High-speed gel microelectrophoresis, a new and easy approach for detection of PCR-amplified microbial DNA from environmental and clinical samples in microgels using conventional equipment.

AIMS: Microelectrophoresis allows the detection of DNA bands using minimal amounts of sample in a short time, but commonly requires the use of special equipment which is not available in all laboratories. This fact has limited the application of this technique in microbiology despite its advantages. In this work, we describe a new approach to perform gel microelectrophoresis, named high-speed gel microelectrophoresis (HSGME), and its application for rapid detection of bacteria, protozoa and viruses in clinical, vegetal and environmental samples. METHODS AND RESULTS: Aliquots of 0.4-1 microl of PCR product were loaded in 2 cm 1% agarose microgels and electrophoresed at high voltage (125 V cm(-1)) in conventional submarine horizontal mini-slabs. By using HSGME, single-DNA bands obtained after specific-PCR useful in diagnosis of different diseases caused by micro-organisms were detected in 5 min. CONCLUSIONS: HSGME is a rapid and easy procedure applicable to detection of microbial genes, which is carried out using conventional equipment and thus can be performed in any research and diagnostic laboratory. SIGNIFICANCE AND IMPACT OF THE STUDY: The performance of HSGME saves up to 90% time, material and energy costs, as well as laboratory hazardous wastes including carcinogenic agents used for visualizing DNA bands.

Endogenous gamma interferon and interleukin-10 in Brucella abortus 2308 infection in mice.

CD-1 mice intravenously infected with the virulent Brucella abortus 2308 strain simultaneously produce significant levels of gamma interferon (IFN-gamma) and interleukin-10 (IL-10) in their spleens between the second and eighth day post-infection with no production of interleukin-4 (IL-4). Endogenous synthesis of IL-10 does not affect the production of IFN-gamma in this organ, while the production of both cytokines during this period of time is accompanied by a statistically significant increase (P < 0.001) in the number of colony forming units (cfu) of B. abortus 2308 present in the organ. These findings suggest that although the endogenous synthesis of IL-10 apparently does not affect IFN-gamma production, it may affect the effector functions of macrophages to control intracellular brucellae. Production of the Th1 cytokine IFN-gamma during B. abortus 2308 infection is also associated with a specific IgG3 and IgG2a response against the B. abortus 2308 lipopolysaccharide (S-LPS) antigen.

Analysis of the immune response to Yersinia enterocolitica serotype-O:9-released proteins by immunoblot and ELISA.

The immune response towards the released Yersinia enterocolitica outer membrane proteins (Yop) was analysed by immunoblotting and ELISA using a rabbit experimental model. Rabbits orogastrically or intravenously infected with the virulent (plasmid-bearing) Y. enterocolitica O:9 W836 strain developed a significant response by (IgG) antibodies to the released proteins having molecular weights of 51 (YopH) and 41 (LcrV) kDa, respectively. However, only in animals infected via the orogastric route were specific antibodies of the IgG class found against plasmid-encoded polypeptides of 35 (YopN) and 20 (YopQ) kDa. These results suggest that the expression of Yop in vivo may be conditioned by the route of infection used. Using ELISA, a significant response by IgG-class antibodies to YopH protein was evident in the sera from rabbits both orogastrically and intravenously infected with the virulent (pYV+) Y. enterocolitica O:9 W836 strain. By contrast, no specific antibodies to this antigen were detected in sera of rabbit infected with an avirulent (plasmid-cured) derivative (pYV-) strain. Accordingly, this protein could be very useful as an antigen in ELISA for serological diagnosis of infections caused by enteropathogenic strains of Yersinia spp.

Characterization of smooth Brucella lipopolysaccharides and polysaccharides by monoclonal antibodies.

Two mouse monoclonal antibodies (mAb) generated to the M antigen of Brucella melitensis 16M were analysed. Binding profiles of both monoclonals were established by a competitive enzyme-linked immunosorbent assay using chemically defined lipopolysaccharides, O polysaccharides and native hapten polysaccharides from B. melitensis 16M, B. abortus 544 and Yersinia enterocolitica O:9. Using this assay, significant differences in the reactivity of both antibodies were found with A and M antigens from Brucella spp. and the O polysaccharide from Y. enterocolitica O:9. These findings are consistent with the simultaneous expression of A and M epitopes on the lipopolysaccharide of all smooth Brucella strains. Quantitatively similar inhibitory powers were established for the native hapten and O polysaccharide from B. melitensis 16M. However, different behaviour was observed between both antigenic preparations obtained from B. abortus 544. The use of lipopolysaccharide-M-specific mAb in different serological tests instead of polyclonal antisera may be of great practical use for minimizing the risk of the appearance of cross-serological reactions between smooth Brucella strains and Y. enterocolitica O:9.

Class-specific immune response to Yersinia enterocolitica serotype O9 antigens as determined by enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) using lipopolysaccharide (S-LPS) as the antigen was used to analyze the antibody response in rabbits orogastrically and intravenously infected with virulent (plasmid-bearing) Yersinia enterocolitica O9 strains (pYV+) and with the avirulent (plasmid-cured) derivatives (pYV-). A significative response of immunoglobulin G (IgG), IgA, and IgM antibodies against the S-LPS antigen was evident in sera from the rabbits orogastrically infected with pYV+ strains. This immune response was stronger and persisted longer than those obtained with the corresponding pYV- strains. In contrast, few differences were observed in the titers and evolution of IgG, IgA, and IgM antibodies against the S-LPS antigen in rabbits intravenously infected with pYV+ and pYV- strains. These results suggest that the necessity of the virulence plasmid for the establishment of infection by Y. enterocolitica serotype O9 is conditioned by the infection route used. When the S-LPS ELISA was compared with the radial immunodiffusion test using the native hapten as the antigen, the results showed that the ELISA technique was more sensitive. However, only those sera obtained between 2 and 8 weeks postinfection from rabbits intravenously infected with plasmid-bearing strains were positive in the radial immunodiffusion test.

Rapid serotyping of enteropathogenic Yersinia enterocolitica strains by co-agglutination.

A rapid co-agglutination test using monospecific antisera was developed for the serological typing of enteropathogenic strains of Yersinia enterocolitica. A total of 70 bacterial strains (17 reference strains and 53 clinical isolates) were examined. Absorption of immune sera against serotypes O:3, O:8 and O:9 with their heterologous antigens (S-LPS) was necessary to avoid the appearance of different cross-reactions, as observed by co-agglutination. The proteins present in the S-LPS preparations obtained from each serotype seemed to be responsible for such cross-reactions. Results obtained with a total of 57 clinical isolates belonging to other members of the family Enterobacteriaceae indicate a high specificity of the assay.