Velikovsky, G. phytopathogens ((have been reported almost exclusively to occur in immunocompromised patients or in those with debilitating underlying diseases or undergoing chemotherapy (2, 3, 21). There are no reports of human disease by and other nodule-forming bacteria. The close genetic and antigenic relationship between and nonpathogenic alphaproteobacteria (NPAP) has been revealed by several studies. A total of 1 1,902 open reading frames are conserved in open Tetrahydrobiopterin reading frames are conserved in at least one of these three genomes (25). Similar relationships have been observed between NPAP and and (13, 17). Few studies have investigated the immunological cross-reactivity between proteins from and those from related alphaproteobacteria. Velasco et al. (30) found Tetrahydrobiopterin an extensive cross-reactivity between cytosolic proteins from and those from and also some cross-reactions at the level of outer membrane proteins and lipopolysaccharide. More recently, we have shown the existence of cross-reactivities between and other NPAP, including and (12). Moreover, we showed that some of these cross-reactivities could potentially be exploited for the development of serological tests for brucellosis based on antigens from NPAP. Live attenuated strains are still widely used for the vaccination of animals against brucellosis. While these vaccines have reduced virulence for animals, they still can produce disease in humans (4, 5, 6, 27). Therefore, large-scale production of such vaccines requires biosafety level 3 facilities (11), which are seldom available in developing countries, where brucellosis is more prevalent. This is one of the reasons that has led many researchers to investigate alternative vaccination strategies for brucellosis, including the use of subunit vaccines based on recombinant proteins (1, 15) or the use of DNA vaccination (9, 20). Taking into account our previous findings that antigens and those from NPAP are cross-recognized by the immune system, we hypothesized that the immune response elicited by immunization with the latter could protect mice from infection. Here we report the results of the studies conducted to address this hypothesis. We show that systemic immunization with heat-killed NPAP or their cytosoluble fractions partially protects mice from challenge. However, protection levels are higher in mice orally immunized with live NPAP and challenged with by the oral route than in mice immunized systemically with NPAP. MATERIALS AND METHODS Bacteria and culture conditions. 2308, H38, were initially grown in tryptic soy agar (TSA) and expanded in tryptic soy broth to obtain inocula for infections or to prepare heat-killed bacteria. and were initially grown on solid TY medium (Bacto tryptone, 5 g; yeast extract, 3 g; CaCl2, 1.3 g; agar, 7 g in 1 liter of distilled water) and expanded in TY broth (same medium but without agar). All liquid cultures were performed overnight at 28C with constant agitation. Antigens. (i) Heat-killed bacteria. After overnight growth, liquid cultures were placed in a water bath at Cxcr4 80C for 4 h. Samples were plated on TSA or solid TY medium to check for sterility. (ii) CYTs. Cytosoluble fractions were obtained as described previously (12). Liquid bacterial cultures were centrifuged at 15,000 for 10 min and washed three times with 10 mM Tris-HCl (pH 8) (Tris buffer). The cells were suspended in Tris buffer (0.1 g of cells [wet weight] per ml) and disrupted with a French press (SIM-AMINCO-Espectronic Instruments). Bacterial cells were broken by two passages and then digested for 1 h at 37C with DNase and RNase (10 g/ml). Unbroken cells were separated by centrifugation. Particulate matter was pelleted by centrifugation at 105,000 for 4 h, and the resulting supernatant (cytosoluble extract [CYT]) was stored at ?20C until use. Systemic immunization and challenge. Mice (five animals per group) were immunized with three doses (at 2-week intervals) of heat-killed bacteria (1 109 CFU per dose) mixed with incomplete Freund adjuvant administered subcutaneously in the back or with three doses of CYTs (40 g per dose) administered intraperitoneally. Control groups received PBS by the same Tetrahydrobiopterin routes and at the same instances. For the experiment with heat-killed bacteria, the group was immunized with H38 since this strain is considered a good laboratory research for evaluating experimental vaccines in mice challenged with or (8, 10, 16, 31). One month after the last immunization, mice.