Trypanosoma cruzi strains from triatomine collected in Bahia and Rio Grande do Sul, Brazil.

OBJECTIVE: Collection of triatomines in domestic, peridomestic and sylvatic environments in states of Bahia and Rio Grande do Sul, Northeastern and Southern Brazil respectively, and isolation of Trypanosoma cruzi strains.
METHODS: First, the captured triatomines were identified using insect identification keys, then their intestinal content was examined by abdominal compression, and the samples containing trypanosomatid forms were inoculated in LIT medium and Swiss mice.
RESULTS: Six triatomine species were collected in cities in Bahia, namely Panstrongylus geniculatus (01), Triatoma melanocephala (11), T. lenti (94), T. pseudomaculata (02), T. sherlocki (26) and T. sordida (460), and two in cities in Rio Grande do Sul, namely T. circummaculata (11) and T. rubrovaria (115). Out of the specimens examined, T. cruzi was isolated from 28 triatomine divided into four different species: T. melanocephala (one), T. lenti (one), T. rubrovaria (16) and T. sordida (10). Their index of natural infection by T. cruzi was 6.4%.
CONCLUSIONS: The isolation of T. cruzi strains from triatomines found in domestic and peridomestic areas shows the potential risk of transmission of Chagas disease in the studied cities. The maintenance of those T. cruzi strains in laboratory is intended to promote studies that facilitate the understanding of the parasite-vector-host relationship.

INTRODUCTION

Chagas disease is estimated to affect around 10 million people worldwide and 25 million people in Latin America are at risk of infection.[7]

In relation to the epidemiology of Chagas disease in Brazil, in 1996 the Chagas Disease Control Program, of the National Foundation for Health/Brazilian Ministry of Health (FNS/MS), collected 290,576 triatomines, of which 201,156 (69.2%) were captured in the Northeast region, where more attention and care are required in view of the presence of sylvatic species that are potentially invasive and difficult to control.[8]

In 1975, the Superintendency for Public Health Campaigns (SUCAM) implemented the Chagas Disease Control Program in the state of Rio Grande do Sul, Southern Brazil. This program was responsible for methodological standardization, supply regulation and rationalization of the activities that provided a basis for vector control.[4] Almeida et al[1] (2009) reported the presence of Triatoma rubrovaria naturally infected by T. cruzi in domestic and peridomestic areas of Rio Grande do Sul, where Rosa et al[18] (2000) collected both T. rubrovaria and Triatoma circummaculata also naturally infected by the parasite. Pena et al[16] (2011) isolated T. cruzi strains that came from patients, reservoirs and vectors during an outbreak of acute Chagas disease in the city of Navegantes, state of Santa Catarina, Southern Brazil, which shows the persistence of transmission of the disease in that region.

Silveira & Pinto Dias[20] (2011) reported the control activities exercised on a regular basis in Brazil, using systematic methodology and epidemiological, entomological and serological surveys. These actions led to depletion of the main vector species, Triatoma infestans, allochthonous and domestic only, and control of household colonization species that are important in transmission. Transmission is now residual in native species such as Triatoma brasiliensis and Triatoma pseudomaculata. There is a risk of domestication of species, previously considered of wild behavior, such as Panstrongylus lutzi and T. rubrovaria, beyond the possibility of human infections, directly related to the zoonotic transmission cycle. Therefore, it is still essential to maintain strict entomological surveillance.[20]

The ecological history of the distribution of the several strains of T. cruzi is marked by important changes starting from the migration of mammals in the Americas, habitat expansion, and destruction and urbanization of the environment by humans.[22]

Collecting species, studying geographic distribution and infection of triatomines by T. cruzi can contribute to the reporting of bugs and surveillance measures.

This study aimed to isolate Trypanosoma cruzi strains from triatomines in domestic, peridomestic and sylvatic environments in states of Bahia and Rio Grande do Sul, Brazil.

METHODS

In Rio Grande do Sul, triatomines were collected in sylvatic environments in fourteen sites in six cities (Figure) in the period between 2002 and 2003. In the state of Bahia, triatomines were captured in sylvatic, peridomestic and domestic areas in nine sites of five cities in 2003, 2006, 2008 and 2009 (Table 1). The Bahia and Rio Grande do Sul State Departments of Health cooperated in the collection activities. The cities were chosen according to the report against triatomine by the health departments of Bahia and Rio Grande do Sul.

Figure

Cities in Bahia and Rio Grande do Sul where triatomines were collected, 2002-2009.

Table 1

Locality, collected species, year and environment of collection. Brazil, 2002-2009.

State	Locality	Triatominae	Year	Ecotope
Rio Grande do Sul	Sao Sepé	T. rubrovaria	2002	S
	Caçapava do Sul	T. rubrovaria/T. circummaculata	2002	S
	Lavras	T. rubrovaria/T. circummaculata	2002	S
	Alegrete	T. rubrovaria	2002	S
	Quaraí	T. rubrovaria	2002	S
	Sta. Livramento	T. rubrovaria	2003	S
Bahia	Santo Inácio	T. sordida/T. sherlocki	2003	P/S
	Santo Inácio	T. sordida/T. sherlocki/ T. pseudomaculata	2006	P/D
	Macaúbas	T. lenti/T. sordida/T. pseudomaculata	2008	P/D
	Macaúbas	T. lenti/T. sordida/T. pseudomaculata/P geniculatus	2009	P/D
	Poções/Jequié/Bom Jesus da Serra	T. melanocephala	2009	P

S: Sylvatic; P: Peridomestic; D: Domestic

Environments of domestic units closed by doors, i.e., houses and annexes, were considered domestic ecotopes. Triatomines were found sheltering in cracks in those environments, under beds or behind objects placed along the walls. The studied peridomestic ecotopes were in a radius of 50 m from the houses, where domestic animals sleep or are bred. In the active researches, triatomines were collected manually with tweezers and placed in individual flasks labeled with information on the place of collection.[12]

The captured triatomines were taken to the Triatominae Insectarium of the Faculdade de Ciências Farmacêuticas of the Universidade Estadual Paulista “Júlio de Mesquista Filho” (FCF/UNESP) at Araraquara, SP, Southeastern Brazil, where they were identified according to morphological characters present in insect identification keys[13] and kept under controlled temperature and humidity (27±2ºC and 60±3% RH). This species are maintained in the colony with controlled conditions in the Laboratory of Parasitology. The capture was conducted by a group containing guides, researchers, and department of health employees and lasted around ten days. The study took place during the day, but there were nocturnal collections that stayed in the environment overnight and used white cloth to attract the insects. Triatominae samples were sought in trees, stones and their attachments.

Natural infection of triatomines by trypanosomatid forms was analyzed in feces obtained by abdominal compression. The natural infection index was obtained from the ratio between the number of infected triatomines and the number of examined triatomines.[12]

In order to isolate and confirm the parasite, samples tested positive for trypanosomatids were diluted with 0.9% saline and examined between slide and coverslip under optical microscopy. Then 0.3 mL was intraperitoneally injected into Swiss mice aged 23 and 35 days, and another 0.3 mL was inoculated in Liver Infusion Tryptose medium. Around 15 days following the inoculation, the peripheral blood of the mice was checked for flagellate forms and the regular growth of the parasite in LIT medium was verified.[17] The 28 strains of T. cruzi are in study by molecular techniques and biological assays such as cellular infection, growth kinetics in LIT medium and parasitemia curve in BALB/c mice. In the present work these data were not included because some trials are ongoing in the laboratory and will be published later.

All the procedures were reported to and approved by the Ethical Committee for Animal Experimentation of the Universidade Estadual Paulista “Júlio de Mesquita Filho” – Araraquara (Protocol 13/2012).

RESULTS

In Rio Grande do Sul, two triatomine species were collected: Triatoma circummaculata, which tested negative for T. cruzi in all 11 samples taken, and Triatoma rubrovaria, with 20 specimens (of the 115) testing positive for T. cruzi (Table 2, Figure).

Table 2

Natural ecotopes investigated in cities in the states of Bahia and Rio Grande do Sul, indicating Triatominae foci, 2002-2009.

Species	Locality	Triatominae examined	Positive	%
T. circummaculata	Caçapava do Sul, RS	8	0	0
T. circummaculata	Lavras, RS	3	0	0
T. sherlocki	Santo Inácio, BA	26	0	0
T. pseudomaculata	Santo Inácio, BA	1	0	0
T. pseudomaculata	Macaúbas, BA	1	0	0
P. geniculatus	Macaúbas, BA	1	0	0
T. sordida	Santo Inácio, BA	1	0	0
T. sordida	Macaúbas, BA	324	0	0
T. rubrovaria	Lavras, RS	36	0	0
T. rubrovaria	Quaraí, RS	53	2	3.7
T. lenti	Macaúbas, BA	90	4	4.4
T. sordida	Macaúbas, BA	100	6	6.1
T. melanocephala	Poções, Jequié, Bom Jesus da Serra, BA	11	1	9.0
T. sordida	Santo Inácio, BA	35	12	34.0
T. rubrovaria	Santana do Livramento, RS	5	3	60.0
T. rubrovaria	Quaraí, RS	21	15	71.0
T. lenti	Macaúbas, BA	4	3	75.0
Total	-	720	46	6.4

In Bahia, six species were collected: Panstrongylus geniculatus, Triatoma melanocephala, Triatoma lenti, Triatoma pseudomaculata, Triatoma sherlocki and Triatoma sordida, summing up 594 captured specimens, 4.4% of them naturally infected (Table 2, Figure).

From the examined specimens (720) a total of 28 T. cruzi strains were isolated: one from the vector T. melanocephala, one from T. lenti, 16 from T. rubrovaria, and 10 from T. sordida with the SIGR3 strain was isolated in 2006 by cat xenodiagnosis in a peridomestic area of Santo Inácio, Bahia.[17] (Table 3).

Table 3

Strains of Trypanosoma cruzi isolated and maintained in laboratory.

Species	Strain	Year of collection
T rubrovaria	QG1	2002
	QG2	2002
	JI	2003
	JIII	2003
	QBI	2003
	QMM1	2008
	QMM2	2008
	QMM3	2008
	QMM4	2008
	QMM5	2008
	QMM6	2008
	QMM7	2008
	QMM9	2008
	QMM10	2008
	QMM11	2008
	QMM12	2008
T. sordida	SI1	2003
	SI2	2003
	SI3	2003
	SI5	2003
	SI7	2003
	SI8	2003
	SI9	2003
	SI11	2003
	SIGR3[a]	2006
	SIGR5[a]	2006
T. lenti	T lenti	2008
T. melanocephala	Tm	2009
Total	28	-

Strains isolated from cats by xenodiagnosis.

Triatoma melanocephala specimens were collected in the cities of Jequié, Poções and Bom Jesus da Serra, BA, and only one specimen provided a T. cruzi strain, named “Tm”.

DISCUSSION

Epidemiological studies on Chagas disease and vector capture have been conducted in both endemic and non-endemic areas.[1,7,8,18] In areas where vector-borne transmission was controlled by measures against the main vector of the disease, namely Triatoma infestans, invasion and colonization by secondary vectors can be observed, and they were sometimes captured inside the houses. Species like T. sordida and P. megistus in Sao Paulo (Southeastern Brazil), T. rubrovaria in Rio Grande do Sul,[2] Southtern Brasil, and T. brasiliensis in Northeastern Brazil[2] are examples of that situation.

Originally, sylvatic specimens of triatomine can adapt to peridomestic and domestic environments occupying the ecological niche of primary species. In this sense, the gathering of the T. sherlocki in sylvatic and domestic environment may indicate that sylvatic populations have invaded and colonized localities, thus the importance of entomological surveys. The presence of triatomines inside houses represents a risk of vector-borne transmission of Chagas disease to humans.[3] In Brazil, there are 52 species, but five of them – Panstrongylus megistus, Triatoma brasiliensis, T. infestans, T. pseudomaculata and T. sordida – have considerable epidemiological importance.[6] Triatoma sordida is one of the most captured species in Brazil and is regarded as mostly peridomiciliary, whereupon it can be found naturally infected by T. cruzi. Therefore, that species has an important role in the maintenance and transmission of Chagas disease.[3]

In order to have a better understanding of the response of that triatomine to the control activities, research was carried out in 12 rural localities in the city of Porteirinha, MG, Southeastern Brazil.[9] A total of 772 specimens of T. sordida were captured, of which 3.6% were infected by T. cruzi. Of the 406 domiciliary units researched, 34.9% were infested. Of the 695 peridomiciliary ecotopes, 27.6% had T. sordida. Triatoma rubrovaria is distributed in a wide area comprehending Argentina, Uruguay and Southern Brazil[13,18] and it lives in holes and cracks in rocky places in fields. It can be captured in domiciles in rural areas next to sylvatic ecotopes, which explains its epidemiological importance.[18] Almeida et al[1] (2000) reported the presence of that species in a domiciliary environment, confirming its ability to invade and colonize houses.[2]

Trypanosoma cruzi has genetically different strains for which more than 100 mammalian species can serve as reservoirs.[7] Geographic distribution in the Americas depends on the interaction of the parasite with the vector, and natural infection can only take place when the trypanosomatid-triatomine system is balanced.[11] So far, there are six groups of parasite – Discrete Typing Units (DTU): TcI, TcII, TcIII, TcIV, TcV and TcVI[14] and biological differences among strains are pointed out by researchers.[5,16,21] Martins et al[15] (2008) performed the molecular and biological characterization of five isolates obtained from T. rubrovaria collected in Rio Grande do Sul wherein 1.6% of the collected specimens were positive for T. cruzi. Rimoldi et al[17] (2012) showed the biological diversity of strains isolated from T. sordida and a domestic cat in Santo Inácio, Bahia.

Freitas et al[10] (2005) collected 921 specimens of T. pseudomaculata in cities in the state of Ceará, Northeastern Brazil. Even though they tested negative for T. cruzi, the presence of that vector in that region is clear. In 1980 Sherlock & Guitton[19] (1980) collected 12 samples of T. lenti in Bahia and all of them were positive for T. cruzi. The fact that the specimens tested positive shows the importance of that vector in the transmission of Chagas disease in the state. Few studies related to T. lenti and T. melanocephala are found in the literature and in this sense this work can contribute.

Chagas disease can be controlled by interruption of the transmission mechanisms, improvement of dwellings and adjacent areas, education, basic sanitation for exposed populations, and treatment. To make control successful, it is essential to completely prevent contact between triatomines and humans in areas surrounding habitations, which could be achieved by the use of residual insecticides, improvement of dwellings, and repair of crevices in walls using plaster, preventing triatomines from having a place to hide.[6]

In view of the 28 strains of T. cruzi collected from species regarded as “secondary”, such as T. sordida and T. rubrovaria, it is necessary to continue entomological surveillance and health education actions in order to minimize the chances of triatomines colonizing domestic and peridomestic areas in cities in the states of Bahia and Rio Grande do Sul. This study continues with biological, morphological and molecular approaches under development.