Characterization of Trypanosoma cruzi Strains Isolated from Chronic Chagasic Patients, Triatomines and Opossums Naturally Infected from the State of Rio Grande do Sul, Brazil

Thirty-five Trypanosoma cruzi strains were isolated from chronic chagasic patients, triatomines and opossums from different municipalities of the State of Rio Grande do Sul. Parasites were characterized by means of mice infectivity, enzyme electrophoresis and randomly amplified polymorphic DNA (RAPD) analysis. Twenty-nine strains were isolated from chagasic patients, 4 from triatomines (2 from Triatoma infestans and 2 from Panstrongylus megistus) and 2 from opossums Didelphis albiventris. Thirty-three T. cruzi strains were of low and 2 strains of high virulence in mice. Both virulent strains were isolated from P. megistus. Isoenzyme analysis of the strains showed 3 different zymodemes. Eleven strains isolated from chagasic patients and 2 from D. albiventris were Z2. Eighteen strains from patients and 2 from T. infestans were ZB and 2 T. cruzi strains isolated from P. megistus were Z1. RAPD profiles obtained with 4 random primers showed a high genetic heterogeneity of the T. cruzi strains. Zymodeme 2 and ZB strains were the more polymorphic. A band sharing analysis of the RAPD profiles of Z2 and ZB strains using 3 primers, showed a very low percentage of shared bands, 20% among 13 ZB strains and 14% among 13 Z2 strains. According to the isoenzyme results, 3 T. cruzi populations were present in State of Rio Grande do Sul. Zymodeme 2 and ZB strains were found infecting man (domiciliar transmission cycle) whereas Z1 strains were found infecting the sylvatic vector P. megistus

Genetic Variability and Microdistribution of Triatoma infestans Genotypes and Trypanosoma cruzi Clones in Arequipa Region (Peru)

The genetic variability of Triatoma infestans and Trypanosoma cruzi populations was studied by isoenzyme analysis in two distinct areas of Arequipa province (Peru); one, Santa Rita de Siguas, being an endemic area for Chagas' disease, the second, Arequipa, recently infected. Analysis of T. infestans genetic variability indicates, (i) temporal stability of genotypes found in Santa Rita de Siguas, (ii) high genetic differences between Arequipa and Santa Rita de Siguas populations suggesting minor contact between them, (iii) multiple origin of the T. infestans population in Arequipa, and (iv) poor dispersal capacity of T. infestans: the panmictic unit could be reduce to a house. Parasite isoenzyme analysis was performed in 29 Peruvian stocks of T. cruzi, mainly isolated from bugs taken in a single locality, Santa Rita de Siguas. The results show, (i) a high genetic polymorphism, (ii) nine different multilocus genotypes were detected and clustered in two different clades, (iii) most of the parasite isolates pertained to one of the clade and were genetically similar to those analyzed 12 years before. This sample allowed the study of the mating system of T. cruzi in strict sympatic conditions and gave more strength to the hypothesis of the clonal structure of T. cruzi populations

First Case of Natural Infection in Pigs: Review of Trypanosoma cruzi Reservoirs in Mexico

An epidemiological research project was performed in the State of Morelos including collection of samples for blood smears and culture, serological tests, and xenodiagnoses from a total of 76 domestic and peridomestic mammals. Two strains of Trypanosoma cruzi were isolated by haemocultures; one from a pig (Sus scrofa), the first case of natural infection reported in Mexico, and the other from a dog (Canis familiaris). This study summarizes current information in Mexico concerning confirmed reservoirs of T. cruzi

Immune Response to Trypanosoma cruzi Shed Acute Phase Antigen in Children from an Endemic Area for Chagas' Disease in Bolivia

A field study of the immune response to the shed acute phase antigen (SAPA) of Trypanosoma cruzi was carried out in the locality of Mizque, Cochabamba department, Bolivia. Schoolchildren (266), with an average of 8.6 ± 3.6 years, were surveyed for parasitological and serological diagnosis, as well as antibodies directed against SAPA using the corresponding recombinant protein in ELISA. The antibodies against SAPA were shown in 82% of patients presenting positive serological diagnosis (IgG specific antibodies). The positive and negative predictive values were 0.88. Antibodies anti-SAPA were shown in 80.8% of the chagasic patients in the initial stage of the infection (positive IgM serology and/or positive buffy coat (BC) test) and in 81.4% of the patients in the indeterminate stage of the infection (positive IgG serology with negative BC and IgM tests). These results show that the anti-SAPA response is not only present during the initial stage of the infection (few months) but extends some years after infection

TcruziDB, an Integrated Database, and the WWW Information Server for the Trypanosoma cruzi Genome Project

Data analysis, presentation and distribution is of utmost importance to a genome project. A public domain software, ACeDB, has been chosen as the common basis for parasite genome databases, and a first release of TcruziDB, the Trypanosoma cruzi genome database, is available by ftp from ftp://iris.dbbm.fiocruz.br/pub/genomedb/TcruziDB as well as versions of the software for different operating systems (ftp://iris.dbbm.fiocruz.br/pub/unixsoft/). Moreover, data originated from the project are available from the WWW server at http://www.dbbm.fiocruz.br. It contains biological and parasitological data on CL Brener, its karyotype, all available T. cruzi sequences from Genbank, data on the EST-sequencing project and on available libraries, a T. cruzi codon table and a listing of activities and participating groups in the genome project, as well as meeting reports. T. cruzi discussion lists (tcruzi-l@iris.dbbm.fiocruz.br and tcgenics@iris.dbbm.fiocruz.br) are being maintained for communication and to promote collaboration in the genome project

Biological Parameters and Molecular Markers of Clone CL Brener - The Reference Organism of the Trypanosoma cruzi Genome Project

Clone CL Brener is the reference organism used in the Trypanosoma cruzi Genome Project. Some biological parameters of CL Brener were determined: (a) the doubling time of epimastigote forms cultured in liver infusion-tryptose (LIT) medium at 28oC is 58±13 hr; (b) differentiation of epimastigotes to metacyclic trypomastigotes is obtained by incubation in LIT-20% Grace´s medium; (c) trypomastigotes infect mammalian cultured cells and perform the complete intracellular cycle at 33 and 37oC; (d) blood forms are highly infective to mice; (e) blood forms are susceptible to nifurtimox and benznidazole. The molecular typing of CL Brener has been determined: (a) isoenzymatic profiles are characteristic of zymodeme ZB; (b) PCR amplification of a 24Sa ribosomal RNA sequence indicates it belongs to T. cruzi lineage 1; (c) schizodeme, randomly amplified polymorphic DNA (RAPD) and DNA fingerprinting analyses were performed

The Trypanosoma cruzi Genome Project: Nuclear Karyotype and Gene Mapping of Clone CL Brener

By using improved pulsed field gel electrophoresis conditions, the molecular karyotype of the reference clone CL Brener selected for Trypanosoma cruzi genome project was established. A total of 20 uniform chromosomal bands ranging in size from 0.45 to 3.5 Megabase pairs (Mbp) were resolved in a single run. The weighted sum of the chromosomal bands was approximately 87 Mbp. Chromoblots were hybridized with 39 different homologous probes, 13 of which identified single chromosomes. Several markers showed linkage and four different linkage groups were identified, each comprising two markers. Densitometric analysis suggests that most of the chromosomal bands contain two or more chromosomes representing either homologous chromosomes and/or heterologous chromosomes with similar sizes

Towards the Physical Map of the Trypanosoma cruzi Nuclear Genome: Construction of YAC and BAC Libraries of the Reference Clone T. cruzi CL-Brener

Strategies to construct the physical map of the Trypanosoma cruzi nuclear genome have to capitalize on three main advantages of the parasite genome, namely (a) its small size, (b) the fact that all chromosomes can be defined, and many of them can be isolated by pulse field gel electrophoresis, and (c) the fact that simple Southern blots of electrophoretic karyotypes can be used to map sequence tagged sites and expressed sequence tags to chromosomal bands. A major drawback to cope with is the complexity of T. cruzi genetics, that hinders the construction of a comprehensive genetic map. As a first step towards physical mapping, we report the construction and partial characterization of a T. cruzi CL-Brener genomic library in yeast artificial chromosomes (YACs) that consists of 2,770 individual YACs with a mean insert size of 365 kb encompassing around 10 genomic equivalents. Two libraries in bacterial artificial chromosomes (BACs) have been constructed, BACI and BACII. Both libraries represent about three genome equivalents. A third BAC library (BAC III) is being constructed. YACs and BACs are invaluable tools for physical mapping. More generally, they have to be considered as a common resource for research in Chagas disease

Parasite Genome Projects and the Trypanosoma cruzi Genome Initiative

Since the start of the human genome project, a great number of genome projects on other "model" organism have been initiated, some of them already completed. Several initiatives have also been started on parasite genomes, mainly through support from WHO/TDR, involving North-South and South-South collaborations, and great hopes are vested in that these initiatives will lead to new tools for disease control and prevention, as well as to the establishment of genomic research technology in developing countries. The Trypanosoma cruzi genome project, using the clone CL-Brener as starting point, has made considerable progress through the concerted action of more than 20 laboratories, most of them in the South. A brief overview of the current state of the project is given

Identification of Transcribed Sequences (ESTs) in the Trypanosoma cruzi Genome Project

Random single pass sequencing of cDNA fragments, also known as generation of Expressed Sequence Tags (ESTs), has been highly successful in the study of the gene content of higher organisms, and forms an integral part of most genome projects, with the objective to identify new genes and targets for disease control and prevention and to generate mapping probes. In the Trypanosoma cruzi genome project, EST sequencing has also been a starting point, and here we report data on the first 797 sequences obtained, partly from a CL Brener epimastigote non-normalized library, partly on a normalized library. Only around 30% of the sequences obtained showed similarity with Genbank and dbEST databases, half of which with sequences already reported for T. cruzi.