Acta Tropica 100 (2006) 110–118

High variability of Colombian Trypanosoma cruzilineage I stocks as revealed by low-stringency

single primer-PCR minicircle signatures

Alexander Salazar a, Alejandro G. Schijman b, Omar Triana-Chavez a,∗a Grupo de Chagas, Corporacion de Patologıas Tropicales, Instituto de Biologıa, Universidad de Antioquia, Medellın, Colombia

b Laboratorio de Biologıa Molecular de la Enfermedad de Chagas, Instituto de Investigaciones en Ingenierıa Genetica yBiologıa Molecular, CONICET, Argentina

Received 19 July 2006; received in revised form 22 September 2006; accepted 4 October 2006Available online 13 November 2006

Abstract

In Colombia, high genetic variability has been found among Trypanosoma cruzi stocks isolated from different vector and hostspecies, using isoenzyme analysis and RFLP of total kinetoplastid DNA (kDNA), suggesting that several genetically related T.cruzi populations might be present within a single geographical area or adjacent ones. The objective of this study was to use thelow-stringency single primer (LSSP)-PCR technique on variable regions of kDNA minicircles of T. cruzi to determine possiblegenetic relationships among stocks from distinct geographical regions of Colombia and different vector species and hosts. AlthoughLSSP-PCR analysis showed a high genetic variability among 30 Colombian T. cruzi stocks, 29 of them belonged to T. cruzi lineageI, confirming that this lineage is predominant in different vector and host species from Colombia. Interestingly, one stock isolatedfrom a Pastrongylus geniculatus bug was identified as T. cruzi lineage IIb, using PCR strategies targeted to the intergenic region ofminiexon genes, a sequence encoding the D7 domain of the 24s� ribosomal genes and the A10 fragment, being this finding, thefirst description of this lineage in Colombia.

The LSSP-PCR signatures allowed correlation of most isolates with their respective geographical origins, and in one case from

host and vector specimens at a same region, suggesting a transmission event. Moreover, variations in LSSP-PCR profiles among T.cruzi I stocks from a same region suggest that they may have a multiclonal character. Our results show that LSSP-PCR is a fast,valuable technique for characterization of intra-lineage polymorphism among T. cruzi stocks.© 2006 Elsevier B.V. All rights reserved.

ity; Lin

Keywords: Chagas; Trypanosoma cruzi; LSSP-PCR; Genetic variabil

1. Introduction

Chagas’ disease caused by Trypanosoma cruzi is animportant public health problem, affecting 16–18 million

∗ Corresponding autor at: Calle 62 No. 52-59, Sede de InvestigacionUniversitaria, SIU, Universidad de Antioquia, Medellın, Colombia.Tel.: +57 4 210 65 20; fax: +57 4 210 65 20.

E-mail address: otriana@gmail.com (O. Triana-Chavez).

0001-706X/$ – see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.actatropica.2006.10.003

eage

people in Central and South America. It has been esti-mated that 1.3 million people in Colombia are infectedand 3.6 million are at risk of acquiring the infection(Moncayo, 2003). The severity and symptoms of Cha-gas’ disease vary among different geographical regions

and it has been suggested that this variability could bedetermined by genetic factors, both host- and parasite-related (Macedo and Pena, 1998). Initial characteriza-tion of natural populations of T. cruzi stocks suggested

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hat they can be classified into three different groupsr “zymodemes” (Miles et al., 1978). However, highenetic heterogeneity was demonstrated by analysis forize polymorphism of kinetoplast DNA restriction frag-ents (Morel et al., 1980), DNA fingerprinting (Macedo

t al., 1992) and molecular karyotyping (Henriksson etl., 1996).

Long-term clonal evolution together with episodesf genetic exchange led to the individualization of sixiscrete genetic subdivisions or lineages, designated as. cruzi I, T. cruzi IIa, T. cruzi IIb, T. cruzi IIc, T. cruziId and T. cruzi IIe, which include all typed strains andloned stocks thus far isolated (Tibayrenc, 2003; Brisset al., 2003).

T. cruzi lineages appear to be distributed differen-ially among triatomine and host species and habitats inifferent geographical areas (Higo et al., 2004; Yeo etl., 2005). Although all T. cruzi populations cause theuman disease, epidemiological studies suggest that T.ruzi IIb, IId and IIe are more related to anthroponoticnvironments and chronic Chagas’ disease patients, T.ruzi lineages IIa and IIc to sylvatic environments, and. cruzi lineage I to both (Yeo et al., 2005; Marcet etl., 2006). These host and geographic specificities aressociated with different biological properties, such asrowth rate, histotropism, antigenicity, pathogenicity,nfectivity of potential insect vectors, drug suscepti-ility, chromosome number, and DNA content, whichre likely to represent key determinants in transmissionnd pathogenesis of Chagas’ disease (Macedo andena, 1998; Macedo et al., 2004). The identificationf lineages, natural isolates and strains of T. cruzi haveeen established in parasite isolates by a plethora ofiochemical and molecular markers (Brisse et al., 2001;acedo et al., 1992; Marcet et al., 2006).High genetic variability of Colombian T. cruzi stocks

as been found based on isoenzyme analysis and restric-ion fragment length polymorphism of kDNA (Saraviat al., 1987; Marquez et al., 1998; Triana et al., 1999;aramillo et al., 1999; Montilla et al., 2002). These workshowed that T. cruzi I (zymodeme I) prevailed in Colom-ia; however, Saravia et al. (1987), also reported thexistence of Zymodeme 3-like stocks and Montilla etl. (2002), the presence of T. cruzi II.

Low-stringency single specific primer-polymerasehain reaction (LSSP-PCR) for amplifying the variableegion of the T. cruzi kDNA minicircles permits transla-ion of the intra-lineage polymorphism in the nucleotide

equence into single reproducible profiles (Vago et al.,996). Changes in a base may alter the multiband pat-ern generated by LSSP-PCR, producing new signa-ures that are diagnostic for specific alterations (Pena

100 (2006) 110–118 111

et al., 1994; Vago et al., 1996, 2000; Andrade et al.,1999).

The objective of the present study was to use LSSP-PCR of kDNA to determine possible genetic associationamong T. cruzi stocks from a geographical region andto evaluate the usefulness of this technique as a molec-ular tool for epidemiological analysis of T. cruzi stocksisolated from different host and vector species in severalgeographical regions of Colombia, representing areas oflow, medium and high endemicity for Chagas’ disease.

2. Material and methods

2.1. Origin of T. cruzi stocks

Thirty Colombian T. cruzi stocks were analysed dur-ing the present study. These were isolated from differenthost species and geographical regions of Colombia, rep-resenting areas of low, medium and high endemicity forthe parasite (Moreno, 1997). The T. cruzi II stocks CLBrener, Noel, Y and JGBH from Brazil, Tulahuen fromChile, and Gal57 of T. rangeli from Colombia were usedas reference strains (Table 1).

2.2. Culture of parasites

Epimastigotes were cultured at 28 ◦C in LIT mediumsupplemented with 5% bovine foetal serum. The stockswere subcultured every 7 days during the exponentialgrowth phase (Camargo, 1964).

2.3. Extraction of DNA

Epimastigotes in logarithmic phase were harvested bycentrifugation at 3000 rpm and washed three times withPBS at pH 7.4. DNA was extracted by the salting-outmethod (Miller et al., 1988).

2.4. PCR-based identification of T. cruziphylogenetic lineages

To distinguish between T. cruzi I and T. cruzi IIb, IIdand IIe phylogenetic lineages, a PCR strategy targetedto the intergenic region of the miniexon genes (SL-DNAPCR) was carried out, using sense oligonucleotides 5-GTGTCCGCCACCTCCTTCGGGCC-3′ (TC1, groupII specific), 5′-CCTGCAGGCACACGTGTGTGTG-3′(TC2, group I specific), and antisense oligonucleotide

5′-CCCCCCTCCCAGGCCACACTG-3′ (TC, commonto groups I and II) (Souto et al., 1996). PCR was per-formed in 25 �l of reaction mixture containing 25 ng ofDNA, 50 mM KCl, 10 mM Tris–HCl (pH 8.0), 200 �M

112 A. Salazar et al. / Acta Tropica 100 (2006) 110–118

Table 1Biological hosts, vectors and geographical origin of Trypanosoma cruzi stocks

International identification Geographical origin Vector/host

TDIM/CO/1999/Lb53 Las Brisas (Sucre) Triatoma dimidiataTPAC/CO/1995/So8 San Onofre (Sucre) Rhodnius pallescensTCUS/CO/1995/So22 San Onofre (Sucre) Eratyrus cuspidatusTPAC/CO/1995/So9 San Onofre (Sucre) R. pallescensMDID/CO/1991/Gal52 Galeras (Sucre) Didelphis marsupialisTPAC/CO/1999/G20 Galeras (Sucre) R. pallescensTPAC/CO/1991/Gal34 Galeras (Sucre) R. pallescensXXXX/CO/1991/Gal61 Galeras (Sucre) RodentTPAC/CO/1991/Gal4 Galeras (Sucre) R. pallescensTGEN/CO/1998/Ov1 Ovejas (Sucre) Pastrongylus geniculatusTGEN/CO/1998/Ov17 Ovejas (Sucre) P. geniculatusMHOM/CO/1998/W3534 Ovejas (Sucre) Homo sapiensTDIM/CO/1999/B94 San Bernardo (Cordoba) T. dimidiataTPRX/CO/2001/Mag1 San Sebastian (Magdalena) R. prolixusTPAC/CO/2002/Mag 4 Guamal (Magdalena) R. pallescensTCUS/CO/2002/Mag3 Chimichagua (Cesar) E. cuspidatusTPAC/CO/1999/Ac17 Acandi (Choco) R. pallescensTPAC/CO/1999/Ac29 Acandi (Choco) R. pallescensTGEN/CO/1993/Af1 Amalfi (Antioquia) P. geniculatusMCAN/CO/1998/Am05 Amalfi (Antioquia) Canis familiarisTGEN/CO/1997/Yg03 Yolombo (Antioquia) P. geniculatusTCOL/CO/1991/Stp3.3 Coyaima (Tolima) R. colombiensisTCOL/CO/1991/Stp2.2 Coyaima (Tolima) R. colombiensisMDID/CO/1991/Coy8 Coyaima (Tolima) D. marsupialisTROB/CO/0000/Put Puerto Asis (Putumayo) R. robustusTROB/CO/2001/Put4 Puerto Asis (Putumayo) R. robustusTPRX/CO/2001/Cas16 Taladro (Casanare) R. prolixusTPRX/CO/1995/Cas7 Paz de Ariporo (Casanare) R. prolixusTPRX/CO/2000/Cas15 Villanueva (Casanare) R. prolixusMHOM/CO/1997/HA Casanare Homo sapiensMHOM/BR/0000/Y Brazil Homo sapiensMHOM/BR/0000/Jgbh Uberaba (Brazil) Homo sapiensMHOM/BR/0000/Noel Brazil Homo sapiensTINF/BR/1963/CL Brener Brazil T. infestans

(Colom

TINF/CH/1956/Tulahuen ChileTPAC/CO/0000/Gal 57 T. rangeli Sucre

each deoxynucleotide triphosphate, 1.5 mM MgCl2,12.5 pmol each primer, and 0.625 U of Taq DNA poly-merase. Amplification was performed on a PTC-100 pro-grammable thermal cycler (MJ Research) programmedfor an initial denaturation step of 94 ◦C for 1 min, fol-lowed by 27 cycles of 94 ◦C for 30 s, 55 ◦C for 30 sand 72 ◦C for 30 s, with a final extension at 72 ◦C for10 min (Souto et al., 1996). Ten microlitres of the prod-ucts were analysed by electrophoresis on 1.5% agarosegels at 100 V. Products were stained by ethidium bromideand visualized under UV light.

A PCR procedure designed to detect putative TCIIa

or TCIIc sublineages, which are not recognized by TC1or TC2 primer sequences, was developed using TCac 5′-CTCCCCAGTGTGGCCTGGG-3′ as sense primer andUTCC 5′-CGTACCAATATAGTACAGAAACTG-3′ as

T. infestansbia) D. marsupialis

antisense oligonucleotide (Burgos et al., 2005). A 50 �ltotal volume PCR reaction containing 1 U of Taq DNApolymerase (Platinum, Invitrogen, USA), 150 pmol ofeach primer, 250 �M dNTPs, 3 mM MgCl2, in buffer 1X.Cycling conditions were the same as SL-DNA PCR butannealing temperatures ranged from 68 to 60 ◦C duringtouch down-amplification.

To distinguish between T. cruzi IIb or IIe from T.cruzi IId lineages a PCR was performed using 5 �Mof oligonucleotides D71 and D76 (Briones et al., 1999)in a 30 �l volume reaction containing 250 �M dNTPs,2 mM MgCl and 0.75 U of Taq Platinum. PCR condi-

2tions were 3 min at 94 ◦C, 3 cycles of 94 ◦C for 1 min,60 ◦C for 1 min and 72 ◦C for 1 min; 3 cycles of 57 ◦C forthe annealing step, 35 cycles of 55 ◦C as annealing tem-perature, and elongation at 72 ◦C for 7 min. Amplified

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A. Salazar et al. / Acta

roducts were analysed in 3% agarose gels and ultra-iolet light (UV) visualization after ethidium bromidetaining.

To differentiate between TCIIb and TCIIe sublin-ages a PCR assay based on the fragment A-10 describedy Brisse et al. (2000) was used to distinguish betweenCIIb (A10 negative) from TCIIe (A10 positive, 657 bproduct). This PCR used primers p3 and p6 in a 50 �l vol-me reaction containing 250 �M dNTPs, 2 mM MgCl2nd 0.75 U of Taq Platinum, under the following cyclingonditions: an initial denaturation step of 3 min at 94 ◦C,ollowed by 40 cycles of 94 ◦C for 1 min, 55 ◦C for 1 minnd 72 ◦C for 1 min and a final step at 72 ◦C.

.5. Amplification of T. cruzi kDNA 330 bp fragmenty PCR

The variable region of the minicircles of kineto-last DNA was amplified with the primers S35 (5′-AATAATGTACGGGT/GGAGATGCATGA-3′) and36 (5′-GGTTCGATTGGGGTTGGTGTAATATA-3′)Sturm et al., 1989). PCR was carried out at a finalolume of 50 �l of reaction mix containing 50 mMCl, 10 mM Tris–HCl, 0.1% Triton X-100, 1 �l ofNA template (10 ng/�l), 37 pmol of each primer,00 �M of dNTPs, 1.5 mM of MgCl2 and 2.5 U of TaqNA polymerase. PCR was carried out at an initial

emperature of 94 ◦C for 3 min, followed by 35 cycles at4 ◦C for 45 s, 63 ◦C for 45 s, 72 ◦C for 45 s and a finalycle at 72 ◦C for 10 min. The amplification productsor each of the stocks were analysed by electrophoresisn 2% agarose gel, stained by ethidium bromide andisualized under UV light (Maniatis et al., 1982).

.6. LSSP-PCR

Ten microlitres of the PCR products were run on 1.5%ow melting point agarose gel and stained with ethid-um bromide. Bands corresponding to 330 bp were cutrom the gel and diluted to 1:10 in double distilled water.ne microlitre of the dilution was used as a template

or the LSSP-PCR reaction (Vago et al., 1996). LSSP-CR was performed in 25 �l of the final volume using20 pmoles of the S35 primer, 4 U of Taq polymerase,00 �M of each dNTP, 1.5 mM of MgCl2, 50 mM ofCl, 10 mM Tris–HCl and 0.1% Triton X-100. Amplifi-

ation was carried out with 3 min of initial denaturing at4 ◦C, followed by 35 cycles of 94 ◦C for 45 s, 30 ◦C for

5 s, 72 ◦C for 45 s and a final cycle at 72 ◦C for 10 minPena et al., 1994). Fifteen microlitres of the amplifica-ion products from each of the stocks were analysed bylectrophoresis in 6% polyacrylamide gel (Maniatis et

100 (2006) 110–118 113

al., 1982) and stained with silver nitrate (Andrade et al.,1999). LSSP-PCR for each of the stocks was performedin duplicate.

2.7. Data analysis

Analysis of the bands was performed using the ImageJsoftware (Rasband, 2004). Molecular weight markerswere used as reference standards to correct the variationsin migration among the different gels. For this, the dig-ital profiles of the molecular markers were divided intosquares of 10 pixels each, this corresponding to the max-imum peak width in most of the T. cruzi bands (99%).The gels were considered to be normalised when thepeak corresponding to each of the bands of the molec-ular weight markers laying in the same vertical square.Similarly, the banding profiles of each T. cruzi stock wereconsidered to be equal when they layed in the same ver-tical square. A data matrix of 0 and 1 was created on thebasis of the absence or presence of the peaks for eachstock signature, respectively. Finally, genetic distancesanalyses were performed using PAUP 4.0 (beta version4.0b7; Swofford, 2002) with the UPGMA option.

3. Results

3.1. PCR-based identification of T. cruziphylogenetic lineages

The amplification of the intergenic region of miniexongenes revealed a 350 bp amplicon in 29 out of 30 anal-ysed parasite stocks (96.67%) indicating T. cruzi lineageI predominance (Fig. 1). Only Af1 stock depicted a300 bp amplicon, corresponding to T. cruzi lineage II.Further characterization of this stock by means of 24s�ribosomal DNA-PCR and A10-PCR allowed assignmentof Af1 as T. cruzi IIb (data not shown). None of the sam-ples amplified miniexon sequences for T. cruzi IIa or IIclineages, corresponding to Zymodeme III.

3.2. LSSP-PCR profiling of T. cruzi stocks

For all stocks, a 330 bp band corresponding to thevariable region of the minicircles of kinetoplast DNAwas amplified. This band was used as a template forthe second PCR reaction with a single primer, in orderto obtain a greater number of bands and observe thegenetic relationship among the stocks (Fig. 2). The elec-

trophoretic profiles obtained with LSSP-PCR (Fig. 2Aand B) showed a high genetic variability among all the T.cruzi stocks analysed. However, similar electrophoreticprofiles were observed among Colombian T. cruzi stocks

114 A. Salazar et al. / Acta Tropica 100 (2006) 110–118

Fig. 1. Two percent agarose gel electrophoresis showing amplification proColombian parasite stocks and reference strains. CN: negative control without100 bp ladder.

Fig. 2. LSSP-PCR profiles of kDNA of T. cruzi from Department ofSucre and other regions, as revealed after 6% polyacrylamide gel elec-trophoresis and silver staining. Lane M: 100 bp ladder.

ducts of the intergenic spacer of the T. cruzi miniexon genes fromDNA; Homo: negative control, Human DNA; PM: Molecular marker,

from a single region, e.g. Ac17 and Ac29 from Choco;Gal52, Lb53, Ov1, G20, Gal4 and W3534 from Sucre;HA, Cas7, Cas15 and Cas 16 from Casanare; and Stp2.2and Stp3.3 from Tolima.

No clear groupings were observed among the stocksin the dendogram generated by the UPGMA method(Fig. 3), but as observed on the gels, a slight associationcould be discerned among stocks from the same region.Af1 was located at a separate branch from the remain-ing Colombian stocks, in agreement with its differentphylogenetic lineage. The reference stocks presentedcompletely different electrophoretic profiles from theColombian stocks.

4. Discussion

The LSSP-PCR technique is capable of detecting sin-gle or multiple variations in fragments of target DNAand has already been applied to the study of genetic dis-eases (Pena et al., 1994), and genetic characterization ofdiverse microorganisms (Villa et al., 1995; Gomes et al.,1997; Oliveira et al., 2003).

This is the first study to genotype Colombian stocks ofT. cruzi by means of LSSP-PCR using the variable regionof kDNA minicircles as a genetic marker. In fact, the highsensitivity of kDNA-PCR allows the direct typing of nat-ural parasite populations in biological and clinical sam-ples (Vago et al., 2000; Mejıa and Triana, 2005; Burgos

et al., 2005). This cannot be achieved using other PCR-based typing techniques such as arbitrarily primed PCR(AP-PCR) and random amplified polymorphic DNA fin-gerprinting (RAPD) which require significant quantities

A. Salazar et al. / Acta Tropica 100 (2006) 110–118 115

F ithmetics replicat

oi

gm((figwic

fpsffs

ig. 3. Dendogram constructed by unweighted pair-group method artocks. Bootstrapping among T. cruzi stocks are lower than 50 of 100

f purified DNA which can only be obtained once thenfecting organism has been isolated and cultured.

LSSP-PCR of T. cruzi kDNA has already shown thatenetic variability is one of the most important deter-inant factors in the pathogenesis of Chagas’ disease

Andrade et al., 1999; Vago et al., 2000). Vago et al.2000) observed similarities among the LSSP-PCR pro-les of T. cruzi kDNA in different patients from the sameeographical region. This suggests that T. cruzi stocksithin a single geographical region may be related genet-

cally, which might play a role in the development of thelinical forms of the disease.

In this paper, the electrophoretic standards obtainedrom LSSP-PCR showed heterogeneity in the bandingattern for each T. cruzi stock. It is important to empha-

ize that the electrophoretic profiles were reproducibleor all the stocks, when a second LSSP-PCR was per-ormed based on the 330 bp DNA as template (data nothown). Interestingly, despite the high minicircle vari-

averages (UPGMA) depicting the genetic distances among T. cruziions (not shown).

ability among the 30 tested stocks, 29 of them belongedto the T. cruzi lineage I, confirming previous reportsshowing the predominance of this lineage in differentvector and vertebrate host species in Colombia (Montillaet al., 2002; Cuervo et al., 2002). In this country, thedescription of T. cruzi I strains circulating in both thedomestic and sylvatic environments may seem to con-trast with findings in Brazil, where T. cruzi I is prefer-entially associated with the sylvatic cycle. Lainson et al.(1979) stated that cases of Chagas’ disease in the Brazil-ian Amazon might be derived from sporadic visits ofwild triatomines to human dwellings near the forests orfrom the eventual contact of the inhabitants of a specificarea with the sylvatic cycle of the parasite. A similarecological setting may occur in Colombia, explaining

the finding of T. cruzi I in both the domestic and sylvatictransmission cycles of the parasite.

The only case in our study belonging to T. cruzi IIwas stock Af1, isolated from a P. geniculatus specimen

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116 A. Salazar et al. / Acta

from the Amalfi locality in Antioquia. Further analysisof this stock, applying PCR targeted to the 24s� riboso-mal DNA and A-10 fragment, allowed identification as T.cruzi IIb lineage. Previous isoenzymes studies showed T.cruzi II isolates from this vector species in the same geo-graphical region (Montilla et al., 2002). The molecularmarkers used in our work allowed further characteriza-tion leading to identification of lineage IIb. This findingopens the question regarding the existence and epidemi-ological relevance of a T. cruzi IIb-related transmissioncycle associated to P. geniculatus in Colombia.

When the electrophoretic profiles obtained by LSSP-PCR were analysed by the neighbor-joining method,the bootstrapping values among the Colombian T. cruzistocks were lower than 50%. However, a possible asso-ciation could be inferred among stocks of a single geo-graphical region and also with a similar biologic origin.This was observed for T. cruzi stocks Ac17 and Ac29,isolated from R. pallescens in the Colombian depart-ment of Choco; Cas7, Cas15 and Cas16, isolated fromR. prolixus in Casanare; G20, Gal34 and Gal4, isolatedfrom R. pallescens in Sucre or Stp2.2 and Stp3.3, iso-lated from R. colombiensis in Tolima. Interestingly, thestocks from Casanare presented similar profiles despiteoriginating from different municipalities; indeed, thereare no geographical barriers among these localities thatcould impede the continuous flow of a predominant cloneamong the wild fauna of the region. It is noteworthythat the HA stock, despite being isolated from a patientwith Chagas’ disease, clustered with the other Casanarestocks. This suggests transmission events between theinsect vector and the human host in this region. Previousstudies in which smaller numbers of stocks were anal-ysed, have also suggested a genetic correlation amongthose from a single geographical region (Saravia et al.,1987; Andrade and Magalhaes, 1997; Jaramillo et al.,1999; Campos et al., 1999; Vago et al., 2000).

On the other hand, the differences in the elec-trophoretic profiles among the stocks from Antioquia,Cesar and Magdalena can be explained by the fact thateach of them belongs to a different locality separatedby geographical barriers. Moreover, such dissimilaritycould be explained by the multiclonal character of eachstock, without a specific clone being predominant and/orby the geographic, ecological and biological diversity ofthe region. This implies that a greater number of T. cruzistocks from each geographic locality need to be studiedto obtain significant comparative patterns.

In Colombia, the study of the genetic variability ofT. cruzi is complicated by the fact that this species pos-sesses a great geographical and ecological variety and alarge number of T. cruzi vector species has been recorded

a 100 (2006) 110–118

(Moreno, 1997). This contrasts with other countries suchas Chile, where T. infestans and T. spinolai are the onlyvectors of T. cruzi, and the Atacama desert presents asingle geographical barrier that divides the country intojust two endemic areas (Solari et al., 1998).

Diverse studies have demonstrated that stocks iso-lated from vectors and sylvatic mammals are muchgenetically complex than those obtained from patientsin the acute phase of Chagas’ disease (Diosque et al.,2003). These stocks, in turn, are more complex than onesfrom patients in the chronic phase (Oliveira et al., 1998;Macedo et al., 2002). Accordingly, the high genetic vari-ability observed among the Colombian parasite stocksmay be due to the biodiversity of triatomine species aswell as to the wide range of vertebrate hosts, whichconstitute the natural sylvatic cycle of transmission inColombia.

It should be emphasized that this study was carried outwith a large number of stocks of T. cruzi isolated fromvectors and vertebrate hosts from different geographi-cal regions of a single country. Additional populationgenetics and micro-evolutionary studies are needed toprovide a better understanding of the nature and fre-quency of molecular events leading to the complexgenetic diversity of the Colombian T. cruzi natural iso-lates. The high sensitivity of the LSSP-PCR allows directtyping of natural parasite populations. Due the simplic-ity of its execution, its speed and capacity to producehighly reproducible and informative genetic signatures,this technique is a useful tool for molecular epidemio-logical studies of Chagas’ disease.

Moreover, as soon as a greater number of stocks canbe collected and characterised from different hosts indifferent endemic areas, a deeper insight of the dynamicsof T. cruzi transmission in Colombia will be achieved.

Acknowledgements

This work was financed by Proyecto de Sostenibil-idad University of Antioquia 2005–2006 to OTC andpartially by Bunge and Born Foundation and PIP 5469CONICET to AGS. We thank the technical assistance ofTomas Duffy (LaBMeCh, INGEBI) in PCR-based char-acterization of AF1 stock.

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