Use of butyric acid-producing enterobacteriaceae as probiotic against eimeria tenella

Abstract

The present application relates to the field of microbial technology, and particularly relates to the application of butyric acid-producing Enterobacter as an anti-Eimeria tenella probiotic. In the present application, the E. tenella infection model induced by an antibiotic cocktail is used to explore the effect of butyric acid-producing Enterobacter DSM 26588 on the development of E. tenella. The butyric acid-producing Enterobacter DSM 26588 inhibits the expression of the EtGFAT gene of the gamete of E. tenella, thereby inhibiting the development of the gamete; improves the intestinal lesions caused by E. tenella infection, reduces the pathological damage to the intestine, and reduces the output of E. tenella oocysts, with an ACI value of 162, and has good anti-coccidial effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to the application of I. butyriciproducens as probiotics against E. tenella. BACKGROUND

[0002] Chicken coccidiosis is an intestinal protozoan disease caused by mixed infection of one or more coccidia of the genus Eimeria. It has strong infectivity and high morbidity. Seven species of Eimeria are known to infect chickens, and their pathogenicity varies. In particular, E. tenella only parasitizes the cecum and has the strongest pathogenicity. The main clinical symptoms after infection are reduced feed intake and diarrhea. Severe infection can cause hemorrhagic diarrhea and even death. The loss of productivity, high mortality and high treatment costs to control the disease have a serious economic impact on the poultry industry.

[0003] I. butyriciproducens belongs to the Intestinimonas genus under the Eubacteriales order, Clostridia class and Bacillota (formerly Firmicutes) phylum in taxonomy. I. butyriciproducens has appeared in many disease studies. Studies have shown that I. butyriciproducens is the only intestinal microbial species with a negative correlation with Candida albicans in relative abundance. Eating aronia melanocarpa fruit can improve the arterial function of prehypertensive middle-aged people, which is believed to be related to the significant increase in the abundance of I. butyriciproducens. Another study found that Cyclocarya paliurus polysaccharide promotes the production of short-chain fatty acids by increasing the production of bacteria such as I. butyriciproducens, thereby reducing the symptoms of type 2 diabetes. Studies have also confirmed that I. butyriciproducens has the potential to be the next generation of probiotics. The above series of related research results show that the abundance of I. butyriciproducens changes due to the occurrence and development of diseases, similar to some disease markers. However, there are few studies on the direct impact of the bacteria on diseases or pathogens, especially on intestinal parasites, and no relevant studies have been found. SUMMARY

[0004] To overcome the above problems, the present application provides the application of I. butyriciproducens as probiotics against E. tenella.

[0005] To achieve the above technical purposes, the present application adopts the following technical solutions:

[0006] In a first aspect, the application provides use of I. butyriciproducens DSM 26588 as probiotics against E. tenella.

[0007] I. butyriciproducens DSM 26588 is purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ).

[0008] In one or more embodiments, the use comprises:

[0009] (A1) use of culture supernatant of I. butyriciproducens DSM 26588 in inhibiting invasion of E. tenella sporozoites;

[0010] (A2) use of culture supernatant of I. butyriciproducens DSM 26588 in preparing a product for inhibiting invasion of E. tenella sporozoites.

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

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

[0013] Further preferably, I. butyriciproducens DSM 26588 is inoculated into anaerobic agar medium at a volume ratio of 1:80-120, anaerobically incubated for 40-50 h until the I. butyriciproducens DSM 26588 is cultured to the stationary phase with OD 600 at 0.8-1.2, centrifuged, and the culture supernatant of the I. butyriciproducens DSM 26588 is obtained.

[0014] In one or more embodiments, the use comprises:

[0015] (B1) use of I. butyriciproducens DSM 26588 in inhibiting expression of the E. tenella gamete EtGFAT gene;

[0016] (B2) Use of I. butyriciproducens DSM 26588 in the preparation of a product for inhibiting the expression of the gamete EtGFAT gene of E. tenella.

[0017] In one or more embodiments, the use comprises:

[0018] (C1) Use of I. butyriciproducens DSM 26588 for increasing the body weight of chicks after infection with E. tenella;

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

[0020] (C3) Use of I. butyriciproducens DSM 26588 for reducing the oocyst excretion of E. tenella in chicks after infection with E. tenella;

[0021] (C4) Use of I. butyriciproducens DSM 26588 in the preparation of a product for reducing the oocyst excretion of E. tenella in chicks after infection with E. tenella;

[0022] (C5) Use of I. butyriciproducens DSM 26588 for reducing the degree of cecal lesions in chicks after infection with E. tenella;

[0023] (C6) Use of I. butyriciproducens DSM 26588 in the preparation of a product for reducing the degree of cecal lesions in chicks after infection with E. tenella.

[0024] In one or more embodiments, the use comprises:

[0025] (D1) Use of I. butyriciproducens DSM 26588 for modulating the immune response of an organism against E. tenella;

[0026] (D2) Use of I. butyriciproducens DSM 26588 in the manufacture of a product for modulating the immune response of an organism against E. tenella.

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

[0028] I. butyriciproducens DSM 26588 increases the number of B lymphocytes in peripheral blood lymphocytes; increases the number of CD8 + T cells; increases the secretion level of IFN-γ; and decreases the secretion level of IL-17 on day 6 of infection.

[0029] In a second aspect of the application, there is provided a probiotic or probiotic formulation against E. tenella, comprising I. butyriciproducens DSM 26588.

[0030] In a third aspect of the application, there is provided a probiotic composition against E. tenella, comprising I. butyriciproducens DSM 26588.

[0031] In a fourth aspect of the application, there is provided an animal feed or animal feed additive against E. tenella, comprising I. butyriciproducens DSM 26588.

[0032] The application has the following beneficial effects:

[0033] In the present application, the model of E. tenella infection induced by antibiotic cocktail is used to explore the effect of I. butyriciproducens DSM 26588 on the development and infection of E. tenella.

[0034] In vitro test proved that the culture supernatant of I. butyriciproducens DSM 26588 can inhibit the invasion of anti-E. tenella sporozoite, in which 2'-O-methyl adenosine is the main effective component, and in the cell model, the bacteria itself cannot directly inhibit the invasion of sporozoite; I. butyriciproducens DSM 26588 has no significant effect on E. tenella schizont; I. butyriciproducens DSM 26588 can stimulate CD8 + T cells to secrete high levels of IFN-γ.

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

[0036] I. butyriciproducens DSM 26588 can inhibit the development of E. tenella gamete by inhibiting the expression of E. tenella gamete EtGFAT gene; improve the intestinal lesions caused by E. tenella infection, reduce the intestinal pathological damage, and reduce the E. tenella oocyst discharge, with an ACI value of 162, and has good anticoccidial effect. BRIEF DESCRIPTION OF DRAWINGS

[0037] The drawings constituting a part of the specification of the present application serve to provide a further understanding of the present application, and the illustrative embodiments of the present application and their description serve to explain the present application, and do not constitute an improper limitation on the present application.

[0038] Figure 1 A difference analysis diagram for detecting the influence of I. butyriciproducens bacteria (BC) of different bacterial numbers on the invasion of E. tenella into DF-1 cells by using flow cytometry;

[0039] Figure 2 A difference analysis diagram for detecting the influence of I. butyriciproducens culture supernatant (CS) of different concentrations on the invasion of E. tenella into DF-1 cells by using flow cytometry;

[0040] Figure 3Figure 6 is a classification pie chart of metabolites detected in samples under positive and negative ion dual modes, wherein a is the substances and proportion detected under positive ion mode, including organic acids and derivatives, lipids and lipid molecules, organic heterocyclic compounds, organic oxidation compounds, benzene compounds, nucleosides, nucleotides and analogues, organic nitrogen compounds, phenylpropanoids and polyketones, alkaloids and derivatives, and lignin / neolignin related compounds; b is the substances and proportion detected under negative ion mode, including lipids and lipid molecules, organic acids and derivatives, nucleosides, nucleotides and analogues, organic heterocyclic compounds, organic oxidation compounds, benzene compounds, phenylpropanoids and polyketones, and organic nitrogen compounds;

[0041] Figure 4 Figure 7 is a principal component analysis (PCA) quality control chart of IB group samples and AB group samples under positive and negative ion dual modes; wherein a is the PCA chart under positive ion mode, and b is the PCA chart under negative ion mode;

[0042] Figure 5 Figure 8 is the influence and difference analysis of the top 6 different metabolites in different concentration gradients on the invasion rate of E. tenella, wherein 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 Figure 9 is the difference analysis of the effect of different numbers of I. butyriciproducens bacterial bodies (BC) on schizonts;

[0044] Figure 7 Figure 10 is the difference analysis of the effect of different concentrations of I. butyriciproducens culture supernatant (CS) on schizonts;

[0045] Figure 8 Figure 11 is the relative expression level of EtGFAT gene in each test group;

[0046] Figure 9 Figure 12 is the PCA score chart of samples in each test group;

[0047] Figure 10 Figure 13 is a clustering heat map of short-chain fatty acid content, wherein the horizontal axis represents sample grouping and number, the vertical axis represents short-chain fatty acid types (including caproic acid, acetic acid, butyric acid, propionic acid, and valeric acid), and the color gradient from red to blue reflects the content from high to low;

[0048] Figure 11 Figure 14 is the difference analysis of the proportion of B lymphocytes in peripheral blood lymphocytes of chicks in each test group at 5-7 days after E. tenella infection;

[0049] Figure 12 Figure 15 is the difference analysis of the proportion of CD4+T cells in peripheral blood lymphocytes of chicks in each test group at 5-7 days after E. tenella infection;+ T lymphocyte proportion difference analysis;

[0050] Figure 13 For E. tenella infection after 5-7 days, the CD8 + T lymphocyte proportion difference analysis;

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

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

[0053] Figure 16 For flow cytometry detection of the effect of I. butyriciproducens on CD4 + T lymphocyte proliferation, wherein a is a flow cytometry contour plot, and b is a difference analysis plot;

[0054] Figure 17 For flow cytometry detection of the effect of I. butyriciproducens on CD8 + T lymphocyte proliferation, wherein a is a flow cytometry contour plot, and b is a difference analysis plot;

[0055] Figure 18 For ELISA detection of IFN-γ level in co-culture supernatant, wherein a is the level of IFN-γ secreted by CD4 + T cells stimulated by I. butyriciproducens, and b is the level of IFN-γ secreted by CD8 + T cells stimulated by I. butyriciproducens;

[0056] Figure 19 For the representative cecal tissue photograph images of each test group. DETAILED DESCRIPTION

[0057] It should be noted that the following detailed description is exemplary in nature and is intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0058] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, devices, components and / or combinations thereof, but do not preclude the presence or addition of one or more other features, steps, operations, devices, components and / or combinations thereof.

[0059] In order for those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in conjunction with specific examples.

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

[0061] The E. tenella strain used in the present application is preserved by the Ministry of Education Engineering Research Center for Animal Major Disease Microecological Vaccine (Drug), and is rejuvenated once every 3 months, stored in a 2.5% (mass fraction) potassium dichromate (207802; Sigma) solution, stored at 4°C, and has good worm morphology and strong virulence. The DF-1 cell line is purchased from ATCC (CRL-3586).

[0062] The 1-day-old healthy white-feather broilers used in the present application are purchased from Changchun Dehui Farm, and the whole feeding process ensures free access to feed and water. Before feeding, 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, the animals are first anesthetized by intraperitoneal injection of 50 mg / kg sodium pentobarbital, and then killed by air embolism of the heart.

[0063] Table 1 lists the main reagents used in the present application

[0064]

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

[0066] Example 1

[0067] Experimental design:

[0068] 1.1 Divide 300 1-day-old white-feather broilers into six groups at random, 50 in each group, namely the MOCK group, the ABX group, the CON group, the FMT group, the IB group (I. butyriciproducens backfilling group), and the NaB group (sodium butyrate group).

[0069] MOCK group: no treatment during the whole experiment, blank control group and FMT donor;

[0070] ABX group: from 1 day old, oral gavage of antibiotic cocktail for 14 consecutive days, then each chicken was infected with 5x10 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 at the same time;

[0072] FMT group: from 1 day old, oral gavage of antibiotic cocktail for 9 consecutive days; at 10 days old, performed flora transplantation with the MOCK group as the donor for 5 consecutive days;

[0073] IB group: from 1 day old, gavage of antibiotic cocktail for 9 consecutive days; at 10 days old, supplemented with I. butyriciproducens for 5 consecutive days;

[0074] NaB group: from 1 day old, oral gavage of antibiotic cocktail for 9 consecutive days; at 10 days old, added 1 g / L sodium butyrate in drinking water for 5 consecutive days;

[0075] Except that the MOCK group was not infected, the other groups were simultaneously infected with E. tenella.

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

[0077] Table 2 Preparation method of antibiotic cocktail

[0078]

[0079] After mixing the 4 antibiotics, the amount was calculated according to the body weight of the chicken, and 10 mL / kg body weight was gavaged, and the gavage system was adjusted with the growth of the age, and 1 g / L of penicillin was added in the drinking water (metronidazole needs to be used with ultrasonic dissolution), and was replaced every time. Continuous 14 days, twice a day.

[0080] 1.2 Purification of sporozoites and invasion test

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

[0082] The purification steps of E. tenella oocysts are as follows: using a 50 mL centrifuge tube, E. tenella stored in 2.5% (mass fraction) potassium dichromate is dispensed, 20 mL per tube. 25 mL of sterile distilled water is added to each tube, centrifuged at 4000 r / min for 10 min for more than 3 times, and the potassium dichromate component is completely washed off; the precipitate after centrifugation is suspended with 2 times the volume of 10% (mass fraction) sodium hypochlorite solution, vortexed and mixed well. Place on ice for 15 min; add sterile distilled water to the tube, centrifuge at 4000 r / min for 10 min; suspend the precipitate after centrifugation with 3 times the volume of 5% (mass fraction) sodium hypochlorite solution, mix well, centrifuge at 3000 r / min for 20 min; collect the uppermost white flocculent material, add sterile distilled water, centrifuge at 4000 r / min for 10 min for 3 times; collect the white precipitate, suspend in PBS, and the purified E. tenella oocysts are obtained.

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

[0084] The purified oocysts are dispensed into a lysis tube preloaded with ceramic beads for oocyst crushing. After precooling on ice for 10 min, crush twice at 6000 r / min for 30 s; collect the liquid mixture containing sporocysts and crushed oocyst walls after crushing, and centrifuge twice at 2500 r / min for 10 min to remove most of the crushed oocyst walls; prepare 50 mL of a trypsin and sodium taurocholate solution containing 0.25% (mass fraction) trypsin and 0.25% (mass fraction) sodium taurocholate, and digest the sporocysts under the following conditions: 42°C for 15 min; centrifuge at 1500 r / min for 10 min, and add cold PBS containing 1% FBS to terminate the digestion; resuspend the precipitate with PBS, make up to 20 mL, filter through a G3 sand core funnel, and use a pipette to repeatedly blow and add an appropriate amount of PBS until no sporozoites are observed under a microscope. Centrifuge the filtrate at 1500 r / min for 5 min to obtain purified sporozoites.

[0085] Flow cytometry is used to detect the effects of I. butyriciproducens bacteria (BC) and culture supernatant (CS) on CFSE-labeled E. tenella invasion of DF-1 cells, respectively. For the amount of I. butyriciproducens used in the present application, 1 mL of bacterial solution with OD 600 = 1 is taken, the number of bacteria after centrifugation is defined as 100% BC, 1 mL of culture supernatant after centrifugation with OD 600 = 1 is taken as 100% CS, and BC or CS is mixed with DMEM medium containing 10% FBS (without penicillin-streptomycin) at concentrations of 50%, 20%, and 10% for testing. DF-1 cells are cultured in a 24-well culture plate to 80%, and then 5 x 105 CFSE-labeled sporozoites were mixed with different concentrations of BC and CS, and incubated at 37 °C for 12 h. Then, the cells were harvested and detected by flow cytometry. CFSE-positive cells were the invaded cells, and the proportion of CFSE-positive cells was the invasion rate of E. tenella.

[0086] 1.3 Purification of E. tenella merozoites and flow cytometry detection:

[0087] 14-day-old healthy chicks were orally infected with 1 x 10 5 On the 5th day after infection, 5 chicks were killed, and the cecal wall was longitudinally opened on the clean bench. The cecal tissue was washed with PBS containing 2% (v / v) penicillin-streptomycin twice to remove the contents.

[0088] The cecal mucosa tissue was scraped with a sterile glass slide, mixed with an appropriate amount of PBS, and aliquoted into lysing tubes containing preloaded zirconia beads for homogenization at 3500 r / min for 45 s. After homogenization, the mixture was centrifuged at 3000 r / min for 10 min.

[0089] The precipitate was collected, and five times the volume of red blood cell lysis solution was added. After mixing, the mixture was lysed at 4 °C for 3 min, and then the lysis was terminated by adding PBS. The mixture was centrifuged at 3000 r / min for 10 min. The lysis was repeated twice.

[0090] A 30% Percoll solution was prepared using sterile PBS, and the precipitate after centrifugation was uniformly dispersed in the solution to obtain a 30% Percoll suspension containing merozoites.

[0091] A 50% Percoll solution was prepared using sterile PBS, and 1 mL of the solution was added to the bottom of each 15 mL centrifuge tube.

[0092] The 30% Percoll suspension containing merozoites was slowly added to the top of the 50% Percoll solution, and 5 mL of the suspension was added to each tube. The mixture was centrifuged at 3000 r / min for 10 min.

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

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

[0095] 1.4 Extraction of total RNA from cecum:

[0096] Put equal mass of cecum tissue of soybean size into a lysis tube preloaded with ceramic beads, add 1 mL of RNAisoPlus for homogenization, repeat 4500 r / min, 30 s twice, complete homogenization of the tissue;

[0097] After homogenization of the sample, stand at room temperature for 5 min, centrifuge at 12000 x g for 5 min at 4℃;

[0098] Transfer the supernatant into a 1.5 mL tube, immediately add 200 μL of chloroform, vortex to mix, stand at room temperature for 5 min, centrifuge at 12000 x g for 15 min at 4℃;

[0099] Carefully pipette the supernatant into a 1.5 mL tube, add 500 μL of isopropanol, mix by inverting, stand at room temperature for 5 min, centrifuge at 12000 x g for 10 min at 4℃;

[0100] Prepare 75% ethanol with DEPC water, add 1 mL per sample. Centrifuge at 7500 x g for 5 min at 4℃;

[0101] Carefully pipette the supernatant, keep the lid of the 1.5 mL centrifuge tube open, dry at room temperature, add 50 μL of DEPC water to dissolve the RNA. Store the RNA at -80℃ for later use.

[0102] 1.5 cDNA synthesis:

[0103] Use the reverse transcription kit of Promega company for cDNA synthesis. According to the instructions, mix 4 μL of DEPC water, 4 μL of buffer containing random primers, and 2 μL of premixed enzyme to obtain 10 μL of reverse transcription system, add 10 μL of RNA system, and make the final system 20 μL. The PCR reaction program is set as follows: 25℃, 5 min, 1 cycle; 42℃, 1 h, 1 cycle; 95℃, 5 min, 1 cycle; 4℃, complete the reaction. Store the obtained cDNA at -20℃ for later use.

[0104] 1.6 qPCR:

[0105] Use SYBR Green dye method for qPCR, and the reaction system is shown in Table 3.

[0106] Table 3 qPCR reaction system

[0107]

[0108] The qPCR reaction program is set as follows:

[0109] (1) pre-denaturation: 95℃ 2min; (2) denaturation: 95℃ 15s; (3) annealing / extension: 60℃ 15-30s; (4) repeat steps 2 and 3 for 40 cycles; (5) melting curve analysis: 95℃ 15s, 60℃ 15s, 95℃ 15s;

[0110] The primers used in 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] The chicken peripheral blood lymphocyte kit was used to isolate peripheral blood lymphocytes, and the steps were as follows:

[0115] The chicken heart blood was collected using a 1 mL syringe and injected into a heparin sodium anticoagulation tube;

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

[0117] Balance the separation solution from a 4℃ environment to room temperature, and add 3 mL of separation solution to a 15 mL centrifuge tube;

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

[0119] Carefully pipette the intermediate lymphocyte layer into a new centrifuge tube, add 5 times the volume of PBS, centrifuge at 500 x 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 the lymphocytes 50 times with PBS, mix well, and take 10 μL under the erythrocyte counting plate for counting, with 1 x 10 6 cells per sample added to the flow tube, with a sample system of 1 x 10 6 cells / 100 μL, two tubes per sample (T cells and B cells labeled separately), 4℃ standby;

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

[0124] Cell typing: all samples were divided into two groups. T cell typing: CD3-FITC, CD4-APC, CD8a-PE antibodies were mixed and diluted, each sample was labeled according to 10 μL of the diluted system; B cell labeling: after dilution, Bu-1 antibody was added to the sample, each sample was labeled according to 10L of the diluted system. Incubate at 4°C for 30 min, after incubation, add 1 mL of PBS, centrifuge at 500 x g for 10 min, after washing off the antibody, add PBS to each sample to 500 μL for machine detection.

[0125] 1.9I. butyriciproducens and lymphocyte co-culture

[0126] CD4 - , CD8 - Cell complex sorting: under sterile conditions, take the spleen of a 14-day-old healthy chick, place it in a 200-mesh cell screen, and use a 5-mL syringe rubber plug to grind it into a spleen cell suspension. Isolate the lymphocytes using a chicken spleen lymphocyte separation kit, according to the instructions, centrifuge and wash the spleen cell suspension, add tissue diluent to 3 mL, similar to the peripheral blood lymphocyte separation step, slowly add 3 mL of the diluted spleen cell diluent to the top of 3 mL of separation solution, centrifuge at 500 x g, with an ascending speed of 0, for 30 min. To preserve antigen-presenting cells, the middle lymphocyte layer and the separation solution layer below it must be carefully aspirated after centrifugation, and 10 times the volume of PBS must be added to wash away the separation solution components, resulting in a final spleen mononuclear cell complex. After labeling with CD4-APC, CD8a-PE antibodies, use flow cytometry negative sorting technology to separate 1 x 10 6 CD4 - , CD8 - cell complexes, respectively.

[0127] Co-culture: mix the two cell complexes with 1 mL of DMEM medium containing 10% FBS, and inoculate 100 μL per well in a 24-well plate. Mix 2 x 10 9 cfu of I. butyriciproducens bacteria with 18 mL of DMEM medium containing only 10% FBS (without penicillin / streptomycin), inoculate 900 μL per well (1 mL of co-culture per well). Incubate at 37°C and 5% CO2 for 48 hours. After incubation, collect the cells and cell supernatant, respectively. The cells are used for flow cytometry detection, and the cell supernatant is centrifuged at 12000 r / min for 10 min and used for ELISA detection of IFN-γ secretion levels.

[0128] 1.10 ELISA

[0129] Freshly collected blood was allowed to stand at room temperature for 10 min, then placed at 37℃ for 1 h, 4000 r / min for 15 min, and the supernatant was collected and stored at -80℃. According to the kit instructions, the kit was equilibrated to room temperature 30 min in advance, and during this period, the standard was prepared according to the progressive dilution method, and the biotinylated antibody, SABC complex working solution, mixed TMB A and B solutions were prepared.

[0130] 50 μL of the standard or sample diluent was added to the blank wells, and 50 μL of the standard or sample to be tested was added to the remaining wells, respectively. The plate was sealed with sealing tape, mixed well, and then placed at 37℃ for 1 h. The plate was washed with 1x washing solution for 3 times, and then dried with filter paper. 100 μL of biotinylated antibody diluent was added to the blank wells, and 100 μL of 1x biotinylated antibody working solution was added to the remaining wells, respectively. The plate was sealed with sealing film, mixed well, and then placed at 37℃ for 1 h. The plate washing process was repeated, 100 μL of SABC complex working solution was added to each well, the plate was sealed with sealing film, mixed well, and then placed at 37℃ for 30 min. After washing the plate, 100 μL of TMB mixed solution was added to each well, mixed well, and then sealed with sealing tape. The plate was placed at 37℃ in the dark for 20 min, 50 μL of stop solution was added to each well, mixed well, and then the absorbance value at 450 nm was detected by an enzyme-labeled instrument.

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

[0132] I. butyriciproducens was inoculated into 3 Hungate tubes containing 10 mL anaerobic agar medium at a volume ratio of 1:100, and the anaerobic agar medium without inoculation was placed in an anaerobic incubator at the same time for 48 hours. After the culture was completed, the bacterial cells and culture supernatant were separated by centrifugation at 10,000 x g for 10 min. The culture supernatant was filtered through a 0.22 μm filter, sterilized again, and the supernatant was collected. Finally, 3 samples were obtained for each of the culture supernatant group (IB) and the anaerobic agar medium group (AB). The samples were quickly frozen in liquid nitrogen and transported on dry ice to Beijing Novogene Biosciences Co., Ltd. for non-targeted metabolomics detection.

[0133] Inhibition of invasion effect evaluation method:

[0134] The proportion of CFSE-positive cells was detected to reflect the invasion rate of E. tenella sporozoites. DF-1 cells were inoculated into a 48-well cell culture plate at a seeding number of 1 x 10 5 The DMEM containing 10% FBS (mass fraction) and 1% (mass fraction) penicillin-streptomycin was used to prepare different concentrations of antibiotic cocktails. When the cells grew to about 80%, the original culture DMEM was replaced.

[0135] The purified E. tenella sporozoites were labeled with CFSE at a concentration of 5 mM, incubated in a 37°C water bath for 20 min. The same number of sporozoites were added to each well, and co-cultured at 37°C in 5% CO2 for 12 h.

[0136] After co-culture, the medium components were washed off with PBS, and the cells were digested with 0.25% (mass fraction) trypsin, collected in 5 mL polystyrene tubes, and detected on a flow cytometer.

[0137] After analysis of the supernatant components, a concentration gradient of 1 mM, 500 mM, 200 mM, 100 mM, 50 mM, 20 mM, 10 mM, and 0 mM 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 the 6th day after E. tenella infection, 50 mg / kg of sodium pentobarbital was injected into the abdominal cavity of the chicken for anesthesia. After anesthesia, the chicken was sacrificed by injecting 5 mL of air into the heart using the heart air embolism method. After surface disinfection with alcohol, the abdominal cavity was opened in a clean bench, and the cecal tissue was sharply separated. The cecal contents were collected in a sterile enzyme-free cryotube, quickly frozen in liquid nitrogen, and transported on dry ice to Suzhou Panomics Biomedical Technology Co., Ltd. for detection.

[0140] 1.13 Relative weight gain rate

[0141] The body weight of the chicks was recorded, and the difference in body 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 test 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 output:

[0144] Oocyst feces per gram (OPG): During the oocyst shedding period of E. tenella (7 dpi), feces from each group were randomly sampled five times, weighed, and then ground using a PBS group mixer. The number of oocysts per gram of feces was finally obtained by counting under a hemocytometer.

[0145] Oocyst reduction rate is the difference between the number of oocysts in the conventional infection group (CON) and each test group, compared with the MOCK group. The result is expressed in the form of percentage. Oocyst value is divided into different ranges according to the oocyst ratio. Different ranges of oocyst ratio are defined as different values, a total of 5. When the oocyst ratio is less than 1%, the oocyst value is 0, when the oocyst ratio is between 1% and 25%, the oocyst value is 5; when the oocyst ratio is between 26% and 50%, the oocyst value is 10; when the oocyst ratio is between 51% and 75%, the oocyst value is 20; when the oocyst ratio is between 76% and 100%, the value is 40.

[0146] 1.15 Cecal lesion score and lesion value

[0147] The lesions of different degrees after coccidiosis infection were scored: 0 points when no obvious lesions were observed in the cecal tissue and the contents were normal; 1 point when the cecal wall was not thickened and only had a few bleeding points, and the contents were normal; 2 points when the cecal wall appeared thickening and there were many bleeding points, and the contents had a small amount of bloody stool; 3 points when the cecal wall was significantly thickened and there were severe deformation and shrinkage, and the contents had obvious blood clots and white block-shaped substances; 4 points when the cecal wall was extremely thickened, the tissue was severely atrophied, and the intestinal cavity was filled with a large amount of blood clots, white block-shaped substances and cheese-like substances. The lesion value is 10 times the lesion score.

[0148] 1.16 Anti-coccidial index (ACI)

[0149] The anti-coccidial effect is mainly reflected by the anti-coccidial index (ACI). The sum of the relative weight gain rate and the survival rate minus the sum of the lesion value and the oocyst value is the anti-coccidial index (ACI). The anti-coccidial index ACI value is divided into 4 ranges, corresponding to 4 different anti-coccidial effects: when the ACI value is less than 120, it indicates no anti-coccidial effect; when the ACI value is between 120 and 160, the anti-coccidial effect is moderate; when the ACI value is between 160 and 180, the anti-coccidial effect is good; when the ACI value is greater than 180, the anti-coccidial effect is excellent.

[0150] 1.17 Data analysis

[0151] GraphPad Prism 9 software was used to analyze the data differences and draw graphs. One-way ANOVA and Ttest statistical analysis 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] Results:

[0153] (1) The effect of I. butyriciproducens on the invasion of E. tenella

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

[0155] However, the culture supernatant of I. butyriciproducens had a good effect on inhibiting the invasion of E. tenella. The results showed that the invasion rate of E. tenella could be reduced to about 1% by adding different concentrations of I. butyriciproducens culture supernatant. This inhibitory effect was dependent on the concentration of the culture supernatant to some extent, and the inhibitory effect on the invasion of E. tenella was significantly reduced as the concentration of the culture supernatant decreased Figure 2 ).

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

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

[0158] In the cation mode, 517 metabolites were identified; in the anion mode, 314 metabolites were identified; organic acids and derivatives, lipids and lipid molecules accounted for the highest proportion, accounting for 53.1% and 58.65% of all metabolites identified in each mode Figure 3 ).

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

[0160] Differential analysis was performed on the components of anaerobic agar medium (AB) and I. butyriciproducens culture supernatant (IB). The significant up-regulated metabolites were screened by setting the threshold value as VIP > 1.0 and FC > 1.2, and P < 0.05. The main metabolites produced after IB culture were screened (Table 5).

[0161] Table 5 Metabolite differential screening results

[0162]

[0163] Nine categories of substances were screened in the IB vs. AB comparison, including 70 differential metabolites. Among them, 19 organic acids accounted for the highest proportion of 27.14%; 10 lipids accounted for 14.29%; 8 nucleosides and 8 organic heterocyclic compounds each accounted for 11.43%; 7 organic oxygen compounds accounted for 10%; 1 benzene, 1 alkaloid and 1 organic nitrogen compound each accounted for 1.43%; and 15 unclassified compounds accounted for 21.43%.

[0164] To further screen, remove Invalid mass metabolites and set the threshold value to FC≥2, finally 12 metabolites were obtained (Table 6), ranked by FC, P value and VIP value, and the top 5 of each order was taken, finally 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 6 metabolites such as lauric acid ethyl ester on E. tenella invasion:

[0168] Through the DF-1 invasion model, 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. Figure 5 The results showed that uridine diphosphate and deoxycholic acid had no effect on sporozoite invasion; 50 μM of lauric acid ethyl ester promoted sporozoite invasion (P<0.01); 20 μM of inosine significantly promoted sporozoite invasion (P<0.01); 1 mM and 500 μM of guanosine significantly inhibited sporozoite invasion (P<0.0001), 200 μM and 100 μM of guanosine also inhibited sporozoite invasion (P<0.05), and among the 6 metabolites, only 2'-O-methyladenosine significantly inhibited E. tenella sporozoite invasion at 8 concentration gradients (P<0.0001). The above results proved that 2'-O-methyladenosine was the key component in the culture supernatant of I. butyriciproducens that inhibited E. tenella invasion.

[0169] (3) The effect of I. butyriciproducens on the schizonts of E. tenella

[0170] The I. butyriciproducens cells and culture supernatant were co-cultured with purified E. tenella schizonts for 12 h. The proportion of schizonts with necrosis was detected by flow cytometry to study the direct effect of I. butyriciproducens on E. tenella schizonts. The results showed that the effect of I. butyriciproducens cells on E. tenella schizonts was not significant (P>0.05) Figure 6 ), and the effect of I. butyriciproducens culture supernatant on E. tenella schizonts was also not significant (P>0.05) Figure 7 ).

[0171] (4) The effect of I. butyriciproducens on the expression of EtGFAT gene

[0172] The relative expression level of EtGFAT gene in cecal tissue of each group at 6 dpi was detected, and the results showed that the expression level of EtGFAT gene in ABX group was significantly lower than that in CON group (P<0.0001). After antibiotic cocktail treatment, the down-regulation of EtGFAT gene expression level was observed in the I. butyriciproducens back-supplemented group, 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 back-supplemented group was significantly lower than that in the CON group and the FMT group (P<0.0001) Figure 8 ).

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

[0174] Considering the butyric acid-producing characteristics of I. butyriciproducens, the contents of isobutyric acid, propionic acid, acetic acid, butyric acid, valeric acid, isovaleric acid, and hexanoic acid in the cecum of CON group, ABX group, FMT group, and I. butyriciproducens back-supplemented group at 6 dpi were detected.

[0175] The results of the quantification of short-chain fatty acids in the cecum showed that the contents of valeric acid and caproic acid were not detectable in the ABX group, and the contents of isobutyric acid, propionic acid, acetic acid, butyric acid, and isovaleric acid were lower than those in the other test groups. The content of butyric acid in the I. butyriciproducens backfill group was higher than that in the other test groups, which proved the excellent performance of I. butyriciproducens in producing butyric acid. It was found through the quantitative results that whether it was fecal microbiota transplantation or I. butyriciproducens single strain backfill, the content of short-chain fatty acids in the cecum would recover to a certain level (Table 7).

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

[0177]

[0178] The principal component analysis (PCA) of the metabolites of each group showed that the ABX group was significantly different from the CON group, the FMT group, and the I. butyriciproducens backfill group ( Figure 9 ).

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

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

[0181] Detection of the number of peripheral blood B lymphocytes:

[0182] On the 5th day of E. tenella infection, the number of B lymphocytes in the I. butyriciproducens backfill group was the highest among all the test groups, which was 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 of E. tenella infection, the number of peripheral blood B lymphocytes in the I. butyriciproducens backfill group remained at a high level, which was significantly higher than that in the MOCK group (P<0.01). On the 7th day of E. tenella infection, the number of B lymphocytes in the I. butyriciproducens backfill group was the highest, which was 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] Detection of the number of peripheral blood T lymphocytes:

[0184] The number of peripheral blood CD4+ T lymphocyte quantity level Figure 12 ) showed that the peripheral blood CD4 + T cell quantity level of the I. butyriciproducens backfill group was significantly higher than that of the ABX group (P<0.0001) and the NaB group (P<0.001) on the 5th day after infection; on the 6th day after infection, the peripheral blood CD4 + T lymphocyte quantity of the I. butyriciproducens backfill group was significantly lower than that of the CON group and the FMT group, and was significantly higher than that of the ABX group and the NaB group (P<0.0001); on the 7th day after infection, the peripheral blood CD4 + T lymphocyte quantity of the I. butyriciproducens backfill group was significantly lower than that of the CON group (P<0.001) and the FMT group (P<0.000).

[0185] The peripheral blood CD8 + T lymphocyte quantity level Figure 13 ) of E. tenella infected chickens from the 5th to the 7th day after infection was studied, and the results showed that the CD8 + T lymphocyte quantity of the I. butyriciproducens backfill group was significantly higher than that of the NaB group (P<0.0001) and the ABX group (P<0.0001) on the 5th day after infection, and was at the highest level; on the 6th day after infection, the peripheral blood CD8 + T lymphocyte quantity of the I. butyriciproducens backfill group was significantly higher than that of the CON group (P<0.05); on the 7th day after infection, the peripheral blood CD8 + T lymphocyte quantity of the I. butyriciproducens backfill group was still at the highest level among all test groups, and was significantly higher than that of the ABX group (P<0.0001) and the NaB group (P<0.01). In summary, I. butyriciproducens can produce immune regulation on the host during the sexual reproduction period of E. tenella.

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

[0187] The IFN-γ and IL-17 secretion levels in serum from the 5th to the 7th day after E. tenella infection were analyzed, and the results showed that the IFN-γ secretion level of the I. butyriciproducens backfill group was significantly higher than that of 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) Figure 14 ) from the 5th to the 7th day after E. tenella infection.

[0188] The results of serum IL-17 secretion level detection indicate that ( Figure 15 On day 5 after E. tenella infection, the IL-17 secretion level in the CON group was significantly lower than that in the ABX group and the I. butyriciproducens replacement group (P < 0.0001), while there was no significant difference between the ABX group and the I. butyriciproducens replacement group. On day 6 after E. tenella infection, the IL-17 secretion level in the CON group was significantly higher than that in the ABX group (P < 0.0001) and the I. butyriciproducens replacement group (P < 0.001). On day 7 after E. tenella infection, the IL-17 secretion level in the I. butyriciproducens replacement group was significantly higher than that in 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] Connect I.butyriciproducens to CD4 respectively. - CD8 - Cell complex co-culture, CD4 count was detected by flow cytometry. + T, CD8 + The number of T cells indicated that I. butyriciproducens stimulated CD4 + The effect of T proliferation was not significant. Figure 16 ), stimulating CD8 + The T proliferation effect was significant (P < 0.001) Figure 17 ).

[0190] The IFN-γ content in the cell supernatant of the co-culture system was detected. Figure 18 The results showed that I. butyriciproducens is associated with CD4+. - CD8 - When the complex is co-cultured, it can stimulate CD8. + T secretes high levels of IFN-γ, which interacts with CD8. + The difference between the T-culture group and the group cultured alone was significant (P < 0.001); I. butyriciproducens could not stimulate CD4. + T secretes high levels of IFN-γ, which interacts with CD4+. + Compared with the T culture group alone, the IFN-γ in the cell supernatant was decreased (P<0.05).

[0191] (7) Evaluation of anticoccidiosis efficacy

[0192] Weight changes:

[0193] The body weight of 10 chickens in each group before infection with E. tenella (14 days old) and 7 dpi (21 days old) was recorded. The results showed that the average weight gain of the FMT group, I. butyriciproducens backfill group, and 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] Oocyst excretion:

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

[0198] Table 9 Oocyst excretion

[0199]

[0200] Cecal lesion score:

[0201] The macroscopic pathological changes of the cecal tissue at 7 dpi after infection with E. tenella showed that Figure 19 ), the cecal length of the I. butyriciproducens backfill group was longer, the intestinal wall thickness was significantly thinner than that of the CON group, there was no obvious blood clot in the intestine, and the overall lesion degree was significantly lower than that of the CON group. The cecum of the NaB group was slightly shrunk, the intestinal wall thickness was slightly thinner than that of the CON group, the intestinal contents were relatively solid, and the overall lesion degree was lower than that of the CON group but higher than that of the I. butyriciproducens backfill group. The cecal lesion score showed that the lesion degree of the I. butyriciproducens backfill group was lower than that of all the infected groups, and the lesion value was 16 (Table 10).

[0202] Table 10 Lesion score and lesion value

[0203]

[0204] Anticoccidial index

[0205] The relative weight gain rate, survival rate, oocyst value, lesion value of each group of chicks were integrated to calculate the ACI index. NaB had a certain degree of anti-coccidial effect, and the ACI value was 149.7, which was slightly lower than that of the I. butyriciproducens backfill group, and the anti-coccidial effect was moderate. The ACI value of the I. butyriciproducens backfill group was 162, and the anti-coccidial effect was good (Table 11).

[0206] Table 11 Anti-coccidial index

[0207]

[0208] Results and discussion:

[0209] In the present application, the effect of I. butyriciproducens on E. tenella was verified from each period of infection of E. tenella, and it was first found that the metabolites of I. butyriciproducens effectively inhibited the invasion of E. tenella sporozoites, and the effective component was 2'-O-methyladenosine. 2'-O-methyladenosine belongs to nucleoside analogs, and nucleoside analogs have been widely used as anti-viral infection. In addition, since protozoa and helminths lack the initial synthesis pathway of purine and cannot synthesize purine by themselves, they must completely rely on the salvage pathway, that is, rely on the rich base and nucleoside in the host to adapt to the purine synthesis pathway, such as trypanosomes, malaria parasites and toxoplasma, and nucleoside analogs are also considered to be promising anti-trypanosomal agents. In the present application, 2'-O-methyladenosine inhibits the invasion of E. tenella sporozoites, which may interfere with the nucleotide salvage pathway of the worm, prevent the synthesis of nucleic acid of the parasite, and the specific mechanism of action still needs further research.

[0210] In addition, the I. butyriciproducens bacteria and culture supernatant were directly acted on the merozoites. The effect was evaluated by Annexin V-PE / 7-AAD cell apoptosis method, and the results showed that I. butyriciproducens had no significant effect on E. tenella merozoites. Considering that there is no effective in vitro culture model of merozoites at present, it is impossible to carry out more convincing research on the process from asexual reproduction to sexual reproduction, and whether this result is also related to the interference of nucleotide salvage pathway still needs further research.

[0211] After the in vitro test preliminarily proved that I. butyriciproducens had no significant effect on sporozoites and merozoites, we further confirmed that I. butyriciproducens could inhibit the expression of E. tenella gamete EtGFAT gene through animal test. The enzyme encoded by GFAT gene is a rate-limiting enzyme that plays a key role in the hexosamine biosynthetic pathway (HBP), which catalyzes the conversion of glutamine and fructose-6-phosphate to glutamine-6-phosphate and fructose-6-phosphate, which is 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, so GFAT is closely related to nucleotide metabolism. In addition, the activity of HBP can be affected by nutritional status and cell signaling, thereby affecting the metabolic state of the whole cell, including nucleotide metabolism. EtGFAT gene is a E. tenella macrogamete-specific gene related to its structure and development, and is a key gene for the synthesis of oocysts. After the complementation of I. butyriciproducens, the EtGFAT gene was significantly down-regulated, which proved that the amino sugar and nucleotide sugar metabolism of the gamete was directly affected, and the metabolic pathway of the gamete itself was inhibited, which was beneficial to prove the inhibitory effect of the bacteria on the development process of E. tenella gamete.

[0212] I. butyriciproducens is a major candidate for developing next-generation probiotics. I. butyriciproducens can not only ferment glucose, galactose and arabinose into butyrate, the main short-chain fatty acid, but also produce butyrate from lysine and even glycolysed lysine. These two pathways are the main metabolic pathways for I. butyriciproducens to produce butyrate. Butyrate is one of the most important short-chain fatty acids related to intestinal health. It is the main energy source for colonocytes, promotes their growth and maintains the integrity of the intestinal barrier, and 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 body weight and feed intake of chickens, and is beneficial to the recovery of intestinal homeostasis after infection. To compare the anti-E. tenella infection effect of I. butyriciproducens and sodium butyrate, the anti-coccidial index was calculated by E. tenella oocyst discharge, cecal lesion value of chicks, mortality rate of chicks and relative weight gain rate of body weight, which confirmed that the anti-coccidial effect of I. butyriciproducens was much better than that of sodium butyrate, that is, the effect of live bacteria on E. tenella was better than that of single metabolic product. Bacteria can have a wide impact on the metabolic health of the host through various metabolic products. Butyrate may not be the optimal main component in this experiment to play an anti-E. tenella role.

[0213] After the replenishment of probiotics, the immune system also has certain regulatory effects. C. butyiricum, as one of the widely used probiotics, has been shown to stimulate immune responses and activate non-specific immunity. Studies have also shown that the combination of C. butyiricum and coccidial vaccine 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 same order of Eubacteriales in Clostridia, and have a relationship in evolution, but it does not mean that they are completely the same in physiological function. There is no related research on I. butyriciproducens regulating the immune system. Based on the above reasons, we evaluated the effect of I. butyriciproducens on regulating the immune response of the body to E. tenella.

[0214] The immune response against E. tenella is dependent on Th1-type immune responses, and T cell immunity dominates in the host after infection. Cellular immunity can provide protection against E. tenella infection for the host, while humoral immunity cannot be ignored. IFN-γ is the main cytokine with anti-coccidial effect, and recombinant IFN-γ can significantly increase body weight and reduce oocyst shedding. The results of related detection in the late stage of E. tenella infection showed that I. butyriciproducens can positively regulate the host immune system during the gametogony and oocyst shedding stages. I. butyriciproducens can significantly increase the number of B cells, up-regulate the number of CD8 + T cells, significantly increase the secretion level of IFN-γ, and stimulate CD8+ T cells to secrete high levels of IFN-γ.

[0215] In addition to the response of Th1 cells mediated by IFN-γ to E. tenella, the role of IL-17 cannot be ignored. As a signature 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, people know little about the exact role of Th17 cells in chickens. Studies have shown that IL-17 isolated from IELs of Eimeria-infected chickens plays a pro-inflammatory role in coccidiosis, and treatment with IL-17 or IL-17F can induce pro-inflammatory cytokine expression in chicken fibroblasts. The dynamic changes in the secretion levels of IL-17 in the I. butyriciproducens-replenished group and the CON group on days 5-7 after E. tenella infection showed that both groups first increased and then decreased, reflecting the dynamic regulatory role of I. butyriciproducens in “pro-inflammatory”.

Claims

1. Enterobacter butyrate-producing bacteria ( I. butyriciproducens Application of DSM 26588 in the preparation of probiotics against Eimeria tenella.

2. Enterobacter butyrate-producing bacteria ( I. butyriciproducens Application of DSM 26588 culture supernatant in the preparation of products that inhibit the invasion of Eimeria tenella sporozoites.

3. The application as described in claim 2, characterized in that, The butyrate-producing Enterobacter ( I. butyriciproducens The culture supernatant of DSM 26588 contained 2'-O-methyladenosine.

4. The application as described in claim 2, characterized in that, Culture of butyrate-producing Enterobacteriaceae ( I. butyriciproducens DSM 26588 was heated to the plateau phase, centrifuged, and the culture supernatant of the butyrate-producing Enterobacter was obtained.

5. The application as described in claim 4, characterized in that, Enterobacter butyrate-producing bacteria ( I. butyriciproducens DSM 26588 was inoculated into anaerobic agar medium at a volume ratio of 1:80-120 and anaerobic cultured for 40-50 h until the *Enterobacter butyrate-producing* bacteria were cultured. I. butyriciproducens DSM 26588 reached a plateau phase with an OD600 of 0.8–1.

2. After centrifugation, the *Enterobacter butyrate-producing* was obtained. I. butyriciproducens DSM 26588 culture supernatant.

6. Enterobacter butyrate-producing bacteria ( I. butyriciproducens Application of DSM 26588 in the preparation of products that increase the weight of chicks infected with Eimeria tenella.

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