CN102847147B

CN102847147B - Combined live vaccine for muscovy duck parvovirus disease and muscovy duck reovirus disease

Info

Publication number: CN102847147B
Application number: CN201210046139.0A
Authority: CN
Inventors: 陈少莺; 程晓霞; 林锋强; 陈仕龙; 胡奇林; 林天龙; 程由铨
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: 2012-02-24
Filing date: 2012-02-24
Publication date: 2014-10-01
Anticipated expiration: 2032-02-24
Also published as: CN102847147A

Abstract

The invention discloses preparation and application of a combined vaccine for a muscovy duck parvovirus disease and a muscovy duck reovirus disease; the combined vaccine adopts a muscovy duck parvovirus attenuated P1 strain (preservation number: CCTCC V201013) and a muscovy duck liver white-spot disease (muscovy duck reovirus disease) attenuated MWCA strain (preservation number: CGMCC 0667) as seed viruses; the seed viruses are proliferated by using muscovy duck embryo fibroblasts and SPF chicken embryo fibroblasts through culture in spinner flasks; the cell viral liquid is gained, mixed according to a proper proportion, and freeze-dried by adding a freeze-drying protective agent to obtain the combined live vaccine; the combined live vaccine is applicable to muscovy duck breeding areas with epidemic muscovy duck three-week diseases and liver white-spot diseases.

Description

The sick bigeminal live vaccine of Muscovy duck parvovirus infection and muscovy duck reovirus

Technical field

The present invention relates to the sick bigeminal live vaccine of Muscovy duck parvovirus infection and muscovy duck reovirus, belong to Preventive Veterinary Medicine field.

Background technology

Muscovy duck parvovirus infection (be commonly called as " three weeks sick ") be within 1987, occur first at our province 1-3 age in week young Muscovy duck to breathe heavily the new epidemic disease of one of rushing down as clinical symptoms.This disease is mainly encroached on the young Muscovy duck of 7-21 age in days, sickness rate 27-62% and case fatality rate 22-43%, and the duck major part of recovering becomes stiff duck, causes very large economic loss to Muscovy duck industry.Muscovy duck reovirus disease (being commonly called as " liver ichthyophthiriasis ", " flower hepatopathy ", " ichthyophthiriasis ", " liver spleen ichthyophthiriasis " and " Muscovy duck hepatitis sequestrans " etc.) has been since 1997, in Putian, Fujian Province, the ground Muscovy duck such as Fuqing, Foochow, permanently happy and Jinhua, Zhejiang, Foshan raises district and one occurs clinically taking weak foot as cardinal symptom, has volume white point, kidney enlargement, hemorrhage, surface to have the infectious disease of yellow streak as main pathological change taking liver, spleen surface.This disease only betides Muscovy duck, and morbidity age in days is 7～45 ages in days, with the young Muscovy duck of 10～30 ages in days for the most very.Sickness rate is 30～90%, and case fatality rate is 60～80%, after the dead duck of morbidity is resistance to, becomes stiff duck, causes very large economic loss to Muscovy duck industry.

At present, Muscovy duck parvovirus infection and muscovy duck reovirus disease are still the Important Infectious Diseases of serious harm Muscovy duck aquaculture, and the immunity inoculation of vaccine can effectively be controlled this two kinds of new epidemic diseases, and repeatedly immunity easily causes bad stress to Muscovy duck group.For this reason, the present invention develops muscovy duck reovirus disease and Muscovy duck parvovirus infection bigeminal live vaccine at home and abroad first.

Summary of the invention

The object of this invention is to provide the sick bigeminal live vaccine of Muscovy duck parvovirus infection and muscovy duck reovirus, the present invention is achieved by the following technical solution:

A, derive from the weak malicious P1 strain (MPV-P1 strain) of the exquisite weak Muscovy duck parvovirus of people, be deposited in Chinese Typical Representative culture collection center (China Center for Type Culture Collection, be called for short: CCTCC), the CCTCC of depositary institution address is in Wuhan University of Wuhan, China city, June 2 2010 preservation day, deposit number: CCTCC NO:V201013;

B, derive from the weak malicious MWCA strain of the exquisite weak foreign duck liver ichthyophthiriasis (muscovy duck reovirus disease) of people, this strain is deposited at China Committee for Culture Collection of Microorganisms's common micro-organisms center and (is called for short: CGMCC), depositary institution address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, calendar year 2001 preservation day December 18 days, deposit number: CGMCC0667;

Malicious P1 strain a little less than malicious MWCA strain and Muscovy duck parvovirus a little less than above-mentioned foreign duck liver ichthyophthiriasis is applied to and prepares Muscovy duck parvovirus infection and the sick bigeminal live vaccine of muscovy duck reovirus.

Compared with prior art, the present invention with Muscovy duck parvovirus a little less than the weak malicious MWCA strain of malicious P1 strain (MPV-P1 strain) and foreign duck liver ichthyophthiriasis (that is: the weak malicious CA strain of muscovy duck reovirus or claim MDRV-CA strain, MDRV is the abbreviation of the English Muscovy Duck of muscovy duck reovirus Reovirus) be kind of a poison, apply respectively Muscovy duck embryo fibroblast and SPF chick embryo fibroblast spinner culture technology virus of proliferation, harvesting venom, after mixing by proper proportion, add freeze drying protectant lyophilization, be developed into genetic stability, safety is good, the bigeminal live vaccine that immunogenicity is strong and cost is low, in the Muscovy duck cultivation area application of three weeks sick regulating liver-QI ichthyophthiriasises of popular Muscovy duck, can reach the object that one shot immunity could prevent simultaneously and control these two kinds of epidemic diseases, the young Muscovy duck immunity of 1-2 age in days once can effectively prevent Muscovy duck parvovirus infection and muscovy duck reovirus disease, manpower and materials are not only reduced, reduce production of vaccine cost, also reduce the distress reaction because repeatedly inoculation causes duck group, the present invention is suitable for the large-scale production under GMP condition.

Detailed description of the invention

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

A. derive from the weak malicious P1 strain (MPV-P1 strain) of the exquisite weak Muscovy duck parvovirus of people, to utilize Muscovy duck embryo and Muscovy duck embryo fibroblast alternately to go down to posterity etc. to form a little less than biotechnology artificially breeding causes, lose pathogenic to the young Muscovy duck of an age in days, and there is following characteristic (1) virus titer: MPV-P1 strain is to Muscovy duck embryo fibroblast cell monolayer TCID ₅₀>=10 ^-5.0/ 0.1ml; (2) safety: with young Muscovy duck 10 plumages of the healthy susceptible of 1 age in days, 0.2 milliliter of every plumage leg muscle injection seed culture of viruses stock solution, observes 20 days, should all survive; (3) immunogenicity: the healthy responsive young Muscovy duck of an age in days, 0.2 milliliter of leg muscle injection, its minimum immunity amount is 100TCID ₅₀.MPV-P1 strain is deposited in CCTCC, June 2 2010 preservation day, deposit number CCTCC V201013;

B. derive from the weak malicious MWCA strain of the exquisite weak foreign duck liver ichthyophthiriasis (muscovy duck reovirus disease) of people, to utilize Muscovy duck embryo fibroblast and chick embryo fibroblast to go down to posterity to form a little less than mutagenic and breeding causes, lose pathogenic to the young Muscovy duck of an age in days, and there is following characteristic (1) virus titer: MWCA strain is to chick embryo fibroblast cell monolayer TCID ₅₀>=10 ^-5.0/ 0.1ml; (2) safety: with young Muscovy duck 10 plumages of the healthy susceptible of 1 age in days, 0.2 milliliter of every plumage leg muscle injection seed culture of viruses stock solution, observes 20 days, should all survive; (3) immunogenicity: the young Muscovy duck of the healthy susceptible of an age in days, lower limb intramuscular injection is penetrated 0.2 milliliter, and its minimum immunity amount is 300TCID ₅₀.This strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, (existing name: Chinese common micro-organisms culture presevation administrative center, China General Microbiological Culture Collection Center, be called for short CGMCC), address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, calendar year 2001 preservation day December 18 days, deposit number: CGMCC0667.(see and authorize China Patent No. 01144485.1, Granted publication CN1179751C; The weak malicious MWCA strain of foreign duck liver ichthyophthiriasis (muscovy duck reovirus disease) also claims in this patent documentation: CGMCC0667 strain);

By weak to malicious P1 strain and foreign duck liver ichthyophthiriasis (muscovy duck reovirus disease) a little less than above-mentioned Muscovy duck parvovirus malicious MWCA strain for the preparation of Muscovy duck parvovirus infection and the sick bigeminal live vaccine of muscovy duck reovirus.

The concrete preparation method of the sick bigeminal live vaccine of above-mentioned Muscovy duck parvovirus infection and muscovy duck reovirus is as follows:

(1) MPV-P1 strain propagation, by MPV-P1 strain kind poison, by the 1% synchronous inoculation Muscovy duck embryo fibroblast (MDEF) of cultivating liquid measure, 37 DEG C of rotating and culturing to cytopathys reach more than 75%, results MPV-P1 cell toxicant.

(2) MDRV-CA strain propagation, by MDRV-CA strain kind poison, by the 1% synchronous inoculation SPF chick embryo fibroblast (CEF) of cultivating liquid measure, 37 DEG C of rotating and culturing to cytopathys reach more than 75%, results MDRV-CA cell toxicant.

(3) viral level TCID ₅₀measure the TCID50 difference>=10 of MDRV-CA and MPV-P1 ^-5.0/ 0.1ml and>=10 ^-4.5when/0.1ml is above, can be used for preparing vaccine.

(5) vaccine preparation, adds freeze drying protectant lyophilization after MPV-P1 and MDRV-CA are mixed by proper proportion and forms.Make the viral level of various compositions in every plumage part vaccine reach respectively MDRV-CA 10 ^3.5tCID ₅₀with MPV-P1 10 ^3.0tCID ₅₀.

Vaccine of the present invention is applied to control Muscovy duck parvovirus infection and muscovy duck reovirus disease, and its amynologic index is determined as follows:

1. steriling test: carry out asepsis growth by existing " People's Republic of China's veterinary drug allusion quotation " annex.

2. Security test: with young Muscovy duck 10 plumages of the healthy susceptible of 1 age in days, every plumage leg muscle is injected 10 multiple dose triple vaccinies, observes 20 days, and inoculation duck is showed no diet wants and psychological problem, and growth promoter is good, all survivals.

3. immunity is measured: get at random young Muscovy duck 20 plumages of an age in days susceptible, lower limb flesh is inoculated 1 plumage part bigeminal live vaccine, wherein 10 plumage immune ducks, in inoculating 0 day, 7 days, 14 days, 21 days, 28 days, 60 days, 90 days, 150 days, 180 days together with not immune contrast duck blood sampling separation of serum, are measured for anti-MPV-LPAI antibody titer; Another 10 plumage immune ducks are attacked MDRV poison by force together with not immune matched group in latter 7 days in inoculation simultaneously, observe 20 days, record Mortality situation.Result: the immunity of the young Muscovy duck bigeminy of one age in days Seedling produces antibody for latter 5 days, and the anti-MPV-LPAI antibody in latter 7 days serum of immunity is 2 ^2.1above, be highly resistant to MPV strong virus attack, extremely the rear antibody horizontal peaking for 28 days of immunity is respectively 2 ^6.0, antibody horizontal still maintains higher level subsequently, and continues to latter 180 days of immunity always, has now exceeded Muscovy duck and has delivered age in days for sale. the immunity of the young Muscovy duck bigeminy of one age in days Seedling is attacked the strong malicious 0.2ml/ plumage of MDRV together with not immune matched group in latter 7 days, the sickness rate 80%(8/10 of contrast counteracting toxic substances duck simultaneously), mortality rate 30%(3/10); And the sickness rate 10%(1/10 of Immunization duck), mortality rate 0%; Show within 7 days, to be highly resistant to the strong malicious attack of MDRV after the young Muscovy duck immunity of age in days bigeminy vaccine. the immunity measurement result of bigeminy vaccine shows, vaccine immunity power is good, and 7 days immune generation phases, immune duration is more than 180 days.Primary immune response can protect young Muscovy duck to ride out vulnerable period; reach 1 inoculation and can prevent Muscovy duck parvovirus infection (three weeks diseases of Muscovy duck) and muscovy duck reovirus disease (foreign duck liver ichthyophthiriasis) simultaneously; not only reduce manpower, material resources and financial resources, also reduce the stress because repeatedly inoculation causes.

Effect evaluation of the present invention: be total to the young Muscovy duck of inoculation plumage more than 6500 part one age in days in three weeks test duck fields sick and foreign duck liver ichthyophthiriasis of Zeng Liuhang Muscovy duck, tracing observation 20d, has no immune duck and occur untoward reaction; And there is not Muscovy duck parvovirus infection and muscovy duck reovirus disease in immune group Muscovy duck, survival rate reaches more than 97.5%; And all to there are foreign duck liver ichthyophthiriasis and three weeks in the matched group Muscovy duck of 650 plumages sick in various degree, survival rate only 73%.Further prove bigeminal live vaccine safety of the present invention, effective, can be used for preventing clinically muscovy duck reovirus disease (foreign duck liver ichthyophthiriasis) and Muscovy duck parvovirus infection (three weeks diseases of Muscovy duck).

Derive from the weak malicious P1 strain (MPV-P1 strain) of the exquisite weak Muscovy duck parvovirus of people, deposit number: CCTCC NO:V201013; (China Center for Type Culture Collection, is called for short: CCTCC), the CCTCC of depositary institution address is in Wuhan University of Wuhan, China city June 2 2010 preservation day to be deposited in Chinese Typical Representative culture collection center; (see and authorize China Patent No. 201010215818.7, Granted publication CN101880651B).

Claims

1. the sick bigeminal live vaccine of Muscovy duck parvovirus infection and muscovy duck reovirus, is characterized in that, the sick bigeminal live vaccine of described Muscovy duck parvovirus infection and muscovy duck reovirus is to adopt:

A. derive from the weak malicious P1 strain (MPV-P1 strain) of the exquisite weak Muscovy duck parvovirus of people, be deposited in CCTCC, June 2 2010 preservation day, deposit number CCTCC V201013;

B. derive from the weak malicious MWCA strain (MDRV-CA strain) of the exquisite weak foreign duck liver ichthyophthiriasis (muscovy duck reovirus disease) of people, this strain preservation CGMCC, calendar year 2001 preservation day December 18 days, deposit number: CGMCC0667;

By weak to malicious P1 strain and foreign duck liver ichthyophthiriasis (muscovy duck reovirus disease) a little less than above-mentioned Muscovy duck parvovirus malicious MWCA strain for the preparation of Muscovy duck parvovirus infection and the sick bigeminal live vaccine of muscovy duck reovirus; Concrete preparation method is as follows:

(1) .MPV-P1 strain propagation, by MPV-P1 strain kind poison, by the 1% synchronous inoculation Muscovy duck embryo fibroblast of cultivating liquid measure, 37 DEG C of rotating and culturing to cytopathys reach more than 75%, results MPV-P1 cell toxicant;

(2) .MDRV-CA strain propagation, by MDRV-CA strain kind poison, by the 1% synchronous inoculation SPF chick embryo fibroblast (CEF) of cultivating liquid measure, 37 DEG C of rotating and culturing to cytopathys reach more than 75%, results MDRV-CA cell toxicant;

(3). viral level TCID ₅₀measure the TCID50 difference>=10 of MDRV-CA and MPV-P1 ^-5.0/ 0.1ml and>=10 ^-4.5when/0.1ml is above, can be used for preparing vaccine;

(4). vaccine preparation, after MPV-P1 and MDRV-CA are mixed by proper proportion, add freeze drying protectant lyophilization and form, make the viral level of various compositions in every plumage part vaccine reach respectively MDRV CA 10 ^3.5tCID ₅₀with MPV-P1 10 ^3.0tCID ₅₀.