Cholera is a severe diarrhoeal disease triggered by a toxin produced by specific biotypes of the bacterium Vibrio cholerae that are pathogenic only to humans. There seems to be no chronic state of the disease and the natural reservoir of the pathogen is environmental1. Here we show that egg masses of the non-biting midge Chironomus sp. (Diptera) harbour V. cholerae and act as its sole carbon source, thereby providing a possible natural reservoir for the cholera bacterium.
Chironomids are the most widely distributed and often the most abundant insect in fresh water2. Females deposit egg masses, each containing hundreds of eggs encased in a layer of gelatin, at the water's edge — a convenient location for bacteria to exploit this nutrient-rich substrate.
Two hundred floating Chironomus egg masses (Fig. 1a, b) collected from a waste-stabilization pond settled out overnight as thousands of individual eggs (Fig. 1c), most of which did not hatch. To test whether bacteria feeding on and destroying the gelatin matrix might account for this phenom enon, we extracted freshly collected egg masses with a minimal salt-solution medium containing no carbon source3 in order to isolate and identify any bacteria present.
Four isolates were selected and identified as Vibrio cholerae non-O1 non-O139 by standard microbiological tests, as well as by their serotype and fatty-acid profiles. We then collected Chironomus egg masses from other waste-stabilization ponds in various regions of Israel and were able to isolate V. cholerae from all of these samples.
We inoculated fresh egg masses in a salt-solution medium with 1 × 103 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 × 103 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 × 106 CFU ml−1. 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 similar4.
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 cholera5,6. Propagules may be carried by marine zooplankton along the continental seashore, aided by climatic events such as the El Niño Southern Oscillation7,8,9. 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.
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