First evidence for the presence of pathogenic serogroups of Vibrio cholerae in chironomids

Vibrio cholerae causes the fatal cholera diarrhea and is a natural inhabitant of aquatic ecosystems. Chironomids (Insecta: Diptera, Chironomidae) are the most abundant macro-invertebrate group in freshwater aquatic habitats and estuaries. They undergo a complete metamorphosis of four life stages; three (eggs, larvae and pupae) take place in the water, while the adults emerge into the air. Chironomids are natural reservoirs of V. cholerae and we commonly isolate V. cholerae from all four life stages of chironomids. However, up until now, only the non-O1/non-O139 serogroups were identified from chironomids. Our aim was to explore the presence of pathogenic strains of V. cholerae in chironomids. To obtain this purpose we sampled all four life stages of chironomids from two rivers in Pune, India. In total, we analyzed 173 chironomid samples. The identity of chironomid species was verified using cytochrome oxidase gene sequences. Seven chironomid species were identified. Of them, Chironomus circumdatus was the most abundant (62%). The presence of V. cholerae in all chironomid life stages was verified by amplifying the OmpW gene (a gene of an outer membrane protein). Moreover, we were able to amplify wbe and/or wbf genes that detect serogroups O1 or O139, respectively (46% and 13% positive samples, respectively). Cholera toxin subunit A (ctxA) was detected in 49% of the samples. This is the first evidence that pathogenic strains of V. cholerae inhabit chironomids. The results of the current study may provide significant novel tools for monitoring and predicting cholera epidemics.