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Access Type

WSU Access

Date of Award

January 2021

Degree Type


Degree Name



Immunology and Microbiology

First Advisor

Jeffrey H. Withey


Vibrio cholerae is the bacterium that causes the diarrheal disease cholera, which is spread through the ingestion of contaminated food or water. Cholera endemics occur largely in developing countries that lack proper infrastructure to treat sewage and provide clean water. One environmental reservoir of V. cholerae is fish. Diarrheal symptoms similar to those seen in humans are seen in zebrafish, a natural host model, as early as 6 hours after exposure. Our understanding of basic zebrafish immunology is currently rudimentary, and no research has been done to date exploring the immune response of zebrafish to V. cholerae infection. Furthermore, the relationship between V. cholerae and select antimicrobial proteins has not been established.

Bacteria were grown in LB broth and were diluted in sterile 1× PBS to an infectious dose of 2.5 × 107 CFU/ml. One mL of bacterial inoculum was added to 400ml water with 4-5 adult wild-type ZDR zebrafish. RNA and protein were extracted from intestinal homogenates. A time-course study was performed to assess mRNA expression of neutrophils & neutrophil associated cytokines, as well as select neutrophilic antimicrobial proteins. Bacterial cultures were grown in the presence of purified antimicrobial proteins to assess inhibitory effects.

During the course of V. cholerae infection, large increases in neutrophils, neutrophil-associated cytokines, and neutrophilic antimicrobial proteins were detected. Addition of purified antimicrobial proteins to bacterial culture significantly or completely inhibited growth. Protein variants with altered metal ion binding capacity highlight the importance of select metal ions in V. cholerae growth, as well as binding site dynamics. Additionally, downstream targets of neutrophil proteins were upregulated, as were humoral and cellular immunity markers and antibodies.

Our study for the first time describes the immune response in zebrafish to V. cholerae infection, as well as establishing relationships between select antimicrobial proteins and V. cholerae. These results provide valuable understanding of the natural life cycle of V. cholerae and its relationship with zebrafish, and help in understanding differences and similarities between the immune systems of zebrafish and our own. In addition to uncovering potential diagnostic markers and areas of therapeutic interest, this work enables the use of the zebrafish in exploring unanswered questions in the cholera immunology field.

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