CA2148829A1 - Avirulent live vaccine and method for immunizing animals against p. multocida pasteurellosis - Google Patents

Abstract

2148829 9411024 PCTABScor01 The invention provides vaccines and methods for protecting an animal against P. multocida associated pasteurellosis. A vaccine of the invention can be comprised of a stable avirulent immunogenic P. multocida mutant or a recombinantly produced P.
multocida) virulence factor. The avirulent immunogenic mutant can be a transposon-mediated mutant or a mutant having at least one genetically modified virulence gene. The methods of the invention include steps of producing an avirulent immunogenic mutant and administering an effective amount of the mutant to protect an animal against P. multocida pasteurellosis.

Description

- !-COMPOSITION PROTECTIVE AGAINST
P. MULTOCIDA PASTEURELLOSIS INFECT:ltON
' .

~ackground of the Invention Pasteurella multocida has been recognized as an important veterinary pathogen in disease processes of a variety of domestic and feral mammals and avian species.
For ~xample, ~. multocida is associated with atrop~
rhinitis and pneumonia of swine and with enzootic pneumonia in cattle. P. multocida is also the etiological agent of fowl cholera in avian species. P.
multocida associated diseases cause major economic losses to the swine, cattle and avian industries. -~
Pasteurellosis or fowl cholera in turkeys is a ~-highly contagious disease which occurs as a hyperacute, acute or chronic form. Pasteurella multocida belonging to different capsule types and somatic serotypes is the e~iological agent of fowl cholera. The hyperacute and -~
acute forms of the disease are characterized by septicemia, irreversible lesi~ns in the lung, liver and ~-spleen, and eventual death caused by the endotoxin of `~
P. multocida. The chronic form is associat~d with high morbidity and the development of a carrier state. ;
The turkey industry in the United States is part of the $26 billion poultry industry and it produced 4.9 billion pounds of live weight in 1988 from 242.47 million turkeys, valued at $1.95 billion. The per capita consumption of turkey meat increased from 6.1 pounds in 1960 to 17.9 pounds in 1990. Death due to diseases has been a major cause of monetary loss to the U.S. turkey industry. In 1988 (the latest year for which statistics are a~ailable), disease cost the U.S.
turkey industry an estimated $222 million, of which 50%
was from respiratory diseases. The National Tur~ey Federation and the American Association of Avian Pathologlsts have recognized avian pasteurellosis, also known as fowl cholera, as one of the three most important diseases wreaking substantial economic losses to the U.S. turkey industry through increased mortality, WO94/11024 PCT/US93/i~60 2 ~ 4~ 8 2 9 2 ^ condemnation and medication costs. Carpenter et al., Avian Dis., 31:16-2i (1988). 1 ~-With the recognition of the involvement of P. multocida in various animal diseases, efforts have 5 been made to prevent this disease by vaccination with an , ,-array of commerc~ial bacterins and attenuated li-ve vaccines. Bierer et al.l Poult. Sci., 5l:408-4l6 (lg72). However, efficacy and epidemiologic data available for these vaccines indicate that they are not ~otally ef~ective in preventing the disease. It is now established that bacterins prepared from strains grown in vitro on artificial media, induce pr~tection only . . .
a~ainst the somatic serotype from which the bacterin is made, i.e., serotype-specific immunity. Heddleston, Avian Dis., 6:315-321 (1962); Heddleston et al., A~ian Dis., l~:626-635 (1970). The immunogen that is responsible for serotype-specific immunity has been identified as the lipopolysaccharide, while the immunogen which induce cross-protective immunity are the ~;
membrane associated proteins, called cross-protection factor (CPF) immunogens. Heddleston e~ al., Poult.
Sci., 54:217-221 (lg74). ~-_. _ ,. .
Live vaccines induce cross-protecti~e immunity against challenge exposure with multiple somatic -serotypes of P. multocida. Bierer et al., cited supra.
A serious disadvantage often encount0red with the available li~e Yaccines is that, in previously compromised turkeys, they actually cause sys~emic in ection and death. Hofacre et al., National Turkey Federation Pasteurellosis SYmposium at pages 12-16 (l~89). Another important disadvantage has been the short duxation of immunity induced by both bacterins and live vaccines. The protection never lasts beyond four weeks. In a recent symposium on fowl cholera disease sponsored by the National Turkey Federation, ss~eral speakers challenged the researchers to develop a new generation of superior vaccines that are safe and yet . .
-".`;
,: .' W0~4/1l0~4 21 4 8829 PCT/US93/~0600 .". ;-.-still provide a broad spectrum of protection against all j 16 somatic serotypes of P. multocida. ¦
Thus, there is also a need for a vaccine ¦
specific for pasteurellosis that is simple to administer, yet provides long-lasting cross-protecti~
immunity without adversely affecting the host. There is a need for a highly immunogenic avirulent live vaccine for fowl cholera which can be administered orally.
- -.
Summary of the Inv~ntion The invention provides vaccines and methods for protecting an animal against P. mul~ocida associated pasteurellosis. A ~accine of the invention can be :~
comprised of an ef~ective amount of a stable avirulent immunogenic P. multocida mutant or a recombinantly produced P. multocida ~irulence factor in a liquid non-toxic carrier. The avirulent immunogenic mutants can be a transposon-mediated mutant or a mutant having at leas~
one genetically modified virulence gene located on a 9.4 kb EcorV fragment of the P. multocida genome. The methods of the invention include the steps of producing an avirulent immunogenic mutant and administering an effective amount of the mutant to protect an animal -~
against pasteurellosis.
~ transposon-mediated mutant can be a transposon insertion or deletion mutant. The mutant can be produced by introducing a transposon into the genome of a virulent strain of P. multocida under conditions fa~oring integration of the transposon. Suitable 3~ tran~posons include Tnl, Tn3, Tn5, TnphoA, Tn7, Tn9, TnlO, and functional fragments thereof. The transposon ~ -insertion mutants are selected for avirulence and the ability to provide immunity against pasteurellosis.
Especially pre~erred mutants are those that provide long-lasting cross-protective immunity against pasteurellosis.
" ~, W094/llU~4 ~`` PCI/US~60ol~ k 8 ~ 9 ';: ~ :
~ 4 1 ~
An avirulent mutant having a genetically modified virulence gene located on a 9.4 kb EcorV l, fragment of the P. multocida genome can be produced by ~;
standard methods of~mutagenesis. The virulence gene can be genetically modified by transposon insertion or deleti.on mutagenesis, chemical mutagenesis, restriction ~-endonuclease and exonuclease mutagenesis, and polymerase --chain reaction mediated mutagenesis. The mutants so -produced can then be selected for avirulence and ~-protection against pas~eurellosis. The genetic modification to a virulence gene located on a 9 kb EcorV
fragment in the selected mutants can be verified by standard methods, such as restriction enzyme mapping. -A gene encoding a virulence factor on a 9.4 kb 15 EcorV fragment can be subcloned and transformed into a -~
suitable host so that a recombinant virulence factor can be produced. The ~irulence gene i5 subcloned under -appropriate transcriptional and translational control regions to provide a high level expression of the ,~-20 virulence factor. The virulence factor can be -identified and purified by standard methods. The ~-virulence factor can then be used to immunize animals -~
and provide protection against pasteurellosis.
The method of the invention provides for administering an effective amount of the avirulenk immunogenic P. multocida mutant to an animal to provide protection against pasteurellosis. The mutant can be administered by several routes, including the parenteral routej nasal drops, aerosol, and preferably in the drinking watar. The effective amount is that amount of the mutan~ that provides for protection against pasteurellosis, and preferably is about 1Oa CFV/ml to about 109 CFU/ml. A wide variety of animals can ~e immunized in the method of the invention including cattle, pigs, ducks, turkeys, and chic~ens. The preferred animal is thç turkey.
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~ ~ w094~0~4 21~8829 PCT/IlS93/l~600 I ;
Brief Description of the Fiqures ' -I
FIGURE 1 shows a restriction enzyme map of TnphoA. ~ ;
FIGURE 2 shows Southern blot analysis of DNA
digests of avirulent transposon mediated mutants of P.
multocida.
:`
Detailed Description_of the In~entio~
~he invention provides vaccines and methods for protecting animals against pasteurellosis including fowl cholera. A vaccine is comprised of avirulent immunogenic mutants of P. multocida that can provide ;~
i~munity against P. multocida associated pasteurellosis.
The vaccine can also be comprised of a recombinantly produced P. multocida factor, and preferably the virulence factor is a gene product encoded on a 9.4 kb EcorV fragment of the genome P. multocid~. Once the avirulent mutant or recombinant virulenc~ factor is produced, an effective amount of the vaccine is administered to the anim~l to provide for immunity against pasteurellosis including fowl cholera.

A. Vaccines An immunogenic bacterium employed as the active componeht of a vaccine is a sta~le live avirulent immunogenic mutant of Pa~teurella_multocida that provides immu~ity against P. multocida. The mutant can be administered to an animal without causing disease or death and preferably provides long-lasting cross- j protective immunity. The immunogenic bacteria can be a transposon mediated mutant or a mutant having at least one genetically modified virulence gene located on a 9.4 kb EcorV fragment of the P. multocida genomeO An ~ -effective amount of the immunogenic avirulent mutant i ~`
3S~ bacteria or the recombinant virulence factor of the inve~tion is combined with a physiologically acceptable ;~
non-toxîc liquid vehicle to form the vaccine.
''~' W094/~l0~4 rCT/US93/~0600 ~ ~
2~ 29 6 ! :::
~ As used herei~;''stable" means that the mutant ll : maintains the desired ~Aaracteristics for multiple passages through an animal or for multiple generations :
of growth. Preferably, the mutant has a reversion 5 frequency of less than about 10-5 to about 10~l, and more ; .;
preferably less than about 10-6 to about 10-8.
As used herein, "cross-protective immunity"
refers to the capacity of the avirulent immunogenic 1.'~ ., mutant to protect the immunized animal from infection by 10 multiple virulent serotypes of P. multocida, and :-preferably the immunogenic mutant protects against all ::~
virulent serotypes.
As us~d herein, "long-lasting immunity" refers to the capability of the immunogenic mutant to generate 15 an immune response, preferably that lasts from at least ;~
about 6 weeks to about 20 weeks, and more preferably for ~ : -the lifetime of ~he animal.
As used herein, "an effecti~e amount" is the amount of immunogenic avirulent mutant or virulence factor that provides protection of the immunized animal against P. mul~ocida associated pasteurellosis.
. ..
As used herein, a "transposon" refers to a DNA
sequence that can move from place to place in a genome by processes which do not re~uire extensive DNA sequence homology between the transposon and the site of insertion nor the recombination enzymes need for classical homologous crossing over.
An immunogenic avirulent mutant bacteria can ~e a transposon-mediated mutant. The transposon-mediated 30 mutants are those mutants in which a transposon has been ~` ~
inserted or deleted from the genome of a virulent strain ~ ~:
of P. multocida. A transposon:insertion mutant is a ~-mutant that has at least one transposon ox a functional fragment thereof inserted in the genome at one or 35 multiple sites. Preferably, the transposon inserts . ~-randomly in the genome. Transposon insertion mutants -are then selected for the presence of transposon encoded -.,.-'.' ,'',`.

21~8829 .. ~ W094/11024 PCT/~S93/10600 -:.

genes. The transposon insertion mutants can then be further selec~ed for avirulence and for providing immunity against P. multocida associated pasteurellosi~s in animals. A transposon deletion mutant can be produced from avirulent transposon insertion mutants by selecting for mutants that ha~e lost the transposon encoded genes but still maintain avirulence and the ~.
a~ility to protect animals against P. multocida .~.
associated pasteurellosis.
The txansposon-mediated mutants can be produced by introduction of a transposon or functional fragment thereof into P. multocida and selecting for avirulent transposon insertion mutants. Suitable transposons are those that encode a marker gene including Tnl, Tn3, Tn5, TnphoA, Tn7, Tn~, and TnlO and functional fragments thereof. The especially preferred transposon is TnphoA.
An avirulent immunogenic mutant can also be a mutant having at least one genetically modified virulence gene located on a 9.4 kb EcorV fragment of the ~
20 P. multocida genome. Virulence genes can be identified .~-:
and mapped by transposon-mediated mutagenesis. A
virulence gene is one that is essentially non~unctional :~
or produces an essentially nonfunctional gene product in an avirulent mutant but is functional in a virulent P. multocida strain. An essentially nonfunctional gene can be one that is not expressed at a level sufficient .to provide the gene-associated function, including virulence, to the mutant and/or one which is expressed ! -~
but produces a nonfunctional gene product. An essentially nonfunctional virulence gene can be identified by assaying for function, including virulence of the gene product, and preferably a gene product ~.. --.`
having at least about 10- to about 1000-fold reduction r 5 in function is essentlally nonfunctional. .`
35 Alternatively, the gene pr~duct encoded by the :~:
essentially nonfunctional virulence gene can be . .
identified by a change in physical characteristics of -~;
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21~8829 PC~/US93/l~6olo 8 ~3 Rec'd ~ 1 9 O~T l99~ , the gene product including molecular weight, isoelectric point, and amino acid composition. A preferred mutant is one that has an essentially nonfunctional virulence gene encoded on a 9 kb EcorV fragment of the P.
multocida genome. ~;
Once identified, ~irulence genes in virulent strain of P. multocida can be rendered nonfunctional by mutations or genetic modifications generated by standard ` :
methods known to those of skill in the art, including transposon-mediated mutagenesis, chemical mutagenesis, restriction enzyme andJor exonuclease-mediated mutagenesis, and the llke. The P. multoclda mutants ~-having at least one genetically modified virulence gene are selected by screening for conversion of the virulent strain of P. multocida into an avirulent strain and for the ability to protect aga1nst pasteurellosis in -~
animals.
Specific examples of the avirulent mutants of ,-~
20 the invention include the avirulent transposon insertion ; -~
P. multocida mutants designated PmTn-294 and PmTn-396. -Both mutants are characterized by expression of alkaline phosphatase activity, loss of resistance to complement ~-mediated killing, and loss of virulence in turkeys.
Preferred mutants of the invention include a mutant having the characteristics of ATCC No. 55394, the mutant is P. mul tocida PmTn 294 deposited with the American `
Type Culture Collection, Rockville, Maryland, on February 17, 1993, and a mutant having the , -, characteristics of ATCC No. 55395, the mutant is P. ~ -mul tocida PmTn 396 deposited with the American Type , ~
Culture Collection, Rockville, Maryland, on February 17, ~ -1993. . ~
A vaccine of the invention can also be -comprised of an effective amount of at least one recombinantly produced virulence factor from P. .
multoclda in a liquid non-toxic vehicle. A virulence - `
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21~8829 ,-CI/US '~ 3 / 1 0 6 O O !~
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factor can be a gene product that is essentially ' nonfunctional in avirulent bacterial strain and functional in a virulent P. multocida strain. The ~ I .
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virulence factor can be identified as the gene p.roduct of a virulence gene of P. multocida by methods known to those of skill in the art including in vitro transcription translation systems. Alternatively, the 5 virulence factor can be identified by a functional assay ~ ~-including virulence or by the ability ~o produce ~ -symptoms and lesions of the disease. A ~irulence factor that i5 essentially nonfunctional in avirulent mutant bacteria has at least about a 10- to 1000-fold reduction in functional activity. Optionally, the virulence factor can be identified by a change in its physical characteristics when the same factor is compared between virulent and avirulent mutants. To produce the recombinant virulence factor, a virulence gene is cloned 15 from virulent P. multocida strains into an appropriate -host organism by standard methods, such as described in Sambrook et al., Nolecular Cloninq- A Laboratory Manual, Cold Sp.ring Harbor Laboratory, Cold Spring, NY (1983), -~
and the recombinantly produced virulence factor is expressed. The ~irulence factor is also preferably selected for long-lasting cross-protective immunity against pasteurellosis in an animal. An effecti~e amount of the recombinant virulence factor is an amount sufficient to provide for protection against pasteurellosis, preferably about 5 mg/kg to about 10 mg/kg. A preferred recombinantly produced Yirulence factor is a gene product encoded by a gene on a 9.4 kb EcorY fragment of the P. multocida genome. `
To use the mutants of the present invention as ~ `
30 a vaccine, cells of the mutant are combined with a ---suitable physiologically cceptable non-toxic liquid vehicle. Specific examples of suitable liquid non-toxic vehicles include buffered salt solutions, 0.85% saline ii and, preferably, drinking water. The amount of cells i --included in a given unit dosage form of the vaccine can vary widely and depends upon factors such as age, weight and physical condition of the animal. Such factors can ........ ..... . ..... . .... . .

~ ~ 4a~2 9 PCT/US93/10600 j,~ ~

be readily determined by the clinician or veterinarian employing animal models or other test systems which are well known to those of skill in the art. Preferably, an effective amount of the mutant will range from about 5 1 X 106 to 1 x lO1l cfu/ml, and more preferably about 1 x 107 to 1 x 101 cfu!ml. A unit dose of the vaccine can be administered parenterally, e.g., by subcutaneous or intramuscular injection, however oral or aerosol ~-delivery is preferred. The preferred vaccine can be administered by mixing the mutant in the drinking water and making the water available to the animals ~lternatively, the vaccine can be administered intra-nasally by dropping into the nares or by aerosol. In a preferred version, a vaccine comprised of 107 cfu/ml of a mutant having the characteristics of ATCC No. 55394 or ATCC No. 55395 is administered in the drinking water to ~-turkeys.
The vaccines can be administered to a variety of animals including cattle, pigs, ducks, turkeys, and 20 chickens to protect against pasteurellosis. The ; -especially preferred animal is the turkey.
, . ....
B. Methods o Produci~g an Avirulent Tra~sposQn 7 '' o f P Multocida a~d~Immunizi~g an Animal 2 5 ~ c~ l ~e ~
ThQ invention also provides a method of `-immunizing an animal against pasteurellosis with a stahle avirulent immunogenic transposon-mediated mutan~ , -of P. multocida. The method involves the steps of producing a stable avirulent immunogenic transposon-mediated mu~ant and administering an effective amount of ~ ;
the mutant to the animal to provide immunity against P.
multocida associated pasteurellosis.
The preferred stable avirulent immunogenic transposon-mediated mutants of P. multocida can be -~
produced by transposon-mediated mutagenesis. -Transposon-mediated mutants include both those that have a transposon inserted into the bacterial genome, known ', ':

, WO94/110~4 2 1 ~ 8 8 2 9 PCT/Us93"0600 1~
J

as insertion mutants, and those wAere the transposon has been inserted and then excised with a portion of the bacterial gene, creating a nonreverting deletion mutant.
Transposon-mediated mutan~s are then selected for S a~irulence and for the ability to protect against pasteurellosis, and preferably for stability.
A transposon insertion mutant of P. multocida can be produced by standard methods known to those of skill in the art and as described by Taylor et al., J.
BacterioloqY, 171:1870 (1989). Briefly, a transposon in ~-a suitable vector is introduced into P. multocida, ;
preferably a virulent strain, under conditions that favor insertion of the transposon into the genome of the bacteria~ For example, a transposon can be placed in a 15 suicide vector. A suicide vector is one that can be ~`
introduced into a wide variety of bacteria but is only ~`
capable of replicating in certain types of bacteria.
The inability of the suicide ~ector to replicate favors selection of bacteria having the transposon inserted "
into the genome. The vector is preferably introduced into the P. multocida by transconjugation but can also ~`
be introduced by other methods known to those of skill 1--in the art, such as electroporation or calcium phosphate ~-precipitation. Once introduced into P. multocida, transposon insertion mutants are selected by the presence of transposon encoded marker genes and further l-selected for avirulence for animals, for protection ~
against pasteurellosis ~nd stability. ; .
As used herein, a "transposon" is a DNA segment , ;
that can move to new locations in DNA molecules by processes which do not require extensive DNA sequence i `~
homology or recombination~enzymes. Transposons can ~-include marker genes encoding antibiotic resistance and transposition enzymes, and are typically bounded by a region of DNA sequences, known as insertion sequences, that mediate insertion of the transposon into DNA. For W0~4/11024 .~ PCT/US93/10600 -~ ~:

12 j example, the DNA sequences at the termini of insertion sequence 50 of t~ Tn5 transposon are~
~ , 5' C~GACTCTTATACACAAGTAGCGTCCTGAACG. . . -3' GACTGAGAATATGTGTTCATCGCAGGACTTGC. . . ~ .

: .
. . .GCGCAGGGGATCAAGATCTGATCAAGAGACAG tSEQ ID .. -NO:l) .
. . .CGCGTCCCCTAGTTCTAGACTAGTTCTCTGTC .

' ' ''"' as reported by Berg et al., Biotechnoloqy, l(5):4l7 (July l983). Transposons can be modified by metho~s known to those of skill in the art, as long as they retain the functional ability to insert into DNA. Some .
modified transposons are known as insertion sequences.
All or portions of ~ransposons can insert into one or . -.
more locations in a bacterial genome and, if they insert into a ~ene, typically form a mutant no longer having the function associated with that gene. Sui*able ,`-transposons include Tnl, Tn3, Tn5, TnphoA, Tn7, Tn9, TnlO, and functional fragments thereof. The especially `~.
preferred transposon is the TnphoA transposon, which contains the lef~ insertion s~equence of Tn5 lin~ed to .
the gene encoding:a marker gene, such as the gene for alkaline phosphatase, and an anti~iotic resistance gene, such as kanamycin resistance and tetracycline .:.~
~esistance. `~.
Suit~ble vectors for introducing the transposon ~-into P. multocida are vectors~that favor integration of 30 the transposon into the bacterial genome. Specific 7r "`', examples include suicide plasmids that can coniugate .-~
with but cannot replicate in P. multocida.
~: Alternati~ely, P. multocida can be co-transfor~ed with a -~
plasmid containing the transpo~on and a plasmid of the '~
35 same incompatibility group so that ~he plasmids will not ..
be able to replicate in the cell. The preferred plasmid is a suicide vector suc:h as pRT733 which can ~::

... ~.. . . . .. .. . .

: W094/11024 PCT/US93/10600 transCQnjUgate but not replicate in P. multocida and ~`
carries the TnphoA transposon.
P. multocida mutants with integrated transposons are selected by identifying those bacteria ' ~ -5 having at least one selectable marker gene encoded by ~`
t-he transposon. The selectab]e marker gene can include antibiotic resistance ~enes, such as kanamycin ; `-~
resistance genes, tetracycline resistance genes, and the `~
like. Other marker genas can include reporter genes, such as the chloramphenicol acetyltransferase gene t the alkaline phosphatase gene, the ~-galactosidase gene, and ,~-the like. The especially preferred marker gene is the alkaline phosphatase gene because this marker gene provides for selection of mutants having modified genes ~-15 encoding membrane or secreted membranes. Alkaline !``
phosphatase is detected as a secreted enzyme and it is believed that mutants secreting alkaline phosphatase have the transposon inserted into a gene that encodes a --membrane or secreted gene product.
The transposon insertion mutants are further s~lected for avirulence. The avirulent mutants can be identified by either in vitro or in vivo methods. -~-~
Avirulent mutants can be identified by in vitro assays that correlate with the in vivo vixulence. A suitable example includes a complement-mediated lysis assay;
virulent strains of P. multocida are resistant to complement-mediated lysis, whereas avirulent strains are susceptible to complement-mediated lysis.
Alternatively, a~irulent mutants can be identified and 30 selected by the inability to cause death or disease in '-the animal by standard methods.
Avirulent mutants of the in~ention can be further selected for the ability to protect animals ;
against pasteur~llosis. Different amounts of an avirulent mutant can be administered to the animal.
~f~er about two weeks, the animals can be examined for the presence of protective îmmunity specific for P. -....;

.

WO94/1102~ PC~/US93/1~60 4~ 8 2 g 14 ~ multocida by standard methods, including detecting antibodies by the ELI~A;~`test. The animals can also be -~
challenged with at least one virulent strain of P. multocida. The avirulent mutants that protect against pasteurellosis caused by virulent P. multocida can be identified as well as the effective amount of the mutant providing protection against the diseas~.
Protection against pasteurellosis can be determined by comparing the percentage of nonimmunized animals which die or show symptoms of the disease after challenge with those that were immunized with the avirulent mutant.
Symptoms of the diseases associated with P. multocida in each species of animal are well known to those of skill in the art. An avirulent mutant that protects about 90-lO0~ of animals from death or the symptoms of the disease is preferred. :
~ An especially preferred avirulent mutant is one - that provides long-lasting cross-protective immunity -against pasteurellosis. An avirulent mutant can be 20 selected for provlding c~oss-protective immunity by 'i`
challenging ani~als immunized with the avirulent mutant -with all of the virulent serotypes and identifying avirulent mutants that provide protection against some or all of the virulent serotypes of P. multocida. Long-lasting immunity can be evaluated by challenging the animals immunized with the avirulent mutants after diferent time periods, from about 2 weeks to about 20 weeks. The preferred avirulent mutants can provide immunity against pasteurellosis from at least about 6 30 weeks up to about 2~ weeks, and the especially preferred -~
mutants provide lifetime Lmmunity against pasteurellosis for the animal. ~ ~
Preferably, the avirulent mutant of P. ~-multocida is also a stable mutant. Stable mutants can -35 be identified by growing the mutant for about lO to -~
50 generations without the loss of desireable characteristics, such as avirulence and protection '''.,'`

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~ WO94/l1024 2 1 ~ 8 8 2 ~ PCT~US93/10600 ~ ~
.... . .

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against pasteurellosis. Reversion frequ.ency can also be measured to determine stability by standard methods known to those of skill in the art. A stable avirulent ~-mutant of the invention preferably has a reversion frequency of less than 10-5 to 10-1 and more pre~erably ~.-of about than 10-6 to about 10-8. Alternatively, the mutant can be passed through animals for about 10 to 20 passages and examined for the rate of the loss of the `;;
desired characteristics of avirulence and protection . .
against pasteurellosis. A stable mutant is one that can be passed through animals for about 10 to 20 passages -and still maintain the desired characteristics.
... .
The transposon-mediated mutant of the invention -;
can also be aj transposon-mediated deletion mutant. A `
transposon-media~ed del~tion mutant can be selected and isolated-from the transposon insertion mutants produc~d and selected as described above. An avirulent ~;, transposon insertion mutant can be grown under conditions no longer selecting for the marker gene ~-20 encoded by the transposon, such as a gene for antibiotic ;~
resistance or for alkaline phosphatase~ Bacteria which have lost these marker g2nes can be further screened for maintenance of the avirulence characteri~tic. It is believed that, at a very low frequency of less than 10-8, `
the transposon can excise from the genome of the bacteria and, if that excision is not perfect, can carry some of the DNA sequence from the gene into which the transposon initially inserted. When the transposon excises in this manner, a tranæposon-mediated deletion mutant is created. Transposon-deletion mutants can be identified and isolated by screening for those mutants that have lost the marker gene encoded by the transposon while still maintaining the avirulence characteristic.
Once identified, the transposon deletion mutants can be further selected for s~ability and for providing long-lasting cross-protecti~e immunity against pasteurellosis, as described above.

. z~48~9 PCT/US93/10600~ - r In a pre~erred version, a transposon-mediated mutant of P. multocida~~is produced. A suicide vector encoding the TnphoA transposon is introduced into a virulent strain of P. multocida by transconjugation.
... ..
5 Transconjugates with the TnphoA transposon inserted into the genome are first selected by ~creening ~or antibiotic resistance and for secretion of alkaline phosphatase. ~utants that are resistant to antibiotics and which secrete alkaline phosphatase are then screened 10 for avirulence in ~i~o and in vitro. An avirulent mu~ant which secretes alkaline phosphatase, isolated as -described herein, has been~deposited with the ATCC on -February 17, 1993 and given Accession No. 55394.
Another avirulent mutant which secretes alkaline -~
15 phosphatase, isolated as described herein, has been -deposited with the ATCC on February 17, 1993 and given --Accession No. 55395. The mutant is then screened for the ability to pro~ide protection against pasteurellosis in an animal. The avirulent mutant also preferably is 20 stable and provides long-lasting cross-protective ~ -immunity against pasteurellosis. `~
Once produced, the transposon-mediated mutants `~
of the invention are administered to the animal.
Administration can occur by any one of several routes 25 including parenteral,nasal drops, aerosol, and/or through the drinking water. An effective dose of each mutant can be determined as described above, but preferably is about 108 to 109 CFUjml. The mutants can be administered to a variety of animal species including 30 cattler pigs, ducks, chickens, and turkeys but is preferably administered to turkeys.
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.'' ~;WO94/11024 2~4~829 PCr/US93/106~0 C. Method of Producing an Avirulent Immunoge~ic Mutant Having at Least One Gen~tically Modif ied Virulence Gene And Immurlizinq Animals A.qainst Pasteurellosis I .
The invention provides a method of immunizing , `
an animal against P. multocida associated pastel1rellosis with a stable a~irulent immunogenic mutant of P.
multocida wherein the mutant has at least one genetically modified virulence gene loca~ed in a 9.4 kb 10 EcorV fragment of the P. multocida genome. The method `
involves the steps of producing a stable a~irulent immunogenic mutant having a genetically modified ~irulence gene and administering an effective amount to ~
an animal to provide immunlty against pasteurellosis. `
The avirulent immunogenic mutants can be produced by first identifying virulence genes and then genetically modifying the ~irulence genes located in a 9.4 kb EcorV fragment of the P. multocida genome. ~-~
Virulence genes of P. multocida can be identified by 20 using transposon insertion mutants to identify and to ;~
map the location of P. multocida virulence genes. It is believed that insertion of a transposon into a gene can result in inactivation of the gene. Transposon insertion mutants ~howing a loss of virulence have 25 transposons inserted in genes required for virulence. - -~
The location of a transposon insertion in avirulent mutants can be detected and mapped by standard methods including Southern blot hybridi~ation and DNA ;-sequencing.
Once identified and mapped, the virulence genes can be genetically modified by standard methods known to ,- -thosa of skill in the art. A virulence gene is genetically modified resulting in the avirulence phenotype of the mutant. The virulence gene can be ~7'- ' 35 genetically modified so that ~ the gene is expressed at a level below that required to produce virulence or it can ~ -be modified to produce an essentially nonfunctional gene ;~
product.

.

WO94/11024 PCT/US93/10600, 2 ~ 4~ ~ 2 9 18 A genetically modified virulence gene can be produced by standard methods of mutagenesis. Suitable methods include transposon-mediated mutagenesis ~insertion or deletion), chemical mutagenesis, restriction enzyme or exonuclease mutagenesis, and polymerase chain reaction medlated mutagenesis. The preferred method for generating the mutants is by transposon-mediated mutagenesis. A preferred mutant of -~
the invention is a mutant having a genetically modified 10 virulence gene located in a 9.4 kb EcorV fragment of the `-P. multocida genome.
A genetically modified virulence gene can be detected by a variety of methods known to those of skill in the art. The genetic modification can be detected by a change in restriction enzyme mapping, ribosomal RNA
profile, or by a change detected by direct DNA
sequencing. Alternatively, the genetically modified virulence gene can be detected by a functional assay for -~
the virulence gene product. A genetically modified virulence gene preferably~expresses a gene product that is essentially nonfunctional in the avirulent mutant. ~;
Essentially nonfunctional refers to at least about l0-to l000-fold reduction of the functional activity of th~
gene product. Optionally, the genetically modified virulence gene can be identified by a change in the physico-chemical characteristics of the gene product, ~-~
such as a change in molecular weight, isoelectric point, or amino acid composition or the like. ~
A mutant having a genetically modified ` ~-30 virulence gene is also further selected for avirulence ~:
and for protection against pasteurellosis, as described herein. In addition, the prefèrred mutant is selected for long-lasting cross-protective immunity against ~ `;~;`
pasteurelloisis as~described~herein.
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~W094/1l024 21~8829 PCT/US93/10600 19 I '.. ' In a preferred version, an avirulent transposon (TnphoA) insertion mutant, produced as described herein, can be used to identify and locate a virulence gene of P~ multocida. A virul~nce gene of P. multocida can be ~`
identified by Southern blot hybridi~ation with th~ pro~e which hybridi~es to TnphoA sequences. A virulence gene located on a 9.4 kb EcorV fragment of P. multocida can be mapped by restriction enzymes and seguenced by direct DNA sequencing methods. The virulence gene located on the 9.4 kb EcorV fragment of a virulent P. multocida strain can then be genetically modified by point `
mutation to generate an a~irulent mu~ant. The mutant is then selected for a~irulencè and protection against -pasteurellosis. The genetic modification of a virulence gene located on the 9.4 kb EcorV fragment can be verified by standard methods, including restriction enzyme mapping or DNA sequencing.
Once produced, an avirulent mutant of P. multocida having at least one genetically modified virulence gene is administered to animals to provide for protection against pasteureIlosis. The mutant can be administered by parenteral route, nasal drops, aerosol, and~preferably in the drinking water. An ef~ective amount can be determined by injecting different amounts of the mutant into~animals and determining the minimum amount that p~otects against the disease. Preferably, the effectiv2 dose is about 108 to 109 CFU/ml. The mutants of P. multocida can be administered to animals such as cattle, pigs~ ducks, turkeys, and chickens. ~he preferred animal is the turkey.

D. Method for Clon~ng a P. multoc~-da V~rulsnc~ Ge~e and ~-Purifvi~q Rec~o~binantly Produced Virulence_Factor The invention provides a vaccine comprised of a recombinant P. multocida virulence factor encoded in a 9.4 kb EcorY fragment of P. multocida. The recombinant ~irulence factor can be produced by cloning a gene :~-L ~
WO94~ 4 PCT/US93/10600 ~
~,~.488~9 encoding a virulence gene into a suitable host by , -.
standard methodsr as described in Sambrook et al., cited ... -supra. The recombinantly produced virulence factor can . ~
then be identified and purified from the host cell.
For example, a virulence gene located on a .. : `
9.4 ~b EcorV fragment,.isolated.as described herein, can be subcloned into a vector such as the plasmid pBR322. .
The virul~nce gene is preferably subcloned at a location ~
in the pBR322 such ~hat it i5 under the control of the lO appropriate transcriptional and translational control :.
regions to provide for a high level of gene expression .. ~:~
in ~he host cell. The subcloned virulence gene can be .~
introduced into a suitable host, such as E. coli.and -~.
expression of the subcloned virulence gene can be monitored by standard methods, including Western blot using an antibody such as pasteurellosis convalescent serum. The recombinant virulence factor can be isolated ~.
and purifîed from E coli cell lysates by standard methods, including affinity, size exclusion, and/or HPLC
chromatography.
The virulence factor can then be tested for the ability to protect against pasteurellosis by immunizing an animal with different amounts of the purified .
xecombinant virulence factor. The immunized animals can .:~
25 be analyzed for the developme~t of protective antibody .~.
response by standard methods, including E~ISA. The immunized animals are also challenged with at least on .virulent serotype of P. multocida to validate whether the virulence factor provides protective immunity . ~.
against pasteurellosis. The virulence fartor of the invention provides for protection against pasteurellosis ~ .
and preferably long-1asting cross protec~ive immunity.
., ~ ..

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,. W094/11024 2148829 PCI/US93/10600 i~

21 1, EXAMPLE 1 ! -Ganerat~ on of TnphoA Muta~ts of Pasteurella multocida ¦ :
Nutants of Pasteurella multocida were generated ~y transposon mutagenesis. The transposon utilized was a modified Tn5 (TnphoA) carrying the left insertion sequence of Tn5 linked to the gene for alkaline phosphatase without the natural promoter or signal sequences for the alkaline phosphatase gene. The transposon is present in a plasmid pRT733 which is a pGM703.1 derivativa carrying the TnphoA and kanamycin resistance gene and is available from J. Mekalanos, Department of Microbiology and Molecular Genetics, -Harvard Medical School. The plasmid pRT733 is a broad host range suicide vector. The plasmid can conjugate with a wide variety of bacteria but is only capable of replicating in those bacterial strains carrying the A-pir transducing phage. The plasmid cannot replicate without a protein encoded by the A-pir transducing phage. The alkaline phosphatase gene, when inserted into the bacterial genome along with the transposon, serves as a marker for genes that encode secreted, excreted and membrane bound proteins. The alkaline phosphatase is only active when excreted and has shown '`~
to be active as a fusion protein. -The pRT733 plasmid was introduced into a -~
virulent complement resistance streptomycin resistant recipient strain of P. multocida designated Pm-P1059tSmR) ` -~
and mutants containing transpositions were selected in a ' ~;~
single step. E. coIi R12SMlO lysogenized with A-pir carrying pRT733 were mated with Pm-P1059(SmR) overnight at 37C on an LB plateO Pm-P1059(Sm~) insertion mutants , were selected on LB plates containing streptomycin (100 ~g/ml) and kanamycin (225 ~g/ml)~ Selected colonies were then incubated on LB plates containing the s antibiotics and 5-bromo-4-chloro-3-indolyl-phospha~e-P-toluidine-(XP) (20 ~g/ml for 18-24 hours. The XP is a chromogenic substrate for the ~4~a~9 2~ PC~/US93/]060~

; j .
alkaline phosphatase e~zyme and indicates the presence ~.... I ,.
of secreted alkaline phosphatase by the mutant. Blue j colonies were indicative of insertion mutants secretinq alkaline phosphatase.
Forty-two TnphoA insertion mutants were isolated. The ~nphoA mutants were screened for alkaline phosphatase activity, expression of fusion proteins, `;
expression of iron-regulated outer mem~rane proteins, i~;~
loss of complement resistance, and loss of virulence for turkeys. Alkaline phosphatase activity as a fusion protein was measured with the chromogenic substrate XP
or P-nitrophenol as described in Taylor et al., Bacteriol., 171:1870 ~1989). The iron-regulated outer membrane proteins having molecular weights of 94 kDa, 84 kDa, and 76 kDa were de~ected by standard Western blot methods using antisera specific for these iron-regulated membrane proteins. `~
Two mutants, designated PmTn-294 and PmTn-396, were positive for alkaline phosphatase activity, 20 expression of fusion proteins and iron-regulated outer `~
membrane proteins. These two mutants were further characterized for virulence in turkeys.

E:XAMPI.E 2 ~.
Ide tification of Av~ rulent TnE~oA Mutants The transposition insertion mutants PmTn-294 and PmTn-396 were screened for resistance to complement~
mediated lysis and for ~irulence in turkeys. Re~istance to complement mediated lysis correlates with ~irulence in vivo~ The parent P-1059 wild strainsl Pm-P1059 and ¦
Pm-P1059(SmR), were resistant to complement-mediated lysis and cau~ed fatal dise~se in 100% of turkeys within 18 hours.
Both PmTn-294 and PmTn-396 were susceptible to I -35 complement mediated lysisO About 1 x 108 cells/ml of :~
PmTn-294 and PmTn-396 cells were incubated with 5 ml of turkey plasma containing complement and incubated for :``' ``

,.

~ W094/11024 2148~29 PCT/US93/1060~ ~

23 j-l hour at 40C. After incubation, a sample of the PmTIl-294 and PmTn-396 cells was serially diluted and plated. After 24 hours of incubation, the number of via~le bacteria present after treatment with complement 5 containing turkey plasma was determined by plate counts. J`` ;
~oth the PmTn~294 and PmTn-396 showed a 3-fold decreas~
in viable cells after treatment with complement when compared tG the control complement resistant Pm-Pl059 strain. -For in vivo virulence testing, groups of five l~week old turkey poults were inoculated intravenously with 5 x 104 colony forming units (CFU) of transposon ~;
insertion mutants or the virulent Pm-Pl053 strain. The poults were observed for 8 weeks for the presence of - ~-;
15 disease. All dead turkeys were subjected to postmortem --and bacteriological examination to establish the presence of pasteurellosis disease. One hundred percent (100%) of the poults infected with the virulent Pm-Pl059 strain died and 100% also showed symptoms of the disease before death. However, infection of poults with either PmTn-294 or PmTn-396 did not result in death or development of the disease. The avirulent mutants are being characterized further to determine the site of the ~-transposon insertion by Southern hybridization. -~
~XAMP~E 3 `~
Ide~tificatio~ of the Locatio~ of a Virule~ce ~ene of P. Multo~ida : -~
:
' To identify the location of a virulence gene 1 -inactivated by insertion of the TnphoA transposon and to confirm the presence of the TnphoA transposon in the two insertion mutants, genomic DNA ~as analyzed by Southern blot hybridization by standard methods.
DNA was obtained from the wild-type virulent P. multocida Pm-Pl059, the recipient streptomycin-resistant P. multocida l059 strain (Pm-Pl059 SmR), the donor E. coli strain carrying pRT733 (TnphoA
:

W094/1]024 PCT/US93/106~
9 1; ~

transposon) r PmTn-29.4~(TnphoA insertion mutant), and PmTn-396 (Tnpho~ ~sertion mutant) was digested with ¦
either KpnI or EcorV. DNA restriction fragments were~
separated by gel electrophoresis and probed using a S EcorI-XhoI digested 1.3 kb fragment or DraI-HpaI
digeE.t~ed 7 kb fragment.from pRT733. The restriction ma? :~
of the TnphoA transposon is shown in Fig. 1. The 1.3 kb probe is a EcorI-XhoI fragment having a DNA sequence -located between two portions of the left insertion 50 ~
10 sequence of the TnphoA transposon. The 7 kb probe is a .. ~.
DraI-HpaI probe encoding portions of the left insertion -.
sequence and the right insertion seguence and the kanamycin resistance gene. ~
The results of the Southern blot hybridization , ~:
15 are shown in Fig. 2. DNA from the Pm-PlOS9, rPcipient ::-Pm-P1059 SmR, and the PmTn-294 digested with KpnI did not ;-hybridize with the 1.3 kb probe. Howe~er, DNA digests .:~
from the donor E. coli carrying pRT733 ~nd PmTn-396 ~
showed identical fragments which hybridized with the ... :
1.3 kb probe. DNA from the Pm-P1059 and rec-ipient Pm-P1059 SmR digested with EcorV also did not hybridize ~.
with the 1.3 kb probe.~ In contrast, DNA from the ..
transconjugant PmTn-396 digested with EcorV showed two !~''' - bands at 10.9 kb and 9.4 Kb, which hybridized with the 1.3 kb probe. One band at 9.4 kb from PmTn-294 also hybridized with the 1.3 kb probe. The DNA EcorV digest `-.
of the pRT733 donor strain showed identical fragments as that of PmTn-396 which hybridized with the probe. The -.
same results were obtained when the digests were probed --:
with the 7 kb DraI-HpaI fragment from pRT733. --The results indicate that avirulence is associated with the insertion of all or a portion of the TnphoA in a 9 . 4 kb EcorV fragment of the genomic DNA of 6 ~ . ViruIence gene or genes present in this . ... :
35 region and inactivated by this insertion will be mapped .. ~-~
by additional restriction enzyme digestion and sequenced :~

, ~ .

21~8829 ; WO94/11024 ^ PCT/US93/10600 . 25 by standard methods, as described in San~rook et al., cited supra. ~ :~
All patents and publications cited herein are hereby incorporated by reference. While the present ~ ~
5 invention has been described in connection with the ~, -preferred embodiment thereof, it will be understood man-y modifications will be readily apparent to those s~illed ..
in the art, and this application is intended to cover any~adaptations o.r variations thereof. It is manifestly ~.
lO intended this invention be limited only by the claims .
and equivalents thereof. ~

~, ,.:

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7 ~ ~
~`

1 ( WO94/11024 PCTIUS93~10600 '~ r ?,~4QoQo~19 ~ ~

~ .~SEQUENCE LISTING

(1) GENERAL INFORMATION:
(i) APPLICANT: Regents of the University of ~inneso~a Morrill: Hall 100 Church Street S.E.
Minneapolis, MN 55455 .
U.S.A. .
(ii) TITLE OF INVENTION: COMPOSITION PROTECTIVE AGAINST
P. ~LTOCIDA PASTEURELLOSIS INFECT10N
(iil) NUMBER OF SEQUENCES: 1 .
(iv) CORRESPONDENCE ADDRESS: -:-(A) ADDRESSEE: ~erchant & Gould -~B) STREET: 3100 Norwest Center ~.:
(C) CITY: Minneapolis (D) STATE: MN
(E) COUNTRY: USA
(F) ZIP: 5S402 l.
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Flop~y disk (B) COMPUTER: IBM PC ~ompatible --.
(C) OPERATING SYSTE~: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version ~1.25 .
(vi) CURRENT APPLICATION DATA~
(A) APPLICATION NUMBER~
(B) FILING DATE: .
(C) CLASSIFICATION: ~-~vii) PRIOR APPLICATION DATA~
(A) APPLICATIOM NUMBER: US 07/973,070 ~:-(B) FILING DATE:: 06-NOV-1992 ~C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION: -(A) NAME: Woessner, Warren D.
(B) REGISTRATION NUMBER.: 30,440 ; -~
(C) REFERENCE/DOCKET NUNBER: 600.256-WO-01 ~ -(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 612-332-5300 ~ :.
(B) TELEFAX: 612-332-9081 ~ -~
i~ -; ~

21488~9 ., WOg4J~1024 - PCT/US93/10~00 (2) INFO~MATION FOR SEQ ID NO.l: ~.
(i) SEQUENCE CHARACTERISTICS~
(A) LENGTH: 64 base pairs . :-~) TYPE: nucLeic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear ;:~
(ii) MOLECULE TYPE: DNA (genomic) (vii) IMMEDIATE SOURCE:
(B) CLONE: Termini of insertion sequence 50 of the Tn5 transposon -~
~.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
CTGACTCTTA TACACAAGTA GCGTCCTGAA CG . . .32 . . . GCGCAGGGGA TCAAGATCTG ATCAAGAGAC AG 64 -:~
.

' .

Claims (14)

WHAT IS CLAIMED IS:

1. A method of immunizing an animal against pasteurellosis comprising:
producing a stable avirulent immunogenic transposon mediated mutant of P. multocida; and administering an effective amount of the stable avirulent mutant to the animal to provide immunity against pasteurellosis.

2. A method according to claim 1, wherein the stable avirulent mutant of P. multocida is P. multocide ATCC No. 55394.

3. The method according to claim 1, wherein the mutant of P. multocida is P. multocida PmTn-396.

4. The method according to claim 1, wherein the stable avirulent mutant of P. multocida is administered orally.

5. The method of claim 1, wherein in the step of producing a stable avirulent immunogenic transposon mediated mutant, the transposon mediated mutant is produced by insertion of a transposon selected from the group consisting of Tn1, Tn3, Tn5, TnphoA, Tn7, Tn9, and Tn10.

6. The method of claim 5, wherein in the step of producing a stable avirulent immunogenic transposon mediated mutant, the mutant is produced with a plasmid encoding the left insertion sequence of Tn5 linked to the gene for alkaline phosphatase.

7. The method according to claim 1, wherein the animal is a turkey.

8. A method of immunizing an animal against pasteurellosis, which comprises:
producing a stable, avirulent immunogenic transposon mediated mutant of P. multocida, wherein the mutant has at least one genetically modified DNA
sequence located in a 9.4 kb EcorV fragment of the P. multocida genome that hybridizes with a 1.3 kb probe as shown in Figure 1; and administering an effective amount of the stable avirulent mutuant to the animal to provide for immunity against pasteurellosis.

9. A vaccine for protecting an animal against pasteurellosis comprising:
an effective amount of a stable avirulent immunogenic transposon mediated mutant of P. multocida;
and a pharmaceutically acceptable carrier.

10. The vaccine according to claim 9, wherein the stable avirulent mutant is P. multocida ATCC No.
55394.

11. The vaccine according to claim 9, wherein the pharmaceutically acceptable carrier is water.

12. An avirulent immunogenic transposon mediated mutant of P. multocida ATCC No. 55395.

13. A vaccine for protecting an animal against pasteurellosis, which comprises-an effective amount of a stable avirulent transposon mediated immunogenic mutant of P. multocida, wherein the mutant has at least one genetically modified DNA sequence located in a 9.4 kb EcorV fragment of the P. multocida genome that hybridizes with a 1.3 kb probe as shown in Figure 1; and a pharmaceutically acceptable carrier.

14. A vaccine for protecting an animal against pasteurellosis, which comprises:
an effective amount of a recombinantly produced virulence factor from P. multocida, wherein the virulence factor is encoded by a 9.4 kb EcorV fragment of the P. multocida genome.