The products of the Brucella abortus virB gene locus, which are highly similar to conjugative DNA transfer system, enable the bacterium to replicate within macrophage vacuoles. The replicative phagosome is thought to be established by the interaction of a substrate of the VirB complex with macrophages, although the substrate and its host cellular target have not yet been identified. We report here that Hsp60, a member of the GroEL family of chaperonins, of B. abortus is capable of interacting directly or indirectly with cellular prion protein (PrPC) on host cells. Aggregation of PrPC tail-like formation was observed during bacterial swimming internalization into macrophages and PrPC was selectively incorporated into macropinosomes containing B. abortus. Hsp60 reacted strongly with serum from human brucellosis patients and was exposed on the bacterial surface via a VirB complex–associated process. Under in vitro and in vivo conditions, Hsp60 of B. abortus bound to PrPC. Hsp60 of B. abortus, expressed on the surface of Lactococcus lactis, promoted the aggregation of PrPC but not PrPC tail formation on macrophages. The PrPC deficiency prevented swimming internalization and intracellular replication of B. abortus, with the result that phagosomes bearing the bacteria were targeted into the endocytic network. These results indicate that signal transduction induced by the interaction between bacterial Hsp60 and PrPC on macrophages contributes to the establishment of B. abortus infection.

Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in professional and nonprofessional phagocytes and cause abortion in domestic animals and undulant fever in humans. The mechanism and factors of virulence are not fully understood. To identify genes related to internalization and multiplication in host cells, Brucella abortus was mutagenized by mini-Tn5Km2 transposon that carryied the kanamycin resistance gene, 4,400 mutants were screened, and HeLa cells were infected with each mutant. Twenty-three intracellular-growth-defective mutants were screened and were characterized for internalization and intracellular growth. From these results, we divided the mutants into the following three groups: class I, no internalization and intracellular growth within HeLa cells; class II, an internalization similar to that of the wild type but with no intracellular growth; and class III, internalization twice as high as the wild type but with no intracellular growth. Sequence analysis of DNA flanking the site of transposon showed various insertion sites of bacterial genes that are virulence-associated genes, including virB genes, an ion transporter system, and biosynthesis-and metabolism-associated genes. These internalization and intracellular-growth-defective mutants in HeLa cells also showed defective intracellular growth in macrophages. These results suggest that the virulence-associated genes isolated here contributed to the intracellular growth of both nonprofessional and professional phagocytes.Brucellosis is a major bacterial zoonosis that causes a serious debilitating disease in humans and abortion and sterility in domestic animals. The etiologic agents of brucellosis are Brucella spp., small gram-negative and facultative intracellular pathogens that can multiply within professional and nonprofessional phagocytes (9, 10). In contrast to other intracellular pathogens, Brucella species do not produce exotoxins, antiphagocytic capsules or thick cell walls, resistance forms, or fimbriae and do not show antigenic variation (16). A key aspect of the virulence of brucella is its ability to proliferate within professional and nonprofessional phagocytic host cells and thereby successfully bypasses the bactericidal effects of phagocytes, and their virulence and chronic infections are thought to be due to their ability to avoid the killing mechanisms within host cells (30, 41). The molecular mechanisms and genetic basis for intracellular survival and replication, however, are not understood completely. Some studies with nonprofessional phagocytes have shown that Brucella invades host cells and is contained within early endosome-like vacuoles. These vacuoles rapidly fuse with early autophagosomes that acquire vacuolar [H ϩ ]ATPase and lysosome-associated membrane proteins (LAMP), mature into a late autophagosome, inhibit fusion with lysosomes, and finally become a replicating vacuole normally associated with the endoplasmic reticulum (5,11,31,32). The genetic basis of Brucella virul...

Enzyme-linked immunosorbent assays using antigens extracted from Brucella abortus with n-lauroylsarcosine differentiated natural Brucella-infected animals from Brucella-vaccinated or Yersinia enterocolitica O9-infected animals. A field trial in Mongolia showed cattle, sheep, goat, reindeer, camel, and human sera without infection could be distinguished from Brucella-infected animals by conventional serological tests.Brucellosis is a worldwide infectious disease of domestic animals, and the causative agent, Brucella spp., is transmitted to humans by contact with infected animals or by contaminated dairy products (4). Serodiagnosis of acute and recent infections with Brucella and Yersinia enterocolitica O9 by using the commonly used microagglutination assay is seriously impaired by the well-documented and strong serological cross-reactivity between these bacteria (2, 6-11, 13, 14). The Rose Bengal test and complement fixation test are the most accepted tests worldwide (5). These tests are based on a reaction between a Brucella whole-cell antigen and antibodies produced in response to the infection. Differentiating between animals infected with Brucella and animals vaccinated against Brucella is too difficult by conventional serological tests, such as the Rose Bengal test, tube agglutination test, and complement fixation test (13), because vaccinated animals have a high titer against Brucella antigens. Therefore, we tried to find an easy serological method to differentiate Brucella-infected animals from vaccinated or Y. enterocolitica O9-infected animals.To differentiate natural Brucella-infected animals from Y. enterocolitica O9-infected animals, antigens extracted from the virulent Brucella abortus strain 544 (15) with n-lauroylsarcosine were used for an enzyme-linked immunosorbent assay (ELISA), and the specificity of the ELISA was tested. The antigens were extracted as follows. B. abortus strain 544 cells were grown to A 600 ϭ 3.0 in brucella broth (Becton Dickinson, Sparks, Md.), and bacterial cells were harvested by centrifugation and washed once with distilled water (DW). For whole bacterial cell antigens, bacteria were inactivated by formalin (0.5% final concentration) and were concentrated to 1.5 (the optical density at 600 nm [OD 600 ]) in DW at this step. For n-lauroylsarcosine-extracted antigens, n-lauroylsarcosine (0.5% final concentration) was added to the bacterial suspension and the cells were incubated at room temperature for 30 min with shaking. The bacterial suspension was centrifuged and filtrated, and then the supernatant was transferred to a new centrifuge tube for use with the antigens. The protein concentration of antigen was checked by Bio-Rad protein assay, and the antigen was also checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. Western blotting was done with anti-B. abortus or anti-Y. enterocolitica O9 rabbit serum for each preparation. To coat the antigens on Immuno plates for the ELISA, 50 l of the antigens (sarcosine extracts; 4 g/ml...

Brucellosis is an important zoonosis, and serological surveillance is essential to its control. However, cross‐reactions of attenuated live cells of Brucella abortus strain S‐19 and B. melitensis strain Rev‐1 with Yersinia enterocolitica O9 or vaccinated animal sera interfere with accurate serological diagnosis by the Rose Bengal test (RBT). Therefore, we used ELISA with sarcosine extracts from the virulent B. abortus strain 544 to eliminate false‐positives among RBT positive‐sera. A total of 697 serum samples were collected in Mongolia from humans and animals in 23 nomadic herds. The herds were classified into three groups as brucellosis‐endemic (BE), brucellosis‐suspected (BS), or Brucella‐vaccinated (BV). The number of 295 animals (43.0%) was positive by RBT, but 206 (69.8%) of these were positive according to ELISA; therefore, 30.2% of the RBT‐positive sera were found to be false positives. The false positive samples for RTB represent 4.1%, 27.4%, and 68.2% of the animals from the BE, BS, and BV herds, respectively. In addition, 32% of RBT‐positive human sera were also false positives. Thus, our ELISA would be more specific than RTB and useful for epidemiological surveillance for brucellosis.

Aims: To detect Bacillus anthracis DNA from soil using rapid and simple procedures. Methods and Results: Various amounts of B. anthracis Pasteur II spores were added artificially to 1 g of soil, which was then washed with ethanol and sterile water. Enrichment of the samples in trypticase soy broth was performed twice. A DNA template was prepared from the second enrichment culture using a FastPrep instrument. The template was then used for nested and real-time polymerase chain reaction (PCR) with B. anthracis-specific primers, to confirm the presence of B. anthracis chromosomal DNA and the pXO1/pXO2 plasmids. Conclusions: One cell of B. anthracis in 1 g of soil could be detected by nested and real-time PCR. The usefulness of the PCR method using field samples was also confirmed. Significance and Impact of the Study: The results indicate that this could be a useful method for detecting anthrax-spore contaminated soil with high sensitivity. Its application could have great impact on the progress of epidemiological surveillance.