Date of Award

5-2004

Level of Access Assigned by Author

Campus-Only Thesis

Degree Name

Master of Science (MS)

Department

Microbiology

Advisor

Carol H. Kim

Second Committee Member

Bruce L. Nicholson

Third Committee Member

Keith W. Hutchison

Abstract

Animal models for infectious diseases are essential for detailed studies of hostpathogen interactions. Based on unique features such as optical clarity of embryos, external fertilization, small size, and large clutches per mating, the zebrafish has become a popular animal model for genetics, development, toxicology, and neurobiology. The increasing availability of information and tools to study the zebrafish also makes it an ideal model animal to study infectious disease pathogenesis and innate immune response. This manuscript describes infectious disease models for the zebrafish using the bacterial pathogen Edwardsiella tarda and the viral pathogen snakehead rhabdovirus (SHRV). Zebrafish embryos (24 hours post fertilization) were exposed to each pathogen by static immersion. Cumulative percent mortality, histopathology, and cytokine expression levels were examined in control and infected embryos. Adult zebrafish (> 5 months old) were exposed to each pathogen by static immersion, static immersion following dermal abrasion, and intraperitoneal (i.p.) injection. Cumulative percent mortality, gross pathology, and histopathology were examined in control and infected fish. We found that zebrafish embryos exposed to E. tarda suffered moderate mortalities. In histological sections, E. tarda infected embryos displayed bacterial sepsis and signs of inflammation. Analysis of expression levels of inflammatory cytokines interleukin-1 fl (IL- 10) and tumor necrosis factor-a (TNFa) revealed that infected fish had significant increases in expression of these cytokines in comparison to control fish. Adult fish immersed in E. tarda were refractory to infection, but when subjected to dermal abrasion prior to immersion, fish became readily infected. Adult fish were also susceptible to i.p. injection of E. tarda and displayed rectal protrusion and hemorrhagic lesions near the site of injection. Histopathology of i.p. injected fish revealed pathology and bacteria in the abdominal cavity. Embryos exposed to SHRV were susceptible to infection and suffered significant mortalities. Histopathology of infected embryos showed that the virus had toxic effects and stimulated the production of macrophages. Analysis of IL- 1 fl and Mx (an antiviral molecule) expression levels demonstrated that both the inflammatory response and antiviral response were stimulated in SHRV infected embryos. Adult fish were not susceptible to immersion in SHRV, even after dermal abrasion. Fish i.p. injected with SHRV suffered high levels of mortality and displayed severe petechial hemorrhaging. Histological sections of i.p. injected fish showed edema and hemorrhaging under the skin, and toxic effects in the developing ova of infected females. The data presented in this paper indicates that both E. tarda and SHRV are able to infect and cause disease in zebrafish embryos and adults, and that the innate immune response is stimulated in infected embryos. The zebrafish disease models introduced in this study will serve as a valuable tools for future investigations into host-pathogen interactions and the development, modulation, and evolution of the vertebrate immune system.

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