CN107569681B - Bivalent inactivated vaccine for bovine pasteurellosis and preparation method thereof - Google Patents
Bivalent inactivated vaccine for bovine pasteurellosis and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a bivalent inactivated vaccine for cattle pasteurellosis, belonging to the technical field of preparation of biological products for livestock. The bivalent inactivated vaccine comprises an antigen and a vaccine adjuvant, wherein the antigen is a bovine pasteurella multocida capsular type A Pm-TJ strain and a bovine pasteurella multocida capsular type B C45-2 strain. The bivalent inactivated vaccine prepared by the invention adopts a concentration and purification process, optimizes a fermentation culture process of a bovine pasteurella multocida capsular type B C45-2 strain, shortens the fermentation time by more than half, improves the production efficiency, can simultaneously prevent bovine cellulosic suppurative pneumonia and bovine hemorrhagic septicemia caused by bovine pasteurella multocida infection through one-needle immunization, and has the characteristics of safety, reliability and good immune effect.
Description
Technical Field
The invention belongs to the technical field of biological products for livestock, and particularly relates to a bivalent inactivated vaccine for bovine pasteurellosis.
Background
Bovine pasteurellosis is an infectious disease caused by infection of cattle with bovine pasteurellosis. Currently, bovine pasteurella multocida diseases prevalent in China are mainly divided into two types, one is bovine cellulous suppurative pneumonia caused by bovine pasteurella multocida capsular type A bacteria, and the other is bovine hemorrhagic septicemia caused by bovine pasteurella multocida capsular type B bacteria. Wherein, the bovine cellulose suppurative pneumonia is a disease seriously invading the respiratory system of the cattle, and the incidence rate can reach 100 percent and the death rate can reach 40 percent under the stimulation of stress conditions such as long-distance transportation and the like after healthy cattle are mixed. The disease belongs to a new infectious disease in China, has been spread to main cattle raising areas of all provinces in China in recent years, and becomes one of the diseases seriously harming cattle raising industries in China. Bovine hemorrhagic septicemia is an infectious disease characterized by hyperpyrexia, pneumonia, acute gastroenteritis and extensive bleeding of internal organs, has already seen a history of prevalence in China for decades, and is still sporadic or endemic at present.
Immunization with vaccines is the primary measure for the prevention of pasteurellosis multocida disease in cattle. However, the existing pasteurella multocida inactivated vaccine (B type single vaccine for short) in China can only prevent bovine hemorrhagic septicemia caused by pasteurella multocida capsular B type bacteria, and no vaccine is available for preventing bovine cellulose suppurative pneumonia caused by pasteurella multocida capsular A type bacteria. The existing B type bacteria single vaccine is prepared by using capsular B type pasteurella multocida C45-2 strain (CVCC 44502), C46-2 strain (CVCC 44602) and C47-2 strain (CVCC 44702) with good immunogenicity, optionally inoculating 1-3 strains to an adaptive culture medium according to needs, inactivating the culture by formaldehyde solution and adding aluminum hydroxide gel; the vaccine is injected intramuscularly or subcutaneously, and the injection dosage is 4ml for cattle with the weight of less than 100kg, and 6ml for cattle with the weight of more than 100 kg. Therefore, the single B-type bacterium vaccine selects three strains, namely C45-2 strain, C46-2 strain and C47-2 strain, to be used for preparing the vaccine, the culture of the first-level seeds and the culture of the second-level seeds need to be respectively cultured in the production process, the operation process is increased undoubtedly, and more procedures are not beneficial to controlling the difference among vaccine batches. In addition, because the injection dosage (4ml-6ml) is large, the operation is inconvenient and the operation time is increased when the injection dosage is used in a clinical large-scale farm; meanwhile, in terms of the loading capacity of manufacturers, the same number of vaccine bottles are required to be filled with the same number of vaccine bottles, so that the packaging cost is increased. The B-type bacterium single vaccine can effectively prevent cattle hemorrhagic septicemia, but in clinical application of nearly two decades, relevant article reports and actual feedback of individual customers exist, and after the B-type bacterium single vaccine is injected into cattle, certain side reaction exists, and the cattle can die under severe conditions; therefore, the safety of the B-type bacterium single seedling is expected to be further improved.
At present, no commercial vaccine aiming at preventing bovine cellulose suppurative pneumonia caused by bovine pasteurella multocida capsular type A bacteria exists in China. In 2006, since the outbreak of the cellulosic suppurative pneumonia of cattle in our country, the harbin veterinary research institute of the Chinese academy of agricultural sciences successfully isolated and identified the pathogen causing the disease: bovine pasteurella multocida capsular type A bacterium (microorganism preservation number: CGMCC No.3619), and a patent of bovine-derived capsular type A pasteurella multocida and separation, identification and application thereof (patent application number CN201010115552.9) is applied in 2 months 2010. The patent describes that after the A-type bacteria are cultured by BHI, the A-type bacteria are prepared into inactivated vaccines by adopting an oil adjuvant, and a mouse model proves that the A-type bacteria can provide complete protection for virulent attack of the A-type bacteria and have no cross protection with the B-type bacteria. In view of poor cross immunity among different serotypes of pasteurella multocida, the existing commercial products cannot effectively prevent and control bovine cellulous suppurative pneumonia caused by capsular type A bacteria, and great difficulty is brought to the prevention and control of bovine pasteurella multocida. Therefore, the best method is to prepare the bivalent vaccine of the bovine pasteurellosis multocida disease, so as to obtain the best prevention effect.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a bivalent inactivated vaccine for bovine pasteurellosis.
In order to realize the purpose of the invention, the technical scheme of the invention is as follows: the bivalent inactivated vaccine for the bovine pasteurella multocida disease contains antigens and adjuvants, wherein the antigens are a bovine pasteurella multocida capsular type A Pm-TJ strain and a bovine pasteurella multocida capsular type B C45-2 strain, the preservation number of the bovine pasteurella multocida capsular type A Pm-TJ strain is CGMCC No.3619, and the preservation number of the bovine pasteurella multocida capsular type B C45-2 strain is CVCC 44502.
The adjuvant is aluminum hydroxide gel.
In the bivalent inactivated vaccine for the bovine pasteurella multocida disease, the effective content of a capsular A type Pm-TJ strain of the bovine pasteurella multocida is 2.0-10.0 multiplied by 109CFU/mL; the effective content of C45-2 strain of bovine pasteurella multocida capsule type B is 2.0-10.0 × 109CFU/mL。
Preferably, the effective content of the Pasteurella multocida capsular type A Pm-TJ strain is 6.0 x 109CFU/mL; the effective content of C45-2 strain of bovine pasteurella multocida capsule type B is 4.0 × 109CFU/mL。
The invention provides a preparation method of the bivalent inactivated vaccine for the bovine pasteurella multocida disease, which comprises the following steps:
(1) fermenting and culturing a bovine pasteurella multocida capsular type A Pm-TJ strain and a bovine pasteurella multocida capsular type B C45-2 strain respectively;
(2) the zymophyte liquid of the two strains is subjected to centrifugation or hollow fiber concentration and purification, and is inactivated;
(3) and (3) inactivating the two bacteria solutions according to a volume ratio of 2:1 mixing, adding adjuvant, mixing, adding thimerosal solution to make its final concentration be 0.005%, and the content of two antigens in the vaccine is not less than 2.0X 109CFU/mL。
In the preparation method, the fermentation culture method of the pasteurella multocida capsular type A Pm-TJ strain in the step (1) comprises the following steps: inoculating Pm-TJ strain at 1-2% inoculation amount to brain heart infusion culture medium for preparing vaccine, culturing at 35-37 deg.C for 6-10 hr with gradually increasing ventilation volume, specifically 0-3 hr ventilation volume of 0.1m3Per hour/10L, and the ventilation volume is 0.3m in 3-6 hours3h/10L, a ventilation of 0.5m after 6 hours3h/10L, and the maximum ventilation amount is not more than 0.5m3/h/10L。
The fermentation culture method of the pasteurella multocida capsular B type C45-2 strain in the step (1) comprises the following steps: inoculating C45-2 strain at 1% -2% inoculum size in Martin broth containing 0.1% lysed blood cell, culturing at 35-37 deg.C under gradually increasing ventilation amount for 6-10 hr, wherein the ventilation amount is 0.1m in 0-3 hr3Per hour/10L, and the ventilation volume is 0.3m in 3-6 hours3h/10L, a ventilation of 0.5m after 6 hours3h/10L, and the maximum ventilation amount is not more than 0.5m3/h/10L。
And (2) culturing the two bacteria in the step (1) in a non-stirring manner in the fermentation culture process.
The centrifugation method in the step (2) is to maintain the flow rate of the bacteria liquid at 3000ml/min and the continuous flow centrifugation at 12000r/min at 8000-.
And (3) the centrifugation method in the step (2) is to maintain the flow rate of the bacterial liquid to be 2500ml/min, perform continuous flow centrifugation at 10000r/min, collect bacterial sludge sediment, and re-suspend the bacterial sludge sediment by using sterile PBS with the pH value of 7.2-7.4 to prepare 10-fold concentrated bacterial liquid.
The hollow fiber concentration and purification method in the step (2) is to concentrate hollow fibers with the aperture of 0.22 mu m, control the pressure within 0.03Mpa and maintain the flow rate within 300ml/min so as to concentrate the hollow fibers into 1/10 of the original bacteria liquid.
The invention also provides application of the mouse in the efficacy test of the bovine pasteurella multocida bivalent inactivated vaccine.
The method for testing the efficacy of the bivalent inactivated vaccine against the bovine pasteurella multocida disease, which is established by the invention, by using the mice comprises the following steps: 5 mice with weight of 18-22 g were injected intramuscularly with vaccine 50 μ l, 21 days later, and 5 control mice with the same conditions were injected intraperitoneally with 1.0 × 104MLD bovine pasteurella multocida Pm-TJ strain virulent strain liquid is observed for 7 days; the control group died entirely and the immune group should have at least 4 protection.
The invention also provides application of the rabbit in efficacy test of the bovine pasteurella multocida disease bivalent inactivated vaccine.
The method for testing the efficacy of the rabbit used for carrying out the bovine pasteurella multocida disease bivalent inactivated vaccine comprises the following steps: 5 rabbits with the weight of 1.5-2.0 kg are injected with 0.5ml of vaccine through each muscle or subcutaneous injection, and after 21 days, 1MLD bovine pasteurella multocida C45-2 virulent strain liquid is injected through each subcutaneous injection together with 5 control rabbits with the same conditions, and the observation is carried out for 8 days. The control group died entirely and the immune group should have at least 3 protections.
The invention has the beneficial effects that: (1) the bivalent inactivated vaccine can provide good immune protection effect on bovine cellulose suppurative pneumonia and bovine hemorrhagic septicemia which are popular in China at present. The injection can be used for immunizing cattle through one injection, and simultaneously preventing two diseases of bovine cellulose suppurative pneumonia and bovine hemorrhagic septicemia, and the vaccine has the advantages of good safety, stable immune effect and long immune duration. (2) The injection dose of the bivalent inactivated vaccine is 2ml, and compared with the traditional B-type bacterium single vaccine injection dose of 4-6ml, the bivalent inactivated vaccine greatly improves the convenience of clinical injection operation and improves the packaging utilization rate of the vaccine. (3) The bivalent inactivated vaccine of the invention adopts a centrifugal or hollow fiber concentration and purification mode in the preparation process, can effectively remove impurity components such as bacterial fragments, culture medium and the like, and reduce the content of bacterial exotoxin, thereby effectively reducing side reaction, having good safety, and having no local or systemic adverse reaction caused by the vaccine. (4) According to the invention, C45-2 is selected as the B type capsular strain antigen of the pasteurella bovis, so that the vaccine production process is simplified compared with the traditional B type strain single vaccine (C45-2 strain, C46-2 strain and C47-2 strain are adopted as antigens), and the complexity of the operation of the vaccine process is reduced. (5) The bivalent inactivated vaccine is optimized aiming at the fermentation culture process of B-type bacteria in the production process, and the ventilation capacity is gradually increased to 0-3 hours and 0.1m in the fermentation process3Per hour/10L, and the ventilation volume is 0.3m in 3-6 hours3h/10L, a ventilation of 0.5m after 6 hours3h/10L, and the maximum ventilation amount is not more than 0.5m3The culture time is greatly shortened to 6-10 hours compared with the fermentation time of single B-type bacteria seedlings by 12-24 hours in a/h/10L mode, and the production efficiency is greatly improved. (6) The invention is in twoThe inactivated vaccine establishes a parallel relation model of the efficacy tests of mice and cattle and rabbits and cattle in the efficacy test, so that the efficacy test of the cattle can be replaced by the mice and the rabbits, the animal welfare is maintained, and the manpower, material resources and financial resources are greatly saved.
Drawings
FIGS. 1A-1C are graphs showing the growth of Pasteurella multocida capsular type A Pm-TJ strain in different batches under different aeration conditions.
FIGS. 2A-2C are graphs showing the growth of C45-2 strain of Pasteurella multocida capsule type B under different aeration conditions for different batches.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental methods used in the examples are conventional methods unless otherwise specified. The materials, reagents and the like used in the examples are commercially available unless otherwise specified.
The Pm-TJ strain of the bovine pasteurella multocida capsule A type is provided by Harbin veterinary research institute of Chinese academy of agricultural sciences, and the preservation number is CGMCC No. 3619. The pasteurella multocida capsule type B C45-2 strain was supplied by the midrange industrial company, inc, luck bio-pharmaceuticals, inc, under accession number CVCC 44502. Other instruments and reagents for preparing the seedlings are provided by Lanzhou biological pharmaceutical factory, a company Limited in the middle-aged and animal husbandry industries. Brain heart infusion medium (BHI) and Martin broth were purchased from Oboxing Biotechnology, Inc., Beijing. Hollow fibers, available from Beijing Asahi Membrane Equipment liability Co., Ltd, with pore sizes of 0.22 μm. Tube centrifuge, available from Liaoyang sun pharmaceutical machinery, Inc.
Example 1 preparation of bivalent inactivated vaccine against bovine pasteurellosis
1. Culture medium
Preparing a brain heart infusion medium (BHI): 37g of BHI powder was weighed and added to 1000ml of distilled water, and after thoroughly mixing, sterilized at 121 ℃ for 15 minutes.
Preparing a martin broth culture medium: 50g of Martin broth culture medium powder is weighed and added into 1000ml of distilled water, after fully and uniformly mixing, the Martin broth culture medium powder is sterilized for 15 minutes at 121 ℃, and after sterilization, 0.1 percent of lysed blood cell whole blood is added according to the volume of the culture medium.
Preparing a brain heart infusion agar culture medium: weighing 37g of BHI powder and 15g of agar powder, adding the BHI powder and the agar powder into 1000ml of distilled water, fully and uniformly mixing, sterilizing at 121 ℃ for 15 minutes, cooling to 50-60 ℃, and pouring the flat plate for later use.
Preparing a Martin broth agar culture medium: weighing 50g of Martin broth culture medium powder and 15g of agar powder, adding into 1000ml of distilled water, fully mixing, sterilizing at 121 ℃ for 15 minutes, cooling to 50-60 ℃, adding 0.1% of lysed blood cell whole blood and 4% of healthy animal serum according to the volume of the culture medium, fully mixing, and pouring the mixture into a flat plate for later use.
2 method
2.1 preparation of bovine Pasteurella multocida capsular type A Pm-TJ strain antigen
First-stage seed propagation and identification, a Pm-TJ strain freeze-dried strain is inoculated in a brain heart infusion culture medium, cultured for 8-12 hours at 37 ℃ at 75r/min, then streaked and inoculated in a brain heart infusion agar plate, cultured for 16-22 hours at 37 ℃, 5-10 typical colonies are selected, a plurality of brain heart infusion agar inclined planes are respectively inoculated, and cultured for 24 hours at 37 ℃ to serve as first-stage seeds. The product is stored at the temperature of 2-8 ℃ and the service life is not more than 20 days.
And (3) second-stage seed propagation and identification, namely, inoculating the first-stage seeds into a brain-heart infusion culture medium, culturing at 37 ℃ for 75r/min for 8-12 hours, and storing at 2-8 ℃ after pure inspection is qualified, wherein the service life is not more than 7 days.
Preparing an antigen for preparing the vaccine: the bacterial liquid culture adopts a microbial fermentation tank to culture pasteurella multocida bacterial liquid. Filling the brain-heart infusion culture medium and a proper amount of antifoaming agent into a fermentation tank, sterilizing at 121 ℃ for 30 minutes, adding secondary seed liquid according to 1-2% of the total amount of the culture medium when the temperature of the culture medium is cooled to about 37 ℃, uniformly mixing, and performing aeration culture at 37 ℃, wherein the aeration culture is performed for 0-3 hoursThe amount is 0.1m3Per hour/10L, and the ventilation volume is 0.3m in 3-6 hours3h/10L, a ventilation of 0.5m after 6 hours3h/10L, and the maximum ventilation amount is not more than 0.5m3h/10L, and the culture time is 6-10 hours.
After the pure inspection and the culture of the viable bacteria count bacterial liquid are finished, sampling and carrying out pure inspection according to the addendum of Chinese animal pharmacopoeia 2010, and the samples are required to be pure; and simultaneously sampling for viable bacteria counting.
And (3) concentrating the bacterial liquid, namely centrifuging the bacterial liquid by using a centrifugal machine or concentrating the bacterial liquid by using hollow fibers, and re-suspending the concentrated bacterial liquid by using PBS (the pH value is 7.2-7.4) to prepare bacterial suspension.
And (3) inactivating, namely adding a formaldehyde solution according to 0.1-0.15% of the amount of the concentrated bacterium solution, and inactivating for 7-12 hours at 37 ℃, wherein the stirring is carried out for a plurality of times. Inactivation test the inactivation test was carried out according to the appendix of the 2010 version of Chinese animal pharmacopoeia, and the cells should be grown aseptically.
2.2 preparation of bovine Pasteurella multocida capsular type B C45-2 strain antigen
First-order seed propagation and identification, inoculating a freeze-dried strain of pasteurella multocida C45-2 strain to Martin broth containing 0.1% of lysed blood cell whole blood, culturing for 8-12 hours at 37 ℃, then streaking and inoculating to Martin agar plates containing 0.1% of lysed blood cell whole blood and 4% of healthy animal serum, culturing for 16-22 hours at 37 ℃, selecting 5-10 typical colonies, inoculating a plurality of fresh blood agar inclined planes, and culturing for 24 hours at 37 ℃ to serve as first-order seeds. The product is stored at the temperature of 2-8 ℃ and the service life is not more than 20 days.
And (3) second-level seed propagation and identification, namely, inoculating the first-level seeds into a martin broth containing 0.1% of whole blood of a lytic blood cell, culturing for 8-12 hours at 37 ℃, sampling, passing a pure inspection, and storing at 2-8 ℃ for no more than 7 days.
Preparing an antigen for preparing the vaccine: the bacterial liquid culture adopts a microbial fermentation tank to culture the pasteurella multocida bacterial liquid. Putting Martin broth culture medium and appropriate amount of antifoaming agent into a fermentation tank, sterilizing at 121 deg.C for 30 min, cooling the culture medium to about 37 deg.C, adding 0.1% of lysed blood cell whole blood, adding 1% -2% of the total amount of the culture medium into the second-stage seed solution, mixing, and culturing at 37 deg.C with aeration amount of 0.1m for 0-3 hr3The ventilation rate is maintained at 0.3m in 3-6 hours per 10L3h/10L, the ventilation after 6 hours was maintained at 0.5m3h/10L, and the culture time is 6-10 hours.
After the pure inspection and the culture of the viable bacteria count bacterial liquid are finished, sampling and carrying out pure inspection according to the addendum of Chinese animal pharmacopoeia 2010, and the samples are required to be pure; and simultaneously sampling for viable bacteria counting.
And (3) concentrating the bacterial liquid, namely centrifuging the bacterial liquid by using a centrifugal machine or concentrating the bacterial liquid by using hollow fibers, and re-suspending the concentrated bacterial liquid by using PBS (the pH value is 7.2-7.4) to prepare bacterial suspension.
And (3) inactivating, namely adding a formaldehyde solution according to 0.1-0.15% of the amount of the concentrated bacterium solution, and inactivating for 7-12 hours at 37 ℃, wherein the stirring is carried out for a plurality of times. Inactivation test the inactivation test was carried out according to the appendix of the 2010 version of Chinese animal pharmacopoeia, and the cells should be grown aseptically.
2.3 vaccine compounding
Adding two antigen solutions in an emulsifying tank according to the volume ratio of 2:1 of bovine pasteurella multocida capsular model A Pm-TJ strain antigen to bovine pasteurella multocida capsular model B C45-2 strain antigen, stirring at low speed for 5 minutes, slowly adding an aluminum hydroxide gel adjuvant according to the volume ratio of 1 part of adjuvant to 5 parts of antigen, fully stirring for 15-20 minutes to fully mix, adding 1% thimerosal solution to make the final concentration of the thimerosal solution be 0.005%, and fully stirring. The final vaccine contains two antigens which are not less than 2.0 x 109CFU/ml。
2.4 inspection of the semi-finished product, performing aseptic inspection according to the appendix of 2010 version of Chinese animal pharmacopoeia, and performing aseptic growth.
2.5, quantitatively subpackaging after the subpackage is qualified through aseptic inspection, stirring at any time during the subpackage period to uniformly mix, plugging and sealing, and sticking a label. Storing at 2-8 ℃.
Example 2 safety testing of vaccines prepared according to the invention
1 Material
1.1 vaccine bovine pasteurella multocida bivalent inactivated vaccine (type A Pm-TJ strain + type B C45-2 strain) prepared by the method of example 1. The batch numbers are ZM201401, ZM201402 and ZM201403 respectively.
1.2 test animals are healthy and susceptible to cattle, 3 months old and above.
2 method
2.1 safety test for one-time Single dose vaccination of target animals
The 12 test cows were randomly grouped into 4 animals each, and 3 batches of the vaccine were each inoculated with a single dose of 2.0ml (1 animal) at one time through the neck muscle to a group of test cows, 3 test cows having the same conditions were additionally provided, and sterilized PBS was inoculated in the same manner as a control. Clinical performance of the test cattle was observed, and body temperature was measured at the same time every day for 14 days.
2.2 safety test for Single dose repeat inoculation of target animals
The 12 test cows were randomly grouped into 4 animals each, and 3 batches of the vaccine were each inoculated with a single dose of 2.0ml (1 animal) at one time through the neck muscle to a group of test cows, 3 test cows having the same conditions were additionally provided, and sterilized PBS was inoculated in the same manner as a control. 14 days after the first inoculation, another inoculation was performed in the same manner. Clinical performance of the test cattle was observed after repeated vaccination, and body temperature was measured at the same time every day for 14 days.
2.3 safety test for one-time overdose inoculation of target animals
Randomly grouping 12 test cows, wherein each group has 4 cows; a group of test cattle was inoculated with 4.0ml (2 heads) of vaccine in 3 batches at one overdose through the neck muscle, and 3 test cattle under the same conditions were inoculated with sterile PBS in the same manner as a control. Clinical performance of the test cattle was observed after inoculation, and body temperature was measured at the same time every day for 14 days.
3 results
3.1 safety test results for one-time Single dose vaccination of target animals
After the vaccine is inoculated by one single dose, the body temperature, the breathing condition, the mental state, the appetite and the like of the test cattle are normal in the observation period, the injection part does not have red swelling and nodule formation, the test cattle do not have clinical symptoms of the cattle pasteurella multocida disease, and the test cattle have no obvious difference with a control group. The results are shown in Table 1.
TABLE 1 safety test results for one single dose vaccination of target animals
3.2 safety test results for Single dose repeat inoculation of target animals
After the vaccine is repeatedly inoculated in a single dose, the body temperature, the breathing condition, the mental state, the appetite and the like of the test cattle are normal in an observation period, the injection part does not have red swelling and nodule formation, the test cattle do not have clinical symptoms of the cattle pasteurella multocida disease, and the clinical symptoms are not obviously different from those of a control group. The results are shown in Table 2.
TABLE 2 safety test results for single dose repeat vaccination of target animals
3.3 safety test results for one-time overdose inoculation of target animals
After the vaccine is inoculated in one-time overdose, the body temperature, the breathing condition, the mental state, the appetite and the like of the test cattle are normal in the observation period, the injection part does not have red swelling and nodule formation, the test cattle do not have clinical symptoms of the cattle pasteurella multocida disease, and the clinical symptoms are not obviously different from those of a control group. The results are shown in Table 3.
TABLE 3 safety test results for one overdose inoculation of target animals
4 conclusion
By carrying out a single-dose inoculation test, a single-dose repeated inoculation test and a single-dose overdose inoculation test on 3 batches of the product for the target animal, the body temperature, the breathing condition, the mental state, the appetite and the like of the tested animal are normal, the injection part does not have red swelling and nodule formation, the clinical symptoms of the bovine pasteurella multocida disease do not appear, and the product is not obviously different from a control group. The above results indicate that the product is safe for cattle.
EXAMPLE 3 potency assay for vaccines prepared according to the invention
1 Material
1.1 vaccine cattle pasteurella multocida bivalent inactivated vaccine (Pm-TJ type A strain + C45-2 type B strain) prepared by the method of example 1. The batch numbers are ZM201401, ZM201402 and ZM201403 respectively. The batch number of the inactivated vaccine for bovine pasteurella multocida (B type bacterial single vaccine, contained in C45-2 strain, C46-2 strain and C47-2 strain) is 1407001.
1.2 test animals are healthy susceptible cattle, 6 months old and above.
2 method
The 3 batches of vaccine prepared by the invention are inoculated to 8 test cattle by muscle with 1 head (2.0ml) dose, the commercial cattle pasteurella multocida disease inactivated vaccine (B type single vaccine) is inoculated to 4 test cattle by muscle with 1 head (4.0ml) dose, and 6 non-immunized cattle are additionally arranged as a control. 21 days after inoculation, performing A-type bacteria challenge on 4 of 8 cattle and 3 of 6 control cattle of each batch of vaccine immunization groups, namely dripping Pm-TJ strain liquid of 2MID into each trachea of each cattle, observing for 10 days, observing clinical symptoms of the cattle, and recording the body temperature at the same time every day; on the 10 th day after the toxin is attacked, the immune cattle and the control cattle are subjected to autopsy, the pathological changes of the lung are observed and recorded, and the morbidity and the protection condition of the cattle are analyzed and judged; the other 4 of 8 cattle immunized by each batch of the vaccine, 4 cattle immunized by a single vaccine and the rest 3 control cattle are subjected to B-type bacteria challenge, namely, each cattle is injected with 10MLD C45-2 virulent strain liquid. The cattle were observed for 14 days and their death was recorded.
3 results
The vaccine product is used for virus challenge 21 days after cattle are immunized, and the protection results of the virus challenge are shown in tables 4 and 5. As can be seen from Table 4, after the A-type bacteria challenge, the 3 vaccine immunization groups prepared by the invention are protected at 3/4 or above, and all the non-immunization control groups are attacked (3/3); as can be seen from Table 5, after challenge with type B bacteria, the 3 vaccine immunization groups prepared by the present invention were protected at 3/4 or more, the single vaccine immunization group was protected at 3/4, and all non-immune control groups died (3/3).
TABLE 4 protective results of A-type bacteria challenge after immunizing cattle with the bivalent vaccine of the present invention
Note: "-" indicates that the item does not have the corresponding symptom, and "+" indicates that the item has the corresponding symptom.
TABLE 5 protective results of B-type bacterial challenge after immunization of cattle with the bivalent vaccine of the present invention
4 conclusion
After 3 batches of vaccines prepared by the invention are applied to vaccinate cattle, the immunized cattle can be effectively protected against virulent attack, and the protection effect is not lower than that of single vaccine of the similar product, which shows that the vaccine prepared by the invention has good immune protection capability on cattle.
EXAMPLE 4 study of the Large Scale culture and concentration purification Process of the vaccine prepared according to the present invention
1. Air flow test
1.1 bovine Pasteurella multocida capsular type A Pm-TJ strain
Inoculating Pm-TJ strain at 2% inoculation amount into 3 batches of 20L reactors filled with brain heart infusion culture medium for preparing seedlings, wherein the culture medium filling amount of each batch is 1.5 ten thousand milliliters, culturing for 14 hours at 37 ℃ by adopting a method of gradually increasing ventilation volume, and the maximum ventilation volume is not more than 0.3, 0.5 and 0.75m respectively3The ventilation was adjusted in a manner of/h/10L, as shown in Table 6. Samples were taken at 4, 6, 8, 10, 12, 14 hours of incubation for pure test and viable count, respectively, and growth curves were plotted. Each culture condition was cultured in 3 batches.
Table 6 Pm-TJ strain bacteria culture test ventilation regulating method
1.2 bovine Pasteurella multocida capsular type B C45-2 strain
Inoculating C45-2 strain at 2% inoculum size in 3 batches of 20L reactor containing Martin broth (containing 0.1% lysed blood cells) for preparing vaccine, wherein the culture medium is 1.5 ten thousand ml each, culturing at 37 deg.C under gradually increasing ventilation rate for 14 hr, and the maximum ventilation rate is not more than 0.3, 0.5, and 0.75m3the/h/10L, the ventilation regulation method is shown in Table 7. Samples were taken at 4, 6, 8, 10, 12, 14 hours of incubation for pure testing and viable count and growth curves were plotted. Each culture condition was cultured in 3 batches.
TABLE 7 aeration regulating method for culture test of C45-2 strain
2. Stirring speed test
2.1 bovine Pasteurella multocida capsular type A Pm-TJ strain
Pm-TJ strain is inoculated into 3 batches of 20L reactors filled with brain core infusion culture medium for preparing the vaccine with the inoculation amount of 2 percent, the loading amount of each batch of culture medium is 1.5 ten thousand milliliters, the optimal aeration condition obtained by 1.1 test is adopted, the culture is carried out at the condition of 37 ℃ by adopting the stirring rotating speed of 0r/min, 75r/min and 150r/min respectively, and sampling is carried out for pure inspection and viable count after the culture is carried out for 8 hours. Each culture condition was cultured in 3 batches.
2.2 bovine Pasteurella multocida capsular type B C45-2 strain
Inoculating 2% inoculum size of C45-2 strain into 3 batches of 20L reactors filled with Martin broth culture medium (containing 0.1% of lysed blood cells), wherein the culture medium content of each batch is 1.5 ten thousand ml, adopting the optimal aeration condition obtained by 1.2 experiments, respectively adopting stirring rotation speeds of 0, 75 and 150r/min for culture at 37 ℃, and sampling for pure inspection and viable count after 8 hours of culture. Each culture condition was cultured in 3 batches.
2.3 research on concentration and purification Process
2.3.1 centrifugal concentration purification method
Respectively taking 3 batches of Pm-TJ strain and C45-2 strain liquid which are subjected to amplification culture, wherein each batch is 10 ten thousand milliliters; maintaining the flow rate of the bacteria liquid at 2500ml/min, performing continuous flow centrifugation at 10000r/min, collecting bacterial sludge precipitate, performing heavy suspension with sterile PBS to prepare 10 times of concentrated bacteria liquid, and counting viable bacteria of the concentrated bacteria liquid.
2.3.2 hollow fiber concentration purification method
3 batches of Pm-TJ strain and C45-2 strain liquid are respectively taken, and each batch is 10 ten thousand milliliters. Injecting the bacteria liquid into a concentration tank, concentrating with hollow fiber with aperture of 0.22 μm, controlling pressure within 0.03Mpa, maintaining flow rate within 300ml/min, concentrating to 1/10 of original bacteria liquid, collecting concentrated bacteria liquid, sampling, and counting viable bacteria.
3 results
3.1 aeration test bovine pasteurella multocida capsular type A and B bacteria were cultured under different aeration conditions, the bacteria liquid at each culture time point was harvested and subjected to a pure test, the results are all pure, the results of viable bacteria counting are shown in tables 8 and 9, respectively, and bacterial growth curves were drawn according to the counting results, and the results are shown in FIGS. 1A-1C and 2A-2C, respectively.
TABLE 8 viable count results of type A bacterium Pm-TJ strain at various time points under different aeration conditions
TABLE 9 viable count results of type B bacterium C45-2 strain at various time points under different aeration conditions
As can be seen from tables 8 and 9 and fig. 1A-1C and fig. 2A-2C, the time at which the peak of the viable count of the bacteria in test group 1 was reached was the latest, and the maximum viable count of the bacteria was the lowest; experiment 3 groups of bacteria have the earliest time of reaching the peak value of the viable count, but the bacteria have shorter time in the stationary phaseIt is difficult to grasp the harvesting time in production; the time for the viable bacteria number of the bacteria in the test 2 group to reach the peak value is between the test 1 group and the test 3 group, and the maximum viable bacteria number of the bacteria is higher than that of the test 1 group and is equivalent to that of the test 3 group; therefore, for the industrial production, the aeration conditions of the test 2 group were determined as the large-scale culture conditions of the A type bacterium Pm-TJ strain and the B type bacterium C45-2 strain, i.e., the aeration amount was gradually increased to 0.5m3Culturing for 6-10 hours at 37 ℃ under the condition of/h/10L to obtain bacterial liquid.
3.2 after obtaining the aeration culture condition of the A type bacteria and the B type bacteria in the stirring rotating speed test, gradually increasing the aeration quantity to 0.5m3The culture conditions of/h/10L, type A bacteria and type B bacteria under different stirring speed for 8 hours, harvest each batch of bacteria liquid and pure test, the results show that all pure, the results of viable count is shown in Table 10 and 11. As can be seen from tables 10 and 11, the bacteria type A and B were cultured under aeration culture conditions at the agitation-free and different agitation speeds (75r/min and 150r/min), and the concentration of the harvested bacteria liquid was not significantly different. In order to avoid the damage of shearing force generated by stirring to bacteria, the A-type bacteria and the B-type bacteria are cultured in a non-stirring mode in large-scale production selection.
TABLE 10 viable count results of bacteria liquid obtained from type A strain Pm-TJ by different stirring speeds
TABLE 11 viable count results of bacterial solutions obtained from type B bacterium C45-2 strain at different stirring speeds
3.2 results of concentration and purification test
The 3 batches of bacterial liquid are concentrated by 10 times by a centrifugal method and a hollow fiber method, the viable count results of the concentrated bacterial liquid are shown in tables 12 and 13, and the concentration of the bacteria concentrated by the hollow fiber is slightly higher than that of the bacteria concentrated by the centrifugal method, but all meet the requirements of seedling preparation. Therefore, depending on the plant conditions, centrifugation or hollow fiber can be selected to concentrate and purify type a and type B bacteria for vaccine preparation.
TABLE 12 bacterial count results for type A bacteria under different concentration and purification conditions (× 10)8CFU/ml)
| Batches of | Before concentration | Concentrating by centrifugation (10 times) | Hollow fiber concentration (10 times) |
| 1 | 45.5 | 348 | 353 |
| 2 | 32.8 | 265 | 272 |
| 3 | 41.2 | 302 | 321 |
TABLE 13 bacterial count results for type B bacteria under different concentration and purification conditions (× 10)8CFU/ml)
| Batches of | Before concentration | Concentrating by centrifugation (10 times) | Hollow fiber concentration (10 times) |
| 1 | 42.9 | 347 | 364 |
| 2 | 47.3 | 361 | 372 |
| 3 | 54.5 | 395 | 412 |
4 conclusion
Through large-scale culture process research, the optimal culture conditions of the culture processes of the type A bacteria Pm-TJ strain and the type B bacteria C45-2 strain of the pasteurella multocida are determined: the inoculation amount is 1 to 2 percent at the culture temperature of 37 ℃; the aeration method comprises culturing type A bacteria and type B bacteria with small aeration amount, and gradually increasing aeration amount, wherein the maximum aeration amount should not exceed 0.5m3h/10L; the optimal culture time is 6-10 hours. As a result, the culture time of the conventional type B bacteria in a single seedling is shortened to 6 to 10 hours (12 to 24 hours) in terms of the culture time of the type B bacteria.
Through research on a concentration and purification process, the bacteria liquid is determined to be concentrated and purified by a centrifugal or hollow fiber method for subsequent inactivation and seedling preparation.
Example 5 comparative testing of vaccines prepared according to the invention with like products
1 Material
1.1 vaccine A bivalent inactivated vaccine against pasteurella multocida disease (model A Pm-TJ strain + model B C45-2 strain) prepared by the method of example 1 was obtained with batch number ZM 201401.
Inactivated vaccine against pasteurella multocida disease (type B bacterial single vaccine): produced by Lanzhou biological pharmaceutical factory, Zhongmu industries, Ltd, with a batch number of 1407001; xinjiang Tiankang animal husbandry biotechnological corporation, lot number 2014001
1.2 healthy susceptible cattle of 6 months old or more and rabbits with the weight of 1.5-2.0 kg are tested animals.
1.3 virulent bovine pasteurella multocida capsule type B C45-2 strain (CVCC 44502) for challenge, provided by Lanzhou biological pharmaceutical factory, Zhongmu industries, Ltd.
2 test method
2.1 immunization and challenge of cattle test cattle were each intramuscularly inoculated with the bivalent inactivated vaccine, a single vaccine of type B bacteria produced by Lanzhou, Zhongsheguo, Lanzhou, and Xinjiang Tiankang, at an injection dose of 1 part (2.0 ml/head), and 1 part (4.0 ml/head) of the other 2 batches of commercial similar products, each 4-head of vaccine was inoculated, and 3 cattle were simultaneously used as a control group. And (3) after 21 days of immunization, all the cattle are detoxified, each cattle is injected with 10MLD C45-2 virulent strain liquid, observed for 14 days, and the death condition of the cattle is recorded.
2.2 immunization and challenge of rabbits 5 rabbits were immunized subcutaneously at a dose of 0.5 ml/rabbit, and after 21 days, 1MLD of virulent C45-2 strain was subcutaneously injected into each of 5 control rabbits under the same conditions, and the rabbits were observed for 8 days to record the death status of the rabbits. Respectively inoculating and immunizing 4 rabbits subcutaneously with B-type bacteria single vaccine produced by Lanzhou factory and Xinjiang Tiankang in the middle grazing range at a dose of 1.0ml, and using the other 2 rabbits as controls; after 21 days of inoculation, 2 control rabbits with the same conditions were injected subcutaneously with 1MLD of C45-2 virulent strain of bacteria for each rabbit, observed for 8 days, and the death of the rabbits were recorded.
3 results
The product and 2 commercial single-seedling products are used for combating poison 21 days later after the cattle are immunized, and the results are shown in a table 14. Compared with the control cattle which are all dead after being attacked by poison, the protective rate of the product to the cattle is 3/4 compared with the B type bacteria single seedling produced by Lanzhou factory and Xinjiang Tiankang, which shows that the product has no obvious difference with the similar product to the immune protection provided by the B type bacteria under the attack of poison.
TABLE 14 comparative potency test results for congener products (bovine potency test)
The product and similar products are used for immunizing rabbits for 21 days and then are subjected to toxicity attack, and the results are shown in a table 15. The protective rate of the product after challenge of the immune rabbits is 4/5, and control rabbits all die; the protection rate of B-type bacteria single vaccine immune rabbits produced by Lanzhou factories and Xinjiang Tiankang in the middle grazing department after toxin attack is 3/4, and control rabbits all die. The results show that the product and 2 similar products all meet the requirements of various effectiveness tests.
TABLE 15 comparative potency test results for congeners (rabbit potency test)
3 conclusion
As the pasteurella multocida capsular type A bacteria and capsular type B bacteria have no cross protection, and the commercialized like products of the product are not available in the current market, the selection of the existing commercialized B type bacteria single vaccine and the product to carry out the immune effect comparison test aiming at the B type bacteria are feasible. The test results show that the bivalent vaccine prepared by the method in the embodiment 1 of the invention has the same immune protection capability on animals under the virus attack of the B-type bacteria as other commercialized B-type bacteria single vaccines.
EXAMPLE 6 correlation of the efficacy test results of the vaccine prepared according to the present invention on the animal and the experimental animal
1 Material
1.1 vaccines
The bovine pasteurella multocida bivalent inactivated vaccine prepared by the method in the example 1 (A type Pm-TJ strain + B type C45-2 strain) has the batch numbers of ZM201401, ZM201402 and ZM 201403. 1.2 test animals
Healthy susceptible cattle of 6 months old or older. SPF-grade BALB/c mice weighing 18-22 g. The weight of the rabbit is 1.5-2.0 kg.
2 method
2.1 this animal efficacy test
The vaccine preparations of 3 batches were each inoculated to 8 test cattle at a dose of 1 part (2.0ml) of the neck muscle per batch, while 6 cattle under the same conditions were used as controls. Performing challenge 21 days after immunization, randomly selecting 4 immune cattle from 8 cattle immunized by each batch of vaccine, performing challenge with 3 control cattle by adopting cattle pasteurella multocida capsular type A Pm-TJ strain, and instilling 2MID type A bacteria liquid in each trachea; in addition, 4 immune cattle and 3 control cattle adopt cattle pasteurella multocida capsular B type C45-2 strain to detoxify, and each cattle is injected with 10MLD B type virulent bacterial liquid. Observing the clinical symptoms of the cattle subjected to type A bacteria challenge, and recording the body temperature at the same time every day; and (5) performing autopsy on the immunized cattle and the control cattle on the 10 th day after the challenge, observing and recording pathological changes of the lung, and analyzing the morbidity and the protection condition of the cattle. And (5) carrying out cattle with B-type bacteria for counteracting the toxin, observing for 14 days after counteracting the toxin, and recording the death condition of the cattle.
2.2 laboratory animals in place of the animal for efficacy testing correlation tests
2.2.1 mouse efficacy test methods study test
3 batches of vaccine preparations were each intramuscularly inoculated to 35 mice, each mouse had 1/40 heads of the bovine immune dose, i.e. 50 μ l/mouse; at the same time, 35 mice with the same conditions were used as controls. At 21 days after immunization, the immunized mice and control mice of each vaccine batch were divided into 7 groups, 5 mice/group, and 10 mice/group1、102、103、104、105、106、1077 gradients of MLD A-type bacterial liquid are respectively injected into the abdominal cavity of a group of mice; the observation period is 7 days, the death condition of each group of mice is recorded, and the vaccine mouse efficacy test method is determined according to the mouse protection result.
2.2.2 mice replacement of cattle efficacy test correlation test
The vaccine of 3 batches was intramuscularly inoculated to 5 mice at three doses of 100. mu.l, 50. mu.l and 25. mu.l, respectively, of the bovine immunizing dose, and 5 mice under the same conditions were set as controls. On 21 days after immunization, the immunized mice and the control mice were injected with the type A bacterial liquid into each abdominal cavity at the toxicity attacking dose determined by the test results of 2.2.1, and after the toxicity attacking, the mice were observed for 7 days, and the death condition of each group of mice was recorded. The relevance of the efficacy test of mice in place of cattle was analyzed based on the test results.
2.2.3 Rabbit surrogate bovine potency assay correlation test
The vaccine of 3 batches was inoculated to 5 rabbits in three doses of 1.0ml, 0.5ml and 0.25ml of the bovine immunizing dose by intramuscular injection and subcutaneous injection, respectively, and 5 rabbits in the same condition were used as a control. On 21 days after immunization, 1MLD of C45-2 virulent strain liquid is injected subcutaneously into each of the immunized rabbits and the control rabbits, and after challenge, the death condition is recorded after 8 days. The correlation of the rabbit replacement cattle efficacy test was analyzed based on the test results.
3 results
3.1 the results of this animal efficacy test
The 3 vaccine preparations were used to immunize cattle at a dose of 1 head (2.0ml) and were challenged 21 days later, and the protection results of the challenge are shown in tables 16 and 17. As can be seen from table 16, after challenge with type a bacteria, 3 vaccine batches of immunized cattle were protected at 3/4 or more, and control cattle were all attacked (3/3); as can be seen from Table 17, after challenge with type B bacteria, 3 vaccine batches of immunized cattle were protected at 3/4 or above, and all control cattle died. The result shows that the product has good immune protection to cattle.
TABLE 16 protective results of A-type bacteria challenge after bovine immunization with the product
Note: "-" indicates that the item does not have the corresponding symptom, and "+" indicates that the item has the corresponding symptom.
TABLE 17 protective results of B-type bacteria challenge after immunizing cattle with the product
| Vaccine lot number | Immunization dose | Number of cattle | Counteracting strain | Number of deaths | Protection rate (survival number/toxic number) |
| ZM201401 | 2.0ml | 4 heads | C45-2 Strain | 1 | 3/4 |
| ZM201402 | 2.0ml | 4 heads | C45-2 Strain | 0 | 4/4 |
| ZM201403 | 2.0ml | 4 heads | C45-2 Strain | 0 | 4/4 |
| Control 2 | 0ml | 3 heads | C45-2 Strain | 3 | 0/3 |
3.2 laboratory animals replace the results of the animal's efficacy test correlation test
3.2.1 mouse efficacy test method findings
Immunizing mice with 3 batches of vaccine products at a dose of 50 μ l for cattle immunization, and performing challenge after 21 days, wherein 7 challenge doses (10 doses) are set1～107MLD), 5 mice were challenged per challenge dose, and the results are shown in table 18, from table 18 it can be seen that mice immunized with 3 vaccine preparations were treated with 101～103After the A-type bacteria of MLD detoxify, the mice are completely protected; 104After the A-type bacteria of the CFU are attacked, the survival number of the mice is all 4 or more; 105After the A-type bacteria of the CFU are attacked, the survival number of the mice is 2-3; 106And 107After the A-type bacteria of CFU are attacked, the survival number of the mice is 1 or less. Based on the above results, the efficacy of the vaccine mice was determined by immunizing 5 mice with 1/40 head (50. mu.l) of bovine immunizing dose, and injecting 1.0X 10 intraperitoneally with 5 mice under the same conditions 21 days after immunization4CFU type a bacteria, observed for 7 days, control mice all died and immunized mice protected at least 4.
TABLE 18 protective results of type A bacterial challenge after immunization of mice with the preparations
3.2.2 mice replacement of cattle efficacy test correlation test results
Immunizing mice with 3 batches of vaccine at different doses for 21 days, and injecting the vaccine into abdominal cavity of each mouse by 1.0 × 104The toxicity counteracting results of the type A bacterial liquid of CFU are shown in Table 19. All control mice died, and the mice immunized with 100. mu.l and 50. mu.l of the 3 batches of vaccine had a protection rate of 4/5 or higher, while the mice immunized with 25. mu.l had a protection rate of 1/5 or lower. The test result shows that the vaccine mouse efficacy test method determined by the test result of 3.2.1 has good correlation with the bovine efficacy test method, and the mouse efficacy test method can replace the bovine efficacy test.
Table 19 protection results of A-type bacteria against challenge after different doses of the product were used to immunize mice
3.2.3 Rabbit surrogate bovine potency assay correlation test results
The 3 batches of vaccines were immunized according to different dosages, and the challenge was carried out 21 days later, and the challenge results are shown in table 20. After the immune rabbits of 3 batches of vaccine products are subjected to B-type bacteria challenge, all the control rabbits die, the survival rates of the immune rabbits inoculated by the muscle and subcutaneous two modes at the doses of 1.0ml and 0.5ml are both 3/5 and above, and the survival rates of the immune rabbits inoculated by the muscle and subcutaneous two modes at the doses of 0.25ml are both 2/5 and below. Therefore, according to the above results, the efficacy test method for rabbits for vaccine was determined as follows: the vaccine is inoculated to 5 rabbits through muscle or subcutaneous inoculation of 0.5ml of bovine immune dose, 1MLD C45-2 virulent strain liquid is injected subcutaneously to each of 5 control rabbits with the same condition 21 days after immunization, after the virulent strain liquid is observed for 8 days, all the control rabbits die, and the immunized rabbits are protected by at least 3. The test result also shows that the rabbit efficacy test method has good correlation with the bovine efficacy test method, and the rabbit efficacy test method can replace the bovine efficacy test.
TABLE 20 protective results of type B bacteria challenge after different dosages of product immunized rabbit
4 conclusion
The present animal efficacy test for the present product can be replaced with mouse and rabbit efficacy tests and the vaccine determined using the criteria of the experimental animal efficacy test as: after 21 days, 5 mice with a weight of 18-22 g were injected intramuscularly with 50. mu.l of each vaccine, and 5 control mice with the same conditions were injected intraperitoneally with 1.0X 104MLD bovine pasteurella multocida Pm-TJ strain virulent strain liquid is observed for 7 days; the control group died entirely and the immune group should have at least 4 protection. 5 rabbits with the weight of 1.5-2.0 kg are injected with 0.5ml of vaccine through each muscle or subcutaneous injection, and after 21 days, 1MLD bovine pasteurella multocida C45-2 virulent strain liquid is injected through each subcutaneous injection together with 5 control rabbits with the same conditions, and the observation is carried out for 8 days. The control group died entirely and the immune group should have at least 3 protections.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (11)
1. The bivalent inactivated vaccine for the bovine pasteurella multocida disease is characterized by comprising an antigen and an adjuvant, wherein the antigen is a bovine pasteurella multocida capsular type A Pm-TJ strain and a bovine pasteurella multocida capsular type B C45-2 strain, the preservation number of the bovine pasteurella multocida capsular type A Pm-TJ strain is CGMCC No.3619, and the preservation number of the bovine pasteurella multocida capsular type B C45-2 strain is CVCC 44502; the bivalent inactivated vaccine for the bovine pasteurellosis multocida disease is prepared by the following method steps:
(1) fermenting and culturing a bovine pasteurella multocida capsular type A Pm-TJ strain and a bovine pasteurella multocida capsular type B C45-2 strain respectively;
the fermentation culture method of the pasteurella multocida capsular B type C45-2 strain in the step (1) comprises the following steps: inoculating the C45-2 strain into a Martin broth culture medium containing 0.1% of lysed blood cell whole blood for preparing vaccine at an inoculation amount of 1-2%, culturing at 35-37 deg.C under gradually increasing ventilation rate for 6-10 hr, wherein the maximum ventilation rate is not more than 0.5m3h/10L; the gradually increased ventilation rate is 0-3 hr and 0.1m3Per hour/10L, and the ventilation volume is 0.3m in 3-6 hours3h/10L, a ventilation of 0.5m after 6 hours3h/10L, and the maximum ventilation amount is not more than 0.5m3/h/10L；
(2) The zymophyte liquid of the two strains is subjected to centrifugation or hollow fiber concentration and purification, and is inactivated;
(3) and (3) inactivating the two bacteria solutions according to a volume ratio of 2:1 mixing, adding adjuvant, mixing, adding thimerosal solution to make its final concentration be 0.005%, and the content of two antigens in the vaccine is not less than 2.0X 109CFU/mL。
2. The bivalent inactivated vaccine for pasteurella multocida disease according to claim 1, wherein the effective content of pasteurella multocida capsular type a Pm-TJ strain is 2.0-10.0 x 109CFU/mL; the effective content of C45-2 strain of bovine pasteurella multocida capsule type B is 2.0-10.0 × 109CFU/mL。
3. The bivalent inactivated vaccine for pasteurella multocida disease according to claim 1, wherein the effective content of pasteurella multocida capsular type a Pm-TJ strain is 6.0 x 109CFU/mL; the effective content of C45-2 strain of bovine pasteurella multocida capsule type B is 4.0 × 109CFU/mL。
4. The preparation method of the bivalent inactivated vaccine for the pasteurellosis multocida disease of the cattle is characterized by comprising the following steps:
(1) fermenting and culturing a bovine pasteurella multocida capsular type A Pm-TJ strain and a bovine pasteurella multocida capsular type B C45-2 strain respectively; the preservation number of the bovine pasteurella multocida capsular type A Pm-TJ strain is CGMCC No.3619, and the preservation number of the bovine pasteurella multocida capsular type B C45-2 strain is CVCC 44502;
the fermentation culture method of the pasteurella multocida capsular B type C45-2 strain in the step (1) comprises the following steps: inoculating the C45-2 strain into a Martin broth culture medium containing 0.1% of lysed blood cell whole blood for preparing vaccine at an inoculation amount of 1-2%, culturing at 35-37 deg.C under gradually increasing ventilation rate for 6-10 hr, wherein the maximum ventilation rate is not more than 0.5m3h/10L; the gradually increased ventilation rate is 0-3 hr and 0.1m3Per hour/10L, and the ventilation volume is 0.3m in 3-6 hours3h/10L, a ventilation of 0.5m after 6 hours3h/10L, and the maximum ventilation amount is not more than 0.5m3/h/10L；
(2) The zymophyte liquid of the two strains is subjected to centrifugation or hollow fiber concentration and purification, and is inactivated;
(3) and (3) inactivating the two bacteria solutions according to a volume ratio of 2:1 mixing, adding adjuvant, mixing, adding thimerosal solution to make its final concentration be 0.005%, and the content of two antigens in the vaccine is not less than 2.0X 109CFU/mL。
5. The preparation method according to claim 4, wherein the fermentation culture method of the pasteurella multocida capsular type A Pm-TJ strain in step (1) comprises the following steps: inoculating Pm-TJ strain into brain heart infusion culture medium for preparing vaccine at 1-2%, culturing at 35-37 deg.C for 6-10 hr with gradually increased ventilation amount not more than 0.5m3/h/10L。
6. The method according to claim 4 or 5, wherein in the step (3), the contents of the two antigens in the vaccine are respectively: effective content of Pasteurella multocida capsular type A Pm-TJ strain is 2.0-10.0 × 109CFU/mL; the effective content of C45-2 strain of bovine pasteurella multocida capsule type B is 2.0-10.0 × 109CFU/mL。
7. The method according to claim 4 or 5, wherein in the step (3), the contents of the two antigens in the vaccine are respectively: the effective content of Pasteurella multocida capsular type A Pm-TJ strain is 6.0 × 109CFU/mL; the effective content of C45-2 strain of bovine pasteurella multocida capsule type B is 4.0 × 109CFU/mL。
8. The process according to claim 4 or 5, wherein both of the bacteria are cultured in the step (1) without agitation during the fermentation culture.
9. The method according to claim 4 or 5, wherein the centrifugation in step (2) is continuous flow centrifugation at 8000-.
10. The method according to claim 4 or 5, wherein the centrifugation in step (2) is performed by continuous flow centrifugation at 10000r/min while maintaining the flow rate of the bacterial suspension at 2500ml/min, collecting bacterial sludge precipitate, and resuspending the precipitate with sterile PBS (pH 7.2-7.4) to obtain 10-fold concentrated bacterial suspension.
11. The method according to claim 4 or 5, wherein the hollow fiber concentration and purification method in step (2) is to concentrate hollow fiber with pore diameter of 0.22 μm, the pressure is controlled within 0.03MPa, and the flow rate is maintained within 300ml/min, so that the hollow fiber is concentrated to 1/10 of original bacterial suspension.
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