Gooling plague and duck virus hepatitis bivalent egg yolk antibody as well as preparation method and application thereof

Abstract

The invention relates to a gosling plague and duck virus hepatitis bivalent egg yolk antibody as well as a preparation method and application thereof. The vaccine is prepared from a gosling plague virus antigen part and a duck virus hepatitis virus antigen part, the gosling plague virus antigen part is a gosling plague virus HVFS22 strain, and the duck virus hepatitis virus antigen part comprises a duck virus hepatitis virus type 1 HVSD20 bead and a duck virus hepatitis virus type 3 HVGZ22 bead. The egg yolk antibody can provide immune protection for gosling plague virus and duck virus hepatitis virus (type 1 and type 3) at the same time, the tedious procedure of inoculating various vaccines is reduced, and the breeding efficiency is improved; a high-purity and high-titer specific antibody is adopted, so that the vaccine can quickly generate immune reaction in an animal body and has relatively strong resistance to infection of corresponding viruses; through strict quality control in the preparation process, possible harmful substances can be effectively removed by adopting an antibody purification technology, so that not only is the emergency treatment effect improved, but also the occurrence rate of adverse reactions after vaccination is greatly reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary vaccines, and in particular relates to a preparation method and application of a bivalent trivalent egg yolk antibody against gosling plague and duck viral hepatitis. Background Art

[0002] Gosling plague is an acute or subacute sepsis of goslings caused by the gosling plague virus. It is highly contagious and lethal. It mainly attacks goslings 4 to 20 days old after hatching, seriously affecting the development of the goose farming industry. Current prevention and control measures have certain limitations, and traditional vaccines need to be further improved in terms of immune effect and immune persistence.

[0003] Duck viral hepatitis is a highly lethal infectious disease of ducks, of which duck viral hepatitis virus type 1 and type 3 (DHV-1 and DHV-3) are the main pathogenic types. After infection, ducks will experience symptoms such as liver enlargement and bleeding, which will lead to growth stunting and even mass death of ducks, causing huge economic losses to the duck farming industry. Existing vaccines for duck viral hepatitis are insufficient in preventing mixed infection of the two types of viruses.

[0004] It can be seen that goose plague and duck viral hepatitis have seriously endangered waterfowl farming, and there is no one-shot prevention and control strategy for the aforementioned viruses on the current market. The bivalent trivalent antibody of the present invention comprises specific antibodies for goose plague virus, specific antibodies for duck viral hepatitis type 1 virus, and specific antibodies for duck viral hepatitis type 3 virus. These antibodies are obtained by immunizing laying hens to obtain highly immune eggs, and through yolk separation, extraction, filtration and purification and other biotechnologies, a tetravalent yolk antibody with high specificity, low side effects, low exogenous pollution, easy preparation and good efficacy and its preparation method are provided for the mixed infection of the two. Summary of the invention

[0005] The purpose of the present invention is to provide a bivalent trivalent egg yolk antibody against gosling plague and duck viral hepatitis, which can effectively prevent and treat the infection of gosling plague and duck viral hepatitis with one injection.

[0006] The technical scheme adopted by the present invention is: a goose plague and duck viral hepatitis bivalent trivalent egg yolk antibody is prepared from a goose plague virus antigen part and a duck viral hepatitis virus antigen part; the goose plague virus antigen part is a goose plague virus HVFS22 strain, and the duck viral hepatitis virus antigen part includes duck viral hepatitis virus type 1 HVSD20 beads and duck viral hepatitis virus type 3 HVGZ22 beads.

[0007] Preferably, the virus content of the goose plague virus HVFS22 strain is 10 5.5 ~10 7.5 EID 50 / 0.1mL, the virus content of duck virus hepatitis virus type 1 HVSD20 beads is 10 6.5 ~10 8.5 EID 50 / 0.1mL, the virus content of duck virus hepatitis virus type 3 HVGZ22 beads is 10 5.5 ~10 7.5 EID 50 / 0.1mL.

[0008] Preferably, the virus content of the goose plague virus HVFS22 strain is 10 6.5 EID 50 / 0.1mL, the virus content of duck virus hepatitis virus type 1 HVSD20 beads is 10 7.5 EID 50 / 0.1mL, the virus content of duck virus hepatitis virus type 3 HVGZ22 beads is 10 6.5 EID 50 / 0.1mL.

[0009] Another object of the present invention is to provide a method for preparing bivalent and trivalent egg yolk antibodies against gosling plague and duck viral hepatitis.

[0010] The preparation method comprises the following steps:

[0011] Step S1, preparing goose plague virus HVFS22 strain antigen liquid:

[0012] The virus seed was diluted 1:50 with sterile saline, and healthy susceptible goose embryos aged 11.5 to 13 days were inoculated into the chorioallantoic cavity at 0.2 mL / embryo, and the cells were incubated at 37 to 38°C for 144 hours, and the dead goose embryos and allantoic fluids were harvested at 48 to 144 hours.

[0013] Step S2, preparing duck viral hepatitis virus type 1 HVSD20 strain antigen liquid;

[0014] The virus seed was diluted 1:100 with sterile saline, and 0.1 mL / embryo was inoculated into the chorioallantoic cavity of 11-12 day old SPF duck embryos. The embryos were incubated at 37-38°C for 120 hours, and the allantoic fluid of dead embryos was harvested at 48-120 hours.

[0015] Step S3, preparing duck viral hepatitis virus type 3 HVSD20 strain antigen liquid;

[0016] The virus seed was diluted 1:50 with sterile saline, and 0.1 mL / embryo was inoculated into the chorioallantoic cavity of 11-12 day old SPF duck embryos. The embryos were incubated at 37-38°C for 120 hours, and the allantoic fluid of dead embryos was harvested at 48-120 hours.

[0017] Step S4, concentrating and inactivating the antigen solution;

[0018] The harvested antigen solution was concentrated 10 times, then inactivated with formaldehyde at a final concentration of 1‰ at 37°C for 24 hours, and stored at 4°C for later use;

[0019] Step S5, emulsifying the antigen concentrate to form an immunogen;

[0020] emulsifying duck viral hepatitis virus type 1 antigen concentrate and duck viral hepatitis virus type 3 antigen concentrate according to a volume ratio of 2-5:5-8, and emulsifying goose plague virus antigen liquid alone;

[0021] Step S6, establishing an immunization program;

[0022] Healthy 120-day-old laying hens were selected from clean farms and immunized four times with multiple injections. Each time, duck viral hepatitis virus immunogens and goose plague virus immunogens were injected simultaneously. The injection doses of the immunogens were 1.0 mL, 1.2 mL, 1.5 mL and 2.0 mL, respectively. Each time was 14 days apart. Eggs were collected 12 to 14 days after the four immunizations. The yolk antibodies in the yolks were immunologically tested. The AGP antibody titer of the goose plague virus HVFS22 strain was required to be ≥1:400, the neutralizing antibody titer of the duck viral hepatitis virus HVSD20 strain was ≥1:1024, and the neutralizing antibody titer of the duck viral hepatitis virus HVGZ22 was ≥1:300. After meeting the requirements, high-immunity eggs were collected.

[0023] Step S7, using highly immune eggs to prepare yolk antibodies.

[0024] Preferably: step S7 includes the following steps,

[0025] Step S71, after sterilizing the highly immune eggs, separating the vitelline membrane and collecting the egg yolk;

[0026] Step S72, preparing an acidified buffer solution, and adjusting the pH value of the acidified buffer solution to between 4.6 and 5.4;

[0027] Step S73, according to the neutralizing antibody titer of the high-immune egg, the egg yolk and the acidified buffer solution are mixed and stirred at a ratio of 1:4 to 8, and then allowed to stand at room temperature for 5 to 12 hours;

[0028] Step S74, filtering the supernatant of the mixed solution, adding 0.1-0.8% of n-octanoic acid, mixing and stirring, and standing at room temperature for 3-7 hours;

[0029] Step S75, taking the clear solution of the mixed solution and filtering it successively with 1.0 μm and 0.45 μm filter elements;

[0030] Step S76, deep filtering the semi-finished egg yolk antibody product using multiple layers of diatomaceous earth;

[0031] Step S77, using a membrane package with a molecular weight cutoff of 100 KD to concentrate the filtrate by 5 to 10 times;

[0032] Step S78, inactivating the aforementioned concentrated solution using formaldehyde at a final concentration of 1‰ for 24 hours at room temperature.

[0033] Another object of the present invention is to provide a gosling plague and duck viral hepatitis bivalent trivalent egg yolk antibody for use in immunization and treatment of gosling plague virus and duck viral hepatitis virus.

[0034] The advantages and positive effects of the present invention are:

[0035] (1) Highly effective prevention: This vaccine can provide immune protection against both goose plague virus and duck viral hepatitis virus (type 1 and type 3), reducing the cumbersome procedure of vaccinating multiple vaccines separately and improving breeding efficiency;

[0036] (2) Good immune effect: Due to the use of high-purity, high-titer specific antibodies, the vaccine can quickly produce an immune response in the animal body and has a strong resistance to infection by the corresponding virus;

[0037] (3) High safety: The vaccine preparation process undergoes strict quality control, and the antibody purification technology used can effectively remove possible harmful substances, which not only improves the emergency treatment effect, but also greatly reduces the incidence of adverse reactions after vaccination. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is a flow chart of the preparation of the quadruple yolk antibody of the present invention. DETAILED DESCRIPTION

[0039] In order to further understand the content, features and effects of the present invention, the following embodiments are given to explain in detail.

[0040] See also Figure 1 The goose plague and duck viral hepatitis bivalent trivalent egg yolk antibody of the present invention is prepared from a goose plague virus antigen portion and a duck viral hepatitis virus antigen portion, and can be used to immunize poultry against goose plague virus and duck viral hepatitis virus.

[0041] The goose plague virus antigen part is the goose plague virus HVFS22 strain, and the duck viral hepatitis virus antigen part includes duck viral hepatitis virus type 1 HVSD20 beads and duck viral hepatitis virus type 3 HVGZ22 beads.

[0042] The bivalent trivalent egg yolk antibody of the present invention, wherein: the virus content of goose plague virus HVFS22 strain is 10 5.5 ~10 7.5 EID 50 / 0.1mL, the virus content of duck virus hepatitis virus type 1 HVSD20 beads is 10 6.5 ~10 8.5 EID 50 / 0.1mL, the virus content of duck virus hepatitis virus type 3 HVGZ22 beads is 10 5.5 ~10 7.5 EID 50 / 0.1mL. Furthermore, the virus content of goose plague virus HVFS22 strain was 10 6.5 EID 50 / 0.1mL, the virus content of duck virus hepatitis virus type 1 HVSD20 beads is 10 7.5 EID 50 / 0.1mL, the virus content of duck virus hepatitis virus type 3 HVGZ22 beads is 10 6.5 EID 50 / 0.1mL.

[0043] The method for preparing the bivalent trivalent egg yolk antibody against gosling plague and duck viral hepatitis of the present invention comprises the following steps:

[0044] Step S1, preparing goose plague virus HVFS22 strain antigen liquid:

[0045] The virus seed was diluted 1:50 with sterile saline, and healthy susceptible goose embryos of 11.5 to 13 days old were taken, and 0.2 mL / embryo was inoculated into the chorioallantoic cavity. The incubation was continued at 37 to 38 °C and observed for 144 hours. The dead goose embryos and allantoic fluid were harvested at 48 to 144 hours.

[0046] Step S2, preparing duck viral hepatitis virus type 1 HVSD20 strain antigen liquid;

[0047] The virus seed was diluted 1:100 with sterile saline. 0.1 mL / embryo was inoculated into the chorioallantoic cavity of 11-12 day old SPF duck embryos. The embryos were incubated at 37-38°C for 120 hours and the allantoic fluid of dead embryos was harvested at 48-120 hours.

[0048] Step S3, preparing duck viral hepatitis virus type 3 HVSD20 strain antigen liquid;

[0049] The virus seed was diluted 1:50 with sterile saline, and 0.1 mL / embryo was inoculated into the chorioallantoic cavity of 11-12 day old SPF duck embryos. The incubation was continued at 37-38°C, and the incubation was observed for 120 hours. The allantoic fluid of dead embryos at 48-120 hours was harvested.

[0050] Step S4, concentrating and inactivating the antigen solution;

[0051] The harvested antigen liquid was concentrated 10 times, then inactivated with formaldehyde at a final concentration of 1‰ at 37°C for 24 hours, and stored at 4°C until use.

[0052] After this step, semi-finished product inspection is carried out, and the inspection method is as follows:

[0053] 1. Sterility test

[0054] Take the inactivated antigen liquid and test it according to the appendix of the current "Chinese Veterinary Pharmacopoeia". It should be free of sterile growth.

[0055] 2. Determination of potency of semi-finished products

[0056] 2.1 Goose plague virus antigen liquid

[0057] The allantoic fluid that passed the bacterial test was diluted in 10 series with sterile saline, and 10 -5 , 10 -6 and 10 -7 There are three dilutions in total, each dilution has 5 12-day-old goose embryos, 0.2 mL / embryo, and the incubation continues at 37-38°C.

[0058] Record the number of goose embryos that died within 24 to 120 hours and calculate ELD 50 , the virus content per mL should be ≥10 6.5 ELD 50 .

[0059] 2.2 Duck viral hepatitis virus antigen liquid

[0060] 2.2.1 Preparation of duck viral hepatitis virus type 1 HVSD20 strain antigen solution;

[0061] The virus solution that passed the bacterial test was diluted in 10 series with sterile saline, and 10 -6 , 10 -7 and 10 -8 There are three dilutions in total. Five 11-day-old SPF duck embryos were inoculated with each dilution, 0.2 mL / embryo, and the incubation was continued at 37-38°C.

[0062] Record the number of dead embryos from 24 to 168 hours and calculate ELD 50 , the virus content per mL should be ≥10 6.5ELD 50 .

[0063] 2.2.2 Preparation of duck viral hepatitis virus type 3 HVSD20 strain antigen solution;

[0064] The virus solution that passed the bacterial test was diluted in 10 series with sterile saline, and 10 -5 , 10 -6 and 10 -7 There are three dilutions in total. Five 11-day-old SPF duck embryos were inoculated with each dilution, 0.2 mL / embryo, and the incubation was continued at 37-38°C.

[0065] Record the number of dead embryos from 24 to 168 hours and calculate ELD 50 , the virus content per mL should be ≥10 5.5 ELD 50 .

[0066] 3. Inactivation test:

[0067] 3.1 Goose plague virus antigen liquid

[0068] Inoculate the inactivated antigen liquid into the allantoic cavity of 10 susceptible goose embryos at 11 days old, 0.2 mL per embryo. Incubate at 37-38℃ for 7 days. Observe the embryos every day. No goose embryos should die.

[0069] 3.2 Duck viral hepatitis virus antigen liquid

[0070] Inoculate the inactivated antigen solution into the allantoic cavity of 10 SPF duck embryos aged 10-11 days, 0.2 mL per embryo. Incubate at 36-37℃ for 7 days. Observe the embryos every day. No duck embryos should die.

[0071] Step S5, emulsifying the antigen concentrate to form an immunogen;

[0072] The duck viral hepatitis virus type 1 antigen concentrate and the duck viral hepatitis virus type 3 antigen concentrate are emulsified at a volume ratio of 2-5:5-8, and the goose plague virus antigen liquid is emulsified separately.

[0073] The specific operation method of this step is as follows:

[0074] 1. Vaccine preparation

[0075] 1.1 Preparation of oil phase: Take 95 parts of white oil, add 1 part of aluminum stearate, stir evenly and heat to 80°C, then add 5 parts of Siben 80, after it is completely dissolved, keep at 121°C for 30 minutes.

[0076] 1.2 Preparation of aqueous phase: Take the inactivated antigen liquid with the titer meeting the standard and prepare it according to the proportion: duck viral hepatitis virus type 1 antigen liquid and duck viral hepatitis virus type 3 antigen liquid are mixed in the volume ratio of 2-5:5-8; goose plague virus antigen liquid is added in the total proportion, and sterile 5% Tween 80 is added and mixed thoroughly.

[0077] 1.3 Emulsification: Take 2 portions of oil phase and place them in an emulsification tank, slowly add 1 portion of water phase, and then emulsify at 8000rpm for 10 minutes. After the emulsification operation is completed, take 10mL and centrifuge at 3000rpm for 15 minutes. The water phase precipitated at the bottom of the tube should not exceed 0.5mL. Carry out quantitative packaging, seal with a cover, and store at a temperature of 2-8℃.

[0078] 1.4 Safety test: Use 5 healthy susceptible goslings of 1 day old, inject 2 mL of the vaccine subcutaneously, and observe for 10 days. All goslings should be alive and well. Use 10 SPF ducks of 7 days old, inject 2.0 mL of the vaccine into the chest muscle of each duck. At the same time, set up a control group of 10 ducks, raise them under the same conditions, and observe for 14 days. There should be no local or systemic adverse reactions caused by the vaccine. All ducks should be killed after 14 days, and all internal organs should be free of lesions.

[0079] Step S6, establishing an immunization program;

[0080] Healthy 120-day-old laying hens were selected from clean farms and immunized four times with multiple injections. Each time, duck viral hepatitis virus immunogen and goose plague virus immunogen were injected simultaneously. The injection doses of the immunogens were 1.0mL, 1.2mL, 1.5mL and 2.0mL, respectively. Each time was 14 days apart. Eggs were collected 12 to 14 days after the four immunizations, and the yolk antibodies in the yolk were immunologically tested. The AGP antibody titer of goose plague virus HVFS22 strain was required to be ≥1:400, the neutralizing antibody titer of duck viral hepatitis virus HVSD20 strain was ≥1:1024, and the neutralizing antibody titer of duck viral hepatitis virus HVGZ22 was ≥1:300. After meeting the requirements, high-immunity eggs were collected.

[0081] The specific operation method of this step is as follows:

[0082] 1. Immunize chickens

[0083] Laying hens around 130 days old were selected for immunization, with a total of 4 immunizations, all using multi-point injection method.

[0084] First immunization: duck viral hepatitis immunogen (type 1 + type 3) and goose plague immunogen are injected in the left and right directions respectively, with an injection volume of 1.0 ml per chicken.

[0085] Secondary immunization: duck viral hepatitis immunogen (type 1 + type 3) and goose plague immunogen were injected in the left and right directions respectively, with an injection volume of 1.2 ml per chicken.

[0086] Three immunizations: duck viral hepatitis immunogens (type 1 + type 3) and goose plague immunogens were injected in the left and right directions respectively, with an injection volume of 1.5 ml per chicken.

[0087] Four immunizations: duck viral hepatitis immunogens (type 1 + type 3) and goose plague immunogens were injected in the left and right directions respectively, with an injection volume of 2.0 ml per chicken.

[0088] Two weeks after the fourth vaccination, the high-immunity eggs can be used normally for workshop production. Later, based on the antibody titer detected, the duck viral hepatitis immunogen (type 1 + type 3) or goose plague immunogen is usually boosted again at an interval of about 4 weeks. The injection volume for each chicken is 1.0 ml each time.

[0089] 2. Collect high-immunity eggs

[0090] After 12-14 days of the four vaccinations, the laying hens were collected for testing the titer of high-immunity eggs. When the titer of AGP antibody of goose plague virus HVFS22 strain was ≥1:400, the titer of neutralizing antibody of duck viral hepatitis virus HVSD20 strain was ≥1:1024, and the titer of neutralizing antibody of duck viral hepatitis virus HVGZ22 was ≥1:300, the high-immunity eggs were collected and stored in a refrigerator for later use.

[0091] Step S7, using highly immune eggs to prepare yolk antibodies.

[0092] This step specifically includes the following steps:

[0093] Step S71, after sterilizing the highly immune eggs, separating the vitelline membrane and collecting the egg yolk;

[0094] Step S72, preparing an acidified buffer solution, and adjusting the pH value of the acidified buffer solution to between 4.6 and 5.4;

[0095] Step S73, according to the neutralizing antibody titer of the high-immune egg, the egg yolk and the acidified buffer solution are mixed and stirred at a ratio of 1:4 to 8, and then allowed to stand at room temperature for 5 to 12 hours;

[0096] Step S74, filtering the supernatant of the mixed solution, adding 0.1-0.8% of n-octanoic acid, mixing and stirring, and standing at room temperature for 3-7 hours;

[0097] Step S75, taking the clear solution of the mixed solution and filtering it successively with 1.0 μm and 0.45 μm filter elements;

[0098] Step S76, deep filtering the semi-finished egg yolk antibody product using multiple layers of diatomaceous earth;

[0099] Step S77, using a membrane package with a molecular weight cutoff of 100 KD to concentrate the filtrate by 5 to 10 times;

[0100] Step S78, inactivating the aforementioned concentrated solution using formaldehyde at a final concentration of 1‰ for 24 hours at room temperature.

[0101] The dilution was adjusted according to the antibody neutralization titer results to obtain bivalent and trivalent egg yolk antibodies against gosling plague and duck viral hepatitis.

[0102] Antibody potency test

[0103] 1. Finished product HI detection

[0104] Determination of the potency of the finished duck virus hepatitis antibody: dilute the antibody twice with physiological saline, take three dilutions of 1:512, 1:1024, and 1:2048 and mix them with type 1 or type 3 duck virus hepatitis liquid (containing 200 ELD 50 / 0.1mL), incubate at 37℃ for 60 minutes, inoculate 5 SPF duck embryos aged 11-12 days in the allantoic cavity at each dilution, 0.2mL per embryo, and set up a virus control group and a healthy control group, 5 embryos each. Continue incubation at 37℃, discard dead chicken embryos within 48 hours, remove duck embryos that died within 48-168 hours, record the death of duck embryos in each group, and calculate the half protective dose (PD 50 ), the titer of the antibody against duck viral hepatitis type 1 was 1:200, and the titer of the antibody against duck viral hepatitis type 3 was 1:80.

[0105] Determination of the potency of finished goose plague products: Add 1g of high-quality agar powder to 100mL of 8.0% sodium chloride solution, heat until the agar powder melts, and then cast a gel plate. Punch holes in the form of 1 hole in the center and 6 holes on the periphery, with a hole diameter of 3mm and a hole spacing of 4mm. The sample volume is controlled at 15-20μl. After the sample is added, place it in a wet box at 37-38℃ for incubation for 24 hours, and then place it at room temperature for 24-48 hours to determine the result. The maximum dilution multiple of the antibody that forms a white precipitate line with the standard antigen is the titer of the egg yolk antibody, and the agar diffusion antibody titer is 1:80.

[0106] 2. Attack and protection test

[0107] 2.1 Curing status of duck viral hepatitis

[0108] 20 ducklings infected with duck viral hepatitis were selected from the market, 10 of which were selected as the blank control challenge group, and the other 10 were injected with 1 mL of the antibody product as the treatment group. At the same time, 10 ducklings were selected as the blank control group, and the incidence and death of ducklings were recorded for 7 days.

[0109] Table 1 Comparison of cure status of duck viral hepatitis

[0110] Group Immunization dose Disease Death Survival Treatment Group 1mL 3 / 10 0 / 10 10 / 10 Blank control group / 10 / 10 4 / 10 6 / 10 Blank control non-challenge group / 0 / 10 0 / 10 10 / 10

[0111] The experimental data showed that none of the non-challenged groups in the blank control group developed the disease, while 10 / 10 of the challenged groups in the blank control group developed the disease; while the incidence of the antibody-treated group was only 3 / 10, and 10 / 10 were alive. This result shows that the antibody can effectively treat the onset of duck viral hepatitis.

[0112] 2.2 Cures of goose plague

[0113] The goslings infected with gosling plague in the market were divided into two groups, a blank control challenge group and a treatment group. At the same time, a blank control non-challenge group was set up, with 10 ducks in each group. The vaccine was then used for emergency injection with an injection volume of 1 mL. The ducks were observed for 7 days and the incidence and mortality of the ducklings were recorded.

[0114] Table 2 Comparison of cure status of goose plague

[0115] Group Immunization dose Disease Death Survival Treatment Group 1mL 3 / 10 0 / 10 10 / 10 Blank control group / 10 / 10 5 / 10 5 / 10 Blank control non-challenge group / 0 / 10 0 / 10 10 / 10

[0116] The experimental data showed that none of the non-challenged groups in the blank control group developed the disease, while 10 / 10 of the challenged groups in the blank control group developed the disease; while the disease rate in the antibody-treated group was only 3 / 10, and 10 / 10 were alive. This result shows that the antibody can effectively treat gosling plague.

Claims

1. A bivalent trivalent egg yolk antibody against goose plague and duck viral hepatitis, characterized in that: The goose plague virus antigen part is prepared from the goose plague virus antigen part and the duck viral hepatitis virus antigen part; the goose plague virus antigen part is the goose plague virus HVFS22 strain, and the duck viral hepatitis virus antigen part includes duck viral hepatitis virus type 1 HVSD20 beads and duck viral hepatitis virus type 3 HVGZ22 beads.

2. The goose plague and duck viral hepatitis bivalent trivalent egg yolk antibody according to claim 1, characterized in that: The virus content of the goose plague virus HVFS22 strain is 10 5.5 ~10 7.5 EID 50 / 0.1mL, the virus content of duck virus hepatitis virus type 1 HVSD20 beads is 10 6.5 ~10 8.5 EID 50 / 0.1mL, the virus content of duck virus hepatitis virus type 3 HVGZ22 beads is 10 5.5 ~10 7.5 EID 50 / 0.1mL.

3. The goose plague and duck viral hepatitis bivalent trivalent egg yolk antibody according to claim 2, characterized in that: The virus content of the goose plague virus HVFS22 strain is 10 6.5 EID 50 / 0.1mL, the virus content of duck virus hepatitis virus type 1 HVSD20 beads is 10 7.5 EID 50 / 0.1mL, the virus content of duck virus hepatitis virus type 3 HVGZ22 beads is 10 6.5 EID 50 / 0.1mL.

4. The method for preparing the bivalent trivalent egg yolk antibody against goose plague and duck viral hepatitis as claimed in claim 3, characterized in that: Includes the following step, Step S1, preparing goose plague virus HVFS22 strain antigen liquid: The virus seed was diluted 1:50 with sterile saline, and healthy susceptible goose embryos aged 11.5 to 13 days were inoculated into the chorioallantoic cavity at 0.2 mL / embryo, and the cells were incubated at 37 to 38°C for 144 hours, and the dead goose embryos and allantoic fluids were harvested at 48 to 144 hours. Step S2, preparing duck viral hepatitis virus type 1 HVSD20 strain antigen liquid; The virus seed was diluted 1:100 with sterile saline, and 0.1 mL / embryo was inoculated into the chorioallantoic cavity of 11-12 day old SPF duck embryos. The embryos were incubated at 37-38°C for 120 hours, and the allantoic fluid of dead embryos was harvested at 48-120 hours. Step S3, preparing duck viral hepatitis virus type 3 HVSD20 strain antigen liquid; The virus seed was diluted 1:50 with sterile saline, and 0.1 mL / embryo was inoculated into the chorioallantoic cavity of 11-12 day old SPF duck embryos. The embryos were incubated at 37-38°C for 120 hours, and the allantoic fluid of dead embryos was harvested at 48-120 hours. Step S4, concentrating and inactivating the antigen solution; The harvested antigen solution was concentrated 10 times, then inactivated with formaldehyde at a final concentration of 1‰ at 37°C for 24 hours, and stored at 4°C for later use; Step S5, emulsifying the antigen concentrate to form an immunogen; emulsifying duck viral hepatitis virus type 1 antigen concentrate and duck viral hepatitis virus type 3 antigen concentrate according to a volume ratio of 2-5:5-8, and emulsifying goose plague virus antigen liquid alone; Step S6, establishing an immunization program; Healthy 120-day-old laying hens were selected from clean farms and immunized four times with multiple injections. Each time, duck viral hepatitis virus immunogens and goose plague virus immunogens were injected simultaneously. The injection doses of the immunogens were 1.0 mL, 1.2 mL, 1.5 mL and 2.0 mL, respectively. Each time was 14 days apart. Eggs were collected 12 to 14 days after the four immunizations. The yolk antibodies in the yolks were immunologically tested. The AGP antibody titer of the goose plague virus HVFS22 strain was required to be ≥1:400, the neutralizing antibody titer of the duck viral hepatitis virus HVSD20 strain was ≥1:1024, and the neutralizing antibody titer of the duck viral hepatitis virus HVGZ22 was ≥1:

300. After meeting the requirements, high-immunity eggs were collected. Step S7, using highly immune eggs to prepare yolk antibodies.

5. The method for preparing the bivalent trivalent egg yolk antibody against gosling plague and duck viral hepatitis as claimed in claim 4, characterized in that: Step S7 includes the following steps, Step S71, after sterilizing the highly immune eggs, separating the vitelline membrane and collecting the egg yolk; Step S72, preparing an acidified buffer solution, and adjusting the pH value of the acidified buffer solution to between 4.6 and 5.4; Step S73, according to the neutralizing antibody titer of the high-immune egg, the egg yolk and the acidified buffer solution are mixed and stirred at a ratio of 1:4 to 8, and then allowed to stand at room temperature for 5 to 12 hours; Step S74, filtering the supernatant of the mixed solution, adding 0.1-0.8% of n-octanoic acid, mixing and stirring, and standing at room temperature for 3-7 hours; Step S75, taking the clear solution of the mixed solution and filtering it successively with 1.0 μm and 0.45 μm filter elements; Step S76, deep filtering the semi-finished egg yolk antibody product using multiple layers of diatomaceous earth; Step S77, using a membrane package with a molecular weight cutoff of 100 KD to concentrate the filtrate by 5 to 10 times; Step S78, inactivating the aforementioned concentrated solution using formaldehyde at a final concentration of 1‰ for 24 hours at room temperature.

6. Use of the bivalent and trivalent egg yolk antibody against gosling plague and duck viral hepatitis according to any one of claims 1 to 3 in immunization and treatment of gosling plague virus and duck viral hepatitis virus.

Images