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Fluorescent markers to distinguish Trypanosoma brucei strains causing Sleeping Sickness

Multiple-genotype infections are increasingly recognized as important factors in disease evolution, parasite transmission dynamics, and the evolution of drug resistance. However, the distinction of co-infecting site genotypes and the tracking of their dynamics have been // ficult with traditional methods based on various genotyping techniques, leaving most questions unaddressed.

Trypanosoma brucei , a protozoan parasite transmitted by the bite of the tsetse fly (Glossina spp.), is of great medical and economic interest in Africa. It causes human sleeping sickness, and Nagana, a cattle disease that prevents cattle farming and the use of work animals over huge areas of sub-Saharan Africa, thus profoundly affecting the economics of the entire continent.

A recent study, to be published in Acta Tropica (available online 4 October 2005) reports new fluorescence markers of various colours that are inserted into the genome of T. brucei to phenotypically label live parasites of all life cycle stages. If different parasite strains are labelled with different colours they can be easily distinguished from each other in experimental studies.

For the study, a total of 10 T. brucei strains were successfully transfected with different fluorescence markers and were monitored in culture, tsetse flies and mice, to demonstrate stability of marker _expression.

The use of fluorescence activated cell sorting (FACS) allowed rapid and accurate identification of parasite strains labelled with different markers. Cell counts by FACS were virtually identical to counts by traditional microscopy but were considerably faster and had a significantly lower sampling error . Co-infecting strains transfected with fluorescence genes of different colour were easily distinguished by eye and their relative and absolute densities were reliably counted by FACS in experimental multiple infections in mice.

Since the FACS can simultaneously determine th e population sizes of differently labelled T. brucei strains or subspecies it allows detailed and efficient tracking of multiple-genotype infections within a single host or vector individual, enabling more powerful studies on parasite dynamics. In addition, it also provides a simple way to separate genotypes after experimental mixed infections, to measure responses of the single strains to an applied treatment, thus eliminating the need for laborious cloning steps.

The markers presented in this study broaden the spectrum of tools available for experimental studies on multiple-genotype infections. They are fundamentally different from isoenzyme analysis and other genotyping approaches in that they allow the distinction of parasite genotypes based on an easily recognizable phenotypic trait.

The authors conclude that the distinction of live parasites based on an easily recognized phenotypic trait and the physical separation of genotypes from mixed infections made possible by live fluorescence markers make trypanosomes an attractive system for experimental studies on parasite population dynamics. The marker described in this study will be especially important by opening new avenues of research on the ecology and evolution of mixed trypanosome infections and their implications for disease dynamics and control. The authors assert that they will be of specific interest to researches addressing ecological, evolutionary and epidemiological questions using trypanosomes as an experimental system.
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