RU2018321C1 - Vaccine against trychophytosis of animals - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: vaccine is additionally treated with antigen of strain Tr. mentagrophytes VGNKI No. 27-DEP. It also contains an antigen of strain Tr. verrucosum 130L and saccharose-gelatin stabilizer in effective proportions. EFFECT: introduction of vaccine into veterinary practice enhances efficiency of fighting trychophytosis in animals. 2 tbl

Description

 The invention relates to biotechnology, in particular to vaccines against trichophytosis of animals.

 The known vaccine LTP-130 and the vaccine TF-130 (K) against trichophytosis of animals, mainly cattle, containing the antigen from strain Trichophyton faviforme N 130 (syn. Tr. Verrucosum) or the antigen from strain Tr. faviforme 130 L and gelatin-sucrose stabilizer [1], [2].

 A disadvantage of the known vaccines is that they prevent the disease in only one animal species - cattle and only one species of the pathogen Tr. faviforme.

 The aim of the invention is to develop a highly immunogenic vaccine against trichophytosis of animals caused by Tr. verrucosum, Tr. verrucosum var. autotrophicum, Tr. mentagrophytes, Tr. equinum, Tr. sarkisovii.

This is achieved by the fact that the vaccine further comprises an antigen from a strain of Tr. mentagrophytes VGNKI N 27 in the following ratio of components (wt./about.%): Antigen from strain Tr. verrucosum 130 L 300-600 million microconidia / cm3 Antigen from strain Tr. mentagrophytes VGNKI N 27 300-600 million microconidia / cm3 Sucrose 10.0-20.0 Gelatin 1.5-4.0 Water Else
For the manufacture of vaccines using strain Tr. mentagrophytes VGNKI N 27.

 Strain Tr. mentagrophytes VGNKI N 27 obtained from epizootic isolated from white mice by the method of directed stepwise selection on the basis of spore formation of the fastest growing colonies with active accumulation of microconidia.

 Strain Tr. mentagrophytes N 27 is deposited in the collection of microorganisms of the All-Russian State Research Institute for Control, Standardization and Certification of Veterinary Medicines and has registration number 27.

 Mushroom Strain Tr. mentagrophytes VGNKI N 27 is characterized by the following features and properties.

 Morphological signs. The mycelium is not colored, even, branched, septate, can decompose into fragments, terminal hyphae do not always form spirals (usually 3-5 curls), the diameter of the mycelium is 2.0-3.0 microns. Microconidia are numerous, round, pear-shaped, elongated, close to rod-shaped, located on the surface of the mycelium in clusters, often separately, the size of 1.5-5.2 microns. Macroconidia are single. The number of partitions 1-5, often 1-2, the size of 10-30 x 3-8 microns. In cultures of 20-30 days of age, round-shaped chlamydospores with a bypass shell can always be observed, always single, 3.0-5.0 microns in diameter. Arthrospores occur in individual sections of the mycelium, thin-walled, in shape resembling oval or rod-shaped microconidia 3.0–9.0 μm. Cultural properties. On wort agar, it grows in the form of a flat white, velvety colony with a slight elevation in the center, the growing edge is even, the reverse is reddish-brown, the diameter of the colony is 50-90 mm (15-20 day old culture).

 On agar, Saburo grows in the form of a flat, white, velvety, cream-colored colony with a slight elevation in the center, the growing edge is flat, the reverse is pale yellow, sometimes darker in the center, the colony size is 50-90 mm (15-20 day culture) .

 On the environment, Genneberg grows in the form of a white, velvety, radially folded colony, a growing cobwebbed edge, a reddish-violet reverse, a colony diameter of 50-80 mm (15-20 day old culture).

 Spore formation. The maximum level of spore formation on wort agar is 198.4 - 259.2 million / cm3.

 Virulent properties. When administered intramuscularly, the culture of strain Tr. mentagrophytes N 27 in guinea pigs, rabbits, sheep, goats, calves 2-3 weeks later, at the injection site, a specific localized surface foci with a diameter of 2.0-2.5 cm develops, which spontaneously heals within 15-20 days. Mycological isolation of the pathogen fails. When a strain of non-immunized animals is applied to a scarified skin at a dose of 0.25 million / cm3 of microconidia, a typical trichophytosis focus develops at the site of application within 10-20 days. The strain isolated from the pathological material corresponds in its properties to the initial one.

 Antigenic properties. On the introduction of the culture of the strain in the body of calves, sheep, rabbits, specific agglutinins are formed in titers 1:40 -1: 1280.

 Immunogenic properties. When administration of a vaccine made from this strain to calves, rabbits twice, in a dose containing 10-20 million live microconidia, the animals develop intense immunity to subsequent infection of trichophythelium with a duration of at least 5 years (observation period).

 Strain Tr. mentagrophytes VGNKI N 27 differs from epizootic by weak virulence, lack of infection upon contact between animals, high activity of microconidia accumulation and immunogenicity after intramuscular administration to animals.

 PRI me R 1. To obtain the vaccine 15-20-day-old culture of strains of Tr. verrucosum 130 L and Tr. mentagrophytes VGNKI N 27 are grown separately in mattress flasks on wort agar (pH 6.2-6.8) at a temperature of 26-28 ° C for 12-20 days.

 Grown mycelium of each culture under aseptic conditions is removed from the surface of the nutrient medium with special scrapers and placed in sterile jars. The mushroom mass of each strain is homogenized in mixers or a colloid mill, and then, using sterile water, the obtained Tr homogenate. verrucosum 130 L is resuspended to a concentration of microconidia of 300 ppm, and the homogenate Tr. mentagrophytes VGNKI N 27-600 million / cm3. Resuspension is carried out in the presence of penicillin or streptomycin at the rate of 100 PIECES per 1 cm3 of homogenate. Homogenate strain of the fungus Tr. verrucosum 130 L is combined in equal volume with the homogenate of the strain of the fungus Tr. mentagrophytes VGNKI N 27. An equal amount of stabilizer (pH 6.2-6.8) containing 10% sucrose and 1.5% gelatin in water is added to the resulting suspension of mushroom mass. All is thoroughly mixed, poured into sterile vials and freeze-dried.

 PRI me R 2. The vaccine is obtained similarly as described in example 1. For its preparation using the homogenate of strains of fungi Tr. verrucosum 130 L and Tr. mentagrophytes VGNKI N 27 with a concentration of microconidia of 450 million / cm3, and as a stabilizer an aqueous solution containing 15% sucrose and 2.5% gelatin.

 PRI me R 3. The vaccine against trichophytosis of animals is prepared in the same way as described in example 1. To obtain it, use the homogenate of the strain of the fungus Tr. verrucosum 130 L with a concentration of microconidia of 600 million / cm3 and the homogenate of strain Tr. mentagrophytes N 27 with a concentration of microconidia of 300 million / cm3. An aqueous solution containing 20% sucrose and 4% gelatin is used as a stabilizer.

 The finished vaccine is a homogeneous porous dry mass in the form of a gray tablet. The vaccine retains its biological properties when stored in closed, dry, dark rooms at a temperature of 2-10 ° C. Shelf life 12 months. Vials with a sterile diluent are included with the vaccine.

 PRI me R 4. The prepared series of the vaccine "Vermet" against trichophytosis of animals according to examples 1, 2, 3 before use is dissolved in a diluent at the rate of 1 dose in 1 cm3. A series of Vermet vaccines is obtained containing the following concentration of antigens in ppm in a vaccination dose (Table 1).

 The vaccine "Vermet" according to examples 1-3 is used to immunize calves, administered intramuscularly twice with an interval of 10-14 days at a dose of 1 cm3.

 As a control, similarly prepared vaccines from the Tr strain are tested. verrucosum 130 L and Tr. mentagrophytes VGNKI N 27 with a concentration of microconidia antigen in a vaccination dose of 7.5 million / cm3. 35 days after the second immunization, calves are infected with virulent cultures of Tr. verrucosum N 153 (TV-153), Tr. mentagrophytes N 251 (TM-251), Tr. sarkicovii N 724 (TC-724), Tr. verrucosum var. autotrophicum 118 (TBA-188). At the same time, six control (non-immunized) calves are infected with the same cultures.

 The results of experimental infection of calves are presented in table. 2.

 The table shows that 1-3 series of the Vermet vaccine in immunized calves develop intense (100%) immunity against trichophytosis during experimental infection. Immunization of calves with monovaccines does not cause a sufficiently stable immunity during subsequent infection with heterologous cultures of trichophytosis pathogens. Calves not previously immunized and not affected by trichophytosis are susceptible to experimental infection with virulent cultures of trichophytosis pathogens.

 PRI me R 5. Three experimental series of the vaccine "Vermet" analogously to example 4, 14 goats are immunized with their subsequent infection with virulent cultures of strains of Tr. verrucosum N 153 and Tr. mentagrophytes N 251. Control animals (2 heads) are not vaccinated.

 All vaccinated animals did not get trichophytosis; all signs of the disease were noted in the control with the release of the indicated strains of virulent cultures from the affected foci.

PRI me R 6. To immunize 10 rabbits of the chinchilla breed with a live weight of 2.5-3.0 kg using the vaccine "Vermet" according to example 2. The vaccine before use is dissolved in a diluent at the rate of 1 dose in 1 cm3. Receive the following concentration of antigens in 1 vaccination dose:
Tr. verrucosum 130 L 7.5 million microconidia
Tr. mentagrophytes VGNKI N 27 7.5 million microconidia The vaccine is administered to rabbits intramuscularly twice at a dose of 0.5 cm3. As a control for vaccination of 10 rabbits using a vaccine from strain Tr. mentagrophytes N 135, containing in 1 vaccination dose of 20 million microconidia in 1 cm3, which is used intramuscularly in the region of the gluteal muscles. After 30 days, the rabbits are cutaneously infected with virulent cultures of Tr. mentagrophytes N 251 and Tr. mentagtophytes N 18. Within 25 days take into account the results of infections. It was established that the use of the Vermet vaccine promotes the formation of reliable immunity in all immunized rabbits, while the use of the well-known Mentavak vaccine does not give a reliable effect - 2 out of 10 rabbits fell ill with trichophytosis, and cultures of virulent strains were isolated from the lesions.

 PRI me R 7. 20 calves are immunized with the Vermet vaccine variants of example 1 and 3 with the concentration of antigens in the vaccination dose of example 4. For each group of calves (10 goals), the vaccine is administered intramuscularly twice in a volume of 1 cm3 with an interval of 10- 14 days. After 4 and 9 months after the second immunization, both groups of calves are infected with virulent cultures of Tr. verrucosum N 153 and Tr. mentagrophytes N 251. As a result, not a single calf became ill. Thus, the Vermet vaccine maintains stable immunity 4 and 9 months after infection.

 The Vermet vaccine is highly effective against trichophytosis of animals. The Vermet vaccine is used prophylactically twice with an interval of 10-14 days in the following doses: calves from 14 days to 4 months 1 cm3, older than 5 months - 2 cm3, and small cattle (sheep, goats) from 3 to 6 months - 0.5 cm3, older than 6 months - 1 cm3. Rabbits weighing 2.5-3.0 kg are administered 0.3-0.5 cm3 for prophylactic purposes. For therapeutic purposes, the vaccine is administered to sick animals twice with an interval of 10-14 days in a double dosage.

 The use of the Vermet vaccine in veterinary practice will increase the effectiveness of measures to combat animal trichophytosis.

Claims (1)

ANIMAL TRICHOFITIA VACCINE, containing an antigen from the Trichophyton verrucosum strain 130 L, sucrose, gelatin and water, characterized in that it additionally contains the antigen from the Trichophyton mentagrophytes strain VGNKI N 27-DEPT in the following ratio of components, wt.%:
Sucrose 10.0 - 20.0
Gelatin 1.5 - 4.0
Water rest,
and also the antigen from the strain Trichophyton verrucosum 130 L and the antigen from the strain Trichophyton mentagrophytes VGNKI N 27-DEPT, taken in equal proportions, in the amount of 300 - 600 million microconidia / cm ³ respectively.

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