RU2218177C2 - Strain of bacterium salmonella enteritidis = 22 used for preparing vaccine and diagnostic preparation - Google Patents

Abstract

FIELD: veterinary microbiology. SUBSTANCE: strain of bacterium Salmonella enteritidis = 22 shows morphological, cultural and enzymatic properties that are typical for genus Salnonella. By serological properties the proposed strain is agglutinated with salmonellosis monoreceptor sera 09 (1 and 5 complex) H-G. The strain is producer of endotoxin and hemolysine, it shows protein structure and nucleoitide composition of its DNA (% GC) - 53.1. It comprises plamids with molecular mass 60 mDa and is lyzed by polyvalent bacteriophage. Virulent properties: value LD₅₀ for white mice in intraperitoneal administration is 2.4 x 10⁶ live microbe cells. The strain is pathogenic for newborn calves and oral infection in the dose 10 billions microbe cells diarrhea is evident in 24 h and death of animal - in 2-3 day. Concentration of microbe cells in culturing the strain is 40-50 billions by optical turbidity standard GISK named after K. A. Tarasevich, protective effect against infection with virulent strains is on 5-7 day. The strain is useful for preparing vaccines against salmonellosis in animals and for preparing diagnostic preparation and hyperimmune curative sera and globulins. EFFECT: valuable biological and medicinal properties of strain. 1 ex

Description

 The present invention relates to the field of veterinary microbiology, in particular to the production of a strain of Salmonella enteritidis, suitable for use in vaccines and diagnostic preparations against animal salmonellosis.

 In acute cases, salmonellosis is characterized by fever, diarrhea and degenerative processes in the small and large intestines, and in chronic cases, pneumonia.

 The causative agent is Salmonella enteritidis.

 Salmonellosis, including that caused by Salmonella enteritidis, is registered in many countries of the world, regions of Russia and the CIS countries. Over the past decade, it has grown into a serious economic and social problem.

Known strain Salmonella enteritidis 204 VGNKI, which is part of the "Vaccines against salmonellosis of chickens" with a concentration of live microbial cells 175-250 • 10 ⁹ cells / cm ³ .

 The strain Salmonella enteritidis 204 has the following properties.

 Morphological signs. Small gram-negative rods without spores and capsules, have active mobility, milling. The size of the sticks is 2-3x0.6-0.7 microns.

Cultural properties. On the BCH, it forms a uniform turbidity after 5-7 hours of cultivation at a temperature of 37 ^o C, then a delicate film is formed and a gray-white easily breaking sediment precipitates. On MPA it forms after 18 .... 20 h of growth at a temperature of 37 ^o C colonies with a diameter of 0.8-1.5 mm in S - shape, gray-white, transparent, round, smooth, fine-grained, convex. After 2-3 days, the size of the colonies increases to 2-3 mm. Optional aerob.

 Enzymatic properties. It ferments glucose, maltose, mannitol, sorbitol, xylose, levulose, galactose, mannose, trihalose, dextrin, glycerin, dulcite, inositol to form acid and gas, forms hydrogen sulfide, does not form sucrose, lactose, arabinose, raffinose, inulinon, salicin, salicin , does not clot milk, does not form indole, nitrites reduces to nitrates.

 Antigenic structure. It is agglutinated by salmonella monoreceptor “O” serums: 1, 9, 12 and “H” serums: g, 1.7. The non-specific phase “H” of the antigen (1 and 7) is intermittently detected.

Genetic markers - the strain is resistant to streptomycin (150 μg / cm ³ ), rifampicin (200 μg / cm ³ ), trimethoprim (200 μg / cm ³ ). Resistance markers stably remain after 10-fold passages on MPA, 5-fold passage through white mice and 5-fold passage through chickens.

Virulence for white mice. The initial Salmonella enteritidis 126 strain has an LD _{50 of} 18 for Walb / s mice, and 5 microbial cells for outbred white mice. The LD _{50 of} the Rif strain is 36 and 14 microbial cells, respectively, and for the attenuated Salmonella enteritidis 204 strain, 1.6 • 10 ⁶ and 5.6 • 10 ⁶ microbial cells.

 Immunogenicity - the survival rate of white mice and chickens immunized with an attenuated strain of 204, after infection with 5-7 LD5o of the virulent initial strain of Salmonella enteritidis, is 80-95% of the number of animals infected with the death of 90-100% in the control (1).

Also known phage-resistant, attenuated strain of Salmonella enteritidis VGNKI 204-R, which is included as a component with a concentration of (150-250) • 10 ⁹ , cells / cm ³ in the "Preparation against chicken salmonellosis."

 The strain Salmonella enteritidis 204-R, has the following properties.

 Morphological signs. Gram-negative bacillus without spores and capsules, 2-3x0.6-0.7 microns in size, milling.

 Enzymatic properties. It ferments glucose, maltose, mannitol, sorbitol, levulose, galactose, dextrin, glycerin, inositol to form acid and gas, forms hydrogen sulfide, does not form indole, and reduces nitrites to nitrates.

 Antigenic structure. It is agglutinated by monoreceptor salmonella "O" sera: 1.9, 12 and "H" sera: g, 1.7.

Genetic markers. Resistant to streptomycin (150 μg / cm ³ ), rifampicin (200 μg / cm ³ ), trimethaprim (200 μg / cm ³ ). Markers are stably preserved after 10-fold passages on MPA and at 5-fold passages on white mice and chickens.

 Resistant to the bacteriophage F-6 strain.

Virulence. LD ₅₀ for white mice of the Valb / s line - 5.6 • 10 ⁶ microbial cells.

Immunogenicity The survival rate of white mice and chickens immunized with an attenuated strain after infection with 5-7 LD _{50 of the} virulent strain is 80-95% of the number of infected animals, with death in the control - 90-100% (2).

 The objective of our research was to obtain a strain of Salmonella enteritidis, which has a stable low residual virulence, high immunogenicity, and is suitable for the manufacture of salmonella vaccines, as well as diagnostic drugs.

 The proposed bacterial strain Salmonella enteritidis 22, the causative agent of salmonellosis, is assigned to the family Enterobacteriaceae of the genus Salmonella, isolated in the National Laboratory of Infectious Pathology and Biotechnology K.I. Scriabin from the small intestine of the piglet in 1988, the farm "Labinsky", Krasnodar Territory.

 The proposed production strain of Salmonella enteritidis 22 was deposited on April 27, 2001, registration number "Salmonella enteritidis strain 22 MGAVMiB-DEP", in the All-Russian state collection of microorganism strains used in veterinary medicine and animal husbandry (123022, Russia, Moscow, 5 Zvenigorodskoye Shosse, VGNKI) .

 The collection of the research laboratory of infectious pathology and biotechnology of MVABMiB named after K.I. Skryabin, 23), duplicate storage of research institutes of microbiology of the Ministry of Defense of the Russian Federation (610024, Russia, Kirov, Oktyabrsky pr-t, 121, military unit 23527).

 The proposed production strain of Salmonella enteritidis 22 has the following properties.

 Morphological, cultural and enzymatic properties.

Gram-negative, motile, polymorphic bacilli form translucent S - colonies after 18 ..... 24 hours of growth in a dense nutrient medium at a temperature of 36 ..... 38 ^o C.

 Enzymatic activity. It ferments with the formation of acid and gas glucose, rhamnose, sorbitol, mannitol, does not ferment lactose, sucrose, maltose, inositol, does not hydrolyze urea, forms hydrogen sulfide, does not form indole and acetylmethylcarbinol in the Voges-Proskauer reaction.

 Serological properties. It agglutinates with salmonella monoreceptor serum 09 (1 and 5 complex) H-g.

 Toxigenicity. The strain is a producer of endotoxin and hemolysin.

Virulence. LD ₅₀ for white mice intraperitoneally 2.4 • 10 ⁶ live m.

 It is pathogenic for newborn calves with oral infection at a dose of 10 billion cu.m., diarrhea is observed after 24 hours, and death of animals is observed for 2-3 days.

 Protein structure.

 The nucleotide composition of DNA (% HC) is 53.1.

 Contains plasmids with m.v. 60 MD.

 Phagocytosis. Lysed with polyvalent bacteriophage.

The applied method of storing and maintaining the strain. In a lyophilized state under vacuum in hermetically sealed amplitudes at minus 18 ... 20 ^o C after passage through the body of white mice.

 The frequency of reseeding on nutrient media is 15-20 days.

 Transportation conditions. In a lyophilized state under vacuum in hermetically sealed ampoules.

 Shelf life 10 years.

Example. The strain Salmonella enteritidis 22 is grown in casein broth reactors. Sowing density 150-250 billion m. per 1 cm ^{3 of} nutrient medium.

Cultivation is carried out at a temperature of 36-37 ^o C with intensive mechanical stirring and aeration with air, based on the volume of sterile air 1 l of nutrient medium in a ratio of 1: 3. After 16 .... 18 hours of cultivation, the concentration of microbial cells reaches 40-50 billion m. according to the optical turbidity standard GISK im. K.A. Tarasevich, which made it possible to apply the strain for the manufacture of a vaccine against salmonellosis of farm animals and fur animals.

 A vaccine made using the proposed strain provides a protective effect against infection with virulent epizootic strains for 5-7 days.

 The vaccine from the production strain Salmonella enteritidis 22 was tested with a positive result from 1995 to 2001. in Russia and the CIS countries: Belarus, Kyrgyzstan, Uzbekistan, etc. on 11550 heads of pregnant cows, 7290 pregnant sows, 4270 ewes, 2290 fur animals.

 The safety of the livestock is 95-99%.

 The proposed strain will be used for the manufacture of salmonella vaccines and diagnosticums, as well as for the preparation of hyperimmune therapeutic serums and globulins.

Sources of information
1. RF patent 2030916, cl. A 61 K 39/112, 1995

 2. RF patent 2026082, cl. A 61 K 39/112, 1995

Claims (1)

The bacterial strain Salmonella enteritidis VGNKI No. 22 MGAVM and B-DEPT used for the manufacture of vaccines and diagnostic drugs.