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Three monoclonal antibodies generated by immunization of mice with Plasmodium berghei-infected red blood cells were found to react with the 75-kDa heat-shock protein (HSP70) present in liver stages and erythrocytic forms of the parasites. These antibodies were shown to react with a recombinant protein encoding the carboxyl terminal half of PfHSP70 (aa 365-681). Differently from earlier results, we clearly demonstrated that HSP70 was also expressed in the sporozoite stage, using these monoclonal antibodies in an immunofluorescence and Western immunoblot assay. These monoclonal antibodies react not only with sporozoites of P. berghei, the parasites originally used for the immunization, but also with sporozoites of several other rodent and human plasmodial species. Passive transfer of these monoclonal antibodies into naive mice, simultaneously injected with sporozoites, failed to neutralize the infectivity of P. berghei sporozoites and to inhibit the development of liver stages of P. yoelii.

A simple method was developed for the characterization of different strains of Tiypanosoma cruzi. T. cruzi stocks isolated from vectors or by hemoculture from patients with Chagas disease could be grouped in subpopulations having similar patterns of restriction endonuclease products of kinetoplast DNA minicircles. We designate such subpopulations by the term "schizodemes." Furthermore, it is shown that, from a given T. cruzi strain, clones with different biological properties can be isolated and identified by their restriction patterns.The hemoflagellate Trypanosoma cruzi is the etiological agent of Chagas disease, a pleomorphic clinical entity that is an important cause of morbidity and mortality in Central and South America. In some patients the infection is devastating from the very beginning with death occurring after a short acute phase; meningoencephalitis and myocarditis are prominent findings. In other cases, however, the acute phase is oligosymptomatic or even silent and may evolve without detectable sequels. Between these two extremes, in most cases the course has a variable acute phase which subsides in a few weeks, to be followed years later by digestive or cardiac symptoms or both (1).The exact causes of this clinical pleomorphism are not known, although evidence suggests that both host and parasite factors may be involved (1). In terms of the parasite, differences have been found among several strains of T. cruzi in regard to morphology, virulence, pathogenicity, and tissue tropism (1). Particularly well documented are the differences between the "polar" Y (2) and CL (3) strains. Isozyme analysis has also been used to distinguish strains of T. cruzi. Miles et al. (4-6) examined T. cruzi stocks isolated from humans and sylvatic animals in Brazil and found three distinct isozyme groups (or zymodemes). Romanha et al. (7,8) studied T. cruzi cultures obtained from humans in the city of Bambui (Minas Gerais, Brazil) and were able to group the stocks into four isozyme groups. Variations in kinetoplast DNA (kDNA) buoyant density between strains of T. cruzi and strains and species of Leishmania have been reported, and this classification method has been used by Chance (9) and Baker et al. (10).Comparison of restriction endonuclease-generated fragments of kDNA has been proposed as another method for the intrinsic classification of trypanosomes (11,12). We show here that stocks of T. cruzi isolated from patients with Chagas disease can be characterized by restriction digests of the kDNA networks. MATERIALS AND METHODSCells. With the exception of the strains Y and CL (13), all the T. cruzi stocks used in this study were obtained from patients who had positive serologic tests against Chagas disease and who were from the city of Bambui (Minas Gerais, Brazil). Stock cultures were initiated with cells-from 30 ml of blood, grown to stationary phase in liver infusion/tryptose medium (14) at 270C, and stored frozen in liquid nitrogen. Clinical histories are available for each human isolate used in this study...

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SummaryHeterochromatin plays a central role in the process of immune evasion, pathogenesis, and transmission of the malaria parasite Plasmodium falciparum during blood stage infection. Here, we use ChIP sequencing to demonstrate that sporozoites from mosquito salivary glands expand heterochromatin at subtelomeric regions to silence blood-stage-specific genes. Our data also revealed that heterochromatin enrichment is predictive of the transcription status of clonally variant genes members that mediate cytoadhesion in blood stage parasites. A specific member (here called NF54varsporo) of the var gene family remains euchromatic, and the resultant PfEMP1 (NF54_SpzPfEMP1) is expressed at the sporozoite surface. NF54_SpzPfEMP1-specific antibodies efficiently block hepatocyte infection in a strain-specific manner. Furthermore, human volunteers immunized with infective sporozoites developed antibodies against NF54_SpzPfEMP1. Overall, we show that the epigenetic signature of var genes is reset in mosquito stages. Moreover, the identification of a strain-specific sporozoite PfEMP1 is highly relevant for vaccine design based on sporozoites.

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To cite this version:Eric Prina, Emeric Roux, Denise Mattei, Geneviève Milon. Leishmania DNA is rapidly degraded following parasite death: an analysis by microscopy and real-time PCR.. Microbes and Infection, Elsevier, 2007, 9 (11) Abstract Control of human leishmaniases relies on appropriate diagnosis and reliable methods for monitoring chemotherapy. The current method used for estimation of parasite burden during chemotherapy patient follow-up as well as in pharmacological studies performed in experimental models involves PCR-based assays. Compared to time-consuming conventional methods, this type of Leishmania DNA detection-based method is extremely sensitive, but could fail in distinguishing viable Leishmania from slowly degenerating ones. We have used an in vitro model to monitor the duration of Leishmania DNA persistence in mouse macrophages following exposure to L-leucine ester, a molecule otherwise known to rapidly kill intracellular Leishmania amazonensis amastigotes. At one hour post L-leucine ester exposure, more than 98% of amastigote-loaded macrophages harbored killed parasites and parasite remnants, as assessed by microscopy. This dramatic decrease in parasite load and the microscopic parasite follow-up over the 120 h time period studied correlated with Leishmania DNA as quantified by real-time PCR. Our results indicate that kinetoplast and nuclear parasite DNA degradation occurs very rapidly after amastigote death. These data add further weight to the argument that PCR assays represent not only a robust method for diagnosis but can also be reliable for monitoring parasite size reduction rate post any intervention (Leishmaniatargeting molecules, immunomodulators…).

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