Composition of bacteriophage-based poultry feed additives

Abstract

This invention aims to provide a composition of bacteriophage-based poultry feed additives that can reduce the chicken mortality rate and improve feeding efficiency, thereby supporting poultry productivity. The composition of the bacteriophage-based poultry feed additives consists of a mixture of T4 Coliphage bacteriophages from the Myoviridae group and Allolevirus bacteriophages from the Leviviridae group, a Coliform bacterial growth media, and Colfag buffer solution, which is composed of tris HCl, MgSO4, Ca(NO3)2, NaCl, and trehalose.

Description

[0001] Description

[0002] COMPOSITION OF BACTERIOPHAGE -BASED POULTRY FEED ADDITIVES

[0003] Technical Field of the Invention This invention relates to a poultry feed additive.

[0004] Specifically, the composition of a bacteriophage-based poultry feed additive, where the bacteriophage consortium consists of a mixture of T4 Coliphage bacteriophages from the Myoviridae group and Allolevirus bacteriophages from the Leviviridae group. The additive reduces poultry mortality rates and increases feed efficiency, thereby supporting poultry productivity.

[0005] Background of the Invention

[0006] Eggs and chicken meat are essential commodities produced by chicken farms. However, there has been a decrease in the productivity of laying hens (layers and meat chickens (broilers) in chicken farms due to the prohibition of antibiotics as growth promoters and a means for preventing infectious diseases. The prohibition is intended to address the health problems regarding increased morbidity and mortality due to infectious diseases caused by pathogen resistance to intensive and uncontrolled use of antibiotics. Efforts to improve chicken productivity with additives as a substitute for antibiotics, such as feed acidifiers, probiotics, and herbal supplements in animal feed, have been unable to increase the production rates in chicken farms. Thus, the additive is considered effective if it can support the chickens’ growth, development, and production.

[0007] A practical method for manufacturing feed additives is needed to replace antibiotics to reduce the chickens' mortality rate and increase feed efficiency (food consumption ratio) , thereby supporting poultry production.

[0008] The use of bacteriophages (bacterial, parasitic viruses) that can control the specific population of the host bacteria, in this case, pathogenic bacteria, can replace the role of antibiotics in preventing infection. The selection and formulation of bacteriophages is essential because of their specific mechanisms. The present patent uses bacteriophages selected based on their interactions with specific pathogenic bacteria targets relevant, to the scope of chicken farming. These bacteriophages vzere selected as they can adapt and be applied in the chicken farming environment. The selected bacteria, T4 Coliphage bacteriophage from the Myoviridae group and Allolevirus bacteriophage from the Levi viridae group were formulated in a consortium to target the Colibacillus pathogenic bacteria. This bacteriophage consortium plays a role in controlling the Escherichia coli bacterial populations and other pathogenic bacteria in the coliform group. It plays an essential role in the growth of DOCs (day-old chicks) and their development, into productive chickens.

[0009] The use of bacteriophages to control pathogenic bacteria in various cases has been widely disclosed and patented. However, most, cases typically use a single bacteriophage and not a consortium. Most cases are also not for the specific scope of chicken farming to improve feed efficiency and production performance. The following is a review of similar patents and their differences with our patent. US Patent 6, 485, 902 B2 of 2002 discloses the use of specific bacteriophages to control Escherichia coli 0157 in the scope of pharmaceutical and ruminant farming. This patent differs from this present invention in the type of specific target bacteria used and the environment in which they are used.

[0010] Patent WO 2005 / 009451 of 2005 discloses the use bacteriophages against a different pathogenic bacterium from our invention, namely Pseudomonas aeruginosa. US Patent 2008 / 0318311 is a bacteriophage encapsulation method not performed in our patent. US Patent 2009 / 0297561 Al of 2009 discloses a method of making bacteriophage lysate for poultry vaccinations. The present patent is different because it is for the immunization of chicken livestock. US Patent 7.625, 741 B2 2009: concerning the application of specific bacteriophages to E. Coli 0157 :H7. Patent WO 2009 / 076668 A2 of 2009 and US 2010 / 0297086 Al of 2010 specifically concerns the use of bacteriophages for Clostridium. Patent 7,758, 856 B2 of 2010 describes using bacteriophages for Pseudomonas aeruginosa bacterial biofilms. Patent WO 2010 / 064044 Al of 2010 describes a method to design bacteriophages to control the infection of various bacteria, including Escherichia coll, Enterobacteri aceae spp., and Salmonella typhimurium. US Patent 8,153,119 B2 of 2012 describes an enzymatic engineering method for degrading Staphylococcus and E. coll bacterial biofilms. US Patent. 8,377, 678 B2 of 2013 is a method for detecting, enumerating and quantifying bacteriophages in water and liquid samples. Patent WO 2013 / 024304 Al of 2013 uses a special bacteriophage for Salmonella .

[0011] Patent EP 2 893 933 Al of 2014 describes a method of bacteriophage therapy for E. coll infection in mammals. US Patent 2017 / 0000831 Al of 2017 also describes a specific bacteriophage therapy for E. coll . Based on Indonesian patent data, several patents originate from abroad using one type of bacteriophage from the Mi crovi ridae group, to control a specific target bacterium. However, this patent differs from ours in that it uses a single type of bacteriophage, not a consortium, to control a single type of bacterial target and is not specific to chicken farms. Patent WO2014133290A1 of 2014, patented in Indonesia as IDP000050119 of 2018, concerning the use of the new bacteriophage ©CJ20, patent IDP000055599 of 2019, on the use of bacteriophage patent IDP000065494 of 2019 concerning the use of bacteriophage patent. IDP000062925 of 2019 on the use of bacteriophage , and patent IDP000059935 of 2019 concerning the use of bacteriophage , all control the enterotoxigenic bacteria E. coll (ETEC) in animals, except humans. Patent WO 20141333010a! of 2014, patented as IDP000053313 of 2018 in Indonesia, concerning the use of a new bacteriophage, , to prevent and treat E. coll pathogenic infections (EPEC) in birds. Patent W02015160164 of 2015, patented as IDP000059808 of 2019 in Indonesia, also has the same purpose but uses bacteriophage . In the patents mentioned above, the avian / bird scope may include chickens, but the patent may not directly be related to chicken feed and production. Patent IDP000063205 of 2019 is a new method of using bacteriophage to prevent and treat Clostridium bacterial infections in animals.

[0012] This patent differs from the present patent regarding the bacteriophage used and their hosts.

[0013] Summary of the Invention

[0014] This invention aims to provide a composition of bacteriophage-based poultry feed additives that, can reduce the chicken mortality rate and improve feeding efficiency, thereby supporting poultry productivity. The bacteriophage-based poultry feed additive comprised:

[0015] • a mixture of T4 Coliphage bacteriophage from the Myoviridae group and Allolevirus bacteriophage from the Levivi ridae group, containing 5.00x1012 - 5.00x1016 plaque forming units of bacteriophage particles for each millimeter (Pfu / ml) of active suspensions;

[0016] • a growth media of the coliform bacteria comprising tryptone, yeast extract, glucose, NaCl, agar-agar, and dH2O;

[0017] • the Colfag buffer solution is composed of tris HC1, MgSO4, Ca(NO3)2, NaCl, and trehalose.

[0018] According to this invention, the mixture of T4 Coliphage and Allolevirus bacteriophage contains 5.00x1014 plaque-forming units of bacteriophage particles for each millimeter (Pfu / ml) of suspension. In addition, the ratio between tryptone, yeast extract, glucose, NaCl, agar-agar, and dH2O in the Coliform bacteria growth medium is 3: 1: 1: 2 : 2: 200. Meanwhile, the ratio between tris HCl, MgSO4, Ca (NO3) 2, NaCl, and trenalose in the Colfag buffer solution is 5: 10: 1: 0.1.

[0019] Brief Description of the Figures Figure 1 shows the observation results of the mortality rate of DOCs kept in cages for 13 weeks.

[0020] Figure 2 shows the observation results of production performance and feed consumption ratio. These results describe the feed additives’ efficiency in laying hens' (layer) products. Figure 3 shows the production performance and feed consumption ratio of me a t chicken (broiler) due to the b a c t. e ri o p h a g e - b a s e d t r e a t m e n t .

[0021] Description of the Invention

[0022] This invention relates to a composition of a consortium and. the use of a mixture of T4 C o l i p h age b a c t. e ri o p h a g es from the

[0023] Myovi ridae group and Allolevirus b a c t. e ri o p h a g e s from the

[0024] L e vi vi ridae g r o up as an additive to the drinking water of chicken livestock. This additive aims to support the growth and development of healthy chickens by inhibiting the growth of targeted diseasecausing pathogenic bacteria, thereby reducing the risk of death.

[0025] Healthy chickens will efficiently use feed to produce high-quality products . The application of this bacteriophage consortium additive is not limited to chickens. It can be given to other types of poultry, such as laying ducks meat ducks, geese, turkeys, and. quails .

[0026] By using an additive containing a consortium of the T4

[0027] Coliphage b a c t e r i o p h a g e from the Myovi ridae group and the

[0028] Allolevirus b a c t e r i o p h a g e from the L e vi vi ri da e group, chicken farms can gain more than one benefit from feed, reduce the use of antibiotics and other drugs. reduce the risk of disease, improve the cleanliness and sanitation ot the farm environment, and increase the quantity and quality of products in the form of eggs and meat, from chickens in a. more sustainable and. environmentally friendly Unlike chemicals and antibiotics that can be degraded and lost in the chicken’s system, this bacteriophage consortium can replicate itself to develop because it is a. parasitic living' creature that develops spec i f i c a l l y i n its host pathogenic bacteria to control it to a relatively safe level for the chicken

[0029] The bacteriophage consortium mentioned in this invention contains at least (1) bacteriophage mixture comprising T4

[0030] Coliphage of the Myovi ri da e group and Allolevirus of the

[0031] Levi vi ridae group containing 5.00xl012- 5. 00 x 1016p 1 a qu e f o rm. i n g unit of bacteriophage particles for each millimeter (Pfu / ml) of active suspension, preferably b. 00x1014plaque forming unit tor each millimeter (Pfu / ml) of active suspension; (2) media in the form of growth broth for the coliform bacteria; and (3) buffer solution for the bacteriophages and its consortium.

[0032] The bacteriophage consortium in this additive is a mixture of T4 Colipnage of the Myoviridae group and Allolevirus of the Leviviridae group bacteriophage particles in a Colifag buffer solution. The viability of the bacteriophage particles in the buffer solution was determined by having them infect Coliform bacteria and counting the number of lysis zones or plaque-forming units in the coliform bacteria grown on the coliform culture media in Petri dishes (Figure 1) .

[0033] In this case, the additive is administered to the chickens through their drinking water so that it can be absorbed into the body of the animal ad libitum in a dilution concentration of 1 / 20 000 to 1 / 100 000 times, but the preferred dilution is 1 / 50 000 times. The additive is administered when raising DOCs and when the chickens begin to mature to produce and move cages. The first administration is done when the DOCs are aged 1-5 days, preferably 3 days since their first entry into the barn. The second administration is given when the chicks are aged 14 - 28 days, preferably 21 days. The third administration is given to chickens aged 12-15 weeks, preferably 13 weeks.

[0034] During the first administration of the feed additives to DOCs, the T4 Colipnage and Allolevirus bacteriophage consortium would control the coliform bacteria population. These pathogenic bacteria play a major role in infecting chicken livestock and facilitating infections with other pathogenic bacteria. Therefore, the bacteriophage consortium’s ability to control the coliform population will help the chickens remain healthy by enabling them to grow and develop and preventing them from contracting infectious diseases that cause death (Figure 2) .

[0035] In the second administration of the feed additives to newly productive chickens, the T4 Colipnage and Allolevirus bacteriophages would control the coliform bacteria population and other infectious pathogens and bacteria that produce ammonia gas. These bacteriophages would also improve the chickens’ probiotic environment and support their health. Healthy chickens can use their feed intake to produce eggs (Figure 3) and better meat quality (Figure 4) .

[0036] The following section describes the composition of the media used in this invention. Coliform Culture Media

[0037] This culture media consists of:

[0038] Tryptone : 15.0 g

[0039] Yeast Extract: 7.0 g

[0040] Glucose : 8.0 9

[0041] NaCl : 10.0 g

[0042] Agar-agar : 1.5 g (for solid media)

[0043] Agar-agar : 0.7 g (for semisolid top media) dH2O : 1000 ml The production process of the host coliform bacteria begins by isolating the bacteria from the chicken’s drinking water and feces. The streak plate method is used to grow the bacteria on

[0044] Eosin Methylene Blue (EMB) agar media for 48 hours. Then, colony is replanted for purification on a solid coliform culture medium for 20-50 hours, preferably 24 hours, at 30°C-40°C; a temperature of 35°C is preferred. Next, one pure colony is grown in liquid coliform culture media for 20-50 hours, preferably for 24 hours at 30°C-40°C, but 35°C is preferred, with a rotation of 80-120 rpm, preferably 100 rpm. Example 2: Colfag butter solution

[0045] The buffer soluti on has the following composition:

[0046] 1. Tris HC1: 20 mM

[0047] 2. MgSO4: 10 mM

[0048] 3. Ca (NO3)2: 10 mM 4. NaCl : 10 mM

[0049] 5. Trehalose: 8 mM

[0050] This solution has a pH of 6.6-7.9, preferably 7.5.

[0051] The T4 Coliphage and Allolevi virus bacteriophages were obtained from culture stocks. These bacteriophacres were isolated from the aquatic and chicken farm environments and selected based on their virulence properties. The bacteriophages were produced by in f e c t ing liquid coliform media cultures ror 20 — 50 hours, preferably 24 hours, at 30°C-40°C, preferably 35°C, with a rotation of 80-120 rpm (revolutions per minute) , preferably 100 rpm. Then, the amount and virulence of the culture filtrate were analyzed by the double layer media method in which 1 ml of the filtrate was diluted in series, and each dilution was added with 0.1 ml of coliform bacteria culture in the stationary phase, or about 107- 109CFU per ml, but preferably 108CFU per ml. The suspension of the bacteriophage and its bacterial host were then added to 2-4 ml, preferably 3 ml, of semisolid coliform agar culture media (0.7%) , which would then be grown on coliform agar culture media (1.5%) for 20-50 hours, preferably 24 hours, at 30°C~40°C, preferably 35°C. The number of plaques in the form of clear zones would then be observed and calculated to obtain the number of bacteriophage titers after considering and calculating the dilution factor.

[0052] The bacteriophage particles were produced from the liquid culture of coliform bacteria infected with T4 bacteriophage or Allolevirus by centrifuging the culture at 7,000 - 10,000 rpm, preferably 8,000 rpm, for 8-12 minutes, preferably 10 minutes. The process is conducted at 2°C-6°C, preferably 4°C. Then, the supernatant formed is filtered with a cellulose acetate membrane filter measuring 0.1 pm - 0.5 pm, preferably 0.22 pm. The bacteriophages in the filtrate were then further purified by the salt precipitation method using NaCl with a concentration of 0.4 M-0.6 M, preferably 0.52 M, and PEG8000 with a concentration of 8% - 12%, preferably 10.5%, for 20 hours-30 hours, preferably 24 hours, at 2°C-6°C, preferably 4°C. Next, the suspension filtrate precipitation was centrifuged at 10,000 - 14,000 rpm, preferably 12,000 rpm, for 8-12 minutes, preferably 10 minutes. The pellets formed are then resuspended in the Colfag buffer solution. The bacteriophage suspension in Colfag buffer is then stored in the refrigerator at 4°C or in the freezer at -20°C.

[0053] The embodiment of this invention can be seen in some examples below. However, the application of this invention is not limited to these embodiment examples. Application Example of the Bacteriophage Consortium on DOCs

[0054] In this case, the additive is administered to the chickens through their drinking water so that it can be absorbed into the body of the animal ad libitum in a dilution concentration of 1 / 20 000 to 1 / 100 000 times, but the preferred dilution is 1 / 50 000 times. The additive is administered when raising DOCs and when the chickens begin to mature to produce and move cages. The first administration is done when the DOCs are aged 1-5 days, preferably 3 days since their first entry into the barn. The second administration is given when the chicks are aged 14 - 28 days, preferably 21 days. The third administration is given to chickens aged 12-15 weeks, preferably 13 weeks. The number of dead DOCs was then calculated and analyzed to determine the effectiveness of the additives. The health of DOCs can be seen from the decrease in death rate (mortality) . Application example of the Bacteriophage Consortium in Productive Chickens

[0055] In the administration of productive chickens, the T4 Coliphage and Allolevirus in the bacteriophage consortium additive are given through the chickens' drinking water so that it would enter the chicken's body freely ad libitum, in a dilution concentration of 1 / 20 000 - 1 / 100 000 times, preferably 1 / 50 000 times. Observations were made on the number of produced eggs and the feed's efficiency. Healthy chickens can utilize feed intake optimally to produce better eggs (laying hens / layers) and meat (meat chickens / broilers ) .

Claims

Claims1. The bacteriophage-based poultry feed additive comprised:• a mixture of the T4 Coliphage bacteriophage from the Myoviridae group and the Allolevirus bacteriophage from theLevi vi ridae group, containing 5.00 xl 012- 5.00x1016plaque forming units of bacteriophage particles for each millimeter (Pfu / ml) of active suspensions;• a growth media of the coliform bacteria comprising tryptone, yeast extract, glucose, NaCl, agar-agar, and dH2O;• a Colfag buffer solution composed of tris HCl, MgSO4, Ca(NO3)2, NaCl, and trehalose.

2. The composition of the bacteriophage-based poultry feed additives in Claim 1 consists of a mixture of T4 Coliphage and Allolevirus bacteriophages, containing 5. OOxlO14plaque-forming units of bacteriophage particles for each millimeter (Pfu / ml) of suspension .

3. The composition of the bacteriophage-based poultry feed additives in Claim 1, where the ratio between tryptone, yeast extract, glucose, NaCl, agar-agar, and dH2O in the Coliform bacterial growth media is 3:1: 1:2:2:

200.

4. The composition of bacteriophage-based poultry feed additives in Claim 1, where the ratio between tris HC1, MgSO4, Ca (NO3)2, NaCl, and trehalose in the Colfag buffer solution is 5:10:1: 0.1.

5. The composition of bacteriophage-based poultry feed additives in Claims 1 and 4, where the Colfag buffer solution has a pH of 6.6 — 7.9.

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