Probiotic bacillus for antagonizing necrotic enteritis of laying hens and application

Abstract

The invention discloses probiotic bacillus for antagonizing necrotic enteritis of laying hens and application of the probiotic bacillus, the bacillus is Sooraia deserticola bacillus CB-6, the classification name is bacillus sonorensis CB-6, the preservation number is CCTCC NO: M20251373, the preservation date is June 12, 2025, the preservation unit is China Center for Type Culture Collection, the preservation number is CCTCC NO: M20251373, the preservation number is CCTCC NO: M20251373, the preservation number is CCTCC NO: M20251373, the preservation number is CCTCC NO: M20251373, and the preservation number is CCTCC NO: M20251373. The address of the preservation unit is Wuhan University Campus, Wuchang District, Wuchang District, Hubei Province, No. 299. The pH value of a sterile fermentation supernatant of the bacillus sorosa CB-6 provided by the invention is neutral, clostridium perfringens can be inhibited with high activity, the bacteriostatic action does not depend on organic acids such as lactic acid and the like, the bacteriostatic effect is remarkable and stable, the immunity of laying hens infected by the clostridium perfringens can be remarkably enhanced, the laying rate can be increased, and the laying hens can be prevented from being infected by the clostridium perfringens. The problems that the laying hens in the egg producing period are prone to necrotic enteritis and antibiotics are not suitable for prevention and control are solved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of agricultural micro-ecological preparations and feed additives, and particularly relates to a probiotic Bacillus antagonistic to necrotic enteritis of laying hens and application thereof. BACKGROUND

[0002] Avian necrotic enteritis is a common intestinal disease in laying hen farming, and Clostridium perfringens is the main pathogenic bacteria causing avian necrotic enteritis. Although Clostridium perfringens naturally exists in the intestines of healthy laying hens, only when the intestinal barrier of the chicken is damaged, the microbial flora is disordered, or the immunity of the body is decreased, the Clostridium perfringens in the intestine will proliferate abnormally and release various toxins (such as alpha toxin, beta toxin, etc.), thereby causing intestinal inflammation, necrosis, diarrhea and production performance decline, and even death, causing serious economic losses to the laying hen farming industry.

[0003] Since the use of antibiotics to prevent and control avian necrotic enteritis is prohibited during the laying period of laying hens, finding a safe and effective alternative has become a research hotspot in recent years. Probiotic intervention as an effective strategy to regulate intestinal flora and enhance the immunity of the body has attracted much attention. Currently, there is no literature report on Bacillus strains that directly inhibit Clostridium perfringens without relying on the production of organic acids, and there is no related research report on Bacillus strains that antagonize avian necrotic enteritis caused by Clostridium perfringens.

[0004] Therefore, a probiotic Bacillus antagonistic to necrotic enteritis of laying hens and application thereof are urgently needed. SUMMARY

[0005] To solve the defects in the prior art, the present application provides a probiotic Bacillus antagonistic to necrotic enteritis of laying hens and application thereof.

[0006] To solve the above technical problems, the present application provides the following technical solutions:

[0007] The first object of the present application provides a probiotic Bacillus antagonistic to necrotic enteritis of laying hens, which is Bacillus sonorensis CB-6, classified and named as Bacillus sonorensis CB-6, with a preservation number of CCTCC NO: M20251373, a preservation date of June 12, 2025, and a preservation unit of China Center for Type Culture Collection (CCTCC) located at Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan, Hubei Province.

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

[0009] The second object of the present application provides a use of the probiotic Bacillus in the preparation of a preparation for preventing and treating necrotic enteritis of laying hens.

[0010] Preferably, the content of Bacillus sonorensis CB-6 in the preparation 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 application has the following beneficial effects:

[0013] The Bacillus sonorensis CB-6 provided by the present application has strong safety and stress resistance, and is beneficial to be prepared into a probiotic product and applied to production practice. The sterile fermentation supernatant of the Bacillus sonorensis CB-6 provided by the present application has a neutral pH value, can inhibit Clostridium perfringens with high activity, and the bacteriostatic effect is not dependent on organic acids such as lactic acid, the bacteriostatic effect is significant and stable, solves the problem that laying hens are prone to necrotic enteritis during the laying period and antibiotics cannot be used for prevention and control, and can significantly enhance the immunity of laying hens after Clostridium perfringens infection and improve the egg production rate.

[0014] Cell preservation:

[0015] The Bacillus sonorensis CB-6 provided by the present application is screened by the inventors of the present application, is classified and named as Bacillus sonorensis CB-6, has a preservation number of CCTCC NO: M20251373, a preservation date of June 12, 2025, and is preserved in the China Center for Type Culture Collection (CCTCC) located at No. 299, Bajiyilu, Wuchang District, Wuhan, Hubei Province. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is the result of the in-vitro bacteriostatic experiment of the fermentation supernatant of Bacillus sonorensis CB-6 after 12-24 h of culture in the present application (Clostridium perfringens ML2 is the indicator bacteria).

[0017] Figure 2 is the growth state of Bacillus sonorensis CB-6 on an LB agar plate in the present application.

[0018] Figure 3 is the result of Gram staining of Bacillus sonorensis CB-6 in the present application (x1000).

[0019] Figure 4is a bacterial phylogenetic tree of Bacillus sonorensis CB-6 in the present application constructed by Neighbor-Joining method.

[0020] Figure 5 is a growth curve of Bacillus sonorensis CB-6 in the present application.

[0021] Figure 6 is the antibiotic sensitivity test result of Bacillus sonorensis CB-6 strain in the present application.

[0022] Figure 7 is a gel map of virulence gene detection of Bacillus sonorensis CB-6 in the present application.

[0023] In the figure, M: DL2000 DNA molecular weight marker; Lane 1: positive control (Bacillus cereus AH7-7); Lane 2: Bacillus sonorensis CB-6 in the present application; Figure 7 A in the figure is the detection result of virulence gene nheA; Figure 7 B in the figure is the detection result of virulence gene nheB; Figure 7 C in the figure is the detection result of virulence gene nheC; Figure 7 D in the figure is the detection result of virulence gene hblD; Figure 7 E in the figure is the detection result of virulence gene bceT; Figure 7 F in the figure is the detection result of virulence gene cytK; Figure 7 G in the figure is the detection result of virulence gene entFM.

[0024] Figure 8 is the detection result of the expression amount of inflammatory factors in the serum of laying hens in the feeding test of Bacillus sonorensis CB-6 in the present application against necrotic enteritis of laying hens. In the figure, Figure 8 A in the figure is the detection result of the expression amount of IL-1β in the serum; Figure 8 B in the figure is the detection result of the expression amount of TNF-α in the serum; Figure 8 C in the figure is the detection result of the expression amount of IL-10 in the serum.

[0025] Figure 9 is the detection result of the content of immunoglobulin in the serum of laying hens in the feeding test of Bacillus sonorensis CB-6 in the present application against necrotic enteritis of laying hens. In the figure, Figure 9 A in the figure is the detection result of the content of IgA in the serum; Figure 9 B in the figure is the detection result of the content of IgM in the serum; Figure 9 C in the figure is the detection result of the content of IgY in the serum. DETAILED DESCRIPTION

[0026] The preferred embodiments of the present application will be described below with reference to the drawings, and it should be understood that the preferred embodiments described herein are intended for explaining and illustrating the present application only, and are not intended to limit the present application.

[0027] Example 1: Isolation and identification of Bacillus sonorensis CB-6

[0028] I. Strain isolation

[0029] (1) Sample collection: In a healthy white-feathered broiler chicken flock, select individuals with strong body type, and collect cecal contents.

[0030] (2) Sample processing: In a clean bench, gradient dilution was performed with sterile distilled water, 100 μL of 10 6 and 10 7 dilution gradient contents were taken and placed on solid LB medium and MRS medium, and evenly smeared with a swab stick. The LB medium was placed in a constant temperature incubator at 37°C and cultured for 24 h, and the MRS medium was placed in an anaerobic incubator and cultured for 24 h. The colony morphology was observed.

[0031] (3) Gram staining: Gram staining was purple, short rod-shaped, single, double or chain arrangement.

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

[0033] II. Strain species identification

[0034] On the basis of the above identification, further 16S rDNA gene sequence detection was performed on the Bacillus sonorensis CB-6 strain in this embodiment to confirm and identify the species to which the Bacillus sonorensis CB-6 belongs. The specific steps are as follows

[0035] (I) Target strain genome extraction

[0036] (1) Take 1 mL of cultured bacterial solution, centrifuge at 10000 rpm for 30 s, and as much as possible to aspirate the supernatant and collect the bacterial body.

[0037] (2) Add 200 μL of buffer RB, 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) 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 by blowing.

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

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

[0041] (6) Add the mixture (including the precipitate that may have appeared) of the previous step to an adsorption column AC (the adsorption column is placed in a collection tube), centrifuge at 13000 rpm for 30-60 s, 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 rinse solution WB (anhydrous ethanol has been added), centrifuge at 12000 rpm for 30 s, and discard the waste liquid.

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

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

[0046] (11) Take out the adsorption column AC and place it in a clean centrifuge tube, add 50 μL of elution buffer EB (the elution buffer has been preheated in a 67-70°C water bath) to the middle part of the adsorption membrane, place at room temperature for 3-5 min, and centrifuge at 12000 rpm for 1 min. Re-add the obtained solution to the centrifugal adsorption column, place at room temperature for 2 min, and centrifuge at 12000 rpm for 1 min.

[0047] (12) Store the DNA sample at -20°C for standby use.

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

[0049] The 16S rRNA sequence of the isolated Bacillus sonorensis CB-6 strain was amplified using bacterial universal primers, the primers were synthesized by Shenguo Bioengineering (Shanghai) Co., Ltd., and the DNA sequences of the primers 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] The G-type Clostridium perfringens ML2 from a typical necrotic enteritis chicken and carrying NetB toxin was used as an indicator strain, and the sterile fermentation supernatant of the strain in this example was used as a bacteriostatic agent to detect the in-vitro bacteriostatic activity of the strain. The specific operation steps are as follows.

[0060] (1) Preparation of indicator strain (G-type Clostridium perfringens ML2) suspension: TSC plate of indicator strain was streaked and cultured in an anaerobic box at 37°C for 20h; a single colony was picked and cultured in RCM liquid medium in an anaerobic box at 37°C for 16h to recover the indicator strain. The cultured indicator strain was adjusted to a bacterial number of 1.0×10 6 CFU / mL.

[0061] (2) Preparation and treatment of CB-6 fermentation supernatant: the strain in this example was streaked on a plate and cultured at 37°C for 16h. The isolated strain was inoculated into 50mL of LB liquid medium and cultured at 37°C and 200r / min for 24h as a seed liquid. The seed liquid was inoculated into 100mL of LB liquid medium at an inoculation amount of 1%, and cultured at 37°C and 200r / min for 12h, 14h, 16h, 18h, 20h, 22h and 24h respectively as fermentation liquid. The prepared fermentation liquid was centrifuged at 10000r / min for 5min, and the supernatant after centrifugation was filtered with a 0.22µm sterile filter and detected for pH value with an acid-base detector.

[0062] (3) Bacteriostatic test: sterilized TSC medium was taken and cooled until it was not solidified, then Clostridium perfringens culture liquid equal to one thousandth of the volume of TSC medium was added, mixed and poured into a culture dish, and dried in a clean bench for 2h. A puncher was used to punch holes in the TSC plate, and a burner was used to burn the bottom to prevent the liquid from leaking to the bottom of the plate. 200µL of CB-6 fermentation supernatant was taken with a micropipette gun and placed in the hole of the TSC plate, which was placed in an anaerobic incubator and cultured for 10h. Then it was taken out and the diameter of the bacteriostatic ring was measured with a vernier caliper.

[0063] The test results showed that the sterile fermentation supernatant of the strain CB-6 in this example was neutral (pH 7) and had strong inhibitory ability on Clostridium perfringens ML2, i.e. it could inhibit Clostridium perfringens without producing acid. The results are shown in Table 2.

[0064] Table 2: Inhibition ring of Bacillus sonorensis 12-24h.

[0065]

[0066] Example 3: Safety detection of Bacillus sonorensis CB-6.

[0067] I. Drug sensitivity test.

[0068] Penicillin, cephalosporin, quinolone, aminoglycoside, tetracycline and other drug sensitive paper (purchased from Hangzhou Microbial Reagent Co., Ltd.) were selected for drug sensitivity test. The test judgment standard refers to the latest version of the standard provided by the World Health Organization (WHO) (2018 edition). The specific steps are as follows:

[0069] (1) The bacteria liquid was added into the LB liquid medium at an amount of 1%, and cultured at 37℃ with 200rpm / min shaking bed for 12h;

[0070] (2) The OD600nm value of the bacteria liquid was adjusted to 1 by OD instrument;

[0071] (3) The bacteria liquid was coated on the LB agar plate with a sterile cotton swab, and each time the plate was rotated by 60 degrees, and finally coated on the inner edge of the plate for two circles, repeated several times to ensure uniform coating;

[0072] (4) After the water on the plate was completely absorbed by the agar, the drug sensitive paper was pasted on the surface of the plate with sterile forceps. Once the paper was pasted, it could not be picked up again. Three pieces of paper were pasted on each plate, and the distance between each piece of paper was not less than 45mm, and the center distance of the paper from the edge of the plate was not less than 20mm;

[0073] (5) The plate was placed in a 37℃ constant temperature incubator for 10-12h to observe the results.

[0074] The test results are shown in Table 3 and Figure 6 In this embodiment, the Sonoran Desert Bacillus CB-6 is sensitive to commonly used antibiotics such as penicillin, ciprofloxacin, gentamicin, enoxacin, cephalexin, neomycin and oxytetracycline, i.e. does not produce drug resistance.

[0075] Table 3: Drug sensitivity test results of Sonoran Desert Bacillus CB-6

[0076]

[0077] In the table, S means sensitive, M means intermediate, and R means resistant.

[0078] II. Virulence gene detection test

[0079] Bacillus cereus AH7-7 was used as a positive control strain to detect the target strain related virulence genes.

[0080] The target strain genome prepared as described above was used as a template, and each virulence gene specific primer was used for PCR amplification. Each primer was synthesized by Shengong Shanghai Bioengineering (Shanghai) Co., Ltd. The PCR reaction conditions were 95℃ for 10min; 95℃ for 15s, 55℃ for 15s, 72℃ for 40s, 35 cycles; 72℃ for 5min.

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

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

[0083]

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

[0085]

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

[0087] The above-mentioned results of drug sensitivity test, virulence gene detection and the like jointly prove that the safety of Bacillus sonorensis CB-6 is good.

[0088] Example 4: Anti-stress characteristics and growth characteristics of Bacillus sonorensis BL-M25

[0089] I. Heat resistance test

[0090] (1) Preparation of seed liquid: a small amount of the isolated and preserved strain was taken with a sterile inoculation loop, streaked on a Bacillus agar plate, and cultured at 37°C for 24 h, and a single colony was picked and inoculated into TSB liquid medium and cultured at 37°C to the logarithmic growth phase of the bacteria.

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

[0092] (3) Heat treatment of bacterial liquid: 3 mL of bacterial liquid was divided into 4 tubes at 500 μL per tube, numbered 1, 2, 3, and 4, and the bacterial liquid was treated in a water bath at 37°C, 50°C, and 70°C for 10 min and in a water bath at 90°C for 5 min, and then immediately placed in an ice box.

[0093] (4) Determination of viable bacterial count: all the bacterial liquids were diluted by 10 times (10 -1 , 10 -2 , 10 -3,10 -4 ,10 -5 ,10 -6 ,10 -7 ,10 -8 ), take 100 μL 10 -6 diluted bacteria liquid with counting agar plate for plate pouring counting. The same heat-treated bacteria liquid needs more than three repeated plates to reduce the error caused by accidental factors, and cultivate in 37℃ incubator for 24h. The results are shown in Table 6.

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

[0095]

[0096] II. Determination of growth curve.

[0097] (1) Preparation of seed liquid: pick single colony and inoculate into 3mL LB liquid medium, cultivate at 37℃ for 24h, and obtain seed liquid;

[0098] (2) Preparation of bacteria liquid: inoculate seed liquid into 100mL LB liquid medium at 1% inoculation amount, cultivate at 37℃ for 24h, and obtain bacteria liquid;

[0099] (3) Determination of growth curve: prepare 13 10mL EP tubes in clean bench, add 4mL liquid LB, pick single colony into 13 EP tubes, cultivate at 37℃, take out one tube every 2h to dilute in clean bench, take 100 μL of bacteria liquid with dilution of 10 2 ,10 3 ,10 4 , and inoculate on solid LB medium, and cultivate in 37℃ constant temperature incubator for 24h, take out single colony to count, and calculate the concentration of bacteria liquid at current time (CFU / mL). Take cultivation time as abscissa, and logarithm of bacteria liquid concentration as ordinate, to draw growth curve of the strain. 5 ,10 6 ,10 7 , and inoculate on solid LB medium, and cultivate in 37℃ constant temperature incubator for 24h, take out single colony to count, and calculate the concentration of bacteria liquid at current time (CFU / mL). Take cultivation time as abscissa, and logarithm of bacteria liquid concentration as ordinate, to draw growth curve of the strain.

[0100] The test shows that the strain isolated in the embodiment has strong reproduction ability, and two strains are in logarithmic growth phase at 2-10h, and grow exponentially, and enter stable phase after 10h. The strain has strong reproduction ability, which is beneficial to industrial large-scale fermentation production, and the growth curve is shown in Figure 5 .

[0101] Example 5: Antagonistic feeding test of egg-laying hens with necrotic enteritis.

[0102] Under the premise of successfully establishing the model of egg-laying hens with necrotic enteritis by Clostridium perfringens ML2 infection, the egg-laying hens were fed with the bacterial solution of Bacillus sonorensis CB-6 in this example, aiming to verify the effect of Bacillus sonorensis CB-6 in antagonizing the infection of Clostridium perfringens in egg-laying hens, and all the egg-laying hens were raised in the same standard environment (23-26℃, 40-60% humidity) during the test.

[0103] I. Test animals and grouping

[0104] (1) Test animals and grouping: 72 white leihang egg-laying hens of 280 days old were purchased from Hubei Jingzhou Zhili egg-laying hen breeding base, and the test was divided into 3 groups: control group, ML2 group, ML2+CB-6. The test period was 27 days, and a total of 72 egg-laying hens were used; single factor completely randomized grouping was adopted, 24 egg-laying hens in each group, 6 repeats were set, and 4 egg-laying hens in each repeat.

[0105] II. Preparation of bacterial solution

[0106] (1) Preparation of Bacillus sonorensis CB-6 bacterial solution: the strain was streaked on LB solid plate and cultured at 37℃ for 24h. The isolated strain was inoculated in 3mL of LB liquid medium, and cultured at 37℃, 200r / min for 12h as seed solution. The seed solution was inoculated into 3mL of LB liquid medium at a inoculation amount of 1%, and cultured at 37℃, 200r / min for 12h, then centrifuged at 8000rpm / min to remove the supernatant, resuspended with sterile PBS, and adjusted to 1×10 8 CFU / mL.

[0107] (2) Preparation of Clostridium perfringens ML2 bacterial solution: Clostridium perfringens was streaked on TSC solid medium, and then a layer of TSC medium was poured to sandwich Clostridium perfringens to make double-layer TSC plate, which was placed in 37℃ anaerobic box for 24h culture. A single colony was inoculated into 3mL of RCM liquid medium, and placed in 37℃ anaerobic box for 12h culture. Again, the strain was streaked on double-layer TSC plate, and inoculated in RCM liquid medium and placed in 37℃ anaerobic box for 24h culture as seed solution. The seed solution was inoculated into 50mL of RCM liquid medium at a inoculation amount of 1%, and cultured at 37℃ under anaerobic conditions for 12h, then centrifuged at 8000rpm / min to remove the supernatant, resuspended with sterile PBS, and adjusted to 1×10 10 CFU / mL.

[0108] III. Test process

[0109] (1) The establishment of Clostridium perfringens infection model; on the basis of ensuring normal daily feeding of laying hens, the ML2 group of laying hens was fed with Clostridium perfringens ML2 bacterial solution (1x10 10 CFU / each) in drinking water every day from the 11th to the 17th day, and was fed with regular feed every day.

[0110] (2) The feeding scheme of the ML2+CB-6 group of laying hens: CB-6 bacterial solution (1x10 8 CFU / each) was fed every day from the 0th to the 27th day, Clostridium perfringens ML2 bacterial solution (1x10 10 CFU / each) was fed every day from the 10th to the 17th day, and regular feed was fed every day.

[0111] (3) The feeding scheme of the control group of laying hens: normal regular feed was fed every day.

[0112] II. Determination index

[0113] 1. Production performance (laying rate)

[0114] The effect of Sonora desert Bacillus CB-6 in the present embodiment on antagonizing Clostridium perfringens infection leading to necrotic enteritis in laying hens was verified by comparing the laying rates of the three groups of laying hens.

[0115] .

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

[0117] A pair of specific primers was designed for the Clostridium perfringens alpha toxin gene cpa sequence, and a fluorescent quantitative PCR detection method was established. The linear correlation of this method was good, R 2 =0.9998, and it could be used for quantitative detection of Clostridium perfringens. After the end of the laying hen feeding test, 6 laying hens were randomly selected from each group for detection of Clostridium perfringens load in cecal contents according to the above fluorescent quantitative detection method.

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

[0119] Gradient dilution of standard mother liquor. Set blank wells (buffer), standard wells (50 μL standard solution) and sample wells (40 μL diluent + 10 μL sample, 5-fold dilution) on the enzyme-labeled plate, incubate at 37°C for 30 min. Wash with 1:19 diluted washing solution for 5 times (discard-liquid-add-30 s soak-pat dry), add 50 μL enzyme-labeled substance to all wells except blank wells, incubate at 37°C for 30 min, and then wash again. Add 50 μL color developing agent A / B solution, avoid light, react at 37°C for 10 min, stop the reaction with 50 μL stop solution (blue to yellow), and measure OD value at 450 nm within 15 min using an enzyme-labeled instrument (zero the blank). Establish a standard curve with OD value of standard wells as abscissa and different concentrations of diluted standard as ordinate, and calculate the corresponding concentration of sample wells according to OD value.

[0120] III. Results analysis

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

[0122] According to the following table, the whole egg laying rate of the control group was 87.02%, that of the ML2 group was 82.38%, and that of the ML2+CB-6 group was 86.30%. The egg laying rate of the ML2+CB-6 group was significantly higher than that of the ML2 group (p<0.05), and there was no significant difference compared with the control group (p>0.05). The post-infection egg laying rate of the control group was 88.75%, that of the ML2 group was 83.75%, and that of the ML2+CB-6 group was 90.63%. The egg laying rate of the ML2+CB-6 group was extremely significantly higher than that of the ML2 group (p<0.01), and the egg laying rate of the ML2+CB-6 group was slightly higher than that of the control group without significant difference (p>0.05).

[0123] Table 7: Egg laying rate of egg-laying hens in animal experiment

[0124]

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

[0126] 2. C. perfringens load in cecal contents

[0127] The C. perfringens load in the cecal contents of the control group was 1×10 3.75 CFU / g, which was at the normal level of the body and would not cause necrotic enteritis in egg-laying hens; the load in the ML2 group was as high as 1×10 5.66 CFU / g, which was extremely significantly higher than that of the control group (p<0.01), indicating that the modeling was successful; the load in the ML2+CB-6 group was 1×10 4.23 CFU / g, which was extremely significantly decreased by 1.43 orders of magnitude compared with the ML2 group (p<0.01).

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

[0129]

[0130] In the table: the same column data with different small letters represent significant difference (p < 0.05), and different capital letters represent extremely significant difference (p < 0.01).

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

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

[0133] 4. Content of immunoglobulin in serum of laying hens

[0134] The content of immunoglobulin IgA, IgY and IgM in serum of the ML2 group was extremely significantly lower than that of the control group (p < 0.01), indicating that the immune ability of the laying hens after ML2 infection was decreased; the content of immunoglobulin IgA, IgY and IgM in serum of the ML2+CB-6 group was extremely significantly higher than that of the ML2 group (p < 0.01), indicating that CB-6 could enhance the immune ability of the laying hens after ML2 infection (see Figure 9 ).

[0135] Finally, it should be noted that the above only describes the preferred embodiments of the present application and is not intended to limit the present application. Although the present application 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 replacements to some technical features. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A probiotic Bacillus sp. that antagonizes necrotic enteritis in laying hens, characterized in that, The Bacillus is Bacillus sonorensis CB-6, the classification name is 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, located at Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan, Hubei Province.

2. The probiotic Bacillus sp. that antagonizes necrotic enteritis in laying hens according to claim 1, characterized by, The Bacillus sonorensis CB-6 is used for inhibiting the activity of avian pathogenic Clostridium perfringens ML2.

3. Use of the probiotic Bacillus antagonizing necrotic enteritis of laying hens according to any one of claims 1-2 in the preparation of a preparation for preventing and treating necrotic enteritis of chickens.

4. Use according to claim 3, characterized in that, The content of Bacillus sonorensis CB-6 in the preparation is 10 8 ~ 10 10 CFU / g.

5. Use according to claim 3, characterized in that, The preparation is used as a feed additive.

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