A cat multi-feline disease prevention and control vaccine composition, a preparation method thereof and application thereof

Abstract

This invention discloses a feline multivalent disease prevention and control vaccine composition, its preparation method, and its application, belonging to the field of feline disease vaccine technology. The feline multivalent disease prevention and control vaccine composition provided by this invention is composed of inactivated feline panleukopenia virus, feline rhinotracheitis virus, and feline calicivirus virus, combined with an adjuvant. Using the method provided by this invention, a feline multivalent disease prevention and control vaccine has been successfully prepared. The virus inactivation is thorough, the safety is high, and the immunogenicity is good. It can simultaneously prevent and control feline panleukopenia, feline rhinotracheitis, and feline calicivirus disease. It can be used to develop clinically applicable vaccines for the prevention and control of feline panleukopenia, feline rhinotracheitis, and feline calicivirus disease, which is of great significance for significantly reducing feline suffering and medical burden.

Description

Technical Field

[0001] This invention relates to the field of feline disease vaccine technology, and in particular to a feline multivalent disease prevention and control vaccine composition, its preparation method, and its application. Background Technology

[0002] The most serious and common infectious diseases in cats include feline panleukopenia (FPV), feline rhinotracheitis (FHV-1), and feline calicivirus (FCV).

[0003] Feline panleukopenia is caused by feline parvovirus, which attacks the rapidly dividing cells in a cat's body: destroying the intestinal lining cells, leading to severe vomiting and diarrhea, causing dehydration and malnutrition; attacking hematopoietic stem cells in the bone marrow, causing a sharp drop in white blood cell, red blood cell, and platelet counts; and destroying immune organs such as lymph nodes, further weakening the immune response. Main symptoms include: initially, lethargy (drowsiness), loss of appetite or complete anorexia, and sudden high fever; persistent vomiting, severe watery diarrhea, bloody stools, abdominal pain, and rapid weight loss. Feline panleukopenia is a highly contagious and extremely deadly infectious disease. Kittens' condition deteriorates very rapidly, and death may occur within 24-48 hours of symptom onset. Currently, there is no specific treatment; supportive care is the primary approach.

[0004] Feline rhinotracheitis, caused by feline herpesvirus (FHV-1), is one of the most common upper respiratory tract infections in cats. It spreads very rapidly, with high morbidity and infectivity rates. The main symptoms resemble a severe cold, such as sneezing, runny nose, conjunctivitis (red, watery eyes, photophobia), and fever. Secondary complications may include oral ulcers, pneumonia, and difficulty breathing. The severity varies depending on the cat's age and health condition, but it is extremely dangerous for kittens, with a mortality rate as high as 100% if left untreated. Feline rhinotracheitis is not completely curable; recovered cats become lifelong carriers, and the virus can reactivate when they experience stress, illness, or a weakened immune system, leading to relapse. Currently, there are no specific medications for feline rhinotracheitis; treatment focuses on improving the cat's own immunity through enhanced daily care.

[0005] Feline calicivirus is one of the main pathogens causing feline calicivirus disease. Typical symptoms of feline calicivirus disease include sneezing, nasal congestion, conjunctivitis, oral ulcers, excessive drooling, and loss of appetite. Some strains may also cause lameness (especially in kittens) and pneumonia. The disease then rapidly progresses to systemic edema, ulcerative dermatitis, and multiple organ failure; the condition deteriorates very quickly and has a very poor prognosis. Currently, there is no specific treatment for feline calicivirus disease; supportive care is the primary approach.

[0006] Therefore, actively developing vaccines that can simultaneously prevent feline panleukopenia, feline rhinotracheitis, and feline calicivirus disease is of great significance in filling the treatment gap where there are no specific antiviral drugs for these three diseases. By injecting vaccines, cats can develop long-lasting neutralizing antibodies and establish an immune barrier, which can significantly reduce the suffering of cats and the medical burden on owners. Summary of the Invention

[0007] The purpose of this invention is to provide a feline multivalent disease prevention and control vaccine composition, its preparation method and application, to fill the treatment gap where there are no specific antiviral drugs for the three diseases. By injecting the vaccine, cats can produce long-lasting neutralizing antibodies, establish an immune barrier, and thus reduce the cat's suffering and the owner's medical burden.

[0008] To achieve the above objectives, the present invention provides a feline multivalent disease prevention and control vaccine composition, wherein the multivalent diseases are feline panleukopenia, feline rhinotracheitis, and feline calicivirus disease; the composition is composed of inactivated virus and adjuvant.

[0009] Preferably, the feline panleukopenia virus is feline parvovirus; the feline rhinotracheitis virus is feline herpesvirus; and the feline calicivirus is feline calicivirus.

[0010] Preferably, the feline parvovirus, feline herpesvirus, and feline calicivirus are all inactivated by β-propiolactone.

[0011] Preferably, the inactivation method of the β-propiolactone is as follows: β-propiolactone with a final concentration (v / v) of 0.01-0.1% is added dropwise under ice bath conditions, and the virus solution is continuously and gently stirred during the dropwise addition process. After the dropwise addition is completed, the solution is placed at 4°C and protected from light for inactivation. During this period, the solution is gently stirred once every 30 minutes for a total of 2-3 times, and then gently stirred once every 6-8 hours for a total of 3-6 times.

[0012] Preferably, after inactivation, the virus is infected into live cells, passaged blindly for 3-5 generations, and if no cytopathic effect is observed under a microscope, the virus is considered to have been successfully inactivated.

[0013] Preferably, the adjuvant is lecithin and / or aluminum hydroxide gel.

[0014] The preparation method of the feline multivalent disease prevention and control vaccine composition as described above involves mixing inactivated feline panleukopenia, feline rhinotracheitis, and feline calicivirus at a total viral infection unit ratio of 1-2:1:1-2, and then adding adjuvants and stirring until homogeneous.

[0015] The application of the feline multivalent disease prevention and control vaccine composition as described above in the prevention and control of feline diseases, wherein the feline multivalent disease prevention and control vaccine composition is administered orally.

[0016] Therefore, the feline multivalent disease prevention and control vaccine composition, its preparation method, and its application provided by the present invention have the following specific technical effects: This invention successfully prepared a feline multivalent disease prevention and control vaccine. The virus is thoroughly inactivated, with high safety and good immunogenicity. It can simultaneously prevent and control feline panleukopenia, feline rhinotracheitis, and feline calicivirus disease. It can be used to develop vaccines for clinical application to prevent and control feline panleukopenia, feline rhinotracheitis, and feline calicivirus disease, which is of great significance for significantly reducing cat suffering and medical burden.

[0017] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention 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.

[0019] Figure 1 These are the ELISA test results from the effectiveness test of this invention. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0021] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0022] The instruments, equipment, reagents and materials used in the embodiments were all obtained through commercial means; the methods and steps not described in detail in the embodiments are all conventional techniques in the art.

[0023] Example 1 The specific steps for culturing CRFK cells are as follows: (1) After taking out the CRFK cells frozen at -80℃, immediately place them in a 37℃ water bath and gently shake them to allow the CRFK cell line to thaw quickly. Then spray the surface of the cryovial with 70% alcohol so that the surface of the cryovial is completely wetted with 70% alcohol.

[0024] (2) Transfer the cryopreservation tubes sterilized with 70% alcohol in step (1) into a clean bench (turn on the UV lamp for sterilization for 30 minutes in advance), slowly drop the cell suspension (about 1 mL) into a centrifuge tube containing 4-6 mL of DMEM complete culture medium (containing 10% fetal bovine serum) preheated at 37°C, and gently shake while adding. After the addition is complete, shake well and then centrifuge at 1000 rpm for 5 minutes.

[0025] (3) Carefully discard the supernatant after centrifugation in step (2), then add 1 mL of fresh DMEM complete medium (containing 10% fetal bovine serum) to gently resuspend the cell pellet. Transfer the resuspended solution to a T25 culture flask, add 8 mL of DMEM complete medium (containing 10% fetal bovine serum), gently shake to mix, and then place in a 37°C, 5% CO2 incubator for culture.

[0026] (4) On the second day of culture in the incubator at 37°C and 5% CO2 in step (3), after the cells adhere to the wall, remove the old culture medium (containing dead cell fragments), add 8 mL of fresh DMEM complete culture medium (containing 10% fetal bovine serum), and continue to culture in the incubator at 37°C and 5% CO2 until the confluence reaches 80-90%.

[0027] (5) Use a sterile pipette to remove the old culture medium from the culture flask that has reached 80-90% confluence in step (4), then add 2 mL of PBS, gently shake to wash the bottom of the flask to remove residual serum. After removing the PBS, add 1 mL of 0.25% trypsin-EDTA, gently shake to completely cover the cell layer, and then place in an incubator at 37°C and 5% CO2 for 1-3 minutes to digest. Observe under an inverted microscope. When most of the cells become round, detach from the flask wall and begin to float, stop digestion immediately, quickly add 3 mL of fresh DMEM complete culture medium (containing 10% fetal bovine serum), and gently blow the bottom of the flask with a pipette to wash off the detached cells.

[0028] (6) Transfer the cell suspension obtained in step (5) to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 3 minutes, carefully aspirate the supernatant from the centrifuge tube, add 2 mL of fresh complete culture medium, and gently pipette the cell pellet to resuspend it into a single cell suspension.

[0029] (7) Take 2 mL of the cell suspension obtained in step (6) and add it to a T25 bottle containing 4 mL of fresh DMEM complete medium (containing 10% fetal bovine serum). Continue to culture in an incubator at 37°C and 5% CO2. Take the cells that have reached 80-90% confluence for later use.

[0030] Example 2 The specific steps for culturing feline parvovirus (which is isolated from infected cats) are as follows: Discard the old culture medium from CRFK cells cultured to a confluence of 80-90% in step (7) of Example 1, and add 4 mL of fresh DMEM complete culture medium (containing 10% fetal bovine serum). Then, inoculate the obtained feline parvovirus into the cell culture flask at a multiplicity of infection (MOI) of 0.1, and place the cell culture flask in an incubator at 37°C and 5% CO2 for 60 minutes, gently shaking it once every 15-20 minutes during this period.

[0031] After adsorption, maintenance medium (DMEM medium containing 2% fetal bovine serum) was added, and the culture was continued in a 37°C, 5% CO2 incubator. Cytopathic effect (CPE) was observed daily. CPE morphological characteristics included: cell rounding, shrinkage, increased refractive index, formation of small clusters of lesions, nucleus condensation, and eventual detachment from the flask wall. When CPE reached 75% or higher (usually 48-96 hours), the culture flask was repeatedly freeze-thawed three times to lyse the cells and release the virus. The supernatant was collected by centrifugation at 2500 rpm for 10 minutes, aliquoted, and stored at -80°C.

[0032] Example 3 The specific steps for culturing feline parvovirus (which is isolated from infected cats) are as follows: Discard the old culture medium from CRFK cells cultured to a confluence of 80-90% in step (7) of Example 1, and add 4 mL of fresh DMEM complete culture medium (containing 10% fetal bovine serum). Then, inoculate the obtained feline parvovirus into the cell culture flask at a multiplicity of infection (MOI) of 0.1, and place the cell culture flask in an incubator at 37°C and 5% CO2 for 90 minutes, gently shaking it once every 15-20 minutes during this period.

[0033] After adsorption, maintenance medium (DMEM medium containing 2% fetal bovine serum) was added, and the culture was continued in a 37°C, 5% CO2 incubator. Cytopathic effect (CPE) was observed daily. CPE morphological characteristics included: cell rounding, shrinkage, increased refractive index, formation of small clusters of lesions, nucleus condensation, and eventual detachment from the flask wall. When CPE reached 75% or higher (usually 48-96 hours), the culture flask was repeatedly freeze-thawed three times to lyse the cells and release the virus. The supernatant was collected by centrifugation at 2500 rpm for 10 minutes, aliquoted, and stored at -80°C.

[0034] Example 4 The specific steps for culturing feline parvovirus (which is isolated from infected cats) are as follows: Discard the old culture medium from CRFK cells cultured to a confluence of 80-90% in step (7) of Example 1, and add 4 mL of fresh DMEM complete culture medium (containing 10% fetal bovine serum). Then, inoculate the obtained feline parvovirus into the cell flask at a multiplicity of infection (MOI) of 0.01, and place the cell flask in an incubator at 37°C and 5% CO2 for 90 minutes, gently shaking it once every 15-20 minutes during this period.

[0035] After adsorption, maintenance medium (DMEM medium containing 2% fetal bovine serum) was added, and the culture was continued in a 37°C, 5% CO2 incubator. Cytopathic effect (CPE) was observed daily. CPE morphological characteristics included: cell rounding, shrinkage, increased refractive index, formation of small clusters of lesions, nucleus condensation, and eventual detachment from the flask wall. When CPE reached 75% or higher (usually 48-96 hours), the culture flask was repeatedly freeze-thawed three times to lyse the cells and release the virus. The supernatant was collected by centrifugation at 2500 rpm for 10 minutes, aliquoted, and stored at -80°C.

[0036] Example 5 The specific steps for culturing feline herpesvirus (which is isolated and identified from infected cats) are as follows: Discard the old culture medium from CRFK cells cultured to a confluence of 80-90% in step (7) of Example 1, and add 4 mL of fresh DMEM complete culture medium (containing 10% fetal bovine serum). Then, inoculate the obtained feline herpesvirus into the cell flask at a multiplicity of infection (MOI) of 0.1, and place the cell flask in an incubator at 37°C and 5% CO2 for 60 minutes, gently shaking it once every 15-20 minutes during this period.

[0037] After adsorption, maintenance medium (DMEM medium containing 2% fetal bovine serum) was added, and the cells were incubated at 37°C with 5% CO2. Cytopathic effect (CPE) was observed daily. The morphological characteristics of CPE were: cells clustered in a grape-like pattern, and cells fused to form syncytia (multinucleated giant cells). When CPE reached 75% or higher (usually 48-72 hours), the culture flask was repeatedly frozen and thawed three times to lyse the cells and release the virus. The supernatant was collected by centrifugation at 2500 rpm for 10 minutes, aliquoted, and stored at -80°C.

[0038] Example 6 The specific steps for culturing feline calicivirus (feline caliciviruses are all identified and isolated from diseased cats) are as follows: Discard the old culture medium from CRFK cells cultured to a confluence of 80-90% in step (7) of Example 1, and add 4 mL of fresh DMEM complete culture medium (containing 10% fetal bovine serum). Then, inoculate the obtained feline calicivirus into the cell flask at a multiplicity of infection (MOI) of 0.1, and place the cell flask in an incubator at 37°C and 5% CO2 for 60-90 minutes, gently shaking it once every 15-20 minutes during this period.

[0039] After adsorption, maintenance medium (DMEM medium containing 2% fetal bovine serum) was added, and the cells were incubated at 37°C with 5% CO2. Cytopathic effect (CPE) was observed daily. The morphological characteristics of CPE were: cells becoming rounded and granular, with obvious cell swelling, and cells agglomerating and then detaching. When CPE reached 75% or higher (usually after 24-48 hours), the culture flask was repeatedly frozen and thawed three times to lyse the cells and release the virus. The supernatant was collected by centrifugation at 2500 rpm for 10 minutes, aliquoted, and stored at -80°C.

[0040] Example 7 The specific steps for inactivating feline parvovirus are as follows: After melting the feline parvovirus solution prepared in Example 1 at 0°C, β-propiolactone with a final concentration (v / v) of 0.1% was added dropwise (1 drop / second) under ice bath conditions. During the addition process, the virus solution was continuously and gently stirred to ensure that β-propiolactone was evenly distributed in the virus solution.

[0041] After the addition is complete, the virus solution is placed at 4°C in the dark for inactivation. After 30 minutes of inactivation, the virus solution is gently stirred, and then placed at 4°C in the dark for further inactivation. After 30 minutes of inactivation, the virus solution is gently stirred again, and then placed at 4°C in the dark for 45 hours, with the virus solution gently stirred once every 6 hours during this period.

[0042] The inactivated virus solution was taken and used to infect new CRFK cells using the method described in Example 1. After four consecutive blind passages, no cytopathic effect (CPE) was observed under a microscope, confirming that the virus had been completely inactivated.

[0043] Example 8 The specific steps for inactivating feline herpesvirus are as follows: After melting the feline herpesvirus solution prepared in Example 5 at 0°C, β-propiolactone with a final concentration (v / v) of 0.1% was added dropwise (1 drop / second) under ice bath conditions. During the addition process, the virus solution was continuously and gently stirred to ensure that β-propiolactone was evenly distributed in the virus solution.

[0044] After the addition is complete, the virus solution is placed at 4°C in the dark for inactivation. After 30 minutes of inactivation, the virus solution is gently stirred, and then placed at 4°C in the dark for further inactivation. After 30 minutes of inactivation, the virus solution is gently stirred again, and then placed at 4°C in the dark for 22 hours, with the virus solution gently stirred once every 6 hours during this period.

[0045] The inactivated virus solution was taken and infected into new CRFK cells using the method described in Example 5. After four consecutive blind passages, no cytopathic effect (CPE) was observed under a microscope, confirming that the virus had been completely inactivated.

[0046] Example 9 The specific steps for inactivating feline calicivirus are as follows: After melting the feline calicivirus cultured in Example 6 at 0°C, β-propiolactone with a final concentration (v / v) of 0.1% was added dropwise (1 drop / second) under ice bath conditions. During the addition process, the virus solution was continuously and gently stirred to ensure that β-propiolactone was evenly distributed in the virus solution.

[0047] After the addition is complete, the virus solution is placed at 4°C in the dark for inactivation. After 30 minutes of inactivation, the virus solution is gently stirred, and then placed at 4°C in the dark for further inactivation. After 30 minutes of inactivation, the virus solution is gently stirred again, and then placed at 4°C in the dark for 44 hours, with the virus solution gently stirred once every 6 hours during this period.

[0048] The inactivated virus solution was taken and infected into new CRFK cells using the method described in Example 6. After four consecutive blind passages, no cytopathic effect (CPE) was observed under a microscope, confirming that the virus had been completely inactivated.

[0049] Example 10 The specific steps for preparing a feline multivalent disease prevention and control vaccine are as follows: Feline parvovirus, feline herpesvirus, and feline calicivirus, which were successfully inactivated in Examples 7-9, were mixed at a total viral infection unit ratio of 1:1:1. Then, 1 / 2 volume of aluminum hydroxide gel was added and stirred until well mixed.

[0050] Example 11 The specific steps for preparing a feline multivalent disease prevention and control vaccine are as follows: The feline parvovirus, feline herpesvirus, and feline calicivirus that were successfully inactivated in Examples 7-9 were mixed at a total viral infection unit ratio of 1:1:1, and then 1 / 2 volume of lecithin was added and stirred until well mixed.

[0051] Example 12 The specific steps for preparing a feline multivalent disease prevention and control vaccine are as follows: The feline parvovirus, feline herpesvirus, and feline calicivirus that were successfully inactivated in Examples 7-9 were mixed at a total viral infection unit ratio of 1.5:1:1.5, and then 1 / 2 volume of lecithin was added and stirred until well mixed.

[0052] Effect test (1) Seven-month-old, 3.5±0.2kg SPF-grade British Shorthair cats were used as experimental animals. After 7 days of acclimatization, they were orally administered 10 7.0 TCID 50 The feline multivalent disease vaccines prepared in Examples 10-12 were administered orally to three cats after 7 days, with an equal dose of the corresponding vaccine given 7 days later. Each example involved three cats receiving the feline multivalent disease vaccine. Cats orally administered an equal volume of lecithin / aluminum hydroxide gel (the same adjuvant used in the vaccine) served as blank controls, and cats orally administered an equal volume of distilled water served as negative controls.

[0053] Whole blood was collected on days 14, 21, and 28 after the first oral vaccination. Serum was separated and analyzed by ELISA using a kit. The mean values ​​for each treatment group are shown in Table 1. Figure 1 As shown, the feline multivalent disease control vaccines prepared orally in Examples 10-12 can induce antibody production in experimental cats.

[0054] Table 1 ELISA test results

[0055] (2) The neutralizing antibody titers against feline parvovirus of the feline multivalent disease control vaccines prepared in Examples 10 and 12 were examined. Details are as follows: After titrating the feline parvovirus, it was diluted with cell maintenance medium to a concentration of 100 TCID10 per 0.1 mL. 50 The working fluid.

[0056] Cat serum from days 14, 21, and 28 following oral vaccination in (1) was inactivated by water bath at 56°C for 30 minutes to remove nonspecific inhibitors. Then, it was serially diluted with cell maintenance medium.

[0057] Add an equal volume of 100 TCID to cat serum at different dilutions. 50The virus working solution was shaken well and incubated in a 37°C water bath for 60 minutes to allow the antibody to fully neutralize the virus. The incubated serum-virus mixture was then seeded at 0.2 mL per well onto a monolayer of FL74 cells in a 96-well plate, with four replicates for each serum dilution. Four wells were set up as a blank control (normal cells) and four wells as a virus-infected control. The culture plate was placed in a 37°C 5% CO2 incubator and observed continuously for 7 days, recording cytopathic effects (CPE) in each well.

[0058] The serum dilution at which 50% of cells do not show lesions was calculated using the Reed-Muench method, and this dilution was taken as the neutralizing antibody titer of the serum. The results are shown in Table 2.

[0059] (3) The method for evaluating the neutralizing antibody titers of feline herpesvirus and feline calicivirus is the same as that for feline parvovirus in (2). The statistical results are shown in Table 2.

[0060] As shown in Table 2, with the increase in the number of immunizations, the neutralizing antibody titers against feline parvovirus, feline herpesvirus, and feline calicivirus of the feline multivalent disease control vaccines prepared in Examples 10 and 12 all increased. This indicates that the feline multivalent disease control vaccine prepared by the method provided in this invention can simultaneously prevent and control feline panleukopenia, feline rhinotracheitis, and feline calicivirus disease, and can be used to develop vaccines for clinical application to prevent and control feline panleukopenia, feline rhinotracheitis, and feline calicivirus disease.

[0061] The results in Table 2 also show that the feline multivalent disease control vaccines prepared in Examples 10 and 12 had higher titers against feline herpesvirus and feline calicivirus than against feline parvovirus. Furthermore, the feline multivalent disease control vaccine prepared in Example 12 showed higher titers than the feline multivalent disease control vaccine prepared in Example 10 at all stages after oral immunization, possibly indicating that lecithin as an adjuvant was more effective than aluminum hydroxide gel. This is consistent with the ELISA analysis results in (1).

[0062] Table 2. Statistical results of neutralizing antibody titers

[0063] Therefore, the method provided by this invention has successfully prepared a feline multivalent disease prevention and control vaccine. The virus is thoroughly inactivated, with high safety and good immunogenicity. It can simultaneously prevent and control feline panleukopenia, feline rhinotracheitis, and feline calicivirus disease. It can be used to develop vaccines for clinical application to prevent and control feline panleukopenia, feline rhinotracheitis, and feline calicivirus disease, which is of great significance for significantly reducing the suffering of cats and the medical burden.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A feline multivalent disease prevention and control vaccine composition, characterized in that: The multivalent disease is feline panleukopenia, feline rhinotracheitis, and feline calicivirus disease; the composition is composed of inactivated virus and adjuvant.

2. The feline multivalent disease prevention and control vaccine composition according to claim 1, characterized in that: The feline panleukopenia virus is feline parvovirus; the feline rhinotracheitis virus is feline herpesvirus; and the feline calicivirus virus is feline calicivirus.

3. The feline multivalent disease prevention and control vaccine composition according to claim 2, characterized in that: The feline parvovirus, feline herpesvirus, and feline calicivirus were all inactivated by β-propiolactone.

4. The feline multivalent disease prevention and control vaccine composition according to claim 3, characterized in that, The inactivation method using the β-propiolactone is as follows: β-propiolactone with a final concentration (v / v) of 0.01-0.1% is added dropwise under ice bath conditions. The virus solution is continuously and gently stirred during the dropwise addition process. After the dropwise addition is completed, the solution is placed at 4°C in the dark for inactivation. During this period, the solution is gently stirred once every 30 minutes for a total of 2-3 times. Then, the solution is gently stirred once every 6-8 hours for a total of 3-6 times.

5. The feline multivalent disease prevention and control vaccine composition according to claim 4, characterized in that: After inactivation, the virus is infected into live cells and passaged blindly for 3-5 generations. If no cytopathic effect is observed under a microscope, the virus is considered to have been successfully inactivated.

6. The feline multivalent disease prevention and control vaccine composition according to claim 1, characterized in that: The adjuvant is lecithin and / or aluminum hydroxide gel.

7. The method for preparing the feline multivalent disease prevention and control vaccine composition according to any one of claims 1-6, characterized in that: Mix inactivated feline panleukopenia, feline rhinotracheitis, and feline calicivirus at a total viral infection unit ratio of 1-2:1:1-2, then add adjuvant and stir until well combined.

8. The use of the feline multivalent disease prevention and control vaccine composition according to any one of claims 1-6 in the prevention and control of feline diseases, characterized in that: The feline multivalent disease prevention and control vaccine composition is administered orally.

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