DESC:METHOD AND FORMULATION FOR PREVENTING SALMONELLA COLONIZATION IN POULTRY
FIELD OF INVENTION
The invention generally relates to the field of preventing Salmonella colonization in poultry and more particularly to providing a method and stabilized formulations for preventing Salmonella colonization in poultry.
BACKGROUND
Salmonella enterica causes severe infections in humans and animals throughout the world. Poultry typhoid is disease of poultry caused by Gram negative bacteria Salmonella Gallinarum. Poultry are natural hosts for S. Gallinarum. While S. Gallinarum is the main pathogen, S. Pullorum is also quite prevalent. During past two decades, the incidence of S. Enteritidis infections in humans has dramatically increased, with contaminated eggs being the major infection source (Chousalkar et al, 2017). The highest numbers of Salmonella outbreaks from the past decade are related to land animals, with poultry as a main reservoir. Poultry typhoid is a highly contagious disease of the poultry. One of the predominant routes of infection is through the droppings of the infected birds. Alternatively, the bacteria may also spread through contaminated food, water, clothing and equipment. During infection, the pathogen colonises in the lower part of intestine. For effective treatment of disease, the drug needs to be delivered to the terminal part of the intestine to aid in colonic release. This requires that the drug pass the acidic gut and reach the point of infection/colonisation.
Numerous methods are known in the art for the control of Poultry typhoid. Examples of control methods include but are not limited to antibiotic administration, vaccination. One such method is discussed in US patent application US20170049872, assigned to Arizona Board of Regents on behalf of Arizona State University. The patent discloses the use of live S. Gallinarum which includes mutation for prevention of Poultry Typhoid in poultry. However, a significant disadvantage of administering Salmonella Gallinarum is the variability in protective efficacy between breeds and the continued persistence of the infection, in immunized chickens, leading to transmission of the infection, through eggs and the residual virulence in some breeds.
Feed additives such as antibiotics, prebiotics, probiotics, and synbiotics that modify the intestinal microflora are part of another field of investigation, and their success depends on the additive used. (Vandeplas et al, 2010). Another method known in the art, discloses Phage therapy as an alternate method of preventing Poultry typhoid. One of the common routes of administration of the phage therapy is to encapsulate the phage in bird feed. In one method, the phages are immobilised and further coated with an anionic copolymer, Eudragit, for colon specific drug delivery. One advantage of the method is the phage recovery in vitro. However, the use of Eudragit coating is expensive.
Yet another method of the present invention is Lyophilisation of phages with and without stabilisers. However, all such methods adopted are not cost effective.
Hence, there is a need for a bacteriophage formulation that is easy to incorporate in the bird feed that renders the formulation effective in treating poultry typhoid but are also inexpensive in production.
BRIEF DESCRIPTION OF DRAWINGS
So that the manner in which the recited features of the invention can be understood in detail, some of the embodiments are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments.
FIG. 1a shows TEM image of phage P16, according to an embodiment of the invention.
FIG. 1b shows TEM image of phage P18, according to an embodiment of the invention.
FIG. 1c shows TEM image of phage AB9, according to an embodiment of the invention.
FIG. 2 shows the initial and final body weights of the birds, according to an embodiment of the invention.
SUMMARY OF THE INVENTION
One aspect of the invention provides a method of obtaining a formulation for preventing Salmonella colonization in poultry. The method includes the steps of selecting a carrier, coating the carrier with a cocktail of bacteriophage and drying the coated carrier to obtain a formulation for preventing Salmonella colonization in poultry.
Another aspect of the invention provides a formulation for preventing Salmonella colonization in poultry. The formulation contains a cocktail of bacteriophage and a carrier coated with the cocktail of bacteriophage.
Yet another aspect of the invention provides a method of obtaining a formulation for preventing Salmonella colonization in Poultry. The method includes selecting starch as a first base and maltodextrin as a second base, and then mixing a cocktail of bacteriophage with the first base to obtain a mixture and finally adding the second base to the mixture to obtaining a formulation for preventing Salmonella colonization in poultry.
Further aspect of the invention provides a formulation for preventing Salmonella colonization in poultry, containing a first base of starch, a second base of maltodextrin and a cocktail of bacteriophage.
DETAILED DESCRIPTION OF THE INVENTION
Various embodiments of the invention provide a method and stabilized formulations for prevention of Salmonella colonization.
In one embodiment of the invention, the method includes the steps of selecting a carrier, coating the carrier with a cocktail of bacteriophage and drying the coated carrier to obtain a formulation for preventing Salmonella colonization in Poultry. The method described hereinabove briefly shall be explained in detail.
The method includes selecting a carrier. Examples of carrier include but are not limited to a soya grit, soybean meal, canola meal, cottonseed meal, sunflower meal, peas, lupins, wheat, corn, sorghum, barley, oats, finger millet/ragi, extruded soybeans, bran, broken rice, cellulose lignin, cellulose chitin, starch, maltodextrin, lactose, poultry enzyme premixes and/or combinations thereof. In one example of the invention, a de-oiled soy grit is used as the carrier.
Preparation of de-oiled soy grit: The de-oiled soy grit is produced from machine cleaned soya bean seeds after following a series of preparatory physical processes (cracking, flaking, grinding & dry extrusion) followed by multistage extraction of oil. It is a free flow granular material. The de-oiled soy grit produced consists of protein content of about 52%, maximum crude fiber content of about 3%, moisture content of about 11%, fat content of about a maximum of 1.5%, sand and silica content of about a maximum of 0.3%, Protein Solubility Index, hereinafter referred to as PSI, of about 80-90%, Nitrogen Solubility Index, hereinafter referred to as NSI, of about 23-28%, and urease activity of about 0.05 – 0.20.
Preparation of cocktail of Bacteriophage: Subsequent to the selection of de-oiled soy grit as the carrier, a cocktail of bacteriophage is prepared for coating onto the soy grit. The preparation of bacteriophage cocktail comprises of the steps of isolating 40 strains of phages from sewage samples. The sewage samples can be collected from the list including but not limited to chicken slaughter houses, poultry farms, domestic sewage, and/or different geographical areas all over India.
Subsequent to collection of phages from the sewage samples, six phages are tested for their host range on Salmonella isolates. Examples of Salmonella isolates include but are not limited to Salmonella Gallinarum, Salmonella Pullorum, Salmonella Enteritidis, Salmonella Hadar, Salmonella Indiana, Salmonella Infantis, Salmonella Typhimurium and/or combinations thereof. Host range studies are carried out using methods known to a person having ordinary skill in the art. Phages P16, P18 and AB9 are found to be broad having broad host range and hence selected for further characterization. The MTCC accession number assigned to P16 is MTCC 25382 and MTCC accession number assigned to AB9 is MTCC 25380.
Bacteriophage characterization: FIG. 1 generally shows TEM images of bacteriophage P16, P18, and AB9, according to an embodiment of the invention. Phages cocktail are centrifuged at 25,000 x g for 60 minutes using a Beckman J2–21 centrifuge in a fixed-angle rotor. The phage-containing pellet is washed in 0.1 M ammonium acetate solution, and the 10 µl of phages are deposited on copper grids with carbon-coated films, stained with 2% potassium phosphotungstate and visualized at 60 kV by transmission electron microscopy at 295,000X magnification. Transmission micrograph of phages reveals that all phage P16, P18 and AB9 belong to the Caudovirales order. FIG. 1a shows TEM image of phage P16, according to an embodiment of the invention. Phage P16 is a T1 type Siphovirus that has an icosahedral head of 58 nm and a very flexible tail of 160 x 9 nm. FIG. 1b shows TEM image of phage P18, according to an embodiment of the invention. Phage P18 is T7 type podovirus that has a head of 58 nm and a tail of 12 x 9 nm. FIG. 1c shows TEM image of phage AB9, according to an embodiment of the invention. Phage AB9 is T5 type Siphovirus. The AB9 phage has an icosahedral head of 74 nm and a long non contractile tail of 187 x10 nm.
The bacteriophage isolated is further characterized for genome sequencing. For isolation of bacteriophage genomic DNA, phage cocktail of concentrations of about 1 ×1010 PFU/ml are treated with DNase I of concentration of about 20 µg/ml and RNase A of concentration of about 30 µg/ml at 37oC overnight. The mixture is adjusted to contain 1% SDS, 20 mM EDTA of pH 8.0, 150 mM NaCl and Proteinase K to a final concentration of 150 µg/ml. Tubes are incubated at 65°C for 120 min with intermittent mixing by inverting. The mixture is then subjected to phenol-chloroform-isoamyl alcohol, in the ratio of 25:24:1, extraction following which the top aqueous phase is separated and an equal volume of absolute ethanol is added. DNA is isolated from resulting solution using a commercial Genomic DNA extraction kit according to the manufacturer’s instructions.
Sequencing of phage DNA is carried using Illumina GAII Analyzer/454 GS FLX/5500 SOLiD. Raw data analysis and QC is done using analysis tools known to a person having ordinary skill in the art. The isolated phages AB9, P16, and P18 are double-stranded DNA phages with genome sizes of ~111,059, 50369, and 37302 bp respectively. Analysis of the genome sequence data, the G+C content of the phages AB9, P16, and P18 is determined to be 39.84%, 41.29%, and 48.78% respectively. Contig assembly and ordering for the phages utilized the homologs identified in the NCBI database. AB9 is a close homolog of T5 like Enterobacteria phage EPS7, reference of which is incorporated herein, with nucleotide identity in range 98% to 81%, (coverage 91%). P16 is a close homolog of T1-like Bacteriophage TLS with nucleotide identity of 94% to 77%, (coverage, 78%), reference of which is incorporated herein. P18 is a close homolog of T7 like Salmonella phage phiSG-JL2 with pair-wise nucleotide identity of 96% to 86%, (coverage 89%), reference of which is incorporated herein. Gene organization in these phages is similar to their respective homologs identified from the database. Neither lysogenic elements nor genes associated with lysogeny such as repressors, integrases or excisionase are detected in these phages by sequence analysis. In addition, no known toxin encoding genes are identified in these phage genomes.
Coating and drying the carrier with bacteriophage cocktail: The cocktail of the three phages P16, P18 and AB9 are used for phage coating. For large scale production of the formulation, the core material is taken in a pan and sprayed with the phage cocktail. The amount of core material can vary from 50 Kg to about 100 Kg. In one example of the invention, 50 Kg of core material is taken in the pan and sprayed with 1.3 L of phage cocktail comprising of ~5 X 1012 PFU of total phage titer, in sterile saline. The coating can be done using but not limited to spraying, blending, and dribbling. In one example of the invention, the coating is done through a nozzle sprayer at low pressure from the distance of 10 to 12 cm, to avoid dusting or aerosalization. The pan is set to rotate at a speed of about ~60 rpm. The pan is allowed to keep rotating at the same speed from about 5 minutes to about 10 minutes to ensure complete drying of the core material. A sample of the final formulation is taken for analysis and the product is packed in aluminum pouches.
Another embodiment of the present invention provides a stabilized formulation for preventing Salmonella colonization in poultry. The formulation comprises of a cocktail of bacteriophage coated onto a carrier. The bacteriophage is selected from the list comprising P16, P18, AB9 and/or combinations thereof. The MTCC accession number assigned to P16 is MTCC 25382 and MTCC accession number assigned to AB9 is MTCC 25380.
The carrier is selected from the list including but not limited to soya grit, soybean meal, canola meal, cottonseed meal, sunflower meal, peas, lupins, wheat, corn, sorghum, barley, oats, finger millet/ragi, extruded soybeans, bran, broken rice, cellulose lignin, cellulose chitin, starch, maltodextrin, lactose, poultry enzyme premixes and/or combinations thereof. The bacteriophage is selected from the list comprising P16, P18, AB9 and/or combinations thereof. The bacteriophage is present in the formulation at a concentration range of about 104 to about 109 PFU. The formulation is water soluble and allows for sustained release of bacteriophages.
The formulation is effective against Salmonella including Salmonella Gallinarum, Salmonella Pullorum, Salmonella Enteritidis, Salmonella Braenderup, Salmonella Hadar, Salmonella Indiana, Salmonella Infantis, Salmonella Mbandaka, and Salmonella Typhimurium.
The formulation is used as a feed additive of the poultry, including but are not limited to are poultry selected from chickens, turkey, ducks, quail and geese. The formulation is lyophilized or freeze dried for storage in powder form.
In another embodiment of the invention, the method for preventing Salmonella colonization in poultry includes selecting a first base; selecting a second base; preparing a bacteriophage cocktail; mixing the prepared cocktail of bacteriophages with the first base to obtain a mixture; and adding the second base to the mixture. The first base is selected from the list including but not limited to starch, maltodextrin, lactose and/or combinations thereof. The second base is selected from the list comprising of maltodextrin.
In one example of the invention, starch is selected as the first base and maltodextrin is selected as second base.
Preparation of bacteriophage cocktail: Subsequent to the selection of the first base, a cocktail of bacteriophage is prepared for coating onto the first base. The preparation of bacteriophage comprises of the steps of isolating 40 strains phages from sewage samples. The sewage samples can be collected from the list including but not limited to chicken slaughter houses, poultry farms, domestic sewage, and/or different geographical areas all over India. Subsequent to collection of phages from the sewage samples, six phages are tested for their host range on Salmonella isolates. Examples of Salmonella isolates include but not limited to Salmonella Gallinarum, Salmonella Pullorum, Salmonella Enteritidis, Salmonella Hadar, Salmonella Indiana, Salmonella Infantis, Salmonella Typhimurium and/or combinations thereof. Host range studies are carried out using methods known to a person having ordinary skill in the art. Phages P16, P18 and AB9 are found to be broad having broad host range and hence selected for further characterization. The bacteriophage is selected from the list comprising P16, P18, AB9 and/or combinations thereof. The MTCC accession number assigned to P16 is MTCC 25382 and MTCC accession number assigned to AB9 is MTCC 25380.
Coating the carrier with bacteriophage cocktail: 1 Litre of Phage cocktail comprising of ~5 X 107 PFU/ml of each phage is added to 20 Kg starch. The phage cocktail is added to the starch slowly and is hand mixed. The mixture is then sieved to remove any lumps formed, the lumps are crushed, uniformly mixed and sieved again until no lumps are observed.
Subsequent to mixing and sieving, the phage coated starch is added to a blender followed by addition of about 80kg of maltodextrin (a total of 100Kg). The mixture of phage coated starch and maltodextrin is then blended for a time ranging from about 10 minutes to 30 minutes (15 min) and is sieved. The samples are collected at every stage for phage analysis.
Yet another embodiment of the invention provides a formulation for preventing Salmonella colonization in Poultry. The formulation contains a first base of starch, a second base of maltodextrin; and a cocktail of bacteriophage.
The bacteriophage is selected from the list comprising P16, P18, AB9 and/or combinations thereof. The MTCC accession number assigned to P16 is MTCC 25382 and MTCC accession number assigned to AB9 is MTCC 25380. The bacteriophage is present in the formulation at a concentration range of about 103 to about 109 PFU. The formulation is water soluble and allows for sustained release of bacteriophages.
The formulation is effective against Salmonella including Salmonella Gallinarum, Salmonella Pullorum, Salmonella Enteritidis, Salmonella Braenderup, Salmonella Hadar, Salmonella Indiana, Salmonella Infantis, Salmonella Mbandaka, Salmonella Typhimurium.
The formulation is used as a feed additive of the Poultry, including but are not limited to are poultry selected from chickens, turkey, ducks, quail and geese. The formulation is lyophilized or freeze dried for storage in powder form.
Phage efficacy trial at poultry: A placebo controlled trial is conducted with the aim of replacing antibiotics in commercial broiler birds. Seven hundred and twenty chicks are randomly distributed into three control group (T1to T3) receiving standard of care antibiotics and three treatment group (T4 to T6) receiving bacteriophage alone or bacteriophage in combination with antibiotics.
Study Groups Group ID Treatments Details
Control Groups T1
(Control group 1) Standard feed with Placebo + vaccination+ Gentamycin Inj on Day . Neodox in drinking water for 3 days (day2-4). Enrocin in drinking water for 4 days (day 21-24).
All medicines are administered as per manufacturer’s instructions.
T2
(Control group 2) Standard feed with Placebo + vaccination + Gentamycin Inj on Day 1.
NO OTHER ANTIBIOTICS
T3
(Control group 3) Standard feed with Placebo + vaccination.
NO ANTIBIOTICS
Treatment Groups T4
(Treatment group 1) Standard feed+ vaccination + Gentamycin Inj on Day 1 + Bacteriophage cocktail (Low Dose – LD).
T5
(Treatment group 2) Standard feed + vaccination+ Gentamycin Inj on Day 1 + Bacteriophage cocktail (High Dose - HD).
T6
(Treatment group 3) Standard feed+ vaccination+ Bacteriophage cocktail (High Dose - HD).
NO ANTIBIOTICS – same as in Control Group 3 (T3).

Requirement of poultry feed for each treatment group for every week is calculated based on the standard practice of poultry farm. To this, soy grit based feed additive is added to achieve 1 x 106 PFU/day per bird (T4, low dose group) and 1 x 107pfu/day per bird (T5 and T6, high dose group). For control group uncoated soya grit, which functions like a placebo, are mixed to poultry feed in the same ratio. Each week, left over feed from feeders of each group are tested for titer check to make sure that phages are present in the feed at expected titer and no cross contamination of phages across various groups (data not shown). Qualitative phage analysis of cloacal swabs is carried out to ascertain absence of inherent phages in a day old chick before study initiation. Daily and weekly data is collected separately for each treatment group and is recorded for various parameters like body weights, feed consumption and clinical signs.
Body weight: FIG. 2 shows the initial and final body weights of the birds, according to an embodiment of the invention. No significant differences in body weights, hereinafter referred to as BW, are observed between the groups on day 1 (p>0.05). No significant differences in final BW are observed between T4 and T1 indicating low dose phage group with Gentamycin is comparable to the standard of care. However, the body weight for high dose bacteriophage (T5) group is significantly lower than T1. The high dose bacteriophage on its own without antibiotic (T6) is comparable to T1 suggesting that the bacteriophage at high dose employed has a beneficial effect on the BW similar to that of the SOC. The bacteriophage high dose on its own (T6) is significantly higher in terms of BW when compared to T3, suggesting that the BP alone has the equivalent beneficial effect of an antibiotic (p ?0.001).
Feed conversion ratio (FCR). For each study group, feed conversion ratio is calculated at the completion of study, as, ratio of total amount of feed consumed to the total weight gained by the birds. Lower FCR value is an indicative of better output and hence a higher economic benefit to the farmers. Bacteriophage cocktail treatment group shows a significantly lower FCR compared to control antibiotic group suggesting a beneficial effect of phage cocktail in poultry feed. No significant differences are observed in FCR between the low dose bacteriophage (T4) and T1, and T2. Bacteriophage cocktail treatment group shows a significantly lower FCR compared to control antibiotic group.
Group T1 T2 T3 T4 T5 T6
FCR (Mean ± SD) 2.02 ± 0.35 2.12 ± 0.35 2.10 ± 0.32 1.96 ± 0.23 1.93 ± 0.22a,c 1.84 ± 0.20b,d
a (p ? 0.05) when compared with T1. Differences between groups are determined by one way ANOVA followed by a post hoc Dunnett’s multiple comparison test.
b (p ? 0.001) when compared with T1.
c (p ? 0.001) when compared with T2.
d (p ? 0.001) when compared with T3.
The FCR is significantly lower in T5 when compared to T1 and T2. The T6 group shows a significantly lower FCR compared to T1 and T3.
The present invention provides cost effective method for producing a bacteriophage formulation. The formulation provides for sustained release of bacteriophages. The formulation is compatible with poultry feed.
The foregoing description of the invention has been set merely to illustrate the invention and is not intended to be limiting. Since modifications of the disclosed embodiments incorporating the spirit and substance of the invention may occur to person skilled in the art, the invention should be construed to include everything within the scope of the appended claims and equivalents thereof.

References:
1. Hong J, Kim KP, Heu S, Lee SJ, Adhya S, Ryu S. 2008. Identification of host receptor and receptor-binding module of a newly sequenced T5-like phage EPS7. FEMS Microbiol Lett 289: 202–209.
2. German GJ, Misra R. 2001 The TolC Protein of Escherichia coli Serves as a Cell-surface Receptor for the Newly Characterized TLS Bacteriophage. J Mol Biol 308:579–585.
3. Kwon HJ, Cho SH, Kim TE, Won YJ, Jeong J, Park SC, Kim JH, Yoo HS, Park YH, Kim SJ. 2008. Characterization of a T7-like lytic bacteriophage (phiSG-JL2) of Salmonella enterica serovar Gallinarum biovar Gallinarum. Appl Environ Microbiol 74:6970–6979.
4. Vandeplas S, Dubois Dauphin R, Beckers Y, Thonart P, Théwis A. Salmonella in chicken: current and developing strategies to reduce contamination at farm level. J Food Prot. 2010 Apr;73(4):774-85.
5. Chousalkar K, Gast R, Martelli F, Pande V. Review of egg-related salmonellosis and reduction strategies in United States, Australia, United Kingdom and New Zealand. Crit Rev Microbiol. 2017 Sep 14:1-14.

,CLAIMS:We Claim:
1. A method of obtaining a formulation for preventing Salmonella colonization in Poultry, the method comprising of:
selecting a carrier;
coating the carrier with a cocktail of bacteriophage containing one or more strains of bacteriophage, wherein the bacteriophages have accession number MTCC 25380 and MTCC 25382; and
drying the coated carrier to obtaining a formulation for preventing Salmonella colonization in Poultry.
2. The method as claimed in claim 1, wherein the Salmonella is selected from Salmonella Gallinarum, Salmonella Pullorum, Salmonella Enteritidis, Salmonella Braenderup, Salmonella Hadar, Salmonella Indiana, Salmonella Infantis, Salmonella Mbandaka, Salmonella Typhimurium.
3. The method as claimed in claim 1, wherein the carrier is selected from soya grit, soybean meal, canola meal, cottonseed meal, sunflower meal, peas, lupins, wheat, corn, sorghum, barley, oats, finger millet/ragi, extruded soybeans, bran, broken rice, cellulose lignin, cellulose chitin, starch, maltodextrin, lactose, poultry enzyme premixes and/or combinations thereof.
4. The method as claimed in claim 1, wherein coating is done by spraying, blending, dribbling and/or combinations thereof .
5. The method as claimed in claim 1, wherein drying is done by air drying, lyophilisation, freeze drying and/or combinations thereof.
6. A formulation for preventing Salmonella colonization in Poultry, comprising
a cocktail of bacteriophage having accession number MTCC 25380 and MTCC 25382; and
at least one carrier;
wherein the cocktail of bacteriophage is coated on the carrier.
7. The formulation as claimed in claim 6, wherein the Salmonella is selected from Salmonella Gallinarum, Salmonella Pullorum, Salmonella Enteritidis, Salmonella Braenderup, Salmonella Hadar, Salmonella Indiana, Salmonella Infantis, Salmonella Mbandaka, Salmonella Typhimurium.
8. The formulation as claimed in claim 6, wherein the carrier is selected from soya grit, soybean meal, canola meal, cottonseed meal, sunflower meal, peas, lupins, wheat, corn, sorghum, barley, oats, finger millet/ragi, extruded soybeans, bran, broken rice, cellulose lignin, cellulose chitin, starch, maltodextrin, lactose, poultry enzyme premixes and/or combinations thereof.
9. The formulation as claimed in claim 6, wherein the poultry is selected from chickens, turkey, ducks, quail, geese.
10. The formulation as claimed in claim 6, wherein the bacteriophage is present at a concentration of about 104 to about 109 PFU.
11. The formulation as claimed in claim 6, wherein the formulation is a feed additive.
12. The formulation as claimed in claim 6, wherein the formulation allows for sustained release of bacteriophages.
13. The formulation as claimed in claim 6, wherein the formulation is water soluble.
14. The formulation as claimed in claim 6, wherein the formulation is lyophilized or freeze dried for storage in powder form.
15. A method of obtaining a formulation for preventing Salmonella colonization in poultry, the method comprising of:
selecting starch, as a first base;
selecting maltodextrin, as a second base;
mixing a cocktail of bacteriophage with the first base to obtain a mixture, wherein the cocktail of bacteriophage contains one or more strains of bacteriophage having accession number MTCC 25380 and MTCC 25382; and
adding the second base to the mixture to obtaining a formulation for preventing Salmonella colonization in Poultry.
16. The method as claimed in claim 15, wherein the Salmonella is selected from Salmonella Gallinarum, Salmonella Pullorum, Salmonella Enteritidis, Salmonella Braenderup, Salmonella Hadar, Salmonella Indiana, Salmonella Infantis, Salmonella Mbandaka, Salmonella Typhimurium.
17. A formulation for preventing Salmonella colonization in Poultry, comprising
a first base of starch;
a second base of maltodextrin; and
a cocktail of bacteriophage having accession MTCC 25380 and MTCC 25382.
18. The formulation as claimed in claim 17, wherein the Salmonella is selected from Salmonella Gallinarum, Salmonella Pullorum, Salmonella Enteritidis, Salmonella Braenderup, Salmonella Hadar, Salmonella Indiana, Salmonella Infantis, Salmonella Mbandaka, Salmonella Typhimurium.
19. The formulation as claimed in claim 17, wherein the poultry selected from chickens, turkey, ducks, quail, geese.
20. The formulation as claimed in claim 17, wherein the bacteriophage is present in the composition at a concentration of about 104 to about 109 PFU.
21. The formulation as claimed in claim 17, wherein the formulation is feed additive.
22. The formulation as claimed in claim 17, wherein the formulation allows for sustained release of bacteriophages.
23. The formulation as claimed in claim 17, wherein the formulation is lyophilized or freeze dried for storage in powder form.
24. The formulation as claimed in claim 17, wherein the formulation is water soluble.