RU2074251C1 - Strain of fungus trichophyton verrucosum used for preparing vaccine against animal dermatophytosis - Google Patents

Abstract

FIELD: biotechnology, mycology. SUBSTANCE: invention proposes the strain Trichophyton verrucosum DUYR N ТВ-201 showing high level of sporogenesis and immunogenic activity. Antigen-base vaccine prepared from this strain induces stable expressing immunity in cattle, sheep, goats and rabbits 20-30 days after its application. Strain can be used for preparing vaccine against animal dermatophytosis. EFFECT: enhanced effectiveness of vaccine. 4 tbl

Description

 The invention relates to veterinary mycology, in particular to the production of a strain of Trichophyton verrucosum used for the manufacture of vaccines against dermatophytosis in animals.

 Known strain of the fungus Trichophyton verrucosum TF-130 L, used for the manufacture of a vaccine against trichophytosis in cattle. This strain is characterized by its inherent cultural-morphological, spore-forming characters, virulent and antigenic-immunogenic properties (Ed. St. USSR N 955571, class A 61 K 39.00, C 12 N 1/14, 1980).

 As a result of prolonged use of this strain, the level of spore formation has decreased, the degree of dissociation has increased in the direction of increasing the content of weakly sporulating morphological types in its population, as a result of which the vaccine's efficiency has decreased and the costs of its production have increased.

 The aim of the invention is to obtain a strain of the fungus Trichophyton verrucosum, which has a high level of immunogenic activity of sporogenesis, a homogeneous composition of the population, and the ability to manufacture vaccines for the prevention and treatment of dermatophytosis in other animal species.

 according to George's classification, the causative agent of trichophytosis in animals is mainly called Trichophyton verrucosum Bodin, 1902 (syn: T.album Sabouraud, 1908; T.ochraceum Sabouraud, 1909; T.discoides Sabouraud, 1909; T.faviforme-med.ac). Leshchenko V.M. Laboratory diagnosis of fungal diseases, M. Medicine, 1982, p. 34).

 A new strain of the fungus Trichophyton verrucosum was obtained from the population of the fungus strain T.verrucosum TF-130 L (T.verrucosum TF-130 L) by the method of stepwise selection on the basis of spore formation of the fastest growing colonies in the population with certain cultural-morphological characteristics and intensive formation of stable immunogenic microconidia cells (Aleuria).

 The strain of the fungus Trichophyton verrucosum is deposited in the collection of the All-Russian State Research Institute for Control, Standardization and Certification of Veterinary Medicines and has a registration number TV-201.

 The strain of the fungus T.verrucosum TV-201 VGNKI is characterized by the following features and properties.

 Morphological signs. The mycelium is not stained, septate branching. Hyphae are even or crimped, diameter 1.5-5.0 microns, microconidia (aleuria) are spherical, oval, pear-shaped, sometimes rod-shaped in size 2.0x2.0 - 2.0x5.0 microns. Thin-walled macroconidia, as a rule, with a blunt apex, with 1-2 partitions, size 4.0-5.0x8.0-10.0 microns. Arthrospores, as well as terminal and intercalary chlamydospores, are found in aging cultures.

 Cultural signs.

 on wort agar grows in the form of a flat or slightly convex, with a white or light cream, velvety or velvety-fluffy column with a bell-shaped elevation in the center, with a pronounced growth zonality in the form of concentric rings (usually 2-5), with the formation of intense warts on the periphery , the growing edge is even or serrated, cobwebbed, the reverse is not painted or yellow, the diameter of the colony is 40-50 mm (20-25 day old culture).

 On carrot-potato agar grows in the form of a flat, even white, velvety colony with an average degree of development of aerial mycelium and with pronounced concentric zonality (up to 5 rings), the growing edge is even or jagged, slightly cobwebbed, the reverse is not painted or light yellow, darker in the center, the diameter of the colony is 35-40 mm (20-25 day old culture).

 On agar, Saburo grows in the form of a flat, with a bell-shaped elevation in the center, a creamy white colony, a cobwebby powder, the reverse is light yellow, darker in the center, the colony diameter is 40-60 mm (20-25 days old culture).

 On agar, Grigoraki grows in the form of a strongly radially folded, creamy color with a more intense color in the center, velvety powder, closer to the edge of a fluffy colony, the growing edge is even or jagged, the reverse is light yellow, with a wide area (10-12 mm) of clarification of the medium around colonies, diameter 40-60 mm (20-25 days old culture).

 Biochemical properties.

 The rate of destruction of the emulsion layer of the film by filtrate of the culture fluid 125 min

 Spore formation.

The maximum level of spore formation on wort agar is 169.2-211.9 million / cm ³ .

 Virulent properties.

When applying the culture of the fungus at a dose of 10-25 million / cm ^{3 of} microconidia on a previously epilated and scarified area of the skin in animals that previously had no dermatophytosis and were not immunized, a specific dermatophytic focus develops on day 6-10, skin hyperemia, an exudatively inflammatory process, the formation of scaly scab, but the clinic of experimental dermatophytosis has been erased. The period of illness of animals (depending on the species) is 15-30 days. The strain of the fungus isolated from pathological material in its characteristics and properties corresponds to the initial one.

 Antigenic properties.

 On the introduction of the culture of the strain of the fungus in the body of animals (calves, rabbits, etc.) specific agglutinins (RA) are formed in titers of 1: 80-1: 640.

 Immunogenic properties.

When the calves are immunized twice with a vaccine made from this strain in a dose containing at least 20 million / cm ^{3 of} live microconidia, in 98-100% of the animals a stable intense immunity to subsequent infection with a pathogenic fungus is formed.

 The invention is illustrated by the following examples.

 PRI me R 1. Study the variability of the population composition of the culture of the strain of the fungus T.verrucosum TF-130 L and the strain of the fungus T.verrucosum TV-201 VGNKI, Suspensions of the grown cultures are seeded on the surface of the wort agar in Petri dishes and after incubation in an incubator calculate the total number of colonies and determine the percentage in the population of colonies of varying shape, morphology and pigmentation.

When studying the cultures of fungal strains, 3 types of colonies are established:
Type I colony is flat or slightly convex, white or light cream, velvety or velvety-fluffy, with a bell-shaped elevation in the center, with pronounced growth zonality in the form of concentric rings (usually 2-5), with the formation of intense wartiness on the periphery, the growing edge is even or toothed, cobwebby, reverse not colored or yellow.

 The type II colony is convex, white or cream, velvety, unflavored, concentric zoning is absent, the periphery is flat, the growing edge is even or jagged, the reverse is not painted or with a yellow tint.

 The type III colony is convex, hemispherical, white, velvety, fluffy, on the 30th day of growth it can be radially folded, the growing edge is even, the reverse is not painted.

 These morphological types of colonies have different spore-forming abilities. The maximum level of spore formation is observed in type I.

 The ratio of morphological types in the population of strains TF-130L and TV-201 VGNKI are given in table. one.

 As can be seen from the table, the predominant morphological type in strain TB-201 VGNKI is I, while in strain TF-130 L II.

 PRI me R 2. The effect of population reseeding (generation) on the spore formation of cultures of the strain of the fungus T.verrucosum TF-130 L and the fungus strain T. verrucosum TV-201 VGNKI. For this purpose, they are inoculated with suspensions of both cultures on wort agar in test tubes, the crops are grown in a thermostat, the concentration of microconidia is calculated and then transferred to new tubes, repeating the passages until a significant decrease in spore formation is observed.

 The effect of population reseeding on the level of spore formation is presented in table. 2.

 as can be seen from the table, the culture of the strain T.verrucosum TV-201VGNKI in the process of 7-9 transfers (generations) retains the ability to spore formation at a fairly high level. The maximum spore formation is observed at 3-5 reseeding.

PRI me R 3. To obtain a vaccine 15-20 day old culture of a strain of the fungus T. verrucosum TV-201 VGNKI grown in mattress flasks on wort agar (pH 6.8-7.0) at a temperature of 26-28 ^o C within 12-20 days.

The grown culture under aseptic conditions is removed from the surface of the nutrient medium with special scrapers and placed in sterile jars. The fungal mass of strain TB-201 VGNKI is homogenized in mixers, or in blenders, or in colloidal mills, and then, using sterile water, the resulting homogenate is resuspended in the presence of penicillin and streptomycin (100 IU / cm ³ ) to a concentration of microconidia of at least 300 million / cm ³ .

When grinding mushroom mass obtained from cultures from 3 mattresses, a homogenate is obtained from strain TB-201 with a concentration of microconidia of 400-600 million / cm ³ , from strain TF-130 L 200-300 million / cm ³ . To the resulting suspension of mushroom mass, after checking for purity, an equal amount of sucrose-gelatin stabilizer is added, thoroughly mixed, poured into sterile vials and freeze-dried.

The finished vaccine is a homogeneous porous dry mass in the form of a gray tablet. The vaccine retains its biological properties for 12 months if stored in closed, dry, dark rooms at a temperature of 2-10 ^o C. Vials with a sterile diluent are attached to the vaccine.

PRI me R 4. Study the antigenic and immunogenic activity of the vaccine against trichophytosis of animals from strain TB-201 VGNKI prepared according to example 3, in comparison with the known vaccine from strain TF-130 L. Vaccines are used in two groups of rabbits, chinchilla breed, live weight of 2.5-3.0 kg, injected intramuscularly from the inside of the thigh, twice with an interval of 7 days at a dose of 0.6 cm ³ (content in a vaccination dose of 10 million viable microconidia).

 The control group of rabbits is not vaccinated. Blood is taken from rabbits before vaccination and from 7 days after it, every 7 days to 42 days, as well as on the 63rd and 98th day of the experiment. Blood serums are obtained in which the level of antibodies in RA is determined.

 The dynamics of antibodies in rabbits immunized with vaccines based on antigens from fungi strains TV-201 VGNKI and TF-130 L are presented in table. 3.

 The table shows that the vaccine from the strain of the fungus TV-201 VGNKI in comparison with the vaccine from the strain TF-130 L causes the formation of more intense and prolonged immunity in rabbits.

PRI me R 5. Prepared according to example 3, a vaccine against trichophytosis of animals used for vaccination of calves 4-45 days of age. Calves are vaccinated twice in the gluteus muscle in a dose of 1 cm ³ for 20 million viable microconidia. The vaccine prepared from the culture of strain TB-201 VGNKI administered with an interval of 10-14 days. The study of post-vaccination immunity in this case is carried out for 3 years. 45-60 days after 1 immunization, some calves are infected with the conventional cutaneous method using a virulent culture of T.verrucosum 153 fungus at a dose of 5.0 million microconidia per 1 cm ^{2 of} skin. At the same time, in each case, 3-5 non-immunized calves that have not previously had trichophytosis are infected (control).

 The results of the study of post-vaccination immunity to trichophytosis in calves are presented in table. 4.

 The table shows that the vaccine based on the antigen from the strain of the fungus TV-201 VGNKI, develops immunity against trichophytosis during experimental infection. Calves not previously immunized and not suffering from trichophytosis are susceptible to experimental infection with a virulent culture of the pathogen.

 The tests showed that the new strain of the fungus T.verrucosum TV-201 VGNKI has high immunogenic activity, causing animals to generate intense and prolonged immunity, which allows us to recommend it for the manufacture of an effective biological product (vaccine) against trichophytosis of animals.

 An animal trichophytosis vaccine made on the basis of an antigen from a strain of the fungus T.verrucosum TB-201 VGNKI can replace the existing vaccine made on the basis of an antigen from a strain of the fungus T.verrucosum TF-130 L as a higher immunogenic one.

 The biological product is ready for use.

Claims (1)

 The strain of the fungus Trichophyton verrucosum VGNKI N TV-201 VGNKI-DEPT used for the manufacture of vaccines against dermatophytosis of animals.

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