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In parasitology, particularly in helminthes studies, several methods have been used to look for the expression of specific molecules, such as RT-PCR, western blot, 2D-electrophoresis, and microscopy, among others. However, these methods require homogenization of the whole helminth parasite, preventing evaluation of individual cells or specific cell types in a given parasite tissue or organ. Also, the extremely high interaction between helminthes and host cells (particularly immune cells) is an important point to be considered. It is really hard to obtain fresh parasites without host cell contamination. Then, it becomes crucial to determine that the analyzed proteins are exclusively from parasitic origin, and not a consequence of host cell contamination. Flow cytometry is a fluorescence-based technique used to evaluate the expression of extra-and intracellular proteins in different type cells, including protozoan parasites. It also allows the isolation and recovery of single-cell populations. Here, we describe a method to isolate and obtain purified helminthes cells.

Toxoplasma gondii proliferates and organizes within a parasitophorous vacuole in rosettes around a residual body and is surrounded by a membranous nanotubular network whose function remains unclear. Here, we characterized structure and function of the residual body in intracellular tachyzoites of the RH strain. Our data showed the residual body as a body limited by a membrane formed during proliferation of tachyzoites probably through the secretion of components and a pinching event of the membrane at the posterior end. It contributes in the intravacuolar parasite organization by the membrane connection between the tachyzoites posterior end and the residual body membrane to give place to the rosette conformation. Radial distribution of parasites in rosettes favors an efficient exteriorization. Absence of the network and presence of atypical residual bodies in a ΔGRA2-HXGPRT knock-out mutant affected the intravacuolar organization of tachyzoites and their exteriorization.

We evaluated the in vitro effects of estradiol, progesterone, and testosterone on the molting process, which is the initial and crucial step in the development of the muscular larvae (ML or L1) to adult worm. Testosterone had no significative effect on the molting rate of the parasite, however, progesterone decreased the molting rate about a 50% in a concentration- and time-independent pattern, while estradiol had a slight effect (10%). The gene expression of caveolin-1, a specific gene used as a marker of parasite development, showed that progesterone and estradiol downregulated its expression, while protein expression was unaffected. By using flow citometry, a possible protein that is recognized by a commercial antiprogesterone receptor antibody was detected. These findings may have strong implications in the host-parasite coevolution, in the sex-associated susceptibility to this infection and could point out to possibilities to use antihormones to inhibit parasite development.