RU2158304C2 - Strain "vnivip-dep" for preparing vaccine against reoviral tendinous synovitis in chickens - Google Patents

Abstract

FIELD: veterinary science, virology. SUBSTANCE: invention relates to production of vaccine preparations against reoviral tendinous synovitis in chickens and can be used for diagnostic preparations making. Strain is deposited in VGNKI collection at number "VNIVIP- -DEP". Vaccine strain "VNIVIP-DEP" is prepared by intermitted passages in developing chicken embryos and in cultured chicken fibroblasts. Virions have spheric form, cubic symmetry, size is 65-70 nm. Nucleocapsid has RNA. In cultured cells the strain induces formation of plaques (size is 1-2 mm). Strain is reproduced in cultured chicken embryos, it induces acute infection form with expressed cytopathic effect in 2-4 days and accumulated at titers 6.0-7.0 lg TCA₅₀. Strain is attenuated, it induces formation of plaques in XAO chicken embryos. Strain does not induce disease in chickens and laboratory animals at pareneteral administration in doses exceeding vaccinial dose by 100-fold. Strain does not show hemagglutinating and heme adsorbing activity. Strain shows resistance to ether and chloroform, it shows stability at 60 C for 1 h. Strain is closely related to antigens of epizootic strains of reovirus inducing diseases in poultry farmings and to the strain "1133". Strain shows enhanced immunogenic activity being independently of method of its use in combination with ability to high level of reproduction in cultured cells, absence of reactivity and retention of increased thermostability. EFFECT: strain indicated above, valuable properties. 5 tbl, 3 ex

Description

 The invention relates to veterinary virology and biotechnology, and more specifically to the production of vaccines against rheovirus tenosynovitis chickens, and may be applicable for the manufacture of diagnostic drugs.

 An important means of combating chickens reovirus tenosynovitis, a disease that causes significant damage to industrial poultry farming, is vaccine prophylaxis. The technology for the production of virus vaccines provides for lyophilization of the drug, so the strains should have a pronounced resistance to temperature effects.

 Strain "S 1133" [1] was selected as a prototype.

 Strain S 1133 was isolated from the tendon of the finger flexor of a 7-week-old broiler in acute tenosynovitis. On the chorioallantoic membrane of SPF - chicken embryos 9-10 days old, the strain causes the formation of grayish plaques, the death of infected chicken embryos and the clinical manifestation of the disease in chickens with morphological changes in the tendons of the finger flexors. The strain forms inside the cytoplasmic viral particles with a double capsid with a diameter of about 70 nm. From infected chickens, the virus is transmitted with the hatching egg within 8-12 days after infection.

 The strain causes a cytopathic effect in the culture of chicken fibroblasts [2]. Physico-chemical, biological and morphological features of the prototype are presented in the table. 1.

 In foreign practice, this strain is used for the production of vaccines against tenosynovitis chickens [3]. It is cultured in cell culture of chicken SPF embryos. However, obtaining SPF-embryos is expensive. The strain has residual reactogenicity. High immunogenic activity of the prototype is manifested during its subcutaneous (individual) administration. The use of a vaccine made from this strain in other ways: with drinking water, on the conjunctiva of the eye or nasal mucosa, - provides low protection against infection control.

 The aim of the invention is to obtain a vaccine strain of chickens tenosynovitis reovirus with increased immunogenic activity, regardless of the method of use, in combination with the ability to a high level of reproduction in cell culture, the absence of reactogenicity and the preservation of increased heat resistance.

 The goal is achieved by the strain "VNIVIP-DEP". The strain is deposited in the collection of the All-Russian State Research Institute for the Control of Standardization and Certification of Veterinary Medicines under the number "VNIVIP-DEP".

 The vaccine strain "VNIVIP-DEP" is obtained by the method of alternating passages in developing chicken embryos and in the culture of chicken fibroblasts.

The strain is attenuated, causes plaque formation on the chorioallantoic membrane of chicken embryos, does not induce disease in chickens and laboratory animals when administered parenterally at doses 100 times higher than the vaccine. The strain does not have hemagglutinating and hemadsorbing activity, has antigenic affinity with epizootic strains of reovirus that cause disease of birds in poultry farms and strain "S 1133"
CHARACTERISTIC OF THE STRAIN "VNIVIP-DEP"
MORPHOLOGICAL CHARACTERISTIC
Virions are spherical, cubic symmetry, with a size of 65-70 nm, the nucleocapsid contains RNA.

Pathogenicity for Chicken Embryos and Chickens
In experiments to determine the pathogenicity of the claimed strain used 7-11-day-old chicken embryos and chickens 1-10-day old. Infection was carried out in the yolk sac of 7-8 day old and in the allantoic cavity of 9-11 day old embryos in doses of 100 and 1000 EID ₅₀ in 0.2 cm ³ , and the chickens were infected subcutaneously, enteral and in the foot pad at a dose of 10000 EID ₅₀ / 0.2 cm ³ . Embryos were incubated for 5 days at a temperature of 37 ^o C. Chickens were observed for 40 days.

 Table 2 presents the results of a study of the pathogenicity of the strain "VNIVIP-DEP" for chicken embryos (death of embryos,%), in table 3 - the same for chickens (sick / sting,%).

 The data in table 2 indicate that when inoculated in the yolk sac, the strain "VNIVIP-DEPT" causes the death of chicken embryos by 7-20% more.

At a temperature of 32 ^o C and 34 ^o C the pathogenicity of the strain "VNIVIP-DEP" for chicken embryos decreases and causes the death of 10 and 55% of infected embryos, respectively.

 The data in table 3 indicate that the strain "VNIVIP-DEPT" does not cause the death of chickens.

REPRODUCTION IN CELL CULTURES
It is reproduced in a culture of chicken embryo cells, causing an acute form of infection with a pronounced cytopathic effect after 2-4 days and accumulates in the titers of 6.0-7.0 lg TCD ₅₀ . In cell cultures KF (primary trypsinized culture of chicken embryo fibroblast cells), KP (culture of liver cells of the chicken embryo) and transplanted lines BHK-21, BGM, Vero multiplies. Infectious virus titer in the culture fluid reaches 5-7 lg TDC ₅₀ at 4-5 days of cultivation at 37 ^o C.

 In CF culture under an agar coating, the VNIVIP-DEP strain forms small plaques up to 2 mm in diameter, colorless with smooth edges with cell detritus inside.

REPRODUCTION IN CHICKEN EMBRYOS
When 9-11-day-old chicken embryos are infected in the allantoic cavity and 7-8-day-old in the yolk sac, the titer of the VNIVIP-DEP virus on the 5th day is 6.25, respectively; 7.0 lg EID ₅₀ at a cultivation temperature of 37 ^o C. Causes the formation of single plaques on the allantoic membrane from the side of the air chamber of the embryo, convex irregular shape, different in size.

THERMAL RESISTANCE
The strain "VNIVIP-DEP" is thermostable. The inactivation kinetics constant (T / P - decrease in the infectious titer when heating the culture virus-containing fluid at 56 and 60 ^o C for 60 min) is 1.

SENSITIVITY TO ETHER AND CHLOROFORM
The strain "VNIVIP-DEP" is not sensitive to the inactivating effect of ether and chloroform (contact with 20% of the reagents for 60 minutes). The inactivation constant is 1.

SPECIFICITY
By the methods of RNGA, ELISA and MFA using type-specific serum to the strain "S 1133" of the reovirus, the strain "VNIVIP-DEP" was identified as chicken tenosynovitis reovirus.

ANTIGENIC ACTIVITY
In chickens 35-40 days old, 21 days after a single enteral immunization with the strain "VNIVIP-DEP" virus-neutralizing antibodies in blood serum are contained in a concentration of 2.0 lg IN. In chickens, on day 21 after three times immunization with the strain "VNIVIP-DEP" virus neutralizing antibodies in the blood serum are contained in a concentration of 2.7 lg IN or in titers of 7.3 log ₂ .

IMMUNOGENICITY
Chickens that were twice enterally immunized at 7–10 and 35–40 days old with the VNIVIP-DEP strain develop resistance to control infection in the paw pad with the pathogenic strain SP-73 reovirus. Immunogenicity is 92-95%. Maximum seroconversion occurs on 21 days. The titer of antibodies in blood serum is 1: 400 - 1: 800 in ELISA.

STABILITY
The strain "VNIVIP - DEL" is stable, its properties remain unchanged for 30 passages in the culture of cells of CF and on chicken embryos.

Antigenic relationship
Antigenic affinity of the strain "VNIVIP-DEPT" was investigated in relation to the epizootic strains "SP-73", "M-4" and "Ros-92" and vaccine "S 1133" in the cross-reaction of neutralization (table 4).

 Experimental data indicate complete antigenic affinity of epizootic strains of reovirus and prototype "S1133".

 EXAMPLE 1

The immunogenic activity was tested on 7-10-day-old chickens without antibodies to chicken tenosynovitis reovirus by oral or subcutaneous administration of the VNIVIP-DEP vaccine strain at a dose of 1000 TCD ₅₀ and determining the level of specific antibodies in the blood serum of chickens 21 days after immunization enzyme immunoassay (ELISA) or neutralization reactions in chicken fibroblast culture. The verification data are shown in Table 5.

 The results: titers of at least 1: 400 in ELISA or 2.0 lg in the neutralization reaction, indicate the formation of intense immunity in birds and the high immunogenic activity of the virus strain.

 EXAMPLE 2

The experiment was carried out on chickens of 4-day-old meat breed. The chickens were infected in the paw pad with virus-containing culture material at a dose of 10,000 TCD ₅₀ in 0.2 ml each.

 The attenuated strain "VNIVIP-DEP" of the chicken tenosynovitis virus was maintained in chicken embryo cell culture. A primary trypsinized cell culture was prepared from skin and muscle tissue of chicken embryos of 9-11 days old using Eagle's medium and medium 199 as a growth medium with the addition of 5-10% serum from the fetal cow, penicillin 100 IU / ml and streptomycin 100 μg / ml

 The experimental data showed that the incubation period during infection in the paw pad was 3-4 days. In this case, the following clinical signs were found: depression, limited movement, weak joint damage with slight limp.

 In pathomorphological studies of digital finger flexors in the metatarsal area on the 4th day after infection, swelling and thickening of the dermis, muscle bundles, tendon sheath walls, intensive infiltration by polymorphic cells, and foci of necrobiosis in the cartilage were noted.

 Maximum seroconversion occurred on day 21. The titer of antibodies in serum was 1: 400 in ELISA.

EXAMPLE 3
The experiment was carried out on chickens of 4-day-old meat breed. The chickens were infected with an enteric culture virus-containing material at a dose of 10,000 TCD ₅₀ , 0.5 ml each.

 The attenuated strain "VNIVIP-DEP" of the chicken tenosynovitis virus was maintained in chicken embryo cell culture. A primary trypsinized cell culture was prepared from skin and muscle tissue of chicken embryos of 9-11 days old using Eagle's medium and medium 199 as a growth medium with the addition of 5-10% serum from the fetal cow, penicillin 100 IU / ml and streptomycin 100 μg / ml

 The experimental data showed that with enteral infection, no clinical signs of the disease were found.

 In pathomorphological studies of digital finger flexors in the metatarsus on the 4th day after infection, no changes were noted.

 Maximum seroconversion occurred on day 21. The titer of antibodies in serum was 1: 400 in ELISA.

 The inventive strain "VNIVIP-DEP" can be widely used in industrial poultry as the main component of the vaccine against reovirus tenosynovitis chickens. At the same time, the revealed properties of the claimed strain make it possible to obtain a high level of immunity during enteral administration of the drug, in contrast to the widely used vaccine preparations based on known strains administered exclusively intramuscularly and subcutaneously.

References
1. L. van der Heide, J Geissler, ES Bryant. Infektious tenosynovitis: Serologic and Histopathologic response after experimental infection with a connecticut isolate.-Avian Dis., 1974, vol. 18, n.3, p. 289-296.

 2. L. van der Heide, M. Kalbac. Infectious tenosynovitis (Viral arthritis): Characterization of a connecticut viral isolant as a reovirus and evidence of viral egg transmission by reovirus-infected broiler breeders.- Avian Dis., 1975, vol. 19, n.4, p. 683-688.

 3. C. S. Eidson, R. K. Page, O.J. Eleccher and I.I. Kleven. Vaccination of Broiler Breeders with a Tenosynovitis Virus Vaccina.- Poultry Science. 1979, n. 58, p. 1490-1497.

Claims (1)

 The strain of hens tenosynovitis virus "VNIVIP-DEP" to obtain a vaccine against chickens reovirus tenosinovitis.

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