RU2092188C1 - Vaccine for specific prophylaxis of chlamydiosis in cattle - Google Patents

Abstract

FIELD: veterinary microbiology. SUBSTANCE: vaccine has an antigen from the strain Chlamydia psittaci-"250" (pathogen inducing abortus in cows). Antigen is inactivated with formaldehyde in the presence of glutaric dialdehyde. Pathogen is grown simultaneously in yolk and allantois envelops of chicken embryos at concentration 15-20 mcg/ml. Pathogen is adsorbed on alumino-potassium alum and emulsified in oil-lanolin mixture. EFFECT: enhanced effectiveness of vaccine, decreased morbidity and death of calves. 4 tbl

Description

 The invention relates to veterinary microbiology and can be used for specific prophylaxis of chlamydial infection in cattle.

 Chlamydia in cattle is an infectious disease, which is manifested mainly by abortion in cows and heifers by the premature birth of weak and non-viable calves, inflammatory phenomena of the organs of the genital system in cows and bulls and the incidence of young cattle with gastroenteritis, pneumonia, polyarthritis, meningoencephalitis and other syndromes. Chlamydia in cattle in recent years has become widespread in livestock complexes and breeding enterprises of the country.

Chlamydial infection as a whole is characterized by a weak immunological reaction, a tendency to a chronic and latent course with preservation of the pathogen in the body even after acute illness and clinical recovery [1]
Until recently, measures to combat cattle chlamydia in our country and abroad were based on the use of medicines, veterinary-sanitary and restrictive measures.

 The disadvantage of this vaccine is its low efficiency for immunization of cattle against chladiosis due to antigenic differences between the chlamydia strains. It is known that antigenic differences between strains of chlamydia significantly affect the effectiveness of immunization against diseases caused by chlamydia (Ata F.A. Storz J. 1973).

The closest in essence to the proposed invention (prototype) is a “vaccine against chlamydia of farm animals”, containing a chlamydial abortion causative agent grown in the yolk membrane of chicken embryos inactivated with merthiolate, and a solution of trihydrooxymethylaminomethane and tetramethyl amide amide amide amide amide amine and tetramethyldiamine methyl bisacrylamide, ammonium persulfate solution and glutaraldehyde solution [2]
The composition of the vaccine, taken as a prototype, the following, ml:
Antigenic liquid from a 20-40% suspension of yolk sacs infected with chlamydia (ELD ₅₀ 10 ^-6.5-7.0 ) 8-10
A mixture of solutions of trihydroxymethylaminomethane (36.3%) with tetramethyldiamine (0.23%) 1-2
A mixture of solutions of acrylamide (30%) and methylbisacrylamide (0.8%) - 3-4
1.4% solution of ammonium persulfate 1-2
25% glutaraldehyde solution to a final concentration of 0.05% - 0.038
A disadvantage of the known vaccine is the lack of purity of the components of the vitelline membrane of the chicken embryos of the original chlamydial biomass, which is used as a 20-40% suspension of the causative agent of chlamydial abortion of cows (strain "250") and a low concentration of chlamydial antigen in an inactivated vaccine and, as a result, weak immunogenic activity vaccines are not providing the formation of long-term and intense immunity against chlamydia in cattle.

 The aim of the invention is the development of a chlamydia vaccine that provides increased tension and duration of immunity due to gentle inactivation of the pathogen, purification, concentration, adsorption on alum-potassium alum and emulsification of chlamydial biomass in oil-lanolin mixture.

 This goal is achieved by the fact that the causative agent of chlamydial abortion of cows (strain "250") is grown simultaneously in the vitelline and chorionallantoic membranes of chicken embryos (according to ed. St. N 1641883 of December 15, 1990), the chlamydia biomass is inactivated by 0.2% formaldehyde in the presence of 0.1% glutaraldehyde for 24 hours, the antigen is purified and concentrated to 30-40 μg / ml chlamydia by differential centrifugation, adsorbed on potassium alum and emulsified in an oil-lanolin mixture in the following ratios to mponentov wt.

Antigen from Chlamydia psittaci strain "250" 45-50
Oil-lanolin adjuvant 45-50
Formaldehyde 0.04-0.06
Comparative analysis with the prototype allows us to conclude that the proposed vaccine against chlamydia in cattle differs from the known new method of growing specific chlamydial antigen simultaneously in the vitelline and allantoic membranes of the chicken embryo, a new accelerated method of inactivation of chlamydia by combined exposure of the pathogen to formaldehyde (0.2% ) in the presence of glutaraldehyde (0.1%), purification of biomass from components of chicken embryos, concentration up to 30-40 μ / ml specific chlamydial antigen and the introduction of new components into the vaccines, namely potassium alum, mineral oil (PES-3) and formaldehyde as a preservative. Thus, the proposed technical solution meets the criterion of "novelty." Analysis of the compositions of known vaccines against chlamydia of farm animals (ed. St. NN 826744, 690128) showed that some of the substances introduced in the proposed solution are known, for example, the causative agent of chlamydial abortion of cows (strain "250"), an oil adjuvant. However, their use in these preparations in combination with other components does not provide vaccines with the properties that they exhibit in the proposed solution, namely, a significant increase in immunogenic properties and, as a result, increased tension and duration of antichlamydial immunity in vaccinated cattle. Thus, this composition of the components gives the vaccine new properties, which allows us to conclude that the proposed solution meets the criterion of "significant differences".

 The strain (Chlamydia psittaci "250", the causative agent of chlamydial abortion of cows, is used as an antigen in the vaccine. The strain is isolated from the aborted cow fetus and deposited in the microorganism collection of the All-Union State Scientific and Control Institute of Veterinary Medicines under number 5 of October 3, 1990).

The strain of Chlamydia psittaci "250" has the following properties:
Morphological signs. The culture of the strain is a small elementary body of chlamydia having a spherical shape with a size of 200-400 nm. which perceive Tsilya carbol-fuchsin well at a dilution of 1: 5 and are stained in bright red color according to the modified Stemp method.

Cultural properties. Chlamydia strain "250" is well cultivated in the vitelline membrane of 6-7 day old and allantoic membrane of 8-9-day old developing chicken embryos. The specific death of chicken embryos infected in the vitelline cavity is observed 5–9 days after infection; the pathogen accumulates in this case in a titer of 10 ^6.5 -10 ^-7.5 ELD ₅₀ in 0.3 ml.

Pathogenic properties. Chlamydia strain "250" has toxicity and high virulence for laboratory and susceptible animals. With intraperitoneal and intranasal infection of white mice and guinea pigs at a dose of 0.5 and 1 ml, respectively, of a 10% freshly obtained suspension of infected yolk membranes, the disease and death of animals occur on 4-16 days after administration of the material. In heifers, after internal infection at a dose of 8 ml of 10 ^-7.5 ELD ₅₀ in 0.3 ml, the strain causes abortion and the birth of an underdeveloped offspring on the 20-60 day after infection.

 Immunogenic properties. The use of biomass prepared from Chlamydia psittaci strain "250" as an antigen in an inactivated adsorbed-emulsified vaccine ensures the formation of intense and prolonged immunity in 100% of vaccinated animals.

 In experimental experiments with double immunization of heifers, the vaccine protected 100% of the animals from the development of chlamydial infection and abortion after artificial infection with their virulent strain of the causative agent of chlamydial abortion of cows.

 The use of the vaccine in field experiments in chlamydia-poor households significantly reduces the number of abortions and stillbirths in cows and heifers, reduces the incidence and death of calves from chlamydial infections, and thereby reduces the loss of young cattle by 4-5 times and reduces the tension of the epizootic situation for chlamydia infections.

For experimental verification of the proposed vaccine, it is prepared as follows. Chicken embryos of 7 days of age are infected into the vitelline cavity with a suspension of a freshly-passaged strain of chlamydia "250" of the causative agent of chlamydial abortion of cows, diluted to 10 ^-4 at a dose of 0.3 ml. Infected embryos are incubated at 37-38 ^o C for two days, then ovoscopy is performed, the dead are taken and destroyed (non-specific death), and live embryos are re-infected in the allantoic cavity of suspensions of strain "250" diluted to 10 ^-3 at a dose of 0, 3 ml Next, infected embryos are incubated at 37-38 ^o C for 7-8 days. The specific death of 90-95% of infected embryos occurs 6–9 days after the initial infection. Fallen embryos are cooled and opened. From each embryo, 4-4.5 ml of chlamydia-containing allantoic fluid is aspirated and 1-2 g of chorionallantoic and vitelline membranes are taken together with the accompanying yolk.

Chlamydol-containing material is stored frozen at minus 20-30 ^o C until use. Allantoic fluid before freezing is clarified by centrifugation at 3 thousand rpm for 30 minutes

 To obtain a semi-finished vaccine from chlamydia-containing material, a 10% initial suspension is prepared in phosphate-buffered saline (FBI) pH 7.2-7.4 as follows: chlamydia-containing membranes are weighed, thermolysed by freezing and thawing three times, and they are suspended up to 20% th concentration in a colloid mill by adding the calculated amount of the FBI. The suspension is centrifuged at 3000 rpm for 30 minutes and the middle layer of the centrifugate is suctioned off a suspension of chlamydia. The tissue sediment and the upper yolk layer are re-homogenized in the same amount of PBS, centrifuged under the same conditions, and the chlamydia suspension is again suctioned off.

Frozen chlamydia-containing allantoic liquid is thawed as quickly as possible in a water bath at a temperature of 37 ^o C intensively shaking the bottle in order to prevent the formation of large flakes. Thawed slightly opalescent allantoic liquid is diluted in a ratio of 1: 1 with the FBI.

Chlamydia-containing suspensions of infected membranes and allantoic fluid are combined in a 1: 1 ratio and glutaraldehyde is added first at a final concentration of 0.1%, then after 30 minutes, formaldehyde at a final concentration of 0.2%. The suspension after adding the reagents is stirred and incubated for 24 hours at temperature plus 20-24 ^o (room temperature) for inactivation of chlamydia.

 Chlamydia from an inactivated suspension is precipitated by centrifuging it at 18-20 thousand rpm for 60 minutes The supernatant together with the upper yolk layer is removed, the precipitate of elementary chlamydial bodies is weighed and resuspended in a ratio of 1:10 with PBS containing 0.1% formaldehyde.

 The standardization of the suspension according to the content of elementary bodies of chlamydia is carried out by determining its optical density. For this, a 1:10 dilution is prepared from the prepared suspension of chlamydia (4.5 ml of PBS are added to 0.5 ml of suspension) and the absorption coefficient is measured on an FEK 56M at a wavelength of 540 nm in cuvettes with a working length of 10 mm. Optimum for obtaining an immunogenic vaccine is an absorption coefficient of 2.4-2.6 units, which corresponds to a content of 30-40 μg of antigen in one ml of suspension.

A standardized suspension of chlamydia is passed through a colloidal mill, potassium alum at a final concentration of 0.5% is added and homogenized again for 5 minutes, then the suspension is poured into glass cylinders and placed in a refrigerator at 4-6 ^° C for 1-2 days. Under these conditions, chlamydia is adsorbed on alum-potassium alum and precipitated in the form of flakes. The supernatant is carefully aspirated and removed, and the precipitate is resuspended to the original volume with a phosphate-buffered saline containing 0.1% formaldehyde. The procedure for washing chlamydia from excess alum-potassium alum is repeated 2-3 times. The resulting prefabricated vaccine with a preservative is stored until use in the refrigerator at 4-6 ^o C.

 Example 1. For the manufacture of 1 liter of vaccine take 500 ml of inactivated antigen Chlamydia psittaci "250" containing 30 μg / ml of chlamydia adsorbed on potassium alum with 0.08% formaldehyde. Add 500 ml of a sterile oil-lanolin mixture in a ratio of 84:16. Pass through a colloidal mill and packaged in sterile vials of 100 ml. The arithmetic average titer of complement-binding antibodies in the blood serum of vaccinated guinea pigs is not less than 1:20.

 Example 2. For the manufacture of 1 liter of vaccine take 450 ml of inactivated antigen Chlamydia psittaci "250" containing 40 μg / ml of chlamydia adsorbed on potassium alum with 0.12% formaldehyde. 550 ml of a sterile oil-lanolin mixture are added in a ratio of 85:15. Pass through a colloidal mill and packaged in sterile vials of 100 ml. The arithmetic average titer of complement-binding antibodies in the blood serum of vaccinated guinea pigs is not less than 1:20.

 When the minimum and maximum changes in the ratios of the components in the manufacture of the vaccine, the emulsion is stratified into water and oil fractions, which reduces the immunogenicity of the emulsion vaccine.

 The vaccine is monitored for sterility, durability of the emulsion and harmlessness according to generally accepted methods. Immunogenicity control is carried out on guinea pigs. Animals are vaccinated at a dose of 0.5 ml subcutaneously in the scapular region on the right and left (0.25 ml each). Vaccination promotes the development of specific chlamydial complement-binding antibodies on days 30-35 in titers of 1: 5 1:80 (average arithmetic titer not lower than 1:20).

 The vaccine is used as a preventive measure in farms and breeding enterprises of dysfunctional or threatened by chlamydia cattle. All seronegative cattle are vaccinated once once a year from three months of age. Heifers of a random age and cows are vaccinated 20-25 days before insemination or mating. Chlamydia patients in animals are vaccinated before treatment with tetracycline antibiotics and vaccinated after recovery.

 The vaccine is administered to cattle under aseptic conditions subcutaneously in the area of the breast or upper third of the neck at the rate of not more than 1.5 ml in one place in the following doses: 1 ml to calves aged 3 to 6 months; 2 ml of calves aged 6 to 12 months; 3 ml of calves older than a year, cows and bulls.

 In vaccinated animals, at the injection site, a seal forms up to 3x4 cm in size, which gradually dissolves over 4-8 months. Immunity in vaccinated animals is created after 30 days. after vaccination and persists for 12 months. The shelf life of the vaccine is 1.5 years.

 Tests of 6 experimental and 3 pilot industrial series of inactivated emulsion vaccine against chlamydia in cattle were tested for antigenic and immunogenic activity.

 The experimental vaccine series were tested in experiments on laboratory animals and heifers. White mice weighing 16-18 g were vaccinated twice subcutaneously with an interval of 7 days at a dose of 0.1-0.15 ml. 21-28 days after vaccination, the mice were infected with the causative agent of chlamydial abortion of cows (strain "250") intraperitoneally at a dose of 0.3-0.5 ml of a 10% suspension of the pathogen. Simultaneously with immunized mice, intact (control) mice of the same age were infected. The results of six series of experiments (Table 1) showed that the survival rate of pre-vaccinated mice after control of infection with a virulent chlamydia culture ranged from 86-92.3% (88.6 ± 1.2%). In the control group, deaths from 66.7 to 88% of mice (78.7%) were noted. Therefore, the vaccine has high immunogenicity and ensures the resistance of vaccinated mice to artificial infection with a virulent chlamydia culture.

 In experiments on heifers under experimental conditions, it was found that double subcutaneous administration of a vaccine in a dose of 1.5 ml of heifers prevents them from developing chlamydial abortion in cattle after infection with a virulent chlamydia strain "250" (Table 2).

 Under the conditions of the Kherson Biofactory, according to the technology developed by us, 3 pilot industrial series of the vaccine against chlamydia in cattle were manufactured. Production control of sterility, safety and immunogenic activity, their acceptance, as well as the conclusion on industrial production was made according to the order of the USSR GUV N 5.06.06. It was found that all three manufactured experimental industrial vaccine series are sterile, harmless and immunogenic preparations (Act of the Kherson Biofactory of May 24, 1990).

 Production tests of a vaccine manufactured at the Kherson Biofactory were carried out in nine chlamydia-poor farms in the TSSR and Vladimir Region. At the same time 6862 goals were vaccinated. cattle, including 3,715 cows, 79 manufacturing bulls and 3,068 calves and heifers of a random age. Field test results of the vaccine are summarized in table. 3 and 4. From the above table. 3 data shows that a month after vaccination in 100% of vaccinated animals revealed seropositivity in CSC to chlamydial antigen. The arithmetic average titer of specific chlamydial antibodies in this case ranged from 1:65 to 1:87. After 6 months, from 90 to 93.6% of vaccinated animals with an arithmetic average titer of 1:12 1:14 reacted positively to chlamydia by CSC. After 12 months, seropositivity for chlamydial antigen persisted in 50-60% of cattle with an average complement-binding antibody of 1: 7 1:10.

 Thus, vaccination of animals determines their seropositivity for chlamydial antigen. The maximum level of antibodies (titers 1:10 1: 160) is formed 30-35 days after vaccination and remains at a high level in 90-93.6% of the animals for 6 months; then there is a gradual decrease in seropositivity by 12 months after immunization.

 The effectiveness of vaccination of cattle in the field was assessed by comparing the number of abortions and stillbirths among cows and heifers, as well as the number of deaths and forced slaughter of calves in dysfunctional farms before vaccination (1989) and after vaccination (1990, 1991). From the above table. Figure 4 shows that the number of abortions and stillbirths in five households before vaccination averaged 6% mortality and forced slaughter 10%. The total loss of young animals averaged 16%. After using the vaccine, the number of abortions and stillbirths in these same households decreased by 5.4 times and amounted to only 1.1%. At the same time, the incidence and forced slaughter of calves from chlamydial infection decreased by 3.7 times. The total loss of young animals after vaccination was 3.7%, which is 4.2 times less than before the vaccine was used in these farms. Consequently, the vaccine under conditions of cattle chlamydia-dysfunctional cattle economy minimizes the number of abortions and stillbirths, significantly reduces the incidence and death of calves from chlamydial infections, improves the epizootic situation for chlamydia, and thus ensures an increase in animal productivity.

 Due to the fact that the present invention has the features of patentability, a vaccine for specific prophylaxis of chlamydia in cattle can be submitted for foreign patenting.

 The introduction of the vaccine in veterinary practice will allow livestock farms to prevent economic losses due to underreporting of up to 20% of calves due to abortion, stillbirths in cows and heifers, mortality and forced slaughter of calves, as well as stabilization of weight gain in animal fattening and maintenance of milk yield by lactating cows.

Claims (1)

 A vaccine for specific prophylaxis of cattle chlamydia containing an inactivated Chlamydia psittaci antigen, an adjuvant and a preservative, characterized in that it contains an inactivated and purified chlamydial antigen from a Chlamydia psittaci strain "250" of a cow abortion pathogen at a concentration of 15 to 20 mg / ml adsorbed alum and emulsified in an oil-lanolin adjuvant, and as a preservative - formaldehyde in the following ratios of components, wt. Inactivated and purified chlamydial antigen from strain Chlamydia psittaci "250" of the causative agent of abortion of cows 45 50
Alum potassium alum 0.2 0.3
Oil-lanolin adjuvant 45 55
Formaldehyde 0.04 0.06 °

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