Triatomines in dwellings and outbuildings in an endemic area of Chagas disease in northeastern Brazil

Introduction: The present study identified the triatomines collected in intra and peri-domestic environments, observed the occurrence of Trypanosoma cruzi infection in triatomines and correlated this information with housing conditions and the fauna associated with the rural areas of the City of Itabaianinha, located in the State of Sergipe, Brazil. Methods: Quarterly visits were conducted between March 2009 and March 2010, and the homes to be visited for the active search of insects were determined by random selection. In each housing unit, the insects were collected by a manual search with a metal clip and flashlight to inspect openings and cavities, with a collection time of one hour/home/individual. The Pirisa® dislodge chemical was used to force the insects to leave their ecotopes. Analysis of the intestinal contents of triatomines was performed in the laboratory to establish the presence of Trypanosomatidae. Results: Of the 103 dwellings surveyed, 17.5% were infested with Panstrongylus megistus. The village of Mutuca exhibited the highest infestation rate (38.1%). All the villages with relevant infestation rates were situated in the northern area of the city. The highest percentage of vector infection was found in the village of Água Boa (56.5%). The rural dwellings were found to be primarily brick or wooden house with or without roughcast or plastered walls, and the outbuilding most frequently associated with triatomines was the chicken run. Conclusions: These results emphasise the need for broader vector control and surveillance and for educational campaigns in the context of the Chagas Disease Control Program.

In Brazil, the vector transmission of human Chagas disease is present in all states of the northeastern region 1 , and its occurrence depends primarily on the distribution of domestic vectors 2 .Vector transmission has always been considered to be the most important mode of transmission in Brazil, corresponding to 80% of cases of parasitic diseases, and is associated with the ability of these triatomines to invade and adapt to homes and outbuildings, especially those of low socioeconomic conditions 3,4 .Carlos Chagas correlated this trypanosomiasis transmission with the presence of triatomines in intra-domestic areas 5 .
Hunger is one of the main factors involved in the spread of these insects to other ecotopes.Consequently, in addition to bird nests, tree hollows, crevices between rocks, fallen trunks, exposed roots, loose tree bark and palm tree and bromeliad leaves, triatomines can also be found in peri-and intra-domestic environments, especially in poorly constructed housing units.Triatomines undergo a domiciliation process when they are forced out of their wild environments due to habitat destruction around dwellings, after which they find shelter in rural constructions [6][7][8] .
In addition, the presence of domestic animals and peri-domestic constructions used for animal maintenance and breeding greatly influences the presence and transmission of parasites because they provide a permanent food source and promote contact between vectors and humans 7,[9][10][11][12] .
The northeastern region of Brazil is still predominantly rural and has the highest indices of low-quality human dwellings that are adequate to shelter triatomines.In 1996, the Programa de Controle da Doença de Chagas (PCDCh -Chagas Disease Control Program) of the Fundação Nacional de Saúde/Ministério da Saúde (FNS/MS -Brazilian Health Ministry National Health Foundation) collected 290576 triatomines in Brazil, the majority (69.2%) collected in the northeastern region of the country 13 .
In recent years, northeastern Brazil has been affected by several actions impacting the environment, including native vegetation deforestation, mangrove destruction, desertification and the concentration of coastal estates that, together with certain economic activities such as cattle raising, citrus fruit cultivation, subsistence agriculture and livestock raising, destroy the environment in areas close to where low-income populations live 14 .
The State of Sergipe has been on constant alert since the first reports of Chagas disease in the northeastern region in 1913, followed by serological surveys conducted in schoolchildren aged between 7 and 14 years in the 1970s and 1990s as well as the Health Ministry's publication of the Consenso Brasileiro em Doença de Chagas 15 , which aimed to guide control activities and classified the state as a highrisk area.Anthropic destruction of the environment, such as that associated with citrus fruit plantations and red ceramic exploration, have occurred in Sergipe, especially in the City of Itabaianinha.The lack of areas dedicated to the production of vegetable species used to make fuel in Sergipe has become a serious problem for red ceramic producers due to the destruction of the native vegetation, especially the caatinga (Brazilian xeric shrubland and thorn forest), causing a relevant impact on the environment.
In the last ten years, the City of Itabaianinha alone included more than 50% of the reported human cases of Chagas disease, and several vector species were found in this location, including Panstrongylus megistus, Panstrongylus lutzi, Triatoma tibiamaculata, Triatoma melanocephala and Triatoma pseudomaculata 16 .
The aim of the present study was to identify the triatomines collected in the intra-and peri-domestic environments and to observe the occurrence of Trypanosoma cruzi infection in these insects' digestive tract, correlating this information with the housing conditions and fauna associated with the rural area of Itabaianinha, State of Sergipe, Brazil.

METHODS
The City of Itabaianinha covers an area of 480.4km 2 and is situated in the south-central area of Sergipe (11°16'26'S and 37°47'24''W), 120km from the capital City of Aracaju.The local population is estimated to be 37,431 inhabitants 17 .The city has a typical dry and sub-humid megathermal climate, with an annual mean temperature of 24.2 o C, annual mean rainfall of 976.9 mm and a rainy season that occurs between March and August.The city's rural area is divided into 72 villages and includes 55.7% of the population.Its economy is based on citrus fruit plantations, ceramic production and small and large animal husbandry.The area's natural vegetation is composed of grasslands, shrublands, capoeira (secondary vegetation composed of grasslands and scattered shrubs), caatinga and traces of vegetation 18 .
The present study was conducted with quarterly visits between March 2009 and March 2010, and the housing units sampled were randomly selected from the rural housing units (intra-and peridomestic environments) situated in the villages that were registered with the City of Itabaiaininha's Chagas Disease Control Program.
First, villages with the highest prevalence of Chagas' disease recorded in the control program were selected.Then, a random selection was held to determine which homes would be visited for the active search of insects, thus establishing an average seek time number of 24 housing units per field trip, for a total of 103 housing units analysed.
In each housing unit, insects were collected using the selective method, i.e., manual search with metallic tweezers and a flashlight to inspect openings and dark places, with a collection time of one hour/housing unit/individual.Pirisa® chemical dislodger was used to force insects to leave their resting places.In the housing units where triatomines were found, a transect extending 50m in all cardinal directions (north, south, east and west) was established, forming a circle around the house.In the area of the transect, a search based on the selective method in natural environments was conducted (burrows, openings in the ground, bird nests, waste materials and tree trunks, among others).
The triatomines collected were identified using the key of Lent and Wygdzinsky 19 .Insects were dissected, and their last abdominal segments were compressed to obtain faecal material, which was observed under light microscopy (at 400 x magnification) to detect Trypanosomatidae 15,20 .Housing units where the searches and triatomine collections were performed were geo-referenced using a GPS (Global Positioning System) receiver (Etrex-Garmin®).
The housing units visited were classified according to the type of construction: Type 1, brick house with roughcast walls, sub-floor and floor, ceiling and clay tile or fibre cement roof; Type 2, brick or wooden house with or without roughcast or plastered walls, sub-floor and floor, without a ceiling or clay tile or fibre cement roof; Type 3, wooden, straw or thatch house without a roof or ceiling, or with a roof of zinc, straw or other material 21 .In addition, outbuildings, such as deposits, waste deposits, chicken runs, pigsties and corrals, and the animals associated with outbuildings were observed.
The following entomological indicators were used to analyse the data, as recommended by the Brazilian Health Ministry 15 : Pearson's correlation coefficient was obtained to associate the number of triatomines infected with Trypanosomatidae with the variety of animal raised.All analyses were performed with a significance level of 5%.In the present study, 103 rural housing units were visited between March 2009 and March 2010, including primary buildings and outbuildings, in the villages of Garangau, Maurício, Carretéis, Mata Verde, Mutuca, Fundão, Antas, Água Boa and Piabas.The dispersion index (DI) was 66.7% among the nine villages, with triatomines found in both the domestic and peri-domestic areas.Of the 103 housing units analysed, 18 were infested with triatomines (HII = 17.5%), and insect colonies were observed in both the peri-and intra-domestic environments.The village of Mutuca exhibited the highest HII (38.1%), followed by the villages of Fundão and Antas (25%) (Table 1).The local profile of the rural housing units revealed a very heterogeneous and precarious pattern, with the majority of houses as type 2 (56.3%), followed by 27.2% as type 3 and 16.5% as type 1.The mean distance of properties to Itabaianinha's urban centre was approximately 11.55km (±2.26; maximum=13.81km and minimum=6.54km),and the mean distance between properties was approximately 4.08km (±2.15; maximum=9.63kmand minimum=0.66km).
In the peri-domestic areas studied in the City of Itabaianinha, there were corrals, waste deposits and chicken runs, many of which had improvised structures.
Of the 131 triatomine specimens analysed, 41 were positive for Trypanosomatidae, which corresponds to a natural infection index (NII) of 31.3% (Table 2).All six villages where triatomines were found exhibited natural infection indices; the lowest vector infection percentage was found in Antas (2.9%), while the highest infection percentage was in Água Boa, where 56.5% of insects studied had Trypanosomatidae in their intestinal contents.
All triatomine specimens collected belonged to the Panstrongylus megistus species.
With regards to the developmental stage of insects collected, 56.4% were in the adult stage, whereas 43.6% were in nymph stages.Associating the developmental stage with the natural infection indices, adult triatomines exhibited the highest rates of infection (63%), followed by third-stage nymphs (30.8%) and fifth-stage nymphs (23.8%) (Table 3).In addition, there were traces of triatomines, such as exuviae, exocoria, faeces and eggs.With regards to sex, the number of females collected was higher than that of males at a ratio of 1.25:1 (F:M) except in September, when the number of males collected was higher.
The colonization index of triatomine P. megistus collected in the region appears to be low due to not having nymphs were found inside the home.However, we emphasize the presence of these developmental stages in household near or adjacent to human dwellings, some of which were found positive (Table 3).
During collections, many housing units included breeding domestic animals and other types of animals that can be a food source for triatomines or act as reservoir hosts.Among these animals, chickens (33.1%), dogs (21.3%), pigs (12.4%) and cats (10.7%) stand out.
In addition, there was a significant correlation between the number of triatomines collected, the number of positive triatomines and the variety of animal present on the property (p< 0.0001; r= 0.605 and r= 0.534, respectively; Figure 1).

DISCUSSION
All villages that had a household infestation index were located in northern rural Itabaianinha in an area bordering the Cities of Riachão do Dantas and Tobias Barreto, both of which are in southern central Sergipe.This region includes citrus plantations that cover an area of 71.31km 2 , which corresponds to 16.2% of the total city area 18 .The changes in the natural landscapes made by agricultural systems intensify the triatomine domiciliation trend, especially where herbicide and pesticide use is frequent 22 .
Houses were situated in the middle of pastures and orange plantations and around small secondary forest fragments.The majority of human dwellings surveyed had a highly heterogeneous structure.Sometimes, these housing units were made of bricks; at other times, they were made of wood, with or without plastered or roughcast walls.The construction of the housing units contributed further to the maintenance of the parasitic infection's domestic cycle and reflects the low socioeconomic conditions of the area studied.The area in question has a human development index (HDI) of 0.590, which is lower than the mean value of 0.682 found in the State of Sergipe 17 .
In addition to poor housing quality, rural Itabaianinha residents frequently hang objects such as paintings on the walls, thus providing insects with shelter 9,11 .A study conducted by Villela et al. 23 investigated what could be done to prevent triatomines from entering houses in rural Bambuí, Minas Gerais (Brazil), a city that has traditionally controlled Chagas disease for more than 50 years.Both adults and children associated anti-triatomine actions with the idea of cleanliness and hygiene, especially by avoiding disorganisation and accumulated objects at home.This reveals a different degree of knowledge of Chagas disease in an area that has been systematically influenced by the Chagas Disease Control Program.
High adult triatomine densities in areas with well-defined dry seasons, such as the State of Sergipe, facilitate insect dispersion to artificial habitats, such as chicken runs and colonisation, especially between October and December for Panstrongylus megistus 24 .
Pigsties, chicken runs and barns were considered to be the main outbuildings colonised by triatomines in the City of Douradoquara, State of Minas Gerais, according to Diotaiuti et al. 25 .Service 26 argued that the maintenance of corrals around houses provides an adequate habitat for Chagas disease vector populations.Dias 27 and Dias et al. 28 reported that such types of peri-domestic outbuildings are used for storage and protection of equipment and tools and create adequate shelter for many insects, which is especially true of triatomines because of their nocturnal habits.
Since the early works of Carlos Chagas, the epidemiological importance of dogs and cats has been emphasised.This is due primarily to the transmission of Trypanosoma cruzi by Triatoma infestans, which also lives in human dwellings 29 .Thus, dogs and cats play an important role in the maintenance of the disease, in addition to other domestic and wild mammals that frequent the areas surrounding homes 3,4,12 .In the case of cats, although infection rates are low, their association with the domestic cycle is closely associated with their wandering habits 30 .Pig and bird breeding was also significant in rural areas and was maintained almost exclusively in the peri-domestic environment.According to Barreto 29 , natural infection of pigs had already been detected in 1940, but their role as domestic reservoir hosts needs to be further studied.Domestic birds represent an important food source, maintaining triatomine colonies 10,30 .Rossell-Reyes 31 suggested that chickens are easily detected due to their high body temperature and high surface/volume ratio.In a rural area of the City of Santiago del Estero, Argentina, Wisnivesky-Colli et al. 30 established that the feeding behaviour of T. infestans populations varied depending on the time of the year.These insects fed predominantly on birds exclusively in the warmer months, a fact that could be attributed to egg incubation occurring in the intra-domestic environment.
The results confirm the occurrence of Panstrongylus megistus in rural Itabaianinha, an area where hills predominate and that is located 225 meters above sea level.Infestation (17.5%) and natural infection indices (31.3%), in addition to the presence of such vectors in both peri-and intra-domestic areas, reveal their ability to explore the human environment and their potential to transmit Trypanosoma cruzi in the region studied.
Dispersion index (DI) = number of locations with triatomines x 100 number of locations surveyed Household infestation index (HII)= number of positive housing units x 100 number of housing units surveyed Natural infection index (NII)= number of triatomines infected x 100 number of triatomines collected Household triatomine density (HTD) = number of triatomine specimens collected x 100 number of housing units surveyed

FIGURE 1 -
FIGURE 1 -Pearson correlation coefficient graphs between the varieties of animals present on the properties and the number of triatomines collected (A) and the number of positive triatomines (B).

TABLE 4 -Household infestation index of peri-domestic outbuildings and natural infestation index of triatomines collected in the outbuildings in the villages of Itabaianinha, State of Sergipe, Brazil between March 2009 and March 2010.
HII: household infestation index; IIN: natural infestation index of triatomines.