**3. Molecular profile of** *T. cruzi* **populations**

Early investigations on the genetic of *T. cruzi* populations are based on electrophoretic profiling of isoenzymes (zimodeme analysis), a technique used to explore the genetic diversity of microorganisms. Enzymatic electrophoresis uses soluble raw-materials and extracts from an organism to assess the activity of a protein, and its product is revealed by means of a colorimetric reaction. Under controlled conditions, differences in isoenzymatic mobility imply genetic differences (Miles, 1985; Miles & Cibulkis, 1986). Toye (1974) was the first to use isoenzymes to classify trypanosomas from the New World, reporting differences among *T. cruzi* samples. By the end of the 70's and beginning of the 80's, several studies on isoenzymatic variability among *T. cruzi* populations were performed in Brazilian Northeast, and later in different regions within the country, by employing six enzymes: ALT (alanine aminotransferase), AST (aspartate aminotransferase), glucose phosphate isomerase (GPI), glucose-6-dehydrogenase phosphate (G6PDH), malic enzyme (ME) and phosphoglucomutase (PGM), characterizing three enzymatic profiles belonging to parasite groups called zymodemes I (Z1), II (Z2) and III (Z3). Z1 and Z3 are related with the sylvatic transmission cycle and Z2 with the domestic transmission cycle of the parasite (Miles et al., 1977, 1978, 1980, 1981a, b). As the number of analyzed isoenzymes has been amplified and sub-populations circulating among domestic and sylvatic vertebrates and invertebrates have been studied, an elevated degree of *T. cruzi* heterogeneity was verified (Miles et al., 1980; Bogliolo et al., 1986; Tibayrenc et al., 1986; Tibayrenc & Ayala, 1988; Barnabe et al., 2000).

forms ingested during hematophagy differentiate into epimastigotes in the digestive tract. Another differentiation occurs in the digestive tract, more specifically in its final portion and in rectus, when epimastigotes transform into metacyclic trypomastigotes, which is infectious for the vertebrate host and eliminated with the feces (Zeledón et al., 1977; Garcia &

*T. cruzi* is found as a parasite in a considerable number of mammals and in a wide range of tissues and niches in these hosts (Deane et al., 1984). Such eclecticism has characterized *T. cruzi* as one of the most successful microorganism in presenting parasitary life (Jansen et al., 1999). Therefore, this protozoan comprises a wide set of heterogeneous populations that circulate through very diverse vertebrate and invertebrate hosts, with a variation of different genotype predominance. The parasite has several morphological, physiological and ecological variations, and also in which refers to its infectivity and pathogenicity (Miles et al., 1978, 1980, 2009), which can warrant the various clinical manifestation forms of Chagas disease observed in different geographic regions (Miles et al., 1981a). Many studies have been performed seeking molecular markers that could correlate the parasite genotype with varying types of this infirmity clinical manifestation. Several works tried to clarify the

*T. cruzi* has a great phenotypic and genotypic variability in its strains, and therefore this protozoan has the ability to perform genetic exchanges through an unusual mechanism of nuclear fusion, forming a polyploidy progeny, which can suffer recombination among alleles, and after losing its chromosome, can return to diploid status. Some studies provided strong evidence that sexual reproduction is absent in *T. cruzi*, and that its population

Early investigations on the genetic of *T. cruzi* populations are based on electrophoretic profiling of isoenzymes (zimodeme analysis), a technique used to explore the genetic diversity of microorganisms. Enzymatic electrophoresis uses soluble raw-materials and extracts from an organism to assess the activity of a protein, and its product is revealed by means of a colorimetric reaction. Under controlled conditions, differences in isoenzymatic mobility imply genetic differences (Miles, 1985; Miles & Cibulkis, 1986). Toye (1974) was the first to use isoenzymes to classify trypanosomas from the New World, reporting differences among *T. cruzi* samples. By the end of the 70's and beginning of the 80's, several studies on isoenzymatic variability among *T. cruzi* populations were performed in Brazilian Northeast, and later in different regions within the country, by employing six enzymes: ALT (alanine aminotransferase), AST (aspartate aminotransferase), glucose phosphate isomerase (GPI), glucose-6-dehydrogenase phosphate (G6PDH), malic enzyme (ME) and phosphoglucomutase (PGM), characterizing three enzymatic profiles belonging to parasite groups called zymodemes I (Z1), II (Z2) and III (Z3). Z1 and Z3 are related with the sylvatic transmission cycle and Z2 with the domestic transmission cycle of the parasite (Miles et al., 1977, 1978, 1980, 1981a, b). As the number of analyzed isoenzymes has been amplified and sub-populations circulating among domestic and sylvatic vertebrates and invertebrates have been studied, an elevated degree of *T. cruzi* heterogeneity was verified (Miles et al., 1980; Bogliolo et al., 1986; Tibayrenc et al., 1986; Tibayrenc & Ayala, 1988;

multiple factors related with population epidemiology and genetics.

structure is clonal (Gaunt et al., 2003; Lewis et al., 2009).

**3. Molecular profile of** *T. cruzi* **populations** 

Azambuja, 2000).

Barnabe et al., 2000).

With technologic advancement and the discovery of new molecular biology tools, it was possible to study the diversity of *T. cruzi* by means of DNA analysis, allowing for molecular characterization of this parasite strains (Devera et al., 2003). Therefore, the genetic diversity was corroborated by randomly amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) analyses, DNA fingerprinting, microsatellites and molecular karyotyping (reviewed by Zingales et al., 1999). Analyses of gene sequences with lowest evaluative rates, such as ribosomal RNA genes, classic evolution markers and mini-exon genes, indicated dimorphism in *T. cruzi* isolates, rating them into two groups (Souto et al., 1996). Mini-exon gene that is present in Kinetoplastid nuclear genome at approximately 200 copies in a tandem type array is composed by three different regions: exon, intron and intergenic regions. Exon is a highly preserved sequence between de order compounds, added to nuclear messenger RNA post-transcription (Devera et al., 2003). Intron is moderately preserved between species of the same genus or sub-genus, and the intergenic region is particularly different among species. In *T. cruzi*, the amplification of mini-exon intergenic region by Polimerase Chain Reaction (PCR) allowed us to classify the different isolates into two main taxonomic groups: *T. cruzi* I and *T. cruzi* II (Fernandes, 1996; Souto et al., 1996; Fernandes et al., 1998). Thereafter, PCR amplification assay were standardized, allowing for rapid molecular typing, which started to be broadly used. Thereby the use of multiplex PCR based on intergenic region allowed us to classify the isolates as *T. cruzi* I, *T. cruzi* II, *T. cruzi* Z3 or *T. rangeli* with 200, 250, 150 pb and 100 pb, respectively (Fernandes et al., 2001a).

Aiming at standardizing double lines and hybrid isolates, a committee settled the lines were referred to as *T. cruzi I* and *T. cruzi II* "groups" (Zingales et al., 1999). Such denomination was not attributed to hybrid isolates, and additional studies are recommended to better characterize them (Zingales, 2011). From hybrid isolate gene sequence analysis, it has been shown that events of genetic exchanges with these parasites originated four distinct isolate groups (Sturm & Campbell, 2009). Thus, by using multilocus enzyme electrophoresis (MLEE) and RAPD markers, it was suggested that the group *T. cruzi* II was divided into five subgroups, including the four hybrid groups (Freitas et al., 2006; Brisse et al., 2000). *T. cruzi III*, a third ancestral group, was proposed from the analysis of microsatellites and mitochondrial DNA.

In 2009, the scientific community felt the need to standardize once again *T. cruzi* groups' nomenclature, aiming at clarifying questions on biology, eco-epidemiology and pathogenicity (Zingales et al., 2009). In this respect, it was recommended that *T. cruzi* was divided into six groups (*T. cruzi I–VI*), and that each group was called Discreet Taxonomic Units (DTUs) I, IIa, IIb, IIc, IId, IIe (Figure 3), defined as groups of isolates that are genetically similar and can be identified through molecular or immune markers (Tibayrenc, 1998), with DTU I corresponding to *T. cruzi* line I and DTU IIb corresponding to *T. cruzi* line II, and sub-lines IIa and IIc-e associated with hybrid strains and those belonging to zymodeme 3 (Brisse et al., 2000). The distribution of haplotypes from five nuclear genes and one satellite DNA was analyzed in isolates that were representative of the six DTUs by net genealogy and Bayesian phylogeny. Such data indicated that DTUs *T. cruzi I* and *T. cruzi II* are monophyletic and the other DTUs have different combinations of *T. cruzi I* and *T. cruzi II*  haplotypes and DTU-specific haplotypes (Tomazi et al., 2009; Ienne et al., 2010). One of the possible interpretations for this observation is that *T. cruzi I* and *T. cruzi II* are two different species and that DTUs II-IV are hybrid resulting from independent hybridization/genomic combination events (Zingales, 2011).

Molecular and Proteolytic Profiles of

Isolates

SMM10 SMM53 SMM88

SMM36 SMM82

influence the parasite-host cell interaction.

SMM (Santa Maria Madalena)

and was done haemoculture.

Rio de Janeiro, Brazil

A A A

B B

**3.2 Molecular profile of** *T. cruzi* **isolates from Rio de Janeiro** 

(Gonçalves, 2000).

*Trypanosoma cruzi* Sylvatic Isolates from Rio de Janeiro-Brazil 165

area A, located at 250-meter altitude and 3.5 km distant from the district headquarters, very modified by deforestation for banana farming; area B, located at 130-meter altitude and 4 km distant from the headquarters, placed in a valley with preserved vegetation (secondary forest). These areas are 2-km distant to each other, separated by a mountain (Figure 3). Area C, the district headquarters, at 40-meter distance, was totally modified by pasture formation, and areas D and E were totally preserved and placed at 10 and 12-km distances from the headquarters, respectively. *T. cruzi* isolates used in this study were extracted from triatomines captured from areas A, B and F (Table 1). Area F was located in Vista Alegre, a city neighboring Conceição de Macabu, at Northern region of Rio de Janeiro State

(Samples) Area Host Geographical

SMM98 A Tv Triunfo

Tv Tv Tv

Tv Tv

SMM1 F HCD Conceição de Macabu

Tv – *Triatoma vitticeps*; HCD (Haemoculture of the swiss mouse) – the parasites were inoculated in mice

Table 1. *Trypanosoma cruzi* samples isolated from *Triatoma vitticeps* captured on the State of

Those *T. cruzi* samples isolated from *Triatoma vitticeps*, collected in Rio de Janeiro State, were classified by our group as Z3 based on mini-exon gene (Santos-Mallet et al., 2008) and showed great heterogeneity regarding growth curve and mouse virulence patterns (Silva, 2006), susceptibility to benznidazole (Sousa, 2009), total protein pattern and proteolytic activity profile (Gomes et al., 2006; Gomes et al., 2009). This heterogeneity observed in samples collected from the same region leads to questionings on how this diversity could

The results obtained by means of molecular analysis revealed that the isolates have similar profiles, except for sample SMM1 (area F). Samples SMM10, SMM53, SMM88, SMM98 (area A), SMM36 and SMM82 (area B) revealed the presence of 150 bp, indicating that they belong to the zymodeme III group (Z3; Figure 4). Likewise, sample SMM1 from area F showed similarity to Z3 (150 bp), but also presented another band that may be related to the TcII profile (250 bp) and was very similar to the reference strain CL Brener (Figure 4). The phylogenetic position of Z3 has been much debated. According to some authors, the numerical taxonomy based on 24 isoenzymatic Z3 profiles is more closely associated with Z1 (TcII) than with Z2 (TcI) (Ready & Miles, 1980). However, other works place Z3 in an intermediate position between Z1 and Z2 (Stothard et al., 1998). Our study revealed one isolate (SMM1) with a hybrid profile associated with Z3 and TcII. This result may corroborate the hypothesis that this isolate is the product of a

origin

Triunfo Triunfo Triunfo

Triunfo Triunfo

In this setting, the characterization of these parasites extracted from different hosts aim at helping clarify the biological meaning and repercussion of this variability for clinics and for Chagas disease epidemiology (Lainson et al., 1979). However, the great majority of studies performed are related to parasite populations belonging to TCI and TCII groups, with scarce works performed with Z3 group.

Fig. 3. General pattern of distribution of *T. cruzi* lineages and sublineages; the sylvatic isolates from Rio de Janeiro (extended map showing in green Triunfo, Santa Maria Madalena municipal district) were typed as *T. cruzi* IIa/Z3. (Adaptated map by Noireau F. Vet. Res. (2009)).

#### **3.1** *T. cruzi* **isolates from Rio de Janeiro**

Therefore, this work was performed from *T. cruzi* samples isolated from *Triatoma vitticeps* (Figure 1) by Gonçalves in 2000, at Triunfo location, 2nd district of Santa Maria Madalena city, Rio de Janeiro state (Figure 2). Four hundred sixty five (465) *Triatoma vitticeps* specimens were collected: 294 females, 156 males, and 15 nymphs from five different areas: area A, located at 250-meter altitude and 3.5 km distant from the district headquarters, very modified by deforestation for banana farming; area B, located at 130-meter altitude and 4 km distant from the headquarters, placed in a valley with preserved vegetation (secondary forest). These areas are 2-km distant to each other, separated by a mountain (Figure 3). Area C, the district headquarters, at 40-meter distance, was totally modified by pasture formation, and areas D and E were totally preserved and placed at 10 and 12-km distances from the headquarters, respectively. *T. cruzi* isolates used in this study were extracted from triatomines captured from areas A, B and F (Table 1). Area F was located in Vista Alegre, a city neighboring Conceição de Macabu, at Northern region of Rio de Janeiro State (Gonçalves, 2000).


SMM (Santa Maria Madalena)

164 Gel Electrophoresis – Advanced Techniques

In this setting, the characterization of these parasites extracted from different hosts aim at helping clarify the biological meaning and repercussion of this variability for clinics and for Chagas disease epidemiology (Lainson et al., 1979). However, the great majority of studies performed are related to parasite populations belonging to TCI and TCII groups, with scarce

Fig. 3. General pattern of distribution of *T. cruzi* lineages and sublineages; the sylvatic isolates from Rio de Janeiro (extended map showing in green Triunfo, Santa Maria Madalena municipal district) were typed as *T. cruzi* IIa/Z3. (Adaptated map by

Therefore, this work was performed from *T. cruzi* samples isolated from *Triatoma vitticeps* (Figure 1) by Gonçalves in 2000, at Triunfo location, 2nd district of Santa Maria Madalena city, Rio de Janeiro state (Figure 2). Four hundred sixty five (465) *Triatoma vitticeps* specimens were collected: 294 females, 156 males, and 15 nymphs from five different areas:

works performed with Z3 group.

Noireau F. Vet. Res. (2009)).

**3.1** *T. cruzi* **isolates from Rio de Janeiro** 

Tv – *Triatoma vitticeps*; HCD (Haemoculture of the swiss mouse) – the parasites were inoculated in mice and was done haemoculture.

Table 1. *Trypanosoma cruzi* samples isolated from *Triatoma vitticeps* captured on the State of Rio de Janeiro, Brazil

Those *T. cruzi* samples isolated from *Triatoma vitticeps*, collected in Rio de Janeiro State, were classified by our group as Z3 based on mini-exon gene (Santos-Mallet et al., 2008) and showed great heterogeneity regarding growth curve and mouse virulence patterns (Silva, 2006), susceptibility to benznidazole (Sousa, 2009), total protein pattern and proteolytic activity profile (Gomes et al., 2006; Gomes et al., 2009). This heterogeneity observed in samples collected from the same region leads to questionings on how this diversity could influence the parasite-host cell interaction.
