Xiaochaihu Decoction Treatment of Chicken Colibacillosis by Improving Pulmonary Inflammation and Systemic Inflammation

Chicken colibacillosis—the most common disease of poultry, is caused mainly by avian pathogenic Escherichia coli (APEC). It has a major impact on the poultry industry worldwide. The present study was conducted to investigate the therapeutic effects of Xiaochaihu Decoction (XCHD) supplementation on clinical manifestation, organ index, bacterial load in organ and inflammatory mediators in a chicken model challenged with APEC. The results showed that all doses of XCHD significantly elevated the survival rate of infected chickens. XCHD improved the clinical signs of infected chickens, reduced the organ index, reduced the bacterial load of organs, and inhibited the secretion of serum and pulmonary inflammatory factors IL-1β, IL-6 and TNF- α. Taken together, this study demonstrates that XCHD had protective effects on APEC-infected chickens. Its mechanism includes anti-inflammatory and antibacterial effects. These findings may contribute to the further study of the mechanism of the formula and the prevention or treatment of colibacillosis in poultry. The significance of this study is that it provides a certain theoretical basis for the replacement of antibiotics by XCHD.


Introduction
Avian pathogenic Escherichia coli (APEC) causes chicken colibacillosis and results in acute and mostly systemic infections in avians. Due to the age, resistance, pathogenicity and infection routes of infected chickens, Chicken Colibacillosis include many clinical signs and different disease types. There is a high incidence of diseases in chicken farming, which is responsible for high economic losses in the chicken industry [1,2]. Chicken colibacillosis is clinically characterized by a variety of disease types, including pericarditis, perihepatitis, peritonitis, air sac inflammation and septicemia [3].
At present, there are more than 60 types of Escherichia coli (E. coli) serotypes in China, including O1, O2, O35 and O78 [4]. Chickens of all ages are susceptible to colibacillosis when kept under poor hygienic conditions and when improperly fed. The usual incidence of chicken colibacillosis is 11% to 69% and the mortality rate is about 3.8% to 72%, which can reach 100% in severe cases [5].
Chicken colibacillosis is often secondary to other respiratory diseases in chickens. APEC is a symbiotic bacterium in the intestinal tract of animals and can become pathogenic through horizontal gene transfer and recombination with other pathogenic bacteria. As a result, the death rate of young birds has increased year by year [6].
The use of antibiotics can be effective in the treatment of chicken colibacillosis, but the misuse of antibiotics promotes resistance to APEC, leading to multidrug resistance and making the disease difficult to control [7]. However, the control of coliforms is complicated by the prohibition of antibiotics in feed. To cope with this, people choose to find natural alternatives with antimicrobial properties to maintain the level of production and product Table 1. Grouping and treatment of animals.

Group Number Treatment
Negative Control (NC) 20 Injected 0.4 mL saline into the pectoral muscle, and then 0.4 mL saline was administered by gavage twice daily for 3 days and followed by 4 days of observation.

Positive Control (PC) 20
Injected 0.4 mL APEC into the chest muscle, and then 0.4 mL saline was administered by gavage twice daily for 3 days and followed by 4 days of observation.
2 g/kg 20 The drug was administered by gavage 3 h after APEC was injected, twice daily for 3 days and followed by 4 days of observation.
1 g/kg 20 The drug was administered by gavage 3 h after APEC was injected, twice daily for 3 days and followed by 4 days of observation.

g/kg 20
The drug was administered by gavage 3 h after APEC was injected, twice daily for 3 days and followed by 4 days of observation.
Antibiotic 20 E. coli was injected and after 3 h, the drug was given once a day, and 0.1 mL/was used continuously for 3 days and followed by 4 days of observation.

Clinical Manifestation
After the establishment of the model, the clinical symptoms, mortality, and survival rate of the experimental animals were observed and recorded, and the dead chickens were examined at the same time.

Body Weight
At the end of the experiment, all chickens were weighed for growth performance calculations.

Organ Index
At the end of the observation, the chickens in each group were weighed, blood samples were collected from the jugular vein, anesthesia was performed, and the chicks were dissected. The pathological changes of chickens in each group were observed after dissection and the heart, liver, spleen, lung, kidney, and bursa of Fabricius were collected. The organ index was calculated after weighing, and the formula was as follows: organ index = organ weight (g)/body weight (kg) [26].

Determination of Bacterial Load in Organs
Establishment of a standard curve. Dilute the prepared 10 8 cfu/mL bacterial solution tenfold to 10 2 cfu/mL, extract DNA from bacterial liquid according to the instructions of the TIANamp Bacteria DNA Kit (Tiangen Biochemical Technology Co., Ltd., Beijing, China), and establish a standard curve using the data detected by qPCR. Take the logarithm of the bacterial load in the reaction as the abscissa and the CT value in the PCR reaction as the ordinate, establish a standard curve and 16s rRNA gene of bacteria was amplified by qPCR. The forward primer was CACAATGGGCGAAAGCCTGA, and the reverse primer was GGCTGCTGGCACGTAGTTAG.
Bacterial load in organ: After grinding the organ (heart, liver, spleen, lung, kidney and bursa of Fabricius, the sample weight of each organ is 0.1 g), the supernatant was obtained after 6000 rpm centrifugation for 10 min. The bacterial genome was extracted from tissues

Blood Biochemical Indicators
After the observation, blood was collected from the chickens of each group through the jugular vein, centrifuged at 3000 rpm for 10 min, serum was taken, and the aspartatealanine were tested according to the instructions of the test kit (Huili Biotechnology Co., Ltd., Changchun, China).

Serum Inflammatory Factors
At the end of the observation period, blood samples were collected from the jugular vein, and 5 chickens in each group were randomly selected. The blood samples were centrifuged at 3500 rpm for 15 min, and the serum was separated into 1.5 mL centrifuge tubes and stored at −20 • C. IL-1, IL-6 and TNF-α were detected by ELISA. (BPRO, Lengton Bioscience Co., Ltd., Shanghai, China).

Lung Tissue Inflammatory Factors
According to the weight/volume ratio of 1:19, the lung tissue was grinded with normal saline, and the supernatant was obtained by 3000 r/min centrifugation for 10 min. According to the instructions of the kit, the expression of IL-1 β, IL-6 and TNF-α in lung tissue was detected. (BPRO, Lengton Bioscience Co., Ltd., Shanghai, China).

Statistical Analysis
Data were expressed as mean ± SEM and analyzed by one-way and two-way ANOVA for single-factor and two-factor designs, respectively, using SPSS (SPSS statistics 20). In the case of the two-way analysis, interactions between treatment factors were also assessed using the SPSS program. The mean differences among different treatments were separated by Duncan's multiple range tests. A level of p < 0.05 was used as the criterion for statistical significance.

Clinical Signs
Chicks infected with APEC showed typical clinical signs, including depression, anorexia, and inactivity, with a 100% incidence rate. Considering the high mortality rate of chicks (60%), the typical clinical signs and the appearance of autopsy changes, it shows that the model used in our study was successful. Gross examination revealed subcutaneous fibrinous exudates, severe pericarditis, perihepatitis, hemorrhagic necrosis of the lungs, enlarged spleen and kidneys and inflated intestine in dead chickens (Figures 1 and 2).
After treatment, the diet and drinking water returned to normal compared to the PC. The therapeutic effects of each group were as follows: Table 2 showed that the survival rate of each group was significantly higher than that of the Antibiotic group. Survival curves are indicative of these results ( Figure 3). Compared to the PC group, chicken mortality was reduced by more than 50% in the XCHD treatment group and 30% in the antibiotic group.

Body Weight
Mean body weights of different groups are illustrated in Figure 4. At the end of the observation, the mean body weight of 2 g/kg group and 1 g/kg group was higher than that of the PC group. The results demonstrate that XCHD can well restore the weight loss caused by APEC infection as 2 g/kg group and 1 g/kg group increased weight compared to the NC group.

Body Weight
Mean body weights of different groups are illustrated in Figure 4. At the end of the observation, the mean body weight of 2 g/kg group and 1 g/kg group was higher than that of the PC group. The results demonstrate that XCHD can well restore the weight loss caused by APEC infection as 2 g/kg group and 1 g/kg group increased weight compared to the NC group.

Body Weight
Mean body weights of different groups are illustrated in Figure 4. At the end of the observation, the mean body weight of 2 g/kg group and 1 g/kg group was higher than that of the PC group. The results demonstrate that XCHD can well restore the weight loss caused by APEC infection as 2 g/kg group and 1 g/kg group increased weight compared to the NC group.

Organ Index
Typical pathological changes of APEC could be seen in the PC group during the treatment, with varying degrees of swelling in the organs, causing an increase in organ index. The examination results were also alleviated after treatment. Organ indices showed a significant reduction in the increase of organ indices caused by APEC in all groups (Table 3).

Determination of Bacterial Load in Organs
The DNA extracts of 10-fold dilutions from 10 8 to 10 2 cfu/mL were used for qPCR assays to establish the standard curve ( Figure 5. A linear relationship between the log10 cfu and the threshold cycle Ct values of (r = 0.99).  Data were presented as mean ± SD (n = 5). * p < 0.05 and ** p < 0.01 are significantly different from the NC group.

Determination of Bacterial Load in Organs
The DNA extracts of 10-fold dilutions from 10 8 to 10 2 cfu/mL were used for qPCR assays to establish the standard curve ( Figure 5. A linear relationship between the log10 cfu and the threshold cycle Ct values of (r = 0.99). Visceral bacterial load in all treatment groups was lower than PC group, the Antibiotic group significantly inhibited the proliferation of APEC in the lung and liver. (Table 4) Visceral bacterial load in all treatment groups was lower than PC group, the Antibiotic group significantly inhibited the proliferation of APEC in the lung and liver (Table 4) Data were presented as mean ± SD (n = 5). ** p < 0.01 are significantly different from the NC group.

Blood Biochemical Indicators
The biochemical index test showed that the biochemical indexes AST and ALT indexes of the chicks in the PC group after infection were significantly higher than NC group. After XCHD treatment, each administration group can significantly reduce the ALT index of the experimental chicks; both the 2 g/kg dose group and the antibiotic group can significantly reduce the AST and ALT index of the experimental chicks ( Figure 6). The results showed that both the XCHD treatment group and the antibiotic group could reduce the liver injury caused by APEC infection.
indexes of the chicks in the PC group after infection were significantly higher than NC group. After XCHD treatment, each administration group can significantly reduce the ALT index of the experimental chicks; both the 2 g/kg dose group and the antibiotic group can significantly reduce the AST and ALT index of the experimental chicks ( Figure 6. The results showed that both the XCHD treatment group and the antibiotic group could reduce the liver injury caused by APEC infection.

Figure 6. Effect of XCHD on APEC-induced blood biochemical indicators production. (A) Effects
of XCHD on serum ALT activities; (B) Effects of XCHD on serum AST activities. Data were presented as mean ± SD (n = 5). ## p < 0.01 is significantly different from the NC group; * p < 0.05 and ** p < 0.01 are significantly different from the PC group.

Serum Inflammatory Factors
The results showed that the levels of IL-1β，IL-6 and TNF-α in the model group were significantly higher than in the NC group. In addition, serum IL-1, IL-6 and TNF-α secretions were inhibited significantly in all XCHD groups, especially in high doses (Figure 7. The results showed that both the XCHD group and the antibiotic group had good anti-inflammatory effects, proving that XCHD could control systemic inflammation A B

Figure 6. Effect of XCHD on APEC-induced blood biochemical indicators production. (A) Effects
of XCHD on serum ALT activities; (B) Effects of XCHD on serum AST activities. Data were presented as mean ± SD (n = 5). ## p < 0.01 is significantly different from the NC group; * p < 0.05 and ** p < 0.01 are significantly different from the PC group.

Serum Inflammatory Factors
The results showed that the levels of IL-1β, IL-6 and TNF-α in the model group were significantly higher than in the NC group. In addition, serum IL-1, IL-6 and TNFα secretions were inhibited significantly in all XCHD groups, especially in high doses (Figure 7). The results showed that both the XCHD group and the antibiotic group had good anti-inflammatory effects, proving that XCHD could control systemic inflammation.

Lung Tissue Inflammatory Factors
The results showed that compared with the control group, model group induced the production ofIL-1 β, IL-6 and TNF-α, in lung tissue. The secretion of IL-1, IL-6 and TNFα in lung tissue was significantly inhibited in all treatment groups, especially in the high dose group. (Figure 8. The results showed that both the XCHD group and the antibiotic group had good anti-pulmonary inflammatory effects, proving that XCHD could control the inflammation of the lung.

Lung Tissue Inflammatory Factors
The results showed that compared with the control group, model group induced the production ofIL-1 β, IL-6 and TNF-α, in lung tissue. The secretion of IL-1, IL-6 and TNF-α in lung tissue was significantly inhibited in all treatment groups, especially in the high dose group. (Figure 8). The results showed that both the XCHD group and the antibiotic group had good anti-pulmonary inflammatory effects, proving that XCHD could control the inflammation of the lung.
The results showed that compared with the control group, model group induced the production ofIL-1 β, IL-6 and TNF-α, in lung tissue. The secretion of IL-1, IL-6 and TNFα in lung tissue was significantly inhibited in all treatment groups, especially in the high dose group. (Figure 8. The results showed that both the XCHD group and the antibiotic group had good anti-pulmonary inflammatory effects, proving that XCHD could control the inflammation of the lung.

Discussion
APEC associated with colibacillosis results in high morbidity and mortality, and severe economic losses to the poultry industry. APEC is a zoonotic pathogen and can infect humans through contaminated poultry products. Vaccination and antibiotic treatment are currently used to control APEC infections; however, the limited effect of vaccines and the emergence of antibiotic-resistant strains have necessitated the development of novel therapeutics.
In a previous study, our team had used either laryngeal inoculation or intraperitoneal injection of APEC and found that the mortality rate of chickens in the laryngeal inoculation model was low at about 20-40%, with mild lesions on gross examination, which did not replicate the chicken colibacillosis model well. The mortality rate of chickens in the intraperitoneal injection model was high at about 80%, and the infected chickens died

Discussion
APEC associated with colibacillosis results in high morbidity and mortality, and severe economic losses to the poultry industry. APEC is a zoonotic pathogen and can infect humans through contaminated poultry products. Vaccination and antibiotic treatment are currently used to control APEC infections; however, the limited effect of vaccines and the emergence of antibiotic-resistant strains have necessitated the development of novel therapeutics.
In a previous study, our team had used either laryngeal inoculation or intraperitoneal injection of APEC and found that the mortality rate of chickens in the laryngeal inoculation model was low at about 20-40%, with mild lesions on gross examination, which did not replicate the chicken colibacillosis model well. The mortality rate of chickens in the intraperitoneal injection model was high at about 80%, and the infected chickens died very rapidly due to acute sepsis. Therefore, in the present study we chose pectoral muscle injection of APEC to establish the model of chicken colibacillosis (Table S3).
To address the serious problem of bacterial resistance, researchers are now seeking new alternative antibiotic drugs. There have been many studies showing many new antibiotic alternatives, such as Schisandrin A, small molecule growth inhibitors (GIs) and quorum sensing autoinducer-2 (QS AI-2) inhibitors [25,27,28]. The main mechanisms include regulation of the hepatic-intestinal axis, anti-inflammatory, reduces bacterial load on organs, etc.
In our study the results showed that XCHD was effective in the treatment of chicken colibacillosis, and the healing rate was higher than that of the antibiotic group. There was no significant difference between groups treated with XCHD, although the cure rate was dose related. In addition, the body weight value of birds treated with XCHD was greater than that of the model group, suggesting that XCHD could reduce the negative effects of APEC infection in chickens. At 36 h after infection by APEC, the mortality rates of the model chickens were highest while the mortality rate and body temperature of XCHD-treated chickens were significantly improved compared with the PC group.
We speculate that animal temperatures may be related to the body's immunity. When the body temperature returns to normal, the body's resistance is stronger and the survival rate of animals is higher. The APEC infection causes inflammation of chicken organs, with swelling and bleeding, which can be well demonstrated by organ index. However, sometimes the organs do not expand, the weight of the bird decreases and the organ index increases. In this study, however, no weight loss was observed in chickens infected with APEC, and the increase in spleen index was significant. The immune organ indexes are essential in evaluating the speed and state of the immune system [29]. As the largest peripheral immune organ, the spleen can produce an immune response to blood antigen. Therefore, LPS can stimulate lymphocyte overgrowth and induce inflammatory cytokine production on a large scale, leading to splenomegaly [30].
After XCHD treatment, the load of APEC in each organ decreased in different degrees compared with the model. Ceftiofur sodium was selected as an antibiotic and is widely used in veterinary clinics. It has good therapeutic effect on APEC. The pharmacokinetics of ceftiofur sodium, which can be absorbed by subcutaneous injection and distributed to various tissues and organs, showed that the main absorption site was the lung. Our experimental results show that after ceftiofur sodium treatment, the bacterial load in the lungs and liver is significantly reduced, therefore, it is speculated that the liver may also be one of its targets. Cephalosporins reduce levels of inflammation throughout the body by inhibiting damage to the liver by APEC to reduce the mortality rate from APEC infection.
After APEC infection, it breaks through host defense systems, causing tissue damage and host inflammation., By testing the AST and ALT indicators, the results showed that the model group chickens were significantly higher than the control group, which may be due to infection-induced perihepatitis. and the secretion of inflammatory cytokines such as IL-6, and TNF-α also supports this phenomenon. IL-6 is a typical inflammatory factor that is highly expressed when tissue cells are infected with the pathogen. Disease conditions, such as local and systemic infections, septic shock, degenerative arthritis and other autoimmune diseases appear to be regulated by TNF-α and IL-1β.
As a unique structure in poultry, air sac is an "open" system from nasal cavity to lungs, abdominal organs, bones and related tissues. At the same time, poultry have no diaphragm, and the chest is connected to the abdominal cavity. These special structures determine that poultry are more prone to lung injury [31,32]. Pathogenic factors of lung injury induce systemic infection and lead to systemic inflammatory reaction. In the process of inflammation, a variety of inflammatory cytokines (IL-1, TNF-α, etc.) play an important role in inducing tissue injury. As the target organ of systemic inflammatory response, inflammatory mediators enter the lungs through blood circulation, inducing related inflammatory cells (such as chemotactic monocytes, lymphocytes, polymorphonuclear cells and platelets) to accumulate in the lungs, which in turn causes inflammatory injury. Clinical inhibition of inflammatory cytokines and reduction of inflammatory response has become one of the important methods to reduce lung injury. Some studies have shown that some drugs can effectively suppress these diseases. For instance, some drugs can regulate TNF-α receptor antagonist, IL-6 receptor antagonist and IL-1 receptor antagonist and reverse endotoxin-mediated death. Therefore, we speculate that XCHD can antagonize these three inflammatory cytokines and prevent the death of infected chickens. In order to test this hypothesis, we measured the levels of TNF-α, IL-1 and IL-6 in serum and lung tissue of chickens at the end of the experiment. The results showed that XCHD could significantly reduce the concentrations of IL-1, IL-6 and TNF-α in infected chicks, indicating that XCHD can treat chicken colibacillosis by inhibiting systemic and pulmonary inflammation.

Conclusions
The results showed that XCHD could improve the survival rate of infected chickens. XCHD can improve the body weight of chickens, reduce the swelling of organs, reduce their bacterial load, reduce liver damage caused by APEC infection and inhibit the secretion of serum and pulmonary inflammatory factors IL-1β, IL-6 and TNF-α. These actions ultimately promote the overall health and growth of the chicken.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/pathogens12010030/s1, Table S1:Therapeutic effect of XCHD in chickens colibacillosis; Figure S1: Mean body weight (g) of chickens in different experimental groups at the end of the experiment; Table S2: Organ index of chickens in each group; Figure S2: Effect of XCHD on APEC-induced Blood biochemical indicators production; Table S3: Effect of different inoculation doses on the establishment of chicken colibacillosis model; Figure S3: Gross examination of infected chickens; Figure S4: Internal organs of treated and non-treated chickens.
Author Contributions: B.F., K.S., P.Y. conceived the idea for this study. J.L., J.Y. and Y.T. interpreted the data and drafted the figures. S.S. and M.L. performed the statistical tests. K.S. conducted the study and wrote the original draft. L.P. made revisions to the manuscript. All authors have read and agreed to the published version of the manuscript. The funder was not involved in the study design, collection, analysis, interpretation of data, writing the paper or decision to submit it for publication.

Institutional Review Board Statement:
The experiments were conducted in a manner that avoided unnecessary discomfort to the animals by the use of proper management and laboratory techniques. All the experimental procedures were approved by the Institutional Animal Care and Use Committee of Jilin University.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author, upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.