A bovine Pasteurella multocida capsule type A capsular polysaccharide vaccine and preparation method thereof

Abstract

The invention discloses a bovine Pasteurella multocida capsule type A capsular polysaccharide vaccine and a preparation method thereof, and belongs to the technical field of veterinary vaccines. The provided capsular polysaccharide vaccine is prepared from bovine Pasteurella multocida capsule type A capsular polysaccharide and a vaccine adjuvant made from raw materials including refined Sperm-80 and refined Tween-80. The capsular polysaccharide vaccine can effectively prevent and control bovine cellulose suppurative pneumonia caused by type A Pm, and has good safety, and can be used to provide safe and effective protection for healthy susceptible calves, healthy pregnant cows and healthy milk-producing cows.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary vaccines, and particularly relates to a bovine Pasteurella multocida capsule type A capsular polysaccharide vaccine and a preparation method thereof. Background Art

[0002] Pasteurella multocida (Pm) is a common pathogen that can cause a variety of diseases such as bovine hemorrhagic septicemia and calf pneumonia. The diseases caused by this bacterium are sporadic or endemic, and are mainly characterized by high fever, pneumonia, acute gastroenteritis and extensive systemic bleeding. Pm can be divided into five serotypes, A, B, D, E, and F, according to the different bacterial capsule antigens. The immune cross-protection between the serotypes is low.

[0003] Type A Pm mainly causes pneumonia in calves, with a mortality rate of up to 40%. It mainly causes respiratory system lesions, and diarrhea often occurs in the later stage. The acute type can be transformed into a chronic type. Cattle of any age may be infected with bovine pneumonia caused by type A Pm. The main sources of infection are carrier animals and sick cattle in the disease stage. Bad weather or seasonal changes may cause the disease. The route of infection is direct or indirect contact, and the incubation period of infection is about one week. The symptoms of sick cattle are mainly fever, severe coughing, which is aggravated when the weather turns cold, and nasal discharge is viscous. Through dissection, it was found that the main lesions of bovine pneumonia are respiratory tract and catarrhal inflammation of the upper respiratory tract mucosa; there are necrotic foci in the lungs, connective tissue capsules surrounding the diaphragm and bronchial lymph nodes, and bleeding; bleeding of the true stomach mucosa, swelling and bleeding of the intestinal mucosa.

[0004] So far, vaccination is still the best way to prevent and treat pneumonia caused by Pasteurella mitis in cattle. However, most of the vaccines currently used in my country's cattle herds are against type B Pm. There is no vaccine product that can effectively prevent and control bovine fibrinous suppurative pneumonia caused by type A Pm. Therefore, it is urgent to develop vaccine products against type A Pm. Summary of the invention

[0005] In view of one or more problems existing in the prior art, one aspect of the present invention provides a bovine Pasteurella multocida capsule type A capsular polysaccharide vaccine, which comprises bovine Pasteurella multocida capsule type A capsular polysaccharide and a vaccine adjuvant;

[0006] The vaccine adjuvant is a water-in-oil-in-water emulsion adjuvant, including: 75wt%-85wt% of injection oil, 1wt%-5wt% of refined Siben-80 and 10wt%-24wt% of refined Tween-80; wherein refining refers to treating Siben-80 or Tween-80 with H2O2 until its color no longer changes from dark to light, removing unreacted H2O2 in the product and then filtering.

[0007] In some embodiments, the concentration of the bovine Pasteurella multocida type A capsular polysaccharide is 20-200 μg / ml.

[0008] In some embodiments, the mass ratio of the bovine Pasteurella multocida type A capsular polysaccharide to the vaccine adjuvant is (1-4):1, optionally (1-2):1, and further optionally 1:1.

[0009] In some embodiments, the filtration during the purification is firstly coarse filtration using a filtration device with a filtration pore size of 0.40 μm-0.65 μm, and then fine filtration of the filtrate using a filtration device with a filtration pore size of 0.10 μm-0.22 μm.

[0010] In some embodiments, the injection oil includes injection mineral oil, injection vegetable oil, or a combination thereof.

[0011] In some embodiments, the mineral oil for injection includes white oil.

[0012] In some embodiments, the white oil includes Marcol-52 white oil, Primol 352 white oil, Total 130# white oil, Total 150# white oil, Total 170# white oil, Drakeol-5 white oil, Drakeol-7 white oil, Sonneborn 4# white oil, Sonneborn 10# white oil PARACOS KF40, PARACOS KF50, squalene, and squalane.

[0013] On the other hand, the present invention provides a method for preparing a bovine Pasteurella multocida capsule type A capsular polysaccharide vaccine, which comprises mixing and emulsifying the bovine Pasteurella multocida capsule type A capsular polysaccharide and a vaccine adjuvant in a mass ratio of (1-4):1, so that the concentration of the bovine Pasteurella multocida capsule type A capsular polysaccharide in the mixed solution is 20-200 μg / ml, and obtaining the bovine Pasteurella multocida capsule type A capsular polysaccharide vaccine.

[0014] In some embodiments, the method for obtaining the bovine Pasteurella multocida capsule type A capsular polysaccharide comprises the following operations:

[0015] S1: using lysozyme to enzymatically hydrolyze the culture solution of bovine Pasteurella multocida capsular type A bacteria, collecting the supernatant after centrifugation to obtain a lysate;

[0016] S2: adding a CTAB solution with a final concentration of 1%-10% to the lysate obtained in step S1, and centrifuging to obtain polysaccharide and protein precipitates;

[0017] S3: adding CaCl2 solution to the polysaccharide and protein precipitates obtained in step S2, mixing, centrifuging and collecting the supernatant;

[0018] S4: adding anhydrous ethanol to the supernatant collected in step S3 to a final concentration of 10%-50%, centrifuging and collecting the supernatant overnight;

[0019] S5: adding anhydrous ethanol to the supernatant collected in step S4 to a final concentration of 75%-85%, mixing and centrifuging to collect the precipitate, thereby obtaining bovine Pasteurella multocida capsule type A capsular polysaccharide.

[0020] In some embodiments, the vaccine adjuvant is made of raw materials including the following components: 75wt%-85wt% of injection oil, 1wt%-5wt% of refined Siben-80 and 10wt%-24wt% of refined Tween-80;

[0021] The refined Siben-80 is a product obtained by treating Siben-80 in the following manner:

[0022] 1) treating Siben-80 with H2O2 to change the color of the Siben-80 from dark to light, and obtaining a first treated product when the color of the Siben-80 no longer changes;

[0023] 2) removing unreacted H2O2 in the first treated product to obtain a second treated product;

[0024] 3) filtering the second treated product to obtain the refined Siben-80;

[0025] The refined Tween-80 is a product obtained by treating Tween-80 in the following manner:

[0026] (1) treating Tween-80 with H2O2 to change the color of the Tween-80 from dark to light, and when the color of the Tween-80 no longer changes, obtaining a third treated product;

[0027] (2) removing unreacted H2O2 in the third treated product to obtain a fourth treated product;

[0028] (3) filtering the fourth treatment product to obtain the refined Tween-80.

[0029] In some embodiments, the process of preparing the vaccine adjuvant comprises:

[0030] T1: heating 75wt%-85wt% of injection oil to 30°C-40°C;

[0031] T2: Add 1wt%-5wt% of refined Sperm-80 and 10wt%-24wt% of refined Tween-80 to the heated injection oil in step T1, mix well and filter to obtain the vaccine adjuvant.

[0032] The capsular polysaccharide vaccine of bovine Pasteurella multocida capsule type A provided based on the above technical solution is prepared using the capsular polysaccharide of bovine Pasteurella multocida capsule type A and a vaccine adjuvant made from raw materials including refined Siben-80 and refined Tween-80. Compared with vaccines prepared using existing aluminum hydroxide gel adjuvants, SEPPIC ISA206 vaccine adjuvants or oil adjuvants (for example, made from raw materials including Siben-80 and Tween-80), the capsular polysaccharide vaccine provided by the present invention can effectively prevent and control bovine fibrinous suppurative pneumonia caused by type A Pm, and has good safety, and can be used to provide safe and effective protection for healthy susceptible calves, healthy pregnant cows, and healthy dairy cows. DETAILED DESCRIPTION

[0033] In view of the defect that there is no vaccine in the prior art that can effectively prevent and control bovine fibrinous suppurative pneumonia caused by type A Pm, the present invention aims to provide a bovine Pasteurella multocida capsule type A capsular polysaccharide vaccine that can effectively prevent and control bovine fibrinous suppurative pneumonia caused by type A Pm, and provides a preparation method of the capsular polysaccharide vaccine.

[0034] The present invention is described in detail by the following specific embodiments. The terms "first", "second", "third" and "fourth" are used to distinguish similar objects, and are not used as a limitation on a specific order or sequence, nor as a limitation on the number of objects.

[0035] The methods used in the following examples are conventional methods unless otherwise specified. The methods for obtaining various biological materials described in the examples are only to provide an experimental method for obtaining them to achieve the specific disclosed purpose, and should not be a limitation on the source of the biological materials of the present invention. In fact, the sources of the biological materials used are extensive, and any biological materials that can be obtained without violating laws and moral ethics can be replaced and used according to the instructions in the examples.

[0036] The embodiments are implemented on the premise of the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. The embodiments will help to understand the present invention, but should not be used as a limitation to the contents of the present invention.

[0037] Example 1: Fermentation and extraction of bovine Pasteurella capsular type A bacteria (strain provided by Jinyu Baoling Biological Pharmaceutical Co., Ltd.) and capsular polysaccharide

[0038] 1.1. Fermentation culture of bovine Pasteurella capsular type A

[0039] Inoculate bovine Pasteurella capsule type A into 4ml Martin broth liquid culture medium and culture at 37℃ on a shaker for 13 hours. After the culture is completed, shake the bacterial solution evenly, divide it into 3 or 4 areas and streak it on the Martin agar plate, and culture it in a greenhouse at 37℃ for 13 hours. Under a fluorescence microscope, select 3-5 colonies with good fluorescence and different colony sizes, dilute them in 3ml PBS solution, pour them on the Martin blood slope of a square bottle, evenly spread them, and culture them in a greenhouse at 37℃ for 13 hours. Add 100ml (normal saline) / PBS liquid buffer to each blood plate, blow it repeatedly, scrape the bacteria on the surface of the plate, and adjust the pH to 5.0-6.0 with saturated potassium dihydrogen phosphate. After the bacterial solution culture is completed, take samples for purity inspection according to the appendix of the 2015 edition of the "Chinese Veterinary Pharmacopoeia", which should be pure.

[0040] 1.2 Capsular polysaccharide extraction

[0041] 1.2.1 Enzymatic hydrolysis of bacterial solution: Add lysozyme to the bacterial solution obtained in step 1.1, with a final concentration of 100-500 μg / ml, at a temperature of 20-25°C, and react for 20-30 minutes.

[0042] 1.2.2 Collect the lysed bacterial solution, centrifuge at 8000rpm for 30min, and collect the supernatant.

[0043] 1.2.3 Precipitation of polysaccharides and proteins: Add 5%-30% CTAB solution to the supernatant collected in step 1.2.2 to a final concentration of 1%-10%, mix thoroughly and let stand to form a precipitate, centrifuge at 8000 rpm for 30 min, and collect the precipitate.

[0044] 1.2.4 Protein removal: Add CaCl2 solution with a final concentration of 1 mol / L to the precipitate collected in step 1.2.3, shake on a shaker for 1 hour, centrifuge at 8000 rpm for 30 minutes, and collect the supernatant.

[0045] 1.2.5 Removal of nucleic acid: Add anhydrous ethanol to the supernatant collected in step 1.2.4 to a final concentration of 10%-50% and incubate at 4°C overnight. Centrifuge at 8000 rpm for 30 min and collect the supernatant.

[0046] 1.2.6 Extraction of capsule polysaccharide: Add pre-cooled (4°C) anhydrous ethanol to the supernatant collected in step 1.2.5 again to a final concentration of 80%, and mix thoroughly. After the bacterial capsule polysaccharide is fully precipitated at room temperature, centrifuge at 8000rpm for 30min, and collect the precipitate. Wash the precipitate repeatedly with anhydrous ethanol for 3-5 times. Freeze-dry and store at -80°C to obtain the bovine Pasteurella capsule type A capsular polysaccharide for later use.

[0047] 1.3. Quantification of capsular polysaccharides—phenol-concentrated sulfuric acid method

[0048] Take a certain amount of the capsule polysaccharide sample extracted in step 1.2 and restore it to room temperature, dissolve it in 2ml of sterile deionized water to obtain a capsule polysaccharide solution, and then add 1ml of 2%-10% phenol solution and 5ml of concentrated sulfuric acid, and shake well. Leave it at room temperature for 40min and observe the color change of the mixed solution. Set 2ml of purified water as a blank control and measure the absorbance value at a wavelength of 490nm. Take 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4ml of standard glucose (sucrose) solution respectively, and add water to make up to 2ml each. Add 1ml of 6% phenol solution and 5ml of concentrated sulfuric acid respectively, let it stand for 10min, shake well, and leave it at room temperature for 20min. Set 2ml of purified water as a blank control and measure the absorbance value at a wavelength of 490nm. After conversion of the standard curve, calculate the content of polysaccharides in the extracted capsule polysaccharide sample.

[0049] Example 2: Preparation of vaccine adjuvant and preparation of bovine Pasteurella multocida capsule type A capsular polysaccharide vaccine

[0050] This example prepares a water-in-oil-in-water adjuvant as a vaccine adjuvant, and uses the vaccine adjuvant and an antigen solution of bovine Pasteurella multocida capsule type A capsular polysaccharide (obtained by dissolving the capsular polysaccharide obtained in Example 1 in sterile deionized water) to prepare a bovine Pasteurella multocida capsule type A capsular polysaccharide vaccine at a mass ratio of antigen to adjuvant of 1:1.

[0051] 2.1. The preparation of vaccine adjuvants includes the following steps:

[0052] S1: heating 80 wt% of Marcol-52 white oil to 35° C.;

[0053] S2: Add 3 wt% of refined Sperm-80 and 17 wt% of refined Tween-80 to the heated injection oil in step S1, mix well, and filter (for example, filter with a 0.22 μm filter membrane) to obtain a vaccine adjuvant. Wherein:

[0054] The refined Siben-80 described in step S2 is obtained by the following method:

[0055] 1) Weigh Siben-80 (purchased from Guangzhou Qiaolinger Biotechnology Co., Ltd.) and put it into a stainless steel reactor. Start stirring until it is evenly stirred, then introduce nitrogen to reduce the pressure and raise the temperature to 80±5℃ / 700mmHg. The pressure in the reactor is 0.15-0.25MPa, and H2O2 is slowly added. To control the amount of H2O2 added, a small amount of H2O2 is measured and slowly dripped into it by connecting a vacuum pump. Observe the color change of Siben-80 in the reactor. When the color changes from deep to shallow, stop adding H2O2 and keep the reaction time for 10-15min.

[0056] 2) When the color of the Siben-80 in the reactor no longer changes and becomes light yellow, clear and transparent, heat the reactor to 110±5℃, evacuate and dehydrate, and extract the unreacted excess H2O2 from the reactor. The evacuation and dehydration time is 3-5 minutes.

[0057] 3) Stop vacuuming, continue to introduce nitrogen to maintain pressure and cool down, and then discharge the material. The discharge material is connected to a 0.40μm-0.65μm filter device to coarsely filter the liquid to remove unreacted substances, and then transfer the filtrate to a sterile environment, and filter and sterilize it again with a 0.10μm-0.22μm filter membrane to obtain refined Siben-80.

[0058] The refined Tween-80 described in step S2 is obtained by the following method:

[0059] (1) Weigh Tween-80 (purchased from Guangzhou Qiaolinger Biotechnology Co., Ltd.) and put it into a stainless steel reactor. Stir until it is evenly stirred. Then, introduce nitrogen to reduce the pressure and raise the temperature to 80±5℃ / 700mmHg. The pressure in the reactor is 0.15-0.25MPa. Slowly add H2O2. To control the amount of H2O2 added, measure a small amount of H2O2 and connect a vacuum pump to slowly drip it in. Observe the color change of Tween-80 in the reactor. When the color changes from deep to shallow, stop adding H2O2 and keep the reaction time for 10-15min.

[0060] (2) When the color of Tween-80 in the reactor no longer changes and becomes slightly yellow, clear and transparent, heat the reactor to 110±5°C, evacuate and dehydrate, and remove the unreacted excess H2O2 from the reactor. The evacuation time is 3-5 minutes.

[0061] (3) Stop vacuuming, continue to introduce nitrogen to maintain pressure and cool down, and then discharge the material. The discharge material is connected to a 0.40μm-0.65μm filter device to coarsely filter the liquid to remove unreacted substances, and then the filtrate is transferred to a sterile environment and sterilized by filtration with a 0.10μm-0.22μm filter membrane again to obtain refined Tween-80.

[0062] 2.2. The preparation method of bovine Pasteurella capsule type A polysaccharide vaccine comprises the following steps:

[0063] Take the capsular polysaccharide obtained in Example 1, add sterile deionized water thereto to dissolve, stir at low speed for 5 minutes to obtain a capsular polysaccharide antigen liquid, and then mix the capsular polysaccharide antigen liquid with the vaccine adjuvant prepared in the above step 2.1 according to the mass ratio of antigen (capsular polysaccharide) to vaccine adjuvant of 1:1, stir for 15-20 minutes to fully mix, and obtain bovine Pasteurella capsule type A capsular polysaccharide vaccine, so that the concentration of bovine Pasteurella multocida capsule type A capsular polysaccharide antigen in the prepared vaccine is between 20-200 μg / ml, and the three batches of bovine Pasteurella multocida type A capsular polysaccharide vaccines obtained are named A21001P, A21002P, and A21003P, respectively.

[0064] In this example, the following control vaccines 1-3 of bovine Pasteurella capsule type A capsular polysaccharide vaccines were also prepared simultaneously. They were prepared according to the steps of preparing bovine Pasteurella capsule type A capsular polysaccharide vaccines in step 2.2 above, and the only difference was that different vaccine adjuvants were used, specifically:

[0065] The raw materials of the vaccine adjuvant used in the control vaccine 1 are: 80wt% Marcol-52 white oil, 3wt% Siben-80 and 17wt% Tween-80, which are prepared according to the method of step 2.1 above;

[0066] The vaccine adjuvant used in control vaccine 2 was aluminum hydroxide gel adjuvant;

[0067] The vaccine adjuvant used in control vaccine 3 is SEPPIC ISA206 vaccine adjuvant.

[0068] 2.3. Semi-finished product inspection

[0069] According to the appendix of the 2015 edition of the Chinese Pharmacopoeia of Veterinary Medicine, the three batches of vaccines prepared in 2.2 (A21001P, A21002P, A21003P) and the control vaccines 1-3 were tested for sterility and no sterile growth was observed.

[0070] 2.4. Packaging

[0071] After passing the above sterility test, quantitative packaging should be carried out. Stirring should be done at any time during packaging to ensure uniform mixing. Seal with a stopper and label. Store at 2-8℃.

[0072] Example 3: Efficacy test of bovine Pasteurella capsular type A polysaccharide vaccine

[0073] The bovine Pasteurella multocida type A capsular polysaccharide vaccine prepared in Example 2 (vaccine batch numbers: A21001P, A21002P, A21003P) was used for efficacy test on SPF mice and healthy susceptible cattle. The control vaccines 1-3 prepared in Example 2 were used as controls.

[0074] 3.1. SPF mouse efficacy test

[0075] The experiment was divided into two groups, namely the two-free group and the three-free group. Each vaccine in the two-free group and the three-free group was set with 8 attack dose groups; each group was randomly divided into 5 SPF mice (experimental SPF mice), and the vaccine obtained in the above Example 2 was inoculated into SPF mice through the inner thigh muscle at a dose of 0.2 ml / mouse, and sterilized PBS was inoculated into the blank control group SPF mice in the same way. Each group of SPF mice in the two-free group was subjected to the second immunization 21 days after the first inoculation, and the bovine Pasteurella multocida type A bacteria attack was carried out 7 days after the second immunization; each group of SPF mice in the three-free group was subjected to the second immunization 21 days after the first inoculation, and the third immunization was carried out 7 days after the second immunization, and the bovine Pasteurella multocida type A bacteria attack was carried out 7 days later. After the two-free attack and the three-free attack, each group of SPF mice was continuously observed for 10 days. The results are shown in Tables 1 and 2 below.

[0076] Table 1: Results of the challenge efficacy immunization test of SPF mice in each group after the second immunization

[0077]

[0078] Table 2: Results of the virus-challenging efficacy immunization test of SPF mice in each group of the three-immunization group after the second immunization

[0079]

[0080]

[0081] From the results recorded in Table 1 and Table 2 above, it can be seen that when the number of challenge bacteria exceeds 50 CFU / mouse, all SPF mice in the blank control group become ill, and the test is established. In the two-immunization group and the three-immunization group, when the number of challenge bacteria of bovine Pasteurella multocida capsule type A is less than 5000 CFU / mouse, the three batches of bovine Pasteurella multocida type A capsule polysaccharide vaccines provided by the present invention (vaccine batch numbers are: A21001P, A21002P, A21003P) all show 5 / 5 immune protection effects, and when the number of challenge bacteria of bovine Pasteurella multocida capsule type A is as high as 10000 CFU / mouse, the three batches of bovine Pasteurella multocida type A capsule polysaccharide vaccines provided by the present invention can still provide 4 / 5 immune protection effects. In the triple-immunization group, when the number of bovine Pasteurella multocida capsule type A challenge bacteria was as high as 20,000 CFU / head, the three batches of bovine Pasteurella multocida type A capsular polysaccharide vaccines provided by the present invention could still provide 4 / 5 immune protection effects. As a control, in the double-immunization group and the triple-immunization group, only when the number of bovine Pasteurella multocida capsule type A challenge bacteria was less than 500 CFU / head, the control vaccine 1 and the control vaccine 3 could achieve 5 / 5 immune protection effects. In the double-immunization group, when the number of challenge bacteria of Pasteurella multocida type A was above 5000 CFU / head, control vaccine 1 and control vaccine 3 could not provide effective immune protection (the protection rate was below 3 / 5); while for control vaccine 2, an immune protection effect of 5 / 5 could only be achieved when the number of challenge bacteria of Pasteurella multocida type A was below 100 CFU / head, and it could not provide effective immune protection (the protection rate was below 3 / 5). In the triple-immunization group, control vaccine 1 can achieve a protective effect of more than 4 / 5 only when the number of attacking bacteria of Pasteurella multocida capsule type A is less than 5000 CFU / head; when the number of attacking bacteria of Pasteurella multocida capsule type A is more than 5000 CFU / head, control vaccine 3 cannot provide effective immune protection effect (the protection rate is less than 3 / 5); and for control vaccine 2, it can only provide effective immune protection effect (the protection rate is above 4 / 5) when the number of attacking bacteria of Pasteurella multocida capsule type A is less than 500 CFU / head.

[0082] The above results show that after SPF mice are inoculated with the bovine Pasteurella multocida type A capsular polysaccharide vaccine prepared by the present invention, when the number of challenge bacteria does not exceed 10,000 CFU / mouse, the immune SPF mice can obtain effective protection against the challenge strain (protection rate is above 4 / 5), and compared with the control vaccines 1-3, the bovine Pasteurella multocida type A capsular polysaccharide vaccine provided by the present invention has better immune effect and stable immune protection effect.

[0083] 3.2. Efficacy test on this animal (referring to animals susceptible to Pasteurella multocida in cattle)

[0084] 24 healthy susceptible cows (4-6 months old) were randomly divided into groups of 4 as test cows. The three batches of vaccines (A21001P, A21002P, A21003P) and control vaccines 1-3 were inoculated into one group of test cows through the neck muscle at a dose of 1 head (2.0 ml). Three healthy susceptible cows with the same conditions were inoculated with sterilized PBS in the same way as blank controls. A second vaccination was performed 21 days after vaccination. 14 days after the second vaccination, the 24 test cows and the three blank control cows were challenged with Pasteurella multocida (5×10 9 CFU / head), followed by 10 consecutive days of observation, observation of clinical symptoms of the test cattle and blank control cattle, measurement of body temperature at the same time every day, and autopsy of the test cattle and blank control cattle on the 10th day after the challenge, observation and record of lung pathological changes, analysis and determination of the disease and protection of the cattle. The test results are shown in Table 3 below.

[0085] Table 3: Results of this animal efficacy test

[0086]

[0087] Note: “-” indicates that the corresponding symptom does not occur for this item, and “+” indicates that the corresponding symptom occurs for this item.

[0088] The results in Table 3 above show that after the cattle in the blank control group were challenged with Pasteurella multocida, all three cattle in the group became ill, and the test was established. After the test cattle were challenged with Pasteurella multocida, the A21003 batch vaccine showed an immune protection effect of 4 / 4, A21001 and A21002 showed an immune protection effect of 3 / 4, while the control vaccine 1 and the control vaccine 3 showed an immune protection effect of 2 / 4, and the control vaccine 2 showed an immune protection effect of 1 / 4. The above results show that the capsular polysaccharide vaccine provided by the present invention can effectively protect the immunized cattle against the challenge strain after inoculation of cattle, and the immune effect of the capsular polysaccharide vaccine is stable.

[0089] The experiment also verified the immune protection efficacy of the following bovine Pasteurella capsule type A capsular polysaccharide vaccine (capsular polysaccharide vaccine AD) on SPF mice and healthy susceptible cattle of the animal, and all of them can achieve similar immune protection effects as the three batches of bovine Pasteurella multocida type A capsular polysaccharide vaccines in 3.1 and 3.2 above (vaccine batch numbers: A21001P, A21002P, A21003P), that is, all of them can enable the immunized cattle to obtain effective protection against the attacking strains, and the immune effect is stable:

[0090] Capsular polysaccharide vaccine A: obtained according to the operation steps of 2.2 in the above Example 2, except that the raw materials of the vaccine adjuvant used are: 75wt% Marcol-52 white oil, 1wt% refined Sperm-80 and 24wt% refined Tween-80;

[0091] Capsular polysaccharide vaccine B: obtained according to the operation steps of 2.2 in the above Example 2, except that the raw materials of the vaccine adjuvant used are: 85wt% Marcol-52 white oil, 5wt% refined Sperm-80 and 10wt% refined Tween-80;

[0092] Capsular polysaccharide vaccine C: obtained according to the operating steps of 2.2 in the above Example 2, except that the mass ratio of capsular polysaccharide to vaccine adjuvant in the prepared capsular polysaccharide vaccine is 4:1;

[0093] Capsular polysaccharide vaccine D: obtained according to the operating steps of 2.2 in the above Example 2, except that the mass ratio of capsular polysaccharide to vaccine adjuvant in the prepared capsular polysaccharide vaccine is 2:1.

[0094] In summary, the bovine Pasteurella capsule type A capsular polysaccharide vaccine provided by the present invention may include a vaccine adjuvant whose raw materials are 75wt%-85wt% of injection oil, 1wt%-5wt% of refined Siben-80 and 10wt%-24wt% of refined Tween-80, and bovine Pasteurella capsule type A capsular polysaccharide as an antigen, wherein the mass ratio of bovine Pasteurella capsule type A capsular polysaccharide to the vaccine adjuvant may be (1-4):1 (usually so that the concentration of the antigen in the capsular polysaccharide vaccine is 20-200μg / ml), which can enable the immunized cattle to obtain effective protection against the attacking strain, and the immune effect is stable.

[0095] Example 4: Safety test of bovine Pasteurella capsular type A polysaccharide vaccine

[0096] The bovine Pasteurella multocida type A capsular polysaccharide vaccine prepared in Example 2 (vaccine batch numbers: A21001P, A21002P, A21003P) was used to conduct safety tests on SPF mice and healthy susceptible cattle.

[0097] 4.1 Safety study of single overdose vaccination in SPF mice

[0098] Fifteen SPF mice (18-22g) were randomly divided into three groups as experimental animals, with 5 mice in each group. The three batches of vaccines were subcutaneously inoculated with an overdose of 0.4 ml / mouse. Another group of mice with the same conditions were inoculated with sterilized PBS in the same way as a control. The clinical manifestations of the experimental mice and the control mice were observed for 14 days. The observation results are shown in Table 4 below.

[0099] Table 4: Observation results of safety trial of one overdose vaccination

[0100]

[0101] From the results recorded in Table 4, it can be seen that after the three batches of vaccines were inoculated with a single overdose, the respiratory condition, mental state, appetite, etc. of the test mice were normal during the observation period, no redness, swelling or ulceration was observed at the injection site, and there was no significant difference between the test mice and the control mice, which preliminarily proves the safety of the bovine Pasteurella multocida type A capsular polysaccharide vaccine provided by the present invention.

[0102] 4.2 Safety test of single-dose vaccination in animals

[0103] Three types of cattle, 12 healthy susceptible calves (1-6 months old), 12 healthy milking cows (6-12 months old), and 12 healthy pregnant cows (6-12 months old), were used as test cattle. Each type of cattle was randomly divided into 3 groups, 4 in each group. The three batches of vaccines were inoculated into the test cattle through the neck muscle with a single dose of 2.0 ml (1 head). Three healthy susceptible cows with the same conditions were inoculated with sterile PBS in the same way as controls. The clinical manifestations of the test cattle and control cattle were observed 2 days before inoculation and 15 days after inoculation, and the body temperature was measured at the same time every day. The milk production of healthy milking cows was monitored.

[0104] The results are shown in Tables 5-8 below.

[0105]

[0106]

[0107]

[0108]

[0109] From the results recorded in Tables 5-8, it can be seen that after a single dose of the three batches of vaccines were inoculated, the temperature, respiration, mental state, appetite, etc. of healthy susceptible calves, healthy pregnant cows and healthy dairy cows during the observation period were normal, no swelling or nodule formation was observed at the injection site, and no clinical symptoms of bovine Pasteurellosis multocida were observed in all types of test cows, and there was no significant difference from the control cows. In addition, no dystocia, abortion, stillbirth, etc. were found in healthy pregnant cows, and the milk production of healthy dairy cows remained basically the same before and after vaccination, without significant changes, and there was no significant difference from the control cows.

[0110] 4.3 Safety test of single-dose repeated vaccination in this animal

[0111] The single-dose inoculated test cattle and control cattle in 4.2 above were inoculated again in the same way 15 days after inoculation, and the clinical manifestations of the test cattle and control cattle were observed 2 days before the second inoculation and 15 days after inoculation, and the body temperature was measured at the same time every day, and the milk production of healthy dairy cows was monitored. The results are shown in Tables 9-12 below.

[0112]

[0113]

[0114]

[0115]

[0116] From the results recorded in Tables 9-12, it can be seen that after repeated single-dose vaccination of the three batches of vaccines, the temperature, respiration, mental state, appetite, etc. of healthy susceptible calves, healthy pregnant cows and healthy dairy cows during the observation period were normal, no redness, swelling or nodule formation was observed at the injection site, and no clinical symptoms of bovine Pasteurellosis of various test cows were observed, and there was no significant difference from the control cows. In addition, no dystocia, abortion, stillbirth, etc. were found in healthy pregnant cows, and the milk production of healthy dairy cows remained basically the same before and after the second vaccination, without significant changes, and there was no significant difference from the control cows.

[0117] 4.4. Safety test of overdose vaccination in animals

[0118] Three types of cattle, 12 healthy susceptible calves (1-6 months old), 12 healthy milking cows (6-12 months old), and 12 healthy pregnant cows (6-12 months old), were used as test cattle. Each type of cattle was randomly divided into 3 groups, 4 in each group. The three batches of vaccines were inoculated into the test cattle through the neck muscle with a single dose of 4.0 ml (2 doses). Three healthy susceptible cows with the same conditions were inoculated with sterile PBS in the same way as controls. The clinical manifestations of the test cattle and control cattle were observed 2 days before inoculation and 15 days after inoculation, and the body temperature was measured at the same time every day. The milk production of healthy milking cows was monitored.

[0119] The results are shown in Tables 13-16 below.

[0120]

[0121]

[0122]

[0123]

[0124] From the results recorded in Tables 13-16, it can be seen that after the three batches of vaccines were inoculated with overdoses, the temperature, respiration, mental state, appetite, etc. of healthy susceptible calves, healthy pregnant cows and healthy dairy cows during the observation period were normal, no swelling or nodule formation was found at the injection site, and no clinical symptoms of bovine Pasteurellosis multocida were found in all types of test cows, and there was no significant difference from the control cows. In addition, no dystocia, abortion, stillbirth, etc. were found in healthy pregnant cows, and the milk production of healthy dairy cows remained basically the same before and after the overdosage vaccination, without significant changes, and there was no significant difference from the control cows.

[0125] In summary, a single-dose vaccination test, a single-dose repeated vaccination test, and an overdose vaccination test were conducted on healthy susceptible calves, dairy cows, and pregnant cows using 3 batches of capsular polysaccharide vaccines prepared in Example 2. The test results all showed that during the observation period, the body temperature, respiratory condition, mental state, appetite, etc. of the test cows were normal, no redness, swelling, or nodule formation was observed at the injection site, and no clinical symptoms of bovine Pasteurellosis multocida appeared, which was not significantly different from the control group. In addition, the milk production of dairy cows was not affected by vaccination, and the milk production remained basically the same before and after vaccination. Pregnant cows did not experience miscarriage, dystocia, etc. after vaccination. The above test results show that the capsular polysaccharide vaccine provided by the present invention is safe for all types of test cows (especially calves, pregnant cows, and dairy cows).

[0126] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A bovine Pasteurella multocida capsule type A capsular polysaccharide vaccine, comprising bovine Pasteurella multocida capsule type A capsular polysaccharide and a vaccine adjuvant; The vaccine adjuvant is a water-in-oil-in-water emulsified adjuvant, comprising: 75wt%-85wt% of injection oil, 1wt%-5wt% of refined Siben-80 and 10wt%-24wt% of refined Tween-80; wherein refining means treating Siben-80 or Tween-80 with H2O2 until its color no longer changes from dark to light, removing unreacted H2O2 in the product and then filtering; The bovine Pasteurella multocida capsule type A polysaccharide is obtained by the following operation: S1: using lysozyme to enzymatically hydrolyze the culture solution of bovine Pasteurella multocida capsular type A bacteria, collecting the supernatant after centrifugation to obtain a lysate; S2: adding a CTAB solution with a final concentration of 1%-10% to the lysate obtained in step S1, and centrifuging to obtain polysaccharide and protein precipitates; S3: adding CaCl2 solution to the polysaccharide and protein precipitates obtained in step S2, mixing, centrifuging and collecting the supernatant; S4: adding anhydrous ethanol to the supernatant collected in step S3 to a final concentration of 10%-50%, centrifuging and collecting the supernatant overnight; S5: adding anhydrous ethanol to the supernatant collected in step S4 to a final concentration of 75%-85%, mixing and centrifuging to collect the precipitate, thereby obtaining bovine Pasteurella multocida capsule type A capsular polysaccharide.

2. The bovine Pasteurella multocida capsule type A capsular polysaccharide vaccine according to claim 1, wherein the concentration of the bovine Pasteurella multocida capsule type A capsular polysaccharide is 20-200 μg / ml.

3. The bovine Pasteurella multocida capsule type A capsular polysaccharide vaccine according to claim 1 or 2, wherein the mass ratio of the bovine Pasteurella multocida capsule type A capsular polysaccharide to the vaccine adjuvant is (1-4):

1.

4. The bovine Pasteurella multocida capsule type A capsular polysaccharide vaccine according to claim 3, wherein the mass ratio of the bovine Pasteurella multocida capsule type A capsular polysaccharide to the vaccine adjuvant is (1-2):

1.

5. The bovine Pasteurella multocida capsule type A capsular polysaccharide vaccine according to claim 4, wherein the mass ratio of the bovine Pasteurella multocida capsule type A capsular polysaccharide to the vaccine adjuvant is 1:

1.

6. The bovine Pasteurella multocida capsule type A polysaccharide vaccine according to claim 1 or 2, wherein the filtration during the purification is firstly coarse filtration using a filtration device with a filtration pore size of 0.40 μm-0.65 μm, and the filtrate is then finely filtered using a filtration device with a filtration pore size of 0.10 μm-0.22 μm.

7. The bovine Pasteurella multocida capsule type A capsular polysaccharide vaccine according to claim 1 or 2, wherein the injection oil is selected from mineral oil for injection, vegetable oil for injection or a combination thereof.

8. The bovine Pasteurella multocida capsule type A capsular polysaccharide vaccine according to claim 7, wherein the mineral oil for injection is white oil.

9. The bovine Pasteurella multocida capsule type A capsular polysaccharide vaccine according to claim 8, wherein the white oil is selected from Marcol-52 white oil, Primol 352 white oil, Total 130# white oil, Total 150# white oil, Total 170# white oil, Drakeol-5 white oil, Drakeol-7 white oil and Sonneborn 4# white oil, Sonneborn 10# white oil PARACOS KF40, PARACOS KF50, squalene, and squalane.

10. A method for preparing a bovine Pasteurella multocida capsule type A capsular polysaccharide vaccine, comprising mixing and emulsifying bovine Pasteurella multocida capsule type A capsular polysaccharide and a vaccine adjuvant in a mass ratio of (1-4):1, so that the concentration of the bovine Pasteurella multocida capsule type A capsular polysaccharide in the mixed solution is 20-200 μg / ml, and obtaining the bovine Pasteurella multocida capsule type A capsular polysaccharide vaccine; wherein the method for obtaining the bovine Pasteurella multocida capsule type A capsular polysaccharide comprises the following operations: S1: using lysozyme to enzymatically hydrolyze the culture solution of bovine Pasteurella multocida capsular type A bacteria, collecting the supernatant after centrifugation to obtain a lysate; S2: adding a CTAB solution with a final concentration of 1%-10% to the lysate obtained in step S1, and centrifuging to obtain polysaccharide and protein precipitates; S3: adding CaCl2 solution to the polysaccharide and protein precipitates obtained in step S2, mixing, centrifuging and collecting the supernatant; S4: adding anhydrous ethanol to the supernatant collected in step S3 to a final concentration of 10%-50%, centrifuging and collecting the supernatant overnight; S5: adding anhydrous ethanol to the supernatant collected in step S4 to a final concentration of 75%-85%, mixing and centrifuging to collect the precipitate, thereby obtaining bovine Pasteurella multocida capsule type A capsular polysaccharide; The vaccine adjuvant is made of raw materials including the following components: 75wt%-85wt% of injection oil, 1wt%-5wt% of refined Siben-80 and 10wt%-24wt% of refined Tween-80, wherein refining refers to treating Siben-80 or Tween-80 with H2O2 until its color no longer changes from dark to light, removing unreacted H2O2 in the product and then filtering.

11. The preparation method according to claim 10, wherein the refined Siben-80 is a product obtained by treating Siben-80 in the following manner: 1) treating Siben-80 with H2O2 to change the color of the Siben-80 from dark to light, and obtaining a first treated product when the color of the Siben-80 no longer changes; 2) removing unreacted H2O2 in the first treated product to obtain a second treated product; 3) filtering the second treated product to obtain the refined Siben-80; The refined Tween-80 is a product obtained by treating Tween-80 in the following manner: (1) treating Tween-80 with H2O2 to change the color of the Tween-80 from dark to light, and when the color of the Tween-80 no longer changes, obtaining a third treated product; (2) removing unreacted H2O2 in the third treated product to obtain a fourth treated product; (3) filtering the fourth treatment product to obtain the refined Tween-80.

12. The preparation method according to claim 10 or 11, wherein the operation of preparing the vaccine adjuvant comprises: T1: heating 75wt%-85wt% of injection oil to 30°C-40°C; T2: Add 1wt%-5wt% of refined Sperm-80 and 10wt%-24wt% of refined Tween-80 to the heated injection oil in step T1, mix well and filter to obtain the vaccine adjuvant.

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