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We have analysed the regulation of histone synthesis in Leishmania infantum following inhibition of DNA replication. Run-on experiments indicated that transcription rates of the genes coding for the four core histones (H2A, H2B, H3 and H4) were not affected by the inhibition with hydroxyurea of DNA synthesis. However, a dramatic decrease was observed in the newly synthesized histones after inhibition of DNA synthesis. Furthermore, the synthesis of both the histones and DNA resumed in promastigotes after removal of hydroxyurea, indicating that inhibition was reversible. Unlike most eukaryotes, in which the replication-dependent histone transcripts decrease upon a replication blockade, the levels of L. infantum histone mRNAs do not change under similar conditions. Thus the present data indicate that histone synthesis in Leishmania is tightly coupled to DNA replication by a mechanism operating at the translational level.

In the present work, we describe the sequence, organization and expression of histone H4 genes in the protozoan parasite Leishmania infantum. The predicted L. infantum histone H4 is a polypeptide of 100 amino acids with a molecular mass of 11.5 kDa. Comparison of the amino acid sequence of Leishmania histone H4 with the rest of histone H4 sequences indicates that this is the most divergent sequence reported to date. The genomic distribution analysis of histone H4 genes indicates that there must be up to seven gene copies. A single size-class histone H4 mRNA of 0.6 kb was detected, whose level dramatically decreases from logarithmic to stationary phase. However, the Leishmania histone H4 mRNAs do not decrease in abundance following treatment with inhibitors of DNA synthesis, suggesting a regulation by a replication-independent mechanism.

Mapping of antigenic determinants of the Leishmania infantum Hsp70 was studied by analysis of the reactivity of sera from dogs with natural visceral leishmaniasis against a collection of peptides representing overlapping sequences of the Hsp70. Despite the considerable variation in the immune response among individuals three immunodominant regions were revealed encompassing residues 241 to 260 (region I), 435 to 469 (region II) and from amino acid 601 to the carboxyl-terminal end of the protein (region III). Since anti-peptide H17 antibodies purified from a pool of leishmaniasis sera recognized the L. infantum Hsp70 and since they did not react with the homologous Hsp70s from other trypanosomatids, such as Trypanosoma cruzi , Trypanosoma rangeli and Leishmania panamensis , it was concluded that peptide H17 (region I) contains an immunodominant B cell species-specific epitope. Our data indicate, however, that the complete recombinant L. infantum Hsp70 protein cannot be used as a disease-specific tool for serodiagnosis since it is also recognized by the sera from patients with Chagas' disease.

Gene-array technologies have been applied in a wide number of organisms to study gene expression profiling under several physiological and experimental conditions. Gene-array implementations combined with the information arising from emerging genome sequencing projects are expected to be in the near future a major tool to characterize genes involved in different processes. So far, gene expression profile studies in trypanosomatids have been performed in microarrays that use a glass support to immobilize fragments of genomic DNA followed by fluorescent detection. Here, we wanted to test the potential of genomic DNA macroarrays of Leishmania infantum using nylon membranes and radioactive detection. Nylon macroarrays present a number of advantages since the processing of the membranes is based on standard Southern blotting protocols familiar to molecular biologists, and the data acquisition equipment is available to most research institutions. Nylon macroarrays were employed to search for genes showing increased mRNA abundance during an axenic differentiation of L. infantum promastigotes to amastigotes. Several clones were rescued and, after validation by Northern blot assays, these L. infantum sequences were used to screen the Leishmania major gene database. The L. major contigs with high homology to the L. infantum sequences allowed a consistent identification of the regulated genes.

All kinetoplastids contain membrane-bound microbodies known as glycosomes,in which several metabolic pathways including part of glycolysis are compartmentalized. Peroxin 2 is essential for the import of many proteins into the microbodies of yeasts and mammals. The PEX2 gene of Trypanosoma brucei was identified and its expression was silenced by means of tetracycline-inducible RNA interference. Bloodstream-form trypanosomes, which rely exclusively on glycolysis for ATP generation, died rapidly upon PEX2 depletion. Insect-form (procyclic) trypanosomes do not rely solely on glycolysis for ATP synthesis. PEX2 depletion in procyclic forms resulted in relocation of most tested matrix proteins to the cytosol, and these mutants also died. Compartmentation of microbody enzymes is therefore essential for survival of bloodstream and procyclic T. brucei. In contrast, yeasts and cultured mammalian cells grow normally in the absence of peroxisomal membranes unless placed on selective media.

The prevalence and importance of Cyclospora cayetanensis as an enteropathogen among 71 patients (22-45 years old) with acquired immunodeficiency syndrome (AIDS) and 132 children with diarrhea (0-12 years old) from Venezuela was assessed retrospectively. Two to three stool samples from each patient attending our parasitology laboratory for parasitologic and medical assistance were examined. For identification of the coccidium, modified Ziehl-Neelsen carbolfuchsin staining of formalin-ether stool concentrates was used, and for other intestinal parasites, iron-hematoxylin-stained smears and formalin-ether concentrates were examined. Cyclospora oocysts were found in seven (9.8%) of 71 AIDS patients and seven (5.3%) of 132 children with diarrhea. Other pathogenic parasites were present in most of the patients (9 of 14, 64.3%) shedding oocysts. Cyclosporiasis predominated in children 2-5 years of age with respect to those < or = one year of age (P < 0.05). The findings suggest that C. cayetanensis is common in diarrheal patients from Venezuela. However, the role of the parasite as the causal agent of diarrhea in these patients is uncertain.

The 70-kDa heat shock protein (Hsp70) is a major target of the humoral immune response during Leishmania infections. In the present paper, it is shown that 84.6% of sera from mucocutaneous leishmaniasis (MCL) patients and 78.9% of sera from visceral leishmaniasis (VL) patients reacted with the L. infantum Hsp70. The mapping of the antigenic determinants indicated that this protein is highly rich in linear B-epitopes. As the complete protein cannot be used for specific serodiagnosis of VL because it is also recognized by the sera from Chagas’ disease patients, a search for specific epitopes recognized by leishmaniasis patients was undertaken. A 50-mer synthetic peptide, located in the most divergent region of the protein, was found to be recognized by a high percentage of leishmaniasis sera and not recognized by chagasic sera. Such a tool would be particularly useful for serodiagnosis of leishmaniasis in geographical areas where mixed infections with Trypanosoma cruzi and Leishmania occurs.

In the present study we show that sera from dogs naturally infected with the protozoan parasite Leishmania infantum contain antibodies that specifically react with the parasite histone H3. Using synthetic peptides covering the complete sequence of the protein we located the linear antigenic determinants within the 40 amino-terminal amino acids of the molecule. In addition to the complete form of the protein (rLiH3), two regions of the Leishmania histone H3 were expressed as recombinant proteins: the rLiH3-Nt fragment containing the 39 amino-terminal amino acids and the rLiH3-Ct fragment containing the 90 carboxyl-terminal residues. Competition experiments using the protein fragment rLiH3-Nt as competitor confirmed that the antigenic determinants of histone H3 are confined to the amino-terminal domain. This domain, which is believed to be exposed on the nucleosome surface, is also the most evolutionarily divergent region of the L. infantum histone H3. Visceral leishmaniasis (VL) sera do not react with mammalian histones, an indication that the anti-histone response elicited during Leishmania infection is triggered by the parasite histone. The results of the prevalence of anti-histone H3 antibodies in canine VL sera together with the sequence-specific characteristics of the amino-terminal region of L. infantum histone H3 indicate that the recombinant protein rLiH3-Nt may be of use for diagnosis of canine VL.

In the present work we show that a high percentage of the sera from dogs naturally affected with viscero-cutaneous leishmaniasis contain antibodies reacting with the Leishmania infantum P0 ribosomal protein. In order to map the antigenic determinants of the LiP0 protein during Leishmania-infection, the complete amino acid sequence of the protein was synthesized as overlapping 20-mer peptides. We have identified the sequence AAKEEPEESDEDDFGMG, located adjacent to the C-terminal end of the protein, as the major antigenic determinant. The anti-LiP0 antibodies present in the sera of the infected dogs do not cross-react with a relatively similar antigenic determinant of the LiP2 acidic proteins as an indication that the Leishmania P0 protein is an independent immunogenically functional antigen in the canine form of the infection.

We studied the prevalence of Cryptosporidium in 29 patients with acquired immunodeficiency syndrome (AIDS) from Zulia State, Venezuela. They ranged in age from five months to 46 years. Two were children and 27 were adults, of which six were women. Of the 21 men, 66.6% reported homosexual behavior. Three stool samples from each patient were examined, and modified Ziehl-Neelsen carbolfuchsin staining of formalinether stool concentrates was used to identify Cryptosporidium oocysts. To detect the presence of other intestinal parasites, direct wet mounts and iron-hematoxylin-stained smears were examined. Cryptosporidium was found in 12 (41.3%) of the patients and was identified as a single parasitic infection in seven of the 12 patients (58.3%). Other pathogenic parasites encountered were Giardia lamblia (3 of 12, 25%), Entamoeba histolytica (1 of 12, 8.3%), Ascaris lumbricoides, Trichuris trichiura, and Strongyloides stercoralis (each 1 of 12, 8.3%). Blastocystis hominis, an organism with an uncertain taxonomic position and pathogenicity, was observed in three of 12 patients (25%). An inflammatory exudate was observed in 10 of 12 patients infected with Cryptosporidium. Most of the patients with this infection presented with chronic watery diarrhea and weight loss. Our results suggest that Cryptosporidium is very common in AIDS patients with diarrhea in Venezuela. However, the role of this parasite as an enteropathogen in these patients is uncertain.

Cryptosporidium is a coccidian parasite found in the intestinal and respiratory epithelia of a wide variety of vertebrate hosts. First reported in 1976 cryptosporidiosis among humans has increased dramatically since 1982. Cryptosporidium has now become one of the most common intestinal protozoa and one of the most frequent opportunistic infections in AIDS patients. Infection causes only self-limited diarrheal illness in immunocompetent persons but severe sometimes life-threatening and protracted diarrhea in immunosuppressed persons. Large-scale surveys of selected populations have found infection prevalence rates of 0.6-20% in developed countries and 4-20% in developing countries. There is however little information about cryptosporidiosis in Venezuela. The authors report findings from a preliminary study designed to evaluate the prevalence and clinical significance of Cryptosporidium in patients with AIDS in Maracaibo. Three stool specimens were collected from each of seven male and three female symptomatic patients. All were residents of Maracaibo aged 7-35 years. All had mild or severe diarrhea and some had one or more of the following manifestations: abdominal pain malaise weight loss cough with expectoration lymphadenopathy candidiasis and herpes. Four patients had Cryptosporidium oocysts associated with Trichuris trichiura and Blastocystis hominis in one case while two had only Giardia lamblia trophozoites. All cases infected with Cryptosporidium presented an inflammatory exudate replete with leukocytes plasma cells and macrophages; two cases had Charcot-Leyden crystals. This frequency of infection with Cryptosporidium is higher than that observed in the US and Brazil and similar to the frequencies reported from Africa and Haiti. The authors note that the main symptom of cryptosporidiosis is a non-inflammatory diarrhea with leukocytes rarely seen. The inflammatory exudate with abundant leukocytes observed in all of these infected patients however suggests the involvement of other associated enteropathogens and warrants further investigation.

The genomic organization and transcription of the genes encoding the histone H3 of the protozoan parasite Leishmania infantum have been studied. It was found that there are multiple copies of the histone H3 genes distributed in chromosomal bands XIX and XIV. The nucleotide sequence of two of the L. infantum H3 genes, each one located in a different chromosome, is reported. Although the nucleotide sequence of the coding region of both genes is identical, the sequence of the 3´ untranslated region is highly divergent. It was found also that there exist two different size classes of histone H3 transcripts, each one derived from a different gene, and that they are polyadenylated. The steady-state level of the transcripts dramatically decreases when the parasites enter the stationary phase of growth, suggesting a mode of regulation which is linked to the proliferation status of the cell. Unlike the replication-dependent histones, the L. infantum H3 mRNA levels do not decrease after treatment with DNA synthesis inhibitors. A comparative analysis of the sensitivity of the histone mRNA levels to DNA inhibition in the parasites L. infantum and Trypanosoma cruzi revealed the existence of different control mechanisms in histone expression in these two phylogenetically related protozoan parasites.

SUMMARYIn this work we show that in the sera from dogs naturally infected with the protozoan parasite Leishmania infantum there are antibodies that react specifically against the parasite acidic ribosomal proteins LiP2a and LiP2b, and that each one of the Leishmania P proteins elicits a specific humoral immune response. Using synthetic peptides, the antigenic epitope of these proteins has been mapped in a single region located adjacent to the C-terminal domain highly conserved among the eukaryotic P proteins. The anti-P antibodies elicited during the Leishmania infection do not recognize the conserved C-terminal domain of the parasite P proteins, in contrast with the findings reported in Chagas’ disease or systemic lupus erythematosus. The antigenic epitopes of the LiP2a and LiP2b are almost identical in amino acid sequence. No reactivity against Trypanosoma cruzi and human P proteins was found in sera from L. infantum-infected dogs.

Leishmania infantum HSP70 has been described as an immunodominant antigen in both humans and dogs suffering from visceral leishmaniasis. In this study, we used L. infantum HSP70 fused to Escherichia coli maltose-binding protein (MBP), as the reporter protein, to analyze the influence of HSP70 on the immunogenicity of MBP in BALB/c mice. Plasmids were constructed to produce the three recombinant proteins used in this study, namely, MBP, L. infantum HSP70, and MBP-HSP70, which consists of MBP fused to the L. infantum HSP70 amino terminus. Immunization of BALB/c mice with the MBP-HSP70 fusion protein elicited humoral and cellular responses against MBP that were higher by an order of magnitude than those elicited by immunization with MBP alone or with a mixture of MBP and HSP70. Covalent linkage of MBP to HSP70 was essential for eliciting a strong anti-MBP immune response. Cytokine secretion and immunoglobulin G isotype analyses indicated that immunization with the MBP-HSP70 fusion protein preferentially induces a Th1 immune response. Immunization of athymic nu/nu mice with the MBP-HSP70 fusion protein unexpectedly gave rise to an anti-MBP humoral response showing features of a T-cell-dependent response. Thus, we present evidence that L. infantum HSP70 demonstrates an adjuvant effect in the immune response against a covalently linked reporter protein.

During recent years, several Leishmania infantum genes have been cloned and characterized. Here, we have summarized the available information on the gene organization and expression in this protozoan parasite. From a comparative analysis, the following outstanding features were found to be common to most of the genes characterized: tandemly organized genes with conserved coding regions and divergent untranslated regions, polycistronic transcription and post-transcriptional regulation of gene expression. The analysis of chromosomes of L. infantum by pulsed-field electrophoresis showed the existence of both size and number polymorphisms such that each strain has a distinctive molecular karyotype. Despite this variability, highly conserved physical linkage groups exists among different strains of L. infantum and even among Old World Leishmania species. Gene mapping on the L. infantum molecular karyotype evidenced a bias in chromosomal distribution of, at least, the evolutionary conserved genes.