Composition for preventing and treating chicken bacterial diarrhea and application thereof

Abstract

The invention relates to the technical field of traditional Chinese medicine compositions, in particular to a traditional Chinese medicine composition for preventing and treating chicken bacterial diarrhea, which is prepared from the following components in parts by weight: 20 to 50 parts of radix curcumae, 20 to 50 parts of cortex fraxini, 20 to 50 parts of honeysuckle stem, 40 to 80 parts of rheum officinale, 20 to 50 parts of fructus gardeniae, 10 to 30 parts of fructus chebulae, 10 to 30 parts of radix paeoniae alba and 10 to 30 parts of pericarpium zanthoxyli seed. The traditional Chinese medicine composition with different doses has a good clinical curative effect on chicken diarrhea caused by escherichia coli O78, and can relieve adverse effects of escherichia coli challenge on chicken by repairing tissue damage, regulating serum biochemical indexes and inflammatory factor content, improving intestinal morphology and reducing large intestine rod bacterium loading amount. According to the characteristics of multiple targets and multiple components of the compound traditional Chinese medicine, the effects of antibiosis, anti-inflammation, immunoregulation and the like are achieved, and meanwhile, the traditional Chinese medicine composition is nontoxic in clinical medication curative effect and safety evaluation and can be used for a long time.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine composition technology, specifically to a composition for preventing and treating bacterial diarrhea in chickens and its application. Background Technology

[0002] Bacterial diarrhea in chickens is a disease caused by various bacteria, including but not limited to Salmonella, Escherichia coli, Campylobacter, and Yersinia. These pathogens easily invade chickens through contaminated feed, water, or the environment, especially when chicken coops are unsanitary and stressful. Bacterial diarrhea in chickens not only severely affects their growth and development, leading to decreased feed conversion rates and increased mortality, but also causes significant economic losses to the poultry industry. Furthermore, if contaminated chicken products enter the human food chain, they can cause food safety problems in humans, such as intestinal diseases and food poisoning. Regarding drug prevention and treatment, failure to use drugs under scientific guidance can lead to antibiotic abuse and misuse, increasing the development of drug resistance in the pathogens causing bacterial diarrhea in chickens.

[0003] Traditional Chinese medicine (TCM) has shown good efficacy in the clinical treatment of livestock and poultry diseases, and compared with conventional antibiotics, it is less likely to induce drug resistance. However, the lack of newly developed antibiotics and vaccines significantly increases the difficulty of preventing and controlling bacterial diarrhea in chickens during the breeding process. Currently, there are relatively few and limited types of bacterial vaccines for chickens, offering limited protection against mixed infections. Furthermore, the extensive use of antibiotics has led to prominent issues of drug residues and drug resistance. In recent years, the Lanzhou Institute of Animal Husbandry and Veterinary Medicine of the Chinese Academy of Agricultural Sciences has conducted extensive research on the clinical differentiation and classification of livestock and poultry diseases, the development of preventive and therapeutic drugs, and clinical treatment, accumulating rich experience. However, the promotion and application of its related technologies and products in chicken farms in our province and city have not yet been fully popularized.

[0004] Yujin San is a classic formula from the *Yuanheng Liaoma Ji* (Collection of Treating Horse Diseases by Yuanheng), composed of herbs such as turmeric, coptis, scutellaria, and phellodendron. It has the effects of clearing heat and detoxifying, dispersing blood stasis, and stopping diarrhea. However, the cost of the herbs in Yujin San is relatively high, placing a significant economic burden on poultry farmers. This invention targets bacterial diarrhea, a disease that severely impacts the chicken industry. Based on the classic formula, it selects more effective and inexpensive traditional Chinese medicine formulations to develop a Chinese medicine product for the prevention and treatment of bacterial diarrhea in chickens. Research on its trial production process and quality standards is being conducted, and the research and application of prevention and treatment technologies for bacterial diarrhea in chickens are being promoted in counties and districts surrounding Lanzhou City. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned shortcomings and provide a composition for preventing and treating bacterial diarrhea in chickens and its application.

[0006] To achieve the above objectives, the following technical solution is provided:

[0007] The primary objective of this invention is to provide a traditional Chinese medicine composition for preventing and treating bacterial diarrhea in chickens. The traditional Chinese medicine composition, by weight, comprises: 20-50 parts of turmeric, 20-50 parts of fraxinus bark, 20-50 parts of honeysuckle vine, 40-80 parts of rhubarb, 20-50 parts of gardenia, 10-30 parts of chebula, 10-30 parts of white peony root, and 10-30 parts of Sichuan pepper seeds.

[0008] Preferably, the traditional Chinese medicine composition consists of 20-50 parts of turmeric, 20-50 parts of fraxinus bark, 20-50 parts of honeysuckle vine, 40-80 parts of rhubarb, 20-50 parts of gardenia, 10-30 parts of chebula, 10-30 parts of white peony root, and 10-30 parts of Sichuan pepper seeds.

[0009] Preferably, the traditional Chinese medicine composition consists of 20-30 parts of turmeric, 20-30 parts of fraxinus bark, 20-30 parts of honeysuckle vine, 40-60 parts of rhubarb, 20-30 parts of gardenia, 10-15 parts of chebula, 10-15 parts of white peony root, and 10-30 parts of Sichuan pepper seeds.

[0010] Preferably, the traditional Chinese medicine composition consists of 30 parts turmeric, 30 parts fraxinus bark, 30 parts honeysuckle vine, 60 parts rhubarb, 30 parts gardenia, 15 parts chebula, 15 parts white peony root, and 30 parts Sichuan pepper seeds.

[0011] A second objective of this invention is to provide a method for preparing the traditional Chinese medicine composition, comprising the following steps:

[0012] (1) Mix the raw materials in proportion and soak them in water;

[0013] (2) Boil the raw materials from step (1) and filter them to obtain the final product.

[0014] A third objective of this invention is to provide the application of the aforementioned traditional Chinese medicine composition in the preparation of a drug for treating bacterial diarrhea in chickens.

[0015] The beneficial effects of this invention are as follows:

[0016] This invention combines turmeric, fraxinus bark, honeysuckle vine, rhubarb, gardenia, chebula, white peony root, and Sichuan pepper seeds in a specific ratio. By leveraging the multi-target and multi-component characteristics of compound traditional Chinese medicine, it achieves antibacterial, anti-inflammatory, and immunomodulatory effects. Furthermore, this formula is derived from the screening and optimization of classic prescriptions. Clinical efficacy and safety evaluations have shown that it is non-toxic and suitable for long-term use. Attached Figure Description

[0017] Figure 1 Graph showing the changes in E. coli content in the feces of chickens treated with different formulations;

[0018] Figure 2 Images of the vent state of chicks after drug treatment in each group following viral challenge;

[0019] Figure 3 Schematic diagram of experimental design and animal treatment;

[0020] Figure 4 Images of chick feces and vent after viral challenge

[0021] Among them: A. No fecal samples were tested; B. No fecal samples were tested; C. No anal samples were tested; D. Anal samples were tested.

[0022] Figure 5 Graphs showing changes in inflammatory factors in chickens of different experimental groups after treatment

[0023] Among them: A: IL-1β; B: IL-6; C: IL-10; D: TNF-α;

[0024] Figure 6 Schematic diagram of liver tissue pathological examination results: HE staining 20×, black arrow: inflammatory cell infiltration; blue arrow: hepatocellular steatosis; A: CON group, B: MOD group, C: 1 / 2TCV group, D: TCV group, E: 2TCV group, F: FFC group;

[0025] Figure 7 Pathological examination results of duodenal tissue

[0026] Among them, HE staining 4×, A: CON group; B: MOD group; C: TCV group; D: FFC group;

[0027] Figure 8 Duodenal bacterial load scoring criteria;

[0028] Among them: bacterial growth scoring criteria A is worth 1 point; B is worth 2 points; and C is worth 3 points. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0030] Example 1, Traditional Chinese Medicine Composition 1

[0031] A traditional Chinese medicine composition for preventing and treating bacterial diarrhea in chickens: 20g of turmeric, 20g of fraxinus bark, 20g of honeysuckle vine, 40g of rhubarb, 20g of gardenia, 10g of chebula, 10g of white peony root, and 10g of Sichuan pepper seeds.

[0032] Example 2, Traditional Chinese Medicine Composition 2

[0033] A traditional Chinese medicine composition for preventing and treating bacterial diarrhea in chickens: 50g of turmeric, 50g of fraxinus bark, 50g of honeysuckle vine, 80g of rhubarb, 50g of gardenia, 30g of chebula, 30g of white peony root, and 30g of Sichuan pepper seeds.

[0034] Example 3, Traditional Chinese Medicine Composition 3

[0035] A traditional Chinese medicine composition for preventing and treating bacterial diarrhea in chickens: 20g of turmeric, 20g of fraxinus bark, 20g of honeysuckle vine, 40g of rhubarb, 20g of gardenia, 15g of chebula, 15g of white peony root, and 15g of Sichuan pepper seeds.

[0036] Example 4, Traditional Chinese Medicine Composition 4

[0037] A traditional Chinese medicine composition for preventing and treating bacterial diarrhea in chickens: 20g of turmeric, 20g of fraxinus bark, 20g of honeysuckle vine, 40g of rhubarb, 20g of gardenia, 10g of chebula, 10g of white peony root, and 10g of Sichuan pepper seeds.

[0038] Preliminary experiments showed that the effects of traditional Chinese medicine composition 3 were similar to those of traditional Chinese medicine composition 1, and the price of traditional Chinese medicine composition 3 was lower. Therefore, traditional Chinese medicine composition 3 was selected for subsequent experiments.

[0039] Comparative Example 1

[0040] A traditional Chinese medicine composition for preventing and treating bacterial diarrhea in chickens: 20g of turmeric, 20g of fraxinus bark, 20g of honeysuckle vine, 40g of rhubarb, 20g of gardenia, 15g of chebula, and 15g of white peony root.

[0041] Comparative Example 2

[0042] A traditional Chinese medicine composition for preventing and treating bacterial diarrhea in chickens: 30g of turmeric, 30g of scutellaria baicalensis, 60g of rhubarb, 30g of coptis chinensis, 30g of phellodendron chinense, 30g of gardenia, 15g of chebula, and 15g of white peony root.

[0043] Example 5: Evaluation of the efficacy of traditional Chinese medicine composition in treating bacterial diarrhea in chickens

[0044] (1) Comparison of cure rate and effectiveness rate

[0045] A bacterial diarrhea model in chickens was established using antibiotics and E. coli challenge. Ninety-six hours after challenge, chickens were treated with decoctions (1 g / mL) of the formulations from Example 3, Comparative Examples 1 and 2, at a dose of 2.7 g / kg per chicken per day for 5 days, followed by a 3-day observation period after treatment.

[0046] Table 1. Cure rate and effectiveness of each group

[0047]

[0048] As shown in Table 1, the cure rate and effectiveness of Example 3 and Comparative Example 2 were significantly higher than those of Comparative Example 1.

[0049] (2) Weight changes in chickens after treatment

[0050] Table 2. Weight changes of chickens in each group after treatment.

[0051]

[0052] Note: *p<0.05 indicates a significant difference; **p<0.01 indicates a highly significant difference.

[0053] Note: Total weight gain and average daily weight gain marked as A are the total weight gain and average daily weight gain of chickens in each group during the 5-day continuous administration of each formula. Total weight gain and average daily weight gain marked as B are the total weight gain and average daily weight gain of chickens in each group during the 3-day observation period after the discontinuation of each formula.

[0054] As shown in Table 2, after 5 days of continuous administration, the diarrhea of ​​chickens in each experimental group improved significantly, with Example 3 and Comparative Example 2 showing better results than Comparative Example 1. During the 5-day period of continuous administration followed by a 3-day observation period after drug withdrawal, the total weight gain and average daily weight gain of chickens in each group during the 3-day observation period after drug withdrawal were significantly higher than the average daily weight gain during the 5-day continuous administration period.

[0055] (3) Changes in the content of Escherichia coli in the feces of chickens after treatment

[0056] like Figure 1 As shown, the image labeled A is an eosin methylene blue agar medium used for determining the Escherichia coli content in the feces of chickens in the model group (antibiotic + E. coli challenge) and the blank group; the image labeled B is a medium used for determining the Escherichia coli content in the feces of chickens in the blank group; and the images labeled 1, 2, and 3 are mediums used for determining the Escherichia coli content in the feces of chickens after treatment in Comparative Example 2, Comparative Example 1, and Example 3, respectively. ① is a dilution of 10... -4 The condition after culturing a 10-fold diluted fecal solution; ② is the result of dilution 10 times. -5 The condition after culturing a diluted fecal solution.

[0057] After 5 days of continuous administration, the E. coli content in the feces of chickens in each experimental group was significantly lower than that in the control group and the chickens after intervention. It can be inferred that the drugs in each group are effective in treating diarrhea after O78 challenge.

[0058] (4) Changes in the anal condition of chickens after treatment in Examples 1-3

[0059] like Figure 2As shown, the anal condition of chicks in each group after drug treatment following O78 challenge is as follows: from left to right, no drug treatment group, Comparative Example 2, Comparative Example 1, and Example 3. After 5 days of continuous drug administration, the anal condition of chicks in each experimental group was significantly improved, and the pasty anal condition was alleviated. It can be inferred that the drugs in each group are effective in treating diarrhea after O78 challenge.

[0060] Evaluation of the therapeutic effects of the formulations in Example 6 and Example 3 on chicken Escherichia coli diarrhea

[0061] 1. Test materials

[0062] 1.1 Main Drugs and Reagents

[0063] The medicinal materials from Example 3 were weighed according to the specified proportion and soaked in 10 times their volume of water for 30 minutes. After boiling over high heat, the mixture was simmered over low heat for 30 minutes and filtered through gauze. The dregs were then simmered again in 6 times their volume of water for 30 minutes. The two filtrates were combined, concentrated under reduced pressure, and brought to a final volume of 1 g / mL. The solution was stored at 4°C for later use. Florfenicol was purchased from Nanjing Rishengchang Biotechnology Co., Ltd., batch number 100582110, in 100g / bag specifications.

[0064] Serum biochemical assay kits: alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), creatinine (CREA), glucose (GLU), total cholesterol (TCH), triglycerides (TG), and urea (UREA) were all purchased from Meikang Biotechnology. Inflammatory factor assay kits: chicken interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor-α (TNF-α) were all purchased from Shanghai Enzyme-Linked Biotechnology.

[0065] 1.1.1 Main Instruments

[0066] The multi-functional microplate reader (Synergy LX) was purchased from Donghe Instruments; the semi-automatic blood biochemistry analyzer (ErbaXL-640) was purchased from Shanghai San Jose Corporation; and the vertical double-layer constant temperature shaking incubator (HZQ-F160) was purchased from Shanghai Jingsheng Scientific Instruments Co., Ltd.

[0067] 1.1.2 Experimental Animals

[0068] 160 healthy 11-day-old Lufeng chickens were purchased from a broiler farm in Yuzhong, Lanzhou. The chickens were kept in separate cages, provided with suitable temperature and humidity, and allowed free access to feed and water. They were given an acclimatization period of 3 days before the start of the experiment.

[0069] 1.1.3 Test strains and preparation of bacterial culture

[0070] Escherichia coli strain O78 (CVCC1555) was purchased from the China Veterinary Microbial Culture Collection Center. The E. coli strain O78, stored at -80℃, was quickly thawed and inoculated onto LB agar plates. It was then incubated overnight at 37℃ to allow for the growth of single colonies of suitable size. Single colonies were picked and inoculated into 100 mL of LB liquid medium with shaking and cultured for 24 h. Colonies were counted using a turbidimetric method, and the medium was diluted with physiological saline to a concentration of 1×10⁻⁶. 9 Prepare a bacterial culture at CFU / mL for later use.

[0071] 1.2 Methods

[0072] 1.2.1 Grouping and treatment of experimental animals

[0073] One hundred and sixty 14-day-old chicks were randomly divided into eight groups, including a blank control group (CON group), a model control group (MOD group), traditional Chinese medicine compound (TCV) groups at 1 / 3, 1 / 2, 1, 2, and 3 times the dosage, and a florfenicol (FFC) group. Figure 3 As shown in Table 3, except for the CON group, chicks in all other groups were given 50 mg / kg doxycycline hydrochloride in their drinking water for two consecutive days at 14 days of age, followed by a two-day withdrawal period for pretreatment. At 18 days of age, each chick was orally administered 0.5 mL of E. coli O78 bacterial solution to establish a bacterial diarrhea model. At 21 days of age, the clinical symptoms of the model chicks were observed, and treatment was initiated after scoring the clinical symptoms. The TCV dosage groups and the FFC group were given corresponding doses of traditional Chinese medicine compound and florfenicol in their drinking water, while the CON and MOD groups were given physiological saline. Treatment was administered once daily for 5 days, followed by a 3-day observation period after the withdrawal period at 25 days of age.

[0074] Table 3. Grouping and Dosage of Experimental Animals

[0075]

[0076] 1.2.2 Sample Collection and Processing

[0077] Relevant samples were collected from chickens in the CON, MOD, 1 / 2 TCV, TCV, 2 TCV, and FFC groups. On day 18 of the experiment (after drug withdrawal observation), blood was collected from the subwing vein of 6 chicks in each group. The collected blood samples were centrifuged at ultra-low temperature and the serum was separated and stored at -80℃. After dissection, the heart, liver, spleen, lungs, kidneys, and bursa of Fabricius were separated, surface fat was removed, and the weight was recorded. Partial liver tissue was collected and fixed in 4% paraformaldehyde solution for preservation. Approximately 1 cm of the middle segment of the duodenum and jejunum from the CON, MOD, TCV, and FFC groups was taken, rinsed with physiological saline to remove chyme, and fixed in 4% paraformaldehyde solution for preservation. Another approximately 1 cm segment of the middle segment of the duodenum was placed in a centrifuge tube containing PBS for preservation.

[0078] 1.2.3 Model Determination

[0079] Referring to relevant research literature and the diagnostic criteria and scoring of the Clinical Research Technical Guidelines for Veterinary Traditional Chinese Medicine for Damp-Heat Diarrhea in Chickens, the clinical symptoms of chickens in each experimental group after Escherichia coli O78 challenge were diagnosed and scored. A chicken E. coli model was considered successfully established when the clinical symptoms of the affected chickens met the criteria listed in Table 4 and the symptom score was ≥5.

[0080] Table 4. Symptom scoring criteria for chicken Escherichia coli model

[0081]

[0082] 1.2.4 Observation of clinical signs after treatment

[0083] During the experiment, the mental state, body temperature, fecal morphology, and vent condition of the chickens in each group were observed and recorded daily. Chicken mortality was also observed and recorded, and dead chickens were necropsied.

[0084] 1.2.5 Determination of growth performance indicators

[0085] After the trial challenge (day 8), before drug treatment (day 11), and after drug withdrawal observation (day 18), the chickens' body weight and feed intake were weighed and recorded. The average daily feed intake (ADFI), average daily weight gain (ADG), and feed conversion ratio (F / G) of each group of chickens were calculated.

[0086] 1.2.6 Efficacy assessment

[0087] After drug treatment (day 18), the clinical symptoms of the sick chickens in each experimental group were scored according to the symptom scoring criteria in 1.2.2, and the symptom score reduction rate of each experimental group was calculated. Referring to Table 5, the therapeutic effect of the drug on the experimental chickens was determined, and the cure rate, significant effect rate, effective rate, ineffective rate, and total effective rate were calculated.

[0088]

[0089] Cure rate (%) = (Number of chickens cured in this group / Number of chickens in this experimental group) × 100%

[0090] Effective rate (%) = (Number of chickens showing significant improvement in this group / Number of chickens in this experimental group) × 100%

[0091] Effective rate (%) = (Number of chickens that responded to treatment in this group / Number of chickens in this experimental group) × 100%

[0092] Ineffectiveness (%) = (Number of chickens in this group whose treatment was ineffective / Number of chickens in this experimental group) × 100%

[0093] Overall effective rate (%) = Cure rate + Significantly effective rate + Effective rate

[0094] Table 5. Criteria for Evaluating the Efficacy of Traditional Chinese Medicine Compound Therapies in the Prevention and Treatment of Chicken Escherichia coli Model

[0095]

[0096] 1.2.7 Detection of blood biochemical indicators and serum inflammatory factor levels

[0097] The levels of blood biochemical indicators were measured using a semi-automated biochemical analyzer according to the instructions of the ALT, AST, ALB, CRE, GLU, TG, TCH, and UREA kits. The levels of inflammatory factors IL-1β, IL-6, IL-10, and TNF-α in the serum of chickens in each experimental group were measured according to the instructions of the ELISA kits.

[0098] 1.2.8 Determination of organ indices

[0099] After a 3-day observation period following the discontinuation of medication, a portion of the chickens were necropsyd and their organs were examined in detail, including the heart, liver, spleen, lungs, kidneys, and bursa of Fabricius. The weight of each organ was measured and its organ index was calculated.

[0100] Organ Index = Organ Weight (g) / Body Weight (kg)

[0101] 1.2.9 Histopathological observation

[0102] A portion of the liver tissue was collected and fixed, embedded in paraffin, and sectioned. After staining with hematoxylin and eosin (HE), the histopathological changes were examined under a microscope, and photographs were taken using a pathological image analysis system.

[0103] 1.2.10 Morphological observation of intestinal tissue

[0104] The duodenum fixed with paraformaldehyde was removed, embedded in paraffin, and sectioned. After staining with hematoxylin and eosin (HE), the histomorphological changes of the duodenal mucosa were observed under a microscope. The height of well-oriented villi (VH) and the depth of crypts (CD) were measured, and the ratio of villi height to crypt depth (V / C) was calculated.

[0105] 1.2.11 Analysis of bacterial load in the duodenum

[0106] Following the method used by Guo Yanna et al. to determine the bacterial load in organs, 0.05g of duodenal tissue was homogenized with 1mL of sterile water. 100μL of the homogenate was spread onto eosin methylene blue agar and incubated at 37℃ for 12h. Bacterial growth was observed. Bacterial growth and quantity were evaluated using a scoring system (0 points - no bacterial growth; 1 point - small amount of bacterial growth; 2 points - large amount of bacterial growth, which can be divided into individual colonies; 3 points - densely packed colonies in the petri dish, invisible to the naked eye). The Escherichia coli load in the duodenum of five chickens in each experimental group was measured and included in the final score.

[0107] 1.3 Data Statistics and Analysis

[0108] Symptom score reduction rate, cure rate, and overall effective rate were presented as percentages, and the chi-square test was used to analyze the clinical efficacy data. SPSS 27.0 software was used to process other data (excluding clinical efficacy data), and one-way ANOVA was performed. The significance of differences between groups was assessed using t-tests. All data are expressed as mean ± standard deviation. P > 0.05 was considered insignificant, P < 0.05 was considered significant, and P < 0.01 was considered highly significant.

[0109] 2 Results and Analysis

[0110] 2.1 Evaluation of the chicken Escherichia coli diarrhea model

[0111] Following the viral challenge (day 11), chickens in the CON group exhibited normal mental state, normal body temperature, normal fecal morphology, clean cloaca, and normal perianal skin color. Chickens in all experimental groups that became ill showed lethargy, drooping wings, and elevated body temperature. Feed intake decreased, and feeding time prolonged. They excreted yellow, watery feces, with large amounts of dried, hardened feces or feces adhering to the perianal area, and in severe cases, pasty feces. Figure 4 The clinical symptom scores of chickens in each experimental group are shown in Table 6. The morbidity rate of chickens challenged with the virus in each experimental group reached 100%, and the clinical symptom scores of all groups were ≥5. There was no significant difference in the clinical symptom scores among the experimental groups (P>0.05). The results indicate that the chicken Escherichia coli model can be successfully established by gavage administration of Escherichia coli O78 bacterial solution after intervention with doxycycline hydrochloride.

[0112] Table 6. Clinical symptom scoring table for chickens in each experimental group after viral challenge.

[0113]

[0114] Note: # and ## indicate significant differences (P<0.05) and highly significant differences (P<0.01) between the group and the CON group, respectively. No annotation indicates no significant differences (P>0.05). * and ** indicate significant differences (P<0.05) and highly significant differences (P<0.01) between the group and the MOD group, respectively.

[0115] 2.2 Changes in clinical symptoms after medication

[0116] After treatment (day 15), the mental state and body temperature of chickens in all treatment groups returned to normal, the yellow watery feces disappeared, the feces morphology returned to normal, and the clinical symptoms of fecal adhesion and pasty vent significantly improved. However, the feces morphology and vent condition of chickens in the MOD group did not show significant improvement. No recurrence or worsening of clinical symptoms occurred in any of the treatment groups during the observation period. The changes in body temperature of chickens in each experimental group are shown in Table 7. Before treatment (day 11), except for the CON group, the body temperature of chickens in all experimental groups was significantly elevated (P<0.05). Three days after treatment (day 13), the body temperature of chickens in the MOD group was still higher than that of all treatment groups and the CON group (P<0.05). During the observation period (days 15-18), there was no significant difference in body temperature among the experimental groups (P>0.05).

[0117] Table 7. Changes in body temperature of chickens in each experimental group before and after treatment.

[0118]

[0119] 2.3 Effects of drugs on the growth performance of experimental chickens

[0120] The changes in average daily weight gain and feed conversion ratio of chickens in each experimental group are shown in Table 8. After challenge (days 8-11), compared with the CON group, ADFI and ADG were significantly lower and F / G significantly higher in all groups (P<0.05). After treatment (days 11-18), ADFI and ADG in the CON group were significantly higher than those in the MOD group (P<0.05), and F / G was significantly lower than that in the MOD group (P<0.05). ADG in each treatment group was significantly higher than that in the MOD group (P<0.05), and F / G was significantly lower than that in the MOD group (P<0.05). Among the traditional Chinese medicine compound groups, ADFI and ADG were significantly higher and F / G significantly lower in the TCV group (P<0.05). Compared with the FFC group, ADFI and ADG were significantly higher and F / G significantly lower in the TCV group (P<0.05).

[0121] Table 8. Measurement of growth performance of chickens in each experimental group.

[0122]

[0123] 2.4 Assessment of Clinical Efficacy of Drugs

[0124] Table 9 shows the clinical efficacy assessment results of chickens in each experimental group after treatment (day 18). Compared with the MOD group, the cure rate and total effective rate of each treatment group were significantly higher (P<0.05). Among the various traditional Chinese medicine compound groups, the cure rate and total effective rate of the TCV group, 2TCV group, and 3TCV group were significantly higher than those of the 1 / 3TCV group and 1 / 2TCV group (P<0.05); there were no significant differences in the cure rate and total effective rate among the TCV group, 2TCV group, and 3TCV group (P>0.05). Compared with the FFC group, the cure rate and total effective rate of the TCV group, 2TCV group, and 3TCV group were significantly higher (P<0.05).

[0125] Table 9. Evaluation of therapeutic effects (%) in chickens of each experimental group

[0126]

[0127] Note: a and A indicate significant differences (P<0.05) and highly significant differences (P<0.01) between the group and the MOD group; b and B indicate significant differences (P<0.05) and highly significant differences (P<0.01) between the group and the drug administration group; no label indicates no significant difference (P>0.05).

[0128] 2.5 Effects of drugs on blood biochemical parameters of experimental chickens

[0129] Table 10 shows the results of blood biochemical index measurements in chickens after treatment (day 18). There were no significant differences in CRE, GLU, TG, TCH, and UREA levels among the experimental groups (P>0.05). Compared with the CON group, the MOD group showed significantly increased ALT and AST levels and significantly decreased ALB levels (P<0.05). Compared with the MOD group, the TCV and 2TCV groups showed significantly decreased ALT and AST levels (P<0.05) and significantly increased ALB levels (P<0.05), while the 1 / 2TCV and FFC groups showed no significant changes in ALT, AST, and ALB levels (P>0.05). Among the treatment groups, the 2TCV and TCV groups showed significantly lower ALT and AST levels than the 1 / 2TCV and FFC groups (P<0.05), and significantly higher ALB levels than the 1 / 2TCV and FFC groups (P<0.05).

[0130] Table 10. Serum biochemical indicators of chickens in each experimental group after treatment.

[0131]

[0132]

[0133] Note: # and ## indicate significant differences (P<0.05) and highly significant differences (P<0.01) between the group and the CON group, respectively. No annotation indicates no significant differences (P>0.05). * and ** indicate significant differences (P<0.05) and highly significant differences (P<0.01) between the group and the MOD group, respectively.

[0134] 2.6 Effects of drugs on serum inflammatory factor levels in experimental chickens

[0135] The results of the measurement of inflammatory factors IL-1β, IL-6, IL-10, and TNF-α in chicken serum after treatment (day 18) are detailed below. Figure 5 Compared with the CON group, the MOD group showed a highly significant increase in serum IL-1β, IL-6, and TNF-α levels (P<0.01), and a highly significant decrease in IL-10 levels (P<0.01). Compared with the MOD group, all treatment groups showed highly significant decreases in serum IL-1β, IL-6, and TNF-α levels (P<0.01), and a highly significant increase in IL-10 levels (P<0.01). Among the treatment groups, the TCV and 2TCV groups showed significantly lower IL-6 and TNF-α levels than the FFC group (P<0.05), and significantly higher IL-10 levels than the FFC group (P<0.05). There were no significant differences among the other treatment groups (P>0.05).

[0136] 2.7 Effects of drugs on organ indices in experimental chickens

[0137] Table 11 shows the results of organ indices measured and dissected chickens in each group after treatment (day 18). There were no significant differences in kidney and lung indices among the experimental groups (P>0.05). Compared with the CON group, the organ indices of the heart, liver, and spleen were significantly increased in the MOD group (P<0.05), while the bursa of Fabricius index was significantly decreased (P<0.05). Compared with the MOD group, the organ indices of the heart, liver, and spleen were significantly decreased in the TCV and 2TCV groups (P<0.05), while the bursa of Fabricius index was significantly increased (P<0.05). There were no significant differences in organ indices of the heart, liver, spleen, and bursa of Fabricius between the 1 / 2TCV and FFC groups (P>0.05). When comparing the different drug administration groups, the organ indices of the heart, liver, and spleen in the TCV group and the 2TCV group were significantly higher than those in the 1 / 2TCV and FFC groups, while the bursa of Fabricius index was significantly lower in the TCV group and the FFC group (P<0.05).

[0138] Table 11. Measurement of organ indices in chickens of each experimental group after treatment.

[0139]

[0140] 2.8 Pathological observation of liver tissue after drug treatment

[0141] The pathological observation results of liver tissue in each experimental group are as follows: Figure 6 As shown, in the CON group, hepatocytes in the chicken liver tissue were arranged radially around the central vein, forming hepatocyte tubes that anastomosed with each other, and no obvious inflammatory cell infiltration was observed. In the MOD group, the central vein of the liver lobules of the chickens was congested, the sinusoids were dilated and congested, and cytoplasmic granular degeneration and fatty degeneration were visible in the hepatocytes; the hepatocyte cords were severely damaged, and there was inflammatory cell infiltration around the liver lobules. Compared with the MOD group, the 1 / 2TCV group, TCV group, 2TCV group, and FFC group showed more significant improvement in liver tissue.

[0142] 2.9 Morphological observation of intestinal tissue after drug treatment

[0143] The results of histological observation of the duodenum are as follows: Figure 7 As shown in Table 12, the results of duodenal villus height, crypt depth, and villus-to-crypt ratio measurements were as follows: Compared with the CON group, the MOD group showed a significantly decreased duodenal villus height, a significantly increased crypt depth, and a significantly decreased villus-to-crypt ratio (P<0.05). Compared with the MOD group, the TCV group showed a significantly decreased duodenal villus height (P<0.05), a highly significantly decreased crypt depth, and a highly significantly increased villus-to-crypt ratio (P<0.01), while the FFC group showed a significantly decreased duodenal crypt depth and a significantly increased villus-to-crypt ratio (P<0.05).

[0144] Table 12 Effects of drug treatment on the morphology of duodenal tissue in chickens in the experimental group

[0145]

[0146] 2.10 Effect of drug treatment on bacterial load in the duodenum of chickens in the experimental group

[0147] The scoring criteria for duodenal bacterial load are as follows: Figure 8 The results of the duodenal bacterial load determination in chickens of each experimental group are shown in Table 13. Compared with the CON group, the duodenal bacterial load in the MOD group was significantly increased (P<0.05). Compared with the MOD group, the duodenal bacterial load in the TCV group was significantly decreased (P<0.01), and the duodenal bacterial load in the FFC group was significantly decreased (P<0.05).

[0148] Table 13 Effects of drug treatment on bacterial load in the duodenum of chickens in the experimental group

[0149]

[0150] In summary, this invention provides a traditional Chinese medicine composition for preventing and treating bacterial diarrhea in chickens. The composition, by weight, comprises: 20-50 parts Curcuma longa, 20-50 parts Fraxinus chinensis bark, 20-50 parts Lonicera japonica vine, 40-80 parts Rheum palmatum, 20-50 parts Gardenia jasminoides, 10-30 parts Terminalia chebula, 10-30 parts Paeonia lactiflora, and 10-30 parts Zanthoxylum bungeanum seeds. The modified formulations of the traditional Chinese medicine composition at different dosages showed cure rates of 40%, 50%, 75%, 80%, and 85%, respectively; and total effective rates of 70%, 75%, 85%, 90%, and 90%, respectively. The cure rate and total effective rate of the FFC group were 60% and 75%, respectively. There was no significant difference in therapeutic effect among the modified formulations at 1-3 times the dosage, and the therapeutic effect was superior to that of florfenicol. After treatment with the aforementioned traditional Chinese medicine composition, the ADG of chickens in all TCV dosage groups and the FFC group significantly increased, and the F / G ratio significantly decreased. Among them, the ADFI and ADG of the TCV group were significantly higher than those of the modified formula and the FFC group at other dosages, while the F / G ratio was significantly lower than that of the modified formula and the FFC group at other dosages; indicating that the modified formula can increase the feed conversion rate of E. coli-infected chicks and improve the growth performance of E. coli-challenged broilers to a certain extent. Treatment with the traditional Chinese medicine combination can alleviate the inflammatory response of the body after E. coli infection. After treatment, the ALT and AST levels of the TCV group and the 2TCV group decreased, while the ALB level increased. There were no significant changes in the ALT, AST, and ALB levels of the 1 / 2TCV group and the FFC group. Different dosages of the modified formula significantly reduced the secretion of serum inflammatory factors IL-1β, IL-6, and TNF-α, while significantly increasing the secretion of serum inflammatory factor IL-10. The levels of IL-6 and TNF-α in the TCV group and the 2TCV group were lower than those in the FFC group, while the level of IL-10 was higher. Studies have shown that *E. coli* alters some serum biochemical indicators and inflammatory factors in chicks, while the modified formula, to a certain extent, regulates the levels of these indicators and inflammatory factors, thus mitigating the adverse effects of *E. coli* on chicks. The traditional Chinese medicine composition has a positive effect on improving liver and spleen damage in chicks challenged by *E. coli*. The heart, liver, and spleen indices of chicks in the TCV and 2TCV groups decreased, while the bursa of Fabricius index increased; no significant changes were observed in the 1 / 2TCV and FFC groups. Research indicates that *E. coli* infection damages the heart, liver, and spleen of chicks, while simultaneously reducing the body's resistance. The traditional Chinese medicine composition can alleviate tissue damage and mitigate the adverse effects of *E. coli* challenge on broilers by regulating the body and enhancing its immunity. After treatment with the traditional Chinese medicine composition, *E. coli*-infected chicks showed increased down feather height, decreased crypt depth, increased down feather-crypt ratio, and decreased tissue bacterial load. Florfenicol treatment was less effective than the traditional Chinese medicine compound group in repairing mucosal function damage and reducing intestinal bacterial load in chicks.Studies have shown that E. coli infection can damage the intestinal mucosa of chicks and increase the colonization rate of E. coli. The modified formula can reduce the damage to the intestinal mucosal structure caused by E. coli infection and decrease the bacterial load of E. coli in the intestinal tissue. Different doses of the modified formula have shown good clinical efficacy in treating diarrhea induced by E. coli O78 in chicks, and can alleviate the adverse effects of E. coli challenge on chicks by repairing tissue damage, regulating serum biochemical indicators and inflammatory factor levels, improving intestinal morphology, and reducing the number of E. coli.

[0151] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

Claims

1. A traditional Chinese medicine composition for preventing and treating bacterial diarrhea in chickens, characterized in that, The traditional Chinese medicine composition, by weight, includes: 20-50 parts turmeric, 20-50 parts fraxinus bark, 20-50 parts honeysuckle vine, 40-80 parts rhubarb, 20-50 parts gardenia, 10-30 parts chebula, 10-30 parts white peony root, and 10-30 parts Sichuan pepper seeds.

2. The traditional Chinese medicine composition as described in claim 1, characterized in that, The aforementioned traditional Chinese medicine composition consists of 20-50 parts of turmeric, 20-50 parts of fraxinus bark, 20-50 parts of honeysuckle vine, 40-80 parts of rhubarb, 20-50 parts of gardenia, 10-30 parts of chebula, 10-30 parts of white peony root, and 10-30 parts of Sichuan pepper seeds.

3. The traditional Chinese medicine composition as described in claim 2, characterized in that, The traditional Chinese medicine composition consists of 20-30 parts of turmeric, 20-30 parts of fraxinus bark, 20-30 parts of honeysuckle vine, 40-60 parts of rhubarb, 20-30 parts of gardenia, 10-15 parts of chebula, 10-15 parts of white peony root, and 10-30 parts of Sichuan pepper seeds.

4. The traditional Chinese medicine composition as described in claim 3, characterized in that, The aforementioned traditional Chinese medicine composition consists of 30 parts turmeric, 30 parts fraxinus bark, 30 parts honeysuckle vine, 60 parts rhubarb, 30 parts gardenia, 15 parts chebula, 15 parts white peony root, and 30 parts Sichuan pepper seeds.

5. The method for preparing the traditional Chinese medicine composition according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Mix the raw materials in proportion and soak them in water; (2) Boil the raw materials from step (1) and filter them to obtain the final product.

6. The use of the traditional Chinese medicine composition according to any one of claims 1-4 in the preparation of a drug for treating bacterial diarrhea in chickens.

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