RU2146288C1 - Method of preparing homoprobiotic bacterial preparation containing living lactic bacteria - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: bacterial preparation is designed for preventing and treating animal and poultry diseases and as well for preparing gnotobiotic organisms. To isolate natural bacterial complexes, native biomaterial is selectively decontaminated by diluting it with liquid lactic bacterium-selective nutrient medium. This procedure allows removing accidentally occurring in biomaterial microorganisms foreign for given animal and poultry species. Subsequent treatment of diluted biomaterial with blood serum (or with immunoglobulin preparations) from the same animal or poultry family enables most complete removal of foreign microflora. Once fried of all contaminating microorganisms, biomaterial is cultured under anaerobic conditions. Resulting biomass is then reseeded, without isolating pure cultures into nutrient medium, and recultured under anaerobic conditions. EFFECT: more efficiently reduced mortality in chickens upon oral administration under industrial poultry keeping. 3 tbl, 4 ex

Description

 The invention relates to biotechnology, in particular the production of bacterial preparations, and can be used for the prevention and treatment of diseases of animals and birds, as well as the production of gnotobiological organisms (SPF-animals and birds).

 The creation of large poultry and livestock enterprises raised a number of new environmental and clinical problems, many of which have not been resolved to date. Among them are such as the need to protect the livestock from epizootics that lead to mortality of young animals and mass death of poultry, protecting consumers of products from infectious agents, the source of which is animal and poultry meat - salmonella, campylobacter, Escherichia coli, Yersinia, Listeria, cryptosporidium and etc., preventing the spread in nature of antibiotic-resistant strains of animal microorganisms, etc. It has been established, for example, that 77% of cases of human salmonellosis are caused by bird salmonella its origin. The annual losses from salmonellosis in humans and birds in the United States alone are at least $ 300 million.

 There is no doubt that the range of potential pathogens of domestic animals and birds will expand steadily, which is associated with both improved diagnostics and a deterioration of the environmental situation in individual regions and in the world as a whole, causing a violation of the protective mechanisms of the macroorganism, primarily its normal microflora . The role of normal microflora in protecting the host organism from the adverse effects of various infectious and non-infectious agents is well known. Under natural conditions, the normal microflora of animals and birds is formed primarily due to their infection with the microflora of their parents, as well as microorganisms from the external environment. However, the modern technology of industrial animal husbandry and especially poultry farming, due to its characteristics, violates the evolutionary relationships between the host organism and its microflora, since this technology virtually eliminates the seeding of offspring with normal microflora of the parents. Therefore, chickens hatched in incubators are seeded by microorganisms randomly from the environment. Their biofilm is inferior in terms of ensuring colonization resistance. In this regard, infection of such chickens even with single cells of potentially pathogenic microorganisms is accompanied by a rapid multiplication of the latter, their dissemination to internal organs with the occurrence of pathological processes and the mass death of young animals or the formation of individuals with prolonged carriage of pathogenic microorganisms.

 Many methods are known for protecting young birds and domestic animals from infectious diseases. First of all, this is the inclusion of a variety of antibacterial drugs and their complexes in the diet of young animals. In addition, various vaccines, bacteriophages and other antimicrobial agents are widely used. However, prolonged use of antibiotics leads to the selection of antibiotic-resistant strains of microorganisms, which leads to an extension of carriage and their dissemination to the environment. The widespread use of antibiotics in poultry also leads to their accumulation in poultry meat with the subsequent entry of these chemical compounds into the consumer's body with the possible development of a variety of toxic, allergic and dysbiotic reactions in the latter.

 Currently, various techniques are used to restore the disturbed microbiocenosis of animals and birds, primarily the introduction of large amounts of antagonistic strains of bacteria - representatives of the normal microflora of the intestines of animals, birds and humans (E. coli, bifidobacteria, lactobacilli, etc.). Numerous compositions of pure cultures of microorganisms isolated from humans and various species of animals and birds are described for the prevention and treatment of dysbacterioses and intestinal diseases in birds and animals, or their combination with bacterial growth stimulants or immunomodulators.

 For example, probiotic preparations for preventing colibacteriosis in chickens made from monoculture of Escherichia coli isolated from chickens or from lactobacilli and propionibacteria cultures isolated from sheep are known. To prevent salmonellosis and campylobacteriosis, it was proposed to feed young monocultures of lactobacilli or enterococci to young animals. Known preparations are bactoferon prepared from living cultures of bifidobacteria and streptococci with the addition of interferon; streptobifid, which is a dry biomass of a mixture of pure cultures of bifidobacteria and streptococci isolated from piglets; SBA preparation containing a mixture of three strains of bacteria of various types - acidophilic lactobacilli, bifidobacteria and fecal streptococci, and intended for the prevention of diseases of piglets; known is a complex bacterial preparation for the treatment and prevention of gastrointestinal diseases of animals - enteric P, containing one strain of acidophilic lactobacilli and fecal enterococci isolated from Arctic fox, the bacterial preparation Biosan for treating bovine endometritis, containing a mixture of two types of lactobacilli strains isolated from a healthy vagina women, and many others.

 However, it was noted that the best protective effect is observed with the introduction of bacterial cultures isolated from an animal or bird of the same species. For example, bacteria isolated from mice, pigs, and other animals were much less likely to protect newborn chickens from infectious diseases than bacteria isolated from chickens. In this regard, the disadvantage of most of the above drugs is that they contain bacteria that are heterogeneous for the recipient's body, and therefore are quickly excreted in transit from the body. In this regard, it is difficult to expect the same positive effect when prescribing any of these drugs to animals and birds of different species.

 Known methods for creating colonization resistance in young chickens by using pure cultures of microorganisms isolated from goiter, cecum and colon of adult chickens. It is also known that the protection of young birds from infectious diseases is more pronounced when using not monocultures, but a mixture of pure cultures of microorganisms, consisting of 2, 6 and even 28 microorganisms isolated from goiter and cecum of healthy adult chickens, and some of them were not identified ( 1). However, with this method of creating a homoprobiotic preparation, that is, a probiotic prepared on the basis of allochthonous microorganisms of a particular animal or bird species and intended for use by animals and birds of the same species, it is highly probable that the isolated cultures are clones of microorganisms heterogeneous for the species belonging to the host, caught in its digestive tract with food and water, contaminated with transient bacteria from the environment or animal organisms of another species th accessory (fish, amphibians, insects and others, including humans). In addition, according to the authors, the effectiveness of the bacterial mixture that was prepared for each test from preserved pure cultures progressively decreased over the course of 20 weeks of testing, while the effectiveness of the bacterial mixture prepared for each test from stored cecal contents remained unchanged.

 There is evidence of the protective effect of not only homoprobiotic preparations prepared from a mixture of pure cultures of fecal bacteria after culturing them under anaerobic conditions, but also the purpose of the native intestinal contents of adult chickens.

 The protective effect against salmonellosis in the oral administration of the intestinal microflora of adult birds has been established in Sweden, where 2.86 million broiler chickens have been injected with the fecal bacterial flora of chickens for 5 years (2). It was also shown that the appointment of the contents of the colon can have a pronounced therapeutic effect in the appointment of a bird infected with pathogenic enterobacteria (3). However, in the intestinal contents of healthy animals and birds, along with a high content of autochthonous bacteria, there is a large number of various allochthonous bacteria, as well as fungi. In this regard, the appointment of young colon contents of the colon of adult animals and birds for prophylactic or therapeutic purposes is associated with a risk of infection of the recipient with random bacterial species, including pathogenic ones.

A known method for producing a homoprobiotic bacterial preparation for the prevention of intestinal diseases of birds, especially salmonellosis (4). The method involves anaerobic cultivation by known methods of the contents of the cecum of the bird in the presence of epithelial cells of the digestive tract, such as goiter, followed by cultivation of the isolation of pure cultures of bacteria, the growth of biomass, its separation from the nutrient medium, followed by freezing or lyophilization. For the preparation of the preparation, only bacterial strains with high adhesive activity (at least 10 bacteria per 1 epithelial cell) selected in the Fuller adhesion test are used. Thus isolated strains were identified as Lactobacillus acidophilus, L.fermentum, L. lactis and Clostridium spp. The final preparation is prepared by separately culturing the selected bacteria on a nutrient medium such as MRS-broth or VL-broth under anaerobic conditions at 35 ^° C. overnight. At the end of the cultivation, a mixture of cultures is prepared by placing several drops of each culture in a nutrient broth and cultivating the mixture under anaerobic conditions for 2 days. Then the bacterial cells are separated from the nutrient broth, dried or frozen. Perhaps co-cultivation of selected strains.

 Thus, the cultivation of the contents of cecum under anaerobic conditions and the subsequent selection of bacteria according to their ability to adhere to it makes it possible to increase the efficiency of the resulting preparation by using bacteria with high adhesive characteristics when preparing the final probiotic preparation. With this method of constructing the drug, the adhesive activity of bacteria to epithelial cells is determining. However, it is known that many bacteria alien to the host, including pathogenic intestinal microorganisms, also have nonspecific adhesive activity. In addition, they can carry plasmid transmissible genes that encode the formation of structures in them that determine increased specific adhesion to epithelial cells (such as K-88 and K-99 antigens in Escherichia isolated from piglets and chickens, surface proteins in streptococci, etc. .).

 The purpose of the invention is the production of a homoprobiotic bacterial preparation containing live lactic acid bacteria (lactic acid bacteria are a large heterogeneous group of gram-positive anaerobic and microaerophilic bacteria that accumulate lactic acid in the medium during carbohydrate utilization). This goal is achieved in that the probiotic is prepared on the basis of natural symbiotic complexes of autochthonous anaerobic microorganisms present in the normal intestinal microflora of healthy animals and / or birds of a particular species, which will subsequently act as a recipient of this probiotic. To isolate the natural complexes of bacteria, the native biomaterial is selectively decontaminated by diluting it with a liquid selective nutrient medium for lactic acid bacteria, which makes it possible to remove microorganisms alien to this species of animals and birds from the biomaterial. Subsequent processing of the diluted biomaterial with blood serum (or immunoglobulin preparations) of the same species of animals or birds allows the most complete removal of extraneous microflora. After releasing the biomaterial from all contaminating microorganisms, it is cultivated under anaerobic conditions, followed by reseeding the resulting biomass without isolation of pure cultures in a nutrient medium suitable for the accumulation of biomass of lactic acid bacteria, and again cultivated under anaerobic conditions.

 Thus obtained biomass can be used in liquid, lyophilized or frozen form as a homoprobiotic to correct intestinal microbiocenosis or to construct the microbial ecology of young animals and birds during their mass cultivation or to obtain SPS gnitobionts.

It was found that the mucous membranes of the gastrointestinal tract begin to respond to orally administered microorganisms by lgA synthesis only if the number of microorganisms is 10 ⁶ or more cells per gram of intestinal contents. Therefore, any microorganisms found in the digestive tract in quantities of less than 10 ⁶ CFU are transient and foreign. In connection with these data, for the mechanical removal of possible contamination of the initial biomaterial by extraneous transient microorganisms, the intestinal contents of the donor are diluted with a selective medium of no less than 10 ^-6 .

It is known that serum immunoglobulins of various classes contribute to the inactivation and elimination of foreign microorganisms. This is confirmed by the fact that 99% of bacteria - representatives of indigenous anaerobic intestinal flora are not covered with secretory immunoglobulins, while enterobacteria, enterococci, other aerobic and anaerobic transient microorganisms are completely covered with IgA. Based on these data, the final removal of extraneous microflora from the diluted biomaterial taken from the donor is carried out by introducing into the diluted up to 10 ^-6 biomaterial of blood serum (or complex immunoglobulin preparation) of the species of animals or birds for which the homoprobiotic manufactured by the present method is intended.

 Example 1. Preparation of the source biomaterial, microbiological evaluation of its composition.

 Freshly taken feces samples of live healthy adult chickens from the breeding farm or the contents of the cecum of slaughtered healthy birds are placed in a freshly reduced thioglycol physiological saline solution for transport to a bacteriological laboratory. Lyophilized or cryopreserved samples can be used as biomaterial.

Serial tenfold dilutions of the medium used for transportation are prepared from the delivered biomaterial, from which the samples are then sown in the amount of 0.05-0.1 ml by the drop method on selective and differential diagnostic media, dense Endo, Ploskireva, Saburo, Rogozy, mannitol salt, azide agar, clostridia isolation medium and semi-liquid BS medium with lithium propionate. Crops are cultured under anaerobic conditions at 42 ^o C for 24-72 hours. The microflora composition of healthy broiler chickens is shown in table 1.

 As can be seen from the table, lactic acid bacteria (enterococci, lactobacilli and bifidobacteria) are present in feces of healthy broiler chickens in significant quantities. The species composition of lactobacilli is characterized by a wide range of species (the species composition of lactobacilli was determined by the spectrum of carbohydrate utilization) - Lactobacillus acidophilus, L.alimentarius, L.fermentum, L.brevis, L.buchneri, L.plantarum, L. salivarius, L. lactis.

 Example 2. Decontamination of biomaterial from allochthonous microflora.

Decontamination of the biomaterial from allochthonous microflora present in the sample in an amount of less than 10 ⁶ CFU / g is carried out by successive ten-fold dilution of the test sample. Two parallel rows of test tubes are prepared with successive ten-fold dilutions of the biomaterial with any liquid nutrient medium suitable for isolation and cultivation of lactobacilli and bifidobacteria, for example, MRS or BS, which are currently optimal for growing lactobacilli and bifidobacteria.

 They take a standard nutrient medium for isolation of dry bifidobacteria (BS) produced by the State Scientific Center for Applied Microbiology of the Ministry of Health of the Russian Federation (Obolensk, Moscow Region) with the following composition in g / l (see Table 2).

50.35 g of dry medium is thoroughly mixed in 1 l of distilled water, heated to boiling, sterilized by autoclaving at a temperature of 110 ^o C for 30 minutes and cooled to a temperature of 50-45 ^o C.

The first row consists of tubes prior to dilution 10 ^-9 and is designed to assess the total quantitative content of lactobacilli and bifidobacteria in the original biomaterial. The second row is designed to obtain biomass of these bacteria and consists of tubes with biomaterial before breeding 10 ^-6 . For the final decontamination of biomaterial from foreign microorganisms present in the biomaterial in amounts comparable to lactic acid bacteria (more than 10 ⁶ CFU / g), and for the production of symbiotic communities of indigenous lactobacilli and bifidobacteria free from similar microorganisms of other origin that have been ingested by donor birds and food, and guaranteed removal of allochthonous, including pathogenic, bacteria, carry out the 2nd stage of decontamination.

In a test tube from the last ten-fold dilution of second-row biomaterial prepared using MRS or BS nutrient medium, a complex immunoglobulin preparation (CIP) prepared from chicken blood is added according to the method described in RF patent N 2050858 10%, in an amount of 5-20% from the volume of the test sample. Instrumentation was prepared from the blood of chickens of the same poultry farm in which the present study was conducted and contained significant quantities of immunoglobulins of all main classes (IgG 40-50%, IgM 25-30%, IgA 25%). Instrumentation is characterized by pronounced specific activity against pathogens of the main infectious diseases of chickens, including salmonella and enteropathogenic Escherichia, rotaviruses, as well as other potentially pathogenic microorganisms. The contents of the tube are thoroughly mixed by shaking, sealed with a stopper and placed in a thermostat at 42 ^o C for 48 hours. Cultivation is carried out under anaerobic conditions.

 Example 3. Preparation of a homoprobiotic preparation.

After cultivation, make control crops of biomaterial to determine the number of microorganisms (in accordance with example 1) and the presence of extraneous microflora. In the absence of growth on Endo, Ploskirev, Saburo, mannitol salt agar media, the material is sowed (1:10) into a fresh liquid nutrient medium for cultivation of lacto- and bifidobacteria (MPC or BS medium) and cultured under anaerobic conditions at 42 ^o C within 48 hours. The resulting biomass is a symbiotic complex of lactobacilli and bifidobacteria of indigenous origin. To preserve the resulting biomass for a long time, it is lyophilized or frozen and used as a homoprobiotic to correct or construct the microbial ecology of newborn chickens, or to prevent and treat infectious diseases of adult chickens.

 In the same way, a probiotic preparation for other animal and bird species can be prepared on the basis of cultivation under anaerobic conditions on selective nutrient media of determinant species of microorganisms - representatives of the normal microflora of this animal species and birds with decontamination with an immunoglobulin preparation prepared from blood serum of the same animal species and birds.

 If necessary, the last tube from the second row can be replaced by two tubes: the first one with Rogosa medium for isolation of symbiotic associations of only lactobacilli, the second one with BS medium with azide and lithium propionate for isolation of only symbiotic associations of bifidobacteria. All subsequent operations are carried out as described above. To prepare a complex probiotic preparation, the obtained biomass of the associations of bifidobacteria and lactobacilli are mixed in a ratio of 1:10 and used as a homoprobiotic.

 Example 4. Control of a probiotic preparation.

 The obtained probiotic is monitored by the conventional method of seeding on Endo and TGS nutrient media to determine extraneous microflora - there is no growth under aerobic conditions on these media.

To assess the quantitative content of lactobacilli and bifidobacteria in the finished homoprobiotic, dilutions are made on semi-liquid media of Rogosa and BS with lithium propionate. The content of bifidobacteria and lactobacilli in the probiotic is at least 10 ⁸ CFU / ml. All definitions are generally accepted methods.

 The study of the species of lactobacilli and bifidobacteria contained in the preparation is carried out on the basis of studying the cultural and enzymatic characteristics of individual strains of lactobacilli and bifidobacteria isolated from the homoprobiotic complex. The isolated strains of lactobacilli were identified as L. acidophilus, L.fermentum, L. lactis, L. plantarum Lactobacillus spp .; bifidobacteria - B. bifidum, B. longum, Bifidobacterium spp.

 Example 5. Evaluation of the effectiveness of oral inoculation of a probiotic preparation.

 The probiotic was made according to the method described above in examples 2 and 3.

 The experiment was conducted on the basis of the VNITIP experimental facility near Moscow.

 13107 one-day-old chickens, after they were placed in an outdoor house, were orally inoculated with drinking water using a homoprobiotic preparation. The first control group consisted of chickens in the amount of 21432 heads, which are in similar conditions in other houses and receive the antibiotic chloramphenicol according to the scheme adopted on the farm. The second control group consisted of 18,747 chickens who received dry bifidumbacterin, prepared on the basis of industrial strains of bifidobacteria of human origin, with drinking water.

 Control over the death of chickens of the experimental and control groups was carried out daily for 30 days.

 The test results are shown in table 3.

 An analysis of the data presented in the table indicates that the oral administration of a homoprobiotic preparation prepared according to this method in industrial poultry farming reduces the death of broiler chickens more efficiently than conventional methods (the use of antibiotics and probiotics based on monocultures of bifidobacteria of human origin). Pathological autopsy and bacteriological examination of dead chickens of the experimental group in the vast majority of cases did not reveal any signs of their death from known infectious agents.

 Thus, a method for producing a homoprobiotic based on indigenous lactobacilli and bifidobacteria present in the intestines of a particular species of animals and birds is proposed.

 List of references.

 1. GIeeson T.M., Starvic S, Blanchfield B. Protection of chicks against Salmonella infection with a mixture of pure cultures of intestinal bacteria. Avian Dis, 1989 Oct-Dec, 33 (4), P. 636-42.

 2. Wierup M., Wold-Troell M, Nurmi E. et al. Epidemiological evaluation of the salmonella-controling effect of a nationwide use of a comprtitive exclusion culture in poultry. Acta Vet. Scand. Suppi., 1988, 84, 309-311.

 3. Weinack O. M Snoeyenbos GH, Soerjadi-Liem AS et al. Therapeutic trials with native intestinal microflora for salmonella typhimurium infections in chickens. Avian Dis., 1985, 29 (4), 1230-1234.

 4. US patent N 4689226. Priority 08.25.87, NKI 424/93. IPC A 01 N 63/00.

Claims (1)

 A method for producing a homoprobiotic bacterial preparation containing live lactic acid bacteria by diluting the native donor biomaterial with subsequent cultivation under anaerobic conditions on selective nutrient media and accumulating their biomass, characterized in that the native donor biomaterial is diluted with a nutrient medium suitable for selection of lactic acid bacteria, subsequent processing of the diluted biomaterial with blood serum or an immunoglobulin preparation prepared from human blood otnyh or birds of the same species as the donor, and the capacity of biomass resulting complex lactic acid bacteria is carried out without isolation of pure cultures.

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