HK1133384A

HK1133384A - Methods and compositions for vaccination of poultry

Info

Publication number: HK1133384A
Application number: HK09111233.5A
Authority: HK
Inventors: 维维安‧W‧杜林; 瑞贝卡‧M‧波斯通; 柴瑞林‧L‧海根-派; 阿兰‧P‧阿维肯; 朱利叶斯‧泰克寇斯科
Original assignee: 恩布里克斯公司
Priority date: 2006-03-30
Filing date: 2007-03-29
Publication date: 2010-03-26

Description

Methods and compositions for poultry vaccination

Priority declaration

Based on 35u.s.c. § 119(e), the present application claims benefit of U.S. provisional application No.60/787,567 filed 3, 30, 2006, which is incorporated herein by reference in its entirety.

Technical Field

The present invention provides methods and compositions for generating an immune response in an avian subject by directly delivering an immune composition to an embryo (embryo) in ovo (inovo) of an avian subject. The invention also provides immunogenic compositions and methods for generating an immune response against Clostridium species in avian subjects, for protecting avian subjects against Clostridium infection, and for protecting avian subjects against related disorders, such as necrotic enteritis.

Background

In ovo vaccination offers several benefits to the poultry industry over existing control methods and possible post-hatch vaccination of birds, including the possibility of uniform, automated delivery; in ovo co-administration with other vaccines, thereby reducing post-hatch handling of the bird; and reduced use of antibiotics.

The discovery that certain vaccines (e.g., inactivated or non-replicating vaccines) can be delivered to the embryo body to elicit a strong immune response (similar or better than that expected from vaccination of birds on the day of hatch) allows the development of automated devices that can target the embryo body (particularly avoiding amniotic fluid surrounding the embryo body) during in ovo application of such vaccines. Furthermore, knowing that inactivated vaccines should be preferentially delivered to the embryo body allows one to develop vaccine compositions that are more compatible with injection into the embryo body. Prior to this finding, it was unknown or unexpected that inactivated vaccines would need to be administered preferentially to the embryo body to elicit a strong immune response.

Thus, the present invention represents an important advance in the art by providing a more efficient method of administering inactivated (killed) vaccines in ovo. Prior to the present invention, no one has suggested any difference between the administration of inactivated vaccine to the blastocyst (amniotic fluid) surrounding the blastocyst. The invention shows that: inactivated vaccines need to be delivered in ovo to the embryo body, rather than to the fluid surrounding the embryo body. Knowing that the embryo body is a suitable target for optimal efficiency allows the development of inactivated vaccines and delivery methods for in ovo routes. The invention encompasses the in ovo administration of immunogenic compositions to poultry or other avian species.

Thus, the present invention meets the need in the art for improved immunogenic compositions for in ovo administration to embryos and methods for inducing an immune response in birds, for protecting birds from infection and/or contamination by avian or other pathogens, and for protecting birds from related conditions.

Clostridium perfringens is associated with several diseases in poultry, the most prominent being necrotic enteritis (type C. perfringens a and C. perfringens C), but also cholangitis, cellulitis, myogastric erosion, and umbilical infections. These bacteria are part of the normal intestinal bacterial flora and a number of factors predispose birds to disease. Such factors include: diets with high levels of fish, wheat, barley or rye, litter with high moisture content, or exposure to diplococcus (cocidiia). Clinically, disease symptoms typically include: diarrhea, decreased appetite, intestinal injury, loss of semen collection, and mortality. Traditionally, antibiotics in feed have been used to control these diseases; however, overuse of antibiotics can lead to the development of antibiotic-resistant bacterial strains, which are significant health risks for humans and animals. Furthermore, in some jurisdictions, the use of antibiotics is highly aversive or even prohibited.

For controllingOther methods of making Clostridium include: diets that avoid components that irritate the intestinal mucosa, e.g., corn-soybean ration; low moisture content litter, having an absorbent material, such as wood chips or rice hulls; competitive exclusion products are used to maintain a healthy balance of intestinal microflora, such as Primalac (Star-Labs/form Research, Inc., Clarksdale, MO), AVIGUARD^TM(Bayer Corporation, Kansas City, MO) and BIO-MOS(Alltech, inc., Nicholasville, KY); and potent prophylactic acidification or disinfection of water to minimize losses during disease. Vaccination has been used in other species, including cattle, sheep, goats and pigs, to control Clostridium-caused disease. Two main vaccine types against c.perfringens have been developed against non-poultry species: toxoid (inactivated toxin) and bacterin-toxoid (inactivated [ "killed"]Bacterial cultures and inactivated toxins). Antitoxins (antibodies specific for toxin (s)) are also used in non-poultry species for the prevention and treatment of clostridial-induced diseases.

Until the present inventors, in ovo administration of c. perfringens vaccine (i.e., administration into eggs containing developing avian embryos) has not been described. The effectiveness of in ovo use of existing toxoid or toxoid-bacterin vaccines is uncertain because such vaccines often require the use of adjuvants, which may be harmful to the embryo, may inactivate live vaccines against other organisms (typically administered in ovo) (e.g., to provide vaccination against Marek's disease), and/or may be incompatible with existing in ovo injection equipment. In ovo vaccination against c.perfringens offers several benefits to the poultry industry over existing control methods and possible post-hatch vaccination of birds, including the possibility of uniform, automated delivery; in ovo co-administration with other vaccines, thereby reducing post-hatch handling of the bird; and reduced use of antibiotics.

Accordingly, there is a need in the art for improved immunogenic compositions and methods for inducing an immune response against Clostridium in birds, for protecting birds against Clostridium infection, and for protecting birds against related disorders, such as necrotic enteritis.

Disclosure of Invention

In some embodiments, the invention provides methods of immunizing an avian against a pathogen (e.g., an avian pathogen or a non-avian pathogen carried by an avian) comprising administering in ovo during the last quarter of incubation an effective immunizing dose of a composition that induces an immune response against the pathogen, wherein the immunogenic composition is administered by direct in ovo injection into an embryo body.

In some other embodiments of the invention, the composition induces an immune response to treat and/or prevent infection and/or contamination of birds exposed to or contacted with pathogens that cause the following diseases, infections and/or conditions, non-limiting examples of which are: coccidiosis, Marek's disease, infectious bursal disease, newcastle disease, fowlpox infection, Clostridium spp, avian influenza, infectious bronchitis, chicken anaemia virus infection, avian laryngotracheitis, avian metapneumovirus infection, avian reovirus infection, avian adenovirus infection, rotavirus infection, astrovirus infection, inclusion body hepatitis, egg drop syndrome, adenovirus infection, Escherichia coli infection, Mycoplasma spp.

In still other embodiments of the invention, the compositions may comprise, consist essentially of, and/or consist of a non-replicative agent capable of inducing an immune response against avian pathogens and/or pathogens that can cause food-borne diseases (e.g., Salmonella spp.

The invention also includes methods wherein an effective immunizing dose of two or more compositions capable of inducing an immune response against an avian pathogen are administered in ovo to an embryo, wherein the two or more compositions are administered simultaneously or sequentially in any order.

The invention also provides methods wherein an effective immunizing dose of two or more compositions capable of inducing an immune response against an avian pathogen are administered in ovo and at least one composition is administered to the embryo, wherein the two or more compositions are administered simultaneously or sequentially in any order.

The present invention provides methods of inducing an immune response against Clostridium species (e.g., Clostridium perfringens) in birds to protect birds from Clostridium infection and/or to protect birds from related disorders (e.g., necrotic enteritis). The methods can be performed in ovo and/or post-hatch. The present invention also provides compositions for inducing an immune response against Clostridium species (e.g., Clostridium perfringens) in birds to protect birds against Clostridium infection and/or to protect birds against related disorders (e.g., necrotic enteritis).

Accordingly, one aspect of the present invention provides a method of immunizing an avian subject (e.g., a chicken) against necrotic enteritis comprising administering in ovo (e.g., during the last quarter of incubation) an effective immunizing dose of an immunogenic composition that induces an immune response against clostridium perfringens, wherein the immunogenic composition is administered by in ovo injection. In certain embodiments, the immunogenic composition is administered to the amniotic membrane or to the embryo. Optionally, the methods may be combined with other immunization protocols (e.g., vaccination against infectious bursal disease, Marek's disease, newcastle disease, and/or coccidiosis) and/or in ovo feeding of nutritional formulas and/or gut modulators (enteromodulators).

Another aspect of the invention provides an immunogenic composition comprising an effective immunizing dose of the attenuated Clostridium species in a pharmaceutically acceptable carrier. In certain embodiments, the immunogenic composition further comprises an adjuvant, which may be, for example, a depot adjuvant. Representative adjuvants of the invention include, but are not limited to: aluminium salts, such as aluminium hydroxide gel (alum), aluminium phosphate or algannmulin, and/or also salts of calcium, magnesium, iron and/or zinc or mineral gels, and/or insoluble suspensions which may be acylated tyrosine or acylated sugars, cationically or anionically derivatized polysaccharides or polyphosphazenes and/or saponins (e.g. Quil-a), and/or oil emulsions, such as water-in-oil and water-in-oil (water-in-oil), and/or complete or incomplete Freund's or any combination thereof. In some representative embodiments, the immunogenic composition comprises an aqueous-in-oil-in-aqueous emulsion. Optionally, the immunogenic composition may further comprise one or more additional agents capable of inducing an immune response against other avian pathogens (e.g., agents that induce an immune response against Eimeria, infectious bursal disease virus, Marek's disease virus, and/or newcastle disease virus) and/or a nutritional formula and/or an enteric modulator. The one or more additional agents may be immunological agents that generate a protective immune response against Eimeria, infectious bursal disease virus, Marek's disease virus, and/or newcastle disease virus.

Yet another aspect of the invention provides an immunogenic composition comprising, in a pharmaceutically acceptable carrier:

(a) an effective immunizing dose of a Clostridium toxoid, a Clostridium bacterin, a Clostridium toxin, or any combination thereof; and

(b) an effective immunizing dose of a coccidiosis vaccine, a Marek's disease vaccine, an infectious bursal disease vaccine, a newcastle disease vaccine, a fowlpox vaccine, or any combination thereof.

The present invention also provides an immunogenic composition comprising, in a pharmaceutically acceptable carrier:

(b) an oil emulsion.

(b) an adjuvant comprising an adjuvant derived from aluminum, a saponin, an oil, or any combination of the foregoing.

In other embodiments, the present invention provides methods of immunizing avian subjects against infection by Clostridium species comprising administering to the avian subject an effective immunizing dose of a Clostridium bacterin-toxoid composition by in ovo injection during the last quarter of incubation. In some embodiments of the invention, the species of Clostridium may be Clostridium perfringens.

The present invention also provides a method of immunizing an avian subject against infection by a Clostridium species, the method comprising: administering to the avian subject an effective immunizing dose of a recombinant toxin of Clostridium species, or an immunogenic fragment thereof, by in ovo injection during the final quarter of incubation. In some embodiments, the toxin or immunogenic fragment thereof is a clostridium perfringens toxin or immunogenic fragment thereof.

These and other aspects of the invention are shown in more detail in the following description of the invention.

Detailed Description

Certain aspects of the present invention are based on the following unexpected findings: certain antigenic or immunogenic compositions, when administered directly to the embryo in ovo, allow for a more effective immune response in the bird.

Accordingly, in one embodiment, the present invention provides a method of immunizing an avian subject against a pathogen, which may be an avian pathogen and/or a non-avian pathogen carried by an avian subject (e.g., a human food-borne pathogen), comprising: administering in ovo during the final quarter of incubation an effective immunizing dose of a composition capable of inducing an immune response against the avian pathogen, wherein the immunogenic composition is administered by direct in ovo injection into the embryo body. In the methods of the invention, the composition may be administered directly to skeletal muscle tissue in the embryo, which may be (but is not limited to): pectoral and pipping muscle tissue. In other embodiments, the composition may be administered directly to the head, neck, shoulders, wings, back, chest, legs, or any combination thereof in the embryo.

Furthermore, in the methods of the invention, the composition may be administered subcutaneously into the embryo body. In other embodiments, the composition can be administered subcutaneously into the head, neck, shoulder, wing, back, chest, leg, or any combination thereof.

Any suitable route of administration into the embryo is suitable for carrying out the method of the invention. For example, the composition can be administered to the embryo subcutaneously, intradermally, intravenously, intramuscularly, intraperitoneally, or any combination thereof.

The avian subject of the present invention can be any avian species, and in certain embodiments, the subject can be a chicken, turkey, duck, goose, pheasant, quail, partridge, guinea fowl, ostrich, emu or peacock, as well as any other commercially processed avian species and/or any avian species for which eggs are available for manipulation in the methods of the present invention.

In embodiments where the individual is a chicken, it may be desirable to administer the compositions of the present invention in ovo over the period of 15 to 20 days of incubation, and in particular embodiments, the compositions may be administered on day 18 or day 19 of incubation. When the individual is a turkey, the composition of the present invention can be administered during the period from day 21 to day 28 of incubation, and in particular embodiments, the composition can be administered on day 24 or day 25 of incubation. In other embodiments where the individual is a goose, the composition of the invention may be administered during the period from day 23 to day 31 of incubation, and in particular embodiments, the composition may be administered on day 28 or day 29 of incubation. In other embodiments where the individual is a duck, the compositions of the invention may be administered during the period from day 21 to day 38 of incubation, and in particular embodiments, the compositions may be administered on day 25 or day 28 of incubation.

For other avian species, the final season of incubation, and thus the optimal date range for in ovo administration of the compositions of the present invention, can be determined according to methods well known in the art. For example, a duck in america has an incubation period in the range of 33-35 days, a pheasant in girdling has an incubation period of 23-24 days, quail in japan has an incubation period of 17-18 days, quail in gable quail has an incubation period of 23 days, chuckar partridge has an incubation period of 22-23 days, guinea fowl has an incubation period of 26-28 days, and peacock has an incubation period of 28 days.

In some particular embodiments of the invention, the composition may comprise, consist essentially of, and/or consist of the immunogenic composition and the adjuvant. Non-limiting examples of adjuvants of the present invention include adjuvants derived from aluminum, saponins, mineral gels, polyanions, pluronic polyols, saponin derivatives, lysolecithins and other similar surface active substances, glycosides, all types of oils and any combination thereof. In some particular embodiments of the invention, the composition may comprise an aqueous-in-oil-in-aqueous emulsion.

As mentioned herein, in some embodiments of the invention, the compositions of the invention comprise an adjuvant, which in some particular embodiments may be an aluminium-derived adjuvant (e.g. aluminium hydroxide), a saponin (e.g. Quil-a, including QuilA QS21) or an oil (e.g. complete or incomplete Freund's adjuvant) in any combination.

Other non-limiting examples of adjuvants of the invention include: mineral salts (e.g., aluminum hydroxide; aluminum phosphate; calcium phosphate), oil emulsions and surfactant-based formulations (e.g., Freund's emulsified oil adjuvant; Arlaceel A; mineral oil; emulsified peanut oil adjuvant (adjuvant 65); MF59 (microfluidized detergent stabilized oil-in-water emulsion); QS21 (purified saponin); AS02[ SBAS2] (oil-in-water + MPL + QS 21); Montanide ISA-51; ISA-720 (stabilized water-in-oil emulsion)), bacterial products and derivatives [ e.g., Bordetella pertussis, P40 component from Corynebacterium grandis ]; lipopolysaccharide (adjuvant against humoral and cell-mediated immunity); mycobacterium and its components (MDPs, an adjuvant unacceptable in humans); cholera toxin (mucosal adjuvant) ], microbial derivatives (natural and synthetic) [ e.g., monophosphoryl lipid a (mpl); detox (MPL + m. pherei cell wall skeleton); AGP [ RC-529] (synthetic acylated monosaccharides); DC-Chol (a fat-like immunostimulant capable of self-organizing into liposomes); OM-174 (lipid a derivative); CpG motifs (synthetic oligonucleotides containing immunostimulatory CpG motifs); modified LT and CT (bacterial toxins genetically modified to provide a non-toxic adjuvant effect) ], endogenous chicken immunomodulators [ cytokines; an antibody; hGM-SCE or HII-12 (cytokines that can be administered as a protein or encoded plasmid); immudaptin (C3d tandem array); squalene ], a particular adjuvant [ viral particles (a unilamellar liposome vehicle comprising an antigen); AS04([ SBAS4] Al salt and MPL); ISCOMs (structured complexes of saponins and lipids); polylactic polyglycolic acid (PLG) ] and inert carriers (gold particles, silver particles).

Other adjuvants of the invention may include mineral gels, polyanions, pluronic polyols, saponin derivatives, lysolecithin and other similar surface active substances. Still other adjuvants may include: toll-like receptor (TLR) agonists, including, for example, agonists of TLR-1 (e.g., triacyl lipopeptides); agonists of TLR-1 [ e.g., peptidoglycans of gram-positive bacteria (e.g., streptococci and staphyloccci); lipoteichoic acid ]; agonists of TLR-3 (e.g., double stranded RNAs and their analogs, e.g., poly 1: C); agonists of TLR-4 [ e.g., lipopolysaccharide (endotoxin) of gram-negative bacteria (e.g., Salmonella and e.coli) ]; agonists of TLR-5 (e.g., flagellin of a motile bacterium (e.g., Listeria)); agonists of TLR-6 [ e.g., TLR-2 peptidoglycans and certain lipids (diacyllipopeptides) ]; agonists of TLR-7 [ e.g., the single stranded rna (ssrna) genome of viruses such as influenza, measles, and mumps; and small synthetic guanosine-based antiviral molecules such as loxoribine (loxoribine) and ssRNA and their analogs ]; agonists of TLR-8 (e.g., binding ssRNA); agonists of TLR-9 (e.g., unmethylated CpG of DNA of pathogens and analogs thereof); agonists of TLR-10 (function undetermined) and TLR-11 (e.g., binding to several heavy infectious protozoan expressed proteins (Apicompplexi)). Chickens have a well-developed TLR system with approximately 10 TLRs similar to those detected extensively in mammals (broadly).

Further examples of adjuvants of the invention include complement receptors (secreted PRRs) in which C3d (complement component) is activated by the microorganism CHO. The complement pathway leads to opsonization (opsonization) and rapid phagocytosis of pathogens.

In other embodiments, an adjuvant of the invention may be an amino acid sequence that is a peptide, protein fragment, or whole protein that functions as an adjuvant, or the adjuvant may be a nucleic acid that encodes a peptide, protein fragment, or whole protein that functions as an adjuvant. As used herein, "adjuvant" describes a substance that can be any immunomodulatory substance that can be combined with a polypeptide or nucleic acid to enhance, improve or modulate an immune response in an individual without deleterious effects on the individual.

Adjuvants of the invention may be, but are not limited to: for example, immunostimulatory cytokines (including, but not limited to, GM/CSF, interleukin-2, interleukin-12, interferon- γ, interleukin-4, tumor necrosis factor- α, interleukin-1, hematopoietic factor flt3L, CD40L, B7.1 co-stimulatory molecule, and B7.2 co-stimulatory molecule), SYNTEX adjuvant formulation 1(SAF-1) consisting of 5% squalene (wt/vol) (DASF, Parsippany, n.j.) in phosphate buffered saline, 2.5% pluronic, L121 polymer (Aldrich Chemical, Milwaukee), and 0.2% sorbitan (Tween80, Sigma). Suitable adjuvants also include aluminium salts such as aluminium hydroxide gel (alum), aluminium phosphate or algannmulin, but may also be salts of calcium, iron or zinc, or may be insoluble suspensions of acylated tyrosines or acylated sugars, cationically or anionically derivatized polysaccharides or polyphosphazenes.

Other adjuvants are well known in the art and include: N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-N-muramyl-L-alanyl-D-isoglutamine (CGP11637, referred to as N-MDP), N-acetyl-muramyl-L-alanyl-D-isoglutamyl-L-alanine-2- (1 '-2' -palmitoyl-sn-glycero-3-hydroxyphosphonoxy) -ethylamine (CGP19835A, referred to as MTP-PE) and RIBI, it contains three components extracted from bacteria, monophosphoryl lipid a, trehalose dimycolate and cell wall skeleton (MPL + TDM + CWS), in a 2% squalene/Tween 80 emulsion.

Other adjuvants may include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL), and an aluminium salt. Enhanced adjuvant systems include combinations of monophosphoryl lipid a and saponin derivatives, in particular, combinations of QS21 and 3D-MPL, as disclosed in PCT publication No. WO94/00153 (the entire contents of which are incorporated herein by reference), or less reactogenic compositions in which QS21 is quenched by cholesterol, as described in PCT publication No. WO96/33739 (the entire contents of which are incorporated herein by reference). One particularly effective adjuvant formulation comprising QS213D-MPL and tocopherol in oil-in-water is described in PCT publication WO95/17210, the entire contents of which are incorporated herein by reference.

The compositions and methods of the invention are useful for inducing immune responses to treat and/or prevent diseases and disorders, for example, coccidiosis, Marek's disease, infectious bursal disease, newcastle disease, fowlpox infection, Clostridium spp infection (e.g. necrotic enteritis, gangrenous dermatitis, cholangitis, cellulite, ulcerative enteritis, botulism, Tyzzer's disease), avian influenza, infectious bronchitis, chicken anaemia virus infection, avian laryngotracheitis, avian metapneumovirus infection, avian reovirus infection, avian adenovirus infection, rotavirus infection, astrovirus infection, inclusion body hepatitis, chicken egg drop syndrome, adenovirus infection, Escherichia coli infection, Mycoplasma spp.

Thus, in certain embodiments, the compositions of the invention may comprise a species from Marek's disease virus, infectious bronchitis virus, Mycoplasma spp, avian leukosis virus, reovirus, poxvirus, adenovirus, cryptosporidium, chicken infectious anemia virus, Pasteurella, avian influenza virus, Newcastle Disease Virus (NDV), Infectious Bursal Disease Virus (IBDV), Rous sarcoma virus, Escherichia coli, Eimeria species such as eimeriatella (causing coccidiosis), Haemophilus species, Mycoplasma, Listeria species, Salmonella species, Campylobacter species, Clostridium species (e.g., c.peringens, c.septicum, c.sordidi, c.difficile, c.novyi, c.bortulinum, c.colinum, c.gene, chaerula, c.c.c.c.c.and antigens or a combination thereof, or consist of antigens or immunogens thereof, or immunogens thereof.

The present invention is intended to include methods and compositions for immunizing birds against pathogens, which may be pathogens that cause disease in birds and/or pathogens that are carried by birds (contaminated birds) and passed to humans and other animals handling or eating such contaminated birds. Accordingly, the present invention provides compositions comprising, consisting essentially of, and/or consisting of a non-replicating agent that induces an immune response against an avian pathogen that is capable of causing disease in other animals by contacting or ingesting an egg or meat or other body part of a contaminated avian. Such pathogens may include, but are not limited to, Salmonella spp.

In some embodiments, the methods of the invention described herein may further comprise administering to the avian subject post-hatch a booster dose of a composition of the invention.

In still further embodiments of the methods of the invention, an effective immunizing dose of two or more compositions capable of inducing an immune response against an avian pathogen is administered in ovo to the embryo, wherein the two or more compositions are administered simultaneously or sequentially in any order, in any order. Thus, the compositions and methods of the present invention can be accomplished using devices and techniques that apply multiple compositions at a single site, multiple compositions at multiple sites, and/or a single composition at multiple sites. Such methods may utilize a single entry site into the egg or multiple entry sites into the egg. Non-limiting examples of such devices and techniques are described in U.S. Pat. No.4,903,635, U.S. Pat. No.5,136,979, U.S. Pat. No. RE35,973, U.S. Pat. No.5,339,766, U.S. Pat. No.6.032,612, U.S. Pat. No.6,286,455, U.S. Pat. No.5,158,038, U.S. Pat. No.6,601,534, and U.S. Pat. No.6,981,470, each of which is incorporated herein by reference in its entirety.

The invention also includes methods wherein an effective immunizing dose of two or more compositions that induce in ovo administration of the compositions against avian pathogens and at least one composition is administered to the embryo, wherein the two or more compositions are administered simultaneously or sequentially in any order. In some embodiments of these methods, the at least one composition may be applied to the amniotic membrane, which includes the amniotic fluid, the embryoid body, and the yolk sac, and/or the at least one composition may be administered directly into the amniotic fluid, the embryoid body, and/or the yolk sac (separately or in any combination).

In some embodiments of the invention, the method further comprises administering an immunostimulant in ovo at any time during the incubation period, wherein the immunostimulant and composition are administered simultaneously or sequentially in any order. The methods of the present invention may further comprise administering a nutritional formula, an enteric modulator, or a combination thereof in ovo at any time during the incubation period, wherein the nutritional formula, the enteric modulator, or a combination thereof and the composition are administered simultaneously or sequentially in any order.

Several aspects of avian embryo development make the embryos an attractive immunological target. First, since the maximum period of embryo development occurs in eggs outside the maternal reproductive tract, it is easy to introduce a composition (e.g., an immunogenic composition of the invention) into the embryo.

Second, the fact that the egg is a multi-compartmentalized unit can be used to deliver biological material to a specific embryo site. For example, the yolk sac functions in the early embryo to produce blood. Just before hatching, the yolk sac provides the basic nutritional function, which is partly taken into the intestinal tract, thereby turning into cecal pits (cecal pits) during and after hatching. Thus, yolk sac administration of the material may result in delivery of the cecum or vasculature of the embryo. In addition, the composition of the present invention can be efficiently administered by injection onto chorioallantoic membrane or injection onto air sac membrane (air cell membrane). Finally, the muscle tissue compartment of the embryo can be accessed by direct embryo injection at the transfer of the last quarter of incubation, which is usually done on days 17 to 19 of incubation in chickens.

The immunogenic composition may be introduced into any region of the egg, including the air sac, albumen, chorioallantoic membrane, yolk sac, yolk, allantois, amnion, or directly into the embryonated bird. In a particular embodiment of the invention, the composition is introduced into muscle tissue of an embryonic bird, and in other embodiments, the composition is introduced into skeletal muscle tissue. In certain embodiments, introduction of a nucleic acid molecule encoding a protein remaining in a muscle cell can be used to administer an immunogenic protein directly, in particular, directly to a muscle cell. Alternatively, nucleic acid molecules encoding proteins to be secreted from muscle cells can be introduced, and the method can be used to deliver proteins to the entire body of a bird via contact between muscle tissue and plasma. Exemplary skeletal muscle tissue introduction sites are pectoral and pipping muscle tissue, which are located near the eggshell, whereby the injection apparatus is relatively easy to reach without damaging other embryonic structures.

Any suitable means may be used for introducing the composition of the invention in ovo, including in ovo injection, high pressure spraying through the eggshell, and biolistic bombardment of the egg with microparticles carrying the composition. In some embodiments, the composition is administered by allowing an aqueous, pharmaceutically acceptable solution (containing the composition to be allowed to settle) to settle into the muscle.

When in ovo injection is used, the mechanism of injection is not critical, but preferably the method does not unduly damage the tissues and organs of the embryo or the extraembryonic membranes surrounding the embryo so that such treatment does not reduce hatchability. Preferred injection sites are intramuscular and subcutaneous. Preferred intramuscular injection sites are skeletal muscles, more particularly the pectoral muscle and the pipping muscle tissue, which are located near the eggshell, whereby the injection apparatus is relatively easy to reach without damaging other embryo structures and without threatening the protection provided by the eggshell. Syringes equipped with needles of approximately 18 to 26 gauge (g) are suitable for this purpose. Depending on the location of the embryo and the precise stage of development, an 3/4 to 4 inch needle will end up in the liquid on the chicken or in the chicken itself. A guide hole may be drilled or punched in the housing prior to insertion of the needle to prevent damage or dulling of the needle. If desired, the egg may be sealed with a highly bacteria-impermeable sealing material, such as wax or the like, to prevent subsequent unwanted entry of bacteria.

In various embodiments of the invention, the composition is applied to the embryo body with a needle having a length of about 3/4 inches to about 4 inches (e.g., 1 inch, 1.25 inches, 1.5 inches, 1.75 inches, 2.0 inches, 2.25 inches, 2.5 inches, 2.75 inches, 3.0 inches, 3.25 inches, 3.5 inches, 3.75 inches, or 4 inches). Further, the gauge of the needle gauge of the present invention may be in the range of 15g to 28g (e.g., 15g, 16g, 17g, 18g, 19g, 20g, 21g, 22g, 23g, 24g, 25g, 26g, 27g, or 28 g). In some embodiments, the needle may have a flat end, in some embodiments, the needle may have a beveled end, and have a bevel angle of about 10 ° to about 45 ° (e.g., 11 °, 12 °,13 °, 14 °,15 °,16 °,17 °, 18 °, 19 °, 20 °,21 °, 22 °, 23 °,24 °, 25 °,26 °, 27 °,28 °,29 °,30 °,31 °,32 °,33 °, 34 °, 35 °, 36 °, 37 °,38 °, 39 °,40 °, 41 °, 42 °, 43 °,44 °, or 45 °).

In some particular embodiments of the invention, in methods of administering a composition of the invention in ovo, a needle may pass through the shell at the large end of the egg at an offset angle of about 1 ° to about 20 ° from the long axis of the egg (e.g., 1 °,2 °,3 °,4 °,5 °,6 °,7 °,8 °,9 °, 10 °, 11 °, 12 °,13 °, 14 °,15 °,16 °,17 °, 18 °, 19 °, or 20 °).

The invention also provides methods wherein the compositions of the invention are administered in ovo using an automated injection device.

It is envisioned that high speed automatic injection systems for avian embryos are particularly well suited for practicing the present invention. A large number of such devices are available, exemplified by EMBREX INOVOJECT^TMSystems (described in U.S. Pat. No.4,681,063 to Hebrank) and U.S. Pat. Nos.4,040,388, 4,469,047, and 4,593,646 to Miller. The disclosures of these references, and all references cited therein, are incorporated herein by reference. All such apparatus, adapted to be suitable for practicing the invention, comprise a syringe containing DNA as described herein, the location of the syringe allowing injection of the device-loaded egg with DNA. In addition, a sealing device may be provided in operative communication with the injection device to seal the aperture of the egg after injection thereof.

Presently preferred apparatus for carrying out the invention are disclosed in U.S. Pat. No.4,681,063 to Hebrank and U.S. Pat. No.4,903,625 to Hebrank, the disclosures of which are incorporated herein by reference. The apparatus comprises injection means for delivering fluid substance into a plurality of eggs, and suction means capable of simultaneously engaging and lifting a plurality of individual eggs from their upper facing portion, which cooperates with the injector to inject the eggs when the eggs are engaged by the suction means. The features of the device may be combined with those of the device described above for carrying out the invention. Those skilled in the art will appreciate that the apparatus may be adapted for injection into any portion of an egg by adjusting the penetration depth of the injector, as will be discussed in more detail below.

Embodiments of injection methods and devices that may be used in the present invention are described in U.S. Pat. No.6,032,612 (multi-site in ovo injection device), U.S. Pat. No.6,244,214 (device for in ovo injection and detection of information related to the interior of an egg), U.S. Pat. Nos.6,176199, 6,510,811, and 6,834,615 (method of confining allantoic fluid within an egg), U.S. Pat. No.7,089,879 (method for operating a balloon in an avian egg), and U.S. Pat. No.7,165,507 (method and device for accurately positioning a device within the subgerminal cavity of an egg), the entire contents of each of which are incorporated herein by reference.

Thus, in some embodiments, the methods and apparatus used are substantially as described in one or more of the previously listed patents, which include positioning an extended syringe (or syringe needle) at the large head end of an egg and an offset angle (a) with respect to the long axis of the egg, the offset angle being selected such that the needle is directed toward the shoulder or chest of the embryo. The needle is then inserted through the egg shell and into the egg along a substantially linear path deep enough to enter the shoulder or chest of the embryo. The material to be deposited in the egg, which may be a liquid or a (e.g. injectable) solid that can be administered by syringe (but is typically an aqueous liquid containing the composition of the invention described herein), is then injected via a needle. In some embodiments, the needle is removed along a substantially linear path, and the step of injecting the substance is performed simultaneously with the step of removing the needle such that the substance is administered along the path within the egg. The offset angle (a) is sufficient to enhance the likelihood of injection into the shoulder or pectoral muscles. Typically, the angle is 1 to 20 degrees, preferably, the angle is 2 to 3 degrees. For example, the needle may be inserted deep enough under the egg shell to enter or enter and pass through the shoulder or chest of the embryo. The apparatus may be modified to include means operatively associated with the apparatus to enable the position of the egg relative to the needle to attain said angle (a), for example by angularly positioning and orienting the needle relative to the suction device.

In one particular example, the methods of the present invention may be practiced with the apparatus described in U.S. Pat. No.6,244,214 to Herbrank, the entire contents of which are incorporated herein by reference, which describes an apparatus (i.e., "smart probe") for identifying specific structures and/or compartments within an egg that are in contact with a needle that penetrates the shell of the egg, and methods of using the apparatus to deliver compositions into specific structures and/or compartments within an egg.

Accordingly, in certain embodiments, the present invention provides a method of introducing a substance into the muscle of a chicken in ovo, the method comprising:

a) obtaining an egg, wherein the egg contains chick embryos in the last season of incubation prior to hatching; b) positioning an extension syringe needle at an offset angle of about 1 to 5 degrees relative to the long axis of the egg to the large end of the egg, the angle selected such that the needle is directed toward the shoulder or chest of the embryo; c) inserting said needle along a substantially linear path through the shell of said egg to a depth of about 7/8 inches to 1.5 inches into the shoulder or chest of said embryo; and d) injecting the substance into the egg via the needle.

In some further embodiments, provided herein are methods of introducing a substance into the muscle of a chicken in ovo, comprising: a) obtaining an egg, wherein the egg comprises 17-19 days old chick embryos; b) positioning an extension syringe needle at an offset angle of about 1 to 5 degrees relative to the long axis of the egg to the large end of the egg, the angle selected such that the needle is directed toward the shoulder or chest of the embryo; c) inserting said needle along a substantially linear path through the shell of said egg to a depth of about 7/8 inches to 1.5 inches into the shoulder or chest of said embryo; and d) injecting the substance into the egg via the needle. In such methods, the needle may be inserted deep enough to enter and pass through the shoulder or chest of the embryo.

Also provided herein is an apparatus for injecting simultaneously in chick embryo muscle tissue of a plurality of eggs during days 17 to 19 of incubation, the apparatus comprising: an engaging device for engaging the plurality of eggs; an injection tool cooperating with the articulating tool for inserting an extension needle through the shell of the egg along a substantially linear path to a depth of about 7/8 inches to 1.5 inches to a shoulder or chest of the embryo; and positioning means for positioning the extended injection needle at an offset angle of about 1 to 5 degrees relative to the long axis of the egg to the large head end of the egg such that the needle is directed toward the shoulder or chest of the embryo. In such devices, the engaging means may comprise suction means for simultaneously lifting a plurality of individual eggs.

In other embodiments of the invention, compositions and methods are provided for inducing an immune response against a Clostridium species in an avian subject. For example, acute enterotoxemia in birds known as necrotic enteritis is the result of Clostridium perfringens (type a and C have been linked to avian disease). When feed and litter contaminated with clostridium are ingested by birds, necrotic enteritis may occur and organisms will grow in the gut and then sporulate. The sporulation process results in the release of alpha and beta toxins, the action of which results in intestinal necrosis (particularly in the jejunum and ileum). Clinical indications include: loss of semen, loss of appetite and diarrhea. Acute death may occur. Predisposing factors for perifringens infection and necrotic enteritis include damage to the diet and intestinal mucosa. In the commercial poultry field, the majority of cases of necrotic enteritis occur in broilers (broiler chicken) at 2 to 5 weeks of age. Thus, some particular embodiments of the invention relate to immunogenic compositions and methods for protecting birds against Clostridium perfringens and necrotic enteritis, for example, by reducing the rate of infection and/or by reducing the severity of infection and/or disease.

The invention will be described below with reference to some particular embodiments thereof. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the specification and the appended claims, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

As used herein, "and/or" means and includes any and all combinations of one or more of its associated listed items and the absence of such combinations when expressed in alternative form ("or").

Furthermore, when referring to a measurable value (e.g., amount, dose, time, temperature, etc. of a compound or agent of the invention), the term "about" as used herein is meant to encompass variations of ± 20%, ± 10%, ± 5%, ± 1%, ± 0.5% or even ± 0.1% of that particular amount.

The terms "avian" and "avian subject" or "bird" or "avian subject" as used herein are intended to include both the male and female of any bird or bird, and in particular, are intended to include poultry raised commercially for eggs, meat or as pets. Thus, the terms "avian" and "avian subject" or "avian subject" include chickens, turkeys, ducks, geese, quail, pheasants, parakeets, parrots, macrocrowigars, macadamias, ostriches, emus, and the like. Commercial poultry includes broiler chickens and egg-laying chickens, which are raised for the production of meat and eggs, respectively.

In some particular embodiments, the subject is one at risk of or suspected of having an infection or disease caused by a Clostridium species (e.g., necrotic enteritis caused by Clostridium perfringens infection). Risk factors for necrotic enteritis are known in the art and include, but are not limited to, dietary factors (e.g., diets high in wheat, barley, rye, or fish), poor litter conditions, and/or exposure to Eimeria (e.g., natural exposure or live Eimeria vaccine). Thus, in some particular embodiments, the compositions and methods of the invention may be advantageously used to reduce the severity and/or incidence (incidence) of necrotic enteritis in birds that have been vaccinated against coccidiosis or in a flock of birds (flock) that are experiencing a coccidiosis outbreak.

Other diseases and disorders caused by Clostridium infection of birds include, but are not limited to: necrotic enteritis, gangrenous dermatitis, cholangiohepatitis, cellulite, ulcerative enteritis, botulism, and Tyzzer's disease. The present invention therefore provides immunogenic compositions and uses thereof to protect avians against infection by, for example, Clostridium perfringens, c.septicum, c.sordelii, c.difficile, c.novyi, c.botulinum, c.coli, c.chauvoei, c.fallax, c.sporogenes and/or c.piliform.

The avian subject of the present invention may be a live in ovo embryonic bird, or may be a hatched bird, including newly hatched (i.e., about 1, 2, or 3 days post-hatch), young and adult birds.

In some special casesIn embodiments, the bird is about 6,5, 4, 3, 2, or 1 week old or less. In other representative embodiments, the avian subject is "tianzhen: (a) ((b))) An "individual, i.e., one that has not been previously exposed to the antigen to which it is desired to immunize.

Unless otherwise indicated, the various parts of the word "administered in ovo" as used herein means: the immunogenic composition (e.g., vaccine) is administered to an avian egg containing a live, developing embryo by any means that penetrates the eggshell and introduces the immunogenic composition. Such modes of administration include, but are not limited to, in ovo injection of the immunogenic composition.

The present invention provides methods of administering an immunogenic composition to an individual to induce an immune response, optionally a protective immune response, against Clostridium species in said individual. The immunogenic composition may be administered to any suitable compartment of an egg (e.g., the allantois, the yolk sac, the amnion, the air sac, and/or into the avian embryo itself), as will be apparent to those skilled in the art. Methods of administration into the embryo include, but are not limited to: parenteral administration, e.g., subcutaneous, intramuscular, intraperitoneal, intravenous, and/or intraarticular administration. In some particular embodiments, the immunogenic composition is administered to the amniotic membrane (e.g., by axial injection through the large end of an egg).

The immunogenic composition can be administered to the egg by any suitable method. In some particular embodiments, the immunogenic composition is administered by injection. The mechanism of egg injection is not critical, but generally should be selected such that the method does not damage the tissues and organs of the embryo or the extraembryonic membrane surrounding the embryo to such an extent that the treatment does not unduly reduce hatchability. Syringes equipped with needles of approximately 18 to 23 gauge are generally suitable for this purpose. Examples of needles suitable for the present invention include needles having the following gauge: 18. 19, 20, 21, 22 or 23 gauge. The needle length of the present invention may be at least 1/2 inches, 5/6 inches, 3/4 inches, 7/8 inches, 1 inch, 1 and 1/4 inches, 1 and 3/8 inches, 1 and 1/2 inches, 1 and 5/8 inches, 1 and 3/4 inches, 1 and 7/8 inches, or 2.0 inches. The beveling of the needle of the present invention ranges from about 5 to about 45 degrees (e.g., 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 45, 56, 37, 38, 39, 40, 41, 42, 43, 44, or 45 degrees). In certain embodiments of the invention, the beveling of the needle of the present invention ranges from about 12 degrees to 20 degrees. A guide hole may be drilled or punched in the housing prior to insertion of the needle to prevent damage or dulling of the needle. If desired, the egg may be sealed with a highly bacteria-impermeable sealing material, such as wax or the like, to prevent subsequent unwanted entry of bacteria.

High speed automatic injection systems for avian embryos are particularly suitable for practicing the present invention. A large number of such devices are available, exemplified by those disclosed in U.S. patent nos.4,681,063 and 4,903,635 to Hebrank and U.S. patent nos.4,040,388, 4,469,047 and 4,593,646 to Miller, each of which is incorporated herein by reference in its entirety. Such apparatus, adapted to be suitable for carrying out the invention, typically comprises a syringe containing the immunogenic composition, the location of the syringe being such that the egg carried by the device is injected with the immunogenic composition. Further, if desired, a sealing device may be provided in operative communication with the injection device to seal the aperture of the egg after injection of the egg.

In one embodiment, the apparatus for practicing the present invention may be as disclosed in U.S. Pat. No.4,681,063 to Hebrank and U.S. Pat. No.4,903,625 to Hebrank, the disclosures of which are incorporated herein by reference. The apparatus comprises an injection device for delivering fluid substance into a plurality of eggs, and a suction device capable of simultaneously engaging and lifting individual eggs from their upper facing portion, which cooperates with the injector to inject the eggs when the eggs are engaged by the suction device. Those skilled in the art will appreciate that the apparatus may be adapted for injection into any portion of an egg by adjusting the penetration depth of the injector (as is known in the art). The methods of the present invention may also be practiced with the devices and methods described in U.S. patent No.6,244,214 to Herbrank, the entire contents of which are incorporated herein by reference, wherein devices for identifying specific structures and/or compartments within an egg that are in contact with a needle that penetrates the shell of the egg (i.e., "smart probes") are described, as well as methods for using such devices to deliver compositions into specific structures and/or compartments within an egg.

The appropriate volume of immunogenic composition to be administered will depend on the size of the egg being treated, and ostrich eggs are significantly capable of accommodating more volume than eggs. In some particular embodiments, the immunogenic composition is administered in a volume of about 10 to about 500, 1000, or 2000 μ Ι or more, including any number between 10 and 2000, even including numbers not explicitly mentioned herein, exemplary volumes include 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 626, 650, 675, 700, 725, 752, 775, 800, 850, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 μ Ι. Other suitable volumes for delivery of the immunogenic composition can be readily determined by one skilled in the art.

According to some particular embodiments of the invention, the egg (i.e., the embryonated bird) to which the immunogenic composition is administered is in the second half or the last quarter of in ovo incubation (i.e., embryo development). For example, for an egg, the second half of incubation begins from about day 12 to 20 of incubation (e.g., E12, E13, E14, E15, E16, E17, E17.5, E18, E18.5, E19, E19.5, and/or E20), and the last quarter of in ovo incubation begins from about day 15 to 20 of incubation (e.g., E15, E15.5, E16, E16.5, E17, E17.5, E18, E18.5, E19, E19.5, and/or E20). In some particular embodiments, the immunogenic composition is administered to the egg on about day 18(E18 or E18.5) or day 19(E19 or E19.5) of in ovo incubation. In other embodiments, the immunogenic composition is administered to the turkey egg on about day 14 to 27 of incubation (E14, E15, E16, E17, E18, E19, E20, E21, E21.5, E22, E22.5, E23, E23.5, E24, E24.5, E25, E25.5, E26, and/or E27), about day 21 to 27 of incubation (e.g., E21, E21.5, E22, E22.5, E23, E23.5, E24, E24.5, E25, E25.2, E26, E26.5, and/or E27), or about day 25(E25 or E25.5). The skilled artisan will appreciate that the invention can be practiced in ovo at any predetermined time, so long as the administration results in a desired immune response to the immunogenic composition without an excessive level of morbidity (morbidity) and/or mortality in the treated individual.

Alternatively or additionally, the invention may be practiced to administer the immunogenic composition to hatched birds, including newly hatched (i.e., about 1, 2, or 3 days post-hatch), young, and/or adult birds. In certain embodiments, administration is performed within the first 6,5, 4, 3, and/or 2 weeks after hatching and/or even within about one week after hatching. According to one aspect of the invention, the administration is performed within the first three weeks after hatching. In other embodiments, the immunogenic composition is administered in ovo (e.g., during the last quarter of incubation in ovo), and the booster dose is administered post-hatch (e.g., within about 1, 2, or 3 days or 1, 2, or 3 weeks post-hatch).

The method of the present invention differs from maternal vaccination, in which older females (e.g., hens aged about 10-15 weeks) are vaccinated with the goal of providing passive immunity to their offspring. Such birds may not be "indigenous", i.e. they have been exposed to Clostridium (e.g. Clostridium perfringens), and are not immunized in order to protect the vaccinated birds, but to protect the offspring by passive transfer of antibodies.

The immunogenic composition of the invention may be administered to the hatched bird by any suitable means. Exemplary means are oral administration (e.g., in feed or drinking water), intramuscular injection, subcutaneous injection, intravenous injection, intraabdominal injection, eye drops, and/or nasal spray. Furthermore, the immunogenic composition may be administered to the bird in the form of a spray booth (i.e., a booth in which the bird is placed and exposed to a mist containing the vaccine), or by a moving spray (coarse spray).

The present invention may be practiced to protect birds against necrotic enteritis. The various morphological forms of "protection" and similar terms mean any level of protection against necrotic enteritis that is beneficial to the population of individuals such that the incidence and/or severity of the disease is reduced in the treated birds, which is manifested in the form of decreased mortality, decreased wounds, increased weight gain, increased feed conversion efficiency, and/or any other deleterious effects of the disease, whether protection is partial or complete. One skilled in the art will appreciate that protection can be determined by comparing a plurality of treated birds to untreated birds, even if individual treated birds are not protected. In some particular embodiments, the invention provides methods of reducing the incidence of necrotic enteritis in a plurality of birds administered an immunogenic composition of the invention. The invention also provides methods of reducing morbidity and/or mortality in a plurality of birds treated according to the invention.

As used herein, "prepared" and its various forms of speech ("prime", or "priming") means eliciting an active immune response that is not sufficiently protective until a second dose (boost) is administered at a later post-hatch time period.

"reduce" and its various parts of the word "reduce" as used herein means a reduction in an indicated infection-related or disease-related parameter (e.g., incidence of necrotic enteritis, infection, morbidity, mortality, wound, etc.), which is of value or benefit (e.g., commercial value) to the user, e.g., a reduction of about 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97% or more, as compared to untreated birds.

The present invention also provides a method of protecting birds against infection by Clostridium species, which results in any level of protection that is beneficial to the population of individuals, such that the incidence and/or severity of Clostridium infection in treated birds is reduced.

The invention may also be practiced to induce an immune response against Clostridium. As used herein, the term "inducing (or grammatical variants thereof) an immune response against Clostridium" is intended to include agents that induce an immune response against the organism itself and/or toxins produced by the organism, either by passive transfer or active immune response. Optionally, the immune response induced is a protective immune response, e.g., in a vaccination method. Protection is not necessary if there are other purposes to induce an immune response, such as for research purposes, or to generate antibodies for passive immunization or as reagents (e.g., to detect, isolate and/or identify Clostridium species).

As used herein, unless otherwise indicated, "c.perfringens" is intended to include c.perfringens type a and/or c.perfringens type C and/or any other c.perfringens type implicated in the etiology of avian necrotic enteritis. In some particular embodiments, the present invention provides methods of protecting birds against infection by C. The invention also provides methods of inducing an immune response against type C. Different types of c. perfringens and strains thereof are well known in the art. See, for example, AMERICAN ASSOCIATION OF AVIAN PATHOLOGISTS, A LABORATORY MANUALFOR THEIOLATION OF PATHOGENS (3 d.ed.1989).

Unless otherwise indicated, the term "effective immunizing dose" as used herein refers to a dose of the immunogenic composition sufficient to induce a protective immune response in the treated bird that is higher than the innate immunity of the non-immunized bird. In the context of in ovo treatment of an avian, "effective immunizing dose" means a dose sufficient to induce a protective immune response in a hatched avian treated in ovo that is greater than the innate immunity of an avian not treated in ovo. In any particular context, effective immunizing doses may be routinely determined using methods known in the art.

An "effective immunizing dose" may comprise one or more (e.g., two or three) doses of the immunogenic composition to achieve the desired level of protection. Each dose may be administered in ovo and/or post-hatch.

As discussed above, the effectiveness of a dose and/or immunogenic composition can be assessed by evaluating the effect of vaccination on the flock as a whole, for a person skilled in the art when treating a plurality of birds (e.g. in commercial poultry production). In other words, an effective immunizing dose or effective vaccine for the avian population as a whole may not induce an immune response and/or provide adequate protection against disease in certain individual birds.

The terms "vaccination" or "immunization" are well known in the art and are used interchangeably herein. For example, the term "vaccination" or "immunization" may be understood as a process in which: it increases the immune response of an individual to an antigen (by providing an active immune response) and thereby increases its ability to fight, overcome, and/or recover from an infection (i.e., a protective immune response).

The term "protective immunity" or "protective immune response" as used herein is intended to mean: the host animal has an active immune response against the immunogenic composition and/or the immunogenic composition provides passive immunity such that upon subsequent exposure or challenge, the animal is able to combat or overcome the infection and/or disease. Thus, a protective immune response will reduce the prevalence and/or mortality caused by subsequent exposure to pathogens in treated birds.

An "active immune response" or "active immunity" is characterized by: "host tissue and cell involvement after encountering an immunogen. This includes differentiation and proliferation of immunoreceptive cells in the tissues of lymphoreticular endothelial cells, which leads to synthesis of antibodies and/or development of cell-mediated reactivity, or both, "Herbert b. herscowitz, Immunophysiology: cell functions and cellular Interactions in Antibody Formation, in IMMUNOLOGY: BASICPROCESSES 117(Joseph A. Bellanti ed., 1985). In other words, an active immune response is possessed by the host upon exposure to an immunogen, either by infection or by vaccination. Active immunity differs from passive immunity, which is the transfer of a substance prepared in advance (antibody, transfer factor, thymic transplant, interleukin-2) from an actively immunized host to a non-immunized host. And Id.

Necrotic Enteritis (NE) models for assessing the efficacy of vaccines and vaccination strategies are known in the art. For example, Hofacre et al (2003, J.appl.Poult.Res.12: 60-64) describes a model in which chickens are fed a corn soybean diet with 26% fish flesh on days 0 to 14 post hatch. Fish meat was removed from the diet starting on day 14. Birds were challenged with diplococcus by oral gavage on day 14 and then with c.perfringens by oral gavage daily from days 17-19. Feed conversion rate, body weight and score for intestinal injury were used to assess the presence and severity of necrotic enteritis in challenged birds. NE wounds were assessed on day 22 or day 28 using the following scores: none, 1 mild, 2 moderate, and 3 significant/severe. Other models known in the art can be used to assess vaccine efficacy and vaccine regimens. In certain embodiments of the invention, administration of a composition of the invention (e.g., an effective amount of a composition of the invention) can result in a reduction in intestinal wound and/or weight change of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 75, 80, 90, or 100% in an animal of the model or other models known or accepted in the art as compared to an unimmunized animal or control animal.

In some other embodiments of the invention, the compositions and methods of the invention can be used to induce an antibody response in an avian that is at least greater than or equal to about 0.5 antitoxin units per mL (e.g., at least about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10a.u. of antitoxin antibody per mL of avian antiserum).

In still other embodiments of using the compositions and methods of the invention, the percentage of eggs in a plurality of eggs in which a composition of the invention is delivered into the embryo body can be from about 70% to about 100% (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) of the total number of eggs in a plurality of eggs to which the composition is administered.

The invention also includes immunogenic compositions which are capable of inducing an active and/or passive immune response against c.perfringens (including type a and/or C), optionally useful for protecting birds against c.perfringens infection and/or protecting them against necrotic enteritis, as described in more detail above.

The immunogenic composition of the invention comprises and/or consists essentially of, and/or consists of an agent capable of inducing an immune response against Clostridium. The immunological agent of Clostridium may be a replicating antigen and/or a non-replicating antigen. Replicative and non-replicative antigens of the invention may be delivered to the amniotic membrane, the embryo and/or both the amniotic membrane and the embryo in ovo.

Furthermore, the immunogenic compositions of the invention comprise and/or consist essentially of, and/or consist of, an effective immunizing dose of Clostridium immunizing agent in a pharmaceutically acceptable carrier. In some representative embodiments, the immunogenic composition can be formulated with Clostridium toxoid and/or bacterin. According to this embodiment, the immunogenic composition optionally further comprises an adjuvant (see below). Toxoids are inactivated toxins, which are obtainable from Clostridium toxins, including: those obtained from c. perfringens, including alpha toxin, beta 2 toxin, enterotoxin,A toxin, iota toxin, kappa toxin, lambda toxin and/or theta toxin; sordelii, including hemorrhagic and/or lethal toxins; those obtained from c.difficile, including a toxin (enterotoxin) and B toxin (cyto-degenerative toxin); those obtained from c.septicum, including alpha toxin; novyi, including alpha toxin and/or beta toxin; and/or those obtained from C. Methods of making toxoids are known in the art and include, for example, formaldehyde treatment or heat treatment of the toxin (see, e.g., Walker, (1992) Vaccine 10: 977-. Bacterins are cellular components of bacteria, which may be derived from Clostridium species, e.g., from c.perfringens type a and/or c.perfringens type c. Perfringens toxoid vaccines are known in the art (see, e.g., U.S. patent No.4,292,306 to Zemlyakova).

In other embodiments, the immunogenic composition comprises, consists essentially of, or consists of a killed (i.e., non-replicating) Clostridium bacterium (i.e., a bacterin) (optionally in a water-in-oil-in-water emulsion, see, e.g., U.S. patent No.5,817,320 to Stone, which describes in ovo immunization of avian embryos with an oil emulsion vaccine, the entire contents of which are incorporated herein by reference), and/or a pharmaceutically acceptable carrier. In other embodiments, the immunogenic composition comprises or consists essentially of killed Clostridium and an adjuvant (e.g., an adjuvant derived from aluminum (e.g., aluminum hydroxide), a saponin (e.g., Quil-a, including QuilA QS21), or an oil, e.g., complete or incomplete Freund's), optionally a water-in-oil emulsion, and/or a pharmaceutically acceptable carrier.

As another alternative, the immunogenic composition comprises, consists essentially of, or consists of a replicating immunological agent of Clostridium, such as live c.perfringens, which is typically live attenuated (i.e., having reduced virulence) c.perfringens (see, e.g., PCT publication No. PCT WO2005/053737, the entire contents of which are incorporated herein by reference for the teaching of producing live attenuated bacteria for vaccine use). Methods for producing attenuated bacteria are known in the art and include, but are not limited to: irradiation, chemical treatment, sequential passage in culture, etc. In certain embodiments of the invention, a live Clostridium bacterium (e.g., c. perfringens) is administered in the presence of an agent that protects the individual against the pathological effects of the organism, for example, by co-administration of a neutralizing factor (as described in U.S. patent No.6,440,408 to Thoma et al) or an interferon (as described in U.S. patent No.6,506,385 to Poston et al). Optionally, Clostridium and a neutralizing factor and/or interferon are administered in the same formulation.

Other examples of immunogenic compositions include those comprising, consisting essentially of, or consisting of antitoxin (i.e., an antibody that provides passive immunity to Clostridium alpha and/or beta toxin, see, e.g., U.S. patent No.5,719,267 to carrolet al.), an antigenic peptide capable of inducing an immune response against Clostridium (including c.perfringens toxin; see, e.g., U.S. patent nos.5,817,317 and 5,851,827 to Titball et al; U.S. patent No.6,610,300 to Segers et al; U.S. patent No.5,695,956 to mccrene et al) and recombinant vaccines comprising a carrier nucleic acid, such as a plasmid or virus, capable of delivering a nucleic acid encoding an antigenic peptide or protein capable of inducing an immune response against Clostridium.

In some representative embodiments, the immunogenic compositions comprise recombinant Clostridium alpha and/or beta toxins, e.g., having the amino acid sequence shown in SEQ ID nos.5,817,317 and 5,851,827, herein and/or, for example, in Titball et al: 2[370 amino acids of the full-length sequence; GenBank accession No.1GYGB (GI: 21730290), the coding sequence of which is set forth herein as SEQ ID NO: 1 providing]、SEQ ID NO：4(Cpa_247-370(ii) a SEQ ID NO: amino acid 247-370 of 2; the coding sequence of which is designated herein as SEQ ID NO: 3), SEQ id no: 6 (amino acids 1-278 of SEQ ID NO: 2; the coding sequence of which is provided herein as SEQ ID NO: 5), SEQ ID NO: 8 (Cpa)_261-300(ii) a SEQ ID NO: amino acid 261- > 300 of 2; the coding sequence of which is set forth herein as SEQ ID NO: 7, or SEQ id no: 10[398 amino acids full length sequence, the coding sequence of which is set forth herein as SEQ id no: 9 supply of]Alpha toxin of the amino acid sequence shown. In some embodiments, the toxin of the invention is an immunogenic composition comprising amino acids 1-278(SEQ ID NO: 6) of the 370 amino acid sequence (SEQ ID NO: 2) of the Clostridium alpha toxin.

Other examples of toxins, including immunogenic fragments thereof, useful in the compositions and methods of the invention include, but are not limited to, C.perfringens toxin [ e.g., alpha toxin, accession number CAA35186(Saint-Joanis et al. mol. Gen. Genet.219 (3): 453-460(1989) 0; beta toxin, accession number CAA58246(Steinthorsdottir et al. FEMSMimcrobiol. Lett.130 (2-3): 273-278 (1995)); beta 2 toxin, accession number NP-150010 (Shimizu et al. Proc. Natl. Acad. Sci. U.S.A.2006 (2): 996-1001 (2002)); enterotoxin, accession number BAE79112(Miyamoto et al. J. Bacteriol.188(4 1585): 1595-1598 (1598));toxin, accession No. AAA23236(Havard et al, FEMSMiicrobiol. Lett.97: 77-82 (1992); iota toxin, accession No. CAA51959(Perelle et al, infection. Immun.61 (12): 5147-; lambda toxin, accession number CAA35187(Saint-Joanis et al. mol. Gen. Genet.219 (3): 453-460 (1989)); and theta toxin, accession number NP-561079 (Shimizu et al Proc. Natl. Acad. Sci U.S.A.99 (2): 996-]Difficile toxins [ e.g., toxin A, accession number A37052(Wren et al. FEMSMICrobiol. Lett 70: 1-6(1990)), and toxin B, accession number CAA43299(von Eichel-Streiber et al. mol. Gen. Genet.233 (1-2): 260-268(1992))]C.septicum toxin [ alpha toxin, accession No. AAB32892(Ballardet al. infection. Immun.63 (1): 340-]And c. novyi toxins [ e.g.,alpha toxin, accession number AAB27213(Ball et al. infection. Immun.61 (7): 2912-2918(1993))]。

The terms "toxin", "alpha toxin", "beta toxin",toxins "(or similar terms) and the like, as used herein, include: full-length toxins as well as antigenic peptides or immunogenic variants thereof (e.g. attenuated) that are capable of inducing an immune response (optionally a protective immune response) against Clostridium in an individual. In some particular embodiments, the antigenic peptide comprises at least about 6,8, 10, 12, 15, 18, 20, 25, 30, 50, 75, or 100 or more contiguous amino acids of a full-length toxin (see, e.g., the full-length alpha toxin sequences shown in SEQ ID NO: 2 and SEQ ID NO: 2 in U.S. Pat. Nos.5,817,317 and 5,851,827).

It is also understood that the immunogenic fragments of the invention may be combined in any order or number. For example, fragments 1-10 can be combined with fragments 10-20 to produce fragments of amino acids 1-20. As another example, fragments 1-20 can be combined with fragments 50-60 to produce a single fragment of the invention having 31 amino acids (AA10-20 and AA 50-60). Furthermore, the fragments may be present in any number of pieces and in any combination of the fragments of the present invention. Thus, for example, fragments 1-150 can be combined with second fragments 1-150 and/or with fragment 400-500 to produce fragments of the invention.

In some embodiments, the antigenic or immunogenic fragment of a Clostridium toxin of the present invention may comprise and/or consist essentially of and/or consist of the amino terminal domain of the alpha toxin of C.perfringens (amino acids 1-246 of SEQ ID NO: 2), the carboxy terminal domain of the alpha toxin of C.perfringens (amino acids 256-370 of SEQ ID NO: 2) and/or a fragment between the above domains (amino acids 247-255 of SEQ ID NO: 2) in any combination and any number of overlaps in the amino acid sequence that result in a fragment with immunogenic activity. This statement is intended to include all possible toxin peptides and fragments as specifically set forth herein (e.g., any peptide or fragment comprising at least about 6,8, 10, 12, 15, 18, 20, 25, 30, 50, 75, or 100 or more contiguous amino acids of SEQ ID NO: 2 of U.S. patent nos.5,817,317 and 5,851,827 and the full-length alpha toxin sequence set forth herein as SEQ ID NO: 10). In some particular embodiments, the antigenic peptide lacks an amino acid sequence having phospholipase C and/or sphingomyelin hydrolyzing activity (i.e., the antigenic alpha toxin peptide may lack amino acids 1-240). The localization of some c. perfringens alpha toxin epitopes has been determined (see, e.g., logane et al, (1992) Infection and Immunity 59: 4338-4382, the entire contents of which are incorporated herein by reference for teaching on alpha toxin epitopes).

Other examples of recombinant Clostridium toxins that can be used in the methods of the present invention include, but are not limited to, Clostridium perfringens beta toxin or a fragment thereof, wherein said beta toxin has the amino acid sequence of seq id NO: 11, or a pharmaceutically acceptable salt thereof. SEQ ID NO: the beta toxin of 11 may also comprise a mutation at one of the regions between amino acids 62, 182, 197 or amino acids 80-103, 145-147, 281-291, 295-299 or downstream of amino acid position 292 (as described in U.S. Pat. No.6,610,300, which is incorporated herein by reference in its entirety), wherein the resulting toxin or fragment thereof has immunogenic activity.

The nucleic acid and amino acid sequences of perfringens alpha and beta toxins are known in the art, see, e.g., GenBank accession nos. dq202275, NP _560952, NC _003366, AY823400, AY277724, AF204209, X17300, X13608, L43548, L43547, L77965, and L13198. See also, Sheedy et al, high ply Conserved Alpha-Toxin Sequences of Avian Isolates of Clostridium perfringens, j.clin.microbiol.42: 1345-1347(2004), which shows the analysis of the alpha toxin sequence of the c. perfringens strain from 25 chickens.

In still other embodiments of the invention, the Clostridium toxin may be cA toxin having the amino acid sequence of SEQ ID NO: 12(328 amino acids) or SEQ id no: 13, or a pharmaceutically acceptable salt thereof. In still other embodiments of the present invention, the substrate may be,the toxin may comprise seq id NO: 13, wherein residue 2 is proline, as described in U.S. patent No.6,403, 094, the entire contents of which are incorporated herein by reference.

In certain embodiments, the present invention provides methods of immunizing an avian subject against Clostridium infection, the method comprising: an effective immunizing dose of the Clostridium bacterin-toxoid composition is administered to the avian subject by in ovo injection during the final quarter of the incubation. The methods of the invention may further comprise the step of administering a booster dose of the Clostridium bacterin-toxoid composition to the avian subject after hatching. Species of Clostridium of the present invention include, but are not limited to Clostridium perfringens. In some particular embodiments of the invention, the composition may comprise VisionA vaccine. In some particular embodiments where the individual is a chicken, the bacterin-toxoid composition may be administered to the amniotic fluid on day 18 of incubation through a 20g, 1.0 inch needle, or during day 18 of incubation through a 22g, 1.0 inch needle.

In some other embodiments of the invention, there is provided a method of immunizing an avian subject against Clostridium infection, the method comprising: administering to the avian subject an effective immunizing dose of recombinant Clostridium toxin or immunogenic fragment thereof by in ovo injection during the final quarter of incubation. In some embodiments, the methods may further comprise the step of administering a booster dose of the recombinant toxin or immunogenic fragment thereof to the avian subject post-hatch. In some particular embodiments, the compositions used in these methods may comprise an adjuvant, which may be Quil a and incomplete Freund's adjuvant. In some embodiments wherein the individual is a chicken, the bacterin-toxoid composition may be administered to the embryo bodies through a23 g, 1.25 inch needle during day 19 of incubation.

In still other embodiments, when the individual is a chicken, the toxin of the invention or immunogenic fragment thereof can be administered to the embryo body through a 20g, 1.5 inch needle on day 19 of incubation. In addition, toxins (e.g., alpha toxin) or immunogenic fragments thereof and/or other subunit proteins or glycoproteins or other types of biomolecules used as vaccines of the invention can be administered in amounts of about 1 μ g to about 1000 μ g per dose, with an exemplary range of about 55 μ g to about 60 μ g per dose.

For compositions of the invention comprising inactivated virus, the virus concentration per dose is about 10³EID₅₀/TCID₅₀To about 10¹²EID₅₀/TCID₅₀(EID ═ egg infection dose; TCID ═ tissue culture infection dose). In embodiments comprising activated virus, the virus concentration per dose may be about 10^0.1EID₅₀/TCID₅₀To about 10¹²EID₅₀/TCID₅₀. In some particular embodiments of the invention, the toxin of the invention comprises SEQ ID NO: 2. 4,6, 8, or 10 (including any combination thereof), consisting essentially of and/or consisting of the amino acid sequence of SEQ ID NO: 2. 4,6, 8, or 10 (including any combination thereof).

As shown herein, in some embodiments of the invention, the compositions of the invention further comprise an adjuvant, which in particular embodiments may be an aluminum-derived adjuvant (e.g., aluminum hydroxide), a saponin (e.g., Quil-a, including QuilA QS21), or an oil (e.g., complete or incomplete Freund's adjuvant) in any combination. Other examples of adjuvants useful in any of the methods of the invention described herein are provided herein.

In some representative embodiments, the immunogenic compositions of the invention comprise cToxin, kappa toxin, lambda toxin, theta toxin and/or iota toxin, optionally in addition to the alpha and/or beta toxins of c.

In other representative embodiments, the immunogenic composition comprises, consists essentially of, or consists of a toxoid or toxoid/bacterin. The bacterins may be type a and/or type C bacterins of C. For example, exemplary immunogenic compositions comprise, consist essentially of, or consist of an alpha toxoid and a c.perfringens type a bacterin. Optionally, the immunogenic composition further comprises an adjuvant, such as an adjuvant derived from aluminum (e.g., aluminum hydroxide), a saponin (e.g., Quil-a, including QuilAQS21), or an oil (e.g., complete or incomplete Freund's adjuvant).

An exemplary immunogenic composition of the invention comprises, or consists essentially of, an effective immunizing dose of a c.perfringens immunizing agent in a water-in-oil-in-water emulsion (see, e.g., U.S. patent No.5,817,320 to Stone), optionally in a pharmaceutically acceptable carrier.

The immunogenic composition may optionally comprise two or more agents (i.e. any combination of the agents described above) capable of inducing an immune response against c.

In some particular embodiments, the agent capable of inducing an immune response against c.perfringens (e.g., a toxoid, a bacterin, an attenuated c.perfringens and/or toxin, etc.) is a c.perfringens strain derived from avian, optionally a c.perfringens strain derived from chicken.

As used herein, the term "consisting essentially of … …" (and grammatical variations) means that the immunogenic composition contains no other immunogenic reagent materials other than the indicated substances. The term "consisting essentially of … …" does not exclude the presence of other components, such as adjuvants, immunomodulators and the like.

By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to an individual without causing appreciable undesirable biological effects. Thus, such pharmaceutical compositions may be used, for example, to prepare compositions for immunization. The physiologically and pharmaceutically acceptable carrier may contain other compounds including, but not limited to: stabilizers, salts, buffers, adjuvants and/or preservatives (e.g., antibacterial, antifungal and antiviral agents) known in the art. The pharmaceutically acceptable carrier need not be sterile, although this will typically be the case for in ovo administration to avian embryos.

In some particular embodiments, the immunogenic composition further comprises an immunostimulant. Alternatively, the immunostimulant may be administered to the individual in a separate formulation. Immunostimulatory agents useful in the invention include, but are not limited to: cytokines, growth factors, chemokines, supernatants of cell cultures of lymphocytes, monocytes or cells from lymphoid organs, cell preparations or cell extracts (e.g., immobilized Staphylococcus aureus or lipopolysaccharide preparations), mitogens or adjuvants, including low molecular weight drugs. The immunostimulant may be administered in ovo at any time during the incubation period. Optionally, the immunostimulant and the agent that induces an immune response against c.

As used herein, the term "simultaneously" means close enough in time to produce a combined effect (i.e., simultaneously may be simultaneous, or it may be two or more events occurring in a short time before or after).

Any suitable vaccine adjuvant may be used in accordance with the present invention, including chemical and polypeptide immunostimulants capable of stimulating the immune system's response to an antigen. Adjuvants include, but are not limited to, adjuvants derived from aluminum (e.g., aluminum hydroxide), aluminum phosphate, vegetable and animal oils (e.g., incomplete or complete Freund's), saponins (e.g., Quil-A, including Quil A QS21),(Intervet), etc. Representative adjuvants of the invention include, but are not limited to: aluminium salts, such as aluminium hydroxide gel (alum), aluminium phosphate or algannmulin, but also salts of calcium, magnesium, iron or zinc or mineral gels, or insoluble suspensions of acylated tyrosines or acylated sugars, cationically or anionically derivatized polysaccharides or polyphosphazenes and/or saponins (e.g. Quil-a), and/or oil emulsions, e.g. water-in-oil and water-in-oil, and/or complete or incomplete Freund's or any combination thereof.

Optionally, the immunogenic composition may contain one or more stabilizers. Any suitable stabilizer may be applied, including: carbohydrates, such as sorbitol, mannitol, starch, sucrose, dextrin or glucose; proteins, such as albumin or casein; and buffering agents such as alkali metal phosphates and the like.

It is often convenient to immunize birds against multiple diseases in a single treatment. Thus, in some particular embodiments, the immunogenic composition comprises one or more additional agents capable of inducing an immune response against other avian pathogens (e.g., viruses, bacteria, or fungi), optionally an immunological agent that generates a protective immune response. For example, the immunogenic composition may further comprise a vaccine against coccidiosis (i.e., Eimeria), infectious bursal disease, Marek's disease, newcastle disease, avian influenza, fowl pox, avian reovirus, avian metapneumovirus, avian adenovirus, infectious bronchitis, Salmonella spp. Avian vaccines suitable for in ovo or post-hatch use are known in the art and are commercially available (e.g., Burserlex for bursal disease)^TMA vaccine; newplex for Newcastle disease^TMA vaccine; and Inovocox for coccidiosis^TMVaccines, all available from Embrex, inc, and Marek's HVT-SB-1 vaccine for Marek's disease, available from Merial). Comprising both coccidiosis (i.e. Eimeria) and necrotic enteritis (i.e. cImmunogenic compositions of vaccine agents are particularly advantageous because Eimeria exposure is known to increase the likelihood of necrotic enteritis in birds by disturbing the gastrointestinal environment.

Thus, in another aspect, the invention comprises co-administering an immunogenic composition comprising (or consisting essentially of, or consisting of) an effective immunizing dose of a c. The multiple immunizing agents may be provided in a single formulation or may be administered simultaneously or sequentially in different formulations in any order. As discussed above, this aspect of the invention is particularly suitable for co-administration of coccidiosis and necrotic enteritis vaccines.

In another representative embodiment, the avian subject is immunized against necrotic enteritis and then coccidiosis, or vice versa. Immunization may be performed in ovo, both after incubation, or may be performed in ovo and one after incubation. For example, in one illustrative embodiment, an avian subject is immunized against coccidiosis in ovo, and then immunized against necrotic enteritis after hatch.

The present invention may also be practiced with the administration of c.perfringens immunoreagents in combination with "in ovo fed" (see, U.S. patent No.6,592,878, incorporated herein by reference in its entirety) avian individuals, either in ovo or post-hatch. For example, according to certain embodiments, the c.perfringens immunoreagent and nutritional formula and/or gut modulator are administered to the avian in ovo, optionally, by delivery to the amniotic membrane. Optionally, vaccines against other infectious agents are also administered in ovo and/or post-hatch (see above). Perfringens immunoreagents and nutritional formulas and/or gut modulators may be administered simultaneously in the same or separate compositions, and/or may be administered sequentially in any order.

Other embodiments of the invention may include compositions comprising an antigen selected from the group consisting of c.perfringens alpha toxin, antigenic fragments of c.perfringens alpha toxin, inactive antigenic fragments of c.perfringens alpha toxin, and any combination thereof; wherein about 0.1 to about 1.0mL (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0) per dose of the one or more doses of the composition is sufficient to induce at least 0.5 antitoxin units (A.U.) of anti-alpha toxin antibodies per mL of antiserum in the vaccinated avian (e.g., chicken). In some embodiments, the composition can induce approximately 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10a.u. anti-toxin antibodies per mL of avian antiserum.

As used herein, the "antitoxin unit" or "a.u." (used interchangeably with the "antitoxin neutralization test" unit or "TNT" unit) of an antitoxin antibody per mL of antiserum is defined by the ability of the serum to neutralize the toxic effects of the toxin in a mouse bioassay. In this test, a known amount of toxin, established by international standards known in the art, is mixed with serial dilutions of serum from vaccinated animals. The mixture was incubated at room temperature for 1 hour and then injected intravenously into mice. If the toxin is completely neutralized by serum, the mice will survive, otherwise they will die. Antitoxin units or titers are determined as the reciprocal of the highest dilution (dilution) of serum capable of neutralizing the toxin.

In other embodiments, the invention may comprise the antigen in a cell-free preparation. In other embodiments, the antigen may be an alpha toxoid in the supernatant of a c. In certain embodiments, the composition may comprise, and/or consist essentially of and/or consist of an antigen which may be a c.perfringens type a alpha toxoid and/or a c.perfringens type C alpha toxoid. In some embodiments, the compositions of the invention may comprise c.perfringens β toxin, c.perfringens β 2 toxin, c.perfringens enterotoxin, c.perfringensToxin, c.perfringens iota toxin, c.perfringens kappa toxin, c.perfringens lambda toxin, c.perfringens theta toxin, c.sordelii hemorrhagic toxin, c.sordelii lethal toxin, c.difficile a toxin, c.difficile B toxin, c.septicum alpha toxin, c.novyi beta toxin, and/or any combination thereof. Such compositions may also comprise, and/or consist essentially of, and/or consist of, one or more viral antigens, one or more bacterial antigens, and/or one or more parasitic antigens, as described herein.

The invention will be further explained in the following non-limiting examples. In these examples, ". μ L" means microliter, ". μ g" means microgram, "mL" means milliliter, "cc" means cubic centimeter, "mM" means millimeter, "mM" means concentration shown in millimole, "mg" means milligram, "° c" means celsius, and E18 and E19 mean days 18 and 19 after initiation of the embryo, respectively.

Examples

Example 1 commercially available vaccines of the types Clostridium perfringens C and D are used (Siteguard G &Vision CD) immune response following in ovo vaccination

Design of experiments

With commercially available Clostridiumperfringens toxoidAnd bacterin-toxoid vaccines (Vaccine) were manually injected in ovo into broiler eggs. Hatched chickens were grown to measure antibody responses. Injection site evaluation was performed. On day 0 (hatch), treatment groups were selected for post-hatch vaccination. All chickens were placed in cages (5 chickens/cage). Each cage was provided with normal broiler initiation (Norma)l Broiler Starter) diet. Chickens were transferred to Broiler growth Feed (Broiler Grower Feed) on day 14. The chickens were bled and tested for antibody response with serum by a serum-toxin neutralization test.

Materials and methods

Injection material

G (adjuvant not known) is the C. perfringens type C and D toxoid vaccines produced by Schering-Plough. It protects cattle and sheep against diseases caused by type C and type D toxins. For immunization, 4.0mL (bovine) or 2.0mL (ovine) of vaccine was administered Subcutaneously (SQ) or Intramuscularly (IM). Booster vaccinations were performed three to four weeks after the initial vaccination and every year.

Injection material

(containing a proprietary adjuvant)) Is a vaccine-toxoid vaccine of types C.perfringens and D produced by Intervet. It protects cattle, sheep and goats against enterotoxemia caused by C. For vaccination, 2.0mL of vaccine was administered subcutaneously to animals (cattle, sheep or goats). The animals received an additional 2.0ml (sq) 3 to 4 weeks after the initial vaccination and were then re-vaccinated annually.

The injection scheme is as follows:

at E19, broiler eggs were injected with test material, targeting either the amniotic fluid or the embryoid body of each egg. In addition, on day 0 post-hatch, some in ovo and non-in ovo injection treatment groups received immunizations or booster immunizations of the test material. For post-hatch immunization or booster immunization, 0.5mL of vaccine was administered by subcutaneous injection behind the neck.

Injection site:

at the time of injection, the eggs assigned to the injection site assessment are injected with a dye. Eggs were then euthanized and necropsy was performed for injection site assessment. The site of injection was analyzed by the probability ratio chi-square test (Table 1).

Blood drawing:

on posthatch days 7, 14, 21 and 28, blood was removed from each chicken and pooled into individual vacutainers (per treatment group). On days 7, 14 and 21, less than or equal to 0.5mL of blood was collected via the wings or jugular vein. On day 28, 0.5mL or more of blood was collected by cardiac puncture. The blood was then incubated at room temperature for 1 hour. The blood samples were then centrifuged on a tabletop centrifuge at 2400RPMs for 10 minutes. Once centrifugation is complete, serum is removed from each blood sample and stored in 96-well storage plates (2-8 ℃ or-70 ℃) for later evaluation of immune responses.

Perfringens type C (β) toxin neutralization test in mice: sample preparation for mouse inoculation:

A. material

C. perfringens type C (β) toxin-CVBL Lot No. irp513(04)

C.perfringens type C (. beta.) antitoxin-CVBL Lot No. IRP486 Receptip

3. Diluent-1% peptone, 0.25% NaCl ph7.2, BBL Lot No.051006, nbref. -NB 140p.87

4. Chicken serum sample-EMHE 1381, NB 140p.80

a. Sample nos.7, 8, 9, 10A, 10B, 11A, 11B, 12A, 12B, 13A, 14A, 15A

5. Vials of 3ml and 1.8ml were sterilized.

B. Method of producing a composite material

1. Standard beta antitoxin was diluted 1:50 in diluent for a total of 10 ml.

a. The beta antitoxin is thawed at room temperature.

b. 200 μ l of beta antitoxin was mixed with 9.8ml of diluent at 1: 50.

c. Kept on ice.

2. The beta toxin was diluted 1:120 in diluent for a total of 12 ml.

a. The beta toxin was thawed at room temperature.

b. 200 μ l of β toxin was mixed with 1.8ml of diluent ═ 1: 10.

c. 1ml of beta toxin diluted 1:10 was mixed with 11ml of diluent 1:120.

d. Kept on ice.

3. Preparation of L_oControl samples.

a. 0.5ml of beta toxin (1:120 dilution) was mixed with 0.5ml of the dilution.

b. 1ml of beta antitoxin (1:50 dilution) was added.

c. Mixed and incubated at room temperature for 1 hour.

d. The samples were kept on ice.

4. Preparation of L₊Control samples.

a. 0.8ml of beta toxin (1:120 dilution) was mixed with 0.2ml of the dilution.

b. 1ml of beta antitoxin (1:50 dilution) was added.

c. Mix and incubate at room temperature for l hours.

d. The samples were kept on ice.

5. 12 test serum samples were prepared.

a. 3.25ml of beta toxin (1:120 dilution) was mixed with 3.25ml of the dilution.

b. To each of 12 tubes of 1.8ml was added 0.5ml of toxin from step 5. a.

c. Each tube is labeled with a sample number and a treatment group number.

d. 0.5ml of each undiluted chicken serum was added to a suitably labelled tube.

e. Mixed and incubated at room temperature for 1 hour.

f. The samples were kept on ice.

6. All unused samples were stored at 2-7 ℃.

Activities before study

78 female Swiss white (CD-1) mice (weighing 16-20 g) were purchased for study. Mice were shipped from a supplier (Charles River Laboratories) and transferred to a Clinical Testing laboratory (Clinical Testing Facility).

The mice were housed in cages, 2 mice per cage in the chicken serogroup, and 5 mice per cage in 4 cages in the control group. Mice remained acclimated for 5 days prior to day 0 of the initial study. Mice were fed following standard procedures and fed standard laboratory diets with unlimited water supply.

Day 0

The mice were subjected to normal health and appearance checks and 76 mice were included in the test. Two mice were not involved in the study and they were euthanized. Each mouse was injected Intravenously (IV) in the tail vein with a 26g x3/8 needle according to the treatment groups described in the study design section. Mice were monitored twice daily for indications of shock, pain or distress as evidenced by:

without generating gas

Curling of

Rough/wrinkled fur

Posture of bow back

Disorder (ataxia)

Anorexia or inability to obtain food and water

According to standard operating procedures, by CO₂Moribund mice were euthanized in excess.

Day 1 (approximately 24 hours after inoculation)

Mice were observed and the number of deaths recorded.

Results

Using a serum-toxin neutralization test, positive specific antibody responses were detected in the sera of chickens vaccinated with a commercial c. These data indicate that in ovo administration of c.perfringens bacterin-toxoid vaccine resulted in partial protection, and that in ovo administration followed by post-hatch intensification conferred complete protection against c.perfringens (table 2).

Example 2 immunization following in ovo vaccination with Experimental vaccine preparations containing recombinant alpha toxin Answering

Design of experiments

The above study was performed to determine if humoral (antibody) immune responses could be detected in broilers after in ovo, in ovo + post hatch or post hatch vaccination with Clostridium perfringens recombinant alpha toxin (SEQ ID NO: 6) (Dr. Glenn singer, depth. of Veterinery Science and microbiology, The University of Arizona) adjuvanted with incomplete Freund's adjuvant and Quil-A. Immunization strategies included in ovo vaccination targeting the embryo at E18 and vaccination at day 7 post-hatch. Antibody responses were assessed at day 28 of age.

At E18, broiler eggs were injected manually in ovo with control material (Quil A; Accurate Chemical & Scientific Corporation, Product # AP04991, adjuvant grade, Batch # L77-238) or C.perfringens recombinant alpha toxin (55 or 60 μ g/dose, adjuvanted with Quil A + IF (13 or 15 μ g/dose)) emulsified with incomplete Freund's adjuvant (IFA; Rockland, Lot # 16235). Injection site evaluation was performed by injection of dye. On day 0 (hatch), the chickens were placed in cage units (5 chickens/cage). The chickens received a Normal broiler starter diet (Normal BroilerStarter). In addition, some chickens were vaccinated (0.2 mL vaccine injected subcutaneously behind the neck) on day 7 or day 17 depending on treatment. Chicken sera were then evaluated for specific antibody responses by western blot.

Materials and methods

And (3) injection:

at E18, broiler eggs were injected with test material (Clostridium perfringens alpha toxin + vaccine adjuvant) targeting the embryo of each egg. In addition, some in ovo and non-in ovo injection groups received vaccination on day 7 or day 17 post-hatch (table 3).

Injection Site (SOI):

at the time of injection, eggs assigned for SOI assessment were injected with dye. Eggs were then euthanized and carcasses were examined for SOI assessment (table 4).

Collecting serum:

on posthatch days 7, 14, 21 and 28, blood was removed from each chicken and pooled into each evacuated blood collection tube. On days 7, 14 and 21, less than or equal to 0.5mL of blood was collected via the wings or jugular vein. On day 28, 0.5mL or more of blood was collected by cardiac puncture. The blood was then incubated at room temperature for 1 hour. Blood samples were centrifuged, serum removed, and stored in 96-well storage plates (2-8 ℃ or-70 ℃) for future immune response assessment.

Western blot assay:

SDS slab gel electrophoresis was performed on 10% acrylamide slab gel (125mm long X150mm wide X0.75mm thick) covered with 25mm layered gel according to Laemmli (Nature 227: 680-. Electrophoresis was performed at 12mAmp for approximately 3.5 hours or until the bromophenol blue front moved to the end of the slab gel. After gel electrophoresis of the plate, the gel used for the hybridization blot was placed in transfer buffer (12.5mM Tris, pH8.8, 96mM glycine, 20% MeOH) and transferred to PVDF membrane overnight at 200mA and approximately 100V/2 piece of gel. The PVDF membrane was then stained with Coomassie blue and dried between filter paper layers.

PVDF membrane was stained with Coomassie Brilliant blue R-250 and subjected to tabletop scanning before and after cutting into individual tracks. Each hybridization blot was placed in a separate container, blocked for 2 hours in 5% skim milk in Tween-20Tris buffered saline (TTBS), and rinsed in TTBS. The hybridization blots were then incubated overnight with primary antibody (2% skim milk powder diluted 1:100 in TTBS) and rinsed in TTBS for 3X10 min.

Hybridization blot 1 (positive control) was placed in a secondary antibody [ rabbit anti-goat IgG-HRP (Sigma Cat. # a-5420 and Batch #034K4858), diluted 1:5,000 in 2% NFDM in TTBS ] for 2 hours, rinsed 3X10 minutes in TTBS, treated with ECL, and exposed to X-ray film.

The remaining hybridization blots were then placed in secondary antibodies [ rabbit anti-chicken IgG-HRP (Bethy Cat. # A30-107P and Batch # A30-107P-3), 1:2,000 diluted in 2% skim milk powder in TTBS ] for 2 hours, rinsed in TTBS for 3X10 minutes, treated with ECL, and exposed to X-ray film.

The results of the Western blot study are shown in Table 5.

Results

Specific antibody responses were detected in chickens after in ovo (targeted embryo) vaccination with recombinant alpha toxin adjuvanted with Quil-a and IFA (with or without post-hatch boost). These data indicate that in ovo vaccination with recombinant alpha toxin can elicit immune responses against c.

Example 3

Commercially available inactivated oil emulsion vaccines against newcastle disease are purchased from Maine biologicalcale laboratories. The vaccine is administered in ovo by the amniotic route at E18, or by the embryonal route at E19. Site-directed application to amniotic fluid and embryoid bodies was verified by injection site analysis using dyes at E18 or E19. Site-directed administration to the amniotic fluid was accomplished using a flat-ended needle (group 2). Site-directed administration to the embryo was performed using a sharp 1.25 inch needle (group 3). Sera were collected at day 14, 21 and 28 and tested against antibodies specific for Newcastle Disease Virus (NDV) using ELISA (Idexx, Inc.). On each day of blood collection, blood was drawn from different chickens.

Analysis of the E18 injection site indicated that 22/24 eggs were injected into the amniotic fluid, 22/24 into the allantoic fluid, and 0/24 into the embryo. Analysis of the E19 injection site showed that 7/10 eggs were injected into the embryo and 3/10 eggs were injected into the amniotic fluid. Table 6 shows the antibody response to newcastle disease virus after in ovo vaccination of chickens. Table 7 shows the percent hatchability data.

Studies have shown that the immune response of developing embryos is strongly influenced by the in ovo site of administration of the killed oil emulsion newcastle disease vaccine (table 6). Embryos vaccinated in amniotic fluid around the embryo bodies did not respond to newcastle disease specific antibodies. On the other hand, embryos vaccinated directly into the embryo body responded with a strong antibody response, which increased to 28 days with age. A total of 34 chickens were bled in this study and 26/34 were positive for newcastle disease virus antibodies (table 6). 26/34 was 76.5%, which is very close to the injection site study where 70% of the E19 immunized embryos were injected into the embryo body. The percent hatchability was within the normal range for both the treated and untreated groups (table 7).

Example 4

Commercially available inactivated oil emulsion vaccines against newcastle disease are purchased from Maine biologicalcale laboratories. At E19, the vaccine is administered in ovo by the embryonal route, or subcutaneously at hatch. Site-directed application to the embryo was performed using a sharp 1.25 inch needle (group 3). Day-of-hatch vaccination was performed by subcutaneous injection of vaccine into the hind neck of newly hatched chickens (group 2). Sera were collected at 21 days of age and tested against antibodies specific for NDV using ELISA (Idexx, Inc.). The results are shown in Table 8.

The data in table 8 show that embryos vaccinated with newcastle disease oil emulsion vaccine in embryoid bodies responded as well as chickens vaccinated by the standard day-of-hatch route. The percent hatchability was within the normal range for both the treated and untreated groups (table 9).

The data shown in examples 3 and 4 above show that the hit embryo is necessary to stimulate an active immune response against an inactivated antigen (in this case newcastle disease virus). These data also indicate that the embryos were not adversely affected by the injection of inactivated antigen in oil emulsion adjuvant into the embryo bodies.

In ovo (prior to hatching) embryo injections may be accomplished manually using a syringe and needle, or may be accomplished by an automated injection device, also using a needle. In the examples given herein, the vaccine was applied manually to the embryo body or amniotic fluid surrounding the embryo body using a syringe and needle (example 3 only). To complete the embryo injection, a needle is inserted through a hole in the shell at the balloon end of the egg. The inserted needle passes through the air sac membrane, the allantoic membrane and the liquid, and finally enters the amnion cavity where the embryo body is located. The needle then pierces the embryo and the vaccine settles. The injection of the embryo can occur at multiple sites in the embryo, including subcutaneous, intradermal, intravenous, intramuscular, and intraabdominal deposition of the vaccine, as well as any combination of these sites. In addition, the embryo injections may occur in the head, neck, shoulders, wings, back, chest, and legs, including any combination. Embryo injections do not involve just depositing the vaccine in the air sac, allantoic cavity, amniotic fluid or albumin.

In ovo injections may be performed using needles having a length in the range of 3/4 inches to up to 4 inches and a gauge in the range of 15 to 28 gauge. The needle tip can range from very sharp (hypodermic) to a flat end.

In examples 3 and 4, newcastle disease virus vaccine was used as a model antigen. However, any suitably formulated oil emulsion vaccine with sufficient antigen content would be expected to be similar to the newcastle disease vaccine tested. Thus, inactivated vaccines against infectious bursal disease, avian influenza, infectious bronchitis, chicken anaemia virus infection, laryngotracheitis, avian reovirus, avian adenovirus, rotavirus, astrovirus, inclusion body hepatitis, egg drop syndrome, Escherichia coli, Mycoplasma spp, Salmonella spp, Campylobacter spp, Clostridium spp, haemaphyseal spp, Pasteurella spp, can be delivered directly in ovo to the embryo according to the methods described herein. Vaccines made from these agents may be whole cells or subunits. Vaccines made from these agents may be produced in culture medium, egg or tissue culture in a conventional manner, and/or may be produced by recombinant means according to methods well known in the art. Furthermore, it is also contemplated that: any disease agent that can be produced to have an amount of antigen sufficient to be able to effectively vaccinate the day-of-hatch chickens when inactivated is also effective to vaccinate the embryo in ovo when delivered directly to the embryo body.

The adjuvants used in the vaccines tested in these examples are typical of commercial oil emulsions. Non-oil emulsion inactivated vaccines with adjuvants other than oil are expected to generate an active immune response when delivered directly to the embryo prior to hatch. Suitable adjuvants will include, but are not limited to: mineral gels, polyanions, pluronic polyols, saponin derivatives, lysolecithin and other similar surface-active substances, glycosides and all types of oils and combinations thereof.

Example 5

As described below, for the primer containing no specificityPathogen (SPF) leghorn (leghorn) vaccination in ovo: group 1: phosphate Buffered Saline (PBS); groups 2 and 3: 0.3 × 10 in PBS⁹Inactivated NDV EID₅₀Dose/dose; group 4: 0.3x10⁹Inactivated NDV EID₅₀Dose, mix alum storage adjuvant (Imject, Pierce; aluminum hydroxide and magnesium hydroxide); group 5: commercial oil emulsion vaccines against NDV. At day 11 of age, individuals of group 3 received a second dose of NDV in PBS injected subcutaneously. In ovo administered vaccines are targeted to the embryo body and injection site analysis using dyes is performed on a separate set of similar eggs to assess the percentage of embryos injected directly into the ligand. In ovo vaccination was performed on day 19 of incubation with a23 gauge, 1.25 inch needle. Each group of 14 chickens was housed and grown to 21 days of age.

Serum samples were collected at 21 days of age and tested for IgG antibodies to NDV by ELISA (Idexx, Inc.). Serum samples from groups 2, 4 and 5 were also tested for NDV specific antibodies by erythrocyte coagulation inhibition (HI) using four HA units. The number of samples tested by HI is different from the number tested by ELISA, as some samples did not collect enough serum to perform the HI test. If the serum sample has an ELISA titer of 200 or more and 3.0log or more₂Is determined to show a measurable antibody response to vaccination (i.e. seroconverted).

Results

Injection site analysis showed that the embryo injections accounted for 78% of all injections (table 10).

The percent hatchability and the ratio of serum conversion to NDV as measured by ELISA are shown in table 11. In table 12, the number of seroconverted chickens tested using HI is reported.

The following key points were noted from these studies. 1) NDV antigen in PBS did not stimulate an antibody response measurable by NDV ELISA, even when inactivated NDV antigen was given twice (once in ovo, again at day 11 age) (e.g. group 3); 2) NDV-alum (group 4) stimulated seroconversion in 8/14 chickens when measured by ELISA, whereas commercial oil emulsion vaccine (group 5) stimulated seroconversion in 10/13 chickens when measured by ELISA; 3) NDV-alum (group 4) stimulated seroconversion in 12/14 chickens when measured with HI, whereas oil emulsion vaccines stimulated seroconversion in 11/11 chickens when measured with HI; and 5) commercial oil emulsion vaccine against Newcastle disease (group 5) stimulated a stronger antibody response than the NDV-alum vaccine (group 4). Example 3 shows that in ovo administration of an antigen in an oil emulsion storage adjuvant requires that the vaccine be delivered to the embryo body to stimulate an antibody response that can be measured by ELISA. In this example, in ovo injection site analysis showed that 78% of eggs received vaccine directly into the embryo.

Example 6

A study was conducted using SPF leghorn chickens to determine whether alum-reservoir adjuvants can stimulate an immune response when administered in ovo. The groups tested are as follows: group 1: phosphate Buffered Saline (PBS), in ovo; groups 2 and 3: 1.2X10 in PBS⁹EID₅₀Beta-propiolactone inactivated NDV/dose, in ovo; group 4: in ovo administration of 1.2x10⁹EID₅₀Beta-propiolactone inactivated NDV/dose, mixed with alum at a ratio of 30% to 70% alum to NDV antigen. The alum used was a commercial solution of aluminum hydroxide and magnesium hydroxide (Imject, Peirce). Group 3 additional doses of NDV antigen (in PBS) were obtained by subcutaneous injection at day 11. In ovo vaccine administration was performed on day 19 of incubation with a23 gauge, 1.25 inch needle. Vaccine was targeted to the embryo body in ovo and injection site analysis was performed on a separate set of similar eggs to assess the percentage of embryos injected directly into the ligand. Serum samples were collected at 21 days of age and tested for antibodies against NDV by ELISA (Idexx, Inc.). If the ELISA had a titer of ≧ 200, the chicken was considered to exhibit a measurable antibody response (i.e., seroconverted).

Results

Injection site analysis showed that 81% of the embryos were injected directly into the embryo body (table 13). The percent hatchability and the degree and proportion of seroconversion against newcastle disease virus are shown in table 14.

These studies provide the following key points: 1) embryos were injected at E19, and the injection site data indicated that 81% of the embryos were injected in the embryo bodies; and 2) in this study, alum was mixed with beta-propiolactone inactivated NDV at a ratio of 30% to 70%, which is different from the study described in example 5 herein, in which example 5, heat inactivated NDV was mixed at a ratio of 50% alum to 50% NDV antigen. The difference in immune responses in the two studies may be due to differences in the NDV antigens used and/or differences in the ratio of alum to test antigen and differences in the ELISA used to measure the antibodies.

Example 3 demonstrates that the presence of antigen in a depot adjuvant oil emulsion requires in ovo delivery of the vaccine to the embryo to stimulate an antibody response that can be measured by ELISA. In this study, injection site analysis indicated that 81% of the embryos were injected in the embryo body, and from these data it is expected that approximately 11 of 14 eggs immunized in ovo will respond to an antibody response. The actual number of replies is 9 out of 14.

Example 7

Commercial oil emulsion newcastle disease vaccines are administered to broiler chickens in ovo. This study determined the ability of broiler chickens to respond to inactivated newcastle disease virus antigens (delivered in ovo via the amniotic and intraembryonic pathways). Chickens were bled at day 13, 21, 26 and 35 and antibody titers against NDV were measured using ELISA (Idexx, Inc.). Injection site analysis was performed using dyes at E18 and E19.

Hatchability data are shown in table 16. Percent hatchability was normal when oil emulsion vaccine was delivered into the embryo body.

The injection site (table 15) was very precise and over 90% of embryos were injected by the route indicated for treatment (table 16).

Newcastle disease virus specific antibody response data are shown in table 17. It can be seen that the chickens respond to newcastle disease antibodies when the vaccine is delivered in ovo to the embryo or subcutaneously at hatch. When the NDV vaccine is delivered to the amniotic fluid in ovo, there is no antibody response, indicating that the vaccine must be administered to the embryo body to stimulate a suitable immune response.

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.

Table 1: percent hatchability and injection site

Eggs from Vision CD or Siteguard G showed somewhat lower hatchability overall (82.5% and 75.0% versus 95.0%, respectively) than eggs from the control material.

Table 2: serum-toxin neutralization assay results:

all sera were collected (approximately 5 birds/group)

● specific positive responses were detected in birds boosted post-hatch after in ovo (embryo-targeted) vaccination with the c. The results suggest that antibody responses were lower (partial protection) in birds vaccinated in ovo only (no post hatch boost).

● no protection was observed in the serum from birds vaccinated with the c.

Table 3: summary of the study

Table 4: percent hatchability and injection site

A percent hatchability of 96% was obtained after in ovo vaccination with recombinant alpha toxin. An embryo targeting rate of 72.16% was obtained using a 20g1.5 "needle.

Table 5: detection of specific antibody responses by western blot

Positive and negative were performed as expected, confirming the validity of the test.

Specific antibody responses were detected in birds vaccinated in ovo (targeted to the embryo) with recombinant alpha toxin formulations (with and without post-hatch boost).

Table 6: antibody response of chickens to newcastle disease virus following site-directed in ovo administration of inactivated oil emulsion newcastle disease vaccine

Table 7: percent hatchability after in ovo spot administration of inactivated oil emulsion newcastle disease vaccine

Table 8: antibody response of chickens to newcastle disease virus following in ovo administration or subcutaneous administration of inactivated oil emulsion newcastle disease vaccine on the day of hatch

Table 9: percent hatchability after in ovo administration of inactivated oil emulsion newcastle disease vaccine

Table 10: injection site using dye

Table 11: percent hatchability and NDV ELISA results for birds vaccinated in ovo with NDV antigen, NDV antigen-alum or oil emulsion NDV vaccine

Group of	Vaccine	Reinforcement on day 11	Total hatchability (%)	Number of seroconverted birds/Total test number	Mean titer of seroconverted birds by ELISA (day 21)
Group of	Vaccine	Reinforcement on day 11	Total hatchability (%)	Number of seroconverted birds/Total test number	Mean titer of seroconverted birds by ELISA (day 21)	1	Is free of	Is free of	96	0/13	n/a
2	NDV	Is free of	77	0/14	n/a	1	Is free of	Is free of	96	0/13	n/a
2	NDV	Is free of	77	0/14	n/a	3	NDV	Is provided with	86	0/14	n/a
4	NDV-Alum	Is free of	88	8/14	1272	3	NDV	Is provided with	86	0/14	n/a
4	NDV-Alum	Is free of	88	8/14	1272	5	NDV-OE^**	Is free of	87	10/13	3038

^**Commercial oil emulsion vaccines of NDV-OE ═ NDV (lahi)

not applicable n/a

Table 12: NDV erythrocyte coagulation inhibition results of broiler chickens vaccinated in ovo with NDV antigen-alum or oil emulsion NDV vaccine

Group of	Treatment of	HI potency (log)₂) Mean value. + -. SD	Number of birds with HI titer ≥ 3/number of birds tested (day 21)
Group of	Treatment of	HI potency (log)₂) Mean value. + -. SD		2	NDV	1.8±0.4	0/13
4	NDV-Alum	3.8±1.1	12/14	2	NDV	1.8±0.4	0/13
4	NDV-Alum	3.8±1.1	12/14	5	NDV-OE^**	8.9±2.2	11/11

^**Commercial oil emulsion vaccines of NDV-OE ═ NDV (lahi)

Table 13: injection site results

Treatment of	Air bag	Allantoic sac	Amniotic fluid	Embryo body	Yolk sac	N
Treatment of	Air bag	Allantoic sac	Amniotic fluid	Embryo body	Yolk sac	N	23G x 1.25＂	0％	2.1％	16.7％	81.3％	0％	48

Table 14: percentage hatchability and ELISA results of broiler chickens vaccinated in ovo with NDV antigen or NDV-Alu

^*Both NDV treatments were from the same group of hatched birds; not applicable n/a

Table 15: injection site for site-specific in ovo delivery of inactivated newcastle disease virus oil emulsion vaccine

Table 16: hatchability of treatment groups and broiler groups given an oil emulsion NDV vaccine in ovo

Group #	Description of the invention¹	Dosage (ml)	Route of the way	# injected/# incubation	Percent hatchability
Group #	Description of the invention¹	Dosage (ml)	Route of the way	# injected/# incubation	Percent hatchability	1	Without vaccination	NA	NA	39/40	97.5
2	NDV oil emulsions	0.1	In the sheep water egg	16/21	76.2	1	Without vaccination	NA	NA	39/40	97.5
2	NDV oil emulsions	0.1	In the sheep water egg	16/21	76.2	3	NDV oil emulsions	0.1	In the embryo egg	21/21	100.0
4	NDV oil emulsions	0.1	When hatching under the skin	20/20	100.0	3	NDV oil emulsions	0.1	In the embryo egg	21/21	100.0

¹NDV oil emulsion-formulated for days old chickens (day of age chicks) 1 dose is 0.1 ml.

Table 17: antibody response to NDV in broiler immunized in ovo with NDV oil emulsion vaccine

¹NDV-prepared for chickens several days old, 1 dose was 0.1 ml.

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Claims

1. A method of immunizing an avian subject against a pathogen, the method comprising administering in ovo during the final quarter of incubation an effective immunizing dose of a composition capable of inducing an immune response against the pathogen, wherein the immunogenic composition is administered by direct injection in ovo into the embryo.

2. The method of claim 1, wherein the composition is administered directly to skeletal muscle tissue in the embryo.

3. The method of claim 2, wherein said skeletal muscle tissue is selected from the group consisting of pectoral muscle tissue and pipping muscle tissue.

4. The method of claim 1, wherein the composition is administered directly into the head, neck, shoulder, wing, back, chest, leg, or any combination thereof of the embryo.

5. The method of claim 1, wherein the composition is administered to the embryo subcutaneously, intradermally, intravenously, intramuscularly, intraperitoneally, or any combination thereof.

6. The method of claim 5, wherein the composition is administered subcutaneously to the embryo.

7. The method of claim 5, wherein the composition is administered to the embryo intraperitoneally.

8. The method of claim 1, wherein the avian subject is a chicken.

9. The method of claim 8, wherein the composition is administered between days 15 and 20 of incubation.

10. The method of claim 8, wherein the composition is administered on day 18 or 19 of incubation.

11. The method of claim 1 wherein said composition comprises a water-in-oil-in-water emulsion.

12. The method of claim 1, wherein the composition is administered in a needle having a length of 3/4 inches to 4 inches.

13. The method of claim 12, wherein the needle has a gauge size between 15 gauge and 28 gauge.

14. The method of claim 12, wherein the needle has a blunt end.

15. The method of claim 12, wherein the needle has a sharp tip.

16. The method of claim 15, wherein the needle has a beveled tip at an angle of from about 10 ° to about 45 °.

17. The method of claim 12, wherein the needle passes through the shell at the large end of the egg at an offset angle of about 1 ° to about 20 ° relative to the long axis of the egg.

18. The method of claim 1, further comprising the step of administering a booster dose of the composition to the avian subject after hatching.

19. The method of claim 1, wherein the composition comprises an adjuvant.

20. The method of claim 19, wherein the adjuvant comprises an aluminum-derived adjuvant, a saponin, a mineral gel, a polyanion, pluronic polyols, saponin derivatives, lysolecithin and other similar surface active substances, glycosides, all types of oils, and any combination thereof.

21. The method of claim 1, wherein the composition induces an immune response to treat and/or prevent coccidiosis, Marek's disease, infectious bursal disease, newcastle disease, fowlpox infection, Clostridium spp.

22. The method of claim 1, wherein the composition comprises a non-replicating agent that induces an immune response against the pathogen.

23. The method of claim 1, wherein two or more effective immunizing doses of the composition that induces an immune response against the pathogen are administered to the embryo in ovo, wherein the two or more compositions are administered simultaneously or sequentially in any order.

24. The method of claim 1, wherein two or more effective immunizing doses of the composition that induces an immune response against the pathogen are administered in ovo and at least one composition is administered to the embryo, wherein the two or more compositions are administered simultaneously or sequentially in any order.

25. The method of claim 24, wherein at least one composition is administered to the amniotic membrane, amniotic fluid, embryoid body, yolk sac, or any combination thereof.

26. The method of claim 1, further comprising administering an immunostimulant in ovo at any time during the incubation period, wherein the immunostimulant and the composition are administered simultaneously or sequentially in any order.

27. The method of claim 1, further comprising administering a nutritional formula, an enteric modulator, or a combination thereof in ovo at any time during the incubation period, wherein the nutritional formula, the enteric modulator, or a combination thereof and the composition are administered simultaneously or sequentially in any order.

28. The method of claim 1, wherein the composition is administered with an automatic injection device.

29. A method of immunizing an avian subject against Clostridium infection, the method comprising: administering in ovo during the final quarter of the incubation an effective immunizing dose of an immunogenic composition capable of inducing an immune response against the Clostridium species, wherein the immunogenic composition is administered by in ovo injection.

30. The method of claim 29, wherein the immunogenic composition is administered to an amniotic membrane.

31. The method of claim 30, wherein the immunogenic composition is administered into the amniotic fluid axially through the large end of the egg.

32. The method of claim 29, wherein the immunogenic composition is administered directly into the embryo body.

33. The method of claim 32, wherein the immunogenic composition is administered to the embryo parenterally.

34. The method of claim 29, wherein the avian subject is a chicken.

35. The method of claim 34, wherein the immunogenic composition is administered between days 15 and 20 of incubation.

36. The method of claim 34, wherein the immunogenic composition is administered on day 18 or 19 of incubation.

37. The method of claim 29, wherein the avian subject is a turkey.

38. The method of claim 29 wherein the immunogenic composition comprises a Clostridiumperfringens toxoid.

39. The method of claim 29 wherein the immunogenic composition comprises a vaccine of clostridium perfringens.

40. The method of claim 29 wherein the immunogenic composition comprises a Clostridium perfringens toxoid and a Clostridium perfringens bacterin.

41. The method of claim 29 wherein the immunogenic composition comprises a clostridium perfringens toxin.

42. The method of claim 41 wherein the Clostridium perfringens toxin is Clostridium perfringens alpha toxin.

43. The method of claim 29, wherein the immunogenic composition comprises an attenuated Clostridium perfringens.

44. The method of claim 29, wherein the immunogenic composition comprises a water-in-oil-in-water emulsion.

45. The method of claim 29, wherein the immunogenic composition comprises an adjuvant.

46. The method of claim 45, wherein the adjuvant comprises an aluminum-derived adjuvant, a saponin, an oil, or any combination of the foregoing.

47. The method of claim 29, further comprising administering the immunostimulant in ovo at any time during the incubation.

48. The method of claim 29, further comprising administering in ovo a coccidiosis vaccine, a Marek's disease vaccine, an infectious bursal disease vaccine, a newcastle disease vaccine, a fowlpox vaccine, or any combination of the foregoing.

49. The method of claim 48, wherein the immunogenic composition and the vaccine are administered simultaneously.

50. The method of claim 48, wherein the immunogenic composition and the vaccine are administered in the same formulation.

51. The method of claim 29, further comprising administering the nutritional formula, the enteric modulator, or a combination thereof in ovo.

52. An immunogenic composition comprising an effective immunizing dose of attenuated Clostridium perfringens in a pharmaceutically acceptable carrier.

53. The immunogenic composition of claim 52, wherein the immunogenic composition comprises a water-in-oil-in-water emulsion.

54. The immunogenic composition of claim 52, wherein the immunogenic composition comprises an adjuvant.

55. The immunogenic composition of claim 52, wherein the immunogenic composition further comprises a coccidiosis vaccine, a Mark's disease vaccine, an infectious bursal disease vaccine, a Newcastle disease vaccine, a fowlpox vaccine, or any combination of the foregoing.

56. The immunogenic composition of claim 52, wherein the immunogenic composition further comprises a nutritional formula, an enteric modulator, or a combination thereof.

57. An immunogenic composition comprising, in a pharmaceutically acceptable carrier:

(a) an effective immunizing dose of a Clostridium perfringens toxoid, a Clostridium perfringens bacterin, a c.perfringens toxin, or any combination thereof; and

58. An immunogenic composition comprising, in a pharmaceutically acceptable carrier:

(b) a water-in-oil-in-water emulsion.

59. An immunogenic composition comprising, in a pharmaceutically acceptable carrier:

60. A method of immunizing an avian subject against Clostridium infection, the method comprising: administering to the avian subject an effective immunizing dose of a Clostridium bacterin-toxoid composition by in ovo injection during the final quarter of incubation.

61. The method of claim 60, further comprising the step of administering a booster dose of the Clostridium bacterin-toxoid composition to the avian subject after hatching.

62. The method of claim 60, wherein said avian subject is a chicken.

63. The method of claim 60, wherein said composition further comprises an adjuvant.

64. The method of claim 60, wherein the composition further comprises VisionA vaccine.

65. A method of immunizing an avian subject against Clostridium infection, the method comprising: administering to the avian subject an effective immunizing dose of a recombinant toxin of Clostridium, or an immunogenic fragment thereof, by in ovo injection during the final quarter of incubation.

66. The method of claim 65 further comprising the step of administering a booster dose of the Clostridium bacterin-toxoid composition to the avian subject after hatching.

67. The method of claim 65, wherein said avian subject is a chicken.

68. The method of claim 65, wherein the toxin is administered with an adjuvant.

69. The method of claim 68, wherein said adjuvant is Quil A and incomplete Freund's adjuvant.

70. A method of immunizing an avian subject against necrotic enteritis infection, the method comprising: administering in ovo during the final quarter of the incubation an effective immunizing dose of an immunogenic composition capable of inducing an immune response against the Clostridium species, wherein the immunogenic composition is administered by in ovo injection.