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In rabbits immunized with intact bacteria, the immune response to the enterobacterial common antigen (ECA) predominantly consists of the production of immunoglobulin M antibodies. This is not dependent on whether the animals are immunized for a short (2 weeks) or a long (3 months) period of time. The highest ECA-specific immunoglobulin G titers were observed after a short immunization with living bacteria. ECA-specific antisera were obtained by absorption with appropriate ECA-negative mutants. The absorbed antisera were then separated on Sephadex G-200. The resulting immunoglobulin G fractions were conjugated to ferritin by glutardialdehyde and used to visualize the distribution of ECA inE. coli . Bacterial strains either possessing the immunogenic form of ECA (F470, 2387) or solely the nonimmunogenic form (F614) or being devoid of both (ECA-negative mutants F1283 and F1327) were labeled with the conjugates. Freezeetchings of ferritin-labeled strains showed a dense labeling of the outer membrane in case of ECA-immunogenic strains, an essentially weaker labeling of the non-immunogenic ECA mutant and, as expected, no labeling of ECA-negative mutants. Comparable results were obtained with the indirect immunofluorescence technique: the whole cell envelope of strain F470 showed a brilliant fluorescence, whereas a much lesser, spotty distribution of fluorescence was noted with strain F614 and none at all was noted with the ECA-negative strains. These data show that ECA is localized in the outer membrane of ECA-containing strains and further demonstrate that there is more in the immunogenic strains than in the nonimmunogenic ones.

Localization of Enterobacterial Common Antigen: Immunogenic and Nonimmunogenic Enterobacterial Common Antigen-Containing Escherichia coli