RU2224019C2 - Strain of bacterium klebsiella pneumoniae = 24 mgavmib-dep for producing vaccine against klebsiellosis in agriculture young stock - Google Patents

Abstract

FIELD: veterinary microbiology. SUBSTANCE: invention proposes new strain of bacterium Klebsiella pneumoniae that is deposited in VGNKI at = 24 MGAVMiB-DEP. The proposed strain is isolated in NIL of infectious pathology and biotechnology MGAVMiB from newborn piglet organs in 1987 year. The strain is a producer of α- and β-hemolysins. The new strain shows high yield, 50-60 billion cells in 1 ml by optical turbidity standard of GISK named for K. A. Tarasevich. The proposed strain can be used in veterinary science for producing vaccines against klebsiellosis in agriculture young stock and for preparing immune preparations used for prophylaxis and treatment of acute intestine diseases in animals. EFFECT: valuable properties of strain.

Description

 The invention relates to the field of veterinary microbiology and biotechnology, in particular to the production of Klebsiella pneumoniae strains for the production of Klebsiella vaccines and immune preparations for the prevention of intestinal diseases of farm animals.

 Currently, special attention is paid to the diagnosis of poorly studied infectious diseases, characterized by a severe course, and often fatal. These include Klebsiella infections, which are usually not recognized and pass under other diagnoses. Klebsiella are found in pneumonia, infections of the genitourinary system, acute gastrointestinal diseases, various purulent-septic complications, as well as in nosocomial infections, meningitis, eye infections, ankylosing spondylitis, polyarthritis, etc.

 Klebsiella are of interest to specialists of the veterinary service, botanists and hygienists, since they are also found in animal diseases (pneumonia, mastitis, septicemia), they are isolated from food products, from the surface of vegetables and seeds, flowers, and from water in various reservoirs. Such a widespread distribution of Klebsiella indicates the importance of this problem not only for medicine, but also for the national economy (1).

 Known bacterial strain Klebsiella pneumoniae GISK 233, used as a reference strain in the determination of hemolysins of enterobacteria. The strain is isolated from the clinical material of a patient with OCI.

 The bacterial strain Klebsiella pneumoniae GISK 233 is characterized by the following properties.

 Morphological signs. Gram-negative short sticks, motionless, does not form flagella and spores. When staining revealed a capsule, the diameter of which is 1/2 the diameter of the body of a bacterial cell.

 Cultural properties. On BCH, after 3 hours it gives a slight homogeneous turbidity, after 18-24 hours - diffuse growth, with pronounced moire waves.

On MPA: light, translucent colonies, with smooth smooth edges, can be easily removed, diameter 1.0-1.5 mm, no signs of dissociation. On the Endo medium: pale pink colonies with a brighter dot in the center when viewed in the light, domed, oily, the diameter of the colonies is 1.5-2.0 mm. There are no signs of dissociation. On K ₂ medium: light translucent colonies, with a slight yellowish tint, oily, colony diameter 1.0-1.5 mm. There are no signs of dissociation.

Physico-biological properties. The optimum temperature growth of 37 ± 1 ^o C; pH of the medium is 7.0-7.2. Optimal conditions are aerobic. Actively breaks down glucose and glycerin with the formation of acid and gas, as well as mannitol, sucrose, maltose, arabinose, adonite, inositol, dulcite, xylose, sorbitol, rhamnose, slow-down lactose. Decarboxylates lysine, utilizes sodium malonate and Kozar citrate, as well as Simons, has a delayed urease activity, gives a positive Foges-Proscauer reaction, i.e. ferments glucose to form acetyl-methylcarbinol (acetoin). Does not form hydrogen sulfide and indole; in the reaction, methyl-mouth is negative. Ornigindecarboxylase, erginine dehydrolase and phenylalanine deaminase are absent. The strain is stationary.

 Antigenic structure of the strain during serotyping with a set of sera TsNIIIViS im. Mechnikov is not installed.

 Sensitivity to antibacterial drugs. Serial dilution studies have shown sensitivity to the following antibiotics: neomycin (10 mg / ml), kanamycin (5 μg / ml), gentamicin 82.5 (μg / ml), sisomycin (1 μg / ml), kefzola (5 μg / ml ), claforan (25 μg / ml), chloramphenicol (10 μg / mm), polymyukin B (5 μg / ml), nalidixic acid (10 μg / mm). Resistant to benzylpenicillin (10 μg / mm), ampicillin (1.5 μg / mm), ampiox (1.5 μg / mm), tetracycline (50 μg / mm), erythromycin (250 μg / mm).

 Biological properties. The strain has the ability to stably produce α- and β-hemolysins.

Virulent properties. LD ₅₀ for white mice 2 • 10 ⁹ mt (2).

 The bacterial strain Klebsiella pneumoniae 140, which has a complex of pathogenicity factors, has a registration strain of 245, is also known. The strain was isolated from a sick child with intestinal infection.

 The strain Klebsiella pneumoniae GISK 245, characterized by the following properties.

 Morphological signs. When stained by Gram - gram-negative sticks are located in a smear separately or in pairs.

Cultural properties. When grown on 1.5% MPA (pH 7.2) at a temperature of 35-37 ^o With the strain grows with the formation of turbid mucous membranes, different in structure of the colonies. When grown in Hottinger broth (pH 7.2), a temperature of 33-36 ^o C for 20 h gives intense turbidity.

Methods for determining the activity of LT enterotoxin:
- subplanar administration to mice weighing 15-16 g of 0.05 ml of a centrifuge of a 20-hour broth culture causes paw edema of at least 90 mg (Aksenova E.V. 1980);
- intranasal administration of 0.05 mg of a 20-hour broth culture causes the death of 95% of male mice weighing 15-16 g for 18 minutes with the phenomena of tonic seizures and asphyxiation;
- intramuscular injection of 300 million microbial cells of a 20-hour broth culture causes necrosis of rabbit skin with a diameter of 3.5 cm;
- in the experiment, the loop of the small intestine of the rabbit with the introduction of 500 million MT 20-hour broth culture gives a positive result (y / 1 = 1.2);
- a centrifugate of a 20-hour broth culture heated at 56 ^° C for 45 minutes causes swelling of the paws of mice up to 30 mg, does not cause necrosis of rabbit skin. Heated at 100 ^o With a 20-hour broth culture does not cause fluid accumulation in an isolated loop of the rabbit small intestine (y / 1 = 0.3).

Storage conditions of the culture after leophilic drying when grown in 0.3% MPA (pH 7.2-7.4), filled with sterile liquid paraffin, at a temperature of 4 ^o C.

Propagation media - Hottinger broth (pH 7.2-7.4) containing 5% rabbit erythrocytes at 35-36 ^o C for 20 hours

 Genetic features. Resistant to ampicillin, erythromycin, oxacillin, methicillin, carbenicillin, tetracycline, streptomycin (3).

 The objective of our research was to obtain a strain of Klebsiella pneumoniae, suitable for the production of immune preparations for the prevention and treatment of Klebsiella in farm animals.

 The proposed strain Klebsiella pneumoniae 24, the causative agent of Klebsiellosis, belongs to the family Enterobacteriaceae, the genus Klebsiella, was isolated from the organs of a newborn piglet at the Alekseevsky pig farm, Samara Region, in 1987, at the Laboratory of Infectious Pathology and Biotechnology of the Moscow State Medical Academy of Medicine and Biotechnology.

 The proposed production strain Klebsiella pneumoniae 24 was deposited on April 27, 2001, the registration number is “strain Klebsiella pneumoniae 24 MGAVMiB-DEP”, in the All-Russian State Collection of Microorganism Strains Used in Veterinary and Animal Husbandry (123022, Russia, Moscow, Zvenigorodskoye Shosse, 5, VGNKI).

 The storage location of the production strain of Klebsiella pneumoniae 24, the causative agent of Klebsiellosis, has been determined by the collection of the Research Laboratory of Infectious Pathology and Biotechnology of MGAVMiB named after K.I. Scriabin, 109472, Moscow, ul. Ak. Skryabin, 23, duplicate storage of the Research Institute of Microbiology of the Ministry of Defense of the Russian Federation (610024, Russia, Kirov, Oktyabrsky pr., 121, military unit 23727).

 The proposed production strain Klebsiella pneumoniae 24 has the following properties.

 Morphological signs: Gram-negative, short rods, does not form spores, when stained according to Burri, the presence of capsules is established, does not have flagella, is motionless.

 Cultural properties. On the BCH, after 5-6 hours of growth, a uniform turbidity of the medium is observed with the subsequent formation of a thin surface film.

 On MPA it forms translucent, with smooth edges, large colonies of mucous consistency.

 On the medium, Endo forms pale pink colonies with a reddish center.

 On Ploskirev there are pink colonies with a yellow center. No signs of dissociation were found.

 On 5% blood agar, lysis zones are noted; it is a producer of α- and β-hemolysins.

 Enzymatic properties. It actively breaks down glucose and glycerin with the formation of acid and gas, as well as sucrose, lactose, rhamnose, maltose, sorbitol, mannitol, inositol, does not form indole, hydrogen sulfide, and forms acetylmethylcarbinol in the Voges-Proskauer reaction.

 Antigenic structure. Serotyped by a set of Klebsiella K-serums NIIVS them. Mechnikov as Klebsiella pneumoniae K 2.

 Genetic signs.

 Sensitivity to antibiotics. According to the results of the study using the standard disc method, the strain is sensitive to gentamicin, pefloxacin, sisomycin and ofloxadine.

 Resistant to benzylpenicillin, erythromycin, streptomycin, roxithromycin, clarithromycin, levomycetin.

 The nucleotide composition. DNA (% HC) - 57.9.

 Contains a plasmid with m.v. 60 MD and 2 MD.

Pathogenic properties. Virulent for white mice. LD ₅₀ for intraperitoneal administration is 3.1 • 10 ⁸ live microcells.

 With oral infection of newborn calves after 10-18 hours, the animals showed clinical signs of a disease of the gastrointestinal tract, which indicates the epizootic significance of the strain Klebsiella pneumoniae 24 in the development of the clinical picture of acute intestinal diseases. When infected in association with other enteropathogenic strains, it caused the death of animals on days 5-6. In experiments on white mice, the presence of a synergistic effect in the influence of pathogenicity factors of the strain Klebsiella pneumoniae 24 with other pathogens of acute intestinal infections was established.

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

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

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

 Shelf life 10 years.

Example. The strain Klebsiella pneumoniae 24 is in a lyophilized state. For cultivation, the ampoule is opened under aseptic conditions, diluted with MPB and grown for 4 hours at 37 ^° C, then transferred to a 20-liter bottle with MPB and grown at a temperature of 36-38 ^° C for 18-20 hours under aeration with sterile air from the calculation of 4-5 volumes per volume of nutrient medium per minute. During deep cultivation, the microbial culture, observing the requirements of sterility, is pumped into casein broth in reactors. Sowing density is 100-300 million microcells per 1 cm ³ .

Deep cultivation is carried out in reactors at 37-38 ^o C with vigorous stirring and aeration with sterile air based on the volume of sterile air per volume of the medium 1: 2.

 During the cultivation process, the pH of the medium is adjusted within 7.6-8.0 units. pH

 The yield of the strain is on average 55-60 billion microns. cells in 1 ml according to the optical turbidity standard GISK them. K.A. Tarasevich, which made it possible to use the strain Klebsiella pneumoniae 24 as an antigen in an inactivated associated vaccine and ensured the creation of active immunity against Klebsiella in young farm animals.

 The vaccine using the production strain Klebsiella pneumoniae 24 was tested with a positive result from 1995 to 2001. in Russia and the CIS countries: Belarus, Kyrgyzstan, Uzbekistan, etc. on 11,550 heads of pregnant cows, 7,290 pregnant sows, 4,270 ewes, 2290 fur animals.

 The safety of the livestock is 96-99%.

 The proposed strain can be used to obtain immune preparations for the prevention and treatment of acute intestinal diseases of animals.

Sources of information
1. Prince V. N. Krasnoloveda and B. S. Kiselyov "Klebsiellosis infections", M .: Medicine, 1996, p. 3.

 2. RF patent 2081168, 1997, cl. C 12 N 1/20.

 3. RF patent 2105809, 1998, cl. C 12 N 1/20.

Claims (1)

The bacterial strain Klebsiella pneumoniae, deposited in VGNKI under No. 24 MGAVMiB-DEPT, for the production of vaccines against Klebsiella infection of young farm animals.