Application of butyric acid producing enterobacter as probiotic for resisting eimeria tenella

Abstract

The invention relates to the technical field of microorganisms, in particular to application of butyric acid producing enterobacter as a probiotic for resisting eimeria tenella. In the invention, an antibiotic cocktail-induced eimeria tenella infection model is utilized to explore the effect of butyric acid-producing enterobacter DSM 26588 on eimeria tenella development. The enterobacter butyricum DSM 26588 inhibits the growth of gametes by inhibiting the expression of the eimeria tenella gamete EtGFAT gene; according to the present invention, the intestinal tract lesion caused by Eimeria tenella infection can be improved, the intestinal tract pathological injury can be alleviated so as to reduce the Eimeria tenella oocyst discharge amount, the ACI value is 162, and the good anti-coccidiosis effect can be provided.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to application of butyric acid-producing Enterobacter as a probiotic against Eimeria tenella. Background Art

[0002] Chicken coccidiosis is an intestinal protozoal disease caused by a mixed infection with one or more coccidia of the genus Eimeria. It is highly contagious and has a high morbidity rate. Seven species of Eimeria are known to infect chickens, each with varying pathogenicity. E. tenella, which parasitizes exclusively in the cecum, is particularly pathogenic. Clinical symptoms of infection primarily include decreased feed intake and diarrhea. Severe infections can lead to bloody diarrhea and even death. Coccidiosis has a severe economic impact on the poultry industry due to productivity losses, high mortality rates, and high treatment costs associated with disease control.

[0003] Butyric acid-producing Enterobacterium (I. butyriciproducens) is taxonomically classified as Intestinimonas in the phylum Bacillota (formerly Firmicutes), class Clostridia, and order Eubacteriales. I. butyriciproducens has been implicated in numerous disease studies. Studies have shown that I. butyriciproducens is the only gut microbial species whose relative abundance is negatively correlated with Candida albicans. Consuming aronia berries can improve arterial function in middle-aged individuals with prehypertension, a phenomenon believed to be related to a significant increase in the abundance of I. butyriciproducens. Other studies have found that Cyclocarya paliurus polysaccharides promote the production of short-chain fatty acids by increasing the production of bacteria such as I. butyriciproducens, alleviating the symptoms of type 2 diabetes. Other studies have also confirmed that I. butyriciproducens has the potential to become a next-generation probiotic. These findings suggest that the onset and progression of disease can lead to changes in the abundance of I. butyriciproducens, similar to certain disease markers. However, there are not many studies on the direct effects of this bacterium on diseases or pathogens, especially its effects on intestinal parasites, and no relevant studies have been found. Summary of the Invention

[0004] In order to overcome the above problems, the present invention provides the use of Enterobacter butyric acid-producing bacteria as a probiotic against Eimeria tenella.

[0005] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides the use of Enterobacterium butyriciproducens DSM 26588 as a probiotic against Eimeria tenella.

[0007] Among them, Enterobacter butyriciproducens (I. butyriciproducens) DSM 26588 was purchased from the German Microorganism Collection Center (DSMZ).

[0008] In one or more embodiments, the application includes:

[0009] (A1) Application of the culture supernatant of Enterobacter butyriciproducens (I. butyriciproducens) DSM 26588 in inhibiting the invasion of Eimeria tenella sporozoites;

[0010] (A2) Use of the culture supernatant of Enterobacterium butyriciproducens (I. butyriciproducens) DSM 26588 in the preparation of a product for inhibiting the invasion of sporozoites of Eimeria tenella (E. tenella).

[0011] Preferably, the culture supernatant of Enterobacterium butyriciproducens (I. butyriciproducens) DSM 26588 comprises 2'-O-methyladenosine.

[0012] Preferably, Enterobacter butyriciproducens (I. butyriciproducens) DSM 26588 is cultured to the stationary phase, and centrifuged to obtain the culture supernatant of Enterobacter butyriciproducens.

[0013] Further preferably, the butyric acid-producing Enterobacter (I.butyriciproducens) DSM 26588 is inoculated into the anaerobic agar medium at a volume ratio of 1:80-120, and the culture is anaerobically statically cultured for 40-50 hours until the butyric acid-producing Enterobacter (I.butyriciproducens) DSM 26588 reaches a plateau OD 600 The pH value was 0.8 to 1.2, and the mixture was centrifuged to obtain the culture supernatant of the butyric acid-producing Enterobacterium (I. butyriciproducens) DSM 26588.

[0014] In one or more embodiments, the application includes:

[0015] (B1) Use of Enterobacterium butyriciproducens DSM 26588 in inhibiting the expression of the EtGFAT gene in Eimeria tenella gametes;

[0016] (B2) Use of Enterobacterium butyriciproducens DSM 26588 in the preparation of a product for inhibiting the expression of the EtGFAT gene in Eimeria tenella gametes.

[0017] In one or more embodiments, the application includes:

[0018] (C1) Use of Enterobacterium butyriciproducens DSM 26588 in increasing the body weight of chicks infected with Eimeria tenella;

[0019] (C2) Use of Enterobacterium butyriciproducens (I. butyriciproducens) DSM 26588 in the preparation of a product for increasing the body weight of chicks infected with Eimeria tenella (E. tenella);

[0020] (C3) Use of Enterobacterium butyriciproducens DSM 26588 in reducing Eimeria tenella oocyst excretion in chicks infected with Eimeria tenella;

[0021] (C4) Use of Enterobacterium butyriciproducens DSM 26588 in the preparation of a product for reducing the amount of Eimeria tenella oocysts excreted in chicks infected with Eimeria tenella;

[0022] (C5) Use of Enterobacterium butyriciproducens DSM 26588 in reducing the severity of cecal lesions in chicks infected with Eimeria tenella;

[0023] (C6) Use of Enterobacterium butyriciproducens (I. butyriciproducens) DSM 26588 in the preparation of a product for reducing the severity of cecal lesions in chicks infected with Eimeria tenella (E. tenella).

[0024] In one or more embodiments, the application includes:

[0025] (D1) Use of Enterobacterium butyriciproducens (I. butyriciproducens) DSM 26588 in regulating the immune response against Eimeria tenella (E. tenella);

[0026] (D2) Use of Enterobacterium butyriciproducens (I. butyriciproducens) DSM 26588 in the preparation of a product for regulating the body's immune response against Eimeria tenella (E. tenella).

[0027] Preferably, the anti-Eimeria tenella (E. tenella) immune response comprises:

[0028] On the 5th to 7th day of Eimeria tenella infection, Enterobacter butyriciproducens DSM26588 increased the number of B lymphocytes in peripheral blood lymphocytes; increased CD8 + T cell number level; increase the secretion level of IFN-γ; reduce the secretion level of IL-17 on the 6th day of infection.

[0029] The second aspect of the present invention provides a probiotic or probiotic preparation for use against Eimeria tenella, comprising Enterobacter butyriciproducens (I. butyriciproducens) DSM 26588.

[0030] The third aspect of the present invention provides a probiotic composition for combating Eimeria tenella, comprising Enterobacter butyriciproducens (I. butyriciproducens) DSM 26588.

[0031] A fourth aspect of the present invention provides an animal feed or animal feed additive for resisting Eimeria tenella, comprising Enterobacter butyriciproducens (I. butyriciproducens) DSM 26588.

[0032] The beneficial effects of the present invention are:

[0033] In the present invention, an Eimeria tenella infection model induced by an antibiotic cocktail was used to explore the effects of Enterobacter butyriciproducens DSM 26588 on the development and infection of Eimeria tenella.

[0034] In vitro experiments have shown that the culture supernatant of I. butyriciproducens DSM 26588 can inhibit the invasion of Eimeria tenella sporozoites, of which 2'-O-methyladenosine is the main active ingredient. However, in cell models, the bacteria themselves cannot directly inhibit the invasion of sporozoites; I. butyriciproducens DSM 26588 has no significant effect on Eimeria tenella merozoites; I. butyriciproducens DSM 26588 can stimulate CD8 + T cells secrete high levels of IFN-γ.

[0035] In vivo experiments showed that I. butyriciproducens DSM 26588 could enhance the host immune response and increase the expression of B lymphocytes, CD8 + T cell number, increase IFN-γ secretion level, reduce IL-17 secretion level, and promote Th1 response.

[0036] Butyriciproducens (I.butyriciproducens) DSM 26588 inhibits the expression of the EtGFAT gene in Eimeria tenella gametes, thereby inhibiting gamete development; it improves intestinal lesions caused by Eimeria tenella infection, alleviates intestinal pathological damage, and reduces the excretion of Eimeria tenella oocysts. Its ACI value is 162, indicating excellent anticoccidial effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0038] Figure 1 This is a differential analysis diagram of the effects of different bacterial counts of I. butyriciproducens (BC) on E. tenella invading DF-1 cells using flow cytometry;

[0039] Figure 2 This is a differential analysis diagram of the effects of different concentrations of I. butyriciproducens culture supernatant (CS) on E. tenella invasion of DF-1 cells detected by flow cytometry;

[0040] Figure 3The pie chart shows the classification of metabolites detected in samples under positive and negative ion dual mode, where a represents the substances and their proportions detected in positive ion mode, including organic acids and derivatives, lipids and lipid molecules, organic heterocyclic compounds, organic oxygen compounds, benzene compounds, nucleosides, nucleotides and analogs, organic nitrogen compounds, phenylpropanoids and polyketides, alkaloids and derivatives, and lignin / neolignin-related compounds; b represents the substances and their proportions detected in negative ion mode, including lipids and lipid molecules, organic acids and their derivatives, nucleosides, nucleotides and analogs, organic heterocyclic compounds, organic oxygen compounds, benzene compounds, phenylpropanoids and polyketides, and organic nitrogen compounds;

[0041] Figure 4 The principal component analysis (PCA) quality control charts of samples in group IB and group AB under positive and negative ion dual mode; a is the PCA chart in positive ion mode, and b is the PCA chart in negative ion mode;

[0042] Figure 5 The effects of the top six differential metabolites at different concentration gradients on the invasion rate of E. tenella and their differential analysis, where a is ethyl laurate, b is 2'-O-methyladenosine, c is uridine diphosphate, d is inosine, e is deoxycholic acid, and f is guanosine;

[0043] Figure 6 This is a differential analysis of the effects of different bacterial numbers of I. butyriciproducens cells (BC) on merozoites;

[0044] Figure 7 This is a differential analysis of the effects of different concentrations of I. butyriciproducens culture supernatant (CS) on merozoites;

[0045] Figure 8 is the relative expression level of EtGFAT gene in each experimental group;

[0046] Figure 9 PCA score diagram of samples in each experimental group;

[0047] Figure 10 This is a cluster heat map of short-chain fatty acid content. The horizontal axis represents the sample group and number, and the vertical axis represents the type of short-chain fatty acids (including caproic acid, acetic acid, butyric acid, propionic acid, and valeric acid). The color gradient from red to blue reflects high to low content.

[0048] Figure 11 This is an analysis of the difference in the proportion of B lymphocytes in the peripheral blood lymphocytes of chicks in each experimental group on days 5-7 after E. tenella infection;

[0049] Figure 12 The CD4 counts in the peripheral blood lymphocytes of the chicks in each experimental group were 5-7 days after infection with E. tenella.+ Analysis of differences in the proportion of T lymphocytes;

[0050] Figure 13 The CD8 expression in peripheral blood lymphocytes of chicks in each experimental group was 5-7 days after infection with E. tenella. + Analysis of differences in the proportion of T lymphocytes;

[0051] Figure 14 The IFN-γ concentration and difference analysis of the serum of chicks in each experimental group on days 5-7 after E. tenella infection;

[0052] Figure 15 The IL-17 concentration and difference analysis of the serum of chicks in each experimental group on days 5-7 after E. tenella infection;

[0053] Figure 16 Flow cytometry was used to detect the effect of I. butyriciproducens on CD4 + The effect of T lymphocyte proliferation, where a is the flow cytometry contour map and b is the differential analysis map;

[0054] Figure 17 Flow cytometry was used to detect the effect of I. butyriciproducens on CD8 + The effect of T lymphocyte proliferation, where a is the flow cytometry contour map and b is the differential analysis map;

[0055] Figure 18 ELISA was used to detect the IFN-γ level in the co-culture supernatant, where a is I. butyriciproducens stimulated CD4 + The level of IFN-γ secreted by T cells, b is the level of IFN-γ secreted by I. butyriciproducens stimulating CD8 + IFN-γ levels secreted by T cells;

[0056] Figure 19 Representative images of cecal tissue from each experimental group were taken. DETAILED DESCRIPTION

[0057] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0058] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0059] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0060] In the following examples, Enterobacterium butyriciproducens (Intestinimonas butyriciproducens) DSM 26588 is referred to as I. butyriciproducens for short.

[0061] The Eimeria tenella strain used in this invention is maintained at the Engineering Research Center for Major Animal Disease Probiotic Vaccines (Drugs) of the Ministry of Education, rejuvenated every three months, and stored in a 2.5% (mass fraction) potassium dichromate solution (207802; Sigma) at 4°C. The strain exhibits good morphology and high virulence. The DF-1 cell line was purchased from ATCC (CRL-3586).

[0062] The present invention uses one-day-old healthy white-feathered broiler chickens purchased from Changchun Dehui Farm, and ensures free access to food and water throughout the entire breeding process. Before breeding, the house is fumigated with formaldehyde, and the metal cages are flame-sterilized to ensure that the environment is free of coccidia and other infectious pathogens. When animal tissues need to be isolated, they are first anesthetized by intraperitoneal injection of 50mg / kg of sodium pentobarbital, and then killed by cardiac air embolism.

[0063] Table 1 shows the main reagents used in the present invention.

[0064]

[0065] Anaerobic bacteria agar medium was purchased from Shandong Top Bioengineering Co., Ltd., product number: M5664.

[0066] Example 1

[0067] Experimental design:

[0068] 1.1 Three hundred one-day-old white-feathered broilers were randomly divided into six groups, with 50 chickens in each group, namely MOCK group, ABX group, CON group, FMT group, IB group (I. butyriciproducens supplementation group), and NaB group (sodium butyrate group).

[0069] MOCK group: No treatment was performed during the entire experiment, serving as the blank control group and FMT donor;

[0070] ABX group: Each chicken was infected with 5×10 4 E. tenella sporulated oocysts;

[0071] CON group: no other treatment, infected with the same number of E. tenella sporulated oocysts as the ABX group;

[0072] FMT group: From 1 day of age, oral gavage of antibiotic cocktail was performed for 9 consecutive days; at 10 days of age, microbiota transplantation was performed using the MOCK group as the donor for 5 consecutive days;

[0073] Group IB: Starting from the first day of age, oral administration of antibiotic cocktail was continued for 9 consecutive days; at the 10th day of age, I. butyriciproducens was supplemented for 5 consecutive days;

[0074] NaB group: oral administration of antibiotic cocktail for 9 consecutive days starting from 1 day of age; at 10 days of age, 1 g / L sodium butyrate was added to the drinking water for 5 consecutive days;

[0075] Except for the MOCK group, all other groups were infected with E. tenella.

[0076] Among them, antibiotic cocktail was prepared with sterile water, and the concentration and usage method are shown in Table 2.

[0077] Table 2 Preparation method of antibiotic cocktail

[0078]

[0079] After mixing the four antibiotics, the dosage is calculated based on the chicken's body weight and administered by gavage at a rate of 10 mL / kg body weight. The gavage system is adjusted with the chicken's age. Add 1 g / L of penicillin (metronidazole requires ultrasound-assisted dissolution) to the drinking water, and replace it each time. This is done twice daily for 14 consecutive days.

[0080] 1.2 Sporozoite purification and invasion assay

[0081] The purification of E. tenella sporozoites is divided into two parts: oocyst purification and sporozoite purification.

[0082] E. tenella oocyst purification steps are as follows: Aliquot E. tenella stored in 2.5% (mass fraction) potassium dichromate into 50 mL centrifuge tubes, 20 mL per tube. Add 25 mL of sterile distilled water to each tube and centrifuge at 4000 rpm for 10 minutes or more to completely remove the potassium dichromate. Resuspend the pellet in 2 volumes of 10% (mass fraction) sodium hypochlorite solution and vortex to mix thoroughly. Place on ice and let stand for 15 minutes. Fill the tube with sterile distilled water and centrifuge at 4000 rpm for 10 minutes. Resuspend the pellet in 3 volumes of 5% (mass fraction) sodium hypochlorite solution, three times the pellet volume, mix thoroughly, and centrifuge at 3000 rpm for 20 minutes. Aspirate the topmost white flocculent layer, add sterile distilled water, and centrifuge at 4000 rpm for 10 minutes three times. Collect the white pellet and suspend it in PBS to obtain the purified E. tenella oocysts.

[0083] The purification steps of E. tenella sporozoites are as follows:

[0084] Purified oocysts were placed in lysis tubes pre-filled with ceramic beads for oocyst disruption. After pre-cooling on ice for 10 minutes, the mixture was disrupted twice at 6000 rpm for 30 seconds. The resulting liquid mixture, containing the sporangia and broken oocyst walls, was collected and centrifuged twice at 2500 rpm for 10 minutes to remove most of the broken oocyst walls. 50 mL of a digestion solution containing 0.25% (mass fraction) pancreatic enzyme and 0.25% (mass fraction) sodium taurocholate was prepared and digested at 42°C for 15 minutes. The solution was centrifuged at 1500 rpm for 10 minutes, and cold PBS containing 1% FBS was added to terminate the digestion. The pellet was resuspended in PBS, made up to 20 mL, and filtered through a G3 sand core funnel. The filtrate was repeatedly pipetted and PBS was continuously added until no sporozoites were found under microscopic examination. The filtrate was centrifuged at 1500 rpm for 5 minutes to obtain purified sporozoites.

[0085] Flow cytometry was used to detect the effects of I. butyriciproducens bacteria (BC) and culture supernatant (CS) on the invasion of CFSE-labeled E. tenella into DF-1 cells. 600 = 1, the number of cells after centrifugation was defined as 100% BC, and the OD 600 = 1, the 1 mL culture supernatant after centrifugation was 100% CS, and BC or CS was mixed with DMEM medium containing 10% FBS (without penicillin-streptomycin) at a concentration gradient of 50%, 20%, and 10% for testing. DF-1 cells were cultured in a 24-well culture plate until 80% of the cells were grown, and then 5×10 cells were added to each well.5 CFSE-labeled sporozoites were mixed with varying concentrations of BC and CS and incubated at 37°C for 12 hours. Cells were then harvested and analyzed by flow cytometry. CFSE-positive cells were considered invasive, and the proportion of CFSE-positive cells was the invasion rate of E. tenella.

[0086] 1.3 E. tenella merozoite purification and flow cytometry detection:

[0087] 14-day-old healthy chicks were orally infected with E. tenella 1×10 5 On the fifth day after infection, five E. tenella sporulated oocysts were taken and killed, and the cecum wall was longitudinally cut open in a clean bench. The cecum tissue was washed more than three times with PBS containing 2% (volume fraction) penicillin-streptomycin to remove the contents.

[0088] Use a sterile glass slide to scrape the cecal mucosal tissue, mix it with an appropriate amount of PBS, and divide it into lysis tubes pre-filled with ceramic beads for homogenization at 3500 rpm for 45 seconds; after homogenization, centrifuge at 3000 rpm for 10 minutes;

[0089] Take the pellet after centrifugation, add five times the volume of red blood cell lysis buffer, mix well, lyse at 4°C for 3 minutes, add PBS to stop lysis, centrifuge at 3000 rpm for 10 minutes; repeat lysis twice;

[0090] Prepare 30% Percoll separation solution with sterile PBS and evenly disperse the precipitate after centrifugation in the previous step to obtain a 30% Percoll suspension containing merozoites;

[0091] Prepare 50% Percoll separation solution with sterile PBS and add 1 mL to the bottom of a 15 mL centrifuge tube;

[0092] Slowly add the 30% Percoll suspension containing merozoites onto the top of the 50% Percoll separation solution, 5 mL per tube, 3000 rpm, and centrifuge for 10 minutes;

[0093] The 50% Percoll solution layer after centrifugation was aspirated into a new centrifuge tube, and 10 times the volume of sterile PBS was added for washing. The tube was centrifuged at 3000 r / min for 10 min. The precipitate was collected, which was the purified merozoite.

[0094] The purified merozoites were co-cultured with bacterial supernatants, labeled with 7-AAD, and detected by flow cytometry.

[0095] 1.4 Extraction of total RNA from cecum:

[0096] Place an equal mass of soybean-sized cecal tissue into a lysis tube pre-filled with ceramic beads, add 1 mL of RNAisoPlus and homogenize at 4500 rpm for 30 seconds, repeat twice to complete the tissue homogenization;

[0097] After the sample was homogenized, it was allowed to stand at room temperature for 5 min and then centrifuged at 12,000 × g for 5 min at 4°C;

[0098] The supernatant was transferred to a 1.5 mL tube, and 200 μL of chloroform was added immediately, vortexed to mix, allowed to stand at room temperature for 5 min, and centrifuged at 12,000 × g for 15 min at 4°C.

[0099] Carefully aspirate the supernatant and transfer it to a 1.5 mL tube. Add 500 μL of isopropanol and mix by inversion. Let it stand at room temperature for 5 min and centrifuge at 12,000 × g for 10 min at 4°C.

[0100] Prepare 75% ethanol with DEPC water and add 1 mL to each sample. Centrifuge at 7500 × g for 5 min at 4°C.

[0101] Carefully remove the supernatant, leaving the 1.5 mL centrifuge tube cap open, dry at room temperature, and add 50 μL of DEPC water to dissolve the RNA. Store the RNA at -80°C until needed.

[0102] 1.5 cDNA synthesis:

[0103] cDNA synthesis was performed using a Promega reverse transcription kit. Following the instructions, 4 μL of DEPC water, 4 μL of buffer containing random primers, and 2 μL of premixed enzyme were mixed to create a 10 μL reverse transcription system. 10 μL of RNA was added to the system, bringing the final volume to 20 μL. The PCR reaction program was as follows: 25°C for 5 minutes, one cycle; 42°C for 1 hour, one cycle; 95°C for 5 minutes, one cycle; and 4°C for completion. The resulting cDNA was stored at -20°C until further use.

[0104] 1.6qPCR:

[0105] qPCR was performed using the SYBR Green dye method, and the reaction system is shown in Table 3 below.

[0106] Table 3 qPCR reaction system

[0107]

[0108] The qPCR reaction setup procedure is as follows:

[0109] (1) Pre-denaturation: 95°C for 2 min; (2) Denaturation: 95°C for 15 s; (3) Annealing / extension: 60°C for 15–30 s; (4) Repeat steps 2 and 3 for a total of 40 cycles; (5) Melting curve analysis: 95°C for 15 s, 60°C for 15 s, and 95°C for 15 s.

[0110] The primers used for qPCR are shown in Table 4 below.

[0111] Table 4 Primers used in qPCR

[0112]

[0113] 1.7 Isolation of chicken peripheral blood lymphocytes:

[0114] Peripheral blood lymphocytes were isolated using the Chicken Peripheral Blood Lymphocyte Kit as follows:

[0115] Use a 1 mL syringe to collect chicken heart blood and inject it into a sodium heparin anticoagulant tube;

[0116] Add whole blood diluent to dilute the blood to 3 mL;

[0117] Equilibrate the separation solution from 4°C to room temperature, and add 3 mL of separation solution to a 15 mL centrifuge tube;

[0118] Slowly add 3 mL of whole blood dilution to the upper layer of separation solution, centrifuge at 500 × g, speed 0, and centrifuge for 30 min;

[0119] Carefully aspirate the middle lymphocyte layer into a new centrifuge tube, add 5 volumes of PBS, and centrifuge at 500 × g for 10 min.

[0120] The white precipitate obtained after centrifugation is the peripheral blood lymphocytes.

[0121] 1.8 Flow cytometry:

[0122] Cell counting: Dilute lymphocytes 50-fold with PBS, mix well, take 10 μL and count on a red blood cell counting plate, with 1×10 per sample. 6 The number of cells was added to the flow tube, and the sample system was 1×10 6 cells / 100 μL, two tubes per sample (T cells and B cells are labeled separately), and stored at 4°C until ready to use;

[0123] Antibody dilution: dilute the antibody with sterile PBS at a dilution factor of 40;

[0124] Cell typing: All samples were divided into two groups. For T cell typing, CD3-FITC, CD4-APC, and CD8α-PE antibodies were diluted and labeled with 10 μL of the diluted solution per sample. For B cell labeling, the diluted Bu-1 antibody was added to the sample and labeled with 10 μL of the diluted solution per sample. Incubate at 4°C for 30 minutes. After incubation, add 1 mL of PBS and centrifuge at 500 × g for 10 minutes to remove the antibodies. After washing, add PBS to each sample to bring the volume up to 500 μL for analysis.

[0125] 1.9 Co-culture of I. butyriciproducens and lymphocytes

[0126] CD4 - 、CD8 - Cell complex sorting: operate under sterile conditions, take the spleen of a 14-day-old healthy chicken, place it in a 200-mesh cell sieve, and grind it with the stopper part of a 5mL syringe to prepare a spleen cell suspension. Use a chicken spleen lymphocyte separation kit to separate lymphocytes. According to the instructions, the spleen cell suspension is centrifuged and washed, and tissue diluent is added to a fixed volume of 3mL. Similar to the peripheral blood lymphocyte separation steps, 3mL of diluted spleen cell diluent is slowly added to the top of the 3mL separation solution, 500×g, acceleration and deceleration 0, centrifugation for 30min. In order to retain antigen-presenting cells, after centrifugation, it is necessary to carefully aspirate the middle lymphocyte layer and the separation liquid layer below it, add 10 times the volume of PBS to wash away the separation liquid components, and finally obtain the spleen monocyte complex. After labeling with CD4-APC and CD8α-PE antibodies, 1×10 6 CD4 - 、CD8 - of cell complexes.

[0127] Co-culture: The two cell complexes were mixed with 1 mL of DMEM medium containing 10% FBS, and inoculated into 24-well plates, with 100 μL per well. The number of bacteria was 2 × 10 9 CFU of I. butyriciproducens cells were mixed with 18 mL of DMEM medium containing only 10% FBS (without penicillin / streptomycin), and 900 μL was inoculated into each well (1 mL per well). The cells were cultured at 37°C and 5% CO₂ for 48 hours. After the incubation period, the cells and supernatant were harvested. The cells were analyzed by flow cytometry, and the supernatant was centrifuged at 12,000 rpm for 10 minutes and then used for ELISA to measure IFN-γ secretion.

[0128] 1.10ELISA

[0129] Freshly collected blood was allowed to stand at room temperature for 10 minutes, then incubated at 37°C for 1 hour, at 4000 rpm for 15 minutes, and the supernatant was collected and stored at -80°C. According to the kit instructions, the kit was equilibrated to room temperature 30 minutes in advance. During this time, the standards were prepared using a progressive dilution method, along with the biotinylated antibody, SABC complex working solution, and mixed TMB solutions A and B.

[0130] Add 50 μL of standard or sample diluent to the blank well, and 50 μL of standard or sample to be tested to each of the remaining wells. Apply sealing tape, mix the reaction plate, and incubate at 37°C for 1 hour. Wash the reaction plate three times with 1× wash buffer and blot dry with filter paper. Add 100 μL of biotinylated antibody diluent to the blank well, and 100 μL of 1× biotinylated antibody working solution to each of the remaining wells. Apply sealing tape, mix, and incubate at 37°C for 1 hour. Repeat the washing process, add 100 μL of SABC complex working solution to each well, apply sealing tape, mix, and incubate at 37°C for 30 minutes. After washing, add 100 μL of TMB mixture to each well, mix, apply sealing tape, and incubate at 37°C in the dark for 20 minutes. Add 50 μL of stop solution to each well, mix, and measure absorbance at 450 nm using a microplate reader.

[0131] 1.11 Component detection of I. butyriciproducens culture supernatant and verification of its invasion inhibition effect:

[0132] I. butyriciproducens was inoculated at a volume ratio of 1:100 into three Hungate tubes pre-filled with 10 mL of anaerobic agar medium. The tubes were then incubated in an anaerobic incubator for 48 hours, along with uninoculated anaerobic agar medium. Following incubation, the cells were separated from the culture supernatant at 10,000 × g for 10 minutes. After centrifugation, the supernatant was filtered through a 0.22 μm filter and sterilized again. The supernatant was collected to obtain three samples each for the culture supernatant group (IB) and the anaerobic agar group (AB). The samples were snap-frozen in liquid nitrogen and transported on dry ice to Beijing Novogene Technology Co., Ltd. for nontargeted metabolomics analysis.

[0133] Evaluation method for suppressing invasion effect:

[0134] The invasion rate of E. tenella sporozoites was reflected by measuring the percentage of CFSE-positive cells. 5 DF-1 cells were seeded into 48-well cell culture plates at a seeding density of 100 cells / well. DMEM containing 10% FBS (mass fraction) and 1% (mass fraction) penicillin-streptomycin was used to prepare an antibiotic cocktail of varying concentrations. When the cells reached approximately 80% growth, the original DMEM was replaced.

[0135] Purified E. tenella sporozoites were labeled with 5 μM CFSE and incubated at 37°C in a water bath for 20 minutes. An equal number of sporozoites were added to each well and incubated at 37°C in 5% CO2 for 12 hours.

[0136] After the co-culture, the culture medium components were washed away with PBS, and the cells were digested with 0.25% (mass fraction) trypsin. The cells were collected into 5 mL polystyrene tubes and detected by flow cytometry.

[0137] After analyzing the supernatant components, a concentration gradient of 1 mM, 500 μM, 200 μM, 100 μM, 50 μM, 20 μM, 10 μM, and 0 μM was set and co-cultured with DF-1 cells and CFSE-labeled sporozoites for 12 h. The proportion of CFSE-positive cells was detected by flow cytometry.

[0138] 1.12 Quantitative detection of short-chain fatty acids based on GC-MS:

[0139] On day 6 after E. tenella infection, chickens were anesthetized by intraperitoneal injection of 50 mg / kg sodium pentobarbital. After anesthesia, they were sacrificed by cardiac air embolism, with a 5 mL injection of air into the heart. After surface disinfection with alcohol, the abdominal cavity was opened in a cleanroom, and the cecal tissue was sharply dissected. The cecal contents were collected and placed in sterile, enzyme-free cryopreservation tubes, snap-frozen in liquid nitrogen, and transported on dry ice to Suzhou Panomik Biopharmaceutical Technology Co., Ltd. for testing.

[0140] 1.13 Relative weight gain rate

[0141] The weight of the chicks was recorded, and the difference in weight before and after E. tenella infection was calculated, which was the average weight gain (g). The relative weight gain rate was the ratio of the average weight gain of each experimental group to the average weight gain of the blank control group, and the results were expressed as a percentage.

[0142] 1.14 Oocyst value

[0143] Calculation method of oocyst excretion:

[0144] Oocyst count per gram (OPG): During the oocyst excretion period of E. tenella (7 dpi), feces were randomly sampled at five points in each group. After weighing, the feces were mixed and ground with PBS within the group. The samples were counted on a hemocytometer to obtain the final number of oocysts per gram (OPG).

[0145] The oocyst reduction rate is the difference between the oocyst counts in the conventional infection group (CON) and each experimental group, then the ratio compared to the MOCK group. The results are expressed as a percentage. The oocyst reduction rate is divided into five ranges based on the oocyst ratio. When the oocyst ratio is less than 1%, the oocyst reduction rate is 0; when the oocyst ratio is between 1% and 25%, the oocyst reduction rate is 5; when the oocyst ratio is between 26% and 50%, the oocyst reduction rate is 10; when the oocyst ratio is between 51% and 75%, the oocyst reduction rate is 20; and when the oocyst ratio is between 76% and 100%, the oocyst reduction rate is 40.

[0146] 1.15 Cecal lesion scoring and lesion values

[0147] Scoring of varying degrees of lesions after coccidia infection: 0 points for no obvious lesions in the cecal tissue and normal-colored contents; 1 point for no thickening of the cecal wall, only a few bleeding spots, and normal-colored contents; 2 points for thickening of the cecal wall, numerous bleeding spots, and a small amount of bloody stool; 3 points for significant thickening of the cecal wall, severe deformation and shrinkage, and the presence of obvious blood clots and white masses; and 4 points for extreme thickening of the cecal wall, severe atrophy, and the intestinal cavity filled with numerous blood clots, white masses, and cheesy material. The lesion score is set at 10 times the lesion score.

[0148] 1.16 Anticoccidial Index (ACI)

[0149] The anticoccidial effect is primarily measured through the anticoccidial index (ACI). The ACI is calculated by subtracting the sum of the lesion and oocyst counts from the sum of the relative weight gain and survival rate. The ACI is divided into four ranges, corresponding to four different levels of anticoccidial effect: an ACI value less than 120 indicates no anticoccidial effect; an ACI value between 120 and 160 indicates moderate anticoccidial effect; an ACI value between 160 and 180 indicates good anticoccidial effect; and an ACI value greater than 180 indicates excellent anticoccidial effect.

[0150] 1.17 Data Analysis

[0151] GraphPad Prism 9 software was used to analyze data differences and plot graphs. One-way ANOVA and T test were used to analyze the differences between the data (ns, P>0.05; *, P<0.05; **, P<0.01; ***, P<0.001, ****, P<0.0001).

[0152] result:

[0153] (1) Effects of I. butyriciproducens on E. tenella invasion

[0154] The results of flow cytometry showed that I. butyriciproducens did not directly inhibit the invasion of E. tenella ( Figure 1 ).

[0155] The culture supernatant of I. butyriciproducens has a good effect in inhibiting the invasion of E. tenella. The results showed that adding different concentrations of the culture supernatant of I. butyriciproducens can reduce the invasion rate of E. tenella to about 1%. This inhibitory effect is dependent on the concentration of the culture supernatant to a certain extent. As the concentration of the culture supernatant decreases, the inhibitory effect on E. tenella invasion also decreases significantly. Figure 2 ).

[0156] (2) Analysis of components in the culture supernatant of I. butyriciproducens and screening of components that inhibit E. tenella invasion:

[0157] Non-targeted metabolomics analysis of I. butyriciproducens culture supernatant components:

[0158] In the cationic mode, a total of 517 metabolites were identified; in the anionic mode, a total of 314 metabolites were identified; organic acids and their derivatives, lipids and lipid molecules accounted for the highest proportion, accounting for 53.1% and 58.65% of all metabolites identified in their respective modes. Figure 3 ).

[0159] The results of principal component analysis showed that the distribution of QC samples was relatively clustered, with small differences and high data quality ( Figure 4 ).

[0160] The components of anaerobic bacteria agar medium (AB) and the culture supernatant components of I. butyriciproducens (IB) were differentially analyzed. The IB vs. AB comparison pair was set to screen significantly upregulated metabolites. The thresholds were set as VIP>1.0 and FC>1.2, and P<0.05 to screen the main metabolites produced after IB culture (Table 5).

[0161] Table 5 Metabolite differential screening results

[0162]

[0163] Nine major categories of substances were screened from the IB vs. AB comparison, encompassing a total of 70 differential metabolites. Among these, 19 organic acids accounted for the largest proportion, at 27.14%, followed by 10 lipids, at 14.29%, 8 nucleosides and 8 organic heterocycles, each accounting for 11.43%, 7 organic oxygen compounds, at 10%, 1 benzene, 1 alkaloid, and 1 organic nitrogen compound, each accounting for 1.43%, and 15 unclassified compounds, accounting for 21.43%.

[0164] For further screening, invalid mass metabolites were removed and the threshold was set to FC ≥ 2. Finally, 12 metabolites were obtained (Table 6). They were ranked by FC, P value, and VIP value, and the top 5 in each order were selected. Finally, a total of 6 differential metabolites were obtained: lauric acid ethyl ester, 2'-O-methyladenosine, uridine diphosphate (UDP), inosine, deoxycholic acid, and guanosine.

[0165] Table 6 Metabolite differential screening results

[0166]

[0167] Effects of six metabolites including lauric acid ethyl ester on E. tenella invasion:

[0168] The effects of 6 metabolites at concentrations of 1 mM, 500 μM, 200 μM, 100 μM, 50 μM, 20 μM, 10 μM, and 0 μM on E. tenella invasion were evaluated using the DF-1 invasion model. Figure 5 The results showed that uridine diphosphate and deoxycholic acid had no effect on sporozoite invasion; 50 μM ethyl laurate promoted sporozoite invasion (P < 0.01); 20 μM inosine significantly promoted sporozoite invasion (P < 0.01); 1 mM and 500 μM guanosine significantly inhibited sporozoite invasion (P < 0.0001), and 200 μM and 100 μM guanosine also inhibited sporozoite invasion (P < 0.05). Among the six metabolites, only 2'-O-methyladenosine had a significant inhibitory effect on E. tenella sporozoite invasion at all eight concentration gradients (P < 0.0001). Taken together, these results demonstrate that 2'-O-methyladenosine is the key component in the culture supernatant of I. butyriciproducens that inhibits E. tenella invasion.

[0169] (3) Effects of I. butyriciproducens on E. tenella merozoites

[0170] I. butyriciproducens bacteria and culture supernatant were co-cultured with purified E. tenella merozoites for 12 hours. The proportion of necrotic merozoites was detected by flow cytometry to study the direct effect of I. butyriciproducens on E. tenella merozoites. The results showed that I. butyriciproducens bacteria had no significant effect on E. tenella merozoites (P>0.05) ( Figure 6 ), and the culture supernatant of I. butyriciproducens had no significant effect on the merozoites of E. tenella (P>0.05) ( Figure 7 ).

[0171] (4) Effect of I. butyriciproducens on EtGFAT gene expression

[0172] The relative expression levels of EtGFAT gene in cecal tissue of each group on the sixth day after infection with E. tenella were detected. The results showed that the expression level of EtGFAT gene in the ABX group was significantly lower than that in the CON group (P < 0.0001). After treatment with the antibiotic cocktail, supplementation with I. butyriciproducens could downregulate the expression level of EtGFAT gene, but there was no statistical difference compared with the ABX group. Compared with the CON group, the expression level of EtGFAT gene in the I. butyriciproducens supplementation group was significantly lower than that in the CON group and the FMT group (P < 0.0001) ( Figure 8 ).

[0173] (5) Effect of I. butyriciproducens on the content of short-chain fatty acids in the cecum:

[0174] Considering the butyric acid production characteristics of I. butyriciproducens, the experiment detected the contents of isobutyric acid, propionic acid, acetic acid, butyric acid, valeric acid, isovaleric acid and hexanoic acid in the cecum of the CON group, ABX group, FMT group and I. butyriciproducens supplementation group at 6 dpi.

[0175] Quantitative analysis of short-chain fatty acids in the cecum revealed that valeric acid and caproic acid were undetectable in the ABX group, while the levels of isobutyric acid, propionic acid, acetic acid, butyric acid, and isovaleric acid were all lower in the cecum than in the other experimental groups. The I. butyriciproducens supplementation group had higher butyric acid levels in the cecum than the other experimental groups, demonstrating the superior butyric acid production of I. butyriciproducens. Quantitative results revealed that both fecal microbiota transplantation and single-strain I. butyriciproducens supplementation restored the levels of short-chain fatty acids in the cecum to a certain level (Table 7).

[0176] Table 7 Contents of 7 short-chain fatty acids in each group of samples

[0177]

[0178] The principal component analysis (PCA) of the metabolites in each group showed that there were significant differences among the ABX group, CON group, FMT group, and I. butyriciproducens supplementation group ( Figure 9 ).

[0179] The results of differential analysis and clustering of metabolites in each group showed that the contents of acetic acid, propionic acid, butyric acid, valeric acid and hexanoic acid in the ABX group were significantly lower than those in the other groups ( Figure 10 ).

[0180] (6) Effects of I. butyriciproducens on host immunity in the late stages of E. tenella infection

[0181] Peripheral blood B lymphocyte count detection:

[0182] On the 5th day after infection with E. tenella, the number of B lymphocytes in the I. butyrici producens complement group was the highest among all experimental groups, significantly higher than that in the NaB group (P < 0.001), the MOCK group (P < 0.01), and the ABX group (P < 0.0001); on the 6th day after infection with E. tenella, the number of B lymphocytes in the I. butyrici producens complement group remained high, significantly higher than that in the MOCK group (P < 0.01); on the 7th day after infection with E. tenella, the number of B lymphocytes in the I. butyrici producens complement group was the highest, significantly higher than that in the NaB group (P < 0.0001), the MOCK group (P < 0.01), and the CON group (P < 0.05) ( Figure 11 ).

[0183] Peripheral blood T lymphocyte count detection:

[0184] The peripheral blood CD4+ T lymphocyte count level ( Figure 12 The results showed that the peripheral blood CD4 + The number of T cells in the I. butyriciproducens replenishment group was significantly higher than that in the ABX group (P < 0.0001) and the NaB group (P < 0.001). + The number of T lymphocytes decreased, significantly lower than that of CON group and FMT group, and significantly higher than that of ABX group and NaB group (P<0.0001); on the 7th day after infection, the CD4 + The number of T lymphocytes decreased and was significantly lower than that in the CON group (P < 0.001) and the FMT group (P < 0.000).

[0185] The peripheral blood CD8 + T lymphocyte count level ( Figure 13 ), the results showed that on the 5th day after infection, the CD8 + The number of T lymphocytes was significantly higher than that in the NaB group (P < 0.0001) and the ABX group (P < 0.0001), and was at the highest level. On the 6th day after infection, the peripheral blood CD8 + The number of T lymphocytes in the CON group was significantly higher than that in the CON group (P<0.05); on the 7th day after infection, the peripheral blood CD8 + The number of T lymphocytes remained the highest in all experimental groups, significantly higher than that in the ABX group (P < 0.0001) and the NaB group (P < 0.01). In summary, I. butyriciproducens can regulate the host's immune function during the sexual reproduction period of E. tenella.

[0186] Detection of IFN-γ and IL-17 secretion levels

[0187] The secretion levels of IFN-γ and IL-17 in serum from the 5th to the 7th day after E. tenella infection were analyzed. The results showed that the secretion levels of IFN-γ in the I. butyriciproducens complement group were significantly higher than those in the CON group (P < 0.001, P < 0.0001, P < 0.05) and the ABX group (P < 0.0001, P < 0.0001, P < 0.01) from the 5th to the 7th day after E. tenella infection. Figure 14 ).

[0188] The results of serum IL-17 secretion level test showed that ( Figure 15 ), on the 5th day after E. tenella infection, the IL-17 secretion level of the CON group was significantly lower than that of the ABX group and the I. butyriciproducens complement group (P < 0.0001), and there was no significant difference between the ABX group and the I. butyriciproducens complement group; on the 6th day after E. tenella infection, the IL-17 secretion level of the CON group was significantly higher than that of the ABX group (P < 0.0001) and the I. butyriciproducens complement group (P < 0.001); on the 7th day after E. tenella infection, the IL-17 secretion level of the I. butyriciproducens complement group was significantly higher than that of the ABX group and the CON group (P < 0.0001), and the CON group was significantly higher than the ABX group (P < 0.001).

[0189] I. butyriciproducens and CD4 - 、CD8 - Cell complexes were co-cultured and CD4 + T, CD8 + T cell numbers, the results showed that I. butyriciproducens stimulated CD4 + The effect of T proliferation was not significant ( Figure 16 ), stimulate CD8 + The effect of T proliferation was significant (P<0.001) ( Figure 17 ).

[0190] Detection of IFN-γ in the cell supernatant of the co-culture system ( Figure 18 ), the results showed that I.butyriciproducens and CD4 - 、CD8 - When the complex is co-cultured, it can stimulate CD8 + T cells secrete high levels of IFN-γ and CD8 + There was a significant difference compared with the T single culture group (P<0.001); I. butyriciproducens could not stimulate CD4 + T secretes high levels of IFN-γ and CD4 + Compared with the T alone culture group, the IFN-γ level in the cell supernatant was decreased (P<0.05).

[0191] (7) Evaluation of anticoccidial effect

[0192] Weight changes:

[0193] The body weights of 10 chicks in each group were recorded before infection with E. tenella (14 days old) and 7 days after infection with E. tenella (21 days old). The results showed that the average weight gain of the FMT group, the I. butyriciproducens supplementation group, and the NaB group was significantly higher than that of the CON group (P < 0.01, P < 0.001) (Table 8).

[0194] Table 8 Body weight changes in each group

[0195]

[0196] Number of oocysts discharged:

[0197] After each group was infected with E. tenella, the number of oocysts in feces was recorded and the number of oocyst reduction was calculated. The results showed that the oocyst reduction rates were highest in the I. butyriciproducens supplementation group and the NaB group, which were 42.45% and 33.05%, respectively; the oocyst ratios were 57.5% and 66.9%, respectively, and the oocyst value was 20 (Table 9).

[0198] Table 9 Oocyst discharge

[0199]

[0200] Cecal lesion scoring:

[0201] The macroscopic pathological changes of cecal tissue on the 7th day after infection with E. tenella showed that ( Figure 19 The I. butyriciproducens-filled group had a longer cecum, significantly thinner intestinal walls, and no obvious blood clots in the intestines compared to the CON group. The overall severity of lesions was significantly lower in the NaB group. The cecum in the NaB group was slightly wrinkled, with slightly thinner intestinal walls and firmer intestinal contents than in the CON group. The overall severity of lesions was lower in the CON group but higher in the I. butyriciproducens-filled group. Cecal lesion scoring showed that the I. butyriciproducens-filled group had the lowest lesion severity among all infection groups, with a lesion score of 16 (Table 10).

[0202] Table 10 Lesion score and lesion value

[0203]

[0204] Anticoccidial index

[0205] The ACI index was calculated based on the relative weight gain, survival rate, oocyst count, and lesion count of each chick group. NaB exhibited a moderate anticoccidial effect, with an ACI value of 149.7, slightly lower than that of the I. butyriciproducens supplementation group, which showed moderate anticoccidial efficacy. The I. butyriciproducens supplementation group had an ACI value of 162, indicating excellent anticoccidial efficacy (Table 11).

[0206] Table 11 Anticoccidial Index

[0207]

[0208] Results and Discussion:

[0209] In the present invention, the effects of I. butyriciproducens on E. tenella were verified at various stages of E. tenella infection. It was first discovered that the metabolites of I. butyriciproducens effectively inhibited the invasion of E. tenella sporozoites, and the active ingredient was 2'-O-methyladenosine. 2'-O-methyladenosine is a nucleoside analogue, and nucleoside analogues have been widely used as antiviral infections. In addition, since protozoa and worms lack the initial synthesis pathway of purines and cannot synthesize purines themselves, they must rely entirely on salvage pathways, that is, rely on bases and nucleosides that are abundant in the host body to adapt to the purine synthesis pathway, such as trypanosomes, plasmodium and toxoplasma. Nucleoside analogues are also considered to be promising anti-trypanosomal agents. In the present invention, 2'-O-methyladenosine inhibited the invasion of E. tenella sporozoites, which may have interfered with the salvage pathway of nucleotides in the parasite, thereby preventing the synthesis of nucleic acids in the parasite. The specific mechanism of action still needs further research.

[0210] In addition, I. butyriciproducens cells and culture supernatant were directly exposed to merozoites. The effect was evaluated using the Annexin V-PE / 7-AAD apoptosis assay, which showed that I. butyriciproducens had no significant effect on E. tenella merozoites. Given the lack of an effective in vitro merozoite culture model, a more convincing study of the transition from asexual to sexual development is impossible. Whether this result is also related to the salvage pathway of interfering nucleotides requires further investigation.

[0211] After preliminary in vitro studies demonstrated that I. butyriciproducens had no significant effect on sporozoites and merozoites, we further confirmed in animal studies that I. butyriciproducens could inhibit the expression of the EtGFAT gene in E. tenella gametes. The enzyme encoded by the GFAT gene is a key, rate-limiting enzyme in the hexosamine biosynthetic pathway (HBP). It catalyzes the conversion of glutamine and fructose-6-phosphate into glutamine-6-phosphate and fructose-6-phosphate, the first step in the synthesis of UDP-GlcNAc (uridine diphosphate N-acetylglucosamine). UDP-GlcNAc is not only an important substrate for protein glycosylation but also participates in nucleotide metabolism, thus closely linking GFAT to nucleotide metabolism. Furthermore, HBP activity can be affected by nutritional status and cell signaling, thereby influencing the overall metabolic state of the cell, including nucleotide metabolism. The EtGFAT gene is specific to E. tenella megagametes, implicated in their structure and development, and is a key gene for oocyst synthesis. After complementation with I. butyriciproducens, the EtGFAT gene was significantly downregulated, proving that the gamete amino sugar and nucleotide sugar metabolism was directly affected and the gamete's own metabolic pathway was inhibited, which favorably demonstrated the inhibitory effect of the bacterium on the E. tenella gamete development process.

[0212] I. butyriciproducens is a leading candidate for the development of next-generation probiotics. It can not only ferment sugars such as glucose, galactose, and arabinose into butyrate, the main short-chain fatty acid, but can also produce butyrate from lysine and even glycated lysine. These two pathways are also the main metabolic pathways for I. butyriciproducens to produce butyrate. Butyrate is one of the most important short-chain fatty acids for intestinal health. It is the main energy source for colonocytes, promoting their growth and maintaining the integrity of the intestinal barrier. It plays a role in gene expression regulation, apoptosis (programmed cell death), and mucin production. Studies have shown that sodium butyrate can improve the disorder of cecal flora after E. tenella infection, increase the weight and feed intake of chickens, and promote the restoration of intestinal homeostasis after infection. To compare the anti-E. tenella efficacy of I. butyriciproducens and sodium butyrate, the anti-coccidial index was calculated based on E. tenella oocyst excretion, cecal lesion counts, chick mortality, and relative weight gain. This study confirmed that I. butyriciproducens exhibited significantly greater anti-coccidial efficacy than sodium butyrate, indicating that supplementation with live bacteria exerted a greater anti-E. tenella effect than a single metabolite. Bacteria can exert a wide range of effects on host metabolic health through the production of multiple metabolites. Butyric acid may not be the optimal primary component for anti-E. tenella efficacy in this study.

[0213] After probiotics are replenished, they also have a certain regulatory effect on the immune system. As one of the widely used probiotics, Clostridium butyricum (C. butyiricum) has been shown to stimulate immune responses and activate nonspecific immunity. Studies have also shown that the combination of Clostridium butyricum and coccidia vaccines can significantly improve the production performance of broilers, significantly reduce OPG, and reduce intestinal lesions and alleviate infection. C. butyiricum and I. butyriciproducens belong to the order Eubacteriales under the class Clostridia. They are evolutionarily related, but this does not mean that their physiological functions are exactly the same. There is no relevant research on I. butyriciproducens regulating the immune system. Based on the above reasons, we evaluated the effect of I. butyriciproducens in regulating the body's anti-E. tenella immune response.

[0214] Immunity against E.tenella depends on the Th1 immune response, and the host's T cell immunity is dominant after infection. Cellular immunity can provide the host with protection against E.tenella infection, but humoral immunity cannot be ignored. IFN-γ is the main cytokine with anti-coccidial effect. Recombinant IFN-γ can significantly increase body weight and reduce the amount of oocysts discharged. Relevant test results in the late stage of E.tenella infection showed that I.butyriciproducens can show a positive regulatory effect on the host immune system during both the gamete reproduction period and the oocyst discharge period. I.butyriciproducens can significantly increase the number of B cells and upregulate the host CD8 + It can also stimulate CD8+ T cells to secrete high levels of IFN-γ.

[0215] In addition to IFN-γ-mediated Th1 cell responses to E. tenella, the role of IL-17 cannot be ignored. As a hallmark cytokine of Th17 cells, its biological activities include recruiting neutrophils, stimulating the production of antimicrobial peptides (such as β-defensins and mucins), and inducing cytokines and chemokines. Due to the lack of immune reagents, the precise role of Th17 cells in chickens is poorly understood. Studies have shown that IL-17 isolated from E. imeria-infected IELs plays a proinflammatory role in coccidiosis, and treatment with IL-17 or IL-17F can induce the expression of proinflammatory cytokines in chicken fibroblasts. Dynamic changes in IL-17 secretion levels in the I. butyriciproducens supplementation and control groups on days 5-7 after E. tenella infection indicate an initial increase followed by a decrease, demonstrating the dynamic regulatory effect of I. butyriciproducens on proinflammatory responses.

Claims

1. Use of Enterobacterium butyriciproducens DSM 26588 as a probiotic against Eimeria tenella.

2. The use according to claim 1, characterized in that The applications include: (A1) Use of the culture supernatant of Enterobacter butyriciproducens (I. butyriciproducens) DSM 26588 in inhibiting invasion of Eimeria tenella sporozoites; (A2) Use of the culture supernatant of Enterobacterium butyriciproducens (I. butyriciproducens) DSM 26588 in the preparation of a product for inhibiting the invasion of Eimeria tenella sporozoites.

3. The use according to claim 2, characterized in that The culture supernatant of the Enterobacterium butyriciproducens (I. butyriciproducens) DSM 26588 contains 2'-O-methyladenosine.

4. The use according to claim 2, characterized in that Cultivating Enterobacter butyriciproducens (I. butyriciproducens) DSM 26588 to a stationary phase, and centrifuging to obtain a culture supernatant of the Bacillus subtilis; Preferably, the butyric acid producing Enterobacter (I.butyriciproducens) DSM 26588 is inoculated into the anaerobic agar medium at a volume ratio of 1:80-120, and anaerobically cultured for 40-50 hours until the butyric acid producing Enterobacter (I.butyriciproducens) DSM 26588 reaches the plateau OD 600 The pH value was 0.8 to 1.2, and the mixture was centrifuged to obtain the culture supernatant of the butyric acid-producing Enterobacterium (I. butyriciproducens) DSM 26588.

5. The use according to claim 1, characterized in that The applications include: (B1) Use of Enterobacterium butyriciproducens DSM 26588 in inhibiting the expression of the EtGFAT gene in Eimeria tenella gametes; (B2) Use of Enterobacterium butyriciproducens DSM 26588 in the preparation of a product for inhibiting the expression of the EtGFAT gene in Eimeria tenella gametes.

6. The use according to claim 1, wherein The applications include: (C1) Use of Enterobacterium butyriciproducens (I. butyriciproducens) DSM 26588 in increasing the body weight of chicks infected with Eimeria tenella; (C2) Use of Enterobacterium butyriciproducens (I. butyriciproducens) DSM 26588 in the preparation of a product for increasing the body weight of chicks infected with Eimeria tenella; (C3) Use of Enterobacterium butyriciproducens (I. butyriciproducens) DSM 26588 in reducing Eimeria tenella oocyst excretion in chicks infected with E. tenella; (C4) Use of Enterobacterium butyriciproducens (I. butyriciproducens) DSM 26588 in the preparation of a product for reducing the amount of Eimeria tenella oocysts excreted in chicks infected with Eimeria tenella; (C5) Application of Enterobacterium butyriciproducens (I. butyriciproducens) DSM 26588 in reducing the severity of cecal lesions in chicks infected with Eimeria tenella; (C6) Use of Enterobacterium butyriciproducens (I. butyriciproducens) DSM 26588 in the preparation of a product for reducing the severity of cecal lesions in chickens infected with Eimeria tenella.

7. The use according to claim 1, wherein The applications include: (D1) application of Enterobacterium butyriciproducens (I. butyriciproducens) DSM 26588 in regulating the body's immune response against Eimeria tenella; (D2) Use of Enterobacterium butyriciproducens (I. butyriciproducens) DSM 26588 in the preparation of a product for regulating the body's immune response against Eimeria tenella; The anti-Eimeria tenella (E.tenella) immune response comprises: On the 5th to 7th day of Eimeria tenella infection, Enterobacter butyriciproducens DSM26588 increased the number of B lymphocytes in peripheral blood lymphocytes; increased CD8 + T cell number level; increase the secretion level of IFN-γ; reduce the secretion level of IL-17 on the 6th day of infection.

8. A probiotic or probiotic preparation for use against Eimeria tenella, characterized in that: Includes I. butyriciproducens DSM 26588.

9. A probiotic composition for resisting Eimeria tenella, characterized in that: Includes I. butyriciproducens DSM 26588.

10. An animal feed or animal feed additive for resisting Eimeria tenella, characterized in that: Includes I. butyriciproducens DSM 26588.

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