A simple method to purify biologically and antigenically preserved bloodstream trypomastigotes of Trypanosoma cruzi using Deae-cellulose columns

A method to purify trypanosomastigotes of some strains of Trypanosoma cruzi (Y, CL, FL, F, "Berenice", "Colombiana" and "São Felipe") from mouse blood by using DEAE-cellulose columns was standardized. This procedure is a modification of the Lanham & Godfrey methods and differs in some aspects from others described to purify T. cruzi bloodstream trypomastigotes, mainly by avoidance of prior purifications of parasites. By this method, the broad trypomastigotes were mainly isolated, accounting for higher recoveries obtained with strains having higher percentages of these forms: processing of infected blood from irradiated mice could be advantageous by increasing the recovery of parasites (percentage and/or total number) and elution of more slender trypomastigotes. Trypomastigotes purified by this method presented normal morphology and motility, remained infective to triatomine bugs and mice, showing in the latter prepatent periods and courses parasitemia similar to those of control parasites, and also reproducing the polymorphism pattern of each strain. Their virulence and pathogenicity also remained considerably preserved, the latter property being evaluated by LD 50 tests, mortality rates and mean survival time of inoculated mice. Moreover, these parasites presented positive, clear and peripheral immunofluorescence reaction at titres similar to those of control organisms, thus suggesting important preservation of their surface antigens.

Astronotus ocellatus (Cichlidae: Pisces) and Macropodus opercularis (Anabatidae: Pisces) as predators of immature Aedes fluviatilis (Diptera: Culicidae) and Biomphalaria glabrata (Mollusca: Planorbidae)

Two fish species, Astronotus ocellatus (Cichlidae) and Macropodus opercularis (Anabatidae) were tested for predacious behavior toward immature mosquitoes (Aedes fluviatili9s, Diptera: Culicidae) and schistosomiasis snail hosts (Biomphalaria glabrata, Mollusca: Planorbidae), in the presence or absence of non-living food and laboratory conditions. A. ocellatus, a species indigenous to Brazil, was a very efficient predator of both organisms (alpha=1,05); M. operculatis, an exotic species, preyed well on immature mosquitoes, but small snails and snail egg-masses were ingested only irregulary. Both fish species seemed to prefer live to non-living food.

Genotoxic effect of alkaloids

Because of the increase use of alkaloids in general medical practice in recent years, it is of interest to determine genotoxic, mutagenic and recombinogenic response to different groups of alkaloids in prokaryotic and eucaryotic organisms. Reserpine, boldine and chelerythrine did not show genotoxicity response in the SOS-Chromotest whereas skimmianine showed genotixicity in the presence of a metabolic activation mixture. Voacristine isolated fromthe leaves of Ervatamia coronaria shows in vivo cytostatic and mutagenic effects in Saccharomyces cerevisiae hapioids cells. The Rauwolfia alkaloid (reserpine) was not able to induce reverse mutation and recombinational mitotic events (crossing-over and gene conversion) in yeast diploid strain XS2316.

Trypanosomatidae codon usage and GC distribution

A study od Typanosomatidae GC distribution and codon usage is presented. The codon usage patterns in coincidence with the phylogenetical data are similar in Crithidia and Leishmania, whereas they are more divergent in Trypanosoma brucei and T. cruzi. The analyisis of the GC mutational pressure in these organisms reveals that T. brucei, and to a lesser extent T. cruzi, have envolved towards a more balanced use of all bases, whereas Leishmania and Crithidia retain features of a primeval genetic apparatus. Tables with approximated GC mutational pressure in homologous genes, and codon usage in Trypanosomatidae are presented.

Vitellogenesis in insects and other groups: a review

The eggs from oviparous organisms contain large amounts of vitellus, or yolk, wich are utilized by the growing embryo. Vitellogenesis is the process of vitellus accumulation and involves massive heterosynthetic synthesis of the protein vitellogenin (Vg) and its deposition in the oocyte. This work summarizes data on Vg structure, synthesis, uptake by oocytes and its fate during embryogenesis. The hormonal control of vitellogenesis and its tissue, sex and temporal regulation are also discussed. Where it is available, data on structure and expression of Vg-coding genes are reviewed. Insect vitellogenesis is priorized although other oviparous animal groups outside insects are also treated.

Visceral leishmaniasis in Teresina, state of Piauí, Brazil: preliminary observations on the detection and transmissibility of canine and sandfly infections

A Leishmania donovani-complex specific DNA probe was usedto confirm the widespread dissemination of amastigotes in apparently normal skinof dogs with canine visceral leishmaniasis. When Lutzomyia longipalpis were fed on abnormal skin of five naturally infected dogs 57 of 163 (35 per cent) fliesbecame infected: four of 65 flies (6 per cent) became infected when fed on apparently normal skin. The bite of a single sandfly that had fed seven days previouslyon a naturally infected dog transmitted the infection to a young dog from a non-endemic area. Within 22 days a lesion had developed at the site of the infectivebite (inner ear): 98 days after infection organisms had not disseminated throughout the skin, bone marrow, spleen or liver and the animal was still serologically negative by indirect immunofluorescence and dot-enzyme-linked immunosorbent assay. When fed Lu. longipalpis were captured from a kennel with a sick dog known to be infected, 33 out of 49 (67 per cent) of flies contained promastigotes. In contrast only two infections were detected among more than 200 sandflies captured in houses. These observations confirm the ease of transmissibility of L.chagasi from dog to sandfly to dog in Teresina. It is likely that canine VL is the major source of human VL by the transmission route dog-sandfly-human. the Lmet2 DNA probe was a useful epidemiological tool for detecting L. chagasi in sandflies.

Some environmental and biological factors influencing the activity of entomopathogenic Bacillus on mosquito larvae in Brazil

The influence of environmental and biological factors on the efficacy of Bacillus thuringiensis serovar israelensis and B. sphaericus as mosquito larvicides are reviewed. The importance of strain dependence, cultivating media/methods, mosquito species/specificity, formulations and their relation to mosquito feeding habits, as well as temperature, solar exposure, larval density and concomitant presence of other aquatic organisms are addressed with reference to the present status of knowledge in Brazil.

Proceedings of the Schistosome Genome Project

"The host-parasite relationship" is a vast and diverse research field which, despite huge human and financial input over many years, remains largely shrouded in mystery. Clearly, the adaptation of parasites to their different host species, and to the different environmental stresses that they represent, depends on interactions with, and responses to, various molecules of host and/or parasite origin. The schistosome genome project is a primary strategy to reach the goal; this systematic research project has successfully developed novel technologies for qualitative and quantitative characterization of schistosome genes and genome organization by extensive international collaboration between top quality laboratories. Schistosomes are a family of parasitic blood flukes (Phylum Platyhelminthes), which have seven pairs of autosomal chromosomes and one pair of sex chromosomes (ZZ for a male worm and ZW for a female), of a haploid genome size of 2.7x108 base pairs (Simpson et al. 1982). Schistosomes are ideal model organisms for the development of genome mapping strategies since they have a small genome size comparable to that of well-characterized model organisms such as Caenorhabditis elegans (100 Mb) and Drosophila (165 Mb), and contain functional genes with a high level of homology to the host mammalian genes. Here we summarize the current progress in the schistosome genome project, the information of 3,047 transcribed genes (Expressed Sequence Tags; EST), complete sets of cDNA and genomic DNA libraries (including YAC and cosmid libraries) with a mapping technique to the well defined schistosome chromosomes. The schistosome genome project will further identify and characterize the key molecules that are responsible for host-parasite adaptation, i.e., successful growth, development, maturation and reproduction of the parasite within its host in the near future

Identification of Transcribed Sequences (ESTs) in the Trypanosoma cruzi Genome Project

Random single pass sequencing of cDNA fragments, also known as generation of Expressed Sequence Tags (ESTs), has been highly successful in the study of the gene content of higher organisms, and forms an integral part of most genome projects, with the objective to identify new genes and targets for disease control and prevention and to generate mapping probes. In the Trypanosoma cruzi genome project, EST sequencing has also been a starting point, and here we report data on the first 797 sequences obtained, partly from a CL Brener epimastigote non-normalized library, partly on a normalized library. Only around 30% of the sequences obtained showed similarity with Genbank and dbEST databases, half of which with sequences already reported for T. cruzi.

Mechanisms of cell accumulation induced by Mycobacterium bovis BCG

Mycobacteria, specially Mycobacterium tuberculosis are among the micro-organisms that are increasing dramatically the number of infections with death, all over the world. A great number of animal experimental models have been proposed to investigate the mechanisms involved in the host response against these intracellular parasites. Studies of airway infection in guinea-pigs and rabbits, as well as, in mice intravenously infected with BCG have made an important contribution to our understanding of the virulence, pathogenesis and the immunology of mycobacterial infections. Although, there are few models to study the mechanisms of the initial inflammatory process induced by the first contact with the Mycobacteria, and the relevance of the acute generation of inflammatory mediators, cytokines and leukocyte infiltration to the development of the mycobacterial infection. In this work we reviewed our results obtained with a model of M. bovis BCG-induced pleurisy in mice, describing the mechanisms involved in the leukocyte influx induced by BCG at 24 hr. Different mechanisms appear to be related with the influx of neutrophils, eosinophils and mononuclear cells and distinct inflammatory mediators, cytokines and adhesion molecules are involved in the BCG-induced cell accumulation.

Programmed Cell Death in Procyclic Form Trypanosoma brucei rhodesiense - Identification of Differentially Expressed Genes during Con A Induced Death

Trypanosoma brucei rhodesiense can be induced to undergo apoptosis after stimulation with Con A. As cell death in these parasites is associated with de novo gene expression we have applied a differential display technique, Randomly Amplified Differential Expressed Sequence-Polymerase Chain Reaction (RADES-PCR) to the study of gene expression during Con A induced cell death in these organisms. Twenty-two differentially displayed products have been cloned and sequenced. These represent the first endogenous genes to be identified as implicated in cellular death in trypanosomatids (the most primitive eukaryote in which apoptosis has been described). Evidence for an ancestral death machinery, `proto-apoptosis' in single celled organisms is discussed.

Characterization of Endotrypanum (Kinetoplastida: Trypanosomatidae), a Unique Parasite Infecting the Neotropical Tree Sloths (Edentata)

This article reviews current concepts of the biology of Endotrypanum spp. Data summarized here on parasite classification and taxonomic divergence found among these haemoflagellates come from our studies of molecular characterization of Endotrypanum stocks (representing an heterogenous population of reference strains and isolates from the Brazilian Amazon region) and from scientific literature. Using numerical zymotaxonomy we have demonstrated genetic diversity among these parasites. The molecular trees obtained revealed that there are, at least, three groups (distinct species?) of Endotrypanum, which are distributed in Central and South America. In concordance with this classification of the parasites there are further newer molecular data obtained using distinct markers. Moreover, comparative studies (based on the molecular genetics of the organisms) have shown the phylogenetic relationships between some Endotrypanum and related kinetoplastid lineages.

A role for extracellular amastigotes in the immunopathology of Chagas disease

In spite of the growing knowledge obtained about immune control of Trypanosoma cruzi infection, the mechanisms responsible for the variable clinico-pathological expression of Chagas disease remain unknown. In a twist from previous concepts, recent studies indicated that tissue parasitism is a pre-requisite for the development of chronic myocarditis. This fundamental concept, together with the realization that T. cruzi organisms consist of genetically heterogeneous clones, offers a new framework for studies of molecular pathogenesis. In the present article, we will discuss in general terms the possible implications of genetic variability of T. cruzi antigens and proteases to immunopathology. Peptide epitopes from a highly polymorphic subfamily of trans-sialidase (TS) antigens were recently identified as targets of killer T cell (CTL) responses, both in mice and humans. While some class I MHC restricted CTL recognize epitopes derived from amastigote-specific TS-related antigens (TSRA), others are targeted to peptide epitopes originating from trypomastigote-specific TSRA. A mechanistic hypothesis is proposed to explain how the functional activity and specificity of class I MHC restricted killer T cells may control the extent to which tissue are exposed to prematurely released amastigotes. Chronic immunopathology may be exacerbated due the progressive accumulation of amastigote-derived antigens and pro-inflammatory molecules (eg. GPI-mucins and kinin-releasing proteases) in dead macrophage bodies.

Microsporidia and acquired immunodeficiency syndrome

Microsporidia is a common term that has been used to refer to a group of eukaryotic, obligate intracellular protozoan parasites belonging to the phylum Microspora. They are important agricultural parasites, contaminating commercial insects; they are also important by infecting laboratory rodents, rabbits and primates. Ever since the early cases found by Magarino Torres, who reported the presence of Encephalitozoon in a patient suffering of a meningoencephalomyelitis, some human pathology caused by microsporidia has been described. However, only after the acquired immunodeficiency syndrome outbreak have these organisms appeared as significant etiological agents in different pathologies. Even so, they remain underestimated. In the present article, the importance of microsporidia for the human pathology in immunocompromised host has been stressed.

Immunocompromised host: from the early events until the impact of acquired immunodeficiency syndrome

The concept that microorganisms can modulate the host resistance was historically reviewed in the present article. The importance of African trypanosomiasis in the development of the research on immunosuppression as well as the impact of human immunodeficiency virus infection are discussed. Each day new opportunistic organisms establish a constant challenge for the correct diagnosis of concomitant infections in acquired immunodeficiency syndrome. The importance of parasite infection in the balance of host resistance in the third world was emphasized. Finally, some aspects of Leishmania as opportunistic organisms were presented.