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Trypanosoma cruzi, the etiological agent of Chagas disease, presents considerable heterogeneity among populations of isolates within the sylvatic and domestic cycle. This study aims to evaluate the genetic diversity of 14 isolates collected from specimens of Triatoma vitticeps from Triunfo, Conceição de Macabu, and Santa Maria Madalena cities (Rio de Janeiro—Brazil). By using PCR based on the mini-exon gene, all isolates showed a profile characteristic of bands zymodeme III and with a lower intensity characteristic of TcII. To verify possible hybrids among the strains analyzed, the polymorphisms analysis of the MSH2 gene was performed. HhaI restriction enzyme digestion products resulted in characteristic TcII fragments only, demonstrating the absence of hybrids strains. In our attempt to characterize isolation in accordance with the reclassification of T. cruzi into six new groups called DTUs (“discrete typing unit”), we genotyped the mitochondrial cytochrome oxidase subunit two gene, ribosomal RNA gen (24Sα rDNA), and the spliced leader intergenic region (SL-IR). This procedure showed that TcII, TcIII, and TcIV are circulating in this area. This highlights the diversity of parasites infecting specimens of T. vitticeps, emphasizing the habit of wild type and complexity of the region epidemiological study that presents potential mixed populations.
Faculty of Pharmacy, Laboratory of Molecular Diagnosis and Hematology, Federal University of Rio de Janeiro-UFRJ, Brazil
Luciana Reboredo de Oliveira da Silva
Central Public Health Laboratory—LACEN-BA, Brazil
Teresa Cristina Monte Gonçalves
Interdisciplinary Laboratory of Entomological Surveillance in Diptera and Hemiptera—LIVEDIH, Oswaldo Cruz Institute-IOC-Fiocruz, Brazil
Renato da Silva Junior
Interdisciplinary Laboratory of Entomological Surveillance in Diptera and Hemiptera—LIVEDIH, Oswaldo Cruz Institute-IOC-Fiocruz, Brazil
Faculty of Medical Sciences of Maricá, Laboratory of Vector Insects, University of Vassouras (Campus Maricá), Brazil
Jacenir R. Santos-Mallet*
Interdisciplinary Laboratory of Entomological Surveillance in Diptera and Hemiptera—LIVEDIH, Oswaldo Cruz Institute-IOC-Fiocruz, Brazil
Fiocruz-Piaui Regional Office, Brazil
Iguaçu University—UNIG, Brazil
*Address all correspondence to: jacenir@ioc.fiocruz.br
1. Introduction
Trypanosoma cruzi (T. cruzi) is a heterogeneous parasite, in which strains are composed of different sub-populations or clones that circulate in nature between triatomine vectors, wild and domestic mammals, including man. The need for adaptation and survival in different hosts appears to be responsible for the high genetic diversity of the parasite [1] and the various clinical manifestations observed in Chagas’ disease [2, 3].
The heterogeneity of strains of this parasite has been demonstrated using different markers: morphological, biological susceptibility to chemotherapeutic agents, immunological, biochemical and molecular [4, 5, 6, 7, 8, 9, 10, 11, 12, 13].
Considerable advances have been made to understand the genetic makeup of T. cruzi and the process that involves the control of gene expression of the parasite. Molecular genetic markers have been used to correlate different strains with their different biological properties, clinical and epidemiological characteristics [14].
Ribosomal gene sequences have been widely used to infer phylogenetic relationships among the trypanosomatids and representatives of other families of the order Kinetoplastida and phylum Euglenozoa. In trypanosomes, the sequence of the 24S subunit is interrupted by an internal spacer generating two molecules, 24Sα and 24Sβ.
The conserved non-transcribed regions of the pre-rRNA correspond to the internal transcribed spacer (ITS) and external (ETS). The presence of several regions, transcribed or not, that display varying degrees of variability, entail a high degree of polymorphism of the ribosomal cistrons and for this reason, have proved to be excellent as a tool for identification and phylogenetic studies of trypanosomes [15]. The ITS spacers are highly variable compared with ITS which, in turn, are much more variable regions of the Small Sub Unit (SSU) and Large Sub Unit (LSU). Analysis of polymorphism of ribosomal sequences has been used in the identification and genotyping of strains.
Souto and cols. [16] standardized a marker based on the region of the LSU 24Sα, which distinguishes the T. cruzi I and T. cruzi II strains. Another excellent marker for the study of diversity in T. cruzi gene is the Mini-Exon. The identification of strains (genotyping) using PCR methods based on gene sequences of mini-exon has been widely used [16, 17, 18, 19, 20].
Due to its organization comprising regions with differing degrees of conservation of the mini-exon genes have been used for diagnosis purposes and taxonomic. Each repeating unit of the Spliced Leader (SL) gene can be basically divided into three parts: a highly conserved exon of 39 nucleotides, an intron moderately conserved nucleotides 50–100 and an intergenic spacer region, which varies in size and sequence among trypanosomes species and strains. There are about 200 repeated copies of the SL gene “in tandem” in the trypanosomes genome which are therefore a good target for diagnosis [21, 22, 23]. The use of PCR methods for genotyping based on the mini-exon and ribosomal genes segregates this parasite into three major lineages: T. cruzi I, T. cruzi II and Z3 [16, 20, 24].
Augusto-Pinto and cols [25] demonstrated that T. cruzi can be divided into three distinct haplogroups called A, B and C, based on an analysis of polymorphisms in the MSH2 gene of several strains of this parasite. It was subsequently found that strains of haplogroups B and C have a lower efficiency of the mismatching error repair (MMR) compared to strains of haplogroup A after treatment with cisplatin hydroxide and hydrogen [26].
These results suggested that the lower efficiency of MMR of haplogroups B and C could be associated with an increased generation of genetic variability in these strains. Thus an analysis of genetic variability by targeting the gene encoding the T. cruzi called TcAg48 is present in a large number of copies in the genome of this parasite. Digestion of the amplified product of a region of this gene with the restriction enzyme HhaI allowed by the group 35 strains in the same haplogroups already described for the analysis of MSH2. It was found even greater genetic variability of this antigen in strains belonging to haplogroups B and C, which showed a less efficient MMR. Some of the haplogroup B strains have a digestion pattern with characteristics of both strains of haplogroup B and C, indicating a hybrid character.
Zingales and cols [13] standardized nomenclature into six groups (T. cruzi I-VI), each group termed DTU (“discrete typing unit”), where DTU is defined by a set of strains that are genetically similar, and that can be identified by molecular markers common or immunological [27]; DTUs T. cruzi I and T. cruzi II respond to two groups originally defined at the first meeting [28]. Although it was evident that the V-VI DTUs correspond to hybrid organisms, their origin swims from different events of genetic exchange.
This study aimed to evaluate the genetic diversity of 14 isolates of specimens of Triatoma vitticeps collected from the locality of Triunfo, 2nd District of the municipality of Santa Maria Madalena and Conceição Macabu from Rio de Janeiro State.
The isolates were obtained from specimens of Triatoma vitticeps captured in the locality of Triunfo whose latitude is 22° 02′52″S and longitude 41° 56′ 32″W, in the Santa Maria Madalena and Conceição Macabu cities, both the State of Rio de Janeiro. In this study, we used a total of 14 samples obtained from triatomines collected in three distinct regions for this study called A, B and C. Six of them (SMM1, SMM10, SMM51, SMM57, SMM88 and SMM98) were isolated from area A, area deforested to make way for banana cultivation, being located at an altitude of 250 m and 3.5 km away from the village, seven samples (SMM9, SMM30, SMM34, SMM36, SMM39, SMM82 and SMM89) from area B, located in a valley with vegetation preserved around thinking to 130 m above sea level and 4 km far from the village. These two areas are 2 km far from each other and separated by mountainous area with altitudes between 400 and 900 m, belonging to the locality of Triunfo, Municipality of Santa Maria Madalena. One sample (SMM106) were isolated from D area, a preserved area of 10 km far from area C.
2.2 Growth of parasite
The samples were maintained in tubes containing NNN medium plus LIT (Liver Infusion Tryptose), as the liquid phase, supplemented 30% fetal calf serum. The tubes were incubated in an oven of the BOD (FANEM) to 27.3°C, subcultured regularly at intervals of 14 days for maintenance of the samples.
2.3 DNA extraction
Cultures of T. cruzi (10 ml) in the exponential growth phase were washed three times by centrifugation in PBS (Phosphate Buffered Saline) at 2,000 rpm for 10 minutes. After removal of the supernatant, the DNA was extracted by DNAzol (Invitrogen) following the manufactured instructions.
The variability of the intergenic region of the gene of the mini-exon of the samples was studied using the technique of multiplex PCR, using primers TcI, TcII, Z3, Tr and ME [24]. These primers generate an amplification product of 200 bp (TcI), 250 bp (TcII), 150 bp (Z3) and 100 bp (T. rangeli). The reaction was conducted in a final volume for each sample of 50 μl containing ~100 ng of DNA template, 1U Taq Gold DNA polymerase (Invitrogen), 0.2mM of dNTPs, 1.5mM MgCl2, buffer 1X (10mM Tris-HCl pH 8.5), 10 pmol of each primer (TcI, TcII, Z3, Tr and ME). Fragment amplification was carried out under the following temperature conditions: initial denaturation at 94°C/5 min and 5 cycles (94°C/1 min, 50°C/1 min, 72°C/1 min), followed by 25 cycles (94°C/30 sec, 55°C/30 sec, 72°C/30 sec) and a final extension at 72°C/5 min. Amplification products were analyzed by electrophoresis on 2.5% agarose gel and visualized under UV light after ethidium bromide staining. Dm 28c (TcI), CL Brener (TcII), 3663 (Z3) and R1625 (Trypanosoma rangeli) strains were used as control.
3.2 MSH2 gene
The study to check possible characters hybrids between the strains analyzed was made based on an analysis of the MSH2 gene polymorphisms with the digestion of the 875bp amplification product of a region of this gene with the restriction enzyme HhaI [26]. The reaction was conducted in a final volume for each sample of 50 μl containing ~100 ng of DNA template, 1U Taq DNA polymerase (Thermo Fisher Scientific), 0.2mM of dNTPs, 1.5mM MgCl2, buffer 1X (10mM Tris-HCl pH 8.8, 50mM KCl, 0.8% Nonidet P40), 10 pmol of each primer (tmuts30 and tmuts41). PCR reaction was carried out under the following conditions: initial denaturation at 94°C/5 min and 30 cycles (94°C/30 sec, 55°C/1 min and 72°C/2 min). The fragment obtained by PCR was then digested with the HhaI restriction enzyme for 16 h at 37°C. The digestion products were analyzed by polyacrylamide gel electrophoresis in 7.5%. The gels were revealed by silver impregnation (DNA Silver Staining Kit /Amersham Biosciences).
Polymorphism in mitochondrial cytochrome oxidase subunit 2 (COII) gene was analyzed using the Tcmit-10 and Tcmit-21 primers that amplified a fragment of approximately 375 bp [29]. The reaction was conducted in a final volume for each sample of 50 μl containing ~100 ng of DNA template, 1U Taq DNA polymerase (Thermo Fisher Scientific), 0.2mM of dNTPs, 1.5mM MgCl2, buffer 1X (10mM Tris-HCl pH 8.8, 50mM KCl, 0.8% Nonidet P40), 10 pmol of each primer (tcmit10 and tcmit21). The reaction was performed under the following temperature conditions: 95°C/5 min and 40 cycles of 95°C/45 sec, 48°C/45 sec and 72°C/1 min and a final extension of 72°C/10 min. The obtained fragments were digested with AluI restriction endonuclease and the polymorphism was analyzed after electrophoresis in 6.5% acrilamide gel. Dm28c (TcI), Y (TcII), 3663 (TcIII), 4167 (TcIV), (TcV) and CL Brener (TcVI) strains were used as control.
3.4 Ribosomal RNA gene (24Sα)
The study segment of the ribosomal RNA gene (DNAr24Sα) T. cruzi was performed using primers D71 and D72 [16]. These primers generate an amplification product of 110 bp and 125 bp for TcI to TcII, respectively. The reaction was conducted in a final volume for each sample of 50 μl containing ~100 ng of DNA template, 1U Taq DNA polymerase (Thermo Fisher Scientific), 0.2mM of dNTPs, 1.5mM MgCl2, buffer 1X (10mM Tris-HCl pH 8.8, 50 mM KCl, 0.8% Nonidet P40), 10 pmol of each primer (D71 and D72). Fragment amplification was carried out under the following temperature conditions: initial denaturation at 94°C/4 min and 30 cycles (94°C/1 min, 62.5°C/1 min, 72°C/1 min), followed by a final extension at 72°C/5 min. The visualization of the amplification product was performed by polyacrylamide gel electrophoresis in 7.5%. The gels were revealed by silver impregnation (DNA Silver Staining Kit/Amersham Biosciences). Dm28c (characterized as TcI) and CL Brener (characterized as TcII) strains were used as controls.
3.5 Spliced leader intergenic region (SL-IRac) gene
The amplification of the spliced intergenic region (SL-IRac) gene was realized with TcIII and UTCC primers in order to distinguish TcIII (fragment of 200 bp) and other DTU (fragment of 150 to 157 bp). The reaction was conducted in a final volume for each sample of 50 μl containing ~100 ng of DNA template, 1U Taq DNA polymerase (Thermo Fisher Scientific), 0,2mM of dNTPs, 1.5mM MgCl2, buffer 1X (10mM Tris-HCl pH 8.8, 50mM KCl, 0.8% Nonidet P40), 10 pmol of each primer (TcIII and UTCC). The reaction was performed under the following temperature conditions: 95°C/5 min, 3 cycles of 94°C/30 sec, touch down 70–64°C/30 sec, 72°C/1 min and 33 cycles of 94°C/30 sec, 62°C/30 sec and 72°C/1 min and final extension of 72°C/10 min. The final products were visualized by electrophoresis in 6.5% acrylamide gel. Dm28c and 3663 strains were used as control.
4.1 Variability of the intergenic region of the mini-exon gene
All 14 isolates of T. cruzi analyzed showed a band of 150 bp characteristic of the zymodeme III. Still in the same analysis also showed bands with a lower intensity of ~ 250pb characteristic of the group TcII, suggesting the presence of possible blends in the isolates (Figure 1).
Figure 1.
Molecular characterization of Trypanosoma cruzi isolates by segment analysis of the non-transcribed spacer of the mini-exon gene obtained by electrophoresis in agarose gel stained with ethidium bromide. M—molecular marker (100bp), lanes 1 and 12—control sample of Tc I (Dm 28c), 2 and 13—control sample of Tc II (CL Brener), 3 and 14—ZIII (3663), 4 and 15—control sample of T. rangeli (R1625), 5—SMM1, 6—SMM9, 7—SMM10, 8—SMM30, 9—SMM34, 10—SMM36, 11—SMM39. 16—SMM51, 17—SMM57, 18—SMM82, 19—SMM88, 20—SMM89, 21—SMM98, 22—SMM106, B—negative control.
4.2 Variability of the segment of ribosomal RNA gene (24Sα)
PCR amplification of ribosomal RNA gene (24Sα) using primers D71 and D72 resulted in fragments 125pb characteristic of lineage TcII (not shown).
4.3 MSH2 gene
The products of digestion with HhaI restriction enzyme resulted in fragments of 173pb, 207pb and 294pb for all isolates, which indicate a characteristic pattern for the TcII, so demonstrating that there is not possibly hybrids between our isolates (Figure 2).
Figure 2.
Electrophoresis in polyacrylamide Gel 7.5% of MSH2 gene after the restriction digestion enzyme HhaI. M—molecular marker (100 bp), 1—SMM1, 2—SMM9, 3—SMM10, 4—SMM30, 5—SMM34, 6—SMM36, 7—SMM39, 8—SMM51, 9—SMM57, 10—SMM82, 11—SMM88, 12—SMM89, 13—SMM98, 14—SMM106.
4.4 DTU genotyping
DTU was determined according to D´Ávila et al. [30] that propose a three-step assay: polymorphism of the mitochondrial cytochrome oxidase subunit 2 (COII) after digestion with restriction enzyme AluI (Figure 3), amplification of the D7 divergent domain of the 24Sα rRNA gene (Figure 4) and amplification of the spliced leader intergenic region (SL-IRac) (Figure 5).
Figure 3.
Electrophoresis in acrylamide gel of COII gene after digestion with AluI restriction enzyme. M—molecular marker (50 bp), 1–6—TcI-TcVI controls (1—haplotype A, 2—haplotype C, 3,5,6—haplotype B, 4—uncutted), 7–15 samples.
Figure 4.
Electrophoresis in acrylamide gel of 24Sα gene. M1—molecular marker (50 bp), 1—TcI, 2—TcII, 3–9—samples, M2 molecular marker (100 bp).
Figure 5.
Electrophoresis in acrylamide gel of SL-IRac gene. M1—molecular marker (100 bp), 1–7—samples, 8 and 9—controls, B—negative control.
According to the results, it was possible to determine DTU. Most of them showed the presence of two mixed T. cruzi populations (Table 1).
Many studies have been performed for the characterization of isolates of Trypanosoma cruzi obtained from human cases, animal reservoirs and triatomines mainly in Latin America, highlighting the heterogeneity of this species [31, 32, 33, 34, 35, 36, 37, 38, 39].
In previous studies using isoenzyme analysis, samples of zymodemes were grouped into three and were related to different isozyme groups found with the epidemiological profile of the isolates. Thus, zymodemes I (Z1) and III (Z3) are related to the sylvatic cycle of the parasite and zymodeme II (Z2) to the domestic cycle [7, 40]. Through molecular biology techniques [10, 12, 16, 20, 41, 42, 43, 44, 45, 46] was able to evidence a clear dimorphism between the isolates of T.cruzi, leading to pooling of samples into two major phylogenetic lineages: Tc I and TcII. Related phylogenetic groups with TcI and TcII zymodemes: TcI was related to the Z1 and TcII be related to the Z2. However, the position of Z3 in relation to TcI and TcII phylogenetic groups remains controversial and is constantly debated. Some authors consider that Z3 is phylogenetically closer than TcI/TcII [19, 47, 48], while other authors consider the opposite [49, 50, 51]. However, other authors have included Z3 in an intermediate position between Z1 and Z2 [52].
In 2009 the scientific community was divided into six groups (Tc I-VI) and each group was termed DTU (“discrete typing unit”), which can be identified by the markers molecular or common immunological [27]. In the 70s and 80s, a large number of “group” was identified, and 90 years in 2000, only two major groups, and currently six groups.
In this study, samples of T. cruzi isolated from Triatoma vitticeps from the municipality of Santa Maria Magadalena, State of Rio de Janeiro, were analyzed by several molecular markers, showing a mixed population. The 14 isolates analyzed for the variability of the intergenic region of the Mini-Exon gene showed a profile of bands with 150 bp characteristic of zymodeme III and bands with a lower intensity with ~ 250pb, also indicating a profile for TcII. The PCR amplification of the ribosomal RNA gene (24Sα) using the primers D71 and D72 resulted in fragments 125pb showing a characteristic line TcII. In order to verify possible hybrid characters among the strains analyzed was done with PCR using specific primers tmuts30 and tmuts41 based on analysis of polymorphisms in the MSH2 gene with the digestion of the amplification product of a region of this gene with the restriction enzyme HhaI (Haemophilus haemolyticus) which resulted in fragments of 173pb, 207 pb and 294pb for each isolate, which also indicated a pattern characteristic for strain Tc II, showing then there is no hybrids between these isolates.
According to a protocol for determining DTU proposed by D´Ávila et al. [30] through the polymorphism analysis of the mitochondrial cytochrome oxidase subunit 2 (COII) gene, amplification of the D7 divergent domain of the 24Sα rRNA gene and amplification of the spliced leader intergenic region (SL-IRac) was possible to found two DTU circulating in the studied area.
Our results corroborate the hypothesis that isolated from T. cruzi infection may be a product of a mixture of populations of parasites as the vector into the wild environment can feed on various mammalian hosts. This complex was demonstrated by Fernandes et al. [18] in a study in the state of Rio de Janeiro showed that the association of two strains (TcI and TcII) with different wild hosts.
Evidence indicates that different populations of T. cruzi may circulate in nature by independent cycles of transmission and that these may, under certain conditions, if overlap. In these cases of overlapping cycles, one must admit that possibly different populations, which remained isolated, start to interact in the same vector and/or host. But while there is that possibility, little is known about these mechanisms between these different populations when they come into sympatry. The remarkable capacity of the parasite to infect several species implies adaptation of the parasite to live in various microhabitats, including the different segments of the intestinal tract of the insect [53, 54], nucleated mammalian cells including macrophages [55], blood and also the discharge of the scent glands some marsupials [56, 57]. Some of these are apparently microhabitats hostile to the development of the parasite [58].
Experimental studies have shown that mixed infection with T. cruzi can have a major impact on the biological properties of the parasite in the host, emphasizing the possible occurrence of natural mixed infections in humans and its consequences on the biological aspects of Chagas disease [2].
Our isolates showed indeed a correlation TcIV (formerly Z3) with TcII, indicating that these locality samples associating both the sylvatic cycle, as the domestic cycle, respectively, confirming the complexity of the sylvatic cycle of the disease. These results suggest that in this area might occur studied cycle T. cruzi epidemiological characteristics proposed by Zingales et al. [12], where both strains circulate in the wild habitat.
As TcV and TcVI, TcII has rarely been recorded in wild cycles and their natural niches are not well defined. Recent studies have demonstrated that TcII strain was isolated from opossums and primates in the wild forest, which led to the suggestion that primates could be the primary mammalian hosts of original TcII [59].
TcIV is relatively the more poorly understood group. It is the type responsible for the cause of Chagas disease in Venezuela [48] and was also responsible for the first record of an outbreak of acute cases simultaneously orally transmitted Chagas disease in the suburb of Canudos, State of Belém do Pará/Brazil [40].
Understanding the distribution and phylogeography of TcIV is complicated by the fact that several genotyping methods can not distinguish this strain from others, particularly TcIII.
It is important to emphasize that, TcIV and TcI is known to be endemic, in, North America, and were associated with raccoons in this region [60, 61]. Moreover, there is evidence that TcIV in North America is quite different from TcIV in South America [49, 62], and the presence of identical sequences of mitochondrial DNA in North America strains TcIV and TcI lineages suggests that genetic exchange has contributed to the diversity of strains seen in North America ([51]; Yeo et al, unpublished data).
The existence of mixed populations isolated from the T. vitticeps may reflect the pressure that these insects are suffering due to human action, prompting them to move into different ecological niches, increasing the possibility of contracting the infection of different hosts [63].
Genotyping demonstrates that the strains have a history that makes biological sense with widely current ecological structure, although the details are not yet well elucidated, but still require further research. The study of the genetic diversity of T. cruzi is of great importance for the control of Chagas disease. As seen, the application of molecular methods has shown that this parasite is possibly a body, but a fascinating complex heterogeneous, which will inevitably have different phenotypes. Molecular epidemiology can reveal the different types of transmission cycles and this is very important to develop strategies for vector control and understand their limitations.
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Written By
Helena Keiko Toma, Luciana Reboredo de Oliveira da Silva, Teresa Cristina Monte Gonçalves, Renato da Silva Junior and Jacenir R. Santos-Mallet
Submitted: 20 January 2022Reviewed: 14 March 2022Published: 18 May 2022
Open access peer-reviewed chapter
