Trypanosoma cruzi: metacyclogenesis in vitro - I. Changes in the properties of metacyclic trypomastigotes maintained in the laboratory by different methods

Abstract

In this work we have studied the modifications in the biological properties of Trypanosoma cruzi when the parasite is maintained for a long time in axenic culture. The studies were done with a clone from an avirulent strain (Dm30L) and a non-cloned virulent strain (EP) of T. cruzi. Both parasites were maintained, for at least three years, by successive triatomine/mouse alternate passage (control condition), or by serial passage in axenic medium (culture condition), or only in the mouse (mouse condition). The comparison between parasites of culture and control condition showed that metacyclogenesis capacity was reduced in the former and that the resulting metacyclics displayed an attenuated virulence. In order to compare the virulence of metacyclics from the urine of the insect vector, Rhodnius prolixus were infected by artificial feeding with parasites of the control or culture condition. After three triatomine/triatomine passages, there was observed an almost identical biological behavior for these parasites, hence indicating that the maintenance of T. cruzi for a long time in axenic culture affects the differentiation capacity and the virulence of the parasite. Additionally, it was demonstrated that it is possible to maintain T. cruzi exclusively through passages in the invertebrate host.

Trypanosoma cruzi: Maintenance in Culture Modify Gene and Antigenic Expression of Metacyclic Trypomastigotes

“…The loss of virulence of EP-strain maintained by long terms in culture could be explained by selection of a low-virulent subpopulation (Deane et al 1984, Garcia et al 1986). These results, taken together with previous evidences showing that metacyclic trypomastigotes of the Dm30L-clone maintained in mice condition had increased virulence (Contreras et al 1994), and knowing that a full avirulent parasite for vertebrate host no may be maintained in nature, suggest that our maintenance conditions in laboratory is affecting intrinsically the biological properties of metacyclic trypomastigotes.…”

“…Both parasites were maintained, for at least seven years, by successive triatomine/ mouse passages (triatomine condition) or by serial passages in axenic medium (culture condition) as previously described (Contreras et al 1994).…”

“…Thereafter, the parasites were incubated in TAU3AAG medium (TAU supplemented with 10 mM L-proline, 50 mM Lglutamate, 2 mM L-aspartate, 10 mM glucose) to a final concentration 3 x 10 6 cell/ml in a final volume of 70 ml in Roux flask. They were incubated at 27°C for 72 hr, then centrifuged at 10,000 xg, resuspended in TAU medium, treated for 30 min at 37°C with fresh guinea pig serum, and separated on DEAE cellulose (Sousa 1983) as previously described (Contreras et al 1994). Metacyclic trypomastigotes from triatomine condition were obtained from epimastigote forms with no more than two months in culture.…”

“…Culture-derived metacyclic forms had decreased their capacity to invade cells and whenever becoming intracellular were destroyed inside phagolysosomes (Contreras et al 1994). There is increasing evidence that the entry of T. cruzi invasive forms into mammalian cells involves glycoconjugates-dependent attachment followed by parasite internalization (Andrews et al 1984, Yoshida et al 1990, 1997, Schenkman et al 1993, Ramirez et al 1993, Vermelho & Meirelles 1994, Burleigh & Andrews 1995.…”

Morphological comparison of axenic amastigogenesis of trypomastigotes and metacyclic forms of Trypanosoma cruzi

“…The parasite was cloned and maintained by alternate triatomine/mouse passages as previously described (Contreras et al 1994). The procedures to obtain in vitro-induced metacyclic and extracelullar-derived metacyclic amastigotes were described in details previously (Contreras et al 2002).…”