VACCINE AGAINST NECROTIC ENTERITIS IN POULTRY

Abstract

An immunogenic polypeptide selected from an isolated Clostridium perfringens pilus polypeptide, a variant of the pilus polypeptide; a fragment of the pilus polypeptide; and a fragment of the variant, is useful for the preparation of a vaccine for the treatment or prevention of enteric necrosis in poultry. The isolated Clostridium perfringens pilus polypeptide includes an assembled pilus or the pilus subunits CnaA, FimA and/or FimB.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent application Ser. No. 14/616,619, filed Nov. 25, 2019; which is a National Stage of International Application No. PCT/CA2018/050643, filed on May 31, 2018; which claims the benefit of U.S. Provisional Application Ser. No. 62/513,001, filed May 31, 2017, all of which are hereby incorporated by reference in their entirety.

SEQUENCE LISTING

[0002] The Sequence Listing for this application is labeled "SeqList-07Apr21-ST25.txt", which was created on Apr. 7, 2021, and is 38 KB. The entire content is incorporated herein by reference in its entirety.

BACKGROUND

[0003] The present application is directed to polypeptides useful in preparing a vaccine against necrotic enteritis in poultry. More specifically, the present application is directed to a Clostridium perfringens pilus polypeptide useful in preparing a vaccine against necrotic enteritis related to Clostridium perfringens infection in poultry.

[0004] Necrotic enteritis is an intestinal disease of poultry such as broiler chickens, that in 2015 was estimated to cost the poultry industry $US 6 billion in losses. Necrotic enteritis is caused primarily by certain Type A strains of Clostridium perfringens that produce the NetB pore-forming toxin, which overgrow and adhere to the intestinal mucosa, eventually causing the characteristic lesions of the disease. Clostridium perfringens is a normal inhabitant of the intestinal tract, and typically only those strains that carry the NetB toxin can cause necrotic enteritis. Necrotic enteritis is primarily controlled by application of in-feed antibiotics, a practice that is increasingly discouraged due to the potential spread of antimicrobial resistance, and which is currently being phased out of production in some countries. It is therefore important, from both a financial and public health perspective, to find alternative approaches to control necrotic enteritis, such as the development of a vaccine.

[0005] A Clostridium perfringens adherence genetic locus (VR-10B) has been recently identified through its association with NetB-positive strains (Lepp D, Gong J, Songer J G, Boerlin P, Parreira V R, Prescott J F. 2013. Identification of Accessory Genome Regions in Poultry Clostridium perfringens Isolates Carrying the netB Plasmid. Journal of Bacteriology 195: 1152-1166). The identified genetic locus was found to be present in 87% of netB-positive and 42% of netB-negative isolates, of 54 poultry isolates examined. This genetic locus (subsequently renamed the collagen adhesion (CA) locus) was later shown to be involved in collagen binding, and required for necrotic enteritis pathogenesis (Wade B, Keyburn A L, Haring V, Ford M, Rood J I, Moore R J: The adherent abilities of Clostridium perfringens strains are critical for the pathogenesis of avian necrotic enteritis. Vet Microbiol 2016, 197: 53-61; Wade B, Keyburn A L, Seemann T, Rood J I, Moore R J: Binding of Clostridium perfringens to collagen correlates with the ability to cause necrotic enteritis in chickens. Vet Microbiol 2015, 180: 299-303.).

[0006] A number of Clostridium perfringens proteins have previously been evaluated as vaccine candidates. However, these proteins offer at best partial protection against necrotic enteritis. In addition, many of these proteins are not specific to necrotic enteritis-causing strains, and are not known to play a role in necrotic enteritis pathogenesis. Therefore, it is desirable to identify alternative Clostridium perfringens polypeptides which may be candidates for producing a vaccine against necrotic enteritis.

SUMMARY

[0007] One aspect of the present invention provides an isolated Clostridium perfringens pilus polypeptide. In another aspect, the present invention provides an immunogenic polypeptide selected from an isolated Clostridium perfringens pilus polypeptide, a variant of the pilus polypeptide; a fragment of the pilus polypeptide; and a fragment of the variant, wherein the pilus polypeptide, the variant, the fragment of the polypeptide and the fragment of the variant are immunogenic in poultry. In at least one embodiment, the pilus polypeptide is a CnaA polypeptide. In at least one embodiment, the pilus polypeptide is a FimA polypeptide. In at least one embodiment, the pilus polypeptide is a FimB polypeptide. In at least one embodiment, the pilus polypeptide is an assembled pilus.

[0008] Another aspect of the present invention provides a polynucleotide comprising a sequence encoding an isolated Clostridium perfringens pilus polypeptide or an immunogenic polypeptide as described herein. In another aspect, the present application provides a vector comprising a polynucleotide having a sequence encoding an isolated Clostridium perfringens pilus polypeptide or an immunogenic polypeptide as described herein, wherein the vector is configured for expression of the isolated Clostridium perfringens pilus polypeptide or immunogenic polypeptide in a host cell.

[0009] In another aspect, the present invention provides a vaccine for the treatment or prevention of necrotic enteritis in poultry, wherein the vaccine comprises an immunogenic polypeptide as described herein. In another aspect, the present application provides a vaccine for the treatment or prevention of Clostridium perfringens infection in poultry, wherein the vaccine comprises an immunogenic polypeptide as described herein.

[0010] In another aspect, the present invention provides the use of an immunogenic polypeptide as described herein in the preparation of a medicament for the treatment or prevention of necrotic enteritis in poultry or for the treatment or prevention of Clostridium perfringens infection in poultry.

[0011] In another aspect, the present invention provides a method of treatment or prevention of necrotic enteritis in poultry or for the treatment or prevention of Clostridium perfringens infection in poultry, the method comprising administering an effective amount of an immunogenic polypeptide as described herein, or a vaccine thereof, to the poultry.

[0012] In another aspect, the present invention provides the use of an immunogenic polypeptide as described herein as a vaccine for the treatment or prevention of necrotic enteritis in poultry or for the treatment or prevention of Clostridium perfringens infection in poultry.

[0013] A further aspect of the present invention provides an antibody which binds selectively to an immunogenic polypeptide as described herein. In another aspect, the present invention provides a method of detecting Clostridium perfringens infection in poultry by obtaining a biological sample from the poultry and detecting in the biological sample the presence of an antibody which binds selectively to an immunogenic polypeptide as described herein. Yet another aspect of the present invention provides a method of detecting an immunogenic polypeptide as described herein comprising exposing the immunogenic polypeptide to an antibody which binds selectively to the immunogenic polypeptide and detecting binding of the immunogenic polypeptide to the antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Further features of the present invention will become apparent from the following written description and the accompanying figures, in which:

[0015] FIG. 1 is a diagrammatic representation of the 5.2 kilobase-pair Clostridium perfringens VR-10B chromosomal locus;

[0016] FIG. 2A is a photograph illustrating separation of recombinant histidine-tagged pilus subunit polypeptide CnaA by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) visualized by COOMASSIE staining;

[0017] FIG. 2B is a photograph illustrating separation of recombinant pilus subunit polypeptide FimA by SDS-PAGE visualized by COOMASSIE staining;

[0018] FIG. 2C is a photograph illustrating separation of recombinant pilus subunit polypeptide FimB by SDS-PAGE visualized by COOMASSIE staining;

[0019] FIG. 2D is a photograph illustrating visualization by SDS-PAGE and COOMASSIE staining of pooled fractions of CnaA, FimA and FimB following concentration and desalting;

[0020] FIG. 3A is a graph illustrating serum IgY antibody response (absorbance at 405 nm) against CnaA recombinant polypeptide from chickens immunized with adjuvant alone, or with CnaA in a first vaccination trial. Each dot represents a single individual and horizontal lines represent means. * indicates a significant difference from the pre-immune sample (d8) from each group at the p<0.05 level, ** indicates a significant difference at the p<0.01 level, and *** indicates a significant difference at the p<0.001 level when measured by the Tukey's test (Tukey, J. "Comparing Individual Means in the Analysis of Variance". Biometrics (1949) 5(2): 99-114);

[0021] FIG. 3B is a graph illustrating serum IgY antibody response (absorbance at 405 nm) against FimA recombinant polypeptide from birds immunized with adjuvant alone, or with FimA in the trial of FIG. 3A. Each dot represents a single individual and horizontal lines represent means. * indicates a significant difference from the pre-immune sample (d8) from each group at the p<0.05 level, ** indicates a significant difference at the p<0.01 level, and *** indicates a significant difference at the p<0.001 level when measured by the Tukey's test;

[0022] FIG. 4 is a graph illustrating necrotic enteritis (NE) lesion scores from groups of chickens immunized with adjuvant alone, or with CnaA or FimA in the trial of FIG. 3A, followed by in-feed challenge with Clostridium perfringens strain CP1. Each dot represents a single individual and horizontal lines represent mean necrotic enteritis lesion score;

[0023] FIG. 5A is a graph illustrating serum IgY antibody response (absorbance at 405 nm) against CnaA recombinant polypeptide from chickens immunized with adjuvant alone, with CnaA, or with a combination of CnaA, FimA and FimB (Comb), in a second vaccination trial. Each dot represents a single individual and horizontal lines represent means. * indicates a significant difference from the pre-immune sample (d7) from each group at the p<0.05 level, ** indicates a significant difference at the p<0.01 level, and *** indicates a significant difference at the p<0.001 level when measured by the Tukey's test;

[0024] FIG. 5B is a graph illustrating serum IgY antibody response (absorbance at 405 nm) against FimA recombinant polypeptide from chickens immunized with adjuvant alone or with a combination of CnaA, FimA and FimB (Comb), in the trial of FIG. 5A. Each dot represents a single individual and horizontal lines represent means. * indicates a significant difference from the pre-immune sample (d7) from each group at the p<0.05 level, ** indicates a significant difference at the p<0.01 level, and *** indicates a significant difference at the p<0.001 level when measured by the Tukey's test;

[0025] FIG. 5C is a graph illustrating serum IgY antibody response (absorbance at 405 nm) against FimB recombinant polypeptide from chickens immunized with adjuvant alone, with FimB, or with a combination of CnaA, FimA and FimB (Comb), in the trial of FIG. 5A. Each dot represents a single individual and horizontal lines represent means. * indicates a significant difference from the pre-immune sample (d7) from each group at the p<0.05 level, ** indicates a significant difference at the p<0.01 level, and *** indicates a significant difference at the p<0.001 level when measured by the Tukey's test;

[0026] FIG. 6 is a graph illustrating necrotic enteritis (NE) lesion scores from groups of chickens immunized with adjuvant alone, CnaA, FimB, or a combination of CnaA, FimA and FimB (Comb), in the trial of FIG. 5A, followed by in-feed challenge with Clostridium perfringens CP1. Each dot represents a single individual and horizontal lines represent mean necrotic enteritis lesion score. Letters (a, b) denote significantly different groups (Tukey's; p<0.01);

[0027] FIG. 7 is a graph illustrating necrotic enteritis (NE) lesion scores from groups of chickens following in-feed challenge with Clostridium perfringens strain CP1 or CP1 null-mutants of the pilus subunit genes fimA and fimB (CP1.DELTA.fimA, and CP1.DELTA.fimB). Lines represent mean necrotic enteritis lesion score;

[0028] FIG. 8A is a photograph illustrating separation by SDS-PAGE of surface polypeptides extracted from Clostridium perfringens strain CP1 or CP1 null mutants of genes for each of the pilus subunits cnaA, fimA and fimB (CP1.DELTA.cnaA, CP1.DELTA.fimA, and CP1.DELTA.fimB), visualized with COOMASSIE stain;

[0029] FIG. 8B is a photograph illustrating a Western blot analysis of SDS-PAGE-separated surface polypeptides extracted from Clostridium perfringens strain CP1 or CP1 null mutants of genes for each of the pilus subunits cnaA, fimA and fimB (CP1.DELTA.cnaA, CP1.DELTA.fimA, and CP1.DELTA.fimB), detected using anti-FimA antibodies obtained from chicken serum as the primary antibody;

[0030] FIG. 8C is a photograph illustrating a Western blot analysis of SDS-PAGE-separated surface polypeptides extracted from Clostridium perfringens strain CP1 or CP1 null mutants of genes for each of the pilus subunits cnaA, fimA and fimB (CP1.DELTA.cnaA, CP1.DELTA.fimA, and CP1.DELTA.fimB), detected using anti-FimA antibodies raised in rabbits as the primary antibody;

[0031] FIG. 9A is a photograph illustrating separation by SDS-PAGE of surface polypeptides extracted from various Clostridium perfringens strains visualized with COOMASSIE stain;

[0032] FIG. 9B is a photograph illustrating a Western blot analysis of SDS-PAGE-separated surface polypeptides extracted from various Clostridium perfringens strains visualized with anti-FimA antibodies obtained from chicken serum as the primary antibody; and

[0033] FIG. 10 is a series of photographs obtained by transmission electron microscopy of cells of Clostridium perfringens strain CP1 or of the CP1 null mutants CP1.DELTA.fimA, and CP1.DELTA.fimB labeled with gold particles using rabbit anti-FimA antibody as a primary antibody and 6 nm immunogold-labelled goat anti-rabbit IgG as a secondary antibody.

DETAILED DESCRIPTION

[0034] It has been found by the present applicants that the VR-10B genetic locus identified in strains of Clostridium perfringens associated with necrotic enteritis in poultry (Lepp D et al, Journal of Bacteriology (2013) 195: 1152-1166) contains six putative genes which have been found to encode an adhesive pilus: three genes (cnaA, fimA and fimB) encoding structural pilus subunits, and genes encoding two sortases and a signal peptidase presumably involved in pilus assembly. A diagrammatic representation of the VR-10B locus is shown in FIG. 1.

[0035] A pilus is a hair-like structure that is present on the surface of many bacteria and is often involved in virulence. This type of pilus is composed of covalently-linked major and minor polypeptide subunits that form a cell surface structure having a length of approximately 1 .mu.m. Pili have been extensively studied in Gram-negative bacteria, but several Gram-positive species, including Corynebacterium diphtheriae, Streptococcus pneumoniae, and Streptococcus pyogenes, have more recently been shown to produce a specific type of pilus that is assembled by sortase enzymes. This type of adhesive Gram-positive pilus is assembled at the cell surface via covalent linkage of pilin subunits by housekeeping and pilin-specific sortase enzymes, and is eventually covalently linked to the cell wall peptidoglycan to form the assembled pilus.

[0036] Without being bound by theory, it is contemplated that the Clostridium perfringens pilus polypeptides described herein may be a viable and promising target for development of a vaccine against necrotic enteritis for a number of reasons. The gene locus is present mainly in necrotic enteritis-causing strains of Clostridium perfringens. Therefore, the immune response elicited by an immunogenic pilus protein is expected to specifically target strains of Clostridium perfringens that cause disease. In addition, pili are present on the surface of the bacterial cell, and are often involved in attachment to the host during the pathogenesis of bacterial infections, which can expose the pili to the host immune system. Furthermore, possibly because of their role in disease and their location on the bacterial cell surface, pili have been successfully used to develop vaccines for a number of other infectious diseases.

[0037] Thus, one aspect of the present application provides an immunogenic polypeptide selected from an isolated Clostridium perfringens pilus polypeptide, a variant of the pilus polypeptide; a fragment of the pilus polypeptide; and a fragment of the variant, wherein the pilus polypeptide, the variant, the fragment of the polypeptide and the fragment of the variant are immunogenic in poultry.

[0038] As used herein, the term "poultry" is used to refer to species of birds or fowl which are raised agriculturally for products including but not limited to meat, eggs and feathers. Poultry include but are not limited to chickens, turkeys, ducks, geese, quail, ostriches, pheasants and other agriculturally relevant birds or fowl. Especially included are poultry which are susceptible to necrotic enteritis caused by Clostridium perfringens infection. In at least one embodiment, the poultry are broilers or chickens raised for meat production.

[0039] As used herein, the term "polypeptide" is intended to mean a compound containing two or more amino acid residues linked together by peptide bonds. Polypeptides include but are not limited to oligopeptides or polypeptides in which two or more amino acid residues are linked together sequentially by covalent peptide bonds to form a single polypeptide strand, and proteins comprising two or more polypeptide strands non-covalently associated with each other or linked with each other by covalent bonds other than peptide bonds, including but not limited to disulfide bonds and isopeptide bonds. As used herein, the term "isopeptide bond" is intended to mean an amide bond formed between an amino group of one amino acid and a carboxyl group of a second amino acid, wherein at least one of the amino group and the carboxyl group is located on the side chain of the respective amino acid.

[0040] As used herein, the term "Clostridium perfringens pilus polypeptide" is intended to mean a polypeptide which has the function of a pilus or a pilus subunit and which is encoded by one or more genes found in a strain of Clostridium perfringens associated with necrotic enteritis in poultry. In at least one embodiment, the gene is the cnaA gene, the fimA gene, or the fimB gene found in the VR-10B genetic locus identified in Lepp D et al, Journal of Bacteriology (2013) 195: 1152-1166, as diagrammatically represented in FIG. 1.

[0041] As used herein, the term "variant" when used in reference to a polypeptide is intended to refer to a polypeptide which differs in its amino acid sequence from the sequence of a reference polypeptide to which the variant is being compared by one or more amino acid residues. The differences between the sequence of the variant and the sequence of the reference polypeptide can include substitution of one or more amino acid residues with different amino acid residues, insertion of additional amino acid residues or deletion of amino acid residues. In certain embodiments, a variant can differ from a reference polypeptide by conservative substitution of one or more amino acid residues with replacement amino acid residues which may have similar properties, including but not limited to charge, size and hydrophilicity, to the amino acid residues which the new residues replace. In certain embodiments, variants may completely or partially retain one or more biological functions of the reference polypeptide, including but not limited to immunogenicity. In at least one embodiment, the reference polypeptide is an isolated Clostridium perfringens pilus polypeptide as described herein.

[0042] In at least one embodiment, the sequence of a variant can have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or at least 99.9% identity to the sequence of a reference polypeptide. As used herein, the term "percent identity" or "% identity" when used in reference to the sequence of a polypeptide or a polynucleotide is intended to mean the percentage of the total number of amino acid or nucleotide residues, respectively, in the sequence which are identical to those at the corresponding position of a reference polypeptide or polynucleotide sequence. In at least one embodiment, when the length of the variant sequence and the length of the reference sequence are not identical, percent identity can be calculated based on the total number of residues in the variant sequence or based on the total number or residues in the reference sequence. Percent identity can be measured by various local or global sequence alignment algorithms well known in the art, including but not limited to the Smith-Waterman algorithm and the Needleman-Wunsch algorithm. Tools using these or other suitable algorithms include but are not limited to BLAST (Basic Local Alignment Search Tool) and other such tools well known in the art.

[0043] As used herein, the term "fragment" when used in relation to a polypeptide or a variant is intended to refer to a smaller polypeptide containing fewer amino acid residues than the polypeptide or variant and having a sequence which is identical to a portion of the sequence of the polypeptide or variant. In at least one embodiment, the fragment retains one or more biological activities of the polypeptide or variant, including but not limited to immunogenicity. In at least one embodiment, the fragment comprises an epitope of the polypeptide or variant. In at least one embodiment, the fragment is at least 6 amino acids in length or at least 7 amino acids in length, or at least 8 amino acids in length or at least 9 amino acids in length or at least 10 amino acids in length.

[0044] As used herein, the term "immunogenic" is intended to refer to an agent, including but not limited to a polypeptide or polynucleotide or a fragment thereof, which is capable of eliciting an immunoprotective response in a subject to which the immunogenic agent is administered. As used herein, the term "immunoprotective response" is intended to refer to an immune response that prevents, reduces or eliminates one or more of the symptoms of disease in an infected subject.

[0045] The present immunogenic polypeptide, including the present isolated Clostridium perfringens pilus polypeptide, the variant of the pilus polypeptide, the fragment of the pilus polypeptide and the fragment of the variant, are immunogenic in poultry. Thus, in at least one embodiment, poultry immunized with any one or more of the present isolated Clostridium perfringens pilus polypeptide, the variant of the pilus polypeptide, the fragment of the pilus polypeptide and the fragment of the variant will show an immunoprotective response to challenge with one or more of a Clostridium perfringens cell, an assembled Clostridium perfringens pilus, a Clostridium perfringens pilus polypeptide, a fragment of a Clostridium perfringens pilus polypeptide, or a portion of a Clostridium perfringens cell, including but not limited to a cell membrane or portion thereof, or a cell wall or a portion thereof, which bears one or more of an assembled Clostridium perfringens pilus, a Clostridium perfringens pilus polypeptide or a fragment of a Clostridium perfringens pilus polypeptide.

[0046] Another aspect of the present application provides a polynucleotide comprising a sequence encoding an isolated Clostridium perfringens pilus polypeptide or an immunogenic polypeptide as described herein. In at least one embodiment, the polynucleotide is messenger RNA (mRNA) having a sequence which can be translated to generate the isolated Clostridium perfringens pilus polypeptide or the immunogenic polypeptide. In at least one embodiment, the polynucleotide is DNA, at least one strand of which can be transcribed to produce mRNA which in turn can be translated to generate the isolated Clostridium perfringens pilus polypeptide or the immunogenic polypeptide. In at least one embodiment, the DNA can be expressed by a biochemical system, including but not limited to a cell, to produce the isolated Clostridium perfringens pilus polypeptide or the immunogenic polypeptide. In at least one such embodiment, the DNA can be incorporated into a vector configured for expression of the DNA in a host cell, as well known in the art.

[0047] In at least one embodiment, the polynucleotide can include a variant polynucleotide sequence which hybridizes to a polynucleotide comprising a sequence encoding an isolated Clostridium perfringens pilus polypeptide or an immunogenic polypeptide as described herein under at least moderately stringent conditions. By "at least moderately stringent hybridization conditions" it is meant that conditions are selected which promote selective hybridization between two complementary nucleic acid molecules in solution. Hybridization may occur to all or a portion of a nucleic acid sequence molecule. The hybridizing portion is typically at least 15 (e.g. 20, 25, 30, 40 or 50) nucleotides in length. Those skilled in the art will recognize that the stability of a nucleic acid duplex, or hybrid, is determined by the melting temperature (T.sub.m), which in sodium-containing buffers is a function of the sodium ion concentration ([Na.sup.+]) and temperature (T.sub.m=81.5.degree. C.-16.6 (Log.sub.10[Na.sup.+])+0.41(% (G+C)-600/l), where % G+C is the percentage of cytosine and guanine nucleotides in the nucleic acid and I is the length of the nucleic acid in base pairs, or similar equation). Accordingly, the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature. In order to identify molecules that are similar, but not identical, to a known nucleic acid molecule, a 1% mismatch may be assumed to result in about a 1.degree. C. decrease in T.sub.m. For example, if nucleic acid molecules are sought that have a >95% identity, the final wash temperature may be reduced by about 5.degree. C. Based on these considerations those skilled in the art will be able to readily select appropriate hybridization conditions.

[0048] In some embodiments, stringent hybridization conditions are selected. By way of example the following conditions may be employed to achieve stringent hybridization: hybridization at 5.times. sodium chloride/sodium citrate (SSC)/5.times.Denhardt's solution/1.0% sodium dodecylsulfate (SDS) at T.sub.m-5.degree. C. based on the above equation, followed by a wash of 0.2.times.SSC/0.1% SDS at 60.degree. C. Moderately stringent hybridization conditions include a washing step in 3.times.SSC at 42.degree. C. It is understood, however, that equivalent stringencies may be achieved using alternative buffers, salts and temperatures. Additional guidance regarding hybridization conditions may be found in: Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 2002, and in: Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001.

[0049] In at least one embodiment, the isolated Clostridium perfringens pilus polypeptide is a CnaA polypeptide. In at least one embodiment, the CnaA polypeptide has an amino acid sequence selected from SEQ ID NO:10 and SEQ ID NO:13. In at least one embodiment, the CnaA polypeptide is encoded by a polynucleotide having a sequence selected from SEQ ID NO:1, SEQ ID NO:4 and SEQ ID NO:7. In at least one embodiment, the CnaA polypeptide is encoded by a polynucleotide which hybridizes under at least moderately stringent conditions to a polynucleotide having a sequence selected from SEQ ID NO:1, SEQ ID NO:4 and SEQ ID NO:7. In at least one embodiment, when the pilus polypeptide is a CnaA polypeptide, the variant has at least 75%, 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity to an amino acid sequence selected from SEQ ID NO:10 and SEQ ID NO:13.

[0050] In at least one embodiment, the isolated Clostridium perfringens pilus polypeptide is a FimA polypeptide. In at least one embodiment, the FimA polypeptide has an amino acid sequence selected from SEQ ID NO:11 and SEQ ID NO:14. In at least one embodiment, the FimA polypeptide is encoded by a polynucleotide having a sequence selected from SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8. In at least one embodiment, the FimA polypeptide is encoded by a polynucleotide which hybridizes under at least moderately stringent conditions to a polynucleotide having a sequence selected from SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8. In at least one embodiment, when the pilus polypeptide is a FimA polypeptide, the variant has at least 75%, 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity to an amino acid sequence selected from SEQ ID NO:11 and SEQ ID NO:14.

[0051] In at least one embodiment, the isolated Clostridium perfringens pilus polypeptide is a FimB polypeptide. In at least one embodiment, the FimB polypeptide has an amino acid sequence selected from SEQ ID NO:12 and SEQ ID NO:15. In at least one embodiment, the FimB polypeptide is encoded by a polynucleotide having a sequence selected from SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:9. In at least one embodiment, the FimB polypeptide is encoded by a polynucleotide which hybridizes under at least moderately stringent conditions to a polynucleotide having a sequence selected from SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:9. In at least one embodiment, when the pilus polypeptide is a FimB polypeptide, the variant has at least 75%, 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity to an amino acid sequence selected from SEQ ID NO:12 and SEQ ID NO:15.

[0052] In at least one embodiment, the isolated Clostridium perfringens pilus polypeptide is an assembled pilus. In at least one embodiment, the assembled pilus comprises one or more subunits, each individually selected from a CnaA polypeptide, a FimA polypeptide, and a FimB polypeptide. In at least one embodiment, the one or more subunits are covalently bonded to each other. In at least one embodiment, the assembled pilus is a pilus isolated from a Clostridium perfringens cell, or a portion thereof including but not limited to a cell membrane or a portion thereof or a cell wall or a portion thereof. In at least one embodiment, the assembled pilus is a fragment of a pilus isolated from a Clostridium perfringens cell, or a portion thereof including but not limited to a cell membrane or a portion thereof or a cell wall or a portion thereof, wherein the fragment comprises one or more subunits, each individually selected from a CnaA polypeptide, a FimA polypeptide, and a FimB polypeptide.

[0053] In at least one embodiment, the isolated Clostridium perfringens pilus polypeptide can be isolated from a culture of Clostridium perfringens. Thus, in at least one embodiment, the isolated Clostridium perfringens pilus polypeptide can be part of a preparation containing one or more portions of a Clostridium perfringens cell, including but not limited to a cell membrane or a portion thereof or a cell wall or a portion thereof, which bears the pilus polypeptide or a fragment thereof as described herein. In at least one embodiment, the isolated Clostridium perfringens pilus polypeptide can be recombinantly produced by expression in a suitable host cell of a vector comprising a polynucleotide having a sequence encoding the pilus polypeptide. In at least one embodiment, when the pilus polypeptide is an assembled pilus, the assembled pilus can be recombinantly produced by expression in a suitable host cell of a vector comprising a polynucleotide having a sequence encoding genes and other nucleotide sequences required for assembly of the assembled pilus. In addition, the isolated Clostridium perfringens pilus polypeptide can be at least partially purified after isolation or recombinant production. Suitable vectors and host cells, including but not limited to prokaryotic and eukaryotic host cells adapted for the production of recombinant polypeptides, and methods of isolating or recombinantly producing such polypeptides, including methods of at least partial purification of such polypeptides, are well known in the art or can be readily identified and selected by the skilled person with no more than routine experimental effort.

[0054] In another aspect, the present application provides a vaccine for the treatment or prevention of necrotic enteritis in poultry, or for the treatment or prevention of Clostridium perfringens infection in poultry, wherein the vaccine comprises at least one immunogenic polypeptide as described herein. As used herein, the term "vaccine" is intended to refer to an immunogenic preparation used to prevent, treat or reduce the effects of infection by Clostridium perfringens. Vaccine formulations typically contain an immunologically effective amount of an immunogenic agent, and may also contain an adjuvant or may be adjuvant-free. In the case of the present vaccine, the immunogenic agent can be an immunogenic polypeptide as described herein.

[0055] As used herein, the term "adjuvant" is intended to refer to an agent which is effective for enhancing an immune response against an immunogenic agent of a subject vaccinated with a vaccine comprising the immunogenic agent. Suitable adjuvants are well known in the art and include but are not limited to inorganic compounds including but not limited to alum, aluminum hydroxide, and other aluminum-containing compounds; saponins including but not limited to Quil-A.TM.; Freund's complete and incomplete adjuvants; lipid or mineral oil-containing adjuvants, including but not limited to oil-in-water emulsions; polysaccharide adjuvants; protein adjuvants; immunomodulators; adjuvants obtained from killed or attenuated bacterial cells; and other suitable adjuvants known in the art.

[0056] Vaccines can be formulated in one or more pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable" is intended to refer to molecular entities and compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to an animal or a human. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals or humans. As used herein, the term "carrier" is intended to refer to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Suitable carriers are well known in the art and, in at least one embodiment, are described in "Remington's Pharmaceutical Sciences" by E. W. Martin, 18th Edition, or other editions.

[0057] The vaccines can be formulated for administration by any convenient route known in the art, including but not limited to orally, rectally, nasally, transmucosally, transdermally, parenterally, intravenously, intramuscularly, subcutaneously, in ovo, or by other known routes. In at least one embodiment, it is contemplated that the vaccine can be administered orally. Without being bound by theory, it is contemplated that oral vaccination can directly target gut-associated lymphoid tissues, at the site of infection by necrotic enteritis-associated strains of Clostridium perfringens. In at least one embodiment, it is contemplated that progeny can be immunized by vaccination of a mother and subsequent transfer of maternal immunity, including but not limited to maternal antibodies, to the progeny.

[0058] In another aspect, the present invention provides the use of an immunogenic polypeptide as described herein in the preparation of a medicament for the treatment or prevention of necrotic enteritis in poultry or for the treatment or prevention of Clostridium perfringens infection in poultry. The medicament can be a vaccine as described herein.

[0059] In another aspect, the present invention provides a method of treatment or prevention of necrotic enteritis in poultry or for the treatment or prevention of Clostridium perfringens infection in poultry, the method comprising administering an effective amount of an immunogenic polypeptide or of a vaccine as described herein to the poultry. Administration can be by routes well known in the art, including but not limited to orally, rectally, nasally, parenterally, intravenously, intramuscularly, subcutaneously or by other routes. In at least one embodiment, administration can be by subcutaneous injection. In at least one embodiment, administration can be oral. In at least one embodiment, the vaccine can be administered more than once to the poultry, to provide an initial immunization followed by one or more booster immunizations, as understood in the art. In at least one embodiment, one or more of the initial immunization and the one or more booster immunizations are administered to the poultry after the disappearance of maternal antibodies in the poultry. In at least one such embodiment, one or more of the initial immunization and the one or more booster immunizations are administered to the poultry no earlier than about 10 days after hatching.

[0060] In another aspect, the present invention provides the use of an immunogenic polypeptide as described herein as a vaccine for the treatment or prevention of necrotic enteritis in poultry or for the treatment or prevention of Clostridium perfringens infection in poultry.

[0061] A further aspect of the present invention provides an antibody which binds selectively to an immunogenic polypeptide as described herein. In at least one embodiment, the antibody is a poultry antibody. In at least one embodiment, the antibody can be a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single chain antibody or an antibody fragment that retains the property of selective binding to an immunogenic polypeptide as described herein. The term "antibody fragment" as used herein is intended to include but not be limited to Fab, Fab', F(ab').sub.2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, and multimers thereof and bispecific antibody fragments. Antibodies can be fragmented using conventional techniques. For example, F(ab').sub.2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab').sub.2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab').sub.2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques.

[0062] Methods of preparing and characterizing such antibodies and fragments thereof are well known in the art and can be readily carried out by the skilled person without undue effort. For example, polyclonal antisera or monoclonal antibodies can be made using standard methods. A mammal, (e.g., a mouse, hamster, or rabbit), bird (e.g. poultry) or other animal can be immunized with an immunogenic form of the present immunogenic polypeptide which elicits an antibody response in the mammal. Techniques for conferring immunogenicity on a peptide include conjugation to carriers or other techniques well known in the art. For example, the peptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassay procedures can be used with the immunogenic agent as antigen to assess the levels of antibodies. Following immunization, antisera can be obtained and, if desired, polyclonal antibodies isolated from the sera.

[0063] To produce monoclonal antibodies, antibody producing cells (lymphocytes) can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells. Such techniques are well known in the art. Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with an immunogenic polypeptide as described herein and the monoclonal antibodies can be isolated. Therefore, the disclosure also contemplates hybridoma cells secreting monoclonal antibodies with specificity for an immunogenic polypeptide as described herein.

[0064] Specific antibodies, or antibody fragments reactive against an immunogenic polypeptide as described herein may also be generated by screening expression libraries encoding immunoglobulin genes, or portions thereof, expressed in bacteria with peptides produced from nucleic acid molecules as described herein. For example, complete Fab fragments, VH regions and FV regions can be expressed in bacteria using phage expression libraries.

[0065] In another aspect, the present invention provides a method of detecting infection of poultry by a strain of Clostridium perfringens associated with necrotic enteritis, wherein the method includes obtaining a biological sample from the poultry and detecting in the biological sample the presence of an antibody which binds selectively to an immunogenic polypeptide as described herein. In at least one embodiment, the biological sample is a blood sample. In at least one embodiment, the sample is a fecal sample. In at least one embodiment, the detection includes measurement of the amount or concentration of antibody present in the biological sample, using methods well known by those skilled in the art.

[0066] Yet another aspect of the present invention provides a method of detecting an immunogenic polypeptide as described herein comprising exposing the immunogenic polypeptide to an antibody which binds selectively to the immunogenic polypeptide and detecting binding of the immunogenic polypeptide to the antibody. In at least one embodiment, the immunogenic polypeptide can be an isolated Clostridium perfringens pilus polypeptide as described herein. In at least one embodiment, the immunogenic polypeptide can be an assembled pilus attached to the surface of a Clostridium perfringens bacterial cell. Such embodiments of the method can be useful for identifying and detecting strains of Clostridium perfringens which are capable of producing necrotic enteritis in poultry.

[0067] As used herein, the terms "about" or "approximately" as applied to a numerical value or range of values are intended to mean that the recited values can vary within an acceptable degree of error for the quantity measured given the nature or precision of the measurements, such that the variation is considered in the art as equivalent to the recited values and provides the same function or result. For example, the degree of error can be indicated by the number of significant figures provided for the measurement, as is understood in the art, and includes but is not limited to a variation of .+-.1 in the most precise significant figure reported for the measurement. Typical exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms "about" and "approximately" can mean values that are within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term "about" or "approximately" can be inferred when not expressly stated.

[0068] As used herein, the term "substantially" refers to the complete or nearly complete extent or degree of an action, characteristic, property, state, structure, item, or result. For example, two substances which have "substantially" the same properties would have completely identical properties or would have properties which are so nearly completely the same that the differences are not measurable or significant. The exact allowable degree of deviation from absolute completeness may in some cases depend on the specific context. However, generally speaking the nearness of completion will be so as to have the same overall result as if absolute and total completion were obtained. The use of "substantially" is equally applicable when used in a negative connotation to refer to the complete or near complete lack of an action, characteristic, property, state, structure, item, or result.

EXAMPLES

[0069] Other features of the present invention will become apparent from the following non-limiting examples which illustrate, by way of example, the principles of the invention.

Example 1: Production of Purified Recombinant Pilus-Associated Polypeptides from Clostridium perfringens

[0070] Coding regions for the three pilus subunits (cnaA, fimA and fimB) were codon-optimized and truncated to exclude the predicted N-terminal signal peptides and C-terminal cell wall sorting signal LPXTG (SEQ ID NO: 16) transmembrane domains. The C-terminal domain is a hydrophobic region predicted to be removed during pilus assembly. The truncated codon-optimized coding regions were synthesized (Integrated DNA Technologies, Coralville, USA), cloned into the pET28a expression vector (MilliporeSigma, Etobicoke, Ontario, Canada) by In-Fusion.TM. cloning according to the manufacturer's instructions (Takara Bio USA, Mountain View, Calif., USA), sequence-verified, and then transformed into E. coli BL21 cells. Transformed colonies were grown at 37.degree. C. for 18 h with shaking in 1 L LB broth supplemented with 50 .mu.g/ml kanamycin and 1 mM IPTG. The culture was pelleted and resuspended in 20 ml of binding buffer (20 mM NaPO.sub.4, 0.5M NaCl, 30 mM imidazole) and lysed by sonication for 10 min on ice (10 s pulses, 20 s pauses). The cell lysate was purified under native conditions on a HisTrap.TM. FF Crude column (GE Healthcare, Montreal, Canada) using a gradient of 50 to 500 mM imidazole on an AKTA.TM. prime plus system. One ml fractions were collected, and fractions exhibiting a 280 nm peak were pooled and concentrated with Pierce.TM. Protein Concentrators (9K molecular weight cut-off) (Fisher Scientific, Unionville, Ontario, Canada) and desalted using Zeba.TM. Spin 7K molecular weight cut-off desalting columns (Fisher Scientific). Quantitation of the purified proteins was performed using the BCA (bicinchoninic acid) Protein Assay kit (Fisher) according to the manufacturer's instructions. Polypeptides were visualized by SDS-PAGE and COOMASSIE staining.

[0071] The truncated sequences resulted in high levels of expression of histidine (His)-tagged polypeptides, as evidenced by the SDS-PAGE gels shown in FIGS. 2A (CnaA), 2B (FimA), 2C (FimB) and 2D (pooled fractions of CnaA, FimA and FimB, following concentration and desalting). These increased levels of expression may be due to increased solubility of the resulting polypeptides.

[0072] Table 1 shows the sequences of the full-length, codon-optimized and truncated gene sequences, along with the sequences of the full-length subunit polypeptides and the expressed truncated and His-tagged subunit polypeptides.

TABLE-US-00001 TABLE 1 Sequences of pilus subunit genes and polypeptides Sequence cnaA fimA fimB Full length ATGAAAATAAATAAAAAAATTTTTAGCATGCTAT ATGATAAACAAGAAAAAA ATGGAAACAAAG gene TTATGGTTATTGTACTTTTTACATGCATATCATC TTAAGTGCATTATTATTA AAAATAAGAAAC AAATTTTTCTGTTTCTGCTTCTTCTATTCAAAGA AGTGGAGCAATGTTTATG AAAATCCTTATG GGAAGAGATATCAGTAATGAGGTACTTACAAGCC AGTATGAATACAAATGTA GCTATCGTAGCA TAGTGGCTACTCCAAATAGTATAAATGATGGTGG TTCGCATCAAATTTACCT TTGAGCTTTATA AAACGTTCAGGTTCGTTTGGAATTTAAAGAAAAT TCTGGAGGGGTAGAAGGT TTGCTTCCAAAC CATCAAAGAAATATACAAAGTGGAGATACTATAA ACAGAACAGAATCCTGCA ACTAGAGTATAT CTGTCAAATGGACAAATTCAGGGGAAGTATTTTT AAAGCAACAATTACAAAG GCTACTGAAAAT TGAAGGATATGAAAAAACAATTCCACTTTATATA AATTTTGAATTTCCAGAA ACAGCAAATATT AAAGACCAAAATGTTGGTCAAGCAGTAATAGAGA GGTATTAATACACCTAGT CCTTTGATAGTT AAACAGGTGCAACACTTACATTTAATGATAAAAT GCAACATTCAAGTTTACA AGACAGGAATTT AGATAAATTAGATGATGTTGGTGGATGGGCAACA GCAGAAAAAATAACTAAT AATGTATATACG TTTACTTTGCAAGGAAGAAACATTACCTCAGGTA GATGCGCCAGATGCAACA AAAGATTCAAAA ATCATGAACACACAGGAATAGCATATATTATATC ATTGGAGATATTAATTAT GCAATAGACATG TGGTTCAAAGCGGGCAGATGTAAATATAACCAAA ACACAAGGGGATAATGGA ATTGGAAAATAC CCAGAATCAGGTACAACTAGTGTATTCTATTATA ACTTTATCAAATGGAAAA GAGCTAAAGGCA AAACAGGTAGTATGTATACCAATGATACAAATCA TATAGTGTAAAGAAAACA ATAAGTGAAAAT TGTCAATTGGTGGTTACTGGTGAATCCAAGCAAG ACTGAAATTACTTTTGGA GCCCCTATGCCA GTATATTCTGAAAAAAACGTTTATATTCAAGATG AATTTCCCACATGCAGGA GAAGAAAGTAAA AAATCCAAGGCGGACAAACATTAGAACCTGATTC GAATATGATTATAATGTA AATGGAAGTTTT TTTTGAAATAGTAGTAACTTGGTATGATGGTTAT AAAGAAACGAATGAGGGA ATCTTTAATATA GTAGAAAAGTTTAAAGGAAAAGAAGCGATAAGGG GTAGGTGGTATTACATAT GATGGAAATGAT AATTCCATAATAAATATCCAAATTCAAATATATC GATACAAAAGAATACAAA AAGCAGTTTACT GGTATCAGAAAATAAAATAACAGTAAACATTTCA GTTCATGTGTATGTTGCA ATTCCATTAGCT CAAGAGGATTCCACACAAAAGTTTATTAATATTT AATAGTAACGCTATGGAT TATACACATGGT TTTATAAAACTAAGATTACAAATCCGAAACAAAA GGAAAAACTTATGTAAAA GGTGTGTATATC AGAATTCGTTAATAATACAAAAGCATGGTTTAAA GCCATTACATCAGAAAAT TATCAAATTCAA GAGTATAATAAGCCAGCTGTAAATGGAGAATCCT GGAGGTGAAAAAGCTCCA CAGATAACGCAA TTAACCATAGCGTACAAAATATTAATGCAGATGC ATTGAGTTTGTTAATACA TCTAAAGATAAT TGGAGTTAATGGAACTGTAAAAGGCGAATTAAAA TATAAAAAGGACACTTCT TACATATATGAT ATCATAAAAACATTAAAAGATAAAAGTATTCCAA TTACTTATAGAAAAGAAT AAAAATAGCTAT TTAAAGATGTTCAGTTTAAGATGAGAAGAGTTGA GTAATAGGAGATTTAGCT AAGATAACTGTA TAATACAGTTATCAAAGATGGTAAAAAAGAATTA GACTTAACAAAACAGTTT TATGTAAAAAAT TTACTAACAACTGATGATAAAGGTATTGCAAATG GAGTTTCAGATTAATTTA GCAGAAAATAAT TAAAAGGTCTTCCTGTAGGAAAATATGAAGTAAA AAAAAATCAGCAACATCT CATTTAATACCA AGAGATTTCAGCTCCAGAATGGATTGCTTTTAAT GACATAACAAAATTCGAA CAAATTATTGTG CCTCTTATTGCACCAAAATTGGAATTCACAATAT GGAAATATTATTAGAAAA AAAAATGAAAAT CAGATCAGGACACAGAAGGCAAATTGTGGGCTGT GATGGTAAAATAGAGCCT AATGAAAAATGT TGAAAATGAATTAAAGACAATTTCAATTCCGGTT GTAACATATACAGCTGAA GAAGAAATATGT GAAAAGGTCTGGGTAGGACAAACTAGTGAACGAG AATACAGAAACTTTTAAA TTTTATAACATT CAGAAATCAAGCTTTTTGCAGATGGTATTGAAGT TTAGCAAATGGAGATAAA TACAAACAAAAA AGACAAAGTGATTTTAAATGCAGATAACAATTGG CTTAAGTTTGAAAGTATT AATAAAATTAAT AAACACACATTTGAAAATAAACCTGAATATAATT CCAGCAGGAACAAAATAT GAGATTTCTAAA CAGAAACAAAACAGAAAATCAATTATTCTGTGTC GAAGTAAAAGAGATAGGT ACACCATATAAG AGAGACAACTATTTCTGGATATGAAAGCAATATC GCTAGTGATGGATATACA CCAAATGGAATA ACAGGCGATGCTAAGAATGGTTTTATTGTAACCA CCTTCTATAACAGTAATT AATGTTCCTAAA ATACAGAACTTCCTGATTTGACTATTGGTAAAGA GAAAATGGAAATGAGACT ACAGGCGATACC AGTTATAGGAGAATTGGGTGACAAGACGAAGGTA TCTAATAATCGTACGGTA ACAAACATTGGA TTTAACTTTGAGCTTACATTAAAGCAAGCAGATG GCTGAAAAAGATGGTATA TTTTATATTGTA GAAAGCCTATCAATGGTAAATTTAATTACATTGG TCATCTAAGTCAAATTCT ATACTTATAATT TAGTGTAGATGACAGGTACAAAAAAGAAAGCATA AATGATAACTTAATTGGT TCACTTGGATTA AAGCCTTCTGATOGTGAGATTACTTTTATAGAAG GAAGGTGAAAACAAAGTA CTTGTGGTATTG GAAAAGCAACTATAACTTTATCACATGGACAAGA ACATTTACAAACACATAT AAATGGAAAGAA GATTACAATCAAGGATTTACCATATGGGGTTACA AATGACAAACCTATCACA TATAAAAAGAGA TATAAAGTTATGGAAAAAGAAGCTAATGAAAATG GGTATTGTTATGAATAAT AAAAAAGAATAA GCTATTTAACTACCTATAATGGAAATAACGAAGT ATTCCATTTATTCTAATG (SEQ ID NO: 3) CACAACAGGTGAATTGAAACAGGATACAAAAGTA ATTAGTTTTGCTGTCCTT CAGGTAGTTAACAACAAAGAGTTTGTTCCAACAA GGATTTGGTGCTTTAGCT CTGGTATATCAACCACAACAGAGCAAGGTACAAT ATTATAAAAAGACGTAAA GGTTGGAATGGTGATTTTTTCTATAGGAATACTT ACTATAAGATAA ATGGTCATGATTGTAGTTCTTTTACAATTGAATA (SEQ ID NO: 2) AAGGACTGAAAAGATGA (SEQ ID NO: 1) Codon- ATGAAGATCAACAAGAAGATCTTCAGCATGTTAT ATGATTAATAAAAAGAAA ATGGCTATTGTT optimized TTATGGTCATTGTGCTGTTCACCTGTATCAGCTC CTGTCGGCGCTGCTCTTA GCTTTGTCArTT gene TAACTTCAGTGTGAGCGCGTCAAGCATCCAGCGC AGCGGGGCCATGTTTATG ATCCTGCTCCCG GGCCGGGACATCAGCAACGAGGTGGTGACATCGC AGCATGAACACGAATGTG AATACCCGGGTC TCGTAGCTACCCCGAATAGCATCAACGATGGTGG TTCGCGTCTAACCTCCCA TATGCGACGGAG TAACGTCCAAGTGCGTCTGGAATTTAAAGAGAAT TCGGGTGGTGTGGAGGGC AACACCGCTAAT CACCAGCGGAACATTCAGTCCGGCGACACGATTA ACCGAACAAAACCCAGCG ATCCCGTTAATT CGGTCAAATGGACTAACTCAGGTGAGGTCTTTTT AAAGCGACAATCACGAAA GTACGCCAAGAA TGAAGGCTACGAAAAAACCATCCCGCTGTATATC AACTTCGAGTTTCCGGAA TTTAATGTTTAC AAGGATCAGAACGTTGGCCAGGCGGTTATTGAAA GGTATTAATACACCCAGC ACTAAAGATTCT AAACCGGTGCAACATTAACATTCAACGATAAGAT GCGACATTCAAATTTACC AAAGCCATTGAC CGACAAATTAGATGATGTCGGCGGCTGGGCCACA GCCGAAAAAATTACCAAC ATGATCGGAAAA TTCACGCTCCAGGGTCGCAATATTACTTCAGGAA GATGCGCCGGATGCTACT TATGAATTAAAA ATCATGAGCATACTGGTATTGCGTACATTATCTC ATTGGCGACATCAATTAT GCCATTTCTGAG GGGTAGCAAACGTGCGGACGTTAACATCACAAAA ACCCAAGGTGATAATGGG AACGCTCCCATG CCTGAATCCGGAACAACGTCTGTGTTTTACTACA ACGTTAAGCAATGGCAAA CCGGAGGAATCA AGACGGGTTCGATGTACACCAATGACACAAATCA TACAGTGTGAAAAAGACT AAAAATGGTAGC TGTGAATTGGTGGCTGCTGGTTAACCCGAGCAAA ACCGAGATTACCTTCGGG TTTATTTTTAAC GTATACTCTGAGAAAAATGTCTATATTCAGGATG AACTTCCCGCATGCTGGT ATCGACGGTAAT AAATTCAAGGCGGTCAGACCCTGGAGCCGGACAG GAGTATGATTATAACGTC GATAAACAGTTT TTTTGAAATCGTCGTTACATGGTACGATGGTTAT AAAGAAACCAATGAAGGC ACTATTCCGCTG GTGGAAAAATTTAAAGGTAAAGAAGCGATCCGGG GTGGGTGGCATTACTTAC GCGTACACTCAC AGTTCCACAATAAATATCCGAATAGTAATATCTC GATACGAAAGAATATAAA GGTGGCGTCTAC GGTCAGTGAAAATAAAATCACGGTAAATATTTCG GTTCATGTGTATGTGGCC ATCTATCAAATC CAAGAAGATTCCACCCAAAAATTCATTAACATCT AACTCAAATGCGATGGAC CAGCAAATTACC TTTACAAGACTAAAATCACCAACCCGAAGCAGAA GGTAAGACATATGTTAAA CAGAGCAAGGAT AGAATTTGTAAACAACACCAAAGCCTGGTTCAAA GCGATTACTAGCGAAAAT AACTACATCTAC GAGTACAATAAGCCGGCGGTTAACGGTGAAAGTT GGCGGGGAAAAAGCACCG GATAAAAACAGC TTAATCACAGTGTGCAGAATATCAACGCAGATGC ATCGAATTCGTTAACACC TATAAAATCACG CGGGGTAAATGGTACTGTTAAAGGTGAATTGAAA TATAAAAAAGATACGTCG GTATATGTCAAG ATTATCAAAACCCTGAAAGATAAAAGTATTCCGA TTACTGATTGAAAAAAAT AACGCAGAAAAC TCAAGGATGTGCAGTTTAAGATGCGCCGCGTGGA GTAATTGGCGATCTGGCA AATCATCTGATC TAATACCGTTATTAAAGACGGCAAGAAAGAGCTG GACCTCACCAAACAGTTT CCGCAGATTATT CTGTTGAGCACAGATGATAAAGGGATTGCAAACG GAGTTTCAAATCAACTTG GTAAAAAATGAG TGAAAGGTCTGCCAGTCGGGAAATACGAAGTCAA AAAAAGAGCGCGACTAGT AACAATGAAAAA AGAAATCAGTGCGCCTGAGTGGATCGCCTTCAAT GATATTACCAAGTTTGAA TGTGAAGAAATC CCACTGATTGCGCCCAAACTTGAATTTACGATCA GGTAACATTATTCGCAAA TGCTTCTACAAT GCGATCAAGACACAGAGGGGAAATTATGGGCAGT GACGGTAAGATTGAACCC ATCTACAAACAG GGAAAACGAACTCAAAACCATCTCGATTCCGGTC GTGACCTATACCGCGGAA AAAAACAAGATC GAAAAAGTCTGGGTAGGTCAGACGAGTGAACGGG AATACCGAGACCTTTAAG AATGAGATCTCT CGGAGATCAAACTGTTTGCGGATGGAATTGAAGT TTAGCCAACGGAGACAAG AAAACCCCCTAT TGATAAGGTGATCCTGAACGCGGATAATAATTGG TTAAAATTCGAGTCCATC AAGCCGAATGGT AAGCACACCTTTGAGAATAAACCCGAATATAACT CCCGCCGGTACAAAATAT ATTAATGTCCCG CCGAGACTAAACAAAAAATCAACTATAGTGTGAG GAAGTCAAGGAAATCGGG AAAACGGGTGAT CGAAACTACCATCAGTGGCTATGAATCAAATATT GCGAGCGATGGGTACACG ACCACGAACATC ACTGGCGATGCGAAAAACGGATTTATTGTCACCA CCCTCAATCACCGTTATC GGATTCTACATT ACACAGAACTOCCTGATTTGACGATCGGGAAAGA GAAAATGGCAACGAAACC GTGATCTTGATT GGTAATCGGCGAACTCGGCGATAAAACCAAGGTA TCAAATAACCGCACTGTA ATTTCCCTGGGC TTCAACTTTGAACTGACACTTAAGCAGGCTGACG GCCGAAAAAGATGGAATC CTGCTGGTGGTC GAAAGCCCATTAACGGGAAATTTAACTATATTGG TCTAGCAAAAGCAACTCG TTGAAGTGGAAA TTCGGTGGATGATCGTTATAAGAAGGAATCGATT AACGACAATTTAATCGGC GAATATAAAAAA AAGCCTAGCGATGOGGAAATTACGTTCATCGAGG GAAGGCGAAAATAAAGTG CGTAAGAAGGAA GAAAAGCAACGATTACCCTCTCCCACGGACAAGA ACCTTTACCAATACGTAC (SEQ ID NO: 6) GATCACCATTAAGGACCTTCCGTATGGTGTGACC AACGATAAACCAATCACG TATAAAGTCATGGAAAAAGAAGCCAACGAGAATG GGAATCGTAATGAATAAT GATATTTAACCACTTACAACGGAAATAACGAAGT ATTCCGTTCATTCTTATG CACCACCGGGGAGTTGAAACAGGATACGAAAGTA ATTAGCTTTGCCGTTCTT CAAGTGGTTAATAATAAAGAATTCGTCCCGACAA GGCTTCGGTGCATTAGCG CCGGGATCAGCACCACCACCGAACAGGGAACCAT ATCATTAAACGCCGCAAA GGTCGGGATGGTGATCTTTAGCATCGGTATTCTC ACCATCCGCCCCATCGAT ATGGTAATGATTGTCGTTCTGCTGCAGCTGAATA ACGCGT AAGGACTGAAACGC (SEQ ID NO: 4) (SEQ ID NO: 5) Truncated TCAAGCATCCAGCGCGGCCGGGACATCAGCAACG TCTAACCTCCCATCGGGT ACGGAGAACACC gene AGGTGGTGACATCGCTCGTAGCTACCCCGAATAG GGTGTGGAGGGCACCGAA GCTAATATCCCG CATCAACGATGGTGGTAACGTCCAAGTGCGTCTG CAAAACCCAGCGAAAGCG TTAATTGTACGC GAATTTAAAGAGAATCACCAGCGGAACATTCAGT ACAATCACGAAAAACTTC CAAGAATTTAAT CCGGCGACACGATTACGGTCAAATGGACTAACTC GAGTTTCCGGAAGGTATT GTTTACACTAAA AGGTGAGGTCTTTTTTGAAGGCTACGAAAAAACC AATACACCCAGCGCGACA GATTCTAAAGCC ATCCCGCTGTATATCAAGGATCAGAACGTTGGCC TTCAAATTTACCGCCGAA ATTGACATGATC AGGCGGTTATTGAAAAAACCGGTGCAACATTAAC AAAATTACCAACGATGCG GGAAAATATGAA ATTCAACGATAAGATCGACAAATTAGATGATGTC CCGGATGCTACTATTGGC TTAAAAGCCATT GGCGGCTGGGCCACATTCACGCTCCAGGGTCGCA GACATCAATTATACCCAA TCTGAGAACGCT ATATTACTTCAGGAAATCATGAGCATACTGGTAT GGTGATAATGGGACGTTA CCCATGCCGGAG TGCGTACATTATCTCGGGTAGCAAACGTGCGGAC AGCAATGGCAAATACAGT GAATCAAAAAAT GTTAACATCACAAAACCTGAATCCGGAACAACGT GTGAAAAAGACTACCGAG GGTAGCTTTATT CTGTGTTTTACTACAAGACGGGTTCGATGTACAC ATTACCTTCGGGAACTTC TTTAACATCGAC CAATGACACAAATCATGTGAATTGGTGGCTGCTG CCGCATGCTGGTGAGTAT GGTAATGATAAA GTTAACCCGAGCAAAGTATACTCTGAGAAAAATG GATTATAACGTCAAAGAA CAGTTTACTATT TCTATATTCAGGATGAAATTCAAGGCGGTCAGAC ACCAATGAAGGCGTGGGT CCGCTGGCGTAC CCTGGAGCCGGACAGTTTTGAAATCGTCGTTACA GGCATTACTTACGATACG ACTCACGGTGGC TGGTACGATGGTTATGTGGAAAAATTTAAAGGTA AAAGAATATAAAGTTCAT GTCTACATCTAT AAGAAGCGATCCGGGAGTTCCACAATAAATATCC GTGTATGTGGCCAACTCA CAAATCCAGCAA GAATAGTAATATCTCGGTCAGTGAAAATAAAATC AATGCGATGGACGGTAAG ATTACCCAGAGC ACGGTAAATATTTCGCAAGAAGATTCCACCCAAA ACATATGTTAAAGCGATT AAGGATAACTAC AATTCATTAACATCTTTTACAAGACTAAAATCAC ACTAGCGAAAATGGCGGG ATCTACGATAAA CAACCCGAAGCAGAAAGAATTTGTAAACAACACC GAAAAAGCACCGATCGAA AACAGCTATAAA AAAGCCTGGTTCAAAGAGTACAATAAGCCGGCGG TTCGTTAACACCTATAAA ATCACGGTATAT TTAACGGTGAAAGTTTTAATCACAGTGTGCAGAA AAAGATACGTCGTTACTG GTCAAGAACGCA TATCAACGCAGATGCCGGGGTAAATGGTACTGTT ATTGAAAAAAATGTAATT GAAAACAATCAT AAAGGTGAATTGAAAATTATCAAAACCCTGAAAG GGCGATCTGGCAGACCTC CTGATCCCGCAG ATAAAAGTATTCCGATCAAGGATGTGCAGTTTAA ACCAAACAGTTTGAGTTT ATTATTGTAAAA GATGCGCCGCGTGGATAATACCGTTATTAAAGAC CAAATCAACTTGAAAAAG AATGAGAACAAT GGCAAGAAAGAGCTGCTGTTGACCACAGATGATA AGCGCGACTAGTGATATT GAAAAATGTGAA AAGGGATTGCAAACGTGAAAGGTCTGCCAGTCGG ACCAAGTTTGAAGGTAAC GAAATCTGCTTC GAAATACGAAGTCAAAGAAATCAGTGCGCCTGAG ATTATTCGCAAAGACGGT TACAATATCTAC TGGATCGCCTTCAATCCACTGATTGCGCCCAAAC AAGATTGAACCCGTGACC AAACAGAAAAAC TTGAATTTACGATCAGCGATCAAGACACAGAGGG TATACCGCGGAAAATACC AAGATCAATGAG GAAATTATGGGCAGTGGAAAACGAACTCAAAACC GAGACCTTTAAGTTAGCC ATCTCTAAAACC ATCTCGATTCCGGTCGAAAAAGTCTGGGTAGGTC AACGGAGACAAGTTAAAA CCCTATAAGCCG AGACGAGTGAACGGGCGGAGATCAAACTGTTTGC TTCGAGTCCATCCCCGCC AATGGTATTAAT GGATGGAATTGAAGTTGATAAGGTGATCCTGAAC GGTACAAAATATGAAGTC GTCCCGAAAACG GCGGATAATAATTGGAAGCACACCTTTGAGAATA AAGGAAATCGGGGCGAGC (SEQ ID NO: 9) AACCCGAATATAACTCCGAGACTAAACAAAAAAT GATGGGTACACGCCCTCA CAACTATAGTGTGAGCGAAACTACCATCAGTGGC ATCACCGTTATCGAAAAT TATGAATCAAATATTACTGGCGATGCGAAAAACG GGCAACGAAACCTCAAAT GATTTATTGTCACCAACACAGAACTGCCTGATTT AACCGCACTGTAGCCGAA GACGATCGGGAAAGAGGTAATCGGCGAACTCGGC AAAGATGGAATCTCTAGC GATAAAACCAAGGTATTCAACTTTGAACTGACAC AAAAGCAACTCGAACGAC TTAAGCAGGCTGACGGAAAGCCCATTAACGGGAA AATTTAATCGGCGAAGGC ATTTAACTATATTGGTTCGGTGGATGATCGTTAT GAAAATAAAGTGACCTTT AAGAAGGAATCGATTAAGCCTAGCGATGGGGAAA ACCAATACGTACAACGAT TTACGTTCATCGAGGGAAAAGCAACGATTACCCT AAACCAATCACG CTCCCACGGACAAGAGATCACCATTAAGGACCTT (SEQ ID NO: 8) CCGTATGGTGTGACCTATAAAGTCATGGAAAAAG AAGCCAACGAGAATGGATATTTAACCACTTACAA CGGAAATAACGAAGTCACCACCGGGGAGTTGAAA CAGGATACGAAAGTACAAGTGGTTAATAATAAAG AATTCGTCCCGACAACC (SEQ ID NO: 7) Full-length MKINKKIFSMLFMVIVLFTCISSNFSVSASSIQR MINKKKLSALLLSGAMFM METKKIRNKILM polypeptide GRDISNEVVTSLVATPNSINDGGNVQVRLEFKEN SMNTNVFASNLPSGGVEG AIVALSFILLPN HQRNIQSGDTITVKWTNSGEVFFEGYEKTIPLYI TEQNPAKATITKNEEFPE TRVYATENTANI KDQNVGQAVIEKTGATLTENDKIDKLDDVGGWAT GINTPSATFKFTAEKITN PLIVRQEFNVYT FTLQGRNITSGNHEHTGIAYIISGSKRADVNITK DAPDATIGDINYTQGDNG KDSKAIDMIGKY PESGTTSVFYYKTGSMYTNDTNHVNWWLLVNPSK TLSNGKYSVKKTTEITFG ELKAISENAPMP VYSEKNVYIQDEIQGGQTLEPDSFEIVVTWYDGY NFPHAGEYDYNVKETNEG EESKNGSFIFNI VEKFKGKEAIREFHNKYPNSNISVSENKITVNIS VGGITYDTKEYKVHVYVA DGNDKQFTIPLA QEDSTQKFINIFYKTKITNPKQKEFVNNTKAWFK NSNAMDGKTYVKAITSEN YTHGGVYIYQIQ EYNKPAVNGESFNHSVQNINADAGVNGTVKGELK GGEKAPIEFVNTYKKDTS QITQSKDNYIYD IIKTLKDKSIPIKDVQFKMRRVDNTVIKDGKKEL LLIEKNVIGDLADLTKQF KNSYKITVYVKN LETTDDKGIANVKGLPVGKYEVKFISAPEWIAFN EFQINEKKSATSDITKFE AENNHLIPQIIV PLIAPKLEFTISDQDTEGKLWAVENELKTISIPV GNIIRKDGKIEPVTYTAE KNENNEKCEEIC EKVWVGQTSERAEIKLFADGIEVDKVILNADNNW NTETFKLANGDKLKFESI FYNIYKQKNKIN KHTFENKPEYNSETKQKINYSVSETTISGYESNI PAGTKYEVKEIGASDGYT EISKTPYKPNGI TGDAKNGFIVTNTELPDLTIGKEVIGELGDKTKV PSITVIENGNETSNNRTV NVPKTGDTTNIG FNFELTLKQADGKPINGKFNYIGSVDDRYKKESI AEKDGISSKSNSNDNLIG FYIVILIISLGL KPSDGEITFIEGKATITLSHGQEITIKDLPYGVT EGENKVTFTNTYNDKPIT LVVLKWKEYKKR YKVMEKEANENGYLTTYNGNNEVTTGELKQDTKV GIVMNNIPFILMISFAVL KKE QVVNNKEFVPTTGISTTTEQGTMVGMVIFSIGIL GFGALAIIKRRKTIR (SEQ ID NO: 12) MVMIVVLLQLNKGLKR (SEQ ID NO: 10) (SEQ ID NO: 11) Expressed MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGS MGSSHHHHHHSSGLVPRG MGSSHHHHHHSS truncated EFSSIQRGRDISNEVVTSLVATPNSINDGGNVQV SHMASMTGGQQMGRGSEF GLVPRGSHMASM polypeptide RLEFKENHQRNIQSGDTITVKWTNSGEVFEEGYE SNLPSGGVEGTEQNPAKA TGGQQMGRGSEF KTIPLYIKDQNVGQAVIEKTGATLTENDKIDKLD TITKNFEFPEGINTPSAT TENTANIPLIVR DVGGWATFTLQGRNITSGNHEHTGIAYIISGSKR FKETAEKITNDAPDATIG QEFNVYTKDSKA ADVNITKPESGTTSVFYYKTGSMYTNDTNHVNWW DINYTQGDNGTLSNGKYS IDMIGKYELKAI LLVNPSKVYSEKNVYIQDEIQGGQTLEPDSFEIV VKKTTEITEGNFPHAGEY SENAPMPEESKN VTWYDGYVEKFKGKEAIREFHNKYPNSNISVSEN DYNVKETNEGVGGITYDT GSFIFNIDGNDK KITVNISQEDSTQKFINIFYKTKITNPKQKEFVN KEYKVHVYVANSNAMDGK QFTIPLAYTHGG NTKAWEKEYNKPAVNGESENHSVQNINADAGVNG TYVKAITSENGGEKAPIE VYIYQIQQITQS TVKGELKIIKTLKDKSIPIKDVQFKMRRVDNTVI FVNTYKKDTSLLIEKNVI KDNYIYDKNSYK KDGKKELLLTTDDKGIANVKGLPVGKYEVKEISA GDLADLTKQFEFQINLKK ITVYVKNAENNH PEWIAFNPLIAPKLEFTISDQDTEGKLWAVENEL SATSDITKFEGNIIRKDG LIPQIIVKNENN KTISIPVEKVWVGQTSERAEIKLEADGIEVDKVI KIEPVTYTAENTETFKLA EKCEEICEYNIY LNADNNWKHTFENKPEYNSETKQKINYSVSETTI NGDKLKFESIPAGTKYEV KQKNKINEISKT SGYESNITGDAKNGFIVTNTELPDLTIGKEVIGE KEIGASDGYTPSITVIEN PYKPNGINVPKT LGDKTKVFNEELTLKQADGKPINGKENYIGSVDD GNETSNNRTVAEKDGISS VDKLAAALEHHH RYKKESIKPSDGEITFIEGKATITLSHGQEITIK KSNSNDNLIGEGENKVTF HHH DLPYGVTYKVMEKEANENGYLTTYNGNNEVTTGE TNTYNDKPITVDKLAAAL (SEQ ID NO: 15) LKQDTKVQVVNNKEFVPTTVDKLAAALEHHHHHH EHHHHHH (SEQ ID NO: 13) (SEQ ID NO: 14)

Example 2: Preparation of Clostridium perfringens Strain CP1 Pilus Subunit Null-Mutants

[0073] The three pilus subunit genes (cnaA, fimA and fimB) were each insertionally inactivated in the virulent Clostridium perfringens strain CP1 by ClosTron mutagenesis (Heap, J. T., et al, Methods Mol. Biol. (2010), 646: 165-182), essentially as described previously (Yu, Q., Lepp, D., Mehdizadeh Gohari, I., Wu, T., Zhou, H., Yin, X., Yu, H., Prescott, J. F., Nie, S. P., Xie, M. Y., Gong, J., 2017. The Agr-like quorum sensing system is required for necrotic enteritis pathogenesis in poultry caused by Clostridium perfringens. Infection and Immunity 85(6): e00975-16), to generate CP1 null-mutants for each of the pilus subunit genes (CP1.DELTA.cnaA, CP1.DELTA.fimA, and CP1.DELTA.fimB). Briefly, ClosTron intron-targeting regions were designed to insert at the following gene positions using the Perutka algorithm implemented at www.clostron.com: base-pair (bp) 183 of the cnaA sense strand, bp 231 of the fimA sense strand, and bp 273 of the fimB sense strand. The intron-targeting regions were synthesized and cloned into ClosTron plasmid pMTL007C-E2 by DNA 2.0 (Menlo Park, Calif., USA). The resultant plasmids were separately electroporated into CP1 as described previously with minor modifications (Jiraskova A, Vitek L, Fevery J, Ruml T, Branny P. 2005. Rapid protocol for electroporation of Clostridium perfringens. J Microbiol Methods 62:125-127). Briefly, after growth at 37.degree. C. anaerobically overnight in 5 ml TGY broth (3% tryptone, 2% glucose, 1% yeast extract), CP1 was subcultured into 50 ml TGY and grown to exponential phase (optical density at 600 nm [OD 600], 0.8). The cells were harvested by centrifugation at 6,000 g for 10 min at 20.degree. C. and washed once in 10 ml sucrose electroporation buffer (SEB) (272 mM sucrose, 1 mM MgCl.sub.2, 5 mM Na.sub.2HPO.sub.4, pH 7.4) and then resuspended in 5 ml SEB. Aliquots (0.2 ml) were mixed with 2 .mu.g concentrated plasmid DNA in prechilled cuvettes (0.2-cm gap), and plasmid DNA was introduced into the cells by electroporation (1,000 V, 25F) using a Bio-Rad GenePulser Xcell apparatus (Bio-Rad, Hercules, Calif., USA). Immediately after transformation, the mixture was transferred into 1 ml of TGY broth and incubated anaerobically at 37.degree. C. for 3 h, followed by plating onto TGY agar containing 15 .mu.g/ml thiamphenicol anaerobically at 37.degree. C. overnight for selecting transformants. The resulting colonies were subcultured onto TGY agar containing 10 .mu.g/ml erythromycin for selecting integrants and then passaged for 10 consecutive days to cure the shuttle vector. Those clones resistant to erythromycin but sensitive to thiamphenicol were chosen for further analysis.

Example 3: Animal Trials

[0074] Two vaccination trials were carried out to assess the ability of the three purified His-tagged recombinant pilus subunits to protect against necrotic enteritis (NE) in a chicken challenge model. Commercial day-old male White Plymouth Rock broiler chickens were randomly divided into experimental groups (n=15-17) and housed in separate rooms within an isolation unit. A summary of the trial designs is shown in Table 2. In addition, the CP1 .DELTA.fimA, and CP1 .DELTA.fimB mutants were assessed for virulence in the same model.

TABLE-US-00002 TABLE 2 Summary of vaccination trial designs Vacci- Injec- Serum CP1 Day of nation tion collection challenge eutha- Trial Antigens tested days site days days nasia 1 CnaA, FimA 8, 20 i.m. 8, 20, 31 28, 29 31 2 CnaA, FimB, 7, 14, 19 s.c. 7, 19, 29 26, 27 29 CnaA + FimA + FimB

[0075] Trial 1:

[0076] The first trial included three groups of 18 birds vaccinated with either adjuvant-only control, CnaA or FimA. Each bird was injected intramuscularly (i.m.) in the pectoral muscle with 200 .mu.l phosphate-buffered saline (PBS) containing Quil-A.TM. adjuvant (50 .mu.g) and recombinant pilus polypeptide (50 .mu.g) at days 8 and 20, and birds were euthanized on day 31.

[0077] Serum was collected from five birds from each group at days 8 (prior to immunization), and at days 20 and 31 (after immunization). Serum IgY titres against CnaA and FimA were determined by ELISA (enzyme-linked immunosorbent assay). C. perfringens recombinant pilus polypeptides were diluted to 10 .mu.g/ml in 50 mM carbonate/bicarbonate coating buffer at pH 9.6, and 100 .mu.l was added to each well of a 96-well MaxiSorp.TM. Immuno plate (Fisher Scientific). Wells were coated for 1 h at 37.degree. C., followed by overnight at 4.degree. C., washed three times with wash buffer (PBS containing 0.05% TWEEN 20), and then blocked in wash buffer containing 1% bovine serum albumin (BSA) (Sigma) for 2 h at 37.degree. C. Two-fold serial dilutions of each serum sample diluted in wash buffer containing 1% BSA (1/64 to 1/65,536) were incubated in separate wells for 2 h at 37.degree. C. and then washed three times in wash buffer. Wells were incubated with goat anti-chicken IgY horseradish peroxidase (HRP)-conjugated polyclonal antibody, diluted 1:5,000 in wash buffer for 1 h at room temperature, and then washed three times in wash buffer. Substrate solution (0.2 mg/ml 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) (Sigma) in 1.times.ABTS buffer (Sigma)) was added to each well and incubated for 30 min at room temperature. After the reaction was stopped with 0.5% sodium dodecyl sulfate (SDS), the absorbance was measured in a BioTek.TM. plate reader at 405 nm. Titers were calculated as the log.sub.2 value of the lowest serum dilution with an absorbance greater than twice that of the background wells, in which PBS containing 1% BSA was used in place of serum. Statistical differences between pre-immune and post-immune titers for each antigen among the different vaccination groups were determined by one-way ANOVA followed by Tukey's post-hoc test.

[0078] The results are shown in FIGS. 3A and B, respectively. The average serum response against CnaA in the CnaA-immunized group was significantly higher at d31 compared to the pre-immune birds (d8), however the overall increase was small. In the FimA-immunized group, the average response against FimA did not significantly increase after immunization. However, two of the birds did exhibit a high titre by d31.

[0079] Birds were fed an antibiotic-free starter ration containing 20% protein until experimental induction of necrotic enteritis (NE). At day 27, birds were fasted for 24 h, and then switched to an antibiotic-free turkey starter ration (28% protein) containing C. perfringens CP1 culture at days 28 and 29 prior to euthanasia on day 31. The infected ration was prepared daily in the morning and afternoon by mixing with C. perfringens CP1 culture, grown in fluid thioglycollate (FTG) (Difco) medium at 37.degree. C. for 15 h or 24 h, respectively, at a 2:1 (v/w) ratio. Following euthanasia, the small intestine (duodenum to ileum) of the bird was examined grossly for necrotic enteritis lesions and scored blindly from 1 to 6 using the system described by Keyburn et al (Keyburn A L, Boyce J D, Vaz P, Bannam T L, Ford M E, Parker D, Di Rubbo A, Rood J I, Moore R J. 2008. NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens. PLoS Pathog. 4:e26) as follows:

[0080] 0, no gross lesions;

[0081] 1, thin or friable walls;

[0082] 2, focal necrosis or ulceration (1-5 foci);

[0083] 3, focal necrosis or ulceration (6-15 foci);

[0084] 4, focal necrosis or ulceration (16 or more foci);

[0085] 5, patches of necrosis 2-3 cm long;

[0086] 6, diffuse necrosis typical of field cases.

[0087] Statistical differences between necrotic enteritis (NE) scores among groups were determined by one-way ANOVA (analysis of variance) followed by Tukey's post-hoc test. The results, shown in FIG. 4, indicate that all groups had similarly high average lesion scores. The average necrotic enteritis scores for the adjuvant-only control, CnaA-immunized and FimA-immunized groups were 3.1, 3.0 and 3.3, respectively.

[0088] Without being bound by theory, it is contemplated that the immunization at day 8 may have been subject to interference from maternal antibodies, and there may not have been time for the immunization at day 20 to elicit sufficient immune response prior to challenge with C. perfringens CP1. Therefore, a second vaccination trial was carried out including an additional immunization prior to challenge with C. perfringens CP1.

[0089] Trial 2:

[0090] The second trial consisted of four groups of 18 birds vaccinated subcutaneously (s.c.) with either adjuvant-only control, CnaA, FimB or a combination of CnaA, FimA and FimB. In this trial, each bird was immunized subcutaneously with 50 .mu.g of recombinant polypeptide combined with 50 .mu.g of Quil-A.TM. adjuvant at days 7, 14 and 19, and serum was collected at days 7, 19 and 29 for measurement of antibody titres. Birds were challenged in-feed with Clostridium perfringens strain CP1 on days 26 and 27 as described for Trial 1, and on day 29, birds were euthanized and intestinal lesions were scored.

[0091] A significant (p<0.001) serum antibody (IgY) response was observed at both days 19 and 29 in all of the immunized groups compared to the pre-immune controls (with the exception of the group immunized with FimB at day 19), and the magnitude of response was also much greater than in Trial 1. The results are shown in FIGS. 5A (anti-CnaA serum response), 5B (anti-FimA serum response) and 5C (anti-FimB serum response).

[0092] In addition, as seen in FIG. 6, both the CnaA- and FimB-immunized groups had significantly lower necrotic enteritis scores (2 and 2.06, respectively) compared to the adjuvant control (3.75), when measured and scored as in Trial 1, indicating these antigens offered at least partial protection against necrotic enteritis. For the FimB antigen, the number of birds with severe disease (necrotic enteritis score >2) was 33.3% compared to 93.7% in the control. Immunization with the combined subunits did not appear to reduce the severity of disease (average necrotic enteritis score=3.7), despite eliciting a strong serum response against all three subunits, as seen in FIGS. 5A-C.

[0093] Challenge of Chickens with Clostridium perfringens Strain CP1 Pilus Subunit Null-Mutants

[0094] Three groups of 18 birds in Trial 2 which had not been immunized were challenged in-feed twice daily on days 26 and 27 with CP1, CP1.DELTA.fimA or CP1.DELTA.fimB prepared as described in Example 2. On day 29, the birds were euthanized and necrotic enteritis lesions were scored as described in Example 3. As seen from the results presented in FIG. 7, neither the CP1.DELTA.fimA nor the CP1.DELTA.fimB mutant strain caused disease in the challenged birds, indicating that a functional pilus appears to be required for necrotic enteritis pathogenesis.

Example 4: Characterization of Clostridium perfringens Pilus Surface Polypeptides

[0095] Clostridium perfringens Strain CP1 and CP1 Pilus Subunit Mutants:

[0096] Surface polypeptides were extracted from Clostridium perfringens strain CP1 and the pilus subunit mutants CP1.DELTA.cnaA, CP1.DELTA.fimA and CP1.DELTA.fimB described in Example 3, using the method of Chang, C., Huang, I.-H., Hendrickx, A. P. A., Ton-That, H. 2013. Visualization of Gram-positive Bacterial Pili, In: Delcour, H. A. (Ed.) Bacterial Cell Surfaces: Methods and Protocols. Humana Press, Totowa, N.J., 77-95. Strains were grown overnight in TGY medium (3% tryptone, 2% glucose, 1% yeast extract) anaerobically at 37.degree. C., subcultured 1:100 into 10 ml TGY medium and grown to an OD.sub.600.about.1. Cells were pelleted at 6,000.times.g for 5 min and washed once in SMM buffer, pH 6.8 (0.5M sucrose, 10 mM MgCl.sub.2, 10 mM maleate). The bacterial pellet was resuspended in 1 ml SMM buffer, to which was added 60 .mu.l of 5 U/.mu.l of mutanolysin (Sigma) in muramidase buffer (2 mM acetic acid, 48 mM sodium acetate) and 10 .mu.l of 0.1M phenylmethylsulfonyl fluoride (PMSF) (Sigma). Following at least 4 h incubation at 37.degree. C. with constant rotation, protoplasts were pelleted at 20,000.times.g for 5 min, and the supernatant fraction containing cell wall proteins was removed. Proteins were precipitated by addition of 81 .mu.l 100% (w/v) trichloroacetic acid (TCA) (Sigma) per ml and incubation at 4.degree. C. overnight. Following centrifugation at 20,000.times.g at 4.degree. C. for 20 min, the protein pellet was washed with acetone and slowly resuspended in 50 .mu.l sample loading buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 20% glycerol, 4% .beta.-mercaptoethanol, 3M urea, 0.01% bromophenol blue) at room temperature for at least 15 min.

[0097] Surface protein extracts (5 .mu.l) were loaded onto Novex.TM. NuPAGE.TM. 3-8% Tris-Acetate gels (Fisher Scientific) and electrophoresed at 150V for 1 h. Gels were stained with Bio-Safe.TM. COOMASSIE stain (BioRad), or were transferred onto a polyvinylene difluoride (PVDF) membrane at 350V for 1 h in 1.times. transfer buffer (48 mM Tris, 39 mM glycine, 20% methanol, 0.1% SDS). Chemiluminescent detection was performed with the WesternBreeze.TM. Chemiluminescent kit (Life Technologies) according to the manufacturer's instructions, using chicken anti-FimA serum (1:200) as primary antibody, and a goat anti-chicken IgY alkaline phosphatase (AP)-conjugated secondary antibody (1:2,000). The serum used as primary Ab was obtained at sacrifice from a FimA-immunized chicken from Trial 1 (Example 3) that subsequently exhibited a high anti-FimA titer or polyclonal antibodies raised in rabbits against the recombinant pilus polypeptides described in Example 1. The results are shown in FIGS. 8A-C.

[0098] It is known that Western blot analysis of SDS-PAGE separated sortase-dependent pili can produce a high-molecular weight (HMW) ladder-like pattern reflecting different polymer lengths, reflective of the mechanism by which the pilus is assembled at the cell surface. Pilin subunits are covalently linked by housekeeping and pilin-specific sortase enzymes, resulting in a growing heteropolymeric structure, which is eventually covalently linked to the cell wall peptidoglycan. The termination of assembly, and hence polymer length, is variable, giving rise to a characteristic high molecular weight ladder-like pattern when these pili are visualized by Western blotting. As seen in FIGS. 8B and C, a ladder-like pattern indicative of a pilus structure was observed in a Western blot of surface polypeptides extracted from Clostridium perfringens strain CP1 but not in a corresponding Western blot of surface polypeptides extracted from the mutant strains, whether visualized with antibodies obtained from chicken serum or raised in rabbits.

[0099] Various Clostridium perfringens Strains:

[0100] Extraction of surface polypeptides from five C. perfringens isolates that originated from poultry (CP1, JGS4141 and JGS4120) or non-poultry (Strain 13, ATCC13124) sources was performed as described above. Surface protein extracts (5 .mu.l) were loaded onto two Novex.TM. NuPAGE.TM. 3-8% Tris-Acetate gels (Fisher Scientific) and electrophoresed at 150V for 1 h. One gel was used for staining with Bio-Safe.TM. COOMASSIE stain (BioRad), and the second gel was transferred onto a polyvinylene difluoride (PVDF) membrane at 350V for 1 h in 1.times. transfer buffer (48 mM Tris, 39 mM glycine, 20% methanol, 0.1% SDS). Chemiluminescent detection was performed with the WesternBreeze.TM. Chemiluminescent kit (Life Technologies) according to the manufacturer's instructions, using chicken anti-FimA serum (1:200) as primary antibody, and a goat anti-chicken IgY alkaline phosphatase (AP)-conjugated secondary antibody (1:2,000). The serum used as primary Ab was obtained at sacrifice from a FimA-immunized chicken that subsequently exhibited a high anti-FimA titer.

[0101] The results are shown in FIGS. 9A-B. The presence (+) or absence (-) of the genetic locus (VR-10B (CA) locus) for the pilus subunit genes cnaA, fimA and fimB in each Clostridium perfringens strain had previously been determined by both microarray analysis and polymerase chain reaction (PCR) methodology (Lepp D et al, Journal of Bacteriology (2013) 195: 1152-1166). As seen in FIGS. 9A-B, strains which carry the pilus genetic locus in their genomes (JGS4141 and CP1) showed the characteristic ladder-like pattern of a pilus structure in extracted surface polypeptides (indicated by the vertical line to the right of the gel image in FIG. 9B), when the Western blot was visualized with chicken anti-FimA antibody, while other strains which do not carry the pilus genetic locus in their genome do not show this pattern. Visualization of smaller molecular weight bands in the extracts are likely due to unrelated antibodies present in the crude chicken serum. None of the extracts showed a band corresponding to the FimA polypeptide itself, whose expected location is indicated by an arrow to the right of the gel image in FIG. 9B. This is not surprising, as surface-associated proteins would not be expected to include the FimA monomer, which is only found within cells.

[0102] Immunogold Labeling of Clostridium perfringens Strain CP1 and CP1 Pilus Subunit Mutants:

[0103] Cells of Clostridium perfringens strain CP1 or of the CP1 null mutants CP1.DELTA.fimA, and CP1.DELTA.fimB were labeled with gold particles using an immunogold technique including rabbit anti-FimA as a primary antibody and 6 nm Colloidal Gold-AffiniPure.TM. Goat Anti-Rabbit IgG (H+L) (min X Hu,Ms,Rat Sr Prot) (Cedarlane) as secondary antibody, and examined by transmission electron microscopy, essentially as described previously (Chang, C., Huang, I.-H., Hendrickx, A. P. A., Ton-That, H. 2013. Visualization of Gram-positive Bacterial Pili, In: Delcour, H. A. (Ed.) Bacterial Cell Surfaces: Methods and Protocols. Humana Press, Totowa, N.J., 77-95). As seen in FIG. 10, cells of the native CP1 strain show the presence of a pilus structure on the cell surface, while cells of the CP1.DELTA.fimA, and CP1.DELTA.fimB mutants lack such structures.

[0104] The embodiments described herein are intended to be illustrative of the present compositions and methods and are not intended to limit the scope of the present invention. Various modifications and changes consistent with the description as a whole and which are readily apparent to the person of skill in the art are intended to be included. The appended claims should not be limited by the specific embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

Sequence CWU 1

1

1612091DNAClostridium perfringens 1atgaaaataa ataaaaaaat ttttagcatg ctatttatgg ttattgtact ttttacatgc 60atatcatcaa atttttctgt ttctgcttct tctattcaaa gaggaagaga tatcagtaat 120gaggtagtta caagcctagt ggctactcca aatagtataa atgatggtgg aaacgttcag 180gttcgtttgg aatttaaaga aaatcatcaa agaaatatac aaagtggaga tactataact 240gtcaaatgga caaattcagg ggaagtattt tttgaaggat atgaaaaaac aattccactt 300tatataaaag accaaaatgt tggtcaagca gtaatagaga aaacaggtgc aacacttaca 360tttaatgata aaatagataa attagatgat gttggtggat gggcaacatt tactttgcaa 420ggaagaaaca ttacctcagg taatcatgaa cacacaggaa tagcatatat tatatctggt 480tcaaagcggg cagatgtaaa tataaccaaa ccagaatcag gtacaactag tgtattctat 540tataaaacag gtagtatgta taccaatgat acaaatcatg tcaattggtg gttactggtg 600aatccaagca aggtatattc tgaaaaaaac gtttatattc aagatgaaat ccaaggcgga 660caaacattag aacctgattc ttttgaaata gtagtaactt ggtatgatgg ttatgtagaa 720aagtttaaag gaaaagaagc gataagggaa ttccataata aatatccaaa ttcaaatata 780tcggtatcag aaaataaaat aacagtaaac atttcacaag aggattccac acaaaagttt 840attaatattt tttataaaac taagattaca aatccgaaac aaaaagaatt cgttaataat 900acaaaagcat ggtttaaaga gtataataag ccagctgtaa atggagaatc ctttaaccat 960agcgtacaaa atattaatgc agatgctgga gttaatggaa ctgtaaaagg cgaattaaaa 1020atcataaaaa cattaaaaga taaaagtatt ccaattaaag atgttcagtt taagatgaga 1080agagttgata atacagttat caaagatggt aaaaaagaat tattactaac aactgatgat 1140aaaggtattg caaatgtaaa aggtcttcct gtaggaaaat atgaagtaaa agagatttca 1200gctccagaat ggattgcttt taatcctctt attgcaccaa aattggaatt cacaatatca 1260gatcaggaca cagaaggcaa attgtgggct gttgaaaatg aattaaagac aatttcaatt 1320ccggttgaaa aggtctgggt aggacaaact agtgaacgag cagaaatcaa gctttttgca 1380gatggtattg aagtagacaa agtgatttta aatgcagata acaattggaa acacacattt 1440gaaaataaac ctgaatataa ttcagaaaca aaacagaaaa tcaattattc tgtgtcagag 1500acaactattt ctggatatga aagcaatatc acaggcgatg ctaagaatgg ttttattgta 1560accaatacag aacttcctga tttgactatt ggtaaagaag ttataggaga attgggtgac 1620aagacgaagg tatttaactt tgagcttaca ttaaagcaag cagatggaaa gcctatcaat 1680ggtaaattta attacattgg tagtgtagat gacaggtaca aaaaagaaag cataaagcct 1740tctgatggtg agattacttt tatagaagga aaagcaacta taactttatc acatggacaa 1800gagattacaa tcaaggattt accatatggg gttacatata aagttatgga aaaagaagct 1860aatgaaaatg gctatttaac tacctataat ggaaataacg aagtcacaac aggtgaattg 1920aaacaggata caaaagtaca ggtagttaac aacaaagagt ttgttccaac aactggtata 1980tcaaccacaa cagagcaagg tacaatggtt ggaatggtga ttttttctat aggaatactt 2040atggtcatga ttgtagttct tttacaattg aataaaggac tgaaaagatg a 209121074DNAClostridium perfringens 2atgataaaca agaaaaaatt aagtgcatta ttattaagtg gagcaatgtt tatgagtatg 60aatacaaatg tattcgcatc aaatttacct tctggagggg tagaaggtac agaacagaat 120cctgcaaaag caacaattac aaagaatttt gaatttccag aaggtattaa tacacctagt 180gcaacattca agtttacagc agaaaaaata actaatgatg cgccagatgc aacaattgga 240gatattaatt atacacaagg ggataatgga actttatcaa atggaaaata tagtgtaaag 300aaaacaactg aaattacttt tggaaatttc ccacatgcag gagaatatga ttataatgta 360aaagaaacga atgagggagt aggtggtatt acatatgata caaaagaata caaagttcat 420gtgtatgttg caaatagtaa cgctatggat ggaaaaactt atgtaaaagc cattacatca 480gaaaatggag gtgaaaaagc tccaattgag tttgttaata catataaaaa ggacacttct 540ttacttatag aaaagaatgt aataggagat ttagctgact taacaaaaca gtttgagttt 600cagattaatt taaaaaaatc agcaacatct gacataacaa aattcgaagg aaatattatt 660agaaaagatg gtaaaataga gcctgtaaca tatacagctg aaaatacaga aacttttaaa 720ttagcaaatg gagataaact taagtttgaa agtattccag caggaacaaa atatgaagta 780aaagagatag gtgctagtga tggatataca ccttctataa cagtaattga aaatggaaat 840gagacttcta ataatcgtac ggtagctgaa aaagatggta tatcatctaa gtcaaattct 900aatgataact taattggtga aggtgaaaac aaagtaacat ttacaaacac atataatgac 960aaacctatca caggtattgt tatgaataat attccattta ttctaatgat tagttttgct 1020gtccttggat ttggtgcttt agctattata aaaagacgta aaactataag ataa 10743660DNAClostridium perfringens 3atggaaacaa agaaaataag aaacaaaatc cttatggcta tcgtagcatt gagctttata 60ttgcttccaa acactagagt atatgctact gaaaatacag caaatattcc tttgatagtt 120agacaggaat ttaatgtata tacgaaagat tcaaaagcaa tagacatgat tggaaaatac 180gagctaaagg caataagtga aaatgcccct atgccagaag aaagtaaaaa tggaagtttt 240atctttaata tagatggaaa tgataagcag tttactattc cattagctta tacacatggt 300ggtgtgtata tctatcaaat tcaacagata acgcaatcta aagataatta catatatgat 360aaaaatagct ataagataac tgtatatgta aaaaatgcag aaaataatca tttaatacca 420caaattattg tgaaaaatga aaataatgaa aaatgtgaag aaatatgttt ttataacatt 480tacaaacaaa aaaataaaat taatgagatt tctaaaacac catataagcc aaatggaata 540aatgttccta aaacaggcga taccacaaac attggatttt atattgtaat acttataatt 600tcacttggat tacttgtggt attgaaatgg aaagaatata aaaagagaaa aaaagaataa 66042088DNAArtificial SequencecnaA codon optimized 4atgaagatca acaagaagat cttcagcatg ttatttatgg tcattgtgct gttcacctgt 60atcagctcta acttcagtgt gagcgcgtca agcatccagc gcggccggga catcagcaac 120gaggtggtga catcgctcgt agctaccccg aatagcatca acgatggtgg taacgtccaa 180gtgcgtctgg aatttaaaga gaatcaccag cggaacattc agtccggcga cacgattacg 240gtcaaatgga ctaactcagg tgaggtcttt tttgaaggct acgaaaaaac catcccgctg 300tatatcaagg atcagaacgt tggccaggcg gttattgaaa aaaccggtgc aacattaaca 360ttcaacgata agatcgacaa attagatgat gtcggcggct gggccacatt cacgctccag 420ggtcgcaata ttacttcagg aaatcatgag catactggta ttgcgtacat tatctcgggt 480agcaaacgtg cggacgttaa catcacaaaa cctgaatccg gaacaacgtc tgtgttttac 540tacaagacgg gttcgatgta caccaatgac acaaatcatg tgaattggtg gctgctggtt 600aacccgagca aagtatactc tgagaaaaat gtctatattc aggatgaaat tcaaggcggt 660cagaccctgg agccggacag ttttgaaatc gtcgttacat ggtacgatgg ttatgtggaa 720aaatttaaag gtaaagaagc gatccgggag ttccacaata aatatccgaa tagtaatatc 780tcggtcagtg aaaataaaat cacggtaaat atttcgcaag aagattccac ccaaaaattc 840attaacatct tttacaagac taaaatcacc aacccgaagc agaaagaatt tgtaaacaac 900accaaagcct ggttcaaaga gtacaataag ccggcggtta acggtgaaag ttttaatcac 960agtgtgcaga atatcaacgc agatgccggg gtaaatggta ctgttaaagg tgaattgaaa 1020attatcaaaa ccctgaaaga taaaagtatt ccgatcaagg atgtgcagtt taagatgcgc 1080cgcgtggata ataccgttat taaagacggc aagaaagagc tgctgttgac cacagatgat 1140aaagggattg caaacgtgaa aggtctgcca gtcgggaaat acgaagtcaa agaaatcagt 1200gcgcctgagt ggatcgcctt caatccactg attgcgccca aacttgaatt tacgatcagc 1260gatcaagaca cagaggggaa attatgggca gtggaaaacg aactcaaaac catctcgatt 1320ccggtcgaaa aagtctgggt aggtcagacg agtgaacggg cggagatcaa actgtttgcg 1380gatggaattg aagttgataa ggtgatcctg aacgcggata ataattggaa gcacaccttt 1440gagaataaac ccgaatataa ctccgagact aaacaaaaaa tcaactatag tgtgagcgaa 1500actaccatca gtggctatga atcaaatatt actggcgatg cgaaaaacgg atttattgtc 1560accaacacag aactgcctga tttgacgatc gggaaagagg taatcggcga actcggcgat 1620aaaaccaagg tattcaactt tgaactgaca cttaagcagg ctgacggaaa gcccattaac 1680gggaaattta actatattgg ttcggtggat gatcgttata agaaggaatc gattaagcct 1740agcgatgggg aaattacgtt catcgaggga aaagcaacga ttaccctctc ccacggacaa 1800gagatcacca ttaaggacct tccgtatggt gtgacctata aagtcatgga aaaagaagcc 1860aacgagaatg gatatttaac cacttacaac ggaaataacg aagtcaccac cggggagttg 1920aaacaggata cgaaagtaca agtggttaat aataaagaat tcgtcccgac aaccgggatc 1980agcaccacca ccgaacaggg aaccatggtc gggatggtga tctttagcat cggtattctc 2040atggtaatga ttgtcgttct gctgcagctg aataaaggac tgaaacgc 208851086DNAArtificial SequencefimA codon optimized 5atgattaata aaaagaaact gtcggcgctg ctcttaagcg gggccatgtt tatgagcatg 60aacacgaatg tgttcgcgtc taacctccca tcgggtggtg tggagggcac cgaacaaaac 120ccagcgaaag cgacaatcac gaaaaacttc gagtttccgg aaggtattaa tacacccagc 180gcgacattca aatttaccgc cgaaaaaatt accaacgatg cgccggatgc tactattggc 240gacatcaatt atacccaagg tgataatggg acgttaagca atggcaaata cagtgtgaaa 300aagactaccg agattacctt cgggaacttc ccgcatgctg gtgagtatga ttataacgtc 360aaagaaacca atgaaggcgt gggtggcatt acttacgata cgaaagaata taaagttcat 420gtgtatgtgg ccaactcaaa tgcgatggac ggtaagacat atgttaaagc gattactagc 480gaaaatggcg gggaaaaagc accgatcgaa ttcgttaaca cctataaaaa agatacgtcg 540ttactgattg aaaaaaatgt aattggcgat ctggcagacc tcaccaaaca gtttgagttt 600caaatcaact tgaaaaagag cgcgactagt gatattacca agtttgaagg taacattatt 660cgcaaagacg gtaagattga acccgtgacc tataccgcgg aaaataccga gacctttaag 720ttagccaacg gagacaagtt aaaattcgag tccatccccg ccggtacaaa atatgaagtc 780aaggaaatcg gggcgagcga tgggtacacg ccctcaatca ccgttatcga aaatggcaac 840gaaacctcaa ataaccgcac tgtagccgaa aaagatggaa tctctagcaa aagcaactcg 900aacgacaatt taatcggcga aggcgaaaat aaagtgacct ttaccaatac gtacaacgat 960aaaccaatca cgggaatcgt aatgaataat attccgttca ttcttatgat tagctttgcc 1020gttcttggct tcggtgcatt agcgatcatt aaacgccgca aaaccatccg ccccatcgat 1080acgcgt 10866624DNAArtificial SequencefimB codon optimized 6atggctattg ttgctttgtc atttatcctg ctcccgaata cccgggtcta tgcgacggag 60aacaccgcta atatcccgtt aattgtacgc caagaattta atgtttacac taaagattct 120aaagccattg acatgatcgg aaaatatgaa ttaaaagcca tttctgagaa cgctcccatg 180ccggaggaat caaaaaatgg tagctttatt tttaacatcg acggtaatga taaacagttt 240actattccgc tggcgtacac tcacggtggc gtctacatct atcaaatcca gcaaattacc 300cagagcaagg ataactacat ctacgataaa aacagctata aaatcacggt atatgtcaag 360aacgcagaaa acaatcatct gatcccgcag attattgtaa aaaatgagaa caatgaaaaa 420tgtgaagaaa tctgcttcta caatatctac aaacagaaaa acaagatcaa tgagatctct 480aaaaccccct ataagccgaa tggtattaat gtcccgaaaa cgggtgatac cacgaacatc 540ggattctaca ttgtgatctt gattatttcc ctgggcctgc tggtggtctt gaagtggaaa 600gaatataaaa aacgtaagaa ggaa 62471887DNAArtificial SequencecnaA truncated 7tcaagcatcc agcgcggccg ggacatcagc aacgaggtgg tgacatcgct cgtagctacc 60ccgaatagca tcaacgatgg tggtaacgtc caagtgcgtc tggaatttaa agagaatcac 120cagcggaaca ttcagtccgg cgacacgatt acggtcaaat ggactaactc aggtgaggtc 180ttttttgaag gctacgaaaa aaccatcccg ctgtatatca aggatcagaa cgttggccag 240gcggttattg aaaaaaccgg tgcaacatta acattcaacg ataagatcga caaattagat 300gatgtcggcg gctgggccac attcacgctc cagggtcgca atattacttc aggaaatcat 360gagcatactg gtattgcgta cattatctcg ggtagcaaac gtgcggacgt taacatcaca 420aaacctgaat ccggaacaac gtctgtgttt tactacaaga cgggttcgat gtacaccaat 480gacacaaatc atgtgaattg gtggctgctg gttaacccga gcaaagtata ctctgagaaa 540aatgtctata ttcaggatga aattcaaggc ggtcagaccc tggagccgga cagttttgaa 600atcgtcgtta catggtacga tggttatgtg gaaaaattta aaggtaaaga agcgatccgg 660gagttccaca ataaatatcc gaatagtaat atctcggtca gtgaaaataa aatcacggta 720aatatttcgc aagaagattc cacccaaaaa ttcattaaca tcttttacaa gactaaaatc 780accaacccga agcagaaaga atttgtaaac aacaccaaag cctggttcaa agagtacaat 840aagccggcgg ttaacggtga aagttttaat cacagtgtgc agaatatcaa cgcagatgcc 900ggggtaaatg gtactgttaa aggtgaattg aaaattatca aaaccctgaa agataaaagt 960attccgatca aggatgtgca gtttaagatg cgccgcgtgg ataataccgt tattaaagac 1020ggcaagaaag agctgctgtt gaccacagat gataaaggga ttgcaaacgt gaaaggtctg 1080ccagtcggga aatacgaagt caaagaaatc agtgcgcctg agtggatcgc cttcaatcca 1140ctgattgcgc ccaaacttga atttacgatc agcgatcaag acacagaggg gaaattatgg 1200gcagtggaaa acgaactcaa aaccatctcg attccggtcg aaaaagtctg ggtaggtcag 1260acgagtgaac gggcggagat caaactgttt gcggatggaa ttgaagttga taaggtgatc 1320ctgaacgcgg ataataattg gaagcacacc tttgagaata aacccgaata taactccgag 1380actaaacaaa aaatcaacta tagtgtgagc gaaactacca tcagtggcta tgaatcaaat 1440attactggcg atgcgaaaaa cggatttatt gtcaccaaca cagaactgcc tgatttgacg 1500atcgggaaag aggtaatcgg cgaactcggc gataaaacca aggtattcaa ctttgaactg 1560acacttaagc aggctgacgg aaagcccatt aacgggaaat ttaactatat tggttcggtg 1620gatgatcgtt ataagaagga atcgattaag cctagcgatg gggaaattac gttcatcgag 1680ggaaaagcaa cgattaccct ctcccacgga caagagatca ccattaagga ccttccgtat 1740ggtgtgacct ataaagtcat ggaaaaagaa gccaacgaga atggatattt aaccacttac 1800aacggaaata acgaagtcac caccggggag ttgaaacagg atacgaaagt acaagtggtt 1860aataataaag aattcgtccc gacaacc 18878894DNAArtificial SequencefimA truncated 8tctaacctcc catcgggtgg tgtggagggc accgaacaaa acccagcgaa agcgacaatc 60acgaaaaact tcgagtttcc ggaaggtatt aatacaccca gcgcgacatt caaatttacc 120gccgaaaaaa ttaccaacga tgcgccggat gctactattg gcgacatcaa ttatacccaa 180ggtgataatg ggacgttaag caatggcaaa tacagtgtga aaaagactac cgagattacc 240ttcgggaact tcccgcatgc tggtgagtat gattataacg tcaaagaaac caatgaaggc 300gtgggtggca ttacttacga tacgaaagaa tataaagttc atgtgtatgt ggccaactca 360aatgcgatgg acggtaagac atatgttaaa gcgattacta gcgaaaatgg cggggaaaaa 420gcaccgatcg aattcgttaa cacctataaa aaagatacgt cgttactgat tgaaaaaaat 480gtaattggcg atctggcaga cctcaccaaa cagtttgagt ttcaaatcaa cttgaaaaag 540agcgcgacta gtgatattac caagtttgaa ggtaacatta ttcgcaaaga cggtaagatt 600gaacccgtga cctataccgc ggaaaatacc gagaccttta agttagccaa cggagacaag 660ttaaaattcg agtccatccc cgccggtaca aaatatgaag tcaaggaaat cggggcgagc 720gatgggtaca cgccctcaat caccgttatc gaaaatggca acgaaacctc aaataaccgc 780actgtagccg aaaaagatgg aatctctagc aaaagcaact cgaacgacaa tttaatcggc 840gaaggcgaaa ataaagtgac ctttaccaat acgtacaacg ataaaccaat cacg 8949468DNAArtificial SequencefimB truncated 9acggagaaca ccgctaatat cccgttaatt gtacgccaag aatttaatgt ttacactaaa 60gattctaaag ccattgacat gatcggaaaa tatgaattaa aagccatttc tgagaacgct 120cccatgccgg aggaatcaaa aaatggtagc tttattttta acatcgacgg taatgataaa 180cagtttacta ttccgctggc gtacactcac ggtggcgtct acatctatca aatccagcaa 240attacccaga gcaaggataa ctacatctac gataaaaaca gctataaaat cacggtatat 300gtcaagaacg cagaaaacaa tcatctgatc ccgcagatta ttgtaaaaaa tgagaacaat 360gaaaaatgtg aagaaatctg cttctacaat atctacaaac agaaaaacaa gatcaatgag 420atctctaaaa ccccctataa gccgaatggt attaatgtcc cgaaaacg 46810696PRTClostridium perfringens 10Met Lys Ile Asn Lys Lys Ile Phe Ser Met Leu Phe Met Val Ile Val1 5 10 15Leu Phe Thr Cys Ile Ser Ser Asn Phe Ser Val Ser Ala Ser Ser Ile 20 25 30Gln Arg Gly Arg Asp Ile Ser Asn Glu Val Val Thr Ser Leu Val Ala 35 40 45Thr Pro Asn Ser Ile Asn Asp Gly Gly Asn Val Gln Val Arg Leu Glu 50 55 60Phe Lys Glu Asn His Gln Arg Asn Ile Gln Ser Gly Asp Thr Ile Thr65 70 75 80Val Lys Trp Thr Asn Ser Gly Glu Val Phe Phe Glu Gly Tyr Glu Lys 85 90 95Thr Ile Pro Leu Tyr Ile Lys Asp Gln Asn Val Gly Gln Ala Val Ile 100 105 110Glu Lys Thr Gly Ala Thr Leu Thr Phe Asn Asp Lys Ile Asp Lys Leu 115 120 125Asp Asp Val Gly Gly Trp Ala Thr Phe Thr Leu Gln Gly Arg Asn Ile 130 135 140Thr Ser Gly Asn His Glu His Thr Gly Ile Ala Tyr Ile Ile Ser Gly145 150 155 160Ser Lys Arg Ala Asp Val Asn Ile Thr Lys Pro Glu Ser Gly Thr Thr 165 170 175Ser Val Phe Tyr Tyr Lys Thr Gly Ser Met Tyr Thr Asn Asp Thr Asn 180 185 190His Val Asn Trp Trp Leu Leu Val Asn Pro Ser Lys Val Tyr Ser Glu 195 200 205Lys Asn Val Tyr Ile Gln Asp Glu Ile Gln Gly Gly Gln Thr Leu Glu 210 215 220Pro Asp Ser Phe Glu Ile Val Val Thr Trp Tyr Asp Gly Tyr Val Glu225 230 235 240Lys Phe Lys Gly Lys Glu Ala Ile Arg Glu Phe His Asn Lys Tyr Pro 245 250 255Asn Ser Asn Ile Ser Val Ser Glu Asn Lys Ile Thr Val Asn Ile Ser 260 265 270Gln Glu Asp Ser Thr Gln Lys Phe Ile Asn Ile Phe Tyr Lys Thr Lys 275 280 285Ile Thr Asn Pro Lys Gln Lys Glu Phe Val Asn Asn Thr Lys Ala Trp 290 295 300Phe Lys Glu Tyr Asn Lys Pro Ala Val Asn Gly Glu Ser Phe Asn His305 310 315 320Ser Val Gln Asn Ile Asn Ala Asp Ala Gly Val Asn Gly Thr Val Lys 325 330 335Gly Glu Leu Lys Ile Ile Lys Thr Leu Lys Asp Lys Ser Ile Pro Ile 340 345 350Lys Asp Val Gln Phe Lys Met Arg Arg Val Asp Asn Thr Val Ile Lys 355 360 365Asp Gly Lys Lys Glu Leu Leu Leu Thr Thr Asp Asp Lys Gly Ile Ala 370 375 380Asn Val Lys Gly Leu Pro Val Gly Lys Tyr Glu Val Lys Glu Ile Ser385 390 395 400Ala Pro Glu Trp Ile Ala Phe Asn Pro Leu Ile Ala Pro Lys Leu Glu 405 410 415Phe Thr Ile Ser Asp Gln Asp Thr Glu Gly Lys Leu Trp Ala Val Glu 420 425 430Asn Glu Leu Lys Thr Ile Ser Ile Pro Val Glu Lys Val Trp Val Gly 435 440 445Gln Thr Ser Glu Arg Ala Glu Ile Lys Leu Phe Ala Asp Gly Ile Glu 450 455 460Val Asp Lys Val Ile Leu Asn Ala Asp Asn Asn Trp Lys His Thr Phe465 470 475 480Glu Asn Lys Pro Glu Tyr Asn Ser Glu Thr Lys Gln Lys Ile Asn Tyr 485 490 495Ser Val Ser Glu Thr Thr Ile Ser Gly Tyr Glu Ser Asn Ile Thr Gly 500 505 510Asp Ala Lys Asn Gly Phe Ile Val Thr Asn Thr Glu Leu Pro Asp Leu 515 520 525Thr Ile Gly Lys Glu Val Ile Gly Glu Leu Gly Asp Lys Thr Lys Val 530 535 540Phe Asn Phe Glu Leu Thr Leu Lys Gln Ala Asp Gly Lys Pro Ile Asn545 550 555 560Gly Lys Phe Asn Tyr Ile Gly Ser Val Asp Asp Arg Tyr Lys Lys Glu 565 570 575Ser Ile Lys Pro Ser Asp Gly Glu Ile Thr Phe Ile Glu Gly Lys Ala 580 585 590Thr Ile Thr Leu Ser His Gly Gln Glu Ile Thr Ile

Lys Asp Leu Pro 595 600 605Tyr Gly Val Thr Tyr Lys Val Met Glu Lys Glu Ala Asn Glu Asn Gly 610 615 620Tyr Leu Thr Thr Tyr Asn Gly Asn Asn Glu Val Thr Thr Gly Glu Leu625 630 635 640Lys Gln Asp Thr Lys Val Gln Val Val Asn Asn Lys Glu Phe Val Pro 645 650 655Thr Thr Gly Ile Ser Thr Thr Thr Glu Gln Gly Thr Met Val Gly Met 660 665 670Val Ile Phe Ser Ile Gly Ile Leu Met Val Met Ile Val Val Leu Leu 675 680 685Gln Leu Asn Lys Gly Leu Lys Arg 690 69511357PRTClostridium perfringens 11Met Ile Asn Lys Lys Lys Leu Ser Ala Leu Leu Leu Ser Gly Ala Met1 5 10 15Phe Met Ser Met Asn Thr Asn Val Phe Ala Ser Asn Leu Pro Ser Gly 20 25 30Gly Val Glu Gly Thr Glu Gln Asn Pro Ala Lys Ala Thr Ile Thr Lys 35 40 45Asn Phe Glu Phe Pro Glu Gly Ile Asn Thr Pro Ser Ala Thr Phe Lys 50 55 60Phe Thr Ala Glu Lys Ile Thr Asn Asp Ala Pro Asp Ala Thr Ile Gly65 70 75 80Asp Ile Asn Tyr Thr Gln Gly Asp Asn Gly Thr Leu Ser Asn Gly Lys 85 90 95Tyr Ser Val Lys Lys Thr Thr Glu Ile Thr Phe Gly Asn Phe Pro His 100 105 110Ala Gly Glu Tyr Asp Tyr Asn Val Lys Glu Thr Asn Glu Gly Val Gly 115 120 125Gly Ile Thr Tyr Asp Thr Lys Glu Tyr Lys Val His Val Tyr Val Ala 130 135 140Asn Ser Asn Ala Met Asp Gly Lys Thr Tyr Val Lys Ala Ile Thr Ser145 150 155 160Glu Asn Gly Gly Glu Lys Ala Pro Ile Glu Phe Val Asn Thr Tyr Lys 165 170 175Lys Asp Thr Ser Leu Leu Ile Glu Lys Asn Val Ile Gly Asp Leu Ala 180 185 190Asp Leu Thr Lys Gln Phe Glu Phe Gln Ile Asn Leu Lys Lys Ser Ala 195 200 205Thr Ser Asp Ile Thr Lys Phe Glu Gly Asn Ile Ile Arg Lys Asp Gly 210 215 220Lys Ile Glu Pro Val Thr Tyr Thr Ala Glu Asn Thr Glu Thr Phe Lys225 230 235 240Leu Ala Asn Gly Asp Lys Leu Lys Phe Glu Ser Ile Pro Ala Gly Thr 245 250 255Lys Tyr Glu Val Lys Glu Ile Gly Ala Ser Asp Gly Tyr Thr Pro Ser 260 265 270Ile Thr Val Ile Glu Asn Gly Asn Glu Thr Ser Asn Asn Arg Thr Val 275 280 285Ala Glu Lys Asp Gly Ile Ser Ser Lys Ser Asn Ser Asn Asp Asn Leu 290 295 300Ile Gly Glu Gly Glu Asn Lys Val Thr Phe Thr Asn Thr Tyr Asn Asp305 310 315 320Lys Pro Ile Thr Gly Ile Val Met Asn Asn Ile Pro Phe Ile Leu Met 325 330 335Ile Ser Phe Ala Val Leu Gly Phe Gly Ala Leu Ala Ile Ile Lys Arg 340 345 350Arg Lys Thr Ile Arg 35512219PRTClostridium perfringens 12Met Glu Thr Lys Lys Ile Arg Asn Lys Ile Leu Met Ala Ile Val Ala1 5 10 15Leu Ser Phe Ile Leu Leu Pro Asn Thr Arg Val Tyr Ala Thr Glu Asn 20 25 30Thr Ala Asn Ile Pro Leu Ile Val Arg Gln Glu Phe Asn Val Tyr Thr 35 40 45Lys Asp Ser Lys Ala Ile Asp Met Ile Gly Lys Tyr Glu Leu Lys Ala 50 55 60Ile Ser Glu Asn Ala Pro Met Pro Glu Glu Ser Lys Asn Gly Ser Phe65 70 75 80Ile Phe Asn Ile Asp Gly Asn Asp Lys Gln Phe Thr Ile Pro Leu Ala 85 90 95Tyr Thr His Gly Gly Val Tyr Ile Tyr Gln Ile Gln Gln Ile Thr Gln 100 105 110Ser Lys Asp Asn Tyr Ile Tyr Asp Lys Asn Ser Tyr Lys Ile Thr Val 115 120 125Tyr Val Lys Asn Ala Glu Asn Asn His Leu Ile Pro Gln Ile Ile Val 130 135 140Lys Asn Glu Asn Asn Glu Lys Cys Glu Glu Ile Cys Phe Tyr Asn Ile145 150 155 160Tyr Lys Gln Lys Asn Lys Ile Asn Glu Ile Ser Lys Thr Pro Tyr Lys 165 170 175Pro Asn Gly Ile Asn Val Pro Lys Thr Gly Asp Thr Thr Asn Ile Gly 180 185 190Phe Tyr Ile Val Ile Leu Ile Ile Ser Leu Gly Leu Leu Val Val Leu 195 200 205Lys Trp Lys Glu Tyr Lys Lys Arg Lys Lys Glu 210 21513680PRTArtificial SequencecnaA expressed His-tagged 13Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro1 5 10 15Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30Gly Ser Glu Phe Ser Ser Ile Gln Arg Gly Arg Asp Ile Ser Asn Glu 35 40 45Val Val Thr Ser Leu Val Ala Thr Pro Asn Ser Ile Asn Asp Gly Gly 50 55 60Asn Val Gln Val Arg Leu Glu Phe Lys Glu Asn His Gln Arg Asn Ile65 70 75 80Gln Ser Gly Asp Thr Ile Thr Val Lys Trp Thr Asn Ser Gly Glu Val 85 90 95Phe Phe Glu Gly Tyr Glu Lys Thr Ile Pro Leu Tyr Ile Lys Asp Gln 100 105 110Asn Val Gly Gln Ala Val Ile Glu Lys Thr Gly Ala Thr Leu Thr Phe 115 120 125Asn Asp Lys Ile Asp Lys Leu Asp Asp Val Gly Gly Trp Ala Thr Phe 130 135 140Thr Leu Gln Gly Arg Asn Ile Thr Ser Gly Asn His Glu His Thr Gly145 150 155 160Ile Ala Tyr Ile Ile Ser Gly Ser Lys Arg Ala Asp Val Asn Ile Thr 165 170 175Lys Pro Glu Ser Gly Thr Thr Ser Val Phe Tyr Tyr Lys Thr Gly Ser 180 185 190Met Tyr Thr Asn Asp Thr Asn His Val Asn Trp Trp Leu Leu Val Asn 195 200 205Pro Ser Lys Val Tyr Ser Glu Lys Asn Val Tyr Ile Gln Asp Glu Ile 210 215 220Gln Gly Gly Gln Thr Leu Glu Pro Asp Ser Phe Glu Ile Val Val Thr225 230 235 240Trp Tyr Asp Gly Tyr Val Glu Lys Phe Lys Gly Lys Glu Ala Ile Arg 245 250 255Glu Phe His Asn Lys Tyr Pro Asn Ser Asn Ile Ser Val Ser Glu Asn 260 265 270Lys Ile Thr Val Asn Ile Ser Gln Glu Asp Ser Thr Gln Lys Phe Ile 275 280 285Asn Ile Phe Tyr Lys Thr Lys Ile Thr Asn Pro Lys Gln Lys Glu Phe 290 295 300Val Asn Asn Thr Lys Ala Trp Phe Lys Glu Tyr Asn Lys Pro Ala Val305 310 315 320Asn Gly Glu Ser Phe Asn His Ser Val Gln Asn Ile Asn Ala Asp Ala 325 330 335Gly Val Asn Gly Thr Val Lys Gly Glu Leu Lys Ile Ile Lys Thr Leu 340 345 350Lys Asp Lys Ser Ile Pro Ile Lys Asp Val Gln Phe Lys Met Arg Arg 355 360 365Val Asp Asn Thr Val Ile Lys Asp Gly Lys Lys Glu Leu Leu Leu Thr 370 375 380Thr Asp Asp Lys Gly Ile Ala Asn Val Lys Gly Leu Pro Val Gly Lys385 390 395 400Tyr Glu Val Lys Glu Ile Ser Ala Pro Glu Trp Ile Ala Phe Asn Pro 405 410 415Leu Ile Ala Pro Lys Leu Glu Phe Thr Ile Ser Asp Gln Asp Thr Glu 420 425 430Gly Lys Leu Trp Ala Val Glu Asn Glu Leu Lys Thr Ile Ser Ile Pro 435 440 445Val Glu Lys Val Trp Val Gly Gln Thr Ser Glu Arg Ala Glu Ile Lys 450 455 460Leu Phe Ala Asp Gly Ile Glu Val Asp Lys Val Ile Leu Asn Ala Asp465 470 475 480Asn Asn Trp Lys His Thr Phe Glu Asn Lys Pro Glu Tyr Asn Ser Glu 485 490 495Thr Lys Gln Lys Ile Asn Tyr Ser Val Ser Glu Thr Thr Ile Ser Gly 500 505 510Tyr Glu Ser Asn Ile Thr Gly Asp Ala Lys Asn Gly Phe Ile Val Thr 515 520 525Asn Thr Glu Leu Pro Asp Leu Thr Ile Gly Lys Glu Val Ile Gly Glu 530 535 540Leu Gly Asp Lys Thr Lys Val Phe Asn Phe Glu Leu Thr Leu Lys Gln545 550 555 560Ala Asp Gly Lys Pro Ile Asn Gly Lys Phe Asn Tyr Ile Gly Ser Val 565 570 575Asp Asp Arg Tyr Lys Lys Glu Ser Ile Lys Pro Ser Asp Gly Glu Ile 580 585 590Thr Phe Ile Glu Gly Lys Ala Thr Ile Thr Leu Ser His Gly Gln Glu 595 600 605Ile Thr Ile Lys Asp Leu Pro Tyr Gly Val Thr Tyr Lys Val Met Glu 610 615 620Lys Glu Ala Asn Glu Asn Gly Tyr Leu Thr Thr Tyr Asn Gly Asn Asn625 630 635 640Glu Val Thr Thr Gly Glu Leu Lys Gln Asp Thr Lys Val Gln Val Val 645 650 655Asn Asn Lys Glu Phe Val Pro Thr Thr Val Asp Lys Leu Ala Ala Ala 660 665 670Leu Glu His His His His His His 675 68014349PRTArtificial SequencefimA expressed His-tagged 14Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro1 5 10 15Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30Gly Ser Glu Phe Ser Asn Leu Pro Ser Gly Gly Val Glu Gly Thr Glu 35 40 45Gln Asn Pro Ala Lys Ala Thr Ile Thr Lys Asn Phe Glu Phe Pro Glu 50 55 60Gly Ile Asn Thr Pro Ser Ala Thr Phe Lys Phe Thr Ala Glu Lys Ile65 70 75 80Thr Asn Asp Ala Pro Asp Ala Thr Ile Gly Asp Ile Asn Tyr Thr Gln 85 90 95Gly Asp Asn Gly Thr Leu Ser Asn Gly Lys Tyr Ser Val Lys Lys Thr 100 105 110Thr Glu Ile Thr Phe Gly Asn Phe Pro His Ala Gly Glu Tyr Asp Tyr 115 120 125Asn Val Lys Glu Thr Asn Glu Gly Val Gly Gly Ile Thr Tyr Asp Thr 130 135 140Lys Glu Tyr Lys Val His Val Tyr Val Ala Asn Ser Asn Ala Met Asp145 150 155 160Gly Lys Thr Tyr Val Lys Ala Ile Thr Ser Glu Asn Gly Gly Glu Lys 165 170 175Ala Pro Ile Glu Phe Val Asn Thr Tyr Lys Lys Asp Thr Ser Leu Leu 180 185 190Ile Glu Lys Asn Val Ile Gly Asp Leu Ala Asp Leu Thr Lys Gln Phe 195 200 205Glu Phe Gln Ile Asn Leu Lys Lys Ser Ala Thr Ser Asp Ile Thr Lys 210 215 220Phe Glu Gly Asn Ile Ile Arg Lys Asp Gly Lys Ile Glu Pro Val Thr225 230 235 240Tyr Thr Ala Glu Asn Thr Glu Thr Phe Lys Leu Ala Asn Gly Asp Lys 245 250 255Leu Lys Phe Glu Ser Ile Pro Ala Gly Thr Lys Tyr Glu Val Lys Glu 260 265 270Ile Gly Ala Ser Asp Gly Tyr Thr Pro Ser Ile Thr Val Ile Glu Asn 275 280 285Gly Asn Glu Thr Ser Asn Asn Arg Thr Val Ala Glu Lys Asp Gly Ile 290 295 300Ser Ser Lys Ser Asn Ser Asn Asp Asn Leu Ile Gly Glu Gly Glu Asn305 310 315 320Lys Val Thr Phe Thr Asn Thr Tyr Asn Asp Lys Pro Ile Thr Val Asp 325 330 335Lys Leu Ala Ala Ala Leu Glu His His His His His His 340 34515207PRTArtificial SequencefimB expressed His-tagged 15Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro1 5 10 15Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30Gly Ser Glu Phe Thr Glu Asn Thr Ala Asn Ile Pro Leu Ile Val Arg 35 40 45Gln Glu Phe Asn Val Tyr Thr Lys Asp Ser Lys Ala Ile Asp Met Ile 50 55 60Gly Lys Tyr Glu Leu Lys Ala Ile Ser Glu Asn Ala Pro Met Pro Glu65 70 75 80Glu Ser Lys Asn Gly Ser Phe Ile Phe Asn Ile Asp Gly Asn Asp Lys 85 90 95Gln Phe Thr Ile Pro Leu Ala Tyr Thr His Gly Gly Val Tyr Ile Tyr 100 105 110Gln Ile Gln Gln Ile Thr Gln Ser Lys Asp Asn Tyr Ile Tyr Asp Lys 115 120 125Asn Ser Tyr Lys Ile Thr Val Tyr Val Lys Asn Ala Glu Asn Asn His 130 135 140Leu Ile Pro Gln Ile Ile Val Lys Asn Glu Asn Asn Glu Lys Cys Glu145 150 155 160Glu Ile Cys Phe Tyr Asn Ile Tyr Lys Gln Lys Asn Lys Ile Asn Glu 165 170 175Ile Ser Lys Thr Pro Tyr Lys Pro Asn Gly Ile Asn Val Pro Lys Thr 180 185 190Val Asp Lys Leu Ala Ala Ala Leu Glu His His His His His His 195 200 205165PRTArtificial SequenceLPXTG motifMISC_FEATURE(3)..(3)any amino acid 16Leu Pro Xaa Thr Gly1 5

Claims

1. A method of treating or preventing necrotic enteritis or Clostridium perfringens infection in poultry, the method comprising administering to the poultry an effective amount of an immunogenic polypeptide or an effective amount of a vaccine comprising the immunogenic polypeptide, wherein the immunogenic polypeptide is selected from the group consisting of an isolated Clostridium perfringens pilus polypeptide, a variant of the pilus polypeptide, a fragment of the pilus polypeptide, and a fragment of the variant of the pilus polypeptide; wherein the pilus polypeptide, the variant of the pilus polypeptide, the fragment of the pilus polypeptide and the fragment of the variant of the pilus polypeptide are immunogenic in poultry; wherein the pilus polypeptide is an assembled sortase-dependent pilus or a subunit thereof, and wherein the pilus polypeptide is encoded by one or more genes in the VR-10B genetic locus.

2. The method according to claim 1 wherein the variant of the pilus polypeptide has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or at least 99.9% sequence identity to the pilus polypeptide.

3. The method according to claim 1, wherein the subunit of the assembled sortase-dependent pilus is selected from the group consisting of a CnaA polypeptide, a FimA polypeptide, and a FimB polypeptide; and wherein the assembled sortase-dependent pilus comprises one or more subunits each individually selected from the CnaA polypeptide, the FimA polypeptide, and the FimB polypeptide.

4. The method according to claim 3, wherein the CnaA polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:10 and SEQ ID NO:13.

5. The method according to claim 3, wherein the CnaA polypeptide is encoded by a polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4 and SEQ ID NO:7.

6. The method according to claim 3, wherein the CnaA polypeptide is encoded by a polynucleotide which hybridizes under at least moderately stringent conditions to a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4 and SEQ ID NO:7.

7. The method according to claim 3, wherein the FimA polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:11 and SEQ ID NO:14.

8. The method according to claim 3, wherein the FimA polypeptide is encoded by a polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8.

9. The method according to claim 3, wherein the FimA polypeptide is encoded by a polynucleotide which hybridizes under at least moderately stringent conditions to a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8.

10. The method according to claim 3, wherein the FimB polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:12 and SEQ ID NO:15.

11. The method according to claim 3, wherein the FimB polypeptide is encoded by a polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:9.

12. The method according to claim 3, wherein the FimB polypeptide is encoded by a polynucleotide which hybridizes under at least moderately stringent conditions to a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:9.

13. A vaccine comprising an effective amount of an immunogenic polypeptide and an adjuvant, wherein the immunogenic polypeptide is selected from the group consisting of an isolated Clostridium perfringens pilus polypeptide, a variant of the pilus polypeptide, a fragment of the pilus polypeptide, and a fragment of the variant of the pilus polypeptide; wherein the pilus polypeptide, the variant of the pilus polypeptide, the fragment of the pilus polypeptide and the fragment of the variant of the pilus polypeptide are immunogenic in poultry; wherein the pilus polypeptide is an assembled sortase-dependent pilus or a subunit thereof, wherein the pilus polypeptide is encoded by one or more genes in the VR-10B genetic locus, and wherein the vaccine is effective to treat or prevent necrotic enteritis or Clostridium perfringens infection in poultry.

14. The vaccine according to claim 13 wherein the variant of the pilus polypeptide has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or at least 99.9% sequence identity to the pilus polypeptide.

15. The vaccine according to claim 13, wherein the subunit of the assembled sortase-dependent pilus is selected from the group consisting of a CnaA polypeptide, a FimA polypeptide, and a FimB polypeptide; and wherein the assembled sortase-dependent pilus comprises one or more subunits each individually selected from the CnaA polypeptide, the FimA polypeptide, and the FimB polypeptide.

16. The vaccine according to claim 15, wherein the CnaA polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:10 and SEQ ID NO:13.

17. The vaccine according to claim 15, wherein the CnaA polypeptide is encoded by a polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4 and SEQ ID NO:7.

18. The vaccine according to claim 15, wherein the CnaA polypeptide is encoded by a polynucleotide which hybridizes under at least moderately stringent conditions to a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4 and SEQ ID NO:7.

19. The vaccine according to claim 15, wherein the FimA polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:11 and SEQ ID NO:14.

20. The vaccine according to claim 15, wherein the FimA polypeptide is encoded by a polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8.

21. The vaccine according to claim 15, wherein the FimA polypeptide is encoded by a polynucleotide which hybridizes under at least moderately stringent conditions to a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8.

22. The vaccine according to claim 15, wherein the FimB polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:12 and SEQ ID NO:15.

23. The vaccine according to claim 15, wherein the FimB polypeptide is encoded by a polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:9.

24. The vaccine according to claim 15, wherein the FimB polypeptide is encoded by a polynucleotide which hybridizes under at least moderately stringent conditions to a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:9.

25. A method of treating or preventing necrotic enteritis or Clostridium perfringens infection in poultry, the method comprising administering to the poultry an effective amount of a vaccine according to claim 13.

26. A method of immunizing a subject, the method comprising administering an effective amount of a vaccine according to claim 13 to a subject.

27. The method of claim 26 wherein the subject is poultry.