Z-011. Real-Time PCR Assay for Brucella suis and a New Strategy for Target Identification

F. F. Roberto, J. M. Barnes, D. T. Newby;
Idaho Natl. Lab., Idaho Falls, ID.

The zoonotic disease brucellosis is caused by the α-2 proteobacterium, Brucella. The genus Brucella has been divided into named species based on origin (B. melitensis, isolated in Malta), primary disease symptom (e.g., B. abortus and abortions in ungulates), or primary host (e.g., B. suis and swine). As molecular tools for discrimination have been employed to study the taxonomy of the Brucella, it has emerged that there is greater than 98% genomic sequence similarity between so-called species. It is apparent that the Brucella are a very homogeneous group of bacterial pathogens, yet cause a disease in their hosts with different etiologies. This host range is complicated by the ability of the various species to cross-infect, or cause disease in other hosts (for example, humans can be infected by B. abortus, B. suis, B. melitensis, B. canis, and B. ovis). In the United States, separate, formal surveillance and control programs exist for brucellosis in cattle and swine caused by B. abortus and B. suis, so it is important to be able to discriminate between pathogenic species when formal diagnosis is made. We report here the development of a real-time PCR assay for B. suis targeting a unique region of Chromosome II in that organism. The assay has been demonstrated to be specific for all B. suis isolates in our collection, and to be capable of detecting as little as 2 fg of genomic DNA from the organism. The assay also works with unpurified DNA released from heat-treated cell suspensions as well as those that have been inactivated through the addition of methanol and heat treatment. It therefore can be used in diagnostic laboratories using protocols that minimize the potential for lab-acquired infections. We will also report on preliminary results of a novel genomic fragment amplification strategy utilizing Brucella insertion sequence IS711 that may be useful not only in identifying novel genomic regions of Brucella strains and isolates, but also as a fingerprinting technique.