RU2051964C1 - Strain of bacterium corynebacterium pseudotuberculosis used as reference and for preparing diagnostic preparations at cavernous lymphadenitis in sheep - Google Patents

Abstract

FIELD: microbiology. SUBSTANCE: preparing strain C. pseudotuberculosis ВГНКИ N 74 R-ДЕП for identification acceleration of cavernous lymphadenitis pathogen in sheep at bacteriological, allergic and serological diagnosis. Molecular properties of strain DNA: spectral indices: A 260/280 = 2.0, A 260/230 = 2.2, concentration - 1200 mcg/ml, melting point is 33.5 C, content of G + C = 47.8 mole%. On the basis of homology of DNA nucleotide sequence strain is concerned to the group III. For disease diagnosis allergen is administrated to sheep by intracutaneous route into subtail wrincle which is prepared from this strain. Reaction recording is carried out after 24 hr. In sick animal inflammatory reaction appeared in the place of diagnosticum administration. No inflammation in health animals. Method ensures to detect additionally up to 80% infected animals as compared with conventional method of diagnosis (by palpation of surface lymphatic nodes). EFFECT: accelerated identification of pathogen.

Description

 The invention relates to veterinary microbiology, epizootology and can be used in the practice of bacteriological laboratories.

 The aim of the invention is to find a strain of bacteria that easily and quickly multiplies in natural and artificial nutrient media and produces a toxin.

Strains of Corynebacterium pseudotuberculosis in domestic collections are absent, although they have been isolated and studied by many researchers [1]
In the foreign literature there is information about strains of C. pseudotuberculosis, from which the Praysnokordin allergen and vaccines were prepared, but due to the low specificity and immunogenicity, they were not used [2-4]
Despite the wide spread of infections in sheep farms, in vivo methods for diagnosing the disease, preventive measures and control measures have not been developed. To a certain extent, this is due to the absence of reference C. pseudotuberculosis strains in domestic collections of microorganisms. In foreign collections of microorganisms, reference strains, judging by the special articles and the Bergi microbial determinant (Hoult J. Bergi Brief bacterium determinant. M. Mir, 1980, pp. 312-319) possess significant variability of phenotic properties, however, information about the genotype is very limited and fits by G + C the content in the DNA of this species is 67.5 mol. and for all pathogens for animals and humans, microorganisms of the genus Corynebacterium ranging from 57 to 60 mol.

 The strain C. pseudotuberculosis N 74R was obtained on March 26, 2017 from the purulent contents of an abscess of the superficial cervical lymph node of a sheep belonging to the Ekaterinoslavsky state farm in the Shcherbakulsky district of the Omsk Region.

 Morphological signs. In smears from cultures of microorganisms in the form of short thick sticks or ovondas, 1.09 ± 0.02 μm in length, 0.68 ± 0.02 μm in thickness. Gram stain. The ends and contour of the cell perceive color more intensely. More often the cells are collected in heaps; in young cultures there are cells located at an angle to each other.

 Cultural, physiological and biochemical characteristics.

 On all culture media, when reseeded from the storage medium, it gives meager growth. With several transfers on MPA, growth becomes more abundant.

On blood agar telluritovom after 2-3 days of incubation at ³⁷ ° C, the organism recovers metal tellurium, as a result of the colony becomes black. They are round in shape, radially striated, with uneven edges, the surface is dull, dry, rough, the texture is crumbly, the colonies move along the surface of the medium when touched.

 On the MPA, R-shaped colonies are gray-white in color, round with uneven edges, with a rough matte surface, fragile, and shift along the surface of the medium.

 On the BCH, the strain grows in the form of a gray-white thin, brittle film, which easily breaks when shaken into individual blocky parietal ring. The environment remains transparent. At the bottom of the tube, a granular precipitate forms, increasing due to a broken film.

 With growth on BCH, the strain produces exotoxin, the maximum amount of which is observed on the third day, which coincides with a decrease in pH.

 The strain is catalytic. Weakly ferment glucose. It does not form indole and hydrogen sulfide, does not dilute gelatin, does not ferment milk, does not convert nitrates to nitrites, does not form urease. Does not ferment sorbitol, maltose, arabinose, mannitol, sucrose, rhamnose, dulcite, raffinose, galactose.

 With intraperitoneal infection of guinea pigs at a dose of 10 MEM / ml, the microorganism causes damage to internal organs: hemorrhagic foci in the lungs, multiple necrotic nodules on the liver, spleen, and intestines. The death of animals occurs in 3-7 days.

Molecular characteristics of DNA strain: 1. Spectral characteristic
A 260/280 2.0
A 260/230 2.2 1200 2. Concentration ug / ml 3. Melting point G + C 73.5 ^C. 4. The value of G + C content of DNA is 47.8 mol 8.

 In the DNA-DNA hybridization reaction with several tens of C. pseudotuberculosis strains, strain N 74R is assigned to group III in terms of the homology of DNA nucleotide sequences (Trofimov I.G. Borisenko M.S. Fomin B.A. Artyushin S.K.).

 Due to the presence in this strain of microorganisms of a number of distinctive properties, from the described analogues, such as the properties of the R-form, the low content of G + C in DNA is 47.8 mol. relation to group III, by the homology of the nucleotide sequences of DNA, it can be used as a reference, as well as for the preparation of diagnosticums (antigens, allergens).

 The strain was assigned the registration number Corynebacterium Pseudotuberculosis N 74R-DEPT in the collection of microorganisms of the laboratory for the control and standardization of drugs against tuberculosis, pseudo- and paratuberculosis of VGNKI veterinary drugs (123022, Moscow, Zvenigorodskoye Shosse, 5), where it is stored.

Claims (1)

 The bacterial stamp of Corynebacterium pseudotuberculosis VGNKI N 74R-DEPT, used as a reference and for the manufacture of diagnostic preparations for sheep caseous lymphadenitis.