Brucella spp. are the causative agents of brucellosis in many different hosts, including humans. Most of the serological methods of diagnosis are based on the detection of antilipopolysaccharide antibodies, which makes the differentiation of vaccinated animals from infected animals difficult. By using molecular biology techniques, a gene that encodes a 26-kDa protein (BP26) was isolated from a Brucella abortus S19 genome gt11 library. This protein is in the periplasm of B. abortus and in transformed Escherichia coli. It is exported to the periplasm via a preprotein of 29 kDa with a signal sequence of 28 amino acids. The nucleotide and amino acid sequences of this gene and protein did not show any similarity with those of previously sequenced genes. The use of this protein in Western blotting allowed the differentiation between vaccinated bovines from infected bovines and the detection of infected rams; on the other hand, sera from human patients with active brucellosis were positive, while sera from human patients with chronic brucellosis or without clinical signs were nonreactive. BP26 might be of value as an antigen for serological diagnosis of brucellosis in different mammals.

Using the shuttle vector pMCO2 and the vaccinia virus wild-type WR strain, we constructed a recombinant virus expressing an 18-kDa outer membrane protein of Brucella abortus. BALB/c mice inoculated with this virus produced 18-kDa protein-specific antibodies, mostly of immunoglobulin G2a isotype, and in vitro stimulation of splenocytes from these mice with purified maltose binding protein-18-kDa protein fusion resulted in lymphocyte proliferation and gamma interferon production. However, these mice were not protected against a challenge with the virulent strain B. abortus 2308. Disruption of the 18-kDa protein's gene in vaccine strain B. abortus RB51 did not affect either the strain's protective capabilities or its in vivo attenuation characteristics. These observations suggest that the 18-kDa protein plays no role in protective immunity.

Brucella abortus is a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. The mechanism of virulence of Brucella spp. is not fully understood yet. Furthermore, genes that allow Brucella to reach the intracellular niche and to interact with host cells need to be identified. Using the genomic survey sequence (GSS) approach, we identified the gene encoding an ATPbinding cassette ( Brucellosis is a major zoonotic disease that causes abortion in domestic animals and undulant fever in humans. Brucella proliferates within macrophages of the host and thereby successfully bypasses the bactericidal effects of phagocytes (26,34). Thus, Brucella virulence is associated with the capacity of the organisms to multiply inside the host cells. Therefore, cell-mediated immunity and subsequent activation of macrophages are essential for host clearance of infection (16). Once inside cells, Brucella prevents fusion of the phagosome with the lysosome by altering the intracellular traffic of the early phagosome vesicle (24). It has recently been demonstrated that brucellae replicate in a vesicle compartment containing reticuloendoplasmic markers reached after fusion between phagosomes and lysosomes is prevented (25). However, the genes that allow Brucella to invade and reach the appropriate intracellular replication niche remain to be identified.

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