WO2004071324A2 - Method to reduce, eliminate, or prevent bacterial pathogen colonization in eggs and/or poultry using bacteriophages - Google Patents

Abstract

Methods for reducing, eliminating, or preventing colonization of bacterial pathogens, such as, for example, Salmonella, in fertilized eggs, shell eggs, and/or poultry are disclosed. The methods are directed to the introduction to the eggs and/or administration to the poultry of bacteriophages effective against the bacterial pathogens.

Description

RELATED APPLICATIONS

[0001] The instant application claims benefit under 35 U.S.C. 119(e) to U.S. Ser. No.

60/446,265 filed February 11, 2003. The application also is a continuation-in-part of U.S. Ser. No. 09/757,687 filed January 11, 2001, which claims benefit to U.S. Ser. Nos. 60/175,415 filed January 11, 2000, 60/175,377 filed January 11, 2000, 60/175,416 filed January 11, 2000 and 60/205,240 filed May 19, 2000. The disclosures of the above-noted applications are incorporated herein by reference in entirety.

BACKGROUND OF INVENTION

[0002] The present invention is directed to methods of reducing, eliminating, or preventing bacterial pathogen colonization in eggs and/or poultry using bacteriophages.

[0003] In addition to causing human disease, bacterial contamination of animals often leads to disease and increases animal morbidity. In commercial animal operations, where animals may be crowded into facilities in which other animals were previously raised, the likelihood of such contamination is often high. This is particularly true of Salmonella contamination in the poultry industry. Suzuki, S., International Journal of Food Microbiology 21:89-105 (1994).

[0004] The USDA estimates that in 50-75% of human Salmonella cases, the bacteria are acquired from meat, including poultry, or eggs, with poultry serving as the primary vehicle of transmission. In fact, studies conducted in the early 1990s by the USDA indicated that 20-25% of broiler carcasses and 18% of turkey carcasses were contaminated with Salmonella prior to sale. Food Safety and Inspection Service, 9 CFR Part 308, Pathogen Reduction, Hazard Analysis and Critical Control Point (HACCP) Systems, Proposed Rule 60 Fed. Reg. 6774-6889 (1995).

[0005] Although Salmonella are part of the normal, colonizing intestinal flora of many animals, including chickens, when homeostasis is disrupted, serious problems can arise. For example, early chick mortality (ECM) is often associated with Salmonella exposure during incubation, hatching, and processing. Such exposure can be caused by Salmonella that reside on and/or within the eggshell. Moreover, Salmonella colonization frequently occurs at hatching through transmission from contaminated eggs. Cason, J.A. et al, Avian Diseases 35:583-88 (1994). Colonized hatchlings then give rise to flocks that are colonized with the bacterial pathogen. When the colonized poultry flocks are processed, further contamination may result from the rupture of the intestinal tract during slaughter. A common source of Salmonella is the skin of the bird, with the feather follicles serving as a reservoir for the bacteria. Chickens are slaughtered "skin on," so that ante mortem contamination of feathers becomes an important element in determining whether Salmonella can be isolated from the carcass. The close quarters in chicken houses and the piling of chicken crates on trucks to slaughterhouses result in frequent contamination of feathers by feces. Further, if members of a flock have high levels of intestinal colonization with Salmonella, then there are multiple opportunities for contamination of feathers and feather follicles with the bacteria, and, in turn, for Salmonella contamination of the final product.

[0006] The rate of Salmonella contamination of poultry carcasses was a major focus of the recently implemented revision of the national food safety regulations, which mandate government testing for Salmonella in all slaughterhouses and processing plants. Regulations now in effect require that poultry products be tested by putting a whole bird carcass in a "baggie" with culture media and shaking. Growth of any Salmonella from broth counts as a positive test. Slaughterhouses and processing plants must meet specific standards for percentage of product contaminated based on national averages. Failure to meet these standards results in slaughterhouse and plant closure. Food Safety Inspection Service, 9 CFR Part 304, et seq., Pathogen Reduction, Hazard Analysis and Critical Control Point (HACCP) Systems, Final Rule 61 Fed. Reg. 38806-989 (1996).

[0007] Concerns about Salmonella contamination have also become a major issue in international trade, countries have embargoed millions of dollars worth of chicken because of Salmonella in the product. [0008] Clearly, there are strong public health, regulatory, and trade incentives for poultry producers to reduce levels of Salmonella contamination in poultry and eggs. Therefore, numerous anti-bacterial substances and procedures have been generated to improve bird mortality and bacterial reduction.

[0009] Irradiation of raw product, e.g., chicken carcass, has been shown to be efficacious, but is expensive and limited by the small number of irradiation facilities and by consumer acceptance. Alternatively, poultry have been treated with antibiotics. However, this treatment does not eradicate colonization, tending to select for resistant organisms instead. Further, antibiotics generally have activity against multiple bacterial species. Thus, their administration can result in serious perturbations in the microbial ecology of the bird's intestinal tract with accompanying loss of "colonization resistance" and overgrowth of bacteria resistant to the antibiotic. Vaccination, another alternative, is only moderately effective in reducing the levels of Salmonella. Hassan, J.O. et al., Avian. Dis. 41:783-91 (1997); Methner, U. et al., Int. J. Food Microbiol. 35:223-30 (1997); Tan, S. et al, Vet. Microbiol. 54:247-54 (1997).

[00010] A fifth alternative, competitive exclusion, i.e., administration of "good" bacteria to "crowd out" Salmonella and other "bad" bacteria, has shown variable success. Palmu, L. et al, Poultry Sci. 76:1501-05 (1997); WO 90/14765; WO 97/13405; WO 96/04364. A commercially available competitive exclusion product known as PREEMPT® (produced by MS Bioscience, Dundee, IL) consists of 29 different bacterial strains. In preliminary testing, it appears to be effective in limiting Salmonella colonization, but its usage is hampered by the cost. Most importantly, its efficacy is significantly decreased if antibiotics are administered to animals as growth additives (a standard practice in the poultry industry).

[00011] Clearly, there continues to be a pressing need to reduce, eliminate, or prevent bacterial colonization and the ensuing infection in poultry and eggs both for the health benefits to the bird as well as to decrease the likelihood of transmitting bacterial pathogens through the food chain to the consumer of retail poultry products.

SUMMARY OF THE INVENTION [00012] It is an object of the invention to provide methods and means for exposing poultry eggs and poultry to bacteriophage.

[00013] It is a further object of the invention to minimize or to remove the likelihood of exposure of poultry eggs and poultry to bacterial pathogens by treating the environment within which the hens, eggs and offspring reside with bacteriophage.

[00014] It is an object of the invention to provide methods of improving poultry health and of reducing, eliminating, or preventing poultry mortality.

[00015] It is also an object of the invention to interrupt or prevent the cycle of bacterial pathogen colonization in poultry by reducing, eliminating, or preventing the transmission of bacterial pathogens during the bird life cycle.

[00016] It is a further object of the invention to reduce, eliminate, or prevent the colonization of eggs by bacterial pathogens.

DETAILED DESCRIPTION OF THE INVENTION

[00017] The bacterial pathogens include, but are not limited to, Escherichia coli; Listeria including, but not limited to, L. monocytogenes; Clostridia including, but not limited to, C. perfringensis such as, for example, C. perfringensis Types A and C; Streptococcus including, but not limited to, S. suis such as, for example, S. suis Types 1 and 2; Mycoplasma including, but not limited to, M. hyopneumoniae; Salmonella including, but not limited to, S. typhimurium such as, for example, S. typhimurium DT 104, S. typhi suis, S. cholerae suis, S. enteriditis, S. newport, S. heidelberg, S. kentucky, S. hadar, S. typhimurium, and S. thomson; Serpulina including, but not limited to, S. hyodysenteria; Isospora including, but not limited to, I. suis; Eimeria including, but not limited to, E. acervulina, E. maxima, and E. tenella; Campylobacter including, but not limited to, C. jejunum; and Chlamydia including, but not limited to, C. psittaci. In a preferred embodiment, the bacterial pathogens are C. perfringensis such as, for example, C. perfringensis Types A and C, Salmonella including, but not limited to, S. typhimurium such as, for example, S. typhimurium DT 104, S. typhi suis, S. cholerae suis, S. enteriditis, S. newport, S. heidelberg, S. kentucky, S. hadar, S. typhimurium, and S. thomson, E. acervulina, E. maxima, E. tenella, C. jejunum, and C. psittaci. In a prefereed embodiment, the bacterial pathogens are from the genus Salmonella. In a preferred embodiment, the bacterial pathogens are from the Salmonella species S. enteriditis, S. newport, S. heidelberg, S. kentucky, S. hadar, S. typhimurium, and S. thomson.

[00018] To meet these objectives, the present invention is directed to the introduction, exposure and/or application of bacteriophages to the production of poultry and/or eggs to reduce, eliminate, or prevent colonization of bacterial pathogens. The term "production" as applied to poultry includes, but is not limited to, breeding, raising, storing, processing and handling poultry and all functions associated therewith. The term "production" as applied to eggs includes, but is not limited to, incubating, storing, and processing eggs for hatch or consumption and all functions associated therewith. The term "poultry" includes, but is not limited to, chickens, roosters, turkeys, ducks, quail, guinea fowl, pheasant, peafowl, pigeon, geese, and other avian species. The tenns "bird" and "birds" are interchangeable with the term "poultry." Bacterial "colonization" relates to the initial exposure of a host to a pathogen, and the initial growth and expansion of the population from the founding microbe or microbes. Colonization precedes infection where the bacterial population precipitates a response by the host. Often the response is of the nature that disrupts homeostasis and yields an adverse symptom in the host.

Treatment of Fertilized Eses [00019] To reduce, eliminate, or prevent the presence of bacterial pathogens, preferably, Salmonella, in and/or on fertilized eggs, the invention contemplates treating fertilized eggs, also known as hatchery eggs, by introducing into and or onto the eggs at least one bacteriophage or, more preferably, at least two bacteriophages or, most preferably, a bacteriophage cocktail of three or more phage strains effective against the bacterial pathogens. A preferred bacteriophage cocktail comprises a plurality of phage strains that lyse the bacterial pathogens of interest. For example, a cocktail can comprise four, five, six or more bacteriophages against Salmonella species. The bacteriophage- treated eggs can be incubated to hatch and the hatched birds can be raised in facilities free of bacterial pathogens, such as Salmonella.

[00020] In the methods of the invention, the eggs are treated by any suitable means, and may be treated using a combination of means. Several techniques known for introducing materials into and/or onto eggs have been described in the art and are applicable to bacteriophage introduction, including, but not limited to, forcing fluid through the eggshell using pressure, physically forming an opening in the shell and then adding the desired material, e.g., by injection using a syringe and needle arrangement, and syringe injecting by hand. The eggs may be sprayed with the desired material. The sprayer may be, inter alia, a pressurized container, e.g., aerosol canister, fogging device, trigger spray device, pump spray device, or even watering can. Alternatively, the eggs may be dipped with or submerged in a bacteriophage solution.

[00021] In one embodiment of the invention, the bacteriophages may be introduced into an egg by any suitable means, but are preferably injected in ovo into any compartment of the egg, including, but not limited to, the body of the embryo, the amniotic fluid near the small end of the egg, and the air sac at the large end of the egg. Physical injection is typically targeted at preferred positions within the egg to administer bacteriophages into specific developing regions of the embryo.

[00022] As understood by those of skill in the art, as the incubation period progresses towards maturity, i.e., hatching, the embryo and its membranes, e.g., the air cell, the allantois, and yolk sac, correspondingly change in both volume and position within the eggshell. Additionally, the quantitative volume of the enclosed fluid varies. Thus, the location of injection varies depending upon the purpose for which the egg is being treated and the time of treatment as is understood by one skilled in the art. For example, when eggs are being incubated to produce live poultry, care must be taken to avoid injuring the embryos during the injection and delivery of the bacteriophages. In addition, injecting bacteriophages into the albumin may increase the risk of leakage of albumin and the ingress of air and contaminants after injection. Thus, when injecting bacteriophages into the albumin of eggs, preferably, means are provided for preventing air and contaminants from entering the albumin, and preferably means are provided for preventing leakage of albumin after injection.

[00023] Moreover, individual eggs can vary widely in size with accompanying associated differences in the distance between the shell and the location to which delivery of the bacteriophages is desired. Due to such size differences, care must be taken to consistently supply the bacteriophages to a particular location within each egg, particularly, when injection occurs at a fast rate of speed due to, for example, automation.

[00024] In addition to selection of the site of injection, as understood by those skilled in the art, the time of injection may impact the effectiveness of the bacteriophages as well as the mortality rate of the treated embryos. Fertilized eggs may be immediately injected with bacteriophages after collection at a fertilized egg collection site or any other site, or, alternatively, the eggs may be incubated and then injected with bacteriophages on any day up to the day of hatching. Preferably, eggs are injected between days 17 and 19 of incubation. Most preferably, eggs are injected on day 18 of incubation. However, the day of injection may vary depending on the poultry breed, the type of incubator used, the incubation conditions, and the injection conditions as would be understood by one skilled in the art.

[00025] Bacteriophages may be introduced into fertilized eggs alone or mixed with at least one biologically active agent, such as, for example, a vaccine, antibiotic, antiviral, antiparasitic and the like using the same method or by different methods. For the purposes of the invention, the biologically active agent is not only an agent that removes or prophylactically protects the egg and/or poultry from a pathogen, but can also be a nutrient or a supplement, such as a vitamin or a mineral, a dye and so on. Suitable examples of vaccines include those for Newcastle, Marek's disease, bronchitis, and/or INDIA vaccines.

[00026] Bacteriophages and/or biologically active agents can be delivered to the eggs by automated injection, such as, for example, the INOVOJECT® system (Embrex, Inc., Research Triangle Park, NC). Additional automated egg injection devices are known in the art. See, e.g., U.S. Pat. No. 2,477,752; U.S. Pat. No. 3,377,989; U.S. Pat. No. 4,040,388; U.S. Pat. No. 4,469,047; U.S. Pat. No. 4,593,645; U.S. Pat. No. 4,681,063; U.S. Pat. No. 4,903,635; U.S. Pat. No. 5,056,464; and U.S. Pat. No. 5,136,979. Some injection devices seal the injection hole after injection to prevent leakage and contamination. See, e.g., U.S. Pat. No. 4,593,646.

Treatment of Hatched Birds

[00027] Bacterial pathogens that may colonize and thus be present in and/or on the surface of the fertilized eggs may cause poultry to become infected on hatching. Thus, according to another embodiment of the invention, a method of treating poultry is disclosed, comprising: administering at least one bacteriophage, at least two bacteriophages or, a bacteriophage cocktail comprising three or more phage strains, to the poultry under conditions whereby the bacteriophages reduce, eliminate, or prevent bacterial pathogen colonization, for example, Salmonella colonization, in the poultry. A bacteriophage cocktail comprising five bacteriophage strains against Salmonella species can be used to remove most common strains of that bacteria.

[00028] In the methods of the invention, poultry may be treated with bacteriophages by any suitable means, practiced alone or in combination, including, but not limited to, providing bacteriophages orally in drinking water or in food, injecting bacteriophages into the birds, instilling eye drops containing phage, spraying bacteriophages on the birds or in their environment, or orally inoculating the birds with bacteriophages using, for example, a gavage. The concepts of providing substances to poultry in water, in food, by injection, by spraying, and by inoculation are well known in the art. See, e.g., U.S. Pat. Nos. 2,851,006 and 6,410,016. The bacteriophages can be sprayed onto the birds before they are transferred to a chicken house, farm, or other poultry containment facility. By spraying the birds, the bacteriophages are ingested as the birds preen their feathers. As noted above, the sprayer may be, twter alia, a pressurized container, e.g., aerosol canister, fogging device, trigger spray device, pump spray device, or even watering can. The phage can be in a liquid vehicle, for example, a suspension, solution, syrup and the like, or on or in a dry vehicle, such as a powder, a fine powder, a dust, a freeze-dried preparation, one that has been fluidized and so on. [00029] The birds may be treated with bacteriophages at any age, for example, within the first five days following hatching. Birds can be treated on the day of hatch since any bacterial pathogen-positive birds, e.g., Salmonella contaminated birds, introduced into a chicken house or other poultry containment facility, rapidly contaminate all other birds in the house or facility. Thus, application of bacteriophages immediately after hatching and before transfer to the chicken house or other containment facility may reduce or eliminate the risk of or prevent the bacteria from spreading between and among birds and or reduce the number of or eliminate the bacteria in pathogen-positive birds during placement and grow-out.

[00030] Optionally, after the birds are hatched, they may be treated with bacteriophages and at least one other biologically active agent including, but not limited to, vaccines, such as Newcastle, Marek's disease, bronchitis, and/or INDIA vaccines, antibiotics, vitamins and the like. Administering biologically active agents, such as vaccines, to birds is a well-known practice in the art. As described above for bacteriophage administration, the additional biologically active agents may be administered by providing biologically active agents in drinking water or in food, injecting biologically active agents into the birds, spraying biologically active agents on the birds or in their environment, or orally inoculating the birds with biologically active agents using the methods described above. Bacteriophages and the other biologically active agents may be administered using the same method, e.g., in combination, or by different methods.

[00031] Alternatively, according to another embodiment of the invention, treatment of the birds may be combined with treatment of the fertilized eggs such that the bacteriophages are introduced into and/or onto the fertilized eggs under conditions whereby the bacteriophages reduce, eliminate, or prevent bacterial pathogen colonization, preferably, Salmonella colonization, as described above; and then the bacteriophages are administered to the hatched birds under conditions whereby the bacteriophages reduce, eliminate, or prevent bacterial pathogen colonization, preferably, Salmonella colonization, in the birds as described above.

Treatment of Shell Eεεs and Ess Contents [00032] In another embodiment of the invention, colonization of bacterial pathogens, such as, for example, Salmonella, in eggs destined for consumption, i.e., shell eggs or table eggs, is reduced, eliminated, or prevented by treating the eggs with at least one bacteriophage. In a preferred embodiment, the eggs are treated with at least two bacteriophages. The eggs can be treated with a bacteriophage cocktail comprising at least three bacteriophage strains. A preferred bacteriophage cocktail comprises five bacteriophages against Salmonella species.

[00033] In one embodiment, bacteriophages can be introduced into and/or onto shell eggs following laying of the eggs, but prior to consumption, using any one or more of the methods of bacteriophage introduction described above. In one embodiment, shell eggs are injected with bacteriophages in the first 48 hours following laying of the eggs.

[00034] In another embodiment, bacteriophages are added to processed egg preparations resulting from the separation of shell egg contents from membranes and shells. The bacteriophage can be added to the egg contents soon after liberation from the intact shell. The phage can be present to act early on bacterial colonization events and to prevent further attempts at colonization.

[00035] In one embodiment, bacteriophages are added to preparations of egg yolks.

[00036] In yet another embodiment, bacteriophages are added to preparations of egg whites.

[00037] In the context of treating egg preparations, the phage can be added at any time during food preparation. It may be beneficial to add the phage soon after release from the egg. However, phage can be added at a later time, for example, just prior to packaging. Moreover, the phage can be added at multiple time points prior to packaging or sale.

Bacteriophage Cocktails

[00038] The invention is also directed to novel bacteriophage cocktails that are used to, inter alia, carry out the various methods set forth above. Bacteriophage cocktails contain at least three bacteriophages and can be custom tailored to the bacterial pathogens that are prevalent in a certain situation. For example, to carry out the various methods set forth above, a bacteriophage cocktail can contain four, five, six or more lytic bacteriophages which specifically target Salmonella species, as described in Example 1 below.

[00039] Bacteriophages are highly host-specific as any one variety of bacteriophage will generally only infect one species of bacterium and, frequently, only selected strains of that species. Thus, to be used most successfully to control bacterial pathogens in eggs and/or poultry, multiple strains of bacteriophages are used which are capable of killing a broad spectrum of bacterial strains within the target strains, species or genus of pathogen. In addition, the use of a cocktail assists in reducing the ability of the bacterial pathogens to develop resistance to bacteriophage infection by a particular phage. Thus, if the target bacterial pathogen develops resistance to one bacteriophage, reduction in, elimination of, or prevention of colonization of the unwanted pathogen still occurs by other phage in the cocktail. Alternatively, the members of the bacteriophage cocktail can be substituted as needed to minimize the risk of resistance developing.

[00040] Since the bacteriophage cocktail members must survive in the egg and/or bird, while retaining the ability to infect and kill the target bacterial pathogen, rational selection of bacteriophages appropriate for inclusion in the cocktail is often made based on information about the types of bacteria to be controlled, the reaction of potential bacteriophages with the bacteria, the activity of the bacteriophages against the target bacterial pathogens in the host, and the stability of the bacteriophages in the host. One skilled in the art can readily determine the appropriate bacteriophage cocktail members by conducting susceptibility testing. For example, in the present invention, Salmonella are isolated from a source of contamination, such as a contaminated bird or environment thereof or feces found in a poultry farm or poultry processing plant, and susceptibility testing of the bacteria to various bacteriophages is performed by methods analogous to anti-microbial susceptibility testing which is well known in the art. (Adams, Mark H., Phages, Interscience Publishers: New York, 1959) [00041] To isolate phage, the environmental sample is mixed with growth medium and a Salmonella strain of interest and grown overnight to increase the number of Salmonella phage in the sample. The sample is filtered through a 0.45 μm filter to remove bacteria and other debris, and then serial dilutions of the putative phage source are mixed with top agar that is then plated over a lawn of Salmonella contained in bottom agar. The plates are incubated overnight and inspected for areas of bacterial lysis, or plaques. Plaques are isolated, eluted, and re-plated serially until clonal as determined by several features including (i) a stable restriction digest pattern, (ii) a stable protein pattern by SDS-PAGE, and (iii) stable and homogeneous morphology by electron microscopy.

[00042] Once the bacteriophage susceptibility profile is determined for each bacterial pathogen, a bacteriophage cocktail can be formulated containing the bacteriophages to which the more prevalent bacteria species present in that environment are susceptible. The cocktail is then administered to eggs and/or poultry and/or applied to the poultry environment as described above. The members of the bacteriophage cocktail may be applied at the same time, i.e., in the same application, or they may be applied in separate applications spaced in time such that they are effective at the same time.

[00043] In one embodiment, the lytic bacteriophage cocktail members are effective against bacterial pathogens including, but not limited to, Escherichia coli; Listeria including, but not limited to, L. monocytogenes; Clostridia including, but not limited to, C. perfringensis such as, for example, C. perfringensis Types A and C; Streptococcus including, but not limited to, S. suis such as, for example, S. suis Types 1 and 2; Mycoplasma including, but not limited to, M. hyopneumoniae; Salmonella including, but not limited to, S. typhimurium such as, for example, S. typhimurium DT 104, S. typhi suis, S. cholerae suis, S. enteriditis, S. newport, S. heidelberg, S. kentucky, S. hadar, S. typhimurium, and S. thomson; Serpulina including, but not limited to, S. hyodysenteria; Isospora including, but not limited to, I. suis; Eimeria including, but not limited to, E. acervulina, E. maxima, and E. tenella; Campylobacter including, but not limited to, C. jejunum; and Chlamydia including, but not limited to, C. psittaci. In a preferred embodiment, the bacterial pathogens are C. perfringensis such as, for example, C. perfringensis Types A and C, Salmonella including, but not limited to, S. typhimurium such as, for example, S. typhimurium DT 104, S. typhi suis, S. cholerae suis, S. enteriditis, S. newport, S. heidelberg, S. kentucky, S. hadar, S. typhimurium, and S. thomson, E. acervulina, E. maxima, E. tenella, C. jejunum, and C. psittaci. In another embodiment, the bacterial pathogens are from the genus Salmonella, the bacterial pathogens are from the Salmonella species S. enteriditis, S. newport, S. heidelberg, S. kentucky, S. hadar, S. typhimurium, and S. thomson. Lytic bacteriophages specific for these bacteria may be isolated by the methods described in U.S. Appl. No. 09/757,687, incorporated by reference herein in entirety.

[00044] The preferred delivery vehicle for the bacteriophage cocktail depends on the manner of administration and includes, but is not limited to, aqueous suspension, gels, sols, tablet or capsule form, powder or coated form, or incorporation in or on material that can be applied to surfaces or ingested. More than one delivery vehicle, or carrier, may be used. For examples of suitable carriers, diluents, excipients, etc., see Remington: The Science and Practice of Pharmacy, Gennaro, A.R. (ed.), Mack Publishing Co., Eaton, PA (2000).

[00045] The concentration or amount of bacteriophage cocktail varies depending on the carrier and method of administration, and can be extrapolated from culture and sensitivity studies. The bacteriophage concentration may range from 1x105 - 1x1010 plaque forming units (pfu)/ml. Because the bacteriophages of interest are replicable entities, specific amounts at administration are not critical. As the phage infect and lyse target bacterial cells, amplification in titer occurs. However, suitable amounts of bacteriophage at the initial exposure time are desirable to ensure the anti-bacterial effects of the phage are obtained in a suitable time frame. That amount, as taught hereinabove, can be obtained by monitoring bacterial population dynamics following exposure to varying amounts of phage, simulating conditions found at the anticipated administering environment.

[00046] The invention also relates to devices for applying bacteriophage to poultry eggs and to poultry, as well as to the environment in which poultry and eggs are produced. The device can be, for example, a container, optionally with a means for dispersing the phage, such as one where the phage contents are under pressure, such as an aerosol canister, a trigger spray device, a pump spray device, a fogging device, a means for injection, such as a needle, and so on.

[00047] As the phage can be applied by other means, such as by spraying, dipping, pouring, brushing, toweling, sponging, wiping, painting and so on, the invention also relates to a device suitable for such means of application, and with a storage container attached thereto containing a reservoir of phage. Thus, a brush may have attached thereto a phage-containing container that releases a tonic amount of phage onto the bristles. Disposable towels or sponges can be impregnated with a phage solution and stored in a container.

[00048] Alternatively, the container may be one for the mere storage of phage for whatever the intended means of application, such as a container that attaches to a water hose, wherein the phage solution is diluted into the water stream emanating from the hose opening. The container may be simply a storage container for housing quantities of virus, such as a metallic can, a bag, a plastic container and the like.

[00049] The container can be configured to maintain the phage intact for long periods of time. The container may have an internal coating to provide an inert surface in direct contact with the phage. The container may be opaque to provide a darkened interior. The container may contain a UV blocking lining or coating.

[00050] The container can be configured to contain a plurality of chambers, wherein the contents thereof are admixed, for example, prior to use.

[00051] The phage preparation can be stored as a liquid, semisolid, gel, sol or solid. The solid may be a residuum, a freeze-dried solid, a desiccated solid and the like. The phage preparation may contain additives to enhance the shelf life of the stored phages, such as buffer salts, stabilizers, preservatives and the like.

[00052] The phage-containing containers of interest also can be those that are useable in existing delivery means, that is, the phage-containing container is a component of an existing device. Thus, the container can be one that fits into an injection device or an automated injection device, such as one of those mentioned hereinabove, such as the device of Embrex. Thus, the container can be one that contains a unit dose or multiple doses for administration.

[00053] The phage can be added to solutions, sprays and the like for use in sanitizing the poultry, the eggs, the environment housing same or devices used in the rearing and processing of the eggs and birds. Thus, equipment, enclosures, pens, food processing equipment, knives, incubators and the like can be treated with such solutions. Alternatively, the spray may be of a dust or other dry formulation wherein the particles can be readily dispersed.

[00054] The invention will now be described in more detail in the following non-limiting examples. The examples are for illustration only and do not limit the scope of the present invention as defined by the claims.

EXAMPLES

Example 1 - Isolation and Purification of Bacteriophages [00055] Salmonella-specific lytic bacteriophages were isolated by standard techniques from various environmental sources in Maryland, for example, using water from the Inner Harbor of Baltimore, MD. Purification was performed by a combination of low-speed and high-speed centrifugation and by sequential fractionation with various chromatographic media. The purified bacteriophages were then buffer-exchanged against physiological phosphate-buffered saline, pH 7.6, and sterilized using a 0.22 micron filter, titered, and stored in sterile glass ampules at 40oC.

[00056] To render newly isolated phage monoclonal, plaques are isolated, eluted, and re- plated serially until clonal as deteπnined by several features including (i) a stable restriction digest pattern, (ii) a stable protein pattern by SDS-PAGE, and (iii) stable and homogeneous morphology by electron microscopy.

[00057] Bacteriophage isolates were tested against a Salmonella strain collection consisting of a number of Salmonella strains, including S. hadar (84 strains), S. typhimurium DT- 104 (42 strains), S. enteriditis (24 strains), S. heidelberg (21 strains), and S. newport (18 strains). Other strains representing other serotypes also were typed. [00058] Seven clones of Salmonella-specific lytic bacteriophages were found to be "tailed phages" of the family Myoviridae and Siphoviridae by electron microscopy. The most active phage clone lysed 220 (90%) of the 245 different Salmonella strains, including all DT-104 (multi-drug resistant) Salmonella isolates. The second most active phage clone lysed 74% of the 245 tested strains.

[00059] The rapid PFGE procedure developed for typing E. coli 0157:H7 strains (Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, and Swaminathan B. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33:2233-2239) was used for PFGE typing of the Salmonella strains. All the Salmonella strains were analyzed after digesting their DNA with-Xb I, and selected strains were also analyzed after digesting their DNA with Avr II and Spe I restriction enzymes. The CDC-standard S. newport strain amOl 144 (Xba I-digested) was used as the reference strain in all experiments. Bacteria sfrains were typed to assure that each host represented an independent strain that would thus contribute to the overall diversity of sfrains target ed by a given phage or phage cocktail.

[00060] Example 2 - Injection of Fertilized Eggs and Treatment of Chicks

[00061] The objective of this example was to determine the effects of injecting fertilized eggs and treating chicks in a commercial setting with a bacteriophage cocktail specific for Salmonella. Bacteriophages employed in this experiment were mixed into a cocktail, which consisted of five clonal bacteriophages directed against the following pathogenic Salmonella species: S. typhimurium, S. enteriditis, S. heidelberg, S. newport, S. hadar, S. kentucky, and S. thomson.

[00062] To conduct this experiment, two egg hatchers were randomly paired and each contributed fertilized eggs. One thousand eggs from one hatcher were processed as usual in the commercial setting, which included injecting the eggs with a vaccine for Marek's disease (Merial Select, Inc.) (the control hatcher) according to the manufacturer's recommendations. The 1000 eggs from the other hatcher were injected with the bacteriophage cocktail mixed with the Marek's vaccine. More specifically, 200 ml of the bacteriophage cocktail at 1010 pfu/ml phage were introduced into a 1600 ml vaccine bag after prior removal of 200 ml of diluent to accommodate the bacteriophage cocktail. Injection of 0.1 ml of the bacteriophage cocktail/vaccine mixture per egg thus resulted in injection of a dose of 1.25x108 pfu/egg.

[00063] At the time of hatching, chicks from the control hatcher (receiving only Marek's vaccine) were processed and moved first. Forty chicks were collected and placed in labeled egg boxes for sampling for Salmonella. The remaining chicks were sprayed with 7.0 ml of Newcastle/Bronchitis vaccine per 100 chicks according to the manufacturer's recommendations, and then taken to the farm for placement. The control chicks remained in the chick boxes until the bacteriophage cocktail/vaccine mixture-treated chicks were delivered to the farm so that they were placed near water and food at approximately the same time.

[00064] Chicks from the second hatcher (bacteriophage cocktail/vaccine mixture-treated eggs) were processed and moved second. Forty chicks were collected and placed in labeled boxes for sampling for Salmonella. The remaining chicks were sprayed with 7.0 ml of Newcastle/Bronchitis vaccine per 100 chicks mixed with a second application of the bacteriophage cocktail. The bacteriophage cocktail concentration was 2.5x109 pfu/ml resulting in a dose of 1.7x106 pfu/chick.

[00065] Chicks housed at the farm were arranged so that bacteriophages would not be tracked into the chambers housing the control chicks. Disinfectant foot pans were used and maintained at each closed chamber door. The traffic flow path was designed to isolate chicks exposed to the bacteriophages. The chicks from each hatcher were placed into the trial house with 60 birds per pen.

[00066] Chicks from both groups were sampled periodically to determine the rate of contamination with Salmonella.

[00067] For sampling of the newly hatched birds collected prior to spray vaccination, the gastrointestinal tracts were collected, added to buffered peptone water (BPW), held at room temperature for 48 hours, and qualitatively assayed for Salmonella using tetrathionate enrichment and supplemental polymerase chain reaction amplification using the BAX® PCR test kit (DuPont Qualicon, Inc., Wilmington, DE) according to the manufacturer's instructions.

[00068] A second group of chicks from both groups was sampled at three weeks of age. Ceca were collected from control and experimental groups. Specimen collection occurred over two days. Chick weights were recorded prior to collecting the ceca. The ceca were assayed qualitatively for the incidence of Salmonella using tetrathionate enrichment and supplemental polymerase chain reaction amplification using the BAX® PCR test kit (DuPont Qualicon, Inc., Wilmington, DE) according to the manufacturer's instructions.

[00069] Any positives from the bacteriophage cocktail/vaccine mixture-treated group were isolated for phage sensitivity testing.

[00070] A third group of chickens from both groups was sampled at market age. (This sampling was done at 39-40 days. Market age can vary and can depend on the target weight of the bird. For example, market age for broilers is between 42-60 days where as market age for roasters can be even later.) Chicken weights were recorded prior to collecting the ceca. The ceca were assayed qualitatively for the incidence of Salmonella using tetrathionate enrichment and supplemental polymerase chain reaction amplification using the BAX^® PCR test kit (DuPont Qualicon, Inc., Wilmington, DE) according to the manufacturer's instructions.

[00071] Any positives from the bacteriophage cocktail/vaccine mixture-treated group were isolated for phage sensitivity testing.

[00072] In ovo application resulted in significant reduction of Salmonella in the GI tract and yolk sac in newly hatched chicks. Incidence was reduced and the log number of bacteria in the ceca was reduced by 50%.

[00073] Additional results of the groups of the experiment where in ovo administration was combined with a post-hatch spray are shown in Tables 1 and 2.

[00074] The data demonstrate that the bacteriophage cocktail/vaccine mixture treatment produced a statistically significant reduction in the incidence of Salmonella. Testing at 3 weeks of age showed that only 0.6% of bacteriophage cocktail/vaccine mixture- treated chicks were Salmonella positive as compared to 11.3% of control chicks. At market age, 1.3% of bacteriophage cocktail/vaccine mixture-treated chickens were Salmonella positive as compared to 16.3% of control chickens. The results indicate that introduction of a bacteriophage cocktail/vaccine mixture to the eggs combined with vaccine spray treatment of the chicks is an effective method of reducing Salmonella colonization during poultry production.

Table 1

Table 2

Example 3 - Injection of Fertilized Eggs and Treatment of Chicks

[00075] The objective of this example was to compare (1) the level of Salmonella reduction obtained by first intentionally contaminating fertilized eggs with Salmonella, then injecting the eggs with a bacteriophage cocktail specific for Salmonella, to (2) the level of Salmonella reduction obtained by spraying chicks with the bacteriophage cocktail; and to compare the reduction levels from each group to the level of Salmonella reduction in a third group treated by injecting the eggs and spraying the chicks.

[00076] A bacteriophage cocktail was employed in this experiment. [00077] Eggs were collected from the hatchery. Forty eggs were randomly collected and sampled for natural Salmonella contamination by crushing the whole egg into 50 ml buffered peptone water and assaying for Salmonella.

[00078] Eggs were contaminated with 107 cfu of a mixture of S. kentucky, S. heidelberg, S. hadar, and S. typhimurium. The Salmonella inoculum was prepared by combining equal aliquots from individual overnight cultures of each strain. Each egg was inoculated with 20 μl (two 10 μl loops) of the Salmonella cocktail at room temperature. The cocktail suspension was spread over the blunt (air cell) end of the eggs and allowed to dry. Following inoculation, the eggs were incubated overnight, then ten eggs were sampled as above to determine the level of Salmonella contamination by preparing a shell rinse sample in 50 ml buffered peptone water, an eggshell/membrane sample in 10 ml buffered peptone water, and an egg contents sample in 50 ml buffered peptone water.

[00079] Each rinse and eggshell sample was serially diluted out to 10-5 and 10-4, respectively, from the initial pre-enrichment (samples are collected prior to an enrichment to detect low amounts of bacteria by culture overnight prior to plating), using buffered peptone water, and all dilutions were incubated for plating. The egg contents samples were diluted to 10-2 in buffered peptone water and incubated for plating. All samples and dilutions were incubated at 37°C for 24 hours. A 10 μl aliquot of each sample was then plated and incubated overnight. The extinction point from each dilution series of Salmonella was recorded to estimate the log number of Salmonella in the original sample.

[00080] On day 18 of incubation, 50 ml of a bacteriophage cocktail at 5x1010 pfu/ml phage were introduced into a 400 ml Marek's disease vaccine bag, (Merial Select Inc.). Injection of 0.1 ml of the bacteriophage cocktail/vaccine mixture per egg thus resulted in injection of a bacteriophage cocktail dose of 5.6 x 108 pfu/egg. A total of 220 eggs were injected with 0.1 ml of the bacteriophage cocktail/vaccine mixture and the eggs were incubated until hatch.

[00081 ] For control eggs, a group of 220 eggs was manually injected with 0.1 ml of the control preparation that was prepared from 50 ml of phosphate-buffered saline solution introduced into a 400 ml Marek's disease vaccine bag.

[00082] On the day of hatch, gastrointestinal tracts, yolk sacs, and whole bird feather rinses were collected from chicks of each group. Each sample was assayed qualitatively for the presence of Salmonella using the following procedure. Each ceca sample was serially diluted out to 10-9 from the initial pre-enrichment, using BPW. Also, serially dilute each rinse sample out to 10-8 from the original rinse sample (100). Incubate all bags and dilution tubes at 370C overnight in the appropriate incubator. After 18-24 hrs, transfer 0.1 ml from each dilution into broth. Incubate each dilution at 420C overnight, in the appropriate incubator. For each sample, drip plate 10 μl aliquots from each tube onto two plates and incubate in the appropriate incubator, overnight. Also, for each sample, streak one plate from the undiluted sample. Record the extinction point from each dilution series of Salmonella to estimate the original log inoculum.

[00083] In addition, 10 gastrointestinal tract samples and 10 feather rinse samples from each group were quantitatively assayed by serial dilution and plated.

[00084] After day-of-hatch sample collection, groups of chicks were sprayed with either 28 ml of the bacteriophage cocktail at 5x109 pfu/ml or 28 ml of a control solution, phosphate-buffered saline.

[00085] At three weeks of age and at market age, ceca and feather rinses were sampled for Salmonella using both quantitative and qualitative assays.

[00086] Sampling of the inoculated eggs at 24 hours demonstrated that 100% of eggs were contaminated with Salmonella recoverable from whole egg washes, shell and membrane fractions, and egg contents.

[00087] The bacteriophage cocktail/vaccine treatment produced a statistically significant reduction of intentional Salmonella contamination in gastrointestinal fracts.

[00088] Substantially less Salmonella colonization occurred in chicks hatched from bacteriophage cocktail/vaccine injected eggs that were subsequently sprayed for birds at 3 weeks of age and at market age. In this experiment, the principal benefit is from injection since spraying of chicks alone had little effect.

[00089] All references, including, patents and patent applications, cited herein are incorporated by reference in their entirety.

[00090] While the invention has been described with reference to specific embodiments thereof, it will be appreciated that numerous variations, modifications, and embodiments are possible, and accordingly, all such variations, modifications, and embodiments are to be regarded as being within the spirit and scope of the invention.

Claims

WE CLAIM:

1. A method of reducing, eliminating, or preventing a bacterial colonization in fertilized eggs, comprising: introducing a bacteriophage cocktail which specifically lyses said bacteria into or onto at least one fertilized egg, wherein introduction of the bacteriophage cocktail is effective in reducing, eliminating, or preventing the bacterial colonization in the at least one egg.

2. The method of claim 1, wherein the bacteriophage cocktail is combined with a poultry biologically active agent prior to introduction into the at least one fertilized egg.

3. The method of claim 1 , wherein the introduction is carried out by injection.

4. The method of claim 1, wherein the introduction is carried out by spraying said at least one egg.

5. The method of claim 1 , wherein the introduction is carried out by dipping said at least one egg in a liquid carrier comprising said cocktail.

6. The method of claim 1, wherein said bacteria is a Salmonella.

7. The method of claim 1, wherein said bacteria is a Campylobacter.

8. The method of claim 1 , wherein said bacteria is a Listeria.

9. The method of claim 1 , wherein said bacteria comprises at least two strains.

10. The method of claim 1 , wherein said bacteria comprises at least two species.

11. A method of reducing, eliminating, or preventing a bacterial colonization in poultry, comprising: administering a bacteriophage cocktail which specifically lyses said bacteria to at least one bird, wherein the administration of the bacteriophage cocktail is effective in reducing, eliminating, or preventing the bacterial colonization in the at least one bird.

12. The method of claim 11 , wherein the bacteriophage cocktail is combined with a poultry biologically active agent prior to administration to the at least one bird.

13. The method of claim 11 , wherein the method of administration is selected from the group consisting of: oral dosing in drinking water, oral dosing in food, injecting, inoculating, and spraying.

14. The method claim 13, wherein the method of administration is spraying.

15. The method of claim 11 , wherein the administration is within the first five days following hatching.

16. The method of claim 15, wherein the adminisfration is on the day of hatch.

17. A method of reducing, eliminating, or preventing bacterial colonization in poultry, comprising: introducing a first bacteriophage cocktail which specifically lyses said bacteria into at least one fertilized egg, and administering a second bacteriophage cocktail which specifically lyses said bacteria to the at least one bird hatched from the at least one bacteriophage-treated fertilized egg, such that bacteria colonization is reduced, eliminated, or prevented in the at least one hatched bird.

18. The method of claim 17, wherein the bacteriophage cocktail is introduced by injection.

19. The method of claim 18, wherein the injection is automated injection.

20. The method of claim 17, wherein the method of administration is selected from the group consisting of: oral dosing in drinking water, oral dosing in food, injecting, inoculating, and spraying.

21. The method claim 20, wherein the method of administration is spraying.

22. The method of claim 17, wherein the poultry is also treated with a vaccine.

23. The method of claim 17, where said first cocktail comprises the same phage strains as said second cocktail.

24. A method of reducing, eliminating, or preventing bacteria colonization in shell eggs, comprising: introducing a bacteriophage cocktail which specifically lyses said bacteria into at least one shell egg, wherein said introduction reduces, eliminates, or prevents bacteria colonization in the at least one shell egg.

25. The method of claim 24, wherein the method of introduction is injection.

26. The method of claim 24, wherein the injection is automated injection.

27. A container for storing a bacteriophage that specifically lyses a poultry bacterial pathogen, wherein said container is a component of an automated poultry egg injection device.

28. The container of claim 27, comprising a plurality of bacteriophage.

29. The container of claim 27, wherein said pathogen is a Salmonella.