Bordetella bronchiseptica strain and application thereof

Abstract

The application discloses a cat bronchial septic Bordetella strain and application thereof, and belongs to the technical field of vaccines.The strain is a cat bronchial septic Bordetella WF202409 strain, which comprises FLA 、 bvgs 、 betA and CyaA virulence genes.The strain has strong virulence and good immunogenicity, and can be used for preparing an inactivated vaccine.The application further discloses an inactivated vaccine comprising the strain and a preparation method of the vaccine.The vaccine is prepared by mixing and emulsifying inactivated bacteria liquid and propolis adjuvant at a volume ratio of 25:1.The animal experiment verifies that the vaccine has good safety and immunogenicity, can effectively induce the body to produce specific antibodies, and has a cross protection effect on heterologous strains.The application provides an effective vaccine candidate strain and technical scheme for preventing cat bronchial septic Bordetella disease, and has important clinical application value.

Description

Technical Field

[0001] This invention relates to the field of vaccine technology, and in particular to a strain of Bordetella feline bronchiseptica and its application. Background Technology

[0002] Bordetella bronchiseptica ( Bordetella bronchiseptica, Bb Bordetella belongs to the class Beta-Proteobacteria, genus Bordetella, and is considered a classic pathogenic species of the genus Bordetella, along with Bordetella pertussis, Bordetella parapertussis, and Bordetella avianis. Feline bronchial septicemia Bordetella infection is caused by... Bb One of the main respiratory diseases in cats, it can cause tracheobronchitis, primarily manifested as sneezing, runny nose, swollen submandibular lymph nodes, and coughing with rales. Mild cases can heal in about 10 days; severe cases can lead to pneumonia, which is common in kittens. Clinical symptoms of pneumonia caused by feline bronchial septicemia are quite severe and can lead to death in kittens. Therefore, feline bronchial septicemia caused by Bordetella bronchiseptica deserves attention. Studies have shown... Bb The ability to survive in the external environment for 10 days creates a potential pathway for continuous transmission via aerosols in confined environments, increasing the risk of indirect contact transmission in multi-cat environments.

[0003] The strains used in foreign vaccines are mostly locally prevalent strains, whose antigenic structures may differ from the Bb strains prevalent in Chinese cat populations. This can lead to poor immunization efficacy or incomplete protection. Furthermore, simply isolating feline Bb strains and preparing inactivated vaccines using conventional methods carries the risk of either excessive virulence leading to poor safety or insufficient virulence resulting in poor immunogenicity. Domestically, there is a lack of domestically sourced feline strains that have undergone systematic screening and validation, possessing both high virulence and excellent immunogenicity. Bb The vaccine candidate strains also lack a complete technical chain from strain screening, vaccine preparation, quality testing to immunization efficacy evaluation, which cannot support the independent research and development and approval of commercial vaccines. Therefore, it is particularly important to develop a vaccine that can be used clinically to prevent feline bronchogenic bortezomib infection. Summary of the Invention

[0004] This application provides a strain of Bordetella feline bronchiseptica and its application, which solves the problems in the prior art of lacking vaccine candidate strains with strong virulence and excellent immunogenicity suitable for the prevalent strains of cats in China, as well as the lack of a complete technical system from strain screening to vaccine preparation, and realizes an inactivated vaccine with cross-protective ability.

[0005] The strain of Bordetella feline bronchiseptica provided in this application, WF202409, was deposited on July 23, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 35353.

[0006] Furthermore, the strain contains FLA , bvgs , betA and CyaA Virulence genes.

[0007] The aforementioned strain of *Bordetella felis* is used to prepare vaccines to prevent respiratory diseases caused by *Bordetella felis*.

[0008] A vaccine containing inactivated Bordetella feline bronchiseptica, comprising an inactivated bacterial solution of a strain of Bordetella feline bronchiseptica.

[0009] Furthermore, the vaccine also contains propolis adjuvant.

[0010] Furthermore, the volume ratio of the inactivated bacterial solution to the propolis adjuvant is 25:1.

[0011] Furthermore, its preparation method specifically includes:

[0012] S1. Bacterial resuscitation and culture: The frozen Bordetella feline bronchiseptica strain was streaked onto TSA plates and incubated in a 37 ℃, 5% CO2 incubator for 24 hours. Single colonies were picked and inoculated into TSB liquid medium and cultured at 37 ℃, 200 rpm / min for 12-16 hours with shaking. S2. Bacterial inactivation: When the OD value of the bacterial culture is measured... 600 When the concentration of bacteria reaches 1.0, the bacterial culture is harvested, centrifuged at 4000 rpm / min for 5 minutes with sterile physiological saline, the bacterial cells are washed 3 times, the bacterial cells are collected, and inactivated with formaldehyde solution. S3. Vaccine preparation: Mix the inactivated bacterial solution with propolis adjuvant, stir thoroughly, let stand at room temperature for 2-3 hours to allow the vaccine to fully emulsify, dispense and store in a 4 ℃ refrigerator for later use; S4. Physical property testing of vaccines.

[0013] Furthermore, the specific inactivation conditions for the formaldehyde solution are as follows: the final concentration of formaldehyde is 0.4%, the inactivation temperature is 37°C, and the inactivation time is 36 hours.

[0014] One or more technical solutions provided in the embodiments of this application have at least the following technical effects or advantages: The WF202409 strain screened from 128 clinical samples in this invention carries a specific combination of virulence genes ( FLA , bvgs , betA , CyaA ), and key virulence genes ( bvgs , CyaAIt possesses unique amino acid mutation sites, which not only have sufficient toxicity to stimulate an immune response, but also maintain good safety and immunogenicity after inactivation.

[0015] The vaccine produced by this strain exhibited good safety, with no local or systemic adverse reactions reported, achieving a balance between high virulence and excellent immunogenicity. The low proportion of mobile elements in the strain's genome suggests a stable genetic background, making it less susceptible to safety risks associated with reversion mutations or horizontal gene transfer when used as a vaccine production strain. This strain carries 31 "hypervirulence-related genes," but the loss of function of these genes leads to increased virulence, indicating that they are negative regulators of virulence. After inactivation, the strain showed good safety, demonstrating a naturally occurring, sophisticated virulence regulation mechanism that avoids excessive pathogenicity.

[0016] Analysis of data from phenotype, sterility testing, safety testing, and immunization efficacy testing showed that all indicators were stable and effective. Efficacy testing results demonstrated that strain WF202409 rapidly produces antibodies and can resist different prevalent strains after immunization. A complete technical system was established, encompassing strain screening, identification, virulence analysis, pathogenicity verification, and inactivated vaccine preparation. The prepared inactivated vaccine exhibits good safety, sterility, stability, and dissolution properties. The vaccine effectively induces specific antibodies in rabbits and cats, with antibody titers reaching up to 2. 5 The vaccine provides cross-protection against both homologous and heterologous strains, and can effectively alleviate clinical symptoms. The inactivated vaccine prepared in this invention exhibits a synergistic effect when mixed with propolis adjuvant at a ratio of 25:1. The presence of extracellular polysaccharide synthesis-related genes in the CAZy annotation suggests that bacterial surface polysaccharides may form a better antigen delivery system with the propolis adjuvant, enhancing the immune response. Its technical efficacy far exceeds the expectations of conventional inactivated vaccines, demonstrating significant cross-protection against heterologous circulating strains and effectively alleviating clinical symptoms after heterologous challenge. This vaccine can cover a wider range of circulating strains and also shows good efficacy against highly variable bacterial diseases.

[0017] A complete technical system based on native Chinese strains, covering strain screening, virulence verification, vaccine preparation and efficacy evaluation, has been constructed, filling the technological gap in this field in China and providing a practical technical solution for the prevention and control of native feline bronchoseptica Bordetella infection. Attached Figure Description

[0018] Figure 1 Colony morphology of Bordetella felineis WF202409 strain, where Figure A shows the colony morphology on TSA agar culture; Figure B shows the colony morphology on MacConkey agar culture. Figure 2Microscopic examination results of Bordetella feline bronchiseptica strain WF202409; Figure 3 PCR amplification diagram of the 16S gene of Bordetella felineis strain WF202409, where M: DNA marker DL2000; 1: 16S rRNA gene of the isolated strain; 2: negative control; Figure 4 Genetic evolutionary analysis diagram of Bordetella feline bronchiseptica strain WF202409, wherein WF20240910 is the isolate of this invention; Figure 5 Growth curve of Bordetella feline bronchiseptica strain WF202409; Figure 6 PCR amplification diagram of the virulence gene of Bordetella feline bronchiseptica strain WF202409, where M: DL2000 Marker; A: 1-5: FLA Virulence genes, DNT Virulence genes, Bvgs Virulence genes, AC-Hly Virulence genes, FHA Virulence genes; B: 1-5: brfZ Virulence genes; Prn Virulence genes; FhaB Virulence genes; betA Virulence genes; CyaA Virulence genes; Figure 7 : Autopsy images of mouse lungs of Bordetella feline bronchiseptica strain WF202409, where A: challenge group; B: control group; Figure 8 Survival curve of mouse challenge with Bordetella felineis WF202409 strain; Figure 9 Images of spleen and lung tissue lesions in kittens with Bordetella felineis strain WF202409; Figure 10 : Antibody titer monitoring chart of Bordetella feline bronchiseptica strain WF202409; Figure 11 Distribution map of gene length of Bordetella bronchiseptica WF202409 in feline bronchitis; Figure 12 Functional distribution map of Bordetella felineis WF202409 GO; Figure 13 Functional distribution diagram of COG in Bordetella feline bronchiseptica WF202409; Figure 14 Functional distribution diagram of Bordetella feline bronchiseptica WF202409 NR; Figure 15 Functional classification and corresponding gene number statistics of Bordetella feline bronchiseptica WF202409 CAZy; Figure 16 Distribution of PHI phenotypic mutation types in Bordetella feline bronchiseptica WF202409. Detailed Implementation

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to limit the invention; the term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0020] Example 1: Isolation and identification of Bordetella feline bronchiseptica strain: Sample collection and bacterial isolation and purification: A total of 128 samples were collected from cats with respiratory symptoms in animal hospitals and catteries in Qingdao, including 12 lung samples, 36 blood samples, and 80 mixed swab samples from the eyes, mouth, and nose. In a clean bench, a sterile inoculation loop was used to collect the swab fluid, which was then streaked in three zones on MacConkey agar and TSA medium. After incubation at 37°C for 36 hours, the culture was inverted. Single colonies with morphology similar to Bb were picked and re-inoculated onto TSA and MacConkey agar medium for purification and culture.

[0021] like Figure 1 The selected strains showed milky-white raised areas on the surface of TSA agar plates, forming medium-sized, opaque colonies with smooth surfaces and regular edges. Figure 1 A). Colonies appear red on MacConkey agar plates with a narrow red ring around them, and the culture medium is stained amber (…). Figure 1 B).

[0022] Bacterial identification: Morphological observation: Purified colonies from the culture medium were picked, Gram-stained, and examined under a microscope. Bacterial morphology was observed using an oil immersion microscope (100×) for preliminary identification of the isolated bacteria. For example... Figure 2 Microscopic examination reveals small, Gram-negative bacilli with blunt ends and bipolar staining, mostly scattered, with a few existing in pairs.

[0023] PCR amplification of the 16S rRNA gene: Picking and purifying BbColonies were inoculated into TSB and cultured at 37°C with shaking for 24 h. Bacterial genomic DNA was extracted from the isolated bacterial culture using a bacterial genomic DNA extraction kit. Amplification was performed using 16S rRNA primers (F-AGAGTTTGATCCTGGCTCAG, R-TACGGCTACCTTGTTACGACTT), with an expected fragment size of 1428 bp. The primers were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0024] The PCR reaction mixture consisted of 25 μL: 12.5 μL of 2×T5 Super PCR Mix, 1 μL each of forward and reverse primers, 2 μL of DNA template, and 8.5 μL of ddH2O. The PCR program was as follows: 98 ℃ pre-denaturation for 5 minutes; 98 ℃ denaturation for 10 seconds, 55 ℃ annealing for 15 seconds, 72 ℃ extension for 30 seconds, for a total of 34 cycles, followed by a final extension at 72 ℃ for 2 minutes. Amplified fragments were observed after 1% agarose gel electrophoresis. The PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0025] like Figure 3 After PCR amplification of the 16S rRNA gene from the isolated strain, the target band appeared at 1428 bp after 1% agarose gel electrophoresis. The sequencing results of the amplified product were then compared with BLAST. Figure 4 One strain of feline bronchogenic bronchitis bacillus was isolated.

[0026] Example 2: Growth curve determination of the selected strains: The isolates were cultured in TSB until OD. 600 =1.0. The bacterial culture was diluted 1:100 and placed back into fresh culture medium. Incubate at 37 °C, 200 rpm / min, and measure OD every 2 hours. 600 And count viable bacteria. Three replicates were set up for each group, and the obtained data were used to plot a growth curve. For example... Figure 5 As can be seen from the growth curve, the isolates were in the growth adaptation period within 4 hours, in the logarithmic growth period from 4 hours to 13 hours, and in the growth stationary period from 13 hours to 24 hours.

[0027] Example 3: Virulence gene analysis: PCR amplification of isolated strains FLA , DNT , Bvgs , AC-Hly , FHA , brfZ , Prn , FhaB , betA , CyaA After identifying 10 virulence genes, the samples were subjected to 1% agarose gel electrophoresis, as follows: Figure 6Detected FLA , bvgs , betA , CyaA Four virulence genes. All four genes are associated with pathogenicity, among which... bvgs Genes can change according to changes in environmental conditions. This characteristic is achieved by regulating the expression of various virulence factors, which mediates host cell adhesion and immune escape. FLA It is a flagella-related gene. Mutants lacking flagella usually have significantly reduced virulence. This gene plays a key role in the early stages of infection, helping to overcome the mucosal barrier and promote colonization. betA Genes can enhance the survival ability of bacteria in the host and are an important supporting factor for chronic infection or systemic spread. CyaA Genes can directly damage host cells, suppress innate immune responses, and promote bacterial colonization and persistent infection by increasing cAMP levels in host cells, thereby disrupting immune cell function.

[0028] Using the MegAlign feature in the DNAstar software, other Bordetella feline bronchiseptica bacteria were analyzed from the NCBI database. bvgs Genes and CyaA Gene sequence analysis was performed, comparing differences in amino acid sequences. The results showed... bvgs The change of amino acid T to A at position 1045 of the gene may affect the function of the BvgAS two-component regulatory system, thereby altering the bacteria's regulation of virulence factors and motility-related genes, and affecting its pathogenicity, colonization and environmental adaptability. CyaA The change of amino acid N to D at position 97 and P to R at position 235 of the gene may alter the spatial structure of adenylate cyclase toxin, affecting its ability to bind to target cells, enzyme activity, and pore-forming activity, thereby affecting the bacteria's ability to evade host immunity and its pathogenicity.

[0029] Example 4: Whole genome sequencing analysis: such as Figure 11 The x-axis represents gene length, and the y-axis represents the number of corresponding genes. The total genome size of the isolated strain WF202409 is 5,233,092 bp, with 4,843 coding genes, 8 gene islands in the non-coding RNA, and 14 prophages. It contains 510 proteins with signal peptide structures, 1,027 proteins with transmembrane structures, 394 predicted as secretory proteins, 147 virulence factors, and 24 drug resistance genes.

[0030] Gene function annotation: GO database annotation: The GO function annotation results for strain WF202409 are as follows: Figure 12The numbers on the bar chart represent the number of annotated genes, and the vertical axis represents the secondary classification of GO functions in the sample gene annotations. This shows that their molecular functions are primarily catalytic and binding activities, with biological processes highly enriched in cellular and metabolic processes. This indicates that the strain possesses efficient material metabolism, energy conversion, and cell proliferation capabilities, which are related to modifications of cell surface polysaccharides or antigenic epitopes, thus affecting immune recognition and cross-protection profiles. Simultaneously, the presence of complete genes related to environmental adaptation, such as signal regulation and stimulus response, endows the strain with excellent environmental awareness and resilience. Cellular component analysis shows that the genes are mainly located in basic cellular structures, with a concise and stable genome structure, no redundant functional interference, and good potential for industrial applications and genetic stability.

[0031] COG database annotations: such as Figure 13 (The horizontal axis represents the COG functional type, and the vertical axis represents the number of genes annotated): COG functional classification of the genome of strain WF202409 shows a high enrichment of core metabolic functional genes, including amino acid transport and metabolism, energy production and conversion, lipid metabolism, and transcriptional regulation, demonstrating efficient material and energy utilization. Simultaneously, genes related to environmental adaptation, such as inorganic ion transport, signal transduction, and defense mechanisms, are complete, endowing the strain with excellent environmental tolerance and survival competitiveness. Furthermore, the strain exhibits a low proportion of genomic mobile elements, high genetic stability, and a large number of genes with unknown functions, possessing good application potential and value for further functional exploration.

[0032] NR database annotations: such as Figure 14 (The horizontal axis represents the species ID, and the vertical axis represents the number of genes annotated.) The NR database annotation results for strain WF202405 show that its coding genes are related to those of the genus Bordetella (…). Bordetella ) Species are highly homologous, among which... Bordetella bronchiseptica The presence of up to 2281 matching genes indicates that this strain belongs to the genus *Bordezoella* taxonomically, and is related to... Bordetella bronchiseptica The closest kinship.

[0033] CAZy database annotations: such as Figure 15 (The horizontal axis represents the types of carbohydrate-related enzymes, and the vertical axis represents the number of genes.) The CAZy database annotation results of strain WF202409 show that its carbohydrate-active enzymes are mainly glycosyltransferases (GT) and glycoside hydrolases (GH), and it is also enriched with carbohydrate binding module (CBM) and carbohydrate esterase (CE) related genes. This indicates that the strain has a high efficiency in carbohydrate synthesis, degradation and utilization, and can participate extensively in polysaccharide metabolism, carbon source utilization and extracellular polysaccharide synthesis. It has good application potential in biomanufacturing, environmental remediation, probiotic development and other fields.

[0034] Pathogen-Host Interaction Database (PHI) Notes: (e.g.) Figure 16 (The horizontal axis represents the phenotypic mutation type, and the vertical axis represents the number of annotated genes.) The PHI database annotation results for strain WF202405 show that it carries 31 hypervirulence-related genes. The loss of function of these genes directly leads to increased virulence levels, indicating that they are natural negative regulators of virulence, playing a crucial role in maintaining appropriate pathogenicity and preventing excessive host damage. The presence of these genes provides the molecular basis for the strain's fine-tuning of virulence, offering precise modification targets for attenuated vaccine development and core resources for elucidating the virulence regulation mechanism of pathogen-host interactions, thus possessing significant application and research value.

[0035] Example 5: Pathogenicity test in mice: Separate the present invention Bb The bacterial strain was inoculated into TSB medium containing 5% fetal bovine serum and cultured in a constant temperature shaking incubator at 37℃ and 180 rpm / min for 20 h. The colony concentration of the cultured bacterial solution was determined by viable cell counting, and the final inoculation concentration was determined through serial dilution. Thirty-six 6-week-old Kunming mice were randomly divided into 6 groups of 6 mice each. Mice in the experimental groups were sequentially injected intraperitoneally with 1×10⁻⁶ oz. 9 CFU / mL ~10 5 Each mouse was injected with 0.2 mL of CFU / mL bacterial suspension; control mice were injected with the same dose of TSB. The mice's mental state and eating habits were observed every 2 hours, and the morbidity and mortality were observed and recorded for 7 consecutive days.

[0036] The results showed that the experimental group mice exhibited lethargy, rough fur, and rapid breathing, but their body temperature and weight did not change significantly, while the control group showed normal behavior. Figure 7 Autopsy revealed pulmonary congestion and edema in the experimental group mice. Figure 8 The mortality rate in mice increased with increasing bacterial concentration. By streaking the lungs of dead mice with TSA solid medium, bacteria could be isolated. Bb The concentration and dosage of the bacterial solution injected into the five groups of mice, as well as the mortality rate, are shown in Table 1. The median LD50 of the mice was calculated using the Reed-Muench method. 50 The value is 3.16 × 10 7 CFU / mL.

[0037] Table 1: Results of Pathogenicity Tests on Isolated Bacteria

[0038] Example 6: Pathogenicity test in kittens: The isolated Bb was inoculated into TSB medium containing 5% fetal bovine serum and cultured at 37°C with shaking at 180 rpm / min for 20 h. Six 3-month-old kittens (negative for common pathogens and Bb antibody) were randomly divided into two groups of three. After slight anesthesia, the kittens in the experimental group were intranasally infected with 1×10⁻⁶ Bb. 9 Each kitten received 500 μL of bacterial suspension at CFU / mL; control kittens received the same dose of TSB via nasal instillation. Clinical symptoms were observed and scored daily after infection, and body temperature and weight were measured. Morbidity and mortality were recorded. Necropsy was performed on deceased experimental kittens during the observation period to observe pathological changes in various tissues and to isolate bacteria from the lungs. The observation period lasted 14 days.

[0039] Three kittens in the experimental group developed fever, nasal and ocular discharge, sneezing, lethargy, and a gradual decrease in appetite and weight three days after infection. The symptoms worsened from day 5 to 7, with purulent nasal and ocular discharge, nasal congestion, sneezing, difficulty breathing, lethargy, and severe loss of appetite. On day 5, a blood test showed a white blood cell count (WBC) of 20.16 ± 3.0 × 10⁻⁶. 9 / L, the total number of neutrophils (NEU) increased to 15.0±2.5 ×10 / L. 9 / L, the total lymphocyte count (LYM) decreased to 2.5±0.6×10. 9 Serum amyloid A (fSAA) concentration (119.01±12 mg / L) showed a significant increase. All three kittens died by day 14. Autopsy revealed pulmonary congestion and slight spleen enlargement. Figure 9 In the experimental group, nasopharyngeal swabs were collected daily for qPCR testing of Bb. Bb was detected in all samples, and the CT value showed a decreasing trend, indicating that the kittens were infected with Bb and could replicate and proliferate in the body and shed the virus. The control group had normal clinical manifestations and all indicators.

[0040] Example 7: Preparation of inactivated Bordetella bronchiseptica vaccine for felines: S1. Strain recovery and culture: Cryopreserved Bordetella felineis strains were streaked onto TSA plates and incubated at 37 ℃ in a 5% CO2 incubator for 24 hours. Single colonies were picked and inoculated onto TSB liquid medium and incubated at 37 ℃ with shaking at 200 rpm for 12-16 hours.

[0041] S2. Inactivation of bacterial suspension: When the OD of the bacterial culture is measured 600When the concentration of the bacterial culture reached 1.0, the bacterial culture was harvested, centrifuged at 4000 rpm / min for 5 minutes with sterile physiological saline, and the cells were washed three times. Formaldehyde solution with a final concentration of 0.4% was added, and the culture was inactivated at 37 ℃ for 36 hours. The inactivated bacterial culture was then inoculated onto TSA plates and TSB liquid medium, and incubated at 37 ℃, 5% CO2 incubator, and 37 ℃ constant temperature incubator for 7 days, respectively. Bacterial growth was observed to verify successful inactivation.

[0042] S3. Vaccine preparation: Take the qualified inactivated bacterial solution and mix it thoroughly with propolis adjuvant at a volume ratio of 25:1. After thorough mixing, let it stand at room temperature for 2-3 hours to allow the vaccine to fully emulsify. Then, dispense and store it in a refrigerator at 4 ℃ for later use.

[0043] S4. Physical property testing of vaccines.

[0044] The tests include: visual inspection, viscosity test, solubility and dispersibility test, sterility test of the vaccine, formaldehyde residue test, and safety test of the vaccine. Appearance inspection: High-quality propolis adjuvant should be relatively clear and transparent. The color of the propolis adjuvant should be a uniform liquid ranging from light yellow to brownish-yellow, without any layering or sedimentation. This is considered qualified.

[0045] Viscosity test: Use a pipette tip to draw 1 mL of the inactivated vaccine to be tested and allow it to flow out naturally at room temperature. If more than 0.4 mL flows out within 8 seconds, it meets the requirements.

[0046] Solubility and dispersibility test: Take a certain amount of vaccine sample, add an appropriate amount of physiological saline solvent, and observe the mixture by stirring at an appropriate speed in a constant temperature water bath at 37 ℃. If the sample can quickly dissolve in the solvent and form a uniform solution, it indicates that the solubility and dispersibility are good.

[0047] Sterility test of the vaccine: The inactivated vaccine was inoculated onto TSA plates and TSB liquid culture medium, and then placed in 37 ℃, 5% CO2 incubator and 37 ℃ constant temperature incubator for 7 days respectively. Sterile growth was observed, which meets the requirements.

[0048] Formaldehyde residue test: The test was conducted according to the method specified in the Chinese Veterinary Pharmacopoeia. The result was 0.16%, which meets the requirements (<0.2%).

[0049] Adjuvant content detection: The adjuvant content was determined according to the method specified in the Chinese Veterinary Pharmacopoeia. The result showed trace residues, which met the requirements.

[0050] Safety testing of the vaccine: Twelve 6-week-old Kunming mice were selected and divided into two groups. One experimental group was injected subcutaneously with 0.2 mL of the vaccine to be tested, while the other control group was injected subcutaneously with an equal volume of PBS. After 14 days of observation, no local or systemic adverse reactions caused by the vaccine were observed in the experimental group, which met the requirements.

[0051] Example 8: Evaluation of the immunization efficacy of inactivated Bordetella bronchiseptica vaccine in felines: Evaluation of the immunization effect of inactivated vaccines on rabbits: Twelve healthy 6-week-old New Zealand white rabbits were randomly divided into two groups of six each. The vaccine-immunized group received a subcutaneous injection of 1 mL of inactivated vaccine per rabbit in the neck, while the control group received a subcutaneous injection of 1 mL of sterile PBS in the neck. Seven days after the first immunization, a second immunization was administered using the same dose and method, followed by a third immunization 14 days later. Blood samples were collected from all rabbits every week, and serum was separated for agglutination testing to determine the Bb antibody titer.

[0052] Serum antibody detection and analysis as follows Figure 10 Antibodies began to develop in the vaccine-immunized group 7 days after the first immunization, and reached their peak 7 days after the third immunization. 5 No effective antibodies were produced in the control immunization group. This indicates that strain WF202409 can produce high levels of antibodies in rabbits.

[0053] Evaluation of the immunization efficacy of inactivated vaccines in cats: Nine healthy 3-month-old kittens were randomly divided into three groups of three each. Group A (homogeneous challenge) and Group B (heterogeneous challenge) received a subcutaneous injection of 1 mL of inactivated vaccine per kitten in the neck, while the control group received a subcutaneous injection of 1 mL of sterile PBS per kitten in the neck. Twenty-one days after the first immunization, a second immunization was administered using the same dosage and method. Twenty-one days after the second immunization, all three groups underwent a challenge test. Group A received nasal drops at an infectious concentration of 1 × 10⁻⁶. 9 CFU bacterial suspension WF202409 500μL / animal, Group B nasal drop infection concentration is 1×10 9 CFU bacterial suspension QD202407 (another feline Bb strain isolated, identified, and preserved in our laboratory) 500 μL / cat, and the blank control group was infected with the same dose of sterile PBS via nasal instillation. After challenge, the clinical symptoms of the three groups of experimental cats were observed daily, and their body temperature and weight were measured. The morbidity and survival rate of kittens were recorded.

[0054] In the blank control group, kittens developed severe respiratory symptoms 3 days after challenge, and all three died. The homologous challenge group (Group A) showed no significant clinical symptoms, while the heterologous challenge group (Group B) only experienced mild, transient runny nose; however, both groups had a 100% survival rate. 3-5 days after challenge, the blank control group showed a significant increase in body temperature (>40°C) and a continuous decrease in weight. Group A did not show a significant increase in body temperature, and its weight steadily increased; Group B experienced a transient increase in body temperature 3 days after challenge, with no significant change in weight. In conclusion, the developed inactivated vaccine not only provides complete protection against homologous strain challenge but also provides significant cross-protection against heterologous strain challenge and effectively reduces clinical symptoms.

[0055] Inactivated vaccines typically have weak immunogenicity, primarily producing type-specific immunity. Their protective effect against heterologous strains is usually poor or nonexistent. The inactivated vaccine against Bordetella bronchiseptica in feline bronchiolitis provides cross-protection against heterologous circulating strains, which is the result of screening for strains carrying specific conserved antigenic epitopes.

[0056] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A strain of Bordetella feline bronchiseptica ( Bordetella bronchiseptica, Bb ), characterized in that, The strain is Bordetella felis bronchiseptica strain WF202409, with accession number CCTCC No.35353.

2. The *Bordetella felis* strain as described in claim 1, characterized in that, The strain contains FLA , bvgs , betA and CyaA Virulence genes.

3. The application of the *Bordetella felis* strain as described in claim 1, characterized in that, Used to prepare vaccines to prevent respiratory diseases caused by Bordetella felineis.

4. A vaccine against inactivated Bordetella feline bronchiseptica, characterized in that, An inactivated bacterial solution containing the strain of Bordetella feline bronchiseptica as described in claim 1.

5. The inactivated Bordetella feline bronchiseptica vaccine as described in claim 4, characterized in that, The vaccine also contains propolis adjuvant.

6. The inactivated Bordetella feline bronchiseptica vaccine as described in claim 5, characterized in that, The volume ratio of the inactivated bacterial solution to the propolis adjuvant is 25:

1.

7. The inactivated Bordetella feline bronchiseptica vaccine as described in claim 4, characterized in that, Its preparation method specifically includes: S1. Bacterial resuscitation and culture: The frozen Bordetella feline bronchiseptica strain was streaked onto TSA plates and incubated in a 37 ℃, 5% CO2 incubator for 24 hours. Single colonies were picked and inoculated into TSB liquid medium and cultured at 37 ℃, 200 rpm / min for 12-16 hours with shaking. S2. Bacterial inactivation: When the OD value of the bacterial culture is measured... 600 When the concentration of bacteria reaches 1.0, the bacterial culture is harvested, centrifuged at 4000 rpm / min for 5 minutes with sterile physiological saline, the bacterial cells are washed 3 times, the bacterial cells are collected, and inactivated with formaldehyde solution. S3. Vaccine preparation: Mix the inactivated bacterial solution with propolis adjuvant, stir thoroughly, let stand at room temperature for 2-3 hours to allow the vaccine to fully emulsify, dispense and store in a 4 ℃ refrigerator for later use; S4. Physical property testing of vaccines.

8. The inactivated Bordetella feline bronchiseptica vaccine as described in claim 7, characterized in that, The specific inactivation conditions for the formaldehyde solution are as follows: the final concentration of formaldehyde is 0.4%, the inactivation temperature is 37°C, and the inactivation time is 36 hours.

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