The taxonomic position and evolutionary relationships of Trypanosoma rangeli.

This paper presents a re-evaluation of the taxonomic position and evolutionary relationships of Trypanosoma (Herpetosoma) rangeli based on the phylogenetic analysis of ssrRNA sequences of 64 Trypanosoma species and comparison of mini-exon sequences. All five isolates of T. rangeli grouped together in a clade containing Trypanosoma (Schizotrypanum) cruzi and a range of closely related trypanosome species from bats [Trypanosoma (Schizotrypanum) dionisii, Trypanosoma (Schizotrypanum) vespertilionis] and other South American mammals [Trypanosoma (Herpetosoma) leeuwenhoeki, Trypanosoma (Megatrypanum) minasense, Trypanosoma (Megatrypanum) conorhini] and an as yet unidentified species of trypanosome from an Australian kangaroo. Significantly T. rangeli failed to group with (a) species of subgenus Herpetosoma, other than those which are probably synonyms of T. rangeli, or (b) species transmitted via the salivarian route, although either of these outcomes would have been more consistent with the current taxonomic and biological status of T. rangeli. We propose that use of the names Herpetosoma and Megatrypanum should be discontinued, since these subgenera are clearly polyphyletic and lack evolutionary and taxonomic relevance. We hypothesise that T. rangeli and T. cruzi represent a group of mammalian trypanosomes which completed their early evolution and diversification in South America.     

Trypanosoma equiperdum: master of disguise or historical mistake?

After 100 years of research, only a small number of laboratory strains of Trypanosoma equiperdum exists, and the history of most of the strains is unknown. No definitive diagnosis of dourine can be made at the serological or molecular level. Only clinical signs are pathognomonic and international screening relies on an outdated cross-reactive serological test (the complement-fixation test) from 1915, resulting in serious consequences at the practical level. Despite many characterization attempts, no clear picture has emerged of the position of T. equiperdum within the Trypanozoon group. In this article, we highlight the controversies that exist regarding T. equiperdum, and the overlap that occurs with Trypanosoma evansi and Trypanosoma brucei brucei. By revisiting the published data, from the early decades of discovery to the recent serological- and molecular-characterization studies, a new hypothesis arises in which T. equiperdum no longer exists as a separate species and in which current strains can be divided into T. evansi (the historical mistake) and Trypanosoma brucei equiperdum (the master of disguise). Hence, dourine is a disease caused by specific host immune responses to a T. b. equiperdum or T. evansi infection.     

A comparison of Glossina morsitans centralis originating from Tanzania and Zambia, with respect to vectorial competence for pathogenic Trypanosoma species, genetic variation and inter-colony fertility

wo laboratory strains of Glossina morsitans centralis originating from different fly-belts (one from Singida, in Tanzania, and the other from Mumbwa, in Zambia) were compared with respect to vectorial competence for pathogenic Trypanosoma species, genetic variation and inter-colony fertility. The vectorial competence of G.m.centralis of Tanzanian origin for Trypanosoma vivax and T.congolense is similar to, whereas for T.brucei brucei it is lower than the colony of Zambian origin. Nevertheless, these two laboratory strains of G.m.centralis showed levels of susceptibility to the three pathogenic Trypanosoma species which were much greater than previously observed in laboratory colonies of other Glossina species. Electrophoresis of fifteen enzymes revealed that the two colonies differ significantly in allele frequencies at only three loci that are relatively close together on one of the autosomes. Hybridization experiments revealed that G.m.centralis from the two fly-belts are consubspecific.     

Randomly Amplified Polymorphic DNA (RAPD) and Isoenzyme Analysis of Trypanosoma rangeli Strains

ABSTRACT. Sixteen Trypanosoma rangeli strains were compared by isoenzyme and randomly amplified polymorphic DNA (RAPD) analysis. Eight strains were isolated from either Rhodnius prolixus or Homo sapiens from Honduras, Colombia and Venezuela. Another eight strains were isolated from either Panstrongylus megistus or the rodent Echimys dasythrix from the State of Santa Catarina, southern Brazil. All six T. rangeli strains isolated from P. megistus were co-infections with Trypanosoma cruzi , demonstrating an overlap of the sylvatic cycles of these parasites and that the accurate identification of species is of utmost importance. Both isoenzyme and RAPD analysis revealed two distinct groups of T. rangeli strains, one formed by the strains from Santa Catarina and the other, by the strains from Honduras, Colombia and Venezuela. With the five enzymes used, all the strains from Santa Catarina had identical profiles which overlapped with those of the other regions only in the pattern obtained with malic enzyme. Analysis of 138 RAPD bands by means of an unweighted pair group method analysis (UPGMA) phenogram using the Dice similarity coefficient allowed the separation of the two groups based on their divergence at a lower level of similarity than the phenon line. We show that the identification of T. cruzi and T. rangeli in naturally mixed infections is readily achieved by either RAPD or isoenzyme analysis.     

Selective pressure can influence the resistance of Trypanosoma congolense to normal human serum

Resistance and sensitivity to normal human serum (NHS) of Trypanosoma congolense, a parasite believed to cause disease in animals only, were investigated in vivo as well as in vitro. Our results indicate that like Trypanosoma brucei, T. congolense can be grouped into three different phenotypes according to its resistance to NHS. Some strains are completely resistant to NHS, like Trypanosoma brucei gambiense and the resistant form of Trypanosoma brucei rhodesiense. Other strains show a very low degree of resistance comparable to the sensitive form of T. b. rhodesiense, and some are completely sensitive to NHS. Continuous passaging in mice in the presence or absence of NHS shows that the resistance and sensitivity of T. congolense can be reversed like in T. b. rhodesiense. Our data suggest that T. congolense might be able to infect man in regions where animals may serve as reservoirs for the infection.     

Differential susceptibility of triatomines of the genus Rhodnius to Trypanosoma rangeli strains from different geographical origins

The susceptibility of four Rhodnius species to different Trypanosoma rangeli strains was evaluated using both intracoelomic inoculation and oral infection. Rhodnius prolixus, Rhodnius domesticus, Rhodnius neglectus and Rhodnius nasutus were infected with Trypanosoma rangeli Macias (Venezuela), Choachi (Colombia) and SC-58 (Brazil) strains, revealing distinct haemolymph and salivary glands infection rates. The obtained infection rates were revealed to be dependent on the method of infection and the triatomine species. Our results suggest the existence of a high adaptation between the strain and the local vector.     

Trypanosoma cruzi: Antigenic invariance of the cell surface glycoprotein

The cell surface antigens of Trypanosoma cruzi have been studied for evidence of antigenic variation. The majority of the cell surface antigens found on epimastigotes were also present on trypomastigote and amastigote forms. Serum absorption studies and peptide mapping of the major cell surface glycoprotein from a series of clones and strains of Trypanosoma cruzi failed to find evidence of antigenic variation. Differences found between geographically distinct strains of Trypanosoma cruzi were minor and not associated with the major glycoprotein. Components present in normal mouse serum were capable of binding to the surface of Trypanosoma cruzi and these components could interfere in subsequent radioimmune assays, particularly with bloodstream derived trypomastigotes.     

Identification of a novel large extrachromosomal DNA (LED) in the Trypanosomatidae

We have identified a novel 75 kbp large extrachromosomal DNA (LED) which is stably maintained during developmental conversion of Trypanosoma cruzi. It has a covalently closed circular conformation and is not derived from the kinetoplast network. In all T. cruzi strains analysed, LED contains 18S rRNA and spliced leader (sl) sequences. LED from the T. cruzi Y strain contains a minimum of 15 copies of the sl repeat arrayed in a head-to-tail configuration and 50 copies of a 196 bp repeat. LED is also present in Trypanosoma dionisii (subgenus Schizotrypanosoma) and in other members of the family Trypanosomatidae. LED from different T. cruzi strains and from other members of the Trypanosomatidae differ in their content of large ribosomal subunit rRNA sequences and the 196 bp repeat. The presence of LED in four evolutionarily distant trypanosomatid species suggests that it plays an important role in the biology of these parasites.     

Analyses of surface membrane carbohydrates in parasitic flagellates of the order kinetoplastida using lectins.

Crithidia fasciculata, Leishmania donovani, Leishmania major, Leishmania mexicana amazonensis, Leishmania tropica, Leishmania tarentolae, Trypanosoma sp. from Formosan bats (Tb), Trypanosoma lewisi, Trypanosoma musculi, and different strains of Trypanosoma cruzi (Tc) were cultivated at 27 degrees C in a liquid culture medium. Flagellates harvested from log phase culture were analyzed for their lectin agglutinating characteristics with concanavalin A (Con A), Peanut agglutinin, Ricinus communis agglutinin 120, soybean agglutinin (SBA), Ulex europeus agglutinin (UEA) and wheat germ agglutinin (WGA). Results indicated that all these flagellates might have D-galactose and methyl- alpha-D-manopyranoside on their surface. The presence of L-Fucose, which complexes specifically with UEA, could not be demonstrated on the surface of these flagellates. Results from quantitative comparison of surface molecules of Tb and the Tulahuen strain of Tc suggested that Tb may have more WGA-binding molecules while Tc may have more ConA-binding molecules. Pretreatment of the flagellates with 0.05% trypsin at 37 degrees C for 30 minutes caused some reduction of agglutination titers. Cell agglutination with lectins was completely inhibited or reversed in the presence of the specific lectin-binding monosaccharides.     

Alternative oxidase (AOX) genes of African trypanosomes: phylogeny and evolution of AOX and plastid terminal oxidase families

To clarify evolution and phylogenetic relationships of trypanosome alternative oxidase (AOX) molecules, AOX genes (cDNAs) of the African trypanosomes, Trypanosoma congolense and Trypanosoma evansi, were cloned by PCR. Both AOXs possess conserved consensus motifs (-E-, -EXXH-). The putative amino acid sequence of the AOX of T. evansi was exactly the same as that of T. brucei. A protein phylogeny of trypanosome AOXs revealed that three genetically and pathogenically distinct strains of T. congolense are closely related to each other. When all known AOX sequences collected from current databases were analyzed, the common ancestor of these three Trypanosoma species shared a sister-group position to T. brucei/T. evansi. Monophyly of Trypanosoma spp. was clearly supported (100% bootstrap value) with Trypanosoma vivax placed at the most basal position of the Trypanosoma clade. Monophyly of other eukaryotic lineages, terrestrial plants + red algae, Metazoa, diatoms, Alveolata, oomycetes, green algae, and Fungi, was reconstructed in the best AOX tree obtained from maximum likelihood analysis, although some of these clades were not strongly supported. The terrestrial plants + red algae clade showed the closest affinity with an alpha-proteobacterium, Novosphingobium aromaticivorans, and the common ancestor of these lineages, was separated from other eukaryotes. Although the root of the AOX subtree was not clearly determined, subsequent phylogenetic analysis of the composite tree for AOX and plastid terminal oxidase (PTOX) demonstrated that PTOX and related cyanobacterial sequences are of a monophyletic origin and their common ancestor is linked to AOX sequences.     

In vitro growth kinetics of two Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae) clones in myocardial cells from rodents of different susceptibility

In vitro growth kinetics of two Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae) clones in myocardial cells from rodents of different susceptibility. Two Trypanosoma cruzi isolates, TCR-4 from Costa Rica and UES-1 from El Salvador, were studied in vitro to compare their infectivity or resistance and intracellular replication in myocardial cells in three strains of mice and rats: NGP white mice, C3 H mice and Sprague Dowley rats. Myocardial cells were cultured on coverslips at 37 degrees C in a humid 10% CO2 atmosphere and then infected at a ratio of one tripomastigote per cell. Samples were studied after 24, 72, 96 and 120 h of infection to determine parasite infection capacity and intracellular multiplication. Both parasites had the highest infection capacity in C3 H mice, followed by NGP mice cells with a very low infection rate. Lastly, almost no Trypanosoma cruzi multiplication was observed in Sprague Dowley rats, suggesting a strong natural resistance in this animal to both strains of the parasite. The UES-1 isolate presented higher multiplication and greater invasion than the TCR-4 strain, showing greater virulence of UES-1 in heart cells, at least in vitro.     

Chromosomal localization of the KMP-11 genes in the KP1(+) and KP1(-) strains of Trypanosoma rangeli

Genes encoding for the KMP-11 protein were localized on the chromosomes of Trypanosoma rangeli. These genes were located in two chromosomes of 3,100 and 3,400 kb in the KP1(-) strain whereas in the KP1(+) H14 and Choachí strains, the genes are located in a chromosome of 1,600 kb. The Choachí strain presents an additional band of 1,400 kb. In the Shubacbarina and Munanta strains of Trypanosoma cruzi, the KMP-11 genes are located on a chromosomal band of 1,490 kb. Therefore, the chromosomal localization of the KMP-11 genes presents a potential tool to differentiate among these parasites.     

The kinetoplast DNA of Trypanosoma equiperdum

We have analyzed the kinetoplast DNA for Trypanosoma equiperdum (American Type Culture Collection 30019) and two dyskinetoplastic strains derived from it. The DNA networks from the kinetoplastic strain are made up of catenated mini-circles and maxi-circles, like the networks from the closely-related Trypanosoma brucei. The mini-circles of T. equiperdum lack the pronounced sequence heterogeneity of T. brucei mini-circles, as shown by the fragment distribution of restriction digests and by the predominance of well-matched duplexes in electron micrographs of renatured DNA. The electrophoretic analysis of kinetoplast DNA digested with various restriction endonucleases shows the maxi-circle of T. equiperdum to consist of circular DNA molecules of 8.4 x 10(6) daltons, without size or sequence heterogeneity or repetitious segments. A comparison of the sequence by restriction endonuclease fragmentation and hybridization shows extensive sequence homology. The size difference between both maxi-circles is due to the deletion of one continuous segment of 5.10(6) daltons. In the two dyskinetoplastic strains, we cannot detect DNA sequences that hybridize with kinetoplast DNA from T. brucei or from the kinetoplastic strain of T. equiperdum. In one of these strains, a 'low-density' DNA fraction contained a simple sequence DNA, cleaved by restriction endonuclease HindIII into fragments of 180 base-pairs and multimers of this. The relation of this DNA to kinetoplast DNA, if any, is unknown.     

Biological characterization of clones of the Y, CL and MR strains of Trypanosoma cruzi in inbred C3H mice

Ten clones of Trypanosoma cruzi isolated from Y, CL and MR strains were studied. The infectivity of culture forms, parasitemia pattern, polymorphism and mortality were studied in C3H inbred mice. Significant intra-group differences among Y and CL clones were found. MR clones showed higher homogeneity. These data indicate that T. cruzi strains can show different degrees of heterogeneity. It is suggested that conditions used to maintain T. cruzi strains may result in a selective advantage for some subpopulations (clones) after many years of laboratory maintenance.     

Variations in maxi-circle and mini-circle sequences in kinetoplast DNAs from different Trypanosoma brucei strains

We have compared a total of 30 recognition sites for eight restriction endonucleases on the 20-kilobase-pair maxi-circle of kinetoplast DNAs from five different Trypanosoma brucei strains. In addition to three polymorphic sites were have found a 5 kilobase-pair region that is not cleaved by any of the eight enzymes and that varies in size over 1 kilobase pair in the strains analysed. Mini-circles from these five strains, digested with endonuclease TaqI or MboII, yield very complex fragment patterns, showing that extensive mini-circle sequence heterogeneity is a common characteristic of these T. brucei strains. The size distribution of mini-circle fragments in these digests was identical for different clones of the 427 strain, but very different for mini-circles from different strains. These results show that maxi-circle sequence is conserved, whereas mini-circle sequence is not. Restriction digests of maxi-circles could be useful in determining how closely two Trypanosoma strains are related, whereas mini-circle digests can serve as sensitive tags for individual strains.     

Non-specific induction of increased resistance in mice to Trypanosoma congolense and Trypanosoma brucei by immunostimulants.

Administration of the immunostimulants Corynebacterium parvum, Bacillus Calmette-Guérin (BCG) or Bordetella pertussis prior to, or at the same time as, challenge with Trypanosoma congolense significantly increased survival times in mice, both of trypano-susceptible (A/J) and trypano-resistant (C57Bl) strains. The increased survival time was associated with significant alterations in parasitaemia, which included lengthening of the pre-patent period, a delay in the time taken to reach the first peak of parasitaemia and a reduction in the level of parasitaemia. Similar results were obtained when these strains of mice were challenged with Trypanosoma brucei following pre-treatment with C. parvum. Thus, by the use of immunostimulants it was possible to reduce the susceptibility of mice to trypanosomiasis and the hope is that this can also be achieved with domestic livestock.     

Genetic Heterogeneity in Trypanosoma cruzi Strains From Naturally Infected Triatomine Vectors in Northeastern Brazil: Epidemiological Implications

Eighteen Trypanosoma cruzi strains isolated from naturally infected triatomines were studied genetically. The majority of the strains were from Triatoma brasiliensis, the principal vector of Chagas disease in the northeast of Brazil. Multilocus enzyme electrophoresis (MLEE) and randomly amplified polymorphic DNA (RAPD) analyses were used to investigate the genotypic diversity and the spread of the T. cruzi genotypes in different environments. MLEE clearly distinguished two distinct isoenzyme profiles, and RAPD analysis revealed 10 different genotypes circulating in rural areas. The strains could be typed as isoenzyme variants of the T. cruzi principal zymodeme Z1 (T. cruzi I). An effective program of epidemiological vigilance is required to prevent the spread of T. cruzi I strains into human dwellings.     

Relationship between biological behaviour and randomly amplified polymorphic DNA profiles of Trypanosoma cruzi strains

Once known some biological characteristics of six Trypanosoma cruzi strains, randomly amplified polymorphic DNA (RAPD) analysis was made. Cluster analysis by UPGMA (unweighted pair group method analysis) was then applied both to biological parameters and RAPD profiles. Inspection of the UPGMA phenograms indicates identical clusters, so supporting that usefulness of biological parameters to characterization of T. cruzi strains still remains.     

Trypanosoma cruzi from the Paraguayan Chaco: isoenzyme profiles of strains isolated at Makthlawaiya

At Makthlawaiya, in the Paraguayan Chaco, the prevalence of Trypanosoma (Schizotrypanum) cruzi infection among both domestic Triatoma infestans and domestic dogs was 38%, and IgG anti-T. cruzi antibody was detected by the quantitative enzyme-linked immunosorbent assay (ELISA) in 80% (105/133) of human sera. Ninety percent (25/28) of T. cruzi strains isolated from both T. infestans and dogs showed heterozygous isoenzyme profiles for glucose phosphate isomerase, phosphoglucomutase and 6-phosphogluconate dehydrogenase. These strains appeared to be closely related to Bolivian zymodeme 2. Three Paraguayan T. cruzi strains showed homozygous isoenzyme profiles, similar to those of major Brazilian zymodemes. It was concluded that T. cruzi strains with heterozygous isoenzyme profiles predominate in domestic transmission cycles in this highly endemic area of the Paraguayan Chaco.     

Trypanocidal and antimicrobial activities of Moquinia kingii.

A chloroform crude extract (aerial part) and two compounds, apigenin (1) and cynaropicrin (2), isolated from Moquinia kingii were evaluated against Trypanosoma cruzi trypomastigotes in vitro. Antimicrobial activity was also screened using twenty-two strains including gram-positive and gram-negative bacteria and the yeasts Candida albicans and C. tropicalis. The chloroform crude extract, fractions and isolated compounds from M. kingii were active for both activities. The IC50 values for trypanocidal activity obtained for cynaropicrin and apigenin were 93.5 microg/ml and 181 microg/ml, respectively, while the minimum inhibitory concentrations (MICs) varied from 100 microg/ml to 2500 microg/ml, against the strains of bacteria and yeasts evaluated.