Egg yolk antibody of goose-derived muscovy duck reovirus as well as preparation method and application of egg yolk antibody

Abstract

The invention discloses an egg yolk antibody of goose-derived muscovy duck reovirus as well as a preparation method and application of the egg yolk antibody, and belongs to the technical field of veterinary biological products. The egg yolk antibody is prepared by the following steps: immunizing a poultry body with a vaccine prepared by taking inactivated goose-derived muscovy duck reovirus with the preservation number of CCTCC NO: V202491 as an antigen, and extracting and purifying from highly-immunized egg yolk. The goose-derived muscovy duck reovirus with the preservation number of CCTCC NO: V202491 is used as a vaccine production strain to prepare an inactivated vaccine for immunizing poultry bodies, the egg yolk antibody can be efficiently obtained, and the egg yolk antibody is good in safety, high in neutralizing titer, good in prevention and treatment effect on goose-derived muscovy duck reovirus infection and wide in commercialization development prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of veterinary biological products, and in particular to an egg yolk antibody of a goose-derived Muscovy duck reovirus and a preparation method and application thereof. Background Art

[0002] Avian orthoreovirus (ARV) is a type of non-enveloped, segmented, icosahedral, double-stranded RNA virus with a double-layer capsid structure and a diameter of 50-80 nm. The International Committee on Taxonomy of Viruses clearly stated in its ninth classification report that the Reoviridae family has two subfamily, Spikereovirinae and Non-spikereovirinae. ARV belongs to the Reoviridae family, Spikereovirinae subfamily, and Orthoreovirus genus.

[0003] Muscovy duck reovirus is a dsRNA virus with no envelope structure, regular icosahedral symmetry, and a double-layer capsid structure. Under a high-power microscope, the virus particle diameter is between 50 and 80 nm and is hexagonal. Since 2021, Muscovy duck reovirus disease has broken out in major goose-raising areas in China. The disease mainly causes large groups of geese to develop in different sizes, be listless, lie on the ground, have movement disorders, have messy and dull feathers, and significantly reduce their water and feed intake, and excrete yellow-green loose feces. The mortality rate can be as high as 50%, causing huge economic losses to the goose industry.

[0004] Yolk antibody is a technology that obtains specific polyclonal antibodies from the yolk of highly immune poultry eggs by injecting specific antigens into the poultry body, thereby preventing and treating corresponding diseases. Compared with serum antibodies, the preparation of yolk antibodies has the advantages of uniform properties, easy extraction, effective avoidance of non-specific immune reactions, low production cost, easy operation, high yield, safety, high efficiency, no residue, mild and environmentally friendly. It has great development potential in immune technology, immune monitoring, human and animal treatment, etc.

[0005] There are currently no commercial vaccines or antibodies for goose-derived Muscovy duck reovirus infection, and conventional antiviral and antibacterial treatments are ineffective. There are no reports of egg yolk antibodies for emergency prevention and early infection treatment of goose-derived Muscovy duck reovirus. Therefore, it is extremely urgent to develop safe and efficient egg yolk antibodies that can effectively prevent goose-derived Muscovy duck reovirus. Summary of the invention

[0006] In view of the above-mentioned prior art, the purpose of the present invention is to provide a yolk antibody of goose-derived Muscovy duck reovirus and its preparation method and application, which are used to prevent and treat outbreaks of goose-derived Muscovy duck reovirus infection with a large number of white necrotic spots and joint cavity bleeding in tissues and organs such as the liver and spleen as the main symptoms, thereby compensating for the serious economic losses caused by the disease.

[0007] To achieve the above object, the present invention adopts the following technical solution: The present invention is based on a goose-derived Muscovy duck reovirus isolated from the liver tissue of diseased goslings. An inactivated vaccine that can prevent and treat the goose-derived Muscovy duck reovirus has been developed. The inactivated vaccine is used to highly immunize healthy poultry eggs, and then the yolk antibodies are extracted and purified from the immunized poultry eggs. The virus has been deposited in the China Center for Type Culture Collection (CCTCC) on October 30, 2024, with the preservation name: Muscovy Duck Reovirus MDRV-JS001, and the classification name: Muscovy Duck Reovirus. The preservation address is: Wuhan University, Wuhan, China, and the preservation number is: CCTCC NO: V202491. Specific: In a first aspect, the present invention provides an egg yolk antibody for preventing and treating goose-derived Muscovy duck reovirus that causes goose "flower liver disease" and joint cavity bleeding. The egg yolk antibody is obtained by immunizing birds with inactivated goose-derived Muscovy duck reovirus with a deposit number of CCTCC NO: V202491 as an immunogen, and then extracting and purifying it from the egg yolk of highly immune poultry eggs.

[0008] Preferably, the poultry is a laying hen, and the high-immunity poultry egg yolk is a high-immunity chicken egg yolk.

[0009] In a second aspect, the present invention provides a method for preparing the above-mentioned egg yolk antibody, the preparation method comprising the following steps: (1) Using the inactivated goose-derived Muscovy duck reovirus with a deposit number of CCTCC NO: V202491 as an immunogen to prepare an inactivated goose-derived Muscovy duck reovirus vaccine; (2) injecting the inactivated vaccine prepared in step (1) into poultry to obtain highly immune poultry eggs; (3) The yolk of highly immune poultry eggs is collected and subjected to primary inactivation, acid extraction, secondary inactivation, coarse filtration, sterile filtration, concentration and tertiary inactivation to prepare yolk antibodies for the prevention and treatment of goose, Muscovy and duck reovirus.

[0010] The neutralizing antibody titer of the prepared egg yolk antibody for preventing and treating goose, Muscovy and duck reovirus is ≥1:1024.

[0011] Preferably, in step (1), the inactivated vaccine is prepared by the following method: 1) The goose-derived Muscovy duck reovirus with the deposit number of CCTCC NO: V202491 was inoculated into LMH cells, and the goose-derived Muscovy duck reovirus was proliferated and cultured, and then the cells were broken by freezing and thawing, and the supernatant was collected and purified to obtain the goose-derived Muscovy duck reovirus liquid; 2) The virus liquid of the goose-derived Muscovy duck reovirus strain was inactivated, Tween-80 was added to the mixture as the water phase, white oil, aluminum stearate and Span-80 were mixed as the oil phase, the oil phase and the water phase were evenly mixed in a ratio of 2:1, and emulsified to obtain an inactivated vaccine for the prevention and treatment of goose, Muscovy duck reovirus.

[0012] The virus titer in the goose-derived Muscovy duck reovirus strain virus liquid is 10 -6.2 TCID 50 / 0.1 mL.

[0013] Preferably, in step (2), the prepared inactivated vaccine is used to immunize laying hens 4 times, with an interval of 2 weeks between each immunization. The immunization route is subcutaneous or intramuscular injection of the inactivated vaccine into the neck.

[0014] Preferably, in step (3), the primary inactivation is specifically as follows: mixing the egg yolk liquid and water in a volume ratio of 1:(0.5-1.5), stirring evenly to obtain an egg yolk dilution, and keeping warm at 60-65°C for 25-35 minutes.

[0015] Preferably, in step (3), the acid extraction is specifically: adding 2.5 to 3.5 times the volume of the egg yolk dilution to the acetate buffer, stirring and filtering to collect the filtrate. Further preferably, the pH value of the acetate buffer is 4.8-5.2; more preferably 5.0.

[0016] Preferably, in step (3), the secondary inactivation is specifically: adding caprylic acid to the filtrate after acidification extraction to a final concentration of 3.5-4.5%, stirring for 30-120 minutes, and then placing at 2-8°C for 4-8 hours.

[0017] Preferably, in step (3), the coarse filtration is specifically: first filtering with a filter cloth, and then filtering with a filter membrane until the filtrate is clarified. Further preferably, the filter cloth is a polypropylene 750B filter cloth.

[0018] Preferably, in step (3), the sterilization filtration is specifically: sterilization by filtration using a 0.22 μm filter membrane.

[0019] Preferably, in step (3), the concentration is specifically: using a 30-50KD PES hollow fiber ultrafiltration membrane to concentrate at 2-8°C until the antibody titer is not less than 1:1024.

[0020] Preferably, in step (3), the three inactivations are specifically: adding formaldehyde solution to a final concentration of 0.1%, and inactivating at 37°C for 16 hours.

[0021] The third aspect of the present invention provides the use of the above-mentioned egg yolk antibody in the preparation of a product for preventing and / or treating goose-derived Muscovy duck reovirus infection; the deposit number of the goose-derived Muscovy duck reovirus is CCTCC NO: V202491.

[0022] In the application, the egg yolk antibody can be used alone or in combination with other immune preparations and / or drugs to prepare a product for preventing and / or treating goose-derived Muscovy duck reovirus infection.

[0023] Beneficial effects of the present invention: (1) The Muscovy duck reovirus with the deposit number of CCTCC NO: V202491 provided by the present invention has the ability of cross-species transmission and can infect goslings. Moreover, the goose-derived Muscovy duck reovirus JS001 has a wide range of cell tropism and can proliferate on LMH cells, which is very suitable for the production of high-quality inactivated vaccines with high antigen titer; it has good immunogenicity for newly-emerged goose-derived Muscovy duck reovirus infection with goslings' "speckled liver disease" and joint cavity hemorrhage as the main symptoms.

[0024] (2) The inactivated vaccine prepared with the goose-derived Muscovy duck reovirus strain with the preservation number CCTCC NO: V202491 has good safety, a protection rate of 100%, and a long duration of immune protection.

[0025] (3) Using the goose-derived Muscovy duck reovirus with a preservation number of CCTCC NO: V202491 as the vaccine production strain to prepare an inactivated vaccine for immunizing laying hens can efficiently obtain egg yolk antibodies. The egg yolk antibodies are safe and have high neutralization titers. They have good preventive and therapeutic effects on goose-derived Muscovy duck reovirus infection and have broad prospects for commercial development. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a lesion image of a virus cultured in LMH cells; Figure 1 A in the figure is a picture of goose-derived Muscovy duck reovirus JS001 inoculated into LMH cells 48 hours later. Figure 1 B in the figure is a picture of control cells that were not inoculated with the virus.

[0027] Figure 2 PCR identification results; in the figure, lane 1 is duck reovirus, lane 2 is duck Tembusu virus, lane 3 is duck astrovirus, lane 4 is duck circovirus, lane 5 is duck plague virus, lane 6 is duck hepatitis A virus, lane 7 is blank control, lane 8 is goose-derived Muscovy duck reovirus, and M is a 2000bp Marker. DETAILED DESCRIPTION

[0028] 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.

[0029] The following detailed description is for illustrative purposes only and is intended to provide further explanation of the present application, rather than to limit the scope of the present invention.

[0030] 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. If the specific experimental conditions are not specified in the embodiments, they are usually in accordance with conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following embodiments, unless otherwise specified, can be obtained through commercial channels. Among them: RNA extraction kit was purchased from Beijing Kangwei Century Co., Ltd., ordinary agarose gel recovery kit was purchased from Beijing Quanshijin Company, reverse transcription kit was purchased from Bao Biotechnology (Dalian) Co., Ltd., 2×Es Taq MasterMix was purchased from Beijing Kangwei Century Co., Ltd., and DL2000 Marker was purchased from Bao Biotechnology (Dalian) Co., Ltd.

[0031] Example 1: Isolation and identification of goose-derived Muscovy duck reovirus 1. Epidemiological survey: Since March 2021, an infectious disease characterized by paralysis and a large number of white necrotic spots in tissues and organs such as the liver and spleen has broken out in goslings in Jiangsu, Liaoning and Henan provinces of my country. The disease mainly occurs in goslings aged 7 to 35 days. The diseased geese have a large number of white necrotic spots in all tissues and organs of the body. The diseased goslings also have clinical symptoms of swollen joints, and the mortality rate can be as high as 80%. The autopsy of the dead goslings showed the following main pathological changes: enlarged liver and spleen, with a large number of white necrotic spots on the surface, and severe bleeding in the joint cavity.

[0032] 2. Sample collection and processing: The liver tissue of the diseased goslings was collected, sterile PBS was added, and the tissue was ground into a homogenate. The tissue was frozen and thawed three times, and the supernatant was centrifuged and sterilized by filtration through a 0.22 μm filter. The supernatant was inoculated onto LMH cells in good growth condition and adsorbed in a cell culture incubator for 30 min. The sample treatment solution was discarded, and 2% cell maintenance solution was added to continue culturing. A negative control was set up with sterile saline and incubated at 37°C. The cell status was observed and recorded daily, and the virus solution was collected when the cell fusion phenomenon reached 80%. The virus solution was blindly propagated for three generations in the same way for subsequent testing. After three generations of subculture, the degree of cell fusion tended to be stable, and 80% of the cells showed typical cytopathic effects 48 h after inoculation ( Figure 1 A in the figure), and the negative control showed no lesions ( Figure 1 B in the figure).

[0033] 3.RT-PCR identification: (1) RNA extraction: The collected cell virus fluid was repeatedly frozen and thawed, and the supernatant was obtained by centrifugation. Viral RNA was extracted according to the instructions of the RNA extraction kit and stored at -20°C for later use.

[0034] (2) Reverse transcription to obtain cDNA: The reverse transcription kit used was PrimeScript™ RT Master Mix with catalog number RR036A from Takara Biotech (Dalian) Co., Ltd. 5× PrimeScript RT Master Mix×2μL was added to a 200 μL PCR reaction tube, and 2 μL of RNA extracted from the virus solution was added, and RNase FreeddH2O was used to supplement the system to 10 μL. The reaction was carried out in a PCR instrument under the following conditions: 37℃ for 15min; 85℃ for 5s, and then stored at 4℃.

[0035] (3) PCR amplification: Muscovy duck reovirus belongs to the genus Orthoreovirus of the family Reoviridae. σC protein is an extremely important structural protein of Orthoreovirus and plays an important role in the pathogenicity of the virus. In addition, the gene encoding σC protein is the gene fragment most susceptible to mutation in the genome. Therefore, in this study, a pair of specific primers for amplifying a 1002 bp fragment were designed based on the σC gene sequence of Muscovy duck reovirus on Genbank, a gene sequence database established by the National Center for Biotechnology Information of the United States, and synthesized by Beijing Qingke Xinye Biotechnology Co., Ltd.

[0036] Upstream primer: 5′- CCGATTGGGCCGACATCTCAT-3′ (SEQ ID NO.1) Downstream primer: 5′-ACTACCTCAAGTGGTCGCAA-3′ (SEQ ID NO.2) A 20 μL system was used for amplification: template cDNA × 2 μL, upstream and downstream primers × 1 μL each, 2 × Es TaqMasterMix × 10 μL, and ddH2O was used to supplement the 20 μL system. After mixing and centrifuging, the mixture was placed in a PCR instrument for reaction. The reaction conditions were 95°C, 5 min, followed by 30 cycles of 95°C, 45 s, 52°C, 30 s, 72°C, 25 s, and 72°C, 10 min, and then stored at 4°C for later use.

[0037] At the same time, the reported specific primers were used to detect conventional duck-origin viruses, including duck reovirus (DRV), duck Tembusu virus (TMUV), duck astrovirus (DAStV), duck circovirus (DuCV), duck plague virus (DPV) and duck hepatitis A virus (DHAV).

[0038] (4) Agarose gel electrophoresis: The PCR amplification products were detected by 1% agarose gel electrophoresis. The results showed that the products amplified by PCR using primers SEQ ID NO.1 and SEQ ID NO.2 showed a specific band with the expected size of 1002 bp after electrophoresis in 1% agarose gel, indicating that the cell culture of the isolated strain contained goose-derived Muscovy duck reovirus ( Figure 2 ), named JS001 strain. In addition, the isolates were tested for routine duck-derived viruses, including DRV, TMUV, DAStV, DuCV, DPV, and DHAV, and the results were all negative, and no other viral contamination was detected.

[0039] 4. Sequencing and sequence analysis: The amplified product was recovered from the gel and sent to Beijing Qingke Xinye Biotechnology Co., Ltd. for sequencing. The sequencing results were spliced ​​and aligned using Blast, and homology analysis was performed with other waterfowl reoviruses.

[0040] The results showed that the σC gene sequence (SEQ ID NO.3) of the JS001 isolate was most closely related to the classic Muscovy duck reovirus ZJ2000M strain, with nucleotide and amino acid homology of more than 95%. It was distantly related to other waterfowl reovirus isolates and was in a different genetic evolutionary branch. Therefore, it can be determined that the JS001 isolate is a goose-derived Muscovy duck reovirus.

[0041] SEQ ID NO.3: .

[0042] The strain was named goose-derived Muscovy duck reovirus MDRV-JS001. The strain was biologically preserved, and the preservation information is as follows: Bacteria name: Muscovy duck reovirus MDRV-JS001 Collection institution: China Center for Type Culture Collection Abbreviation of depository institution: CCTCC Address: Wuhan University, Wuhan, China Deposit date: October 30, 2024 Classification name: Muscovy Duck Reovirus The registration number of the Collection Center is: CCTCC NO: V202491.

[0043] Example 2: Preparation of inactivated vaccines (1) Virus proliferation and harvesting: The isolated and identified goose-derived Muscovy duck reovirus JS001 was inoculated into well-grown LMH cells at an inoculation volume ratio of 1:100; adsorbed at 37°C for 30 min, and then cultured with DMEM containing 2% newborn calf serum at 37°C and 5% CO2. When 80% of the cells showed pathological changes, the cells were harvested to obtain the cell virus fluid. The obtained sufficient cell virus fluid was frozen at -20°C, and after two freeze-thaw cycles, the supernatant was collected by centrifugation to obtain the virus fluid. Calculate TCID 50 The virus content in each 0.1ml virus liquid is 10 -6.2 TCID 50 .

[0044] (2) Virus purification: Use established PCR, RT-PCR and other detection methods to detect whether the obtained virus liquid contains other common viruses. The detection items include DRV, TMUV, DAStV, DuCV, DPV, DHAV, etc., and purify the seed virus.

[0045] (3) Inactivation of virus fluid: The virus solution that passed the bacterial test was inactivated with formaldehyde. The best inactivation condition was to add formaldehyde to a final concentration of 0.2% and stir at 37°C for 16 hours.

[0046] (4) Vaccine preparation: ① Preparation of oil phase: Take No. 10 medicinal white oil and Span-80 in a ratio of 94:6 and mix them evenly. Then add 2% aluminum tristearate and stir until it becomes light yellow and clear. Sterilize with high-pressure steam at 121℃ for later use.

[0047] ② Preparation of aqueous phase: Mix the completely inactivated virus solution and sterile Tween-80 at a ratio of 96:4 by oscillation to completely dissolve Tween-80.

[0048] ③ Emulsification: The oil phase and the water phase are mixed in a ratio of 2:1. Add 2 parts of the oil phase into a tissue homogenizer in an ultra-clean bench, and slowly add 1 part of the water phase while stirring continuously. After all the water phase is added, mix at 6000 rpm for 10 min, then emulsify at 8000 r / min for 20 min, and divide into portions for use. The inactivated vaccine is prepared.

[0049] The prepared inactivated vaccine is subjected to quality inspections including dosage form, centrifugal stability, viscosity, sterility and shelf life. For specific methods, refer to the "Pharmacopoeia of Veterinary People's Republic of China" (2015 edition).

[0050] The results showed that the dosage form of the inactivated vaccine prepared by the present invention was water-in-oil (W / O); the centrifugal stability, viscosity and sterility tests were in compliance with the requirements of the Veterinary Pharmacopoeia of the People's Republic of China (2015 edition).

[0051] Example 3: Preparation of goose-derived Muscovy duck reovirus yolk antibodies 1. Immunize laying hens with inactivated vaccines: The goose-derived Muscovy duck reovirus inactivated vaccine prepared in Example 2 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 goose-derived Muscovy duck reovirus neutralizing antibody titer, which should be ≥1:1024.

[0052] 2. Yolk antibody production: (1) Eggshell disinfection: Collect high-immunity eggs and soak them in 1% chlorhexidine solution for 15 minutes. After taking them out, let them dry naturally or blow dry, and then spray 75% alcohol to disinfect the eggshell surface for later use.

[0053] (2) Yolk separation: Mechanical egg beating is used to fully remove the egg white, germinal disc and frenulum, and collect the yolk.

[0054] (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 volume of the egg yolk (the injection water should be sterilized at 80℃ for 30 min and cooled to below 65℃). Stir and mix thoroughly, and then keep warm at 65℃ (inactivation) for 30 min.

[0055] (4) Acid extraction: First, add acetic acid buffer with a pH value of 5.0 equivalent to 3 times the volume of the original yolk into the isolation reaction tank, then add the egg yolk liquid, turn on the mixer, and stir thoroughly for 30 minutes.

[0056] (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°C for 4 h.

[0057] (6) Coarse filtration: Filter with polypropylene 750B filter cloth, then filter with a column filter until clear.

[0058] (7) Sterilization and filtration: Sterilize with a 0.22 μm filter membrane. Store at 2°C for no more than 14 days. At the same time, take samples to test the neutralizing antibody titer of goose-derived Muscovy duck reovirus.

[0059] (8) Concentration: If the neutralizing antibody titer of the detected goose-derived Muscovy duck reovirus is lower than 1:1024, the sterilized and filtered egg yolk antibody should be ultrafiltered and concentrated at an appropriate multiple using a 30 KD concentration membrane package at 2°C. The neutralizing antibody titer of the goose-derived Muscovy duck reovirus after concentration should be ≥1:1024.

[0060] (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.

[0061] (10) Packaging and storage: Under sterile conditions, pack the inactivated filtrate into sterile glass bottles, cover with rubber stoppers, roll aluminum caps, attach labels, and store for future use at -20°C.

[0062] Example 4: Yolk antibody quality inspection 1. Safety inspection: Ten 5-day-old susceptible goslings (goose-derived Muscovy duck reovirus maternal antibodies were all <1:4) were injected with 2.0 mL of the yolk antibody prepared in Example 3 of the present invention at each muscle point. After 14 days of observation, all the susceptible goslings survived healthily, indicating that the yolk antibody of the present invention has good safety.

[0063] 2. Sterility test: The test was carried out in accordance with the "Pharmacopoeia of Veterinary Medicine of the People's Republic of China" (2015 edition), and the results were: no bacterial, mycoplasma and exogenous virus contamination.

[0064] 3. Efficacy test (immune challenge method): 40 5-day-old susceptible goslings (maternal antibodies to goose-derived Muscovy duck reovirus were all <1:4) were randomly divided into four groups, A and B, with 20 in each group. A was the immunization group, and the yolk antibody prepared in Example 3 was injected subcutaneously or intramuscularly in the neck, 0.5 mL per gosling; B was the challenge control group, and physiological saline was injected subcutaneously or intramuscularly, 0.5 mL per gosling. They were kept in isolation.

[0065] After 16 hours, the goslings in groups A and B were injected intramuscularly with 0.5 mL of goose-derived Muscovy duck reovirus diluted 10 times with normal saline. The goslings were observed for 10 days after the challenge, and the incidence and mortality of the goslings in each group were recorded.

[0066] Results: Group A was 20 / 20 protected after challenge; Group B began to develop the disease 12 hours after challenge, and 13 died within 10 days. This shows that the egg yolk antibody prepared by the present invention has a good immune protection effect against goose-derived Muscovy duck reovirus infection.

[0067] Example 5: Application of yolk antibodies 1. Application in the prevention of goose-derived Muscovy duck reovirus Sixty 5-day-old susceptible goslings (maternal antibodies to goose-derived Muscovy duck reovirus were all <1:4) were randomly divided into three groups, with 20 in each group. Experimental group: subcutaneous or intramuscular injection of the egg yolk antibody prepared in Example 3, 0.5 mL per mouse; Control group: using duck reovirus with NCBI number MK749407 as the immunogen, according to the inactivated vaccine preparation method described in Example 2 of the present application, an inactivated duck reovirus vaccine was prepared to immunize laying hens, and egg yolk antibodies were prepared according to the method of Example 3, and the egg yolk antibodies were injected subcutaneously or intramuscularly into the neck, 0.5 mL per laying hens; Blank control group: subcutaneous or intramuscular injection of normal saline, 0.5 mL per mouse.

[0068] The three groups of goslings were isolated and raised. After 16 hours, 0.5 mL of goose-derived Muscovy duck reovirus diluted 10 times with normal saline was injected intramuscularly. The goslings were observed for 10 days after the challenge, and the incidence and mortality of each group of goslings were recorded. The results are shown in Table 1.

[0069] Table 1: Note: Preventive protection is the number of healthy surviving goslings after challenge / total number of goslings.

[0070] It can be seen from Table 1 that the egg yolk antibody of the present invention has an excellent preventive protective efficacy and can provide 100% preventive protection against the goose-derived Muscovy duck reovirus. The protective efficacy is better than that of the egg yolk antibody prepared by the existing inactivated vaccine.

[0071] 2. Application in the treatment of goose-derived Muscovy duck reovirus In the goose-derived Muscovy duck reovirus infection outbreak area, the symptoms are a large number of white necrotic spots and joint cavity bleeding in the liver, spleen and other tissues and organs. Thirty goslings with similar disease courses were used as test subjects and randomly divided into three groups, with 10 in each group, all of which were kept in isolation, including: Experimental group: subcutaneous or intramuscular injection of the egg yolk antibody prepared in Example 3, 1.0 mL per mouse; Control group: Laying hens were immunized with the commercially available inactivated duck reovirus vaccine, and egg yolk antibodies were prepared according to the method of Example 3. The egg yolk antibodies were injected subcutaneously or intramuscularly into the neck, 1.0 mL per egg; Blank control group: subcutaneous or intramuscular injection of normal saline, 1.0 mL per mouse.

[0072] The observation was continued for 10 days, and the illness and mortality of each group of goslings were recorded.

[0073] The results were as follows: 2 days after the injection of yolk antibodies in the experimental group, the feed intake of the sick goslings began to increase, their mental state improved significantly, and no goslings died within 10 days; 10 days later, 1 / 2 of the goslings were killed and dissected, and the results showed that the white necrotic spots in the liver and spleen of the sick goslings were significantly reduced, and the amount of joint bleeding was small. After the injection of yolk antibodies in the control group, the feed intake and mental state of the sick goslings did not improve significantly. Sick goslings began to die from the 4th day of the injection of yolk antibodies, and the mortality rate of sick goslings within 10 days was 30%; 10 days later, the surviving goslings were killed and dissected, and the results showed that the white necrotic spots in the liver and spleen of the sick goslings did not decrease significantly, and there were obvious symptoms of joint bleeding. The mortality rate of sick goslings in the blank control group reached 45% within 10 days.

[0074] 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 goose-derived Muscovy duck reovirus infection, and has significant economic and social benefits.

[0075] The above description is only a 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 made within the spirit and principle of the present application shall be deemed to be appropriate. Equivalent replacements, improvements, etc. should all be included in the protection scope of this application.

Claims

1. An egg yolk antibody for preventing and treating goose-derived Muscovy duck reovirus that causes goose "speckled liver disease" and joint cavity bleeding, characterized in that: The egg yolk antibody is obtained by immunizing birds with inactivated goose-derived Muscovy duck reovirus with a preservation number of CCTCC NO: V202491 as an immunogen, and then extracting and purifying it from the egg yolk of highly immune poultry eggs.

2. The method for preparing the egg yolk antibody according to claim 1, characterized in that: The preparation method comprises the following steps: (1) Using the inactivated goose-derived Muscovy duck reovirus with a deposit number of CCTCC NO: V202491 as an immunogen to prepare an inactivated goose-derived Muscovy duck reovirus vaccine; (2) injecting the inactivated vaccine prepared in step (1) into poultry to obtain highly immune poultry eggs; (3) The yolk of highly immune poultry eggs is collected and subjected to primary inactivation, acid extraction, secondary inactivation, coarse filtration, sterile filtration, concentration and tertiary inactivation to prepare yolk antibodies for the prevention and treatment of goose, Muscovy and duck reovirus.

3. The preparation method according to claim 2, characterized in that: The neutralizing antibody titer of the prepared egg yolk antibody for preventing and treating goose, Muscovy and duck reovirus is ≥1:1024.

4. The preparation method according to claim 2, characterized in that: In step (1), the inactivated vaccine is prepared by the following method: 1) The goose-derived Muscovy duck reovirus with the deposit number of CCTCC NO: V202491 was inoculated into LMH cells, and the goose-derived Muscovy duck reovirus was proliferated and cultured, and then the cells were broken by freezing and thawing, and the supernatant was collected and purified to obtain the goose-derived Muscovy duck reovirus liquid; 2) The virus liquid of the goose-derived Muscovy duck reovirus strain was inactivated, Tween-80 was added to the mixture as the water phase, white oil, aluminum stearate and Span-80 were mixed as the oil phase, the oil phase and the water phase were evenly mixed in a ratio of 2:1, and emulsified to obtain an inactivated vaccine for the prevention and treatment of goose, Muscovy duck reovirus.

5. The preparation method according to claim 3, characterized in that: The virus titer in the goose-derived Muscovy duck reovirus strain virus liquid is 10 -6.2 TCID 50 / 0.1 mL.

6. Use of the egg yolk antibody according to claim 1 in the preparation of a product for preventing and / or treating goose-derived Muscovy duck reovirus infection; the deposit number of the goose-derived Muscovy duck reovirus is CCTCC NO: V202491.

7. The use according to claim 6, characterized in that: In the application, the egg yolk antibody can be used alone or in combination with other immune preparations and / or drugs to prepare a product for preventing and / or treating goose-derived Muscovy duck reovirus infection.

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