Abstract.
Chagas disease affects between six and seven million people. Its etiological agent, Trypanosoma cruzi, is classified into six discrete typing units (DTUs). The biological study of 11 T. cruzi strains presented here included four parameters: growth kinetics, parasitemia curves, rate of macrophage infection, and serology to evaluate IgM, total IgG, IgG1, IgG2a, and IgG3. Sequencing of small subunit of ribosomal RNA (SSU rRNA)was performed and the T. cruzi strains were classified into three DTUs. When their growth in liver infusion tryptose medium was represented in curves, differences among the strains could be noted. The parasitemia profile varied among the strains from the TcI, TcII, and TcIII groups, and the 11 T. cruzi strains produced distinct parasitemia levels in infected BALB/c. The TcI group presented the highest rate of macrophage infection by amastigotes, followed by TcII and TcIII. Reactivity to immunoglobulins was observed in the TcI, TcII, and TcIII; all the animals infected with the different strains of T. cruzi showed anti-T. cruzi antibodies. The molecular study presented here resulted in the classification of the T. cruzi strains into the TcI (Bolivia, T lenti, Tm, SC90); TcII (Famema, SC96, SI8, Y); and TcIII (QMM3, QMM5, SI5) groups. These biological and molecular results from 11 T. cruzi strains clarified the factors involved in the biology of the parasite and its hosts. The collection of triatomine (vector) species, and the study of geographic distribution, as well as biological and molecular characterization of the parasite, will contribute to the reporting and surveillance measures in Brazilian states.
INTRODUCTION
Chagas disease presents an annual incidence of 28,000 cases in the Americas. It is estimated that 65,000,000 people live in areas of risk.1 The study of the biochemical, genetic, and biological markers in Trypanosoma cruzi has allowed for the separation of the parasite into six discrete typing unit (DTU) groups known as TcI to TcVI.2,3
The genus Triatoma is of epidemiological importance because most vector species transferring T. cruzi to humans belong to this genus, an example is Triatoma infestans, considered the main domiciled species.4,5 In Latin American countries, Chagas disease is considered an important cause of death and affects between six and seven million people.6 Vector-borne transmission of T. cruzi may occur through one of the 148 triatomine species.7 For this reason, epidemiological studies are performed in both endemic and nonendemic areas, and triatomines are also captured for analysis.8–14
Many studies have been performed in an attempt to describe the genetic structure of the parasites.15 These efforts have included the sequencing of the genome of T. cruzi (CL Brener), which was published in conjunction with the genome sequences of Leishmania major and Trypanosoma brucei. The current classification of T. cruzi proposes a triple assay using rDNA polymerase chain reaction (PCR)16 and PCR–restriction fragment length polymorphism of the HSP60 and GPI loci17 to type the six T. cruzi DTUs2,3 and suggests that the TcI and TcII groups are molecularly more divergent. The phylogenetic relationship between TcIII and TcIV may be interpreted as a hybridization event between the TcI and TcII groups (“Two-Hybridization” model) and in another model TcIII is also ancestral (“Three Ancestor model”).18
Both Two-Hybridization model17 and Three Ancestor model18 incorporate two hybridization events. In the Three Ancestor model, two recent genetic exchange events between TcII and TcIII yield TcV and TcVI. The Two-Hybridization model invokes one ancient genetic exchange event between TcI and TcII, with loss of heterozygosity among progeny to produce TcIII and TcIV, followed by a second more recent hybridization event between TcII and TcIII to yield both TcV and TcVI.18
The major difference between the Two-Hybridization and the Three Ancestor models is, therefore, whether TcV and TcVI are progeny from a single hybridization event incorporating TcI alleles acquired via TcIII17 or progeny of two hybridization events excluding TcI.19 The TcI group is more abundant and widespread; there have been recent attempts to characterize it into sub-DTUs.20–24 The ability to isolate multiple T. cruzi clones from a given host explains the existence of intrapopulation patterns within the TcI strains.25 The TcI group is of ecological and evolutionary importance, particularly in terms of the sylvatic cycle of infection.26
TcII is found predominantly in the southern and central regions of South America, but its true extent is not yet clear. It has been isolated mostly from domestic transmission cycles. The natural hosts and vectors of TcII have proven elusive and most of the reported isolations have been made in the remaining fragments of the Atlantic forest of Brazil, from primates and sporadically from other mammalian species.27–29 The divergence date between TcI and TcII is ill defined; it is estimated as being between 88 and 37 million years ago, based on small subunit rDNA,30,31 and between 16 and 3 million years ago, based on dihydrofolate reductase–thymidylate synthase and trypanothione reductase genes.32
Since TcIII is present in the domestic cycle, this group is responsible for human cases of Chagas disease, as has already been described.33 TcIII participation in sylvatic cycles has also been described in different biomes and involves infection of several wild hosts, ranging from bats to carnivores.34 TcIV shows a similar pattern of distribution in South America to TcIII. Unlike TcIII, TcIV occurs fairly frequently in humans and is a secondary cause of Chagas disease in Venezuela.35 Further research is required to understand the history of TcIV and these complex ecological associations.18
TcV and TcVI are two similar hybrid DTUs associated with Chagas disease in southern and central South America. The understanding of the ecology of TcII, TcV, and TcVI is as yet vulnerable to the limited sampling of sylvatic hosts and vectors. The paradigms may change. It has been suggested that TcII, TcV, and TcVI are more widespread geographically than is currently acknowledge and might be found much further north.36 A recent study demonstrated the finding of TcII infecting triatomine bugs and mammals in two different areas of the Brazilian Amazon, a biome always before quoted as free from TcII.37 If the known TcV and TcVI hybrids are also found in Central and North America, it will most likely imply recent migration with humans or other carriers; if genetically distinct TcII/TcIII hybrids are observed, hybridization may be an ongoing phenomenon where such mixed infections occur.18
The biological and molecular characterization of T. cruzi strains aid in the understanding of parasite–host interactions. This elucidation is seen in the study by Zingales et al.,18 who discuss associations between vectors, breeding sites, hosts, and the six distinct T. cruzi groups (DTUs). Although the division of T. cruzi into six groups (TcI–TcVI) makes it easier to understand the diversity of this protozoan (a diversity that, it is important to mention, was already noted in the description by Chagas in 190938), the artificial nature of this classification became apparent in the study by Pena et al.39 in which mixed TcI/TcII T. cruzi populations that had been isolated from Triatoma tibiamaculata were identified.
This investigation was conducted to provide a biological and molecular characterization of T. cruzi isolated from triatomine specimens collected in Brazilian states, as the understanding of the T. cruzi DTUs and their epidemiological implications could provide new insights to guide research and interventions against this devastating infectious disease.
To clarify the biological and molecular parameters of T. cruzi using in vivo and in vitro studies, this work presents the parasite’s rate of macrophage infection, growth kinetics based on liver infusion tryptose (LIT) culture, parasitemia curves using BALB/c mice and reactivity to immunoglobulins, and the phylogeny of 11 strains (TcI, TcII, and TcIII) isolated in the Brazilian states of Bahia (Triatoma sordida/Triatoma melanocephala/Triatoma lenti), Rio Grande do Sul (Triatoma rubrovaria), Santa Catarina (Didelphis aurita/Homo sapiens), and São Paulo (H. sapiens). The study of strains isolated from the five species of triatomines belonging to the genus Triatoma is needed and would complement data on the biological and molecular characterization of the 11 strains of T. cruzi belonging to DTUs TcI, TcII, and TcIII. The results achieved with this work can be used to detect ecological factors associated with T. cruzi, which are part of a complex epidemiological context, and help to increase the knowledge of Chagas disease in the regions from which the triatomines were collected.
MATERIALS AND METHODS
Strains of T. cruzi.
A total of 11 strains were characterized in the present study, six strains were isolated from the posterior intestine of triatomine species12: Triatoma melanocephala from Bahia-BR—Tm (TcI); T. lenti from Bahia-BR—T lenti strain (TcI); T. sordida from Bahia-BR—SI5 (TcIII) and SI8 (TcII) strains; and T. rubrovaria from Rio Grande do Sul-BR—QMM3 and QMM5 strains (TcIII). Five further strains of T. cruzi were included in the biological assays as reference strains: Bolivia40 from Vitichi-BO (T. infestans—TcI); Famema41 from São Paulo-BR (H. sapiens—TcII); SC9042 from Santa Catarina-BR (D. aurita—TcI); SC9642 from Santa Catarina-BR (H. sapiens—TcII); and Y43 from São Paulo-BR (H. sapiens—TcII). A total of 44 sequences were studied in the phylogenetic analysis. Sequences from 26 T. cruzi isolates and three sequences of Trypanosoma cruzi marinkellei from GenBank were included in the phylogenetic analysis (with the accession numbers shown in Table 1). The 18 sequences that were determined in this study and deposited in GenBank are underlined (Table 1).
Table 1.
Isolate | GenBank accession number† | |||||
---|---|---|---|---|---|---|
Host origin | Geographic Origin (state) | DTU* | V7V8 SSU rRNA | |||
Trypanosoma cruzi | ||||||
Tm | Triatomine | Triatoma melanocephala | Bahia | BR | TcI | xxxxxx |
Bolivia cl3 | Triatomine | Triatoma infestans | Vitichi | BO | TcI | xxxxxx |
Bolivia cl4 | Triatomine | T. infestans | Vitichi | BO | TcI | xxxxxx |
SC90 | Opossum | Didelphis aurita | Santa Catarina | BR | TcI | xxxxxx |
G | Opossum | Didelphis marsupialis | Amazonas | BR | TcI | AF239981 |
TCC45 | Opossum | D. aurita | São Paulo | BR | TcI | FJ183394 |
Sylvio X10 | Human | Homo sapiens | Para | BR | TcI | AF303659 |
TCC269 | Wild primate | Saguinus midas | Amazonas | BR | TcI | EU755221 |
T lenti | Triatomine | Triatoma lenti | Bahia | BR | TcI | xxxxxx |
SC96 cl3 | Human | H. sapiens | Santa Catarina | BR | TcII | xxxxxx |
SC96 cl4 | Human | H. sapiens | Santa Catarina | BR | TcII | xxxxxx |
SI8 cl1 | Triatomine | Triatoma sordida | Bahia | BR | TcII | xxxxxx |
SI7 | Triatomine | T. sordida | Bahia | BR | TcII | xxxxxx |
FAMEMA | Human | H. sapiens | Sao Paulo | BR | TcII | xxxxxx |
Esmeraldo clone 3 | Human | H. sapiens | Bahia | BR | TcII | AY785564 |
TCC139 | Opossum | D. aurita | São Paulo | BR | TcII | FJ001616 |
Y | Human | H. sapiens | São Paulo | BR | TcII | AF301912 |
SIGR3 cl1 | Cat | Felis silvestris catus | Bahia | BR | TcII | xxxxxx |
QMM3 | Triatomine | Triatoma rubrovaria | Rio Grande do Sul | BR | TcIII | Xxxxxx/xxxxxx |
QMM5 | Triatomine | T. rubrovaria | Rio Grande do Sul | BR | TcIII | Xxxxxx/xxxxxx |
QMM12 | Triatomine | T. rubrovaria | Rio Grande do Sul | BR | TcIII | xxxxxx |
SI5 cl1 | Triatomine | T. sordida | Bahia | BR | TcIII | Xxxxxx/xxxxxx/xxxxxx |
Arma13 cl1 | Armadillo | Dasypus novemcinctus | Boqueron | PY | TcIII | FJ549385 |
TCC2557 | Bat | Phyllostomus hastatus | São Paulo | BR | TcII | KT305894 |
TCC863 | Armadillo | Euphractus sexcinctus | Rio Grande do Norte | BR | TcIII | FJ549376 |
MT3869 | Human | H. sapiens | Amazonas | BR | TcIII | AF303660 |
MT3663 | Triatomine | Panstrongylus geniculatus | Amazonas | BR | TcIII | AF288660 |
Can III | Human | H. sapiens | Para | BR | TcIV | AJ009148 |
TCC206 | Carnivore | Nasua nasua | Para | BR | TcIV | FJ555615 |
TCC1441 | Human | H. sapiens | Para | BR | TcIV | EU755247 |
TCC1446 | Human | H. sapiens | Para | BR | TcIV | EU755248 |
M6241 cl6 | Human | H. sapiens | Para | BR | TcIII | AY785578 |
Sc43 cl1 | Triatomine | T. infestans | Santa Cruz | BO | TcV | AF232214 |
TCC186 | Triatomine | T. infestans | – | BO | TcV | FJ001630 |
CL Brener | Triatomine | T. infestans | Rio Grande do Sul | BR | TcVI | AF245383 |
TCC1122 | Bat | Myotis albescens | São Paulo | BR | Tcbat | FJ001628 |
TCC294 | Bat | Myotis levis | São Paulo | BR | Tcbat | FJ001634 |
TCC1994 | Bat | M. levis | São Paulo | BR | Tcbat | FJ900241 |
TCC597 | Bat | Myotis nigricans | Mato Grosso do Sul | BR | Tcbat | FJ001623 |
NR cl3 | Human | H. sapiens | Salvador | CL | TcV | AF228685 |
Trypanosoma cruzi marinkellei | ||||||
TCC421 | Bat | Phyllostomus discolor | Amazonas | BR | – | FJ001637 |
TCC424 | Bat | P. discolor | Amazonas | BR | – | FJ001639 |
TCC501 | Bat | Carollia perspicillata | Rondonia | BR | – | FJ001665 |
BO = Bolivia; BR = Brazil; CL = Chile; DTU = discrete typing unit; PY = Paraguay; SSU = small subunit.
Lineages determined based on mini-exon markers and phylogenetic analyses inferred in this study.48
Sequences determined in this study and deposited in GenBank are underlined.
Small subunit rRNA gene amplification (V7V8 region) of T. cruzi.
DNA was extracted according to the methods of Sambrook et al.,44 with modifications. The amount of DNA was estimated through comparisons to known standards in 1% (w/v) agarose gel stained with GelRed®, as well as through the use of spectrophotometry. The PCRs contained 100 ng of genomic DNA, 100 ng of each primer, 200 mM of each dNTP, 5 μL of a buffer solution (Tris-HCl [pH 8.4], 500 mM KCl, and 1.5 mM MgCl2), 2.5 U of Taq DNA polymerase, and double distilled deionized and autoclaved water (in a final volume of 50 μL). The amplification cycle and the annealing temperatures were defined according to the oligonucleotide primers used, which were 609F (5′-GAT CCG CGG TAA TTC CAG C-3′) and 706R (5′-TTG AGG TTA CAG TCT CAG-3′).
Nucleotide sequencing.
The DNA fragments that were amplified using PCR were purified and then underwent sequencing reactions using a Big Dye Terminator kit (Perkin Elmer; Applied Biosystems Inc., Austin, TX) according to the manufacturer’s instructions. The reactions were performed with an initial cycle of 1 minute at 96°C, followed by 30 cycles of 15 seconds at 96°C, 15 seconds at 50°C, 4 minutes at 60°C. The following oligonucleotide primers were used: 1156F (5′-CGT ACT GGT GCG TCA AGA GG-3′); 1156R (5′-CCT CTG ACG CAC CAG TCA G-3′); 609F (5′-GAT CCG CGG TAA TTC CAG C-3′); and 706R (5′-TTG AGG TTA CAG TCT CAG-3′).
Nucleotide sequencing alignment.
The sequencing chromatograms were analyzed with the Seqman-DNAstar program (DNASTAR Inc., Madison, Wisconsin).45 Both the nucleotide sequences determined in this study and those obtained from GenBank (http://www.ncbi.nlm.nih.gov/) were aligned using the Clustal X program.46 The nucleotide alignments were manually adjusted in the GeneDoc program, version 2.7.000.45
Phylogenetic analyses.
Phylogenetic interferences were determined using the maximum likelihood (ML) method and Bayesian analysis. The tree was developed using the PAUP program, version 4.0b1047 through the heuristic search with 100 random addition replicates of the terminals, followed by branch breaking. One hundred replicates of the bootstrap support analyses were performed with the same parameters used in the search.
The ML analyses were performed using the RAxML program, version 7.0.4.48 A total of 500 replicates were used using the generalized time reversible analysis as a substitution model, and four gamma categories, and diagrams obtained through parsimony were used as initial trees. The parameters of the substitution model were estimated during the search. Branch support was estimated using 500 bootstrap replicates in the RAxML program.
The Bayesian analyses were performed in the MrBayes program, version 3.1.2.49 A total of 500,000 generations were used using generalized time reversible analysis as the substitution model, and four gamma categories, plus a proportion of invariant sites. Only the diagrams obtained from the last 75 replicates were used to build the final cluster tree. The genealogies of the nucleotide sequences were inferred using network analysis in the Splitstree program, version 4.11.3.50 The NeighborNet method was used, and the support values were estimated through the completion of the 100 bootstrap replicas.
Parasitemia curve.
Trypanosoma cruzi trypomastigote counts were performed based on the method of Brener.51 To study the parasitemia curve, BALB/c mice—five animals for each group (22-day-old males weighing between 25 and 30 g)—were intraperitoneally inoculated with 5 × 103 trypomastigote forms of T. cruzi. To establish the infection pattern, 5 μL of blood obtained from the tails of the mice was examined microscopically and the number of forms was established. Counts were performed in all the strains studied on alternate days, starting from the second day after the initial inoculation until the 60th day of the infection. The number of animals that died during the course of the infection was observed on a daily basis. The animals were maintained under temperature- and light-controlled conditions. The experiments were approved by the Research Ethics Committee of the School of Pharmaceutical Sciences within São Paulo State University, Araraquara, Brazil (CEUA/FCF/CAr no 13/2012).
Growth kinetics.
Trypanosoma cruzi epimastigote growth dynamics were studied using the Bolivia, Famema, QMM3, QMM5, SC96, SC90, SI5, SI8, Tl, Tm, and Y strains. The study was performed by inoculating 5 × 106 parasites per mL in 5 mL of LIT medium. Cell counts were measured five times over 10 days in a Neubauer chamber using an optic microscope.
Trypanosoma cruzi assay in peritoneal macrophages.
The invasion assay was performed according to Muelas-Serrano et al.52 The macrophages were grown at 37°C in Roswell Park Memorial Institute media with 5% CO2. For the assay, 5 × 105 cells were seeded in 24-well plates. The cells were incubated at 37°C for 4 hours and then infected with 5 × 106 T. cruzi trypomastigotes. (These trypomastigotes had been obtained in stationary phase, washed in phosphate-buffered saline (PBS), and the concentration adjusted using a Neubauer chamber.) At 24 and 72 hours after infection, the cells were washed in PBS, fixed in methanol, and stained using Giemsa. The rate of infection was determined by counting 200 macrophages that contained internalized amastigote forms. The counts were repeated three times and the parasite duplication time of the 11 T. cruzi strains in macrophages was determined using the formula reported by Jawetz et al.53
where dt = doubling time; t1 = 72 hours; t0 = 24 hours; N1 = mean number of parasites per infected cell at 72 hours of infection; and N0 = at 24 hours of infection.
Determination of the presence of anti-T. cruzi antibodies.
An enzyme-linked immunosorbent assay (ELISA) was used to determine the presence of anti-T. cruzi antibodies (IgM, IgG, IgG1, IgG2a, and IgG3) in the serum of mice infected with the TcI, TcII, and TcIII genotypes of T. cruzi. Mice (N = 3 per strain) were injected intraperitoneally with 1 × 105 trypomastigote forms of T. cruzi, strain Bolívia, Y, and QMM5, and killed at 30, 55, and 70 days after infection. Blood samples were collected by cardiac puncture, without anticoagulant, and the sera obtained were stored at −20°C. Healthy mice were used as a negative control (N = 3). For the positive control, a polyclonal anti-T. cruzi serum was produced by inoculation of a crude antigen extract of T. cruzi epimastigotes. CD1 mice were inoculated with 100 μL of the T. cruzi extracts by the intraperitoneal route. After the immunization period, the animals were euthanized, and blood samples were then collected for serum fractionation, and stored at −20°C. To determine the presence of anti-T. cruzi antibodies, 100 ng/mL of the T. cruzi epimastigote antigen was diluted in a bicarbonate buffer (0.1 M and pH 8.5) and incubated overnight at 5°C in microplates. The microplates were washed with wash buffer (PBS–Tween-20, 0.05% v/v), and incubated with a Bovine Serum Albumin blocking buffer (PBS–BSA–Tween-20, 0.05% ) for 1 hour, at which point they were washed again. The plates were incubated for 1 hour with 100 μL diluted serum (1:400) and washed five times with the wash buffer. To determine the presence of anti-T. cruzi IgG antibodies, anti-IgG secondary antibody solution conjugated with enzyme horseradish peroxidase (HRP) 1:4,000 anti-mouse IgG (Sigma-Aldrich, St. Louis, MO) was added to the plates and incubated for 1 hour. The other antibody subclasses were determined with the following conjugated antibodies: IgG1 (50 ng of anti-mouse IgG1:HRP; AbD Serotec); IgG2a (25 ng anti-mouse IgG2a:HRP; AbD Serotec); IgG3 (25 ng anti-mouse IgG3:HRP; AbD Serotec); and IgM (1:400 anti-mouse IgM:HRP; Sigma-Aldrich). Next, five washes were performed and the microplate was incubated with a substrate solution for 30 minutes (10 mL of citrate buffer, 10 mg of o-phenylenediamine, and 5 μL of hydrogen peroxide 3% [v/v]). In addition, 4 N sulfuric acid was used to stop the reaction, and absorbance was measured at 490 nm. The assay was performed in duplicate and the optical density data were presented as means and standard deviations, using the software Instat (Graphpad, San Diego, CA). The analysis of variance for all the data was carried out by two-way analysis of variance.
RESULTS
Molecular characterization.
In this work, T. cruzi strains were separated into TcI (Bolivia, Tm, T lenti, and SC90); TcII (Y, FAMEMA, SI8, and SC96); and TcIII (SI5, QMM3, and QMM5). The origins, hosts, and DTUs of the T. cruzi strains are reported in Table 1, and the phylogenetic relationships among the 11 strains are shown in Figure 1. In all analyses (ML and Bayesian), T. cruzi formed a monophyletic assemblage.
Parasitemia profile.
It was found that the 11 T. cruzi strains resulted in distinct parasitemia levels in infected BALB/c mice, despite the fact that the inoculum (5 × 103) had been standardized in the TcI, TcII, and TcIII groups. The parasitemia profile of the TcI, TcII, and TcIII groups during the acute phase of the experimental infection by T. cruzi is presented in Figure 2.
In the TcI group (Bolívia, SC90, T lenti, and Tm), subpatent infection was observed for the SC90 and T lenti strains, although T. cruzi was not found in the bloodstream. In the case of the Tm strain, peak parasitemia occurred around the 15th day, with a mortality rate of 20% .
In the TcII group (Famema, SC96, SI8, and Y), the Famema strain had more trypomastigote forms and a parasitemic peak around the 27th day. The TcIII group of T. cruzi, represented by SI5, QMM3, and QMM5, showed the SI5 strain with more trypomastigotes and a parasitemic peak on the 52th day. The SI5 (TcIII) and SI8 (TcII) T. cruzi strains presented differences in the prepatent period, in the peak parasitemia levels, and in the decreases in blood trypomastigote populations, although the SI5 and SI8 have been isolated from T. sordida. After that period, the number of blood trypomastigotes started to decrease, with trypomastigotes disappearing from the bloodstream around the 60th day of the infection.
Biological variability of T. cruzi strains in LIT medium.
When T. cruzi growth in LIT medium was represented by curves, differences among the strains could be noted (Figure 3). Maximum parasite multiplication occurred at around 5–10 days (10.9 × 106–8.7 × 106 parasites) for the TcI group, 6–9 days (13.2 × 106–14.8 × 106 parasites) for the TcII group, and 7–8 days (13.2 × 106–11 × 106 parasites) for the TcIII group. Together, these results are suggestive that the maintenance of populations of T. cruzi may be related to intrinsic characteristics of the parasite, such as its infection ability.
Infection rate of different strains of T. cruzi in peritoneal macrophages.
The mean amastigote counts of the 11 T. cruzi strains in 200 peritoneal macrophages are represented in Figure 4. The T. cruzi groups were separated into TcI (Bolivia, Tm, T lenti, and SC90); TcII (Y, FAMEMA, SI8, and SC96); and TcIII (SI5, QMM3, and QMM5). The TcI group presented the following values for 24 and 72 hours of infection: 479–751 amastigotes/200 macrophages (Bolívia); 453–858 amastigotes/200 macrophages (Tm); 391–702 amastigotes/200 macrophages (T lenti); and 230–501 amastigotes/200 macrophages (Sc90). The TcII group had the values: 415–702 amastigotes/200 macrophages (Y); 490–702 amastigotes/200 macrophages (FAMEMA); 392–822 amastigotes/200 macrophages (SI8); and 611–799 amastigotes/200 macrophages (SC96). The TcIII group had the values: 546–682 amastigotes/200 macrophages (SI5); 388–610 amastigotes/200 macrophages (QMM3); and 335–603 amastigotes/200 macrophages (QMM5). In T. cruzi, the times required for the multiplication of amastigote forms were TcI, between 1.7 and 3 days; TcII, between 1.8 and 5.1 days; TcIII, between 2.3 and 6.2 days. So the TcI group showed the least time required for the multiplication of the parasite, followed by TcII and TcIII.
The strains with the highest rates of infection in macrophages were isolated from T. melanocephala (Tm) and T. sordida (SI5/SI8). The insects were collected in the state of Bahia, Brazil, data reinforcing the parasite–host–environment association, as well as the adaptation of T. cruzi TcI, TcII, and TcIII to their vertebrate or invertebrate host.
Determination of anti-T. cruzi antibodies in mice infected with different strains of T. cruzi.
All the animals infected with different strains of T. cruzi showed anti-T. cruzi antibodies (Figures 5 and 6). There was a decreased production of anti-T. cruzi IgM antibody in all groups 30 days after infection, due to the time-dependent decrease of infection (Figure 5A). The IgM decrease 30 and 70 days after infection was significantly different in Y and QMM5 strain groups, using Tukey’s multiple comparison test (P < 0.0001). The Y strain triggered increased IgM production and QMM5 less antibody production. A progressive increase of the production of anti-T. cruzi total IgG was verified at 30, 55, and 70 days postinfection (Figure 5B), with significant differences in all groups (P < 0.001). The comparison among the groups demonstrated more pronounced differences compared with Y and QMM5 strains (Figure 5B). The analysis of the anti-T. cruzi antibody subclasses IgG1, IgG2a, and IgG3 demonstrated an increase of the titers followed by an increasing time of infection (Figure 6), which was most significant at 70 days postinfection (P < 0.0001). The Y strain showed higher titers of anti-T. cruzi IgG1 (Figure 6A), with significant differences compared with strain QMM5 (P value < 0.01) and no differences compared with Bolivia. Seventy days after infection, IgG2a and IgG3 antibody production was significantly more pronounced compared with production at 30 days after infection (P < 0.0001). The Y strain demonstrated higher titers compared with the other strains at 70 days after infection (Figure 6B and C). As shown in Figure 6C, although there was an increase over the duration of the infection, the titers of IgG3 showed it was virtually absent from or only present at very low levels in the group infected with strain QMM5 compared with the other groups infected with strains Bolivia and Y (P <0.0001).
DISCUSSION
Molecular characteristics of the T. cruzi parasite, such as the diversity of the DTUs, can explain the peculiarities of Chagas disease, as the inherent characteristics of the parasite result in different clinical manifestations.54 The results of this study reflect T. cruzi diversity, which was first mentioned by Chagas.38
The association between the TcI group (Tm, T lenti, and SC90) and wild reservoir hosts, such as T. melanocephala and T. lenti, is reported for the first time herein, since no studies were found in the literature. The data from Câmara et al.55 showed the interaction between TcII and humans, and the involvement of the TcIII group in the wild environment was also considered in their work. The QMM3 and QMM5 strains isolated from T. rubrovaria were placed in the TcIII group; therefore, given this species’ wide range in the Brazilian state of Rio Grande do Sul, it must still be monitored because the species has the ability to spread into domestic environments.13
In ML and Bayesian analyses, TcI, TcII, and TcIII were clearly separated and the distribution found herein is consistent with the literature.18,22 The findings are indicative that TcII is phylogenetically separated from TcI. The TcIII group developed later and was the basis of the hybridization event in T. cruzi.18 The TcI group was placed as a sister group to Tcbat,56 which has been established as a T. cruzi ancestor, forming a monophyletic assemblage and clarified the evolutionary process of the taxon. As expected, the sequences of the hybrid TcIII strains were clustered very closely with strains of the TcV group, used as a control (Sc43c11, 186, NRc13).
Trypanosoma cruzi strains SI5 (Bahia—peridomestic), QMM3, and QMM5 (Rio Grande do Sul—wild environment) belonged to TcIII, highlighting the originality and importance of the work since triatomines collected in the peridomestic environment rarely belong to the TcIII group, because generally T. cruzi-TcIII is reported in the wild environment and only sporadically has been isolated from domestic transmission cycles. Generic identification methods based on SSU rRNA sequences can be used to identify trypanosome species, as well as to infer phylogenetic relationships in trypanosomes and vectors.57
Some authors58–60 have mentioned the variation of parasitemia in animals inoculated with T. cruzi strains, either for samples collected from humans, wild animals, or the vector itself. The parasitemia curves of the TcI (Bolivia, Tm, T lenti, and SC90), the TcII (Y, FAMEMA, SI8, and SC96), and the TcIII group (SI5, QMM3, and QMM5) showed differences in the prepatent period, stationary phase, and a decrease in parasitemia levels. Together these results show that the biological variability between TcI, TcII, and TcIII groups of T. cruzi can be explained by the host–parasite association and the geographical region that the Triatominae were collected from.
That variation is frequently seen when a new strain of the parasite is isolated and it is directly related to the stabilization of the relationship between the parasite and the vertebrate host.61 Biological differences between the TcI and TcII groups were observed by Lisboa et al.29 and in this work the population dynamics of the TcI, TcII, and TcIII groups presented culture differences, which may be linked to the parasite’s adaptation to the axenic environment.
The establishment of T. cruzi infection depends on a series of events, involving interactions between the molecules of the parasite and the host.62 In the case of macrophages, most parasites are internalized via the phagocytic mechanism, involving polymerization of actin filaments required for the formation of membrane projections.63,64 This study devoted to new strains of T. cruzi aimed to create ideal conditions for the growth and differentiation of the parasite and as a result, the biological characteristics of T. cruzi became important in the interaction with the host cells during the infection process.
Despite advancements in the diagnosis of Chagas disease, new antigens are still needed.65 During infection in the murine model, the humoral response to T. cruzi is mainly due to IgM and IgG2a antibodies for acute infection.66 The low fraction of anti-T. cruzi IgM after 30 days of infection verified in this study was similar to results found in other studies, indicating that anti-T. cruzi IgM does not protect in the most advanced stage of infection.67 The analysis in this study confirmed isotype switching of anti-T. cruzi IgM class to IgG.
The analysis of IgG antibodies, specifically anti-T. cruzi IgG1, in the present study demonstrated low levels during the acute phase, with the sharpest increase 70 days after infection, indicating a lack of protective ability of IgG1 in the acute phase. The results of this study are in accordance with studies reported in the literature, where it was found that during experimental infection, the immune response is a detectable high concentration of IgG2a anti-T. cruzi antibodies.67,68 In this study, IgG1 and IgG3 antibodies were present only at very low levels during the acute phase 30 days after infection. Moreover, IgG2a is associated with protection and is considered more effective against T. cruzi.69 Anti-T. cruzi IgG3 increased 70 days after infection with TcII compared with the other groups.
The heterogeneity of T. cruzi infection results in various aspects of immune responses, including inflammatory parameters and the development of tissue damage. The smaller profile of the production of anti-T. cruzi antibodies was verified in the QMM5 group and could be correlated with the genetic characteristics of the group. The ELISA technique complemented the biological results and provided information on the TcI and TcII groups, which were the most divergent in molecular terms. The biological and molecular results from these 11 T. cruzi strains clarified key factors involved in the biology of the parasite and its hosts. These factors included the classification of the strains into the TcI, TcII, and TcIII groups, the finding that TcI presented the highest rate of macrophage infection, and the higher growth kinetic values in the TcII group. These findings underscore the complexity of the pathogenesis of Chagas disease and the influence of the heterogeneity of T. cruzi strains on the pathophysiology of disease. Improving the comprehension of these processes will lead to improvements in drug development and the treatment of the disease.
Acknowledgments
We thank Julio César Rente Ferreira Filho and Vagner José Mendonça, who collected Triatoma lenti, Eliane Góes Nascimento, from the Health Department of the State of Bahia/SESAB—Entomology Division, who sent us specimens of Triatoma melanocephala.
REFERENCES
- 1.Organização Pan-Americana da Saúde , 2017. Neglected Infectious Diseases. Available at: http://www.paho.org/hq/index.php?option=com_topics&view=article&id=10&Itemid=40743. Accessed February 21, 2017.
- 2.Zingales B, et al. 2009. A new consensus for Trypanosoma cruzi intraspecific nomenclature: second revision meeting recommends TcI to TcVI. Mem Inst Oswaldo Cruz 104: 1051–1054. [DOI] [PubMed] [Google Scholar]
- 3.Lewis MD, Ma J, Yeo M, Carrasco HJ, Llewellyn MS, Miles MA, 2009. Genotyping of Trypanosoma cruzi: systematic selection of assays allowing rapid and accurate discrimination of all known lineages. Am J Trop Med Hyg 81: 1041–1049. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Obara MT, Rosa JA, Silva NN, Ceretti W, Jr, Urbinatti PR, Barata JMS, Jurberg J, Galvão C, 2007. Estudo morfológico e histológico dos ovos de seis espécies do gênero Triatoma (Hemiptera:Reduviidae). Neotrop Entomol 36: 798–806. [DOI] [PubMed] [Google Scholar]
- 5.Araujo CAC, Waniek PJ, Jansen AM, 2009. An overview of Chagas disease and the role of triatomines on its distribution in Brazil. Vector Borne Zoonotic Dis 9: 227–234. [DOI] [PubMed] [Google Scholar]
- 6.World Health Organization , 2017. Chagas Disease (American Trypanosomiasis). Available at: http://www.who.int/mediacentre/factsheets/fs340/en/. Accessed February, 21, 2017. [PMC free article] [PubMed]
- 7.Galvão C, (Organizador), 2014. Vetores da doença de chagas no Brasil [online]. Zoologia: Guias e Manuais de Identificação Series. Curitiba, Paraná: Sociedade Brasileira de Zoologia, 289 ISBN 978-85-98203-09-6 Available at: http://books.scielo.org. Accessed February 21, 2017. [Google Scholar]
- 8.Almeida CE, Vinhaes MC, Almeida JR, Silveira AC, Costa J, 2000. Monitoring the domiciliary and peridomiciliary invasion process of Triatoma rubrovaria in the state of Rio Grande do Sul, Brazil. Mem Inst Oswaldo Cruz 95: 761–768. [DOI] [PubMed] [Google Scholar]
- 9.Carvalho DB, Almeida CE, Rocha CS, Gardim S, Mendonça VJ, Ribeiro AR, Alves ZC, Ruellas KT, Vedoveli A, Rosa JA, 2014. A novel association between Rhodnius neglectus and the Livistona australis palm tree in an urban center foreshadowing the risk of Chagas disease transmission by vectorial invasions in Monte Alto City, São Paulo, Brazil. Acta Trop 130: 35–38. [DOI] [PubMed] [Google Scholar]
- 10.Coura JR, Viñas PA, 2010. Chagas disease: a new worldwide challenge. Nature 465: S6–S7. [DOI] [PubMed] [Google Scholar]
- 11.Dias JCP, 2000. Epidemiological surveillance of Chagas disease. Cad Saude Publica 16: 43–59. [PubMed] [Google Scholar]
- 12.Ribeiro AR, Mendonça VJ, Alves RT, Martinez I, Araújo RF, Mello F, Rosa JA, 2014. Trypanosoma cruzi strains from triatomine collected in Bahia and Rio Grande do Sul, Brazil. Rev Saude Publica 48: 295–302. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Rosa JA, Barata JMS, Santos JLF, Cilense M, 2000. Morfologia de ovos de Triatoma circummaculata e Triatoma rubrovaria (Hemiptera, Reduviidae). Rev Saude Publica 34: 538–542. [DOI] [PubMed] [Google Scholar]
- 14.Rosa JA, et al. 2012. Description of Rhodnius montenegrensis n. sp. (Hemiptera: Reduviidae: Triatominae) from the state of Rondônia, Brazil. Zootaxa 3478: 62–76. [Google Scholar]
- 15.Ramírez JD, Duque MC, Montilla M, Cucunubá Z, Guhl F, 2012. Natural and emergent Trypanosoma cruzi I genotypes revealed by mitochondrial (Cytb) and nuclear (SSU rDNA) genetic markers. Exp Parasitol 132: 487–494. [DOI] [PubMed] [Google Scholar]
- 16.Souto RP, Fernandes O, Macedo AM, Campbell DA, Zingales B, 1996. DNA markers define two major phylogenetic lineages of Trypanosoma cruzi . Mol Biochem Parasitol 83: 141–152. [DOI] [PubMed] [Google Scholar]
- 17.Westenberger SJ, Barnabé C, Campbell DA, Sturm NR, 2005. Two hybridization events define the population structure of Trypanosoma cruzi . Genetics 171: 527–543. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Zingales B, et al. 2012. The revised Trypanosoma cruzi subspecific nomenclature: rationale, epidemiological, relevance and research applications. Infect Genet Evol 12: 240–253. [DOI] [PubMed] [Google Scholar]
- 19.Freitas JM, et al. 2006. Ancestral genomes, sex, and the population structure of Trypanosoma cruzi . PLoS Pathog 2: e24. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Herrera C, Bargues MD, Fajardo A, Montilla M, Triana O, Vallejo GA, Guhl F, 2007. Identifying four Trypanosoma cruzi I isolate haplotypes from different geographic regions in Colombia. Infect Genet Evol 7: 535–539. [DOI] [PubMed] [Google Scholar]
- 21.Llewellyn MS, et al. 2009. Genome-Scale multilocus microsatellite typing of Trypanosoma cruzi discrete typing unit I reveals phylogeographic structure and specific genotypes linked to human infection. PLoS Pathog 5: e1000410. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Guhl F, Ramírez JD, 2011. Trypanosoma cruzi I diversity: towards the need of genetic subdivision? Acta Trop 119: 1–4. [DOI] [PubMed] [Google Scholar]
- 23.Ocaña-Mayorga S, Llewellyn MS, Costales JA, Miles MA, Grijalva MJ, 2010. Sex, subdivision, and domestic dispersal of Trypanosoma cruzi lineage I in Southern Ecuador. PLoS Negl Trop Dis 4: 1–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Ramírez JD, Duque MC, Guhl F, 2011. Phylogenetic reconstruction based on cytochrome b (Cytb) gene sequences reveals distinct genotypes within Colombian Trypanosoma cruzi I populations. Acta Trop 119: 61–65. [DOI] [PubMed] [Google Scholar]
- 25.Llewellyn MS, Rivett-Carnac JB, Fitzpatrick S, Lewis MD, Yeo M, Gaunt MW, Miles MA, 2011. Extraordinary Trypanosoma cruzi diversity within single mammalian reservoir hosts implies a mechanism of diversifying selection. Int J Parasitol 41: 609–614. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Zumaya-Estrada FA, et al. 2012. North American import? Charting the origins of an enigmatic Trypanosoma cruzi domestic genotype. Parasit Vectors 5: 1–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Fernandes O, et al. 1998. Brazilian isolates of Trypanosoma cruzi from humans and triatomines classified into two lineages using mini-exon and ribosomal RNA sequences. Am J Trop Med Hyg 58: 807–811. [DOI] [PubMed] [Google Scholar]
- 28.Zingales B, Stolf BS, Souto RP, Fernandes O, Briones MR, 1999. Epidemiology, biochemistry and evolution of Trypanosoma cruzi lineages based on ribosomal RNA sequences. Mem Inst Oswaldo Cruz 1: 159–164. [DOI] [PubMed] [Google Scholar]
- 29.Lisboa CV, Pinho AP, Monteiro RV, Jansen AM, 2007. Trypanosoma cruzi (kinetoplastida Trypanosomatidae): biological heterogeneity in the isolates derived from wild hosts. Exp Parasitol 116: 150–155. [DOI] [PubMed] [Google Scholar]
- 30.Briones MRS, Souto RP, Stolf BS, Zingales B, 1999. The evolution of two Trypanosoma cruzi subgroups inferred from rRNA genes can be correlated with the interchange of American mammalian faunas in the Cenozoic and has implications to pathogenicity and host specificity. Mol Biochem Parasitol 104: 219–232. [DOI] [PubMed] [Google Scholar]
- 31.Kawashita SY, Sanson GF, Fernandes O, Zingales B, Briones MR, 2001. Maximum-likelihood divergence date estimates based on rRNA gene sequences suggest two scenarios of Trypanosoma cruzi intraspecific evolution. Mol Biol Evol 18: 2250–2259. [DOI] [PubMed] [Google Scholar]
- 32.Machado CA, Ayala FJ, 2001. Nucleotide sequences provide evidence of genetic exchange among distantly related lineages of Trypanosoma cruzi . Proc Natl Acad Sci USA 13: 7396–7401. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Monteiro WM, Magalhães LK, Santana Filho FS, Borborema M, Silveira H, Barbosa Md, 2010. Trypanosoma cruzi TcIII/Z3 genotype as agent of an outbreak of Chagas disease in the Brazilian western Amazonia. Trop Med Int Health 15: 1049–1051. [DOI] [PubMed] [Google Scholar]
- 34.Jansen AM, Xavier SC, Roque AL, 2015. The multiple and complex and changeable scenarios of the Trypanosoma cruzi transmission cycle in the sylvatic environment. Acta Trop 151: 1–15. [DOI] [PubMed] [Google Scholar]
- 35.Miles MA, Cedillos RA, Póvoa MM, de Souza AA, Prata A, Macedo V, 1981. Do radically dissimilar Trypanosoma cruzi strains (zymodemes) cause Venezuelan and Brazilian forms of Chagas’ disease? Lancet 20: 1338–1340. [DOI] [PubMed] [Google Scholar]
- 36.Zafra G, Mantilla JC, Valadares HM, Macedo AM, González CI, 2008. Evidence of Trypanosoma cruzi II infection in Colombian chagasic patients. Parasitol Res 103: 731–734. [DOI] [PubMed] [Google Scholar]
- 37.Lima VS, Xavier SCC, Maldonado IFR, Roque ALR, Vicente ACP, Jansen AM, 2014. Expanding the knowledge of the geographic distribution of Trypanosoma cruzi TcII and TcV/TcVI Genotypes in the Brazilian Amazon. PLoS One 9: e116137. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Chagas C, 1909. Nova tripanozomase humana. Estudos sobre a morfologia e o ciclo evolutivo do Schizotrypanum cruzi n. gen. n. gen. n. sp, agente etiológico de nova entidade mórbida do homem. Mem Inst Oswaldo Cruz 1: 159–218. [Google Scholar]
- 39.Pena DA, Eger I, Nogueira L, Heck N, Menin Á, Báfica A, Steindel M, 2012. Selection of TcII Trypanosoma cruzi population following macrophage infection. J Infect Dis 204: 478–486. [DOI] [PubMed] [Google Scholar]
- 40.Funayama GK, Prado Júnior JC, 1974. Estudo dos caracteres de uma amostra boliviana do Trypanosoma cruzi . Rev Soc Bras Med Trop 8: 75–81. [Google Scholar]
- 41.Martins LPA, Castanho REP, Rosa JA, Silva LC, Godoy CAP, Rosa RM, 2003. Caracterização biológica, histopatológica e análise de ácido nucléico de uma cepa Trypanosoma cruzi da região de Marília, SP. Rev Soc Bras Med Trop 36: 35–39. [DOI] [PubMed] [Google Scholar]
- 42.Steindel M, et al. 2008. Characterization of Trypanosoma cruzi isolated from humans, vectors, and animal reservoirs following an outbreak of acute human Chagas disease in Santa Catarina State, Brazil. Diagn Microbiol Infect Dis 60: 25–32. [DOI] [PubMed] [Google Scholar]
- 43.Silva LHP, Nussenzweig V, 1953. Sobre uma cepa de Trypanosoma cruzi altamente virulenta para o camundongo branco. Folia Clin Biol (Sao Paulo) 20: 191–208. [Google Scholar]
- 44.Sambrook J, Russell DW, 2001. Molecular Cloning: A Laboratory Manual, 3rd edition, Vols 1, 2, and 3. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, 2100. [Google Scholar]
- 45.Nicholas KB, Nicholas HBJ, Deerfield DW, 1997. GeneDOC: Analysis and Visualization of Genetic Variation. EMBNEW News 4: 4–14. [Google Scholar]
- 46.Thompson JD, Gibson TJ, Plewnia KF, 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25: 4876–4882. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 47.Swofford DL, PAUP* , 2002. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4. Sunderland, MA: Sinauer Associates. [Google Scholar]
- 48.Stamatakis A, 2006. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22: 2688–2690. [DOI] [PubMed] [Google Scholar]
- 49.Ronquist F, Huelsenbeck JP, 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574. [DOI] [PubMed] [Google Scholar]
- 50.Huson D, Bryant D, 2006. Application of phylogenetic networks in evolutionary studies. Mol Biol Evol 23: 254–267. [DOI] [PubMed] [Google Scholar]
- 51.Brener Z, Chiari E, 1963. Variações morfológicas observadas em diferentes amostras de Trypanosoma cruzi . Rev Inst Med Trop Sao Paulo 5: 220–224. [PubMed] [Google Scholar]
- 52.Muelas-Serrano S, Le-Senne A, Fernandez-Portillo C, Nogal JJ, Ochoa C, Gomez-Barrio A, 2002. In vitro and vivo anti-Trypanosoma cruzi activity of a novel nitro-derivative. Mem Inst Oswaldo Cruz 97: 553–557. [DOI] [PubMed] [Google Scholar]
- 53.Jawetz E, Melnick JL, Adelberg EA, 1991. Microbiologia médica. In: O crescimento, a sobrevida e a morte de microorganismos, 18th edition, Vol. 1 Rio de Janeiro, Brazil: Guanabara Koogan. [Google Scholar]
- 54.Burgos JM, et al. 2010. Molecular Identification of Trypanosoma cruzi discrete typing units in end-stage chronic Chagas heart disease and reactivation after heart transplantation. Clin Infect Dis 51: 485–495. [DOI] [PubMed] [Google Scholar]
- 55.Câmara AC, Lages-Silva E, Sampaio GH, D’Ávila DA, Chiari E, da Cunha Galvão LM, 2013. Homogeneity of Trypanosoma cruzi I, II, and III populations and the overlap of wild and domestic transmission cycles by Triatoma brasiliensis in northeastern Brazil. Parasitol Res 112: 1543–1550. [DOI] [PubMed] [Google Scholar]
- 56.Marcili A, et al. 2009. Comparative phylogeography of Trypanosoma cruzi TCIIc: new hosts, association with terrestrial ecotopes, and spatial clustering. Infect Genet Evol 9: 1265–1274. [DOI] [PubMed] [Google Scholar]
- 57.Lima L, Álvarez OEI, Hamilton PB, Neves L, Takata CSA, Campaner M, Attias M, Souza W, Camargo EFP, Teixeira MMG, 2013. Trypanosoma livingstonei: a new species from African bats supports the bat seeding hypothesis for the Trypanosoma cruzi clade. Parasit Vectors 6: 221. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 58.Barreto MP, 1965. Tripanossomos semelhantes ao Trypanosoma cruzi em animais silvestres e sua identificação com o agente etiológico da doença de Chagas. Rev Inst Med Trop Sao Paulo 7: 305–315. [PubMed] [Google Scholar]
- 59.Belda Neto FM, 1973. Estudos Sobre a Existência de Correlação Entre Os Dados Biométricos e o Grau de Patogenicidade de Amostras Humanas do Trypanosoma cruzi Chagas, 1909. Tese Doutorado; Faculdade de Farmácia e Odontologia de Araraquara. São Paulo, Brasil: Universidade Estadual Júlio de Mesquita Filho, Araraquara. [Google Scholar]
- 60.Andrade SG, 1974. Caracterização de cepas do Trypanosoma cruzi isoladas no Recôncavo Baiano. Rev. Patol. Trop 3: 65–121. [Google Scholar]
- 61.Albuquerque S, 2001. Considerações Relativas ao Comportamento Biológico de Amostras de Uma Cepa de Trypanosoma cruzi, Obtidas Por Centrifugação Diferencial. Tese Livre-Docente; Faculdade de Ciências Farmacêuticas de Ribeirão Preto. São Paulo, Brasil: Universidade de São Paulo, Ribeirão Preto. [Google Scholar]
- 62.Yoshida N, 2006. Molecular basis of mammalian cell invasion by Trypanosoma cruzi . An Acad Bras Cienc 78: 87–111. [DOI] [PubMed] [Google Scholar]
- 63.Nogueira N, Cohn Z, 1976. Trypanosoma cruzi: mechanism of entry and intracellular fate in mammalian cells. J Exp Med 1: 1402–1420. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 64.Meirelles MN, Araújo-Jorge TC, Souza W, 1982. Interaction of Trypanosoma cruzi with macrophages in vitro: dissociation of the attachment and internalization phases by low temperature and cytochalasin B. Z Parasitenkd 68: 7–14. [DOI] [PubMed] [Google Scholar]
- 65.Campos Y, Briceño L, Reina K, Figarella K, Pérez JL, Mosca W, 2009. Serological diagnosis of Chagas disease: evaluation and characterisation of a low cost antigen with high sensitivity and specificity. Mem Inst Oswaldo Cruz 104: 914–917. [DOI] [PubMed] [Google Scholar]
- 66.Bouhdidi A, Truyens C, Rivera MT, Bazin H, Carlier Y, 1994. Trypanosoma cruzi infection in mice induces a polyisotypic hypergammaglobulinaemia and parasite-specific response involving high IgG2a concentrations and highly avid IgG1 antibodies. Parasite Immunol 16: 69–76. [DOI] [PubMed] [Google Scholar]
- 67.Takehara HA, Perini A, da Silva MH, Mota I, 1981. Trypanosoma cruzi: role of different antibody classes in protection against infection in the mouse. Exp Parasitol 52: 137–146. [DOI] [PubMed] [Google Scholar]
- 68.Brodskyn CI, Silva AM, Takehara HA, Mota I, 1989. IgG subclasses responsible for immune clearance in mice infected with Trypanosoma cruzi . Immunol Cell Biol 67: 343–348. [DOI] [PubMed] [Google Scholar]
- 69.Jorge TCA, Castro SL, (orgs) 2000. Doença de chagas: manual para experimentação animal [online]. Antropologia e Saúde Collection. Rio de Janeiro, Brazil: Editora FIOCRUZ, 368 Available at: http://books.scielo.org. Accessed February 21, 2017. [Google Scholar]