CS254937B1 - Bivalent vaccine against Chlamydial and Parathyroid Disease and its production - Google Patents

Abstract

Bivalent vaccine against contagious chlamydial and paratyphoid fever of sheep with oil adjuvant is intended for immunization of sheep and goats against these two diseases. It contains the strain of Chlamydia psittaci, which was isolated in the territory of the Czechoslovak Socialist Republic, and the strain of Salmonella abortus ovis isolated in Great Britain. The oil adjuvant suitably potentiates the effectiveness of the vaccine. The Chlamydia psittaci antigen is prepared by multiplying this microbe on the yolk sacs of chicken embryos, the Salmonella abortus ovis antigen by multiplying salmonella in a liquid medium using submerged cultivation. Formaldehyde is used to inactivate both microbial species in an amount of 0.5 ml per 100 ml of culture. One vaccination dose, which represents 4 ml of vaccine, contains the following amounts of active substances: Chlamydia psittaci 50 Salmonella abortus ovis 2X 5.6 . 109 microbes

Description

The invention relates to a bivalent vaccine against contagious chlamydial and paratyphoid sheep disease with an oil solvent and a method of its preparation. In connection with the construction of enterprises with a large concentration of sheep, effective preventive measures against infectious diseases affecting this species of animals are necessary. These two infections, which are among the zoonoses, cause abortions in adult sheep, and septiform forms of the disease in lambs with subsequent deaths. Chlamydia psittaci also causes orchitis, epididymitis and vesiculitis in rams, while breeding rams secrete the virulent bacteria Chl. psittaci. Chlamydia psittaci represents one of two representatives of the genus Chlamydia, with some strains being characterized by a strong affinity for the pregnant uterus [STORZ, J.: Chlamydia and ChlamydiaInduced Diseases, Charles C., Thomas Publisher, Springfield, Illinois, USA, 1971, pp. 171 to 207], where they cause inflammation of the placenta, which results in abortion in most infected animals (STAMP, J. T. et al.: Enzootic abortion in ewes. I. Transmission of the disease. Vet. Rec., 62, 1950, pp. 251—254; TRÁVNIČEK, M. et al.: Veterinářství, XXVIII, 1978, No. 12, p. 541). The economic importance of chlamydial abortions is high. In freshly infected herds, 56.9% of pregnant ewes may abort (WACHENDORFER, G., VALDER, W. A.: Der Praktische Tierarzt, 1976, No. 7, pp. 422—426] or up to 80% of ewes (HIEPE, Th.: Schafkrankheiten, VEB Gustav Fischer Verlag Jena, 1970, p. 119). Live-born lambs from infected mothers are characterized by low viability, most of them die within 48 hours, or suffer from pneumonia, polyarthritis and keratoconjunctivitis. Chlamydia psittaci strains that cause abortions in sheep can also cause abortions in pregnant women (JOHNSON, W. A. et al.: Brit. Med. J. Febrary, 1985, 290, pp. 592—594], or systemic and organ diseases (atypical pneumonia, thrombophlebitis) especially in professionally exposed workers (Surveillance zoonoses, ORNITHOSES, SLOVAKIA, 1978, pp. 1—6, published by the Commission of the Government of the SSR for the Coordination of Work in the Field of Combating Diseases Transmissible from Animals to Humans). Salmonella abortus ovis causes paratyphoid fever in sheep, and in an infected herd 40—60% of sheep can abort (VRZGULA, L. et al.: Diseases of Sheep, Nature, Bratislava, 1973, p. 21; HIEPE, Th.: Schafkrankheiten, VEB Gustav Fischer Verlag Jena, 1970, p. 142). As a result of complications after abortion (retention of the secondaries, endometritis), up to 5% of adult sheep die in the herd. The infection also affects newborn lambs, which die of sepsis. Humans can also become infected. Currently, a monovalent chlamydial vaccine without adjuvant is being produced in the Czechoslovak Socialist Republic to prevent infectious abortions in sheep, as well as a vaccine against paratyphoid contagious abortion of mares and sheep. It is a broth culture of two bacterial strains Salmonella abortus equi and Salmonella abortus ovis, inactivated by heat and preserved with phenol. The vaccine is adjuvant-free and is used for the protective vaccination of breeding mares and sheep against paratyphoid abortion. Vaccines against chlamydial abortion of sheep are produced in the following European countries: Hungary — the company Phylaxia produces a monovalent vaccine with aluminum hydroxygel. The vaccine in a dose of 2 ml s. c. is used to prevent chlamydial abortions of sheep. France — the company IST (Institut de Sérothérapie de Toulouse] produces the vaccine Cila mystan. It is a monovalent vaccine with an oil adjuvant, which is used to prevent chlamydial infections! υ sheep in a dose of 2 ml s. c. and in cattle in a dose of 5 ml s. c. England — Wellcome produces a monovalent vaccine with an oil adjuvant, intended for the prevention of chlamydial abortion in sheep in a dose of 1 ml s. c. GDR — from the literature it is known (VALDER,

A., WACHENDORFER, G.: Untersuchungen zur Wirksamkeit der Vakzination gegen den Chlamydien abort des Schafer, Deutsch. Tierárztl. Wschr., 82, 1975, 6, p. 220 -225), that in the GDR a monovalent chlamydial vaccine without adjuvant produced in laboratory conditions was experimentally used. A monovalent vaccine against paratyphoid fever in sheep is produced in the GDR and the Federal Republic of Germany, in both cases it is a mass culture of strains of Salmonella abortus ovis, killed with formalin without adjuvant. It is used for the prevention of paratyphoid fever in a dose of 5 and 10 ml s. c. According to available information, a vaccine against contagious chlamydial and paratyphoid fever in sheep with an oil adjuvant is not produced in the world.

Chlamydia psittaci in the sheep causes inflammation of the amniotic sac in the pregnant uterus, resulting in a disruption of the connection between the mother and the fetus and subsequent abortion. Salmonella abortus ovis causes inflammation of the uterus, a general septic condition in the mother and fetus, resulting in the death of the fetus and its expulsion from the mother's body.

The composition of the oil solvent used in the bivalent vaccine is given in the following text on page 6. The oil solvent from Bioveta Nitra also has a similar formula. The exact composition of the oil solvents used in the vaccines of the Wellcome and I. S. T companies is not stated in the instructions for use, or they keep it secret for commercial reasons. The Wellcome company describes the oil adjuvant used in its vaccine as light, liquid paraffin, the I. S, T. company only as an oil adjuvant.

The invention is a bivalent vaccine against contagious chlamydial and paratyphoid sheep disease, characterized in that it contains inactivated elementary bodies of the Chlamydia psittaci strain in an amount of 50 µg per 4 ml of vaccine and inactivated bacterial bodies of the Salmonella abortus ovis strain in an amount of at least 2X 5.6 .10 ⁹ micrograms per 4 ml of vaccine. The bivalent vaccine is characterized in that it contains the Chlamydia psittaci CAMP R-24 strain and the Salmonella abortus ovis strain CCM 5611, which are stored in the Collection of Pathogenic Microorganisms, Research Institute, Veterinary Medicine, Brno, Hudcova 70. This bivalent vaccine is further characterized in that it contains a diluent in the form of a lipoid adjuvant, buffered saline solution and a preservative. The invention also provides a method of vaccine production, consisting in that the Chlamydia psittaci strain is propagated on the yolk sacs of chicken embryos for 4 to 7 days, after removal of the yolk sacs, inactivation with formaldehyde and homogenization are followed, and after centrifugation, purification of the antigen with ether. The Salmonella abortus ovis strain is propagated in meat peptone broth at a temperature of 37 °C, then inactivated with formaldehyde, and then the inactivated Calmonella abortus ovis culture is mixed with an adjuvant and with a concentrated Chlamydia psittaci antigen and, after homogenization, it is expanded. The production method is characterized in that the oil adjuvant is mixed in the same volume ratio with the inactivated Salmonella abortus ovis culture.

The vaccine according to the invention is intended for active immunoprophylaxis of pregnant ewes and active immunoprophylaxis of breeding rams. The advantages of the vaccine include:

— simultaneous action against serious infectious diseases of the farm, which are classified as zoonoses, — improved vaccine stability due to the use of an oil adjuvant, — higher level of protection of animals against both infections due to the use of an oil solvent, — radical reduction in the incidence of abortions in sheep caused by chlamydia and salmonella and a simultaneous reduction in lamb deaths within 48 hours after birth, — reduction in the possibility of traumatization of pregnant sheep, because the number of manipulations with animals is reduced from 4 for monovalent vaccines to 2 for bivalent vaccines, — compared to the costs of producing monovalent vaccines, the costs of producing bivalent vaccines are lower.

The vaccine is a suspension of inactivated elementary bodies of Chlamydia psittaci and inactivated bacterial bodies of Salmonella abortus ovis in buffered saline, preserved with formaldehyde and an oil diluent. Components for the preparation of a bivalent vaccine against contagious chlamydial and paratyphoid distemper in sheep with an oil adjuvant:

1. Chlamydia psittaci CAMP R-24 strain, isolated in Czechoslovakia, flood designation EPá 4/85 Debrad.

2. Strain Salmonella abortus ovis CCM 5611 isolated in the UK, accession number: NCTC 10241, J. Taylor, CPHL London.

3. Chicken embryos 5-7 days old, obtained from hatching eggs in accordance with ČSN.

4. Buffered saline pH 7.2, 0.25 sucrose solution in phosphate buffer, 1 M sodium chloride solution.

A. Buffered saline solution pH 7.2

a) preparation of phosphate buffer:

1. 0.15 M sodium hydrogen phosphate solution (Na2H2O)

53.79 g distilled water 1,000.0 ml

2. 0.15 M potassium dihydrogen phosphate solution 20.41 g distilled water 1,000.00 ml

To achieve a buffer of pH 7.2, mix solutions No. 1 and 2 in the following ratio:

solution no. 1 14.3 ml solution no. 2 5.7 ml

20.0 ml of pH 7.2 buffer

b) preparation of physiological solution:

NaC' (sodium chloride) 8.5 g distilled water 1,000.0 ml

The physiological solution is adjusted with buffer until pH 7.2 is reached.

B. 0.25 M sucrose solution in phosphate buffered saline — preparation:

sucrose 85.6 g buffered saline solution pH 7.2

000.0 ml

C. 1 M sodium chloride solution (Načl) — preparation:

sodium chloride 58.44 g distilled water 1,000.00 ml

5. Streptomycin 200 IU per 1 ml of buffered saline,

Gentamicin 0.2 mg per 1 ml of buffered saline.

6. Formaldehyde according to ČSN (36-38% solution p. a.) 686236.

7. Diluent and solvent — composition:

Medical Vaseline Oil AI Csl 3

000 grams

Lanolin Czech Republic 3 8,000 g

Tween 80 (Polyoxyethylene sorbitan monooleate, n ca.20) HLB 15.0; d20 1.07 MWca 1,300 1,600 g

Redistilled water 66,400 g

Description of the design example:

For the propagation of Chlamydia psittaci (elementary bodies) embryonated chicken eggs of 5-7 days old are used. Embryos are infected with a 20% wt. suspension prepared from vein sacs infected with the Chlamydia psittaci strain in 0.25 M sucrose buffered physiological solution with pH 7.2 into the yolk sacs. The amount of inoculum is 0.2 to 0.4 ml per 1 embryo. Embryos that die in the first 3 days are discarded. Embryos that die on the 4th, 4th or 6th day (all embryos are removed on the 7th day) are processed as follows:

The yolk sacs are selected and weighed. A 20% wt. suspension of the yolk sacs is prepared with 1 M NaCl solution, adding 0.5 ml of formaldehyde for every 100 ml of suspension. The material is homogenized in a blender for 3 minutes. The homogenized yolk sacs are placed in a refrigerator at +4 degrees Celsius for 72 hours. This is followed by centrifugation at 15,000 rpm for 30 minutes. The fat and supernatant are removed. The sediment is resuspended in a sufficient amount of physiological solution (V of the initial volume) by mixing. The suspension is poured into a separating funnel and 1.5 times the volume of ether is added. It is shaken well and placed in the refrigerator overnight. Phase differentiation occurs. The aqueous phase containing elementary bodies of Chlamydia psittaci is used. The ether residues are removed by bubbling air through. This is followed by another centrifugation at 15,000 rpm for 30 minutes. The sediment is resuspended in the original volume in physiological saline. The suspension in the ^separating funnel is mixed again with ether. 2-3 hours are now sufficient for the separation of the individual layers. This is followed by bubbling out the ether and a final centrifugation at 12,000 rpm for 30 minutes. The sediment is diluted with physiological saline so that a slightly cloudy solution is formed. From the antigen diluted in this way, 3 to 5 dilutions are prepared in the ratios 1 : 4, 1 : 8, 1 : 16, 1 : 32 and 1 : 64. The extinction values are then measured on a spectrophotometer! at a wavelength of 420. The antigen is measured against buffered saline solution at pH 7.2. Subsequent calculation using the coefficient for Chlamydia psittaci sets the active substance content to 1 mg per 1 ml. The active substance content in the vaccination dose — 50 µg per 4 ml is achieved by subsequent dilution with solvents.

Preparation of Salmonella abortus ovis antigen. After opening the ampoule, the lyophilized vaccine strain of Salmonella abortus ovis is rehydrated in 0.2 ml of massopeptone broth and inoculated with a brush onto massopeptone agar and onto Endo agar, where it is incubated for 24 hours at 37 ^C C. For further propagation of the Salmonella abortus ovis strain, a liquid propagation medium of the following composition is used:

Enzymatic casein hydrolysate

Imun 10.0 g

Acid casein hydrolysate 10.0 g

Soy peptone 5.0 g

Sodium chloride 5.0 g

Distilled water 1,000.0 ml

From Endo agar, the culture is transferred with a funnel into flasks containing 50 ml of the above-described liquid propagation medium. After 6 hours of incubation at 37 ^° C, the flask is inoculated into 10-liter bottles containing 3,003 ml of liquid propagation medium under strictly sterile conditions (pouring over a flame), the flasks are carefully closed after annealing the neck. After mixing, they are cultured statically for 2 hours at 37 °C and then submerged in a shaker for 18 hours at 37 °C. After cultivation, samples are taken from each culture flask to determine the number of microbes and to check the purity of the culture. After sampling, the grown culture is inactivated with formaldehyde in the amount of 0.5 ml per 100 ml of culture for at least 24 hours at 37 ^C C. Determination of the purity of the culture is made by inoculation onto massopstone agar, Endo agar, brilliant blue and galactose agar. Determination of the number of microbes is made using a spectrophotometer. 1 ml of grown culture contains at least 5.6 . . 10 ⁹ individuals of Salmonella abortus ovis.

Mixing of Chlamydia psittaci and Salmonella abortus ovis antigens and adding oil solvent. Inactivated Salmonella abortus ovis culture, which contains in 1 ml at least 5.6 . 10 ⁹ microbes, is mixed with the same volume of oil solvent (ratio 1 : 1). For each liter of the vaccine prepared in this way, 12.5 ml of concentrated Chlamydia psittaci antigen is added (active substance content 1 mg in 1 ml), thus achieving a final concentration of 50 ug per 4 ml of vaccine. The final concentration of antigens will be in 1 vaccination dose, i.e. in 4 ml, as follows:

Salmonella abortus ovis

2X 5.6 . 10 ⁹ microbes

Chlamydia psittaci 50 μ%

Checking the finished vaccine: a) Sterility test:

The vaccine is inoculated onto blood agar, Endo agar and thioglycolate broth, which must remain sterile during a 48-hour incubation at 37°C.

b) Inactivation test:

A 0.2 ml vaccine is injected into the yolk sacs of 5-7 day old chicken embryos, and no growth of Chlamydia psittaci is allowed for 5 days. The vaccine is inoculated 2X onto a slant of meat peptone agar, 0.5% wt. beef broth and glucose agar, which are incubated for 7 days at 37 °C and simultaneously for 10 days at room temperature. No growth of Salmonella abortus ovis is allowed during this incubation.

c) Harmlessness test:

rabbits are administered s. c. a double dose, i.e. 8 ml of vaccine. Assessment after 10 days. The vaccine must be harmless. A reaction may occur at the site of application, which disappears within 14 days. The general health condition must not be impaired. Results so far:

Rabbits

Number Dose Method of administration Days of pieces

123456789 10

8 ml s. c.

' 4 ml s. c.

Sheep

4 ml s. c.

8 ml s. c.

neg.

neg.

neg.

neg.

The safety test is based on the increase in antibody levels in healthy rabbits against the Chlamydia psittaci and Salmonella abortus ovis antigens. Rabbits are injected with 4 ml of vaccine s. c. After 10 days, a booster dose is given with the same dose. After another 14 days, blood is taken by cardiac puncture and antibody levels are determined. For the detection of chlamydia antibodies, we use the complement fixation reaction method, for the detection of salmonella antibodies, the slow tube agglutination method. We consider an antigen to be effective if antibodies are formed against it at a dilution of at least 1:16, or the difference from the initial titers must be on average 2 dilutions. Results so far:

Rabbits — complete vaccine administered

Rabbit number Dose Method of administration Cold soap before the vaccine. — antibodies after vaccination. before the holidays after vacation 1 4 ml p. c. 0 32 0 160 2 4 ml p. c. 0 32 0 80 3 4 ml p. c. 0 32 0 40 4 4 ml p. c. 0 32 0 80

Chlamydia antigen alone applied with oil adjuvant —

1 2 ml p. c. 0 32 0 10 2 2 ml p. c. 0 64 0 10 Applied only salmonella antigen ,éo with oil and help — 1 2 ml p. c. 0 8 0 40 2 2 ml p. c. 0 8 0 20

Sheep — fully applied vaccine

Sheep number Dose Method of administration Chlamydia before the vaccine. 1 4 ml p. c. 16 2 4 ml p. c. 8 3 4 ml p. c. 16 4 4 ml p. c. 16 5 4 ml p. c. 8 6 4 ml p. c. 8 7 4 ml p. c. 16 8 4 ml p. c. 0 9 4 ml p. c. 8 10 4 ml p. c. 16 11 4 ml p. c. 32 12 4 ml p. c. 16 e) Determination of pH is set to 7.0 +0.3. f) Relative vaccines: Vaccine represents

Milky white cloudy oily liquid without sediment and flakes.

The vaccine was experimentally produced and verified in laboratory validation in rabbits and sheep (see results above). Vaccination results were used in laboratory validation). Vaccination doses of 2, 4 and 8 ml s. c. were used in laboratory validation to determine vaccine tolerance and monitor post-vaccination titers. No post-vaccination reactions were detected in either sheep or rabbits during validation. The increase in antibodies in sheep against both antigens indicates good protection induced by the vaccine. In addition, a cellular component of immunity can also be expected for the chlamydial antigen. The increase in antibodies in rabbits indicates the suitability of these animals for testing the efficacy of the vaccine.

From the challenge trials, a preliminary trial was conducted to determine the dose for the actual challenge. In the preliminary trial, a homologous strain was used, which was administered iv in a dose of 2 and 4 cm ³ and sc in a dose of 2 and 4 cm ³ . The trial is not yet completed, it will continue in the next sheep admission period. In the preliminary trial, the following indicators were monitored: TRIAS, serological examination using RVK, heniatological examination, Skin-test and clinical monitoring.

Salmonella antibodies — antibodies after vaccination. before vacation after vacation

64 0 40 32 10 40 64 10 20 32 10 20 32 10 40 64 20 40 256 10 40 16 0 20 128 10 80 64 10 40 128 10 80 64 10 40

Clinical verification of the vaccine against chlamydial and paratyphoid sheep distemper was carried out according to the methodology approved by the Institute of Veterinary Medicine in Brno. The verification was actually carried out in two sheep farms, namely at JRD Velký Lipník, district Stará Lubovňa and JRD Drienov, district Prešov. The verification results are as follows:

JRD Vel'ký Lipník, Stará Lubovňa district

Biological experiment on 20 sheep performed on 20. 5. 1986. The vaccine was administered according to the instructions. No local or general changes in health status were observed. A dose of 10 ml was administered s. c. Vaccination — 200 ewes were vaccinated after synchronization. Vaccination was performed on 28. 5. 1986, and blood samples were taken to determine the initial antibody levels in 30 sheep. Revaccination — was performed on 18. 6. 1986 also in all 200 ewes and blood samples were taken from 30 individually marked animals. Further blood samples were taken 2 and 4 months after revaccination.

The results of serological tests (titer) using KFR and Chl. psittaci antigen are presented in a separate Report on the clinical validation of the vaccine.

The control group consisted of 312 sheep, which were given placebo (antigen-free adjuvant) and were kept separately from the experimental group. The incidence of abortions in the groups was as follows:

Number of sheep Number of abortions % Cause group vacation 200 4 2.0 non-infectious group contr. 312 27 8.33 ChL psittaci Comparison of incidence abortions with a past the season is as follows: Number of sheep Number of abortions % Cause 1985-86 557 39 7.0 Chl. psittaci 1986-87 312 27 8.33 Chl. psittaci Control group 200 4 2.0 non-infectious Rating new 17. 6. 1986. It was vaccine administered according to

The dynamics of antibody formation after administration of chlamydial antigen had a significant increase after the first dose. The second dose partially maintained these changes. However, the levels of antibodies in chlamydial vaccines are not directly proportional to the protective effect of the vaccine, because the cellular component of immunity plays the main role here. In antibodies to the salmonella antigen, the increase in antibodies was highest after the 1st dose, while 2 months after revaccination we recorded a slight increase.

The vaccine itself was tolerated by the sheep without any local or general reactions. Any health problems were not related to the administration of the vaccine. The incidence of abortions in the vaccinated group and the control group, respectively. in the breeding in the previous season indicates a positive effect after vaccination. JRD Drienov, Prešov district

A biological experiment was carried out on 20 sheep. No local or general changes in health status were observed.

Vaccination — 274 ewes were vaccinated after synchronization. Vaccination was performed on June 26, 1986, and blood samples were taken to determine initial antibody levels in 30 individually marked sheep.

Revaccination was carried out on 21 July 1986 for all vaccinated animals. At the same time, blood samples were taken from 30 individually marked sheep.

The results of serological tests (titer) using KFR and the Chl. psittaci antigen are presented in a separate Report on the Clinical Verification of the Vaccine.

The control group consisted of 270 sheep, which were given placebo (adjuvant without antigen) and were kept separately from the experimental group. The incidence of abortions in the groups was as follows:

Number of sheep Number of abortions % Cause group vacation (fawn) 174 2 1.14 non-infectious group contr. 270 13 4.81 undetermined inf. etiology

Comparing the incidence of abortions with last season is as follows:

Number of sheep Number of abortions % Cause 1985-86 464 26 5.60 Chl. psittaci 1986-87 270 13 4.81 undetermined inf. cause control group 174 2 1.14 non-infectious Rating we recorded none locally or overall

The increase in antibodies to the Ohi. psittaci antigen was more pronounced after the 2nd dose. Antibody levels decreased significantly 4 months after revaccination. The protective effect of chlamydia vaccines is based primarily on the cellular component of immunity. We noted an increase in antibodies to the salmonella antigen after the first dose of the vaccine.

The vaccine was tolerated by sheep very well, similar to that in the Vel'ký Lipník locality. Non-reactions. The reduction in the incidence of abortions in the vaccinated group compared to the control group and the previous season indicates the protective effect of the vaccine.

Postvaccination cellular immunity was evaluated only on a small number of animals and so the results of the present study are not authoritative. The results of testing 4 antigens on model animals (guinea pig, mouse) using the Skin-test according to FAYE et al. (1974) are as follows: in the skin test of the four tested strains, the vaccine strain reacted most intensely — the width of the induration was 6—9 mm, which represents the 4th degree of the Faye scale. The following were used as controls:

1. Tuberculin allergen in an amount of 50 |Ug.

2. Antigen prepared from a non-infected seed sac.

Claims (7)

A bivalent vaccine against infectious chlamydial and parataphic sheep sweeping, characterized in that it contains inactivated elemental bodies of Chlamydia psittaci in an amount of 50 µg per 4 ml of vaccine and inactivated bacterial bodies of Salmonella abortus ovis in an amount of at least 2X 5.6. 10 ⁹ microbes per 4 ml vaccine and adjuvant at 2 ml per 4 ml vaccine. 2. The bivalent vaccine of claim 1 comprising a Chlamydia psittaci CAPM R-24 strain that causes inflammation of the fetal membranes in the sheep, resulting in abortion. 3. The bivalent vaccine of claim 1, which comprises a strain of Salmonella abortus ovis CCM 5611 which causes a maternal and fetal septic condition in the ovine organism, resulting in abortion. 4. The bivalent vaccine of claims 1 to 3 comprising a buffered saline and a preservative and a diluent in the form of a lipoid adjuvant. 5. A method of producing a bivalent vaccine for the invention of a contagious chlamydial and parathyphic sweeping of sheep according to items 1 to 4, characterized in that the Chlamydia psittaci strain is propagated on the embryo smoking pouch bags for 4 to 7 days after removal. the bag is followed by formaldehyde inactivation and homogenization and after removal of the antigen purification with ether and the Salmonella abortus ovis strain is propagated in a liquid propagation pod using submerged culture at 37 ° C, then inactivated with formaldehyde followed by inactivated salmonella abortus adjuvant culture with concentrated Chlamydia psittaci antigen and is homogenized after homogenization. 6. The production method according to claim 5, wherein 10-20% by weight of the embryo is used to infect 5-7 day-old smokers. suspension of infected rye bags at a dose of 0.2 ml per chicken embryo. 7. The process of claim 5, wherein the oil adjuvant is mixed in an equal volume ratio with an inactivated culture of Salmonella abortus ovis. Severography, n. p. Race 7, Most Price 2,40 Kčs