CS209379B1 - Vaccination against infectious bursitidis from attenuated strain and method of its preparation - Google Patents

Description

(5 4) Attenuated cinema infectious bursitis vaccine and method for its preparation

The invention relates to a vaccine against infectious bursal disease of chickens (Gumboro disease) and a process for its preparation from a virus strain isolated in Slovakia and attenuated by serial passages in cell cultures.

Infectious bursitis (Gumboro disease) is a viral disease of chickens, described for the first time in 1957, which occurs very often subclinically, but causes a decrease in weight gain and a slowdown in growth, which, even at low mortality, causes economically significant losses.

The causative agent of infectious bursitis is a virus which is extremely resistant in the external environment to the exposure to physical and chemical agents. It persists in contaminated areas and this is the cause of recurrent infection of the following poultry arrest. The virus particle lacking the envelope has a diameter of 60 nm. It contains a single-stranded ribonucleic acid composed of two segments. The molecular weight of the virus genome is 1.75 to 1.8 x 10 ⁶ .

Infection of susceptible chickens up to 14 weeks of age causes degeneration and necrosis of immature B lymphocytes in the Fabricius Exchange, resulting in a selective decrease in immunoglobulin G production and thus irreversible weakening of immune mechanisms. Necrotic changes in the thymus and spleen also adversely affect the development of a cellular immune response. Increased susceptibility is particularly evident in Newcastle disease viruses, infectious bronchitis, E. coli, salmonella, Mark's disease virus and infectious bronchitis and adenovirus infection during the laying period.

The virus can be isolated only a few days after infection in non-specified pathogenic germline (SPF) germs and passaged in SPF chickens or in cell cultures prepared from SPF eggs.

In 1978, between 60% and 100% of reproductive poultry farms were infested in Western Europe. In the CSSR, according to preliminary results of serological surveys and virus isolation, a high degree of infectious bursitis virus infestation was found in the breeding of both meat and carrier breeds.

Prophylaxis of infection is based on the following principles:

- the virus is very widespread in the population, is extremely resistant and has long-term survival in the environment;

- the disease is transmitted horizontally and can also infect freshly hatched chickens;

- chickens may have maternal antibodies that provide temporary protection but at the same time hinder the development of active immunity.

Non-specific prophylaxis is based on the general principles of zoohygiene: simultaneous installation and emptying of halls, limitation of material movements and passage of persons, and effective disinfection with formalin vapors.

Specific prophylaxis is based on passive protection of chickens with maternal antibodies that lasts for 2-3 weeks and on active immunization of chickens and reproductive laying hens. For vaccination, they use live vaccines from a virus of varying degrees of virulence, multiplied in hatching vaccines from non-specified pathogenic germs (SPF). (Becht, H, Rapp. OIE, 1977, No 201, p. 9; BENNEJEAN, G., Rapp. OIE, 1977, No 216, p. 8; Cessi, D. - Gualandi, GL, Rapp. OIE 1977, No 211, p. 10; Cullen, GA-Wyeth, PJ, Vet. Rec., 99, 1976, No 21, p. 418; Thomton, DH, Rapp. OIE, 1977, No 200, Yadin, H. - Hoekstra, J., Rapp. OIE, 1977, No. 212, p. 7, Zanella, A. - et al., Avian Pathol., 6, 1977, No. 1, p. 8). .

A vaccine from a virus adapted to cell cultures was also prepared and tested; embryonic bursa, embryonic kidneys and broblasts and expanded in cultures of SPF egg fibroblasts (Skeeles, J.K. et al., Avian Dis., 23, 1979, No 2, p. 456).

The infectious bursitis virus strain Z-2037 was isolated on cell cultures of embryonic fibroblast smokers in 1977 from lymphatic ajbarate of infected Shaver line chickens at Turcianske Teplice. Using 5-iodo-2-de-zenoxyuridine, the isolate was confirmed to belong to the ribovirus family. The virus is not enveloped because the chloroform tests for the presence of essential lipids are negative. It is thermostable, withstands a temperature of 56 ° C for 30 minutes without reducing the infection titer for chicken fibroblasts. It is cytopathogenic, replicates and propagates on smoking embryonic cells (KEB) and embryonic kidney cells (KEOB) to produce a tiny-spherical cytopathic effect characteristic of infectious bursitis viruses. The isolated virus strain was compared to the reference strain Stuttgart (prof. Woernle). In the nuclei of infected cells, they do not form inclusion bodies. Cross-neutralization assays and agar-gel precipitation assays with antisera against the Stuttgart reference strain and against the Z-2037 isolate are positive, suggesting their antigenic identity.

The infectious titer of isolate in the third passage on KEB was relatively low: 10 ³ TKID 50. ml ^-1 . Chicken embryos without maternal antibodies infected with the first 2 to 3 passages of the virus did not show macroscopic changes and their mortality did not exceed 20%. Chickens without maternal antibodies infected with infectious bursitis virus (0.1 ml of virus adjusted to 1000 TKID50) by the intraconjunctival and oral route at 5 days of age did not become clinically ill. They had morphologically enlarged Fabricius exchanges with specific histological changes indicative of bursitis with reticulocyte hyperplasia in part of the bursal follicles. Also in the liver of infected chickens, lymphocytic infiltration of perivascular spaces and sporadic lesions with vacuolar hepatocyte degeneration, respectively, were observed. hemorrhage.

The isolate passed 10 blind passages at four-day intervals on smoking germs without maternal antibodies against infectious bursal disease (ROSS line) in the usual virological manner. The presence of infectious bursitis virus in passaged suspensions was monitored on cell cultures. The virus titer in the mixed suspensions of the smoking germ organs reached 10 ² to 10 ³ TKID _S0 . ml ^-1 .

In addition, the Z-2037 virus strain was serially passaged on primary cells of embryonic smoker cells, according to virological methods. The presence of the virus was demonstrated by cytopathic changes. The virus content was determined by reum calculation according to Reed and Muench. After 25 to 27 passages on KEB, the virus titer reached at least 5 x 10 ⁶ TKID ₅₀ . ml ¹ .

The procedure for attenuating virulence of infectious bursitis virus was controlled by intraconjunctival and oral administration of the virus at a dose of approximately 5 x 10 ⁴ TKID ₅₀ five-day-old chickens without antibodies against infectious bursitis virus. The infected animals were monitored clinically, pathohistologically, in particular by examining the Fabricius bourse and liver, and by experimental vaccination of such inoculated chickens against Newcastle disease 7 days after the application of the infectious bursitis virus. In the serological examination of chickens for pseudomorphic antibodies, there could be no significant difference between the test and control groups. The attenuated Z-2037 virus was tested for pathogenicity for various age groups of poultry without antibodies to infectious bursalitis, namely chicken embryos, day-old chicks, 5 and 10-day-old chickens after 10 passages in chicken embryos and 27 passages in embryonic cell culture. - day broilers and 25 weeks laying hens. Clinical or pathomorphological changes were not detected in any of the experimental groups, therefore the attenuated strain was considered harmless for all age groups of chickens. The experimental groups responded 7 to 14 days after application of the Z-2037 virus with the production of Specific Antibodies whose specificity was verified by the reference infectious bursitis virus, Stuttgart strain.

Virus Z-2037, attenuated and adapted to cultured smoking embryonic cells as described above, is deposited in Cs. Collection of Microorganisms, Collection of Zoopathogenic Microorganisms, Veterinary Research Institute, Brno, under number CAPM V-260.

SUMMARY OF THE INVENTION The vaccine against infectious bursitis is prepared from strain Z-2037 (CAPM V-260), isolated in Slovakia and attenuated and adapted through serial passages for embryonic smoking cell cultures. The vaccine virus is propagated and titrated on primary or secondary cultures of smoking embryonic cells prepared from conventional eggs. The attenuated and adapted vaccine virus of infectious bursitis Z-2037 reaches a titer of 10 ⁷ TKID _SO . ml ¹ and is harmless to all ages of chickens.

Example 1

Maintaining the production batch

The production strain Z-2037 is kept in a lyophilized state in ampoules sealed under vacuum even at +2 to +8 ° C. Cell culture passages are performed once a year: Two vials of virus containing 0.3 ml of lyophilized material are opened and dissolved in 4 ml of culture medium (Hanks' lactalbumin hydrolyzate solution according to PNL 01-63-74). The diluted virus is infected with a culture of smoking embryonic cells in 2-4 ml Roux flasks of 1200 ml. The inoculated Roux flasks were incubated at 37 ° C. After detecting specific cell degeneration, after 24 to 96 hours, the culture is frozen at -20 ° C, thawed, filtered through sterile gauze and titrated in tube cultures. After bacterial sterility control and virus assay, the stock stock is ready for lyophilization with the addition of 40% protection medium (95% inactivated horse serum and 5% glucose) or stored frozen at -20 ° C and used to inoculate cell cultures. cultures in vaccine production.

Example 2

Vaccine preparation on primary cell cultures. , j

To produce the vaccine, stocks of chicken embryonic cells in Roux flasks are inoculated with stock stock at 1 ml of viral suspension per 1200 ml Roux flask containing 100 ml culture medium. The inoculated Roux flasks were incubated at 37 ° C. After detecting specific cell degeneration, after 24 to 96 hours, cultures are frozen at -20 ° C, thawed, filtered through sterile gauze and titrated in tube cell cultures. After checking the bacterial sterility and determining the virus content, the virus suspension is filled into vials as a liquid vaccine, or filled into ampoules with the addition of 20% protective lyophilic medium (3 sticks of 20% Lactin solution and 1 part 40% glucose) and lyophilized. . The liquid vaccine is stored frozen, lyophilized at +2 to +8 ° C.

Example 3 ', _f

Vaccine preparation on secondary cell cultures

Well grown cultures of smoking embryonic cells in Roux flasks of 1200 ml volume are released with a 37 ° C pre-warmed solution of versintrypsin. Suspension of released saprenesia cells. into culture medium and is seeded in Roux flasks as the primary cell culture. Secondary cultures are inoculated and incubated as primary cultures, and the virus suspension is processed as described in Example 2.

Example 4

Control of the vaccine by titration

Cultures of smoking embryonic cells for virus titration are plated in 14/15 X 90 mm tubes at 1.0 mL culture medium. Cell cultures are infected with 0.1 ml. vaccine virus, diluted 10-fold from 10 ^-3 to 10 ^-8 , in succession, each dilution into at least 4 tubes. The inoculated tube cultures are incubated for 96 to 120 hours at 37 ° C and the virus titer is read microscopically for cytopathic effect. virus on cells. The TKID _{50 of the} virus is determined by the accumulation calculation method of Reed and Muench. Titer. the virus in the vaccine must be at least 10 ⁵ 'TKID ₅₀ .mL ¹ .

Príidad 5

Determination of neutralizing antibodies

Test cultures of smoking embryonic cells are prepared as. Example 4. Threat _'r suspension containing 100 to 1000 Tkid _{5 ()} of the virus' of 0.1. ml are mixed with an equal volume of the material to be examined (serum), diluted ten times successively from 10 to ¹ to ⁵ , and allowed to stand at room temperature for 60 minutes. The tube cultures are inoculated with 0.2 ml of the viral suspension / serum mixture, each serum dilution into at least 4 tubes. The tube cultures are incubated at 37 ° C for 96 to 120 hours. The neutralizing antibody titer is read microscopically for lack of cytopathic effect of the virus on the cells. The 50% neutralization dose (ND ₅₀ ) is determined by an accumulation calculation according to Reed and Muench.

Claims (3)

3 tier 107 TKIDS0. ml 1 and is harmless to all age categories of chickens. Example 1 Maintaining a Production Strain The production strain Z-2037 is maintained in a lyophilized state in ampoules sealed under vacuum at +2 to +8 ° C. Once a year cell culture passage: Two vials of virusus each containing 0.3 ml of lyophilized material are opened and dissolved in 4 ml of culture medium (Hanks' solution with lactalbumin hydrolysis according to PNL 01-63-74). Virus diluted in this way infects cultures of embryonic cells in 2 to 4 Roux bottles of 1200 ml. Inoculated Roux flasks are incubated at 37 ° C. After detecting specific cell degeneration at 24-96 hours, the culture is frozen at -20 ° C, thawed, filtered through sterile gauze and tube culture titrated. After bacterial sterility control and virus determination, a single stock prepared for lyophilization by addition of 40% protection medium (95% inactivated horse serum and 5% glucose) or frozen at -20 ° C and used to inoculate cellular cultures in the production of vaccine. Example 2 Preparation of a vaccine on primary cell cultures. In the manufacture of a vaccine, stock strains of embryo cell cultures in Roux flasks were inoculated with 1 ml of viral suspension per Roux bottle with a volume of 1200 ml and 100 ml of culture medium. Inoculated Roux flasks were incubated at 37 ° C. After specific cell degeneration was detected, 24-96 hours, the cultures were frozen at -20 ° C, pulverized, filtered through sterile gauze and titrated in assay cell cultures. After bacterial sterility control and virus determination, the viral suspension is filled into vials as a liquid vaccine or filled into ampoules and lyophilized with the addition of 20% flowholyophilic medium (3 parts 20% Lactin and 1 part 40% glucose). The liquid vaccine is stored frozen, lyophilized at +2 to +8 ° C. 209379 Example 3 ', f Preparation of a Vaccine on Secondary Cell Cultures Good growth cultures of embryonic cell smokers in Roux bottles of 1200 ml were released with a pre-warmed solution of 37 ° C. A suspension of released cells with saprenesis into the culture medium and is based on the Roux fliax primary cell culture. Secondary cultures are inoculated and incubated as primary cultures, and the virus suspension is treated as described in Example 2., Example 4 Vaccine control by titration Embryonic cell smoker cultures for virus titration are loaded into 14/15 X 90 mm tubes in an amount of 1.0 ml culture medium. Cell cultures are infected with 0.1 ml. vaccine virus, diluted ten-fold from 10 -3 to 10 -8, each dilution into at least 4 tubes. The inoculated tube cultures are incubated for 96-120 hours at 37 ° C and the virus titer is counted microscopically according to the cytopathic effect. virus to cells. Virus TKID50 is determined by accumulation analysis according to Reed and Muench. Titer. virus in the vaccine must be at least 105 K TKID 50 .mU1. Example 5 Determination of Neutralizing Antibody Tubal cultures of embryonic cell smokers are prepared as. in Example 4. Virus Suspension containing 100 to 1000 TKID5 () virus in 0.1. ml is mixed with the same volume of test material (serum) diluted ten-fold from 10 -1 to 10 -5, and allowed to stand for 60 minutes at room temperature. Tube cultures are inoculated with 0.2 ml of a serum suspension of virus suspension, each serum dilution to at least 4 tubes. Tube cultures are incubated for 96-120 hours at 37 ° C. The neutralizing antibody titer is subtracted microscopically due to the lack of cytopathic effect of the virus on the cells. The 50% neutralization dose (ND50) is determined by the accumulation calculation according to Reed and Muench. OBJECT What is claimed is: 1. A vaccine against infectious bursitis of chickens, in a liquid or lyophilized state, characterized in that it contains 105 to 107TKID50 virus infectious bursal disease CAPM V-260 in 1 ml. 2. A method of preparing a vaccine as claimed in claim 1, wherein the attenuated strain of infectious bursal virus CAPM V-260 is propagated in primary cultures of embryonic smokers in cells prepared from conventional eggs at a temperature of 36-39 ° C for 24-96. hours. 3. A method of preparing a vaccine according to claim 1, wherein the attenuated strain of infectious bursal virus CAPM V-260 propagates subcultures of embryonic cells prepared from conventional eggs at a temperature of 36-39 ° C for 24-96 hours.