Attenuated Mannheimia haemolytica Vaccines and Methods of Making and Use

Abstract

The present invention provides attenuated M. haemolytica strains that elicit an immune response in animals against M. haemolytica, compositions comprising said strains, methods of vaccination against M. haemolytica, and kits for use with such methods and compositions. The invention further provides multi-valent vaccines, which provide protective immunity when administered in an effective amount to animals susceptible to "shipping fever" or bovine respiratory disease.

Description

[0001] This application incorporates by reference the contents of a 61.8 kb text file created on Sep. 30, 2016 and named "00848800006sequencelisting.txt," which is the sequence listing for this application.

FIELD OF THE INVENTION

[0002] The present invention relates generally to attenuated bacterial vaccines, particularly those providing broad, safe, and effective protection to production animals against infections/disease caused by gram-negative bacteria, including Mannheimia (Pasteurella) haemolytica. The invention further relates to methods of producing the attenuated bacteria, and to PCR methods for differentiating among M. haemolytica serotypes A1 and A6, in vivo.

[0003] The invention accordingly relates to immunogenic or vaccine compositions comprising the bacteria of the invention; e.g., live attenuated bacteria. The bacteria also could be inactivated in the compositions, but it may be advantageous that the bacteria are live attenuated M. haemolytica bacteria, either alone, or combined with other bacteria such as Haemophilus somnus and/or Pasteurella multocida. The invention therefore further relates to methods for preparing and/or formulating such compositions; e.g., culturing or growing or propagating the bacteria on or in suitable medium, harvesting the bacteria, optionally inactivating the bacteria, and optionally admixing the bacteria with a suitable veterinarily or pharmaceutically acceptable carrier, excipient, diluent or vehicle and/or an adjuvant and/or stabilizer. Thus, the invention also relates to the use of the bacteria in formulating such compositions.

BACKGROUND OF THE INVENTION

[0004] M. haemolytica is a gram negative bacterium normally found in the upper respiratory tract of healthy cattle, sheep and wild sheep. M. haemolytica descends into the lungs when cattle experience stress such as shipping, weaning, overcrowding, or viral infections and causes fibrinous and necrotizing bronchopneumonia, a chief component of the bovine respiratory disease complex (BRDC). Economic losses due to BRDC in North America is >$1 billion annually (Bowland and Shewen, 2000). M. haemolytica is the bacterium most commonly isolated from the lungs of cattle affected with BRDC. M. haemolytica serotype AI is responsible for approximately 60% of shipping fever, whereas serotypes A6 and A2 account for 26% and 7% respectively (Al-Gharndi et al., 2000; Purdy et al., 1997). Both M. haemolytica A1 and A6 account for >85% of BRDC cases involving bacterial pathogens.

[0005] The vaccines currently available in the market against M. haemolytica infections are only moderately protective against shipping fever of beef cattle but generally ineffective against neonatal dairy calf pneumonia (Virtala et al., 1996; Rice et al., 2007). The major cause of severe bacterial pneumonia in feedlot and neonatal dairy cattle is M. haemolytica serotype A1 followed by serotype A6 (Schreuer et al., 2000, Rice et al., 2007).

[0006] Experimental evaluation of all the commercial M. haemolytica A1 vaccines used in feedlot showed only partial protection in 50% of the studies (Perino and Hunsaker, 1997). Furthermore, cross-protection against M. haemolytica serotypes (either A6 or A2) has been difficult to achieve using conventional vaccine preparations (Purdy et al., 1993; Sabri et al., 2000). Therefore, an efficacious vaccine against M. haemolytica serotypes A1 and A6 could significantly improve dairy/beef production.

[0007] Effective immunity against M. haemolytica is multifaceted. Neutralizing Antibodies against exotoxin leukotoxin A (LktA) and surface antigens are necessary for protective immunity against M. haemolytica (Shewen and Wilkie, 1988). Due to the complex genetic machinery involved in controlling the expression of various M. haemolytica virulence factors, the specific surface antigens that are important in stimulating immunity have not been clearly determined (Lawrence et al, 2010). However, M. haemolytica outer membrane proteins (OMPs) have been implicated in stimulating immunity against surface antigens (Confer et al., 2003, Morton et al., 1995; Potter et al., 1999).

[0008] Intranasal immunization of cattle has been pursued for a while using bovine herpesvirus-1 (BoUV-1), bovine respiratory syncytial virus (BRSV) and infectious bovine rhinotracheitis virus (IBR) (Ellis et al., 2007; Muylkens et al., 2007). Commercially available Pfizer's INFORCE 3 when administered intranasally claims to prevent BRSV and also aids in the prevention of respiratory disease caused by IBR and bovine parainfluenza virus type 3 (PI3).

[0009] In an experimental study when a modified live leukotoxin deficient M. haemolytica mutant was administered intranasally in weaned beef feedlot calves, it resulted in reduced nasopharyngeal colonization with wild type M. haemolytica compared to non-vaccinated control calves (Frank et al., 2003). Although intranasal vaccination and leukotoxin deficient M. haemolytica are known, inventors are aware of no M. haemolytica vaccines successfully combining these concepts.

SUMMARY OF THE INVENTION

[0010] An object of the present disclosure is to provide effective vaccines comprising attenuated M. haemolytica serotypes A1 & A6. Relative to a parent M haemolytica serotype AI or A6 strain, the attenuated strains may have genomic modifications, including deletions, substitutions, and additions, and whose presence (or absence) is associated with reduced virulence. In an embodiment, a wildtype M. haemolytica (serotype AI DI53) may be modified to contain a partial gene deletion of the leukotoxin CA (IktCA) genomic locus, resulting in an attenuated bacterium, which secretes a truncated, noncytotoxic form of LktA protein. The vaccines ideally provide safe, effective, and broad protective immunity.

[0011] Another object of the disclosure is to provide multi-valent vaccines, comprising the attenuated M haemolytica in combination with other bacteria, including P. multocida, M. haemolytica serotype A6, and Histophilus somni (H. somni). Thus, the invention encompasses a 4-way avirulent, modified live vaccine useful against bovine respiratory disease.

[0012] A further object of this invention is to provide methods for treatment and prophylaxis of infection bovine respiratory disease, comprising the steps of administering effective amounts of the inventive vaccines to susceptible bovine animals.

[0013] In one embodiment, the attenuated vaccines further comprises an adjuvant. The adjuvant may be any substance which increases and/or augments the elicited immune response, as compared to attenuated vaccine alone. Mucosal adjuvants, including chitosans and derivatives thereof, are particularly useful for the disclosed oral attenuated vaccines.

[0014] The invention further provides methods for inducing an immunological (or immunogenic) or protective response against M. haemolytica, as well as methods for preventing or treating M. haemolytica, or disease state(s) caused by M. haemolytica, comprising administering the attenuated bacteria, or a composition comprising the attenuated bacteria to animals in need thereof.

[0015] In addition, the disclosure provides PCR methods and reagents useful for diagnosing and/or discriminating between M. haemolytica serotypes A1 and A6. Comparative genomic sequence analysis, further described below, revealed A1- and A6-specific genes, which provide the basis for the methods and reagents provided in this disclosure.

[0016] Kits comprising at least the attenuated M. haemolytica strain and instructions for use are also provided.

[0017] These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] A full and enabling disclosure of the present invention, including the best mode thereof, to one of ordinary skill in the art, is set forth more particularly in the remainder of the specification, including reference to the accompanying figures, wherein:

[0019] FIG. 1 presents the scheme used to produce the pCT109GA189.DELTA.lktCA-Kan plasmid (replacement plasmid). The final product for vaccine manufacture incorporated a consensus ribosome-binding site (AGGAGG, rbs) upstream of the start codon which replaced the poor lktC rbs and increased expression of leukotoxoid. The native lktA gene, deleted in the vaccine strain, uses a strong rbs (AGGAGA). For this product, lktRBSr primer was used in-lieu of lktCAdelr primer. The consensus site is underlined;

[0020] FIG. 2 illustrates integration of the replacement plasmid into the bacterial genome;

[0021] FIG. 3 depicts resolution/excision of the replacement plasmid, leaving behind only the desired .DELTA.lktCA sequence, stably integrated into the bacterial genome, and encoding the truncated LktA protein;

[0022] FIG. 4A agarose gel electrophoresis of PCR products from M. haemolytica LktCABD operon showing truncated LktCA (lane 2) and wildtype LktCA (lane 3);

[0023] FIG. 4B Western blot analysis of truncated LktA expressed by M. haemolytica D153.DELTA.-1-PKL, vaccine strain. Lanes: 1- marker; 2-5 .mu.l of culture supernatant containing truncated LktA (*=27 kDa, M. haemolytica D153.DELTA.-1-PKL); 3-5 .mu.l of culture supernatant containing wildtype LktA (*=102 kDa, M. haemolytica D153 parent strain);

[0024] FIG. 5 is a Venn diagram representing the unique and overlapping genes present in five M. haemolytica isolates.

DETAILED DESCRIPTION OF TIRE INVENTION

[0025] The present invention relates to a M. haemolytica vaccine or composition which may comprise an attenuated M. haemolytica strain and a pharmaceutically or veterinarily acceptable carrier, excipient, or vehicle, which elicits, induces or stimulates a response in an animal.

[0026] In order to develop an effective M. haemolytica intranasal vaccine, which protects bovines against serogroups A1/A6, inventors used M. haemolytica having a partially deleted LktA gene. This bacterium does not cause cytolysis, but is able to elicit neutralizing antibodies. Prior to the instant disclosure, it was not known whether intranasal administration (or administration via any route) would elicit in bovines a protective immune response.

[0027] Although there are serological methods to distinguish M. haemolytica A1 and A6 these methods are not always reliable and developing strong antisera against A6 is particularly difficult. To overcome this problem, inventors sequenced both A1 and A6 genomes, performed a comparative genomic analysis and developed a real time quantitative polymerase chain reaction (RT-QPCR) method to distinguish between A1 and A6 field isolates and to track our intranasal vaccine combination (M. haemolytica, M. somnus, and P. multocida).

[0028] Thus, an embodiment of this disclosure provides useful RT-QPCR methods, which enable at least the following activities: a) identification of field isolates of M. haemolytica A1 and A6 quickly and screen large number of colonies; b) monitoring of vaccination/colonization of A1 and A6 in nasal cavities; c) elimination of the need for developing high titer antisera; and d) development of rapid, automated diagnostic test kits.

[0029] The present invention further provides attenuated M. haemolytica strains having a deletion in at least one virulence gene. In an embodiment, the deletion is within LktCA, a locus that encodes an enzyme acylase (LktC) and leukotoxin A (LktA), the chief cytotoxin. This deletion may be amplified by polymerase chain reaction (PCR) and the secretion of a truncated LktA can be detected on a Western blot to determine if the bacterium is the mutant or wildtype.

[0030] Deletion of genomic sequence(s) from virulent parental bacteria to produce avirutent, attenuated mutant bacteria is accomplished through novel and non-Obvious inventive activity. Such mutant bacteria, also referred to herein as modified-live microorganisms (MLNI) are useful for the production of immunogenic compositions or vaccines having both a high degree of immunogenicity and a low (to non-existent) degree of pathogenicity.

[0031] These mutants are also useful as vectors which can be useful for expression in vitro of expression products, as well as for reproduction or replication of nucleotide sequences e.g., replication of DNA), and for in vivo expression products.

[0032] Engineering of the deletion mutations provides novel and nonobvious nucleotide sequences and genes, as well as novel and nonobvious gene products encoded by the nucleotide sequences and genes. Such gene products provide antigens, immunogens and epitopes, and are useful as isolated gene products. Such isolated gene products, as well as epitopes thereof, are also useful for generating antibodies, which are useful in diagnostic applications.

[0033] Such gene products, which can provide or generate epitopes, antigens or immunogens, are also useful for immunogenic or immunological compositions, as well as vaccines.

[0034] In an aspect, the invention provides bacteria containing an attenuating mutation in a nucleotide sequence or a gene wherein the mutation modifies, reduces or abolishes the expression and/or the biological activity of a polypeptide or protein encoded by a gene, resulting in attenuated virulence of the bacterium. In a particular embodiment, the mutation is an in-frame deletion resulting in the bacterium secreting a truncated leukotoxin. In a particular embodiment, the truncated leukotoxin migrates at about 27 kD on a typical SDS gel.

[0035] Attenuation reduces or abolishes the pathogenicity of the bacteria and the gravity of the clinical signs or lesions, decreases the growth rate of the bacteria, and prevents the death from the bacteria.

[0036] In particular, the present invention encompasses attenuated M. haemolytica strains and vaccines comprising the same, which elicit an immunogenic response in an animal, particularly the attenuated M. haemolytica strains that elicit, induce or stimulate a response in a bovine.

[0037] Particular M. haemolytica attenuated strains of interest have mutations in genes, relative to wild type virulent parent strain, which are associated with virulence. It is recognized that, in addition to strains having the disclosed mutations, attenuated strains having any number of mutations in the disclosed virulence genes can be used in the practice of this invention.

[0038] In another aspect, the novel attenuated M. haemolytica strains are formulated into safe, effective vaccine against M. haemolytica and infections/diseases cause by M. haemolytica.

[0039] In an embodiment, the M. haemolytica vaccines further comprise an adjuvant. In a particular embodiment, the adjuvant is a mucosal adjuvant, such as chitosan, methylated chitosan, trimethylated chitosan, or derivatives or combinations thereof.

[0040] As defined herein, the term "gene" will be used in a broad sense, and shall encompass both coding and non-coding sequences 6.c upstream and downstream regulatory sequences, promoters, 5'/3' UTR, introns, and exons). Where reference to only a gene's coding sequence is intended, the term "gene's coding sequence" or "CDS" wilt be used interchangeably throughout this disclosure.

[0041] By "antigen" or "immunogen" means a substance that induces a specific immune response in a host animal. The antigen may comprise a whole organism, killed, attenuated or live; a subunit or portion of an organism; a recombinant vector containing an insert with immunogenic properties; apiece or fragment of DNA capable of inducing an immune response upon presentation to a host animal; a potypeptide, an epitope, a hapten, or any combination thereof. Alternately, the immunogen or antigen may comprise a toxin or antitoxin.

[0042] The terms "protein", "peptide", "polypeptide" and "polypeptide fragment" are used interchangeably herein to refer to polymers of amino acid residues of any length. The polymer can be linear or branched, it may comprise modified amino acids or amino acid analogs, and it may be interrupted by chemical moieties other than amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling or bioactive component.

[0043] The term "immunogenic or antigenic polypeptide" as used herein includes polypeptides that are immunologically active in the sense that once administered to the host, it is able to evoke an immune response of the humoral and/or cellular type directed against the protein. Preferably the protein fragment is such that it has substantially the same immunological activity as the total protein. Thus, a protein fragment according to the invention comprises or consists essentially of or consists of at least one epitope or antigenic determinant An "immunogenic" protein or polypeptide, as used herein, includes the full-length sequence of the protein, analogs thereof or immunogenic fragments thereof. By "immunogenic fragment" is meant a fragment of a protein which includes one or more epitopes and thus elicits the immunological response described above. Such fragments can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996). For example, linear epitopes may be determined by e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known in the art and described in, e.g., U.S. Pat. No. 4,708,871; Geysen et al., 1984; Geysen et al,, 1986. Similarly, conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, supra. Methods especially applicable to the proteins of T. parva are fully described in PCT/US2004/022605 incorporated herein by reference in its entirety.

[0044] As discussed herein, the invention encompasses active fragments and variants of the antigenic polypeptide. Thus, the term "immunogenic or antigenic polypeptide" further contemplates deletions, additions and substitutions to the sequence, so long as the polypeptide functions to produce an immunological response as defined herein. The term "conservative variation" denotes the replacement of an amino acid residue by another biologically similar residue, or the replacement of a nucleotide in a nucleic acid sequence such that the encoded amino acid residue does not change or is another biologically similar residue. In this regard, particularly preferred substitutions will generally be conservative in nature, i.e., those substitutions that take place within a family of amino acids. For example, amino acids are generally divided into four families: (1) acidic--aspartate and glutamate; (2) basic--lysine, arginine, histidine; (3) non-polar--alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar--glycine, asparagine, glutamine, cystine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. Examples of conservative variations include the substitution of one hydrophobic 3( )residue such as isoleucine, valine, leucine or methionine for another hydrophobic residue, or the substitution of one polar residue for another polar residue, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, and the like; or a similar conservative replacement of an amino acid with a structurally related amino acid that will not have a major effect on the biological activity. Proteins having substantially the same amino acid sequence as the reference molecule but possessing minor amino acid substitutions that do not substantially affect the immunogenicity of the protein are, therefore, within the definition of the reference polypeptide. All of the polypeptides produced by these modifications are included herein. The term "conservative variation" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide.

[0045] The term "epitope" refers to the site on an antigen or hapten to which specific B cells and/or T cells respond. The term is also used interchangeably with "antigenic determinant" or "antigenic determinant site". Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.

[0046] An "immunological response" to a composition or vaccine is the development in the host of a cellular and/or antibody-mediated immune response to a composition or vaccine of interest. Usually, an "immunological response" includes but is not limited to one or more of the following effects: the production of antibodies, B cells, helper T cells, and/or cytotoxic T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest. Preferably, the host will display either a therapeutic or protective immunological response such that resistance to new infection will be enhanced and/or the clinical severity of the disease reduced. Such protection will be demonstrated by either a reduction or lack of symptoms and/or clinical disease signs normally displayed by an infected host, a quicker recovery time and/or a lowered viral titer in the infected host.

[0047] By "animal" is intended mammals, birds, and the like. Animal or host as used herein includes mammals and human. The animal may be selected from the group consisting of equine (e.g., horse), canine (e.g., dogs, wolves, foxes, coyotes, jackals), feline (e.g., lions, tigers, domestic cats, wild cats, other big cats, and other felines including cheetahs and lynx), ovine (e.g., sheep), bovine (e.g., cattle), porcine (e.g., pig), avian (e.g., chicken, duck, goose, turkey, quail, pheasant, parrot, finches, hawk, crow, ostrich, emu and cassowary), primate (e.g., prosimian, tarsier, monkey, gibbon, ape), ferrets, seals, and fish. The term "animal" also includes an individual animal in all stages of development, including newborn, embryonic and fetal stages.

[0048] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a", "an", and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicate otherwise.

[0049] It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as "comprises", "comprised", "comprising" and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean "includes", "included", "including", and the like; and that terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

[0050] The term "nucleic acid" and "polynucleotide" refers to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA/DNA hybrids. The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars and linking groups such as fluororibose and thiolate, and nucleotide branches. The sequence of nucleotides may be further modified after polymerization, such as by conjugation, with a labeling component. Other types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides or solid support. The polynucleotides can be obtained by chemical synthesis or derived from a microorganism.

[0051] The term "gene" is used broadly to refer to any segment of polynucleotide associated with a biological function. Thus, genes include introns and exons as in genomic sequence, or just the coding sequences as in cDNAs and/or the regulatory sequences required for their expression. For example, gene also refers to a nucleic acid fragment that expresses mRNA or functional RNA, or encodes a specific protein, and which includes regulatory sequences.

[0052] An "isolated" biological component (such as a nucleic acid or protein or organelle) refers to a component that has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, for instance, other chromosomal and extra-chromosomal DNA and RNA, proteins, and organelles. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant technology as well as chemical synthesis.

[0053] The term "conservative variation" denotes the replacement of an amino acid residue by another biologically similar residue, or the replacement of a nucleotide in a nucleic acid sequence such that the encoded amino acid residue does not change or is another biologically similar residue. In this regard, particularly preferred substitutions will generally be conservative in nature, as described above.

[0054] The term "recombinant" means a polynucleotide with semisynthetic, or synthetic origin which either does not occur in nature or is linked to another polynucleotide in an arrangement not found in nature.

[0055] "Heterologous" means derived from a genetically distinct entity from the rest of the entity to which it is being compared. For example, a polynucleotide may be placed by genetic engineering techniques into a plasmid or vector derived from a different source, and is a heterologous polynucleotide. A promoter removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous promoter.

[0056] The polynucleotides of the invention may comprise additional sequences, such as additional encoding sequences within the same transcription unit, controlling elements such as promoters, ribosome binding sites, 5'UTR, 3'UTR, transcription terminators, polyadenylation sites, additional transcription units under control of the same or a different promoter, sequences that permit cloning, expression, homologous recombination, and transformation of a host cell, and any such construct as may be desirable to provide embodiments of this invention.

Methods of Use and Article of Manufacture

[0057] The present invention includes the following method embodiments. In an embodiment, a method of vaccinating an animal comprising administering a composition comprising an attenuated M. haemolytica strain and a pharmaceutical or veterinarily acceptable carrier, excipient, or vehicle to an animal is disclosed. In one aspect of this embodiment, the animal is a bovine.

[0058] The dose volume of compositions for target species that are mammals, e.g., the dose volume of pig or swine compositions, based on bacterial antigens, is generally between about 0.1 to about 2.0 ml, between about 0.1 to about 1.0 ml, and between about 0.5 ml to about 1.0 ml.

[0059] The efficacy of the vaccines may be tested about 2 to 4 weeks after the last immunization by challenging animals, such as bovine, with a virulent strain of M. haemolytica. Both homologous and heterologous strains are used for challenge to test the efficacy of the vaccine. The animal may be challenged by IM or SC injection, spray, intra-nasally, infra-ocularly; intra-tracheally, and/or orally. Samples from joints, lungs, brain, and/or mouth may be collected before and post-challenge and may be analyzed for the presence of M. haemolytica-specific antibody.

[0060] The compositions comprising the attenuated bacterial strains of the invention used in the prime-boost protocols are contained in a pharmaceutically or veterinary acceptable vehicle, diluent or excipient. The protocols of the invention protect the animal from M. haemolytica and/or prevent disease progression in an infected animal.

[0061] The various administrations are preferably carried out 1 to 6 weeks apart. Preferred time interval is 3 to 5 weeks, and optimally 4 weeks according to one embodiment, an annual booster is also envisioned. The animals, for example pigs, may be at least 3-4 weeks of age at the time of the first administration.

[0062] It should be understood by one of skill in the art that the disclosure herein is provided by way of example and the present invention is not limited thereto. From the disclosure herein and the knowledge in the art, the skilled artisan can determine the number of administrations, the administration route, and the doses to be used for each injection protocol, without any undue experimentation.

[0063] Another embodiment of the invention is a kit for performing a method of eliciting or inducing an immunological or protective response against M. haemolytica in an animal comprising an attenuated M. haemolytica immunological composition or vaccine and instructions for performing the method of delivery in an effective amount for eliciting an immune response in the animal.

[0064] Another embodiment of the invention is a kit for performing a method of inducing an immunological or protective response against M. haemolytica in an animal comprising a composition or vaccine comprising an attenuated M. haemolytica strain of the invention, and instructions for performing the method of delivery in an effective amount for eliciting an immune response in the animal.

[0065] Yet another aspect of the present invention relates to a kit for prime-boost vaccination according to the present invention as described above. The kit may comprise at least two vials: a first vial containing a vaccine or composition for the prime-vaccination according to the present invention, and a second vial containing a vaccine or composition for the boost-vaccination according to the present invention. The kit may advantageously contain additional first or second vials for additional prime-vaccinations or additional boost-vaccinations.

[0066] The pharmaceutically or veterinarily acceptable carriers or vehicles or excipients are well known to the one skilled in the art. For example, a pharmaceutically or veterinarily acceptable carrier or vehicle or excipient can be a 0.9% NaCl (e.g., saline) solution or a phosphate buffer. Other pharmaceutically or veterinarily acceptable carrier or vehicle or excipients that can be used for methods of this invention include, but are not limited to, poly-(L-glutamate) or polyvinylpyrrolidone. The pharmaceutically or veterinarily acceptable carrier or vehicle or excipients may be any compound or combination of compounds facilitating the administration of the vector (or protein expressed from an inventive vector in vitro); advantageously, the carrier, vehicle or excipient may facilitate transfection and/or improve preservation of the vector (or protein). Doses and dose volumes are herein discussed in the general description and can also be determined by the skilled artisan from this disclosure read in conjunction with the knowledge in the art, without any undue experimentation.

[0067] The immunological compositions and vaccines according to the invention may comprise or consist essentially of one or more adjuvants. Suitable adjuvants for use in the practice of the present invention are (1) polymers of acrylic or methacrylic acid, maleic anhydride and alkenyl derivative polymers, (2) immunostimulating sequences (ISS), such as oligodeoxyribonucleotide sequences having one or more non-methylated CpG units (Klinman et al., 1996; WO98/16247), (3) an oil in water emulsion, such as the SPT emulsion described on page 147 of "Vaccine Design, The Subunit and Adjuvant Approach" published by M. Powell, M. Newman, Plenum Press 1995, and the emulsion MF59 described on page 183 of the same work, (4) cationic lipids containing a quaternary ammonium salt, e.g., DDA (5) cytokines, (6) aluminum hydroxide or aluminum phosphate, (7) saponin or (8) other adjuvants discussed in any document cited and incorporated by reference into the instant application, or (9) any combinations or mixtures thereof.

[0068] In an embodiment, adjuvants include those which promote improved absorption through mucosal linings. Some examples include MPL, LTK63, toxins, PLG microparticles and several others (Vajdy, M. Immunology and Cell Biology (2004) 82, 617-627). In an embodiment, the adjuvant may be a chitosan (Van der Lubben et al. 2001; Patel et al. 2005; Maj et al. 2008; U.S. Pat. No. 5,980.912).

[0069] In an embodiment, the adjuvant may be inactivated bacteria, an inactivated virus, fractions of inactivated bacteria, bacterial lipopolysaccharides, bacterial toxins, or derivatives or

REFERENCES

[0070] Ackermann, M. R, Brogden, K. A. 2000. Response of the ruminant respiratory tract to Mannheimia (Pasteurella) haemolytica. Microbes Infect. 2:1079-1088. [0071] Al-Ghamdi, G. M., et al, 2000, Serotyping of Mannheimia (Pasteurella) haemolytica isolates from the upper Midwest United States. J. Vet. Diagn. Invest. 12, 576-578. [0072] Bowland, S., Shewen, P., 2000. Bovine respiratory disease: commercial vaccines currently available in Canda. Can. Vet. J. 41, 33-38. [0073] Briggs R. E, Tatum F. M. 2005. Generation and molecular characterization of new temperature-sensitive plasmids intended for genetic engineering of Pasteurellaceae. Appl Environ Micobiol 71:7187-7195. [0074] Burriel, A. R. 1997. News & Notes: Isolation of Pasteurella haemolytica from Grass, Drinking Water, and Straw Bedding Used by Sheep. Curr. Microbiol. 35: 316-318. [0075] Confer, A. W., et al., 2003. immunogenicity of recombinant Mannheimia haemolytica serotype 1 outer membrane protein PlpE and augmentation of a commercial vaccine. Vaccine 21, 2821-2829. [0076] Davies, R. L, et al. 2002. Mosaic structure and molecular evolution of the leukotoxin operon (IktCABD) in Mannheimia (Pasteurella) haemolytica, Mannheimia glueosida, and Pasteurella trehalosi. J Bacteriol. 184(1):266-277. [0077] Davies, R. L, et al. 2001. Sequence diversity and molecular evolution of the leukotoxin (lktA) gene in bovine and ovine strains of Mannheimia (Pasteurella) haemolytica. J Bacteriol. 183(4):1394-1404. [0078] Ellis, J., et al., 2007. Response of calves to challenge exposure with virulent bovine respiratory syncytial virus following intranasal administration of vaccines formulated for parenteral administration. J. Am. Vet. Med. Assoc. 230, 233-243. [0079] Frank, G. H, et al. 2003. Effect of intranasal exposure to leukotoxin-deficient Mannheimia haemolytica at the time of arrival at the feedyard on subsequent isolation of M. haemolytica from nasal secretions of calves. Am J Vet Res. 64(5):580-585. [0080] Gioia, J. et al. 2006. The genome sequence of Mannheimia haemolytica A1: insights into virulence, natural competence, and Pasteurellaceae phylogeny. J Bacteriol. 188(20):7257-7266. [0081] Lawrence, P. K., et al., 2010. Three-way comparative genomic analysis of two Mannheimia haemolytica isolates. BMC Genomics, 11:535 (Open access) [0082] Morton, R. J., et al., 1995. Vaccination of cattle with outer membrane protein-enriched fractions of Pasteurella haemolytica and resistance against experimental challenge exposure. Am. J. Vet. Res. 56, 875-879. [0083] Miller, M. W. 2001. Pasteurellosis, In E. S. Williams and I. K. Barker (ed.), Infectious diseases of wild mammals. Iowa State University. Press, Ames, Iowa. p. 330-339 [0084] Mosier, D. A. 1997. Bacterial pneumonia, Vet. Clin, N. Am. Food Anim, Pract, 13:483-493. [0085] Muylkens, B., et al., 2007. Bovine herpesvirus 1 infection and infectious bovine rhinotracheitis. Vet. Res. 38, 181-209. [0086] Potter, A. A., et al., 1999. Protective capacity of the Pasteurella haemolytica transferrin-binding proteins TbpA and TbpB in cattle. Microb Pathog 27, 197-206. [0087] Perino, L. J., Hunsaker, B. D., 1997. A review of bovine respiratory disease vaccine field efficacy. The Bovine Practitioner 31, 59-66. [0088] Purdy, C. W., et al, 1993. Efficacy of Pasteurella haemolytica subunit antigens in a goat model of pasteurellosis. Am. J. Vet. Res. 54, 1637-1647. [0089] Purdy, C. W., et al., 1997. Efficacy of a subcutaneously administered, ultraviolet light-killed Pasteurella multocida A:3-containing bacterin against transthoracic challenge exposure in goats. Am. J. Vet. Res. 58, 841-847. [0090] Rehmtulla, A. J, Thomson, R. G. 1981. A review of the lesions in shipping fever of cattle. Can. Vet. J. 22:1 [0091] Rice, J. A., et al., 2007. Mannheimia haemolytica and bovine respiratory disease. Anim. Health Res. Rev. 8, 117-128. [0092] Sabri, M. Y., et al., 2000. Efficacy of an outer membrane protein of Pasteurella haemolytica A2, A7 or A9-enriched vaccine against intratracheal challenge exposure in sheep. Vet. Microbiol. 73, 13-23.

[0093] Schreuer, D., et al. 2000. Evaluation of the efficacy of a new combined (Pasteurella) Mannheimia haemolytica serotype A1 and A6 vaccine in pre-ruminant calves by virulent challenge. Journal Cattle Practice Vol. 8 No. 1 pp. 9-12 [0094] Shewen, P. E., Wilkie, B. N., 1988. Vaccination of calves with leukotoxic culture supernatant from Pasteurella haemolytica. Can. J. Vet. Res. 52, 30-36. [0095] Virtala, A. M., et al., 1996. Epidemiologic and pathologic characteristics of respiratory tract disease in dairy heifers during the first three months of life. J. Am. Vet. Med. Assoc. 208, 2035-2042.

[0096] The invention will now be further described by way of the following non-limiting examples.

EXAMPLES

Example 1

Production of Attenuated M. haemolytica

[0097] M. haemolytica is a commensal organism of the upper respiratory tract of calves and other ruminants. Under stress and in immunocompromised animals M. haemolytica descends into lungs and causes severe systemic disease resulting in pneumonic pasteurellosis or "shipping fever". The pathogen can be spread by nose to nose contact. To attenuate the bacterium, we deleted nucleotides within the LktCA locus, which encodes an enzyme acylase (LktC) and leukotoxin A (LktA), the bacterium's chief cytotoxin. This deletion can be amplified by polymerase chain reaction (PCR) and the secretion of a truncated LktA can be detected on a Western blot to determine if the bacterium is the mutant or wildtype. The genetic engineering is summarized in FIGS. 1-3. All reagents, including the shuttle vectors pCR2.1, pBC SK, pSK, and pCT109GA189 ts ori, and the E. coli DH11S host cell, are well-known to and accessible by persons skilled in the art.

[0098] Construction of lktCA deletion. pCT109GA189-Kan.DELTA.lktCA and pCT109GA189-Kan.DELTA.lktCA-rbs were constructed as outlined in FIGS. 1-3. Briefly, two DNA fragments, upstream (1.06 kb, SEQ ID NO:6) and downstream (1.29 kb, SEQ ID NO:7) were PCR amplified from M. haemolytica strain NADC D153 (FIG. 1). Whole cells were used as template using the primer sets, lktCAf (SEQ ID NO:1)/lktCAdelr (SEQ ID NO:4) and lktCAr (SEQ ID NO:2)/lktCAdelf (SEQ ID NO:3). The PCR products were phenol-chloroform-extracted to inactivate Taq polymerase and then digested with Muni prior to ligation. The ligation products were PCP, amplified with primer pair lktCAf/lktCAr and the products were cloned using a commercially available vector (PCR2.1, invitrogen, Carlsbad, Calif.) according to manufacturer instructions.

[0099] A product containing an approximately 2.3 kb insert was selected and proper sequence across the deletion was confirmed by DNA sequencing and designated pTA.DELTA.lktCA. A kanamycin cassette derived from pUC4K was placed into the SalI site of pBC SK-(Stratagene Inc.) to generate pBCKan. The 2.3 kb deleted leukotoxin insert in pTAAIktCA was transferred into pBCKan by digestion with EcoRI and ligation into the unique EcoRI site to form pBCKan.DELTA.lktCA This product was amplified by PCR using primer pair lktCAdelf (SEQ ID NO:3) and lktRBSr (SEQ ID NO:5) to replace the native lktC ribosome binding site (RBS) with a consensus RBS (FIG. 1). The product was digested with Muni and ligated onto itself to form pBCKan.DELTA.lktCArbs. Proper sequence adjacent to the deletion was confirmed by DNA sequencing. Finally the pBC plasmid backbone of both pBCKan.DELTA.lktCA and pBCKan.DELTA.lktCArbs was replaced with the temperature-sensitive plasmid origin of replication from pCT109GA189 (Briggs and Tatum, 2005) by ligating BssHII-digested preparations of each to generate pCT109GA189Kan.DELTA.lktCA and pCT109GA89Kan.DELTA.lktCArbs.

[0100] Electrocompetent M. haemolytica serotype AI D153 cella (virulent parental strain were transformed with pCT109GA189Kan lktCA and pCT109GA189Kan.DELTA.lktCArbs by previously described methods except unmethylated ligation product was directly introduced into the competent cells, (Briggs and Tatum, 2005) Briefly, cells were made electrocompetent by growing them to logarithmic phase in 100 mi of Columbia broth (Difco Laboratories, Detroit, Mich.) at 37.degree. C. with gentle shaking. The cells were pelleted by centrifugation at 5,000 .mu.g and washed in 100 ml of 272 mM sucrose at 0.degree. C., and the pellet was suspended in an equal volume of 272 mM sucrose at 0.degree. C. After electroporation, cells recovered overnight in 10 ml Columbia broth at 30.degree. C. Growth (50 .mu.l) was spread onto Columbia agar plates containing 50 .mu.g/ml kanamycin, which were then incubated 36 hours at 30.degree. C. Individual colonies were passed to broth containing 50 .mu.g/ml kanamycin and incubated overnight at 30.degree. C. Growth (100 .mu.l) was passed again to Columbia agar plates with kanamycin which were incubated overnight at 39.degree. C.

[0101] Individual colonies were passed to trypticase soy agar (TSA) plates containing 5% defibrinated sheep blood (BA plates, incubated overnight at 39.degree. C.) and to Columbia broth without selection (incubated overnight at 30.degree. C.). Growth in broth was streaked for isolation on BA plates and passed again in broth at 30.degree. C. Non-hemolytic colonies which were kanamycin-sensitive were detected on BA plates after 1 to 3 passages without selection. Representative colonies from each recipient strain and replacement plasmid were selected for further study.

[0102] Because the temperature-sensitive plasmid origin functions poorly in E. coli cloning hosts, these final ligation products were introduced directly into M. haemolytica. Prior cloning steps used E. coil DH11S (Life Technologies, Rockville, Md.) as the cloning host.

[0103] Non-hemolytic mutants were grown in Columbia broth at 37.degree. C. for 3 hours and harvested in late logarithmic growth. Supernatants were dotted onto nitrocellulose along with supernatants from the wild-type parent and a leukotoxin-negative isogenic mutant. After appropriate blocking and washing, the blot was probed with monoclonal anti-leukotoxin antibody 2C9-1E8 (neutralizing antibody produced by NADC, Ames, Iowa), Mutant products containing the native ribosome binding site were found to express low levels of protein reactive to monoclonal antibody, less than that produced by the wild-type parent strain. Products which contained the new ribosome binding site produced much higher levels of reactive protein. Supernatants of two products expressing high levels of leukotoxin were concentrated I5-fold on a 10,000 MW filter (Centriprep, Amicon). The concentrates (1.5 .mu.l) were subjected to SDS-PAGE, blotted to nitrocellulose, and probed with antibody 2C9-1E8. Western blot analysis indicated a new protein reactive with neutralizing anti-leukotoxin monoclonal antibody at an apparent molecular weight consistent with the 27 kDa predicted protein (truncated LktA) product. These representative mutants and single-crossover controls were analyzed by PCR to demonstrate the absence of temperature-sensitive origin and kanamycin-resistance cassette (Step G). The mutant M. haemolytica serotype A1 was designated as D153.DELTA.lktCA4-707, which refers to the amino acid positions in LktC and LktA respectively where the deleted region begins and ends. Gene insertion was characterized by PCR amplification using LktCAf (SEQ ID NO:1) and LktCAr (SEQ ID NO:2) primers, which flank the deletion site. As indicated by the gel image, PCR amplification yielded the expected 2.3 kb for truncated LktCA, and 5.0 :kb for the wildtype bacterium (FIG. 4A). Finally, PCR performed with primers (SEQ ID NOs: 1 & 2) flanking ts ori and kanamycin resistance genes confirmed those elements were no longer present in the final LktCA mutant for Master Seed (MS). Five microliters of the concentrated culture supernatant as run on a SDS-PAGE system, blotted onto PVDF membrane and probed using mouse anti-LktA, neutralizing antibody 2C9-IE8 (1:1000) as primary antibody. Goat anti-mouse IgG (1:4000) coupled with alkaline phosphatase was used as secondary antibody and developed in a substrate solution containing NBT/BCIP for I-5 min (FIG. 4B). The lack of functional acylase prevents the activation of LktA, and furthermore, the N-terminal deletion of LktA prevents it from forming pores on host animal neutrophils or macrophages.

Example 2

Efficacy of Attenuated M. haemolytica in Calves

[0104] Calves were randomly assigned to one of three groups, each receiving either 10.sup.6 or 10.sup.7 CFU of the MHA A1+A6 vaccine, or the control RPMI (diluent). Lyophilized Mannheimia haemolytica (MH) serotypes A1 and A6 were resuspended and administered intranasally, I mL to each nostril, of nine calves, aged 5-6 weeks. The calves were observed for feed intake and rectal temperatures taken morning and evening for 3 days post vaccination. Nasal colonization of M. haemolytica A1 and A6 following vaccination was analyzed by RT-QPCR. (differentiated among M. haemolytica A1 and A6 throughout the study). Vaccines were plated on TSA for exact CFU/ml count on each vaccine the following day.

[0105] Challenge. A fresh glycerol stock of virulent MH A1 was grown O/N in BHI medium, plated (TSA) the next day and incubated at 37.degree. C. The following day, plates were scraped and diluted into RPMI medium supplemented with 2% inactivated fetal bovine serum. The inoculum was grown at 37.degree. C./200 rpm until desired O.D.sub.600 was achieved, and the culture was diluted to the desired CFU/challenge dose and dilution plated to enumerate the exact CFU/ml the following day. The remaining inoculum was immediately dilution plated in the lab. Calves were challenged on DAY via trans-tracheal administration of 2.4.times.10.sup.9 CPU in 20 ml RPMI, chased with 60 ml RPMI. The calves were monitored for change in behavior including lethargy, coughing, nasal discharge and scored as shown in Table 3. Rectal. temperatures were monitored for calves showing clinical signs. The lungs were scored for pneumonic lesions and recorded as % lesion on each lobe, and lung tissue was also collected for histopathology. Swabs were taken from lung lesions and trachea to recover the challenge organism. Table 1 presents the study schedule.

TABLE-US-00001 TABLE 1 Study schedule Age Day Event 5-6 0 Day 0-Bleed, Swab and vaccinate intra-nasally weeks 7 7 days post vax-Bleed and swab old 14 14 days post vax-Bleed and swab 21 21 days post vax-Bleed and swab 22 22 days post vax-Bleed, swab & Challenge with M. haemolytica A1 22-29 Observe clinical signs starting 8/7, euthanize any calves if necessary. Euthanize and necropsy all on 8/13 *Calves were observed for feed intake and rectal temperatures (morning/evening) for 3 days, post vaccination.

Samples from each calf were tested using whole cell, Lkt ELISA and RT-QPCR.

TABLE-US-00002 TABLE 2 Clinical signs criteria 0 = Normal 1 = Depression, Anorexia, Cough, Nasal Discharge, Dyspnea 2 = Severely Depressed, Unable to Rise or Walk, Euthanized for Humane Reasons 3 = Dead On Arrival (DOA)

[0106] Results. Three days post challenge one of the control calves showed severe signs of pneumonia and was euthanized (36.92% typical M. haemolytica lesions). The remaining 8 calves were euthanized on day 6 and their percent lung involvement is described in Table 3. The results clearly indicate that the vaccine affords protection when administered intranasally. As indicated in table 4 intranasal vaccination of M. haemolytica A1/A6 combo significantly reduced (62.0% and 76.7% for 6 log and 7 log group respectively) the lung lesions when compared to sham. Furthermore, histopathological analysis clearly indicated typical necrotizing bronchopneumonia characteristic of M. haemolytica.

TABLE-US-00003 TABLE 4 Average % reduction in Average lung lesion Actual vaccine dose Lung lung compared Animal # A1/A6 CFU/animal lesion (%) lesion (%) to sham 125 1.19 .times. 10.sup.6/9.2 .times. 10.sup.5 24.03* 176 1.19 .times. 10.sup.6/9.2 .times. 10.sup.5 0.0 188 1.19 .times. 10.sup.6/9.2 .times. 10.sup.5 6.40 10.43 62.0 179 1.19 .times. 10.sup.7/9.2 .times. 10.sup.6 0.87 185 1.19 .times. 10.sup.7/9.2 .times. 10.sup.6 1.837 189 1.19 .times. 10.sup.7/9.2 .times. 10.sup.6 14.91* 6.48 76.7 122 Sham 8.85 177 Sham 37.75 182 Sham 36.92 27.84 *The lesions (gross pathology) were due to typical Mycoplasma bovis chronic infection

Example 3

Development of RT-QPCR Method for Distinguishing Between A1 /A6 Serotypes

[0107] The efficacy of intranasal colonization of M. haemolytica A1/A6 was followed during the course of experiment by a novel QPCR method. Briefly, the genomes of above-described A1 and A6 serotype bacteria were compared against one A1 and two A2 genomes available in GenBank. The comparison revealed 63 genes specific for A1 (D153) and 42 genes specific for A6 (D174). Out of these 105 genes we picked a S6 family IgA-specific metalloendopeptidase (SEQ ID NO:14) specific for A1 and BCCT family betaine/camitinelcholine transporter gene (SEQ ID NO:12) specific for A6 respectively for differential real time PCR. These gene sequences were amplified by using gene specific primers, sequenced by standard Sanger method and verified. Next, we designed real time PCR primers and tagged the probes with two different dyes (A1-5'6 FAM/ZEN/3 and A6-5'Cy5/3'IBRQ) within each gene. To verify the efficacy our assay method we picked M. haemolytica colonies from nasal swabs obtained from calves maintained in our facilities 7 days post vaccination. The individual colonies were amplified by multiplex real time colony PCR using QuantiTect Probe PCR kit mastermix (Qiagen) following the manufacturer's instruction in a MX3000P qPCR machine (Stratagen). A1 and A6 colonies verified by serotyping were used as positive controls for multiplex real time quantitative PCR (RT-QPCR). The et values were set at machine default setting and each colony verified by multiplex real time PCR was confirmed by leukotoxin (LktA) specific PCR. The RT-QPCR results 7 days post vaccination indicated a preferential colonization of A1 over A6 (Table 5), Which was further confirmed by leukotoxin gene specific deletion PCR (Table 6). But 14 and 21 days post vaccination indicated essentially exclusive colonization of A1 (Tables 7 & 8).

TABLE-US-00004 TABLE 5 RT-QPCR results for nasal swabs from D7 Post Vaccination ID A1 A6 .DELTA.Lkt 151-1 17 11 + 151-2 15 - + 151-3 16 - + 151-4 17 - + 151-5 15 - + 154-1 - - 154-2 - 39 154-3 - - 154-4 - - 154-5 - 22 157-1 15 - + 157-2 22 - + 157-3 17 - + 157-4 15 33 + 157-5 16 - + 160-1 18 13 + 160-2 - 12 + 160-3 - 12 + 160-4 - 12 + 160-5 - 11 + 178-1 - - 178-2 - - 178-3 - - 178-4 - 24 178-5 - 31 181-1 15 15 + 181-2 17 - + 181-3 - 13 + 181-4 17 - + 181-5 15 - + 183-1 16 12 + 183-2 - 35 183-3 17 - + 183-4 16 - + 183-5 - 17 + 186-1 - 42 186-2 - 43 186-3 - - 186-4 - - 186-5 - 20 190-1 - - 190-2 - - 190-3 - 10 190-4 - - 190-5 - - 193-1 15 38 + 193-2 15 - + 193-3 - 36 193-4 16 20 + 193-5 - - A1 mut. Vx 15 - + A6 mut. Vx - 11 + Neg - -

TABLE-US-00005 TABLE 6 PCR results for nasal swabs from D7 Post Vaccination ID/colony A1 A6 Lkt .DELTA. ~2300 bp 122-1 - - 122-2 - - 122-3 - - 122-4 - - 122-5 - - 125-1 16 - + Y 125-2 17 - + Y 125-3 17 - + Y 125-4 16 - + Y 125-5 17 - + Y 176-1 17 - + Y 176-2 17 - + Y 176-3 16 - + Y 176-4 16 - + Y 176-5 16 - + Y 177-1 - - 177-2 - - 177-3 - - 177-4 - - 177-5 - - 179-1 17 - + Y 179-2 16 - + Y 179-3 - - 179-4 16 - + Y 179-5 29 - + Y 182-1 - - 182-2 - - 182-3 - - 182-4 - - 182-5 - - 185-1 - 15 + Y 185-2 18 - + Y 185-3 16 - + Y 185-4 - - + Y 185-5 22 - + Y 188-1 - - 188-2 - - 188-3 - - 188-4 - - 188-5 - - 189-1 16 - + Y 189-2 16 - + Y 189-3 21 - + Y 189-4 16 - + Y 189-5 17 - + Neg - -

TABLE-US-00006 TABLE 7 PCR results for nasal swabs from D14 Post Vaccination ID-colony # A1 A6 Lkt .DELTA. PCR Lkt .DELTA. 122-1 (Con. 0 0 Neg 122-2 (Con. 0 0 Neg 122-3 (Con. 0 0 Neg 125-1 (6 log) 15 0 Pos Y 125-2 (6 log) 16 0 Pos Y 125-3 (6 log) 16 0 Pos Y 176-1 (6 log) 0 0 Neg 176-2 (6 log) 0 0 Neg 176-3 (6 log) 0 0 Neg 177-1 (Con. 0 0 Neg 177-2 (Con. 0 0 Neg 177-3 (Con. 0 0 Neg 179-1 (7 log) 0 0 Neg 179-2 (7 log) 0 0 Neg 179-3 (7 log) 0 0 Neg 182-1 (Con.) 0 0 Neg 182-2 (Con.) 0 0 Neg 182-3 (Con.) 0 0 Neg 185-1 (7 log) 0 0 Neg 185-2 (7 log) 0 0 Neg 185-3 (7 log) 0 0 Neg 188-1 (6 log) 0 0 Neg 188-2 (6 log) 0 0 Neg 188-3 (6 log) 0 0 Neg 189-1 (7 log) 15 0 Pos Y 189-2 (7 log) 15 0 Pos Y 189-3 (7 log) 15 0 Pos Y A1 Mutant Pos 15 0 Pos Y A6 Mutant Pos 0 16 Pos Y Neg Con. 0 0 Neg

TABLE-US-00007 TABLE 8 PCR results for nasal swabs from D21 Post Vaccination ID-colony # A1 A6 Lkt .DELTA. PCR Lkt .DELTA. 122-1 (Con.) 0 0 .DELTA. 122-2 (Con.) 0 0 122-3 (Con.) 0 0 125-1 (6 log 14 0 + Y 125-2 (6 log 15 0 + Y 125-3 (6 log 15 0 + Y 176-1 (6 log 15 0 + Y 176-2 (6 log 15 0 + Y 176-3 (6 log 15 0 + Y 177-1 (Con.) 0 0 177-2 (Con.) 0 0 177-3 (Con.) 0 0 179-1 (7 log 0 0 179-2 (7 log 0 0 179-3 (7 log 0 0 182-1 (Con.) 0 0 182-2 (Con.) 0 0 182-3 (Con.) 0 0 185-1 (7 log) 15 0 + Y 185-2 (7 log) 14 0 + Y 185-3 (7 log) 15 0 + Y 188-1 (6 log) 14 0 + Y 188-2 (6 log) 15 0 + Y 188-3 (6 log) 14 0 + Y 189-1 (7 log) 16 0 + Y 189-2 (7 log) 17 0 + Y 189-3 (7 log) 15 0 + Y A1 Mutant Pos 15 0 + Y A6 Mutant Pos 0 16 + Y Neg Control 0 0 neg Pre Challenge A1 Wt 15 0 + WT Post Challenge A1 Wt 16 0 + WT

Example 3

Intranasal Vaccination of Calves Using Mannheimia haemolytica A1 & A6 Vaccines Followed By Virulent Challenge

[0108] Fifteen calves, 4 weeks of age and housed in 3 different pens/5 calves per pen, were randomly assigned to one of the two treatment groups. Calves were vaccinated intranasally with modified live Mannheimia haemolytica serotypes A1 and A6 (reconstituted from lyophilized, Table 9), and intranasal colonization of A1 and A6 was monitored by real time PCR. Calves were finally challenged with virulent M. haemolytica A6 (wild type) to determine vaccine efficacy.

TABLE-US-00008 TABLE 9 Treatment Groups. Total Dose/CFU Group Treatment per animal Route/volume Calf Id # 1 M. haemolytica A1 + A6 10.sup.7 (1.43 .times. 10.sup.6 + Intranasal 1 ml 2, 4, 6 8, 10 8.63 .times. 10.sup.5)* per nostril 2 M. haemolytica A1 + A6 10.sup.8 (1.43 .times. 10.sup.7 + Intranasal 1 ml 1, 3, 5, 7, 9 8.63 .times. 10.sup.6)* per nostril 3 Control-Lyophilized control Intranasal 1 ml 162, 166, 170, RPMI + stabilizer per nostril 174, 175 *Actual CFU/ml based on plate count

[0109] Vaccination, Lyophilized cultures of M. haemolytica A1 and A6 were enumerated from a batch stored at 4.degree. C. On vaccination day, the vaccines were diluted in. RPMI (colorless) to required CFU/ml for each isolate. Similarly, the sham vaccine (lyophilized RPMI in stabilizer) was diluted in RPMI. The vaccines were plated on TSA to determine the exact CFU/ml count on each vaccine the following day. The vaccines were mixed and administered 1 ml/nostril using a repeat syringe attached with a cannula according to the dose in Table 9. The control group was vaccinated first, followed by the lowest to highest log group. Following vaccination, the samples were collected as described in Table 10, and the calves were observed for feed intake and rectal temperatures taken morning and evening for 3 days post vaccination. Nasal colonization of M. haemolytica A1 and A6 following vaccination was analyzed by Q-PCR as described above.

[0110] M. haemolytica A6 challenge culture. A fresh glycerol stock of M. haemolytica A6 was grown O/N in BM medium, plated (TSA) the next day and incubated at 37.degree. C. The following day, plates were scraped and diluted into RPMI medium supplemented with 2% inactivated fetal bovine serum. The inoculum was grown at 37.degree. C./200 rpm until desired OD.sub.600 was achieved. The culture was diluted to desired CFU/challenge dose and dilution plated to enumerate the exact CFU/ml the following day. The inoculum was transported on ice and kept on ice during challenge, and administered trans-tracheally using a 14G.times.1 inch needle. The dose was 1.09.times.10.sup.9 CFU/animal in 20 ml RPMI, chased with 60 ml RPMI. Once completed, the remaining inoculum was immediately dilution plated. The calves were monitored for behavior changes including lethargy, coughing, and nasal discharge and scored as shown in Table 11. Rectal temperatures were monitored for calves showing clinical signs. The lungs were scored for pneumonic lesions and recorded as % lesion on each lobe, and tissues were collected for histopathology. Swabs were also taken from lungs (lesions) and trachea to recover the challenge organism.

TABLE-US-00009 TABLE 10 Study Schedule. Age Date Event 4 weeks old 0 Day 0-Bleed, Swab and vaccinate intra-nasally 7 days post vax 7 days post vax-Bleed and swab 15 days post vax 15 days post vax-Bleed and swab & Challenge with M. haemolytica 15 to 20 days post Observe clinical signs starting day 15; vax euthanized any calves when necessary. Euthanized and necropsy all on day 20 *Feed intake (daily) and rectal temperatures (twice daily) were monitored or 3 days post vaccination.

TABLE-US-00010 TABLE 11 Clinical signs. Criteria for Post Challenge Observations 0 = Normal 1 = Depression, Anorexia, Cough, Nasal Discharge, Dyspnea 2 = Severely Depressed, Unable to Rise or Walk, Euthanized for Humane Reasons 3 = Dead On Arrival (DOA)

[0111] Results. Two days post challenge calf# 5 and 174 showed severe signs of pneumonia and were euthanized. Calf #7 died on day 3, post challenge. The remaining 12 calves were euthanized on day 5 and their % lung involvement is described in Table 4. The results indicate that 80% of vaccinates were protected by the modified live M. haemolytica Al! A6 vaccine. From the 7 log group, three (1, 3 and 9) animals were protected while the other two animals (5, 7) had significantly large lesions compared to controls. The large lesions could have been caused by an existing Mannheimia, mycoplasma or viral infection, which had been exacerbated by challenge. Overall, 80% of vaccinates (1, 2, 3, 4, 6, 8, 9 and 10) had significantly (89.55% reduction) reduced lung lesion as compared to control, and histopathological analysis indicated typical necrotizing bronchopneumonia in the control animals.

TABLE-US-00011 TABLE 12 Dosage groups. Actual A1/A6 Lung Average Average % reduction in vaccine dose lesion lung lesion lung lesion compared to Group Animal # CPU/animal (%) (%) sham vaccine 10.sup.7 2 1.43 .times. 10.sup.6/8.63 .times. 10.sup.5 0.0 4 1.43 .times. 10.sup.6/8.63 .times. 10.sup.5 8.67 6 1.43 .times. 10.sup.6/8.63 .times. 10.sup.5 5.92 8 1.43 .times. 10.sup.6/8.63 .times. 10.sup.5 4.83 10 1.43 .times. 10.sup.6/8.63 .times. 10.sup.5 0.0 3.88 85.04 10.sup.8 1 1.43 .times. 10.sup.7/8.63 .times. 10.sup.6 0.0 3 1.43 .times. 10.sup.7/8.63 .times. 10.sup.6 0.0 5 1.43 .times. 10.sup.7/8.63 .times. 10.sup.6 41.58 7 1.43 .times. 10.sup.7/8.63 .times. 10.sup.6 64.47 9 1.43 .times. 10.sup.7/8.63 .times. 10.sup.6 2.295 21.66 14.47 162 Sham 37.11 166 Sham 29.82 170 Sham 11.235 174 Sham 25.54 175 Sham 25.97 25.93

[0112] The efficacy of intranasal colonization of M. haemolytica A1/A6 was followed during the course of experiment by above-described QPCR methods. Results for 7 and 15 days post-vaccination indicated vaccinates had a preferential colonization of A1 over A6 which was further confirmed by leukotoxin gene specific deletion PCR (Tables 13 & 14).

TABLE-US-00012 TABLE 13 Day 7 Post Vaccination Sample # Animal # FAM MHA1 MHA1? CY5 MHA6 MHA6? 1 1 No Ct 16.5 + 2 1 No Ct 38.26 + 3 1 No Ct 16.53 + 4 1 No Ct 25 + 5 2 No Ct No Ct 6 2 No Ct No Ct 7 2 No Ct No Ct 8 2 17.01 + No Ct 9 3 No Ct 15.87 + 10 3 25.11 + 20.81 + 11 3 21.91 + 19.69 + 12 3 22.35 + 21.8 + 13 4 16.52 + No Ct 14 4 17.11 + No Ct 15 4 16.26 + No Ct 16 4 16 + No Ct 17 5 39.07 + 41.17*Plot was bad ~NEG 18 5 15.98 + No Ct 19 5 16.4 + No Ct 20 5 16.44 + No Ct 21 6 17.08 + No Ct 22 6 18.24 + No Ct 23 6 16.8 + No Ct 24 6 17.94 + No Ct 25 7 17.98 + No Ct 26 7 No Ct 16.34 + 27 7 26.57 + 15.46 + 28 7 16.7 + 17.52 + 29 8 16.7 + No Ct 30 8 16.71 + No Ct 31 8 16.1 + No Ct 32 8 15.16 + No Ct 33 9 16.32 + No Ct 34 9 17.03 + No Ct 35 9 16.63 + No Ct 36 9 16.04 + No Ct 37 10 No Ct No Ct 38 10 No Ct No Ct 39 10 No Ct No Ct 40 10 No Ct No Ct 41 162 No Ct No Ct 42 162 No Ct No Ct 43 162 No Ct No Ct 44 162 No Ct No Ct 45 166 No Ct No Ct 46 166 No Ct No Ct 47 166 No Ct No Ct 48 166 No Ct No Ct 49 170 No Ct No Ct 50 170 No Ct No Ct 51 170 No Ct No Ct 52 170 No Ct No Ct 53 174 No Ct No Ct 54 174 No Ct No Ct 55 174 No Ct No Ct 56 174 No Ct No Ct 57 175 No Ct No Ct 58 175 No Ct No Ct 59 175 No Ct No Ct 60 175 No Ct No Ct 61 A1 mut + 16.66 No Ct 62 A6 mut + No Ct 13.85 63 A1 Wt + 15.87 No Ct 64 Neg 40.77 No Ct

TABLE-US-00013 TABLE 14 Day 15 Post Vaccination Animal # FAM MHA1 MHA1? CY5 MHA6 MHA6? Lkt del PCR 1 No Ct 40.53 1 No Ct No Ct 1 No Ct No Ct 1 No Ct No Ct 1 No Ct No Ct 2 No Ct No Ct 2 No Ct No Ct 2 No Ct No Ct 2 No Ct No Ct 2 No Ct No Ct 3 No Ct 15.1 + Mutant 3 No Ct 15.08 + Mutant 3 No Ct 15.19 + Mutant 3 No Ct 15.3 + Mutant 3 No Ct 15.1 + Mutant 4 15.82 No Ct Mutant 4 No Ct No Ct 4 No Ct No Ct 4 No Ct No Ct 4 No Ct No Ct 5 16.13 + No Ct Mutant 5 15.27 + No Ct Mutant 5 17.03 + No Ct Mutant 5 16.49 + No Ct Mutant 5 18.06 + No Ct Mutant 6 No Ct No Ct 6 No Ct No Ct 6 No Ct No Ct 6 40.05 No Ct 6 No Ct No Ct 7 No Ct 16.83 + Mutant 7 No Ct No Ct + 7 No Ct 14.92 + Mutant 7 No Ct 15.21 + Mutant 7 No Ct 16.16 + Mutant 8 No Ct No Ct 8 No Ct No Ct 8 No Ct No Ct 8 No Ct No Ct 8 No Ct No Ct 9 No Ct No Ct 9 No Ct No Ct 9 No Ct No Ct 9 No Ct No Ct 9 No Ct No Ct 10 15.94 + No Ct Mutant 10 No Ct + No Ct 10 No Ct + No Ct 10 23.82 + No Ct Mutant 10 30.04 + No Ct Mutant 162 No Ct No Ct 162 No Ct No Ct 162 No Ct No Ct 162 No Ct No Ct 162 No Ct No Ct 166 No Ct No Ct 166 No Ct No Ct 166 No Ct No Ct 166 No Ct No Ct 166 No Ct No Ct 170 No Ct No Ct 170 No Ct No Ct 170 No Ct No Ct 170 No Ct No Ct 170 No Ct No Ct 174 No Ct No Ct 174 No Ct No Ct 174 No Ct No Ct 174 No Ct No Ct 174 No Ct No Ct 175 16.24 + No Ct Mutant 175 No Ct + No Ct 175 16.54 + No Ct Mutant 175 No Ct + No Ct Mutant 175 23.06 + No Ct Mutant ********

[0113] Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

Sequence CWU 1

1

26126DNAArtificial SequencelktCAf primer 1gcattgaatt gatcaactaa tacttg 26225DNAArtificial SequencelktCAr primer 2caaggtttct agaaagattt ttcgg 25331DNAArtificial SequencelktCAAdelf primer 3gatcaattga aagctgttga agaaattatc g 31429DNAArtificial SequencelktCAAdelr primer 4atacaattga ttcataattt gcactcgat 29548DNAArtificial SequencelktRBSr primer 5caacaattga ttcataattt gcctcctata attattctaa attaggtc 4861068DNAArtificial Sequence5' deltalktCA PCR fragment 6gcattgaatt gatcaactaa tacttggttt ttcaagtgag ttgcaatgcc taaaccatca 60ccaaaatagt ttggattatt gattttctcc cctacaaaat ctagcccttc gtgttttctt 120gccatctcag ccaataccgg cacatcgcca aaaatagcat caattcgccc attttgcaca 180tctaaaatag cattttgata agaggcataa gatttcacat tgtactcttt tttctctttt 240gctaaatagt gttggtaagt agtcccattt tgcacaccaa tcgttttcac cttagcaaaa 300tctgtatctt ttttcgcaat gaaggcagca gagcttggaa agtaaggctc gctaaataat 360acttgtttct tacgtggttc cgtaataccc atacctgaaa ttgcagcatc aaattgtttt 420tgttttaggc tttggattaa gctatcaaaa ggttggctat ggaatgtaca atttgcattc 480atctctttac agatagcatt tgcaatatcc acatcaaaac cgataatttc tcccttctct 540tcggtcattt caaatggagg atagcttggc tccatcacaa atttgatatc ttgtgcctgc 600gcagtaacca cacacccgaa taaaagggtc aaaagtgttt ttttcataaa aagtccctgt 660gttttcatta taaggattac cactttaacg cagttacttt cttaaaaaaa gtcttctttt 720cataaagttt gttttatgtc atacaaacac atcaaattga gatgtagttt ctcaatcctc 780ttgattcctc tatctcaaaa aaacaaccca aaagaaaaaa gaaaagtata tgttacatta 840atattacaat gtaattattt tgtttaattt ccctacattt tgtataactt taaaacactc 900ctttttctct tctgattata taaaagacaa aaaatacaat ttaagctaca aaaaacaaca 960aaaaacaaca aaaaacacga caataagatc gagtaatgat tatattatgt tataattttt 1020gacctaattt agaataatta tcgagtccaa attatgaatc aattgtat 106871295DNAArtificial Sequence3' deltalktCA PCR fragment 7caattgaaag ctgttgaaga aattatcggt acatcacata acgatatctt taaaggtagt 60aagttcaatg atgcctttaa cggtggtgat ggtgtcgata ctattgacgg taacgacggc 120aatgaccgct tatttggtgg taaaggcgat gatattctcg atggtggaaa tggtgatgat 180tttatcgatg gcggtaaagg caacgaccta ttacacggtg gcaagggcga tgatattttc 240gttcaccgta aaggcgatgg taatgatatt attaccgatt ctgacggcaa tgataaatta 300tcattctctg attcgaactt aaaagattta acatttgaaa aagttaaaca taatcttgtc 360atcacgaata gcaaaaaaga gaaagtgacc attcaaaact ggttccgaga ggctgatttt 420gctaaagaag tgcctaatta taaagcaact aaagatgaga aaatcgaaga aatcatcggt 480caaaatggcg agcggatcac ctcaaagcaa gttgatgatc ttatcgcaaa aggtaacggc 540aaaattaccc aagatgagct atcaaaagtt gttgataact atgaattgct caaacatagc 600aaaaatgtga caaacagctt agataagtta atctcatctg taagtgcatt tacctcgtct 660aatgattcga gaaatgtatt agtggctcca acttcaatgt tggatcaaag tttatcttct 720cttcaatttg ctagagcagc ttaattttta atgattggca actctatatt gtttcacaca 780ttatagagtt gccgttttat tttataaaag gagacaatat ggaagctaac catcaaagga 840atgatcttgg tttagttgcc ctcactatgt tggcacaata ccataatatt tcgcttaatc 900cggaagaaat aaaacataaa tttgatcttg acggaaaagg gctttcttta actgcttggc 960ttttagctgc aaaatcgtta gcgttgaaag cgaaacacat taaaaaagag atttcccgct 1020tacacttggt gaatttaccg gcattagttt ggcaagataa cggtaaacat tttttattgg 1080taaaagtgga taccgataat aaccgctatt taacttacaa tttggaacaa gatgctccac 1140aaattctgtc acaagacgaa tttgaagcct gctatcaagg gcagttaatt ttggtcacgt 1200ccagagcttc cgtagtaggt caattagcaa agttcgattt cacctggttt attccggcgg 1260tgatcaaata ccgaaaaatc tttctagaaa ccttg 1295827DNAArtificial SequenceBCCT FAMILY-BETAINE-CARNITINE-CHOLINE Transporter forward primer 8atgttattcg ccgccggaat ggggatc 27925DNAArtificial SequenceBCCT FAMILY-BETAINE-CARNITINE-CHOLINE Transporter reverse primer 9acctgcatca ccccaaagcc aagtg 251026DNAArtificial SequenceIGA SPECIFIC SERINE METALLO-ENDOPEPTIDASE forward primer 10atgaagacca aaacatttac tcgttc 261125DNAArtificial SequenceIGA SPECIFIC SERINE METALLO-ENDOPEPTIDASE reverse primer 11agcgcttgtg tccctgaacc agcac 25122007DNAP. multocida 12ttggatttaa tcaaaaaatt aaacacagga agtaccttta gggtaccgat tttcctaccg 60agtttactct ttgtcagctt tgttgccgtt ttctgtatca tctttccaca gcaagcacaa 120acctcacttg ataccatcaa aaatagtctc ttccaacatt ttagctggtt ctatattttt 180gcaggctcta tctttttcct gtttctaatt tttctctctt tcagccgatt gggtgatatt 240aaattagggg cagataccga tgagcctgaa tttggttttg gctcttggat tgcgatgtta 300ttcgccgccg gaatggggat cgggttaatg tattttgggg tagcagaacc tattttgcat 360taccttaaac ccgtccaaca aaatttaact gagccggagc gtatgaaaga agcgatgatg 420acaacgttct atcattgggg tattcacgct tgggcaattt atggtgtgat tgccttagct 480cttgcttatt ttggcttcag atataagtta gcactcacta ttcgttccgg attttatccc 540ttactaaaac atcgtatttc aggcttctgg gggcatttaa ttgatattat tgccctttgt 600agcacgattt tcggtttaac gactacactt ggctttgggg tgatgcaggt cagtgctggc 660tttaacaatc taggtttaat tgaacagagc aattttactg ttcttgcgat tatcgtaaca 720gtagcaatgg ctcttgccgt gttatctgcc gtttcgggcg taggcaaagg ggttaaaatc 780ttaagtgaaa tcaatctcac attagccgga ttgctactta tttttgtgat aatcaccggc 840ccaactctat tacttttctc aagcttcacc gaaaatttag gctattattt tagctcgctg 900cttgagatga gtttccgtac cttcgcttat gaaccggaac atcaaggctg gctaagcggc 960tggacggtcc tttattgggc atggtgggca tcttgggcgc catttgttgg tttgtttatt 1020gccaagatct ctaaaggcag aaccattcgt gaatttattt taggggtgct atttgttcca 1080tcgctgttta acattttatg gatgaccagc ttcggcagct ctgccatttg gttcgatcaa 1140caaactgccg gtgctttagc tgaagtcagc ggcaataccg aacaactgtt atttaccttt 1200tttgagcaat taccgtttgg ctctattgcc tctttcgttg ccgtcattgt tatcagtatt 1260ttctttatca cctctgccga ctcggggatt tttgttctca acagcattgc ttcacaaggc 1320gaagaaaatg caccgaaatg gcaaagcgtg ctttggggag cattattagc catcttagcg 1380ttatcactac tctattcggg tggcttggct tctctgcaaa caatgacact gattatcgcc 1440ttaccattta ccttcattat gctgattctc tgtatcggct tatggaaagg attaatggta 1500gataaccaat acttcaacaa aaaattctcg caaggtagcc aacattgggc gggtaaagat 1560tggaaacaac gcttggagaa aatcatcaac ccaagcaata agcaagatgt ccgtcacttc 1620tttattaaag ttgccagacc agcattttta gaacttatcg aggaatttga aagctatggc 1680ttaatcgcta aaatgaattt caccaacgaa caaaacccga aattagagtt tgaagtggtg 1740aaagaaaatt tacgcaattt catttacggc attgaaagtg tgccacggga attatcggat 1800ttggtggtag gtgacgacaa cctaccgaac attgagcaaa ataccattta cgagccgatt 1860acttatttct tagacgggcg gaaaggttat gatgtgcaat atatgaccaa agaagagttg 1920attgccgacg tgctgcaaca gtatgaacgc tttatcaatt tagcgatgga caactcgcac 1980gacttaatga cggctgattt caatcac 200713669PRTP. multocida 13Leu Asp Leu Ile Lys Lys Leu Asn Thr Gly Ser Thr Phe Arg Val Pro1 5 10 15Ile Phe Leu Pro Ser Leu Leu Phe Val Ser Phe Val Ala Val Phe Cys 20 25 30Ile Ile Phe Pro Gln Gln Ala Gln Thr Ser Leu Asp Thr Ile Lys Asn 35 40 45Ser Leu Phe Gln His Phe Ser Trp Phe Tyr Ile Phe Ala Gly Ser Ile 50 55 60Phe Phe Leu Phe Leu Ile Phe Leu Ser Phe Ser Arg Leu Gly Asp Ile65 70 75 80Lys Leu Gly Ala Asp Thr Asp Glu Pro Glu Phe Gly Phe Gly Ser Trp 85 90 95Ile Ala Met Leu Phe Ala Ala Gly Met Gly Ile Gly Leu Met Tyr Phe 100 105 110Gly Val Ala Glu Pro Ile Leu His Tyr Leu Lys Pro Val Gln Gln Asn 115 120 125Leu Thr Glu Pro Glu Arg Met Lys Glu Ala Met Met Thr Thr Phe Tyr 130 135 140His Trp Gly Ile His Ala Trp Ala Ile Tyr Gly Val Ile Ala Leu Ala145 150 155 160Leu Ala Tyr Phe Gly Phe Arg Tyr Lys Leu Ala Leu Thr Ile Arg Ser 165 170 175Gly Phe Tyr Pro Leu Leu Lys His Arg Ile Ser Gly Phe Trp Gly His 180 185 190Leu Ile Asp Ile Ile Ala Leu Cys Ser Thr Ile Phe Gly Leu Thr Thr 195 200 205Thr Leu Gly Phe Gly Val Met Gln Val Ser Ala Gly Phe Asn Asn Leu 210 215 220Gly Leu Ile Glu Gln Ser Asn Phe Thr Val Leu Ala Ile Ile Val Thr225 230 235 240Val Ala Met Ala Leu Ala Val Leu Ser Ala Val Ser Gly Val Gly Lys 245 250 255Gly Val Lys Ile Leu Ser Glu Ile Asn Leu Thr Leu Ala Gly Leu Leu 260 265 270Leu Ile Phe Val Ile Ile Thr Gly Pro Thr Leu Leu Leu Phe Ser Ser 275 280 285Phe Thr Glu Asn Leu Gly Tyr Tyr Phe Ser Ser Leu Leu Glu Met Ser 290 295 300Phe Arg Thr Phe Ala Tyr Glu Pro Glu His Gln Gly Trp Leu Ser Gly305 310 315 320Trp Thr Val Leu Tyr Trp Ala Trp Trp Ala Ser Trp Ala Pro Phe Val 325 330 335Gly Leu Phe Ile Ala Lys Ile Ser Lys Gly Arg Thr Ile Arg Glu Phe 340 345 350Ile Leu Gly Val Leu Phe Val Pro Ser Leu Phe Asn Ile Leu Trp Met 355 360 365Thr Ser Phe Gly Ser Ser Ala Ile Trp Phe Asp Gln Gln Thr Ala Gly 370 375 380Ala Leu Ala Glu Val Ser Gly Asn Thr Glu Gln Leu Leu Phe Thr Phe385 390 395 400Phe Glu Gln Leu Pro Phe Gly Ser Ile Ala Ser Phe Val Ala Val Ile 405 410 415Val Ile Ser Ile Phe Phe Ile Thr Ser Ala Asp Ser Gly Ile Phe Val 420 425 430Leu Asn Ser Ile Ala Ser Gln Gly Glu Glu Asn Ala Pro Lys Trp Gln 435 440 445Ser Val Leu Trp Gly Ala Leu Leu Ala Ile Leu Ala Leu Ser Leu Leu 450 455 460Tyr Ser Gly Gly Leu Ala Ser Leu Gln Thr Met Thr Leu Ile Ile Ala465 470 475 480Leu Pro Phe Thr Phe Ile Met Leu Ile Leu Cys Ile Gly Leu Trp Lys 485 490 495Gly Leu Met Val Asp Asn Gln Tyr Phe Asn Lys Lys Phe Ser Gln Gly 500 505 510Ser Gln His Trp Ala Gly Lys Asp Trp Lys Gln Arg Leu Glu Lys Ile 515 520 525Ile Asn Pro Ser Asn Lys Gln Asp Val Arg His Phe Phe Ile Lys Val 530 535 540Ala Arg Pro Ala Phe Leu Glu Leu Ile Glu Glu Phe Glu Ser Tyr Gly545 550 555 560Leu Ile Ala Lys Met Asn Phe Thr Asn Glu Gln Asn Pro Lys Leu Glu 565 570 575Phe Glu Val Val Lys Glu Asn Leu Arg Asn Phe Ile Tyr Gly Ile Glu 580 585 590Ser Val Pro Arg Glu Leu Ser Asp Leu Val Val Gly Asp Asp Asn Leu 595 600 605Pro Asn Ile Glu Gln Asn Thr Ile Tyr Glu Pro Ile Thr Tyr Phe Leu 610 615 620Asp Gly Arg Lys Gly Tyr Asp Val Gln Tyr Met Thr Lys Glu Glu Leu625 630 635 640Ile Ala Asp Val Leu Gln Gln Tyr Glu Arg Phe Ile Asn Leu Ala Met 645 650 655Asp Asn Ser His Asp Leu Met Thr Ala Asp Phe Asn His 660 665144017DNAP. multocida 14atgaagacca aaacatttac tcgttcttat cttgcttctt ttgtaacaat cgtattaagt 60ttacctgctg tagcatctgt tgtacgtaat gatgtggact atcaatactt ccgcgatttt 120gccgaaaata aaggaccatt ttcagttggt tcaatgaata ttgatattaa agacaacaat 180ggacaacttg taggcacgat gcttcataat ttaccaatgg ttgattttag tgctatggta 240agaggtggat attctacttt aattgcacca caatatttag ttagtgttgc acataatact 300ggatataaaa atgttcaatt tggtgctgca ggttataacc ctgattcaca tcactatact 360tataaaattg ttgaccgcaa tgattatgaa aaggttcaag gagggttgca cccagactat 420catactcctc gattaaataa attagtaaca gaagttgtgc ctgccgcagt caccaatgca 480ggtacatcta ttaaacccta cttaaatgaa gaacgcttcc ctatgtttct tcgtgctggt 540tcagggacac aagcgctaag aggaaaagaa agtaataaaa caactggaat cgctggtgct 600tatgaatatc ttactggcgg taccacatta caattatcta aaagctcccc tgatcactgg 660ttagattatt caagtaacct ttatcaagta agctatggac cactttcaac ctatgcacta 720cctggtgata gtggttcagg ttcttacgcc tatgatatga acgaaaaacg atgggtatta 780gttggtgtgc tcaatttcta taatggtatg gataatcaat tcaaccgctc tgcgattatc 840cgtaaagatt tccacgagaa aaaatttgcc gaagatattg caggaacaat caataatacc 900gtacaaaatg cacaattcaa ttggactgct caaggtaaat ccagctctct tagtcaatca 960aataatgtgc aaaaactcaa cgttgatcta aaagatagta gcattgcaaa ccaaaacact 1020tctctgccac aagaaaatca cggtaaaacc attaatttta atggtaaaga tgcaactatt 1080gtactaaaac aggatattga ccaaggtgca ggtgcattaa atctgaacgc taatctcact 1140attcgtcctg aaacagacca aacttggcaa ggtgcaggta ttatcgtcgg taaagataaa 1200aaagtgaatt ggcaagtaaa aaatccacaa ggcgatcgtt tatctaaact cggggaagga 1260acactctatg taaatggacg tggacagaat cttggcgata tcagtgtagg tgatggtatt 1320gtaatactta accagcaagc cgatcaccaa ggaagaaaac aggcctttaa tacagtagga 1380atcgtaagtg gtcgccccac tgttgtgcta ggtagtgcag atcaagttaa tcccgataat 1440atttactttg gatttcgcgg aggtcgttta gacctaaacg gtaacagcat cgcctttaaa 1500cgtattcaaa acagcgataa acatgctcgt attgtaaacc acaatcgcga tcacatttct 1560accttaataa tacaaggcca agatcctctc actagtaatg atcttatatg gggaaaatgg 1620gcaagtaata gcccagcaga catttacgaa tataccaatc cttatcaaaa taaacgcaaa 1680gattacttcc gtctgaaagg taattcgaga gtatattatc caacgaatgc tacaagtaac 1740gatcactggg aatttctttc cagtaaccgc gagcaagcaa tacagaaaat cctagatgcc 1800aaaaacttaa gacagcgcta tgacacgttt aatggtttta taggggaaga tgcttccaat 1860aaaactaatg ggatattaaa tgtcgtgttt gatacaaaaa cagaagtaaa tacagaacaa 1920gataaattaa agaatatcta cacaatgtcg ggaggattta accttaatgg tgaactcacc 1980cttaaaggtg gtacattgtt gctttctggt cacccaacgc cacacgctta tgatattaag 2040aataagcatg atgttgtgcg tgaaaacgat tggcaagaca gccattttac tgctaaaaat 2100atcacggtaa ataaaatggc acaactctat atcgggagaa atgtcaatga agtaaatagt 2160cactttactg cgactgataa agccaaactc aatttaggat ttattaatcg ttcaacgcca 2220agttgctatg attctgaata cacaggcact acacattgtg aagtgcaagc ggtcatttcc 2280gataatattt ttgcaaatct agcaacaacc gccattaaag gtaatgttaa attacaaaac 2340catagccaat taaatttagg caaagcaaac ctcactggtt ctgtacaagc tgatcaaaca 2400actcatatca ctttagcaaa tcacagtcac tggttaaaca atggtacgag ccagattggg 2460catcttacaa tggaaaaagg gtcgatcctt agcctaaacg ataaatttgc taccacggaa 2520atcccagtcc gattcaacaa gatgatcatc caaggtaatc taaaaggtaa tggacgaatt 2580aactataccg caaatttagc caagggcgaa tctgatcatc tccaagttga cggtattgct 2640gaaggaaatt ttgtccttgc cgttagaaat agcacaactg aagcaaatcc aaaaagctca 2700ttaaacctac taagcttaaa aaatagcaac caagaaggca ataaagcttc tatttctcta 2760gaaaataatt atgttgatct aggtacttat cgttatgtat tagaaaatcg taatcacaat 2820taccatttat ttaatccatt aataccaaat tcaacctcta aagagatgaa tgctacatct 2880gtatcctcta ttccaaaaaa ggaatctgtt actaatgttc ctactttaga taagaaagaa 2940actgaacaaa atcttactca actacaaaaa gatttttcag cacaccaatt agaaaatcaa 3000aaagcaaaac aatctatgat aaatgctcaa tctgagctaa gacgactcaa ttcacaactg 3060aatgtattgc aaaaatatgt gaattctcgt cgcttaggtt actatactca gcaggcagtt 3120ttagaacaaa ttagcattat tcaaaataaa attaaacaaa cacaaacaat atttaatgac 3180gctaatgcaa ctgtaaaact cacagatcaa aagctagaag aagccaaatt agctctaggc 3240tctgtaaacg atcttgtatt aataaaagcc tctgctccag caatgcaagc aactaatcaa 3300gatacgagta tgatgaatat tattcaagca gattggataa gccaatacgc taacacagca 3360ctttctgaac tctcggcaca ggctaattct gctctgcaaa tcagtaatag cttagatcgc 3420caactcttca aacaaagcga taaattcaac gtatggagca gcgtcgaaca tcagaaaacc 3480gagcataaat cagatttata ccgcccgtat aaacaacaaa ccaacctgac ccaactgggc 3540atacaaatgc cgatagataa cggtttaatg tttggagttg cattatctaa aaaccacgct 3600aacgcggaat ttaacgaggg tgtaaacggt aaatcgaatc tactaatggc aagcctatat 3660ggtaagtggc aatctcaaca aggcactttt atcagccttg atggcagcta cggtaaagca 3720aaaaaccaac tctacctatt tggtgaaaac cactttaccc gccgaatttc ctctattggt 3780gctaacattg gacatcaatt tgacctcgca ggagttcaaa ttcagccaac aataggagca 3840agatactacc atttcagcgg ccaagactat acactaggag gagcgaaaat cagctcacca 3900aatacccact ttatgacata tcaagcgggt ctaaaagcta gtaaaacttt tcattggatg 3960actggaaagt tgaaccaagc attacaaccc actatgtgga tgcaagtaac aaacgct 4017151339PRTP. multocida 15Met Lys Thr Lys Thr Phe Thr Arg Ser Tyr Leu Ala Ser Phe Val Thr1 5 10 15Ile Val Leu Ser Leu Pro Ala Val Ala Ser Val Val Arg Asn Asp Val 20 25 30Asp Tyr Gln Tyr Phe Arg Asp Phe Ala Glu Asn Lys Gly Pro Phe Ser 35 40 45Val Gly Ser Met Asn Ile Asp Ile Lys Asp Asn Asn Gly Gln Leu Val 50 55 60Gly Thr Met Leu His Asn Leu Pro Met Val Asp Phe Ser Ala Met Val65 70 75 80Arg Gly Gly Tyr Ser Thr Leu Ile Ala Pro Gln Tyr Leu Val Ser Val 85 90 95Ala His Asn Thr Gly Tyr Lys Asn Val Gln Phe Gly Ala Ala Gly Tyr 100 105 110Asn Pro Asp Ser His His Tyr Thr Tyr Lys Ile Val Asp Arg Asn Asp 115 120 125Tyr Glu Lys Val Gln Gly Gly Leu His Pro Asp Tyr His Thr Pro Arg 130 135 140Leu Asn Lys Leu Val Thr Glu Val Val Pro Ala Ala Val Thr Asn Ala145 150 155 160Gly Thr Ser Ile Lys Pro Tyr Leu Asn Glu Glu Arg Phe Pro Met Phe 165 170 175Leu Arg Ala Gly Ser Gly Thr Gln Ala Leu Arg Gly Lys Glu Ser Asn 180 185 190Lys Thr Thr Gly Ile Ala Gly Ala Tyr Glu Tyr Leu Thr Gly Gly Thr 195 200 205Thr Leu Gln Leu

Ser Lys Ser Ser Pro Asp His Trp Leu Asp Tyr Ser 210 215 220Ser Asn Leu Tyr Gln Val Ser Tyr Gly Pro Leu Ser Thr Tyr Ala Leu225 230 235 240Pro Gly Asp Ser Gly Ser Gly Ser Tyr Ala Tyr Asp Met Asn Glu Lys 245 250 255Arg Trp Val Leu Val Gly Val Leu Asn Phe Tyr Asn Gly Met Asp Asn 260 265 270Gln Phe Asn Arg Ser Ala Ile Ile Arg Lys Asp Phe His Glu Lys Lys 275 280 285Phe Ala Glu Asp Ile Ala Gly Thr Ile Asn Asn Thr Val Gln Asn Ala 290 295 300Gln Phe Asn Trp Thr Ala Gln Gly Lys Ser Ser Ser Leu Ser Gln Ser305 310 315 320Asn Asn Val Gln Lys Leu Asn Val Asp Leu Lys Asp Ser Ser Ile Ala 325 330 335Asn Gln Asn Thr Ser Leu Pro Gln Glu Asn His Gly Lys Thr Ile Asn 340 345 350Phe Asn Gly Lys Asp Ala Thr Ile Val Leu Lys Gln Asp Ile Asp Gln 355 360 365Gly Ala Gly Ala Leu Asn Leu Asn Ala Asn Leu Thr Ile Arg Pro Glu 370 375 380Thr Asp Gln Thr Trp Gln Gly Ala Gly Ile Ile Val Gly Lys Asp Lys385 390 395 400Lys Val Asn Trp Gln Val Lys Asn Pro Gln Gly Asp Arg Leu Ser Lys 405 410 415Leu Gly Glu Gly Thr Leu Tyr Val Asn Gly Arg Gly Gln Asn Leu Gly 420 425 430Asp Ile Ser Val Gly Asp Gly Ile Val Ile Leu Asn Gln Gln Ala Asp 435 440 445His Gln Gly Arg Lys Gln Ala Phe Asn Thr Val Gly Ile Val Ser Gly 450 455 460Arg Pro Thr Val Val Leu Gly Ser Ala Asp Gln Val Asn Pro Asp Asn465 470 475 480Ile Tyr Phe Gly Phe Arg Gly Gly Arg Leu Asp Leu Asn Gly Asn Ser 485 490 495Ile Ala Phe Lys Arg Ile Gln Asn Ser Asp Lys His Ala Arg Ile Val 500 505 510Asn His Asn Arg Asp His Ile Ser Thr Leu Ile Ile Gln Gly Gln Asp 515 520 525Pro Leu Thr Ser Asn Asp Leu Ile Trp Gly Lys Trp Ala Ser Asn Ser 530 535 540Pro Ala Asp Ile Tyr Glu Tyr Thr Asn Pro Tyr Gln Asn Lys Arg Lys545 550 555 560Asp Tyr Phe Arg Leu Lys Gly Asn Ser Arg Val Tyr Tyr Pro Thr Asn 565 570 575Ala Thr Ser Asn Asp His Trp Glu Phe Leu Ser Ser Asn Arg Glu Gln 580 585 590Ala Ile Gln Lys Ile Leu Asp Ala Lys Asn Leu Arg Gln Arg Tyr Asp 595 600 605Thr Phe Asn Gly Phe Ile Gly Glu Asp Ala Ser Asn Lys Thr Asn Gly 610 615 620Ile Leu Asn Val Val Phe Asp Thr Lys Thr Glu Val Asn Thr Glu Gln625 630 635 640Asp Lys Leu Lys Asn Ile Tyr Thr Met Ser Gly Gly Phe Asn Leu Asn 645 650 655Gly Glu Leu Thr Leu Lys Gly Gly Thr Leu Leu Leu Ser Gly His Pro 660 665 670Thr Pro His Ala Tyr Asp Ile Lys Asn Lys His Asp Val Val Arg Glu 675 680 685Asn Asp Trp Gln Asp Ser His Phe Thr Ala Lys Asn Ile Thr Val Asn 690 695 700Lys Met Ala Gln Leu Tyr Ile Gly Arg Asn Val Asn Glu Val Asn Ser705 710 715 720His Phe Thr Ala Thr Asp Lys Ala Lys Leu Asn Leu Gly Phe Ile Asn 725 730 735Arg Ser Thr Pro Ser Cys Tyr Asp Ser Glu Tyr Thr Gly Thr Thr His 740 745 750Cys Glu Val Gln Ala Val Ile Ser Asp Asn Ile Phe Ala Asn Leu Ala 755 760 765Thr Thr Ala Ile Lys Gly Asn Val Lys Leu Gln Asn His Ser Gln Leu 770 775 780Asn Leu Gly Lys Ala Asn Leu Thr Gly Ser Val Gln Ala Asp Gln Thr785 790 795 800Thr His Ile Thr Leu Ala Asn His Ser His Trp Leu Asn Asn Gly Thr 805 810 815Ser Gln Ile Gly His Leu Thr Met Glu Lys Gly Ser Ile Leu Ser Leu 820 825 830Asn Asp Lys Phe Ala Thr Thr Glu Ile Pro Val Arg Phe Asn Lys Met 835 840 845Ile Ile Gln Gly Asn Leu Lys Gly Asn Gly Arg Ile Asn Tyr Thr Ala 850 855 860Asn Leu Ala Lys Gly Glu Ser Asp His Leu Gln Val Asp Gly Ile Ala865 870 875 880Glu Gly Asn Phe Val Leu Ala Val Arg Asn Ser Thr Thr Glu Ala Asn 885 890 895Pro Lys Ser Ser Leu Asn Leu Leu Ser Leu Lys Asn Ser Asn Gln Glu 900 905 910Gly Asn Lys Ala Ser Ile Ser Leu Glu Asn Asn Tyr Val Asp Leu Gly 915 920 925Thr Tyr Arg Tyr Val Leu Glu Asn Arg Asn His Asn Tyr His Leu Phe 930 935 940Asn Pro Leu Ile Pro Asn Ser Thr Ser Lys Glu Met Asn Ala Thr Ser945 950 955 960Val Ser Ser Ile Pro Lys Lys Glu Ser Val Thr Asn Val Pro Thr Leu 965 970 975Asp Lys Lys Glu Thr Glu Gln Asn Leu Thr Gln Leu Gln Lys Asp Phe 980 985 990Ser Ala His Gln Leu Glu Asn Gln Lys Ala Lys Gln Ser Met Ile Asn 995 1000 1005Ala Gln Ser Glu Leu Arg Arg Leu Asn Ser Gln Leu Asn Val Leu Gln 1010 1015 1020Lys Tyr Val Asn Ser Arg Arg Leu Gly Tyr Tyr Thr Gln Gln Ala Val1025 1030 1035 1040Leu Glu Gln Ile Ser Ile Ile Gln Asn Lys Ile Lys Gln Thr Gln Thr 1045 1050 1055Ile Phe Asn Asp Ala Asn Ala Thr Val Lys Leu Thr Asp Gln Lys Leu 1060 1065 1070Glu Glu Ala Lys Leu Ala Leu Gly Ser Val Asn Asp Leu Val Leu Ile 1075 1080 1085Lys Ala Ser Ala Pro Ala Met Gln Ala Thr Asn Gln Asp Thr Ser Met 1090 1095 1100Met Asn Ile Ile Gln Ala Asp Trp Ile Ser Gln Tyr Ala Asn Thr Ala1105 1110 1115 1120Leu Ser Glu Leu Ser Ala Gln Ala Asn Ser Ala Leu Gln Ile Ser Asn 1125 1130 1135Ser Leu Asp Arg Gln Leu Phe Lys Gln Ser Asp Lys Phe Asn Val Trp 1140 1145 1150Ser Ser Val Glu His Gln Lys Thr Glu His Lys Ser Asp Leu Tyr Arg 1155 1160 1165Pro Tyr Lys Gln Gln Thr Asn Leu Thr Gln Leu Gly Ile Gln Met Pro 1170 1175 1180Ile Asp Asn Gly Leu Met Phe Gly Val Ala Leu Ser Lys Asn His Ala1185 1190 1195 1200Asn Ala Glu Phe Asn Glu Gly Val Asn Gly Lys Ser Asn Leu Leu Met 1205 1210 1215Ala Ser Leu Tyr Gly Lys Trp Gln Ser Gln Gln Gly Thr Phe Ile Ser 1220 1225 1230Leu Asp Gly Ser Tyr Gly Lys Ala Lys Asn Gln Leu Tyr Leu Phe Gly 1235 1240 1245Glu Asn His Phe Thr Arg Arg Ile Ser Ser Ile Gly Ala Asn Ile Gly 1250 1255 1260His Gln Phe Asp Leu Ala Gly Val Gln Ile Gln Pro Thr Ile Gly Ala1265 1270 1275 1280Arg Tyr Tyr His Phe Ser Gly Gln Asp Tyr Thr Leu Gly Gly Ala Lys 1285 1290 1295Ile Ser Ser Pro Asn Thr His Phe Met Thr Tyr Gln Ala Gly Leu Lys 1300 1305 1310Ala Ser Lys Thr Phe His Trp Met Thr Gly Lys Leu Asn Gln Ala Leu 1315 1320 1325Gln Pro Thr Met Trp Met Gln Val Thr Asn Ala 1330 1335162354DNAArtificial SequenceComplete deltaLKTCA with original RBS 16gcattgaatt gatcaactaa tacttggttt ttcaagtgag ttgcaatgcc taaaccatca 60ccaaaatagt ttggattatt gattttctcc cctacaaaat ctagcccttc gtgttttctt 120gccatctcag ccaataccgg cacatcgcca aaaatagcat caattcgccc attttgcaca 180tctaaaatag cattttgata agaggcataa gatttcacat tgtactcttt tttctctttt 240gctaaatagt gttggtaagt agtcccattt tgcacaccaa tcgttttcac cttagcaaaa 300tctgtatctt ttttcgcaat gaaggcagca gagcttggaa agtaaggctc gctaaataat 360acttgtttct tacgtggttc cgtaataccc atacctgaaa ttgcagcatc aaattgtttt 420tgttttaggc tttggattaa gctatcaaaa ggttggctat ggaatgtaca atttgcattc 480atctctttac agatagcatt tgcaatatcc acatcaaaac cgataatttc tcccttctct 540tcggtcattt caaatggagg atagcttggc tccatcacaa atttgatatc ttgtgcctgc 600gcagtaacca cacacccgaa taaaagggtc aaaagtgttt ttttcataaa aagtccctgt 660gttttcatta taaggattac cactttaacg cagttacttt cttaaaaaaa gtcttctttt 720cataaagttt gttttatgtc atacaaacac atcaaattga gatgtagttt ctcaatcctc 780ttgattcctc tatctcaaaa aaacaaccca aaagaaaaaa gaaaagtata tgttacatta 840atattacaat gtaattattt tgtttaattt ccctacattt tgtataactt taaaacactc 900ctttttctct tctgattata taaaagacaa aaaatacaat ttaagctaca aaaaacaaca 960aaaaacaaca aaaaacacga caataagatc gagtaatgat tatattatgt tataattttt 1020gacctaattt agaataatta tcgagtccaa attatgaatc aattgaaagc tgttgaagaa 1080attatcggta catcacataa cgatatcttt aaaggtagta agttcaatga tgcctttaac 1140ggtggtgatg gtgtcgatac tattgacggt aacgacggca atgaccgctt atttggtggt 1200aaaggcgatg atattctcga tggtggaaat ggtgatgatt ttatcgatgg cggtaaaggc 1260aacgacctat tacacggtgg caagggcgat gatattttcg ttcaccgtaa aggcgatggt 1320aatgatatta ttaccgattc tgacggcaat gataaattat cattctctga ttcgaactta 1380aaagatttaa catttgaaaa agttaaacat aatcttgtca tcacgaatag caaaaaagag 1440aaagtgacca ttcaaaactg gttccgagag gctgattttg ctaaagaagt gcctaattat 1500aaagcaacta aagatgagaa aatcgaagaa atcatcggtc aaaatggcga gcggatcacc 1560tcaaagcaag ttgatgatct tatcgcaaaa ggtaacggca aaattaccca agatgagcta 1620tcaaaagttg ttgataacta tgaattgctc aaacatagca aaaatgtgac aaacagctta 1680gataagttaa tctcatctgt aagtgcattt acctcgtcta atgattcgag aaatgtatta 1740gtggctccaa cttcaatgtt ggatcaaagt ttatcttctc ttcaatttgc tagagcagct 1800taatttttaa tgattggcaa ctctatattg tttcacacat tatagagttg ccgttttatt 1860ttataaaagg agacaatatg gaagctaacc atcaaaggaa tgatcttggt ttagttgccc 1920tcactatgtt ggcacaatac cataatattt cgcttaatcc ggaagaaata aaacataaat 1980ttgatcttga cggaaaaggg ctttctttaa ctgcttggct tttagctgca aaatcgttag 2040cgttgaaagc gaaacacatt aaaaaagaga tttcccgctt acacttggtg aatttaccgg 2100cattagtttg gcaagataac ggtaaacatt ttttattggt aaaagtggat accgataata 2160accgctattt aacttacaat ttggaacaag atgctccaca aattctgtca caagacgaat 2220ttgaagcctg ctatcaaggg cagttaattt tggtcacgtc cagagcttcc gtagtaggtc 2280aattagcaaa gttcgatttc acctggttta ttccggcggt gatcaaatac cgaaaaatct 2340ttctagaaac cttg 2354172354DNAArtificial SequenceComplete deltalktCA (with consensus RBS) 17gcattgaatt gatcaactaa tacttggttt ttcaagtgag ttgcaatgcc taaaccatca 60ccaaaatagt ttggattatt gattttctcc cctacaaaat ctagcccttc gtgttttctt 120gccatctcag ccaataccgg cacatcgcca aaaatagcat caattcgccc attttgcaca 180tctaaaatag cattttgata agaggcataa gatttcacat tgtactcttt tttctctttt 240gctaaatagt gttggtaagt agtcccattt tgcacaccaa tcgttttcac cttagcaaaa 300tctgtatctt ttttcgcaat gaaggcagca gagcttggaa agtaaggctc gctaaataat 360acttgtttct tacgtggttc cgtaataccc atacctgaaa ttgcagcatc aaattgtttt 420tgttttaggc tttggattaa gctatcaaaa ggttggctat ggaatgtaca atttgcattc 480atctctttac agatagcatt tgcaatatcc acatcaaaac cgataatttc tcccttctct 540tcggtcattt caaatggagg atagcttggc tccatcacaa atttgatatc ttgtgcctgc 600gcagtaacca cacacccgaa taaaagggtc aaaagtgttt ttttcataaa aagtccctgt 660gttttcatta taaggattac cactttaacg cagttacttt cttaaaaaaa gtcttctttt 720cataaagttt gttttatgtc atacaaacac atcaaattga gatgtagttt ctcaatcctc 780ttgattcctc tatctcaaaa aaacaaccca aaagaaaaaa gaaaagtata tgttacatta 840atattacaat gtaattattt tgtttaattt ccctacattt tgtataactt taaaacactc 900ctttttctct tctgattata taaaagacaa aaaatacaat ttaagctaca aaaaacaaca 960aaaaacaaca aaaaacacga caataagatc gagtaatgat tatattatgt tataattttt 1020gacctaattt agaataatta taggaggcaa attatgaatc aattgaaagc tgttgaagaa 1080attatcggta catcacataa cgatatcttt aaaggtagta agttcaatga tgcctttaac 1140ggtggtgatg gtgtcgatac tattgacggt aacgacggca atgaccgctt atttggtggt 1200aaaggcgatg atattctcga tggtggaaat ggtgatgatt ttatcgatgg cggtaaaggc 1260aacgacctat tacacggtgg caagggcgat gatattttcg ttcaccgtaa aggcgatggt 1320aatgatatta ttaccgattc tgacggcaat gataaattat cattctctga ttcgaactta 1380aaagatttaa catttgaaaa agttaaacat aatcttgtca tcacgaatag caaaaaagag 1440aaagtgacca ttcaaaactg gttccgagag gctgattttg ctaaagaagt gcctaattat 1500aaagcaacta aagatgagaa aatcgaagaa atcatcggtc aaaatggcga gcggatcacc 1560tcaaagcaag ttgatgatct tatcgcaaaa ggtaacggca aaattaccca agatgagcta 1620tcaaaagttg ttgataacta tgaattgctc aaacatagca aaaatgtgac aaacagctta 1680gataagttaa tctcatctgt aagtgcattt acctcgtcta atgattcgag aaatgtatta 1740gtggctccaa cttcaatgtt ggatcaaagt ttatcttctc ttcaatttgc tagagcagct 1800taatttttaa tgattggcaa ctctatattg tttcacacat tatagagttg ccgttttatt 1860ttataaaagg agacaatatg gaagctaacc atcaaaggaa tgatcttggt ttagttgccc 1920tcactatgtt ggcacaatac cataatattt cgcttaatcc ggaagaaata aaacataaat 1980ttgatcttga cggaaaaggg ctttctttaa ctgcttggct tttagctgca aaatcgttag 2040cgttgaaagc gaaacacatt aaaaaagaga tttcccgctt acacttggtg aatttaccgg 2100cattagtttg gcaagataac ggtaaacatt ttttattggt aaaagtggat accgataata 2160accgctattt aacttacaat ttggaacaag atgctccaca aattctgtca caagacgaat 2220ttgaagcctg ctatcaaggg cagttaattt tggtcacgtc cagagcttcc gtagtaggtc 2280aattagcaaa gttcgatttc acctggttta ttccggcggt gatcaaatac cgaaaaatct 2340ttctagaaac cttg 235418249PRTArtificial SequenceTranslation of deltaLKTCA 18Met Asn Gln Leu Lys Ala Val Glu Glu Ile Ile Gly Thr Ser His Asn1 5 10 15Asp Ile Phe Lys Gly Ser Lys Phe Asn Asp Ala Phe Asn Gly Gly Asp 20 25 30Gly Val Asp Thr Ile Asp Gly Asn Asp Gly Asn Asp Arg Leu Phe Gly 35 40 45Gly Lys Gly Asp Asp Ile Leu Asp Gly Gly Asn Gly Asp Asp Phe Ile 50 55 60Asp Gly Gly Lys Gly Asn Asp Leu Leu His Gly Gly Lys Gly Asp Asp65 70 75 80Ile Phe Val His Arg Lys Gly Asp Gly Asn Asp Ile Ile Thr Asp Ser 85 90 95Asp Gly Asn Asp Lys Leu Ser Phe Ser Asp Ser Asn Leu Lys Asp Leu 100 105 110Thr Phe Glu Lys Val Lys His Asn Leu Val Ile Thr Asn Ser Lys Lys 115 120 125Glu Lys Val Thr Ile Gln Asn Trp Phe Arg Glu Ala Asp Phe Ala Lys 130 135 140Glu Val Pro Asn Tyr Lys Ala Thr Lys Asp Glu Lys Ile Glu Glu Ile145 150 155 160Ile Gly Gln Asn Gly Glu Arg Ile Thr Ser Lys Gln Val Asp Asp Leu 165 170 175Ile Ala Lys Gly Asn Gly Lys Ile Thr Gln Asp Glu Leu Ser Lys Val 180 185 190Val Asp Asn Tyr Glu Leu Leu Lys His Ser Lys Asn Val Thr Asn Ser 195 200 205Leu Asp Lys Leu Ile Ser Ser Val Ser Ala Phe Thr Ser Ser Asn Asp 210 215 220Ser Arg Asn Val Leu Val Ala Pro Thr Ser Met Leu Asp Gln Ser Leu225 230 235 240Ser Ser Leu Gln Phe Ala Arg Ala Ala 24519372DNAP. multocida 19atgctactta tagataacgg tattccgatc gcttattgta gttgggcaga tttaaacctt 60gagactgagg tgaaatatat taaggatatt aattcgttaa caccagaaga atggcagtct 120ggtgacagac gctggattat tgattgggta gcaccattcg gacattctca attactttat 180aaaaaaatgt gtcagaaata ccctgatatg atcgtcagat ctatacgctt ttatccaaag 240cagaaagaat taggcaaaat tgcctacttt aaaggaggta aattagataa aaaaacagca 300aaaaaacgtt ttgatacata tcaagaagag ctggcaacag cacttaaaaa tgaatttaat 360tttattaaaa aa 37220124PRTP. multocida 20Met Leu Leu Ile Asp Asn Gly Ile Pro Ile Ala Tyr Cys Ser Trp Ala1 5 10 15Asp Leu Asn Leu Glu Thr Glu Val Lys Tyr Ile Lys Asp Ile Asn Ser 20 25 30Leu Thr Pro Glu Glu Trp Gln Ser Gly Asp Arg Arg Trp Ile Ile Asp 35 40 45Trp Val Ala Pro Phe Gly His Ser Gln Leu Leu Tyr Lys Lys Met Cys 50 55 60Gln Lys Tyr Pro Asp Met Ile Val Arg Ser Ile Arg Phe Tyr Pro Lys65 70 75 80Gln Lys Glu Leu Gly Lys Ile Ala Tyr Phe Lys Gly Gly Lys Leu Asp 85 90 95Lys Lys Thr Ala Lys Lys Arg Phe Asp Thr Tyr Gln Glu Glu Leu Ala 100 105 110Thr Ala Leu Lys Asn Glu Phe Asn Phe Ile Lys Lys 115 120212859DNAP. multocida 21atgggaacta gacttacaac cctatcaaat gggctaaaaa acactttaac ggcaaccaaa 60agtggcttac ataaagccgg tcaatcatta acccaagccg gcagttcttt aaaaactggg 120gcaaaaaaaa ttatcctcta tattccccaa aattaccaat atgatactga acaaggtaat 180ggtttacagg atttagtcaa agcggccgaa gagttgggga ttgaggtaca aagagaagaa 240cgcaataata ttgcaacagc tcaaaccagt ttaggcacga ttcaaaccgc tattggctta 300actgagcgtg gcattgtgtt atccgctcca caaattgata aattgctaca gaaaactaaa 360gcaggccaag cattaggttc tgccgaaagc attgtacaaa atgcaaataa agccaaaact 420gtattatctg gcattcaatc tattttaggc tcagtattgg

ctggaatgga tttagatgag 480gccttacaga ataacagcaa ccaacatgct cttgctaaag ctggcttgga gctaacaaat 540tcattaattg aaaatattgc taattcagta aaaacacttg acgaatttgg tgagcaaatt 600agtcaatttg gttcaaaact acaaaatatc aaaggcttag ggactttagg agacaaactc 660aaaaatatcg gtggacttga taaagctggc cttggtttag atgttatctc agggctatta 720tcgggcgcaa cagctgcact tgtacttgca gataaaaatg cttcaacagc taaaaaagtg 780ggtgcgggtt ttgaattggc aaaccaagtt gttggtaata ttaccaaagc cgtttcttct 840tacattttag cccaacgtgt tgcagcaggt ttatcttcaa ctgggcctgt ggctgcttta 900attgcttcta ctgtttctct tgcgattagc ccattagcat ttgccggtat tgccgataaa 960tttaatcatg caaaaagttt agagagttat gccgaacgct ttaaaaaatt aggctatgac 1020ggagataatt tattagcaga atatcagcgg ggaacaggga ctattgatgc atcggttact 1080gcaattaata ccgcattggc cgctattgct ggtggtgtgt ctgctgctgc agccggctcg 1140gttattgctt caccgattgc cttattagta tctgggatta ccggtgtaat ttctacgatt 1200ctgcaatatt ctaaacaagc aatgtttgag cacgttgcaa ataaaattca taacaaaatt 1260gtagaatggg aaaaaaataa tcacggtaag aactactttg aaaatggtta cgatgcccgt 1320tatcttgcga atttacaaga taatatgaaa ttcttactga acttaaacaa agagttacag 1380gcagaacgtg tcatcgctat tactcagcag caatgggata acaacattgg tgatttagct 1440ggtattagcc gtttaggtga aaaagtcctt agtggtaaag cctatgtgga tgcgtttgaa 1500gaaggcaaac acattaaagc cgataaatta gtacagttgg attcggcaaa cggtattatt 1560gatgtgagta attcgggtaa agcgaaaact cagcatatct tattcagaac gccattattg 1620acgccgggaa cagagcatcg tgaacgcgta caaacaggta aatatgaata tattaccaag 1680ctcaatatta accgtgtaga tagctggaaa attacagatg gtgcagcaag ttctaccttt 1740gatttaacta acgttgttca gcgtattggt attgaattag acaatgctgg aaatgtaact 1800aaaaccaaag aaacaaaaat tattgccaaa cttggtgaag gtgatgacaa cgtatttgtt 1860ggttctggta cgacggaaat tgatggcggt gaaggttacg accgagttca ctatagccgt 1920ggaaactatg gtgctttaac tattgatgca accaaagaga ccgagcaagg tagttatacc 1980gtaaatcgtt tcgtagaaac cggtaaagca ctacacgaag tgacttcaac ccataccgca 2040ttagtgggca accgtgaaga aaaaatagaa tatcgtcata gcaataacca gcaccatgcc 2100ggttattaca ccaaagatac cttgaaagct gttgaagaaa ttatcggtac atcacataac 2160gatatcttta aaggtagtaa gttcaatgat gcctttaacg gtggtgatgg tgtcgatact 2220attgacggta acgacggcaa tgaccgctta tttggtggta aaggcgatga tattctcgat 2280ggtggaaatg gtgatgattt tatcgatggc ggtaaaggca acgacctatt acacggtggc 2340aagggcgatg atattttcgt tcaccgtaaa ggcgatggta atgatattat taccgattct 2400gacggcaatg ataaattatc attctctgat tcgaacttaa aagatttaac atttgaaaaa 2460gttaaacata atcttgtcat cacgaatagc aaaaaagaga aagtgaccat tcaaaactgg 2520ttccgagagg ctgattttgc taaagaagtg cctaattata aagcaactaa agatgagaaa 2580atcgaagaaa tcatcggtca aaatggcgag cggatcacct caaagcaagt tgatgatctt 2640atcgcaaaag gtaacggcaa aattacccaa gatgagctat caaaagttgt tgataactat 2700gaattgctca aacatagcaa aaatgtgaca aacagcttag ataagttaat ctcatctgta 2760agtgcattta cctcgtctaa tgattcgaga aatgtattag tggctccaac ttcaatgttg 2820gatcaaagtt tatcttctct tcaatttgct agagcagct 285922953PRTP. multocida 22Met Gly Thr Arg Leu Thr Thr Leu Ser Asn Gly Leu Lys Asn Thr Leu1 5 10 15Thr Ala Thr Lys Ser Gly Leu His Lys Ala Gly Gln Ser Leu Thr Gln 20 25 30Ala Gly Ser Ser Leu Lys Thr Gly Ala Lys Lys Ile Ile Leu Tyr Ile 35 40 45Pro Gln Asn Tyr Gln Tyr Asp Thr Glu Gln Gly Asn Gly Leu Gln Asp 50 55 60Leu Val Lys Ala Ala Glu Glu Leu Gly Ile Glu Val Gln Arg Glu Glu65 70 75 80Arg Asn Asn Ile Ala Thr Ala Gln Thr Ser Leu Gly Thr Ile Gln Thr 85 90 95Ala Ile Gly Leu Thr Glu Arg Gly Ile Val Leu Ser Ala Pro Gln Ile 100 105 110Asp Lys Leu Leu Gln Lys Thr Lys Ala Gly Gln Ala Leu Gly Ser Ala 115 120 125Glu Ser Ile Val Gln Asn Ala Asn Lys Ala Lys Thr Val Leu Ser Gly 130 135 140Ile Gln Ser Ile Leu Gly Ser Val Leu Ala Gly Met Asp Leu Asp Glu145 150 155 160Ala Leu Gln Asn Asn Ser Asn Gln His Ala Leu Ala Lys Ala Gly Leu 165 170 175Glu Leu Thr Asn Ser Leu Ile Glu Asn Ile Ala Asn Ser Val Lys Thr 180 185 190Leu Asp Glu Phe Gly Glu Gln Ile Ser Gln Phe Gly Ser Lys Leu Gln 195 200 205Asn Ile Lys Gly Leu Gly Thr Leu Gly Asp Lys Leu Lys Asn Ile Gly 210 215 220Gly Leu Asp Lys Ala Gly Leu Gly Leu Asp Val Ile Ser Gly Leu Leu225 230 235 240Ser Gly Ala Thr Ala Ala Leu Val Leu Ala Asp Lys Asn Ala Ser Thr 245 250 255Ala Lys Lys Val Gly Ala Gly Phe Glu Leu Ala Asn Gln Val Val Gly 260 265 270Asn Ile Thr Lys Ala Val Ser Ser Tyr Ile Leu Ala Gln Arg Val Ala 275 280 285Ala Gly Leu Ser Ser Thr Gly Pro Val Ala Ala Leu Ile Ala Ser Thr 290 295 300Val Ser Leu Ala Ile Ser Pro Leu Ala Phe Ala Gly Ile Ala Asp Lys305 310 315 320Phe Asn His Ala Lys Ser Leu Glu Ser Tyr Ala Glu Arg Phe Lys Lys 325 330 335Leu Gly Tyr Asp Gly Asp Asn Leu Leu Ala Glu Tyr Gln Arg Gly Thr 340 345 350Gly Thr Ile Asp Ala Ser Val Thr Ala Ile Asn Thr Ala Leu Ala Ala 355 360 365Ile Ala Gly Gly Val Ser Ala Ala Ala Ala Gly Ser Val Ile Ala Ser 370 375 380Pro Ile Ala Leu Leu Val Ser Gly Ile Thr Gly Val Ile Ser Thr Ile385 390 395 400Leu Gln Tyr Ser Lys Gln Ala Met Phe Glu His Val Ala Asn Lys Ile 405 410 415His Asn Lys Ile Val Glu Trp Glu Lys Asn Asn His Gly Lys Asn Tyr 420 425 430Phe Glu Asn Gly Tyr Asp Ala Arg Tyr Leu Ala Asn Leu Gln Asp Asn 435 440 445Met Lys Phe Leu Leu Asn Leu Asn Lys Glu Leu Gln Ala Glu Arg Val 450 455 460Ile Ala Ile Thr Gln Gln Gln Trp Asp Asn Asn Ile Gly Asp Leu Ala465 470 475 480Gly Ile Ser Arg Leu Gly Glu Lys Val Leu Ser Gly Lys Ala Tyr Val 485 490 495Asp Ala Phe Glu Glu Gly Lys His Ile Lys Ala Asp Lys Leu Val Gln 500 505 510Leu Asp Ser Ala Asn Gly Ile Ile Asp Val Ser Asn Ser Gly Lys Ala 515 520 525Lys Thr Gln His Ile Leu Phe Arg Thr Pro Leu Leu Thr Pro Gly Thr 530 535 540Glu His Arg Glu Arg Val Gln Thr Gly Lys Tyr Glu Tyr Ile Thr Lys545 550 555 560Leu Asn Ile Asn Arg Val Asp Ser Trp Lys Ile Thr Asp Gly Ala Ala 565 570 575Ser Ser Thr Phe Asp Leu Thr Asn Val Val Gln Arg Ile Gly Ile Glu 580 585 590Leu Asp Asn Ala Gly Asn Val Thr Lys Thr Lys Glu Thr Lys Ile Ile 595 600 605Ala Lys Leu Gly Glu Gly Asp Asp Asn Val Phe Val Gly Ser Gly Thr 610 615 620Thr Glu Ile Asp Gly Gly Glu Gly Tyr Asp Arg Val His Tyr Ser Arg625 630 635 640Gly Asn Tyr Gly Ala Leu Thr Ile Asp Ala Thr Lys Glu Thr Glu Gln 645 650 655Gly Ser Tyr Thr Val Asn Arg Phe Val Glu Thr Gly Lys Ala Leu His 660 665 670Glu Val Thr Ser Thr His Thr Ala Leu Val Gly Asn Arg Glu Glu Lys 675 680 685Ile Glu Tyr Arg His Ser Asn Asn Gln His His Ala Gly Tyr Tyr Thr 690 695 700Lys Asp Thr Leu Lys Ala Val Glu Glu Ile Ile Gly Thr Ser His Asn705 710 715 720Asp Ile Phe Lys Gly Ser Lys Phe Asn Asp Ala Phe Asn Gly Gly Asp 725 730 735Gly Val Asp Thr Ile Asp Gly Asn Asp Gly Asn Asp Arg Leu Phe Gly 740 745 750Gly Lys Gly Asp Asp Ile Leu Asp Gly Gly Asn Gly Asp Asp Phe Ile 755 760 765Asp Gly Gly Lys Gly Asn Asp Leu Leu His Gly Gly Lys Gly Asp Asp 770 775 780Ile Phe Val His Arg Lys Gly Asp Gly Asn Asp Ile Ile Thr Asp Ser785 790 795 800Asp Gly Asn Asp Lys Leu Ser Phe Ser Asp Ser Asn Leu Lys Asp Leu 805 810 815Thr Phe Glu Lys Val Lys His Asn Leu Val Ile Thr Asn Ser Lys Lys 820 825 830Glu Lys Val Thr Ile Gln Asn Trp Phe Arg Glu Ala Asp Phe Ala Lys 835 840 845Glu Val Pro Asn Tyr Lys Ala Thr Lys Asp Glu Lys Ile Glu Glu Ile 850 855 860Ile Gly Gln Asn Gly Glu Arg Ile Thr Ser Lys Gln Val Asp Asp Leu865 870 875 880Ile Ala Lys Gly Asn Gly Lys Ile Thr Gln Asp Glu Leu Ser Lys Val 885 890 895Val Asp Asn Tyr Glu Leu Leu Lys His Ser Lys Asn Val Thr Asn Ser 900 905 910Leu Asp Lys Leu Ile Ser Ser Val Ser Ala Phe Thr Ser Ser Asn Asp 915 920 925Ser Arg Asn Val Leu Val Ala Pro Thr Ser Met Leu Asp Gln Ser Leu 930 935 940Ser Ser Leu Gln Phe Ala Arg Ala Ala945 950232124DNAP. multocida 23atggaagcta accatcaaag gaatgatctt ggtttagttg ccctcactat gttggcacaa 60taccataata tttcgcttaa tccggaagaa ataaaacata aatttgatct tgacggaaaa 120gggctttctt taactgcttg gcttttagct gcaaaatcgt tagcgttgaa agcgaaacac 180attaaaaaag agatttcccg cttacacttg gtgaatttac cggcattagt ttggcaagat 240aacggtaaac attttttatt ggtaaaagtg gataccgata ataaccgcta tttaacttac 300aatttggaac aagatgctcc acaaattctg tcacaagacg aatttgaagc ctgctatcaa 360gggcagttaa ttttggtcac gtccagagct tccgtagtag gtcaattagc aaagttcgat 420ttcacctggt ttattccggc ggtgatcaaa taccgaaaaa tctttctaga aaccttgatt 480gtttcgatct ttttgcaaat ttttgcccta attacaccgc tattcttcca agttgttatg 540gataaagtac tggtgcatcg aggtttttca accttgaata tcattacggt tgccttagct 600attgtgatca tctttgaaat tgtactaagt ggtttgagaa cctatgtttt ttctcatagc 660actagccgta ttgatgttga attaggcgct aaattatttc gacatttatt atcactaccc 720atttcttatt ttgaaaacag acgagttgga gatacagtcg ctagggttag agaattagat 780caaattcgta atttccttac cggacaagca ttaacctcgg tgttagatct cttattctct 840tttatctttt ttgccgtaat gtggtattac agcccaaaat taaccttggt aattcttggt 900tcattgccct gctatatttt atggtcaatt tttattagtc cgattttaag acggcgttta 960gatgagaaat ttgcccgaag tgctgataac caagcattct tagttgagtc ggtaacagcc 1020atcaatatga ttaaagcgat ggcggttgct ccacaaatga cggatacatg ggataaacag 1080ctggcaagct atgtttcatc aagtttccgt gtcaccgtat tagcaaccat tgggcaacaa 1140ggtgtacaac ttattcaaaa aaccgttatg gtgattaacc tttggttagg ggcacactta 1200gttatttcag gcgatctgag tattgggcaa ttaattgcct ttaatatgct atcagggcaa 1260gtgattgcac cggtgattcg gctggctcag ctctggcaag atttccaaca agttgggatt 1320tccgtcactc gcttaggtga tgttttaaac tctccaaccg aacaatatca aggcaaatta 1380tcactaccag aaataaaagg cgatatctca tttaaaaata tccgctttag atataaacca 1440gatgcaccaa ctattttaaa taatgtgaat ttagaaatta ggcaaggaga agtgattggg 1500attgttggac gttccggttc aggcaaaagt actctgacta aattactgca acgtttttat 1560attcctgaaa atgggcaggt tttgattgat ggacatgatc tagccttagc tgatccaaac 1620tggctacgcc gtcaaatagg tgtagtgctg caagataatg tgttattaaa ccgcagtatc 1680cgagaaaata ttgcgctatc agatccagga atgccaatgg agcgagtaat ttatgcagca 1740aaattagcag gggctcacga ttttatttca gaattgcgtg aaggttataa caccattgtg 1800ggtgaacaag gagcggggct ttcaggcggg caacgccaac ggattgcgat tgctcgagct 1860ttggtaaaca acccgaaaat cctgattttt gatgaggcaa ccagtgccct cgattacgaa 1920tctgagcata ttattatgca aaatatgcaa aaaatatgcc aaggcagaac cgtgattttg 1980attgcacatc gtttatcgac cgtcaaaaat gcggatcgaa ttattgtgat ggaaaagggg 2040gaaattgttg agcaaggcaa gcaccacgaa ttactgcaaa acagtaacgg actttattcc 2100tacttacacc aattacaact taat 212424708PRTP. multocida 24Met Glu Ala Asn His Gln Arg Asn Asp Leu Gly Leu Val Ala Leu Thr1 5 10 15Met Leu Ala Gln Tyr His Asn Ile Ser Leu Asn Pro Glu Glu Ile Lys 20 25 30His Lys Phe Asp Leu Asp Gly Lys Gly Leu Ser Leu Thr Ala Trp Leu 35 40 45Leu Ala Ala Lys Ser Leu Ala Leu Lys Ala Lys His Ile Lys Lys Glu 50 55 60Ile Ser Arg Leu His Leu Val Asn Leu Pro Ala Leu Val Trp Gln Asp65 70 75 80Asn Gly Lys His Phe Leu Leu Val Lys Val Asp Thr Asp Asn Asn Arg 85 90 95Tyr Leu Thr Tyr Asn Leu Glu Gln Asp Ala Pro Gln Ile Leu Ser Gln 100 105 110Asp Glu Phe Glu Ala Cys Tyr Gln Gly Gln Leu Ile Leu Val Thr Ser 115 120 125Arg Ala Ser Val Val Gly Gln Leu Ala Lys Phe Asp Phe Thr Trp Phe 130 135 140Ile Pro Ala Val Ile Lys Tyr Arg Lys Ile Phe Leu Glu Thr Leu Ile145 150 155 160Val Ser Ile Phe Leu Gln Ile Phe Ala Leu Ile Thr Pro Leu Phe Phe 165 170 175Gln Val Val Met Asp Lys Val Leu Val His Arg Gly Phe Ser Thr Leu 180 185 190Asn Ile Ile Thr Val Ala Leu Ala Ile Val Ile Ile Phe Glu Ile Val 195 200 205Leu Ser Gly Leu Arg Thr Tyr Val Phe Ser His Ser Thr Ser Arg Ile 210 215 220Asp Val Glu Leu Gly Ala Lys Leu Phe Arg His Leu Leu Ser Leu Pro225 230 235 240Ile Ser Tyr Phe Glu Asn Arg Arg Val Gly Asp Thr Val Ala Arg Val 245 250 255Arg Glu Leu Asp Gln Ile Arg Asn Phe Leu Thr Gly Gln Ala Leu Thr 260 265 270Ser Val Leu Asp Leu Leu Phe Ser Phe Ile Phe Phe Ala Val Met Trp 275 280 285Tyr Tyr Ser Pro Lys Leu Thr Leu Val Ile Leu Gly Ser Leu Pro Cys 290 295 300Tyr Ile Leu Trp Ser Ile Phe Ile Ser Pro Ile Leu Arg Arg Arg Leu305 310 315 320Asp Glu Lys Phe Ala Arg Ser Ala Asp Asn Gln Ala Phe Leu Val Glu 325 330 335Ser Val Thr Ala Ile Asn Met Ile Lys Ala Met Ala Val Ala Pro Gln 340 345 350Met Thr Asp Thr Trp Asp Lys Gln Leu Ala Ser Tyr Val Ser Ser Ser 355 360 365Phe Arg Val Thr Val Leu Ala Thr Ile Gly Gln Gln Gly Val Gln Leu 370 375 380Ile Gln Lys Thr Val Met Val Ile Asn Leu Trp Leu Gly Ala His Leu385 390 395 400Val Ile Ser Gly Asp Leu Ser Ile Gly Gln Leu Ile Ala Phe Asn Met 405 410 415Leu Ser Gly Gln Val Ile Ala Pro Val Ile Arg Leu Ala Gln Leu Trp 420 425 430Gln Asp Phe Gln Gln Val Gly Ile Ser Val Thr Arg Leu Gly Asp Val 435 440 445Leu Asn Ser Pro Thr Glu Gln Tyr Gln Gly Lys Leu Ser Leu Pro Glu 450 455 460Ile Lys Gly Asp Ile Ser Phe Lys Asn Ile Arg Phe Arg Tyr Lys Pro465 470 475 480Asp Ala Pro Thr Ile Leu Asn Asn Val Asn Leu Glu Ile Arg Gln Gly 485 490 495Glu Val Ile Gly Ile Val Gly Arg Ser Gly Ser Gly Lys Ser Thr Leu 500 505 510Thr Lys Leu Leu Gln Arg Phe Tyr Ile Pro Glu Asn Gly Gln Val Leu 515 520 525Ile Asp Gly His Asp Leu Ala Leu Ala Asp Pro Asn Trp Leu Arg Arg 530 535 540Gln Ile Gly Val Val Leu Gln Asp Asn Val Leu Leu Asn Arg Ser Ile545 550 555 560Arg Glu Asn Ile Ala Leu Ser Asp Pro Gly Met Pro Met Glu Arg Val 565 570 575Ile Tyr Ala Ala Lys Leu Ala Gly Ala His Asp Phe Ile Ser Glu Leu 580 585 590Arg Glu Gly Tyr Asn Thr Ile Val Gly Glu Gln Gly Ala Gly Leu Ser 595 600 605Gly Gly Gln Arg Gln Arg Ile Ala Ile Ala Arg Ala Leu Val Asn Asn 610 615 620Pro Lys Ile Leu Ile Phe Asp Glu Ala Thr Ser Ala Leu Asp Tyr Glu625 630 635 640Ser Glu His Ile Ile Met Gln Asn Met Gln Lys Ile Cys Gln Gly Arg 645 650 655Thr Val Ile Leu Ile Ala His Arg Leu Ser Thr Val Lys Asn Ala Asp 660 665 670Arg Ile Ile Val Met Glu Lys Gly Glu Ile Val Glu Gln Gly Lys His 675 680 685His Glu Leu Leu Gln Asn Ser Asn Gly Leu Tyr Ser Tyr Leu His Gln 690 695 700Leu Gln Leu Asn705251434DNAP. multocida 25atgaaaatat ggcttagtgg tatttatgaa tttttcctac gctataaaaa catttgggca 60gaagtatgga aaattcgtaa agaattagac cacccaaaca gaaaaaaaga cgaaagtgaa 120tttttaccgg cacatttaga actgattgaa accccggttt ctaaaaaacc acgtctaatt 180gcttatttga ttatgctatt tttagttgtg gcaattgtgc ttgccagtgt aagcaaagtt 240gaaattgtgg cgactgctcc cggtaaatta acttttagtg gcagaagtaa agaaattaaa 300ccgattgaaa acgccattgt

acaagaaatt ttcgttaaag atgggcagtt tgtggaaaaa 360gggcaattat tagtcagctt aactgcattg ggttctgatg cagatatcaa aaagaccatg 420gcttcacttt ctttagctaa actggagaac tatcgctacc aaactttgct tactgccatt 480gaaaaagagt ccttgccggt gattgattta tctagaaccg aatttaaaga ttcatcggaa 540gaagatcgac tacgtattaa acacttaatt gaggagcaat acaccacttg gcaaaaacaa 600aaaacacaga aaactttagc gtataagcgt aaagaggctg aaaaacaaac aatatttgcc 660tatgtccgta aatatgaagg tgcaacacgt attgaacaag aaaaattaaa agactttaag 720gcactttata aacagaagtc tttatctaag cacgaacttc ttgcgcaaga aaataaatta 780attgaggctc agaatgagct agctgtttat cgctcaaaat taaatgaatt agaaaatgat 840ctactcaatg taaaagaaga acttgaattg atcacgcaat tctttaaaag cgatgtgttg 900gaaaaattaa agcaacatat tgaaaatgaa cgccaacttc ggctcgagtt agaaaaaaat 960aatcaacgca gacaggcctc gatgatcaga gcaccggttt ccggtacggt tcagcaactg 1020aaaattcaca ctataggtgg tgttgttacg actgctgaaa ccttgatgat cattgtgccg 1080gaagacgatg tgttagaggc caccgctctg gttccaaaca aagatatcgg ctttgttgca 1140gcagggcagg aggtgattat taaagtggaa actttccctt atacacgcta tggttatcta 1200actggtcgaa ttaaacatat tagcccggat gcgattgaac aacctaatgt aggcttagtt 1260tttaatgcaa ctatagctat agataggaag aatctaacat cgcctgatgg gcgaaaaatt 1320gatttgagtt caggtatgac aataactgct gaaatcaaaa ccggtgaacg gagtgtaatg 1380agttatttac tcagcccatt agaagaatct gtcacagaaa gtttaaggga acgc 143426478PRTP. multocida 26Met Lys Ile Trp Leu Ser Gly Ile Tyr Glu Phe Phe Leu Arg Tyr Lys1 5 10 15Asn Ile Trp Ala Glu Val Trp Lys Ile Arg Lys Glu Leu Asp His Pro 20 25 30Asn Arg Lys Lys Asp Glu Ser Glu Phe Leu Pro Ala His Leu Glu Leu 35 40 45Ile Glu Thr Pro Val Ser Lys Lys Pro Arg Leu Ile Ala Tyr Leu Ile 50 55 60 Met Leu Phe Leu Val Val Ala Ile Val Leu Ala Ser Val Ser Lys Val65 70 75 80Glu Ile Val Ala Thr Ala Pro Gly Lys Leu Thr Phe Ser Gly Arg Ser 85 90 95Lys Glu Ile Lys Pro Ile Glu Asn Ala Ile Val Gln Glu Ile Phe Val 100 105 110Lys Asp Gly Gln Phe Val Glu Lys Gly Gln Leu Leu Val Ser Leu Thr 115 120 125Ala Leu Gly Ser Asp Ala Asp Ile Lys Lys Thr Met Ala Ser Leu Ser 130 135 140Leu Ala Lys Leu Glu Asn Tyr Arg Tyr Gln Thr Leu Leu Thr Ala Ile145 150 155 160Glu Lys Glu Ser Leu Pro Val Ile Asp Leu Ser Arg Thr Glu Phe Lys 165 170 175Asp Ser Ser Glu Glu Asp Arg Leu Arg Ile Lys His Leu Ile Glu Glu 180 185 190Gln Tyr Thr Thr Trp Gln Lys Gln Lys Thr Gln Lys Thr Leu Ala Tyr 195 200 205Lys Arg Lys Glu Ala Glu Lys Gln Thr Ile Phe Ala Tyr Val Arg Lys 210 215 220Tyr Glu Gly Ala Thr Arg Ile Glu Gln Glu Lys Leu Lys Asp Phe Lys225 230 235 240Ala Leu Tyr Lys Gln Lys Ser Leu Ser Lys His Glu Leu Leu Ala Gln 245 250 255Glu Asn Lys Leu Ile Glu Ala Gln Asn Glu Leu Ala Val Tyr Arg Ser 260 265 270Lys Leu Asn Glu Leu Glu Asn Asp Leu Leu Asn Val Lys Glu Glu Leu 275 280 285Glu Leu Ile Thr Gln Phe Phe Lys Ser Asp Val Leu Glu Lys Leu Lys 290 295 300Gln His Ile Glu Asn Glu Arg Gln Leu Arg Leu Glu Leu Glu Lys Asn305 310 315 320Asn Gln Arg Arg Gln Ala Ser Met Ile Arg Ala Pro Val Ser Gly Thr 325 330 335Val Gln Gln Leu Lys Ile His Thr Ile Gly Gly Val Val Thr Thr Ala 340 345 350Glu Thr Leu Met Ile Ile Val Pro Glu Asp Asp Val Leu Glu Ala Thr 355 360 365Ala Leu Val Pro Asn Lys Asp Ile Gly Phe Val Ala Ala Gly Gln Glu 370 375 380Val Ile Ile Lys Val Glu Thr Phe Pro Tyr Thr Arg Tyr Gly Tyr Leu385 390 395 400Thr Gly Arg Ile Lys His Ile Ser Pro Asp Ala Ile Glu Gln Pro Asn 405 410 415Val Gly Leu Val Phe Asn Ala Thr Ile Ala Ile Asp Arg Lys Asn Leu 420 425 430Thr Ser Pro Asp Gly Arg Lys Ile Asp Leu Ser Ser Gly Met Thr Ile 435 440 445Thr Ala Glu Ile Lys Thr Gly Glu Arg Ser Val Met Ser Tyr Leu Leu 450 455 460Ser Pro Leu Glu Glu Ser Val Thr Glu Ser Leu Arg Glu Arg465 470 475

Claims

1. A vaccine comprising an attenuated Mannheimia haemolytica (M. haemolytica) A1 strain and an attenuated M. haemolytica A6 strain, which vaccine provides a safe and protective immune response in a bovine against both M. haemolytica strain A1 and M. haemolytica strain A6, or diseases caused by M. haemolytica strains A1 and A6; and wherein both the A1 and A6 strains contain nucleic acid deletions in their respective leukotoxin A (lktA) genes, which deletions have rendered the strains attenuated relative to the virulent parental strains A1 and A6 from which the attenuated strains A1 and A6 were produced.

2. The vaccine of claim 1, consisting essentially of the attenuated strains.

3. The vaccine of claim 1, further comprising an adjuvant.

4. The vaccine of claim 1, wherein a safe and protective intranasal dose of the vaccine comprises from about 1.19.times.10.sup.6 to 1.19.times.10.sup.7 CFU of the attenuated A1 strain and from about 9.2.times.10.sup.5 to 9.2x 10.sup.6 CFU of the attenuated A6 strain.

5. The vaccine of claim 4, further comprising a pharmaceutically or veterinary acceptable vehicle, diluent or excipient and from about 1.19.times.10.sup.6 to 1.19.times.10.sup.7 CFU of the attenuated A1 strain and from about 9.2.times.10.sup.5 to 9.2.times.10.sup.6 CFU of the attenuated A6 strain.

6. The vaccine of claim 5, further comprising an adjuvant.

7. The vaccine of claim 6, wherein the adjuvant is inactivated bacteria, inactivated virus, fractions of inactivated bacteria, bacterial lipopolysaccharides, bacterial toxins, or derivatives or combinations thereof.

8. The vaccine of claim 4, which provides a protective immune response in a bovine against an experimental challenge of about 2.4.times.10.sup.9 CFU of virulent M. haemolytica strain A1.

9. The vaccine of claim 1, further comprising at least one additional antigen associated with a bovine pathogen other than M. haemolytica.

10. A method of vaccinating an animal comprising administering at least one dose of the vaccine of claim 1.

11. The method of claim 10, wherein the animal is a bovine.

12. The method of claim 11, wherein the bovine is a calf that is 28 days or older.

13. An immunological composition suitable for the prevention of bovine respiratory disease caused by M. haemolytica, comprising the vaccine of claim 1, and further comprising an immunologically effective amount of attenuated Pasteurella multocida and Histophilus somni.

14. The method of claim 10, wherein the vaccine is administered intranasally.