CN115058360A - Preparation method of salmonella equine abortion live vaccine - Google Patents
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Abstract
The invention provides a preparation method of a salmonella horse abortion live vaccine, belonging to the technical field of preparation of veterinary vaccines. The method comprises the following steps: inoculating salmonella equi abortus C355 attenuated bacteria on a common agar slant for slant culture to obtain first-grade seeds; inoculating the first-stage seeds into a salmonella liquid culture medium to perform second-stage seed culture to obtain second-stage seeds; inoculating the secondary seeds into a salmonella liquid culture medium for fermentation to obtain a fermentation liquid, and collecting salmonella in the fermentation liquid; and mixing the obtained salmonella with a freeze-drying protective agent to obtain the salmonella equine abortion live vaccine. The preparation method of the invention has the advantages of short vaccine preparation period, low production intensity, and safety and immunity superior to those of the original production process.
Description
Technical Field
The invention belongs to the technical field of preparation of veterinary vaccines, and particularly relates to a preparation method of a salmonella equine abortion live vaccine.
Background
Salmonella abortus disease is an infectious disease caused by Salmonella abortus (Salmonella abortus equi) and is characteristic of abortions in equine animals, and is pathogenic only to equine animals. The phenomenon of massive horse abortion occurs in European and American areas as early as the end of 18 th century and at the beginning of 19 th century, and the disease can occur all the year round, but mainly occurs in spring and autumn, and most of the diseases are sporadic and sometimes have local popularity. The salmonella abortus of horses is susceptible to infection of the first pregnant horses, the abortion is mainly performed in the later period of pregnancy, most horses have no obvious clinical symptoms before abortion, the abortion occurs suddenly, most horses are dead fetuses, a few live fetuses exist, and the death also occurs after several days of birth. During abortion of pregnant mares, pathogenic bacteria are discharged out of the body along with aborted fetuses, fetal membranes, amniotic fluid and vaginal secretions, and sick male mares can discharge bacteria along with semen. Horses infected with salmonella abortus in horses are often carriers of the bacteria and are also generally considered to be the primary source of infection in non-epidemic areas.
In order to prevent and treat the equine abortion salmonellosis, the salmonella equine abortion live vaccine is prepared, but the prior production technology adopts solid culture of the salmonella equine abortion live vaccine, does not control pH, does not add supplementary materials, does not control DO, keeps temperature after inoculation, and harvests within a specified time. The existing method has the advantages of long period, high working strength, no intermediate control and difficult large-scale production.
Disclosure of Invention
In view of the above, the invention aims to provide a preparation method of a salmonella equine abortion live vaccine, which is short in period and low in working strength and is suitable for large-scale production.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a preparation method of a salmonella horse abortion live vaccine, which comprises the following steps:
1) inoculating salmonella equi abortus C355 attenuated bacteria on a common agar slant for slant culture to obtain first-grade seeds;
2) inoculating the primary seeds obtained in the step 1) into a salmonella liquid culture medium for secondary seed culture to obtain secondary seeds;
3) inoculating the secondary seeds obtained in the step 2) into a salmonella liquid culture medium for fermentation to obtain fermentation liquor, and collecting salmonella in the fermentation liquor;
4) mixing the salmonella obtained in the step 3) with a freeze-drying protective agent to obtain the salmonella equine abortion live vaccine.
Preferably, the inoculation amount of the secondary seeds in the step 3) is 1-2%.
Preferably, the fermentation conditions in step 3) include: the temperature is 35-37 ℃, the pH value is 7.4-7.6, the dissolved oxygen is controlled to be 40% -80%, the stirring speed and the ventilation volume are increased when the dissolved oxygen is reduced, and the stirring speed is not higher than 150 rmp; and (3) supplementing nutrient substances every 2h from the 2 nd hour of fermentation, wherein the nutrient substances are glucose solutions with the mass percentage of 50%, the supplementing amount of each time is 0.5% of the total volume of the fermentation liquid, and the total fermentation time is 5-6 h.
Preferably, the conditions for the secondary seed culture of step 2) include: the temperature is 35-37 ℃, the rotating speed is 150rpm, and the culture time is 12 h.
Preferably, the salmonella in the fermentation liquor collected in the step 3) is collected by using a high-speed freezing tubular centrifuge or a hollow fiber concentration system.
Preferably, when the salmonella is collected using a high-speed refrigerated tubular centrifuge, the conditions include: the centrifugal speed of 48HZ is 2L/min, and the temperature is 2-8 ℃.
Preferably, when the salmonella is collected using the hollow fiber concentration system, the conditions include: and (3) carrying out high-pressure sterilization on the whole concentration system, starting a power pump after the system is cooled after the sterilization is finished, controlling the membrane pressure of the hollow fibers to be below 0.1Mpa in the system operation process, and finally collecting concentrated bacteria liquid according to the concentration proportion.
Preferably, the freeze-drying protective agent in the step 4) contains gelatin and sucrose, wherein the mass percentage of the gelatin is 1-5%, and the mass percentage of the sucrose is 10-50%.
Preferably, the bacteria content of the salmonella in the salmonella equine abortion live vaccine is 1 x 10 10 CFU/first share is more than.
Preferably, the condition of the slant culture in step 1) comprises: the temperature is 35-37 ℃, and the time is 18-20 h.
The invention has the beneficial effects that:
1. the production cycle is as follows: the original production process produces a batch which requires at least 7.5 days from the process flow to the end of the freeze-drying process, and comprises the following steps: preparing the first-stage strain for 3 days, preparing the second-stage strain for 1 day, preparing a bacterial solution for preparing the seedlings for 2.5 days, and precipitating the bacterial solution for 1 day; the invention only needs 4.5 days, which comprises the following steps: preparing the first-level strain for 3 days, preparing the second-level strain for 0.5 day, and performing bacterial fermentation, concentration and counting result judgment for 1 day.
2. Production strength: the primary process adopts solid fermentation, a large amount of personnel is needed to operate simultaneously, aseptic operation is achieved, operation difficulty is high, a large amount of flat bottles are needed, the primary seed preparation is basically consistent with the primary production process, fermentation tank production concentration equipment is used for concentrating in the production stage of the bacteria for seedling production, the working intensity of personnel is reduced, and the probability of pollution in production is greatly reduced (the secondary seed of the primary process is subjected to standing culture for 1-2 days in the middle of shaking culture, and the shaking culture is only needed for 0.5 day at present).
3. Yield: the original production process needs 25m of surface area for every 10000 head parts produced 2 The common agar flat bottles used in the production are 0.025m each 2 The total amount of 1000 flat bottles is needed, the workload is extremely huge, and the method adopts liquid fermentation and then concentration, and only 40L of fermentation medium is needed.
4. The vaccine prepared by the invention is superior to the original production process in safety and immune effect.
Drawings
FIG. 1 shows the relationship between time and viable cell count in the fermentation process of example 1.
Detailed Description
The invention provides a preparation method of a salmonella horse abortion live vaccine, which comprises the following steps:
1) inoculating salmonella equi abortus C355 attenuated bacteria on a common agar slant for slant culture to obtain first-grade seeds;
2) inoculating the primary seeds obtained in the step 1) into a salmonella liquid culture medium for secondary seed culture to obtain secondary seeds;
3) inoculating the secondary seeds obtained in the step 2) into a salmonella liquid culture medium for fermentation to obtain fermentation liquor, and collecting salmonella in the fermentation liquor;
4) mixing the salmonella obtained in the step 3) with a freeze-drying protective agent to obtain the salmonella equine abortion live vaccine.
The invention inoculates salmonella on the common agar slant for slant culture to obtain the first-class seed. In the present invention, the preparation time of the primary seed is 3 d. The invention has no special limitation on the common slant culture medium, and the culture medium for culturing salmonella is adopted. In the invention, the salmonella is preferably salmonella abortus C355 attenuated bacterium, which is derived from the Chinese veterinary medicine inspection institute. In the present invention, the conditions for the slant culture preferably include: the temperature is 35-37 ℃, and the time is 18-20 h.
The obtained first-stage seeds are inoculated into a salmonella liquid culture medium for second-stage seed culture to obtain second-stage seeds. In the present invention, the preparation time of the secondary seed is 0.5 d. The salmonella liquid medium is not particularly limited, and those skilled in the art can use a conventional salmonella culture medium. In the present invention, the conditions for the secondary seed culture preferably include: the temperature is 35-37 ℃, the rotating speed is 150rpm, and the culture time is 12 h.
Inoculating the obtained secondary seeds into a salmonella liquid culture medium for fermentation to obtain fermentation liquor, and collecting salmonella in the fermentation liquor. In the invention, the total time for fermentation, collection of salmonella and determination of viable count results is 1 d. In the invention, the inoculation amount of the secondary seeds is preferably 1-2%. In the present invention, the conditions of the fermentation preferably include: the temperature is 35-37 ℃, the pH value is 7.4-7.6, the dissolved oxygen is controlled to be 40% -80%, the stirring speed and the ventilation volume are increased when the dissolved oxygen is reduced, and the stirring speed is not higher than 150 rmp; and (3) supplementing nutrient substances every 2h from the 2 nd hour of fermentation, wherein the nutrient substances are glucose solutions with the mass percentage of 50%, the supplementing amount of each time is 0.5% of the total volume of the fermentation liquid, and the total fermentation time is 5-6 h. The fermentation liquor is preferably concentrated, and the salmonella is collected after the concentration.
In the present invention, the collection of salmonella in the fermentation broth is preferably performed using a high-speed freezing tubular centrifuge or a hollow fiber concentration system. In the present invention, when collecting salmonella using a high-speed freezing tube centrifuge, the conditions preferably include: the centrifugal speed of 48HZ is 2L/min, and the temperature is controlled to be 2-8 ℃. In the present invention, when collecting salmonella using the hollow fiber concentrating system, the conditions preferably include: and (3) carrying out high-pressure sterilization on the whole concentration system, starting a power pump after the system is cooled after the sterilization is finished, and controlling the membrane pressure of the hollow fibers to be below 0.1Mpa in the system operation process. And finally, collecting concentrated bacteria liquid according to a concentration ratio, centrifuging according to 1/50 (namely diluting the concentrated bacteria liquid to one fiftieth of the volume of the original fermentation medium by using a freeze-drying protective agent), and obtaining 1/10 hollow fibers (namely concentrating the hollow fibers to 1/10 of the volume of the original fermentation medium and then adding the corresponding freeze-drying protective agent). The invention has no special limitation on the high-pressure sterilization condition, and the sterilization can be carried out conventionally, such as sterilization at 121 ℃ for 30 min.
The salmonella and the freeze-drying protective agent are mixed to obtain the salmonella equine abortion live vaccine. In the invention, the freeze-drying protective agent (the solvent is water for injection) preferably contains gelatin and sucrose, the mass percentage of the gelatin is preferably 1-5%, and the mass percentage of the sucrose is preferably 10-50%. In the invention, when a high-speed freezing tubular centrifuge is adopted to collect salmonella, the mass percentage of gelatin in the selected freeze-drying protective agent is 1 percent, and the mass percentage of sucrose is 10 percent. In the invention, when the hollow fiber concentration system pipeline is adopted to collect salmonella, the selected freeze-drying protective agent contains 5 percent of gelatin and 50 percent of sucrose by mass.
In the present invention, the bacteria content of salmonella in the live salmonella equine abortion vaccine is preferably 1 × 10 10 CFU/first share is more than.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The preparation method of the salmonella equine abortion live vaccine comprises the following steps:
(1) preparing first-level seeds: diluting salmonella equi abortus C355 attenuated bacteria (from Chinese veterinary drug monitoring institute) with common broth, inoculating a common agar plate, culturing at 37 ℃ for 18-20 hours (totally requiring 2 days), selecting a plurality of medium-sized colonies, streaking and inoculating on a common agar slant, culturing at 35-37 ℃ for 18-20 hours (totally requiring 1 day), and passing through pure inspection to serve as a primary seed;
(2) preparing secondary seeds: inoculating one first-stage seed into a salmonella culture medium, culturing for 12 hours (0.5 days) at 35-37 ℃ under 150rmp, sampling and purely checking, and taking the qualified first-stage seed as a second-stage seed;
(3) bacterial liquid culture: before inoculation, adjusting the temperature of a salmonella liquid culture medium to 35-37 ℃, and adjusting the pH to 7.4-7.6; inoculating the secondary seeds of the salmonella according to 1-2% of the total amount of the culture medium, and performing fermentation culture, wherein in the process of fermentation culture, the temperature is controlled at 35-37 ℃, the pH is controlled at 7.4-7.6, and nutrient substances are supplemented every two hours from the 2 nd hour. The nutrient substance is glucose solution with the mass percentage content of 50 percent, the adding amount of each time is 0.5 percent of the total volume of the fermentation liquor, and the salmonella zymophyte liquid is obtained after 5 hours of culture;
(4) concentrating bacterial liquid: after fermentation is finished, reducing the temperature of the fermentation tank to 2-8 ℃, simultaneously reducing the stirring speed to 50rmp, and connecting a pipeline of a high-speed freezing tubular centrifuge or a pipeline of a hollow fiber concentration system (the total time of fermentation, concentration and counting is 1 day);
high-speed refrigerated centrifugation is used: and the centrifugation speed of 48HZ is 2L/min, after the centrifugation is finished, salmonella thalli are collected under the A-level condition, and are diluted by a freeze-drying protective agent (1% of gelatin and 10% of cane sugar which are both in percentage by mass), and finally the volume of the obtained product is 1/50 of the volume of the fermentation medium. Sampling for pure inspection and viable count;
② concentrating by using hollow fiber: concentrating to 1/10 of fermentation medium volume, collecting concentrated thallus under A-grade condition, and adding lyophilized protectant (5% gelatin and 50% sucrose). Sampling for pure inspection and viable count;
(5) seedling preparation: according to the viable bacteria counting result, adding a freeze-drying protective agent (the volume ratio of the hollow fiber concentrated bacterial liquid to the freeze-drying protective agent is 1:5) (the final concentration is 1% gelatin and 10% cane sugar, and the mass percentage content is the same) into the concentrated bacterial liquid, and adjusting the number of bacteria in the concentrated bacterial count. The live vaccine (C355 strain) for salmonella equine abortions of different specifications is formulated as shown in table 1 below;
TABLE 1 Salmonella equine abortions live vaccine (C355 strain) vaccine preparation Standard
(6) And (3) vaccine subpackaging: mixing the prepared vaccine liquid, batching, quantitatively subpackaging, stirring at any time during subpackaging, and uniformly mixing;
(7) after the freeze-drying is finished (the specific bacteria content of the freeze-dried powder can reach the first designed number according to the viable bacteria counting result and the vaccine preparation standard), viable bacteria counting, water content inspection, vacuum degree inspection, safety inspection and efficacy inspection are carried out.
The original process condition is as follows: (1) preparing first-level seeds: diluting Salmonella abortus C355 attenuated bacteria (purchased from Chinese veterinary medicine inspection institute) with common broth, inoculating common agar slant tubules and common broth tubules for propagation, inoculating common agar plates with common broth cultures, culturing at 37 ℃ for 18-20 hours, observing colony morphology, selecting a plurality of medium-sized colonies, inoculating common agar slant, culturing at 37 ℃ for 24 hours, and obtaining the first-grade seeds after pure inspection qualification. The product can be stored at 2-8 ℃ for no more than 2 months, and can be transplanted for 1-2 times (3 days).
(2) And (3) secondary seed propagation: inoculating the first-stage seeds into a common broth or a Martin broth with the pH value of 7.4-7.6, culturing for 24 hours, and shaking for 1-2 times. The broth cultures were subjected to a pure test (1 day) per flask using ordinary agar plates and slants. Storing at 2-8 deg.C for no more than 2 days.
(3) Preparing a bacterial liquid for preparing the seedlings: inoculating the seed solution into a common agar large flat bottle with the pH value of 7.4-7.6, uniformly shaking each bottle (the flat bottle is placed at 37 ℃ for 2-4 days before inoculation, and aseptic growth is carried out through visual inspection), enabling the seed solution to be full of agar, flatly culturing for 5-7 hours at 37 ℃, and then lifting the bottle mouth for slant culture for 36-48 hours (2-2.5 days). And (3) checking the pure state through naked eyes, adding a stabilizer (containing 1% of gelatin and 10% of cane sugar which are all in mass percentage and have the pH value of 7.4-7.6), washing the lawn, filtering the lawn in groups by using three layers of gauze in a sterilized empty bottle, and standing and precipitating the lawn at the temperature of 2-8 ℃.
(4) Seedling preparation and subpackaging: after standing for 24 hours (1 day) and examining the pure bacterial solution, 1/3 of the supernatant was aspirated from each bottle and mixed well. Quantitatively subpackaging according to the specified first batch, wherein the fungus content of each batch is 100 hundred million per batch. Vacuum drying is carried out quickly after subpackaging.
From example 1 and comparative example 1, it can be seen that: production cycle: the production process of the present invention needs at least 7.5 days, and the present invention needs only 4.5 days. Production strength: the original process adopts solid fermentation, needs a large amount of personnel to operate simultaneously, has aseptic operation, high operation difficulty and needs a large amount of flat bottles, and the bacteria for preparing the seedlings are concentrated by adopting fermentation tank production concentration equipment in the production stage, so that the working intensity of the personnel is reduced, and the pollution probability in the production is greatly reduced. Yield: the original production process needs 25m of surface area for every 10000 head parts produced 2 The common agar flat bottles used in the production are 0.025m each 2 The total amount of 1000 flat bottles is needed, the workload is extremely large, the invention adopts liquid fermentation and concentration, and only the liquid fermentation and concentration are carried out40L of fermentation medium is required to have the number of fermentation bacteria of 80-100 CFU/ml, a high-speed refrigerated centrifuge is adopted, and the number of bacteria after concentration and dilution is 4000 x 10 8 ～4500*10 8 CFU/ml, volume of 800 ml-1000 ml, and standard of 200 x 10 for each part of the vaccine 8 The CFU is calculated according to the above minimum value, and the finished product inspection should be 16 parts per bottle. The 10-header specification can be reported. (the counting results of the viable bacteria of the three batches S2022003-S2022006 are respectively 14.8 head parts/bottle, 15.1 head parts/bottle and 16.9 head parts/bottle)
Test examples
And (3) checking the safety of a finished product: the vaccines of example 1 and comparative example 1 were diluted with Martin broth or normal broth with the label indicating the head, and 10 mice weighing 18-20 g were intraperitoneally injected, 0.2ml each (containing 0.015 head), and observed for 14 days, and at least 8 mice should survive.
And (3) testing the efficacy: the first part is marked on a bottle label, the vaccine is diluted by 20% of aluminum hydroxide gel normal saline, 15 mice with the weight of 18-20 g are injected with 0.2ml (containing 0.015 part) into each abdominal cavity of 10 mice, and the other 5 mice are used as controls. After 21 days of inoculation, each mouse was intraperitoneally injected with 0.2-0.3 ml (4-5 MLD) of a 24-hour culture of Salmonella abortus C77-1 strain (CVCC79001) in a common broth, and observed for 14 days. Control mice should die entirely, and immunized mice are protected by at least 8 mice, with the results shown in table 2.
TABLE 2 comparison of Security and Effect tests
| Batch number | Specification (first part/bottle) | Security inspection | Effect testing |
| S2022001 | 10 | Survived 8 | 8/10 protection |
| S2022002 | 10 | |
The security inspection is not qualified and no effect inspection is carried out |
| S2022003 | 10 | Survived 8 | 8/10 protection |
| S2022004 | 10 | Survived 10 cells | 9/10 protection |
| S2022005 | 10 | Survived 9 pieces | 9/10 protection |
| S2022006 | 10 | Survived 10 cells | 10/10 protection |
Note: the above tables S2022001 to S2022003 show the vaccine prepared in comparative example 1, and the tables S2022004 to S2022006 show the vaccine prepared in the present invention, and it can be seen from Table 2 that the safety and the immune effect of the conventional production process are inferior to those of the present invention.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.
Claims (10)
1. The preparation method of the salmonella equine abortion live vaccine is characterized by comprising the following steps:
1) inoculating salmonella equi abortus C355 attenuated bacteria on a common agar slant for slant culture to obtain first-grade seeds;
2) inoculating the primary seeds obtained in the step 1) into a salmonella liquid culture medium for secondary seed culture to obtain secondary seeds;
3) inoculating the secondary seeds obtained in the step 2) into a salmonella liquid culture medium for fermentation to obtain fermentation liquor, and collecting salmonella in the fermentation liquor;
4) mixing the salmonella obtained in the step 3) with a freeze-drying protective agent to obtain the salmonella equine abortion live vaccine.
2. The method according to claim 1, wherein the amount of the secondary seeds used in step 3) is 1-2%.
3. The method according to claim 1, wherein the fermentation conditions of step 3) include: the temperature is 35-37 ℃, the pH value is 7.4-7.6, the dissolved oxygen is controlled to be 40% -80%, the stirring speed and the ventilation volume are increased when the dissolved oxygen is reduced, and the stirring speed is not higher than 150 rmp; and (3) supplementing nutrient substances every 2h from the 2 nd hour of fermentation, wherein the nutrient substances are glucose solutions with the mass percentage of 50%, the supplementing amount of each time is 0.5% of the total volume of the fermentation liquid, and the total fermentation time is 5-6 h.
4. The method according to claim 1, wherein the conditions for the secondary seed culture of step 2) include: the temperature is 35-37 ℃, the rotating speed is 150rpm, and the culture time is 12 h.
5. The method as claimed in claim 1, wherein the salmonella in the fermentation broth collected in step 3) is collected using a high-speed freezing tube centrifuge or a hollow fiber concentration system.
6. The method of claim 5, wherein when the Salmonella is collected using a high-speed refrigerated tubular centrifuge, the conditions include: the centrifugal speed of 48HZ is 2L/min, and the temperature is 2-8 ℃.
7. The method of claim 5, wherein when collecting Salmonella using a hollow fiber concentration system, the conditions include: and (3) carrying out high-pressure sterilization on the whole concentration system, starting a power pump after the system is cooled after the sterilization is finished, controlling the membrane pressure of the hollow fibers to be below 0.1Mpa in the system operation process, and finally collecting concentrated bacteria liquid according to the concentration proportion.
8. The preparation method according to claim 1, wherein the cryoprotectant in the step 4) contains gelatin and sucrose, wherein the mass percentage of the gelatin is 1-5%, and the mass percentage of the sucrose is 10-50%.
9. The method of claim 1, wherein the live Salmonella equine abortion vaccine contains Salmonella in an amount of 1X 10 10 CFU/first share is more than.
10. The method according to claim 1, wherein the conditions for the step 1) of slant culture include: the temperature is 35-37 ℃, and the time is 18-20 h.
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| RU2124366C1 (en) * | 1997-10-03 | 1999-01-10 | Ярцев Михаил Яковлевич | Method of preparing vaccine against salmonellosis in agriculture animals and poultry |
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| CN111100817A (en) * | 2019-11-26 | 2020-05-05 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Equine abortion salmonella equine origin strain and application thereof in preparation of inactivated vaccine for equine abortion salmonella |
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