Compound preparation containing bacillus velezensis and piper betel for preventing and treating avian colibacillosis and application thereof

Abstract

The present application belongs to the technical field of biological agents, and discloses a compound preparation containing bacillus velezensis and piper betel for preventing and treating avian colibacillosis and application. The mass ratio of the bacillus velezensis complex bacteria and the piper betel extract is 2-4:0.02-0.03; the bacillus velezensis is named as BV-838 and BV-839. It is proved by tests that the bacillus velezensis and the piper betel compound preparation have potential interaction, can cooperate with each other, play synergistic effect, can effectively prevent and treat avian colibacillosis, have the effect of enhancing immunity and promoting growth, are novel, efficient, green and environmentally friendly, and have important significance for the development of non-antibiotic breeding industrialization.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological agents, and particularly relates to a compound preparation containing Bacillus velezensis and Piper longum for synergistically preventing and treating avian colibacillosis. BACKGROUND

[0002] Avian colibacillosis is an important infectious disease caused by pathogenic Escherichia coli, which can be transmitted through respiratory tract, digestive tract and reproductive tract, etc., causing acute septicemia, perihepatitis, pericarditis, peritonitis, etc. in chickens. At present, it is widely prevalent in the poultry industry, ranks first among bacterial diseases, and is seriously mixed or secondary infected, causing economic losses to the poultry industry.

[0003] Due to the complex and diverse serotypes of E. coli, there is no effective vaccine for prevention and control, and antibiotics are often used for treatment in clinic. With the overuse or even abuse of antibiotics, the drug resistance rate of E. coli is increasing, the drug resistance spectrum is expanding, and the multiple drug resistance is increasing, which brings great challenges to the prevention and treatment of the disease. At the same time, plasmids containing drug resistance genes can also be conjugated and transferred between bacteria, spread among them, and transmitted to humans through the food chain, seriously threatening public health and environmental safety, so it is particularly important to find new effective means for treatment.

[0004] With the advent of the era of reducing and replacing antibiotics, the development and application of antibiotic substitutes have become a hot issue in the research and production of animal husbandry. The combination of probiotics and traditional Chinese medicine has the advantages of both probiotics and traditional Chinese medicine, and the combination of the two can produce a synergistic effect, that is, it can enhance the immunity of the body and promote growth, and also inhibit pathogenic microorganisms. Therefore, the development of a new type of high-efficiency, green and environmentally friendly compound preparation is the current industrial demand. SUMMARY

[0005] In view of the problems in the prior art, the present application aims to provide a compound preparation containing Bacillus velezensis and Piper longum for preventing and treating avian colibacillosis, which comprises Bacillus velezensis complex bacteria: BV-838 and BV-839, with preservation numbers of CCTCC NO: M 2025082 and CCTCC NO: M 2025083, respectively, and Piper longum extract.

[0006] Another object of the present application is to provide the use of the above-mentioned compound preparation in the preparation of a medicine for treating or preventing E. coli infection.

[0007] To solve the above technical problems, the present application provides the following technical solutions:

[0008] The application screens out two strains of Bacillus velezensis with antagonistic Escherichia coli and synergistic effect, the two strains are sent to China General Microbiological Culture Collection Center on January 10, 2025, the address is: Wuhan University, Wuhan, China, wherein the classification of strain BV-838 is named as Bacillus velezensis BV-838, the preservation number is CCTCC NO: M 2025082; the classification of strain BV-839 is named as Bacillus velezensis BV-839, the preservation number is CCTCC NO: M 2025083.

[0009] The protection scope of the application includes:

[0010] The compound preparation for preventing and treating avian colibacillosis contains Bacillus velezensis and Boerhaave, the compound preparation includes Bacillus velezensis complex bacteria: BV-838 and BV-839, the preservation numbers are CCTCC NO: M 2025082 and CCTCC NO: M 2025083 respectively, and the Boerhaave is Boerhaave extract.

[0011] The compound preparation is preferably that the Bacillus velezensis and the Boerhaave extract are both spray dried, and the mass ratio of the Bacillus velezensis complex bacteria and the Boerhaave extract is 2-4:0.02-0.03, and the more preferable ratio is 2:0.03;

[0012] The compound preparation is preferably that the effective bacteria concentrations of the BV-838 and the BV-839 are the same, and the two are compounded according to the mass ratio of 1:1;

[0013] The compound preparation is preferably that the effective bacteria concentration of the Bacillus velezensis complex bacteria is 5.0x10 9 CFU / g, and the Boerhaave extract is Boerhaave water extract.

[0014] The application of the compound preparation in preparing a medicine for treating or preventing colibacillosis.

[0015] The application is that the serotypes of the colibacillus include O2, O18 and / or O78.

[0016] The application is that the dosage form of the medicine is all dosage forms acceptable in pharmacy, including but not limited to tablets, capsules, granules, injections, powders or drops, etc.

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

[0018] The application provides a compound preparation containing bacillus velezensis and piper longum, which combines microecological preparation and Chinese veterinary medicine preparation, wherein the bacillus velezensis can regulate metabolism, enhance immunity, promote feed digestion and absorption, and has good inhibitory effect on main epidemic serotypes of avian pathogenic E. coli (O2, O18, O78); the piper longum has the effects of removing blood stasis, dispelling wind, detoxifying, relieving pain, inhibiting bacteria and expelling parasites. The test proves that the prevention and treatment effect of the compound preparation on avian colibacillosis can reach 90% to 100%, and the compound preparation has the effects of promoting growth and enhancing immunity on animal body, which has important significance for the development of non-antibiotic breeding industry. BRIEF DESCRIPTION OF DRAWINGS

[0019] The application is further described below in combination with the drawings and examples:

[0020] Figure 1 It is the colony morphology, gram staining microscopic examination and scanning electron microscope results of the strain BV-838;

[0021] Among them: A: macroscopic morphology of strain BV-838 NA medium; B: optical microscope examination results (10x100) of strain BV-838; C: electron microscope examination results of strain BV-838.

[0022] Figure 2 It is the colony morphology, gram staining microscopic examination and scanning electron microscope results of the strain BV-839;

[0023] Among them: A: macroscopic morphology of strain BV-839 NA medium; B: optical microscope examination results (10x100) of strain BV-839; C: electron microscope examination results of strain BV-839.

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

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

[0026] Among them: A: acid tolerance results of BV-838 and BV-839; B: bile salt tolerance results of BV-838 and BV-839.

[0027] Figure 5 It is the anatomical diagram of animal test;

[0028] Among them: A: blank control group; B: challenge control group; C: BV-838 group; D: BV-839 group; E: bacillus velezensis complex bacteria group; F: piper longum 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 objectives, technical solutions and advantages of the present application clearer, the present application will be further described below in conjunction with embodiments. It should be understood that the embodiments described herein are only used to explain the present application and should not be used to limit the present application.

[0030] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0031] The materials, reagents and the like used in the following examples can be obtained from commercial channels unless otherwise specified.

[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 breeding farms in multiple provinces across the country. 1 g or 1 ml of the sample was dissolved in 100 mL of sterile normal saline, gradient diluted, and then spread on NB solid medium (10 g of proteose peptone, 3 g of beef extract powder, 5 g of sodium chloride, 15 g of agar, 1000 ml of distilled water, 121℃ high pressure sterilization for 15 min) and incubated at 37℃ for 24 h. The bacteria were isolated and purified according to the morphological characteristics such as colony size, color, transparency and edge. Avian E. coli was used as an indicator for preliminary screening and multiple rescreening, and finally two strains with strong bacteriostatic effect were obtained, which were named as 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 characteristics and molecular biology detection results, it was identified as Bacillus velezensis. The specific technical solutions are as follows:

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

[0038] On NB solid medium, the colony was milky white or white with a slight yellowish color, early stage was concave round or round, surface was moist and opaque, edge was neat, and later stage was rough, edge was irregular and opaque colony; a thin film was formed on the surface of NB liquid medium, and the liquid was uniformly turbid; after Gram staining and microscopic examination, the result was Gram-positive bacteria, the cell was short rod-shaped, wrapped with mucus, could produce spores, and the central spore was full (such as Figure 1 and Figure 2 ).

[0039] (2) Physiological and biochemical characteristics identification

[0040] The single colony was inoculated in a bacterial micro-detection tube for physiological and biochemical index determination. Referring to the "Berger's Bacteriology Identification Manual" and "Common Bacterium System Identification Manual", the classification and determination were carried out, and the results are shown in Tables 1 and 2. It is preliminarily judged that the strains BV-838 and BV-839 belong to Bacillus.

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

[0042] Physiological and biochemical indexes Results Physiological and biochemical indexes Results 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: + represents positive, and - represents negative.

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

[0045] Physiological and biochemical indexes Results Physiological and biochemical indexes Results 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 +

[0046] Note: + represents positive, and - represents negative.

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

[0048] The strain BV-838 was cultured in NB liquid medium (10 g of proteose peptone, 3 g of beef extract powder, 5 g of sodium chloride, 1000 ml of distilled water, 121 ℃ high pressure sterilization for 15 min) at 37 ℃ with 220 r / min shaking for 8 h, then centrifuged at 12000 rpm for 5 min to collect the bacterial body. After the DNA quality detection was qualified, the sequencing analysis was entrusted to Wuhan Fisagen Information Co., Ltd. The whole genome shotgun (WGS) strategy was adopted to construct different insert libraries, and the second generation sequencing technology (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 for sequencing analysis of these libraries.

[0049] The genome size of strain BV-838 is 3938728 bp, the G+C content is 46.65%, there are 4207 genes, the coding region accounts for 88.06% of the genome, including 27 ribosomal RNA (rRNA) operons and 88 transfer RNAs (tRNA) (as shown in Table 3).

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

[0051] Item Results Genome size 3938728 bp G+C content % 46.65％ Number of genes encoding 4207 Average size of genes encoding 870.9 Total length of coding region / genome length 88.06％ Number of rRNA operons 27 tRNA 88

[0052] The genomic sequence was compared with the Nucleotide Sequence Database (NT) to obtain the species information alignment result TOP3 of the genome, as shown in Table 4.

[0053] Table 4. NT library species information alignment result TOP3 of strain BV-838

[0054]

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

[0056] BLAST analysis was performed on 16S rDNA of strains BV-838 and BV-839, and standard strains with higher homology were selected to construct a phylogenetic tree by Neighbor-Joining method using MEGA7.0 (as shown in FIG. 2). Figure 3

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

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

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

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

[0061] (1) Protease activity

[0062] ​2μL BV-838 and BV-839 bacterial liquid were vertically dropped on NB solid medium containing 10% skim milk powder, 10 min later, 24 h and 5 d were respectively placed at 37℃, whether the obvious protein dissolution ring appeared around the strain was observed, and the diameter of the dissolution ring was measured to evaluate the ability of the strain to produce protease. The results showed that the dissolution ring diameter (D) of BV-838 was 13.99±0.56 mm, 35.66±0.32 mm, respectively, and the dissolution ring diameter (D) of BV-839 was 13.62±0.78 mm, 35.93±0.65 mm, respectively, indicating that the bacillus velezensis BV-838 and BV-839 could produce protease, and the protease production characteristics were prominent, which could significantly improve the utilization rate of feed protein as a feed additive.

[0063] (2) Amylase activity

[0064] 2μL BV-838 and BV-839 bacterial liquid were vertically dropped on NB solid medium containing 1% soluble starch, 1 mL iodine solution was added after 24 h of culture at 37℃, and whether the obvious starch dissolution ring appeared around the strain was observed within 1 min, and the diameter of the dissolution ring was measured to evaluate the ability of the strain to produce amylase. The dissolution ring diameter (D) of BV-838 and BV-839 was 13.48±0.89 mm, 35.66±0.76 mm, respectively, indicating that the bacillus velezensis BV-838 and BV-839 could produce amylase.

[0065] 1.4 Anti-stress characteristics of bacillus velezensis BV-838 and BV-839

[0066] (1) Cholate salt tolerance test

[0067] Strains BV-838 and BV-839 were cultured on NB solid medium for 3 d, 6 mL of physiological saline was used to elute the colonies and mix thoroughly, and the bacterial liquid was inoculated into NB liquid medium with concentrations of 0.0%, 0.1%, 0.2%, 0.3% and 0.4% (10% inoculation amount, the initial bacterial concentration was 5×10 8 CFU / mL), each group was repeated 3 times, and the growth was observed and the colony count was performed after 2 h of culture at 37℃.

[0068] The results showed that Figure 4 In the bile salt concentration of 0.1%-0.4% (mass percentage) liquid, after 2 h of tolerance, the viable bacterial count of strain BV-838 and zero bile salt group (bile salt concentration of 0.0%) was above 3×10 9 CFU / mL, and the viable bacterial count of strain BV-839 and zero bile salt group (bile salt concentration of 0.0%) was above 1×10 9CFU / mL, and no significant difference, indicating that Bacillus velezensis BV-838 and BV-839 can tolerate high bile salt environment.

[0069] (2) Acid resistance test

[0070] Strains BV-838 and BV-839 were cultured on NB solid medium for 3 days, 6 mL of physiological saline was used to elute the colonies and fully mixed, and the bacterial solution was inoculated into NB liquid medium with pH values of 2, 3, 4, 5, 6 and 7 respectively (the initial bacterial concentration was 5×10 8 CFU / mL), 3 repeats were made for each group, and the growth was observed and colony counting was performed after 2h of 37℃ shaking culture.

[0071] The results show that Figure 4 CFU / mL, and no significant difference, indicating that Bacillus velezensis BV-838 and BV-839 can tolerate high bile salt environment. 9 CFU / mL, and no significant difference, indicating that Bacillus velezensis BV-838 and BV-839 can tolerate high bile salt environment. 9 CFU / mL, and no significant difference, indicating that Bacillus velezensis BV-838 and BV-839 can tolerate high bile salt environment.

[0072] Example 2:

[0073] Fermentation process of Bacillus velezensis BV-838 and BV-839

[0074] After activation of Bacillus velezensis BV-838 and BV-839, single colonies were picked and inoculated into NB liquid medium, and after 12h of 37℃ 220r / min shaking culture, seed liquid was obtained. The seed liquid was inoculated into fermentation medium (corn starch 25g, peptone 10g, soybean meal powder 2g, calcium chloride 3g, sodium chloride 2g, potassium hydrogen phosphate 2g, magnesium sulfate 0.5g, manganese sulfate 0.3g, defoaming agent 0.002g, 1000ml distilled water, sodium hydroxide was used to adjust pH to 7.0, 121℃ high pressure sterilization for 15min) at a ratio of 3%-5%, and 37℃ constant temperature culture was carried out, the pH was 6.5±0.2, the initial rotation speed was 75rpm, and the stirring rotation speed was gradually increased with the decrease of dissolved oxygen value, and finally increased to 200rpm; the initial aeration amount was 1m 3 / h, and the aeration amount was gradually increased when the fermentation density increased and the dissolved oxygen decreased, and the DO was controlled at 5%-10%, and the aeration amount reached 8m 3 / h, and the aeration amount was gradually increased when the fermentation density increased and the dissolved oxygen decreased, and the DO was controlled at 5%-10%, and the aeration amount reached 8m 9CFU / mL, the effective bacteria number of BV-839 can reach 5.0 x 10 9 CFU / mL.

[0075] Example 3

[0076] Preparation of compound preparation containing bacillus velezensis and pachysandra terminalis and other control groups:

[0077] Compound preparation group:

[0078] (1) Bacillus velezensis BV-838 and BV-839 fermentation liquor are respectively spray dried (the effective bacteria concentration after spray drying is 5.0 x 10 9 CFU / g), and then mixed according to a mass ratio of 1:1 to obtain bacillus velezensis compound bacteria; the total colony number is 5.0 x 10 9 CFU / g.

[0079] (2) The dried stems and leaves of pachysandra terminalis are crushed and sieved, extracted twice according to a solid-liquid (i.e. pure water) ratio of 1:10, the filtrates are combined and heated to reflux, and then filtered to obtain an extract, which is concentrated and then spray dried to obtain the pachysandra terminalis extract.

[0080] (3) The bacillus velezensis compound bacteria and the pachysandra terminalis extract are mixed according to a mass ratio of 2:0.03, 4:0.02 or 3:0.025 to obtain the bacillus velezensis and pachysandra terminalis compound preparation of the application, which are respectively named as compound preparation group 1, compound preparation group 2 and compound preparation group 3.

[0081] Comparative example 1

[0082] Bacillus velezensis compound bacteria group: only the bacillus velezensis compound bacteria prepared in the above step (1) is contained in the comparative example, and the total bacteria concentration of the compound bacteria is 5.0 x 10 9 CFU / g.

[0083] Comparative example 2

[0084] Pachysandra terminalis extract group: only the pachysandra terminalis extract is contained in the comparative example, and the preparation method of the extract is as described above.

[0085] Comparative example 3

[0086] BV-838 group: only the bacillus velezensis BV-838 spray dried bacteria agent prepared in the above step (1) is contained, and the bacteria concentration of BV-838 is 5.0 x 10 9 CFU / g.

[0087] Comparative example 4

[0088] The BV-839 group only contains the spray-dried Bacillus velezensis bacterial agent prepared in step (1) above, and the concentration of the BV-839 bacteria is 5.0 x 10 9 CFU / g.

[0089] Example 4

[0090] Preparation of animal models and animal tests

[0091] 1.1 The test animals were 11-day-old male Arbor Acres (AA) broilers, which were randomly divided into 7 groups, with 20 broilers in each group. All the broilers were raised in the same environmentally controlled facility (clean cages equipped with glass fiber feeders and plastic mesh floors), and were allowed to freely eat and drink, and were given the same light period (16 hours of light: 8 hours of darkness), relative humidity (60%-70% for 1-7 days, 50%-60% for 8-42 days), and room temperature (33°C±2°C for 1-7 days, gradually reduced to 24°C for 8-16 days, and 24°C for 17-42 days), and were adaptively fed for one week.

[0092] 1.2 The test was set up with a blank control group, an infection control group, a compound preparation group 1, a compound preparation group 2, a compound preparation group 3, a Boerhaave extract group, a Bacillus velezensis complex bacteria group, a BV-838 group, and a BV-839 group (see Example 3 for details).

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

[0094] The feeding was continued until the broilers were 28 days old, at which time each group was injected with avian pathogenic E. coli (O2 and O78 serotypes) in the abdominal cavity, with 10 chickens in each group being infected, at 5 x 10 7 CFU per chicken. After the infection, each group was continued to be fed according to the above method for 3 days, and necropsy was performed when the broilers were 31 days old. During the test period, the mental state of the test animals was observed at any time, and the incidence of death was recorded; after the test was completed, 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 challenge control group reached 20% after challenge, and the surviving chickens showed typical symptoms of avian E. coli infection, such as listlessness, scattered feathers, and yellowish green watery feces. The acute mortality rate of the single bacteria groups BV-838 and BV-839 reached 15% after challenge, and the surviving chickens also showed typical symptoms of avian E. coli infection. The acute mortality rate of the complex bacteria group and the Boerhaave group reached 10% after challenge, and the symptoms of the surviving chickens were improved compared with the challenge group and the single bacteria group. The compound preparation groups 1, 2, and 3, and the blank control group did not show acute death after challenge, and the surviving chickens grew well.

[0098] The results of the autopsy showed that all the chickens in the challenge control group showed severe pericarditis and peritonitis symptoms after dissection. The single bacteria groups BV-838 and BV-839 showed obvious pericarditis and peritonitis symptoms, which were slightly improved compared with the challenge group. Nearly half of the chickens in the complex bacteria group and the Boerhaave group showed mild pericarditis symptoms, which were improved compared with the challenge control group. About 10% of the chickens in the compound preparation group 1 showed mild pericarditis symptoms, and the other chickens had normal dissection results, basically reaching the dissection 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 pericarditis symptoms, and the other chickens had normal dissection results, basically reaching the dissection level of the blank control group, which was significantly improved compared with the challenge control group (see Table 4). Figure 5

[0099] The survival rate of the challenge control group after challenge reached 80%. The survival rate of the single bacteria groups BV-838 and BV-839 after challenge reached 85%. The survival rate of the complex bacteria group and the Boerhaave group after challenge reached 90%. The survival rates of the compound preparation groups 1, 2, and 3, and the blank control group reached 100%. The experimental results confirmed that the combination of Bacillus velezensis and Boerhaave extract was more effective than a single substance intervention, and had a synergistic effect.

[0100] The survival rates with the number of days are shown in the following table:

[0101] Table 5 Acute mortality rate and survival rate of broilers

[0102] Acute mortality rate (%) Survival rate (%) Blank control group 0 100 Challenge control group 20 80 Compound preparation group 1 0 100 Compound preparation group 2 0 100 Compound preparation group 3 0 100 Boerhaavia diffusa extract group 10 90 Bacillus velezensis complex bacteria group 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 the feed intake from 11 to 28 days of age and from 28 to 31 days of age was recorded. The average body weight (BW), average daily gain (ADG), average daily feed intake (ADFI), and feed to meat ratio (F / G) were calculated.​

[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 experimental period / (number of experimental days × number of chickens in each group)

[0108] Feed to Grain Ratio (F / G) = Average Daily Feed Intake / Average Daily 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 to the control group (P < 0.05). Broilers in compound preparation group 1 gained 160 g more weight than the challenge control group. During the late growth period and the entire growth period, the average daily 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 an average weight was selected from each group. The intestinal tissue of about 0.5 cm in the middle 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 tissue 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 Table 7, compared with the challenge control group, the addition of the compound preparation in the feed can significantly improve the villus height of jejunum and the ratio of villus height and crypt depth (V / C) (P<0.05). The higher the villus height, the greater the V / C value, and the stronger the digestive and absorptive function of the intestinal tract. The addition of the compound preparation in the diet can enhance the digestive and absorptive capacity of the jejunum.

[0120] In summary of the test results, the compound preparation can effectively prevent and treat avian colibacillosis, and has the effects of improving the intestinal flora of animals, enhancing the immunity of the body and promoting growth. The combination of Bacillus velezensis and extract of Boerhaave has a better effect than that of a single substance intervention, and has a synergistic effect.

[0121] The above only describes the embodiments of the present application, and does not limit the patent scope of the present application, and any equivalent structure or equivalent process transformation using the content of the present application specification, or direct or indirect application in other related technical fields, are also included in the patent protection scope of the present application.

Claims

1. A compound preparation for preventing and treating avian colibacillosis, the compound preparation being prepared from a Bacillus velezensis complex and a Macleaya cordata aqueous extract; the Bacillus velezensis complex being BV-838 and BV-839, with deposit numbers CCTCC NO: M 2025082 and CCTCC NO: M 2025083, respectively; BV-838 and BV-839 having the same effective bacterial concentration and being compounded in a mass ratio of 1:1; the Bacillus velezensis complex and the Macleaya cordata aqueous extract being spray-dried, with the mass ratio of the Bacillus velezensis complex to the Macleaya cordata aqueous extract being 2-4:0.02-0.

03.

2. The compound preparation according to claim 1, wherein the mass ratio of the Bacillus Velezii composite bacteria to the Macleaya cordata aqueous extract is 2:0.

03.

3. The compound preparation according to claim 1, wherein the effective bacterial concentration of the Bacillus Velezii complex is 5.0×10 9 CFU / g.

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

5. The use according to claim 4, wherein the serotype of Escherichia coli includes O2, O18 and / or O78.

6. The use according to claim 4, wherein the dosage form of the drug is any pharmaceutically acceptable dosage form.

7. The use according to claim 6, wherein the dosage form is selected from tablets, capsules, granules, injections, powders or drops.

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