RU2741097C1 - Mycelial fungi microsporum canis and trichophyton mentagrophytes strains to control nutrient medium growth properties - Google Patents

Abstract

FIELD: veterinary medicine; medicine.

SUBSTANCE: invention refers to veterinary and medical mycology and can be used for assessing nutrient medium growth properties. To control growth properties of nutrient media for diagnosing dermatophytosis caused by mycelial fungi Microsporum canis and Trichophyton mentagrophytes, Microsporum canis strain "FL 79-18 Viev" is proposed, deposited in the Russian National Collection of Pathogenic and Vaccine Strains of Microorganisms-Exciters of Infectious Diseases of Animals of the Federal State Budgetary Scientific Institution Federal Scientific Center - All-Russian Scientific Research Institute of Experimental Veterinary Medicine of the Russian Academy of Sciences under number F-81, as well as strain Trichophyton mentagrophytes "CN 38-18 Viev", deposited in the Russian National Collection of Pathogenic and Vaccine Strains of Microorganisms-Exciters of Infectious Animal Diseases of the Federal State Budgetary Scientific Institution Federal Scientific Centre - All-Russian Scientific Research Institute of Experimental Veterinary Medicine of the Russian Academy of Sciences under the number F-82. Strains are weakly pathogenic for laboratory animals and have typical for the type of cultural properties, method enables to evaluate growth properties of the used nutrient medium, thereby confirming its suitability for use, improving reliability of the obtained results.

EFFECT: advantage of the invention is higher stability and reliability of laboratory diagnostics results of dermatophytosis, as well as reduced time of diagnosing animal dermatophytosis.

2 cl, 9 ex

Description

The invention relates to the field of veterinary and medical mycology, as well as to clinical microbiology, and can be used by a laboratory assistant or physician researcher to assess the growth properties of the nutrient medium.

Dermatophytosis of animals is most often a chronic, contagious disease of the skin surface caused by pathogenic dermatophyte fungi of the genus Microsporum spp. And Trichophyton spp. About 20% of all dermatological diseases are dermatophytosis. The infection is widespread, especially in areas with a humid and warm climate [Savinov, V.A. Prevalence of dermatophytosis in small domestic animals / V.A. Savinov // Advances in medical mycology. 2018.Vol. 19.P. 373-375].

There are more than 40 types of pathogens of dermatophytosis, but the main and most common pathogens among small domestic animals are Microsporum earns and Trichophyton mentagrophytes [Savinov, V.A. Rapid diagnostics of animal dermatophytosis / V.A. Savinov, R.S. Ovchinnikov, A.V. Kapustin, A.A. Gainullina // Agrarian Science. 2019. No. 10. S. 20-24].

Diagnosis of dermatophytosis involves several stages, on the basis of which the final diagnosis is established. There is an initial diagnosis using a Wood's filter fluorescent lamp, which detects M. canis metabolites on the surface of the hair. The research is based on the luminescence of waste products, which have an emerald glow under the rays of the lamp. Another diagnostic method is direct microscopy of the affected hair. The coat is examined for the presence of hyphae and arthrospores located at the hair root. Both methods have a rather low efficiency for a number of reasons, however, they are often limited to only one of the above methods. The gold standard for diagnosing dermatophytosis is sowing the affected hair on nutrient media to isolate a pure culture and identify the pathogen. This method differs in the duration of the determination, however, it allows you to establish the pathogen to the species. For accurate identification, it is necessary to have control strains that will have all the typical properties of pathogens [Ovchinnikov, R.S. Mycological screening of domestic animals is an important way to prevent human dermatophytosis / R.S. Ovchinnikov, P.P. Ershov, A.V. Kapustin, V.A. Savinov, A.G. Gainullina // Advances in medical mycology. 2019. T. 20. S. 712-716].

State of the art

Currently, the following nutrient media are used to diagnose dermatophytosis: Sabouraud agar with chloramphenicol and cycloheximide (http://www.himedialabs.ru/m664-m286), Myobio agar (http://www.himedialabs.ru/m475) , Sabouraud agar with glucose and chloramphenicol (http://www.himedialabs.ru/ml067-m063), Kimmig agar base for mushrooms (http://www.himedialabs.ru/ml232), agar base for dermatophytes - DTM Agar ( http://www.himedialabs.ru/ml88), heart-heart agar CC (http://www.himedialabs.ru/m209), Littman agar base (http://www.himedialabs.ru/m373-m663) , Ulrich agar for the isolation of pathogenic fungi (http://www.himedialabs.ru/arap-ulrich- for- isolation of pathogenic- fungi_t246), a medium for the differentiation of trichophytons (http://www.himedialabs.ru/m531-m532- m533-m534-m535-m536-ml52), mycological agar (http://www.himedialabs.ru/m094-m264-m095-m265), etc.

The manufacturer proposes to use the following dermatophyte strains to control the growth properties of these funds: Trichophyton mentagrophytes ATCC 9533; Trichophyton rubrum ATCC 28191; Trichophyton equinum ATCC 22443; Trichophyton verrucosum ATCC 36058; Microsporum audouinii ATCC 9079; Trichophyton megninii ATCC 12106; Trichophyton tonsurans ATCC 10220. According to the above data, for the control of the commercial media for the diagnosis of dermatophytosis, there is not a single proposed and studied strain of Microsporum canis. This nuance can negatively affect the quality of the diagnostics performed, and lead to false results. In addition, all the above cultures of filamentous fungi are located in a foreign collection, which in turn undermines the principles of import substitution on the territory of the Russian Federation, since it makes all domestic laboratories and testing centers dependent on foreign suppliers.

Known "The strain of the fungus Microsporum canis used to control the immunogenicity of vaccines against animal dermatophytosis" [patent RU 2074252 A publ. 1995.09.27], as well as "The strain of the fungus Trichophyton mentagrophytes, used to control the immunogenic activity of vaccines against animal trichophytosis" [patent RU 2013444 C1 publ. 1994.05.30], as well as "The strain of the fungus Trichophyton mentagrophytes used for the manufacture of a vaccine against trichophytosis of animals" [patent RU 2013445 C1 publ. 1994.05.30]. Since the above strains are intended for the control of immunogenicity or the production of vaccines, they have high virulence, which is not suitable for control cultures used in control culture media, since the test cultures should be non-virulent or weakly virulent.

The technical problem to be solved by this invention is the need to increase the stability and reliability of laboratory diagnostic studies for dermatophytosis through the use of control test cultures of Microsporum canis and Trichophyton mentagrophytes with known properties, carried out using routine diagnostic methods, the need to expand the arsenal of strains, designed to control the growth properties of nutrient media.

Disclosure of the essence of the invention

The technical result of the invention is to increase the stability and reliability of the results of laboratory diagnosis of dermatophysosis in animals and humans, as well as the timing of the diagnosis of dermatophytosis in animals, expanding the arsenal of strains designed to control the growth properties of nutrient media.

To control the growth properties of nutrient media intended for the diagnosis of dermatophytosis caused by filamentous fungi of the species Microsporum canis and Trichophyton mentagrophytes, we offer well-studied and characterized strains of Microsporum canis "FL 79-18 Viev" deposited in the All-Russian State Collection of Pathogenic and Vaccine Pathogens of Infectious Diseases animals FGBNU FSC VIEV RAS under the number F-81, as well as Trichophyton mentagrophytes "CN 38-18 Viev" deposited in the All-Russian state collection of pathogenic and vaccine strains of microorganisms-causative agents of infectious animal diseases FGBNU FSC VIEV RAS under the number F-82. These strains are weakly pathogenic for laboratory animals and have cultural properties typical of the species.

Thanks to the use of the proposed invention, a laboratory assistant or a medical researcher can evaluate the growth properties of the nutrient medium used, thereby confirming its suitability for use, increasing the reliability of the results obtained.

Implementation of the invention

The examples below are illustrative and thus do not limit the scope of the present invention.

Example # 1. Author's strain of the microorganism Microsporum canis "FL 79-18 Viev"

1. Type of microorganism: Anamorph: Microsporum canis (Bodin) Bodin Teleomorph: Arthroderma otae (Hasegawa & Usui) McGinnis Strain FL 79-18 Viev is anamorphic

2. The strain was deposited in the All-Russian state collection of pathogenic and vaccine strains of microorganisms-causative agents of infectious animal diseases of the Federal State Budgetary Scientific Institution FSC VIEV RAS on December 13, 2019 under the number F-81.

3. Basis for deposition: a control strain to assess the growth properties of the nutrient medium.

4. Pathogenicity: in accordance with the classification of microorganisms - causative agents of human infectious diseases, protozoa, helminths and poisons of biological origin according to pathogenicity groups of sanitary rules SP 1.3.2322-08 "Safety of work with microorganisms of III-IV groups of pathogenicity (danger) and causative agents of parasitic diseases »Fungal agents of the genus Microsporum belong to the fourth group of danger and are zoopathogenic.

5. Method of obtaining the strain (when, where and by whom it was obtained): the strain was isolated from a natural host during a mycological study of clinical material from a cat with signs of dermatophytosis (Moscow), in October 2018, in the laboratory of mycology and antibiotics named after V.I. OH. Sarkisov FNTS VIEV RAS, the authors of the strain.

6. Where and by whom the culture was identified: in the laboratory of mycology and antibiotics. OH. Sarkisov FNTS VIEV RAS, the authors of the strain.

7. Methods of identification: the strain was identified on the basis of the studied morphological and physiological properties in accordance with the mycological determinants of clinically significant fungi.

8. Cultural features: the culture of the strain grows on solid nutrient media: Sabouraud's medium, wort-agar.

9. Morphological features:

A. Macromorphological features: colonies on Sabouraud's medium are beige-white, flat, velvety-woolly, in the center there is a bulging eminence. The growing edge is smooth, ciliate. The reverse is ocher yellow. Aerial mycelium is moderately developed, white. Colony diameter on the 10th day of growth is 50-55 mm. Pigment and exudate does not form. As the culture ages, the reverse acquires a darker color.

B. Micromorphological features: mycelium is even, branching, septate, colorless, hyphae width 1-2 microns. Macroconidia are numerous, multicellular, fusiform with a coracoid tip, have from 4 to 8 septa. Microconidia are few, oval, pear-shaped, 1-3 × 1.5-5 microns in size, located directly on undifferentiated hyphae.

B. Additional signs (presence of chlamydospores, arthrospores, sclerotia, etc.) were not identified.

Cultural and morphological characteristics of the strain are typical for the species Microsporum canis.

10. Molecular genetic characteristics of the strain: the strain has been sequenced by the regions of the internal transcribed spacer of the ribosomal RNA gene (ITS) and the P-tubulin gene. Phylogenetic BL AST analysis confirmed that the strain belongs to the species Microsporum canis.

11. Cultivation conditions: the culture of the strain is grown on Sabouraud agar or wort agar (pH 6.2-6.8) at a temperature of 26-28 ° C. Incubation period 7-14 days.

12. Storage conditions: cultures of the strain are stored in a lyophilized state under vacuum in ampoules at a temperature of 4 to 6 ° C or in test tubes on Sabouraud agar under cotton-gauze plugs at a temperature of 4 to 6 ° C. The frequency of reseeding from test tubes once every 6 months, from ampoules - once every 5 years.

Example # 2. Author's strain of the microorganism Trichophyton mentagrophytes "CN 38-18 Viev"

1. Type of microorganism:

Anamorph: Trichophyton mentagrophytes (Robin) Blanchard Teleomorph: Arthroderma benhamiae (Ajello et Cheng) Graser et de Hoog CN 38-18 Viev strain is anamorphic.

2. The strain was deposited in the All-Russian state collection of pathogenic and vaccine strains of microorganisms-causative agents of infectious animal diseases of the Federal State Budgetary Scientific Institution FSC VIEW RAS on December 13, 2019 under the number F-82.

3. Basis for deposition: a control strain to assess the growth properties of the nutrient medium.

4. Pathogenicity: in accordance with the classification of microorganisms - causative agents of human infectious diseases, protozoa, helminths and poisons of biological origin according to pathogenicity groups of sanitary rules SP 1.3.2322-08 "Safety of work with microorganisms of I1I-IV groups of pathogenicity (danger) and causative agents of parasitic diseases »Fungal agents of the genus Trichophyton belong to the fourth hazard group and are zoopathogenic.

5. Method of obtaining the strain (when, where and by whom it was obtained): the strain was isolated from a natural host during a mycological study of clinical material from a dog with signs of dermatophytosis (Moscow region), in June 2018, in the laboratory of mycology and antibiotics named after V.I. OH. Sarkisov FNTS VIEV RAS, the authors of the strain.

6. Where and by whom the culture was identified: in the laboratory of mycology and antibiotics. OH. Sarkisov FNTS VIEV RAS, the authors of the strain.

7. Methods of identification: the strain was identified on the basis of the studied morphological and physiological properties in accordance with the mycological determinants of clinically significant fungi.

8. Cultural features: the culture of the strain grows on solid nutrient media: Sabouraud's medium, wort-agar.

9. Morphological features:

A. Macromorphological features: colonies on Sabouraud's medium are white, convex, velvety-fluffy, in the center there is a crater-like depression. The growing edge is smooth, ciliate. The reverse is ocher brown. Aerial mycelium is well developed, white. Colony diameter on the 10th day of growth is 60-65 mm. Pigment and exudate does not form. As the culture ages, the relief becomes flatter.

B. Micromorphological signs: curved mycelium, septate, branching, colorless. Microconidia are numerous, oval or clavate, located directly on the hyphae, singly or in clusters. Macroconidia are single, are rare, cigar-shaped.

B. Additional signs (presence of chlamydospores, arthrospores, sclerotia, etc.) were not identified.

The cultural and morphological characteristics of the strain are typical for the Trichophyton mentagrophytes species.

10. Molecular genetic characteristics of the strain: the strain was sequenced by the regions of the internal transcribed spacer of the ribosomal RNA gene (ITS) and the gene (3-tubulin. Phylogenetic BLAST analysis confirmed the belonging of the strain to the Trichophyton mentagrophytes species.

11. Cultivation conditions: the culture of the strain is grown on Sabouraud agar or wort agar (pH 6.2-6.8) at a temperature of 26-28 ° C. Incubation period 7-14 days.

12. Storage conditions: stored in a lyophilized state under vacuum in ampoules at a temperature of 4 to 6 ° C or in test tubes on Sabouraud agar under cotton-gauze plugs at a temperature of 4 to 6 ° C. The frequency of reseeding from test tubes once every 6 months, from ampoules - once every 5 years.

Example No. 3. Method for preparing cultures from a lyophilized state for use as test objects

0.3-1.0 ml of sterile saline solution or Sabouraud's liquid medium is added to an ampoule or vial with a lyophilized culture of the strain, stirred until completely dissolved. The resulting mixture is plated on 2-3 dishes with a solid nutrient medium such as Sabouraud agar or wort agar, 0.2 ml for each Petri dish, making injections in three different places in the form of a triangle. Crops are cultivated at a temperature of 28 ° C for 7-14 days. In parallel, inoculation is carried out on control media: MPA, MPB, MPPB to exclude contamination of the material by extraneous microflora.

Example No. 4. Method for preparing cultures from a native state for use as test objects

Using a mycological hook or needle, a sample is taken from the surface of the native culture, trying to capture aerial mycelium. Then the selected material is transferred onto a pure solid culture medium Sabouraud or wort agar and injected at three points in a Petri dish in the form of a triangle.

The culture is able to maintain its viability for more than a month.

Example No. 5. An example of crop growth on Saburo's environment

On the nutrient medium of Sabouraud, M. canis grows quite quickly - on the 7th day, the diameter of the colony reaches 30 mm under the condition of incubation at 28 ° C. Morphology woolly, fluffy, radial growth, colonies prostrate, white-gray or yellowish. The reverse is ocher yellow. By 5-7 days, folds are formed. The edge of the colony is ciliated.

Tr. mentagrophytes on Sabouraud's medium is characterized by intensive growth - on the 7th day, the diameter reaches 30-35 mm at 28 ° C. Colonies are fluffy, round, convex, white, cream or yellowish brown in color. The edge of the colony is flat. The reverse is ocher to red-brown, sometimes yellow.

Example No. 6. An example of culture growth on Sabouraud's medium with chloramphenicol and cycloheximide

On the nutrient medium of Sabouraud, M. canis grows quite quickly - on the 7th day, the diameter of the colony reaches 30 mm under the condition of incubation at 28 ° C. Morphology woolly, fluffy, radial growth, colonies prostrate, white-gray or yellowish.

The reverse is ocher yellow. By 5-7 days, folds are formed. The edge of the colony is ciliated.

Tr. mentagrophytes on Sabouraud's medium is characterized by intensive growth - on the 7th day, the diameter reaches 30-35 mm at 28 ° C. Colonies are fluffy, round, convex, white, cream or yellowish brown in color. The edge of the colony is flat. The reverse is ocher to red-brown, sometimes yellow.

Example No. 7. An example of the growth of cultures on the Sabouraud medium with glucose and chloramphenicol On the Sabouraud medium, M. canis grows quite quickly - on the 7th day, the colony diameter reaches 30 mm under the condition of incubation at 28 ° C. Morphology woolly, fluffy, radial growth, colonies prostrate, white-gray or yellowish. The reverse is ocher yellow. By 5-7 days, folds are formed. The edge of the colony is ciliated.

Tr. mentagrophytes on Sabouraud's medium is characterized by intensive growth - on the 7th day, the diameter reaches 30-35 mm at 28 ° C. Colonies are fluffy, round, convex, white, cream or yellowish brown in color. The edge of the colony is flat. The reverse is ocher to red-brown, sometimes yellow.

Example No. 8. An example of crop growth on DTM medium

On a nutrient medium DTM, a colony of M. canis is characterized by moderate growth. On the 7th day, the diameter of the colony reaches 20-25 mm with an incubation mode of 28 ° C. Colonies are white, round in shape, velvety surface. The morphology is friable, translucent. Radial folds rarely form. The color change of the medium occurs on the 4th-5th day.

Colony Tr. mentagrophytes is of moderate growth. The colony is white, round, the surface is velvety, slightly convex. Has a cotton-like consistency. Color change occurs on 4-5 days.

Example No. 9. An example of the growth of crops on the environment DTM expert

On a nutrient medium DTM, a colony of M. canis is characterized by moderate growth. On the 7th day, the diameter of the colony reaches 20-25 mm with an incubation mode of 28 ° C. Colonies are white, round in shape, velvety surface. The morphology is friable, translucent. Radial folds rarely form. A change in the color of the medium occurs within 3-5 days.

Colony Tr. mentagrophytes is of moderate growth. The colony is white, round, the surface is velvety, slightly convex. Has a cotton-like consistency. The color change occurs within 3-5 days.

Claims (2)

1. Strain of filamentous fungus Microsporum canis "FL 79-18 Viev" for control of growth properties of nutrient media, deposited in the All-Russian state collection of pathogenic and vaccine strains of microorganisms-causative agents of infectious diseases of animals FGBNU FSC VIEV RAS under number F-81. 2. Strain of the filamentous fungus Trichophyton mentagrophytes "CN 38-18 Viev" for control of the growth properties of nutrient media, deposited in the All-Russian State Collection of Pathogenic and Vaccine Microorganisms-Pathogenic Strains of Animal Infectious Diseases FGBNU FSC VIEV RAS under number F-82.