CS201481B1 - Live lyofilized vaccine against the horned cattle trichofytose - Google Patents

Description

(54) A live lyophilized vaccine against bovine trichophytosis and a process for its manufacture

The present invention relates to a live lyophilized vaccine against bovine trichophytosis and to a method thereof

preparation from a live virulent culture of Trichophyton verrucosum Bodin 1902 (strain Bi-O36-š Strážnice) and intended for the prophylaxis and therapy of bovine trichophytosis.

Bovine trichophytosis is a very serious infectious disease -anthropozoonosis, which has spread in the Czech Republic especially with the increase of the concentration of cattle in large-capacity calves and in large cattle herds. Trichophytosis is transmissible to humans, and treatment of one infected person is costly. In addition to widespread damage to the leather industry (unusable leather for leather processing), the disease also causes other economic losses, especially in agriculture - by preventing modern breeding and the possibility of moving animals in large farms.

A number of therapeutic agents against the disease are described in the literature. However, these preparations are usually inefficient, costly and labor intensive and time consuming. A number of attempts to develop a vaccine against bovine trichophytosis have been unsuccessful, and the vaccines prepared have been insufficiently effective. Only in the USSR was recently prepared an effective vaccine for the prophylaxis of trichophytosis in cattle. It is a suspension of microconidia of the immunogenic Trichophyton faviforme strain in saline

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201 481 at a concentration of 6 to 9 million in 1 cm \ The vaccine was obtained by propagation of the Soviet production strain δ. 130 on malt agar, homogenizing the grown culture in saline and diluting the cell suspension to the above concentration of microconidia. The preparation is administered to animals at intervals of 10 to 14 days in doses ranging from .5 to 10 cm @ 3. Sarkisov: Prophylaxis specific to triehophyton des jeunes bovides. Bull, OIB, 85, 1976: 481-488 and U.S. Pat. Certificate No 160324 /.

The essence of our invention is the production of live lyophilized vaccine against bovine trichophytosis from Triehophyton verrucosum strain obtained in the territory of the CSB. The production of the vaccine is carried out according to our technological process, so that the production strain is propagated on liquid Sabouraud soil, liquid sweet soil or on solid soils Sabeuraud glucose agar and sweeten agar (soil enriched against the usual thiamine and glutamic acid regulations). grown cultures in a buffered saline medium, buffered lyophilization medium with neomycin is added and the number of microconidia in a vaccine unit is microscopically determined and the vaccine concentration is filled to 10-20 million based on the concentration of microconidia. Hozplněná. the vaccine is frozen at -40 ° C and dried in a lyophilizer.

The lyophilization-stabilized immunogenic strain Triehophyton verrucosum becomes part of this invention and is deposited in Cs. collection of microorganisms - collection of zoopathogenic microorganisms - Brno, Class of Defenders of Peace 10, namely the culture Triehophyton verrucosum, strain Bi-036-S / CCM-E-650 / used for the production of Czechoslovak vaccine against bovine trichophytosis isolated from District of Hodonín. Cultivation on malt agar shows growth as early as 2-3 days after inoculation. The surface of the culture is bright white, without pigments, the center of the colonies is raised, the edges are broadly star-shaped. The culture stand for vaccine production (14 to 21 days old) covers continuously the culture medium, is white, the surface is fine, fibrous, slightly scored. Microscopy reveals microconidia (almost round, pear or clublike, 1.5 to 2 x 2 to 5 µm large), macroconidia (thin-walled club spores with numerous baffles, 4 to 6 x 10 to 50 large), mycelium and chlamydospores . The optimum pH for growth and sporulation is 6.0 to 6.9.

Experimental verification of the efficacy of vaccine against bovine trichophytosis of Czechoslovak origin gave very good results / Komárek Surveillance of dermatomycoses of domestic animals 1976, 102 to 112, Nos. enlightenment Pardubice. When using the vaccine in large calves of calves, the therapeutic effect was 98%, prophylactic 97.5% / Jaroš: Experienced Trichophytosis Suppression in Large-calf Calf Trutnov, Veterinary 26, 1976 »256 / , she did not become ill with this disease. Vaccination of sick pieces led to their recovery. The vaccine is harmless, it was not potentiated by trichophytosis in healthy animals.

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The use of a vaccine against bovine trichophytosis brings considerable economic benefits, facilitates the work of the veterinary service and has a positive impact on the health sector. The use of this vacoin in cattle breeding has already led to a reduction in infestations of districts, municipalities and outbreaks in Czechoslovakia when comparing the epidemiological situation in 1977 and 1978, as can be seen from the table:

Infested districts Difference 1977/1978 absolutely -9 in% -14.6 municipalities -156 38.1 outbreaks -168 -38.7

The essence of the technological process of the invention lies in the fact that MS. the Trichophyton cerrucosum production strain propagated on a suitable culture medium is homogenized in a buffered saline medium for 5 to 10 minutes, buffered lyophilization medium with neomycin at a concentration of 0.2 g / l is added, the number of germs is determined microscopically, the amount of concentrate is determined filling so that the number of microconidia in 1 cm @ 2 of vaccine was 15 + 5 million, vialing and lyophilizing the vaccine were performed. The vaccine is dispensed into vials of 50 or 100 cc and buffered saline is added to each vial as a diluent.

The vaccine dosage is as follows:

a) prophylactic doses: 2 x 2.5 cm ^ calves 1 to 2 months 2x5 em ^ cattle over 2 months b / treatment doses: twice the prophylactic doses according to age and animal category,

An additional revaccination dose can be used safely in animals severely affected.

The vaccine is administered deep intramuscularly in the lumbar, sacral or thigh landscape, the vaccine dose in the left, the revaccination dose in the right half of the animal. There is a range of 10 to 14 days between vaccination and revaccination.

Examples

Example 1 - Multiplication of Trichophyton verrucosum production culture in Sabouraud soil - Production of 20,000 doses of vaccine.

Sabouraud's solution of 10 liters of distilled water, 400 g of maltose, 100 g of peptone is used as propagation medium. The pH is adjusted to 6.6 to 6.9. The soil is autoclaved for 30 minutes at 0.05 MPa. The soil is then dispensed into Roux 2 L bottles of 150 to 200 cm @ 3 per bottle and further autoclaved at the same ratios. After cooling, the soil is inoculated with Trichophyton verrucosum. The inoculum was prepared from a 14-35 day culture of Trichophyton verrucosum (production strain Bi-036-5) propagated on solid soil. The crop growth in a total amount of about 3 to 6 × 10 ¹⁰ microconidia was homogenized in 1000 cm 3 of buffered saline in a mixer for 5 to 10 minutes. 0.2 g of neomycin was added to the inoculum thus prepared. Inoculation was carried out with a syringe by using 8 shots using 10 enr inoculum (about 3 to 6 x 10 micro201481 conidia of the fungus) per culture flask. After 14-21 days of dark growth at 28-30 ° C, the grown 5-6 bottle culture was transferred to a mixer, 0.5 L buffered saline was added, and the culture was homogenized for 5-10 minutes. The homogeneous culture was filtered through gauze into a 20 liter stock bottle to which buffered lyophilized media was added in the same amount as buffered saline.

Composition of buffered saline:

distilled water 1000 cnp sodium chloride p.a. 3 8 Potassium chloride p.a. 0.2 g secondary sodium phosphate p, a. (12 HgO) 2.38 g potassium phosphate acid p.a. 0.2 g

Composition of buffered lyophilization medium:

redistilled water 1000 cm gelatin for bacteria. 50 g sucrose 75 g secondary sodium phosphate p.a. (12 H 2 O) 3.82 g of potassium potassium phosphate p.a. 0.36 g

Neomycin at a concentration of 0.2 g / l is added to the homogenate thus prepared.

All work on vaccine production should be done under sterile environmental conditions, and the material and supplies used to prepare the vaccine must be autoclaved and burnt over a gas burner before use. A sample of about 5 is taken from the vaccine homogenate stock bottle to perform control tests. The number of microconidia in a vaccine sample is microscopically determined using a Btlrker counting chamber. Based on the result obtained, the loading ratio of the vaccine is calculated so that its final concentration is 15 + 5 million microconidia per cm @ -1 of vaccine.

The vaccine is dispensed into 50 or 100 cm vials. Following freezing to -40 ° C for at least 4 to 5 hours and drying in a lyophilizer for 24 to 48 hours with reheating to 25 ° C, the lyophilization is terminated when the temperature of the product reaches the temperature of the drying boiler and does not change for at least 4 hours . After drying, the vacuum in the apparatus is canceled with nitrogen. The dried vaccine vials are closed with rubber stoppers under vacuum.

Before use, the lyophilized vaccine is diluted with a diluent. For lyophilized vaccine /

the following control tests are performed: the purity and bacterial sterility test is performed by sowing the vaccine on blood agar, masopepton agar, masopepton broth, glucose agar and Sabouraud agar. The determination of bacterial sterility at 37 ° C lasted for 72 hours and 7 days and at 20 ° C for 9 days. Monitoring of the absence of foreign fungi on Sabouraud agar was carried out for 12 days at a temperature of 28-30 ° C. The determination of hydrogen ion concentration was carried out at pH meter. The permissible pH of the diluted vaccine is 6.0 to 7.5. The potency test is performed by determining the number of viable germs. It is determined by the plate method on Sabouraud agar. The vaccine should contain at least 10 to 20 million germs live zárod201 481 ing Trichophyton verrucosum in 1 cm ^3, to test the vacuum prováůí pomooí high-frequency electrical tester vacuum. The harmlessness test is carried out on 2 rabbits weighing around 3 kg. The rabbits are injected deeply intramuscularly with a 2.5 cm ³ vaccine into the hind limb muscle. Batches with unsatisfactory parameters are not suitable for immunization and must be inactivated by autoclaving.

Example 2 - Enhancement of Trichophyton verrucosum production culture on thiamine-enriched liquid wort broth.

For culturing the Trichophyton verrucosum culture liquid liquid soils of the following composition may be used: 10 liters of distilled water, 1 kg solid brew obtained by drying liquid brewing brew in an atomizer, 50 g peptone, 0.2 g thiamine.

This soil is autoclaved at a pressure of 0.05 MPa for 30 minutes in an autoclave, the pH is adjusted to 6.0-6.6 and the soil is filled into Roux bottles of 2 liters of 150-200 cm ³ per bottle. After closing the flasks with cotton plugs, autoclaving is performed at the same ratios. The inoculation of the soil is carried out with a syringe, with a 3 x 6 x 10 x Trichophyton verrucosum germ per flask. The cultivation takes place in a dark chamber at a temperature of 28 to 30 ° C. After 14-21 days of growth, the culture is transferred to a mixer where it is homogenized in a buffered saline medium for 5-10 minutes. Per 500 cm ^{3 of} buffered saline, there are 5 to 6 culture flasks. After completion of the homogenization of the culture, the mixer is rinsed with 0.2 g / l of neomycin buffered lyophilization medium. The amount of lyophilization medium was the same as the amount of saline. The germ counting in the homogenate is performed microscopically. Vaccination is performed on the basis of a calculation so that there are 15 + 5 million microconidia of Trichophyton verrucosum per cm ^{3 of} vaccine.

Freezing and lyophilization of the filled formulation as well as control tests were performed as described in Example 1. A total of 20,000 doses of lyof bovine trichophytosis vaccine were obtained.

Example 3 - Multiplication of Trichophyton verrucosum production culture on diluted wort agar enriched with thiamine and glutamic acid.

The nutrient medium used was dilute and enriched malt agar enriched with thiamine and glutamic acid as follows: 10 liters of liquid brewery malt, 5 liters of distilled water, 225 g of agar, 75 g of peptone, 0.3 g of thiamine and 0.3 g of acid glutamic.

The medium was autoclaved at 0.05 MPa for 30 minutes, the pH adjusted to 6.0-6.6 and the soil expanded by about 400 cm ³ into 2 liter Roux bottles. The bottles were sealed with cotton plugs and autoclaved again at the same ratios. After the medium had solidified, the bottles were

201,481 were transferred to a chamber at 28-30 ° C, where they were left for 5 to 9 days to determine the sterility of the medium. Inoculation was by syringe, per culture flask with about 3 to 6 x 10 microconidia of Trichophyton verrucosum. After inoculation, the bottles were placed in a dark chamber at 28-30 ° C with culture medium down. After 1 to 2 days after attachment of the culture to the nutrient medium, the flasks were inverted with the culture medium. The culture was examined macroscopically daily for the absence of foreign flora. After 14 to 21 days of growth, the grown culture was separated from the culture medium by means of a metal loop and transferred to a mixer using metal hoes. The growth from 4 culture flasks was used per 500 cm < 3 > of buffered saline, and the homogenization time was 5-10 minutes. The homogenate was filtered through gauze into a 20 liter glass bottle and rinsed with 0.2 g / L buffered lyophilization medium with neomycin. Total amount of buffered lyophilization; the medium was the same as the total amount of buffered saline. In the obtained homogenate, the number of microconidia was determined microscopically in a Btirker counting chamber. On the basis of the value determined, the ratio of the vaccine to 50 or 100 cm 2 vials was calculated to contain the finished product; 15 + 5 million microconidia in 1 cm \

The freezing and freeze-drying of the product and the control tests are the same as in Example 1.

About 16,000 doses of bovine trichophytosis vaccine are obtained as described above.

All work on the preparation of ihoculum, inoculation, vaccine production, filling and lyophilization, as well as closure of vials, should be done in a sterile manner. Laboratories are sterilized by UV radiation and disinfectants. The solutions and aids needed to prepare the product must be autoclaved twice for 30 minutes at 0.05 MPa. E-filling the concentrate into vials before lyophilizaoi‘se going in a stream of laminar sterile air. Workers are required to wear protective clothing, rubber gloves, a face mask, and a headgear when working with the Trichophyton verrucosum culture. Used culture broths and other aids contaminated with Trichophyton verrucosum must be autoclaved for 1 hour at 0.2 MPa. Similarly, inadequate batches of vaccine or empty vials are inactivated after vaccination in the field.

The product has an expiry date of 1 year. The product should only be stored at 4 ° C and in the dark.

Claims (6)

SUBJECT OF THE INVENTION A live lyophilized vaccine against bovine trichophytosis for the prophylaxis and therapy of bovine trichophytosis, characterized in that it comprises a lyophilized multiplied strain of Trichophyton verrucosum Bodin / BI-036-S / CCM E-650 in an amount of 10 to 20 x 10 6 microconidia in 1 cm 2 vaccine and diluent. 201 481 2. The vaccine of claim 1, wherein the diluent is a buffered saline solution containing 8 g of sodium chloride, 12 g of potassium chloride, 2.38 g of secondary sodium phosphate, 0.2 g of potassium potassium phosphate and 1 liter of distilled water. of 3. The vaccine production method of claim 1, wherein the Trichophyton verrucosum Bodin CCM E-650 strain is cultured in a culture medium for 14 to 21 days, the vegetation obtained is homogenized for 5 to 10 minutes in a buffered saline medium. buffered lyophilization medium is added, the concentration of microconidia is determined microscopically and filled to 1 cm ^{J of} vaccine containing 10 to 20 x 10 microconidia. 4. A method according to claim 3, wherein the amount of buffered lyophilization medium is the same as the amount of buffered saline. 5. A method according to any one of claims 3 and 4, characterized in that the filled vaccine is frozen at -40 ° C for at least 4 to 5 hours, dried in a lyophilizer for 25 to 48 hours at a temperature of up to 25 ° C. C. 6. A method according to claim 3, characterized in that neomycin is added to the prepared homogenate at a concentration of 0.2 g / l homogenate prior to filling.