RU2005781C1 - Strain of bacterium brucella melitensis biovara i used for preparing of polyvalent hyperimmune serum to brucellas - Google Patents

Abstract

FIELD: microbiology. SUBSTANCE: strain B. melitensis 1-297 is isolated from patient. Strain does not dissociate, it is agglutinated with polyvalent brucellosis serum (up to titer 1: 3200), monoserum antimelitensis (1: 160), but not with monoserum antiabortus. Antigenic properties: in rabbits immunization strain induces formation of specific antibodies. Serum shows positive reaction in IEA: 1: 1827889 1:1827889 ± 155887 155887. Strain B. melitensis ! -297 is deposited in Collection of living culture at No. 791. EFFECT: isolation of strain indicated above.

Description

 The invention relates to microbiology and may find application in the preparation of immunoglobulin from a diagnostic brucellosis dry peroxidase used in an enzyme immunoassay.

 The reference strain of Brucella melitensis 16M is known, which has cultural-morphological and biological properties characteristic of this genus.

 The closest to the proposed technical essence and the achieved positive effect is a strain of Brucella melitensis 565, previously used in combination with a strain of Brucella abortus 146 to obtain polyvalent serum.

 The disadvantage of the above strains is the content of rough colonies in them of more than 8%, which does not allow them to be used to obtain serum to S-antigens.

 The aim of the invention is to find a strain that is in a stable S-form with stable virulence to replace the strain of Brucella melitensis 565.

 The strain Brucella melitensis N I-297 was isolated from the patient, deposited in the collection of live cultures of the brucellosis laboratory of the Scientific Research Institute of Epidemiology and Microbiology named after NF Gamalei Academy of Medical Sciences of the USSR (NIIEM) under N 791 and is characterized by the following cultural, morphological and physiological and biochemical characteristics.

Cultural and morphological characters. Small polymorphic coccobacilli 0.4-0.8 x 0.4-2.2 microns in size, occur one at a time, less often in short chains. Spore and capsules do not form. Motionless. Gram-negative. Martin broth pH 6.8 at ³⁷ ° C over 48 hours give a stable uniform turbidity. On Marten agar with the addition of 1% glucose and 1.5% glycerol, pH 6.8 form convex colonies of regular round shape with equal edges, a smooth surface, moist, oily, whitish in incident light and amber-yellow in transmitted light, with a diameter of 2-3 mm

Physiological and biochemical characteristics. Aerobe. Temperature growth range of 20-40 ^° C Optimum growth temperature 37 ^° C Optimum pH 6.6-7.4. Catalytic. Indole does not form. Nitrates reduces to nitrites. It has high urease activity (reddening of Christensen's medium occurs after 5 min), does not produce hydrogen sulfide, does not lyse with the TB phage (RTD˙10 ⁴ RTD), does not dissociate, is in a stable S-form, resistant to dilutions of thiotine 1: 25, 1: 50, 1: 100 thousand) and fuchsin (1: 50 and 1: 100 thousand). It does not require high content of CO ₂ to culturing. Agglutinable - agglutinated with polyvalent serum to a titer of 1: 3200 (p. 33; 1267, valid. 06.91 g) and antimelitensis monoserum; monoserum antiabortus does not agglutinate.

 The strain is stored in freeze-dried state in ampoules. After lyophilization, all strain properties are preserved. Repeated reseeding and lyophilization do not affect the antigenic properties of the strain (observation period is seven years).

 Antigenic properties. Immunization of rabbits causes the formation of specific antibodies.

 The strain of Brucell melitensis N I-297 differs from the strain of the prototype (Brucella melitensis N 565) in the absence of rough colonies in the suspension and stable virulence.

 The invention is illustrated by the following specific example of the use of strain Brucella melitensis N And-297.

Chinchilla rabbits weighing 2.5-3 kg are immunized with a suspension of living cells in a 0.85% sodium chloride solution of a 48-hour agar culture of Brucella strains Brucella melitensis N I-297 and Brucella abortus 146. Animals are injected subcutaneously three times at four points of the back along the spine, 0.5 ml (2 × ^{10 9} microbes), 0.75 ml (3 × 10 ⁹ microbes), 1.0 ml (4 × ^{10 9} microbes) of the mixture in equal amounts of cultures with an equal volume of Freund's complete adjuvant with a weekly interval. Two weeks after the last injection, 1.0 ml (4 × ^{10 9} microbes) of the culture mixture in a 0.85% sodium chloride solution is injected subcutaneously into the region of the inner upper third of the thigh surface. 14 days after the end of immunization, rabbits are bled. Sera were inactivated by heating at ⁵⁶ ° C for 30 minutes and adding sodium merthiolate 1: 10,000 agglutinating serum colorless, slightly opalescent, has high activity and specificity. It stored at ⁴ ° C in lyophilized form in sealed ampoules. The specific titer of serum with ELISA is 1: 1827889 ± 155817.

 The strain Brucella melitensis N I-297 and Brucella abortus 146 were used to obtain twenty series of rabbit polyvalent hyperimmune serum to brucella.

 Serum is used to obtain immunoglobulin from diagnostic brucellosis peroxidase dry. (56) Joint FAO / WHO Expert Committee on Brucellosis. - Sixth Report World Health Organization. Technical Services 740, World Health Organization, Geneva, 1986. - 133 p.

 Guidelines for the detection of the causative agent of brucellosis by the enzyme immunoassay (S.P. Merinov, T. Yu. Zagoskina, E.P. Golubinsky, O.D. Zakhlebnaya, etc. - Irkutsk. - 1984. - 7 p.

Claims (1)

 The bacterial strain Brucella melitensis biovar I N I-297 GISK N 791, used to obtain multivalent hyperimmune serum to brucella.