|Ewalt, Darla - USDA,NVSL, AMES, IA|
Submitted to: Book Chapter
Publication Type: Book / Chapter
Publication Acceptance Date: July 27, 2005
Publication Date: January 1, 2006
Citation: Bricker, B.J., Ewalt, D.R. 2006. Hoof-prints: Brucella Strain Typing by PCR Amplification of Multilocus Tandem-Repeat Polymorphisms. In: O'Connor, L., editor. Methods in Molecular Biology, Vol. 345, Diagnostic Bacteriology Protocols. 2nd Edition. Totowa, NJ: Humana Press. p. 141-173. Interpretive Summary: Brucellosis is a disease of livestock. It causes abortion in cattle and can make humans sick. When there is an outbreak of the disease, it is important to find out where the infection came from originally to prevent further spread. This paper describes step by step how to perform a procedure for differentiating the bacterial strains that cause brucellosis. This is crucial for showing that the outbreak strain and the proposed source strain are (or are not) the same strain.
Technical Abstract: Infectious diseases of livestock have a negative impact on the agricultural economy. A critical component of limiting disease outbreaks is the ability to trace new outbreaks to their original sources so that further spread from the index populations is prevented. Trace back requires some type of bacterial identification system that will reliably discriminate between related and unrelated bacterial populations. This is difficult to do for pathogenic bacteria with highly conserved genomes, since distinguishing markers or traits are hard to find. Brucella species are one example of disease causing bacteria with highly conserved genomes. Complete genome sequencing of three Brucella species has revealed remarkable genetic homology despite differences in host preference. Although some phenotypic characteristics and genetic features have been identified to distinguish among the species, highly discriminating methods are not available. Recently, small tandemly repeated DNA sequences have become popular sites for finding genetic polymorphisms in bacteria. This is because these short tandemly repeated DNAs mutate at a much higher level than the normal mutation rate caused by slip-strand mispairing and recombination. This phenomenon has been known for a long time in eukaryotic organisms and is the basis for human DNA identification, but it has only recently been applied to typing pathogenic bacteria. This paper describes in detail the procedure for identifying Brucella strains based on the high mutation rates associated with the tandemly repeated DNA sequences found at ten independent genomic loci. Because all ten loci contain 8-bp tandem repeats, we have named this genotyping technique "HOOF-Prints" for Hypervariable Octameric Oligonucleotide Finger-Prints. The procedure presented here is designed for medium to high throughput analyses (at least 10 strains or bacterial colonies) and takes advantage of available technologies for rapid and sensitive genetic analyses, including PCR and capillary electrophoresis with fluorescent detection. However, all of the methods described can be scaled down to characterize a smaller number of samples, and can be performed in a modestly equipped laboratory. A section included at the end of the chapter discusses the application of this technology to low throughput analyses.