Selection of Trypanosoma cruzi clonal genotypes (clonet 20 and 39) isolated from Bolivian triatomines following subculture in liquid medium

Previous studies showed that two groups of Trypanosoma cruzi clonal genotypes named clonet 20 and clonet 39 were predominant in Triatoma infestans, the unique vector of Chagas disease in Bolivia. These groups of clones correspond to distinct genetic clusters. These clonets were detected in T. infestans and Rhodnius pictipes fecal samples before isolation and after culture by kDNA PCR (polymerase chain rreaction) and hybridization of the amplified products with clonet specific kDNA probes named 20 and 39 as previously reported. Forty eight T. infestans and three R. pictipes infected insects captured at random in different Bolivian departments were proceeded. As previously reported the direct identification of the two major clonets in fecal samples allowed the detection of abundant mixed infections: 41% in the original sample, however after culture, only 6% of mixed infections were detected. Among the 21 parasite stocks isolated from digestive tracts where mixed infections were initially detected (clonet 20 + 39) clonet 20 alone was detected in 81% of them. This result clearly showed that the culture step selected clonet 20 parasites over those belonging to clonet 39. The taxonomic status of the isolated stocks was also confirmed by isoenzyme typing, and correlation was observed between clustering topology and hybridization patterns with the probes 20 and 39.

Trypanosoma cruzi infection in Didelphis marsupialis in Santa Catarina and Arvoredo Islands, southern Brazil

Between 1984 and 1993 the prevalence of the Trypanosoma cruzi infection in opossums (Didelphis marsupialis) was studied in Santa Catarina and Arvoredo Islands, State of Santa Catarina, Brazil. The association of the triatomine bug Panstrongylus megistus with opossums nests and the infection rate of these triatomines by T. cruzi was also studied. Thirteen different locations were studied in Santa Catarina Island (SCI), in which 137 D. marsupialis were collected. Sixty two opossums were collected at the Arvoredo Island (AI), located 12 miles north from SCI. All captured animals were submitted to parasitological examinations that revealed the presence of T. cruzi in 21.9% of the opossums captured in SCI and 45.2% among opossums captured in the AI. The presence of P. megistus was detected in most of the D. marsupialis nests collected in the SCI, however, in the non-inhabited AI only eight triatomines were collected during the whole study. The presence of T. cruzi-infected D. marsupialis associated with P. megistus in human dwellings in the SCI, and the high infection rate of D. marsupilais by T. cruzi in the absence of a high vector density are discussed.

A simplified method for sample collection and DNA isolation for polymerase chain reaction detection of Trypanosoma rangeli and Trypanosoma cruzi in triatomine vectors

Due to the overlapping distribution of Trypanosoma rangeli and T. cruzi in Central and South America, sharing several reservoirs and triatomine vectors, we herein describe a simple method to collect triatomine feces and hemolymph in filter paper for further detection and specific characterization of these two trypanosomes. Experimentally infected triatomines feces and hemolymph were collected in filter paper and specific detection of T. rangeli or T. cruzi DNA by polymerase chain reaction was achieved. This simple DNA collection method allows sample collection in the field and further specific trypanosome detection and characterization in the laboratory.

Studies on the virulence and attenuation of Trypanosoma cruzi using immunodeficient animals

Tissue invasion and pathology by Trypanosoma cruzi result from an interaction between parasite virulence and host immunity. Successive in vivo generations of the parasite select populations with increasing ability to invade the host. Conversely, prolonged in vitro selection of the parasite produces attenuated sublines with low infectivity for mammals. One such subline (TCC clone) has been extensively used in our laboratory as experimental vaccine and tested in comparative experiments with its virulent ancestor (TUL). The experiments here reviewed aimed at the use of immunodeficient mice for testing the infectivity of TCC parasites. It has not been possible to obtain virulent, revertant sublines by prolonged passaged in such mice.

Population genetic analysis of Colombian Trypanosoma cruzi isolates revealed by enzyme electrophoretic profiles

Although Colombia presents an enormous biological diversity, few studies have been conducted on the population genetics of Trypanosoma cruzi. This study was carried out with 23 Colombian stocks of this protozoa analyzed for 13 isoenzymatic loci. The Hardy-Weinberg equilibrium, the genetic diversity and heterogeneity, the genetic relationships and the possible spatial structure of these 23 Colombian stocks of T. cruzi were estimated. The majority of results obtained are in agreement with a clonal population structure. Nevertheless, two aspects expected in a clonal structure were not discovered in the Colombian T. cruzi stocks. There was an absence of given zymodemes over-represented from a geographical point of view and the presumed temporal stabilizing selective phenomena was not observed either in the Colombian stocks sampled several times through the years of the study. Some hypotheses are discussed in order to explain the results found.

Biological characterization of Trypanosoma cruzi strains

Biological parameters of five Trypanosoma cruzi strains from different sources were determined in order to know the laboratory behaviour of natural populations. The parameters evaluated were growth kinetics of epimastigotes, differentiation into metacyclic forms, infectivity in mammalian cells grown in vitro and parasite susceptibility to nifurtimox, benznidazole and gentian violet. Differences in transformation to metacyclic, in the percentage of infected cells as well as in the number of amastigotes per cell were observed among the strains. Regarding to pharmacological assays, Y strain was the most sensitive to the three assayed compounds. These data demonstrate the heterogeneity of natural populations of T. cruzi, the only responsible of infection in humans.

Molecular karyotype and chromosomal localization of genes encoding ß-tubulin, cysteine proteinase, hsp 70 and actin in Trypanosoma rangeli

The molecular karyotype of nine Trypanosoma rangeli strains was analyzed by contour-clamped homogeneous electric field electrophoresis, followed by the chromosomal localization of ß-tubulin, cysteine proteinase, 70 kDa heat shock protein (hsp 70) and actin genes. The T. rangeli strains were isolated from either insects or mammals from El Salvador, Honduras, Venezuela, Colombia, Panama and southern Brazil. Also, T. cruzi CL-Brener clone was included for comparison. Despite the great similarity observed among strains from Brazil, the molecular karyotype of all T. rangeli strains analyzed revealed extensive chromosome polymorphism. In addition, it was possible to distinguish T. rangeli from T. cruzi by the chromosomal DNA electrophoresis pattern. The localization of ß-tubulin genes revealed differences among T. rangeli strains and confirmed the similarity between the isolates from Brazil. Hybridization assays using probes directed to the cysteine proteinase, hsp 70 and actin genes discriminated T. rangeli from T. cruzi, proving that these genes are useful molecular markers for the differential diagnosis between these two species. Numerical analysis based on the molecular karyotype data revealed a high degree of polymorphism among T. rangeli strains isolated from southern Brazil and strains isolated from Central and the northern South America. The T. cruzi reference strain was not clustered with any T. rangeli strain.

Role of two Triatoma (Hemiptera: Reduviidae: Triatominae) species in the transmission of Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae) to man in the West Coast of Mexico

From August 1997 to August 1998, 334 specimens of Triatoma longipennis and 62 of T. picturata were collected in four groups of localities placed in the zone from Guadalajara, Jalisco to Tepic, Nayarit, in the West Coast of Mexico. Most T. longipennis were collected outdoors (69.2%) while most T. picturata (58.1%) were collected indoors. All collected specimens were examined for Trypanosoma cruzi infection, which was detected on 98 (29.3%) T. longipennis and 17 (27.4%) T. picturata. This study confirms the role of T. longipennis and T. picturata as some of the main T. cruzi vectors to humans in Mexico. Habitation Infestation Rate with T. longipennis was of 0.09 and with T. picturata was of 0.03 and the predominating ecotopes were pile of blocks, chicken coops, pigsties, wall crawls and beds.

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.

Usefulness of microsatellite typing in population genetic studies of Trypanosoma cruzi

Through microsatellite analysis of 53 monoclonal populations of Trypanosoma cruzi, we found a remarkable degree of genetic polymorphism with no single multilocus genotype being observed more than once. The microsatellite profile proved to be stable during 70 generations of the CL Brener clone in culture. The microsatellite profiling presented also high diagnostic sensitivity since DNA amplifications could be achieved with less than 100 fg DNA, corresponding to half parasite total DNA content. Based on these technical attributes the microsatellite assay turns out to be an important tool for direct typing T. cruzi in biological samples. By using this approach we were able to type T. cruzi in feces of artificially infected bugs and in single cells sorted by FACS. The microsatellites have shown to be excellent markers for T. cruzi phylogenetic reconstruction. We used maximum parsimony based on the minimum number of mutational steps to build an unrooted Wagner network, which confirms previous conclusions based on the analysis of the D7 domain of the LSU rDNA gene that T. cruzi is composed by two major groups. We also obtained evidence that strains belonging to rRNA group 2 are subdivided into two genetically distant clusters, and that one of these clusters is more related to rRNA group 1/2. These results suggest different origins for these strains.

Comparison of polymerase chain reaction on fresh tissue samples and fecal drops on filter paper for detection of Trypanosoma cruzi in Rhodnius prolixus

PCR detection of Trypanosoma cruzi in Rhodnius prolixus using fresh tissue or fecal drops on filter paper showed comparable results: 38.7% infection rate using the fresh tissue sample and 37.9% by dried fecal drop.

Isoenzyme profiles of Trypanosoma cruzi stocks from different areas of Paraguay

Twenty one Trypanosoma cruzi stocks from humans, domiciliary triatomines and one sylvatic animal of different areas of Paraguay were subjected to isoenzyme analysis. Thirteen enzyme systems (15 loci in total) were studied. MN cl2 (clonets 39) and SO34 cl4 (clonets 20) were used as references. Relationships between stocks were depicted by an UPGMA dendrogram constructed using the Jaccard´s distances matrix. Among the Paraguayan stocks 14 zymodemes were identified (Par1 to Par14), Par 5 being the most frequent. Polymorphism rate and clonal diversity were 0.73 and 0.93, respectively. Average number of alleles per polymorphic locus was 2.5 (range 2-4). These measurements show a high diversity, which is confirmed by the dendrogram topology. All stocks belong to the same lineage, as MN cl2 reference strain (T. cruzi II). Moreover three distinct subgroups were identified and two of them correspond to Brazilian and Bolivian zymodemes, respectively. The third subgroup, the most common in Paraguay, is related to Tulahuen stock. The large geographical distribution of some zymodemes agrees with the hypothesis of clonality for T. cruzi populations. However sample size was not adequate to detect genetic recombination in any single locality.

Morphometry of submucous and myenteric esophagic plexus of dogs experimentally reinfected with Trypanosoma cruzi

We carried out a morphometric study of the esophagus of cross-bred dogs experimentally infected or consecutively reinfected with Trypanosoma cruzi 147 and SC-1 strains, in order to verify denervation and/or neuronal hypertrophy in the intramural plexus. The animals were sacrificed in the chronic stage, 38 months after the initial infection. Neither nests of amastigotes, nor myositis or ganglionitis, were observed in all third inferior portions of esophageal rings analyzed. No nerve cell was identified in the submucous of this organ. There was no significant difference (p>0.05) between the number, maximum diameter, perimeter, or area and volume of the nerve cells of the myenteric plexus of infected and/or reinfected dogs and of the non-infected ones. In view of these results we may conclude that the 147 and SC-1 strains have little neurotropism and do not determine denervation and/or hypertrophy in the intramural esophageal plexuses in the animals studied, independent of the reinfections.

Trypanosoma evansi control and horse mortality in the Brazilian Pantanal

The impact of three treatment strategies for Trypanosoma evansi control on horse mortality in the Brazilian Pantanal based on four size categories of cattle ranches is explored. The region's 49,000 horses are indispensable to traditional extensive cattle ranching and T. evansi kills horses. About 13% of these horses would be lost, annually, due to T. evansi if no control were undertaken. One preventive and two curative treatment strategies are financially justifiable in the Pantanal. The best available technology for the treatment of T. evansi from a horse mortality perspective is the preventive strategy, which spares 6,462 horses, annually. The year-round cure spares 5,783 horses, and the seasonal cure saves 5,204 horses on a regional basis relative to no control strategy. Regardless of the strategy adopted, 39% of the costs or benefits fall to the largest ranches, while 18% fall to the smallest ranches.

Diagnostic performance characteristics of rapid dipstick test for Plasmodium vivax malaria

We compared the diagnostic performance characteristics of newly developed method, the rapid dipstick test, which provides colorimetric determination by developing antibody to the lactate dehydrogenase enzyme of parasites, with conventional standard thick-blood film examination. For the rapid test, OptiMAL commercial kits were used. The results were also evaluated with clinical findings from patients. The parasites were determined by microscopic examination of thick-blood films from 81 patients with vivax malaria from southeastern Anatolia, Turkey. The OptiMAL test results were found to be negative in five patients who were diagnosed clinically and through thick-film testing as having vivax malaria. There was no false positivity observed with the OptiMAL test. We concluded that this rapid malaria test has a lower level of sensitivity than the classical thick-blood-film test for malaria, but that these methods have equal specificity.