Construction of Pasteurella haemolytica vaccines

Abstract

Methylation of DNA can be a critical step in the introduction of DNA into P. haemolytica . A methyltransferase has been isolated and molecularly cloned for this purpose. Use of the methyltransferase has allowed construction of defined, attenuated mutants for use as vaccines to protect cattle.

1 .- 11 . (canceled)  
     
     
         12 . An isolated and purified gene encoding PhaI methyltransferase.  
     
     
         13 . An isolated and purified gene encoding PhaI restriction endonuclease.  
     
     
         14 . A preparation of PhaI methyltransferase free from PhaI restriction endonuclease.  
     
     
         15 . A preparation of PhaI endonuclease free from PhaI methyltransferase.  
     
     
         16 . The preparation of  claim 14  which is free from all other  P. haemolytica  proteins.  
     
     
         17 . The preparation of  claim 15  which is free from all other  P. haemolytica  proteins.  
     
     
         18 .- 33 . (canceled)  
     
     
         34 . A vaccine to induce protective immunity against  Pasteurella haemolytica  infection, comprising an isolated  Pasteurella haemolytica  bacterium which comprises a mutation in a leukotoxin B gene, wherein the mutation attenuates the bacterium.  
     
     
         35 . The vaccine of  claim 34  which contains an adjuvant.  
     
     
         36 . The vaccine of  claim 34  which is formulated for a route of administration selected from the group consisting of intranasal, intratracheal, intramuscular, and intravenous routes of administration.  
     
     
         37 . The vaccine of  claim 34  which is formulated for subcutaneous injection.  
     
     
         38 . The vaccine of  claim 34  wherein the mutation is a deletion mutation.  
     
     
         39 . The vaccine of  claim 38 , wherein the deletion mutation is introduced into the leukotoxin B gene by a method comprising the steps of: 
 isolating a region of a  P. haemolytica  genome comprising the leukotoxin B gene;    introducing a deletion into the leukotoxin B gene to form a mutated DNA region comprising a mutated leukotoxin B gene;    introducing the mutated DNA region into  P. haemolytica  bacteria that do not express a PhaI restriction endonuclease to form transformants; and    screening the transformants to identify  P. haemolytica  bacteria whose chromosomal DNA contains the mutated leukotoxin B gene.    
     
     
         40 . A vaccine to induce protective immunity against  Pasteurella haemolytica  infection, comprising an isolated  Pasteurella haemolytica  bacterium which comprises a deletion mutation in a leukotoxin B gene, wherein the deletion mutation is introduced into the leukotoxin B gene by a method comprising the steps of: 
 isolating a region of a  P. haemolytica  genome comprising the leukotoxin B gene;    introducing a deletion into the leukotoxin B gene to form a mutated DNA region comprising a mutated leukotoxin B gene;    introducing the mutated DNA region into  P. haemolytica  bacteria that do not express a PhaI restriction endonuclease to form transformants; and    screening the transformants to identify  P. haemolytica  bacteria whose chromosomal DNA contains the mutated leukotoxin B gene,    wherein the mutation attenuates the bacterium.    
     
     
         41 . A vaccine to induce protective immunity against  Pasteurella haemolytica  infection, comprising an isolated  Pasteurella haemolytica  bacterium which comprises a mutation in a leukotoxin D gene, wherein the mutation attenuates the bacterium.  
     
     
         42 . The vaccine of  claim 41  which contains an adjuvant.  
     
     
         43 . The vaccine of  claim 41  which is formulated for a route of administration selected from the group consisting of intranasal, intratracheal, intramuscular, and intravenous routes of administration.  
     
     
         44 . The vaccine of  claim 41  which is formulated for subcutaneous injection.  
     
     
         45 . The vaccine of  claim 41  wherein the mutation is a deletion mutation.  
     
     
         46 . The vaccine of  claim 45 , wherein the deletion mutation is introduced into the leukotoxin D gene by a method comprising the steps of: 
 isolating a region of a  P. haemolytica  genome comprising the leukotoxin D gene;    introducing a deletion into the leukotoxin D gene to form a mutated DNA region comprising a mutated leukotoxin D gene;    introducing the mutated DNA region into  P. haemolytica  bacteria that do not express a PhaI restriction endonuclease to form transformants; and    screening the transformants to identify  P. haemolytica  bacteria whose chromosomal DNA contains the mutated leukotoxin D gene.    
     
     
         47 . A vaccine to induce protective immunity against  Pasteurella haemolytica  infection, comprising an isolated  Pasteurella haemolytica  bacterium which comprises a deletion mutation in a leukotoxin D gene, wherein the deletion mutation is introduced into the leukotoxin B gene by a method comprising the steps of: 
 isolating a region of a  P. haemolytica  genome comprising the leukotoxin B gene;    introducing a deletion into the leukotoxin D gene to form a mutated DNA region comprising a mutated leukotoxin D gene;    introducing the mutated DNA region into  P. haemolytica  bacteria that do not express a PhaI restriction endonuclease to form transformants; and    screening the transformants to identify  P. haemolytica  bacteria whose chromosomal DNA contains the mutated leukotoxin D gene,    wherein the mutation attenuates the bacterium.    
     
     
         48 . An isolated  P. haemolytica  bacterium comprising a mutation in a leukotoxin B gene, wherein the mutation attenuates the bacterium.  
     
     
         49 . The bacterium of  claim 48  wherein the mutation is a deletion mutation.  
     
     
         50 . The bacterium of  claim 49 , wherein the deletion mutation is introduced into the leukotoxin B gene by a method comprising the steps of: 
 isolating a region of a  P. haemolytica  genome comprising the leukotoxin B gene;    introducing a deletion into the leukotoxin B gene to form a mutated DNA region comprising a mutated leukotoxin B gene;    introducing the mutated DNA region into  P. haemolytica  bacteria that do not express a PhaI restriction endonuclease to form transformants; and    screening the transformants to identify  P. haemolytica  bacteria whose chromosomal DNA contains the mutated leukotoxin B gene.    
     
     
         51 . An isolated  P. haemolytica  bacterium comprising a mutation in a leukotoxin D gene, wherein the mutation attenuates the bacterium.  
     
     
         52 . The bacterium of  claim 51  wherein the mutation is a deletion mutation.  
     
     
         53 . The bacterium of  claim 52 , wherein the deletion mutation is introduced into the leukotoxin B gene by a method comprising the steps of: 
 isolating a region of a  P. haemolytica  genome comprising the leukotoxin D gene;    introducing a deletion into the leukotoxin B gene to form a mutated DNA region comprising a mutated leukotoxin D gene;    introducing the mutated DNA region into  P. haemolytica  bacteria that do not express a PhaI restriction endonuclease to form transformants; and    screening the transformants to identify  P. haemolytica  bacteria whose chromosomal DNA contains the mutated leukotoxin D gene.