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.

On the molecular taxonomy of Trypanosoma cruzi using riboprinting.

In order to investigate the molecular taxonomy within Trypanosoma cruzi, the ribosomal small subunit (18S) gene was amplified by polymerase chain reaction (PCR) from a selection of 21 stocks and 3 outgroup taxa. Amplification products were digested with 10 restriction enzymes; restriction fragments were separated by polyacrylamide gel electrophoresis and profiles were visualized by silver staining. Upon analysis of such riboprint profiles, an estimate of pairwise phenetic distance between stocks of T. cruzi was calculated. Upon principal coordinate analysis of this data matrix, a tendency towards a bi-polar grouping of stocks was observed. These 2 groups were predominantly either zymodeme 1 stocks or zymodeme 2 stocks. The position of zymodeme 3 stocks remained intermediate between the 2 groups but did not form a coherent group by themselves. It would therefore appear premature to warrant division of T. cruzi into 2 discrete taxa or subspecies until the relationships of further zymodeme 3 stocks are elucidated.

Temperature gradient gel electrophoresis (TGGE) analysis of riboprints from Trypanosoma cruzi.

To test the homogeneity of 18S sequences within Trypanosoma cruzi, riboprint profiles were separated by temperature gradient gel electrophoresis (TGGE). Upon interpretation of melting curves of fragments within a riboprint profile, there appeared to be two 18S sequence types within each stock examined. Two similar types were also observed within outgroup taxa Trypanosoma conorhini, Trypanosoma rangeli and Leishmania braziliensis. From DNA hybridization studies, these fragments were shown to have homology to the 18S V1 region. There are therefore two 18S V1 regions, differing in sequence, present in all taxa examined. When only a single 18S sequence is used to represent each taxa for phylogenetic inference, comparisons may be between paralogous and not orthologous copies of this region, such that, inferred relationships may merely reflect a gene history. This seriously questions the current molecular phylogeny of these protozoa using 18S data.

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.

Use of polymerase chain reaction-based single strand conformational polymorphism and denaturing gradient gel electrophoresis methods for detection of sequence variation of ribosomal DNA of Trypanosoma cruzi.

Polymerase chain reaction (PCR) was used to amplify the V1 region of the small sub-unit (18S) ribosomal DNA gene from representative strains of Trypanosoma cruzi. In order to screen for sequence variation, amplification products were subsequently analysed by single strand conformational polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) techniques. SSCP could not detect sequence variation within T. cruzi, although electrophoretic profiles were clearly distinct from both Leishmania donovani and Leishmania braziliensis. DGGE could differentiate strains of T. cruzi and it appears that there are at least 2 18S V1 sequences of this multi-gene family within each strain examined, contrasting with Leishmania spp. where only 1 was identified. This is the first application of PCR-linked SSCP and DGGE analysis for differentiating parasitic protozoa.

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.

The role of dogs in the epidemiology of human visceral leishmaniasis in northern Pakistan.

Infantile visceral leishmaniasis (VL) was first reported from north Pakistan over 3 decades age in the remote valleys of the western Himalayas. These foci were reported as being completely devoid of domestic dogs. The later emergence of sporadic cases of infantile VL in the sub-Himalayan region of the country, where dogs are abundant, enabled us to investigate the prevalence of canine disease and study its relation with disease in humans. A serological survey in dogs by direct agglutination test (DAT) and enzyme-linked immunosorbent assay (ELISA) indicated that 18% (DAT) and 26.6% (ELISA) harboured anti-Leishmania antibodies, with older dogs showing higher prevalence; 10% of the infected dogs had no clinical signs of leishmaniasis. Deoxyribonucleic acid (DNA) probing by 32P-labelled Lmet 2 cDNA probe showed high sensitivity with aspirates obtained from the popliteal lymph nodes of dogs but not with skin snips. Parasites isolated from dogs in these foci were identified as L. infantum by isoenzyme characterization.

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.

Variability of in vitro culture characteristics, including metacyclic trypomastigote production, in different stocks of Trypanosoma congolense.

Six cloned stocks of Trypanosoma congolense, isolated from the same area of Eastern Zambia, were maintained in vitro as insect form cultures producing infective metacyclic trypanosomes. Although the same general culture conditions were applied, different handling regimes were required for optimum growth of each stock. During primary isolation, many differences were found in the culture characteristics of the stocks. The time taken for cytoadherence to occur varied from 14 to 62 days, while the interval between attachment and the appearance of infective metacyclic trypanosomes ranged from 9 to 94 days. There was a 10-fold difference in the numbers of metacyclic forms produced by different stocks. Time in culture appeared to have little effect on the production of metacyclic forms, and it is probable that in vitro characteristics of T. congolense depend on the genetic constitution of individual stocks or clones.

Characterization of Trypanosoma congolense serodemes in stocks isolated from Chipata District, Zambia.

Six stocks of Trypanosoma congolense were cloned from 17 stocks isolated from Eastern Zambia and used to initiate insect-form in vitro cultures producing metacyclic trypanosomes. Serological assays were then developed using these in vitro-derived metacyclics as a reference collection of antigens. Monoclonal antibodies recognized 8 metacyclic variable antigen types (M-VATs) of one stock, T. congolense TREU 1885, representing 70-80% of that stock's M-VAT repertoire, and in an indirect fluorescent antibody test (IFAT) there were no cross-reactions between them and the metacyclic trypanosomes of the other 5 stocks. Cross-protection assays between the 6 stocks in mice showed that the stocks cultured in vitro were serologically distinct. In order to facilitate serological typing for serodeme characterization, an IFAT was developed using formalin-fixed metacyclic trypanosomes to identify VAT specific immune responses using 21 day post-infection antisera. The cultured stocks reacted only with their homologous antisera thus confirming the results obtained in the cross-protection assays. No cross-reactions were observed with the 6 cloned stocks and antisera against the 11 stocks of T. congolense isolated in the same area at the same time suggesting that these stocks were different from the reference collection of cultured metacyclics. Hence, at least 7 serodemes of T. congolense have been identified from the 17 stocks isolated.

Analysis of trypanosome variable antigen types in cultures of metacyclic and mammalian forms of Trypanosoma congolense.

Cultured metacyclic forms of Trypanosoma congolense display a characteristic repertoire of metacyclic variable antigen types (M-VATs) similar to that exhibited in vitro in the tsetse fly. There appeared to be no change in expression of M-VATs in cultures of two stocks of T. congolense even after several passages, cryopreservation or long-term cultivation in vitro. Metacyclic forms transformed into mammalian forms when transferred to cultures of bovine aorta endothelial cells and whilst one stock retained expression of M-VATs without change even after 4 months, the other stock underwent antigenic variation within 14 days of transfer. Analysis of the M-VAT composition of mammalian forms of this stock using monoclonal antibodies showed that although the proportion of mammalian forms expressing certain M-VATs declined considerably, trypanosomes expressing one M-VAT increased proportionally to comprise 50% of the population. In contrast, only small changes were seen in antigen expression in cultures of metacyclic trypanosomes from which mammalian-form cultures were derived. It was possible to produce in vitro, loss and reacquisition of variable antigen surface coat, similar to the differentiation process occurring when bloodstream trypanosomes are ingested by the tsetse fly and eventually develop into metacyclic forms.