RU2166323C2 - Veterinary probiotic preparation - Google Patents

Abstract

FIELD: veterinary science, microbiology. SUBSTANCE: probiotic preparation has the following components: dry bacterial mass of bifidobacteria, dry bacterial mass of streptococci, dry bacterial mass of recombinant strain of Bacillus subtilis producing human alpha- 2-interferon, carbohydrates, aluminium oxide, aluminium hydroxide and filling agent. Preparation can contain additionally dry bacterial mass of acidophilic bacteria also. The preparation composition can contain any strains of bifidobacteria, streptococci and lactic acid bacteria showing high adhesive ability and antagonistic activity. Also, any recombinant strains of Bacillus subtilis that are able to produce human alpha-2-interferon can be used. In part, the known strains of Bifidobacterium globosum VGNKI N BF-4-DEP, Streptococcus faecium VGNKI N 27-B- DER, Lactobacillus acidophilus VGNKI and industrial strain of Bacillus subtilis B-7092 can be used. Preparation diminishes time of animal treatment, enhances the level of natural resistance and stimulates immune processes. Invention is designated for preparing the biopreparation showing simultaneous antibacterial and antiviral activities that is able to enhance immune status of animal and poultry body and recover their normal intestinal microflora. EFFECT: enhanced effectiveness, improved properties of preparation. 5 cl, 2 tbl, 5 ex

Description

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

 Probiotic preparations based on live microbial cultures, such as lactobacilli, bifidobacteria, streptococci, are widely used in veterinary medicine and medicine for the prevention of gastrointestinal diseases (Antipov V.A. Biological preparations of symbiont microorganisms and their use in veterinary medicine // Agriculture Abroad, 1981, N 2, pp. 43-47).

 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. A wide range of antagonistic activity of probiotics along with complete harmlessness make them an effective tool for the treatment and prevention of gastrointestinal infections, purulent wounds, restoration of the immune status of patients. Probiotics are also used to correct intestinal microflora in case of dysbacteriosis resulting from treatment with antibiotics and chemotherapy.

 The composition of probiotics may include one bacterial strain or, most often, several strains of bacteria of different types in various combinations.

 Known biological product "Streptobifid", which includes dry bacillus bifidobacteria strain Bifidobacterium globosum VGNKI N BF-4 / DEPT, dry bakmassa streptococcus strain Streptococcus faecium VGNKI N 27/11-B-DEPT, aluminum hydroxide, aluminum hydroxide RU N 2086348, A 61 K 35/74, publ. 10.08.97).

 The known drug "Enteracide P" for the prevention and treatment of gastrointestinal diseases of animals, which includes a strain of Lactobacillus acidophilus VKPM B-6535 and a strain of Enterococcus faecium VKPM B-2990 (RU N 2091075, A 61 K 35/74, publ. 27.09.09 .97).

 The probiotic "Bifacid" is also known, containing strains of bifidobacteria and lactobacilli, which is used for human dysbiosis (Ganina V.I., Semenikhina V.F., Inozemtseva N.F., Sundukova M. B. Bifacid - a new domestic biological preparation. // All-Russian scientific-practical conference "Dysbacteriosis and Eubiotics", March 26-28, 1996, Abstracts of Moscow, Moscow, 1996, p. 12).

 However, known probiotics do not have pronounced antiviral activity. As a result, they are not very effective, since most gastrointestinal diseases have a mixed bacterial-viral etiology (Timoshko M.A., Kholmetskaya V.G., Bursuk N.F. Bacteriocinosis of the digestive tract of piglets. Chisinau, Shtiintsa, 1983).

 In addition, gastrointestinal diseases are often accompanied by immunodeficiency states, which sharply reduce the prophylactic effect of probiotics (Klemparskaya N. N. Some results of applying the method of studying the species composition and the number of microbes of autoflora as an indicator of the state of reactivity of an organism. // Math. Conf.: " Autoflora of a healthy and diseased organism ", Tallinn, 1972, p. 3-75; Lenzer A.A. Lactoflora of the animal organism and its protective function. // Theor. And practical. Fundamentals of gnotobiology. M.: Agropromizdat, 1986, p. 195 -200).

 It has been established that the effectiveness of probiotics can be significantly increased if they are used in combination with immunoregulatory proteins of the cytokine class, in particular with interferons (Sat. Actual Problems of Chemotherapy of Bacterial Infections, // Tez. Dokl. All-Union Conf., Moscow, All-Union The Center for Antibiotics, October 22-24, 1991; Bondarenko V.M., Gorskaya E.M. New approaches to modeling, diagnosis and treatment of intestinal dysbiosis // In collection "Medical aspects of microbial ecology", Moscow, 1992, vol. 6, p. 23-25).

 In this regard, the most promising are probiotic preparations, which include strains capable of producing cytokines directly in the intestine of the animal. In this case, the likelihood of a rapid loss of cytokine activity due to digestion by digestive enzymes decreases and their path to target organs - to lymphoid formations in the intestine and Kupffer liver cells is shortened (Belyavskaya V.A. Probiotics from recombinant bacilli - a new class of therapeutic and prophylactic drugs and a method for the delivery of drug proteins into the body // Collection of scientific papers NIKTI BAS SSC WB "Vector", Berdsk, 1996, pp. 190-197).

 Known systems for expressing the artificial human leukocyte interferon gene in Bacillus subtilis cells and a method for constructing a plasmid encoding the synthesis of human alpha-2 leukocyte interferon (V.N. expression of the artificial gene of human leukocyte interferon // Biotechnology, 1988, T. 4, N 5, S. 609-617).

 Known recombinant strains of Bacillus subtilis carrying plasmids encoding the synthesis of intracellular and extracellular interferon (Krasnykh V.N., Danilyuk N.K., Lomakin A.I., Schelkunov S.N. // In the book Hybrid plasmids and plasmid expression genes, 1984, pp. 45-46).

 Known prophylactic biological product "Subalin" based on a recombinant strain of Bacillus subtilis VKPM B-4759, capable of producing alpha-2-interferon person (RU N 2035185, publ. 20.05.95).

 Also known is the biological product "Vetom-1,1", containing the recombinant strain of Bacillus subtilis VKPM B-7092, carrying the plasmid producing alpha-2-interferon person (TU 9382-002-23609643-95 "Instructions for use of the drug Vetom-1,1 in veterinary medicine ", approved by the Department of Veterinary Medicine of the Russian Federation on December 23, 1995).

However, the use of known preparations from aerobic spore-forming bacteria does not fully restore normal intestinal microbiocenosis and requires the additional use of probiotics. The level of production of interferon in the culture medium does not exceed 10 ⁴ • M.E. / ml.

 The closest analogue is the complex probiotic preparation "Bactoferon" for veterinary use, containing dry bacillus of bifidobacteria, streptococci, recombinant interferon alpha and / or gamma, aluminum oxide, carbohydrate lactose and / or sucrose and excipient (RU N 2084234, publ. 20.07.97) .

 The combined use of cytokines with probiotics leads to stimulation of hellers, the removal of the immunosuppressive state in newborn animals, the release of the receptor fields of cells from enteropathogens and the sanitation of mucosal cells from viruses.

 The disadvantage of the drug "Bactoferon" is the instability of cytokines to the action of amylolytic and proteolytic enzymes of the gastrointestinal tract.

 The objective of the invention is the creation of a comprehensive probiotic preparation, characterized simultaneously by antibacterial and antiviral effects with higher antagonistic activity, capable of increasing the immune status of animals and farm birds and resistant to the action of enzymes of the gastrointestinal tract.

 The technical result of the invention is to obtain a probiotic preparation that contains a recombinant strain of Bacillus subtilis that produces human alpha-2 interferon, thereby stimulating local immune responses to the intestinal mucosa and having an antiviral effect, and probiotic bacteria that can correct intestinal biocenosis due to antagonistic and the colonization properties of these strains and displacement from the intestinal biocenosis of opportunistic microflora.

SUMMARY OF THE INVENTION The probiotic preparation contains a dry bacmid of bifidobacteria, a dry bacmass of streptococci, a dry bacmass of a recombinant strain of Bacillus subtilis producing human alpha-2 interferon, carbohydrates, aluminum oxide, aluminum hydroxide and a filler, in the following ratio of components per 1 kg of the drug:
Dry bacillus of bifidobacteria with a content of 10-3000 billion microbial cells (m.c.) in 1 g - 2.0 - 4.0 g
Dry Bakmass streptococci with a content of 10-3000 billion m. in 1 g - 2.0 - 4.0 g
Dry bacmass of the recombinant strain of Bacillus subtilis producing human alpha-2-interferon, containing 10-3000 billion spores in 1 g - 2.0 - 4.0 g
Carbohydrate - 20 - 40 g
Alumina - 10 - 30 g
Aluminum hydroxide - 100 - 300 g
Filler - Else
From carbohydrates, the preparation may contain lactose and / or sucrose, and from the fillers, starch, milk powder, milk whey, a substitute for whole milk, powdered sugar or edible sugar.

 The preparation may also additionally contain dry backass of acidophilic bacteria with a content of 10-3000 billion m. in 1 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-B-DEPT and the strain of acidophilic bacteria Lactobacillus acidophilus VGNKI can be used.

 Of the strains producing human alpha-2-interferon, any recombinant Bacillus subtilis strains capable of producing human alpha-2-interferon can be used, including the well-known production strain of Bacillus subtilis VKPM B-7092.

 The synergism of the action of this combination lies in the fact that recombinant strains produce human alpha-2-interferon, which, acting on and through the intestinal mucosa, has antiviral properties, stimulates the functioning of immunocompetent cells and the production of secretory immunoglobulin A by the local lymphoid organs of the intestine, which is responsible for the adhesion of enteropathogens , carries out regulatory functions between various links of the immunological chain, increases macrophage activity, complement activation, promotes comfort increases the resistance of the mucosa to contamination with viruses and bacteria and provides protection of the mucosa from viruses and rapid colonization of the intestine with strains of probiotic bacteria.

 The strains of bifidobacteria, streptococci and acidophilic bacteria that are part of the drug have a high growth rate, adhesive and antagonistic activity against opportunistic microflora, take part in microbial digestion, produce enzymes, acids, hydrogen peroxide and other biologically active substances that increase non-specific body resistance.

 The drug can be used as an independent tool in the prevention of gastrointestinal diseases or in combination with antibiotics.

The use of the drug inhibits the reproduction of hemolytic opportunistic bacteria and stimulates the growth of normal intestinal microflora. The number of lactobacilli and bifidobacteria in animals treated with the drug significantly exceeds the control values and can be used as a diagnostic sign of the restoration of intestinal biocenosis
The drug does not have a negative effect on the animal organism, convenient and easy to use.

 In experiments on laboratory animals, the harmlessness of the drug and the absence of acute and chronic toxicity were confirmed. Morphological and biochemical studies of blood and its serum did not reveal a negative effect of the drug on blood-forming organs, metabolic processes, and liver and kidney functions.

 The most effective application regimen is its use, starting from the first day of the animal’s life every day for 5-10 days.

 The drug reduces the time of treatment of animals, increases the level of natural resistance and stimulates the immune processes.

 The invention is illustrated by the following examples.

 Example 1. A probiotic preparation is obtained by separate deep cultivation of strains of bifidobacteria, streptococci and a recombinant strain on liquid nutrient media adopted for the cultivation of these types of microorganisms.

 Use strains of Streptococcus faecium VGNKI N 27/11 B-DEPT, Bifidobacterium globosum VGNKI N BF / 4-DEPT and the recombinant strain Bacillus subtilis VKPM B-7092.

 During the cultivation of bifidobacteria and streptococci for 16 + 2 h, a biomass yield of each bacterial species of at least 10 billion live microbial cells in 1 ml of culture suspension is achieved.

A method of cultivating a recombinant strain and a method of obtaining a dry bacterial mass of the strain is the subject of "know-how"
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 semi-finished products of each strain contain at least 100 billion live microbial cells in one gram.

Next, the dry Bakmass of each strain is mixed in equal proportions and carbohydrate, aluminum oxide and hydroxide and a filler (for example, starch or food sugar, powdered sugar) are added, with the following ratio of components per 1 kg:
dry Bakmass of a strain of streptococcus with a content of 10 - 3000 billion live microbial cells in 1 g - 2 g
dry bacillus of a strain of bifidobacteria containing 10-3000 billion living microbial cells in 1 g - 2 g
dry bakmassa strain of spore-forming bacteria with a content of live spores of 10-3000 billion in 1 g - 2 g
carbohydrate (lactose or sucrose) - 20 g
aluminum oxide - 10 g
aluminum hydroxide - 100 g
filler - up to 1 kg
The specified composition allows you to get a series of the drug with a total CFU of streptococci, bifidobacteria and spore-forming bacteria (with an equal quantitative ratio) from 60 million to 6 billion in one gram.

 Example 2. Take strains of Streptococcus faecium VGNKI N 27/11 B-DEPT, Bifidobacterium globosum VGNKI N BF / 4-DEPT, Lactobacillus acidophilus VGNKI and the recombinant Bacillus subtilis strain VKPM B-7092. The drug is prepared analogously to example 1.

Get the drug with the following ratio of components per 1 kg:
dry Bakmass of a strain of streptococcus with a content of 10 - 3000 billion m. in 1 g - 3 g
dry bakmassa strain of bifidobacteria with a content of 10-3000 billion m. in 1 g - 3 g
dry Bakmass of a strain of spore-forming bacteria with a content of live spores of 10-3000 billion in 1 g - 3 g
dry backbase of acidophilic bacteria with a content of 10-3000 billion m. in 1 g - 3 g
carbohydrate (lactose or sucrose) - 30 g
aluminum oxide - 20 g
aluminum hydroxide - 200 g
filler - up to 1 kg
The specified composition allows you to get a series of the drug with a total value of CFU of streptococci, bifidobacteria, acidophilus bacteria and spore-forming bacteria (with an equal quantitative ratio) from 90 million to 9 billion m. In one gram.

 Example 3. Obtained in examples 1 and 2 preparations with a total bacterial concentration of 90 million m. and 1.2 billion m. in one gram, respectively, are used to restore the intestinal biocenosis of carnivores.

 As a biological model, 57 puppies of dogs of various breeds were admitted to the clinic with a diagnosis of gastrointestinal diseases of viral etiology (viral enteritis, viral hepatitis).

 For testing puppies are divided into three groups. The animals of the first group (24 puppies), along with symptomatic treatment, are prescribed a drug with a total concentration of live bacteria of 90 million cubic meters. in grams based on one gram of the drug per head for 5 days. The animals of the second group (16 puppies) are given a drug with a total concentration of bacteria of 1.2 billion cubic meters in one gram is similar. Puppies of experimental groups are additionally prescribed general strengthening therapy. Puppies of the third group are used as control.

 A pronounced clinical effect is observed in puppies of the first experimental group on the third to fifth day after the start of the drug. In puppies of the second experimental group, a pronounced clinical effect is observed already on the second day of its giving. The act of defecation in puppies is restored, behavioral reactions are activated, appetite is restored, the gag reflex disappears. In the first experimental group 3 puppies fell, in 3 puppies the disease turned into a chronic form. In the second experimental group, there was no case, the disease turned into a chronic form in 1 puppy. In the control group receiving symptomatic, passive immunizing therapy and antibiotics, 2 puppies fell, in 5 the disease turned into a chronic form. The course of restorative aftercare of puppies in both experimental groups was 10-18 days, while in the control from 12-14 to one month.

 Thus, the drug significantly accelerates the normalization of the intestinal biocenosis of dogs.

 Example 4. Obtained in examples 1 and 2 preparations with a total concentration of bacteria of 90 million m. K. and 12 billion m. in one gram, respectively, they are used for the prevention and treatment of gastrointestinal diseases in poultry at a poultry farm unsuccessful for gastrointestinal diseases (average safety of the bird is 87-89%).

 Before the test, the case with signs of colibacteriosis, according to the pathological and anatomical autopsy of the dead bird, makes up from 28 to 40% in the general structure of the case, digestive diseases (hepatitis, enteritis) up to 20-25%. Salmonella, Escherichia, staphylococci are sown from the bodies of dead birds. The peak of death occurs in the first 10 days of chickens and in the first days after routine vaccinations with live attenuated viral vaccines.

 Testing the prophylactic effectiveness of the preparations is carried out in two workshops of the poultry farm on broiler chickens, starting from the daily to 10 days of age.

 The test drugs are used with water or food at the rate of 0.01 g per head once a day for 5-10 days, depending on the indications.

 Chickens are divided into three groups: two experimental and one control. The test results are given in table. 1.

 The results of the pathoanatomical autopsy of the dead bird showed a decrease in the mortality rate from gastrointestinal diseases in the overall mortality structure by 86.9% compared with the control group.

 For therapeutic purposes, the drug is used for young laying hens at the age of 64 days to eliminate the vaccine complication of the vaccine against infectious laryngotracheitis.

The drug is set similarly, at the rate of 0.05 doses per head, dividing the workshop into two halves - the experimental and control. The test drug was mixed with food and asked once a day, starting from the second day after vaccination. The results of the use of the drug are given in table. 2
Example 5. Conduct a test of the therapeutic effectiveness of the drug in experiments on calves with a clinic of diarrheal syndrome.

 The disease of calves with acute gastrointestinal diarrhea in the early postnatal period of life is associated with the birth of young animals with reduced resistance and with overload of colostrum of the mother with antigens during unbalanced feeding. In some households, the disease is registered in 100% of newborn calves.

 The use of various pharmacological agents for the treatment of gastrointestinal diseases in calves did not give positive results.

 The drug is prescribed for sick calves in combination with symptomatic therapy and the use of cardiovascular therapy, drinking plenty.

 The calf population is divided into three groups: two experimental and one control. In the first experimental group, only the drug is used; in the second experimental group, the drug is used in conjunction with antibiotics. In the control group, only antibiotics are used. Record the number of calves recovered, the duration of their treatment and death.

 As shown by the research results, the calves of the first experimental group fully recovered within 5-6 days.

 The greatest effect is observed in the second experimental group when using the drug in conjunction with antibiotics. This effect is due to the preventive effect of antibiotics on the secondary microflora and suggests that the strains included in the drug do not lose their activity during antibiotic therapy.

 When antibiotics were used (control group), complete recovery was observed only in 40% of calves; in other calves, treatment after antibiotic use was continued by other drugs.

 The results of bacteriological studies of intestinal microflora in calves of the experimental and control groups showed a significant effect of the drug on the formation of intestinal biocenosis. In the contents of the intestine, the number of lactose-positive and lactose-negative bacteria of the group of Escherichia coli decreases, the number of representatives of lacto- and bifidoflora increases.

 Marked changes in the composition of the intestinal biocenosis of calves indicate that the drug helps to protect the intestines from colonization by its opportunistic microflora.

 The results of an immunological study of blood and its serum showed that the calves of the experimental groups increase the activity of lysozyme, beta-lysines and phagocytic activity. These indicators indicate a shift in the immune system towards its activation. In the lymphocyte population, the content of T-helper cells increases, the activity of T-suppressors decreases. Studies have established that the drug does not cause negative changes in the white blood cell count with a 10-day use.

 The results obtained confirm the immunostimulating effect of the drug and its participation in the formation of intestinal biocenosis.

Claims (4)

1. A probiotic veterinary preparation containing dry bacillus of bifidobacteria, streptococci, recombinant interferon, alumina, carbohydrate lactose and / or sucrose and an excipient, characterized in that it additionally contains aluminum hydroxide, and as a recombinant interferon contains dry bacmass of recombinant strain Bac capable of producing alpha-2-interferon, in the following ratio of components per 1 kg of the drug:
Dry bacillus of bifidobacteria with a content of 10 - 3000 billion microbial cells (m.c.) in 1 g - 2.0 - 4.0 g
Dry Bakmass streptococci with a content of 10 - 3000 billion m.k. in 1 g - 2.0 - 4.0 g
Dry bacmass of the recombinant strain of Bacillus subtilis producing human alpha-2-interferon, containing 10 - 3000 billion spores in 1 g - 2.0 - 4.0 g
Carbohydrate - 20 - 40 g
Alumina - 10 - 30 g
Aluminum hydroxide - 100 - 300 g
Filler - Else
2. 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  3. 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, and from recombinant strains of Bacillus subtilis that can produce alpha -2-interferon, a strain of Bacillus subtilis VKPM B-7092.  4. The drug according to claim 2, characterized in that from acidophilic bacteria it contains a strain of Lactobacillus asidophilus VGNKI.  5. The drug according to claim 1, characterized in that of the fillers it contains starch, milk powder, milk whey powder, whole milk replacer, powdered sugar or edible sugar.

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