Goose reovirus freeze-dried egg yolk antibody as well as preparation method and application thereof

Abstract

The invention discloses a goose reovirus freeze-dried egg yolk antibody as well as a preparation method and application thereof, and belongs to the technical field of bioengineering. The goose reovirus strain YJ24X disclosed by the invention is preserved in the China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, China, the preservation date is August 29, 2024, and the preservation number is CCTCC NO: V202477. The invention also discloses an application of the goose reovirus strain YJ24X in preparation of the goose reovirus egg yolk antibody. The invention also discloses an application of the goose reovirus freeze-dried egg yolk antibody in preparation of a combined vaccine with a duck infectious serositis bivalent inactivated vaccine. The goose reovirus strain YJ24X has high toxicity to goslings, and the freeze-dried egg yolk antibody prepared from the goose reovirus strain YJ24X is safe, effective and high in protection capacity; a combined vaccine is prepared from the vaccine and a duck infectious serositis bivalent inactivated vaccine, so that mixed infection epidemic situations can be effectively handled, and the disease prevention and control pressure is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a goose reovirus freeze-dried egg yolk antibody and a preparation method and application thereof. Background Art

[0002] my country has a huge waterfowl breeding and consumption market. The waterfowl breeding industry, especially the breeding of waterfowl such as ducks and geese, has a long history. With the advancement of agricultural modernization, it has gradually realized the transformation from free-range breeding to large-scale and intensive breeding. This transformation has not only significantly improved production efficiency, but also promoted the prosperity and diversification of the waterfowl product market. However, with the continuous expansion of waterfowl breeding scale and the increase in breeding density, a series of challenges have also arisen, among which the most urgent is the prevention and control of waterfowl diseases. In recent years, waterfowl diseases have shown a trend of increasing year by year. Old diseases still exist stubbornly, and new diseases continue to emerge, bringing unprecedented pressure to the breeding industry.

[0003] Avian orthoreoviruses (ARV) are members of the Orthoreovirus genus of the Reoviridae family. They can infect different types of poultry, including common chickens, turkeys, ostriches, waterfowl, wild ducks, etc. After infection, they can cause diseases such as malabsorption syndrome, tenosynovitis, viral arthritis, dwarfism syndrome and immunosuppression in poultry. This type of virus has become an important factor affecting the healthy development of the global poultry industry with its complex pathogenic mechanism and diverse clinical manifestations.

[0004] Specifically, different subtypes of ARVs cause different diseases in waterfowl. For example, duck reovirus (DRV) targeting ducks can cause a series of serious clinical symptoms, affecting the growth performance and health status of ducks; Muscovy duck reovirus (MDRV) and the newly discovered goose reovirus (GRV) in recent years should not be underestimated either. They can cause immunosuppression, growth retardation and even death in geese, posing a major threat to the goose industry.

[0005] In addition, there is no report on the use of goose reovirus freeze-dried egg yolk antibody and its combined use with vaccine. Therefore, it is particularly important to develop a goose reovirus freeze-dried egg yolk antibody and prepare a combined vaccine with a co-infection virus vaccine for goose disease prevention and control. Summary of the invention

[0006] In order to meet the needs of the above-mentioned field, the present invention provides a goose reovirus freeze-dried egg yolk antibody and a preparation method and application thereof.

[0007] One of the purposes of the present invention is to provide a goose reovirus, named goose reovirus strain YJ24X, which is deposited in China Center for Type Culture Collection, with a deposit address of Wuhan University, Wuhan, China, a deposit date of August 29, 2024, and a deposit number of CCTCC NO: V202477.

[0008] The second object of the present invention is to provide the use of the goose reovirus strain YJ24X in the preparation of goose reovirus egg yolk antibodies.

[0009] The third object of the present invention is to provide a method for preparing freeze-dried egg yolk antibodies of goose reovirus, comprising the following steps:

[0010] (1) inactivating the goose reovirus strain YJ24X, preparing it into an immunogen and then immunizing laying hens;

[0011] (2) When the agar titer of the yolk antibody of the laying hens immunized in step (1) is ≥1:26, eggs are collected;

[0012] (3) Using the eggs collected in step (2) to prepare freeze-dried egg yolk antibodies against goose reovirus.

[0013] Preferably, the immunization process in step (1) is:

[0014] The first immunization: 4 to 6 weeks before laying, 0.5 mL / feather;

[0015] Second immunization: 14 days after the first immunization, booster immunization 1.0 mL / bird;

[0016] The third immunization: 14 days after the second immunization, booster immunization 1.5 mL / bird;

[0017] Nth immunization: After the third immunization, booster immunizations of 1.5 mL / bird are performed every 2 to 3 months.

[0018] The fourth object of the present invention is to provide freeze-dried egg yolk antibodies against goose reovirus prepared by the above-mentioned preparation method of freeze-dried egg yolk antibodies against goose reovirus.

[0019] The fifth object of the present invention is to provide the use of the above-mentioned goose reovirus freeze-dried egg yolk antibody in the preparation of a combined vaccine with a duck infectious serositis bivalent inactivated vaccine.

[0020] Preferably, the use of freeze-dried egg yolk antibody of goose reovirus in preparing a combined vaccine with a bivalent inactivated duck infectious serositis vaccine comprises the following steps:

[0021] The combined vaccine was prepared by diluting the freeze-dried egg yolk antibody of goose reovirus with a diluent and then mixing it with the bivalent inactivated duck infectious serositis vaccine.

[0022] The beneficial effects of the technical scheme adopted in the present invention are as follows: after the freeze-dried egg yolk antibody of goose reovirus and the inactivated vaccine are fully dissolved and mixed with a diluent, the freeze-dried egg yolk antibody of goose reovirus and the inactivated vaccine are not affected when used in combination; it can achieve the effect of multiple protections with one shot, and while preventing multiple diseases at the same time, it also reduces the immunization cost and immune stress; the use process is safe and reliable, green and environmentally friendly, no antibiotics are added, and antibiotic reduction and limitation are done well.

[0023] More preferably, the diluent comprises the following components in parts by weight: 1 to 3 parts of white oil, 0.5 to 2 parts of Span 80, 0.3 to 0.8 parts of Tween-80 and 4 to 6 parts of transfer factor.

[0024] More preferably, the diluent comprises the following components in parts by weight: 2 parts of white oil, 1 part of Span 80, 0.5 parts of Tween-80 and 5 parts of transfer factor.

[0025] More preferably, each milliliter of transfer factor includes: 0.5-1.5 mg of polypeptide, 25-35 μg of nucleotide and 1-1.5 mg of polysaccharide.

[0026] More preferably, each milliliter of transfer factor includes: 1 mg of polypeptide, 30 μg of nucleotide and 1.3 mg of polysaccharide.

[0027] More preferably, the polypeptide is Pueraria peptide extract, and the polysaccharide is Astragalus polysaccharide.

[0028] The present invention has the following beneficial effects:

[0029] The goose reovirus provided by the present invention is named goose reovirus strain YJ24X, which is deposited in China Center for Type Culture Collection, Wuhan University, Wuhan, China, on August 29, 2024, and with a deposit number of CCTCC NO: V202477. 50 For 10 -3.6 / 0.1mL; the gosling regression experiment showed that the strain attacked 1-7 day old goslings with a morbidity rate of 100% and a mortality rate of 50%. The goose reovirus strain YJ24X is highly toxic to goslings. It is prepared as a freeze-dried egg yolk antibody, which has good solubility, is safe and effective, can produce a high level of antibodies, and has strong protective ability. In addition, the present invention also prepares a combined vaccine with the above-mentioned goose reovirus freeze-dried egg yolk antibody and the duck infectious serositis bivalent inactivated vaccine, which can effectively improve the immune effect of goslings against reovirus and serositis, reduce mortality and morbidity during the protection period, effectively respond to mixed infection epidemics, reduce disease prevention and control pressure, and significantly reduce the economic losses caused to the waterfowl farming industry by goose reovirus disease and mixed virus diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The results of RT-PCR of F3 generation allantoic fluid; M: DL 2000 Marker; 1: F3 generation allantoic fluid sample; 2: negative control;

[0031] Figure 2 This is the result diagram of the genetic evolution tree of goose reovirus strain YJ24X;

[0032] Figure 3 RT-PCR identification results of the samples; M: DL 2000 Marker; 3: challenge control group; 4: negative control. DETAILED DESCRIPTION

[0033] The following examples are only used to explain the present invention and are not used to limit the scope of the present invention. If the specific conditions are not specified in the examples, they shall be carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially. Pueraria peptide extract, purity 98%, is provided by Lanzhou Waters Biotechnology Co., Ltd.; nucleotides, purity 99%, are provided by Hubei Xinkang Pharmaceutical Chemical Co., Ltd.; astragalus polysaccharide, purity 99%, is provided by Hubei Changao Pharmaceutical Co., Ltd.

[0034] Example 1: Isolation and Identification of Goose Reovirus Strain YJ24X

[0035] 1Isolation and preliminary identification of goose reovirus strain YJ24X

[0036] 1.1 Sample collection and processing

[0037] The diseased geese in Jiangsu Province with white loose feces and soft feet as the main symptoms were used as the diseased materials. The autopsy showed that the liver was hemorrhagic and yellowish in color. Yellow-white necrotic foci were visible on the livers of some diseased geese. The spleen was enlarged, hemorrhagic and dark red, and the kidneys were enlarged and hemorrhagic. The liver, spleen and kidney diseased tissues of the diseased materials were collected, mixed and ground in a volume ratio of 1:3 between the diseased tissue and normal saline, frozen and thawed three times, and centrifuged at 12000rpm for 10 minutes. The supernatant was treated with 1 / 10 chloroform, and centrifuged at 12000rpm for 10 minutes. The supernatant was filtered through a 0.22μm filter membrane for sterilization, and stored in aliquots for later use.

[0038] 1.2 Embryo test

[0039] The isolated virus was inoculated into the chorioallantoic membrane of 11-day-old non-immune goose embryos. Death began to occur on the 4th day after inoculation. The embryo grafting situation is shown in Table 1. The mortality rate of F1 generation was 16.7% (10 / 60). The allantoic fluid of F1 generation was harvested and blindly propagated for 3 generations. Death began to occur on the 3rd day of F2 generation, with a mortality rate of 28.1% (9 / 32). The mortality rate of F3 generation from the 2nd to the 7th day was 100% (50 / 50). Compared with healthy goose embryos of the same embryonic age, the dead embryos showed systemic bleeding. ELD was calculated according to the Reed-Muench method 50 , ELD 50 For 10 -3.6 / 0.1mL.

[0040] Table 1 Embryo grafting after inoculation of 11-day-old non-immune goose embryos

[0041]

[0042] 1.3 PCR identification

[0043] PCR identification was performed on the allantoic fluid of the isolated strain F3, and the specific PCR primer sequences are shown in Table 2.

[0044] Table 2 Specific PCR primers

[0045]

[0046] The PCR reaction program was 3 μL RNA template, 0.5 μL upstream and downstream primers, 12.5 μL 2×1Step Buffer, 1 μL PrimeScript 1Step Enzyme Mix, and ddH2O was added to make up to 25 μL. The PCR reaction program was: reverse transcription at 50°C for 40 min; pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, 38 cycles; final extension at 72°C for 10 min.

[0047] After the reaction is completed, take 5 μL of the PCR product and run it on 1% agarose gel for gel electrophoresis. The results are as follows: Figure 1 As shown. Figure 1 It can be seen that the F3 generation allantoic fluid of the isolated strain was identified by RT-PCR, and the target band consistent with the expected one was observed, and it was preliminarily identified as goose reovirus.

[0048] 2 Analysis of genetic phylogenetic tree of goose reovirus strain YJ24X

[0049] The genetic evolution tree was constructed based on the gene of isolate YJ24X by the neighbor-joining method. Figure 2As shown, the results showed that the isolate was in the same evolutionary branch as the goose reovirus, with a homology of 92.6%; the isolate was identified as the goose-derived Muscovy duck reovirus. The present invention named the isolate as goose reovirus strain YJ24X, which was deposited in the China Center for Type Culture Collection, with a deposit address of Wuhan University, Wuhan, China, a deposit date of August 29, 2024, and a deposit number of CCTCC NO: V202477.

[0050] 3 Cell assay

[0051] The isolate was inoculated into LMH cells at a concentration of 1%, and obvious CPE was observed after 48 to 96 hours, and the virus solution was harvested. TCID 50 The titer is 10 4.5 .

[0052] 4 Animal regression test

[0053] Thirty healthy susceptible goslings aged 1 to 7 days were randomly selected, of which 10 were selected as blank control group geese and 20 as challenge group geese. The challenge group geese were inoculated intramuscularly with the strain, and the inoculation dose was 1 mL / geese. The disease appeared 2 days after the infection. The sick geese were listless, had soft feet, and excreted white loose feces. They died 3 days after the infection. After a 10-day observation period, the challenge group geese were dissected, and the autopsy revealed hemorrhage, swelling, necrotic foci in the liver, and enlarged spleen. The morbidity rate of 1-7 day old goslings attacked by the strain was 20 / 20 (100%), and the mortality rate was 10 / 20 (50%); liver, spleen and other pathological samples were collected for RT-PCR identification, and the results are as follows Figure 3 As shown, a positive band of 349 bp appeared, and during the 10-day observation period in the blank control group, 10 / 10 had no clinical symptoms.

[0054] 5 Virus culture characteristics

[0055] 5.1 Inoculation of healthy susceptible goose embryos

[0056] The virus was inoculated into 30 healthy susceptible goose embryos at 10 to 13 days of age, of which 20 were selected as the challenge group. The virus-inoculated group was inoculated with 0.3 mL of virus solution per embryo through the chorioallantoic membrane, and the blank control group (10) was inoculated with saline under the same conditions, incubated at 37°C, and observed continuously for 168 hours. During the observation period, goose embryos in the virus-inoculated group died and the dead embryos hemorrhaged; all goose embryos in the blank control group were alive.

[0057] 5.2 Cell inoculation

[0058] The virus was inoculated into LMH cells at a concentration of 1%, cultured at 37°C, and observed for 120 hours until CPE appeared in the cells.

[0059] Example 2: Preparation of freeze-dried egg yolk antibodies against goose reovirus

[0060] 1. Virus isolation and identification

[0061] The diseased material and normal saline were mixed and ground in a volume ratio of 1:3, freeze-thawed three times, and centrifuged at 12000rpm for 10 minutes. The supernatant was treated with 1 / 10 chloroform, centrifuged at 12000rpm for 10 minutes, and the supernatant was filtered and sterilized through a 0.22μm filter membrane. The healthy susceptible goose embryos of 10 to 13 days old were inoculated through the allantoic cavity at 0.3mL / piece. After 6 days, the allantoic fluid of dead goose embryos was collected and blindly propagated for 5 generations. The allantoic fluid F5 was collected, which could reach a mortality rate of 92%. Embryonic bleeding was visible in dead goose embryos. All goose embryos in the blank control group were healthy.

[0062] 2. Preparation of immunogen

[0063] (1) Inoculate the virus seed into healthy susceptible goose embryos of 10 to 13 days old with good development via chorioallantoic membrane, 0.3 mL per embryo; incubate at 37°C, collect the allantoic fluid of goose embryos that died after 48 to 120 hours, and store at -20°C;

[0064] (2) Inoculate chicken liver cancer cells (LMH cells) with 0.2% virus seed, supplement with DMEM nutrient solution, incubate at 37°C, collect CPE virus solution after 96-120 hours, and centrifuge to obtain the supernatant;

[0065] (3) The two culture solutions were mixed at a volume ratio of 1:1, and a formaldehyde solution with a final concentration of 0.2% was added, and the mixture was inactivated by shaking at 37°C for 48 hours; samples were taken for inactivation test, and after sterile growth, the samples were emulsified into an oil-in-water type immunogen with an aqueous phase and an oil phase at a volume ratio of 1:2.

[0066] 3 Immunization procedures

[0067] Select healthy laying hens before laying, and inject the prepared immunogen subcutaneously in the neck:

[0068] The first immunization: 4 to 6 weeks before laying, 0.5 mL / feather;

[0069] Second immunization: 14 days after the first immunization, booster immunization 1.0 mL / bird;

[0070] The third immunization: 14 days after the second immunization, booster immunization 1.5 mL / bird;

[0071] Nth immunization: After the third immunization, booster immunizations of 1.5 mL / bird are performed every 2 to 3 months.

[0072] Starting from 2 weeks after the third immunization, 10 high-immunity eggs were collected every 3 days, and the antibody agar titer was measured after the egg yolks were mixed. When the titer was not less than 1:26, the high-immunity eggs were collected.

[0073] 4 Preparation and effectiveness of freeze-dried purified egg yolk antibodies

[0074] 4.1 Preparation of freeze-dried egg yolk antibodies against goose reovirus strain YJ24X

[0075] The preparation method in this embodiment comprises the following steps:

[0076] (1) Eggshell disinfection: The highly immune eggs obtained in Example 2 were soaked in a 0.1% chlorhexidine aqueous solution at 42° C. for 15 min to disinfect the eggshells, and the yolks were collected;

[0077] (2) Inactivation I: The collected egg yolks were inactivated at 60°C for 30 min;

[0078] (3) Acid extraction: First, add 6 times the volume of egg yolk in water for injection (pre-adjust the pH to 3.0 with hydrochloric acid and cool to 4°C) into the sandwich reaction tank; then add the inactivated egg yolk solution while stirring. When the temperature of the egg yolk solution drops to 4°C, keep it warm and let it stand for 4 hours. After the acidification is completed, centrifuge and take the supernatant;

[0079] (4) Inactivation II: Add 0.2% octanoic acid as an inactivator and extractant, stir, and place at room temperature for 2 h;

[0080] (5) Coarse filtration and deep filtration: filter with cartridge filter elements with pore sizes of 5 μm and 1 μm respectively;

[0081] (6) Ultrafiltration concentration: Concentrate by 50-200 times through a hollow fiber ultrafiltration column with a molecular weight cutoff of 100 kDa, so that the titer of each feather is not less than 1:26;

[0082] (7) Sterile filtration: sterilize and filter using 0.45 μm and 0.22 μm cartridge filters in sequence;

[0083] (8) Packaging and freeze-drying: Add freeze-dried sucrose protective agent (1% gelatin and 5% sucrose), pack into 10 mL / bottle, and vacuum freeze-dry at -50 to -85°C for 96 hours to obtain goose reovirus freeze-dried egg yolk antibody.

[0084] 4.2 Effectiveness of freeze-dried egg yolk antibodies against goose reovirus

[0085] This embodiment also provides a method for testing the effectiveness of freeze-dried egg yolk antibodies against goose reovirus strain YJ24X, comprising the following steps:

[0086] Thirty healthy susceptible geese aged 1 to 5 days were randomly divided into three groups, with 10 geese in each group. Group 1 was a blank control group (injected with the same dose of sterile saline); Group 2 was a challenge control group; and Group 3 was an antibody group. The antibody group was subcutaneously injected with 1.0 mL of antibody per bird. 24 hours later, the challenge control group and antibody group geese were injected with 2×10 YJ24X embryo fluid toxin. 5.0 ELD 50After the injection, each group was isolated and kept for 30 days. On the last day of the observation period, the body weights of the groups were measured and all surviving geese were dissected.

[0087] The results showed that all the geese in the blank control group were alive and well, and no lesions were found in the autopsy. The incidence rate of the virus-challenged control group was 100%. The geese in the diseased group were listless, had soft feet, and excreted white loose feces. The geese in the control group had no clinical symptoms, and the autopsy revealed an enlarged liver with necrotic foci; the spleen was enlarged and dark red; the average weight of the geese in the antibody group was equivalent to that of the blank control group, and significantly higher than that of the virus-challenged control group, and the relative protection rate was up to 90%, and no liver and spleen lesions were found in the autopsy.

[0088] Example 3: Application of freeze-dried egg yolk antibody of goose reovirus in preparation of combined vaccine with bivalent inactivated duck infectious serositis vaccine

[0089] The freeze-dried egg yolk antibody of goose reovirus of the present invention has the characteristics of high titer, good stability, low stress and flexible compatibility. Nowadays, there are many types of waterfowl diseases, serious mixed infections, and huge pressure on disease prevention and control. It is difficult to cope with complex epidemics by relying solely on vaccines or egg yolk antibodies. Based on this, the present invention also provides the use of the freeze-dried egg yolk antibody of goose reovirus in preparing a combined vaccine with a bivalent inactivated vaccine of duck infectious serositis. The following provides a method and effect of preparing a combined vaccine with the freeze-dried egg yolk antibody of goose reovirus of the present invention and a bivalent inactivated vaccine of duck infectious serositis from different sources. The diluent used to dilute the freeze-dried egg yolk antibody of goose reovirus includes the following components in parts by weight: 2 parts of white oil, 1 part of Span 80, 0.5 parts of Tween-80 and 5 parts of transfer factor; wherein, 10mL of transfer factor contains 10mg of pueraria peptide extract, 300μg of nucleotides and 13mg of astragalus polysaccharide.

[0090] 1. Safety test of diluent

[0091] SPF chickens aged 7 to 14 days were selected and administered the diluted solution at 10 times the dose for 3 consecutive days and observed for 14 days. After 14 days, all the SPF chickens were alive and well, and the diluted solution was safe.

[0092] After the freeze-dried egg yolk antibody of goose reovirus was used in combination with the bivalent inactivated duck infectious serositis vaccine, the geese were immunized at a dose of 0.5mL / feather, 5 times the dosage. After 10 days of feeding, the immunized geese showed no abnormalities, their spirits and appetite were good, and the absorption was good. The immunization sites were examined 3 days after the immunization of the geese, and no abnormal bulges were found.

[0093] 2 Preparation of combined vaccine with Tianjin Ruipu Biological Duck Infectious Serositis Bivalent Inactivated Vaccine

[0094] Use 10mL of diluent to dilute 1000 copies of goose reovirus freeze-dried egg yolk antibody, and then shake with the duck infectious serositis bivalent inactivated vaccine (type 1 RAf63 strain + type 2 RAf34 strain) (approval number: veterinary medicine 020302202; production batch number: 20240201) of Tianjin Ruipu Biotechnology Co., Ltd. Airport Economic Zone Branch for 3 minutes, and then immunize 7-day-old geese in three goose farms A, B, and C with a scale of 5000 geese, and inject subcutaneously in the neck at 0.2mL / feather. After 3 days, 10 geese were randomly selected and the injection site was checked. The absorption was good and there was no abnormal bulge. Until the 55th day of slaughter, there were no symptoms of goose reovirus, goose plague, and serositis in the three farms, and the slaughter rate was 92%.

[0095] 3 and Zhaofenghua Biological Duck Infectious Serositis Bivalent Inactivated Vaccine to Prepare Combined Vaccine

[0096] The experimental animals were 2200 healthy 3-day-old susceptible goslings randomly selected from a farm in Rongchang, Chongqing, and divided into antibody group, vaccine group, and combined application group 1 and combined application group 2. Among them, the antibody group was subcutaneously injected with freeze-dried egg yolk antibody of goose reovirus (diluted by feather with sterile saline); the vaccine group was subcutaneously injected with the divalent inactivated vaccine of duck infectious serositis (type 1 RAf63 strain + type 2 RAf34 strain) (approval number: 100992202, production batch number: 24222049) of Zhaofenghua Biotechnology (Nanjing) Co., Ltd.; the combined application group 1 was subcutaneously injected with a mixture of freeze-dried egg yolk antibody of goose reovirus, divalent inactivated vaccine of duck infectious serositis of Zhaofenghua Biotechnology (Nanjing) Co., Ltd. and saline; the combined application group 2 was subcutaneously injected with a mixture of freeze-dried egg yolk antibody of goose reovirus, divalent inactivated vaccine of duck infectious serositis of Zhaofenghua Biotechnology (Nanjing) Co., Ltd. and diluent. On the 7th, 10th, 14th and 21st days after immunization, 10 goslings were randomly selected from each group for subcutaneous injection of virus. Goslings in the antibody group and the combined application group 1 and the combined application group 2 were challenged with the reoentery of duck embryos and allantoic fluid, and goslings in the vaccine group and the combined application group 1 and the combined application group 2 were challenged with Riemerella anatipestifer. After the challenge, the goose was observed for 10 days, and the dead geese and the geese that survived after the challenge observation period were dissected. The specific experimental grouping scheme is shown in Table 3 below, the challenge protection results are shown in Tables 4 and 5, and the death and morbidity of the immunized goslings that were not challenged within one month are shown in Table 6.

[0097] Table 3 Experimental grouping scheme

[0098]

[0099] Table 4 Goose reovirus challenge protection results

[0100]

[0101]

[0102] Table 5 Riemerella anatipestifer challenge protection results

[0103]

[0104] Table 6 Death and morbidity of immunized goslings during the protection period

[0105]

[0106] From the above results, it can be seen that the use of diluent can make the freeze-dried egg yolk antibody of goose reovirus and the oil-emulsion inactivated vaccine fully and evenly mixed, and at the same time, the immune period of the freeze-dried egg yolk antibody of goose reovirus can be extended by more than 10 days; immunizing goslings with a mixture of freeze-dried egg yolk antibody of goose reovirus, bivalent inactivated duck infectious serositis vaccine and diluent can effectively improve the immune effect of goslings against reovirus and serositis, reduce mortality and morbidity during the protection period, effectively respond to mixed infection epidemics, and reduce the pressure of disease prevention and control.

[0107] The goose reovirus freeze-dried egg yolk antibody provided by the present invention can effectively resist infection and death caused by goose reovirus. Its combined use with the duck infectious serositis bivalent inactivated vaccine can achieve joint prevention and control of complex epidemics, and can significantly reduce the economic losses caused by goose reovirus disease and mixed virus diseases to the waterfowl breeding industry.

[0108] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A goose reovirus, named goose reovirus strain YJ24X, deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, the deposit date is August 29, 2024, and the deposit number is CCTCC NO: V202477.

2. Use of the goose reovirus strain YJ24X according to claim 1 in the preparation of goose reovirus egg yolk antibodies.

3. A method for preparing freeze-dried egg yolk antibody of goose reovirus, characterized in that: The following steps are involved: (1) inactivating the goose reovirus strain YJ24X described in claim 1, preparing an immunogen from the inactivated virus and then immunizing laying hens with the inactivated virus; (2) When the agar titer of the yolk antibody of the laying hens immunized in step (1) is ≥1:26, eggs are collected; (3) Using the eggs collected in step (2) to prepare freeze-dried egg yolk antibodies against goose reovirus.

4. The preparation method according to claim 3, characterized in that: The immunization process in step (1) is: The first immunization: 4 to 6 weeks before laying, 0.5 mL / feather; Second immunization: 14 days after the first immunization, booster immunization 1.0 mL / bird; The third immunization: 14 days after the second immunization, booster immunization 1.5 mL / bird; Nth immunization: After the third immunization, booster immunizations of 1.5 mL / bird are performed every 2 to 3 months. 5 . The freeze-dried egg yolk antibody against goose reovirus obtained according to the method for preparing freeze-dried egg yolk antibody against goose reovirus according to any one of claims 3 to 4 .

6. Use of the freeze-dried egg yolk antibody of goose reovirus according to claim 5 in preparing a combined vaccine with a bivalent inactivated duck infectious serositis vaccine.

7. The use according to claim 6, characterized in that The following steps are involved: The combined vaccine was prepared by diluting the freeze-dried egg yolk antibody of goose reovirus with a diluent and then mixing it with the bivalent inactivated duck infectious serositis vaccine.

8. The use according to claim 7, characterized in that The diluent comprises the following components in parts by weight: 1 to 3 parts of white oil, 0.5 to 2 parts of Span 80, 0.3 to 0.8 parts of Tween-80 and 4 to 6 parts of transfer factor.

9. The use according to claim 8, characterized in that Each milliliter of the transfer factor includes: 0.5-1.5 mg of polypeptide, 25-35 μg of nucleotide and 1-1.5 mg of polysaccharide.

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