RU2279892C1 - Cultural vaccine against fowl chlamydiosis (psittacosis) - Google Patents

Abstract

FIELD: veterinary virology.

SUBSTANCE: claimed vaccine contains strain Chlamydia psittary (var.avis) cultured on monolayer of primary cell culture such as avian embrio fibroblast with titer not less than 10^-7.5 TCD_50/ml as antigen; phenol as inactivator; and polyvinylpyrrolidone as adjuvant in the next ratio (mass %): Chlamydia psittary (var.avis) with titer not less than 10^-7.5 TCD_50/ml 99.1-99.3; phenol 0.3-0.5; polyvinylpyrrolidone 0.4-0.6.

EFFECT: harmless vaccine with high specificity and immunogenic properties.

4 ex, 1 tbl

Description

The invention relates to specific prophylaxis and therapy of chlamydia (ornithosis) in birds. The vaccine contains a cytopathogenic strain of Chlamydia psittaci var. avis "J-87" VGNKI No. 23-DEP grown on a monolayer of the primary culture of chicken embryo fibroblast cells (FEC) with a titer of at least 10 ^-7.5 TCD _{50 / ml} . The vaccine also contains an inactivator - phenol and polyvinylpyrrolidone, used for the first time as an adjuvant. The vaccine is specific, highly immunogenic and has a low reactivity. 6 tab. The invention relates to veterinary microbiology and virology, in particular to the production of vaccines used for specific prophylaxis and therapy of farm animals, for example birds, from chlamydia disease.

Known vaccine against enzootic abortion of sheep inactivated, made from chlamydia-infected chicken embryos (Franc et al. Am. J. Vet. Pec., Vol.29, p. 1441, 1968). The disadvantages of this vaccine are its high cost, its manufacture requires a lot of time, a high content of ballast proteins, reducing its immunogenicity and increasing reactogenicity. This vaccine does not contain a specific antigen and therefore is not immunogenic for birds. We selected this vaccine as a prototype. The closest in technical essence is US patent No. 4386065 A 61 K 39/118, 1983. "Vaccine against enzootic abortion of sheep", which includes a mixture of an aqueous suspension, formalin-inactivated chlamydial elementary bodies obtained from Chlamydia SP organisms ranging from 2 · 10 ⁶ to 10 ⁹ ELD _{50 / ml} propagated in cell culture, and an equal amount of an oil adjuvant, which is a mixture of light mineral oil and lanolin.

The disadvantage of this vaccine is that the strain of chlamydia as a producer does not have a cytopathic effect. As a result, to control the accumulation of chlamydia, it is necessary to prepare smears daily, stain them and count infected cells, and when collecting raw materials, it is necessary to separate the infected monolayer from the growth surface using trypsin with versts. To create the desired concentration of chlamydia in the vaccine, the culture suspension is centrifuged. In addition, one stationary monolayer is used as seed for only five pure monolayers or their equivalents. All these operations complicate the manufacturing process and reduce productivity in the manufacture of the vaccine, which makes it more expensive. In addition, the vaccine only prevents enzootic chlamydial abortion in sheep and does not prevent chlamydia in birds.

SUMMARY OF THE INVENTION

The objective of the invention is the creation of a specific, highly immunogenic vaccine for the prevention of chlamydia (ornithosis) of birds, which has low reactogenicity and is obtained in a simpler (compared to the prototype) technological method.

The task is achieved due to the fact that the vaccine culture, inactivated against chlamydia (ornithosis) of birds, as an antigen contains a cytopathogenic strain of Chlamydia psittaci "DZH-87" VGNKI No. 23-DEP, grown on a monolayer of the primary culture of chicken embryo fibroblast cells with a titer of at least 10 ^-7.5 TCD _{50 / ml} , first used as an adjuvant - polyvinylpyrrolidone, phenol in the following percentages:

1. Suspension of FEC cells (fibroblasts of chicken embryos) infected with chlamydia of the Chlamydia psittaci strain "J-87" VGNKI No. 23-DEP with a titer of at least 10 ^-7.5 TCD _{50 / ml} - 99.1-99.3.

2. Phenol - 0.3-0.5.

3. Polyvinylpyrrolidone - 0.4-0.6.

A significant difference between this vaccine and its prototype is that the cytopathogenic strain of Chlamydia psittaci var. Avis "ДЖ-87" VGNKI №23-ДЭП, deposited in the collection of VGNKI veterinary products. Its cultivation is carried out on a monolayer of the primary culture of FEC. The strain Chlamydia psittaci "DZH-87" VGNK No. 23-DEP was isolated in 1987 from the liver and spleen of a turkey poultry farm Mrzytayskaya, a patient of ornithosis, in the Jambyl region. Isolation was carried out on developing 6-7-day-old chicken embryos by infection in the yolk sac. Then the strain was adapted to the primary cell culture of FEC. The strain passed passage 124 to the FEC culture under the conditions of a roller; as a result, it acquired the ability to exert a high cytopathogenic effect on the monolayer of the FEC cell culture. Along with this, its immunogenic activity steadily increased and reactogenicity decreased.

The strain Chlamydia psittaci "J-87" VGNKI No. 23-DEP is a collection of VGNKI standardization and certification of veterinary drugs and is stored in the museum of living cultures SKZNIVI.

MORPHOLOGICAL SIGNS. The elementary mature particles are round in shape, with a diameter of 180-200 nm, the initial intermediate forms - up to 500-600 nm are stained in pink-red color according to Romanovsky-Giemsa in dark blue color according to the Stamp method.

CULTURAL PROPERTIES.

Chlamydia is cultivated in the vitelline membranes of 7-8-day-old chicken embryos. Strain Chlamydia psittaci var. Avis "J-87" VGNKI No. 23-DEP adapted to the FEC culture, causes the formation of cytoplasmic inclusions after 48 hours of cultivation, has a cytopathic effect.

ADAPTOGENIC PROPERTIES. Strain Chlamydia psittaci var. Avis "J-87" VGNKI No. 23-DEP, the causative agent of chlamydia (ornithosis) of birds, adapted to 6-7-day-old chicken embryos, and culture of FEC, while maintaining pathogenicity and immunogenicity. The titer of chlamydia grown on chicken embryos is 10 ^6.5 -10 ^7.5 ELD _{50/03 ml} , the titer of chlamydia grown on cell culture FEC, 10 ^-7.0 -10 ^-8.0 TCD _{50 / ml.}

PATHOGENIC PROPERTIES. The strain is pathogenic for turkey poults, guinea pigs and white mice - it causes disease and death in them. Cytoplasmic inclusions and elementary particles of chlamydia with parenteral (for birds), intranasal, intraperitoneal, introperitoneal (for mice) and intraperitoneal infections for guinea pigs are found in smears from the organs (liver, spleen, brain) of dead and sick animals.

ANTIGENIC PROPERTIES. In the formulation of the cross-reaction of complement binding (CSC) with homologous antigens, a high antigenic relationship to the homologous antigen was established in a titer of 1: 80-1: 160. Antigenic affinity for other Chlamydia antigens is four dilutions further.

TAXONOMIC CHARACTERISTIC. According to the decision of the Legal Commission of the International Association of Microbiological Societies (MAMO), from January 1, 1980, the definition of the microorganisms in question as Chlamydia became mandatory. According to "The Shorter Bergey's manual of determinative" (1980), chlamydias are assigned to the kingdom of Procaryotae, a division of 2 prokaryotes indifferent to light, i.e. "cattle bacteria", class 2 Ricketsia. The Ricketsia class includes orders of 1 Ricketsialesu 2 Chamydiales. The family of Chlamydiaceae, the genus Chlamydia, which describes 4 species: Chlamydia psittaci, Chlamydia trachomatis, Chlamydia pecorum, Chlamydia Pneumoniae. All types of chlamydia are heterogeneous.

IMMUNOGENOUS PROPERTIES. 48 turkey poults of 3 weeks of age weighing 250-300 g were taken. 24 turkey was vaccinated with a culture inactivated vaccine made from the Chlamydia psittaci strain (var. Avis) VGNKI No. 23 - DEP, subcutaneously at a dose of 0, 5 ml The remaining 24 turkeys made up the control (unvaccinated) group.

14 days after the introduction of the vaccine prepared from the titrated control strain of Chlamydia psittaci (var. Avis) "J-87" VGNK No. 23 - DEPT with a titer of 10 ^-7.5 TCD _{50 / ml} , 12 experimental and 12 control turkey poultices were infected subcutaneously in dose of 1 ml. Then, 20 days after the vaccine was administered, 12 vaccinated and 12 unvaccinated turkeys were also infected. The results are presented in table. one.

Table 1
Immunogenic properties of the strain Chlamydia psittaci "DZH-87" VGNKI No. 23-DEP Strain Group name Reactogenicity Infection Result Immunity 14 days 20 days 14 days 20 days J-87 Vaccinated Absent 3/9 0/12 Partial Full VGNKI No. 23-DEP Control Absent 12/0 12/0 Absent Absent

Note. In columns 4.5, the numerator indicates the number of turkeys that have died, the denominator is the number of survivors.

Thus, as can be seen from the description and table, the strain Chlamydia psittaci (var. Avis) "J-87" VGNKI No. 23 - DEP is specific for birds and has high immunogenic properties, which allows us to carry out the task.

The second significant difference is that polyvinylpyrrolidone (PVP) is administered as an adjuvant to the vaccine. Polyvinylpyrrolidone - a white powder, highly soluble in water, is used as a drug prolongator. It has an inherent detoxification and anti-inflammatory effect. In our preparation, PVP significantly increases the immunogenicity of the vaccine due to the fact that it binds (adsorbs) antigenic proteins.

Comparative analysis with the prototype allows us to conclude that the claimed composition of the vaccine differs from the known introduction of new components, namely the new strain of Chlamydia psittaci (var. Avis) "DZH-87" VGNKI No. 23-DEP with a titer of at least 10 ^-7.5 TCD _{50 / ml} and polyvinylpyrrolidone as adjuvant, as well as the ratio of components. Thus, the claimed technical solution is new. The introduction of polyvinylpyrrolidone as an adjuvant significantly increases the immunogenicity of the vaccine. Compared with a vaccine containing vaseline oil, hydroxyl or polymer No. 1 as adjuvant. To prove the following experiment was carried out. Four groups of eight pigeons were formed. Inactivated culture vaccine made from the Chlamydia psittaci strain (var. Avis) "J-87" VGNKI No. 23 DEP containing vaccine in the 1st group - paraffin oil, the 2nd group - polyvinylpyrrolidone, the 3rd group was injected into each group. group — hydroxyl; group 4 — polymer No. 1. The vaccine was administered subcutaneously, three times with an interval of 7 days at a dose of 0.2 ml. The immunogenicity of the vaccine was evaluated by the results of CSC (with the blood serum of experimental pigeons taken 20 days after the last injection of the vaccine). As follows from the results of the experiment, polyvinylpyrrolidone as an adjuvant most effectively increases the protective properties of the chlamydia vaccine.

Thus, this composition of the components gives the vaccine new properties, which allows us to conclude that the claimed technical solution meets the criterion of "inventive step".

Example 1. (The best option).

Preparation of a cell culture of FEC and infection with its chlamydia was carried out according to the generally accepted technique (L.P. Dyakonov et al., Cultivation of animal cells and tissues. Part 1, Stavropol, 1986). Then, a suspension of FEC cells infected with chlamydia of the Chlamydia psittaci strain (var. Avis) "JJ-87" VGNKI No. 23 - DEP, was subjected to control seeding for bacterial and fungal contamination. After that, a clean, non-contaminated suspension was taken to determine the titer of TCD _{50 / ml} . The titer was determined according to the generally accepted method and was calculated according to the method of Reed and Mench. In this example, it is 10 ^-7.5 TCD _{50 / ml} .

Then the components were mixed in the following ratios of a suspension of cells of FEC infected with chlamydia - 99.2 wt.%, Phenol - 0.4 wt.%, Polyvinylpyrrolidone - 0.5 wt.%. The resulting mixture was kept for 96 hours at a temperature of + 10 ° C.

Then, selective immunogenicity control was performed. For this, eight pigeons were taken and a vaccine was administered to them according to the following scheme:

1st introduction - subcutaneous 0.2 ml;

2nd introduction - subcutaneously after 7 days, 0.2 ml;

3rd introduction - subcutaneously after 14 days with 0.2 ml.

The control was four pigeons to which the vaccine was not administered. Three weeks after the last injection of the vaccine, the level of accumulation of antibodies in the blood serum of pigeons in the complement binding reaction (CSC) was determined. Result: four crosses at a dilution of 1: 5, four at a dilution of 1:10, two at a dilution of 1:20.

Similarly done for a titer of 7.5; 7.75; 7.0; 6.5; 6.0; 5.5 TCD _{50 / ml} . Thus, experience has shown that the titer of Chlamydia strain 7.5 is optimal and provides a high accumulation of antibodies in the blood serum of pigeons. In this experiment, the dependence of the titer of antibody in the blood serum of pigeons on the titer of the strain Chlamydia psittaci (var. Avis) "J-87" VGNKI No. 23-DEP is clearly visible. This test was taken as a control of the immunogenicity of the vaccine in its manufacture.

Example 2. The preparation of the cell culture FEC, infection with its chlamydia, growing and accumulating them was carried out according to the standard method. Then, a suspension of FEC cells infected with chlamydia of the Chlamydia psittaci strain (var. Avis) "JJ-87" VGNKI No. 23-DEP was subjected to control seeding to check for bacterial and fungal contamination, the titer TCD _{50 / ml} in this example is 10 ^{-7, 5} TCD _{50 / ml} .

A pure, non-contaminated bacterial and fungal flora cell chlamydial suspension was combined into a common sterile container and chlamydia phenol was inactivated. To do this, the components were mixed in a container in the following ratio: suspensions of PEC cells infected with chlamydia were taken 99.1 wt.%, And inactivator (phenol) was added 0.3 wt.%. The resulting mixture was kept for 98 h at a temperature of + 10 ° C. Then added 0.6 wt.% Adjuvant (polyvinylpyrrolidone). After that, selective control was carried out for the completeness of inactivation. For this, 10 developing chicken embryos of 7-day incubation were sterilely injected with 0.3 ml of the vaccine into the cavity of the yolk sac and incubated at 37.0-37.5 ° for 12 days. As a control, 10 embryos were used, which were injected with an inactivated cell suspension containing chlamydia strain "ДЖ-87" VGNKI No. 23-DEP. The incubation conditions are the same. The experimental embryos remained alive for 12 days. Chlamydia was not found in smear prints made from the shells of the yolk sac, stained by the Romanovsky-Giems method. Control embryos fell on the 5-7th day. This result corresponds to the established control of the vaccine for completeness of inactivation.

Similarly done for the following amounts of phenol: 0.2 wt.%; 0.3 wt.%; 0.5 wt.%; 0.6 wt.%. It has been experimentally established that the amount of inactivator in the range from 0.3 wt.% To 0.5 wt.% Ensures the complete inactivation of chlamydia in suspension. Above 0.5 wt.% Phenol is not added in order to save it, because it does not improve the quality of the vaccine. The inactivator concentration below 0.3 wt.% Is not allowed, because this does not ensure complete inactivation of chlamydia.

Example 3. Preparation of the FEC culture, infection with its chlamydia, their cultivation and accumulation was carried out according to the generally accepted method. Then, a suspension of PEC cells infected with chlamydia of the Chlamydia psittaci strain (var. Avis) “J-87” of the All-Russian Scientific Research Institute No. 23-DEP was subjected to control seeding to check for bacterial and fungal contamination. If the test turned out to be negative, then the titer of TCD _{50 / ml was} selectively determined, which in this example is 7.5 TCD _{50 / ml} . Then the components were mixed in the following proportions: a suspension of cells infected with chlamydia was taken 99.3 wt.%, Phenol 0.3 wt.% (The mixture was kept for 10 ° hours at -6 ° C). Polyvinylpyrrolidone was administered 0.4 wt.%. The process was carried out as follows: a weighed portion of the adjuvant and an inactivated cell suspension containing the antigen of the chlamydial strain "J-87" VGKNKI No. 23-DEP, and was at room temperature until it was completely dissolved - 24 hours. Then, the immunogenicity control was carried out. The result is as follows: at a dilution of 1: 5 - ++++; at a dilution of 1:10 - ++++; at a dilution of 1:20 - ++.

Example 4. Preparation of a cell culture of FEC, infection with chlamydia, their growth and accumulation was carried out according to the generally accepted method. Then, a suspension of PEC cells infected with chlamydia of the Chlamydia psittaci strain (var. Avis) “J-87” of the All-Russian Scientific Research Institute No. 23-DEP was subjected to control seeding to check for bacterial and fungal contamination. With a negative test result, the average titer of TCD _{50 / ml was} determined, which in this example is 7.5 TCD _{50 / ml} . Then the components were mixed in the following proportions: suspensions of FEC cells infected with chlamydia were taken 99.3 wt.%; phenol - 0.4 wt.%, polyvinylpyrrolidone - 0.3 wt.%. Then, the immunogenicity control was carried out as in Example 1. The results of CSC are as follows: at a dilution of 1: 5 - ++++; at a dilution of 1:10 - ++++; at a dilution of 1:20 - ++.

Thus, a vaccine containing a suspension of FEC cells infected with chlamydia strain "J-87" VGNKI No. 23-DEP in the range from 99.1 to - 99.3 wt.%, Phenol in the amount of 0.3-0.5 wt. %, polyvinylpyrrolidone in an amount of 0.4-0.6 wt.%, provides the immunogenicity of the vaccine in accordance with its control and causes the development of reliable immunity in birds.

TECHNICAL RESULT

The vaccine prevents the occurrence of chlamydia in birds: pneumonia, arthritis, aerosacculitis. It increases the percentage of hatching of chicks by 10-12%, increases the safety of young animals by 18-24%. Egg production increases, weight gain increases. In addition, the vaccine reduces the environmental pollution of chlamydia, which are anthropozoonoses, and also reduce the costs of a dysfunctional farm for ineffective treatment and preventive measures using antibiotics and other therapeutic agents. The vaccine is harmless. In general, the effectiveness of the vaccine is 90-93%.

Claims (1)

Inactivated culture vaccine against chlamydia (ornithosis) of birds, including a suspension containing chlamydia antigen, adjuvant and inactivator, characterized in that it contains the Chlamydia psittaci strain (var. Avis) "J-87" VGNKI No. 23-DEP grown as an antigen in monolayer culture of primary chicken embryo fibroblast cells with a titer of not lower than 10 ^-7 'TTsD5o ⁵ / ml, as an adjuvant - polyvinyl pyrrolidone, and phenol as an inactivator of the following ratio, wt.%: Chlamydia psittaci (var. Avis) "J-87" VGNKI No. 23-DEP with a titer of at least 10 ^-7.5 TCD _{50 / ml} 99.1-99.3 Phenol 0.3-0.5 Polyvinylpyrrolidone 0.4-0.6