RU2084240C1 - Vaccine "microderm" for dermatophytosis control in animals - Google Patents

Abstract

FIELD: biotechnology, veterinary mycology. SUBSTANCE: vaccine has antigen from the strain Microsporum canis VGNKI N 2293 and antigen from the strain Trichophyton mentagrophytes VGNKI N 27 and sucrose-gelatin stabilizing agent taken at effective ratios. Vaccine shows high immunogenic activity, harmless and high protective effect with respect to both Microsporum canis and Trichophyton mentagrophytes. EFFECT: enhanced effectiveness of vaccine.

Description

 The invention relates to veterinary mycology, in particular to vaccines against dermatophytosis of animals, mainly carnivores and rodents.

 Also known is a vaccine against dermatophytosis of farm animals, in particular cattle, as well as rabbits, containing an antigen from the strain of the fungus Trichophyton mentagrophytes VGNKI N 27, sucrose, gelatin, distilled water / cm. RF patent N 2013445, according to the application N 92015463, cl. C 12 N 1/14, A 61 K 39/00, 1992 /.

 A disadvantage of the known vaccines is that they are intended mainly for the prevention of certain infectious diseases, only trichophytin or microsporia, and with a mixed course of the disease they do not provide the appropriate preventive and therapeutic effect.

 It is known that representatives of carnivores / cats, dogs, etc. / and rodents / rabbits, nutria, etc. / are affected by various types of dermatophytes (Trichophyton and Microsporum), while in one animal in a dermatophytic focus or individual foci with their multiplicity can be observed as Trichophyton mentagrophytes and Microsporium canis / cm. Dvorak F.A. Otenaeek M. Natural relationahip of dermatophytos to the millen of their enistance. A review. Mykosen, 1982, v. 25, Neft 4, p, 197-209.

 The aim of the invention is a vaccine with high immunogenic activity, safety and high protective effect against T. mentagrophytes, also against M. canis.

This goal is achieved in that it additionally contains an antigen from a strain of the fungus Microsporum canis VGNKI N 2293 with a concentration of 70-90 million microconidia / cm ³ in the following ratio / wt./:
Antigen from the strain of the fungus T. mentagrophytes VGNKI N 27 with a concentration of 70-90 million microconidia / cm ³ 20-30
Antigen from the strain of the fungus M. canis VGNKI N 2293 with a concentration of 70-90 million microconidia / cm ³ 20-30
sucrose 10-20
gelatin 1.5-4.0
distilled water the rest.

 For the manufacture of the vaccine using the strain of the fungus Microsporum canis VGNKI N 2293.

 The strain of the fungus Microsporum canis isolated from a dog with dermatophytosis. The properties of the strain are stabilized by the method of stepwise selection according to the growth rate, uniformity of the composition of the population, cultural-morphological and other characteristics.

 The strain M. canis N 2293 was deposited in the collection of the All-Russian State Research Institute for Control, Standardization and Certification of Veterinary Medicines and assigned the number 2293.

 Strain M. canis N 2293 is characterized by the following features and properties.

 Morphological signs. The mycelium is not colored, smooth, branched, septate, with a diameter of 2.0-2.5 microns. Microconidia are numerous, often oval, spherical, pear-shaped, located on the surface of the mycelium in clusters. Size 1.5x4.0 microns. Microconidia with a pronounced prickly bypass shell, spindle-shaped, multicellular / up to 12 cells /. Size 10-25x50-100 microns. Relatively rarely observed are terminal, intercalar, lateral chlamydospores. The size is 3-5 microns in diameter. They usually form in aging cultures or under adverse cultivation conditions.

 Cultural properties. On wort agar it grows in the form of a flat white with a creamy tint, creamy, velvety fluffy, powdery colony, flat on the periphery / closer to the growing edge /, in the center it is strongly folded, deeply growing into agar, cracking is observed along the edges of the folds, a growing edge arachnoid, reverse from dark yellow to brown, darker in the center, colony diameter 82-86 mm / 20-day culture /.

 On agar, Grigoraki grows in the form of a white with a creamy tint, radially folded, velvety powder colony, closer to the growing spider web edge with poorly developed aerial mycelium, the spider web is growing, the reverse is colored, the colony diameter is 72-80 mm / 20-day culture /.

 On agar with wheat flour, it grows in the form of a flat, powdery-powdery, cream-colored colony with a medium developed aerial mycelium, the growing edge is arachnoid, the reverse is not visible, the diameter of the colony is 65-70 mm / 20 day culture /.

 It grows on a potato agar in the form of a flat white color of a spider web colony, with separate sections of fluffy mycelium in the center and around the periphery (the mycelium is mainly substrate /, the growing edge is spider, reverse yellowish, brighter in the center, colony diameter 64-72 mm / 20 daytime culture.

 On Chapek-Doks agar, it grows in the form of a flat white translucent cobwebly colony, in the center is stellate, with poorly developed aerial mycelium, on the periphery the mycelium is substrate, the reverse is not colored around the periphery, in the center is yellow, the colony diameter is 76-78 mm / 20 day culture / .

Spore formation: the maximum level of sporogenesis on wort agar is 66.8-146.8 million / cm ³ . Biochemical properties. The destruction rate of the emulsion layer of the film by the filtrate of the culture fluid after growing the culture of the strain of the fungus 235-290 minutes

Virulent properties. When applying the culture of M. canis N 2293 at a dose of 0.25 million / cm ^{3 of} microconidia on a previously epilated and scarified area of the skin of animals that had not previously had dermatophytosis / microsporia / and were not immunized with vaccines against this disease, specific dermatophytosis develops on days 5-10 hearth. The clinic of experimental dermatophytosis is old, an inflammatory process is noted with varying degrees of skin hyperemia and the formation of exudate, the formation of flaky crusts or scabs.

 Natural disease of animals lasts for 15-40 days, depending on the species. The subcultures of the fungal strain isolated from the experimental focus, according to their characteristics and properties, correspond to the initial culture taken for infection.

 Antigenic properties. With intramuscular administration of a suspension of strain culture in animals / rabbits, nutria, guinea pigs / specific agglutinins (RA) are formed in titers of 1: 80-1: 160.

Immunogenic properties. With a double intramuscular injection of the vaccine from M. canis strain VGNKI N 2293 to rabbits at a dose of 0.5-1.0 cm ³ in 95-100% of the animals after 20-30 days the formation of persistent and intense immunity is observed. Duration two years / observation period /.

 The invention is illustrated by the following examples.

Example 1. For the manufacture of a vaccine culture T. mentagrophytes VGNKI N 27 and M, canis VGGKI N 2293 grown separately on wort agar in test tubes for 12-20 days. Then, a spore suspension is prepared from the cultures of each strain and the beveled wort agar is seeded in the mattresses. Cultures are grown for 12-20 days at a temperature of 26-28 ^o C. After growing cultures of fungal strains are removed from the surface of the nutrient medium with special scrapers in compliance with strict aseptic conditions and placed in sterile jars. The resulting mass of T. mentagrophytes VNGKI N 27 is homogenized in a colloid mill or mixers, adding sterile water to a concentration of 80 million microconidia / cm ³ . Similarly homogenize the mass of the fungus M. canis VGNKI N 2293 mixed in an equal volume. To the resulting mixture of mushroom homogenates add an equal amount of an aqueous solution of sucrose with gelatin, which are thoroughly mixed, packaged in styryl vials and freeze-dried.

The resulting vaccine before freeze drying has the following component composition / wt./:
Antigen from the strain of fungus T. mentagrophytes VGNKI N 27 with a concentration of 80 million microconidia / cm ³ 25,0
Antigen from the strain of the fungus M. canis VGNKI N with a concentration of 80 million microconidia / cm ³ 25.0
Sucrose 10.0
Gelatin 1.5
Distilled water rest.

 Example 2

The vaccine against dermatophytosis of animals is prepared analogously to example 1, using for its preparation a mixture of homogenates from strains of T. mentagrophytes VGNKI N 27 and M. canis VGNKI N 2293 with a concentration of 70 million microconidia / cm ³ taken in a ratio of 1: 1.5.

The resulting vaccine before freeze drying has the following component composition / wt./:
Antigen from the strain of fungus T. mentagrophytes VGNKI N 27 with a concentration of 70 million microconidia / cm ³ 20.0
Antigen from a strain of the fungus M. canis VGNKI N 2293 with a concentration of 70 million microconidia / cm ³ 30,0
Sucrose 15.0
Gelatin 2.8
Distilled water rest.

 Example 3

The vaccine against dermatophytosis of animals is prepared analogously to example 1, using for its preparation a mixture of strains T. mentagrophytes VGNKI N 27 and M. canis VGNKI N 2293 with a concentration of 90 million microconidia / cm ³ taken in a ratio of 1.5: 1.

The resulting vaccine before freeze drying has the following component composition / wt./:
Antigen from the strain of fungus T. mentagrophytes VGNKI N 27 with a concentration of 90 million microconidia / cm ³ 30.0
Antigen from a strain of the fungus M. canis VGNKI N 2293 with a concentration of 90 million microconidia / cm ³ 20.0
Sucrose 20.0
Gelatin 4.0
Distilled water Else.

 The finished lyophilized vaccine is a homogeneous dry porous mass in the form of a gray-beige tablet.

The vaccine retains its biological properties when stored in a dry, dark room at a temperature of 2-10 ^o C. The Microderm vaccine has a shelf life of 12 months.

 Example 4

3 world series of the Microderm vaccine are tested, manufactured according to example 1, according to example 2, previously diluted with a diluent 2 times / 1 series /, 4 times / 2 series / and 6 times / 3 series /, containing 1 vaccination dose / 1 cm ³ / respectively 20 million, 10 million and 6.6 microconidium / cm ³ .

Chinchilla rabbits 265-3,0 kg body weight were vaccinated intramuscularly in the thigh, twice with an interval of 8-10 days in a dose of 0.5 cm ^3/1, Series 2 /, and in a dose of 1.0 cm ^3/1, 2 and 3 series /, using 2 animals goals to test each dose.

30 days after immunization, rabbits are infected with a cutaneous virulent culture of fungi T. mentagrophytes VGNKI N 18 in the scapular region, and a virulent culture of the fungus M. canis VGNKI N s. 20 in the sacral region. 2 weeks after infection, the animals report an allergic reaction in the form of hyperemia and the formation of weak dermatophytic crusts at the site of application. During a clinical examination 19 days after infection, the following findings are established: rabbits immunized with vaccine of the 1st series at a dose of 0.5 and 1 cm ³ did not get sick, from rabbits immunized with the vaccine of series 2 at a dose of 0.5 cm ³ , 1 got sick from the fungus culture M canis N p.20 with a picture of weak dermatophytosis / weak scales are noted at the localization site /, from rabbits immunized with vaccine 3 series 1, the rabbit got sick from a culture of the fungus M. canis N p.20 with a picture of weak dermatophytosis / thin crusts and hair growth under them. In control rabbits, the immunization of which was not carried out, hyperemia, flaky gray crusts, purulent exudate under crusts are noted on the site infected with both cultures.

Thus, for rabbits, the optimal immunizing dose, providing reliable immunity, is a dose of 1 cm ³ with a concentration of 10 million microconidia / cm ³ .

 Example 5. Conduct a clinical examination of the affected skin in 2 dogs and 11 cats. Affected areas of the skin of animals are irradiated with a fluorescent lamp with a Wood filter / OLD 40 /, samples of pathological material are taken from all animals for mycological studies. In 12 animals with skin lesions, an emerald-green glow characteristic of the fungal disease was established, and cultures of M. canis fungus were isolated from pathological material during mycological examination. Diagnosed with microsporia.

Patients and suspected animals are inoculated with the new Microderm vaccine against dermatophytosis of animals according to Example 2 intramuscularly in the thigh area at a dose of 0.5 to 1.0 cm ³ depending on the age and weight of the animals, twice with an interval of 7-10 days.

 All animals recovered. The therapeutic effect occurred on the 15-25th day after repeated administration of the vaccine. At the injection site, there were no reactions in animals, the mycotic focus was loosened, the crusts were separated, and under them a new hair growth was noted. Repeated mycological findings were negative.

 Example 6

Microderm vaccine against dermatophytosis of animals is used for 25 nutrias for preventive purposes, 25 for patients with dermatophytosis for nutrias. The vaccine is used according to example 3, introducing it intramuscularly into the debris, twice with an interval of 7-12 days at a dose of 1.0 cm ³ .

 The vaccine had a high therapeutic and prophylactic effect: all the animals that were given the vaccine for the preventive purpose did not get sick, and in patients one week after the second vaccination their hair began to grow and almost 40 days after it they recovered and became covered with new healthy hair.

 Microderm vaccine is highly effective against trichophytosis and microsporia of carnivores and rodents.

The Microderm vaccine is administered to animals intramuscularly with an interval of 7-12 days: for prophylactic purposes in doses from 0.5 to 2.0 cm ³ depending on the age and type of animals, and for therapeutic purposes from 1.0 to 2.0 cm ³ .

 The use of the Microderm vaccine in veterinary practice will improve the effectiveness of measures to combat dermatophytosis of carnivores and rodents.

Claims (1)

Dermatophytosis vaccine of animals, mainly carnivores and rodents, containing an antigen from the strain of the fungus Trichophyton mentagrophytes VGNKI N 27, sucrose, gelatin and distilled water, characterized in that it additionally contains an antigen from the strain of the fungus Microsporum canis VGNKI N 2293 with a concentration of 70 90 million micronid / cm ³ in the following ratio of components, wt. Antigen from the strain of the fungus Trichophyton mentagrophytes VGNKI N 27 with a concentration of 70 90 million microconidia / cm ³ 20.0 30.0
Antigen from the strain of the fungus Microsporum canis VGNKI N 2293 with a concentration of 70 90 million micronidium / cm ³ 20.0 30.0
Sucrose 10.0 20.0
Gelatin 1.5 4.0
Distilled water Else2