CN102492661B

CN102492661B - Preparation method and application of immunization preparation for controlling novel duck reovirus

Info

Publication number: CN102492661B
Application number: CN201110461064.8A
Authority: CN
Inventors: 陈仕龙; 陈少莺; 林锋强; 程晓霞; 朱小丽; 王劭; 李兆龙
Original assignee: Institute of Animal Husbandry and Veterinary of Fujian Academy of Agricultural Sciences
Current assignee: Institute of Animal Husbandry and Veterinary of Fujian Academy of Agricultural Sciences
Priority date: 2011-12-29
Filing date: 2011-12-29
Publication date: 2014-01-08
Anticipated expiration: 2031-12-29
Also published as: CN102492661A

Abstract

The invention discloses a novel duck reovirus strain NDRV-JM85 with a preservation number of CCTCC NO: V201127. The novel duck reovirus strain NDRV-JM85 is used as a virus seed to inoculate an allantoic cavity of a muscovy duck embryo and obtain a virus-containing allantoic fluid, or inoculate an MDEF cell to obtain cytotoxin when a CPE reaches 75%. The virus liquid is inactivated by formaldehyde and added with a white oil adjuvant, and the mixture is mixed thoroughly to from a safe and effective oil emulsion inactivated vaccine for novel duck reovirus. The vaccine is used to immunize a laying hen three times, with an immunization interval of 21 days and 1.0 ml for each hen at each time. Eggs laid 21-63 days after the three immunizations are gained, and extracted yolk and a proper amount of disinfected normal saline is prepared into a hyperimmune antibody preparation for the novel duck reovirus. A duckling without an NDRV maternal antibody is injected with the antibody preparation at 1-2 days old for 0.5ml / duckling, or a duckling with a maternal antibody is injected with the antibody preparation at 10 days old for 1.0 ml / duckling, so as to prevent and control the novel duck reovirus. The invention applies to large-scale production under a GMP condition.

Description

Preparation method and the application of control Novel duck reovirus disease immunological reagent

Technical field

The present invention relates to a kind of preparation method and application that prevents and treats Novel duck reovirus disease immunological reagent.

Background technology

Novel duck reovirus disease (being commonly called as duck hemorrhagic necrosis hepatitis) has been since 2005, in the provinces such as Fujian China, Guangdong and Zhejiang kind duck, half kind of duck and sheldrake mass-sending, give birth to a kind of take the irregular necrosis of liver and hemorrhagely mix, cardiac muscle is hemorrhage, spleen enlargement patch shape downright bad, kidney and the fabricius bursa epidemic disease as principal character such as hemorrhage, this disease is without obvious seasonality, various kind ducks (as a kind duck, half kind of duck, sheldrake, Beijing duck etc.) all can occur, and the trend increased is year by year arranged; The morbidity age in days is about 3～25 ages in days, wherein in the majority with 5～10 ages in days, the course of disease 5～7d, and sickness rate 5%～20%, mortality ratio 2%～15%, when age in days is healed little or accompanying infection, its sickness rate mortality ratio is higher, and provisions duck industry causes certain financial loss.Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute confirms that from viral physics and chemistry biology, animal Orthogonal Rotational Regressive Tests, genomic characterization and molecular level the cause of disease of duck hemorrhagic necrosis hepatitis is a kind of Novel duck reovirus (Novel Duck Reovirus, NDRV) that is different from muscovy duck reovirus (MDRV), Avianreovirus (ARV) first.Up to now, this disease there is no the effectively preventing method.

Summary of the invention

One of purpose of the present invention is to provide duck reovirus NDRV-JM85 strain:

Duck reovirus NDRV-JM85 strain, be that the Novel duck reovirus forms through the seed selection of biotechnology Attenuation, be deposited in Chinese Typical Representative culture collection center (China Center for Type Culture Collection, be called for short CCTCC), July 29 2011 preservation day, deposit number: CCTCC NO:V201127.The CCTCC of depositary institution address is in Wuhan, China city Wuhan University.

Two of purpose of the present invention is to provide the method for preparing control Novel duck reovirus disease immunological reagent with duck reovirus NDRV-JM85 strain.Described preparation method is as follows:

Duck reovirus NDRV-JM85 strain is cultivated in a kind DEF (MDEF) or 11 ages in days kind duck embryo, after the deactivation of results virus liquid, with oily adjuvant, prepares Novel duck reovirus inactivated vaccine.Apply 3 Immune Laying Hens of this vaccine, gather in the crops 3 and exempt from the egg of latter 21 days to 63 days, extract yolk and be prepared into Novel duck reovirus disease immunological reagent with appropriate sterile saline.

Three of purpose of the present invention is to provide the application of the control Novel duck reovirus disease immunological reagent prepared with duck reovirus NDRV-JM85 strain:

Without the duckling of NDRV maternal antibody, at the 1-2 age in days, every bipennate muscle is annotated the immunological reagent of 05ml; Contain the duckling of NDRV maternal antibody at 10 age in days left and right injecting immune preparations, 1.0ml/ plumage; Duckling can be resisted the infection of NDRV after the injecting immune preparation.

The appropriate immunological reagent of the present invention of duckling inoculation, can preventing duck hemorrhagic necrosis hepatitis (Novel duck reovirus disease)

Advantage of the present invention: the present invention makes first the Novel duck reovirus and prepares immunological reagent, and said preparation prepares easy, and cost is low, and security is good, for anti-Novel duck reovirus disease processed provides a kind of method safely and effectively.

Embodiment

Below in conjunction with specific embodiment, the present invention is further described.(but not being limitation of the present invention).

Duck reovirus NDRV-JM85 strain is that the Novel duck reovirus forms through the seed selection of biotechnology Attenuation.Be deposited in CCTCC, July 29 2011 preservation day, deposit number: CCTCC NO:V201127.

By above-mentioned duck reovirus NDRV-JM85 strain, be kind of a poison, inoculation kind duck embryo allantoic cavity, allantoic cavity is inoculated 11 ages in days kind duck embryo virus of proliferation, gathers in the crops toxic allantoic fluid; Or inoculation kind DEF individual layer, when cytopathy reaches 75% poison of harvested cell when above; Above-mentioned virus, with after formalin-inactivated, is mixed in suitable proportion (as 1: 1 ratio) and oily adjuvant, be developed into inactivated vaccine.The inactivated vaccine immunity is opened to laying hen, the 1.0ml/ plumage, immunity is 3 times altogether, every minor tick 21 days.Results 3 are exempted from the egg of latter 21 days to 63 days, and aseptic extraction yolk mixes and filter packing with appropriate sterile saline, is Novel duck reovirus disease immunological reagent.

Concrete grammar is as follows:

(1) antigen preparation: by kind duck embryo of JM85 virus strain inoculation kind DEF (MDEF) or 11 ages in days, the harvested cell pathology reaches the allantoic fluid poison of dead kind duck embryo after 75% cell toxicant or 24h, and-20 ℃ frozen.As the TCID of virus to a kind DEF (MDEF) ₅₀>=10 ^-60during/0.1ml, can be used for viral deactivation.Allantoic fluid or cell toxicant by results, add pure formaldehyde by 0.06% of cumulative volume, 37 ℃ of deactivation 24h.The virus liquid of getting after deactivation is inoculated 9 age in days SPF chicken embryos or 11 ages in days kind duck embryo, and chicken embryo and a kind duck embryo should all be survived.

(2) vaccine preparation: inactivation of viruses liquid is mixed by 1: 1 with oily adjuvant, stir and make the water-in-oil emulsion vaccine.This vaccine steriling test is answered asepsis growth.The healthy susceptible kind ducks of 7 ages in days, after the vaccine of every plumage neck subcutaneous injection 1.0ml, the inoculation duck should all survive, and searches for food, drinks water, grows etc. and be all normal; Cut open at random and kill duck, the internal organ such as the heart, liver, spleen are answered no abnormality seen.

(3) preparation of immunological reagent: the vaccine immunity laying hen of the above preparation of application 3 times, every minor tick 21 days, every plumage per injection 1.0ml.Collecting 3 exempts to exempt from egg containing the height of Novel duck reovirus antibody in latter 21 days to 63 days, after the eggshell washing and sterilizing, aseptic technique, separate yolk, adds a certain proportion of sterile saline (determining according to the antibody titer in yolk) to carry out homogenate, and every milliliter add each 1000 units of penicillin, Streptomycin sulphate, Thiomersalate 1.0/ ten thousand, filter, in the packing sterilizing bottle,-20 ℃ of refrigerations are standby, are prepared into the yolk antibody immunological reagent.

(4) mensuration of immunological reagent index of correlation

4.1 steriling test: undertaken by the existing People's Republic of China (PRC) " veterinary drug allusion quotation ", all without growths such as bacterium, mould, mycoplasmas.

4.2 safety examination: with young kind duck 10 plumages of the healthy susceptible of 1 age in days, 1.0 milliliters of every plumage leg muscle injections, observe 20 days, all strong living.

4.3 attack poison protection check: a 1-2 age in days kind duck is divided into 2 groups, every group of 10 plumages, and wherein preparation 0.5ml/ plumage is penetrated in the intramuscular injection of immunological reagent group leg, and sterilizing hanks liquid 0.5ml/ plumage is penetrated in the intramuscular injection of control group leg; After injection the 3rd day, inoculate respectively NDRV poison by force for two groups, the 0.5ml/ plumage, situations such as observing the searching for food of each group test kind duck, drink water, fall ill, be dead, and after attacking poison the 7th day every group cut open at random and kill 3 plumages, the pathologies such as the observation heart, liver, spleen.Result: in the whole observation period, search for food, drinking-water etc. is all normal after the strong poison of immunological reagent group artificial challenge, and cut open at random and kill 3 plumages, each organ is showed no extremely.After the strong poison of control group artificial challenge, a kind duck shows as material feeding, drinking-water reduces or death, and whole viewing duration body weight is significantly less than the preparation group; Pathologies such as cuing open and examine visible liver, spleen is significantly hemorrhage and downright bad, and heart is hemorrhage.

(5) application of immunological reagent:

Without the duckling of NDRV maternal antibody, at the 1-2 age in days, every bipennate muscle is annotated the preparation of 0.5ml; There is the duckling of NDRV maternal antibody can be at 10 age in days left and right injecting immune preparations, 1.0ml/ plumage.

(6) clinical application effect test-results:

Select the control test that 3 meat duck plants carry out immunological reagent in the popular epidemic-stricken area of NDRV.Duckling is at the 1-2 age in days, and every plumage is inoculated NDRV preparation 0.5ml, tracing observation 45 days, and untoward reaction does not appear in the test duckling, duckling all infects without NDRV in whole feeding period.And periphery does not take the plant of above measure to have NDRV morbidity and dead always.Further proof said preparation safety, effective, can be used for preventing duck hemorrhagic necrosis hepatitis (Novel duck reovirus disease) clinically.

Claims

1. duck reovirus NDRV-JM85 strain, be deposited in CCTCC, preservation date on July 29th, 2011, deposit number: CCTCC NO:V201127.

2. the application of duck reovirus NDRV-JM85 according to claim 1 strain in preparation control Novel duck reovirus disease immunological reagent.