RU2202367C2 - Method for preparing inactivated vaccine against cattle leucosis - Google Patents

Abstract

FIELD: veterinary science. SUBSTANCE: the present method deals with cultivation of virus-containing peripheral blood leucocytes in leucosis-suffering cattle, inactivation and antigen obtaining. Cultivation of leucocytes is carried out for 24-36 h at inoculation concentration equal to 5-10×10⁶ cells/ml medium. Then leucocytes should be deposited due to centrifuging. Suspension should be prepared upon Eagle's medium at concentration of 10 mln cells/ml medium, leucocytes are fixed to inactivate the virus inside by adding 5 volumes of 2-3%-glutaric aldehyde solution. Incubation is carried out at room temperature for 30-40 min. Leucocytes should be thrice washed in buffered physiological solution at pH=7.2-7.4, resuspended in Eagle's medium at 100 mln leucocytes/ml to be used as immunizing agent. Vaccine obtained due to the suggested method provides accelerated terms of sanitation in leucosis-suffering farms by 2-3 times and decreased expenses necessary for sanitation. EFFECT: higher efficiency. 2 ex, 1 tbl

Description

 The invention relates to the field of veterinary virology, in particular to methods for the production of vaccines against hemoblastosis in cattle.

 Leukemia of cattle is widespread in our country, and in most regions of the world, causing great damage to livestock.

 Currently known methods for producing vaccines against leukemia diseases in various animal species.

 There is a method of obtaining a vaccine for immunizing birds against lymphoid leukemia and erythroblastosis by obtaining a mixture of living cells and ground tissues infected with this virus in a pharmaceutical liquid, the concentration of the mixture being such that 1 g of the mixture is contained in 10-200 ml of liquid. As the source material used tissue of the kidney, spleen or liver of a sick animal [1].

 A known method of preparing a vaccine against feline leukemia obtained from cells of a culture infected with a cat leukemia virus. The vaccine consists of cells infected with cat leukemia virus, or from inactivated particles of leukemia virus isolated from culture cells [2].

 There is also known a method for producing a vaccine against cattle leukemia, including culturing virus-containing cells, isolating cells and viral material from the culture fluid, destroying cells and viral material while simultaneously inactivating the nucleic acids contained therein and then isolating the p25 and gp70 protein fractions used as an immunizing agent [3].

The objective of our research was to develop a method for producing an environmentally friendly inactivated vaccine with immunogenicity against cattle leukemia
The essence of the proposed method lies in the fact that the leukocyte peripheral blood leukemia leukocytes carrying both cattle leukemia virus and tumor and transplant antigens cultured for 24-36 hours in a nutrient medium consisting of Medium Needle or RPMI 1640 - 90% and 10% of inactivated serum of cow or sheep serum embryos. Cultured cells and the viral material contained in them, tumor and transplantation antigens are fixed with a 2-3% glutaraldehyde solution for 30-40 minutes while the virus is inactivated and resuspended in buffered saline solution, pH 7.2-7.4, based on 10 • 10 ⁷ cells in 1 ml. One immunizing dose is 50-100 • 10 ⁶ cells.

 Adjuvant, colloidal alumina hydrate according to GOST 18287-81, is attached to the vaccine. Immediately before use, the vaccine is mixed with adjuvant in a ratio of 1: 1. The vaccine is administered subcutaneously in the neck.

 Immunization is carried out three times with an interval of 10-14 days in the above dose.

 The inactivated vaccine against cattle leukemia, prepared according to the proposed method, was tested with a positive result in laboratory and industrial conditions.

 The vaccine is harmless and nonreactogenic when administered three times subcutaneously for cattle aged 2 months to 2 years (table).

 Example 1

Leukocytes are isolated from peripheral blood by the method of hemolytic shock of red blood cells by adding two volumes of distilled water to 1 volume of stabilized blood, followed by restoration of the isotonicity of the solution or two volumes of a 0.85% solution of ammonium chloride. The isolated cells are resuspended in a culture medium of the following composition: inactivated serum of cow embryos or sheep serum - 10%, Medium Needle or RPMI 1640 - 90% based on 10 • 10 ⁶ cells per 1 ml of medium. After 24 hours of cultivation at 37 ^° C, the leukocytes are pelleted by centrifugation at 1000 rpm for 20 minutes and resuspended in Eagle or RPMI 1640 medium at the rate of 10 million cells per ml of medium.

 To 100 ml of the resulting suspension, add 500 ml of a 3.0% glutaraldehyde solution and incubate at room temperature for 30 minutes. Then the cells are precipitated by centrifugation at 1000 rpm for 10 min, the pellet is washed three times in buffered saline solution with a pH of 7.2-7.4 and resuspended in Eagle's medium at the rate of 100 million cells per 1 ml.

 Immunization is carried out three times with an interval of 14 days.

 During the first immunization, the resulting material was injected subcutaneously at a dose of 100 million cells in the neck of the calves at the age of 2-3 months with an adjuvant in a ratio of 1: 1.

 The second and third immunization was carried out without adjuvant in the same doses.

 2 weeks after the third injection of the drug, a control infection of the cattle leukemia virus was carried out for all vaccinated and unvaccinated animals of the control group. Infection was carried out by leukocytes isolated from the blood of a patient with an animal leukemia, at a dose of 250,000 cells per animal, which is 100 infectious doses.

 All animals in the control group were infected with the leukemia virus, while vaccinated animals remained uninfected. Immunity is 8 months.

 Example 2

Cells are isolated from the peripheral blood of a patient with cattle leukemia in an animal, as described in Example 1. Cells are cultured in RPMI 1640 medium containing 10% inactivated sheep serum at a rate of 5.0 x 10 ⁶ cells per ml of medium. After 36 hours of cultivation, the leukocytes are fixed with a 2% glutaraldehyde solution for 40 minutes.

 The resulting vaccine with adjuvant is administered subcutaneously to heifers of 20-24 months of age according to a scheme similar to Example 1. Vaccinated heifers are placed in a herd unsuccessful for leukemia (with an infection level of 70%) under close contact.

 After 12 months of observation, 85% of vaccinated animals remained uninfected.

 The vaccine made according to the proposed method will find application in farms that are disadvantageous for leukemia, will allow to speed up the recovery time of these farms by 2–3 times and reduce the cost of rehabilitation.

Sourse of information
1. US patent 3590128, CL 424-89, 1973.

 2. French patent 2243702, cl. A 61 K 39/12, 1975.

 3. Auto USSR 820015, class A 61 K 39/12, 1980.

Claims (1)

A method of preparing an inactivated vaccine against leukemia in cattle, including cultivating the virus-leukocytes of peripheral blood of cattle of a patient with leukemia, inactivating and obtaining antigen, characterized in that the cultivation of leukocytes is carried out 24-26 hours at an inoculum concentration of 5-10 • 10 ⁶ cells per 1 ml of medium, then white blood cells are precipitated by centrifugation, a suspension is prepared on Eagle’s medium with a concentration of 10 million cells per 1 ml of medium, white blood cells are fixed and the virus contained in them is inactivated. by adding 5 volumes of a 2-3% solution of glutaraldehyde, they are incubated at room temperature for 30-40 minutes, the leukocytes are washed three times in buffered saline with a pH of 7.2-7.4, and they are resuspended in Eagle’s medium at the rate of 100 million leukocytes in 1 ml and use them as an immunizing agent.

Images