Post-mortem examination showed that E. coli O78 infection led to severe perihepatitis, pericarditis, granuloma, and bleeding spots in the small intestine (Fig. 1). E. coli O78 infection reduced average daily gain (ADG) between 1 and 32 days of age (p <0.05), which was reversed by HQD (Fig. 2A). There was no significant difference between HQD-HQ, HQD-DZ, HQD-GC, HQD-SY and ENR supplementation compared with HQD. E. coli O78 infection increased mortality at day 5 post E. coli O78 challenge, which was reversed by HQD, 500 mg/kg HQD-GC and 500 mg/kg HQD-SY (Fig. 2B). Moreover, there was no effect on mortality in chicks challenged with E. coli O78 at the dose of 250 mg/kg except for the HQD group. Therefore, the dose of 500 mg/kg groups were more worthy of further study.
3.2. HQD changed the visceral index in chicks with colibacillosis
In contrast, the heart index and the liver index were increased in chicks challenged with E. coli O78, as compared with control group, which was reversed by HQD and ENR. Chicks with supplementation challenged with E. coli O78 led to reduced visceral index in the bursal, which was reversed by HQD and ENR. Chicks challenged with E. coli O78 were no effect on spleen index, which was increased by HQD. Compared with HQD, HQD-DZ and HQD-SY have the same effect as HQD, HQD-GC resulted in decreased spleen index, while HQD-HQ resulted in increased liver index in chicks challenged with E. coli O78. The results showed that HQD exerted significant protective effects against the organ index of artificial induced colibacillosis, in which HQ played the main role (Table 2).
Table 2
Visceral index of chicks in Control group (CG), Model group (MG), Huangqin Decoction group (HQD); and that in HQD group of DZ absent (HQD-DZ), GC absent (HQD-GC), SY absent (HQD-SY), HQ absent (HQD-HQ); and in Enrofloxacin group (ENR). The different letter superscript in the same column indicates that the difference is significant (p<0.05).
|
Spleen
|
Bursal
|
Heart
|
Liver
|
CG
|
1.89±0.44b
|
5.84±0.25a
|
6.91±0.54c
|
26.20± 0.40c
|
MG
|
2.68±0.84b
|
2.77±1.21b
|
15.05±1.64a
|
72.43±16.63a
|
HQD
|
3.79±0.37a
|
4.67±2.12ab
|
7.83±1.43bc
|
37.71± 9.08c
|
HQD-DZ
|
3.04±0.44ab
|
3.14±0.88b
|
7.14±0.89c
|
34.41± 7.05c
|
HQD-GC
|
2.55±0.91b
|
2.13±1.06b
|
8.12±1.37bc
|
42.55± 3.00bc
|
HQD-SY
|
3.31±0.90ab
|
3.28±1.22b
|
7.90±0.61bc
|
39.25± 2.38bc
|
HQD-HQ
|
2.81±0.68ab
|
3.16±1.23b
|
9.27±0.64b
|
49.78± 4.12b
|
ENR
|
3.29±0.83ab
|
5.36±1.92a
|
8.02±1.12bc
|
34.71± 5.44c
|
3.3. Effect of HQD on E. coli O78-induced lysozyme in serum
E. coli O78 infection increased the protein levels of lysozyme in serum, as compared with that of control group, which was significantly reversed by HQD and ENR administration. HQD-GC and HQD-SY have the same effect as HQD, but HQD-DZ and HQD-HQ couldn’t reverse the increase in lysozyme protein level. The results showed that HQ and DZ in HQD played an important role in eliminating the effect of E. coli O78 on lysozyme (Fig. 3).
3.4. Effect of HQD on E. coli O78-induced inflammatory cytokines in serum
ELISA assay was conducted to determine protein level of inflammatory cytokines in serum. E. coli O78 infection increased the protein levels of IL-1β, TNF-α and IL-10 in serum, as compared with control group, indicating activation of inflammatory response following E. coli O78 infection. These alterations were significantly reversed by HQD and ENR administration. The protein level of IL-6 was increased by E. coli O78 challenged in serum of chicks, as compared with control group, however, HQD reduced its protein levels to even below control level. The results showed that HQD could reduce the inflammatory reaction in chicks challenged by E. coli O78 for 5 days. Compared with the HQD group, the protein levels of IL-1β and IL-10 were not different, while the levels of IL-6 protein were significantly increased in all herbs absent HQD. And the level of TNF-α protein in HQD-SY and HQD-HQ groups were significantly higher. The results indicated that the herbs in HQD have the effect of down-regulating pro-inflammatory factors IL-6. Meanwhile, HQ and SY play a key role in down-regulating TNF-α (Table 3).
Table 3
The level of inflammatory factor in serum of chicks in Control group (CG), Model group (MG), Huangqin Decoction group (HQD); and that in HQD group of DZ absent (HQD-DZ), GC absent (HQD-GC), SY absent (HQD-SY), HQ absent (HQD-HQ); and in Enrofloxacin group (ENR). The different letter superscript in the same column indicates that the difference is significant (p<0.05).
|
IL-1β
|
IL-6
|
TNF-α
|
IL-10
|
CG
|
48.65±8.61c
|
30.82±2.91c
|
26.77±2.10d
|
33.93±4.82bc
|
MG
|
106.71±5.38a
|
70.72±2.39a
|
72.82±3.58a
|
66.96±2.39a
|
HQD
|
60.77±14.61bc
|
20.11±1.60e
|
24.38±5.93d
|
37.95±7.91bc
|
HQD-DZ
|
47.67±7.22c
|
38.29±2.40b
|
30.39±3.17cd
|
24.68±7.17c
|
HQD-GC
|
52.73±25.10c
|
36.77±3.73b
|
35.62±7.79cd
|
30.90±13.98c
|
HQD-SY
|
70.92±29.76b
|
24.58±2.86d
|
49.89±11.95b
|
43.69±28.43b
|
HQD-HQ
|
43.26±10.91c
|
38.78±3.04b
|
38.40±2.57c
|
20.61±8.13c
|
ENR
|
61.75±20.39bc
|
28.33±2.61c
|
27.21±12.38d
|
31.99±5.98bc
|
3.5. Effect of HQD on mRNA level of Toll-like receptors in spleen and bursal of chicks
Further to explore the effect of HQD on the immune function of chicks challenged with E. coli O78, the mRNA levels of TLR4, TLR5 and TLR15 in spleen and bursal were detected. Activation of Toll-like receptor signaling pathway plays an important role in defense against invading pathogens. E. coli O78 challenge enhanced mRNA level of TLR4, TLR5 and TLR15 in spleen, as compared with control group, indicating activation of the innate immune system in the spleen following E. coli O78 infection. And this alteration was significantly reversed by all administration. Compared with CG, E. coli O78 challenge had no effect on mRNA level of TLR4, TLR5 and TLR15 in bursal, but the mRNA levels were significantly increased after administration. The results indicated that the administration groups could regulate the anti-infection ability of chicks suffered E. coli O78 by increasing the mRNA levels of TLR4, TLR5 and TLR15 in the bursal. There was no significant difference between the results of other herb absent formulae and those of HQD group, indicating that HQD acted indirectly on immune organs (Table 4).
Table 4
The mRNA level of Toll-like receptor (TLR) in spleen and bursal of chicks in Control group (CG), Model group (MG), Huangqin Decoction group (HQD); and that in HQD group of DZ absent (HQD-DZ), GC absent (HQD-GC), SY absent (HQD-SY), HQ absent (HQD-HQ); and in Enrofloxacin group (ENR). The different letter superscript in the same column indicates that the difference is significant ( p<0.05).
|
TLR4
|
TLR5
|
TLR15
|
|
spleen bursal
|
spleen bursal
|
spleen bursal
|
CG
|
1.00±0.05b
|
1.03±0.26b
|
1.02±0.20b
|
1.02±0.22b
|
1.00±0.13b
|
1.02±0.23b
|
MG
|
5.46±1.55a
|
0.98±0.39b
|
10.36±2.02a
|
0.67±0.31b
|
5.88±2.21a
|
1.11±0.60b
|
HQD
|
1.51±0.53b
|
5.75±2.67a
|
1.66±0.41b
|
3.29±1.71a
|
1.51±0.54b
|
6.54±3.92a
|
HQD-DZ
|
1.22±0.44b
|
7.11±3.93a
|
1.20±0.52b
|
3.13±1.52a
|
1.19±0.42b
|
6.42±3.26a
|
HQD-GC
|
1.37±0.40b
|
6.96±4.08a
|
1.41±0.54b
|
3.07±2.64a
|
1.40±0.53b
|
6.50±3.95a
|
HQD-SY
|
1.66±0.74b
|
6.00±3.16a
|
1.73±0.68b
|
3.33±2.51a
|
1.74±0.68b
|
6.70±4.15a
|
HQD-HQ
|
1.18±0.46b
|
7.19±4.41a
|
1.18±0.49b
|
3.10±1.15a
|
1.07±0.45b
|
6.47±4.07a
|
ENR
|
1.51±0.46b
|
6.14±3.92a
|
1.60±0.43b
|
3.25±1.83a
|
1.51±0.49b
|
6.62±3.90a
|
3.6. The effect of HQD on gut microbiota in chick feces
3.6.1 HQD altered overall structural modulation of gut microbiota
Structural changes in the gut microbiota have been reported in E. coli O78 models (Muegge et al., 2011). Alpha diversity analysis showed a clear decline in bacterial diversity in response to E. coli O78 treatment, and a clear increase in diversity in response to HQD treatment. Compared with HQD group, the Shannon index of HQD-DZ group was significantly decreased, indicating that DZ had an important role in maintaining the intestinal microflora structure of chicks challenged with E. coli O78 (Table 5).
A plateaued rarefaction curve of OTUs indicated that the sequencing depth covered all the species in the samples. E. coli O78 infection increased the OTUs, as compared with control group, which was reversed by HQD and ENR administration. Principal coordinate analysis (PCoA) showed similarity among samples, with similarity indicated by distance in the diagrams. Treatment with E. coli O78 altered the composition and structure of the gut microbiota according to PCoA. Treatment with HQD partially inhibited E. coli O78-induced changes in the gut microbiota (Fig. 4).
Table 5
The diversity of microbial in group of chicks in Control group (CG), Model group (MG), Huangqin Decoction group (HQD); and that in HQD group of DZ absent (HQD-DZ), GC absent (HQD-GC), SY absent (HQD-SY), HQ absent (HQD-HQ); and in Enrofloxacin group (ENR). The different letter superscript in the same column indicates that the difference is significant (p<0.05).
|
Chao1
|
Shannon
|
CG
|
995.70±133.14a
|
8.79±0.26a
|
MG
|
638.46±43.52b
|
6.87±0.13b
|
HQD
|
884.87±119.90a
|
8.55±1.26ab
|
HQD-DZ
|
518.60±42.76b
|
2.78±0.77c
|
HQD-GC
|
823.83±268.85ab
|
8.53±0.78ab
|
HQD-SY
|
455.77±127.88b
|
6.76±1.06b
|
HQD-HQ
|
565.00±86.09b
|
7.38±2.01ab
|
ENR
|
338.13±35.36b
|
5.03±0.76b
|
3.6.2 HQD regulated structure of the gut microbiota
The gut microbiota community structure was reported using histograms at the phylum and genus levels. All samples contained abundant Firmicutes, Bacteroidetes and Proteobacteria. Compared with the control group, MG decreased the relative abundance of Bacteroidetes and Firmicutes and increased the proportion of Proteobacteria. These alterations were significantly reversed by HQD administration. Compared with HQD group, the HQD-DZ significantly increased in the abundance of Proteobacteria, which is a marker of intestinal microbial dysregulation, and other herb absent formulae had varying degrees of influence on the three phylum levels. These results indicate that each herb in HQD influences structural segregation of the gut microbiota, in which DZ plays an important role. The ENR group can’t recover the changes in the abundance of dominant bacteria caused by E. coli O78. In contrast, the abundance of Proteobacteria was further increased. The results suggest that HQD is more beneficial than ENR in the treatment of E. coli O78 (Fig. 5).
More than 30 genera were identified in all samples. Bacteroides, Faecalibacterium, Lactobacillus and Prevotella were dominant communities in the control group but were reduced by E. coli O78 treatment. Compared with the control group, Escherichia-Shigella becomes the dominant genus, but were reduced by HQD treatment. The results showed that the proportions of most bacteria returned to control levels following HQD treatment.
The gut microbiota community structure of HQD, HQD-GC and HQD-SY were like that of CG group, indicating that HQD, HQD-GC and HQD-SY had protective effect on the structural changes caused by E. coli O78 Challenge. Compared with HQD group, the proportion of Escherichia-Shigella in HQD-DZ and HQD-HQ groups were increased, indicating that DZ and HQ in HQD had a certain regulating effect on the increase Escherichia-Shigella (Fig. 6).