CA1340712C - Leukotoxin gene of pasturella haemolytica - Google Patents

Abstract

The gene coding for Pasteurella haemolytica leukotoxin can be cloned in a plasmid expressed in Escherichia coli. The leukotoxin gene can be isolated from a clone bank of P. haemolytica. The clone bank is constructed by partial digestion of genomic DNA. The resultant 5 to 10 kilobase-pair fragments are ligated into plasmid vector pBR322. The resultant clones are screened for the production of P. haemolytica soluble antigens by a colony enzyme-linked immunosorbent assay blot method with a rabbit antiserum raised against the soluble antigens. The clones producing P. haemolytica soluble antigens are then analyzed for the production of the leukotoxin by a cytotoxicity assay with cells from a bovine leukemia-derived B-lymphocyte cell line as the target cells. Positive clones are identified, and subsequent restriction analysis of the recombinant plasmids shows the same insert DNA is cloned in the plasmid vector. The DNA sequence analysis of the insert DNA reveals regions coding for the leukotoxin.

Description

h LEUKOTOXIN GENE OF PASTEURELLA HAEMOLYTICA
Field of the Invention This invention relates to the cloning and expression of the leukotoxin gene of Pasteurella haemolytica in a suitable host microorganism.
Background of the Invention Bovine pneumonic pasteurellosis associated with Pasteurella haemolytica A1 is a major cause of sickness and death in feedlot cattle. Although vaccination with this organism might be expected to produce immunity to the disease, experimental trials and field studies using conventional formal.inized bacterins have failed to show a protective effect, in fact, vaccinated animals are frequently more susceptible to the disease than their non-vaccinated counterparts.
Immunization with live P. haemolytica has been shown to protect cattle against experimental challenge exposure to the bacterium. Live P. haemolytica produces a cytotoxin with specificity for ruminant leukocytes. This may contribute to the pathogenesis of pneumonic pasteurellosis by impairing primary lung defense and subsequent immune response or by induction of inflammation as a consequence of leukocyte lysis.
The protective capability of cytotoxic supernate from P. haemolytica has been used as a vaccine. This preparation contains numerous soluble antigens from the bacterium which may be important in protection. These soluble antigens include a ruminant-leukocyte-specific cytotoxin, serotype-specific soluble surface antigens, neuraminidase and protease. An example of such vaccine and its development is disclosed in applicant's copending Canadian Patent application Serial No. 499,833 filed January 17, 1986. Developments of vaccines from the crude cytotoxic supernate requires the purification and characterization of these antigens which can become a difficult and costly process.
Recent advances in molecular biology have provided a new approach in the characterization of bacterial determinants involved in pathogenicity. The particular genes which code for bacterial antigens can be isolated by molecular cloning using various recombinant DNA
techniques. For example, it is known the genes which code for the heat-labile enterotoxins of E. coli of porcine and human origin have been isolated. The cholera toxin genes from Vibrio cholera have also been isolated using the cloned E-. coli elt genes as a hybridization probe. Genes encoding the enzymatic moiety of the exotoxin A from Pseudomonas aeruginosa and the phospholipase C determinant of P. aeruqinosa have also been successfully cloned and expres sed in E. coli.
These cloned genes greatly facilitate the analyses of the pathogenic and immunogenic characteristics of the toxin protein products as well as enabling the characterization of the genetic organization and regulation of expression of these bacterial toxins. Hence, an alternative method involving the isolating o~ genes coding for the soluble antigens of P. haemolytica would be an important advance over existing vaccine developed from culture supernatant.
Summary of the Invention According to an aspect of the invention a nucleotide sequence coding for leukotoxin which is a protein produced by tlhe metabolism of Pasteurella haemolytica is provided.
According to another aspect of the invention a recombinant plasmid may be developed which includes the nucleotide sequence coding for the leukotoxin.
According to another aspect of the invention a phage may be developed having the nucleotide sequence.
According to another aspect of the invention the nucleotide sequence is a DNA fragment of approximately 8.7 kbp.
According to another aspect of the invention a micro-organism may be transformed with the recombinant plasmid and, in particular, may be that having the identifying characteristics of ATCC deposition #68025.
According to another aspect of the invention, the nucleotide sequence of the leukotoxin may be altered to produce modified derivatives of the leukotoxin.

According to another aspect of the invention a process for producing a foreign proteinaceous leukotoxin having a molecular weight of approximately 100,000 daltons comprises transforming a micro-organism-with the nucleotide sequence. The transformed micro-organism is then cultured under suitable conditions to yield the leukotoxin. Accordingly, the leukotoxin may be isolated and a vaccine may be developed against Pasteurella haemolytica by mixing isolated leukotoxin and/or its derivatives with suitable carriers.
According to another aspect of the invention polyclonal and monoclonal antibodies may b~ raised to the leukotoxin and/or derivatives.
According to another aspect of the invention the entire nucleotide sequence or a portion thereof may be used as a DNA probe.
According to another aspect of the invention, a process for making a vaccine effective against Pasteurella haemolytica comprising mixing:
A. (i) transforming a microorganism with a DNA sequence encoding for leukotoxin which is a protein produced by Pasteurella haemolytica, wherein said leukotoxin exhibits cytotoxic activity specific against leukocytes;
(ii) culturing the resulting transformed microorganism; and (iii) obtaining the resulting leukotoxin produced from said cultured transformed microorganism;
with B. a vaccine carrier.
According t:o another aspect of the invention, a vaccine effective against Pasteurella haemolytica comprising:
(i) a leuk:otoxin which is produced by:
(a) transforming a microorganism with a DNA
~~equence encoding for leukotoxin which is r, " a protein produced by Pasteurella 1..~~~~r~~

haemolytica, wherein said leukotoxin exhibits cytotoxic activity specific against leukocytes;
(b) culturing the resulting transformed microorganism; and (c) obtaining the resulting leukotoxin produced from said cultured transformed microorganism; and (ii) a vaccine carrier.
Brief Description of the Drawings Preferred embadiments of the invention are exemplified in the drawings wherein:
Figure 1 is an Agarose gel electrophoresis profile of the Pasteurella haemolytica plasmid clone bank;
Figure 2 are restriction maps of the recombinant plasmids which contain the Pasteurella haemo ytica leukotoxin gene;
Figure 3 is a Southern blot hybridization analysis of insert DNA. from the plasmid pPHS against total Pasteurella haemolytica genomic DNA;
Figure 4 is a Western blot analysis of the cellular proteins from. the E. coli clones carrying pPHS, pPHlO and the vector pHR322; and Figure 5 are restriction maps of additional recombinant plasmids which contain the P. haemolytica leukotoxin gene.
Detailed Description of the Preferred Embodiments Based on. the work disclosed in applicant's copending Canadian Patent application Serial No. 499,833 non-viable vaccines protecting calves against experimental challenge by intrabronchular inoculation with live P. haemolytica can be developed from the culture supernatant.
Vaccinated calves had significantly lower pneumonic scores based on clinical signs and postmortem lesions compared to unvaccinated calves or bacterin vaccinated calves. As noted, however, such vaccines include other antigens aside from the leukotoxin present in the culture supernate. I:n an attempt to locate a DNA sequence which might code for the leukotoxin, as expressed in P.

haemolytica, a clone bank of P. haemolytica A1 genomic DNA was constructed in Escherichia coli using the plasmid vector pBR322;. From this clone bank, a collection of recombinant plasmids coding for the soluble antigens of P. haemolytic:a A1 were isolated. The F. @1 clones were screened for the presence of P. haemolytica antigens by the colony EhISA technique (enzyme-linked immunosorbant assay) using a rabbit antiserum raised to the soluble antigens of _F!. haemolytica A1. From this collection, plasmids coding for the leukotoxin were identified by screening protein preparations from the E. coli clones for leukotoxi.n activity using the neutral red assay. The presence of t:he leukotoxin in the E_. coli cells was confirmed by serum neutralization using both rabbit and calf sera. The cloned leukotoxin was identified as a protein having a molecular weight of approximately 100,000 daltons. Such identification was through SDS-PAGE and Western blot analysis of the cytoplasmic proteins from the E. coli clones. DNA sequence analysis revealed the complete sequence of the leukotoxin gene on the cloned fragment in the plasmid.
Looking specifically at the methodology and techniques for isolating and identifying the leukotoxin gene, the fo7.lowing demonstrates a preferred embodiment thereof. In the following procedures and examples, the materials and methods were:
Bacteria and plasmid strains. P. haemolytica A1 ATCC 43270 (biotype A, serotype 1) was originally obtained from E.L. Biberstein, University of California, Davis. Stocl~: organisms were maintained as lyophilized cultures after freeze-drying in distilled water containing (wt/vol) 5% dextran (molecular weight, 70,000), 7% sucrose, and 1% monosodium glutamate.
Escheric:hia coli HB101 was used as the recipient, and plasmid pBR322 was used as the vector for the construction of the clone bank. The E. coli HB101 is characterized by deposit at ATCC 33694. Their genotypes and characteristics were described by Maniatis et al (1982, molecular cloning: a laboratory manual; Cold ~~~Q~1~

Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) and Bolivar et al (1977, Construction and characterization of new cloning vehicles. II. A multipurpose cloning system; Gene 2:95-113), respectively.
Media, enzymes and chemicals. P. haemolytica was cultured in brain heart infusion broth (Difco Laboratories, Detroit, Mich.) while _E. coli cultures were grown in LT medium as described by R.Y.C. Lo and L.A.
Cameron, A Simple Immunological Detection Method for the Direct Screening of Genes from Clone Banks (Can. J. of Biochem. and Cell Biol., Vol. 64, No. 1, 73-76, 1986).
Where appropriate the antibiotics ampicillin and tetracycline were supplemented at 100 and 12.5 mg/liter, respectively, after the medium was autoclaved.
Restriction endonuclease, T4 DNA ligase, E. coli DNA
polymerase I, and bacterial alkaline phosphate were purchased from Bethesda Research Laboratories, Inc., Gaithersburg, Md.; GIBCO, Burlington, Ontario, Canada; or Boehringer Mannheim, Dorval, Quebec, Canada and used as described by the suppliers. Low-melting-point agarose was from Bethesda Research Laboratories. [alpha- 32 P]DATP (3,000 Ci/mmol) was purchased from @lew England Nuclear Corp., Lachine, Quebec.
Procedure 1 Construction of a clone bank of P. haemolytica DNA
in E.coli A clone bank of P. haemolytica A1 was constructed by the method of Nasmyth and Reed (1980, Isolation of genes by com~~lementation in yeast: molecular cloning of a cell=cycle gene. Proc. Natl. Acad. Sci. USA
77:2119-2123). Genomic DNA was extracted from P.
haemolytica P,1 by sodium dodecyl sulfate (SDS) lysis of the bacterial. cells and phenol extraction (Marmer, J.
1961. A procedure for the isolation of deoxyribonucleic acid from microorganisms. J. Mol. Biol. 3:208-218).
The DNA was then partially digested by the restriction endonuclease Sau3A, and fragments of 5 to 10 kilobase pairs were recovered by a sucrose gradient centrifugation by the method of Maniatis et al (1978. The isolation of structural genes from libraries of eukaryotic DNA. Cell 15: 687-701). 'These fragments were inserted into the unique BamHI site of the plasmid vector pBR322 which had been dephosphor;ylated by bacterial alkaline phosphatase.

The ligated DNA mixture was transformed into E. coli competent cells, and recombinants were recovered by selecting for ampicillin-resistant (Amp r ) colonies.

The recombinant; were pooled and amplified in broth cultures, and p:lasmid DNA was prepared by cesium chloride-ethidium bromide centrifugation (Thompson, R., S.G. Hughes, an<i P. Broda. 1974. Plasmid identification using specific E~ndonucleases. Mol. Gen. Genet.

133:141-149) anti stored at -20C.

Procedure 2 Preparation of Rabbit antiserum to soluble antigens and screening of the clone bank New Zealand White rabbits (Riemens Fur Ranches Ltd., St. Agatha, Ontario, Canada) were immunized with lyophilized P. haemolytica culture supernatant prepared essentially as described previously (Shewen, P.E., and B.N. Wilkie. 19:2. Cytotoxin of Pasteurella haemolytica acting on bovine: leukocytes. Infec. Immun. 35:91-94) except that 7o a.utologous rabbit serum was substituted for fetal bovine: serum in the growth medium. Each rabbit received a total of seven inoculations of 10 mg of crude supernatant in 1 ml of= saline, divided equally intradermally and intravenously, at intervals of 10 to 12 days. The first two intradermal inoculations were emulsified in complete Freund adjuvant (Difco).

Serum was collected 1 week after the seventh immunization. This serum had a titer to P. haemolytica surface antigens of 1/32, as measured by direct bacterial agglutination (Shewen, P.E., and B.N. Wilkie. 1982.

Antibody titer to Past.eurella haemolytica Al in Ontario beef cattle. Can. J. Comp. Med. 46:354-356), and a neutralizing titer to P. haemolytica leukotoxin of 1/640, as measured in the microplate assay described below.

The antibody preparation was used to screen the clone bank for production of P. haemolytica antigens by the colony enzyme-linked immunosorbent assay (ELISA) blot method (Lo and Cameron, supra). Briefly, the _E. coli recombinant clones were grown on nitrocellulose paper, and the cells were lysed with chloroform vapor. The nitrocellulose paper was immersed in a blocking solution of TBS buffer (20 mM Tris hydrochloride [pH 7.5], 500 mM
NaCl) containing 3% gelatin, 150 mM NaCl, 5 mM MgC2, 1 ug of DNase I per ml, and 40 ug of lysozyme per ml for 1 h and then inoculated with the rabbit antiserum (1/200 dilution) in TBS buffer overnight. After being washed twice in TBS buffer, the nitrocellulose paper was reacted with goat anti-rabbit immunoglobulin G (IgG) conjugated with horseradish peroxidase (Bio-Rad Laboratories, Mississauga, Ontario, Canada) at a 1/2,000 dilution in TBS buffer for 1 h. The blots were washed twice in TBS
buffer and developed in horseradish peroxidase color development reagents (Bio-Rad).
Procedure 3 Aaarose gel electrophoresis and Southern blot analysis The restriction fragments of the recombinant plasmids were: analyzed by agarose gel electrophoresis as previously described by Lo and Cameron, (supra). For Southern blot. analysis (Southern, E.M. 1975. Detection of specific ~:equences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517), P_.
haemolytica g~enomic DNA was digested with an appropriate restriction endonuclease, electrophoresed on an agarose gel, and blotaed onto nitrocellulose paper by electrophoret:ic transfer. The DNA fragments to be used as probes were recovered from the recombinant plasmids after enzyme digestion and purification on low-melting-~>oint agarose gels by the method of Wieslander (Hiieslander, L. 1979. A simple method to recover intact high molecular weight RNA and DNA after electrophoret;ic separation in low gelling temperature agarose gels. Anal. Biochem. 98:305-309) and labeled with [alpha-3ZP]dATP by nick-translation by the procedure of Rigby et al (Rigby, P.W.J., M. Dieckmann, C. Rhodes, and P. Berg. 1977. Labelling deoxyribonucleic acid to ~~3~07~~

high specific activity in vitro by nick translation with DNA polymerase I.J. Mol. Biol. 113:237-251).
Procedure 4 preparation of periplasmic and cellular proteins The ~. co ' cells carrying the recombinant plasmids were subjected to osmotic shock treatment by the method of Neu and Heppel (1965. The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. J. Biol. Chem.
240:3685-3692). The resulting cell suspension was stirred for 10 min on ice and then centrifuged, and the supernatant was recovered. The supernatant was adjusted to 0.01 M Tris hydrochloride (pH 8), concentrated by centrico ~units (Amicon Corp., Oakville, Ontario, Canada), stored at 4°C, and designated as the periplasmic protein preparatian. The cell pellet was suspended in 10 ml of 0.01 M Tris hydrochloride (pH 8), sonicated at 100 W for 1 min, and centrifuged at 100,000 x g for 1 h at 4°C. The sonicated supernatant recovered was designated as the cellular protein preparation. The enzymes cyclic phosphodiesterase and beta-galactosidase were assayed as markers for periplasmic and cellular proteins, respectively, as described previously (Neu, H.C., and L.A. Heppel. 1965. The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. J.Biol. Chem.
240:3685-3692). The protein concentrations of the preparations were determined by the method of Lowry (1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275).
Procedure 5 SDS-polyacrvlamide gel electro~lhoresis and Western blot analysis The periplasmic and cellular proteins from the ~.
coli recombinant clones were analyzed by SDS-polyacryl-amide gel electrophoresis by the method of Hancock and Carey (1979. Outer membrane of Pseudomonas aeruqinosa:
heat- and 2-mercaptoethanol-modifiable proteins. J.
Bacteriol. 140:902-910). The separating gel consisted of 153~0'~1~
to 9% (wt/vol) acrylamide (acrylamide/bisacrylamide ratio, 40:0.8) in 0.4 M Tris hydrochloride (pH 8.8), 0.09 M
NaCl-1% SDS, while the stacking gel consisted of 3%
(wt/vol) acrylamide (acrylamide/bisacrylamide ratio, 20:0.8) in 0.13 M Tris hydrochloride (pH 7), 1% SDS. For each lane, approximately 15 ~,g of protein was mixed with the solubilization reduction mixture (Hancock, R.E.W., and A.M. Ca:rey. 1979. Outer membrane of Pseudomonas aeruqinosa: heat- and 2-mercaptoethanol-modifiable proteins. .J. Bacteriol. 140:902-910) and electrophoresed at 150 V with 0.025 M Tris, 0.2 M glycine (pH 8.4), 1%
SDS as the :running buffer. Protein bands were visualized by staining the 0.05% Coomassie blue 8250 (wt/vol) in 10%
acetic acid, 14% methanol.
For Weatern blot analysis (Burnette, W.N. 1981.
"Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem.
112:195-203), the proteins were transferred to nitrocellulose paper after SDS-polyacrylamide gel electrophoresis in a TRANS-BLOT~ ell apparatus (Bio-Rad) with 0.025 1K Tris, 0.2 M glycine (pH 8.4), 20% methanol as the blotting buffer. The P. haemolytica proteins were then detected by ELISA with the rabbit antiserum preparation described above.
Proce~ire 6 Evaluation of leukotoxic activity The leukotoxic activities of periplasmic and cellular protein preparations were measured in a microplate cytotoxicity assay C.N. Greer and P.E. Shewen "Automated Colourimetric Assay for Detection of Pasteurella haemolytica Leukotoxin" Vet. Micro. 12, 31-42, 1986 with BL-3 cells, a bovine leukemia-derived B-lymphocyt~a cell line (originally obtained from G.
Theilen, University of California, Davis), as targets.
Cell viability, measured as the uptake of the vital dye neutral red, was determined by reading the optical density of each well at 540 nm with an automated .a_ 134I~'~12 spectrophotometer. After overnight dialysis against RPMI
1640 medium, ~xuadruplicate 200 ~1 samples (at protein concentrations of 6 mg/ml) were incubated with 2 x 105 cell in each of four wells of a microtiter plate for 1 h at 37°C. Lyo~>hilize~d P. haemolytica culture supernatant (Shewen, P.E., and B.N. Wilkie. 1982. Cytotoxin of Pasteurella h<~emolLrtica acting on bovine leukocytes.
Infec. Immun. 35:9 1-94), reconstituted at 3 mg/ml in RPMI
1640 medium, was used as the positive control for toxic activity. Percent toxicity was calculated as the percent loss of viabi=Lity by comparing the mean optical density in test wells wit=h that in control wells containing cells incubated with RPMI 1640 medium only. The heat lability of toxic activity was determined by preheating an aliquot of each samples at 56°C for 30 min. before testing. Host species specificity in toxic activity was confirmed by retesting toxic samples with canine, porcine, and human peripheral blood lymphocytes purified by density gradient centrifugation on Ficoll-Hypaque~ (Shimizu, M., I.C. Pan, and W.R. Hess. 1976., T and B lymphocytes in porcine blood. Am. J. Vet. Res. 36:309-317) as targets. In addition, the rabbit, antiserum and a bovine serum with antitoxic activity, obtained from an infected calf, were tested in serial twofold dilutions for the ability to neutralize to~;icity in one of the clone-derived samples.
In accordance with the above procedures the following methodology was used to achieve the results noted with reference to t:he fo7.lowing figures .
Method No. 1 Construction of a clone bank of P. haemolytica About 4 }: 103 E. coli colonies were recovered after transformation with the P. haemolytica DNA fragments and the pBR322 DNF, ligat:ion mixture, of which less than 1~
were also tetracycline resistant. The transformants were pooled from the agar plates and amplified in broth cultures in L'f medium containing ampicillin, and plasmid DNA was prepared by cesium chloride-ethidium bromide centrifugation for :storage (-20°C) as the clone bank.

Plasmid DNA of the clone bank was analyzed by agarose gel electrophoresis, which showed that it contained plasmid s larger than the vector pBR322. In Figure 1 lane a is plasmid vector pBR322; lane b is plasmid DNA from the P.
haemolytica clone bank. Shown are covalently closed circular (ccc), and (oc) open circular forms of pBR322.
Method No. 2 Isolation of recombinant plasmids coding' the P.
haemolytica soluble antigens Plasmid DNA from the clone bank was transformed into E. coli competent cells, and the transformants were screened for P. haemolytica antigens by the colony ELISA
blot method to detect the production of P. haemolytica antigens, Lo and Cameron, (supra). Twenty-seven positive recombinants were identified. Periplasmic and cellular proteins were prepared from the positive clones to assay for the P. haemolytica leukotoxin.
Method No. 3 Characterization of the recombinant plasmids Plasmid DNA from the positive recombinant clones was analyzed by restriction endonuclease mapping, and the results indicated that some of the recombinant plasmids had the same insert DNA. Four recombinant plasmids, 10, 11, 1.6, and 18, were found to have the same restriction map in that a 6.3-kilobase-pair (kbp) insert was cloned in the vector pBR322 as shown in Figure 2. The heavy lines represent pBR322 sequences and light lines represent insert sequences.
(a) Plasmid pBR322 is represented linearly at the coordinates of 3 kdbp (b) Recombinant plasmids pPH 5 and pPH 6 (c) Recambinant plasmids pPH 10, pPH 11, pPH
16, and pPH 18 The nomenclature used in Figure 2 has the following meaning:
P is equivalent to restriction site PstI H is equivalent to restriction site HincII C is equivalent to restriction site ClaI A is equivalent to restriction site AvaI PI is equivalent to restriction site PvuI B is equivalent to restriction site BamHI PII is equivalent to restriction site PvuII B/S is equivalent to restriction site BamHI-Sa:u3A junctions In this restriction mapping there are no restriction sites for the: endonucleases on the insert DNA of EcoRI, BamHI, HindIII, SaII, NdeI, KpnI, SmaI and XbaI. More interestingly, the same 6.3-kbp insert DNA was also cloned in pla.smids 5 and 6 in the opposite orientation (as shown in Figure 3).
To demonstrate that the insert DNA was of P.
haemolytica origin, the PstI-AvaI fragment from plasmid 5 was purified, nick-translated with [alpha- 32 P]DATP, and used as a probe in Southern blot analysis against _P.
haemolytica genomic DNA. The results (shown in Figure 3) indicate that the insert DNA hybridized to unique fragments of the P. haemolytica DNA digest.
Method 4 Testing for the P. haemoljrtic heukotoxin in the E coli recombinant clones Periplasmic and cellular proteins from the E. coli clones carrying the recombinant plasmids 1, 5, 81, 9, 10, 11 and 13 as well a.s plasmid pBR322 were assayed for leukotoxin activity. None of the periplasmic protein preparations showed significant cytotoxic activity.
Cellular proteins recovered after sonication from three recombinant clones were found to be toxic for BL-3 cells.
These were clones carrying plasmids 5, 10, and 11, which showed 95.5, 53.1, and 55.5% toxicity, respectively. For plasmids 10 and 11, all activity was heat labile (56~C, 1 h), while 26.1% of the toxicity in the plasmid 5 preparation was heat stable. In comparison, the P.
haemolytica culture supernatant (Shewen, P.E., and B.N.
Wilkie. 1982,. Cytotoxin of Pasteurella haemolytica acting on bovine leukocytes. Infec. Immun. 35:91-94) used as a toxicity control was 93.4% and 19.6% toxic after heating.
Cellular proteins from the clone bearing plasmid 5 showed no toxicity for canine, porcine or human peripheral blood lymphocites when tested. Likewise, the ~~~0~112 P. haemolytica control was not toxic for these non-ruminant cells.
Both bovine and rabbit antitoxic sera neutralized the toxic activity of cellular proteins from the clone bearing plasmid 5 at a 1/256 dilution, the highest dilution tested. No neutralization occurred at any dilution with the normal rabbit serum pool or the fetal calf serum pool, which were used as controls.
Method 5 SDS-polyacrylamide gel electrophoresis and Western blot analysis The protein preparations from the recombinant clones carrying plasmid 5 and plasmid 10 were analyzed by SDS-polyacrylamide gel electrophoresis followed by electrophoretic transfer to nitrocellulose paper and ELISA. Results from Coomassie blue staining of the gels indicated no ne~a protein bands in the periplasmic protein preparations. However, an additional protein band was detected after «estern blot analysis of the cellular protein preparai~ion from the recombinant clone carrying plasmid 5 (as shown in Figure 4). This additional protein band migrated to a position which corresponds to one of the soluble antigens in the P. haemolytica culture supernatant. The molecular weight of the extra protein was estimated to be about 100,000.
In the Western blot analysis (as shown in Figure 4), several other bends were detected in all of the protein preparations. ~~ince these bands were present also in the control sample, E. Co:Li carrying pBR322, they are probably E. coli. proteins which react with the antibody preparation and do not affect the present interpretation.
With reference to Figure 5, the plasmid pPHS is one of the recombinant plasmids isolated in accordance with the above procedures. Upon further characterization of the insert DNA of pPHS by DNA sequencing analysis it became apparent that t:wo open reading frames (ORF, and ORFZ were identi:Eied. It was found, however, that the ORF1 was incomplE~te in that it was lacking a termination ~~~~'~12 signal. By subsequent probing of the P. haemolytica A1 clone bank a second plasmid pPHSA was recovered. Such probe was accomplished on the basis of using the 1.7 kbp BamHI-PstI fragment from the pPHS DNA sequence which was 5 purified and used as a probe to screen the P_. haemolytica A1 bank. This plasmid contains insert sequences further to the left of the BamHI site on the pPH5. With reference to Figure 5, however, the pPHSA was lacking sequences to the right of the PvuI site on the pPH5 10 sequence. The sequence from the PvuI site to EcoRV on the pPHS was determined to be part of the coding region of ORF2. From this information a new plasmid was constructed using sequences from pPHS and pPHSA to produce the plasmid pLKTS. This plasmid contains all the 15 insert sequences covering the regions of ORFZ and ORF1.
The legend for Figure 5 is as follows:
P is equivalent to the restriction site PstI
P, is equivalent to the restriction site PvuI
H is equivalent to the restriction site HincIII
Ev is equivalent to the restriction site EcoRV
B is equivalent to the restriction site BamHI
PZ is equivalent to the restriction site PvuII
BII is equivalent to the restriction site B_glII .
B/S is equivalent to the restriction site BamHI-Sau3A junction The ORFZ is a coding sequence of 498 nucleotides which codes for 166 amino acids. The deduced molecular weight then of the resultant protein is 19,820 daltons.
ORF1 is a coding sequence of 2856 nucleotides which in turn codes for protein having 952 amino acid groups with a deduced molecular. weight of 101,883 daltons. This is the leukotoxi.n which is produced on expression of the plasmid pLKTS~ in the suitable host microorganism such as E. coli.
The identifying characteristics of pBR322 and _E.
coli HB101 are already reported as noted above. The leukotoxin gene as found in the recombinant plasmid pLKTS
and as transformed in host E. coli HB101 is characterized as deposited at ATCC, deposition No. 68025.

.....
1,~ ~ 0 '~ .~

V~lhen the protein preparation from a number of the E.
coli recombin~~nt clones were assayed for leukotoxic activity, thr~se preparations were found to be toxic for the BL-3 cell;. These were the cellular proteins from the clones carrying plasmids 5, 10, and 11.
Interestingly, they all carried the same P. haemolytica insert DNA in either of the two orientations (Figure 2).
After the inil~ial toxicity assay, three more recombinant plasmids, 6, :L6 and 18, were also identified as carrying the same inse~_t DNA. These results suggest that the leukotoxin gene is carried on the 6.3-kbp insert DNA.
Furthermore, i:he 6.:3-kbp DNA must also carry the necessary regulatory regions for the expression of the leukotoxin.
The leukotoxic activity of these protein preparations was heat labi:Le, which is characteristic of the P.
haemolytica le~ukoto:~in. Furthermore, similar to the P.
haemolytica lE~ukotoain, the preparation from the recombinant c=Lones carrying pPHS was found to be non-toxic for canine, porcine, and human peripheral blood lymphocytes (Shewen" P.E., and B.M. Wilkie. 1982.
Cytotoxin of I?asteurella haemolytica acting on bovine leukocytes. 7=nfect.. Immun. 35:91-94). In addition, both rabbit and bovine antitoxic sera neutralized the toxicity in the pPH5 preparation. Therefore, the P. haemolytica leukotoxin gene is <:arried in this 6.3-kbp DNA fragment.
Results from :3outhern blot analysis, in which the insert DNA hybridized to unique DNA fragments of the P.
haemolytica gE~nome, indicate that there is only one copy of the leukotoxin gene in P. haemolytica.
The leukotoxic activity of the protein preparation from the E. coli clones carrying pPH5 was found to be twice that of the pi°otein preparations from the clones carrying pPHlC) and pPHll. Furthermore, an additional protein band was found in the pPH5 protein preparation after Western blot analysis (Figure 4). This additional band was absent in t:he pPHlO and the control pBR322 protein prepax-ations. We suggest that there is a higher level of leukotoxin produced when the 6.3-kbp insert DNA
' '.

is cloned in the orientation seen in pPHS. This increased level of leukotoxin production may be a result of expression from both the leukotoxin promoter and the tetracycline promoter on pBR322 in pPHS, while in the other orientation, i.e., pPHlO, the leukotoxin can be expressed only from its own promoter.
The migration of the P. haemolytica leukotoxin produced in the _E. coli recombinant clone carrying pPH5 during SDS-polyacrylamide gel electrophoresis corresponded to one of the soluble antigens present in the P. haemolYtica culture supernatant (Figure 4), confirming that the molecular weight of the leukotoxin is About 100,000. other researchers, by using physical techniques for purification, have reported molecular weights for the P. haemolytica leukotoxin of 150,000 (Himmel, M.E., M.D. Yates, L.H. Lauerman, and P.G.
Squire. 1982. Purification and partial characterization of a macrophage cytotoxin from Pasteurella haemolytica.
Am. J. Vet. Res. 43:764-767) and 300,000 (Baluyut, C.S., R.R. Simonson, W.J. Bemrick, and S.K. Maheswaran. 1981.
Interaction of Pasteurella haemolytica with bovine neutrophils: identification and partial characterization of cytotoxin. Am. J. Vet. Res. 42:1920-1926). These may be overestimated due to the aggregation of binding of serum materials with the leukotoxin. A more accurate determination of the molecular weight of the leukotoxin is possible from the DNA sequence analysis of the cloned gene.
During the initial screening of leukotoxic activity from the recombinant clones, toxic activities were observed only with the cellular protein preparations from E. coli containing pPHS, pPHlO or pPHll. No leukotoxic activity was found in the respective periplasmic protein preparations. The failure of the _P. haemolytica leukotoxin to be excreted by the E. coli cells is not unexpected since it. has been observed on a number of occasions that foreign proteins produced in _E. coli from cloned genes were not exported (Coleman, K., G. Dougan, and J,P. Arbuthnot.t. 1983. Cloning, and expression in ~~340'r.~?

Escherichia coli K-12, of the chromosomal hemolysin (phospholipase C) determinant of Pseudomonas aeruginosa.
J. Bacteriol. 153:909-915); (Gray, G.L., D.H. Smith, J.S.
Baldridge, R.N. Haskins, M.L. Vasil, E.Y. Chen, and H.L.
Heyneker. 1984. Cloning, nucleotide sequence and expression in Escherichia coli of the exotoxin A
structural gene of Pseudomonas aeruqinosa. Proc. Natl.
Acad. Sci. USA 81.:2645-2649).
By way of DNA sequencing analysis in accordance with standard procedures, the nucleotide sequence of ORF 1 and ORF 2 is as follows:
The sequence is numbered from -469 at the rightmost EcoRV site of Figure 5 on the pLKT4 plasmid and counting leftward. Nucleotide No. I is the first nucleotide of the coding region of ORF 2 at approximately nucleotide 510 there is the break between ORFZ and ORF1.

19 i~3~ U ~rl~

GATATCITG 'IGCA GTAACCACAC ACCCGAATAA i'~AGGGTCAAA AG'I~'IT

TCATAAAAAG TCCCTGZ'GIT T1'CATTATAA GGATTACCAC TTTP~CGCAG TTACTh!'CIT

pp~AAAAGTC TIC.TITI'CAT P~P~IT~ TTATGTCATA C'F~AACACATC AAATrGAGAT

GTAGITrCrC ,AAZC'.CICZZG ATTCCI~'!'AT C~~CAAAAAAA CAACCCAAAA GAAAAAAGAA

AAGTATATGT '~CATTAATA TTACAATGTA ATTATITTGT TTAATITCCC TACATTTTGT
-170 ~-160 -150 -140 -130 -120 i'~1TAACTITAA i'~A~CP~CICI~IT 'l.'ITCrCITCT GATTATATAA AACii!~AAAA ATACAATTTA
?_ ti -110 ~-100 -90 -80 -70 -60 i'~GCTACAAAA i~ACAACAAAA A~1CAACAAAA AACACGACAA TAAGATCGAG TAATGATTAT

2 5 ATT~AT AATICTAATITAGA ATAATTATCG AGTGCAAATT ATG AAT CAA
Met Asn Gln 'hC'I' TAT TTT AAC TTA CTA GGA AAC ATT i'~CI' TGG CTA TGG ATG AAC TCC TCC CTC
30 ~ ~ phe Asn Leu Leu Gly Asn Ile Thr Trp Leu Trp Met Asn Ser Ser Leu CAC AAA GF~A TGG i'~C-~C: TGT GAA CTA CTA GCA CGC i'~AT GTG ATT CCT GCS AT'r GAA
His Lys Glu Trp Sex- Cys Glu Leu Leu Ala Arg Asn Val Ile Pro Ala Ile Glu AAT GAA CAA TAT ATG CTA CTT ATA GAT AAC GGT ATT CCG ATC GCr TAT TGT AGT
Asn Glu Gln Tyr Met Leu Leu Ile Asp Asn Gly Ile Pro Ile Ala Tyr Cys Ser 20 ~~~orr~~

TGG GCA GAT TrA AAC CTT GAG ACT GAG GTG AAA TAT ATT AAG GAT ATT AAT TCG
Trp Ala Asp Leu Asn Leu Glu Thr Glu Val Lys Tyr Ile Lys Asp Ile Asn Ser TTA ACA CCA GAA GAA TGG CAG TCT GGT GAC AGA CGC TGG ATT ATT GAT TGG G'LA
Leu Thr Pro Glu Glu Trp Gln Ser Gly Asp Arg Arg Trp Ile Ile Asp Trp Val GCA aCA TTC GGA CAT TCT CAA TTA CTT TAT AAA AAA ATG TGT CAG AAA TAC CCT
Ala Pro Phe Gly His Ser Gln Leu Leu Tyr Lys Lys Met Cys Gln Lys Tyr Pro GAT ATG ATC GTC AGA TCT ATA CGC TTT TAT CCA AAG CAG AAA GAA TTA GGC AAA
Asp Met Ile Val Arg Ser Ile Arg Phe Tyr Pro Lys Gln Lys Glu Leu Gly Lys ATT GCC TAC TTT AAA GGA GGT AAA TTA GAT AAA AAA ACA GCA AAA AAA CGT TTT
Ile Ala Tyr Phe Lys Gly Gly Lys Leu Asp Lys Lys Thr Ala Lys Lys Arg Phe GAT ACA TAT CAA GAA GAG CTG GCA ACA GCA CTT AAA AAT C'TAA TTT AAT TTT ATT
Asp Thr Tyr Gln Glu Glu Leu Ala Thr Ala Leu Lys Asn Glu Phe Asn Phe Ile AAA AAA TAG AAC~AG ACATCCCrT ATG GGA ACT AGA CTT ACA ACC CTA 'ICA AAT
Lys Lys * Met Gly Thr Arg Leu Thr Thr Leu Ser Asn GGG CTA AAA AAC ACI' TTA ACG GCA AC7C AAA AGT GGC TTA CAT AAA GCC GGT CAA
Gly Leu Lys Asn Thr Leu Thr Ala Thr Lys Ser Gly Leu His Lys Ala Gly G1n Ser Leu Thr Gln Ala Gly Ser Ser Leu Lys Thr Gly Ala Lys Lys Ile Ile Leu TAT ATT CCC CAA AAT TAC CAA TAT GAT ACT GAA CAA GGT AAT GGT TTA CAG GAT
Tyr Ile Pro Gln Asn Tyr Gln Tyr Asp Thr Glu Gln Gly Asn Gly Leu Gln Asp 21 1~~0?1~

TTA GTC AAA. GCG GCC GAA GAG TTG GGG ATT GAG GTA CAA AGA GAA GAA CGC AAT
Leu Val Lys Ala A.la Glu Glu Leu Gly Ile Glu Val Gln Arg Glu Glu Arg Asn AAT ATT GCA.ACA GCr CAA ACC AGT TTA GGC ACG ATT CAA ACC GCT ATT GGC TTA
Asn Ile Ala Thr Ala Gln Thr Ser Leu Gly Thr Ile Gln Thr Ala Ile Gly Leu ACT GAG CGT GGC ATT GTG TTA TCC C~C.'I' CCA CAA ATT GAT AAA TTG CTA CAG AAA
Thr Glu Arg Gly Ile Val Leu Ser Ala Pro G1n Ile Asp Lys Leu Leu Gln Lys ACT AAA GCA GGC CAA GCA TTA GGr TCT GCC GAA AGC ATT GTA CAA AAT GCA AAT
Thr Lys Ala Gly G1n Ala Leu Gly Ser Ala Glu Ser Ile Val Gln Asn Ala Asn AAA GCC AAA ACT GTA TTA TCT GGC ATT CAA TCT ATT T?'A GGC TCA GTA TTG GCT
?_0 Lys Ala Lys Thr Val Leu Ser Gly Ile Gln Ser Ile Leu Gly Ser Val Leu Ala GGA ATG GAT TTA GAT GAG GCC TTA CAG AAT AAC AGC AAC CAA CAT GGT CTT GCT
Gly Met Asp Leu Asp Glu Ala Leu Gln Asn Asn Ser Asn Gln His Ala Leu Ala AAA GCT GGC TTG GAG CTA ACA AAT TCA TTA ATT GAA AAT ATT GCT AAT TCA GTA
Lys Ala Gly Leu Glu Leu Thr Asn Ser Leu Ile Glu Asn Ile Ala Asn Ser Val AAA ACA CTT GAC GAA TTT GGT GAG CAA ATT AGT CAA TTT GGT TCA AAA CTA CAA
Lys Thr Leu Asp Glu Phe Gly Glu Gln Ile Ser Gln Phe Gly Ser Lys Leu G1n AAT ATC AAA GGC TTA GGG ALT TTA GGA GAC AAA CTC AAA AAT ATC GGT GGA CTT
Asn Ile Lys Gly Leu Gly Thr Leu Gly Asp Lys Leu Lys Asn Ile Gly Gly Leu GAT AAA GCT GGC CZ'.~ GGT TTA GAT GTT ATC TCA GGG CTA TTA TCG CSC GCA ACA
Asp Lys Ala Gly Leu Gly Leu Asp Val Ile Ser Gly Leu Leu Ser Gly Ala Thr GCT GCA CTT GTA CTT GCA GAT AAA AAT GCT TCA ACA GCT AAA AAA GTG GGT GCG
Ala Ala Leu Val Leu Ala Asp Lys Asn Ala Ser Thr Ala Lys Lys Val Gly Ala GGr TTT GAA TTG GC'~. AAC CAA GTT GIT GGT AAT ATT ACC AAA GCC GTT TCT TCT
Gly Phe Glu Leu Ala Asn Gln Val Val Gly Asn Ile Thr Lys Ala Val Ser Ser TAC ATT TTA GCC CAA CGT GTT GCA GCA GGT TTA TCT TCA ACT GGG CCT GTG GCT
Tyr Ile Leu Ala G1n Arg Val Ala Ala Gly Leu Ser Ser Thr Gly Pro Val Ala GCT TTA ATT GCT TCT A~.'I'_ GTT TCT CTT GCG ATT AGC CCA TTA GCA TTT GCC GGT
A7_a Leu Ile Ala Ser Thr Val Ser Leu Ala Ile Ser Pro Leu Ala Phe Ala Gly ATT GCC GAT AAA TTT AAT CAT GCA AAA AGT TTA) GAG AGT TAT GCC GAA CGC TTr Ile Ala Asp Lys Phe A.sn His Ala Lys Ser Leu Glu Ser Tyr Ala Glu Arg Phe ?_U
153() 1545 1560 1575 AAA AAA TTA GGC TAT GAC GGA GAT AAT TTA TTA GCA GAA TAT CAG CGG GGA ACA
Lys Lys Leu Gly Tyr Asp GIy Asp Asn Leu Leu Ala Glu Tyr Gln Arg Gly Thr GGG ACT ATT GAT GCA TCG GTT ACT GC~1 ATT AAT ACC GCA TTG GCC GCT ATT GCT
Gly Thr Ile Asp Ala Ser Val Thr Ala Ile Asn Thr Ala Leu Ala Ala Ile Ala Gly Gly Val Ser Ala Ala Ala Ala GIy Ser Val Ile Ala Ser Pro Ile Ala Leu TTA GTA TCT GGG ATT ACC GGT GTA ATT TCT AC;G ATT CTG CAA TAT.TCT AAA CAA
35 Leu Val Ser Gly Ile Thr Gly Val Ile Ser Thr Ile Leu Gln Tyr Ser Lys Gln GCA ATG TTT GAG CAC: GTT GrA AAT AAA ATT CAT AAC AAA ATT GTA GAA TGG GAA
Ala Met Phe Glu His Val Ala Asn Lys Ile His Asn Lys Ile Val Glu Trp Glu 23 Z3~0"l1~

AAA AAT AAT CAC GGT AAG AAC TAC TTT GAA AAT GGT TAC GAT GCC CGT TAT CTT
Lys Asn Asn His Gly Lys Asn Tyr Phe Glu Asn Gly Tyr Asp Ala Arg Tyr Leu GCG AAT TTP~ CAA GAT AAT ATG AAA TTC TTA CTG AAC TTA AAC AAA GAG TTA CAG
Ala Asn Leu Gln Asp Asn Met Lys Phe Leu Leu Asn Leu Asn Lys Glu Leu Gln ~ ~ ~~ ~ A~ ~ ATT ACT CAG CAG CAA TGG CAT AAC AAC ATT GGT GAT
Ala Glu Arg Val Il.e Ala Ile Thr Gln Gln G1n Trp Asp Asn Asn Ile Gly Asp TTA GCT GGr ATT AQC CGT TTA GGI' GAA AAA GTC CTT AGT GGT AAA GCC TAT GZG
Leu A1a Gly Ile Ser Arg Leu Gly Glu Lys Val Leu Ser Gly Lys Ala Tyr Val GAT GCG TTT GAA GAA GGC AAA CAC ATT AAA GCC GAT AAA TTA GTA CAG TTG GAT
Asp Ala Phe Glu Glu Gly Lys His Ile Lys Ala Asp Lys Leu Val Gln Leu Asp ?_U
2070 . 2085 2100 2115 TCG GCA AAC GGT ATT ATT GAT GTG AGT AAT TCG GGT AAA GCG AAA ACT CAG CAT
Ser Ala Asn Gly Ile Ile Asp Val Ser Asn Ser Gly Lys Ala Lys Thr Gln His 2 5 21.30 2145 2160 ATC TTA TTC AGA ACG CCA TTA TTG ACG CCG GGA ACA GAG CAT OGT GAA CGC GTA
Ile Leu Phe Arg Thr Pro Leu Leu Thr Pro Gly Thr Glu His Arg Glu Arg Val 3 0 ~ AC,a, p~~I p~ Tp,T ~, TAT ATT ACC AAG CTC AAT ATT AAC CGT GTA GAT AGC
Gln Thr Gly Lys Tyr.Glu Tyr Ile Thr Lys Leu Asn Ile Asn Arg Val Asp Ser TC-G AAA ATT ACA GAT GV"r GCA GCA AGT TCT ACC TTT GAT TTA ACT AAC GTT GTT
35 Trp Lys Ile Thr Asp Gly Ala Ala Ser Ser Thr Phe Asp Leu Thr Asn Val Val CAG CGT ATT GGr AT'I GAA TTA GAC AAT GCT GGA AAT GTA ACT AAA ACC AAA GAA
Gln Arg Ile Gly Ile Glu Leu Asp Asn Ala Gly Asn Val Thr Lys Thr Lys Glu 239:0 2355 2370 2385 ACA AAA ATT' ATT GCC AAA CTT GGr GAA GGT GAT GAC AAC GTA TTT GTZ' GGT TCT
Thr Lys Ile: Ile Ala Lys Leu Gly Glu Gly Asp Asp Asn Val Phe Val Gly Ser GGT ACG ACG GAA ATT GAT GGC GGT GAA GGZ' TAC GAC CGA GTT CAC TAT AGC CGr Gly Thr Thr Glu Ile Asp Gly Gly Glu Gly Tyr Asp Arg Val His Tyr Ser Arg (~Gp, AAC TAT GGT GCT TTA ACT ATT GAT GCA ACC AAA GAG ACC GAG CAA GGT AG'I' Gly Asn Tyr Gly Ala Leu Thr Ile Asp Ala Thr Lys Glu Thr Glu Gln Gly Ser TAT AOC GTA AAT CGr TTC GTA GAA ACC GGT AAA GCA CTA CAC GAA GTG ACT TCA
1 S ~ Thr Val Asn Arg Phe Val Glu Thr Gly Lys Ala Leu His Glu Val Thr Ser ACC CAT ACC GCA TTA GTG GGC AAC OGT GAA GAA AAA ATA GAA TAT CGT CAT AGC
Thr His Thr Ala Leu Val Gly Asn Pirg Glu Glu Lys Ile Glu Tyr Arg His Ser ?_U

AAT AAC CAG CAC CAT GCC GG'r TAT TAC ACC AAA GAT ACC TTG AAA GCT GTT GAA
Asn Asn Gln His His Ala Gly Tyr Tyr Thr Lys Asp Thr Leu Lys Ala Val Glu GAA ATT A'IC GGT ACA TCA CAT AAC GAT ATC TTT AAA GGT AGT AAG TTC AAT GAT
Glu Ile Ile Gly Thr Ser His Asn Asp Ile Phe Lys Gly Ser Lys Phe Asn Asp 30 GCC TTT AAC GGT GGZ' GAT GGr GIC GAT ACT ATT GAC CST AAC GAC GGC AAT GAC
Ala Phe Asn Gly Gly Asp Gly Val Asp Thr Ile Asp Gly Asn Asp Gly Asn Asp 35 ~ TTA TTT GGT GGT AAA GGC GAT GAT ATT CTC C,AT GGT GGA AAT GGT GAT GAT
Arg Leu Phe Gly Gly Lys Gly Asp Asp Ile Leu Asp Gly Gly Asn Gly Asp Asp TTT ATC GAT GGC GGT AAA GGC AAC GAC C'IA TTA CAC GGT GGC AAC GGC GAT GAT
Phe Ile Asp Gly Gly Lys Gly Asn Asp Leu Leu His Gly Gly Lys Gly Asp Asp ATT TTC GTrC CAC CGT AAA GGC GAT GGT AAT GAT ATT ATT ACC GAT TCT GAC GGC
Ile Phe Va:L His Ar_g Lys Gly Asp Gly Asn Asp Ile Ile Thr Asp Ser Asp Gly AAT GAT AAF~ TTA TC~1 TTC TCT GAT TCG AAC TTA AAA GAT TTA ACA TTT GAA AAA
Asn Asp Ly~s Leu Ser Phe Ser Asp Ser Asn Leu Lys Asp Leu Thr Phe Glu Lys ~ AAA CAT AAT CZT GTC ATC ACG AAT AGC AAA AAA GAG AAA GTG ACC ATT CAA
Val Lys His. Asn Leu Val Ile Thr Asn Ser Lys Lys Glu Lys Val Thr Ile Gln AAC TGG 'ITC' CGA GAG GGT GAT TTT GCT AAA GAA GTG CGT AAT TAT AAA GCA ACT
p~ T~ phe Arg Glu Ala Asp Phe Ala Lys Glu Val Pro Asn Tyr Lys Ala Thr AAA GAT GAG AAA ATC GAA GAA ATC ATC GGT CAA AAT GGC GAG CGG ATC ACC TCA
Lys Asp Glu Lys Ile Glu Glu Ile Ile Gly Gln Asn Gly Glu Arg Ile Thr Ser ?_ (i AAG CAA GTT GAT GAT CTT A'IC GCA AAA GGr AAC GGC AAA ATT ACC CAA GAT GAG
Lys Gln Val Asp Asp Leu Ile Ala Lys Gly Asn Gly Lys Ile Thr Gln Asp Glu CTA TCA AAA GIT GTT GAT AAC TAT GAA TTG CTC AAA CAT AGC AAA AAT GTG ACA
Leu Ser Lys Val VaL Asp Asn Tyr Glu Leu Leu Lys His Ser Lys Asn Val Thr ~ AGC TTA GAT AAG TTA ATC TCA TCT GTA AGT GCA TTT ACC TCG TCT AAT GAT
Asn Ser Leu Asp Lys Leu Ile Ser Ser Val Ser Ala Phe Thr Ser Ser Asn Asp TCG AGA AAT GTA TTA GTG GCT CCA ACT TCA ATG TTG GAT CAA AGT TTA TCT TCT
~r p~.g ~n Val Leu Val Ala Pro Thr Ser Met Leu Asp Gln Ser Leu Ser Ser CTT CAA TTT GCT AG~1 GCA GCT TAA TTTTTAATG ATTGGCAACT CTATATTGTr Leu Gln Phe Ala Arg Ala Ala 26 l~~o~ri~

TCACACATTA 'rAGAGl'IGCC GTT'ITATTTT ATAAAAGGAG ACAATATGGA AGCTAACCAT

CAAAGGAATG i~TLTIGG.rIT AGIT~OCTC ACTATGI~C; CACAATACCA TAATATTTCG

CITAATCCGG ~~AGAAAT~1AA ACATAAATIT GATGTrGACG GAAi9AGGGC'I' T~AACT

GCTTGGCTZT ~CAGCTGC~~AA ATGGTTAGCG TTGAAAGOGA AACAGATTAA AAAAGCGCIT

2CCCGCITAC it~,TIGCTGAA Tr~ACCGGCA TTAGZTt~GGC AAGATAACGG TAAAGA~

TTATTuGTAA AAGTGGATAC CGAT

~~~~~1~~
Beneath the codons of the DNA sequence, the corresponding amino acid has been identified. It is appreciated that the letters indicating the nucleic acids of t:he DNA sequence and the expressed amino acids are in accordance with International lettering which is well understood by those skilled in the art.
Having isolated the gene coding for the leukotoxin of P. haemolytica it is appreciated that many steps may be taken in improving animal health care. Based on this information and in the expression of the gene, a suitable vaccine is prepared without the impurities of a vaccine prepared from the culture supernatant. The DNA
sequence may be used in its entirety or fragment or fragments thereof as a DNA probe. The expressed leukotoxin protein may be used as the pure antigen in developing poly- and monoclonal antibodies. It is appreciated that the DNA sequence coding for the leukotoxin may be transformed into a suitable host micro-organism by either a suitable plasmid such as pBR322 or a suitable phage lamda.
The suitable host micro-organism such as the _E.
coli may bas cultured under suitable conditions to produce the leukotoxin. The leukotoxin may be isolated from the cells after culture is complete. A sonicated host cell ~~rotein preparation can be fractionated by preparativ~s polyacrylamide gel electrophoresis using a denaturing system at pH 9.7 where the leukotoxin has been shown to migrate at a low rate. Using _in vivo expression of the leukotoxin in the E, coli mini cell system, then position of the leukotoxin on a non-denaturing system by PAGE and Western blot analysis is achieved. Using the cloned protein, rabbits can be immunized by subcutaneous and intravenous inoculation and evaluai:ed for development of antibodies to the cloned ant~Lgen by micro immunodiffusion and dot blot analysis. The antibodies are tested for reactivity with P. haemolyt:ica surface antigens by micro-agglutination and for the' ability to neutralize the cytotoxin.

In establishing that the cloned leukotoxin antigen proves to be immunogenic in rabbits, tests may then be conducted for immunogenicity and protective capabilities in challenge experiments involving calves. Such analysis E~stabl:ishes the effectiveness of vaccines prepared from the genetically produced leukotoxin against pneumonia in calves.
Whiles preferred embodiments of this invention have been described and illustrated herein, the person skilled in. the art will appreciate that changes and modifications may be made therein without departing from the spirit and scope of this invention as defined in the appended claims.

Claims (18)

1. A process for making a vaccine effective against Pasteurella haemolytica comprising mixing:
A. a leukotoxin which is produced by (i) transforming a microorganism with a DNA sequence encoding leukotoxin which is a protein produced by Pasteurella haemolytica, said DNA
sequence having the restriction site map of plasmid pLKT4 shown in Figure 5C and is approximately 8.7 Kbp in size, said leukotoxin exhibiting cytotoxic activity specific against leukocytes;
(ii) culturing the resulting transformed microorganism; and (iii) obtaining the resulting leukotoxin produced from said cultured transformed microorganism; with B. a vaccine carrier.

2. A vaccine effective against Pasteurella haemolytica comprising:
(i) a leukotoxin which is a produced by:
(a) transforming a microorganism with a DNA
sequence encoding for leukotoxin which is a protein produced by Pasteurella haemolytica, said DNA sequence having the restriction site map of plasmid pLKT4 shown in Figure 5C and is approximately 8.7 Kbp in size, said leukotoxin exhibiting cytotoxic activity specific against leukocytes;
(b) culturing the resulting transformed microorganism; and (c) obtaining the resulting leukotoxin produced from said cultured transformed microorganism; and (ii) a vaccine carrier.

3. The process as claimed in claim 1, wherein said DNA comprises the following DNA sequence for ORF1, wherein T represents a thymine nucleotide, G represents a guanine nucleotide, A represents an adenine nucleotide, and C represents a cytosine nucleotide:
ATG GGA ACT AGA CTT ACA ACC CTA TCA AAT GGG CTA AAA AAC
ACT TTA ACG GCA ACC AAA AGT GGC TTA CAT AAA GCC GGT CAA
TCA TTA ACC CAA GCC GGC AGT TCT TTA AAA ACT GGG GCA AAA
AAA ATT ATC CTC TAT ATT CCC CAA AAT TAC CAA TAT GAT ACT
GAA CAA GGT AAT GGT TTA CAG GAT TTA GTC AAA GCG GCC GAA
GAG TTG GGG ATT GAG GTA CAA AGA GAA GAA CGC AAT AAT ATT
GCA ACA GCT CAA ACC AGT TTA GGC ACG ATT CAA ACC GCT ATT
GGC TTA ACT GAG CGT GGC ATT GTG TTA TCC GCT CCA CAA ATT
GAT AAA TTG CTA CAG AAA ACT AAA GCA GGC CAA GCA TTA GGT
TCT GCC GAA AGC ATT GTA CAA AAT GCA AAT AAA GCC AAA ACT
GTA TTA TCT GGC ATT CAA TCT ATT TTA GGC TCA GTA TTG GCT
GGA ATG GAT TTA GAT GAG GCC TTA CAG AAT AAC AGC AAC CAA
CAT GCT CTT GCT AAA GCT GGC TTG GAG CTA ACA AAT TCA TTA
ATT GAA AAT ATT GCT AAT TCA GTA AAA ACA CTT GAC GAA TTT
GGT GAG CAA ATT AGT CAA TTT GGT TCA AAA CTA CAA AAT ATC
AAA GGC TTA GGG ACT TTA GGA GAC AAA CTC AAA AAT ATC GGT
GGA CTT GAT AAA GCT GGC CTT GGT TTA GAT GTT ATC TCA GGG
CTA TTA TCG GGC GCA ACA GCT GCA CTT GTA CTT GCA GAT AAA
AAT GCT TCA ACA GCT AAA AAA GTG GGT GCG GGT TTT GAA TTG
GCA AAC CAA GTT GTT GGT AAT ATT ACC AAA GCC GTT TCT TCT
TAC ATT TTA GCC CAA CGT GTT GCA GCA GGT TTA TCT TCA ACT
GGG CCT GTG GCT GCT TTA ATT GCT TCT ACT GTT TCT CTT GCG
ATT AGC CCA TTA GCA TTT GCC GGT ATT GCC GAT AAA TTT AAT
CAT GCA AAA AGT TTA GAG AGT TAT GCC GAA CGC TTT AAA AAA
TTA GGC TAT GAC GGA GAT AAT TTA TTA GCA GAA TAT CAG CGG
GGA ACA GGG ACT ATT GAT GCA TCG GTT ACT GCA ATT AAT ACC
GCA TTG GCC GCT ATT GCT GGT GGT GTG TCT GCT GCT GCA GCC

GGC TCG GTT ATT GCT TCA CCG ATT GCC TTA TTA GTA TCT GGG
ATT ACC GGT GTA ATT TCT ACG ATT CTG CAA TAT TCT AAA CAA
GCA ATG TTT GAG CAC GTT GCA AAT AAA ATT CAT AAC AAA ATT
GTA GAA TGG GAA AAA AAT AAT CAC GGT AAG AAC TAC TTT GAA
AAT GGT TAC GAT GCC CGT TAT CTT GCG AAT TTA CAA GAT AAT
ATG AAA TTC TTA CTG AAC TTA AAC AAA GAG TTA CAG GCA GAA
CGT GTC ATC GCT ATT ACT CAG CAG CAA TGG GAT AAC AAC ATT
GGT GAT TTA GCT GGT ATT AGC CGT TTA GGT GAA AAA GTC CTT
AGT GGT AAA GCC TAT GTG GAT GCG TTT GAA GAA GGC AAA CAC
ATT AAA GCC GAT AAA TTA GTA CAG TTG GAT TCG GCA AAC GGT
ATT ATT GAT GTG AGT AAT TCG GGT AAA GCG AAA ACT CAG CAT
ATC TTA TTC AGA ACG CCA TTA TTG ACG CCG GGA ACA GAG CAT
CGT GAA CGC GTA CAA ACA GGT AAA TAT GAA TAT ATT ACC AAG
CTC AAT ATT AAC CGT GTA GAT AGC TGG AAA ATT ACA GAT GGT
GCA CCA AGT TCT ACC TTT GAT TTA ACT AAC GTT GTT CAG CGT
ATT GGT ATT GAA TTA GAC AAT GCT GGA AAT GTA ACT AAA ACC
AAA GAA ACA AAA ATT ATT GCC AAA CTT GGT GAA GGT GAT GAC
AAC GTA TTT GTT GGT TCT GGT ACG ACG GAA ATT GAT GGC GGT
GAA GGT TAC GAC CGA GTT CAC TAT AGC CGT GGA AAC TAT GGT
GCT TTA ACT ATT GAT GCA ACC AAA GAG ACC GAG CAA GGT AGT
TAT ACC GTA AAT CGT TTC GTA GAA ACC GGT AAA GCA CTA CAC
GAA GTG ACT TCA ACC CAT ACC GCA TTA GTG GGC AAC CGT GAA
GAA AAA ATA GAA TAT CGT CAT AGC AAT AAC CAG CAC CAT GCC
GGT TAT TAC ACC AAA GAT ACC TTG AAA GCT GTT GAA GAA ATT
ATC GGT ACA TCA CAT AAC GAT ATC TTT AAA GGT AGT AAG TTC
AAT GAT GCC TTT AAC GGT GGT GAT GGT GTC GAT ACT ATT GAC
GGT AAC GAC GGC AAT GAC CGC TTA TTT GGT GGT AAA GGC GAT
GAT ATT CTC GAT GGT GGA AAT GGT GAT GAT TTT ATC GAT GGC
GGT AAA GGC AAC GAC CTA TTA CAC GGT GGC AAG GGC GAT GAT
ATT TTC GTT CAC CGT AAA GGC GAT GGT AAT GAT ATT ATT ACC
GAT TCT GAC GGC AAT GAT AAA TTA TCA TTC TCT GAT TCG AAC
TTA AAA GAT TTA ACA TTT GAA AAA GTT AAA CAT AAT CTT GTC
ATC ACG AAT AGC AAA AAA GAG AAA GTG ACC ATT CAA AAC TGG
TTC CGA GAG GCT GAT TTT GCT AAA GAA GTG CCT AAT TAT AAA
GCA ACT AAA GAT GAG AAA ATC GAA GAA ATC ATC GGT CAA AAT
GGC GAG CGG ATC ACC TCA AAG CAA GTT GAT GAT CTT ATC GCA
AAA GGT AAC GGC AAA ATT ACC CAA GAT GAG CTA TCA AAA GTT
GTT GAT AAC TAT GAA TTG CTC AAA CAT AGC AAA AAT GTG ACA

AAC AGC TTA GAT AAG TTA ATC TCA TCT GTA AGT GCA TTT ACC
TCG TCT AAT GAT TCG AGA AAT GTA TTA GTG GCT CCA ACT TCA
ATG TTG GAT CAA AGT TTA TCT TCT CTT CAA TTT GCT AGA GCA
GCT TAA.

4. The process as claimed in claim 1, wherein said DNA comprises DNA having the following DNA sequence for ORF1 and ORF2, wherein T
represents a thymine nucleotide, G represents a guanine nucleotide, A
represents an adenine nucleotide, and C represents a cytosine nucleotide:
ATG AAT CAA TCT TAT TTT AAC TTA CTA GGA AAC ATT ACT TGG
CTA TGG ATG AAC TCC TCC CTC CAC AAA GAA TGG AGC TGT GAA
CTA CTA GCA CGC AAT GTG ATT CCT GCA ATT GAA AAT GAA CAA
TAT ATG CTA CTT ATA GAT AAC GGT ATT CCG ATC GCT TAT TGT
AGT TGG GCA GAT TTA AAC CTT GAG ACT GAG GTG AAA TAT ATT
AAG GAT ATT AAT TCG TTA ACA CCA GAA GAA TGG CAG TCT GGT
GAC AGA CGC TGG ATT ATT GAT TGG GTA GCA CCA TTC GGA CAT
TCT CAA TTA CTT TAT AAA AAA ATG TGT CAG AAA TAC CCT GAT
ATG ATC GTC AGA TCT ATA CGC TTT TAT CCA AAG CAG AAA GAA
TTA GGC AAA ATT GCC TAC TTT AAA GGA GGT AAA TTA GAT AAA
AAA ACA GCA ARA AAA CGT TTT GAT ACA TAT CAA GAA GAG CTG
GCA ACA GCA CTT AAA AAT GAA TTT AAT TTT ATT AAA AAA TAG
AAGGAG ACATCCCTT ATG GGA ACT AGA CTT ACA ACC CTA TCA AAT
GGG CTA AAA AAC ACT TTA ACG GCA ACC AAA AGT GGC TTA CAT
AAA GCC GGT CAA TCA TTA ACC CAA GCC GGC AGT TCT TTA AAA
ACT GGG GCA AAA AAA ATT ATC CTC TAT ATT CCC CAA AAT TAC
CAA TAT GAT ACT GAA CAA GGT AAT GGT TTA CAG GAT TTA GTC
AAA GCG GCC GAA GAG TTG GGG ATT GAG GTA CAA AGA GAA GAA
CGC AAT AAT ATT GCA ACA GCT CAA ACC AGT TTA GGC ACG ATT
CAA ACC GCT ATT GGC TTA ACT GAG CGT GGC ATT GTG TTA TCC
GCT CCA CAA ATT GAT AAA TTG CTA CAG AAA ACT AAA GCA GGC
CAA GCA TTA GGT TCT GCC GAA AGC ATT GTA CAA AAT GCA AAT
AAA GCC AAA ACT GTA TTA TCT GGC ATT CAA TCT ATT TTA GGC
TCA GTA TTG GCT GGA ATG GAT TTA GAT GAG GCC TTA CAG AAT
AAC AGC AAC CAA CAT GCT CTT GCT AAA GCT GGC TTG GAG CTA
ACA AAT TCA TTA ATT GAA AAT ATT GCT AAT TCA GTA AAA ACA
CTT GAC GAA TTT GGT GAG CAA ATT AGT CAA TTT GGT TCA AAA

CTA CAA AAT ATC AAA GGC TTA GGG ACT TTA GGA GAC AAA CTC
AAA AAT ATC GGT GGA CTT GAT AAA GCT GGC CTT GGT TTA GAT
GTT ATC TCA GGG CTA TTA TCG GGC GCA ACA GCT GCA CTT GTA
CTT GCA GAT AAA AAT GCT TCA ACA GCT AAA AAA GTG GGT GCG
GGT TTT GAA TTG GCA AAC CAA GTT GTT GGT AAT ATT ACC AAA
GCC GTT TCT TCT TAC ATT TTA GCC CAA CGT GTT GCA GCA GGT
TTA TCT TCA ACT GGG CCT GTG GCT GCT TTA ATT GCT TCT ACT
GTT TCT CTT GCG ATT AGC CCA TTA GCA TTT GCC GGT ATT GCC
GAT AAA TTT AAT CAT GCA AAA AGT TTA GAG AGT TAT GCC GAA
CGC TTT AAA AAA TTA GGC TAT GAC GGA GAT AAT TTA TTA GCA
GAA TAT CAG CGG GGA ACA GGG ACT ATT GAT GCA TCG GTT ACT
GCA ATT AAT ACC GCA TTG GCC GCT ATT GCT GGT GGT GTG TCT
GCT GCT GCA GCC GGC TCG GTT ATT GCT TCA CCG ATT GCC TTA
TTA GTA TCT GGG ATT ACC GGT GTA ATT TCT ACG ATT CTG CAA
TAT TCT AAA CAA GCA ATG TTT GAG CAC GTT GCA AAT AAA ATT
CAT AAC AAA ATT GTA GAA TGG GAA AAA AAT AAT CAC GGT AAG
AAC TAC TTT GAA AAT GGT TAC GAT GCC CGT TAT CTT GCG AAT
TTA CAA GAT AAT ATG AAA TTC TTA CTG AAC TTA AAC AAA GAG
TTA CAG GCA GAA CGT GTC ATC GCT ATT ACT CAG CAG CAA TGG
GAT AAC AAC ATT GGT GAT TTA GCT GGT ATT AGC CGT TTA GGT
GAA AAA GTC CTT AGT GGT AAA GCC TAT GTG GAT GCG TTT GAA
GAA GGC AAA CAC ATT AAA GCC GAT AAA TTA GTA CAG TTG GAT
TCG GCA AAC GGT ATT ATT GAT GTG AGT AAT TCG GGT AAA GCG
AAA ACT CAG CAT ATC TTA TTC AGA ACG CCA TTA TTG ACG CCG
GGA ACA GAG CAT CGT GAA CGC GTA CAA ACA GGT AAA TAT GAA
TAT ATT ACC AAG CTC AAT ATT AAC CGT GTA GAT AGC TGG AAA
ATT ACA GAT GGT GCA GCA AGT TCT ACC TTT GAT TTA ACT AAC
GTT GTT CAG CGT ATT GGT ATT GAA TTA GAC AAT GCT GGA AAT
GTA ACT AAA ACC AAA GAA ACA AAA ATT ATT GCC AAA CTT GGT
GAA GGT GAT GAC AAC GTA TTT GTT GGT TCT GGT ACG ACG GAA
ATT GAT GGC GGT GAA GGT TAC GAC CGA GTT CAC TAT AGC CGT
GGA AAC TAT GGT GCT TTA ACT ATT GAT GCA ACC AAA GAG ACC
GAG CAA GGT AGT TAT ACC GTA AAT CGT TTC GTA GAA ACC GGT
AAA GCA CTA CAC GAA GTG ACT TCA ACC CAT ACC GCA TTA GTG
GGC AAC CGT GAA GAA AAA ATA GAA TAT CGT CAT AGC AAT AAC
CAG CAC CAT GCC GGT TAT TAC ACC AAA GAT ACC TTG AAA GCT
GTT GAA GAA ATT ATC GGT ACA TCA CAT AAC GAT ATC TTT AAA
GGT AGT AAG TTC AAT GAT GCC TTT AAC GGT GGT GAT GGT GTC

GAT ACT ATT GAC GGT AAC GAC GGC AAT GAC CGC TTA TTT GGT
GGT AAA GGC GAT GAT ATT CTC GAT GGT GGA AAT GGT GAT GAT
TTT ATC GAT GGC GGT AAA GGC AAC GAC CTA TTA CAC GGT GGC
AAG GGC GAT GAT ATT TTC GTT CAC CGT AAA GGC GAT GGT AAT
GAT ATT ATT ACC GAT TCT GAC GGC AAT GAT AAA TTA TCA TTC
TCT GAT TCG AAC TTA AAA GAT TTA ACA TTT GAA AAA GTT AAA
CAT AAT CTT GTC ATC ACG AAT AGC AAA AAA GAG AAA GTG ACC
ATT CAA AAC TGG TTC CGA GAG GCT GAT TTT GCT AAA GAA GTG
CCT AAT TAT AAA GCA ACT AAA GAT GAG AAA ATC GAA GAA ATC
ATC GGT CAA AAT GGC GAG CGG ATC ACC TCA AAG CAA GTT GAT
GAT CTT ATC GCA AAA GGT AAC GGC AAA ATT ACC CAA GAT GAG
CTA TCA AAA GTT GTT GAT AAC TAT GAA TTG CTC AAA CAT AGC
AAA AAT GTG ACA AAC AGC TTA GAT APG TTA ATC TCA TCT GTA
AGT GCA TTT ACC TCG TCT AAT GAT TCG AGA AAT GTA TTA GTG
GCT CCA ACT TCA ATG TTG GAT CAA AGT TTA TCT TCT CTT CAA
TTT GCT AGA GCA GCT TAA.

5. The process as claimed in claim 1, wherein said DNA sequence encodes for leukotoxin protein of ORF and having the following amino acid sequence:
Met Gly Thr Arg Leu Thr Thr Leu Ser Asn Gly Leu Lys Asn Thr Leu Thr Ala Thr Lys Ser Gly Leu His Lys Ala Gly Gln Ser Leu Thr Gln Ala Gly Ser Ser Leu Lys Thr Gly Ala Lys Lys Ile Ile Leu Tyr Ile Pro Glu Asn Tyr Gln Tyr Asp Thr Glu Gln Gly Asn Gly Leu Gln Asp Leu Val Lys Ala Ala Glu Glu Leu Gly Ile Glu Val Gln Arg Glu Glu Arg Asn Asn Ile Ala Thr Ala Gln Thr Ser Leu Gly Thr Ile Gln Thr Ala Ile Gly Leu Thr Glu Arg Gly Ile Val Leu Ser Ala Pro Gln Ile Asp Lys Leu Leu Gln Lys Thr Lys Ala Gly Gln Ala Leu Gly Ser Ala Glu Ser Ile Val Gln Asn Ala Asn Lys Ala Lys Thr Val Leu Ser Gly Ile Gln Ser Ile Leu Gly Ser Val Leu Ala Gly Met Asp Leu Asp Glu Ala Leu Gln Asn Asn Ser Asn Gln His Ala Leu Ala Lys Ala Gly Leu Glu Leu Thr Asn Ser Leu Ile Glu Asn Ile Ala Asn Ser Val Lys Thr Leu Asp Glu Phe Gly Glu Gln Ile Ser Gln Phe Gly Ser Lys Leu Gln Asn Ile Lys Gly Leu Gly Thr Leu Gly Asp Lys Leu Lys Asn Ile Gly Gly Leu Asp Lys Ala Gly Leu Gly Leu Asp Val Ile Ser Gly Leu Leu Ser Gly Ala Thr Ala Ala Leu Val Leu Ala Asp Lys Asn Ala Ser Thr Ala Lys Lys Val Gly Ala Gly Phe Glu Leu Ala Asn Gln Val Val Gly Asn Ile Thr Lys Ala Val Ser Ser Tyr Ile Leu Ala Gln Arg Val Ala Ala Gly Leu Ser Ser Thr Gly Pro Val Ala Ala Leu Ile Ala Ser Thr Val Ser Leu Ala Ile Ser Pro Leu Ala Phe Ala Gly Ile Ala Asp Lys Phe Asn His Ala Lys Ser Leu Glu Ser Tyr Ala Glu Arg Phe Lys Lys Leu Gly Tyr Asp Gly Asp Asn Leu Leu Ala Glu Tyr Gln Arg Gly Thr Gly Thr Ile Asp Ala Ser Val Thr Ala Ile Asn Thr Ala Leu Ala Ala Ile Ala Gly Gly Val Ser Ala Ala Ala Ala Gly Ser Val Ile Ala Ser Pro Ile Ala Leu Leu Val Ser Gly Ile Thr Gly Val Ile Ser Thr Ile Leu Gln Tyr Ser Lys Gln Ala Met Phe Glu His Val Ala Asn Lys Ile His Asn Lys Ile Val Glu Trp Glu Lys Asn Asn His Gly Lys Asn Tyr Phe Glu Asn Gly Tyr Asp Ala Arg Tyr Leu Ala Asn Leu Gln Asp Asn Met Lys Phe Leu Leu Asn Leu Asn Lys Glu Leu Glu Ala Glu Arg Val Ile Ala Ile Thr Gln Gln Gln Trp Asp Asn Asn Ile Gly Asp Leu Ala Gly Ile Ser Arg Leu Gly Glu Lys Val Leu Ser Gly Lys Ala Tyr Val Asp Ala Phe Glu Glu Gly Lys His Ile Lys Ala Asp Lys Leu Val Gln Leu Asp Ser Ala Asn Gly Ile Ile Asp Val Ser Asn Ser Gly Lys Ala Lys Thr Gln His Ile Leu Phe Arg Thr Pro Leu Leu Thr Pro Gly Thr Glu His Arg Glu Arg Val Gln Thr Gly Lys Tyr Glu Tyr Ile Thr Lys Leu Asn Ile Asn Arg Val Asp Ser Trp Lys Ile Thr Asp Gly Ala Ala Ser Ser Thr Phe Asp Leu Thr Asn Val Val Gln Arg Ile Gly Ile Glu Leu Asp Asn Ala Gly Asn Val Thr Lys Thr Lys Glu Thr Lys Ile Ile Ala Lys Leu Gly Glu Gly Asp Asp Asn Val Phe Val Gly Ser Gly Thr Thr Glu Ile Asp Gly Gly Glu Gly Tyr Asp Arg Val His Tyr Ser Arg Gly Asn Tyr Gly Ala Leu Thr Ile Asp Ala Thr Lys Glu Thr Glu Gln Gly Ser Tyr Thr Val Asn Arg Phe Val Glu Thr Gly Lys Ala Leu His Glu Val Thr Ser Thr His Thr Ala Leu Val Gly Asn Arg Glu Glu Lys Ile Glu Tyr Arg His Ser Asn Asn Gln His His Ala Gly Tyr Tyr Thr Lys Asp Thr Leu Lys Ala Val Glu Glu Ile Ile Gly Thr Ser His Asn Asp Ile Phe Lys Gly Ser Lys Phe Asn Asp Ala Phe Asn Gly Gly Asp Gly Val Asp Thr Ile Asp Gly Asn Asp Gly Asn Asp Arg Leu Phe Gly Gly Lys Gly Asp Asp Ile Leu Asp Gly Gly Asn Gly Asp Asp Phe Ile Asp Gly Gly Lys Gly Asn Asp Leu Leu His Gly Gly Lys Gly Asp Asp Ile Phe Val His Arg Lys Gly Asp Gly Asn Asp Ile Ile Thr Asp Ser Asp Gly Asn Asp Lys Leu Ser Phe Ser Asp Ser Asn Leu Lys Asp Leu Thr Phe Glu Lys Val Lys His Asn Leu Val Ile Thr Asn Ser Lys Lys Glu Lys Val Thr Ile Gln Asn Trp Phe Arg Glu Ala Asp Phe Ala Lys Glu Val Pro Asn Tyr Lys Ala Thr Lys Asp Glu Lys Ile Glu Glu Ile Ile Gly Gln Asn Gly Glu Arg Ile Thr Ser Lys Gln Val Asp Asp Leu Ile Ala Lys Gly Asn Gly Lys Ile Thr Gln Asp Glu Leu Ser Lys Val Val Asp Asn Tyr Glu Leu Leu Lys His Ser Lys Asn Val Thr Asn Ser Leu Asp Lys Leu Ile Ser Ser Val Ser Ala Phe Thr Ser Ser Asn Asp Ser Arg Asn Val Leu Val Ala Pro Thr Ser Met Leu Asp Gln Ser Leu Ser Ser Leu Gln Phe Ala Arg Ala Ala.

6. The process as claimed in claim 1, wherein said DNA sequence encodes for leukotoxin protein of ORF and ORF2 and having the following amino acid sequence:
Met Asn Gln Ser Tyr Phe Asn Leu Leu Gly Asn Ile Thr Trp Leu Trp Met Asn Ser Ser Leu His Lys Glu Trp Ser Cys Glu Leu Leu Ala Arch Asn Val Ile Pro Ala Ile Glu Asn Glu Gln Tyr Met Leu Leu Ile Asp Asn Gly Ile Pro Ile Ala Tyr Cys Ser Trp Ala Asp Leu Asn Leu Glu Thr Glu Val Lys Tyr Ile Lys Asp Ile Asn Ser Leu Thr Pro Glu Glu Trp Gln Ser Gly Asp Arg Arg Trp Ile Ile Asp Trp Val Ala Pro Phe Gly His Ser Gln Leu Leu Tyr Lys Lys Met Cys Gln Lys Tyr Pro Asp Met Ile Val Arch Ser Ile Arg Phe Tyr Pro Lys Gln Lys Glu Leu Gly Lys Ile Ala Tyr Phe Lys Gly Gly Lys Leu Asp Lys Lys Thr Ala Lys Lys Arg Phe Asp Thr Tyr Gln Glu Glu Leu Ala Thr Ala Leu Lys Asn Glu Phe Asn Phe Ile Lys Lys Met Gly Thr Arg Leu Thr Thr Leu Ser Asn Gly Leu Lys Asn Thr Leu Thr Ala Thr Lys Ser Gly Leu His Lys Ala Gly Gln Ser Leu Thr Gln Ala Gly Ser Ser Leu Lys Thr Gly Ala Lys Lys Ile Ile Leu Tyr Ile Pro Gln Asn Tyr Gln Tyr Asp Thr Glu Gln Gly Asn Gly Leu Gln Asp Leu Val Lys Ala Ala Glu Glu Leu Gly Ile Glu Val Gln Arg Glu Glu Arg Asn Asn Ile Ala Thr Ala Gln Thr Ser Leu Gly Thr Ile Gln Thr Ala Ile Gly Leu Thr Glu Arg Gly Ile Val Leu Ser Ala Pro Gln Ile Asp Lys Leu Leu Gln Lys Thr Lys Ala Gly Gln Ala Leu Gly Ser Ala Glu Ser Ile Val Gln Asn Ala Asn Lys Ala Lys Thr Val Leu Ser Gly Ile Gln Ser Ile Leu Gly Ser Val Leu Ala Gly Met Asp Leu Asp Glu Ala Leu Gln Asn Asn Ser Asn Gln His Ala Leu Ala Lys Ala Gly Leu Glu Leu Thr Asn Ser Leu Ile Glu Asn Ile Ala Asn Ser Val Lys Thr Leu Asp Glu Phe Glu Glu Gln Ile Ser Gln Phe Gly Ser Lys Leu Gln Asn Ile Lys Gly Leu Gly Thr Leu Gly Asp Lys Leu Lys Asn Ile Gly Gly Leu Asp Lys Ala Gly Leu Gly Leu Asp Val Ile Ser Gly Leu Leu Ser Gly Ala Thr Ala Ala Leu Val Leu Ala Asp Lys Asn Ala Ser Thr Ala Lys Lys Val Gly Ala Gly Phe Glu Leu Ala Asn Gln Val Val Gly Asn Ile Thr Lys Ala Val Ser Ser Tyr Ile Leu Ala Gln Arg Val Ala Ala Gly Leu Ser Ser Thr Gly Pro Val Ala Ala Leu Ile Ala Ser Thr Val Ser Leu Ala Ile Ser Pro Leu Ala Phe Ala Gly Ile Ala Asp Lys Phe Asn His Ala Lys Ser Leu Glu Ser Tyr Ala Glu Arg Phe Lys Lys Leu Gly Tyr Asp Gly Asp Asn Leu Leu Ala Glu Tyr Gln Arch Gly Thr Gly Thr Ile Asp Ala Ser Val Thr Ala Ile Asn Thr Ala Leu Ala Ala Ile Ala Gly Gly Val Ser Ala Ala Ala Ala Gly Ser Val Ile Ala Ser Pro Ile Ala Leu Leu Val Ser Gly Ile Thr Gly Val Ile Ser Thr Ile Leu Gln Tyr Ser Lys Gln Ala Met Phe Glu His Val Ala Asn Lys Ile His Asn Lys Ile Val Glu Trp Glu Lys Asn Asn His Gly Lys Asn Tyr Phe Glu Asn Gly Tyr Asp Ala Arg Tyr Leu Ala Asn Leu Gln Asp Asn Met Lys Phe Leu Leu Asn Leu Asn Lys Glu Leu Gln Ala Glu Arg Val Ile Ala Ile Thr Gln Gln Gln Trp Asp Asn Asn Ile Gly Asp Leu Ala Gly Ile Ser Arg Leu Gly Glu Lys Val Leu Ser Gly Lys Ala Tyr Val Asp Ala Phe Glu Glu Gly Lys His Ile Lys Ala Asp Lys Leu Val Gln Leu Asp Ser Ala Asn Gly Ile Ile Asp Val Ser Asn Ser Gly Lys Ala Lys Thr Gln His Ile Leu Phe Arg Thr Pro Leu Leu Thr Pro Gly Thr Glu His Arg Glu Arg Val Gln Thr Gly Lys Tyr Glu Tyr Ile Thr Lys Leu Asn Ile Asn Arg Val Asp Ser Trp Lys Ile Thr Asp Gly Ala Ala Ser Ser Thr Phe Asp Leu Thr Asn Val Val Gln Arcs Ile Gly Ile Glu Leu Asp Asn Ala Gly Asn Val Thr Lys Thr Lys Glu Thr Lys Ile Ile Ala Lys Leu Glu Glu Gly Asp Asp Asn Val Phe Val Gly Ser Gly Thr Thr Glu Ile Asp Gly Gly Glu Gly Tyr Asp Arg Val His Tyr Ser Arg Tyr Thr Val Asn Arg Phe Val Glu Thr Gly Lys Ala Leu His Glu Val Thr Ser Thr His Thr Ala Leu Val Gly Asn Arg Glu Glu Lys Ile Glu Tyr Arg His Ser Asn Asn Glu His His Ala Gly Tyr Tyr Thr Lys Asp Thr Leu Lys Ala Val Glu Glu Ile Ile Gly Thr Ser His Asn Asp Ile Phe Lys Gly Ser Lys Phe Asn Asn Ala Phe Asn Gly Gly Asp Gly Val Asp Thr Ile Asp Gly Asn Asp Gly Asn Asp Arg Leu Phe Gly Gly Lys Gly Asp Asp Ile Leu Asp Gly Gly Asn Gly Asp Asp Phe Ile Asp Gly Gly Lys Gly Asn Asp Leu Leu His Gly Gly Lys Gly Asp Asp Ile Phe Val His Arg Lys Gly Asp Gly Asn Asp Ile Ile Thr Asp Ser Asp Gly Asn Asp Lys Leu Ser Phe Ser Asp Ser Asn Leu Lys Asp Leu Thr Phe Glu Lys Val Lys His Asn Leu Val Ile Thr Asn Ser Lys Lys Glu Lys Val Thr Ile Gln Asn Trp Phe Arg Glu Ala Asp Phe Ala Lys Glu Val Pro Asn Tyr Lys Ala Thr Lys Asp Glu Lys Ile Glu Glu Ile Ile Gly Gln Asn Gly Glu Arg Ile Thr Ser Leu Ser Lys Val Val Asp Asn Tyr Glu Leu Leu Lys His Ser Lys Asn Val Thr Asn Ser Leu Asp Lys Leu Ile Ser Ser Val Ser Ala Phe Thr Ser Ser Asn Asp Ser Arg Asn Val Leu Val Ala Pro Thr Ser Met Leu Asp Gln Ser Leu Ser Ser Leu Gln Phe Ala Arg Ala Ala.

7. A leukotoxin protein of ORF1 and having the following amino acid sequence:
Met Gly Thr Arg Leu Thr Thr Leu Ser Asn Gly Leu Lys Asn Thr Leu Thr Ala Thr Lys Ser Gly Leu His Lys Ala Gly Gln Ser Leu Thr Gln Ala Gly Ser Ser Leu Lys Thr Gly Ala Lys Lys Ile Ile Leu Tyr Ile Pro Glu Asn Tyr Gln Tyr Asp Thr Glu Gln Gly Asn Gly Leu Gln Asp Leu Val Lys Ala Ala Glu Glu Leu Gly Ile Glu Val Gln Arg Glu Glu Arg Asn Asn Ile Ala Thr Ala Gln Thr Ser Leu Gly Thr Ile Gln Thr Ala Ile Gly Leu Thr Glu Arg Gly Ile Val Leu Ser Ala Pro Gln Ile Asp Lys Leu Leu Gln Lys Thr Lys Ala Gly Gln Ala Leu Gly Ser Ala Glu Ser Ile Val Gln Asn Ala Asn Lys Ala Lys Thr Val Leu Ser Gly Ile Gln Ser Ile Leu Gly Ser Val Leu Ala Gly Met Asp Leu Asp Glu Ala Leu Gln Asn Asn Ser Asn Gln His Ala Leu Ala Lys Ala Gly Leu Glu Leu Thr Asn Ser Leu Ile Glu Asn Ile Ala Asn Ser Val Lys Thr Leu Asp Glu Phe Gly Glu Gln Ile Ser Gln Phe Gly Ser Lys Leu Gln Asn Ile Lys Gly Leu Gly Thr Leu Gly Asp Lys Leu Lys Asn Ile Gly Gly Leu Asp Lys Ala Gly Leu Gly Leu Asp Val Ile Ser Gly Leu Leu Ser Gly Ala Thr Ala Ala Leu Val Leu Ala Asp Lys Asn Ala Ser Thr Ala Lys Lys Val Gly Ala Gly Phe Glu Leu Ala Asn Gln Val Val Gly Asn Ile Thr Lys Ala Val Ser Ser Tyr Ile Leu Ala Gln Arg Val Ala Ala Gly Leu Ser Ser Thr Gly Pro Val Ala Ala Leu Ile Ala Ser Thr Val Ser Leu Ala Ile Ser Pro Leu Ala Phe Ala Gly Ile Ala Asp Lys Phe Asn His Ala Lys Ser Leu Glu Ser Tyr Ala Glu Arg Phe Lys Lys Leu Gly Tyr Asp Gly Asp Asn Leu Leu Ala Glu Tyr Gln Arg Gly Thr Gly Thr Ile Asp Ala Ser Val Thr Ala Ile Asn Thr Ala Leu Ala Ala Ile Ala Gly Gly Val Ser Ala Ala Ala Ala Gly Ser Val Ile Ala Ser Pro Ile Ala Leu Leu Val Ser Gly Ile Thr Gly Val Ile Ser Thr Ile Leu Gln Tyr Ser Lys Gln Ala Met Phe Glu His Val Ala Asn Lys Ile His Asn Lys Ile Val Glu Trp Glu Lys Asn Asn His Gly Lys Asn Tyr Phe Glu Asn Gly Tyr Asp Ala Arg Tyr Leu Ala Asn Leu Gln Asp Asn Met Lys Phe Leu Leu Asn Leu Asn Lys Glu Leu Gln Ala Glu Arg Val Ile Ala Ile Thr Gln Gln Gln Trp Asp Asn Asn Ile Gly Asp Leu Ala Gly Ile Ser Arg Leu Gly Glu Lys Val Leu Ser Gly Lys Ala Tyr Val Asp Ala Phe Glu Glu Gly Lys His Ile Lys Ala Asp Lys Leu Val Gln Leu Asp Ser Ala Asn Gly Ile Ile Asp Val Ser Asn Ser Gly Lys Ala Lys Thr Gln His Ile Leu Phe Arg Thr Pro Leu Leu Thr Pro Gly Thr Glu His Arg Glu Arg Val Gln Thr Gly Lys Tyr Glu Tyr Ile Thr Lys Leu Asn Ile Asn Arg Val Asp Ser Trp Lys Ile Thr Asp Gly Ala Ala Ser Ser Thr Phe Asp Leu Thr Asn Val Val Gln Arg Ile Gly Ile Glu Leu Asp Asn Ala Gly Asn Val Thr Lys Thr Lys Glu Thr Lys Ile Ile Ala Lys Leu Gly Glu Gly Asp Asp Asn Val Phe Val Gly Ser Gly Thr Thr Glu Ile Asp Gly Gly Glu Gly Tyr Asp Arg Val His Tyr Ser Arg Gly Asn Tyr Gly Ala Leu Thr Ile Asp Ala Thr Lys Glu Thr Glu Gln Gly Ser Tyr Thr Val Asn Arg Phe Val Glu Thr Gly Lys Ala Leu His Glu Val Thr Ser Thr His Thr Ala Leu Val Gly Asn Arg Glu Glu Lys Ile Glu Tyr Arg His Ser Asn Asn Gln His His Ala Gly Tyr Tyr Thr Lys Asp Thr Leu Lys Ala Val Glu Glu Ile Ile Gly Thr Ser His Asn Asp Ile Phe Lys Gly Ser Lys Phe Asn Asp Ala Phe Asn Gly Gly Asp Gly Val Asp Thr Ile Asp Gly Asn Asp Gly Asn Asp Arg Leu Phe Gly Gly Lys Gly Asp Asp Ile Leu Asp Gly Gly Asn Gly Asp Asp Phe Ile Asp Gly Gly Lys Gly Asn Asp Leu Leu His Gly Gly Lys Gly Asp Asp Ile Phe Val His Arg Lys Gly Asp Gly Asn Asp Ile Ile Thr Asp Ser Asp Gly Asn Asp Lys Leu Ser Phe Ser Asp Ser Asn Leu Lys Asp Leu Thr Phe Glu Lys Val Lys His Asn Leu Val Ile Thr Asn Ser Lys Lys Glu Lys Val Thr Ile Gln Asn Trp Phe Arg Glu Ala Asp Phe Ala Lys Glu Val Pro Asn Tyr Lys Ala Thr Lys Asp Glu Lys Ile Glu Glu Ile Ile Gly Gln Asn Gly Glu Arg Ile Thr Ser Lys Gln Val Asp Asp Leu Ile Ala Lys Gly Asn Gly Lys Ile Thr Gln Asp Glu Leu Ser Lys Val Val Asp Asn Tyr Glu Leu Leu Lys His Ser Lys Asn Val Thr Asn Ser Leu Asp Lys Leu Ile Ser Ser Val Ser Ala Phe Thr Ser Ser Asn Asp Ser Arg Asn Val Leu Val Ala Pro Thr Ser Met Leu Asp Gln Ser Leu Ser Ser Leu Gln Phe Ala Arg Ala Ala.

8. A leukotoxin protein of ORF1 and ORF2 and having the following amino acid sequence:
Met Asn Gln Ser Tyr Phe Asn Leu Leu Gly Asn Ile Thr Trp Leu Trp Met Asn Ser Ser Leu His Lys Glu Trp Ser Cys Glu Leu Leu Ala Arg Asn Val Ile Pro Ala Ile Glu Asn Glu Gln Tyr Met Leu Leu Ile Asp Asn Gly Ile Pro Ile Ala Tyr Cys Ser Trp Ala Asp Leu Asn Leu Glu Thr Glu Val Lys Tyr Ile Lys Asp Ile Asn Ser Leu Thr Pro Glu Glu Trp Gln Ser Gly Asp Arg Arg Trp Ile Ile Asp Trp Val Ala Pro Phe Gly His Ser Gln Leu Leu Tyr Lys Lys Met Cys Gln Lys Tyr Pro Asp Met Ile Val Arg Ser Ile Arg Phe Tyr Pro Lys Gln Lys Glu Leu Gly Lys Ile Ala Tyr Phe Lys Gly Gly Lys Leu Asp Lys Lys Thr Ala Lys Lys Arg Phe Asp Thr Tyr Gln Glu Glu Leu Ala Thr Ala Leu Lys Asn Glu Phe Asn Phe Ile Lys Lys Met Gly Thr Arg Leu Thr Thr Leu Ser Asn Gly Leu Lys Asn Thr Leu Thr Ala Thr Lys Ser Gly Leu His Lys Ala Gly Gln Ser Leu Thr Gln Ala Gly Ser Ser Leu Lys Thr Gly Ala Lys Lys Ile Ile Leu Tyr Ile Pro Gln Asn Tyr Gln Tyr Asp Thr Glu Gln Gly Asn Gly Leu Gln Asp Leu Val Lys Ala Ala Glu Glu Leu Gly Ile Glu Val Gln Arg Glu Glu Arg Asn Asn Ile Ala Thr Ala Gln Thr Ser Leu Gly Thr Ile Gln Thr Ala Ile Gly Leu Thr Glu Arg Gly Ile Val Leu Ser Ala Pro Gln Ile Asp Lys Leu Leu Gln Lys Thr Lys Ala Gly Gln Ala Leu Gly Ser Ala Glu Ser Ile Val Gln Asn Ala Asn Lys Ala Lys Thr Val Leu Ser Gly Ile Gln Ser Ile Leu Gly Ser Val Leu Ala Gly Met Asp Leu Asp Glu Ala Leu Gln Asn Asn Ser Asn Gln His Ala Leu Ala Lys Ala Gly Leu Glu Leu Thr Asn Ser Leu Ile Glu Asn Ile Ala Asn Ser Val Lys Thr Leu Asp Glu Phe Gly Glu Gln Ile Ser Gln Phe Gly Ser Lys Leu Gln Asn Ile Lys Gly Leu Gly Thr Leu Gly Asp Lys Leu Lys Asn Ile Gly Gly Leu Asp Lys Ala Gly Leu Gly Leu Asp Val Ile Ser Gly Leu Leu Ser Gly Ala Thr Ala Ala Leu Val Leu Ala Asp Lys Asn Ala Ser Thr Ala Lys Lys Val Gly Ala Gly Phe Glu Leu Ala Asn Gln Val Val Gly Asn Ile Thr Lys Ala Val Ser Ser Tyr Ile Leu Ala Gln Arg Val Ala Ala Gly Leu Ser Ser Thr Gly Pro Val Ala Ala Leu Ile Ala Ser Thr Val Ser Leu Ala Ile Ser Pro Leu Ala Phe Ala Gly Ile Ala Asp Lys Phe Asn His Ala Lys Ser Leu Glu Ser Tyr Ala Glu Arg Phe Lys Lys Leu Gly Tyr Asp Gly Asp Asn Leu Leu Ala Glu Tyr Gln Arch Gly Thr Gly Thr Ile Asp Ala Ser Val Thr Ala Ile Asn Thr Ala Leu Ala Ala Ile Ala Gly Gly Val Ser Ala Ala Ala Ala Gly Ser Val Ile Ala Ser Pro Ile Ala Leu Leu Val Ser Gly Ile Thr Gly Val Ile Ser Thr Ile Leu Gln Tyr Ser Lys Gln Ala Met Phe Glu His Val Ala Asn Lys Ile His Asn Lys Ile Val Glu Trp Glu Lys Asn Asn His Gly Lys Asn Tyr Phe Glu Asn Gly Tyr Asp Ala Arg Tyr Leu Ala Asn Leu Gln Asp Asn Met Lys Phe Leu Leu Asn Leu Asn Lys Glu Leu Gln Ala Glu Arg Val Ile Ala Ile Thr Gln Gln Gln Trp Asp Asn Asn Ile Gly Asp Leu Ala Gly Ile Ser Arg Leu Gly Glu Lys Val Leu Ser Gly Lys Ala Tyr Val Asp Ala Phe Glu Glu Gly Lys His Ile Lys Ala Asp Lys Leu Val Gln Leu Asp Ser Ala Asn Gly Ile Ile Asp Val Ser Asn Ser Gly Lys Ala Lys Thr Gln Hip Ile Leu Phe Arg Thr Pro Leu Leu Thr Pro Gly Thr Glu His Arg Glu Arg Val Gln Thr Gly Lys Tyr Glu Tyr Ile Thr Lys Leu Asn Ile Asn Arg Val Asp Ser Trp Lys Ile Thr Asp Gly Ala Ala Ser Ser Thr Phe Asp Leu Thr Asn Val Val Gln Arg Ile Gly Ile Glu Leu Asp Asn Ala Gly Asn Val Thr Lys Thr Lys Glu Thr Lys Ile Ile Ala Lys Leu Glu Glu Gly Asp Asp Asn Val Phe Val Gly Ser Gly Thr Thr Glu Ile Asp Gly Gly Glu Gly Tyr Asp Arg Val His Tyr Ser Arg Tyr Thr Val Asn Arg Phe Val Glu Thr Gly Lys Ala Leu His Glu Val Thr Ser Thr His Thr Ala Leu Val Gly Asn Arg Glu Glu Lys Ile Glu Tyr Arg His Ser Asn Asn Gln His His Ala Gly Tyr Tyr Thr Lys Asp Thr Leu Lys Ala Val Glu Glu Ile Ile Gly Thr Ser His Asn Asp Ile Phe Lys Gly Ser Lys Phe Asn Asn Ala Phe Asn Gly Gly Asp Gly Val Asp Thr Ile Asp Gly Asn Asp Gly Asn Asp Arg Leu Phe Gly Gly Lys Gly Asp Asp Ile Leu Asp Gly Gly Asn Gly Asp Asp Phe Ile Asp Gly Gly Lys Gly Asn Asp Leu Leu His Gly Gly Lys Gly Asp Asp Ile Phe Val His Arg Lys Gly Asp Gly Asn Asp Ile Ile Thr Asp Ser Asp Gly Asn Asp Lys Leu Ser Phe Ser Asp Ser Asn Leu Lys Asp Leu Thr Phe Glu Lys Val Lys His Asn Leu Val Ile Thr Asn Ser Lys Lys Glu Lys Val Thr Ile Gln Asn Trp Phe Arg Glu Ala Asp Phe Ala Lys Glu Val Pro Asn Tyr Lys Ala Thr Lys Asp Glu Lys Ile Glu Glu Ile Ile Gly Gln Asn Gly Glu Arg Ile Thr Ser Leu Ser Lys Val Val Asp Asn Tyr Glu Leu Leu Lys His Ser Lys Asn Val Thr Asn Ser Leu Asp Lys Leu Ile Ser Ser Val Ser Ala Phe Thr Ser Ser Asn Asp Ser Arg Asn Val Leu Val Ala Pro Thr Ser Met Leu Asp Gln Ser Leu Ser Ser Leu Gln Phe Ala Arg Ala Ala.

9. A polyclonal antibody raised to antigenic determinants of the leukotoxin protein of claim 7 or 8.

10. A process for making a vaccine effective against Pasteurella haemolytica comprising mixing:
A. a leukotoxin which is produced by (i) transforming a microorganism with a DNA sequence encoding leukotoxin comprising the following amino acid sequence:
Met Gly Thr Arg Leu Thr Thr Leu Ser Asn Gly Leu Lys Asn Thr Leu Thr Ala Thr Lys Ser Gly Leu His Lys Ala Gly Gln Ser Leu Thr Gln Ala Gly Ser Ser Leu Lys Thr Gly Ala Lys Lys Ile Ile Leu Tyr Ile Pro Glu Asn Tyr Gln Tyr Asp Thr Glu Gln Gly Asn Gly Leu Gln Asp Leu Val Lys Ala Ala Glu Glu Leu Gly Ile Glu Val Gln Arg Glu Glu Arg Asn Asn Ile Ala Thr Ala Gln Thr Ser Leu Gly Thr Ile Gln Thr Ala Ile Gly Leu Thr Glu Arg Gly Ile Val Leu Ser Ala Pro Gln Ile Asp Lys Leu Leu Gln Lys Thr Lys Ala Gly Gln Ala Leu Gly Ser Ala Glu Ser Ile Val Gln Asn Ala Asn Lys Ala Lys Thr Val Leu Ser Gly Ile Gln Ser Ile Leu Gly Ser Val Leu Ala Gly Met Asp Leu Asp Glu Ala Leu Gln Asn Asn Ser Asn Gln His Ala Leu Ala Lys Ala Gly Leu Glu Leu Thr Asn Ser Leu Ile Glu Asn Ile Ala Asn Ser Val Lys Thr Leu Asp Glu Phe Gly Glu Gln Ile Ser Gln Phe Gly Ser Lys Leu Gln Asn Ile Lys Gly Leu Gly Thr Leu Gly Asp Lys Leu Lys Asn Ile Gly Gly Leu Asp Lys Ala Gly Leu Gly Leu Asp Val Ile Ser Gly Leu Leu Ser Gly Ala Thr Ala Ala Leu Val Leu Ala Asp Lys Asn Ala Ser Thr Ala Lys Lys Val Gly Ala Gly Phe Glu Leu Ala Asn Gln Val Val Gly Asn Ile Thr Lys Ala Val Ser Ser Tyr Ile Leu Ala Gln Arg Val Ala Ala Gly Leu Ser Ser Thr Gly Pro Val Ala Ala Leu Ile Ala Ser Thr Val Ser Leu Ala Ile Ser Pro Leu Ala Phe Ala Gly Ile Ala Asp Lys Phe Asn His Ala Lys Ser Leu Glu Ser Tyr Ala Glu Arg Phe Lys Lys Leu Gly Tyr Asp Gly Asp Asn Leu Leu Ala Glu Tyr Gln Arg Gly Thr Gly Thr Ile Asp Ala Ser Val Thr Ala Ile Asn Thr Ala Leu Ala Ala Ile Ala Gly Gly Val Ser Ala Ala Ala Ala Gly Ser Val Ile Ala Ser Pro Ile Ala Leu Leu Val Ser Gly Ile Thr Gly Val Ile Ser Thr Ile Leu Gln Tyr Ser Lys Gln Ala Met Phe Glu His Val Ala Asn Lys Ile His Asn Lys Ile Val Glu Trp Glu Lys Asn Asn His Gly Lys Asn Tyr Phe Glu Asn Gly Tyr Asp Ala Arg Tyr Leu Ala Asn Leu Gln Asp Asn Met Lys Phe Lets Leu Asn Leu Asn Lys Glu Leu Glu Ala Glu Arg Val Ile Ala Ile Thr Gln Gln Gln Trp Asp Asn Asn Ile Gly Asp Leu Ala Gly Ile Ser Arg Leu Gly Glu Lys Val Leu Ser Gly Lys Ala Tyr Val Asp Ala Phe Glu Glu Gly Lys His Ile Lys Ala Asp Lys Leu Val Gln Leu Asp Ser Ala Asn Gly Ile Ile Asp Val Ser Asn Ser Gly Lys Ala Lys Thr Gln His Ile Leu Phe Arg Thr Pro Leu Leu Thr Pro Gly Thr Glu His Arg Glu Arg Val Gln Thr Gly Lys Tyr Glu Tyr Ile Thr Lys Leu Asn Ile Asn Arg Val Asp Ser Trp Lys Ile Thr Asp Gly Ala Ala Ser Ser Thr Phe Asp Leu Thr Asn Val Val Gln Arg Ile Gly Ile Glu Leu Asp Asn Ala Gly Asn Val Thr Lys Thr Lys Glu Thr Lys Ile Ile Ala Lys Leu Gly Glu Gly Asp Asp Asn Val Phe Val Gly Ser Gly Thr Thr Glu Ile Asp Gly Gly Glu Gly Tyr Asp Arg Val His Tyr Ser Arg Gly Asn Tyr Gly Ala Leu Thr Ile Asp Ala Thr Lys Glu Thr Glu Gln Gly Ser Tyr Thr Val Asn Arg Phe Val Glu Thr Gly Lys Ala Leu His Glu Val Thr Ser Thr His Thr Ala Leu Val Gly Asn Arg Glu Glu Lys Ile Glu Tyr Arg His Ser Asn Asn Gln His His Ala Gly Tyr Tyr Thr Lys Asp Thr Leu Lys Ala Val Glu Glu Ile Ile Gly Thr Ser His Asn Asp Ile Phe Lys Gly Ser Lys Phe Asn Asp Ala Phe Asn Gly Gly Asp Gly Val Asp Thr Ile Asp Gly Asn Asp Gly Asn Asp Arg Leu Phe Gly Gly Lys Gly Asp Asp Ile Leu Asp Gly Gly Asn Gly Asp Asp Phe Ile Asp Gly Gly Lys Gly Asn Asp Leu Leu His Gly Gly Lys Gly Asp Asp Ile Phe Val His Arg Lys Gly Asp Gly Asn Asp Ile Ile Thr Asp Ser Asp Gly Asn Asp Lys Leu Ser Phe Ser Asp Ser Asn Leu Lys Asp Leu Thr Phe Glu Lys Val Lys His Asn Leu Val Ile Thr Asn Ser Lys Lys Glu Lys Val Thr Ile Gln Asn Trp Phe Arg Glu Ala Asp Phe Ala Lys Glu Val Pro Asn Tyr Lys Ala Thr Lys Asp Glu Lys Ile Glu Glu Ile Ile Gly Gln Asn Gly Glu Arg Ile Thr Ser Lys Gln Val Asp Asp Leu Ile Ala Lys Gly Asn Gly Lys Ile Thr Gln Asp Glu Leu Ser Lys Val Val Asp Asn Tyr Glu Leu Leu Lys His Ser Lys Asn Val Thr Asn Ser Leu Asp Lys Leu Ile Ser Ser Val Ser Ala Phe Thr Ser Ser Asn Asp Ser Arg Asn Val Leu Val Ala Pro Thr Ser Met Leu Asp Gln Ser Leu Ser Ser Leu Gln Phe Ala Arg Ala Ala (ii) culturing the resulting transformed microorganism; and (iii) obtaining the resulting leukotoxin produced from said cultured transformed microorganism; with B. a vaccine carrier.

11. A process for making a vaccine effective against Pasteurella haemolytica comprising mixing:
A. a leukotoxin which is produced by (i) transforming a microorganism with a DNA sequence encoding leukotoxin comprising the following amino acid sequence:
Met Asn Gln Ser Tyr Phe Asn Leu Leu Gly Asn Ile Thr Trp Leu Trp Met Asn Ser Ser Leu His Lys Glu Trp Ser Cys Glu Leu Leu Ala Arg Asn Val Ile Pro Ala Ile Glu Asn Glu Gln Tyr Met Leu Leu Ile Asp Asn Gly Ile Pro Ile Ala Tyr Cys Ser Trp Ala Asp Leu Asn Leu Glu Thr Glu Val Lys Tyr Ile Lys Asp Ile Asn Ser Leu Thr Pro Glu Glu Trp Gln Ser Gly Asp Arg Arg Trp Ile Ile Asp Trp Val Ala Pro Phe Gly His Ser Gln Leu Leu Tyr Lys Lys Met Cys Gln Lys Tyr Pro Asp Met Ile Val Arg Ser Ile Arg Phe Tyr Pro Lys Gln Lys Glu Leu Gly Lys Ile Ala Tyr Phe Lys Gly Gly Lys Leu Asp Lys Lys Thr Ala Lys Lys Arg Phe Asp Thr Tyr Gln Glu Glu Leu Ala Thr Ala Leu Lys Asn Glu Phe Asn Phe Ile Lys Lys (ii) culturing the resulting transformed microorganism; and (iii) obtaining the resulting leukotoxin produced from said cultured transformed microorganism; with B. a vaccine carrier.

12. A process for making a vaccine effective against Pasteurella haemolytica comprising mixing:
A. a leukotoxin which is produced by (i) transforming a microorganism with a DNA sequence encoding leukotoxin comprising the following amino acid sequence:
Met Asn Gln Ser Tyr Phe Asn Leu Leu Gly Asn Ile Thr Trp Leu Trp Met Asn Ser Ser Leu His Lys Glu Trp Ser Cys Glu Leu Leu Ala Arg Asn Val Ile Pro Ala Ile Glu Asn Glu Gln Tyr Met Leu Leu Ile Asp Asn Gly Ile Pro Ile Ala Tyr Cys Ser Trp Ala Asp Leu Asn Leu Glu Thr Glu Val Lys Tyr Ile Lys Asp Ile Asn Ser Leu Thr Pro Glu Glu Trp Gln Ser Gly Asp Arg Arg Trp Ile Ile Asp Trp Val Ala Pro Phe Gly His Ser Gln Leu Leu Tyr Lys Lys Met Cys Gln Lys Tyr Pro Asp Met Ile Val Arg Ser Ile Arg Phe Tyr Pro Lys Gln Lys Glu Leu Gly Lys Ile Ala Tyr Phe Lys Gly Gly Lys Leu Asp Lys Lys Thr Ala Lye; Lys Arg Phe Asp Thr Tyr Gln Glu Glu Leu Ala Thr Ala Leu Lys Asn Glu Phe Asn Phe Ile Lys Lys and Met Gly Thr Arch Leu Thr Thr Leu Ser Asn Gly Leu Lys Asn Thr Leu Thr Ala Thr Lys Ser Gly Leu His Lys Ala Gly Gln Ser Leu Thr Gln Ala Gly Ser Ser Leu Lys Thr Gly Ala Lys Lys Ile Ile Leu Tyr Ile Pro Glu Asn Tyr Gln Tyr Asp Thr Glu Gln Gly Asn Gly Leu Gln Asp Leu Val Lys Ala Ala Glu Glu Leu Gly Ile Glu Val Gln Arg Glu Glu Arg Asn Asn Ile Ala Thr Ala Gln Thr Ser Leu Gly Thr Ile Gln Thr Ala Ile Gly Leu Thr Glu Arg Gly Ile Val Leu Ser Ala Pro Gln Ile Asp Lys Leu Leu Gln Lys Thr Lys Ala Gly Gln Ala Leu Gly Ser Ala Glu Ser Ile Val Gln Asn Ala Asn Lys Ala Lys Thr Val Leu Ser Gly Ile Gln Ser Ile Leu Gly Ser Val Leu Ala Gly Met Asp Leu Asp Glu Ala Leu Gln Asn Asn Ser Asn Gln His Ala Leu Ala Lys Ala Gly Leu Glu Leu Thr Asn Ser Leu Ile Glu Asn Ile Ala Asn Ser Val Lys Thr Leu Asp Glu Phe Gly Glu Gln Ile Ser Gln Phe Gly Ser Lys Leu Gln Asn Ile Lys Gly Leu Gly Thr Leu Gly Asp Lys Leu Lys Asn Ile Gly Gly Leu Asp Lys Ala Gly Leu Gly Leu Asp Val Ile Ser Gly Leu Leu Ser Gly Ala Thr Ala Ala Leu Val Leu Ala Asp Lys Asn Ala Ser Thr Ala Lys Lys Val Gly Ala Gly Phe Glu Leu Ala Asn Gln Val Val Gly Asn Ile Thr Lys Ala Val Ser Ser Tyr Ile Leu Ala Gln Arg Val Ala Ala Gly Leu Ser Ser Thr Gly Pro Val Ala Ala Leu Ile Ala Ser Thr Val Ser Leu Ala Ile Ser Pro Leu Ala Phe Ala Gly Ile Ala Asp Lys Phe Asn His Ala Lys Ser Leu Glu Ser Tyr Ala Glu Arg Phe Lys Lys Leu Gly Tyr Asp Gly Asp Asn Leu Leu Ala Glu Tyr Gln Arg Gly Thr Gly Thr Ile Asp Ala Ser Val Thr Ala Ile Asn Thr Ala Leu Ala Ala Ile Ala Gly Gly Val Ser Ala Ala Ala Ala Gly Ser Val Ile Ala Ser Pro Ile Ala Leu Leu Val Ser Gly Ile Thr Gly Val Ile Ser Thr Ile Leu Gln Tyr Ser Lys Gln Ala Met Phe Glu His Val Ala Asn Lys Ile His Asn Lys Ile Val Glu Trp Glu Lys Asn Asn His Gly Lys Asn Tyr Phe Glu Asn Gly Tyr Asp Ala Arg Tyr Leu Ala Asn Leu Gln Asp Asn Met Lys Phe Leu Leu Asn Leu Asn Lys Glu Leu Glu Ala Glu Arg Val Ile Ala Ile Thr Gln Gln Gln Trp Asp Asn Asn Ile Gly Asp Leu Ala Gly Ile Ser Arg Leu Gly Glu Lys Val Leu Ser Gly Lys Ala Tyr Val Asp Ala Phe Glu Glu Gly Lys His Ile Lys Ala Asp Lys Leu Val Gln Leu Asp Ser Ala Asn Gly Ile Ile Asp Val Ser Asn Ser Gly Lys Ala Lys Thr Gln His Ile Leu Phe Arg Thr Pro Leu Leu Thr Pro Gly Thr Glu His Arg Glu Arg Val Gln Thr Gly Lys Tyr Glu Tyr Ile Thr Lys Leu Asn Ile Asn Arg Val Asp Ser Trp Lys Ile Thr Asp Gly Ala Ala Ser Ser Thr Phe Asp Leu Thr Asn Val Val Gln Arg Ile Gly Ile Glu Leu Asp Asn Ala Gly Asn Val Thr Lys Thr Lys Glu Thr Lys Ile Ile Ala Lys Leu Gly Glu Gly Asp Asp Asn Val Phe Val Gly Ser Gly Thr Thr Glu Ile Asp Gly Gly Glu Gly Tyr Asp Arg Val His Tyr Ser Arg Gly Asn Tyr Gly Ala Leu Thr Ile Asp Ala Thr Lys Glu Thr Glu Gln Gly Ser Tyr Thr Val Asn Arg Phe Val Glu Thr Gly Lys Ala Leu His Glu Val Thr Ser Thr His Thr Ala Leu Val Gly Asn Arg Glu Glu Lys Ile Glu Tyr Arg His Ser Asn Asn Gln His His Ala Gly Tyr Tyr Thr Lys Asp Thr Leu Lys Ala Val Glu Glu Ile Ile Gly Thr Ser His Asn Asp Ile Phe Lys Gly Ser Lys Phe Asn Asp Ala Phe Asn Gly Gly Asp Gly Val Asp Thr Ile Asp Gly Asn Asp Gly Asn Asp Arg Leu Phe Gly Gly Lys Gly Asp Asp Ile Leu Asp Gly Gly Asn Gly Asp Asp Phe Ile Asp Gly Gly Lys Gly Asn Asp Leu Leu His Gly Gly Lys Gly Asp Asp Ile Phe Val His Arg Lys Gly Asp Gly Asn Asp Ile Ile Thr Asp Ser Asp Gly Asn Asp Lys Leu Ser Phe Ser Asp Ser Asn Leu Lys Asp Leu Thr Phe Glu Lys Val Lys His Asn Leu Val Ile Thr Asn Ser Lys Lys Glu Lys Val Thr Ile Gln Asn Trp Phe Arg Glu Ala Asp Phe Ala Lys Glu Val Pro Asn Tyr Lys Ala Thr Lys Asp Glu Lys Ile Glu Glu Ile Ile Gly Gln Asn Gly Glu Arg Ile Thr Ser Lys Gln Val Asp Asp Leu Ile Ala Lys Gly Asn Gly Lys Ile Thr Gln Asp Glu Leu Ser Lys Val Val Asp Asn Tyr Glu Leu Leu Lys His Ser Lys Asn Val Thr Asn Ser Leu Asp Lys Leu Ile Ser Ser Val Ser Ala Phe Thr Ser Ser Asn Asp Ser Arg Asn Val Leu Val Ala Pro Thr Ser Met Leu Asp Gln Ser Leu Ser Ser Leu Gln Phe Ala Arg Ala Ala (ii) culturing the resulting transformed microorganism; and (iii) obtaining the resulting leukotoxin produced from said cultured transformed microorganism; with B. a vaccine carrier.

13. A process for making a vaccine effective against Pasteurella haemolytica comprising mixing:
A. a leukotoxin which is produced by (i) transforming a microorganism with a DNA sequence comprising the following nucleic acid sequence:
ATG GGA ACT AGA CTT ACA ACC CTA TCA AAT GGG CTA AAA AAC
ACT TTA ACG GCA ACC AAA AGT GGC TTA CAT AAA GCC GGT CAA
TCA TTA ACC CAA GCC GGC AGT TCT TTA AAA ACT GGG GCA AAA
AAA ATT ATC CTC TAT ATT CCC CAA AAT TAC CAA TAT GAT ACT
GAA CAA GGT AAT GGT TTA CAG GAT TTA GTC AAA GCG GCC GAA
GAG TTG GGG ATT GAG GTA CAA AGA GAA GAA CGC AAT AAT ATT
GCA ACA GCT CAA ACC AGT TTA GGC ACG ATT CAA ACC GCT ATT
GGC TTA ACT GAG CGT GGC ATT GTG TTA TCC GCT CCA CAA ATT
GAT AAA TTG CTA CAG AAA ACT AAA GCA GGC CAA GCA TTA GGT
TCT GCC GAA AGC ATT GTA CAA AAT GCA AAT AAA GCC AAA ACT
GTA TTA TCT GGC ATT CAA TCT ATT TTA GGC TCA GTA TTG GCT
GGA ATG GAT TTA GAT GAG GCC TTA CAG AAT AAC AGC AAC CAA
CAT GCT CTT GCT AAA GCT GGC TTG GAG CTA ACA AAT TCA TTA
ATT GAA AAT ATT GCT AAT TCA GTA AAA ACA CTT GAC GAA TTT
GGT GAG CAA ATT AGT CAA TTT GGT TCA AAA CTA CAA AAT ATC
AAA GGC TTA GGG ACT TTA GGA GAC AAA CTC AAA AAT ATC GGT
GGA CTT GAT AAA GCT GGC CTT GGT TTA GAT GTT ATC TCA GGG
CTA TTA TCG GGC GCA ACA GCT CCA CTT GTA CTT GCA GAT AAA

AAT GCT TCA ACA GCT AAA AAA GTG GGT GCG GGT TTT GAA TTG
GCA AAC CAA GTT GTT GGT AAT ATT ACC AAA GCC GTT TCT TCT
TAC ATT TTA GCC CAA CGT GTT GCA GCA GGT TTA TCT TCA ACT
GGG CCT GTG GCT GCT TTA ATT GCT TCT ACT GTT TCT CTT GCG
ATT AGC CCA TTA GCA TTT GCC GGT ATT GCC GAT AAA TTT AAT
CAT GCA AAA AGT TTA GAG AGT TAT GCC GAA CGC TTT AAA AAA
TTA GGC TAT GAC GGA GAT AAT TTA TTA GCA GAA TAT CAG CGG
GGA ACA GGG ACT ATT GAT GCA TCG GTT ACT GCA ATT AAT ACC
GCA TTG GCG GCT ATT GCT CGT GGT GTG TCT GCT GCT GCA GCC
GGC TCG GTT ATT GCT TCA CCG ATT GCC TTA TTA GTA TCT GGG
ATT ACC GGT GTA ATT TCT ACG ATT CTG CAA TAT TCT AAA CAA
GCA ATG TTT GAG CAC GTT GCA AAT AAA ATT CAT AAC AAA ATT
GTA GAA TGG GAA AAA AAT AAT CAC GGT AAG AAC TAC TTT GAA
AAT GGT TAC GAT GCC CGT TAT CTT GCG AAT TTA CAA GAT AAT
ATG AAA TTC TTA CTG AAC TTA AAC AAA GAG TTA CAG GCA GAA
CGT GTC ATC GCT ATT ACT CAG CAG CAA TGG GAT AAC AAC ATT
GGT GAT TTA GCT GGT ATT AGC CGT TTA GGT GAA AAA GTC CTT
AGT GGT AAA GCC TAT GTG CAT CCG TTT GAA CAA GGC AAA CAC
ATT AAA GCC GAT AAA TTA GTA CAG TTG GAT TCG GCA AAC GGT
ATT ATT GAT GTG AGT AAT TCG GGT AAA GCG AAA ACT CAG CAT
ATC TTA TTC AGA ACG CCA TTA TTG ACG CCG GGA ACA GAG CAT
CGT GAA CGC GTA CAA ACA GGT AAA TAT GAA TAT ATT ACC AAG
CTC AAT ATT AAC CGT GTA GAT AGC TGG AAA ATT ACA GAT GGT
GCA GCA AGT TCT ACC TTT GAT TTA ACT AAC GTT GTT CAG CGT
ATT GGT ATT GAA TTA GAC AAT GCT GGA AAT GTA ACT AAA ACC
AAA GAA ACA AAA ATT ATT GCC AAA CTT GGT GAA GGT GAT GAC
AAC GTA TTT GTT GGT TCT GGT ACG ACG GAA ATT GAT GGC GGT
GAA GGT TAC GAC CGA GTT CAC TAT AGC CGT GGA AAC TAT GGT
GCT TTA ACT ATT GAT GCA ACC AAA GAG ACC GAG CAA GGT AGT
TAT ACC GTA AAT CGT TTC GTA GAA ACC GGT AAA GCA CTA CAC
GAA GTG ACT TCA ACC CAT ACC GCA TTA GTG GGC AAC CGT GAA
GAA AAA ATA GAA TAT CGT CAT AGC AAT AAC CAG CAC CAT GCC
GGT TAT TAC ACC AAA GAT ACC TTG AAA GCT GTT GAA GAA ATT
ATC GGT ACA TCA CAT AAC GAT ATC TTT AAA GGT AGT AAG TTC
AAT GAT GCC TTT AAC GGT GGT GAT GGT GTC GAT ACT ATT GAC
GGT AAC GAC GGC AAT GAC CGC TTA TTT GGT GGT AAA GGC GAT
GAT ATT CTC GAT GGT CGA AAT GGT GAT GAT TTT ATC GAT GGC
GGT AAA CCC AAC GAC CTA TTA CAC GGT GGC AAG GGC GAT GAT

ATT TTC GTT CAC CGT AAA GGC GAT GGT AAT GAT ATT ATT ACC
GAT TCT GAG GGC AAT GAT AAA TTA TCA TTC TCT CAT TCG AAC
TTA AAA GAT TTA ACA TTT GAA AAA GTT AAA CAT AAT CTT GTC
ATC ACG AAT AGC AAA AAA GAG AAA GTG ACC ATT CAA AAC TGG
TTC CGA GAG GCT GAT TTT GCT AAA GAA GTG CCT AAT TAT AAA
GCA ACT AAA GAT GAG AAA ATC GAA GAA ATC ATC GGT CAA AAT
GGC GAG CGG ATC ACC TCA AAG CAA GTT GAT GAT CTT ATC GCA
AAA GGT AAC GGC AAA ATT ACC CAA GAT GAG CTA TCA AAA GTT
GTT GAT AAC TAT GAA TTG CTC AAA CAT AGC AAA AAT GTG ACA
AAC AGC TTA GAT AAG TTA ATC TCA TCT GTA AGT GCA TTT ACC
TCG TCT AAT GAT TCG AGA AAT GTA TTA GTG GCT CCA ACT TCA
ATG TTG GAT CAA AGT TTA TCT TCT CTT CAA TTT GCT AGA GCA
GCT TAA
(ii) culturing the resulting transformed microorganism; and (iii) obtaining the resulting leukotoxin produced from said cultured transformed microorganism; with B. a vaccine carrier.

14. A process for making a vaccine effective against Pasteurella haemolytica comprising mixing:
A. a leukotoxin which is produced by (i) transforming a microorganism with a DNA sequence comprising the following nucleic acid sequence:
ATG AAT CAA TCT TAT TTT AAC TTA CTA GGA AAC ATT ACT TGG
CTA TGG ATG AAC TCC TCC CTC CAC AAA GAA TGG AGC TGT GAA
CTA CTA GCA CGC AAT GTG ATT CCT CCA ATT GAA AAT GAA CAA
TAT ATG CTA CTT ATA GAT AAC GGT ATT CCG ATC GCT TAT TGT
AGT TCG GCA GAT TTA AAC CTT GAG ACT GAG GTG AAA TAT ATT
AAG GAT ATT AAT TCG TTA ACA CCA GAA GAA TCG CAG TCT GGT
GAC AGA CGC TGG ATT ATT GAT TGG GTA GCA CCA TTC GGA CAT
TCT CAA TTA CTT TAT AAA AAA ATG TGT CAG AAA TAC CCT GAT
ATG ATC GTC AGA TCT ATA CGC TTT TAT CCA AAG CAG AAA GAA
TTA GGC AAA ATT GCC TAC TTT AAA GGA GGT AAA TTA GAT AAA
AAA ACA GCA AAA AAA CGT TTT GAT ACA TAT CAA GAA GAG CTG

GCA ACA GCA CTT AAA AAT GAA TTT AAT TTT ATT AAA AAA TAG
(ii) culturing the resulting transformed microorganism; and (iii) obtaining the resulting leukotoxin produced from said cultured transformed microorganism; with B. a vaccine carrier.

15. A process for making a vaccine effective against Pasteurella haemolytica comprising mixing:
A. a leukotoxin which is produced by (i) transforming a microorganism with a DNA sequence comprising the following nucleic acid sequence:
ATG GGA ACT AGA CTT ACA ACC CTA TCA AAT GGG CTA AAA AAC
ACT TTA ACG GCA ACC AAA AGT GGC TTA CAT AAA GCC GGT CAA
TCA TTA ACC CAA GCC GGC AGT TCT TTA AAA ACT GGG GCA AAA
AAA ATT ATC CTC TAT ATT CCC CAA AAT TAC CAA TAT GAT ACT
GAA CAA GGT AAT GGT TTA CAG GAT TTA GTC AAA GCG GCC GAA
GAG TTG GGG ATT GAG GTA CAA AGA GAA GAA CGC AAT AAT ATT
GCA ACA GCT CAA ACC AGT TTA GGC ACG ATT CAA ACC GCT ATT
GGC TTA ACT GAG CGT GGC ATT GTG TTA TTC GCT CCA CAA ATT
GAT AAA TTG CTA CAG AAA ACT AAA GCA GGC CAA GCA TTA GGT
TCT GCC GAA AGC ATT GTA CAA AAT GCA AAT AAA GCC AAA ACT
GTA TTA TCT GGC ATT CAA TCT ATT TTA GGC TCA GTA TTG GCT
GGA ATG GAT TTA GAT GAG GCC TTA CAG AAT AAC AGC AAC CAA
CAT GCT CTT GCT AAA GCT GGC TTG GAG CTA ACA AAT TCA TTA
ATT GAA AAT ATT GCT AAT TCA GTA AAA ACA CTT GAC GAA TTT
GGT GAG CAA ATT AGT CAA TTT GGT TCA AAA CTA CAA AAT ATC
AAA GGC TTA GGG ACT TTA GGA GAC AAA CTC AAA AAT ATC GGT
GGA CTT GAT AAA GCT GGC CTT GGT TTA GAT GTT ATC TCA GGG
CTA TTA TCG GGC GCA ACA GCT CCA CTT GTA CTT GCA GAT AAA
AAT GCT TCA ACA GCT AAA AAA GTG GGT GCG GGT TTT GAA TTG
GCA AAC CAA GTT GTT GGT AAT ATT ACC AAA GCC GTT TCT TCT
TAC ATT TTA GCC CAA CGT GTT GCA GCA GGT TTA TCT TCA ACT
GGG CCT GTG GCT GCT TTA ATT GCT TCT ACT GTT TCT CTT GCG
ATT AGC CCA TTA GCA TTT GCC GGT ATT GCC GAT AAA TTT AAT

CAT GCA AAA AGT TTA GAG AGT TAT GCC GAA CGC TTT AAA AAA
TTA GGC TAT GAC GGA GAT AAT TTA TTA GCA GAA TAT CAG CGG
GGA ACA GGG ACT ATT GAT GCA TCG GTT ACT GCA ATT AAT ACC
GCA TTG GCG GCT ATT GCT CGT GGT GTG TCT GCT GCT GCA GCG
GGC TCG GTT ATT GCT TCA CCG ATT GCC TTA TTA GTA TCT GGGG
ATT ACC GGT GTA ATT TCT ACG ATT CTG CAA TAT TCT AAA CAA
GCA ATG TTT GAG CAC GTT GCA AAT AAA ATT CAT AAC AAA ATT
GTA GAA TGG GAA AAA AAT AAT CAC GGT AAG AAC TAC TTT GAA
AAT GGT TAC GAT GCC CGT TAT CTT GCG AAT TTA CAA GAT AAT
ATG AAA TTC TTA CTG AAC TTA AAC AAA GAG TTA CAG GCA GAA
CGT GTC ATC GCT ATT ACT CAG CAG CAA TGG GAT AAC AAC ATT
GGT GAT TTA GCT GGT ATT AGC CGT TTA GGT GAA AAA GTC CTT
AGT GGT AAA GCC TAT GTG CAT CCG TTT GAA GAA GGC AAA CAC
ATT AAA GCC GAT AAA TTA GTA CAG TTC GAT TCG GCA AAC GGT
ATT ATT GAT GTG AGT AAT TCG GGT AAA GCG AAA ACT CAG CAT
ATC TTA TTC AGA ACG CCA TTA TTG ACG CCG GGA ACA GAG CAT
CGT GAA CGC GTA CAA ACA GGT AAA TAT GAA TAT ATT ACC AAG
CTC AAT ATT AAC CGT GTA GAT AGC TGG AAA ATT ACA GAT GGT
GCA GCA AGT TCT ACC TTT GAT TTA ACT AAC GTT GTT CAG CGT
ATT GGT ATT GAA TTA GAC AAT GCT GGA AAT GTA ACT AAA ACC
AAA GAA ACA AAA ATT ATT GCC AAA CTT GGT GAA GGT GAT GAC
AAC GTA TTT GTT GGT TCT GGT ACG ACG GAA ATT GAT GGC GGT
GAA GGT TAC GAC CGA GTT CAC TAT AGC CGT GGA AAC TAT GGT
GCT TTA ACT ATT GAT GCA ACC AAA GAG ACC GAG CAA GGT AGT
TAT ACC GTA AAT CGT TTC GTA GAA ACC GGT AAA GCA CTA CAC
GAA GTG ACT TCA ACC CAT ACC GCA TTA GTG GGC AAC CGT GAA
GAA AAA ATA GAA TAT CGT CAT AGC AAT AAC CAG CAC CAT GCC
GGT TAT TAC ACC AAA GAT ACC TTG AAA GCT GTT GAA GAA ATT
ATC GGT ACA TCA CAT AAC GAT ATC TTT AAA GGT AGT AAG TTC
AAT GAT GCC TTT AAC GGT GGT GAT GGT GTC GAT ACT ATT GAC
GGT AAC GAC GGC AAT GAC CGC TTA TTT GGT GGT AAA GGC GAT
GAT ATT CTC GAT GGT CGA AAT GGT GAT GAT TTT ATC GAT GGC
GGT AAA CCC AAC GAC CTA TTA CAC GGT GGC AAG GGC GAT GAT
ATT TTC GTT CAC CGT AAA GGC GAT GGT AAT GAT ATT ATT ACC
GAT TCT GAG GGC AAT GAT AAA TTA TCA TTC TCT CAT TCG AAC
TTA AAA GAT TTA ACA TTT GAA AAA GTT AAA CAT AAT CTT GTC
ATC ACG AAT AGC AAA AAA GAG AAA GTG ACC ATT CAA AAC TGG
TTC CGA GAG GCT GAT TTT GCT AAA GAA GTG CCT AAT TAT AAA

GCA ACT AAA GAT GAG AAA ATC GAA GAA ATC ATC GGT CAA AAT
GGC GAG CGG ATC ACC TCA AAG CAA GTT GAT GAT CTT ATC GCA
AAA GGT AAC GGC AAA ATT ACC CAA GAT GAG CTA TCA AAA GTT
GTT GAT AAC TAT GAA TTG CTC AAA CAT AGC AAA AAT GTG ACA
AAC AGC TTA GAT AAG TTA ATC TCA TCT GTA AGT GCA TTT ACC
TCG TCT AAT GAT TCG AGA AAT GTA TTA GTG GCT CCA ACT TCA
ATG TTG GAT CAA AGT TTA TCT TCT CTT CAA TTT GCT AGA GCA
GCT TAA
and ATG AAT CAA TCT TAT TTT AAC TTA CTA GGA AAC ATT ACT TGG
CTA TGG ATG AAC TCC TCC CTC CAC AAA GAA TGG AGC TGT GAA
CTA CTA GCA CGC AAT GTG ATT CCT CCA ATT GAA AAT GAA CAA
TAT ATG CTA CTT ATA GAT AAC GGT ATT CCG ATC GCT TAT TGT
AGT TCG GCA GAT TTA AAC CTT GAG ACT GAG GTG AAA TAT ATT
AAG GAT ATT AAT TCG TTA ACA CCA GAA GAA TCG CAG TCT GGT
GAC AGA CGC TGG ATT ATT GAT TGG GTA GCA CCA TTC GGA CAT
TCT CAA TTA CTT TAT AAA AAA ATG TGT CAG AAA TAC CCT GAT
ATG ATC GTC AGA TCT ATA CGC TTT TAT CCA AAG CAG AAA GAA
TTA GGC AAA ATT GCC TAC TTT AAA GGA GGT AAA TTA GAT AAA
AAA ACA GCA AAA AAA CGT TTT GAT ACA TAT CAA GAA GAG CTG
GCA ACA GCA CTT AAA AAT GAA TTT AAT TTT ATT AAA AAA TAG
(ii) culturing the resulting transformed microorganism; and (iii) obtaining the resulting leukotoxin produced from said cultured transformed microorganism; with B. a vaccine carrier.

16. A process for making a vaccine effective against Pasteurella haemolytica comprising mixing:
A. a leukotoxin which is produced by (i) transforming a microorganism with a DNA sequence comprising the following nucleic acid sequence:

ATG AAT CAA TCT TAT TTT AAC TTA CTA GGA AAC ATT ACT TGG
CTA TGG ATG AAC TCC TCC CTC CAC AAA GAA TGG AGC TGT GAA
CTA CTA GCA CGC AAT GTG ATT CCT CCA ATT GAA AAT GAA CAA
TAT ATG CTA CTT ATA GAT AAC GGT ATT CCG ATC GCT TAT TGT
AGT TCG GCA GAT TTA AAC CTT GAG ACT GAG GTG AAA TAT ATT
AAG GAT ATT AAT TCG TTA ACA CCA GAA GAA TCG CAG TCT GGT
GAC AGA CGC TGG ATT ATT GAT TGG GTA GCA CCA TTC GGA CAT
TCT CAA TTA CTT TAT AAA AAA ATG TGT CAG AAA TAC CCT GAT
ATG ATC GTC AGA TCT ATA CGC TTT TAT CCA AAG CAG AAA GAA
TTA GGC AAA ATT GCC TAC TTT AAA GGA GGT AAA TTA GAT AAA
AAA ACA GCA AAA AAA CGT TTT GAT ACA TAT CAA GAA GAG CTG
GCA ACA GCA CTT AAA AAT GAA TTT AAT TTT ATT AAA AAA TAG
AAGGAG ACATCCCTT ATG GGA ACT AGA CTT ACA ACC CTA TCA AAT
GGG CTA AAA AAC ACT TTA ACG GCA ACC AAA AGT GCC TTA CAT
AAA GCC GGT CAA TCA TTA ACC CAA GCC GGC AGT TCT TTA AAA
ACT GGG GCA AAA AAA ATT ATC CTC TAT ATT CCC CAA AAT TAC
CAA TAT GAT ACT GAA CAA GGT AAT GGT TTA CAG GAT TTA GTC
AAA GCG GCC GAA GAG TTG GGC ATT GAG GTA CAA AGA GAA GAA
CGC AAT AAT ATT GCA ACA GCT CAA ACC AGT TTA GGC ACG ATT
CAA ACC GCT ATT GGC TTA ACT GAG CGT GGC ATT GTG TTA TCC
GCT CCA CAA ATT GAT AAA TTG CTA CAG AAA ACT AAA GCA GGC
CAA GCA TTA GGT TCT GCC GAA AGC ATT GTA CAA AAT GCA AAT
AAA GCC AAA ACT GTA TTA TCT GGC ATT CAA TCT ATT TTA GGC
TCA GTA TTG GCT GCA ATG GAT TTA GAT GAG GCC TTA CAG AAT
AAC AGC AAC CAA CAT GCT CTT GCT AAA GCT GGC TTG GAG CTA
ACA AAT TCA TTA ATT GAA AAT ATT GCT AAT TCA GTA AAA ACA
CTT GAC GAA TTT GGT GAG CAA ATT AGT CAA TTT GGT TCA AAA
CTA CAA AAT ATC AAA GGC TTA GGG ACT TTA GGA GAC AAA CTC
AAA AAT ATC GGT GGA CTT GAT AAA GCT GGC CTT GGT TTA GAT
GTT ATC TCA GGG CTA TTA TCG GGC GCA ACA GCT GCA CTT GTA
CTT GCA GAT AAA AAT GCT TCA ACA GCT AAA AAA GTG GGT GCG
GGT TTT GAA TTG GCA AAC CAA GTT GTT GGT AAT ATT ACC AAA
GCC GTT TCT TCT TAC ATT TTA GCC CAA CGT GTT GCA GCA GGT
TTA TCT TCA ACT GGG CCT GTG GCT GCT TTA ATT GCT TCT ACT
GTT TCT CTT GCG ATT AGC CCA TTA GCA TTT GGC GGT ATT GCC
GAT AAA TTT AAT CAT GCA AAA AGT TTA GAG AGT TAT GCC GAA
CGC TTT AAA AAA TTA GGC TAT GAC GGA GAT AAT TTA TTA GCA
GAA TAT CAG CGG GGA ACA GGG ACT ATT GAT GCA TCG GTT ACT

GCA ATT AAT ACC GCA TTG GCC GCT ATT GCT GGT GGT GTG TCT
GCT GCT GCA GCC GGC TCG GTT ATT GCT TCA CCG ATT GCC TTA
TTA GTA TCT GGG ATT ACC GGT GTA ATT TCT ACG ATT CTG CAA
TAT TCT AAA CAA GCA ATG TTT GAG CAC GTT GCA AAT AAA ATT
CAT AAC AAA ATT GTA GAA TGG GAA AAA AAT AAT CAC GGT AAG
AAC TAC TTT GAA AAT GGT TAC GAT GCC CGT TAT CTT CCG AAT
TTA CAA GAT AAT ATG AAA TTC TTA CTG AAC TTA AAC AAA GAG
TTA CAG GCA GAA CGT GTC ATC GCT ATT ACT CAG CAG CAA TGG
GAT AAC AAC ATT GGT GAT TTA GCT GGT ATT AGC CGT TTA GGT
GAA AAA GTC CTT AGT GGT AAA GCC TAT GTG CAT CCG TTT GAA
GAA GGC AAA CAG ATT AAA GCC GAT AAA TTA GTA CAG TTG GAT
TCG GCA AAC GGT ATT ATT GAT GTG ACT AAT TCG CGT AAA GCG
AAA ACT CAG CAT ATC TTA TTC AGA ACG CCA TTA TTG ACG CCG
GGA ACA GAG CAT CGT GAT GGC GTA CAA ACA GGT AAA TAT GAA
TAT ATT ACC AAG CTC AAT ATT AAC CGT GTA GAT AGC TGG AAA
ATT ACA GAT GGT GCA GCA AGT TCT ACC TTT GAT TTA ACT AAC
GTT GTT CAG CGT ATT GGT ATT GAA TTA GAC AAT GCT GGA AAT
GTA ACT AAA ACC AAA GAA ACA AAA ATT ATT GCC AAA CTT GGT
GAA GGT GAT GAC AAC GTA TTT GTT GGT TCT GGT ACG ACG GAA
ATT GAT GGC GGT GAA GGT TAC GAC CGA GTT CAC TAT AGC CGT
GGA AAC TAT GGT GCT TTA ACT ATT GAT GCA ACC AAA GAG ACC
GAG CAA GGT AGT TAT ACC GTA AAT CGT TTC GTA GAA ACC GGT
AAA GCA CTA CAC GAA GTC ACT TCA ACC CAT ACC GCA TTA GTG
GGC AAC CGT GAA GAA AAA ATA GAA TAT CGT CAT AGC AAT AAC
CAG CAC CAT GCC GGT TAT TAC ACC AAA GAT ACC TTG AAA GCT
GTT GAA GAA ATT ATC GGT ACA TCA CAT AAC GAT ATC TTT AAA
GGT AGT AAG TTC AAT GAT GCC TTT AAC GGT GGT GAT GGT GTC
GAT ACT ATT GAC GGT AAC GAC CGC AAT GAC CGC TTA TTT GGT
GGT AAA GGC GAT GAT ATT CTC GAT GGT GGA AAT GGT GAT GAT
TTT ATC GAT GGC GGT AAA GGC AAC GAC CTA TTA CAC GGT GGC
AAC GGC GAT GAT ATT TTC GTT CAC CGT AAA GGC GAT GGT AAT
GAT ATT ATT ACC GAT TCT GAC GGC AAT GAT AAA TTA TCA TTC
TCT GAT TCG AAC TTA AAA GAT TTA ACA TTT GAA AAA GTT AAA
CAT AAT CTT GTC ATC ACG AAT AGC AAA AAA GAG AAA GTG ACC
ATT CAA AAC TGG TTC CGA GAG GCT GAT TTT GCT AAA GAA GTG
CCT AAT TAT AAA GCA ACT AAA GAT GAG AAA ATC GAA GAA ATC
ATC GGT CAA AAT GGC GAG CGG ATC ACC TCA AAG CAA GTT GAT
GAT CTT ATC GCA AAA GGT AAC GGC AAA ATT ACC CAA GAT GAG

CTA TCA AAA GTT GTT GAT AAC TAT GAA TTG CTC AAA CAT AGC
AAA AAT GTG ACA AAC AGC TTA GAT AAG TTA ATC TCA TCT GTA
AGT GCA TTT ACC TCG TCT AAT GAT TCG AGA AAT GTA TTA GTG
GCT CCA ACT TCA ATG TTG GAT CAA AGT TTA TCT TCT CTT CAA
TTT GCT AGA GCA GCT TAA
(ii) culturing the resulting transformed microorganism; and (iii) obtaining the resulting leukotoxin produced from said cultured transformed microorganism; with B. a vaccine carrier.

17. A process as claimed in any one of claims 10 to 16, wherein in A. (i) the microorganism is transformed with a sequence which is homologous to the DNA sequence or is a fragment of the DNA sequence.

18. A vaccine effective against Pasteurella haemolytica produced in accordance with a process as claimed in any one of claims 10 to 16.