Analysis of genetic diversity of Trypanosoma cruzi: an application of riboprinting and gradient gel electrophoresis methods.

Analysis of restriction fragment length polymorphism (RFLP) profiles derived from digestion of polymerase chain reaction (PCR) products of the ribosomal 18S from Trypanosoma cruzi yields a typical 'riboprint' profile that can vary intraspecifically. A selection of 21 stocks of T. cruzi and three outgroup taxa: T. rangeli, T. conorhini and Leishmania braziliensis were analysed by riboprinting to assess divergence within and between taxa. T. rangeli, T. conorhini and L. braziliensis could be easily differentiated from each other and from T. cruzi. Phenetic analysis of PCR-RFLP profiles indicated that, with one or two exceptions, stocks of T. cruzi could be broadly partitioned into two groups that formally corresponded to T. cruzi I and T. cruzi II respectively. To test if ribosomal 18S sequences were homogeneous within each taxon, gradient gel electrophoresis methods were employed utilising either chemical or temperature gradients. Upon interpretation of the melting profiles of riboprints and a section of the 18S independently amplified by PCR, there would appear to be at least two divergent 18S types present within T. cruzi. Heterogeneity within copies of the ribosomal 18S within a single genome has therefore been demonstrated and interestingly, this dimorphic arrangement was also present in the outgroup taxa. Presumably the ancestral duplicative event that led to the divergent 18S types preceded that of speciation within this group. These divergent 18S paralogues may have, or had, different functional pressures or rates of molecular evolution. Whether or not these divergent types are equally transcriptionally active throughout the life cycle, remain to be assessed.

Genetic diversity and genetic exchange in Trypanosoma cruzi: dual drug-resistant progeny from episomal transformants

Extensive characterisation of Trypanosoma cruzi by isoenzyme phenotypes has separated the species into three principal zymodeme groups, Z1, Z2 and Z3, and into many individual zymodemes. There is marked diversity within Z2. A strong correlation has been demonstrated between the strain clusters determined by isoenzymes and those obtained using random amplified polymorphic DNA (RAPD) profiles. Polymorphisms in ribosomal RNA genes, in mini-exon genes, and microsatellite fingerprinting indicate the presence of at least two principal T. cruzi genetic lineages. Lineage 1 appears to correspond with Z2 and lineage 2 with Z1. Z1 (lineage 2) is associated with Didelphis. Z2 (lineage 1) may be associated with a primate host. Departures from Hardy-Weinberg equilibrium and linkage disequilibrium indicate that propagation of T. cruzi is predominantly clonal. Nevertheless, two studies show putative homozygotes and heterozygotes circulating sympatrically: the allozyme frequencies for phosphoglucomutase, and hybrid RAPD profiles suggest that genetic exchange may be a current phenomenon in some T. cruzi transmission cycles. We were able to isolate dual drug-resistant T. cruzi biological clones following copassage of putative parents carrying single episomal drug-resistant markers. A multiplex PCR confirmed that dual drug-resistant clones carried both episomal plasmids. Preliminary karyotype analysis suggests that recombination may not be confined to the extranuclear genome.

Genetic diversity and genetic exchange in Trypanosoma cruzi: dual drug-resistant "progeny" from episomal transformants.

Extensive characterisation of Trypanosoma cruzi by isoenzyme phenotypes has separated the species into three principal zymodeme groups, Z1, Z2 and Z3, and into many individual zymodemes. There is marked diversity within Z2. A strong correlation has been demonstrated between the strain clusters determined by isoenzymes and those obtained using random amplified polymorphic DNA (RAPD) profiles. Polymorphisms in ribosomal RNA genes, in mini-exon genes, and microsatellite fingerprinting indicate the presence of at least two principal T. cruzi genetic lineages. Lineage 1 appears to correspond with Z2 and lineage 2 with Z1. Z1 (lineage 2) is associated with Didelphis. Z2 (lineage 1) may be associated with a primate host. Departures from Hardy-Weinberg equilibrium and linkage disequilibrium indicate that propagation of T. cruzi is predominantly clonal. Nevertheless, two studies show putative homozygotes and heterozygotes circulating sympatrically: the allozyme frequencies for phosphoglucomutase, and hybrid RAPD profiles suggest that genetic exchange may be a current phenomenon in some T. cruzi transmission cycles. We were able to isolate dual drug-resistant T. cruzi biological clones following copassage of putative parents carrying single episomal drug-resistant markers. A multiplex PCR confirmed that dual drug-resistant clones carried both episomal plasmids. Preliminary karyotype analysis suggests that recombination may not be confined to the extranuclear genome.

Random amplification of polymorphic DNA as a tool for taxonomic studies of triatomine bugs (Hemiptera: Reduviidae).

Eleven of 27 decameric primers were found to be suitable for random amplification of polymorphic DNA (RAPD) from triatomine bugs on the basis that they produced discrete profiles and distinguished among Panstrongylus megistus (Burmeister), Rhodnius prolixus Stål, and Triatoma infestans (Klug). The legs, or single leg segments, of individual bugs were used as the source of DNA so that the taxonomic value of the bug was conserved. Within the scope of the specimens studied, RAPD profiles allowed assignment to species even when bugs were kept dry for up to 12 mo. Profiles for individuals within a species were not identical. RAPD profiles, with the specimens tested, distinguished among species of 3 pairs considered to be morphologically similar and closely related, namely, Rhodnius ecuadorensis Lent & León and Rhodnius pictipes Stål; Rhodnius nasutus Stål, and Rhodnius neglectus Lent; Rhodnius prolixus Stål and Rhodnius robustus Larrousse. RAPD data conformed with the perceived affinities among these species. RAPD polymorphisms were seen with T. infestans from 3 different localities, but none of the polymorphisms was confined to 1 source. RAPD provided a molecular basis to reassess taxonomic relationships within the Triatomine subfamily. The accurate distinction of triatomine species and of intraspecific bug populations may contribute to elimination of vector-borne Chagas disease from the Americas.

Genetic exchange as a possible source of genomic diversity in sylvatic populations of Trypanosoma cruzi.

Thirty six stocks of Trypanosoma cruzi isolated from sylvatic mammals (32 Didelphis marsupialis and one Philander opossum) and triatomine bugs (Rhodnius robustus and one unidentified bug) in the Amazonian forest by Carajas, Brazil were characterized by isoenzyme and random amplified polymorphic DNA (RAPD) analysis as belonging to principal zymodeme '1 (Z1). Two different homozygous phenotypes and the corresponding heterozygous phenotype were found for phosphoglucomutase with an observed frequency almost identical with that predicted by the theoretical Hardy-Weinberg distribution. Parental and hybrid profiles were also suggested by RAPD analysis, which allowed exclusion of mixed parental strains from the hybrids: isoenzyme and RAPD profiles of biological clones were also indistinguishable from those of uncloned stocks. Trypanosoma cruzi stocks from widely separated geographic origins in Central and South America gave similar RAPD profiles that allowed them to be recognized as being Z1. These results indicate that genetic exchange could contribute to the generation of genetic diversity during the sylvatic cycle of T. cruzi, and this may have epidemiologic and taxonomic implications.

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

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 used to confirm the widespread dissemination of amastigotes in apparently normal skin of dogs with canine visceral leishmaniasis. When Lutzomyia longipalpis were fed on abnormal skin of five naturally infected dogs 57 of 163 (35%) flies became infected: four of 65 flies (6%) became infected when fed on apparently normal skin. The bite of a single sandfly that had fed seven days previously on 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 infective bite (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%) 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.

Visceral leishmaniasis in Teresina, n. e. Brazil: towards a DNA probe kit and its adaptation to processing blood-contaminated samples.

The Lmet2 chemiluminescent DNA probe is a valuable tool for identifying parasites of the Leishmania donovani -complex in sand flies, dogs and human samples. Recent blood meals in sand flies or blood contamination of tissue samples inhibited probe sensitivity, whether radiolabelled or chemiluminescent detection systems were used. Treatment of membranes with protease before hybridisation restored positive signal. Alternatively samples could be lysed with protease and applied to membranes with a vacuum blotting apparatus. The Lmet2 protocol provides the basis for a DNA probe kit that is adaptable for use with a wide range of other probes.