Compound preparation containing bacillus velezensis and macleaya cordata and capable of preventing and treating avian colibacillosis and application of compound preparation

Abstract

The invention belongs to the technical field of biological agents, and discloses a compound preparation containing bacillus velezensis and macleaya cordata and capable of preventing and treating avian colibacillosis and application. The mass ratio of the bacillus velezensis compound bacteria to the macleaya cordata extract is (2-4): (0.02-0.03); the bacillus velezensis is named as BV-838 and BV-839, and the bacillus velezensis is named as Tests prove that the bacillus velezensis and the macleaya cordata recovery formula preparation have potential interaction, can be matched with each other to play a synergistic interaction role, can effectively prevent and treat avian colibacillosis, also have the effects of enhancing the immunity of the organism and promoting growth, are novel, efficient, green and environment-friendly, and can be widely applied to prevention and treatment of avian colibacillosis. And the method has important significance on the industrial development of antibiotic-free breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of biological preparations, and particularly relates to a Bacillus Velezii and Bolufu Fang preparation capable of synergistically preventing and treating avian Escherichia coli disease. Background Art

[0002] Avian colibacillosis is an important infectious disease caused by pathogenic Escherichia coli. It can be transmitted through multiple pathways such as the respiratory tract, digestive tract and reproductive tract, causing acute septicemia, perihepatitis, pericarditis, peritonitis, etc. in chickens. It is currently widely prevalent in the poultry industry, ranking first among bacterial diseases, and mixed or secondary infections are serious, causing economic losses to the poultry industry.

[0003] Due to the complexity and diversity of E. coli serotypes and the lack of effective vaccines, antibiotics are often used for clinical treatment. With the overuse and even abuse of antibiotics, the resistance rate of E. coli has increased, the resistance spectrum has continued to expand, and multidrug resistance has worsened, posing a huge challenge to the prevention and control of the disease. At the same time, plasmids containing drug-resistant genes can also be transferred by conjugation between bacteria, spread between each other, and passed to humans through the food chain, seriously threatening public health and environmental safety. It is particularly important to find new and effective means of treatment.

[0004] With the advent of the "antibiotic reduction / antibiotic replacement era", the development and application of antibiotic substitutes have become a hot topic in animal husbandry research and production. Among them, probiotic-traditional Chinese medicine compositions have the advantages of both probiotics and traditional Chinese medicine. The combination of the two can play a synergistic role, which can enhance the body's immunity and promote growth, and inhibit pathogenic microorganisms. Therefore, the development of a new, efficient, green and environmentally friendly compound preparation is the current industry demand. Summary of the invention

[0005] In view of the problems existing in the prior art, the object of the present invention is to provide a compound preparation containing Bacillus Velezii and Macleaya cordata for preventing and treating avian Escherichia coli disease. The compound preparation comprises Bacillus Velezii composite bacteria: BV-838 and BV-839, whose deposit numbers are CCTCC NO: M 2025082 and CCTCC NO: M 2025083, respectively. The Macleaya cordata is a Macleaya cordata extract.

[0006] Another object of the present invention is to provide the use of the above compound preparation in the preparation of drugs for treating or preventing Escherichia coli infection.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0008] The present invention screened out two strains of Bacillus velezensis that have antagonistic effects on avian Escherichia coli and have synergistic effects. The two strains were sent to the China Center for Type Culture Collection for preservation on January 10, 2025, with the address: Wuhan University, Wuhan, China. The classification name of strain BV-838 is: Bacillus velezensis BV-838, and the preservation number is CCTCCNO: M 2025082; the classification name of BV-839 is: Bacillus velezensis BV-839, and the preservation number is CCTCCNO: M 2025083.

[0009] The protection scope of the present invention includes:

[0010] The invention discloses a compound preparation containing Bacillus Velezii and Macleaya cordata for preventing and treating avian Escherichia coli disease. The compound preparation comprises Bacillus Velezii compound bacteria: BV-838 and BV-839, whose deposit numbers are CCTCC NO: M 2025082 and CCTCC NO: M 2025083 respectively. The Macleaya cordata is a Macleaya cordata extract.

[0011] In the above-mentioned composite preparation, preferably, the Bacillus Velezii and Macleaya cordata extract are both spray-dried, and the mass ratio of the Bacillus Velezii composite bacteria to the Macleaya cordata extract is 2-4:0.02-0.03, and the more preferred ratio is 2:0.03;

[0012] In the above-mentioned compound preparation, preferably, the effective bacterial concentration of BV-838 and BV-839 is the same, and they are compounded in a mass ratio of 1:1;

[0013] In the above-mentioned compound preparation, preferably, the effective bacterial concentration of the Bacillus Velezii compound bacteria is 5.0×10 9 CFU / g, the Macleaya cordata extract is a Macleaya cordata water extract.

[0014] Application of the compound preparation in the preparation of medicines for treating or preventing Escherichia coli infection.

[0015] In the above application, the serotype of Escherichia coli includes O2, O18 and / or O78.

[0016] In the above-mentioned applications, the dosage form of the drug is all pharmaceutically acceptable dosage forms, including but not limited to tablets, capsules, granules, injections, powders or drops, etc.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention provides a compound preparation containing Bacillus velez and Macleaya cordata, combined with microecological preparations and Chinese veterinary medicine preparations, wherein the Bacillus velez can regulate body metabolism, enhance immunity, promote feed digestion and absorption, and has a good inhibitory effect on the main popular serotypes of avian pathogenic Escherichia coli (O2, O18, O78); Macleaya cordata has the effects of dispersing blood stasis, dispelling wind, detoxifying, relieving pain, and inhibiting bacteria and expelling insects. It is confirmed by experiments that the compound preparation of the present invention can achieve a preventive and therapeutic effect of 90% to 100% on avian Escherichia coli disease, and has the effects of promoting growth and enhancing immunity on animal bodies, which is of great significance to the industrial development of antibiotic-free breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present invention will be further described below in conjunction with the accompanying drawings and embodiments:

[0020] Figure 1 The colony morphology, Gram staining and scanning electron microscopy results of strain BV-838;

[0021] Among them: A: macroscopic morphology of strain BV-838 in NA culture medium; B: optical microscopic examination results of strain BV-838 (10×100); C: electron microscopic examination results of strain BV-838.

[0022] Figure 2 The colony morphology, Gram staining and scanning electron microscopy results of strain BV-839;

[0023] Among them: A: macroscopic morphology of strain BV-839 in NA culture medium; B: optical microscopic examination results of strain BV-839 (10×100); C: electron microscopic examination results of strain BV-839.

[0024] Figure 3 This is the phylogenetic tree of the 16S rDNA sequences of strains BV-838 and BV-839.

[0025] Figure 4 These are the stress resistance results of strains BV-838 and BV-839;

[0026] Among them: A: is the acid resistance result of BV-838 and BV-839; B: is the bile resistance result of BV-838 and BV-839.

[0027] Figure 5 Anatomical diagrams for animal testing;

[0028] Among them: A: blank control group; B: challenge control group; C: BV-838 group; D: BV-839 group; E: Bacillus Velezii complex bacteria group; F: Macleaya cordata extract group; G: compound preparation group 1; H: compound preparation group 2; I: compound preparation group 3. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the present invention clearer, the present invention is further described below in conjunction with embodiments. It should be understood that the embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0031] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0032] Example 1: Strain screening and identification

[0033] 1.1 Isolation and culture of strains BV-838 and BV-839

[0034] Feces, milk and intestinal samples were collected from farms in many provinces across the country. 1g or 1ml of the sample was dissolved in 100mL sterile saline, and after gradient dilution, it was spread on NB solid medium (10g peptone, 3g beef extract powder, 5g sodium chloride, 15g agar, 1000ml distilled water, 121℃ autoclave for 15min) and cultured at 37℃ for 24h. The bacteria were separated and purified according to morphological characteristics such as colony size, color, transparency, and edge. Avian Escherichia coli was used as an indicator bacteria for primary screening and multiple rescreening, and finally two bacterial strains with strong antibacterial effects were obtained, which were named BV-838 and BV-839.

[0035] 1.2 Identification of strains BV-838 and BV-839

[0036] Based on the colony characteristics, physiological and biochemical properties, and molecular biological test results, they were all identified as Bacillus velezensis. The specific technical plan is as follows:

[0037] (1) Colony characteristics and bacterial cell morphology

[0038] In NB solid culture medium, the colonies are milky white or slightly yellowish white. In the early stage, they are concave or round, with a moist surface, opaque, and neat edges. In the later stage, they grow into rough, irregular-edged, non-transparent colonies. A thin film is formed on the surface of NB liquid culture medium, and the liquid is turbid and uniform. After Gram staining and microscopic examination, the results are Gram-positive bacteria, the bacteria are short rod-shaped, wrapped in mucus, and can produce spores. The mid-terminal spores are full (such as Figure 1 and Figure 2 ).

[0039] (2) Identification of physiological and biochemical characteristics

[0040] Single colonies were picked and inoculated into bacterial micro-test tubes for determination of physiological and biochemical indices. Classification was performed with reference to Bergey's Manual of Bacteriological Identification and Manual of Common Bacterial Systematic Identification. The results are shown in Tables 1 and 2. It was preliminarily determined that strains BV-838 and BV-839 belonged to the genus Bacillus.

[0041] Table 1 Physiological and biochemical test results of strain BV-838

[0042] Physiological and biochemical indicators result Physiological and biochemical indicators result Escin + Lysine + Ornithine + Malonic acid + gelatin + glucose + MR-VP + Hydrogen sulfide - Rhamnose - Mannitol + Propionate - Growth at pH 5.7 - Xylose - Arabinose - Nitrate reduction - Starch hydrolysis + 7% Sodium Chloride +

[0043] Note: + indicates positive, – indicates negative.

[0044] Table 2 Physiological and biochemical test results of strain BV-839

[0045] Physiological and biochemical indicators result Physiological and biochemical indicators result Esculin + Lysine + Ornithine + Malonic acid + gelatin + glucose + MR-VP + Hydrogen sulfide - Rhamnose - Mannitol + Propionate - Growth at pH 5.7 - Xylose - Arabinose - Nitrate reduction - Starch hydrolysis + 7% Sodium Chloride +

[0046] Note: + indicates positive, – indicates negative.

[0047] (3) Whole genome sequencing of strain BV-838

[0048] The strain BV-838 was cultured in NB liquid medium (10g peptone, 3g beef extract powder, 5g sodium chloride, 1000ml distilled water, autoclaved at 121℃ for 15min) at 37℃ 220r / min for 8h, and then centrifuged at 12000rpm for 5min to collect the bacteria, extract genomic DNA, and after the DNA quality test was qualified, Wuhan Fraser Gene Information Co., Ltd. was commissioned for sequencing analysis. The whole genome shotgun (WGS) strategy was used to construct libraries with different insertion fragments, and the second-generation sequencing technology (Next-Generation Sequencing, NGS) was used based on the Illumina NovaSeq sequencing platform, and the third-generation single-molecule sequencing technology was used based on the Oxford Nanopore ONT sequencing platform to sequence and analyze these libraries.

[0049] The genome size of strain BV-838 is 3938728 bp, with a G+C content of 46.65%. It has a total of 4207 genes, with coding regions accounting for 88.06% of the genome, including 27 ribosomal RNA (rRNA) operons and 88 transfer RNAs (tRNAs) (Table 3).

[0050] Table 3 Genome information of strain BV-838

[0051] project result Genome size 3938728bp G+C content% 46.65％ Gene code number 4207 Average size of coding genes 870.9 Total length of coding region / genome length 88.06％ rRNA operon number 27 tRNA 88

[0052] The genome sequence was compared with the Nucleotide Sequence Database (NT) to obtain the top 3 species information comparison results of the genome as shown in Table 4.

[0053] Table 4 TOP3 results of NT library species information comparison of strain BV-838

[0054]

[0055] (4) Phylogenetic tree of strains BV-838 and BV-839

[0056] The 16S rDNA of strains BV-838 and BV-839 were selected for BLAST analysis, and the standard strains with high homology were selected to construct a phylogenetic tree using MEGA7.0 with the Neighbor-Joining method (e.g. Figure 3 ).

[0057] The nucleotide sequence of 16S rDNA of BV-838 is shown in SEQ ID NO.1, and the nucleotide sequence of 16S rDNA of BV-839 is shown in SEQ ID NO.2.

[0058] According to the phylogenetic tree and physiological and biochemical characteristics analysis results of 16S rDNA, combined with the genome species information comparison results of strain BV-838, strains BV-838 and BV-839 were identified as Bacillus velezensis.

[0059] These two strains were sent to the China Center for Type Culture Collection for preservation on January 10, 2025, with the address being Wuhan University, Wuhan, China. The classification name of strain BV-838 is Bacillus velezensis BV-838, and the preservation number is CCTCC NO: M 2025082; the classification name of strain BV-839 is Bacillus velezensis BV-839, and the preservation number is CCTCC NO: M 2025083.

[0060] 1.3 Enzyme production capacity of Bacillus velez BV-838 and BV-839

[0061] (1) Protease activity

[0062] 2 μL of BV-838 and BV-839 bacterial liquid were dropped vertically on NB solid medium containing 10% skim milk powder. After 10 minutes, they were cultured at 37°C for 24 hours and 5 days respectively. The obvious protein dissolution zone around the strain was observed, and the diameter of the dissolution zone was measured to evaluate the protease production ability of the strain. The results showed that the diameters of the dissolution zone (D) of BV-838 were 13.99±0.56mm and 35.66±0.32mm, respectively, and the diameters of the dissolution zone (D) of BV-839 were 13.62±0.78mm and 35.93±0.65mm, respectively, indicating that Bacillus Velezii BV-838 and BV-839 can produce protease, and the protease production characteristics are more prominent. As feed additives, they can significantly improve the utilization rate of feed protein.

[0063] (2) Amylase activity

[0064] Take 2 μL of BV-838 and BV-839 bacterial liquid and drop them vertically on NB solid medium containing 1% soluble starch. After 24 hours of static culture at 37°C, add 1 mL of iodine solution and observe whether there is an obvious starch dissolution zone around the strain within 1 minute. The diameter of the dissolution zone (D) of BV-838 and BV-839 was measured to be 13.48±0.89mm and 35.66±0.76mm, respectively, indicating that Bacillus Velezii BV-838 and BV-839 can produce amylase.

[0065] 1.4 Stress resistance of Bacillus velezinii BV-838 and BV-839

[0066] (1) Bile salt tolerance test

[0067] The strains BV-838 and BV-839 were cultured on NB solid medium for 3 days, 6 mL of physiological saline was taken to elute the colonies and mixed thoroughly, and the bacterial solution was inoculated into NB liquid medium with concentrations of 0.0%, 0.1%, 0.2%, 0.3% and 0.4% at a 10% inoculum (the initial bacterial concentration was 5×10 8 CFU / mL), with 3 replicates per group, and the growth was observed and the colony counts were performed after shaking culture at 37°C for 2 h.

[0068] The results show that Figure 4 B) After tolerating for 2 h in a liquid with a bile salt concentration of 0.1%-0.4% (mass percentage), the number of viable bacteria of strain BV-838 and the zero bile salt group (bile salt concentration of 0.0%) were both 3×10 9 CFU / mL, the viable counts of strain BV-839 and the zero bile salt group (bile salt concentration was 0.0%) were both above 1×10 9There was no significant difference above CFU / mL, indicating that both Bacillus Velezii BV-838 and BV-839 could tolerate the high bile salt environment.

[0069] (2) Acid resistance test

[0070] The strains BV-838 and BV-839 were cultured on NB solid medium for 3 days, 6 mL of physiological saline was taken to elute the colonies and mixed thoroughly, and the bacterial solution was inoculated into NB liquid medium with pH values ​​of 2, 3, 4, 5, 6, and 7 at a 10% inoculum (the initial bacterial concentration was 5×10 8 CFU / mL), with 3 replicates per group, and the growth was observed and the colony counts were performed after shaking culture at 37°C for 2 h.

[0071] The results show that Figure 4 A) In the pH range of 2 to 7, the number of viable bacteria of strain BV-838 was 3×10 9 CFU / mL, the number of viable bacteria of strain BV-839 was 1×10 9 There was no significant difference above CFU / mL, indicating that the survival rate of Bacillus Velezii BV-838 and BV-839 in the present invention was very high in the low pH gastric acid environment, and enough live bacteria could pass through the stomach and reach the intestine to exert their effects.

[0072] Embodiment 2:

[0073] Fermentation Technology of Bacillus Velezii BV-838 and BV-839

[0074] After activating Bacillus Velezii BV-838 and BV-839, single colonies were picked and inoculated into NB liquid culture medium, and the culture was shaken at 37°C and 220r / min for 12h to obtain seed liquid. The seed liquid was inoculated into fermentation medium (25g corn starch, 10g peptone, 2g soybean meal powder, 3g calcium chloride, 2g sodium chloride, 2g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, 0.3g manganese sulfate, 0.002g defoamer, 1000ml distilled water, sodium hydroxide to adjust pH7.0, and autoclaved at 121°C for 15min) at a ratio of 3%-5%, cultured at 37°C, pH 6.5±0.2, initial speed 75rpm, and the stirring speed was gradually increased as the dissolved oxygen value decreased, and finally increased to 200rpm; the initial ventilation volume was 1m 3 / h, as the fermentation time increases, the fermentation density increases, and the dissolved oxygen decreases, the ventilation volume is gradually increased to control the DO at 5%-10% and the ventilation volume reaches 8m 3 / h, and then the tank was released after culturing for 24h under this condition. When the conidial rate reached 90%, the fermentation liquid of Bacillus Velezii BV-838 and BV-839 was obtained. The effective bacterial count of BV-838 could reach 7.8×10 9CFU / mL, the effective bacterial count of BV-839 can reach 5.0×10 9 CFU / mL.

[0075] Embodiment 3:

[0076] Preparation of the compound preparation containing Bacillus velezensis and Macleaya cordata and other control groups:

[0077] Compound preparation group:

[0078] (1) The fermentation broth of Bacillus Velezii BV-838 and BV-839 was spray dried (the effective bacterial concentration after spray drying was 5.0×10 9 CFU / g) were mixed at a mass ratio of 1:1 to obtain the composite bacteria of Bacillus Velez; the total colony count was 5.0×10 9 CFU / g.

[0079] (2) The dried stems and leaves of Macleaya cordata are crushed and sieved, and extracted twice at a feed-liquid (i.e., pure water) ratio of 1:10, the combined filtrates are heated under reflux, and the extract is filtered to obtain an extract, and the obtained extract is concentrated and spray-dried to obtain the Macleaya cordata extract.

[0080] (3) The Bacillus velezensis compound bacteria and the Macleaya cordata extract were mixed in a mass ratio of 2:0.03, 4:0.02 or 3:0.025 to obtain the Bacillus velezensis and Macleaya cordata preparations of the present invention, which were named compound preparation group 1, compound preparation group 2 and compound preparation group 3, respectively.

[0081] Comparative Example 1:

[0082] Velez Bacillus composite bacteria group: This comparative example only contains the Velez Bacillus composite bacteria prepared in the above step (1), and the total bacterial concentration of the composite bacteria is 5.0×10 9 CFU / g.

[0083] Comparative Example 2

[0084] Macleaya cordata extract group: The Macleaya cordata extract group in this comparative example only contains Macleaya cordata extract, and the preparation method of the extract is as described above.

[0085] Comparative Example 3

[0086] BV-838 group: containing only the spray-dried bacterial agent of Bacillus Velezii BV-838 prepared in step (1) above, with a bacterial concentration of 5.0×10 9 CFU / g.

[0087] Comparative Example 4

[0088] BV-839 group: containing only the spray-dried bacterial agent of Bacillus Velezii BV-839 prepared in step (1) above, with a bacterial concentration of 5.0×10 9 CFU / g.

[0089] Embodiment 4:

[0090] Animal model preparation and animal experiments

[0091] 1.1 Experimental Animals 11-day-old male Arbor Acres (AA) broilers were randomly divided into 7 groups, each with 20 chickens. All broilers were housed in the same environmentally controlled facility (clean cages equipped with fiberglass feeders and plastic mesh floors), with free access to food and water, and given the same photoperiod (16 hours of light: 8 hours of darkness), relative humidity (1-7 days, 60%-70%; 8-42 days, 50%-60%) and room temperature (1-7 days, 33℃±2℃; 8-16 days gradually reduced to 24℃; 17-42 days, 24℃), and adaptive feeding for one week.

[0092] 1.2 The experiment set up a blank control group, a toxic attack control group, a compound preparation group 1, a compound preparation group 2, a compound preparation group 3, a Macleaya cordata extract group, a Bacillus Velezii composite bacteria group, a BV-838 group and a BV-839 group (see Example 3 for details).

[0093] The blank control group and the challenge control group (CON) were fed a corn-soybean-based diet that met the nutritional requirements of broilers for pelleted feed; the compound preparation group 1 was fed 2000 g / T feed of the compound preparation group 1 of Example 3; the compound preparation group 2 was fed 2000 g / T feed of the compound preparation group 2 of Example 3; the compound preparation group 3 was fed 2000 g / T feed of the compound preparation group 3 of Example 3; the Macleaya cordata extract group was fed 30 g / T feed of the Macleaya cordata extract of Comparative Example 2 of Example 3 (this dose has been widely proven to be effective); the Bacillus velezii composite bacteria group was fed 2000 g / T feed of Comparative Example 1 of Example 3; the BV-838 group was fed 2000 g / T feed of Comparative Example 3 of Example 3; and the BV-839 group was fed 2000 g / T feed of Comparative Example 4 of Example 3.

[0094] When the chickens were fed to 28 days of age, each group was intraperitoneally injected with avian pathogenic Escherichia coli (serotypes O2 and O78), with 10 chickens in each group challenged with the virus, and 5×10 7 CFU, each group continued to be fed according to the above method for 3 days after the virus attack, and an autopsy was performed at 31 days of age. During the experiment, the mental state of the experimental animals was observed at any time, and the incidence and death were recorded; after the end of the experiment, the relevant indicators were calculated to obtain Tables 5-7.

[0095] 1.3 Measurement indicators

[0096] (1) Clinical symptoms and pathological examination

[0097] The results showed that the acute mortality rate of the control group after the virus challenge reached 20%, and the surviving chickens showed typical symptoms of avian Escherichia coli infection such as lethargy, ruffled feathers, and yellow-green loose feces; the acute mortality rate of the single bacteria groups BV-838 and BV-839 after the virus challenge reached 15%, and the surviving chickens also showed typical symptoms of avian Escherichia coli infection; the acute mortality rate of the compound bacteria group and the Macleaya group after the virus challenge reached 10%, and the symptoms of the surviving chickens were improved compared with the virus challenge group and the single bacteria group; there was no acute death in the compound preparation group 1, compound preparation group 2, compound preparation group 3 and the blank control group after the virus challenge, and the surviving chickens were in good growth condition.

[0098] The autopsy results showed that all the chickens in the challenge control group showed severe symptoms of pericardium, perihepatitis and peritonitis after autopsy; the single bacteria groups BV838 and BV839 showed obvious symptoms of pericardium, perihepatitis and peritonitis, which were slightly improved compared with the challenge group; nearly half of the chickens in the composite bacteria group and the Macleaya group showed mild pericardium and perihepatitis symptoms, which were improved compared with the challenge control group; about 10% of the chickens in the compound preparation group 1 showed mild pericardium symptoms, and the autopsy results of the other chickens were normal, basically reaching the autopsy level of the blank control group, which was significantly improved compared with the challenge control group; about 20% of the chickens in the compound preparation groups 2 and 3 showed mild pericardium symptoms, and the autopsy results of the other chickens were normal, basically reaching the autopsy level of the blank control group, which was significantly improved compared with the challenge control group (see Figure 5 ).

[0099] The survival rate of the control group after the challenge reached 80%; the survival rate of the single bacteria group BV838 and BV839 after the challenge reached 85%; the survival rate of the compound bacteria group and the Macleaya group after the challenge reached 90%; the survival rate of the compound preparation group 1, compound preparation group 2, compound preparation group 3 and the blank control group reached 100%. The experimental results confirmed that the combination of Bacillus Velezii and Macleaya extract is better than the intervention effect of a single substance, and has a synergistic effect.

[0100] The specific survival rates by day are shown in the following table:

[0101] Table 5 Acute mortality and survival rate of broiler chickens

[0102] Acute mortality (%) Survival rate (%) Blank control group 0 100 The challenge control group 20 80 Compound preparation group 1 0 100 Compound preparation group 2 0 100 Compound preparation group 3 0 100 Macleaya cordata extract group 10 90 Bacillus Velez complex 10 90 BV-838 Group 15 85 BV-839 Group 15 85

[0103] (2) Growth performance

[0104] All broilers were weighed on an empty stomach at 11, 28 and 31 days of age, and their feed intake from 11 to 28 days and from 28 to 31 days of age was recorded to calculate average body weight (BW), average daily weight gain (ADG), average daily feed intake (ADFI) and feed-to-meat ratio (F / G).

[0105] Average body weight (BW) = body weight at the end of the experiment / number of chickens in each group;

[0106] Average daily gain (ADG) = (weight at the end of the experiment - weight at the beginning of the experiment) / (number of experimental days × number of chickens in each group);

[0107] Average daily feed intake (ADFI) = feed intake during the test period / (number of test days × number of chickens in each group)

[0108] Feed to meat ratio (F / G) = average daily feed intake / average daily weight gain

[0109] Table 6 Effects on broiler growth performance

[0110]

[0111] Note: * indicates significant difference compared with the challenge control group (p < 0.05), no mark indicates no significant difference (p > 0.05).

[0112] As shown in Table 6, at 31 days of age, the body weight (BW) of broilers in the compound preparation group was significantly increased compared with the control group (P<0.05), and the broilers in the compound preparation group 1 gained 160g more than the challenge control group. In the late growth period and the entire growth period of broilers, the average daily weight gain (ADG) and average daily feed intake (ADFI) of broilers in the compound preparation group were significantly increased (P<0.05).

[0113] (3) Intestinal morphology and structure

[0114] After the experiment, one broiler chicken with a weight close to the average was selected from each group. The intestinal tissue of about 0.5 cm in the middle section of the jejunum was taken and immersed in 4% paraformaldehyde solution for fixation. The solution was changed after 24 hours. After fixation for half a month, the chicken was embedded in paraffin and sectioned. HE staining was performed, and the villus height (VH) and crypt depth (CD) of the jejunum were observed and measured under a microscope, and the ratio of villus height to crypt depth (V / C) was calculated.

[0115] Table 7 Intestinal morphology and structure

[0116]

[0117]

[0118] Note: * indicates significant difference compared with the challenge control group (p < 0.05), no mark indicates no significant difference (p > 0.05).

[0119] As shown in the results in Table 7, compared with the challenge control group, the addition of the compound preparation to the diet can significantly increase the height of the jejunal villi and the ratio of the villi height to the crypt depth (V / C) (P<0.05). The higher the villi height, the greater the V / C value, and the stronger the digestion and absorption function of the intestine. Adding the compound preparation to the diet can enhance the digestion and absorption capacity of the jejunum.

[0120] In summary, the compound preparation of the present invention can effectively prevent and treat avian Escherichia coli disease, and at the same time has the effects of improving the intestinal flora of animals, enhancing the body's immunity and promoting growth. The combined effect of Bacillus velezensis and Macleaya cordata extract is better than the intervention effect of a single substance, and has a synergistic effect.

[0121] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A compound preparation containing Bacillus Velezii and Macleaya cordata for preventing and treating avian Escherichia coli disease, wherein the compound preparation comprises Bacillus Velezii composite bacteria: BV-838 and BV-839, with the deposit numbers being CCTCC NO: M 2025082 and CCTCC NO: M 2025083, respectively, and the Macleaya cordata is a Macleaya cordata extract.

2. The compound preparation according to claim 1, wherein the Bacillus Velezii composite bacteria and the Macleaya cordata extract are both spray-dried, and the mass ratio of the Bacillus Velezii composite bacteria to the Macleaya cordata extract is 2-4:0.02-0.

03.

3. The compound preparation according to claim 2, wherein the mass ratio of the Bacillus Velezii composite bacteria to the Macleaya cordata extract is 2:0.

03.

4. The compound preparation according to claim 1, wherein the effective bacterial concentrations of BV-838 and BV-839 are the same and are compounded in a mass ratio of 1:

1.

5. The compound preparation according to claim 2, wherein the effective bacterial concentration of the Bacillus Velezii compound bacteria is 5.0×10 9 CFU / g, the Macleaya cordata extract is a Macleaya cordata water extract.

6. Use of the compound preparation according to claim 1 in the preparation of a medicament for treating or preventing Escherichia coli infection.

7. The use according to claim 6, wherein the serotype of Escherichia coli includes O2, O18 and / or O78.

8. The use according to claim 6, wherein the dosage form of the drug is any pharmaceutically acceptable dosage form.

9. The use according to claim 8, wherein the dosage form comprises tablets, capsules, granules, injections, powders or drops.

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