RU2051966C1 - Strain of bacterium corynebacterium pseudotuberculosis used as reference and for preparing diagnostic preparations at cavernous lymphadenitis in sheep - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: preparing strain C. pseudotuberculosis ВГНКИ N 70 R-ДЕП for identification acceleration of cavernous lymphadenitis pathogen in sheep at bacteriological, allergic and serological diagnosis of infection. Molecular properties of strain DNA: spectral indices: A 260/280 = 2.0, A 260/230 = 2.4, concentration is 1400 mcg/ml, melting point is 83.3 C, content of G + C = 71.8 mole%. On the basis of homology DNA nucleotide sequence strain is concerned to the group I. For diagnosis allergen prepared of this strain is administrated to sheep by intracutaneous route into subtail wrincle. Reaction recording is carried out after 24 hr. Inflammatory reaction appeared in sick animals at the place of diagnosticum administration. No inflammation in health animals. Method ensured 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, epizotology 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 pseudotuberсulosis in domestic collections are absent, although they have been isolated and studied by many researchers, but they have not found application [1, 4]
In the foreign literature there is information about strains of C. pseudotuberculosis isolated by Carne HR Onon EO Actipn of Cor. ovis exotoxin on ondotelial calls of blod vessels // Nature (London). 1978, 271, p. 246-248. Cassamagnaghi A. The Diagnosis of coseous Lymphadenitis in sheep buthe Intradermal heaction to Preiznocardin // Bull. Acod. Vet. de France. 1931, 4, p. 328. Cameron CM Buchan L. Identification of protective od toxic Antigens of Corynebacterium pseudotuberculosis // Onderstepord I. Vet. Res. 1966, 33, p. 39-48.

 Preisnocordin allergen and vaccines were prepared from them, but because of their high specificity and immunogenicity, they were not used.

 In domestic collections of microorganisms, this causative agent of C preudotuberculosis infection is absent. In special domestic and foreign literature there is information about this type of microorganism, which indicate the huge variability of its properties. In this regard, the determinant of Bergi microbes can serve as a rather good example, where four of the five properties are not constant [3] According to the same determinant of microbes, the content of G + C in the DNA of this species is 67.5 mol. and for pathogenic microorganisms of the genus corynebacteria from 57 to 60 mol.

 The strain was obtained on November 6, 855 from the purulent contents of the superficial cervical lymph node of a ram belonging to the Yekaterinoslavsky state farm in the Shcherbakulsky district of the Omsk Region.

 Morphological signs. The strain has a pronounced polymorphism. In smears from cultures, the microorganism is present in the form of small cocci, ovoids, short thick sticks with rounded ends. The average cell size in length is 0.865 ± 0.03, and the thickness is 0.488 ± 0.02 mmk.

 In smears from young cultures, cells are often located at an angle to each other, indicating a breakable type of division.

 Cultural, physiological and biochemical characteristics. On blood tellurite agar, the strain of microorganisms restores metallic tellurium as a result of the colony becoming black, dull, brittle, are shifted to the surface of the medium.

 On the MPA, R-type colonies are gray-white in color (when serum with a high carotene content is added to the medium, they turn yellow), round, with uneven edges, with a matte rough surface, brittle are shifted along the surface of the medium.

 In older cultures, colonies become radially and concentrically striated with a center raised in the form of a dome.

 On the BCH it grows in the form of gray-white, thin, brittle, film. At the bottom, forms a granular precipitate, increasing due to the frayed and settled film.

Maximum increase in nutrient media celebrated on 2-3 day incubation at ³⁷ ° C under aerobic conditions.

With growth on the BCH, the microorganism strain produces exotoxin, which has strong lethal and hemolytic properties. When cultured with aeration for BCH coefficient of 0.96 and a temperature of 37 ^{° C} for 3-4 hours at reduced pH 0,76-1,0, this coincides with maximum exotoxin accumulation in the medium, 1 ml of which when administered intraperitoneally causes destruction of marine mumps after 8-10 days, and in the rabbit hypersensitivity of the skin, in a dilution of the centrifugal medium in a ratio of 1 to 4.

 The strain contains catalase, does not contain urease and gelatinase, reduces nitrates to nitrites, ferments glucose.

 When intraperitoneal infection of guinea pigs at a dose of 10 MEM / ml causes damage to internal organs: hemorrhagic foci in the lungs, necrotic nodules on the liver and spleen, orchitis.

 In susceptible animal sheep (with subcutaneous infection) in the neck, at a dose of 125 million microbial bodies, after 4 months on slaughter, specific necrotic foci (with greenish-yellow, creamy contents, odorless) can be found in the regional infection site of the lymph nodes, lungs, liver and subcutaneous tissue.

 Additional information about the strain. Microbial cells per gram stain positively, however, the color of the ends is more intense than its middle part.

Molecular characteristics of the strain. 1. Spectral characteristic
A 260/280 2.0
A 260/230 2.4 2. Concentration 1400 μg / ml 3. Melting point / T. square / 83.3 ^about C 4. The value of the content of G + C 71.8 mol.

 A feature of the strain is the difficulty of isolating nucleic acids from DNA.

When studying the homology of the nucleotide DNA sequences of several tens of C. pseudotuberculosis strains isolated from sick sheep of the Omsk region, strain N 70 is assigned to group I [1]
Repeated passages on artificial nutrient media of strain N 70 leads to its transformation into S-form, however, this does not affect the characteristics of DNA.

Strain relatively poorly resistant to elevated temperatures at 55 ° ^C retains its viability for 10 min at 60 ° ^C 0.5 min with a biological protection in 20% serum withstands 55 ^{° C} for 15 min, 60 ^{° C} 3.2 min

 The strain is not sensitive in vitro to polymyxin, but it is detrimental: benzylpenicillin, methicillin, kanamycin, streptomycin, tetracycline, erythromycin, gentamicin, oxacillin, ristomycin, carbenicillin, oleandomycin, monomycin, neomycin, amphetamine.

 In view of the presence of the proposed strain of a number of distinctive properties, from analogues described in the foreign literature, such as: properties of the R-form, a high content of G + C in the DNA of 71.8 mol. relation to a specific (I) group of microorganisms of a given species by the homology of nucleotide DNA sequences; it can be used as a reference strain.

 In addition, strain 70 can be used to prepare diagnosticums (antigens and allergens).

 The strain was assigned the registration number Corunebacterium pseudotuberculosis 70 R-DEPT in the collection of microorganisms of the laboratory for the control and standardization of drugs against tuberculosis, pseudo- and paratuberculosis of VGNKI veterinary preparations (123022, Moscow, Zvenigorodskoye Shosse, 5), where it is stored.

Claims (1)

 The bacterial stamp Corynebacterium pseudotubeculosis VGNKI N 70R-DEPT, used as a reference and for the manufacture of diagnostic drugs for caseous lymphadenitis of sheep.