Preparation method of an immune liquid for preventing and treating broiler coccidiosis and application thereof

Abstract

The application discloses a preparation method and application of an immune liquid for preventing and treating broiler coccidiosis. The method comprises the following steps: firstly, extracting sugar liquid and pectin liquid from beet roots; then taking the two liquids as a matrix to load coccidiosis-trapping protein and antibacterial enzyme liquid to form an immune adjuvant liquid; taking rabbit liver Eimeria oocysts to prepare a cross-species low-toxicity vaccine; and suspending the vaccine in the immune adjuvant liquid; and finally, using a drinking water immunization method to immunize broilers three times in an expanded field management mode, so that the broilers have high resistance to coccidiosis infection.

Description

Technical Field

[0001] The invention belongs to the technical field of poultry breeding, and particularly relates to a preparation method and application of an immune solution for preventing and treating broiler coccidiosis. Background Art

[0002] Coccidia is a major cause of intestinal disease in broiler chickens. Coccidia infect and colonize the intestines, causing fatal diarrhea, intestinal bleeding, anorexia, and intestinal damage. Chickens infected with the disease exhibit ruffled feathers, lethargy, a significant decrease in appetite and feed intake, and symptoms of emaciation, anemia, and dehydration. The mortality rate can exceed 50%.

[0003] Antibiotics and chemical synthetic drugs have become the main means of controlling coccidia in broiler chickens, but long-term and high-dose use of these methods can lead to drug resistance in coccidia strains and drug residues in corresponding meat products, which seriously restricts the healthy development of the broiler farming industry.

[0004] Therefore, coccidiosis immunology of broiler chickens has attracted much attention in the prevention and control of coccidiosis. Using live coccidia as immune vaccines is a common method in the current market, but this method has problems such as vaccine instability and difficulty in controlling dosage.

[0005] Therefore, there is an urgent need in the art for a stable, widely applicable, and non-toxic attenuated immune solution. Summary of the Invention

[0006] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of this application to avoid obscuring the purpose of this section, the abstract and the title of the invention, and such simplifications or omissions should not be used to limit the scope of the present invention.

[0007] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0008] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing an immune solution for preventing and treating broiler coccidiosis.

[0009] In order to solve the above technical problems, the present invention provides the following technical solutions: a method for preparing an immune solution for preventing and treating broiler coccidiosis, comprising:

[0010] Preparation of beetroot extract: washing beetroot tubers and shredding them, grinding the tuber shreds in phosphate buffer, incubating and then extracting the juice, separating the sugar solution and the waste meal, immersing the waste meal in phosphate buffer and adding cellulase, incubating and then separating to obtain a pectin filtrate;

[0011] Preparation of immune adjuvant: After starving the earthworms, clean the body surface and immerse the earthworms in concentrated sugar solution to induce osmotic pressure stress, then ultrasonicate them, filter out the mucin solution and the earthworm body, immerse the earthworm body in pectin filtrate, grind it, and homogenize it to obtain fibrinolytic enzyme solution;

[0012] Preparation of gangrene bacteria immune solution: Mix sugar solution, fibrinolytic enzyme solution and Corynebacterium glucuronolyticum solution, incubate at 30-37°C for 2-3 days, then inoculate Bifidobacterium solution, Lactobacillus plantarum solution and Clostridium butyricum solution, and store for later use;

[0013] Preparation of species-crossed vaccine: Compounding low-toxicity oocyst vaccines of Eimeria tenella, Eimeria precocious, and other strains with Eimeria skrjabini oocysts to prepare a compounded immune vaccine, soaking the compounded immune vaccine in a mucin solution, spreading the mixture into a thin layer of 2 to 4 mm, and irradiating with ultraviolet light for 60 to 120 minutes to obtain a species-crossed low-toxicity vaccine;

[0014] The pectin filtrate, mucin solution and low-toxicity vaccine are mixed into a suspension to prepare a first immunization solution for preventing and treating broiler coccidiosis;

[0015] The mucin solution, fibrinolytic enzyme solution and gangrene bacterial group immune agent are compounded to prepare a three-immunity solution for preventing and treating broiler coccidiosis.

[0016] As a preferred embodiment of the method for preparing the immune solution for preventing and treating broiler coccidiosis according to the present invention, the first immune solution comprises, by weight, 100 to 200 parts of pectin filtrate, 100 to 200 parts of mucin solution, and 1 to 3 parts of low-toxic vaccine; the third immune solution comprises, by weight, 1 to 3 parts of mucin solution, 1 to 3 parts of fibrinolytic enzyme solution, and 1 to 3 parts of gangrene bacterial immune agent.

[0017] As a preferred embodiment of the method for preparing the immune solution for preventing and treating broiler coccidiosis of the present invention, the beetroot extract comprises beetroot sugar solution and beetroot pectin filtrate, wherein the preparation method of the beetroot extract comprises:

[0018] The beetroot tubers are cleaned and shredded, and 1 to 3 parts by weight of the tuber shreds are immersed in 1 to 3 parts of a phosphate buffer solution having a pH of 6.5 for grinding, and the pulp is incubated at 60 to 70° C. for 12 to 24 hours, and then the juice is squeezed to separate the sugar liquid and the waste meal;

[0019] 100-300 parts of waste meal are immersed in 100-300 parts of phosphate buffer with pH=6.5, and 3-5 parts of cellulase are added, and incubated at 50-60°C for 3-5 hours to separate and obtain pectin filtrate, wherein the cellulase activity is ≥4400U / g.

[0020] As a preferred embodiment of the method for preparing the immune solution for preventing and treating broiler coccidiosis of the present invention, the immune adjuvant is composed of earthworm mucin solution and earthworm fibrinolytic enzyme solution. The method for preparing the immune adjuvant comprises:

[0021] The earthworms were starved for 24 hours, their bodies cleaned, and 1 to 3 parts of the earthworms were immersed in 5 to 7 parts of concentrated sugar solution for 3 to 5 hours of osmotic stress. The earthworms were then sonicated 10 to 20 times at a frequency of 30 to 35 kHz, a treatment time of 3 seconds, and a treatment interval of 30 seconds to filter out the mucin solution and the earthworm body.

[0022] Soak 1-3 parts of earthworm body in 5-7 parts of pectin filtrate, grind and homogenize to obtain fibrinolytic enzyme solution;

[0023] Among them, the sugar content of the sugar solution is 30-40%.

[0024] As a preferred embodiment of the method for preparing the immune solution for preventing and treating broiler coccidiosis of the present invention, the method for preparing the immune solution for gangrene bacteria comprises:

[0025] By weight, 1 to 3 parts of sugar solution, 5 to 7 parts of fibrinolytic enzyme solution and 0.01 to 0.05 parts of glucuronidase solution were mixed evenly, and the mixture was incubated at 30 to 37°C for 2 to 3 days, and then 0.01 parts of Bifidobacterium solution, 0.01 parts of Lactobacillus plantarum solution and 0.01 parts of Clostridium butyricum solution were added and stored at 4°C for later use; wherein,

[0026] Corynebacterium glucuronidase solution CFU≥10 9 , Bifidobacterium liquid CFU≥10 9 , Lactobacillus plantarum liquid CFU≥10 9 , Clostridium butyricum liquid CFU ≥ 10 9 .

[0027] As a preferred embodiment of the method for preparing the immune solution for preventing and treating broiler coccidiosis of the present invention, the species-crossed vaccine is composed of 5×10 low-toxicity oocyst vaccines of chicken coccidia: Eimeria tenella, Eimeria precocious and other insect strains. 4 strain, with rabbit coccidia: 4×10 Eimeria skrjabini oocysts 4 It is made by compounding plants.

[0028] As a preferred embodiment of the method for preparing the immune solution for preventing and treating broiler coccidiosis of the present invention, the method for weakening the toxicity of the species-crossed vaccine comprises:

[0029] One portion of the compounded immune vaccine is immersed in three portions of mucin solution, the mixture is spread into a 2 mm thin layer, and irradiated with ultraviolet light for 120 minutes to obtain a species-crossed low-toxicity vaccine.

[0030] Another object of the present invention is to overcome the deficiencies in the prior art and provide a product obtained by a method for preparing an immune solution for preventing and treating broiler coccidiosis.

[0031] Another object of the present invention is to overcome the deficiencies in the prior art and provide an application of the product in preventing and treating broiler coccidiosis, comprising:

[0032] Broiler drinking water immunization: Place the first immunization solution in a pen of 1,500 to 2,000 broilers aged 4 to 8 days for free feeding. After 5 to 7 hours, replace the drinking water with fresh drinking water to stop the drinking water immunization.

[0033] Multiple vaccination and expansion management: expand the pen to 30-40 chickens / m2 for broilers that have received their first vaccination 5-7 days after drinking water vaccination 2 The litter is thickened to 7-9 cm and the water content of the litter is maintained at 20-30%. The two-free broiler chickens are obtained after 3-5 days of breeding.

[0034] Then the litter is turned and raked, and the drinking water of the broilers is replaced with the three-free solution according to the weight ratio. After free diet for 12 to 24 hours, normal drinking water is provided to obtain the three-free broilers;

[0035] Remove the litter from the chicken pen and rinse it with boiling water. Re-lay the litter to 3-5cm and expand the pen to 10-13 chickens / m 2 Raise until the broiler chickens are ready for slaughter.

[0036] Beneficial effects of the present invention:

[0037] (1) The present invention provides a method for preparing an immune solution for preventing and treating broiler coccidiosis, wherein osmotic stress is a method for efficiently extracting earthworm mucin. Compared with traditional electric stimulation and microwave treatment, the advantage of sugar solution osmotic stress is the slow and mild nature of the stress. On the one hand, earthworms can continue to secrete anti-adversity mucus under weak stress without dying prematurely; on the other hand, mild stress is conducive to the secretion of earthworm mucus tending to escape mucin rather than death stress hormone.

[0038] (2) The addition of gangrene bacteria immune solution to the third drinking water immunization of the present invention can effectively regulate the probiotic flora of the broiler intestinal tract, and successfully plan glucuronidase-degrading bacteria, Bifidobacterium and Lactobacillus plantarum as the dominant flora of the broiler intestinal tract; among them, glucuronidase-degrading bacteria can effectively degrade the capsule of the typical gangrene bacteria Clostridium perfringens, thereby playing a role in immunizing the broiler intestinal tract and preventing tissue necrosis, and under the intervention of probiotics, the pathogenic bacteria Clostridium perfringens, Enterococcus faecalis and Staphylococcus aureus become non-dominant flora and are inhibited.

[0039] (3) The present invention introduces cross-species and cross-organ coccidia eggs based on the traditional weakly toxic coccidia eggs as immune vaccines. Under this condition, based on the non-pathogenicity of cross-species and cross-organ coccidia, the abundance of coccidia in the broiler intestine can be guaranteed and the toxicity caused by the immune vaccine can be reduced.

[0040] (4) The coccidia control method of the present invention achieves the optimal anticoccidial index after three drinking water immunizations. Mucin serves as a targeted trapping site for coccidia, preventing them from infecting the intestines. The fibrinolytic enzyme decomposes the coccidia capsule and stress barrier, reducing the stress resistance of the coccidia, thereby achieving the optimal anticoccidial effect. The gangrene bacterial group immune agent helps to coordinate the toxicity of the immune vaccine and prevent intestinal tissue gangrene caused by excessive toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort. Among them:

[0042] Figure 1 This is a diagram showing the effect of sugar solution stress on the extraction of earthworm mucin in an example of the present invention.

[0043] Figure 2 This is a diagram showing the intervention of the gangrene bacteria immune solution on the intestinal flora of broiler chickens in an embodiment of the present invention.

[0044] Figure 3 This is a graph showing the effect of the proportion of live coccidia egg vaccine on the first drinking water immunization in the examples of the present invention. DETAILED DESCRIPTION

[0045] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0046] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0047] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive of other embodiments.

[0048] Raw materials used in the present invention:

[0049] Qiannan broiler chickens: Institute of Animal Science, Guangdong Academy of Agricultural Sciences;

[0050] Earthworms: Chenggong Earthworm Farm in Dongli District, Tianjin;

[0051] Fresh beetroot: Xichuan County Xuyu Agricultural Products Sales Department;

[0052] Corynebacterium glucuronolyticum (DSM 100936), Bifidobacterium BMZ132587, Lactobacillus plantarum BMZ239542, and Clostridium butyricum BMZ134196: Ningbo Mingzhou Biotechnology Co., Ltd.;

[0053] Eimeria tenella, numbered bio-70778; Eimeria precocious, numbered bio-70847; Eimeria skrjabini, numbered bio-70858: China Microbial Strain Query Network, commonly available commercially;

[0054] Other raw materials are commercially available unless otherwise specified.

[0055] Example 1

[0056] (1) Preparation of Beetroot Extract: Wash and shred beetroot tubers. 1 part of the tuber shreds was immersed in 3 parts of phosphate buffer (pH = 6.5) for grinding, incubated at 70°C for 24 hours, and then juiced to separate the sugar solution and waste meal. 100 parts of the waste meal was immersed in 200 parts of phosphate buffer (pH = 6.5), and 5 parts of cellulase (≥ 4400 U / g) were added. The mixture was incubated at 60°C for 5 hours to separate the pectin filtrate.

[0057] (2) Preparation of immune adjuvant: 3 parts of earthworms were starved for 24 hours. After washing the body surface, 3 parts of earthworms were immersed in 5 parts of concentrated sugar solution (sugar content 40%) to induce osmotic stress for 5 hours. Subsequently, they were sonicated 20 times at a 35 kHz ultrasonic frequency, 3 seconds for 3 seconds, and 30 seconds between treatments. The mucin solution and earthworm bodies were filtered out. 3 parts of earthworm bodies were immersed in 5 parts of pectin filtrate, ground, and homogenized to obtain a fibrinolytic enzyme solution.

[0058] (3) Preparation of gangrene bacteria immune solution: According to weight, 2 parts of sugar solution, 6 parts of fibrinolytic enzyme solution and Glucuronidobacterium Glucuronidum solution (CFU≥10 9 ) 0.05 parts were mixed and incubated at 30-37℃ for 3 days before inoculating with Bifidobacterium solution (CFU≥10 9 )0.01 parts, Lactobacillus plantarum liquid (CFU≥10 9 ) 0.01 parts and Clostridium butyricum solution (CFU≥10 9) 0.01 portion was stored at 4℃ for future use.

[0059] (4) Preparation of species-crossed vaccines: 5×10 low-toxicity oocyst vaccines of Eimeria tenella, Eimeria precocious, and other strains 4 strains, and 4×10 Eimeria skrjabini oocysts 4 The strains were compounded, and one part of the compounded immune vaccine was immersed in three parts of mucin solution by weight. The mixture was spread into a 2 mm thin layer and irradiated with ultraviolet light for 120 minutes to obtain a species-interlaced low-toxicity vaccine.

[0060] (5) Broiler drinking water immunization: According to weight, 100 parts of pectin filtrate, 200 parts of mucin solution, and 3 parts of low-toxicity vaccine were mixed to form a suspension. The suspension was placed in a pen of 2,000 6-day-old broilers (70 birds / m2). 2 ) and provided free access to feed for broilers. After 6 hours, fresh drinking water was replaced and water immunization was stopped.

[0061] (6) Multiple vaccination and expansion management: expand the pen of broilers that received the first vaccination 7 days after drinking water vaccination to 40 / m 2 The litter was thickened to 8 cm and the moisture content of the litter was maintained at 30% for 5 days to obtain two-free broiler chickens.

[0062] The litter was then turned and raked, and the broiler drinking water was replaced with a three-immunity solution consisting of 3 parts of mucin solution, 3 parts of fibrinolytic enzyme solution, and 1 part of gangrene bacteria immune agent, according to weight. After 12 hours of free diet, normal drinking water was provided to obtain the three-immunity broiler chickens.

[0063] Remove the litter from the chicken pen and rinse it with boiling water. Re-lay the litter to 5cm and expand the pen to 10 chickens / m 2 Raise until the broiler chickens are ready for slaughter.

[0064] Example 2

[0065] Compared with Example 1, the preparation method of the immune adjuvant in this example is as follows:

[0066] The earthworms were starved for 24 hours by weight, and after cleaning their body surfaces, 3 parts of earthworms were immersed in 5 parts of water for 5 hours. They were then ultrasonically treated 20 times at a 35 kHz ultrasonic frequency, 3 s treatment time, and 30 s treatment intervals to filter out the mucin liquid and earthworm body.

[0067] Soak 3 parts of earthworm bodies in 5 parts of pectin filtrate, grind and homogenize to obtain fibrinolytic enzyme solution.

[0068] Other preparation process conditions are the same as those in Example 1.

[0069] Example 3

[0070] Compared with Example 1, the preparation method of the immune adjuvant in this example is as follows:

[0071] According to the weight ratio, the earthworms were starved for 24 hours. After cleaning the body surface, one part of the earthworm was immersed in 7 parts of concentrated sugar solution (sugar content 40%) to induce osmotic pressure stress for 5 hours. Then, the earthworms were ultrasonically treated 20 times at a frequency of 35kHz, a treatment time of 3s, and a treatment interval of 30s to filter out the mucin solution and the earthworm body.

[0072] 3 parts of earthworm bodies were immersed in 5 parts of pectin filtrate, ground and homogenized to obtain a fibrinolytic enzyme solution. Other preparation process conditions were the same as those in Example 1.

[0073] Example 4

[0074] Compared with Example 1, the preparation method of the immune adjuvant in this example is as follows:

[0075] Earthworms were starved for 24 hours. After washing their bodies, two parts of the earthworms were immersed in six parts of concentrated sugar solution (40% sugar content) to induce osmotic stress for five hours. The earthworms were then sonicated 20 times at a 35 kHz ultrasonic frequency, with a treatment time of 3 seconds and a 30-second interval. The mucin solution and the earthworm bodies were filtered out. Three parts of the earthworm bodies were immersed in five parts of the pectin filtrate, ground, and homogenized to obtain a fibrinolytic enzyme solution. All other preparation conditions were the same as in Example 1.

[0076] The mucin solution of the example was subjected to polyacrylamide gel electrophoresis to separate the proteins in the mixture. The collected tape proteins were digested with trypsin, and the digested product was centrifuged at 16,900 × g for 30 minutes. The resulting peptide components were injected into a fused quartz trapping column with reversed-phase silica gel, and then the peptides were separated on a C18 column with 1% formic acid aqueous solution and 1% acetonitrile solution as mobile phases A and B, and eluted at a linear gradient of 300 nL / min.

[0077] Liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS, SCIEX Triple Quad TMProteomic analysis was performed using a 7500 system (Thermo Fisher Scientific). The mass spectrometer used a source temperature of 90°C and a capillary voltage of 3.5 kV. The instrument was calibrated using fragments of the doubly protonated ion [glu1]-fibropeptide B (m / z 785.84), and the lock mass used during acquisition was the intact ion. LC single-strand MS / MS was performed according to a data-dependent acquisition method, selecting doubly or triply charged precursor ions for MS / MS. Ions were fragmented by collision-induced dissociation using argon as the collision gas and ramped collision energies that varied according to the charge state of the selected precursor ions. Data acquisition was performed over the m / z range of 300–2100 for MS searches (1 scan / s), and over the m / z range of 50–2500 for MS / MS. Data were collected using MassLynx 4.1 software and processed and converted into peak list text files for database searching using Protein Lynx Global Server 2.4. For peptide identification, the processed spectra were searched for MS / MS ions against the NCBIprot and SwissProt databases using MASCOT Daemon and search engine (Matrix Science Inc., USA). After data quality control and filtering, relative quantitative analysis of mucins was performed.

[0078] like Figure 1 Comparison of Examples 1 and 2 demonstrates that osmotic stress is a highly effective method for extracting earthworm mucin. The optimal stress method is to immerse 3 parts of earthworms in 5 parts of concentrated sugar solution (40% sugar) for 5 hours. Compared to traditional electrostimulation and microwave treatments, sugar solution osmotic stress offers the advantage of a gentle, slow stress response. This allows earthworms to continuously secrete stress-resistant mucus under mild stress, preventing premature death. Furthermore, this mild stress response promotes the secretion of escape-promoting mucins, rather than death-promoting stress hormones.

[0079] Example 5

[0080] (1) Preparation of Beetroot Extract: Wash and shred beetroot tubers. 1 part of the tuber shreds was immersed in 3 parts of phosphate buffer (pH = 6.5) for grinding, incubated at 70°C for 24 hours, and then juiced to separate the sugar solution and waste meal. 100 parts of the waste meal was immersed in 200 parts of phosphate buffer (pH = 6.5), and 5 parts of cellulase (≥ 4400 U / g) were added. The mixture was incubated at 60°C for 5 hours to separate the pectin filtrate.

[0081] (2) Preparation of immune adjuvant: 3 parts of earthworms were starved for 24 hours. After washing the body surface, 3 parts of earthworms were immersed in 5 parts of concentrated sugar solution (sugar content 40%) to induce osmotic stress for 5 hours. Subsequently, they were sonicated 20 times at a 35 kHz ultrasonic frequency, 3 seconds for 3 seconds, and 30 seconds between treatments. The mucin solution and earthworm bodies were filtered out. 3 parts of earthworm bodies were immersed in 5 parts of pectin filtrate, ground, and homogenized to obtain a fibrinolytic enzyme solution.

[0082] (3) Preparation of species-crossed vaccines: 5×10 low-toxicity oocyst vaccines of Eimeria tenella, Eimeria precocious, and other strains 4 strains, and 4×10 Eimeria skrjabini oocysts 4 The strains were compounded, and one part of the compounded immune vaccine was immersed in three parts of mucin solution by weight. The mixture was spread into a 2 mm thin layer and irradiated with ultraviolet light for 120 minutes to obtain a species-interlaced low-toxicity vaccine.

[0083] (4) Broiler drinking water immunization: Mix 100 parts pectin filtrate, 200 parts mucin solution, and 3 parts low-toxicity vaccine by weight to form a suspension. Place the suspension in a pen of 2,000 6-day-old broilers (70 birds / m2) and allow the broilers to feed ad libitum. After 6 hours, replace the drinking water with fresh drinking water and discontinue drinking water immunization.

[0084] (5) Multiple vaccination and expansion management: expand the pen of broilers that received the first vaccination 7 days after drinking water vaccination to 40 / m 2 The litter was thickened to 8 cm and the moisture content of the litter was maintained at 30% for 5 days to obtain two-free broiler chickens.

[0085] The litter was then turned and raked, and the broiler drinking water was replaced with a three-free solution made of 3 parts of mucin solution and 3 parts of fibrinolytic enzyme solution by weight. After free diet for 12 hours, normal drinking water was provided to obtain the three-free broiler chickens.

[0086] Remove the litter from the chicken pen and rinse it with boiling water. Re-lay the litter to 5cm and expand the pen to 10 chickens / m 2 Raise until the broiler chickens are ready for slaughter.

[0087] Feces from three-free broiler chickens were collected for total bacterial DNA extraction. After quality control and filtration, the intestinal bacterial genome was amplified using 16S primers, followed by gene sequencing using a NovaSeq 6000 high-throughput sequencer. The sequences were quality-controlled, assembled, clustered, and annotated to obtain information on all root rot fungi in the soil environment.

[0088] like Figure 2As shown, adding necrotic bacteria immunization solution to the third drinking water immunization effectively modulates the probiotic flora in broiler chickens' intestinal tract, successfully establishing Corynebacterium Glucuronidase, Bifidobacterium, and Lactobacillus plantarum as the dominant intestinal flora. Corynebacterium Glucuronidase can effectively degrade the capsule of Clostridium perfringens, a typical necrotic bacteria, thereby providing immunity to intestinal damage in broiler chickens and preventing tissue necrosis. Furthermore, under the intervention of probiotics, the pathogenic bacteria Clostridium perfringens, Enterococcus faecalis, and Staphylococcus aureus became non-dominant and were suppressed.

[0089] Example 6

[0090] Compared with Example 1, the species-crossed vaccine in this example is a low-toxic oocyst vaccine of Eimeria tenella, Eimeria precocious and other strains, 1×10 4 strains, and 9×10 4 The other preparation process conditions were the same as those in Example 1.

[0091] Example 7

[0092] Compared with Example 1, the species-crossed vaccine in this example is composed of 3×10 4 strains, and 7×10 4 The other preparation process conditions were the same as those in Example 1.

[0093] Example 8

[0094] Compared with Example 1, the species-crossed vaccine in this example is a low-toxic oocyst vaccine of 7×10 4 strains, and 3×10 Eimeria skrjabini oocysts 4 The other preparation process conditions were the same as those in Example 1.

[0095] Example 9

[0096] Compared with Example 1, the species-crossed vaccine in this example is a low-toxic oocyst vaccine of 9×10 4 strains, and 1×10 Eimeria skrjabini oocysts 4 The other preparation process conditions were the same as those in Example 1.

[0097] The ceca of broiler chickens were collected 3 days after the first vaccination to compare the intensity of the first vaccination.

[0098] Depend on Figure 3 It can be seen that when the species-crossed vaccine is composed of 5×10 low-toxic oocyst vaccines of Eimeria mitis, Eimeria harveyi, Eimeria precocious and other strains, 4strain, and 4×10 rabbit liver Eimeria oocysts 4 When the strains are combined, the primary immune response is most effective, with small blood clots appearing in the cecum, but the cecal structure is intact, indicating optimal broiler immunity. However, when the proportion of Eimeria rabbidum oocysts is too high, coccidia infection is poor and broiler immunity is inducible, as Eimeria rabbidum oocysts do not cross species and infect the broiler intestine. However, when the proportion of Eimeria rabbidum oocysts is too low, coccidia infection is significantly enhanced, with noticeable blood clots in the cecum and severe damage to the intestinal mucosa, indicating overinfection.

[0099] Example 10

[0100] Compared with Example 1, the water source for the third drinking water immunization in this example is prepared by compounding 1 part of mucin solution, 3 parts of fibrinolytic enzyme solution and 3 parts of gangrene bacteria immune agent, and the other preparation process conditions are the same as those in Example 1.

[0101] Example 11

[0102] Compared with Example 1, the water source for the third drinking water immunization in this example is prepared by compounding 2 parts of mucin solution, 2 parts of fibrinolytic enzyme solution and 3 parts of gangrene bacteria immune agent, and the other preparation process conditions are the same as those in Example 1.

[0103] Example 12

[0104] Compared with Example 1, the water source for the third drinking water immunization in this example is prepared by compounding 2 parts of mucin solution, 3 parts of fibrinolytic enzyme solution and 2 parts of gangrene bacteria immune agent, and the other preparation process conditions are the same as those in Example 1.

[0105] Example 13

[0106] Compared with Example 1, the water source for the third drinking water immunization in this example is prepared by compounding 3 parts of mucin solution, 2 parts of fibrinolytic enzyme solution and 2 parts of gangrene bacteria immune agent, and the other preparation process conditions are the same as those in Example 1.

[0107] Broiler samples were collected 2-3 days before slaughter. The lesion value was scored based on the cecal lesion phenomenon: 0 points: normal, no macroscopic lesions; ±1 point: a small number of scattered petechiae on the cecal wall, no intestinal wall thickening, and normal contents; ±2 points: a large number of lesions, obvious blood in the cecal contents, slight cecal wall thickening, and normal contents; ±3 points: a large amount of blood or cecal core in the cecum, obvious cecal wall hypertrophy, and little fecal content in the cecum; ±4 points: the cecum is enlarged due to a large amount of blood or intestinal core (dead chickens are scored ±4 points). Oocyst value conversion method: the number of oocysts excreted in feces is <0.1×10 5 The corresponding oocyst value is 0; the number of oocysts excreted in feces is 0.1-1.0×10 5 The corresponding oocyst value is 1; the number of oocysts excreted in feces is 2.0-5.0×10 5The corresponding oocyst value is 10; the number of oocysts excreted in feces is 6.0-10.0×10 5 The corresponding oocyst value is 20; the number of oocysts excreted in feces is >11.0×10 5 The corresponding oocyst value is 40.

[0108] Calculation of the anticoccidial index for broiler chickens:

[0109] Anti-coccidial index = (weight gain rate + survival rate) × 100 - (lesion value + coccidia egg number)

[0110] Table 1 Comparison of anticoccidial index under various drinking water immunization formulas

[0111]

[0112] Regarding the immune adjuvant extraction method, as shown in Examples 1-4 in Table 1, the core component of the immune adjuvant is earthworm fibrinolytic enzyme, which can destroy the surface resistance barrier of coccidia, making them easier to kill. The second core component is beetroot pectin, which has antibacterial properties. The extraction of earthworm fibrinolytic enzyme is based on the fact that earthworms are forced to produce enzymatic mucus under the high osmotic pressure brought by a high-concentration sugar solution. When the sugar solution concentration is too high (Examples 3-4) or the osmotic pressure is insufficient to coerce the earthworms (Example 2), the earthworms are unable to produce high-quality fibrinolytic enzyme.

[0113] The necessity of the gangrene bacteria immune solution is shown in Examples 1 and 5 in Table 1, and Figure 2 As shown, after the gangrene bacteria immune solution was added, intestinal probiotics such as Corynebacterium Glucuronidiolyticum, Bifidobacterium and Lactobacillus plantarum replaced pathogenic bacteria such as Clostridium perfringens, Enterococcus faecalis and Staphylococcus aureus to become the dominant intestinal bacteria. Therefore, the relative weight gain value of Example 1 was higher than that of Example 5, and the lesion value was significantly reduced.

[0114] For the coccidial vaccine composite amount, as shown in Example 1 and Example 6-9 in Table 1, when the strain amounts such as chicken coccidial ovum and rabbit coccidial ovum are composited to form coccidial vaccine, the coccidial vaccine effect is best. When the ratio is destroyed, as the chicken coccidial ovum amount is greater than the rabbit coccidial ovum (embodiment 8-9), now young broiler chickens are persecuted too seriously by coccidia, and this causes the relative weight gain rate of broiler chickens to decline and pathological changes value and oocyst value significantly rise; As the rabbit coccidial ovum amount is greater than the chicken coccidial ovum (embodiment 6-7), due to cross-species coccidia infection efficiency low, so cause chicken coccidial immune response deficiency, the final coccidial immunity of broiler chickens is not strong, therefore pathological changes value and oocyst value significantly rise relative to Example 1, and broiler chickens are first significantly reduced to weight gain rate.

[0115] Table 2 Comparison of anticoccidial index under various drinking water immunization formulas

[0116]

[0117] As shown in Table 2, the non-drug immune enhancement method for preventing and controlling coccidiosis in broiler chickens can maintain the survival rate of broiler chickens at 100%, and the third drinking water immunization composed of 3 parts of mucin solution, 3 parts of fibrinolytic enzyme solution and 1 part of gangrene bacterial group immune agent can achieve the optimal anticoccidial index.

[0118] Mucin acts as a targeted trapping site for coccidia, preventing them from infecting the intestines. Fibrinolytic enzyme breaks down the coccidia capsule and stress barrier, reducing their resistance to stress and achieving optimal anticoccidial efficacy. Necrotic bacteria immunization agents help balance the toxicity of vaccines, preventing intestinal gangrene caused by excessive toxicity.

[0119] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for preparing an immune solution for preventing and treating coccidiosis in broiler chickens, characterized in that: include, Preparation of beetroot extract: washing beetroot tubers and shredding them, grinding the tuber shreds in phosphate buffer, incubating and then extracting the juice, separating the sugar solution and the waste meal, immersing the waste meal in phosphate buffer and adding cellulase, incubating and then separating to obtain a pectin filtrate; Preparation of immune adjuvant: After starving the earthworms, clean the body surface and immerse the earthworms in concentrated sugar solution to induce osmotic pressure stress, then ultrasonicate them, filter out the mucin solution and the earthworm body, immerse the earthworm body in pectin filtrate, grind it, and homogenize it to obtain fibrinolytic enzyme solution; Preparation of gangrene immune solution: Mix sugar solution, fibrinolytic enzyme solution and Corynebacterium glucuronolyticum solution, incubate at 30-37°C for 2-3 days, then inoculate Bifidobacterium solution, Lactobacillus plantarum solution and Clostridium butyricum solution and store for future use; wherein the Corynebacterium glucuronolyticum is numbered DSM 100936, the Bifidobacterium is numbered BMZ132587, the Lactobacillus plantarum is numbered BMZ239542, and the Clostridium butyricum is numbered BMZ134196, all of which are purchased from Ningbo Mingzhou Biotechnology Co., Ltd. Preparation of species-crossed vaccine: 5×10 4 strains, and 4×10 Eimeria skrjabini oocysts 4 The strains were compounded to prepare a compounded immune vaccine, which was immersed in a mucin solution, and the mixture was spread into a thin layer of 2 to 4 mm, and irradiated with ultraviolet light for 60 to 120 minutes to obtain a species-crossed low-toxicity vaccine. The pectin filtrate, mucin solution and low-toxicity vaccine are mixed into a suspension to prepare a first immunization solution for preventing and treating broiler coccidiosis; The mucin solution, fibrinolytic enzyme solution and gangrene bacterial group immune agent are compounded to prepare a three-immunity solution for preventing and treating broiler coccidiosis. The first immunization solution comprises, by weight, 100 to 200 parts of pectin filtrate, 100 to 200 parts of mucin solution, and 1 to 3 parts of low-toxicity vaccine; the third immunization solution comprises, by weight, 1 to 3 parts of mucin solution, 1 to 3 parts of fibrinolytic enzyme solution, and 1 to 3 parts of gangrene bacterial immune agent.

2. The method for preparing an immune solution for preventing and treating broiler coccidiosis according to claim 1, wherein: The beetroot extract comprises beetroot sugar solution and beetroot pectin filtrate, wherein the preparation method of the beetroot extract comprises: Wash and shred the beetroot tubers. 1-3 parts by weight of the tuber shreds are immersed in 1-3 parts of phosphate buffer at pH 6.5 for grinding. The pulp is incubated at 60-70°C for 12-24 hours and then the juice is extracted to separate the sugar solution and the waste meal. 100-300 parts of waste meal are immersed in 100-300 parts of phosphate buffer solution with a pH of 6.5, and 3-5 parts of cellulase are added. The mixture is incubated at 50-60°C for 3-5 hours to separate a pectin filtrate, wherein the cellulase activity is ≥4400 U / g.

3. The method for preparing an immune solution for preventing and treating broiler coccidiosis according to claim 1 or 2, wherein: The immune adjuvant is composed of earthworm mucin solution and earthworm fibrinolytic enzyme solution. The preparation method of the immune adjuvant is as follows: include, The worms were starved for 24 hours, cleaned, and then immersed in 5-7 parts of concentrated sugar solution for 3-5 hours. The worms were then sonicated 10-20 times at a frequency of 30-35 kHz, a treatment time of 3 seconds, and a treatment interval of 30 seconds. The mucin solution and worm bodies were filtered out. Soak 1-3 parts of earthworm body in 5-7 parts of pectin filtrate, grind and homogenize to obtain fibrinolytic enzyme solution; Among them, the sugar content of the sugar solution is 30~40%.

4. The method for preparing an immune solution for preventing and treating broiler coccidiosis according to claim 1 or 2, wherein: The method for preparing the gangrene bacteria immune solution comprises: By weight, 1-3 parts of sugar solution, 5-7 parts of fibrinolytic enzyme solution and 0.01-0.05 parts of glucuronidase solution were mixed evenly, and incubated at 30-37°C for 2-3 days, and then 0.01 parts of Bifidobacterium solution, 0.01 parts of Lactobacillus plantarum solution and 0.01 parts of Clostridium butyricum solution were added and stored at 4°C for later use; wherein, Corynebacterium glucuronidase solution CFU≥10 9 , Bifidobacterium liquid CFU≥10 9 , Lactobacillus plantarum liquid CFU≥10 9 , Clostridium butyricum liquid CFU ≥ 10 9 .

5. The method for preparing an immune solution for preventing and treating broiler coccidiosis according to claim 1, wherein: Methods for reducing the toxicity of cross-species vaccines include: One portion of the compounded immune vaccine was immersed in three portions of mucin solution, the mixture was spread into a 2 mm thin layer, and irradiated with ultraviolet light for 120 minutes to obtain a species-crossed low-toxicity vaccine.

6. The product obtained by the method for preparing the immune solution for preventing and treating broiler coccidiosis according to any one of claims 1 to 5.

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