RU2180575C2 - Bacillus subtilis x-15 strain used to prevent and treat endometritis in cows - Google Patents

Abstract

FIELD: veterinary medicine. SUBSTANCE: the strain is characterized by a complex of morphological and cultural-biochemical features. It represents straight and slightly curved rods with rounded edges 1X3-6 mcm being single, in pairs and in chains. Upon a broth, long and coiled filaments are formed consisted of several indivisible cells. It is characterized by polymorphism. Oval spores are developed which take up 1/2 - 2/3 cell size. They are located centrally or subterminally with no distinct sporangium bulging. Aerobes are mobile at 38 C optimal growth temperature. Rods are dyed positively according to Gram. Upon a meat-peptone agar, they are growing as grayish-white dull nontransparent flat folded dry colonies of rounded shape and average size (D=5- 10 mm). Colonies are tightly united with a substrate, at 2nd-3d having a yellow- colored shade. It releases catalase, acetoin and acethylmethylcarbon. It releases no indole and hydrogen sulfide. It reduces nitrates and nitrites, hydrolizes a starch. It hydrolizes no hippurate. It disintegrates glucose, saccharose, lactose and mannitol by developing an acid, not a gas. It releases proteolytic ferments, gelatin is diluted by a funnel, then - by layers. EFFECT: decreased average duration of therapy against the known "Sporobacterine" preparation. 2 ex _

Description

 The invention relates to veterinary medicine and is a new strain of Bacillus subtilis X-15, used as a probiotic preparation for the prevention and treatment of endometritis in cows.

 One of the means to increase the effectiveness of the prevention and treatment of endometritis is the use of drugs containing live cultures of symbiotic microorganisms. The cultures used for this purpose should have a strong antagonistic effect on a wide range of pathogenic and conditionally pathogenic microorganisms that cause endometritis. They should also be harmless to the animal, not have acute and chronic toxicity, and not cause adverse side effects, such as decreased reproductive ability, teratogenicity and oncogenicity. The strains used should be characteristic of the existing microbiocenosis of the genital organs of cows and take root for a long time on their mucous membranes. It is also desirable to have high resistance to widely reconciled antibiotics and unpretentiousness to cultivation conditions.

 Currently, in veterinary medicine, a strain of Bacillus subtilis 534, VKM B-1666D, is used to successfully prevent and treat endometritis in cows (see 1,2,3). However, this strain obtained from a purulent wound of a person is not characteristic of microbiocenosis of the uterus and vagina of cows. It does not have good survival on the mucous membranes of the genital tract and is sensitive to most antibiotics. The antagonistic properties of this strain with respect to pathogenic and conditionally pathogenic microflora in endometritis are also not strong enough. The biotechnological properties of the strain Bacillus subtilis 534, VKM B-1666D do not allow to achieve a high concentration of microbial bodies when cultured in a nutrient broth, which makes the technology of making a probiotic preparation difficult and expensive from it (see 4,5).

 The problem to which the invention is directed, is to obtain a strain of a microorganism that has all of the above properties, and the technical result is to increase the effectiveness of prevention and treatment of endometritis in cows. (Increased antagonistic properties, better adaptation to the environment of the reproductive organs of cows, improved survival on the mucous membranes, increased resistance to antibiotics and improved biotechnological properties).

 The obtained strain of Bacillus subtilis X-15 has the following characteristics: morphological properties: a 24-hour culture on meat and peptone agar is a straight and slightly curved sticks with rounded edges 1X3-6 microns, located individually, in pairs and in chains. Long convoluted filaments are formed on the broth, consisting of several undivided cells. With a lack of oxygen, they form coccal forms. It is characterized by polymorphism. They form oval spores, occupying 1 / 2-2 / 3 cells, located centrally and subterminally; there is no distinct swelling of sporangium.

Biological properties. Spore formation occurs on agar media on the first day, on broths on the first or second day, with the threads breaking down. Aerobes are mobile, the optimum growth temperature is 38 ^o C, the growth temperature range is from 6 to 50 ^o C. The sticks stain Gram positively, however, under certain conditions they can become gram-negative (the first few generations after prolonged storage, after being passed through the animal’s body).

 Cultural properties. On meat and peptone agar, they grow in the form of gray-white opaque opaque flat folded dry colonies of rounded shape of medium size (D = 5-10 mm). The colonies are tightly fused with the substrate, the edge is indented, on the second or third day they become yellow. With abundant sowing, they form a continuous coating that resembles hoarfrost. When sowing on meat-peptone agar from the broth culture, in addition to the standard ones, two more types of revertant colonies are formed: loose moist consistency and mucous membranes. On the meat and peptone broth on the first day they form a gray-white delicate wrinkled film on the surface. The film does not break, sticks to the loop entirely, on the second or third day it becomes loose, a precipitate appears. On the plates of potatoes on the first day forms a creamy white or yellowish, wrinkled coating. On the second or third day, a red-brown pigment is formed. On gelatin, a plate forms gray rounded, depressed in the middle of the colony with a dilution of the medium. Milk turns and peptones, the environment is neutral. In the thickness of anaerobic agar does not grow.

 Biochemical properties. It releases catalase, acetoin and acetylmethylcarbon, indole and hydrogen sulfide does not. Reduces nitrates to nitrites, hydrolyzes starch, does not hydrolyze hippurate. It decomposes glucose, sucrose, lactose and mannitol to produce acid, but not gas. It releases proteolytic enzymes, liquefies gelatin with a funnel, then in layers.

 Biological properties. Not pathogenic for white mice, rats, guinea pigs and rabbits.

 Additional information. It grows at a concentration of sodium chloride 7%, ethyl alcohol 10%, sucrose 10%. Preserves properties during prolonged cultivation.

 To obtain the strain, the microflora of the vagina and cervix of healthy cows in the Vologda region was studied.

 As a result of selection and research in the Belozersky district veterinary laboratory of the Vologda region, the strain Bacillus subtilis X-15 was isolated from the cervical canal of a healthy deep-shelled cow.

 In a laboratory test of antagonistic properties, it was found that the strain of Bacillus subtilis X-15 inhibits the development of strains of E.colli, Staph albus, Staph. flavus, Staph. aureus. Salmonella sp., Clostridium sporogenes, Clostridium oedematiens, Clostridium perfringens, Peptostreptococcus anaerobius isolated from cows with a clinical form of endometritis.

 The strain Bacillus subtilis X-15 is well adapted to the environment of the genital organs of cows and takes root for a long time on their mucous membranes.

 The strain Bacillus subtilis X-15 is sensitive to chloramphenicol, gentamicin, neomycin and ampicillin, insensitive to streptomycin, polymyxin and tetracycline, not sensitive to benzylpenicillin, erythromycin and lincomycin.

EXAMPLE 1. When cultivating the strain in a composition medium: 10 g peptone, 20 g glucose, 50 ml yeast hydrolyzate, cabbage broth 1:10 to 1000 ml, at a temperature of 38 ^o C and pH = 7.0 under aerobic conditions for three days a concentration of 55 billion mt / ml is achieved.

 EXAMPLE 2. In a production test of the resulting broth culture for prophylactic purposes with intrauterine administration at a dose of 20 ml, it was once possible to reduce the incidence by 35.6% and increase fertility by 25.3% compared with the use of the drug "Sporobacterin" containing the Bacillus strain subtilis 534, VKM B-1666D. When using a culture of a new strain for the treatment of endometritis (intrauterine administration at a dose of 20 ml once a day, daily, for 5-8 days), it was possible to reduce the average duration of treatment by 4.2 days compared with the drug "Sporobacterin".

Sources of information
1. Nikitenko V.I. Bacteria instead of drugs // Science in the USSR, 1991, 4, p. 116-121.

 2. Podberezny V.V., Parikov V.A., Polyantsev N.I. Preventive therapy for postpartum diseases and mastitis of cows // Veterinary medicine, 1996, 2, p. 40-42.

 3. Ivanovsky A.A. A new probiotic - bactocellolactin for various pathologies in animals // Veterinary Medicine, 1996, 11, p. 34-35.

 4. Zhdanov P.I. Biological and epizootological aspects of the production and use of a new probiotic from bacteria of the genus Bacillus in pig husbandry: Dis .... Dr. Vet. Sciences .- Orenburg, 1997, 332 p.

 5. RF patent 5015232/13, priority 02.12.91, class 6 A 61 K 35/74, C 12 1/20. The method of obtaining the drug "Sporobacterin." Mikhailova N.A., Kuznetsova G.P., Shayakhmetov A.Sh., Kunyagana O.V., Shaimukhametov F.A. publ. 1996, Bull. 9, p. 163.

Claims (1)

 The bacterial strain Bacillus subtilis X-15, deposited in the collection of pure cultures of microorganisms of the Department of Epizootology and Microbiology of the Faculty of Veterinary Medicine of the Vologda State Dairy Academy named after N.V. Vereshchagin, used for the prevention and treatment of endometritis in cows.