Diagnosis of human and animal brucellosis using extracts of bacteria phylogenetically related to Brucella

DELPINO MV,; WALLACH JC,; FOSSATI CA,; PABLO CESAR BALDI

Pamplona

Congreso; Brucellosis 2003 International Research Conference; 2003

Members of the genus Brucella are gram negative alpha-proteobacteria that cause brucellosis, an infectious disease affecting livestock and humans. The production of antigens for the serological diagnosis of brucellosis implies the handling of live Brucella, which is a dangerous pathogen. We speculated that cytoplasmic proteins from other alpha-proteobacteria (Agrobacterium sp, Sinorhizobium sp and Ochrobactrum sp) would show cross-reactivity with Brucella antigens and could be used to diagnose brucellosis in humans and animals. Proteins from A. tumefaciens, S. meliloti and O. anthropi were obtained by French press disruption, followed by ultracentrifugation and DNAse and RNAse digestion. Indirect ELISAs were designed in which the plates were coated with the corresponding cytoplasmic proteins at 0.5 µg/well. Cut-off values were calculated as mean ± SD of specific optical densities (sOD= OD with antigen ? OD without antigen) obtained with normal sera (humans, 20; sheep, 20; cows, 36; dogs, 34). These tests were used to assay sera from humans (n=32), sheep (n=75), cows (n=59), and dogs (n=61) infected with different Brucella species. These sera were all positive for antibodies against lipopolysaccharides and cytoplasmatic proteins of Brucella sp by previously described ELISA systems. Canine infection by B. canis was detected with high specificity (97,6% for Agrobacterium, 93,3% for Sinorhizobium, 100% for Ochrobactrum) and sensitivity (32% for Agrobacterium, 77% for Sinorhizobium, 100% for Ochorbactrum) by all the ELISAs tested, and it was possible to diagnose the disease shortly after the exposure to the pathogen (15 days). In contrast, normal sera from humans, sheep, and cattle yielded high sOD with all the antigens, which resulted in high cut-off values and, consequently, in low sensitivities. For human sera, cut-off values were 2.550 for Agrobacterium, 1.820 for Sinorhizobium, 1.500 for Ochrobactrum; for sheep, cut off values were 1.821, 1.255, and 0.856, respectively; for cattle, cut off values were 0.822, 0.930, and 0.665, respectively.These results show that antibodies to cytoplasmatic proteins from related proteobacteria have no diagnostic role in ovine, bovine and human brucellosis. In contrast, they allow the specific and sensitive diagnosis of canine brucellosis and the detection of the infection by B. canis shortly after the exposure to the pathogen.