Probiotic preparation for veterinary use

 

(57) Abstract:

The invention is intended to obtain a biological product that has both antibacterial and antiviral activities, can improve the immune status of the animals and birds and to restore the normal microflora. Probiotic product contains dry backass bifidobacteria, dry backass streptococcal dry backass recombinant strain of Bacillus subtilis that produce alpha-2-interferon man, carbohydrates, aluminum oxide, aluminum hydroxide and the filler. The drug may also optionally contain dry backass acidophilus bacteria. In the composition of the drug can be used any strains of bifidobacteria, streptococci and lactic acid bacteria, characterized by high adhesion power and antagonistic activity. Can be used any recombinant strains of Bacillus subtilis are capable of producing alpha-2-interferon-man. In particular, there can be used known strains of Bifidobacterium qlobosum VGNKI BF-4-DEPT, Streptococcus faecium VGNKI 27 IN-DEPT, Lactobacillus acidophilus VGNKI and production strain Bacillus subtilis VKPM B-7092. The drug reduces the time of treatment of animals, increases the level of natural and biotechnology and veterinary medicine, namely the creation of a biological product with antibacterial and antiviral activities, but can also increase the immune status of the animals and birds and to restore normal flora.

Probiotic preparations on the basis of live microbial cultures, such as lactobacilli, bifidobacteria, streptococci, are widely used in veterinary medicine and medicine for the prevention of gastrointestinal diseases (Antipov Century A. Biological products, symbiotic microorganisms and their application in veterinary medicine // Agriculture abroad, 1981, No. 2, S. 43-47).

The mechanism of action of probiotics is based on adhesive and antagonistic properties of bacterial probionts, displacing from the composition of the intestinal population of conditionally pathogenic microorganisms and nonspecific controlling the redundancy of their growth. A wide spectrum of antagonistic activity of probiotics along with the complete harmlessness make them effective for the treatment and prevention of gastrointestinal infections, purulent wounds, restoration of the immune status of the patients. Probiotics are also used for correction of the intestinal microflora in dysbacterioses, resulting from antibiotic treatment and himiob what about the bacterial strains of different species in various combinations.

Known biological product "Streptavidin", which includes dry backmask Bifidobacterium Bifidobacterium globosum VGNKI N CF-4/DEPT, dry backmask streptococcal strain of Streptococcus faecium VGNKI N 27/11-IN-DEPT, carbohydrate, aluminum oxide, aluminum hydroxide and the filler (RU N 2086348, A 61 K 35/74, publ. 10.08.97).

Known drug "Interacid P" for the prevention and treatment of gastrointestinal diseases in animals, which includes Lactobacillus acidophilus VKPM B-6535 and strain Enterococcus faecium VKPM B-2990 (RU N 2091075, A 61 K 35/74, publ. 27.09.97).

Also known probiotic "Biface" containing strains of bifidobacteria and lactobacilli, which is used when the dysbacteriosis in humans (Ganina Century. And. , Semenikhina C. F., Inozemtseva N. F., Sundukova M. B. Biface - new domestic biological drug. // The scientifically conference. scientific-practical. proc. "Dysbacteriosis and eubiotics", March 26-28, 1996, proc.Dokl., Moscow, 1996, S. 12).

However, the known probiotics do not have a pronounced antiviral activity. As a result, they are not effective, since the majority of gastrointestinal diseases have mixed bacterial-viral etiology (Timoshka M. A. , Holmetskaya Century, Borsuk N. F. Bacteriocins the digestive tract of piglets. Chisinau,diversified States, drastically reducing the preventive effect of probiotics (Lamparska N. N. Some results of applying the method to the study of species composition and microbial autoflora as an indicator of the reactivity of the organism. // Mat. scient. Conf.: "Autoflora healthy and diseased organism", Tallinn, 1972, S. 3-75; Lentser A. A. Lactiflora animal organism and its protective function. // Tiora. and practical use. fundamentals of gnotobiology. M: Agropromizdat, 1986, S. 195-200).

It is established that the effectiveness of probiotics can be significantly enhanced if they are used in combination with immunoregulatory proteins class of cytokines, in particular interferon (Sat. "Actual problems of chemotherapy of bacterial infections"// proc.Dokl. Vsesojuzn. Conf., Moscow, Vsesojuzn. Center for antibiotics, 22-24 Oct., 1991; Bondarenko, C. M., E. Gorsky M. New approaches to modeling, diagnosis, and treatment of dysbacteriosis//In: "Medical aspects of microbial ecology", Moscow, 1992, vol. 6, S. 23-25).

In this regard, the most promising probiotic preparations, which include strains capable of cytokine production directly in the gut of the animal. In this case decreases the probability of rapid rubbed the targets - to lymphoid formations in the gut and copperskin liver cells (Belyavskaya Century A. Probiotics from recombinant bacilli - a new class of preventive and curative drugs and method of drug delivery of proteins in the body// Sat. the scientific. Tr. Nikti BAS SRC VB "Vector",, Berdsk, 1996, S. 190-197).

Known systems for the expression of synthetic gene leukocyte interferon in human cells of Bacillus subtilis and method of constructing a plasmid that encodes the synthesis of alpha-2-leukocyte interferon man (C. N. Red and other Chemical-enzymatic synthesis and cloning of the gene signal peptide-amylase of Bacillus amylolitquefaciens for the expression of synthetic gene leukocyte interferon/ human/ Biotechnology, 1988, T. 4, N 5, S. 609-617).

Known recombinant strains of Bacillus subtilis carrying plasmids encoding the synthesis of intra - and extracellular interferon (Red C. N., Danyluk N. K. , Lomakin, A. I., Shchelkunov, S. N. // In the book. Hybrid plasmids and expression of plasmid genes, 1984, S. 45-46).

Known preventive biological preparation "Subalign" on the basis of recombinant Bacillus subtilis strain VKPM B-4759, capable of producing alpha-2-interferon person (RU N 2035185, publ. 20.05.95).

Also known biological product "Wloclawek (TU 9382-002-23609643-95 "Guidance on the use of drug Council-1.1 in veterinary medicine", decl. The veterinary Department of the Russian Federation of 23 December 1995).

However, the use of known products of aerobic spore-forming bacteria does not provide full recovery of normal intestinal microbiocenosis and requires additional use of probiotics. The level of interferon production in the culture medium does not exceed 104M. E./ml.

The closest analogue is a complex probiotic preparation "Lactoferrin" for veterinary use containing dry backass bifidobacteria, streptococci, recombinant interferon alpha and/or gamma alumina, carbohydrate lactose and/or sucrose and filler (RU N 2084234, publ. 20.07.97).

The combined use of cytokines with probiotics leads to stimulation of Hellerau, withdrawal of immunosuppressive conditions in newborn animals, the release of the receptor fields of cells from enteropathogenic and rehabilitation of mucosal cells from viruses.

Lack of preparation "Lactoferrin" is the volatility of cytokines to the action of amylolytic and proteolytic enzymes of the gastrointestinal tract.

The objective of the invention is to create a comprehensive probiotic preparation, kharakterizuyushchego to improve the immune status of animals and poultry and resistant to the action of enzymes of the gastrointestinal tract.

The technical result of the invention consists in obtaining a probiotic product containing recombinant strain of Bacillus subtilis that produce alpha-2-interferon person, thereby stimulating a local immune response of the intestinal mucosa and has antiviral action, and bacteria-probeonly capable korrigirovat intestinal biocenosis due to antagonistic and colonization properties of these strains and displacement of intestinal biocenosis of conditionally pathogenic microflora.

The essence of the invention. Probiotic product contains dry backass bifidobacteria, dry backass streptococcal dry backass recombinant strain of Bacillus subtilis that produce alpha-2-interferon man, carbohydrates, aluminum oxide, aluminum hydroxide and a filler, in the following ratio of components per 1 kg of the drug:

Dry backmask bifidobacteria content 10-3000 billion microbial cells (M. K.) 1 g - 2,0 - 4,0 g

Dry backmask streptococcal content 10-3000 billion, M. K. 1 g - 2,0 - 4,0 g

Dry backmask recombinant strain of Bacillus subtilis that produce alpha-2-interferon person, with the content of 10-3000 billion spores per 1 g - 2,0 - 4,0 g

Carbohydrate - 20 - 40 g

Aluminum oxide is 10 to avoid lactose and/ or sucrose, and fillers - starch, milk powder, whey milk, whole milk powder, powdered sugar or food sugar.

The drug may also optionally contain dry backass acidophilus bacteria content 10-3000 billion, M. K. 1,

In the composition of the drug can be used any strains of bifidobacteria, streptococci and lactic acid bacteria, characterized by high adhesion power and antagonistic activity. In particular, there can be used known strains of Bifidobacterium globosum VGNKI N BF-4-DEPT, Streptococcus faecium VGNKI N 27 IN-DEPT and strain acidophilus bacteria Lactobacillus acidophilus VGNKI.

From strains producing alpha-2-interferon may be used any recombinant strains of Bacillus subtilis are capable of producing alpha-2-interferon person, including the well-known production strain Bacillus subtilis VKPM B-7092.

The synergistic actions of this combination lies in the fact that recombinant strains produce alpha-2-interferon person who, acting on and through the intestinal mucosa, has antiviral properties, stimulates the immune cells and the production of local lymphoid organs to the major tasks between different parts of immunological circuit, increases the activity of macrophages, activation of complement, enhance the resistance of the mucosa to contamination by bacteria and viruses and protects the mucosa from viruses and rapid colonization of the intestine by the bacterial strains of probionts.

Included in the drug strains of bifidobacteria, streptococci and acidophilic bacteria have a high rate of growth, adhesion and antagonistic activity against pathogenic microflora, participate in the processes of microbial digestion, produce enzymes, acids, hydrogen peroxide and other biologically active substances which increase nonspecific resistance of the organism.

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

The drug inhibits the propagation of hemolytic conditionally pathogenic bacteria and stimulates the growth of normal intestinal microflora. The number of lactobacilli and bifidobacteria in animals treated with the drug, significantly greater than control values and can be used as a diagnostic sign of restoration of intestinal biocenosis is"ptx2">

In experiments on laboratory animals confirmed the safety of the drug and the absence of the acute and chronic toxicity. Morphological and biochemical studies of blood and blood serum revealed no negative effects of the drug on blood-forming organs, metabolism, liver and kidneys.

The most effective use of the scheme is its use, starting with the first day of life of the animal daily 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. Probiotic preparation is produced by a separate submerged cultivation of strains of bifidobacteria, streptococci and recombinant strains in liquid nutrient media adopted for the cultivation of these species of microorganisms.

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

During cultivation of bifidobacteria and streptococci for 16+2 h biomass yield of each species of bacteria of at least 10 billion live micro is Holocene dry bacterial mass strain is the subject of know-how

The obtained culture was concentrated in 10-15 times depending on the density of the culture of sediments washed from the products of metabolism in the separators or microfiltration units.

Concentrated bacterial suspension is dried by sublimation method according to the conventional technology.

In the resulting dry semi-finished products of each strain contains at least 100 billion living microbial cells per gram.

Next dry backass each strain are mixed in equal proportions and add carbohydrate, oxide and hydroxide of aluminum and the filler (e.g., starch or sugar food, powdered sugar), with the following ratio of 1 kg:

dry backmask strain of Streptococcus content of 10 to 3000 billion living microbial cells in 1 g - 2 g

dry backmask strain of bifidobacteria content 10-3000 billion living microbial cells in 1 g - 2 g

dry backmask strains of spore-forming bacteria content of live spores 10-3000 billion in 1 g - 2 g

carbohydrate (lactose or sucrose 20 g

aluminum oxide 10 grams

aluminium hydroxide - 100 g

filler - up to 1 kg

The composition allows to obtain a series of drug with a total value of SOME streptococcal Vivienne.

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

Receiving this medicine with the following ratio of components per 1 kg:

dry backmask strain of Streptococcus content of 10 to 3000 billion, M. K. 1 g - 3 g

dry backmask strain of bifidobacteria content 10-3000 billion, M. K. 1 g - 3 g

dry backmask strains of spore-forming bacteria content of live spores 10-3000 billion in 1 g - 3 g

dry backmask acidophilus bacteria content 10-3000 billion, M. K. 1 g - 3 g

carbohydrate (lactose or sucrose) - 30 grams

aluminum oxide - a 20 g

aluminium hydroxide - 200 grams

filler - up to 1kg

The composition allows to obtain a series of drug with a total value of SOME streptococci, bifidobacteria, acidophilic bacteria and spore-forming bacteria (when equal to their proportions) from 90 million to 9 billion m K. in one gram.

Example 3. Obtained according to examples 1 and 2 drugs total concentration of bacteria 90 million M. K., and 1.2 billion m K. in one gram respectively used for restoration of intestinal biocenosis carnivorous.

As Biol is cnyh diseases of viral etiology (viral enteritis, viral hepatitis).

For testing puppies are distributed into three groups. Animals of the first group (24 puppy) along with symptomatic treatment is prescribed a drug with a total concentration of live bacteria 90 million M. K. gram one gram of a preparation per head for 5 days. Animals of the second group (16 puppies) specify the medication with a total concentration of bacteria 1.2 billion m K. in one gram similarly. Puppies experimental groups appoint restorative therapy. Puppies of the third group used as control.

Pronounced clinical effect observed in pups of the first experimental group on the third or fifth day after the beginning of the drug. In puppies the second experimental group expressed clinical effect observed on the second day of his villas. The defecation puppies restored, behavioral responses are activated, appetite disappears gag reflex. In the first experimental group fell 3 puppies, 3 puppies disease from becoming chronic. In the second experimental group case is not marked, the disease has become chronic in 1 puppy. In the control group treated with symptomatic, passive immunization therapy and antibiotics, the experimental groups was 10-18 days while in the control from 12-14 to one month.

Thus, the drug significantly speeds up the process of normalization of intestinal biocenosis dogs.

Example 4. Obtained according to examples 1 and 2 drugs total concentration of bacteria 90 million M. K., and 12 billion m K. in one gram respectively used for the prevention and treatment of gastrointestinal diseases in poultry at the poultry farm prone to gastro-intestinal diseases (medium safety of the birds is 87-89%).

To test the case with signs of colibacillosis, according to the anatomic dissection of dead birds, ranging from 28 to 40% of the total mortality, digestive diseases (hepatitis, enteritis) to 20-25%. From the corpses of dead birds seeded Salmonella, Escherichia, Staphylococcus. Peak mortality occurs within the first 10 days of life the Chicks and in the first days after routine vaccination live attenuated viral vaccines.

Test the prophylactic efficacy of drugs is carried out in two workshops poultry chickens-broilers, starting with a daily and up to 10 days of age.

Tested preparations used with water or feed rate of 0.01 g on the head once in de the control. The results are given in table. 1.

The results of the anatomic dissection of dead birds showed a decrease in mortality from gastrointestinal diseases in the General structure of mortality at 86.9 per cent in comparison with the control group.

With the medical purpose the drug is administered to young laying hens at the age of 64 days for the elimination of post-vaccination complications vaccine against infectious laryngotracheitis.

The drug is set similarly, the rate of 0.05 dose on the head, dividing the plant into two halves - experimental and control. The test drug is mixed with food and asked once a day, starting from the second day after vaccination. The results of the drug are given in table. 2

Example 5. Conduct the test therapeutic efficacy of the drug in the experiments on calves with clinical diarrhea syndrome.

Disease of calves acute gastrointestinal diarrhea in early postnatal life is associated with the birth of calves with low resistance and overload colostrum mother antigens when unbalanced feeding. In some farms, the disease is recorded at 100% of newborn calves.

The use of different CLASS="ptx2">

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

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

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

The greatest effect observed in the second experimental group 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 belonging to the drug, do not lose their activity during antibiotic treatment.

When the use of antibiotics (control group) full recovery see only 40% of calves, the rest of the calves treatment after the use of antibiotics was continued other drugs.

The results of bacteriological studies of the intestinal microflora in Tosa. In the contents of the intestine decreases the number ectoparasiticide and lactosaminated a group of bacteria Escherichia coli, an increasing number of representatives of lacto - and bifidoflora.

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

The results of immunological studies of blood and blood serum showed that calves experimental groups increases the activity of lysozyme, beta-lysine and phagocytic activity. These figures indicate the shift of the immune system in the direction of its activation. In a population of lymphocytes increases the amount of T-helper cells, decreases the activity of T-suppressor. Research has shown that the drug does not cause negative changes in the leukocyte formula blood with a 10-day it is applied.

These results confirm the immunostimulatory effect of the drug and its involvement in the formation of intestinal biocenosis.

1. Probiotic preparation for veterinary use containing dry backass bifidobacteria, streptococci, recombinant interferon, aluminum oxide, from what I and as recombinant interferon contains dry backass recombinant Bacillus subtilis strain capable of producing alpha-2-interferon, in the following ratio of components per 1 kg of the drug:

Dry backmask bifidobacteria content of 10 to 3000 billion microbial cells (M. K.) 1 g - 2,0 - 4,0 g

Dry backmask streptococci with a content of 10 to 3000 billion, M. K. 1 g - 2,0 - 4,0 g

Dry backmask recombinant strain of Bacillus subtilis that produce alpha-2-interferon man, with a content of 10 to 3000 billion spores per 1 g - 2,0 - 4,0 g

Carbohydrate - 20 - 40 g

Aluminum oxide is 10 to 30 grams

Aluminium hydroxide - 100 - 300 g

Filler - Rest

2. The drug under item 1, characterized in that it further comprises a dry backass acidophilus bacteria content of 30 to 1000 billion, M. K. 1,

3. The drug under item 1, characterized in that it contains bifidobacteria strain Bifidobacterium globosum VGNKI BF-4/DEPT of streptococcal strains of Streptococcus faecium VGNKI 27/11 IN-DEPT and of recombinant strains of Bacillus subtilis are capable of producing alpha-2-interferon, a strain of Bacillus subtilis VKPM B-7092.

4. The drug under item 2, characterized in that acidophilus bacteria it contains Lactobacillus asidophilus VGNKI.

5. The drug under item 1, tlicho milk, powdered sugar or food sugar.

 

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