Experimental American leishmaniasis and Chagas'' disease in the Brazilian squirrel monkey: cross immunity and electrocardiographic studies of monkeys infected with Leishmania braziliensis and Trypanosoma cruzi

, and 1988. Experimental American leishmaniasis and Chagas' disease in the Brazilian squirrel monkey: cross immunity and electrocardiographic studies of monkeys infected with Leishmania braziliensis and Trypanosoma cruzi. International Journal for Parasitology 18: 1053–1059. Adult, laboratory-bred squirrel monkeys (Saimiri sciureus) previously infected with either Leishmania braziliensis braziliensis or L. b. panamensis were challenge infected with blood-form trypomastigotes of Trypanosoma cruzi (Brazil strain). Monkeys previously infected with T. cruzi were challenged with stationary phase promastigote forms of L. b. braziliensis. Monkeys were examined during the course of challenge for evidence of infection, electrocardiographic alterations and parasite-specific antibody responses. T. cruzi epimastigotes were cultured from the blood of monkeys up to 3 months after challenge with this parasite. Unulcerated cutaneous lesions appeared and persisted in monkeys challenged with L. b. braziliensis. The formation of satellite lesions was observed in one monkey. Increased QRS intervals were not observed in T. cruzi challenged monkeys without prior cardiac irregularities and QRS left axis shifts were observed in only two of these monkeys. Elevated titers of parasite binding IgM and IgG specific for both T. cruzi and L. braziliensis were observed in all monkeys following challenge. These results indicate that prior infection with T. cruzi or L. braziliensis does not protect against heterologous challenge infection with these organisms. However, prior infection with Leishmania parasites may provide some protection against chagasic cardiopathies.     

Genotypic variation among lineages of Trypanosoma cruzi and its geographic aspects

Isozyme analysis with 18 enzyme loci was conducted on 146 isolates of Trypanosoma cruzi from Mexico, Guatemala, Colombia, Ecuador, Peru, Brazil, Bolivia, Paraguay and Chile. Forty-four different MLGs (groups of isolates with identical multilocus genotypes) were identified and a phylogeny was constructed. The phylogenetic tree consisted of two main groups (T. cruzi I, T. cruzi II), and the latter was further divided into two subgroups (T. cruzi IIa, T. cruzi IIb–e). Evidence of hybridization between different MLGs of T. cruzi II was found, which means that genetic exchanges seem to have occurred in South American T. cruzi. On the other hand, the persistence of characteristic T. cruzi I and T. cruzi II isozyme patterns in single small villages in Bolivia and Guatemala suggested that genetic exchange is very rare between major lineages. A significant difference in genetic diversity was shown between T. cruzi I and T. cruzi II from several indices of population genetics. Two possibilities could explain this genetic variation in the population: differences in evolutionary history and/or different tendencies to exchange genetic material. Broad-scale geographic distributions of T. cruzi I and T. cruzi IIb–e were different; T. cruzi I occurred in Central America and south to Bolivia and Brazil, while T. cruzi IIb–e occurred in the central and southern areas of South America, overlapping with T. cruzi I in Brazil and Bolivia.     

Genetic profiling of Trypanosoma cruzi directly in infected tissues using nested PCR of polymorphic microsatellites

The investigation of the importance of the genetics of Trypanosoma cruzi in determining the clinical course of Chagas disease will depend on precise characterisation of the parasites present in the tissue lesions. This can be adequately accomplished by the use of hypervariable nuclear markers such as microsatellites. However the unilocal nature of these loci and the scarcity of parasites in chronic lesions make it necessary to use high sensitivity PCR with nested primers, whose design depends on the availability of long flanking regions, a feature not hitherto available for any known T. cruzi microsatellites. Herein, making use of the extensive T. cruzi genome sequence now available and using the Tandem Repeats Finder software, it was possible to identify and characterise seven new microsatellite loci – six composed of trinucleotide (TcTAC15, TcTAT20, TcAAT8, TcATT14, TcGAG10 and TcCAA10) and one composed of tetranucleotide (TcAAAT6) motifs. All except the TcCAA10 locus were physically mapped onto distinct intergenic regions of chromosome III of the CL Brener clone contigs. The TcCAA10 locus was localised within a hypothetical protein gene in the T. cruzi genome. All microsatellites were polymorphic and useful for T. cruzi genetic variability studies. Using the TcTAC15 locus it was possible to separate the strains belonging to the T. cruzi I lineage (DTU I) from those belonging to T. cruzi II (DTU IIb), T. cruzi III (DTU IIc) and a hybrid group (DTU IId, IIe). The long flanking regions of these novel microsatellites allowed construction of nested primers and the use of full nested PCR protocols. This strategy enabled us to detect and differentiate T. cruzi strains directly in clinical specimens including heart, blood, CSF and skin tissues from patients in the acute and chronic phases of Chagas disease.     

Early evolution within kinetoplastids (euglenozoa), and the late emergence of trypanosomatids

Many important relationships amongst kinetoplastids, including the position of trypanosomatids, remain uncertain, with limited taxon sampling of markers other than small subunit ribosomal RNA (SSUrRNA). We report gene sequences for cytosolic heat shock proteins 90 and/or 70 (HSP90, HSP70) from the potentially early-diverging kinetoplastids Ichthyobodo necator and Rhynchobodo sp., and from bodonid clades ‘2’ (Parabodonidae) and ‘3’ (Eubodonidae). Some of the new cytosolic HSP70 sequences represent a distinct paralog family (HSP70-B), which is related to yet another paralog known from trypanosomatids (HSP70-C). The (HSP70-B, HSP70-C) clade seemingly diverged before the separation between kinetoplastids and diplonemids. Protein phylogenies support the basal placement of Ichthyobodo within kinetoplastids. Unexpectedly, Rhynchobodo usually forms the next most basal group, separated from the clade ‘1’ bodonids with which it has been allied. Bootstrap support is often weak, but the possibility that Rhynchobodo represents a separate early-diverging lineage within core kinetoplastids deserves further testing. Trypanosomatids always fall remote from the root of kinetoplastids, forming a specific relationship with bodonid clades 2 (and 3), generally with strong bootstrap support. These protein trees with improved taxon sampling provide the best evidence to date for a ‘late’ emergence of trypanosomatids, contradicting recent SSUrRNA-based proposals for a relatively early divergence of this group.     

Ribosomal RNA phylogeny of bodonid and diplonemid flagellates and the evolution of euglenozoa

Euglenozoa is a major phylum of excavate protozoa (comprising euglenoids, kinetoplastids, and diplonemids) with highly unusual nuclear, mitochondrial, and chloroplast genomes. To improve understanding of euglenozoan evolution, we sequenced nuclear small-subunit rRNA genes from 34 bodonids (Bodo, Neobodo, Parabodo, Dimastigella-like, Rhynchobodo, Rhynchomonas, and unidentified strains), nine diplonemids (Diplonema, Rhynchopus), and a euglenoid (Entosiphon). Phylogenetic analysis reveals that diplonemids and bodonids are more diverse than previously recognised, but does not clearly establish the branching order of kinetoplastids, euglenoids, and diplonemids. Rhynchopus is holophyletic; parasitic species arose from within free-living species. Kinetoplastea (bodonids and trypanosomatids) are robustly holophyletic and comprise a major clade including all trypanosomatids and most bodonids ('core bodonids') and a very divergent minor one including Ichthyobodo. The root of the major kinetoplastid clade is probably between trypanosomatids and core bodonids. Core bodonids have three distinct subclades. Clade 1 has two distinct Rhynchobodo-like lineages; a lineage comprising Dimastigella and Rhynchomonas; and another including Cruzella and Neobodo. Clade 2 comprises Cryptobia/ Trypanoplasma, Procryptobia, and Parabodo. Clade 3 is an extensive Bodo saltans species complex. Neobodo designis is a vast genetically divergent species complex with mutually exclusive marine and freshwater subclades. Our analysis supports three phagotrophic euglenoid orders: Petalomonadida (holophyletic), Ploeotiida (probably holophyletic), Peranemida (paraphyletic).     

Anti-plasmodial and anti-trypanosomal activity of synthetic naphtho[2,3-b]thiophen-4,9-quinones

Naphtho[2,3-b]thiophen-4,9-quinone and five derivatives were prepared using the Friedel-Crafts reaction and tandem-lithiation of aromatic diethylamides. These quinones were evaluated for their trypanocidal and anti-plasmodial activities by their effects on: (1) growth of epimastigote forms of Trypanosoma cruzi in vitro, (2) lysis of trypomastigote forms of T. cruzi in murine blood, (3) growth of Plasmodium falciparum in vitro, and (4) inhibition of the recombinant enzyme trypanothione reductase. The parent compound, naphtho[2,3-b]thiophen-4,9-quinone (3a), was among the most active quinone tested in vitro against P. falciparum at 0.2 μM. However, it was inactive against P. berghei-infected mice treated with 2.3 mmol/kg daily for 5 days. Most of the quinones prepared were active against T. cruzi epimastigotes in culture but exhibited weak activity at 4 °C against trypomastigotes in murine blood as well against the enzyme trypanothione reductase. Further structural modifications will be necessary to improve the in vivo activity of the naphthothiophenquinones.     

Time-dependent mode of immunization augments suppressed antibody responses in spleen cell cultures from mice experimentally infected with Trypanosoma cruzi

Mice infected with Trypanosoma cruzi develop immunosuppressed responses to heterologous antigens. Experiments were performed using infected mice in the acute stage of infection to assess immunoregulatory activities during induction of direct plaque-forming cells (DPFC) to sheep erythrocytes (SRBC). After normal or infected mice were primed with SRBC, their spleen cells were restimulated 4 days later with SRBC in Mishell-Dutton cultures and found to mount hyperaugmented IgM anti-SRBC responses. It was also demonstrated that T-cells derived from normal mice primed in vivo 4 days previously with SRBC, and subsequently added to cultures of spleen cells from T. cruzi-infected mice, enhanced anti-SRBC DPFC responses in a dose-dependent fashion. These results show that functional help provided by T-cells activated during an in vivo priming and exposed to an in vitro challenge dose of antigen (SRBC) in a time-dependent mode can overcome the effect of immunosuppression in the spleen cell cultures from T. cruzi-infected mice.     

Correlation between conformation and antibody binding: NMR structure of cross-reactive peptides from T. cruzi, human and L. braziliensis

The structure of peptides corresponding to the C-terminal residues from Trypanosoma cruzi (R13), human (H13) and Leishmania braziliensis (A13) ribosomal proteins were determined using nuclear magnetic resonance. Although there is only one amino acid difference between them, the peptides present distinct structures in solution: R13 adopts a random coil conformation while H13 and A13 form a bend. Interaction of these peptides with polyclonal antibodies from chronic Chagas’ disease patients and a monoclonal antibody raised against T. cruzi ribosomal P2β protein was probed by transferred NOE. The results show that the flexibility of R13 is fundamental for the binding to the antibody.     

Phylogenomic analysis of kinetoplastids supports that trypanosomatids arose from within bodonids

Kinetoplastids are a large group of free-living and parasitic eukaryotic flagellates, including the medically important trypanosomatids (e.g., Trypanosoma and Leishmania) and the widespread free-living and parasitic bodonids. Small subunit rRNA- and conserved protein-based phylogenies support the division of kinetoplastids into five orders (Prokinetoplastida, Neobodonida, Parabodonida, Eubodonida, and Trypanosomatida), but they produce incongruent results regarding their relative branching order, in particular for the position of the Trypanosomatida. In general, small subunit rRNA tends to support their early emergence, whereas protein phylogenies most often support a more recent origin from within bodonids. In order to resolve this question through a phylogenomic approach, we carried out massive parallel sequencing of cDNA from representatives of three bodonid orders (Bodo saltans -Eubodonida-, Procryptobia sorokini -Parabodonida-, and Rhynchomonas nasuta -Neobodonida-). We identified 64 well-conserved proteins shared by these species, four trypanosomatids, and two closely related outgroup species (Euglena gracilis and Diplonema papillatum). Phylogenetic analysis of a concatenated data set yielded a strongly supported tree showing the late emergence of trypanosomatids as a sister group of the Eubodonida. In addition, we identified homologues of proteins involved in trypanosomatid mitochondrial mRNA editing in the three bodonid species, suggesting that editing may be widespread in kinetoplastids. Comparison of expressed sequences from mitochondrial genes showed variability at U positions, in agreement with the existence of editing activity in the three bodonid orders most closely related to trypanosomatids (Neobodonida, Parabodonida, and Eubodonida). Mitochondrial mRNA editing appears to be an ancient phenomenon in kinetoplastids.     

Interaction with host factors exacerbates Trypanosoma cruzi cell invasion capacity upon oral infection

Outbreaks of severe acute Chagas’ disease acquired by oral infection, leading to death in some cases, have occurred in recent years. Using the mouse model, we investigated the basis of such virulence by analyzing a Trypanosoma cruzi isolate, SC, from a patient with severe acute clinical symptoms, who was infected by oral route. It has previously been shown that, upon oral inoculation into mice, T. cruzi metacyclic trypomastigotes invade the gastric mucosal epithelium by engaging the stage-specific surface glycoprotein gp82, whereas the surface molecule gp90 functions as a down-modulator of cell invasion. We found that, when orally inoculated into mice, metacyclic forms of the SC isolate, which express high levels of gp90, produced high parasitemias and high mortality, in sharp contrast with the reduced infectivity in vitro. Upon recovery from the mouse stomach 1 h after oral inoculation, the gp90 molecule of the parasites was completely degraded, and their entry into HeLa cells, as well as into Caco-2 cells, was increased. The gp82 molecule was more resistant to digestive action of the gastric juice. Host cell invasion of SC isolate metacyclic trypomastigotes was augmented in the presence of gastric mucin. No alteration in infectivity was observed in T. cruzi strains CL and G which were used as references and which express gp90 molecules resistant to degradation by gastric juice. Taken together, our findings suggest that the exacerbation of T. cruzi infectivity, such as observed upon interaction of the SC isolate with the mouse stomach components, may be responsible for the severity of acute Chagas’ disease that has been reported in outbreaks of oral T. cruzi infection.     

Evaluation of the trypanocidal and leishmanicidal in vitro activity of the crude hydroalcoholic extract of Pfaffia glomerata (Amarathanceae) roots

Three different concentrations (1, 10 and 50 μg/ml) of lyophilized hydroalcoholic crude extract of Pfaffia glomerata roots were assayed in vitro against strains of Trypanosoma cruzi (Y) and Leishmania braziliensis. It was observed that P. glomerata hydroalcoholic extract was relatively active within the tested concentrations for L. (V.) braziliensis, but inactive against T. cruzi. Despite the fact that both protozoans belong to the Trypanosomatidae family, we suggest that the difference observed for activity should be related to the biological differences between the two parasite species.     

Trypanosoma cruzi infection by oral route: how the interplay between parasite and host components modulates infectivity

Trypanosoma cruzi infection by oral route constitutes the most important mode of transmission in some geographical regions, as illustrated by reports on microepidemics and outbreaks of acute Chagas' disease acquired by ingestion of food contaminated with parasites from triatomine insects. In the mouse model, T. cruzi metacyclic trypomastigotes invade the gastric mucosal epithelium, a unique portal of entry for systemic infection. High efficiency of metacyclic forms in establishing infection by oral route is associated with expression of gp82, a stage-specific surface molecule that binds to gastric mucin and to epithelial cells. Gp82 promotes parasite entry by triggering the signaling cascades leading to intracellular Ca²⁺ mobilization. T. cruzi strains deficient in gp82 can effectively invade cells in vitro, by engaging the Ca²⁺ signal-inducing surface glycoprotein gp30. However, they are poorly infective in mice by oral route because gp30 has low affinity for gastric mucin. Metacyclic forms also express gp90, a stage-specific surface glycoprotein that binds to host cells and acts as a negative regulator of invasion. T. cruzi strains expressing gp90 at high levels, in addition to gp82 and gp30, are all poor cell invaders in vitro. Notwithstanding, their infectivity by oral route may vary because, unlike gp82 and gp30, which resist degradation by pepsin in the gastric milieu, the gp90 isoforms of different strains have varying susceptibility to peptic digestion. For instance, in a T. cruzi isolate, derived from an acute case of Chagas' disease acquired by oral route, gp90 is extensively degraded by gastric juice in the mouse stomach and this renders the parasite highly invasive towards target cells. If such an exacerbation of infectivity occurs in humans, it may be responsible for the severity of the disease reported in outbreaks of oral infection.     

Synthesis and in vitro evaluation of leishmanicidal and trypanocidal activities of N-quinolin-8-yl-arylsulfonamides

In the present paper 12 N-quinolin-8-yl-arylsulfonamides synthesized by coupling 8-aminoquinolines with various arylsulfonylchlorides were assayed in vitro against Leishmania amazonensis, Leishmania chagasi and Trypanosoma cruzi strains. This series of new compounds were found to be selective for Leishmania spp. promastigote and amastigote forms. The most active compound was the N-(8-quinolyl)-3,5-difluoro-benzenesulfonamide 10 with an IC₅₀ against L. amazonensis and L. chagasi of 2.12 and 0.45 μM, respectively. The less cytotoxic biphenyl derivative 7 was very effective against intracellular L. amazonensis with a reduction of macrophage cell infection of 82.1% at 25 μM. In addition, a copper complex 17 of an inactive ligand was readily synthesized and showed high leishmanicidal and trypanocidal activity against both extra and intracellular forms.     

The evolutionary history of kinetoplastids and their kinetoplasts

Despite extensive phylogenetic analysis of small subunit ribosomal RNA (SSUrRNA) genes, the deep-level relationships among kinetoplastids remain poorly understood, limiting our grasp of their evolutionary history, especially the origins of their bizarre mitochondrial genome organizations. In this study we examine the SSUrRNA data in the light of a new marker--cytoplasmic heat shock protein 90 (hsp90) sequences. Our phylogenetic analyses divide kinetoplastids into four main clades. Clades 1-3 include the various bodonid kinetoplastids. Trypanosomatids comprise the fourth clade. SSUrRNA analyses give vastly different and poorly supported positions for the root of the kinetoplastid tree, depending on the out-group and analysis method. This is probably due to the extraordinary length of the branch between kinetoplastids and any out-group. In contrast, almost all hsp90 analyses place the root between clade 1 (including Dimastigella, Rhynchomonas, several Bodo spp., and probably Rhynchobodo) and all other kinetoplastids. Maximum likelihood and maximum likelihood distance analyses of hsp90 protein and second codon-position nucleotides place trypanosomatids adjacent to Bodo saltans and Bodo cf. uncinatus (clade 3), as (weakly) do SSUrRNA analyses. Hsp90 first codon- plus second codon-position nucleotide analyses return a slightly different topology. We show that this may be an artifact caused, in part, by the different evolutionary behavior of first- and second-codon positions. This study provides the most robust evidence to date that trypanosomatids are descended from within bodonids and that B. saltans is a close relative of trypanosomatids. A total reevaluation of the high-level systematics within kinetoplastids is needed. We confirm that the interlocking network organization of kinetoplast DNA seen in trypanosomatids is a derived condition within kinetoplastids but suggest that open-conformation minicircles may have arisen early in kinetoplastid evolution. Further understanding of the evolution of kinetoplast structure and RNA editing is hampered by a paucity of data from basal (i.e., clade 1) bodonids.     

Synthesis and biological activivity of juvenele hormone analogues (JHA) for Trypanosoma cruzi

Several derivatives of geraniol, geranylacetone, and farnesol bearing carbonate and thiolcarbonate functional groups as well as several derivatives of 4-phenoxyphenol were synthesized and tested for their respective biological activity as growth inhibitors for Trypanosoma cruzi, and for inhibition of tritium-labelled thymidine incorporation in T. cruzi cells. The results indicated that some JHA showed important activity against the development of the cells.     

RNA editing and mitochondrial genomic organization in the cryptobiid kinetoplastid protozoan Trypanoplasma borreli.

The bodonids and cryptobiids represent an early diverged sister group to the trypanosomatids among the kinetoplastid protozoa. The trypanosome type of uridine insertion-deletion RNA editing was found to occur in the cryptobiid fish parasite Trypanoplasma borreli. A pan-edited ribosomal protein, S12, and a novel 3'- and 5'-edited cytochrome b, in addition to an unedited cytochrome oxidase III gene and an apparently unedited 12S rRNA gene, were found in a 6-kb fragment of the 80- to 90-kb mitochondrial genome. The gene order differs from that in trypanosomatids, as does the organization of putative guide RNA genes; guide RNA-like molecules are transcribed from tandemly repeated 1-kb sequences organized in 200- and 170-kb molecules instead of minicircles. The presence of pan-editing in this lineage is consistent with an ancient evolutionary origin of this process.     

The circadian system of Trypanosoma cruzi-infected mice

The effects of Chagas disease on the mammalian circadian system were studied in Trypanosoma cruzi-infected C57-Bl6J mice. Animals were inoculated with CAI or RA strains of T. cruzi or vehicle, parasitism confirmed by blood specimen visualization and locomotor activity rhythms analyzed by wheel-running recording. RA-strain infected mice exhibited significantly decreased amplitude of circadian rhythms, both under light-dark and constant dark conditions, probably due to motor deficiencies. CAI-treated animals showed normal locomotor activity rhythms. However, in these mice, reentrainment to a 6h phase shift of the LD cycle took significantly longer than controls, and application of 15min light pulses in DD produced smaller phase delays of the rhythms. All groups exhibited light-induced Fos expression in the suprachiasmatic nuclei. We conclude that the main effect of T. cruzi infection on the circadian system is an impairment of the motor output from the clock toward controlled rhythms, together with an effect on circadian visual sensitivity.     

Plastome phylogenomic analysis of Torreya (Taxaceae)

Torreya Arn., a small genus of Taxaceae, consists of six species occurring in North America and eastern Asia. Several phylogenetic studies have previously been undertaken to reveal relationships within this genus, although only a few DNA segments or species were used. In the present study, we sequenced five Torreya plastomes and combined these with two existing plastomes from the genus to investigate plastome evolution and phylogenetic relationships within Torreya. All sequenced Torreya plastomes shared the same complement of 82 protein‐coding genes, 4 ribosomal RNA genes, and 31 transfer RNA genes. Phylogenetic inference using a maximum likelihood framework consisted of an 82‐gene, 17‐taxon dataset, including all species of Torreya, resolved Torreya as a monophyletic clade. Strongly supported relationships within the genus include the position of the early diverging T. jackii Chun, the two sister pairs T. fargesii Franch.–T. nucifera (L.) Siebold & Zucc. and T. grandis Fortune ex Lindl.–T. californica Torr., and the monophyly of the clade including T. fargesii var. yunnanensis, T. fargesii, and T. nucifera. In addition to the inference of species relationships, divergence time estimation and biogeographical analysis were carried out. The diversification of Torreya was estimated to be approximately 8.9 Ma. Ancestral state reconstruction of the geographical area suggested China/eastern North America as the most likely ancestral region for the six extant Torreya species.     

Trypanosoma cruzi: skin-penetration kinetics of vector-derived metacyclic trypomastigotes

In order to investigate the natural route of infection of nude and normal BALB/c mice with Trypanosoma cruzi via the skin, a drop of vector faeces/urine containing metacyclic trypomastigotes was placed onto the puncture site of a bite from Triatoma infestans. The periods of exposure, i.e. until removal of flagellates from the skin, and the time elapsed until surgical removal of the skin around the puncture were varied. After 15 min of exposure, T. cruzi developed in all nude mice without surgery, and in four of 10 mice if the puncture region of the skin was removed directly after exposure. In a shaved puncture region, 5 min of exposure were sufficient to infect all normal BALB/c mice without surgery and one of four mice with direct removal of the puncture region. Longer periods of exposure or time until removal of the skin only sometimes resulted in higher infection rates. Prepatent periods and the development of parasitaemia varied irrespective of the period of exposure or the period until skin removal at the puncture site. The importance of these findings is that they clearly prove that T. cruzi can rapidly invade the host via the puncture site of the bite of the vector and that at least some parasites are immediately transported away from this site.