Identification of virulence determinants

A method for identifying virulence determinants in Mycobacterium paratuberculosis comprising:

• introducing at least one random mutation into the genome of a M. paratuberculosis bacteria by introduction of a transposon;
• infecting macrophages with said mutated bacteria culturing the macrophages containing said mutated bacteria in the presence of a fluoroquinolone or D-cycloserine;
• selecting surviving bacteria;
• testing the selected surviving bacteria for virulence in an animal;
• selecting the non virulent organisms;
• sequencing genetic material from said selected non virulent bacteria;
• determining the site of mutation;
• and
• comparing the sequence at the mutated site to the corresponding wild type sequence.

A composition for immunizing an animal against bacterial infection comprising:

• a pharmaceutically acceptable carrier, diluent or excipient;
• and at least one non-virulent strain of bacteria produced by the process comprising:
• introducing at least one mutation into the genome of a bacteria;
• culturing the mutated bacteria in the presence of an antimicrobial agent that kills growing but not non-growing bacteria;
• selecting surviving bacteria;
• testing the selected surviving bacteria for virulence;
• and selecting the non-virulent strains.

A composition for immunizing an animal against Mycobacterium paratuberculosis comprising:

• a pharmaceutically acceptable carrier, diluent or excipient;
• and at least one non-virulent strain of M. paratuberculosis produced by the process comprising:
• introducing at least one random mutation into the genome of a strain of M. paratuberculosis by insertion of a transposon;
• infecting macrophages with the mutated strain;
• culturing the infected macrophages in the presence of a fluoroquinolone or D-cycloserine;
• selecting surviving M. paratuberculosis organisms;
• testing the selected surviving organisms for virulence in an animal;
• and
• selecting the non-virulent strains.

A composition for immunizing an animal against a bacteria comprising:

• a pharmaceutically acceptable carrier diluent or excipient;
• and at least one bacterial virulence determinant, the determinant identified by a process comprising;
• introducing at least one mutation into the genome of a bacteria;
• culturing the mutated bacteria in the presence of an antimicrobial agent that kills growing but not non-growing bacteria;
• selecting surviving bacteria;
• testing the selected surviving bacteria for virulence;
• selecting the non-virulent strains;
• sequencing genetic material from the selected non-virulent bacteria to determine the site of the mutation;
• and
• identifying the virulence determinant based on the site of the mutation.

A composition for immunizing an animal against Mycobacterium paratuberculosis comprising:

• a pharmaceutically acceptable carrier diluent or excipient;
• and at least one Mycobacterium paratuberculosis virulence determinant, the determinant identified by a process comprising;
• introducing at least one mutation into the genome of a strain of Mycobacterium paratuberculosis by insertion of a transposon;
• infecting macrophages with the mutated strain;
• culturing the infected macrophages in the presence of a fluoroquinolone or D-cycloserine;
• selecting surviving bacteria;
• testing the selected surviving bacteria for virulence in an animal;
• selecting the non-virulent bacteria;
• sequencing genetic material from the selected non-virulent bacteria to determine the site of the mutation;
• and
• determining the virulence determinant based on the site of the mutation.