CA2156090A1 - Method of treating immature birds with il-2 - Google Patents

Abstract

A method of combatting bacterial infections, including Escherichia coli and Salmonella infections, in immature birds by adminstering to said immature bird a T-cell growth factor comprising Interleuikin-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.

Description

~WO 94/~g~ 21~ ~ ~ g ~ PCT~S94/01179 Field of the Invention This invention relates to the treatment of birds by the a~r;n;ctration of Interleukin-2 to combat bacterial infections.
Backqround 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 reduced 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 W094/1g9~ 215 6 0 9 ~ PCT~S94/01179 ~

in 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.
~umm~ry 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 immature 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 a~m; n; ~tration 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 ~WO g411Y9~ 21 5609 0 PC~S94l0ll79 been 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 Invention 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 (IL-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, turkeys, ducks, geese, quail, and pheasant. Various species of avian IL-2 are known. See. e.q., U.S.
Patents Nos. 5,028,421 and 5,106,617. In brief, avian 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) cont~; n; ng 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 W094/l9g~ 21 5 ~ n g ~ PCT~S94/~1179 administer 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 cross-reactivity of IL-2 of various avian species can be routinely determined with known bioassay procedures employing IL-2 responder cells, see e.q., Schnetzler et al., supra at 561, or can simply be screened by administering to a sample group of birds n 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 a~r;ni~tered 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 cross-reactivities of IL-2 and simple screening tests known to those skilled in the art. The quantity of IL-2 a~ministered 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 3OK species. See Fredericksen and Sharma, supra; Schnetzler, suPra at 565. The 30K

~0 ~4119~ ~9~ PCT~S~4/01179 species is believed to be the native form. A 3OK 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 performance 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 A~-; n; stered 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 a~r;~i~tered 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 ~m; n;~tered contains avian IL-2 in an amount of from about .0035 activity units per egg. More preferably, the IL-2 cond tioned 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 non-avian species, it is expected that synthetic analogs of avian IL-2, when available, can be screened for wo 94,lgg~ ~G~ PCT~S94/01179 ~
' .

activity 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, Pseudom~n~c species, and Salmonella species.
3 0 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 09~9~ ~ PCT~S94/01179 able 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 longit-1~;n~1 axis. A vaccine may be administered by the same means and to the same location as the IL-2.
The me~hAn;sm 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 WO94/199~ 2~ ] ~ 9~0 PCT~S94/01179 ~
., ., , ~ . , ~

chick 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 bacteria-impermeable sealing material such as wax or the like toprevent 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 contA;n;ng 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.

094/199~ 21 ~ PCT~S94/01179 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, ~ means micron, mm means millimeter, cm means centimeter, cc means cubic centimeter, ml means milliliter, ~l means microliter, g means gram, mg means milligram, ~g means mi~o~Lam, n means number, oz means ounce, M ~^~nC
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, CO2 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, Preparation of Spleen Cells Spleens were obtained from Hyline, SC
chickens between 4 and l0 weeks of age, and a single cell suspension prepared from decapsulated spleen.
Specifically, the spleen was placed in phosphate buffered saline (PBS) (pH =7.4), 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.

W094/1g9~ 2~Q~b PCT~S94/01179 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 milliliter)(Sigma, St.
Louis, M0).
Single-cell suspensions from spleen were placed into 75 square centimeter flasks with 50 milliliters of RPMI-G medium at a cell concentration of 6 x 1o6 cells per milliliter and a Concanavalin A (Sigma Chemical Co., St. Louis, M0) concentration of 4 micrograms per milliliter. IL-2 Conditioned Medium (IL2CM) was obtained from these cultures following incubation at 40 C in a humidified atmosphere with 5%
C02 for 64-96 hours, with peak production being obtained after 96 hours.

IL-2 Assay Avian IL-2 activity is defined by in vitro proliferative responses of T-cells which have been pre-exposed to a mitogenic agent. The proliferative response is indicated by mitochondrial incorporation of a specific dye (MTT, or 3-t4,5-dimethylthiazolyl-2]-2,5-diphenyl tetrazolium bromide). An activity unit is defined as the amount of material which causes 50% or one-half ~ 1 proliferative response as measured in the following cell proliferation assay.
1. Preparation of lym~hocYte suspensions Chickens are sacrificed and excised spleens are placed in cold PBS containing gentamicin (2 ml at 25 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 50 ml with cold PBS.

-11- PCT~S94/01179 To remove cell stroma and excess tissue, the tissue slurry is allowed to settle for seven minutes;
35 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 rpm for five minutes to remove excess RBCs. The supernatant is then centrifuged at 1500 rpm for 15 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 ml 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 prepared using 12 ml of 60% Percoll in PBS as the bottom layer and 15 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 15C 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-750s RPMI 1640 W094/199~ 2~1S6~ PCT~S94/01179 without phenol red) is combined with 5ml lOOX L-Glutamine and lml Gentamicin (25 mg/ml).
Cells are counted at a dilution factor of 1:100 in 4% Trypan Blue (i.e., 450 ~1 Trypan Blue + 50 ~1 cells = 1:10; 50 ~1 of 1:10 + 450 ~1 Trypan Blue =
1:100). Cells are seeded at 15 x 106/cm2 in RPMI
culture medium. Free Con-A is added to stimulate lymphoblast transformation. Tissue culture grade Concanavalin-A (Sigma, Catalog Number C-5275), 2 ~g/ml of media is re~uired.
The cell suspensions are incubated for lymphoblast transformation by placing 100 ml of cell suspension in a flask. Each flask is gassed with 5% C02 in air for thirty seconds, and placed on its side in 5%
C02 at 37C. 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 (5) 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.

094/lgg~ t S~OD~ PCT~S94/01179 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 ~1 of PBS per well. The remaining wells are loaded with 100 ~1 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 lOOx/500 ml;
Gibco Cat. #321-5030A6); 2% H.I. Chicken Serum (Sigma, Cat. #C5405); 0.1 M Mannopyranoside (3.884 g/200 ml)(Siqma 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 ~1 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 #lo. After column #10 has been mixed 100 ~1 is discarded, leaving 100 ~1 in each of the wells. To each well is added 100 ~1 of cell suspension. Column #2 and Column ~11 are control wells (cells only without sample). Microtiter plates are placed into 5% C02, 37C
incubator for not less than 46 hours or more than 50 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 ~ sterile filter unit, and 25 ~1 is added to each well of each plate; plates are incubated in a 5~
C02, 37C incubator for 60 minutes. At the end of the 60 minute incubation, 150 ~1 is removed from each well and 100 ~1 of acid-isopropanol (3.3 ml 12M HCl 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.

WO94/199~ ,~215 ~ 9 ~ PCT~S94/01179 ~

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 OD 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) = ~ OD value % change = ~ OD x 100 x Control OD

The percent change for each log dilution is plotted. The intercept reference line at 50% of the max;~rll~ 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.
E)CAMPLE 4 ~aterials and Methods Eqq Injection and Husbandry. Fertile Hubbard X Hubbard broiler breeder eggs were obtained from a commercial hatchery and stored at 16C. 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 094/199~ S6090 PCT~S94/01179 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 20 Escherichia coli bacterial culture (serogroup O1) or a nalidixic novobiocin resistant strain of Salmonella ty~h;~rium were grown overnight at 37C in trypticase soy broth (TSB). Confluent cultures were diluted in O.9% saline as needed. Preparation of oral E. coli challenge inoculum doses required a lOX concentration by centrifugation. Pellets were resuspended in O.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.

Oral E. Col i Challen~e 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 lOO ~l PBS containing 8.8 X 1O8 CFU E. coli . An additional 40 chicks which received WO94/199~ ~ PCT~S94/01179 PBS in ovo were gavaged with l00 ~l PBS. Chicks were commingled within one floor pen and given feed and water ad l ibitum . Daily mortality was recorded for five consecutive days at which time all chicks were weighed and the study terminated.
Resul~s. In ovo IL2CM significantly reduced 5 day mortality of broiler chicks challenged orally with E. coli from l0 to 2.5% (Table l). 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).

In Ovo IL2CM Efficacy Against Oral E. coli Challenge of Day Old Broiler Chicks Daysp~,~",~
In Ovo ChallengedIlalcl, ' '~y 20T,t:dl",e"l % Mortality Average Chicks % BodyWeight (n) (g) Saline No 92 2.5 92 40 Saline Yes 92 10.0 97 40 IL2CM Yes 90 2.5 99 40 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.

Salmonella typhimurium Hatcher ana Posthatch Seeder ~ransmission 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 094/199~ 21 S6 PCT~S94/01179 each 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 ty~hi~l~rium 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 cont~m;n~ted. 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 was 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.

WO94/199~ ~ ~S~`~9~ PCT~S94101179 Efficacy of In Ovo IL2CM Administration Against Sal monel l a typh i ml ~ ri um In Ovo Bacterial Mortality Average Chicks T~ ,.. el~l Exposure % Body Weight (n) (9) PBS Hatcher 36 222 50 IL 2CM Hatcher 28 197 50 PBS Posl~ 20 222 50 IL2CM Po~l,~ 12 217 50 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.

Intramuscular ~. col i Challenqe 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 l00 ~l PBS containing either 104 or 106 CFU E. coli. Additionally, 30 negative control birds treated in ovo with PBS were injected IM with l00 ~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 l0,000 CFUs of E. coli exhibited little mortality with only two birds dying following challenge regardless of in ovo treatment (Table 3). Although mortality was equivalent across in ovo ~WO 94/lgg44 21S~'0 PCT/US94/01179 treatments, 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.
col i exhibited reduced mortality if IL2CM had been administered in ovo (Table 3). Body weights of the remaining birds were numerically decreased.

Efficacy of IL2CM In Ovo Against E. col i IM Challenge at Day 18 In Ovo Ch- - lged Mortali~y Average Birds Treal",enl (%) Body Weight (n) (oz.) 106 Challenge:
Saline No o 31.4 30 Saline Yes 20.0 26.8 30 15IL2CM Yes 6.7 25.9 30 104 Challenge:
Saline No o 28.9 30 Saline Yes 6.7 27.9 30 IL2CM Yes 6.7 28.7 30 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 (10)

THAT WHICH IS CLAIMED IS:

1. The use of avian Interleukin- 2 (IL-2) for the preparation of a medicament for inhibiting a bacterial infection in a non-adult bird by administering to said bird avian IL-2 in an effective bacterial inhibiting amount, wherein said bacterial infection is selected from the group consisting of Escherichia coli infections and Salmonella infections.

2. The use according to claim 1, wherein said IL-2 is chicken IL-2.

3. The use according to claim 1, wherein said avian IL-2 is 30K chicken IL-2.

4. The use according to Claim 1, wherein said bird is administered said avian IL-2 in ovo.

5. The use according to Claim 1, wherein said avian IL-2 is administered to said bird in ovo during about the last quarter of in ovo incubation.

6. The use of avian Interleukin-2 (IL-2) for the preparation of a medicament for inhibiting an Escherichia coli infection in a non-adult bird by administering to said bird avian IL-2 in an effective Escherichia coli inhibiting amount.

7. The use according to claim 6, wherein said avian IL-2 is chicken IL-2.

8. The use according to claim 6, wherein said avian IL-2 is 30K chicken IL-2.

9. The use according to claim 6, wherein said bird is administered said avian IL-2 in ovo.

10. The use according to Claim 6, wherein said avian IL-2 is administered to said bird in ovo during about the last quarter of in ovo incubation.