A new consensus for Trypanosoma cruzi intraspecific nomenclature: second revision meeting recommends TcI to TcVI.

In an effort to unify the nomenclature of Trypanosoma cruzi, the causative agent of Chagas disease, an updated system was agreed upon at the Second Satellite Meeting. A consensus was reached that T. cruzi strains should be referred to by six discrete typing units (T. cruzi I-VI). The goal of a unified nomenclature is to improve communication within the scientific community involved in T. cruzi research. The justification and implications will be presented in a subsequent detailed report.

Trypanosomatid biodiversity in Costa Rica: genotyping of parasites from Heteroptera using the spliced leader RNA gene.

The biodiversity of insect trypanosomes is largely unknown, resulting in significant gaps in the understanding of pathogen evolution. A culture-independent preliminary survey of trypanosomatid fauna was conducted for the parasites of Heteroptera (Hemiptera) from several localities in Costa Rica. Trypanosomatid infections were detected by light microscopy of smeared gut contents. Out of 257 insects representing 6 families, infections were found in 62 cases; cultures were obtained for 29 new isolates. Gut material from infected hosts was preserved in the field using an SDS-EDTA buffer solution for subsequent DNA extraction in the laboratory. PCR amplification of the trypanosomatid-specific spliced leader (SL) RNA gene repeats was successful for 60 field samples. Eighteen distinct SL RNA typing units were identified in a set of 28 samples analysed in detail. Cluster analysis indicated that these typing units were unique and thus could represent new species and, in some cases, new genera. This study reveals only a minor fraction of the trypanosomatid biodiversity, which is anticipated to be high.

A new species of trypanosome, Trypanosoma desterrensis sp. n., isolated from South American bats.

Trypanosomes isolated from South American bats include the human pathogen Trypanosoma cruzi. Other Trypanosoma spp. that have been found exclusively in bats are not well characterized at the DNA sequence level and we have therefore used the SL RNA gene to differentiate and characterize kinetoplastids isolated from bats in South America. A Trypanosoma sp. isolated from hats in southern Brazil was compared with the geographically diverse isolates T. cruzi marinkellei, T. vespertilionis, and T. dionisii. Analysis of the SL RNA gene repeats revealed size and sequence variability among these bat trypanosomes. We have developed hybridization probes to separate these bat isolates and have analysed the DNA sequence data to estimate their relatedness. A new species, Trypanosoma desterrensis sp. n., is proposed, for which a 5S rRNA gene was also found within the SL RNA repeat.

The genetic diversity of Brazilian Trypanosoma cruzi isolates and the phylogenetic positioning of zymodeme 3, based on the internal transcribed spacer of the ribosomal gene.

There is a high level of genetic diversity among the clinical isolates of the taxon Trypanosoma cruzi. The results of recent studies on well defined gene loci have revealed that T. cruzi can be divided into two major phylogenetic lineages, designated T. cruzi I and T. cruzi II. Further intra-lineage polymorphisms were explored, in clinical isolates from five representative regions of Brazil, using the internal transcribed spacers that flank the 5.8S ribosomal RNA gene. These regions were amplified in a PCR and the products were digested with six restriction enzymes before being subjected to RFLP analysis in polyacrylamide gels. The results were used to construct a complex evolutionary tree, in which the isolates were clustered into two major phylogenetic lineages with a low level of similarity. To investigate the evolutionary relationships between the isolates, the PCR products were cloned and sequenced. The results of the subsequent phylogenetic analysis confirmed the presence of the two major groups of T. cruzi and showed that zymodeme 3, a third iso-enzymatic sub-group, is closer to T. cruzi I than to T. cruzi II.

The spliced leader RNA gene array in phloem-restricted plant trypanosomatids (Phytomonas) partitions into two major groupings: epidemiological implications.

The arbitrary genus Phytomonas includes a biologically diverse group of kinetoplastids that live in a wide variety of plant environments. To understand better the subdivisions within the phytomonads and the variability within groups, the exon, intron and non-transcribed spacer sequences of the spliced leader RNA gene were compared among isolates of the phloem-restricted members. A total of 29 isolates associated with disease in coconut, oil palm and red ginger (Alpinia purpurata, Zingibreaceae) were examined, all originating from plantations in South America and the Caribbean over a 12-year period. Analysis of non-transcribed spacer sequences revealed 2 main groups, I and II; group II could be further subdivided into 2 subgroups, IIa and Ilb. Three classes of spliced leader (SL) RNA gene were seen, with SLI corresponding to group I, SLIIa to group lIa, and SLIIb to group IIb. Two isolates showed some characteristics of both major groups. Group-specific oligonucleotide probes for hybridization studies were tested, and a multiplex amplification scheme was devised to allow direct differentiation between the 2 major groups of phloem-restricted Phytomonas. These results provide tools for diagnostic and molecular epidemiology of plant trypanosomes that are pathogenic for commercially important flowers and palms.

Mini-exon gene sequences define six groups within the genus Crithidia.

To develop molecular markers for lower trypanosmatids, we have examined the mini-exon gene repeats of 17 isolates that were classified as Crithidia by traditional methods. Representative repeats were amplified by polymerase chain reaction and the amplification products were cloned and used as hybridization probes against genomic DNA. Six hybridization groups of Crithidia were defined on the basis of the DNA blotting experiments. The three endosymbiont-bearing species (C. deanei, C. desouzai and C. oncopelti) and C. acanthocephali each belonged to single-member hybridization groups, while the C. fasciculata group contained additional named and undesignated species. The Crithidia lucilae thermophila probe hybridized to multiple undesignated isolates. The DNA sequence of the cloned products revealed that the specificity of the hybridization probes was due to substantial differences in the intron and the nontranscribed spacer regions. These data indicate substantial heterogeneity within the mini-exon gene locus of the taxon Crithidia.