Parasitol Res (2008) 103:1041–1045 DOI 10.1007/s00436-008-1089-y
Molecular characterization of Trypanosoma cruzi sylvatic isolates from Rio de Janeiro, Brazil Jacenir R. Santos-Mallet & Cristina S. Silva & Suzete A. O. Gomes & Daise L. Oliveira & Cristina L. Santos & Daniele M. Sousa & Nadja L. Pinheiro & Angela C. V. Junqueira & Teresa Cristina M. Gonçalves
Received: 11 April 2008 / Accepted: 10 June 2008 / Published online: 12 July 2008 # Springer-Verlag 2008
Abstract Trypanosoma cruzi, the etiologic agent of Chagas disease, presents considerable heterogeneity between isolated populations within the wild and domestic cycles. By using multiplex polymerase chain reaction based on the mini-exon gene, characterization was performed on seven samples isolated from specimens of Triatoma vitticeps that had been collected from the locality of Triunfo in the municipality of Santa Maria Madalena, state of Rio de Janeiro, Brazil. The samples SMM10, SMM53, SMM88, and SMM98 (area A) and SMM36 and SMM82 (area B) revealed the presence of 150 base pairs, corresponding to the zymodeme III (Z3). Our study suggested that one isolate (SMM1) presents a mixed genotype associated with Z3 and TcII. The typing of isolates of T. cruzi has the main aim of identifying strains with different epidemiological and/or clinical characteristics of Chagas disease. Our results corroborate other descriptions in the literature and contribJ. R. Santos-Mallet (*) : C. S. Silva : S. A. O. Gomes : D. M. Sousa : T. C. M. Gonçalves Setor de Morfologia, Ultra-estrutura e Bioquímica de Artrópodes e Parasitos/Laboratório de Transmissores de Leishmanioses, Instituto Oswaldo Cruz (FIOCRUZ), Av. Brasil 4365, CEP: 21040-900 Rio de Janeiro, Brazil e-mail: [email protected]
D. L. Oliveira : C. L. Santos : A. C. V. Junqueira Laboratório de Doenças Parasitárias, Instituto Oswaldo Cruz (FIOCRUZ), Av. Brasil 4365, CEP: 21040-900 Rio de Janeiro, RJ, Brazil N. L. Pinheiro Programa de Pós-graduação em Biologia Animal, Instituto de Biologia, Universidade Federal Rural do Rio de Janeiro (UFRRJ), Seropédica, RJ, Brazil
ute towards the knowledge and records of the profile of some additional wild isolates of T. cruzi in regions not yet affected by the disease.
Introduction Trypanosoma cruzi, the etiologic agent of Chagas disease, presents considerable heterogeneity between isolated populations within the wild and domestic cycles (Fernandes et al. 2001a, b). In the 1960s, when the present-day molecular methods did not exist, Coura et al. (1966) already advocated the use of the term “cruzi complex”, based on morphological variations, immunological characteristics, different types of virulence and differences in the parasite’s regional patterns, and the individual patterns of Chagas disease. Today, it is known that T. cruzi is a heterogeneous species composed of several parasite subpopulations that circulate among various wild and domestic vertebrate and invertebrate hosts (Morel et al. 1986; Zingales et al. 1998). Several studies have pointed out intra-species variations between different strains of T. cruzi (Andrade 1974, 1985; Miles et al. 1977; Morel et al. 1980; Steindel et al. 1993). Isoenzymatic differences between isolates of the parasite were first demonstrated by Toyé (1974) and confirmed by Miles et al. (1980). The latter authors proposed dividing the different strains into three main zymodemes, Z1, Z2, and Z3, in which Z1 would be of wild origin, with circulation among wild triatomines, and would be infective towards man; Z2 would be of domestic origin and would include strains isolated from patients with acute or chronic Chagas disease and from domestic animals; and Z3 would be parasites isolated from autochthonous human cases and would include the wild cycle, since it had been isolated
from armadillos and from some human cases with the acute form of the disease. Souto et al. (1996) and Fernandes et al. (1998, 1999), using molecular techniques such as analysis of the gene responsible for coding ribosomal RNA and the mini-exon gene, demonstrated clear dimorphism between the populations of T. cruzi corresponding to two of the main zymodemes proposed by Miles et al. (1980). These two phylogenetic lineages were named TcI and TcII, corresponding to Z2 and Z1, respectively. Lineage 1 related to isolates from the domestic cycle and lineage 2 to the wild cycle, preferentially. However, the position of Z3 is still under review, and it has been described as isolates of T. cruzi that present a hybrid profile since they show genotypic characteristics of both TcI and TcII. Additional studies identified around six lineages of T. cruzi, including TcI and TcII (Brisse et al. 2000). The high polymorphism of T. cruzi and the significance of its genetic variability in the pathogenesis of Chagas disease have been addressed (Macedo et al. 2004). Its heterogeneity may be related to the varied types of clinical manifestations of Chagas disease and the regional differences in the morbidity due to this disease (Devera et al. 2003). The mini-exon gene is present in the nuclear genome of Kinetoplastida, in around 200 arrangements of the repeated sequences, thus constituting three distinct regions: exon, intron, and intergene region. The exon is the only sequence of 39 base pairs (bp) that is highly conserved among the components of this order and is transported to the RNA (mRNAs). The intron, which is moderately conserved among species in the same genus, subgenus, and intergene region or in the untranscribed spacer, may be amplified by means of polymerase chain reaction (PCR), a technique that makes it possible to classify different isolates of T. cruzi taxonomically into two distinct groups: T. cruzi I and T. cruzi II (Fernandes 1996; Souto et al. 1996; Fernandes et al. 1998). A more practical, recently suggested, division consists of typification as T. cruzi I, T. cruzi II, or Trypanosoma rangeli by means of multiplex PCR, which conserved the isolate for subsequent characterization (Fernandes et al. 2001a, b).
Materials and methods By using multiplex PCR based on the mini-exon gene, characterization was performed on seven samples isolated from specimens of Triatoma vitticeps that had been collected from the locality of Triunfo, at latitude 22°02′ 52″ S and longitude 41°56′32″ W, in the municipality of Santa Maria Madalena, state of Rio de Janeiro, Brazil (Gonçalves et al. 1998). The samples SMM10, SMM53, SMM88, and SMM98 came from area A, which had been deforested for banana cultivation; it was at an altitude of 250 m and was 3.5 km away from human settlement.
Parasitol Res (2008) 103:1041–1045
ˆ Oceano Atlantico
ESTADO DO RIO DE JANEIRO
ˆ Oceano Atlantico
ÁREAS DE CAPTURA
Parasitol Res (2008) 103:1041–1045
1 Studied area and sites of capture of Triatoma vitticeps in Triunfo, Santa Maria Madalena, municipal district, state of Rio de Janeiro, Brazil
SMM36 and SMM82 came from area B, which was located in a valley with preserved vegetation, at an altitude of 130 m and 4 km away from the settlement. The seventh sample, SMM1, came from a triatomine captured in the settlement of Vista Alegre, in the neighboring municipality of Conceição de Macabu, which was named area F (Fig. 1). The T. cruzi strain CL Brener (coming from a human case—TcII), T. rangeli strain H 14 (coming from blood culturing of a human case), T. cruzi DM28c (coming from Didelphis marsupialis—TcI), and T. cruzi strain JJ (coming from a human case—Z3) were used as the reference strains (Table 1). The samples were grown and maintained in NNN medium (Novy and McNeal 1904; Nicolle 1908) with the addition of liver infusion tryptose (Camargo 1964) as a liquid phase, supplemented with 10% bovine fetal serum. Parasites at a concentration of 2×109 were centrifuged (2,500×g at 4°C for 10 min) and washed for DNA extraction. The integrity of the DNA was checked on 1% agarose gel and its concentration was estimated using a spectrophotometer at 260 nm. Multiplex PCR (as adapted by Fernandes et al. 2001a, b) of the isolates was performed using 25 ng of genomic DNA extracted using the phenol– chloroform method. Five primers were used: for Tc1 (5′-TTG CTC GCA CAC TCG GCT GCAT-3′), for Tc2 (5′-ACA CTT TCT GTG GCG CTG ATC G-3′), for Z3 (CCG CGW ACA ACC CCT MAT AAA AAT G-3′), for Tr (CCT ATT GTG ATC CCC ATC CCC ATC TTC G3′), and for the mini-exon (5′ TAC CAA TAT AGT ACA GAA ACT G-3′). The lyophilized primers were made up in 10 mM Tris pH 8.0, EDTA 1 mM. The amplification product was checked by means of 2% agarose gel electrophoresis in TBE (0.5×) using a molecular weight marker at the scale of 100 bp. The standard fragments obtained presented the following sizes: Tc1, 200 bp; Tc2,
Fig. 2 PCR multiplex—mini-exon. The gel of agarose for electrophoresis was amplified using isolates of Trypanosoma cruzi of reference that possess approach bands of TCI, compared to TCII, Z3, and T. rangeli and with isolated sylvatics of the state of Rio de Janeiro. Lane 1 Molecular weight marker (100 bp DNA ladder), 2 SMM98, 3 SMM36, 4 SMM82, 5 T. rangeli, 6 CL Brener, 7 DM28c, 8 JJ, 9 molecular weight marker (100 bp DNA ladder), 10 SMM1, 11 SMM10, 12 SMM53, 13 SMM88, 14 T. rangeli, 15 CL Brener, 16 DM28c, 17 JJ, 18 molecular weight marker (100 bp DNA ladder), 19 negative control (no DNA added). bp base pairs
250 bp; Z3, 150 bp; and Tr, 100 bp. For the negative control, no DNA was added to the reaction.
Results and discussion The results obtained by means of molecular analysis revealed that the isolates had similar profiles, except for the sample SMM1 (area F). The samples SMM10, SMM53, SMM88, and SMM98 (area A) and SMM36 and SMM82 (area B) revealed the presence of 150 bp, thus indicating that they belonged to the zymodeme III (Z3; Fig. 2). Likewise, the sample SMM1 from area F showed similarity to Z3 (150 bp), but also presented another band that may possibly have been related to the TcII profile (250 bp) and was very similar to the reference strain CL Brener (Fig. 2). The phylogenetic position of Z3 has been constantly debated. According to some authors, the numerical taxonomy based on 24 isoenzymatic Z3 profiles is more
Table 1 Molecular characterization of Trypanosoma cruzi derived from Triatoma vitticeps captured on the state of Rio de Janeiro, Brazil Isolates
SMM10 SMM53 SMM88 SMM98 SMM36 SMM82 SMM1
A A A A B B F
XN Tv HM Tv Tv Tv HM
Triunfo Triunfo Triunfo Triunfo Triunfo Triunfo Conceição de Macabu
Z3 Z3 Z3 Z3 Z3 Z3 TII/Z3
JJ JJ JJ JJ JJ JJ CL Brener/JJ
Tv Triatoma vitticeps, HM hemoculture of the Swiss mouse—the parasites were inoculated in mice and was done hemoculture, XN xenodiagnosis— bug sucking the Swiss mouse inoculating the parasites; later the blood of this mouse was collected and was cultivated in vitro
associated with Z1 (TcII) than with Z2 (TcI; Ready and Miles 1980). However, others have included 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 provide corroboration for the hypothesis that this isolate may be the product of a mixture of parasite populations since the vector in wild environments may feed on several vertebrate hosts. This complexity was demonstrated in Rio de Janeiro state by Fernandes et al. (1999) that showed preferential association of the two lineages of T. cruzi with different hosts. They suggested that the vector T. vitticeps is involved in the transmission cycle among the lineage 2 infected mammals in Teresopolis municipality and with the transmission cycle of the primates in Silva Jardim municipality. The hybrid profile found in these samples may indicate the possibility of the vector T. vitticeps not to participate only in the wild cycle of the disease. T. vitticeps may be considered a sylvatic species by the low prevalence of human infection (Santos et al. 1969a, c; Pinto et al. 1969, 1986; Barros et al. 1975; Peçanha et al. 1983; Silveira et al. 1983; Dias et al. 1989). However, it is beginning to colonize in some localities (Sessa and Carias 1986; Dias et al. 1989; Santos et al. 1969b; Silveira et al. 1983) suggesting a wide ecological valence which was previously low (Forattini et al. 1979). In Rio de Janeiro state, T. vitticeps were collected in the locality of Triunfo, Santa Maria Madalena municipal district, in the domicile with 79% of T. cruzi infection by Gonçalves et al. (1998). The same authors registered fifth instar nymphs infected with T. cruzi in the bedroom, possibly acquired outside the domicile. Lorosa et al. (2003) reported triatomine nymphs, eggs, and 46 specimens of T. vitticeps inside a house in Arcadia, Miguel Pereira, state of Rio de Janeiro, with 13% of T. cruzi infection rate. Both results emphasized that an epidemiological vigilance is necessary to know the behavior of this species following the continuous modifications promoted by the presence of man. The typing of isolates of T. cruzi has the main aim of identifying strains with different epidemiological and/or clinical characteristics of Chagas disease. Our results corroborate other descriptions in the literature and contribute towards the knowledge and records of the profile of some additional wild isolates of T. cruzi in regions not yet affected by the disease. Added to the complexity observed between the isolates is the finding of the division of Z3 profile into two groups called Z3a and Z3b (Mendonça et al. 2002). Our laboratory is interested in investigating whether a dichotomy occurs among the Z3 isolates obtained from T. vitticeps in this studied area. Acknowledgments We would like to thank the Laboratório de Doenças Parasitarias and Departamento de Medicina Tropical,
Parasitol Res (2008) 103:1041–1045 Instituto Oswaldo Cruz (FIOCRUZ). This study is supported by grants from Fundação de Amparo a Pesquisa do Rio de Janeiro (FAPERJ) and FIOCRUZ, Rio de Janeiro, Brazil.
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