Lactobacillus reuteri from chicken for improving the development of gut microbiota in native animals and application thereof

Abstract

The application discloses a chicken-derived Lactobacillus rhamnosus S3 capable of improving the development of intestinal microorganisms of native animals and application thereof. Limosilactobacillus reuteri The chicken-derived Lactobacillus rhamnosus S3 can be used for preparing probiotic additives for chicks, for improving the growth performance of chicks, for enhancing the development of immune organs, for promoting the colonization of beneficial bacteria in chicks and for enhancing the diversity of intestinal flora, and for reducing the harm of chicken coccidiosis. The strain is safe and harmless to chicks when continuously fed to the chicks, is green and environment-friendly, has a significant growth-promoting effect on chicks, and can promote the development of thymus, thereby enhancing the immunity of the chicks. The strain can effectively reduce the harm of Eimeria tenella, Eimeria maxima and Eimeria necatrix infection to chicks, which is manifested in that the clinical symptoms and intestinal lesions caused by coccidiosis can be reduced after the Lactobacillus rhamnosus S3 is supplemented, the harm of coccidiosis to the weight gain of chicks can be reduced, and the excretion of coccidial oocysts can be reduced, thereby having a significant prevention and control effect on chicken coccidiosis.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to a strain of Lactobacillus reuteri from chickens that improves the development of intestinal microbiota in native animals and its application. Background Technology

[0002] Lactobacillus reuteri is widely distributed in the gastrointestinal tracts of mammals and birds. It is a strain with important probiotic functions and plays a key role in maintaining intestinal microecological balance, inhibiting the growth of harmful bacteria, and promoting host health. According to the Ministry of Agriculture and Rural Affairs Announcement No. 2045, "Catalogue of Feed Additive Varieties (2013)," Lactobacillus reuteri has been approved as a feed microbial additive for clinical animal production. Lactobacillus reuteri exerts its probiotic effects through multiple mechanisms, including: (1) physical competition and repulsion: it can occupy the adhesion sites of intestinal epithelial cells and prevent the adhesion of pathogenic bacteria, thereby exerting a competitive repulsion effect; (2) antibacterial effect of metabolites: it can produce a variety of antibacterial metabolites such as lactic acid and acetic acid, which inhibit the growth of acid-sensitive pathogens by lowering the intestinal pH value and maintaining intestinal health; (3) immunomodulatory function: its cell surface components can promote the immune response of the host's intestine. Studies have shown that Lactobacillus reuteri can effectively reduce the diarrhea rate of piglets, increase daily weight gain, and improve the diversity of the intestinal microbial community, thereby enhancing the health level of piglets. Adding Lactobacillus reuteri to feed can improve feed conversion rate, promote growth and development in broilers, and reduce the risk of intestinal pathogen infection.

[0003] Lactobacillus reuteri is widely used in health care and disease management as a functional food and biological additive. However, the function and safety of Lactobacillus reuteri are highly strain-specific, therefore it is necessary to determine the safety of Lactobacillus reuteri isolates before clinical application.

[0004] The chicken's gastrointestinal tract is home to a complex and dynamic microbial community, containing a wide variety and a large number of microorganisms. There are approximately 500 to 1,000 species of bacteria in the chicken's gastrointestinal tract, with a total bacterial count reaching trillions. There are extensive and complex interactions between the chicken's intestinal flora and the host, including: (1) promoting the digestion and absorption of nutrients in the chicken's intestines, converting unusable polysaccharides into short-chain fatty acids, and enhancing intestinal immune function; (2) promoting intestinal development, as the short-chain fatty acids produced by microbial metabolism can stimulate the proliferation of intestinal epithelial cells and promote the development and maturation of intestinal structure; (3) maintaining the intestinal mucosal barrier function, by competitively rejecting pathogen adhesion and enhancing the integrity of the mucosal barrier, which helps maintain the homeostasis of the intestinal environment; (4) participating in systemic physiological regulation, as some metabolites such as lactic acid and bacteriocins can participate in various physiological processes of the host through blood circulation, and have a systemic regulatory effect. However, the interaction between the gut microbiota and the host is a double-edged sword. A healthy, dynamically balanced microbiota structure provides the host with many physiological benefits, but when the gut ecology is imbalanced, the disordered microbiota can have harmful effects on the host, including: (1) affecting the chicken's digestive function, leading to incomplete nutrient absorption and thus affecting production performance; (2) weakening the chicken's immune response, making it more susceptible to pathogens; (3) causing the overgrowth of harmful bacteria, such as Salmonella and Escherichia coli, leading to diarrhea and intestinal diseases; and (4) increasing the chicken's stress response. In addition, chicks gradually develop their own unique gut microbiota from the time they hatch by contacting the microorganisms on the eggshell surface, and this microbiota changes with age, feed intake, and growth environment, gradually maturing. The diversity of the chicken's gut microbiota plays an important role in immune function and disease resistance. Therefore, maintaining the balance of gut microbiota during the brooding period and promoting the colonization and reproduction of beneficial bacteria is conducive to the gut health of chicks, thereby promoting a smooth transition in early growth and development and improving growth performance.

[0005] Coccidiosis in chickens is a serious protozoan disease caused by Eimeria species parasitizing the intestinal mucosal epithelial cells of chickens. Seven pathogens are recognized, with *Eimeria tenella* and *Eimeria virulence* being the most pathogenic, and *Eimeria giant* being the most widespread. It is estimated that coccidiosis causes economic losses of up to $3 billion annually worldwide, and the direct and indirect economic losses are difficult to accurately estimate. Currently, the control of chicken coccidiosis mainly relies on anticoccidial drugs and live vaccines. However, long-term drug use has led to increasingly serious drug resistance in coccidia, and improper drug use can leave residues in meat and eggs. Live vaccines can produce good immune protection, but improper use can cause coccidiosis, and there is a risk of vaccine strain virulence reversion and shedding of the virus during use. Therefore, there is an urgent need for new methods to control chicken coccidiosis in clinical practice.

[0006] Coccidiosis infection not only directly damages the intestinal mucosa but also severely disrupts the composition and functional homeostasis of the chicken's intestinal microbiota. Studies have found that infection with *Eimeria tenella* causes significant changes in the intestinal flora structure, primarily manifested as a dual imbalance of a sharp decrease in beneficial bacteria and abnormal proliferation of pathogenic bacteria. Specifically, the relative abundance of important probiotics such as *Lactobacillus* and *Bifidobacterium* decreases dramatically, while the abundance of potentially pathogenic bacteria such as *Escherichia coli* and *Clostridium* increases significantly. *Eimeria virulentis* infection can also lead to a significant decrease in the abundance of *Lactobacillus* and promote the proliferation of opportunistic pathogens, including *Shigella* and *Escherichia coli*, ultimately causing severe intestinal dysbiosis. This imbalance of pathogenic flora is not only a consequence of coccidiosis infection but also a significant factor in exacerbating intestinal inflammation, prolonging the course of the disease, and hindering host recovery, creating a vicious cycle. Eimeria giantiform infection can affect the microbial diversity and abundance in the small and cecum of chickens, and mixed infection with Clostridium perfringens can induce necrotizing enteritis, accompanied by typical intestinal lesions and a significant decrease in the percentage of relative body weight gain. Notably, different species of Eimeria exhibit significant species-specific differences in their effects on the gut microbiota, a phenomenon that profoundly reflects the unique pathogenic mechanisms of each coccidia species and the complex and delicate interaction patterns between them and their hosts.

[0007] Probiotics have garnered widespread attention as a green and safe regulatory tool for addressing the pathological changes and gut microbiota dysbiosis caused by coccidiosis infection. Studies have found that adding Bacillus subtilis to feed can alleviate gut microbiota dysbiosis in chickens caused by Eimeria tenella infection, thereby reducing intestinal tissue damage caused by Eimeria tenella. Adding Weizmannii coagulans (SANK70258) to feed can alleviate clinical symptoms caused by Eimeria giantiformis infection and enhance intestinal barrier function by regulating the intestinal immune response and microbial community structure in broilers. However, research reports on the anticoccidial effects of Lactobacillus reuteri are extremely limited, and those reports are of non-chicken strains. In fact, most of the probiotics reported in existing research literature are non-chicken strains, which to some extent limits their adaptability and efficacy in chickens. Given that host specificity is a key factor affecting probiotic colonization and function, it can be inferred that chicken-derived probiotic strains have better survival adaptability and stronger biological activity in the chicken gut compared to non-chicken strains. Therefore, the rational application of specific probiotic strains derived from chickens can not only maintain the balance of the chicken gut microbiota but also effectively reduce the harm of coccidiosis, providing an important technical solution for the prevention and control of chicken coccidiosis. Thus, the development and application of *Lactobacillus reuteri* derived from chickens has significant innovative value and broad application prospects. However, developing multifunctional *Lactobacillus reuteri* derived from chickens, possessing anti-coccidiosis functions and enhancing chicken immune function, requires long-term arduous efforts from researchers and some fortunate coincidences. Summary of the Invention

[0008] Purpose of the invention: The technical problem to be solved by the present invention is to provide a strain of Limosilactobacillus reuteri S3 that can alleviate intestinal lesions caused by various coccidiosis infections, is safe and harmless to chicks, can significantly promote weight gain, improve immune organ index, and promote the colonization of various beneficial bacteria in the chicken intestine to improve the intestinal flora.

[0009] Another technical problem that the present invention also solves is to provide a probiotic preparation containing the aforementioned Lactobacillus reuteri S3.

[0010] Another technical problem to be solved by the present invention is to provide the application of the aforementioned Limosilactobacillus reuteri S3 or probiotic agent in the preparation of anticoccidia drugs.

[0011] Another technical problem to be solved by the present invention is to provide the application of the aforementioned Limosilactobacillus reuteri S3 or probiotic agent in the preparation of drugs for the prevention and treatment of coccidiosis.

[0012] Another technical problem to be solved by the present invention is to provide the application of the aforementioned Limosilactobacillus reuteri S3 or probiotic agent in the preparation of products that improve the growth performance of chicks.

[0013] Another technical problem to be solved by the present invention is to provide the application of the aforementioned Limosilactobacillus reuteri S3 or probiotic agent in the preparation of products that enhance chicken immunity.

[0014] The technical problem that this invention also aims to solve is to provide the application of the aforementioned Limosilactobacillus reuteri S3 or probiotic agent in the preparation of products that promote the colonization of beneficial bacteria in chicks and improve the intestinal flora.

[0015] The final technical problem to be solved by this invention is to provide the application of the aforementioned Limosilactobacillus reuteri S3 or probiotic agent in the preparation of chicken feed additives.

[0016] Technical solution: A strain of *Limosilactobacillus reuteri* S3, which is deposited at the China Center for Type Culture Collection (CCTCC) on June 9, 2025, with accession number CCTCC NO: M 20251305.

[0017] The present invention also includes a probiotic agent containing the aforementioned *Limosilactobacillus reuteri* S3.

[0018] The present invention also includes the application of the aforementioned Limosilactobacillus reuteri S3 or the aforementioned probiotic agent in the preparation of drugs for treating coccidiosis.

[0019] The present invention also includes the application of the aforementioned Limosilactobacillus reuteri S3 or the aforementioned probiotic agent in the preparation of drugs for the prevention and treatment of coccidiosis.

[0020] The coccidia include one or more of Eimeria tenella, Eimeria giantiformis, and Eimeria toxicis.

[0021] The present invention also includes the application of the aforementioned Limosilactobacillus reuteri S3 or the aforementioned probiotic agent in the preparation of products that improve the growth performance of chicks.

[0022] The present invention also includes the application of the aforementioned Limosilactobacillus reuteri S3 or the aforementioned probiotic agent in the preparation of products that enhance chicken immunity.

[0023] The present invention also includes the application of the aforementioned Limosilactobacillus reuteri S3 or the aforementioned probiotic agent in the preparation of products that promote the colonization of beneficial bacteria in chicks and improve the intestinal flora.

[0024] The present invention also includes the application of the aforementioned Limosilactobacillus reuteri S3 or the aforementioned probiotic agent in the preparation of chicken feed additives.

[0025] The drug, product, or additive mentioned herein is an oral preparation.

[0026] Beneficial Effects: Compared with existing technologies, this invention has the following advantages: The *Lactobacillus reuteri* S3 used in this invention is derived from the small intestine of Leghorn SPF chickens. It has strong DF1 cell adhesion ability and exhibits significant inhibitory effects on common pathogenic bacteria (such as *Salmonella pullorum*, *Escherichia coli*, and *Staphylococcus aureus*). This strain can be used to prepare probiotic additives for chicks to improve chick growth performance, enhance immune organ development, promote the colonization of beneficial bacteria in chicks, enhance intestinal flora diversity, and reduce the harm of coccidiosis infection. This strain is safe and harmless for continuous feeding to chicks, is environmentally friendly, and has a significant growth-promoting effect on chicks. It can also promote thymus development, thereby enhancing the chicken's immunity. This strain can promote the colonization of various beneficial bacteria in the chick's intestines and improve the balance of intestinal flora. This strain can effectively reduce the harm of Eimeria tenella, Eimeria giantiformis, and Eimeria toxicis infections to chicks. This is demonstrated by the fact that supplementing with Lactobacillus reuteri S3 can reduce the clinical symptoms and intestinal lesions caused by coccidiosis, reduce the harm of coccidiosis to chick weight gain, and reduce the excretion of coccidia oocysts. It has anti-coccidiosis ability, indicating that this strain has a preventive and control effect on chicken coccidiosis infection. Attached Figure Description

[0027] Figure 1 Colony diagram of Lactobacillus reuteri S3 on MRS agar medium;

[0028] Figure 2 Gram-stained microscopic image of Lactobacillus reuteri S3;

[0029] Figure 3 The graph shows the tolerance results of Lactobacillus reuteri isolates to artificial gastric fluid (A) and artificial intestinal fluid (B);

[0030] Figure 4 Figure showing the tolerance of Lactobacillus reuteri isolates to fresh chicken bile;

[0031] Figure 5 The image shows the adhesion ability of Lactobacillus reuteri isolates to DF-1 cells.

[0032] Figure 6 Figure 1 shows the effects of Lactobacillus reuteri S3 on body weight gain and different organ indices in SPF chickens. (A) Average daily weight gain; (B) Liver; (C) Spleen; (D) Kidney; (E) Thymus; (F) Bursa of Fabricius.

[0033] Figure 7 The results show the effects of Lactobacillus reuteri S3 on the gut microbiota diversity (A, B) and community structure composition (C, D) of SPF chickens.

[0034] Figure 8 This is a graph showing the results of blood stool counts in each group after the insect attack. Detailed Implementation

[0035] The applicant will now provide a detailed description of the preparation and application processes of the product of this invention, using specific embodiments, to facilitate a clear understanding of the invention by those skilled in the art. However, it should be understood that the following embodiments should not be construed as limiting the scope of protection claimed in this application.

[0036] Example 1: Isolation, identification, screening, and preservation of Limosilactobacillus reuteri S3

[0037] 1. Isolation and taxonomic identification of *Lactobacillus reuteri*

[0038] In June 2023, intestinal contents were collected from White Leghorn SPF chickens for the isolation of *Lactobacillus reuteri*. The specific procedure was as follows: Intestinal contents were scraped off using a sterile scalpel under sterile conditions. 1 g of the contents was diluted in 10 mL of phosphate buffer (PBS) and thoroughly vortexed. 1 mL of the sample suspension was serially diluted with PBS, and 100 µL of each suspension was evenly spread onto sterile lactic acid bacteria medium (MRS medium) agar plates. The plates were incubated anaerobicly at 37°C for 24 h. Single colonies exhibiting typical lactic acid bacteria characteristics—medium-sized, slightly white, round, slightly raised, and with regular edges—were selected and purified by streak subculturing. The results are shown in the table below. Figure 1 The PBS buffer used in this invention has a concentration of 0.01M and a pH of 7.4.

[0039] The isolated strain was purified four times until the colonies on the plates exhibited a uniform morphology. Single colonies were selected and inoculated into MRS liquid medium, and anaerobically cultured at 37°C for 24 h to preserve the culture. Gram staining and microscopic examination of the isolated strain revealed blue-purple, short rod-shaped cells resembling *Lactobacillus*. The results are shown in the table below. Figure 2 Single colonies were selected and cultured overnight in MRS liquid medium. Genomic DNA was extracted from the bacterial suspension using a DNA extraction kit. The genomic DNA was amplified by PCR using universal 16S rDNA primers, and then the 16S rDNA gene sequence was obtained by sequencing. The sequence was entered into NCBI for BLAST comparison and phylogenetic tree construction. The results showed that the isolated strain had >99% similarity to Lactobacillus reuteri, and the isolated strain was identified as Lactobacillus reuteri.

[0040] 2. Screening and preservation of Lactobacillus reuteri S3

[0041] In vitro resistance tests to artificial gastric and intestinal fluids: Artificial gastric and intestinal fluids were prepared according to the 2010 edition of the Chinese Pharmacopoeia and filtered through a 0.22 µm microporous sterile membrane (aqueous). 1 mL of overnight culture was centrifuged (6000 rpm, 5 min). The supernatant was discarded, and the culture was resuspended in 1 mL of sterile PBS and mixed well. The solution was then diluted to a concentration of 1×10⁻⁶. 8 CFU / mL, 100 µL of bacterial suspension was mixed with 900 µL of simulated gastric fluid or 900 µL of intestinal fluid, and anaerobically cultured at 37℃ for 0 and 3 h, respectively. The tolerance of the isolated strain to simulated gastric and intestinal fluids was assessed and compared with two commercially available lactic acid bacteria strains, CLV (Limosilactobacillus vaginalis ATCC 55739) and CLP (Lactobacillus plantarum ATCC8014). All commercially available lactic acid bacteria were purchased from Ningbo Mingzhou Biotechnology Co., Ltd. Results are shown below. Figure 3 Of the eight isolated strains (numbered S2, S3, S4-2, C3, C3-2, C-4, C4-2, and 9), four strains (S2, S3, C3, and C3-2) showed resistance to artificial gastric fluid ( Figure 3 A in the middle) and intestinal fluid ( Figure 3 Both of the B) showed high tolerance.

[0042] In vitro test for tolerance to fresh bile: Take 1 mL of a solution with a concentration of 1×10 8 CFU / mL bacterial suspensions were added to fresh chicken bile at final volumes (v / v, bile / (bacterial suspension + bile)) of 40%, 60%, and 80%, respectively. Strains without added bile (CLV, CLP, S2, S3, C3, C3-2) served as control groups. The cultures were anaerobically incubated at 37°C for 3 h, serially diluted, and plated onto MRS agar plates. After overnight incubation, colonies were counted. Survival rate (%) = number of colonies in experimental groups / number of colonies in control groups. Each group was replicated three times. This method was used to evaluate the tolerance of commercially available lactic acid bacteria CLV, CLP, and isolated strains (S2, S3, C3, and C3-2) to fresh chicken bile. Results are shown below. Figure 4 Of the four isolated strains, three (S2, S3, and C3) showed high and stable tolerance to fresh chicken bile.

[0043] In vitro antibacterial test: Salmonella Pullorum ATCC19945, Escherichia coli ATCC 25922, and Staphylococcus aureus ATCC 25923, preserved by the Department of Veterinary Parasitology, College of Veterinary Medicine, Yangzhou University, were selected as pathogen indicator bacteria to test the antibacterial ability of the isolated strains. The "cup-and-dish method" was used, with 100 µL of a 1×10⁻⁶ concentration of [unspecified bacteria] being placed in a clean bench. 8 CFU / mL of pathogen indicator bacteria were evenly spread on the surface of LB agar plates. Autoclaved Oxford cups were placed on the plates containing the indicator strains. The tested Lactobacillus strains (CLV, CLP, S2, S3, and C3) were centrifuged at 6000 rpm for 5 min to remove bacteria. The supernatant was injected into the Oxford cups containing the different indicator strains. The plates were then incubated at 37℃ for 24 h. The diameter of the inhibition zone (mm) was measured using calipers, with each group repeated three times. The results are shown in Table 1. All three isolated strains (S2, S3, and C3) showed the ability to inhibit the pathogen, with strains S3 and C3 exhibiting stronger inhibitory effects.

[0044] Table 1. Inhibitory effect of isolated strains on indicator pathogens

[0045]

[0046] Note: For data in the same column, different letters indicate significant differences (P < 0.05), and the same letters indicate no significant differences (P > 0.05). The same applies to the following table.

[0047] Lactobacillus cell adhesion assay: DF-1 cells were placed at 10 cells per well. 5 / cell suspension was added to 24-well plates (with cell spreaders) and incubated at 37°C in a 5% CO2 cell culture incubator for 24 h; after the cells had completely adhered, the plates were washed twice with sterile PBS, and 1 mL of 10-cell suspension was added to each well. 8 CFU / mL suspensions of Lactobacillus (CLV, CLP, S3, and C3) were prepared, with three replicates per strain, and incubated for 3 h. The coverslips were then removed and washed three times with sterile PBS. The bacteria were fixed with 4% paraformaldehyde for 30 min, dried, and stained with crystal violet. The adhesion of Lactobacillus cells was observed using a biological microscope (oil immersion), and the number of bacteria adhering to the cells was counted in five randomly selected fields. Results are shown below. Figure 5 Both isolated strains (S3 and C3) had the ability to adhere to DF-1 cells, with S3 exhibiting the best adhesion performance.

[0048] Based on the overall performance of the isolates in terms of resistance to gastric juice, intestinal juice, and fresh bile, as well as their in vitro antibacterial and cell adhesion abilities, isolate S3 exhibits superior comprehensive performance. Therefore, isolate S3 was deposited at the China Center for Type Culture Collection on June 9, 2025, with accession number CCTCC M 20251305, and classified as Limosilactobacillus reuteri S3.

[0049] Example 2 Safety test of Lactobacillus reuteri S3

[0050] Using Leghorn SPF chickens (purchased from Zhejiang Lihua Agricultural Technology Co., Ltd.) as target animals, an oral toxicity test was conducted to verify the safety of *Lactobacillus reuteri* S3. Under standard sanitary conditions, SPF chickens were weighed at 3 days of age, and chickens of similar weight were randomly divided into 4 groups of 10 each. The average weights of the groups were 47.62±1.21 g, 47.58±1.19 g, 47.54±1.13 g, and 47.63±1.16 g, respectively. The three experimental groups were administered a low dose (2×10⁻⁶ g) orally. 8 CFU / mL (Low), Medium dose (6×10) 8 CFU / mL (Middle) and high dose (1×10) 9 200 µL of *Lactobacillus reuteri* S3 at a concentration of CFU / mL (High) was administered orally to the control group (PBS) at 200 µL PBS. After 18 days of continuous oral administration of *Lactobacillus reuteri* S3, routine health parameters of chicks, including average daily gain (ADG) and organ index, were calculated to assess the safety of the isolate. Chicks were euthanized by CO2 inhalation and cervical dislocation, and liver, spleen, kidney, thymus, and bursa of Fabricius were collected and weighed. The organ index was measured as follows: Organ index = organ weight / chick body weight. Results are shown in […]. Figure 6 Compared with the control group, the low-dose and medium-dose groups showed significantly increased mean daily weight gain (ADG) (P < 0.05) and significantly increased thymus development index (P < 0.05). However, no significant differences were observed in other organ development indices among the different groups.

[0051] Example 3: Effects of Lactobacillus reuteri S3 on the gut microbiota of chicks

[0052] On day 7 following oral administration of *Lactobacillus reuteri* S3 for 18 consecutive days according to the high, medium, and low dose groups in Example 2, three chickens were randomly selected from each dose group and the control group. After euthanasia by CO2 inhalation and cervical dislocation, microorganisms were collected from the jejunum (High-s-7 in the high-dose group, Middle-s-7 in the medium-dose group, Low-s-7 in the low-dose group, and Uc-s-7 in the control group) and cecum (High-c-7, Middle-c-7, Low-c-7, and Uc-c-7) for 16S rRNA full-length amplicon sequencing. The results are as follows: Figure 7 As shown, the low-dose group of *Lactobacillus reuteri* S3 increased jejunal (… Figure 7 A in the middle) and the cecum ( Figure 7 The microbial diversity of B) and the optimization of the structure and composition of the gut microbiota in chicks by oral administration of low and medium doses of Lactobacillus reuteri S3 (B) Figure 7 The C (jejunum) and D (cecum) in the intestine increase the number of beneficial bacteria.

[0053] Example 4: Efficacy of *Lactobacillus reuteri* S3 against *Eimeria tenella*, *Eimeria giant*, and *Eimeria virulence* infections.

[0054] 1. Chick grouping and experimental procedures

[0055] One-day-old Suqin green-shelled chickens were purchased from the Jiangsu Institute of Poultry Science, Chinese Academy of Agricultural Sciences. They were transported back to the laboratory immediately after hatching and fed chick feed produced by Jiangsu Xiehe Pharmaceutical Biotechnology Co., Ltd., which contained no anticoccidial or antibiotic drugs. At three days of age, each chick was weighed, and 70 healthy chicks of similar weight were randomly divided into seven groups: S3 / Eimeria tenella group, S3 / Eimeria macrocarpa group, S3 / Eimeria toxicis group, PBS / Eimeria tenella group, PBS / Eimeria macrocarpa group, PBS / Eimeria toxicis group, and PBS / uninfected coccidia group. Each group consisted of 10 chickens. From four days of age, the first three groups of chickens were orally administered 200 µL of Lactobacillus reuteri S3 (2×10⁻⁶) daily. 8 (CFU / mL), the chickens in the latter four groups were orally administered the same amount of PBS daily. At 14 days of age, each chicken in the S3 / Eimeria tenella group and the PBS / Eimeria tenella group was orally infected with 5 × 10 CFU / mL. 4 Each chicken was orally infected with 5 × 10tender Eimeria sporulated oocysts; in the S3 / Giant Eimeria group and the PBS / Giant Eimeria group. 4 Giant Eimeria sporulated oocysts; S3 / Eimeria toxicans group and PBS / Eimeria toxicans group, each chicken was orally infected with 1.5 × 10⁻⁶ oocysts. 4Eimeria tenella sporulated oocysts were administered orally to each chicken in the PBS / uninfected coccidia group. The *Eimeria tenella*, *Eimeria giant*, and *Eimeria tenella* strains used in this invention were derived from an article published by our research group (Li Jianmei. ITS region sequence analysis of four chicken *Eimeria* strains and the immune interaction between *Eimeria tenella* and *Eimeria giant* or *Eimeria tenella* [D]. Yangzhou: Yangzhou University, 2011). After infection with coccidia, the chickens' mental state, appetite, water intake, and mortality were observed daily. After the appearance of bloody stools, the number of bloody stools was counted every 12 hours. After the appearance of oocysts in the feces, feces from each experimental group were collected every 12 hours, and the oocysts were counted using the McMaster counting method. The experiment ended on the 8th day after infection, and all chickens were euthanized and dissected for intestinal lesion scoring. Simultaneously, cecal contents were collected from the S3 / Eimeria tenella group, PBS / Eimeria tenella group, S3 / Eimeria toxicis group, and PBS / Eimeria toxicis group for oocyst counting. Experimental groups and treatments are shown in Table 2.

[0056] Table 2 Experimental Groups and Treatments

[0057]

[0058] 2. Evaluation Indicators and Results

[0059] (1) Clinical symptoms

[0060] The results showed that on the 4th day after coccidiosis infection, chickens in all infected groups began to exhibit symptoms such as lethargy, reduced appetite and water intake, and ruffled feathers. However, compared with the group that did not receive oral Lactobacillus reuteri S3, the clinical symptoms were milder in the group that received oral Lactobacillus reuteri S3. The number of bloody stools in the S3 / Eimeria tenella group was significantly less than that in the PBS / Eimeria tenella group, and similarly, the number of bloody stools in the S3 / Eimeria toxoid group was significantly less than that in the PBS / Eimeria toxoid group. (See attached results). Figure 8 Neither the S3 / Eimeria giantis group nor the PBS / Eimeria giantis group showed bloody stools, but both excreted cotton-like feces, with the former producing fewer feces than the latter.

[0061] (2) Survival rate (%)

[0062] Survival rate (%) = (Number of surviving chickens at the end of the experiment / Number of chickens at the beginning) × 100%

[0063] The results showed that no chickens died in any of the experimental groups, and the survival rate was 100%.

[0064] (3) Average weight gain and relative weight gain rate (%)

[0065] Chickens were weighed individually at the time of grouping, before the insect invasion, and at the end of the experiment, and the average weight gain and relative weight gain rate were calculated.

[0066] Average weight gain 1 = body weight before insect invasion - body weight at the time of grouping; relative weight gain rate 1 = (weight gain of each experimental group before insect invasion / weight gain of the control group) × 100%; average weight gain 2 = body weight at the end of the experiment - body weight before insect invasion; relative weight gain rate 2 = (weight gain of each experimental group at the end of the experiment / weight gain of the control group) × 100%.

[0067] The results showed that in the pre-infection stage (average weight gain 1), the average weight gain of chickens in the three experimental groups orally administered *Lactobacillus reuteri* S3 was higher than that of the other four experimental groups, indicating that oral administration of *Lactobacillus reuteri* S3 can promote weight gain in chickens. In the infection stage (average weight gain 2), the relative weight gain rate of each group orally administered *Lactobacillus reuteri* S3 was significantly higher than that of the corresponding control group, indicating that oral administration of *Lactobacillus reuteri* S3 can reduce weight loss caused by coccidiosis infection. Specific results are shown in Table 3.

[0068] Table 3. Changes in weight gain of chicks after parasite treatment

[0069]

[0070] (4) Lesion score and lesion score reduction rate (%)

[0071] Lesion scoring: All chickens were euthanized on the 8th day after infection, and the intestinal lesions were scored according to the Johnson and Reid (1970) criteria (0-4 points).

[0072] The reduction rate of lesion score (%) = (PBS / lesion score of coccidia-infected group - S3 / lesion score of coccidia-infected group) / PBS / lesion score of coccidia-infected group × 100%.

[0073] The results showed that the average intestinal lesion scores in the three experimental groups treated with oral administration of *Lactobacillus reuteri* S3 were all lower than those in the corresponding control groups, but the differences between the groups were not statistically significant (P > 0.05). The reduction rates of lesion scores in the S3 / *Eimeria tenella* group, the S3 / *Eimeria giantiella* group, and the S3 / *Eimeria toxicae* group were 23.53%, 31.25%, and 50.00%, respectively, indicating that oral administration of *Lactobacillus reuteri* S3 can alleviate intestinal damage caused by coccidia parasitism. The intestinal lesion score in the negative control group was 0. Detailed results are shown in Table 4.

[0074] Table 4. Intestinal lesion scores and reduction rates after worm infection in chicks.

[0075]

[0076] (4) Oocyte reduction rate

[0077] Oocyst counting. After oocysts appear in the feces of each test group, all feces of each group are collected every 12 hours, and the oocyst counting is performed using the McMaster method. After the intestinal lesion scoring is completed, the cecal contents and mucosa are taken, digested with 2% pepsin solution, and then subjected to oocyst counting. Finally, the number of fecal oocysts and the number of cecal oocysts are summed to obtain the total oocyst amount of each test group.

[0078] Oocyst reduction rate (%) = (Oocyst yield of PBS / coccidia-infected group - Oocyst yield of S3 / coccidia-infected group) ÷ Oocyst yield of PBS / coccidia-infected group × 100%

[0079] The results showed that the oocyst reduction rates of the S3 / Eimeria tenella group, the S3 / Eimeria maxima group, and the S3 / Eimeria necatrix group were 29.16%, 25.37%, and 28.57%, respectively, indicating that oral administration of Limosilactobacillus reuteri S3 can inhibit the growth and development of coccidia in the chicken intestine and reduce oocyst formation. The specific results are shown in Table 5.

[0080] Table 5 Oocyst reduction rate of chicks after challenge with coccidia

[0081]

[0082] (5) Anticoccidial Index (ACI)

[0083] ACI = (Relative weight gain rate + Survival rate) - (Lesion value + Oocyst value).

[0084] Lesion value = Average intestinal lesion score of each group × 10.

[0085] Oocyst value. When the relative oocyst yield is 0 to 1%, the oocyst value is 0; when the relative oocyst yield is 1 to 25%, the oocyst value is 5; when the relative oocyst yield is 26 to 50%, the oocyst value is 10; when the relative oocyst yield is 51 to 75%, the oocyst value is 20; when the relative oocyst yield is 76 to 100%, the oocyst value is 40.

[0086] Relative oocyst yield (%) = (Oocyst yield of PBS / coccidia-infected group ÷ Oocyst yield of S3 / coccidia-infected group) × 100%.

[0087] Effect judgment: ACI>180 indicates high anticoccidial efficacy; 160<ACI<180 indicates medium anticoccidial efficacy; 120<ACI<160 indicates low anticoccidial efficacy; ACI<120 indicates no anticoccidial efficacy.

[0088] Table 6 Anticoccidial Index (ACI)

[0089]

[0090] The results showed that the ACI values ​​of the S3 / Eimeria tenella group, the S3 / Eimeria giant group, and the S3 / Eimeria toxicae group were 130.67, 148.06, and 163.40, respectively, all of which demonstrated anti-coccidial ability, indicating that Lactobacillus reuteri S3 has a control effect on coccidiosis in chickens.

Claims

1. A strain of *Lactobacillus reuteri* ( Limosilactobacillus reuteri S3, characterized in that, The Lactobacillus reuteri ( Limosilactobacillus reuteri S3 is deposited at the China Center for Type Culture Collection on June 9, 2025, with accession number CCTCC NO: M 20251305.

2. A probiotic preparation, characterized in that, The probiotic agent contains *Lactobacillus reuteri* as described in claim 1. Limosilactobacillus reuteri S3.

3. The *Lactobacillus reuteri* as described in claim 1 (… Limosilactobacillus reuteri The use of the probiotic agent according to claim 3 or claim 2 in the preparation of a drug for treating chicken coccidia, wherein the chicken coccidia is one or more of Eimeria tenella, Eimeria giantiformis, and Eimeria toxicis.

4. The *Lactobacillus reuteri* as described in claim 1 (… Limosilactobacillus reuteri The use of the probiotic agent according to claim 3 or claim 2 in the preparation of a drug for preventing coccidiosis in chickens, wherein the coccidia is one or more of Eimeria tenella, Eimeria giantiformis, and Eimeria toxicis.

5. The *Lactobacillus reuteri* as described in claim 1 (… Limosilactobacillus reuteri The application of the probiotic agent as described in claim 3 or claim 2 in the preparation of products that improve the growth performance of chicks.

6. The *Lactobacillus reuteri* as described in claim 1 (… Limosilactobacillus reuteri The use of the probiotic agent as described in S3 or claim 2 in the preparation of products that enhance chicken immunity.

7. The *Lactobacillus reuteri* as described in claim 1 ( Limosilactobacillus reuteri The use of the probiotic agent as described in S3 or claim 2 in the preparation of products that promote the colonization of beneficial bacteria in chicks and improve the intestinal flora.

8. The *Lactobacillus reuteri* as described in claim 1 (… Limosilactobacillus reuteri The application of the probiotic agent described in S3 or claim 2 in the preparation of chicken feed additives.

9. The application according to any one of claims 3 to 8, characterized in that, The drug, product, or additive is an oral preparation.

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