AU677040B2 - Method of treating immature birds with IL-2 - Google Patents

Description

OPI DATE 26/09/94 APPLN. ID AOJP DATE 24/11/94 PCT NUMBER 61005/94 (Ili f fillillil lli9l600 lli PCT/US94/01194 1111111 111111111111111 111111111ll li illiii AU9461005 (51) International Patent Classification 5 (11) International Publication Number: WO 94/19944 A01N 37/18, A61K 37/00, 45/05, C07K Al 3/00, 13/00, 15/00, 17/0 (43) International Publication Date: 15 September 1994 (15.09.94) (21) International Application Number: PCT/US94/01179 (81) Designated States: AT, AU, BB, BG, BR, BY, CA, CH, CN, CZ, DE, DK, ES, FI, GB, HU, JP, KP, KR, KZ, LK, LU, (22) International Filing Date: 2 February 1994 (02.02.94) LV, MG, MN, MW, NL, NO, NZ, PL, PT, RO, RU, SD, SE, SK, UA, US, UZ, VN, European patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), Priority Data: OAPI patent (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, 029,040 10 March 1993 (10.03.93) US NE, SN, TD, TG).

Parent Application or Grant Published (63) Related by Continuation With international search report.

US 029,040 (CON) Filed on 10 March 1993 (10.03.93) (71) Applicant (for all designated States except US): EMBREX, INC. [US/US]; 1035 Swabia Court, P.O. Box 13989, Research Triangle Park, NC 27709-3989 (US).

(72) Inventors; and Inventors/Applicants (for US only): RICKS, Catherine, A.

[US/US]; 3336 Manor Ridge Drive, Raleigh, NC 27603 PHELPS, Patricia, V. [US/US]; 2514 Anderson Drive, Raleigh, NC 27608 (US).

(74) Agents: SIBLEY, Kenneth, D. et al.; Bell, Seltzer, Park Gibson, P.O. Drawer 34009, Charlotte, NC 28234 (US).

(54) Title: METHOD OF TREATING IMMATURE BIRDS WITH IL-2 (57) Abstract A method of combatting bacterial infections, including Escherichia coli and Salmonella infections, in immature birds by administering to said immature bird a T-cell growth factor comprising Interleukin-2 (IL-2) in an amount effective to combat bacterial infections in immature birds is disclosed. The method is preferably carried out on chickens on about the eighteenth day of incubation.

~a WO 94/19944 PCT/US94/01179 METHOD OF TREATING IMMATURE BIRDS WITH IL-2 Field of the Invention This invention relates to the treatment of birds by the administration of Interleukin-2 to combat bacterial infections.

Background of the Invention Hatching birds are exposed to pathogenic bacteria liberated from the broken shells and shell membranes. Bacteria inhaled or ingested can colonize respiratory or gastrointestinal mucosa and may penetrate the epithelial barriers. Hatchlings are initially protected against bacterial pathogens by maternally derived antibodies. However, maternal antibodies provide only temporary protection during the period before and after hatching when the bird's own immune system is immature. Often maternal antibody levels wane before natural antibody synthesis can adequately protect the immature bird against pathogens, causing r auced antibody levels. This period of reduced antibody levels occurs between weeks two and three posthatch and is often associated with bacterial pathogen-associated peaks in mortality, specifically colisepticemia, at three to four weeks posthatch.

U.S. Patent No. 4,775,621 to Berkhoff and Vinal discusses diagnosing colisepticemia in birds by determining the presence or absence of invasive E. coli

,I

WO 94/19944 PCT/US94/01179 -2in an incubator, but does not discuss how colisepticemia might be treated.

In view of the foregoing, there is a continued need for new means of controlling bacterial infections in birds.

Summary of the Invention A method of treating immature birds is disclosed herein. The method comprises administering to an immature bird Interleukin-2 (IL-2) in an amount effective to combat a bacterial infection in said bird.

Also disclosed is a method of treating immature birds to combat an Escherichia coli infection in said birds, comprising administering to an inm.-aure bird Interleukin-2 (IL-2) in an amount effective to combat E. coli infection in said bird.

A particular embodiment of the foregoing provides a method of combatting colisepticemia infections in a flock of birds. The method comprises administering IL-2 to said birds in ovo in an amount effective to combat colisepticemia, incubating said birds to hatch together in a common incubator, and then raising the birds to at least two weeks of age.

Also disclosed is a method of treating immature birds to combat Salmonella infection in said birds, comprising administering to an immature bird IL- 2 in an amount effective to combat Salmonella infection in said bird.

Another aspect of the present invention is the use of IL-2 for the preparation of a medicament for carrying out the treatments described herein.

U.S. Patents No. 5,028,421 discloses the administration of IL-2 to a bird in ovo and to promote weight gain after hatch, and the administration of IL-2 in ovo in combination with a vaccine. U.S. Patent No.

5,106,617 discloses a method of inducing an immature bird to gain weight by administering IL-2 to the immature bird after hatch. It has now unexpectedly I=J L__ WO 94/19944 PCT/US94/01179 -3been found that IL-2 administered to birds in ovo combats bacterial infections, particularly Escherichia coli and Salmonella infections. The ability of IL-2 to produce such an effect has not heretofore been suggested.

Detailed Description of the Ifvention The term "birds" as used herein, is intended to include males or females of any avian species, but is primarily intended to encompass poultry which are commercially raised for eggs or meat. Accordingly, the term "bird" is particularly intended to encompass hens, cocks and drakes of chickens, turkeys, ducks, geese, quail and pheasant. Birds are preferred. The term "immature as used herein, is intended to include any bird not yet of adult age, including birds in ovo.

The term "interleukin-2 as used herein, refers to IL-2 of any species, including bovine, ovine, murine, human or avian IL-2. Numerous types of IL-2 are known, as described in U.S. Patent No. 5,106,617 and 5,028,421 (applicants specifically intend that the disclosure of all U.S. Patent references cited herein be incorporated herein by reference).

The term "avian IL-2," as used herein, means IL-2 corresponding to IL-2 produced by any avian species. The term "avian" is intended to encompass all avian species, including, but not limited to, chickens, turk.,ys, ducks, geese, quail, and pheasant. Various jes of avian IL-2 are known. See, U.S.

,nts Nos. 5,028,421 and 5,106,617. In brief, avi.an IL-2 may be obtained by collecting lymphocytes from an avian donor (most conveniently from the spleen of an avian donor), growing the lymphocytes in a medium (preferably a serum-free medium) containing a T-cell mitogenic agent such as Concanavalin A, and, optionally, recovering the IL-2 from the medium. Thus, the IL-2 conditioned media itself may be used to L WO 94/19944 PCT/US94/01179 -4administer IL-2. When avian IL-2 is used in practicing the present invention, the degree of purity of the avian IL-2 is not critical, although it is preferably at least substantially serum free and mitogen free. A crude IL-2 preparation may be purified by any of a variety of known separation procedures, with various fractions from these procedures being screened for IL-2 activity by IL-2 assay procedures known in the art. The IL-2 may be provided in any suitable pharmaceutically acceptable carrier, but is preferably provided in an aqueous carrier such as a phosphate-buffered saline solution.

The coss-reactivity of IL-2 of various avian species can be routinely determined with known bioassay procedures employing IL-2 responder cells, see, e.g., Schnetzler et al., supra at 561, or can simply be screened by administering to a sample group of birds in ovo an IL-2 for which activity in that species is to be determined. In general, it is to be expected that, the closer the species of origin of the IL-2 administered to the avian species being treated in any one case, the greater the biological activity of the IL-2 in that subject. Hence, it is preferred, but not essential, that the IL-2 being administered in the method disclosed herein correspond in species of origin to the avian subject being treated. Those skilled in the art will be able to select an appropriate IL-2 composition for the bird being treated based on the known crossreactivities of IL-2 and simple screening tests known to those skilled in the art. The quantity of IL-2 administered per bird will vary according to the species being treated, the species of IL-2 origin, the subject's age, the site of injection in the bird, and the purity of the IL-2.

Chicken IL-2, as discussed above, exists as a 14K species and a 30K species. See Fredericksen and Sharma, supra; Schnetzler, supra at 565. The WO 94/19944 PCT/US94/01179 species is believed to be the native form. A 30K avian IL-2 preparation which is prepared according to Example 3 of U.S. Patent No. 5,028,421 (the disclosures of all patent references cited herein are to be incorporated herein by reference) and which is determined to be 97% pure by high perfo:.-mance liquid chromatography (HPLC) provides about 100-300 activity units of 30K avian IL-2 per milligram of protein. For practicing the present invention, avian IL-2 is administered in an amount of from about .0035 activity units per egg. More preferably, avian IL-2 is administered in an amount of from about .035 activity units per egg. For practicing the present invention, avian IL-2 is administered in an amount to about 35 activity units per egg. More preferably, avian IL-2 is administered in an amount to about 3.5 activity units per egg.

In practicing the present invention the IL-2 may be administered in the form of IL-2 conditioned media. For example, positive results with the method of the present invention are expected in chickens administered IL-2 conditioned media on about the 18th day of incubation, where the IL-2 conditioned media administered contains avian IL-2 in an amount of from about .0035 activity units per egg. More preferably, the IL-2 conditioned media administered contains avian IL-2 in an amount of from about .035 activity units per egg. For practicing the present invention, IL-2 conditioned media administered contains avian IL-2 in an amount to about 35 activity units per egg. More preferably, IL-2 conditioned media administered contains avian IL-2 in an amount to about 3.5 activity units per egg.

Insofar as this applicant is aware, analogs of avian IL-2 have not yet been synthesized. However, based on the cross-reactivity of various IL-2s in nonavian species, it is expected that synthetic analogs of avian IL-2, when available, can be screened for WO 94/19944 PCT/US94/01179 -6activity in the present invention in a routine manner, and should function in the present invention in substantially the same way as the naturally occurring IL-2s.

The term "in ovo," as used herein, refers to birds contained within an egg prior to hatch. Thus, the present invention may be conceived of as both a method of treating eggs and a method of treating birds. The present invention may be practiced with any type of bird egg, including chicken, turkey, duck, goose, quail, and pheasant eggs. Chicken and turkey eggs are preferred, with chicken eggs most preferred. Eggs treated by the method of the present invention are fertile eggs which are preferably in the fourth quarter of incubation. Chicken eggs are treated on about the fifteenth to nineteenth day of incubation, and are most preferably treated on about the eighteenth day of incubation (the eighteenth day of embryonic development). Turkey eggs are preferably treated on about the twenty-first to twenty-sixth day of incubation, and are most preferably treated on about the twenty-fifth day of incubation.

The present invention may be used to combat both gram negative and gram positive bacterial infections. Such gram positive bacteria include, but are not limited to, Pasteurella species, Staphylococci species, and Streptococcus species. Gram negative bacteria include, but are not limited to, Escherichia coli, Pseudomonas species, and Salmonella species.

Salmonella enteriditis is an important pathogen in the commercial layer industry, as ovarian colonization of layers may result in maternally transmitted Salmonella in table eggs.

The term "colisepticemia" as used herein refers to septicemic E. coli infections caused by invasive E. coli bacteria. While some strains of E.

coli are noninvasive, invasive strains exist that are II h WO 94/19944 PCTIUS94/01179 -7able to invade host tissue and cause severe disease.

The manner by which colisepticemia is transmitted among poultry is not yet well understood.

In the present invention, administration of IL-2, including administration using IL-2 conditioned media, to immature birds may be carried out by any suitable means, such as by in ovo injection, intravenous injection, intraperitoneal injection, and subcutaneous injection. Eggs may be administered IL-2 by any means which transports the compound through the shell. The preferred method of administration is, however, by injection. The site of injection is preferably within either the region defined by the amnion, including the amniotic fluid and the embryo itself, in the yolk sac, or in the air cell. By the beginning of the fourth quarter of incubation, the amnion is sufficiently enlarged that penetration thereof is assured nearly all of the time when the injection is made from the center of the large end of the egg along the longitudinal axis. A vaccine may be administered by the same means and to the same location as the IL-2.

The mechanism of injection is not critical, but it is preferred that the method not unduly damage the tissues and organs of the embryo or the extraembryonic membranes surrounding it so that the treatment will not decrease hatch rate. A hypodermic syringe fitted with a needle of about 18 to 22 gauge is suitable for the purpose. To inject into the air cell, the needle need only be inserted into the egg by about two millimeters. A one inch needle, when fully inserted from the center of the large end of the egg, will penetrate the shell, the outer and inner shell membranes enclosing the air cell, and the amnion.

Depending on the precise stage of development and position of the embryo, a needle of this length will terminate either in the fluid above the chick or in the -~py I WO 94/19944 PCT/US94/01179 -8chick itself. A pilot hole may be punched or drilled through the shell prior to insertion of the needle to prevent damaging or dulling of the needle. If desired, the egg can be sealed with a substantially bacteriaimpermeable sealing material such as wax or the like to prevent subsequent entry of undesirable bacteria.

It is envisioned that a high speed automated injection system for avian embryos will be particularly suitable for practicing the present invention.

Numerous such devices are available, exemplary being those disclosed in U.S. Patent Nos. 4,903,635 and 4,681,063 to Hebrank, U.S. Patent No. 5,056,464 to Lewis, and U.S. Patents Nos. 4,040,388, 4,469,047, and 4,593,646 to Miller (the disclosures of all U.S. patent references cited herein are to be incorporated herein by reference). All such devices, as adapted for practicing the present invention, comprise an injector containing avian IL-2 as described herein, with the injector positioned to inject an egg carried by the apparatus with the avian IL-2. Other features of the apparatus are discussed above. In addition, if desired, a sealing apparatus operatively associated with the injection apparatus may be provided for sealing the hole in the egg after injection thereof.

Preferred apparatus for practicing the present invention is disclosed in U.S. Patent No.

4,903,635 to Hebrank and U.S. Patent No. 5.056,464 to Lewis, the disclosures of which are incorporated herein by reference. These devices comprise an injection apparatus for delivering fluid substances into a plurality of eggs and apparatus for aligning the eggs in relation to the injection apparatus. The features of these apparatus may be combined with the features of the apparatus described above for practicing the present invention. In practicing the present invention, injected eggs are incubated to hatch and the birds are raised to at least 2 weeks of age.

B re .b IA WO 94/19944 PCT/US94/01179 -9- The following examples are provided to more fully illustrate the present invention, and are not to be taken as restrictive thereof. In the following examples, °C means degree Centigrade, g means micron, mm means millimeter, cm means centimeter, cc means cubic centimeter, ml means milliliter, 41 means microliter, g means gram, mg means milligram, Ag means microgram, n means number, oz means oun M means molar, mM means millimolar, TSB means trypticase soy broth, PBS means phosphate buffered saline (pH 7.4), IL-2 means avian interleukin 2, IL2CM means avian interleukin-2 conditioned media, Con-A means concanavalin-A, RBC means red blood cell, rpm means revolutions per minute, CO 2 means carbon dioxide, CFU means colony forming unit, IM means intramuscular, MTT means 3-[4,5-dimethylthiazolyl-2]-2,5-diphenyl tetrazolium bromide, RPMI means Roswell Park Memorial Institute, OD means optical density, EXAMPLE 1 Preparation of Spleen Cells Spleens were obtained from Hyline, SC chickens between 4 and 10 weeks of age, and a single cell suspension prepared from decapsulated spleen.

Specifically, the spleen was placed in phosphate buffered saline (PBS) (pH passaged through a five millimeter syringe barrel, and then passaged through an eighteen gauge needle. The suspension was held at room temperature for five minutes to allow the stroma to settle and the supernatant was collected. An additional five milliliters of PBS was added to the settled stroma, mixed, held for five minutes, and the supernatant was collected and pooled with the first supernatant. These cells were then pelleted and washed twice with PBS before adding RPMI-G medium, as explained in Example 2 below. Cell viability was greater than 95% as measured by trypan blue dye exclusion.

l WO 94/19944 PCT/US94/01179 EXAMPLE 2 Preparation of IL-2 Conditioned Medium RPMI-G medium consisted of RPMI-1640 media supplemented with L-glutamine (2 mM), and gentamicin sulfate (50 micrograms per millilit( (Sigma, St.

Louis, MO).

Single-cell suspensions from spleen were placed into 75 square centimeter flasks with milliliters of RPMI-G medium at a cell concentration of 6 x 106 cells per milliliter and a Concanavalin A (Sigma Chemical Co., St. Louis, MO) concentration of 4 micrograms per milliliter. IL-2 Conditioned Medium (I2CM) was obtained from these cultures following '".jubation at 40' C in a humidified atmosphere with 1 ',02 for 64-96 hours, with peak production being obtained after 96 hours.

EXAMPLE 3 IL-2 Assay Avian IL-2 activity is defined by in vitro proliferative responses of T-cells which have been preexposed to a mitogenic agent. The proliferative response is indicated by mitochondrial incorporation of a specific dye (MTT, or 3-[4,5-dimethylthiazolyl-2]tetrazolium bromide). An activity unit is defined as the amount of material which causes 50% or one-half maximal proliferative response as measured in the following cell proliferation assay.

1. Preparation of lymphocyte suspensions Chickens are sacrificed and excised spleens are placed in cold PBS containing gentamicin (2 ml at mg/ml per 1000 ml PBS). The spleens are decapsulated and extruded through a sterile syringe, and the tissue is minced. The minced tissue slurry from three spleens is forced through an 18 gauge needle into a 50 ml test tube, and the volume is brought to ml with cold PBS.

I WO 94/9944 PCT/US94/01179 -11- To remove cell stroma and excess tissue, the tissue slurry is allowed to settle for seven minutes; ml of the cell suspension from the top of each tube is saved. The remaining stroma in each tube is brought to a total volume of about 47 ml with PBS and allowed to settle for five minutes. The cell suspension above the 15 ml graduation mark is removed and pooled with the first collection. Cell suspensions are centrifuged at 200 rnm for five minutes to remove excess RBCs. The supernatant is then centrifuged at 1500 rpm for minutes. Supernatants are decanted (the pellet saved), and centrifuged again at 1500 rpm for 15 minutes.

Each pellet is resuspended in 50 ml cold PBS and centrifuged with the supernatants (1500 rpm for fifteen minutes). Supernatants are then discarded and the cell pellets pooled into 50 ,l or less with cold PBS. The cell suspension is divided into two equal parts and each is brought to a total volume of 37 ml with PBS.

Percoll density gradients are repared using 12 ml of 60% Percoll in PBS as the bottom layer and ml of 20% Percoll in PBS as the top layer. Six ml of the cell suspension is loaded on the top of each gradient and centrifuged at approximately 15"C at 2000 rpm for 25 minutes. The white cells are removed from each gradient and placed into a separate 50 ml tube.

Each tube is brought to a total volume of 50 ml with cold PBS and centrifuged at 1600 rpm for 10 minutes.

Supernatants are discarded and the cell pellets are pooled and resuspended in a total volume of 35 to 40 ml of cold PBS, and centrifuged at 1200 rpm for ten minutes.

The cell pellet from Example 1, above, is resuspended in a total volume of 35 to 45 ml of RPMI culture medium. For each 500 ml total volume of RPMI culture medium, RPMI (Sigma Catalog #R-7509 RPMI 1640 I ii L r I WO 94/19944 W C'-rPVS94 11 79 -12without phenol red) is combined with 5ml 100X L- Glutamine and 1ml Gentamicin (25 mg/ml).

Cells are counted at a dilution factor of 1:100 in 4% Trypan Blue 450 Al Trypan Blue Al cells 1:10; 50 pl of 1:10 450 l1 Trypan Blue 1:100). Cells are seeded at 15 x 10 6 /cm 2 in RPMI culture medium. Free Con-A is added to stimulate lymphoblast transformation. Tissue culture grade Concanavalin-A (Sigma, Catalog Number C-5275), 2 gg/ml of media is required.

The cell suspensions are incubated for lymphoblast transformation by placing 100 ml of cell suspension in a flask. Each flask is gassed with 5% CO 2 in air for thirty seconds, and placed on its side in

CO

2 at 37°C. The flask caps are loosened to allow for gas exchange and flasks are incubated for 44 to 48 hours.

2. Harvest of Cells for Assay Cells and media are centrifuged at 1200 rpm/10 minutes at 50 ml/tube. Cell pellets are pooled in a total volume of 25 ml of RPMI media, and incubated at 40*C for 15 minutes. The cell suspensions are mixed and placed in a centrifuge, brought to 400 rpm, and the centrifuge turned off. Supernatant is saved (pellet is discarded) and centrifuged at 1000 rpm for five minutes. Supernatant is discarded and the cell pellet is resuspended in 10 ml RPMI media in a 15 ml tube.

The cell suspension is placed on ice for 30 minutes to allow aggregates to settle. Approximately 9 ml of cell suspension is pipetted from the tube; remaining aggregates are discarded.

The cell suspension is centrifuged at 400 rpm for five minutes; supernatant is discarded. The cell pellet is resuspended in five to seven ml of RPMI media at 3 x 106 cells/ml.

WO 94/19944 PCT/US94/01179 -13- 3. Cell Proliferation Assay Ninety-six well microtiter plates are used.

Single wells on the outside of the top, bottom and sides of each plate are loaded with 200 pl of PBS per well. The remaining wells are loaded with 100 gl RPMI media (RPMI 1640, Sigma Cat. #R-7509 without phenol red) with the following additives: 1 ml Gentamicin (Gentamicin sulfate, Cat. #16051, United States Biochemical Corp.); L-Glutamine (5 ml of 100x/500 ml; Gibco Cat. #321-5030A6); 2% H.I. Chicken Serum (Sigma, Cat. #C5405); 0.1 M Mannopyranoside (3.884 g/200 ml)(Sigma Cat. #M-6882).

The samples to be tested are loaded into column #3 in duplicate (3 samples per plate can be assayed). Wells are loaded with 100 pl sample per well, two wells per sample. Serial dilutions are made by first mixing the sample with the media in column #3.

Serial dilutions extend from column #3 through column After column #10 has been mixed 100 Al is discarded, leaving 100 Al in each of the wells. To each well is added 100 gl of cell suspension. Column #2 and Column #11 are control wells (cells only without sample). Microtiter plates are placed into 5% C0 2 37°C incubator for not less than 46 hours or more than hours.

For the MTT dye uptake, 5 mg of MTT (United States Biochemical Corp., Catalog #19265) is dissolved in each ml of PBS. The dissolved MTT is filtered with a 0.45 p sterile filter unit, and 25 pl is added to each well of each plate; plates are incubated in a

CO

2 37°C incubator for 60 minutes. At the end of the minute incubation, 150 pl is removed from each well and 100 Al of acid-isopropanol (3.3 ml 12M HC1 in 1000 ml isopropanol) is added; each plate is mixed on a microtiter plate shaker at high speed for two minutes.

Plates are read at a wavelength of 570 nanometers on a microtiter-plate analyzer.

s ~I WO 94/19944 PCT/US94/10179 -14- The average optical density (OD) for background wells (wells containing only PBS) is calculated for each microtiter plate. The average background OD is subtracted from the OD reading for each control and sample well. The average GD is calculated from duplicate wells (minus background) for each control and each sample well at each dilution.

Average control (cells without sample) OD is calculated for each plate and average control values are calculated for all plates used in the assay.

The percent change of sample at each serial dilution is calculated against the average control value.

x unknown value for change (x sample OD) (x control OD) A OD value change A OD x 100 x Control OD The percent change for each log dilution is plotted. The intercept reference line at 50% of the maximum response for standard reference material above control value is calculated. The log value at intercept is determined and the inverse log is calculated to determine the dilution titer for each sample. A unit is defined as the amount of sample material which causes 50% or one-half maximal proliferative response.

EXAMPLE 4 Materials and Methods EQ Injection and Husbandry. Fertile Hubbard X Hubbard broiler breeder eggs were obtained from a commercial hatchery and stored at 16°C. On Day 0 of incubation, eggs were randomly set in a single stage Jamesway 252 forced draft incubator. Temperature and humidity conditions closely adhered to manufacturer's guidelines. On Day 18 of incubation, eggs were removed

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WO 94/19944 PCT/US94/01179 from incubators and candled for viability. All infertile, malpositioned, and nonviable eggs were withdrawn from the study.

Embryonated eggs were randomly divided into treatment groups, labeled, injected, transferred into hatching baskets, and returned to the incubator. Egg injection consisted of shell penetration 5 mm through the top or bottom of the egg, utilizing an 18 gauge needle. Next, a 23 gauge needle attached to a 1 cc tuberculin syringe was inserted 22 mm through the punched hole, delivering the test article. Injection sites were neither sealed nor sanitized.

All unhatched eggs were broken out and categorized as either late deads, live pips, dead pips, dead chicks, or culls. Malpositioned and malformed embryos were eliminated from the hatchability analysis.

Viable chicks were counted, weighed, and banded. Birds were color coded by treatment group.

Bacterial Challenge Material. The Escherichia coli bacterial culture (serogroup 01) or a nalidixic novobiocin resistant strain of Salmonella typhimurium were grown overnight at 37°C in trypticase soy broth (TSB). Confluent cultures were diluted in 0.9% saline as needed. Preparation of oral E. coli challenge inoculum doses required a 10X concentration by centrifugation. Pellets were resuspended in 0.9% saline. Bacterial suspensions remained in crushed ice until challenge. Challenge preparations were diluted serially and plated in triplicate onto Trypticase Soy agar to determine actual dose.

EXAMPLE Oral E. Coli Challenge at Hatch Day 18 embryonated broiler eggs were injected with either PBS or 0.35 activity units of IL2CM. On the day of hatch 40 chicks from each treatment group were orally gavaged with 100 Al PBS containing 8.8 X 108 CFU E. coli. An additional 40 chicks which received I I I WO 94/19944 1'CTIUS94/01179 -16- PBS in ovo were gavaged with 100 Al PBS. Chicks were commingled within one floor pen and given feed and water ad libitum. Daily mortality was recorded for five consecutive days at which time all chicks were weighed and the study terminated.

Results. In ovo IL2CM significantly reduced day mortality of broiler chicks challenged orally with E. coli from 10 to 2.5% (Table Moreover, body weights of birds receiving IL2CM in ovo were numerically higher than both nonchallenged and challenged controls indicating no morbidity of remaining birds. Effects of in ovo IL2CM on hatchability were not evident. Hatchability is based on the number of chicks hatched from eggs which were alive at injection (Day 18 of incubation).

TABLE 1 In Ovo IL2CM Efficacy Against Oral E. coli Challenge of Day Old Broiler Chicks Day 5 Parameters In Ovo Challenged Hatchability Treatment Mortality Average Chicks Body Weight (n) (9) Saline No 92 2.5 92 Saline Yes 92 10.0 97 IL2CM Yes 90 2.5 99 These results indicate that a single in ovo administration of IL2CM to day 18 embryonated eggs protects newly hatched chicks against oral exposure to E.

coli. Protection was afforded immediately upon hatching.

EXAMPLE 6 Salmonella typhimurium Hatcher and Posthatch Seeder Transmission One hundred fifty (150) Day 18 embryonated broiler eggs were injected with either PBS or 0.35 activity units of IL2CM. Seventy-five (75) eggs from WO 94119944 I'CT/US94/O 1179 -17each treatment group were returned to a control incubator. Another 75 eggs per treatment group were placed in a hatcher contained in an isolation room.

Additionally, 170 CFU of Salmonella typhimurium were injected into the air space of 25 eggs. These eggs were placed in a hatcher tray at the top of the incubator and utilized only for horizontal transmission within the hatcher. This hatcher will heretofore be designated contaminated. Fifteen (15) minute aerosol plate counts taken within the contaminated hatcher on Day 20 of incubation averaged 169 CFU. Selective agar containing both novobiocin and nalidixic acid was utilized. Counts from plates positioned in each hatching basket were uniform. The test strain was not isolated from the control hatcher. At hatch 25 chicks per treatment group from both the control and the contaminated hatcher were placed in each of two floor pens. Commingling birds from the control hatcher with birds from the contaminated hatcher in a 1:1 ratio resulted in posthatch horizontal transmission from hatcher exposed to non-exposed chicks.

Chicks were given feed and water ad libium. Mortality was measured daily. At 12 days posthatch, birds were weighed and the study was terminated.

Results. Hatchability equivalent among both in ovo treatments and hatcher treatments.

Hatchability of eggs injected with Salmonella was 72%.

These chicks were not placed in the pens. In ovo IL2CM protected against horizontal Salmonella transmission both within the hatcher and posthatch. Twelve day mortality of chicks exposed to Salmonella within the hatcher was significantly reduced from 36 to 28% (Table 2).

Mortality of chicks commingled with birds contaminated within the hatcher was reduced from 20 to 12% (Table 2).

The numerically lower body weights of IL2CM treated birds suggests Salmonella colonization of the gastrointestinal tract is not affected but blood borne pathogenesis is reduced.

id -I WO 94/19944 I'CT/US94/01179 -18- TABLE 2 Efficacy of In Ovo IL2CM Administration Against Salmonella typhimurium In Ovo Bacterial Mortality Average Chicks Treatment Exposure Body Weight (n) (g) PBS Hatcher 36 222 IL2CM Hatcher 28 197 PBS Posthatch 20 222 IL2CM Posthatch 12 217 These results indicate that a single in ovo administration of IL2CM to day 18 embryonated eggs protected chicks from Salmonella infection pre-hatch and post-hatch.

EXAMPLE 7 Intramuscular E. coli Challenge on Day 18 Posthatch Day 18 embryonated broiler eggs were injected with either PBS or 0.35 activity units of IL2CM. At hatch, chicks were wingbanded by treatment and commingled within floor pens. On Day 18 posthatch 30 birds of uniform size per treatment group were weighed, injected into the breast muscle with 100 il PBS containing either 104 or 106 CFU E. coli. Additionally, 30 negative control birds treated in ovo with PBS were injected IM with 100 l PBS. Birds were returned to floor pens and daily mortality was monitored for seven days at which time birds were reweighed and the study terminated.

Results. Challenge was administered at day 18 posthatch, a time of depleted maternal antibody levels and inadequate natural antibody synthesis, and therefore a time of increased susceptibility to bacterial challenge. Birds challenged with 10,000 CFUs of E. coli exhib.ced little mortality with only two birds dying following challenge regardless of in ovo treatment (Table Although mortality was equivalent across in ovo

I

s- WO 94/19944 PCT/US94/01179 -19treatments, numerically increased body weights of IL2CM treated birds suggest less morbidity of birds remaining.

In contrast, birds challenged with 1,000,000 CFUs of E.

coli exhibited reduced mortality if IL2CM had been administered in ovo (Table Body weights of the remaining birds were numerically decreased.

TABLE 3 Efficacy of IL2CM In Ovo Against E. coli IM Challenge at Day 18 .0 In Ovo Challenged Mortality Average Birds Treatment Body Weight (n) (oz.) 106 Challenge: Saline No 0 31.4 Saline Yes 20.0 26.8 IL2CM Yes 6.7 25.9 104 Challenge: Saline No 0 28.9 Saline Yes 6.7 27.9 IL2CM Yes 6.7 28.7 !0 These results indicate that a single in ovo administration of IL2CM to day 18 embryonated eggs provided prolonged protection to chicks, protecting chicks at day 18 posthatch against intramuscular E. coli challenge.

The foregoing examples are illustrative of the present invention, and are not to be taken as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims (22)

1. A method of combating a bacterial infection in an immature bird at risk thereof, comprising administering to said bird Interleukin-2 (IL-2) in an effective bacterial combatting amount.

2. A method according to Claim 1, wherein said IL-2 is avian IL-2.

3. A method according to Claim 1, wherein said infection is a Salmonella infection.

4. A method according to Claim 1, wherein said infection is an Escherichia coli infection.

A method according to Claim 1, wherein said bird is selected from the group consisting of chickens, turkeys, ducks, geese, quail and pheasant.

6. A method according to Claim 1, wherein said bird is selected from the group consisting of chickens and turkeys.

7. A method according to Claim 1, wherein said administering step is carried out by means selected from the group consisting of in ovo injection, intravenous injection, intraperitoneal injection and subcutaneous injection.

8. A method according to Claim 1, wherein said bird is administered said IL-2 in ovo.

9. A method according to Claim 1, wherein said IL-2 is administered to said bird in ovo by injecting said IL-2 into an egg in which the bird is contained.

A method according to Claim 1, wherein said IL-2 is administered to said bird in ovo during about the last quarter of in ovo incubation.

11. A method according to Claim 1, wherein said bird is a chicken and said IL-2 is administered to said chicken in ovo on about the fifteenth to nineteenth day of incubation.

12. A method according to Claim 1, wherein said bird is a turkey and said IL-2 is administered to said turkey S'R sta keepspeci6100594 27.197 T- O LU .JT s~ sg 2 in ovo on about the twenty-first to twenty-sixth day of incubation.

13. A method according to Claim 1, wherein said administering step is carried out by administering said IL-2 into the region defined by the amnion, the yolk sac, or the air cell.

14. A method of combatting colisepticemia infections in a plurality of birds at risk thereof, comprising: administering IL-2 to each of said birds in ovo in an amount effective to combat colisepticemia; then incubating said birds to hatch together in a common incubator; and then raising each of said birds to at least two weeks of age.

15. A method according to Claim 14, wherein said IL-2 is avian IL-2.

16. A method according to Claim 14, wherein said Sbirds are selected from the group consisting of chickens, turkeys, ducks, geese, quail and pheasant.

17. A method according to Claim 14, wherein said birds are selected from the group consisting of chickens and turkeys.

18. A method according to Claim 14, wherein said IL-2 .is administered to said bird in ovo by injecting said IL-2 into an egg in which the bird is contained.

19. A method according to Claim 14, wherein said IL-2 is administered to said bird in ovo during about the last quarter of in ovo incubation.

A method according to Claim 14, wherein said bird is a chicken and said IL-2 is administered to said chicken in ovo on about the fifteenth to nineteenth day of incubation.

21. A method according to Claim 14, wherein said bird is a turkey and said IL-2 is administered to said turkey in ovo on about the twenty-first to twenty-sixth day of incubation. stafvukepspea6105 94 27.197 L 2*2-

22. A method according to Claim 14, wherein said administering step is carried out by administering said IL-2 into the region defined by the amnion, the yolk sac or the air cell. DATED this 5th day of December 1995 EMBREX, INC. By Their Patent Attorneys: GRIFFITH HACK CO Fellows Institute of Patent Attorneys of Australia 0 9 0 *0 0 0 000 0 0000 0 *000 0000 0 0000 000000 00 *0*0 00 0 0 *00 0 000* 00 Oi 0 00 0 00 00 000009 0 *0 0 0 0000 000 stat lalkeep/RETYPESG 100594 5 12 -1 4:N INTERNATIONAL SEARCH REPORTI If It, tiosial nplietion No,. PCTIUS94/Ol 179 A. CLASSIFICATION OF SUBJECT MATITER :Please See Extra Sheet. US CL :424/85.2; 514/2; 530/351 According to International Patent Classification (IPC) or to both national classification and IPC B. FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols) U.S. :424/85.2; 51412; 530/351 Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) APS, BIOSIS s-trrh terms: interleukin-2, avian, chicken#, antibiotic, bacterial infection, e. col, salmonella, colisepticemia C. DOCUMENTS CONSIDERED TO BE RELEVANT Category* Citation of document, with indication, where appropriate, of the relevant passages f Relevant to claim No. X US, A, 5,106,617 (FREDERICKSEN ET AL) 21 April 1992, 1-18 see entire document. Further documents are listed in the continuation of Box C. El See patent family annex. Specia categories of cited documents: r7 later document publishedl after the internationa fiing date or priority dtie and not in conflict with the application butcited to underst"n the doctsmenttdefin the general state of the int which is not considered principle or theory unterlying the invention to be pant of paticular relevance; arler ocuentpubishd o oraftr te iteratinalfilng ale X. document of partcular relevance: the claime invention cannot be arler ocuentpubishd o or fte th inerntioal ingdamconsidered novel or cannot be considered to involve an inventive step .I document which may tlu-, doubts on priority clatim(s) or which is when the document is taken alone cited to establish publication date of another citation or other dcmnofpriuarevne-:i liednetoniik b specal easn (a spcifed)considered to involve an inventive step when the document is document referring to an oral disclosure, use. exhibition or other combined with one or more other such documents, such combinatico mans being obvious to a person skilled in the PAt TP document pubtliuhed prior to the international filing date hut later than document member of the same patent fxmily the priority date claimed Date of the actual completion of the international search FDate of mailin of the international search report 17 March 1994 MAO 919 94 Name and mailing address of the ISA/US IAuthorized officer Commissioner of Patents and Trademarks I36v Box PCT HYSU KIM) Washington, D.C. 20231 HOU Facsimile No. (703) 305-=320 Teephone No. (703 308-0196 Form PCT/15A1210 (second sheet)(July 1992)* INT1ERN ATIONAL SE~ARCH R EPORT' In nutonal aipplication No. PCT/US94/O1 179 A. CLASSIFICATION OF SUBJECT MATTER: IPC A01N 37118; A61K 37/00, 45/05; C07K 3/00, 13/00, 15100, 17/00 Form PCTII'SAJ21O (extra sh=e)(July 1992)*