regions of Israel and were able to isolate V. cholerae from all of these samples.We inoculated fresh egg masses in a saltsolution medium with 1 ǂ10 3 V. cholerae per ml. Two controls were run concurrently: egg masses incubated alone under the same conditions, and medium without egg masses inoculated with 1 ǂ10 3 per ml V. cholerae. We determined growth of V. cholerae after 24 h by plating samples on thiosulphate-citrate-bile salt substrate.The number of bacterial colony-forming units (CFUs) that developed in the first control was always less than 0.1% of that recovered in the treated samples; CFUs recovered in the second control did not change. In the medium containing egg masses as the sole carbon source, V. cholerae reached 2 ǂ10 6 CFU ml ǁ1 . Similar results were obtained when sterilized egg masses were provided as a carbon source. These findings show that egg masses can provide a carbon source to support the development and multiplication of V. cholerae.Although the V. cholerae biotypes isolated here are non-pathogenic, it is likely that chironomid egg masses would also be a suitable (and abundant) substrate for the pathogenic V. cholerae O1 and O139, assuming that the microhabitat of the pathogenic biotypes is similar 4 .An association has been noted between both viable and 'viable but non-culturable' V. cholerae and zooplankton, and copepods have been implicated in the spread of cholera 5,6 . Propagules may be carried by marine zooplankton along the continental seashore, aided by climatic events such as the El Niño Southern Oscillation 7-9 . These results are relevant to the dispersion of pandemics and to the autochthonous existence of V. cholerae in endemic locales during periods between epidemics -when there is local build-up of the bacterium but no outbreak of disease. Our findings indicate that chironomid egg masses may serve as an intermediate 'host' reservoir for V. cholerae, facilitating its survival and multiplication in freshwater bodies.

Cholera is a diarrheal disease caused by the gram-negative bacterium Vibrio cholerae, and an estimated 120,000 deaths from cholera occur globally every year. The natural reservoir of the bacterium is environmental. A recent report indicated an association between V. cholerae and chironomid egg masses. Chironomids, the "non-biting midges" (Diptera; Chironomidae), are the most widely distributed and frequently the most abundant insects in freshwater. Females attach egg masses, each containing hundreds of eggs encased in a layer of gelatin, to the water's edge where bacteria are abundant and may encounter the nutrient-rich substrate. Here we report the isolation of non-O1 and non-O139 V. cholerae from chironomid egg masses from different freshwater bodies in Israel, India, and Africa. In a yearly survey in Israel, chironomid populations were found to peak biannually, and it seemed that those peaks were followed by subsequent bacterial growth and disappearance during the winter in the Mediterranean region. The bacterial population rose as water temperature surpassed 25 degrees C. Thirty-five different serogroups of V. cholerae were identified among the bacteria isolated from chironomids, demonstrating population heterogeneity. Two strains of V. cholerae O37 and O201 that were isolated from chironomid egg masses in Zanzibar Island were NAG-ST positive. Our findings support the hypothesis that the association found between chironomids and the cholera bacteria is not a rare coincidence, indicating that chironomid egg masses may serve as yet another potential reservoir for V. cholerae.

Cholera is a severe diarrheal disease caused by specific serogroups of Vibrio cholerae that are pathogenic to humans. The disease does not persist in a chronic state in humans or animals. The pathogen is naturally present as a free-living organism in the environment. Recently, it was suggested that egg masses of the nonbiting midge Chironomus sp. (Diptera) harbor and serve as a nutritive source for V. cholerae, thereby providing a natural reservoir for the organism. Here we report that V. cholerae O9, O1, and O139 supernatants lysed the gelatinous matrix of the chironomid egg mass and inhibited eggs from hatching. The extracellular factor responsible for the degradation of chironomid egg masses (egg mass degrading factor) was purified from V. cholerae O9 and O139 and was identified as the major secreted hemagglutinin/protease (HA/P) of V. cholerae. The substrate in the egg mass was characterized as a glycoprotein. These findings show that HA/P plays an important role in the interaction of V. cholerae and chironomid egg masses.Cholera is a severe diarrheal disease that causes the death of many thousands of people each year and affects the lives of millions. This disease is caused by specific serogoups of Vibrio cholerae that are pathogenic to humans (22). Since 1991, the world has witnessed extension of the seventh pandemic into South America and South Africa, as well as the appearance of a previously unknown pathogenic serogroup of V. cholerae (O139) (23). The disease is not found in a chronic state in humans or animals, indicating that its natural reservoir is environmental (11).Chironomids (Diptera; Chironomidae), the nonbiting midges, are the most widely distributed and frequently the most abundant insects in freshwater (1). Females lay egg masses on the water's edge, and each mass contains hundreds of eggs encased in a layer of gelatinous material. The presence of several thousand egg masses at one site is not unusual. In extreme cases, gelatinous layers several centimeters thick are formed (3,18).Recently, we suggested that chironomid egg masses are an intermediate host reservoir for V. cholerae. Chironomus egg masses collected from a waste stabilization pond settled out overnight as thousands of individual eggs, most of which did not hatch. V. cholerae O9 was isolated from the degraded egg masses. When new freshly collected egg masses were reinfected with V. cholerae O9, the egg masses were destroyed. V. cholerae grew on the chironomid egg masses as a nutritive source (4).Hemagglutinin/protease (HA/P) is one of the main secreted proteases of V. cholerae, and it is usually associated with the stationary phase and starvation (2). HA/P was purified from V. cholerae O1 (7) and O139 strains (17), as well as from non-O1 strains (10, 16). All HA/P purified enzymes showed both hemagglutination and proteolytic activities. Honda et al. (10) compared the purified HA/P from V. cholerae O1 and non-O1 strains. They found that the proteases (or hemagglutinins) derived from V. cholerae O1 and non-O1 strains were immunolog...