A recombinant Muscovy duck parvovirus, vaccine, egg yolk antibody and application thereof

Abstract

The present invention discloses a recombinant Muscovy duck parvovirus, a vaccine, an egg yolk antibody and an application, and belongs to the field of microbial technology. The present invention utilizes a recombinant Muscovy duck parvovirus rMDPV-GD23 isolated from a diseased Muscovy duck, with a deposit number of CGMCC No.46206. The virus has the ability to spread across species between Muscovy ducks and geese, and an inactivated vaccine and an egg yolk antibody are prepared therefrom, which has a preventive and therapeutic effect on the currently popular recombinant Muscovy duck parvovirus with a high mortality rate that can cause "feather loss" symptoms and can spread across species, changing the situation that there is currently no vaccine for prevention and conventional antiviral and antibacterial treatment methods have little effect, and has a good prospect for commercial development.

Description

Technical Field

[0001] The invention relates to the field of microbial technology, and in particular to a recombinant Muscovy duck parvovirus, a vaccine, an egg yolk antibody and applications. Background Art

[0002] The virus particles of the genus Parvovirus are non-enveloped, have strong resistance to the outside world, are resistant to alcohol, ether, and chloroform, and have a certain tolerance to temperature. Waterfowl parvovirus disease belongs to the genus Parvovirus. It is an acute, highly contagious disease that can cause high morbidity and mortality in waterfowl, causing huge economic losses to the waterfowl farming industry. Waterfowl parvovirus is divided into goose parvovirus (GPV)-related groups and Muscovy duck parvovirus (MDPV)-related groups. Recombinant Muscovy duck Parvovirus (r-MDPV) is derived from the recombination of Muscovy duck Parvovirus (MDPV) and goose parvovirus (GPV) genes. It belongs to the genus Dependent virus of the subfamily Parvovirinae. It is a single-stranded DNA virus. The virus particles are spherical and have a regular icosahedral symmetric structure.

[0003] In terms of infection characteristics, both goose parvovirus and duck parvovirus can infect Muscovy ducks, but duck parvovirus cannot infect geese. It is generally believed that the recombinant duck parvovirus (r-MDPV) formed by the recombination of duck parvovirus (MDPV) and goose parvovirus (GPV) also mainly infects ducklings, and is not infectious to geese or other Anseriformes. In terms of prevention and control, although the recombinant duck parvovirus is formed by the recombination of duck parvovirus (MDPV) and goose parvovirus (GPV), it is impossible to obtain a comprehensive effect by injecting two vaccines (MDPV vaccine, GPV vaccine) separately to prevent and control the recombinant duck parvovirus. Because the recombination site of the recombinant duck parvovirus is complex, the antibodies that can be bound by its capsid protein are not simply a mixture of GPV or MDPV, but require antibodies stimulated by a completely new capsid protein to be effective. Even ducklings immunized with the classic MDPV attenuated vaccine strain are still susceptible to rMDPV, making prevention and control difficult. Therefore, it is necessary to develop products that can prevent and treat diseases caused by specific recombinant Muscovy duck parvoviruses. Summary of the invention

[0004] In view of the above-mentioned prior art, the purpose of the present invention is to provide a recombinant Muscovy duck parvovirus, a vaccine, an egg yolk antibody and applications.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] In a first aspect of the present invention, a recombinant Muscovy duck parvovirus is provided. The recombinant Muscovy duck parvovirus is a recombinant Muscovy duck parvovirus ( Anas platyrhynchos recombinatus parvovirus ) rMDPV-GD23 was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (referred to as CGMCC, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​October 24, 2024, with the deposit number CGMCC No.46206.

[0007] The recombinant Muscovy duck parvovirus can infect young Muscovy ducks, which will show characteristic lesions of intestinal mucosal shedding and intestinal embolism, with obvious "feather loss" symptoms; infected goslings will experience feather loss on their necks at around 14 days of age, and a small amount of feather loss on their backs at 4 weeks of age. The virus will seriously affect the overall down quality of the geese. The feathers of the infected geese are of poor quality and feel defective and uneven, and the down production is greatly reduced.

[0008] Compared with other recombinant Muscovy duck parvoviruses, the recombinant Muscovy duck parvovirus rMDPV-GD23 of the present invention has the ability to spread across species.

[0009] Homologous recombination analysis revealed that the recombinant Muscovy duck parvovirus rMDPV-GD23 of the present invention recombined a segment in the P9 promoter region and the VP structural protein region.

[0010] The second aspect of the present invention provides the use of the recombinant Muscovy duck parvovirus in the preparation of a product for preventing and treating a disease caused by the recombinant Muscovy duck parvovirus.

[0011] Furthermore, the products include live vaccines, subunit vaccines, recombinant gene vaccines, nucleic acid vaccines and inactivated vaccines.

[0012] The vaccine of the present application can be formulated as a medicament for systemic or local administration, and can contain a pharmaceutically acceptable carrier, such as a phosphate-buffered saline solution, distilled water, a water / milk emulsion, a wetting agent, an emulsifier, a pH adjuster, and the like.

[0013] In some embodiments, the method for preparing an inactivated vaccine comprises the following steps:

[0014] (1) Preparation of oil phase: Take 94 parts of veterinary white oil and 2 parts of aluminum stearate, place them in an oil phase preparation tank and heat to 80°C, then add 6 parts of Span-80, and maintain the temperature until it reaches 115°C for 30 minutes. Then cool and set aside.

[0015] (2) Preparation of aqueous phase: Add 4 parts of sterile Tween-80 to 96 parts of the inactivated virus solution that has passed the inspection, and start the homogenizer to stir for 20 to 30 minutes to completely dissolve the Tween-80.

[0016] (3) Emulsification: The volume ratio of the oil phase to the water phase is 2:1. First, introduce the oil phase into the colloid mill, stir at 2500 rpm, slowly add the water phase, and emulsify at 10000 rpm for 5 minutes after adding. Before stopping the emulsification, add 0.01% of the total amount of the vaccine to thimerosal and shake thoroughly to mix. After emulsification, take 10mL and centrifuge at 3000r / min for 15 minutes. No water phase precipitates at the bottom of the tube. Obtain the inactivated vaccine, aseptically package the inactivated vaccine, roll the cap and store at 2~8℃.

[0017] The third aspect of the present invention provides a recombinant Muscovy duck parvovirus vaccine, wherein the recombinant Muscovy duck parvovirus vaccine comprises the recombinant Muscovy duck parvovirus.

[0018] Furthermore, adjuvants are also included.

[0019] The adjuvants include aluminum adjuvants, oil emulsion adjuvants, microbial adjuvants, propolis adjuvants, levamisole adjuvants, liposome adjuvants, traditional Chinese medicine adjuvants and small peptide adjuvants. In some embodiments, the adjuvant is white oil.

[0020] The fourth aspect of the present invention provides the use of the recombinant Muscovy duck parvovirus vaccine in the preparation of a product for preventing and treating a disease caused by the recombinant Muscovy duck parvovirus.

[0021] The fifth aspect of the present invention provides a recombinant Muscovy duck parvovirus vaccine composition, comprising the recombinant Muscovy duck parvovirus vaccine.

[0022] In the recombinant Muscovy duck parvovirus vaccine composition, the recombinant Muscovy duck parvovirus vaccine can also be used in combination with at least one of a goose parvovirus vaccine, a Muscovy duck parvovirus vaccine or other recombinant Muscovy duck parvovirus vaccines.

[0023] The sixth aspect of the present invention provides an egg yolk antibody, wherein the egg yolk antibody is prepared using the recombinant Muscovy duck parvovirus vaccine as an antigen.

[0024] In some embodiments, the method of preparation comprises the following steps:

[0025] (1) Eggshell disinfection: Collect high-immunity eggs and soak them in 1% chlorhexidine solution for 15 minutes. Take them out and dry them, then spray the surface of the eggshell with 75% alcohol for disinfection and set aside.

[0026] (2) Yolk separation: Mechanical egg beating is used to fully remove the egg white, germinal disc and frenulum, and collect the yolk.

[0027] (3) Inactivation I: Stir the collected egg yolks thoroughly to make them into a uniform paste. Start the peristaltic pump to pump the egg yolk liquid into the sandwich reaction tank. Add an injection water volume equal to the egg yolk volume (the injection water should be sterilized at 80℃ for 30 minutes and cooled to below 65℃). Stir and mix thoroughly, and then keep warm (inactivation) at 60-65℃ for 30 minutes.

[0028] (4) Acidification extraction: First, add acetic acid buffer with a pH value of 5.0 equivalent to 3 times the volume of the original egg yolk into the isolation reaction tank, then add the egg yolk liquid, turn on the mixer, and stir thoroughly for 30 minutes.

[0029] (5) Inactivation II: Add 4% caprylic acid (V / V) as an inactivator and extractant to the egg yolk solution, stir vigorously for 90 min, and place at 2-8°C for 4-8 hours.

[0030] (6) Coarse filtration: Filter with polypropylene 750B filter cloth, then filter with a column filter until clear.

[0031] (7) Sterilization and filtration: Sterilize with a 0.22 μm filter membrane. Store at 2-8°C for no more than 14 days. At the same time, take samples to test the neutralizing antibody titer of Muscovy duck parvovirus.

[0032] (8) Concentration: If the neutralizing antibody titer of the detected Muscovy duck parvovirus is lower than 1:512, the sterilized and filtered egg yolk antibody should be ultrafiltered and concentrated at an appropriate multiple using a 30-50KD concentration membrane package at 2-8°C. The neutralizing antibody titer of the concentrated Muscovy duck parvovirus should not be lower than 1:512.

[0033] (9) Inactivation III: Pour the concentrated solution into the inactivation tank, add 10% formaldehyde solution, turn on the stirrer to stir and mix thoroughly. The final concentration (V / V) of the formaldehyde solution is 0.1%. Inactivate at 37°C for 16 hours.

[0034] (10) Packaging and storage: The neutralizing antibody titer of the inactivated filtrate should not be less than 1:512. The filtrate should be packaged into sterile glass bottles under sterile conditions, covered with rubber stoppers, rolled aluminum caps, labeled, and stored for future use at -20°C.

[0035] The seventh aspect of the present invention provides the use of the egg yolk antibody in the preparation of a product for preventing and treating diseases caused by the recombinant Muscovy duck parvovirus.

[0036] The eighth aspect of the present invention provides a product for preventing and treating recombinant Muscovy duck parvovirus disease, wherein the product contains the egg yolk antibody at a pharmacologically effective concentration.

[0037] Beneficial effects of the present invention:

[0038] (1) The present invention isolates a recombinant Muscovy duck parvovirus rMDPV-GD23 from a diseased Muscovy duck. The strain can cause growth and development retardation in young Muscovy ducklings. At the same time, the virus can achieve cross-species infection, causing growth retardation and "feather loss" in geese. The whole genome of rMDPV-GD23 was analyzed, and the strain was named rMDPV-GD23, which was deposited in the General Microbiology Center of the China Microbiological Culture Collection Committee with a deposit number of CGMCC No.46206.

[0039] (2) The present invention further uses the strain rMDPV-GD23 to prepare an inactivated vaccine. After testing, the dosage form of the inactivated vaccine prepared by the present invention is a water-in-oil type (W / O); the centrifugal stability, viscosity and sterility tests meet the requirements of the "Pharmacopoeia of the People's Republic of China" (2020 edition). The vaccine was further tested: the safety test results proved that the trial vaccine was safe and harmless and had no effect on animal growth; the protective test results showed that the immune protection rate of the inactivated vaccine could reach 80%. In summary, the inactivated vaccine of recombinant Muscovy duck parvovirus rMDPV-GD23 prepared by the present invention is an inactivated vaccine with relatively ideal immune effect, which can effectively prevent the occurrence of diseases caused by recombinant Muscovy duck parvovirus with geese developmental delay and "feather loss" as the pathological characteristics.

[0040] (3) The present invention further utilizes the inactivated vaccine to prepare recombinant Muscovy duck parvovirus yolk antibodies, and conducts quality inspection on the yolk antibodies. The results confirm that the yolk antibodies have passed safety inspections, sterility inspections, and efficacy inspections (immunoassay method). The yolk antibodies are used to prevent and treat diseases caused by recombinant Muscovy duck parvovirus, and are effective for both Muscovy ducks and goslings. The present invention has a preventive and therapeutic effect on the currently prevalent diseases caused by recombinant Muscovy duck parvovirus, which can cause "feather loss" symptoms and can be transmitted across species with a high mortality rate. This changes the situation where there is currently no vaccine for prevention and conventional antiviral and antibacterial treatments have little effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a picture of the lesions of geese infected with rMDPV-GD23, causing "feather loss".

[0042] Figure 2 This is a homology analysis diagram of the recombinant Muscovy duck parvovirus rMDPV-GD23.

[0043] Figure 3 This is a diagram of the recombination analysis of the recombinant Muscovy duck parvovirus rMDPV-GD23.

[0044] Figure 4 This is the evolutionary tree diagram of the recombinant Muscovy duck parvovirus rMDPV-GD23.

[0045] Figure 5This is a picture of the lesions of Muscovy duck primary fibroblasts (MDEF) infected with rMDPV-GD23 for 36 hours. DETAILED DESCRIPTION

[0046] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0047] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0048] The experimental materials used in the examples of the present invention but not specifically described are all conventional experimental materials in the art and can be purchased through commercial channels.

[0049] Example 1: Discovery and identification of recombinant Muscovy duck parvovirus

[0050] The inventors isolated a recombinant Muscovy duck parvovirus strain from a diseased Muscovy duck with intestinal mucosal shedding to form intestinal plugs and neck feather shedding. The specific isolation and identification steps are as follows:

[0051] (1) The cell line selected was Muscovy duck primary fibroblasts (MDEF) which are more susceptible to Muscovy duck parvovirus. The MDEF frozen in the laboratory were revived and cultured until the cells were in good condition and suitable for virus inoculation.

[0052] (2) The liver and spleen of the dead Muscovy duck with severe hemorrhage and necrosis were taken and ground and homogenized with sterile saline at a ratio of 1:5 (W / V). After repeated freezing and thawing for 3 times, the mixture was centrifuged at 8000 rpm for 15 min and the supernatant was taken for use. 8000 U / mL of double antibiotics (penicillin and streptomycin) were added to the supernatant and stored at 4°C overnight. The supernatant was filtered to remove bacteria and other substances to obtain the original virus mixture. The mixture was inoculated into MDEF cells for passage. After the 4th passage, the virus liquid with cytopathic effect was isolated. The strain in the virus liquid was subjected to whole genome analysis (GenBank accession number is PP763298), and it was identified as recombinant Muscovy duck parvovirus by the laboratory.

[0053] The recombinant duck parvovirus strain was named rMDPV-GD23, and the strain was deposited. The deposit information is as follows:

[0054] Reference biological material: rMDPV-GD23;

[0055] Classification and nomenclature: Recombinant Muscovy duck parvovirus Anas platyrhynchos recombinatus parvovirus ;

[0056] Deposit number: CGMCC No.46206;

[0057] Deposit date: October 24, 2024;

[0058] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[0059] Deposit location: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0060] Example 2: Investigation of virus characteristics

[0061] 1. Verification of the cross-species transmission characteristics of the virus:

[0062] Forty one-day-old goslings that were not immunized with gosling plague antibodies were randomly divided into two groups, with 20 in each group. The first group was the experimental group, which was inoculated with 1 mL of the recombinant Muscovy duck parvovirus rMDPV-GD23 isolated in Example 1 through drinking water to simulate natural infection; the second group was the control group, which drank 1 mL of sterile PBS. After inoculation, the mental state of the animals in each group was observed daily, and the number of cases of feather shedding and growth retardation in each group was counted. The observation was continued for 2 weeks. After 2 weeks, the experimental animals were dissected to observe the lesions. The condition of the sick goslings is shown in the figure. Figure 1 The statistical results are shown in Table 1, among which, the statistical method for feather shedding and growth retardation is the number of diseased goslings after vaccination / the total number of goslings.

[0063] Table 1 Verification of cross-species transmission characteristics of the virus

[0064]

[0065] As can be seen from Table 1, the recombinant Muscovy duck parvovirus rMDPV-GD23 isolated in Example 1 achieved cross-species transmission.

[0066] 2. Homologous recombination analysis:

[0067] Homology analysis of the recombinant duck parvovirus was performed. Figure 2 .

[0068] Depend on Figure 2 It can be seen that the homology of this strain with GPV currently registered on NCBI is between 84.4 and 89.4, the homology with MDPV is between 93.3 and 93.9, and the homology with NGPV is between 84.3 and 84.9, among which the homology with MDPV is higher. The virus has the ability to spread across species, which is something that none of the Muscovy duck parvovirus strains introduced so far have mentioned.

[0069] The strain was further recombined by RDP4, and the results are shown in Figure 3 .

[0070] Depend on Figure 3 It can be seen that a segment was recombined in the P9 promoter region and the VP structural protein region. The virus capsid protein is composed of VP3. In our recombination analysis, the VP3 region of the virus contains a small part of the MDPV gene and most of the GPV gene. This is a new capsid protein. The antibodies that this new capsid protein can bind to are not simply a mixture of GPV or MDPV, but require antibodies stimulated by this new capsid protein to be effective.

[0071] Based on the 3121 to 4251 positions of the VP1 segment of the recombination region, a phylogenetic tree was constructed using this recombination region ( Figure 4 ), according to the recombinant evolutionary tree we constructed, it can be found that the strains that provide the recombinant templates in this type of virus are basically wild strains of GPV, and the parent strains are wild strains of MDPV. However, the recombinant virus can cause the "feather loss" characteristics of goslings, which is unique to this virus. This also means that the recombinant Muscovy duck parvovirus strains obtained from different types of major parent strains and secondary parent strains are inconsistent in infection characteristics, and this strain has evolved new cross-species infection characteristics.

[0072] Example 3: Preparation of recombinant Muscovy duck parvovirus inactivated vaccine

[0073] The preparation steps of inactivated vaccine are as follows:

[0074] 1. Virus proliferation procedure: The recombinant duck parvovirus rMDPV-GD23 strain isolated in Example 1 was inoculated into the well-growing primary duck fibroblasts MDEF, and the virus was massively propagated to obtain sufficient cell fluid. The MDEF cells were infected with rMDPV-GD23 for 36 hours. Figure 5 The obtained sufficient cell fluid was frozen at -80°C, and after three freeze-thaw cycles, the virus fluid was collected;

[0075] 2. Virus liquid concentration: The harvested virus liquid is concentrated to 1 / 3 of the original volume using an ultrafiltration concentrator at 2-8°C, and sterility tests are performed according to the current appendix of the Chinese Pharmacopoeia of Veterinary Medicine. Sterile growth is detected, and samples are kept for testing of virus titer. The virus content of each 0.1 mL of virus liquid should be ≥10 5.0 TCID 50 The concentrated virus solution is then inactivated.

[0076] 3. Inactivation: Place the concentrated virus solution into an inactivation tank, add 10% formaldehyde solution to make the final concentration 0.1%. After adding the formaldehyde solution, place it into another inactivation tank to prevent the virus adhering near the tank mouth from contacting the inactivator. Seal and inactivate at 37℃ for 24 hours (starting from when the temperature in the tank reaches 37℃), stir every 4~6 hours, and store at 2~8℃ for later use after inactivation.

[0077] 4. Preparation of inactivated vaccines:

[0078] (1) Preparation of oil phase: Take 94 parts of veterinary white oil and 2 parts of aluminum stearate, place them in an oil phase preparation tank and heat to 80°C, then add 6 parts of Span-80, and maintain the temperature until it reaches 115°C for 30 minutes. Then cool and set aside.

[0079] (2) Preparation of aqueous phase: Add 4 parts of sterile Tween-80 to 96 parts of the inactivated virus solution that has passed the inspection, and start the homogenizer to stir for 20 to 30 minutes to completely dissolve the Tween-80.

[0080] (3) Emulsification: The volume ratio of the oil phase to the water phase is 2L:1L. First, introduce the oil phase into the colloid mill, stir at 2500 rpm, slowly add the water phase, and emulsify at 10000 rpm for 5 minutes after adding. Before stopping the emulsification, add 0.01% of the total amount of the vaccine to thimerosal, and shake and mix thoroughly. After emulsification, take 10mL and centrifuge at 3000r / min for 15 minutes. No water phase precipitates at the bottom of the tube. Obtain the inactivated vaccine, aseptically package the inactivated vaccine, roll the cap and store at 2~8℃.

[0081] The prepared inactivated vaccine is subjected to quality inspections including dosage form, centrifugal stability, viscosity and sterility. For specific methods, please refer to the "Pharmacopoeia of Veterinary People's Republic of China" (2020 edition).

[0082] After inspection, the dosage form of the inactivated vaccine prepared by the present invention is water-in-oil (W / O); the centrifugal stability, viscosity and sterility tests comply with the provisions of the "Pharmacopoeia of Veterinary People's Republic of China" (2020 edition).

[0083] Example 4: Vaccine safety testing

[0084] Take 20 6-day-old goslings and randomly divide them into 2 groups, namely the gosling test group and the gosling control group, with 10 goslings in each group. Take 20 6-day-old Muscovy ducklings and randomly divide them into 2 groups, namely the Muscovy duck test group and the Muscovy duck control group, with 10 ducklings in each group. The gosling test group and the Muscovy duck test group: inject the recombinant Muscovy duck parvovirus inactivated vaccine prepared in Example 3 into the leg muscle, 0.5 mL / bird. The gosling control group and the Muscovy duck control group: inject an equal amount of sterile white oil adjuvant into the leg muscle. After vaccination, observe the mental state of the animals in each group every day, and observe whether local inflammatory reactions such as redness, swelling, heat and pain occur at the injection site. The observation is continued for 2 weeks. After 2 weeks, the experimental animals are dissected to observe the absorption of the vaccine at the injection site;

[0085] Results: The experimental group experienced a brief mental depression, but recovered quickly. After two weeks of observation, the experimental and control groups grew and developed normally, and their mental states were good. The autopsy of the experimental group found that the vaccine was well absorbed at the injection site, without redness, swelling, tissue necrosis, and other inflammatory reactions. The results proved that the trial vaccine was safe and harmless, and had no effect on animal growth.

[0086] Example 5: Testing the protective properties of vaccines

[0087] Twenty 6-day-old goslings were randomly divided into two groups, a gosling test group and a gosling control group, with 10 goslings in each group. Twenty 6-day-old Muscovy ducklings were randomly divided into two groups, a Muscovy duck test group and a Muscovy duck control group, with 10 ducklings in each group. The gosling test group and the Muscovy duck test group: the recombinant Muscovy duck parvovirus inactivated vaccine prepared in Example 3 was injected into the leg muscle, 0.5 mL / bird. The gosling control group and the Muscovy duck control group: an equal amount of sterile white oil adjuvant was injected into the leg muscle. Seven days after vaccination, the two groups of goslings and two groups of Muscovy ducklings were injected with 0.2 mL of the rMDPV-GD23 virus solution prepared in Example 3 with a content of 100 TCID50 into the legs. The TCID50 of the virus is 10 -6.8 / 0.1mL, and continued to be isolated and raised in groups for 14 days, and the mental state and mortality of the goslings and ducklings were observed. The results are shown in Table 2:

[0088] Table 2 Vaccine protection test

[0089]

[0090] After the virus attack, the subjects in the test group showed short-term mental depression, 1 gosling died, and 2 ducklings died; the remaining experimental subjects in the duckling test group did not show clinical symptoms of recombinant duck parvovirus disease, and 2 ducklings in the gosling test group showed growth retardation. After the virus attack, the duckling control group showed obvious clinical symptoms and 8 deaths, and the 2 surviving ducks showed typical lesions such as beak development disorders; 5 ducklings died in the gosling control group, and the 5 surviving goslings showed obvious developmental retardation and "feather loss" in the later stage of growth. The formula for calculating the immune protection rate is as follows:

[0091] Immune protection rate = (number of deaths and cases in the control group / number of samples in the control group - number of deaths and cases in the experimental group / number of samples in the experimental group) × 100%

[0092] The results showed that the inactivated vaccine has an immune protection rate of 90% for Muscovy ducklings and 60% for goslings.

[0093] In summary, the inactivated vaccine of recombinant Muscovy duck parvovirus rMDPV-GD23 prepared by the present invention is an inactivated vaccine with relatively ideal immune effect, which can effectively prevent the occurrence of recombinant Muscovy duck parvovirus with developmental delay and "feather loss" as the pathological characteristic of geese.

[0094] Example 6: Preparation of recombinant duck parvovirus yolk antibody

[0095] In order to further develop the virus, the recombinant Muscovy duck parvovirus inactivated vaccine prepared in Example 3 was used to prepare recombinant Muscovy duck parvovirus yolk antibody, and the method was as follows:

[0096] 1. Immunize laying hens with inactivated vaccines:

[0097] The recombinant Muscovy duck parvovirus inactivated vaccine prepared in Example 3 was used to immunize laying hens. For the first immunization, 2.0 mL of the inactivated vaccine was injected subcutaneously into the neck of each chicken. The second immunization was performed 14 days later, and 2.0 mL of the inactivated vaccine was injected subcutaneously into the neck of each chicken. The third immunization was performed 14 days after the second immunization, and 2.0 mL of the inactivated vaccine was injected subcutaneously into the neck of each chicken. A booster immunization was performed 14 days after the third immunization, and 2.0 mL of the inactivated vaccine was injected subcutaneously into the neck of each chicken. 14 days after the booster immunization, the yolk was collected to determine the recombinant Muscovy duck parvovirus neutralizing antibody titer, which should be ≥1:1024.

[0098] 2. Yolk antibody production:

[0099] (1) Eggshell disinfection: Collect high-immunity eggs and soak them in 1% chlorhexidine solution for 15 minutes. Take them out and dry them, then spray the surface of the eggshell with 75% alcohol for disinfection and set aside.

[0100] (2) Yolk separation: Mechanical egg beating is used to fully remove the egg white, germinal disc and frenulum, and collect the yolk.

[0101] (3) Inactivation I: Stir the collected egg yolks thoroughly to make them into a uniform paste. Start the peristaltic pump to pump the egg yolk liquid into the sandwich reaction tank. Add an injection water volume equal to the egg yolk volume (the injection water should be sterilized at 80℃ for 30 minutes and cooled to below 65℃). Stir and mix thoroughly, and then keep warm (inactivation) at 60-65℃ for 30 minutes.

[0102] (4) Acidification extraction: First, add acetic acid buffer with a pH value of 5.0 equivalent to 3 times the volume of the original egg yolk into the isolation reaction tank, then add the egg yolk liquid, turn on the mixer, and stir thoroughly for 30 minutes.

[0103] (5) Inactivation II: Add 4% caprylic acid (V / V) as an inactivator and extractant to the egg yolk solution, stir vigorously for 90 min, and place at 2-8°C for 4-8 hours.

[0104] (6) Coarse filtration: Filter with polypropylene 750B filter cloth, then filter with a column filter until clear.

[0105] (7) Sterilization and filtration: Sterilize with a 0.22 μm filter membrane. Store at 2-8°C for no more than 14 days. At the same time, take samples to test the neutralizing antibody titer of Muscovy duck parvovirus.

[0106] (8) Concentration: If the neutralizing antibody titer of the detected Muscovy duck parvovirus is lower than 1:512, the sterilized and filtered egg yolk antibody should be ultrafiltered and concentrated at an appropriate multiple using a 30-50KD concentration membrane package at 2-8°C. The neutralizing antibody titer of the concentrated Muscovy duck parvovirus should not be lower than 1:512.

[0107] (9) Inactivation III: Pour the concentrated solution into the inactivation tank, add 10% formaldehyde solution, turn on the stirrer to stir and mix thoroughly. The final concentration (V / V) of the formaldehyde solution is 0.1%. Inactivate at 37°C for 16 hours.

[0108] (10) Packaging and storage: The neutralizing antibody titer of the inactivated filtrate should not be less than 1:512. The filtrate should be packaged into sterile glass bottles under sterile conditions, covered with rubber stoppers, rolled aluminum caps, labeled, and stored for future use at -20°C.

[0109] Example 7: Yolk antibody quality test

[0110] 1. Safety inspection:

[0111] Ten susceptible Muscovy ducks (Muscovy duck parvovirus maternal antibodies <1:4) aged 1-3 days were injected intramuscularly with 2.0 mL of the egg yolk antibody prepared in Example 6 of the present invention.

[0112] After 14 days of observation, all susceptible Muscovy ducks survived in good health, indicating that the egg yolk antibody of the present invention has good safety.

[0113] 2. Sterility test:

[0114] According to the Pharmacopoeia of the People's Republic of China (2020 edition),

[0115] Results: No bacterial, mycoplasma and exogenous virus contamination.

[0116] 3. Efficacy test (immune challenge method):

[0117] Twenty one-day-old susceptible ducklings (Muscovy duck parvovirus maternal antibodies were all <1:4) were randomly divided into two groups, A and B, with 10 ducks in each group. A was the immunization group, and the egg yolk antibody prepared in Example 6 was injected intramuscularly, 0.5 mL per duck; B was the challenge control group, and physiological saline was injected intramuscularly, 0.5 mL per duck. They were isolated and raised.

[0118] After 24 hours, the ducklings in groups A and B were injected intramuscularly with a 10-fold dilution of the recombinant duck parvovirus rMDPV-GD23 with saline. The TCID50 of the diluted virus was 10 -6.8 / 0.1mL, 0.5mL per duck. Observe after the challenge and record the incidence and mortality of each group of ducklings.

[0119] Results: After infection with the virus in group A, the immune protection rate of the experimental animals was 100%. After infection with the virus in group B, the experimental animals began to become ill within 24 hours, and 9 died within 14 days.

[0120] Example 8: Use of egg yolk antibodies in the prevention of diseases caused by recombinant Muscovy duck parvovirus

[0121] 1. Preventive effect on Muscovy ducks

[0122] Sixty one-day-old susceptible Muscovy ducks (recombinant Muscovy duck parvovirus maternal antibodies were all <1:4) were used as test subjects and randomly divided into three groups, with 20 ducks in each group.

[0123] Experimental group: intramuscular injection of the egg yolk antibody prepared in Example 6, 0.5 mL per mouse;

[0124] Control group: The currently available inactivated Muscovy duck parvovirus vaccine was used to immunize laying hens, and egg yolk antibodies were prepared according to the method of Example 6. The egg yolk antibodies were injected intramuscularly, 0.5 mL per hens;

[0125] Blank control group: intramuscular injection of normal saline, 0.5 mL per mouse.

[0126] The three groups of ducks were isolated and raised. After 16 hours, they were injected intramuscularly with a 10-fold dilution of the recombinant duck parvovirus rMDPV-GD23 with saline. The TCID50 of the diluted virus was 10 -6.8 / 0.1mL, 0.5mL per duck. After 14 days of observation after the challenge, the ducks were autopsied and the incidence and mortality of each group of ducks were recorded.

[0127] The results are shown in Table 3, where the statistical method for preventive protection is the number of healthy surviving Muscovy ducklings after infection / the total number of Muscovy ducklings.

[0128] Table 3 The preventive effect of egg yolk antibody in Muscovy ducks

[0129]

[0130] The observation and record results are as follows: After the test group was intramuscularly injected with the egg yolk antibody prepared in Example 6, the group did not show symptoms of disease, and there was no acute death. The growth and development and psychomotor status of the ducks were good, and there was no appearance of feather loss, beak development disorder, etc. After autopsy, there was no bleeding in various organs, and no specific lesions such as intestinal embolism and thymus hemorrhage were found.

[0131] The control group was challenged with the virus after intramuscular injection of egg yolk antibodies made from the currently commercially available Muscovy duck parvovirus inactivated vaccine. Six ducks died acutely, and two of the remaining 14 ducks had dysplasia and ruffled feathers on the back and abdomen. After autopsy, the liver and spleen were hemorrhaged, the cyst of Fabricius was enlarged, the intestinal mucosal hemorrhage at the junction of the jejunum and ileum, and the thymus hemorrhage were specific lesions. No specific lesions of virus infection were found in the autopsy of the remaining Muscovy ducks.

[0132] The blank control group was challenged with the virus after intramuscular injection of normal saline, with a morbidity rate of 100% and a mortality rate of 95%, with only one duck surviving. Autopsy of the dead ducks revealed diffuse bleeding in various organs, as well as specific lesions such as intestinal embolism.

[0133] It can be seen from Table 3 that the egg yolk antibody of the present invention has an excellent preventive protective efficacy and can provide 100% preventive protection against the recombinant Muscovy duck parvovirus. The protective efficacy is better than that of the egg yolk antibody prepared by the existing inactivated vaccine.

[0134] 2. Preventive effect on goslings

[0135] 60 one-day-old geese (goose parvovirus maternal antibodies <1:4) were used as test subjects and randomly divided into three groups, 20 in each group.

[0136] Experimental group: intramuscular injection of the egg yolk antibody prepared in Example 6, 0.5 mL per mouse;

[0137] Control group: The currently available inactivated Muscovy duck parvovirus vaccine was used to immunize laying hens, and egg yolk antibodies were prepared according to the method of Example 6. The egg yolk antibodies were injected intramuscularly, 0.5 mL per hens;

[0138] Blank control group: intramuscular injection of normal saline, 0.5 mL per mouse.

[0139] The three groups of goslings were isolated and raised. After 16 hours, they were injected intramuscularly with a 10-fold dilution of the recombinant Muscovy duck parvovirus rMDPV-GD23 with saline. The TCID50 of the diluted virus was 10 -6.8 / 0.1mL, 0.5mL per gosling. After the infection, the goslings were observed for 14 days, and the incidence and mortality of each group of goslings were recorded by autopsy.

[0140] The results are shown in Table 4, where the preventive protection statistical method is the number of healthy surviving goslings after infection / the total number of goslings.

[0141] Table 4 The preventive effect of yolk antibodies in goslings

[0142]

[0143] The observation and record results are as follows: After the test group was intramuscularly injected with the egg yolk antibody prepared in Example 6, the group did not show symptoms of disease and there was no acute death. The geese had good growth and development and psychomotor status, and there was no appearance of feather loss, growth retardation, etc. After autopsy, no specific lesions such as bleeding were found in various organs.

[0144] The control group was injected intramuscularly with egg yolk antibodies made from the currently available Muscovy duck parvovirus inactivated vaccine, and then challenged with the virus. Two geese died acutely, and 12 of the remaining 18 geese showed growth and development disorders and messy feathers. After autopsy of the geese with "feather loss", it was found that their livers and spleens had bleeding, the bursa of Fabricius was obviously edematous, and the thymus had punctate hemorrhages and other specific lesions.

[0145] The blank control group was challenged with the virus after intramuscular injection of normal saline. The growth and development disorder rate of the geese reached 95%. After autopsy, diffuse bleeding in various organs was found, and a large number of "feather loss" occurred in the geese.

[0146] It can be seen from Table 4 that the egg yolk antibody of the present invention has excellent preventive protection efficacy and can provide 100% preventive protection for goslings infected with the recombinant Muscovy duck parvovirus. The protective efficacy is better than that of the egg yolk antibody prepared by the existing inactivated vaccine.

[0147] Example 9: Use of egg yolk antibodies in the treatment of diseases caused by recombinant Muscovy duck parvovirus

[0148] 1. Therapeutic effect on Muscovy ducks

[0149] In the area where recombinant Muscovy duck parvovirus infection occurs, the symptoms are depression and stunted growth and development. Thirty Muscovy ducks with similar disease course and age were used as test subjects. They were randomly divided into three groups, with 10 ducks in each group. All were kept in isolation, including:

[0150] Experimental group: intramuscular injection of the egg yolk antibody prepared in Example 6, 1.0 mL per mouse;

[0151] Control group: Laying hens were immunized with the currently available inactivated Muscovy duck parvovirus vaccine, and egg yolk antibodies were prepared according to the method of Example 6. The egg yolk antibodies were injected intramuscularly, 1.0 mL per egg;

[0152] Blank control group: intramuscular injection of normal saline, 1.0 mL per mouse.

[0153] The ducks were observed for 14 days and the disease and mortality of each group were recorded.

[0154] The results showed that 2 days after the injection of yolk antibodies in the experimental group, the feed intake of the diseased ducks began to increase, their mental state improved significantly, and no ducks died within 10 days; 10 days later, 1 / 2 of the ducks were killed and dissected, and the results showed that the thymus and cysts of Fabricius of the diseased ducks were enlarged, and there were no bleeding spots in the liver, spleen and other tissues and organs. After the injection of yolk antibodies in the control group, the feed intake and mental state of the diseased ducks did not improve significantly. From the 5th day of the injection of yolk antibodies, some ducks died, and the mortality rate of the diseased ducks within 10 days was 60%; 10 days later, 1 / 2 of the surviving ducks were killed and dissected, and the results showed that the bursa of Fabricius and thymus of the diseased ducks were significantly enlarged and bleeding, and there were a small number of necrotic spots in the pancreas and other tissues and organs, and the intestinal mucosa was severely red and swollen. The mortality rate of the diseased ducks in the blank control group reached 90% within 10 days.

[0155] 2. Therapeutic effect on goslings

[0156] In the area where recombinant Muscovy duck parvovirus infection occurs, the symptoms are depression and stunted growth and development. Thirty geese with similar disease course and age were used as test subjects. They were randomly divided into three groups, with 10 geese in each group. All were kept in isolation, including:

[0157] Experimental group: intramuscular injection of the egg yolk antibody prepared in Example 6, 1.0 mL per mouse;

[0158] Control group: Laying hens were immunized with the currently available inactivated Muscovy duck parvovirus vaccine, and egg yolk antibodies were prepared according to the method of Example 6. The egg yolk antibodies were injected intramuscularly, 1.0 mL per egg;

[0159] Blank control group: intramuscular injection of normal saline, 1.0 mL per mouse.

[0160] The geese were observed for 14 days and the illness and mortality of each group were recorded.

[0161] The results are as follows: 2 days after the injection of yolk antibodies in the experimental group, the feed intake of the sick geese began to increase, and their mental state improved significantly. No goose died within 10 days; 10 days later, 1 / 2 of the geese were killed and dissected, and the results showed that the thymus and cysts of Fabricius of the sick geese were enlarged, and there were no bleeding spots in the liver, spleen and other tissues and organs. After the injection of yolk antibodies in the control group, the feed intake and mental state of the sick geese did not improve significantly. Sick geese began to die from the 5th day after the injection of yolk antibodies, and the mortality rate of sick geese within 10 days was 20%; 10 days later, 1 / 2 of the surviving geese were killed and dissected, and the results showed that the bursa of Fabricius and thymus of the sick geese were significantly enlarged and bleeding, and there were a small number of necrotic spots in the pancreas and other tissues and organs, and the intestinal mucosa was severely red and swollen. The mortality rate of sick geese in the blank control group reached 30% within 10 days.

[0162] The above test results show that the egg yolk antibody of the present invention has good safety, good preventive effect and high cure rate, can be used for the prevention and treatment of recombinant Muscovy duck parvovirus infection, and has significant economic and social benefits.

[0163] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A recombinant Muscovy duck parvovirus ( Anas platyrhynchos recombinatus parvovirus ) rMDPV-GD23, characterized in that It is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC No.46206.

2. Use of the recombinant Muscovy duck parvovirus according to claim 1 in the preparation of products for preventing and treating diseases caused by the recombinant Muscovy duck parvovirus.

3. The use according to claim 2, characterized in that: The product described is an inactivated vaccine.

4. A recombinant Muscovy duck parvovirus vaccine, characterized in that: The recombinant Muscovy duck parvovirus vaccine comprises the inactivated virus of the recombinant Muscovy duck parvovirus according to claim 1.

5. The recombinant Muscovy duck parvovirus vaccine according to claim 4, characterized in that: Adjuvants are also included.

6. Use of the recombinant Muscovy duck parvovirus vaccine according to claim 4 or 5 in the preparation of products for preventing and treating diseases caused by the recombinant Muscovy duck parvovirus.

7. A recombinant Muscovy duck parvovirus vaccine composition, characterized in that: Comprising the recombinant Muscovy duck parvovirus vaccine according to claim 4 or 5.

8. An egg yolk antibody, characterized in that: The egg yolk antibody is prepared using the recombinant duck parvovirus vaccine according to claim 4 or 5 as an antigen.

9. Use of the egg yolk antibody according to claim 8 in the preparation of a product for preventing and treating diseases caused by recombinant Muscovy duck parvovirus.

10. A product for preventing and treating recombinant Muscovy duck parvovirus disease, characterized in that: The product contains the egg yolk antibody according to claim 8 at a pharmacologically effective concentration.

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