Sheep-derived pasteurella multocida and mannheimia haemolytica combined inactivated vaccine

Abstract

The invention relates to the technical field of biology, and particularly discloses a sheep-derived pasteurella multocida and mannheimia haemolytica bivalent inactivated vaccine which is technically characterized in that the bivalent inactivated vaccine comprises an antigen, and the antigen is composed of an inactivated PM-D strain, an MH-2 strain and an aluminum hydroxide adjuvant. The antigen is prepared by uniformly mixing bacterial solutions of an inactivated PM-D strain and an inactivated MH-2 strain with an aluminum hydroxide adjuvant according to a ratio of 1: 1. The invention solves the problem that there is no effective vaccine prevention method for pneumonia caused by pasteurella multocida and mannheimia haemolytica. The bivalent vaccine provided by the scheme of the invention can effectively prevent and control two bacterial diseases at the same time and effectively reduce the infection risk; moreover, according to the health state and visceral organ slice observation, the bivalent vaccine disclosed by the invention can effectively protect host animals; meanwhile, as an inactivated vaccine, the preparation process and technology are convenient and rapid, the cost is low, and the inactivated vaccine is suitable for large-scale production and use.

Description

Technical Field

[0001] The patent of this invention relates to the field of biotechnology, and specifically, to a sheep-derived combined inactivated vaccine against Pasteurella multocida and Mannheimia hemolytica. Background Art

[0002] In order to ensure the basic consumption of residents, it is necessary to accelerate the expansion of beef and mutton production. Under this circumstance, it is particularly important to reduce the occurrence of epidemics and improve breeding efficiency. At present, there are relatively few large-scale sheep farming industries, and they are still mainly free-range. Therefore, it is difficult to implement the scientific breeding concept of prevention over treatment in clinical production. Sheep diseases occur frequently, and bacterial resistance is increasing, causing significant economic losses to farms. Shaanxi is an important goat breeding base in the country with a large number of goats. In recent years, respiratory diseases have become one of the most important diseases that endanger the sheep industry in Shaanxi. Respiratory diseases are not only rapid in onset, highly contagious, but also extremely high in mortality. Among them, Pasteurella multocida and Mannheimia hemolytica are both important pathogens that endanger sheep, greatly increasing the mortality rate of sheep. There is currently no specific two-in-one vaccine available in China, and the prevention and control situation is extremely severe.

[0003] Pasteurella multocida (Pm) belongs to the family Pasteurellaceae, a Gram-negative bacterium with a size of 0.5-2.5 μm × 0.2-0.4 μm. Under an optical microscope, Gram staining shows short rod-shaped or cocciform red bacteria with blunt ends and a slightly convex center. They can exist not only individually, but also in pairs, chains, or even clusters. Wright staining shows strong staining at both poles, no flagella, no spores, and some have peritrichous fimbriae and capsules. The capsule has a clear boundary and is a mucus-like substance. Newly isolated virulent strains have capsules. As the number of passages increases, the capsule disappears and the toxicity decreases accordingly. The capsule colonies are mucous and shiny. After losing the capsule, they become rough colonies. The culture conditions of Pasteurella multocida are relatively strict. It is an aerobic or facultative anaerobic bacterium. The optimal growth conditions are pH: 7.2-7.4 and temperature: 37°C. The bacteria can grow into small gray-white colonies with neat edges on nutrient agar medium containing 5% serum, and can grow into small white colonies without hemolysis on blood agar, and will not grow on MacConkey medium. After 36 hours of culture, Pasteurella multocida can decompose galactose, mannitol, sorbitol, glucose, fructose and sucrose, but mostly does not decompose inositol, rhamnose, salicin and lactose. The methyl red test and VP are negative, and the oxidase, indigo matrix, catalyst, ornithine decarboxylase and catalase tests are positive, and hydrogen sulfide is produced.

[0004] Mannheimia haemolytica (Mh) belongs to the genus Pasteurella, a gram-negative bacterium that is a facultative anaerobic or aerobic bacterium. Its optimal growth temperature is 37°C. Mannheimia haemolytica is polymorphic, and its size and shape can change with the environment. The size is 0.5-2.5μm×0.2-0.4μm. When the environment is suitable, Mannheimia haemolytica is coccobacillus-shaped; when the environment is bad, most of them are linear or strip-shaped. Under an optical microscope, Wright stains the two poles. Gram staining shows short rod-shaped, coccobacillus-shaped red bacteria with blunt ends and a slightly convex center. It can exist not only individually, but also in pairs, chains, or even clusters. It has no flagella and spores but has a capsule. After culturing on BHI agar plates at 37°C for 16 hours, medium-sized grayish-white colonies can be seen; it grows more slowly on MacConkey plates; after growing on 5% sheep blood agar plates for 16 hours, smooth white droplet-like colonies and β-hemolysis can be seen. Hemolytic Mannheimia can ferment sugars such as lactose, glucose, sucrose and maltose after 48 hours of culture; it does not ferment mannose, but ferments mannitol; it does not produce hydrogen sulfide; the urea test is negative, the citrate test is negative, the urease test is negative, and the oxidase test is positive. Hemolytic Mannheimia has poor resistance and can be killed by conventional disinfectants in a few dozen minutes, such as 0.5% formaldehyde, which can kill it in 15 minutes; 0.001% iodine solution, which can kill it in 15 minutes; 0.001% peracetic acid, which can kill it in 10 minutes; 0.05% sodium hydroxide, which can kill it in 20 minutes.

[0005] Since Pasteurella multocida is widely distributed throughout the world, it can infect most mammals, such as humans, goats, seals, sheep, cats, pigs, cattle, rabbits, chickens, sea lions and dogs. The symptoms of infection with this pathogen in different animals are similar but not exactly the same. Infection with Pasteurella multocida in humans mainly causes pneumonia, arthritis, cerebral neuritis and urinary tract infection; infection with Pasteurella multocida in pigs mainly causes swine plague; infection with Pasteurella multocida in cattle mainly causes hemorrhagic septicemia and fibrinous pleuropneumonia; infection with Pasteurella multocida in sheep mainly causes dyspnea, septicemia and pneumonia. At present, the sheep Pasteurella multocida disease reported in the prior art is mostly type A and D, and is closely related to the sheep house environment. Pasteurella multocida is a conditional pathogen that is widely present in the environment. In healthy animals, Pasteurella multocida is widely present in their respiratory tract, but it usually does not cause disease. Animals are prone to infection with Pasteurella multocida only when their immunity is reduced due to stress, sudden temperature changes, or infection with other diseases. After infection, it can cause secondary infection, thus causing serious harm to the animal body. Therefore, when the pathogen is isolated from the animal, it cannot be diagnosed. Only when the characteristic symptoms of Pasteurella multocida occur can it be diagnosed as Pasteurella multocida infection. The disease has no seasonality and is prone to infection in damp and dirty places. It is sporadic or regionally prevalent. Sheep of all ages will be infected, causing serious losses to the sheep industry.

[0006] The susceptible animals of Mannheimia haemolytica are mainly ruminants, such as cattle and sheep. Studies have shown that calves and lambs are more susceptible to the bacteria; light-weight cattle and sheep are more susceptible than heavy-weight cattle and sheep. The epidemic of this disease has no obvious seasonality and is local and sporadic, with a high mortality rate. Ruminants often suffer from Mannheimia haemolytica infection when their immunity is low due to factors such as fatigue, stress and sudden changes in temperature after long-distance transportation. Therefore, Mannheimia haemolytica is also called "transport fever". However, transportation is not considered to be an infectious factor that causes the disease. Dehydration of the animal body during transportation is considered to be an important cause of Mannheimia haemolytica infection. According to research, when cattle from different groups are mixed, the incidence of the disease in the cattle herd is greatly increased, so mixed group feeding is also considered to be one of the important factors that induce the disease. The incubation period of Mannheimia haemolytica is generally 1 to 3 days, and typical pneumonia symptoms appear in about 1 to 2 weeks.

[0007] When sheep are infected with Pasteurella multocida, they will develop sepsis and respiratory diseases. According to the different clinical symptoms of sheep, the infection of this bacteria is usually divided into the most acute type, acute type and chronic type. The three symptoms are also different. The most acute type often occurs in lactating lambs within 3 months of age. They often develop symptoms such as whole body chills, difficulty breathing and weakness, and die quickly within minutes and hours; sheep with acute type are first depressed and then lose their appetite, their respiratory rate accelerates, they cough violently, their body temperature rises to 41-42℃, their conjunctiva is flushed and there is sticky secretion, and bloody secretion will also appear in the nostrils of sick sheep. They will be constipated in the early stage and then start to have diarrhea with watery feces, and eventually die of dehydration; sheep with chronic type usually have loss of appetite and extreme weight loss, edema in the neck and back, sticky nasal discharge, cough, diarrhea and foul-smelling feces, and they are extremely weak before death, with a drop in body temperature and cold limbs. Pasteurella multocida infection in cattle can cause hemorrhagic septicemia in cattle, with an incubation period of 2 to 5 days. It can be divided into three types: pneumonia, edema and septicemia. The pneumonia type mainly manifests as the clinical symptoms of fibrinous pleuropneumonia, mainly manifested as severe coughing in the morning and evening, and the course of the disease is about 5 days; the edema type generally has edema in the throat, neck and chest, and mucosal cyanosis due to difficulty in breathing, and often suffocates to death; the septicemia type often has blood in the urine, feces and nasal fluid, and the body temperature is as high as 42°C. The sick cattle have diarrhea that is initially porridge-like and then watery diarrhea, and the temperature drops accordingly, usually within 1 day. After sheep are infected with Pasteurella multocida, autopsy can reveal light yellow and reddish-yellow exudates in the chest cavity, severe lung bleeding with peanut-sized purulent foci, fibrinoid exudates in the bronchi, bronchioles and lungs, and the cross-section of the lungs is dark red, grayish white and grayish-yellow marble-like in appearance; the spleen becomes soft and enlarged, with small hemorrhages on the surface, and there are yellow-white soybean-sized necrotic foci in the splenic medulla; the liver is swollen and congested, and necrotic foci can be seen when it is opened.

[0008] Mannheimia hemolytica is an important conditional pathogen that often invades the upper respiratory tract of ruminants. In severe cases, it may also cause secondary lower respiratory tract infections. Sick sheep often show symptoms such as depression, decreased appetite, fever, cough, runny nose, purulent eye secretions and difficulty breathing. The bacteria mainly invade the animal lungs and can also cause putrefactive mastitis in sheep, causing necrosis, bleeding and decay of mammary tissue. In the late stage of the disease, systemic toxic complications often occur and may even lead to secondary infection of other diseases, resulting in the death of sheep. It can also cause fibrinous pneumonia and acute hemorrhagic pneumonia in cattle and sheep. The respiratory rate of sick animals increases, their breathing becomes shallow, and they become sluggish after their breathing begins to become disordered. Their body temperature quickly rises to above 42°C, and fibrinous secretions appear, as well as purulent eye secretions. The course of the disease is relatively short, generally 2 to 3 days, and therefore it often causes a high morbidity and mortality rate. Autopsy of the dead sheep showed yellowish effusion in the abdominal and thoracic cavities, severe lung hemorrhage with a clear boundary between the bleeding and non-bleeding areas, lesions often appeared in the lower part of the bilateral pulmonary lobes and apical lobes, the texture became hard and pulmonary edema was present, the cross-section of the lungs was meaty, necrotic foci were obvious, there were fibrinous exudates and neutrophil infiltration in the lungs, bronchioles and bronchi, and fibrin deposition on the pleura.

[0009] Regarding inactivated vaccines, also known as dead vaccines, they are vaccines prepared by culturing a large number of strong or weak strains with immunogenicity and then undergoing special inactivation treatment. After inactivation, the bacteria can lose their infectivity and toxicity, but their immunogenicity can be retained. Inactivated vaccines generally require the addition of adjuvants to better exert the immune effect. According to the addition of different adjuvants, they can be divided into oil adjuvant vaccines, aluminum gel adjuvant vaccines and propolis adjuvant vaccines. Inactivated vaccines usually cause humoral immunity but almost no cellular immunity. Inactivated vaccines are less affected by maternal antibodies, have higher safety, are easy to prepare, store, and transport, and are easy to prepare polyvalent vaccines and combined vaccines, but they require multiple immunizations and immunity is slow to develop.

[0010] Making inactivated vaccines is mainly divided into the following steps. First, a large amount of target pathogens need to be cultivated, usually in a cell culture system or animal model suitable for its growth. For the present invention, a special culture medium needs to be used to cultivate and amplify two pathogens under specific culture conditions. Then, the pathogen is inactivated by chemical methods (such as formaldehyde, β-propiolactone) or physical methods (such as heat treatment, radiation). This process requires accurate control of time and dosage to ensure thorough inactivation without destroying its antigenic determinant. Then, the culture medium residues and other impurities are removed by process steps such as centrifugation, filtration, purification, etc. to improve the purity and safety of the vaccine. In order to enhance immunogenicity, inactivated vaccines are usually mixed with appropriate adjuvants, which play a role in enhancing immune response. Finally, according to strict aseptic operation standards, the vaccine is formulated into a stable preparation, loaded into a predetermined container, and quality control and stability tests are carried out to ensure that the vaccine remains effective during storage and use.

[0011] At present, there is no effective vaccine to prevent sheep respiratory diseases, especially pneumonia caused by Pasteurella multocida and Mannheimia haemolytica. Therefore, this application aims to establish a bivalent inactivated vaccine that can effectively prevent and control diseases caused by these two pathogens at the same time. Summary of the invention

[0012] The purpose of the present invention is to solve the problem that there is no effective vaccine prevention method for pneumonia caused by Pasteurella multocida and Mannheimia haemolytica, and to provide a sheep-derived Pasteurella multocida and Mannheimia haemolytica combined inactivated vaccine.

[0013] The above-mentioned purpose of the present invention is achieved like this:

[0014] The scheme of the present invention provides a sheep-derived combined inactivated vaccine of Pasteurella multocida and Mannheimia haemolytica, wherein the combined inactivated vaccine comprises antigens, which are composed of inactivated PM-D strains, MH-2 strains and aluminum hydroxide adjuvant.

[0015] Furthermore, the antigen is composed of a mixture of inactivated PM-D strain and inactivated MH-2 strain bacterial liquid and an aluminum hydroxide adjuvant in a ratio of 1:1.

[0016] Furthermore, the preparation method of the bivalent inactivated vaccine comprises the following steps:

[0017] S1. Bacteria recovery and culture:

[0018] PM-D and MH-2 strains frozen in a -80°C refrigerator were taken out, thawed, and three-zoned on a 5% blood plate. After constant temperature culture at 37°C for 24 hours, single colonies were picked and inoculated into brain heart infusion (BHI) containing 5% calf serum, and cultured for 12 hours as the primary seed liquid. After that, 10 μL of the primary seed liquid was taken and placed in 500 mL of BHI containing 5% serum, and the plate was strictly sealed and placed in an air shaker at 37°C for 18 hours.

[0019] S2. Preparation of inactivated vaccines:

[0020] Take out the cultured bacterial solution from the 37°C shaker, adjust the centrifuge speed to 7000r / min, centrifuge for 10min, discard the supernatant, resuspend three times with sterile PBS, and adjust the bacterial solution concentration to 2×10 9cfu / mL, add 0.3% formaldehyde solution, strictly seal and place in an air shaker at 37℃, take 100μL of bacterial solution and spread it on the blood plate at 5h, 10h, 15h, 20h, 25h, 30h, 35h and 40h respectively to observe whether there is bacterial growth; take 100μL of bacterial solution and spread it on the blood plate every 5h, and the inactivation is complete if there is no sterile growth on the blood plate; it is determined that the inactivation time of PM-D strain with 0.3% formaldehyde is 25h, and the inactivation time of MH-2 strain with 0.3% formaldehyde is 20h. To ensure the safety of the experiment, the inactivation time is increased by 5h; PM-D strain is inactivated with 0.3% formaldehyde for 30h, and MH-2 strain is inactivated with 0.3% formaldehyde for 25h. After the inactivation is completed, the bacterial solution is evenly mixed with aluminum hydroxide adjuvant in a ratio of 1:1, and the vaccine test is carried out. After passing the test, the inactivated vaccine is prepared.

[0021] The present invention also provides the use of the above-mentioned dual inactivated vaccine in the preparation of goat respiratory disease prevention and control drugs.

[0022] The difficulty and significance of the technical problem solved by the present invention are:

[0023] There are many serological typings of Pasteurella multocida and Mannheimia haemolytica. The serotypes of sheep infected in different regions are also different. There is no vaccine on the market to prevent the epidemic serotypes of these two bacteria. The present invention collects clinical sheep pneumonia samples and separates pathogenic bacteria, cultures the serotype strains with more infections and stronger toxicity, screens the strong strains of the epidemic serotype as vaccine strains, and prepares a combined inactivated vaccine of Pasteurella multocida and Mannheimia haemolytica to respond to the epidemic dominant serotypes, so as to protect production and improve the breeding efficiency of sheep farms.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The solution of the present invention overcomes the problem that there is no combined prevention method for two pathogens, Pasteurella multocida and Mannheimia hemolytica. The specific advantages of the present invention are:

[0026] 1. The bivalent vaccine provided in the solution of the present invention can effectively prevent and control two bacterial diseases at the same time and effectively reduce the risk of infection;

[0027] 2. Based on the observation of health status and organ sections, the bivalent vaccine of the present invention can effectively protect the host animals;

[0028] 3. As an inactivated vaccine, the present invention has a convenient and fast production process and technology with low cost, and is suitable for large-scale production;

[0029] 4. The dual vaccine regimen of the present invention reduces the frequency of vaccination, reduces the stress response and pain of animals, and improves the compliance and acceptance of vaccination;

[0030] 5. The inactivated vaccine of the present invention has lost its pathogen activity and will not reproduce in the host body, so it is safer and suitable for animals with various immune conditions;

[0031] 6. The inactivated vaccine provided in the solution of the present invention can induce a strong and lasting immune response and ensure long-term protection by optimizing the inactivation and adjuvant formula;

[0032] 7. The inactivated vaccine of the present invention has good tolerance to temperature fluctuations, is easy to store and transport, and is suitable for use in areas with limited resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is the antibody level of the mouse test group and the control group in the embodiment of the present invention (ns: no significant difference; *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001);

[0034] Figure 2 is the antibody level of the goat test group and the control group in the embodiment of the present invention (ns: no significant difference; *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001);

[0035] Figure 3 These are organ slices of mice in the control group (HE20×10) in the present invention ((A): lung slice; (B): spleen slice; (C): liver slice; (D): kidney slice);

[0036] Figure 4 These are organ slices of mice in the inactivated vaccine group (HE 20×10) in the embodiment of the present invention ((A): lung slice; (B): spleen slice; (C): liver slice; (D): kidney slice);

[0037] Figure 5 The temperature changes of goats within three weeks after the virus attack in the embodiment of the present invention;

[0038] Figure 6 These are photos of goat organs and corresponding sections (HE 40×10) in the control group of the present invention (A: liver section; B: spleen section; C: lung section; D: kidney section; E: mesenteric lymph node section);

[0039] Figure 7 The photographs of goat organs and corresponding sections (HE 40×10) of the inactivated vaccine group in the embodiment of the present invention (A: liver section; B: spleen section; C: lung section; D: kidney section; E: mesenteric lymph node section). DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0041] The following is combined with Figure 1-7 The implementation of the present invention is described in detail with reference to the accompanying drawings and specific embodiments.

[0042] The embodiment of the present invention provides a sheep-derived Pasteurella multocida and Mannheimia hemolytica combined inactivated vaccine, which includes an antigen. The antigen is composed of a bacterial solution of an inactivated PM-D strain and an inactivated MH-2 strain and an aluminum hydroxide adjuvant mixed in a ratio of 1:1. The combined inactivated vaccine provided by the embodiment of the present invention can be used in the preparation of goat respiratory disease prevention and control drugs.

[0043] Clinical practice shows that the number of pneumonia cases caused by Pasteurella multocida and Mannheimia haemolytica infections is increasing, but there is currently no commercial vaccine available in my country. In addition, there are many serotypes of the two pathogens, and the prevalence of serotypes of Pasteurella multocida and Mannheimia haemolytica varies between different regions. The cross-protection between different serotypes of the two bacteria is poor, and the prevention and control situation is extremely severe.

[0044] In recent years, respiratory pathogens headed by Pasteurella multocida and Mannheimia hemolytica have been rampant in the northwest region. The first step of the present invention is the isolation and identification of pathogens. A large number of sheep in a sheep farm in Yulin City, Shaanxi Province have respiratory diseases, and the onset is extremely rapid. The sick sheep in the farm have symptoms such as accelerated breathing, severe coughing and diarrhea. Some sick sheep die acutely, and 5 sheep die within one day. Autopsy of the dead sheep revealed that there was light yellow liquid exudation in the chest cavity, severe lung bleeding, and the cut surface was dark red, grayish white and grayish yellow marble-like appearance; there were fibrinous exudates in the bronchi and trachea; the spleen was enlarged with necrotic foci; the liver was enlarged with slight congestion, and necrotic foci were also visible. The lungs of the dead sheep were sent back to the laboratory for testing. Through bacterial isolation and purification, Gram staining microscopy, biochemical tests, 16SrRNA gene sequencing and serotype PCR testing, it was finally determined that the cause of the disease in the sheep was infection with type D Pasteurella multocida and type A2 hemolytic Mannheimia, which was consistent with the orange serotype in sheep reported in the literature. A total of 3 strains of type D Pasteurella multocida and 2 strains of type A2 hemolytic Mannheimia were isolated from 5 lung tissues, and no cross-infection of the two pathogens in the same sheep was found.

[0045] The following is a specific experiment of the implementation scheme of the present invention:

[0046] 1 Materials required for invention

[0047] 1.1 Strains

[0048] The strains used in the present invention are all highly toxic epidemic strains separated from naturally infected goats. Pasteurella multocida is numbered as PM-D, and Mannheimia hemolytica is numbered as MH-2.

[0049] 1.2 Experimental animals

[0050] The mice used in the present invention are SPF-grade female Kunming mice weighing 18 to 22 g, and the goats used are 6-8 month-old female goats raised by the Experimental Animal Center of Northwest Agriculture and Forestry University.

[0051] 1.3 Test consumables

[0052] The 4% tissue cell fixative used in the present invention was purchased from Beijing Solebow Technology Co., Ltd.; the formaldehyde solution was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; the aluminum hydroxide adjuvant was purchased from Beijing Biolabo Biotechnology Co., Ltd.; the disposable sterile dispensing syringe was purchased from Pingdingshan Shengguang Medical Products Co., Ltd.; the 96-well plate was purchased from Guangzhou Jiete Biofiltration Products Co., Ltd.; the ELISA antigen coating solution was purchased from Changsha Green Leaf Technology Co., Ltd.; the TMB single-component colorimetric solution, HRP-labeled mouse anti-sheep IgG antibody, washing solution, antibody diluent and stop solution were all purchased from Beijing Solebow Biotechnology Co., Ltd.

[0053] 1.4 Instruments and Equipment

[0054] The micro-volume oscillator was purchased from Qilin Bell Instrument Manufacturing Co., Ltd.; the dissecting instruments were purchased from Shanghai Jinzhong Medical Equipment Co., Ltd.; the high-speed refrigerated centrifuge was purchased from Hunan Michael Experimental Instrument Co., Ltd.; the veterinary electronic thermometer was purchased from Shandong Shangnong Electronic Technology Co., Ltd.; the Nanodrop One ultra-micro-volume spectrophotometer was purchased from Gene Co., Ltd.; the desktop high-speed micro-volume refrigerated centrifuge was purchased from SCILOGEX; the ultrasonic disruptor was purchased from Ningbo Xinzhi Biotechnology Co., Ltd.; and the ELISA reader was purchased from Shanghai Thermo Fisher Scientific Instrument Co., Ltd.

[0055] 2 Vaccine preparation

[0056] 2.1 Bacterial recovery and cultivation

[0057] PM-D and MH-2 strains frozen in a -80°C refrigerator were taken out, and after thawing, three-zone lines were drawn on a 5% blood agar plate. After constant temperature culture at 37°C for 24 hours, single colonies were picked and inoculated into brain heart infusion (BHI) containing 5% calf serum and cultured for 12 hours as the primary seed solution. After that, 10 μL of the primary seed solution was aspirated and placed in 500 mL of BHI containing 5% serum. After strictly sealing, it was placed in an air shaker at 37°C for 18 hours.

[0058] 2.2 Preparation of inactivated vaccines

[0059] Take out the cultured bacterial solution from the 37℃ shaker, adjust the centrifuge speed to 7000r / min, centrifuge for 10min, discard the supernatant, resuspend three times with sterile PBS, adjust the bacterial solution concentration to 2 109CFU / mL, add 0.3% formaldehyde solution, strictly seal and place in a 37℃ air shaker, take 100μL of bacterial solution and apply it to the blood plate at 5h, 10h, 15h, 20h, 25h, 30h, 35h and 40h, and observe whether there is bacterial growth. Take 100μL of bacterial solution and apply it to the blood plate every 5h. If there is no sterile growth on the blood plate, the inactivation is complete. It was determined that the inactivation time of PM-D strain with 0.3% formaldehyde is 25h, and the inactivation time of MH-2 strain with 0.3% formaldehyde is 20h. To ensure the safety of the experiment, the inactivation time is increased by 5h. The PM-D strain was inactivated with 0.3% formaldehyde for 30 hours, and the MH-2 strain was inactivated with 0.3% formaldehyde for 25 hours. After the inactivation was completed, the bacterial solution was mixed with aluminum hydroxide adjuvant in a ratio of 1:1 and the vaccine was tested. If the test was qualified, the inactivated vaccine was prepared.

[0060] 3. Vaccine quality inspection

[0061] 3.1 Shelf life inspection

[0062] The vaccine was placed at room temperature for three months and its properties were observed. The vaccine bacteria still showed stratification and no flocs appeared.

[0063] 3.2 Safety Inspection

[0064] Take 100 μL of the vaccine with adjuvant and spread it on the blood plate to observe whether there is bacterial growth. After confirming the sterile growth, divide the 10 mice evenly into 2 groups, 5 mice in each group, 0.2 mL / mouse. After the inactivated vaccine and sterile PBS were injected into the mice, the mice were slightly depressed one day after vaccination, but quickly returned to normal, and no deaths occurred within 14 days. The inactivated vaccine and sterile PBS were injected into 3 sheep, 2 mL / mouse. Two days before the injection of the vaccine, the body temperature of some sheep rose slightly, but quickly returned to normal, and the rest were normal. In summary, the shelf life test and safety test of the vaccine are qualified.

[0065] 4. Evaluation of immune effect

[0066] 4.1 Animal immunization procedures

[0067] 40 mice were divided into 2 groups, 20 mice in each group. One group was subcutaneously injected with sterile PBS, 0.2 mL / mouse, as the blank control group; the other group was subcutaneously injected with PM-D and MH-2 dual inactivated vaccine, 0.2 mL / mouse. The second immunization was performed on the 14th day after the first immunization, and the vaccine injection dose and method were still 0.2 mL / mouse, subcutaneously injected.

[0068] When the vaccine's immune effect on mice is within a reasonable range, sheep are immunized to determine the specific effect. Ten goats were evenly divided into two groups, with 5 goats in each group. One group was subcutaneously injected with inactivated vaccine in the neck, 2 mL / goat, and the other group was subcutaneously injected with sterile PBS in the neck as a blank control, 2 mL / goat. A second immunization was performed 14 days after the first immunization, and the vaccine injection dose and method were still 2 mL / goat, subcutaneously injected in the neck.

[0069] 4.2 Determination of serum antibodies

[0070] 4.2.1 Serum preparation

[0071] Use a 1mL syringe to collect blood from the mouse heart, 0.4mL / mouse; use a 2mL syringe to collect blood from the goat jugular vein, 1mL / mouse. After the needle hole is removed, gently push the blood into a 1.5mL centrifuge tube, place the centrifuge tube in a 37℃ constant temperature box for 1h, centrifuge at 3000r / min for 10min, aspirate the upper light yellow serum and put it in a new 1.5mL centrifuge tube for use. The serum collected in the early stage is stored at -20℃ and taken out for use after all the serum is collected.

[0072] 4.2.2 Antigen preparation

[0073] PM-D and MH-2 strains were inoculated into 30 mL of brain heart infusion (BHI) containing 5% calf serum, cultured at a constant temperature for 18 h in an air shaker, centrifuged at 6000 r / min for 10 min, the supernatant discarded, and washed three times with sterile PBS. Finally, 10 mL of PBS was added and the bacteria were fully disrupted with an ultrasonic disruptor. After complete disruption, the bacterial protein concentration was determined with an ultra-micro spectrophotometer.

[0074] 4.2.3 ELISA experimental steps

[0075] (1) Add 100 μL of diluted antigen to a 96-well plate, incubate in a 37°C incubator for 1 h, and then transfer to a refrigerator at 4°C for coating for 12 h.

[0076] (2) Shake out the coated antigen, add 300 μL 1× PBST to each well, wash on a microplate oscillator for 1 min, and pat dry. Repeat 5 times.

[0077] (3) Add 100 μL skim milk powder (blocking solution) to each well to block the wells. Place the wells in a 37°C incubator for 1 h. Wash the wells 5 times with 1×PBST and pat dry.

[0078] (4) Add 100 μL of diluted serum to be tested to each well and incubate in a 37°C incubator for 1 h. Wash 5 times with 1× PBST and pat dry.

[0079] (5) Add 100 μL of diluted rabbit anti-sheep HRP-labeled IgG to each well and incubate in a 37°C incubator for 1 h. Wash 5 times with 1× PBST and pat dry.

[0080] (6) Add 50 μL of color development solution to each well, wrap it with tin foil, and place it in a 37°C incubator away from light for 15 min.

[0081] (7) Add 50 μL of stop solution to each well and immediately measure the OD450 value in a microplate reader.

[0082] 4.2.4 Microplate agglutination test

[0083] (1) Heat the serum to be tested in a 56°C constant temperature water bath for 30 min.

[0084] (2) Add 50 μL of sterile PBS to a 96-well plate.

[0085] (3) Add 50 μL of positive serum, negative serum and test serum to a 96-well plate in sequence and dilute them in multiples to arrange the serum concentrations in a gradient of 1:2-1:256. Do not perform this operation on the last column.

[0086] (4) Add 50 μL of diluted antigen into a 96-well plate and set the last column as the antigen control.

[0087] (5) Place the 96-well plate on a microplate shaker for 30 seconds, mix well, and place in a 37°C constant temperature incubator for 20 hours. After taking out, place in 4°C for 1 hour and observe the results.

[0088] (6) No agglutination or a small amount of agglutination (no obvious agglutination) is judged as negative, and most agglutination or complete agglutination is judged as positive.

[0089] (7) Normal standard: There should be no agglutination in the antigen control well, the antibody titer of negative serum should not be higher than 1:4, and the antibody titer of positive serum should not be lower than 1:64; the standard for the serum to be tested to be antibody negative is that the antibody titer is no higher than 1:4, and the antibody titer of positive serum is the maximum dilution multiple for complete agglutination.

[0090] 4.2.5 Mouse antibody level test results

[0091] Blood was collected from the heart and serum was separated on the 7th, 14th, 21st, 28th, 35th and 42nd day after the first immunization. The serum separated in the early stage was stored at -20℃ for later use. After all the serum was collected, the OD of the mouse serum was determined by enzyme-linked immunosorbent assay. 450The results showed that the antibody level after immunization showed a slow growth trend. On the 14th day after immunization, the antibody levels of mice in the test group and the control group began to show significant differences (P < 0.05); and the difference was extremely significant from the 42nd day (P < 0.0001).

[0092] 4.2.6 Goat antibody level test results

[0093] Venous blood was collected and serum was separated on the 0th, 7th, 14th, 21st and 28th day after the first immunization. The serum separated in the early stage was stored at -20℃ for future use. After all the serum was collected, the antibody titer was determined by microplate agglutination test. The results showed that there was no significant difference between the experimental group and the control group on the 7th day after the first immunization; from the 14th day to the 28th day of the first immunization, the difference between the experimental group and the control group was extremely significant (P < 0.001). The antibody level of sheep showed a slow growth after the first immunization, and the antibody level increased rapidly after the second immunization. After reaching a certain value, it tended to a stable level.

[0094] 4.3 Immune protection test

[0095] In the immune protection test of mice on the 45th day after the first immunization, the concentration of D-type Pasteurella multocida and A2-type hemolytic Mannheimia was adjusted to 5×LD 50 , respectively, the inactivated vaccine group and the blank control group were challenged with bacteria, 0.2mL / mouse. The mental state of the mice was observed daily, and the death of the mice within 14 days was recorded. The mice were dissected on the 14th day after the challenge, and the lungs, spleens, livers and kidneys of the mice were collected and fixed with 4% paraformaldehyde. Then they were made into paraffin sections, and the differences in the tissue structures of the lungs, spleens, livers and kidneys between the vaccine group and the control group were observed under a microscope after HE staining. Vaccine protection rate = (1-mortality rate of mice in the vaccine group / mortality rate of mice in the control group) × 100%. The inactivated vaccine has a strong protective effect, with a mortality rate of 15% and a protection rate of 85%. All mice in the blank control group died (as shown in Table 1 below).

[0096] Table 1 Protection of mice by inactivated vaccines

[0097]

[0098] After the infection, all the mice in the control group that were not vaccinated died within one day. After autopsy, red and yellow exudates were found in the chest cavity, and the lungs were severely hemorrhaged. The liver was enlarged, and the texture became brittle and reddish brown. The spleen was enlarged and had bleeding spots. The kidneys were slightly hemorrhaged. The lungs, spleen, liver and kidneys were made into paraffin sections, and the tissues of each organ were subjected to histological examination. Microscopic observation showed that the capillaries were obviously congested, the normal morphology of the alveoli was no longer obvious, and the alveolar cavity was filled with pink serous exudates, red blood cells, lymphocytes and a small amount of detached alveolar epithelial cells; the spleen was congested, hemorrhagic and contained a large amount of inflammatory exudates, and the spleen parenchymal cells, blood vessels and supporting cells were degenerated and necrotic; a large number of red blood cells were also exuded in the liver, and red blood cells accumulated in the hepatic sinusoids and veins; the kidneys were severely congested and hemorrhaged, and a large amount of serous fluid, fibrin and red blood cells were accumulated. The lungs of the dead mice in the blank control group were collected for bacterial isolation and identification, and Pasteurella multocida and Mannheimia hemolytica were detected again.

[0099] On the 14th day after challenge, the mice in the inactivated vaccine group were dissected, and the lungs, spleen, liver and kidneys were made into paraffin sections for histological observation. It was shown that all organs were normal without pathological changes, indicating that the vaccine of the present invention produced a good immune protection effect on mice.

[0100] In the 30th day after the first immunization, the goats were subjected to immune protection test. The concentration of the bacterial solution of D-type Pasteurella multocida and A2-type hemolytic Mannheimia was adjusted to 1×10 9 cfu / mL, and the inactivated vaccine group and the blank control group were challenged with bacteria, 0.2mL / goat. The mental state and death of the goats were observed daily. On the 45th day after the challenge, all the goats in the blank group and the test group were dissected, and the lungs, spleens, livers and kidneys were collected and fixed with 4% paraformaldehyde, and then made into paraffin sections. After HE staining, the differences in the tissue structures of the lungs, spleens, livers and kidneys between the vaccine group and the control group were observed under a microscope (as shown in Table 2 below).

[0101] Table 2 Protection of goats by combined inactivated vaccine

[0102]

[0103] After the virus attack, the body temperature of the goats in the experimental group and the control group was measured every other day and the average value of each group was recorded. The test was continued for three weeks. The goats in the blank control group had an increase in body temperature and respiratory symptoms on the second day after the virus attack, and the temperature dropped on the fourth day. All died within 5 days. The body temperature of the goats in the bivalent inactivated vaccine group began to rise slightly on the fourth day after the virus attack, but began to drop again after 2 days and gradually stabilized.

[0104] After the infection, the goats in the control group developed obvious respiratory symptoms such as coughing and runny nose, and all died within 5 days. After autopsy, yellow jelly-like exudates were found under the skin of the abdomen and chest, and the lungs were suppurated and bleeding; the liver became brittle and reddish-brown; the spleen was enlarged and bleeding; the kidneys were slightly bleeding, and the goats in the immunization group had no obvious symptoms. The organ tissues of the control group were subjected to histological examination. The alveolar cavity was filled with red serous exudates, red blood cells, lymphocytes, and a small amount of detached alveolar epithelial cells, which could not maintain normal morphology; the spleen was severely hemorrhagic, and the interstitium contained a large amount of inflammatory exudates and red blood cells; the liver lesions were not obvious, and a small amount of red blood cells were exuded; the kidneys were severely congested and hemorrhaged, and a large amount of red blood cells were accumulated; the mesenteric lymph nodes were enlarged and had a small amount of bleeding. The lungs of the dead blank control group goats were collected for bacterial isolation and identification, and Pasteurella multocida and hemolytic Mannheimia were re-detected.

[0105] On the 45th day after infection, the goats in the test group were dissected and the lungs, spleen, liver, kidneys and mesenteric lymph nodes were made into paraffin sections for histological observation. The results showed that the goats in the two-combination inactivated vaccine group had no lesions in their organs and no abnormalities in the section observation.

[0106] In the above-mentioned embodiment of the present invention, the chemical method adopted by the present invention inactivates pathogens. The chemical method includes not only the formaldehyde inactivation method provided by the present invention, but also the β-propiolactone inactivation method. In addition, there are also physical fire extinguishing methods such as heat treatment or radiation treatment, but they are relatively dangerous.

[0107] In addition, the vaccine adjuvant used in the present invention is aluminum gel adjuvant, because the adjuvant has low cost, simple mixing method, enhances long-lasting immune response, and is suitable for large-scale production. In addition, there are oil-emulsion adjuvants, but the production cost is relatively high and requires a complex preparation process.

[0108] In summary, through the above-mentioned embodiments of the present invention, the strain selected by the present invention is a highly toxic and common epidemic serotype in sheep farms that is clinically isolated in this laboratory. It is unique to this laboratory and has a very high research value. In addition, the present invention is the first to use an inactivated vaccine method to jointly prevent and treat two important pathogens of goat respiratory diseases, Pasteurella multocida and Mannheimia hemolytica, filling the gap in the prevention and control of the disease in China. In addition, the scheme of the present invention explores and explores the optimal concentration and inactivation time of formaldehyde in the inactivation process, which is convenient for large-scale production. At the same time, clinical trials have shown that the two-unit inactivated vaccine of the present invention has a high antibody level after immunization and an excellent clinical protection effect, can effectively protect susceptible animals, and has a high immune efficacy. In addition, this vaccine has been tested for safety and has high safety.

[0109] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A sheep-derived combined inactivated vaccine against Pasteurella multocida and Mannheimia hemolytica, characterized in that: The dual inactivated vaccine comprises antigens, which are composed of inactivated PM-D strain, MH-2 strain and aluminum hydroxide adjuvant.

2. The sheep-derived combined inactivated vaccine against Pasteurella multocida and Mannheimia hemolytica according to claim 1, characterized in that: The antigen is prepared by uniformly mixing the bacterial liquid of the inactivated PM-D strain and the inactivated MH-2 strain with an aluminum hydroxide adjuvant in a ratio of 1:

1.

3. The sheep-derived combined inactivated vaccine against Pasteurella multocida and Mannheimia hemolytica according to claim 1, characterized in that: The preparation method of the bivalent inactivated vaccine comprises the following steps: S1. Bacteria recovery and culture: PM-D and MH-2 strains frozen in a -80°C refrigerator were taken out, thawed, and three-zoned on a 5% blood plate. After constant temperature culture at 37°C for 24 hours, single colonies were picked and inoculated into brain heart infusion (BHI) containing 5% calf serum, and cultured for 12 hours as the primary seed liquid. After that, 10 μL of the primary seed liquid was taken and placed in 500 mL of BHI containing 5% serum, and the plate was strictly sealed and placed in an air shaker at 37°C for 18 hours. S2. Preparation of inactivated vaccines: Take out the cultured bacterial solution from the 37°C shaker, adjust the centrifuge speed to 7000r / min, centrifuge for 10min, discard the supernatant, resuspend three times with sterile PBS, and adjust the bacterial solution concentration to 2×10 9 cfu / mL, add 0.3% formaldehyde solution, strictly seal and place in an air shaker at 37℃, take 100μL of bacterial solution and spread it on the blood plate at 5h, 10h, 15h, 20h, 25h, 30h, 35h and 40h respectively to observe whether there is bacterial growth; take 100μL of bacterial solution and spread it on the blood plate every 5h, and the inactivation is complete if there is no sterile growth on the blood plate; it is determined that the inactivation time of PM-D strain with 0.3% formaldehyde is 25h, and the inactivation time of MH-2 strain with 0.3% formaldehyde is 20h. To ensure the safety of the experiment, the inactivation time is increased by 5h; PM-D strain is inactivated with 0.3% formaldehyde for 30h, and MH-2 strain is inactivated with 0.3% formaldehyde for 25h. After the inactivation is completed, the bacterial solution is evenly mixed with aluminum hydroxide adjuvant in a ratio of 1:1, and the vaccine test is carried out. After passing the test, the inactivated vaccine is prepared.

4. The sheep-derived combined inactivated vaccine against Pasteurella multocida and Mannheimia hemolytica according to any one of claims 1 to 3, characterized in that: The application of the bivalent inactivated vaccine in the preparation of drugs for the prevention and control of goat respiratory diseases.

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