RU2013444C1 - Strain of trichophyton mentagrophytes used for control of immunogenic activity of animal trichophytosis vaccines - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: new strain of trichophyton mentagrophytes No. 18 possesses high conidiferous characteristics, homogeneous population, stable virulence properties to ensure efficient control of immunogenetic properties of biopreparation against animal trichophytosis. Offered strain is deposited in microorganism collection, in the VGNKI research institute, of veterinary preparations under No. 18. EFFECT: higher stability and confidence of results as compared with known strains. 3 tbl

Description

 The invention relates to veterinary mycology, in particular to the production of Trichophyton methagrophytes strain used to control the immunogenic activity of vaccines against trichophytosis of animals.

 Known strain Tryhophyton mentagrophytes N 251, used to control the immunogenic activity of the vaccine against trichophytosis of animals.

 The disadvantages of this strain are a decrease in its virulent properties due to its cleavage with a predominance of weakly virulent variants in the population, as well as the variability of its characteristics according to cultural properties, spore formation, which leads to a biased assessment of the immunogenic properties of the animal trichophytosis vaccine.

 The aim of the invention is to obtain a reference strain of Trishophyton mentagrophytes with abundant conidization, uniform population composition, high stable virulent properties, providing control of the immunogenic activity of vaccines against trichophytosis of animals.

 The strain of Trishophyton mentagrophytes was obtained from an epizootic isolated from a guinea pig trichophytosis patient by increasing conid formation by stepwise selection of colonies that are stable according to cultural and morphological characteristics.

 The strain of fungus Trichophyton mentagrophytes is deposited in the collection of microorganisms of the All-Russian State Research Institute for Control, Standardization and Certification of Veterinary Medicines and has a registration number of 18.

 The strain of the fungus Trichophyton mentagrophytes N 18 is characterized by the following features and properties.

 Morphological signs.

 The mycelium is not colored, even, branched, septate, terminal hyphae form half-helices or spirals (up to 5 curls), the diameter of the mycelium is up to 3.0 μm. Macroconidia are numerous oval or elongated, 1.5-5.0 microns. Single macroconidia with a number of partitions up to 7, usually 2-4, size 10-30 × 3-8 microns. Chlamydospores are generally absent. Arthrospores occur in individual sections of the mycelium, not numerous.

 Cultural properties.

 On wort agar grows in the form of flat creamy, powdery colonies, the growing edge is even or cobwebby, the reverse is brown-red, along the periphery with a yellowish tinge. The diameter of the colonies is 80-90 mm (25 day growth).

 On agar, Saburo grows in the form of flat creamy-colored, highly powdery, asteroid-shaped colonies with a small center-like elevation, individual segments of the substrate mycelium are purple, the reverse in the center is creamy with a yellowish tint, and segments are purple. Diameter is 70 mm.

 Genneberg agar grows in the form of flat creamy yellow color of fluffy-powdery colonies, the growing edge is cobwebby, the center reverse is purple-red, yellow along the periphery, diameter 90 mm.

 On Geneneberg agar with BVK, it grows in the form of flat, fluffy white or creamy-white colonies with a bell-shaped elevation in the center, the growing edge is flat, the reverse is not visible, the diameter is 70 mm.

 On agar, Appel and Wollenweber grows in the form of flat, fluffy cream-colored colonies with a bell-shaped elevation in the center, the growing edge is cobwebbed, the reverse in the center is dark yellow, cream-yellow on the periphery, 90 mm in diameter.

 On agar, Grigoraki grows in the form of a dark cream color of strongly folded velvety, powdery colonies, the edge is smooth, the reverse is not visible (dark brown), diameter 90 mm.

 On potato-glycerin agar, it grows in the form of white, flat in the center, slightly folded colonies, aerial mycelium is developed unevenly (sectors), the growing edge is uneven, a ring of less developed aerial mycelium on the periphery, reverse yellow, diameter 68 mm.

 On agar, Plauta grows in the form of flat cream-colored powdery colonies, the growing edge is cobwebbed, the reverse is not painted, sometimes with a purple tint, diameter is 70 mm.

 On agar, Czapek grows in the form of cobwebbed colonies, the mycelium is mostly substrate, in the center is a purple pigment, diameter 70 mm.

Spore formation. The sporogenesis level of strain 18 on wort agar is from 20.8 to 90.8 million / cm ³ .

 Virulent properties.

When applying a homogenized culture of strain Tr. mentagrophytes N 18 together with the growth medium (wort agar) on a scarified and depilated skin area (3 x 3 cm) in the lumbosacral region in rabbits and guinea pigs causes a typical clinic of trichophytosis. The infecting dose for guinea pigs and rabbits is 0.25-0.50 million / cm ^{3 of} microconidia. At the site of application of the culture (5-8 days), a pronounced exudative-inflammatory reaction is noted, the skin surface is eroded with layering of crusts more than 0.5 cm thick on it. Self-recovery of rabbits within 28-50 days.

 Proteolytic activity.

 Strain Tr. mentagrophytes N 18 actively forms a complex of exoproteases when grown for 30-45 days on Saburo's liquid medium or on Chapek's medium with human hair.

PRI me R 1. The culture of strain Tr. mentagrophytes N 18 is grown in wort agar in test tubes at 26 ± 1 ^about C for 14-20 days. 5 cm ^{3 of} sterile distilled water is poured into test tubes with the grown culture, and a spore suspension is prepared using a micro-hook. A 1 cm ^{3 of} the suspension obtained is taken with a pipette, which is subsequently diluted to the required concentration of microconidia, established by counting using a Goryaev camera.

A rabbit test of 5 infecting doses of the culture of the grown Tr strain is carried out. mentagrophytes N 18 containing 0.5; 0.25; 0.125; 0.0625; 0.0312 million / cm ³ microconidia.

 Rabbits are infected with the cutaneous method in the lumbosacral region.

 Virulence is assessed by the duration of the incubation period, the nature and severity of the infectious process, the nature of skin and hair damage (micro picture), and the duration of the disease of the animals.

 The intensity of the manifestations of the clinic of the disease is evaluated in points: 5 points - a pronounced exudative-inflammatory reaction, the formation of crusts with a thickness of 0.5 cm, the formation of erosion; 4 points - the inflammatory-infiltrative reaction is less pronounced, the thickness of the crust is 0.3-0.5 cm; 3 points - the reaction is exudative-inflammatory moderate, the skin is strongly hyperemic with thickening. Crust 0.3 cm thick, no erosion formed; 2 points - hyperemia and puffiness of the skin are poorly expressed, abundant peeling is observed at the places of rubbing the culture, crusts up to 0.3 cm thick.

 The infection results are presented in table. 1.

 From the table. 1 shows that the strain Trichophyton mentagrophytes N 18 shows the correspondence of the severity of the clinical signs of trichophytosis depending on the infectious dose and can serve to control the immunogenicity of vaccines against trichophytosis of animals.

 PRI me R 2. Study the variability of the composition of the population of culture of strain Tr. mentagrophytes N 18 and strain Tr. mentagrophytes N 251 (control). Suspensions of the grown cultures are seeded on the surface of the wort agar in Petri dishes and, after incubation in a thermostat, the total number of grown colonies is counted and the percentage in the population of colonies that differ in shape, morphology and pigmentation is determined.

 The results are presented in table. 2.

 From the table. 2 it follows that the culture of the fungus Tr. mentagrophytes N 18 compared with the culture of the fungus Tr. mentagrophytes N 251 has a fairly homogeneous composition of the fungus population, which remains at a high level until passage 12.

PRI me R 3. Study the virulence of the grown cultures of strains of fungi Tr. mentagrophytes N 18 and N 251 on 12 rabbits weighing 2.5-3.0 kg. Assessment of virulence is carried out according to the initial population as a whole. To do this, the cultured mushroom cultures, after flushing, are diluted with physiological saline, and the concentration of microconidia is calculated using a Goryaev chamber. The suspension is diluted with sterile saline to a concentration of 0.0625-1,000 million / cm ³ and wort-agar is added in an equal volume. A suspension of each strain is rubbed into a pre-clipped and scarified area of the skin in the lumbosacral region. 5 infectious doses containing respectively 0.5 are tested; 0.25; 0.125; 0.0625 and 0.0312 million microconidia. The development of infection and the process of self-healing in animals is observed until complete recovery.

 When determining the virulence of cultures take into account: the timing of self-recovery of animals; the nature of the fungus infection of the skin (macroscopically) and hair (microscopically). The data are presented in table. 3.

 From the table. 3 it follows that the culture of the fungus strain Tr. mentagrophytes N 18 is more virulent for rabbits than the culture of strain N 251. Trichophytosis in rabbits, caused by the culture of the strain of fungus N 18, proceeds on average 10 days longer and in a more severe manifestation of the disease, and on the 35th day of observation in control animals note the absence of skin and hair lesions, the excretion of the fungus from the investigated substrate.

PRI me R 4. Determine the effectiveness of cultures of strains Tr. mentagrophytes N 251 and N 18 to control the immunogenic activity of the Mentavak vaccine. For this, the Mentavak vaccine immunize 9 rabbits intramuscularly, twice with an interval of 9 days at a dose of 1 cm ³ per injection. 6 control rabbits do not immunize. On the 42nd day after the second immunization, 5 vaccinated rabbits and 3 control were infected with a culture of strain Tr in the lumbosacral region cutaneously. mentagrophytes N 251, and 4 other vaccinated and 3 control - culture strain Tr. mentagrophytes N 18.

 On the 32nd day after experimental infection, a clinical examination of the rabbits is carried out and the effectiveness of the control strains is evaluated. It was established: from the number of vaccinated and infected with the culture of strain Tr. mentagrophytes N 251 all 5 rabbits were clinically healthy, and out of 4 vaccinated and infected with culture strain Tr. mentagrophytes N 18, two at the site of infection had typical trichophytosis lesions in the form of thick crusts with severe skin hyperemia, and in the other two, skin hyperemia was weaker, there were no crusts, but peeling was noted, the scales were plentiful in the form of dandruff. In the group of control animals infected with strain N 251 culture, only one rabbit showed severe hyperemia and the presence of trichophytosis crusts at the site of infection. In the second rabbit, lesions were characterized by profuse peeling and weak focal hyperemia. The third rabbit was clinically healthy. All rabbits (control) infected with culture of strain N 18 turned out to be sick and had typical skin lesions for trichophytosis: swelling, hyperemia, formed thick crusts up to 0.5 cm.

 Thus, the use of culture strain Tr. mentagrophytes N 18 when evaluating the immunogenic activity of the test vaccine gives more stable and reliable results.

PRI me R 5. Immunize 10 goals of calves 3-4 months of age with the associated vaccine "Vermet" intramuscularly, twice, with an interval of 10 days, at a dose of 2 cm ³ . 2 months after immunization of these calves, as well as non-immunized (5 animals), calves of the same age are infected with a virulent culture of strain Tr. mentagrophytes N 18 in the middle third of the neck on the left side in the amount of 2-4 million microconidia per infection site. The results of experimental infection take into account after 25 days. It was established: all 10 immunized calves were clinically healthy, and dense, grayish-colored flaky trichophytic foci were found in the calves of the control group at the site of experimental infection.

 The use of culture of the strain of the fungus Tr. mentagrophytes N 18 in the biological industry to control the immunogenic activity of vaccines against trichophytosis of animals will make it possible to more accurately judge the quality of manufactured biological products.

Claims (1)

 The strain of the fungus Trichophyton mentagrophytes VGNKI N 18-DEPT used to control the immunogenic activity of vaccines against trichophytosis of animals.

Images