RU2264453C2 - Veterinary biopreparation - Google Patents

Abstract

FIELD: biotechnology, in particular production of new probiotic preparation based on bacteria of genus Bacillus useful in prophylaxis and treatment of invective diseases in animals and poultry.

SUBSTANCE: New strains B.subtilis 07 and B.licheniformis 09 have wide range of antagonistic activity, high proteolytic and amylase activity, and lysozyme producing ability without competition. Furthermore said strains have synergic action, namely are capable to increase antagonistic action of biopreparation. Biopreparation contains effective amount of strains Bacillus subtilis and Bacillus licheniformis and protective medium. Optionally biopreparation contains solvent and/or filler. Biopreparation has higher antagonistic activity in relation to wide range of pathogenic and opportunistic microorganisms and is resistant to various antibiotics. Claimed preparation inhibits pathogenic Escherichia, provides optimal microbial balance in digestive tract, increases non-specific animal resistance and is useful in treatment of diarrhoea, prophylaxis of dysbacteriosis, and antimicrobial therapy due to strain chromosome resistance to antibiotics.

EFFECT: high effective biopreparation for breeding of domestic animals and poultry.

12 cl, 8 tbl, 9 ex

Description

The invention relates to biotechnology and veterinary medicine and for the creation of new probiotic preparations based on bacteria of the genus Bacillus, which can be used for non-specific prevention and treatment of infectious diseases and dysbiosis of animals and birds.

One of the reasons for the difficult epizootic situation in animal husbandry is the uncontrolled use of antibiotics and chemotherapeutic agents. Their unreasonably wide application causes the appearance of pathogens with increased and multidrug resistance. The treatment of animals is reduced and delayed. Often there is a need for corrective use of probiotic drugs. To compensate for physiological bacteriosis and possibly the early establishment of colonization resistance of the intestine, newborns are prescribed probiotics - preparations containing living normal intestinal microflora or its metabolites and stimulators of the development of their own normal microflora in the intestine. Such, for example, are bifidumbacterin, lactobacterin, bifilact and bifimol, lactoamilovarin, and the like.

Known probiotics based on lactic acid bacteria in some cases have pronounced therapeutic and prophylactic effectiveness in gastrointestinal diseases of calves. This increases the safety and weight gain, reduces the duration of the disease, reduces the severity of its course and the severity of technological stresses. However, the use of probiotics is not always accompanied by a positive effect. The conflicting results obtained in a number of studies indicate an unsuccessful selection of the strains used for their preparation, a violation of the production technology and other reasons that reduce the quality of the drugs, as well as incompatibility with antimicrobial agents, which is a deterrent in the treatment of dyspeptic disorders in newborn calves. In addition, although bifidobacteria and lactobacilli inhibit the development of pathogenic microflora through the formation of organic acids and other antibiotic substances and normalize the activity of the gastrointestinal tract, under the conditions of the existence of newborn calves, the formation of bifidoflora occurs, as a rule, after the appearance of Escherichia coli in the intestinal tract, staphylococci , Proteus and other representatives of conditionally pathogenic and pathogenic microflora, which leads to intestinal dysbiosis.

Currently, biological products from spore-forming bacteria are widely used. The composition of probiotic preparations based on bacteria of the genus Bacillus includes strains with a different spectrum of antibacterial and antimycotic activity. The effectiveness of the drugs is determined by the breadth of the spectrum and the strength of the antibacterial activity of the strains included in the drug.

A known strain of Bacillus subtilis X-15, used for the prevention and treatment of endometritis in cows (Patent RU No. 2180575, C 12 N 1/20, publ. 20.03.2002).

The disadvantage of the drug based on the strain of Bacillus subtilis X-15 is the limited spectrum of action, since it is active only against a narrow group of microorganisms that cause endometritis in cows.

The probiotic preparation “Subtilis” is also known on the basis of a liquid or lyophilically dried culture of the Bacillus subtilis strain VKM No. B-2250 D. The drug is intended for the prevention and treatment of gastrointestinal diseases of animals, birds and fish, helps to increase the digestibility of feed, increase productivity and weight gain ( Patent RU No. 2184774, C 12 N 1/20, publ. 10.07.2002).

The disadvantage of the drug "Subtilis" is a limited spectrum of antagonistic activity.

The closest analogue is a complex probiotic preparation containing the following components, vol.%: A mixture of Bacillus subtilis VKPM - 4759 biomass with a titer of 1 × 10 ⁷ -10 × 10 ⁹ live microbial cells and Bacillus biomass. Licheniformis 2336/105 with a titer of 1 × 10 ⁸ -10 × 10 ⁹ live microbial cells in 1 ml of physiological saline - 92-98%, and a stabilizer based on the blood serum of cattle, or milk, or a mixture thereof, sucrose and gelatin - 2-8%. The drug has both antibacterial and antiviral activities, is capable of prolonged synthesis of interferon in various parts of the open cavities of the body (Patent RU No. 2159625, A 61 K 35/66, publ. 27.11.2000).

The objective of the invention is the creation of a probiotic biological product for veterinary use, characterized by a more pronounced antagonistic activity against opportunistic microorganisms due to the high lysozyme, proteolytic and amylase activities of the Bacillus subtilis and Bacillus licheniformis strains that are convenient in use and have different formulations.

The technical result of the invention is to increase the effectiveness of the prevention and treatment of infectious diseases and dysbiosis of animals, including birds, by expanding the spectrum of antagonistic activity of bacterial strains included in the biological product of Irilis, increasing the resistance of the biological product to the action of enzymes of the gastrointestinal tract and ease of use, and also increasing the productivity and weight gain of animals and birds.

The invention consists in the following:

The new strains of B. subtilis 07 (VKPM No. B-8611) and B. licheniformis 09 (VKPM No. B-8610) that are part of the Irilis biological product are characterized by a wide spectrum of antagonistic activity, high proteolytic and amylase activity (B. subtilis 07) and pronounced ability to the production of lysozyme (B.licheniformis 09), do not compete with each other, but enter into a synergistic relationship, which is manifested in an increase in the antagonistic effect of the biological product.

The strains B.subtilis 07 (VKPM No. B-8611) and B.licheniformis 09 (VKPM No. B-8610) were isolated from a healthy wheat plant, deposited in the All-Russian collection of industrial microorganisms and are characterized by the following properties:

Bacillus subtilis VKPM No. B-8611 - gram-positive aerobic spore-forming bacilli, size 2.7-0.6 × 0.8-0.7 microns, located singly or in chains. Cells are mobile, form aerobic spores of an oval shape, which are located centrally in the cell. When spore formation, the cells do not swell.

On Gauze No. 2 medium, wort-agar, Gromyko medium, the strain grows abundantly, forms matte folded colonies of flesh-colored with rugged edges, easily removed by loop from agar.

In the protoplasm of the strain after growth on glucose agar, poly-β-hydroxybutyric acid inclusions were not detected. On the BCH, the culture forms a film.

It does not grow under anaerobic conditions, does not hydrolyze urea, does not form gas from nitrates under anaerobic conditions, does not produce arginine dihydrolase. Culture forms catalase. Gives a positive reaction of Voges-Proskauer, grows in the presence of 7% NaCl. Hydrolyzes starch and casein, liquefies gelatin. Ferments glucose, arabinose, xylose, mannitol with the formation of acid without gas. Reduces nitrates, bleaches methylene blue. It does not have coagulase and lecithinase activity, it has high proteolytic and amylase activity.

The main physical and biochemical properties of B.subtilis VKPM No. B-8611 are presented in Table 1 and Table 2.

Bacillus licheniformis VKPM No. B-8610 - gram-positive spore-forming bacilli, size 2.6-0.7 × 0.5-06 microns. The cells are mobile, peritrichia, located mainly in the form of chains. The spores are oval, located centrally in the cell. Cells do not swell during spore formation. After growth on glucose agar, protoplasm does not show inclusions of poly-β-hydroxybutyric acid.

On the MPA, it forms colonies with a dull rough surface, opaque, tightly attached to agar: mucus is often found on the surface. A wrinkled film forms on the BCH, sometimes with a creamy tint.

The culture forms catalase, characterized by the ability to grow on agar under anaerobic conditions. Gives a positive Foges-Proscauer reaction, grows at 7% NaCl. Hydrolyzes starch, casein, does not hydrolyze urea. Gelatin liquefies slowly. Ferments glucose, arabinose, xylose, mannitol with the formation of acid without gas. Reduces nitrates, in anaerobic conditions it forms gas from nitrates. It produces arginine dihydrolase, lysozyme. It does not have coagulase and lecithinase activity.

The main properties of the strain B.licheniformis VKPM No. B-8610 are presented in table. 1 and table 2.

Strains B.subtilis VKPM No. B-8611 and B.licheniformis VKPM No. B-8610 are characterized by high antagonistic activity against a wide range of pathogenic and opportunistic microorganisms and resistance to a number of antibiotics

In the composition of the proposed biological product Irilis, these strains can be used in various combinations, for example, in equal proportions (according to cell titer): biomass of B. subtilis strain VKPM No. B-8611 in a titer of 1 · 10 ⁹ CFU / ml and biomass of B.licheniformis strain VKPM No. B-8610 in the titer of 1 · 10 ⁹ CFU / ml or in any ratios within the range (1-100) :( 100-1), for example, the biomass of the strain B. subtilis VKPM No. B-8611 in the titer of 5 · 10 ⁹ CFU / ml and biomass of B.licheniformis strain VKPM No. B-8610 in a titer of 1 · 10 ⁹ CFU / ml, or biomass of B. subtilis strain VKPM No. B-8611 in a titer of 1 · 10 ¹⁰ CFU / ml and biomass of B.licheniformis strain №V VKPM-8610 at a titer of 5 × 10 ⁹ cfu / m Or a biomass of B. subtilis VKPM B-8611 №V titer 1 × 10 ⁹ cfu / ml and the biomass of B. licheniformis VKPM B-8610 №V titer of 1 × 10 ⁷ cfu / ml, etc.

The biological product Irilis also contains a protective environment to ensure the safety of bacteria in the process of technological processing, obtaining the final prescription form and subsequent storage. As a protective medium, it may contain, for example, sucrose-gelatinous medium, milk powder, gelatose, lactose, sucrose and the like.

The biological product Irilis may additionally contain a solvent. As a solvent, for example, distilled or boiled water, physiological saline, and the like can be used.

The biological product Irilis may additionally also contain a filler, usually used in the manufacture of various prescription forms. When forming tablets, it may contain, for example, dextrans, polyglucin, starch, polyvinylpyrrolidone, sucrose, lactose, calcium stearate, glucose, sodium hydrogen carbonate, aluminum hydroxide, methyl cellulose, talc, and the like. Upon receipt of a suppository or suppository as a filler, it contains, for example, confectionery fat, paraffin, lanolin, cocoa butter, aluminum hydroxide gel, and the like.

The biological product can be encapsulated or immobilized on various types of carriers or sorbents, for example, aerosil, cellulose, activated carbon, carboxymethyl cellulose, hydroxyethyl cellulose, chitosan, and the like.

The drug may also be lyophilized.

Biological product "Irilis" can be used for oral or vaginal or rectal or external use in the form of an aqueous suspension. The mechanism of action of the biological product Irilis is based on the adhesive and antagonistic activity of probiotic bacteria that produce various physiologically active substances and displace pathogenic and conditionally pathogenic microorganisms from the digestive tract of the macroorganism and have a stimulating effect on specific and non-specific protective reactions of the macroorganism.

The invention is illustrated by the following examples.

Example 1. Obtaining a liquid preparation.

The strains are cultivated individually in either solid or liquid culture media.

With stationary technology, the strains are cultured on dense agarized media in mattresses, or in glass bottles on a thermostatically controlled rocking chair at a temperature of 22 to 38 ° C for 12-16 hours to 7 days. At the end of the incubation, the biomass grown on the surface of the nutrient medium is washed off with a stabilizer, a protective medium containing 5% lactose solution, brought together in a ratio of 10: 1, poured into bottles. Get biological product containing biomass of strain B. subtilis VKPM No. B-8611 in a titer of 1 · 10 ¹⁰ CFU / ml and biomass of strain B. licheniformis VKPM No. B-8610-1 · 10 ⁹ CFU / ml

In industrial technology, the strains are cultured in a reactor / fermenter with a culture medium at a temperature of 35-38 ° C for 10-18 hours. The process is considered complete if the cell concentration is 4-5 billion / ml and the ratio of spores and vegetative cells 1: 1. At the end of the incubation, separately grown cultures are brought together in a 1: 1 ratio and the sucrose-gelatin protective medium is added and poured into vials. Get a biological product containing biomass of strain B. subtilis VKPM No. B-8611 in a titer of 5 · 10 ⁹ CFU / ml and biomass of strain B.licheniformis VKPM No. B-8610 - 5 · 10 ⁹ CFU / ml.

Example 2. Obtaining the drug in the form of a lyophilisate.

With stationary technology, the strains are cultured on dense agarized media in mattresses, or in glass bottles on a thermostatically controlled rocking chair at a temperature of 22 to 38 ° C for 12-16 hours to 7 days. At the end of the incubation, the biomass grown on the surface of the nutrient medium is washed off with a protective medium containing a 10% glycerol solution, poured into ampoules (vials) or stainless steel cassettes, after which it is frozen and dehydrated in a vacuum dryer or spray dried way.

In industrial technology, the strains are cultured in a reactor / fermenter with a culture medium at a temperature of 35-38 ° C for 10-18 hours. The process is considered complete if the cell concentration is 4-5 billion / ml and the ratio of spores and vegetative cells 1: 1. At the end of the incubation, separately grown cultures are brought together in a 2: 1 ratio and the stabilizer is added, poured into ampoules (vials) or stainless steel cassettes, after which they are frozen and dehydrated in a vacuum dryer or spray dried.

Example 3. Obtaining the drug in tablet form.

Cultured cultures of B. subtilis strains VKPM No. B-8611 and B.licheniformis VKPM No. B-8610 after adding the components of the suspension medium are dehydrated on a vacuum dryer or spray dryer, combined with sugar granulate, glidants (starch or calcium stearate) and pressed on rotary presses.

For stability, 10 experimental-production series of a tabletted preparation were studied. As the results show, immediately after pressing, the content of living B. subtilis VKPM No. B-8611 and B.licheniformis VKPM No. B-8610 in tablets is not less than 10 ^9-7 -10 ^8-6 CFU / dose (respectively). After storing tablets for 12 months. the content of live microbial cells in no batch was below 10 ^9-7 -10 ^8-6 CFU / dose.

Example 4. Obtaining the drug in the form of suppositories.

Cultured strains of B. subtilis VKPM No. B-8611 and B.licheniformis VKPM No. B-8610 strains after adding the components of the suspension medium are dehydrated on a vacuum dryer or spray dryer, combined with fillers (confectionery grease or paraffin) and cast on a special machine suppositories.

On stability studied 10 experimental-production series of the drug. As the results show, immediately after pressing, the content of live B. subtilis VKPM No. B-8611 and B.licheniformis VKPM No. B-8610 in suppositories is at least 10 ^{9-7-10 8-6} CFU / suppository (respectively). After storing suppositories for 12 months, the content of live microbial cells in no batch was lower than 10 ^9-7 -10 ^8-6 CFU / suppository.

Example 5. All obtained in examples 1, or 2, or 3, or 4 variants and forms of the biological product Irylis checked for harmlessness in laboratory animals, specific antagonistic activity against test cultures of various groups of pathogenic and opportunistic microorganisms and resistance to antibiotics. Biological product Irilis is characterized by harmlessness. To determine the safety, the contents of the vial are diluted in 0.5 ml of physiological saline and this dose is administered orally to mice. For each experiment, at least 10 mice weighing 15-16 g are used. The drug is considered harmless if all the mice remain alive for 5 days of observation and no disease is detected.

The biological product Irilis is characterized by a wide spectrum of antagonistic activity against test strains of cultures of pathogenic microorganisms. The characteristics of the antagonistic activity of the drug are presented in table.3.

To determine the specific activity, the antagonistic activity of the drug variants against test cultures is examined. The study is carried out by the method of delayed antagonism. For this, the contents of the vial are dissolved in 1 ml of physiological saline. The resulting suspension is seeded with a stroke along the diameter of the Petri dish with agar medium Gause No. 2. Crops are incubated in a thermostat at 37 ° C for 72 hours. Then, test microorganisms (500 millionth suspension of daily cultures in physiological saline) are inoculated with the culture on the grown culture. Results are taken into account after 18 hours of incubation at 37 ° C according to the size of the zones of no growth test -cultures.

The control of the growth of test cultures is their parallel growing on plates with agar medium Gause No. 2 without the studied culture.

From the data in Table 3 it follows that the optimal number of living cells in one dose of the drug is 1-5 × 10 ⁹ . A further increase in the number of microbial cells does not significantly change the antagonistic activity of the drug against test cultures of microorganisms.

Example 6. To determine the therapeutic efficacy obtained from Example 1, or 2, or 3 of the probiotic Irilis, in comparison with the use of the drug pectosorbin, in a farm that is long-term inadequate for gastrointestinal diseases of piglets with a symptom complex of diarrhea, three groups of 81 piglets 1- are formed 2 days of age with clinical signs of dyspepsia (diarrhea, feces liquefied, feces had an acidic smell, contained gas bubbles, no appetite, dryness and pallor of visible mucous membranes, heart rate increased, heart sounds deaf, breathing speeded up).

After a clinical examination, it was found that 69 piglets - simple dyspepsia, 12 - toxic.

In bacteriological studies of fecal samples in all patients, piglets secrete large amounts of pathogenic Escherichia (10 ⁸ -10 ¹¹ bacteria per 1 g), while Escherichia strains - normal representatives of the large intestine are isolated only in 8 piglets (10 ³ -10 ⁴ bacteria in 1 g).

Pathogenic Escherichia, unlike normal representatives of the intestine, as a rule, do not ferment lactose, cause the death of white mice with intraperitoneal administration. When serotyping the strains isolated from piglets, 11 strains are assigned to enteropathogenic serogroup 0141, the remaining 40 strains were not agglutinated with type-specific sera. The research results indicate a violation of the microflora of the large intestine of patients with dyspepsia of piglets (dysbiosis), which is the cause of their incidence.

Piglets of the first and second groups for the first time after a 3-4-hour water-hungry diet for 20-30 minutes before feeding drink Irilis in the amount of 2 and 4 doses, respectively 2 times a day with an interval of 12 hours before clinical recovery. Piglets of the third group, three times a day before clinical recovery with colostrum, use the drug Pectosorbin at a dose of 0.26 g per kg of live weight in accordance with the instructions for use. Piglets are under clinical observation, taking into account the number of recovered animals, the duration of treatment. Recovery is judged by the disappearance of signs of diarrhea, pathological impurities in feces, flatulence, cramping, improvement in general condition, and the appearance of appetite. The results are presented in table 4.

Table 4. Comparative efficacy of the use of Irilis and Pectosorbin for the treatment of gastrointestinal diseases of piglets caused by pathogenic microflora Indicators Group of piglets First, n = 27 Irilis 2 doses The second, n = 27 Irilis 4 doses Third, n = 27 Pectosorbin Recover, goal. 24 26 twenty Fallen and forced to kill, goal. 3 1 7 The course of treatment, days 4,5 ± 0,4 2.0 ± 0.6 6.5 ± 0.6 Therapeutic efficacy,% 88.9 96.3 74.1

As can be seen from the table. 4, the probiotic Irilis provided high efficiency when applied in the amount of 4 doses 2 times a day. Recovery occurs after 4-6 times the use of Irilis in compliance with the diet. Normalization of intestinal microflora eliminates dehydration, which eliminates the need for rehydration measures. After discontinuation of the drug, the disease is not recorded.

Example 7. To determine the effectiveness of the drug Irilis in the treatment of calves with a symptom complex of diarrhea and to evaluate the preventive effect on animals until they reach 2 months of age, three groups of 100 healthy newborn calves of 1-2 days of age, weighing 28-30 kg at birth, were formed .

Calves of all groups are fed in accordance with the recommendations on growing technology: 1.5-2 liters of colostrum (milk) for each feeding three times a day. The first time they drink colostrum no later than 1.5 hours after birth. The calves of the first two groups (40 goals each) in 20-30 minutes before the second feeding for five days in two courses with a break of 3-4 days between them give the drug Irilis 1 and 2 doses, respectively. Before the introduction of the drug, the contents of one ampoule (vial) are dissolved in 100 ml of boiled and cooled to a temperature of 38-39 ° C. Calves of the first experimental group are given 50 ml each, calves of the second experimental group 100 ml each. The third group is the control (20 goals), the drug Irilis does not receive.

Calves are weighed at birth and at 2 months of age. Daily take into account the physical condition, appetite and functioning of the digestive system (the nature of the stool, flatulence), the behavior of the animal. At the end of the experiment, blood samples were taken from five calves of each group for hematological studies. In the blood, the hemoglobin content is determined using a Sali hemometer, blood cells in the Goryaev's counting chamber, the leukocyte formula is the usual method, the concentration of total protein in the blood serum is refractometric, the content of immunoglobulins using the zinc sulfate test, the lysozyme content according to E.V. Ermolaeva, bactericidal activity according to S.V.Smirnova and T.A. Kuzmina. The research results are presented in table.5.

As can be seen from the table. 5, the use of the probiotic Irilis does not significantly affect the concentration of hemoglobin in the blood, the number of blood cells and the leukocyte formula. However, in the blood of calves treated with the drug, the level of total protein in blood serum, immunoglobulins, lysozyme and bactericidal activity of blood serum increases significantly.

It was found that Irilis does not adversely affect the health of animals, but significantly increases their resistance to gastrointestinal diseases, improves the quality of life of calves, they do not lose weight. (Table 6).

As can be seen from table 6, in the control group that did not receive Irilis, 15 out of 20 calves fell ill, which is 75% of the number of animals in the group, and in the first experimental group (1 dose of Irilis 1 time per day) - 11 out of 40 calves (27.5%), in the second experimental group (2 doses of Irilis 1 time per day) - 7 out of 40 calves (17.5%). The preventive efficacy of the probiotic Irilis is 72.5% and 82.5%, respectively. In this case, the calves of the control group get sick already on days 2 and 9, and the disease proceeds mainly in a severe form, as a result, the waste is 30%, and the safety is 70%. The use of the drug Irilis in an amount of at least 2 doses is impractical, as evidenced by the high percentages of calf preservation and average daily weight gain, respectively equal to 95% and 9%.

Table 5 Studies of the main blood counts of 2-month-old calves after preventive measures Indicators Calf groups Experienced 1, n = 40 Experimental 2, n = 40 Control 3, n = 20 Hemoglobin, g / l 127.0 ± 13.6 132.7 ± 4.0 126.0 ± 3.0 Red blood cells 7.2 ± 0.2 7.4 ± 0.4 7.2 ± 0.2 White blood cells 6.4 ± 1.0 6.3 ± 0.9 6.8 ± 0.7 Total protein, g / l 62.1 ± 1.6 70.5 ± 1.7 61.0 ± 2.6 General immunoglobulins, g / l 29.4 ± 1.9 38.0 ± 2.1 27.4 ± 1.7 Lysozyme, mcg / l 770.0 ± 31.0 980.0 ± 22.0 765.0 ± 33.0 Bactericidal activity,% 67.0 ± 0.5 86.5 ± 2.0 60.2 ± 1.7 Leukocyte formula,%: Basophils - - - Eosinophils 4 3 3 Neutrophils: - young - - - - stab 3 3 2 - segmented 25 23 24 Lymphocytes 66 68 69 Monocytes 2 3 2

Table 6 The preventive efficacy of Irylis in the cultivation of milkman calves Indicators Calf groups First, n = 40 The second, n = 40 Third, n = 20 Ill, goal. (%) 11 (27.5) 7 (17.5) 15 (75.0) Aged, days 3.5 ± 0.3 3.5 ± 0.1 1.5 ± 0.2 (6 goals)
8.5 ± 0.8 (9 goals) The course of the disease, goal. (%) - easy 4 (10.0) 5 (12.5) 5 (25.0) - heavy 7 (17.5) 2 (5.0) 10 (50.0) Dropped during the observation period, goal. (%) 2 (5.0) - 4 (20.0) Forced killed goal. (%) 6 (20.0) 2 (5.0) 2 (10.0) Safety, goal. (%) 32 (80.0) 38 (95.0) 14 (70.0) The average daily weight gain, g (%) 665.0 ± 39.0 (104.0) 700.0 ± 27.0 (109.4) 640.0 ± 25.0 (100.0)

Example 8. The effectiveness of the use of the drug Irilis for the prevention of gastrointestinal diseases with a symptom complex of diarrhea is determined in a farm that is long-term unsuccessful for these diseases, in comparison with pectosorbin and immunoglobulin preparations. Of 105 newborn calves, three groups of 35 goals are formed. in each. All animals are healthy, normally developed, and the difference between the groups in terms of average live weight does not exceed 2%.

The calves of the first group are given the drug Irilis (2 doses) 20-30 minutes before the second colostrum, the calves of the second group are given pectosorbin in accordance with the instructions for its use, the calves of the third group are given intramuscularly non-specific immunoglobulin according to the instructions for its use.

The prevention course in groups is: in the first group - 10 days, in the second - 10 days, in the third - 50 days. The observation period is 60 days. Calves are weighed at birth, after one month and at the end of the observation period. The clinical condition is taken into account daily, paying attention to the number of diseased animals, the course and outcome of the disease. The research results are presented in table.7

As can be seen from table 7, the effectiveness of the probiotic Irilis in comparison with the known drugs pectosorbin and immunoglobulin is much higher, which is confirmed by the safety and average daily growth of body weight.

Table 7. The comparative effectiveness of the use of Irilis and known drugs for the prevention of gastrointestinal diseases caused by "stable" microflora in newborn calves Indicators Calf groups First, n = 35 The second, n = 35 Third, n = 35 Prevention Method irilis pectosorbine Immunoglobulin The calves got sick, a goal. 2 8 7 Of these, the goal fell. - 5 3 Diagnosis Dyspepsia Toxic dyspepsia Toxic dyspepsia The course of the disease, goal: - heavy - 5 4 - easy 2 3 3 Preventive efficacy,% 94.3 77.1 80.0 Preservation,% 100.0 80.0 91.4 The average daily weight gain, g 820.0 680.0 660.0

Example 9. Carry out tests to include in the traditional feeding scheme of the probiotic preparation Irilis in poultry when growing broiler chickens. 4 groups of chickens are formed, which receive:

1 g - Vetom preparation manufactured by NPF-IC Koltsovo,

2 gr. - drug Irilis,

3 gr. - STF preparation manufactured by Bioavtomatika LLC,

4 gr. - the drug "Enterosporin".

The experimental preventive regimen according to the days of cultivation is as follows:

1 day - probiotics with food,

1-3 days - fluoroquinolone,

4-13 days - probiotics with food.

Further - according to the preventive scheme operating in farms.

The results are taken into account according to the main technological indicators: the weight of chickens on days 1, 7, 14, 28, 35 and 42, and when they are delivered for slaughter; registration of the case every week and the general - when handing over for slaughter. The results are presented in table.8.

The data presented in Table 8 indicate that the use of the Irilis preparation in the process of raising chickens allows to obtain a high result on the safety of the livestock, the Irilis preparation has a positive effect on the body of chickens, providing a greater increase in live weight compared to the control.

Thus, the tests of the drug Irilis showed the possibility and high efficiency of its use in growing animals and birds. It provides inhibition of pathogenic Escherichia, maintaining optimal microbial balance in the digestive tract, increasing non-specific resistance of animals, their safety and weight gain, has a prophylactic therapeutic effect in diseases accompanied by diarrhea, and ensures the prevention of dysbiosis. The advantage of the drug Irilis is the chromosomal resistance to the antibiotics of its constituent strains, which makes it possible to use it in the complex of antimicrobial therapy.

Table 1 Cultural, morphological and biochemical properties Properties B.subtilis VKPM No. B-8611 B.licheniformis VKPM No. B-8610 Growth under anaerobic conditions - + Fermentation glucose + + arabinose + + xylose + - mannitol + + Citrate disposal + + Propionate Disposal - + Hydrolysis of starch + + Urea hydrolysis - - Nitrate reduction + + The formation of gas from NO ₃ ^- under anaerobic conditions - + Methylene Blue discoloration + + Arginine dihydrolase - + Lecithinase - - Hyaluronidase - - Hemolytic activity - - The formation of poly-β-hydroxybutyric acid globules on glucose agar

table 2 Antibiotic sensitivity of strains of the genus Bacillus Study drug The diameter of the zones of growth inhibition of crops, mm B.subtilis 07 B.licheniformis 09 Penicillins Azlocillin 19 ± 0.1 16 ± 0.1 Amoxicillin 19 ± 0.2 16 ± 0.1 Ampicillin 11 ± 0.2 0 Carbenicillin 23 ± 0.5 20 ± 0.1 Meslocillin 21 ± 0.2 15 ± 0.2 Methicillin 20 ± 0.1 10 ± 0.1 Oxacillin 15 ± 0.3 11 ± 0.1 Benzylpenicillin 7 ± 0.1 3 ± 0.2 Piperacillin 15 ± 0.3 11 ± 0.1 Ticarcillin 25 ± 0.4 21 ± 0.2 Carbapenems Imipenem 38 ± 0.2 30 ± 0.1 Cephalosporins Moxalactam 11 ± 0.2 12 ± 0.3 Cephalotin 35 ± 0.1 21 ± 0.3 Cefazolin 25 ± 0.2 19 ± 0.2 Cefamandol 35 ± 0.3 15 ± 0.1 Cefoxitin 18 ± 0.1 13 ± 0.1 Cefoperazone 15 ± 0.2 13 ± 0.1 Cefotaxime 15 ± 0.1 9 ± 0.1 Ceftazidime 6 ± 0.3 13 ± 0.2 Ceftizoxime 3 ± 0.1 11 ± 0.1 Ceftriaxone 20 ± 0.4 12 ± 0.2 Cefuroxime 2 ± 0.1 3 ± 0.1 AMINOGLYCOSIDES Amikacin 22 ± 0.3 17 ± 0.2 Gentamicin 25 ± 0.3 19 ± 0.2 Kanamycin 22 ± 0.2 21 ± 0.1 Tobramycin 25 ± 0.1 20 ± 0.2 OTHER Vancomycin 12 ± 0.1 10 ± 0.1 Clindamycin 9 ± 0.2 12 ± 0.1 Tetracycline 26 ± 0.1 24 ± 0.1 Chloramphenicol 18 ± 0.3 10 ± 0.1 Polymyxin E 11 ± 0.1 10 ± 0.2 Nitrofurantoin 15 ± 0.2 20 ± 0.1 Trimethoprim 23 ± 0.1 25 ± 0.2 Bactrim 29 ± 0.2 31 ± 0.3 Norfloxacin 24 ± 0.2 25 ± 0.2 Nalidixic acid 21 ± 0.1 15 ± 0.1

Table 3 Antagonistic activity of the biologics Irilis Test culture Biological product options (ratio
B.subtilis: B.licheniformis) × 10 ^8-6 CFU / ml 1: 1 2: 1 5: 1 1:10 10: 1 Staphylococcus aureus (n = 20) 10-15 15-20 15-20 15-20 18-20 Proteus vulgaris (n = 18) 10-15 15-20 15-20 15-20 15-20 Proteus mirabilis (n = 15) 15-18 18-20 18-20 18-20 18-20 Candida albicans (n = 20) 25-30 25-30 25-30 25-30 25-30 Escherihia coli (n = 18) 20-22 22-25 22-25 22-25 22-25 Shigella sonne (n = 25) 10-12 12-15 12-15 12-15 12-15 Streptococcus (n = 17) 10-12 12-15 12-15 12-15 12-15

Claims (12)

1. Veterinary product containing live microbial mass of strains of Bacillus subtilis, Bacillus licheniformis and a protective medium, characterized in that among the strains of Bacillus subtilis it contains a strain of Bacillus subtilis VKPM No. B-8611, and of the strains of Bacillus licheniformis Bacillus licheniformis VKPM VKPM VKPM -8610 taken in effective amounts. 2. The biological product according to claim 1, characterized in that it further comprises a filler. 3. The biological product according to claim 2, characterized in that it contains protein, an organic polymer, milk, whey, albumin, sucrose, lactose, calcium stearate, glucose, sodium bicarbonate, aluminum hydroxide, methyl cellulose, talc, confectionery fat, as a filler, paraffin, lanolin, cocoa butter, aluminum hydroxide gel. 4. The biological product according to claim 3, characterized in that it contains dextrans, polyglucin, starch, polyvinylpyrrolidone from organic polymers. 5. The biological product according to claim 1, characterized in that it further comprises a solvent. 6. The biological product according to claim 5, characterized in that as a solvent it contains distilled or boiled water, physiological saline. 7. The biological product according to any one of claims 1 to 6, characterized in that it is freeze-dried. 8. The biological product according to any one of claims 1 to 6, characterized in that it is granular. 9. The biological product according to any one of claims 1 to 6, characterized in that it is enclosed in a polymer capsule. 10. The biological product according to any one of claims 1 to 6, characterized in that it is tableted. 11. The biological product according to any one of claims 1 to 6, characterized in that it is presented in the form of suppositories or suppositories. 12. The biological product according to any one of claims 1 to 10, characterized in that it is mixed with the components of the feed.