Clostridium perfringens compositions and method of use for preventing necrotizing enteritis

Abstract

Provided are compositions, feeds, rations, drinking water and methods of use thereof for inducing protective immunity against or preventing Clostridium perfringens infection and / or necrotic enteritis in poultry, optionally as single dose vaccines. The composition comprises live virulent Clostridium perfringens (CP) and a mucosal adjuvant, such as cholera toxin (CT), which is for administered at or after hatch by for example oral delivery. The composition can be used with an immunomodulatory composition administered in ovo before hatching or a boost composition. The composition can be administered as a single dose.

Description

TITLE: CLOSTRIDIUM PERFRINGENS COMPOSITIONS AND METHOD OF USE FOR PREVENTING NECROTIZING ENTERITISCROSS REFERENCE

[0001] This application claims priority to United States Provisional Patent App No.63 / 726,407 filed November 29, 2024 which is incorporated by reference in its entirety.INCORPORATION OF SEQUENCE LISTING

[0002] A computer readable form of the Sequence Listing “P93249644WO01 Sequence Listing” (2,846 bytes) created on November 27, 2025, is herein incorporated by reference.FIELD

[0003] The present disclosure relates to compositions and methods of use for inducing immunity against or preventing Clostridium perfringens infection and / or necrotic enteritic, and, in particular, compositions comprising live Clostridium perfringens and at least one adjuvant.BACKGROUND

[0004] Necrotic enteritis (NE) caused by Clostridium perfringens (CP) is a complex enteric disease of broiler chickens. Overgrowth of CP in the gut produces wide array of enzymes and exotoxins which are responsible for the necrosis and acute death of broiler chickens (2). Mortality could range from 2 to 50% (5,6) leading to severe production and economic losses in terms of poor weight gain, increased feed conversion ratio and increased mortality. It was estimated that global production losses due to NE was approximately $6 billion USD annually (3,4). Broiler chickens between 3-6-weeks of age are most susceptible to NE (5) which might be due to the reduction of maternal antibodies (Ab) by 2-3 weeks of age (2,6,7) and naive immune system of young birds. Abrupt increase in the protein content of the feed, increase amount of fiber, wheat-based diet, immunosuppressive viral agents such as infectious bursal disease virus (IBDV), raised without antibiotics (RWA) and concurrent infections such as coccidia are the major predisposing factors for NE in broiler chickens. Antibiotic growth promoters (AGPs) have been an effective tool to control NE however, withdrawal of AGPs presented an enormous challenge to control NE. Studies have been conducted to find alternatives to antibiotics such as probiotics, prebiotics and organic acids against NE but effectiveness is limited (8-10).

[0005] It has been demonstrated that mucosal vaccinations induce both local and systemic immune responses including cytotoxic T cell responses (11,12). However, immune tolerance is a limitation in mucosal vaccination (13). Oral vaccination using a non-virulent CP isolate with necrotic enteritis -B like toxin (NetB) gene at various ages, for example the age of 8, 9, 10, 11 and 12 days by the oral gavage or in feed vaccination have shown to elicit some mucosal and systemic antibody response in broiler chickens (15), however even with multiple administrations, efficacy was only partial. In another study, oral delivery of a virulent CP was delivered for five consecutive days in one week old broiler chickens followed by bacitracin treatment for 9 days significantly reduced the NE lesions when birds were challenge 2 days later with a virulent CP (16). However vaccination protocols that require multiday administration is impractical and costly. Various studies have been carried out using live attenuated CP, vectored vaccines expressing CP proteins, inactivated vaccines, protein-based toxin, and toxin based to prevent NE in broiler chickens and breeder hens. These vaccines have shown to reduce the severity of NE lesions and provided only partial protection against NE under experimental conditions (15-19).

[0006] Multiple approaches have been explored to control NE. Types of vaccines ranged from live attenuated CP, non-inactivated CP culture supernatants, crude toxoid vaccines, recombinant proteins of CP, bacterial vectored vaccine and modified toxins. A number of vaccine delivery systems have been explored such as oral, IM and SC delivery. However, these vaccines provided only partial protection particularly those using protein antigens or toxoid vaccines (16,17,29-33). Administration of recombinant proteins expressing perfringolysin (PFOR ) at day 1 and day 14 did not protect broiler chickens against NE (17).

[0007] Developing an effective vaccination strategy against NE, preferably delivering a single dose of vaccine at hatch, to protect broiler chickens against NE without a booster vaccine is an enormous challenge. Protection of broiler chickens against NE at 3-4 weeks of age when maternal Ab decline by inducing immunity with a single vaccine at hatch is a practical solution for the broiler chicken industry. Moreover, immune system is still developing at hatch so it is a challenge to induce immunity in a bird with developing immune system (6). Several other challenges are posed with live vaccine development for the oral route in broiler chickens such as identification of a suitable oral adjuvant while avoiding oral tolerance.

[0008] Various adjuvants have been tested without success.

[0009] CT has been used as a mucosal adjuvant in mice (37). CT has high affinity to bind ganglioside receptors GM1. CT has been studied extensively in human research but there is only one study in broiler chickens demonstrating antibody response against NE using CT as an oral adjuvant with non-pathogenic CP carrying netB. In this study, CT vaccine was administered as an esophageal gavage for several consecutive days at 8, 9, 10, 11 and 12 days of age in feed (15) which is not a practical solution.

[0010] A need for effective and / or practical vaccines remains.SUMMARY

[0011] Disclosed herein for example are methods to (1) induce immunity in broiler chickens against necrotic enteritis (NE) by oral delivery of a live virulent Clostridium perfringens (CP) vaccine adjuvanted with cholera toxin (CT) at hatch optionally following in ovo administration of an immunomodulator such as cytosine phosphodiester guanine oligodeoxynucleotides (CpG-ODN); as well as (2) to protect broiler chicken against NE by immunizing their broiler breeder parents by oral delivery of live CP-CT vaccine. This disclosure demonstrates protection against NE in broiler progeny by delivering a live CP vaccine adjuvanted with CT by for example the oral route in broiler breeders.

[0012] An aspect includes a composition, comprising: live virulent Clostridium perfringens (CP), a mucosal adjuvant, and optionally a pharmaceutically acceptable carrier or diluent.

[0013] In an embodiment, the mucosal adjuvant is cholera toxin (CT) mucosal adjuvant.

[0014] Another aspect is a combination comprising a composition described herein and one or more of an immunomodulator composition and a boost composition.

[0015] In an embodiment, the immunomodulator composition comprising a cytosine- phosphorothioate-guanine oligodeoxynucleotide or a cytosine-phosphodiester-guanine oligodeoxynucleotide (CpG-ODN), optionally wherein CpG-ODN has a sequence 5’- TCGTCGTTGTCGTTTTGTCGTT-3’(SEQ ID NO: 1) or comprising 5’-TCGTCGTTGTCGTT-3’ (SEQ ID NO 2), optionally wherein the CpG-ODN with the sequence 5’-TCGTCGTTGTCGTTTTGTCGTT-3’ (SEQ ID NO: 1) or comprising 5’-TCGTCGTTGTCGTT-3’ (SEQ ID NO 2) has a phosphorothioate backbone.

[0016] In an embodiment, a composition described herein or a combination described herein, is for use in inducing protective immunity against or preventing Clostridium perfringens infection and / or necrotic enteritis in one or more subject(s).

[0017] The composition or combination for use described herein, wherein the one or more subject(s) are poultry.

[0018] A further aspect is a feed, ration or drinking water comprising a composition described herein or a boost composition described herein.

[0019] Yet a further aspect is a kit comprising a composition described herein, a combination described herein, a feed, ration or drinking water described herein, optionally further comprising one or more of instructions for use, or one or more vials, containers, and / or other suitable packaging comprising one or more of the composition, immunomodulator composition or boost composition, or the feed, ration or drinking water.

[0020] Also provided in another aspect is a method of inducing protective immunity against or preventing Clostridium perfringens infection and / or necrotic enteritis, the method comprising administering to one or more subject(s) an immunologically effective amount of a composition comprising live virulent Clostridium perfringens (CP), a mucosal adjuvant, e.g, CT and optionally a pharmaceutically acceptable carrier or diluent, optionally where the composition is orally administered.

[0021] In an embodiment, the method comprises using a composition, feed ration or drinking water or kit described herein.

[0022] In some embodiments, the composition is administered within 18 weeks after hatching, wherein the composition is administered within 12 weeks after hatching, wherein the composition is administered within 8 weeks after hatching, wherein the composition is administered within 4 weeks after hatching.

[0023] In some embodiments, the composition is administered within 5 days after hatching.

[0024] In some embodiments, wherein the composition is administered within 24 hours after hatching or wherein the composition is administered at hatch.

[0025] In some embodiments, the composition is orally administered, optionally in a feed, a ration, a drinking water or a gel composition.

[0026] In some embodiments, the method further comprises administration of a boost composition to the one or more subject(s) after administration of the composition, wherein the boost composition comprises Clostridium perfringens, at least one adjuvant, and optionally a pharmaceutically acceptable carrier or diluent.

[0027] In some embodiments, the composition is administered as a single dose.

[0028] In some embodiments, the plurality of subjects comprise poultry in a hatchery.

[0029] Also provided are uses of a composition, combination, feed, ration or drinking water or kit described herein in the manufacture of a medicament and / or vaccine for the treatment against and / or to prevent Clostridium perfringens infection and / or necrotic enteritis in one or more subject(s).

[0030] Other features and advantages of the present application will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating embodiments of the application, are given by way of illustration only and the scope of the claims should not be limited by these embodiments, but should be given the broadest interpretation consistent with the description as a whole.BRIEF DESCRIPTION OF DRAWINGS

[0031] The embodiments of the application will now be described in greater detail with reference to the attached drawings in which:

[0032] Fig. 1A-C are gross and histopathological lesions of NE in broiler chickens following CP challenge (A) Necrotic areas (arrows) of the jejunal mucosa through serosal surface. (B) Severe, diffuse, acute necrosis (stars) of the jejeunal mucosa on gross examination. (C) Histopathological appearance of the jejunum depicting severe, diffuse, acute necrosis of the mucosa (arrow).

[0033] Fig. 2A-D are survival, histopathological score and antibody response against NE in broiler chickens following CP vaccination by the oral route at hatch. (A) Birds challenged with CP with no vaccine had 30% mortality in contrast, birds vaccinated with CP by the oral route following in ovo CpG-ODN or saline had no mortality. (B) Groups vaccinated with CP by the oral route with or without in ovo CpG-ODN, had significantly low histopathological lesions of NE (p<0.0001). (C) and (D) Groups vaccinated with CP by the oral route with or without CpG-ODN by the in ovoroute had significantly high IgY and IgA compared to the group not vaccinated with CP (pO.OOOl) [**p<0.01, ***p<0.001, ****p<0.0001, CT-cholera toxin, ns-not significant],

[0034] Fig. 3A-D are survival, histopathological score and antibody response following CP challenge in birds vaccinated by the oral route with live CP adjuvanted with CT (A) Birds challenged with CP with no vaccine had 20% mortality in contrast, birds vaccinated with CP by the oral route following in ovo CpG-ODN or saline had no mortality. (B) Groups vaccinated with CP by the oral route with or without in ovo delivery of CpG-ODN, had significantly low histopathological lesions of NE (p<0.0001). Groups vaccinated with CP by the oral route, with or without in ovo delivery of CpG-ODN, had significantly high IgY (C) or IgA (D) compared to the group challenged with CP but not vaccinated with CP in a homologous and heterologous challenge study (pO.OOOl) [**p<0.01, ***p<0.001, ****p<0.0001],

[0035] Fig. 4A-B are survival and histopathology lesion score of NE in broiler progeny following their parents were vaccinated against NE by an oral CP vaccine at 13 and 16 weeks of age (A) Broiler breeder progeny had 100% survival following their parents were vaccinated against NE in contrast, 10% mortality in broilers when their parents were not vaccinated against NE. (B) Broiler progeny had significantly low NE lesions when their parents were vaccinated against NE by an oral CP vaccine [****p<0.0001],

[0036] Fig. 5A-F are a set of bar charts with Gl(zw ovo saline + no vaccine), G2(in ovo CpG-ODN + CP-CT oral vaccine) and G3(in ovo saline + CP-CT oral vaccine); (A) CD8+ T cells, CD4+ T cells and monocytes / macrophages in the PBMC and jejunum two days following CP-CT vaccination by the oral route following in ovo delivery of CpG-ODN or saline. (A, B) PBMCs and jejunum had a significantly high CD8+ T cells in birds vaccinated with CP-CT vaccine by the oral route following in ovo delivery of CpG-ODN or saline. (C) PBMCs had a significantly low CD 4+ T cells in birds vaccinated with CP-CT vaccine by the oral route following in ovo delivery of CpG-ODN or saline. (D) Jejunum had a significantly high CD 4+ T cells in birds vaccinated with CP-CT vaccine by the oral route following in ovo delivery of CpG-ODN (E, F) PBMCs and jejunum had a significant high monocytes and macrophages following delivery of CP-CT by the oral route following in ovo delivery of CpG-ODN or saline [**p<0.01, ***p<0.001, [****p<o oooi ]DETAILED DESCRIPTION

[0037] The following is a detailed description provided to aid those skilled in the art in practicing the present disclosure. Unless otherwise defined, 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 disclosure belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the disclosure. All publications, patent applications, patents, figures and other references mentioned herein are expressly incorporated by reference in their entirety.I. Definitions

[0038] As used herein, the following terms may have meanings ascribed to them below, unless specified otherwise. However, it should be understood that other meanings that are known or understood by those having ordinary skill in the art are also possible, and within the scope of the present disclosure. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0039] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the description. Ranges from any lower limit to any upper limit are contemplated. The upper and lower limits of these smaller ranges which may independently be included in the smaller ranges is also encompassed within the description, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the description.

[0040] As used in this application and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "include" and "includes") or "containing" (and any form of containing, such as "contain" and "contains"), are inclusive or open- ended and do not exclude additional, unrecited elements or process steps.

[0041] The term “consisting” and its derivatives as used herein are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, andalso exclude the presence of other unstated features, elements, components, groups, integers and / or steps.

[0042] The term “consisting essentially of’, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel character! stic(s) of these features, elements, components, groups, integers, and / or steps.

[0043] The terms "about", “substantially” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies or unless the context suggests otherwise to a person skilled in the art.

[0044] As used in the present application, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. For example, an embodiment including “a compound” should be understood to present certain aspects with one compound, or two or more additional compounds.

[0045] In embodiments comprising an “additional” or “second” component, the second component as used herein is different from the other components or first component. A “third” component is different from the other, first, and second components, and further enumerated or “additional” components are similarly different.

[0046] The term “and / or” as used herein means that the listed items are present, or used, individually or in combination. In effect, this term means that “at least one of’ or “one or more” of the listed items is used or present.

[0047] Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0048] The term “composition of the application” or “composition of the present application” and the like as used herein refers to a composition comprising one or more compounds of the application.

[0049] The term “suitable” as used herein means that the selection of the particular composition or conditions would depend on the specific steps to be performed, the identity of the components to be transformed and / or the specific use for the compositions, but the selection would be well within the skill of a person trained in the art.

[0050] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from anyone or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.

[0051] The term “adjuvant” as used herein refers to a substance that enhances or modifies the immune response to one or more antigens when co-administered so as to improve the immune recognition of the one or more antigens, which antigens can comprise a live, weakened, inactivated or virulent pathogen, or a protein or a fragment thereof, oil-in water emulsion” (Emulsigen) use in almost all poultry vaccines, used in IM route Exemplary adjuvants include oil-in water emulsion (e.g., Emulsigen™) used in many poultry vaccines, particularly for intramuscular injection, cholera toxin or CpG-ODN.

[0052] The term “mucosal adjuvant” as used herein refers to a substance that enhances the immune response to one or more antigens when administered via a mucosal route so as to boost the immunogenicity at mucosal surfaces, such as the gastrointestinal, respiratory and urogenital tracts. Exemplary mucosal adjuvants include for example cholera toxin.

[0053] The term “cholera toxin” as used herein as a mucosal adjuvant refers to at least the B subunit of the cholera toxin ABs multimeric protein complex. Cholera toxin is secreted by the bacterium Vibrio choleras, which is a member of the heat-labile enterotoxin family. The ABs cholera toxin comprises a hexamer structure comprising a single copy of a subunit A which activates a G protein and 5 copies of a subunit B which bind a G protein. Recombinant or isolatedforms can be used, including recombinant cholera toxin B subunit (rCTB) which can be produced by Bacillus brevis carrying pNU212- CTB and AB5 for example, from Sigma Aldrich C8052. The CT can also be obtained from LubioScience GmbH Baumackerstrasse 24 8050 Zurich. CT and cholera toxin (CT) mucosal adjuvant are used interchangeably herein.

[0054] The term “at hatch” as used herein refers to the moment when a subject such as a bird hatches, i.e. emerges from its egg, to up to 12 hours after hatching of said subject, optionally before first drinking or eating.

[0055] The term “CP” as used herein refers to Clostridium perfringens, an anaerobic gram-positive bacterium of the Clostridium genus. CP includes for example, all CP strains, optionally type G CP, including for example strains that produce toxinotype A, toxinotype B, toxinotype C, toxinotype D, toxinotype E, toxinotype F and / or toxinotype G of Clostridium Perfringens. Based on the toxin genes carriage, CP has been classified into 7 toxinotypes, including CP type G which among multiple other toxinotypes induces NE (1). CP type G is characterized by the production of the alpha toxin (a-toxin) and the necrotic enteritis B-like toxin (NetB). Type G can also carry beta2 toxin (CPB20 and / or large cytotoxin (TepL) as well as others. The CP can comprise for example cpa, netB, cpb2 and tpeL genes, cpa, cpb2 and netB genes or cpa and cpb2 genes

[0056] The term “live virulent Clostridium perfringens", “pathogenic Clostridium perfringens” or variations thereof as used herein, refers to Clostridium perfringens (CP) bacteria that are viable e.g. under normal growth conditions and that comprise on or more toxin genes, for example two or more or three or more, and produce the one or more toxins, and induce necrotizing enteritis optionally under permissive conditions (e.g. predisposing factors like coccidiosis, dietary changes such as increased protein consumption or stress), which can be characterized by depression, dehydration, diarrhea and / or death in an infected host. The term includes wild type or naturally occurring CP that is pathogenic as well as mutated virulent CP. In some embodiments, the live virulent Clostridium perfringens can belive attenuated Clostridium perfringens. Suitable CP strains are available from ATCC, for example ATCC13124 (cpa(alpha toxin), pfoA (perfringolysin O)), ATCC27324 (cpa(alpha toxin), cpb2 (beta2 toxin), cpe (enterotoxin), pfoA (perfringolysin O)), ATCC 19574 (cpa(alpha toxin), cpb2 (beta2 toxin), pfoA (perfringolysin O)), ATCC 10543 ((cpa(alpha toxin), cpb2 (beta2 toxin), pfoA (perfringolysin O)), or ATCC27060.

[0057] The term “homologous” with respect to bacterial, refers to bacterium, such as Clostridium perfringens herein, that are genetically similar or identical and belong to the same strain. For example, a composition may comprise a pathogenic isolate of CP and a boost may comprise a homologous strain (e.g. homologous to the pathogenic isolate in the composition).

[0058] The term “heterologous” with respect to bacteria, refers to bacterium isolate, such as Clostridium perfringens isolate herein described, that is genetically distinct from a specified or reference strain. For example, if a reference strain is administered, a genetically distinct strain, e.g. producing a different toxin or different combination of toxins, would be heterologous. For example, a composition may comprise an isolate of CP and a boost may comprise a heterologous strain (e.g. heterologous to the isolate in the composition).

[0059] The term "immunologically effective amount" of a composition or vaccine of the disclosure comprising for example a live virulent C. perfringens and CT mucosal adjuvant, is a quantity sufficient to, when administered to a subject, elicit an immune response to the C. perfringens and / or induce protective immunity, including homologous immunity and / or heterologous immunity, in the subject(s). For example, an immunologically effective amount will produce IgA detectable at mucosal surfaces and IgY that is detectable in blood.

[0060] The term “use for preventing Clostridium perfringens infection and / or necrotic enteritis” as used herein means the use is for preventing Clostridium perfringens infection and / or necrotic enteritis, and includes where Clostridium perfringens infection and / or necrotic enteritis is prevented, partially prevented or reduced in severity in a subject or a plurality of subjects. For example, it can mean, when referring to a plurality of subjects, reduction in the percentage of the plurality infected or exhibiting NE and / or reduction in the severity of the NE symptoms, such as decreased lesion rates or death rates in the plurality.

[0061] The term “CpG-ODN” refers to a strand of single-stranded synthetic nucleic acid molecule comprising at least one cytosine triphosphate deoxynucleotide followed by a guanine triphosphate deoxynucleotide motif connected through a phosphorothioate linkage or equivalent functional group excluding phosphodiester and naturally occurring groups , wherein the CpG is unmethylated. The strand can be between 6 to 55, for example between 12 to 24, or between 18 to 24, nucleotides long. For example, the nucleic acid molecule can be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides long or longer. All three classes of CpG-ODNare encompassed, class A, class B and C. Also encompassed are hybrid structures comprising CpG- ODN nucleic acid molecules. Class B CpG-ODNs include a phosphorothioate backbone and one or more CpG dinucleotides, but no poly G motifs. Encompassed by this term are related Class B CpG-ODNs including ODN 2006, ODN 2007, ODN 1668, ODN 1862, ODN BW006, and ODN D-SL01. Class C CpG-ODNs include a phosphorothioate backbone, one or more CpG dinucleotides and a CpG-containing palindromic motif. The CpG-ODN can be ODN 2007.

[0062] The term “CpG-ODN 2007” as used herein refers to an oligonucleotide of at least 14 nucleotides and up to 22 nucleotides, and comprising the sequence TCGTCGTTGTCGTT (SEQ ID NO: 2), optionally a 22-mer having the sequence 5'-TCGTCGTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 1) or any part thereof comprising TCGTCGTTGTCGTT (SEQ ID NO: 2) having a phosphorothioate backbone.

[0063] The terms “DC” or “DCs” refer to dendritic cells, which are antigen-presenting cells.

[0064] The term “DW” refers to distilled water.

[0065] The term “EDTA” refers to ethylenediaminetetraacetic acid.

[0066] The term “ELISA” refers to enzyme linked immunosorbent assay, which is an assay to detect the presence of a ligand in a sample via the use of selective antibodies towards the selected analyte.

[0067] The term “FACS” refers to fluorescence-activated cell sorting, which is cell sorting method to isolate cell populations via the cells’ fluorescence that is indicative of the presence or absence of specific characteristics.

[0068] The term “FTG” refers to fluid thioglycolate media, which is microbiological growth medium used to cultivate anaerobic or microaerophilic bacteria.

[0069] The term “IL” refers to interleukin or interleukins, which are a group of signaling cytokine proteins primarily produced by white blood cells.

[0070] The term “Ig” refers to immunoglobulin, which is also known as antibody. The term “IgY” refers to immunoglobulin Y, which is the major antibody in birds, primary located in serum and egg yolk, that exhibits main systemic functions and provides passive immunity to offspring. The term “IgA” refers to immunoglobulin A, which is an antibody primary located in mucosal membranes, such as the gastrointestinal tract.

[0071] The term “IM” refers to intramuscular, as in the intramuscular injection of a vaccine as a method of delivery.

[0072] The term “LT” refers to labile toxin, which is a protein toxin generated by some strains of Escherichia coli bacterium as is part of the heat-labile enterotoxin family. These toxins consist of an AB5 multimer structure, comprising an A chain and 5 B chains in which the pentamer of B chains has a membrane-binding function.

[0073] The term “NE” refers to necrotic enteritis, which is a gastrointestinal disease caused by the bacteria Clostridium perfringens that affects poultry. The toxins produced by the bacteria induce significant inflammation and subsequent necrosis of the intestinal lining.

[0074] The term “OD” refers to optical density, which is a parameter used in spectrophotometry to detect and quantify the concentration of cells or solutes in a solution.

[0075] The term “PBMC” refers to peripheral blood mononuclear cells, which broadly refer to peripheral blood cells that have a single nucleus, such as lymphocytes and monocytes.

[0076] The term “PBS” refers to phosphate buffered saline, which is a buffer solution that is isotonic and non-toxic to a wide variety of cells.

[0077] The term “PBST” refers to phosphate buffered saline with Tween 20, which is a buffer solution that is isotonic and non-toxic to a wide variety of cells, supplemented with polysorbate 20 detergent, also known as Tween 20.

[0078] The term “RPMI” refers to Roswell Park Memorial Institute media, which is a cell culture medium.

[0079] The term “RWA” refers to organisms raised without antibiotics, as in the raising of poultry without administration of antibiotics.

[0080] The term “SC” refers to subcutaneous, as in the subcutaneous injection of a vaccine as a method of delivery.

[0081] The term “TMB” refers to tetramethylbenzidine substrate, which is a chromogenic substance used in immunochemistry such as in ELISA to visualize the presence of specific proteins, such as a target antigen or antibody.

[0082] The term “SWNT” refers to small wall carbon nanotube.

[0083] The term “LSC” refers to liquid surfactant composite.II. Compositions

[0084] An aspect of the disclosure includes a composition comprising live virulent Clostridium perfringens, a mucosal adjuvant, e.g. cholera toxin, and optionally a pharmaceutically acceptable carrier or diluent.

[0085] The compositions as demonstrated herein can be used to induce protective immunity against or prevent Clostridium perfringens infection and / or necrotic enteritis in subjects such as poultry.

[0086] In some embodiments, the live virulent Clostridium perfringens comprises one or more, optionally 2 or more toxin genes, such as, for example, Clostridium perfrigens toxinotype A, B, C, D, E , F and / or G.

[0087] In some embodiments, the live virulent Clostridium perfringens is a pathogenic strain of Clostridium perfringens toxinotype A, B, C, D, E , F and / or G.. The live virulent Clostridium perfringens can be a single pathogenic strain or a combination of pathogenic strains. In some embodiments, the live virulent Clostridium perfringens is live virulent non-attenuated Clostridium perfringens. In some embodiments, the live virulent Clostridium perfringens is fluid thioglycollate (FTG) grown CP. Other culture mediums can also be used.

[0088] In some embodiments, the live virulent Clostridium perfringens comprises cpa, netB, cpb2 and / or tpeL toxin genes.

[0089] In an embodiment, the live virulent Clostridium perfringens comprises at least two or two toxin genes, optionally selected from cpa, netB, cpb2 and / or tpeL toxin genes. In an embodiment, Clostridium perfringens comprises netB and tpeL toxin genes. In another embodiment, live virulent Clostridium perfringens comprises at least three or three toxin genes. In other embodiments, the live virulent Clostridium perfringens comprises any number of toxin genes, e.g.2, 3, 4 or 5 toxin genes.

[0090] It was surprisingly found that live virulent CP in combination CT protected birds against CP as well as other diseases such as E. Coli. It was also found that CT on its own had protective effect.

[0091] Accordingly, in some embodiments, including some embodiments of compositions, methods and uses described herein, cholera toxin is the active component e.g. the sole active component. In some embodiments cholera toxin or a composition comprising cholera toxin as the active component (e.g. without any subunit, or any live or attenuated bacteria or other source of antigen) is for use for inducing protective immunity against CP and / or other organisms.

[0092] In some embodiments, the pharmaceutically acceptable diluent is water or saline. For example, the water can be substantially chlorine free (e.g., less than 0.02 mg / mL, less than 0.01 mg / mL or undectable). Suitable diluents and / or pharmaceutically acceptable diluents include for example water, including sterile water or distilled water, or saline . Pharmaceutically acceptable carriers and diluents include for example carriers and diluents that are suitable for animal administration, for example phosphate buffered saline (PBS) or those which have been filtered for sterility. For example, the carrier can be sucrose.

[0093] In some embodiments, the composition is freeze dried.

[0094] In some embodiments, the composition is an oral formulation, for example a composition that is reconstituted for oral delivery (e.g. with a diluent). For example, the oral formulation, can be a drinking water formulation, a feed formulation or a gel formulation. The oral formulation can also for example be part of a combination or kit described herein.

[0095] Any of the compositions described herein can also comprise a preservative such as thimerosal, phenol or 2 -phenoxy ethanol. In some embodiments, the composition is formulated for oral delivery, e.g. an oral delivery composition. In some embodiments, the composition is formulated for delivery via the respiratory system, e.g. an intranasal delivery composition, a nebulization delivery composition. In another embodiment, the composition is an eye drops formulation.

[0096] In some embodiments, the composition is a liquid formulation. The composition can also be a lyophilized composition, for example to be reconstituted with a diluent such as water, optionally distilled and / or sterile water or saline, optionally sterile saline or water lacking chlorine.

[0097] As described herein, the compositions described herein can be used for inducing protective immunity against or preventing Clostridium perfringens infection and / or necrotic enteritis in one or more subjects.

[0098] In some embodiments the composition is formulated to provide live virulent Clostridium perfringens in a dose amount from about IxlO5colony forming units (CFU) to about IxlO11CFU per subject. In some embodiments, the composition is formulated to provide live virulent Clostridium perfringens in a dose amount of about IxlO5CFU per subject. In some embodiments, the composition is formulated to provide live virulent Clostridium perfringens in a dose amount of about IxlO6CFU per subject. In some embodiments, the composition is formulated to provide live virulent Clostridium perfringens in a dose amount of about IxlO7CFU per subject. In some embodiments, the composition is formulated to provide live virulent Clostridium perfringens in a dose amount of about IxlO8CFU per subject. In some embodiments, the composition is formulated to provide live virulent Clostridium perfringens in a dose amount of about IxlO9CFU per subject. In some embodiments, the composition is formulated to provide live Clostridium perfringens in a dose amount og about IxlO10CFU per subject. In some embodiments, the composition is formulated to provide live virulent Clostridium perfringens in a dose amount of about IxlO11CFU per subject. The dose amount of CP can be any IxlO3CFU increment between IxlO5CFU and IxlO11CFU. For example the dose amount of CP per subject can be 2xl05CFU, or about 2.8 xlO6CFU or about 6.8 xlO7CFU or about 5.4 xlO8CFU.

[0099] In some embodiments, the composition is formulated to provide a dose amount of cholera toxin between about 0.01 pg / g of body weight to about 3 pg / g of body weight (i.e. subject body weight). In some embodiments, the amount of cholera toxin in the composition is sufficient to provide between about 0.05 pg / g of body weight to about 2 pg / g of body weight. In some embodiments, the amount of cholera toxin in the composition is sufficient to provide between about 0.1 pg / g of body weight to about 1 pg / g of body weight. In some embodiments, the amount of cholera toxin in the composition is sufficient to provide between about 0.3 pg / g of body weight to about 0.7 pg / g of body weight. In some embodiments, the composition is formulated to provide a dose amount of cholera toxin of 0.5 pg / g of body weight. For example, a chick at hatch can weigh 20-60 grams and a breeder chicken can weigh about 5 pounds to about 8 pounds. The dose amount of cholera toxin can be any 0.01 pg increment between 0.01 pg and 3 pg. For example, the dose amount can be 0.05 pg, or for example 0.1 pg. As another example it could be 0.71 pg. As a further example it could be 1.5 pg.

[0100] The composition can comprise any number of doses. The composition can be formulated to provide 100, 200 or any number of doses.

[0101] In some embodiments, the composition is formulated for administration to poultry, wherein the poultry is / are breeders. In some embodiments, the poultry is / are chicks.

[0102] The compositions described herein can also be used for inducing protective immunity against or preventing Clostridium perfringens infection and / or necrotic enteritis

[0103] Accordingly, another aspect of the disclosure includes a composition comprising live virulent Clostridium perfringens, a mucosal adjuvant, and optionally a pharmaceutically acceptable carrier or diluent, for use in induction of protective immunity in one or more subject(s).

[0104] Another aspect is use of a composition described herein in the manufacture of a medicament and / or vaccine for the treatment against and / or to prevent Clostridium perfringens infection and / or necrotic enteritis.

[0105] The one or more subjects can be adults such as breeders. It is demonstrated herein that the offspring of vaccinated breeders are also provided protective immunity.

[0106] In some embodiments, the one or more subjects are poultry. In some embodiments, the poultry is / are breeder(s).

[0107] In some embodiments, the composition is for administration within 24 weeks after hatching. In some embodiments, the composition for use comprises administration within 16 weeks after hatching. In some embodiments, the composition for use comprises administration within 8 weeks after hatching. In some embodiments, the composition for use comprises administration within 4 weeks after hatching.

[0108] Each of the compositions or combinations described herein can be administered or is for administration any 3 hour (or .125 day) increment between at hatch and 24 weeks after hatching, for example 2.5 days or 3.25 days, 15.125 days etc.

[0109] The one or more subjects can also be young. As demonstrated herein the compositions are particularly useful for vaccinating birds at hatch. In some embodiments, the one or more subjects are chicks.

[0110] In some embodiments, the composition for use comprises administration within 5 days after hatching. In some embodiments, the composition for use comprises administration within 72 hours after hatching.

[0111] In some embodiments, the composition for use comprises administration at hatch.

[0112] The composition can be formulated as a spray vaccine (e.g. the dosage form can be a spray vaccine).

[0113] The composition can also be formulated as a gel e.g. the dosage form can be a gel delivery system.

[0114] In some embodiments, the composition is formulated as a gel or a composition such as a powder that can be reconstituted as a gel composition. The composition, e.g. the gel composition, can be coloured which increases uptake and can be administered using various gel application technologies. Examples of gel delivery systems include Gel-Pac™ available from Animal Science Products which can be used to deliver vaccines to newly hatched chicks and poults. Such gels can be provided as a source of or via drinking water.

[0115] The compositions can additionally comprise one or more prebiotics, phytochemicals, immune modulators or stabilizers, for example Vac-Pac™, Vac-Pac Plus™, Spray-Vac™ vaccine stabilizer available from Animal Science Products. Spray-Vac for example allows spraying of vaccines without the cost and inconvenience of using distilled water.

[0116] The composition can be formulated as an additive for addition to feed or rations and / or drinking water.

[0117] Accordingly, another aspect of the disclosure comprises a feed or ration, comprising a composition described herein. Another aspect includes a drinking water comprising a composition as described herein.

[0118] In some embodiments, the drinking water comprises a composition described herein and lacks chlorine. For example, it may be distilled water comprising the composition described herein.

[0119] In some embodiments, the feed is a poultry feed. In some embodiments, the poultry feed comprises from 20% to 40% protein. In some embodiments, the feed comprises fluid thioglycollate (FTG) grown CP, optionally wherein the CP to feed ratio is about 1 : 1 (v / w).

[0120] Another aspect of the disclosure includes a boost composition comprising Clostridium perfringens, at least one adjuvant, and optionally a pharmaceutically acceptable carrier or diluent.The boost composition can be provided for example in a separate vial from the compositions described herein. In some embodiments, the boost composition is provided in drinking water.

[0121] In some embodiments, the boost composition comprises live or inactivated Clostridium perfringens. In some embodiments, the boost composition comprises a heterologous or homologous Clostridium perfringens, either live or inactivated, e.g. heterologous or homologous with respect to the CP in the composition.

[0122] In some embodiments, the boost composition is formulated to provide a dose amount of the live or inactivated Clostridium perfringens from about IxlO5to about IxlO11CFU per subject. In some embodiments, the amount of the live or inactivated Clostridium perfringens is from about IxlO6per subject. In some embodiments, the amount of the live or inactivated Clostridium perfringens is from about IxlO7per subject. In some embodiments, the amount of the live or inactivated Clostridium perfringens is to about IxlO8CFU per subject. In some embodiments, the amount of the live or inactivated Clostridium perfringens is to about IxlO9CFU per subject. In some embodiments, the amount of the live or inactivated Clostridium perfringens is to about IxlO10CFU per subject.

[0123] In some embodiments, the boost composition is formulated to provide a dose amount of the live or inactivated Clostridium perfringens of about 2xl08CFU per subject.

[0124] In some embodiments, the boost composition optionally comprising inactivated Clostridium perfringens is formulated for subcutaneous delivery. In some embodiments, the boost composition comprising inactivated Clostridium perfringens comprises an adjuvant such as an oil- in-water adjuvant. In some embodiments the oil-in-water adjuvant is 20% Emulsigen™ .

[0125] In some embodiments, the boost composition comprises an adjuvant. In some embodiments, the adjuvant comprises cholera toxin.

[0126] In some embodiments, the boost composition is formulated to provide a dose amount of cholera toxin from about 0.01 pg / g of body weight to about 3 pg / g of body weight per subject. In some embodiments, the amount of cholera toxin is from about 0.05 pg / g of body weight per subject. In some embodiments, the amount of cholera toxin is from about 0.1 pg / g of body weight per subject. In some embodiments, the amount of cholera toxin is from about 0.3 pg / g of body weight per subject. In some embodiments, the amount of cholera toxin is to about 2 pg / g of body weight per subject. In some embodiments, the amount of cholera toxin is to about 1 pg / g of bodyweight per subject. In some embodiments, the amount of cholera toxin is to about 0.7 pg / g of body weight per subject.

[0127] In some embodiments, the boost composition is formulated to provide an amount of cholera toxin of 0.5 pg / g of body weight.

[0128] In some embodiments, the adjuvant in the boost composition comprises a cytosine- phosphorothioate-guanine oligodeoxynucleotide or a cytosine-phosphodiester-guanine oligodeoxynucleotide. These compounds can be abbreviated as CpG-ODN.

[0129] In some embodiments, the CpG-ODN is or comprises the sequence

[0130] 5’-TCGTCGTTGTCGTTTTGTCGTT-3’ (SEQ ID NO: 1) ) or comprising 5’- TCGTCGTTGTCGTT-3’ (SEQ ID NO 2). In some embodiments, the CpG-ODN comprises a phosphorothioate backbone. In some embodiments, the boost composition is formulated to provide an amount of CpG-ODN from about 0.5 pg to about 100 pg per subject. In some embodiments, the amount of the CpG-ODN is from about 1 pg per subject. In some embodiments, the amount of the CpG-ODN is from about 3 pg per subject. In some embodiments, the amount of the CpG-ODN in the boost composition is from about 5 pg per subject. In some embodiments, the amount of the CpG-ODN in the boost composition is to about 70 pg per subject. In some embodiments, the amount of the CpG-ODN in the boost composition is to about 50 pg per subject. In some embodiments, the amount of the CpG-ODN in the boost composition is to about 20 pg per subject.

[0131] In some embodiments, the boost composition is formulated to provide an amount of CpG-ODN of 10 pg per subject.

[0132] Also provided in an immunomodulator composition that can for example be part of a combination of separate compositions with the composition comprising live Clostridium perfringens, a mucosal adjuvant, and optionally a pharmaceutically acceptable carrier or diluent, alone or in combination with boost composition.

[0133] The immunomodulator composition comprises CpG-ODN. The immunomodulator can be formulated for example for in ovo delivery.

[0134] In some embodiments, the immunomodulator composition is formulated to provide a dose amount of CpG-ODN from about 5 pg to about 200 pg per egg. In some embodiments, the doseamount of CpG-ODN is from about 10 pg per egg. In some embodiments, the dose amount of CpG-ODN is from about 30 pg per egg. In some embodiments, the dose amount of CpG-ODN is from about 40 pg per egg. In some embodiments, the dose amount of CpG-ODN is up to about 100 pg per egg. In some embodiments, the dose amount of CpG-ODN is up to about 50 pg per egg. In some embodiments, the dose amount of CpG-ODN is up to about 60 pg per egg.

[0135] In some embodiments, the dose amount of CpG-ODN is about 50 pg per egg.

[0136] In some embodiments, the composition or combination, is a single dose vaccine. In some embodiments the composition or combination is for use as a single dose vaccine, optionally a single dose oral vaccine.

[0137] In some embodiments, the composition or combination, is a single dose vaccine for delivery at hatch. In some embodiments the composition or combination is for use as a single dose vaccine, optionally a single dose oral vaccine, for delivery at hatch.

[0138] The compositions, and one or more of the immunomodulator composition and the boost compositions can be used in the methods and kits described herein.III. Vials and Kits

[0139] An aspect includes a vial or container comprising a composition, feed or drinking water described herein. The vial can be sized to allow reconstitution with a diluent. It can for example comprise a lyophilized formulation for reconstitution or a concentrated liquid for reconstitution. The container can for example be a bag or box comprising feed or ration.

[0140] Another aspect of disclosure includes a kit comprising at least any of the compositions, feeds or rations, drinking waters described herein. The kit comprises for example the composition comprising live Clostridium perfringens, a mucosal adjuvant, and optionally a pharmaceutically acceptable carrier or diluent optionally in combination with one or more components, such as a vial, other container etc.

[0141] The composition in the kit can be provided as a gel composition or a composition that be reconstituted as a gel delivery system, optionally in combination with a diluent for reconstituting the gel composition.

[0142] The kit can for example comprise the additive composition and one or more of a diluent and a measuring device, for example for measuring the amount of additive or diluent to use. Theadditive can be for addition to feed or ration or drinking water and the kit may comprise the feed or ration or drinking water.

[0143] In some embodiments, the kit comprises an immunomodulator that can be delivered in ovo.

[0144] In another embodiment, the kit further comprises the boost composition.

[0145] In some embodiments, the kit further comprises instructions for use. In some embodiments, the kit further comprises one or more vials comprising the composition and / or boost composition, containers, or other suitable packaging. In some embodiments, the kit further comprises an oral delivery apparatus. In some embodiments, the kit further comprises the diluent and / or a measuring device. In some embodiments, the kit further comprises a diluent for use in reconstituting the composition. In some embodiments, the diluent is saline.

[0146] The kit can comprise the composition in a vial, for example lyophilized with a carrier such as sucrose. The kit can for example comprise a number of doses for example for a flock. The doses can be a plurality of single doses, e.g. single dose vaccine. In some embodiments, the kit is sold comprising a number of vials comprising the composition. The composition may be lyophilized. Each vial may comprise a single dose or a plurality of single doses as described herein.

[0147] In some embodiments, the composition, combination, or kit is a single dose vaccine, optionally a single dose oral vaccine.

[0148] In some embodiments, the composition, combination, or kit is for use as a single dose vaccine, optionally a single dose oral vaccine.

[0149] In some embodiments, the composition, combination, or kit is a single dose vaccine, optionally a single dose oral vaccine for delivery at hatch.

[0150] In some embodiments, the composition, combination, or kit is for use as a single dose vaccine, optionally a single dose oral vaccine for delivery at hatch.IV. Methods and Uses

[0151] An aspect of the disclosure includes a method of inducing protective immunity against or preventing Clostridium perfringens infection and / or necrotic enteritis, the method comprising administering to one or more subject(s) an immunologically effective amount of a compositioncomprising live virulent Clostridium perfringens (CP), a mucosal adjuvant and optionally a pharmaceutically acceptable carrier or diluent.

[0152] Also provided in another aspect is use of an immunologically effective amount of a composition comprising live virulent Clostridium perfringens (CP), a mucosal adjuvant and optionally a pharmaceutically acceptable carrier or diluent for inducing protective immunity against or preventing Clostridium perfringens infection and / or necrotic enteritis in one or more subjects.

[0153] As shown herein, IgA increased by oral delivery of a live CP-CT in broilers at hatch with or without in ovo CpG -ODN and without a booster vaccine provided a significant protection against NE (pO.OOOl).

[0154] Any of the compositions described herein can be used or administered.

[0155] The composition can be administered in feed or rations or drinking water. For example, an additive can be added to feed, rations and / or drinking water and the feed, rations and / or drinking water can be provided the one or more subjects or feed, rations and / or drinking water comprising the composition can be obtained and administered to the one or more subjects.

[0156] The one or more subjects can for example be breeders that are of breeding age. Protective immunity can be induced in offspring when one or both parents are administered the composition described herein. The one or more subjects can be chicks. As disclosed therein, immunity can be obtained by administration of a single dose of the compositions described herein.

[0157] The composition in some embodiments is administered as a single dose.

[0158] In some embodiments, the composition is administered in multiple doses.

[0159] As demonstrated herein, the live virulent Clostridium perfringens included in the composition can produce homologous or heterologous protective immunity. Accordingly, the compositions can be used for homologous or heterologous protection.

[0160] In some embodiments, the composition is administered orally. In some embodiments, the composition is administered via the respiratory system.

[0161] A dose amount of live virulent Clostridium perfringens is for example administered to the one or more subjects. In some embodiments, the dose amount of live virulent Clostridiumperfringens administered is from about IxlO5colony forming units (CFU) to about IxlO11CFU per subject. In some embodiments, the dose amount is about IxlO5CFU per subject. In some embodiments, the dose amount from about IxlO6CFU per subject. In some embodiments, the dose amount is about IxlO7CFU per subject. In some embodiments, the dose amount is about IxlO8CFU per subject. In some embodiments, the dose amount is about IxlO9CFU per subject. In some embodiments, the dose amount to about IxlO10CFU per subject. In some embodiments, dose amount to about IxlO11CFU per subject.

[0162] A dose amount of cholera toxin is for example administered to the one or more subjects. In some embodiments, the dose amount of cholera toxin administered is from about 0.01 pg / g of body weight to about 3 pg / g of body weight per subject. In some embodiments, the dose amount of cholera toxin administered is from about 0.05 pg / g of body weight per subject. In some embodiments, the dose amount of cholera toxin administered is from about 0.1 pg / g of body weight per subject. In some embodiments, the dose amount of cholera toxin administered is from about 0.3 pg / g of body weight per subject. In some embodiments, the dose amount of cholera toxin administered is about 2 pg / g of body weight per subject. In some embodiments, the dose amount of cholera toxin administered is about 1 pg / g of body weight per subject. In some embodiments, the dose amount of cholera toxin administered is about 0.7 pg / g of body weight per subject.

[0163] In some embodiments, the dose amount of cholera toxin administered is about 0.5 pg / g of body weight per subject.

[0164] In some embodiments, the one or more subject(s) can be inoculated with an immunomodulator composition before administration of the composition.

[0165] In some embodiments, the one or more subject(s) can be inoculated with an immunomodulator composition in ovo before hatch. Any of the immunomodulator compositions described herein can be used.

[0166] In some embodiments, the immunomodulator composition comprises a cytosine- phosphorothioate-guanine oligodeoxynucleotide or a cytosine-phosphodiester-guanine oligodeoxynucleotide. These derivatives can both be abbreviated as CpG-ODN.

[0167] In some embodiments, the dose amount of CpG-ODN administered in ovo is from about 5 pg to about 200 pg per egg. In some embodiments, the dose amount of CpG-ODN administeredin ovo is from about 10 pg per egg. In some embodiments, the dose amount of CpG-ODN administered in ovo is from about 30 pg per egg. In some embodiments, the dose amount of CpG- ODN administered in ovo is from about 40 pg per egg. In some embodiments, the dose amount of CpG-ODN administered in ovo is up to about 100 pg per egg. In some embodiments, the dose amount of CpG-ODN administered in ovo is up to about 50 pg per egg. In some embodiments, the dose amount of CpG-ODN administered in ovo is up to about 60 pg per egg.

[0168] In some embodiments, the dose amount of CpG-ODN administered in ovo is about 50 pg per egg.

[0169] In some embodiments, the CpG-ODN is administered in ovo at day 18 of incubation.

[0170] In some embodiments, the one or more subject(s) are poultry. In some embodiments, the poultry is chicken, turkey, duck, goose, quail or pheasant. The one or more subjects can be a combination of any thereof. In some embodiments, the poultry comprises chicken. In some embodiments, the chicken comprises broiler chicken or layer hen.

[0171] In some embodiments, the poultry is a breeder that produces offspring. In some embodiments, the poultry that is administered the composition is a breeder. As demonstrated herein a breeder that is administered the composition can confer immunity to its offspring.

[0172] In some embodiments, the method comprises administration of the composition within 18 weeks after hatching. For example, breeder vaccinations can be completed by 18 weeks of age. In some embodiments, the method comprises administration of the composition within 12 weeks after hatching. In some embodiments, the method comprises administration of the composition within 8 weeks after hatching. In some embodiments, the method comprises administration of the composition within 4 weeks after hatching. In some embodiments, the method comprises administration of the composition within 5 days after hatching. In some embodiments, the method comprises administration of the composition within 72 hours after hatching.

[0173] In some embodiments, the method comprises administration of the composition at hatch. In some embodiments, the administration is within 24 hours of hatch.

[0174] In some embodiments, the method comprises mass administration of the composition to a plurality of subjects, e.g the one or more subjects is a plurality of subjects. In some embodiments, the plurality of subjects are poultry in a hatchery.

[0175] In some embodiments, administration of the composition comprises or is by oral delivery.

[0176] In some embodiments, administration of the composition comprises or is by oral delivery. In some embodiments, the administration of the composition comprises or is by intranasal, pulmonary or ocular delivery. For example, the compositions can be administered by nebulizer or eye drops.

[0177] In some embodiments, oral administration of the composition comprises incorporation of the composition in a feed, a ration, a drinking water or the composition is a gel composition. In some embodiments, intranasal or pulmonary administration of the composition comprises incorporation of the composition in a nasal spray or a nebulizer. In some embodiments, ocular administration of the composition comprises incorporation of the composition in eye drops.

[0178] In some embodiments, the method comprises administration of a boost to the one or more subject(s) after administration of the composition. In some embodiments, administration of the boost comprises or is by oral delivery, intramuscular delivery or subcutaneous delivery.

[0179] In some embodiments, the boost composition is for administration from 10 to 24 weeks of age to subjects who have been administered the composition comprising live CP and mucosal adjuvant. In some embodiments, the boost composition is for administration from 10 to 20 weeks of age to subjects who have been administered the composition. In some embodiments, the boost composition is for administration from 13 to 17 weeks of age to subjects who have been administered the composition.

[0180] In some embodiments, the boost composition includes live or inactivated Clostridium perfringens. In some embodiments, the boost composition includes a heterologous or homologous Clostridium perfringens, either live or inactivated.

[0181] In some embodiments, the dose amount of the live or inactivated Clostridium perfringens administered is from about IxlO5to about IxlO11CFU per subject. In some embodiments, the amount of the live or inactivated Clostridium perfringens administered is from about IxlO6CFU per subject. In some embodiments, the amount of the live or inactivated Clostridium perfringens administered is from about IxlO7CFU per subject. In some embodiments, the dose amount of the live or inactivated Clostridium perfringens administered is about 2xl08CFU per subject. In some embodiments, the amount of the live or inactivated Clostridium perfringens administered is toabout IxlO9CFU per subject. In some embodiments, the amount of the live or inactivated Clostridium perfringens administered is to about IxlO10CFU per subject.

[0182] In some embodiments the dose amount of the live or inactivated Clostridium perfringens administered is about IxlO8CFU per subject.

[0183] In some embodiments, administration of the boost composition comprises oral delivery, intramuscular delivery or subcutaneous delivery.

[0184] In some embodiments, the boost composition comprising inactivated Clostridium perfringens is administered by subcutaneous delivery.

[0185] In some embodiments, the boost composition includes an adjuvant. In some embodiments, the adjuvant comprises CpG-ODN. In some embodiments, the adjuvant comprises cholera toxin. In some embodiments, the subcutaneous delivery of the boost composition comprising inactivated Clostridium perfringens comprises an oil-in-water adjuvant, for example wherein the oil-in-water adjuvant is 20% Emulsigen™ . Any of the boost compositions described can be administered.

[0186] In some embodiments, the dose amount of CpG-ODN administered in the boost is from about 5 pg to about 100 pg per bird. In some embodiments, the dose amount of CpG-ODN administered in the boost is from about 10 pg per bird. In some embodiments, the dose amount of CpG-ODN administered in the boost is to about 70 pg per bird. In some embodiments, the dose amount of CpG-ODN administered in the boost is to about 50 pg per bird. In some embodiments, the dose amount of CpG-ODN administered in the boost is to about 30 pg per bird.

[0187] In some embodiments, the dose amount of CpG-ODN administered in the boost is of about 10 pg per bird.

[0188] In some embodiments, the boost composition is administered to provide a dose amount of cholera toxin from about 0.01 pg / g of body weight to about 3 pg / g of body weight per subject. In some embodiments, the boost composition is administered to provide a dose amount of cholera toxin from about 0.05 pg / g of body weight per subject. In some embodiments, the boost composition is administered to provide a dose amount of cholera toxin from about 0.1 pg / g of body weight per subject. In some embodiments, the boost composition is administered to provide a dose amount of cholera toxin from about 0.3 pg / g of body weight per subject. In some embodiments, the boost composition is administered to provide a dose amount of cholera toxin to about 2 pg / gof body weight per subject. In some embodiments, the boost composition is administered to provide a dose amount of cholera toxin to about 1 pg / g of body weight per subject. In some embodiments, the boost composition is administered to provide a dose amount of cholera toxin to about 0.7 pg / g of body weight per subject.

[0189] In some embodiments, the boost composition is formulated to provide a dose amount of cholera toxin of about 0.5 pg / g per subject.

[0190] In some embodiments, the boost is administered to provide a dose amount of cholera toxin of 0.5 pg / g of body weight.

[0191] The following paragraphs enumerated consecutively from 1 through 55 provide for various aspects of the present invention. In one embodiment, in a first paragraph (1), the present invention provides:1. A composition, comprising: live virulent Clostridium perfringens (CP), a mucosal adjuvant, and optionally a pharmaceutically acceptable carrier or diluent.2. The composition according to paragraph 1, wherein the live Clostridium perfringens comprises cpa, netB, cpb2 and / or tpeL toxin genes, optionally at least two toxin genes, optionally netB and tpeL toxin genes.3. The composition according to paragraph 1 or 2, wherein the live Clostridium perfringens is toxinotype A, B, C, D, E , F and / or G..4. The composition according to any of paragraphs 1 to 3, wherein the mucosal adjuvant is a toxin.5. The composition according to paragraph 4, wherein the toxin mucosal adjuvant is cholera toxin (CT).6. The composition according to any of paragraphs 1 to 5, wherein the composition is formulated for oral delivery.7. The composition according to any of paragraphs 1 to 6, wherein the composition is a liquid formulation, gel formulation or a lyophilized formulation, optionally a lyophilized gel formulation.8. The composition according to any of paragraphs 1 to 7, wherein the composition comprises or is formulated to provide the live virulent Clostridium perfringens in a dose amount from about IxlO5CFU to about IxlO11CFU per subject, from about IxlO6CFU to about IxlO10CFU per subject, or from about IxlO7CFU to about IxlO9CFU per subject, optionally wherein the composition comprises the live virulent Clostridium perfringens in a dose amount of about IxlO8CFU per subject.9. The composition according to any of paragraphs 5 to 9, wherein the composition comprises or is formulated to provide the cholera toxin in a dose amount from about 0.01 pg / g of body weight to about 3 pg / g of body weight of a subject, in a dose amount from about 0.05 pg / g of body weight to about 2 pg / g of body weight per subject, in a dose amount from about 0.1 pg / g of body weight to about 1 pg / g of body weight per subject, in a dose amount from about 0.3 pg / g of body weight to about 0.7 pg / g of body weight per subject, or in a dose amount of about 0.5 pg / g of body weight per subject.10. The composition according to any of paragraphs 1 to 9, wherein the composition comprises about 50, 100, 200, 300 or more doses.11. A combination comprising the composition according to any of paragraphs 1 to 10 and one or more of an immunomodulator composition and a boost composition, optionally wherein the immunomodulator composition comprises a cytosine-phosphorothioate-guanine oligodeoxynucleotide or a cytosine-phosphodiester-guanine oligodeoxynucleotide (CpG-ODN), optionally wherein the CpG-ODN has a sequence 5’-TCGTCGTTGTCGTTTTGTCGTT-3’ (SEQ ID NO: 1) ) or comprising 5’-TCGTCGTTGTCGTT-3’ (SEQ ID NO 2), optionally wherein the CpG- ODN with the sequence 5’-TCGTCGTTGTCGTTTTGTCGTT-3’ (SEQ ID NO: 1) ) or comprising 5’-TCGTCGTTGTCGTT-3’ (SEQ ID NO 2) has a phosphorothioate backbone; optionally wherein the boost composition comprises live or inactivated Clostridium perfringens, an adjuvant, and optionally a pharmaceutically acceptable carrier or diluent, optionally wherein the boost composition comprises a heterologous or homologous Clostridium perfringens compared to the composition.12. The composition according to any of paragraphs 1 to 10 or the combination according to paragraph 11, wherein the composition or combination is for use in inducing protective immunity against or preventing Clostridium perfringens infection and / or necrotic enteritis in one or more subject(s), optionally in a subject’s progeny.13. The composition or combination for use according to paragraph 12, wherein the one or more subject(s) is / are poultry.14. The composition or combination for use according to paragraph 13, wherein the poultry is chicken, turkey, duck, goose, quail or pheasant.15. The composition or combination for use according to paragraph 13, wherein the poultry is chicken.16. The composition or combination for use according to paragraph 15, wherein the chicken is broiler chicken or layer hen.17. The composition or combination for use according to any one of paragraphs 13 to 16, wherein the poultry is a breeder.18. A feed, ration or drinking water comprising the composition according to any one of paragraphs 1 to 10 or the boost composition as defined in paragraph 12 or herein.19. The feed according to paragraph 18, wherein the feed is a poultry feed.20. The feed according to paragraph 19, wherein the poultry feed comprises from 20% to 40% protein.21. The feed according to paragraph 19 or 20, wherein the poultry feed comprises Clostridium perfringens at a ratio of 1 : 1 (v / w) with the feed, optionally wherein the Clostridium perfringens is thioglycollate (FTG) grown.22. A kit comprising the composition according to any one of paragraphs 1 to 10, the combination according to paragraph 11, the feed, ration or drinking water according to any one of paragraphs 18 to 21, optionally further comprising one or more of instructions for use, or one or more vials, containers, and / or other suitable packaging comprising one or more of the composition, immunomodulator composition or boost composition, or the feed, ration or drinking water.23. The kit according to paragraph 22, wherein the kit comprises an oral delivery apparatus and / or a diluent for use in reconstituting the composition.24. The kit according to paragraph 23, wherein the diluent is saline or water, optionally sterile water.25. A method of inducing protective immunity against or preventing Clostridium perfringens infection and / or necrotic enteritis, the method comprising administering to one or more subject(s) an immunologically effective amount of a composition comprising live virulent Clostridiumperfringens (CP), a mucosal adjuvant and optionally a pharmaceutically acceptable carrier or diluent, optionally where the composition is orally administered.26. The method according to paragraph 25, wherein the composition is according to any one of paragraphs 1 to 10, the feed ration or drinking water according to any one of paragraphs 18-21 or the kit according to any one of paragraphs 22 to 24.27. The method according to paragraph 25 or 26, wherein the one or more subject(s) are inoculated with an immunomodulator composition before administration of the composition, optionally using the combination according to paragraph 12 or the feed ration or drinking water according to any one of paragraphs 18-21 or the kit according to any one of paragraphs 22 to 24.28. The method according to paragraph 27, wherein the one or more subject(s) is / are inoculated with the immunomodulator composition in ovo before hatch.29. The method according to paragraph 27 or 28, wherein the immunomodulator is a cytosine- phosphorothioate-guanine oligodeoxynucleotide or a cytosine-phosphodiester-guanine oligodeoxynucleotide (CpG-ODN).30. The method according to paragraph 29, wherein the CpG-ODN has a sequence5 ’ -TCGTCGTTGTCGTTTTGTCGTT-3 ’ (SEQ ID NO: 1) ) or comprising 5’- TCGTCGTTGTCGTT-3’ (SEQ ID NO 2), optionally wherein the CpG-ODN with the sequence 5 ’ -TCGTCGTTGTCGTTTTGTCGTT-3 ’ (SEQ ID NO: 1) ) or comprising 5’- TCGTCGTTGTCGTT-3’ (SEQ ID NO 2) comprises a phosphorothioate backbone.31. The method according to paragraph 29 or 30, wherein the dose amount of the CpG-ODN administered in ovo is from about 5 pg to about 200 pg per egg, optionally wherein the dose amount of the CpG-ODN administered in ovo is from about 10 pg to about 100 pg per egg, the dose amount of the CpG-ODN administered in ovo is from about 30 pg to about 70 pg per egg, wherein the dose amount of the CpG-ODN administered in ovo is from about 40 pg to about 60 pg per egg or wherein the dose amount of the CpG-ODN administered in ovo is 50 pg per egg.32. The method according to any one of paragraphs 25 to 31, wherein the one or more subject(s) is / are poultry.33. The method according to paragraph 32, wherein poultry is chicken, turkey, duck, goose, quail and pheasant, optionally wherein the poultry is chicken.34. The method according to paragraph 33, wherein the chicken is broiler chicken or layer hen.35. The method according to paragraph 32, wherein the poultry is a breeder.36. The method according to any one of paragraphs 25 to 35, wherein the composition is administered within 18 weeks after hatching, wherein the composition is administered within 12 weeks after hatching, wherein the composition is administered within 8 weeks after hatching, wherein the composition is administered within 4 weeks after hatching.37. The method according to any one of paragraphs 25 to 34, wherein the composition is administered within 5 days after hatching.38. The method according to any one of paragraphs 25 to 34, wherein the composition is administered within 24 hours after hatching or wherein the composition is administered at hatch.39. The method according to any one of paragraphs 25 to 38, wherein the composition is orally administered, optionally in a feed, a ration, a drinking water or a gel composition.40. The method according to any one of paragraphs 25 to 39, wherein the method further comprises administration of a boost composition to the one or more subject(s) after administration of the composition, wherein the boost composition comprises Clostridium perfringens, at least one adjuvant, and optionally a pharmaceutically acceptable carrier or diluent.41. The method according to paragraph 40, wherein the boost comprises live or inactivated Clostridium perfringens.42. The method according to paragraph 41, wherein the live or inactivated Clostridium perfringens are heterologous or homologous to the Clostridium perfringens in the composition.43. The method according to any one of paragraphs 40-42, wherein the boost composition is administered from 10 to 24 weeks of age, wherein the boost composition is administered from 10 to 20 weeks of age or wherein the boost composition is for administration from 13 to 17 weeks of age.44. The method according to any one of paragraphs 40 to 43, wherein boost composition is administered in a dose amount from about IxlO5to about IxlO11CFU per subject, in a dose amount from about IxlO6to about IxlO10CFU per subject, in a dose amount from about IxlO7to about IxlO9CFU per subject.45. The method according to any one of paragraphs 40 to 44, wherein the boost composition is administered by oral delivery, intramuscular delivery or subcutaneous delivery.46. The method according to paragraph 45, wherein the boost composition is administered by subcutaneous delivery and comprises an oil-in-water adjuvant.47. The method according to any one of paragraphs 40 to 44, wherein the boost composition comprises an adjuvant, optionally wherein the adjuvant is cholera toxin.48. The method according to paragraph 47, wherein the dose amount of cholera toxin administered is from about 0.01 pg / g of body weight to about 3 pg / g of body weight per subject, from about 0.05 pg / g of body weight to about 2 pg / g of body weight per subject, from about 0.1 pg / g of body weight to about 1 pg / g of body weight per subject or about 0.5 pg / g per subject.49. The method according to any one of paragraphs 47 to 48, wherein the adjuvant comprises a cytosine-phosphorothioate-guanine oligodeoxynucleotide or a cytosine-phosphodiester-guanine oligodeoxynucleotide (CpG-ODNs).50. The method according to paragraph 49, wherein the boost composition is formulated to provide a dose amount of CpG-ODN from about 0.5 pg to about 100 pg per subject, from about 1 pg to about 70 pg per subject, from about 3 pg to about 50 pg per subject, from about 5 pg to about 20 pg per subject or about 10 pg per subject.51. The method according to any one of paragraphs 40 to 50, wherein the composition is administered as a single dose.52. The method according to any one of paragraphs 40 to 50, wherein the one or more subjects is a plurality of subjects.53. The method according to paragraph 52, wherein the plurality of subjects comprise poultry in a hatchery.54. Use of the composition according to any one of paragraphs 1-10, the combination according to paragraph 11, the feed, ration or drinking water according to any one of paragraphs 18-21, the kit according to any one of paragraphs 22-25 in the manufacture of a medicament and / or vaccine for the treatment against and / or to prevent Clostridium perfringens infection and / or necrotic enteritis in one or more subject(s).55. The use according to paragraph 54 wherein the composition, immunomodulatory composition, boost composition is defined according to any one of paragraphs 40 to 53.

[0192] It should also be understood that, in certain methods described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise.

[0193] The following non-limiting examples are illustrative of the present application:EXAMPLESExample 1

[0194] Experiments testing oral delivery of a live CP vaccine adjuvanted with CT (CP-CT) against homologous and heterologous CP isolates with and without a booster vaccination were conducted. Protection was measured by mortality, pathology, IgY and IgA level against CP.

[0195] Oral delivery of a live CP-CT in broilers at hatch with or without in ovo CpG -ODN and without a booster vaccine provided a significant protection against NE (p<0.0001). Immunoprotection of broilers against NE was correlated with reduction of mortality, pathology, and increased levels of IgY and IgA. Immune cell profile of the intestine and peripheral blood at 2 days post vaccination revealed a significant influx of CD8+ T cells peripheral blood and in the jejunum indicating no induction of immune tolerance by this CP-CT vaccine. Furthermore, protection of broilers against NE was demonstrated by vaccinating broiler breeder parents by oral delivery of CP-CT vaccine. This disclosure provides utility of CT as a potent mucosal adjuvant with live CP against NE in broilers and broiler breeders.Materials and methodsAnimal housing and in ovo delivery of CpG-ODN

[0196] All animal experiments were approved by the Animal Research Ethics Board, University of Saskatchewan and adhered to the Canadian Council on Animal Care guidelines for humane animal use. For all the experiments, embryonated chicken eggs from Ross broiler breeders were obtained from a commercial broiler breeder operation in Saskatchewan, Canada (Prairie Pride Chicks Sales, Grandora, SK) and incubated at the Animal Care Unit (ACU) at the Western College of Veterinary Medicine (WCVM), University of Saskatchewan, Canada. Incubating eggs were candled at days 11 and 18 to confirm the viability. At day 18 of incubation, viable eggs were randomly divided into groups and CpG-ODN (50 pg / egg) delivered by the in ovo route (27). The sequence of CpG-ODN (CpG-ODN 2007) used was 5 ’ -TCGTCGTTGTCGTTTTGTCGTT-3 ’ (SEQ ID NO: 1) ) or comprising 5’-TCGTCGTTGTCGTT-3’ (SEQ ID NO 2). ODNs were produced with a phosphorothioate backbone (Operon Biotechnologies, Inc. Huntsville, AL). CpG-ODN formulations were prepared with either single wall carbon nanotubes (SWNT) or liquid surfactant composite. Synthetic CpG-ODNs were diluted in sterile, pyrogen-free saline or formulationsubstrates and administered in a 100-pL volume in ovo into the amniotic cavity through the air cell of the egg using a 22-gauge, 1-inch hypodermic needle. The volume of the injection and the length of the needle were selected to simulate the in ovo injection technology used in the poultry industry. At hatch, broiler chicks were transferred to an experimental room, with air exhausted through high efficiency particulate air (HEPA) filter at rate of 15-20 air changes per hour. Each group was housed in separate pens with 3-5 cm of wood shavings as litter. Continuous lighting was provided at 30-40 lux for the first 2 days post-hatch, then lux and duration decreased until 10-20 lux and 7 hours (h) of darkness was achieved. Room temperature was 32 °C for the first 7 days post-hatch, then decreased by 0.5 °C per day until 20°C. Water was provided ad libitum for the entire trial. A commercial RWA broiler ration was provided ad libitum until 19 days post-hatch.Animal model of NE in broiler chickens

[0197] Day-old broiler chickens were fed with antibiotic free chick starter ration containing 20% protein (Farm Choice™ RWA, Masterfeeds, Canada) until day 18 of age followed by feed withdrawal at day 19 of age for 18-24 h. Then introduced a new feed ration containing 28% protein at day 20 of age. Broiler chickens were fed with ration containing 28% protein with fluid thioglycollate (FTG) grown CP isolate containing cpa, netB, cpb2 and tpeL toxin genes, at a ratio of 1 : 1 (v / w) of FTG grown CP isolate bacterial broth to feed ad libitum for 3 days. Birds were observed for clinical signs and mortality thrice daily until termination of the experiment at day 23 of age. At day 23 of age following euthanasia of birds, the entire length of intestine was examined for gross lesions of NE. Typical gross and histopathological lesion of NE in this animal model is shown in Figure 1. Mortality, gross and histopathological scoring of the intestine was conducted (28).CP isolates and CP vaccine preparation for oral delivery

[0198] In order to perform homologous and heterologous vaccine protection, two CP isolates were selected for oral vaccine preparation. For homologous challenge protection, a CP isolate from a field case of NE containing cpa, cpb2, netB and tpeL genes were used to immunize birds as well as to challenge, while for heterologous challenge protection, a different CP isolate containing cpa, cpb2, netB and tpeL was utilized.

[0199] To prepare oral CP vaccine, CP isolates was streaked on blood agar plates (Columbia Blood Agar with 5% defibrinated sheep blood, ThermoScientific, Canada) in duplicate and incubatedunder anaerobic conditions in an anaerobic chamber (BD Gas Pak EZ chamber with 2 sachets of AnaeroGen 3.5L, ThermoScientific, Canada) at 37 °C overnight. A single colony of CP was inoculated in fluid thioglycollate media (FTG, Sigma-Aldrich, Canada) and incubated anaerobically at 37 °C for 14 h. Following incubation, CP culture contained approximately l x 108colony forming units (CFU) per mL of culture. CFU were determined by plating CP culture on 5% Columbia blood agar in duplicate and incubated anaerobically for 18-24 h. Then CP culture was centrifuged at 3399 g for 20 minutes to obtain a bacterial pellet. Then bacterial pellet was washed twice with phosphate buffered saline (PBS, pH-7.4, 0.01M).

[0200] Cholera toxin (C8052, Sigma- Aldrich, Canada CO.), a mucosal adjuvant, was formulated with live CP vaccine for the oral immunization. The amount of CT was calculated based on the mean body weight of birds at the time of vaccination at hatch. CT was reconstituted according to the manufacturer’s instructions; briefly 1 mL sterile water was used per vial (lOmg / mL) and stored at 4 °C. Then CT was mixed with CP pellet and CP-CT to make vaccine preparation. CT was assigned at the rate of 0.5pg / g body weight of chicks with live CP (IxlO8CFU / bird) and sterile water was added to reach the final volume of 200 pl / bird.Experimental design(A) Homologous protection against NE by oral delivery of a live CP vaccine adjuvanted with CT in broiler chickens at hatch

[0201] Fertilized eggs (n=300) were incubated at the ACU, WCVM, University of Saskatchewan, Canada. To confirm the viability of the eggs, candling was performed at day 11 and 18 of the incubation. After candling at day 18, fertile eggs were randomly assigned to six groups (n=40 eggs / per group). At hatch birds were randomly assigned to groups (n=20): (1) no vaccine, no challenge; (2) CP challenge only; (3) in ovo CpG-ODN + CP challenge; (4) in ovo saline + adjuvant only (CT, 0.5 pg / g body weight) + CP challenge; (5) in ovo CpG-ODN + oral CP-CT + CP boost + CP challenge; (6) in ovo saline + oral CP-CT + CP boost + CP challenge. Groups 5 and 6 boosted with live CP adjuvanted with CT [CP 1 xlO8CFU / bird + CT, 0.5 pg / g body weight] by the oral route at day 10 of age. At day 20, 21 and 22 vaccinated birds were challenged with CP and observed for development of clinical signs and mortality as described previously (28). Six subgroups (n=10 / group) were treated as above but were not challenged with CP and at day 23 of age were used to collect sera and mucosal scapings to detect antibodies (Ab) IgY and IgA against CP.(B) Homologous and heterologous protection against NE by oral delivery of a live CP vaccine adjuvanted with CT in broiler chickens at hatch.

[0202] To confirm the viability of the eggs, candling was performed at day 11 and 18 of the incubation. After candling at day 18, fertile eggs were randomly assigned to 10 groups (n=35 eggs / per group). At hatch birds were randomly assigned to groups (n=20): (1) no vaccine, no challenge; (2) CP challenge only; (3) in ovo CpG-ODN + live CP (homologous) with CT + CP boost + CP challenge; (4) in ovo saline + live CP (homologous) with CT + CP boost + CP challenge; (5) in ovo CpG-ODN + live CP (heterologous) with CT + CP boost + CP challenge; (6) in ovo saline + live CP (heterologous) with CT + CP boost + CP challenge; (7) in ovo CpG-ODN + live CP (homologous) with CT + no CP boost + CP challenge; (8) in ovo saline + live CP (homologous) with CT + no CP boost + CP challenge. (9) in ovo CpG-ODN + live CP (heterologous) with CT + no CP boost + CP challenge; (10) in ovo saline + live CP (heterologous) with CT + no CP boost + CP challenge. Oral CP vaccination at hatch with respective homologous or heterologous CP isolate, each bird received 1 xlO8CFU adjuvanted with CT (0.5 pg / g body weight) by the oral route. Groups of birds boosted at 10 days of age with 0.04% formaldehyde inactivated 1 xlO8CFU of respective homologous or heterologous CP isolate adjuvanted with 20% Emulsigen™ (Emulsigen- D, MVP adjuvants, Omaha, Nebraska 68117, USA) by the SC route. At day 20, 21 and 22 vaccinated birds were challenged with CP and observed for development of clinical signs and mortality as described above. Ten subgroups (n=10 / group) were treated as above but were not challenge with CP and at day 23 of age were used to collect sera and mucosal scapings to detect IgY and IgA against CP.(C) Oral immunization of broiler breeders with a live CP vaccine adjuvanted with CT to protect their progeny broiler chickens against NE

[0203] Day-old broiler breeder parents were obtained from Aviagen Inc., Huntsville, AL. At hatch, all chicks were vaccinated against Marek’s disease (Intervet Rismavac, Summit, NJ; Select HVT, West Perth, WA) by the in ovo and Newcastle Disease (Bl, SB1, Ceva Biomune, Lenexa, KS). Birds were housed at the ACU, WCVM, University of Saskatchewan. Feeding and lighting programs were operated according to Aviagen guidelines. Males (n=4) and females (n=18) were reared separately until 16 weeks of age. Thereafter, broiler breeder parents were divided (n=18) into two groups. At 13 weeks of age, birds were vaccinated with CP-CT vaccine to protect theirprogeny against NE: (1) no CP vaccination; (2) oral live CP-CT (CP-2xlO8CFU / bird + CT-0.5pg per gram body weight). At 16 weeks of age group 2 was administered with booster vaccine by SC route contained inactivated CP (2xl08CFU / bird) adjuvanted with CpG-ODN (lOpg / bird). Eggs from broiler breeders were collected at 58 weeks of age from group 1 and incubated (n=40) and divided into two groups (n=20 / group): (1) no vaccination, no challenge; (2) no CP vaccine, CP challenge; (3) CP vaccine, CP challenge. Broiler progeny was challenged with CP at day 20, 21 and 22 (28).Detection anti-IgA and anti-IgY against CP by indirect ELISA

[0204] An indirect ELISA was used to detect serum IgY and intestinal IgA antibodies against the whole cell of CP. Briefly, to measure mucosal IgA, sections of jejunum (approximately 8-9 cm in length) were carefully opened, and intestinal contents were removed. Jejunum mucosa was gently scrapped and stored in an Eppendorf tube containing 500 pL of 0.1% ethylenediaminetetraacetic acid (EDTA) solution on ice. Then, jejunal contents were vortexed and centrifuged at 2000 rpm and the supernatant was stored at -20 °C until testing. Briefly, 96-well microtiter plates were coated with 100 pL of heat inactivated CP (0.5 OD) and kept at 4 °C overnight. Coated wells were blocked by addition of 100 pL of 10% skim milk diluted in PBS (pH-7.4, 0.1M), then incubated for 30 min at room temperature. Blocked plates were then washed four times with distilled water (DW). Sera was tested at 1 : 100 dilutions and mucosal scrapings at 1 : 10 dilutions, in duplicates. The serum and mucosal supernatant from each bird were separately diluted in 1% skim milk (Sigma-Aldrich, Canada) diluted in PBST (pH -7.4, Tween 20). Plates were then washed 6x with DW thereafter 100 pL of goat anti-chicken IgA (GeneTex, Canada) (1 :2000) or IgY (GeneTex, Canada) was added. Plates were then incubated for 2 h at room temperature. Plates were washed 6x with DW and lOOpL of TMB substrate (3, 3', 5, 5' tetramethylbenzidine, Molecular innovations, Cedarlane) added. The plates were then incubated for 5 min. Negative control consisted of triplicate wells with all reagents except either antigen, or primary, or secondary antibodies. The OD values at 450 nm were obtained using a SpectraMax Plus 340 PC Microplate Reader. Corrected antibody values for each bird was calculated as the average of OD450 values from duplicate wells minus the average OD450 values of the negative controls.Immune cell profile of the PBMC and jejunum of neonatal broiler chickens two days following oral vaccination of CP-CT by flow cytometry.

[0205] Flow cytometry was conducted in birds from the experiment (B). Experimental Groups: (1) no vaccine, no challenge; (2) in ovo CpG-ODN + live CP [oral,l xlO8CFU / bird, homologous, (CT, 0.5 pg / g body weight)]; and (3) in ovo saline + live CP [oral,l xlO8CFU / bird, homologous, (CT, 0.5 pg / g body weight)]. PBMC and sections of jejunum were collected from birds vaccinated with live CP-CT by the oral route at 2 days post-vaccination. The jejunum was collected and placed on cold PBS containing penicillin and streptavidin. Intestines were washed with PBS to remove mucus and fat and incised as 1 cm sections. Then, sections of intestines were placed in a 15 mL centrifuge tube containing collagenase (800 U / mL) and DNase I (125 pg / mL) and incubated for 30 min at 37 °C for digestion. Digested jejunum was pushed through a cell strainer using a syringe plunger and layered on 4 mL Histopaque®-1077in 15 mL centrifuge tubes. Tubes were then centrifuged at 400G at room temperature for 20 min, then the buffy coat collected and collated in Roswell Park Memorial Institute (RPMI, Sigma-Aldrich, Canada) media. Cells were washed thrice with RPMI by pelleting at 400g for 5 min at 4 °C.

[0206] Heparinized blood (3 mL) was collected from each bird (n=5 / group) and kept at room temperature until processing. Afterwards, blood and PBS were pipet mixed at a ratio of 1 : 1 then layered on 3 mL of Histopaque® - 1077, and centrifuged for 45 min at 400g at 25 °C. The peripheral blood mononuclear cells (PBMC) layer was collected and washing steps were repeated. Further cell counting was done for all the samples using trypan blue dye. After counting, each sample was divided into two: (1). a[3 T cells panel [CD3-BIOT (CT-3, Southern Biotech, 8200-08) & Streptavidin APC; CD4-PE (CT-4, Southern Biotech, 8210-09); CD8-FITC (CT-8, Southern Biotech, 8220-02) and 7- Aminoactinomycin D (7-AAD, Invitrogen)], and (2). Monocyte / macrophage panel (MoMa) [Monocyte / Macrophage-PE (KUL01, Southern Biotech, 8420-09); MHC-II-FITC (2G11 -Southern Biotech, 8350-02) and 7-AAD (Invitrogen)] staining. Further, 0.5-1 million cells were counted for each sample and seeded in a 96-well plate. Washing steps were repeated twice with fluorescence-activated cell sorting (FACS) buffer by gentle pipet mixing. The primary antibody mixture (50 pL) was added to each well and incubated for 20 min on ice in the dark. Washing steps were repeated using 150-200 pL of FACS buffer and centrifuged at 400G for 5 min at 4 °C. The secondary antibody mixture was added to the a[3 T cells panelsamples and again incubated for 20 min on ice in dark. Washing steps were repeated and cells were resuspended in 200 pL of FACS buffer.Statistical analysis

[0207] Survival, gross and histopathological data were analyzed using GraphPad Prism V6.0 (Dotmatics Inc.) with a significance level of p<0.05. Survival and mortality data were compared using the long-rank (Mantel-cox) and chi-square tests. Microscopic lesions of the intestine were analyzed between groups using non -parametric one-way ANOVA and Tukey’s multiple comparison. Comparison between two groups is performed using Mann Whitney test (nonparametric test, p<0.0001). Corrected duplicate OD values of antibodies were analyzed between the groups by non-parametric Ordinary one-way ANOVA and Tukey’s multiple comparison (p<0.0001). Comparison between the two groups OD values was analyzed using Mann Whitney test (non-parametric test, p<0.0001). Flow cytometry data were acquired by Epics XL (Beckman Coulter) and FACS Caliber (BD Bioscience), were analyzed with FlowJo (Tree Star).RESULTS(A) Homologous protection against NE by oral delivery of a live CP vaccine adjuvanted with CT in broiler chickens at hatch

[0208] No mortality was observed in groups 1, 5 and 6. Group 2 had a total of 30% mortality; 3 birds were dead at 2 days and 3 birds were dead at 3 days post-challenge (Figure 2A). Group 3 had 3 dead birds at 3 days post-challenge (15%). Group 4 had 2 dead birds at 3 days post-challenge (10%). No gross or microscopic lesions were observed in group 1. Group 2 had 85% of birds with NE lesions [55% of birds with macroscopic lesions (score 3) and 10% of birds with microscopic lesions (score 2 in 10% birds and score 1 in 20% of birds)]. Group 3 had 60% birds with NE lesions (score 1 in 25% birds, score 2 in 5% birds and score 3 in 15% birds). Group 4 had 45% of birds with NE lesions (score 1 in 20% birds, score 2 in 15% birds and score 3 in 10% birds). Group 5 had 35% of birds with NE lesions (score 3 in 15% birds and score 2 in 20% birds). Group 6 had 20% of birds with NE lesions (score 3 in 20% of birds) (Figure 2B). Significantly high levels of IgY and IgA were observed in both groups vaccinated with live CP adjuvanted with CT with or without in ovo delivery of CpG-ODN compared to group 2 (p<0.0001) (Figure 2C, 2D).(B) Homologous and heterologous protection against NE by oral delivery of a live CP vaccine adjuvanted with CT in broiler chickens at hatch.

[0209] There was no mortality in the group 1 and all the groups vaccinated with CP-CT. Group 2 had 20% mortality (Figure 3A). There was no gross or microscopic NE lesions (0%) in group 1 (Figure 3B). Group 2 had 100% birds with NE lesions (score 3 lesions in 90% birds and score 2 lesions in 10% birds). Group 3 had 20% birds with NE lesions (score 1 in 10% birds and score 2 in 10% birds). Group 4 had 30% of birds with NE lesions (score 1 in 15% birds, score 2 in 10% birds and score 3 in 5%). Group 5 had 5% birds with NE lesions (score 1 in 5%). Group 6 had 5% birds with NE lesions (score 2). Group 7 had 15% birds with NE lesions (score 1 in 5%, score 2 in 5% and score 3 in 5%). Group 8 has 20% of birds with NE lesions (score 1 in 10%, score 2 in 5% and score 3 in 5%). Group 9 had 20% birds with NE lesions (score 2 in 5% and score 1 in 15%). Group 10 had 10% of birds with NE lesions (score 1). All the groups vaccinated with either homologous or heterologous CP vaccine with or without in ovo CpG-ODN had significantly higher levels of IgY (pO.OOOl) and IgA (pO.OOOl) compared to group 1 (Figure 3C, 3D).(C) Oral immunization of broiler breeders with a live CP vaccine adjuvanted with CT to protect their progeny broiler chickens against NE

[0210] No mortality was observed in group 1 originated from their broiler breeder parents not vaccinated with CP-CT and broiler progeny was not challenged with CP. Two birds were dead in group 2 at 2 days post-challenge originated from their broiler breeder parents not vaccinated with CP-CT but broiler progeny was challenged with CP. Group 3 had no mortality originated from their broiler breeder parents vaccinated with CP-CT and broiler progeny was challenged with CP (Figure 4A). No macroscopic or microscopic lesions were observed in group 1. Group 2 had 80% of birds with NE lesions (score 1 in 10%, score 2 in 10% and score 3 in 60% birds)). Group 3 had 35% birds with NE lesions (score 1 in 15% birds, score 2 in 10% and score 3 in 10%] (Figure 4B).Immune cell profile in PBMC of neonatal broiler chickens two days following oral vaccination of CP-CT by flow cytometry.

[0211] A significant increase of CD8+ T cells (pO.OOOl) (Figure 5A) and decrease of CD4+ T (pO.Ol) (Figure 5C) cells was observed in PBMC of birds vaccinated with live CP by the oral route with or without in ovo CpG-ODN administration compared to the birds that did not receive a CP vaccine. In the jejunum of birds vaccinated with CP-CT by the oral route had a significant increase in CD8+ T cells with or without in ovo CpG-ODN compared to the group that did not receive a CP vaccine (pO.001) (Figure 5B). There was a significant increase (p=0.0197) (Figure5D) in CD4+T cells of the jejunum of groups vaccinated live CP-CT by the oral route with in ovo CpG-ODN administration, while it was non-significant without in ovo CpG-ODN administration in vaccinated group. Also, a significant difference was detected in the population of monocyte in PBMC and macrophages in jejunum of birds vaccinated with live CP-CT by the oral route with or without in ovo CpG-ODN administration compared to the birds that did not receive a CP vaccine (Figure 5E, 5F).

[0212] The objective of the first experiment was to study the immunoprotective effects of a live CP vaccine delivered by the oral route at the day of hatch against homologous CP challenge associated with NE in broiler chickens following in ovo administration of CpG-ODN as an immune modulator. Immunoprotective effects of a live CP vaccine delivered by the oral route was compared with and without CpG-ODN. A booster dose of CP adjuvanted with CT was administered by the oral route at day 10 of age prior to challenging birds with CP at 20, 21 and 22 days of age. A significant reduction of pathological lesions of NE was observed in both vaccinated groups but the highest protection was achieved in the group received no CpG-ODN by the in ovo route followed by live CP vaccine by the oral route. CT adjuvant alone followed by CP challenge had a significant reduction (p<0.01) in NE lesions, which proves CT as an efficient adjuvant against NE. Previous study with delivery of live attenuated CP vaccine by the oral route without an adjuvant have shown protection against NE however, required multiple doses of CP for 5 consecutive days followed by antibiotic treatment (16). In another study, it has been demonstrated delivery of non-virulent CP strain adjuvanted with CT induced antibody response against NE, but required multiple dose administration at 8, 9, 10, 11 and 12 days of age in feed as an esophageal gavage (15). Administration of CpG-ODN at day 18 of incubation followed by live CP vaccine adjuvanted with CT by the oral route at hatch protected birds significantly against NE. In the second experiment, the ability of oral delivery of a live CP vaccine adjuvanted with CT against heterologous and homologous isolates of a NE with or without administering a booster vaccine was demonstrated. In this experiment the booster dose of CP vaccine was a formulation of inactivated CP with 20% Emulsigen™ and delivered by the SC route at day 10 of age. Here, protection of birds against NE at a significant level by oral delivery of live CP-CT vaccine against heterologous CP with or without in ovo delivery of CpG-ODN and without a booster CP vaccination was demonstrated. This CP-CT vaccination at hatch increased IgY in serum and IgA in the jejunum at a significant level and protected birds against NE.

[0213] There was an influx of CD8+T in peripheral blood and jejunum with or without in ovo delivery of CpG-ODN in birds that had oral delivery of CP-CT two days following vaccination while a significant decline in CD4+ T cells in PBMC and no significant change of CD4+ T cells in the jejunum. No T cell responses along with diminished systemic antibody response are the hallmark of oral tolerance or systemic unresponsiveness (42). Here, it is shown that live CP-CT vaccine was capable of eliciting CD8+T cell response in PBMC and jejunum, which provides clear evidence of lack of induction of oral tolerance following CP-CT oral vaccination.

[0214] The protection of breeder progeny has also been demonstrated herein by vaccinating their broiler breeder parents with oral delivery of a CP-CT vaccine. There was a significant reduction in NE lesions in broiler progeny following CP challenge when broiler breeder parents were vaccinated against NE using CP-CT vaccine by the oral route followed by a CP booster vaccination. It has been reported that there is a significant reduction in mortality due to NE in broiler progeny when broiler breeder parents were vaccinated against NE with a CP type A alpha toxoid vaccine by the IM route (Netvax™, Intervet / Schering-Plough Animal Health, Summit, NJ, USA) at 11 and 18 weeks of age (18). It has been demonstrated that there is a protection of broiler progeny against NE by vaccination of broiler breeder parent with type A and C CP adjuvanted with aluminium hydroxide by IM route and the protection of broilers against NE was better when parents were vaccinated with type A CP than type C CP vaccine (19).

[0215] In conclusion, protection of broiler chickens against NE by a single live CP vaccine adjuvanted with CT by the oral route with or without in ovo delivery of CpG-ODN without a booster vaccination was demonstrated. This vaccination strategy was able to protect broiler chickens against homologous and heterologous isolates of CP at a statistically significant level. Oral delivery of live CP vaccine was able to produce both systemic IgY and mucosal IgA against CP with or without in ovo CpG-ODN and without a booster CP vaccine. Protection of broiler chickens against NE by vaccinating their broiler breeder parents with a live CP vaccine adjuvanted with CT by the oral route at 13 weeks of age followed by booster CP-CT vaccine by the oral route at 16 weeks of age was also demonstrated. This disclosure demonstrated utility of CT as a potent mucosal adjuvant with live CP against NE in broilers and broiler breeders.Example 2Vaccine composition comprising live virulent CP and CT mucosal adjuvant

[0216] ACP isolate such as ATCC13124 or isolated from a CP outbreak, is streaked on blood agar plates (Columbia Blood Agar with 5% defibrinated sheep blood, ThermoScientific, Canada) and incubated under anaerobic conditions in an anaerobic chamber (BD Gas Pak EZ chamber with 2 sachets of AnaeroGen 3.5L, ThermoScientific, Canada) at 37 °C overnight. A single colony of CP is inoculated in fluid thioglycollate media (FTG, Sigma-Aldrich, Canada) and incubated anaerobically at 37 °C for about 14 h until the culture comprises about approximately l x 108colony forming units (CFU) per mL of culture. Then CP culture is centrifuged to obtain a bacterial pellet which is washed with phosphate buffered saline (PBS, pH-7.4, 0.01M).

[0217] Cholera toxin (CT) was obtained (C8052, Sigma-Aldrich, Canada CO.) and formulated with live CP vaccine. The amount of CT is calculated based on the mean body weight of birds at the time of vaccination., for example at hatch. CT is reconstituted in 1 mL sterile water per vial (lOmg / mL) and stored at 4 °C. Then CT was mixed with CP pellet and CP-CT to make vaccine preparation. The amount of CT is about 0.5pg / g body weight of chicks the amount of live CP is about IxlO8CFU / bird and sterile water was added to reach the final volume of 200 pl / bird..

[0218] The preceding section is provided by way of example only and is not intended to be limiting on the scope of the present disclosure and appended claims. Additional objects and advantages associated with the compositions and methods of the present disclosure will be appreciated by one of ordinary skill in the art in light of the instant claims, description, and examples. For example, the various aspects and embodiments of the disclosure may be utilized in numerous combinations, all of which are expressly contemplated by the present description. These additional advantages objects and embodiments are expressly included within the scope of the present disclosure. The publications and other materials used herein to illuminate the background of the disclosure, and in particular cases, to provide additional details respecting the practice, are incorporated by reference, and for convenience are listed in the appended reference section.REFERENCES1. Rood JI, Adams V, Lacey J, Lyras D, McClane BA, Melville SB, Moore RJ, Popoff MR, Sarker MR, Songer JG, et al. Expansion of the Clostridium perfringens toxin-based typing scheme. Anaerobe. 53:5-10; 2018.2. Helmboldt CF, Bryant E %J AD. The pathology of necrotic enteritis in domestic fowl.775-780; 1971.3. Wade B, Keybum A. The true cost of necrotic enteritis. World Poult. 31: 16-7; 2015.4. McDevitt R, Brooker J, Acamovic T, Sparks N. Necrotic enteritis; a continuing challenge for the poultry industry. Worlds’ Poultry Science Journal. 62:221-247; 2006.5. Cooper KK, Songer JG. Virulence of Clostridium perfringens in an experimental model of poultry necrotic enteritis. Veterinary microbiology . 142:323-328; 2010.6. Mot D, Timbermont L, Haesebrouck F, Ducatelle R, Van Immersed F. Progress and problems in vaccination against necrotic enteritis in broiler chickens. Avian pathology. 43:290-300; 2014.7. Ulmer-Franco A, Cherian G, Quezada N, Fasenko G, McMullen L. Hatching egg and newly hatched chick yolk sac total IgY content at 3 broiler breeder flock ages. Poultry science. 91:758-764; 2012.8. Cox CM, Dalloul RA. Immunomodulatory role of probiotics in poultry and potential in ovo application. Beneficial Microbes . 6:45-52; 2015.9. M’Sadeq SA, Wu S, Swick RA, Choct M %J AN. Towards the control of necrotic enteritis in broiler chickens with in-feed antibiotics phasing-out worldwide. 1: 1-11; 2015.10. Lee K, Lillehoj H, Jeong W, Jeoung H, An D. Avian necrotic enteritis: experimental models, host immunity, pathogenesis, risk factors, and vaccine development. Poultry Science. 90: 1381-1390; 2011.11. Belyakov IM, Derby MA, Ahlers JD, Kelsall BL, Earl P, Moss B, Strober W, Berzofsky JA. Mucosal immunization with HIV- 1 peptide vaccine induces mucosal and systemic cytotoxic T lymphocytes and protective immunity in mice against intrarectal recombinant HIV-vaccinia challenge. Proceedings of the National Academy of Sciences. 95: 1709-1714; 1998.12. Gallichan WS, Rosenthal KL. Long-lived cytotoxic T lymphocyte memory in mucosal tissues after mucosal but not systemic immunization. The Journal of experimental medicine. 184: 1879-1890; 1996.Holmgren J, Adamsson J, Anjuere F, Clemens J, Czerkinsky C, Eriksson K, Flach C-F, George- Chandy A, Harandi AM, Lebens M. Mucosal adjuvants and anti-infection and anti- immunopathology vaccines based on cholera toxin, cholera toxin B subunit and CpG DNA. Immunology letters. 97: 181-188; 2005. Holmgren J, Lycke N, Czerkinsky C. Cholera toxin and cholera B subunit as oral — mucosal adjuvant and antigen vector systems. Vaccine. 11: 1179-1184; 1993. Mishra N, Smyth JA %J V. Oral vaccination of broiler chickens against necrotic enteritis using a non-virulent NetB positive strain of Clostridium perfringens type A. 35:6858-6865; 2017. Thompson DR, Parreira VR, Kulkami RR, Prescott JF. Live attenuated vaccine-based control of necrotic enteritis of broiler chickens. Veterinary Microbiology . 113:25-34; 2006. Kulkami RR, Parreira VR, Jiang Y-F, Prescott J %J C, Vaccine Immunology. A live oral recombinant Salmonella enterica serovar Typhimurium vaccine expressing Clostridium perfringens antigens confers protection against necrotic enteritis in broiler chickens. 17:205-214; 2010. Crouch CF, Withanage GSK, De Haas V, Etore F, Francis M %J A pathology. Safety and efficacy of a maternal vaccine for the passive protection of broiler chicks against necrotic enteritis. 39:489- 497; 2010. Lovland A, Kaldhusdal M, Redhead K, Skjerve E, Lillehaug A %J AP. Maternal vaccination against subclinical necrotic enteritis in broilers. 33:81-90; 2004. Shirota H, Tross D, Klinman DM. CpG Oligonucleotides as Cancer Vaccine Adjuvants. Vaccines. 3:390-407; 2015. Kumar H, Kawai T, Akira S. Pathogen recognition by the innate immune system. International reviews of immunology . 30: 16-34; 2011. Shirota H, Klinman DM. Recent progress concerning CpG DNA and its use as a vaccine adjuvant. Expert review of vaccines. 13:299-312; 2014. Gomis S, Babiuk L, Allan B, Willson P, Waters E, Hecker R, Potter A. Protection of Chickens Against a Lethal Challenge of Escherichia coli by a Vaccine Containing CpG Oligodeoxynucleotides as an Adjuvant. Avian Diseases. 51:78-83; 2007. Hartley C, Salisbury A-M, Wigley P. CpG oligonucleotides and recombinant interferon-y in combination improve protection in chickens to Salmonella enterica serovar Enteritidis challenge as an adjuvant component, but have no effect in reducing Salmonella carriage in infected chickens. Avian pathology. 41:77-82; 2012.Cox JC, Coulter AR. Adjuvants — a classification and review of their modes of action. Vaccine. 15:248-256; 1997. Ishida Y, Sakai E, Sato K, Sugiyama E, Mima K, Taneno A, Shimomura H, Cui L, Hirai Y. Induction of Mucosal Humoral Immunity by Subcutaneous Injection of an Oil-emulsion Vaccine against Salmonella enterica subsp. enterica serovar Enteritidis in Chickens. Food Safety. 6: 151-155; 2018. Gunawardana T, Foldvari M, Zachar T, Popowich S, Chow-Lockerbie B, Ivanova MV, Tikoo S, Kurukulasuriya S, Willson P, Gomis S. Protection of neonatal broiler chickens following in ovo delivery of oligodeoxynucleotides containing CpG motifs (CpG-ODN) formulated with carbon nanotubes or liposomes. Avian diseases. 59:31-37; 2014. Gautam H, Ayalew LE, Shaik NA, Subhasinghe I, Popowich S, Chow-Lockerbie B, Dixon A, Ahmed KA, Tikoo SK, Gomis S. Exploring the predictive power of jejunal microbiome composition in clinical and subclinical necrotic enteritis caused by Clostridium perfringens: insights from a broiler chicken model. Journal of Translational Medicine . Tl'. 1-18; 2024. Jang SI, Lillehoj HS, Lee S-H, Lee KW, Lillehoj EP, Hong YH, An D-J, Jeong W, Chun J-E, Bertrand F, et al. Vaccination with Clostridium perfringens recombinant proteins in combination with Montanide™ ISA 71 VG adjuvant increases protection against experimental necrotic enteritis in commercial broiler chickens. Vaccine. 30:5401-5406; 2012. Keybum AL, Portela RW, Sproat K, Ford ME, Bannam TL, Yan X, Rood JI, Moore RJ. Vaccination with recombinant NetB toxin partially protects broiler chickens from necrotic enteritis. Vet Res. 44:54; 2013. Kulkami RR, Parreira VR, Sharif S, Prescott J %J C, vaccine immunology. Clostridium perfringens antigens recognized by broiler chickens immune to necrotic enteritis.2006. Wilde S, Jiang Y, Tafoya AM, Horsman J, Yousif M, Vazquez LA, Roland KL %J P one. Salmonella-vectored vaccine delivering three Clostridium perfringens antigens protects poultry against necrotic enteritis . 14:2019. Zekarias B, Mo H, Curtiss R %J CVI. Recombinant attenuated Salmonella enterica serovar Typhimurium expressing the carboxy-terminal domain of alpha toxin from Clostridium perfringens induces protective responses against necrotic enteritis in chickens. 15:805-816; 2008. Plotkin SA, Plotkin SL. The development of vaccines: how the past led to the future. Nature Review s Microbiology . 9:889-893; 2011.Witer R, Hunt H. Poultry vaccines of the future. Poultry science. 73: 1087-1093; 1994. Jiang Y, Kulkami RR, Parreira VR, Poppe C, Roland KL, Prescot JF. Assessment of 2 Salmonella enterica serovar Typhimurium-based vaccines against necrotic enteritis in reducing colonization of chickens by Salmonella serovars of different serogroups. Canadian Journal of Veterinary Research. 74:264-270; 2010. Lycke N, Holmgren J. Strong adjuvant properties of cholera toxin on gut mucosal immune responses to orally presented antigens. Immunology . 59:301; 1986. Gagliardi MC, De Magistris MT. 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Mucosal immunity and tolerance: relevance to vaccine development. Immunological reviews. 170: 197-222; 1999.

Claims

1. CLAIMS:

1. A composition, comprising: live virulent Clostridium perfringens (CP), a cholera toxin (CT) mucosal adjuvant, and optionally a pharmaceutically acceptable carrier or diluent.

2. The composition of claim 1, wherein the live virulent Clostridium perfringens comprises cpa, netB, cpb2 and / or tpeL toxin genes, optionally at least two toxin genes, optionally netB and tpeL toxin genes and / or wherein the live virulent Clostridium perfringens is toxinotype A, B, C, D, E , F and / or G.

3. The composition of any one of claims 1 or 2, wherein the composition is formulated for oral delivery and / or is a single dose, optionally single dose oral vaccine.

4. The composition of any one of claims 1 to 3, wherein the composition is a liquid formulation, gel formulation or a lyophilized formulation, optionally a lyophilized gel formulation.

5. The composition of any one of claims 1 to 4, wherein the composition is formulated to provide the live virulent Clostridium perfringens in a dose amount from about IxlO5CFU to about IxlO11CFU per subject, from about IxlO6CFU to about lxlOloCFU per subject, or from about IxlO7CFU to about IxlO9CFU per subject, optionally wherein the composition is formulated to provide the live virulent Clostridium perfringens in a dose amount of about IxlO8CFU per subject; and / or wherein the composition is formulated to provide the cholera toxin in a dose amount from about 0.01 pg / g of body weight to about 3 pg / g of body weight of a subject, in a dose amount from about 0.05 pg / g of body weight to about 2 pg / g of body weight per subject, in a dose amount from about 0.1 pg / g of body weight to about 1 pg / g of body weight per subject, in a dose amount from about 0.3 pg / g of body weight to about 0.7 pg / g of body weight per subject, or in a dose amount of about 0.5 pg / g of body weight per subject.; and / or wherein the composition comprises about 50, 100, 200, 300 or more doses.

6. A combination comprising the composition of any one of claims 1 to 5 and one or more of an immunomodulator composition and a boost composition, optionally wherein theimmunomodulator composition comprises a cytosine-phosphorothioate-guanine oligodeoxynucleotide or a cytosine-phosphodiester-guanine oligodeoxynucleotide (CpG-ODN), optionally wherein the CpG-ODN has a sequence 5’-TCGTCGTTGTCGTTTTGTCGTT-3’ (SEQ ID NO: 1) ) or comprising 5’-TCGTCGTTGTCGTT-3’ (SEQ ID NO 2), optionally wherein the CpG- ODN with the sequence 5’-TCGTCGTTGTCGTTTTGTCGTT-3’ (SEQ ID NO: 1) ) or comprising 5’-TCGTCGTTGTCGTT-3’ (SEQ ID NO: 2) has a phosphorothioate backbone; optionally wherein the boost composition comprises live or inactivated Clostridium perfringens, an adjuvant, and optionally a pharmaceutically acceptable carrier or diluent, optionally wherein the boost composition comprises a heterologous or homologous Clostridium perfringens compared to the composition.

7. The composition of any one of claims 1 to 5 or the combination of claim 6, wherein the composition or combination is for use in inducing protective immunity against or preventing Clostridium perfringens infection and / or necrotic enteritis in one or more subject(s).

8. The composition or combination for use of claim 7, wherein the one or more subject(s) is / are poultry, optionally wherein the poultry is chicken, turkey, duck, goose, quail or pheasant.

9. The composition or combination for use of claim 8, wherein the poultry is chicken, optionally a broiler chicken or layer hen.

10. The composition or combination for use of any one of claims 8 or 9, wherein the poultry is a breeder.

11. A feed, ration or drinking water comprising the composition of any one of claims 1 to 5 or the boost composition as defined in claim 6.

12. The feed of claim 11, wherein the feed is a poultry feed, preferably wherein the poultry feed comprises from 20% to 40% protein and / or wherein the poultry feed comprises Clostridium perfringens at a ratio of 1 : 1 (v / w) with the feed, optionally wherein the Clostridium perfringens is thioglycollate (FTG) grown.

13. The drinking water of claim 11, wherein the drinking water is substantially chlorine free, and / or wherein the drinking water comprising the composition or the boost composition is made by a method comprising mixing distilled and / or sterile water with the composition or the boost composition.

14. A kit comprising the composition of any one of claims 1 to 5, the combination of claim 6, the combination or composition for use of any one of claims 7 to 10, the feed, ration or drinking water of any one of claims 11 to 13, comprising one or more of instructions for use, or one or more vials, containers, and / or other suitable packaging housing one or more of the composition, immunomodulator composition or boost composition, or the feed, ration or drinking water.

15. The kit of claim 14, wherein the kit comprises an oral delivery apparatus and / or a diluent for reconstituting the composition or boost composition, optionally wherein the diluent is saline or water, optionally substantially chlorine free, sterile and / or distilled water.

16. A method of inducing protective immunity against or preventing Clostridium perfringens infection and / or necrotic enteritis, the method comprising administering to one or more subject(s) an immunologically effective amount of a composition comprising live virulent Clostridium perfringens (CP), a cholera toxin (CT) mucosal adjuvant and optionally a pharmaceutically acceptable carrier or diluent, optionally where the composition is orally administered.

17. The method of claim 16, wherein the composition is any one of claims 1 to 5, the combination of claim 6, the feed ration or drinking water of any one of claims 11-13 or the kit of any one of claims 14 or 15.

18. The method of claim 16 or 17, wherein the one or more subject(s) are inoculated with an immunomodulator composition before administration of the composition, optionally using the combination of claim 6 or the feed ration or drinking water of any one of claims 11-13 or the kit of any one of claims 14 or 15.

19. The method of claim 18, wherein the one or more subject(s) is / are inoculated with the immunomodulator composition in ovo before hatch, optionally wherein theimmunomodulator is a cytosine-phosphorothioate-guanine oligodeoxynucleotide or a cytosine- phosphodiester-guanine oligodeoxynucleotide (CpG-ODN).

20. The method of claim 19, wherein the CpG-ODN has a sequence5 ’ -TCGTCGTTGTCGTTTTGTCGTT-3 ’ (SEQ ID NO: 1) ) or comprising 5’- TCGTCGTTGTCGTT-3’ (SEQ ID NO 2), optionally wherein the CpG-ODN with the sequence 5 ’ -TCGTCGTTGTCGTTTTGTCGTT-3 ’ (SEQ ID NO: 1) ) or comprising 5’- TCGTCGTTGTCGTT-3’ (SEQ ID NO 2) comprises a phosphorothioate backbone, or wherein the dose amount of the CpG-ODN administered in ovo is from about 5 pg to about 200 pg per egg, optionally wherein the dose amount of the CpG-ODN administered in ovo is from about 10 pg to about 100 pg per egg, the dose amount of the CpG-ODN administered in ovo is from about 30 pg to about 70 pg per egg, wherein the dose amount of the CpG-ODN administered in ovo is from about 40 pg to about 60 pg per egg or wherein the dose amount of the CpG-ODN administered in ovo is 50 pg per egg.

21. The method of any one of claims 16 to 20, wherein the one or more subject(s) is / are poultry, optionally wherein poultry is chicken, turkey, duck, goose, quail and pheasant.

22. The method of claim 21, wherein the poultry is a chicken, optionally wherein the chicken is broiler chicken or layer hen and / or wherein the poultry is a breeder.

23. The method of claim 22, wherein the poultry is a breeder.

24. The method of any one of claims 16 to 23, wherein the composition is administered to the subject(s) within 18 weeks after hatching, wherein the composition is administered within 12 weeks after hatching, wherein the composition is administered within 8 weeks after hatching, or wherein the composition is administered within 4 weeks after hatching.

25. The method of any one of claims 16 to 22, wherein the composition is administered within 5 days after hatching, wherein the composition is administered within 24 hours after hatching or wherein the composition is administered at hatch.

26. The method of any one of claims 16 to 25, wherein the composition is orally administered, optionally in a feed, a ration, a drinking water or a gel composition.

27. The method of any one of claims 16 to 26, wherein the method further comprises administration of a boost composition to the one or more subject(s) after administration of the composition, wherein the boost composition comprises Clostridium perfringens, at least one adjuvant, and optionally a pharmaceutically acceptable carrier or diluent.

28. The method of claims 27, wherein the boost composition comprises live or inactivated Clostridium perfringens.

29. The method of claim 28, wherein the live or inactivated Clostridium perfringens are heterologous or homologous to the Clostridium perfringens in the composition.

30. The method of any one of claims 27-29, wherein the boost composition is administered to the subject(s) at 10 to 24 weeks of age, wherein the boost composition is administered to the subject(s) at 10 to 20 weeks of age or wherein the boost composition is administered to the subject(s) at 13 to 17 weeks of age; and / or wherein boost composition is administered in a dose amount from about IxlO5to about IxlO11CFU per subject, in a dose amount from about IxlO6to about IxlO10CFU per subject, in a dose amount from about IxlO7to about IxlO9CFU per subject .

31. The method of any one of claims 27 to 30, wherein the boost composition is administered by oral delivery, intramuscular delivery or subcutaneous delivery; or wherein the boost composition is administered by subcutaneous delivery and comprises an oil-in-water adjuvant.

32. The method of any one of claims 16 to 31, wherein the composition is an oral formulation, optionally a drinking water formulation, or a gel formulation.

33. The method of any one of claims 27 to 30, wherein the boost composition comprises an adjuvant, optionally wherein the adjuvant is cholera toxin.

34. The method of claim 33, wherein the dose amount of cholera toxin administered is from about 0.01 pg / g of body weight to about 3 pg / g of body weight per subject,from about 0.05 pg / g of body weight to about 2 pg / g of body weight per subject, from about 0.1 pg / g of body weight to about 1 pg / g of body weight per subject or about 0.5 pg / g per subject; wherein the adjuvant comprises a cytosine-phosphorothioate-guanine oligodeoxynucleotide or a cytosine-phosphodiester-guanine oligodeoxynucleotide (CpG-ODNs).

35. The method of claim 34, wherein the boost composition is formulated to provide a dose amount of CpG-ODN from about 0.5 pg to about 100 pg per subject, from about 1 pg to about 70 pg per subject, from about 3 pg to about 50 pg per subject, from about 5 pg to about 20 pg per subject or about 10 pg per subject.

36. The method of any one of claims 27 to 36, wherein the composition is administered as a single dose; or wherein the one or more subjects is a plurality of subjects.

37. The method of claim 36, wherein the plurality of subjects comprise poultry in a hatchery.

38. Use of the composition of any one of claims 1-5, the combination of claim 6, the feed, ration or drinking water of any one of claims 11-13, the kit of any one of claims 14 or 15 in the manufacture of a medicament and / or vaccine for the treatment against and / or to prevent Clostridium perfringens infection and / or necrotic enteritis in one or more subject(s).

39. The use of claim 38 wherein the composition, immunomodulatory composition, boost composition is defined as in any one of claims 27 to 38.

40. The composition of any one of claims 1 to 5, the combination of claim 6, the composition or combination for use of any one of claims 7 to 10, the feed, ration or drinking water of any one of claims 11 to 13, the kit of claim 14 or 15, the method of any one of claims 16- 37 , or the use of claim 38 wherein the composition, combination, composition or combination for use, the feed, ration or drinking water, or the kit is a single dose vaccine, optionally a single dose oral vaccine, or the method or the use, wherein the composition or combination is a single dose vaccine, optionally a single dose oral vaccine.

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