Application of Brucella Romummii metagen in preparation of medicine or feed additive for treating or preventing chicken necrotic enteritis

Abstract

The invention discloses an application of Brucella Romummiana metaplasm in preparation of a medicine or a feed additive for treating or preventing necrotic enteritis, the medicine or the feed additive is oral liquid, tablets, granules and powder prepared by taking Brucella Romummiana metaplasm or Brucella Romummiana metaplasm as an effective component and adding a solvent, an auxiliary material or a killing agent, and the Brucella Romummiana metaplasm can be used for treating or preventing chicken necrotic enteritis. The use dosage is as follows: the content of the post-element of the Brucella Romummii is 200-800mg / kg of chicken body weight; or the Brucella Romummiana metagen or the preparation thereof is directly added into the feed, and the addition amount is that the content of the Brucella Romummiana metagen is 0.02-0.08% of the total weight of the chicken feed. The application finds that the Brucella Romummii metagen as a medicine or feed additive is high in safety and free of toxic and side effects, and can effectively relieve the reduction of chicken production performance, the increase of proinflammatory cytokines and the damage of intestinal absorption and barrier functions caused by necrotic enteritis.

Description

Technical Field

[0001] This application relates to the field of chicken enteritis prevention and control technology, specifically to the application of Rombutz bacteria postbiotics in the preparation of drugs or feed additives for the treatment or prevention of necrotizing enteritis in chickens. Background Technology

[0002] my country is a major producer and consumer of chicken meat, and ensuring the healthy and efficient breeding of broilers to improve chicken yield and quality is crucial to the national economy and people's livelihood. However, necrotic enteritis (NE) severely restricts the healthy and efficient breeding of broilers. NE is one of the major gastrointestinal diseases endangering the healthy breeding of broilers worldwide, mainly caused by infection with Clostridium perfringens types A and G. Abnormal environmental factors (such as heat stress), pathogenic factors (such as coccidiosis) infection, and dietary types (such as wheat-based diets) during broiler production provide conditions for CP infection. NE mainly occurs in the small intestine, primarily the ileum, leading to impaired physical and immune barrier function of the broiler intestine, dysbiosis of the intestinal flora, and consequently, inflammatory damage to the intestine.

[0003] Previously, the use of antibiotics was the main measure to control non-steroidal anti-inflammatory disease (NE) in broilers. However, excessive use of antibiotics has brought many harms: (1) Increased drug resistance: Long-term use of antibiotic additives can lead to drug resistance in microorganisms, causing some pathogenic microorganisms to develop drug resistance through mutation, resulting in 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 emissions of metabolites from poultry fed with antibiotics can damage the ecological environment and pollute drinking water or other agricultural products; (4) Impact on human health: Antibiotic residues in livestock and poultry products, if ingested by humans, can 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 lead to an imbalance in the normal microbial system in chickens, causing a decline in the immune function and resistance of chickens. 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 broilers have been proliferating rapidly, which inevitably damages their intestinal immune barrier and exacerbates the negative effects of norepinephrine (NE) on broilers.

[0004] It is evident that non-steroidal anti-inflammatory disease (NE) is a key issue that urgently needs to be addressed for the healthy and efficient breeding of broilers in the post-antibiotic era. Controlling NE through nutritional measures has gradually become one of the key topics of concern in broiler farming. Summary of the Invention

[0005] In view of this, the purpose of this application is to provide the use of Rombutz metabiotics in the preparation of drugs or feed additives for the treatment or prevention of necrotic enteritis in chickens. Rombutz metabiotics have high safety, stable efficacy, no toxic side effects, and a significant preventive and control effect on necrotic enteritis in chickens. Therefore, Rombutz metabiotics can be used in the preparation of drugs or feed additives for the treatment or prevention of necrotic enteritis in chickens.

[0006] To achieve, or at least partially achieve, the above objectives, this application provides the following technical solution:

[0007] Application of Rombutz bacteria metabiotics in the preparation of drugs or feed additives for the treatment or prevention of necrotizing enteritis in chickens.

[0008] The technical solution provided in this application has at least the following advantages compared with the prior art:

[0009] Rombutz bacteria postbiotics overcome the drawbacks of difficulty in colonizing live bacteria and easy degradation by gastric acid and bile salts, thus targeting the gut to improve intestinal health. They are highly safe, have no toxic side effects, and can be used long-term as a feed additive to prevent necrotic enteritis in chickens. Long-term use of Rombutz bacteria postbiotics does not lead to the development of drug-resistant strains, an advantage not possessed by other anti-disease drugs. Furthermore, in vivo studies have shown that Rombutz bacteria postbiotics can inhibit the expression of pro-inflammatory cytokines, promote the expression of intestinal barrier-related genes, improve intestinal health, and thus improve feed conversion efficiency in broilers with necrotic enteritis. Detailed Implementation

[0010] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0011] Those skilled in the art will understand that, unless otherwise stated, the terms "the," "the," and "the foregoing" used in this application may also include plural forms. It should be further understood that the word "comprising" as used in the specification of this application means the presence of the stated features, steps, or operations, but does not exclude the presence or addition of one or more other features, integers, or steps.

[0012] Those skilled in the art will understand that, where specific experimental steps or conditions are not specified in the embodiments, they can be performed according to the conventional experimental steps or conditions described in the literature in this field; and where the manufacturers of the raw materials or instruments and equipment used are not specified, they are all conventional products that can be obtained commercially.

[0013] Those skilled in the art will understand that, unless otherwise stated in this application, when numerical ranges are given in the embodiments, 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, as well as the prior art known to those skilled in the art and the descriptions in this application, can be implemented using any prior art methods, devices, and materials similar to or equivalent to those described, used, or made by means of methods, devices, and materials in the embodiments of this application.

[0014] Romboutsia (RB) primarily colonizes the intestinal lumen and mucosa of the ileum in hosts. Its metabolism produces various bioactive substances, including β-glucan, functional amino acids, vitamins, propionic acid, and butyric acid. In recent years, due to its high correlation with bodily health, RB has attracted widespread attention in the field of biomedicine. For example, studies have found a significant decrease in the relative abundance of RB in the intestinal lumen of patients with autism, hypertension, and diabetes. In the succession of broiler gut microbiota, RB begins to proliferate at 7–10 days of age and develops into the dominant gut microbiota at 21 days of age. In the later stages of growth, the relative abundance of RB in the broiler gut is second only to Lactobacillus, and in adult turkeys, the relative abundance of RB is even higher than that of Lactobacillus. However, there are currently few reports on the effects of RB on the physiological function of the broiler gut. In fact, RB may play a crucial role in improving the intestinal immune barrier and enhancing the gut health of broilers. Studies have found that RB (reticulobacillus rubrum) is extremely sensitive to the occurrence and development of necrotic enteritis in broilers. Specifically, the relative abundance of RB in the ileum is significantly reduced during the initial stage of infection and the acute inflammatory phase of necrotic enteritis in broilers, and gradually increases after the intestinal inflammation subsides. Therefore, RB may serve as a novel indicator bacterium for assessing the occurrence and development of necrotic enteritis in broilers.

[0015] The RB-based postbiotic is rich in β-glucan, functional amino acids, vitamins, propionic acid, and butyric acid, and also contains a high content of bacterial protein. Compared with live bacteria alone, the RB postbiotic overcomes the drawbacks of live bacteria such as difficulty in colonization and easy degradation by gastric acid and bile salts, and targets the intestine to improve intestinal health. Currently, there are no reports of RB postbiotics in poultry production. Based on this, the embodiments of this application show through in vivo experiments that the Rombutz bacteria postbiotic can inhibit the expression of pro-inflammatory cytokines, promote the expression of intestinal barrier-related genes, improve intestinal health, and thus improve the feed conversion efficiency of broilers with necrotizing enterocolitis. These results can provide a theoretical basis for the development of Rombutz bacteria postbiotics as targeted drugs or feed additives for the prevention and control of necrotizing enterocolitis in broilers.

[0016] Based on this, embodiments of this application provide the use of Rombutz bacteria postbiotics in the preparation of drugs or feed additives for the treatment or prevention of necrotizing enteritis in chickens.

[0017] In some embodiments, the necrotizing enteritis in chickens is caused by Clostridium perfringens infection alone or in combination with coccidia.

[0018] In some embodiments, the drug or feed additive is a Rombutz postbiotic, meaning that the Rombutz postbiotic can be directly used as a drug or feed additive to treat or prevent necrotic enteritis in chickens.

[0019] In some embodiments, the drug is a formulation made by adding a solvent to a probiotic containing Rombutzella metabiotic as the active ingredient. The solvent is at least one of anhydrous ethanol, propylene glycol, dimethyl sulfoxide, edible oil, and water. The formulation is an oral liquid.

[0020] In some preferred embodiments, the oral solution is a mixed solution of Rombutz bacteria metatrophic agent, anhydrous ethanol, and water, wherein the concentration of Rombutz bacteria metatrophic agent is 80-120 mg / mL.

[0021] In some embodiments, the drug is a formulation made with Rombutzella postbiotic as the active ingredient and excipients, wherein the excipients are at least one of lactose, maltose, mannitol, wheat bran, fumed silica, and soluble starch, and the formulation is at least one of granules, tablets, and powders.

[0022] In some preferred embodiments, the granules are a granular mixture prepared by mixing Rombutz metabiotics with lactose or maltose and soluble starch, wherein the mass fraction of Rombutz metabiotics in the granular mixture is 40% to 60%.

[0023] In some preferred embodiments, the tablet is a tablet mixture prepared by mixing Rombutz metabiotic with lactose or maltose and soluble starch, wherein the mass fraction of Rombutz metabiotic in the tablet mixture is 40% to 60%.

[0024] In some preferred embodiments, the powder is a powdered mixture prepared by mixing Rombutz metabiotic with wheat bran and soluble starch, wherein the mass fraction of Rombutz metabiotic in the powdered mixture is 40% to 60%.

[0025] In some embodiments, the strain of Romboutsia ilealis is commercially available Romboutsia ilealis DSM25109.

[0026] In some embodiments, the *Rombutz* postbiotic contains ≥33% bacterial cell protein and ≥10% *Rombutz*. 8 cfu / g, β-glucan content ≥5mg / g, sodium butyrate content ≥10mg / g.

[0027] In some embodiments, the feed additive is a formulation made by adding Rombutz bacteria postbiotic as an active ingredient carrier, wherein the carrier is at least one of montmorillonite, maifanite, vermiculite, zeolite powder, precipitated silica, defatted rice bran, corn cob powder, starch, and wheat bran.

[0028] In some embodiments, the method of using the drug or feed additive includes at least one of the following methods:

[0029] (1) Administer the drug orally to chickens at a dose of 200-800 mg / kg of chicken body weight of Rombutz bacteria postbiotic content;

[0030] (2) The content of Rombutz bacteria postbiotics is 0.02% to 0.08% of the weight of chicken feed; Rombutz bacteria postbiotic granules, powders or tablets are added to chicken feed as feed additives.

[0031] (3) When used for daily prevention of chicken enteritis, add it to feed or drinking water at a dose of 200 mg / kg chicken body weight of Rombutz bacteria postbiotic content and use it throughout the feeding cycle;

[0032] (4) When used for the clinical treatment of chicken enteritis: administer the medicine orally to chickens at a dose of 800 mg / kg chicken body weight of Rombutz bacteria postbiotic content; or crush the granules, powder or tablets and feed them directly at a dose of 800 mg / kg chicken body weight of Rombutz bacteria postbiotic content; or crush the granules, powder or tablets and add them to the feed at a Rombutz bacteria postbiotic content of 0.08% of the weight of the chicken feed; or crush the granules, powder or tablets and use them at a Rombutz bacteria postbiotic content of 0.08% of the weight of the drinking water, adding them three times a day in the morning, noon and evening for one week.

[0033] The technical solution and its effects described in this application are further illustrated below with more specific embodiments. The preparation method of the Rombutz bacteria postbiotic in the following embodiments is as follows:

[0034] A single colony of Romboutsia ilealis DSM 25109 (purchased from Beijing Bioway Biotechnology Co., Ltd.) was inoculated into 10 mL of modified GAM liquid medium (purchased from Shanghai Jinpan Biotechnology Co., Ltd.) and anaerobically cultured at 37°C for 24 hours. At this time, the colony count was approximately 1 × 10⁻⁶. 8 cfu / mL; then, 1 mL of the bacterial culture was inoculated into 100 mL of modified GAM liquid medium, at which point the colony count was approximately 1 × 10⁻⁶. 9cfu / mL; then 100mL of bacterial culture was placed in a 500L fermenter for 24 hours of expansion culture, the temperature of the fermenter was kept constant at 37℃, and N2 was introduced to establish anaerobic fermentation conditions; after the culture was completed, 10% maltodextrin was added to the fermentation broth for adsorption, and spray drying was used to prepare Rombutz postbiotic.

[0035] Example 1

[0036] This embodiment provides a method for preparing a Rombutz bacteria postbiotic oral liquid, the specific steps of which are as follows:

[0037] Weigh 10g of Rombutz bacteria postbiotic and 30mL of anhydrous ethanol; dissolve the Rombutz bacteria postbiotic in anhydrous ethanol and mix well, then slowly add water for injection to make up to 100mL, to obtain a Rombutz bacteria postbiotic solution with a concentration of 100mg / mL, namely Rombutz bacteria postbiotic oral solution L1.

[0038] When using the above oral solution for routine prevention of chicken enteritis, add it to the feed or drinking water at a dose of 200 mg / kg chicken body weight, and use it throughout the rearing cycle. When using the above oral solution for clinical treatment of chicken enteritis, administer the oral solution orally at a dose of 800 mg / kg chicken body weight; or mix the oral solution with the daily diet at a dose of 800 mg / kg chicken body weight and add it to the feed or drinking water. Add it three times a day, morning, noon and evening, for one week.

[0039] Example 2

[0040] This embodiment provides a method for preparing Rombutz bacteria post-biotic powder, the specific steps of which are as follows:

[0041] Weigh out 50g of Rombutz bacteria post-biotic, 20g of wheat bran, and 30g of soluble starch; mix the Rombutz bacteria post-biotic, wheat bran, and soluble starch evenly, and pass them through a 60-mesh sieve to obtain a powdery mixture, namely Rombutz bacteria post-biotic powder L2.

[0042] When using the above powder for routine prevention of chicken enteritis, add it to the feed or drinking water at a dose of 200 mg / kg chicken body weight, and use it throughout the rearing cycle. When using the above powder for clinical treatment of chicken enteritis, dissolve the powder in water at a dose of 800 mg / kg chicken body weight and administer orally; or mix the powder at a dose of 800 mg / kg chicken body weight with the daily diet and add it to the feed or drinking water. Add it three times a day (morning, noon, and evening) for one week.

[0043] Example 3

[0044] This embodiment provides a method for preparing Rombutz-derived postbiotic tablets, the specific steps of which are as follows:

[0045] Weigh out 50g of Rombutz bacteria post-biotic, 20g of lactose, and 30g of soluble starch; mix the Rombutz bacteria post-biotic, lactose, and soluble starch evenly, compress them into tablets using a tablet press, and dry them to obtain a tablet-shaped mixture, namely Rombutz bacteria post-biotic tablet L3.

[0046] When using the above tablets for routine prevention of chicken enteritis, crush the tablets and add them to the feed or drinking water at a dose of 200 mg / kg chicken body weight, and use them throughout the rearing cycle. When using the above tablets for clinical treatment of chicken enteritis, crush the tablets and dissolve them in water at a dose of 800 mg / kg chicken body weight, then administer orally; or crush the tablets and mix them with the diet at a dose of 800 mg / kg chicken body weight, then add them to the feed or drinking water. Add three times a day (morning, noon, and evening) for one week.

[0047] Example 4

[0048] This embodiment provides a method for preparing Rombutz bacteria post-natal granules, the specific steps of which are as follows:

[0049] Weigh out 50g of Rombutz bacteria post-biotic, 20g of maltose, and 30g of soluble starch; mix the Rombutz bacteria post-biotic, maltose, and soluble starch evenly, granulate using a granulator, and dry to obtain a granular mixture, namely Rombutz bacteria post-biotic granules L4.

[0050] When using the above-mentioned granules for routine prevention of chicken enteritis, add the granules to the feed or drinking water at a dosage of 200 mg / kg of chicken body weight, and use throughout the rearing cycle. When using the above-mentioned granules for clinical treatment of chicken enteritis, dissolve the granules in water at a dosage of 800 mg / kg of chicken body weight and administer orally; or mix the granules with the daily diet at a dosage of 800 mg / kg of chicken body weight and add them to the feed or drinking water. Add three times a day (morning, noon, and evening) for one week.

[0051] The formulations and parameters of the Rombutz bacteria postbiotic preparations prepared in the above embodiments are shown in Table 1 below:

[0052] Table 1

[0053] Example number Formulation number Components Rombutz bacteria postbiotic concentration Formulation type Example 1 L1 Rombutz bacteria postbiotics, anhydrous ethanol, water for injection 100mg / mL oral liquid Example 2 L2 Rombutz bacteria post-biotics, wheat bran, soluble starch 50% (mass fraction) powder Example 3 L3 Rombutz bacteria post-biotics, lactose, soluble starch 50% (mass fraction) tablet Example 4 L4 Rombutz bacteria post-biotics, maltose, soluble starch 50% (mass fraction) Granules

[0054] Effects of Rombutzella postbiotics on growth performance and gut health in broilers infected with Clostridium perfringens

[0055] This application's embodiments verified the effects of Rombutzella postbiotics on the growth performance and gut health of broilers infected with Clostridium perfringens. The verification method is as follows:

[0056] 1. Materials and Methods

[0057] The experiment used 480 healthy AA (Animal Humidity) animals of uniform weight at one day old.+ Broiler male chicks were randomly assigned to three treatment groups, each with eight replicates, and each replicate consisting of 20 chicks. The three treatment groups were: a control group, a Clostridium perfringens (CP) infection group, and a Rombutz bacillus postbiotic + CP infection group. The control and CP infection groups were fed a corn-soybean meal basal diet formulated according to the Chinese broiler nutritional requirements standard (NY / T33-2004). The Rombutz bacillus postbiotic + CP infection group received a diet supplemented with 0.02% (by weight) of Rombutz bacillus postbiotic (with bacterial protein ≥33% and Rombutz bacillus ≥10%) in addition to their basal diet. 8 (CFU / g, β-glucan content ≥5 mg / g, sodium butyrate content ≥10 mg / g) or preparations with the same Rombutz bacteria postbiotic content; all broilers were vaccinated against Newcastle disease on day 7 of the experiment (via eye drops or nasal drops). From day 14 to day 21 of the experiment, 1 mL of CP (CVCC2030 strain, dose 1.0 × 10⁻⁶) was administered daily to broilers in the CP infection group and the Rombutz bacteria postbiotic + CP infection group. 9 The control group was fed an equal volume of CP medium (cfu / mL). During the experiment, the broilers had free access to feed and water and were under a 24-hour light regime. On day 22, all broilers were fasted for 8 hours, then weighed and their feed consumption was recorded. Growth performance indicators such as average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR) were calculated. Subsequently, one broiler of uniform weight was selected from each replicate group to collect blood from the wing vein and separate the serum for testing.

[0058] 2. Data Analysis

[0059] The experimental data were analyzed by one-way ANOVA in SPSS 23.0 statistical software. Duncan's method was used to perform multiple comparisons between groups. P < 0.05 was used as the significance criterion, and P < 0.01 was used as the extremely significant criterion. The results are expressed as mean ± standard deviation.

[0060] 3. Test Results

[0061] 3.1 Effects of Rombutzella postbiotics on growth performance of broilers infected with Clostridium perfringens

[0062] Table 2 shows the effect of Rombutz's postbiotic on the growth performance of broilers infected with Clostridium perfringens. In the table, different letters under the data indicate significant differences (P<0.05).

[0063] Table 2

[0064] Testing items control group CP infection group Rombutzolium postbiotic + CP infection group P-value Average body weight (g) on ​​day 21 <![CDATA[683.06±10.39 a ]]> <![CDATA[577.51±31.77 b ]]> <![CDATA[653.64±27.78 a ]]> <0.001 ADFI(g) 45.06±1.39 43.27±3.89 44.39±3.05 0.708 ADG(g) <![CDATA[34.15±0.52 a ]]> <![CDATA[28.81±1.59 b ]]> <![CDATA[32.65±2.89 a ]]> 0.024 FCR <![CDATA[1.32±0.05 b ]]> <![CDATA[1.53±0.05 a ]]> <![CDATA[1.38±0.06 b ]]> <0.001

[0065] Table 2 shows that Clostridium perfringens infection reduced the average body weight and average daily weight gain of broilers, and increased the feed conversion ratio. However, the addition of Clostridium perfringens postbiotics to the diet could alleviate the reduction in average body weight and average daily weight gain of broilers caused by Clostridium perfringens infection, and improve the feed conversion ratio. These results indicate that Clostridium perfringens postbiotics have a protective effect against Clostridium perfringens infection in broilers.

[0066] 3.2 Effects of Rombutzella postbiotics on serum Newcastle disease vaccine antibody levels, D-xylose and diamine oxidase content in broiler chickens infected with Clostridium perfringens

[0067] Table 3 shows the effects of Rombutz's postbiotic on serum Newcastle disease vaccine antibody levels, D-xylose, and diamine oxidase (DAO) content in broiler chickens infected with Clostridium perfringens. Different letters under the data in the table indicate significant differences (P < 0.05). The Newcastle disease vaccine antibody levels are the results obtained after analyzing data read from an ELISA reader at OD600 parameters.

[0068] Table 3

[0069] Testing items control group CP infection group Rombutzolium postbiotic + CP infection group P-value D-xylose, mmol / L <![CDATA[1.06±0.31 a ]]> <![CDATA[0.70±0.13 b ]]> <![CDATA[1.13±0.34 a ]]> 0.003 DAO,U / L <![CDATA[5.81±1.78 b ]]> <![CDATA[20.60±6.57 a ]]> <![CDATA[9.17±2.78 b ]]> <0.001 Newcastle disease vaccine antibody level OD600 <![CDATA[0.76±0.12 a ]]> <![CDATA[0.35±0.07 c ]]> <![CDATA[0.53±0.02 b ]]> 0.045

[0070] D-xylose can be absorbed by the intestines but cannot be utilized by the body. Therefore, evaluating the D-xylose content in the blood can assess intestinal absorption function. In a healthy state, DAO exists in the intestines; when the intestines are damaged, it is released into the bloodstream. Therefore, the higher the DAO activity in the serum, the more significant the intestinal damage. Table 3 shows that adding *R. romobutzin* postbiotics to the diet can alleviate the decrease in D-xylose content and the increase in DAO levels in the blood of broilers caused by *Clostridium perfringens* infection. The results indicate that *R. romobutzin* postbiotics can protect broilers from the decrease in intestinal absorption function and barrier disruption caused by *Clostridium perfringens* infection. Furthermore, adding *R. romobutzin* postbiotics to the diet can alleviate the decrease in Newcastle disease vaccine antibody levels in the serum of broilers caused by *Clostridium perfringens* infection, thereby improving the humoral immune function of broilers.

[0071] 3.3 Effects of Rombutzella postbiotics on serum inflammatory cytokine levels in broiler chickens infected with Clostridium perfringens

[0072] Table 4 shows the effect of Rombutzella postbiotics on the serum inflammatory cytokine levels in broiler chickens infected with Clostridium perfringens. Different letters under the data in the table indicate significant differences (P<0.05). IL-1β and IL-6 represent interleukin-1β and interleukin-6, respectively, and IFN-γ represents interferon-γ. The pro-inflammatory cytokines were detected using a chicken ELISA kit from Beijing Solarbio Co., Ltd.

[0073] Table 4

[0074] Testing items control group CP infection group Rombutzolium postbiotic + CP infection group P-value IL-1β, pg / mL <![CDATA[89.13±8.97 c ]]> <![CDATA[153.66±30.18 a ]]> <![CDATA[111.32±9.93 b ]]> 0.021 IL-6, pg / mL <![CDATA[101.77±17.54 c ]]> <![CDATA[198.33±21.76 a ]]> <![CDATA[132.35±25.58 b ]]> <0.001 IFN-γ, pg / mL <![CDATA[77.71±8.53 b ]]> <![CDATA[188.65±21.11 a ]]> <![CDATA[187.27±16.88 a ]]> 0.017

[0075] Table 4 shows that Clostridium perfringens infection caused an upregulation of pro-inflammatory cytokines such as IL-1β, IL-6, and IFN-γ. However, the addition of Rombutz postbiotics to the diet could alleviate the increase in serum IL-1β and IL-6 in broilers caused by Clostridium perfringens infection. The results indicate that Rombutz postbiotics have a mitigating effect on the inflammatory response in broilers caused by Clostridium perfringens infection. Treatment of inflammatory response in broilers caused by coccidia and Clostridium perfringens co-infection with Rombutzella metabiotic Therapeutic effects

[0076] This application's embodiments validated the therapeutic effect of Rombutzella postbiotics on inflammatory responses in broilers caused by coccidia and Clostridium perfringens co-infection. The validation method is as follows:

[0077] 1. Materials and Methods

[0078] The experiment used 450 healthy AA (Animal Crossings) of uniform weight at one day old. + Broiler male chicks were randomly divided into three treatment groups, each with ten replicates, and each replicate consisting of 15 chicks. The three treatment groups were: a control group, a coccidia and Clostridium perfringens co-infection group (CCP), and a Rombutz bacillus postbiotic + CCP group. All broilers were fed the same corn-soybean meal basal diet, formulated according to the Chinese broiler nutritional requirements standard (NY / T33-2004). On days 8 and 10 of the experiment, the CCP and Rombutz bacillus postbiotic + CCP groups were administered vaccines. The CCP group was vaccinated with an ultra-high dose of coccidia vaccine (purchased from Foshan Zhengdian Company, at 30 times the recommended dose) to establish intestinal coccidia infection. From days 14 to 21 of the experiment, the CCP group and the Rombutz bacillus postbiotic + CCP group were gavaged daily with 1 mL of CP (CVCC2030 strain, dose 1.0 × 10⁻⁶). 9 The control group was fed an equal volume of CP culture medium (cfu / mL). During the experiment, the broilers had free access to feed and water and were under a 24-hour light regime. On day 21 of the experiment, the Rombutz bacteria postbiotic (with bacterial protein ≥33%, Rombutz bacteria ≥10⁸ CFU / g, β-glucan content ≥5 mg / g, and sodium butyrate content ≥10 mg / g) oral solution L1 described in Example 1 was added to the feed of broilers in the Rombutz bacteria postbiotic + CCP group at an addition amount of 0.08% of the total feed weight. It was added three times a day, morning, noon and evening, for one week. The basal diet of the other two groups was not changed. On day 28 of the experiment, all broilers were fasted for 8 hours, and blood was collected from the wing vein of all broilers and the serum was separated for testing. Further anesthesia and slaughter were achieved by injecting sodium pentobarbital (dose of 50 mg / kg live body weight) into the wing vein. Ileal tissue was dissected and sectioned for observation. Ileal samples were taken for subsequent analysis.

[0079] 2. Data Analysis

[0080] The experimental data were analyzed by one-way ANOVA in SPSS 23.0 statistical software. Duncan's method was used to perform multiple comparisons between groups. P < 0.05 was used as the significance criterion, and P < 0.01 was used as the extremely significant criterion. The results are expressed as mean ± standard deviation.

[0081] 3. Test Results

[0082] 3.1 Effects of *Romebutzimia rubella*, *Coccidioidomyces romuli*, and *Clostridium perfringens* infection on broiler ileum morphology and structure

[0083] Table 5 shows the effects of Rombutzella roximatei and Clostridium perfringens on the morphology of the ileum in broilers. Different letters under the superscript 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.

[0084] Table 5

[0085] Testing items control group CCP Group Rombutz bacteria postbiotic + CCP group P-value VH,μm <![CDATA[731.71±68.81 a ]]> <![CDATA[621.20±49.87 b ]]> <![CDATA[718.63±93.66 a ]]> 0.022 CD, μm <![CDATA[81.68±16.18 b ]]> <![CDATA[103.71±11.81 a ]]> <![CDATA[80.09±11.89 b ]]> <0.001 VH / CD <![CDATA[9.09±1.15 a ]]> <![CDATA[6.02±1.28 b ]]> <![CDATA[8.98±1.76 a ]]> 0.012

[0086] The higher the intestinal villi height, the larger the contact area with nutrients in the intestinal lumen, and the better the absorption efficiency. Therefore, a higher intestinal villi height and a higher villi height to crypt depth ratio indicate better intestinal absorption function. Table 5 shows that adding *Romebutzimia ulmoides* postbiotics to the diet can alleviate the decrease in ileal villi height and villi height to crypt depth ratio in broilers caused by co-infection with coccidia and *Clostridium perfringens*. Table 5 results indicate that *Romebutzimia ulmoides* postbiotics can alleviate the negative impact of co-infection with coccidia and *Clostridium perfringens* on the intestinal absorption function of broilers.

[0087] 3.2 Effects of *Romebutzimia ulmoides* and *Clostridium perfringens* infection on serum inflammatory cytokine levels in broiler chickens

[0088] Table 6 shows the effects of *Romebutzella* metastrophic coccidia and *Clostridium perfringens* infection on serum inflammatory cytokine levels in broiler chickens. Different letters under the data in the table indicate significant differences (P < 0.05). IL-1β and IL-6 represent interleukin-1β and interleukin-6, respectively, and IFN-γ represents interferon-γ. Pro-inflammatory cytokines were detected using a chicken ELISA kit from Beijing Solarbio Co., Ltd.

[0089] Table 6

[0090]

[0091] Table 6 shows that co-infection with coccidia and Clostridium perfringens caused an upregulation of pro-inflammatory cytokines such as IL-1β, IL-6, and IFN-γ. However, the addition of Rombutz postbiotic to the diet could alleviate the increase in serum IL-1β and IFN-γ in broilers caused by co-infection with coccidia and Clostridium perfringens. The results indicate that Rombutz postbiotic has a mitigating effect on the inflammatory response in broilers caused by Clostridium perfringens infection.

[0092] 3.3 Effects of *Romebutzimia rubella* and *Clostridium perfringens* infection on gene expression in the ileum of broiler chickens

[0093] Table 7 shows the effects of *Romebutzella* metagenic coccidia and *Clostridium perfringens* infection on gene expression in the ileum of broiler chickens. Different letters under the superscript indicate significant differences (P < 0.05). IL-1β and IL-6 represent interleukin-1β and interleukin-6 genes, respectively; TNF-α represents tumor necrosis factor-α gene; IFN-γ represents interferon-γ gene; AQP-3 represents aquaporin-3 gene; and NHE3 represents Na+. + H + The three transport vector genes are: Pept1 (Oligopeptide transferase 1), ZO-1 (Blocking band protein 1), Mucin-2 (Mucin 2), and Claudin-1 (Tight junction protein 1).

[0094] Table 7

[0095] Testing items control group CCP Group Rombutz bacteria postbiotic + CCP group P-value IL-1β <![CDATA[1.00±0.26 b ]]> <![CDATA[2.56±0.76 a ]]> <![CDATA[0.82±0.27 b ]]> <0.001 IL-6 <![CDATA[1.00±0.59 b ]]> <![CDATA[2.33±0.69 a ]]> <![CDATA[1.29±0.36 b ]]> <0.001 TNF-α <![CDATA[1.00±0.30 b ]]> <![CDATA[1.59±0.47 a ]]> <![CDATA[0.88±0.22 b ]]> <0.001 IFN-γ <![CDATA[1.00±0.54 c ]]> <![CDATA[20.71±6.65 a ]]> <![CDATA[7.49±1.20 b ]]> <0.001 AQP3 <![CDATA[1.00±0.22 a ]]> <![CDATA[0.28±0.07 b ]]> <![CDATA[0.35±0.09 b ]]> <0.001 NHE3 1.00±0.38 1.25±0.48 1.39±0.42 0.132 Pept1 <![CDATA[1.00±0.74 a ]]> <![CDATA[0.13±0.10 b ]]> <![CDATA[0.87±0.88 a ]]> 0.009 ZO-1 <![CDATA[1.00±0.23 a ]]> <![CDATA[0.61±0.17 b ]]> <![CDATA[0.61±0.16 b ]]> <0.001 Claudin1 <![CDATA[1.00±0.28 a ]]> <![CDATA[0.43±0.02 b ]]> <![CDATA[1.16±0.13 a ]]> <0.001 Mucin2 <![CDATA[1.00±0.18 a ]]> <![CDATA[0.45±0.09 b ]]> <![CDATA[1.21±0.30 a ]]> 0.003

[0096] Table 7 shows that co-infection with coccidia and Clostridium perfringens upregulated the transcriptional levels of pro-inflammatory cytokine-related genes IL-1β, IL-6, TNF-α, and IFN-γ; downregulated the transcriptional levels of ion transport channel protein-related genes such as AQP3 and Pept1; and downregulated the transcriptional levels of intestinal barrier-related genes ZO-1, Claudin1, and Mucin2. However, adding Rombutz postbiotic to the diet alleviated the upregulation of IL-1β, IL-6, TNF-α, and IFN-γ transcriptional levels and the downregulation of Pept1, Claudin1, and Mucin2 transcriptional levels caused by co-infection with coccidia and Clostridium perfringens. These results indicate that Rombutz postbiotic has a therapeutic effect on intestinal inflammatory damage in broilers co-infected with coccidia and Clostridium perfringens.

[0097] Effects of Rombutzella postbiotics on broiler production performance under production conditions

[0098] This application's embodiments verified the effect of Rombutz bacteria postbiotics on broiler production performance under production conditions. The verification method is as follows:

[0099] 1. Materials and Methods

[0100] The experiment used 2400 healthy AA birds of uniform weight at one day old. + Male broiler chicks were randomly assigned to two treatment groups, each with 10 replicates. Each replicate group consisted of 1200 chicks. The broilers were raised in May 2023 at a broiler farm in Weifang, Shandong Province. The two treatment groups were a control group and a *Rombutz* postbiotic group. The control group broilers were fed a corn-soybean meal basal diet formulated according to the Chinese broiler nutritional requirements standard (NY / T33-2004). The *Rombutz* postbiotic group broilers were fed a diet supplemented with *Rombutz* postbiotic at 0.02% of the total feed weight (with bacterial protein ≥33% and *Rombutz* ≥10%). 8 The feed intake was measured for 35 days, during which broilers had free access to feed and water and were exposed to a 24-hour light regime. On the 35th day of the experiment, all broilers were fasted for 8 hours, then weighed and their feed consumption was recorded. Average daily gain (ADG), average daily feed intake (ADFI), feed conversion ratio (FCR), and other growth performance-related indicators were calculated.

[0101] 2. Data Analysis

[0102] The experimental data were analyzed using one-way ANOVA in SPSS 23.0 statistical software. The significance of the difference was P<0.05 and the extreme significance of the difference was P<0.01. The results are expressed as mean ± standard deviation.

[0103] 3. Test Results

[0104] Table 8 shows the effects of Rombutz bacteria postbiotics on the growth performance of broilers. Different letters under the data in the table indicate significant differences (P<0.05). Survival rate is expressed as 100 multiplied by the number of chickens slaughtered at 35 days of age and divided by the number of chickens at the start of 1 day of age.

[0105] Table 8

[0106] Testing items control group Rombutzion postgenetic group P-value D35 - Average body weight, g 2322.53±78.32 2401.57±96.82 0.089 ADFI,g 90.06±2.13 89.62±2.28 0.822 ADG,g <![CDATA[68.31±1.21 b ]]> <![CDATA[70.67±1.35 a ]]> 0.031 FCR <![CDATA[1.32±0.02 a ]]> <![CDATA[1.28±0.02 b ]]> <0.001 Survival rate, % 98.54±1.12 99.40±0.69 0.545

[0107] As shown in Table 8, under production conditions, the addition of Rombutz bacteria to the diet significantly increased the average daily feed intake of broilers, improved the feed conversion ratio, and increased the survival rate by about 1%, which has important application value for farmers or enterprises.

[0108] In summary, *Rombutzella* metabiotics can be used to prepare drugs or feed additives for the treatment or prevention of necrotic enteritis in chickens. The prepared drugs or feed additives possess resistance to various pathogens causing necrotic enteritis in chickens, including *Clostridium perfringens*. The dosage forms of these drugs and feed additives include oral liquids, granules, powders, and tablets.

[0109] The present application has been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present application. The descriptions of the embodiments above are only for the purpose of helping to understand the present application and its core ideas. It should be noted that those skilled in the art can make several improvements and modifications to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. Application of Rombutsiella postbiotics in the preparation of drugs or feed additives for treating or preventing necrotic enteritis in chickens.

2. The use according to claim 1, characterized in that: The chicken necrotic enteritis is caused by infection of Clostridium perfringens alone or in combination with coccidia.

3. The use according to claim 1, characterized in that: The medicine or feed additive is a Rombutsiella postbiotic.

4. The use according to claim 1, characterized in that: The medicine is a preparation prepared by adding a solvent to the postbiotics of Rombutsiella as an effective ingredient, wherein the solvent is at least one of anhydrous ethanol, propylene glycol, dimethyl sulfoxide, edible oil and water, and the preparation is an oral solution.

5. The use according to claim 4, characterized in that: The oral liquid is a mixed solution of Rombutsiella postbiotics, anhydrous ethanol and water, wherein the concentration of Rombutsiella postbiotics is 80-120 mg / mL.

6. The use according to claim 1, characterized in that: The medicine is a preparation prepared by adding auxiliary materials with Rombutsiella postbiotics as an effective ingredient, wherein the auxiliary materials are at least one of lactose, maltose, mannitol, bran, white carbon black and soluble starch, and the preparation is at least one of granules, tablets and powders.

7. The use according to claim 6, characterized in that: The drug is one of the following preparations: Granules, a granular mixture prepared by mixing Rombutsiella postbiotics with lactose or maltose and soluble starch, wherein the mass fraction of Rombutsiella postbiotics in the granular mixture is 40% to 60%; Tablets, a flaky mixture prepared by mixing Rombutsiella postbiotics with lactose or maltose and soluble starch, wherein the mass fraction of Rombutsiella postbiotics in the flaky mixture is 40% to 60%; Powder, a powdery mixture prepared by mixing Rombutsiella postbiotics with wheat bran and soluble starch, wherein the mass fraction of Rombutsiella postbiotics in the powdery mixture is 40% to 60%.

8. The use according to claim 1, characterized in that: The strain of Rombutsiella is the commercially available Romboutsia ilealis DSM 25109; the bacterial protein in the Rombutsiella postbiotics is ≥33%, and the Rombutsiella ilealis is ≥10 8 cfu / g, β-glucan content ≥5mg / g, sodium butyrate content ≥10mg / g.

9. The use according to claim 1, characterized in that: The feed additive is a preparation prepared by adding a carrier with Rombutsiella postbiotics as an effective ingredient, and the carrier is at least one of montmorillonite, medical stone, vermiculite, zeolite powder, white carbon black, defatted rice bran, corn cob powder, starch and bran.

10. The use according to claim 1, characterized in that: The method for using the drug or feed additive includes at least one of (1) to (4): (1) Orally administering the drug to chickens at a dosage of 200 to 800 mg / kg of chicken body weight of Rombutsiella postbiotics; (2) The content of Rombutsiella postbiotics is 0.02% to 0.08% by weight of chicken feed; the Rombutsiella postbiotics granules, powder or tablets are added to the chicken feed as a feed additive; (3) When used for daily prevention of chicken enteritis, the Rombutsiella postbiotic content is 200 mg / kg chicken body weight and added to feed or drinking water for the entire feeding cycle; (4) When used for clinical treatment of chicken enteritis: orally administer the drug to chickens at a dosage of 800 mg / kg of chicken body weight of Rombutsiella postbiotics; or grind the granules, powder or tablets into powder and feed directly to chickens at a dosage of 800 mg / kg of chicken body weight of Rombutsiella postbiotics; or grind the granules, powder or tablets into powder and add it to the feed at a dosage of 0.08% of the weight of chicken feed; or grind the granules, powder or tablets into powder and add it to the drinking water at a dosage of 0.08% of the weight of drinking water, three times a day in the morning, noon and evening for one week.

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