EP0777733A1 - Method of treating birds with avian myelomonocytic growth factor - Google Patents

Abstract

A method of treating birds by administering to birds in ovo, avian myelomonocytic growth factor is disclosed. The method is preferably carried out on chickens with chicken MGF on about the eighteenth day of incubation.

Description

METHOD OF TREATING BIRDS WITH AVIAN MYELOMONOCYTIC GROWTH FACTOR

Related Applications This application is a continuation-in-part application of U.S. Serial No. 08/292,854 filed August 19, 1994 for "Method of Treating Birds with Avian Myelomonocytic Growth Factor" by Paul Johnson, et al.

Field of the Invention The present invention relates to the treatment of birds by the in ovo administration of an avian myelomonocytic growth factor such as chicken myelomonocytic growth factor (cMGF) .

Background of the Invention Chicken myelomonocytic growth factor (cMGF) is an avian hematopoietic cytokine which stimulates bone marrow cells to proliferate and produce cells of the monocyte/macrophage lineage. cMGF was originally identified in lectin stimulated spleen cultures and purified from conditioned media produced by an LPS stimulated chicken macrophage cell line (HD11) by Leutz et al., EMBO J. 3, 3191-3197 (1984) . cMGF is a glycoprotein which stimulates the growth of virally transformed chicken myeloid cell lines and formation of macrophage and granulocyte colonies in normal bone marrow cultures in vi tro (Leutz et al . , supra) . cMGF has been cloned by Leutz et al. , EMBO J 8, 175-181 (1989) , but sufficient pure cMGF, either native or recombinant, has not been available to characterize possible uses thereof.

U.S. Patent No. 5,028,421 to Fredericksen discloses a method for increasing the weight of treated birds after hatch by introducing a T-cell growth factor into eggs on about the eighteenth day of incubation.

U.S. Patent Application Serial No. 07/947,035, filed 17

September 1992, discloses a method of enhancing the growth of treated birds by introducing an insulin-like growth factor into eggs. Neither disclose nor suggest the use of a myelomonocytic growth factor to treat birds.

Summary of the Invention A method of treating birds is disclosed herein. The method carried out by administering to a bird in ovo a biologically active amount of avian myelomonocytic growth factor (MGF) .

A second aspect of the present invention is a method of making purified recombinant avian myelomonocytic growth factor. The method includes culturing host cells (e.g., Escherichia coli , Pichia pastoris , and the like) which contain and express a recombinant DNA construct encoding MGF in a culture media, collecting culture media containing recombinant MGF from the host cells, and isolating the MGF from the culture media.

A third aspect of the present invention is a method of enhancing the growth of birds. This method is carried out by administering avian MGF to a bird in ovo in an amount effective to enhance the growth of the bird after hatch.

A fourth aspect of the present invention is a method of enhancing bone marrow proliferation in birds.

The method is carried out by administering avian MGF to a bird in ovo in an amount effective to enhance bone marrow proliferation in the bird. A fifth aspect of the present invention is a method of inhibiting the progression of a bacterial infection such as an Escheri chia coli infection or Salmonella infection in a bird. The method comprises administering to said bird in ovo a avian myelomonocytic growth factor (MGF) in an amount effective to inhibit the progression of the bacterial infection.

A sixth aspect of the present invention is a method of inhibiting the progression of a viral, fungal, or protozoal infection in a bird by administering to the bird in ovo a avian myelomonocytic growth factor (MGF) in an amount effective to inhibit the progression of the viral, fungal, or protozoal infection.

The foregoing and other objects and aspects of the present invention are explained in detail in the specification set forth below.

Brief Description of the Drawings Figure 1 is an illustration of Coomassie blue stained proteins of IPTG-induced or non-induced transformed BL21(DE3) Escherichia coli lysates separated and analyzed by 12% SDS-PAGE.

Figure 2 is a graphical illustration of bone marrow proliferation induced by cMGF eluted from non- reducing SDS-PAGE gels. Figure 3 is a graphical illustration of bone marrow proliferation induced by the growth media of pPIC9-cMGF and pHIL-SI-cMGF transformed Pichia pastoris versus the GS115/His*Mut^~ albumin secreting stain.

Figure 4 is a graphical illustration of Fc- receptor mediated phagocytosis of sheep red blood cells opsonized with rabbit anti-SRBC IgG by bone marrow cells cultured +/-20 μg/ml cMGF.

Figure 5 is a graphical illustration of IS- induced nitrite production by bone marrow cells cultured +/-20 g/ml cMGF. Figure 6 is a graphical illustration of Phorbol ester induced superoxide production by bone marrow cells cultured +/-20 μg/ml cMGF.

Figure 7 is a graphical illustration of Fc- receptor phagocytosis of sheep red blood cells opsonized with rabbit anti-SRBC IgG by bone marrow cells cultured for 48 Hours with the indicated doses of cMGF.

Figure 8 is a graphical illustration of IS (PA-14A, 2.5 ug/ml) induced nitrite production by bone marrow cells cultured for 48 hours with the indicated doses of cMGF.

Figure 9 is a graphical illustration of PMA- inducible superoxide production by adherence purified peripheral blood mononuclear leukocytes isolated from the pooled blood of chicks bled on day 4 post hatch of day 18 injected eggs receiving 2.5 ug/egg yeast expressed albumin or 2.5, 0.25 and 0.025 ug/egg Pichia pas tor is expressed cMGF.

Figure 10 is a graphical illustration of total pooled bone marrow cell counts obtained from 12 femurs removed from day old chicks hatched from egg injected with 0.1 U of IS, 0.25 ug of yeast expressed albumin, and either 0.25ug or 0.05ug of cMGF.

Figure 11 is a graphical illustration of Phorbol ester induced superoxide production by adherence purified bone marrow cells obtained from 12 femurs removed from day old chicks hatched from egg injected with 0.1 U of IS, 0.25 ug of yeast expressed albumin, and either 0.25ug or 0.05ug of cMGF.

Detailed Description of the Invention

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 eggs are preferred. Eggs treated by the method of the present invention are fertile eggs. The eggs may be treated at any point during incubation, although it is preferable to treat the eggs in the fourth quarter of incubation, and most preferably to treat the eggs on about the eighteenth day of incubation (i.e., the eighteenth day of embryonic development) .

The term "avian MGF, " as used MGF corresponding to MGF 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 MGF are known. This term is also intended to include active fragments and analogs thereof. The MGF 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 administration of MGF in ovo provides a variety of useful results. For example, the administration of MGF in ovo may be used to enhance growth of the hatched chick. In addition, the administration of MGF in ovo may be used to enhance the proliferation of bone marrow cells, resulting in a more fully developed immune system at an earlier stage ( i . e . , accelerate the onset of immune competence in the bird) .

The administration of MGF in ovo provides, as noted above, a method of inhibiting the progression of bacterial infections in a bird, such as Escherichia coli infections (i.e., avian colisepticemia) or Salmonella infections. The administration of MGF in ovo provides, as also noted above, a method of inhibiting the progression of viral infections in a bird (e.g., infectious bursal disease virus infections, Newcastle's disease virus, Marek's disease virus, etc.) , of inhibiting the progression of fungal infections, and of inhibiting the progression of protozoal infections { e . g. , Eimeria species such as Eimeria tenella in avian coccidiosis) . The MGF may be administered concurrently with a vaccine ( e . g. , a live vaccine or a nonreplicating immunogen) effective for protecting the bird against the aforesaid infection.

MGF may be administered to eggs by any means which transports the compound through the shell. The preferred method of administration is, however, by injection. MGF may be injected into the egg at any site. Preferably, the site of injection is 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 .

Dosages of MGF used to carry out the methods described herein are not critical and can be determined in a routine manner. In general, the dosages will vary with the species of bird being treated, the time and site of administration, and the desired effect. The upper limit of the dosage can be routinely determined, but in general will be as much as 10 or 100 μg per subject ( i . e . , per in ovo injection; per egg) or more. The lower limit of the dosage likewise can be routinely determined, but can be as little as 1000 or 1 ng per subject or less.

The mechanism of injection is also not critical. Preferably, the method employed does 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. 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 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 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. Patents Nos. 4,903,635 and 4,681,063 to Hebrank, U.S. Patent No. 5,056,464 to Lewis, and U.S. Patent Nos. 4,040,388, 4,469,047, and 4,593,646 to Miller, the disclosures of which are incorporated by reference herein in their entirety. All such devices, as adapted for practicing the present invention, include an injector containing avian MGF as described herein, with the injector positioned to inject an egg carried by the apparatus with the avian MGF. 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 in their entirety. 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.

Chicken myelomonocytic growth factor (cMGF) and DNA encoding the same is known (Leutz et al . , EMBO J. 3, 3191-3197 (1984) ; Leutz et al . , EMBO J 8, 175-181 (1989) ) . The MGF proteins used to carry out the present invention may accordingly be made with techniques known in the art, or by variations thereof which will be readily apparent to those skilled in the art. The production of recombinant DNA, vectors, host cells, and proteins by genetic engineering techniques is well known. See, e . g. , U.S. Patent No. 4,761,371 to Bell et al . at Col. 6 line 3 to Col. 9 line 65; U.S. Patent No. 4,877,729 to Clark et al . at Col. 4 line 38 to Col. 7 line 6; U.S. Patent No. 4,912,038 to Schilling at Col. 3 line 26 to Col. 14 line 12; and U.S. Patent No. 4,879,224 to Wallner at Col. 6 line 8 to Col. 8 line 59.

DNA sequences encoding MGF proteins may be recovered by use of the polymerase chain reaction (PCR) procedure and splicing by overlap extension (SOE) , as is known in the art. See U.S. Patents Nos. 4,683,195 to Mullis et al. and 4,683,202 to Mullis.

The MGF proteins may be synthesized in host cells transformed with vectors containing DNA encoding the MGF proteins. A vector is a replicable DNA construct. Vectors are used herein either to amplify DNA encoding the MGF protein and/or to express DNA which encodes the MGF protein. An expression vector is a replicable DNA construct in which a DNA sequence encoding the MGF protein is operably linked to suitable control sequences capable of effecting the expression of the MGF protein in a suitable host. The need for such control sequences will vary depending upon the host selected and the transformation method chosen. Generally, control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation. Amplification vectors do not require expression control domains. All that is needed is the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants.

Vectors useful for practicing the present invention include plasmids, viruses (including phage) , retroviruses, and integratable DNA fragments (i.e., fragments integratable into the host genome by homologous recombination) . The vector replicates and functions independently of the host genome, or may, in some instances, integrate into the genome itself. Suitable vectors will contain replicon and control sequences which are derived from species compatible with the intended expression host. Transformed host cells are cells which have been transformed or transfected with the MGF protein vectors constructed using recombinant DNA techniques. Transformed host cells ordinarily express the MGF protein, but host cells transformed for purposes of cloning or amplifying the MGF protein DNA need not express the MGF protein.

DNA regions are operably linked when they are functionally related to each other. For example: a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. Generally, operably linked means contiguous and, in the case of leader sequences, contiguous and in reading frame.

Suitable host cells include prokaryotes, yeast cells or higher eukaryotic cells. Prokaryotes include gram negative or gram positive organisms, for example Escherichia coli {E. coli ) or Bacilli. Higher eukaryotic cells include established cell lines of mammalian origin as described below. Exemplary host cells are E. coli W3110 (ATCC 27,325) , E. coli B, E. coli X1776 (ATCC 31,537) , and E. coli 294 (ATCC 31,446) . Pseudomonas species, Bacillus species, and Serra tia marcesans are also suitable. A broad variety of suitable microbial vectors are available. Generally, a microbial vector will contain an origin of replication recognized by the intended host, a promoter which will function in the host and a phenotypic selection gene such as a gene encoding proteins conferring antibiotic resistance or supplying an auxotrophic requirement. Similar constructs will be manufactured for other hosts. E. coli is typically transformed using pBR322. See Bolivar et al . , Gene 2 , 95

(1977) . pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells.

Expression vectors should contain a promoter which is recognized by the host organism. This generally means a promoter obtained from the intended host. Promoters most commonly used in recombinant microbial expression vectors include the beta-lactamase

(penicillinase) and lactose promoter systems (Chang et al . , Nature 275, 615 (1978) ; and Goeddel et al . , Nature

281, 544 (1979) ) , a tryptophan (trp) promoter system (Goeddel et al . , Nucleic Acids Res . 8, 4057 (1980) and EPO App. Publ . No. 36,776) and the tac promoter (H. De Boer et al . , Proc . Na tl . Acad . Sci . USA 80, 21 (1983)) . While these are commonly used, other microbial promoters are suitable. Details concerning nucleotide sequences of many have been published, enabling a skilled worker to operably ligate them to DNA encoding the MGF protein in plasmid or viral vectors (Siebenlist et al . , Cell 20, 269

(1980) ) . The promoter and Shine-Dalgarno sequence (for prokaryotic host expression) are operably linked to the DNA encoding the MGF protein, i.e., they are positioned so as to promote transcription of the MGF protein messenger RNA from the DNA. Eukaryotic microbes such as yeast cultures may be transformed with suitable MGF protein-encoding vectors. See, e . g. , U.S. Patent No. 4,745,057. Saccharomyces cerevisiae is the most commonly used among lower eukaryotic host microorganisms, although a number of other strains are commonly available. Yeast vectors may contain an origin of replication from the 2 micron yeast plasmid or an autonomously replicating sequence (ARS) , a promoter, DNA encoding the MGF protein, sequences for polyadenylation and transcription termination, and a selection gene. An exemplary plasmid is YRp7, (Stinchcomb et al . , Na ture 282, 39 (1979) ; Kingsman et al . , Gene 7, 141 (1979) ; Tschemper et al . , Gene 10, 157 (1980)) . This plasmid contains the trpl gene, which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics 85, 12 (1977)) . The presence of the trpl lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Suitable promoting sequences in yeast vectors include the promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al . , J. Biol . Chem. 255, 2073 (1980) or other glycolytic enzymes (Hess et al . , J. Adv. Enzyme Reg. 7, 149 (1968) ; and Holland et al . , Biochemistry 17, 4900 (1978)) , such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase , phosphofructokinase , glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Suitable vectors and promoters for use in yeast expression are further described in R. Hitzeman et al . , EPO Publn. No. 73,657. A particularly preferred yeast host cell is

Pichia pastoris, which is described along with suitable transformation vectors in U.S. Patents Nos. 4,683,293; 4,808,537; 4,812,405; 4,818,700; 4,837,148 4,855,231 4,857,467; 4,879,231; 4,882,279; 4,885,242 4,895, 800 4,929,555; 5,002,876; 5,004,688; 5,032,516 5,122,465 5,135,868; and 5,166,329, the disclosures of which applicant specifically intends to be incorporated herein by reference.

Cultures of cells derived from multicellular organisms are a desirable host for recombinant MGF protein synthesis. In principal, any higher eukaryotic cell culture is workable, whether from vertebrate or invertebrate culture, including insect cells. However, mammalian cells are preferred, as illustrated in the Examples. Propagation of such cells in cell culture has become a routine procedure. See Tissue Culture, Academic Press, Kruse and Patterson, editors (1973) . Examples of useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and WI138, BHK, COS-7, CV, and MDCK cell lines. Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located upstream from the gene to be expressed, along with a ribosome binding site, RNA splice site (if intron-containing genomic DNA is used) , a polyadenylation site, and a transcriptional termination sequence. The transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells are often provided by viral sources. For example, commonly used promoters are derived from polyoma, Adenovirus 2, and Simian Virus 40 (SV40) . See, e . g. , U.S. Patent No. 4,599,308. The early and late promoters are useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication. See Fiers et al. , Na ture 273, 113 (1978) . The vaccinia virus may be used as a vector, as described in the Examples. Further, the MGF protein promoter, control and/or signal sequences, ay also be used, provided such control sequences are compatible with the host cell chosen.

An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral source (e.g. Polyoma, Adenovirus, VSV, or BPV) , or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter may be sufficient. Rather than using vectors which contain viral origins of replication, one can transform mammalian cells by the method of cotransformation with a selectable marker and the MGF protein DNA. An example of a suitable selectable marker is dihydrofolate reductase (DHFR) or thymidine kinase. See U.S. Pat. No. 4,399,216. Such markers are proteins, generally enzymes, that enable the identification of transformant cells, i.e., cells which are competent to take up exogenous DNA. Generally, identification is by survival of transformants in culture medium that is toxic, or from which the cells cannot obtain critical nutrition without having taken up the marker protein.

Host cells such as insect cells (e.g., cultured Spodoptera frugiperda cells) and expression vectors such as the baculovirus expression vector (e.g., vectors derived from Autogrrapha calif ornica MNPV, Trichoplusia ni MNPV, Rachiplusia ou MNPV, or Galleria ou MNPV) may be employed in carrying out the present invention, as described in U.S. Patents Nos. 4,745,051 and 4,879,236 to Smith et al . In general, a baculovirus expression vector comprises a baculovirus genome containing the gene to be expressed inserted into the polyhedron gene at a position ranging from the polyhedron transcriptional start signal to the ATG start site and under the transcriptional control of a baculovirus polyhedron promoter.

The following examples are provided to illustrate the present invention, and should not be construed as limiting thereof. In these examples, bp means base pairs, hr means hour, kDa mean kilodaltons, M means molar, μg means micrograms, ml means milliliters, SDS means sodium dodecyl sulfate, IPTG means Isopropyl-β- D-thiopyranoside, RPMI means Rosewell Park Memorial Institute, FBS means fetal bovine serum, HPLC means high pressure liquid chromatography, RP-HPLC means reverse phase high pressure liquid chromatography, ng means nanograms, YEA means yeast expressed albumin, U means units, IM means intramuscular, μL means microliters, mg means milligrams, cfu means colony forming units and °F means degrees Fahrenheit .

EXAMPLE 1 Cloning of Chicken Myelomonocytic Growth Factor This example describes the isolation of the DNA encoding cMGF described in Leutz et al . , EMBO J. 8, 175- 181 (1989) , except that a C was found at nucleotide 114 rather than a T as reported by Leutz.

Total RNA was prepared from guanadinium isothiocyanate lysed Concanavalin A (Con A) -stimulated splenocytes which had been isolated from 6 week leghorns . Reverse transcription and polymerase chain reaction (PCR) amplification of the thus prepared total RNA with cMGF- specific oligonucleotide primers gave a band of the expected size of -560 bp and a larger -800 bp band. Both bands were specific to splenocytes stimulated with Con A for 48 hrs and were not amplified in the controls or in splenocytes stimulated for only 24 hrs. A second round of PCR amplification of the 560 bp band using an internal cMGF primer 4 with cMGF primer 6 gave a product of the expected size. The 560 bp band was subcloned into the Invitrogen pCRII vector. DNA sequence analysis of three clones containing the 560 bp PCR product confirmed that the authentic cMGF cDNA had been cloned. The sequence varies from the published sequence by one base pair (the -_> C transition noted above) , which does not result in a change in the amino acid level, indicating that it is probably a naturally occurring genetic polymorphism among the chicken lines.

EXAMPLE 2 Expression of Chicken MGF in Escherichia coli

The 560 base pair PCR product encoding the cDNA for the mature cMGF protein was subcloned in the Escherichia coli (E. coli) expression vectors pET3A, B & C. Isopropyl-β-D-thiopyranoside (IPTG) was added to a growing culture of the BL21(DE3) E. coli host strain, to induce T7 polymerase, which in turn transcribes the target DNA in the plasmid. None of the cMGF recombinants cloned into pET3A, B & C using the Ndel and BamHI sites exhibited any IPTG inducible cMGF expression. Use of the BamHI site allows the translation of the cMGF protein as a fusion with the first 11 amino acids of the gene-10 protein, which may translate more efficiently and confer stability on the expressed protein. The cMGF clone was reamplified with cMGF primers 1+6 to engineer the BamHI sites and subclone it into pCRII. cMGF cDNA was subcloned into the BamHI site into the pET3A vector. Recombinants, in the correct orientation, were then identified by Aval digestion. An IPTG inducible band ~ 21 kDa in BL21(DE3) cells transformed with the pET3A- cMGF-BamHI plasmid was observed. Log phase cultures of the three clones were induced by the addition of IPTG and grown for 3 hrs under conditions previously found to be suitable for expression of the positive control protein gCap41 cloned into the BamHI site of the pET3A vector. The E. coli were pelleted, solubilized in SDS sample buffer and the proteins were analyzed by SDS-PAGE (Figure 1) .

During in vi tro translation of mRNA derived from the complete cMGF coding sequence, a precursor of 26.5 kDa is synthesized whose 23 amino acid signal sequence is cleaved off and, in the presence of membranes, cMGF becomes glycosylated at two N- glycosylation sites to generate a 28 kDa mature cMGF form. The calculated molecular weight of the mature cMGF protein is ~ 20.014 kDa, which is about 4 kDa smaller than that determined by SDS-PAGE.

The cells were broken open by sonication or mild detergent lysis with deoxycholate, followed by centrifugation to separate soluble and insoluble fractions. Samples were then analyzed by SDS-PAGE to determine whether the putative cMGF protein is expressed in the soluble or insoluble fraction. The results from both cell lysis methods indicated that cMGF is preferentially contained in the insoluble fraction, presumably in the inclusion bodies. Natural cMGF and the rcMGF-trpE eluted from non-reducing SDS-PAGE gels stimulate growth of hematopoietic colonies in chick bone marrow cells, indicating that biological activity is not destroyed by SDS but was found to be sensitive to exposure to reducing reagents. rcMGF-glO targeted to inclusion bodies was recovered by washing inclusion bodies with 50% glycerol and solubilizing the inclusion bodies in 5% SDS. Several methods were attempted to refold putative cMGF-glO protein after removing SDS and contaminating bacterial LPS by HPLC and DETOXI-GEL™ columns from Pierce

(Rockford, Illinois, USA) . The existence of a 21 kDa putative cMGF band was confirmed by SDS-PAGE gels for each procedure, but none of the samples exhibited activity in bone marrow proliferation assays. Elution of the 21 kDa putative cMGF band from non-reducing SDS-PAGE gels, by overnight incubation of 2- 3 mm² gel slices in RPMI 1640 media + gentamicin, however, did demonstrate activity in the bone marrow proliferation assay while the 41 kDa gCAP band eluted from the same gel did not. See Figure 2. 0.1% Triton X-100 washed inclusion bodies containing the 21 kDa cMGF protein were solubilized in 5% SDS, 8 M urea and 6 M guanidinium-HCl and analyzed by RP-HPLC. Identical chromatography profiles were obtained for all solubilization methods and the 21 kDa cMGF protein was identified by SDS-PAGE. The dried RP-HPLC purified cMGF protein was solubilized by different techniques including SDS, Triton X-100, dipotassium phosphate and octyl-β-glucoside . Detergent was determined to be the most effective method of solubilization. Size exclusion analysis of solubilized cMGF-inclusion bodies indicated that the protein was eluting as large aggregates, but that the addition of 10% ethanol to the mobile phase of the column significantly changed the elution profile of the cMGF to that more approximating a monomer.

EXAMPLE 3 Expression of Chicken MGF in Pichia pastoris

Pichia pastoris is methylotropic, and therefore capable of metabolizing methanol as a sole carbon source, and producing protein products with glycosylation similar to mammalian systems. Plasmids are available which direct protein products to be intracellular or secreted. Heterologous protein expression in this system is tightly regulated and induced to very high levels by the addition of methanol to the growth media. The Pichia pastoris expression kit available from INVITROGEN^® was used (telephone number 602-748-4400) .

The cMGF clone was reamplfied with primers 7+6 to engineer in BamHI and EcoRI sites, and subcloned into two Pichia expression vectors, pPIC9 and pHIL-Sl both of which produce secreted fusion protein products with signal peptides from the α-mating factor or acid phosphatase genes respectively. Initially the PCR product was subcloned into the pCR™II vector. Digestion with EcoRI/BamHI or EcoRI alone resulted in the correct size inserts. The cMGF inserts were isolated from a low melting point agarose gel using the phenol/chloroform method and ligated into the EcoRI site of pPIC9 vector or the EcoRI/BamHI sites of pHIL-SI. The ligation was transformed into DH5αf' and plated onto selective media. Potential cMGF subclones were verified by restriction digests of the DNA. One clone of each type was selected for further analysis. The correct orientation of cMGF insert in the pPIC9-cMGF and the pHIL-SI-cMGF subclones were checked by digestion with the enzymes Aval and Bgl II. DNA sequencing of the 5' end of the clones confirmed that the clones were subcloned correctly and the cMGF cDNA is in-frame with the Pichia vector signal sequences for secretion.

Spheroplasts were prepared from the GS115 (His-) strain by treatment with zymolase. The Pichia spheroplasts were transformed with Bgll l digests of cMGF subcloned into pPIC9 and pHILSl . The transformed yeast were plated onto selective media along with positive controls for transformation and secretion provided by Invitrogen. The Pichia GS115 (His-) strain had a defect in the histidinal dehydrogenase activity coded for the gene HIS4. This defect enabled the strain to grow on complex media or minimal media supplemented with histidine. When GS115 (His-) is transformed with plasmids containing the HIS4 gene, the transformants are selected on their ability to grow on minimal media not supplemented with histidine. Transformants were then plated on selective media that allowed the identification of recombinants that have stably integrated the cMGF gene into the yeast genome.

Seven potential cMGF recombinant clones were selected from the transformation with the pHILSl-cMGF and pPIC9-cMGF vectors. These clones were grown in liquid culture in the presence of methanol to induce expression and secretion of the recombinant protein into the media. The culture media supernatant from each recombinant clone was collected and analyzed by SDS-PAGE. The results revealed five potential cMGF clones expressing two predominant proteins of -25 and 29 kDa, the size of the glycosylated forms of native cMGF.

The five potential positive samples were tested for in vi tro activity in the bone marrow proliferation assay and were found to be biologically active. Protein expression was scaled-up to a 40 mL, 2 day-induced culture with four of the five previously identified cMGF- expressing clones and the albumin-expressing transformant included as a negative control. Since the yeast methanol growth media inhibited bone marrow cell proliferation, the samples were processed using 10 k and 100 k CENTRIPREP™ ultrafilters from AMICON™ to exchange the yeast media with RPMI media and concentrate the samples. The results of the bone marrow proliferation assay are reported in Figure 3.

All of the cMGF transformed yeast samples exhibited activity in the bone marrow proliferation assay while the albumin control exhibited no proliferative response. The four clones which had shown activity after the first 2 day induction period, were induced for an additional 2 days and the supernatants were processed by ultrafiltration, as described above. No difference in activity was observed for samples induced for the first 2 day period compared to those induced between 2 and 4 days. In addition, the 100 k retentate and 10 k filtrate fractions were analyzed by SDS-PAGE and in the bone marrow proliferation assay. To recover biologically active secreted cMGF from the transformed yeast growth media, the buffer exchange and concentration procedure were modified, eliminating the 100 k filter and utilizing 10% ethanol in the early wash steps. This new procedure produced material containing the 25 and 29 kDa putative cMGF bands when analyzed by SDS-PAGE, and can be resolved on RP-HPLC. One of the fractions collected after RP-HPLC was active in the bone marrow proliferation assay and contained the two cMGF bands. In the Pichia expression system, multiple copies of the gene integrated into the yeast chromosome often results in higher levels of protein expression. Therefore, the Southern blotting technique was used to determine the cMGF gene copy number in each positive clone. The results indicated that all of the cMGF expressing clones contained one copy of the gene cassette. Focus was also directed towards optimizing cell culture conditions to increase production of the cMGF product and one of the pPIC9-cMGF expressing clones was chosen for scale up to a 40.0 mL culture. Gentamicin was added to prevent bacterial contamination. The results obtained appeared to indicate that a 2-day methanol induction was optimal.

EXAMPLE 4 cMGF Induced Enhancement of Macrophage Functional Responses in Cultured Bone Marrow Cells

Chicken bone marrow cells were cultured in RPMI 1640 media + 5% FBS ± 20 μg/ml cMGF and assayed for their ability to perform Fc-receptor mediated phagocytosis, IS- induced nitrite production and phorbol ester induced superoxide production at time 0 and after 1, 2, 3, and 4 days in culture. The results are reported in Figures 4- 6. The acquisition of Fc-receptor mediated phagocytosis and IS-induced nitrite production are significantly accelerated and enhanced by the presence of cMGF in the culture media. See Figures 4 and 5. However, phorbol ester induced superoxide production is unaffected by the presence of cMGF. See Figure 6. These results indicate that pichia pastoris produced recombinant cMGF stimulates both the proliferation of bone marrow cells and the acquisition of macrophage functional responses. EXAMPLE 5 Recovery of cMGF for In vivo and In ovo Use

To recover biologically active Pichia pastoris expressed cMGF from spent transformed yeast growth media, samples were processed using CENTRIPREP™ 10K filters (Amicon) to exchange the yeast media and concentrate the proteins therein. Yeast media was exchanged by ultrafiltration using phosphate buffered saline (PBS) containing 10% ethanol in the early wash steps, followed by PBS alone in the final washes. The resulting product was used in the in vivo and in ovo experiments described below, with amounts indicating the amount of total protein in the preparation.

EXAMPLE 6 In vi tro cMGF Activity

The previous data described the cloning and expression of cMGF and described initial experiments on the effects of treatment of bone marrow cells in vi tro with Pichia pastoris expressed cMGF which demonstrated proliferation of these cells and acquisition of mature macrophage function responses. These studies have been extended and some preliminary in vivo data has been collected.

The acquisition of Fc-receptor mediated phagocytosis and IS-induced nitrite production was significantly accelerated and enhanced by the presence of micro-gram quantities of cMGF (20 ug/ml) in the media over the first two days of BM cell culture. Effects of cMGF on phorbol ester induced superoxide production appears more complicated and is still under evaluation. To determine the dose response for the cMGF induced acquisition of mature macrophage functional responses in BM cultures, cells were incubated in RPMI 1640 media + 5% FBS plus the indicated doses of cMGF for 48 hours and assayed for their ability to perform Fc-receptor mediated phagocytosis (Figure 7) , IS-induced nitrite production (Figure 8) and phorbol ester induced superoxide production (data not shown) .

The cMGF induced acquisition of Fc-receptor mediated phagocytosis and IS-induced nitrite production in BM cells after 48 hours of culture was dose dependent, beginning in the 10-100 ng/ml range and approaching maximal stimulation in the 1-2 micro-gram/ml range of cMGF concentration. cMGF effects on cultured BM cell PMA-induced superoxide production were once again more complex with stimulation in the 10-100 ng/ml range and inhibition at doses of 500 ng/ml or greater. cMGF induced proliferation of BM cells is also initiated in the nanogra range (data not shown) .

Pichia pastoris produced recombinant cMGF therefore stimulates both the proliferation of bone marrow cells and the acquisition of mature macrophage functional responses. Concanavalin A stimulated splenocyte conditioned media (IS) , which contains cMGF in addition to other cytokines, also stimulates the proliferation of bone marrow cells. Comparison of the effects of 48 hour culture of BM cells in media alone or media + 2.5 μg/ml of either IS, cMGF or yeast expressed albumin indicated that both cMGF and IS induced proliferation when compared to media alone, but that albumin at high doses also stimulated some proliferation (data not shown) . A comparison of the effects of cMGF, IS, and yeast expressed albumin on the acquisition of mature macrophage functional responses in BM cultures was performed. cMGF was the most efficient at enhancing Fc- receptor mediated phagocytosis 37% versus 16% for Albumin, 5% for media alone and 2.5% for IS (data not shown) . Both cMGF and IS treatment significantly stimulated IS-induced nitrite production relative to albumin and media cultured cells (data not shown) . cMGF, IS, and albumin treatment all enhanced the PMA-induced superoxide response relative to media cultured cells (data not shown) . Pichia pastoris produced recombinant cMGF therefore stimulates both the proliferation of bone marrow cells and the acquisition of mature macrophage functional responses. IS, which contains cMGF in addition to other cytokines (IL-2 & IFN, etc.), also stimulates both the proliferation of bone marrow cells and the acquisition of mature macrophage functional responses. However, treatment of BM with IS had either no effect or inhibited acquisition of Fc-receptor mediated phagocytosis, an observation made previously with peritoneal elicited macrophages cultured with IS. It is therefore likely that in ovo delivery of cMGF will result in chicks hatching with greater numbers of functionally activated macrophages that are better able to cope with bacterial infections and perform their central role in the modulation of the immune response.

EXAMPLE 7

In ovo cMGF: Effects on Peripheral Blood Mononuclear Leucocytes Post Hatch Eggs were injected on day 18 of incubation with either 2.5 μg/egg of yeast expressed albumin or 0.025, 0.25 or 2.5 μg/egg of yeast expressed cMGF and the chicks hatched from these eggs were housed and 5 chicks from each treatment group were bled at 1, 4, and 7 posthatch. Blood smears were prepared to evaluate leucocyte differential counts. The results are presented in Table 1 below.

TABLE 1. The effects of in ovo cmgf on white blood cell differential counts of chicks on days 1, 4, and 7 posthatch.

DAY 1 POSTHATCH

Treatment in ovo Lymphocytes Monocytes Granulocytes

Vehicle 68 +/- 27.0 7 +/- 4.0 23 +/- 24.9

0.025 ug cMGF 39 +/- 15.9 12 +/- 1.8 47 +/- 15.3

0.25 ug cMGF 80 +/- 12.4 8 +/- 5.4 12 +/- 10.9

2.5 ug cMGF 59 +/- 29.5 18 +/- 5.8 24 +/- 24

DAY 4 POSTHATCH

Treatment in ovo Lymphocytes Monocytes Granulocytes

Vehicle 83 +/- 10.6 14 +/- 8.3 3 +/- 3.0

0.025 ug MGF 81 +/- 6.3 12 +/- 4.1 8 +/- 6.1

0.25 ug cMGF 88 +/- 5.9 11 +/- 5.1 3 +/- 3.1

2.5 ug cMGF 93 +/- 2.4 4 +/- 1.5 2 +/- 1.26

DAY 7 POSTHATCH

Treatment in ovo Lymphocytes Monocytes Granulocytes

Vehicle 83 +/- 6.8 13 +/- 6.9 4 +/- 1.5

0.025 ug cMGF 87 +/- 5.0 10 +/- 2.9 3 +/- 2.4

0.25 ug cMGF 88 +/- 3.0 13 +/- 5.8 3 +/- 2.1

2.5 ug cMGF 85 +/- 6.9 11 +/- 4.7 2 +/- 2.2

Each mean represents 5 chicks within one trial . One hundred cells were counted for each chick sampled.

On day 1 posthatch abnormal leukocyte ratios were evident in 1 or 5 chicks for the vehicle (2.5 μg albumin) , 0.25 μg cMGF and 2.5 ug cMGF treatments and 5 of 5 of the 0.025 μg cMGF treatment. Granulocytes were elevated in the 0.025 μg cMGF in ovo had elevated levels of erythroblasts . No effect on leukocyte differential counts was evident on days 4 and 7 posthatch. Peripheral blood mononuclear leukocytes (PBL's) were prepared from the pooled blood by centrifugation over percoll gradients, about 5 chicks per treatment group, and the resulting PBL's placed into tissue culture and allowed to adhere for two hours. Non-adherent cells were removed by vigorous washing and the resulting adherent population of leukocytes assayed for their ability to perform Fc-receptor mediated phagocytosis, IS- induced nitrite production and phorbol ester induced superoxide production. There was no detectable IS or LPS inducible nitrite production and Fc-receptor mediated phagocytosis (opsonized-SRBC s) at any of the time points examined. Interestingly, leukocytes were able to phagocytose non-opsonized SRBC's, which may be a reflection of the normal homeostatic functions of blood monocytes, that is the recognition and phagocytosis of senescent, damaged or foreign RBC's or other cells. At 1, 4 and 7 days post hatch adherence purified leukocytes generated measurable PMA-induced superoxide and those isolated from the cMGF injected groups on day 4 post hatch produced more that those form the albumin control group (Figure 9) .

PBL's from the 0.25 & 0.025 μg cMGF/egg groups produced more 0₂ in response to PMA than albumin controls on days 1, 4, and 7. PBL's from the 2.5 μg cMGF/egg group produced more 0₂ in response to PMA than albumin controls on day 4 post hatch but not on days 1 or 7.

In a similar experiment, bone marrow cells were isolated from the femurs of day old chicks hatched from eggs injected with yeast expressed cMGF or albumin and

IS. The number of cells isolated (Figure 10) and PMA- inducible superoxide production of adherence purified bone marrow cells is presented (Figure 11) .

In ovo cMGF significantly increased the number of bone marrow cells isolated from the femurs of day old chicks. Similar to the effects of in ovo cMGF on PBL functional responses, bone marrow cells from cMGF treatment groups produced significantly more superoxide in response to PMA stimulation.

In ovo cMGF injection therefore increases the number and functional activation of bone marrow cells available, affects leukocyte blood differential counts (early on) and PMA-inducible superoxide production by blood leukocytes isolated from the chicks hatched form these eggs. Since superoxide production and it's reactive oxygen intermediate derivatives are one of the major anti-microbial products of monocyte/macrophages and granulocytes, which together with antibodies and complement form the first line of defense against bacterial pathogens, chicks hatching from eggs injected with cMGF may get fewer bacterial infections during this period of high susceptibility. The efficacy of in ovo cMGF administration in vivo E. coli challenge models is under investigation.

EXAMPLE 8 Hatchability Studies Eggs obtained from and incubated at a commercial hatchery (Rose Hill) were candled to detect live embryos. Viable eggs were injected in ovo on day 18 of incubation with a variety of test articles, placed in hatching baskets and returned to the incubator. The resulting hatchability on day 21 was recorded (Table 2) .

Table 2. Hatchability following in ovo cMGF administration

Treatment in ovo Hatchability % (S.D.)

Yeast Expressed Albumin 94.0 (2.1)

Immunomodulator 94.2 (2.8)

0.025 μg cMGF 94.9 (3.2)

0.25 μg cMGF 95.4 (2.4)

2.5 μg cMGF* 91.1 (4.1)

2.0 mg Gentamicin 96.1 (1.6)

Noinjected Controls 95.1 (2.1)

Hatchability means are based on 9 replicates of 108 live embryonated eggs (n=972).* Treatment tested positive for bacterial contamination postinjection but not prior to injection, suggesting that the injectable may have became contaminated during the filling of syringes.

With the exception of the 2.5 μg dose of cMGF, which may have become contaminated, in ovo injection of the test articles did not significantly affect hatchability.

EXAMPLE 9

Hatcher Contamination E. coli Challenge Screening Models The hatcher contamination model involves placing eggs (typically 36 eggs) which have been injected into the aircell with the VPI E. coli strain (typically 10⁴ CFU's) in the top tray of the hatcher. The chicks that hatch from these infected eggs then transmit the pathogen to the experimental chicks hatching from eggs incubated in the same hatcher. The various treatment groups are represented in two separate locations within the hatcher, and the locations are randomly varied from trial to trial. On the day of hatch, chicks are pulled, wingbanded, commingled, placed in floor pens and monitored for mortality twice daily for two weeks. At two weeks, chicks are weighed and counted. A series of three separate E. coli hatcher contamination challenge models were performed (Table 3) .

Table 3 : Efficacy of in ovo cMGF in the Hatcher Contamination E. coli Challenge Model.

In ovo Expt. 1 Expt. 2 Expt. 3 Mean SEM Treatment % % % % Mortality Mortality Mortality mortality

PBS 14.3 8.3 14.5 12.4 A ±3.5

Gentamicin 5.7 5.8 0.83 4.1 C ±2.8 0.2 mg cMGF 14.3 7.5 12.5 11.4 AB ±3.5 8 U cMGF 4.3 10 10.8 8.4 ABC ±3.5 80 U cMGF 5.7 2.5 10.1 6.1 BC ±3.8 800 U

10 min 280 247 CFU's 208 CFU's

Hatcher CFU's

CFU's

# Birds 70 120 120 310 per treatment

Means were partitioned by Duncan's Multiple Range Test. Means with no common postscript A, B or C differ significantly (p≤0.05).

These models demonstrated efficacy of 0.2 mg gentamicin, YEA (yeast expressed albumin) , and 800 U rcMGF injected in ovo . The 800 U rcMGF treatment and the 0.2 mg Gentamicin were efficacious when compared to PBS but not relative to the YEA control. 80 units of cMGF was efficacious in Trial 1 but not in Trials 2 and 3. It is unclear why the YEA exhibits comparable efficacy to 800 units of cMGF. In ovo cMGF consistently produces greater increases in both the number of bone marrow cells in chick femurs at hatch and the PMA inducible superoxide production from peripheral blood lymphocytes collected from Day 4 chicks when compared to YEA injected controls. In addition, YEA does not stimulate bone marrow cells to proliferate in vi tro . The YEA is dosed at an equivalent protein concentration to the 800 unit cMGF dose.

Two additional E. coli hatcher contamination challenge trials were conducted to evaluate the efficacy of cMGF and 10 U of IM with or without gentamicin. The results are presented in Table 4.

Table 4: Efficacy of cMGF and IM with or without .05 mg gentamicin in the hatcher contamination model.

Treatment in ovo Day 14 Mortality % Day 14 Body Wei ght (g)

Ex. 1 Ex. 2 Mean Ex. 1 Ex. 2 Mean

PBS 4.2 3.8 4.0 303 320 312

.05 mg Gentamicin 0 0 0 305 322 314

10 U IM (9416) 2.9 1.0 2.0 319 310 315

40 U cMGF 3.8 5.7 4.8 299 299 299

200 U cMGF 3.3 1.0 2.2 313 310 312

800 U cMGF 1.1 2.9 2.0 308 305 307

10 U IM + .05 6.8 0 3.4 304 304 304 mg gentamicin

40 U cMGF + .05 1.9 1.0 1.5 298 316 307 mg Gentamicin

200 U cMGF + 3.8 1 2.4 310 309 310

.05 mg

Gentamicin

800 U cMGF + 0 0 0 305 311 308

.05 mg

Gentamicin

Nonchallenged 1 1 1 314 311 313 Controls

The mortality was low in both trials when compared to previous tests, however the positive (.05 mg gentamicin) and negative controls (PBS) were probably significantly different from each other. Treatments which numerically reduced mortality were 200 and 800 U of cMGF, 10 U of IM and the combination of 40, 200 or 800 U of cMGF with .05 mg gentamicin. 40 U cMGF was not efficacious. Although the level of mortality was low compared to previous trials (perhaps due to the use of different flock as the source of eggs and housing the chicks in a different grow out facility) , these data are consistent with previous data which showed that cMGF exhibited a dose response.

To further evaluate the efficacy of YEA in the hatcher contamination E. coli challenge model, a YEA dose-response trial was performed (Table 5) .

Table 5. Efficacy of YEA against E. coli associated early chick mortality

Treatment in Hatchability (%) Two Week Two Week ovo Mortality (%) Body Weight (g)

PBS 92 7.0 289

0.2 mg 96 8.7 300 Gentamicin

.2 μg YEA 92 7.0 299

. l g YEA 93 12.2 291

.04 μg YEA 98 8.7 311

.02 ug YEA 92 14.8 288

.002 μg YEA 94 12.2 300

500 U cMGF 95 8.7 279 (.2 μg protein)

The gentamicin treatment used as a positive control failed to reduce mortality below PBS injected controls, contrary to our previous data (Tables 3 and 4, above) . The reason for this is unknown, although the chicks were held in the hatcher an additional day in an attempt to elevate early chick mortality so that differences between the efficacy of various YEA doses could be better discriminated. It may be that this additional stress may have overwhelmed the efficacy of the articles tested. In order to correctly discriminate between the relative efficacies of rcMGF and YEA, it may first be necessary to purify these proteins away from potential contaminants present as a component of the yeast media or produced by the yeast during production.

EXAMPLE 10

Intramuscular E. coli Challenge Screening Models

The efficacy of in ovo administration of IM and cMGF against an E. coli intramuscular challenge on Day 3 and Day 7 posthatch was evaluated (Tables 6 and 7) . This is a new model which evaluates cytokine efficacy against a systemic infection, bypassing the protective barriers of the skin, gut and lung epithelia and protection afforded by GALT or BALT. It also begins to evaluate the duration of efficacy as birds are not challenged until Day 3 or 7 posthatch. The Day 3 challenge experiment compared the efficacy of in ovo versus intramuscular hatch-day injection of cytokines.

Birds were injected with test articles either in ovo on day 18 or intramuscularly (left leg) at day of hatch. Chicks were wingbanded, commingled and placed in floor pens. On day 3 or 7 chicks were injected intramuscularly with the appropriate dose of E. coli CQR strain into the right leg. Chicks were replaced in the floor pens and mortality was monitored for seven days post challenge (Tables 6 and 7) . Table 6: Efficacy of in ovo or day of hatch cMGF and IM administration against a Day 3 Intramuscular E. coli challenge.

Treatment % Mortality % Mortality (10⁵ cfu) (IO⁴ cfu)

Ex. 1 Ex. 2 Mean Ex. 1 Ex. 2 Mean

PBS in ovo 10.3 19.4 14.9 5.6 8.3 7.0

.05 mg Gentamicin 15.1 13.6 14.4 3.9 5.0 4.5 in ovo

10 U IM in ovo 10.8 12.7 11.8 5.0 5.0 5.0

40 U cMGF in ovo 6.9 11.1 9.0 5.2 10.0 7.6

200 U cMGF in 11.9 18.3 15.1 5.6 8.3 7.0 ovo

800 U cMGF in 5.0 16.7 10.9 5.1 8.3 6.7 ovo

PBS at hatch 10.3 8.7 9.5 6.3 3.3 4.8

.05 mg gentamicin 9.5 11.7 10.6 3.9 1.7 2.8 at hatch

10 U IM at hatch 9.5 8.7 9.1 4.7 1.7 3.2

40 U cMGF at 5.6 17.5 11.6 3.2 8.3 5.8 hatch

200 U cMGF at 13.5 15.1 14.3 3.9 5.0 4.5 hatch

800 U cMGF at 16.7 18.3 17.5 3.2 11.7 7.5 hatch

Treatment means based on approximately 105 chicks.

The results of the Day 3 challenge model were inconsistent (Table 6) . In the first trial 40 U of cMGF reduced mortality following both in ovo and day of hatch injection and challenge with 10^s CFU's. However this finding was not repeated at the lower challenge level (10" CFU's) . In trial two, 40 U of cMGF reduced mortality following the 10^s challenge when injected in ovo but not posthatch. Table 7 : Efficacy of in ovo cMGF and IM against a Day 7 intramuscular E. coli challenge.

Treatment in ovo One Week Mortality Two Week Body Weight

% (g)

IO⁶ 10^s 10⁶ IO⁵

PBS 43.3 5.0 309 285

.05 mg Gentamicin 28.3 6.7 237 279

10 U IM (9416) 26.7 8.3 294 296

40 U cMGF 33.3 3.3 312 295

200 U cMGF 43.3 8.3 259 270

800 U cMGF 28.3 13.3 304 277

In the first trial the challenge levels chosen,

10⁴ and IO⁵ CFU's, produced no significant mortality (data not shown) . In the second trial, only the IO⁶ CFU challenge dose resulted in a high level of mortality in which Gentamicin, 10 U of IM and both 40 U and 800 U of cMGF numerically reduced mortality.

EXAMPLE 11 Oral Gavage E. coli Challenge Screening Models

In this model chicks are orally gavaged with approximately IO⁹ CFU's of E. coli VPI strain immediately prior to placement in floor pens on the day of hatch.

The challenge culture is maintained on ice during the challenge procedure. Equal numbers of wingbanded chicks from each treatment group are commingled and randomly gavaged with 100 μL of the challenge culture prior to placement into floor pens. Chicks are monitored for mortality twice daily for 10 days.

The results of the two trials evaluating the efficacy of in ovo cMGF and IM administration with or without gentamicin against a hatch day oral E. coli challenge are presented below (Table 8) . This trial was designed to evaluate an IM dose of 10 U and the efficacy of cMGF, and to determine if there were any added benefits when cytokines were injected with 0.05 mg gentamicin.

Table 8: Efficacy of in ovo cMGF and IM with or without gentamicin against an oral E. coli challenge at hatch.

Treatment in ovo Mortality %

Trial 1 Trial 2 Mean

PBS 13.4 8.9 11.1

.05 mg Gentamicin 5.0 3.3 4.2

10 U IM (9416) 6.7 3.3 5.0

40 U cMGF 16.7 7.8 12.3

200 U cMGF 8.3 12.2 10.3

800 U cMGF 11.7 6.7 9.2

10 U IM + .05 mg 5.0 3.3 4.2 Gentamicin

40 U cMGF + .05 mg 4.2 3.3 3.8 Gentamicin

200 U cMGF + .05 mg 9.2 8.9 9.1 Gentamicin

800 U cMGF + .05 mg 7.5 2.2 4.9 Gentamicin

PBS nonchallenged controls 8.3 1.1 4.7

Two pens of the eight pens were deleted from study two. These pens had excessive mortality levels of 50% .

In this model, both 10 U IM and .05 mg gentamicin clearly demonstrated efficacy against E. coli . Neither 40 U or 200 U of cMGF demonstrated efficacy, although 800 U numerically reduced mortality. There was no additive effect when IM was added to gentamicin. The two trials were similar in their results except that in trial 1 non-challenged controls had a surprisingly high level of mortality, perhaps due to horizontal transmission within pens as treatment groups were commingled and equally represented. In other E. coli hatcher contamination challenge trials (see Example 9 above) , yeast expressed albumin (YEA) has been used as a vehicle control for testing cMGF efficacy. However, although YEA fails to exhibit any of the other in vitro or in vivo effects of cMGF on bone marrow and immune cell populations, YEA has consistently demonstrated efficacy in the hatcher contamination model when it was dosed on an equivalent protein basis as 800 U cMGF. The two preparations were tested in an oral challenge model to further evaluate and compare the efficacy of YEA to cMGF and also to determine if the protection could be attributed to in ovo delivery of protein (BSA) or products produced by the yeast expression system (YEM is the media derived from a transformed Pichia pastoris strain expressing intracellular 3-galactosidase) . The results are presented in Table 9.

Table 9: Efficacy of YEA, YEM, cMGF and BSA in the oral Day of Hatch Gavage E. coli challenge model.

Treatment in ovo Mortality % Body Weight (g)

Trial Trial Mean Trial Trial Mean

1 2 1 2

PBS 13.7 10.8 12.3 222 175 199

.05 mg Gentamicin 10.0 5.8 7.9 220 173 197

Yeast Expressed 7.6 1.7 4.7 218 177 198 Albumin

Yeast Extract Media 7.5 8.3 7.9 230 172 204

800 U CMGF 10.8 5.8 8.3 221 179 200

BSA (equivalent 11.7 7.5 9.6 226 174 200 protein)

Mean Mortality based on 240 chicks per treatment (120 in each of two independent trials). YEM, cMGF, YEA, .05 g gentamicin and BSA all reduced mortality, but YEA was the most efficacious. Equivalent body weights of birds suggest birds which did not die in the treated groups are not sick or depressed in growth. EXAMPLE 12 Dietary Stress -E coli Challenge Model

The model is based on Leitner and Heller's finding (1992: Avian Diseases 36:211-220) that stress, (specifically 36 hour food and water deprivation) resulted in E. coli colonization of the liver, spleen, and blood of young turkeys. The turkeys were orally gavaged with E. coli on hatch day, withdrawn from feed 5 days later and then organs harvested to evaluate colonization 36 hours later. In the first experiment broiler chicks were administered either PBS, YEA (yeast expressed albumin) , 5, 50 or 500 U of cMGF in ovo, orally gavaged with IO⁸ CFU E. coli (VPI 1990 strain) at hatch, removed from feed and water for 36 hours beginning on Day 4 posthatch and then either evaluated for tissue (liver and blood) colonization or Day 12 mortality. The results are presented in Table 10.

Table 10: Efficacy of cMGF administration in ovo against E. coli organ invasion and early chick mortality.

Treatment in ovo Percent chicks with Day 12 Mortality tissue colonization n = 15 n = 5

PBS 0 16.7%

YEA 0 0%

5 U cMGF 20% 14.3%

50 U cMGF 20% 25%

500 U cMGF 0 7.1 %

There was very little organ invasion, making it difficult to discern any effects due to in ovo cMGF administration. The highest dose of cMGF and an equivalent amount of YEA protein both reduced mortality. A second study was conducted to evaluate the efficacy of in ovo cMGF administration in this model, The results of both trials are presented in Table 11.

Table 11. Efficacy of cMGF in ovo against E. coli organ invasion and mortality following oral gavage of E. coli at hatch followed by food and water deprivation four days posthatch.

Treatment in Percent of chicks Day 12 ovo exhibiting organ Mortality (%) invasion

Trial Trial Mean Trial Trial Mean Mn) 2(n) l(n) 2(n)

PBS 0 (5) 0 (5) 0 11.8 16.0 13.9

(17) (25)

YEA ( .2 μg 0 (1) 0 (5) 0 0 (17) 11.0 5.6 protein) (27)

5 U cMGF 16.6 20 18.3 10.0 21.4 15.7 (6) (5) (19) (28)

50 U cMGF 20 40 30.0 11.8 26.9 19.4 (10) (5) (17) (26)

500 U CMGF 0 (5) 10 10.0 5.3 32.1 18.7 _.. ( .2 ug protein) (5) (19) (28)

There was no evidence of organ invasion in the PBS injected controls. In the first trial feed and water were deprived for 36 hours beginning on Day 4 posthatch and the chicks were orally gavaged with 100 μL of a IO⁹ cfu/mL E. coli culture. This challenge regimen resulted in no colonization of the liver or blood of PBS injected chicks. In trial two the period of feed and water deprivation was increased to 48 hours and the chicks were orally gavaged with 200 μl of a IO⁹ cfu/mL E. coli culture; however, there was still no evidence of organ invasion in the PBS injected chicks. Chicks receiving YEA at 0.2 μg also failed to exhibit any organ colonization. Interestingly, however, chicks which received cMGF in ovo did exhibit organ invasion, and the results were somewhat consistent in both trials, with the 500 U dose having the least. Perhaps cMGF increases the number of cells of the monocyte/macrophage lineage which can phagocytose and internalize bacteria, but these cells are not sufficiently activated to be biocidal and serve as reservoir of bacteria in the form of tissue macrophages. A second signal such as cIFN may be required for full activation and the destruction of intracellular bacteria. Twelve day mortality was increased in the second trial due to either the increased challenge dose, the increased period of feed and water deprivation, or both. Food and water deprivation did increase two week mortality above levels normally seen in the hatcher contamination and oral gavage models. There was also considerable variability in the mortality data between the two trials, but cMGF appeared to have no effect. It is unclear whether organ colonization and mortality may be correlated since both PBS and YEA groups had no detectable organ colonization but had considerably different mortality levels.

EXAMPLE 13

Day 0 in ovo cMGF Injection and Day 13 in ovo E. coli Challenge Model In ovo cMGF injection on Day 0 of incubation was evaluated to determine if it would increase the number of functional responsiveness of embryo phagocytes that defend against bacterial pathogens beginning the second week of egg incubation. Klasing (1991) surmised opsonin mediated phagocytosis could sufficiently defend the embryo against bacterial pathogens by the second week of incubation. Phagocytic cells protect the embryo during the critical period after the chorioallantoic membrane fuses with the shell membrane, permitting dissemination of pathogens on the shell to the embryo. cMGF was injected into the albumin at either 0.25, 2.5, 25 or 250 U on Day 0 of incubation. Eggs were removed directly from the incubator (99°F) and dipped in an E. coli broth at 40°F for four minutes on Day 13 of incubation. The temperature differential assists in the transport of the bacteria across the shell and shell membranes. A similar E. coli challenge on Day 18 of incubation resulted in a 7% decrease in hatchability and a 7% increase in two week mortality (Reid et al . , 1961) . A separate group of cMGF injected eggs were not exposed to E. coli to evaluate the effects of Day 0 cMGF injection on hatchability. The results are presented in Tables 12 and 13.

Table 12. Hatchability following Day 0 cMGF injection.

Treatment in ovo Hatchability % Infertile, Early and Middle Embryonic Mortality (%)

PBS 84 11.8

0.25 U cMGF 91 5.9

2.5 U cMGF 84 11.8

25 U cMGF 91 4.4

250 U cMGF 85 8.8

Hatchability was not consistently affected by

Day 0 cMGF administration (Table 12) , although the data is variable because only one replicate of 68 eggs was evaluated for each treatment group.

Table 13. Efficacy of Day 0 cMGF injection against an E. coli challenge on Day 13 of incubation: Hatchability results.

Treatment in ovo Hatchability % Embryonic Mortality

After E. coli Challenge

(%)

PBS 92 8.7

0.25 U cMGF 83 14.5

2.5 U cMGF 87 12.7

25 U cMGF 83 16.9

250 U cMGF 90 10.2

The model used to evaluate bacterial challenge during incubation worked well . Hatchability of eggs alive at the time of challenge was 87.3% compared to 94.5% for eggs which were not challenged. The percent of late embryonic mortality and dead chicks was significantly increased above normal unchallenged eggs. Day 0 cMGF administration numerically increased embryonic mortality and demonstrated no efficacy in this model.

EXAMPLE 14

Salmonella twhimurium Hatcher Contamination and Posthatch Seeder Screening Challenge Models

The hatcher contamination model is very similar to the E. coli hatcher contamination model described previously (see Example 9 above) except that the contaminated eggs (typically 15 eggs) placed in the top hatcher basket and used to deliver the challenge were injected with Salmonella typhimurium. The posthatch seeder challenge model involves the commingling of Salmonella exposed and unexposed birds in floor pens simulating the horizontal transmission of pathogens from chick to chick. cMGF and IM were all tested in both a Salmonella typhimurium hatcher contamination model and a posthatch seeder model. These models were conducted to evaluate the efficacy of these cytokine products against a different bacterial pathogen that is intracellular in nature. The Day 10 mortality results for the Salmonella typhimurium Hatcher contamination model and Posthatch Seeder Model are presented below (Table 14) . Results are presented as percent of controls.

Table 14: Efficacy of cMGF and IM against Salmonella typhimuri urn .

Treatment in ovo Hatcher Model Seeder Model

Ex. 1 Ex. 2 Mean Ex. l Ex. 2 Mean

PBS 100 100 100 100 100 100 (62) (62) (47) (43)

0.5 mg Baytril 62 81 72 138 42 90

10 U IM 85 116 101 106 74 90

.01 U IM 81 140 111 113 93 103

800 U cMGF 75 141 108 113 69 91

Each treatment mean based on 120 chicks (15 in each of 4 pens in each of two trials).

The challenge was very robust and produced higher levels of mortality than previously conducted trials of these models. The only treatment that demonstrated efficacy was 0.5 mg Baytril in the hatcher contamination model, and even this exhibited a lower level of efficacy than in previous trials. It may be that the level of challenge may have overwhelmed the efficacy of the products tested.

EXAMPLE 15 Coccidiosis Challenge Screening Model The efficacy of cMGF and IM were evaluated in a coccidiosis E. tenella oocyst challenge model (Table 15) . The model measures fecal oocyst output after a low level oral challenge and evaluates the ability of the parasite to either infect or replicate within the host. Efficacy, measured by a reduction in the number of oocysts shed, suggests that either the parasite was unable to invade or infect cells within the subepithelial layer of the ceca, or that the parasite was unable to replicate once inside the cells and sexual reproduction or production of oocysts was inhibited. Chicks were challenged orally on day 1 or day 7 post-hatch with 1000 oocysts, fecal output was collected during a period of 72 hours post challenge, and the number of oocysts shed were enumerated (Table 15) .

Table 15: Efficacy of cMGF and IM against an -Eimeria tenella challenge.

Treatment in ovo Day chicks were Oocyst Production (X Day 18 of incubation challenged with 1000 IO⁶) per bird

Eimeria tenella oocysts Mean +/- S.D.

PBS Not Challenged 0

PBS Day 1 8.1 +/- 1.9 AB

10 U IM (9416) Day 1 11.8 +/- 1.3 A

0.01 U IM (9416) Day 1 12.3 +/- 0.8 A

800 U cMGF Day 1 8.1 +/- 2.7 AB

40 U cMGF Day 1 6.9 +/- 1.9 AB

PBS Not Challenged 0

PBS Day 7 15.1 +/- 3.1 AB

10 U IM Day 7 17.3 +/- 4.4 A

800 U cMGF Day 7 7.1 +/- 0.6 BC

40 U cMGF Day 7 3.5 +/- 1.5 C

Birds were challenged either 1 or 7 days posthatch. The oocyst output following challenge on Day 1 posthatch was unaffected by in ovo administration of cMGF or IM. For 40 U of cMGF, the mean oocyst production was numerically reduced but not significantly, and IM actually increased the number of oocysts produced. The oocyst output following challenge on Day 7 posthatch was significantly reduced by in ovo administration of 40 U of cMGF. 800 U cMGF numerically reduced the oocyst output, while IM numerically increased oocyst output .

The efficacy of in ovo cMGF administration against coccidiosis was evaluated in a second Eimeria tenella oocyst production challenge model. Eggs were injected with either PBS, YEA, 5, 10, 50, 100, 200 or 800 U cMGF on day 19 of incubation. Broiler chicks were then challenged with 1000 Eimeria tenella oocysts on Day 7 posthatch and fecal output was collected during a period of 72 hours post challenge, and the number of oocysts shed enumerated (Table 16) .

Table 16. Efficacy of in ovo cMGF against Eimeria tenella oocyst production following Day 7 oral challenge with 1000 oocysts.

Treatment in ovo USDA Trial Embrex Trial Pooled Results oocysts produced per bird X 10⁶

PBS 3.06 4.26 3.67 ± 0.6

YEA 3.89 4.13 4.01 ± 0.12

5 U cMGF 1.16 3.10 2.13 ± 0.97

10 U cMGF 2.29 2.70 2.50 ± 0.2

50 U cMGF 1.74 3.20 2.47 ± 0.73

100 U cMGF 3.51 2.24 2.89 ± 0.64

200 U cMGF 1.17 5.13 3.15 ± 1.98

800 U cMGF 3.58 4.90 4.24 ± 0.66

Three pools of feces from five birds were counted in each trial (n = 6).

Eimeria tenella oocyst production was numerically reduced after in ovo cMGF administration at

5, 10, 50, 100 and 200 U cMGF. 800 U cMGF was ineffective. These results are consistent with a previous study which demonstrated efficacy of 40 but not 800 U cMGF in an oocyst production challenge trial. Future studies need to evaluate YEA at equivalent protein levels to protective cMGF doses, the kinetics and dose response of cMGF and efficacy against other coccidial strains.

It should be noted that the challenge models described in Examples 8 - 15 were initiated for this evaluation of cMGF, and additional development of these models would likely lead to more consistent data. For example, the dose of E. coli used to produce mortality in the first day 7 intramuscular challenge model were too low to be effective, and the Salmonella typhimurium challenges may have been so robust that any efficacy was obscured. Nevertheless, the data from these preliminary challenge models indicate that cMGF has consistently demonstrated a dose-dependent efficacy against a hatcher contamination E. coli challenge and has also exhibited a dose dependent efficacy against Coccidiosis E. tenella oocyst challenge. The data from the other models tested were less consistent in that, while cMGF at various doses may have demonstrated efficacy in individual trials, the data was not always reproduced in a second experiment . Overall, however, these data would be consistent with the hypothesis that in ovo administration of recombinant cMGF provides a benefit to hatching chicks which makes them less susceptible to infectious pathogens during the first few weeks post hatch.

Yeast expressed albumin (YEA) , at an equivalent protein concentration to cMGF at 800 U, exhibits comparable efficacy to 800 units of cMGF. In ovo cMGF administration has produced increases in both the number of bone marrow cells in chick femurs at hatch, and the PMA inducible superoxide production from peripheral blood leukocytes collected from Day 4 chicks, when compared to YEA injected controls. In addition, YEA does not stimulate bone marrow cells to proliferate in vi tro . It is therefore likely that the YEA efficacy in the E. coli hatcher contamination model is due to some alternate, as yet unknown, mechanism.

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

Claims

That Which Is Claimed Is:

1. A method of making purified recombinant avian myelomonocytic growth factor (MGF) , comprising: culturing host cells which contain and express a recombinant DNA construct encoding MGF in a culture media; collecting culture media containing recombinant MGF from said host cells; and isolating said MGF from said culture media.

2. A method according to Claim 1, wherein said MGF is chicken MGF.

3. A method according to Claim 1, wherein said host cell is Escherichia coli .

4. A method according to Claim 1, wherein said host cell is Pichia pastoris .

5. A method according to Claim 3, wherein said MGF is glycosylated MGF.

6. Avian MGF produced by the method of Claim

7. A method of treating a bird, comprising administering to said bird in ovo a biologically active amount of avian Myelomonocytic Growth Factor.

8. A method according to Claim 7, wherein said bird is selected from the group consisting of chickens, turkeys, ducks, geese, quail and pheasant.

9. A method according to Claim 7, wherein said birds is selected from the group consisting of chickens and turkeys .

10. A method according to Claim 7, wherein said avian MGF is administered to said bird during about the last quarter of in ovo incubation.

11. A method according to Claim 7, wherein said bird is a chicken and said avian MGF is administered to said chicken on about the fifteenth to nineteenth day of incubation.

12. A method according to Claim 7, wherein said bird is a turkey and said avian MGF is administered to said turkey on about the twenty-first to twenty-sixth day of incubation.

13. A method according to Claim 7, wherein said administering step is carried out by injecting the avian MGF into the egg in which the bird is contained.

14. A method according to Claim 7, wherein said administering step is carried out by injecting said MGF into the region defined by the amnion, the yolk sac, or the air cell.

15. A method according to Claim 7, wherein said bird is a chicken and said avian MGF is chicken MGF.

16. A method according to Claim 7, wherein a vaccine is administered to said bird concurrently with said avian MGF.

17. A method according to Claim 16, wherein said vaccine is a live vaccine.

18. A method according to Claim 16, wherein said vaccine is a nonreplicating vaccine.

19. A method of inhibiting the progression of bacterial infections in a bird, comprising administering to said bird in ovo a bacterial infection-inhibiting amount of avian myelomonocytic growth factor (MGF) .

20. A method according to Claim 19, wherein said bird is selected from the group consisting of chickens, turkeys, ducks, geese, quail and pheasant.

21. A method according to Claim 19, wherein said birds is selected from the group consisting of chickens and turkeys.

22. A method according to Claim 19, wherein said avian MGF is administered to said bird during about the last quarter of in ovo incubation.

23. A method according to Claim 19, wherein said bird is a chicken and said avian MGF is administered to said chicken on about the fifteenth to nineteenth day of incubation.

24. A method according to Claim 19, wherein said bird is a turkey and said avian MGF is administered to said turkey on about the twenty-first to twenty-sixth day of incubation.

25. A method according to Claim 19, wherein said administering step is carried out by injecting the avian MGF into the egg in which the bird is contained.

26. A method according to Claim 19, wherein said administering step is carried out by injecting said MGF into the region defined by the amnion, the yolk sac, or the air cell.

27. A method according to Claim 19, wherein said bird is a chicken and said avian MGF is chicken MGF.

28. A method according to claim 19, wherein said bacterial infection is Escherichia coli infection.

29. A method according to claim 19, wherein said bacterial infection is a Salmonella infection.

30. A method according to Claim 19, wherein a vaccine is administered to said bird concurrently with said avian MGF.

31. A method according to Claim 30, wherein said vaccine is a live vaccine.

32. A method according to Claim 30, wherein said vaccine is a nonreplicating vaccine.