Publications by authors named "Luis Eduardo Ramirez"

36 Publications

28Sβ Ribosomal Gene Allows Intra and Interspecific Molecular Differentiation.

Vector Borne Zoonotic Dis 2020 02 22;20(2):117-124. Epub 2019 Oct 22.

Centro de Educação Profissional (CEFORES), Universidade Federal do Triângulo Mineiro (UFTM), Uberaba, Brasil.

is an avirulent flagellate protozoan that could mislead correct diagnosis of infection, the causative agent of Chagas' disease, given their high similarity. Besides, presents two genetic groups, whose differentiation is achieved mainly by molecular approaches. In this context, ribosomal DNA (rDNA) is a useful target for intra and interspecific molecular differentiation. Analyzing the rDNA of and comparison with other trypanosomatid species, two highly divergent regions (Trβ1 and Trβ2) within the 28Sβ gene were found. Those regions were amplified and sequenced in KP1(+) and KP1(-) strains of , revealing group-specific polymorphisms useful for intraspecific distinction through restriction fragment length polymorphism technique. Also, amplification of Trβ1 allowed differentiation between and Trβ2 predicted restriction length profile, allowed differentiation between , , , and , increasing the use of Trβ1 and Trβ2 beyond a molecular approach for genotyping, but also as a useful target for trypanosomatid classification.
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http://dx.doi.org/10.1089/vbz.2019.2496DOI Listing
February 2020

Evaluation of the multispecies coalescent method to explore intra-Trypanosoma cruzi I relationships and genetic diversity.

Parasitology 2019 07 3;146(8):1063-1074. Epub 2019 May 3.

Grupo de Investigaciones Microbiológicas-UR (GIMUR), Programa de Biología, Facultad de Ciencias Naturales y Matemáticas,Universidad del Rosario,Bogotá,Colombia.

Chagas Disease is a zoonosis caused by the parasite Trypanosoma cruzi. Several high-resolution markers have subdivided T. cruzi taxon into at least seven lineages or Discrete Typing Units (DTUs) (TcI-TcVI and TcBat). Trypanosoma cruzi I is the most diverse and geographically widespread DTU. Recently a TcI genotype related to domestic cycles was proposed and named as TcIDOM. Herein, we combined traditional markers and housekeeping genes and applied a Multispecies Coalescent method to explore intra-TcI relationships, lineage boundaries and genetic diversity in a random set of isolates and DNA sequences retrieved from Genbank from different countries in the Americas. We found further evidence supporting TcIDOM as an independent and emerging genotype of TcI at least in Colombia and Venezuela. We also found evidence of high phylogenetic incongruence between parasite's gene trees (including introgression) and embedded species trees, and a lack of genetic structure among geography and hosts, illustrating the complex dynamics and epidemiology of TcI across the Americas. These findings provide novel insights into T. cruzi systematics and epidemiology and support the need to assess parasite diversity and lineage boundaries through hypothesis testing using different approaches to those traditionally employed, including the Bayesian Multispecies coalescent method.
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http://dx.doi.org/10.1017/S0031182019000428DOI Listing
July 2019

Human leishmaniasis in Brazil: A general review.

Rev Assoc Med Bras (1992) 2018 Mar;64(3):281-289

Department of Immunology, Microbiology and Parasitology, Instituto de Ciências Biológicas e Naturais, Universidade Federal do Triângulo Mineiro, Uberaba, MG, Brazil.

Leishmaniasis is a disease with ample clinical spectrum and epidemiological diversity and is considered a major public health problem. This article presents an overview of the transmission cycles, host-parasite interactions, clinical, histological and immunological aspects, diagnosis and treatment of various forms of the human disease.
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http://dx.doi.org/10.1590/1806-9282.64.03.281DOI Listing
March 2018

Identification of bat trypanosomes from Minas Gerais state, Brazil, based on 18S rDNA and Cathepsin-L-like targets.

Parasitol Res 2018 Mar 16;117(3):737-746. Epub 2018 Jan 16.

Instituto de Ciências Biológicas e Naturais, Universidade Federal do Triângulo Mineiro, Uberaba, Minas Gerais, Brazil.

Several bat species can be infected by trypanosomes, but there is not much information about which of these parasites infect bats from Triângulo Mineiro and Alto Paranaíba, Minas Gerais state, Brazil, a formerly endemic region for Trypanosoma cruzi, the causative agent of Chagas disease. The aim of this study was to describe, characterize, and identify the presence of trypanosomes in bats. The captured bats (448) belong to four families and to 19 different species. Of those, 37 bats were found to be positive for trypanosomes by microhematocrit, (infection rate 8.3%) and 27 were positive after hemoculture analysis. Initially, the isolates were identified by PCR (18S rDNA, 24Sα rDNA, spliced leader, COII RFLP-PCR) using primers originally designed for T. cruzi. PCRs (18S rDNA, 24Sα rDNA) showed compatible bands for TcI, whereas COII RFLP-PCR showed a similar pattern associated to TcII. However, there was no DNA amplification using spliced leader as a target, revealing a discrepancy between the results. Phylogenetic analysis of Cathepsin L-like and 18S rDNA sequences proved that 15 of the isolates corresponded to Trypanosoma cruzi marinkellei and one to Trypanosoma dionisii. These results revealed that the diversity of trypanosome species in a region considered endemic for Chagas disease is greater than previous descriptions. All this can confirm the necessity of using DNA sequencing approaches in order to determinate trypanosomes species isolated from bats.
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http://dx.doi.org/10.1007/s00436-017-5744-zDOI Listing
March 2018

DNA content analysis allows discrimination between Trypanosoma cruzi and Trypanosoma rangeli.

PLoS One 2017 19;12(12):e0189907. Epub 2017 Dec 19.

Departamento de Bioquímica, Farmacologia e Fisiologia, Instituto de Ciências Biológicas e Naturais, Universidade Federal do Triângulo Mineiro, Uberaba, Brasil.

Trypanosoma cruzi, a human protozoan parasite, is the causative agent of Chagas disease. Currently the species is divided into six taxonomic groups. The genome of the CL Brener clone has been estimated to be 106.4-110.7 Mb, and DNA content analyses revealed that it is a diploid hybrid clone. Trypanosoma rangeli is a hemoflagellate that has the same reservoirs and vectors as T. cruzi; however, it is non-pathogenic to vertebrate hosts. The haploid genome of T. rangeli was previously estimated to be 24 Mb. The parasitic strains of T. rangeli are divided into KP1(+) and KP1(-). Thus, the objective of this study was to investigate the DNA content in different strains of T. cruzi and T. rangeli by flow cytometry. All T. cruzi and T. rangeli strains yielded cell cycle profiles with clearly identifiable G1-0 (2n) and G2-M (4n) peaks. T. cruzi and T. rangeli genome sizes were estimated using the clone CL Brener and the Leishmania major CC1 as reference cell lines because their genome sequences have been previously determined. The DNA content of T. cruzi strains ranged from 87,41 to 108,16 Mb, and the DNA content of T. rangeli strains ranged from 63,25 Mb to 68,66 Mb. No differences in DNA content were observed between KP1(+) and KP1(-) T. rangeli strains. Cultures containing mixtures of the epimastigote forms of T. cruzi and T. rangeli strains resulted in cell cycle profiles with distinct G1 peaks for strains of each species. These results demonstrate that DNA content analysis by flow cytometry is a reliable technique for discrimination between T. cruzi and T. rangeli isolated from different hosts.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0189907PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5736184PMC
January 2018

Use of Tc-rCRP as a target for lytic antibody titration after experimental Trypanosoma cruzi infection.

Exp Parasitol 2018 Jan 13;184:103-108. Epub 2017 Dec 13.

Programa de Pós-graduação em Medicina Tropical e Infectologia, Universidade Federal do Triângulo Mineiro, Uberaba, MG, Brazil. Electronic address:

Experimental Chagas disease has been used as a model to identify several host/parasite interaction factors involved in immune responses to Trypanosoma cruzi infection. One of the factors inherent to this parasite is the complement regulatory protein (Tc-CRP), a major epitope that induces production of lytic antibodies during T. cruzi infections. Previous studies have evaluated the function of Tc-CRP as an antigenic marker via ELISAs, which demonstrated high sensitivity and specificity when compared to other methods. Therefore, this study aimed to assess and compare the levels of lytic antibodies induced by this protein following experimental infection using different T. cruzi strains. Our results demonstrated that infections induced by strains isolated from vectors resulted in subpatent parasitaemia and low reactivity, as assessed by Tc-rCRP ELISAs. On the other hand, mice inoculated with T. cruzi strains isolated from patients developed patent parasitaemia, and presented elevated lytic antibodies titres, as measured by Tc-rCRP ELISA. In addition, comparison between different mouse lineages demonstrated that Balb/c mice were more reactive than C57BL/6 mice in almost all types of infections, except those infected by the AQ-4 strain. Parasites from the Hel strain generated the greatest lytic antibody response in all evaluated models. Therefore, application of sensitive techniques for monitoring immune responses would enable us to establish growth curves for lytic antibodies during the course of the infection, and allow us to discriminate between T. cruzi strains that originate from different hosts.
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http://dx.doi.org/10.1016/j.exppara.2017.12.003DOI Listing
January 2018

Amiodarone and itraconazole improve the activity of pentavalent antimonial in the treatment of experimental cutaneous leishmaniasis.

Int J Antimicrob Agents 2017 Aug 28;50(2):159-165. Epub 2017 Jun 28.

Laboratório de Parasitologia, Universidade Federal do Triângulo Mineiro, Av Getúlio Guarita S/N, Abadia, 38001-970, Uberaba, Minas Gerais, Brazil.

Leishmaniasis affect millions of people, causing morbidity and mortality, especially in developing tropical and subtropical countries. Unfortunately, the possibilities of treatment for these infections are still quite limited and most of the available drugs present serious side effects. The objective of this paper was to evaluate the therapeutic role of amiodarone and itraconazole in the treatment of cutaneous leishmaniasis caused by Leishmania (Leishmania) amazonensis. In order to perform this evaluation, hamsters were infected with 1 × 10 metaciclic promastigotes of the parasite in the hind footpad and, after the onset of the lesions, were treated with glucantime, amiodarone, itraconazole, glucantime and amiodarone, glucantime and itraconazole or amiodarone and itraconazole. The treatments' efficacy was evaluated per analysis of the size of the cutaneous lesions and by parasitic investigation of the infected foot (by histopathological examination and PCR) and possible side effects were analyzed taking into account the weight of the animals and some biochemical and metabolic parameters (glucose, urea, creatinine, AST, ALT and ALP). The results have shown that, in hamsters, amiodarone and itraconazole, either used isolated or in combination, are unable to stop the development of cutaneous lesions caused by L. (L.) amazonensis, but improve the activity of glucantime in the treatment of these lesions and seem to present no evident side effects. More studies are necessary in order to investigate the clinical potential of these combinations, so there can be the possibility of broadening the therapeutic options available, especially in resistant cases.
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http://dx.doi.org/10.1016/j.ijantimicag.2017.06.007DOI Listing
August 2017

Effect of the saliva from different triatomine species on the biology and immunity of TLR-4 ligand and Trypanosoma cruzi-stimulated dendritic cells.

Parasit Vectors 2016 12 9;9(1):634. Epub 2016 Dec 9.

Laboratory of Immunology, Federal University of Triângulo Mineiro, Uberaba, Minas Gerais, Brazil.

Background: Triatomines are blood-sucking vectors of Trypanosoma cruzi, the causative agent of Chagas disease. During feeding, triatomines surpass the skin host response through biomolecules present in their saliva. Dendritic cells (DCs) play a crucial role in the induction of the protection to aggressive agents, including blood-sucking arthropods. Here, we evaluated if salivary components of triatomines from different genera evade the host immunity by modulating the biology and the function of LPS- or T. cruzi-stimulated DCs.

Methods: Saliva of Panstrongylus lignarius, Meccus pallidipennis, Triatoma lecticularia and Rhodnius prolixus were obtained by dissection of salivary glands and the DCs were obtained from the differentiation of mouse bone marrow precursors.

Results: The differentiation of DCs was inhibited by saliva of all species tested. Saliva differentially inhibited the expression of MHC-II, CD40, CD80 and CD86 in LPS-matured DCs. Except for the saliva of R. prolixus, which induced IL-6 cytokine production, TNF-α, IL-12 and IL-6 were inhibited by the saliva of the other three tested species and IL-10 was increased in all of them. Saliva per se, also induced the production of IL-12, IL-6 and IL-10. Only the saliva of R. prolixus induced DCs apoptosis. The presence of PGE was not detected in the saliva of the four triatomines studied. Finally, T. cruzi invasion on DCs is enhanced by the presence of the triatomine saliva.

Conclusions: These results demonstrate that saliva from different triatomine species exhibit immunomodulatory effects on LPS and T. cruzi-stimulated DCs. These effects could be related to hematophagy and transmission of T. cruzi during feeding.
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http://dx.doi.org/10.1186/s13071-016-1890-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5148907PMC
December 2016

Correlation between the virulence of T. cruzi strains, complement regulatory protein expression levels, and the ability to elicit lytic antibody production.

Exp Parasitol 2016 Nov 7;170:66-72. Epub 2016 Sep 7.

Programa de Pós-graduação em Medicina Tropical e Infectologia, Universidade Federal do Triângulo Mineiro, Uberaba, MG, Brazil. Electronic address:

Trypanosoma cruzi trypomastigotes are able to resist lysis via the complement system, which involves many surface proteins including the complement regulatory protein (CRP). To examine the diversity in CRP recognition among strains of T. cruzi, the expression levels of the translated protein on trypomastigote surfaces were analyzed by flow cytometry, and associations between protein expression and the biological behavior of these strains, especially the ability to induce lytic antibodies in animal models, were assessed. The highly virulent T. cruzi strains Ninoa, INC-5, and Colombiana and the less virulent strains CL-Brener, LGB-231, and JG were used in the experiments. An expression profile analysis showed that the Colombiana and INC-5 strains have higher translated protein levels and induced higher production of antibodies in mice than the other strains. Our results indicated that there are differences in the surface expression of CRP between parasite strains, with a tendency for the most virulent strains to have higher expression levels. Combined, these results contribute to a better understanding of CRP functions and the complexity of host-parasite interactions, considering the large number of virulence factors involved in the process.
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http://dx.doi.org/10.1016/j.exppara.2016.09.001DOI Listing
November 2016

Semisolid liver infusion tryptose supplemented with human urine allows growth and isolation of Trypanosoma cruzi and Trypanosoma rangeli clonal lineages.

Rev Soc Bras Med Trop 2016 May-Jun;49(3):369-72

Instituto de Ciências Biológicas e Naturais, Universidade Federal do Triângulo Mineiro, Uberaba, Minas Gerais, Brasil.

Introduction: This work shows that 3% (v/v) human urine (HU) in semisolid Liver Infusion Tryptose (SSL) medium favors the growth of Trypanosoma cruzi and T. rangeli.

Methods: Parasites were plated as individual or mixed strains on SSL medium and on SSL medium with 3% human urine (SSL-HU). Isolate DNA was analyzed using polymerase chain reaction (PCR) and pulsed-field gel electrophoresis (PFGE).

Results: SSL-HU medium improved clone isolation. PCR revealed that T. cruzi strains predominate on mixed-strain plates. PFGE confirmed that isolated parasites share the same molecular karyotype as parental cell lines.

Conclusions: SSL-HU medium constitutes a novel tool for obtaining T. cruzi and T. rangeli clonal lineages.
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http://dx.doi.org/10.1590/0037-8682-0190-2015DOI Listing
March 2017

The Driving of Immune Response by Th1 Adjuvants in Immunization of Mice with Trypanosoma cruzi marinkellei Elicits a Controversial Infection Control.

Vector Borne Zoonotic Dis 2016 05 9;16(5):317-25. Epub 2016 Mar 9.

2 Department of Microbiology, Immunology and Parasitology, Institute of Biological and Natural Sciences, Federal University of Triângulo Mineiro (UFTM) , Uberaba, Brazil .

In previous studies, we have demonstrated that inoculation with a Trypanosoma cruzi marinkellei (avirulent RM1 strain) was able to reduce parasitemia in mice challenged with T. cruzi, although it was not able to prevent histopathological lesions. Th1 response stimulation by immunization is necessary for T. cruzi infection control, but the resistance is also dependent on immunoregulatory mechanisms, which can be induced by adjuvants. Thus, we evaluated whether inoculation of T. cruzi marinkellei associated with administration of different adjuvants would be capable of inducing different patterns of immune response to maximize the immune response against T. cruzi (virulent Romildo strain) infection. Two hundred eighty nonisogenic mice were divided into 14 groups according to the immunization scheme and the subsequent challenge with virulent Romildo T. cruzi strain. Nonimmunized groups and animals inoculated without adjuvants were also included. Immune protection was not observed with Th2 adjuvants (incomplete Freund's adjuvant [IFA] and Alum) due to high parasitemia. Th1/Th2-polarizing adjuvants also did not induce immune protection because inulin was unable to maintain survival, and immune-stimulating complexes induced intense inflammatory processes. Animals sensitized with RM1 strain without adjuvants were able to reduce parasitemia, increase survival, and protect against severe histological lesions, followed by adequate cytokine stimulation. Finally, our results demonstrate that the early and balanced IFN-γ production becomes critical to promote protection and that Th1 adjuvant elicited a controversial infection control due to increased histopathological damage. Therefore, the host's immunomodulation remains one of the most important challenges in the research for effective protection against T. cruzi infection. Similarly, the identification of protective antigens in the RM1 strain of T. cruzi marinkellei may contribute to further studies on vaccine development against human Chagas disease.
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http://dx.doi.org/10.1089/vbz.2015.1874DOI Listing
May 2016

Immunomodulation by Trypanosoma cruzi: toward understanding the association of dendritic cells with infecting TcI and TcII populations.

J Immunol Res 2014 13;2014:962047. Epub 2014 Oct 13.

Postgraduate Course of Tropical Medicine and Infectology, Laboratory of Parasitology, Federal University of Triângulo Mineiro, Uberaba, MG, Brazil.

Dendritic cells (DCs) are major immune components, and depending on how these cells are modulated, the protective host immune response changes drastically. Trypanosoma cruzi is a parasite with high genetic variability and modulates DCs by interfering with their capacity for antigen recognition, migration, and maturation. Despite recent efforts, the association between DCs and T. cruzi I (TcI) and TcII populations is unknown. Herein, it was demonstrated that AQ1.7 and MUTUM TcI strains present low rates of invasion of bone marrow-derived DCs, whereas the 1849 and 2369 TcII strains present higher rates. Whereas the four strains similarly induced the expression of PD-L1, the production and expression of IL-10 and TLR-2, respectively, in DCs were differentially increased. The production of TNF-α, IL-12, IL-6, and CCL2 and the expression of CD40, CD80, MHC-II, CCR5, and CCR7 changed depending on the strain. The 2369 strain yielded the most remarkable results because greater invasion correlated with an increase in the levels of anti-inflammatory molecules IL-10 and PD-L1 but not with a change in the levels of TNF-α, MHC-II, or CD40 molecules. These results suggest that T. cruzi strains belonging to different populations have evolved specific evasion strategies that subvert DCs and consequently the host response.
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http://dx.doi.org/10.1155/2014/962047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4211313PMC
June 2015

High similarity of Trypanosoma cruzi kDNA genetic profiles detected by LSSP-PCR within family groups in an endemic area of Chagas disease in Brazil.

Rev Soc Bras Med Trop 2014 Sep-Oct;47(5):653-6

Disciplina de Parasitologia, Departamento de Ciências Biológicas, Universidade Federal do Triângulo Mineiro, Uberaba, MG, Brazil.

Introduction: Determining the genetic similarities among Trypanosoma cruzi populations isolated from different hosts and vectors is very important to clarify the epidemiology of Chagas disease.

Methods: An epidemiological study was conducted in a Brazilian endemic area for Chagas disease, including 76 chronic chagasic individuals (96.1% with an indeterminate form; 46.1% with positive hemoculture).

Results: T. cruzi I (TcI) was isolated from one child and TcII was found in the remaining (97.1%) subjects. Low-stringency single-specific-primer-polymerase chain reaction (LSSP-PCR) showed high heterogeneity among TcII populations (46% of shared bands); however, high similarities (80-100%) among pairs of mothers/children, siblings, or cousins were detected.

Conclusions: LSSP-PCR showed potential for identifying similar parasite populations among individuals with close kinship in epidemiological studies of Chagas disease.
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http://dx.doi.org/10.1590/0037-8682-0255-2013DOI Listing
July 2015

Effects of cholinergic stimulation with pyridostigmine bromide on chronic chagasic cardiomyopathic mice.

Mediators Inflamm 2014 24;2014:475946. Epub 2014 Aug 24.

Natural and Biological Sciences Institute, Triangulo Mineiro Federal University, Praca Manoel Terra 330, Centro, 38025-015 Uberaba, MG, Brazil.

The aim of the present study was to assess the effects of an anticholinesterase agent, pyridostigmine bromide (Pyrido), on experimental chronic Chagas heart disease in mice. To this end, male C57BL/6J mice noninfected (control:Con) or chronically infected (5 months) with Trypanosoma cruzi (chagasic:Chg) were treated or not (NT) with Pyrido for one month. At the end of this period, electrocardiogram (ECG); cardiac autonomic function; heart histopathology; serum cytokines; and the presence of blood and tissue parasites by means of immunohistochemistry and PCR were assessed. In NT-Chg mice, significant changes in the electrocardiographic, autonomic, and cardiac histopathological profiles were observed confirming a chronic inflammatory response. Treatment with Pyrido in Chagasic mice caused a significant reduction of myocardial inflammatory infiltration, fibrosis, and hypertrophy, which was accompanied by a decrease in serum levels of IFNγ with no change in IL-10 levels, suggesting a shift of immune response toward an anti-inflammatory profile. Lower nondifferent numbers of parasite DNA copies were observed in both treated and nontreated chagasic mice. In conclusion, our findings confirm the marked neuroimmunomodulatory role played by the parasympathetic autonomic nervous system in the evolution of the inflammatory-immune response to T. cruzi during experimental chronic Chagas heart disease in mice.
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http://dx.doi.org/10.1155/2014/475946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4158292PMC
May 2015

Immunopathological aspects of experimental Trypanosoma cruzi reinfections.

Biomed Res Int 2014 24;2014:648715. Epub 2014 Jun 24.

Laboratory of Immunology, Federal University of Triângulo Mineiro, Cefores, Frei Paulino Street, 30, 38025-180 Uberaba, MG, Brazil.

Chagas disease is caused by Trypanosoma cruzi infection. Besides the host-related factors, such as immune response and genetic background, the parasite, strain, and occurrences of reinfection episodes, may influence disease outcome. Our results demonstrate that both the primary infection and the reinfection with the Colombiana strain are connected with lower survival rate of the mice. After reinfection, parasitaemia is approximately ten times lower than in primary infected animals. Only Colombiana, Colombiana/Colombiana, and Y/Colombiana groups presented amastigote nests in cardiac tissue. Moreover, the mice infected and/or reinfected with the Colombiana strain had more T. cruzi nests, more intense inflammatory infiltrate, and higher in situ expression of TNF-α and IFN-γ than Y strain. Antigen-stimulated spleen cells from infected and/or reinfected animals produced higher levels of TNF-α, IFN-γ, and IL-10. Our results reinforce the idea that Chagas disease outcome is influenced by the strain of the infective parasite, being differentially modulated during reinfection episodes. It highlights the need of control strategies involving parasite strain characterization in endemic areas for Chagas disease.
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http://dx.doi.org/10.1155/2014/648715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4094717PMC
March 2015

Species-specific markers for the differential diagnosis of Trypanosoma cruzi and Trypanosoma rangeli and polymorphisms detection in Trypanosoma rangeli.

Parasitol Res 2014 Jun 12;113(6):2199-207. Epub 2014 Apr 12.

Instituto de Ciências Biológicas e Naturais, Universidade Federal do Triângulo Mineiro, Avenida Frei Paulino, 30, Bairro Abadia, Uberaba, Minas Gerais, 38025-180, Brazil.

Trypanosoma cruzi and Trypanosoma rangeli are kinetoplastid parasites which are able to infect humans in Central and South America. Misdiagnosis between these trypanosomes can be avoided by targeting barcoding sequences or genes of each organism. This work aims to analyze the feasibility of using species-specific markers for identification of intraspecific polymorphisms and as target for diagnostic methods by PCR. Accordingly, primers which are able to specifically detect T. cruzi or T. rangeli genomic DNA were characterized. The use of intergenic regions, generally divergent in the trypanosomatids, and the serine carboxypeptidase gene were successful. Using T. rangeli genomic sequences for the identification of group-specific polymorphisms and a polymorphic AT(n) dinucleotide repeat permitted the classification of the strains into two groups, which are entirely coincident with T. rangeli main lineages, KP1 (+) and KP1 (-), previously determined by kinetoplast DNA (kDNA) characterization. The sequences analyzed totalize 622 bp (382 bp represent a hypothetical protein sequence, and 240 bp represent an anonymous sequence), and of these, 581 (93.3%) are conserved sites and 41 bp (6.7%) are polymorphic, with 9 transitions (21.9%), 2 transversions (4.9%), and 30 (73.2%) insertion/deletion events. Taken together, the species-specific markers analyzed may be useful for the development of new strategies for the accurate diagnosis of infections. Furthermore, the identification of T. rangeli polymorphisms has a direct impact in the understanding of the population structure of this parasite.
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http://dx.doi.org/10.1007/s00436-014-3872-2DOI Listing
June 2014

Trypanosoma cruzi experimental congenital transmission associated with TcV and TcI subpatent maternal parasitemia.

Parasitol Res 2013 Feb 18;112(2):671-8. Epub 2012 Nov 18.

Discipline of Parasitology, Department of Biological Sciences, Universidade Federal do Triângulo Mineiro, Rua Frei Paulino, 30, Bairro Abadia, 38025-180, Uberaba, Minas Gerais, Brazil.

The congenital transmission of Chagas disease is associated with an increase in parasitemia during pregnancy, maternal and fetal immunity, and populations of Trypanosoma cruzi. In this study, the biological behavior of TcI and TcV (isolated from a human congenital case) strains and their potential for experimental congenital transmission were evaluated in female BALB/C mice. Parasitemia was estimated by fresh blood examination, semiquantitative microhematocrit, and hemoculture, while congenital transmission was evaluated by culture in the liver infusion tryptose medium and by polymerase chain reaction (PCR) of the pups' tissues on postnatal day 7 and of the pups' blood sample at 30 days after birth. Infection was detected in 100 % of the females. Both strains showed subpatent parasitemia, which was higher for TcV infection. The presence of amastigote nest was detected only in an animal infected with TcI. The inflammatory process was more frequent (p = 0.001) in the tissues of the animals infected with TcV (58.6 %) than TcI (31.1 %). The fertility rates of females mated after 35 days postinfection were similar (90 % for TcV, 88.9 % for TcI; p = 0.938). Parasitemia did not change during pregnancy. The average number of pups/female was greater (p = 0.03) in mice with TcV infection (8.30) than in those with TcI infection (4.78). Congenital transmission was detected exclusively by PCR in 50.9 % of the pups, 46.6 % for TcV and 58.1 % for TcI. The PCR positivity for TcI was higher in the blood than in the tissue (p = 0.003). These results demonstrate the T. cruzi experimental congenital infection associated with subpatent maternal parasitemia of TcI and TcV.
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http://dx.doi.org/10.1007/s00436-012-3184-3DOI Listing
February 2013

Autonomic nervous system modulation affects the inflammatory immune response in mice with acute Chagas disease.

Exp Physiol 2012 Nov 15;97(11):1186-202. Epub 2012 Jun 15.

Biological Sciences Institute, Triangulo Mineiro Federal University, Uberaba, MG, Brazil.

The aim of the present study was to evaluate the effects of changes to the autonomic nervous system in mice during the acute phase of Chagas disease, which is an infection caused by the parasite Trypanosoma cruzi. The following types of mice were inoculated with T. cruzi (CHG): wild-type (WT) and vesicular acetylcholine transporter knockdown (KDVAChT) C57BL/6j mice; wild-type non-treated (NT) FVB mice; FVB mice treated with pyridostigmine bromide (PYR) or salbutamol (SALB); and β(2)-adrenergic receptor knockout (KOβ2) FVB mice. During infection and at 18-21 days after infection (acute phase), the survival curves, parasitaemia, electrocardiograms, heart rate variability, autonomic tonus and histopathology of the animals were evaluated. Negative control groups were matched for age, genetic background and treatment. The KDVAChT-CHG mice exhibited a significant shift in the electrocardiographic, autonomic and histopathological profiles towards a greater inflammatory immune response that was associated with a reduction in blood and tissue parasitism. In contrast, the CHG-PYR mice manifested reduced myocardial inflammation and lower blood and tissue parasitism. Similar results were observed in CHG-SALB animals. Unexpectedly, the KOβ2-CHG mice exhibited less myocardial inflammation and higher blood and tissue parasitism, which were associated with reduced mortality. These findings could have been due to the increase in vagal tone observed in the KOβ2 mice, which rendered them more similar to the CHG-PYR animals. In conclusion, our results indicate a marked immunomodulatory role for the parasympathetic and sympathetic autonomic nervous systems, which inhibit both the inflammatory immune response and parasite clearance during the acute phase of experimental Chagas heart disease in mice.
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http://dx.doi.org/10.1113/expphysiol.2012.066431DOI Listing
November 2012

Molecular characterization of Trypanosoma cruzi Mexican strains and their behavior in the mouse experimental model.

Rev Soc Bras Med Trop 2011 Nov-Dec;44(6):684-90. Epub 2011 Nov 21.

Programa de Pós-Graduação em Medicina Tropical e Infectologia, Departamento de Clínica Médica, Universidade Federal do Triângulo Mineiro, Uberaba, MG, Brasil.

Introduction: For a long time, the importance of Chagas disease in Mexico, where many regarded it as an exotic malady, was questioned. Considering the great genetic diversity among isolates of Trypanosoma cruzi, the importance of this biological characterization, and the paucity of information on the clinical and biological aspects of Chagas disease in Mexico, this study aimed to identify the molecular and biological characterization of Trypanosoma cruzi isolates from different endemic areas of this country, especially of the State of Jalisco.

Methods: Eight Mexican Trypanosoma cruzi strains were biologically and genetically characterized (PCR specific for Trypanosoma cruzi, multiplex-PCR, amplification of space no transcript of the genes of the mini-exon, amplification of polymorphic regions of the mini-exon, classification by amplification of intergenic regions of the spliced leader genes, RAPD (random amplified polymorphic DNA).

Results: Two profiles of parasitaemia were observed, patent (peak parasitaemia of 4.6×10(6) to 10(7) parasites/mL) and subpatent. In addition, all isolates were able to infect 100% of the animals. The isolates mainly displayed tropism for striated (cardiac and skeletal) muscle. PCR amplification of the mini-exon gene classified the eight strains as TcI. The RAPD technique revealed intraspecies variation among isolates, distinguishing strains isolated from humans and triatomines and according to geographic origin.

Conclusions: The Mexican T. cruzi strains are myotrophic and belong to group TcI.
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http://dx.doi.org/10.1590/s0037-86822011005000058DOI Listing
September 2012

Sequencing and analysis of chromosomal extremities of Trypanosoma rangeli in comparison with Trypanosoma cruzi lineages.

Parasitol Res 2011 Feb 1;108(2):459-66. Epub 2010 Oct 1.

Discipline of Parasitology, Department of Biological Sciences, Federal University of Triângulo Mineiro, 38025-180, Uberaba, Minas Gerais, Brazil.

The aim of this study was to investigate the genetic variability of sequences present in the chromosome ends of Trypanosoma rangeli strains defined by the presence (+) or absence (-) of KP1 minicircles, and to compare the mean terminal restriction fragment (TRF) lengths to those of Trypanosoma cruzi populations representative of groups TcI, TcII, TcIV, and TcVI. Southern blots containing RsaI-digested genomic DNA of T. rangeli KP1(+) strains, T. rangeli KP1(-) strains, and T. cruzi strains were probed with the previously described subtelomeric sequences (170 bp) of T. rangeli and with telomeric hexamer repeats. Mean TRF length analysis showed that the chromosome ends of T. rangeli are distinctly organized, with TRFs ranging from 1.3 to 9 kb for KP1(+) strains and from 0.3 to 5.0 kb for KP1(-) strains. In T. cruzi, TRF length ranged from 0.2 to 9 kb and no association with the genotype of the parasite could be established. Sequence analysis of the 170-bp amplicons revealed the occurrence of sequence polymorphisms in the subtelomeric region between and within KP1(+) and KP1(-) strains. The GTT triplet was detected in all KP1(+) strains, except for strain Cas4, but not in any of the KP1(-) strains. The dendrogram constructed by alignment of all T. rangeli strains showed the division into two main groups, mainly related to the presence or absence of the KP1 minicircle. In conclusion, the present results extend the genotype differences demonstrated by kDNA and karyotype analysis in T. rangeli to the chromosome ends of the parasite.
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http://dx.doi.org/10.1007/s00436-010-2087-4DOI Listing
February 2011

Acute Chagas' disease in postrenal transplant and treatment with benzonidazole.

Ann Diagn Pathol 2010 Jun 31;14(3):199-203. Epub 2009 Oct 31.

Discipline of General Pathology, Triângulo Mineiro Federal University, Uberaba, the State of Minas Gerais, Brazil.

Transplanted organs may act as a route of transmission of infectious diseases, such as Chagas' disease. The aim of this study was to describe the transmission of the Trypanosoma cruzi through a renal transplant and the anatomo-clinical evolution of the patient after treatment with benzonidazole. The patient was a 31-year-old white male from the State of Minas Gerais in Brazil. He had renal failure secondary to diabetes and later received a kidney from a cadaveric donor. The patient was undergoing immunosuppression therapy with azathioprine, cyclosporine A, and prednisone. After the transplant, he developed an acute phase of Chagas' disease and complications from diabetes and died 2 months later. In the autopsy, T cruzi amastigotes were found in the transplanted kidney, heart, bladder, liver, and pancreas. An important reduction in the parasitemia was obtained through the treatment of the infection with benzonidazole; however, the patient died due to complications from diabetes associated with tissue lesions caused by T cruzi.
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http://dx.doi.org/10.1016/j.anndiagpath.2009.06.008DOI Listing
June 2010

Immunization of mice with a Trypanosoma cruzi-like strain isolated from a bat: predictive factors for involvement of eosinophiles in tissue damage.

Vector Borne Zoonotic Dis 2010 Dec 10;10(10):989-97. Epub 2010 May 10.

Parasitology Division, Department of Biological Sciences, Universidade Federal do Triângulo Mineiro, Uberaba, Brazil.

The granules of eosinophiles are cytotoxic to Trypanosoma cruzi trypomastigote and amastigote forms and to several cell types of the host, revealing their role in either parasite elimination or the production of tissue lesions. In this study, we evaluated the biological characteristics of T. cruzi infection that are responsible for the increase in tissue eosinophile levels in mice previously immunized with a bat isolated T. cruzi-like strain that does not infect mice. Nonisogeneic mice were divided into 24 groups that received from zero to three inoculations of T. cruzi-like RM1 strain, with or without adjuvant, followed by challenge with T. cruzi VIC or JG strains. Uni- and multivariate comparisons were performed comparing the tissue eosinophile levels with the parasitemia peak, severity of myositis in skeletal muscle, phase of infection, and the immunization strategies induced by the T. cruzi-like strain (adjuvant, number of reinoculations, and parasites). Although the severity of inflammation was higher in the acute phase, the score of tissue eosinophiles was similar in the acute and chronic phases of infection. In addition, there was a positive correlation among eosinophile levels and parasitemia peak. In the chronic phase, a greater eosinophile count was accompanied by an augmentation of myositis. Regardless of the phase of infection, we observed a positive correlation between the intensity of eosinophile infiltration and the number of sensitizations with T. cruzi-like strain. The multivariate analysis showed that the peak of parasitemia, number of inoculations with the T. cruzi-like strain, and severity of myositis were associated with greater tissue eosinophilia, in comparison with adjuvant, T. cruzi strains used in the challenge or tissue parasitism. Therefore, tissue eosinophile levels proved to be an important parameter in the pathogenesis of experimental Chagas disease in the acute and chronic phases of infection and might be related to reinfections, parasite multiplication ability, and severity of inflammatory process.
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http://dx.doi.org/10.1089/vbz.2009.0185DOI Listing
December 2010

Karyotype variability in KP1(+) and KP1(-) strains of Trypanosoma rangeli isolated in Brazil and Colombia.

Acta Trop 2009 Apr;110(1):57-64

Departamento de Ciências Biológicas, Universidade Federal do Triângulo Mineiro, Av. Frei Paulino 30, Uberaba, Minas Gerais, Brazil.

In the present study, the molecular karyotypes of 12 KP1(+) and KP1(-) Trypanosoma rangeli strains were determined and 10 different molecular markers were hybridized to the chromosomes of the parasite, including seven obtained from T. rangeli [ubiquitin hydrolase (UH), a predicted serine/threonine protein kinase (STK), hexose transporter, hypothetical protein, three anonymous sequences] and three from Trypanosoma cruzi [ubiquitin-conjugating enzyme E2 (UBE2), ribosomal RNA methyltransferase (rRNAmtr), proteasome non-ATPase regulatory subunit 6 (PSMD6)]. Despite intraspecific variation, analysis of the karyotype profiles permitted the division of the T. rangeli strains into two groups coinciding with the KP1(+) and KP1(-) genotypes. Southern blot hybridization showed that, except for the hexose transporter probe, all other probes produced distinct patterns able to differentiate the KP1(+) and KP1(-) genotypes. The UH, STK and An-1A04 probes exclusively hybridized to the chromosomes of KP1(+) strains and can be used as markers of this group. In addition, the UBE2, rRNAmtr and PSMD6 markers, which are present in a conserved region in all trypanosomatid species sequenced so far, co-hybridized to the same T. rangeli chromosomal bands, suggesting the occurrence of gene synteny in these species. The finding of distinct molecular karyotypes in KP1(+) and KP1(-) strains of T. rangeli is noteworthy and might be used as a new approach to the study of genetic variability in this parasite. Together with the Southern blot hybridization results, these findings demonstrate that differences at the kDNA level might be associated with variations in nuclear DNA.
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http://dx.doi.org/10.1016/j.actatropica.2009.01.004DOI Listing
April 2009

[Prevalence of triatomines (Hemíptera: Reduviidae: Triatominae) infected by Trypanosoma cruzi: seasonality and distribution in the Ciénega region of the State of Jalisco, Mexico].

Rev Soc Bras Med Trop 2008 May-Jun;41(3):257-62

Laboratório de Parasitologia, Universidade Federal do Triângulo Mineiro, Uberaba, MG, Brazil.

The physical and geographical characteristics of the Ciénega region, Jalisco, Mexico make it suitable for transmission of Trypanosoma cruzi, the causative agent for Chagas disease. This study characterizes the prevalence of triatomines infected by this parasite, their seasonality and their distribution in this region. A total of 328 triatomines were evaluated between January 2005 and June 2007, from 13 municipalities in the region. April, May and June were the months with the highest capture levels. Among the triatomines examined, 57.3% were positive for Trypanosoma cruzi, corresponding to 15.4% in urban areas and 84.6% in rural areas. The species with greatest prevalence was Triatoma longipennis and the species with the highest parasitism rate was Triatoma barberi, with an infection rate of 83.3%, whereas the rate for Triatoma longipennis was 67.5% (p<0.05). This natural infection in the captured vectors may indicate that individuals in this region have high exposure to Trypanosoma cruzi. The recent findings of positive Triatoma dimidiata in this region suggest that new species are becoming adapted to the ecological conditions of these populations.
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http://dx.doi.org/10.1590/s0037-86822008000300007DOI Listing
January 2009

Interstitial cells of Cajal in chagasic megaesophagus.

Ann Diagn Pathol 2008 Aug 18;12(4):271-4. Epub 2008 Apr 18.

Parasitology Division, Universidade Federal do Triângulo Mineiro, Uberaba, Minas Gerais 38025-180, Brazil.

Chagasic visceromegalies are the most important digestive manifestations of Chagas disease and are characterized by motor disorders and dilation of organs such as esophagus and colon. One of the theories raised to explain the physiopathogenesis of chagasic megas is the plexus theory. Recent studies have shown a reduction of interstitial cells of Cajal (ICCs) in the colon of chagasic patients. These cells are present throughout the gastrointestinal tract and are considered to be pacemaker cells, that is, they are responsible for coordinating peristalsis and for mediating nerve impulses. In view of the lack of studies on these cells in megaesophagus and the previous observation of a reduction of ICCs in chagasic megacolons, we compared the distribution of ICCs in the esophagus of chagasic and nonchagasic patients to contribute to a better understanding of the physiopathogenesis of this esophageal disease. Esophageal biopsy samples from 10 chagasic and 5 nonchagasic patients were used. Cells were identified with the anti-CD117 antibody. The number of ICCs was quantified in longitudinal and circular muscle layers and myenteric plexus. The results were analyzed statistically by comparison of means. An intense reduction in the number of ICCs was observed in muscle layers and in the myenteric plexus of patients with megaesophagus. We conclude that there is an intense reduction of ICCs in the esophagus of chagasic patients when compared to nonchagasic patients, a finding supporting the important role of these cells in gastrointestinal tract motility. A deficiency in these cells might be implied in the genesis of megaesophagus.
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http://dx.doi.org/10.1016/j.anndiagpath.2007.12.002DOI Listing
August 2008

Absence of experimental cross-protection induced by a Trypanosoma cruzi-like strain isolated from bats.

Rev Soc Bras Med Trop 2008 Mar-Apr;41(2):152-7

Departamento de Ciências Biológicas, Universidade Federal do Triângulo Mineiro, Uberaba, MG.

This study evaluated the possibility of inoculation and reinoculation with a trypanosomatid isolated from bats that is morphologically, biologically and molecularly similar to Trypanosoma cruzi, to protect against infection by virulent strains. Non-isogenic mice were divided into 24 groups that received from zero to three inoculations of Trypanosoma cruzi-like strain RM1, in the presence or absence of Freunds adjuvant, and were challenged with the VIC or JG strains of Trypanosoma cruzi. Parasitemia and survival were monitored and animals were sacrificed for histopathological analysis. Animals immunized with Trypanosoma cruzi-like strain RM1 presented decreased parasitemia, independently of the number of inoculations or the presence of adjuvant. In spite of this reduction, these animals did not present any protection against histopathological lesions. Severe eosinophilic infiltrate was observed and was correlated with the number of inoculations of Trypanosoma cruzi-like strain RM1. These findings suggest that prior inoculation with this strain did not protect against infection but, rather, aggravated the tissue inflammatory process.
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http://dx.doi.org/10.1590/s0037-86822008000200004DOI Listing
November 2008

[Population parameters for Triatoma sordida Stal, 1859: the most frequent vector for Chagas disease in the Triângulo Mineiro (Heteroptera, Triatominae)].

Rev Soc Bras Med Trop 2007 Jan-Feb;40(1):25-8

Departamento de Ciências Biológicas, Universidade Federal do Triângulo Mineiro, Uberaba, MG.

Triatoma sordida is the most frequent vector for Trypanosoma cruzi, Chagas, 1909, in Uberaba, State of Minas Gerais. The objective of this study was to construct a dynamic life table for Triatoma sordida with the aim of supplying support data for controlling its populations.
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http://dx.doi.org/10.1590/s0037-86822007000100005DOI Listing
July 2007

Variability of kinetoplast DNA gene signatures of Trypanosoma cruzi II strains from patients with different clinical forms of Chagas' disease in Brazil.

J Clin Microbiol 2006 Jun;44(6):2167-71

Departamento de Ciências Biológicas, Universidade Federal do Triângulo Mineiro, Uberaba, Brazil.

The clinical course of Chagas' disease varies widely among different patients and geographic regions. For reasons that are not completely understood but involve host and parasite factors, some patients never develop the disease while others present cardiac and/or gastrointestinal symptoms. Many studies have been conducted in order to correlate the genetic variability of the parasites with the clinical forms of the disease, but no conclusive data have been obtained. Our research aims at characterizing the genetic profiles of Trypanosoma cruzi isolates recently obtained from 70 chagasic patients who either showed pathological lesions with symptoms of various intensities or were asymptomatic. All patients came from an area where Chagas' disease is endemic in southeast Brazil where vectorial transmission has been controlled and different clinical forms of the disease can be found. The molecular characterization of parasites evaluated the polymorphisms of the 3' region of the 24Salpha rRNA gene and the variability of kinetoplast DNA (kDNA) minicircles of T. cruzi populations by low-stringency single specific primer PCR. Data presented here provide a strong correlation between T. cruzi II and human infection in this region. However, a high degree of variability was observed within T. cruzi II, as demonstrated by intense kDNA polymorphism among all clinical forms and also within each of them, irrespective of the intensity of pathological processes.
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http://dx.doi.org/10.1128/JCM.02124-05DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1489452PMC
June 2006

Susceptibility of different triatomine species to Trypanosoma rangeli experimental infection.

Vector Borne Zoonotic Dis 2006 ;6(1):50-6

Disciplina de Parasitologia, Departamento de Ciências Biológicas, Universidade Federal do Triângulo Mineiro, Uberaba, Minas Gerais, Brazil.

Trypanosoma rangeli is a kinetoplastid protozoan parasite that has been found in the majority of Latin American countries, overlapping its distribution area with that of Trypanosoma cruzi, the causative agent of Chagas disease. This parasite shares the same reservoirs and vectors as T. cruzi. Triatomines from genus Rhodnius are considered the most susceptible hosts to infection. In this work, we report the susceptibility of different triatomine species (Rhodnius neglectus, Panstrongylus megistus, Triatoma infestans, T. sordida, T. braziliensis, and T. vitticeps) to experimental infection by T. rangeli isolated from Didelphis albiventris in a highly endemic region for Chagas disease. An intense parasitism was evidenced in feces (56% to 81%) of the majority of the species studied on the 10th day after infection, decreasing during the period of the experiment (30 days). T. vitticeps did not present parasites in feces at any time. All triatomine species presented parasites in the hemolymph. In T. vitticeps and P. megistus, this parasitism was scarce (6.3% and 6.6%, respectively). In the other species, the parasitism was variable (62.5% to 100%). Triatomine mortality varied between 3% to 40%, increasing during the infection in all species studied. The lowest mortality was observed for T. infestans. Also, we showed that only trypomastigotes forms from salivary glands, and hemolymph were infective for mice. We conclude that all triatomine species used were susceptible to infection by T. rangeli at different levels. There was no direct correlation between intensity of parasitism and mortality.
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http://dx.doi.org/10.1089/vbz.2006.6.50DOI Listing
May 2007

Functional and histopathological study of the pancreas in hamsters (Mesocricetus auratus) infected and reinfected with Trypanosoma cruzi.

Parasitol Res 2004 Sep;94(2):125-33

Department of Internal Medicine, Faculdade de Medicina do Triângulo Mineiro, Uberaba, Minas Gerais, Brazil.

Histopathological and functional changes in the pancreas were studied in 94 hamsters infected and reinfected with Trypanosoma cruzi VIC strain and in 73 non-infected normal controls. Infection in each animal was verified by microhematocrit, hemoculture, specific peroxidase anti-peroxidase, polymerase chain reaction and seroagglutination. Blood glucose and insulin were determined. The number of islets per section and the number of islet cells marked with antibodies were counted. Insulitis, neuritis, fibrosis, atrophy and inflammatory infiltrates were evaluated. Experimental chagasic infection caused pancreatitis similar to human Chagas' disease, involving acini, islets and nerves, with atrophy and fibrosis, although without correlation to the number of reinfections. Erratic blood glucose levels and a tendency to hypoinsulinemia were observed in infected animals. During the acute phase, the number of somatostatin and pancreatic polipeptide producer islet cells was lower in infected hamsters, which was eventually related to changes in blood sugar levels and hypoinsulinemia. Our findings favor the hypothesis of the existence of an endocrine form of chronic chagasic infection.
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http://dx.doi.org/10.1007/s00436-004-1183-8DOI Listing
September 2004