A method for isolating and sequencing trypanosome cells to investigate species associations in multiple morphotype infections

J Parasitol. 2023 Dec 1;109(6):592-602. doi: 10.1645/23-34.ABSTRACTTrypanosome infections containing multiple morphologies have been described from all classes of vertebrates, including mammals, birds, non-avian reptiles, amphibians, and fish. These mixed infections make it challenging to evaluate trypanosome diversity, as it is not immediately clear whether the forms present in the bloodstream represent different species or a single pleomorphic species. Amphibians are common hosts for trypanosomes and are often infected by multiple trypanosome morphologies in the bloodstream. Based on morphological observations and life cycle studies, many authors have considered multiple trypanosome morphotypes found infecting the same frogs to be a single pleomorphic species. However, molecular evidence supporting pleomorphic trypanosome species in amphibians is lacking, primarily because linking sequence data to bloodstream trypanosome morphology in mixed infections is extremely challenging. Here we present methods to isolate individual trypanosome cells of 6 morphotypes from frog blood for nested PCR of the 18S rRNA and gGAPDH genes. Single trypanosome cells were isolated by dilution and 3 DNA extraction methods, and 5 nested PCR primer regimes were utilized to optimize amplification from very low starting concentrations. The success rates of extraction methods ranged from 29 to 50% with the use of a Direct PCR kit having the highest success rate. Although the success rate varied in the ...
Source: The Journal of Parasitology - Category: Parasitology Authors: Source Type: research