WO2007101337A1

WO2007101337A1 - Methods and compositions comprising bacterial type hi secreted proteins for mucosal immunization of animals

Info

Publication number: WO2007101337A1
Application number: PCT/CA2007/000354
Authority: WO
Inventors: Shawn Babiuk; Andrew A. Potter; Dragan Rogan; Lorne Babiuk
Original assignee: Bioniche Life Sciences Inc.
Priority date: 2006-03-06
Filing date: 2007-03-06
Publication date: 2007-09-13

Abstract

The present invention relates to vaccines comprising bacterial type III proteins or fragments thereof. Also provided is a method of preventing attachment, colonization and/or shedding of bacteria in an animal by administering a vaccine comprising a bacterial type III secreted protein, or fragment thereof to an animal.

Description

METHODS AND COMPOSITIONS COMPRISING BACTERIAL TYPE HI SECRETED PROTEINS FOR MUCOSAL IMMUNIZATION OF ANIMALS

FIELD OF INVENTION

[0001] The present invention relates to methods and compositions for immunizing animals.

BACKGROUND OF THE INVENTION

[0002] Enterohemorrhagic Escherichia coli is an important human pathogen, causing diarrhea and, in some cases, haemolytic-uremic syndrome (HUS) leading to kidney failure and death (1, 2). There are an estimated 75,000 human cases of enterohaemorrhagic E. coli infection per year in the United States with serotype Ol 57:H7 causing the majority of human illness, although other serotypes are more prevalent elsewhere in the world (15). Antibiotics increase the risk of HUS and currently there are no treatments available besides supportive care (24).

[0003] E. coli O157:H7 is a zoonotic pathogen and infection is often associated with the consumption of contaminated food or water. Although E. coli O157:H7 can cause diarrhea in calves, the organism does not cause disease in adult cattle and is considered part of their normal flora (7). Cattle are a major reservoir for E. coli O157:H7 since it can be found in approximately 80% of some cattle populations and is also found on 49% of beef carcasses (9). There is an association between the incidences of E. coli Ol 57:H7 infections in humans and the intensity of beef cattle operations (20). An increased level of shedding of the organism during the summer months is correlated with an increased incidence of disease (21). Shedding of the organism by cattle is intermittent in nature and it is not known if the organism persists during periods when it cannot be detected in feces.

[0004] It has been proposed that since cattle are associated with the majority of cases of E. coli Ol 57:H7 infection in humans, reducing levels of the organism in cattle would be an attractive strategy to reduce the risk of human infection. Computer simulations have suggested that the greatest potential impact on levels of the organism in cattle would be achieved either through vaccination or treatment with an agent that reduces the number of bacteria in feces (1 1). Several different approaches have been proposed to achieve this goal, including modification of feed (8), probiotics, (25), as well as vaccination. With respect to the latter, a number of different antigens have been tested for their ability to induce immune responses which block colonization, including lipopolysaccharide (4, 5), intimin (12), and bacterial type III secreted proteins (16).

[0005] The type III secretion system of E. coli O157:H7 is involved in colonization of mammalian hosts by the organism. The Tir protein is inserted into the mammalian host cell plasma membrane and acts as the receptor for intimin, a bacterial outer membrane protein (13). EspA forms a linkage between the bacterium and the host cell. The interaction of Tir with intimin may be necessary for bacterial adherence to host cells as antibodies against intimin can reduce E. coli O157:H7 adherence in cell culture models (3). In addition, enterohemorrhagic Escherichia co//-derivatives with deletions in the tir gene have been shown to colonize animals less effectively than the wild type strain (18).

[0006] Thus, bacterial type III secreted proteins are good candidate antigens for the prevention of E. coli O157:H7 attachment, colonization, and shedding in cattle. Alternatively, if animals already carry the organism, it may be possible to generate specific immune responses that would allow the host to clear it, a clear advantage for cattle that are going to slaughter. Systemic vaccination of cattle with bacterial type III secreted proteins, including Tir and EspA, has been shown to result in decreased E. coli O157:H7 shedding following both experimental infection as well as under field conditions (16). However, an increased understanding of the immunological mechanisms by which this vaccine worked is an important issue that needs to be addressed in order to enhance mucosal responses.

[0007] There is a need in the art for novel methods and compositions for immunizing animals. There is also a need in the art for novel mucosal vaccines and methods of inducing mucosal immunity. There is also a need in the art for compositions and methods for preventing attachment, colonization and shedding of microorganisms in an animal. Further, there is a need in the art for novel methods and compositions for immunizing animals such that protective immunity is obtained thus resulting in a reduction of the pathogenic bacterial load [0008] SUMMARY OF THE INVENTION

[0009] The present invention relates to methods and compositions for immunizing animals.

[0010] In an embodiment of the present invention, there is provided a vaccine or immunogenic composition comprising a bacterial type III secreted protein or a fragment thereof. Preferably, the bacterial type III protein comprises an amino acid sequence from an organism within the Family Enterobacteriaceae including, but not limited to Escherichia spp., Bordetella spp., Salmonella spp., Shigella spp., Yersinia spp., Ralstonia spp., Serratia spp., Proteus spp., Citrobacter spp., Campylobacter spp., Edwardsiella spp., Klebsiella spp., Campylobacter spp., Pseudomonas ssp., or Chlamydia spp. In an embodiment of the present invention, the organism is Escherichia coli, Bordetella brochiseptica, Bordetella pertussis, Shigella species, Salmonella enterica including all subspecies thereof such as, but not limited to S. typhimurium, S. enteriditis and the like, Yersinia species including, but not limited to Y. pestis and Y. enter ocolitica, Pseudomonas aeruginosa, Campylobacter species, Klebsiella pneumoniae, Citrobacter species or Chlamydia species.

[001 1] In a preferred embodiment, the bacterial type III protein comprises an amino acid sequence from E. coli. The E. coli may be a verocytotoxin-producing or veroto xin-producing E. coli, entero-pathogenic E. coli, entero-hemorrhagic E. coli, entero-toxigenic E. coli, avian pathogenic E. coli, or uropathogenic E. coli, for example, but not limited to 0157, 026, 0111, O103, 0126, Ol 13, 0145 or Ol 16. In an embodiment of the invention, the E. coli is O157:H7, O26:H11, O103:H2, or Ol 1 INM. In a preferred embodiment, which is not meant to be limiting, the E. Coli is O157.Η7.

[0012] Proteins that may be employed in the present invention also include without limitation, the LcrD family of inner membrane transport proteins, the YscN family of cytoplasmic ATPases, Flagellar Export Apparatus Proteins, YscO, YscP and similar proteins, the YscF, Yscl, YscK, and YscL families, YopN and similar proteins, the YscC family and its homologs in phage extrusion and type II secretion.

[0013] The vaccine or immunogenic composition as provided above may comprise an adjuvant. In a preferred embodiment, the adjuvant is a mucosal adjuvant, for example, but not limited to a cholera toxin (CT) subunit, a bacterial cell wall extract, a bacterial cell wall extract complexed with bacterial DNA, a mycobacterial cell wall extract (MCW), a mycobacterial cell wall-DNA complex (MCC), an immunostimulatory oligonucleotide including, but not limited to a CpG containing oligonucleotide (ODN) a non-CpG containing oligonucleotide, any other other well-recognized vaccine adjuvant, or a combination thereof.

[0014] Non-limiting examples of immunostimulatory oligonucleotides that may be employed in the compositions and methods of the present invention are described in WO03089642, US20040058883 and EP 1497424 (which are herein incorporated by reference). The oligonucleotides may contain a phosphodiester backbone or a modified phosphate backbone such as a phosphorothioate backbone, or a combination thereof. Preferred oligonucleotides include, but are not limited to 5OTGTGT3' (SEQ ID NO: 1), 5OGTGGG3' (SEQ ID NO:2), 5'GGGTGG3' (SEQ ID NO:3), 5'GGCCGG3' (SEQ ID NO:4), 5'GGGGGG3' (SEQ ID NO:5), 5'GGGAGG3' (SEQ ID NO:6) and 5OGGCGG3' (SEQ ID NO:7). In a preferred embodiment, which is not meant to be limiting the immunosimulatory oligonucleotide comprises SEQ ID NO: 1 comprising a phosphodiester backbone, SEQ ID NO:2 comprising a phosphorothioate backbone, SEQ ID NO: 3 comprising a phosphorothioate backbone, or SEQ ID NO: 5 comprising a phosphorothioate backbone. More preferably still, the immunostimulatory oligonucleotide is SEQ ID NO:3 comprising a phosphorothioate backbone or SEQ ID NO:5 comprising a phosphorothioate backbone.

[0015] The present invention also contemplates a vaccine or immunogenic composition as provided above, wherein the bacterial type III secreted protein or fragment thereof is EspA, Tir, EspB, EspD, EspP, Shiga toxin 1 , Shiga toxin 2, intimin or a combination thereof. [0016] There is also provided a method of preventing attachment, colonization and/or shedding of bacteria in an animal comprising,

administering a vaccine comprising a bacterial type III secreted protein, or fragment thereof to the animal.

Preferably, the vaccine composition comprises a mucosal adjuvant as described herein. Also, it is preferred that the vaccine composition be delivered via at least one mucosal surface.

[0017] The present invention also contemplates that prior to administering, the animal may be selected. For example, there is provided a method of preventing attachment, colonization and/or shedding of bacteria in an animal comprising,

selecting the animal for administration of a vaccine comprising a bacterial type III secreted protein, or fragment thereof, and;

administering the vaccine to the animal.

[0018] The present invention also contemplates a method as defined above wherein the vaccine is administered via at least one mucosal surface, for example, but not limited to via intranasal, oral, ocular, lung, uterus, rectal, intravaginal, intramammary, intraperitoneal administration, or any combination thereof.

[0019] Also provided is a method of inducing secretory IgA in the gut of an animal comprising,

administering a vaccine comprising a bacterial type III secreted protein or fragment thereof and a mucosal adjuvant to the animal.

[0020] In the above method, the vaccine is preferably administered via at least one mucosal surface as described herein.

[0021 ] This summary of the invention does not necessarily describe all features of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:

[0023] FIGURE 1 shows results depicting Tir- and EspA-specific serum IgG and fecal IgA following intranasal immunization. Mice were intranasally immunized with type III secreted proteins alone, type III secreted proteins with CpG ODNs or type III secreted proteins with CT. Immunizations were performed on day 1 and three weeks later. Two weeks following the secondary immunization sera was assessed for (A) Tir-specific IgG and (B) EspA-specific IgG using ELISA. Fecal samples were also assessed for (C) Tir-specific IgA and (D) EspA-specific IgA using ELISA. N=6 for all groups except antigen + CpG where N=I 1. The error bar is standard deviation. *P<0.05 immunized mice versus naϊve for Tir- and EspA-specific serum IgG using a one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. There were no significant differences in antibody titres between the immunized groups.

[0024] FIGURE 2 shows results of E. coli O157:H7 shedding in feces following oral administration in mice. Mice were immunized with type III secreted proteins alone, type III secreted proteins with CpG ODNs or type III secreted proteins with CT. Immunizations were performed on day 1 and three weeks later. Two weeks following the secondary immunization mice were orally fed 10¹⁰ E. coli O157:H7 and E. coli Ol 57:H7 shedding was monitored in the feces of individual mice for two weeks. N=6 for all groups except antigen + CpG where N=I 1. The error bar is standard deviation and differences in outcome were assessed using a Wilcoxon Rank Sum Test. Differences were considered significant whenever P<0.05. The limit of detection for plating was 100 colony forming units (CFU) per 0.1 g feces. Data have been repeated in a separate experiment. The naϊve mice (diamonds) and mice which received antigen alone shed significantly more E. coli O157:H7 than mice which received antigen formulated with CT or CpG (PO.05).

[0025] FIGURE 3 shows results depicting Tir- and EspA-specific serum IgG following subcutaneous immunization. Mice were immunized with type III secreted proteins with no adjuvant. Immunizations were performed on day 1 and three weeks later. Two weeks following the secondary immunization sera was assessed for (A) Tir-specific IgG and (B) EspA-specific IgG. N=6 for both groups and the error bar is standard deviation. *P<0.05 immunized mice vs naϊve mice for Tir- and EspA- specific serum IgG using a t-test.

DETAILED DESCRIPTION

[0026] The present invention relates to methods and compositions for immunizing animals.

[0027] The following description is of a preferred embodiment.

[0028] In an embodiment of the present invention, there is provided a vaccine or immunogenic composition comprising a bacterial type III secreted protein or a fragment thereof. The bacterial type III protein or fragment thereof may comprise an amino acid sequence or fragment of an amino acid sequence found in a bacterial type III secreted protein, for example, but not limited to Escherichia spp., Salmonella spp., Shigella spp., Yersinia spp., Ralstonia spp., Serratia spp., Proteus spp., Citrobacter spp.. Edwardsiella spp., Klebsiella spp., Campylobacter spp.. or Chlamidia spp., or any other bacterium known in the art that produces a type III secreted protein. In a preferred embodiment, the bacterium is a verocytotoxin-producing or verotoxin- producing ii.co//, entero-pathogenic E. coli, entero-hemorrhagic E. coli, enterotoxigenic E.coli, avian pathogenic E. coli, uropathogenic E. coli or other bacterium comprising a type III secretion system. In a specific embodiment, which is not meant to be limiting in any manner, the bacterium is E. coli Ol 57, O26, Ol 11, Ol 03, 0126, Ol 13, O145 or Ol 16. In a further embodiment of the invention, the E. coli is O157:H7, O26:H11, O103:H2, or Ol HNM. In a preferred embodiment, which is not meant to be limiting, the E. Coli is O157:H7.

[0029] Without limitation, representative bacterial type III secreted proteins known in the art include EspA, Tir, EspB, EspD, EspP, Shiga toxin 1 , Shiga toxin 2, and intimin. For example, but not to be considered limiting in any manner, US 6,355,254 entitled "Pathogenic Escherichia Coli Associated Protein EspA", WO 99/24576 entitled "HP90: Host Membrane receptor for Pathogenic Bacteria, Encoded by the bacterial TIR Gene", WO02053181 entitled "Enterohemorrhagic Escherichia Coli Vaccine", and WO 05042746 entitled "Bacterial Virulence Factors and Uses Thereof, disclose various bacterial type III secreted proteins and fragments thereof (the disclosures and references of which are herein incorporated by reference in their entirety). [0030] Additional proteins that maybe employed in the present invention include without limitation, the LcrD family of inner membrane transport proteins, the YscN family of cytoplasmic ATPases, Flagellar Export Apparatus Proteins, YscO, YscP and similar proteins, the YscF, Yscl, YscK, and YscL families, YopN and similar proteins, the YscC family and its homologs in phage extrusion and type II secretion.

[0031 ] The present invention also contemplates that the vaccine or immunogenic composition may comprise two or more bacterial type III secreted proteins, or fragments thereof. Further, the vaccine or immunogenic composition may comprise a first bacterial type III secreted protein, or fragment thereof as produced in a first type of bacterium and a second bacterial type III secreted protein, or fragment thereof as produced in a second type of bacterium. Further, it is contemplated that two or more bacterial type III secreted proteins may be the same or different. In the event that the two or more proteins or fragments thereof are obtained from two or more bacteria, for example, by purifying the proteins or fragments therefrom, it is also contemplated that the two or more bacteria producing the proteins may be the same or different.

[0032] In still a further embodiment, the bacterial type III protein or fragment thereof may comprise a portion of a larger molecule. For example, but not wishing to be limiting in any manner, the bacterial type III secreted protein or fragment thereof may be attached to a heterologous carrier protein, non-protein carrier or some other amino acid sequence.

[0033] As described previously, it is contemplated that the bacterial type III secreted protein may be obtained from the respective bacterium or bacterial strain that produces it in nature, for example, by purifying it therefrom. Alternatively, the proteins may be produced in a suitable recombinant system as would be known to a person of skill in the art. It is also possible that the bacterial type III secreted proteins, or fragments thereof may be synthesized chemically according to well known methods known in the art.

[0034] A bacterial type III secreted protein, or fragment thereof is employed in a vaccine or immunogenic composition. As used herein, the term "vaccine" or "immunogenic composition" relates to a component or a composition comprising a component that is capable of eliciting an immunological response to the bacterial type III secreted protein or fragment thereof in an animal. Usually, an "immunological response" includes, but is not limited to, one or more of the following effects: the production of antibodies, the production and/or activation of B-cells, the production and/or activation of helper T-cells, the production and/or activation of suppressor T- cells, the production and/or activation of cytotoxic T-cells, the production and/or activation of M cells, the production or activation of dendritic cells, or a combination thereof. In a preferred embodiment, administration of the vaccine or immunogenic composition results in induction of immunity at one or more mucosal surfaces. Further, this may involve a local immunological response that includes both humeral and well as cellular immune responses.

[0035] The vaccine or immunogenic composition may further comprise an adjuvant. By the term "adjuvant" or "immunological adjuvant" it is meant an agent that acts to enhance the immune response to one or more antigens in the vaccine or immunogenic composition. Any adjuvant known in the art may be employed in vaccines and immunogenic compositions of the present invention. Without limitation, such adjuvants may include one or more emulsifϊers including natural, synthetic, anionic, cationic and non-ionic emulsifϊers, aluminum containing adjuvants including, but not limited to aluminum hydroxide, saponins, avridine, oils, bacterial products including but not limited to lipopolysaccharide, cell wall extracts including but not limited to Mycobacterial phlei (M. phlei) cell wall extract, bacterial or mycobacterial cell wall compositions containing complexed immunostimulatory DNA such as, but not limited to mycobacterial cell wall-DNA complex from M. phlei, bacterial DNA, CpG containing oligonucleotides, non-CpG motif containing oligonucleotides, synthetic oligonucleotides and the like, Amphigen, cetyltrimethylammonium bromide, glyceryl esters, polyoxyethylene glycol esters and ethers, sorbitan fatty acid esters and polyoxyethylene derivatives thereof, lecithin, cholesterol, dimethyldioctadecyl ammonium bromide (DDA), or a combination thereof. Further, the vaccine or immunogenic composition may comprise liposomes, nanoparticles, microparticles, or an immunostimulating complex known in the art as ISCOMs.

[0036] While any adjuvant known in the art may be employed in the vaccine or immunogenic composition of the present invention, in an embodiment of the present invention, the adjuvant is preferably a mucosal adjuvant. Any mucosal adjuvant that has the ability to elicit an immunological response to the added antigen once combined therewith, may be employed in the vaccine or immunogenic composition of the present invention. Without limitation, mucosal adjuvants include cholera toxin, heat labile enterotoxin, mammalian immunoglobulin A inducing protein as described in US 6,930,167, mutants of pertussis toxin that lacks ADP-ribosyltransferase activity, CpG-containing oligonucleotides, non-CpG-containing oligonucleotides, bacterial cell wall extracts, bacterial cell wall extracts complexed with bacterial DNA, a mycobacterial cell wall extract (MCW), a mycobacterial cell wall-DNA complex (MCC) and the like. Other mucosal adjuvants are described by Elson, C. O., and Dertzbaugh, M. T. (1994) Mucosal adjuvants. In: Handbook of Mucosal Immunology (Eds. Ogra, P. L., et al.) Academic Press, San Diego.

[0037] The vaccine or immunogenic composition of the present invention may comprise one or more additional components, for example, an excipient or the like as provided in Remington's Pharmaceutical Sciences (Mack Pub. Co., N. J. current edition).

[0038] The present invention also contemplates a DNA based vaccine comprising a nucleotide sequence encoding one or more bacterial type III secreted proteins, or fragments thereof which are capable of being expressed in the animal, or any other sequence that encodes for a protective antigen. As will be evident to a person of skill in the art, a nucleotide sequence encoding one or more bacterial type III secreted proteins or one or more fragments thereof may form part of a nucleotide construct, for example, but not limited to a vector or the like. Such a construct may comprise a promoter, terminator and/or one or more regulatory sequences, as well as immunostimulatory sequences, as would be known in the art.

[0039] In an embodiment of the present invention there is also provided a method of preventing attachment, colonization, and/or shedding in an animal comprising,

administering a vaccine or immunogenic composition comprising a bacterial type III secreted protein, or fragment thereof to the animal. [0040] In a preferred embodiment, the vaccine or immunogenic composition is administered via at least one mucosal surface of the animal. In a more preferred embodiment, which is not meant to be limiting in any manner, the vaccine or immunogenic composition comprises a mucosal adjuvant and is administered via at least one mucosal surface of the animal.

[0041] The method may also comprise selecting the animal prior to the step of administering as provided above. For example, there is provided a method of preventing attachment, colonization, and/or shedding in an animal comprising,

selecting and animal to receive a vaccine or immunogenic composition, and;

administering the vaccine or immunogenic composition comprising a bacterial type III secreted protein, or fragment thereof to the animal.

[0042] In a preferred embodiment, the vaccine or immunogenic composition is administered via at least one mucosal surface of the animal. In a more preferred embodiment, which is not meant to be limiting in any manner, the vaccine or immunogenic composition comprises a mucosal adjuvant and is administered via at least one mucosal surface of the animal.

[0043] The method of the present invention may be practiced on or in an animal, for example, but not limited to any mammal, bird, fish, crustacean, reptile, or amphibian. Without wishing to be limiting in any manner, the compositions and methods of the present invention may be used with rodents including, but not limited to mice, rats, hamsters, guinea pigs, rabbits, and the like, reptiles and amphibians including turtles, tortoises, snakes, salamanders, lizards, dragons, goannas and the like, ungulates including but not limited to horses, donkeys, swine, elephants, ruminants including, but not limited to cattle, sheep, goats, deer, elk, bison or the like, camelids including but not limited to Bactrian camels, dromedaries, alpacas, llamas, guanacos and vicunas, poultry including, but not limited to chickens, ducks, turkeys, pheasants, quail, emus, ostriches, wild game birds, domestic fowl, caged birds and the like, ferrets, cats, dogs, apes, gorillas, chimpanzees, humans or any combination thereof. [0044] It is to be understood that by preventing attachment, colonization and/or shedding in an animal the method of the present invention may prevent, inhibit or reduce attachment of bacteria to host cells. The method may prevent, inhibit or reduce colonization of bacteria in the host animal. Further, the method may prevent, inhibit or reduce shedding of bacteria in the animal. Moreover, any one or combination thereof may be observed in the animal.

[0045] By the term "colonization", it is meant the continued presence and/or growth of bacteria in the intestinal tract of an animal.

[0046] By the term "shedding", it is meant the presence of bacteria in the animal's feces.

[0047] In a further embodiment of the present invention there is provided a method of preventing attachment, colonization, and/or shedding in an animal comprising,

administering a therapeutically effective amount of one or more vaccines or immunogenic compositions comprising a bacterial type III secreted protein, or fragment thereof to the animal.

[0048] By the term "therapeutically effective amount", it is meant an amount of vaccine or immunogenic composition effective to elicit an immune response against an antigen or epitope in the bacterial type III secreted protein thereby a) inhibiting, reducing or preventing attachment of bacteria to animal host cells, b) inhibiting, reducing or preventing colonization of the bacteria in the animal, c) inhibiting, reducing or preventing shedding of bacteria in the animal, or any combination thereof.

[0049] The compositions and methods as described herein may be used to treat or prevent disease in an animal. For example, there is provided a method of treating or preventing disease in an animal by administering one or more vaccines or immunogenic compositions as defined herein. Further, the present invention also contemplates compositions and methods for anti-zoonotic immunization to prevent and/or treat: (1) direct, (2) food-borne, and (3) fomite transferred infections in animals including humans. For example, the compositions and methods of the present invention may be employed to prevent direct transfer of infections in humans by, for example, contacts with pets including, but not limited to mice, rats, hamsters, guinea pigs, turtles, tortoises, snakes, salamanders, lizards, dragons, goannas, fish, aquarium crustaceans, birds, dogs, cats and the like. Similarly, the compositions and methods may be used to prevent indirect contamination, for example, but not limited to via food and food contamination, for example, but not limited to, by cattle, sheep, goats, deer, elk, bison, pigs, rabbits, guinea pigs, chickens, ducks, turkey, pheasants, quail, emu, wild game birds and the like. The compositions and methods of the present invention may be employed to prevent disease via transfer of pathogenic organisms from infected water, foods, including, but not limited to, via fruits and vegetables, and food processing implements and objects, for example, but not limited to knives, cutting boards, counters, packaging and the like.

[0050] It is also contemplated that the compositions and methods as described herein may provide cross-protection to other types of bacteria, for example but not limited to other type III secretory organisms or E. coli.

[0051 ] The vaccine or immunogenic composition of the present invention may be administered by any route and/or technique known in the art. Routes of administration include, but are not limited to topical, intracutaneous, intradermal, transdermal, subdermal, intraocular, rectal, intrarectal, intravaginal, parenteral, intravenous, intramammary, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, or oral administration. Formulations may be in the form of liquid solutions or suspensions, tablets, pessaries, or capsules, powders, nasal drops, nasal sprays, eye drops or aerosols, or incorporated into foods and food-product formulations that will by-pass the gastric stomach.

[0052] Depending on the route of administration, the volume per dose is preferably about 0.001 to 10 ml, more preferably about 0.01 to 5 ml, and most preferably about 0.1 to 3 ml. However, doses of 0.001, 0.005, 0.01, 0.05, 0.1, 0.25, 0.5, 0.75, 1, 2, 2.5, 3, 4, or 5ml are contemplated. It is also contemplated that the volume per dose may be defined by a range of any two of the values listed above. [0053] The vaccine or immunogenic composition may be administered in a single dose or in multiple doses, for example over a period of time. The timing and total dose(s) administered may be determined based on characteristics of the animal including species, sex, age, weight, health, any livestock management practice, and the like, the particular vaccine or immunogenic composition used, and the route of administration. In a preferred embodiment, at least two doses are administered to the animal.

[0054] Any suitable pharmaceutical delivery means known in the art may be employed to deliver the compositions to the animal. For example, but not wishing to be limiting in any manner, injection with a needle, particle or microprojectile injector, aerosol, liposome, spring or compressed gas injector, liquid jet injector or any combination thereof may be employed in the method of the present invention.

[0055] In an embodiment, which is not meant to be limiting, there is provided a method of stimulating secretory IgA in the gut of an animal comprising,

administering a vaccine or a composition comprising a bacterial type three secreted protein and a mucosal adjuvant to the animal.

[0056] As illustrated by the results and examples provided herein, type III secreted proteins from Escherichia coli O157:H7 are involved in the attachment of the organism to mammalian cells and have been shown to be effective vaccine components capable of reducing colonization of cattle by the organism. In the current study, we used a streptomycin-treated mouse model to subcutaneous versus intranasal administration of the vaccine. Following immunization, mice were infected with E coli O157:H7 and feces were monitored for shedding. Immune responses against EspA and Tir were also monitored. Subcutaneous immunization of mice with type III secreted proteins induced significant EspA- and Tir-specific serum IgG antibodies but did not significantly induce any antigen-specific IgA in feces, whereas intranasal immunization elicited significant EspA- and Tir-specific serum IgG antibodies with some animals developing antigen-specific IgA in feces. Only mice that were immunized intranasally with formulations containing mucosal adjuvants, for example, but not limited to either cholera toxin or CpG containing oligonucleotides, showed decreased E. coli O157:H7 shedding following experimental infection. Mice immunized subcutaneously with type III secreted proteins did not shed E. coli in feces. These results demonstrate the potential for the use of type III secreted proteins in mucosal vaccine formulations to prevent colonization and shedding of E. coli O157:H7.

[0057] The data described herein indicate that immunization with bacterial type III secreted proteins off. coli O157:H7 induce antibodies capable of preventing colonization of streptomycin-treated mice, presumably but not wishing to be bound by theory or limiting in any manner, by blocking the interactions of the organism with host cells. Following immunization, Tir- and EspA-specific antibody responses were generated in serum following both subcutaneous and intranasal administration. These proteins elicit antibodies in humans following natural exposure to E. coli O157:H7 (14) whereas calves usually do not mount significant antibody titres to these antigens following exposure to the organism. Unvaccinated mice behaved similar to cattle in that they did not generate high serum antibody titres to Tir or EspA following exposure to the organism. Again, without wishing to be limiting or bound by theory in any manner, for this reason, it is believed that the mouse model is appropriate for screening vaccine formulations ultimately to be used in cattle.

[0058] Conlan et al. (4, 6) demonstrated that both parenteral and oral immunization of mice with glycoconjugate vaccines containing the Ol 57 antigen did not protect against subsequent challenge, whereas immunization via the oral route with live Salmonella landau did reduce the load of E. coli O157:H7 following challenge (5). However, the model that was used in those studies was one that resulted in transient colonization for a period of approximately two weeks, whereas the streptomycin- treated mice used in these studies were colonized rapidly for a period extending out to at least 8 weeks. Thus, it is difficult to compare the results of the two studies. However, the results do suggest that mucosal immune responses can protect against colonization by bacteria, for example, but not limited to E. coli O157:H7, and the observation that although antigen alone can elicit immune responses following intranasal administration, there is an absolute requirement for the presence of a mucosal adjuvant to protect against colonization. [0059] Mucosal immunity, especially the production of IgA antibodies, is thought to be important for the blocking the attachment of E. coli Ol 57:H7 to epithelial cells. However, it has been demonstrated with Citrobacter rodentium that secretory IgA is not necessary for preventing bacterial colonization in mice using intimin as a vaccine antigen (10). The data presented here indicates that secretory IgA is not absolutely necessary for the prevention of E. coli O157:H7 colonization of streptomycin-treated mice since animals immunized subcutaneously with bacterial type III secreted proteins elicited Tir- and EspA-specific IgG without any detectable antigen-specific IgA and were not colonized following experimental infection. However, without wishing to be bound by theory, it is likely that IgA antibodies are important in blocking the interaction of bacterial type III secreted proteins with host cells when the antigens are delivered to mucosal surfaces, since intranasally-immunized mice did not shed E. coli Ol 57. Η7 following infection only if a mucosal adjuvant was used in the formulation. The lack of TTSP-specific IgA in feces can be overcome by either high Tir- and EspA-specific serum IgG or systemic priming of the immune system, resulting in an anamnestic response following challenge. Without wishing to be bound by theory or bound limiting in any manner, presumably, transduction of systemic IgG antibodies was responsible for this protection.

[0060] The present invention will be further illustrated in the following examples.

Examples.

[0061] Example 1

[0062] Materials and Methods

[0063] Bacterial strains. The E. coli O157:H7 strain that was used for production of bacterial type III secreted proteins and for colonization studies was obtained from Li et al. (14) and Tarr et al. (19). Methods for the growth of the organism and production of bacterial type III secreted proteins were as previously described (16).

[0064] Animals. Five to six week old female BALB/C mice used in this study were obtained from the Animal Resource Centre at the University of Saskatchewan (Saskatoon, SK.). Animals were housed and treated in compliance with regulations of the Canadian Council on Animal Care.

[0065] Immunization. Mice were immunized on day 1 and received a secondary immunization 3 weeks later regardless of the route of vaccine administration. Mice were immunized by the intranasal route with either bacterial type III secreted proteins alone [0.5μg of total bacterial type III secreted proteins containing 44ng Tir and 25ng EspA], bacterial type III secreted proteins combined with lμg CpG-containing oligonucleotide (CpG ODN) 1826 5'-TCCATGACGTTCCTGACGTT-S ' containing a phosphorothioate backbone (Qiagen, Mississauga, ON.), or bacterial type III secreted proteins combined with 1 μg Cholera toxin (CT) subunit B (List Biologies, Campbell, CA). All intranasal immunizations were administered in a volume of 10 μl per nostril. Age matched naive non- immunized mice were used as negative controls. For subcutaneous immunizations, mice were immunized with 0.5 μg of bacterial type III secreted proteins in 100 μl of phosphate buffered saline on day 1 and received a secondary immunization 3 weeks later. Serum samples were collected from the mice at weeks 0, 3 and 5.

[0066] Enterohemorrhagic colonization model. Mice were given drinking water containing streptomycin sulfate (5g/liter) to reduce the normal bacterial flora of the mice (23). Following one day of treatment with streptomycin, mice were fasted overnight, and subsequently fed 10¹⁰ colony forming units (CFU) of a nalidixic acid- resistant strain of E. coli O157:H7 in a volume of 100 μl. The bacterial suspension was prepared by collecting by centrifugation (6,000 g for ten minutes), washing with phosphate buffered saline, and resuspension in 20% sucrose. Animals were individually housed and permitted unlimited access to food and water containing streptomycin (5g/liter). Fecal samples from individual mice were collected following infection at two-day intervals over the following two weeks. E. coli O157:H7 fecal shedding was monitored by adding approximately 0.1 g to ImI of LB broth followed by incubation at room temperature for 2-4 hours to allow the fecal pellets to soften. The mixture was then vortexed until the pellets were no longer visible. Serial dilutions of the supernatant were plated onto MacConkey agar plates containing cefiximine, tulurite and nalidixic acid (22). Plates were incubated overnight at 37⁰C and E. coli Ol 57:H7 colonies were enumerated the following day. Bacterial colonies were tested for the 0157 antigen by latex agglutination (16). The limit of detection for determination of E. coli O157:H7 using this procedure was 100 CFU per 0.1 g feces.

[0067] Determination of Serum IgG and Fecal IgA antibody responses. Antibody responses specific for Tir and EspA were determined using an enzyme-linked immunosorbent assay (ELISA) as described (17). Immunlon 2 ELISA plates (DYNEX, Chantilly, VA) were coated with either Tir or EspA proteins (lμg/ml in coating buffer) overnight at 4°C and were washed three times with phosphate buffered saline containing 0.05% Tween 20 (PBST) (Sigma, St. Louis, MO). Mouse serum samples were serially diluted in PBST-0.5% gelatin (Sigma) and then added to the ELISA plates and incubated overnight at 4⁰C. In order to recover IgA from feces, samples were frozen and then mixed with 500μl of PBS. The fecal samples were vortexed until the pellets were not visible and then centrifuged. The supernatant was serially diluted in PBST-0.5% gelatin and added to the ELISA plates which were incubated overnight at 4°C. The following day, plates were washed 3 times in PBST and biotinylated goat anti-mouse IgG or biotinylated goat anti-mouse IgA antibodies (1/10,000 in PBST-0.5% gelatin) (Caltag Laboratories, Burlingame, CA) were added to the washed ELISA plates. Plates were incubated at room temperature for 1 hour and then washed 3 times in PBST. Streptavidin-alkaline phosphatase (1/10,000 in PBST- 0.5% gelatin) (Jackson ImmunoResearch labs, Westgrove, PA) was added to the ELlSA plates which were incubated at room temperature for 1 hour. Plates were washed 6 times with PBST and the alkaline phosphatase activity was determined by p- nitrophenol phosphate (PNPP) (Sigma). The absorbance was read after 15-20 minutes at 405nm (Bio-Rad Laboratories, Hurcules, CA). Antibody titres were calculated using a cut off of two standard deviations over values obtained from naϊve mice.

[0068] Statistics. Differences in immune responses among vaccine groups were analysed by Prism Graphpad statistical software (GraphPad Software, Inc., San Diego, CA), using a one way analysis of variance (ANOVA) followed by Tukey's multiple comparison test and t-test for antibody responses.

[0069] Results [0070] Immune responses elicited following intranasal immunization with type IH secreted proteins. We had previously demonstrated that bacterial type III secreted proteins of E. coli O157:H7 were immunogenic following both natural exposure to the organism (14) as well as subcutaneous immunization of cattle (16). To determine if bacterial type III secreted proteins were immunogenic in mice following intranasal immunization, IgG antibodies in serum specific for Tir and EspA were determined following administration of antigen formulated with a CpG oligonucleotide or cholera toxin B subunit. Two weeks following the secondary intranasal immunization, mice elicited significant Tir- and EspA-specific IgG antibodies in serum (Figure IA). There were no significant differences in IgG antibody titre specific for either Tir or EspA between any of the immunized groups. In addition, secretory IgA specific for Tir or EspA was determined using ELISA. Two weeks following the second immunization both Tir- and EspA-specific IgA were detected in the feces of some mice (Figure IB). However, there were no significant differences in IgA antibody titres between mice immunized with any of the formulations.

[0071] Infection of intranasallv immunized mice with E. coli Ol 57:H7. Two weeks following the secondary immunization, all mice were orally infected with 10¹⁰ CFU of E. coli O157:H7 and feces were collected to determine the level of E. coli O157:H7 shedding every two days over the following two weeks. No mice displayed any adverse reactions such as diarrhea following infection. All control mice shed the organism on all sampling days following infection, and mice immunized only with unadjuvanted bacterial type III secreted proteins shed E. coli O157:H7 at levels similar to non-immunized control mice (Figure 2). Mice immunized with bacterial type III secreted proteins combined with a CpG ODN showed a significant reduction in E. coli O157:H7 shedding with 5 out of 11 mice having no detectable shedding and the remaining 6 mice shedding intermittently on one or two of the sampling days. Mice immunized by the intranasal route with bacterial type III secreted proteins formulated with CT shed no detectable E. coli in their feces on any day over the entire sampling period.

[0072] Immune responses elicited following subcutaneous immunization with type III secreted proteins. Mice were subcutaneously immunized with bacterial type III secreted proteins to determine if serum IgG as well as secretory IgA in feces would be elicited. Two weeks following the secondary immunization, significant Tir- and EspA-specific IgG antibodies were detected in serum using ELISA (Figure 3). However, no Tir- or EspA-specific IgA was detectable in feces following subcutaneous administration (data not shown). Two weeks after infection with E. coli O157:H7, non-immunized mice did not develop significant levels of Tir or EspA specific serum IgG or fecal IgA following E. coli administration (data not shown).

[0073] Challenge of subcutaneously immunized mice with E. coli Ol 57:H7. In order to determine if Tir and EspA specific IgG antibodies in serum could reduce or prevent E. coli O157:H7 shedding in feces following oral administration of the organism, mice immunized subcutaneously with bacterial type III secreted proteins as well as age matched non-immunized control mice were infected orally with 10 CFU of E. coli O157:H7. Shedding of the organism was monitored in feces as described above. None of the immunized mice shed the organism whereas all non-immunized control mice shed high levels of E. coli Ol 57:H7 in their feces over the two-week sampling period (data not shown).

[0074] Example 2 - Induction of Mucosal Immune Response in Calves

[0075] Materials and Methods

[0076] Bacterial strains. The E. coli O157:H7 strain that was used for production of bacterial type III secreted proteins and for colonization studies was obtained from Li et al. (14) and Tarr et al. (19). Methods for the growth of the organism and production of bacterial type III secreted proteins were as previously described (16).

[0077] Animals. Eleven month old beef calves used in this study were obtained from the Vaccine and Infectious Disease Organization (VIDO) at the University of Saskatchewan (Saskatoon, SK.). Animals were housed and treated in compliance with regulations of the Canadian Council on Animal Care.

[0078] Immunization. Six calves were immunized on day 1 and will receive a booster dose on days 21 and 42. Calves were immunized by the intranasal route with 2 mL of vaccine containing 50 μg bacterial type III secreted proteins combined with 10 μg Cholera toxin (CT) subunit B (List Biologies, Campbell, CA). Serum and nasal secretion samples were collected from the calves on days 0, 21, 42, and 56.

[0079] Determination of Serum IgG and Nasal Secretion IgA antibody responses. Antibody responses specific for Tir and EspA are determined using an enzyme-linked immunosorbent assay (ELISA) as described (17). Immunlon 2 ELISA plates (DYNEX, Chantilly, VA) are coated with either Tir or EspA proteins (l μg/ml in coating buffer) overnight at 4°C and are washed three times with phosphate buffered saline containing 0.05% Tween 20 (PBST) (Sigma, St. Louis, MO). Calf serum samples are serially diluted in PBST-0.5% gelatin (Sigma) and then added to the ELISA plates and incubated overnight at 4°C. Calf nasal secretion samples are serially diluted in PBST-0.5% gelatin (Sigma) and then added to the ELISA plates and incubated overnight at 4⁰C. The following day, plates are washed 3 times in PBST and biotinylated goat anti-mouse IgG or biotinylated goat anti-mouse IgA antibodies (1/10,000 in PBST-0.5% gelatin) (Caltag Laboratories, Burlingame, CA) are added to the washed ELISA plates. Plates are incubated at room temperature for 1 hour and then washed 3 times in PBST. Streptavidin-alkaline phosphatase (1/10,000 in PBST- 0.5% gelatin) (Jackson ImmunoResearch labs, Westgrove, PA) is added to the ELISA plates which are incubated at room temperature for 1 hour. Plates are washed 6 times with PBST and the alkaline phosphatase activity is determined by p-nitrophenol phosphate (PNPP) (Sigma). The absorbance is read after 15-20 minutes at 405nm (Bio-Rad Laboratories, Hurcules, CA). Antibody titres are calculated using a cut off of two standard deviations over values obtained from naϊve calves.

[0080] Statistics. Differences in immune responses among vaccine groups is analysed by Prism Graphpad statistical software (GraphPad Software, Inc., San Diego, CA), using a one way analysis of variance (ANOVA) followed by Tukey's multiple comparison test and t-test for antibody responses.

[0081] Results

[0082] Immune responses elicited following intranasal immunization with type III secreted proteins. We had previously demonstrated that bacterial type III secreted proteins of E. coli Ol 57:H7 were immunogenic following both intranasal and subcutaneous immunization of mice (Example 1). To determine if bacterial type III secreted proteins were immunogenic in calves following intranasal immunization, IgG and IgA antibodies specific for Tir and EspA are determined by ELISA following administration of antigen formulated with cholera toxin B subunit. It is expected that in two weeks following the third intranasal immunization, calves will elicit significant Tir- and EspA-specific IgG antibodies in serum and significant Tir- and EspA-specific IgA antibodies in nasal mucosal secretions.

[0083] References

1. Armstrong, G. L., J. Hollingsworth and J.G. Morris, Jr.. 1996. Emerging foodborne pathogens: Escherichia coli O157:H7 as a model of entry of a new pathogen into the food supply of the developed world. Epidemiol. Rev. 18:29-51.

2. Besser, R. E., P. M. Griffin and L. Slutsker. 1999 Escherichia coli O157:H7 gastroenteritis and the hemolytic uremic syndrome: an emerging infectious disease. Annu. Rev. Med. 50:355- 367.

3. Carvalho, H. M., L. D. Teel, J. F. Kokai-Kun, and A. D. O'Brien. 2005 Antibody against the carboxyl terminus of intimin alpha reduces enteropathogenic Escherichia coli adherence to tissue culture cells and subsequent induction of actin polymerization. Infect Immun. 73:2541- 2546.

4. Conlan W.J., A.D. Cox, R. KuoLee, A. Webb, and M.B. Perry. 1999. Parenteral immunization with a glycoconjugate vaccine containing the 0157 antigen of Escherichia coli O157:H7 elicits a systemic humoral immune response in mice, but fails to prevent colonization by the pathogen. Can J Microbiol. 45:279-286.

5. Conlan, W. J., R. KuoLee, A. Webb and M.B. Perry. 1999. Salmonella landau as a live vaccine against E. coli O157:H7 investigated in a mouse model of intestinal colonization. Can. J. Microbiol. 45:723-731. 6. Conlan, W., R. KuoLee, A. Webb, A.D. Cox and M.B. Perry. 2000 Oral immunization of mice with a glycoconjugate vaccine containing the O157 antigen of E coli O157 H7 admixed with cholera toxm fails to elicit protection against subsequent colonization by the pathogen Can J Microbiol 46 28^-290

7. Dean-Nystrom, E. A., B.T. Bosworth, W.C. Cray, Jr. and H. W. Moon. 1997 Pathogenicity of E coli 0157 H7 in the intestines of neonatal calves Infect Immun 65 1842- 1848

8. Diez-Gonzalez, F., T.R. Callaway, M.G. Kizoulis, and J.B. Russell. 1998 Gram feeding and the dissemination of acid-resistant E cob from cattle Science 281 1666-1668

9. Elder, R. O., J. E. Keen, G. R. Siragusa, G. A. Barkocy-Gallagher, M. Koohmaraie, and W. W. Laegreid. 2000 Correlation of enterohemorrhagic Escheyichia coli 0157 prevalence in feces, hides, and carcasses of beef cattle during processing Proc Natl Acad Sci USA 97:2999-3003

10. Ghaem-Maghami, M, C. P. Simmons, S. Daniell, M. Pizza, D. Lewis, G. Frankel, and G. Dougan. 2001 Intimin-specific immune responses prevent bacterial colonization by the attachmg-effacmg pathogen Citrobacter rodentmm Infect Immun 69 5597-5605

11. Jordan, D., S. A. McEwen, A. M. Lammerding, W. B. McNab, and J. B. Wilson. 1999 Pre slaughter control of Escherichia coli 0157 in beef cattle a simulation study Prev Vet

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12. Judge N. A., H. S. Mason, and A. D. O'Brien. 2004 Plant cell-based intimin vaccine given orally to mice primed with intimm reduces time of Escherichia coli 0157 H7 shedding m feces Infect Immun 72 168-175 13. Kenny, B, R. DeVinney, M. Stein, D. J. Reinscheid, E. A. Frey, and B. B. Finlay. 1997 Enteropathogenic E coli (EPEC) transfers its receptor for intimate adherence into mammalian cells Cell 91 51 1-520

14. Li, Y., E. Frey, A. M. Mackenzie, and B. B. Finlay. 2000 Human response to Escherichia 60// 0157 HV iIIfCCtIOn antibodies to secreted virulence factors Infect Immun 68:5090-5095

15. Mead, P. S., L. Slutsker, V. Dietz, L. F. McCaig, J. S. Bresee, C. Shapiro, P. M. Griffin, and R. V. Tauxe. 1999 Food-related illness and death in the United States Emerg Infect Dis 5:607-625

16. Potter A. A., S. Klashinsky, Y. Li, E. Frey, H. Townsend, D. Rogan, G. Erickson, S. Hinkley, T. Klopfenstein, R. A. Moxley, D. R. Smith, and B.B. Finlay. 2004 Decreased shedding of Escherichia coli 0157 H7 by cattle following vaccination with type III secreted proteins Vaccine 22 362-369

17. Potter, A. A., A. B. Schryvers, J. A. Ogunnariwo, W.A. Hutchins, R.Y. Lo, and T. Watts.

1999 Protective capacity of the Pasteurella haemolytica transferrin-binding proteins TbpA and TbpB in cattle Microb Pathog 27 197-206

18. Ritchie J. M., C. M. Thorpe, A. B. Rogers, and M. K. Waldor. 2003 Critical roles for stx2, eae, and Ur in enterohemorrhagic Escherichia coli-mduced diarrhea and intestinal inflammation in infant rabbits Infect Immun 2003 71 7129-7139

19. Tarr, P.I., M.A. Neill, CR. Clausen, J. W. Newland, R.J. Neill and S.L. Moseley. 1989

Genotypic variation in pathogenic E coli 0157 H7 isolated from patients in Washington 1984-1987 J Infect Dis 159 344-347

20. Valcour, J. E., P. Michel, S. A. McEwen, and J. B. Wilson. 2002 Associations between indicators of livestock farming intensity and incidence of human Shiga toxin-producmg Escherichia coll infection Emerg Infect Dis 8:252-257

21. VanDonkersgoed, J., J. Berg, and A. Potter. 2001 Environmental sources and transmission of E coh 0157 in feedlot cattle Can Vet J 42 714-720

22. J. VanDonkersgoed, T. Graham and V. Gannon. The prevalence of verotoxms. E coh 0157 H7, and Salmonella in the feces and rumen of cattle at processing Can Vet J 40 5 (1999), pp 332-338

23. Wadolkowski, E. A., J. A. Burris, and A. D. O'Brien. 1990 Mouse model for colonization and disease caused by enterohemorrhagic Escherichia coh 0157 H7 Infect Immun 58:2438- 2445

24. Wong, C.S., Jelacic, S., Habeeb, R.L., Watkins S.L., and P.I. Tarr, 2000 The risk of the hemolytic-uremic syndrome after antibiotic treatment of Escherichia coh 0157 H7 infections N Engl J Med 342 1930-1936

25. Zhao, T., M. P. Doyle, B. G. Harmon, C. A. Brown, P. O. Mueller, and A. H. Parks.

1998 Reduction of carriage of enterohemorrhagic Escherichia coh 0157 H7 m cattle by inoculation with probiotic bacteria J CIm Microbiol 36:641 -647

[0084] All citations are hereby incorporated by reference.

[0085] The present invention has been described with regard to one or more embodiments. However, it will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.

Claims

WHAT IS CLAIMED IS:

1. A vaccine comprising a bacterial type III secreted protein or a fragment thereof derived from entero-hemorrhagic E. coli 0157 and a mucosal adjuvant.

2. The vaccine of claim 1, wherein the mucosal adjuvant is a cholera toxin (CT) subunit, an immunostimulatory oligonucleotide, a bacterial cell wall extract, a bacterial cell wall extract complexed with bacterial DNA, a mycobacterial cell wall extract (MCW), a mycobacterial cell wall-DNA complex (MCC), bacterial or mycobacterial cell wall compositions containing complexed immunostimulatory DNA such as mycobacterial cell wall-DNA complex from M. phlei, bacterial DNA, or any combination thereof.

3. The vaccine of claim 1 , wherein the bacterial type III secreted protein or fragment thereof is EspA, Tir, EspB, EspD, EspP, Shiga toxin 1, Shiga toxin 2, intimin, a bacterial secreted protein, a bacterial structural protein, or a combination thereof.

4. A method of preventing attachment, colonization and/or shedding of bacteria in an bovine animal comprising,

administering the vaccine as defined in claim 1 to the animal via at least one mucosal surface.

5. The method of claim 4, wherein the vaccine is administered intranasally.

6. A vaccine comprising a bacterial type III secreted protein or a fragment thereof and a mucosal adjuvant.

7. The vaccine of claim 6, wherein the bacterial type III protein comprises an amino acid sequence from Escherichia spp., Bordetella spp., Salmonella spp., Shigella spp., Yersinia spp.. Ralstonia spp., Serratia spp., Proteus spp., Citrobacter spp., Campylobacter spp., Edwardsiella spp., Klebsiella spp., Campylobacter spp., Pseudomonas ssp., or Chlamydia spp.

8. The vaccine of claim 7, wherein the bacterial type III protein comprises an amino acid sequence from Escherichia coli, Bordetella brochiseptica, Bordetella pertussis, Salmonella typhimurium, Salmonella enteriditis, Yersinia pestis, Yersinia enter ocolitica, Pseudomonas aeruginosa, or Klebsiella pneumoniae.

9. The vaccine of claim 8, wherein the bacterial type III protein comprises an amino acid sequence from Escherichia coli and said E. coli is a verocytotoxin-producing or verotoxin-producing £^'.cø//, entero-pathogenic E. coli, entero-hemorrhagic E. coli, entero-toxigenic E. coli, avian pathogenic E. coli, or uropathogenic E. coli.

10. The vaccine of claim 9, wherein the E. coli is O157, 026, 0111, O103, Ol 16 O126, O113 or O145.

1 1. The vaccine of claim 10, wherein the E. coli is O157:H7, O26:H11, O103:H2 or Ol l hNM.

12. The vaccine of claim 6, wherein the mucosal adjuvant is a cholera toxin (CT) subunit, an immunostimulatory oligonucleotide, a bacterial cell wall extract, a bacterial cell wall extract complexed with bacterial DNA, a mycobacterial cell wall extract (MCW), a mycobacterial cell wall-DNA complex (MCC), bacterial or mycobacterial cell wall compositions containing complexed immunostimulatory DNA such as mycobacterial cell wall-DNA complex from M. phlei, bacterial DNA, or any combination thereof.

13. The vaccine of claim 6, wherein the bacterial type III secreted protein or fragment thereof is EspA, Tir, EspB, EspD, EspP, Shiga toxin 1, Shiga toxin 2, intimin, a bacterial secreted protein, a bacterial structural protein, or a combination thereof.

14. A method of preventing attachment, colonization and/or shedding of bacteria in an animal comprising,

15. The method of claim 14, wherein the vaccine is administered via at least one mucosal surface.

16. The method of claim 19, wherein vaccine is administered intranasally.

17. A method of inducing secretory IgA in the gut of an animal comprising,

18. The method of claim 17, wherein said vaccine is administered via at least one mucosal surface.

19. A method of inducing IgG at one or more mucosal surfaces of an animal comprising,

administering a vaccine composition comprising a bacterial type III secreted protein or fragment thereof and a mucosal adjuvant to the animal.

20. The method of claim 19, wherein said vaccine composition is administered via at least one mucosal surface of or on the animal.

21. The vaccine of claim 6, wherein the bacterial type III protein or fragment thereof comprises an amino acid sequence from the LcrD family of inner membrane transport proteins, the YscN family of cytoplasmic ATPases, a Flagellar Export Apparatus Protein, YscO, YscP, the YscF, Yscl, YscK, and YscL families, YopN, the YscC family and its homologs in phage extrusion and type II secretion, or a combination thereof.