Cholera is caused only by O1 and O139 Vibrio cholerae strains. For diagnosis, 3 working days are needed for bacterial isolation from human feces and for biochemical characterization. Here we describe the purification of bacterial outer membrane proteins (OMP) from V. cholerae O1 Ogawa, O1 Inaba, and O139 strains, as well as the production of specific antisera and their use for fecal Vibrio antigen detection. Anti-OMP antisera showed very high reactivity and specificity by enzyme-linked immunosorbent assay (ELISA) and dot-ELISA. An inmunodiagnostic assay for V. cholerae detection was developed; this assay avoids preenrichment and costly equipment and can be used for epidemiological surveillance and clinical diagnosis of cases, considering that prompt and specific identification of bacteria is mandatory in cholera.Cholera is an acute intestinal disease with watery diarrhea, vomiting, high dehydration, acidosis, and circulation disorders caused by Vibrio cholerae. In case of erratic treatment, death can occur during the first day (12, 13). In spite of the appearance of more than 100 V. cholerae serogroups, only O1 and O139 induce disease (12,16,17). Etiological diagnosis of V. cholerae is based on the isolation of the bacteria from human feces and their biochemical characterization (5a, 20). Vomiting material, water sources, and food can also be used to isolate V. cholerae (5a). Cholera is an infection of pandemic magnitude, and thus prompt and specific identification of bacteria is mandatory; nevertheless, routine microbiological and biochemical analyses need 3 working days (5a, 8). In spite of many publications related to immunological and molecular methods for cholera diagnosis (1,2,6,7,10,11,14,19), most assays require enrichment by previous culture of bacteria, which increases the time needed or involves the use of costly equipment and reagents. In this paper we describe the purification of bacterial outer membrane proteins (OMP) and the production of specific antisera and their use in a detection assay for fecal antigens that does not require preenrichment.
MATERIALS AND METHODSSelection and evaluation of bacteria. Four Vibrio strains were used for antibody production and as controls for the assays: V. cholerae O1 Inaba (CDC13), V. cholerae O1 Ogawa (CDC12), V. cholerae O139, and Vibrio alginolyticus. Bacteria were plated and grown in thiosulfate-citrate-bile salts-sucrose agar (TCBS; Dibico-Mexico) for 18 h at 37°C. Colonies were tested by biochemical methods (5a, 9, 13) and by agglutination using polyclonal antibodies against V. cholerae O1 prepared with Roshka antigens in accordance with Centers for Disease Control and Prevention protocols (20 Isolation of OMP. Vibrios for antibody production were grown in TSB at 37°C for 6 h in a humid chamber; 1 ml was transferred to Erlenmeyer flasks in TSB and grown overnight to induce logarithmic-phase growth. Bacteria were processed as described by Pruzzo et al. (18) and Tarsi and Pruzzo (23). Briefly, the culture was washed three times by centrifugation at 10,000 ϫ ...