Swine-derived pasteurella multocida type A strain and application thereof

Abstract

The invention belongs to the field of vaccine microorganisms and application thereof, and particularly relates to screening of an A-type pasteurella multocida vaccine strain and application of the strain. According to the invention, a strain of Pasteurella multocida PmA-115 is separated and screened, and the Pasteurella multocida PmA-115 is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number of the Pasteurella multocida PmA-115 is CCTCC NO: M 20242428. The strain can be applied to preparation of pig pasteurella multocida vaccines.

Description

Technical Field

[0001] The invention belongs to the field of vaccine microorganisms, relates to the screening of a type A Pasteurella multocida vaccine strain, and also relates to the application of the strain. Technical Background

[0002] Pasteurella multocida (Pm) is an important zoonotic pathogen that can infect a variety of animals. It is an important pathogen that causes respiratory diseases in pigs and can cause progressive atrophic rhinitis (porcine atrophic rhinitis) and pneumonia (swine plague). Pneumonia is the main cause of death in nursery and fattening pigs, while porcine atrophic rhinitis can also reduce growth efficiency and feed conversion rate, causing significant economic losses to the pig industry. Its source of infection is difficult to find, and it is generally believed that it is already a carrier before the onset of the disease. Pasteurella multocida mainly includes five capsular serotypes: A, B, D, E, and F. In China, porcine pasteurellosis is mainly caused by type A and type D Pasteurella multocida. In recent years, the number of type F Pasteurella multocida isolated from tissues of pigs with porcine respiratory diseases has gradually increased.

[0003] Although there have been reports on the development of vaccines against Pasteurella multocida type A and type D, the cross-protection effect between the two serotypes of Pasteurella multocida vaccines is poor, and the cross-protection effect between it and other serotypes (such as Pm F type) is still unknown, which brings great difficulties to the prevention and control of porcine pasteurellosis.

[0004] Based on the above reasons, the present invention screened out a highly virulent vaccine strain from the clinical isolates of Pasteurella multocida type A isolated and identified to prepare an inactivated vaccine, and proved that the vaccine has cross protection between porcine type A and type F Pasteurella multocida by immunizing mice. In addition, through the immune protection test on pigs, it was found that the vaccine can protect pigs against the attack of a lethal dose of Pasteurella multocida type A strain, and can also provide partial protection for type F strain, which provides a basis for the clinical prevention and control of diseases caused by porcine Pasteurella multocida. Summary of the invention

[0005] In view of the above problems, one of the purposes of the present invention is to provide a porcine Pasteurella multocida type A strain, which has a strong virulence, 0.8 CFU can completely kill mice, 1×10 8 CFU can completely kill 9-10 week old pigs. The antibodies produced by this strain in mice have cross-protection against the two serotypes of Pasteurella multocida, A and F, and can provide immune protection against both serotypes A and F. At the same time, the inactivated vaccine prepared by this strain can provide complete protection against type A strain (6 / 6, 100%) and partial protection against type F strain (3 / 6, 50%) after immunizing pigs.

[0006] The applicant named the strain Pasteurella multocida, and after identification, the strain was named Pasteurella Sp. PmA-115, and was deposited in China. Wuhan. Wuhan University, China Center for Type Culture Collection on November 4, 2024, with a deposit number of CCTCC NO: M 20242428. The strain was identified as (deposited) alive on November 11, 2024.

[0007] The second object of the present invention is to provide a porcine Pasteurella multocida vaccine, which is an inactivated vaccine containing the above-mentioned strain and Summit Poly Solution water-soluble adjuvant.

[0008] The third object of the present invention is to provide an application method for preparing the porcine Pasteurella multocida vaccine, which comprises: culturing the above-mentioned strain into a bacterial liquid using a TSA medium containing 5% volume of newborn calf serum, then adding 0.3% volume of formaldehyde, inactivating at 37°C for 24 hours, and adding an immune adjuvant after passing the inspection.

[0009] The mouse immune protection test showed that the vaccine provided by the present invention can provide immune protection for mice infected with both serotypes of Pasteurella multocida, Pm A (8 / 8, 100%) and PmF (6 / 8, 75%).

[0010] The immune protection test on pigs showed that the vaccine provided by the present invention can provide complete protection for Pm A (6 / 6, 100%) and 50% (3 / 6) protection for Pm F. However, another inactivated vaccine of a virulent strain of Pm F (such as Pm F-19) can only provide immune protection for Pm F serotype.

[0011] For more detailed schemes, please refer to the specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 PCR identification result of Pasteurella multocida. Explanation of the accompanying symbols: Wherein: M: DL2000maker; 1-4: single colonies suspected to be Pm; +: positive control; -: negative control; positive colonies can amplify a 460bp band.

[0013] Figure 2 PCR identification result diagram of capsular type A Pasteurella multocida. Explanation of the accompanying symbols: Wherein: M: DL2000maker; 1-9: clinically isolated Pasteurella multocida; +: positive control; -: negative control; Type A is Pasteurella multocida, which can amplify a 1044 bp band.

[0014] Figure 3Results of antibody identification in mouse serum after immunization with Pm A-114. Explanation of the accompanying figures: "***" indicates that the p value is <0.001, which indicates an extremely significant statistical difference.

[0015] Figure 4 Results of serum antibody determination of mice after immunization with Pm A-115. Explanation of the accompanying figures: "***" indicates that the p value is <0.001, which indicates an extremely significant statistical difference.

[0016] Figure 5 Results of antibody identification in mouse serum after immunization with Pm F-19. Explanation of the accompanying figures: "***" indicates that the p value is <0.001, which indicates an extremely significant statistical difference.

[0017] Figure 6 Results of serum antibody determination of pigs after immunization with Pm A-115. Explanation of the accompanying figures: "***" indicates that the p value is <0.001, which indicates an extremely significant statistical difference.

[0018] Figure 7 Results of identification of serum antibodies in pigs after immunization with Pm F-19. Explanation of the accompanying figures: "***" indicates that the p value is <0.001, which indicates an extremely significant statistical difference. DETAILED DESCRIPTION

[0019] The present invention is described in detail below with reference to specific embodiments.

[0020] In the following examples, the strains involved are all strains isolated by our laboratory and submitted for legal deposit.

[0021] In the following examples, the formula of TSA solid culture medium (with 5% newborn calf serum) is as follows: 16 g of TSA powder is weighed and dissolved in 400 mL of single distilled water, sterilized by high pressure steam at 121° C. for 15 min, cooled to 50° C., 20 mL of newborn calf serum is added, mixed, and poured into a sterile plate for later use.

[0022] The formula of TSB liquid culture medium is as follows: weigh 12g TSB powder and dissolve it in 400mL single distilled water, sterilize it with high pressure steam at 121℃ for 15min, store it at room temperature after sterilization, and add newborn calf serum to a final concentration of 5% when using it.

[0023] PBS buffer: 8.0 g NaCl, 0.2 g KCl, 1.56 g Na2HPO4, 0.2 g KH2PO4, add them to 800 mL of double distilled water in sequence. After they are completely dissolved, dilute to 1000.0 mL with double distilled water. Add HCl to adjust the pH to 7.8 and store at 4°C for later use.

[0024] PBST buffer: Add Tween-20 to a final concentration of 0.05% to the PBS buffer, mix well and store at room temperature for later use.

[0025] Blocking solution: Weigh 5 g of skim milk powder and completely dissolve it in 100 mL of PBST buffer.

[0026] Coating solution: Weigh 1.59 g Na2CO3 and 2.93 g NaHCO3, add them into 800 mL double distilled water in sequence, and after they are completely dissolved, make up to 1000.0 mL and store at 4°C for later use.

[0027] TMB colorimetric solution and stop solution were purchased from Beijing Solebow Technology Co., Ltd.

[0028] Enzyme-labeled secondary antibodies were purchased from Wuhan Sanying Biotechnology Co., Ltd.

[0029] Summit Poly Solution adjuvant was donated by Wuhan Keqian Biological Co., Ltd. and is a commonly used water adjuvant.

[0030] Example 1

[0031] Screening of Pm A type candidate vaccine strains:

[0032] 1 Isolation and identification of Pasteurella multocida from pigs

[0033] Thirteen Pm A clinical isolates and one Pm F clinical isolate were isolated and identified by our laboratory from lung or liver tissues of pigs with suspected respiratory disease symptoms collected from various regions. The information of the strains is listed in Table 1.

[0034] Table 1 Information on the strains of porcine Pasteurella multocida type A and type F used in the present invention

[0035]

[0036] For sick pigs with suspected symptoms of respiratory diseases, tissue samples were inoculated on TSA plates (with 5% newborn calf serum) under a sterile environment, and divided and marked with a sterile inoculation loop and cultured in a 37°C incubator for 12-24 hours. Single colonies that were grayish white, translucent, round, smooth in surface, neatly edged, moist and sticky on the plate were picked for purification and culture.

[0037] Use a sterile inoculation loop to pick up a single colony suspected of being Pm and place it in TSB (with 5% newborn calf serum) liquid culture medium, then place it in a 37°C shaker and shake at 180r / min for 12-24h. Extract the genomic DNA of the isolated strain, and use primers of the kmt1 gene specific to Pasteurella multocida and the capsule synthesis gene (hyaD-hyaC) of Pasteurella multocida type A to identify the isolated bacteria and the capsule serotype, respectively. The primer sequences for identifying the kmt1 gene are: kmt1-SP6: 5'-GCTGTAAACGAACTCGCCAC-3'; kmt1-T7: 5'-ATCCGCTATTTACCCAGTGG-3', and the expected PCR amplification product size is 460bp. The primer sequences for the hyaD-hyaC gene are: Forward: 5'-TGCCAAAATCGCAGTCAG-3', Reverse: 5'-TTGCCATCATTGTCAGTG-3', and the expected size of the PCR amplification product is 1044 bp.

[0038] The PCR reaction system was as follows: 2×Rapid Taq Master Mix 10μL, sterile single distilled water 7μL, primers Pm-F and Pm-R 1μL each, template 1μL. Reaction conditions: 95℃ pre-denaturation for 5min; 94℃ denaturation for 15s; 56℃ annealing for 15s; 72℃ extension for 30s, 30 cycles; then 72℃ extension for 10min, and finally lowering to 4℃. The PCR product was stored in a 4℃ refrigerator for later use.

[0039] The PCR product was electrophoresed on a 1% agarose gel at 120V for 25 minutes, stained with Goldview, and scanned with a gel imaging system. The image was exported and saved. The results showed that the positive colonies among the four suspected Pm single colonies could amplify a 460bp band. The results are as follows Figure 1 As shown, M: DL2000maker; 1-4: single colonies suspected to be Pm; +: positive control; -: negative control. After the clinical isolates were identified with Pasteurella multocida capsule type A specific primers, a 1044 bp band was amplified, as shown in Figure 2 As shown. Figure 2 M in the table: DL2000maker; 1-9: clinically isolated Pasteurella multocida; +: positive control; -: negative control.

[0040] 2 Virulence test of Pasteurella multocida on mice

[0041] In the early stage, through preliminary experiments, 13 strains were screened out from 80 strains of porcine Pasteurella multocida type A that could 3The test was carried out using the 13 strains of type A and 1 strain of type F porcine Pasteurella multocida in Table 1, of which the LD of type F Pasteurella multocida Pm F-19 has been determined. 50 The dose of the challenge strain in the virulence test was set at 100 CFU based on the results of the preliminary experimental study. After the virulence test, the strains with strong virulence to mice were tested for virulence in pigs. The specific implementation method is as follows.

[0042] The correctly identified strain was revived in TSA solid medium (containing 5% newborn calf serum), cultured in a 37°C incubator for 16 hours, and then a single colony was picked and inoculated in TSB liquid medium containing 5% newborn fetal calf serum, placed in a 37°C shaker, cultured at 180r / min for 12 to 15 hours, and then added to the newly prepared TSB liquid medium (with 5% newborn fetal calf serum added) at a volume ratio of 1:100, and cultured at 37°C, 180r / min for 12 to 15 hours. The bacterial solution was centrifuged (6000r / min, 8min) and resuspended in sterile PBS as the infection bacterial solution, and then diluted in turn to adjust the bacterial amount to 100CFU.

[0043] 70 female BALB / C mice (SPF) aged 6-8 weeks were selected and randomly divided into 14 groups, with 5 mice in each group, of which groups 1-13 were experimental groups and group 14 was the control group. The experimental group was infected by intraperitoneal injection of 0.2 mL of the challenge bacterial solution, and the control group was injected with 0.2 mL of PBS in the same way. After 7 days of infection, the virulence of the strain was judged by the death of mice.

[0044] The results showed (see Table 2) that mice began to die from the 12th hour after the infection. The infected mice showed hemorrhagic inflammatory changes throughout the body. Acutely dead mice showed sepsis changes, with congestion, edema and bleeding in various tissues and organs. The main symptoms of chronically dead mice were depression, slow movement, loss of appetite, inflammatory secretions in the eye sockets, and black congestion in the toes. After PmA-114, Pm A-115, Pm A-193, and Pm A-K12 infected mice, the mice died within 36 hours, so these four strains were selected for the next step of the virulence test of Pasteurella multocida on pigs.

[0045] Table 2 Death of mice infected with Pasteurella multocida

[0046]

[0047] 3 Virulence test of Pasteurella multocida on pigs

[0048] Twenty healthy piglets (conventional breed) aged 8-10 weeks were randomly divided into 5 groups (4 pigs / group), of which groups 1-4 were challenge test groups and group 5 was a blank control group. Before the test, nasal swabs were collected to detect Pm and other respiratory pathogens (HPS, APP, SS, PRV, etc.), blood was collected for Pm antibody detection, and pigs with negative antibodies were selected.

[0049] Single colonies of four strains of type A Pasteurella multocida were inoculated into TSB liquid medium containing 5% fetal bovine serum, and cultured at 37°C, 180r / min for 15h to obtain seed solution. Then, they were inoculated into new TSB liquid medium containing 5% fetal bovine serum at a ratio of 1%, and cultured at 37°C, 180r / min for 6h to obtain the challenge solution. According to the results of the preliminary test, piglets in groups 1-4 were injected with 1×10 9 The fifth group was injected with an equal amount of PBS into the ear vein. After the piglets were challenged with the virus, they were fed with normal piglet feed, and the clinical symptoms and morbidity of the piglets, such as spirit, appetite, hair, joints, excretion, etc., were observed daily for 14 days. The dead piglets were immediately autopsied, and their lungs, livers, spleens, tonsils and other organ tissues were taken for histopathological section analysis.

[0050] The results show (see Table 3) that with 1×10 9 The virus was challenged with the virus liquid of each strain of A-type Pasteurella multocida containing CFU. PmA-114 and Pm A-115 showed strong virulence, and both groups of pigs died within 48 hours after the virus was challenged. The sick pigs showed obvious symptoms of swine plague, including dyspnea, whistling due to poor breathing, swollen throat, cyanosis of skin, and yellow secretions flowing out of the mouth and nose. Obvious symptoms of sepsis were present, with bleeding in multiple tissues and organs, bleeding in lymph nodes; enlarged kidneys, hemorrhage in coronary fat, swollen lungs, black necrotic spots on the surface, and abdominal effusion. After Pm A-193 and Pm A-K12 infected pigs, the main manifestations were anorexia, depression, swollen joints, and lameness of the hind legs, but no death occurred. The results of histopathological examination showed that the serosal membrane on the surface of the lung tissue of the diseased pigs was thickened, accompanied by a small number of inflammatory foci; a large range of alveolar wall thickening was seen, the alveolar structure was unclear, and there was a small amount of inflammatory cell infiltration and shedding of bronchial epithelial cells. Based on the results of virulence test, Pm A-114 and Pm A-115 were determined as candidate vaccine strains for porcine pasteurellosis inactivated vaccine.

[0051] Table 3 Results of the toxicity test of Pasteurella multocida on pigs

[0052]

[0053] Determination of lethal doses of candidate vaccine strains for mice and pigs

[0054] Through virulence tests on mice and pigs, vaccine candidate strains Pm A-114 and Pm A-115 were screened. The minimum lethal dose or median lethal dose of the two strains for mice and pigs was determined through virulence tests. The mouse virulence test method was the same as the "virulence test of Pasteurella multocida on mice". Each group had 5 female BALB / C mice (SPF) aged 6-8 weeks, and the amount of challenge bacteria was adjusted to 10CFU, 0.8CFU, and 0.4CFU. The virulence test on pigs was the same as the "virulence test of Pasteurella multocida on pigs". Each group had 4 healthy piglets aged 8-10 weeks, and the amount of challenge bacteria was adjusted to 2×10 8 CFU, 1×10 8 CFU, 2×10 7 The results showed (Table 4) that the minimum lethal doses of Pm A-114 and Pm A-115 for mice were both lower than 1 CFU. The minimum lethal doses of Pm A-114 and Pm A-115 for pigs were both lower than 1×10 8 CFU (Table 5).

[0055] Table 4 Determination of the minimum lethal dose of candidate vaccine strains to mice

[0056]

[0057] Table 5 Determination of the minimum lethal dose of candidate vaccine strains for pigs

[0058]

[0059] Example 2

[0060] Preparation of inactivated vaccines

[0061] Preparation of bacterial antigens: The correctly identified strains were revived in TSA solid medium (containing 5% newborn calf serum), placed in a 37°C incubator for 16 hours, and then a single colony was picked and inoculated in TSB liquid medium containing 5% newborn fetal calf serum, placed in a 37°C shaker, and cultured at 180r / min for 12 to 15 hours, and then added to the newly prepared TSB liquid medium (with 5% newborn fetal calf serum) at a volume ratio of 1:100, and placed at 37°C, 180r / min for 12 to 15 hours. Then the viable bacteria were counted, and then a formaldehyde solution with a final content of 0.3% was added for inactivation for 24 hours. After the inactivation was completed, an appropriate amount of the inactivated bacterial solution was applied to TSA solid medium (containing 5% newborn calf serum), and the inactivation was determined by observing whether the solid medium had colony growth. Then the bacterial solution was centrifuged at 4°C and the supernatant was discarded (8000r / min), and the precipitate after re-suspension in PBS and centrifugation was the obtained bacterial antigen.

[0062] Vaccine preparation and safety test: According to the content of bacteria before inactivation, add the corresponding volume of PBS to resuspend the bacteria, add 1 portion of Summit Poly Solution adjuvant to every 4 portions of antigen suspension, and stir at a speed of 500-800r / min for 30min until completely mixed. The prepared vaccine was stored at 4°C for use. Then the vaccine was subcutaneously inoculated into 10 6-8 week old SPF grade healthy female BALB / C mice with a dose of 0.2mL. Two weeks after the first vaccination, the immunization route and dose were the same as the first vaccination. At the same time, 10 mice were set as non-immunized control groups to observe the mental state and survival of mice in each group within 14 days.

[0063] The results showed that the sterility test results showed that the Pasteurella multocida could be completely inactivated by inactivating it with formaldehyde at a final concentration of 0.3% for 24 hours. The prepared vaccine was evenly spread on TSA solid culture medium (containing 5% newborn calf serum) and placed in an incubator for 48 hours. There were no colonies in the culture medium, indicating that the vaccine was sterile. The prepared vaccine was inoculated into the subcutaneous tissue of the back of the neck. Within 28 days after immunization, all signs of the mice were normal, and there were no other obvious symptoms such as hair loss and ulceration at the inoculation site, indicating that the prepared vaccine was safe.

[0064] Example 3

[0065] Indirect ELISA to detect antibody titer:

[0066] Preparation of whole bacterial protein: The bacterial solution grown to the stable phase was centrifuged at 8000r / min for 10min, and sterilized PBS with pH 7.4 was added at a volume ratio of 1:10 to the bacterial solution for mixing, and the whole bacteria were broken and lysed by ultrasonic disruption. The lysed bacteria were centrifuged at 6000r / min for 10min, the supernatant was taken, and its protein concentration was detected using a BCA protein concentration determination kit (commercially available), and stored in an ultra-low temperature refrigerator.

[0067] Determine the optimal coating concentration of antigen and the optimal dilution of serum: Use the conventional matrix titration method to select a series of protein coating concentrations and positive and negative antibody dilutions, perform indirect ELISA, and select the positive OD 450nm / Negative OD 450m The antigen coating concentration and serum dilution corresponding to the hole with the largest (P / N value) are the optimal antigen coating concentration and serum dilution.

[0068] Through the square array titration method, the optimal coating concentration of whole bacterial protein was determined to be 0.5 μg / mL, 100 μl / well, the optimal dilution of pig serum was 1:1600, and the dilution multiple of HRP goat anti-pig secondary antibody was 1:5000. The laboratory had previously determined that the optimal dilution of mouse serum was 1:200, and the dilution multiple of HRP goat anti-mouse secondary antibody was 1:5000. Then, this method was used for mouse serum antibody and pig serum antibody detection.

[0069] Table 6 Results of optimal antigen coating concentration

[0070]

[0071] Table 7 Optimal serum dilution results

[0072]

[0073] Example 4

[0074] Mouse immune protection test:

[0075] The experiment was divided into 12 groups, each with 8 female BALB / C mice aged 6-8 weeks. Groups 1-3, 4-6, and 7-9 were immunized with 0.2 mL of Pm A-114, Pm A-115, and Pm F-19 inactivated vaccines, respectively, and vaccinated again after an interval of 21 days; Groups 10-12 were injected with the same dose of vaccine adjuvant (the challenge control group), and Group 13 was injected with the same dose of PBS (phosphate buffered saline, conventional) (the control group without immunization and challenge). Mice in different experimental groups were kept in different cages and isolated from each other. 24 hours after vaccination, the vaccinated mice were checked for adverse reactions or signs of disease.

[0076] Groups 1-3, 4-6, 7-9, and 10-12 were all injected intraperitoneally with a lethal dose of Pm A-114, PmA-115, and Pm F-19 strains on the 35th day after immunization. To detect the antibody titer level, venous blood samples were collected from mice before the first immunization, before the second immunization, and one day before the challenge. The daily symptoms and survival of the mice were observed until 14 days after the challenge, and the daily survival rate of the mice after the parental and heterologous strains were recorded. The dead mice were immediately dissected, and the parenchymal organs were collected to isolate and identify bacteria, and fixed with paraformaldehyde. The remaining surviving mice were euthanized after the observation period, and the parenchymal organs were collected and fixed with paraformaldehyde.

[0077] The results showed that the protection rate of the inactivated vaccine of Pm A-115 strain against the parent strain was 100%, and the protection effect against homologous and heterologous strains was stronger. The protection rate against the homologous strain A type Pm A-114 strain and the heterologous strain F type Pm F-19 strain was 75%. The protection rate of the inactivated vaccine of Pm A-114 strain against the parent strain, homologous and heterologous strains was 63%; the protection rate of F type Pm F-19 strain against the parent strain was 100%, and the protection rates against the heterologous strains A type Pm A-114 strain and Pm A-115 strain were 50% and 25%, respectively. The serum antibodies of mice were tested 14 days and 35 days after immunization, and it was found that the three vaccines could stimulate the body to produce high levels of antibodies after immunization of mice (see Figure 3 , Figure 4 and Figure 5 ). Based on the above test results, Pm A-115 strain was selected as the inactivated vaccine production strain.

[0078] Table 8 Protection rate of each immune group against Pm A-114, Pm A-115, and Pm F-19

[0079]

[0080] Note: “ / ” means not applicable.

[0081] Example 5

[0082] Immunoprotective test of Pasteurella multocida on pigs:

[0083] Thirty-eight healthy pigs (conventional breeds) aged 28-35 days were randomly divided into 7 groups (6 pigs / group, including 2 pigs in the non-immunized and non-challenged control group). Groups 1-2 and 3-4 were injected with Pm A-115 and Pm F-19 inactivated vaccines for swine pasteurellosis through the neck muscle at a dose of 2 mL / head, respectively. Groups 5-7 were the control groups, of which Group 5 was the Pm A-115 challenge control group, Group 6 was the Pm F-19 challenge control group, and Group 7 was the non-immunized and non-challenged control group (2 pigs). Each pig in Groups 5 and 6 was injected with 2 mL of PBS (phosphate buffered saline, conventional). The second immunization was performed in the same way 3 weeks after the first immunization. In the second week after the second immunization, pigs in Groups 1, 4, and 5 (without feed and water for 4 hours before vaccination) were injected with 1.5 lethal doses of 2 mL of swine-derived Pasteurella multocida Pm A-115 strain (live bacteria amount was 1.5×10 8 CFU); the remaining 2, 3, and 6 groups of piglets were inoculated with 2 mL of 1.5 lethal doses of Pm F-19 strain solution (live bacteria volume 1.5×10 9CFU). Observe and record the disease and weight changes daily until 14 days after the challenge. Dead pigs were immediately autopsied and the bacteria were isolated and identified. Finally, statistical analysis showed that the vaccine provided more than 80% protection rate for immunized pigs.

[0084] The results of the immune protection test on pigs showed that the monovalent inactivated vaccines of type A and type F had no obvious side effects on the vaccinated pigs and were highly safe in pigs. The protection rates of monovalent inactivated vaccines of type A and type F against the parent strains were both 100% (6 / 6). The protection rate of monovalent inactivated vaccine of type A against the heterologous strain Pm F-19 was 50% (3 / 6), and the protection rate of monovalent inactivated vaccine of type F against the heterologous strain Pm A-115 was 0 (0 / 6). All the pigs in the control group died within one week after the infection, showing typical symptoms of porcine pasteurellosis. Some pigs in the immunized group showed fever, loss of appetite, and depression, but they returned to normal within 3 days and no deaths occurred (Table 10). The dead pigs were autopsied and showed typical pathological changes of porcine pasteurellosis, characterized by pleural and abdominal effusions, pneumonia, and sepsis. After immunization, both vaccines produced high levels of antibodies in the pigs ( Figure 6 , Figure 7 ). These results show that the vaccine prepared by the Pm A-115 strain of Pasteurella multocida can provide good protection against pasteurellosis in pigs, which can protect against the lethal dose of the virulent strain of type A and can also partially protect against the lethal dose of the virulent strain of type F.

[0085] Table 9 Results of the immune protection test of porcine Pasteurella multocida on pigs

[0086]

[0087] Note: “ / ” means not applicable.

[0088] Table 10 Disease incidence of pigs in each group in the immune protection test of porcine Pasteurella multocida

[0089]

[0090] Note: Body temperature rise: "-" means normal body temperature, "+" means body temperature exceeds 40.0℃ for less than 1 day, "++" means 2-3 days, "+++" means 3-4 days, "++++" means more than 4 days; Mental state: "-" means normal, "+" means slightly worse, "++" means worse, "+++" means very worse; Appetite: "-" means normal food intake, "+" means less food intake, "++" means very little food intake; Shortness of breath / difficulty (48 hours after infection): "-" means normal, "+" means mild, “++” means more serious, “+++” means extremely serious; Cough: “-” means no cough, “+” means cough; Runny nose: “-” means no runny nose, “+” means runny nose; Swollen joints: “-” means basically normal, “+” means swollen joints; The degree of pneumonia: “-” means basically normal, “+” means mild pneumonia, “++” means serious, with necrotic areas on the lung surface, but the area is less than 10%, and “+++” means extremely serious; the area of ​​lesions on the lung surface exceeds 10%; Mortality: “-” means no death, “+” means death.

[0091] “ / ” means not applicable.

Claims

1. A strain of Pasteurella multocida PmA-115, deposited in China Center for Type Culture Collection with the deposit number of CCTCC NO: M 20242428.

2. Use of the strain according to claim 1 in the preparation of a porcine Pasteurella multocida vaccine.

3. Use of the Pasteurella described in claim 1 in the preparation of an inactivated vaccine against porcine Pasteurella multocida, wherein the vaccine further contains a Summit Poly Solution water-soluble immune adjuvant.

4. A method for preparing the porcine Pasteurella multocida vaccine according to claim 2 or 3, characterized in that: The strain of claim 1 is cultured into a bacterial liquid using TSA medium containing 5% volume of newborn bovine serum, and then 0.3% volume of formaldehyde is added and inactivated at 37°C for 24 hours. After passing the inspection, Summit PolySolution water-soluble immune adjuvant is added at a ratio of 4:1 in volume of bacterial liquid to adjuvant.

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