CS246791B1 - Avirulent vaccine against trichofytozis of cattle and method of its production - Google Patents

Description

The present invention relates to an avululent vaccine against bovine trichophytosis and to a process for its manufacture. The vaccine is intended for the prophylaxis of cattle trichophytosis.

TriChofytosis of cattle is a skin disease affecting mainly young cattle up to the age of one year. The average monthly number of animals affected by this disease in the Czechoslovakia ranged from 1970 to 1976 around 10 thousand to 14 000 pieces, which caused our national economy large losses. After the introduction of blanket vaccination of calves in 1976, the number of outbreaks of trichophytosis and the number of sick animals decreased significantly each year, until the end of 1983 the disease was managed in the Czechoslovakia as the first country in the world in cattle breeding. The main contributor to the control of bovine trichophytosis in the Czech Republic was undoubtedly a live vaccine against bovine trichophytosis (Iyophilized). and in the case of improper application and animal health, the Soviet vaccine LTF-130 (US Patent No. 160,324), which contains a live virulent strain Trichophyton faviforme, is similarly disadvantaged.

100 953), although it is prepared from the virulent strain Trichophyton verrucosum, which, however, has a significantly lower yield compared to the production strains and our avirulent strains CCM F-751 and CCM F-764. Also the expiration time of the Polish vaccine (4 weeks) is considerably shorter compared to our avirulent vaccine (the expiration time of our vaccine is 1 year).

The object of our invention, avirulent vaccine against bovine trichophytosis, avoids the risk to animal and, most likely, to human health, since it is made from an avirulent strain of Trichophyton verrucosum. Compared to the previously produced Czech and foreign vaccine, the vaccine represents a qualitatively higher level of this product. The method of its production is also simplified and improved compared to the previous ones. Experiments on calves and guinea pigs have shown that the protective properties of an avirulent vaccine are fully comparable to those obtained from a virulent strain of T. verrucosum.

The avirulent strain Trichophyton verrucosum CCM F-751, which is part of the vaccine, is characterized by faster growth, a faster onset of microconidial formation, higher sporulation and higher ability to survive lyophilization than the virulent strain T. verrucosum CCM F-650 contained in the tri246791 chophytosis vaccine. Live bovine livestock (cf. author's certificate no. 201 481). These features, and in particular the higher yield of the M-9A strain, make it possible to use a younger culture from one day of age to manufacture the vaccine, facilitating production technology (diluted culture medium, lower inoculum density, shorter culture homogenization times) etc. j. At the same time there is an increase in labor productivity.

Similar properties to strain M-9A are also characterized by the avululent strain T. verrucosum TV-M-8 (= CCM F-764), which can also be used to produce a vaccine.

The present invention is based on an avululent vaccine against bovine trichophytosis and a process for its manufacture. The avianulent vaccine against bovine trichophytosis is characterized by containing 2.5 to 10 million viable microconidia of the avirulent strain Trichophyton verrucosum Bodin 1902 CCM F-751 or CCM F-764 per cm ^{3 of the} diluted vaccine. The production method is characterized in that the virulent strain of T. verrucosum is propagated on a suitable medium at pH = 6.6 to 6.9 for 11 to 18 days, at a temperature of 26 to 28 ° C, the grown culture is homogenized in a sterile protective medium, filled into vials and lyophilized.

The experience of Soviet authors shows that after the interruption of vaccination in areas where cattle trichophytosis has been suppressed, the infection has again started. The disease agent, the fungus Trichophyton, is able to survive in the stable environment for 7 to 10 years while maintaining pathogenicity. Therefore, it is recommended to vaccinate all calves born for 10 years after the healing of the last infected animal on the holding.

Vaccination of cattle with virulent trichophytosis vaccine is of epidemiological importance since it prevents the introduction of live viruletous T. verrucosum elements into livestock farming, as is the case with calves vaccinated with a vaccine prepared from a highly virulent T. verrucosum strain (T. faviforme). This is because a virulent vaccine injection site develops a crust of 1 to 3 cm in diameter, which can become a potential source of infection for susceptible animals and for humans because it contains infectious fungal elements. No such crusts are formed after vaccination with the virulent vaccine; culture of the skin at the site of injection of the vaccine showed no vaccine strain.

The practical use of avululent vaccine against bovine trichophytosis creates a prerequisite for maintaining the disease-free state in our cattle breeding as regards trichophytosis and at the same time a prerequisite for reducing the incidence of trichophytosis in humans. There are also opportunities to offer this product to foreign customers.

The use of an avirulent strain will reduce the cost of disinfectants that would have to be incurred in producing a virulent vaccine and in its practical application.

The method of vaccinating animals with a virulent vaccine is the same as that of live lyophilized cattle trichophyte vaccine (AO No. 201 481); administration of two doses deep intramuscularly in the lumbar or cruciform region with an interval of 10 to 14 days between vaccination and revaccination. Vaccination is recommended in the left and revaccination in the right half of the body. Prophylactic doses for calves aged 1 to 3 months are twice 2.5 cm ³ and for cattle over 3 months twice 5 cm ³ vaccine.

Example 1

AND

Preparation of 160,000 doses of vaccine

The propagation of the production culture takes place on a nutrient medium of the following composition: 10 liters of fresh brewers, 30 l of distilled water, 600 g of agar, 200 g of petone, 800 mg of thiamine and 800 mg of L-glutamic acid. Sterilization is carried out at an overpressure of 60 to 80 kPa for 30 minutes. The hydrogen ion concentration of the broth is adjusted to a pH of 6.6 to 6.9. Diluted wort agar is poured about 300 cm ³ into 2 liter Roux culture flasks. The flasks with broth, closed with cotton plugs wrapped with parchment paper or cellophane, are again outoclaved at 60 to 80 kPa overpressure for 30 minutes. To confirm microbial sterility, the culture flasks with solidified nutrient medium are incubated for 3 to 5 days in a thermostat at +37 ° C.

The inoculum of the production strain CCM F-751 (or CCM F-764) maintained in a lyophilized or frozen state is used to inoculate the culture broth. Inoculation is by pipetting syringe, using 2 to 20X10 ⁷ viable vegetative forms applied per 10 cm ^{3 of} saline per culture flask. Immediately after inoculation, the inoculum should be distributed over the entire surface of the culture medium by circular movements of the culture flasks. Incubation of the production culture takes place at a temperature of +26 to +28 ° C in the position of the culture flasks downwards. The 11 to 18-day culture of the production strain is separated from the broth with metal loops and hoes and homogenized in a homogenizer. Use 1 liter of preservation medium (500 cm ^{3 of} saline + 500 cm ^{3 of} lyophilization medium composition: distilled water 1 liter, gelatine for bacto 50 g, sucrose 75 g) to homogenize the stand from 2 to 4 cultivation bottles. The homogenization time for a stepwise increase in the speed of the homogenizer (6,500 to 10,000 rpm) is 3 to 5 minutes. The vaccine concentrate is filtered through gauze into a storage bottle. The number of microconidia in the collected concentrate sample is microscopically determined and the doses determined for filling the vaccine concentrate are determined to a final concentration of 2.5 to 10 million microconidia per cm ³ .

The vaccine concentrate is dispensed into vials of 20 cm ³ and frozen for at least 6-12 hours at -40 ° C or 4-8 hours at -50 ° C. The first drying stage in the lyophilizer is carried out at a maximum plate temperature of 4-50 ° C and a maximum product temperature of 0 ^° C for 6 to 15 hours. In the second drying phase (18 to 30 h), the maximum plate temperature is - (- 28 ^° C and the maximum product temperature is -25 ° C.) Drying is terminated when the plate and material temperature curves coincide for 12 to 14 hours The dried vaccine vials are closed with rubber stoppers under vacuum and are sealed with aluminum caps.

The vaccine is manufactured under conditions of sterile environment, material and equipment.

The lyophilized vaccine is diluted with diluent A (buffered saline) immediately prior to use.

The following control tests are carried out with the vaccine:

1. The culture purity and bacterial sterility test shall be carried out with 0,5 cm ^{3 of} vaccine and seeded on blood agar, masopepton agar, masopepton broth, glucose agar and Sabouraud agar. The bacterial sterility test is assessed for 72 h (Petri dishes and 7 days (test tubes) at -j-37 ° C and for 9 days at + 20 ° C (test tubes). The culture purity test is terminated after 10 days of incubation. at +26 to +28 ° C.

2. The vaccine harmlessness test is carried out by inoculating 3 cm ^{3 of the} vaccine to two rabbits (2.5 to 3.5 kg) deep intramuscularly. No death or local or general changes in animal health may occur within 14 days after vaccine administration.

3. Test aVirulence of the vaccine. Inject into a shaved, scarified, healthy skin of three guinea pigs (250-350 g) to an area of about 3 x 4 cm 0.2 cm ³ vaccine. Typical trichophytic lesions must not develop at the injection site after inoculation. Observation period: 14 days.

4. Vaccine efficacy assay by determining the number of viable vegetative forms of the production strain. This is done by sowing the diluted vaccine (10 ^-5 and 10 ⁶ dilutions) on Sabouraud agar followed by counting the grown production strain colonies after 9 days incubation at + -26 to +28 ° C. The potency test is satisfactory if it contains 1 cm ^{3 of the} diluted vaccine 2.5 to 10x10 ^with viable vegetative forms of the production strain.

'5. The determination of the concentration of hydrogen ions is carried out by measuring the pH on a pH meter. The pH of the vaccine is 6.0 to 7.5.

6. Determination of residual moisture of the lyophilisate: the maximum permissible value is up to 3%. It is determined from the weight loss of the lyophilisate after drying.

7. The vacuum test shall be carried out by means of a high-frequency electrical vacuum tester. The vacuum is satisfactory if the pressure is less than 2.66 Pa.

Example 2

Preparation of the vaccine using a simple lyophilization medium

Malt extract agar (Imuna, n. P, Saris Michalany) is used as culture medium. Sterilization of the nutrient broth and filling was performed as in Example 1. Inoculation of the nutrient broth with CCM F-751 (or CCM F-764) production strain is performed in an amount of 2 to 20X10 ⁷ viable vegetative forms per culture flask. The culture is incubated at a temperature of +26 to +28 ° C. Cultivation flasks with a growing culture are inspected daily and the purity of the culture is evaluated macroscopically. Cultivation is complete after 11 to 18 days of growth. The culture grown in two to four culture flasks is homogenized in 500 cm ^{3 of} protective lyophilization medium (7.5% or 15% sucrose solution in distilled water) and the suspension is poured over a thin gauze into a stock bottle. The vaccine bottle is sealed with a rubber stopper with parchment paper and placed for 24 hrs in a cold room at +2 to +8 ^° C, or placed in a freezer at -18 ^° C for several days. according to the results of culture purity and bacterial sterility tests (see above), the vaccine concentrate was filled, frozen and lyophilized. Dilution of the concentrate is carried out so that there are 2.5 to 10 million microconidia in 1 cm ^{3 of} vaccine after dilution.

In this way, the lyophilization capacity is saved because the drying time of the lyophilisate is shortened, a smaller amount of the vaccine concentrate is filled into the vials and the contaminated vaccine is not used for lyophilization. The number of batches of product from the same amount of culture medium is the same as in Example 1.

Vaccine freezing and lyophilization are performed at the same ratios as described in Example 1. After drying, the vials are vacuum sealed and tested for vaccine as in Example 1.

The period of expiration of the vaccine is 12 months from the completion of the control tests, in compliance with the principles of storage of organic products (in the dark, dry, at a temperature of +2 to +8 ° C).

Claims (7)

An avirulent vaccine against bovine trichophytosis, characterized in that it contains 2.5 to 10 million viable micro-congenies of the avirulent strain Trichophyton versucosum Bodin 1902 per cm ^{3 of the} diluted vaccine. 2. The vaccine of claim 1 comprising microconidia of an avirulent strain of Trichophyton verrocosum CCM F-751 or CCM F-764. 3. The method of claim 1, wherein the avirulent strain of Trichophyton verrucosum is propagated on a culture medium customary in the propagation of the Trichophyton strain, at a pH of 6.6 to 6.9, at a temperature of 26 to 28 ° C, for a period of time. 11-18 days, the grown culture is separated from the culture invention and homogenized in a sterile protective medium. 4. The method of claim 3, wherein the homogenization in the sterile protective medium is carried out for 3 to 5 minutes. 5. The method of claim 3, wherein the sterile preservative medium comprises saline with gelatin and sucrose or sucrose solution. 6. The method of claims 3 to 5, wherein the vaccine concentrate is filled and subsequently frozen and lyophilized. 7. A process according to claim 6, wherein the drying takes place in two phases, the first drying phase lasting 6 to 15 hours at a maximum product temperature of 0 ° C and the second drying phase lasting 18 to 30 hours at a maximum product temperature of 25 ° C. .