**2. Triatomines**

The first report of triatomine existence was recorded by the Spanish Francisco López de Gomara, in 1514, when mentioning Darién region he said: "Hay muchas garrapatas y chinches com alas", apparently referring to *Rhodnius prolixus* (Stål, 1859) (León, 1962). *Cimex rubrofasciatus* (*Triatoma rubrofasciata*), was described in 1773 by De Geer, and later assigned by Laporte as the type species of *Triatoma* genus (Lent & Wygodzinsky, 1979). In Brazil, the first report of triatomine in domicile was possibly *Panstrongylus megistus* (Burmeister, 1835) (Gardner, 1942). However, the identification of *Trypanosoma cruzi* sylvatic isolates is contemporary to the discovery of this parasite and Chagas disease by Carlos Chagas in 1909. When they went to Lassance, Minas Gerais, Brazil, for malaria epidemics study, he identified flagellated forms in the intestine of triatomine of *Conorhinus megistus* (*Panstrongylus megistus*) in humans and cats, referring to them as *Schizotrypanum cruzi* (Chagas, 1909). Later Chagas (1912) isolated the parasite in armadillos (*Tatusia novemcincta*, now called *Daysipus novemcinctus*), identifying the *T. cruzi* sylvatic reservoirs, and in the

Therefore, the transmission cycle of *T. cruzi* is comprised by a sylvatic cycle, in which the parasite circulates among mammals and sylvatic vectors, and a domiciliary cycle, in which the infection is ensued by the contact of mammals, sylvatic vectors and sinantropic animals

Human Chagas disease, an antropozoonosis that evolved from a zoonosis, is strongly related with men's social class, type of work and habitation (Dias, 2000). During the 70's, the disease endemic area achieved at least 2,450 Brazilian cities, 771 of which were detected to have *Triatoma infestans*, the main disease vector in Brazil. At that time, there were over five million people affected by the disease in the country, with an incidence of approximately one hundred thousand new cases yearly and mortality above ten thousand deaths yearly. Less than five percent of blood banks used to control donors and over seven hundred cities had their homes infected by *T. infestans*. This situation led scientists to press the government to prioritize a national program against the disease. Homes from endemic areas were sprinkled with the appropriate insecticide and, in accordance with law; mandatory screening of blood donors was implemented throughout the country (Dias et al., 2002). The control program of the main vector in Brazil was recognized in 2006, with a certificate from the World Health Organization (WHO) for virtual elimination of *T. infestans* in Brazil (Dias, 2006). As the main vector was eliminated, currently there is a concern that other Triatominae species, formerly deemed secondary in the disease transmission, such as *Triatoma brasiliensis*, *Triatoma pseudomaculata* and *Panstrongylus megistus*, take the place of *T. infestans* in some

with domestic and domiciled animals, including men (Barretto, 1979).

locations, therefore becoming potential disease vectors in Brazil (Coura, 2009).

February 21, 2006.

**2. Triatomines**

Despite the great progress in controlling vector and transfusion transmission in the countries from the Southern Cone, transmission is ongoing in other parts of the continent, and the issue of already infected people, most of whom are in the chronic phase of the disease, is still a challenge to public health (Urbina, 1999). Currently Chagas disease affects between twelve and fourteen million people in Latin America, and at least 60 million people live in areas with transmission risk (WHO, 2002). In Brazil, the disease notification became compulsory as per Ordinance V of Health Surveillance Secretary of Ministry of Health dated

The first report of triatomine existence was recorded by the Spanish Francisco López de Gomara, in 1514, when mentioning Darién region he said: "Hay muchas garrapatas y chinches com alas", apparently referring to *Rhodnius prolixus* (Stål, 1859) (León, 1962). *Cimex rubrofasciatus* (*Triatoma rubrofasciata*), was described in 1773 by De Geer, and later assigned by Laporte as the type species of *Triatoma* genus (Lent & Wygodzinsky, 1979). In Brazil, the first report of triatomine in domicile was possibly *Panstrongylus megistus* (Burmeister, 1835) (Gardner, 1942). However, the identification of *Trypanosoma cruzi* sylvatic isolates is contemporary to the discovery of this parasite and Chagas disease by Carlos Chagas in 1909. When they went to Lassance, Minas Gerais, Brazil, for malaria epidemics study, he identified flagellated forms in the intestine of triatomine of *Conorhinus megistus* (*Panstrongylus megistus*) in humans and cats, referring to them as *Schizotrypanum cruzi* (Chagas, 1909). Later Chagas (1912) isolated the parasite in armadillos (*Tatusia novemcincta*, now called *Daysipus novemcinctus*), identifying the *T. cruzi* sylvatic reservoirs, and in the same ecotope he found infected *Triatoma geniculata* (*Panstrongylus geniculatus*) specimens, establishing the disease sylvatic cycle (Coura & Dias, 2009).

Between 1913 and 1924 it became evident that the disease was not restricted to Brazil, being diagnosed in other countries in Central and South Americas, such as El Salvador, Venezuela, Peru and Argentina (Talice et al., 1940; Zeledón, 1981). In subsequent studies, Coura & Dias, 2009 mentions that Chagas (1924) demonstrated *T. cruzi* transmission cycle in the Amazon region with the identification of this parasite in monkeys of *Saimiri scirius* species.

In Rio de Janeiro state, the first Triatominae occurrence dated 1859, when Stal described *Conorhinus vitticeps* species, now called *Triatoma vitticeps*. At that time, Rio de Janeiro was assigned as type location, without defining whether it referred to the city or state.

Following this finding, Neiva (1914) recorded the occurrence of *T. vitticeps* in Conceição de Macabu, formerly Macaé city district, presently Conceição de Macabu city. Due to information accuracy, Lent (1942) suggested it would be considered as the type location of *T. vitticeps*.

Subsequently, Pinto (1931, as cited in Lent, 1942) pointed out its presence in Magé, and Lent (1942) in Nova Friburgo, at Secretario location in Petrópolis city and at Federal District, which was Rio de Janeiro at that time. In Minas Gerais state, it was observed by the first time by Martins et al (1940), and in Espírito Santo state, as mentioned by Lent (1942).

In Rio de Janeiro state other species were also found. Guimarães and Jansen (1943) collected *Panstrongylus megistus* specimens in a building by the hill, and identified *Trypanosoma cruzi* sylvatic reservoir (skunk), but did not find the sylvatic focus. Dias (1943) listed Chagas disease transmitters in Rio de Janeiro as being *Panstrongylus megistus*, *Panstrongylus geniculatus* (Latreille, 1811), *Triatoma vitticeps* (Stal, 1859), *Triatoma oswaldoi* (Neiva & Pinto, 1923), *Triatoma infestans* (Klug) and *Triatoma rubrofasciata* (De Geer, 1773), first recording the occurrence of *Schizotrypanum sp*-infected *P. megistus* in two districts in the capital of Republic (Santa Tereza and Botafogo). In 1953, in a survey performed at Araruama and Magé, Dias stated it was a relevant issue for the State, while Bustamante & Gusmão 1953 pointed out the presence of *T. infestans* at Resende and Itaverá cities. New findings have been identified, such as that of Coura et al. (1966), who found *P. megistus*, *Triatoma tibiamaculata* and *T. rubrofasciata* in three districts at Rio de Janeiro city, and that of Aragão & Souza (1971), who signalized the presence of *T. infestans* colonizing domiciles at two cities in Baixada Fluminense. In the same year, Coura et al. (1966) described some autochthonous instances of *T. infestans*-transmitted Chagas disease at Baixada Fluminense, and Becerra-Fuentes et al. (1971) recorded *T. rubrofasciata* occurrence at Morro do Telégrafo in the former Guanabara state. Silveira et al. (1982) performed an entomologic inquiry at Duque de Caxias and Nova Iguaçu cities (RJ), and only found *T. infestans* species. Ferreira et al. (1986) verified the occurrence of *T. vitticeps*, and positivity for *T. cruzi*-like forms, in 12 cities, of which the one with the highest incidence for both observations was Triunfo location at Santa Maria Madalena city. In 1989, a *P. geniculatus* specimen was found in a domicile at São Sebastião do Alto city (RJ) (personal communication with Teresa Cristina M. Gonçalves). The occurrence of *Rhodnius prolixus* (Stål, 1859) in Teresópolis was pointed out by Pinho et al. (1998), which caused questioning, once this species was restricted to the northern region of the country. Nowadays it is known this species does not occur in Brazil (Monteiro et al., 2000, 2003). *T. vitticeps* was found in Poço das Antas, Silva Jardim city, by Lisbôa et al. (1996), and in Santa Maria Madalena by Gonçalves et al. (1998). In both

Molecular and Proteolytic Profiles of

**2.1** *Trypanosoma cruzi*

and differential expression of surface enzymes.

evolution forms of the parasite (Vickerman, 1985).

*Trypanosoma cruzi* Sylvatic Isolates from Rio de Janeiro-Brazil 161

were isolated, which showed heterogeneity in which refers to biology, histopathogenesis

*Trypanosoma cruzi* (Figure 2) is a flagellated protozoan belonging to Trypanosomatidae family (Kent, 1880), Kinetoplastida order, *Trypanosoma* genus (Chagas, 1909a; Coura, 2006). Kinetoplastida order was established as a function of the presence of a single cytoplasmic structure, the kinetoplast (Wallace, 1966), where mitochondrial DNA or k-DNA is concentrated. Its form, size, and position are important for characterizing the different

Fig. 2. Epimastigote (1) and tripomastigote (2) forms of *Trypanosoma cruzi* sylvatic isolates from Trinfo, Santa Maria Madalena municipal district, State of Rio de Janeiro – Brazil.

It is a euryxene and digenetic trypanosomatid, since part of its life cycle occurs inside a vertebrate or invertebrate host (Hoare, 1964). Vertebrate and invertebrate hosts are represented, respectively, by domiciled or domestic mammals and sylvatic triatomines.

The parasite cycle can be summarized as follows: the triatomine vector usually defecates during or at the end of blood sucking, eliminating metacyclic trypomastigote forms of *T. cruzi* on the vertebrate hosts. These forms found in dejections can penetrate the host through a continuity skin solution or skin mucosa. Inside the host cell, trypomastigotes transform into amastigotes and, approximately 35 hours later, the binary division begins. After five days, amastigotes transform into trypomastigotes, and as soon as they have long flagella, the cell disrupts releasing these forms into the bloodstream, so that they infect other cells or achieve different organs (Sousa, 2000). In triatomines, the blood-sucking trypomastigote

locations, biological and morphological characterization of *T. cruzi* isolates, obtained for both triatomine bugs and vertebrate hosts, confirmed the maintenance of enzootic disease form. In the period from 2008 to 2010 *T. vitticeps* was pointed out at Cantagalo, Tanguá, Trajano de Morais, and São Fidélis cities (Oliveira et al., 2010).

In Espírito Santo, where *T. vitticeps* incidence was also signalized, the rates of infection by *T. cruzi*-like forms were assessed in specimens collected in the domicile: 4% by Santos et al. (1969) at Alfredo Chaves (ES); 25.2% by Silveira et al. (1983) at Cachoeiro do Itapemirim and Guarapari (ES); 35.2% by Ferreira et al. (1986) in 12 cities from Rio de Janeiro state; 64.70% by Sessa & Carias (1986) in 19 cities from Espírito Santo state; and 70.2% and 51.8%, respectively, for females and males, by Dias et al. (1989).

Fig. 1. Studied area and sites of capture of *Triatoma vitticeps* in Triunfo, Santa Maria Madalena, Municipal district, State of Rio de Janeiro, Brazil.

Data from National Health Foundation ("FUNASA") signalized *T. vitticeps* presence in the northern region of Rio de Janeiro state, and the number of notifications on adult form occurrence was increasing (Lopes et al., 2009; Dias et al., 2010). Although studies regarding *T. vitticeps* biology have suggested that this species would not represent a major concern from epidemiologic point of view (Dias, 1956; Heitzmann-Fontenelle, 1980; Silva, 1985; Diotaiuti et al., 1987; Gonçalves et al., 1988, 1989), reports of this species frequently invading the domicile with high *T. cruzi* infection rates (Gonçalves et al., 1998, Gonçalves, 2000) indicated its study was required. With sylvatic habit and unknown habitat, this species ecobiology was studied in further details at Triunfo district, Santa Maria Madalena city (RJ), in three areas (A, B and C) (Figure 1). Of the triatomine bugs collected, 68 *T. cruzi* samples

were isolated, which showed heterogeneity in which refers to biology, histopathogenesis and differential expression of surface enzymes.

### **2.1** *Trypanosoma cruzi*

160 Gel Electrophoresis – Advanced Techniques

locations, biological and morphological characterization of *T. cruzi* isolates, obtained for both triatomine bugs and vertebrate hosts, confirmed the maintenance of enzootic disease form. In the period from 2008 to 2010 *T. vitticeps* was pointed out at Cantagalo, Tanguá,

In Espírito Santo, where *T. vitticeps* incidence was also signalized, the rates of infection by *T. cruzi*-like forms were assessed in specimens collected in the domicile: 4% by Santos et al. (1969) at Alfredo Chaves (ES); 25.2% by Silveira et al. (1983) at Cachoeiro do Itapemirim and Guarapari (ES); 35.2% by Ferreira et al. (1986) in 12 cities from Rio de Janeiro state; 64.70% by Sessa & Carias (1986) in 19 cities from Espírito Santo state; and 70.2% and 51.8%,

Fig. 1. Studied area and sites of capture of *Triatoma vitticeps* in Triunfo, Santa Maria

Data from National Health Foundation ("FUNASA") signalized *T. vitticeps* presence in the northern region of Rio de Janeiro state, and the number of notifications on adult form occurrence was increasing (Lopes et al., 2009; Dias et al., 2010). Although studies regarding *T. vitticeps* biology have suggested that this species would not represent a major concern from epidemiologic point of view (Dias, 1956; Heitzmann-Fontenelle, 1980; Silva, 1985; Diotaiuti et al., 1987; Gonçalves et al., 1988, 1989), reports of this species frequently invading the domicile with high *T. cruzi* infection rates (Gonçalves et al., 1998, Gonçalves, 2000) indicated its study was required. With sylvatic habit and unknown habitat, this species ecobiology was studied in further details at Triunfo district, Santa Maria Madalena city (RJ), in three areas (A, B and C) (Figure 1). Of the triatomine bugs collected, 68 *T. cruzi* samples

Madalena, Municipal district, State of Rio de Janeiro, Brazil.

Trajano de Morais, and São Fidélis cities (Oliveira et al., 2010).

respectively, for females and males, by Dias et al. (1989).

*Trypanosoma cruzi* (Figure 2) is a flagellated protozoan belonging to Trypanosomatidae family (Kent, 1880), Kinetoplastida order, *Trypanosoma* genus (Chagas, 1909a; Coura, 2006). Kinetoplastida order was established as a function of the presence of a single cytoplasmic structure, the kinetoplast (Wallace, 1966), where mitochondrial DNA or k-DNA is concentrated. Its form, size, and position are important for characterizing the different evolution forms of the parasite (Vickerman, 1985).

Fig. 2. Epimastigote (1) and tripomastigote (2) forms of *Trypanosoma cruzi* sylvatic isolates from Trinfo, Santa Maria Madalena municipal district, State of Rio de Janeiro – Brazil.

It is a euryxene and digenetic trypanosomatid, since part of its life cycle occurs inside a vertebrate or invertebrate host (Hoare, 1964). Vertebrate and invertebrate hosts are represented, respectively, by domiciled or domestic mammals and sylvatic triatomines.

The parasite cycle can be summarized as follows: the triatomine vector usually defecates during or at the end of blood sucking, eliminating metacyclic trypomastigote forms of *T. cruzi* on the vertebrate hosts. These forms found in dejections can penetrate the host through a continuity skin solution or skin mucosa. Inside the host cell, trypomastigotes transform into amastigotes and, approximately 35 hours later, the binary division begins. After five days, amastigotes transform into trypomastigotes, and as soon as they have long flagella, the cell disrupts releasing these forms into the bloodstream, so that they infect other cells or achieve different organs (Sousa, 2000). In triatomines, the blood-sucking trypomastigote

Molecular and Proteolytic Profiles of

mitochondrial DNA.

combination events (Zingales, 2011).

*Trypanosoma cruzi* Sylvatic Isolates from Rio de Janeiro-Brazil 163

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

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

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 & Azambuja, 2000).

*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 multiple factors related with population epidemiology and genetics.

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