Bivalent bacterial ghost vaccine and preparation method thereof

Abstract

The invention relates to the field of vaccine preparation, in particular to a bivalent bacterial ghost vaccine and a preparation method thereof.The method comprises the steps that a mixed viable bacterium suspension of pasteurella multocida and mannheimia haemolytica is obtained; adding sodium hydroxide into the mixed bacterial liquid; standing for a period of time, and centrifugally precipitating to obtain a bigeminy bacterial ghost; and finally, uniformly mixing the bivalent bacterial ghost with an aluminum hydroxide adjuvant to obtain the bivalent bacterial ghost vaccine. The method is simple to operate, low in cost and convenient to popularize. On the basis of collecting a clinical sheep pneumonia sample and separating pathogenic bacteria, a serotype strain with more infection and stronger toxicity is subjected to thallus culture, a virulent strain of an epidemic serotype is screened as a vaccine strain, and a pasteurella multocida and mannheimia haemolytica bigeminy ghost vaccine for coping with an epidemic predominant serotype is prepared; production is protected, and the breeding benefits of the sheep farm are improved.

Description

Technical Field

[0001] The present invention relates to the field of vaccine preparation, and in particular to a combined bacterial vaccine and a preparation method thereof. Background Art

[0002] There are relatively few large-scale sheep farming industries in my country, and they are still mainly free-range. Therefore, it is difficult to implement the scientific farming concept of prevention over treatment in clinical production. Sheep diseases frequently occur, and bacterial resistance is increasing, causing significant economic losses to farms. In recent years, respiratory diseases have become one of the most important diseases that endanger the sheep industry. Respiratory diseases are not only rapid in onset, highly contagious, but also extremely high in mortality. Among them, Pasteurella multocida and Mannheimia haemolytica are important pathogens that endanger sheep, greatly increasing the mortality rate of sheep. However, there is a lack of specific bivalent vaccines on the market to prevent the prevalent serotypes of these two bacteria, and the serotypes of the above two pathogens are more diverse. The prevalence of Pasteurella multocida and Mannheimia haemolytica serotypes varies between different regions, and the cross-protection between different serotypes of the two bacteria is poor. Summary of the invention

[0003] The purpose of the present invention is to provide a dual bacterial ghost vaccine and a preparation method thereof in view of the problem that there is a lack of dual bacterial ghost vaccines for Pasteurella multocida and Mannheimia hemolytica in the existing technology. The present invention, based on the collection of clinical sheep pneumonia samples and the isolation of pathogenic bacteria, cultivates the bacterial bodies of serotype strains with more infections and stronger toxicity, screens the highly toxic strains of the prevalent serotype as vaccine strains, and prepares dual bacterial ghost vaccines for Pasteurella multocida and Mannheimia hemolytica to respond to the prevalent dominant serotypes, thereby protecting production and improving the breeding efficiency of sheep farms.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is:

[0005] A method for preparing a combined bacterial vaccine comprises the following steps:

[0006] Step 1, obtaining a mixed live bacterial suspension of Pasteurella multocida and Mannheimia hemolytica;

[0007] Step 2, adding sodium hydroxide to the mixed bacterial solution;

[0008] Step 3, standing for a period of time, centrifuging and precipitating to obtain the double bacterial shadow;

[0009] Step 4: Evenly mix the combined bacterial ghosts and aluminum hydroxide adjuvant to obtain a combined bacterial ghost vaccine.

[0010] The present invention collects clinical sheep pneumonia samples and separates pathogenic bacteria, cultures the serotype strains with more infections and stronger toxicity, screens the highly toxic strains of the prevalent serotypes as vaccine strains, and prepares a combined vaccine of Pasteurella multocida and Mannheimia hemolytica to respond to the prevalent dominant serotypes, thereby protecting production and improving the breeding efficiency of sheep farms.

[0011] The present invention provides a preparation method of a combined bacterial ghost vaccine, comprising obtaining a mixed live bacterial suspension of Pasteurella multocida and Mannheimia hemolytica; adding sodium hydroxide to the mixed bacterial suspension; standing for a period of time, centrifuging and precipitating to obtain a combined bacterial ghost; and finally uniformly mixing the combined bacterial ghost with an aluminum hydroxide adjuvant to obtain a combined bacterial ghost vaccine. The method is simple to operate, low in cost, and easy to promote.

[0012] Furthermore, in step 1, the number of Pasteurella multocida in the mixed live bacterial suspension is at least 2×10 9 CFU / mL; the number of hemolytic Mannheimia is at least 2×10 9 CFU / mL.

[0013] Furthermore, in step 1, the number of Pasteurella multocida in the mixed live bacterial suspension is 2×10 9 ~3×10 9 CFU / mL; the number of hemolytic Mannheimia is 2×10 9 ~3×10 9 CFU / mL.

[0014] Furthermore, in step 2, the concentration of sodium hydroxide in the mixed bacterial solution is adjusted to 3.75 mg / mL.

[0015] Furthermore, in step 3, the standing time is 60 to 90 minutes.

[0016] Furthermore, in step 4, the volume ratio of the bigelobial shadow to the aluminum hydroxide adjuvant is 1:1.

[0017] Another object of the present invention is to provide a product prepared by the preparation method of the above-mentioned combined bacterial vaccine.

[0018] Such as the bivalent bacterial ghost vaccine prepared by the above-mentioned method for preparing the bivalent bacterial ghost vaccine.

[0019] Furthermore, 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 pili 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 is reduced 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.

[0020] 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 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 cow has diarrhea that is initially porridge-like and then watery, and the temperature drops accordingly, and usually dies 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.

[0021] 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. In humans, infection with Pasteurella multocida mainly causes pneumonia, arthritis, brain 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 my country is mostly type A and D, and is closely related to the environment of the sheep house. 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0026] The present invention relates to the field of vaccine preparation, and specifically to a dual bacterial ghost vaccine and a preparation method thereof, the method comprising obtaining a mixed live bacterial suspension of Pasteurella multocida and Mannheimia hemolytica; adding sodium hydroxide to the mixed bacterial liquid; standing for a period of time, centrifuging and precipitating to obtain the dual bacterial ghost; finally, mixing the dual bacterial ghost with an aluminum hydroxide adjuvant to obtain the dual bacterial ghost vaccine. The method is simple to operate, low in cost, and easy to promote. Based on the collection of clinical sheep pneumonia samples and the isolation of pathogenic bacteria, the present invention cultivates the bacterial bodies of serotype strains with more infections and stronger toxicity, screens the strong strains of the prevalent serotype as vaccine strains, and prepares the dual bacterial ghost vaccine of Pasteurella multocida and Mannheimia hemolytica to respond to the prevalent dominant serotype, so as to protect production and improve the breeding efficiency of sheep farms. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Scanning electron microscopy and transmission electron microscopy images of bacilli (left: scanning electron microscopy; right: transmission electron microscopy).

[0028] Figure 2 These are scanning electron micrographs of bacterial shadows of Pm-D and MH-2 (left: Pm-D; right: MH-2).

[0029] Figure 3 Transmission electron microscopy images of bacterial shadows of Pm-D and MH-2 (left: Pm-D; right: MH-2).

[0030] Figure 4 This is a graph showing the antibody level test results for the mouse experimental group and the control group.

[0031] Figure 5 This is a graph showing the antibody level test results for the goat test group and the control group.

[0032] Figure 6 These are organ sections of mice in the control group (HE 20×10), (A): lung section; (B): spleen section; (C): liver section; (D): kidney section.

[0033] Figure 7 These are organ sections of mice in the bacterial ghost vaccine group (HE 10×100); (A): lung section; (B): spleen section; (C): liver section; (D): kidney section.

[0034] Figure 8 This is a chart showing changes in body temperature in goats within three weeks after infection.

[0035] Fig. 9 The photos of goat organs in the control group and the corresponding sections (HE 40×10); A: liver section; B: spleen section; C: lung section; D: kidney section; E: mesenteric lymph node section.

[0036] Fig.10 The photos of goat organs and corresponding sections in the bacterial shadow vaccine group (HE 40×10); A: liver section; B: spleen section; C: lung section; D: kidney section; E: mesenteric lymph node section. DETAILED DESCRIPTION

[0037] The present invention will be described in detail below in conjunction with the accompanying drawings.

[0038] 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 used to limit the present invention.

[0039] Example

[0040] 1. Prepare the materials

[0041] In recent years, respiratory pathogens headed by Pasteurella multocida and Mannheimia hemolytica have been rampant in the northwest region. The present invention first separated and identified the pathogens. A large number of sheep in a sheep farm in YL City had respiratory diseases, and the onset was extremely rapid. The sick sheep in the farm had symptoms such as accelerated breathing, severe coughing and diarrhea. Some sick sheep died acutely, and 5 died 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.

[0042] The lungs of dead sheep were sent back to the laboratory for testing, through bacterial isolation and purification, Gram staining microscopy, biochemical tests, 16S rRNA gene sequencing and serotype PCR detection.

[0043] Bacterial isolation and purification:

[0044] Place the lung tissue of the dead sheep in a biosafety cabinet, cut the lesion tissue with sterile scissors, and dip the fresh cut surface into the first zone of the blood plate, and then draw the second and third zones. After the drawing is completed, place the blood plate in a 37℃ constant temperature incubator for 24 hours. After the colony grows, take a single colony on the blood plate for purification culture.

[0045] The results of bacterial isolation and culture showed that the lung samples of dead sheep were cultured in three zones on blood plates containing 5% sheep blood. Two forms of bacteria were cultured under aerobic conditions. The growth was relatively slow. Only pin-tip-sized colonies were visible after 24 hours of culture. Normal-sized colonies were visible after 48 hours of culture in a 37°C constant temperature incubator. There was no difference in the shape of the colonies, all of which were smooth and round small colonies. The color of the colonies was slightly different, namely white translucent small colonies and white slightly transparent small colonies. Both colonies had wire drawing phenomenon, and the degree of wire drawing was different. Gram staining microscopy showed that they were all Gram-negative bacteria, and they were all club-shaped and short rod-shaped. Some existed in pairs, clusters or chains, and some existed alone.

[0046] Gram stain microscopy:

[0047] (1) Smear fixation: drop 10 μL of sterile saline solution on a glass slide, use an inoculation loop to pick up a small amount of bacteria, and evenly spread it in the saline solution (thin and even). After it dries naturally, place it above an alcohol lamp and fix it 2 to 3 times (it is best not to get hot).

[0048] (2) Primary staining: Add ammonium oxalate crystal violet solution on top of the fixed bacteria, leave it for 1 min, and then slowly rinse it off with distilled water (be gentle when rinsing with distilled water).

[0049] (3) Mordanting: Add one drop of iodine solution on the bacterial cells, leave it on for 1 minute, and then slowly rinse it off with distilled water.

[0050] (4) Decolorization: Add 95% ethanol on top of the bacteria for decolorization, leave it for 30 seconds, and then slowly rinse it off with distilled water.

[0051] (5) Re-staining: Add a drop of safranin solution on top of the bacteria for re-staining, leave it on for 1 min, and then slowly rinse it off with distilled water.

[0052] After staining, the slides were placed under a microscope for observation, and all were found to be Gram-negative bacteria, all in the shape of clubs or short rods, some in pairs, clusters or chains, and some in singles.

[0053] Biochemical tests:

[0054] Take 16 biochemical reaction tubes (oxidase, esculin, catalase, indole, urease, citrate, ornithine decarboxylase, sorbitol, β-galactoside, glucose, lactose, sucrose, indigo matrix, maltose, urea and mannitol) and inoculate the purified single colonies into the 16 biochemical reaction tubes respectively. Seal the biochemical reaction tubes with sterile liquid paraffin, place them vertically in an incubator at 37°C and culture for 24 hours. Compare the test results according to the Bergey Manual of Bacterial Identification. By isolating, purifying and culturing the lung samples, and observing the colony morphology and Gram-stained bacterial characteristics of the colonies on the blood agar plate of 5% sheep blood, it was preliminarily suspected that the isolated strains were Pasteurella multocida and Mannheimia hemolytica. They were verified by biochemical tests. Single colonies of the five strains were inoculated into biochemical test tubes and cultured at 37°C for 24 hours. The results were then judged according to the "Bacterial Microbiochemical Reaction Tube Result Judgment Table". Some strains met the biochemical characteristics of Pasteurella multocida, and some strains met the biochemical characteristics of Mannheimia hemolytica.

[0055] 16S rRNA gene sequence sequencing:

[0056] Bacterial DNA was extracted using a bacterial genomic DNA extraction kit purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. The procedure was performed according to the instructions attached to the kit.

[0057] Synthesize 16S rRNA universal primers, upstream primer: 5′-AGAGTTTGATCCTGGCTCAG-3′; downstream primer: 5′-GGTTACCTTGTTACGACTT-3′. The size of the target fragment of the strain used in this test is 1386 bp.

[0058] The elution buffer TE was used as a template for gene amplification. The total volume of the system was 20 μL (see Table 2-2 for specific ratios). After the ratios were completed, PCR detection was performed. The PCR program was: ① 94°C, 5 min; ② 94°C, 30 s; ③ 55°C, 30 s; ④ 72°C, 1 min; ⑤ 72°C, 10 min. ②-④ were cycled 36 times. After the polymerase chain reaction was completed, 10

[0059] 10 μL of the sample was used for gel electrophoresis test. After the gel was run, the results were observed using the Geno Sens 2000 gel imaging system. After confirming the presence of bands, the remaining 10 μL was sent to Xi'an Qingke Biotechnology Co., Ltd. for testing. After the test results were fed back, the gene sequence was pasted into the BLAST section of NCBI for comparison to determine the type of the tested bacteria.

[0060] Serological PCR test:

[0061] According to the published literature, the identification primers of the serotypes of Pasteurella multocida PM-D and Mannheimia haemolytica MH-2 were designed and synthesized. The primer sequences are PM-D: upstream primer (5′-TTACAAAAGAAAGACTAGGAGCCC-3′), downstream primer (5′-CATCTACCCACTCAACCATATCAG-3′); MH-2: upstream primer (5′-GGCATATCCTAAAGCCGT-3′), downstream primer (5′-AGAATCCACTTATTGGGCACC-3′).

[0062] It was finally determined that the cause of the disease in the sheep on this farm was infection with Pasteurella multocida type D and Mannheimia hemolytica type A2, which was consistent with the orange serotype in sheep reported in the literature.

[0063] 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.

[0064] 1.2 Experimental animals

[0065] 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.

[0066] 1.3 Test consumables

[0067] 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.

[0068] 1.4 Instruments and Equipment

[0069] 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.

[0070] 2 Vaccine preparation

[0071] 2.1 Bacterial recovery and cultivation

[0072] 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.

[0073] 2.2 Preparation of bacterial ghost vaccine

[0074] 2.2.1 Determination of the minimum inhibitory concentration (MIC) of NaOH

[0075] First, the concentration of the PM-D and MH-2 bacterial solutions was adjusted to 2×109 CFU / mL, NaOH particles were weighed and added to PM-D and MH-2 respectively, and the concentration of NaOH particles in the bacterial solution was adjusted to 30 mg / mL, 15 mg / mL, 7.5 mg / mL, 3.75 mg / mL, 1.875 mg / mL, and 0.9375 mg / mL respectively. They were placed in a 96-well plate in sequence, and each group was repeated 3 times. After being cultured in an incubator at 37°C for 24 hours, the results were taken out for observation, and blood plate smear live bacteria detection was performed to ensure the accuracy of the test.

[0076] The minimum inhibitory concentration of NaOH for PM-D and MH-2 was 3.75 mg / mL, and the time for complete lysis was 75 min. The bacterial cells were lysed into bacterial shadows and observed under scanning and transmission electron microscopes. Figure 1-3 As shown in the figure, under the scanning electron microscope, the bacteria have changed from complete bacilli to bacteria shadows with holes, most of which are in the middle and at the two poles; under the transmission electron microscope, the bacteria have changed from bacilli filled with contents that cannot transmit light to bacteria shadows that are almost transparent with contents flowing out. The lysed bacteria shadows are tested for sterility. If there is no bacterial growth, they can be mixed evenly with aluminum hydroxide adjuvant in a 1:1 ratio. If all indicators are qualified, the bacteria shadow vaccine is successfully prepared.

[0077] 2.2.2 Calculation of bacterial ghost lysis rate

[0078] The concentration of the bacterial solution was adjusted to 2×109 CFU / mL with physiological saline, and then resuspended three times with sterile PBS. The amount of NaOH MIC determined was added to the bacterial solution of PM-D and MH-2, respectively, and cultured at 37°C for 2 hours. During this period, the control group (untreated group) and the NaOH-treated group were taken out from the constant temperature incubator every 20 minutes to calculate the bacterial shadow lysis rate, lysis rate = (1-NaOH-treated bacterial count / control group bacterial count) × 100%. To ensure more accurate test results, each group of experiments was repeated three times.

[0079] 2.2.3 Scanning electron microscopy and transmission electron microscopy observations

[0080] The bacteria of the control group and the bacterial shadows of the vaccine group were fixed with 2.5% glutaraldehyde at 4°C overnight; washed three times with sterile PBS buffer with a pH of 7.2 and a concentration of 0.1 M, each washing for 10 min; dehydrated with 30%, 50%, 70%, 80%, 90% and 100% ethanol, 10 min / time, 100% anhydrous ethanol was repeated twice, and the rest were dehydrated once. The action should be quick, and multiple samples should be operated one by one. Batch operation should not be avoided; after drying and spraying treatment, the samples were observed with a scanning electron microscope.

[0081] Take a clean sterile autoclave bag, draw 10 μL of PM-D and MH-2 bacteria from the control group and 10 μL of PM-D and MH-2 shadows from the vaccine group, and place them on the sterile autoclave bag. Lightly cover the copper mesh surface containing the carbon film above the liquid surface and adsorb for 20 minutes. Wet the edge of the filter paper with pure water, lightly clamp the edge of the copper mesh with tweezers, and lightly touch the wet filter paper to the edge of the copper mesh to absorb excess sample. Take a clean sterile autoclave bag, draw 10 μL of pure water and place them on the sterile autoclave bag. Lightly cover the copper mesh surface containing the carbon film above the liquid surface and place for 1 m. in (repeat twice); wet the edge of the filter paper with pure water, lightly clamp the edge of the copper mesh with small tweezers, touch the wet filter paper to the edge of the copper mesh, and absorb excess water (repeat twice); take a clean sterile autoclave, and in the absence of light, take 20 μL of uranyl acetate and place it on the sterile autoclave, lightly cover the copper mesh containing the carbon film above the liquid surface, and stain for 30 seconds; wet the edge of the filter paper with pure water, lightly clamp the edge of the copper mesh with small tweezers, and touch the wet filter paper to the edge of the copper mesh to absorb excess uranyl acetate; dry the copper mesh and observe it with a transmission electron microscope.

[0082] The bacteria were observed by scanning electron microscopy and transmission electron microscopy to ensure that the bacterial shadows were successfully prepared and then mixed evenly with aluminum hydroxide adjuvant in a 1:1 ratio for vaccine testing. If the test was qualified, the preparation of the bacterial shadow vaccine was complete.

[0083] 3. Vaccine quality inspection

[0084] 3.1 Shelf life inspection

[0085] 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.

[0086] 3.2 Safety Inspection

[0087] Take 100 μL of the adjuvanted vaccine and spread it on the blood plate to observe whether there is bacterial growth. After confirming the sterile growth, the 10 mice were evenly divided into 2 groups, 5 mice in each group, 0.2 mL / mouse. After the mice were injected with the two-combination bacterial shadow vaccine and sterile PBS, the mice were slightly depressed one day after vaccination, but quickly returned to normal, and no deaths occurred within 14 days. The bacterial shadow 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.

[0088] 4. Evaluation of immune effect

[0089] 4.1 Animal immunization procedures

[0090] 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 bacterial 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.

[0091] 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 injected with bacterial shadow vaccine subcutaneously in the neck, 2 mL / goat, and the other group was injected with sterile PBS subcutaneously 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.

[0092] 4.2 Determination of serum antibodies

[0093] 4.2.1 Serum preparation

[0094] 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.

[0095] 4.2.2 Antigen preparation

[0096] 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.

[0097] 4.2.3 ELISA experimental steps

[0098] (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.

[0099] (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.

[0100] (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.

[0101] (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.

[0102] (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.

[0103] (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.

[0104] (7) Add 50 μL of stop solution to each well and immediately measure the OD450 value in a microplate reader.

[0105] 4.2.4 Microplate agglutination test

[0106] (1) Heat the serum to be tested in a 56°C constant temperature water bath for 30 min.

[0107] (2) Add 50 μL of sterile PBS to a 96-well plate.

[0108] (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.

[0109] (4) Add 50 μL of diluted antigen into a 96-well plate and set the last column as the antigen control.

[0110] (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.

[0111] (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.

[0112] (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.

[0113] 4.2.5 Mouse antibody level test results

[0114] like Figure 4 As shown, cardiac blood was collected and serum was separated on the 7th, 14th, 21st, 28th, 35th and 42nd day after the first immunization, and the serum separated in the early stage was stored at -20℃ for later use. After all the serum was collected, the OD450 of the mouse serum was determined by enzyme-linked immunosorbent assay. The results showed that the antibody level showed a slow growth trend after immunization. On the 14th day of the first immunization, the antibody levels of the experimental group and the control group mice began to show significant differences (P < 0.05); starting from the 21st day, the difference was extremely significant (P < 0.01). Among them, Figure 4 In the table, ns: not significantly different; *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001.

[0115] 4.2.6 Goat antibody level test results

[0116] like Figure 5 As shown in the figure, (Note: ns: no significant difference; *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001) venous blood was collected and serum was separated on the 0th, 7th, 14th, 21st and 28th day after the first immunization, and the serum separated in the early stage was stored at -20℃ for standby use. After all the serum was collected, the antibody titer was determined by microplate agglutination test. The results showed that on the 7th day after the first immunization, there was no significant difference between the experimental group and the control group; 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.

[0117] 4.3 Immune protection test

[0118] On the 45th day after the first immunization of mice, the immune protection test was carried out. The concentration of the bacterial solution of D-type Pasteurella multocida and A2-type hemolytic Mannheimia was adjusted to 5×LD50, and the bacteria were respectively administered to the bacterial shadow vaccine group and the blank control group, 0.2mL / mouse. The mental state of the mice was observed daily, and the death of the mice within 14 days was recorded. On the 14th day after the infection, the mice were dissected, and the lungs, spleens, livers and kidneys of the mice were collected and fixed with 4% paraformaldehyde. Then, paraffin sections were made, 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 vaccine protection of the bacterial shadow vaccine was stronger, with a mortality rate of 15% and a protection rate of 90%. All mice in the blank control group died. The specific data are shown in Table 1.

[0119] Table 1 Protection of mice by bacterial ghost vaccine

[0120] Blank control group Bacterial ghost vaccine group Survival 0 18 Deaths 20 2 Mouse mortality 100％ 10％ Vaccine protection rate Comparison 90％

[0121] 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 hemorrhagic, and a large amount of serous fluid, fibrin and red blood cells were accumulated.

[0122] like Figure 6 and Figure 7 As shown, on the 14th day after the challenge, the mice in the 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.

[0123] On the 30th day after the first immunization of goats, the immune protection test was carried out. The concentration of the bacterial solution of D-type Pasteurella multocida and A2-type hemolytic Mannheimia was adjusted to 1×109 cfu / mL, and the two-bacterial shadow vaccine group and the blank control group were challenged with 0.2 mL / 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 structure 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.

[0124] Table 2 Protection of goats by the combined bacterial vaccine

[0125]

[0126]

[0127] like Figure 8 As shown, 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 body temperature dropped on the fourth day. All died within 5 days. The body temperature of the goats in the combined bacterial vaccine group began to rise slightly after the virus attack and on the sixth day, and then dropped and gradually stabilized.

[0128] like Fig. 9 and Fig.10 As shown in the figure, 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 exudate was 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 exudate, red blood cells, lymphocytes and a small amount of desquamated alveolar epithelial cells, which could not maintain normal morphology; the spleen was severely hemorrhaged, and the interstitium contained a large amount of inflammatory exudate 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.

[0129] On the 45th day after infection, the goats in the experimental 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 combined vaccine group had no lesions in their organs and no abnormalities in the section observation.

[0130] 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 method for preparing a combined bacterial vaccine, characterized in that: The following steps are involved: Step 1, obtaining a mixed live bacterial suspension of Pasteurella multocida and Mannheimia hemolytica; Step 2, adding sodium hydroxide to the mixed bacterial solution; Step 3, standing still, centrifuging and precipitating to obtain a double bacterial shadow; Step 4: Evenly mix the combined bacterial ghosts and aluminum hydroxide adjuvant to obtain a combined bacterial ghost vaccine.

2. The method for preparing the combined bacterial ghost vaccine according to claim 1, characterized in that: In step 1, the number of Pasteurella multocida in the mixed live bacterial suspension is at least 2×10 9 CFU / mL; the number of hemolytic Mannheimia is at least 2×10 9 CFU / mL.

3. The method for preparing the combined bacterial ghost vaccine according to claim 2, characterized in that: In step 1, the number of Pasteurella multocida in the mixed live bacterial suspension is 2×10 9 ~3×10 9 CFU / mL; the number of hemolytic Mannheimia is 2×10 9 ~3×10 9 CFU / mL.

4. The method for preparing the combined bacterial ghost vaccine according to claim 1, characterized in that: In step 2, the concentration of sodium hydroxide in the mixed bacterial solution was adjusted to 3.75 mg / mL.

5. The method for preparing the combined bacterial ghost vaccine according to claim 1, characterized in that: In step 3, the standing time is 60 to 90 minutes.

6. The method for preparing the combined bacterial ghost vaccine according to any one of claims 1 to 5, characterized in that: In step 4, the volume ratio of the bigelobial shadow to the aluminum hydroxide adjuvant is 1:

1.

7. A combined bacterial ghost vaccine prepared by the method for preparing a combined bacterial ghost vaccine according to any one of claims 1 to 6.

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