RU2218395C2 - Strain of bacterium proteus vulgaris = 25 used for preparing immune preparations - Google Patents

Abstract

FIELD: veterinary bio- technology. SUBSTANCE: strain of bacterium Proteus vulgaris = 25 shows morphological and cultural properties that are typical for genus Proteus. The strain ferments glucose, rhamnose, maltose and sucrose with formation of acid and gas, it doesn't ferment sorbitol, inositol and mannitol. The strain hydrolyzes urea, forms indol and hydrogen sulfide. Foges-Proscower's reaction is negative. By serological properties the strain is agglutinated by adsorbed proteus O- and H-sera as Proteus vulgaris O-16 and H-6. Value LD₅₀ for white mice in intraperitoneal administration is 5.8 x 10⁸ live microbe cells. In oral administration to newborn calves in the concentration 10 billions of microbe cells symptoms of diarrhea are evident in 2-3 days and in 4 day - death of 40% of infected animals. The strain is lyzed by coli-bacterial bacteriophage. The strain Proteus vulgaris = 25 is resistant to: tetracycline, penicillin, streptomycin, polymyxin, levomycetin, gentamycin, erythromycin, pefloxacin. Th strain is sensitive to: sisomicin, ofloxacin, piperacillin, roxithromycin. The yield of microbe mass is 25-30 billions of microbe cells per 1 ml. The strain is useful for preparing immune preparations against proteus infections in agriculture animals. EFFECT: valuable properties of strain. 1 ex

Description

 The present invention relates to veterinary biotechnology and can be used in the preparation of immune preparations against pathogens of acute intestinal infections of farm animals.

 Among opportunistic enterobacteria, the most common causative agents of acute intestinal infections (acute intestinal infections) are Proteus mirabilis and Proteus vulgaris.

 Conditionally pathogenic enterobacteria are the natural inhabitants of the human intestines and many animals, including agricultural ones.

 Conditionally pathogenic enterobacteria are widespread in nature: their constant presence in the water of open reservoirs, especially proteas, has been proved.

 By susceptibility, one can distinguish - the risk group is newborns, weakened children, people with chronic ailments, patients who underwent antibacterial therapy or surgical interventions. From the foregoing, the problem of combating enterobacteria of the genus Proteus (1) becomes apparent.

 A known bacterial strain Proteus vulgaris 154, used as a test object to determine the toxin-forming ability of bacteria of the genus Proteus.

 The bacterial strain Proteus vulgaris 154 is characterized by the following properties.

 Cultural and morphological characters. On the environment of Ploskirev, isolated, translucent colonies of a yellowish hue, with smooth edges, a creamy consistency are formed. The diameter of the colonies is 2-3 mm.

 On the media of Levin, Endo, Wilson-Blair, blood agar, slightly alkaline MPA grows in the form of a gentle translucent coating (the phenomenon of swarming).

 On liquid media gives a homogeneous turbidity of the medium.

 Polymorphic gram-negative motile bacillus. Spore and capsules do not form.

Physico-biochemical properties. The optimum growth is 37 ^o C, the optimum pH is 7.2-7.4.

 Enzymatic properties. It oxidizes and ferments glucose, cleaves urea, forms indole and hydrogen sulfide, does not ferment lactose and dulcite, does not cleave sodium malonate, cleaves maltose and sucrose, does not decarboxylate ornithine, does not have cytochrome oxidase activity. It gives a positive reaction with methyl red and a negative Voges-Proskauer reaction, deaminates phenylalanine, does not form arginine hydrolase and lysine decarboxylase.

 Antigenic formula 047: H1 (Kaufman-Perch scheme).

 Biological properties.

 It is capable of causing early (after 6 hours) death of white mice with intranasal infection as a result of serous-hemorrhagic pulmonary edema.

LD ₅₀ when administered intraperitoneally to white mice in semi-liquid agar is 625 thousand microns. tel.

 The strain causes lamb erythrocytes hemolysis in agar with a hemolysis zone diameter of up to 10 mm.

 Storage of culture - in the thickness of 1% hungry agar under a layer of sterile paraffin oil or in a Dorsi medium.

Biological properties are completely lost after heating for 30 minutes at 56-60 ^o C.

 It produces thermolabile substances such as cholera-like enterotoxin (2).

 The objective of our research was to obtain a strain of Proteus vulgaris, intended for obtaining immune preparations.

 A bacterial strain Proteus vulgaris 25, a causative agent of proteus infection, is proposed, assigned to the family Enterobacteriaceae, to the genus Proteus, isolated in the National Laboratory of Infectious Pathology and Biotechnology, Moscow K.I. Scriabin from the organs of a newborn calf in 1987.

 The proposed production strain of Proteus vulgaris 25 was deposited on April 27, 2001. The registration number is “Proteus vulgaris strain 25 MGAVMiB-DEP” in the All-Russian State Collection of Microorganisms Used in Veterinary Medicine and Animal Husbandry (123022, Russia, Moscow, 5 Zvenigorodskoye Shosse, VGNKI).

 The storage location of the industrial strain Proteus vulgaris 25 of the causative agent of Proteus infection has been determined by the collection of the Research Laboratory of Infectious Pathology and Biotechnology of MGAVMiB named after K.I. Scriabin (109472, Moscow, Scriabin, 23), duplicating storage of the Research Institute of Microbiology of the Ministry of Defense of the Russian Federation (610024, Russia, Kirov, Oktyabrsky Prospect, 121. military unit 23527).

 The proposed industrial strain of bacteria Proteus vulgaris 25 has the following properties.

 Morphological signs. Gram-negative sticks located singly. Are mobile.

 Cultural properties. On dense nutrient media forms a characteristic growth. On the MPA, it has the property of “swarming”, on Ploskirev, it has translucent pinkish colonies with a pearly shade. On the BCH, uniform turbidity with the formation of a supernatant film. On 5% blood agar forms hemolysis zones. Enzymatic properties. It ferments with the formation of acid and gas glucose, rhamnose, maltose, sucrose, does not ferment sorbitol, inositol, mannitol, hydrolyzes urea, forms indole and hydrogen sulfide, the Voges-Proskauer reaction is negative.

 Serological properties. Agglutinated with adsorbed proteic O and H serum produced by NIIVS them. Mechnikov as Proteus vulgaris O16 and H6.

Pathogenic properties. LD ₅₀ for white mice intraperitoneally 5.8 • 10 ⁸ live microbial cells.

 The strain Proteus vulgaris 25, pathogenic for calves, when orally infected with 10 billion suspensions of microbial cells 8-10 hours after infection, general inhibition was noted, body temperature increased and diarrhea of varying degrees was observed in the animal for 2-3 days, and in some profuse diarrhea and on day 4, 40% of infected animals fell.

 Lysed by colibacilli bacteriophage.

 Genetic features.

 Resistant: to tetracycline, penicillin, streptomycin, polymyxin, chloramphenicol, gentamicin, erythromycin, pefloxacin.

 Sensitive: to sisomycin, ofloxacin, piperacillin, roxithromycin.

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

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

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

 Shelf life 10 years.

Example. The preparation of the seed subculture is carried out in two stages. First, they are grown on MPA for 48 hours at a temperature of 36 ... 38 ^o С, then they are transferred to a liquid nutrient medium based on an enzymatic hydrolyzate of meat in a bottle and cultivated at a temperature of 36 ... 38 ^o С with aeration with sterile air at the rate of 4 -6 volumes per volume of nutrient medium for 24-48 hours.

 The biomass yield in this case is 20 ... 30 billion microbial cells.

 Cultivation in reactors is carried out in casein broth (casein hydrolyzate with a content of 200-220 mg% of amine nitrogen, peptone 0.2-0.4%).

 A vaccine made using the Proteus vulgaris 25 strain in it provides a protective effect against infection with virulent epizootic strains on days 5-7.

 The vaccine was tested with a positive result from 1995-2001 in the territory of Russia: Stavropol, Krasnodar Territory, Moscow and Tula Regions and the CIS countries: Belarus, Kyrgyzstan, Uzbekistan.

 The safety of the livestock is 95-99%.

 The proposed strain Proteus vulgaris 25 has a high immunogenic activity and can be used as one of the antigenic components in the associated vaccine against acute intestinal infections of young farm animals.

Sources of information
1. Prince "Guide to the Epidemiology of Infectious Diseases", ed. "Medicine", vol. 2, 1993, pp. 78-80.

2. MKI ⁴ A.S. RF 1497210, C 12 N 1/20; C 12 R 1/37, 1989.

Claims (1)

The bacterial strain Proteus vulgaris VGNKI No. 25 MGAVM and B-DEPT, used for the manufacture of immune preparations.