RU2102075C1 - Method to obtain bacterial preparation - Google Patents

Abstract

FIELD: veterinary science. SUBSTANCE: method deals with cultivating two lactobacillus species upon hepatic beef-extract broth with addition of 1-2% glucose, 0.12% sodium chloride at 37 C and 6-7 pH for 48 h. EFFECT: higher efficiency.

Description

 The present invention relates to the field of veterinary medicine and is intended, in particular, for the treatment of endometritis in cows.

 Known methods for treating endometritis in cows with antibiotics and complex preparations consisting of various antibiotics, sulfonamide and other medicinal substances. However, the wide and prolonged use of such drugs in veterinary medicine and medicine leads to the emergence of resistance to them in microbes that cause inflammatory processes. The effectiveness of the use of drugs decreases, and the duration of treatment increases [5, 6] The antibiotics and sulfonamides used in the treatment of endometritis in cows impair the quality of milk and dairy products. Therefore, the development and use in veterinary medicine of new drugs that do not contain chemotherapeutic agents is an important task.

 Closest to the mechanism of action of the present invention are bacterial preparations lactobacterin and bifidumbacterin. Lactobacterin is a dried mass of live lactobacillus antagonists of Shigella, enteropathogenic Escherichia coli, hemolytic streptococcus and Proteus. Bifidumbacterin is a dried suspension of live antagonistically active bifidobacteria. Both drugs are used in medicine in the treatment of acute intestinal infections, intestinal dysfunctions, chronic colitis, enterocolitis and for genital tract sanitation in inflammatory diseases of the female genital organs [1, 4] The technology for preparing lactobacterin and bifidumbacterin is complex, lengthy and requires expensive equipment for growing, freezing and drying used microbes. This technology has no resemblance to the method of obtaining liquid biosan.

 The purpose of the invention is to develop a method for producing the bacterial preparation "Biosan", which allows to increase the efficiency of treatment of endometritis in cows, reduce the treatment time and use chemotherapeutic agents for this purpose.

 This goal is achieved by the fact that the method includes the separate cultivation on MPPB of two strains of vaginal lactobacilli that have not previously been used in veterinary medicine, and mixing them before packaging in bottles. Biosan (from the words "biological sanitation") includes strains of Lactobacillus plantarum (Kirov-1) and Lactobacillus buchneri (Kirov-4) grown on a liquid nutrient medium. The lactobacilli used are obtained from the natural habitat of the vagina of a healthy woman with a first degree of purity [2, 3]. Both strains exhibit high antagonistic properties against opportunistic microbes that cause endometritis in cows.

 To prepare the biosan, take fresh cattle liver, cut it into pieces, fill it with an equal amount of water and boil for an hour. The resulting extract is filtered through cotton wool and mixed with meat and peptone broth at the rate of 1 part of liver extract in 3 parts of meat and peptone broth. Then, sodium chloride (1.25 g per 1 liter), 2% glucose are added to the medium and the pH is adjusted to 6-7.

The prepared medium is boiled for 15 minutes, filtered, poured into flasks (0.5-2 L), slices of boiled liver (1 g per 2 ml) are added, the medium is autoclaved at 0.5 atm. within 30-40 minutes After that, the uterine culture of lactobacillus plantarum (Kirov-1) and separately lactobacillus buchneri (Kirov-4) are added to the flasks, at the rate of 1 ml of culture per 100 ml of medium, lactobacilli are cultured in an incubator for 48 h at a temperature of 37 ^o C and the amount is determined grown microbes in 1 ml of the drug by serial dilutions.

To prepare uterine broods, 8–9 ml of medium is poured into test tubes, then 1 ml of saline solution from agar with a lactobacillus plantarum strain is added to one of them, and microbes are also grown in an incubator at 37 ^° C in others with a lactobacillus buchneri strain. within 48 hours

The prepared cultures are poured equally under sterile conditions into 12, 50 or 100 ml bottles and closed with rubber stoppers and metal caps using a rolling device. 1 ml of biosan contains 3-3.5 billion lactobacilli. Both strains are stored separately in test tubes in the refrigerator at a temperature of plus 2-4 ^o C and monthly passaged on a jamb of MPA with 1% glucose.

The resulting preparation is a clear light brown liquid with a grayish precipitate at the bottom of the vial. The precipitate breaks easily with shaking and causes turbidity of the drug [3]
There are no analogues to the developed method for preparing the liquid bacterial preparation "Biosan" in veterinary medicine. Studies have shown that lactobacillus biosan is able to take root on the mucous membranes of the genitals in cows for 15 or more days and inhibit the development of conditionally pathogenic microbes (intestinal and Pseudomonas aeruginosa, staphylococci, streptococci, protea) and some pathogenic microbes ( causative agents of dysentery). Store the drug in the refrigerator at a temperature of plus 2-4 ^o C. The shelf life of the prepared drug is 20 days.

When treating 84 cows with acute purulent-catarrhal endometritis with biosan, 82 heads recovered after 1-3 times intrauterine administration of the drug 4 ml each containing 3-3.5 billion lactobacilli (12-15 billion in a dose) (97.6%), of which 72 goals were fertilized (83.3%). The chronic form of endometritis was treated in 168 cows. Moreover, all cows were cured (100%), 139 cows were fertilized (82.7%) [2]
The data presented indicate a high therapeutic efficacy of the use of biosan in the treatment of endometritis in cows. The course of treatment in this case consists of 1-3 intrauterine injections of the drug, which is 2-3 times less than in the treatment of endometritis with chemotherapeutic agents. The product is environmentally friendly and does not lead to a deterioration in the quality of milk.

Sources of information
1. Bakuleva L.P. Zhelnina R.K. Nesterova A.A. A method for the treatment of inflammatory diseases of the female genital area. Description of the invention to the copyright certificate N 1156693. Published on 05.23.85. Bulletin N 19.

 2. Varganov A.I. Guardians K.A. Treatment of endometritis of cows with a vaginal stick. Reports of the Supreme Agricultural Academy, 1983, N 8, p. 38-39.

 3. Varganov A.I. Specifications for Biosan N 10.07.200-91. Approved by the Main Veterinary Administration of the Ministry of Agriculture of the USSR on 10/30/91

 4. Veltishchev E. V. Komarov F.I. Navashin S.M. Handbook of a practitioner. M. Medicine, 1992, volume 2, p. 324-328.

 5. Zavarzin G.A. Microbiology twenty first century. M. Knowledge, 1981, p. 58-59.

 6. Lebedev A.A. The current state of the issue of inflammatory diseases of the female genitalia. Sat "Issues of the pathogenesis and therapy of inflammatory diseases of the female genitalia", Moscow, 1970, v. 5, p. 13.

Claims (1)

A method of obtaining a bacterial preparation from cultures of lactobacilli, including the cultivation of lactobacilli on an artificial nutrient medium, characterized in that strains of lactobacilli Lactobaccillus plantarum (Kirov-1), Lactobacillus buchneri (Kirov-4) are used as lactobacilli, and meat liver is used as nutrient medium broth with the addition of 1 2% glucose, 0.12% sodium chloride, and the cultivation of the strains is carried out at a temperature of 37 ^o C and pH 6 7 for 48 hours, after which the resulting culture is mixed in equal parts.