Philip H. Elzer and Sue D. Hagius

The bacterial genus Brucella includes six recognized species. They are characterized by the animals that they preferentially infect. Three of these bacteria were classified by the Centers for Disease Control as “agents of mass destruction” after the Sept. 11, 2001, tragic events in this country. They are B. abortus, B. melitensis and B. suis.

Brucellae cause abortion and infertility in wild and domestic animals. Several of the brucellae can be trans-mitted from animals to humans. There is no approved vaccine for human use, and most of the animal vaccines are virulent to humans. Thus, we need to find a safe and efficacious vaccine that can be used in humans.

With the concentration of livestock, lack of genetic diversity, increased farm sizes, importation of animals and increased international travel, agriculture around the world could be vulnerable to a terrorism attack through the use of brucellae. Brucellae are highly infectious, can be easily aero-solized and are stable during production. Because of their sensitivity to direct sunlight, these bacteria can be destroyed in the environment over time. Since there are no human vaccines against brucellosis, most, if not all populations, have little or no natural immunity to this organism.

Human infection can be caused by ingestion of Brucella-infected raw milk products, exposure to infected animals and aerosolization of the organism. Brucellosis in humans is characterized by a cyclical fever that starts two to three weeks after exposure. Night sweats, headaches, backaches and general malaise are symptoms associated with acute infection. Chronic brucellosis can lead to arthritis, dementia and even death.

Of all of the brucellae, B. melitensis best fits the criteria of a biological and agricultural weapon because little is needed to infect humans. Low doses infect most mammals. These brucellae account for tremendous economic loss in agriculture worldwide. This microbe was found in biological warheads during the Gulf War, and the U.S. Army has had an active vaccine development program since that time. So far, these efforts have produced no efficacious vaccines for military or civilians against brucellosis.

Spurred by the lack of knowledge of the molecular biology of the genus Brucella, which is necessary for the development of vaccines, LSU AgCenter scientists were part of a project to sequence the genome of B. melitensis. A genome is the entire complement of genetic material present in each cell of an organism. In cooper-ation with scientists at the University of Scranton in Pennsylvania, the AgCenter role was to work with the B. melitensis strain 16M. We have been doing this for the past eight years using the goat as a model system. Strain 16M causes disease in goats and humans.

The genome of B. melitensis strain 16M was sequenced. Further investigation has led to the discovery of genes that influence the ability of the bacteria to establish infections and cause disease. This was the first Brucella species sequenced and published in the scientific literature. It was a template for the sequencing of two other Brucella species: B. suis and B. abortus.

With the sequence completed and the knowledge interpreted, the next step in the process was to examine the Brucella proteome. The genomic sequence allows for the comprehensive and rapid analysis of the organism’s proteome. The proteome is defined as the entire set of proteins.

More than 500 proteins have been identified in B. melitensis using the genomic sequence, and experiments studying the up and down regulation of these proteins under different conditions are under way. We have compared the proteome of three Brucella strains, including strain 16M, grown under experimental conditions; and we have found many differences in their proteomes. We are now looking at the proteome expressed in goats infected with strain 16M versus the proteins expressed in the laboratory-grown cultures. We predict that we will find different numbers or levels of proteins needed for survival in the host.

Using this information, proteins of interest can be modified or deleted from virulent strains. One such protein is OMP25, a major structural outer membrane protein, which we have studied extensively. Genetic mutants lacking OMP25 were made in virulent strain 16M. By studying the proteomes of these mutants, the up and down regulation of other proteins can be determined. Comparing these differ-ences can lead to knowledge about pathogenicity, virulence and potential diagnostic tools. The OMP25 mutants show promise as potential animal vaccines.

Beyond these direct scientific observations, results of genomic and proteomic studies may be used in the development of rapid detection methodology, finger printing, new diagnostics and the development of more potent animal and potential human vaccines. To date there is no safe human vaccine available against B. melitensis infection, and it is our hope to find one in the near future.