Differential infectivity and immunopathology in murine experimental infections by two natural clones belonging to the Trypanosoma cruzi I lineage.

Immunopathology of Chagas' disease in Balb/c mice infected with 2 Trypanosoma cruzi clones, belonging to the T. cruzi I lineage and presenting different in vitro virulence (P/209 cl1 > SO34 c14) was compared. In the acute phase, evading mechanisms such as parasite-induced lymphocyte polyclonal activation and T cell immunosuppression were higher in mice infected with the clone giving a higher parasitaemia (P/209 cl1). A similar increase of non-specific isotypes was observed in both infections with IgG2a prevalence. Interestingly, CD8+ cell hypercellularity and lymphocyte immunosuppression were observed during the chronic phase (245 days post-infection) in mice infected by the most virulent clone. In the same way, the parasite-specific antibody response was more intense in P/209 cl1-infected mice over the acute phase. During the chronic phase this response remarkably dropped down in SO34 cl4-infected mice exclusively. Finally, P/209 cl1-infected mice presented a more severe inflammation and tissue damage in heart and quadriceps than SO34 cl4-infected mice. This comparative study showed differences between the two clones: a higher virulence in vivo being clearly associated with a greater ability to induce evasion mechanisms and severe tissue damage.

Impact of number of isoenzyme loci on the robustness of intraspecific phylogenies using multilocus enzyme electrophoresis: consequences for typing of Trypanosoma cruzi.

Thirty-one stocks of Trypanosoma cruzi, the agent of Chagas disease, representative of the genetic variability of the 2 principal lineages, that subdivide T. cruzi, were selected on the basis of previous multilocus enzyme electrophoresis analysis using 21 loci. Analyses were performed with lower numbers of loci to explore the impact of the number of loci on the robustness of the phylogenies obtained, and to identify the loci that have more impact on the phylogeny. Analyses were performed with numerical (UPGMA) and cladistical (Wagner parsimony analysis) methods for all sets of loci. Robustness of the phylogenies obtained was estimated by bootstrap analysis. Low numbers of randomly selected loci (6) were sufficient to demonstrate genetic heterogeneity among the stocks studied. However, they were unable to give reliable phylogenetic information. A higher number of randomly selected loci (15 and more) were required to reach this goal. All loci did not convey equivalent information. The more variable loci detected a greater genetic heterogeneity among the stocks, whereas the least variable loci were better for robust clustering. Finally, analysis was performed with only 5 and 9 loci bearing synapomorphic allozyme characters previously identified among larger samples of stocks. A set of 9 such loci was able to uncover both genetic heterogeneity among the stocks and to build robust phylogenies. It can therefore be recommended as a minimum set of isoenzyme loci that bring maximal information for all studies aiming to explore the phylogenetic diversity of a new set of T. cruzi stocks and for any preliminary genetic typing. Moreover, our results show that bootstrap analysis, like any statistics, is highly dependent upon the information available and that absolute bootstrap figures should be cautiously interpreted.

First evidence of transmission of Leishmania (Viannia) lainsoni in a Sub Andean region of Bolivia.

Using ubiquitous primers which amplify the variable parts of kDNA minicircle of all Leishmania spp, we obtained for Leishmania (viannia) lainsoni a major band of 605 bp (band 1) shared with L. V. braziliensis and a minor 524 bp band (band 2) specific of L. V. lainsoni. The specificity of the two bands was examined through Southern blot hybridization of kDNA PCR obtained from reference strains belonging to L. braziliensis, L. mexicana, L. donovani complexes with L. V. lainsoni species. Band 1 was not specific of L. V. lainsoni since it hybridized with some isolates belonging to L. braziliensis complex. In contrast, band 2 was L. V. lainsoni specific. PCR-based detection followed by hybridization with the new L. V. lainsoni probe (Band 2) and L. V. braziliensis probe (564 bp), was assayed using sample from a pool of 25 females of Lutzomiya nuneztovari anglesi, blood, skin and liver samples of 18 mammals, spinal cords of four mammals and blood and cutaneous ulcers aspirates from 95 patents from Sub Andean region of La Paz, Bolivia. We observed a ositive hybridization of four patients lesions and the pool of L. nuneztovari anglesi with the L. V. lainsoni probe. It is the first time that L. V. lainsoni is observed in a cycle of transmission in Bolivia. PCR products of three patients lesions and the pool of L. nuneztovari anglesi were also hybridized with the specific probe of L. V. braziliensis suggesting mixed infection in this focus.

Trypanosoma cruzi: data supporting clonality in Mexican stocks.

To further study genetic heterogeneity of Mexican stocks of Trypanosoma cruzi, genomic Southern analyses from 54 Mexican isolates and 5 South American reference stocks were carried out. The membranes were hybridized with a homologous cDNA clone from the ribosomal protein S4 that identifies allelic bands from a single gene type locus. These allelic bands were sequentially numbered depending on their relative size. Mexican T. cruzi stocks were quite homogeneous: 31 cases (57%) showed a homozygous genotype 3/3, and 21 isolates (38%) exhibited the heterozygote genotype 2/3. Just 2 Mexican stocks (3%) showed a different genotype 2/5, but the potential parental homozygous 2/2 was never observed. Being that T. cruzi is a diploid organism, the apparent absence of the presumptive parental homozygous genotype 2/2 argues against sexual reproduction within the population, at least as a common event. Therefore, these data support a clonal population structure of T. cruzi in Mexico.

Sylvatic triatominae of the phyllosoma complex (Hemiptera: Reduviidae) around the community of Carrillo Puerto, Nayarit, Mexico.

Research on domestic and sylvatic triatomines within the community of Carrillo Puerto and neighboring areas of Nayarit, Mexico, documented that Triatoma longipennis (Usinger) and Triatoma picturata (Usinger) were infected with Trypanosoma cruzi (L.) in both habitats. T. picturata was the predominant species in both habitats. Mouse baited-traps increased the effectiveness of collecting sylvatic triatomines, which were difficult to sample by inspecting habitats such as burrows, caves, and cliffs. The colonization of sylvatic and peridomestic habitats by Triatoma, the occurrence of high rates of infection with T. cruzi and the possibility that bugs move between habitats may require modification of current control strategies in Mexico.

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.

Isoenzyme variability of five principal triatomine vector species of Chagas disease in Mexico.

Triatoma barberi, T. dimidiata, T. longipennis, T. pallidipennis and T. picturata, all key Chagas disease vectors in Mexico, were analysed by multilocus enzyme electrophoresis (MLEE) at 17 putative loci. The majority of insect specimens studied were collected from domestic and peridomestic structures from multiple geographic locations while others were collected from sylvatic areas. T. barberi was the least polymorphic species (P(0.95)=0.18), with polymorphism rates of the other species ranging from 0.29 to 0.50. T. barberi, a member of the protracta complex, clustered apart from the other studied species by Nei's genetic distance with >1.36, and at least eight loci were found to be diagnostic for this species. T. dimidiata was more related to T. longipennis, T. pallidipennis and T. picturata (phyllosoma complex) than to T. barberi, with a genetic distance averaging 0.36 with the phyllosoma complex species. In contrast, the genetic distances between the three phyllosoma complex species were not significantly different from zero, and there were no species-specific loci differentiating among them. The results strongly support the grouping of these three species in one complex, separate from the two other species studied.

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 reaction) 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.

Mexican Trypanosoma cruzi stocks: analysis of minicircle kDNA homologies by cross-hybridization.

Homologies of minicircle kDNA of 27 Mexican stocks were studied by cross-hybridization with four kDNA probes derived from three reference stocks belonging to groups Trypanosoma cruzi I (SO34 cl4 and Silvio) and T. cruzi II (MN) and one Mexican stock. High homologies were only observed with Silvio (six stocks) and Mexican probes (11 stocks). After 30 min exposure (low homology) additional stocks were recognized with SO34 cl4 (three stocks) and Silvio (six stocks) probes; with the Mexican probe only five stocks remained non-reactive. All the stocks were typed by isoenzyme (16 loci) and Mexican stocks belonged to T. cruzi I. Hybridization patterns were not strictly correlated with the observed clustering and cross-hybridization of kDNA minicircles is not available to distinct Mexican stocks.

Polymerase chain reaction-based identification of New World Leishmania species complexes by specific kDNA probes.

Here we define a new approach for the detection and characterisation of Leishmania complexes by polymerase chain reaction (PCR) and specific hybridisation. The first step consists of PCR amplification of kDNA minicircles using general kinetoplastid primers, which generate a polymorphic multi-banding pattern for all Leishmania species and other Kinetoplastidae. The second step is the identification of the Leishmania species complexes by hybridisation of the PCR products with specific kDNA probes. Polymorphic PCR-products from a genetically diverse set of Leishmania species were analysed by electrophoresis and the banding patterns compared with multi-locus enzyme electrophoresis (MLEE) data. The banding patterns produced by Leishmania species were very heterogeneous, making kDNA-PCR useful for determining closely related strains and for fingerprinting individual strains. The degree of kDNA-PCR and MLEE polymorphism was compared using UPGMA dendrograms. Three complex-specific probes were generated from major PCR bands of reference stocks belonging to the Leishmania mexicana, Leishmania donovani and Leishmania braziliensis complexes, and hybridisation of these probes to membrane-bound PCR products could reliably identify the strain to a complex level. A combination of kDNA-PCR fingerprinting and hybridisation with kDNA probes was found to be useful for both sensitive detection and direct identification of Leishmania species complexes.

Restriction fragment length polymorphism of 195 bp repeated satellite DNA of Trypanosoma cruzi supports the existence of two phylogenetic groups.

The restriction fragment length polymorphism of the 195 bp repeated DNA sequence of Trypanosoma cruzi was analyzed among 23 T. cruzi stocks giving a reliable picture of the whole phylogenetic variability of the species. The profiles observed with the enzymes Hinf I and Hae III were linked together and supported the existence of two groups. Group 1 shows a 195 bp repeated unit (Hinf I) and high molecular weight DNA (Hae III), while group 2 presents a ladder profile for each enzyme, which is a characteristic of tandemly repeated DNA. The two groups, respectively, clustered stocks pertaining to the two principal lineages evidenced by isoenzyme and RAPD markers. The congruence among these three independent genomic markers corroborates the existence of two real phylogenetic lineages in T. cruzi. The specific monomorphic profiles for each major phylogenetic lineage suggest the existence of ancient sexuality and cryptic biological speciation.

Baja probabilidad de transmisión de trypanosoma cruzi a humanos por triatoma sordida domiciliado en el departamento de Santa Cruz / Low probability of transmission od Trypanosoma cruzi to humans by triatoma sordid domiciled in the department of Santa Cruz

Entre más de 100 especies de triatominae (hemiptera: ruduviidae) descritas en el Nuevo Mundo, triatoma sordida es considerada de significancia epidemiológica como vector de trypanosoma cruzi por su amplia área de distribución y su tendencia para invadir ambientes domésticos.

Genética de las poblaciones de trypanosoma cruzi: conocimientos actuales / Genetica of the populations of Trypanosoma cruzi: current knowledge

El conocimiento de las enfermedades por transmisión vectorial se basa en varios aspectos de los cuales la variabilidad genética de los organismos puede jugar un papel preponderante en la epidemiología.

Eco-distribución de los clones de trypanosoma cruzi / Echo-distribution of the clones of trypanosoma cruzi

El análisis de la repartición geográfica de los clones de T. cruzi en base a una revisión bibliografía general se dificulta por falta de normalización de las técnicas, por estudios con némeros reducidos de loci, por la ausencia de análisis filogenéticos en muchos trabajos y la falta del uso de las mismas cepas de referencias.

Identificación de los clonet 20 y 39 en heces de triatoma infestans por la reacción de la polimerasa en cadena (PCR) / Identification of the clonet 20 and 39 in stools of triatoma infestans by the reaction of the chain polymerase (PCR)

Actualmente, la caracterización genética de las cepas de Trypanosoma cruzi consiste en el análisis isoenzimático o análisis del ADN del genoma e implica el aislamiento y cultivo masivo de cada cepa.

Distribución de los clones de trypanosoma cruzi en vectores secundarios en Bolivia / Distribution of the clones of trypanosoma cruzi in secondary vectors in Bolivia

Triatoma infestans es el vector principal de la enfermedad de Chagas en Bolivia y, excepto algunos focos silvestres de esta especie descrito en el departamento de Cochabamba, existen hasta ahora pocos estudios de las otras especies silvestres.

Selección de clones de trypanosoma cruzi por aislamiento y cultivo / Clone selection of Trypanosoma cruzi by isolation and culture

Las cepas que infectan a los huéspedes vectores y mamíferos son poblaciones heterogéneas compuestas de varios clones de T. cruzi; la caracterización genética clásica de las cepas de T. cruzi (electriforesis de isoenzimas, análisis del ADN) requiere cantidades importantes de parásitos y necesita el aislamiento y el cultivo masivo de las cepas.

En Bolivia, los pacientes chagasicos son mas infectados por el clonet 39 de trypanosoma cruzi / In Bolivia, the Chagas patients are but infected by the clonet 39 of trypanosoma cruzi

Los clones de trypanosoma cruzi exhiben una gran heterogeneidad biológica y se propuso que sus propiedades biológicas puedan estar relacionadas a su contitución genética.

Smallness of the panmictic unit of Triatoma infestans (Hemiptera: Reduviidae).

The population genetic structure of Triatoma infestans (Klug), the principal vector of the causative agent of Chagas disease in Bolivia, was investigated by enzyme electrophoresis at 15 loci, of which 3 were polymorphic. A total of 1,286 adults and nymphs was collected from 19 localities of the Cochabamba (high endemicity) and La Paz (low endemicity) departments. Previous results were confirmed, including a low level of polymorphism (0.20), low genetic distance between geographic areas, and a population structure compatible with an isolation by distance model. However, a high proportion (26.3%) of the surveyed localities showed a significant excess of homozygotes, disputing previous conclusions that considered the village as the probable panmictic unit. The excess of homozygotes was reduced when smaller subunits, such as individual houses or chicken coops, were considered, indicating a Wahlund effect.