RU2166324C2 - Veterinary probiotic preparation - Google Patents

Abstract

FIELD: biotechnology, veterinary science. SUBSTANCE: preparation has bacteria of the starin Bifidobacterium globosum ВГНКИ БФ-4-ДЕП, strain Streptococcus faecium ВГНКИ 27/11-В-ДЕП and combination of virulent polyreceptor staphylococcus bacteriophages. Preparation has aluminium oxide, aluminium hydroxide, carbohydrate and filling agent also. Among staphylococcus strains the preparation has strains ВГНКИ 89/ЦМЭИ-FS-1-ДЕП and ВГНКИ 90/ЦМЭИ-BS-2- ДЕП. Among carbohydrates the preparation can contain lactose and/or sucrose and among filling agents it can contain starch, dry milk, dry whey, whole milk substitute, sugar powder or food sugar. Preparation can contain additionally also dry bacterial mass of acidophilic bacteria and/or dry culture of bacteriophages of pseudomonade strains ВГНКИ 86/ЦМЭИ-AI-ДЕП, ВГНКИ 87/ЦМЭИ-B2-ДЕП, ВГНКИ 88/ЦМЭИ- -C1-ДЕП taken in different combinations. Preparation shows simultaneous prophylactic and curative efficiency in intestine disbacteriosis, it promotes to sanitation of animal intestine from staphylococci and Bacillus pyocyaneus, it shows broad spectrum of antibacterial and phagolytic activity and enhances immune status of animal body. EFFECT: enhanced effectiveness of preparation. 6 cl, 2 tbl, 4 ex

Description

 The invention relates to the field of biotechnology and veterinary medicine, in particular to the creation of a biological product having a wide spectrum of antibacterial and phagolytic activity, as well as capable of increasing the immune status of animals and birds and restoring their normal microflora.

 The known probiotic preparations SBA, STF-1/56, designed to restore intestinal biocenosis due to the antagonistic properties of strains of probiotic bacteria (Antipov V.A., Ermakova T.N., Kosheleva E.K., Badalyan K. M. "Application SBA as a growth-stimulating agent // All-Union Conf. "Microorganisms - stimulants and inhibitors of plants and animals" October 3 - 5, 1989: Abstract, Part 1, Tashkent, 1989, p. 14; Mishurnova V.N. , Kirzhaev F.S., Antagonistic properties of the drug STF-1/56 relative to salmonella and E. coli // Veterinary medicine, 1988, 10, p. 34- 36).

 The mechanism of action of probiotics is based on the adhesive and antagonistic properties of probiotic bacteria, which displace conditionally pathogenic microorganisms from the intestinal population and non-specifically control their growth redundancy.

 However, known probiotic preparations have a limited spectrum of action and have weak antagonistic activity against staphylococci and practically do not inhibit the growth of Pseudomonas aeruginosa (Antipov A.V., Prozorovskaya K.N., Ivanina E.K. et al. // Problems of staphylococcal infections ". Saratov, 1986, part 1. P. 9 - 10).

 However, it is known that intestinal dysbiosis is accompanied by a general decrease in the body's natural resistance and is often complicated by infections caused by staphylococci, pseudomonas and enteropathogenic bacteria (Blokhina I.N., Dorofeychuk V.G. Dysbacteriosis. M., 1979; Korshunov V.M. , Pinegin BV, Volodin NN et al. - Study of intestinal microflora in the formation of postradiation and chemotherapeutic dysbacterioses. // In collection "Theoretical and practical problems of gnotobiology", M., 1986, p. 190).

 The excessive presence in the intestinal biocenosis of representatives of these groups of microorganisms complicates the process of prevention and treatment of gastrointestinal diseases and reduces the effectiveness of probiotics.

 Known medicinal preparations of bacteriophages used for sanitation of the gastrointestinal tract from staphylococci and Pseudomonas aeruginosa (In the book: Handbook of a practitioner. // Veltishchev Yu.I., Komarov F.I., Navashin S.M. et al. Ed. A.I. Vorobeva. - 7th ed., Moscow: ONIKS Publishing House, 1998; Actual Issues of Bacteriophagy and Applied Immunology. - Tbilisi, 1984, p. 168-266; G.G. Bogovazova, N .N. Voroshilova, V.M. Bondarenko et al. - Immunobiological properties and therapeutic efficacy of Klebsiella bacteriophage preparations. // ZhMEI, 1992. N 3, p.31-33).

However, known preparations of bacteriophages do not have a sufficiently wide spectrum of lytic activity. The possibility of phagoresistant forms appearing in the population of pathogenic microorganisms is of the order of 10 ^(-7) - 10 ^(-8) and reduces the effectiveness of phage therapy.

 The closest analogue is the biological product "Streptobifid", which includes dry bacillus of bifidobacteria strain Bifidobacterium globosum VGNKI N BF-4 / DEP, dry bacillus streptococcus strain Streptococcus faecium VGNKI N 27/11-V-alumina, carbohydrate, and filler (RU N 2086248, A 61 K 35/74, publ. 10.08.97).

 The disadvantage of the drug "Streptobifid" is low efficiency in relation to staphylococci and Pseudomonas aeruginosa.

 The objective of the invention is to provide a highly effective agent with a wide spectrum of action for the treatment and prevention of dysbiosis and intestinal dysfunctions of the body of animals and poultry.

 The technical result of the invention is to obtain a comprehensive probiotic and bacteriophage preparation, which allows to reduce treatment time, to obtain a more stable and pronounced therapeutic and prophylactic effect, which can increase the immune status of the organism of animals and poultry and resistant to the action of enzymes of the gastrointestinal tract.

 The preparation contains a combination of polyreceptor virulent staphylococcal and pseudomonas bacteriophages with various receptors on the bacterial surface, which reduce the incidence of resistant forms of microorganisms with a wide spectrum of lytic action, and probiotic bacteria that can correct intestinal biocenosis due to the antagonistic and colonization properties of the strains used.

SUMMARY OF THE INVENTION The probiotic preparation contains a dry bacmid of bifidobacteria, a dry bacmass of streptococci, a dry culture of staphylococcal bacteriophages of strains VGNKI N 89 / TsMEI-FS-1-DEP and VGNKI N 90 / TsMEI-BS-2-DEP, carbohydrates, aluminum oxide, aluminum hydroxide and aluminum hydroxide in the following ratio of components, g / kg of the drug:
Dry bacillus of bifidobacteria with a content of 10-3000 billion microbial cells (m.c.) in 1 g - 2.04 - 4.0
Dry Bakmass streptococci with a content of 10-3000 billion m. in 1 g - 2.0 - 4.0
Dry culture of staphylococcal bacteriophages of strains VGNKI N 89 / TsMEI-FS-1-DEP and VGNKI N 90 / TsMEI-BS-2-DEPT with activity 10 ^(-9) - 10 ^(-12) PFU / g - 0.5 - 2 0
Carbohydrate - 20-40
Alumina - 30-60
Aluminum hydroxide - 100-300
Filler - Else
Of the carbohydrates, the preparation may contain lactose and / or sucrose, and of the fillers, starch, milk powder, milk whey, a substitute for whole milk, powdered sugar or edible sugar.

The drug may also additionally contain in various combinations dry backass of acidophilic bacteria with a content of 10-3000 billion m. in 1 g and / or dry culture of pseudomonas pseudomonas strains VGNKI N 86 / ЦМЭИ-A1-ДЭП, VGNKI N 87 / ЦМЭИ-В2-ДЭП, VGNKI N 88 / ЦМЭИ-С1-ДЭП with activity of each strain 10 ^(-9) - 10 ^(-12) PFU / g.

 The composition of the drug can be used any strains of bifidobacteria, streptococci and lactic acid bacteria, characterized by high adhesive ability and antagonistic activity. In particular, known strains of Bifidobacterium globosum VGNKI N BF-4-DEPT, Streptococcus faecium VGNKI N 27/11-V-DEPT and the acidophilic bacteria strain Lactobacillus acidophilus N A-105-DEPT / VGNKI can be used.

 The strains of bifidobacteria and streptococci that are part of the drug have adhesive and antagonistic activity against opportunistic microflora, take part in the processes of intestinal microbial digestion, and produce enzymes, acids and other biologically active substances.

 The highly virulent polyreceptor bacteriophages included in the preparation are new strains and were obtained from various sources on livestock farms (manure, sewage, equipment washes, intestinal contents, purulent external and internal foci).

 The strains of Pseudomonas aeruginosa and staphylococcal bacteriophages are deposited in the collection of microorganisms VGNKI under the following numbers: strains of Pseudomonas phages N 86 / TsMEI-A1-DEPT, N 87 / TsMEI-B2-DEPT, N 88 / TsMEI-C1-DEPT, strains of staphylococcal bacteriophage numbers 89 / TsMEI-FS-1-DEP, N 90 / TsMEI-BS-2-DEP.

 Strains of bacteriophages have a wide range of lytic activity against a wide range of pathogens of staphylococcosis and animal pseudomonoses. The main methods used to study the properties of isolated bacterial viruses are described in Adams' monograph (Adams M. Bacteriophages, M., Mir, 1961).

The strains of bacteriophages of Pseudomonas aeruginosa VGNKI N 86 / TsMEI-A1-DEP, VGNKI N 87 / TsMEI-B2-DEPT and VGNKI N 88 / TsMEI-C1-DEP are characterized by the following properties:
Morphology and size of phage particles. Electron microscopy data. The strain of phage VGNKI N 86 / TsMEI-A1-DEP has an isometric head with a diameter of 62 nm and a contracting caudal process (145 x 20 nm). The phage adsorption apparatus is a basal plate and 6 (six) tail fibrils. The length of the fibrils is 50 nm.

 The phage strain consists of an elongated head (92 x 58 nm) and a non-contracting caudal process (170 x 12 nm). The phage adsorption apparatus includes two types of organelles. Lateral phage adsorption organelles are fixed on the saws (fimbriae) of Pseudomonas aeruginosa. In addition, three terminal fibrils (12–13 nm) are distinguishable at the end of the caudal process of the phage. Using these fibrils, the phage interacts with the outer membrane of P. aeruginosa after translocation of the phage by drank to the cell surface.

 The strain of phage VGNKI N 88 / TsMEI-C1-DEP consists of an isometric head (64 nm) and a short, non-contracting caudal process 10-11 nm long. No additional structural elements were detected in phage N 88 / TsMEI-C1-DEPT.

 All three strains of bacteriophages contain DNA.

 The strain of the phage VGNKI N 86 / TsMEI-A1-DEPT, the strain of the phage VGNKI N 87 / TsMEI-B2-DEPT and the strain of the phage N 88 / TsMEI-C1-DEPT are virulent bacterial viruses. Strains of P. aeruginosa lysogenic in these phages were not detected.

 The lytic properties of phages. Two collections of Pseudomonas aeruginosa strains were used to evaluate the lytic spectra of bacterial viruses. One collection included 40 Pseudomonas aeruginosa isolates isolated from mastitis cows in various regions of the Russian Federation. The second collection included 28 isolates of Pseudomonas aeruginosa isolated from soil and dung samples of the Chelyabinsk region.

 The strain of bacteriophage VGNKI N 86 / TsMEI-A1-DEPT lyses 48 strains of Pseudomonas aeruginosa from 68.

 The host spectrum of the phage VGNKI N 87 / TsMEI-B2-DEP - 47 cultures of P. aeruginosa.

 Phage VGNKI N 88 / TsMEI-C1-DEPT is active against 43 strains from the same sample (68 isolates).

 The combined preparation of three phages lyses all 68 cultures of Pseudomonas aeruginosa.

 The biological properties of the phages are presented in table. 1. The strain P. aeruginosa PAO1 was used as the host cell.

 Physico-chemical properties of phages. All three phages retain lytic activity in solutions with pH from 4 to 10.5.

Incubation of the suspension of phage VGNKI N 87 / TsMEI-B2-DEPT at 60 ^o C for 30 min leads to a decrease in the titer of the phage by 2 lg. Phage VGNKI N 86 / TsMEI-A1-DEPT is more resistant to such treatment (decrease in concentration by 1 lg). The lytic properties of the phage VGNKI N 88 / TsMEI-C1-DEN do not change when the sample is heated to 60 ^o C.

 A single freeze-thaw practically does not affect the biological properties of phages.

When stored in the cold (4-8 ^o C), phage preparations do not lose lytic activity during the year. The strains of staphylococcal bacteriophages VGNKI N 89 / TsMEI-FS-1-DEP and VGNKI N 90 / TsMEI-BS-2-DEP are characterized by the following properties:
Morphology and size of phage particles. Electron microscopy data. The strain of staphylococcal bacteriophage VGNKI N 89 / TsMEI-FS-1-DEP consists of an icosahedral head with a diameter of 85 nm and a contracting caudal process 200 nm long and 15 nm wide. After reduction, the sheath of the process has a length of 100 nm and a width of 20 nm. The phage adsorption apparatus is a basal plate with fimbriae. The basal plate has a diameter of 45-50 nm.

 The strain of staphylococcal bacteriophage VGNKI N 90 / TsMEI-BS-2-DEP consists of an icosahedral head with a diameter of 50 nm and a long non-contracting caudal process with a length of 240 nm.

 Other morphological details have not been investigated. The lytic properties of phages. To assess the lytic properties of the phages, we used a collection of VGNKI from 117 coagulase-positive pathogenic strains of Staphylococcus aureus isolated from cows with mastitis, from purulent contents of external wounds, fecal contents from various livestock farms in the Moscow region, from domestic animals and birds in the Chelyabinsk region, the Republic of Udmurtia, Kirov region.

 The strain of staphylococcal bacteriophage VGNKI N 89 / CMEI-FS-1-DEPT lyses 102 strains from this collection.

 The strain of phage VGNKI N 90 / TsMEI-B8-2-DEP lyses 97 strains from this collection.

 The combination of these two phages lyzes all 117 strains of staphylococci.

 Biological properties of phages. The strain of staphylococcal bacteriophage VGNKI N 89 / TsMEI-FS-1-DEPT has a latent period of the infection process of 25-30 minutes. Productivity - 60 particles per cell.

 When reproducing the strain of staphylococcal bacteriophage VGNKI N 90 / TsMEI-BS-2-DEPT in host cells, the latent period is 40-45 minutes, the yield is 40-45 particles per cell. Physico-chemical properties of phages. Both phages retain lytic activity in solutions with pH from 4.2 to 9.7.

Incubation of the suspension of the phage strain VGNKI N 89 / TsMEI-FS-1-DEPT at a temperature of 60 ^o C for 30 min leads to a decrease in the phage titer by 1 lg. The lytic properties of the phage VGNKI N 90 / TSMEI-BS-2-DEP do not change when heated to 60 ^o C for 30 minutes

 The strain VGNKI N 89 / TsMEI-FS-1-DEPT has a floating density of 1.47 g / ml. The content of HC pairs of nucleotides in double-stranded linear DNA is 32%.

 The strain of staphylococcal bacteriophage VGNKI N 90 / TsMEI-BS-2-DEPT has a floating density of 1.48 g / ml. The content of HC pairs in double-stranded linear DNA is 35%.

When stored in the cold (4-10 ^o C), the phages do not lose lytic activity for 18 months.

 The invention is illustrated by the following examples.

Example 1. The preparation "Fagosan" containing a culture of a streptococcus strain Streptococcus faecium VGNKI N 27/11-B-DEPT (or any other culture of streptococcal strains used in probiotic preparations), a culture of a strain of Bifidobacterium globosum VGNKI N BF-4-DEPT (or any other culture of bifidobacteria), a combination of the polyreceptor staphylococcal bacteriophages of the strains VGNKI N 89 / TsMEI-FS-1-DEPT, VGNKI N 90 / TsMEI-BS-2-DEPT is prepared as follows:
The microorganism strains used in the preparation are separately cultivated on liquid nutrient media used for the cultivation of bifidobacteria and streptococci, or on media based on enzymatic casein hydrolyzate with the addition of yeast autolysate. During cultivation for (16 ± 2) h, the biomass yield of each bacterial species is not less than 10 billion live microbial cells in 1 ml of culture suspension. The resulting cultures are concentrated 10-15 times depending on the density of the culture suspension, washed from metabolic products on separators or microfiltration plants. Concentrated bacterial suspensions are freeze-dried using conventional technology.

 The resulting dry convenience foods contain at least 500 billion live microbial cells per gram.

 Separately, dry biomass of staphylococcal bacteriophages of strains VGNKI N 89 / TsMEI-FS-1-DEP and VGNKI N 90 / TsMEI-BS-2-DEPT is obtained. For this, cultures of host cells are pre-grown in a liquid medium, infected with phages and cultured until the microorganisms are completely lysed. The resulting phagolysates are purified. The concentration of phages in lysates is determined by titration using the method of agar layers (Adams, 1961).

 The technology of obtaining phages, the number of strains of the host cells, the purification method are the subject of know-how.

Purified phage cultures are dried by freeze-drying to achieve an active phage value in 1 gram of the obtained dry semi-finished product of at least 10 ^(-9) - 10 ^(-12) PFU / g.

 The activity of each bacteriophage after lyophilization is determined according to the "Guidelines for the use of staphylococcal bacteriophages for phagotyping strains isolated from cattle", approved by the GUV of the USSR Ministry of Agriculture on September 16, 1976 on isolates of staphylococcal cultures isolated from purulent foci, internal organs and intestinal contents of different animal species and test strains.

 The dry Bakmass of each strain is mixed with each other in a ratio of 1: 1.

 Dry semi-finished products of bacteriophages are mixed with each other in a ratio of 1: 1.

Next, a dry mixture of strains is mixed with dry bacteriophages in a 1: 1 ratio, carbohydrate, aluminum oxide and hydroxide and a filler (starch, powdered sugar, milk powder, whole milk replacer) are added in the following ratio of components, g / kg:
Dry Bakmass of a streptococcus strain with a content of 30-1000 billion live microbial cells / g - 3
Dry bacillus of a strain of bifidobacteria with a content of 30-1000 billion live microbial cells / g - 3
Dry culture of bacteriophages of staphylococcal VGNKI N 89 / TsMEI-FS-1-DEP, VGNKI N 90 / TsMEI-BS-2-DEPT with activity 10 ^(-9) - 10 ^(-12) PFU / g - 1
Carbohydrate (lactose or sucrose) - 30
Alumina - 50
Aluminum hydroxide - 200
Filler - Up to 1000
The specified composition allows you to get a series of the drug with a total KOE of streptococci and bifidobacteria (with an equal quantitative ratio) from 180 million to 6 billion in one gram and bacterial staphylococcal phages with activity from 10 ^(-6) to 10 ^(-9) PFU in one gram.

 Example 2. Take strains of bacteria Streptococcus faecium VGNKI N 27/11-B-DEPT, Bifidobacterium globosum VGNKI N BF / 4-DEPT, Lactobacillus acidophilus N A-105-DEPT / VGNKI and strains of staphylococcal bacteriophages VGNKI N 89-TsM 1-DEP and VGNKI N 90 / TSMEI-BS-2-DEP.

 A probiotic preparation is obtained by separate deep cultivation of strains of bifidobacteria, streptococci and acidophilus bacteria on liquid nutrient media adopted for the cultivation of these types of microorganisms. Get dry Bakmass of each strain separately as in example 1.

 Next, the dry Bakmass of each strain is mixed in equal proportions.

 Dry semi-finished products of bacteriophages obtained in example 1 are mixed together in a ratio of 1: 1.

Next, the dry mixture of strains is mixed with dry bacteriophages in a 1: 1 ratio, carbohydrate, aluminum oxide and hydroxide and a filler (for example, starch or food sugar, powdered sugar) are added, in the following ratio of components, g / kg:
Dry Bakmass of a streptococcus strain with a content of 10 - 3000 billion live microbial cells in 1 g - 2
Dry bacillus of a strain of bifidobacteria containing 10-3000 billion living microbial cells in 1 g - 2
Dry Bakmass of a strain of acidophilic bacteria with a content of 30-1000 billion living microbial cells in 1 g - 2
Dry culture of bacteriophages of staphylococcal strains VGNKI N 89 / TsMEI-FS-1-DEPT, VGNKI N 90 / TsMEI-BS-2-DEPT with activity 10 ^(-9) - 10 ^(-12) PFU / g - 1
Carbohydrate (lactose or sucrose) - 30
Alumina - 50
Aluminum hydroxide - 200
Filler - Up to 1000
The specified composition allows you to get a series of the drug with a total CFU of streptococci, bifidobacteria and acidophilus bacteria (with an equal quantitative ratio) from 180 million to 6 billion in one gram, bacterial staphylococcal phages with activity from 10 ^(-6) to 10 ^{(- 9)} PFU in one gram.

 Example 3. Take bacterial strains of Streptococcus faecium VGNKI N 27/11-B-DEPT, Bifidobacterium globosum VGNKI N BF / 4-DEPT, Lactobacillus acidophilus N A-105-DEN / VGNKI, strains of staphylococcal bacteriophages VGNKI N 89-TsM 1-DEPH, VGNKI 90 / TSMEI-BS-2-DEPT and strains of pseudomonas bacteriophages VGNKI N 86 / TsMEI-A1-DEPH, VGNKI 87 / TsMEI-B2-DEN, VGNKI N 88 / TsMEI-C1-DEPT. Dry semi-finished products of the used strains of bacteria and bacteriophages are prepared analogously to example 1 and 2.

The activity of the obtained dry semi-finished products of staphylococcal and pseudomonas bacteriophages is at least 10 ⁽¹²⁾ PFU in one gram.

A dry mixture of strains of bifidobacteria, streptococci, acidophilus bacteria is mixed with dry staphylococcal and pseudomonas bacteriophages in a 1: 1 ratio and carbohydrate, aluminum oxide and hydroxide and filler (starch, powdered sugar, milk powder, whole milk replacer) are added in the following ratio of components, g / kg:
Dry Bakmass of a streptococcus strain with a content of 30-1000 billion live microbial cells / g - 3
Dry bacillus of a strain of bifidobacteria with a content of 30-1000 billion live microbial cells / g - 3
Dry Bakmass of the strain of acidophilic bacteria with a content of 30-1000 billion living microbial cells / g - 3
Dry culture of bacteriophages of staphylococcal strains VGNKI N 89 / TsMEI-FS-1-DEPT, VGNKI N 90 / TsMEI-BS-2-DEPT with activity 10 ^(-9) - 10 ^(-12) PFU / g - 1
Dry culture of bacteriophages of the pseudomonas strains VGNKI N 86 / ЦМЭИ-A1-ДЭП, VGNKI N 87 / ЦМЭИ-В2-ДЭП, VGNKI N 88 / ЦМЭИ-С1-ДЭП with activity 10 ^(-9) - 10 ^(-12) PFU / g - 1
Carbohydrate (lactose or sucrose) - 30
Alumina - 50
Aluminum hydroxide - 200
Filler - Up to 1000
The specified composition allows you to get a series of the drug with a total CFU of streptococci, bifidobacteria and acidophilus bacteria (with an equal quantitative ratio) from 180 million to 6 billion in 1 g of bacterial staphylococcal and pseudomonas phages (with equal activity) from 10 ^{(-6 )} to 10 ^(-9) PFU in 1 g.

 Example 4. The drug is used for the prevention and treatment of gastrointestinal diseases of mixed etiology.

The prophylactic effectiveness of the series of the drug "Fagosan" is tested:
- obtained according to example 3, with a total concentration of bacteria in one gram of 180 million cubic meters, activity of phages 2x10 ^(-6) PFU in one gram;
- obtained according to example 3, with a total concentration of bacteria in one gram of 600 million MK, the concentration of active phages 10 ^(-9) PFU in one gram;
- obtained according to example 2 with a total concentration of bacteria in one gram of 180 million m. and activity of phages 1 • 10 ^(-6) PFU / g;
- obtained according to example 1 with a total concentration of bacteria in one gram of 180 million m.k. and activity of phage 1 • 10 ^(-6) PFU / g.

 As a biological model for testing the drug "Fagosan" use puppies of 3-3.5 months of age from a nursery that is dysfunctional for staph infections. In a laboratory study of feces according to the generally accepted methodology, staphylococci and bacteria of the Escherichia coli group were sown in puppies at concentrations exceeding accepted physiological norms, as well as Pseudomonas aeruginosa.

 On visual examination, the puppies lag behind in growth and development, the appetite is perverted or absent, upon palpation the abdominal wall is tense, painful, swollen, defecation is frequent, involuntary, feces are fluid, bubbling, sometimes with an admixture of blood.

 For testing, 5 groups of puppies of 3-3.5 months of age are formed.

The first group of puppies is given orally with food once a day for 5 days, 1 g of the preparation prepared according to Example 1, with a concentration of CFU of bacteria of 180 million, and a total activity of bacteriophages of 2 • 10 ^(-6) PFU / g.

In the second group of puppies, one gram of the preparation prepared in accordance with Example 1 is set similarly for 5 days, with a total concentration of CFU of bacteria of 600 million, and a total activity of bacteriophages of 2 • 10 ⁽⁹⁾ PFU / g.

In the third group of puppies, one gram of the preparation prepared according to Example 2 is similarly administered for 5 days with a total concentration of CFU of bacteria of 180 million, total activity of staphylococcal bacteriophages of 1 • 10 ^(-6) PFU / g.

For the fourth group of puppies, 1 g of the preparation prepared according to Example 1 is similarly administered for 5 days with a total concentration of CFU of bacteria of 180 million, staphylococcal bacteriophage activity of 1 • 10 ^(-6) PFU / g.

 The fifth group of puppies is used as a control and they are fed twice a day with 20 ml of a commercial preparation of staphylococcal bacteriophage together with symptomatic and antibacterial therapy.

 Take into account the effectiveness of the test drug 5 days after the start of treatment by comparing the clinical results obtained in all three groups.

In puppies of the first group, the beginning of the restoration of the activity of the gastrointestinal tract is observed on the second or third day after the start of the use of the drug. The bowel movement normalized, feces without admixture of mucus and without ichorotic odor. Behavioral reactions of puppies intensified. At the end of the drug administration, by the 5th day of its use, the puppies' behavioral reactions fully recovered, and their appetite was normal. According to bacteriological studies, the value of CFU of lactobacilli in puppies of this group is log 6.5 + 0.8. The value of CFU of staphylococci decreases to the boundaries of the physiological norm and amounts to log 3.2 + 0.2. Three out of 6 puppies in the intestinal biocenosis have no staphylococci at a dilution of 10 ^(-3) . Coagulase-positive staphylococci and Pseudomonas aeruginosa are absent in all puppies of this group.

 In puppies of the second group, the activity of the gastrointestinal tract is fully restored by 3-5 days of giving the drug. According to bacteriological studies, the value of CFU of staphylococci of puppies of this group is log 2.4 + 0.3. Coagulase-positive staphylococci and Pseudomonas aeruginosa are absent. The value of CFU of lactobacilli in the intestinal contents is lg 7.9 + 0.6, the bacteria of the group of Escherichia coli lg 8.9 + 0.3, which corresponds to the boundaries of the physiological norm. In puppies of the third and fourth experimental groups, intestinal biocenosis was restored by 4-7 days after the start of the drug administration.

In puppies of both experimental groups, at the end of giving the test drug in the contents of the gastrointestinal tract at a dilution of 10 ^{(-2) there are} no staphylococci and Pseudomonas aeruginosa.

 In puppies of the control group receiving a commercial preparation of bacteriophages, antibacterial and symptomatic therapy, the restoration of the gastrointestinal tract is observed 12-14 days after the start of treatment. In this case, the bowel movement is not fully restored. In bacteriological studies of the contents of the gastrointestinal tract, the CFU value of lactobacilli in puppies of this group on day 14 of treatment is lg 5.8 + 0.2, which is significantly lower than the physiological norm. The value of CFU of staphylococci is log 4.5 + 0.7. In 2 out of 5 puppies of this group, coagulase-positive staphylococci and Pseudomonas aeruginosa are sown. The number of bacteria of the Escherichia coli group is lg 9.4 + 0.7.

 The use of drugs helps to restore intestinal biocenosis, intestinal sanitation from staphylococci and Pseudomonas aeruginosa, from excessive colonization by Escherichia coli bacteria and stimulates the growth and development of puppies.

 Example 5. Test the effectiveness of the drug "Fagosan" for the prevention of gastrointestinal diseases in suckling pigs under lactating sows with mastitis metritis agalactia syndrome (MMA).

 For testing, 6 groups of lactating sows with a diagnosis of MMA are formed with approximately the same number of piglets under a lactating sow.

In the first group, the preparation is prepared according to Example 3, with a total bacterial concentration of 180 million m.k., a total phage concentration of 2 • 10 ^(-6) PFU / g, sows orally individually once a day for 5 days, 2 g the drug, previously diluted with boiled warm water.

The second group of sows is given orally individually once a day for 5 days, 2 g of the preparation prepared according to Example 3, with a total concentration of CFU of probiotic bacteria of 600 million, total bacteriophage activity of 2 • 10 ^(-9) PFU / g, previously diluted with warm boiled water.

 The third group of sows is used as a control and for the prevention of gastrointestinal diseases in suckling piglets, they are treated once with antibiotics.

In the fourth group, the preparation made according to example 3, with a total bacterial concentration of 180 million m.k., a total phage activity of 2 • 10 ^(-6) PFU / g, is given to suckling piglets, starting from 3 days of age, individually, orally once a day for 5 days, 0.5 g per head, pre-diluted with warm boiled water.

In the fifth group, the preparation made according to example 3, with a total bacterial concentration of 600 million m.k., a total phage activity of 2 • 10 ^(-9) PFU / g, is given to suckling pigs, starting from 3 days of age, for 5 days individually, orally, 0.5 g per head, pre-diluted with warm boiled water.

 Piglets in the 6th group are used as control and for the prevention of gastrointestinal diseases, a single treatment with antibiotics is performed.

 The accounting of indicators is carried out 12 days after the end of the dacha preparations according to the general safety and growth of live weight of piglets.

 The results are presented in table 2.

 The data obtained show that the use of the drug "Fagosan" in pregnant sows with mastitis-metritis-agalactia syndrome helps to increase the safety of suckling pigs. The use of the drug for suckling pigs under lactating sows with mastitis-metritis-agalactia syndrome increases the natural resistance of piglets and helps to increase their safety.

 Thus, the drug has prophylactic and therapeutic effectiveness in intestinal dysbiosis, promotes sanitation of the intestines of animals from staphylococci and Pseudomonas aeruginosa, reduces the number of enterobacteria in the group of Escherichia coli and increases the natural resistance of animals.

Claims (5)

1. A probiotic veterinary preparation containing dry bacillus of bifidobacteria, streptococci, aluminum oxide, aluminum hydroxide, carbohydrate lactose and / or sucrose and an excipient, characterized in that it further comprises a dry culture of staphylococcal bacteriophages of VGNKI 89 / TsMEI-FS-1-DEPT strains and VGNKI 90 / TsMEI-BS-2-DEPT in the following ratio of components, g / kg of the drug:
Dry bacillus of bifidobacteria with a content of 10 - 3000 billion m.k. in 1 g - 2.0 - 4.0
Dry Bakmass streptococci with a content of 10 - 3000 billion m.k. in 1 g - 2.0 - 4.0
Dry culture of staphylococcal bacteriophages of strains VGNKI 89 / TsMEI-FS-1-DEP and VGNKI 90 / TsMEI-BS-2-DEPT with activity 10 ^(-9) - 10 ^(-12) PFU-g - 0.5 - 2.0
Carbohydrate - 20 - 40
Alumina - 30 - 60
Aluminum hydroxide - 100 - 300
Filler - Else
2. The drug according to claim 1, characterized in that from bifidobacteria it contains a strain of Bifidobacterium globosum VGNKI BF-4 / DEPT, from streptococci - a strain of Streptococcus faecium VGNKI 27/11-B-DEPT.  3. The drug according to claim 1, characterized in that of the fillers it contains starch, milk powder, milk whey powder, a substitute for whole milk, powdered sugar or edible sugar.  4. The drug according to claim 1, characterized in that it further comprises a dry backass of acidophilic bacteria with a content of 30 - 1000 billion m.k. in 1 g in an amount of 2 to 4 g per 1 kg of the drug.  5. The drug according to claim 4, characterized in that from acidophilic bacteria it contains a strain of Lactobacillus acidophilus A-105-DEP / VGNKI. 6. The drug according to claim 4, characterized in that it further comprises a dry culture of bacteriophages of pseudomonas strains VGNKI 86 / CMEI-A1-DEPT, VGNKI 87 / TsMEI-V2-DEPT, VGNKI 88 / TsMEI-C1-DEPT with activity of each strain 10 ^(-9) - 10 ^(-12) PFU-g.

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