RU2560572C2 - Method of producing vaccine for prevention and therapy of trichophytosis in cattle - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to field of biotechnology and deals with method of producing vaccine for prevention and therapy of trichophytosis in cattle. Characterised solution includes inoculation of matrasses with culture of fungus Trichopyton verrucosum strain TF-130 L VGNKI on solid nutritional medium, placement of inoculated matrasses in thermostat in horizontal position with nutritional medium upward, collection of culture and realisation of fungal mass homogenisation, its connection with stabiliser in ratio 1:1 and prepackaging into flasks, with cultivation of fungus being realised within 8-10 days at temperature (27±0.5)°C.

EFFECT: claimed invention makes it possible to reduce duration of vaccine production and increase viability of micronidia in finished vaccine.

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Description

The invention relates to the field of biotechnology, in particular to the production of a vaccine for the prevention and treatment of cattle dermatophytosis dry live "LTF-130".

According to the requirements of the current scientific and technical documentation on trichophytosis vaccines, it is necessary that at least 20 million / cm ^{3 of} homogeneous suspension of a culture of the genus Trichophyton verrucosum be administered intramuscularly to the animal for prophylactic purposes. To obtain such a suspension, it is necessary to have a concentration of microconidia in the range of 250-320 million / cm ³ in the initial culture homogenate. In practice, this is achieved in two ways:

- optimization of the regime and time of cultivation of strains of the genus Trichopyton verrucosum;

- the search for a nutrient medium on which strains of the genus Trichophyton verrucosum form the maximum number of microconidia with a high degree of survival.

A known technology for the production of vaccines for the prevention and treatment of trichophytosis of cattle dry live "LTF-130". The technology provides for the inoculation of opaque flasks with solid nutrient medium with a Trichopyton verrucosum fungus culture, laying the seeded flasks in a thermostat in a horizontal (nutrient medium up) position, keeping at a temperature of 26-28 ° C for 11-15 days, removing the culture, homogenizing the mushroom mass , its connection with the stabilizer in a ratio of 1: 1 and packaging in bottles (Instructions for the manufacture and control of the vaccine LTF-130 (VIEV) for the prevention and treatment of trochophytosis (ringworm) in cattle).

A significant drawback of the technology for the production of vaccines against trichophytosis is the long cycle of cultivation of generations (up to 60 days) and the low viability of microconidia (65-70%).

The technical result of the claimed invention is to reduce the time of production of the vaccine and increase the viability of microconidia.

The technical result is achieved by optimizing the temperature regime within (27 ± 0.5) ° C, which allows to reduce the time of cultivation of the fungus from 11-15 days to 8-10 days, obtaining homogenate from a younger culture, with highly sporulating properties that increase the viability of microconidia in finished vaccine.

A method of obtaining a vaccine is as follows.

Studies were performed using the vaccine strain Trichophyton verrucosum TF-130 L VGNKI.

Visual control over the growth of the culture showed that both the 11-15-day culture, aged at a temperature of 26-28 ° C, and the 8-11-day culture, aged at a temperature of (27 ± 0.5) ° C, look like even creeping powdery white mass. Morphological elements are represented by mycelium of various thicknesses and a large number of oval-elongated microconidia in the form of single and clustered clusters. Single macroconidia with transverse septa inside may occur.

As a result of comparative experiments on the 8-10th day of cultivation, high sporogenesis and maximum accumulation of the Trichophyton verrucosum culture were noted.

This is because on the 8-10th day of cultivation in the nutrient medium and in the flask itself there is enough moisture, which means that the nutrient medium retains all its properties and conditions for growing this culture. From 11-15 days there is a dehydration of the nutrient medium - the culture has to survive and its viability decreases. In addition, an 8-10-day culture is removed more easily from the surface of the agar than an 11-15-day culture due to the smaller growth of mycelium in the medium.

Testing the immunogenic activity of Trichophyton verrucosum cultures of strain TF-130 L of VGNKI grown in shortened cycles showed that the experimental vaccine series made from these cultures had the same therapeutic and prophylactic effect with the control series made according to the previously used scheme.

Experience 1.

Inoculated with 250 culture mattresses of the fungus Trichopyton verrucosum strain TF-130 L VGNKI. Cultivated at a temperature of (27 ± 0.5) ° C. Removal and homogenization were carried out on days 8, 9, 10, 11, 15 in an amount of 50 mattresses. The homogenate was combined with a stabilizer in a ratio of 1: 1, 2.5 cm each was packaged in bottles and freeze-dried. After lyophilization, the vaccine was put under control, which was carried out in accordance with the requirements of STO 00482861-0071-2012. The total concentration and concentration of viable microconidia was determined in each of the three bottles of controlled series. The control results are presented in table 1.

Experience 2.

Inoculated with 250 culture mattresses of the fungus Trichopyton verrucosum strain TF-130 L VGNKI. Cultivated at a temperature of (27 ± 0.5) ° C. Removal and homogenization were carried out on days 8, 9, 10, 11, 15 in an amount of 50 mattresses. The homogenate was combined with a stabilizer in a ratio of 1: 1, 2.5 cm. Were put into a vial. After lyophilization, the vaccine was put under control, which was carried out in accordance with the requirements of STO 00482861-0071-2012. The total concentration and concentration of viable microconidia was determined in each of the three bottles of controlled series. The control results are presented in table 2.

Experience 3.

Inoculated with 250 culture mattresses of the fungus Trichopyton verrucosum strain TF-130 L VGNKI. Cultivated at a temperature of (27 ± 0.5) ° C. Removal and homogenization were carried out on days 8, 9, 10, 11, 15 in an amount of 50 mattresses. The homogenate was combined with a stabilizer in a ratio of 1: 1, 2.5 cm. Were put into a vial. After lyophilization, the vaccine was put under control, which was carried out in accordance with the requirements of STO 00482861-0071-2012. The total concentration and concentration of viable microconidia was determined in each of the three bottles of controlled series. The control results are presented in table 3.

The cultivation scheme of the production strain of Trichopyton verrucosum TF-130 L VGNKI was optimized.

An analysis of the results shows that this development reduces the vaccine production cycle by 12–20 days (previously the generation cultivation cycle was 60 days, the proposed scheme was 40–48 days), and it allowed increasing the concentration of viable microconidia by 10–20% (previously the number of viable microconidia was 65-70%, and in the proposed scheme 75-89%).

Claims (1)

A method for the production of a vaccine for the prevention and treatment of cattle trichophytosis, including sowing mattresses with a Trichopyton verrucosum fungus strain TF-130 L VGNKI on solid nutrient medium, laying the seeded mattresses in a thermostat in a horizontal position with the nutrient medium up, harvesting and carrying out the homogenization of the mushroom mass, its connection with the stabilizer in a ratio of 1: 1 and packaging in bottles, characterized in that the cultivation of the fungus is carried out within 8-10 days at a temperature of (27 ± 0.5) ° C.