[show abstract][hide abstract] ABSTRACT: A single polymerase chain reaction (PCR) reaction targeting the spliced-leader intergenic region of Trypanosoma cruzi I was standardised by amplifying a 231 bp fragment in domestic (TcIDOM) strains or clones and 450 and 550 bp fragments in sylvatic strains or clones. This reaction was validated using 44 blind coded samples and 184 non-coded T. cruzi I clones isolated from sylvatic triatomines and the correspondence between the amplified fragments and their domestic or sylvatic origin was determined. Six of the nine strains isolated from acute cases suspected of oral infection had the sylvatic T. cruzi I profile. These results confirmed that the sylvatic T. cruzi I genotype is linked to cases of oral Chagas disease in Colombia. We therefore propose the use of this novel PCR reaction in strains or clones previously characterised as T. cruzi I to distinguish TcIDOMfrom sylvatic genotypes in studies of transmission dynamics, including the verification of population selection within hosts or detection of the frequency of mixed infections by both T. cruzi I genotypes in Colombia.
Memórias do Instituto Oswaldo Cruz 09/2013; · 1.36 Impact Factor
[show abstract][hide abstract] ABSTRACT: Spliced leader intergenic region (SL-IR) sequences from 23 Trypanosoma rangeli strains isolated from the salivary glands of Rhodnius colombiensis, R. ecuadoriensis, R. pallescens and R. prolixus and two human strains revealed the existence of 4 genotypes with CA, GT, TA, ATT and GTAT microsatellite repeats and the presence of insertions/deletions (INDEL) and single nucleotide polymorphism (SNP) characterizing each genotype. The strains isolated from the same vector species or the same Rhodnius evolutionary line presented the same genotypes, even in cases where strains had been isolated from vectors captured in geographically distant regions. The dendrogram constructed from the SL-IR sequences separated all of them into two main groups, one with the genotypes isolated from R. prolixus and the other group containing three well defined sub-groups with the genotypes isolated from R. pallescens, R. colombiensis and R. ecuadoriensis. Random amplified polymorphic DNA (RAPD) analysis showed the same two main groups and sub-groups supporting strict T. rangeli genotypes' association with Rhodnius species. Combined with other studies, these results suggest a possible co-evolutionary association between T. rangeli genotypes and their vectors.
[show abstract][hide abstract] ABSTRACT: Previous studies have shown a differential response of triatomines towards the invasion of Trypanosoma cruzi and T. rangeli due to the fact that the immune response of the insect facilitates or limits the development of some parasite genotypes. Also, it has been evidenced the existence of trypanolytic factors in the hemolymph of Rhodnius prolixus that lyse the subpopulation KP1 (-) of T. rangeli , but not the subpopulation KP1 (+). The objective was to verify the presence of trypanolytic factors in other triatomine species; onsequently, the hemolymph of R. robustus, R. stali, R. pallescens II, R. colombiensis, T. dimidiata, T. infestans and T.maculata were incubated with different subpopulations of T. cruzi and T. rangeli.
One hundred and twenty μl of hemolymph were extracted from each vector and mixed with 120 μl parasite suspension at a concentration of 9x10 6 parasites/ml. As a negative control, LIT medium was used. Hemocytometer counts were performed every 2 hours during a 10-hour period. The results showed the presence of trypanolytic factors in the hemolymp of Rhodnius prolixus, R. stali and R. robustus against T. rangeli KP1 (-), T. cruzi II and T. cruzi V . There was agglutination in T. cruzi III epimastigotes at the moment of been incubated with the hemolymph of R. colombiensis, Periplaneta americana and Conocephalus sp . These results suggest that trypanolytic factors of R. prolixus would be responsible for the fact that this species is not involved in the transmission of T. rangeli KP1 (-) and T. cruzi II and V in Colombia, Venezuela and Central American countries
Revista de la Asociación Colombiana de Ciencias Biológicas. 01/2011; 23:134-143.
[show abstract][hide abstract] ABSTRACT: The majority of individuals in the chronic phase of Chagas disease are asymptomatic (indeterminate form, IF). Each year, approximately 3% of them develop lesions in the heart or gastrointestinal tract. Cardiomyopathy (CCHD) is the most severe manifestation of Chagas disease. The factors that determine the outcome of the infection are unknown, but certainly depend on complex interactions amongst the genetic make-up of the parasite, the host immunogenetic background and environment. In a previous study we verified that the maxicircle gene NADH dehydrogenase (mitochondrial complex I) subunit 7 (ND7) from IF isolates had a 455 bp deletion compared with the wild type (WT) ND7 gene from CCHD strains. We proposed that ND7 could constitute a valuable target for PCR assays in the differential diagnosis of the infective strain. In the present study we evaluated this hypothesis by examination of ND7 structure in parasites from 75 patients with defined pathologies, from Southeast Brazil. We also analysed the structure of additional mitochondrial genes (ND4/CR4, COIII and COII) since the maxicircle is used for clustering Trypanosoma cruzi strains into three clades/haplogroups. We conclude that maxicircle genes do not discriminate parasite populations which induce IF or CCHD forms. Interestingly, the great majority of the analysed isolates belong to T. cruzi II (discrete typing unit, (DTU) IIb) genotype. This scenario is at variance with the prevalence of hybrid (DTU IId) human isolates in Bolivia, Chile and Argentina. The distribution of WT and deleted ND7 and ND4 genes in T. cruzi strains suggests that mutations in the two genes occurred in different ancestrals in the T. cruzi II cluster, allowing the identification of at least three mitochondrial sub-lineages within this group. The observation that T. cruzi strains accumulate mutations in several genes coding for complex I subunits favours the hypothesis that complex I may have a limited activity in this parasite.
International journal for parasitology 08/2009; 39(9):963-73. · 3.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: In trypanosomatids the involvement of mitochondrial complex I in NADH oxidation has long been debated. Here, we took advantage of natural Trypanosoma cruzi mutants which present conspicuous deletions in ND4, ND5 and ND7 genes coding for complex I subunits to further investigate its functionality. Mitochondrial bioenergetics of wild type and complex I mutants showed no significant differences in oxygen consumption or respiratory control ratios in the presence of NADH-linked substrates or FADH(2)-generating succinate. No correlation could be established between mitochondrial membrane potentials and ND deletions. Since release of reactive oxygen species occurs at complex I, we measured mitochondrial H(2)O(2) formation induced by different substrates. Significant differences not associated to ND deletions were observed among the parasite isolates, demonstrating that these mutations are not important for the control of oxidant production. Our data support the notion that complex I has a limited function in T. cruzi.
Journal of Bioenergetics 07/2009; 41(3):299-308. · 1.60 Impact Factor
[show abstract][hide abstract] ABSTRACT: Rhodnius colombiensis is a sylvatic triatomine associated with wine palm trees (Attalea butyracea) in the high basin of the Magdalena river (Colombia). The frequent invasion of these vectors into human dwellings and the high prevalences of natural infection with Trypanosoma cruzi of these insects suggest an important role in the transmission of Chagas disease.
The length of the life cycles of R. colombiensis and R. prolixus under laboratory conditions were compared.
Ninety-two individuals for each species were studied. The mean duration time of each stage, the number of bloodmeals for each stage, the percentage of mortality, the cause of death, the mean of eggs laid by female, the number of fertile eggs and the longevity of adults were recorded.
The mean duration time of all stages of R. colombiensis was higher than in R. prolixus, producing significant differences in the overall time from egg to adult. The mean of total eggs and fertile eggs showed significant differences, being higher in R. prolixus than in R. colombiensis. The total mortality was 31.5% for R. colombiensis and 6.5% for R. prolixus. The longevity of females was higher in R. prolixus.
The stages of R. prolixus are of relatively short duration. In general, the nymphs take fewer bloodmeals than R. colombiensis, the adults take more bloodmeals and oviposit a larger number of fertile eggs, and females have a greater longevity. These parameters indicated that R. prolixus has superior reproductive success in comparison with R. colombiensis under the experimental conditions used. These new life cycle data of R. colombiensis will be useful for maintenance of laboratory colonies.
Biomédica: revista del Instituto Nacional de Salud 02/2007; 27 Suppl 1:119-29. · 0.32 Impact Factor
[show abstract][hide abstract] ABSTRACT: Trypanosoma rangeli is a species of trypanosome second to T. cruzi, that is infective to humans in Latin America. Variability in the biological, biochemical and molecular characteristics between different isolates isolates of this parasite have been recorded.
Morphological and molecular characteristics were recorded from strains of T. rangeli that were isolated from different species of Rhodnius and maintained in different vertebrate species.
Nineteen strains of T. rangeli were isolated from R. prolixus, R. pallescens and R. colombiensis in Colombia, R. ecuadoriensis in Peru and R. pallescens in Panama. Polymorphism of blood trypomastigotes in ICR mice was evaluated and pleomorphism of P53 strain of T. rangeli KP1(-) inoculated in mouse, marsupial and canine was studied. RAPD analysis (randomly amplified polymorphic DNA analysis) of 12 strains isolated from four species of Rhodnius was performed.
Based on the total length of blood trypomastigotes, three discrete groups were observed. The P53 strain showed significant differences in the size of blood trypomastigotes in mouse, marsupial and canine. RAPD analysis showed that the strains segregated into two branches corresponding to strains of T. rangeli KP1(+) and T. rangeli KP1(-). All strains of T. rangeli KP1(-) clustered according to the species of Rhodnius from which they were isolated.
These data reveal, for the first time, a close association amongst T. rangeli strains and Rhodnius species, confirming that each species of Rhodnius transmits to vertebrate hosts a parasite population with clear phenotypic and genotypic differences. This is further evidence that supports the concept of clonal evolution of these parasites.
Biomédica: revista del Instituto Nacional de Salud 02/2007; 27 Suppl 1:110-8. · 0.32 Impact Factor
[show abstract][hide abstract] ABSTRACT: Salivary hemeprotein electrophoresis in starch gels is a recent technique used for differentiation of Rhodnius species with great phenotypic similarity. Furthermore, populations of the same species can be differentiated from geographically separated locales. Of the 15 described Rhodnius species in Latin America, at least eight have been reported in Colombia.
To use the salivary hemeproteins electrophoresis for R. prolixus and R. colombiensis identification. These two species are phenotypically similar and have overlapping domestic and sylvatic cycles where they occur in the upper basin of the Magdalena river, Central Colombia.
The content of salivary glands of each insect was subjected to starch gel electrophoresis using glycine buffer, and the bands were revealed with 3,3',5,5'-tetramethylbenzidine. Band patterns were photographically recorded.
Electrophoretic patterns of salivary hemeproteins of R. prolixus and R. colombiensis were able to unequivocally differentiate the two species.
The usefulness of the starch gel technique for distinguishing between R. prolixus and R. colombiensis was demonstrated as an additional tool to the morphometric and molecular methods already in use for differentiation of these two species.
Biomédica: revista del Instituto Nacional de Salud 02/2007; 27 Suppl 1:137-42. · 0.32 Impact Factor
[show abstract][hide abstract] ABSTRACT: Rhodnius colombiensis occasionally comes into human dwellings and consequently its role as an important potential vector in the transmission of American trypanosomiasis has been suggested.
The potential role of R. colombiensis as vector was defined by comparing the feeding and defecation patterns between R. colombiensis and R. prolixus, the main domiciliary vector of Trypanosoma cruzi in Colombia. MATERIALS AND METHODS. For each developmental stage of R. colombiensis and R. prolixus the following data were collected: (1) time of feeding initiation, (2) the time for reaching the repletion, (3) the number of interruptions and defecations during the feeding, (4) the time between the end of the feeding and the first defecation, (5) the number of defecations during 10, 60 and 95 minutes of observation after feeding, and (6) the quantity of blood ingested.
The mean time of feeding initiation of the fifth instar nymphs, males and females, showed significant differences between the two species. The average of insects that defecated within 10 minutes after feeding was higher for each successive stage of R. prolixus and showed significant differences with Rhodnius colombiensis. In contrast, the mean weight of blood ingested by each stage of R. colombiensis and R. prolixus was significantly different between the N1, N2, N5 and females of these species.
Rhodnius colombiensis produced fewer defecations than R. prolixus during feeding. A higher percentage of R. prolixus defecated within 10, 60 and 95 minutes after feeding. However, R. colombiensis remains a longer time in contact with the vertebrate host, thus raising the probability of its role in transmission considering its occasional entry to human dwellings and its higher prevalences of infection withT. cruzi and T. rangeli.
Biomédica: revista del Instituto Nacional de Salud 02/2007; 27 Suppl 1:101-9. · 0.32 Impact Factor
[show abstract][hide abstract] ABSTRACT: The majority of individuals in the chronic phase of Chagas disease are asymptomatic (indeterminate form). Every year 2-3% of these individuals develop severe clinical manifestations (cardiac and digestive forms). In this study a Trypanosoma cruzi DNA microarray was used to compare the transcript profiles of six human isolates: three from asymptomatic and three from cardiac patients. Seven signals were expressed differentially between the two classes of isolates, including tryparedoxin, surface protease GP63, cyclophilin, some hypothetical proteins and the pre-edited maxicircle gene NADH dehydrogenase subunit 7 (ND7). The approximately 30-fold greater signal in cardiac strains for ND7 was the most pronounced of the group, and differential levels of pre-edited ND7 transcript confirmed the microarray analysis. The ND7 gene from asymptomatic isolates showed a deletion of 455bp from nt 222 to nt 677 relative to ND7 of the CL Brener reference strain. The ND7 gene structure correlated with disease manifestation for 20 isolates from clinically characterised, chronic phase patients. The ND7 lesion produces a truncated product that could impair the function of mitochondrial complex I. Possible links between the integrity of the electron transport chain and symptom presentation are discussed. We propose that ND7 and other genes of the pathway constitute valuable targets for PCR assays in the differential diagnosis of the infective T. cruzi strain. While this hypothesis requires validation by the examination of additional recent parasite isolates from patients with defined pathologies, the identification of specific molecular markers represents a promising advance in the association between parasite genetics and disease pathology.
Molecular and Biochemical Parasitology 01/2007; 150(2):236-48. · 2.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: We present data on the molecular characterisation of strains of Trypanosoma rangeli isolated from naturally infected Rhodnius ecuadoriensis in Peru, from Rhodnius colombiensis, Rhodnius pallescens and Rhodnius prolixus in Colombia, and from Rhodnius pallescens in Panama. Strain characterisation involved a duplex PCR with S35/S36/KP1L primers. Mini-exon gene analysis was also carried out using TrINT-1/TrINT-2 oligonucleotides. kDNA and mini-exon amplification indicated dimorphism within both DNA sequences: (i) KP1, KP2 and KP3 or (ii) KP2 and KP3 products for kDNA, and 380 bp or 340 bp products for the mini-exon. All T. rangeli strains isolated from R. prolixus presented KP1, KP2 and KP3 products with the 340 bp mini-exon product. By contrast, all T. rangeli strains isolated from R. ecuadoriensis, R. pallescens and R. colombiensis, presented profiles with KP2 and KP3 kDNA products and the 380 bp mini-exon product. Combined with other studies, these results provide evidence of co-evolution of T. rangeli strains associated with different Rhodnius species groups east and west of the Andean mountains.
Infection Genetics and Evolution 04/2005; 5(2):123-9. · 2.77 Impact Factor
[show abstract][hide abstract] ABSTRACT: The Federal District of Brazil (DF) lies within the Cerrado biome, where open shrubland (savannas) is interspersed with riverside gallery forests and permanent swamps (veredas). Trypanosoma cruzi-infected native triatomines occur in the area, but the enzootic transmission of trypanosomatids remains poorly characterized. A parasitological survey involving sylvatic triatomines (166 Rhodnius neglectus collected from Mauritia flexuosa palms) and small mammals (98 marsupials and 70 rodents, totaling 18 species) was conducted in 18 sites (mainly gallery forests and veredas) of the DF. Parasites were isolated, morphologically identified, and characterized by PCR of nuclear (mini-exon gene) and kinetoplast DNA (kDNA). Six R. neglectus, seven Didelphis albiventris and one Akodon cursor were infected by trypanosomes; wild reservoir infection is documented for the first time in the DF. kDNA PCR detected T. cruzi in five R. neglectus and mini-exon gene PCR revealed T. cruzi I in isolates from D. albiventris. Parasites infecting one bug yielded T. rangeli KP1+ kDNA amplicons. In spite of the occurrence of T. cruzi-infected D. albiventris (an important wild and peridomestic reservoir) and R. neglectus (a secondary vector displaying synanthropic behavior), a low-risk of human Chagas disease transmission could be expected in the DF, considering the low prevalence infection recorded in this work. The detection of T. rangeli KP1+ associated with R. neglectus in the DF widens the known range of this parasite in Brazil and reinforces the hypothesis of adaptation of T. rangeli populations (KP1+ and KP1-) to distinct evolutionary Rhodnius lineages.
Revista do Instituto de Medicina Tropical de São Paulo 01/2004; 46(6):323-30. · 0.96 Impact Factor
[show abstract][hide abstract] ABSTRACT: Trypanosoma rangeli are kinetoplastid protozoa which have been largely recognized and defined in several Latin American countries in relation to T. cruzi, because the two trypanosome species are frequently found in mixed infections in triatominae vectors, humans and a variety of wild and domestic mammals. We report the molecular characterization of 18 T. rangeli strains isolated from the salivary glands of naturally infected Rhodnius colombiensis, R. pallescens and R. prolixus by using two independent set of molecular markers. kDNA and mini-exon amplification indicated dimorphism within both DNA sequences: KP1, KP2 and KP3 or KP2 and KP3 products for kDNA mini-circles and 380 or 340bp products for the mini-exon. One of two associations was observed within individual strains: KP1, KP2 and KP3 kDNA products with the 340bp mini-exon product and the KP2 and KP3 kDNA products with the 380bp mini-exon product. Independent mitochondrial and nuclear molecular markers showed a clear division of T. rangeli into two major phylogenetic groups associated with specific vectors in Colombia and in other Latin America countries. These results support either clonal evolution or speciation in T. rangeli populations, probably derived as a secondary adaptation to their parasitic condition in triatomine vectors.
Infection Genetics and Evolution 06/2003; 3(1):39-45. · 2.77 Impact Factor
[show abstract][hide abstract] ABSTRACT: Trypanosoma rangeli is a hemoflagellate parasite of man, domestic and wild animals in Central and South America. The genus Rhodnius is particularly susceptible to infection by T. rangeli and transmission by salivary inoculation has been demonstrated in 12 of 14 nominal species of naturally and experimentally infected insects. This report describes the molecular characterization of 37 strains of T. rangeli isolated from vertebrate and invertebrate hosts. Strains were analyzed by hybridization with kinetoplast DNA (kDNA) probes, polymerase chain reaction (PCR) amplification of kDNA minicircles and random amplification polymorphic DNA (RAPD). Strains isolated from Rhodnius prolixus present KP1, KP2 and KP3 minicircle amplification products but strains isolated from R. colombiensis or Panstrongylus megistus present amplification products derived only from KP2 and KP3 minicircles. The two T. rangeli groups defined as KP1(+) and KP1(-) present a high genetic divergence as they have probably been co-evolutioned with different adaptive radiated lines of the genus Rhodnius in Latin-America. The data obtained from insects with intestinal and salivary glands infections confirm that each Rhodnius species select the sub-population of T. rangeli KP1(+) or KP1(-) which is susceptible to transmit it by salivary inoculation to the vertebrate host.
[show abstract][hide abstract] ABSTRACT: The parasitic protozoan Trypanosoma cruzi infects an estimated 16 million individuals in Latin America. In a variable proportion of patients, this infection can result in a life-threatening cardiac or digestive pathology recognized as Chagas disease. In the majority of cases, the parasitemic phase of infection is transient and often goes unnoticed against the high background of endemic diseases present in the low-income groups usually affected by T. cruzi infection. Consequently, diagnosis of the infection by direct microscopic examination is rarely possible; therefore, routine serologic procedures as well as modern molecular techniques provide the most sensitive indicators of human infection.
Archives of Medical Research 01/2002; 33(4):362-70. · 2.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Vargas, N., Souto, R. P., Carranza, J. C., Vallejo, G. A., and Zingales, B. 2000. Amplification of a specific repetitive DNA sequence for Trypanosoma rangeli identification and its potential application in epidemiological investigations. Experimental Parasitology 96, 147– 159. Trypanosoma rangeli can infect humans as well as the same domestic and wild animals and triatomine vectors infected by Trypano soma cruzi in Central and South America. This overlapping distribution complicates the epidemiology of American trypanosomiasis due to the cross-reactivity between T. rangeli and T. cruzi antigens and the pres ence of conserved DNA sequences in these parasites. We have isolated a T. rangeli-specific DNA repetitive element which is represented in approximately 103 copies per parasite genome and is distributed in several chromosomal bands. The 542-bp nucleotide sequence of this element, named P542, was determined and a PCR assay was standard ized for its amplification. The sensitivity of the assay is high, allowing the detection of one tenth of the DNA content of a single parasite. The presence of the P542 element was confirmed in 11 T. rangeli isolates from mammalian hosts and insect vectors originating from several countries in Latin America. Negative amplification was ob served with different T. cruzi strains and other trypanosomatids. The potential field application of the P542 PCR assay was investigated in simulated samples containing T. rangeli and/or T. cruzi and intestinal tract and feces of Rhodnius prolixus. Epidemiological studies were conducted in DNA preparations obtained from the digestive tracts of 12 Rhodnius colombiensis insects collected in a sylvatic area in Colombia. Positive amplification of the P542 element was obtained in 9/12 insects. We have also compared in the same samples the diagnostic performance of two PCR assays for the amplification of the variable domain of minicircle kinetoplast DNA (kDNA) and of the large subunit (LSU) of the ribosomal RNA gene of T. cruzi and T. rangeli. Data indicate that the kDNA PCR assay does not allow diagnosis of mixed infections in most insects. On the other hand, the PCR assay of the LSU RNA gene showed lower sensitivity in the detection of T. rangeli than the PCR assay of the P542 element. It is predicted that the use of sensitive detection techniques will indicate that the actual distribution of T. rangeli in America is wider than presumed.