Metabolic and functional consequences of cytosolic 5′-nucleotidase-IA overexpression in neonatal rat cardiomyocytes

Adenosine exerts a spectrum of energy-preserving actions on the heart negative chronotropic effects. The pathways leading to adenosine formation have remained controversial. In particular, although cytosolic 5′-nucleotidases can catalyze adenosine formation in cardiomyocytes, their contribution to the actions of adenosine has not been documented previously. We recently cloned two closely related AMP-preferring cytosolic 5′-nucleotidases (cN-IA and -IB); the A form predominates in the heart. In this study, we overexpressed pigeon cN-IA in neonatal rat cardiomyocytes using an adenovirus. cN-IA overexpression increased adenosine formation and release into the medium caused by simulated hypoxia and by isoproterenol in the absence and presence of inhibitors of adenosine metabolism. Adenosine release was not affected by an ecto-5′-nucleotidase inhibitor, α,β-methylene-ADP, but was affected by a nucleoside transporter, dipyridamole. The positive chronotropic effect of isoproterenol (130 ±3 vs. 100 ±4 beats/min) was inhibited (107 ±3 vs. 94 ±3 beats/min) in cells overexpressing cN-IA, and this was reversed by the addition of the adenosine receptor antagonist 8-(p-sulfophenyl)theophilline (120 ± 3 vs. 90 ± 4 beats/min). Our results demonstrate that overexpressed cN-IA can be sufficiently active in cardiomyocytes to generate physiologically effective concentrations of adenosine at its receptors.Fil: Sala-Newby, Graciela B.. University of Bristol; Reino UnidoFil: Freeman, Nicola V. E.. University of Bristol; Reino UnidoFil: Curto, Maria de Los Angeles. University of Bristol; Reino Unido. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Newby, Andrew C.. University of Bristol; Reino Unid

Uroguanylin regulates net fluid secretion via the NHE2 isoform of the Na+/H+ exchanger in an intestinal cellular model

Uroguanylin (UGN) has been proposed as a key regulator of salt and water intestinal transport. Uroguanylin (UGN) activates cell-surface guanylate cyclase C receptor (GC-C) and modulates cellular function via cyclic GMP (cGMP), thus increasing electrolyte and net water secretion. It has been suggested that the action of UGN could involve the Na+/H+ exchanger, but the actual contribution of this transporter still remains unclear. The objective of our study was to investigate the putative effects of UGN on some members of the Na+/H+ exchanger (NHE) family, as well as to clarify its consequences on transepithelial fluid flow in T84 cells. In order to do so, transepithelial fluid flow (Jv) was studied by optic techniques and intracellular pH (pHi) was measured with a fluorescence method. Results showed that NHE2 is found at the apical membrane and has a major role in Na+ absorption; NHE1 and NHE4 are localized at the basolateral membrane with a house-keeping role in steady state pHi. Cell exposure to apical UGN increases net secretory Jv, without changing short-circuit currents nor transepithelial resistance, and reduces NHE2 activity. Therefore, at physiological pH, the effect on net Jv was produced mainly by a reduction in normal Na+ absorption through NHE2, rather than by the stimulation of electrolyte secretion. Our study shows that the effect of UGN on pHi is GC-C/cGMP-mediated and enhanced by sildenafil, thus involving PDE5 enzyme. Additionally, cell exposure to apical UGN results in intracellular alkalinization, probably due to indirect effects on basolateral NHE1 and NHE4, which have a major role in pHi regulation.Fil: Toriano, Roxana Mabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Biomembranas; ArgentinaFil: Ozu, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Biomembranas; ArgentinaFil: Politi, María T.. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Biomembranas; ArgentinaFil: Dorr, Ricardo Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Biomembranas; ArgentinaFil: Curto, Maria de Los Angeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Capurro, Claudia Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Biomembranas; Argentin

Analytical sensitivity and specificity of a loop-mediated isothermal amplification (LAMP) kit prototype for detection of Trypanosoma cruzi DNA in human blood samples

This study aimed to assess analytical parameters of a prototype LAMP kit that was designed for detection of Trypanosoma cruzi DNA in human blood. The prototype is based on the amplification of the highly repetitive satellite sequence of T.cruzi in microtubes containing dried reagents on the inside of the caps. The reaction is carried out at 65°C during 40 minutes. Calcein allows direct detection of amplified products with the naked eye. Inclusivity and selectivity were tested in purified DNA from Trypanosoma cruzi stocks belonging to the six discrete typing units (DTUs), in DNA from other protozoan parasites and in human DNA. Analytical sensitivity was estimated in serial dilutions of DNA samples from Sylvio X10 (Tc I) and CL Brener (Tc VI) stocks, as well as from EDTA-treated or heparinized blood samples spiked with known amounts of cultured epimastigotes (CL Brener). LAMP sensitivity was compared after DNA extraction using commercial fiberglass columns or after ?Boil & Spin? rapid preparation. Moreover, the same DNA and EDTA-blood spiked samples were subjected to standardized qPCR based on the satellite DNA sequence for comparative purposes. A panel of peripheral blood specimens belonging to Chagas disease patients, including acute, congenital, chronic and reactivated cases (N = 23), and as well as seronegative controls (N = 10) were evaluated by LAMP in comparison to qPCR. LAMP was able to amplify DNAs from T. cruzi stocks representative of the six DTUs, whereas it did not amplify DNAs from Leishmania sp, T. brucei sp, T. rangeli KPN+ and KPN-, P. falciparum and non-infected human DNA. Analytical sensitivity was 1x10-2fg/μL of both CL Brener and Sylvio X10 DNAs, whereas qPCR detected up to 1x 10−1fg/μL of CL Brener DNA and 1 fg/μl of Sylvio X10 DNA. LAMP detected 1x10-2parasite equivalents/mL in spiked EDTA blood and 1x10-1par.eq/mL in spiked heparinized blood using fiberglass columns for DNA extraction, whereas qPCR detected 1x10-2par.eq./mL in EDTA blood. Boil & Spin extraction allowed detection of 1x10-2par.eq /mL in spiked EDTA blood and 1 par.eq/ml in heparinized blood. LAMP was able to detect T.cruzi infection in peripheral blood samples collected from well-characterised seropositive patients, including acute, congenital, chronic and reactivated Chagas disease. To our knowledge, this is the first report of a prototype LAMP kit with appropriate analytical sensitivity for diagnosis of Chagas disease patients, and potentially useful for monitoring treatment response.Fil: Besuschio, Susana Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Llano Murcia, Mónica. Pontificia Universidad Javeriana; ColombiaFil: Benatar, Alejandro Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Monnerat, Severine. Foundation for Innovative New Diagnostics; SuizaFil: Cruz, Israel. Foundation for Innovative New Diagnostics; SuizaFil: Picado, Albert. Foundation for Innovative New Diagnostics; SuizaFil: Curto, Maria de Los Angeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Kubota, Yutaka. Eiken Chemical Company; JapónFil: Wehrendt, Diana Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Pavia, Paula. Pontificia Universidad Javeriana; ColombiaFil: Mori, Yasuyoshi. Eiken Chemical Company; JapónFil: Puerta, Concepción. Pontificia Universidad Javeriana; ColombiaFil: Ndung'u, Joseph M.. Foundation for Innovative New Diagnostics; SuizaFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

A flow cytometer-based method to simultaneously assess activity and selectivity of compounds against the intracellular forms of Trypanosoma cruzi

Chagas disease is a major unsolved health issue in Latin America and an emerging threat worldwide. New drugs are urgently needed for chemotherapy as those available (benznidazole and nifurtimox) have variable efficacy and elevated toxicity. Efforts are actually oriented to improve tools and technologies (e.g. transgenic parasites, flow cytometry or image-based systems) for the screening of large numbers of candidate compounds for their activity against Trypanosoma cruzi (T. cruzi). Methods that test drug efficacy and selectivity in the same assay are suitable to accelerate the process of drug discovery. Here, we developed a GFP expressing T. cruzi from a moderate virulence stock and confirmed that the transgenic parasite retained the biological characteristics of the parental strain. With this tool, we established a flow cytometer-based method to simultaneously test drug activity against intracellular amastigotes and toxicity to the host cell. This one-step procedure allows determining the selectivity index of the tested compound in a sensitive and accurate manner even with low infection rates. This method can provide additional information on the interactions between drug, parasites and host cell and could be adapted to other trypanosomatids and protozoa with intracellular multiplication.Fil: Miranda, Cristian Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Solana, Maria Elisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Curto, Maria de Los Angeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Lammel, Estela Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Alba Soto, Catalina Dirney. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentin

F8 intron 22 inversions and SNP rs73563631 in unrelated families with severe haemophilia A: clinical features and gene testing implications

F8 intron 22 inversions (INV22), the commonest cause of severe haemophilia A (HA), are mostly represented by type I (INV22-1) and type II (INV22-2) patterns (4:1) using BclI-fragments´ based techniques such as inverse shifting-PCR (IS-PCR/2008). Using IS-PCR/2008, we identified unusual patterns of the INV22-1 and INV22-2 (characterised by no signals in the diagnostic test and conventional INV22-1/-2 patterns in the complementary test) in patients and carriers from two (0.6%) out of 308 Argentinean families with severe-HA. A theoretical analysis of the 85 SNPs embedded in the relevant BclI-fragments followed by PCR-BclI-RFLP analysis allowed identification of the SNP rs73563631*G allele associated with a new BclI-restriction site in all four patients of Family 1 and 2. Linkage analysis using seven F8-linked-STRs in the two families confirmed two unrelated haplotypes in phase with INV22-1/-2. Screening of 404 X-chromosomes from the Argentinean general population failed to detected SNP rs73563631*G allele (q<0.24%). A new version of the IS-PCR/2008 adding the genotyping of the new patterns (x) of INV22-1/-2 was developed. Clinical/biochemical characteristics (severity and inhibitor risks) of patients with INV22-1x/-2x were virtually equal to canonical INV22s. Our estimations predict the involvement of INV22-1x/-2x in about 2.7% (4/149) of INV22-affected cases worldwide.Fil: Abelleyro, Miguel Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Rossetti, Liliana Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Curto, Maria de Los Angeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular ; ArgentinaFil: Radic, Claudia Pamela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Marchione, Vanina Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: de Brasi, Carlos Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas ; Argentin

Differential infectivity of two Trypanosoma cruzi strains in placental cells and tissue

Congenital Chagas disease, caused by Trypanosoma cruzi (T. cruzi), has become epidemiologically relevant. The probability of congenital transmission depends on the maternal and developing fetal/newborn immune responses, placental factors and importantly, the virulence of the parasite. It has been proposed, that different genotypes of T. cruzi and their associated pathogenicity, virulence and tissue tropism may play an important role in congenital infection. Since there is no laboratory or animal model that recapitulates the complexities of vertical transmission in humans, here we studied parasite infectivity in human placental explants (HPE) as well as in the human trophoblast-derived cell line BeWo of the Y(DTU II) and the VD (TcVI) T. cruzi strains; the latter was isolated from a human case of congenital infection. Our results show that the VD strain is more infective and pathogenic than the Y strain, as demonstrated by qPCR and cell counting as well as by histopathological analysis. The present study constitutes the first approach to study the relationship between parasite two parasite strains from different genotypes and the infection efficiency in human placenta.Fil: Medina, Lisvaneth. Universidad de Chile; ChileFil: Castillo, Christian. Universidad de Chile; ChileFil: Liempi, Ana. Universidad de Chile; ChileFil: Herbach, Mathias. Universidad de Chile; ChileFil: Cabrera, Gonzalo. Universidad de Chile; ChileFil: Valenzuela, Lucía. Universidad de Chile; ChileFil: Galanti, Norbel. Universidad de Chile; ChileFil: Curto, Maria de Los Angeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Kemmerling, Ulrike. Universidad de Chile; Chil

Cloning and expression of transgenes using linear vectors in Trypanosoma cruzi

The identification of new targets for vaccine and drug development for the treatment of Chagas' disease is dependent on deepening our understanding of the parasite genome. Vectors for genetic manipulation in Trypanosoma cruzi basically include those that remain as circular episomes and those that integrate into the parasite's genome. Artificial chromosomes are alternative vectors to overcome problematic transgene expression often occurring with conventional vectors in this parasite. We have constructed a series of vectors named pTACs (Trypanosome Artificial Chromosomes), all of them carrying telomeric and subtelomeric sequences and genes conferring resistance to different selection drugs. in addition, one pTAC harbours a modified GFP gene (pTAC-gfp), and another one carries the ornithine decarboxilase gene from Crithidia fasciculata (pTAC-odc). We have encountered artificial chromosomes generated from pTACs in transformed T. cruzi epimastigotes for every version of the designed vectors. These extragenomic elements, in approximately 6-8 copies per cell, remained as linear episomes, contained telomeres and persisted after 150 and 60 generations with or without selection drugs, respectively. the linear molecules remained stable through the different T. cruzi developmental forms. Furthermore, derived artificial chromosomes from pTAC-odc could complement the auxotrophy of T. cruzi for polyamines. Our results show that pTACs constitute useful tools for reverse functional genetics in T. cruzi that will contribute to a better understanding of T. cruzi biology. (C) 2014 Published by Elsevier B.V. on behalf of Australian Society for Parasitology Inc.World Health OrganizationAgencia Nacional de Investigaciones Cientificas y Tecnologicas (Argentina)Consejo Nacional de Investigaciones Cientificas y Tecnologicas (CONICET,Argentina)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)United Nations University-Biotechnology for Latin America and the CaribbeanInst Invest Ingn Genet & Biol Mol INGEBI CONICET, Lab Biol Mol Enfermedad Chagas, Buenos Aires, DF, ArgentinaUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, Escola Paulista Med, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, Escola Paulista Med, São Paulo, BrazilWeb of Scienc

A loop-mediated is othermal amplification (LAMP) kit for molecular detection of Trypanosoma cruzi dna: A feasibility study

Loop-mediated isothermal amplification (LAMP) tests have been developedas molecular tests for neglected parasitic diseases such as leishmaniasisor sleeping sickness. A LAMP test for Trypanosoma cruzi, the etiologicalagent of Chagas Disease (ChD), would allow a rapid and reliable diagnosis,in particular in cases of acute and congenital ChD (CChD). We evaluatedthe performance a Trypanosoma cruzi LAMP kit using purified DNA, spikedblood and clinical specimens. Quantitative PCR (qPCR) was used as areference. Different extraction methods for LAMP were also evaluated. TheLAMP reaction was performed at 62.5°C for 45 min. Analytical sensitivitywas measured in ten-fold dilutions of CL Brener (TcVI) and Silvio X10(TcI) DNA. Analytical specificity was measured using ten-fold dilutions ofdifferent Leishmania species and Trypanosoma rangeli DNAs as well asnon-infected human DNA. Seronegative blood in EDTA (EB) or heparin (HB)was spiked with ten-fold dilutions of CL Brener. EB spiked blood was alsoused as dried blood spot (DBS). Stored DNA from EB clinical samples wastested, including 4 Congenital ChD cases, 5 Chronic ChD cases with lowparasite loads, 10 immunosuppressed ChD patients and 5 seronegativecontrols. DNA extraction was done with a commercial kit (EB, HB and DBSsamples) and using the boil & spin (B&S) method (HB samples only). TheT. cruzi LAMP kit showed better analytical sensitivity than qPCR in purifiedDNA specimens, especially for TcI DNA. Analytical sensitivity was 10-2 and10-1 par.eq/mL from spiked EB and HB extracted by columns, respectively,and 10-2 par.eq/mL from HB using B&S. The analytical sensitivity in DBSsamples was 10-2 par.eq/mL. T. cruzi LAMP was positive in congenital andimmunosuppressed ChD samples spanning from 4.8 to 3,684 par.eq/ml,in agreement with qPCR. Chronic ChD samples were only detectable byqPCR, with Ct values below the limit of quantification. The kit was specificfor T. cruzi DNA and samples from seropositive patients.Fil: Besuschio, Susana Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Llano Murcia, Mónica. Pontificia Universidad Javeriana; ColombiaFil: Benatar, Alejandro Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Curto, Maria de Los Angeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Cruz, Israel. Foundation For Innovative Diagnostics; SuizaFil: Monnerat, Séverine. Foundation For Innovative Diagnostics; SuizaFil: Picado, Albert. Foundation For Innovative Diagnostics; SuizaFil: Puerta, Concepción. Pontificia Universidad Javeriana; ColombiaFil: Ndungu, Joseph. Foundation For Innovative Diagnostics; SuizaFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaThe 65th American Society of Tropical Medicine and Hygiene Annual MeetingAtlantaEstados UnidosAmerican Society of Tropical Medicine and Hygien

Development of duplex TaqMan PCR assays for detection and quantification of Trypanosoma cruzi infection in wild and domestic reservoirs

A major question of epidemiological relevance in Chagas disease studies is to understand the dynamics ofT.cruzi infection in sylvatic and domestic transmission cycles and to trace the origins of (re)emerging casesin areas under vector or disease surveillance. Conventional parasitological methods lack sensitivity and molecularapproaches, such as the polymerase chain reaction may ll in this gap, provided that a standardizedmethod can be developed and validated.We have developed two duplex Real Time PCR assays for sensitive detection of T.cruzi satellite or minicirclerepetitive sequences in blood samples from mammal reservoirs, incorporating an internal amplicationstandard that allows distinction of false negative PCR ndings due to inadequate conditions of storageand transport of samples, DNA degradation during nucleic acid purication and/or inhibition of PCR byinterfering substances present in the sample. The housekeeping gene that encodes the interphotoreceptorretinoid-binding protein (IRBP) has been selected as internal standard, because it is highly conserved amongall mammal species and its usefulness as a DNA integrity control was previously reported in a conventionalPCR protocol. Based on the alignment of the IRBP sequence available for several domestic and wild reservoirspecies, we designed primers and a TaqMan probe to a highly conserved region. The analytical sensitivitywas 0.01 par. eq/mL and 0.1 par.eq/mL for satDNA/IRBP duplex and kDNA/IRBP duplex, respectively, astested with two series of canine blood spiked with known concentrations of Silvio X10 (Tc I) and CL-Brener(Tc VI) cultured epimastigotes. The assays were evaluated in DNA extracts from blood samples of 87 wildand 147 domestic animals. Our DNA integrity control worked well for wild reservoir species including smallrodents (Akodon toba, Galea leucoblephara, Rattus rattus), opossums (Didelphis virginiana, D. marsupialis),bats (Tadarida brasiliensis, Promops nasutus, Desmodus rotundus) and other mammals such as the skunk,viscacha, wildcat, brown brocket deer, hare and Pampas fox. Mean Ct values for IRBP amplication variedamong species between 24 and 33. For domestic reservoirs, IRBP amplied in dog, cat, cow, sheep, goat,horse and mule specimens with mean Ct values between 23 and 25. In T.cruzi infected cases the assaysallowed quantication of parasitic loads.Our results promote the use of these methods for rapid and accurate screening of T.cruzi infection in dierentspecies of reservoirs.Fil: Wehrendt, Diana Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Gómez Bravo, Andrea. Fundación Mundo Sano; ArgentinaFil: Pech May, Angélica del Rosario. Instituto Nacional de Salud Pública; México. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ramsey, Janine. Instituto Nacional de Salud Pública; MéxicoFil: Cura, Carolina Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Curto, Maria de Los Angeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Abril, M.. Fundación Mundo Sano; ArgentinaFil: Guhl, F.. Universidad de los Andes; ColombiaFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaXXX Reunión Anual de la Sociedad Argentina de ProtozoologíaResistenciaArgentinaSociedad Argentina de Protozoologí