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WO2004071324A2 - Procede permettant de reduire, eliminer ou prevenir la colonisation des oeufs et/ou de la volaille par des pathogenes bacteriens a l'aide de bacteriophages - Google Patents

Procede permettant de reduire, eliminer ou prevenir la colonisation des oeufs et/ou de la volaille par des pathogenes bacteriens a l'aide de bacteriophages Download PDF

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WO2004071324A2
WO2004071324A2 PCT/US2004/003789 US2004003789W WO2004071324A2 WO 2004071324 A2 WO2004071324 A2 WO 2004071324A2 US 2004003789 W US2004003789 W US 2004003789W WO 2004071324 A2 WO2004071324 A2 WO 2004071324A2
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poultry
bacteria
bacteriophage
cocktail
eggs
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WO2004071324A3 (fr
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Intralytix Inc
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K45/00—Other aviculture appliances, e.g. devices for determining whether a bird is about to lay
    • A01K45/007—Injecting or otherwise treating hatching eggs

Definitions

  • the present invention is directed to methods of reducing, eliminating, or preventing bacterial pathogen colonization in eggs and/or poultry using bacteriophages.
  • 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.
  • ECM early chick mortality
  • 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.
  • 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.
  • 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).
  • 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.
  • the bacterial pathogens are C. perfringensis such as, for example, 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.
  • 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.
  • 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.
  • production as applied to poultry includes, but is not limited to, breeding, raising, storing, processing and handling poultry and all functions associated therewith.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the eggs may be dipped with or submerged in a bacteriophage solution.
  • 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.
  • the embryo and its membranes e.g., the air cell, the allantois, and yolk sac
  • the quantitative volume of the enclosed fluid varies.
  • 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.
  • injecting bacteriophages into the albumin may increase the risk of leakage of albumin and the ingress of air and contaminants after injection.
  • means are provided for preventing air and contaminants from entering the albumin, and preferably means are provided for preventing leakage of albumin after injection.
  • 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.
  • 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.
  • eggs are injected between days 17 and 19 of incubation.
  • eggs are injected on day 18 of incubation.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a method of treating poultry 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.
  • 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.
  • suitable means 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 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.
  • 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.
  • 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.
  • any bacterial pathogen-positive birds e.g., Salmonella contaminated birds
  • 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.
  • the birds 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.
  • 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.
  • 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.
  • colonization of bacterial pathogens such as, for example, Salmonella
  • 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.
  • 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.
  • shell eggs are injected with bacteriophages in the first 48 hours following laying of the eggs.
  • 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.
  • bacteriophages are added to preparations of egg yolks.
  • bacteriophages are added to preparations of egg whites.
  • 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 contain at least three bacteriophages and can be custom tailored to the bacterial pathogens that are prevalent in a certain situation.
  • a bacteriophage cocktail can contain four, five, six or more lytic bacteriophages which specifically target Salmonella species, as described in Example 1 below.
  • 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.
  • 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.
  • the members of the bacteriophage cocktail can be substituted as needed to minimize the risk of resistance developing.
  • bacteriophage cocktail members 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.
  • 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, 1958) [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 ⁇ m 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.
  • 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.
  • 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.
  • S. typhimurium such as, for example, S. typhimurium DT 104, S.
  • 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.
  • 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. thom
  • 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.
  • 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.
  • suitable carriers, diluents, excipients, etc. see Remington: The Science and Practice of Pharmacy, Gennaro, A.R. (ed.), Mack Publishing Co., Eaton, PA (2000).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the container can be configured to contain a plurality of chambers, wherein the contents thereof are admixed, for example, prior to use.
  • 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.
  • 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.
  • 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.
  • the container can be one that contains a unit dose or multiple doses for administration.
  • 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.
  • 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.
  • equipment, enclosures, pens, food processing equipment, knives, incubators and the like can be treated with such solutions.
  • the spray may be of a dust or other dry formulation wherein the particles can be readily dispersed.
  • Example 1 Isolation and Purification of Bacteriophages
  • 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.
  • plaques are isolated, eluted, and re-plated serially until clonal as dete ⁇ nined 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.
  • 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.
  • 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.
  • 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.
  • chicks from the control hatcher 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a bacteriophage cocktail was employed in this experiment.
  • 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.
  • 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 ⁇ l (two 10 ⁇ l 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.
  • 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.
  • 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 ⁇ l 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.
  • 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.
  • the bacteriophage cocktail/vaccine treatment produced a statistically significant reduction of intentional Salmonella contamination in gastrointestinal fracts.

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  • Life Sciences & Earth Sciences (AREA)
  • Environmental Sciences (AREA)
  • Birds (AREA)
  • Animal Husbandry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Fodder In General (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des procédés permettant de réduire, éliminer ou prévenir la colonisation d'oeufs fécondés, d'oeufs de consommation et/ou de volaille par des pathogènes bactériens tels que Salmonella. Les procédés de l'invention permettent d'introduire dans les oeufs et/ou d'administrer à la volaille des bactériophages efficaces contre les pathogènes bactériens.
PCT/US2004/003789 2003-02-11 2004-02-11 Procede permettant de reduire, eliminer ou prevenir la colonisation des oeufs et/ou de la volaille par des pathogenes bacteriens a l'aide de bacteriophages Ceased WO2004071324A2 (fr)

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US60/446,265 2003-02-11

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Publication number Priority date Publication date Assignee Title
CN101195803B (zh) * 2007-12-25 2011-07-27 浙江大学 一种厌氧氨氧化菌富集装置
WO2012031210A1 (fr) * 2010-09-02 2012-03-08 Biova, L.L.C. Séparation de membrane de coquille d'œuf
US10104903B2 (en) 2009-07-31 2018-10-23 Mars, Incorporated Animal food and its appearance
CN109735506A (zh) * 2019-01-02 2019-05-10 江苏省农业科学院 李斯特菌噬菌体组合物及其应用
KR20190099074A (ko) * 2017-02-07 2019-08-23 조에티스 서비시즈 엘엘씨 가금류 부화장 작업에서 병원체를 감소시키기 위한 방법
US11154077B2 (en) 2009-07-31 2021-10-26 Mars, Incorporated Process for dusting animal food
US11388914B2 (en) 2015-04-28 2022-07-19 Mars, Incorporated Process of preparing a wet pet food, wet pet food produced by the process and uses thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BARROW ET AL.: 'Use of lytic bacteriophage for control of experimental Escherichia coli septicemia and Meningitis in chicken and calves' CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY vol. 5, no. 3, May 1998, pages 294 - 298, XP002983441 *
BERCHIERI JR ET AL.: 'The activity in the chicken alimentary tract of bacteriophages lytic for Salmonella typhimurium' RESEARCH IN MICROBIOLOGY vol. 142, 1991, pages 541 - 549, XP001024374 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8418943B2 (en) 2005-01-18 2013-04-16 Biova, L.L.C. Egg shell membrane separation
CN101195803B (zh) * 2007-12-25 2011-07-27 浙江大学 一种厌氧氨氧化菌富集装置
US10104903B2 (en) 2009-07-31 2018-10-23 Mars, Incorporated Animal food and its appearance
US11154077B2 (en) 2009-07-31 2021-10-26 Mars, Incorporated Process for dusting animal food
WO2012031210A1 (fr) * 2010-09-02 2012-03-08 Biova, L.L.C. Séparation de membrane de coquille d'œuf
US11388914B2 (en) 2015-04-28 2022-07-19 Mars, Incorporated Process of preparing a wet pet food, wet pet food produced by the process and uses thereof
KR20190099074A (ko) * 2017-02-07 2019-08-23 조에티스 서비시즈 엘엘씨 가금류 부화장 작업에서 병원체를 감소시키기 위한 방법
KR102459253B1 (ko) 2017-02-07 2022-10-26 조에티스 서비시즈 엘엘씨 가금류 부화장 작업에서 병원체를 감소시키기 위한 방법
CN109735506A (zh) * 2019-01-02 2019-05-10 江苏省农业科学院 李斯特菌噬菌体组合物及其应用

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