CS248872B1 - Tissue vaccine against infection jaundice of ducks and method of its preparation - Google Patents

Abstract

Tissue vaccine against infectious hepatitis ducks is intended for active immunoprophylaxis infectious hepatitis ducks. Contains inactivated duck hepatitis virus CAPM V-296 in lyophilized state. Vaccine is prepared for use by dissolution in lipoid adjuvant. Hepatitis Tissue Vaccine ducks are produced by propagation of the virus strain infectious hepatitis ducks on primary culture kidney epithelial cells in growth medium for 70 to 74 hours at 35-39 ° C until miniLDg is reached · 105 / 0.1 ém3, 11 days intraalantoid / then titers chicken embryo virus inactivates with glutaraldehyde a It is lyophilized.

Description

The invention relates to a tissue vaccine against infectious duck hepatitis and to a method for its preparation

In intensive large-scale farming conditions, effective disease prevention measures are necessary. Infectious liver inflammation is one of the diseases that cause large losses in ducklings. This disease mainly affects ducklings in the first days after hatching. Infectious duck hepatitis virus (IHK) has been classified as duck picorna virus / Taurasso N, Μ., G Goghill β. E., Klutch M.J. Proportions of the attenuated vaccine strain of duck hepatitis virus. Avian D. S. 13, 1969, pp. 321-329].

The greatest losses due to IHK infection occur in the first two weeks after hatching. Schoop, Staub: Ober die Virushepatitis der Enten 3. Mitt. Die Bekampfung.

Mh. Tierheilhde, 10, 1958, pp. 29-35]. As the age of ducklings increases, resistance to the disease increases and deaths are significantly lower. Other authors agree with this data. According to various authors, mortality varies between 3 and 95% during the disease. Schoop, Staub: Virushepatitis of ducks. Mh. Tierheilhde, 9, 1957, pp. 317-322,

These data are the reason for the specific form of the fight against this infection, which was targeted immunization and timely administration of hyperimmune serum. Active immunization can be performed in breeding ducks with live or inactivated vaccine, or vaccination can be used in freshly hatched ducks with live vaccine.

In the sixties a live vaccine against IHK was prepared in the Czechoslovakia, prepared from the attenuated strain TN 62 / žuffa A., Černík Κ. , Rajtár V., Žuffová J .: Immunoprophylaxis of Infectious Inflammation of the Duck. Veterinar., 11, 1966, pp. 490-495]. The vaccine was intended solely to control the disease situation in breeds where a virulent virus was present.

At the same time, an adsorbate inactivated vaccine against IHK was introduced in the 1960s. The production strain Valdíkov 530 is of domestic origin and is antigenically identical with the terrain strains Isolated in Czechoslovakia and with TN 60 strain originating from England / Kožušník Z., Dvořáková D .: Isolation and properties of strains of viral inflammation of duck liver in the CSSR. Acta vet., 39, 1970, pp. 143-150]. The virus multiplies and passages on 10 to 11 days of chicken germs and, when administered intraalantoidly, causes germs to die 2 to 5 days after inoculation.

At present, 2 types of vaccines are provided in the Czechoslovak Socialist Republic against immunoprophylaxis against IHK.

a / Vaccine against infectious liver inflammation of ducks b / Vaccine against infectious liver inflammation of ducks lyophilized ad a / The IHK vaccine is a suspension of IHK virus propagated in chicken embryos inactivated by beta-propiolactone, adsorbed on aluminum hydroxide gel, preserved with formalin.

ad b) The vaccine is a suspension of IHK virus propagated on chicken embryos, inactivated by glutaraldehyde and lyophilized. The lyophilisate is dissolved in an oil solvate prior to use.

These two organic preparations are currently used for regular immunization of breeding flocks in large-scale farms. The resulting antibodies in the breeding duck organism after vaccination are transmitted transovarially to ducks hatched from the eggs of the immunized ducks. Ducklings are protected by these acquired antibodies in the most endangered period of their life.

Despite the undoubtedly good results obtained with these two types of IHK vaccines produced in Bioveta Ivanovice na Hané, these vaccines have some drawbacks. The use of chicken embryos for vaccine production is relatively disadvantageous due to operational reasons (considerable number of eggs, multiple handling, etc.). Because of the impossibility of obtaining SPF eggs, eggs from large-scale breeding hens are used for production, which does not guarantee complete infectiousness and remains a risk of transmission of various infectious agents by embryonic vaccines / mycoplasmas, viruses, etc./. The manufactured vaccines in the form of an embryonic suspension are biopreparations with a high content of ballasts, which complicates the creation of solid immunity against the desired viral agent. These drawbacks lead to the development of new IHK vaccines where the virus is propagated in a suitable culture substrate. Of particular importance are the findings that IHK virus multiplies in tissue cultures / Richard A. Mason, Nicola M. Taurasso, Robert K. Ginn: Growth of Duck Hepatitis in Chicken Embryos and Cell Cultures derived from infected Embryos. Avian Deseases, Vol. 6, p. 5, 1972 (or work) Morris Pollard, Theodore J. Starr: Propagation of Duck Hepatitis Virus in Tissue Culture. Reprinted from Avian Diseases 23, NO. 3, July-September, 1979].

According to available information, the IHK vaccine is not produced abroad on tissue cultures. Abroad, immunoprophylaxis against infectious duck hepatitis is provided by inactivated vaccines for immunization of breeding flocks of ducks and thus ensuring protection of ducks by transovariously passed antibodies from immunized mothers, or using vaccines made from attenuated strains of infectious duck hepatitis viruses. freshly hatched ducklings.

The essence of our invention is a tissue vaccine against infectious duck hepatitis and a method for its preparation. The IHK tissue vaccine is intended for active immunoprophylaxis against IHK by vaccination of breeding ducks. The susceptible ducklings in the youngest age are protected by the transovarian transmission of IHC post-vaccination antibodies from immunized mothers. 1 cm ³ of the vaccine contains at least 1 million LD _5Q inactivated virus duck hepatitis CAPM V-296 in a lyophilized state.

A method for preparing a tissue vaccine against infectious duck hepatitis is characterized in that the IHK vaccine virus is obtained by cultivation and propagation in a primary duck kidney cell culture, is inactivated by glutaraldehyde after harvest and lyophilized. The immunogenic efficacy of the IHK vaccine, containing the virus obtained by propagation on tissue cultures, is stimulated diluted in the form of a lipoid adjuvant.

The vaccine is a suspension of inactivated duck hepatitis virus, a growth medium, a lyophilization medium, and an oil diluent which also serves as a vaccine adjuvant.

Components for the preparation of a tissue vaccine against infectious duck hepatitis:

1. Duck infectious hepatitis virus strain - deposited in the Czechoslovak Collection of Microorganisms, Collection of Zoopathogenic Microorganisms of the Research Institute of Veterinary Medicine, Brno 21, Medlánky, under the designation CAPM V - 296.

2. Cell culture for IHK virus propagation

3. Growth and maintenance medium

4. Antibiotics

5. An inactive agent and method for performing inactivation of the IHK virus

6. Buffered, concentrated lyophilization protective medium

7. Vaccine Diluent and Adjuvant Ad 1 - For the production of a tissue vaccine against infectious liver inflammation of ducks, a strain of infectious duck hepatitis virus isolated from the territory of Czechoslovakia under the designation Valdikov 530, deposited in the collection of microorganisms under the designation CAPM V - 296, is used.

ad 2 - The cell culture for cultivation of the above strain is a culture of primary duck kidney epithelial cells.

ad 3 - Composition of growth medium:

M-199 Parker medium - modified without purines, pyrimidines, vit. D2 and cholesterol / PNY 30-259-79 PN ÚSOL Prague /, Tryptose phosphate Broth - Difco / catalog number 0060-01-6 / Bovine serum ultrafiltered, inactivated / PN Bl 182/85 / Sodium bicarbonate solution 7,5% NaHCO ^ pa (CSL 3). The individual components in the order are: 90: 10: 5: 1.

Maintenance medium composition:

M - 199 Parker medium - modified without purines, pyrimidines, vit. D ₂ and cholesterol / PNY 30-259-79 PN ÚSOL Prague / Tryptose phosphate Broth - Difco / catalog number 0060-01-6 / Bovine serum ultrafiltered, inactivator '/ PN Bl 182/85 / Solution 7,5 * sodium bicarbonate NaHCO 3 (Cs 3). The components are in the order of: 90: 10: 1: 1.

ad 4 - Antibiotics are used for growth and maintenance media in the following concentrations: Penicillin stock solution: Penicillin G 10 million IU.

Streptomycin sulfate 10 g sterile water, 300 g stock solution: Streptomycin sulfate 10 g

Water tri-distilled sterile ad 300 cm ³ Fungioidin stock solution: Pungioidin 20 g

Ethylene glycol p.a. ad 2.000 cm ad 5 - Infectious duck hepatitis virus is inactivated as follows:

glutaraldehyde is used for inactivation at a final concentration of 0.15 to 0.20%, for 1 hour at 34 to 38 ° C. Inactivation takes place in a water bath. After inactivation, the pH is adjusted to 6.4 to 7.8.

ad 6 - Composition of the buffered, concentrated lyophilization protective medium: Gelatin 250 g Sucrose 375 g

Sodium hydrogen phosphate / Na ₂ HPO ₄ x 12H ₂ O / 12 g

Potassium dihydrophosphate (ΚΗ ,, ΡΟ ^) / 1,125 g

Redistilled water ad 1.000 cm ³ ad 7 - Solvents and adjuvants:

Oil vaseline medical Al 24.000 g Lanolin 8.000 g / Tween 80 / polyethylene glycol ether sorbitan monoolejanester 1.600 g Water redistilled 66.400 g

Example:

Trypsinizing the kidneys of young ducks yields primary epithelial kidney cells.

The suspension of these cells is resuspended in growth medium to a density of 300,000 to 400,000 cells of > 33 in 1 cm and filled into Roux bottles and 1,200 cm in 120 cm cell suspensions. Completely grown monolayers of primary cultures 4-5 days old are infected with IHK virus. On

6 3 monolayer weave 15 cm of IHK virus with a titre of at least LD _5Q = 10 / 0,1 cm 11 day chicken embryo, intraalar.toid /. Allow to adsorb for 2 to 3 hours at 35 to 39 ° C.

After this time, sieve excess virus and add 120 cm of maintenance medium.

Virus harvest is performed 72 hours after infection of the cell culture. For the production of vaccine 5 3 collections of virus used a minimum titer LD _5Q - 10 / 0.1 cm 11 day chicken embryo, intraulantoídně /. A suitable harvested virus is inactivated with glutaraldehyde at a final concentration of 0.15-0.20% for 1 hour at 35-39 ° C. The inactivated IHK virus is mixed in a proportion of 9 parts of the virus with 1 part of the lyophilized protective medium buffered, concentrated. The inactivated IHK virus with the lyophilization medium is filled into vials and frozen. Thereafter, the vaccine is lyophilized in the most gentle manner (during lyophilization the temperature of the product must not exceed 25 ° C).

The required residual humidity must be less than 3%. The lyophilized vaccine is sealed with a stopper in a vacuum tube. The vaccine is prepared by a lipoid adjuvant, which is also a dilution vaccine. Said components of the solvens are emulsified and filled into vials. Medicine bottles with neuron ;. They are closed and its contents sterilized by autoclaving. The vaccine is prepared for application by dissolving the ivilnate in iipi.c.in ad j'j · t.ns ..

For each batch of vaccine produced, a final control is performed: Sterility, harmlessness, efficacy, pH, moisture, appearance and vacuum tests in the vaccine.

Possibilities of using the invention:

Tissue vaccine against infectious duck hepatitis has been experimentally produced and verified as part of laboratory and clinical testing in breeding and hatched ducks. The following immunization schedule, dosages and monitoring in ducks were used.

The vaccine dose for ducks is 2 cm ^{3 of} reconstituted vaccine administered intramuscularly. The vaccine is intended for the active immunization of adult ducks before laying. Vaccination is carried out 6 weeks before laying, revaccination is just before the start of laying, and further revaccination is during the laying, ie 2 to 3 months after the start of laying. Transovarian transmission gives the ducks a high degree of protection lasting up to 28 days after hatching from the vaccinated ducks. When administered at the above dosage and procedure, the immunization ability of the vaccine detected in vaccinated ducks and their ducks by a neutralization test in chicken embryos was determined. / Tab. no. 1./.

Post-vaccination reactions following vaccination or subsequent local or generalized revaccination were not observed in the vaccinated animals during verification.

Table 1

Observed mean PDs in sera of vaccinated ducks and their offspring (log 10).

Adult ducks:

1. One month after vaccination, before revaccination (before the start of laying period -50 ⁼ 5.5).

2. In the middle of the laying period PD _5Q = 4.0

3. End of laying period PD _{& 0} = 3.3

Ducklings: / Transovarian transmission of antibodies from vaccinated mothers /:

1 day - ^PD 50 = 4.13 Day 7 - -50 = 4.34 Day 14 - ^PD 50 = 3.29 Day 28 - -50 = 2.85 Day 32 - -50 = 2,0

These results confirm the good immunization effect of the vaccine against infectious duck hepatitis in adult ducks and the high protection of their offspring. Still on day 32 after hatching from vaccinated duck mothers, duck serum protects against 100 id _5Q IHK virus.

Claims (6)

Tissue vaccine against infectious duck hepatitis for the immunoprophylaxis of infectious duck hepatitis by transovarian transmission of post-vaccination antibodies to ducks from immunized mothers, characterized in that the 1 cm vaccine contains at least 1 million LD ^ ₀ inactivated duck hepatitis virus CAPM V-296 in lyophilized condition. 2. The vaccine against infectious duck hepatitis according to claim 1, which comprises a diluent in the form of a lipid adjuvant. 3. A method for producing a tissue vaccine against infectious duck hepatitis according to claim 1 or 2, characterized in that the infectious duck hepatitis virus is propagated on primary cultures of duck kidney epithelial cells in growth medium for 70-74 hours at 35-39 [deg.] C, until a minimum LDg titer of ₀ -10 10 is reached, then the harvested virus is inactivated by glutaraldehyde. 4. The process according to claim 3, characterized in that during production is used to infect the primary cultured kidney cells, epithelioid Infectious hepatitis ducks at a dose of 15 to 20 cm ³ minimum titer of virus by LD _5Q "10 ⁶ per one Roux bottle and 1 200 cm ³ and allowed to adsorb for 2 to 3 hours at 35 to 39 ° C. 5; The method of claim 3 wherein glutaraldehyde is used to inactivate the harvested duck hepatitis virus at a final concentration of 0.15 to 0.20% for 1 hour at 34 to 38 ° C in a water bath followed by pH adjustment. to 6.4 to 7.8. 6. The method of claim 3 wherein the inactivated infectious duck hepatitis virus is mixed with a lyophilization medium containing 3.75% gelatin and 2.5% sucrose in a final blend, filled into vials, frozen and lyophilized.