A probiotic bacillus antagonistic to clostridium perfringens type g and application thereof

Abstract

The application discloses antagonistic Bacillus of Clostridium perfringens type G and application, the Bacillus is Bacillus sonorensis CB-6, and is classified and named as Bacillus sonorensis CB-6, the preservation number is CCTCC NO: M20251373, the preservation date is June 12, 2025, and the preservation unit is China Center for Type Culture Collection, which is located at Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.The sterile fermentation supernatant of Bacillus sonorensis CB-6 provided by the application has neutral pH value, can inhibit Clostridium perfringens with high activity, the bacteriostatic effect is not dependent on organic acid such as lactic acid, the bacteriostatic effect is significant and stable, can significantly enhance the immunity of laying hens after Clostridium perfringens infection and improve the egg production rate, and solves the problem that laying hens are prone to necrotic enteritis during the egg production period and are not suitable for using antibiotics for prevention and control.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microecological preparations and feed additives, specifically relating to a probiotic Bacillus that antagonizes Clostridium perfringens type G and its application. Background Technology

[0002] Necrotic enteritis in poultry is a common intestinal disease in laying hen farming, and *Clostridium perfringens* is the main causative agent. Although *Clostridium perfringens* is naturally present in the intestines of healthy laying hens, it only proliferates abnormally and releases various toxins (such as alpha toxins and beta toxins) when the chicken's intestinal barrier is damaged, its microbiome is disrupted, or its immunity is weakened. This leads to intestinal inflammation, necrosis, diarrhea, decreased production performance, and even death, causing serious economic losses to the laying hen farming industry.

[0003] Since the use of antibiotics to control necrotic enteritis in laying hens is prohibited during the egg-laying period, finding safe and effective alternatives has become a research hotspot in recent years. Probiotic intervention has attracted considerable attention as an effective strategy for regulating gut microbiota and enhancing immunity. Currently, there are no literature reports of Bacillus strains that directly inhibit Clostridium perfringens without relying on organic acid production, nor are there any research reports on Bacillus strains that antagonize Clostridium perfringens-induced necrotic enteritis in laying hens.

[0004] Therefore, a probiotic Bacillus antagonizing Clostridium perfringens type G and its application are urgently needed. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a probiotic Bacillus that antagonizes Clostridium perfringens type G and its application.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0007] The first objective of this invention is to provide a probiotic Bacillus that antagonizes Clostridium perfringens type G. This Bacillus is Bacillus sonorensis CB-6, classified as Bacillus sonorensis CB-6, with accession number CCTCC NO: M20251373, accession date June 12, 2025, and deposited at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, within the campus of Wuhan University.

[0008] Preferably, the Sonora desert Bacillus CB-6 is used to inhibit the activity of avian pathogenic Clostridium perfringens ML2.

[0009] The second objective of this invention is to provide the application of a probiotic Bacillus antagonizing Clostridium perfringens type G in the preparation of a formulation for preventing and treating necrotizing enteritis in chickens.

[0010] Preferably, the content of Sonora desert Bacillus CB-6 in the formulation is 10. 8 ~10 10 CFU / g.

[0011] Preferably, the preparation is used as a feed additive.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The Sonora desert bacillus CB-6 provided by this invention exhibits strong safety and resilience, making it suitable for preparation into probiotic products for practical production. The aseptic fermentation supernatant of Sonora desert bacillus CB-6 provided by this invention has a neutral pH, enabling it to highly inhibit Clostridium perfringens. This antibacterial effect is independent of organic acids such as lactic acid, and its antibacterial effect is significant and stable. It solves the problem of necrotizing enteritis in laying hens during the laying period, which is difficult to control with antibiotics. It can significantly enhance the immunity of laying hens infected with Clostridium perfringens and increase egg production.

[0014] Cell Preservation:

[0015] The Sonora desert Bacillus CB-6 provided in this invention was obtained by the inventors themselves through screening. It is classified and named Bacillus sonorensis CB-6, with accession number CCTCC NO: M20251373, accession date of June 12, 2025, and deposited at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, within the campus of Wuhan University. Attached Figure Description

[0016] Figure 1 This is the result of an in vitro antibacterial experiment on the fermentation supernatant of Sonora desert Bacillus CB-6 after 12-24 h of culture (Clostridium perfringens ML2 is the indicator bacterium).

[0017] Figure 2 This is the growth status of Sonora desert Bacillus CB-6 on LB agar plates in this invention.

[0018] Figure 3 This is the Gram staining result (×1000) of Sonora desert Bacillus CB-6 in this invention.

[0019] Figure 4This is the bacterial phylogenetic tree constructed by Sonora desert Bacillus CB-6 in this invention using the neighbor-joining method.

[0020] Figure 5 This is the growth curve of Sonora desert Bacillus CB-6 in this invention.

[0021] Figure 6 This is the result of the antibiotic susceptibility test of the Sonora desert Bacillus CB-6 strain in this invention.

[0022] Figure 7 This is a gel image of the virulence gene detection of Bacillus desertica CB-6 in this invention.

[0023] Wherein, M: DL2000 DNA molecular weight standard; Lane 1: positive control (Bacillus cereus AH7-7); Lane 2: Sonora desert Bacillus CB-6 in this invention; Figure 7 In this context, A represents the result of detecting the virulence gene nheA; Figure 7 In the figure, B represents the result of detecting the virulence gene nheB; Figure 7 C in the figure represents the result of detecting the virulence gene nheC; Figure 7 The "D" in the figure represents the result of detecting the virulence gene hblD. Figure 7 In the figure, E represents the result of detecting the virulence gene bceT; Figure 7 F in the figure represents the result of detecting the virulence gene cytK; Figure 7 G in the figure represents the result of detecting the virulence gene entFM.

[0024] Figure 8 This is the result of detecting the expression levels of inflammatory factors in the serum of laying hens during a feeding experiment using Sonora desert Bacillus CB-6 to antagonize necrotizing enteritis in laying hens, as described in this invention. Figure 8 In this context, A represents the result of serum IL-1β expression level detection. Figure 8 B in the figure represents the result of serum TNF-α expression level detection; Figure 8 In the figure, C represents the result of serum IL-10 expression level detection.

[0025] Figure 9 This invention relates to the detection results of immunoglobulin content in the serum of laying hens during a feeding trial of Sonora desert Bacillus CB-6 antagonizing necrotizing enteritis in laying hens. Among other things, Figure 9 The A in the figure represents the result of serum IgA content detection; Figure 9 B in the figure represents the result of serum IgM content detection; Figure 9 In the figure, C represents the result of serum IgY content detection. Detailed Implementation

[0026] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0027] Example 1: Isolation and identification of Sonora desert Bacillus CB-6.

[0028] I. Strains Isolation.

[0029] (1) Sample collection: Select robust individuals from a flock of healthy white-feathered broiler chickens and collect cecal contents.

[0030] (2) Sample preparation: In a laminar flow hood, perform serial dilution with sterile distilled water, and take 100 μL of 10 6 and 10 7 The contents of the dilution gradient were placed on solid LB medium and MRS medium and spread evenly with a spreader. The LB medium was incubated at 37°C for 24 h, and the MRS medium was incubated in an anaerobic incubator for 24 h. The colony morphology was then observed.

[0031] (3) Gram staining: Gram staining is purple, short rod-shaped, and can be single, in pairs or in chains.

[0032] (4) One strain of Bacillus was obtained with an average inhibition zone diameter of 21.94 mm in the fermentation broth after 12-24 h at a pH of 6-7 (see Figure 1 ).

[0033] II. Identification of strain species.

[0034] Based on the above identification, the 16S rDNA gene sequence of *Bacillus desertica* CB-6 in this embodiment was further detected to confirm the species to which *Bacillus desertica* CB-6 belongs. The specific steps are as follows:

[0035] (a) Extraction of the target strain genome.

[0036] (1) Take 1 mL of culture medium, centrifuge at 10000 rpm for 30 s, remove the supernatant as much as possible, and collect the bacterial cells.

[0037] (2) Add 200 μL of buffer RB to resuspend, centrifuge at 10000 rpm for 30 s, and discard the supernatant.

[0038] (3) Add 120 μL of lysozyme (20 mg / mL in 10 mM Tris-HCl, pH 8.0), invert and mix well, incubate at 37°C for 30-60 min. Centrifuge at 12000 rpm for 2 min, discard the supernatant and resuspend in 180 μL of buffer RB.

[0039] (4) Add 20 μL of proteinase K (20 mg / mL) solution, mix thoroughly, then add 200 μL of binding solution CB, vortex immediately to mix thoroughly, and place at 70°C for 10 min.

[0040] (5) After cooling, add 100 μL of isopropanol and immediately vortex to mix thoroughly. At this time, flocculent precipitate may appear.

[0041] (6) Add the mixture from the previous step (including any possible precipitate) to an adsorption column AC (place the adsorption column in the collection tube), centrifuge at 13000 rpm for 30-60 seconds, and discard the waste liquid in the collection tube.

[0042] (7) Add 500 μL of inhibitor removal solution IR, centrifuge at 12000 rpm for 30 s, and discard the waste liquid.

[0043] (8) Add 700 μL of washing buffer WB (with anhydrous ethanol added), centrifuge at 12000 rpm for 30 s, and discard the waste liquid.

[0044] (9) Add 500 μL of washing buffer WB (with anhydrous ethanol added), centrifuge at 12000 rpm for 30 s, and discard the waste liquid.

[0045] (10) Place the adsorption column AC back into the empty collection tube, centrifuge at 13000 rpm for 2 min, and remove as much of the washing liquid as possible to avoid the residual ethanol in the washing liquid inhibiting the downstream reaction.

[0046] (11) Remove the adsorption column AC and place it in a clean centrifuge tube. Add 50 μL of elution buffer EB (preheated in a 67-70℃ water bath beforehand) to the middle of the adsorption membrane. Let it stand at room temperature for 3-5 min, then centrifuge at 12000 rpm for 1 min. Add the resulting solution back to the adsorption column, let it stand at room temperature for 2 min, then centrifuge at 12000 rpm for 1 min.

[0047] (12) Store the DNA sample at -20℃ for later use.

[0048] (II) Amplification of the 16S rRNA gene:

[0049] The 16S rRNA sequence of the isolate *Bacillus desertica* CB-6 was amplified using universal bacterial primers. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and their DNA sequences are as follows:

[0050] Forward primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3, as shown in SEQ ID NO:1;

[0051] Reverse primer 1492R: 5'-GGTTACCTTGTTACGACTT-3', as shown in SEQ ID NO:2;

[0052] The PCR amplification system is shown in Table 1. Amplification conditions: 94℃ pre-denaturation for 5 min, 94℃ for 1 min, 50℃ for 30 s, 72℃ for 1.5 min, 30 cycles, followed by a 72℃ extension for 10 min. The PCR amplification products were analyzed by electrophoresis on a 0.8% agarose gel (containing ethidium bromide). The amplified fragment size was consistent with expectations, approximately 1500 bp.

[0053] Table 1: PCR reaction system.

[0054]

[0055] (III) Constructing a bacterial evolutionary tree to determine species and genus.

[0056] Bacterial genomic DNA was extracted and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were compared using BLAST on NCBI. A bacterial phylogenetic tree was constructed using MEGA 7.0 software via neighbor-joining. Analysis showed that the 16S rDNA sequence of strain CB-6 had the highest homology (99.72%) with *Bacillus desertica* HBUAS67343 (see [link to analysis]). Figure 4 The specimen was named *Bacillus desertis* CB-6, classified as *Bacillussonorensis* CB-6, with accession number CCTCC NO: M20251373, accession date June 12, 2025, and deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0057] Sonora desert bacillus CB-6 forms colonies approximately 3 mm in size on LB solid medium. These colonies are white, with a crater-like central protrusion, a dry surface, and are round or irregular in shape with slightly wavy edges (see...). Figure 2 This bacterium is Gram-positive and appears as a bluish-purple rod-shaped bacterium under a microscope. It grows singly or in short chains, with a round or oval shape in the center or at the tip, consistent with the morphological characteristics of the genus *Bacillus* (see...). Figure 3 ).

[0058] Example 2: Detection of the activity and stability of Sonora desert Bacillus CB-6 in inhibiting Clostridium perfringens.

[0059] Using Clostridium perfringens ML2, a type G bacterium from laying hens with typical necrotizing enteritis and carrying NetB toxin, as an indicator bacterium, and the sterile fermentation supernatant of the strain in this embodiment as an antibacterial agent, the in vitro antibacterial activity of the strain was tested. The specific operation steps are as follows.

[0060] (1) Preparation of indicator bacteria (Clostridium perfringens ML2, type G): The indicator bacteria were streaked on TSC plates and incubated at 37°C for 20 h in an anaerobic incubator; single colonies were picked and placed on RCM liquid medium and incubated at 37°C for 16 h in an anaerobic incubator to revive the indicator bacteria. The bacterial count of the cultured indicator bacteria was adjusted to 1.0 × 10⁻⁶. 6 CFU / mL.

[0061] (2) Preparation and treatment of CB-6 fermentation supernatant: The strain of this embodiment was streaked on a plate and cultured at 37℃ for 16h. The isolated strain was picked and cultured in 50mL of LB liquid medium at 37℃ and 200r / min for 24h to obtain the seed culture. The seed culture was inoculated into 100mL of LB liquid medium at an inoculation rate of 1% and cultured at 37℃ and 200r / min for 12h, 14h, 16h, 18h, 20h, 22h, and 24h respectively to obtain the fermentation broth. The prepared fermentation broth was centrifuged at 10000r / min for 5min. The supernatant after centrifugation was filtered through a 0.22µm sterile filter and its pH was measured using an acid-base analyzer.

[0062] (3) Antibacterial test: Take sterilized TSC medium and, when it has cooled but not solidified, add Clostridium perfringens culture medium equal to one-thousandth of the TSC medium volume. Mix well and pour into a petri dish. After air drying in a clean bench for 2 hours, punch holes in the TSC plate with a punch and seal the bottom with an alcohol lamp to prevent the bacterial solution from leaking to the bottom of the plate. Use a micropipette to draw 200 μL of CB-6 fermentation supernatant into the wells of the TSC plate, place it in an anaerobic incubator, and incubate for 10 hours. After incubation, remove the plate and measure the diameter of the inhibition zone with calipers.

[0063] The experimental results show that the aseptic fermentation supernatant of strain CB-6 in this embodiment is neutral (pH 7) and has a strong inhibitory effect on Clostridium perfringens ML2, that is, it can inhibit Clostridium perfringens without acid production. The results are shown in Table 2.

[0064] Table 2: Inhibition zone of Sonora desert Bacillus within 12-24 h.

[0065]

[0066] Example 3: Safety test of Sonora desert Bacillus CB-6.

[0067] I. Drug sensitivity test.

[0068] Antimicrobial susceptibility testing was conducted using antimicrobial susceptibility test discs (purchased from Hangzhou Microbial Reagent Co., Ltd.) for penicillins, cephalosporins, quinolones, aminoglycosides, and tetracyclines. The interpretation criteria were based on the latest version of the NCCLS standards provided by the World Health Organization (WHO) (2018 edition). The specific steps are as follows:

[0069] (1) The bacterial culture was inoculated into LB liquid medium at a rate of 1% and cultured on a shaker at 37°C and 200 rpm / min for 12 h;

[0070] (2) Use an OD meter to adjust the OD600nm value of the bacterial solution to 1;

[0071] (3) Use a sterile cotton swab to dip the bacterial solution and spread it on the LB agar plate. Rotate the plate 60 degrees each time, and finally spread it around the inner edge of the plate twice. Repeat this several times to ensure that it is spread evenly.

[0072] (4) After the moisture on the plate has been completely absorbed by the agar, use sterile tweezers to pick up the drug sensitivity test strips and stick them on the plate surface. Once the test strips are stuck on, they should not be picked up again. Stick 3 test strips on each plate, with a spacing of not less than 45 mm between each test strip and a center distance of not less than 20 mm from the edge of the plate.

[0073] (5) Place the plate in a 37℃ constant temperature incubator for 10-12 hours and observe the results.

[0074] The test results are shown in Table 3 and Figure 6 In this embodiment, Sonora desert bacillus CB-6 is sensitive to commonly used antibiotics such as penicillin, ciprofloxacin, gentamicin, enrofloxacin, cephalexin, neomycin, and oxytetracycline, meaning it does not develop drug resistance.

[0075] Table 3: Results of drug susceptibility testing of Sonora desert Bacillus CB-6.

[0076]

[0077] In the table: S indicates sensitive, M indicates moderately sensitive, and R indicates resistant.

[0078] II. Virulence gene detection test.

[0079] Using Bacillus cereus AH7-7 as a positive control strain, the virulence genes related to the target strain were detected.

[0080] Using the genome of the target strain prepared above as a template, PCR amplification was performed using specific primers for each virulence gene. Each primer was synthesized by Sangon Biotech (Shanghai) Co., Ltd. The PCR reaction conditions were 95℃ for 10 min; 95℃ for 15 s, 55℃ for 15 s, 72℃ for 40 s, 35 cycles; 72℃ for 5 min.

[0081] The primer sequences for the virulence gene and the expected size of the PCR product are shown in Table 4, and the PCR amplification system for the virulence gene is shown in Table 5.

[0082] Table 4: Primer sequences for PCR amplification of virulence genes.

[0083]

[0084] Table 5: PCR amplification system for virulence genes.

[0085]

[0086] PCR products were electrophoresed on a 1.0% agarose gel (containing ethidium bromide), and observed and photographed using a gel imaging system. The electrophoresis results showed that the positive strain amplified seven virulence genes (entFM, nheA, nheB, nheC, hblD, bceT, and cytK), and the sizes of the amplified fragments were consistent with expectations. (See attached image). Figure 7 No related virulence genes were detected in strain CB-6; results are shown below. Figure 7 .

[0087] The results of the above-mentioned drug sensitivity tests and virulence gene detection collectively demonstrate that Sonora desert Bacillus CB-6 has good safety.

[0088] Example 4: Stress resistance and growth characteristics of Sonora desert Bacillus BL-M25.

[0089] I. Heat resistance test.

[0090] (1) Preparation of seed culture: A small amount of isolated and preserved strains were picked up with a sterile inoculation loop, streaked on a Bacillus agar plate, and incubated at 37°C for 24 hours. A single colony was picked and inoculated into TSB liquid medium and cultured at 37°C until the bacterial logarithmic growth phase.

[0091] (2) Preparation of bacterial culture: The seed culture was inoculated into 3 mL of TSB liquid medium at an inoculation rate of 1%, and cultured at 37°C with shaking until the logarithmic growth phase of the bacteria was reached to obtain the bacterial culture.

[0092] (3) Heat treatment of bacterial solution: Take 3 mL of bacterial solution and dispense 4 tubes of 500 μL each, numbered 1, 2, 3 and 4. Place the bacterial solutions in water baths pre-adjusted to 37℃, 50℃ and 70℃ for 10 min and in water baths at 90℃ for 5 min respectively. After treatment, place them in an ice box immediately.

[0093] (4) Determine the number of viable bacteria: Dilute all bacterial solutions 10 times (10 -1 10 -2 10 -310 -4 10 -5 10 -6 10 -7 10 -8 Take 100 μL of 10 -6 The diluted bacterial suspension was counted using counting agar plates. At least three replicate plates were needed for the same heat-treated bacterial suspension to minimize errors caused by random factors. The plates were incubated at 37°C for 24 hours. The results are shown in Table 6.

[0094] Table 6: Heat-resistant survival rate of Bacillus CB-6.

[0095]

[0096] II. Determination of growth curves.

[0097] (1) Preparation of seed culture: Pick a single colony and inoculate it into 3 mL of LB liquid medium, and culture at 37℃ with shaking for 24 h to obtain seed culture;

[0098] (2) Preparation of bacterial culture: The seed culture was inoculated into 100 mL of LB liquid medium at an inoculation rate of 1%, and cultured at 37 °C with shaking for 24 h to obtain bacterial culture;

[0099] (3) Determination of growth curve: Prepare 13 10 mL EP tubes in a clean bench, add 4 mL of liquid LB, pick single colonies and transfer them to the 13 EP tubes respectively, and incubate at 37℃ on a shaker. Every 2 h, take out one tube of bacterial solution and perform serial dilution in the clean bench. Take 10 mL of bacterial solution from the first 6 h. 2 10 3 10 4 For each diluted bacterial culture, 100 μL is spread onto solid LB medium and incubated at 37°C for 24 h. The bacterial culture after 6-24 h is then used to determine the 10% dilution. 5 10 6 10 7 For each diluted bacterial culture, 100 μL was spread onto solid LB medium and incubated at 37°C for 24 h. Single colonies were then counted to calculate the bacterial concentration (CFU / mL) at that time. A growth curve was plotted with incubation time on the x-axis and the logarithm of the bacterial concentration on the y-axis.

[0100] Experiments show that the strains isolated in this example have strong reproductive capacity. Both strains were in the logarithmic growth phase from 2 to 10 hours, exhibiting exponential rapid growth, and entered the stationary phase after 10 hours. This strong reproductive capacity is beneficial for large-scale industrial fermentation production. Its growth curves are shown in [Figure showing growth curves]. Figure 5 .

[0101] Example 5: Feeding trial to antagonize necrotizing enteritis in laying hens.

[0102] In this experiment, laying hens were infected with Clostridium perfringens ML2 to successfully establish a necrotic enteritis model. In this case, Sonora desert Bacillus CB-6 was fed to the laying hens to verify the effect of Sonora desert Bacillus CB-6 on antagonizing Clostridium perfringens infection in laying hens. All laying hens were raised in the same standard environment during the experiment (23-26℃, 40-60% humidity).

[0103] I. Experimental Animals and Grouping

[0104] (1) Experimental animals and grouping: 72 white Leghorn laying hens aged 280 days were purchased from Zhili Laying Hen Breeding Base in Jingzhou, Hubei Province. The experiment was divided into 3 groups: control group, ML2 group, and ML2+CB-6 group. The experimental period was 27 days, with a total of 72 laying hens. The single-factor completely randomized grouping was adopted, with 24 hens in each group and 6 replicates of 4 hens each.

[0105] II. Preparation of bacterial culture.

[0106] (1) Preparation of Sonora desert Bacillus CB-6 bacterial culture: The strain was streaked on LB agar plates and cultured at 37℃ for 24 h. The isolated strain was picked and cultured in 3 mL of LB liquid medium at 37℃ and 200 r / min for 12 h to obtain the seed culture. The seed culture was inoculated into 3 mL of LB liquid medium at a 1% inoculation rate and cultured at 37℃ and 200 r / min for 12 h. Then, the supernatant was removed by centrifugation at 8000 rpm / min, and the culture was resuspended in sterile PBS to adjust the viable count to 1×10⁻⁶. 8 CFU / mL.

[0107] (2) Preparation of Clostridium perfringens ML2 bacterial culture: Clostridium perfringens was streaked on TSC solid medium, and then another layer of TSC medium was poured in to sandwich Clostridium perfringens in the middle to make a double-layer TSC plate. The plate was placed in an anaerobic incubator at 37℃ for 24 h. A single colony was picked and inoculated into 3 mL of RCM liquid medium and placed in an anaerobic incubator at 37℃ for 12 h. The plate was streaked again with TSC double-layer plates, and the strain was picked and placed in RCM liquid medium and placed in an anaerobic incubator at 37℃ for 24 h to obtain the seed culture. The seed culture was inoculated into 50 mL of RCM liquid medium at an inoculation rate of 1% and cultured under anaerobic conditions at 37℃ for 12 h. Then, the supernatant was removed by centrifugation at 8000 rpm / min, and the culture was resuspended in sterile PBS to adjust the viable count to 1×10⁻⁶. 10 CFU / mL.

[0108] III. Experimental Procedure.

[0109] (1) Establishment of Clostridium perfringens infection model; On the basis of ensuring normal daily feeding of laying hens, laying hens in group ML2 were fed Clostridium perfringens ML2 bacterial solution (1×10⁻⁶) in drinking water daily from day 11 to day 17. 10 CFU / animal), and fed with regular feed daily.

[0110] (2) Feeding program for laying hens in the ML2+CB-6 group: Feed CB-6 bacterial solution (1×10) daily from day 0 to day 27. 8 CFU / animal), feed Clostridium perfringens ML2 bacterial solution (1×10) daily for 10-17 days. 10 CFU / animal), and fed with regular feed daily.

[0111] (3) Control group laying hens feeding program: normal routine feed every day.

[0112] II. Measurement Indicators

[0113] 1. Production performance (egg production rate)

[0114] The effect of Sonora desert bacillus CB-6 on antagonizing necrotizing enteritis caused by Clostridium perfringens infection in laying hens was verified by comparing the egg production rates of three groups of laying hens in this embodiment.

[0115] .

[0116] 2. Detection of Clostridium perfringens bacterial load in cecal contents

[0117] A pair of specific primers was designed targeting the cpa gene sequence of Clostridium perfringens α-toxin, and a real-time quantitative PCR method was established for its detection. This method exhibits excellent linearity and R0. 2 =0.9998, which can be used for the quantitative detection of Clostridium perfringens. After the laying hen feeding trial, six laying hens were randomly selected from each group according to the above-mentioned fluorescence quantitative detection method to detect the Clostridium perfringens bacterial load in the cecal contents.

[0118] 3. Detection of inflammatory factors and immunoglobulins in laying hen serum

[0119] Serial dilutions of the standard stock solution were prepared. The microplate was set up with blank wells (buffer), standard wells (50 μL standard solution), and sample wells (40 μL diluent + 10 μL sample, 5-fold dilution), and incubated at 37°C for 30 min. Five washes were performed with a 1:19 diluted washing buffer (discard buffer - add buffer - soak for 30 s - pat dry). Except for the blank wells, 50 μL of enzyme-labeled protein was added, and the plates were incubated at 37°C for 30 min, followed by another wash. 50 μL of chromogenic reagent A / B was added, and the reaction was carried out at 37°C in the dark for 10 min. The reaction was stopped with 50 μL of stop solution (the blue color turned yellow). The OD value was measured at 450 nm using a microplate reader within 15 min (zeroing the blank well). A standard curve was constructed with the OD value of the standard wells on the x-axis and the different concentrations of the diluted standard solution on the y-axis. The corresponding concentrations of the sample wells were calculated based on their OD values.

[0120] III. Results Analysis.

[0121] 1. Production performance (egg production rate)

[0122] The table below shows that the overall egg production rate of the control group was 87.02%, the ML2 group was 82.38%, and the ML2+CB-6 group was 86.30%. The egg production rate of the ML2+CB-6 group was significantly higher than that of the ML2 group (p < 0.05), but there was no significant difference compared with the control group (p > 0.05). After infection, the egg production rate of the control group was 88.75%, the ML2 group was 83.75%, and the ML2+CB-6 group was 90.63%. The egg production rate of the ML2+CB-6 group was extremely significantly higher than that of the ML2 group (p < 0.01), and the egg production rate of the ML2+CB-6 group was slightly higher than that of the control group, but there was no significant difference (p > 0.05).

[0123] Table 7: Egg production rate of laying hens in animal experiments.

[0124]

[0125] In the table: different lowercase letters in the same column indicate significant differences (p < 0.05), and different uppercase letters indicate extremely significant differences (p < 0.01).

[0126] 2. Clostridium perfringens bacterial load in cecal contents

[0127] The bacterial load of Clostridium perfringens in the cecal contents of the control group was 1×10⁻⁶. 3.75 The CFU / g level was within the normal range for laying hens and would not cause necrotic enteritis; the bacterial load in group ML2 was as high as 1×10⁻⁶. 5.66 The CFU / g of the ML2+CB-6 group was significantly higher than that of the control group (p<0.01), indicating successful model establishment; the bacterial load in the ML2+CB-6 group was 1×10⁻⁶. 4.23 The CFU / g level was significantly lower than that of the ML2 group by 1.43 orders of magnitude (p < 0.01).

[0128] Table 8: Clostridium perfringens bacterial load in the cecal contents of laying hens.

[0129]

[0130] In the table: different lowercase letters in the same column indicate significant differences (p < 0.05), and different uppercase letters indicate extremely significant differences (p < 0.01).

[0131] 3. Expression levels of inflammatory factors in laying hen serum

[0132] The expression levels of pro-inflammatory factors (IL-1β and TNF-α) in the serum of laying hens in the ML2 group were significantly higher than those in the control group (p < 0.001), while the expression level of the anti-inflammatory factor (IL-10) in the serum was significantly lower than that in the control group (p < 0.001), indicating that the systemic inflammation level of laying hens was increased after ML2 infection. The expression levels of pro-inflammatory factors (IL-1β and TNF-α) in the serum of laying hens in the ML2+CB-6 group were significantly lower than those in the ML2 group (p < 0.01), while the expression level of the anti-inflammatory factor (IL-10) was significantly higher than that in the ML2 group (p < 0.05), indicating that CB-6 can inhibit systemic inflammation in laying hens after ML2 infection (see...). Figure 8 ).

[0133] 4. Immunoglobulin content in laying hen serum

[0134] The levels of immunoglobulins IgA, IgY, and IgM in the serum of laying hens in the ML2 group were significantly lower than those in the control group (p < 0.01), indicating a decrease in the immunity of laying hens after ML2 infection. The levels of immunoglobulins IgA, IgY, and IgM in the serum of laying hens in the ML2+CB-6 group were significantly higher than those in the ML2 group (p < 0.01), indicating that CB-6 can enhance the immunity of laying hens after ML2 infection (see...). Figure 9 ).

[0135] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A probiotic Bacillus antagonizing Clostridium perfringens type G, characterized in that, The Bacillus species is Sonora desert Bacillus CB-6, classified and named... Bacillus sonorensis CB-6, accession number CCTCC NO: M20251373, accession date June 12, 2025, deposited at China Center for Type Culture Collection, located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. The probiotic Bacillus antagonizing Clostridium perfringens type G according to claim 1, characterized in that, The Sonora desert Bacillus CB-6 is used to inhibit the activity of avian pathogenic Clostridium perfringens ML2.

3. The use of a probiotic Bacillus antagonizing Clostridium perfringens as described in any one of claims 1-2 in the preparation of an anti-Clostaphylococcus perfringens preparation.

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