Composition for resisting necrotic enteritis and application thereof

Abstract

The invention discloses a composition for resisting necrotic enteritis and application thereof, and the composition is prepared from the following components in parts by mass: 5 parts of paeonol, 5 parts of farnesol and 1 part of soybean saponin. The composition can also be prepared into oral liquid, tablets, granules and powder by taking paeonol, farnesol and soyasaponin as effective components and adding auxiliary materials, and the use dosage is 100-1000mg / kg of body weight. Or the composition is directly added into the feed, and the addition amount is 0.1-1% of the total weight of the chicken feed. The composition formed by paeonol, farnesol and soyasaponin is used as a medicine or a feed additive, is high in safety and free of toxic and side effects, can effectively inhibit proliferation of chicken-derived pathogenic bacteria such as clostridium perfringens, salmonella and escherichia coli, effectively improves the antibody titer of a newcastle disease vaccine, and has a good application prospect. And the effects of reducing chicken production performance, increasing proinflammatory cytokines and damaging intestinal absorption and barrier functions caused by chicken coccidiosis and necrotizing enteritis can be relieved.

Description

Technical Field

[0001] The present application relates to the technical field of prevention and treatment of chicken enteritis, and in particular to a composition for resisting chicken necrotic enteritis and its application. Background Art

[0002] my country is a major producer and consumer of chicken. Ensuring the healthy and efficient breeding of broilers to improve chicken production and quality is related to the national economy and people's livelihood. However, necrotic enteritis (NE) caused by Clostridium perfringens (CP) infection has seriously restricted the healthy and efficient breeding of broilers. In addition, since the ban on antibiotics in feed, pathogens in the gastrointestinal tract of broilers have taken the opportunity to proliferate in large numbers, which is bound to destroy their intestinal immune barrier and aggravate the negative impact of NE on broilers. It can be seen that NE is a key issue that needs to be urgently addressed in the healthy and efficient breeding of broilers in the post-antibiotic era.

[0003] NE is one of the major gastrointestinal diseases that endanger the healthy breeding of broiler chickens worldwide. It is mainly caused by infection with type A and type G CP. In the process of broiler production, some abnormal environmental factors (such as heat stress), infection with pathogens (such as coccidia), and dietary types (such as wheat-based diets) provide conditions for CP infection. NE mainly occurs in the small intestine, mainly in the ileum, which leads to damage to the physical and immune barrier function of the broiler intestine, imbalance of intestinal flora structure, etc., and then causes intestinal inflammatory damage.

[0004] Previously, the use of antibiotics was the main measure to prevent and control NE in broiler chickens. However, excessive use of antibiotics has brought many hazards: (1) Increased drug resistance: Long-term use of antibiotic additives can cause microorganisms to develop drug resistance, which can cause some pathogenic microorganisms to develop drug resistance through mutation, leading to a sharp increase in the number of antibiotic-resistant bacteria worldwide; (2) Excessive veterinary drug residues in animal-derived foods: Excessive use of veterinary drugs can cause excessive veterinary drug residues in animal-derived foods, posing a potential threat to human health; (3) Damage to the ecological environment: The discharge of metabolites from poultry that eat antibiotic-containing feed can damage the ecological environment and pollute drinking water or other agricultural products; (4) Impact on human health: If antibiotics remaining in livestock and poultry products are ingested by the human body, they will increase the burden on the liver and kidneys during metabolism, and long-term intake may cause lesions; (5) Reduced immunity: The abuse of antibiotics may cause an imbalance in the normal microbial system in the chicken, resulting in a decrease in the chicken's immune function and resistance. In view of the serious consequences of antibiotics, antibiotics have been banned on a large scale. However, since the ban on antibiotics in feed, pathogens in the gastrointestinal tract of broiler chickens have taken the opportunity to proliferate rapidly, which is bound to destroy their intestinal immune barrier and aggravate the negative impact of NE on broiler chickens.

[0005] At present, the use of antibiotics is the main measure to prevent and control NE in broiler chickens. However, due to the prohibition of antibiotics in feed, the prevention and control of NE through nutritional measures has gradually become one of the key topics of concern in broiler farming. Summary of the invention

[0006] In view of this, the purpose of the present application is to provide a composition for preventing necrotic enteritis in chickens and its application. The composition comprises 5 parts of farnesol, 5 parts of paeonol and 1 part of soybean saponin by mass. The composition has the characteristics of high safety, stable efficacy and no toxic side effects, and has obvious preventive and control effects on necrotic enteritis in chickens. The composition can be used to prepare drugs or feed additives for treating or preventing necrotic enteritis in chickens.

[0007] In order to achieve or at least partially achieve the above objectives, the present application provides the following technical solutions:

[0008] In a first aspect, the present application provides a composition for preventing necrotic enteritis in chickens, the composition comprising paeonol, farnesol and soybean saponin.

[0009] In a second aspect, the present application provides the use of the aforementioned composition in the preparation of a drug or feed additive for treating or preventing necrotic enteritis in chickens.

[0010] Compared with the prior art, the technical solution provided by this application has at least the following beneficial effects:

[0011] 1. Paeonol, farnesol and soybean saponin in the composition provided by the present application are all derived from natural plants, are highly safe, have no toxic side effects, and can be used as a feed additive for a long time to prevent necrotic enteritis in chickens. Long-term use will not lead to the production of drug-resistant strains, which is an advantage that other anti-disease drugs do not have.

[0012] 2. The present application shows through in vitro tests that the composition formed by paeonol, farnesol and soybean saponin can inhibit the proliferation of common pathogenic bacteria in chickens, such as Clostridium perfringens, Escherichia coli, Salmonella, etc.

[0013] 3. The present application shows through in vivo experiments that the composition formed by paeonol, farnesol and soybean saponin can promote the increase of antibody levels of Newcastle disease vaccine, inhibit the expression of pro-inflammatory cytokines, enhance intestinal health, and thus improve the feed conversion efficiency of broilers with necrotic enteritis. DETAILED DESCRIPTION

[0014] In order to make the purpose, technical solution and advantages of the present application more clear, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described here are only used to explain the present application and are not used to limit the present application.

[0015] Those skilled in the art will appreciate that, unless otherwise stated, the words "said", "the", and "the aforementioned" used in the text of the present application may also include plural forms. It should be further understood that the wording "including" used in the specification of the present application means the presence of the features, steps, and operations, but does not exclude the presence or addition of one or more other features, integers, and steps.

[0016] In the text of this application, the terms "first", "second" and the like are used to distinguish between identical or similar items with substantially the same functions and effects. This is only for the purpose of clearly describing the technical solutions of the embodiments of this application, and shall not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features.

[0017] Those skilled in the art will appreciate that, for any specific experimental steps or conditions not specified in the examples, the experiments may be carried out according to the conventional experimental steps or conditions described in the literature in the art; any raw materials or instruments used without specifying the manufacturer are conventional products that can be purchased commercially.

[0018] Those skilled in the art will understand that, unless otherwise specified in the present application, when a numerical range is given in an embodiment, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this application are consistent with the prior art mastery of those skilled in the art and the records of this application, and any methods, equipment and materials of the prior art similar or equivalent to the methods, equipment and materials described in the embodiments of this application may also be used to implement this application.

[0019] Paeonol is a Chinese herbal medicine extracted from the root bark of peony flowers, and can also be extracted from the dried root bark of the ranunculaceae plant peony. Paeonol is a white or slightly yellow needle-shaped crystal with a melting point of about 49-51°C. It is easily soluble in ethanol and methanol and slightly soluble in water. Paeonol can be distilled with water vapor and has strong absorption in the ultraviolet region; the energy of paeonol at a wavelength of 274nm is 862, so it can be measured using a spectrophotometer. Current studies have found that paeonol has analgesic, anti-inflammatory, antipyretic and allergic effects. Specifically, paeonol can relieve pain reactions caused by stimulation by substances such as tail pressure and acetic acid; it can relieve inflammatory reactions caused by egg white, formaldehyde, histamine, 5-hydroxytryptamine, bradykinin and endotoxin; it can relieve fever caused by vaccine immunization. In addition, paeonol also has a variety of biological effects such as antibacterial, anti-inflammatory, antioxidant, and regulation of blood pressure and blood lipids. Due to its green, safe and efficient characteristics, paeonol is used as a clinical drug in human medicine, such as paeonol ointment; however, there are no reports on the use of paeonol in livestock production.

[0020] Farnesol is an active ingredient extracted from the bark of the acacia tree. Its main components are flavonoids and triterpenoids. Farnesol can also be found in citronella, lemongrass, tuberose, cyclamen, rose, orange blossom, balsam and musk. Its chemical formula is C 15 H 25 OH, molecular weight is 223.72g / mol. Studies have found that farnesol has a variety of biological effects such as antibacterial, anti-inflammatory, antioxidant, immune regulation and lipid metabolism. In clinical practice, farnesol is used to treat allergic asthma, diabetes, atherosclerosis, obesity and hyperlipidemia. Due to its green and safe characteristics, farnesol is widely used in food, skin care products, medicine and other fields. In livestock and poultry production, there are few reports on farnesol, and there are no reports on the effect of farnesol on necrotic enteritis in broilers.

[0021] Soya saponin (SS) is a pentacyclic triterpenoid substance naturally present in soybeans. Due to its good immunomodulatory effect, it has been widely studied in the medical field in recent years. SS is composed of soybean saponins and some glycosides, uronic acid, etc. Due to the differences in glycosides, SS is divided into four types: A, B, E, and DDMP. Studies have found that SS is difficult to be absorbed by intestinal epithelial cells and is generally degraded by intestinal microorganisms into soybean saponin or other small molecules before being absorbed and utilized. The structure of group B soybean saponin is simpler than that of group A and is generally easier to absorb. In addition, the more complex the structure of SS, the more obvious its immunomodulatory effect. Existing studies have shown that SS has antioxidant, lipid metabolism improvement, anti-tumor, antiviral, and immune regulation effects, and these biological effects are almost all related to SS immune regulation. Although soybean saponin has been proven to have a variety of biological effects, its application in the prevention and control of broiler enteritis has rarely been reported.

[0022] At present, there are few reports on the application of soybean saponins, farnesol and paeonol in the prevention and control of necrotic enteritis in broilers, and there has been no report on the application of the three as a combination in broiler production. On this basis, the embodiment of the present application forms a composition by mixing paeonol, farnesol and soybean saponins in a certain proportion. In vitro and in vivo tests show that the composition can inhibit the proliferation of common chicken pathogens, such as Clostridium perfringens, Escherichia coli, Salmonella, etc.; promote the increase of antibody levels of Newcastle disease vaccine, inhibit the expression of pro-inflammatory cytokines, and enhance intestinal health, thereby improving the feed conversion efficiency of broilers with necrotic enteritis. The above results provide basic data for the use of the composition in the prevention and treatment of necrotic enteritis in chickens and the development of functional feed additives.

[0023] Based on this, an embodiment of the present application provides a composition for preventing necrotic enteritis in chickens, wherein the composition comprises paeonol, farnesol and soybean saponin.

[0024] In certain preferred embodiments, the anti-necrotic enteritis composition for chickens comprises, by weight, 5 parts of paeonol, 5 parts of farnesol and 1 part of soybean saponin.

[0025] In certain embodiments, the composition for preventing necrotic enteritis in chickens further comprises auxiliary materials, and the auxiliary materials comprise at least one of formic acid, acetic acid, anhydrous ethanol, propylene glycol, dimethyl sulfoxide, Tween 80, water, lactose, mannitol, soluble starch, montmorillonite, vermiculite, zeolite powder, white carbon black, defatted rice bran, corn cob powder, starch, and bran. Among them, at least one of formic acid, acetic acid, anhydrous ethanol, propylene glycol, dimethyl sulfoxide, Tween 80, and water can be used as a solvent to dissolve the composition of paeonol, farnesol, and soybean saponin to prepare an oral liquid preparation; at least one of lactose, mannitol, soluble starch, montmorillonite, vermiculite, zeolite powder, white carbon black, defatted rice bran, corn cob powder, starch, and bran is mixed with the composition of paeonol, farnesol, and soybean saponin to form a solid mixture, which is further prepared into granules, tablets, powders, and other preparations.

[0026] In certain embodiments, the anti-necrotic enteritis composition for chickens is a solid mixture of three active ingredients, namely, paeonol, farnesol, and soybean saponin, and montmorillonite, wherein the mass fraction of the three active ingredients, namely, paeonol, farnesol, and soybean saponin, in the solid mixture is 40% to 60%.

[0027] In certain embodiments, the anti-necrotic enteritis composition for chickens is a mixed solution formed by mixing three active ingredients of paeonol, farnesol, and soybean saponin with acetic acid, anhydrous ethanol, Tween-80, and water, wherein the concentration of the three active ingredients of paeonol, farnesol, and soybean saponin in the mixed solution is 80 to 120 mg / mL.

[0028] In certain embodiments, the anti-necrotic enteritis composition for chickens is a powdery mixture of three active ingredients, namely, paeonol, farnesol, and soybean saponin, mixed with wheat bran and soluble starch, wherein the mass fraction of the three active ingredients, namely, paeonol, farnesol, and soybean saponin, in the powdery mixture is 40% to 60%.

[0029] In certain embodiments, the anti-necrotic enteritis composition for chickens is a flaky mixture formed by mixing three active ingredients of paeonol, farnesol, and soybean saponin with lactose or maltose, and soluble starch, wherein the mass fraction of the three active ingredients of paeonol, farnesol, and soybean saponin in the flaky mixture is 40% to 60%.

[0030] In certain embodiments, the anti-necrotic enteritis composition for chickens is a granular mixture of three active ingredients, namely, paeonol, farnesol, and soybean saponin, with lactose or maltose, and soluble starch, wherein the mass fraction of the three active ingredients, namely, paeonol, farnesol, and soybean saponin, in the granular mixture is 40% to 60%.

[0031] Based on this, the embodiments of the present application provide the use of the aforementioned composition in the preparation of a drug or feed additive for treating or preventing necrotic enteritis in chickens.

[0032] In certain embodiments, the necrotic enteritis in chickens is caused by infection with Clostridium perfringens alone or with coccidia.

[0033] In certain embodiments, the method of using the composition as a medicine or feed additive comprises at least one of the following methods:

[0034] (1) Orally administering the drug to chickens at a dosage of 100 to 1000 mg / kg of chicken body weight of the three active ingredients of paeonol, farnesol, and soybean saponin;

[0035] (2) The active ingredients of paeonol, farnesol and soybean saponin are contained in an amount of 0.1% to 1% by weight of chicken feed; the composition is added to chicken feed as a feed additive;

[0036] (3) When used for daily prevention of chicken enteritis, the three active ingredients of paeonol, farnesol and soybean saponin are added to feed or drinking water at a dosage of 100 mg / kg chicken body weight and used throughout the entire breeding cycle;

[0037] (4) When used for clinical treatment of chicken enteritis: orally administer the composition to chickens at a dosage of 1000 mg / kg of chicken body weight of the three active ingredients of paeonol, farnesol and soybean saponin; or grind the composition into powder and mix it with the daily diet at a dosage of 1000 mg / kg of chicken body weight of the three active ingredients of paeonol, farnesol and soybean saponin, and add it to feed or drinking water three times a day in the morning, noon and evening for one week.

[0038] The technical solution of the present application and the technical effects achieved are described in detail below through more specific embodiments.

[0039] Example 1

[0040] This embodiment provides a method for preparing a composition for resisting necrotic enteritis in chickens, and the specific steps are as follows:

[0041] Weigh 5 g of farnesol and 11 g of montmorillonite, mix them evenly to form a first mixture; weigh 5 g of paeonol and 1 g of soybean saponin, add them to the first mixture, mix them evenly to form a second mixture, i.e., the anti-necrotic enteritis composition Z1.

[0042] Example 2

[0043] This embodiment provides a method for preparing a composition for resisting necrotic enteritis in chickens, and the specific steps are as follows:

[0044] Weigh 5 g of farnesol, 5 g of paeonol, and 1 g of soybean saponin, and mix them evenly to form a mixture; weigh 10 g of the above mixture and mix it with 5 mL of 5% acetic acid, 15 mL of anhydrous ethanol, and 30 mL of Tween-80, and then add water for injection to 100 mL to form a uniform mixed solution, i.e., the anti-necrotic enteritis composition Z2 for chickens.

[0045] Example 3

[0046] This embodiment provides a method for preparing a composition for resisting necrotic enteritis in chickens, and the specific steps are as follows:

[0047] Weigh 50 g of farnesol, 50 g of paeonol, and 10 g of soybean saponin, mix them evenly to form a first mixture; weigh 50 g of the first mixture, mix it evenly with 20 g of wheat bran and 30 g of soluble starch, and pass it through a 60-mesh sieve to form a powdery second mixture, i.e., the anti-necrotic enteritis composition Z3.

[0048] Example 4

[0049] This embodiment provides a method for preparing a composition for resisting necrotic enteritis in chickens, and the specific steps are as follows:

[0050] Weigh 50 g of farnesol, 50 g of paeonol and 10 g of soybean saponin, mix them evenly to form a first mixture; weigh 50 g of the first mixture, mix it evenly with 20 g of lactose and 30 g of soluble starch to form a second mixture; press the second mixture into tablets using a tablet press and then dry them to obtain a composition Z4 for resisting necrotic enteritis in chickens.

[0051] Example 5

[0052] This embodiment provides a method for preparing a composition for resisting necrotic enteritis in chickens, and the specific steps are as follows:

[0053] Weigh 50 g of farnesol, 50 g of paeonol and 10 g of soybean saponin, mix them evenly to form a first mixture; weigh 50 g of the first mixture, mix it evenly with 20 g of maltose and 30 g of soluble starch to form a second mixture; granulate and dry the second mixture to obtain the anti-necrotic enteritis composition Z5.

[0054] The formula and parameters of the anti-necrotic enteritis composition prepared in the above embodiment are shown in Table 1 below:

[0055] Table 1

[0056]

[0057] The composition can be used as a medicine or feed additive to prevent or treat necrotic enteritis in chickens. When used for the prevention of chicken diseases, the composition can be added to the feed at a dosage of 100 mg / kg of the chicken's body weight of the three active ingredients of paeonol, farnesol and soybean saponin, and used throughout the entire breeding cycle; when used for clinical treatment of chicken diseases, the composition can be orally administered at a dosage of 1000 mg / kg of the chicken's body weight of the three active ingredients of paeonol, farnesol and soybean saponin; or the composition can be dissolved in water and administered or mixed with the daily diet at a dosage of 1000 mg / kg of the chicken's body weight of the three active ingredients of paeonol, farnesol and soybean saponin, and added to the feed for use, three times a day in the morning, noon and evening, for continuous use for one week; the dosage of the composition can also be adjusted according to the severity of the chicken disease, and the dosage range is controlled within a range of 100 to 1000 mg / kg of the chicken's body weight of the three active ingredients of paeonol, farnesol and soybean saponin.

[0058] In vitro inhibitory effect of the composition on chicken-derived pathogenic bacteria

[0059] The present application example verifies the in vitro inhibitory effect of the composition of paeonol, farnesol and soybean saponin on Clostridium perfringens, Escherichia coli and Salmonella, and the verification method is as follows:

[0060] 1. Materials and Methods

[0061] 1.1 Bacteria:

[0062] Chicken pathogenic bacteria (Clostridium perfringens, Escherichia coli, and Salmonella) were isolated, preserved, and donated by the National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University.

[0063] 1.2 Trial Drugs

[0064] Weigh 0.5 g of farnesol (purchased from Aladdin, purity > 90%), 0.5 g of paeonol (purchased from Aladdin, purity > 90%), and 0.1 g of soybean saponin (purchased from Sigma, purity > 90%), and dissolve them in 108.9 mL of normal saline to obtain a mother solution of the composition of farnesol, paeonol and soybean saponin (concentration of 10 g / L), and then dilute the mother solution into 6 composition solutions with concentration gradients of 1000 mg / L, 500 mg / L, 250 mg / L, 100 mg / L, 50 mg / L, and 25 mg / L, respectively.

[0065] 1.3 Experimental process

[0066] The Oxford cup method was used to measure the diameters of the inhibition zones of different concentrations of the composition solution against Clostridium perfringens, Escherichia coli, and Salmonella to evaluate the antibacterial effect of the composition. The experiment used physiological saline as a blank control group, and enramycin (purchased from Sinopharm Group, purity>99%) as a positive control group. In the inhibition zone test, enramycin was dissolved in physiological saline, and its dosage was 0.1 μg / mL; the concentrations of the composition solutions of the 6 experimental groups were set as above; and each treatment group was set up with 3 replicates.

[0067] In the specific test process, Escherichia coli and Salmonella were cultured in LB medium and the concentration was 1×10 5 CFU / mL of bacterial suspension was used to prepare LB solid culture plates and LB semi-solid culture medium. When the LB semi-solid culture medium was cooled to about 37°C, 100 μL of 1×10 6 The Escherichia coli and Salmonella liquids with CFU / mL were added to the LB semi-solid medium and shaken well, and then quickly poured onto the LB solid culture plate. After that, the Oxford cup was evenly placed on the culture plate. After the semi-solid medium solidified, the Oxford cup was taken out and 50 μL of the above-mentioned treatment liquids were added into the ring hole, and then placed in a 37°C constant temperature incubator for 24 hours; then the diameter of the inhibition zone was measured with a ruler (accuracy of 0.01 cm); Clostridium perfringens was cultured with cooked meat medium, and the concentration was 1×10 5 CFU / mL of bacterial liquid; prepare cooked meat medium solid culture plates and cooked meat semi-solid culture medium, and when the cooked meat semi-solid culture medium is cooled to about 37°C, take 100 μL of the concentration of 1×10 5 CFU / mL of Clostridium perfringens solution was added to the cooked meat semi-solid culture medium and shaken well, and then quickly poured onto the cooked meat culture medium solid culture plate, and then Oxford cups were evenly placed on the culture plate. After the semi-solid culture medium solidified, the Oxford cups were taken out and 50 μL of the above-mentioned treatment solutions were added to the ring holes, and the plates were placed in a 37°C constant temperature incubator for 24 hours; then the diameter of the inhibition zone was measured using a ruler (accuracy of 0.01 cm).

[0068] 2. Test results

[0069] The test results are shown in Table 2 below.

[0070] Table 2

[0071]

[0072] From the results in Table 2, it can be seen that the composition formed by paeonol, farnesol and soybean saponin showed antibacterial effects on three chicken-derived pathogens, namely, Clostridium perfringens, Escherichia coli and Salmonella, and the diameter of the inhibition zone increased with the increase of the dosage of the composition, indicating that the antibacterial effect of the composition is proportional to the dosage of the composition. Among the three chicken-derived pathogens, the antibacterial effect of the composition on Clostridium perfringens was the most obvious.

[0073] Effects of the combination on growth performance and intestinal health of broilers infected with Clostridium perfringens

[0074] The present application example verifies the effect of a composition formed by farnesol, paeonol and soybean saponin on the growth performance and intestinal health of broilers infected with Clostridium perfringens. The verification method is as follows:

[0075] 1. Materials and Methods

[0076] The experiment involved 720 healthy AA + Broiler male chicks were evenly divided into 3 treatment groups, each of which was divided into 8 replicate groups, and each replicate group included 30 chickens; the 3 treatment groups were: control group, Clostridium perfringens (CP) infection group, and combination + CP infection group. The broilers in the control group and CP infection group were fed with a "corn-soybean meal" type basal diet, and the diet formula was formulated with reference to the nutritional requirements standard of Chinese broilers (NY / T33-2004); the broiler diet of the combination + CP infection group was supplemented with a combination or a combination preparation in the basal diet according to the three active ingredients of farnesol, paeonol and soybean saponin at 0.1% of the weight of the feed (the weight ratio of farnesol, paeonol and soybean saponin was 5:5:1); on the 7th day of the experiment, all broilers were immunized with Newcastle disease vaccine (immunization was performed by eye drops and nasal drops), and on the 14th to 21st days of the experiment, 1 mL of CP (CVCC2030 strain, dose of 1.0×10 9 CFU / mL), and the control group broilers were gavage-fed with an equal volume of CP medium; during the experiment, broilers were free to eat and drink, and a 24-h lighting system was adopted; on the 22nd day of the experiment, all broilers were fasted for 8 h, then weighed and feed consumption was counted, and growth performance-related indicators such as average daily gain (ADG), average daily feed intake (ADFI), and feed-to-weight ratio (FCR) were calculated; thereafter, one broiler with uniform weight was selected from each replicate group to collect subwing venous blood and separate serum for testing.

[0077] 2. Data Analysis

[0078] The experimental data were analyzed by one-way ANOVA in SPSS23.0 statistical software, and Duncan's method was used for multiple comparisons between groups. P < 0.05 was regarded as the standard of significant difference, and P < 0.01 was regarded as the standard of extremely significant difference. The results were expressed as mean ± standard deviation.

[0079] 3. Test results

[0080] 3.1 Effect of the composition on the growth performance of broilers infected with Clostridium perfringens

[0081] Table 3 shows the effects of the composition on the growth performance of broilers infected with Clostridium perfringens, wherein a, b, and c are marks of significant difference, and data with different letters on their shoulders in the table indicate significant differences P<0.05.

[0082] Table 3

[0083] Test items Control group CP infection group Combination+CP infection group P-value d21 average body weight (g) <![CDATA[719.89±31.01 a ]]> <![CDATA[589.93±37.71 c ]]> <![CDATA[653.36±22.21 b ]]> <0.001 ADFI(g) 56.13±3.33 54.31±2.19 55.76±1.91 0.632 ADG(g) <![CDATA[35.95±3.35 a ]]> <![CDATA[29.45±2.19 c ]]> <![CDATA[32.65±1.03 b ]]> <0.001 FCR <![CDATA[1.61±0.04 c ]]> <![CDATA[1.86±0.07 a ]]> <![CDATA[1.71±0.05 b ]]> 0.021

[0084] As shown in Table 3, Clostridium perfringens infection reduces the body weight and average daily weight gain of broilers at day 21, and improves the feed-to-weight ratio; while adding a composition formed by farnesol, paeonol and soybean saponin to the diet can alleviate the negative effect of Clostridium perfringens infection on the growth performance of broilers.

[0085] 3.2 Effect of the composition on serum D-xylose, diamine oxidase content and Newcastle disease vaccine antibody level in broilers infected with Clostridium perfringens

[0086] Table 4 shows the effect of the composition on the serum D-xylose and diamine oxidase (DAO) content of broilers infected with Clostridium perfringens, wherein a, b, and c are significant difference markers, and data with different letters on the shoulders in the table indicate significant differences P<0.05; the Newcastle disease vaccine antibody level was obtained by analyzing the data read under the OD600 parameter in the microplate reader.

[0087] Table 4

[0088]

[0089] Xylose can be absorbed by the intestine but cannot be used by the body. Therefore, by evaluating the content of D-xylose in the blood, the intestinal absorption function can be evaluated. DAO exists in the intestine in a healthy state and is released into the blood when the intestine is damaged. Therefore, the higher the activity of DAO in the serum, the more obvious the degree of intestinal damage. As shown in Table 4, the composition formed by adding farnesol, paeonol and soybean saponin to the diet can alleviate the decrease in D-xylose content and the increase in DAO level in the blood of broilers caused by Clostridium perfringens infection, indicating that the composition formed by farnesol, paeonol and soybean saponin can protect broilers from the decrease in intestinal absorption function and barrier damage caused by Clostridium perfringens infection. In addition, the composition formed by adding farnesol, paeonol and soybean saponin to the diet can alleviate the decrease in the level of Newcastle disease vaccine antibodies in broiler serum caused by Clostridium perfringens infection, thereby improving the humoral immune function of broilers.

[0090] 3.3 Effect of the composition on the levels of inflammatory cytokines in serum of broilers infected with Clostridium perfringens

[0091] Table 5 shows the effects of the composition on the levels of serum inflammatory cytokines in broilers infected with Clostridium perfringens, wherein a, b, and c are marks of significant difference, and data with different letters on their shoulders in the table indicate significant differences P<0.05; IL-1β and IL-6 represent interleukin 1β and interleukin 6, respectively, TNF-α represents tumor necrosis factor-α, and IFN-γ represents interferon-γ; the detection of pro-inflammatory cytokines was carried out using the chicken ELISA kit of Beijing Solebow Company.

[0092] Table 5

[0093] Test items Control group CP infection group Paeonol + CP infection group P-value IL-1β, pg / mL <![CDATA[176.12±24.31 c ]]> <![CDATA[267.34±19.89 a ]]> <![CDATA[198.15±22.17 b ]]> 0.013 TNF-α, pg / mL <![CDATA[71.12±5.32 b ]]> <![CDATA[123.18±21.77 a ]]> <![CDATA[111.57±39.11 a ]]> 0.024 IL-6, pg / mL <![CDATA[101.84±10.02 c ]]> <![CDATA[284.17±22.79 a ]]> <![CDATA[198.55±29.87 b ]]> <0.001 IFN-γ, pg / mL <![CDATA[171.03±26.12 b ]]> <![CDATA[401.08±69.18 a ]]> <![CDATA[298.03±43.19 a ]]> 0.013

[0094] As can be seen from Table 5, Clostridium perfringens infection causes an increase in the content of pro-inflammatory cytokines, such as IL-1β, TNF-α, IL-6, and IFN-γ; and the addition of a composition formed by farnesol, paeonol and soybean saponin to the diet can alleviate the increase in serum IL-1β, IL-6 and IFN-γ in broiler chickens caused by Clostridium perfringens infection. The above results indicate that the composition formed by farnesol, paeonol and soybean saponin has an alleviating effect on the inflammatory response of broiler chickens caused by Clostridium perfringens infection.

[0095] Effects of combination therapy on growth performance and intestinal health of broiler chickens infected with coccidia and Clostridium perfringens

[0096] The present application example verifies the effect of the composition formed by farnesol, paeonol and soybean saponin on the growth performance and intestinal health of broilers infected with coccidia and Clostridium perfringens. The verification method is as follows:

[0097] 1. Materials and Methods

[0098] The experiment involved 288 healthy AA + Broiler male chicks were evenly divided into 3 treatment groups, each of which was divided into 8 replicate groups, and each replicate group included 12 chickens; the 3 treatment groups were: control group, coccidia and Clostridium perfringens combined infection group (CCP), combination + CCP group. The broilers in the control group and CCP group were fed with a "corn-soybean meal" type basal diet, and the diet formula was formulated according to the nutritional requirements standard of Chinese broilers (NY / T33-2004); the broiler diet of the combination + CCP group was supplemented with farnesol, The three active ingredients of paeonol and soybean saponin were added to the feed at 0.1% of the weight of the composition or the composition preparation (the weight ratio of farnesol, paeonol and soybean saponin was 5:5:1); on the 8th and 10th days of the experiment, the broilers in the CCP group and the combination + CCP group were vaccinated with an ultra-high dose of coccidiosis vaccine (purchased from Foshan Zhengdian Company, the dose was 30 times the recommended dose) to establish the intestinal coccidiosis infection state; on the 14th to 21st days of the experiment, the broilers in the CCP group and the combination + CCP group were gavaged with 1 mL of CP (CVCC2030 strain, the dose was 1.0×10 9 CFU / mL), and the control group broilers were gavage-fed with an equal volume of CP medium; during the experiment, broilers were allowed to eat and drink freely, and a 24-h lighting system was adopted; on the 22nd day of the experiment, all broilers were fasted for 8 h, and then weighed and the feed consumption was counted, and growth performance-related indicators such as average daily gain (ADG), average daily feed intake (ADFI), and feed-to-weight ratio (FCR) were calculated; thereafter, one broiler with uniform weight was selected from each replicate group to collect subwing venous blood and separate serum for testing; further, sodium pentobarbital (at a dose of 50 mg / kg live weight) was injected into the subwing vein for anesthesia, and the broilers were slaughtered, and the ileum tissue was dissected to make slices for observation, and ileum samples were taken for subsequent analysis.

[0099] 2. Data Analysis

[0100] The experimental data were analyzed by one-way ANOVA in SPSS23.0 statistical software, and Duncan's method was used for multiple comparisons between groups. P < 0.05 was regarded as the standard of significant difference, and P < 0.01 was regarded as the standard of extremely significant difference. The results were expressed as mean ± standard deviation.

[0101] 3. Test results

[0102] 3.1 Effect of the combination on the growth performance of broilers infected with coccidia and Clostridium perfringens

[0103] Table 6 shows the effects of the composition on the growth performance of broilers co-infected with coccidia and Clostridium perfringens, wherein a, b, and c are significant differences. Data in the table with different letters on their shoulders indicate significant differences, P<0.05.

[0104] Table 6

[0105] Test items Control group CCP Group Combination + CCP group P-value d21 average body weight (g) <![CDATA[775.31±13.28 a ]]> <![CDATA[615.93±27.66 c ]]> <![CDATA[698.15±19.36 b ]]> <0.001 ADFI(g) 58.17±1.32 56.33±4.76 57.11±5.71 0.867 ADG(g) <![CDATA[38.75±1.98 a ]]> <![CDATA[30.79±1.77 c ]]> <![CDATA[34.89±2.81 b ]]> <0.001 FCR <![CDATA[1.53±0.06 c ]]> <![CDATA[1.87±0.11 a ]]> <![CDATA[1.68±0.04 b ]]> 0.002

[0106] As shown in Table 6, the combined infection of coccidia and Clostridium perfringens reduced the body weight and average daily weight gain of broilers at day 21, and increased the feed-to-weight ratio; while adding a combination of farnesol, paeonol and soybean saponin to the diet could alleviate the negative effect of the combined infection of coccidia and Clostridium perfringens on the growth performance of broilers.

[0107] 3.2 Effect of the combination on the ileum morphology of broiler chickens infected with coccidia and Clostridium perfringens

[0108] Table 7 shows the effect of the composition on the developmental parameters of the duodenum, jejunum, ileum and cecum of broilers co-infected with coccidia and Clostridium perfringens (expressed as the ratio of intestinal length to the corresponding broiler weight), wherein a and b are marks of significant difference, and data with different letters on the shoulders in the table indicate significant differences P<0.05.

[0109] Table 7

[0110]

[0111]

[0112] As can be seen from Table 7, the combined infection of coccidia and Clostridium perfringens reduces the relative lengths of the duodenum, ileum and cecum, and the addition of a composition of farnesol, paeonol and soybean saponin to the diet can alleviate the reduction in the relative lengths of the duodenum and ileum caused by the combined infection of coccidia and Clostridium perfringens. The results show that the composition formed by farnesol, paeonol and soybean saponin can promote the development of the intestine of broilers and alleviate the adverse effects of the combined infection of coccidia and Clostridium perfringens on the intestinal development of broilers.

[0113] Table 8 shows the effect of the composition on the morphological structure of the ileum of broiler chickens infected with coccidia and Clostridium perfringens, where a, b, and c are significant differences. Data with different letters on the shoulders in the table indicate significant differences (P<0.05); VH represents villus height, CD represents crypt depth, and VH / CD represents the ratio of villus height to crypt depth.

[0114] Table 8

[0115] Test items Control group CCP Group Combination + CCP group P-value VH,μm <![CDATA[796.55±45.80 b ]]> <![CDATA[718.80±61.32 c ]]> <![CDATA[1015.80±179.02 a ]]> 0.004 CD,μm 163.07±53.56 171.16±21.82 174.03±23.52 0.889 VH / CD <![CDATA[4.88±0.75 b ]]> <![CDATA[4.19±1.05 b ]]> <![CDATA[6.27±1.69 a ]]> 0.004

[0116] The higher the height of the intestinal villi, the larger the contact area with the nutrients in the intestinal cavity, and the better the absorption efficiency. Therefore, the higher the intestinal villi height and the ratio of villi height to crypt depth, the better the intestinal absorption function. As shown in Table 8, the addition of a composition formed by farnesol, paeonol and soybean saponin in the diet can alleviate the decrease in the height of the ileal villi of broilers caused by the combined infection of coccidia and Clostridium perfringens, and increase the ratio of villi height to crypt depth. The results further confirm that the composition formed by farnesol, paeonol and soybean saponin has a beneficial effect on the intestine of broilers, and can reduce the negative impact of the combined infection of coccidia and Clostridium perfringens on the intestine of broilers.

[0117] 3.3 Effect of the combination on ileal gene expression in broiler chickens infected with coccidia and Clostridium perfringens

[0118] Table 9 shows the effects of the composition on ileal gene expression in broiler chickens infected with coccidia and Clostridium perfringens, wherein a, b, and c are significant difference markers, and data with different letters on the shoulders in the table indicate significant differences P<0.05; TNF-α represents tumor necrosis factor-α gene, IFN-γ represents interferon-γ gene, and IL-1β represents interleukin-1β gene.

[0119] Table 9

[0120] Detection of genes Control group CCP Group Combination + CCP group P-value TNF-α <![CDATA[1.00±0.23 b ]]> <![CDATA[1.77±0.19 a ]]> <![CDATA[1.25±0.34 b ]]> 0.007 IFN-γ <![CDATA[1.00±0.26 b ]]> <![CDATA[2.68±0.38 a ]]> <![CDATA[0.61±0.18 c ]]> <0.001 IL-1β <![CDATA[1.00±0.28 b ]]> <![CDATA[2.43±0.67 a ]]> <![CDATA[0.66±0.18 b ]]> <0.001

[0121] As shown in Table 9, the combined infection of coccidia and Clostridium perfringens up-regulated the transcriptional levels of pro-inflammatory cytokines such as TNF-α, IFN-γ, and IL-1β in the ileum of broilers, indicating that the combined infection of coccidia and Clostridium perfringens caused inflammatory response in the ileum, and the addition of a composition formed by farnesol, paeonol, and soybean saponin to the diet could alleviate the up-regulation of TNF-α, IFN-γ, and IL-1β gene transcription levels in the ileum caused by the combined infection of coccidia and Clostridium perfringens. The above results indicate that the composition formed by farnesol, paeonol, and soybean saponin can alleviate the inflammatory response of the intestine of broilers caused by the combined infection of coccidia and Clostridium perfringens.

[0122] Therapeutic effect of the composition on inflammatory response in broilers caused by combined infection of coccidia and Clostridium perfringens

[0123] The present application example verifies the therapeutic effect of the composition formed by farnesol, paeonol and soybean saponin on the inflammatory response of broilers caused by the combined infection of coccidia and Clostridium perfringens, and the verification method is as follows:

[0124] 1. Materials and Methods

[0125] The experiment involved 90 healthy AA +Broiler male chicks were evenly divided into three treatment groups, each with 30 replicates, and each replicate group included one chicken; the three treatment groups were control group, coccidia and Clostridium perfringens combined infection group (CCP), and combination + CCP group; all broilers were fed the same "corn-soybean meal" basal diet, and the diet formula was formulated according to the nutritional requirements standard of Chinese broilers (NY / T33-2004); on the 8th and 10th days of the experiment, broilers in the CCP group and combination + CCP group were vaccinated with ultra-high dose coccidia vaccine (purchased from Foshan Zhengdian Company, the dose was 30 times the recommended dose) to establish intestinal coccidiosis infection state, and on the 14th to 21st days of the experiment, broilers in the CCP group and combination + CCP group were gavaged with 1 mL CP (CVCC2030 strain, the dose was 1.0×10 9 CFU / mL), and the control group broilers were gavage-fed with an equal volume of CP culture medium; during the experiment, the broilers were free to eat and drink, and a 24-h lighting system was adopted; on the 21st day of the experiment, the composition preparation Z1 prepared in the above Example 1 was added to the broiler feed of the composition+CCP group according to the three active ingredients of paeonol, farnesol, and soybean saponin at a content of 1% of the total weight of the feed, and added three times a day in the morning, noon and evening for one week, and the basic diet of the other two groups was not changed; on the 28th day of the experiment, all broilers were fasted for 8h, and the subwing venous blood of all broilers was collected and the serum was separated for testing.

[0126] 2. Data Analysis

[0127] The experimental data were analyzed by one-way ANOVA in SPSS23.0 statistical software, and Duncan's method was used for multiple comparisons between groups. P < 0.05 was regarded as the standard of significant difference, and P < 0.01 was regarded as the standard of extremely significant difference. The results were expressed as mean ± standard deviation.

[0128] 3. Test results

[0129] Table 10 shows the effects of the composition on the content of inflammatory cytokines in the blood of broilers infected with coccidia and Clostridium perfringens, wherein a, b, and c are marks of significant difference, and data with different letters on the shoulders in the table indicate significant differences P<0.05; IL-1β and IL-6 represent interleukin 1β and interleukin 6, respectively, TNF-α represents tumor necrosis factor-α, and IFN-γ represents interferon-γ; the detection of pro-inflammatory cytokines was carried out using the chicken ELISA kit of Beijing Solebow Company.

[0130] Table 10

[0131]

[0132]

[0133] As can be seen from Table 10, the combined infection of coccidia and Clostridium perfringens upregulated the levels of pro-inflammatory cytokines in the serum of broilers, such as IL-1β, IL-6 and IFN-γ, while the addition of a composition formed by farnesol, paeonol and soybean saponin to the diet could alleviate the upregulation of IL-1β and IFN-γ levels in the serum of broilers caused by the combined infection of coccidia and Clostridium perfringens, indicating that the composition of farnesol, paeonol and soybean saponin can treat the inflammatory response of broilers caused by the combined infection of coccidia and Clostridium perfringens.

[0134] In summary, the composition formed by farnesol, paeonol and soybean saponin provided in the present application can be used to prepare a medicine or feed additive for treating or preventing necrotic enteritis in chickens, and the composition has the ability to resist various pathogens that cause necrotic enteritis in chickens, such as Clostridium perfringens. The medicine or feed additive includes dosage forms such as oral liquid, granules, powder, and tablets.

[0135] The present application is described in detail above. Specific examples are used in this article to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the present application and its core ideas. It should be pointed out that for ordinary technicians in this technical field, without departing from the principles of the present application, several improvements and modifications can be made to the present application, and these improvements and modifications also fall within the scope of protection of the claims of the present application.

Claims

1. A composition for preventing necrotic enteritis in chickens, characterized in that: The composition comprises paeonol, farnesol and soybean saponin.

2. The composition according to claim 1, characterized in that Calculated by weight, it includes 5 parts of paeonol, 5 parts of farnesol and 1 part of soybean saponin.

3. The composition according to claim 1, characterized in that The composition also includes auxiliary materials, which include at least one of formic acid, acetic acid, anhydrous ethanol, propylene glycol, dimethyl sulfoxide, Tween 80, water, lactose, mannitol, soluble starch, montmorillonite, vermiculite, zeolite powder, white carbon black, defatted rice bran, corn cob powder, starch, and bran.

4. The composition according to claim 1, characterized in that The composition is a solid mixture formed by mixing three active ingredients of paeonol, farnesol and soybean saponin with montmorillonite, wherein the mass fraction of the three active ingredients of paeonol, farnesol and soybean saponin in the solid mixture is 40% to 60%; or A mixed solution formed by mixing three active ingredients, namely, paeonol, farnesol and soybean saponin, with acetic acid, anhydrous ethanol, Tween-80 and water, wherein the concentration of the three active ingredients, namely, paeonol, farnesol and soybean saponin, in the mixed solution is 80-120 mg / mL; or A powdery mixture formed by mixing three active ingredients, namely, paeonol, farnesol and soybean saponin, with wheat bran and soluble starch, wherein the mass fraction of the three active ingredients, namely, paeonol, farnesol and soybean saponin, in the powdery mixture is 40% to 60%; or A flaky mixture formed by mixing three active ingredients of paeonol, farnesol and soybean saponin with lactose or maltose and soluble starch, wherein the mass fraction of the three active ingredients of paeonol, farnesol and soybean saponin in the flaky mixture is 40% to 60%; or The invention is a granular mixture formed by mixing three active ingredients, namely paeonol, farnesol and soybean saponin, with lactose or maltose and soluble starch, wherein the mass fraction of the three active ingredients, namely paeonol, farnesol and soybean saponin, in the granular mixture is 40% to 60%.

5. Use of the composition according to claim 1 in the preparation of a medicine or feed additive for treating or preventing necrotic enteritis in chickens.

6. The use according to claim 5, characterized in that: The chicken necrotic enteritis is caused by infection of Clostridium perfringens alone or in combination with coccidia.

7. The use according to claim 5, characterized in that: The method for using the composition as a medicine or feed additive includes at least one of (1) to (4): (1) Orally administering the drug to chickens at a dosage of 100 to 1000 mg / kg of chicken body weight of the three active ingredients of paeonol, farnesol, and soybean saponin; (2) The content of the three active ingredients of paeonol, farnesol and soybean saponin is 0.1% to 1% by weight of chicken feed; the composition is added to chicken feed as a feed additive; (3) When used for daily prevention of chicken enteritis, the three active ingredients of paeonol, farnesol and soybean saponin are added to feed or drinking water at a dosage of 100 mg / kg chicken body weight and used throughout the entire breeding cycle; (4) When used for clinical treatment of chicken enteritis: orally administer the composition to chickens at a dosage of 1000 mg / kg of chicken body weight of the three active ingredients of paeonol, farnesol and soybean saponin; or grind the composition into powder and mix it with the daily diet at a dosage of 1000 mg / kg of chicken body weight of the three active ingredients of paeonol, farnesol and soybean saponin, and add it to feed or drinking water three times a day in the morning, noon and evening for one week.

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