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    Agarose gel electrophoresis of PCR products corresponding to ME: a hypervariable region of the mini-exon gene non-transcribed spacer that is able to characterize T. cruzi I (200 bp), II (250 bp), and Z3 (150 bp); and to 24Sα: the D7 domain of the 24Sα rDNA with primers D71 and D72 that are able to characterize T.cruzi I (110 bp), TCIIb (125 bp), TCIIa (117 bp), and TCIIc (110 bp). Lanes: molecular DNA marker; T. cruzi reference isolates: TcG (TCI), TcY(TCIIb), TcJJ (TCIIa), and Tc3663 (TCIIc); stocks of T. cruzi isolated from Triatoma rubrovaria: QBI, QJI, QJIII, QMI, and QMII.

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    Phylogenetic tree based on the rRNA V7-V8 900-bp sequence. All isolates typed by TCI and TCII were clustered into two strongly supported groups (100% of bootstrap). Z3 lineages were segregated in two subgroups: Z3B composed of isolates that clustered with the reference strains José Júlio, CANIII; and Z3A composed by all T. cruzi isolates from T. rubrovaria, RS and by the reference isolates from Amazonas, 3663 and 3869 from Panstrongylus geniculatus and human, respectively (100% of bootstrap). Reference strains: T. cruzi G, Colombiana, Dm28c (TCI); TcY, Peru (TCIIb); CANIII, M2574, JJulio (TCIIa); 3869 and 3663 (TCIIc); isolates of T. cruzi obtained from Triatoma rubrovaria: QBI and QMI.

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    Parasitemic levels in mice infected with different isolates of T. cruzi obtained from T. rubrovaria. The mean values were obtained from five independent experiments and SDs are represented by bars.

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    Histopathologic aspects observed in Swiss mice infected with T. cruzi isolates obtained from T. rubrovaria. A–C, Acute phase of infection. A, Section of mouse skeletal muscle infected with isolate QMI presenting mononuclear inflammatory infiltration; H&E, ×400. B, Myocardium of mouse infected with isolate QMII presenting myocarditis, amastigote nests, and disruption of muscle fibers; H&E, ×400. C, Skeletal muscle of mouse infected with QMII isolate presenting amastigote nests; H&E, ×400. D–F, Chronic phase of infection. D, Skeletal muscle of mouse infected with QJIII isolate presenting chronic myositis and cellular disruption; H&E, ×400. E, Skeletal muscle of mouse infected with QJI isolate presenting intense chronic inflammatory infiltration; H&E, ×400. F, Section of colon of mouse infected with QMII isolate presenting chronic inflammation in the mesenteric plexus; H&E, ×400. H&E, slides stained with hematoxylin and eosin.

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Rural Triatoma rubrovaria from Southern Brazil Harbors Trypanosoma cruzi of Lineage IIc

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  • 1 Discipline of Parasitology, Marília Medical School, São Paulo, Brazil; Department of Parasitology, Institute of Medical Sciences, University of São Paulo, São Paulo, Brazil; Discipline of Pathology, Marília Medical School, São Paulo, Brazil; Department of Biological Sciences, School of Pharmaceutical Sciences, São Paulo State University, São Paulo, Brazil; Discipline of Molecular Biology, Marília Medical School, São Paulo, Brazil

Triatoma infestans, the main vector of Chagas disease, has nearly been eliminated from Brazil. Nevertheless, other triatominae species are involved in the domiciliation process, including Triatoma rubrovaria in Rio Grande do Sul State (RS). Previous studies showed that 1.6% of the T. rubrovaria specimens collected at the rural district of Quaraí, RS, were naturally infected by Trypanosoma cruzi. In this study, five T. cruzi isolates obtained from infected triatomines were characterized molecularly and biologically. Genotyping of the T. cruzi isolates showed that they belong to lineage IIc of T. cruzi (TCIIc). Biological characterization showed miotropism and myositis during acute and chronic phases of infection, respectively. Virulence and mortality rates were variable among isolates. To our knowledge, this study corresponds to the first characterization of T. cruzi isolates from T. rubrovaria and the first description of TCIIc in the sylvatic cycle of T. cruzi from the southern region of Brazil.

INTRODUCTION

Trypanosoma cruzi is the etiologic agent of Chagas disease (American trypanosomiasis), a complex zoonosis that affects 16–18 million people in Latin America.1 Parasite routes of infection include exposure of abraded skin to feces of infected triatomine bugs, ingestion of food contaminated with infected insects, blood transfusion, and congenital contagion. Also, transmission of T. cruzi can occur as domestic, peridomestic, or sylvatic cycles, depending on the ecologic behavior of the triatomine and host species involved.2

Clinical presentation of Chagas disease is highly variable, involving expression as negligible, acute, indeterminate, and chronic.3 Pathogenesis, response to chemotherapy, geographic variation, clinical presentation, and morbidity of Chagas disease have been linked to biological, biochemical, and genetic characteristics of T. cruzi strains.4 The biological behavior of T. cruzi strains in laboratory animals has allowed the distinction of three different biodemes.5 Specific electrophoretic patterns of different enzymes (zymodemes), which may be associated with biological characteristics,6,7 differentiated T. cruzi strains in three major zymodemes: zymodeme 1 (Z1), Z2, and Z3.8 Analysis of the 24Sα rRNA and the mini-exon gene non-transcribed spacer sequences by polymerase chain reaction (PCR) enabled the identification of two main groups of T. cruzi: T. cruzi I (TCI) and TCII, correlated to Z1 and Z2, respectively.9,10 Parasite isolates characterized as Z3 and hybrid strains included in to rDNA group 1/211,12 could not be classified as TCI or TCII. More recently, genetic approaches grouped T. cruzi in to discrete typing units (DTUs),13,14 DTUI (TCI), and DTUII (TCII), which is subdivided in to DTUs/TCIIa to IIe. By this classification,1517 the Z3 group is associated with TCIIa and TCIIc, and hybrid groups of T. cruzi are included in TCIId and TCIIe. TCIIa and TCIIc correspond, respectively, to the Z3B and Z3A subdivisions described by Mendonça and others.18

TCI, TCIIa, and TCIIc predominate in sylvatic transmission cycles from the Amazon basin northward, being the main cause of Chagas disease in countries such as Venezuela and Mexico.19,20 TCIIb, TCIId, and TCIIe groups are mainly represented by parasites related to the domestic transmission cycles in Southern Cone countries of South America including Argentina, Brazil, Bolivia, Chile, Paraguay, and Uruguay,21 where chagasic megasyndromes are common by way of contrast with their virtual absence to the north of the Amazon, where TCI strains predominate. Despite many studies, differences in pathology between TCI and TCII infections remain enigmatic. Thus, genetic study of the T. cruzi isolates can help to clarify the intraspecific heterogeneity of the parasites, whereas studies on its biological behavior in experimental animals can shed light on the relationship of parasite genetic diversity with its pathogenicity and virulence in the host.

Vectorial transmission of T. cruzi in Brazil has decreased substantially since 1983, because of the chemical control of Triatoma infestans, its major domestic vector. However, the risk for domiciliation of sylvatic triatomine species that were not targets of control actions has increased.22,23 Surveillance data obtained by the Brazilian National Health Foundation (Funasa) during the Chagas Disease Control Program have shown that Triatoma rubrovaria (Blanchard, 1843) (Hemiptera, Reduviidae, Triatominae) is taking over the niches of T. infestans and gradually invading houses in southern Brazil.24

Triatoma rubrovaria, an exclusively sylvatic species of triatomine, is found in rocky areas and peridomestic habitats in Rio Grande do Sul (RS) State of Brazil and also is widespread in Uruguay and in parts of northeastern Argentina. It is considered a generalist species, feeding on a wide variety of invertebrate and vertebrate hosts, including humans.2529 Vectorial capability of T. rubrovaria to transmit T. cruzi has been shown experimentally30 and by studies on its patterns of feeding and defecation.31,32 Natural infection of T. rubrovaria by T. cruzi has been shown in Uruguay at a rate of 0.3%,25 and more recently, our research group showed that 1.6% of T. rubrovaria nymphs from a rural district of RS, Brazil, were naturally infected by T. cruzi.29

Therefore, taking into account that the sylvatic transmission cycle of T. cruzi in the rural district of Quaraí, RS, Brazil, is established29 and considering the domiciliation risk of T. rubrovaria,24,33 to know the profile of the T. cruzi strains circulating in the sylvaltic environment of the south of Brazil, we genetically and biologically characterized five T. cruzi isolates obtained from T. rubrovaria collected in Quaraí, RS.

MATERIALS AND METHODS

Trypanosoma cruzi isolates.

The five isolates of T. cruzi used in this study were obtained from fecal samples of T. rubrovaria collected from 23 to 25 April 2003 in rural locales of the district of Quarai (30°22′52″S, 56°25′31″W), situated in the west bordering of the Brazilian State of Rio Grande do Sul and including Quaraí-Branquilhos I (QBI), Quaraí-Macarrão I (QMI), Quaraí-Macarrão II (QMII), Quaraí-Jarau I (QJI), and Quaraí-Jarau III (QJIII). Isolation, identification, and geographic distribution of the triatomines and precipitin tests with several vertebrate antisera on its feces have been described previously.29

Genotyping of T. cruzi isolates.

For preparation of DNA templates, cultured epimastigotes were lysed with digestion buffer (2 mol/L NaCl, 2 mol/L Tris-HCl, pH 8.0, 0.5 mol/L EDTA, pH 8.0, 10% SDS, and 10 mg/mL Proteinase K) for 2 hours at 65°C, and DNA was extracted using the GFX DNA extraction kit (GE Healthcare UK Limited, England). Two PCR assays were used to genotype the T. cruzi isolates: one based on the divergent domain D7 of the 24Sα rDNA9 and a multiplex method based on the non-transcribed spacer of the miniexon gene.34 The T. cruzi reference strains used in the PCR assays were G, Y, and JJ for the lineages TCI, TCIIb, and TCIIa (Z3B), respectively. T. cruzi isolates 3663 and 3869 were used as reference strains for the TCIIc lineage (Z3A).3436

Sequencing of partial SSU rRNA gene and phylogenetic inferences.

A 900-bp DNA fragment containing a partial SSU rRNA sequence (V7–V8 regions) was amplified by PCR and sequenced as previously described37 from the isolates of T. cruzi obtained from T. rubrovaria included in this study. Sequences of the isolates QBI and QMI were aligned among other trypanosome sequences from GenBank (accession number): T. cruzi Peru (X53917); T. cruzi G (AF239981); T. cruzi Y (AF301912); T. cruzi Colombiana (AF239980); T. cruzi Dm28c (AF245382); T. cruzi JJ (AY491761); and T. cruzi CANIII (AJ009148), 3663 (AF288660), and 3869 (AF303660). Alignments were made using the general alignment in the rRNA database (http://rrna.uia.ac.be/) as a guide and manually refined. A dendrogram was inferred using maximum parsimony analysis, whereas bootstrap analyses with 100 replicates and a similarity matrix were performed as before.37 T. cruzi marinkellei (AJ009150) was used as out-group for T. cruzi isolates.

Biological characterization of T. cruzi.

Biological characterization of the five T. cruzi isolates QBI, QMI, QMII, QJI, and QJIII was performed on Swiss mice using infectivity, parasitemia, and mortality parameters. The maintenance and care of animals complied with the National Institutes of Health guidelines for the human use of laboratory animals. To carry out the parasitemic curve of each T. cruzi isolate, groups of five old Swiss mice were intraperitoneally inoculated with 1,800 trypomastigotes obtained from the blood of a previous infected mouse. Parasitemia was evaluated three times a week by microscopic examination of peripheral blood after the third day after infection for 68 days38 and expressed as logarithms of the media of parasites in peripheral blood of five mice for each T. cruzi isolate. Considering the parasitemic peak, parasitemia was classified as low (1,000 tripomastigotes/5 μL of total blood); intermediate (varied from 1,000 to 10,000 tripomastigotes/5 μL of total blood); and high (> 10,000 tripomastigotes/5 μL of total blood).

Histopathologic characterization of T. cruzi.

For tissue tropism and histopathologic studies, for each T. cruzi isolate, groups of 21 Swiss mice, 20 days old, were intraperitoneally inoculated with 0. 1 mL of whole infected blood presenting at concentrations of 34,220, 2,000, 2,000, 3,820, and 92,000 trypomastigotes of QBI, QJI, QJIII, QMI, and QMII, respectively. Heart, skeletal muscles, esophagus, liver, spleen, and colon of groups of three mice7 infected with each T. cruzi isolate were collected at acute (7, 10, 14, 21, and 30 days) and chronic (150 and 180 days) phases of infection after CO2 euthanasia. Organs were fixed in 10% formalin, included in paraffin blocks, serially sectioned (4 μm thick), and stained with hematoxylin and eosin (H&E).39 Three histologic sections of each organ per mouse were analyzed for inflammation process, which was quantitatively classified as discrete (+), moderate (++), and intense (+++) by the presence of focal, multifocal, and diffuse inflammatory infiltrate, respectively.40 For the parasite load, the following criteria was adopted: discrete (+) when the parasites were rare; moderate (++) when they were detected in some focuses; and intense (+++) when they were seen diffusively.40 Classification of the isolates in biodemes was performed according to Andrade.41

RESULTS

Molecular characterization of T. cruzi isolates from T. rubrovaria.

PCR genotyping of the five T. cruzi isolates from RS showed a 110-bp DNA fragment corresponding to the D7 domain of the 24Sα rDNA amplified with primers D71 and D729 and a DNA fragment of 150 bp corresponding to a fragment of the non-transcribed spacer of the mini-exon gene.18,34 These results indicated that the five T. cruzi isolates belong to lineage TCIIc (Z3A) (Figure 1).

Comparison of V7–V8 rRNA sequences from T. cruzi isolates obtained from T. rubrovaria showed 100% of sequence similarity. Sequences from these isolates shared 93.6%, 95.4%, and 96.8% of sequence similarity, respectively, with sequences from the reference strains of T. cruzi TCI (G), TCIIb (Y), and TCIIa (JJ). Highest similarity (~99%) of these new isolates was shared with the reference strains of TCIIc (isolates 3663 and 3869). Therefore, the T. cruzi isolates of T. rubrovaria from southern Brazil proved to be very similar to Amazonian isolates from humans (3869) and Panstrongylus geniculatus (3663).3436

The genetic relatedness inferred in the dendrogram based on sequences of V7–V8 rRNA segregated isolates of T. cruzi in branches corresponding to each T. cruzi lineage: TCI, TCIIa, TCIIb, and TCIIc (Figure 2). According to the branching pattern, the triatomine isolates from T. rubrovaria collected in the state of RS, southern Brazil, clustered with isolates from human and P. geniculatus from the state of Amazonas (100% of bootstrap) (Figure 2).

Biological behavior of T. cruzi isolates.

Virulence of T. cruzi isolates was studied through determination of the parasitemic curve, mortality rate, and pre-patent period. Pre-patent period was short for all strains, occurring at Day 5 after infection for the isolates QJI, QJII, QMII, and QBI and at Day 7 after infection for the QMI isolate. Parasitemia was highly variable (Figure 3), being considered low for isolate QBI, intermediate for isolates QJI, QJIII, and QMI, and high for isolate QMII. Parasitemic peak was reached in 21 days for isolates QMI and QMII and after 31 days for stocks QBI, QJI, and QJIII. Thus, the most virulent T. cruzi isolate was QMII, followed by QMI. During the studied period, one mouse infected with isolate QMI and four mice infected with QMII isolate died in the parasitemic peak.

Tissue tropism and histopathologic lesions during the acute and chronic phases of infection of all five T. cruzi isolates are summarized in Table 1. Fifteen mice infected with each T. cruzi isolate were examined during acute phase, and all isolates presented a discrete to intense inflammatory process in liver lobular portion (60%; 45/75), in liver portal system (30.7%; 23/75), in skeletal muscle (37.3%; 28/75), and in heart (33.3%; 25/75). Amastigote nests were observed in hearts of mice infected with QJIII (4.7%), QMI (9.5%), and QMII (27.8%); in skeletal muscle of mice infected with QMI (33.3%) and QMII (38.9%); and in livers of mice infected with QMII (5.5%; Figure 4A–C). Inflammatory process and amastigote nests were not observed in the colon, esophagus, and spleen of infected mice collected in the acute phase.

During the chronic phase of infection, a total of 27 mice were analyzed (3 mice infected with isolate QMII died before 150 days of infection). Mice infected with each T. cruzi isolate showed a discrete to intense inflammatory process in the liver lobular portion (74.1%; 20/27), in the liver portal system (11.1%; 3/27), in skeletal muscle (44.4%; 12/27), and in the heart (40.7%; 11/27; Figure 4D–F). Amastigote nests were absent in all tissues studied. All isolates with the exception of QJI produced inflammatory infiltration in the colon.

Histopathologic lesions observed during the acute and chronic phases of infection were more intense in animals infected with the QMII isolate. According to the virulence and pathologic characteristics of T. cruzi isolates, QJI, QJIII, QMI, and QBI were classified as biodeme III and QMII as biodeme II as described by Andrade.41

DISCUSSION

Molecular characterization of the T. cruzi isolates by PCR analysis of the 24Sα rRNA D7 domain and of the non-transcribed spacer of the mini-exon genes showed that all the isolates belonged to the TCIIc (Z3A) group. The lineages IIa and IIc of T. cruzi are predominantly sylvatic and present a wide distribution in the Amazon region, being also reported in the Brazilian state of Bahia42; Venezuela19; Colombia43; and in the north up to the United States.44 As far as we know, this is the first description of IIc T. cruzi lineage in the southern region of Brazil. There are indications that these T. cruzi lineages evolved in a terrestrial habitat in burrows and in rocky locations with the triatomine tribe Triatomine, which include bugs from genera Panstrongylus and Triatoma, in association with edentates and/or ground dwelling marsupials.45 Thus, the ecologic behavior of T. rubrovaria is in accordance with its association with the TCIIc group.

Very little data on characterization of T. cruzi isolates obtained from the sylvatic T. cruzi transmission cycle in the countries of the southern region of South America are available. Evidence for the occurrence of TCIIc in this region have been shown in one specimen of domestic T. infestans21 and more recently in armadillos and in a short-tailed opossum but not in T. infestans from the Paraguayan Chaco region.46 Also, in Argentina, TCIIc was found in one skunk specimen, in a few T. infestans specimens, and in dogs from a sylvatic environment.47,48 Thus, the finding of TCIIc in T. rubrovaria indicates that this and other triatomines species with terrestrial ecologic behavior can participate in the sylvatic T. cruzi cycle involving the TCIIc group in the Southern Cone countries of America.

There is evidence that TCIIa (Z3B) and TCIIc (Z3A) have emerged from hybridization events between lineages TCIIb and TCI.49 Subdivision of the Z3 stocks in the clusters Z3A and Z3B could be performed because of the genetic outline of the ribosomal cistron.18 Use of other genetic parameters such as the recent study on chromosome size polymorphism showed intragroup T. cruzi Z3 variability such as the clustering of TCIIc 3663 with hybrid stocks of T. cruzi.50 To verify the relatedness of the TCIIc isolates from T. rubrovaria with representatives of TC groups I and II based on the ribosomal small subunit, a sequence of a 900-bp fragment was compared among the T. cruzi strains described in Figure 2. Using this strategy, we observed that all isolates from the Brazilian South showed high similarity to TCIIc isolates 3663 and 3869 from the Amazon Basin,3436 suggesting a common origin. Geographic distance of these isolates in Brazil could be caused by a lack of information about T. cruzi sylvatic isolates from different animal and bug groups of others Brazilian localities. Thus, to understand the epidemiologic characteristics of the TCIIc group, more studies on T. cruzi isolates from various species of vertebrates and bugs with different ecologic behaviors must be performed, including specimens from the southern region of Brazil.

In the RS, Fernandes and others51 studied 35 T. cruzi strains on the basis of biological behavior in mice and isoenzyme electrophoresis patterns. The authors found that T. cruzi isolates obtained from chagasic patients (domiciliary transmission cycle) were characterized as Z2 and ZB (TCIIb) and strains isolated from the sylvatic vector P. megistus were Z1 (TCI). In this work, we showed the presence of T. cruzi IIc. Therefore, study of T. cruzi parasite–host interaction from domestic and sylvatic cycles in RS State can shed light on the evolution, population structure, and epidemiology of Chagas disease.

Biological characteristics of T. cruzi isolates assessed through experimental infection in laboratory animals represent an important tool for understanding different aspects of the epidemiology of Chagas disease.7 According to Andrade,5 pathogenicity and virulence patterns of T. cruzi enable its classification in biodeme I, II, or III. Furthermore, a specialist committee recommendation52 established that T. cruzi isolates typed as biodeme III are equivalent to T. cruzi I and isolates typed as biodeme II are equivalent to T. cruzi II. However, in the Andrade’s biodeme proposal, there is no prototype of T. cruzi IIc. Therefore, to verify the occurrence of specific biological characteristics of the five TCIIc isolates, infectivity, histopathology, tissue tropism, and mortality rates were evaluated in experimental animals according to An-drade’s classic studies. T. cruzi isolates QJI, QJIII, QMI, and QBI showed low parasitemia and mortality rates characteristic of biodeme III, whereas isolate QMII induced more intense inflammatory infiltration and a higher mortality rate characteristic of biodeme II. These data indicate that in Quaraí, RS, the circulating T. cruzi parasites present heterogeneous biological behavior that may underlie the genetic intravariability of the T. cruzi Z3 group.18

Comparison of tissue tropism of T. cruzi strains with specific kinetoplast DNA profiles in different inbred mice (BALB/c, DBA2, and C57BL/6) and the outbred Swiss mice showed that differential biological behavior of T. cruzi isolates are attributed to both parasite and host genetic background,53,54 leading to experimental evidence of the “clonalhistotropic” model of Chagas disease.55 Thus, T. cruzi natural isolates correspond to a population of parasites composed of distinct clones, which are selected according to the genetic potential of each vertebrate host.56,57 In the chronic phase of infection, the TCIIc isolates analyzed in this work showed tissue tropism in heart, skeletal muscles, liver, and colon, suggesting the presence of different parasite clones. To verify this premise, further analysis of cloned strains from each isolate of T. cruzi obtained from T. rubrovaria in Quarai should be done to study intraspecific variations at the biological, immunologic, biochemical, and genetic levels.

Triatoma rubrovaria presents pre-adaptative characteristics to the domiciliary ecotope because adult bugs have been found in the peridomestic and intradomestic habitats.24 Recently, the potential adaptation of this bug to new environments was suggested by its genomic plasticity shown by the high intraspecific genetic variability detected in populations of T. rubrovaria from Brazil, Uruguay, and Argentina through molecular markers.58 In our previous study, precipitin assays performed on the collected bugs showed one specimen of T. rubrovaria positive to human antiserum in Branquillos, a settlement closer to the urban perimeter of Quaraí.29 This study on the genetic and biological characterization of T. cruzi isolates obtained from T. rubrovaria showed its pathogenicity and virulence in experimental animals. Together these data show a need for permanent entomologic surveillance in the south region of Brazil to prevent re-introduction of the urban Chagas disease. Furthermore, recently, in Santa Catarina, a state in southern Brazil, there was an outbreak of acute Chagas disease with three fatal human victims caused by the ingestion of sugar cane juice containing T. tibiamaculata, an exclusive sylvatic triatomine species, contaminated with TCI and TCII isolates.59 Thus, in addition to the domiciliation possibility of T. rubrovaria, entomologic surveillance may also avert episodes of acute Chagas disease infection by oral transmission through ingestion of food contaminated with infected bugs.

Table 1

Degree of histopathologic lesions and parasitism in mice infected with T. cruzi isolates obtained from T. rubrovaria

QJIQJIIIQMIQMIIQBI
ACPar*ACPar*ACPar*ACPar*ACPar*
% (15)% (6)% (21)% (15)% (6)% (21)% (15)% (6)% (21)% (15)% (3)% (18)% (15)% (6)% (21)
* Parasitism was observed only in the acute phase of infection; QJI, QJIII, QMI, QMII, and QBI correspond to the isolates of T. cruzi: +, discrete; ++, moderate; +++, intense.
A = acute phase of infection; C = chronic phase of infection; Par = parasitism; % = percentage of mice; () = number absolute of mice observed.
+16.7 (1)16.7 (1)20.0 (3)33.3 (2)4.7 (1)26.7 (4)33.3 (1)27.8 (5)40.0 (6)66.7 (4)
Skeletal++13.3 (2)13.3 (2)16.7 (1)13.3 (2)46.7 (7)6.7 (1)
muscle+++33.3 (2)6.7 (1)14.3 (3)11.1 (2)
+20.0 (3)33.3 (2)20.0 (3)50.0 (3)4.7 (1)20.0 (3)66.7 (4)9.5 (2)73.3 (11)33.3 (1)27.8 (5)6.7 (1)
++6.7 (1)16.7 (1)6.7 (1)
Heart+++13.3 (2)
+13.3 (2)26.7 (4)16.7 (1)26.4 (4)16.7 (1)53.3 (8)20.0 (3)16.7 (1)
Liver++6.7 (1)6.7 (1)
porta+++
+60.0 (9)66.7 (4)66.7 (10)83.3 (5)20.0 (3)83.3 (5)66.7 (10)33.3 (1)5.5 (1)73.3 (11)83.3 (5)
++6.7 (1)6.7 (1)
Liver+++
+50.0 (3)16.7 (1)100.0 (3)66.7 (4)
++
Colon+++
Figure 1.
Figure 1.

Agarose gel electrophoresis of PCR products corresponding to ME: a hypervariable region of the mini-exon gene non-transcribed spacer that is able to characterize T. cruzi I (200 bp), II (250 bp), and Z3 (150 bp); and to 24Sα: the D7 domain of the 24Sα rDNA with primers D71 and D72 that are able to characterize T.cruzi I (110 bp), TCIIb (125 bp), TCIIa (117 bp), and TCIIc (110 bp). Lanes: molecular DNA marker; T. cruzi reference isolates: TcG (TCI), TcY(TCIIb), TcJJ (TCIIa), and Tc3663 (TCIIc); stocks of T. cruzi isolated from Triatoma rubrovaria: QBI, QJI, QJIII, QMI, and QMII.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 79, 3; 10.4269/ajtmh.2008.79.427

Figure 2.
Figure 2.

Phylogenetic tree based on the rRNA V7-V8 900-bp sequence. All isolates typed by TCI and TCII were clustered into two strongly supported groups (100% of bootstrap). Z3 lineages were segregated in two subgroups: Z3B composed of isolates that clustered with the reference strains José Júlio, CANIII; and Z3A composed by all T. cruzi isolates from T. rubrovaria, RS and by the reference isolates from Amazonas, 3663 and 3869 from Panstrongylus geniculatus and human, respectively (100% of bootstrap). Reference strains: T. cruzi G, Colombiana, Dm28c (TCI); TcY, Peru (TCIIb); CANIII, M2574, JJulio (TCIIa); 3869 and 3663 (TCIIc); isolates of T. cruzi obtained from Triatoma rubrovaria: QBI and QMI.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 79, 3; 10.4269/ajtmh.2008.79.427

Figure 3.
Figure 3.

Parasitemic levels in mice infected with different isolates of T. cruzi obtained from T. rubrovaria. The mean values were obtained from five independent experiments and SDs are represented by bars.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 79, 3; 10.4269/ajtmh.2008.79.427

Figure 4.
Figure 4.

Histopathologic aspects observed in Swiss mice infected with T. cruzi isolates obtained from T. rubrovaria. A–C, Acute phase of infection. A, Section of mouse skeletal muscle infected with isolate QMI presenting mononuclear inflammatory infiltration; H&E, ×400. B, Myocardium of mouse infected with isolate QMII presenting myocarditis, amastigote nests, and disruption of muscle fibers; H&E, ×400. C, Skeletal muscle of mouse infected with QMII isolate presenting amastigote nests; H&E, ×400. D–F, Chronic phase of infection. D, Skeletal muscle of mouse infected with QJIII isolate presenting chronic myositis and cellular disruption; H&E, ×400. E, Skeletal muscle of mouse infected with QJI isolate presenting intense chronic inflammatory infiltration; H&E, ×400. F, Section of colon of mouse infected with QMII isolate presenting chronic inflammation in the mesenteric plexus; H&E, ×400. H&E, slides stained with hematoxylin and eosin.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 79, 3; 10.4269/ajtmh.2008.79.427

*

Address correspondence to Márcia A. Sperança, Discipline of Molecular Biology, Marilia Medical School, Av. Monte Carmelo 800, CEP 17519f-030, Marília SP, Brazil. E-mail: speranca@famema.br

Authors’ addresses: Luciamá re P. A. Martins, Roberto E. P. Castanho, and Janaína C. P. de Oliveira, Discipline of Parasitology, Marília Medical School, Av. Monte Carmelo, 800, CEP 17519-030, Marília, São Paulo, Brazil, Tel: 55-14-3433-1744, E-mails: luciapam@famema.br and castanho@famema.br. Arlei Marcili and Marta M. G. Teixeira, Department of Parasitology, Institute of Medical Sciences, University of São Paulo, Av. Prof. Lineu Prestes 1374, CEP 05508-900, São Paulo, Brazil, Tel: 55-11-3091-7331, E-mails: amarcili@usp.br and mmgteix@icb.usp.br. Altino L. S. Therezo, Discipline of Pathology, Marília Medical School, Av. Monte Carmelo, 800, CEP 17519-030, Marília, Sã o Paulo, Brazil, Tel: 55-14-3433-1744, E-mail: altinoth@hotmail.com. Rodrigo B. Suzuki and Márcia A. Sperança, Discipline of Molecular Biology, Marilia Medical School, Av. Monte Carmelo 800, CEP 17519-030, Marília, SP, Brazil, Tel: 55-14-3433-1235, Fax: 55-14-3413-4187, E-mails: speranca@famema.br and rbsuzuki@gmail.com. João A. da Rosa, Department of Parasitology, Pharmaceutical Sciences Faculty, University of the State of São Paulo, Rodovia Araraquara-Jaú Km 01, CEP 14801-902, Caixa postal 502, Araraquara, São Paulo, Brazil, Tel: 55-16-3301-6943, E-mail: rosaja@fcfar.unesp.br.

Financial support: This work was supported by Fundação para o Desenvolvimento da UNESP (FUNDUNESP), Secretaria de Saúde do Estado do Rio Grande do Sul, CNPq, and FAPESP.

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