阅读说明：本技术 牛病毒性腹泻、牛传染性鼻气管炎二联活疫苗及其制备方法 (Bivalent live vaccine for bovine viral diarrhea and bovine infectious rhinotracheitis and preparation method thereof ) 是由 闫喜军 孟庆森 李真光 和彦良 陈超阳 宋妮 季烨 王宇菲 武华 于 2020-10-19 设计创作，主要内容包括：本发明公开了一种牛病毒性腹泻、牛传染性鼻气管炎二联活疫苗的制备及其应用,属于动物疫苗与兽用生物制品技术领域。该疫苗包含活的牛病毒性腹泻病毒NMM株和牛传染性鼻气管炎病毒JSM株。制备该疫苗的步骤包括,1)两种有效毒株的获得和致弱培养；2)MDBK细胞悬浮培养分别制备病毒抗原；3)以滴度为10～(4.7)TCID-(50)的浓度进行配比、分装,添加20%的冻干保护剂后冷冻干燥。所制备牛病毒性腹泻、牛传染性鼻气管炎二联活疫苗病毒滴度在10～(3.5)TCID-(50)的浓度下可获得病毒滴度分别为10～(7.3)TCID-(50)和10～(7.8)TCID-(50)的灭活疫苗同样的免疫效果,超剂量接种安全性评价及效力试验评价结果显示,本发明所述牛病毒性腹泻、牛传染性鼻气管炎二联活疫苗具有低成本、高功效及接种安全的特点。(The invention discloses a preparation method and application of a bovine viral diarrhea and bovine infectious rhinotracheitis bivalent live vaccine, and belongs to the technical field of animal vaccines and veterinary biological products. The vaccine comprises a live bovine viral diarrhea virus NMM strain and a bovine infectious rhinotracheitis virus JSM strain. The preparation method of the vaccine comprises the steps of 1) obtaining and weakening culture of two effective strains; 2) respectively preparing virus antigens by MDBK cell suspension culture; 3) to a titer of 10 4.7 TCID 50 The concentration of the active ingredients is proportioned and subpackaged, and then the active ingredients are frozen and dried after 20 percent of freeze-drying protective agent is added. The virus titer of the prepared bovine viral diarrhea and bovine infectious rhinotracheitis bivalent live vaccine is 10 3.5 TCID 50 At a concentration of 10 virus titers respectively 7.3 TCID 50 And 10 7.8 TCID 50 The inactivated vaccine has the same immune effect, and the results of the overdose inoculation safety evaluation and the efficacy test evaluation show that the bovine viral diarrhea and bovine infectious rhinotracheitis bivalent live vaccine has the characteristics of low cost, high efficacy and safe inoculation.)

1. The combined live vaccine for the bovine viral diarrhea and the bovine infectious rhinotracheitis is characterized in that the effective components of the vaccine comprise live bovine viral diarrhea type I virus antigen and bovine infectious rhinotracheitis virus antigen; the content of type I virus in bovine viral diarrhea is not less than 10^3.5TCID₅₀Per ml, the content of infectious bovine rhinotracheitis virus is not less than 10^3.5TCID₅₀Preparing and mixing the mixture evenly under the condition of/ml to form virus mixed liquor, and adding freeze-drying protective solution to mix evenly to obtain the virus-free antiviral vaccine.

2. The combined live vaccine for bovine viral diarrhea and bovine infectious rhinotracheitis according to claim 1, wherein the active ingredients are bovine viral diarrhea type I virus NMM strain and bovine infectious rhinotracheitis virus JSM strain.

3. The combined live vaccine for bovine viral diarrhea and infectious bovine rhinotracheitis according to claim 2, wherein the bovine viral diarrhea type I virus is a continuously subcultured attenuated virus strain NMM strain, and the infectious bovine rhinotracheitis virus is a continuously subcultured attenuated virus JSM strain.

4. The combined live vaccine for bovine viral diarrhea and infectious bovine rhinotracheitis according to claim 1, wherein the virus antigen production method is obtained by inoculating MDBK cells cultured in suspension for two viruses respectively.

5. The combined live vaccine for bovine viral diarrhea and infectious bovine rhinotracheitis according to claim 4, wherein the suspension-cultured MDBK cells are prepared from 0.25% pancreatin digested single layer MDBK cells.

6. The combined live vaccine for bovine viral diarrhea and infectious bovine rhinotracheitis according to claim 1, wherein the titer of the two virus solutions of the live vaccine is not less than 10^4.7TCID₅₀/ml。

7. The combined live vaccine for bovine viral diarrhea and infectious bovine rhinotracheitis according to claim 1, wherein the freeze-drying protective agent is a gelatin and sucrose protective agent, and comprises 2% of gelatin, 5% of sucrose and 1% of thiourea.

8. The bivalent live vaccine for bovine viral diarrhea and bovine infectious rhinotracheitis according to claim 1, wherein the mixing ratio of the virus mixture to the lyoprotectant is 4: 1.

9. Use of the live combination vaccine of claim 1 for the prevention of bovine viral diarrhea and infectious bovine rhinotracheitis.

Technical Field

The invention belongs to the technical field of animal vaccines and biological products for livestock, and particularly relates to a bovine viral diarrhea and bovine infectious rhinotracheitis bivalent live vaccine as well as a preparation method and application thereof.

Background

Bovine viral diarrhea/Mucosal Disease (BVD/MD) is caused By Viral Diarrhea Virus (BVDV), and is an infectious Disease with diarrhea, fever, leucopenia, Mucosal erosion ulcer, diarrhea or cough as main characteristics clinically, BVDV persistent infection can be caused to cattle, most persistent infection animals have healthy appearance, but the production performance is reduced, the infectious Disease becomes an important infectious source, the threat to the breeding industry of China is large, and serious economic loss is caused, the infectious Disease is one of important pathogens which endangers the cattle industry, is discovered in the United states in 1946 at the earliest, and then presents an explosive global epidemic. The disease is reported to be epidemic in China in the 80 th century, serological investigation shows that the average BVDV infection rate in China is 56.5%, the province with the highest positive rate is as high as 84.9%, the province with the lowest positive rate is 5.2%, the epidemic range of the disease is almost spread all over China, and huge harm and economic loss are caused to cattle raising industry in China. The 96 th bulletin issued by Ministry of agriculture defines bovine viral diarrhea as three types of epidemic diseases. The world animal health Organization (OIE) lists that epidemic diseases must be reported.

Infectious Bovine Rhinotracheitis (IBR) is an acute, febrile and contact Infectious disease of cattle caused by bovine herpes virus type I (BHV-I) virus, which is manifested by inflammation of upper respiratory tract and tracheal mucosa, dyspnea, rhinorrhea, lacrimation and the like, and can also cause damage to reproductive system, and secondary bacterial infection can cause more serious respiratory diseases. The disease can be manifested in various types, including respiratory tract type, genital tract infection type, meningitis type and ophthalmia type. The disease causes great economic loss to the cattle industry. It can delay the growth and weight gain of fattening cattle; after infection, calves develop symptoms such as encephalitis, and sometimes conjunctivitis and keratitis occur. After the sick dairy cattle suffer from diseases, the milk production amount is greatly reduced, and even the milk production is completely stopped. The disease was first seen in the united states in the early 50 s of the twentieth century and is currently prevalent worldwide. China discovers the disease from imported New Zealand cattle for the first time in 1980, and most of provincial cows, local yellow cattle and buffalo cattle are infected with IBRV at present. The research on the seroepidemiological investigation shows that the seropositive rate of the infectious bovine rhinotracheitis in China is different from 3 to 70 percent, and the average positive rate reaches 38 percent. With advances in etiology, IBRV has been isolated from many regions in recent years, and dairy cows in infected cattle farms have decreased milk production and increased mortality, resulting in significant economic losses. With the development of cattle raising industry in China, the disease has greater and greater influence on the dairy industry and the beef cattle industry in China.

At present, the vaccine inoculation is mostly adopted for preventing bovine viral diarrhea/mucosal disease and infectious bovine rhinotracheitis, and the commercial vaccines mainly comprise attenuated live vaccines and inactivated vaccines which have advantages and disadvantages respectively. The inactivated vaccine induces immune response with short duration, needs multiple vaccinations and cannot induce cytotoxic T lymphocyte reaction. At present, the commercialized vaccine in China is few, the cattle raising industry faces huge threat, the imported vaccine is expensive, and the vaccine cannot be obtained quickly when epidemic situation occurs. In clinical studies, BVDV and IBDV are often found as mixed infections. Therefore, the development of the bovine viral diarrhea/mucosal disease and bovine infectious rhinotracheitis bivalent live vaccine is urgently needed in China to achieve the purposes of preventing two diseases by one injection, improving the immune effect, saving labor and reducing the cost.

Disclosure of Invention

In order to solve the technical problems, the invention provides a method for producing a combined live vaccine for bovine viral diarrhea and bovine infectious rhinotracheitis by using MDBK cell suspension.

The invention provides a combined live vaccine for bovine viral diarrhea and infectious bovine rhinotracheitis, the effective components of the vaccine comprise live bovine viral diarrhea type I virus antigen and infectious bovine rhinotracheitis virus antigen; the content of I-type virus antigen of bovine viral diarrhea is not less than 10^3.5TCID₅₀Per ml, the content of infectious bovine rhinotracheitis virus antigen is not less than 10^3.5TCID₅₀Preparing and mixing the mixture evenly under the condition of ml to form virus mixed liquor, adding freeze-drying protective solution, mixing evenly and packaging.

Further, the active ingredients are bovine viral diarrhea type I virus NMM strain and bovine infectious rhinotracheitis virus JSM strain.

Furthermore, the bovine viral diarrhea type I virus is a continuously subcultured attenuated virus strain NMM strain, and the bovine infectious rhinotracheitis virus is a continuously subcultured attenuated virus JSM strain.

Further, the virus antigen production method is that two viruses are respectively inoculated to MDBK cells cultured in suspension.

Further, MDBK cells cultured in suspension were prepared from 0.25% trypsinized monolayer MDBK cells.

Further, the titer of the live vaccine prepared from two virus solutions is not less than 10^4.7TCID₅₀/ml。

Further, the freeze-drying protective agent is a gelatin and sucrose protective agent and contains 2% of gelatin, 5% of sucrose and 1% of thiourea respectively.

Further, the mixing ratio of the virus mixed solution to the freeze-drying protective agent is 4: 1.

According to the technical scheme of the combined live vaccine for bovine viral diarrhea and bovine infectious rhinotracheitis, the detailed preparation method comprises the following steps:

1. antigen production strains: the pathogen production strains of the bivalent live vaccine for the bovine viral diarrhea and the bovine infectious rhinotracheitis are a Bovine Viral Diarrhea Virus (BVDV) NMM strain and a bovine infectious rhinotracheitis virus (IBRV) JSM strain. The two strains for preparing the vaccine are respectively F85 and F111, can be neutralized by specific positive serum, and have specific fluorescence in an immunofluorescence test, and qualified and pollution-free attenuated or low-toxicity virus strains with good immunogenicity are detected by bacteria, moulds, mycoplasma and exogenous viruses.

2. Culturing the host cell; well-grown MDBK cells were seeded into a cell culture flask, and when the cells grew into a dense monolayer, they were digested into single cells with 0.25% trypsin and counted.

3. And (3) production of bovine viral diarrhea virus venom: inoculating into a first-stage bioreactor according to a certain cell inoculation density, and culturing when the cell density reaches the standard (not less than 2.5 × 10)⁶One/ml) (with the MOI of 0.04-0.06) is inoculated with bovine viral diarrhea virus to produce a virus strain NMM strain, the strain NMM strain is cultured for a certain time under specific conditions (the strain is cultured for 48-60 hours under the conditions of 37 ℃, 40-60 r/min, the pH value of 7.2 and 50% dissolved oxygen), virus liquid is obtained, the virus content is measured and subjected to sterile inspection, and the strain NMM strain is stored at the temperature below 15 ℃ for later use.

4. Production of infectious bovine rhinotracheitis virus venom: according to the MDBK cell culture procedure, inoculating the infectious bovine rhinotracheitis virus to produce a virus JSM strain (with the MOI of 0.01-0.03), culturing for a certain time under specific conditions (culturing for 24-36 hours under the conditions of 37 ℃, 40-60 r/min, the pH value of 7.2 and 50% dissolved oxygen), harvesting virus liquid, measuring the virus content, performing sterile inspection, and storing at the temperature below-15 ℃ for later use.

5. Seedling preparation and subpackaging: the ratio of the two virus antigens is determined that the content of the I type bovine viral diarrhea virus in each vaccine is not less than 10^4.7TCID₅₀Per ml, the content of infectious bovine rhinotracheitis virus is not less than 10^4.7TCID₅₀Per ml, calculating the dosage of the two components for preparing the seedlingAccurately measuring and then uniformly mixing, and supplementing the required antigen agent with diluent; uniformly mixing the mixed solution of the bovine viral diarrhea virus I and the infectious bovine rhinotracheitis virus with a freeze-drying protective agent according to a ratio of 4:1 to obtain a vaccine stock solution, and quantitatively subpackaging after uniformly mixing.

6. Bigeminal live vaccine overdose safety test: 20 BVD and IBR antigen-antibody which are 3-7 months old and are negative healthy calves are used and divided into 4 groups, 5 calves in each group are respectively inoculated with 3 batches of products, 10 calves/calve (the normal dose is 1 calve/ml), a control group is inoculated with 1ml of PBS solution in each calve, and temperature measurement and clinical symptom observation are carried out on the test animals for 14 consecutive days after vaccination.

7. Bivalent live vaccine efficacy test: 40 negative healthy calves with BVD and IBR antigen and antibodies of 3-7 months old are used, the test is divided into 4 groups, 10 calves in each group are respectively inoculated with 3 batches of the vaccine product, and the injection dose is 1 part/ml/head. Control groups were injected with PBS. On 28 days after immunization, 5 calves were randomly selected from each of the immunized group and the control group, each calf was intranasally sprayed with 6ml of BVDV JL strain F7 virus, after challenge, rectal temperature was measured daily, blood was taken every other day after challenge, white blood cell count and virus isolation were measured, and the process was terminated on 8 days after challenge. 5 calves of each immunization group and control group were kept, each calf was intranasally sprayed with 4ml of IBRV LN 01/08F 3 virus, the body temperature of the calves was measured daily, clinical symptoms including appetite, respiratory symptoms, mental state, nasal and ocular changes were observed daily, and viral isolation was performed on nasal swab samples until 7 days after challenge. Observing and recording the physiological condition of each experimental calf.

8. The efficacy comparison test of the bivalent live vaccine and the inactivated vaccine comprises the following steps: 30 negative healthy calves with BVD and IBR antigen antibodies of 3-7 months of age are used, the test is divided into 3 groups, 10 groups of each group and 1 st group are inoculated with the bivalent live vaccine, and the virus content titer in each group of vaccine is not less than 10^3.5TCID₅₀. Group 2 is inoculated with BVD-IBR combined inactivated vaccine, and the virus titer of each vaccine is 10 respectively^7.3TCID₅₀And 10^7.8TCID₅₀. Group 3 was a PBS control group. On 28 days after immunization, 5 calves were randomly selected from each of the immunized group and the control group, and 6ml of BVDV JL strain F7 was intranasally sprayed to each calfAfter the toxic treatment, the rectal temperature is measured every day, blood is collected every other day after the toxic treatment, the number of leucocytes is measured, and virus separation is carried out until 8 days after the toxic treatment. 5 calves of each immunization group and control group were kept, each calf was intranasally sprayed with 4ml of IBRV LN 01/08F 3 virus, the body temperature of the calves was measured daily, clinical symptoms including appetite, respiratory symptoms, mental state, nasal and ocular changes were observed daily, and viral isolation was performed on nasal swab samples until 7 days after challenge. The results show that 28 days after the vaccination, the combined live vaccine and the BVD-IBR combined inactivated vaccine achieve the same effect, and the control group has corresponding disease symptoms.

The invention has the beneficial effects that:

1. the combined inactivated vaccine for bovine viral diarrhea and infectious bovine rhinotracheitis is produced by a suspension cell culture technology without an attached carrier;

2. the bovine viral diarrhea virus NMM strain and the bovine infectious rhinotracheitis JSM strain are attenuated strains for continuous passage, and can exert efficient immune function under the condition that the virus content is far lower than the titer of an inactivated vaccine;

3. the method effectively reduces the production cost;

4. the produced bivalent live vaccine is safe and effective after being used for immunizing animals.

Drawings

FIG. 1 MDBK cells in suspension culture.

FIG. 2 shows the isolated fluorescent photographs of the leukoviruses after challenge with the BVDV JL strain. Wherein, a, the immune group is negative; b. the control group was positive.

Detailed Description

The present invention will be described in detail with reference to the accompanying drawings and examples.

First, vaccine preparation

1. Vaccine-preparing strain

Bovine viral diarrhea/Mucosal Disease virus (BVD/MD) NMM strain and Bovine Infectious rhinotracheitis (IBR) JSM strain, both of which were maintained by Wagner biopharmaceutical Co., Ltd.

Wherein, the I type cattleThe virus diarrhea virus strain is collected from suspected diseased bovine serum (positive PCR detection) in a certain cow farm of inner Mongolia, 1 virus is obtained by separating after MDBK cells are inoculated to the serum, and the virus is proved to be a BVDVI type virus through RT-PCR detection, gene sequencing, neutralization test and immunofluorescence test and is named as BVDV1 NM 01. In order to obtain virus seeds for vaccine preparation, the separated BVDV1 NM01 strain is continuously passaged and weakened on MDBK cells, cloning and purification are carried out once every 8-10 generations, and the virus titer can reach 10 along with passage adaptation of the virus on the cells^7.0 TCID₅₀And/ml, continuously subculturing the separated strain until F80 generations, and neutralizing viruses of F35 and F80 generations by BVDV positive serum. Animal experiment results show that the F35 and F80 generation viruses have no pathogenicity to cattle and have good immunogenicity. Through sequence analysis, the homology of the F3 gene and the F80 virus E2 gene is 97.8%. The subcultured attenuated strain of the isolated virus is selected as a vaccine strain and is named as BVDV1 NMM strain. As an original virus seed for preparing the vaccine, the purity detection shows that the virus strain is sterile, has no mycoplasma and other exogenous virus pollution, has good adaptability to MDBK cells, and the virus content is 10 after being detected^7.6 TCID₅₀The TCID50 is stable but has weak toxicity to cattle, can be neutralized by specific positive serum of the type I BVDV, has specific fluorescence in an immunofluorescence test, has good safety and immunogenicity, and has no other obvious pathological changes except water samples or viscous secretion in nostrils of virus infected healthy cattle.

The JSM strain is obtained by collecting nasal swabs of cattle with cough, high body temperature and nasal discharge of purulent nasal fluid in a cattle farm in Zhenjiang, Jiangsu province (PCR detection for positive IBRV antigen), and performing MDBK cell subculture. The identification results of a specific neutralization test, an immunofluorescence test, PCR, gene sequencing and the like show that the virus is bovine infectious tracheitis virus (IBRV) and is named as IBRV JS01 strain, the IBRV JS01 strain F3 is cloned and purified on MDBK cells and continuously passaged to F105 generations, and the titer of the F10 generation virus can reach more than 108.0 TCID50/ml along with the increase of the cell passage times of the virus. The pathogenicity research results of different generations of viruses show that the temperature of 1 cow in the F70 generation virus inoculation group is increased by more than 40.0 ℃, the temperature is only raised for 1 time, the body temperature of the F105 generation virus inoculation cow is normal, and the F70 and the F105 generation virus inoculation cow do not show clinical symptoms, which shows that the F70 and the F105 generation virus have no pathogenicity. After challenge, the protection rate of animals inoculated with the F70 and F105 generation viruses is 5/5, and the viruses of each generation can be neutralized by IBRV specific positive serum, which shows that the F70 and F105 generation viruses have good immunogenicity. Therefore, we continued to passage the isolated virus strain to a attenuated strain of F105 generation as an original strain for producing a vaccine, and named it as IBRV JSM strain. The purity detection shows that the strain is sterile, free of mycoplasma and other exogenous virus pollution, good in adaptability to MDBK cells, tested that the virus content is 108.5 TCID50/ml, capable of being neutralized by IBRV specific positive serum, good in safety and immunogenicity, and free of clinical symptoms after virus infection of healthy cattle, and specific fluorescence is generated in an immunofluorescence test.

2. Antigen production

(1) Virus host cell culture: inoculating well-grown MDBK cells into a cell culture bottle, culturing at 37 ℃ for 48-72 hours, and controlling the rotating speed to be 8-12 r/h; when the cells grew into a dense monolayer, they were digested into single cells with 0.25% pancreatin and counted as a host for inoculation of the reactor.

(2) Preparing I type bovine viral diarrhea virus liquid for preparing vaccine: well-grown MDBK cells were taken at 1.2X 10⁵2.0X 10 units/ml⁵Inoculating the cells/ml into a primary cell bioreactor to culture for 48-72 hours, and culturing when the cell density is not less than 2.5 multiplied by 10⁶When the strain is used per ml, the strain is inoculated with bovine viral diarrhea virus with the MOI of 0.04-0.06 to produce a virus strain NMM, and the strain is cultured for 48-60 hours at the temperature of 37 ℃, the temperature of 40-60 r/min, the pH value of 7.2 and the dissolved oxygen of 50 percent. Harvesting virus liquid, storing at-15 deg.C, measuring virus content, and performing aseptic inspection.

(3) Preparing infectious bovine rhinotracheitis virus for vaccine preparation: well-grown MDBK cells were taken at 1.2X 10⁵2.0X 10 units/ml⁵Inoculating the cells/ml into a primary cell bioreactor to culture for 48-72 hours, and culturing when the cell density is not less than 2.5 multiplied by 10⁶Taking the MOI of 0.01-0.03 to receive the infectious bovine rhinotracheitis per mlThe virus JSM strain is produced by the tracheitis virus, and cultured for 24-36 hours under the conditions of 37 ℃, 40-60 r/min, pH value 7.2 and dissolved oxygen of 50%. Harvesting virus liquid, storing at-15 deg.C, determining virus content, and performing aseptic inspection.

3. Determination of viral content

(1) The virus content of the type I bovine viral diarrhea virus NMM strain is determined: diluting the virus seed or vaccine (the vaccine is diluted to 1 part/ml by diluent) with DMEM medium containing 3.5% horse serum to 10 times serial dilution, and collecting 10^-2、10^-3、10^-4、10^-54 dilution, inoculating and culturing MDBK cells for 16-20 hours in a 96-well culture plate, inoculating 8 wells and 0.1 ml/well in each dilution, setting normal cell control wells, placing at 37 ℃, and containing 5% CO₂Culturing in incubator, observing for 4 days, and calculating TCID by Reed-Muench method formula₅₀。

(2) Measuring the virus content of the JSM strain of the infectious bovine rhinotracheitis virus: diluting the virus seed or vaccine (the vaccine is diluted to 1 part/ml by diluent) with DMEM medium containing 2% horse serum to 10 times serial dilution, and collecting 10^-2、10^-3、10^-4、10^-54 dilution, inoculating and culturing MDBK cells for 16-20 hours in a 96-well culture plate, inoculating 8 wells and 0.1 ml/well in each dilution, setting normal cell control wells, placing at 37 ℃, and containing 5% CO₂Culturing in incubator, observing for 4 days, and calculating TCID by Reed-Muench method formula₅₀。

4. Preparing seedlings and subpackaging

The qualified virus liquid is used as virus liquid for vaccine preparation, and the ratio of the two virus antigens is determined that the virus content of the bovine viral diarrhea virus type I in each vaccine is not less than 10^4.7TCID₅₀Per ml, the content of infectious bovine rhinotracheitis virus is not less than 10^4.7TCID₅₀Calculating the dosage of the two components for preparing the vaccine, mixing uniformly, and supplementing the required antigen agent by using a diluent; uniformly mixing the mixed solution of the bovine viral diarrhea virus I and the infectious bovine rhinotracheitis virus with a freeze-drying protective agent according to the proportion of 4:1 to obtain a vaccine stock solution, quantitatively packaging after uniformly mixing, and covering.

Second, vaccine application test

1. Bovine viral diarrhea and bovine infectious rhinotracheitis bigeminal live vaccine overdose inoculation safety test

For evaluating the inoculation safety test of the bovine viral diarrhea and infectious rhinotracheitis bivalent live vaccine (NMM strain + JSM strain, suspension culture), 20 BVD and IBR antigen antibodies of 3-7 months old are used as negative healthy calves, the calves are divided into 4 groups, 5 calves in each group are inoculated into 3 groups, the 1 st group to the 3 rd group are used as vaccination groups, 3 batches of products (batch numbers: 2016001, 2016002 and 2016003) are respectively inoculated into the groups, 10 batches of products are inoculated into the groups per head (normal dose is 1 batch per ml), the 4 th group is used as a control group, PBS solution with corresponding volume is inoculated into each batch, and temperature measurement and clinical symptom observation are carried out on test animals for 14 days continuously after vaccination. Experiments show that no obvious abnormality is found after the vaccine is inoculated. The result shows that the 10-time dose inoculation of the bivalent live vaccine (type I, NMM strain and JSM strain, suspension culture) for bovine viral diarrhea and infectious bovine rhinotracheitis is safe for calves.

2. Bivalent live vaccine efficacy test for bovine viral diarrhea and bovine infectious rhinotracheitis

40 negative healthy calves with BVD and IBR antigen and antibody of 3-7 months old are used, the test is divided into 4 groups, 10 groups are used, 3 batches of the vaccine products (batch numbers: 2016001, 2016002 and 2016003) are respectively inoculated from the 1 st group to the 3 rd group, and the injection dose is 1 batch/batch. Group 4 was a PBS control group. On 28 days after immunization, 5 calves were randomly selected from each of the immunized group and the control group, each calf was intranasally sprayed with 6ml of BVDV JL strain F7 virus, after challenge, rectal temperature was measured daily, blood was taken every other day after challenge, white blood cell count and virus isolation were measured, and the process was terminated on 8 days after challenge. 5 calves of each immunization group and control group were kept, each calf was intranasally sprayed with 4ml of IBRV LN 01/08F 3 virus, the body temperature of the calves was measured daily, clinical symptoms including appetite, respiratory symptoms, mental state, nasal and ocular changes were observed daily, and viral isolation was performed on nasal swab samples until 7 days after challenge.

The test is finished 14 days after challenge, the protection condition of each group of animals is counted after the challenge test is finished, and the results show that the protection rates of 3 batches of vaccine products after the BVDV JL strain is attacked to strong virus by a bigeminy vaccine group 28 days after the vaccination are respectively as follows: 80%, 80% and 100%, wherein the incidence rate of the BVDV JL strain after the BVDV negative control group attacks the virulent strain is 100%; the protection rates of 3 batches of vaccine products after the bivalent vaccine group attacks the IBRV LN01/08 strain virulent virus are respectively as follows: 80%, 100% and 100% of the incidence rate of IBRV negative control group after attacking IBRV LN01/08 strain virulent strain.

3. Comparative test of bovine viral diarrhea and bovine infectious rhinotracheitis bivalent live vaccine and inactivated vaccine

BVD and IBR antigen antibodies of 3-7 months old are 30 negative healthy calves, the test is divided into 3 groups, 10 groups of each group and 1 group are inoculated with bivalent live vaccines, and the virus content of an NMM (bovine viral diarrhea virus) strain of I and a JSM (infectious bovine rhinotracheitis virus) strain in each group of bivalent live vaccines is not lower than 10^3.5TCID₅₀. The 2 nd group inoculation market of a certain brand BVD-IBR combined inactivated vaccine has a virus titer of 10 for each vaccine^7.3TCID₅₀And 10^7.8TCID₅₀. Group 3 was a PBS control group. On 28 days after immunization, 5 calves were randomly selected from each of the immunized group and the control group, each calf was intranasally sprayed with 6ml of BVDV JL strain F7 virus, after challenge, rectal temperature was measured daily, blood was taken every other day after challenge, white blood cell count and virus isolation were measured, and the process was terminated on 8 days after challenge. 5 calves of each immunization group and control group were kept, each calf was intranasally sprayed with 4ml of IBRV LN 01/08F 3 virus, the body temperature of the calves was measured daily, clinical symptoms including appetite, respiratory symptoms, mental state, nasal and ocular changes were observed daily, and viral isolation was performed on nasal swab samples until 7 days after challenge.

The test is finished 14 days after the challenge, the protection condition of each group of animals is counted after the challenge test is finished, and the result shows that 28 days after the vaccination, the combined live vaccine for the bovine viral diarrhea and the bovine infectious rhinotracheitis and the combined inactivated vaccine for the BVD-IBR of a certain brand in the market have the same effect, and the control group has corresponding disease symptoms. The virus content of live vaccines is much lower than that of inactivated vaccines (table 1). Therefore, the application of the combined live vaccine for bovine viral diarrhea and bovine infectious rhinotracheitis can greatly reduce the cost for producing the vaccine.

TABLE 1 comparison of live and inactivated vaccines for bovine viral diarrhea and infectious bovine rhinotracheitis and test results