Trypanosoma cruzi isolated from a triatomine found in one of the biggest metropolitan areas of Latin America

Introduction: To characterize Trypanosoma cruzi (TcI) isolated from a Panstrongylus megistus specimen found in one of the biggest metropolitan areas of Latin America, the relationship between the TcI group of T. cruzi and the transmission cycle in the urban environment was studied. Methods: The T. cruzi strain, Pm, was isolated in a culture medium from the evolutionary forms present in the hindgut of a live male specimen of P. megistus found in the Jabaquara subway in São Paulo City. The sample from the triatomine showed trypomastigote forms of Trypanosomatidae, which were inoculated in the peritoneum of Balb/c mice. The sample was then inoculated in Liver Infusion Tryptose medium and J774 cells for the molecular identification and characterization of the parasite. The Pm strain of T. cruzi was identified by isolation in axenic culture medium, and based on the morphology, cell infection, growth kinetics, and molecular characterization. Results: After isolation, the protozoan was identified as T. cruzi. No parasites were detected in the peripheral blood of the animal, which can be a characteristic inherent to the strain of T. cruzi that was isolated. Cell invasion assays were performed in triplicate in the J774 cell line to confirm the invasive ability of the Pm strain and revealed amastigote forms of the parasite within macrophages. Conclusions: Our biological and molecular characterizations helped understand parasite-host interactions and their evolutionary history in context of the associations between vectors, ecotopes, hosts, and groups of the parasite.

Triatomine species are vectors of Trypanosoma cruzi, the etiological agent of Chagas disease, and so far, 148 Triatomine species have been described (1) (2) .Since the description of new human trypanosomiasis in 1909 by Chagas (3) , who showed that T. cruzi infection in Panstrongylus megistus, the protozoan has been considered epidemiologically important owing to its wide distribution; the Atlantic Forest is considered its center of dispersion (4) .According to Galvão et al. (5) , Panstrongylus megistus species have been found in the States of Alagoas, Bahia, Ceará, Espírito Santo, Goiás, Maranhão, Mato Grosso, Mato Grosso do Sul, Minas Gerais, Pará, Paraíba, Paraná, Pernambuco, Piauí, Rio de Janeiro, Rio Grande do Norte, Rio Grande do Sul, Santa Catarina, São Paulo, and Sergipe.
Epidemiological surveillance in the State of São Paulo focuses on control actions based on the infestation levels registered in systematic searches in infested areas and residences of people who have reported the presence of triatomines (6) .Vector control in São Paulo has been successful since 1990, particularly for Triatoma infestans (7) .However, Leite et al. (8) have identified 109 specimens of T. infestans in a peridomestic area in the City of Paulínia, which indicates the possibility of reintroduction of species such as T. infestans, Triatoma sordida, and P. megistus, which showed T. cruzi infection rates of 2.7%, 0.6%, and 6.4%, respectively, from 1968 to 2007 (7) .The presence of T. sordida and P. megistus in the State of São Paulo results from their ability to invade and colonize human habitations 4) .After the control of T. infestans infection, P. megistus stands out in epidemiological terms as an entomological indicator of home infestation, and studies on the density (5) (9) showed that, in the State of São Paulo, about 1% of the T. sordida and 9% of the P. megistus species tested positive for T. cruzi infection.According to Silva et al. (10) , Panstrongylus megistus is an important vector for T. cruzi infection in the state, with high rates of natural transmission.In addition, given that P. megistus is native to the Atlantic Forest where Forattini (11) and De Paula et al. (12) examined specimens with high (8.3%)rates of T. cruzi infection, surveillance on this species cannot be ignored.

METHODS
According to Barreto 13 , most triatomines retain their primitive wild habits.Although they are often found to be naturally infected, triatomines are rarely or never exposed to humans, and, therefore, play a prominent role in the sylvatic cycle of T. cruzi, but have a small epidemiological significance to humans.On the other hand, domiciled or synanthropic species of triatomines live in close contact with humans, making them responsible for the domestic cycle of T. cruzi infection; such triatomines are therefore considered important from an epidemiological point of view.Between these two extreme scenarios, there is a series of gradual transitions, as observed in P. megistus.
Factors such as species diversity, environmental changes, and residence invasion allow vector-mediated transmission of Chagas disease (14) .For example, in their study in Columbia, Cordovez et al. (15) showed that climatic changes can interfere with the route of vector-mediated transmission of the disease.The domiciliation of triatomines is essentially a dynamic phenomenon and can be explained by the relationship between some species and their natural and artificial ecotopes (13) .The domiciliation of P. megistus, which is in line with the adaptability of this species could be the reason for the high rate of T. cruzi infection (5) .Panstrongylus megistus and small mammals have been found to cohabitate since the beginning of the last century, and the first occurrence of P. megistus in natural environments was reported in the State of São Paulo (16) .The presence of P. megistus in natural environments represents possible sources of infestation and re-infestation and contributes to the maintenance of the life cycle of T. cruzi (17) .Panstrongylus megistus was found in the City of São Paulo, which has been known to have a higher rate of infection in triatomines associated with opossums and rodents (18) .Although P. megistus can adapt to human habitation and its dependencies, it can also be found in different natural ecotopes participating in the sylvatic cycle of T. cruzi owing to its involvement in the transport of the parasite to artificial ecotopes and in the initial infestation or reinfestation of these ecotypes (19) .Forattini et al. (11) found P. megistus foci in hollow trees inhabited by bats, rodents, birds and, mainly, opossums.In artificial ecotopes, P. megistus feeds on humans, domestic animals (especially dogs and cats), commensal mammals (rats), synanthropic mammals (bats and possums), and birds (20) (21) , which results in high infection rates and is also an important link in the domestic transmission cycle of T. cruzi (13) .
In this study, we report T. cruzi infection in a P. megistus specimen found in the Jabaquara subway yard in the City of São Paulo.Our finding raises the possibility that this P. megistus specimen came from the Fontes do Ipiranga State Park (23°38'55.40"Sto 46°37'18.21"O),which is 2km away from the place where the triatomine was found.Our results reemphasize the need for constant surveillance on T. cruzi infection in P. megistus.

Isolation
The Trypanosoma cruzi strain, Pm, was isolated in culture medium from the evolutionary forms present in the hindgut of a live male specimen of P. megistus.The triatomine was found in the Jabaquara subway, more specifically on the mezzanine hallway, Block H "23°38'41.94 S to 46°38'29.46O"(Office for Logistics and Storage of Products of the Subway, Responsible -Rosane Correa de Oliveira), which is an urban area in the City of São Paulo.The biological material obtained by abdominal compression was diluted in 0.9% saline and observed between the slide and the cover slip under an optical microscope (22) .The sample showed trypomastigote forms of Trypanosomatidae, which were inoculated (0.3mL) in the peritoneum of 23 to 35-day-old Balb/c mice and in Liver Infusion Tryptose (LIT) axenic culture medium for the molecular identification and characterization of the parasite.After isolation, the parasite was identified as T. cruzi, whose strain was preserved by repeated subculturing in LIT culture medium.

Growth kinetics
The growth dynamics of the epimastigote forms of the Pm strain of T. cruzi were studied by inoculating 5 × 10 6 parasites/mL in 5mL of LIT medium (23) .Triple counting was performed over 10 days on a Neubauer chamber under an optical microscope.

Cell invasion
The assay for parasite invasion in the J774 cells was performed in triplicate (24) .Cells were cultivated in Roswell Park Memorial Institute (RPMI) medium 1640 supplemented with 20% fetal bovine serum (FBS), streptomycin (100µg/mL), and penicillin (100U/mL) in a humid incubator (5% CO 2 ).For the invasion assay, 1 × 10 6 parasites were seeded onto a 24-well plate.After 72 hours, the plate was stained with Giemsa.The invasion of the J774 cell line by the Pm strain of T. cruzi was observed based on the presence of amastigote forms of the parasite within macrophages.

Molecular characterization of the Pm strain of Trypanosoma cruzi and evolutionary placement
After deoxyribonucleic acid (DNA) extraction, polymerase chain reaction was performed using 100ng genomic DNA; 100ng of each primer; 200mM of each deoxynucleotide triphosphate (dNTP); 5µL buffer solution (200mM Tris-HCl, pH 8.4, 500mM KCl, and 1.5mM MgCl2); 2.5U Taq DNA polymerase and deionized with bidistilled water (qsp 50µL).The amplification cycle and the temperatures were defined in accordance with the primers used.DNA fragments amplified using polymerase chain reaction (PCR) were subjected to agarose gel electrophoresis (1.5%) and then to sequencing reactions using the Big Dye Terminator kit (Perkin Elmer) in an automated sequencer (25) .The nucleotide sequences obtained from GenBank (http://www.ncbi.nlm.nih.gov/) were used as reference.The San Agustin strain of Trypanosoma rangeli was included as an external group.

Ethical considerations
This study was carried out in accordance with the recommendations of the Ethical Committee for Animal Experimentation.The protocol was approved by the Ethics Committee on Animal Use [Comissão de Ética no Uso de Animais (CEUA)] of the

RESULTS DISCUSSION
Universidade Estadual Júlio de Mesquita Filho (UNESP) at Araraquara, SP (Permit Number: 13/2012).Three Balb/c mice were studied in a controlled light and temperature environment.After the study, the mice were euthanized by CO 2 and the appropriate disposal of the animals was assured by the Faculdade de Ciências Farmacêuticas de Araraquara.

Isolation of the Pm strain of Trypanosoma cruzi
The Pm strain of T. cruzi was identified by isolation in axenic culture medium, as well as based on its morphology (Figures 1A  and B), cell infection (Figure 1C), growth kinetics (Figure 2), and molecular characterization (Figure 3).Andrade (26) classified T. cruzi strains according to biological parameters such as the morphology of trypomastigotes and the mortality rate of the infected animals.The Pm strain of T. cruzi was inoculated in the peritoneum of Balb/c mice.No parasites were detected in the peripheral blood of the animals, which can be a characteristic inherent to the TcI group of T. cruzi.To evaluate the invasive ability of the Pm strain, cell invasion assays were performed in triplicate in J774 cells, which revealed amastigote forms of the parasite within macrophages.

Growth kinetics
A growth kinetics study of the Pm strain of T. cruzi was conducted in triplicate, and the data are shown in Figure 2. The of this study indicate a growth similar that of to the Bolivia strain of T. cruzi, which belongs to the TcI group, and different from that of the Y strain of T. cruzi, which has a higher ability to multiply and belongs to the TcII group.

Molecular characterization and evolutionary placement
To complete the biological characterization and to identify the group that the Pm strain of T. cruzi isolated from the P. megistus specimen in this study belongs to, the small subunit ribosomal ribonucleic acid (SSU rRNA) gene was amplified, and its initial placement was studied.The amplification of the V7V8 region of the SSU rRNA gene resulted in products of around 900 bp (25) (Figure 3).By using a dendrogram built after alignment of the SSU rRNA gene sequences, it was found that the Pm strain belongs to the discrete typing unit (DTU) TcI of T. cruzi.

A B C
Chagas disease has become a zoonosis since triatomines were brought from their wild ecotopes to human environments (12) .The presence of residual foci of T. infestans in some Brazilian states and the large number of native species of potential vectors of Chagas disease (Panstrongylus megistus, Triatoma brasiliensis, Triatoma pseudomaculata and T. sordida) found in homes are risk factors for the transmission of the disease (4) (27) .
This work reports the finding of a live male specimen of P. megistus containing potential reservoirs of T. cruzi in November 2011 in the Jabaquara subway yard located in the urban area of the City of São Paulo (Figure 4) next to the State Park (Table 1).In  1), suggesting different centers of dispersion for the species.The molecular characterization (Figure 3) performed in this study showed that the Pm strain of T. cruzi belongs to group I-TcI.Therefore, we then focused on the TcI group of T. cruzi and its transmission cycle in the urban environment.Previously, Alvarado-Otegui et al. (28) identified the TcI and TcIII groups of T. cruzi in 22.7% of the studied mammals using xenodiagnosis and polymerase chain reaction in a rural area in Argentina, confirming the presence of TcI in natural wild environments.The invasive ability of T. was observed in the J774 cell line with amastigote forms of the parasite found within macrophages (Figure 1C) (23) .Complementing the results of the biological analysis, the molecular characterization of the Pm strain showed that it belongs to DTU-TcI (Figure 3), which led us to hypothesize on the origin of the sample, given that the TcI group is characteristic of wild environments.Understanding the T. cruzi-DTU groups can lead to the elucidation of the epidemiological implications and to the complete understanding of the biology of the parasite (29) .
The decrease in the frequency of collection of specimens in the state of São Paulo is probably due to the captures carried out during 1984-1988 and 1992-1993 and the control actions taken along the way (30) .Martins et al. (27) performed searches in two experimental captive sites installed in Marília, São Paulo, for a year and four months, but without successful colonization of wild triatomines.However, the capture of species infected by T. cruzi in the urban environment is a reason for concern,

FIGURE 1 -
FIGURE 1 -Identification of a Trypanosoma cruzi strain isolated from Panstrongylus megistus.A, B: Trypomastigote forms of the Pm strain of Trypanosoma cruzi observed in axenic culture medium and in the intestinal content of the Panstrongylus megistus specimen.C: Arrows show the kinetoplast of the parasite.Amastigote forms of Trypanosoma cruzi in the J774 cell line after 72 hours of infection.Bars represent 10µm.

RibeiroFIGURE 2 -
FIGURE 2 -Growth kinetics of the Pm strain of Trypanosoma cruzi in Liver Infusion Tryptose culture medium.Pm: strain of T. cruzi TcI; Bolívia: strain of T. cruzi TcI; Y: strain of T. cruzi TcII.