RU2705302C1 - Antibacterial composition based on strains of bacteriophages for preventing or treating salmonellosis and / or escherichiosis of farm animals or birds or humans - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention represents an antibacterial composition of medical and preventive purpose, which involves a combination of sterile, purified from toxins filtrate phage lysates with titre of each bacteriophage of not less than 10¹⁰ PFU/ml Gratia with non-overlapping spectrum of lytic activity with respect to Salmonella and Escherichiosis pathogens, capable of causing mass diseases of birds, farm animals and humans. Created composition has no risk of toxic or side effects for humans and animals, provides stability of bacteriophage titre and effectiveness of application. Antibacterial activity of the composition is manifested in relation to 83.72 % of salmonella isolates isolated from birds and in relation to 90.63 % of salmonella isolates isolated from pigs, which is a lytic spectrum. In the case of paired meat treatment with bacteriophages, meat salmonella contamination rate decreased by 99.95 %, and by 99.98 % by the Escherichia. Dissemination of samples of fodder with Escherichia after treatment with antibacterial composition is 88.4 times lower than when using fodder without it.

EFFECT: percentage survival rate of animals of test groups at pig-breeding enterprises was 90.38 %, provided that the non-use for treatment of any additional antibacterial agents and preparations.

3 cl, 12 tbl, 13 ex

Description

The invention relates to biotechnology and veterinary medicine, namely to the creation of an antibacterial composition for the prevention or treatment of salmonellosis and / or escherichiosis in farm animals and birds, as well as humans.

Bacterial infections in the poultry industry are a global economic and health problem. The prevalence of salmonellosis in poultry ranges from 75 to 90%, which leads to clinical or subclinical infections in humans. In the United States, annual economic losses associated with salmonella infection range from 1.188 billion to more than 11.588 billion US dollars [Wernicki A., Nowaczek A., Urban-Chmiel R. Bacteriophage therapy to combat bacterial infections in poultry // Virology journal . - 2017. - T. 14. - No. l. - C. 179.].

Some strains of E. coli pathogenic for birds are causative agents of colibacillosis, one of the leading causes of poultry morbidity and mortality worldwide [Joshi S., Singh R., Singh SP Antibiotic resistance profile of Escherichia coli isolates from colibacillosis in and around Pantnagar , India // Veterinary World. - 2012. - T. 5. - No. 7. - C. 405.]. In addition, strains of E. coli colonizing poultry are a potential source of antibiotic resistance genes that can be transmitted to humans [Overdevest I. et al. Extended-spectrum β-lactamase genes of Escherichia coli in chicken meat and humans, The Netherlands // Emerging infectious diseases. - 2011. - T. 17. - No. 7. - C. 1216 .; Kheiri R., Akhtari L. Antimicrobial resistance and integron gene cassette arrays in commensal Escherichia coli from human and animal sources in IRI // Gut pathogens. - 2016. - T. 8. - No. 1. - C. 40.].

The regular use of antibiotics to improve poultry and as growth stimulants is considered a major factor in the development of antibiotic-resistant strains in zoonotic bacteria [Aarestrup, F.M. The livestock reservoir for antimicrobial resistance: a personal view on changing patterns of risks, effects of interventions and the way forward. Philos Trans R Soc Lond In Biol Sci. 2015; Seiffert, S.N., Hilty, M., Perreten, V., and Endimiani, A. Extended-spectrum cephalosporin-resistant gram-negative organisms in livestock: an emerging problem for human health ?. Drug Resist Updat. 2013; 16: 22-45].

The requirements of regulatory authorities to reduce the use of antibiotics in feed, and subsequently their complete prohibition, led to the search for alternative means for controlling pathogenic bacteria. Bacteriophages - strictly specific antibacterial agents capable of infecting only one species or strain of bacteria, are becoming a promising alternative to antibacterial drugs, vaccines and probiotics for biocontrol for mass diseases of birds.

The well-known drug "Streptophagin" for livestock, including a mixture of 4 bacteriophages Streptococcus bovis in liquid or dry forms. The liquid form contains a mixture of 4 sterile phagolysates, the dry form contains freeze-dried phagolysates with fillers, preferably with corn flour or barley bran. The drug is intended for feeding to lactating cows (RU No. 2059723, C12N 7/00, publ. 05/10/1996). The disadvantage of the drug "Streptofagin" is the limited spectrum of action, since it is directed against a narrow group of diseases caused by the causative agent of Streptococcus bovis.

An anti-salmonella preparation for oral or aerosol administration is also known, containing a filtrate of the culture fluid of the bacteriophage strain Bacteriophagum salmonellae of the State Research Institute of Highly Purpose Preparations NCF-17, aminopeptide and bacteriostatic (chinosole, metacid, ampicillin). The drug lyse salmonella serological groups A, B, C, D, E (RU No. 2080384, C12N 7/00, publ. 05.27.1997). The disadvantage of the drug is the lack of stable forms of the drug, as a result of which the activity of the drug may decrease in the body.

Also known is a biological product based on bacteriophages for the prevention and treatment of animal salmonellosis, containing 11 strains of bacteriophages, chinosol and a stabilizer. The biological product is active against Salmonella enteritidis, Salmonella typhimurium, Salmonella heidelberg, Salmonella choleraesuis, Salmonella gallinarum-pullorum (RU No. 2232808 C12N 7/00, publ. 20.07.2004). The disadvantage of the drug is the accumulation of bacteriophage filtrate in the nutrient broth without further purification, as a result of which a significant amount of endotoxin is present in the obtained filtrates.

A known bacteriophage preparation for the treatment and prevention of calf colibacteriosis containing a mixture of suspensions of phage strains of Phagum coli VGNKI No. 132, Phagum coli VGNKI No. 844, Phagum coli VGNKI No. C-1-272, Phagum coli VGNKI No. K-3-273, Phagum coli VGNKI No. F-1-06, Phagum coli VGNKI No. F-2-05, quinosol, phenol and distilled water (RU No. 1533330, C12N 7/00, publ. 10.10.1995). However, this preparation has a low titer of batirophage and contains significant amounts of endotoxin, which negatively affects the productivity of the bird.

Known biological product based on bacteriophages for the prevention and treatment of colibacteriosis (Escherichiosis) of animals, containing strains of phages EcO21-DEPT, and / or Phagum Escherichia coli Ec0782-DEPT, and / or Phagum Escherichia coli EcO781-DEPT, and / or Phagum Escherichia coli Escherichia coli Escherichia coli Escherichia coli 1-DEPT, and / or Phagum Escherichia coli EPZ-2-DEPT, and / or Phagum Escherichia coli EG-5-DEPT, and / or Phagum Escherichia coli BC-1-DEPT, and / or Phagum Escherichia coli M78-DEPT, and / or Phagum Escherichia coli Sheksna 2k-DEPT, antiseptic and stabilizer (RU No. 2244747 C2). The disadvantage of this drug is a narrow action against one type of pathogen and a high content of endotoxin.

Bacteriophage-based preparations have been registered in Europe and the USA that are used before packaging products for monitoring foodborne bacterial pathogens on the surface of convenience foods, vegetables, fruits, lettuce, etc. [Sulakvelidze A. Using lytic bacteriophages to eliminate or significantly reduce contamination of food by foodborne bacterial pathogens // Journal of the Science of Food and Agriculture. - 2013. - T. 93. - No. 13. - C. 3137-3146; Teufer, T., Von Jagow, C, Which Path To Go ?; European Food and Feed Law Review (EFFL), June 2007, Vol 3 pp136-145].

Currently, the following names of these drugs are presented on the market:

Secure Shield E1™ - натуральный препарат, содержащий 6 бактериофагов в готовой форме коммерческого продукта, лизирующих шига-токсин продуцирующую Е. coli О157:Н7 и не О157:Н7 штаммы Е. coli, производимый компанией FINK TEC GmbH (Hamm, Germany). [https://www.finktec.de/]

Secure Shield E1 ™ is a natural preparation containing 6 bacteriophages in the finished form of a commercial product, ligating a Shiga toxin producing E. coli O157: H7 and non O157: H7 E. coli strains manufactured by FINK TEC GmbH (Hamm, Germany). [https://www.finktec.de/]

EcolicidePX™ - натуральный препарат, содержащий 7 штаммов бактериофагов, лизирующих шига-токсин продуцирующую Е. coli О157:Н7, производимый компанией Intralytix Inc. USA. [http://www.intralytix.com/]

EcolicidePX ™ is a natural preparation containing 7 strains of bacteriophages lysing Shiga toxin producing E. coli O157: H7, manufactured by Intralytix Inc. USA [http://www.intralytix.com/]

EcoShield™ - натуральный, не содержащий химических добавок препарат, состоящий из 3 вирулентных бактериофагов лизирующих Escherichia coli О157:Н7, производимый компанией Intralytix Inc. USA. [http://www.intralytix.com/]

EcoShield ™ is a natural, chemical-free preparation consisting of 3 virulent bacteriophages lysing Escherichia coli O157: H7, manufactured by Intralytix Inc. USA [http://www.intralytix.com/]

Finalyse® - препарат содержащий специфичный бактериофаг против Е. coli О157:Н7, 0,2% м-крезола и 0,07% фенола качестве консерванта, производимый компанией Passport Food Safety Solutions (West Des Moines, IA, USA). [http://www.passportfoodsafety.com]

Finalyse® is a preparation containing a specific bacteriophage against E. coli O157: H7, 0.2% m-cresol and 0.07% phenol as a preservative, manufactured by Passport Food Safety Solutions (West Des Moines, IA, USA). [http://www.passportfoodsafety.com]

SalmoFresh™ - натуральный, не содержащий химических добавок препарат литических бактериофагов активных в отношении Salmonella enterica, производимый компанией Intralytix Inc. USA. [http://www.intralytix.com/]

SalmoFresh ™ is a natural, chemical-free preparation of lytic bacteriophages active against Salmonella enterica, manufactured by Intralytix Inc. USA [http://www.intralytix.com/]

SALMONELEX ™ - натуральный препарат, содержащий бактериофаги активные в отношении ряда серотипов сальмонелл, [http://www.micreos.com/]

SALMONELEX ™ - a natural preparation containing bacteriophages active against a number of Salmonella serotypes, [http://www.micreos.com/]

Biotector - натуральный препарат, содержащий бактериофаги активные в отношении ряда серотипов сальмонелл, в частности Salmonella gallinarum и Salmonella pullorum, производимый компанией Cheil Jedang Co. Korea. [http://www.cj.co.kr/cj-en/index]

Biotector is a natural preparation containing bacteriophages active against a number of Salmonella serotypes, in particular Salmonella gallinarum and Salmonella pullorum, manufactured by Cheil Jedang Co. Korea [http://www.cj.co.kr/cj-en/index]

The disadvantage of these drugs is their narrowly targeted action against a specific type of pathogen, while other pathogens can continue to multiply, causing an infectious process.

The most significant bacterial pathogens of farm animals and birds that cause irreparable economic damage and increase the risk of foodborne infections in humans include Salmonella spp., Shiga-toxin producing E. coli O157: H7 and not O157: H7 shiga- toxin-producing E. coli. We have designed a cocktail of bacteriophages against a wide range of strains of these pathogens.

The technical problem solved by the invention is the expansion of the spectrum of specific activity of a therapeutic composition based on strains of bacteriophages due to the inclusion of a polyvalent cocktail of virulent bacteriophages with a non-overlapping spectrum of lytic activity against pathogens of salmonella and Escherichia infections in farm animals, birds and humans. This composition is aimed not only at treating already ill individuals, but also at preventing and combating the carriage of causative agents of colibacteriosis and salmonellosis in farm animals and birds.

The task is achieved due to the fact that the claimed antibacterial composition includes a combination of sterile, toxin-purified phagolysate filtrates with a titer of each of the bacteriophages of at least 10 ¹⁰ PFU / ml according to Grazia in relation to the most frequently isolated epidemiologically significant microorganisms from poultry farms and pig-breeding enterprises that can cause mass diseases of birds, farm animals and humans.

The composition includes the filtrate of the salmonella Enteritidis phagolysate obtained using the bacteriophage strain Salmonella Enteritidis BF-1354, deposited in the "All-Russian State Collection of Pathogenic and Vaccine Strains of Microorganisms-Pathogens of Infectious Diseases of Animals" (Federal Research Center - All-Russian Scientific Research Institute of Experimental KI Skryabin and Ya.R. Kovalenko of the Russian Academy of Sciences) under the number BF - 1354; Salmonella Infantis phagolysate filtrate obtained using the Salmonella Infantis BF-1355 bacteriophage strain deposited in the “All-Russian State Collection of Pathogenic and Vaccine Strains of Microorganisms-Pathogens of Infectious Diseases of Animals” (Federal Scientific Center - All-Russian Scientific Research Institute of Experimental Veterinary Medicine named after K.I. Scriabin and Ya.R. Kovalenko of the Russian Academy of Sciences) under the number BF - 1355; Salmonella Typhimurium phagolysate filtrate obtained using the bacteriophage strain Salmonella Typhimurium BF-1356, deposited in the “All-Russian State Collection of Pathogenic and Vaccine Strains of Microorganisms-Pathogens of Infectious Diseases of Animals” (Federal Scientific Center - All-Russian Scientific Research Institute of Experimental Veterinary Medicine. Scriabin and Ya.R. Kovalenko of the Russian Academy of Sciences) under the number BF - 1356; Escherichia coli phagolysate filtrate obtained using the bacteriophage strain Escherichia coli BF-1352, deposited in the “All-Russian State Collection of Pathogenic and Vaccine Strains of Microorganisms-Pathogens of Infectious Diseases of Animals” (Federal Scientific Center - All-Russian Scientific Research Institute of Experimental Veterinary Medicine named after K.I. Scriabin and YR Kovalenko of the Russian Academy of Sciences) under the number BF - 1352; Escherichia coli phagolysate filtrate obtained using the bacteriophage strain Escherichia coli BF-1353, deposited in the "All-Russian State Collection of Pathogenic and Vaccine Strains of Microorganisms-Pathogens of Infectious Diseases of Animals" (Federal Scientific Center - All-Russian Scientific Research Institute of Experimental Veterinary Medicine named after K.I. Scriabin and Ya.R. Kovalenko of the Russian Academy of Sciences) under the number BF - 1353; Escherichia coli phagolysate filtrate obtained using the bacteriophage strain Escherichia coli BF-1351, deposited in the “All-Russian State Collection of Pathogenic and Vaccine Strains of Microorganisms-Pathogens of Infectious Diseases of Animals” (Federal Scientific Center - All-Russian Scientific Research Institute of Experimental Veterinary Medicine named after K.I. Scriabin and Ya.R. Kovalenko of the Russian Academy of Sciences) under the number BF - 1351.

The content of species-specific active bacteriophages in the finished product should be at least 10 ¹⁰ PFU / ml. The concentration of viral particles was selected on the basis of data from foreign scientific literature and laboratory and industrial tests carried out on our own, economic feasibility, taking into account the subsequent dilution of not less than 100 times. The composition may include targeted additives in an amount of 99-99.99 wt. % by weight of the composition in the form of a mixture of ingredients selected depending on the purpose. This composition, in connection with a sufficient spectrum of lytic activity and a stable titer of bacteriophages, can be used to prevent or treat salmonella and Escherichia infection in farm animals, birds or humans and is presented as a liquid feed additive, aerosol, spray, etc.

The technical result of the claimed invention is the creation of an antibacterial composition in the form of a multivalent cocktail of bacteriophages with a non-overlapping spectrum of lytic activity against bacteria that are most often isolated from poultry enterprises and pig breeding complexes, as well as the development of an integrated approach for preventive and therapeutic measures to eliminate salmonella and escherichiosis infections. The created composition does not have a risk of toxic or side effects for humans and animals, ensures the stability of the titer of bacteriophages and the effectiveness of use. The antibacterial activity of the composition is manifested in respect of 83.72% of Salmonella isolates isolated from birds, and in respect of 90.63% of Salmonella isolates isolated from pigs, which makes up the spectrum of lytic action.

In the case of processing fresh meat with bacteriophages, the salmonella seeding rate decreased by 99.95%, and by Escherichia by 99.98%. Treatment with chilled meat by bacteriophages led to a decrease in the number of Salmonella cells by 99.99%, and Escherichia by 99.78%.

The seeding of food samples with Escherichia prior to the introduction of bacteriophages taken directly from the animal feeder was 4.6 * 10 ⁵ CFU / g, while in the feed enriched with the composition, this indicator did not exceed 5.2 * 10 ³ CFU / g, which in turn is 88.4 times lower than when using feed without it.

The survival rate of the animals of the experimental groups at pig enterprises amounted to 90.38%, provided that no antibacterial agents and preparations other than bacteriophages were used for treatment.

It is these data that indicate the possibility of the effective use of the proposed composition in industrial poultry and pig farming, as well as a prophylactic with these infections when a person consumes the appropriate food.

According to the invention, the selection of 6 virulent strains of bacteriophages with the widest possible spectrum of lytic activity was carried out using our own bank of pathogenic and opportunistic microorganisms isolated from poultry and pig breeding enterprises of the Russian Federation.

Bacterial culture media and conditions

Bacterial cultures were grown on liquid and solid nutrient media for general purposes: meat and peptone broth; Mueller-Hinton agar (HiMedia, India); 2% meat peptone agar (MPA).

Assessment of the lytic activity of bacteriophages

The spectrum of lytic activity of bacteriophages was evaluated:

- the method of applying phage (spot test) on the lawn of a bacterial culture. 1.5% MPA was poured into Petri dishes. After hardening and drying of the agar, 0.1 ml of a 16-18 hour broth culture of microorganisms was applied to Petri dishes, triturated with a spatula over the entire surface of the plate to obtain uniform continuous growth of the culture. After the culture was absorbed and the plates dried, bacteriophages were applied dropwise (spot test) on them. After the liquid was absorbed into the medium, the plates were turned upside down and placed in a thermostat at 37 ° C for 18-24 hours. The next day, the results were recorded - the presence or absence of a "lysis spot".

Obtaining individual phagolysates and their combinations

4.5 ml of an 18-hour bacterial culture of the host strain in a titer of 10 ⁹ CFU / ml are sown in a cultivation mattress - a beveled solid nutrient medium with a layer thickness of 10 mm to 25 mm, cultivated for 3-3.5 hours at the optimum temperature , to grow the culture of the host strain, then the uterine bacteriophage in the titer of 10 ⁶ -10 ⁷ PFU / ml is sown on the obtained culture law of the host strain, and the culture vessel is sealed. Cultivate for 15-18 hours at the optimum temperature for the growth of the culture of the bacteriophage strain and the thickness of the air layer above the surface of the dense nutrient medium from 25 mm to 40 mm, a phagolysate is obtained by suspending the bacteriophage from the surface of the dense nutrient medium with saline or buffer solution with pH 7 0-7.2 in the amount of 9-10 ml, the phagolysate is transferred to a sterile container, centrifuged for 30 minutes at 5000 rpm./min, the supernatant is passed through a bacterial filter with a pore diameter of 0.22 μm. To obtain an antibacterial composition, filtered liquids containing bacteriophages are mixed and the filtrate obtained is passed through a column containing an endotoxin affinity agent. As a result, we obtain a combination of sterile filtrates of phagolysates purified from toxins containing at least 10 ¹² PFU / ml of each bacteriophage.

Safety Assessment of Antibacterial Composition

The acute toxicity of the antibacterial composition was evaluated in accordance with the Guidelines for preclinical studies of drugs / Ed. A.N. Mironova - M .: Grif and K, 2013. V. 2. Immunobiological medicines ”on 10 healthy white mice of both sexes, weighing 19-21 g after a single intraperitoneal injection for 7 days. The control group received 0.9% sodium chloride solution in the same way. As a result of the experiments, it was shown that with a single injection of the maximum dose (1.0 ml - intraperitoneally) of the test product, no signs of intoxication were observed in mice.

Evaluation of the chronic (subacute) toxicity of the antibacterial composition was carried out on 10 healthy white mice of both sexes weighing 20-21 g. The feed supplement was administered to animals intragastrically at 0.5 ml once a day for 14 days. Mice from the control group were given 0.5 ml intragastrically of a 0.9% sodium chloride solution also for 14 days. All animals were recorded daily body weight, the presence of clinical symptoms of general intoxication, as well as possible death. A study of the chronic toxicity of the antibacterial composition also did not reveal its negative effect on the body of experimental mice.

Strains of virulent bacteriophages used to obtain sterile filtrates of phagolysates purified from toxins.

1. The strain of bacteriophage BF-1354, effective against bacteria of the serotype Salmonella Enteritidis. It was isolated from a soil sample taken in the Moscow region and deposited in the “All-Russian State Collection of Pathogenic and Vaccine Strains of Microorganisms-Pathogens of Infectious Diseases of Animals” (Federal Scientific Center - All-Russian Scientific Research Institute of Experimental Veterinary Medicine named after KI Skryabin and I .R. Kovalenko of the Russian Academy of Sciences) under the number BF - 1354. The bacteriophage is well cultivated, reaching a reproduction of 10 ¹¹ -10 ¹² PFU / ml on the indicator culture Salmonella Enteritidis B-1024. Negative colonies are transparent, rounded, 3.0-4.0 mm in diameter.

The assessment of the spectrum of lytic activity of phage was determined on 34 indicator strains of Salmonella enterica serovars Enteritidis by the spot test method. The accumulated phagolysate filtrate was applied onto freshly planted lawns of Salmonella Enteritidis. The sensitivity of Salmonella Enteritidis strains to phage was judged by the appearance of a sterile spot on the lawn of the bacterial strain tested after 16-18 hours. The results of studying the spectrum of the lytic action of the bacteriophage are presented in table. one.

Note: "+" - the presence of a sterile spot on the bacterial lawn, reflecting the lysis of the bacterial culture; “-” - lack of a sterile spot.

As follows from the data obtained, the lytic spectrum of the bacteriophage BF1354 is 85.2%.

The bacteriophage BF-1354 is resistant to chloroform. So, when treated with chloroform for 30 minutes, the phage did not lose its activity.

When studying the viability of the bacteriophage BF-1354, its resistance to high temperatures was revealed: only heating the bacteriophage for 15 minutes at a temperature above 65 ° completely inactivated it. The optimal storage temperature to maintain the initial phage titer throughout the year is from 2 to 8 ° C.

2. The bacteriophage strain BF-1355, effective against bacteria Salmonella Infantis. It was isolated from cattle excrement in one of the farms in the Moscow region and deposited in the “All-Russian State Collection of Pathogenic and Vaccine Strains of Microorganisms-Pathogens of Infectious Diseases of Animals” (Federal Scientific Center - All-Russian Scientific Research Institute of Experimental Veterinary Medicine named after K.I. Scriabin and Ya.R. Kovalenko of the Russian Academy of Sciences) under the number BF - 1355. The bacteriophage is well cultivated, reaching a reproduction of 10 ¹¹ -10 ¹² PFU / ml on the indicator culture of S. Infantis B-1026. Negative colonies are transparent, rounded, 1.0-2.0 mm in diameter.

The assessment of the spectrum of lytic activity of phage was determined on 22 indicator strains of Salmonella enterica serovars Infantis by the spot test method. The accumulated phagolysate filtrate was applied to freshly planted lawns of Salmonella Infantis. The sensitivity of Salmonella Infantis strains to phage was judged by the appearance of a sterile spot on the lawn of the bacterial strain tested after 16-18 hours. The results of studying the spectrum of the lytic action of the bacteriophage are presented in table. 2.

Note: "+" - the presence of a sterile spot on the bacterial lawn, reflecting the lysis of the bacterial culture; “-” - lack of a sterile spot.

As follows from the data obtained, the lytic spectrum of the bacteriophage BF-1355 is 77.2%.

The bacteriophage BF-1355 is resistant to chloroform. So, when treated with chloroform for 30 minutes, the phage did not lose its activity.

When studying the viability of the bacteriophage BF-1355, its resistance to high temperatures was revealed: only heating the bacteriophage for 10 minutes at a temperature above 60 ° C completely inactivated it. The optimal storage temperature to maintain the initial phage titer throughout the year is from 2 to 8 ° C.

3. The strain of bacteriophage BF-1356, effective against bacteria Salmonella Typhimurium. It was isolated from chicken feces from one of the poultry farms in the Kaluga Region and deposited in the “All-Russian State Collection of Pathogenic and Vaccine Strains of Microorganisms-Pathogens of Infectious Diseases of Animals” (Federal Scientific Center - All-Russian Scientific Research Institute of Experimental Veterinary Medicine named after K.I. Scriabin and YR Kovalenko of the Russian Academy of Sciences) under the number BF - 1356. The bacteriophage is well cultivated, reaching a reproduction of 10 ¹¹ -10 ¹² PFU / ml in the indicator culture of S. Typhimurium B-1025. Negative colonies are transparent, rounded, 1.0-3.0 mm in diameter.

The assessment of the spectrum of lytic activity of the phage was determined on 22 indicator strains of Salmonella enterica of Typhimurium serovars by the spot test method. The accumulated phagolysate filtrate was applied to freshly planted lawns of Salmonella typhimurium. The sensitivity of Salmonella Typhimurium strains to phage was judged by the appearance of a sterile spot on the lawn of the bacterial strain tested after 16-18 hours. The results of studying the spectrum of the lytic action of the bacteriophage are presented in table. 3.

Note: "+" - the presence of a sterile spot on the bacterial lawn, reflecting the lysis of the bacterial culture; “-” - lack of a sterile spot.

As follows from the data obtained, the lytic spectrum of the bacteriophage BF-1356 is 86.3%.

The bacteriophage BF-1356 is resistant to chloroform. So, when treated with chloroform for 30 minutes, the phage did not lose its activity.

When studying the viability of the bacteriophage BF-1356, its resistance to high temperatures was revealed: only heating the bacteriophage for 15 minutes at a temperature above 65 ° C completely inactivated it. The optimal storage temperature to maintain the initial phage titer throughout the year is from 2 to 8 ° C.

4. The strain of bacteriophage BF-1352 was isolated from chicken feces on a culture of Escherichia coli strain O104: H4, which caused an outbreak of hemorrhagic colitis among residents of Germany and France in the spring and summer of 2011. This bacteriophage has lytic activity against Shiga-toxin-producing strains of Escherichia coli O104: H4; 0157: H7, as well as the clinically significant E. coli of other serogroups: O26, O126, O25. The bacteriophage has been deposited in the “All-Russian State Collection of Pathogenic and Vaccine Strains of Microorganisms-Pathogens of Infectious Diseases of Animals” (Federal Scientific Center - All-Russian Scientific Research Institute of Experimental Veterinary Medicine named after KI Skryabin and Ya.R. Kovalenko of the Russian Academy of Sciences) under the number BF -1352. The bacteriophage is well cultured, reaching a reproduction of 10 ¹¹ -10 ¹² PFU / ml in a non-pathogenic culture of E. coli B-1027. Negative colonies are transparent, rounded, 1.0-2.0 mm in diameter. The colonies are small, clear, with smooth edges.

The assessment of the spectrum of lytic activity of phage BF-1352 was determined on 51 pathogenic Escherichia coli strain using the spot test method. The accumulated phagolysate filtrate was applied onto freshly sown lawns of Escherichia coli. The sensitivity of Escherichia coli strains to phage was judged by the appearance of a sterile spot on the lawn of the bacterial strain tested after 16-18 hours. The results of studying the spectrum of the lytic action of the bacteriophage are presented in table. four.

Note: "+" - the presence of a sterile spot on the bacterial lawn, reflecting the lysis of the bacterial culture; “-” - lack of a sterile spot.

As follows from the data obtained, the lytic spectrum of the bacteriophage BF-1352 is 64.7%.

The bacteriophage BF-1352 is resistant to chloroform. So, when treated with chloroform for 30 minutes, the phage did not lose its activity.

When studying the viability of the bacteriophage BF-1352, its resistance to high temperatures was revealed: only heating the bacteriophage for 10 minutes at a temperature above 65 ° C completely inactivated it. The optimal storage temperature to maintain the initial phage titer throughout the year is from 2 to 8 ° C.

5. The strain of the bacteriophage BF-1353 isolated from the wastewater of one of the barn located in the Moscow region. It exhibits specific activity against Escherichia coli O157: H7. The bacteriophage has been deposited in the “All-Russian State Collection of Pathogenic and Vaccine Strains of Microorganisms-Pathogens of Infectious Diseases of Animals” (Federal Scientific Center - All-Russian Scientific Research Institute of Experimental Veterinary Medicine named after KI Skryabin and Ya.R. Kovalenko of the Russian Academy of Sciences) under the number BF -1353. The bacteriophage is well cultured, reaching a reproduction of 10 ¹¹ -10 ¹² PFU / ml in a non-pathogenic culture of E. coli B-1027. Negative colonies are transparent, rounded, 2.0-3.0 mm in diameter. Colonies are rounded, clear, with smooth edges.

The assessment of the spectrum of lytic activity of phage BF-1353 was determined on 51 pathogenic Escherichia coli strain using the spot test method. The accumulated phagolysate filtrate was applied onto freshly sown lawns of Escherichia coli. The sensitivity of Escherichia coli strains to phage was judged by the appearance of a sterile spot on the lawn of the bacterial strain tested after 16-18 hours. The results of studying the spectrum of the lytic action of the bacteriophage are presented in table. 5.

Note: "+" - the presence of a sterile spot on the bacterial lawn, reflecting the lysis of the bacterial culture; “-” - lack of a sterile spot.

As follows from the data obtained, the lytic spectrum of the bacteriophage BF-1353 is 72.5%.

The bacteriophage BF1353 is resistant to chloroform. So, when treated with chloroform for 30 minutes, the phage did not lose its activity.

When studying the viability of the bacteriophage BF-1353, its resistance to high temperatures was revealed: only heating the bacteriophage for 10 minutes at a temperature above 65 ° C completely inactivated it. The optimal storage temperature to maintain the initial phage titer throughout the year is from 2 to 8 ° C.

6. The strain of bacteriophage BF-1351 isolated from wastewater in the Moscow region. It exhibits specific activity against Escherichia coli O157: H7. The bacteriophage has been deposited in the “All-Russian State Collection of Pathogenic and Vaccine Strains of Microorganisms-Pathogens of Infectious Diseases of Animals” (Federal Scientific Center - All-Russian Scientific Research Institute of Experimental Veterinary Medicine named after KI Skryabin and Ya.R. Kovalenko of the Russian Academy of Sciences) under the number BF - 1351. The bacteriophage is well cultivated, reaching a reproduction of 10 ¹¹ -10 ¹² PFU / ml in a non-pathogenic culture of E. coli B-1027. Negative colonies are transparent, rounded, 1.0 mm in diameter.

The nucleotide sequence of the genome of the strain SEQ ID NO: 1 corresponding to that on the attached computer-readable medium was obtained.

The assessment of the spectrum of lytic activity of phage BF-1351 was determined on 112 pathogenic Escherichia coli strains using the spot test method. The accumulated phagolysate filtrate was applied onto freshly sown lawns of Escherichia coli. The sensitivity of Escherichia coli strains to phage was judged by the appearance of a sterile spot on the lawn of the bacterial strain tested after 16-18 hours. The results of studying the spectrum of the lytic action of the bacteriophage are presented in table. 6.

» - наличие зоны гемолиза, «отсут» -отсутствие зоны гемолиза.* “-” - absence of a lysis zone, “+” - presence of a lysis zone, “gender” - lactose cleavage, “neg” - absence of lactose cleavage, “

"- the presence of a hemolysis zone," absent "- the absence of a hemolysis zone.

As follows from the data obtained, the lytic spectrum of the bacteriophage BF-1351 is 62.5%.

The bacteriophage BF-1351 is resistant to chloroform. So, when treated with chloroform for 30 minutes, the phage did not lose its activity.

When studying the viability of the bacteriophage BF-1351, its resistance to high temperatures was revealed: only heating the bacteriophage for 15 minutes at a temperature above 60 ° C completely inactivated it. The optimal storage temperature to maintain the initial phage titer throughout the year is from 2 to 8 ° C.

The composition of the antibacterial composition

The active substance of the antibacterial composition is a mixture of sterile filtrate phagolysates active against Salmonella spp. And Shiga toxin producing E. coli including E. coli O157: H7 and E. coli O154: H4. The content of species-specific active bacteriophages in the finished product is at least 10 ¹⁰ PFU / ml. The concentration of phage particles was selected based on the data of foreign scientific literature and laboratory and industrial tests carried out on our own, economic feasibility, taking into account the subsequent dilution of not less than 100 times. As an auxiliary component, 0.9% NaCl was used. Table 7. shows the composition of the antibacterial composition.

The invention is illustrated by the following examples:

Example No. 1. Obtaining filtrates of phagolysates for the production of antibacterial compositions.

4.5 ml of an 18 hour bacterial culture of the host strain (from the series: S. enteritidis B-1024; S. typhimurium B-1025; S. infantis B-1026; E. coli B-1027; E. coli B-1028) in a titer of 10 ⁹ CFU / ml are sown in a cultivation mattress - a beveled solid nutrient medium with a layer thickness of 10 mm to 25 mm, cultivated for 3-3.5 hours at 37 ° C, then sown on the obtained grass lawn of the host strain uterine bacteriophage (homologous to bacterial culture) in a titer of 10 ⁶ -10 ⁷ PFU / ml, tightly close the vessel for cultivation. Cultivate for 14-16 hours at a temperature of 37 ° C and a thickness of the air layer above the surface of a dense nutrient medium from 25 mm to 40 mm, a phagolysate is obtained by suspending a bacteriophage from the surface of a dense nutrient medium with physiological saline or buffer solution with a pH of 7.0-7 2 in the amount of 9-10 ml, the phagolysate is suctioned off in a sterile container, chloroform is added in an amount of 1:10, incubated for 30 minutes with continuous shuttling of 170 rpm, centrifuged for 30 minutes at 5000 rpm. After centrifugation to obtain an antibacterial composition, supernatants containing bacteriophages of S. enteritidis BF-1354, S. typhimurium BF-1356, S. infantis BF-1355, E. coli BF-1352, E. coli BF-1353, E. coli are mixed BF-1351, then the mixture is sterilized by filtration through a filter with a polyethersulfone membrane with a pore diameter of 0.2-0.22 microns, then the filtrate obtained is passed through a column containing an endotoxin affinity ligand. As a result, we obtain an antibacterial composition containing a combination of sterile phagolysate filtrates of S. enteritidis BF-1354, S. typhimurium BF-1356, S. infantis BF-1355, E. coli BF-1352, E. coli BF-1353, E. coli BF -1351 purified from toxins with a titer of each bacteriophage of at least 10 ¹² PFU / ml.

Example No. 2. Determination of the spectrum of the lytic action of bacteriophages that make up the antibacterial composition in relation to museum and field (epizootic) isolates of S. Typhimurium, S. Enteritidis, S. Infantis.

The spectrum of the lytic action of strains of bacteriophages that are part of the antibacterial composition was checked by the spot test method using museum cultures, as well as field isolates of Salmonella isolated in 2016-2018 in poultry and pig breeding enterprises in the Russian Federation at the Federal State Budget Scientific Institution Federal Research Center for the Exploration and Epidemiology of the Russian Academy of Sciences. The spot test was carried out according to the following steps:

1) prepared Petri dishes with 1.5% meat peptone agar (20 cm ³ agar per cup);

2) after hardening and drying the cups with MPA, the lawn of the tested cultures was inoculated in a volume of 0.1 cm ³ per cup, followed by uniform distribution of this culture with a spatula;

3) after the inoculated suspension was absorbed, and the cups with the lawn dried up, the tested bacteriophages were introduced in drops at a concentration of 10 ⁸ PFU / ml in a volume of 20 μl. After the droplets of the suspension of the bacteriophage were absorbed, the plates were placed on cultivation in a thermostat with a temperature of 37 ° C, the cultivation duration was 18 hours;

4) after 18 hours of cultivation, the presence of lysis zones is checked according to the principle: "+" - the presence of a sterile spot on the bacterial lawn reflecting the lysis of the bacterial culture; “-” - the absence of a sterile spot, indicating that the bacteriophage is not active in relation to this culture.

The results of determining the spectrum of the lytic action of bacteriophages S. enteritidis BF-1354, S. typhimurium BF-1356, S. infantis BF-1355, with respect to Salmonella strains of epizootic significance are presented in table No. 8.

* The number of strains of a certain serotype isolated from a specific species of animals and birds is indicated.

In the course of the work, it was found that despite the possibility of participation in the pathological process of phagoresistant cultures of Salmonella, the antibacterial activity of the composition is manifested in respect to 83.72% of Salmonella isolates isolated from birds, and in respect of 90.63% Salmonella isolated from pigs, respectively, which and makes up a spectrum of lytic action. These data indicate the possibility of effective use of the proposed composition in industrial poultry farming. In addition, the recorded activity of phages against salmonella isolated from pigs allows us to consider their use in the pig industry expedient and promising, despite the fact that the bacteriophage BF-1355 demonstrated the ability to lyse not only Salmonella serovar Infantis cultures, but also Choleraesuis - the most adaptive one for piglets.

Example No. 3. Determination of the spectrum of the lytic action of bacteriophages that are part of the antibacterial composition in relation to museum and field (epizootic) isolates of various E. coli pathotypes.

The study of the lytic action of Escherichia phages was carried out by the spot test method described in 2 example.

The results of determining the spectrum of the lytic action of bacteriophages E. coli BF-1351, E. coli BF-1352 and E. coli BF-1353, which are part of the antibacterial composition, in relation to strains of Escherichia epizootic values are presented in table No. 9.

As can be seen from the data presented in table No. 9, the lytic spectrum of the three Escherichia bacteriophages BF-1351, BF-1352, and BF-1353, which are part of the antibacterial composition, with respect to epizootic isolates of various Escherichia patotypes, is 71.8; 78.1 and 73.9%, respectively. Since all three Escherichia bacteriophages are an integral part of one final product, we can calculate the final antibacterial effectiveness of the claimed composition based on the maximum number of detected phagosensitive cultures. Thus, the full spectrum of the lytic effect of Escherichia bacteriophages that make up the claimed composition is 89.8%.

Example No. 4. The principles of application and dosage regimens of the antibacterial composition for poultry and pig enterprises.

One dose is 0.1 cm ³ , which contains at least 10 (PFU) of each bacteriophage. The antibacterial composition can be used in the following ways:

4.1) Oral drink. This approach is described in examples No. 4-7;

4.2) Aerosol treatment. This approach is described in detail in example No. 8.

4.3) Airborne drip irrigation. This approach is described in detail in example No. 9.

4.4) Biological disinfection of hatching and marketable eggs. This approach is described in detail in example No. 10.

4.5) Decontamination of poultry and livestock products by immersion and irrigation. This approach is described in detail in example No. 11.

4.6) The use of the composition for the biological preservation and neutralization of feed from pathogens of salmonellosis and escherichiosis for pigs with the liquid type of feeding. This approach is described in detail in example No. 12.

4.7) Combined use. This approach is described in detail in example No. 13 and No. 14.

On the territory of enterprises unsuccessful for salmonellosis and escherichiosis, the antibacterial composition is used by alternating the drink with aerosol and / or airborne treatment in order to destroy the stationary nature of the focus of the infectious process and environmental sanitation. In this case, the following scheme has proven itself most effectively:

The first week is a drink of the drug;

The second week - aerosol and / or airborne droplet treatment;

The third week - drinking the drug;

Fourth week - aerosol and / or airborne droplet treatment;

etc. until the destruction of the focus of the infectious process and / or until the termination of bacterial carriage among birds and animals.

4.1) Oral drink.

One dose is 0.1 cm ³ , which contains at least 10 ⁸ (PFU) of each bacteriophage.

The composition can be used for birds and animals of any age category and physiological condition. Oral use of the composition for prophylactic purposes is carried out according to the following principle and dosage regimen:

For chickens:

In directional birds in pedigree, commercial farms and second-order reproducers, the composition is mandatory for 1 dose at the age of 3-5, 55-60, 140-150 days, then on a quarterly basis. In each case, the drink is made twice with an interval of 48-72 hours.

For chickens of 3-5 days of age, the composition is drunk twice in 0.5 doses, in all subsequent periods (as intended) - 1 dose per booze.

3-5 day old broiler chickens, the drug is drunk twice with an interval of 48-72 hours at a dose of 0.5 dose per chicken. 5 days before the slaughter, broilers are treated with 1 dose per head twice with an interval of 48-72 hours, which causes sanitation of the poultry from salmonella and Escherichia and preventing their ingestion into food products.

For quail:

For quail eggs, the composition is prescribed at 3-5 days of age, drinking is done twice at 0.25 doses with an interval of 48-72 hours, then at 0.5 doses twice at the age of 40-45 days, 140-150 days, etc. d. quarterly.

Quail broilers are drunk twice with an interval of 48-72 hours: 0.25 doses at the age of 3-5 days, 0.5 doses 5 days before broiler slaughter.

For geese and ducks:

the composition is used from 3-5 days of age at 1 dose twice with an interval of 48-72 hours, then at 40-45 and 130-140 days of age at 2 doses, then - on a quarterly basis 3 doses twice with an interval of 48-72 hours . 5 days before slaughter, birds are prescribed 3 doses per head twice with an interval of 48-72 hours.

For turkeys:

the composition is used from 3-5 days of age at 1 dose twice with an interval of 48-72 hours, then at 40-45 days of age and quarterly at 2 doses twice with an interval of 48-72 hours. 5 days before slaughter, birds are treated in an amount of 2 doses per head twice with an interval of 48-72 hours.

For pheasants, guinea fowl:

the composition is used from 3-5 days of age at 0.5 doses twice with an interval of 48-72 hours, then at 40-45 days of age and quarterly 1 dose twice with an interval of 48-72 hours. 5 days before slaughter, birds are treated in an amount of 1 dose per head twice with an interval of 48-72 hours.

For pigeons:

the composition is used from 2 days of age at 0.25 doses twice with an interval of 48-72 hours, then at 40-45 days of age, before egg-laying, vaccination and rearrangement, 0.5 doses twice with an interval of 48-72 hours.

For decorative and other birds:

the composition is used from 3-5 days of age at 0.25-0.5 doses twice with an interval of 48-72 hours, then at 40-45 days of age and quarterly 1 dose to a bird weighing up to 2 kg, 2 doses to a bird weighing 2-4 kg, 3 doses for a bird weighing 4-8 kg, 4 doses for a bird weighing more than 8 kg, twice with an interval of 48-72 hours.

For the pigs.

The antibacterial composition for piglets can be used in any age category and in any physiological state, if necessary, to normalize the activity of the gastrointestinal tract, in case of development of lesions caused by salmonella and enteropathogenic Escherichia, as well as for the rehabilitation of the organism of animal carriers.

For piglets located on the lactation of the uterus, the bacteriophage-based composition is used from the first days of feeding and / or, in case of manifestation of the corresponding signs of disturbance of the gastrointestinal tract at the rate of 5 * 10 ⁸ (PFU) of each bacteriophage, i.e. 0.5 ml of a commercial preparation per head. The course of use of the drug should be 3-5 days, depending on the severity of the infectious process.

When individually drinking young animals, the composition can be administered through the mouth with a syringe with a rubber tube. In this case, its effectiveness is increased by preventing losses that occur during group drinking. With an individual method of processing, the composition can be assigned to young animals from the first day of life.

Subsequently, after the transfer to rearing, the composition is introduced to the piglets mixing with water. Dosage regimens for the composition for pigs should be as follows:

For pigs 30-80 days of life - 10-20 doses, a course of 3-5 days;

For pigs 81-180 days of life - 20-30 doses, a course of 3-5 days;

For adult animals (boars, repair pigs, sows) - 30-40 doses, a course of 3-5 days.

Additionally, the drug can be prescribed at any moment in the life of the bird and pigs in the event of a need for normalization of the digestive system, as well as in case of risks of an outbreak of infection as a preventive measure.

Example No. 5. Determination of the required volume of the composition for oral drinking by a group method.

Previously, a day before the start of the appointment of the composition to birds or animals, in a group method, the amount of water consumed by the livestock to be treated is determined within 2-4 hours. As a note, it is worth noting that it is best to evaporate the composition within 2-4 hours after the preparation of the mother liquor, while it is necessary to constantly stir the concentrate.

For example, for a housing with a parent livestock of laying hens at the age of 352 days, with a total of 11882 heads, the volume of water consumed in four hours was 1340 liters of water, which in terms of each head is 28.1 cm ^{3 of} water per hour. As a note, it is worth noting that the volume of water consumed depends on many factors, such as the age of the bird, its physiological condition, indoor temperature, diet, humidity, etc., due to which, in other conditions, the volume of water consumed by the bird may differ . Having determined the required number of doses of the composition (11882) and the amount of water that the bird needs for 4 hours, a mother solution is prepared. In the case when the mediator is set to 1% solution, we need to prepare 13.4 liters of the mother liquor of the composition, which in turn is 1% of the volume consumed by the bird in 4 hours of water. In this case, it was required to use 1223.8 ml of the composition (taking into account 3% of losses to the watering system, etc.) which was resuspended in 12.2 liters of water. Before using the composition, 2-3 hours before drinking stop the flow of water. (Composition: 0.1 wt.% By weight of the composition — a cocktail of bacteriophages, 99.9 wt.% By weight of the composition — purified water).

The calculation of the required volume of the composition for pigs and its feeding is carried out in the same way as for poultry.

Example No. 6. The dynamics of the persistence of phage particles that make up the composition at different time intervals after a single and double evaporation of a bird.

In order to determine the dynamics of the distribution of salmonella and Escherichia phages in the digestive system of a bird previously infected with a mixture of virulent cultures of Salmonella and Escherichia, a study of feces for the presence of previously drunk phages was carried out using the Grazia method.

In the experiment, 14 day old chickens were used in the amount of 40 animals, of which 2 experimental groups of 20 animals each were formed.

Infection of chicks at the age of 14 days of life was carried out by oral infection with cultures of three serotypes of Salmonella and three cultures of Escherichia, namely S. enteritidis: No. R-6 LD ₅₀ 3,5 * 10 ⁶ microns. class .; S. typhimurium: 3 LD ₅₀ 1 * 10 ⁶ μR. class .; S. infantis: No. 1451 LD ₅₀ 9.6 * 10 ⁶ μR. class .; E. coli ETEC: No. 320 (O78: K80), LD ₅₀ 1.2 * 10 ⁷ μR. class .; E. coli EPEC: No. 733 (O119: K69: H4), LD ₅₀ 4.5 * 10 ⁸ μR. class .; E. coli EAEC: No. 857 (O86: K61: H32), LD ₅₀ 3.1 * 10 ⁸ μR. class at a dose equal to the value of 1 LD ₅₀ , in the form of a suspension of daily agar culture based on sterile saline, in an amount equal to 0.2 cm ³ for each strain. The final volume of the infectious suspension for the chicks was 1.2 cm ³ . Inoculation of the infectious suspension was carried out individually using a syringe with a catheter.

Bacteriophage-based composition was drunk 12 hours after infection. So, in the case of experience, if necessary, treatment of 2 experimental groups consisting of 40 heads of chickens at the age of 14 days, with a minimum consumption of 4.7 cm ^{3 of} water by one chicken, 0.1 ml of a composition containing no less than 10 ⁹ (PFU) of each bacteriophage. In other words, 2 ml of the composition was dissolved and carefully juggled for 5 minutes in 188 cm ^{3 of} water, which is enough for a single drink.

The test results are shown in table No. 10. A sample of feces from birds collected at an interval of two hours was examined.

As a result of the experiment, it was found that salmonella bacteriophages are determined in the highest concentration in the excrement of chickens that orally received the composition. Thus, the maximum titer of salmonella phages in the study of feces of both experimental groups was 8.9 lg PFU / g, while the maximum titer of Escherichia phages was 6.7 lg PFU / g. It should be noted that during the experiment we did not separate the Escherichia phages, therefore, in table No. 10, the results of the persistence of all three cultures are presented. In addition, from the data in table No. 10 it is seen that the maximum concentration of phage particles BF-1356 and BF-1354 was reached by 10 hours after the cocktail was drunk, and BF-1355 by 10-12 hours, respectively. Concerning Escherichia bacteriophages, a similar result was established when the maximum titer of phages was reached by 10 hours after the composition was baked. A decrease in phage titer after these periods should be considered as a decrease in the concentration of the pathogen in the body.

The second drink of the composition was made 48 hours after the first previously indicated method. At the same time, the chickens of both experimental groups did not show any mass clinical manifestations of salmonella and Escherichia infection. The results of determining the dynamics of persistence of phage particles in the gut of a bird at different time intervals after twice feeding are given in table No. 11.

Analyzing the data shown in table No. 11, we can conclude that after 48 hours after the first drinker, salmonella phages continue to circulate in the body, while the titer of Escherichia phages becomes minimal. Due to the second drink, it is possible to increase the titer of phages in the intestine of the bird after 6-10 hours, after which the titer of the phages drops. This phenomenon can be explained by a decrease in the number of pathogens in the body, which is the main goal of watering birds using the composition.

Example No. 7. The use of an antibacterial composition based on bacteriophages for the treatment of combined salmonella and coli infection in piglets.

In order to determine the effectiveness of using a cocktail of bacteriophages for the treatment of piglets, an acute experiment was reproduced. Previously, before the animals were infected, their feces were examined for the presence of salmonella and pathogenic Escherichia and the determination of the lytic activity of the phages that make up the cocktail. As a result, it was found that animal feces did not contain bacteria of the species Salmonella sp. And pathogenic Escherichia, and isolated non-pathogenic cultures of E. coli were not sensitive to phages.

In the experiment, 28 day old piglets were used in the amount of 12 animals, of which 2 experimental groups of 5 animals each and one control group of 2 animals were formed (not subjected to treatment with a cocktail of bacteriophages in order to confirm infection).

The choice of this age category of piglets was due to the fact that for this period in piglets obtained from previously vaccinated uterus, colostral immunity against Escherichiosis decreases and / or ceases to act, which reduces the body's natural resistance to the action of pathogenic Escherichia, as well as this period, weaning from the uterus and / or transfer of animals to rearing takes place, which is a stress factor provoking the disease. In addition, during this period, piglets are most often susceptible to colibacillosis infection, which occurs in the form of diarrhea of weaned piglets (PWD - postweaning disease) and edema disease (ED - edema disease) caused by ETEC, EPEC, EDEC.

Infection of piglets at 28 days of age was carried out by oral infection with cultures of three serotypes of Salmonella and three cultures of Escherichia (as in the experiment described in example No. 6), but at a dose of 10 LD ₅₀ , in the form of a suspension of daily agar culture based on sterile saline, in a volume equal to 5.0 cm ³ for each strain. The final volume of the infectious suspension for piglets was 30.0 cm ³ . Inoculation of the infectious suspension was carried out individually using a syringe without a needle.

Drinking of the composition based on bacteriophages began from the moment of manifestation of clinical signs characteristic of salmonellosis and / or escherichiosis, namely, an increase in temperature and the development of diarrhea. So, the first signs of infection appeared in infected animals 36 hours after oral feeding of the infectious suspension. In this case, according to the dosage regimen given in paragraph 4.1.

the dosage of the drug for animals is 10 doses (i.e. 1 ml with a titer of 10 ⁹ PFU of each bacteriophage). Drinking the composition of bacteriophages was carried out individually, over a period of 5 days. Using the composition allowed to stop the development of the pathogenesis of associated pathology in three days, so the diarrhea in piglets stopped after 2-3 drinks. Despite the fact that during the experiment we were able to stop diarrhea, we could not unequivocally say that we stopped the isolation of cultures in the external environment, which is why we continued the course for up to 5 days according to the dosage regimen.

During this test, on a daily basis, feces were selected from the animals of the experimental group in order to isolate previously introduced infectious cultures. Determination of the presence of exactly the previously introduced Escherichia cultures in the feces was carried out by serotyping of E. coli isolates with “O” serums - if they are agglutinating. Salmonella serotyping was carried out with sets of salmonella O-complex and monoreceptor O- and H-agglutinating serums. As a result of the work done, it was found that on the 4th day after the start of the use of the composition based on the cocktail of bacteriophages, the isolation of salmonella cultures, which were previously isolated during the first three days, ceased. The allocation of pathogenic serogroups of Escherichia (O78, O119, O86) ceased on the 5th day. To confirm the effectiveness of the cocktail of bacteriophage feces from animals, another 5 days were studied, during which the excretion of salmonella and pathogenic Escherichia was also not repeated.

In the animals of the control group, the bacteriophage did not evaporate, against which the associated infection progressed and led to the death of one of the two infected animals with dehydration and debilitating diarrhea: the skin turned gray, eyes became sunken for 4 days after infection. The second control pig died on the 6th day with similar symptoms. A pathological study of the corpses of dead animals revealed signs characteristic of Escherichia and salmonella etiology, namely, tissues and organs were dehydrated, the intestinal lumen was enlarged and filled with feces with a large amount of mucus and flakes of casein, the mucous membrane of the small intestine was edematous (typical for Escherichiosis). In addition, hemorrhages were noted under the epicardium, pleura, on the mucous membranes of the stomach, under the capsule of the kidneys, necrotic foci were found on the liver (typical for salmonellosis). In order to confirm the participation in the development of these pathological changes in previously introduced infectious cultures, sectional material was selected from the dead animals in order to isolate them with routine bacteriological methods. As a result, all previously introduced cultures were recorded in various locations, namely, in the thin and thick intestines and liver.

Example No. 8. The use of the claimed composition for the rehabilitation of livestock and poultry facilities, by aerosol aeration.

Aerosol treatment of poultry and animal housing premises with bacteriophage compositions is an effective means of prevention and control of salmonellosis and escherichiosis, the main effect of which is achieved by destroying the focus of infection, interrupting the transmission and distribution of the pathogen. In our opinion, in order to prevent the development of infectious pathology in poultry and livestock enterprises, it is advisable to exclude the following pathways of transmission and distribution of pathogens, namely: 1) from feed - to the bird / animal; 2) from poultry / animals - to the litter; 3) from the litter - to the bird / animals; 4) from a bird / animals - to a bird / animals; 5) from a bird / animal - to a person; 6) from person to bird / animal; 7) from slaughter products - to humans; 8) from a person to slaughter products. By destroying the designated paths, we can control and prevent the development of infection, as well as the circulation of the pathogen in the form of bacterial carriage.

The complexity of the implementation of the indicated task lies in the presence of various methods, as well as methods for keeping poultry and animals, where the use of preparations based on bacteriophages exclusively in the form of an oral drink is not a full-fledged preventive measure, since the focus of infection is often not in the macroorganism itself, but in its environment. This phenomenon should be considered as an example of the poultry industry, when the bird can be kept on the floor and in cages. The pattern of use of bacteriophages in these two cases cannot be the same, because with the floor content of the bird, the pathogen can be in the litter, which is why even after drinking using a composition based on bacteriophages, the bird will be re-infected after some time, since constantly in contact with the litter; and the longer the period of keeping the bird, the higher the risk of infection or the formation of a carrier. For example, in the premises for keeping hens of the parent herd, after 350-400 days, the layer of the litter in which the pathogenic microorganism can be located can reach 30-45 cm. In the second variant, when the bird is grown in cells, the risk of reinfection due to contact with the infected there is no litter.

An additional advantage of aerosol treatment with preparations based on bacteriophages of poultry and livestock enterprises lies in the possibility of carrying out this rehabilitation in the presence of birds and animals. In addition, aerosol treatment allows bacteriophages to spread and penetrate into the difficultly accessible places of localization of the infectious agent, increasing the effectiveness of rehabilitation. As an example, it is worth considering a situation when disinfection and sanitation of premises for keeping poultry and animals cannot be carried out, since the technological scheme of production does not allow to free these areas. So, in pig-breeding enterprises there are insemination sites where maintenance and main sows are kept. The problem of the processing and disinfection of these areas is that they are never exempted from animals, and therefore, neither a complete mechanical washing of the premises nor a complete disinfection can be carried out. In such cases, washing is done locally, i.e. sections, at the same time once every couple of months, which leads to total bacterial carriage among the parent population.

The principle of aerosol treatment using the claimed composition should be as follows:

1) it is necessary to determine the possibility of sealing the ventilation system. If it is not possible to ensure the tightness of the treated rooms, then it is more expedient to reorganize them not by aerosol treatment, but by airborne irrigation, due to the use of appropriate generators (for example SAG), which is described in the following example;

2) it is not advisable to carry out aerosol treatment with a composition based on bacteriophages at high ambient temperatures, when the ventilation system cannot cope with cooling and / or its shutdown will lead to a significant violation of the microclimate. In view of the indicated, aerosol treatment should be used in cool conditions, otherwise it can lead to thermal shock and reduced productivity. For sanitation of premises in a hot period of time, it is worth using irrigation by airborne droplets, as described in example No. 8;

3) the volume of the composition necessary for processing the room is calculated. So, for rooms containing birds and animals, the consumption of the composition with a concentration of 10 ⁹ PFU of each phage in 1 ml is 100 cm ³ per 1000 m ³ . Moreover, due to possible losses of the composition used at the preparatory stage of dilution, as well as due to losses in the aerosol generator, it is advisable to take this into account when calculating the needs of the drug, adding 3-5%. So, according to the technological passport of the housing for poultry, its volume is 8376 m ³ , which is why the volume of composition used for processing this room should be 862.7 cm ³ (taking into account a possible 3% loss).

4) preparation of the composition for aerosol aeration using an aerosol generator (for example, SAG). For aerosol treatment with a composition of livestock buildings, it is recommended to use at least one SAG installation for every 3000 m ³ , which reduces the processing time of the premises and increases its efficiency. In addition to SAG, other types of aerosol generators can be used, and work with them should be carried out in accordance with the instructions. In our example, for a room with a volume of 8376 m ³ , three SAG units were used, in each of which 287.5 ml of a composition was added for aeration, previously dissolved and thoroughly mixed in 6.7 liters of water, so that the final solution volume was 7 liters (full SAG installation volume).

Complete aerosol treatment was carried out for 30 minutes, of which 15 were required to spray the drug, and the remaining 15 minutes was exposure exposure for more efficient distribution of phage particles throughout the room.

The effectiveness of this method of sanitizing the premises from the pathogen as a prophylaxis of salmonellosis and escherichiosis was confirmed by the fact that sprayed bacteriophages in laboratory studies were found in step samples, wall washings, take-offs and nest covers.

To enhance the effect of aerosol treatment, you can use a mixture of an aqueous solution of the composition with glycerin prepared in a ratio of 1: 1. Thanks to glycerin, the suspension in the treated room is longer and maintained in the air. (Composition: 0.12 wt% - a cocktail of bacteriophages; 49.88 wt% - 0.9% NaCl; 50 wt% - glycerin).

Example No. 9. The use of the composition for the rehabilitation of livestock and poultry facilities by airborne irrigation (spray treatment).

Airborne irrigation of livestock and poultry facilities is an alternative method of their rehabilitation from bacterial pathogens compared with aerosol aeration. As already mentioned in the previous example, it is more expedient to use airborne irrigation in the case when it is impossible to seal these rooms at high ambient temperatures, as well as in the absence of aerosol generators. In other cases, airborne irrigation using the claimed composition based on bacteriophages is an effective means of interrupting the transmission of the infectious process of bacterial etiology.

The principle of airborne irrigation of poultry and livestock sites using an antibacterial composition should be as follows:

1) airborne irrigation is carried out using appropriate equipment, for example, portable gasoline generators;

2) it is possible to process both empty rooms after mechanical cleaning and disinfection with chemical reagents, and in the presence of animals and birds in the housing;

3) pre-calculated the volume of the drug required for processing the premises. So, for rooms containing birds and animals, the consumption of the composition with a concentration of 10 ⁹ PFU of each phage in 1 ml is 100 cm ³ per 1000 m ² . Moreover, due to possible losses of the drug at the preparatory stage of breeding and other losses, it is advisable to lay an additional 3-5% of the drug, in the calculations;

4) when calculating the area intended for processing, not only the useful area is taken into account, but also the walls with a height of at least 2 meters from the level of the litter, the entire surface of the take-off and the nest covers.

An example of calculating the area of the treated surface in the case:

* Useful area - 1152 m ² ;

* walls (N - 2 m, L - 168 m) - 336 m ² ;

* takeoffs -180 m ² ;

* nest covers - 270 m ² ;

Total: the area of the treated surface in a particular case is 1938 m ² for the processing of which 199.6 ml of the composition is necessary (taking into account 3% of possible losses).

5) the required volume of the composition is diluted in water 1000 times by serial dilution with mandatory juggling. Irrigation is carried out according to the instructions for the equipment used, so that the drug gets on all surfaces in the housing.

The essence of aerosol aeration using the claimed composition, as well as airborne irrigation, is aimed at suppressing the pathogen in the litter and the environment. Provided that the premises are constantly treated with the above methods, a bacteriophage reservoir is created and maintained in them. (Composition: 0.12 wt% - a cocktail of bacteriophages; 49.88 wt% - 0.9% NaCl; 50 wt% - glycerin).

Example No. 10. The use of the composition to disinfect the incubation and commercial (food) eggs against salmonellosis and escherichiosis.

In order to strengthen the control of salmonella infection, many poultry producers are interested in the biological protection of eggs, including through the use of preparations based on bacteriophages. The effectiveness of using the claimed composition for decontamination of an incubation and marketable egg against salmonellosis and escherichiosis was determined on the basis of the Federal State Budget Scientific Institution of the Federal Scientific Research Center of the Russian Academy of Sciences of the Russian Academy of Sciences, where the experience of processing a pre-seeded egg with the composition was implemented.

The progress of the study was as follows:

1) chicken, goose, duck and quail eggs in the amount of 15 pieces of each species, in the laboratory were seeded with a suspension of bacterial cells S. enteritidis No. R-6; S. typhimurium 3; S. infantis No. 1451; E. coli ETEC No. 320 (O78: K80); E. coli EPEC No. 733 (O119: K69: H4); E. coli EAEC No. 857 (O86: K61: H32) with a concentration of 1 * 10 ⁶ microns. class each strain by complete immersion for 1 minute;

2) after seeding, the egg was dried in a laminar box until completely dried (it was exposed to ultraviolet radiation) for 1 hour;

3) after drying, 5 eggs of each species were used as a positive control, to confirm the seeding of eggs with bacteriological methods. The remaining eggs were completely immersed in a composition solution prepared at the rate of 10 cm ^{3 of the} preparation with a concentration of 10 ¹¹ PFU / ml of each bacteriophage per 10 liters of water, i.e. the concentration of each phage in the working solution was 10 ⁸ PFU / ml. The exposure of the treatment was 3 minutes; (Composition: cocktail of bacteriophages - 1 wt.%; Purified water - 99 wt.%).

4) after treatment with the composition, the eggs were re-dried under laminar conditions for 1 hour;

5) an hour after drying, swabs were made from half of the eggs (5 of each species) for bacteriological research in order to identify cultures that had previously been seeded with eggs. Also, the contents of the egg were subjected to bacteriological examination. In addition, a solution of the composition in which the eggs were immersed for decontamination was subjected to bacteriological investigation, according to the third paragraph of the present experiment. All samples were examined individually. The second part of the eggs was packed in airtight containers and placed in a thermostat at a temperature of 37.5 ° C in order to simulate the incubation procedure during the day, after which the eggs were also subjected to bacteriological examination.

The result of the experiment was the proof of the high efficiency of using the composition for decontamination of the hatching and marketable eggs from salmonella and Escherichia, since in no case were isolates of Salmonella and Escherichia isolated from the surface of the eggs, as well as from the contents of the eggs. In addition, bacteriological examination of samples of the composition taken after immersion of seeded eggs in them also did not reveal the presence of salmonella and Escherichia in it. A positive control in all cases demonstrated the presence on the surface of eggs, cultures of Salmonella and enteropathogenic Escherichia.

In addition to the treatment of seeded eggs by immersion, the experiment was repeated on testing the method of airborne treatment due to spraying from a spray gun. This method did not show absolute effectiveness, since after its reproduction, salmonella were isolated in 67.5% of cases, and Escherichia in 35%, respectively.

Example No. 11. The use of the composition for decontamination, seeded with salmonella and Escherichia poultry.

The use of the composition allows decontamination of poultry and livestock products. To do this, in laboratory conditions, the experience of determining the effectiveness of processing a composition based on bacteriophages of pre-seeded poultry meat was reproduced. Meat samples, 20 pieces of 5 × 5 cm chicken fillet, sterilized in 70% alcohol, then washed in sterile physiological saline, were contaminated by irrigation with salmonella and Escherichia cultures, namely S. enteritidis No. R-6; S. typhimurium 3; S. infantis No. 1451; E. coli ETEC No. 320 (O78: K80); E. coli EPEC No. 733 (O119: K69: H4); E. coli EAEC No. 857 (O86: K61: H32) with a concentration of 1 * 10 ⁶ CFU / ml of each strain. After the samples were seeded, they were dried in sterile trays for 1 hour, to adapt the bacterial cells on the surface of the pieces of meat, then 5 pieces of fillet were packed in an individual package and placed in a refrigerator for 4 days at a temperature of 4 ± 2 ° C ( positive control No. 1 - imitation of chilled meat). 5 pieces of meat of each type were used for bacteriological research immediately after drying (positive control No. 2 - imitation fresh meat). The remaining samples were treated with the composition by complete immersion in a solution with a concentration of 10 ⁸ PFU / ml of each phage, with an exposure of 30 seconds. After immersion in the claimed composition and subsequent drying for 1 hour, 5 meat samples were individually packed and placed in the refrigerator for a day (prototypes No. 1 - imitation of chilled meat). The remaining 5 samples after processing and drying were sent for bacteriological examination (Experimental samples No. 2 - imitation of fresh meat).

The results of bacteriological studies of meat samples after immersion in the claimed composition are shown in table No. 12.

As can be seen from the data given in table No. 12, the use of a composition based on bacteriophages for decontamination of poultry products by pathogens of salmonellosis and escherichiosis is highly effective. So, in the case of processing fresh meat with bacteriophages, the salmonella contamination of meat decreased by 99.95%, and by Escherichia by 99.98%. Chilled meat treated with bacteriophages reduced the number of Salmonella cells by 99.99%, and Escherichia by 99.78%.

Example No. 12. The use of the composition for the biological preservation and neutralization of feed from pathogens of salmonellosis and escherichiosis of pigs in the liquid type of feeding.

Since in some cases the source of the pathogen is feed that has not undergone or undergone insufficient heat treatment, the search for new effective means of protecting these feeds is an urgent task for animal husbandry. In practice, it was possible to establish the high efficiency of using the composition as a means of biological conservation and disinfection of liquid feed used in pig farming.

The technological stages of liquid feeding pigs imply continuous cycles of preparation and feeding of feed, in which dry feed is placed in the mixing tanks, which is subsequently mixed with water at a rate of 1: 4; those. 300 kg of feed + 900 liters of tap water. Thanks to the mixer, a homogeneous mass of feed is obtained, which is subsequently transmitted through pipes to the feeders. After certain periods, the feed remaining in the pipes ends up in a settling tank, which subsequently returns to the newly mixed batch of feed. The problem with this type of feeding is that the feed with this method of preparation can become a factor in the spread of pathogens. As a means of neutralizing such feeds, we conducted tests in which a composition in the volume of 0.6 liters was introduced into the specified volume of liquid feed (Composition: 1 wt% - cocktail of bacteriophages, 99 wt% - purified water) per 1200 kg of feed, or 0.5 liters per 1 ton. The result of this experiment was a decrease in the dissemination of feed by Escherichia, expressed in terms of CFU (colony forming units). Thus, the dissemination of food samples by Escherichia before the introduction of bacteriophages taken directly from the animal feeder was 4.6 * 10 ⁵ CFU / g, while in feed enriched with the composition, this indicator did not exceed 5.2 * 10 ³ CFU / g, which in turn is 88.4 times lower than when using feed without it.

Example No. 13. The effectiveness of the claimed composition in industrial poultry conditions.

The determination of the effectiveness of the use of the composition was confirmed in the conditions of the poultry enterprise of the Russian Federation, in which S. enteritidis and S. infantis were found during 2017. In the tests, the parent livestock of laying hens kept in the amount of 120,000 heads (10 bodies of 12 thousand poultry each) was used, starting from the landing, before which complete mechanical cleaning and disinfection was carried out. The tests were carried out for 6 months. Processing of the bird was carried out by evaporating the composition (Composition: 0.01 wt% - a cocktail of bacteriophages, 99.99 wt% - water) with a titer of 10 ⁹ PFU / ml, a single dose per bird was 10 ⁸ PFU. Since the bird was kept in an outdoor manner, for the purpose of decontamination of the litter, alternating with watering, aerosol treatment with the composition was recommended (Composition: 0.12 wt% - cocktail of bacteriophages, 49.88 wt% - 0.9% NaCl, 50 wt% - glycerin) of all rooms containing birds. The principle of an integrated approach used at the enterprise was described in the fourth example, namely:

the first week - drinking the drug;

second week - aerosol and / or airborne droplet treatment;

third week - drink the drug;

fourth week - aerosol and / or airborne drip treatment;

etc. until the destruction of the focus of the infectious process and / or until the termination of bacterial carriage among birds.

The use of antibiotics that affect gram-negative flora during the tests was minimized, and they were used only in extreme cases in order to prevent losses among the livestock.

Evaluation of the effectiveness of the use of the composition was carried out thanks to monthly bacteriological monitoring, for which the following samples were taken: cloacal swabs - 5 swabs from the body (5 heads per swab); step tests - 2 pairs from the body; body dust - 1 combined sample from the body; swabs from floor and hatching eggs - 1 sample (from ten eggs); swabs from the egg collection tape - 1 sample from the body.

The result of using the composition began to be observed at the 3rd month from the start of use. So, in the first two months, bacteriological monitoring confirmed the presence of S. enteritidis and S. infantis in cloacal swabs, step samples and body dust. Starting from the third month of applying the composition, the release of salmonella completely stopped and was not confirmed until the end of the experiment; in other words, the release of salmonella was not confirmed during the next 4 months.

Example No. 14. The effectiveness of the claimed composition in industrial pig breeding conditions.

The effectiveness of the use of the composition was confirmed in the conditions of a pig-breeding enterprise located in the Pskov region, on livestock breeding sites of which there was a problem of escherichia etiology. The main problem for the company was diarrhea of newborn piglets (neonatal diarrhea) caused by enterotoxigenic strains (ETEC) of E. Coli, in particular, serotypes O8, O149 were isolated from animals. Pathology developed in animals, starting from the first day of life and in 70% of cases passed independently in a mild form by 4-5 days. In other cases, the infection was severe and was accompanied by vomiting in sick pigs, debilitating diarrhea, which provoked dehydration. The color of feces in such animals had a gray-white and / or yellow hue.

An antibacterial composition based on a cocktail of bacteriophages at the rate of 5 * 10 ⁸ (PFU) of each bacteriophage was used as a means of eliminating this pathology, i.e. 5 doses were used for each head (single drink). The drug was used individually by oral drinking. For ease of drinking, it was previously resuspended in water (composition: 0.1 wt% - a cocktail of bacteriophages, 99.9 wt% - purified water). Then, through the use of a syringe dispenser, on the cannula which was wearing a rubber hose, the drug was poured into the mouth of the animal. The duration of the course of therapy was determined by the results of the effectiveness of the use of the drug, but did not exceed 5 days.

Thus, course therapy treated animals of 4 different lots with a total of 926 animals, initially showing signs of neonatal diarrhea that did not pass on its own during the first 4-5 days of life. Clinical signs predominantly disappeared after the first three drinks, but in severe cases after 4-5. The survival rate of animals in the experimental groups was 90.38%, provided that no additional antibacterial agents and drugs were used for treatment. The results indicate a high therapeutic efficacy of using an antibacterial composition based on strains of bacteriophages when used in industrial pig-breeding enterprises.

Claims (3)

1. An antibacterial composition based on strains of bacteriophages for the prevention or treatment of salmonellosis and / or escherichiosis of farm animals or birds, or humans, characterized in that it has a titer of each of the bacteriophages of at least 10 ¹⁰ PFU / ml according to Grazia in relation to isolated from clinical material bacterial isolates and includes the salmonella Enteritidis phagolysate filtrate obtained using the bacteriophage strain Salmonella Enteritidis BF-1354, deposited in the All-Russian State Collection of Pathogenic and Vaccine Staff mm of microorganisms causative agents of animal infectious diseases ”(Federal Scientific Center - All-Russian Scientific Research Institute of Experimental Veterinary Medicine named after K.I. Scriabin and Ya.R. Kovalenko of the Russian Academy of Sciences) under number BF-1354; Salmonella Infantis phagolysate filtrate obtained using the Salmonella Infantis BF-1355 bacteriophage strain deposited in the “All-Russian State Collection of Pathogenic and Vaccine Strains of Microorganisms-Pathogens of Infectious Diseases of Animals” (Federal Scientific Center - All-Russian Scientific Research Institute of Experimental Veterinary Medicine named after K.I. Scriabin and YR Kovalenko of the Russian Academy of Sciences) under the number BF-1355; Salmonella Typhimurium phagolysate filtrate obtained using the bacteriophage strain Salmonella Typhimurium BF-1356, deposited in the “All-Russian State Collection of Pathogenic and Vaccine Strains of Microorganisms-Pathogens of Infectious Diseases of Animals” (Federal Scientific Center - All-Russian Scientific Research Institute of Experimental K. Veterinary. Scriabin and Ya.R. Kovalenko of the Russian Academy of Sciences) under the number BF-1356; Escherichia coli phagolysate filtrate obtained using the bacteriophage strain Escherichia coli BF-1352, deposited in the “All-Russian State Collection of Pathogenic and Vaccine Strains of Microorganisms-Pathogens of Infectious Diseases of Animals” (Federal Scientific Center - All-Russian Scientific Research Institute of Experimental Veterinary Medicine named after K.I. Scriabin and Ya.R. Kovalenko of the Russian Academy of Sciences) under the number BF-1352; Escherichia coli phagolysate filtrate obtained using the bacteriophage strain Escherichia coli BF-1353, deposited in the "All-Russian State Collection of Pathogenic and Vaccine Strains of Microorganisms-Pathogens of Infectious Diseases of Animals" (Federal Scientific Center - All-Russian Scientific Research Institute of Experimental Veterinary Medicine named after K.I. Scriabin and Ya.R. Kovalenko of the Russian Academy of Sciences) under the number BF-1353; Escherichia coli phagolysate filtrate obtained using the bacteriophage strain Escherichia coli BF-1351, deposited in the “All-Russian State Collection of Pathogenic and Vaccine Strains of Microorganisms-Pathogens of Infectious Diseases of Animals” (Federal Scientific Center - All-Russian Scientific Research Institute of Experimental Veterinary Medicine named after K.I. Scriabin and Ya.R. Kovalenko of the Russian Academy of Sciences) under the number BF-1351, and target additives in the amount of 99-99.99 wt. % by weight of the composition. 2. The strain of bacteriophage BF-1351, used to obtain the antibacterial composition according to claim 1, deposited in the "All-Russian State Collection of Pathogenic and Vaccine Strains of Microorganisms-Pathogens of Infectious Diseases of Animals" (Federal Scientific Center - All-Russian Scientific Research Institute of Experimental Veterinary Medicine named after K. . I. Skryabin and Ya.R. Kovalenko of the Russian Academy of Sciences) under the number BF-1351. 3. The nucleic acid molecule corresponding to the bacteriophage genome according to claim 2, presented by SEQ ID NO: 1.

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