Diluent of Marek's disease live vaccine and preparation method thereof

Abstract

The invention relates to a diluent of a Marek's disease live vaccine and a preparation method of the diluent, and aims to improve the immune effect of the vaccine and relieve the immune stress reaction after inoculation. The diluent takes water for injection as a solvent and comprises the following components: cane sugar, L-sodium glutamate, dipotassium phosphate, monopotassium phosphate, sodium chloride, beef extract, phenol red, water-soluble yeast glucan, meglumine and the like. The mass stability and the biological safety of the diluent are ensured by accurately controlling the proportion of each component and strict preparation processes including raw material weighing, dissolving and mixing, pH adjusting, filtering and purifying and storing. Experimental results show that the vaccine diluted according to the optimized formula can effectively improve immune protection, meanwhile, the influence on the weight and immune organs of test chickens is reduced, the obvious anti-stress characteristic is shown, and the vaccine has a good application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of animal vaccine diluent, in particular to a diluent of a Marek's disease live vaccine and a preparation method thereof. Background Art

[0002] Marek's disease (MD) is a common lymphoproliferative tumor disease in chickens. This disease is an infectious tumor disease caused by cell-associated herpes virus. Sick chickens often suffer from emaciation, limb paralysis, and often acute death. Since the 1970s, there have been vaccines to prevent this disease, which have gradually developed into the current Marek's vaccine. Due to its good immune effect, Marek's vaccine has been widely promoted and applied, but due to various factors, such as stress-induced immunosuppression, some chickens still suffer varying degrees of losses caused by MD. There are many factors that cause fluctuations in the immune effect of MD, so the vaccine diluent is also crucial. Currently, commonly used diluents include SPG, SPGA, etc. This paper experimentally studies the effects of adding water-soluble yeast glucan and meglumine to the diluent on the immune effect of the vaccine and the immune stress response of chickens. Summary of the invention

[0003] In order to solve the above technical problems, the present invention provides a diluent of a live Marek's disease vaccine which can reduce the immune stress of chickens without affecting the immune effect of the live Marek's disease vaccine and a preparation method thereof.

[0004] The diluent of a Marek's disease live vaccine of the present invention uses water for injection as a solvent and comprises the following components:

[0005] sucrose

[0006] Sodium L-Glutamate

[0007] Dipotassium hydrogen phosphate

[0008] Potassium dihydrogen phosphate

[0009] Sodium chloride

[0010] Beef extract

[0011] Phenol red

[0012] Water soluble yeast glucan

[0013] Meglumine.

[0014] Preferably, the components contained in each liter of the diluent are specifically:

[0015]

[0016] The balance is water for injection.

[0017] Preferably, the water-soluble yeast glucan is specifically set to 10g.

[0018] Preferably, the meglumine is specifically set to 2g.

[0019] A method for preparing a diluent of a Marek's disease live vaccine comprises the following steps:

[0020] S1: Accurate weighing of raw materials

[0021] S2: Raw material dissolution and mixing

[0022] S3: Volume setting and homogenization

[0023] S4: PH value detection and adjustment

[0024] S5: Filtration and purification

[0025] S6: Storage

[0026] Preferably, step S1 specifically includes using a high-precision electronic scale with a resolution of at least 0.001 g, and using an analytical balance to weigh the trace component phenol red. When weighing, the electronic scale should be placed on a stable horizontal surface to avoid vibration interfering with the reading.

[0027] Preferably, the step S2 specifically includes putting all the components weighed in step 1 into a stainless steel liquid preparation tank, adding an appropriate amount of water for injection, which is preheated to 40 degrees Celsius, and starting a stirring device, which is specifically configured as a magnetic stirrer or a mechanical stirring paddle, so that the components are fully mixed and heated until completely dissolved, and the stirring device can control the temperature.

[0028] Preferably, step 3 specifically includes, after all solid components have been completely dissolved, adding additional cooled injection water to adjust to the desired final volume, and continuously stirring for at least 15 minutes to ensure that the solution is uniform; step 4 specifically includes, measuring the pH value of the diluent during stirring to ensure the uniformity of the entire solution, if the pH value is not between 6.8-7.0, using a dropper to slowly add 1 mol / L sodium hydroxide solution, the amount added each time not exceeding 0.1 mL, and stirring thoroughly after each addition, and measuring the pH value again until the target range is reached.

[0029] Preferably, the step 5 specifically includes aseptically filtering the prepared dilution through a microporous filter membrane with a pore size of 0.22 μm.

[0030] Preferably, the step 6 specifically includes: the diluted solution after filtering in step S5 is immediately transferred to a pre-sterilized sterile container and stored in a refrigerated environment to avoid direct sunlight. The storage container is labeled with the production date, batch number and expiration date. The storage environment is regularly checked to ensure that the temperature and humidity meet the requirements to prevent contamination.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1. Adding a specified amount of water-soluble yeast glucan and meglumine to the special diluent for Marek's disease live vaccine can significantly improve the immune effect of the vaccine. The experimental results show that the optimized diluent can not only enhance the activity of immune cells, improve the protection and durability of the vaccine, but also significantly reduce the immunosuppression phenomenon produced by the test chickens after vaccination. In addition, the diluent ensures the stability of the live vaccine and improves the activity of cells in its environment, thereby further enhancing the overall immune effect of the Marek's disease live vaccine.

[0033] 2. The preparation method provided by the present invention ensures the quality consistency and reliability of the diluent through the steps of accurate raw material weighing, sufficient dissolution and mixing, constant volume homogenization, pH value detection and adjustment, and filtration purification. In particular, the method of gradually adding a trace amount of alkali solution to finely control the pH value of the solution ensures the stability and applicability of the final product. At the same time, strict aseptic operation and safe storage conditions provide guarantees for the effective application of the diluent, avoid contamination risks, and ensure its efficiency and safety in actual use. DETAILED DESCRIPTION

[0034] The following examples are used to further describe the specific embodiments of the present invention. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0035] Example 1

[0036] Formula 1: Weigh 70g sucrose, 0.5g L-sodium glutamate, 1.5g potassium dihydrogen phosphate, 0.5g potassium dihydrogen phosphate, 0.2g sodium chloride, 0.3g beef extract, 0.02g phenol red into the liquid preparation tank, add a certain amount of water for injection, stir continuously, heat to dissolve, and after fully dissolved, add water for injection to the prepared amount, continue stirring until the solution is evenly mixed. Detect the pH value of the solution, and adjust the pH value of the diluent to 6.8-7.0 with 1mol / L sodium hydroxide solution. Finally, filter the prepared vaccine-specific diluent through a 0.2μm microporous filter membrane.

[0037] Recipe 2: Weigh 70g sucrose, 0.5g sodium L-glutamate, 1.5g dipotassium hydrogen phosphate, 0.5g potassium dihydrogen phosphate, 0.2g sodium chloride, 0.3g beef extract, 0.02g phenol red, 5g water-soluble yeast glucan, and 1g meglumine into a liquid preparation tank. The remaining preparation steps are the same as those of Recipe 1 above.

[0038] Example 2

[0039] The diluents of the two formulations in Example 1 were used to compare the animal evaluation effects of the Marek's disease live vaccine (CVI988 / Rispens strain) and the potency test results of the Marek's disease turkey herpes virus live vaccine (FC-126 strain):

[0040] 1. Animal evaluation of Marek's disease live vaccine (CVI988 / Rispens strain)

[0041] After diluting the vaccine with the special diluents for the vaccine of Formula 1 and Formula 2 prepared in Example 1, the treated samples were taken and injected into 40 1-day-old SPF chicks intramuscularly (chest muscle or lateral leg muscle), 0.2 ml per chick (containing 1 feather portion). Another 40 1-day-old SPF chicks were taken as the control group and were not injected with the vaccine.

[0042] At 7d, 14d, 21d, and 42d, 10 chickens were randomly sampled from each group, and the weight, thymus, and bursa of each group were weighed, and the mean percentage of the weight, thymus, and bursa of each group was compared. The test results are shown in Table 1:

[0043] Table 1 Effect of diluents used in animal evaluation of Marek's disease live vaccine (CVI988 / Rispens strain)

[0044]

[0045] As can be seen from Table 1, compared with the control group, both Formula 1 and Formula 2 inhibited the body weight of the experimental chickens, and caused varying degrees of atrophy of the thymus and bursa of Fabricius of the experimental chickens (especially significant at 7 and 14 days of age), but Formula 1 was more significant, while Formula 2 had a significantly smaller inhibitory effect on the immune organs of the experimental chickens.

[0046] 2. Evaluation of the efficacy of Marek's disease turkey herpes virus live vaccine (FC-126 strain)

[0047] (1) Plaque count: Use the special diluents for the vaccine of Formula 1 and Formula 2 prepared in Example 1 to dilute 3 bottles of freeze-dried live vaccine of Marek's disease turkey herpes virus, respectively, melt in a 37°C water bath, oscillate and mix, and continue to perform 10-fold serial dilutions to 10-4. Take the final dilution of each bottle of sample and inoculate 5 chicken embryo fibroblast bottles that have grown into a good monolayer, inoculate 0.2ml per bottle, place at 37°C for adsorption for 1 hour, add 6.0ml cell maintenance solution to each cell bottle, and set up 2 blank control cell bottles. Cultivate for 6 days at 37°C and 5% CO2. According to the test results, calculate the average number of plaques of the 3 bottles of cell bottles corresponding to each bottle of sample and the number of plaques per feather. The results are shown in Table 2:

[0048] Table 2 Plaque count results in different groups

[0049]

[0050] As shown in Table 2, the plaque count results of the Marek's disease freeze-dried vaccine diluted in Formula 2 were significantly higher, indicating that adding water-soluble yeast glucan and meglumine to the diluent can effectively improve the immune effect of the Marek's disease turkey herpes virus live vaccine (FC-126 strain).

[0051] (2) Serum antibody detection: Dilute the vaccine with the special diluents of Formula 1 and Formula 2 prepared in Example 1, and then take the treated samples. Inject 20 chickens into the muscle (chest muscle or outer leg muscle), 0.2 ml per chicken (including 1 dose). Take 20 chickens as the control group, and do not inject the vaccine. At 42 days of age, venous blood was collected from each group of test chickens, and an agar diffusion test of MDV was performed. The test results are shown in Table 3:

[0052] Table 3 Results of agar diffusion test for MDV serum antibody detection

[0053]

[0054] As can be seen from Table 3, the antibody levels in the test chickens injected with the Marek's disease freeze-dried live vaccine diluted with Formula 2 were significantly higher, indicating that compared with Formula 1, the addition of water-soluble yeast glucan and meglumine to the vaccine diluent can improve the immune efficacy of the Marek's disease turkey herpes virus live vaccine (FC-126 strain).

[0055] Example 3

[0056] Formula 2: Same as Formula 2 in Example 1

[0057] Formulation 3: 70 g sucrose, 0.5 g sodium L-glutamate, 1.5 g dipotassium hydrogen phosphate, 0.5 g potassium dihydrogen phosphate, 0.2 g sodium chloride, 0.3 g beef extract, 0.02 g phenol red, 5 g water-soluble yeast glucan, and 2 g meglumine were weighed and put into a liquid preparation tank, and the remaining preparation steps were the same as those of Formulation 1 in Example 1 above.

[0058] Recipe 4: Weigh 70g sucrose, 0.5g sodium L-glutamate, 1.5g dipotassium hydrogen phosphate, 0.5g potassium dihydrogen phosphate, 0.2g sodium chloride, 0.3g beef extract, 0.02g phenol red, 10g water-soluble yeast glucan, and 1g meglumine into a liquid preparation tank, and the remaining preparation steps are the same as those of Recipe 1 in Example 1 above.

[0059] Formulation 5: 70 g sucrose, 0.5 g sodium L-glutamate, 1.5 g dipotassium hydrogen phosphate, 0.5 g potassium dihydrogen phosphate, 0.2 g sodium chloride, 0.3 g beef extract, 0.02 g phenol red, 10 g water-soluble yeast glucan, and 2 g meglumine were weighed and put into a liquid preparation tank. The remaining preparation steps are the same as those of Formulation 1 in Example 1 above.

[0060] 1. Animal evaluation of Marek's disease live vaccine (CVI988 / Rispens strain):

[0061] After diluting the vaccine with the special diluents for the second, third, fourth and fifth vaccines prepared in Example 2, the treated samples were taken and injected into the muscles (chest muscle or outer leg muscle) of 40 chickens, 0.2 ml per chicken (including 1 dose). Another 40 chickens were taken as the control group and were not injected with the vaccine.

[0062] At 7d, 14d, 21d, and 42d, 10 chickens were randomly sampled from each group, and the weight, thymus, and bursa of each group were weighed, and the mean percentage of the weight, thymus, and bursa of each group was compared. The test results are shown in Table 4:

[0063] Table 4 Effect of different component dilutions on animal evaluation of Marek's disease live vaccine (CVI988 / Rispens strain)

[0064]

[0065] As can be seen from Table 4, compared with the control group, the vaccines diluted with Formulas 2, 3, 4, and 5 all inhibited the body weight of the test chickens, and caused varying degrees of atrophy of the thymus and bursa of Fabricius of the test chickens (especially significant at 7 and 14 days of age), but Formula 5 had the least effect, indicating that Formula 5 had a significantly smaller inhibitory effect on the immune organs of the test chickens.

[0066] 2. Evaluation of the efficacy of Marek's disease turkey herpes virus live vaccine (FC-126 strain)

[0067] (1) Plaque count: Dilute 3 bottles of Marek's disease freeze-dried live vaccine with the special diluents for the vaccines of formula 2, 3, 4 and 5 prepared in Example 2, thaw in a water bath at 37°C, oscillate and mix, and continue to perform 10-fold serial dilutions to 10-4. Take the final dilution of each bottle of sample and inoculate 5 chicken embryo fibroblast bottles that have grown into a good monolayer, inoculate 0.2ml per bottle, place at 37°C for adsorption for 1 hour, add 6.0ml of cell maintenance solution to each cell bottle, and set up 2 blank control cell bottles. Cultivate at 37°C and 5% CO2 for 6 days. According to the test results, calculate the average number of plaques of the 3 bottles of cell bottles corresponding to each bottle of sample and the number of plaques per feather. The experimental results are shown in Table 5:

[0068] Table 5 Plaque counting results of different groups

[0069]

[0070] As can be seen from Table 5, compared with Formulas 2, 3, and 4, the plaque count results of the Marek's freeze-dried vaccine diluted in Formula 5 are higher, indicating that Formula 5 can significantly improve the immune effect of the Marek's freeze-dried vaccine.

[0071] (2) Serum antibody detection: Dilute the vaccine with the special diluents for the second, third, fourth and fifth vaccines prepared in Example 2, and then take the treated samples. Inject 20 chickens into the muscle (chest muscle or outer leg muscle), 0.2 ml per chicken (including 1 dose). Take another 20 chickens as the control group, and do not inject the vaccine. At 42 days of age, venous blood was collected from each group of test chickens, and the MDV agar diffusion test was performed. The test results are shown in Table 6:

[0072] Table 6 MDV serum antibody detection agar diffusion test results

[0073]

[0074] As can be seen from Table 6, compared with other formulations, the antibody level in the test chickens injected with the Marek's disease freeze-dried live vaccine diluted in formulation 5 was the highest, indicating that adding 10g of water-soluble yeast glucan and 2g of meglumine to the vaccine diluent can significantly improve the immune efficacy of the Marek's disease freeze-dried vaccine.

[0075] Conclusion: Adding 10g water-soluble yeast glucan and 2g meglumine to the Marek's disease diluent can significantly improve the immune effect of Marek's disease live vaccine and significantly reduce the immunosuppression produced by the experimental chickens after vaccination.

[0076] The advantages of the diluent of the Marek's disease live vaccine and the preparation method thereof of the present invention are:

[0077] 1. Enhance immune effect:

[0078] By adding water-soluble yeast glucan and meglumine as adjuvant components to the diluent, the immune effect of Marek's disease live vaccine (such as CVI988 / Rispens strain) and Marek's disease turkey herpes virus live vaccine (FC-126 strain) was significantly improved. The experimental results show that the vaccine diluted with the optimized formula (especially formula 5) can more effectively stimulate the body to produce antibodies and improve immune protection.

[0079] 2. Reduce immune stress response:

[0080] Experimental data show that compared with traditional diluents, the diluent provided by the present invention can significantly reduce the weight suppression phenomenon of test chickens after vaccination, and has less impact on immune organs such as the thymus and bursa of Fabricius, thereby effectively alleviating the immune stress response caused by vaccination.

[0081] 3. Accurate ratio ensures stability:

[0082] The precise ratio of each component in the diluent (e.g. 70 g sucrose, 0.5 g sodium L-glutamate, 1.5 g dipotassium hydrogen phosphate, etc.) not only ensures the chemical stability and physical properties of the diluent itself, but also helps maintain the stability of the active ingredients of the vaccine and prolong its shelf life.

[0083] 4. Efficient preparation process:

[0084] A complete set of diluent preparation process is proposed, from precise weighing of raw materials to sterile filtration of the final product, each step is carefully designed to ensure the consistency and reliability of product quality. In particular, the gradual addition of alkali solution in the pH adjustment step can more finely control the solution pH to ensure that it is within the most suitable range.

[0085] 5. Safe storage conditions:

[0086] It is clearly stipulated that the filtered diluent should be stored in a refrigerated environment, and a series of measures are taken to prevent contamination (such as pre-sterilizing containers, labeling and indicating information, etc.). These measures provide guarantees for the safe storage of the diluent and ensure its effectiveness in practical applications.

[0087] In summary, the special diluent for Marek's disease live vaccine provided by the present invention and its preparation method, through scientific and reasonable formula design and rigorous and meticulous operation specifications, improves the immune effect of the vaccine while reducing the stress response of the vaccinated animals, and has important practical value and social and economic benefits. In addition, the diluent can also be used in the prevention and treatment of other similar diseases, further expanding its scope of application.

[0088] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A diluent of a Marek's disease live vaccine, characterized in that: The diluent uses water for injection as solvent and comprises the following components: sucrose Sodium L-Glutamate Dipotassium hydrogen phosphate Potassium dihydrogen phosphate Sodium chloride Beef extract Phenol red Water soluble yeast glucan Meglumine.

2. The diluent of a Marek's disease live vaccine according to claim 1, characterized in that: The components contained in each liter of the diluent are specifically:

3. The diluent of a Marek's disease live vaccine according to claim 2, characterized in that: The water-soluble yeast glucan is specifically set to 10g.

4. The diluent of a Marek's disease live vaccine according to claim 2, characterized in that: The meglumine is specifically set to 2g.

5. A method for preparing a diluent of a Marek's disease live vaccine according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1: Accurate weighing of raw materials S2: Raw material dissolution and mixing S3: Volume setting and homogenization S4: PH value detection and adjustment S5: Filtration and purification S6: Storage.

6. The method for preparing a diluent of a Marek's disease live vaccine according to claim 5, characterized in that: The step S1 specifically includes using a high-precision electronic scale with a resolution of at least 0.001 g, and using an analytical balance to weigh the trace component phenol red. When weighing, the electronic scale should be placed on a stable horizontal surface to avoid vibration interfering with the reading.

7. The method for preparing a diluent of a Marek's disease live vaccine according to claim 5, characterized in that: The step S2 specifically includes putting all the components weighed in step 1 into a stainless steel liquid preparation tank, adding an appropriate amount of water for injection, which is preheated to 40 degrees Celsius, and starting a stirring device, which is specifically configured as a magnetic stirrer or a mechanical stirring paddle, so that the components are fully mixed and heated until they are completely dissolved. The stirring device can control the temperature.

8. The method for preparing a diluent of a Marek's disease live vaccine according to claim 5, characterized in that: The step 3 specifically includes, after all solid components have been completely dissolved, adding additional cooled injection water to adjust to the required final volume, and continuously stirring for at least 15 minutes to ensure that the solution is uniform; the step 4 specifically includes, measuring the pH value of the diluent during stirring to ensure the uniformity of the entire solution, if the pH value is not between 6.8-7.0, using a dropper to slowly add 1 mol / L sodium hydroxide solution, the amount added each time not exceeding 0.1 mL, and stirring thoroughly after each addition, and measuring the pH value again until the target range is reached.

9. The method for preparing a diluent of a Marek's disease live vaccine according to claim 5, characterized in that: The step 5 specifically includes aseptically filtering the prepared dilution through a microporous filter membrane with a pore size of 0.22 μm.

10. The method for preparing a diluent of a Marek's disease live vaccine according to claim 5, characterized in that: The step 6 specifically includes: the diluted liquid filtered in step S5 is immediately transferred to a pre-sterilized sterile container and stored in a refrigerated environment to avoid direct sunlight. The storage container is labeled with the production date, batch number and expiration date, and the storage environment is regularly checked to ensure that the temperature and humidity meet the requirements to prevent contamination.

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