Date of Award:

5-1-2005

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Biology

Department name when degree awarded

Life Sciences: Biology

Committee Chair(s)

Daryll B. DeWald

Committee

Daryll B. DeWald

Committee

Nabil N. Youssef

Committee

Karen E. Mock

Abstract

Twenty-five monoclonal antibodies produced against myxospores and triactinomyxon spores of M. cerebralis, the causative agent of whirling disease of salmonids, were selected in the initial screening for their reactivity to myxospores. Twelve were found to be effective and accordingly they were characterized fully for their potential as diagnostic reagents for isolated myxospores, pre-spore stages, and spores in frozen sections. Comparative detection assays using enzyme linked immunosorbent assay (ELISA), immunofluorescent assay (IFA), and immuno dot-blot were conducted. Six antibodies bound to developing stages and mature spores of the parasite. Localization of antibody binding in IFA was affirmed by electron microscopy. Different concentrations of antibodies were tested to find the optimal activity of each antibody at the highest possible dilution. Antibodies were also tested for specificity to M. cerebralis by an IFA with a neurotropic myxosporean. Three antibodies reacted with the neurotropic myxospore, while nine antibodies were specific to M. cerebralis. Furthermore, the 12 antibodies were characterized as either binding to carbohydrate or protein epitopes. Seven antibodies bound to carbohydrate epitopes, two antibodies appeared to bind to periodate-resistant carbohydrate complexes, and three bound to protein epitopes. It is concluded that nine antibodies show strong activity and specificity to M. cerebralis. They provided specific staining in IFA, immunohistochemistry, and immuno dot-blot assays. We propose that a cocktail of these antibodies could be used in conjunction with microscopy and/or immuno dot-blotting for detection of whirling disease in infected fish even at the pre-spore developmental stages and in "lightly" infected fish. To compliment the diagnostic study, we localized chitin in spores and we explored the possibility of using lectin binding as a more specific stain for myxosporeans in histological practices. The results indicate that the lectin wheat germ agglutinin (WGA and s-WGA) bound to myxospore and triactinomyxon (TAM) polar capsules and cell walls while the lectin ulex europaeus (UEA-II) failed to bind to any structure.

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