Meeting Abstract
The genus Trypanosoma consists of flagellated protozoans that infect the circulatory system of all classes of vertebrates. Trypanosomes display a high level of morphological diversity, both between species and within the life cycle of a single trypanosome species. Additionally, a single host individual can be infected with multiple trypanosome morphotypes, and it is unclear whether these morphotypes represent distinct species or a single polymorphic species. To address this, we sequenced the 18s rRNA gene from 5 trypanosome morphotypes infecting amphibians in Oklahoma, USA. However, because the 18s rRNA gene is a multiple copy gene, it is unclear whether we are recovering genes from different trypanosome species, or if we are sequencing different copies of the 18s rRNA gene within a single trypanosome genome. In order to definitively determine the species associations, we need to isolate individual trypanosome morphotypes, and sequence a single copy gene from each morphotype. DEAE cellulose is an anion exchange chromatography resin that can be used to separate trypanosomes from whole blood samples based on differences in their cell surface charge. Trypanosomes are more positively charged than host blood cells, and when blood is added to the chromatography column, trypanosomes pass through the column, while the negatively charged blood cells stick to the positively charged column. Here we describe the use of anion exchange chromatography to isolate trypanosome morphotypes from whole blood. We are in the process of refining the technique and amplifying DNA from separated trypanosome morphotypes, with the overall goal of sequencing the gGAPDH gene of 11 different trypanosome morphotypes infecting Oklahoma amphibians.
