Date of Award

2014

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biology

First Advisor

White, Catherine D. Dr.

Abstract

Haemophilus ducreyi is the causative agent of the genital ulcerative disease chancroid. Chancroid is an important facilitator for increased transmission of HIV amongst heterosexuals. Chancroid is endemic to Asia, Africa, and Latin America, but there have been several outbreaks within the United States. Chancroidal ulcers are categorized as soft, painful, bloody ulcers that may remain chronic if untreated. H. ducreyi produces and secretes a cytolethal distending toxin (CDT) that causes various cell types to undergo cell cycle arrest or apoptosis. The CDT holotoxin consists of three genes: cdtA, cdtB, and cdtC. In 2006, the first report of nonsexual transmission of H. ducreyi was discovered in Samoa. Studies indicate the three children visiting the island had chronic lower extremity ulceration from the gram negative bacteria (Ussher, Wilson, Campanella, Taylor, & Roberts, 2007). The objective of this study therefore was to determine the presence, genetic variability, and functionality of cytolethal distending toxin in Samoa strains of H. ducreyi. To examine the presence of CDT, strains SB 5755, SB 5756, SB 57575, BE 3145, and 35000HP were subjected to Polymerase Chain Reaction (PCR). This technique concluded that 100% of the strains produced cdtA, cdtB, and cdtC. Gel electrophoresis determined approximate sizes of DNA fragments to be 750, 950, and 700 bp respectively. Each gene was cloned independently and transformed into pCR2.1-TOPO plasmid vectors. These genes were successfully amplified into chemically competent Mach1-T1 Escherichia coli cells. Sequence analysis was performed to verify the presence of cdtA, cdtB, and cdtC and to examine genetic differences amongst the strains. The Samoa strains were tested on horse blood agar plates (HBAP) to determine its ability to cause DNAse damage and lyses horse red blood cells. H. ducryei strain 35000HP demonstrated large zones of lysis. The Samoa strains all exhibited clear zones of lysis, but were not as prominent as the wild- type. Increasing knowledge of CDT activity solidifies it as a virulence factor and as a possible target for a novel toxoid vaccine against CDT producing bacteria.

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