Abstract
Trypanosoma cruzi, the etiological agent of Chagas disease, is a polymorphic species. Evidence suggests that the majority of the T. cruzi populations isolated from afflicted humans, reservoir animals, or vectors are multiclonal. However, the extent and the complexity of multiclonality remain to be established, since aneuploidy cannot be excluded and current conventional cloning methods cannot identify all the representative clones in an infection. To answer this question, we adapted a methodology originally described for analyzing single spermatozoids, to isolate and study single T. cruzi parasites. Accordingly, the cloning apparatus of a Fluorescence-Activated Cell Sorter (FACS) was used to sort single T. cruzi cells directly into 96-wells microplates. Cells were then genotyped using two polymorphic genomic markers and four microsatellite loci. We validated this methodology by testing four T. cruzi populations: one control artificial mixture composed of two monoclonal populations – Silvio X10 cl1 (TcI) and Esmeraldo cl3 (TcII) – and three naturally occurring strains, one isolated from a vector (A316A R7) and two others derived from the first reported human case of Chagas disease. Using this innovative approach, we were able to successfully describe the whole complexity of these natural strains, revealing their multiclonal status. In addition, our results demonstrate that these T. cruzi populations are formed of more clones than originally expected. The method also permitted estimating of the proportion of each subpopulation of the tested strains. The single-cell genotyping approach allowed analysis of intrapopulation diversity at a level of detail not achieved previously, and may thus improve our comprehension of population structure and dynamics of T. cruzi. Finally, this methodology is capable to settle once and for all controversies on the issue of multiclonality.
Highlights
Chagas disease, an American protozoonosis caused by Trypanosoma cruzi, is characterized by various clinical manifestations ranging from asymptomatic to severe cardiac and/or digestive injuries
Most of the putative multiclonal populations have been suggested based on indirect evidence, or current conventional cloning methods that may favor some individuals’ growth
In an effort to elucidate the complexity of T. cruzi isolates we adapted a methodology originally described for analyzing single spermatozoids, to sort single parasites
Summary
An American protozoonosis caused by Trypanosoma cruzi, is characterized by various clinical manifestations ranging from asymptomatic to severe cardiac and/or digestive injuries. Most of the putative multiclonal populations were suggested based on the identification of more than two alleles for different markers [7,8,9]. Whether these reported populations are really multiclonal remains controversial because we cannot exclude the possibility of aneuploidy [7,8,10,11]. It is not possible to identify all the clones that constitute a given multiclonal population because current
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
