Bovine Viral Diarrhea Virus, Infectious Bovine Rhinotracheitis Virus, Parainfluenza Virus Type 3 and Mycoplasma Quadrivalent Inactivated Vaccine

Abstract

The present invention discloses a quadruple inactivated vaccine against bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, parainfluenza virus and mycoplasma. The active ingredients of the quadruple inactivated vaccine are inactivated bovine viral diarrhea virus, inactivated infectious bovine rhinotracheitis virus, inactivated bovine parainfluenza virus type 3 and inactivated bovine mycoplasma. The quadruple inactivated vaccine is prepared by propagating BVDV HH03 strain virus, IBRV TY01 strain virus and BPIV3 LH01 strain virus by means of suspension culture of MDBK cells; and culturing bovine mycoplasma CHF01 strain by using Hayflick liquid medium. This method can well culture the virus on suspension-cultured cells, solve the problems of uneven virus inoculation and even difficulty in obtaining a virus solution with a relatively high titer during the process of propagating the virus with adherent cells, and its TCID 50 is greater than 8.0, reducing the production cost, with uniform cell growth, and it is a method worthy of popularization and application.

Description

Technical Field

[0001] The invention relates to a bovine viral diarrhea virus, infectious rhinotracheitis virus, parainfluenza virus type 3 and mycoplasma quadruple inactivated vaccine in the field of veterinary biological products and a preparation method thereof. Background Art

[0002] Bovine viral diarrhea (BVD) belongs to the Flaviviridae family, the genus Pestivirus. Bovine viral diarrhea virus / mucosal disease virus (BVDV) causes subclinical infection, acute infection, mucosal disease and persistent infection in cattle. Acute infection often manifests as diarrhea, abortion and respiratory diseases. It is an important infectious disease with diarrhea as the main symptom, and cattle of all ages are susceptible. The main symptoms of bovine viral diarrhea are: diarrhea, acute and chronic mucosal diseases, persistent infection and immune tolerance, abortion, stillbirth and deformity in female animals. In addition to infecting cattle, it can also infect pigs, sheep, deer, camels and other wild animals.

[0003] Infectious bovine rhino tracheitis (IBR), commonly known as "red nose disease" or "necrotizing rhinitis", is an acute, febrile, contact-transmitted, highly contagious disease caused by bovine herpesvirus type 1 (BHV-1) and infectious bovinerhinotracheitis virus (IBRV). The World Health Organization OIE lists it as one of the diseases that must be reported, and my country lists it as a Category II infectious disease. Cattle and pigs are susceptible and carriers of the virus, and can also infect animals such as pigs and goats. The virus infects the respiratory system, reproductive system, nervous system, conjunctiva, and fetus, and after the virus infects the cow, it can lurk in the nervous system, making the infection intermittent and persistent, and the sick cows will carry the virus for a long time or for life. Viral infection can also induce a decrease in milk production and abortion, and lead to slow growth of beef cattle and convalescent cattle, causing great losses to the cattle industry.

[0004] Bovine parainfluenza (BPI) is an acute respiratory disease caused by bovine parainfluenza virus type 3 (BPIV3), which belongs to the Paramyxoviridae family and the Respiratory virus genus. The disease is bovine parainfluenza, also known as "shipping fever", and is an acute contagious disease. BPIV3 has low pathogenicity to cattle, and single infection only causes mild clinical symptoms. However, under the combined action of secondary bacteria, mycoplasma and external incentives, severe respiratory symptoms can occur, and even cause the death of diseased cattle. BPI, together with BVD and IBR, constitutes the main pathogens of the Bovine respiratory disease complex (BRDC). At present, BRDC is the main cause of morbidity and mortality of feeding cattle worldwide, seriously endangering the healthy development of the cattle industry. Mycoplasma bovis is widespread worldwide and is one of the main pathogens causing various diseases such as bovine pneumonia, arthritis, mastitis, abortion and infertility. Summary of the Invention

[0005] The problem to be solved by the present invention is how to better propagate the virus and obtain a virus solution with a relatively high titer, and / or prevent four diseases with one injection, simplify the immunization program, and reduce the local and systemic adverse reactions of immunized cattle using the quadrivalent vaccine.

[0006] The first object of the present invention is to provide a quadrivalent inactivated vaccine against bovine viral diarrhea virus, bovine infectious rhinotracheitis virus, bovine parainfluenza virus type 3 and Mycoplasma bovis.

[0007] The active ingredients of the quadrivalent inactivated vaccine against bovine viral diarrhea virus, bovine infectious rhinotracheitis virus, bovine parainfluenza virus type 3 and Mycoplasma bovis provided by the present invention are inactivated bovine viral diarrhea virus, inactivated bovine infectious rhinotracheitis virus, inactivated bovine parainfluenza virus type 3 and inactivated Mycoplasma bovis.

[0008] In the above quadrivalent inactivated vaccine, the bovine viral diarrhea virus can be bovine viral diarrhea virus HH03 strain.

[0009] In the above quadrivalent inactivated vaccine, the bovine infectious rhinotracheitis virus can be bovine infectious rhinotracheitis virus TY01 strain.

[0010] In the above quadrivalent inactivated vaccine, the bovine parainfluenza virus type 3 can be bovine parainfluenza virus type 3 LH01 strain.

[0011] In the above quadrivalent inactivated vaccine, the Mycoplasma bovis can be Mycoplasma bovis CHF01 strain.

[0012] In the above-mentioned quadruple inactivated vaccine, the ratio of inactivated bovine viral diarrhea virus, inactivated bovine infectious rhinotracheitis virus, inactivated bovine parainfluenza virus type 3, and inactivated Mycoplasma bovis can be as follows: the inactivated bovine viral diarrhea virus can be 10 8.0 TCID 50 / mL based on the bovine viral diarrhea virus before inactivation; the inactivated bovine infectious rhinotracheitis virus can be 10 8.0 TCID 50 / mL based on the bovine infectious rhinotracheitis virus before inactivation; the inactivated bovine parainfluenza virus type 3 can be 10 8.0 TCID 50 / mL based on the bovine parainfluenza virus type 3 before inactivation; the inactivated Mycoplasma bovis can be 1.0×10 10 ccu / mL based on the Mycoplasma bovis before inactivation.

[0013] The above-mentioned quadruple inactivated vaccine can be composed of active ingredients and adjuvants.

[0014] The above-mentioned adjuvant can be adjuvant 206.

[0015] The second object of the present invention is to provide a method for preparing the above-mentioned quadruple inactivated vaccine.

[0016] The method for preparing the above-mentioned quadruple inactivated vaccine provided by the present invention includes the following steps:

[0017] 1) Bovine viral diarrhea virus, bovine infectious rhinotracheitis virus, and bovine parainfluenza virus type 3 are respectively cultured with suspension MDBK cells to obtain bovine viral diarrhea virus liquid, bovine infectious rhinotracheitis virus liquid, and bovine parainfluenza virus type 3 liquid; the bovine viral diarrhea virus liquid, the bovine infectious rhinotracheitis virus liquid, and the bovine parainfluenza virus type 3 liquid are respectively inactivated to obtain inactivated bovine viral diarrhea virus antigen liquid, inactivated bovine infectious rhinotracheitis virus antigen liquid, and inactivated bovine parainfluenza virus type 3 antigen liquid;

[0018] Mycoplasma bovis is cultured with a liquid medium to obtain Mycoplasma bovis culture solution, and the Mycoplasma bovis culture solution is inactivated to obtain inactivated Mycoplasma bovis antigen liquid;

[0019] 2) The above-mentioned inactivated bovine viral diarrhea virus antigen liquid, the above-mentioned inactivated bovine infectious rhinotracheitis virus antigen liquid, the above-mentioned inactivated bovine parainfluenza virus type 3 antigen liquid, and the above-mentioned inactivated Mycoplasma bovis antigen liquid are mixed as active ingredients to obtain a quadruple inactivated vaccine of bovine viral diarrhea virus, bovine infectious rhinotracheitis virus, bovine parainfluenza virus type 3, and Mycoplasma bovis.

[0020] In the above preparation method, the bovine viral diarrhea virus can specifically be HH03 strain, and / or the bovine infectious rhinotracheitis virus can specifically be TY01 strain, and / or the bovine parainfluenza type 3 can specifically be LH01 strain, and / or Mycoplasma bovis can specifically be CHF01 strain.

[0021] In the above preparation method, the antigen inactivation is to inactivate the three prepared virus antigens and mycoplasma bacterial solution with formaldehyde.

[0022] The present invention provides a quadruple inactivated vaccine against bovine viral diarrhea, bovine infectious rhinotracheitis, bovine parainfluenza virus type 3 and Mycoplasma bovis and a preparation method thereof. By using MDBK fully suspended cells to propagate bovine viral diarrhea virus, bovine infectious rhinotracheitis virus, bovine parainfluenza virus type 3 and propagating Mycoplasma bovis on a culture medium, this method can well culture the virus on suspended cells, solve the problem that the virus inoculation is uneven during the process of propagating the virus with adherent cells and even it is difficult to obtain a virus solution with a relatively high titer. Its TCID 50 is greater than 8.0, reducing the production cost, the cells grow evenly, and the quadruple vaccine of the present invention is safe and reliable, simplifies the immunization program, and can achieve the purpose of "one injection for four preventions". BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It shows the growth status after 24 hours of suspension cell culture.

[0024] Figure 2 It shows the growth status after 48 hours of suspension cell culture.

[0025] Figure 3 It shows the growth status after 72 hours of suspension cell culture.

[0026] Figure 4 It shows the growth status when BVDV is inoculated on suspension cells for virus harvest.

[0027] Figure 5 It shows the growth status when IBRV is inoculated on suspension cells for virus harvest.

[0028] Figure 6 It shows the growth status when BPIV3 is inoculated on suspension cells for virus harvest.

[0029] Figure 7 It shows the growth status of Mycoplasma bovis on a solid culture medium. DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention will be further described in detail below in combination with the specific embodiments. The given embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0031] In the following experimental methods of the examples, unless otherwise specified, they are all conventional methods, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0032] In the following examples, the bovine viral diarrhea virus HH03 strain, bovine infectious rhinotracheitis virus TY01 strain, bovine parainfluenza virus type 3 LH01 strain, and Mycoplasma bovis CHF01 strain have been recorded in Wu Zhiqiang, Shi Yuemeng, Kou Zhaoting, etc. Isolation and Identification of Bovine Respiratory Disease Pathogens BVDV, IBRV, BPIV3 and Mycoplasma bovis [J]. Contemporary Animal Husbandry and Aquaculture, 2022(5): 12-16. The public can obtain this biological material from the applicant in accordance with the relevant regulations on national biosafety. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0033] Example 1. Preparation of a quadrivalent inactivated vaccine against bovine viral diarrhea virus, bovine infectious rhinotracheitis virus, bovine parainfluenza virus type 3 and Mycoplasma bovis

[0034] 1. Isolation and identification of BVDV-HH03 strain, IBRV-TY01 strain, BPIV3-LH01 strain, and Mycoplasma bovis CHF01 strain

[0035] (1) For the bovine viral diarrhea virus strain HH03, cytopathic effect can be produced after inoculating MDBK cells. According to the gene sequence of the BVDV international standard strain, a pair of primers was designed and synthesized at the 5'-untranslated region sequence. The primer sequences are as follows:

[0036] BVDV-F: 5'-CATGCCCATAGTAGGAC-3' (Sequence 1)

[0037] BVDV-R: 5'-CCATGTGCCATGTACAG-3' (Sequence 2)

[0038] A 288bp specific fragment was amplified by RT-PCR.

[0039] (2) For the bovine infectious rhinotracheitis virus TY01 strain, cytopathic effect can be produced after inoculating MDBK cells. Specific primers were designed, and the primer sequences are as follows:

[0040] IBRV-F: 5'-TGACGGTAGCCTGGGACTGG-3' (Sequence 3)

[0041] IBRV-R: 5'-CCGGAAGGCCACGACAAA-3' (Sequence 4)

[0042] PCR can amplify a 311bp specific fragment.

[0043] (3) Bovine parainfluenza virus type 3 strain LH01 can cause cytopathic effect when inoculated into MDBK cells. The primer sequences are as follows:

[0044] BPIV3-F: 5'-GGATGTTTGGGAGTGATCTTGAGTA-3' (Sequence 5)

[0045] BPIV3-R: 5'-TGTGTTGAAAAATGAAGCAAGACCT-3' (Sequence 6)

[0046] RT-PCR can amplify a 425bp specific fragment.

[0047] (4) Mycoplasma bovis strain CHF01. The primer sequences are as follows:

[0048] MB-F: 5'-TATTGGATCAACTGCTGGAT-3' (Sequence 7)

[0049] MB-R: 5'-AGATGCTCCACTTATCTTAG-3' (Sequence 8)

[0050] PCR can amplify a 447bp specific fragment.

[0051] Purity detection shows that the above three strains are free of bacteria, mycoplasma and exogenous virus contamination, with good purity. The three virus strains have good adaptability to MDBK cells, and the TCID 50 is stable; the Mycoplasma bovis strain has good viable cell titer, virulence, immunogenicity and cross-immunoprotection.

[0052] 2. Antigen preparation

[0053] To solve a series of problems that occur during the adaptation of BVDV, IBRV, and BPIV3 to the suspension-cultured bovine kidney cell line MDBK, the present invention provides a method for preparing BVDV HH03 strain, IBRV TY01 strain, and BPIV3 LH01 strain by suspension culture technology; at the same time, inoculate Mycoplasma bovis strain CHF01 into a suitable medium for culture and harvest the culture.

[0054] a. Prepare antigens of BVDV HH03 strain, IBRV TY01 strain, and BPIV3 LH01 strain using the full suspension culture technology

[0055] (1) Cell resuscitation and domestication: Take out the bovine kidney cell line MDBK (from Shenzhen Yishengke Biotechnology) cells from the liquid nitrogen tank, quickly thaw them in a 37°C water bath, and use a pipette to add the cell suspension to the prepared 10 mL MDBK cell complete suspension culture medium (Shenzhen Yishengke Biotechnology, pH value between 7.0 and 7.2), and gently mix. Centrifuge at 1000 rpm for 5 min, discard the supernatant. Take 15 mL of the culture medium to resuspend the cells, transfer the cell suspension to a 125 mL shake flask, and place it in a 37°C, 5.0% CO2 incubator for suspension culture at 130 r / min. Passage every 72 h, passage continuously for multiple times for cell domestication, and observe regularly. The results show that after the 4th passage, the number of cell growth can basically increase to 6×10 6 cells to 7×10 6 cells / mL or more, and the mortality rate is basically lower than 3% (survival rate higher than 97%), the growth state is stable, there is no cell aggregation phenomenon, and the cells of the 4th to 10th generations are the domesticated MDBK cells.

[0056] (2) Reactor scale-up culture of cells: Inoculate the domesticated MDBK cells into a 15 L bioreactor, increase the rotation speed by 150 r / min to fully suspend the cells, then reduce the rotation speed to 50 - 80 r / min, add an antifoaming agent (volume ratio 0.1%), culture for 24 h, and when the cell density reaches 2×10 6 cells / mL, scale up to a 1000 L reactor.

[0057] (3) Suspension culture for propagating virus seeds and harvesting virus: When the MDBK cell density reaches 2.5×10 6 cells / mL, inoculate the BVDV HH03 strain, IBRV TY01 strain, and BPIV3 LH01 strain respectively according to the inoculation amount of MOI = 0.01 - 0.1, and add the MDBK cell complete suspension culture medium for culture. Control the culture conditions in the reactor: dissolved oxygen content is 30 - 50%, temperature is 36.7°C, stirring speed is 60 r / min, and pH is 7.2. Take samples every 6 h to detect cell morphology, and harvest the virus solution at 48 - 72 h. Use a high-pressure homogenizer to physically break the harvested virus solution to release virus particles. The results show that: 72 - 96 h after inoculating the BVDV HH03 strain, the virus content of the harvested BVDV antigen solution reaches a peak of 10 8.0 TCID 50 / mL; 72 - 96 h after inoculating the IBRV TY01 strain, the virus content of the harvested IBRV antigen solution reaches a peak of 10 8.0 TCID 50 / mL; 72 - 96 h after inoculating the BPIV3 LH01 strain, the virus content of the harvested BPIV3 antigen solution reaches a peak of 10 8.0 TCID 50 / mL. The prepared antigen solution was subjected to purity inspection in accordance with the appendix of the Pharmacopoeia of Veterinary Drugs of the People's Republic of China, and should be free from bacterial, fungal, and mycoplasma contamination. The results showed that the harvested virus solutions of BVDV HH03 strain, IBRV TY01 strain, and BPIV3 LH01 strain were all free from bacteria, fungi, and mycoplasmas.

[0058] (4) Inactivation of virus solution

[0059] To the antigen solutions of BVDV HH03 strain, IBRV TY01 strain, and BPIV3 LH01 strain in step (3), add a sterile-filtered formaldehyde solution with a final concentration of 0.1%, and inactivate at room temperature for 48 - 72 hours to obtain inactivated antigen solutions of BVDV HH03 strain, inactivated antigen solutions of IBRV TY01 strain, and inactivated antigen solutions of BPIV3 LH01 strain, respectively.

[0060] Dilute the above inactivated antigen solutions of BVDV HH03 strain, inactivated antigen solutions of IBRV TY01 strain, and inactivated antigen solutions of BPIV3 LH01 strain by 100 times, take samples and inoculate MDBK cells at a 10% inoculation amount, and blindly passage for three generations for inactivation detection. The results showed that no cytopathic effect (CPE) occurred.

[0061] b Preparation of Mycoplasma bovis antigen using the plate culture method technology

[0062] (1) Culture of Mycoplasma bovis: The bacterial solution of Mycoplasma bovis CHF01 strain was evenly inoculated on a Hayflick solid plate (PPLO agar, glucose, sodium pyruvate, 25% yeast extract powder, 100,000 units of penicillin, fetal bovine serum; pH value 7.6 - 7.8), and cultured at 37°C for 4 - 5 days under airtight conditions. Typical "fried egg-shaped" colonies could be seen. The center of the colony was thick and dense, the periphery was a thin transparent granular area, the surface was smooth, the edge was neat, round, growing embedded in the medium, and a β-hemolysis ring could be seen (see attachment Figure 7 ). Inoculate Hayflick liquid medium (PPLO Broth, glucose, sodium pyruvate, 25% yeast extract powder, 100,000 units of penicillin, fetal bovine serum; pH value 7.6 - 7.8), and culture at 37°C for 3 - 4 days. The medium was clear, and when the culture flask was shaken, flocculent substances could be seen floating in a spiral shape at the bottom.

[0063] (2) Inoculation and harvest: Inoculate the secondary production seed of Mycoplasma bovis CHF01 strain into the pre-warmed Hayflick liquid medium at 37°C at a ratio of 5%, culture at a constant temperature of 37°C, 100 r / min, and harvest the fermentation broth after 72 - 120 h to obtain a Mycoplasma bovis antigen solution. The viable count showed that the viable bacteria content in the antigen solution was not less than 1.0×10 10 ccu / mL.

[0064] (3)Inactivation of Mycoplasma bovis: Add a formaldehyde solution with a final concentration of 0.2% that has been sterile filtered to the Mycoplasma bovis antigen solution in step (2), inactivate at 25°C for 10 h. After inactivation is completed, take samples for sterility testing and inactivation testing: Take the inactivated Mycoplasma bovis antigen solution, inoculate it into Hayflick liquid medium for subculture, and blindly passage it continuously for 3 generations. No growth of Mycoplasma bovis or bacteria is found.

[0065] 3. Quadrivalent inactivated vaccine against bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, bovine parainfluenza virus type 3, and Mycoplasma bovis Preparation of the emulsified quadrivalent inactivated vaccine

[0066] Mix the inactivated BVDV HH03 strain, inactivated IBRV TY01 strain, inactivated BPIV3 LH01 strain, and inactivated Mycoplasma bovis antigen solution prepared in step 2 evenly to form the aqueous phase. Use 206 adjuvant (purchased from Seppic, product number 36022E) after autoclaving. Add the inactivated antigen solution to the oil adjuvant that has been sterilized and cooled to about 30°C, and mix and emulsify it at a ratio of aqueous phase:oil phase volume ratio of 46:54, stir for 30 min to prepare a quadrivalent inactivated vaccine against bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, bovine parainfluenza virus type 3, and Mycoplasma bovis. In this quadrivalent inactivated vaccine against bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, bovine parainfluenza virus type 3, and Mycoplasma bovis, the BVDV antigen content is 10 8.0 TCID 50 / mL, the IBRV antigen content is 10 8.0 TCID 50 / mL, the BPIV3 antigen content is 10 8.0 TCID 50 / mL, and the Mycoplasma bovis antigen content is 1.0×10 10 ccu / mL.

[0067] 4. Quality inspection of the quadrivalent inactivated vaccine against bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, bovine parainfluenza virus type 3, and Mycoplasma bovis

[0068] 4.1. Physical properties

[0069] (1) The appearance should be a milky white or light pink slightly viscous homogeneous emulsion.

[0070] (2) The dosage form is water-in-oil-in-water type. Take a clean pipette, suck a small amount of the vaccine and drop it on the surface of clean cold water, and it should diffuse in a cloud-like shape.

[0071] (3) Stability: Transfer 10 mL of the inactivated vaccine into a centrifuge tube and centrifuge at 3000 r / min for 15 min. The amount of water separated at the bottom of the tube should not exceed 0.5 mL.

[0072] (4) Viscosity: Test according to the appendix of the current edition of the Chinese Veterinary Pharmacopoeia. The viscosity should not exceed 200 cP.

[0073] (5) Fill volume: Test according to the appendix of the current edition of the Chinese Veterinary Pharmacopoeia. The fill volume should comply with the regulations.

[0074] 4.2 Sterility test: Conduct the sterility test according to the appendix of the current edition of the Chinese Veterinary Pharmacopoeia. No growth of microorganisms should be observed.

[0075] The physical properties and sterility test results of the quadruple inactivated vaccine against bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, bovine parainfluenza virus type 3, and Mycoplasma bovis are shown in Table 1.

[0076] Table 1. Physical properties and sterility test results

[0077]

[0078]

[0079] 4.3 Safety test

[0080] (1) Test with small animals (substitute animal method): Select 6 guinea pigs weighing 350 - 400 g. Inject 2 mL of the quadruple inactivated vaccine against bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, bovine parainfluenza virus type 3, and Mycoplasma bovis subcutaneously into the back of each guinea pig in one injection, divided into two injection points, 1 mL at each point. Select 5 BALB / c mice weighing 18 - 22 g. Inject 1 mL of the quadruple inactivated vaccine subcutaneously into the back of each mouse in one injection, divided into two injection points, 0.5 mL at each point. Observe continuously for 7 days. No death, obvious local reaction, or systemic adverse reaction caused by the injection of the vaccine should occur.

[0081] (2) Test with cattle (target animal method): Use 4 healthy susceptible cattle at 4 - 6 months of age that are negative for BVDV, IBRV, BPIV3, mycoplasma antigen, and antibody (the serum neutralization antibody titer of BVDV, IBRV, and BPIV3 is not higher than 1:2, and the antibody titer of Mycoplasma bovis detected by indirect hemagglutination test < 1:4). Inject 4.0 mL of the quadruple inactivated vaccine against bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, bovine parainfluenza virus type 3, and Mycoplasma bovis intramuscularly into the neck of each cattle in one injection. Observe continuously for 14 days. No obvious local or systemic adverse reaction caused by the injection of the vaccine should occur.

[0082] The safety test results of the quadruple inactivated vaccine against bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, bovine parainfluenza virus type 3 and Mycoplasma bovis are shown in Table 2.

[0083] Table 2. Safety test results

[0084]

[0085] 4.4. Immune potency test

[0086] (1) Guinea pig neutralizing antibody assay: Twelve SPF female guinea pigs weighing 350 - 400 g (negative for BVDV, IBRV, and BPIV3 serum neutralizing antibody titers; negative for Mycoplasma bovis detected by indirect hemagglutination) were used, with 10 in the immunization group and 2 in the control group. Guinea pigs in the immunization group were each injected intramuscularly in the leg with 1.0 mL of the quadruple inactivated vaccine against bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, bovine parainfluenza virus type 3 and Mycoplasma bovis, and guinea pigs in the control group were each injected intramuscularly in the leg with 1.0 mL of PBS (8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, 0.24 g of KH2PO4, made up to 1 L with water). Three weeks after immunization, a booster immunization was carried out in the same manner and at the same dose. Twenty-one days after the second immunization, blood was collected from the 10 immunized guinea pigs and the 2 control guinea pigs, and the serum was separated to determine the neutralizing antibody level. (The inactivated test samples, positive control serum, and negative control serum were serially diluted 2-fold in a 96-well U-shaped plate with DMEM cell culture medium containing 2% fetal bovine serum (purchased from Gibco). The diluted neutralizing antigen solution was added to the 96-well U-shaped plate containing serum at 0.1 mL per well, and neutralized at 37°C in an incubator containing 5% CO2 for 1 hour. The cells in the 96-well culture plate cultured for 16 - 24 hours were taken, the growth medium was discarded, and the mixture of virus and serum neutralized for 1 hour was transferred to the corresponding wells of the 96-well cell culture plate at 0.1 mL per well, and cultured and observed in an incubator at 37°C containing 5% CO2 for 4 days. The serum neutralizing antibody titer was calculated by the Reed-Muench method.) The BVDV neutralizing antibody titer of at least 8 guinea pigs should not be lower than 1:64, the IBRV neutralizing antibody titer of at least 8 guinea pigs should not be lower than 1:64, the BPIV3 neutralizing antibody titer of at least 8 guinea pigs should not be lower than 1:64, and the neutralizing antibody titers of the negative controls should all be less than 1:2. For Mycoplasma bovis, the serum antibody was detected by ELISA, and the relative potency (RP) value was calculated. The RP value should be ≥1, and all the control group should be antibody negative.

[0087] The potency test results (guinea pigs) of the quadruple inactivated vaccine against bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, bovine parainfluenza virus type 3 and Mycoplasma bovis are shown in Table 3.

[0088] Table 3. Potency test results (guinea pigs)

[0089]

[0090] (2) Bovine immunization and challenge method: Use 40 healthy susceptible cattle (Holstein cattle) at 4 - 6 months of age that are double negative for BVDV, IBRV, BPIV3, mycoplasma antigen and antibody (the serum neutralization antibody titer of BVDV, IBRV, and BPIV3 is not higher than 1:2, and the antibody titer of bovine mycoplasma detected by indirect hemagglutination test < 1:4). Among them, 20 cattle are in the immunization group and 20 cattle are in the challenge control group. Each cattle in the immunization group is intramuscularly injected with 2 mL of the vaccine in the neck. After 3 weeks, a booster immunization is given at the same route and the same site once. 21 days after the second immunization, the cattle in the immunization group are divided into BVDV, IBRV, BPIV3, and mycoplasma groups according to the immunization program, with 5 cattle in each group. A total of 10 cattle including 5 cattle in the BVDV group and 5 challenge control cattle are challenged with BVDV virulent strain. Each cattle is inoculated intranasally with 10.0 mL of the virulent BVDV HH01 strain for testing (the virus content is 10 7.0 TCID 50 / mL), of which 3.0 mL / nostril and 4.0 mL by oral injection; A total of 10 cattle including 5 cattle in the IBRV group and 5 challenge control cattle are challenged with IBRV virulent strain TY01. Each cattle is intranasally challenged with 10.0 mL of the virulent IBRV TY01 strain (the virus content is 10 7.0 TCID 50 / mL), and the challenge is carried out by nasal drip in two times (once in the morning and once in the afternoon), with 2.5 mL in each nostril each time; A total of 10 cattle including 5 cattle in the BPIV3 group and 5 challenge control cattle are challenged with BPIV3 virulent strain. Each cattle is intranasally challenged with 10.0 mL of the virulent BPIV3 LH01 strain (the virus content is 10 7.0 TCID 50 / mL), and the challenge is carried out by nasal drip in two times (once in the morning and once in the afternoon), with 2.5 mL in each nostril each time; A total of 10 cattle including 5 cattle in the mycoplasma group and 5 challenge control cattle are injected intratracheally with 5 mL of fresh bacterial suspension of bovine mycoplasma CHF01 strain per cattle (the viable bacteria content is 5.0×10 10 ccu). After 2 days, the second challenge is carried out in the same manner and dose. Observe continuously for 14 days, and measure the body temperature, virus excretion titer of the test cattle, observe the clinical symptoms and collect nasal swabs for pathogen detection. After challenge with BVDV, IBRV, BPIV3, and bovine mycoplasma, at least 3 cattle in the control group should show symptoms of disease, and at least 4 cattle in the immunization group should be protected.

[0091] Table 4. Results of BVDV potency test (cattle)

[0092]

[0093]

[0094] Note: If three of the following conditions are met: continuous body temperature increase for 3 days, runny nose, tearing, and positive nasal swab PCR, it is judged as showing symptoms of disease.

[0095] Table 5. Results of IBRV Potency Test (Cattle)

[0096]

[0097] Note: If three of the following conditions are met: continuous fever for 3 days, runny nose, tearing, and positive nasal swab PCR, it is judged as diseased.

[0098] Table 6. Results of BPIV3 Potency Test (Cattle)

[0099]

[0100]

[0101] Note: If three of the following conditions are met: continuous fever for 3 days, runny nose, tearing, and positive nasal swab PCR, it is judged as diseased.

[0102] Table 7. Results of Mycoplasma bovis Potency Test (Cattle)

[0103]

[0104]

[0105] Note: If three of the following conditions are met: continuous fever for 3 days, coughing, runny nose, and positive nasal swab PCR, it is judged as diseased.

[0106] The test results (Tables 4 - 7) show that after immunizing cattle with the quadrivalent inactivated vaccine of bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, bovine parainfluenza virus type 3, and Mycoplasma bovis, and inoculating with the virulent strain 21 days after the second immunization, the protection rates against each virulent strain are 100%, 80%, 100%, and 80% respectively. The safety test and potency test of the developed bovine quadrivalent inactivated vaccine are qualified. It shows that the MDBK full suspension culture technology established in this study for preparing BVDV, IBRV, and BPIV3 antigen solutions and the process of preparing the quadrivalent inactivated vaccine by inoculating Mycoplasma bovis with Hayflick liquid medium are stable, have strong repeatability, reduce batch - to - batch differences, and have good value for popularization and application.

[0107] The above has described the present invention in detail. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any changes, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application but are made using conventional techniques known in the art. Some basic features can be applied within the scope of the following appended claims.

Claims

1. A quadrivalent inactivated vaccine against bovine viral diarrhea virus, bovine infectious rhinotracheitis virus, bovine parainfluenza virus type 3 and Mycoplasma bovis, wherein the active ingredients of the quadrivalent inactivated vaccine are inactivated bovine viral diarrhea virus, inactivated bovine infectious rhinotracheitis virus, inactivated bovine parainfluenza virus type 3 and inactivated Mycoplasma bovis; The bovine viral diarrhea virus is bovine viral diarrhea virus strain HH03; The bovine infectious rhinotracheitis virus is bovine infectious rhinotracheitis virus strain TY01; The bovine parainfluenza virus type 3 is bovine parainfluenza virus type 3 strain LH01; The Mycoplasma bovis is Mycoplasma bovis strain CHF01; In the quadruple inactivated vaccine, the ratio of the inactivated bovine viral diarrhea virus, the inactivated bovine infectious rhinotracheitis virus, the inactivated bovine parainfluenza virus type 3, and the inactivated Mycoplasma bovis satisfies: the inactivated bovine viral diarrhea virus is 10 8.0 TCID 50 / mL based on the bovine viral diarrhea virus before inactivation, the inactivated bovine infectious rhinotracheitis virus is 10 8.0 TCID 50 / mL based on the bovine infectious rhinotracheitis virus before inactivation, the inactivated bovine parainfluenza virus type 3 is 10 8.0 TCID 50 / mL based on the bovine parainfluenza virus type 3 before inactivation, and the inactivated Mycoplasma bovis is 1.0×10 10 ccu / mL based on the Mycoplasma bovis before inactivation.

2. The quadruple inactivated vaccine according to claim 1, wherein: The quadrivalent inactivated vaccine is composed of the active ingredients and an adjuvant.

3. A method for preparing the quadrivalent inactivated vaccine according to claim 1 or 2, the method comprising: 1) Culturing bovine viral diarrhea virus, bovine infectious rhinotracheitis virus and bovine parainfluenza virus type 3 respectively with suspension MDBK cells to obtain bovine viral diarrhea virus liquid, bovine infectious rhinotracheitis virus liquid and bovine parainfluenza virus type 3 liquid respectively; inactivating the bovine viral diarrhea virus liquid, the bovine infectious rhinotracheitis virus liquid and the bovine parainfluenza virus type 3 liquid respectively to obtain inactivated bovine viral diarrhea virus antigen liquid, inactivated bovine infectious rhinotracheitis virus antigen liquid and inactivated bovine parainfluenza virus type 3 antigen liquid respectively; Culturing Mycoplasma bovis with a liquid medium to obtain a Mycoplasma bovis culture solution, and inactivating the Mycoplasma bovis culture solution to obtain an inactivated Mycoplasma bovis antigen liquid; 2) Mixing the inactivated bovine viral diarrhea virus antigen liquid, the inactivated bovine infectious rhinotracheitis virus antigen liquid, the inactivated bovine parainfluenza virus type 3 antigen liquid and the inactivated Mycoplasma bovis antigen liquid as active ingredients to obtain a quadrivalent inactivated vaccine against bovine viral diarrhea virus, bovine infectious rhinotracheitis virus, bovine parainfluenza virus type 3 and Mycoplasma bovis; The bovine viral diarrhea virus is bovine viral diarrhea virus strain HH03, the bovine infectious rhinotracheitis virus is bovine infectious rhinotracheitis virus strain TY01, the bovine parainfluenza virus type 3 is bovine parainfluenza virus type 3 strain LH01, and the Mycoplasma bovis is Mycoplasma bovis strain CHF01.

4. The method according to claim 3, wherein: The inactivation is carried out using formaldehyde.

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