CS254937B1 - Bivalent vaccine against sheep's contagious chlamydo and typhus disease and method of its production - Google Patents

Abstract

The bivalent vaccine against contagious chlamydial and parathyroid flocking of sheep with an oil adjuvant is intended to immunize sheep and harans against these two diseases. It contains the Chlamydia strain, which was isolated in the territory of Czechoslovakia and the Salmonella abortus ovis strain isolated in the UK. An oil adjuvant suitable potentiates the efficacy of the vaccine. The Chlamydia psittaci antigen is prepared by propagating this microbe on the Tomato Bags of Smokers of the Embryo, Salmonella abortus antigen by Salmonella propagation in the liquid loading pod using submerged culture. 0.5 ml per 100 ml culture is used to inactivate both microbial species. One vaccine dose, which is 4 ml of vaccine, contains the following amounts of active ingredients: Chlamydia psittaci 50 Salmonella abortus ovis 2X 5.6. 109 microbes

Description

The present invention relates to a bivalent vaccine against infectious chlamydial and parathytic sweeping of sheep with an oil solvens, and a process for its preparation. Effective preventive measures against infectious diseases affecting this species of animals are necessary in the context of the establishment of high-concentration sheep. These two diseases, which are zoonoses, cause aortic disease in adult sheep and sepia in lambs with the following deaths. In addition, Chlamydia psaci taci causes orchids, pididymitis and vesiculitis in rams, while the rearing brood excludes foamy virulence of Chl. psittaci. Chlamydia psittaci is one of two representatives of the genus Chlamydia, with some strains having a pronounced affinity for pregnant uterus (STORZ, J .: Chlamydia and Chlamydia Induced Diseases, Charles C., Thomas Publisher, Springfield, Illinois, USA, 1971, pp. 171 to 171). 207], where they induce inflammation of the placenta, resulting in miscarriage in most infected animals (STAMP, JT et al .: Enzootic Abortion in Ewes. I. Transmission of the disease. Vet. Rec., 62, 1950, pp. 251-254) TRÁVNIČEK, M. et al .: Veterinary Science, XXVIII, 1978, No. 12, pp. 541. The economic severity of chlamydial aboroles is high, 56.9% of pregnant ewes can be aborted in freshly infected herds (WACHENDORFER, G., VALDER , WA: Der Praktische Tierarzt, 1976, No 7, pp. 422–426] and up to 80% of ewes (HIEPE, Th .: Schafkrankheiten, VEB Gustav Fischer Verlag Jena, 1970, p. 119). mothers are characterized by low viability, sac another die within 48 hours, respectively. suffering from pneumonia, polyarthritis and keratoconjunctivitis. Chlamydia psittaci strains that evoke! abortions in sheep may also induce abortions in pregnant women (JOHNSON, WA et al .: Brit. Med. J. Febrary, 1985, 290, pp. 592–594), and systemic and organ diseases (atypical pneumonia, thrombophlebitis, in particular for professionally exposed workers (Surveillance Zoonoses, ORNITOSES, SLOVAKIA, 1978, pp. 1-6), the Commission of the Government of the CSF coordinates the work in the field of combating animal-borne diseases. Salmonella abortus ovis induces parathy sweeping in sheep, whereas 40-60% of sheep can be aborted in the infected herd (VRZGULA, L. et al .: Sheep diseases, Nature, Bratislava, 1973, p. 21; HIEPE, Th .: Schafkrankheiten, VEB Gustav Fischer Verlag Jena, 1970, p. As a consequence of complication, up to 5% of adult sheep die in the herd after retention (second retention, endometritis), and the birth of lambs that die of sepsis is also affected by infection. NYCHA abortion sheep producing monovalent chlamydia vaccine without adjuvant and vaccine PARATYPHOID virulent in abortion mares and sheep. This is a broth! culture of two bacterial strains Sclmonella abortus equi and Salmonella abortus ovis, heat-inactivated and phenol-preserved. The vaccine is non-adjuvanted and is used to protect the breeding mares and sheep against parathytic sweeping. Chlamydia sweep vaccines are produced in the following European countries: Hungary - Phylaxia produces a monovalent aluminum hydroxide gel gel vaccine. 2 ml vaccine p. c. is used to prevent chlamydial abortion of sheep. France - IST (Institut de Sérothérapie de Toulouse) produces Clila mystan, an oil-adjuvanted non-valency vaccine used to prevent chlamydial infections! Υ sheep at 2 ml sc and bovine at 5 ml sc Wellcome manufactures a monovalent oil adjuvant vaccine for the prevention of chlamydial abortion of sheep at a dose of 1 ml sc GDR - it is known from literary data (VALDER,

A., Wachendorfer, G .: Untersuchungen zur Wirksamkeit der Vakzination gegen den Chlamydien abort des Schafer, Deutsch. Tierärztl. Wschr., 82, 1975, 6, p. 220-225) that a non-adjuvanted raonovalent chlamydial vaccine produced under laboratory conditions was used experimentally in the GDR. A monovalent vaccine in the GDR and NSR is produced against parathyphalic sweeping of sheep, both of which are a Hahn culture of Salmonella abortus ovis strains killed with adjuvanted formalin. It is used to prevent parathytic sweeping at 5 and 10 ml s. c. According to available information, a vaccine against contagious chlamydial and parathytic sweeping of oil adjuvanted sheep is not produced worldwide.

Chlamydia psittaci in sheep causes inflammation of the fetal membranes in the pregnant uterus, resulting in a break in the connection between the mother and the fetus and subsequent abortion. Salmonella abortus ovis causes inflammation of the uterus, the overall septic state in the organ of the lower mother and fetus, which in turn cause the fetus to die and its expulsion from the mother's body.

The composition of the oily solvens used in the bivalent vaccine is set forth below. 6. Oil recipe from Bioveta Nitra has similar formula. The exact composition of the oil solvens used in Wellcome and I.S. vaccines, these companies do not disclose, respectively, in the instructions for use. they conceal them from commercial reasons. Wellcome describes me oil adjuvant used in its vaccine as a light, liquid paraffin, firm I. S, T. only as an oil adjuvant.

SUMMARY OF THE INVENTION The present invention provides a bivalent vaccine against infectious chlamydial and parathytic sweeping of sheep comprising 50% of inactivated Chlamydia psittaci elementary bodies per 4 ml of vaccine and 10% of inactivated bacterial bodies of Salmonella abortus ovis at least 2 * 5.6. ⁹ microns per 4 ml vaccine. The bivalent vaccine is characterized in that it contains the Chlamydia psittaci CAMP R-24 strain and the Salmo nella abortus ovis strain CCM 5611, which are deposited in the Collection of Pathogenic Microorganisms, Research Institute, Veterinary Medicine, Brno, Hudcova 70. The composition of claim 1, wherein the diluent in the form of a lipoid adjuvant comprises buffered saline and a preservative. It is also an object of the present invention to provide a vaccine method by multiplying the Chlamydia psittaci strain on yellow embryo smoking bags for 4 to 7 days, removing the yellow bags followed by formaldehyde inactivation and homogenization, and centrifuging the antigen purification with ether. The strain of Salmonella abortus ovis is propagated in a masopeptone broth at 37 ° C, then inactivated with formaldehyde, and then the inactivated culture of Calmonella abortus ovis is admixed with adjuvant and concentrated Chlamydia psittaci antigen and is homogenized after homogenization. The production method is characterized in that the oil adjuvant is mixed in an equal volume ratio with an inactivated culture of Salmonella abortus ovis.

The vaccine according to the invention is intended for the active immunoprophylaxis of pregnant sheep and for the active immunoprophylaxis of rams. Advantages of the vaccine include:

- simultaneous action against the Court of serious infectious diseases classified as zoonoses, - improved vaccine stability due to the use of an oil adjuvant, - increased animal protection against both infections due to the use of an oil solvens, - radically reduced abortions in sheep caused by chlamydia, and salmonella and a simultaneous reduction in lamb mortality within 48 hours after birth, - a reduction in the possibility of traumatization of pregnant sheep, as the number of animal handling will be reduced from 4 monovalent vaccines to 2 for bivalent vaccine, - production costs compared to monovalent vaccines bivalent vaccine below.

The vaccine is a suspension of inactivated Chlamydia psittaci elemental bodies and inactivated Salmonella abortus ovis bacterial bodies in buffered saline, preserved with formaldehyde and an oil diluent. Components for the preparation of a bivalent vaccine against contagious chlamydial and parataphic 6 ointment with adjuvant oil:

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

2. Salmonella abortus ovis strain CCM 5611 isolated in the UK, flood designation: NCTC 10241, J. Taylor, CPHL London.

3. Chicken embryos 5-7 days, obtained from hatching eggs according to ČSN.

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

A. Buffered saline pH 7.2

(a) preparation of phosphate buffer:

1. 0.15 M sodium hydrogen phosphate solution (NaHPCH. I2H2O)

53.79 g distilled water 1000.0 ml

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

To achieve a pH of 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) saline preparation:

NaCl (sodium chloride) 8.5 g distilled water 1000.0 ml

Saline is buffered until pH 7.2 is reached.

B. 0,25 M sucrose solution in phosphate buffered saline - preparation:

sucrose 85.6 g buffered physiol. solution at 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 m. j. per ml of buffered saline,

Gentamycin 0.2 mg per ml of buffered saline.

6. Formaldehyde according to CSN (36-38% solution p. A.) 686236.

7. Diluent and solvens - composition:

Vaseline Medical AI Csl 3

000 g

Lanolin Čsl 3 8 000 g

Tween 80 (Polyoxyethylene sorbitan monooleate, n ca.20) HLB 15.0; d20 1,07 MWca 1,300 1,600 g

Water redistilled 66 400 g

Description of example:

Embryonated chicken eggs of 5-7 days are used to multiply Chlamydia psittaci (elemental bodies). Embryos are infected with 20 wt. a suspension prepared from vein bags infected with Chlamydia psittaci strain in 0.25 M sucrose buffered saline pH 7.2 into the rye bags. The amount of inoculum is 0.2-0.4 ml per embryo. We discard embryos that have died in the first 3 days. Embryos died in the 4th, 4th and 4th respectively. On day 6 (on day 7 all embryos are removed), the following are processed:

The vein bags are selected and weighed. 20 wt. a vein bag suspension with 1 M NaCl solution, with 0.5 ml of formaldehyde added for each 100 m < -1 > suspension. The material is homogenized in a mixer for 3 minutes. The homogenized yellow bags are stored in a refrigerator at +4 degrees Celsius for 72 hours. This is followed by centrifugation at 15,000 rpm. for 30 minutes. Remove fat and supernatant. The residue is resuspended in a sufficient quantity of saline (vii of feed volume) by stirring. The suspension is poured into a separatory funnel and 1.5 times the volume of ether is added. Shake well and store in the refrigerator overnight. Phase differentiation will occur. An aqueous phase is used which contains the elemental bodies of Chlamydia psittaci. The ether residue is removed by bubbling air. Further centrifugation at 15,000 rpm follows. for 30 minutes. The sediment is resuspended in V1 of the original volume in saline. A suspension in a ^separatory: ieviku was again treated with ether. 2-3 hours are now sufficient to separate the individual layers. This is followed by ether bubbling and final centrifugation at 1200 rpm. for 30 minutes. The sediment is diluted with saline to give a slightly turbid solution. From this diluted antigen we prepare 3 to 5 dilutions in ratios of 1: 4, 1: 8, 1: 16, 1: 32 and 1: 64. at a wavelength of 420. The antigen is measured against a buffered saline solution at pH 7.2. The subsequent calculation using the coefficient for Chlamydia psittaci sets the active substance content to 1 mg per ml. The active ingredient content in a vaccine dose of - 50 µg per 4 ml is achieved by subsequent dilution with solvens.

Preparation of Salmonella ahortus ovis antigen. The lyophilized vaccine strain Salmonella abortus ovis is rehydrated after opening the ampoule in 0.2 ml masopepton broth and seeded with a masopepton agar and End's agar, where it is incubated for 24 hours. at 37 ^° C. For further propagation of the Salmonella abortus ovis strain a liquid propagation medium of the following composition is used:

Enzymatic casein hydrolyzate

Immune 10.0 g

Acidic casein hydrolyzate 10.0 g

Soy peptone 5.0 g

Sodium chloride 5.0 g

Distilled water up to 1000.0 ml

The culture is transferred from Endo agar to a batch containing 50 ml of the liquid propagation described above. After incubation at 37 ^° C for 6 hours, the batch is inoculated into 10 liter bottles containing 3,003 milliliters of liquid propagation medium under stringent sterile conditions (pouring through the flame), the bottles are carefully closed after neck annealing. After mixing, they are cultured for 2 hours statically at 37 ° C, followed by submerged shaker culture for 18 hours at 37 ° C. After cultivation, samples were taken from each culture flask to determine microbial counts and to check culture purity. The grown culture is inactivated with formaldehyde 0.5 ml per 100 ml culture for a lower 24 hours at 37 ^° C after sampling. The culture culture purity is determined by inoculation on Masopspton agar, Endova agar, brilliant blue agarose and galactose. The determination of the number of microbes is carried out by means of a spectrophotometer. 1 ml of the grown culture contains at least 5.6. . 10 ⁹ Salmonella abortus ovis.

Mixing the Chlamydia psittaci and Salmonella abortus ovis antigens and adding an oil solvens. Inactivated culture of Salmonella abortus ovis containing at least 5,6 per ml. 10 ⁹ microbes are mixed with an equal volume of oil solvens (1: 1 ratio). For each liter of vaccine thus prepared, 12.5 ml of concentrated Chlamydia psittaci antigen (active ingredient content of 1 mg per ml) was added to give a final concentration of 50 µg per 4 ml of vaccine. The final concentration of antigens in 1 vaccine dose, ie in 4 ml, will be as follows:

Salmonella abortus ovis

2X 5,6. 10 ⁹ microbes

Chlamydia psittaci 50 μ%

Control of the finished vaccine: (a) Sterility test:

The vaccine is inoculated for blood agar, End's agar and thioglycolate broth, which must remain sterile for 48 hours at 37 ° C.

(b) Inactivation test:

The 0.2 ml vaccine is injected into the yolk bags of 5-7 day-old embryonic smokers and Chlamydia psittaci must not multiply within 5 days. The vaccine is seeded 2X on sloping masopeptone agar, 0.5 wt. bovine broth and glucose agar are incubated for 7 days at 37 ° C and simultaneously for 10 days at room temperature. Salmonella abortus ovis must not grow during this incubation.

(c) Harmlessness test:

rabbit is administered with. c. double dose, t. j. 8 ml 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 state of health must not be impaired. Past results:

rabbits

Number Dose Method of administration Days days

123456789 10

8 ml s. c.

4 ml s. c.

sheep

4 ml s. c.

8 ml s. c.

neg.

neg.

neg.

neg.

The harmlessness test is based on an increase in antibody levels in a healthy rabbit against the Chlamydia psitlaci and Salmonella abortus ovis antigen. The rabbits receive 4 ml of the vaccine. c. After 10 days, revaccination was made with the same dose. After another 14 days, blood is drawn by cardiac injection and antibody levels are collected. For detection of chlamydial antibodies we use the method of complement binding reaction, for detection of salmonella antibodies we use the method of slow tube agutation. We consider the antigen to be effective against at least a 1: 16 dilution, resp. the difference from the initial coatings must be an average of 2 dilutions. Past results:

Rabbits - applied complete vaccine

rabbit number dose cause filing Chlamiddle before the bag. - post-vaccine antibodies. před vakc. po vakc. 1 4 ml with. c. 0 32 0 160 2 4 ml with. c. 0 32 0 80 3 4 ml with. c. 0 32 0 40 4 4 ml with. c. 0 32 0 80

Applied only Chlamydia antigen with oil adjuvant -

1 2 ml with. c. 0 32 0 10 2 2 ml with. c. 0 64 0 10 Only salmonella antigen applied , oo with oil and djuvansom - 1 2 ml with. c. 0 8 0 40 2 2 ml with. c. 0 8 0 20

Sheep - applied completely vaccine

Sheep number dose sposob filing Chlamydia in front of vacc. 1 4 ml with. c. 16 2 4 ml with. c. 8 3 4 ml with. c. 16 4 4 ml with. c. 16 5 4 ml with. c. 8 6 4 ml with. c. 8 7 4 ml with. c. 16 8 4 ml with. c. 0 9 4 ml with. c. 8 10 4 ml with. c. 16 11 4 ml with. c. 32 12 4 ml with. c. 16 (e) Determination of pH sa sets to 7.0 +0.3. f) Appearance vaccines: vaccine presents

milky white turbid oil without sediment and flakes.

The vaccine has been experimentally manufactured and verified as part of laboratory testing in rabbits and sheep (see results above). Vaccination results were used in the laboratory verification). For laboratory testing, vaccine doses of 2, 4 and 8 ml s were used. c. to determine the tolerability of the vaccine and to monitor post-vaccination titers. No post-vaccination reactions were detected in sheep or rabbits during the verification. The rise of antibodies in sheep against both antigens indicates the good protection afforded by the vaccine. In addition, the cellular component of immunity can be envisaged for the chlamydial antigen. The rise in antibodies in rabbits indicates the suitability of these animals in testing the efficacy of the vaccine.

The challenge trials have so far been presumed to determine the dose for the challenge itself. A homologous strain was administered in the prescription and was administered iv at 2 and 4 cm ³ and sc at 2 and 4 cm ³ . The attempt is not yet complete and will continue in the next mating period of sheep. The following indicators were followed in advance: TRIAS, RVK serological examination, heniatological examination, Skin test and clinical follow-up.

Salmonella antibodies - post-vaccine antibodies. před vakc. po vakc.

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 parataphic sweeping of sheep was performed according to the methodology approved by the ISCVBM in Brno. Actually verification was done in two sheep breeding and JRD Velky Lipnik, okr. Stará Lubovňa and JRD Drienov, okr. Presov. The results of the verification are as follows:

JRD Vel'ky Lipnik, okr. Stará Lubovňa

Biological experiment on 20 sheep performed on May 20, 1986. The vaccine was administered according to the instructions. None locally resp. overall changes in health, or observed. A dose of 10 ml s was administered. c. Vaccination - 200 ewes were vaccinated after synchronization. Vaccination was performed on May 28, 1986, at the same time blood samples were taken to determine the initial antibody levels in 30 pieces. Revaccination - was also carried out on 18 June 1986 in all 200 ewes and at the same time blood was collected from 30 individually marked animals. Further blood samples were taken 2 and 4 months after revaccination.

Serological results (titers) using KFR and Chl antigen. psittaci are listed in a special report on clinical vaccine validation.

The control group consisted of 312 placebo-treated sheep (adjuvant without antigen) and kept separately from the experimental group. The occurrence of abortions in the above groups was as follows:

Number of sheep Number of aborts % Cause gr. vaccinations. 200 4 2.0 non-infectious gr. controls. 312 27 8.33 ChL psittaci Incidence comparison aborts with the past the season is as follows: Number of sheep Number of aborts % Cause 1985-86 557 39 7.0 Chl. psittaci 1986-87 312 27 8.33 Chl. psittaci Controls. gr. 200 4 2.0 non-infectious Rating June 17. 1986. Bola the vaccine administered according to the invention.

The dynamics of antibody production after administration of the chlamydial antigen had a marked increase after the first antibody. A second dose partially retained these liladins. However, the level of antibody levels in chlamydial vaccines is not in direct proportion to the protective effect of the vaccine since the cellular component of the immune system plays a major role here. For antibodies to the saimonel antigen, the increase in antibodies was highest after the first dose, while a slight increase was observed 2 months after revaccination.

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

Biological experiment on 20 sheep. None locally resp. overall changes in health, or observed.

Vaccination - 274 ewes were vaccinated after synchronization. Vaccination was performed on June 26, 1986, at the same time blood samples were taken to detect initial antibody levels in 30 individually labeled sheep.

Revaccination - was performed on 21.7.1986 on all vaccinated pieces. At the same time, blood samples were collected from 30 individually labeled sheep.

Serological results (titers) using KFR and Chl antigen. psittaci are listed in a separate report on clinical vaccine validation.

Control! the group consisted of 270 sheep treated with placebo (adjuvant without antigen) and kept separately from the experimental group. The occurrence of abortions in the above groups was as follows:

Number of sheep Number of aborts % Cause gr. vaccinations. (Okotené) 174 2 1.14 non-infectious gr. controls. 270 13 4.81 nezistená inf. etiology

Comparing abortion rates to last season is as follows:

Number of sheep Number of aborts % Cause 1985-86 464 26 5.60 Chl. psittaci 1986-87 270 13 4.81 nezistená inf. cause controls. gr. 174 2 1.14 non-infectious Rating we noticed none locally or overall

Rise of antibodies to Ohi antigen. psittaci was more pronounced after the 2nd dose. Antibody levels decreased significantly after 4 months after revaccination. The protective effect of chlamydial vaccines is based primarily on the cellular component of the immune system. For antibodies against salmonella antigen, we have seen an increase after the element dose of the vaccine.

The vaccine was tolerated by sheep very similar to that of Vel'ký Lipník. Non-reacting. The reduction in abortions in the vaccinated group compared to the control and previous season groups is indicative of the protective effect of the vaccine.

Post-vaccination cell immunity was evaluated only in a small set of animals and thus the results of t. no. they are not authoritative. Results of testing 4 antigens on model animals (guinea pig, mouse) using the Skin test of FAYE et al. (1974) are as follows: in the skin test of the four strain 254937 tested, the vaccine strain responded most intensively - induration width 6-9 mm, which is the fourth grade of the Faya scale. The controls used were:

1. Tuberculin allergen in an amount of 50 µg.

2. An antigen prepared from an uninfected rye bag.

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