Term of Award

Summer 2001

Degree Name

Master of Science in Biology

Document Type and Release Option

Thesis (restricted to Georgia Southern)

Department

Department of Biology

Committee Chair

Frank E. French

Committee Member 1

Daniel V. Hagan

Committee Member 2

William S. Irby

Committee Member 3

Laura B. Regassa

Abstract

Spiroplasma bacteria isolates are classified or identified through a series of serological tests that normally consists of screening, one-way deformation tests, cloning, antisera production, and reciprocal deformation tests. Serological tests on the spiroplasmas are followed by molecular analysis. The standard molecular analysis used for spiroplasmas has been 16S rRNA sequencing. The primary goal ofmy research was to evaluate serologically Group VIII spiroplasmas isolated from tropical Costa Rican tabanids (Diptera: Tabanidae) and to compare them to the temperate North American Group VIII spiroplasmas. A secondary goal was to evaluate both the temperate and tropical Group VIII strains by sequencing the 16S-23S rRNA intergenic spacer region.

Spiroplasma cultures were obtained from Costa Rican tabanids and serologically screened. This screening procedure placed ten of the 72 cultures (GSU 5367, 5401, 5404, 5408, 5429, 5431, 5436, 5437, BARC 4898, and BARC 4899) in Group VIII. Further serological procedures including one-way deformation tests, serocloning, dilution cloning, antisera production, and reciprocal deformation tests indicated that the isolates are serologically related to five serovars previously reported for temperate North American Spiroplasma. Five ofthe strains (GSU 5401, 5404, 5408, 5436, and BARC 4899) are serologically related to TAAS-1 and GSU 5367 is closely related to BARC 2649 of the southern United States. Four of the strains, GSU 5429, 5431, 5437, and BARC 4898, form a cluster of intermediate strains, linking TAAS-1 and BARC 1357. The frequency ofthe serovar TAAS-1 (5/10) in the Costa Rican sample was notably higher than the frequency of 7.7% in Tabanus lineola oftemperate North America.

Since 16S rRNA analyses were found to be too conservative in Group VIII spiroplasmas, attempts were made to sequence the 16S-23S rRNA intergenic spacer region in an effort to separate this group. DNA was extracted from six isolates, two from the Costa Rican sample and four temperate North American Group VIII strains. Primers were developed for the 16S-23S rRNA spacer region and used in PCR amplification. Amplification was not achieved with the first set of primers. Consequently, a second set of primers has been designed for PCR amplification and will be used for future research.

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