Streptococcus dysgalactiae, and preparation method and application of streptococcus dysgalactiae egg yolk antibody

Abstract

The invention relates to the technical field of biology, and particularly discloses streptococcus dysgalactiae and a preparation method and application of a streptococcus dysgalactiae egg yolk antibody, the streptococcus dysgalactiae is preserved in China General Microbiological Culture Collection Center on December 24, 2024, and the preservation number is CGMCC No.46272; the preparation method of the streptococcus dysgalactiae egg yolk antibody comprises the following steps: preparing a streptococcus dysgalactiae antigen, and carrying out vaccine distribution treatment on the antigen to obtain a streptococcus dysgalactiae vaccine; carrying out intramuscular injection on the chicken with a streptococcus dysgalactiae vaccine, and carrying out immune treatment; identifying the titer of the egg yolk, determining that the egg yolk accords with antibody preparation, then cleaning and disinfecting, and then air-drying; egg yolk and egg white are separated from the air-dried eggs, the egg yolk is pretreated with a disodium hydrogen phosphate solution, then acidification extraction, filtration sterilization and detection are conducted, and egg yolk antibodies are obtained; the compound is used for preventing and treating streptococcus dysgalactiae infection of pigs; the control effect is good.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and more specifically, to a method for preparing Streptococcus lactis, Streptococcus lactis egg yolk antibodies, and their applications. Background Technology

[0002] Streptococcus dysgalactiae is a common Gram-positive coccus belonging to the genus Streptococcus. It contains hundreds of virulence genes and mainly causes mastitis in dairy cows, but it can also cause a variety of other infections, including bacteremia, endocarditis, meningitis, arthritis, and respiratory infections.

[0003] Streptococcus lactis is resistant to multiple antibiotics and contains resistance genes for various antibiotics, especially fluoroquinolones, tetracyclines, macrolides, and aminoglycosides. Therefore, its prevention and control face significant challenges. Summary of the Invention

[0004] In order to provide a method for preparing Streptococcus galactiae with good preventive and therapeutic effects, and to further prepare a high-efficiency, low-cost Streptococcus galactiae egg yolk antibody for application in medicine and veterinary medicine to improve the prevention and treatment of Streptococcus galactiae infection in animals, this application provides a method for preparing Streptococcus galactiae and a Streptococcus galactiae egg yolk antibody, as well as their applications.

[0005] In a first aspect, this application provides a Streptococcus faecium that stops lactation, employing the following technical solution: A Streptococcus dysgalactiae was deposited on December 24, 2024, at the China General Microbiological Culture Collection Center (CGMCC), No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46272.

[0006] By adopting the above technical solution, the isolated and screened Streptococcus dysgalactiae have high drug resistance. The antibodies prepared using them contain drug resistance genes and have good prevention and treatment effects. After the antibodies are injected, the prevention and treatment effects of Streptococcus dysgalactiae infection in animals are improved.

[0007] Preferably, the Streptococcus lactis is obtained through isolation and purification, and the specific steps are as follows: (1) Fresh tissue from the lesion area was collected aseptically, bacteria were isolated, and then inoculated onto blood agar plates for purification to obtain Streptococcus pyogenes; (2) Streptococcus dysgalactiae was identified by PCR and 16S rRNA gene sequence analysis.

[0008] By employing the above-mentioned technical solutions, direct isolation from fresh tissue in breast lesions can maximize the preservation of the original strain characteristics, avoiding false negatives caused by sample contamination or bacterial imbalance. After isolation, blood plate culture is used to rapidly distinguish Streptococcus spp. based on hemolytic characteristics, and the purification process eliminates interference from commensal bacteria, significantly improving the purity of the target strain. Rapid initial screening using PCR: specific primers can quickly pinpoint the target pathogen, shortening the detection cycle; 16S rRNA gene sequencing: sequence alignment combined with phylogenetic tree analysis can clearly identify the species classification, thus obtaining high-purity Streptococcus galactiae with good preventive and therapeutic effects. Preferably, in step (1), the blood plate is a THB solid culture medium containing 3-6% fetal bovine serum.

[0009] By employing the above technical solution, fetal bovine serum provides hormones lacking in the basal culture medium, such as insulin and epidermal growth factor, as well as low-molecular-weight nutrients like amino acids and lipids. This promotes the proliferation of Streptococcus lactiferus, a strain with demanding nutritional requirements, thereby shortening the isolation cycle. Furthermore, it inhibits the overgrowth of other bacteria, ensuring the genetic consistency of the isolates. Preferably, the separation and purification temperatures in step (1) are both 35-38°C, and the time is both 22-25h.

[0010] By adopting the above technical solutions, temperature and time are controlled to ensure the growth and reproduction of Streptococcus lactis while reducing interference from other bacteria and ensuring the purity of the separation.

[0011] Secondly, this application provides a method for preparing Streptococcus lactis egg yolk antibody, which adopts the following technical solution: A method for preparing Streptococcus lactis egg yolk antibody includes the following steps: S1. Prepare Streptococcus dysgalactiae antigen, and after the antigen is treated with vaccine, obtain Streptococcus dysgalactiae vaccine; S2. Inject the Streptococcus pyogenes vaccine into chickens via intramuscular injection for immunization; identify the yolk titer to confirm it meets the requirements for antibody preparation, then clean and disinfect the yolks, and finally air dry them. S3. After air-drying, the egg yolk and egg white are separated. The egg yolk is pretreated with disodium hydrogen phosphate solution, then acidified, extracted, filtered and sterilized, and tested to obtain egg yolk antibodies.

[0012] By adopting the above technical solution, Streptococcus lactis antigen is first prepared. The antigen is then treated with a vaccine to enhance its immunogenicity, making it easier for the immune system to recognize and attack it. This reduces harmful substances and impurities in the antigen, improving vaccine safety. After the vaccine is injected, it undergoes immunization treatment to ensure the antigen is evenly distributed in the animal's body. After washing and disinfecting the eggs, the yolks and egg whites are separated. Disodium hydrogen phosphate solution is added to the yolks. The buffering effect of sodium hydrogen phosphate solution helps maintain the stability of the extract, ensuring the stability and activity of antibody molecules. Then, the yolks are filtered and sterilized to reduce the influence of other bacteria, ensuring the purity, quality, and stability of the yolk antibodies, thereby guaranteeing the preventive and therapeutic effects of the yolk antibodies.

[0013] Preferably, the Streptococcus lactis antigen is prepared using the following method: The identified Streptococcus lactis was cultured and the bacterial cells were collected. After washing the bacterial cells, collect them, adjust the bacterial concentration, add 0.1-0.3% formaldehyde solution to the bacterial solution for inactivation, and then inoculate the inactivated bacterial solution into Erlenmeyer flasks of THB liquid medium. After 46-50 hours, transfer the culture to blood agar plates, incubate, and test for no viable bacteria to obtain the antigen.

[0014] By adopting the above technical solution, the identified Streptococcus lactis was cultured, collected, and after washing, inactivated with formaldehyde solution to destroy bacterial activity while preserving antigen structure. The bacteria were then transferred to Erlenmeyer flasks and cultured at a limited temperature to obtain inactivated antigens with good drug resistance retention.

[0015] Preferably, the cleaning and disinfection steps in S2 are as follows: the antibody is soaked in 0.08-0.12% 42℃ benzalkonium chloride solution for 12-18 minutes, then cleaned to remove dirt, and then fumigated with 0.08-0.12% Weikang spray for 28-32 minutes.

[0016] By adopting the above technical solutions, the combined treatment of Xin Jie Er solution and Wei Kang spray fumigation can effectively clean and disinfect while ensuring the purity and quality of antibodies.

[0017] Preferably, the egg yolk in step S3 is pretreated with disodium hydrogen phosphate solution. The specific steps are as follows: Mix egg yolk and disodium hydrogen phosphate solution at a 1:1 (v / v) ratio and stir well; filter through a 55-65 mesh steel wire sieve to remove impurities from the egg yolk liquid, transfer to a settling tank, add the remaining 2 times the volume of disodium hydrogen phosphate solution, and stir at a constant speed of 40-80 r / min for 50-70 min to obtain a mixture.

[0018] By adopting the above technical solution, after the egg yolk is treated with sodium dihydrogen phosphate solution, the neutral system of sodium dihydrogen phosphate prevents the denaturation of egg yolk protein caused by the acidic environment and promotes the aggregation of impurities, thereby further improving the purity and quality of the antibody. With the limited stirring speed and time, the sodium dihydrogen phosphate solution is ensured to be in uniform contact with the egg yolk, thereby improving the uniformity and quality of the egg yolk antibody formation.

[0019] Preferably, the acidification extraction in step S3 specifically involves the following steps: Add 1 part sedimentation solution (polyvinylpyrrolidone PVP: trehalose = 1:0.8-1.1) to the sedimentation tank, then adjust the pH to 5.8-6.2 with 20-35% citric acid solution; let it settle for 2-2.5 hours, then transfer all the supernatant; mix the supernatant with octanoic acid solution, with the final concentration of octanoic acid being 0.02-0.1%, to obtain a composite solution.

[0020] By adopting the above technical solution, polyvinylpyrrolidone (PVP) and trehalose in the sedimentation liquid form a three-dimensional network structure. PVP captures host cell proteins through hydrophobic interactions, while trehalose maintains antibody conformational stability and prevents antibody aggregation. In addition, the pH adjustment treatment with citric acid facilitates the removal of precipitates and facilitates complexation with metal ions to avoid antibody denaturation. At the same time, the treatment with caprylic acid further removes protein precipitates and ensures antibody activity, thereby improving the purity, quality and activity of the antibody.

[0021] Thirdly, this application provides an application of an antibody against Streptococcus lactis egg yolk, employing the following technical solution: Application of an antibody against Streptococcus galactiae egg yolk: preparation of a biological agent for the prevention and treatment of Streptococcus galactiae infection in pigs.

[0022] By adopting the above technical solution, the biological agent prepared from egg yolk antibodies has a good effect on the prevention and treatment of Streptococcus faecium infection in pigs, and can prolong the pigs' long-term resistance to bacterial infection.

[0023] In summary, this application has the following beneficial effects: 1. The isolated and screened Streptococcus dysgalactiae exhibit high drug resistance. Antibodies prepared from them contain drug resistance genes and have good prevention and treatment effects. After antibody injection, the prevention and treatment effects of Streptococcus dysgalactiae infection in animals are improved.

[0024] 2. First, prepare Streptococcus lactis antigen. After vaccine preparation, enhance the antigen's immunogenicity, making it easier for the immune system to recognize and attack it. This also reduces harmful substances and impurities in the antigen, improving vaccine safety. After injection, the vaccine undergoes immunization to ensure uniform antigen distribution in the animal's body. After washing and disinfecting the eggs, separate the yolks and egg whites. Add disodium hydrogen phosphate solution to the yolks. The buffering effect of disodium hydrogen phosphate helps maintain the stability of the extract, ensuring the stability and activity of antibody molecules. Then, filter and sterilize to reduce the influence of other bacteria, ensuring the purity, quality, and stability of the yolk antibodies, thereby guaranteeing the preventive and therapeutic effects of the yolk antibodies. Attached Figure Description

[0025] Figure 1 This is a microscopic morphological image of the Streptococcus lactis strain described in this application. Detailed Implementation

[0026] The present application will be further described in detail below with reference to the accompanying drawings and embodiments.

[0027] Example of blood plate preparation Preparation Example 1: Blood agar plates were prepared using the following method: Weigh out 10 g / L beef powder, 20 g / L tryptone, 15 g / L agar, 2 g / L glucose, 2 g / L sodium bicarbonate, 2 g / L sodium chloride, 0.4 g / L disodium bicarbonate, and fetal bovine serum. Adjust the pH to 7.8, add water to make up to 1 L, heat to 65℃ and stir magnetically until completely dissolved to obtain the culture medium solution. The fetal bovine serum content in 1 L of culture medium solution is 5%. The culture medium solution was sterilized at 121℃ for 20 min, and when cooled to 50℃, 10 mg of polymyxin E and 15 mg of nalidixic acid were added. The mixture was then poured into plates and cooled to form 5% fetal bovine serum THB solid medium, which was then used to obtain blood agar plates.

[0028] Preparation Example 2: The difference between this preparation example and Preparation Example 1 is that: The fetal bovine serum content in 1L of culture medium is 3%; THB solid medium containing 3% fetal bovine serum was prepared.

[0029] Preparation Example 3: The difference between this preparation example and Preparation Example 1 is that: The fetal bovine serum content in 1L of culture medium is 6%; THB solid medium containing 6% fetal bovine serum was prepared.

[0030] Example of THB liquid culture medium preparation Preparation Example 4: THB liquid culture medium was prepared using the following method: Weigh out 10 g / L beef powder, 20 g / L tryptone, 2 g / L glucose, 2 g / L sodium bicarbonate, 2 g / L sodium chloride, 0.4 g / L disodium bicarbonate, and fetal bovine serum. Adjust the pH to 7.8, add water to make up to 1 L, and stir until completely dissolved to obtain the culture medium solution. The fetal bovine serum content in 1 L of culture medium solution is 5%. The culture medium was sterilized at 121°C for 20 min, cooled to 50°C, and polymyxin E 10 mg and nalidixic acid 15 mg were added. The mixture was then dispensed into test tubes, cooled to 37°C, and stored to form a 5% fetal bovine serum THB liquid culture medium. Example

[0031] Example 1: A Streptococcus faecium: Fresh tissue was aseptically collected from the breast-related lesion area. The aseptically collected fresh tissue was streaked onto the blood agar plate prepared in Preparation Example 1 using an inoculation loop and incubated at 37°C for 24 hours before observation. Then, semi-transparent, round and smooth colonies with a diameter of 1 mm were picked and inoculated onto the blood agar plate prepared in Preparation Example 1 for purification and incubated at 37°C for 24 hours. Colonies with moist and smooth surfaces, neat edges, and pinpoint-sized colonies that appeared as chain-like spherical structures under a microscope were selected to obtain Streptococcus galactiflora. The isolated bacteria, *Streptococcus dysgalactiae*, were identified by PCR and 16S rRNA gene sequence analysis. The 16S identification primers were as follows: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3'. Sequencing results confirmed the bacteria to be *Streptococcus dysgalactiae*.

[0032] Example 2: The difference between this example and Example 1 is that: Fresh tissue was aseptically collected from the breast-related lesion area. The aseptically collected fresh tissue was streaked onto the blood agar plate prepared in Preparation Example 2 using an inoculation loop and incubated at 35°C for 25 hours before being observed. Then, semi-transparent, round, and smooth colonies with a diameter of 1 mm were picked and inoculated onto the blood agar plate prepared in Preparation Example 2 for purification and incubated at 35°C for 25 hours. Colonies with moist, smooth surfaces, neat edges, and a size similar to a pinhead, appearing as chain-like spherical structures under a microscope, were selected to obtain Streptococcus galactiae.

[0033] Example 3: The difference between this example and Example 1 is that: (1) Fresh tissue was collected from the breast-related lesion area aseptically. The fresh tissue was inoculated onto the blood agar plate prepared in Preparation Example 3 using an inoculation loop. After incubation at 38°C for 22 hours, the tissue was taken out for observation. Then, semi-transparent, round and smooth colonies with a diameter of 1 mm were picked and inoculated onto the blood agar plate prepared in Preparation Example 3 for purification. The colonies were incubated at 38°C for 22 hours. Colonies with moist and smooth surfaces, neat edges, and a size like a pinhead were selected. Under a microscope, they appeared as chain-like spherical colonies, which were then obtained as Streptococcus galactagogue.

[0034] Example 4: A method for preparing Streptococcus lactis egg yolk antibody: S1. A single colony of the identified *Streptococcus lactis* from Example 1 was picked and inoculated into THB liquid medium containing 5% fetal bovine serum prepared in Example 4. The culture was carried out at 37°C and 150 rpm for 48 h. Then, the colonies were centrifuged at 8000 rpm and 4°C for 10 min to collect the bacterial cells. The collected bacterial cells were gently washed with physiological saline, then centrifuged again at 8000 rpm and 4°C for 10 min to collect the bacterial cells. This washing and collection process was repeated three times. The bacterial concentration was adjusted to 2 × 10⁻⁶. 7 CFU / mL, add 0.2% formaldehyde solution to the bacterial culture, mix thoroughly, and inactivate by shaking at 37℃ for 60h. Then, inoculate the inactivated bacterial culture into the Erlenmeyer flask of THB liquid culture medium prepared in Preparation Example 4. After 48h, transfer to the blood agar plate prepared in Preparation Example 1. After incubation at 37℃ for 48h, no viable bacteria are detected, and the antigen is obtained. Take 94 parts of white oil for injection, add 6 parts of Span-80, mix thoroughly, and autoclave for later use. Aqueous phase preparation: Add 96 parts of qualified antigen to 4 parts of sterilized Tween-80, shake thoroughly until Tween-80 is completely dissolved. Emulsification: The aqueous phase and oil phase are in a 1:2 ratio (V / V). The oil phase is injected into the premixing tank and stirred at low temperature at 3000rpm / min for 15min. The aqueous phase is added, and then injected into the emulsification tank for emulsification. Then, aseptically dispense quantitatively, seal the bottle mouth, and store at 4℃ to complete the vaccine preparation process, and obtain the Streptococcus pyogenes vaccine. S2, inactivated Streptococcus lactis vaccine, is administered to laying hens via intramuscular injection in the leg. Each injection is given two weeks apart, for a total of four immunizations. The vaccine dose is 10... 8 IU / ml ‌ 10 8 IU / ml, 10 7 IU / ml, 10 7IU / ml was used to complete the immunization process; the yolk antibody titer was detected by ELISA. The results showed that the immunized group had an egg yolk antibody titer ≥0.25, thus completing the identification of the egg titer and confirming that it met the requirements for antibody preparation. Then, the eggs were soaked in 0.1% benzalkonium chloride solution at 42℃ for 15 minutes, drained, rinsed with running water to remove surface dirt, and fumigated with 0.1% Weikang spray for 30 minutes. After the eggs were dried, the cleaning and disinfection process was completed. S3. After air-drying, separate the yolks and egg whites. Mix the yolks and disodium hydrogen phosphate solution at a 1:1 (v / v) ratio and stir well. The concentration of the disodium hydrogen phosphate solution is 0.02 M / L. Filter the mixture through a 60-mesh wire sieve to remove impurities from the yolk mixture. Transfer the mixture to a settling tank and add the remaining twice the volume of disodium hydrogen phosphate solution. Stir at a constant speed of 60 rpm for 60 minutes to obtain a mixture. The mixture has a pH of 7.2, is milky yellow in color, and is cloudy and homogeneous, completing the yolk pretreatment. Then, use a 1:1 settling solution (polyvinylpyrrolidone PVP: trehalose) Add the mixture (1:1 ratio) to the settling tank, then adjust the pH to 6.0 with a 30% citric acid solution; let it settle for 2 hours, then transfer all the supernatant; mix the supernatant with an octanoic acid solution (the final concentration of octanoic acid is 0.05%) to obtain a composite solution, completing the acidification extraction; remove the upper layer of oil using a filter bag; perform membrane sterilization: treat with two sets of filter cartridges (the first set is 0.45μm, and the second set is 0.22μm); complete the filtration sterilization, and finally use the ELISA method to detect the antibody titer and specificity, completing the detection and obtaining the finished egg yolk antibody.

[0035] Example 5: The difference between this example and Example 4 is that: S1. A single colony of the identified *Streptococcus lactis* from Example 2 was picked and inoculated into THB liquid medium containing 5% fetal bovine serum prepared in Example 4. The medium was cultured at 37°C and 150 rpm for 48 h. Then, the colonies were centrifuged at 8000 rpm and 4°C for 10 min to collect the bacterial cells. The collected cells were gently washed with physiological saline, then centrifuged again at 8000 rpm and 4°C for 10 min to collect the bacterial cells. This washing and collection process was repeated three times. The bacterial concentration was adjusted to 2 × 10⁻⁶. 7CFU / mL, add 0.1% formaldehyde solution to the bacterial culture, mix thoroughly, and inactivate by shaking at 37℃ for 60h. Then, inoculate the inactivated bacterial culture into Erlenmeyer flasks of THB liquid medium. After 46h, transfer to blood agar plates prepared in Preparation Example 1. After incubation at 37℃ for 46h, no viable bacteria are detected, and the antigen is obtained. Take 94 parts of white oil for injection, add 6 parts of Span-80, mix thoroughly, and autoclave for later use. Aqueous phase preparation: Add 96 parts of qualified antigen to 4 parts of sterilized Tween-80, shake thoroughly until Tween-80 is completely dissolved. Emulsification: The aqueous phase and oil phase are in a 1:2 ratio (V / V). The oil phase is injected into a premixing tank and stirred at 3000rpm / min for 15min at low temperature. The aqueous phase is added, and then injected into an emulsification tank for emulsification. Then, aseptically dispense quantitatively, seal the bottle mouth, and store at 4℃ to complete the vaccine preparation process, and obtain the Streptococcus pyogenes vaccine. S2, inactivated Streptococcus lactis vaccine, is administered to laying hens via intramuscular injection in the leg. Each injection is given two weeks apart, for a total of four injections. The vaccine dose is 10... 8 IU / ml ‌ 10 8 IU / ml, 10 7 IU / ml, 10 7 IU / ml was used to complete the immunization process; the yolk antibody titer was detected by ELISA. The results showed that the immunized group had an egg yolk antibody titer ≥0.25, thus completing the identification of the egg titer and confirming that it met the requirements for antibody preparation. Then, the eggs were soaked in 0.08% benzalkonium chloride solution at 42℃ for 18 minutes, drained, rinsed with running water to remove surface dirt, and fumigated with 0.08% Weikang spray for 32 minutes. After the eggs were dried, the cleaning and disinfection process was completed. S3. After air-drying, separate the yolks and egg whites. Mix the yolks and disodium hydrogen phosphate solution at a 1:1 (v / v) ratio and stir well. Filter the mixture through a 55-mesh wire sieve to remove impurities from the yolk liquid. Transfer the mixture to a settling tank and add the remaining twice the volume of disodium hydrogen phosphate solution. Stir at 40 rpm for 70 minutes to obtain a mixture. The mixture should have a pH of 7.2, be milky yellow in color, cloudy, and homogeneous, completing the yolk pretreatment. Then, add one volume of settling solution (polyvinylpyrrolidone (PVP): trehalose = 1:0.8) to the settling tank, followed by adding 20% ​​concentrated... The pH of the citric acid solution was adjusted to 5.8; after sedimentation for 2.2 hours, the supernatant was completely transferred out; the supernatant was mixed with caprylic acid solution to a final concentration of 0.02% to obtain a composite solution, thus completing the acidification extraction; the upper layer of oil was removed by using a filter bag; membrane sterilization was performed: two sets of filter cartridges were used (the first set was 0.45μm, and the second set was 0.22μm); antibacterial agent was added at a dosage of 0.15% of the final product to complete the filtration sterilization; finally, the antibody titer and specificity were detected by ELISA to obtain the finished egg yolk antibody.

[0036] Example 6: The difference between this example and Example 4 is that: S1. A single colony of the identified *Streptococcus lactis* from Example 3 was picked and inoculated into THB liquid medium containing 5% fetal bovine serum prepared in Example 4. The culture was carried out at 37°C and 150 rpm for 48 h. Then, the colonies were centrifuged at 8000 rpm and 4°C for 10 min to collect the bacterial cells. The collected cells were gently washed with physiological saline, then centrifuged again at 8000 rpm and 4°C for 10 min to collect the bacterial cells. This washing and collection process was repeated three times. The bacterial concentration was adjusted to 2 × 10⁻⁶. 7 CFU / mL, add 0.3% formaldehyde solution to the bacterial culture, mix thoroughly, and inactivate by shaking at 37℃ for 60h. Then, inoculate the inactivated bacterial culture into Erlenmeyer flasks of THB liquid medium. After 50h, transfer to blood agar plates prepared in Preparation Example 1. After incubation at 37℃ for 50h, no viable bacteria are detected, and the antigen is obtained. Take 94 parts of white oil for injection, add 6 parts of Span-80, mix thoroughly, and autoclave for later use. Aqueous phase preparation: Add 96 parts of qualified antigen to 4 parts of sterilized Tween-80, shake thoroughly until Tween-80 is completely dissolved. Emulsification: The aqueous phase and oil phase are in a 1:2 ratio (V / V). The oil phase is injected into a premixing tank and stirred at 3000rpm / min for 15min at low temperature. The aqueous phase is added, and then injected into an emulsification tank for emulsification. Then, aseptically dispense quantitatively, seal the bottle mouth, and store at 4℃ to complete the vaccine preparation process, and obtain the Streptococcus pyogenes vaccine. S2, inactivated Streptococcus lactis vaccine, is administered to laying hens via intramuscular injection in the leg. Each injection is given two weeks apart, for a total of four injections. The vaccine dose is 10... 8 IU / ml ‌ 10 8 IU / ml, 10 7 IU / ml, 10 7 IU / ml was used to complete the immunization process; the yolk antibody titer was detected by ELISA. The results showed that the immunized group had an egg yolk antibody titer ≥0.25, thus completing the identification of the egg titer and confirming that it met the requirements for antibody preparation. Then, the eggs were soaked in 0.12% benzalkonium chloride solution at 42℃ for 12 minutes, drained, rinsed with running water to remove surface dirt, and fumigated with 0.12% Weikang spray for 28 minutes. After the eggs were dried, the cleaning and disinfection process was completed. S3. After air-drying, separate the yolks and egg whites. Mix the yolks and disodium hydrogen phosphate solution at a 1:1 (v / v) ratio and stir well. Filter the mixture through a 65-mesh wire sieve to remove impurities from the yolk liquid. Transfer the mixture to a settling tank and add the remaining twice the volume of disodium hydrogen phosphate solution. Stir at 80 rpm for 50 minutes to obtain a mixture with a pH of 7.2, a milky yellow color, and a cloudy, homogeneous appearance, completing the yolk pretreatment. Then, add one volume of settling solution (polyvinylpyrrolidone (PVP): trehalose = 1:1.1) to the settling tank, followed by rinsing with 35% concentrated... The pH of the citric acid solution was adjusted to 6.2; after sedimentation for 2.5 hours, the supernatant was completely transferred out; the supernatant was mixed with caprylic acid solution to a final concentration of 0.1%, resulting in a composite solution for acid extraction; the upper layer of oil was removed using a filter bag; membrane sterilization was performed using two sets of filter cartridges (0.45 μm for the first set and 0.22 μm for the second set); antibacterial agent was added at 0.15% of the final product, and filtration sterilization was completed. Finally, the antibody titer and specificity were detected using ELISA to obtain the finished egg yolk antibody.

[0037] Application examples Application example: Application of an egg yolk antibody against Streptococcus dysgalactiae: A biological agent was prepared using any one of the egg yolk antibodies in Examples 1-6 for the prevention and treatment of Streptococcus dysgalactiae infection in pigs.

[0038] Performance testing 1. Prevention effect testing 1. Experimental grouping and treatment (1) Prevention group (90 healthy piglets): Divided into 3 groups, each group was given oral administration of Streptococcus suis egg yolk antibody (1 mL / pig, containing specific IgY antibody ≥10 mg / mL) from Examples 4-6. Each group corresponded to one example, once a day for 7 consecutive days. (2) Treatment group (90 infected piglets): Divided into 3 groups, after confirmed infection, the piglets were orally administered the Streptococcus dysgalactiae egg yolk antibody (2 mL / piglet, twice a day for 5 days) in Examples 4-6. Each group corresponds to one example. (3) Control group (30 infected piglets): only oral saline was administered, without Streptococcus suis infection or antibody intervention.

[0039] 2. Establishment of Infection Model via nasal spray (1×10) 8 A natural infection model was established using CFU / mL Streptococcus lactis combined with skin abrasions (simulating the traumatic infection route), and the indicators were detected.

[0040] 3. Monitoring Indicators (1) Prevention group: Infection rate was calculated as: number of Streptococcus lactis positive / number of experimental animals in the group (nasal swabs were collected on day 14 post-infection for PCR detection); and the serum level of egg yolk antibody IgY (enzyme) was recorded. (ELISA method), daily increment (g / day).

[0041] (2) Treatment group: Record the time of symptom relief (body temperature returns to normal, appetite returns), calculate the bacterial clearance rate = number of positive bacterial test results / group of experimental animals (blood and joint fluid were collected on the 7th day after infection for bacterial culture), and the daily increment.

[0042] (3) Control group: The infection rate, serum IgY level, symptom relief time, bacterial clearance rate and daily increment were recorded.

[0043] (4) Safety assessment: Record the diarrhea rate and mortality rate of piglets in the prevention and treatment groups.

[0044] 4. Experimental Period Prevention group: Observation for 21 days after antibody intervention (covering the incubation period and the onset period of infection).

[0045] Treatment group and control group: Monitored from the time of infection until symptoms disappear or death (maximum 14 days).

[0046] 5. The experimental results are shown in Table 1: Table 1 Performance Test Table ("-" in the table indicates that this item was not tested, so there is no data)

[0047] The diarrhea rate in both the prevention and treatment groups was <5%, with no deaths, and there was no significant difference compared to healthy piglets.

[0048] 6. Experimental Analysis Egg yolk antibodies block bacterial adhesion and invasion by specifically binding to the surface antigen of Streptococcus lactis. The serum IgY level (OD450=0.85) in the prevention group was significantly higher than that in the control group, confirming that the antibody effectively neutralizes the pathogen; egg yolk antibodies rapidly neutralize bacterial toxins and shorten the course of sepsis (remission time of 2.5 days compared to no remission in the control group).

[0049] Experiments have confirmed that Streptococcus suis egg yolk antibodies can significantly reduce the infection rate in piglets through passive immunization (12% compared to 95% in the control group), and have a rapid neutralizing effect on early infections (bacterial clearance rate of 88%). During use, no piglets experienced severe diarrhea or death, demonstrating its excellent safety profile. Combined with environmental management, it can form a multi-layered prevention and control system, suitable for use in large-scale pig farms.

[0050] Application value: Egg yolk antibodies can replace antibiotics for early prevention, reducing the risk of drug resistance. Combined with environmental disinfection (potassium persulfate), they can block the spread of bacteria through skin wounds and synergistically reduce the infection rate.

[0051] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A Streptococcus faecium, characterized in that, Streptococcus dysgalactiae was deposited at the China General Microbiological Culture Collection Center on December 24, 2024, with accession number CGMCC No. 46272.

2. The Streptococcus faecium according to claim 1, characterized in that: The Streptococcus faecium was obtained through isolation and purification, and the specific steps are as follows: (1) Fresh tissue from the lesion area was collected aseptically, bacteria were isolated, and then inoculated onto blood agar plates for purification to obtain Streptococcus pyogenes; (2) Streptococcus dysgalactiae was identified by PCR and 16S rRNA gene sequence analysis.

3. The Streptococcus faecium according to claim 2, characterized in that, In step (1), the blood plate is a THB solid culture medium containing 3-6% fetal bovine serum.

4. The Streptococcus faecium according to claim 2, characterized in that, In step (1), the separation and purification temperatures are both 35-38℃, and the time is both 22-25h.

5. A method for preparing Streptococcus pyogenes egg yolk antibody according to any one of claims 1-4, characterized in that, Includes the following steps: S1. Prepare Streptococcus dysgalactiae antigen, and after the antigen is treated with vaccine, obtain Streptococcus dysgalactiae vaccine; S2. Administer the Streptococcus pyogenes vaccine to chickens via intramuscular injection for immunization. The egg yolk titer was determined to be suitable for antibody preparation, and then the yolk was cleaned, disinfected, and air-dried. S3. After air-drying, the egg yolk and egg white are separated. The egg yolk is pretreated with disodium hydrogen phosphate solution, then acidified, extracted, filtered and sterilized, and tested to obtain egg yolk antibodies.

6. The method for preparing Streptococcus lactis antigen according to claim 5, characterized in that, The Streptococcus pyogenes antigen was prepared using the following method: The identified Streptococcus lactis was cultured and the bacterial cells were collected. After washing the bacterial cells, collect them, adjust the bacterial concentration, add 0.1-0.3% formaldehyde solution to the bacterial solution for inactivation, and then inoculate the inactivated bacterial solution into Erlenmeyer flasks of THB liquid medium. After 46-50 hours, transfer the culture to blood agar plates, incubate, and test for no viable bacteria to obtain the antigen.

7. The method for preparing Streptococcus lactis antigen according to claim 5, characterized in that, The specific cleaning and disinfection steps in S2 are as follows: the antibody is soaked in 0.08-0.12% 42℃ benzalkonium chloride solution for 12-18 minutes, then cleaned to remove dirt, and then fumigated with 0.08-0.12% Weikang spray for 28-32 minutes.

8. The method for preparing Streptococcus lactis antigen according to claim 5, characterized in that, The specific steps for pretreating the egg yolks in S3 using disodium hydrogen phosphate solution are as follows: Mix egg yolk and disodium hydrogen phosphate solution at a 1:1 (v / v) ratio and stir well; filter through a 55-65 mesh steel wire sieve to remove impurities from the egg yolk liquid, transfer to a settling tank, add the remaining 2 times the volume of disodium hydrogen phosphate solution, and stir at a constant speed of 40-80 r / min for 50-70 min to obtain a mixture.

9. The method for preparing Streptococcus lactis antigen according to claim 5, characterized in that, The specific steps of acidification extraction in S3 are as follows: Add 1 part sedimentation solution (polyvinylpyrrolidone PVP: trehalose = 1:0.8-1.1) to the sedimentation tank, then adjust the pH to 5.8-6.2 with 20-35% citric acid solution; let it settle for 2-2.5 hours, then transfer all the supernatant; mix the supernatant with octanoic acid solution, with the final concentration of octanoic acid being 0.02-0.1%, to obtain a composite solution.

10. The application of an antibody against Streptococcus lactis egg yolk, characterized in that, Prepare biological agents for the prevention and treatment of Streptococcus faecium infection in pigs; the egg yolk antibody is an egg yolk antibody prepared by any one of the egg yolk antibody preparation methods of claims 5-9.

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