Trypanosoma cruzi: Biological characterization of lineages I and II supports the predominance of lineage I in Colombia

Experimental Parasitology 121 (2009) 83–91

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Trypanosoma cruzi: Biological characterization of lineages I and II supports the predominance of lineage I in Colombia Ana María Mejía-Jaramillo, Víctor Hugo Peña, Omar Triana-Chávez * Grupo de Chagas, Universidad de Antioquia, Calle 67 No. 53-108, AA 1226, Medellín, Antioquia, Colombia

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Article history: Received 18 February 2008 Received in revised form 10 September 2008 Accepted 1 October 2008 Available online 8 October 2008 Keywords: Chagas disease Trypanosoma cruzi (Kinetoplastida Trypanosomatidae) Rhodnius prolixus Rhodnius pallescens Rhodnius robustus Panstrongylus geniculatus Triatoma dimidiata Triatoma infestans (Reduviidae: Triatominae) Colombia TcI, T. cruzi I TcIIb, T. cruzi IIb Z1, zymodeme 1 Z2, zymodeme 2 Z3, zymodeme 3 BNZ, benznidazole LIT, liver infusion tryptose FCS, fetal calf serum DNA, deoxyribonucleic acid SL, spliced leader PCR, polymerase chain reaction bp, base pair dNTP, deoxyribonucleotide triphosphate TBE, 89 mM Tris borate, 2 mM EDTA [pH 8.3] UV, ultraviolet kDNA, kinetoplast DNA LSSP-PCR, low stringency single primer-PCR PBS, phosphate-buffered saline EC50, effective concentration 50 %I, percentage of inhibition MTT, 3(4.5-dimethylthiazol-2-yl)2,5diphenyltetrazolium bromide

a b s t r a c t The causes of the particular distribution of both Trypanosoma cruzi lineages throughout the American continent remain unknown. In Colombia, T. cruzi I is the predominant group in both domestic and sylvatic cycles. Here, we present the biological characterization of T. cruzi parasites belonging to both T. cruzi I and T. cruzi IIb groups. Our results show the inability of the T. cruzi IIb clones to infect mammalian cells, produce trypomastigotes and replicate in Rhodnius prolixus, the main vector species in this country. Moreover, this result was confirmed when other species from the same genus, such as R. pallescens and R. robustus, were infected with the same TcIIb clone and its parental strain, while the infection in other genera such as Triatoma and Panstrongylus was successful. Furthermore, the growth kinetics and duplication time in vitro suggest that the high prevalence of T. cruzi I in Colombia results from more successful interactions between parasite lineage, vector, and host species. This type of study may help to understand the factors influencing the particular epidemiological patterns of Chagas disease transmission in different endemic regions. Ó 2008 Elsevier Inc. All rights reserved.

1. Introduction Chagas disease affects 18 million people from different regions in Central and South America. In Colombia, this disease affects * Corresponding author. Fax: +57 4 219 65 20. E-mail address: [email protected] (O. Triana-Chávez). 0014-4894/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2008.10.002

900,000 people, with a total of 3.0 million at risk of infection. The infection is mainly transmitted by contaminated feces of triatomine insect species that act as vectors of the parasite Trypanosoma cruzi (Moncayo, 2003). Rhodnius prolixus and Triatoma dimidiata are the main vectors in Colombia (Guhl and Nicholls, 2001; Guhl, 2007; Guhl et al., 2007). However, Panstrongylus geniculatus is an important wild vector since it has been observed spo-

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radically invading human dwellings (Corredor et al., 1990; Wolff et al., 2001). The ability of P. geniculatus to infest household environments is a risk factor that contributes to maintaining domiciliary transmission and establishing new T. cruzi infections; what has been reported to be highly infected with this parasite (Carrasco et al., 2005; Wolff and Castillo, 2000). Trypanosoma cruzi is characterized by great genetic and phenotypic diversity among isolates (Tibayrenc and Ayala, 1991). Natural infections are constituted by multiple clones with different biological properties such as virulence, growth rate, pathogenicity, tissue tropism and sensitivity to anti-trypanocidal drugs (Macedo and Pena, 1998). Based on biochemical and molecular markers, T. cruzi has been classified into three different groups or zymodemes (Miles et al., 1978) and more recently into two major phylogenetic groups, designated as TcI, corresponding to zymodeme 1 (Z1), and TcII, corresponding to zymodeme 2 (Z2) and 3 (Z3) (Souto et al., 1996; Fernandes et al., 1998). Five sublineages have been defined within lineage II (named IIa–e), associated with the domestic (IIb, IId and IIe) or wild environments (IIa plus IIc) (Brisse et al., 2000; Tibayrenc, 2003). T. cruzi I was initially recognized in wild mammals, mainly in marsupials and sylvatic triatomines in the southern cone of South America (Fernandes et al., 1999; Jansen et al., 1999; Ceballos et al., 2006); however, it is the predominant lineage in the Andean region, Central America and Mexico, where it has been associated with both sylvatic and domestic cycles (Saravia et al., 1987; Bosseno et al., 2002; Anez et al., 2004; Carrasco et al., 2005; Cortez et al., 2006; Salazar et al., 2006; Black et al., 2007; Brenière et al., 2007; Samudio et al., 2007). On the other hand, T. cruzi II has also been reported to be associated with wild vectors and mammals in Colombia (Montilla et al., 2002; Salazar et al., 2006), and more recently the sublineage IIb has been associated with human infection in an endemic area of the country where T. dimidiata is the main vector and P. geniculatus has been found inside dwellings (Corredor et al., 1990; Guhl et al., 2007). The existence of a preferential association of certain parasite lineages with certain vector and mammalian species has been described (Azambuja et al., 1989; Garcia et al., 1989; Garcia and Azambuja, 1991; Pinto et al., 1998, 2000; Gaunt and Miles 2000; Yeo et al., 2005; Araujo et al., 2007). However, in Colombia little is known regarding the relationships of each parasite group with the local vector species and other biological parameters. The natural resistance of T. cruzi strains to chemotherapeutic drugs has been reported (Andrade et al., 1985; Filardi and Brener, 1987). Drug resistance has a strong impact on chemotherapy for Chagas disease, increasing the number of treatment failures in patients and greatly limiting the treatment options. Thus, the analysis of T. cruzi lineages would be relevant for studies focused on the correlation between the prevalence of susceptible or resistant parasite clones and resistance or re-infection (Coronado et al., 2006). In this context, the aims of this study were to evaluate the behavior of T. cruzi parasites belonging to different lineages (TcI and TcIIb) in axenic culture, production of trypomastigote forms, infection to vector species, and finally in responses to benznidazole (BNZ), following the dynamics of T. cruzi growth kinetics in experimental models. Our results support the predominance of T. cruzi I in Colombia, an aspect of great importance to the epidemiology of Chagas disease in the country. 2. Materials and methods 2.1. Parasites The TcI strain SN-5 (I.RHO/CO/02/SN-5.GUAJ) was obtained from a naturally infected R. prolixus in the northern state of Guajira,

while the TcIIb strain, AF-1 (I.PANS/CO/93/AF-1.ANT), was obtained from naturally infected P. geniculatus in the state of Antioquia. Both strains were cryopreserved until they were used to obtain the clones, SN5cl8 (TcI clone) and AF-1cl7 (TcIIb clone), by micromanipulation and to characterize the clones genetically and biologically. Additionally, the reference clone, Tu18cl2 (TcIIb), was also used to carry out the same experiments. Finally, the TcI strain, Sebas16 (I.RHO/CO/06/Sebas16.MAG), isolated from R. pallescens, and its clone, cl17, were used in some insect infections. Parasites were maintained at 28 °C in LIT medium supplemented with 10% FCS by passages every 7 days (Camargo, 1964). 2.2. DNA purification Nucleic acids from epimastigote forms were obtained by means of the Salting-out method (Miller et al., 1988). DNA from triatomine feces was purified using DNAzol reagent (Gibco–BRL, USA), as recommended by the manufacturer, with 25 ll of the feces. 2.3. PCR amplification of the intergenic regions of the SL DNA genes (SL DNA) PCR amplification was performed in 0.2-ml microcentrifuge tubes containing 25 ll of reaction mixture. Primers for amplification of the intergenic region of T. cruzi SL DNA genes were: 50 -GTGTCCGCCACCTCCTTCGGGCC-30 (TC1, group II-specific), 50 CCTGCAGGCACACGTGTGTGTG-30 (TC2, group I-specific) and 50 CCCCCCTCCCAGGCCACACTG-30 (TC, common to groups I and II) (Souto et al., 1996). Amplicons of 350 bp for TcI and 300 bp for TcII were expected. The reaction mixture contained 25 ng of DNA isolated from cultured parasites, 50 mM KCl, 10 mM Tris–HCl (pH 8.0), 0.1% Triton X-100, 200 lM of each dNTP, 1.5 mM MgCl2, 12.5 pmol of each primer, and 0.625 U of Taq DNA polymerase. PCR was carried out at an initial temperature of 94 °C for 3 min, followed by 27 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, with a final extension at 72 °C for 10 min (Souto et al., 1996). The amplification products were analyzed by electrophoresis on 2% agarose gels in 1 TBE, stained by ethidium bromide and detected by UV light (Maniatis et al., 1982). 2.4. PCR amplification of T. cruzi kDNA 330-bp fragment The variable region of the minicircles of kDNA was amplified with the primers 121 (50 -AAATAATGTACGGGT/GGAGATGCATG A-30 ) and 122 (50 -GTTCGATTGGGG TTGGTGTAATATA-30 ) (Sturm et al., 1989). PCR was carried out at a final volume of 50 ll of reaction mix containing 50 mM KCl, 10 mM Tris–HCl, 0.1% Triton X-100, 2.5 ll of DNA from feces or 25 ng of DNA from cultured parasites, 37 pmol of each primer, 200 lM of dNTP, 1.5 mM of MgCl2 and 2.5 U of Taq DNA polymerase. PCR was carried out at an initial temperature of 94 °C for 3 min, followed by 35 cycles at 94 °C for 45 s, 63 °C for 45 s, 72 °C for 45 s and a final cycle at 72 °C for 10 min. The amplification products for each sample were analyzed by electrophoresis on 2% agarose gel in 1 TBE, stained by ethidium bromide and visualized under UV light (Maniatis et al., 1982). 2.5. Detection of PCR inhibition The presence of PCR inhibitors was examined in PCR-negative feces. In brief, 2.5 ll of DNA from feces were mixed with 10 ng of DNA from a reference T. cruzi strain in the same reaction tube to perform the amplification of T. cruzi kDNA by PCR, as described above. The PCR was inhibited when the 330-bp fragment was not detected.

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2.6. LSSP-PCR of kDNA

2.10. Parasite quantification

Fifteen microliters of the PCR products from cultured parasites were run on 1.5% low-melting-point agarose gel and stained with ethidium bromide. Bands corresponding to 330 bp for kDNA were cut from the gel and diluted to 1:10 in double distilled water. One microliter of the dilution was used as a template for the LSSP-PCR (Vago et al., 1996). LSSP-PCR was performed in 25 ll of the final volume using 120 pmol of the 121 primer, 4 U of Taq polymerase, 200 lM of each dNTP, 1.5 mM of MgCl2, 50 mM of KCl, 10 mM Tris–HCl and 0.1% Triton X-100. Amplification was carried out with 3 min of initial denaturing at 94 °C, followed by 35 cycles at 94 °C for 45 s, 30 °C for 45 s, 72 °C for 45 s and a final cycle at 72 °C for 10 min (Pena et al., 1994). Fifteen-microliter samples of the amplification products from each of the stocks were analyzed by electrophoresis on 3% agarose gel in 0.5 TBE, stained with ethidium bromide and visualized under UV light (Maniatis et al., 1982).

For parasite quantification in feces, the abdomen of newly fed insects with defibrinated human blood was carefully squeezed and feces homogenized in 50 ll of PBS. The number of parasites was determined using Neubauer hemocytometers at days 15, 30, 45, 60, 75 and 90 after infection. In addition, the rectum of insect species infected with trypomastigotes and R. prolixus, R. pallescens, and R. robustus infected with the TcIIb Colombian clone and its parental strain were quantified in the same way as feces. Finally, biological efficacy was calculated using the number of total parasites present in each sample.

2.7. Growth curves To determine growth kinetics, 5  105 epimastigotes of each parasite culture were grown in 2.0 ml of LIT medium with 10% FBS at 28 °C in 30 different tubes. The concentration of the parasites was estimated daily from two tubes in a hemocytometer chamber up to the 30th day in culture. The generation number and doubling time were calculated for each curve, on the days at start up the log phase, as follows: the generation number was calculated using log fc–log ic/0.30 (fc, final concentration; ic, initial concentration of parasites counted). The doubling time equaled 24 h per generation number (Frobisher, 1968). Moreover, a mixed curve containing equal proportions of each of the two Colombian clones (TcI and TcIIb) was made to determine the presence of both groups when they were cultured together (Araujo et al., 2007). This curve was characterized by conventional PCR every 7 days using the intergenic spacer of the miniexon gene. 2.8. Production of trypomastigote forms on cultured mammalian cells Trypomastigotes were obtained by infecting Vero cells with parasites from cultures of the three clones at the stationary growth phase. The infected Vero cell monolayers were cultured in RPMI1640 media supplemented with 2.5% FBS (Moore et al., 1967). Cells were cultured at 37 °C, 5% CO2 until the trypomastigotes forms were obtained. 2.9. Insect infections A total of 20 fourth-instar larvae of R. prolixus, R. pallescens, R. robustus, P. geniculatus, T. dimidiata and T. infestans were artificially fed through latex membranes using defibrinated human blood without complement mixed with 5  106 epimastigotes/ml from LIT culture medium near the end of the log phase. Due the differences between the sizes of the triatomine species used in this study, the number of ingested parasites (Ip) by each insect was normalized by weighing each individual before (wb) and after (wa) feeding, which was calculated as follows:

  wa  wb  Cp Ip ¼ dblood where the constant dblood is the blood density equal to 1.050 g/ml and the constant C p represents the concentration of parasites present in the blood. Additionally, 20 fourth-instar larvae of R. prolixus and T. infestans were infected as previously described, using trypomastigotes from the TcI clone.

2.11. Determination of the effective concentration 50 (EC50) and the percentage of inhibition (%I) to BNZ EC50 and %I to BNZ for the three clones studied were determined using an enzymatic micromethod, previously described by Muelas (1999) and Zapata (2004). Briefly, 1  107 epimastigotes at log phase were cultured in triplicate in 96-well plates (Falcon, Ref. 353072) with concentrations of BNZ ranging from 10 lg/ml to 0.16 lg/ml and incubated for 96 h at 28 °C. Untreated parasites were used as positive controls. The plates were incubated with MTT 10 mg/ml for 90 min at 28 °C and the reduction of MTT to Formazan’s crystals was measured to 595 nm in an Enzyme-Linked Immuno Sorbent Assay (ELISA) reader (Bio-Rad). The EC50 and %I corresponding to the percentage of dead parasites at the higher evaluated concentration was calculated using the SPSS program v14 (Zapata, 2004). 2.12. Statistical analysis The growth kinetics was analyzed by nonlinear regression of fixed effects, fitting data to a logistic curve that is defined by the equation:

y ¼ f ðtÞ ¼

a 1 þ expðb  ctÞ

where a defines the maximum growth rate, b the latency phase period, c the change in growth rate by population unit time (slope) and t time (Guisande et al., 2006). All analyses were performed using the statistical Kruskal–Wallis test (SPSS program, v.14) to compare the growth kinetics between the different experiments. Biological efficacy was compared for the different species and clones using the Kruskal–Wallis test. EC50 and %I were compared using one-way analysis of variance (ANOVA). Means were compared using the Tukey test and the results were considered significant when p < 0.05.

3. Results 3.1. Genetic characterization of the parasites by amplification of the intergenic regions of the SL DNA genes and LSSP-PCR of kDNA Parasite lineages were confirmed by the amplification of the intergenic regions of the SL DNA genes. The results obtained corroborated the classification for each of the phylogenetic groups. The parasites belonging to TcI and TcII amplified 350 bp and 300 bp products, respectively (Fig. 1A). To further establish the genetic profile of each clone, LSSP-PCR of kDNA was applied. The signatures obtained were variable among the clones, even for those belonging to the same phylogenetic group (Af1cl7 and Tu18cl2) (Fig. 1B). However, the strains and their derived clones presented the same profile (Fig. 1B).

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Fig. 2. Growth curves obtained for the strains (str) and clones (cl) belonging to T. cruzi I and II. Insert graph: beginning of the log phase to calculate the doubling time of each strain or clone.

Fig. 1. Genetic characterization of the parasites by the amplification of the intergenic regions of the SL DNA genes and LSSP-PCR of kDNA. (A) About 1.5% Agarose gel electrophoretic analysis of the amplification products of the intergenic regions of the SL DNA genes from strains and clones from the two phylogenetic groups of T. cruzi. M: 100-bp ladder molecular-weight marker. (B) Three percent Agarose gel electrophoretic analysis of the signatures obtained by kinetoplastid (kDNA)-LSSP-PCR from strains and clones from the two phylogenetic groups of T. cruzi. M: 50-bp ladder molecular-weight marker.

3.2. Determination of growth kinetics of T. cruzi I and II The growth kinetics in vitro and the generation numbers at the beginning of the log phase for each of the evaluated strains and clones are illustrated in Fig. 2. These data were used to calculate the population doubling time. The results showed the highest doubling time (44 h) for the reference clone Tu18cl2 (TcIIb), while the Colombian strains and their clones shared lower doubling times (between 25 h and 30 h) (Fig. 2, insert). When the parameters from the logistic curve were analyzed, we found that the TcI clone presented the maximum growth rate (parameter a) followed by the TcIIb clone. In addition, the TcI clone showed a significantly different growth rate from its corresponding strain. In contrast, the period of latency (parameter b) and slope (parameter c) were longer for the strains and significantly different from their clones. The analysis of each parameter did not show a correlation with the phylogenetic group (Fig. 3A–C). On the other hand, the PCR amplification of the intergenic region of the SL DNA genes of the mixed curve containing the two Colombian clones (TcI and TcIIb) showed the presence of both 300- and 350-bp products corresponding to the amplification of both clones until day 28 post-inoculation (data not shown).

Fig. 3. Parameters from the logistic curve analyzed for the strains and clones of T. cruzi I and II. (A) Maximum growing rate (parameter a). (B) Latency phase (parameter b) period. (C) Change in rate by population unit of time (parameter c). * p < 0.05.

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3.3. Production of trypomastigote forms and infectivity of cells The capability of the clones to infect different cells and to produce trypomastigotes was also evaluated. The results showed differences between the clones studied associated with the phylogenetic group. The TcI clone was able to infect the Vero cells detected by the presence of intracellular amastigotes and a high number of trypomastigotes after 8 days post-infection, while the clones belonging to TcIIb were not able to infect these cells despite evaluation of a longer period of incubation. 3.4. Biological efficacy of T. cruzi I and II in different vector species To determine if there were differences in biological efficacy between infections using epimastigote or trypomastigote forms, R. prolixus and T. infestans specimens were infected with the TcI clone, the only one able to produce both parasitic forms. In this case, the number of parasites in feces was higher for both vector species when they were infected with trypomastigotes (Fig. 4). However, this experiment showed that epimastigotes could efficiently infect vector species, even though they are not the natural infective form, as was shown previously (Mello et al., 1996; Garcia and Azambuja, 1997; Azambuja et al., 2004; Araujo et al., 2007), and therefore they could be used to initiate the infections in different vector species. When the average number of parasites ingested by each species was calculated, the genus Rhodnius appeared to be the species that fed with one of the largest burdens of parasites (Fig. 5A), even though it was among the smallest species. To calculate the biological efficacy of T. cruzi I and T. cruzi IIb in each epidemiologically important triatomine genus, R. prolixus, P. geniculatus and T. infestans infected with epimastigotes were used. When parasites were quantified in the feces of each vector species, no parasites were detected in the R. prolixus insects infected with the TcIIb clones (Fig. 5B). On the contrary, both the Colombian and the reference clones from this genotype successfully developed, particularly in P. geniculatus and T. infestans, respectively, the species from which the original strains had been isolated (Fig. 5B). The inability of these clones to infect R. prolixus insects was confirmed by microscopic analysis of the rectum, which had shown the greatest number of parasites in the trypomastigotes assay and by means of a more sensitive technique such as kDNAPCR carried out from fecal and rectum material (data not shown). In contrast, the TcI clone was able to infect the three vector species; interestingly, the highest burden of parasites was detected in T. infestans insects (Fig. 5C). In fact, this clone was the only one able

Fig. 4. Biological efficacy of epimastigotes (epis) and trypomastigotes (trypos) of the TcI clone in the feces of R. prolixus and T. infestans.

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to infect R. prolixus, the species from which the original strain had been isolated (Fig. 5C). To confirm the result obtained in R. prolixus with the TcIIb clones, other infections were initiated using more species from the same genus, such as R. pallescens and R. robustus, and from another, such as T. dimidiata. In these experiments it was impossible to detect parasites in either of the Rhodnius species, whereas in T. dimidiata infection could be achieved (Fig. 5B). Moreover, when the TcIIb parental strain was used to infect the same three species of Rhodnius, no parasites could be detected in the feces or rectum of these species (data not shown). In addition, the Sebas16 strain and its clone cl17 were used to infect R. prolixus and R. pallescens in order to increase the parasite’s variability. In this case, both strain and clone infected the two triatomine species successfully (Fig. 5C), as was shown for the TcI clone in R. prolixus. Finally, the post-feeding follow-up in all triatomines showed the highest parasite load at 75 days post-infection (data not shown). 3.5. Determination of EC50 and %I to BNZ To assess whether there were differences in the resistance to BNZ of the studied clones, EC50 and %I were evaluated. The Colombian TcI and TcIIb and the reference TcIIb clone presented high susceptibility to BNZ because the EC50 values were very low: 1.4505, 0.7326 and 2.2075, respectively. Both TcIIb clones showed statistically significant differences in drug susceptibility (p < 0.05). Additionally, %I was very high in all cases, with values ranging from 94.85% to 100%. The greatest differences were found between the TcIIb clones. Therefore, no correlation between the phylogenetic group of T. cruzi and the susceptibility to this drug was found.

4. Discussion Natural strains of T. cruzi are multiclonal. However, the number of clones could change drastically in different environments as a result of either their inability to compete and propagate in the hosts or the action of the host defense mechanisms, decreasing the complexity of the infecting strains. Thus, due to the high genetic diversity of clones that conform a strain, it has been suggested that the biological characteristics of each group of T. cruzi must be studied separately for each natural clone (Macedo and Pena, 1998). Although phylogenetic groups and subgroups have been defined for this parasite (Souto et al., 1996; Brisse et al., 2000), it has not been possible to correlate this genetic variability with different biological parameters such as growth dynamics in axenic cultures, production of trypomastigote forms, infectivity of different vector species and susceptibility to BNZ. Trypanosoma cruzi lineages have a particular distribution throughout the American continent, with some of them predominant in certain geographical areas and transmission cycles. However, the mechanisms leading to this differential geographical distribution remain unknown. In Colombia, T. cruzi I (Z1) is the predominant group in both domestic and sylvatic cycles. The biological parameters evaluated in T. cruzi parasites belonging to the TcI and TcIIb lineages support the argument for the geographical predominance of T. cruzi I in Colombia. First, the highest production of trypomastigote forms from TcI and the lack of infectivity of TcIIb in Vero cells. Mammalian cell invasion by T. cruzi is a complex process in which various parasite and host cell components interact, triggering the activation of signaling cascades and Ca2+ mobilization in both cells. Metacyclic trypomastigotes use surface glycoproteins with dual Ca2+ signaling activity, in a manner dependent on the T. cruzi strain and also highly dependent on the host cell type (Mortara et al., 1999; Calderon-Arguedas et al.,

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Fig. 5. Biological efficacy of T. cruzi I and IIb clones in different vector species. (A) Mean of the number of parasites ingested by the vector species R. prolixus (Rp), R. pallescens (Rpa), R. robustus (Rr), P. geniculatus (Pg), T. dimidiata (Td) and T. infestans (Ti). (B) Number of parasites found in the feces of the R. prolixus (Rp), R. pallescens (Rpa), R. robustus (Rr), T. dimidiata (Td), T. infestans (Ti) and P. geniculatus (Pg) insect vectors infected with T. cruzi IIb parasites. (C) Number of parasites found in the feces of the R. prolixus (Rp), R. pallescens (Rpa), P. geniculatus (Pg) and T. infestans (Ti) insect vectors infected with T. cruzi I parasites. *p < 0.05.

2002). The binding of gp82 or the mucin-like gp35/50 to its receptors triggers a signaling cascade involving protein tyrosine kinase, phospholipase C and the production of inositol 1,4,5-triphosphate or the route in which adenylate cyclase, generation of cAMP and Ca2+ mobilization from acidocalcisomes are implicated, respectively (Burleigh and Andrews, 1995; Burleigh and Woolsey, 2002). In this sense, it had been demonstrated that the failure to infect cells from clones belonging to TcII was caused by the low expression of surface proteins such as gp82 (Atayde et al., 2004). Additionally, other authors corroborated that the interaction between parasite and host cells could be significantly different in the clones but not necessarily the intracellular replication of these (Mortara et al., 1999). This result opens the hypothesis that the predominance of lineage I in Colombia is related to its higher efficacy in invading the host cell. This was reported recently in an endemic area in Colombia where, despite the presence of TcIIb in humans, TcI was the predominant group (Zafra et al., 2008). Finally, this requires evaluating the expression of the different surface

proteins involved in cell infection as well as the other mechanisms involved using other cell lines. Second, the ability of the clones to proliferate in different vector species that are epidemiologically important for Chagas disease in Colombia also differed between the two phylogenetic groups. Indeed, the reference clone Tu18cl2, the TcIIb parental strain and its clone were unable to infect R. prolixus, whereas the latter two were incapable of infecting R. pallescens and R. robustus insects, even though this genus was fed with one of the highest number of parasites. In this case, it was important to control the number of parasites ingested by each one of the vectors, because the results could be biased by the volume of the ingested blood depending on the body size of the species. Additionally, it was necessary to test that the epimastigote forms could infect as well as the trypomastigotes, which was proved using the TcI clone. Moreover, the results found in T. infestans showed that this species was infected with both groups, as was previously reported in natural and artificial infections (Pinto et al., 2000; Marcet et al., 2006). The result

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obtained with the TcIIb strain and clones in R. prolixus was in agreement with those reported by other authors who had shown negative selection of strains, belonging to the same subgroup in this vector (Azambuja et al., 1989; Garcia et al., 1989; Mello et al., 1996; Azambuja et al., 2004). This behavior has been attributed to diverse factors such as the presence of stomach lytic factor, lectins, bacterial flora, the strain or clones and the vector species used, and the metacyclogenesis process of the infecting parasites (Garcia and Azambuja, 1991; Garcia et al., 2007), which should be further characterized in future studies. On the other hand, the TcI clone, Sebas16 and its clone proliferated very well in all the triatomine species tested, proving that T. cruzi I from Colombia could infect not only wild but also domestic vector species. This provides evidence of the capability of this phylogenetic group to interact successfully with multiple hosts and vectors, which has been demonstrated in Colombia and other countries where this group also prevails with the isolate obtained from different hosts (Saravia et al., 1987; Bosseno et al., 2002; Anez et al., 2004; Carrasco et al., 2005; Cortez et al., 2006; Salazar et al., 2006; Black et al., 2007; Brenière et al., 2007; Samudio et al., 2007). This is a problem for the epidemiological control of Chagas disease transmission in Colombia, because wild vector species could always infect or re-infect human and mammal reservoirs, complicating the success of the insecticide actions, focused on domestic habitats, aiming at eradicating transmission. This was demonstrated in a region from Colombia, where occasionally the wild vector E. cuspidatus invades houses. In this region, Dib et al. (2005) identified a common T. cruzi genotype for parasites isolated from this vector and humans using RAPDs, pointing toward the significant epidemiological relevance of E. cuspidatus in the vectorial transmission cycle of T. cruzi. The biological efficacy found for the TcIIb clones in the different triatomine species showed that they proliferated better in those species from which their original parasite strains had been isolated. This reflects the varying co-evolution in this group with specific vectors and hosts in particular zones, as has been suggested by various authors (Gaunt and Miles, 2000; Yeo et al., 2005), and it has been partially proved with the reports of isolations of the parasite from different vector species (Brisse et al., 2000; Yeo et al., 2005), where there are no strains belonging to TcIIb isolated from R. prolixus. Nevertheless, wild vectors such as P. geniculatus were successfully infected with T. cruzi I and T. cruzi IIb parasites (Fig. 5B and C), in agreement with previous reports of natural infections (Carrasco et al., 2005; Salazar et al., 2006), and domestic vectors such as T. dimidiata were infected with the TcIIb clone. The fact that these wild and domestic vectors can become infected with both parasite groups complicates the epidemiological picture and the success of chemotherapy in combating Chagas disease in Colombia. Furthermore, it will be necessary to consider the risks in certain zones of this country, where wild vectors such as P. geniculatus, infected with both T. cruzi groups, are responsible for the transmission of the parasite (Moreno and Jaramillo, 1995; Jaramillo et al., 2000; Wolff et al., 2001; Dib et al., 2005). It will also be important to consider places where T. dimidiata is the main domestic vector, where the subgroup TcIIb has recently been reported infecting humans (Zafra et al., 2008). The assays performed with insects infected with the trypomastigote form of T. cruzi I showed a higher number of parasites in both feces and rectum in R. prolixus compared to the experiments using epimastigotes. This result opens the possibility that when trypomastigotes of TcIIb are used, a positive infection in this vector can be obtained, which remains to be studied. In sum, we found that TcIIb clones could not infect mammalian cells, produce trypomastigotes and replicate in R. prolixus, the main vector in Colombia. Thus, the predominance of T. cruzi I in Colombia is closely related to the predominance of R. prolixus.

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In addition, the other parameters studied (growth kinetics and duplication time) support the hypothesis that the TcI–R. prolixus interaction is the factor responsible for the high prevalence of the TcI group in Colombia, rather than intrinsic parasitic factors. Indeed, when the in vitro growth dynamics of TcI and TcIIb strains and clones was investigated, no correlations between growth and phylogenetic group could be established. Despite this lack of correlation, individual differences between the strains and their clones were observed in all parameters tested, probably because of the multiclonal nature of these strains. The maximum rate of growth (parameter a) was smaller for the strains than for the clones. This could be explained by a competition among the different clones of each strain that generated a more rapid reduction of the nutrients leading to growth inhibition, whereas monoclonal populations could proliferate more successfully. In addition, the longer period of latency (parameter b) for the strains could be due to the heterogeneous periods of adaptation and replication rates of the individual clones that conform a strain. Although the profiles from the LSSP-PCR of the kDNA were identical between the strains and their clones, the differences found in this biological trait demonstrate that the strains are multiclonal. Hence, the homogeneity among strains and clones in their LSSP-PCR profiles could be explained by the predominance of a major clone, as has been described by Devera et al. (2003). Additionally, mathematical models have suggested that clones that multiply faster will be the predominant ones in a multiclonal strain (Finley and Dvorak, 1987). In this study, no differences in the doubling time for the Colombian strains and clones were found, showing that both groups of T. cruzi can succeed in hosts and vectors in natural infections, thus supporting our hypothesis. To further confirm this result, a mixed curve containing both clones from each group was constructed. The amplification of the intergenic regions of the SL DNA genes by PCR showed the presence of both groups when they were cultured together. In this case, PCR was a useful tool to detect both clones over time. The lack of a relationship between the phylogenetic group and the susceptibility to BNZ detected in our study is supported by results obtained by Villarreal et al. (2004). In our case, the EC50 values obtained were very low, in comparison with the values reported for other Colombian TcI strains, but they were in agreement with the susceptibility reported for TcIIb (Zapata, 2004; Triana et al., 2005). Our results support the hypothesis that variable degrees of susceptibility to the drug could be attributed to the multiclonality of the strains (Veloso et al., 2001) and that different clones that conform the same strain and/or that belong to the same phylogenetic group can display variable degrees of susceptibility (Triana et al., 2005). Finally, the biological characterization presented herein contributes to explaining the predominance of T. cruzi I in Colombia, corroborating the relevance of the parasite–host and parasite–vector interactions. This type of study may help to understand the factors influencing the particular epidemiological pictures of Chagas disease transmission in different endemic regions. Acknowledgments This work was financed by Banco de la República of Colombia, Proyect 2.020 and partially by the Sustainability project, University of Antioquia 2007–2008. References Andrade, S.G., Magalhaes, J.B., Pontes, A.L., 1985. Evaluation of chemotherapy with benznidazole and nifurtimox in mice infected with Trypanosoma cruzi strains of different types. Bulletin of the World Health Organization 63, 721–726.

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