Date of Award

1-1-2014

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Environmental Health Sciences

Content Description

1 online resource (xiv, 180 pages) : illustrations (some color)

Dissertation/Thesis Chair

Ellen B Braun-Howland

Committee Members

David Spink, Kimberlee Musser, James Webber, Nicholas Mantis, Lloyd Wilson

Keywords

Vibrio cholerae, Copepoda, Pathogenic microorganisms

Subject Categories

Environmental Health

Abstract

Vibrio cholerae O1 concentrate in copepods by attaching to their chitinous exoskeleton and gut, making these planktonic crustaceans participants in the spread of cholera in marine and estuarine environments. The role of the symbiosis in the spread of gastrointestinal disease caused by non-O1 serotypes has not been well-studied in fresh water systems. The goal of these studies was to determine whether symbiotic association of V. cholerae with copepods affects virulence of hemolysin/cytolysin toxin, suspected in V. cholerae non-O1 related morbidity. The reverse-transcription-polymerase chain reaction was used to evaluate the relative transcriptional response of hlyA, encoding hemolysin, by a non-O1 strain isolated from the NY City water supply system. In comparison with free-living organisms, colloidal chitin-bound V. cholerae exhibited significantly elevated hlyA transcript levels that were not attributable to biofilm formation. In vitro hemolysis studies using sheep erythrocytes demonstrated that active hemolysin was also produced at elevated levels as a result of the bacterial association with chitin. These results imply that the stimulation of hemolysin production in chitin-associated V. cholerae non-O1 increases the risk of illness upon ingestion of copepods, e.g., in unfiltered water or during recreational activities. Microcosm studies suggesting that copepods protect V. cholerae non-O1 from disinfectant chlorine at levels typical of public water distribution systems underscore the importance of these findings. To permit evaluation of autochthonous V. cholerae-infected copepods, a V. cholerae specific 16S rRNA-targeted oligonucleotide probe was designed for microscopical detection using fluorescent in situ hybridizations. The probe was used in microcosm studies to demonstrate that attachment of V. cholerae non-O1 to copepods occurred prior to transcriptional and translational upregulation of hemolysin. Use of the probe in combination with fluorescently-tagged V. cholerae O1-specific antibodies permitted the differentiation of O1 and non-O1 serotypes in environmental samples. Studies conducted on zooplankton collected in Haiti during the cholera epidemic demonstrated the utility of the developed protocol in investigating the potential role of copepods as reservoirs for future cholera outbreaks. The results indicated that V. cholerae was not yet established in local copepod populations. Overall, these studies emphasize the potential public health impact of symbiotic relationships of V. cholerae with copepods.

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