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Access Type

WSU Access

Date of Award

January 2022

Degree Type


Degree Name



Immunology and Microbiology

First Advisor

Jeffrey H. Withey


Vibrio cholerae is a Gram-negative aquatic bacterium that is responsible for cholera, an acute intestinal infection in humans characterized by profuse watery diarrhea. Cholera is spread through ingestion of contaminated food or water, primarily in developing countries that lack the infrastructure for proper water and sewage treatment. The WHO estimates that there are at least 5 million new cases of cholera every year, which lead to thousands of deaths. Prior work has shown that V. cholerae has several environmental reservoirs, including shellfish, fish egg masses, copepods, and, more recently, vertebrate fish. Several animal models for V. cholerae are currently in use. The most commonly used are the infant mouse and adult rabbit. However, neither of these species is a natural host for cholera, nor does the bacterium produce a disease state resembling cholera in either of these models. In an effort to develop a natural host model for V. cholerae, we investigated the zebrafish (Danio rerio), a small freshwater fish whose habitat range overlaps significantly with regions where V. cholerae is endemic—particularly India and Bangladesh. Since other vertebrate fish have been found to harbor V. cholerae, and zebrafish are a well-described model system for increasing numbers of infectious diseases, development of a zebrafish model would allow for investigation of natural colonization and disease processes of V. cholerae. Our results indicate that zebrafish become colonized specifically in the intestinal tract when exposed to pandemic V. cholerae strains in water. No manipulation of the animal is required, and the zebrafish have an intact microbiota. Additionally, we sought to develop a method of quantitating pathogenic effects on the zebrafish as well as investigating whether V. cholerae can be transmitted between different fish populations. Chapter one describes the use of zebrafish to model the infectious life cycle of V. cholerae in a natural host. Included are details on how the fish are infected and colonized by V. cholerae without any manipulation to their existing microbiota. This is followed by results demonstrating the transmissibility of V. cholerae between infected and naïve fish. Chapter two concerns the development of a combination of simple assays to quantitatively measure components of excretions (diarrhea) from V. cholerae-infected zebrafish. The ability to measure zebrafish diarrhea will allow us to assess the effects of virulence factors and accessory toxins on the fish. We showed that the measurements of OD600, mucin, and total protein were indicative of V. cholerae pathogenesis in zebrafish. Chapter three investigates potential colonization genes using INSeq to create single-gene deletion mutants en masse. Input DNA libraries were compared against output DNA libraries collected from V. cholerae-infected zebrafish. The mutant abundance was calculated before and after infection to identify genes that might be involved in intestinal colonization. We were able to find a number of colonization genes that had already been identified in other animal models, as well as some novel genes to be further investigated.

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