RU2097066C1 - Vaccine ovac for control over syndrome of egg yield decrease - 76 poultry - Google Patents

Abstract

FIELD: biotechnology, veterinary virology. SUBSTANCE: vaccine has the inactivated antigen from the strain of virus of egg yield decrease - 76 poultry VGNKI N 98 "B-93"-DEP at titer of infectious activity before inactivation 10^6,0-10^8,0 EID_50/0,2 sm³ and with activity in RGA before and after inactivation 16-12 log², saline diluting agent-stabilizing agent: phosphate-buffer solution at pH = 7.2-7.4, or Henx or Earl solution and adjuvant - silicon dioxide or aluminium hydroxide. Dimerethyleneimine is used for virus inactivation and sodium thiosulfate is used for its neutralization. Preparation is used intramuscularly, at a single dose to chicken (age is 110-120 days) at volume 0.5 sm³. The use of vaccine promotes the egg yield increase (by 27.2 eggs for productive period) and decreases amount of shellless eggs (by 5.5%). EFFECT: enhanced effectiveness of vaccine. 3 tbl

Description

 The invention relates to veterinary virology and biotechnology and relates to the creation of a new vaccine for specific prophylaxis of egg-laying syndrome (SSW-76) in threatened and dysfunctional farms.

Known vaccine against the syndrome of reducing egg production-76 birds, including the inactivated formalin antigen from the strain of the virus syndrome of reducing egg production-76 birds VS-14 and an oil adjuvant of undisclosed composition [1]
The disadvantage of this vaccine is its low immunogenicity and stability. In addition, the use of formalin for inactivation of solutions does not ensure the absence of residual infectivity in it.

Also known is a vaccine against avian egg-reducing syndrome-76, including the antigen inactivated by ethyleneiminoethyl oxamide-antigen from the B8 / 78 avian virus-reducing virus-76 strain, a salt stabilizer and an oil adjuvant based on soybean oil, lecithin and sorbitol [2]
The disadvantage of this vaccine is also not high immunogenicity and stability, higher cost when it is received and higher labor costs when using it.

 The aim of the present invention is to increase the immunogenicity of the vaccine, increase its stability, as well as reduce the cost of its production and reduce labor costs when using it.

This goal is achieved by the fact that the vaccine against the bird egg reduction syndrome-76 contains, as an inactivated antigen, from the strain of the egg-laying syndrome virus-76 birds inactivated by dimeretylenimine antigen from the bird egg-reducing virus strain-76 strain VGNKI N 98 "B-93" DEPT with a titer infectious activity before inactivation 10 ^6.0 -10 ^8.0 EID _{50 / 0.2} cm ³ and with activity in RGA before and after inactivation 16-12 log ₂ , as a salt diluent stabilizer: phosphate-buffered solution with pH 7 , 2-7,4, or Hanks's solution or Earl's solution as an adjuvant suspension of 5% silica and 6% suspension of aluminum hydroxide in the following ratio (in wt.):
Inactivated dimethyleniminimine antigen from a strain of the virus of egg laying down syndrome-76 birds VGNKI N 98 "B-93" -DEP with a titer of infectious activity before inactivation 10 ^6.0 -10 ^8.0 EID _{50 / 0.2} cm ³ and with activity in the RGA before and after inactivation 16-12 log ₂ 16.6 33.2
Phosphate buffer solution with a pH of 7.2-7.4, or Hanks's solution or Earl's solution 66.4 82.8
5th suspension of silica or 6th suspension of aluminum hydroxide - 0.4 0.6
For the manufacture of the vaccine, a strain of the B-93 bird egg-reducing syndrome virus 76 is used.

 The vaccine strain B-93 was isolated from diseased chickens and obtained as a result of adaptation to duck embryos.

 The strain of the virus egg-reduction syndrome-76 birds "B-93" was deposited in the virus collection of the All-Russian State Research Institute for Control, Standardization and Certification of Veterinary Medicines, has a registration N 98.

 The virus strain against SSN-76 VGNKI N 98 "B-93" DEPT has the following characteristics and properties.

 Morphological signs. Electron-scopic examination of strain N 98 "B-93" VGNKI is represented by icosahedral virions, which are devoid of a supercapsid membrane and do not contain lipids. Sedimentation coefficient 550.

 The presence of virions morphologically different from the virus is not established. Adenoviruses.

Cultural properties. Strain N 98 "B-93" VGNKI multiplies well in 10-12-day-old duck embryos infected in the allantoic cavity. Infectious activity of the strain after 120 hours at a temperature of 37.5 ^o C is not less than 10 ^6.0 EID _{50 / 0.2} cm ³ .

 The strain is also cultivated in chicken embryos and the primary duck fibroblast cell culture, causing a cytopathic effect.

Hemagglutinating properties. Strain N 98 "B-93" VGNKI has pronounced hemagglutinating properties. Red blood cells of chickens and ducks agglutinate, causing the formation of a medium-grained hemagglutination reaction with a limit titer of 12-16 log ₂ .

Antigenic properties. 14-21 days after infection with 120-day-old chickens, the appearance of virus-neutralizing and hemagglutinating antibodies is noted. The titer of anti-hemagglutinating antibodies is 7.0 log ₂ or more with their subsequent increase.

 Infectious properties. To strain N 98 "B-93" VGNKI are susceptible chickens, ducks and geese with intratracheal, oral, intramuscular infections. starting from the 14th day after the introduction of the virus, 120-day-old chickens show the appearance of depigmented and shellless eggs. Within four weeks, the percentage of eggshells and shells is about 30% or more.

 The virus does not cause clinical signs of the disease during experimental and natural infections. Pathological changes are not expressed and are determined only histologically in the form of intranuclear inclusions in the epithelial cells of the glandular stomach.

Stability of the strain. Strain N 98 "B-93" VGNKI is stable at a pH in the range of 6.0-9.0. Resistant to fat solvents. Inactivated at 56 ^o C.

Example 1. The strain of the virus SSN-76 "B-93" is propagated on duck embryos infected in the allantoic cavity at a dose of 2.0 3.0 lg EID _{50 / 0.2 ml} Embryos are incubated for 96 120 hours at 37.0 ± 0.5 ^o C. After 120 hours, the embryos are cooled at a temperature of +4 +8 ^o C for 24-36 hours and used to collect the virus-containing extraembryonic fluid. The liquid is collected in bottles with a capacity of 200 to 500 cm ³ . From each vial, a sample of 0.8 cm ^{3 is} taken to test for sterility, in a volume of 0.2 cm ³ by plating on nutrient media: MPA, MPB, MPPB, under paraffin oil and Saburo medium (if there is growth, the virus-containing material is destroyed). After checking for sterility, the infectious activity of the virus is determined by titration on 10-12 day old duck embryos at a temperature of 37 ± 0.5 ^o C for 120 hours. The infectious activity is set equal to 10 ^6.0 EID _{50 / 0.2} cm ³ . Hemagglutinating activity is determined in an RGA with a 1.0% chicken erythrocyte suspension. Set GA activity equal to 12 log ₂ .

To inactivate the virus, dimeretylenimine is added to the extraembryonic virus-containing liquid to a final concentration of 0.2% in the reaction mixture and incubated at a temperature of 37 ± 0.5 ^o C for 4-5 days. Then, the residual inactivator is neutralized by adding sodium thiosulfate to a final concentration in the reaction mixture of 0.03 M. The mixture is kept for 2 hours at a temperature of 20-22 ^o C. The hemagglutinating activity of the inactivated virus in RGA with 1% chicken erythrocyte suspension is re-controlled. In this case, the RGA activity corresponds to the initial one and is 12 log ₂ .

Check the completeness of virus inactivation on duck embryos 10-12 days of age for 3 consecutive passages. In the absence of specific death of embryos and negative RGA in extraembryonic fluid, the inactivated virus is used to prepare a standard vaccine preparation. For the preparation of 1 liter of the OVAC vaccine against the egg laying down syndrome, 76 birds take 332 cm ^{3 of the} antigen inactivated by dimethylethyleneimine from the virus of the bird laying down syndrome 76 birds VGNKI N 98 "B-93" -DEP to which 664 cm ³ of stabilizer diluent is added (phosphate buffered saline) with a pH of 7.2 and 7.4 and stirred. To the resulting suspension of inactivated virus of standard activity add 4 cm ³ 5% suspension of silicon dioxide and again mixed to obtain a uniform suspension.

After checking the completeness of adsorption in the RGA, the vaccine is packaged in sterile 500 cm ³ bottles, tightly closed with sterile rubber stoppers and sealed with aluminum caps.

Example 2. The vaccine "OVAC" against the syndrome of reducing egg laying-76 birds is prepared analogously to example 1, using for its preparation inactivated dimethylenimine antigen from the strain of the virus of the syndrome of reducing egg laying-76 birds VGNKI N 98 "B-93" DEPT with a titer of infectious activity before inactivation 10 ^7.0 EID _{50 / 0.2} cm ³ and with activity in the RGA before and after inactivation 14 log ₂ . To prepare 1 liter of vaccine, 249 cm ^{3 of} inactivated antigen are taken to which 746 cm ^{3 of} diluent-stabilizer Hanks solution is added and after mixing 5 cm ^{3 of} 5% suspension of silica are added.

Example 3. The vaccine "OVAC" against the syndrome of reducing egg laying-76 birds is prepared analogously to example 1, using for its preparation inactivated dimethylenimine antigen from the strain of the virus of the syndrome of reducing egg laying-76 birds VGNKI N 98 "B-93" DEPT with a titer of infectious activity before inactivation 10 ^8.0 EID _{50 / 0.2} cm ³ and with activity in the RGA before and after inactivation 16 log ₂ . To prepare 1 liter of vaccine, 166 cm ^{3 of} inactivated antigen is taken to which 828 cm ^{3 of} diluent-stabilizer Earl solution is added and after mixing 6 cm ^{3 of a} 6% suspension of aluminum hydroxide is added.

 The finished vaccine is a colorless liquid with a white, easy-whipping sediment.

The vaccine retains its biological properties for 12 months if stored in a dry, dark room at a temperature of -20 ^o C.

 Example 4. Form four groups of chickens 110-120 days of age, 40 thousand goals in each.

 Chickens of groups 1-3 are inoculated with the OVAC vaccine against SSN-76 birds from the strain VGNKI N 98 "B-93" DEPT made according to examples 1,2 and 3. A fourth group of chickens are inoculated with the inactivated oil vaccine against SSN-76 from the strain " B8 / 78 "manufactured by IHP" Virion ".

The vaccine is administered intramuscularly once in a volume of 0.5 cm ³ . After 10, 13, 16, 23 and 29 days and then every month, 25 blood serum samples for the presence of post-vaccination antibodies in rtga are examined from the chickens of each group.

 During the monthly productive period, birds of groups 1 and 4 carry out an egg test. Comparative antigenic activity of vaccine samples is presented in table 1.

 The results of the experiments indicate a higher antigenic activity of the vaccine "OVAC" from the strain VGNKI N 98 "B-93" DEPT.

From the data table. Figure 1 shows that 10 days after immunization, the antibody level in groups of chickens vaccinated with the Ovak vaccine reached 7.2 or more log ₂ with maximum accumulation by day 23 and remained at 5.23 log ₂ during the observation period ( 6 months).

In the group of chickens vaccinated with the vaccine against SSV-76 from strain B8-78, the antibody level 10 days after vaccination was 2.8 log lower. By day 23, the difference decreased to 0.52 log ₂ and after 6 months remained at 5.00 log ₂ .

 Indicators of egg productivity of experimental groups of birds are presented in table.2.

 As can be seen from the table. 2, the average number of eggs obtained from chickens vaccinated with the vaccine against SSN-76 from strain B8 / 78 was 76 thousand less than in the group immunized with the vaccine Ovak against SSN-76 from strain VGNKI N 98 "B-93" DEP (as in example 1). In percentage terms, the difference in indicators is 8.6% in favor of the Ovak vaccine.

Example 5. Form two groups of chickens 110-120 days of age. The first group of chickens (34 thousand heads) is inoculated with the Ovak vaccine against a decrease in egg production of 76 birds from VGNKI strain N 98 "B-93" DEPT, according to Example 2, intramuscularly, once in a volume of 0.5 cm ³ . The second group of chickens (6 thousand heads) unvaccinated, serves as a control. In the period from 5 to 15 months of age, the egg laying rate of laying hens in both groups is monitored monthly.

 Indicators of egg productivity of vaccinated and unvaccinated birds are presented in table 3.

 The data table. 3 indicate an increase in the egg productivity of chickens vaccinated with the Ovak vaccine from 5 to 15 months of age compared with an unvaccinated chicken population.

The proposed vaccine "OVAC" against a decrease in egg production-76 birds from the strain VGNKI N 98 "B-93" DEPT compared with existing vaccines has the following advantages:
has a higher antigenic and immunogenic activity;
quickly forms a reliable immunity;
complete inactivation of the virus in the vaccine eliminates the possibility of the spread of the disease in poultry farms during preventive vaccinations;
used adjuvant helps maintain high stability and immunogenic activity of the vaccine.

The use of the OVAC vaccine in poultry farms showed a significant increase in egg production per laying hen for the productive period by 27, 2 eggs and a decrease in the number of laid eggshells by 5.5%
The use of the OVAC vaccine against avian egg-lowering syndrome-76 from the strain VGNKI N 98 "B-93" DEPT in veterinary practice will expand the arsenal of highly effective biological products against this disease of birds and reduce the cost of preventive measures.

Claims (1)

Vaccine against a bird egg reduction syndrome-76, including an inactivated antigen from a strain of a bird egg reduction syndrome-76 virus, a salt diluent stabilizer and an adjuvant, characterized in that it contains an inactivated antigen from a virus strain reduction syndrome of a bird egg-76 virus inactivated dimeretylenimin antigen from a virus strain syndrome Xingzheng-76 egg laying birds VGNKI N 98 "B-93" DEP with a titer of infectious activity prior to inactivation June ¹⁰ August ¹⁰ EID _{50 / cm} 3 and _0.2 with activity before and after WGA inactive novation December 16 log ₂ as saline diluent stabilizer of phosphate buffer solution pH 7,2 7,4, or Hank's solution, or solution Earle, as an adjuvant suspension of 5% silica and 6% suspension hydroxide aluminum in the following ratio of components, wt. Inactivated dimethyleniminimine antigen from a strain of the virus of egg laying down syndrome-76 birds VGNKI N 98 "B 93" DEPT with a titer of infectious activity before inactivation 10 ⁶ 10 ⁸ EID _{50 / 0.2 cm} 3 and with activity in the RGA before and after inactivation 16 12 log ₂ 16.6 33.2
Phosphate buffered saline with a pH of 7.2 7.4, or Hanks's solution, or Earl's solution 66.4 82.8
5% Suspension of silica or 6% suspension of aluminum hydroxy - 0.4 0.6a

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