Strain univap-dept" to get the vaccine reoviruses tenosynovitis chickens

 

(57) Abstract:

The invention is intended for the production of vaccines against reoviruses tenosynovitis chickens and may be applicable for the manufacture of diagnostic products. The strain is deposited in the collection of the all-Russian state research Institute for control, standardisation and certification of veterinary preparations (VGNKI) under the number "UNIVAP-DEPT". Vaccinal strain "UNIVAP-DEPT" get method of intermittent passages on developing chicken embryos and in the cultures of chicken fibroblasts. Virions are spherical, cubic symmetry, with the size of 65-70 nm. Nucleocapsid contains RNA. In cell culture induces the formation of plaques in size 1-2 mm Reproducerea in cell cultures of chicken embryo. Causes an acute form of infection with a pronounced cytopathic effect in 2-4 days and accumulates in the credits 6,0-7,0 lg TCD50. The attenuated strain, causes the formation of plaques on HAO chick embryos. Does not induce disease in chickens and laboratory animals when parenteral injection in doses 100 times greater than vaccination. Has no hemagglutinine and hemadsorbing activity. Sustainable is the relationship with epizootic strains of reovirus, cause disease in poultry farms, strain "1133". The strain has a high immunogenic activity regardless of the method use in combination with the ability to a high level of reproduction in cell culture, lack of reactogenicity and maintaining high thermal stability. 5 table.

The invention relates to veterinary Virology and biotechnology, and more specifically to the production of vaccines against reoviruses tenosynovitis chickens, and can be applicable for the manufacture of diagnostic products.

An important means of struggle against reoviruses tenosynovitis chickens, disease, causing significant damage to industrial poultry, vaccines is. Technology VirusWall provides for the lyophilization of the drug, therefore, the strains must have expressed resistance to temperature influences.

As a prototype of the selected Strain "S 1133" [1].

The strain "S 1133" isolated from the flexor tendons 7-week-old broiler acute during tenosynovitis. On the chorioallantoic membrane of SPF chicken embryos 9-10-days-old strain causes the formation of a grayish plaques death for Elijah finger flexors. The strain forms inside cytoplasmic viral particles with double capsid with a diameter of about 70 nm. From infected chickens the virus is transmitted with hatching eggs for 8 to 12 days after infection.

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

In foreign practice specified strain used for vaccine production against tenosynovitis chickens [3] . It is cultivated in cell cultures SPF chicken embryos. However, obtaining SPF embryos require large expenditures. The strain has a residual reactogenicity. High immunogenic activity of the prototype manifests itself in its subcutaneous (individual) introduction. The use made of this vaccine strain in other ways: drinking water, on the conjunctiva of the eye or mucous membranes of the nose, provides minimal protection from the control of infection.

The purpose of the invention to provide a vaccine strain of reovirus tenosynovitis chickens with enhanced immunogenic activity, regardless of method of application, combined with the ability to a high level of reproduction in the culture of cells is attained strain "UNIVAP-DEPT". The strain is deposited in the collection of the all-Russian state research Institute for control, standardisation and certification of veterinary preparations under UNIVAP-DEPT".

Vaccinal strain "UNIVAP-DEPT" get method of intermittent passages on developing chicken embryos and in the cultures of chicken fibroblasts.

The attenuated strain causes the formation of plaques on the chorioallantoic membrane of chick embryos, does not induce disease in chickens and laboratory animals when parenteral injection in doses 100 times greater than vaccination. The strain does not possess hemagglutinine and hemadsorbing activity has antigenic relationship with epizootic strains of reovirus causing disease in birds in poultry farms and strain "S 1133"

THE CHARACTERISTIC STRAIN "UNIVAP-DEPT"

MORPHOLOGICAL CHARACTERISTICS

Virions are spherical, cubic symmetry, with the size of 65-70 nm, nucleocapsid contains RNA.

PATHOGENICITY FOR CHICKEN EMBRYOS AND CHICKS

In experiments to determine the pathogenicity of the proposed strain used 7-11-day chicken embryos and Chicks 1-10 days of age. Infection conducted the m3and chickens were infected subcutaneously, enterline and in the ball of the foot at a dose of 10000 EID50/0.2 cm3. The embryos were incubated for 5 days at a temperature of 37oC. For chickens watched 40 days.

Table 2 presents the results of a study of the pathogenicity of the strain "UNIVAP-DEPT" for chick embryos (embryos death, %), table 3 - the same for chickens (ill/Palo %).

The data in table 2 indicate that inoculation in yolk SAC strain "UNIVAP-DEPT" cause the death of chick embryos more 7-20%.

When the temperature of the 32oC and 34oC pathogenicity strain "UNIVAP-DEPT" for chick embryos falls and causes the death of 10 and 55% of infected embryos, respectively.

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

REPRODUCTION IN CELL CULTURE

Reproducerea in cell cultures of chicken embryo, causing acute infection with a pronounced cytopathic effect in 2-4 days and accumulates in the credits 6,0-7,0 lg TCD50. In cell cultures, KF (primary trypsinization cell culture of chicken embryo fibroblasts), KP (cell culture of chicken embryo liver) and transplantable lines VNCI cultivation at 37oC.

In culture KF agar under coating strain "UNIVAP-DEPT" forms small plaques up to 2 mm in diameter, colorless with smooth edges with detritus cells inside.

REPRODUCTION IN CHICKEN EMBRYOS

While infecting 9-11-day-old chick embryos in allantoin cavity and 7-8-day-old in the yolk SAC, the titer of the virus "UNIVAP-DEPT" on the 5th day, respectively 6.25; 7.0 lg EID50when the temperature of the cultivation of the 37oC. Causes the formation of single plaques on allantoine shell-side air chamber of the embryo, convex, irregular, of different size.

THE RESISTANCE

Strain UNIVAP-DEPT" thermostable. The constant of inactivation kinetics (T/R - reduction in infectious titer when applied to the cultural vaccinated liquid at 56 and 60oC for 60 min) is equal to 1.

SENSITIVITY TO ETHER AND CHLOROFORM

Strain UNIVAP-DEPT" not sensitive to the inactivating action of ether and chloroform (contact with 20% reagent for 60 min). Constant of inactivation is 1.

SPECIFICITY

Methods RNA, ELISA and IFA using typespecific serum to the strain "S 1133" reovirus, strain "UNIVAP DE is th age through 21 days after a single oral immunization with strain "UNIVAP-DEPT" neutralizing antibody in the serum is contained in a concentration of 2.0 lg JN. In chickens on day 21 after three immunizations strain "UNIVAP-DEPT" neutralizing antibody in the serum is contained in a concentration of 2.7 lg IN or in the credits of 7.3 log2.

IMMUNOGENICITY

In chickens twice enterline immunized 7-10 and 35-40 days of age, strain "UNIVAP-DEPT" develops resistance to infection control in the ball of the foot pathogenic strain SP-73" reovirus. Immunogenicity is 92-95%. Maximum seroconversion occurs on day 21. The antibody titer in the serum is 1:400 - 1:800 in ELISA.

STABILITY

Strain UNIVAP - AFFAIRS" stable, its properties remain unchanged for 30 passages in cell culture KF and chicken embryos.

Antigenic relationship

Antigenic relatedness of strain "UNIVAP-DEPT investigated against epizootic strains of "SP-73", "M-4" and "ROS-92" and the vaccine "S 1133" in cross-neutralization (table 4).

Experimental data indicate full antigenic relationship of epizootic strains reovirus and prototype "S1133".

EXAMPLE 1.

Check immunogenic activity conducted at 7-10-day-old chickens that do not have antibodies to reovirus tenosynovectomy level of specific antibodies in the serum of chickens through 21 days after inoculation enzyme-linked immunosorbent assay (ELISA) or the neutralization in the cultures of chicken fibroblasts. Validation data are given in Table 5.

Results: the titers of at least 1: 400 in ELISA or 2.0 lg in the neutralization indicate the formation of a busy immunity in birds and high immunogenic activity of the strain of the virus.

EXAMPLE 2.

The experiment was carried out on Chicks 4-day old meat breed. Chickens were infected in foot pad cultural vaccinated material at a dose of 10000 TCD500.2 ml.

Attenuated strain "UNIVAP-DEPT" virus tenosynovitis hens maintained in culture cells of chicken embryos. Primary trypsinization culture cells were prepared from skin and muscle tissue of chicken embryos 9-11-day-old age, using as the growth environment environment the Needle and medium 199 supplemented with 5-10% serum fetal cow, penicillin 100 U/ml and streptomycin 100 µg/ml.

These experiments showed that the incubation period for infection in the foot pad was equal to 3-4 days. When this was discovered the following clinical signs: depression, limited movement, weak damage to the joint with a slight limp.

When pathomorphological studies of finger flexors in the area of the metatarsus on the 4th day after the litraly polymorphic cells, foci of necrobiosis in the cartilage.

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

EXAMPLE 3

The experiment was carried out on Chicks 4-day old meat breed. Chickens infected enterline cultural vaccinated material at a dose of 10000 TCD500.5 ml.

Attenuated strain "UNIVAP-DEPT" virus tenosynovitis hens maintained in culture cells of chicken embryos. Primary trypsinization culture cells were prepared from skin and muscle tissue of chicken embryos 9-11-day-old age, using as the growth environment environment the Needle and medium 199 supplemented with 5-10% serum fetal cow, penicillin 100 U/ml and streptomycin 100 µg/ml.

These experiments showed that enteric infection clinical signs of disease were not found.

When pathomorphological studies of finger flexors in the area of the metatarsus on the 4th day after infection changes were noted.

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

The inventive strain "UNIVAP-DEPT may find wide application in commercial poultry production as the basis of the MMA allows to obtain a high level of immunity in the enteral administration of the drug in contrast to commonly used vaccines on the basis of known strains, introduced exclusively intramuscularly and subcutaneously.

Bibliography

1. L. van der Heide, J Geissler, E. S. 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, A. J. Eleccher and I. Kleven. Vaccination of Broiler Breeders with a Tenosynovitis Vaccina Virus.- Poultry Science. 1979, n.58, p. 1490-1497.

The strain of virus tenosynovitis chickens "UNIVAP-DEPT" to get the vaccine reoviruses tenosynovitis chickens.

 

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FIELD: biotechnology, veterinary medicine.

SUBSTANCE: invention relates to the development of biological preparation for prophylaxis and treatment of colibacillosis (escherichiosis) and for control of carriage of escherichious infections pathogens in animals and poultries also. Also, invention can be used in producing curative fodders and ecologically pure human foodstuffs. Biopreparation for prophylaxis and treatment of escherichiosis in animals and poultries comprises strains of bacteriophages Phagum Escherichia coli Ec022-DEP and/or Phagum Escherichia coli Ec021-DEP, and/or Phagum Escherichia coli Ex0782-DEP, and/or Phagum Escherichia coli Ec0781-DEP, and/or Phagum Escherichia coli EPZ-1-DEP, and/or Phagum Escherichia coli EPZ-2-DEP, and/or Phagum Escherichia coli EG-5-DEP, and/or Phagum Escherichia coli BC-1-DEP, and/or Phagum Escherichia coli M78-DEP, and/or Phagum Escherichia coli Sheksna 2k-DEP taken in the effective amount. The biopreparation comprises also antiseptic, for example, quinosol and a stabilizing agent. Protein (for example, soybean protein), vegetable meal, organic polymer, milk, serum, albumin can be used as a stabilizing agent. Among organic polymers can be used: dextran, polyglucin, starch, polyvinylpyrrolidone. The biopreparation can be dried by lyophilization, granulated and placed in polymeric matrix. The biopreparation has no toxic properties on animals, it shows good hygroscopicity and can be good dispersed in water. The biopreparation can be used in liquid and dry prescription formulations and in different methods of its administrations: both by subcutaneous, intraperitoneal, intramuscular injections and as an aerosol, by administration of phage particles into lung compartments including applying as curative fodder and supplement to fodder, and by applying on surface of cutaneous integuments. Invention provides enhancing the effectiveness of treatment of animals and poultries with gastroenteric infections due to reducing treatment period, expanding spectrum of lytic effect of the biopreparation, resistance to effect of digestive tract enzymes and convenience in using.

EFFECT: valuable veterinary properties of biopreparation.

9 cl, 5 tbl, 7 ex

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