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US 2008/0166321 A1Published application
Method for Identification of Virulence Determinants
AssigneeBoard of Regents of the University of Nebraska
InventorsRaul G. Barletta, N. Beth Harris
Life of this patent
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Abstract
Disclosed are methods for the determination of virulence determinants in bacteria and in particular bacteria of the genus Mycobacterium. Also disclosed are compositions and methods for stimulating an immune response in an animal using bacteria and virulence determinants identified by the methods of the present invention.
Claims25
A method for identifying virulence determinants of a bacteria 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 bacteria;
- 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.
5 more elements
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.
7 more elements
A method for isolating a non-virulent mutant bacteria 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 mutant bacteria.
3 more elements
Dependencies are inferred from the claim text.