A pentavalent vaccine for mink canine distemper virus, mink parvovirus, pseudomonas aeruginosa, klebsiella pneumoniae and clostridium botulinum, and a preparation method and application thereof

Abstract

The application discloses a five-component vaccine for mink canine distemper virus, mink parvovirus, pseudomonas aeruginosa, klebsiella pneumoniae and clostridium botulinum as well as a preparation method and application thereof, and belongs to the field of veterinary biological products. A mink parvovirus strain MEV-WH12 and a klebsiella pneumoniae strain K2-SD14 with a serum type of K2 are respectively obtained through self-isolation and systematic cultivation, and combined with an existing canine distemper virus CDV-OR12 strain, a pseudomonas aeruginosa DL-15 strain and a clostridium botulinum C62-4 strain to prepare a five-component vaccine. The five-component vaccine can prevent five diseases at the same time through one-time inoculation, significantly reduces the number of immunization, and reduces the stress of minks and the breeding cost. The protection rate of the vaccine after 21 days of immunization is 100%, the protection period is 5 months, the vaccine is safe for minks of different ages and pregnant minks, the storage period is 18 months, and the vaccine has a good commercial application prospect.

Description

Technical Field

[0001] This invention relates to the field of veterinary biological products, and in particular to a pentavalent vaccine for mink canine distemper virus, mink parvovirus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Clostridium botulinum, as well as its preparation method and application. Background Technology

[0002] Canine distemper, mink parvovirus enteritis, mink hemorrhagic pneumonia, mink pneumonia Klebsiella pneumoniae infection, and mink botulism are five major diseases that seriously threaten the mink farming industry. Among them, canine distemper virus is characterized by attacking the mucous membrane system, often causing biphasic fever and catarrhal pneumonia, with a mortality rate of up to 80%; mink parvovirus enteritis is characterized by severe diarrhea and is caused by mink parvovirus, which was first discovered in 1949 and was subsequently proven to be caused by parvovirus; mink hemorrhagic pneumonia has a rapid onset and high mortality rate, often occurring in local outbreaks, and is caused by Pseudomonas aeruginosa infection; Klebsiella pneumoniae, as an opportunistic pathogen, proliferates rapidly under stress conditions such as regrouping, heat, feed changes, and sudden weather changes that can lead to a decrease in the animal's resistance, causing acute abscesses, cellulitis, and septicemia in mink, often forming mixed infections with Pseudomonas aeruginosa; botulism is closely related to spoiled fish and poultry by-products in mink feed, and mink are listed as one of the susceptible animals for botulism toxin, with a mortality rate close to 100% after poisoning. Currently, vaccination remains the primary means of preventing the aforementioned diseases. Existing products are mainly monovalent or bivalent inactivated vaccines, such as mink viral enteritis inactivated vaccine, mink hemorrhagic pneumonia bivalent inactivated vaccine, and botulism inactivated vaccine.

[0003] However, because each of the five diseases mentioned above requires separate or combined immunization, mink farmers have to administer multiple vaccinations in actual production. Due to the sensitive nature of mink, repeated capture and injections can easily cause severe stress reactions, manifesting as loss of appetite, lethargy, and in severe cases, immunization failure or even death. Simultaneously, multiple immunizations significantly increase the labor intensity and immunization costs for farmers, reducing production efficiency and animal welfare. Although existing bivalent inactivated vaccines targeting two of these diseases exist, they still cannot simultaneously cover the five major pathogens: canine distemper virus, mink parvovirus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum, making it difficult to achieve the goal of "one injection for multiple protections." Developing a multivalent vaccine capable of simultaneously preventing these five diseases has become a pressing technical problem to be solved in this field.

[0004] The main challenges in developing multivalent vaccines lie in the significant differences in optimal culture conditions, inactivation processes, antigen ratios, and adjuvant compatibility among various pathogens (viruses, Gram-negative bacteria, and anaerobic spores). Simply mixing individual vaccines may lead to antigenic interference, decreased immunogenicity, or enhanced local adverse reactions. Furthermore, existing vaccine products have significant limitations in their applicability. Most publicly available technical solutions only evaluate efficacy in adult healthy mink, lacking systematic studies on the immunogenicity and safety of mink at different ages (especially young and old), and failing to address the safety assessment of pregnant mink as a special group. Due to the unique physiological state of pregnant mink, their tolerance to immune stimulation is low, while the immune systems of young mink are not yet fully developed; both groups are considered high-risk groups for vaccine application. Currently, there are no reports on the safety verification of pentavalent vaccine products simultaneously covering five antigens—canine distemper virus, parvovirus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum—in mink of different ages and in pregnant mink, leaving a technological gap for safe clinical application. Therefore, developing a pentavalent vaccine with a scientific formulation, stable process, reliable immunization effect, and safety for mink of different ages and pregnant mink is of great practical significance for achieving simultaneous prevention of five diseases with a single immunization, reducing mink stress, and lowering breeding costs. Summary of the Invention

[0005] The purpose of this invention is to provide a pentavalent vaccine against canine distemper virus, mink parvovirus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum, along with its preparation method and application, to address the problems existing in the prior art. The pentavalent mink vaccine provided by this invention achieves "multiple protections with one injection," simultaneously preventing five diseases with a single vaccination, significantly reducing the number of immunizations and lowering mink stress and breeding costs. The vaccine achieves 100% protection against challenge 21 days after immunization, with a protection period of 5 months. It is safe for mink of different ages and pregnant mink, and has a shelf life of up to 18 months, demonstrating promising commercial application prospects.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a pentavalent vaccine against canine distemper virus, mink parvovirus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum. The pentavalent vaccine comprises an inactivated component and an attenuated component. The inactivated component consists of inactivated mink parvovirus antigen, inactivated Pseudomonas aeruginosa antigen, inactivated Klebsiella pneumoniae antigen, and inactivated Clostridium botulinum toxin antigen. The attenuated component is a freeze-dried antigen of an attenuated strain of canine distemper virus. The mink parvovirus antigen is the mink parvovirus MEV-WH12 strain antigen, and the preservation number of the mink parvovirus MEV-WH12 strain is CGMCC No. 47099. The Klebsiella pneumoniae antigen is Klebsiella pneumoniae (Klebsiella pneumoniae K2-SD14 strain antigen, the Klebsiella pneumoniae K2-SD14 strain has the accession number CGMCC No.38101; The Pseudomonas aeruginosa antigen is Pseudomonas aeruginosa ( Pseudomonas aeruginosa DL-15 strain antigen; The attenuated strain of canine distemper virus is canine distemper virus (Canine distemper virus) Canine Distemper Virus ) CDV-OR12 strain.

[0007] Optionally, the content of the mink parvovirus antigen is not less than 10. 5.73 TCID 50 / mL; the content of the Pseudomonas aeruginosa antigen is not less than 2.0 × 10⁻⁶. 9 CFU / mL; the content of the Klebsiella pneumoniae antigen is not less than 2.0 × 10⁻⁶ CFU / mL. 9 CFU / mL; the content of the botulinum toxin antigen is not less than 21440 MLD / mL; the content of the attenuated strain of canine distemper virus is not less than 10 3.90 TCID 50 / mL.

[0008] Optionally, the pentavalent vaccine also contains adjuvants and preservatives.

[0009] Optionally, the adjuvant is aluminum hydroxide gel adjuvant; the preservative is thimerosal.

[0010] The present invention also provides a method for preparing the aforementioned pentavalent vaccine, comprising the following steps: (1) Prepare inactivated mink parvovirus antigen, inactivated Pseudomonas aeruginosa antigen, inactivated Klebsiella pneumoniae antigen, inactivated Clostridium botulinum toxin antigen and attenuated strain of canine distemper virus respectively. (2) Dilute the inactivated mink parvovirus antigen, inactivated Pseudomonas aeruginosa antigen, inactivated Klebsiella pneumoniae antigen, and inactivated Clostridium botulinum toxin antigen prepared in step (1) to the target concentration and mix them; the target concentration is: mink parvovirus antigen solution not less than 10 6.40 TCID 50 / mL, Pseudomonas aeruginosa antigen solution not less than 9.4×10 9 CFU / mL, Klebsiella pneumoniae antigen solution not less than 9.4 × 10⁻⁶. 9 CFU / mL, the toxicity of the botulinum toxin antigen solution is not less than 1.0 × 10⁻⁶. 5 MLD / mL; (3) The diluted mink parvovirus antigen solution, Pseudomonas aeruginosa antigen solution, Klebsiella pneumoniae antigen solution and Clostridium botulinum toxin antigen solution were mixed in a volume ratio of 1:1:1:1 to obtain a mixed antigen solution. Adjuvants and preservatives were added as inactivation components.

[0011] (4) After diluting the attenuated strain of canine distemper virus, freeze-drying and reconstitute it to ensure that the viral load of canine distemper virus is not less than 10. 3.90 TCID 50 / mL, used alone as a weakly toxic component; (5) The inactivated component is reconstituted with the attenuated component to obtain the pentavalent vaccine.

[0012] Optionally, in step (3), the volume ratio of the mixed antigen solution to the adjuvant is 6:1.

[0013] Optionally, in step (3), the amount of preservative added is 0.005% of the total volume of the mixed antigen solution and the adjuvant.

[0014] The present invention also provides the application of the pentavalent vaccine described above or the pentavalent vaccine prepared by the described preparation method in the preparation of products for the prevention of canine distemper, mink parvovirus enteritis, mink hemorrhagic pneumonia, mink Klebsiella pneumoniae infection, and mink botulism.

[0015] The present invention discloses the following technical effects: The pentavalent mink vaccine provided by this invention achieves "one-shot protection against multiple diseases" for five major diseases in mink farming. Compared with existing single or bivalent vaccines that require multiple immunizations, this invention can simultaneously prevent canine distemper, parvovirus enteritis, hemorrhagic pneumonia, Klebsiella pneumoniae infection, and botulism with a single vaccination. This significantly reduces the number of captures and injections, lowers stress responses in mink, and reduces the labor intensity and immunization costs for farmers. Experimental verification shows that the vaccine achieves 100% protection against viral challenge 21 days after immunization, and the protection period lasts for 5 months, effectively solving the practical problems caused by multiple immunizations in existing technologies.

[0016] Furthermore, through systematic safety verification, this invention has confirmed that the vaccine has good safety for mink of different ages (2-4 months and 8-10 months) as well as pregnant mink. Post-vaccination, only a transient increase in body temperature occurred, with no local or systemic adverse reactions. Simultaneously, this invention clarifies the optimal ratio and content of each antigen component, ensuring the vaccine's immunizing efficacy and batch-to-batch stability. The vaccine has a shelf life of 18 months and shows promising commercial application prospects, providing the mink farming industry with a safe, efficient, and convenient combined immunization solution. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The results are PCR identification results for mink parvovirus; where M: Marker; 1: Parvovirus-specific primer identification in cell culture; 2: Canine distemper virus-specific primer identification in cell culture; 3: Parvovirus positive sample; 4: Canine distemper virus positive sample; 5: Parvovirus negative sample; 6: Canine distemper virus negative sample. Figure 2 The colony morphology of Klebsiella pneumoniae on TSA agar medium (A) and the results of Gram staining microscopy (B). Figure 3 The results of PCR identification (A) and typing (B) of Klebsiella pneumoniae are shown; where M: Marker; 1: Positive control; 2, 4: Klebsiella pneumoniae; 3: Negative control; Figure 4 The results show the body temperature monitoring of mink after immunization with a single dose (A), a repeated single dose (B), and a super-dose (C) of the mink pentavalent vaccine. Figure 5 The results are monitoring of the injection site of mink after a single dose of the pentavalent vaccine; A: Anatomical diagram of the injection site of mink aged 2-4 months; B: Anatomical diagram of the injection site of mink aged 8-10 months; C: stained pathological section of the injection site of mink aged 2-4 months; D: stained pathological section of the injection site of mink aged 8-10 months. Figure 6 The results are monitoring of the injection site in mink after a single dose of the pentavalent vaccine; A: Anatomical diagram of the injection site in mink aged 2-4 months; B: Anatomical diagram of the injection site in mink aged 8-10 months; C: stained pathological section of the injection site in mink aged 2-4 months; D: stained pathological section of the injection site in mink aged 8-10 months. Figure 7 The results show the monitoring of the injection site in mink after overdose immunization with the pentavalent vaccine; A: Anatomical diagram of the injection site in mink aged 2-4 months; B: Anatomical diagram of the injection site in mink aged 8-10 months; C: stained pathological section of the injection site in mink aged 2-4 months; D: stained pathological section of the injection site in mink aged 8-10 months. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] This invention independently isolated and systematically cultured a mink parvovirus strain MEV-WH12 and a Klebsiella pneumoniae strain K2-SD14 with serotype K2, which were then combined with existing canine distemper virus (…). Canine Distemper Virus CDV-OR12 strain (strain preservation number: GMCC NO.13793, published in Chinese patent application number 201810182784.2), Pseudomonas aeruginosa ( Pseudomonas aeruginosa DL-15 strain (culture preservation number: CGMCC No. 3811, published in Chinese patent application number 201010203879.1) and Clostridium botulinum ( Clostrdium botulinumStrain C62-4 (culture preservation number: CVCC60353, from the National Veterinary Microbiology Culture Collection Center) was used to determine the content of each antigen component in the mink pentavalent vaccine through minimum immunization dose testing and antibody matching experiments. Specifically, the mink parvovirus content in the inactivated vaccine components should not be less than 10... 5.73 TCID 50 / mL, the content of Pseudomonas aeruginosa should not be less than 2.0×10 9 The CFU / mL and Klebsiella pneumoniae content should not be less than 2.0 × 10⁻⁶. 9 The CFU / mL and botulinum toxin content should be no less than 21440 MLD / mL; the canine distemper virus content in the attenuated vaccine components should be no less than 10 MLD / mL. 3.90 TCID 50 / mL.

[0025] The prepared pentavalent vaccine containing canine distemper virus, mink parvovirus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum was used to immunize mink. Good immunity was achieved 21 days after vaccination, with a 100% protection rate against challenge with virulent strains. The immunity period is 5 months, and the vaccine has a shelf life of 18 months. Furthermore, the vaccine demonstrates good safety in mink of different ages and in pregnant mink.

[0026] The present invention will now be described in detail with reference to specific embodiments.

[0027] Example 1: Isolation, Identification, and Cultivation of Mink Parvovirus Strains for Seed Production I. Experimental Methods 1. Isolation of mink parvovirus Samples clinically diagnosed as positive for mink parvovirus were collected, mixed with a small amount of sterile saline, and homogenized. Sterile saline was then added at a 1:5 mass-to-volume ratio (g / mL), and the mixture was thoroughly mixed. The mixture was centrifuged at 5000 rpm for 10 min at 4°C. The supernatant was filtered through a 0.1 µm filter to remove bacteria and mycoplasma. Simultaneously, 1 mL of the supernatant was inoculated into a cat kidney cell suspension (CRFK). Uninoculated normal cells were included as a control. The cells were cultured at 37°C and 5% CO2 for 24 h, then replaced with cell maintenance medium. Cytopathic effects were observed daily. After 3–5 days of culture, the virus was harvested after one freeze-thaw cycle and labeled as the first generation of mink parvovirus cell culture. The culture was passaged four times in CRFK cells and stored at -20°C.

[0028] 2. Identification of mink parvovirus Cell cultures stored at -20℃ were used to extract viral genomic DNA using a DNA extraction kit and stored at -20℃. Viral genomic RNA was extracted using an RNA extraction kit, reverse transcribed into cDNA, and stored at -20℃. Mink parvovirus-specific primers and canine distemper virus-specific primers were used for identification.

[0029] 3. Cultivation of Mink Parvovirus Strains for Vaccination 3.1 Mycoplasma removal passage First-generation mink parvovirus cell culture was collected, centrifuged at 10,000 rpm and 4°C for 10 min to remove cell debris, and the supernatant was filtered through a 0.1 μm filter to remove mycoplasma. The filtrate was simultaneously inoculated into CRFK cell suspension (prepared with mycoplasma-removed medium) and cultured at 37°C and 5% CO2 for 24 h. The culture was then replaced with mycoplasma-removed medium (containing 2% FBS), and cytopathic effects were observed daily. Virus was harvested when more than 90% of cells showed cytopathic effects, and this culture was labeled as second-generation mink parvovirus cell culture and stored at -20°C. The mink parvovirus was passaged twice more using the same method to obtain fourth-generation mink parvovirus cell culture.

[0030] 3.2 Mycoplasma suppression of passage Mink parvovirus (MEV) generation 4 cell culture was simultaneously inoculated into CRFK cell suspension (prepared with mycoplasma inhibition medium) and cultured at 37°C and 5% CO2 for 24 hours. The culture was then replaced with mycoplasma inhibition medium (containing 2% FBS), and cytopathic effects were observed daily. When more than 90% of the cells showed cytopathic effects, the virus was harvested and labeled as mink parvovirus generation 5 cell culture, which was stored at -20°C. The mink parvovirus was passaged twice more using the same method to obtain MEV-WH12 generation 7 cell culture.

[0031] 4. Mycoplasma test for strains used in mink parvovirus vaccine production Cell cultures of mink parvovirus at passages 5, 6, and 7 were used for mycoplasma detection according to the methods shown in Appendix III of the Chinese Veterinary Pharmacopoeia.

[0032] 5. Immunogenicity determination of mink parvovirus strains used for vaccine production 5.1 Virus propagation and harvest Mink parvovirus passage 7 cell culture was simultaneously inoculated into prepared CRFK cell suspension at a ratio of 1% (v / v). After culturing at 37°C and 5% CO2 for 24 hours, the culture was replaced with cell maintenance medium. Cytopathic effects were observed daily. When more than 90% of the cells showed cytopathic effects, the virus was harvested, which was the mink parvovirus passage 8 cell culture, and stored at -20°C. The mink parvovirus was passaged to 30 generations using the same method, and each generation of cell culture was stored at -20°C.

[0033] 5.2 Immunogenicity test Cell cultures from passages 5, 8, 11, 14, 17, 20, 23, 26, and 29 were collected, and the viral load was adjusted to 10-1. 6.00 TCID 50 / mL, after inactivation with formaldehyde, was mixed with an appropriate volume of aluminum glue adjuvant to achieve a final aluminum glue adjuvant concentration of 2%. Each generation of the inactivated vaccine was administered to five healthy, negative mink, 1mL per mink, via intramuscular injection on the medial side of the hind limb. Five unvaccinated mink were also included. Twenty-one days after vaccination, the mink, along with the control group, were orally administered 100 IDs. 50 A virulent strain of mink parvovirus was used for testing, and the disease incidence in challenged mink was observed for 10 consecutive days.

[0034] II. Experimental Results 1. Molecular biological identification results of mink parvovirus Mink parvovirus cell cultures were identified using specific primers for both mink parvovirus and canine distemper virus. A specific 573 bp band was amplified using the corresponding primers for canine distemper virus; however, no band was amplified using the corresponding primers for canine distemper virus. The results indicated a positive result for parvovirus and a negative result for canine distemper virus. Figure 1 This indicates that mink parvovirus was successfully isolated and was free from canine distemper virus contamination.

[0035] 2. Mycoplasma test results of the strain used for mink parvovirus vaccine production. Mycoplasma testing was performed on the 5th, 6th, and 7th generation cell cultures of mink parvovirus, and the results were all negative, indicating that the strains used for mink parvovirus vaccine production were free of mycoplasma contamination after mycoplasma removal.

[0036] 3. Immunogenicity test results of the strain used for mink parvovirus vaccine production Immunogenicity assays of different generations of mink parvovirus vaccine strains showed that inactivated vaccine strains from different generations protected mink from virulent infection, preventing disease and death, while all mink in the unvaccinated control group developed the disease. This indicates that mink parvovirus strains from generations 5 to 29 possess good immunogenicity.

[0037] In this embodiment, a mink parvovirus vaccine strain with good purity, stable passage, high virus content, and good immunogenicity was successfully isolated and cultured, designated as Mink Parvovirus MEV-WH12. This strain was deposited on March 19, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 47099.

[0038] Example 2: Isolation, Identification, and Cultivation of Klebsiella pneumoniae Strains for Vaccine Preparation I. Experimental Methods 1. Isolation and identification of Klebsiella pneumoniae Lung tissue was collected from diseased mink exhibiting typical symptoms of hemorrhagic pneumonia. An incision was made in the lung tissue using sterile scissors, and an inoculation loop was inserted into the incision to collect the sample. The sample was then streaked onto TSA agar medium and incubated at 37°C for 18–24 h. Single colonies with typical morphology were selected for subculturing and purification. The purified colonies were subjected to Gram staining and microscopic examination. Suspected Klebsiella pneumoniae strains were identified by PCR and serotyped using capsular serology.

[0039] 2. Cultivation of Klebsiella pneumoniae The isolated *Klebsiella pneumoniae* was streaked onto TSA agar medium and incubated at 37°C for 18–24 hours. Typical colonies were selected and inoculated into TSB liquid medium and incubated at 36–37°C for 18–24 hours. This constitutes the first generation culture of *Klebsiella pneumoniae*. After counting the first generation cultures, a portion was lyophilized and stored. The first generation culture of *Klebsiella pneumoniae* was inoculated into TSB liquid medium and incubated at 36–37°C for 18–24 hours. This constitutes the second generation culture of *Klebsiella pneumoniae*. After counting the second generation cultures, a portion was lyophilized and stored. *Klebsiella pneumoniae* was passaged to 24 generations using the same method, and each generation of cultures was lyophilized and stored separately.

[0040] 3. Virulence determination of Klebsiella pneumoniae Freeze-dried cultures of Klebsiella pneumoniae (generations 1, 12, and 24) were recovered and inoculated onto TSA plates. After incubation at 37°C for 18–24 h, single colonies were picked and transferred to TSB liquid medium. The medium was then incubated at 37°C and 220 rpm for 18–24 h. After bacterial counting, the cultures were appropriately diluted to a concentration of 1.5 × 10⁻⁶. 3 CFU / mL. 1.0 mL of the 1st, 12th, and 24th generation bacterial cultures were inoculated into 5 healthy, susceptible mink aged 2–6 months. A control group of 5 mink was also established, inoculated with 1.0 mL of sterile saline. Disease incidence and mortality were observed and recorded for 14 consecutive days.

[0041] 4. Immunogenicity assay of Klebsiella pneumoniae Cultures of Klebsiella pneumoniae from the 1st, 12th, and 24th generations, preserved by freeze-drying, were inoculated onto TSA plates after resuscitation and cultured at 37°C for 18–24 hours. Single colonies were then picked and cultured in TSB liquid medium at 37°C and 220 rpm for 18–24 hours. After bacterial counting, the cultures were inactivated with formaldehyde. The inactivated bacterial solution was mixed with aluminum glue adjuvant to prepare a monovalent vaccine. Mink were immunized intramuscularly with 1.0 mL per mink, with 5 mink per group. A control group without the vaccine was also included. Twenty-one days later, the mink, along with the control group (5 mink), were challenged with a virulent strain of Klebsiella pneumoniae. Disease incidence and mortality were observed and recorded for 14 consecutive days.

[0042] II. Experimental Results 1. Isolation and identification of Klebsiella pneumoniae Klebsiella pneumoniae forms grayish-white, round, raised, smooth colonies with neat edges on the culture medium, which can be pulled into filaments when picked up; Gram staining and microscopic examination show that the isolated strain is a Gram-negative bacillus with a distinct capsule, no spores, blunt ends, scattered singly, with a few cells connected in pairs, and medium-sized, short, stout bacilli. Figure 2 The 16S rRNA gene was amplified using universal and typing primers. The universal primers amplified a DNA band of approximately 1500 bp, while the typing primers amplified a corresponding specific DNA band of 641 bp. The results indicated that the isolated Klebsiella pneumoniae was of type K2. Figure 3 ).

[0043] 2. Virulence test results of Klebsiella pneumoniae Mink inoculated with different generations of Klebsiella pneumoniae (generations 1, 12, and 24) all showed typical symptoms, with a morbidity rate of 100%. The main symptoms in the affected mink were elevated body temperature (above 40.5°C), lethargy, loss of appetite, severe difficulty breathing (abdominal breathing), rough and disheveled fur, and frothy bright red blood flowing from the nostrils or mouth.

[0044] 3. Immunogenicity test results of Klebsiella pneumoniae Different generations of Klebsiella pneumoniae (generations 1, 12, and 24) were used to prepare monovalent vaccines, which were intramuscularly inoculated into mink. A challenge experiment was conducted 21 days later. The results showed that immunization with different generations of Klebsiella pneumoniae protected mink from infection by the virulent bacterial solution, with a protection rate of 100%. All mink in the control group developed the disease. This indicates that Klebsiella pneumoniae maintains good immunogenicity from generation 1 to generation 24.

[0045] In this embodiment, a K2 strain of Klebsiella pneumoniae with good purity, stable passage, and good immunogenicity was successfully isolated and cultured, and is designated as Klebsiella pneumoniae (K2). Klebsiella pneumoniaeThe strain K2-SD14 was deposited on March 19, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 38101.

[0046] Example 3: Study on the antigen ratio of a five-in-one vaccine containing canine distemper virus, mink parvovirus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum. I. Experimental Methods 1. Determination of the minimum immunization dose (MID) of mink parvovirus MEV-WH12 strain Mink parvovirus MEV-WH12 strains from different culture passages were taken and serially diluted 10-fold with sterile physiological saline. 10 samples were then taken. -1 10 -2 Two dilutions of the virus solution were inactivated with formaldehyde. After verification that inactivation was complete, they were mixed thoroughly with 25% aluminum glue adjuvant at a volume ratio of 6:1. Healthy susceptible mink were immunized with 0.5 mL and 1.0 mL doses, respectively, with 5 mink in each group. A control group of 5 mink was also included. Twenty-one days post-immunization, the mink were challenged with a virulent strain of the test virus. After 10 days of observation, the minimum immunization dose of mink parvovirus MEV-WH12 strain was determined.

[0047] 2. MID determination of Pseudomonas aeruginosa strain DL-15 Pseudomonas aeruginosa strain DL-15 from different culture passages was cultured, counted, and then inactivated with formaldehyde. After verification that inactivation was complete, it was mixed with aluminum hydroxide gel adjuvant at a volume ratio of 6:1 to prepare an immunoantigen. The bacterial content in the finished antigen was 4.0 × 10⁻⁶. 9 CFU / mL. Healthy susceptible mink were inoculated with the finished antigen at doses of 0.125 mL (with aluminum hydroxide saline added to 1 mL), 0.25 mL (with aluminum hydroxide saline added to 1 mL), 0.5 mL, and 1 mL, with 10 mink inoculated at each dose. Twenty-one days after immunization, the mink, along with five control mink, were challenged with a virulent bacterial solution. They were observed for 7 days to determine the minimum immunizing dose of Pseudomonas aeruginosa strain DL-15 against mink.

[0048] 3. MID determination of Klebsiella pneumoniae K2-SD14 strain Klebsiella pneumoniae strain K2-SD14 from different culture passages was cultured, counted, and then inactivated with formaldehyde. After verification that inactivation was complete, it was mixed with aluminum hydroxide gel adjuvant at a volume ratio of 6:1 to prepare an immunoantigen. The bacterial content in the finished antigen was 2.0 × 10⁻⁶. 9CFU / mL. Healthy susceptible mink were inoculated with the finished antigen at doses of 0.125 mL (with aluminum hydroxide saline solution added to 1 mL), 0.25 mL (with aluminum hydroxide saline solution added to 1 mL), 0.5 mL, and 1 mL, with 5 mink inoculated at each dose. 21 days after immunization, the mink, along with 5 control mink, were challenged with a potent bacterial solution. They were observed for 14 days to determine the minimum immunizing dose of the bacterial strain for the mink.

[0049] 4. MID determination of Clostridium botulinum strain C62-4 Clostridium botulinum C62-4 strains from different culture passages were used to determine their virulence (MLD) in mice, diluted with sterile physiological saline to 10⁻⁶. 5 MLD / mL, then inactivated with formaldehyde. After verification of complete inactivation, it was mixed with aluminum glue adjuvant at a volume ratio of 6:1. Healthy susceptible mink were inoculated with the inactivated antigen at doses of 0.0625 mL (replenished to a final concentration of aluminum glue saline to 1 mL), 0.125 mL (replenished to a final concentration of aluminum glue saline to 1 mL), 0.25 mL (replenished to a final concentration of aluminum glue saline to 1 mL), 0.5 mL (replenished to a final concentration of aluminum glue saline to 1 mL), and 1 mL, with 10 mink inoculated at each dose. 21 days post-immunization, mink were injected intramuscularly with 10 MLD of the virulent virus along with the control group. After 14 days of observation, the minimum immunization dose of Clostridium botulinum C62-4 strain against mink was determined.

[0050] 5. MID determination of canine distemper virus CDV-OR12 strain Canine distemper virus CDV-OR12 strains from different culture passages were collected, and the virus content was determined by dividing the samples into 10 groups. -1 10 -2 10 -3 Dilute to a certain degree, take 10 -2 10 -3 Healthy mink were immunized with two dilutions at intramuscular doses of 1 mL and 2 mL, respectively, with 5 mink in each group. A control group of 5 mink was also included. Twenty-one days post-immunization, the mink were challenged with a virulent strain of canine distemper virus for testing. After 14 days of observation, the minimum immunization dose of canine distemper virus CDV-OR12 strain for mink was determined.

[0051] 6. Antigen ratio of the five-in-one vaccine containing mink distemper virus, mink parvovirus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum. Based on the minimum immunizing doses of mink parvovirus MEV-WH12 strain, Pseudomonas aeruginosa DL-15 strain, Klebsiella pneumoniae K2-SD14 strain, and Clostridium botulinum C62-4 strain, four groups of quadrivalent inactivated vaccine components containing different antigen doses were prepared. Each dose (1 mL) contained antigen doses of 0.5 MID, 1 MID, 2 MID, and 3 MID, respectively. Simultaneously, based on the minimum immunizing dose of canine distemper virus CDV-OR12 strain, four groups of attenuated vaccine components containing different antigen doses were prepared and freeze-dried. After freeze-drying, the virus content per dose (1 mL) was 1 MID, 2 MID, 4 MID, and 6 MID, respectively. The attenuated vaccine components were reconstituted with the inactivated vaccine components and immediately inoculated into healthy susceptible mink. Each mink was injected intramuscularly with one dose (1 mL). Challenge was performed at 21 and 150 days post-immunization to determine the vaccine's protective rate at different immunization times.

[0052] II. Experimental Results 1. MID assay results of mink parvovirus MEV-WH12 strain When the parvovirus content in the inoculation is 10 5.43 TCID 50 When the above conditions were met, the protection rate against challenge 21 days after inoculation with mink was 100%; when the parvovirus content of the inoculated mink was 10... 5.13 ~10 5.30 TCID 50 Between these values, the protection rate of mink ranges from 20% to 60%. Therefore, the minimum immunization dose of mink parvovirus MEV-WH12 strain for mink was determined to be 10. 5.43 TCID 50 This translates to a viral load of 10 before inactivation. 5.49 TCID 50 .

[0053] 2. MID determination results of Pseudomonas aeruginosa strain DL-15 When inoculated with 1 mL (containing 4×10⁻⁶ bacteria) 9 CFU), 0.5 mL (containing 2×10⁻⁶ bacteria). 9 At a dose of CFU, all mink received 100% protection; the inoculation dose was 0.25 mL (containing 1×10⁻⁶ bacteria). 9 When CFU was administered, the protection rate of mink was 80%–100%; the inoculation dose was 0.125 mL (containing 0.5 × 10⁻⁶ bacteria). 9 When CFU is used, the protection rate for mink is 60%–80%. Therefore, the minimum immunization dose of Pseudomonas aeruginosa strain DL-15 against mink is determined to be 0.25 mL (containing 1.0 × 10⁻⁶ CFU). 9 CFU, which is the live bacteria content before seedling inoculation, is 1.17 × 10⁻⁶. 9 CFU / mL.

[0054] 3. Results of MID assay for Klebsiella pneumoniae strain K2-SD14 Inoculate with 1 mL (containing 2.0 × 10⁻⁶ bacteria). 9 CFU), 0.5 mL (1.0 × 10⁻⁶) 9 Mink immunized with CFU (0.25 mL, 0.5 × 10⁻⁵) all achieved 100% (5 / 5) protection; 9 CFU), 60% (3 / 5) to 80% (4 / 5) of mink were protected after vaccination; immunization with 0.125 mL (0.25 × 10⁻⁶) 9 The protection rate was below 20% (1 / 5). All mink in the control group became ill and died. Based on the experimental results, the minimum immunization dose of Klebsiella pneumoniae strain K2-SD14 against mink was determined to be 0.5 mL (1.0 × 10⁻⁶ CFU). 9 CFU, which is the live bacteria content before seedling inoculation, is 1.17 × 10⁻⁶. 9 CFU / mL.

[0055] 4. Results of MID assay for Clostridium botulinum strain C62-4 When the immunization dose exceeds 0.125 mL (approximately 10,720 mouse MLDs), mink vaccination provides 100% protection; at an immunization dose of 0.0625 mL (approximately 5,360 mouse MLDs), the protection rate in mink vaccination ranges from 30% to 50%. Therefore, the minimum immunization dose of Clostridium botulinum strain C62-4 against mink is determined to be 0.125 mL (approximately 10,720 mouse MLDs), which translates to a virulence of approximately 12,500 MLD / mL for the uninactivated Clostridium botulinum culture in mice.

[0056] 5. Results of MID assay for canine distemper virus CDV-OR12 strain When the immunization dose exceeds 10 3.30 TCID 50 At that time, mink vaccination provides 100% protection; the immunization dose is between 10 2.80 ~10 3.10 TCID 50 At that time, immunized animals achieved 60-80% protection, while all control group animals developed the disease, exhibiting typical canine distemper symptoms. Therefore, the minimum immunization dose of canine distemper virus CDV-OR12 strain against mink was determined to be 10. 3.30 TCID 50 .

[0057] 6. Antigen ratio results of the five-in-one vaccine containing mink distemper virus, mink parvovirus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum. 21 days after immunization, when the antigen dose of the inactivated vaccine component exceeded one MID, the mink protection rate against parvovirus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum toxin was 100%; when the antigen dose of the attenuated vaccine component exceeded one MID, the mink protection rate against canine distemper virus was 100%. 150 days after immunization, when the antigen dose of the inactivated vaccine component exceeded two MIDs, the mink protection rate against parvovirus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum toxin was 100%; when the antigen dose of the attenuated vaccine component exceeded four MIDs, the mink protection rate against canine distemper virus was 100%. Therefore, the antigen dose of the inactivated vaccine component in the mink pentavalent vaccine was determined to be two MIDs, and the antigen dose of the attenuated vaccine component to be four MIDs, meaning the mink parvovirus content in the inactivated vaccine component should be no less than 10 mg / L. 5.73 TCID 50 The concentration of Pseudomonas aeruginosa fraction should not be less than 2.0 × 10⁶ / mL. 9 The CFU / mL concentration and the content of Klebsiella pneumoniae components should not be less than 2.0 × 10⁻⁶. 9 The CFU / mL and botulinum toxin component content should be no less than 21440 MLD / mL; the canine distemper virus content in the attenuated vaccine component should be no less than 10 MLD / mL. 3.90 TCID 50 / mL.

[0058] Example 4: Preparation of a pentavalent vaccine containing mink distemper virus, mink parvovirus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum. I. Experimental Methods 1. Preparation and testing of inactivated vaccine components in mink pentavalent vaccine 1.1 Preparation of virus solution for mink parvovirus MEV-WH12 strain vaccine production Reconstituted mink parvovirus (MEV-WH12 strain) and simultaneously inoculate it into CRFK cell suspension at a ratio of 0.1–0.2% (v / v). After mixing, incubate at 37°C for 4–5 days. When more than 80% of the cells show cytopathic effects (CPE), discard contaminated or abnormally growing cells. Harvest the virus using a repeated freeze-thaw cycle and determine the virus content. Add formaldehyde solution at 0.2% of the total virus volume while shaking for 20 min. Inactivate the virus at 37°C for 24 h, shaking 2–3 times for 10 min each time.

[0059] 1.2 Preparation of bacterial culture for seedling production of Pseudomonas aeruginosa strain DL-15 Reconstitute the basic strain of *Pseudomonas aeruginosa* (DL-15 strain) and inoculate it into PYG agar medium, incubating at 36–37°C for 18–24 h. Select typical colonies and inoculate them into PYG liquid medium, incubating at 36–37°C for 18–24 h. Then, inoculate a larger volume of PYG liquid medium at a ratio of 3% (v / v), incubating at 36–37°C for 18–24 h, and determine the bacterial count. Add formaldehyde solution at 0.2% of the total bacterial volume while stirring, and inactivate the bacteria at 37°C for 24 h.

[0060] 1.3 Preparation of bacterial suspension for Klebsiella pneumoniae vaccine production Reconstitute the basic Klebsiella pneumoniae strain (K2-SD14) and inoculate it onto TSA agar medium. Incubate at 37°C for 18–24 h. Select typical colonies and inoculate them onto TSB liquid medium. Incubate at 36–37°C for 18–24 h. Inoculate a larger volume of TSB liquid medium at a ratio of 2–3% (v / v) and incubate at 36–37°C for 18–24 h. Determine the bacterial count. Add formaldehyde solution at 0.5% of the total bacterial volume while stirring, and inactivate the bacteria at 37°C for 48 h.

[0061] 1.4 Preparation of bacterial culture for Clostridium botulinum strain C62-4 seedling production The basic Clostridium botulinum strain (C62-4) was inoculated into a semi-solid broth (containing 0.05% agar and 0.5% gelatin) and cultured at 35–37°C for 3 days. Then, 1–2% (v / v) of the culture medium was inoculated into a larger volume of broth and cultured at 35–37°C for 3–5 days. Finally, 1–2% (v / v) of the culture medium was inoculated into a larger volume of broth and cultured statically at 35°C for 5–7 days. The virulence of the bacterial culture was then determined. Formaldehyde solution was added at 0.8% of the bacterial culture volume, and the culture was detoxified at 37°C for 10 days, with stirring three times daily.

[0062] 1.5 Preparation and Packaging of Inactivated Vaccine Components in Mink Pentavalent Vaccine Based on the virus content, viable bacteria count, and virulence of the pre-inactivated virus (bacterial) solution used for vaccine preparation, the mink parvovirus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum virus (bacterial) solutions were diluted with physiological saline to a concentration of not less than 10. 6.40 TCID 50 / mL, 9.4×10 9 CFU / mL, 9.4×10 9 CFU / mL and 1.0×10 5MLD / mL. The diluted mink parvovirus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum vaccine preparation solutions were mixed in a 1:1:1:1 ratio, then thoroughly mixed with 25% aluminum hydroxide gel solution at a 6:1 volume ratio. Finally, 0.005% of the total volume of thimerosal solution was added, and the mixture was thoroughly mixed. The mixture was then quantitatively dispensed into 100mL sterile vaccine vials and stored at 2–8℃.

[0063] 1.6 Detection of inactivated vaccine components in mink pentavalent vaccine The properties of the inactivated vaccine components were tested using conventional methods, and the fill weight, sterility, and residual formaldehyde and mercury preservatives were determined according to the appendix of Part III of the current Chinese Veterinary Pharmacopoeia.

[0064] 2. Preparation and testing of attenuated live vaccine components in mink pentavalent vaccine 2.1 Preparation of virus solution for canine distemper virus CDV-OR12 strain vaccine production The preserved canine distemper virus CDV-OR12 strain was reconstituted and inoculated onto Vero cells for virus amplification culture via adsorption. The virus status of the cells was observed daily. When the cytopathic effect reached about 80%, the virus fluid was harvested by repeated freeze-thaw cycles, the virus content was measured, and the virus was stored at -20℃.

[0065] 2.2 Freeze-drying of attenuated live vaccine components in mink pentavalent vaccine Based on the viral load of canine distemper virus CDV-OR12 strain, after appropriate dilution with physiological saline, 1-2 parts of lyophilization protectant were added to the virus solution at a ratio of 3-5, quantitatively dispensed into sterile 100mL blue bottles, freeze-dried, and stored below -15℃.

[0066] 2.3 Detection of attenuated vaccine components in mink pentavalent vaccine (1) The characteristics of the attenuated vaccine components were tested according to conventional methods, and the sterility, mycoplasma and exogenous virus tests were conducted according to the appendix of Part III of the current Chinese Pharmacopoeia.

[0067] (2) Identification test The attenuated vaccine components were reconstituted with sterile water to achieve a viral load of 200 TCID50 for canine distemper virus. 50 / 0.1mL. Take 1.0mL of virus dilution, add 1.0mL of canine distemper virus positive serum, mix well, neutralize at 37℃ for 1 hour, and seed 0.1mL into 4 wells of a 24-well cell plate. Add 0.9mL of culture medium containing Vero cells. Also include 4 wells each of normal cell control and virus dilution control. Incubate at 37℃ in a 5% CO2 incubator for 5 days. All virus dilution control wells should show CPE, while the neutralization test wells and normal cell control wells should not show CPE.

[0068] (3) Virus content testing The attenuated vaccine components were reconstituted with 100 mL of sterile water, and the viral load of canine distemper virus CDV-OR12 strain was determined using standard methods. The viral load should be within 10%. 3.90 TCID 50 / mL or more.

[0069] (4) The remaining moisture and vacuum degree shall be tested and determined in accordance with the appendix of Part III of the current Chinese Veterinary Pharmacopoeia.

[0070] 3. Inspection of mink pentavalent vaccines 3.1 Safety Inspection of Five-in-One Mink Vaccines The inactivated vaccine component was reconstituted with the attenuated vaccine component, and after being mixed evenly, 5 healthy and susceptible mink were selected. Each mink was injected intramuscularly with 3 mL of vaccine at 3 injection points. The mink were observed for 14 days, and their mental state, appetite, breathing, feces, body temperature, injection site, morbidity and mortality were observed daily.

[0071] 3.2 Efficacy Test of Mink Pentavalent Vaccine Reconstitute the attenuated vaccine component with the inactivated vaccine component, mix thoroughly, and set aside.

[0072] (1) Mink parvovirus Five healthy, susceptible mink were each injected intramuscularly with 1 mL of vaccine, while five unvaccinated control mink were also included. Twenty-one days post-immunization, the serum titer of mink parvovirus hemagglutination inhibition antibodies was measured.

[0073] (2) Part of Pseudomonas aeruginosa Five healthy, susceptible mink were injected intramuscularly with 1 mL of vaccine. Twenty-one days later, along with five control mink, they were challenged with a potent bacterial solution (containing one minimum lethal dose) and observed for seven consecutive days.

[0074] (3) Klebsiella pneumoniae Five healthy, susceptible mink were injected intramuscularly with 1 mL of vaccine. Twenty-one days later, these mink, along with five control mink, were challenged with a potent bacterial solution (containing 1.5 × 10⁻⁶ ppm). 3 (CFU / mL), observed for 14 consecutive days.

[0075] (4) Clostridium botulinum Four healthy, susceptible mink were each injected intramuscularly with 1 mL of the vaccine. Twenty-one days later, the mink, along with two control mink under the same conditions, were each injected intramuscularly with 1 mL of botulinum toxin (containing 10 MLDs), and observed for 14 consecutive days.

[0076] (5) Canine distemper virus part Five healthy, susceptible mink were each injected intramuscularly with 1 mL of vaccine, while five unvaccinated control mink were also included. Twenty-one days post-immunization, the titer of neutralizing antibodies against canine distemper virus in their serum was measured.

[0077] II. Experimental Results 1. Test results of the characteristics of inactivated vaccine components in mink pentavalent vaccine Physical properties of the inactivated vaccine component from the mink pentavalent vaccine were observed. The results showed that after standing for 24 hours, the upper layer was a light yellowish-brown clear liquid, and the lower layer was a grayish-white precipitate. After shaking, it became a uniform suspension.

[0078] 2. Test results of the packaging quantity of inactivated vaccine components in the mink pentavalent vaccine The dosage of the inactivated vaccine component in the mink pentavalent vaccine was tested, and the results showed that the dosage of each component was not less than 100.0 mL.

[0079] 3. Aseptic test results of inactivated vaccine components in mink pentavalent vaccine The inactivated vaccine components from the mink pentavalent vaccine were subjected to sterility testing according to the appendix of Part III of the current Chinese Veterinary Pharmacopoeia. The results showed that the components did not grow sterilely after being inoculated into the culture medium.

[0080] 4. Results of determination of formaldehyde and mercury preservative residues in inactivated vaccine components of mink pentavalent vaccine The inactivated vaccine components of the mink pentavalent vaccine were tested for formaldehyde and mercury preservative residues according to the appendix of Part III of the current Chinese Veterinary Pharmacopoeia. The results showed that the formaldehyde residue in the components was less than 0.5% and the thimerosal residue was less than 0.01%, which met the requirements.

[0081] 5. Results of phenotypic testing of attenuated live vaccine components in mink pentavalent vaccine The live attenuated vaccine components in the mink pentavalent vaccine are milky white or slightly yellow spongy loose clumps that easily detach from the bottle wall and dissolve rapidly after adding diluent.

[0082] 6. Results of purity test for attenuated live vaccine components in mink pentavalent vaccine After reconstituted the attenuated vaccine components from the mink pentavalent vaccine, sterility, mycoplasma, and exogenous virus tests were conducted according to the appendix of Part III of the current Chinese Veterinary Pharmacopoeia. The results showed that the sterility, mycoplasma, and exogenous virus tests were all negative.

[0083] 7. Identification test results of attenuated live vaccine components in mink pentavalent vaccine The attenuated vaccine components from the mink pentavalent vaccine were subjected to identification tests. The results showed that CPE appeared in all control wells of the virus dilution solution, while no CPE appeared in the neutralization test wells and normal cell control wells, which met the requirements.

[0084] 8. Test results of virus content in attenuated vaccine components of mink pentavalent vaccine The mink pentavalent live attenuated vaccine components were reconstituted with 100 mL of sterile water, and the viral load of canine distemper virus CDV-OR12 strain was determined using standard methods. The results showed that the viral load in the live attenuated vaccine components was 10. 4.00 TCID 50 / mL, which meets the requirements.

[0085] 9. Results of residual moisture determination of attenuated live vaccine components in mink pentavalent vaccine The residual moisture content of the attenuated live vaccine component in the mink pentavalent vaccine was determined, and the results showed that the residual moisture content of the component did not exceed 4%, which met the requirements.

[0086] 10. Vacuum degree test results of attenuated live vaccine components in mink pentavalent vaccine Vacuum degree testing was performed on the attenuated live vaccine component from the mink pentavalent vaccine, and the results showed that the vacuum degree of the component met the requirements.

[0087] 11. Safety test results of mink pentavalent vaccine Five healthy, susceptible mink were injected intramuscularly with 3 mL of vaccine at three injection sites. All vaccinated mink showed normal mental state, appetite, respiration, feces, and body temperature. No visible swelling or ulceration was observed at the injection sites, and all vaccinated mink survived. The results indicate that the mink pentavalent vaccine is safe for mink.

[0088] 12. Results of efficacy test for mink pentavalent vaccine Healthy, susceptible mink were vaccinated with a pentavalent vaccine. Twenty-one days after immunization, the serum of vaccinated mink showed parvovirus hemagglutination inhibition antibody titers above 1:64 and canine distemper virus neutralizing antibody titers above 1:45. In contrast, the control group showed parvovirus hemagglutination inhibition antibody titers and canine distemper virus neutralizing antibody titers not exceeding 1:4. Challenge experiments with Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum toxins were conducted on the immunized mink. The results showed a 100% protection rate in all immunized mink, while all control group mink developed the disease and died.

[0089] This embodiment successfully prepared a pentavalent vaccine against canine distemper virus, mink parvovirus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum. Testing showed that the vaccine's characteristics, dosage, sterility, safety, efficacy, and preservative residue levels all met the relevant standards of the current Chinese Veterinary Pharmacopoeia, and it can be used for subsequent clinical trials and widespread application.

[0090] Example 5: Safety testing of a five-in-one vaccine against canine distemper virus, parvovirus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum. I. Experimental Methods 1. Safety test of single-dose inoculation of mink with a five-in-one mink vaccine A pentavalent vaccine containing canine distemper virus, mink parvovirus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum, prepared according to the method in Example 4, was administered intramuscularly in single doses to mink aged 2–4 months and 8–10 months, 1 mL per animal. The mink were observed for 14 consecutive days post-inoculation, and changes in body temperature, mental state, appetite, and feces were observed and recorded, as well as any morbidity or mortality. Changes at the injection site and overall body condition were also recorded. On day 14, three inoculated mink were euthanized, and muscle tissue from the injection site of each mink was collected for pathological examination to check for histopathological changes at the injection site.

[0091] 2. Safety testing of mink vaccinated with a single dose of the five-in-one vaccine. A pentavalent vaccine containing canine distemper virus, mink parvovirus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum, prepared according to the method in Example 4, was administered intramuscularly in single doses to mink aged 2–4 months and 8–10 months, 1 mL per animal. A second intramuscular injection of 1 mL per animal was administered 14 days later. The mink were observed and recorded for 14 consecutive days post-inoculation, noting changes in body temperature, mental state, appetite, and feces, as well as any morbidity or mortality. Changes at the injection site and overall health were also recorded. On the 14th day, three inoculated mink were euthanized, and muscle tissue from the injection site of each mink was collected for pathological examination to check for histopathological changes at the injection site.

[0092] 3. Safety testing of mink vaccinated with overdose of the mink pentavalent vaccine The five-in-one vaccine containing canine distemper virus, mink parvovirus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum, prepared according to the method in Example 4, was administered intramuscularly to mink aged 2-4 months and 8-10 months, respectively. 3 mL (3 times the immunization dose) was injected into each mink at 3 injection points. The mink were observed for 14 consecutive days after vaccination, and changes in body temperature, mental state, appetite, and feces were observed and recorded, as well as any morbidity or mortality. Changes at the injection site and overall body condition were also recorded. On the 14th day, three inoculated mink were euthanized, and muscle tissue from the injection site of each mink was collected for pathological examination to check for histopathological changes at the injection site.

[0093] 4. Safety testing of mink pentavalent vaccine administered to pregnant mink The mink-canine distemper virus, mink parvovirus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum pentavalent vaccine prepared according to the method in Example 4 was administered intramuscularly to gestational mink aged 30-35 days, 1 mL per animal. The mink were observed for 14 consecutive days after vaccination, and changes in their mental state, appetite, and feces were observed and recorded, as well as whether any animals became ill or died. The occurrence of abortion, stillbirth, and the number of offspring in pregnant mink were also recorded.

[0094] II. Experimental Results 1. Safety test of single-dose inoculation of mink with a five-in-one mink vaccine Following single intramuscular injection of the pentavalent mink vaccine into mink aged 2–4 months and 8–10 months, the vaccinated mink exhibited normal behavior, appetite, and fecal condition, with no cases of illness or death. No redness, swelling, inflammation, or ulceration occurred at the vaccination site or throughout the body. A transient increase in body temperature occurred 1–2 days post-vaccination, subsequently returning to normal. Figure 4 On day 14, histopathological changes at the inoculation site of the mink were observed. The results showed no obvious lesions at the inoculation site, the morphology of myofibroblasts and nuclei was normal, and no inflammatory cells or other purulent exudates were found. Figure 5 ).

[0095] 2. Safety testing of mink vaccinated with a single dose of the five-in-one vaccine. Following repeated intramuscular injections of the pentavalent mink vaccine at single doses into mink aged 2–4 months and 8–10 months, the vaccinated mink exhibited normal behavior, appetite, and fecal condition, with no cases of illness or death. No redness, swelling, inflammation, or ulceration occurred at the vaccination site or throughout the body. A transient increase in body temperature occurred 1–2 days post-vaccination, subsequently returning to normal levels. Figure 4 On day 14, histopathological changes at the inoculation site of the mink were observed. The results showed no obvious lesions at the inoculation site, the morphology of myofibroblasts and nuclei was normal, and no inflammatory cells or other purulent exudates were found. Figure 6 ).

[0096] 3. Safety testing of mink vaccinated with overdose of the mink pentavalent vaccine Following intramuscular administration of overdose of the pentavalent mink vaccine to mink aged 2–4 months and 8–10 months, the vaccinated mink exhibited normal mental state, appetite, and fecal condition, with no cases of illness or death. No redness, swelling, inflammation, or ulceration occurred at the vaccination site or throughout the body. A transient increase in body temperature occurred 1–2 days post-vaccination, subsequently returning to normal levels. Figure 4 On day 14, histopathological changes at the inoculation site of the mink were observed. The results showed no obvious lesions at the inoculation site, the morphology of myofibroblasts and nuclei was normal, and no inflammatory cells or other purulent exudates were found. Figure 7 ).

[0097] 4. Safety testing of mink pentavalent vaccine administered to pregnant mink Following single-dose vaccination of mink at 30-35 days of gestation with the pentavalent vaccine, the vaccinated mink exhibited normal behavior, appetite, and fecal condition, with no cases of illness or death. The number of offspring produced by the vaccinated mink all reached normal levels, with no abortions, stillbirths, or abnormal fetal development observed after birth.

[0098] This embodiment demonstrates through safety experiments involving single-dose vaccination, repeated single-dose vaccination, and overdose vaccination that the mink canine distemper virus, mink parvovirus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum pentavalent vaccine prepared in this invention is safe for mink of different ages and has no side effects.

[0099] Single-dose vaccination experiments on pregnant mink have shown that the mink-canine distemper virus, parvovirus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum pentavalent vaccine is safe for pregnant mink and their offspring.

[0100] Example 6: Determination of the immunogenicity period, duration of immunity, and antibody fluctuations of a five-in-one vaccine containing canine distemper virus, mink parvovirus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum. I. Experimental Methods Healthy susceptible mink were vaccinated with a pentavalent vaccine, with each mink receiving 1 mL. Blood samples were randomly collected from five mink in each group (including the control group) at 7, 14, 21, 2, 4, 5, 6, and 7 months post-immunization. Serum titers of parvovirus hemagglutination inhibition antibodies and canine distemper virus neutralizing antibodies were measured. Simultaneously, at 4, 5, and 6 months post-immunization, the mink in each group (including the control group) were challenged with Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum toxin, respectively, and the results were recorded.

[0101] II. Experimental Results 1. Detection results of mink parvovirus hemagglutination inhibition antibody Seven days after immunization with the pentavalent vaccine, mink can produce hemagglutination inhibition antibodies against mink parvovirus. By day 14, the antibody level has reached 1:32. By day 21, the antibody level is above 1:64. Five months after immunization, the serum antibody level of mink is still above 1:32. Six months after immunization, the serum antibody titer of some mink is lower than 1:32.

[0102] 2. Results of canine distemper virus neutralizing antibody test Mink can produce neutralizing antibodies against canine distemper virus 7 days after receiving the pentavalent vaccine. The antibody level is above 1:45 21 days after immunization. Five months after immunization, the neutralizing antibody level against canine distemper virus in mink serum is still above 1:45. Six months after immunization, the antibody titer in the serum of some mink is lower than 1:45.

[0103] 3. Results of protection against challenge with Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum toxin. Four, five, and six months after immunization, patients were challenged with Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum toxin. Results showed that the protection rates against these three strains were 100% at four and five months. At six months, the protection rate of the pentavalent vaccine slightly decreased, but the protection rates against Pseudomonas aeruginosa and Klebsiella pneumoniae remained above 60%, while the protection rate against Clostridium botulinum toxin remained 100%.

[0104] The above results indicate that 5 months after immunization with the mink pentavalent vaccine, the mink parvovirus hemagglutination inhibition antibody ratio remained above 1:32, the canine distemper virus neutralizing antibody ratio remained above 1:45, and the challenge protection rates against Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum toxin were all 100%. Therefore, the protection period of the mink pentavalent vaccine against canine distemper virus, parvovirus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum toxin is determined to be 5 months.

[0105] Example 7: Shelf life experiment of a pentavalent vaccine containing mink distemper virus, mink parvovirus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum. I. Experimental Methods 1. Safety testing of mink pentavalent vaccines stored for different periods The inactivated and attenuated components of the mink pentavalent vaccine were stored at 2–8℃ and below -15℃, respectively. Mink pentavalent vaccines stored for 3, 9, 15, 18, and 21 months were used to inoculate mink intramuscularly. Each mink received 3 mL of the vaccine at 3 injection points, with 5 mink per group. The inoculated mink were observed for 14 consecutive days for 14 days. The mental state, appetite, respiration, feces, body temperature, injection site, morbidity, and mortality of the inoculated mink were also observed.

[0106] 2. Efficacy testing of mink pentavalent vaccines stored for different periods The inactivated and attenuated components of the mink pentavalent vaccine were stored at 2–8℃ and below -15℃, respectively. Healthy susceptible mink were intramuscularly inoculated with 1 ml of the vaccine at 3, 9, 15, 18, and 21 months of storage. Blood was randomly collected from five mink in each group (including the control group) 21 days and 5 months after immunization. Serum titers of parvovirus hemagglutination inhibition antibodies and canine distemper virus neutralizing antibodies were measured. Simultaneously, the mink were challenged with Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum toxin, and the results were recorded.

[0107] II. Experimental Results 1. Safety test results of mink pentavalent vaccines stored for different periods Tests showed that the mink pentavalent vaccine, after being stored for 3, 9, 15, 18, and 21 months, exhibited normal mental state, appetite, respiration, feces, and body temperature. No visible swelling or ulceration was observed at the injection site, and all the mink survived. This indicates that the mink pentavalent vaccine, stored for different periods, is safe for mink.

[0108] 2. Results of efficacy testing of mink pentavalent vaccines stored at different times Twenty-one days after intramuscular inoculation with mink pentavalent vaccines stored for different periods, serum parvovirus hemagglutination inhibition antibody titers were all above 1:32, and canine distemper virus neutralizing antibody titers were all above 1:45. The protection rate was 100% after challenge with Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum toxins. Five months after immunization with pentavalent vaccines stored for 3–18 months, serum parvovirus hemagglutination inhibition antibody titers were not lower than 1:32, and canine distemper virus neutralizing antibody titers were not lower than 1:45. The protection rate was 100% after challenge with Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum toxins. Five months after immunizing mink with a 21-month-old pentavalent vaccine, the serum hemagglutination inhibition antibody titer for parvovirus ranged from 1:8 to 1:64, and the neutralizing antibody titer for canine distemper virus ranged from 1:8 to 1:32. The protection rate of mink vaccinated against Pseudomonas aeruginosa and Klebsiella pneumoniae ranged from 20% to 60%, and the protection rate against botulinum toxin challenge was 100%.

[0109] In summary, after verification, the shelf life of the mink canine distemper virus, mink parvovirus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum pentavalent vaccine has been determined to be 18 months.

[0110] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A pentavalent vaccine against canine distemper virus, mink parvovirus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Clostridium botulinum, characterized in that, The pentavalent vaccine comprises an inactivated component and an attenuated component; the inactivated component consists of inactivated mink parvovirus antigen, inactivated Pseudomonas aeruginosa antigen, inactivated Klebsiella pneumoniae antigen, and inactivated Clostridium botulinum toxin antigen; the attenuated component consists of freeze-dried antigen of an attenuated strain of canine distemper virus. The mink parvovirus antigen is the mink parvovirus MEV-WH12 strain antigen, and the preservation number of the mink parvovirus MEV-WH12 strain is CGMCC No. 47099. The Klebsiella pneumoniae antigen is Klebsiella pneumoniae ( Klebsiella pneumoniae K2-SD14 strain antigen, the Klebsiella pneumoniae K2-SD14 strain has the accession number CGMCC No.38101; The Pseudomonas aeruginosa antigen is Pseudomonas aeruginosa ( Pseudomonas aeruginosa DL-15 strain antigen; The attenuated strain of canine distemper virus is canine distemper virus (Canine distemper virus) Canine Distemper Virus ) CDV-OR12 strain.

2. The pentavalent vaccine according to claim 1, characterized in that, The content of the mink parvovirus antigen is not less than 10. 5.73 TCID 50 / mL; the content of the Pseudomonas aeruginosa antigen is not less than 2.0 × 10⁻⁶. 9 CFU / mL; the content of the Klebsiella pneumoniae antigen is not less than 2.0 × 10⁻⁶ CFU / mL. 9 CFU / mL; the content of the botulinum toxin antigen is not less than 21440 MLD / mL; the content of the attenuated strain of canine distemper virus is not less than 10 3.90 TCID 50 / mL.

3. The pentavalent vaccine according to claim 1, characterized in that, The pentavalent inactivated vaccine also contains adjuvants and preservatives.

4. The pentavalent vaccine according to claim 3, characterized in that, The adjuvant is aluminum hydroxide gel adjuvant; the preservative is thimerosal.

5. A method for preparing the pentavalent vaccine according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Prepare inactivated mink parvovirus antigen, inactivated Pseudomonas aeruginosa antigen, inactivated Klebsiella pneumoniae antigen, inactivated Clostridium botulinum toxin antigen and attenuated strain of canine distemper virus respectively. (2) Dilute the inactivated mink parvovirus antigen, inactivated Pseudomonas aeruginosa antigen, inactivated Klebsiella pneumoniae antigen, and inactivated Clostridium botulinum toxin antigen prepared in step (1) to the target concentration and mix them; the target concentration is: mink parvovirus antigen solution not less than 10 6.40 TCID 50 / mL, Pseudomonas aeruginosa antigen solution not less than 9.4×10 9 CFU / mL, Klebsiella pneumoniae antigen solution not less than 9.4 × 10⁻⁶. 9 CFU / mL, the toxicity of the botulinum toxin antigen solution is not less than 1.0 × 10⁻⁶. 5 MLD / mL; (3) The diluted mink parvovirus antigen solution, Pseudomonas aeruginosa antigen solution, Klebsiella pneumoniae antigen solution and Clostridium botulinum toxin antigen solution were mixed in a volume ratio of 1:1:1:1 to obtain a mixed antigen solution. Adjuvants and preservatives were added as inactivation components. (4) After diluting the attenuated strain of canine distemper virus, freeze-drying and reconstitute it to ensure that the viral load of canine distemper virus is not less than 10. 3.90 TCID 50 / mL, used alone as a weakly toxic component; (5) The inactivated component is reconstituted with the attenuated component to obtain the pentavalent vaccine.

6. The preparation method according to claim 5, characterized in that, In step (3), the volume ratio of the mixed antigen solution to the adjuvant is 6:

1.

7. The preparation method according to claim 5, characterized in that, In step (3), the amount of preservative added is 0.005% of the total volume of the mixed antigen solution and the adjuvant.

8. The use of a pentavalent vaccine according to any one of claims 1-4 or a pentavalent vaccine prepared according to any one of claims 5-7 in the preparation of products for the prevention of canine distemper, mink parvovirus enteritis, mink hemorrhagic pneumonia, mink Klebsiella pneumoniae infection, and mink botulism.

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