Porcine F-type pasteurella multocida and application thereof as vaccine strain

Abstract

The invention belongs to the technical field of preparation of microorganisms and vaccines thereof, and relates to an F-type pasteurella multocida strain and an application of the F-type pasteurella multocida strain in preparation of vaccines, the strain is classified and named as pasteurella PmF-19 (Pasteurella sp.F-19), the strain is preserved in China Center for Type Culture Collection on October 30, 2025, and the preservation number is CCTCC NO: M 20252391. The pig pasteurella multocida provided by the invention has very strong toxicity, the vaccine prepared by inactivation of the pig pasteurella multocida has good safety, the vaccine generates high-level antibodies after immunizing mice and pigs, and the vaccine has cross protection force on F and D serotype pasteurella multocida infection in the mice, and can be used for immunizing the mice and pigs. Meanwhile, complete protection of F-type strains can be provided for pigs, and the pigs can resist attack of F-type Pm lethal dose.

Description

Technical Field

[0001] This invention belongs to the field of microbial and vaccine preparation technology, and relates to a strain of Pasteurella multocida F and its application in preparing vaccines. Background Technology

[0002] Pasteurella multocida (Pm) is an important zoonotic pathogen capable of infecting a variety of animals. In the swine industry, it primarily causes swine pneumonia and atrophic rhinitis. Severe swine pneumonia can lead to a 100% mortality rate in pig herds, causing significant economic losses to the swine industry. In China, swine pasteurellosis is mainly caused by capsular serotypes A and D of Pasteurella multocida. In recent years, the number of Pm serotype F isolated from tissues of pigs with respiratory diseases has shown a gradual increasing trend. As a newly emerging serotype in the swine industry, its detection rate is around 5%. However, laboratory results indicate that this strain is highly virulent in pigs; therefore, prevention and control of pasteurellosis caused by this serotype should also be considered.

[0003] Some domestic scholars have found that *Pasteurella multocida* type F has strong pathogenicity in mice and pigs, falling between that of *Pasteurella aeruginosa* type A and type D. Previous studies have reported poor cross-protection between *Pasteurella aeruginosa* vaccines of types A, B, and D. Currently, commercially available pasteurellosis vaccines mainly use *Pasteurella aeruginosa* type A and type B strains as the vaccine strain, and the cross-protection effect of these vaccines against *Pasteurella multocida* type F is unclear. This poses certain challenges to the clinical control of pasteurellosis caused by *Pasteurella multocida* type F.

[0004] Based on the above reasons, this study screened a highly virulent and immunogenic strain of *Pasteurella multocida* type F clinical isolates to prepare an inactivated vaccine. Mouse challenge experiments revealed that the vaccine exhibited good cross-protective activity against both *Pasteurella multocida* type F and D. Furthermore, immunization protection experiments in pigs showed that the vaccine prepared from this strain could protect pigs against lethal doses of *Pasteurella multocida* type F, providing a reference for the clinical prevention and control of diseases caused by porcine *Pasteurella multocida* type F. Summary of the Invention

[0005] To address the above-mentioned problems, one of the objectives of this invention is to provide a porcine Pasteurella multocida strain F, which is classified and named Pasteurella sp. F-19. It was deposited at the China Center for Type Culture Collection on October 30, 2025, with accession number CCTCC NO: M 20252391.

[0006] The second objective of this invention is the application of the porcine Pasteurella multocida F strain described herein in the preparation of an inactivated vaccine against porcine Pasteurella multocida disease.

[0007] A third objective of this invention is to provide an inactivated vaccine against porcine Pasteurella multocida infection, comprising an inactivated strain of Pasteurella multocida F as described in this invention.

[0008] Preferably, the inactivated swine pasteurellosis vaccine further includes an immune adjuvant.

[0009] Preferably, the immune adjuvant includes one or more of aluminum salt adjuvants, polymer nanoadjuvants, and liposome adjuvants.

[0010] Preferably, the immune adjuvant is ISA206 adjuvant.

[0011] Preferably, the inactivated bacterial count of the *Pasteurella multocida* F strain is not less than 3.0 × 10⁻⁶ cells. 9 CFU / ml.

[0012] The fourth objective of this invention is to provide a method for preparing the porcine Pasteurella multocida inactivated vaccine of the present invention. The method includes: culturing the porcine Pasteurella multocida of the present invention into a bacterial suspension using TSB medium containing 5% volume of newborn calf serum, then adding 0.3% volume of formaldehyde, inactivating at 37°C for 24 hours, resuspending the bacterial cells in PBS, and adding an adjuvant at a ratio of bacterial cells to adjuvant of 1:1.

[0013] The fifth objective of this invention is the application of the porcine Pasteurella multocida inactivated vaccine described herein in the preparation of a drug for preventing porcine Pasteurella multocida infection.

[0014] Preferably, the diseases caused by the *Pasteurella multocida* infection include porcine pasteurellosis and atrophic rhinitis.

[0015] Compared with the prior art, the main advantages of this invention are: The *Pasteurella multocida* strain provided by this invention has strong virulence; 24 CFU can completely kill mice, and 1.0 × 10⁻⁶ CFU is highly lethal. 9 CFU can completely kill 9-10 week old pigs. The vaccine prepared by inactivating it has good safety. After immunizing mice and pigs, the vaccine produced high levels of antibodies. The vaccine has cross-protection against infection with both F and D serotypes of Pasteurella multocida in mice and can provide immune protection against infection with both F and D serotypes at the same time. At the same time, after immunizing pigs with the inactivated vaccine prepared by this strain, it can provide complete protection against the F strain and enable pigs to resist the lethal dose of F strain Pm.

[0016] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0017] Figure 1 This is a graph showing the PCR identification results of Pasteurella multocida.

[0018] Figure 2 This is a graph showing the PCR identification results of Pasteurella multocida capsulatum.

[0019] Figure 3 This is the survival curve of mice infected with Pasteurella multocida Pm F-19.

[0020] Figure 4 This is the survival curve of mice infected with Pasteurella multocida Pm F-46.

[0021] Figure 5 These are the results of serum antibody detection in mice after immunization with an inactivated vaccine prepared from the Pm F-19 strain.

[0022] Figure 6 These are the results of serum antibody detection in mice after immunization with an inactivated vaccine prepared from the Pm F-46 strain.

[0023] Figure 7 These are the results of antibody testing in pig serum after immunization with Pm F-19. Detailed Implementation

[0024] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0025] To better explain the present invention, the main contents of the present invention are further illustrated below with reference to specific embodiments.

[0026] In the following examples, all the strains involved were isolated in our laboratory and submitted for statutory preservation.

[0027] In the following example, the formula for TSA solid culture medium (with 5% newborn calf serum) is as follows: Weigh 16g of TSA powder and dissolve it in 400mL of single-distilled water. Autoclave at 121℃ for 15min. After cooling to 50℃, add 20mL of newborn calf serum, mix well, and pour into a sterile Petri dish for later use.

[0028] The formula for TSB liquid culture medium is as follows: Weigh 12g of TSB powder and dissolve it in 400mL of single-distilled water. Autoclave at 121℃ for 15min. Store at room temperature after sterilization. When using, add 5% newborn calf serum to the solution.

[0029] PBS buffer: 8.0g NaCl, 0.2g KCl, 1.56g Na2HPO4, and 0.2g KH2PO4 are added sequentially to 800 mL of double-distilled water. After complete dissolution, the volume is adjusted to 1000.0 mL. HCl is added to adjust the pH to 7.8. Store at 4℃ for later use.

[0030] PBST buffer: Add Tween-20 to PBS buffer to a final concentration of 0.05%, mix well, and store at room temperature for later use.

[0031] Blocking solution: Weigh 5g of skim milk powder and dissolve it completely in 100mL of PBST buffer.

[0032] Coating solution: 1.59g Na2CO3 and 2.93g NaHCO3 were added sequentially to 800 mL of double-distilled water. After complete dissolution, the solution was brought to a final volume of 1000.0 mL and stored at 4℃ for later use.

[0033] The TMB developer and stop solution were purchased from Beijing Solarbio Technology Co., Ltd.

[0034] The enzyme-labeled secondary antibody was purchased from Wuhan Sanying Biotechnology Co., Ltd.

[0035] ISA206 adjuvant was purchased from Seppic S.p.A. in France and is a water-in-oil-in-water adjuvant.

[0036] Example 1: Screening of F-type Pm vaccine candidate strains

[0037] 1. Isolation and identification of porcine Pasteurella multocida Thirteen clinical isolates of Pm F and one each of Pm A and Pm D were isolated and identified by our laboratory from lung tissue of pigs with suspected respiratory symptoms. Among them, Pm A-115 (CCTCC NO: M 20242428) and Pm D-135 (CCTCC NO: M 20242429) have been deposited in the China Center for Type Culture Collection. The strain information is listed in Table 1.

[0038] Table 1. Information on Pasteurella multocida strains A, D, and F used in this study.

[0039] For pigs with suspected respiratory symptoms, tissue samples were inoculated onto TSA (with 5% newborn calf serum) plates under sterile conditions. After streaking with a sterile inoculation loop, the plates were incubated at 37°C for 12-24 hours. Single colonies that were grayish-white, translucent, round, smooth, with neat edges, and moist and viscous were picked from the plates for purification culture.

[0040] Single colonies suspected to be Pm were picked using a sterile inoculation loop and placed in TSB (with 5% newborn calf serum) liquid medium, then incubated at 37°C and 180 r / min for 12–16 h. Genomic DNA was extracted from the isolates and analyzed using the specific method of Pasteurella multocida. kmt1 Gene and Pasteurella multocida F-type capsule synthesis gene ( fcbD Primers were used to identify the bacterial species and capsular serotype of the isolated strains. kmt1 The gene primer sequences are as follows: kmt1-SP6 :5'-GCTGTAACGAACTCGCCAC-3'; kmt1-T7 : 5'-ATCCGCTATTTACCCAGTGG-3', the expected PCR amplification product size is 460bp. fcbD The gene primer sequences are: Forward: 5'-TCGGAGAACGCAGAAATCAG-3', Reverse: 5'-TTCCGCCGTCAATTACTCTG-3', and the expected PCR amplification product size is 852bp.

[0041] The PCR reaction system was as follows: 10 µL of 2×Rapid Taq Master Mix, 7 µL of sterile distilled water, 1 µL each of primers Pm-F and Pm-R, and 1 µL of template. Reaction conditions: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 15 s; 54℃ annealing for 15 s; 72℃ extension for 30 s, 30 cycles; followed by another 72℃ extension for 5 min, and finally cooling to 4℃. PCR products were stored at 4℃ for later use.

[0042] The PCR products were electrophoresed on a 1% agarose gel at 120 V for 25 min, stained with Goldview, and then scanned and imaged using a gel imaging system. The images were exported and saved.

[0043] The results showed that positive colonies from two suspected Pm strains could amplify a 460bp band, such as... Figure 1 As shown, M: DL2000 maker; 1-2: single colonies suspected to be Pm; +: positive control; -: negative control. After identification with Pasteurella multocida capsularis-specific primers, clinical isolates were able to amplify an 852 bp band, as shown... Figure 2As shown, M: DL2000maker; 1: clinically isolated type F Pasteurella multocida; +: positive control; -: negative control.

[0044] 2. Mouse virulence test of Pasteurella multocida The mouse toxicity test was conducted using the 13 strains of Pasteurella multocida F listed in Table 1. Previous laboratory studies had already determined the LD50 of Pasteurella multocida F strain HN07. 50 The number is 117 CFU. The specific implementation method is as follows.

[0045] The correctly identified bacterial strain was revived in TSA solid medium (containing 5% fetal bovine serum) and incubated at 37°C for 16 hours. A single colony was then picked and inoculated into TSB liquid medium containing 5% fetal bovine serum and incubated at 37°C and 180 rpm for 12-15 hours. Then, the culture was added at a 1:100 ratio to freshly prepared TSB liquid medium (containing 5% fetal bovine serum) and incubated at 37°C and 180 rpm for 12-15 hours. The bacterial culture was centrifuged (6000 rpm, 8 min) and resuspended in sterile PBS as the infection solution. This solution was then serially diluted to adjust the bacterial load to the target challenge dose.

[0046] The experiment was conducted in multiple rounds to screen for highly virulent strains (Pm). Taking the first round of virulence testing as an example, 65 female BALB / c mice (SPF) aged 6-8 weeks were randomly divided into 13 groups of 5 mice each. Groups 1-12 were the experimental groups, and group 13 was the control group. The experimental groups were infected by intraperitoneal injection of 0.2 mL (500 CFU of live bacteria) of challenge solution, while the control group was injected with 0.2 mL of PBS in the same manner. Mice were observed for 7 days post-infection, and the virulence of the strain was determined by the mortality rate of the mice. Subsequent virulence tests were designed based on the results of the first round of virulence testing. The strains that completely killed mice in the first round were used in the second round of virulence testing at a reduced challenge dose, and subsequent tests were conducted in the same manner.

[0047] The results (see Table 2) showed that mice began dying 12 hours after challenge. Infected mice primarily exhibited hemorrhagic inflammation throughout their bodies. Mice that died acutely showed signs of septicemia, with congestion, edema, and hemorrhage in various tissues and organs. Mice that died chronically showed symptoms of depression, lethargy, loss of appetite, and inflammatory secretions covering the eye sockets. In the first round of virulence testing, mice infected with Pm F-19, Pm F-45, Pm F-46, and Pm F-119 at 500 CFU died within 48 hours; therefore, these four strains were selected for the next round of virulence testing. In the second round of virulence testing, mice were challenged with 200 CFU of bacteria. It was found that Pm F-19 and Pm F-46 were completely lethal at this dose. The third round of virulence testing investigated the LD50 of these two strains in mice after challenge with different bacterial doses. 50 The LD value of Pm F-19 was ultimately measured. 50 12.3 CFU, Pm F-46 LD 50 66.7 CFU ( Figure 3 , Figure 4 These two strains were then selected for virulence testing of Pasteurella multocida in pigs.

[0048] Table 2. Mortality of mice after infection with Pasteurella multocida.

[0049] 3. Virulence test of Pasteurella multocida in pigs Twenty-eight healthy piglets aged 8-10 weeks were randomly divided into seven groups (4 piglets per group). Groups 1-6 were the challenge test groups, and group 7 was the blank control group. Nasal swabs were collected before the experiment to detect Pm and other respiratory pathogens (HPS, APP, SS, PRV, etc.), and blood samples were collected for Pm antibody detection. Pigs that were negative for Pm antibodies were selected for the experiment.

[0050] Single colonies of two strains of Pasteurella multocida (type F) were inoculated separately into TSB liquid medium containing 5% fetal bovine serum and cultured at 37°C with shaking at 180 r / min for 15 h to obtain seed culture. Then, 1% of the seed culture was inoculated into fresh TSB liquid medium containing 5% fetal bovine serum and cultured at 37°C with shaking at 180 r / min for 6 h. The OD values ​​of the bacterial culture were measured using a spectrophotometer. 600 The concentration was adjusted to 0.6, at which point the bacterial concentration was 2.0 × 10⁻⁶. 9 CFU / ml. The bacterial suspension was resuspended in sterile PBS, and piglets in groups 1-3 were injected intravenously via the ear vein at a concentration of 2×10⁻⁶ CFU / ml. 9 CFU, 1×10 9 CFU and 5×10 8 CFU Pm F-19 challenge solution was administered to piglets in groups 4-6 via marginal ear vein at a dose of 2×10⁻⁶. 9CFU, 1×10 9 CFU and 5×10 8 CFU-treated Pm F-46 bacterial challenge solution was administered to piglets in group 7 via intravenous injection of an equal volume of PBS. Piglets were fed normal piglet feed after challenge, and their mental state, appetite, coat, joint health, excretion, and other clinical symptoms and disease progression were observed daily for 14 days. Dead piglets were immediately necropsized, and their lungs, liver, spleen, tonsils, and other organ tissues were collected for histopathological analysis.

[0051] The results (see Table 3) showed that PmF-19 exhibited strong virulence after being challenged with two strains of Pasteurella multocida of type F at three different concentrations. Infected pigs displayed obvious symptoms of swine pneumonia, including difficulty breathing, whistling sounds during respiration, swollen throat, cyanosis of the skin, congestion and necrosis of the limbs and ear tips, and yellow or white discharge from the mouth and nose. They also exhibited clear septicemia symptoms, with hemorrhage in multiple organs and tissues, including lymph node hemorrhage; enlarged kidneys, coronary fat hemorrhage, swollen lungs with black necrotic spots on the surface, ascites, yellow pericardial effusion, and white fibrinous exudate on the surface of the heart; swollen joints of the limbs, with yellowish-white purulent discharge upon incision. Histopathological results showed thickened serosal membranes on the surface of the lung tissue of infected pigs, accompanied by a small number of inflammatory foci; extensive thickening of the alveolar walls, unclear alveolar structure, accompanied by a small amount of inflammatory cell infiltration and bronchial epithelial cell shedding.

[0052] Table 3. Results of toxicity tests of Pasteurella multocida in pigs.

[0053] Example 2: Preparation of inactivated vaccine

[0054] Preparation of bacterial antigen: The correctly identified bacterial strain was revived in TSA solid medium (containing 5% newborn calf serum) and incubated at 37°C for 16 hours. A single colony was then picked and inoculated into TSB liquid medium containing 5% newborn calf serum, and incubated at 37°C and 180 rpm for 12-15 hours. Then, the inoculum was added at a 1:100 ratio to freshly prepared TSB liquid medium (containing 5% newborn calf serum) and incubated at 37°C and 180 rpm for 12-15 hours. Viable cell counts were then performed, followed by inactivation with 0.3% formaldehyde solution for 24 hours. After inactivation, an appropriate amount of the inactivated bacterial solution was spread onto TSA solid medium (containing 5% newborn calf serum), and the presence or absence of colony growth on the solid medium was used to determine complete inactivation. The bacterial solution was then centrifuged at 4°C (8000 rpm), the supernatant was discarded, and the precipitate after resuspending in PBS and centrifuging again was the obtained bacterial antigen.

[0055] Vaccine preparation and safety testing: Based on the pre-inactivation bacterial cell count, the cells were resuspended in PBS at an appropriate volume (3.0 × 10⁻⁶ cells / mL). 9 Add the bacterial cells (CFU / ml) to the adjuvant at a 1:1 ratio and mix thoroughly. Stir at 500–800 rpm for 30 minutes until completely mixed. Store the prepared vaccine in aliquots at 4°C for later use. Then, subcutaneously inoculate 10 healthy female 6-8 week old SPF-grade BALB / c mice with a dose of 0.2 mL. Two weeks after the initial immunization, administer two more immunizations at the same route and dosage. Simultaneously, set up 10 mice as a non-immunized control group. Observe the mental status and survival of mice in each group over 14 days.

[0056] The results showed that the sterility test indicated that Pasteurella multocida could be completely inactivated by formaldehyde at a final concentration of 0.3% for 24 hours. The prepared vaccine was evenly spread on TSA solid medium (containing 5% newborn calf serum) and incubated in an incubator for 48 hours; no colonies were found in the medium, indicating that the vaccine was sterile. When the prepared vaccine was injected into the subcutaneous tissue of the neck and back, the mice showed normal physical signs within 28 days post-immunization, with no hair loss, ulceration, or other obvious symptoms at the injection site, indicating that the prepared vaccine had good safety.

[0057] Example 3: Indirect ELISA detection of antibody titer

[0058] Whole-cell protein preparation: The bacterial culture that had reached the stationary phase was centrifuged at 8000 r / min for 10 min. Sterile PBS (pH 7.4) was added at a ratio of 1:10 to the bacterial culture, and the mixture was thoroughly mixed by pipetting. Simultaneously, the whole bacterial cells were lysed using ultrasonication. The lysed bacterial cells were centrifuged at 6000 r / min for 10 min, and the supernatant was collected. The protein concentration was determined using a BCA protein concentration assay kit and stored in an ultra-low temperature freezer.

[0059] Determine the optimal antigen coating concentration and serum dilution: Using a checkerboard titration method, select a series of protein coating concentrations and positive and negative antibody dilutions, perform indirect ELISA, and select the antigen coating concentration and serum dilution corresponding to the well with the largest positive OD450nm / negative OD450nm (P / N value) as the optimal antigen coating concentration and serum dilution.

[0060] The optimal coating concentration of whole bacterial protein was determined to be 1 μg / mL, 100 μl / well, using a square matrix titration method. The optimal dilution of porcine serum was 1:1600, and the optimal dilution of HRP goat anti-porcine secondary antibody was 1:5000. Previously, the optimal dilution of mouse serum was determined to be 1:200, and the optimal dilution of HRP goat anti-mouse secondary antibody was determined to be 1:5000. This method was then used to detect antibodies in mouse and porcine serum.

[0061] Example 4: Mouse Immunoprotection Test

[0062] The experiment consisted of 13 groups, with 8 female BALB / c mice aged 6-8 weeks in each group. Groups 1-4 were immunized with 0.2 mL of PmF-19 inactivated vaccine, and groups 5-8 were immunized with 0.2 mL of PmF-46 inactivated vaccine. They were immunized again after 21 days. Groups 9-12 received the same volume of vaccine adjuvant (challenge control group), and group 13 received the same volume of PBS (no immunization, no challenge control group). Mice in different groups were housed in separate cages and isolated from each other. 24 hours after vaccination, the mice were examined for adverse reactions or signs of disease.

[0063] Groups 1, 5, and 9; groups 2, 6, and 10; groups 3, 7, and 11; and groups 4, 8, and 12 were intraperitoneally injected with a lethal dose of PmF-19, PmF-46, PmA-115, and PmD-135 bacterial challenge solution on day 35 post-immunization. To detect antibody titers, venous blood samples were collected from mice before the first immunization, before the second immunization, and one day before challenge. Daily symptoms and survival were observed in mice up to day 14 post-challenge, and the daily survival rates of mice challenged with parental and heterologous strains were recorded. Dead mice were immediately dissected, and parenchymal organs were collected for bacterial isolation and identification, and fixed with paraformaldehyde. The remaining surviving mice were euthanized at the end of the observation period, and parenchymal organs were collected and fixed with paraformaldehyde.

[0064] The results showed that the Pm F-19 inactivated vaccine provided 100% protection against both the parental strain and homologous strains, and exhibited strong protection against heterologous type D strains (88%). The F-type Pm F-46 strain provided 88% protection against the homologous Pm F-19 strain and 75% protection against heterologous type D strains. Neither F-type strain showed good cross-protection against type A strains. Serum antibody levels in mice at 14 and 35 days post-immunization were measured, revealing that both vaccines stimulated high levels of antibodies in immunized mice. Figure 5 , Figure 6 Based on the above experimental results, Pm F-19 strain was selected as the inactivated vaccine strain.

[0065] Table 4. Protection rates of two strains of Pasteurella f. f. against highly virulent strains of F, A, and D.

[0066] Example 5: Immunoprotective test of Pasteurella multocida in pigs

[0067] Twenty-six healthy pigs aged 28-35 days were randomly divided into five groups (6 pigs per group, including a non-immunized, non-challenged control group of 2 pigs). Groups 1 and 2 received a 2 mL / pig intramuscular injection of Pm F-19 porcine pasteurellosis inactivated vaccine via the neck. Groups 3 and 4 served as the Pm F-19 and Pm A-115 challenge control groups, and Group 5 was a non-immunized, non-challenged control group (2 pigs). Pigs in groups 3 and 4 received 2 mL of PBS per pig. A second immunization was administered three weeks after the first immunization using the same method. Two weeks after the second immunization, pigs in groups 1 and 3 (withheld feed and water for 4 hours prior to vaccination) received a lethal dose of 1 mL of porcine Pasteurella multocida strain Pm F-19 (1.0 × 10⁻⁶ viable cells) via the ear vein. 9 CFU); Following the same method, the remaining groups 2 and 4 piglets were inoculated with 1 mL of lethal dose of Pm A-115 bacterial suspension (live count of 1.0 × 10⁻⁶ CFU). 8 (CFU). Daily observation and recording of disease progression and weight changes were maintained until 14 days after challenge. Dead pigs were immediately necropsed for bacterial isolation and identification. Final statistical analysis showed the vaccine provided over 80% protection to immunized pigs.

[0068] Immunoprotective trials on pigs showed that the F-type Pm inactivated vaccine had no significant adverse reactions in vaccinated pigs and was highly safe in pigs. The F-type Pm inactivated vaccine provided 100% protection against the parent strains (6 / 6), but had no protective effect against the type A strain Pm A-115. All pigs in the control group died within one week of challenge, exhibiting typical symptoms of porcine pasteurellosis. Some pigs in the immunized group showed fever, loss of appetite, and lethargy, but quickly recovered without death (Tables 5 and 6). Autopsy of the dead pigs also revealed typical pathological changes of porcine pasteurellosis, characterized by pleural and peritoneal effusion, pneumonia, and septicemia. After vaccination, high levels of antibodies were produced in the pigs. Figure 7 These results indicate that the vaccine prepared from Pasteurella multocida Pm F-19 provides good protection against porcine pasteurellosis, protecting against lethal doses of the highly virulent F strain.

[0069] Table 5. Results of immunoprotective trials against porcine Pasteurella multocida.

[0070] Note: " / " indicates that it is not applicable.

[0071] Table 6. Disease incidence in pigs of different groups in the immunoprotective test against Pasteurella multocida.

[0072] Note: Elevated body temperature: "-" indicates normal body temperature, "+" indicates body temperature exceeding 40.0℃ for less than 1 day, "++" indicates for 2-3 days, "+++" indicates for 3-4 days, and "++++" indicates for more than 4 days; Mental state: "-" indicates normal, "+" indicates slightly poor, "++" indicates poor, and "+++" indicates very poor; Appetite: "-" indicates normal appetite, "+" indicates low appetite, and "++" indicates very low appetite; Shortness of breath / difficulty breathing (after 48 hours of challenge): "-" indicates normal, "+" indicates mild, "++" indicates moderate, and "+++" indicates very severe; Cough: "-" indicates no cough, and "+" indicates cough; Runny nose: "-" indicates no runny nose, and "+" indicates runny nose; Joint swelling: "-" indicates mostly normal, and "+" indicates joint swelling; Severity of pneumonia: "-" indicates mostly normal, "+" indicates mild pneumonia, and "++" indicates mild pneumonia. Severe: Necrotic areas are present on the surface of the lungs, but the area is less than 10%; "+++" indicates extremely severe; the lesion area on the surface of the lungs exceeds 10%. Death status: "-" indicates not dead, "+" indicates dead. " / " indicates not applicable.

[0073] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A strain of Pasteurella multocida F, characterized in that, The strain was classified and named Pasteurella sp. F-19, and was deposited at the China Center for Type Culture Collection on October 30, 2025, with accession number CCTCC NO: M 20252391.

2. The use of the porcine Pasteurella multocida F strain as described in claim 1 in the preparation of an inactivated vaccine against porcine Pasteurella multocida disease.

3. An inactivated vaccine against porcine Pasteurella multocida infection, characterized in that, It contains an inactivated strain of Pasteurella multocida F as described in claim 1.

4. The inactivated vaccine against porcine Pasteurella multocida according to claim 3, characterized in that, It also includes immune adjuvants.

5. The inactivated vaccine against porcine Pasteurella multocida according to claim 4, characterized in that, The immune adjuvant includes one or more of aluminum salt adjuvants, polymer nanoadjuvants, and liposome adjuvants.

6. The inactivated vaccine against porcine Pasteurella multocida according to claim 4, characterized in that, The immune adjuvant is ISA206 adjuvant.

7. The inactivated vaccine against porcine pasteurellosis according to any one of claims 3-6, characterized in that, The inactivated bacterial count of the *Pasteurella multocida* F strain is not less than 3.0 × 10⁻⁶ cells. 9 CFU / ml.

8. The method for preparing the inactivated vaccine against porcine Pasteurella multocida as described in any one of claims 4-7, characterized in that, include: The porcine Pasteurella multocida F strain described in claim 1 was cultured in TSB medium containing 5% volume of newborn calf serum, and then 0.3% volume of formaldehyde was added. The culture was inactivated at 37°C for 24 hours. After resuspending the bacteria in PBS, an immune adjuvant was added at a ratio of 1:1 between the bacteria and the adjuvant.

9. The use of the porcine Pasteurella multocida inactivated vaccine as described in any one of claims 3-7 in the preparation of a medicament for preventing porcine Pasteurella multocida infection.

10. The application according to claim 9, characterized in that, The diseases caused by Pasteurella multocida infection in pigs include pasteurellosis and atrophic rhinitis.

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